Synthesis, molecular modeling of N-acyl benzoazetinones and their docking simulation on fungal modeled target

Article abstract of DOI:10.1080/00397911.2017.1328514

A series of stable N-acyl benzoazetinones have been synthesized in moderate to good yields (58–85%) from easily available substrates such as 2-(N-acyl) amino benzoic acids through intramolecular amidation under mild conditions. These geometry-optimized benzoazetinones were docked in the model target of P450, class CYP53A15, a benzoate 4-monooxygenase abundantly found in the genome of ascomycetes and Basidiomycetes classes of pathogenic fungi. Low per residue root-mean-square deviation (RMSD) of modeled structure of the enzyme indicated similar topology as template (4D6Z.pdb). Observed score judges site-specific docking, and the interaction of quantum mechanically optimized benzoazetinone derivatives with the target enzyme. These results suggest that 3i is the best antifungal agent. The specific hydrophobic substituent in the benzoazetinones contributed to the stability of ligand–target complex. Overall, the study provided insight into the specificity of the site-specific interactions, thereby, facilitating the possibility of development of broad-spectrum antifungal agents against opportunistic and infectious fungi.

Full text of DOI:10.1080/00397911.2017.1328514

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Synthesis, Molecular Modeling of N-acyl
Benzoazetinones and their Docking Simulation on
Fungal Modeled Target
Inul Ansary, Arijit Das, Parth Sarthi Sen Gupta & Amal Kumar
Bandyopadhyay
To cite this article: Inul Ansary, Arijit Das, Parth Sarthi Sen Gupta & Amal Kumar Bandyopadhyay
(2017): Synthesis, Molecular Modeling of N-acyl Benzoazetinones and their Docking Simulation on
Fungal Modeled Target, Synthetic Communications, DOI: 10.1080/00397911.2017.1328514
To link to this article: http://dx.doi.org/10.1080/00397911.2017.1328514
View supplementary material
Accepted author version posted online: 17
May 2017.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=lsyc20
Download by: [Mount Sinai Health System Libraries]
Date: 17 May 2017, At: 09:51

Synthesis, Molecular Modeling of N-acyl Benzoazetinones and their Docking Simulation
on Fungal Modeled Target
Inul Ansary1, Arijit Das1, Parth Sarthi Sen Gupta2, Amal Kumar Bandyopadhyay2
1Department of Chemistry, The University of Burdwan, Burdwan, W.B, India,
2Department of Biotechnology, The University of Burdwan, Burdwan, W.B, India
Corresponding to authors: Inul Ansary, Department of Chemistry, The University of
Burdwan, Burdwan 713104, W.B, India; E-mail: iansary@chem.buruniv.ac.in; Amal
Kumar Bandyopadhyay, Department of Biotechnology, The University of Burdwan,
Burdwan 713104, W.B, India; E-mail: akbanerjee@biotech.buruniv.ac.in
Abstract
A series of stable N-acyl benzoazetinones have been synthesized in moderate to good
yields (58-85%) from easily available substrates such as 2-(N-acyl) amino benzoic acids
via intramolecular amidation under mild conditions. These geometry-optimized
benzoazetinones were docked in the model target of P450, class CYP53A15, a benzoate
4-monooxygenase abundantly found in the genome of ascomycetes and basidiomycetes
classes of pathogenic fungi. Low per residue RMSD of modeled structure of the enzyme
indicated similar topology as template (4D6Z.pdb). Observed score judges site-specific
docking, and the interaction of quantum mechanically optimized benzoazetinone
derivatives with the target enzyme. These results suggest that 3i is the best antifungal
1

agent. The specific hydrophobic substituent in the benzoazetinones contributed to the
stability of ligand-target complex. Overall, the study provided insight into the specificity
of the site-specific interactions, thereby, facilitating the possibility of development of
broad-spectrum antifungal agents against opportunistic and infectious fungi.
GRAPHICAL ABSTRACT:
KEYWORDS: 2-(N-acyl) amino benzoic acids; EDC.HCl, HOBt; N-acyl
benzoazetinones; molecular modeling; molecular docking
INTRODUCTION
Benzoazetinones is composed of β-lactam fused with a benzene ring. In recent years, β-
lactam and its derivatives have been studied for their role in antibacterial activity[1],
synthesis of bioactive compounds[2], enzymatic inhibitions,[3] therapeutic applications,[4, 5]
and antifungal activities.[6] Drug resistance[7] led to the discovery of new drugs against
invasive fungal infections[8,9] caused by Candida,[10] Aspergillus[11] and Colchilobus,[12]
especially in immunocompromised patients.[13] In this context, derivatives of amines,
amides, and azoles were investigated for their antifungal activities against the target
cytochrome-P450, CYP families.[14] The latter are highly diverse in the genome of
2

fungi.[15, 16] It is more so for the class CYP53A15 in the genome of pathogenic fungi and
therefore, has been explored as a target for a wide variety of phenolic compounds.[17] As
a result, intensive investigations have been focused on β-lactam and its derivatives.[18-20]
Several methods of synthesis of β-lactams have been reported in the recent past.[21, 22]
Since the synthesis of benzoazetinones by photolysis[23] and thermolysis[24, 25] were
reportedly unstable, either tertiary butyl[26] or pivaloyl[27, 28] was incorporated as an N-
substituted group to improve its stability. However, the low yield of benzoazetinones[28,
29] and the presence of a by-product were also reported[28]. Recently, Cioffi CL, et al.
isolated stable benzoazetonone as an intermediate starting from 2-(4-(2-
(trifluoromethyl)phenyl)piperidine-1-carboxamido)benzoic acid during the synthesis of
N-(2-(methylsulfonylcarbamoyl)phenyl)-4-(2-(trifluoromethyl)phenyl)piperidine-1-
carboxamide.[30]
Here, we report the synthesis of benzoazetinones from easily available substrates 2-(N-
acyl) amino benzoic acids via intramolecular amidation under mild conditions. Further,
we carried out extensive investigations on optimization, three-dimensional structure,
docking and site-specific interactions of these benzoazetinones. The study also highlights
the potentiality of CYP53A15 as a target for these compounds, thereby, allowing in
gaining insight into the interactions at its active site. Since the crystal structure of
CYP53A15 from Cochliobolus lunatus is unavailable, detailed homology modeling of
CYP53A15 has also been worked out in the present study.
3

RESULTS AND DISCUSSION
Chemistry
The starting precursors, 2-(N-acyl)amino benzoic acids 2a-k, were synthesized in 70-94%
yields from the corresponding 2-amino benzoic acids 1a-j by acylating with acyl chloride
in the presence of Et3N in THF at room temperature for 2 h (supporting information
file).[31]
We initiated our investigation with substrate 2a, and the results are summarized in Table
1. When the substrate 2a was subjected to react with 1.5 equiv of coupling reagent
HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide
hexafluorophosphate) in the presence of 3.0 equiv of DIPEA (N,N-
diisopropylethylamine) in DMF (N,N-dimethylformamide) at room temperature for 12
hours, only 15% yield of benzoazetinone 3a was obtained (Table 1, entry 1)[30]. To
improve the yield of 3a, the reaction was again carried out in the presence of Et3N and
pyridine, respectively. The former improved the yield compared with the latter (Table 1,
entries 2 and 3). The yield of 3a was further improved when the solvent DMF was
substituted with THF (tetrahydrofuran) but not with DCM (dichloromethane) (Table 1,
entries 4 and 5). Increase in HATU did not improve the yield of the product (Table 1,
entry 6). Other coupling reagents such as DCC (dicyclohexylcarbodiimide), EDC.HCl (1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), TBTU (2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate) and HBTU (2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) were also tested, but
the yield of 3a was unsatisfactory (Table 1, entries 7-10). No product was obtained when
4

the coupling reagent PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate) was used for an extended period of time (Table 1, entry 11).
However, when the reaction was carried out with 1.5 equiv of DCC in the presence of 1.0
equiv of an additive HOBt (1-hydroxybenzotriazole) and 3.0 equiv of Et3N in THF at
room temperature for 2 h, the yield of the product 3a was found to increase sharply up to
1.5 equiv of the additive used (Table 1, entries 12-14). Notably, DCC was substituted by
EDC.HCl as the former led major problem of separation of the product from the reaction
mixture due to the formation of water insoluble by-product dicyclohexyl urea (DCU)
(Table 1, entry 15). Low yield was obtained when the reaction was carried out with
coupling reagent HATU/TBTU/HBTU in the presence of the additive (Table 1, entries
16-18). Again, no product was found with PyBOP as coupling reagent in the presence of
HOBt (Table 1, entry 19). Next, the effect of solvent on the yield of benzoazetinone 3a
was studied. Solvents such as DCM, DMF, pyridine, and NMM provided lower yield
(Table 1, entries 20-23), whereas DIPEA was found to be a more effective base for the
reaction (Table 1, entry 24). Overall, the optimized reaction condition seems to be the use
of 1.5 equiv of EDC.HCl, 1.5 equiv of HOBt and 3.0 equiv of Et3N in THF at room
temperature (Table 1, entry 15).
To test the generality of the reaction, other substrates 2-(N-acyl)amino benzoic acids 2b-
k were reacted under the optimized reaction conditions, and N-acyl benzoazetinones 3b-k
were obtained in 58-80% yields (Table 2). In Table 2, it is observed that the substrates 2b
and 2f, having benzoyl and pivaloyl group at the N-center, obtained lower yields of the
5

benzoazetinone derivatives 3b and 3f, respectively. This might be due to the steric effect
exerted by the bulky group.
The structure of benzoazetinone derivatives 3 was determined from their
analytical and spectral data exclusively (supporting information file).
However, when optimized conditions (Table 2, entry 15) were used with the substrates
1b and 4, the desired benzoazetinones 5 and 6 were not found (Scheme 1). This might be
due to the instability of the aforesaid benzoazetinone derivatives, which indicated the
importance of acyl group.
MOLECULAR MODELING OF BENZOAZETINONES
Global optimization of benzoazetinones was followed prior to flexible docking of these
compounds onto the active site (heme binding site) of the target-model, CYP53A15. The
use of properly minimized three-dimensional structure is a necessity for an effective
assessment of flexible ligand-target interactions. We used semi-empirical procedure
followed by ab initio procedure of Gaussian G03W for the purpose.[32] Two-step
procedures were necessary as only the semi-empirical method was seen to be ineffective
for meeting the convergence criteria of some of these compounds (e.g. 3k; Table 3).
Again, application of ab initio method as sole scheme poses the problem of meeting
convergence criteria. Energies of each of these structures are seen to be very large and
negative, indicative of a well-optimized global minimal structure. The energy of 3g is
highly negative and may be due to the substitution of R3 by Br (Table 3).
6

High negative scores for site-specific docking of this group of compounds against the
model structure indicate well-formed complexes. The nature of the substituent at R3-site
of ligand seems to contribute to the complex formation and hence, the effectiveness of
interactions. Bulky aromatic ring along with non-polar substitution at the site (Table 2) is
seen to produce more negative docking scores (as seen in the case of 3i; Table 3). For
example, compounds 3g to 3j differ only by R3 substitutions. The non-polar and polar
natures of R3-substituent are more in 3i and 3g, respectively. The former has the highest
and the latter has the lowest docking score (Table 3).
HOMOLOGY MODELING OF FUNGAL TARGET
Model structure of CYP53A15 is highly optimized and validated using 4-point criteria[33,
34] and judged by per residue RMSD comparison (Figure 1).
Accuracy in homology model depends on many factors such as choice of template, their
alignment, loop optimization, and energy minimization of the initial model as obtained by
Modeller software.[36] Overall, per residue RMSD of the present model is seen to be
improved in comparison with an earlier model PM0075149[17] (that uses similar template
set) against the reference template structure 4D6Z.pdb (Figure 1). We used high-
resolution X-ray crystallographic structure 4D6Z.pdb instead of 4LXJ.pdb as the
template. There are a few advantages associated with the former. First, it belongs to the
same class (4-monooxygenase) as the query sequence (CYP53A15). Second, query
sequence has a higher identity (26%) with 4D6Z_A than 4LXJ.pdb (19%). Third,
7

4LXJ.pdb, although belonging to the same genera, participates in demethylation reaction.
Further, we used multiple templates’ criteria[36] as 4D6Z_A has missing residues. The
missing region was modeled using the structural information of other relatively low-
resolution template structures (1Z10.pdb and 4NY4.pdb), keeping the rest of the
structural information from 4D6Z_A.
DOCKING STUDIES
To identify and understand the potential antifungal behavior of benzoazetinones, docking
simulation and virtual screening were performed using Autodock 4.2 and vina 1.1.2
tools,[37] respectively. Target (model structure of CYP53A15) and ligand were prepared
for virtual screening using Autodock tools.[37] The scores, thus, obtained are presented in
Table 3. Figure 2 shows stabilizing interactions of CYP53A15 and one of these
representative ligands (3i) at the heme-binding pocket. Ligand-protein interactions are
largely stabilized by hydrogen bonding, and van der Waals, hydrophobic and π-alkyl
interactions.[38] In the heme-binding pocket of the target, 3i is seen to establish a number
of stabilizing interactions that include i] amide-π-stacking with Ala284; ii] alkyl and π-
alkyl interactions with Val429 and Ala430, respectively; iii] hydrogen bonding with
Gly426; iv] covalent bond with Phe108; and v] van der Waals’ interactions with Thr288,
Val425, Cys424, Phe108, Gln281, Leu255, and Glu433 (Figure 2).
Figure 3 (A) shows the docking complex between the target protein and the ligand 3i,
which has maximum interaction energy (Table 3). It is seen that the binding pocket is
appropriately fits by the ligand 3i with its hydrophobic group MeC6H4 directed deep near
8

the floor of the cavity. This region of the cavity is also seen to be hydrophobic [Figure 3
(B), SAS scale]. Interestingly, the methyl group of MeC6H4 (R3 substituent) is also in
contact with the hydrophobic part of the pocket. Such hydrophobic stabilization is not
possible with 3g, 3h and 3j structures that have Br, OMeC6H4 and C6H5 groups,
respectively. Thus, additional stabilization might be related to the methyl group in 3i at
its R3 site. Further, ligand structures 3a to 3f have hydrogen atom at the R3 site (Table 2).
The docking scores of these ligands are much lower than that of 3i except 3b. The R5
substituent of 3i and 3b are Me and C6H5 group, respectively. Overall, it seems the
hydrophobic nature of R3 and also R5 bring stability to the docking complex with higher
effective contribution from the earlier.
CONCLUSIONS
In conclusion, we have successfully developed an inexpensive and efficient method for
the synthesis of stable N-acyl benzoazetinones in moderate to good yields (58-85%) from
easily available starting materials 2-(N-acyl)amino benzoic acids under mild conditions,
which is simply intramolecular amidation. Here, both the EDC.HCl and corresponding
by-product urea are water soluble so that the benzoazetinone derivatives can easily be
isolated from the reaction mixture, i.e., the procedure is simple. Moreover, the N-acyl
functional group present in the benzoazetinone derivatives can be further functionalized.
These benzoazetinones undergo interactions in the heme binding site of CYP53A15, a
fungal P450 isozyme. 3i has the most effective interaction possibly due to hydrophobic
stabilization and better packing at the interaction site. Hydrophobic nature of R3
substituent (also R5) helps to form a stable complex. The score of interaction not only
9

shows this list of compounds to be potential antifungal agents but also raises the
possibility of development of potential antifungal agents specifically targeting the CYP53
class of P450.
Experimental Section
All chemicals and reagents used in current study were commercially available analytical
or chemically pure grade reagents, unless otherwise indicated. Melting points were
determined in open capillaries and are uncorrected. All the reactions were monitored by
analytical thin-layer chromatography (TLC) using 0.25 mm E.Merk precoated silica gel
plates (60F254). Silica gel (100-200 mesh) was used for chromatographic separation. All
the 1H NMR and 13C NMR spectra were recorded using a model DPX400 spectrometer
(TOP Spin 2.1 Ultra shieldTM) in CDCl3/DMSO-d6 and chemical shifts were reported in
(ppm) units relative to the internal standard tetramethylsilane (TMS). LCMS spectra,
GCMS spectra and HRMS spectra were recorded on QTRAP Applied
Biosystem/Shimadzu Autosampler, Agilent 6890 series with 5973 Mass selective
detector and Qtof Micro instrument respectively. CHN were recorded on a Perkin-Elmer
2400 series II CHN-analyzer.
General Synthetic Procedure for the Title Compounds 3
To a stirred solution of 2-Acetylamino-6-methoxy-benzoic acid 2a (200 mg, 0.95 mmol)
in dry THF (10 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC.HCl) (274 mg, 1.43 mmol), 1-hydroxybenzotriazole (HOBt) (193
mg, 1.43 mmol) and triethylamine (0.4 mL, 2.85 mmol) at 0 oC. Then it was stirred at
10

room temperature for 2 h. The reaction mixture was diluted to 50 mL with ethyl acetate
and then the organic phase was washed successively with water (2 x 15 mL), brine (2 x
10mL) and was dried over anhydrous Na2SO4. The organic phase was concentrated under
reduced pressure to get the crude product which was purified by column chromatography
over silica gel (100-200 mesh) with 2:3 ethylacetate/hexane (v/v) eluant to afford the
benzoazetinone 3a as pure product. Accordingly, other products 3b-k were synthesized.
1-Acetyl-3-Methoxybenzo[B]Azet-2(1H)-One (3a)
Yield: 85%; white solid; m.p. 190 oC; 1H NMR (400 MHz, CDCl3) δ: 2.41 (s, 3H,
COCH3), 4.00 (s, 3H, OCH3), 6.93 (d, J = 8.4 Hz, 1H, ArH), 7.09 (d, J = 8.0 Hz, 1H,
ArH), 7.68 (t, J = 8.2 Hz, 1H, ArH); 13C NMR (100 MHz, DMSO-d6) δ: 20.6, 56.2,
105.0, 110.2, 117.6, 137.3, 148.2, 155.2, 160.3, 160.7; HRMS (ESI) m/z: Calcd for
C10H9NNaO3 [M + Na]+: 214.0480; Found: 214.0286; LCMS m/z: 192.1 [M + H]+; Anal.
Calcd. for C10H9NO3: C, 62.82; H, 4.74; N, 7.33. Found: C, 62.69; H, 4.91; N, 7.19.
Optimization Procedure for Benzoazetinone
Chemical structures of benzoazetinones were drawn using Gauss View 3.0. Each of this
structure was subjected to two successive optimization schemes. Semi-empirical, PM3
was used first, followed by ab initio HF/STO-3G* methods of Gaussian 03 package
program. Lowest energy structure was stored for docking studies.
Homology Modeling
11

Three templates for CYP53A15 (UNIPROT ID B8QM33) were chosen using E-value
and sequence identity criteria. Full length of 4D6Z_A (resolution 1.93 A, UNIPROT ID
P08684 and length 23-503) along with part of 1Z10_A (1.90 A, UNIPROT ID P11509)
and 4NY4_A (2.95 A, UNIPROT ID P08684) were used to obtain the topology
information for missing residues in the former using multiple template procedures of
Modeler 9v11.[36] Heteroatoms of template were transfer using advanced modeling
method of Modeler. Five models were generated of which the best model was selected
based on Discrete Optimized Protein Energy (DOPE) score.[36] The model was refined
using conjugate gradient energy minimization scheme of NAMD in presence of explicit
water box for 5000 steps with an interval of 200 steps and thus a total of 25 frames were
collected. Lowest potential energy frame was taken as final model.
Structural evaluation, validation and stereo chemical analyses of the model was done by
PROCHECK, ANOLEA, ERRAT and VERIFY in SAVES v4 web server
(https://services.mbi.ucla.edu/SAVES/).
Docking and Virtual Screening
Autodock 4.2[37] of the Scripps Research Institute was used for docking and preparation
of ligand and target protein respectively. To identify the interaction zone, we perform
docking, at the site of one of the three heteroatom (Heme, GOL and PK9) at a time of the
template. The target (CYP53A15) and one of our compounds (3i) were prepared with
MGLtools. Gasteiger partial charges were added. Non-polar hydrogen atoms were
merged and rotatable bonds were defined. The site of the target intended for docking was
12

placed in a grid box with appropriate dimension and center along with 0.375 Å grid
spacing. Torsions were allowed to the long side chains of the amino acid residues in the
vicinity of the ligand. Docking simulations were performed using the Lamarckian genetic
algorithm using default parameters. Initial position, orientation, and torsions of the ligand
were set randomly. The best interacting site (Heme binding pocket of active site) thus
obtained was then used for docking of all of our compounds (Table 2) using virtual
screening procedure of Vina 1.1.2 (May, 2011).[39]
ACKNOWLEDGEMENTS
We thank UGC (New Delhi) for providing Shimadzu UV-Vis, FTIR and GCMS
spectrometers. We also thank DST (New Delhi) for providing Bruker single crystal XRD
and Thermo Scientific HRMS instruments under the DST-PURSE and DST-FIST
programme respectively. We (IA & AD) are also grateful to The University of Burdwan
for financial support under the University Level Research Assistance programme. AKB
like to thank Prof. Anirvan Misra, Department of Chemistry, North Bengal University,
for allowing us to use his Gaussian software.
REFERENCES
[1]
(a) Ceric, H.; Sindler-Kulyk, M.; Kovacevic, M.; Peric, M.; Zivkovic, A. Bioorg.
Med. Chem. 2010, 18, 3053; (b) Webber, J. A.; Wheeler, W. J. In Chemistry and Biology
of β-Lactam Antibiotics; (ed. Morin, R. B. and Gorman, M.); Academic Press: New York,
1982, pp. 372-436; (c) Long, T. E.; Turos, E. Curr. Med. Chem. 2002, 1, 251; (d)
Cromwell, N. H.; Philips, B. Chem. Rev. 1979, 79, 331; (e) De Kimpe, N. In
13

Comprehensive Heterocyclic Chemistry II; (ed. Padwa, A.); Elsevier Science, 1996, Vol.
1B, pp. 508-545; (f) Synthesis of β-Lactam Antibiotics; (ed. Bruggink, A.); © Springer
Science & Business Media Dordrecht, 2001; (g) Sundberg, R. J. In Comprehensive
Heterocyclic Chemistry II; (ed. Katritzky, A. R.; Rees, C. W. and Scriven, E. F. V.);
Pergamon Press: Oxford, 1996, p. 149; (h) Georg, G. I.; Ravikumar, V. T. The Organic
Chemistry of β-Lactams; (ed. Georg, G. I.); VCH: New York, 1993, pp. 295-368.
[2]
(a) Suffness, M. (Eds.); Taxol Science and Applications; CRC Press; Boca Raton,
FL, USA: 1995, pp. 379-415; (b) Bose, A. K.; Mathur, C.; Wagle, D. R.;. Manhas, M. S.
Tetrahedron Symposium in Print 2000, 56, 5603; (c) Alcaide, B.; Almendros, P.;
Aragoncillo, C. Chem. Rev. 2007, 107, 4437.
[3]
[4]
Buynak, J. Curr. Med. Chem. 2004, 11, 1951.
(a) Burnett, D. A. Curr. Med. Chem. 2004, 11, 1873; (b) Clader, J. W. J. Med.
Chem. 2004, 47, 1.
[5]
Finke, P. E.; Shah, S. K.; Fletcher, D. S.; Ashe, B. M.; Brause, K. A.; Chandler,
G. O.; Dellea, P. S.; Hand, K. M.; Maycock, A. L.; Osinga, D. G.; Underwood, D. J.;
Weston H.; Davies, P.; Doherty, J. B. J. Med. Chem. 1995, 38, 2449.
[6]
O’Driscoll, M.; Greenhalgh, K.; Young, A.; Turos, E.; Dickey, S.; Lim, D. V.
Bioorg. Med. Chem. 2008, 16, 7832.
[7]
[8]
Kanafani, Z. A.; Perfect, J. R. Clinic. Infect. Dis. 2008, 46, 120.
Limper, A. H.; Knox, K. S; Sarosi, G. A.; Ampel, N. M.; Bennett, J. E.;
Catanzaro, A.; Davies, S. F.; Dismukes, W. E.; Hage, C. A.; Marr, K. A.; Mody, C. H.;
Perfect, J. R.; Stevens, D. A. Am. J. Respir. Crit. Care Med. 2011, 183, 96.
[9]
Andriole, V. T. Int. J. Antimicrob. Agents. 2000, 16, 317.
14

[10] Giraud, F.; Guillon, R.; Logé, C.; Pagniez, F.; Picot, C.; Le Borgne, M.; Le Pape,
P. Med. Chem. Lett. 2009, 19, 301.
[11] Vanden, B. H.; Dromer, F.; Improvisi, I.; Lozano-Chiu, M.; Rex, J. H.; Sanglard,
D. Med. Mycol. 1997, 36, 119.
[12] Perfect, J. R. Med. Myco. 2009, 47, S324.
[13] Masubuchi, M.; Ebiike, H.; Kawasaki, K. I.; Sogabe, S.; Morikami, K.; Shiratori,
et. al. Bioorg. Med. Chem. 2003, 11, 4463.
[14] Saha, S.; Priyadharshini, A.; Dhanasekaran, D.; Thajuddin, N.; Chandraleka, S.;
Chandramohan, G.; Panneerselvam, A. Comput. Boil. Med. 2012, 42, 542.
[15] Crešnar, B.; Petric, S. Biochim. Biophys. Acta. 2011, 1814, 29.
[16] Kelly, S. L.; Kelly, D. E. Phil. Trans. R. Soc. 2013, 368, 1612.
[17] Podobnik, B.; Stojan, J.; Lah, L.;
ner, T. L.;
Rozman, D.; Komel, R. J. Med. Chem. 2008, 51, 3480.
[18] Sandanayaka,V. P.; Prashad, A. S.; Yang, Y.; Williamson,T.; Lin, Y. I.;
Mansour,T. S. J. Med. Chem. 2003, 46, 2569.
[19] Buynak, J. D.; Rao, A. S.; Fod, G. P.; Carver, C.; Adam, G.; Geng, B.;
Bachmann, B.; Shobassy, S.; Lackey, S. J. Med. Chem. 1997, 40, 3423.
[20] Bonneau, P. R.; Hasani, F.; Plouffe, C.; Malenfant, E.; Laplante, S. R.; Guse, I.;
Ogilvie, W. W.; Plante, R.; Davidson, W. C.; Hopkins, J. L.; Morelock, M. M.;
Cordingley, M. G. Deziel, R. J. Am. Chem. Soc. 1999, 121, 2965.
[21] (a) Pitts, C. R.; Lectka, T. Chem. Rev. 2014, 114, 7930; and references there in.
(b) Banik, B. K.; Ghatak, A.; Becker, F. F. J. Chem. Soc., Perkin Trans. 2000, 1, 2179;
(c) Banik, B. K.; Becker, F. F. Tetrahedron Lett. 2000, 41, 6551; (d) Ng, S.; Banik, I.;
15

Okawa, A.; Becker, F. F.; Banik, B. K. J. Chem. Res.(S) 2001, 118; (e) Banik, B. K.;
Samajdar, S.; Banik, I. Tetrahedron Lett. 2003, 44, 1699; (f) Banik, B. K.; Adler, D.;
Nguyen, P.; Srivastava, N. Heterocycles 2003, 61, 101; (g) Banik, B. K.; Lecea, B.;
Arrieta, A.; Cozar, A.; Cossio, F. P. Angew. Chem. Int. Ed. 2007, 46, 3028. (h) Banik, B.
K.; Aguilar, H.; Cordova, D. Heterocycles 2008, 11, 2321.
[22] (a) Fodor, L.; Csomos, P.; Holczbauer, T.; Kalman, A.; Csampai, A.; Sohar, P.
Tetrahedron Lett. 2011, 52, 224; (b) Janikowska, K.; Pawelska, N.; Makowiec, S.
Synthesis 2011, 1, 69; (c) Dubey, A.; Srivastava, S. K.; Srivastava, S. D. Bioorg. Med.
Chem. Lett. 2011, 21, 569; (d) Feroci, M.; Chiarotto, I.; Orsinib, M.; Inesi, A. Chem.
Commun. 2010, 46, 4121; (e) Keri, R. S.; Hosamani, K. M.; Shingalapur, R. V.; Reddy,
H. R. S. Eur. J. Med. Chem. 2009, 44, 5123; (f) Dekeukeleire, S.; D’hooghe, M.; Muller,
C.; Vogt, D.; De Kimpe, N. New J. Chem. 2010, 34, 1079; (g) Jarrahpour, A.; Zarei, M.
Molecules 2006, 11, 49; (h) Jarrahpour, A.; Khalili, D. Tetrahedron Lett. 2007, 48, 7140;
(i) Jarrahpour, A.; Ebrahimi, E. Molecules 2010, 15, 515.
[23] Burgess, E. M.; Milne, G. Tetrahedron Lett. 1966, 7, 93.
[24] Smalley, R. K.; Suschitzky, H.; Tanner, E. M. Tetrahedron Lett. 1966, 7, 3465.
[25] Chiu, S.-J.; Chou, C.-H. Tetrahedron Lett. 1999, 40, 9271.
[26] Olofson, R. A.; Vander Meer, R. K.; Stournas, S. J. Am. Chem. Soc. 1971, 93,
1543.
[27] Dai, W-M.; Cheung, Y. K.; Tang, K. W.; Choi, P. Y.; Chung, S. L. Tetrahedron
1995, 51, 12263.
[28] Nowak, M. Malinowski, Z.; Jozwiak, A.; Fornal, E.; Blaszczyk, A.; Kontek, R.
Tetrahedron 2014, 70, 5153.
16

[29] Kayarmar, R.; Nagaraja, G. K; Bhat, M.; Naik, P.; Rajesh, K. P.; Shetty, S.;
Arulmoli, T. Med. Chem. Res. 2014, 23, 2964.
[30] Cioffi, C. L.; Dobri, N.; Freeman, E. E.; Conlon, M. P.; Chen, P.; Stafford, D. G.;
Schwarz, D. M. C.; Golden, K. C.; Zhu, L.; Kitchen, D. B.; Barnes, K. D.; Racz, B.; Qin,
Q.; Michelotti, E.; Cywin, C. L.; Martin, W. H.; Pearson, P. G.; Johnson, G.; Petrukhin,
K. J. Med. Chem. 2014, 57, 7731.
[31] Emenike, B. U.; Liu, A. T.; Naveo, E. P.; Roberts, J. D. J. Org. Chem. 2013, 78,
11765.
[32] Alam, A.; Pal C.; Goyal, M.; Kundu, M. K.; Kumar, R, Iqbal, M. S.; Dey, S.;
Bindu, S.; Sarkar, S; Pal, U.; Maiti, N. C. Bioorg. Med. Chem. 2011, 19, 7365.
[33] Mondal, S.; Mondal, B.; Bandyopadhyay, A. K. Int. J. Eng. Sci. Technol. 2013, 5,
992.
[34] Mondal, B.; Sen Gupta, P. S.; Roy, C.; Islam, R. N. U.; Hazra, N.;
Bandyopadhyay, A. K. Int. J. Pharm. Bio Sci. 2016, 7, 406.
[35] Humphrey, W.; Dalke, A.; Schulten, K. J. mol. graphs. 1996, 29, 33.
[36] Webb, B.; Sali, A. Curr. Protoc. Bioinformatics. 2014, 8, 5.
[37] Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell,
D. S.; Olson, A. J. J. Comp. Chem. 2009, 16, 2785.
[38] Wang, H. C.; Yan, X. Q.; Yan, T. L.; Li, H. X.; Wang, Z. C.; Zhu, H. L.
Molecules. 2016, 21, 1012.
[39] Trott, O.; Olson, A. J. Comp. Chem. 2010, 31, 455.
17

Table 1. Optimization of reaction condition for the synthesis of benzoazetinone 3a.
O
O
O
coupling reagent,
additive,
O
OH
NH
N
base, solvent, rt
O
2a
3a
O
Entry Coupling reagent
(equiv)
Additive
(equiv)
––
Base
Solventa Time
(h)
Yield
(%)b
15
(equiv)a
DIPEA
(3.0)
1
HATU (1.5)
DMF
12
2
HATU (1.5)
HATU (1.5)
HATU (1.5)
HATU (1.5)
HATU (2.0)
DCC (1.5)
––
Et3N (3.0) DMF
Py (3.0) DMF
12
12
12
12
12
12
12
12
12
18
2
25
10
10
45
45
17
52
46
49
n.r.
70
73
73
3
––
4
––
Et3N (3.0) DCM
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) THF
5
––
6
––
7
––
8
EDC.HCl (1.5)
TBTU (1.5)
HBTU (1.5)
PyBOP (1.5)
DCC (1.5)
––
9
––
10
11
12
13
14
––
––
HOBt (1.0)
HOBt (1.5)
HOBt (2.0)
DCC (1.5)
2
DCC (1.5)
2
18

15c
16
17
18
19
20
21
22
23
24
EDC.HCl (1.5)
HATU (1.5)
HOBt (1.5)
HOBt (1.5)
HOBt (1.5)
HOBt (1.5)
HOBt (1.5)
HOBt (1.5)
HOBt (1.5)
HOBt (1.5)
HOBt (1.5)
HOBt (1.5)
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) THF
Et3N (3.0) DCM
Et3N (3.0) DMF
2
2
2
2
2
2
2
2
2
2
85
58
53
50
n.r.
60
37
32
40
76
TBTU (1.5)
HBTU (1.5)
PyBOP (1.5)
EDC.HCl (1.5)
EDC.HCl (1.5)
EDC.HCl (1.5)
EDC.HCl (1.5)
EDC.HCl (1.5)
Py (3.0)
THF
NMM (3.0) THF
DIPEA
(3.0)
THF
aDry solvents and bases were used; bIsolated yield of the compound 3a; cOptimized
reaction conditions; n.r. = No reaction
19

Table 2. Synthesis of N-acyl benzoazetinones 3.
R1
O
R1
R2
R3
EDC.HCl (1.5 equiv),
HOBt (1.5 equiv),
O
R2
R3
OH
N
NH
R5
Et3N (3.0 equiv),
THF, rt, 2 h
O
R4
R4
3a-k
R5
O
2a-k
Entry
R1
OMe
OMe
H
R2
H
R3
R4
R5
Compound Yield (%)a
1
H
H
H
H
H
H
H
H
H
H
H
Me
Ph
85
60
72
78
79
58
73
69
70
74
80
3a
3b
3c
3d
3e
3f
2
H
3
H
Me
Me
Me
4
H
Me
OMe
H
5
H
6
H
OMe tBu
7
H
OMe Br
H
H
H
H
H
Me
Me
Me
Me
Me
3g
3h
3i
8
H
OMe o-OMeC6H4
OMe o-MeC6H4
OMe Ph
9
H
10
11
H
3j
H
-CH=CH-CH=CH-
3k
aIsolated yield of the compounds 3a-k
20

Table 3. Details of optimization and docking score of benzoazetinones.
Compounds
Structure
Energy∞
-654.50
Docking score$
-28.56
3a
-892.67
-542.10
-34.44
-28.14
3b
3c
-580.66
-654.50
-770.23
-31.51
-28.98
-31.54
3d
3e
3f
-3198.71
-993.67
-29.45
-32.34
3g
3h
-919.83
-881.25
-654.29
-37.38
-34.86
-31.53
3i
3j
3k
∞ hartree (a.u.); $ kj mol-1
21

Scheme 1. Reaction of substrates 1b and 4 under optimized condition.
22

Figure 1. Per residue RMSD of 4D6Z (Blue line; Template, used as reference), present
model (red line) and PM0075149 (green). Alignment of three structures were done using
STRAMP structural alignment in VMD interface.[35]
23

Figure 2. 2D model of interactions between the ligand 3i with target protein; for clarity,
only interacting residues are displayed. Presence of blue sphere around residues indicates
accessibility.
24

Figure 3. (A) Docking complex of model structure of CYP53A15 and geometry
optimized ligand, 3i; (B) Ligand (red) binding pocket, interacting residues, and their
solvent accessible surface (SAS) are shown along with SAS scale; (C) A copy of ligand
at identical orientation as it is present in the binding pocket to guess positions of invisible
atoms. Residues labels, written in blue color are not visible in the front view.
25