Synthesis and structure–activity relationship study of novel quinazolinone-based inhibitors of MurA

Article abstract of DOI:10.1016/j.bmcl.2017.05.064

MurA is an intracellular bacterial enzyme that is essential for peptidoglycan biosynthesis, and is therefore an important target for antibacterial drug discovery. We report the synthesis, in silico studies and extensive structure–activity relationships of a series of quinazolinone-based inhibitors of MurA from Escherichia coli. 3-Benzyloxyphenylquinazolinones showed promising inhibitory potencies against MurA, in the low micromolar range, with an IC₅₀ of 8?μM for the most potent derivative (58). Furthermore, furan-substituted quinazolinones (38, 46) showed promising antibacterial activities, with MICs from 1?μg/mL to 8?μg/mL, concomitant with their MurA inhibitory potencies. These data represent an important step towards the development of novel antimicrobial agents to combat increasing bacterial resistance.

Full text of DOI:10.1016/j.bmcl.2017.05.064

Accepted Manuscript
Synthesis and structure–activity relationship study of novel quinazolinone-
based inhibitors of MurA
Martina Hrast, Kaja Rožman, Marko Jukič, Delphine Patin, Stanislav Gobec,
Matej Sova
PII:
S0960-894X(17)30562-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.064
BMCL 25009
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
25 March 2017
18 May 2017
21 May 2017
Please cite this article as: Hrast, M., Rožman, K., Jukič, M., Patin, D., Gobec, S., Sova, M., Synthesis and
structure–activity relationship study of novel quinazolinone-based inhibitors of MurA, Bioorganic & Medicinal
Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.05.064
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Synthesis and structure–activity relationship study of novel quinazolinone-based inhibitors of
MurA
Martina Hrasta, Kaja Rožmana, Marko Jukiča, Delphine Patinb, Stanislav Gobeca, Matej Sovaa*
aFaculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia
bBacterial Cell Envelopes and Antibiotics Group, Institute for Integrative Biology of the Cell (I2BC),
CEA, CNRS, University of Paris Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette cedex, France
*Corresponding author. E-mail: matej.sova@ffa.uni-lj.si (M. Sova)
Keywords: Peptidoglycan, MurA enzyme, antibacterial agents, quinazolinones
Abbreviations: SAR, structure-activity relationship; UDP-GlcNAc, UDP-N-acetylglucosamine.
Abstract
MurA is an intracellular bacterial enzyme that is essential for peptidoglycan biosynthesis, and is
therefore an important target for antibacterial drug discovery. We report the synthesis, in-silico studies
and extensive structure–activity relationships of a series of quinazolinone-based inhibitors of MurA
from Escherichia coli. 3-Benzyloxyphenylquinazolinones showed promising inhibitory potencies
against MurA, in the low micromolar range, with an IC50 of 8 µM for the most potent derivative (58).
Furthermore, furan-substituted quinazolinones (38, 46) showed promising antibacterial activities, with
MICs from 1 µg/mL to 8 µg/mL, concomitant with their MurA inhibitory potencies. These data
represent an important step towards the development of novel antimicrobial agents to combat
increasing bacterial resistance.

Antimicrobial agents have saved countless lives, with significantly improved quality of life achieved
since the discovery of sulphonamides and β-lactams in the first half of the 20th century.1 However,
extensive use, and frequent misuse and abuse, of antibiotics have caused widespread emergence of
antimicrobial-resistant pathogenic bacteria, which now present a serious threat to public health.
Therefore, there is an urgent need for the discovery and development of new antibacterial agents,
preferably with novel mechanisms of action.2, 3
The bacterial cell wall provides the necessary structural integrity for bacteria to withstand the osmotic
pressure between the cytoplasm and the cell exterior. The enzymes involved in peptidoglycan
biosynthesis are among the most important targets for antibacterial drug discovery, as inhibition of
these enzymes can trigger cell lysis.4, 5 The MurA enzyme (UDP-GlcNAc enolpyruvyl transferase)
catalyses the first committed step of the intracellular stage of peptidoglycan biosynthesis: the transfer
of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine. The product, UDP-GlcNAc-
enolpyruvate, is then reduced by the MurB enzyme to UDP-acetylmuramic acid (UDP-MurNAc). In
the next steps, four different ligases, MurC-F, sequentially add amino acids to finally form UDP-
MurNAc-pentapeptide, which is one of the main building blocks of the peptidoglycan cell wall.6 The
Mur enzymes are essential and highly conserved in both Gram-negative and Gram-positive bacteria.
Furthermore, eukaryotic cells do not have MurA-F,7 thereby providing inhibitors of these enzymes
with the desired selectivity for bacteria.
Fosfomycin (Figure 1, 1) is a well-known naturally occurring inhibitor of MurA.8 It mimics
phosphoenolpyruvate and forms a covalent adduct with the cysteine residue within the active site of
MurA from different bacteria. Resistance to fosfomycin frequently occurs due to reduced uptake of
antibiotics, (metabolic) modification of antibiotics, overexpression of MurA, or modification of the
MurA active site.9 Therefore, to overcome this resistance, the development of structurally diverse
MurA inhibitors with novel mechanism(s) of action is needed. However, although numerous inhibitors
have been discovered in recent years, mainly through various high-throughput screening campaigns or
classical medicinal chemistry approaches,10 none of these has reached clinical trials or been developed
for therapeutic use.

8
Figure 1. Fosfomycin 1 and quinazolinone 2 11, as inhibitors of MurA, and two representative quinazolinone
derivatives 3 12 and 4 13 that show antimicrobial activity.
In a recent virtual-screening campaign, some quinazolinone-based compounds (e.g., Figure 1, 2) were
identified as potential MurA inhibitors.11 Quinazolinone is a well-known scaffold in medicinal
chemistry that is part of a diverse set of compounds that show numerous therapeutic and
pharmacological properties14, including antimicrobial activity (e.g., Figure 1, representative
compounds 3,12 413). Therefore, the aim of the present study was to explore the structure–activity
relationships (SARs) of this quinazolinone-based hit compound (2) from virtual screening, and to
improve its antibacterial activity. A series of novel quinazolinone derivatives was synthesised and then
evaluated for inhibitory activity against Escherichia coli MurA, and antibacterial activity against two
representative bacterial strains: E. coli and Staphylococcus aureus.
There are many methods available for the synthesis of the quinazolinone scaffold.14 In the present
study, quinazolinones 9-63 were prepared from anthranilamide (5) and different aldehydes, in
dimethylsulphoxide (DMSO) via aerobic oxidative cyclisation (Scheme 1).15 After construction of the
quinazolinone core (9-41, 49-63), alkylation was carried out using various alkyl bromides in
dimethylformamide (DMF) under basic conditions. Furthermore, when an ester group was attached to
the quinazolinone core via alkylation (47), further hydrolysis in alkaline medium was performed, to
obtain carboxylic acid 48. Quinazolinones with the benzyloxyphenyl moiety and a carboxylic group
(64-69) were prepared from the corresponding 3- or 4-hydroxy aldehydes, via alkylation with methyl
3- or 4-bromomethyl benzoate and subsequent reaction with anthranilamide in DMSO (64-66).
Deprotection in alkaline medium yielded the corresponding benzoic acid derivatives 67-69. As
possible isosteres of quinazolinones, thienopyrimidinones 70-73 were synthesised from 3-
aminothiophene-2-carboxamide (6) and the corresponding aldehydes, according to the same general
procedure used for compounds 9-63.

Scheme 1. Synthesis of quinazolinones 9-69 and thienopyrimidinones 70-73. Reagents and conditions: (a)
aldehyde (R1CHO or R3CHO), DMSO, 100 °C, 16-24 h; (b) K2CO3, DMF, R2Br; (c) K2CO3, DMF,
BrCH2COOMe; (d) NaOH, acetone/water; (e) K2CO3, acetone, methyl 3- or 4-bromomethyl benzoate; (f)
anthranilamide (5), DMSO, 100 °C, 16-24 h.
The synthesised quinazolinones 9-69 (Table 1, Supporting Information Table S1) and
thienopyrimidinones 70-73 (Table 2) were evaluated biochemically for their inhibition of E. coli
MurA, using fosfomycin as the positive control. The first set of compounds with the substituted
phenyl (9-23) or pyridine (24-28) ring and the condensed aromatic systems (29-34) attached to
position 2 of the quinazolinone scaffold (R1) were inactive against MurA, whereas the 2-furano
substituted quinazolinones (35-41) showed inhibitory activities, with IC50 values from 47 µM to 101
µM. Substitution with the electron-withdrawing nitro group (Table 1, 38) gave the most potent
inhibition. To further examine the structural features of furan-substituted quinazolinones, alkylation of
the lactam nitrogen was performed (compounds 42-47). However, with the exception of methyl
substituted derivative 42, the inhibition of MurA was diminished for most of these synthesised
analogues. A two-fold decrease in inhibitory potencies (e.g., IC50, 87 µM for 46) was also observed
following alkylation of compound 38 with the isopropyl moiety. On the other hand, replacement of the
furan ring of compound 35 with 5-phenylthiophene resulted in a potent MurA inhibitor 50 (IC50, 17
µM), albeit quinazolinone with the unsubstituted thiophene 49 showed only weak inhibitory activity,
similar to the imidazole derivative 51. Encouraged by the promising MurA inhibitory potency of 2-(5-

phenylthiophen-2-yl)quinazolin-4(3H)-one 50, compounds containing the biphenyl (52), 4-(pyridine-
4-yl)-phenyl (53) and styryl (54) moieties were synthesized. 2-([1,1'-Biphenyl]-4-yl)quinazolin-4(3H)-
one (52) showed similar inhibitory potency (IC50, 21 µM) as its bioisosteric analogue 50, whereas the
styryl derivative 54 showed only moderate inhibition of MurA. Interestingly, the substitution of a
benzene ring with a long alkyl chain (55) also led to moderate inhibition of MurA (IC50, 84 µM).
Table 1: IC50 values of the most potent quinazolinones against E. coli MurA, and their antibacterial activities
against E. coli and S. aureus.
IC50 vs. MurA
MIC
(µM)a
E. coli, S. aureus
[µg/mL])
Compound
R1
R2
101
H
H
H
H
>128, both
>128, both
>128, both
35
36
37
38
42
45
46
50
81
56
47
1 (E. coli)
8 (S. aureus)
72
>128, both
RA, 93%b
>128 (E. coli)
32 (S. aureus)
87
2 (E. coli)
(2, 8)
8 (S. aureus)
17
21
51
H
H
>128, both
>128, both
52
54
H
>128, both

84
23
H
H
>128, both
>128, both
55
56
36
8
H
H
H
H
>128, both
>128, both
>128, both
>128, both
57
58
59
66
19
21
aData are means of two independent experiments. Standard deviations were within 10% of the means. bResidual
activity of MurA at 100 µM tested compound.
In addition, a series of benzyloxyphenyl substituted quinazolinones (56-69) was also prepared and
evaluated biochemically on E. coli MurA. Some structure–activity trends were evident in this series,
as the compounds with the 3-benzyloxyphenyl moiety showed high inhibitory potencies, whereas their
4-benyzloxyphenyl counterparts were less potent, or even inactive. Furthermore, the SAR analysis
highlighted the key role of the para substitution on the 3-benzyloxyphenyl moiety (58, 59, 66), as the
corresponding meta analogues showed no inhibition of MurA. The 4-chloro derivative 58 showed the
highest inhibition, with an IC50 of 8 µM, and it thus represents the most promising quinazolinone-
based inhibitor of MurA. The replacement of chlorine with fluorine (59) or the COOMe group (66)
retained most of the inhibitory activity, whereas introduction of the polar carboxylic acid (69) led to an
inactive compound.
To further examine the SARs of quinazolinones, a novel series of thienopyrimidinones 70-73 was
prepared (Table 2), as bioisosteres of quinazolinones. As expected, compound 70 was inactive, as was
its quinazolinone analogue 11. Nevertheless, compounds 71-73 showed very similar inhibitory
potencies against MurA as their corresponding quinazolinone counterparts 35, 50 and 57. As no

significant improvements in the potencies were obtained with this series, no further analogues were
synthesized.
Table 2: Inhibitory activities of the thienopyrimidinones against E. coli MurA, and their antibacterial activities
against E. coli and S. aureus.
MIC
R3
IC50 vs. MurA (µM)
E. coli, S. aureus
Compound
[µg/mL])
RA, 80%a
>128, both
70
94
22
>128, both
>128, both
71
72
30
128 (E. coli)
73
aResidual activity of MurA at 100 µM tested compound
In order to inspect the binding modes of synthesized quinazolinones, we conducted an in-silico
docking study. MurA-TAV crystal model (E. coli, PDB ID: 1UAE) was selected and docking
performed using OpenEye OEDocking 3.0.1 software package (OpenEye Scientific Software, Inc.,
Santa Fe, NM, USA; www.eyesopen.com) 16-18. Docking experiment calculations exposed compounds
59, 56, 55, 66 and 58 as top-scoring according to Chemgauss 4 scoring function. Inspection of
calculated docking poses revealed a distinct binding mode that is shared amongst top scoring
compounds (Figure 2). In all calculated poses, quinazolinone moiety can be observed in a polar pocket
defined by Arg91, Arg120, Asp123, Hsd125 and Ser162 (MurA PDB ID: 1UAE numbering). Central
meta-substituted ring serves as a linker enabling the positioning of the terminal moiety into the large
cavity defied by Trp95 and Val163 on one side, with Asn23, Glu190 and Arg232 on the other.
Observed geometry is apparently necessary for activity on the isolated enzyme system as truncated
analogues or central-linker para-substituted compounds are devoid of activity. Truncated analogues
mainly occupy the large cavity space while para-substituted compounds cannot adopt the observed
common binding mode due to steric hindrance. Calculated docking scores also correlate with the
measured activity versus MurA enzyme from E. coli as most active compounds 56, 58, 59, 66 display
IC50 values in the range 8-23 µM.

Figure 2. Left: calculated binding mode of compound 59 in MurA (E. coli, PDB ID: 1UAE); Right: Schematic
2D superimposition of top-scoring compounds 59, 56 and 58 exposing a common binding mode.
Antibacterial activities against E. coli and S. aureus were tested according to the Clinical Laboratory
Standards Institute protocol.19 In general, most of the quinazolinones showed poor antibacterial
activities against both E. coli and S. aureus. However, compound 45 had a MIC of 32 µg/mL against
S. aureus. Furthermore, compounds 38 and 46, which had the 5-nitrofuran moiety, showed the most
promising activities against E. coli and S. aureus, with MICs from 1 mg/mL to 8 mg/mL. As these two
compounds also inhibited MurA in the micromolar range (IC50 values, 47 µM, 87 µM for 38, 46,
respectively), these represent important compounds for further development of novel antimicrobial
agents.
To conclude, a comprehensive series of new quinazolinone derivatives was prepared and evaluated as
inhibitors of MurA, which is one of the essential enzymes involved in bacterial cell wall biosynthesis.
A set of 3-benzyloxyphenylquinazolinones (56, 58, 59, 66) showed potent inhibitory activities against
MurA in the low micromolar range. The most promising compound of this series was 3-(4-
chlorobenzyloxy)phenyl, as derivative 58, with an IC50 of 8 µM. This thus represents the most potent
of these quinazolinone-based inhibitors of MurA, and an important starting point for further
optimisation. In the series of furan-substituted quinazolinones, 38 and 46 were the derivatives with the
greatest potential. Docking studies indicated common binding mode shared by most active compounds
56, 58, 59, 66. Concurrent with their inhibition of MurA, compounds 38 and 46 also showed
antimicrobial activities against both E. coli and S. aureus, with MICs from 1 mg/mL to 8 mg/mL.
Taken together, the SARs of these quinazolinone-based inhibitors of MurA reveal some potent
inhibitors that have antibacterial activity. These compounds thus provide the greatest interest for
further development of novel antimicrobial agents to combat increasing bacterial resistance.
Author contributions

The manuscript was written through contributions of all of the authors. All of the authors gave their
approval to the final version of the manuscript. All of the authors have read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
The authors acknowledge the financial support from the Slovenian Research Agency (research core
funding No. P1-0208). The authors acknowledge the project (Combating bacterial resistance:
optimisation of bacterial cell wall biosynthesis inhibitors, ID L1-6745 (B)) was financially supported
by the Slovenian Research Agency. We thank Dr David Roper for providing the bacterial strains for
microbiological evaluation. We thank Dr Chris Berrie for critical reading of the manuscript.
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Highlights
•
•
•
•
synthesis and extensive SAR of a series of quinazolinone-based inhibitors of MurA
potent MurA inhibitors with IC50 in low micromolar range were discovered
some quinazolinone-based MurA inhibitors possess antibacterial activity
important compounds in the development of novel antimicrobial agents