Synthesis, biological evaluation and molecular docking of 2-phenyl-benzo[d]oxazole-7-carboxamide derivatives as potential Staphylococcus aureus Sortase A inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.06.074

A series of novel 2-phenyl-benzo[d]oxazole-7-carboxamide derivatives were designed, synthesized and evaluated for their in vitro inhibitory activities against Staphylococcus aureus Sortase A with known Sortase A inhibitor pHMB as positive compound (IC₅₀?=?130?μM). Most compounds exhibited excellent inhibitory activity (IC₅₀?=?19.8–184.2?μM). Structure–activity relationship studies demonstrated that substitution at 7-position and 2-position of benzoxazole had great influence on the activities. Specifically, the substituent at 7-position is indispensable for inhibitory activity. The molecular docking studies revealed the i-butyl amide group went towards the β6/β7 loop-β8 substructure of the protein and the benzoxazole core lied in a hydrophobic pocket composed of Ala118, Val166, Val168, Val169 and Ile182, shaping the whole molecule into a L-shape mode to be recognized by Sortase A.

Full text of DOI:10.1016/j.bmcl.2016.06.074

Accepted Manuscript
Synthesis, Biological Evaluation and Molecular Docking of 2-Phenyl-Ben-
zo[d]oxazole-7-Carboxamide Derivatives as Potential Staphylococcus aureus
Sortase A Inhibitors
Yong Zhang, Jian Bao, Xin-Xian Deng, Wan He, Jia-Jun Fan, Fa-Qin Jiang, Lei
Fu
PII:
S0960-894X(16)30685-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.074
BMCL 24027
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 March 2016
9 June 2016
25 June 2016
Please cite this article as: Zhang, Y., Bao, J., Deng, X-X., He, W., Fan, J-J., Jiang, F-Q., Fu, L., Synthesis, Biological
Evaluation and Molecular Docking of 2-Phenyl-Benzo[d]oxazole-7-Carboxamide Derivatives as Potential
Staphylococcus aureus Sortase A Inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.06.074
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Synthesis, Biological Evaluation and Molecular Docking of
2-Phenyl-Benzo[d]oxazole-7-Carboxamide Derivatives as Potential
Staphylococcus aureus Sortase A Inhibitors
Yong Zhanga, Jian Baob, Xin-Xian Dengb, Wan Heb, Jia-Jun Fanb, Fa-Qin Jiangb, *, Lei Fub, *
a
School of Science and Biotechnology, Shanghai Jiao Tong University, No. 800 Dongchuan
Rd.,Shanghai 200240, PR China;
b
School of Pharmacy, Shanghai Jiao Tong University, No. 800 Dongchuan Rd.,Shanghai 200240,
PR China;
*Correspondence:jfq2008@sjtu.edu.cn (F.-Q.J.); leifu@sjtu.edu.cn(L.F.); Tel./Fax:
+86-21-3420-4787(F.-Q.J.); Tel./Fax.: +86-21-3420-4791 (L.F.);
Abstract: A series of novel 2-phenyl-benzo[d]oxazole-7-carboxamide derivatives were designed,
synthesized and evaluated for their in vitro inhibitory activities against Staphylococcus aureus Sortase A
with known Sortase A inhibitor pHMB as positive compound (IC50=130 μM). Most compounds
exhibited excellent inhibitory activity (IC50=19.8 μM-184.2 μM). Structure–activity relationship
studies demonstrated that substitution at 7-position and 2-position of benzoxazole had great influence on
the activities. Specifically, the substituent at 7-position is indispensable for inhibitory activity. The
molecular docking studies revealed the i-butyl amide group went towards the β6/β7 loop-β8
substructure of the protein and the benzoxazole core lied in a hydrophobic pocket composed of Ala118,
Val166, Val168, Val169 and Ile182, shaping the whole molecule into a L-shape mode to be recognized
by Sortase A.
Keywords: Sortase A; anti-infective; benzo[d]oxazole
The emergence of multidrug-resistant bacterial strains has limited the clinical efficacy
of most of the currently marketed antibiotics.1 The strong need of new antibiotics has
stimulated research into the development of new virulence target.2,3 Sortase A transpeptidase
is a therapeutically important membrane-bound enzyme in Gram-positive bacteria. It
catalyzes the cell wall anchoring of important virulence factors to the surface of the
organism, by identifying the conserved characteristic amino acid motif LPXTG (LPXTG,
leucine, proline, X, threonine, and glycine, where X is any amino acid) of C-thermal of
virulence factors. If Sortase A of bacteria is blocked, virulence factors cannot be anchored to
the cell wall, resulting in significant decreased infection of the pathogens.4 Several studies
have confirmed the important role of Sortase A in bacterial pathogenesis and considered a
promising target for the development of new anti-infective drugs. 5,6
As clinical importance of Sortase A has begun to be fully considered, scientists have

highlighted the discovery of new compounds as potential Sortase A inhibitors. A large
number of compounds were screened for Sortase A inhibitors, but only a few compounds
exhibited excellent inhibitory activity at the low micromolar range.7-10 It is probably related
to specific recognition of Sortase A to its substrate (LPXTG). Usually, Sortase A can
specifically recognize substrate with L-shape mode, which is afforded by Leu and Pro side
chains, with the Leu side chain directing the conformational change of the enzyme and the
Pro side chain stabilizing the conformation of the substrate. 11,12
Rationally, the compounds with kink forming propensity to L-shape like Leu/Pro
residues can be tolerated by Sortase A. It provides the theoretical basis to discover new and
highly active inhibitors against Sortase A. In searching for novel Sortase A inhibitors, we
designed a series of 2-phenyl-benzo[d]oxazole-7-carboxamide derivatives with functional
module arrangement similar to that of Sortase A substrate LPATG (Figure 1). The skeleton
nucleus of moiety A, B and C in 2-phenyl-benzo[d]oxazole-7-carboxamide mimic the Leu,
Pro and Ala of Sortase A substrate, respectively. The whole molecule will be shaped into a
kinked pattern like LPXTG.
Leu
Moiety A
R1
HN
O
H2N
O
O
O
N
O
N
R2
O
N
H
NH
OH
N
Pro
H
Ala
O
HO
Moiety C
Moiety B
Sortase A substrate LPXTG,
here x was drawn as Ala
2-phenyl-7-carboxide-benzo[d]oxade
Target compounds
(1)2-phenyl-7-carboxide-benzo[d]oxade, R1 and R2 will afford L-shape like Leu and pro side chain in substrate.
(2)2-substituted phenyl will produce a various space to mimick the X amino acid in the substrate.
(3)R2 can be some special group to bind with the Sortase A active site.
Figure 1. Design of 2-phenyl-benzo[d]oxazole-7-carboxamide as novel Sortase A inhibitors.
The synthetic routes for the novel benzo[d]oxazole derivatives are outlined in
Schemes 1 and Schemes 2. In general, compounds 8 and compounds 9 were prepared in
seven steps starting from commercially available salicylic acid 1 (Scheme 1). Treatment of
salicylic acid 1 with methanol in H2SO4 afforded hydroxybenzoic ester 2 according to the
modified known method.13 Methyl 2-hydroxy-3-nitrobenzoate 3 was produced by nitration
in mixture of HNO3 and H2SO4.14 Reduction of methyl 2-hydroxy-3-nitrobenzoate 3 in the
15
presence of hydrogen with Pd/C gave methyl 3-amino-2-hydroxybenzoate 4, which was

reacted with 4-methy benzoyl bromide via intermolecular cyclization to afford the
important skeleton benzo[d]oxazole 5 in high yield. 7 was obtained from 5 via hydrolysis
and condensation reaction with different kind of amines. Finally, compounds 8 were
yielded by coupling reaction of 7 with various amines. Compounds 9 were yielded by
coupling reaction of 7 with various acids.
Compound 12 was synthesized in two steps starting from commercially available
2-aminophenol 10 (Scheme 2). Specifically, 2-aminophenol 10 was intermolecular cyclized
with 4-methy benzoyl bromide to afford 11, which was further coupled with various acid or
amines to give compound 12. The structures of the synthesized compounds were confirmed
by analytical and spectral data (1H, 13C NMR and ESI-HRMS).16
COOH
OH
COOCH3
OH
COOCH3
OH
COOCH3
OH
COOCH3
O
a
c
b
d
NO2
3
NH2
N
5
Br
2
4
1
H
H
COOH
O
N
R1
N
O
R1
O
e
O
f
g
O
N
Br
HN R2
N
7
Br
N
6
7a R1=i-propyl
7b R1=i-butyl
7c R1=n-butyl
8
8a R1
2
=i-b
utyl
, R =
butyl
h
8b R1
1
2
=i-b
utyl,
R =b
enzy
l
2=4-m
i-bu
tyl, R
etho
xylp
8
c R =
heny
l
H
N
O
R1
O
O
N
O
R2
9
9l R1=i-butyl, R2=2,4-dihydroxylphenyl
9m R1=i-butyl, R2=3-hydroxyl4-nitrophenyl
9n R1=i-butyl, R2=4-nitrophenyl
9a R1=i-butyl, R2=4-hydroxylbenzyl
9b R1=i-butyl, R2=3-hydroxylbenzyl
9c R1=i-butyl, R2=3,4-dihydroxylbenzyl
9d R1=i-butyl, R2=3-hydroxyl-4-nitrobenzyl
9e R1=i-butyl, R2=2-nitrophenyl
9f R1=i-butyl, R2=phenyl
9g R1=i-butyl, R2=3,4-dihydroxylphenyl
9h R1=i-butyl, R2=3-hydroxylphenyl
9o R1=i-butyl, R2=4-hydroxyl-styrene
9p R1=i-propyl, R2=4-hydroxylbenzyl
9q R1=i-propyl, R2=3,4-dihydroxylbenzyl
9r R1=i-propyl, R2=3-hydroxylphenyl
9s R1=i-propyl, R2=4-hydroxylphenyl
9t R1=n-butyl, R2=4-hydroxylbenzyl
9u R1=n-butyl, R2=3-hydroxylbenzyl
9v R1=n-butyl, R2=3-hydroxylphenyl
9w R1=n-butyl, R2=4-hydroxylphenyl
9x R1=n-butyl, R2=4-hydroxyl-styrene
9i R1=i-butyl, R2=4-hydroxylphenyl
9j R1=i-butyl, R2=3,5-dimethoxylphenyl
9k R1=i-butyl, R2=2,3,4-trihydroxylphenyl
Scheme 1. Synthesis of Compounds 8 and 9. Reagents and conditions: a CH3OH, H2SO4, reflux, 18
h, yield 98%; b. HNO3, H2SO4, CH2Cl2, 0 oC, 20 min, yield 35.3%; c. Pd/C, H2, CH3OH, r.t. 20 h, yield
89.8%; d. 1)4-(bromomethyl)benzoyl chloride, Et3N, CH2Cl2, r.t. 1h; 2)TsOH, xylene, reflux, 20 h, yield
84%; e. LiOH, THF, H2O, r.t. 4h, yield 87%; f. 1) SOCl2, reflux 2h; 2) amine, Et3N, CH2Cl2, r.t. 2h, yield
o
69.3%-76.4%; g. amines, NaOH, DMF, 45 C, 6h, yield 24.4%-26.5%; h. acid, NaOH, DMF, 45 oC, 6h,
21.7%-76.4%.

O
OH
a
Br
O
N
O
O
N
b
R3
NH2
10
11
12
12 a, R3=4-hydroxylphenyl
12b, R3=3,4-dihydroxylbenzyl
12c, R3=3-hydroxylphenyl
12d, R3=3-hydroxylbenzyl
Scheme 2. Synthesis of compounds 12. Reagents and conditions. (a) 1)4-(bromomethyl)benzoyl
chloride, pyridine, toluene, 80 oC, 1h. 2)TsOH, toluene, reflux, 20 h, yield 91%; (b) Carboxylic acid,
NaOH, DMF, 45 oC, 3h, yield 31%-76%.
The half maximal inhibitory (IC50) assays of all target compounds were conducted
against Sortase A with known Sortase A inhibitor pHMB as positive control according to
previously reported fluorescence resonance energy transfer (FRET) assay.17,18 This FRET
assay employs the use of the donor and quencher pair, Abz and Dnp, respectively.
Abz-LPETG-Dnp peptide cleavage was monitored by fluorescence at 420 nm after
excitation at 320 nm. The concentration of inhibitor where 50% of this maximum velocity
is observed is taken to be the IC50. Each experiment was repeated at least three times. Their
IC50 values are listed in Table 1 and Table 2.
Table 1. Inhibitory activity of the target compounds 8a-9x against Sortase A.
H
O
N
R
1
O
N
R
2
Compound
R1
R2
IC50(μM)a
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
136±1.3
8a
8b
N
H
N
127.6±1.8
65.5±0.7
73.2±0.7
81.4±0.3
64.4±1.4
H
O
8c
9a
9b
9c
N
H
OH
OH
O
O
O
O
O
OH
OH
O

NO
OH
2
O
O
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-propyl
i-propyl
i-propyl
i-propyl
n-butyl
n-butyl
n-butyl
n-butyl
n-butyl
87.4±0.9
154.8±6.9
161.1±8.6
20.6±0.2
26.1±0.5
24.8±0.7
83.2±1.8
19.8±0.4
22.4±1.1
71.8±0.4
158.6±1.4
87.4±4.8
116.3±5.9
74.8±0.5
144±3.1
9d
9e
O
O
O2N
O
9f
O
O
9g
O
OH
OH
O
9h
O
OH
O
O
9i
O
O
O
9j
O
NO
2
OH
O
O
OH
OH
9k
HO
O
O
O
OH
9l
HO
O
9m
9n
OH
O
O
NO
2
O
O
9o
OH
OH
O
9p
O
O
OH
OH
O
O
9q
9r
O
OH
OH
OH
O
46.3±0.6
181.3±1.2
177.2±2.9
161.3±1.9
154.8±1.5
184.2±3.0
130±6.1
9s
O
O
9t
O
O
O
9u
OH
O
9v
O
OH
OH
O
9w
9x
O
O
O
OH
Positiveb
pHMB
a
The compounds were tested in at least three independent experiments by fluorescence resonance
energy transfer (FRET) assay.
b Positive control (pHMB) was purchased.

Table 2. Inhibitory activity of the target compounds 12a-12d against Sortase A.
O
R
N
2
Compound
12a
R2
IC50(μM)a
O
O
O
OH
>200
OH
OH
O
>200
>200
12b
O
O
12c
O
OH
OH
>200
12d
O
Positiveb
pHMB
130±6.1
a
The compounds were tested in at least three independent experiments by fluorescence resonance
energy transfer (FRET) assay.
b Positive control (pHMB) was purchased.
As shown in Table 1 and Table 2, seventeen compounds showed higher inhibitory
activity than positive the positive control (130 μM), indicating that the synthesized
compounds are good Sortase A inhibitors. Several compounds (9g, 9h, 9i, 9k and 9l)
exhibited excellent inhibitory activity with IC50 values of 19.8 μM-26.1 μM.
Observing the results, we could deduce some valuable structure activity relationship
(SAR) of the tested benzo[d]oxazole derivatives. Firstly, variations (R1) on the 7-position
of benzo[d]oxazole in this study include i-butyl, i-propyl and n-butyl substitution. The
substitution at 7-position of benzo[d]oxazole has great influence on inhibition, as expected.
Compounds 9a to 9x (R1=i-butyl, IC50=19.8 μM-184.2 μM) display obviously higher
inhibitory activity, as compared to those of 12a to 12d (no substituent at 7-position,
IC50>200 μM), indicating that amide group at 7-position is necessary for the activity, which
is consistent with our design. Secondly, variations (R2) at 2-position of benzo[d]oxazole
comprise benzoate, phenyl acetate, phenyl acrylate and various amines. Substitution on R2
has a great influence on the inhibitory activity. Introduction of –OCH3, -NO2 group instead

of hydroxyl group is significantly detrimental to the activity (9i vs.9j and 9n). The activity
is proportional to the number of hydroxyl groups (9k with three hydroxyl groups vs. 9g
with two vs. 9i with one).
Experimental observations were followed up with molecular docking studies, in which
the most active compounds 9k was docked into the active site of Staphylococcus aureus
Sortase A. Molecular docking studies of 9k were performed on the X-ray crystal structure
of Sortase A and co-crystallized LPATG (PDB code: 2KID) using Gold Suite v5.0.1
(CCDC, Cambridge, U.K., 2010). It has been a consensus that peptide substrates such as
LPXT series bind to Sortase A with a special L-shape mode.4 A ~90o kink at the
Alanine-Proline peptide bond shapes the conformation. Along with the kink pattern comes
the binding of isoleucine (Ile) side chain to the channel comprised of β6/β7 loop and β8.
The Ile binding exerts decisive effect for compound activity.11 Therefore, we design our
ligands with building blocks mimicking the peptide substrates in order to fit in the binding
pocket. Data obtained from docking as depicted in Figure 2A shows that the i-butyl amide
group goes towards the β6/β7 loop-β8 substructure of the protein. Along with our
bio-activity assay, we consider the i-butyl amide binding plays a crucial part for the
inhibitory activity. The benzo[d]oxazole core lies in a hydrophobic pocket composed of
Ala118, Val166, Val168, Val169 and Ile182. Its H-bond with the key residue Arg197 helps
to stabilize the binding conformation. Together with the above interactions, the
benzo[d]oxazole is a key component shaping the whole molecule into a kink pattern. The
linker (benzol group) of 9k is lying on the β strands where a π-π interaction is formed with
Tyr194. Another crucial factor for the LPATG binding is the disulfide linkage between the
Cys184 of the Sortase A and the cysteine of the peptide. We believe the formation of such a
covalent bond is the mechanism of enzyme catalysis. Accordingly, we design our
compounds bearing various functional groups towards the catalytic pocket composed of
His120, Cys184 and Tyr194 to inhibit the enzyme activity. Indeed, as depicted on Figure
2A, the three phenolic hydroxyl groups of 9k form a strong H-bond network with Cys184.
In such way, 9k efficiently blocks the Sortase A enzyme with IC50 of 19.8 μM.

A
B
Figure 2. A: Docking of compound 9k to Sortase A (PBD code: 2KID). The ligand is colored by
cyan, the protein is colored by white and the key residues of the binding pocket are colored by gray; B:
Overlap of the LPAT and compound 9k in the binding pocket of Sortase A. The protein is indicated with
electrostatic potential surface, Compound 9k is colored by cyan and LPAT is colored by violet.
Overlay of compound 9k and LPATG in the binding pocket (Figure 2B) indicates that
these two molecules share similar binding conformation with Sortase A. This includes the
i-butyl stretching, the L-shape pattern and the strong interaction with Cys184. However,
our ligand stretch through the active site while LPATG binds to the pocket with a more
proper pattern. Further modifications on our ligands shall focus on the substituted benzoate
group for more efficient inhibition and the linker of the compound.
In summary, a serial of 2-phenyl-benzo[d]oxazole-7-carboxamide derivatives were
synthesized and screened in-vitro inhibitory activity against Sortase A. Seventeen
compounds showed higher inhibitory activity than positive control. Several compounds (9g,
9h, 9i, 9k and 9l) exhibited remarkable inhibitory activity with IC50 value arranging from
19.8 μM to 26.1 μM. Substitution at 7-position and 2-position of benzo[d]oxazole affected
the inhibitory activities, specially, the i-bubyl group at 7-position was indispensable for
activity. The inhibitory activity was proportional to the number of hydroxyl groups on R2,
and compounds 9k (IC50=19.8 μM) with three hydroxyl groups showed the highest
inhibitory activity.

A docking study of compound 9k was performed on the X-ray crystal structure of Srt
A and co-crystallized LPATG (PDB code: 2KID). The i-butyl amide group went towards
the β6/β7 loop-β8 substructure of the protein and the benzoxazole core lied in a
hydrophobic pocket composed of Ala118, Val166, Val168, Val169 and Ile182, shaping the
whole molecule into a L-shape mode. It presented a rationale for the activity of these
compounds.
Acknowledgements
This research was supported by the National Natural Science Foundation, Beijing,
China (to Dr. Jiang, No: 81202397), the Shanghai Natural Science Foundation Project,
Shanghai, China (to Dr. Jiang, No: 12ZR1415400), and the International S&T Cooperation
Program of China (to Dr. Fu, No. 2014DFA41360).
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16. General procedure for synthesis of 2-phenyl-benzo[d]oxazole-7-carboxamide 8 and 9. To a solution
of compound 7(0.258 mmol), amines or aromatic acids (0.774 mmol) and sodium hydroxide (31mg,
o
0.774 mmol) in DMF (3 mL) were added and the reaction was heated to 45 C and stirred for 3 h. Then
the mixture was poured into H2O (20 mL) and EtOAc(15 mL), the organic layer was washed by water
(20 mL), saturated sodium bicarbonate solution (20 mL), brine (10 mL), and finally dried by anhydrous
Na2SO4. The organic layer was concentrated under vacuum and residue was purified by silica gel
column eluting with dichloromethane/methanol (100:1) to give compound 8 or 9. 8a: pale yellow
solid(yield 26.5%), m.p. 165-167 oC.1H NMR (DMSO-d6) δ 8.46 (s, 1H), 8.18 (d, J=8.2 Hz, 2H), 7.92 (d,
J=7.8 Hz, 1H), 7.72 (d, J=6.8 Hz, 1H), 7.59 (d, J=8.2 Hz, 2H), 7.47 (t, J=7.8 Hz, 1H), 3.80 (s, 2H), 3.22
(t, J = 6.4 Hz, 2H), 1.96 (dt, J=13.6, 6.4 Hz, 1H), 1.49–1.18 (m, 6H), 1.00 (d, J=6.7 Hz, 6H), 0.86 (t,
J=7.2 Hz, 3H). 13C NMR (DMSO-d6) δ 167.59, 162.75, 148.87, 141.63, 140.27, 128.91, 128.40, 128.26,
127.39, 127.28, 123.37, 115.85, 49.29, 47.50, 46.28, 32.72, 28.69, 20.21, 20.08, 12.92. HRMS-ESI
C23H29N3O2 calcd [M+H] 380.2338, found 380.2351. 9a: white solid (yield 56.3%), m.p. 180-182oC. 1H
NMR (DMSO-d6) δ 9.30(s, 1H), 8.44 (s, 1H), 8.18 (d, 2H, J=8.0 Hz), 7.91 (d, 1H, J=7.6 Hz), 7.71 (d,
1H, J=7.6 Hz), 7.53 (d, 2H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.06 (d, 2H, J=8.4 Hz), 6.69 (d, 2H, J=8.4
Hz), 5.18 (s, 2H), 3.62 (s, 2H), 3.19 (t, 2H, J=6.4 Hz), 1.94 (m, 1H), 0.97 (d, 6H, J=6.4 Hz). 13C NMR
(DMSO-d6) δ 171.83, 163.54, 162.84, 156.72, 147.77, 142.59, 140.99, 130.77, 128.66, 127.99, 126.04,
125.50, 125.22, 124.69, 122.64, 120.40, 115.58, 65.41, 47.07, 28.59, 20.57. HRMS-ESI C27H26N2O5,
calcd [M+H] 459.1914, found 459.2005.
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2010, 45, 3752.

Highlight
 Potent Staphylococcus aureus Sortase A inhibitors were designed and synthesized.
 Most compounds exhibited excellent inhibitory activity (IC50=19.8 μM-184.2 μM).
 The structural activity relationship (SAR) and molecular docking studies were
revealed.

Graphical Abstract
Thirty-one 2-phenyl-benzo[d]oxazole-7-carboxamide derivatives were designed and synthesized as
potentially potent Staphylococcus aureus Sortase A inhibitors. Most compounds exhibited excellent
inhibitory activity (IC50=19.8 μM-184.2 μM). The structural activity relationship (SAR) of these
compounds demonstrated that substitution at 7-position and 2-position of benzoxazole has great
influence on the inhibitory activities. The molecular docking studies revealed the i-butyl amide group
and the benzoxazole core shaped the whole molecule into a L-shape mode to be recognized by Sortase
A.
R1
HN
O
O
R2
N
2-Phenyl-Benzo[d]oxazole-7-Carboxamide Derivatives as potential Sortase A inhibitors