Synthesis and SAR studies of 1,4-benzoxazine MenB inhibitors: Novel antibacterial agents against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmcl.2010.08.076

Menaquinone is an essential component of the electron transport chain in many pathogens and consequently enzymes in the menaquinone biosynthesis pathway are potential drug targets for the development of novel antibacterial agents. In order to identify lea

Full text of DOI:10.1016/j.bmcl.2010.08.076

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6306–6309
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR studies of 1,4-benzoxazine MenB inhibitors: Novel
antibacterial agents against Mycobacterium tuberculosis
Xiaokai Li a, Nina Liu a, Huaning Zhang a, Susan E. Knudson b, Richard A. Slayden b, , Peter J. Tonge a,
⇑
⇑
a Institute for Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, USA
b Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Menaquinone is an essential component of the electron transport chain in many pathogens and
consequently enzymes in the menaquinone biosynthesis pathway are potential drug targets for the
development of novel antibacterial agents. In order to identify leads that target MenB, the 1,4-dihy-
Received 28 April 2010
Revised 13 August 2010
Accepted 17 August 2010
Available online 21 August 2010
droxy-2-naphthoyl-CoA synthase from Mycobacterium tuberculosis,
a high-throughput screen was
performed. Several 1,4-benzoxazines were identified in this screen and subsequent SAR studies resulted
in the discovery of compounds with excellent antibacterial activity against M. tuberculosis H37Rv with
Keywords:
Mycobacterium tuberculosis
MenB
MIC values as low as 0.6
lg/ml. The 1,4-benzoxazine scaffold is thus a promising foundation for the
development of antitubercular agents.
Ó 2010 Elsevier Ltd. All rights reserved.
Menaquinone
1,4-Dihydroxy-2-naphthoyl-CoA synthase
1,4-Benzoxazine
The emergence of multi-drug resistant (MDR-TB) and exten-
sively-drug resistant (XDR-TB) strains of Mycobacterium tuberculo-
sis represents a severe threat to human health. Consequently, novel
drugs are needed that are active against drug resistant bacteria and
that also shorten the course of chemotherapy. In addition, novel
agents are also required that are active against latent, non-replicat-
ing populations of bacteria since M. tuberculosis can persist in this
state for many years.1,2 Since it seems likely that latent M. tubercu-
losis bacteria must respire in order to remain viable, compounds
that target respiration are promising candidates for the develop-
ment of drugs that are active against both replicating and non-rep-
licating bacterial populations. Currently we are pursuing the
hypothesis that menaquinone biosynthesis is essential for bacterial
viability in vivo, and are actively engaged in identifying compounds
that inhibit enzymes in this pathway.
Menaquinone (vitamin K1) (Fig. 1) is a polyisoprenylated naph-
thoquinone that shuttles electrons between membrane-bound
protein complexes in the electron transport chain. In mammalian
cells this function is performed by ubiquinone (Fig. 1), and
although menaquinone is required for blood clotting, the biosyn-
thetic pathway for this vitamin is absent in humans. This has re-
sulted in the proposal that enzymes involved in menaquinone
biosynthesis may be promising targets for drug discovery.
O
O
O
R1
R1
H
n
R2
O
R2=
Ubiquinone R1=CH3O, n=6-10 R2=
Plastoquinone R1=CH3, n=9
H
n
H
3
Menaquinone, n=3-8
(Vitamin K2)
Phylloquinone
(Vitamin K1)
Figure 1. Structures of the benzoquinone and naphthoquinone redox cofactors.
The biosynthesis of menaquinone in M. tuberculosis is thought
to mirror the pathway found in organisms such as Escherichia coli,
Bacillus subtilis, and Mycobacterium phlei (Fig. 2),3–5 where it is
known that the men genes are essential for survival.6 Previous
inhibitor discovery efforts have focused on the o-succinylbenzoate
synthase from E. coli and Bacillus anthracis (MenE),7,8 while Crick
and co-workers have demonstrated the antibacterial activity of
inhibitors of the polyprenyl transferase (MenA) from M. tuberculo-
sis,9 validating this pathway as a target for tuberculosis drug
discovery.
MenB, the 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA) syn-
thase catalyzes an intramolecular Claisen condensation leading to
the formation of DHNA from o-succinylbenzoate (Fig. 2).5 In order
to provide a foundation for the discovery of compounds that target
MenB, we used a coupled assay10 to screen 105,091 small drug-like
molecules that contained a large variety of chemical structures.
⇑
Corresponding author. Tel.: +1 970 491 2902; fax: +1 970 491 1815 (R.A.S.); tel.:
+1 631 632 7907; fax: +1 631 632 7960 (P.J.T.).
E-mail addresses: Richard.Slayden@colostate.edu (R.A. Slayden), peter.tonge@
sunysb.edu (P.J. Tonge).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.076

X. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6306–6309
6307
COO-
Table 1
OH
Enzyme inhibition data for 1,4-benzoxazines identified in the high-throughput
H
8
screena
O
COO-
Compound
% Inhibition of MenB at 125 lg/ml
OH
Chorismate
OH
1482I15
1486C16
1615I20
1515O10
68.4 7.3
98.0 1.2
60.3 10.3
77.3 5.2
Menaquinone-8
MenF, MenD
MenH, MenC
MenA, UbiE
a
MenB concentration was 150 nM and OSB concentration was 30 lM.
OH O
SCoA
COO-
O
COO-
O
O
MenB
SCoA
MenE
CoA, ATP
OH
O
OR3
O
OSB
OH
DHNA-CoA
R1
R2
N
N
R1
R2
X
OSB-CoA
R
N
N
A
Figure 2. The menaquinone biosynthesis pathway in E. coli showing the reactions
catalyzed by MenE and MenB.
X
C
O
O
B
Lindsley et al.
anticancer12
Seitz et al.
antibacterial13
Babenysheva et al.
antibacterial18
R1
Following the primary screen, in which compounds were tested at
a single concentration of 125 lg/ml, we obtained 455 hits that had
R2
R3
Y
N
X
R
N
O
O
R
at least 30% enzyme inhibition relative to control (data not shown).
Within these hits, we identified four compounds based on the 1,4-
benzoxazine scaffold (Fig. 3, Table 1). Since a coupled assay was
used for screening, the ability of each compound to inhibit MenE
was also evaluated by directly monitoring the formation of PPi.11
However, none of the cherry-picked compounds affected MenE
R4
He et al.
E
D
Fringuelli et al.
antifungal14
tyrosine kinase
inhibitor15
Figure 4. Representative structures of existing benzoxazines and quinoxalines.
activity at a concentration of 50 lM.
Benzoxazines and quinoxalines (Fig. 4) are privileged ring sys-
tems that are found in a broad range of biologically active mole-
cules with anticancer,12 antibacterial,13 and antifungal activity,14
and also serve as scaffolds for kinase inhibitors.15,16 In order to ex-
plore the utility of this ring system as a starting point for develop-
ing MenB inhibitors, we synthesized 13 compounds as shown in
Scheme 1. In this procedure, the keto group is replaced by an ester
for convenience and the 1,4-benzoxazines are generated by mixing
commercially available 2-aminophenols with dimethyl but-2-yne-
dioate. After 1 h at room temperature the product precipitates and
is recrystallized from MeOH/EtOAc.
O
O
R1
R1
O
O
H
N
R2
R3
NH2
OH
R2
R3
MeOH
RT, 1h
+
O
O
O
O
1-13
Scheme 1. Synthetic route for the (Z)-methyl 2-(2-oxo-2H-benzo[b][1,4]oxazin-
3(4H)ylidene)acetates.
stituent and not from electronic effects. Interestingly, a series of
benzoxazines bearing an ethylsulfonyl group (similar to compound
7), had poor antibacterial activity against E. coli and Staphylococcus
aureus,17 raising the possibility that a reduction in substituent size
in the latter case might lead to an increase in compound activity.
The data in Table 2 also demonstrate that the benzoxazine core
is essential for antibacterial activity as well as enzyme inhibition,
since compounds with quinoxaline (12) or benzothiozine (13)
cores had greatly reduced MIC and IC50 values compared to the
parent compounds. This result is in agreement with studies by Rey-
nolds and co-workers who observed moderate MIC values
The synthesized compounds were evaluated for their ability to
inhibit the reaction catalyzed by MenB as well as the growth of M.
tuberculosis H37Rv (Table 2). Several compounds had MIC values of
less than 1 lg/ml in which there was either no substituent on the
benzoxazine core (1), or in which R2 = F (4) or R3 = F or Cl (9, 10).
However introduction of a methyl group (2, 3, 8) resulted in a
greater than 100-fold reduction in antibacterial activity. In addi-
tion, for substituents at R2 and R3, introduction of larger electron
withdrawing groups (R2 = Cl (5), R2 = NO2 (6), R2 = EtSO2 (7),
R3 = NO2 (11)) also caused a dramatic reduction in antibacterial
activity. Since the methyl group is electron donating, we speculate
that the effect on activity results primarily from the size of the sub-
(6.25
pounds also had poor activity against M. tuberculosis H37Ra and
Mycobacterium avium (>64
g/ml),13 while in separate studies it
lg/ml) against H37Rv for a series of quinoxalines. These com-
l
was also shown that quinoxalines had low activity against both
E. coli and S. aureus.18
O
O
Although substitution of the benzoxazine core generally had a
dramatic effect on the MIC values, the impact of the inhibition of
MenB was much less pronounced. Although it can be seen from
Table 2 that the benzoxazine core is essential for MenB inhibition,
the IC50 values for the benzoxazine analogues only varied by
4–5 fold. These data suggest that other factors such as altered cell
permeability and/or evasion of detoxification strategies may also
modulate antibacterial activity, together with the possibility that
MenB might not be the only target in the cell. To gain further in-
sight into the mechanism of enzyme inhibition, detailed kinetic
studies revealed that compounds 1, 4, and 5 are non-competitive
inhibitors of MenB with Ki and Ki0 values that were similar to the
IC50 values. These compounds can thus bind to the enzyme both
H
N
H
N
Cl
O
O
O
O
O
O
1486C16
1482I15
O
O
H
N
O
S
H
N
O
O
O
1515O10
1615I20
Figure 3. Chemical structures of hits from the HTS that have the common 1,4-
benzoxazine backbone.

6308
X. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6306–6309
Table 2
In vitro activity of the (Z)-methyl 2-(2-oxo-2H-benzo[b][1,4]oxazin-3(4H)ylidene)acetatesa
O
O
R1
H
N
R2
R3
O
O
Compound
R1
R2
R3
IC50b
(l
M)
Ki (
lM)
Ki0
(
lM)
MICc
(lg/ml)
1
2
3
4
5
6
7
8
9
H
Me
H
H
H
H
H
H
H
H
H
H
H
Me
F
Cl
NO2
EtSO2
H
H
H
H
H
H
H
H
H
H
Me
F
10.0 1.0
24.1 1.8
23.1 1.0
27.0 3.0
46.3 3.5
28.2 4.4
17.9 3.0
18.2 2.8
30.0 3.7
35.7 4.8
20.3 1.8
9.1 1.2
67.0 7.9
0.64
25
>100
0.63
5
11.5 1.5
22.5 1.1
10.1 0.9
18.5 2.6
50
>100
100
0.63
0.63
>100
10
11
Cl
NO2
H
O
O
O
O
H
N
12
13
>122
>140
>100
>100
N
H
O
O
H
N
S
a
b
c
MenB concentration was 150 nM.
Compound concentration giving 50% inhibition of enzyme activity.
The lowest concentration of compound that inhibited visible growth of M. tuberculosis H37Rv in all replicates.
Table 3
before and after the varied substrate (OSB-CoA). Since MenB is a
hexamer, it is possible that binding of inhibitor to one subunit af-
fects the ability of substrate to bind to an adjacent monomer. Alter-
natively, there must be a distinct binding site for the benzoxazines
in the MenB monomer separate from the active site, occupation of
which perturbs catalysis. X-ray crystallography is currently being
used to distinguish between these possibilities.
To further explore the antibacterial properties of the 1,4-ben-
zoxazines, we therefore synthesized a second series of compounds
in which the methyl ester was replaced with a substituted phenyl
ring (Scheme 2). In this procedure the commercially available
20-chloroacetophenone was converted to the corresponding
(Z)-ethyl 2-hydroxy-4-oxo-4-(20-chlorophenyl)-but-2-enoate by
treating with diethyl oxalate at room temperature overnight. The
but-2-enoate was then refluxed with a selection of substituted
2-aminophenols in acetic acid for 2 h in order to generate the
1,4-benzoxazine. Interestingly, none of these compounds (14-20)
In vitro activity of the (Z)-3-(2-aryl-2-oxoethylidene)-3,4-dihydro-2H-benzo[b][1,4]
oxazin-2-ones
O
H
N
R2'
R3'
Cl
O
O
0
0
Compound
R2
R3
MICa
(lg/ml)
14
15
16
17
18
19
20
H
Me
F
Cl
H
H
H
H
H
H
3.13
100
3.13
12.5
>100
1.56
3.13
Me
F
H
Cl
a
The lowest concentration of compound that inhibited visible growth of M.
tuberculosis H37Rv in all replicates.
were able to inhibit MenB up to concentrations of 100 lM. Thus,
introduction of a bulky group into the side chain significantly per-
turbs the ability of the 1,4-benzoxazines to inhibit MenB. In addi-
tion, the MIC values increased 5 fold for 14 and 16, and 2.5-fold for
17, (Table 3), compared to the analogous compounds in Table 2,
suggesting that while a portion of their antibacterial activity could
stem from an ability to inhibit MenB, there must be additional
targets for these compounds in the cell. Many antibacterial
benzoxazines previously reported also have bulky side chains
and relatively poor activity against M. tuberculosis19 as well as
other bacteria.17,20 Thus, again we would suggest that activity
might be improved in the latter cases by reducing the size of the
side chain.
The compounds in Table 3 also inhibit the growth of E. coli
M
17 and S. aureus P-209,20 and the SAR data presented here follows
a similar trend to that reported previously in which halogen
O
Cl
O
Cl
O
2-aminophenol
Diethyl oxalate
O
H
R2'
R3'
N
Cl
AcOH, reflux
Benzene, RT,
overnight
O
1h
OH
O
O
14-20
Scheme 2. Synthetic route for the (Z)-3-(2-aryl-2-oxoethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-ones.

X. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6306–6309
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6309
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pounds 3 and 8). Thus, while the introduction of a bulky side chain
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0
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benzoxazines and identifying the proteins in bacteria to which
they bind.
In summary, we have identified a group of 1,4-benzoxazines
with promising in vitro antibacterial activity toward M. tuberculosis
H37Rv. These compounds were identified by screening a compound
library against the 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA)
synthase (MenB) in the M. tuberculosis menaquinone biosynthesis
pathway. However the current SAR data suggest that these com-
pounds act by binding to additional targets within the cell. These
molecules provide a foundation for the development of novel anti-
mycobacterial agents that may ultimately lead to the discovery of
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This work was supported in part by National Institutes of
Health Grants AI044639, AI070383, and AI058785 to P.J.T. High
throughput screening was performed at The National Screening
Laboratory for the Regional Centers of Excellence in Biodefense
and Emerging Infectious Diseases (NSRB) with the support of Na-
tional Institutes of Health Grant U54AI057159. We thank members
of the NSRB and ICCB-Longwood for their help with compound
screening.
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Supplementary data
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Supplementary data (procedures for compound synthesis, en-
zyme assays, and antibacterial activity together with H NMR data
for the synthesized compounds) associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.2010.08.076.
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