Development of improved inhibitors of wall teichoic acid biosynthesis with potent activity against Staphylococcus aureus

Article abstract of DOI:10.1016/j.bmcl.2010.01.036

A small molecule (1835F03) that inhibits Staphylococcus aureus wall teichoic acid biosynthesis, a proposed antibiotic target, has been discovered. Rapid, parallel, solution-phase synthesis was employed to generate a focused library of analogs, providing detailed information about structure-activity relationships and leading to the identification of targocil, a potent antibiotic.

Full text of DOI:10.1016/j.bmcl.2010.01.036

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1767–1770
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of improved inhibitors of wall teichoic acid biosynthesis with
potent activity against Staphylococcus aureus
Kyungae Lee a, Jennifer Campbell b, Jonathan G. Swoboda b, Gregory D. Cuny c, Suzanne Walker b,
*
a The New England Regional Center of Excellence in Biodefense and Emerging Infectious Diseases (NERCE/BEID), Harvard Medical School, Boston, MA 02115, USA
b Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
c Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women’s Hospital and Harvard Medical School, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A small molecule (1835F03) that inhibits Staphylococcus aureus wall teichoic acid biosynthesis, a pro-
posed antibiotic target, has been discovered. Rapid, parallel, solution-phase synthesis was employed to
generate a focused library of analogs, providing detailed information about structure–activity relation-
ships and leading to the identification of targocil, a potent antibiotic.
Received 3 November 2009
Revised 4 January 2010
Accepted 6 January 2010
Available online 20 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Wall teichoic acids (WTAs)
Staphylococcus aureus
Antibiotic
Bacteriostatic
Structure–activity relationship (SAR)
Triazoloquinazoline
Targocil
More people now die from hospital-acquired methicillin-resis-
tant Staphylococcus aureus (MRSA) infections in the United States
than from HIV/AIDs.1 Until recently, vancomycin was known as
the ‘last line of defense’ against these infections, but high-level
vancomycin resistance has now begun to appear in S. aureus.2–4
Although two new classes of antibiotics to treat S. aureus infections
have been introduced in the past decade, clinical resistance to each
has already appeared and can be expected to spread.5–7 For these
reasons, a deep pipeline of novel antibiotics is required to combat
MRSA infections.8
One suggested but as yet unexploited antibiotic target in S. aur-
eus is wall teichoic acid (WTA) biosynthesis.9,10 WTAs are anionic,
phosphate-rich, carbohydrate-based polymers that are synthesized
on a lipid carrier inside the bacterial cell before being transported
to the cell surface where they are covalently linked to peptidogly-
can.11 Their biological functions are diverse, ranging from cation
homeostasis to critical roles in host colonization.12 They are pro-
posed virulence factors since deleting the biosynthetic pathway
by knocking out the first gene (tarO) prevents infection. The down-
stream enzymes in this pathway are potential antibiotic targets
since initiating flux into the biosynthetic pathway without com-
pleting it is deleterious to bacterial viability.13
We have discovered the first small molecule that specifically
inhibits WTA biosynthesis using a cell-based, pathway-specific
high-throughput screen that reports only on the antibiotic targets
in the pathway. From a screen of 55,000 compounds, we identified
1835F03 (1) as a WTA-active antibiotic.14 Compound 1 has good
antibiotic activity (low
lM) against all S. aureus strains tested,
including both hospital- and community-acquired MRSA isolates.
We identified the target of 1 as TarG, the transmembrane compo-
nent of the two component ABC transporter that exports WTAs
from the cytoplasm to the external surface of the bacterial mem-
brane where they are attached to peptidoglycan (Fig. 1).14 Two
other compounds (1856A19 and 1856K21) that share a similar
core with 1 were also identified as confirmed ‘hits’ from the screen,
but their potencies were lower (Fig. 2). Moreover, commercial ana-
logs of this class of inhibitors (1856A19 and 1856K21) appeared to
have diminished target specificity relative to 1 since they showed
partial growth inhibitory activity against the
DtarO mutant strain
(see Supplementary Table 1). Therefore we focused on 1 as a lead
for optimization.
The commercially available analogs of compound 1 had limited
structural variations and all lacked activity (see Supplementary
Figs. 1 and 2). Therefore, we prepared a focused library of com-
pound 1 analogs in order to identify sites on the scaffold that could
be altered to improve potency without sacrificing selectivity for
the target. The studies reported below provide key information
about where this class of compounds is amenable to modification
* Corresponding author. Tel.: +1 617 432 5488.
E-mail address: Suzanne_Walker@hms.harvard.edu (S. Walker).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.036

1768
K. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1767–1770
Figure 1. Poly(ribitol-phosphate) wall teichoic acids are constructed on a bactoprenol carrier lipid (C55–P; partial structures shown here) embedded in the cytoplasmic
membrane in S. aureus. TarGH is the ABC transporter that exports WTAs and is the target of the class of small molecule antibiotics described here. X and Y denote tailoring
modifications of the WTA polymer (e.g., alanylation and glycosylation).
However, use of potassium carbonate in place of sodium methoxide
(as previously published) resulted in moderate improvement. Chlo-
OMe
H
rination of the resulting quinazolone using phosphorus oxychloride
in the presence of tetramethylammonium chloride afforded the
chloroquinazoline, which was converted in a straightforward man-
ner to the corresponding aminoquinazoline derivatives. Whenever
possible, the synthesis was carried out in a parallel format where
the reactions were conducted in vials and the products were worked
up by parallel solid phase extraction (SPE) using strong cation ex-
change (SCX) cartridges. Interestingly, attempts to remove the
methyl ethers of 38 (in order to install solubilizing functional
groups) utilizing boron tribromide caused ring opening of the tria-
zole with concomitant loss of nitrogen, yielding inactive compounds
(see SI for details).
N
N
Cl
N
N
N
O
S
A
B
O
O
NNC
N
N
D
O
R
1835F03 (1)
1856A19 R = H
1856 K21 R = OMe
Figure 2. Chemical structures of the most active hits discovered in our high-
throughput screen, with ‘common’ core structures shown in bold.
All compounds were tested for antibacterial activity against a
common laboratory strain of S. aureus, RN4220, and against the
and have led to the identification of an analog 10 times more
potent than 1 and is non-toxic in mice at doses of 75 mg/kg.
Based on the structure of 1 (1835F03), libraries of compounds
with varying core substitution and side chains were prepared. The
triazoloquinazoline core was synthesized following previously pub-
lished methods (Scheme 1).15 The 2-azidobenzoic acid or ester, pre-
pared from the corresponding anthranilic acid derivative, was
heated with an arylsulfonylacetonitrile in the presence of base to
furnishthetriazoloquinazoloneframeworkin onestep. Thisreaction
is believed to proceed via 1,3-dipolar cycloaddition of the azide to
the enolate form of the nitrile, followed by cyclocondensation of
the resulting aminotriazole. The yields of the triazoloquinazolones
varied depending on the substituents on the azidobenzoic acid.
While the cyclization proceeded smoothly with electron-deficient
ring systems, such as chloro- or nitro-substituted azidobenzoic
acids, yields were low to moderate with electron-rich substrates.
isogenic RN4220
DtarO strain, which lacks the first gene in the bio-
synthetic pathway and is thus not susceptible to WTA inhibitors.
This latter strain allows for assessment of compound specificity
since any toxic effects against it must be due to mechanisms other
than WTA inhibition. The minimum inhibitory concentrations
(MIC) of all compounds are reported against both strains in Tables
1 and 2 and Figure 3. Active compounds were subsequently tested
against clinical S. aureus isolates (see Table 3).
The first analog library was designed to investigate the posi-
tions, sizes and electronics of the A-ring substituents. Commercial
starting materials were chosen to probe the amenability of this
area of the molecule to derivatization. The chloro group was placed
at all possible locations on the A-ring (2–4), but activity was lost
when this functionality was displayed anywhere other than C2.
Therefore, we sought to optimize the substituent at this position.
Modest changes at the C2 position (i.e., F, Me, Br, NO2, CN; 5–9)
gave compounds with lower activity than the parent C2 chloro
compound (1). Replacement of the chloro group with a methyl
(6), bromo (7), or methoxy (10) substituent was tolerated, while
a fluoro (5), nitro (8), or cyano (9) group abolished activity. From
these data, it appears that size rather than electronics is the deter-
mining factor for activity. Likewise, substituents displayed at the
C3 position resulted in inactive compounds 11–14. Surprisingly,
however, 2,3-dimethoxy substitution on the A-ring yielded a po-
tent compound (15). Since both the mono-methoxy derivatives
(10 and 14) are less active than the parent compound 1, the effects
of changing these positions were not additive. The benzodioxane
CO2R
NH2
CO2R
N3
NaNO2
NaN3
ArSO2CH2CN
X
X
NaOMe
R: H or Me
NR2
N
O
1. POCl3, Me4NCl
NH
N
2. R2NH
X
X
SO2Ar
SO2Ar
N
N
N
N
N
Scheme 1. Synthesis of triazoloquinazoline analogs.

K. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1767–1770
1769
Table 1
A-ring derivatives and their corresponding minimum inhibitory concentrations (MIC) against Staphylococcus aureus RN4220
N
1
2
R
3
N
O
O
S
N
N
4
N
Compd
R
Wildtype S. aureus MIC (
lM)
DtarO S. aureus MIC (lM)
1
2
2-Cl
1-Cl
3.6
na
naa
na
3
3-Cl
na
na
4
4-Cl
na
na
5
6
2-F
2-Me
>100
25
na
na
7
8
9
2-Br
2-NO2
2-CN
12.5
>100
na
>100
na
na
10
11
12
13
14
15
16
2-OMe
3-Me
3-Br
3-NO2
3-OMe
2,3-di-OMe
2,3-OCH2CH2O–
25
na
na
na
100
50
>100
na
>100
100
>100
1.8
na
na
a
na (not active) indicates <30% growth inhibition at 100 lM. MICs are represented as >100 lM if the compound gave between 31% and 89% growth inhibition at the highest
concentration tested (100 lM).
Table 2
D-ring substitutions can enhance biological activity. The lack of activity against the
D
tarO strain indicates specificity for the WTA biosynthetic pathway
N
R1
N
O
O
O
O
S
N
N
N
R2
Compd
R1
R2
Wildtype S. aureus MIC (
l
M)
DtarO S. aureus MIC (lM)
15
37
38
39
H
H
H
CH3
H
CH3
Cl
1.8
2.5
0.3
2.5
naa
na
na
na
Cl
a
na [not active] indicates <30% growth inhibition at 100
lM.
analog (16) was also prepared, but found to be inactive, suggesting
Finally, we explored changes to the D-ring. Compounds 37 and
38 were made, which contain a para-methyl and para-chloro sub-
stituent on the D-ring, respectively (Table 2). The 4-chlorophenyl-
sulfone analog (38) exhibited the highest antibacterial activity of
that some flexibility of the substituents may be important. The 2,3-
dimethoxy substitution on the A-ring was incorporated into addi-
tional analogs in anticipation that it would give improved physico-
chemical properties (e.g., cLog P) compared to the C2 chloro.
With the A-ring substitution fixed, variations to the amine side
chains of the B-ring (Fig. 3) were examined, including several pri-
mary and secondary amines (17–36), but few variants matched the
potency of the parent diethylamine side chain (15). From the MIC
data, it was clear that neither a hydrophilic moiety (e.g., 21, 27, 28,
30) nor a larger, hydrophobic group (e.g., 19, 20, 33–36) was toler-
ated. Only the acyclic secondary amines of small alkyl groups im-
parted reasonable antibacterial activity (i.e., 24–26). It is
surmised that this residue binds to a hydrophobic pocket of limited
size and depth so that it can only accommodate C2–C4 alkyl groups
at the nitrogen atom. Therefore, additional optimization was pur-
sued retaining the diethylamine group on the B-ring.
all compounds made, with an MIC of 0.3 lM against the S. aureus
lab strain (RN4220), which shows that modifying the D-ring can
have beneficial effects. A broader exploration of D-ring substitu-
tions was hampered by the lack of commercially available aryl-
sulfonyl-acetonitriles. However, since the biological activity of 38
is sufficient (see below) we did not pursue other synthetic ap-
proaches to make D-ring analogs. Efforts to combine the best fea-
tures from each sublibrary (namely, the 2,3-di-OMe on the A-ring,
ethylisopropylamine on the B-ring and para-chloro on the D-ring)
generated 39. However, its activity was not improved over 38.
The original inhibitor 1 and two representative active analogs
(15 and 38) were tested against four additional S. aureus strains
(Table 3). All showed growth inhibitory activities against both

1770
K. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1767–1770
R
N
quency estimated for compound 1.14,16 The measured frequency of
O
O
resistance to targocil (38) in vitro is still higher than is typically
desirable, butmanyof theresistantmutantsarepathwaynulls. Since
these mutants are avirulent, they are not expected to contribute to
resistance in vivo, and the compound is thus highly promising.9,10
The acute toxicity of targocil (38) is acceptable. Doses of 75 mg/
kg administered via tail vein injection (see SI for details) caused no
adverse effects in mice after 24 h. Although the MIC of targocil (38)
shifts by a factor of eight in the presence of 50% fetal bovine serum,
its tolerated dose in mice is greater than 100-fold the serum MIC.
In conclusion, a structure–activity relationship study of the
WTA inhibitor 1 identified positions that are tolerant of substitu-
tion and led to the discovery of an analog (targocil, 38) that is
10-fold more potent than the original screening hit while also hav-
ing a 10-fold reduced mutational frequency. Targocil (38) is active
against methicillin-sensitive and resistant clinical strains, and
analysis of resistant mutants shows that it inhibits the same target
as 1 (TarG). Its properties suggest that it is a candidate for proof of
concept studies to address whether inhibiting WTA biosynthesis is
a promising strategy for treating bacterial infections in vivo.
N
O
S
O
N
N
N
HN
N
N
N
N
N
Ph
CO Me
2
17
18
19
20
21
22
23
>100 µM
N
N
N
N
N
HN
OMe
EtO
OEt
N
24
25
26
27
28
29
1.6 µM
3.1 µM
6.3 µM
>100 µM
>100 µM
N
O
N
N
N
N
N
N
N
N
N
Ph
Ac
Ph
Bn
30
31
32
33
34
35
36
>100 µM
>100 µM
Acknowledgments
Figure 3. SAR of the B-ring amine side chains. MICs are given as the lowest
concentration of compound (tested) that gave >90% growth inhibition in wildtype
This work was supported by the NIH (1P01AI083214 and
5R01GM078477 to S.W., F32AI084316 to J.C., and F3178727 to
J.G.S.) and support to K.L. and G.D.C. was provided by the National
Screening Laboratory for the Regional Centers of Excellence in Bio-
defense and Emerging Infectious Diseases (NIAID U54 AI057159).
We would like to thank Su Chiang and Gerald Beltz for helpful
discussions.
S. aureus RN4220. MICs are represented as >100
31% and 89% growth inhibition at the highest concentration tested (100
Compounds 33 and 36 showed greater than 70% growth inhibition in the
mutant at 100 M, whereas the remaining derivatives gave less than 30% growth
l
M if the derivative gave between
M).
tarO
l
D
l
inhibition (data not shown).
Table 3
MICs of the original inhibitor and two analogs against several S. aureus strains
Compds RN6390 MICa
M)
Newman MIC
M)
COL MIC
M)
MW2 MIC
(lM)
Supplementary data
(
l
(
l
(
l
1
15
38
6.3
3.1
0.3
12.5
3.1
0.3
6.3
3.1
0.6
6.3
3.1
0.6
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.01.036.
a
MICs are given as the lowest concentration of compound (tested) that gave
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Compound 38, which we have named targocil, has a bacterio-
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activity depends on flux into the WTA biosynthetic pathway since
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biosynthetic pathway as 1. For example, targocil (38) shows
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