Structure-activity relationships of bioisosteres of a carboxylic acid in a novel class of bacterial translation inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.04.074

The discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents which suffered from extensive protein binding has been previously reported. The structure-activity relationships around the carboxylic acid substituent are described herein. This acid was replaced by several alternative functional groups in attempts to retain bioactivity while reducing protein binding. Only groups with an acidic proton retained activity, and analogs containing those groups maintained the protein binding inherent to this class of antibacterial agents.

Full text of DOI:10.1016/j.bmcl.2007.04.074

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4040–4043
Structure–activity relationships of bioisosteres of a
carboxylic acid in a novel class of bacterial translation inhibitors
J. Craig Ruble,a Brian D. Wakefield,a Gregg M. Kamilar,a Keith R. Marotti,b
Earline Melchior,b Michael T. Sweeney,b Gary E. Zurenkob and Donna L. Romeroa,*
aMedicinal Chemistry, Pharmacia Corporation, 301 Henrietta Street, Kalamazoo, MI 49001, USA
bInfectious Diseases Biology, Pharmacia Corporation, 301 Henrietta Street, Kalamazoo, MI 49001, USA
Received 3 April 2007; accepted 24 April 2007
Available online 27 April 2007
Abstract—The discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents which suffered from
extensive protein binding has been previously reported. The structure–activity relationships around the carboxylic acid substituent
are described herein. This acid was replaced by several alternative functional groups in attempts to retain bioactivity while reducing
protein binding. Only groups with an acidic proton retained activity, and analogs containing those groups maintained the protein
binding inherent to this class of antibacterial agents.
Ó 2007 Elsevier Ltd. All rights reserved.
In efforts to overcome the rising problem of bacterial
resistance,1–3 our antibacterial program has focused on
identifying lead compounds with novel mechanisms of
action. We successfully identified a novel class of trans-
lation inhibitors through the use of high throughput
screening and medicinal chemistry optimization
(Fig. 1).4 Advanced compounds in this series failed to
show activity in a standard mouse bacteremia model
of infection, likely due to their high protein binding.
Subsequent optimization efforts focused on further
improving the potency and reducing the affinity of these
leads for serum proteins and led to the synthesis of ana-
log 3.5,6 Since these compounds contain a carboxylic
acid and since human serum albumin is known to bind
aromatic carboxylic acids such as salicylates and ibupro-
fen,7 we began systematically replacing the carboxylic
acid in our series with alternative functional groups in
an effort to reduce the extent of protein binding. This
paper describes the results of these efforts.
large quantities and easily stored (Scheme 1). Conver-
sion of acid intermediate 4 to the acid chloride with oxa-
lyl chloride in methylene chloride with catalytic DMF
afforded 5. Reaction of 5 with commercially available
methyl 2-amino-5-bromobenzoate followed by hydroly-
sis yielded carboxylic acid 6 which we used as our stan-
dard for activity comparisons. Reaction of 5 with other
appropriately substituted anilines afforded the desired
analogs of interest, 7–19.
In general, the requisite bromoanilines were easily pre-
pared via bromination of the 2-substituted anilines with
NBS in DMF at room temperature. The non-commer-
cially available trifluoromethyl-sulfonamido(aniline)
was synthetically accessible via monosulfonylation of
Br
CO2H
NC
CO2H
CO2H
NH
O
NH
O
NH
O
Initial attempts to replace the 2-carboxylic acid focused
on replacing the acid with a variety of other functional
groups to ascertain the impact on bioactivity. Synthesis
of the requisite analogs was accomplished through the
use of common intermediate 4 which can be made in
N
O
O2S
N
O2S
N
3
BnS
2
1
MIC
SAUR 9218
0.125 μg/mL (no serum)
4 μg/mL (10% serum)
Keywords: Bioisostere; Antibacterial; Translation inhibitor; Protein
binding; Carboxylic acid.
Cl
*
Figure 1. Evolution of translation inhibitor lead series.
Corresponding author. E-mail: donna.romero@sbcglobal.net
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.074

J. C. Ruble et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4040–4043
4041
H
CH3
N
COOH
HOOC
NEt3
Br
Br
Cl
OH
NH
O
O
(COCl)2
+
S
NH
NaBH4
MeOH
N
CH2Cl2, cat. DMF
O
4
CH3
O
O
Cl
SO2Cl
O
O
S
O
CH3
Br
R
S
O
CH3
18
N
13
N
Br
R
ClOC
NH
O
NH2
S
CH3
CH3
O
OH
NH
N
O
O
O
Cl
Cl
Br
CH2Cl2
pyridine
Br
S
CH3
O
N
O
5
6-19
NH
LiBH4
Cl
O
O
O
O
Cl
S
O
CH3
S
CH3
21
19
N
N
O
Scheme 1. Preferred synthesis of 5-bromo aryl analogs containing
carboxyl replacements.
Cl
Cl
1,2-phenylenediamine (Scheme 2). Some of the desired
analogs were not synthesized utilizing the route shown
in Scheme 1 either because the functional groups were
not compatible with the amide bond-forming condi-
tions, or because they were more readily synthesized
via an alternate route. For example, reduction of the
methylketone 13 or the methyl ester 21 led to the sec-
ondary or primary alcohols 18 and 19, respectively
(Scheme 3).
Scheme 3. Alternate synthesis of analogs via direct elaboration of
2-substituent.
Table 1. Activity of compounds synthesized according to Schemes 1–3
Compound
R
% T/T inhibitiona SAUR MICb
at 100 lM
(lg/mL)
6
7
–COOH
–H
–CH3
79–82
11
12
6
8–16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
Analogs synthesized by these routes and the correspond-
ing bioassay data in the transcription/translation (T/T)
inhibition assay8 and the antibacterial assay versus
Staphylococcus aureus are shown in Table 1. Substitu-
tion of the carboxylic acid moiety by most functional
groups resulted in analogs devoid of activity (7–16;
18–19). The only analog showing significant activity in
both the T/T inhibition assay and the antibacterial assay
was the 2-(trifluoromethyl)sulfonamido analog 17, how-
ever it was less potent in the antibacterial assay. These
initial results indicated that the acidic moiety is critical
for activity (Table 1). The pKa’s of the only two active
compounds are calculated to be 2.96 for the acid com-
pound 6 and 4.28 for the (trifluoromethyl)sulfonamido
analog 17.9
8
9
–Br
–CN
10
11
12
13
14
15
16
17
18
19
0
–NO2
0
–SO2NH2
–COCH3
–CONH2
–CONHOCH3
–NHSO2CH3
–NHSO2CF3
–CH(OH)CH3
–CH2OH
0
0
13
7
19
72
7
>128
>128
4
a Staphylococcus aureus coupled transcription–translation assay (Ref.
8).
b Minimum inhibitory concentration versus Staphylococcus aureus
UC9218.
In addition to their high overall lipophilicity and molec-
ular weight, we continued to believe that analogs in this
series suffered from extensive protein binding in part due
to the presence of the carboxylic acid. After confirming
that many of the active analogs were bound tightly to
human serum albumin (HSA), which accounts for 60%
of the total mass of plasma proteins, we felt that there
was still a compelling reason to investigate acid replace-
OH
NH2
Br
NH2
Br
N
O
N
NH2
Br
N
(EtO)2CO
CN
HO-NH2
O
NH2
H
24
23
22
NH2
NH2
OH
CF3
CF3
NH2
CF3(CO)CF3
TsOH (cat.)
NH2 H
N
NH2 H
N
NH2
ClSO2CF3
SO2CF3
NBS
SO2CF3
Br
Br
xylenes
120 oC
26
Br
25
Scheme 2. Synthesis of a non-commercially available aniline.
Scheme 4. Synthesis of specialized anilines bearing bioisosteres.

4042
J. C. Ruble et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4040–4043
Table 2. Activity of compounds synthesized according to Scheme 1
ments. Therefore, we next turned our attention to the
synthesis of a variety of known carboxylic acid bioisos-
teric replacements10 to explore their activity and protein
binding properties.
b
Compound
R
% T/T Inhib. SAUR 9218
MICa (lg/mL)
pKa
at 100 lM
6
–COOH
79–82
8–16
2.96
3.75
4.85
6.37
6.55
8.50
Some of the requisite analogs (32,33) were prepared via
Scheme 1 using the appropriately substituted anilines
synthesized as depicted in Scheme 4. For example, nitrile
22 was treated with hydroxylamine to afford amidoxime
23, which when treated with diethyl carbonate afforded
the elaborated aniline 24. Similarly, treatment of 4-bro-
moaniline 25 with hexafluoroacetone and catalytic tolu-
enesulfonic acid provided elaborated aniline 26.
Tetrazoles 27 and 29 were prepared via elaboration of
the corresponding nitriles 10 and 28 (Scheme 5). Oxa-
diazolone 31 was prepared from the corresponding
hydrazide 30 with CDI.
N
N
N
27
29
31
32
33
96
46
60
98
41
16
>128
2
N
H
N
N
N
N
H
N
NH
O
O
O
O
N
8–16
0.5
N
H
Table 2 contains the results of the T/T inhibition assay
and the antibacterial activity of these bioisostere ana-
logs. The extended tetrazole analog 29 exhibited the
poorest activity from this group. Tetrazole 27 and oxa-
diazolone 32 exhibited good T/T inhibition and antibac-
terial activity comparable to the carboxylic acid (MICs
ꢀ16 lg/mL). Oxadiazolone 31 and the hexafluoro-iso-
propanol 33 exhibited more modest T/T inhibition but
good antibacterial activities (MICs of 2 and 0.5 lg/
mL, respectively).11 The calculated pKa’s for the bioisos-
teres are also shown in Table 2. Even though the bioisos-
teres’ calculated pKa’s are not as low as that of the
carboxylic acid, several appear to be suitable replace-
–C(OH)(CF3)2
a Minimum inhibitory concentration. Staphylococcus aureus UC9218.
b pKa calculated using PALLAS.
ments in terms of potency and seemed to offer promise
in terms of reducing the protein binding.
To determine whether the bioisosteres’ performance in
the presence of serum was superior to that of the parent
acid, they were tested in the antibacterial assay in the ab-
sence and presence of both 5% and 10% serum (Table 3).
By evaluating the ratio of MICs in the presence and
absence of 5% serum (column entitled ‘Ratio’) it is
apparent that relative to the acid, the bioisosteres have
a similar (compound 32) or even greater degree (com-
pound 33) of protein binding. The increased antibacte-
rial potency11 of compound 33 is helpful in that some
antibacterial activity in the presence of 5% HSA is
retained. Vancomycin and novobiocin were used as
standards to gauge what level of antibacterial activity
in the presence of HSA was needed to observe an
in vivo effect. Clearly, MICs in the presence of 10%
HSA for both these standards were significantly lower
N
N
N
Br
Br
CN
NH
N
H
NH
NaN3
CH3
N
CH3
N
O
O
10
S
27
S
O
O
O
O
Cl
Cl
N N
N
N
H
CN
Br
NaN3
Br
Table 3. Activity of selected bioisosteres in the presence of serum
NH
Compound
MIC
Ratioc
MIC
NH
SAURa
5% HSAb
10% HSAd
CH3
CH3
O
O
N
O
N
O
6
31
8–16
2
128
—
8–16
—
>128
>128
>128
>128
1
S
S
29
28
O
O
Cl
Cl
32
33
8–16
0.5
>128
64
>8–16
128
1
H
Vancomycine
1
>0.125
1
0.5
N N
O
Br
CDI
Novobiocin
—
1
Br
CONHNH2
NH
O
a Minimum inhibitory concentration (lg/mL). Staphylococcus aureus
UC9218.
NH
CH3
N
b Staphylococcus aureus UC9218 + 5% pooled human serum. Human
serum (male, from Sigma) was thawed at room temperature, then
placed in a 56 °C water bath for 30 min. The serum was filtered using
a 0.2 lm filtration system.
CH3
N
O
O
S
S
O
O
O
O
Cl
Cl
30
31
c Ratio = MIC (5% HSA)/MIC (0% HSA).
Scheme 5. Synthesis of bioisosteres via direct elaboration of 2-
substituent.
d Staphylococcus aureus UC9218 + 10% pooled human serum.
e Positive control vancomycin.

J. C. Ruble et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4040–4043
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http://www.acdlabs.com/.
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11. The lack of correlation of T/T activity with antibacterial
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suggest that compound 33 may act via a different
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Fiziologicheski Aktivnye Veshchestva 1972, 4, 41.
than the acid or its bioisosteres. For the translation
inhibitor compounds, even the addition of just 10%
HSA caused all antibacterial activity to be lost.
Conclusion. In an effort targeted at reducing the protein
binding of this series of translation inhibitors, the SAR
surrounding the acid substituent was explored to probe
whether it could be replaced with functional groups less
likely to bind to HSA. A trifluoromethylsulfonamide
replacement (17) was the only functional group that
came close to the activity of the carboxylic acid. Our re-
sults indicate that a functional group containing an
acidic proton is required for good potency. In addition,
some bioisosteres, such as a oxadiazolone (32), could re-
place the acid and offered comparable bioactivity. The
bioisosteres did not offer any advantages over the acid
in terms of reducing protein binding. These results led
to a future chemistry strategy which focused on the
use of the carboxylic acid moiety in the design of new
analogs.
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