Preparation of novel anthranilic acids as antibacterial agents: Extensive evaluation of structural and physical properties on antibacterial activity and human serum albumin affinity

Article abstract of DOI:10.1016/j.bmcl.2007.03.036

In the past few years a significant effort has been devoted by Pharmacia toward the discovery of novel antibiotics. We have recently described the identification of an anthranilic acid lead 1 and the optimization resulting in the advanced lead 2. In this

Full text of DOI:10.1016/j.bmcl.2007.03.036

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3113–3116
Preparation of novel anthranilic acids as antibacterial
agents: Extensive evaluation of structural and physical properties
on antibacterial activity and human serum albumin affinity
Atli Thorarensen,*, Jianke Li, Brian D. Wakefield, Donna L. Romero, Keith R. Marotti,
Michael T. Sweeney, Gary E. Zurenko and Ronald W. Sarver
Medicinal Chemistry and Infectious Diseases Biology, Pharmacia Corporation, 301 Henrietta Street, Kalamazoo, MI 49001, USA
Received 19 February 2007; accepted 12 March 2007
Available online 15 March 2007
Abstract—In the past few years a significant effort has been devoted by Pharmacia toward the discovery of novel antibiotics. We
have recently described the identification of an anthranilic acid lead 1 and the optimization resulting in the advanced lead 2. In this
report, we describe the preparation of several selected analogs to probe the dependency of this template for antibacterial activity and
the affinity these compounds have for human serum albumin (HSA). These analogs illustrate that decreased affinity for HSA can be
achieved while retaining relevant antibacterial activity. The most important factor for reduced HSA affinity is decrease in logP
rather than a structural change.
Ó 2007 Elsevier Ltd. All rights reserved.
Emergence of bacterial resistance is a significant prob-
lem in the treatment of bacterial infections,1 and this
has fueled a continual search for novel antibiotics result-
ing in numerous commercially available products. We
recently reported2 on the optimization of acid 1 resulting
in the discovery of lead compound 2 which displayed
potent broad-spectrum antibacterial activity (Fig. 1).
Extensive results from biological assays indicated that
high affinity to human serum albumin3 (HSA) was the
main reason for lack of in vivo activity of 2. An obser-
vation in the literature describes the utility of anthranilic
acids as high affinity ligands for HSA.4 There are
numerous ways to evaluate the affinity of different li-
gands for HSA such as chromatographic, spectroscopic,
and displacement of a high affinity ligand from HSA.5 A
very common approach described in the literature is the
addition of serum to the biological assay. The difference
of activity in the presence and absence of serum can be
related to HSA affinity.6 The affinity to HSA can be
O
CO2H
Br
CO2H
A
B
NC
OH
NH
NH
O
NH
O
N
O
O
O2S
N
O2S
N
2
BnS
3
1
Cl
Figure 1. Progression in lead optimization.
described as binding constant (Kd), % free fraction, or
a ratio of activity in the presence or absence of serum.
The difficulty inherent in protein binding can be illus-
trated by looking at the case of a compound with a Kd
of 1 lM for HSA at a fixed compound concentration.
At the biologically relevant concentration of 600 lM
HSA, the free fraction will only be 0.2%. A decrease in
affinity for HSA such as an increase in the Kd to
10 lM results in a free fraction of 1.6%. An early exam-
ple of the evaluation of antibiotics’ affinity to HSA was
the measurement of MIC activity of a range of commer-
cially available antibiotics in the absence and presence of
Keywords: Antibacterial; HSA; Human serum albumin; Protein
binding.
*
Corresponding author. Tel.: +1 636 247 3962; fax: +1 636 247
0250; e-mail: atli.thorarensen@pfizer.com
Present address: Pfizer, 700 Chesterfield Parkway West AA2G,
Chesterfield, MO 63017, USA.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.036

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A. Thorarensen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3113–3116
100% serum.7 Highly bound drugs such as Dicloxacillin
require good pharmacokinetic properties and signifi-
cantly more potency to exert the same effect as less pro-
tein bound drugs.8 In general, it is only the free fraction
of a drug that is of biological consequence (or rele-
vance?). A rule of thumb has been proposed that in
order to observe in vivo activity with 80% certainty,
Cfmreaex=MIC P 8:1.8b We elected to utilize the primary
MIC assay in the presence and absence of serum as a
primary indicator for monitoring improvements in
potency and decreases in HSA affinity. The major
advantage of the MIC assay was high throughput,
acceptable accuracy, and a measurement of overall
HSA affinity. Utilizing this approach we recently
reported on optimization of 2 targeting the reduction
of its affinity for human serum albumin (HSA) which re-
sulted in advanced compound 3.9 A detailed description
of the SAR with regard to structural tolerance correla-
tion with lipophilicity and how that relates to HSA affin-
ity are presented in this report.
CN
O
i), ii), iii)
O
Br
OH
N
H
7
N
8
N
O
OH
Scheme 2. Reagents: (i) (COCl)2, DCM, pyridine, t-butyl-2-amino-4-
cyano-benzoate (61%); (ii) 4-t-butyl-benzene boronic acid, tetrakis(tri-
phenylphosphine) palladium, Na2CO3, THF/water (75%); (iii) TFA
(57%).
Table 1. Antibacterial activity of new probe analogs
Compound
MICa (lg/mL)
SAURb
SAURc
SAURd
6
8
16
32
>128
>128
>128
>128
a Minimal inhibitory concentration.
b Staphylococcus aureus UC 9218.
c Staphylococcus aureus UC 9218 + 5% 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 then filtered using a 0.2 lm
filtration system.
The potential for rational design of new anthranilic ana-
logs with decreased HSA affinity. A rational approach
is the utilization of protein crystallography to engineer
in non-favorable interaction. A few structures of HSA
have been determined10 and a recent report for a suc-
cessful rational design of non-favorable interaction has
been described for Diflunisal analogs.11 In order to eval-
uate if this would be a feasible approach here we elected
to prepare analogs that were confirmationally rigid, dis-
playing a bulky group where SAR indicated that substi-
tution was allowed. This could be accomplished on each
periphery of the core such as example 4 (Fig. 2).
d Staphylococcus aureus UC 9218 + 10% serum.
antibacterial agents (Table 1). Despite our efforts to
bump these analogs out of their HSA binding site via
their large linear size, they were still excellent substrates
for HSA. These observations diminished our hope that
rational design via steric interaction with HSA would
be a viable path forward in new analog design. This
was not surprising since HSA binding characterization
of the deschloro analog of 2 was found to have high
affinity to both the ibuprofen (Kd ꢀ 1 lM) and the war-
farin site (Kd < 1 lM).12 Because of the high affinity for
multiple binding sites (vide supra), structural alteration
for affinity for one site could be tolerated by the other
and vice versa.13 The net result is still high overall affin-
ity for HSA.
The analogs were prepared by sequential amide coupling
followed by a Suzuki reaction and finally a TFA depro-
tection in modest overall yield, Schemes 1 and 2. This
approach allowed us to rapidly prepare numerous ana-
logs in each series (data not shown). The key observa-
tion is that compounds 6 and 8 were active as
Systematic evaluation of physical properties with antibac-
terial activity and HSA affinity. A less direct approach to
reducing protein binding is to guide SAR based on the
correlation of physical parameters with bioactivity and
HSA affinity. This has been a very common approach
since the initial disclosure by Hansch applied to a series
of penicillins.14 There are numerous reports on the cor-
relation of physical parameters with activity of antibac-
terial agents mainly for penicillins, and in general, the
major correlation is between lipophilicity and HSA
affinity.15 In addition, acidic groups are known to in-
crease affinity for HSA. A recent publication describes
a detailed analysis where the major factor for HSA affin-
ity appears to be the compound’s lipophilicity.16 In
addition to lipophilicity, the analysis indicated that cyc-
lic structures especially six-membered rings have high
affinity for HSA. Affinity was decreased by branching
or conversion to acyclic systems.
O
OH
A
NH
O
B
4
Figure 2. Design of probe analogs.
Cl
O
OH
CO2H
H
Cl
N
O
i), ii), iii)
N
S
O
O
N
6
S
5
O
O
In order to evaluate these possibilities, we selected to
prepare a series of pyridyl-thioethers, where we could
modulate both the logP as well as the nature of the sub-
stituent in a systematic fashion. Our initial approach to
Scheme 1. Reagents: (i) (COCl)2, DCM, pyridine, t-butyl-2-amino-4-
iodo-benzoate (55%); (ii) 4-t-butyl-benzene boronic acid, tetrakis(tri-
phenylphosphine) palladium, Na2CO3, THF/water (61%); (iii) TFA
(75%).

A. Thorarensen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3113–3116
3115
the synthesis of these compounds was to prepare the in-
CN
O
i), ii), iii)
O
tact extended analog and displace a chloride with a thio-
late in the last step. This worked very well for methyl
and ethyl thiolates, Scheme 3. However, when the chain
length was increased to propyl none of the desired mate-
rial was obtained despite several optimization attempts.
An alternative approach was designed that relied on the
reduction of disulfide followed by alkylation of the de-
rived anion with an alkyl halide, Scheme 4. The acid
was then coupled to the amine. In this instance, acid
chloride formation required the harsher conditions of
thionyl chloride in order to provide a reliable yield of
the desired amide. This alternative approach allowed
us to prepare a significant number of structurally diverse
thioether analogs.
10a R = Me
10b R = Et
OH
N
H
R2
9
Cl
N
S
N
O
OH
Scheme 3. Reagents: (i) (COCl)2, DCM, pyridine, t-butyl-2-amino-4-
cyano-benzoate (55%); (ii) Sodium thiolate, dioxane, reflux, (R = Me
86%, R = Et 59%); (iii) TFA (70–90%).
R1
O
i), ii), iii)
O
OH
11
N
H
R2
S
N
S
N
O
OH
2
12 a-u
R1 = CN, Cl, Br
Scheme 4. Reagents: (i) NaBH4, alkyl halide (25–100%); (ii) SOCl2,
DCM, pyridine t-butyl-2-amino-4-bromo (or chloro, or cyano)-
benzoate (17–74%); (iii) TFA (52–92%).
In general, the thioether analogs are very active against
Staphylococcus aureus with numerous analogs having
MIC of 0.125 lg/mL, Table 2. Numerous analogs are
very potent in the presence of added serum in the S. aur-
eus MIC assay. In most instances, as the performance in
presence of serum improves the spectrum of activity
deteriorates (data not shown). There is not a linear
correlation between decrease in ClogP and improved
performance in the presence of serum. That is to some
extent contradictory to numerous observations in the lit-
erature.15,16 Rather, it appears that improvement in po-
tency with increase in ClogP is greater than increase in
Table 2. Antibacterial activity of selected pyridyl thioether analogs in the presence and absence of serum
R2
Compound
R1
MIC SAUR 9218 (lg/mL)
0% serum 10% serum
R (10%)a
Me
Et
10a
10b
12a
12b
12c
12d
12e
12f
12g
12h
12i
CN
CN
CN
CN
CN
CN
Br
4
32
8
8
16
0.5
0.5
0.25
0.125
0.125
0.125
0.125
1
n-Pr
n-Bu
32
16
4
64
64
n-C5H11
n-C6H13
n-C6H13
n-C6H13
n-C9H19
n-C3H6OC2H4OCH3
i-Pr
32
64
8
64
64
>128
4
512
512
>128
4
Cl
CN
CN
CN
Br
1
1
16
32
8
16
32
i-Pr
i-Bu
12k
12l
1
0.5
CN
16
12m
CN
0.125
16
128
12n
CN
0.125
16
128
12o
12p
12r
CN
CN
Br
0.25
0.5
1
8
32
64
32
64
64
12s
12t
CN
CN
0.5
16
4
32
32
0.125
O
O
12u
Br
1
32
32
a R = [(MIC 10% serum)/(MIC 0% serum)].

3116
A. Thorarensen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3113–3116
observed any correlation with other physical parameters
such as topical polar surface area or MW.
Conclusion. In this paper, we described some of the key
observations we made regarding antibacterial SAR of
this novel template and the properties influencing its
affinity for HSA. Based on the synthesis of compounds
6 and 8, we believe that rational design is unlikely to
guide the design of less protein bound analogs. On the
other hand, the close correlation of ClogP with HSA
affinity provides a strategy for designing new analogs.
These compounds bind to multiple sites on HSA and
the ClogP modification has dramatic effect on affinity
of the compounds for the ibuprofen site. The overall ef-
fect on decreased HSA affinity is more modest. The fu-
ture focus is on improving potency in new compounds
while retaining a low ClogP. Potency enhancements
which are the results of ClogP increases are unlikely
to provide analogs with the desired properties.
Figure 3. Plot of ClogP of prepared analogs vs. ratio (ratio defined as
MIC 10% serum/MIC 0% serum).
HSA affinity. This trend is well exemplified by the linear
series C1–C9 (10a–12g), where it appears that a C5 tether
is optimal in 12c. It is difficult to compare the acyclic
series with the cyclic series (12p–u), but in general it ap-
pears that the cyclic series does not perform as well. A
more interesting observation is that branched acyclic
compounds appear to be performing the best in both
the C3 tether (12a vs. 12i) and the C4 tether (12b vs.
12l). The branched analog is more potent and performs
better in the presence of serum as judged by MIC or the
R-value (ratio). Therefore, analogs 12o, 12n, and 12m
were prepared. Their disappointing performance dem-
onstrates the difficulty in analyzing and predicting bio-
logical activity in the presence of serum. The addition
of oxygen atoms in the tether in general improves the
performance in the presence of serum.
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