Elaboration of a fragment library hit produces potent and selective aspartate semialdehyde dehydrogenase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.09.017

Aspartate-β-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action. The ASADH enzyme family shares the same substrate binding and active site catalytic groups; however, the enzymes from representative bacterial and fungal species show different inhibition patterns when previously screened against low molecular weight inhibitors identified from fragment library screening. In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor docking, kinetic, and structure-activity relationship (SAR) studies have been used to guide ASADH inhibitor development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromolar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development of inhibitors against this validated drug-target enzyme.

Full text of DOI:10.1016/j.bmc.2015.09.017

Bioorganic & Medicinal Chemistry 23 (2015) 6622–6631
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Elaboration of a fragment library hit produces potent and selective
aspartate semialdehyde dehydrogenase inhibitors
y
y,à
⇑
Bharani Thangavelu , Pravin Bhansali , Ronald E. Viola
Department of Chemistry & Biochemistry, University of Toledo, Toledo, OH 43606, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2015
Revised 1 September 2015
Accepted 8 September 2015
Available online 9 September 2015
Aspartate-b-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate meta-
bolic pathway which leads to the biosynthesis of several essential amino acids and some important
metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria
and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway
are attractive potential targets for development of new antibiotics with novel modes of action. The
ASADH enzyme family shares the same substrate binding and active site catalytic groups; however,
the enzymes from representative bacterial and fungal species show different inhibition patterns when
previously screened against low molecular weight inhibitors identified from fragment library screening.
In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor dock-
ing, kinetic, and structure–activity relationship (SAR) studies have been used to guide ASADH inhibitor
development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromo-
lar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and
Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development
of inhibitors against this validated drug-target enzyme.
Keywords:
Aspartate semialdehyde dehydrogenase
Enzyme inhibitors
Fragment based drug discovery
Structure–activity relationships
Ó 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
microbes, with genetically-modified bacterial strains lacking the
4
,5
asd gene no longer viable. In addition to the synthesis of four
essential amino acids, a variety of important metabolites that are
required for microbial growth and survival are also produced by
the aspartate pathway. Methylation reactions which are crucial
for cell growth and viability are mediated by S-adenosyl methion-
The growing threat of microbial infections caused by increased
microbial resistance to existing antibiotics has led to a renewed
and urgent need for new antibiotics.1 The expanding population
of microbes that have developed resistance to many of the avail-
able front-line antibiotics require new drugs which can act through
novel mechanisms to thwart this increasing resistance.
6
,7
ine (AdoMet), one of the key end products of this pathway. Addi-
tionally, 4,5-dihydroxy-2,3-pentanedione and acyl homoserine
lactones produced from this pathway are two classes of signaling
2
The aspartate pathway in microorganisms and plants controls
3
8
one-quarter of de novo amino acid biosynthesis. ASADH, coded by
molecules used in bacterial quorum sensing. These quorum sens-
the asd gene, catalyzes the production of aspartate semialdehyde
ing molecules control the expression of a large number of bacterial
genes, including those that produce virulence factors such as
secreted toxins, proteases and hemolysins that cause disease
(
ASA) that is located at a critical junction in this pathway. Numer-
ous studies have shown that the deletion of this gene is fatal to
9
pathology. Furthermore, this pathway furnishes components
required for the assembly of the polysaccharide matrix of biofilms
that protect microbes against phagocytes and antibiotics. Because
of these many important microbial events that are controlled by
the aspartate pathway, it is clear why blockage of this pathway
would be fatal to microorganisms. The identification of effective
inhibitors against this target enzyme (ASADH) will provide lead
compounds for the development of new biocides with unique
mechanisms of action. In addition, achieving selective inhibition
of ASADHs from different microorganisms can lead to species-
Abbreviations: AdoMet, S-adenosyl-L-methionine; ASA, aspartate semialdehyde;
DCM, dichloromethane; FBDD, fragment-based drug discovery; HEPES, 2-[4-(2-
hydroxyethyl)piperazin-1-yl]ethane-sulfonic acid; NBS, N-bromosuccinimide; spA-
SADH, Streptococcus pneumoniae aspartate semialdehyde dehydrogenase; vcASADH,
Vibrio cholerae aspartate semialdehyde dehydrogenase.
⇑
Corresponding author. Tel.: +1 419 530 1582; fax: +1 419 530 1583.
E-mail address: ron.viola@utoledo.edu (R.E. Viola).
B.T. and P.B. contributed equally to this work.
Present address: School of Medicine, Johns Hopkins University, Baltimore, MD
y
à
2
1287, USA.
http://dx.doi.org/10.1016/j.bmc.2015.09.017
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0

B. Thangavelu et al. / Bioorg. Med. Chem. 23 (2015) 6622–6631
6623
specific biocides that should further delay the development of drug
resistance.
chain amine nitrogen and the 2-carboxyl group. Blocking this
nitrogen by methylation did allow completion of this synthesis
(Scheme 2), but this compound (9) showed only weak inhibition
(K = 1.8 mM) of spASADH and no inhibition of vcASADH, similar
i
to the affinities that were observed with compound 4.
Previous work against this target enzyme has utilized an exten-
sive background of structural information1
0–14
to employ various
1
5
approaches, including structure-guided design, library screening,
and fragment based drug discovery (FBDD)1
6,17
to identify and
develop lead compounds. Several substrate analog inhibitors have
been found to exhibit good selectivity between the Gram-negative
and Gram-positive bacterial orthologs and the fungal forms of
2.3. Introduction of substituted benzyl groups
Varying the nature of the N-carboxymethyl side chain through
the introduction of an a-L-methyl group (5) or an a-L-hydrox-
1
8
ASADH. In addition, phthalate and benzene tricarboxylate deriva-
tives identified from fragment library screening were shown to
ymethyl group (6) had no appreciable effect on the binding affinity
to either ASADH ortholog (Table 1). To explore the improved affin-
ity of the parent compound 3 to the ASADHs several additional
derivatives were synthesized through coupling with various
halides at the secondary amine position. However, the introduction
of simple alkyl, allyl, nitrile, aryl or aldehyde groups (10–13, 37)
did not alter the inhibitory properties of these compounds
(Table 1). Next, in an attempt to improve the binding affinity, a ser-
ies of substituted benzyl substituents were introduced at this
amine position. Now significant changes were observed in both
binding affinity and in binding selectivity between the two forms
of ASADH, with the improvement in activity highly dependent both
on the nature and on the position of the aromatic substitutions.
Coupling with benzyl halides substituted with either bromo (18
and 19) or methyl groups (21 and 22) at the ortho- or meta-posi-
tions did not yield any improvement in affinity, but similar substi-
tutions at the para-position of the benzyl ring (20 and 23) resulted
1
9
occupy unique binding orientations at the active site of ASADH.
However, extension of the phthalate core structure into a fortu-
itous acetate binding site did not yield the enhanced affinity that
was expected to be achieved through the combined affinities at
these multiple binding sites.20 All of the inhibitors that have been
developed so far show only modest affinity against these target
enzymes.
In the present work, we have reexamined the structure–activity
properties of these earlier inhibitors, and have now applied a sys-
tematic approach to elaborate this phthalate core structure. This
approach has resulted in the synthesis of a series of enzyme inhi-
bitors, with the most effective compounds inhibiting the ASADH
from Streptococcus pneumoniae with significantly enhanced selec-
tivity and with greater affinity than previously observed.
2
2
. Results
i
in a 10-fold improvement in the K values with spASADH (Table 2).
Surprisingly, these para-substituted derivatives did not show any
improved affinity for vcASADH.
.1. General chemistry
Based on the numerous productive interactions that have been
2
.4. Incorporation of hydrophobic benzyl substituents
observed with the phthalate derivatives bound at the active site of
1
9
ASADH, analogs were designed in which a heteroatom was intro-
duced into the side chain of this core structure to allow additional
structural elaborations. To produce the parent compounds the car-
boxylates of commercially available 4-methylphthalic acid (1)
were protected by esterification (1Me), followed by free radical
bromination (2Me). Coupling to either 2-aminoacetate methyl
ester or 3-aminopropionate methyl ester by nucleophilic displace-
ment of the introduced bromine, followed by base-catalyzed
hydrolysis yielded the corresponding amine-containing 4-(car-
boxymethylaminomethyl) and 4-(2-carboxyethylaminomethyl)
phthalate analogs 3 and 4, respectively, (Scheme 1). Coupling to
alanine methyl ester or serine methyl ester followed by hydrolysis
produced analogs 5 and 6, respectively, (Scheme 1). The affinities
of these parent compounds for representative Gram-negative (Vib-
rio cholerae) and Gram-positive (Streptococcus pneumoniae) forms
of ASADH were examined, and then systematically improved
through the production of an extensive series of derivatives at
the introduced secondary amine.
Next, the effect of changes in the methyl substituent was exam-
ined. Replacement of the o-methyl or m-methyl groups with either
trifluoromethyl (24 and 25) or trifluoromethoxy groups (27 and
2
8) did not alter the affinity of these derivatives for either spASADH
or vcASADH. However, replacing the p-methyl group with these
same substituents (26 and 29) led to a two-fold additional
improvement in binding affinity for spASADH (Table 2). Changing
p-trifluoromethoxy (29) to p-difluoromethoxy (30) reversed this
improvement, while an additional two-fold improvement
(K
i
= 16
lM) was observed for the p-tert-butylbenzyl derivative
(
33) as an inhibitor of spASADH (Table 2). To further enhance the
hydrophobic nature of the p-benzyl substituent a perfluorinated
-propyl derivative was prepared (34). This compound shows the
most potent inhibition among this family of structures, with a K
of for spASADH and over 70-fold selectivity for this
2
i
9 lM
enzyme form relative to the Gram-negative ASADH ortholog from
V. cholerae. As was seen for the parent compounds, for each of
these synthesized derivatives no significant changes were
observed in their affinity for the Gram-negative vcASADH.
2
.2. Preliminary evaluation of enzymatic activity
2
.5. Examination of additional hydrophobic derivatives
Unfortunately compound 4, with the longer N-carboxyethyl
side chain, did not show appreciable inhibition of vcASADH, but
does inhibit spASADH with a relatively weak K of 2.4 mM. These
Now that the importance of para-hydrophobic substituents has
i
been established some additional groups were incorporated in
place of the benzyl group to examine the nature of this hydropho-
bic interaction. Substitution with a naphthyl group coupled at
either the 1- or 2-position (14 and 15) resulted in slightly worse
inhibition, while the replacement of the benzyl group with a mor-
pholine (16) gave only a slight improvement. The greatest change
was observed with the incorporation of a biphenyl group (17),
resulting in 25-fold improved inhibition of spASADH relative to
the parent benzyl derivative (Table 1).
values are comparable to those that were observed for the inhibi-
tion by 3-(3-carboxypropyl)phthalate,19 despite this longer side
chain, the introduction of a nitrogen heteroatom in the side chain,
and the shift of the side chain from the 3- to the 4-position.
However, the one-carbon shorter homolog (3) shows some
improvement, functioning as a 0.5 mM inhibitor of vcASADH and
a two-fold stronger inhibitor of spASADH (Table 1).
Attempts to synthesize the corresponding 3-position analogs to
compounds 3 and 4 were thwarted by cyclization between the side

6
624
B. Thangavelu et al. / Bioorg. Med. Chem. 23 (2015) 6622–6631
COOH
COOH
COONa
COONa
NaOOC
N
H
MeOOC NH2
COOMe
1
3
c,d
a
NaOOC
NH
COONa
N
H
COOMe
COOMe
Br
MeOOC
COOMe
c,d
2
COONa
b
4
2
Me
1Me
OH
NH
MeOOC NH
2
c,d
c,d
MeOOC
2
COONa
COONa
NaOOC
N
H
OH
COONa
COONa
5
NaOOC
N
H
6
Scheme 1. Synthetic route to 4-aminomethylphthalate derivatives. Reagents and conditions: (a) H
2
SO
4
/MeOH,
l
wave, 80 °C, 8 h; (b) NBS/DCM, light, 3 h; (c) NaHCO
3
/DMF,
rt, 8 h; (d) 1 N NaOH, rt, 3 h.
Table 1
Synthesis and kinetic evaluation of 4-aminomethylphthalate derivatives
R1
O
O
R1
O
R1
O
O
NaHCO3
R2-X
O
O
NaO
N
R2
ONa
ONa
N
H
O
O
N
R2
O
O
1N NaOH
THF:H2O
O
O
CH3CN:H2O
O
O
Compd
R
1
R
2
i
K (lM)
spASADH
vcASADH
3
4
5
6
H
H
(S)-CH
(S)-CH
H
H
H
H
H
H
H
H
H
H
H
H
H
CH
3
Allyl
246 ± 20
2400 ± 180
296 ± 26
324 ± 24
296 ± 36
303 ± 22
220 ± 14
297 ± 14
396 ± 42
329 ± 22
176 ± 22
12 ± 1.2
528 ± 32
n.i.
a
b
3
2
609 ± 23
654 ± 22
675 ± 54
663 ± 36
498 ± 27
696 ± 33
749 ± 54
724 ± 47
692 ± 39
634 ± 29
655 ± 51
OH
10
11
12
13
14
15
16
17
37
Propionitrile
Benzyl
1-Naphthyl
2-Naphthyl
N-Ethylmorpholino
4-Biphenyl
Acetaldehyde
276 ± 16
a
N-Carboxylmethyl group replaced with N-carboxyethyl.
No inhibition observed at concentrations up to 2 mM.
b
2
.6. Exploration of a structure–activity outlier
The only outlier from this correlation between higher affinity
observed for the p-methylbenzyl derivative (23). It seems reason-
able to suggest that the improved affinity for this charged benzyl
derivative is likely due to the orientation of this side chain in a dif-
ferent binding position, perhaps making an electrostatic interac-
tion with a positively-charged amino acid side chain near the
and increased para-hydrophobicity was the observation that the
p-carboxylbenzyl derivative (31) has comparable affinity to that
Br
H
MeOOC
c
N
COOH
COOH
COOMe
COOMe
COOMe
COOMe
CH
3
a
b
7
7Me
8Me
CH3
N
CH3
N
MeOOC
NaOOC
COOMe
COOMe
COONa
COONa
d
9
Me
9
2 4 3
Scheme 2. Synthetic route to 3-aminomethylphthalate derivatives. Reagents and conditions: (a) H SO /MeOH, lwave, 80 °C, 8 h; (b) NBS/DCM, light, 3 h; (c) NaHCO /DMF,
rt, 8 h; (d) 1 N NaOH, rt, 3 h.

B. Thangavelu et al. / Bioorg. Med. Chem. 23 (2015) 6622–6631
6625
Table 2
Synthesis and kinetic evaluation of 4-(N-substituted)benzylaminoalkylphthalates
O
O
O
O
O
NaHCO3
O
NaO
O
1N NaOH
THF:H O
N
Bz-R
O
O
N
Bz-R
ONa
ONa
N
H
O
O
R-Bz-X
CH CN:H O
n
n
n
O
2
O
O
3
2
O
Compda
R
i
K (lM)
spASADH
vcASADH
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
38
35
36
2-Bromo
3-Bromo
4-Bromo
2-Methyl
3-Methyl
4-Methyl
2-Trifluroromethyl
3-Trifluoromethyl
4-Trifluoromethyl
2-Trifluoromethoxy
3-Trifluoromethoxy
4-Trifluoromethoxy
4-Difluoromethoxy
4-Carboxy
529 ± 38
442 ± 29
57 ± 5
524 ± 27
430 ± 22
63 ± 7
509 ± 29
441 ± 27
24 ± 3
540 ± 32
427 ± 31
36 ± 6
54 ± 5
72 ± 7
229 ± 32
16 ± 2
9 ± 0.2
737 ± 49
665 ± 37
629 ± 53
721 ± 46
690 ± 41
608 ± 42
727 ± 60
678 ± 36
628 ± 48
708 ± 47
684 ± 35
663 ± 39
698 ± 44
635 ± 55
649 ± 25
648 ± 32
639 ± 37
1100 ± 200
4-Carboxamide
4-tert-Butyl
4-(2-Perfluoropropyl)
4-((2-Benzyl)vinyl)
4-Bromo
476 ± 52
3600 ± 230
3800 ± 220
⁄
⁄
b
n.i.
b
4-Trifluoromethyl
n.i.
a
⁄
n = 1 for all entries, except where n = 2.
b
No inhibition observed at concentrations up to 4 mM.
active site. To test this idea the p-carboxyl group was replaced with
a polar but uncharged substituent, a carboxamide group (32). As
predicted, this derivative shows a 3-fold loss of affinity to a value
that is now comparable to that of the underivatized parent com-
pound (3) (Table 2).
Now that the significance of these p-substituted benzyl
derivatives as potent and selective ASADH inhibitors has been
demonstrated, several longer homologs of these derivatives
were synthesized starting from parent compound 4 (Scheme 1)
to determine if the improved affinities of these derivatives
would be able to overcome the lower affinity of this parent
compound. However, in these cases neither the p-bromobenzyl
selective drugs that can function through a novel mechanism of
action against this new target.
Fragment library screening against this enzyme target had pre-
viously led to the identification of several non-substrate-like struc-
tures that function as modest inhibitors of the ASADHs.16 Among
these compounds were some aromatic inhibitors that offered the
possibility of additional structural elaborations. Subsequent
molecular modeling and docking studies starting with these core
structures led to the identification of aryl di- and trianionic com-
pounds that were determined to be low millimolar inhibitors of
both Gram-negative and Gram-positive forms of ASADH.17 Extend-
ing one of the carboxylate side chains into an adjacent anionic
binding pocket was then explored to improve inhibitor affinity.
Unfortunately, some steric clashes coupled with multiple possible
nor the p-trifluoromethylbenzyl derivatives of compound
4
gave better than low millimolar inhibition of spASADH, and
no inhibition of vcASADH was observed with these derivatives
1
9
binding orientations blocked the further development of this
core structure into more potent inhibitors. What was needed to
realize the objective of potent inhibition by this class of structures
was the introduction of additional functionality that would favor
the adoption of a single orientation when bound to the enzyme.
(
data not shown).
3
3
. Discussion
.1. Target validation and preliminary inhibitor identification
3
.2. Identification and optimization of aryl dicarboxylate
inhibitors
The past few decades have seen increased attention of the drug
development community focusing on diseases such as cancer and
cardiovascular diseases, the leading causes of human mortality.
However, the development of an increasingly diverse set of defen-
sive mechanisms by microbes against most of the current antibi-
otics demands a renewed focus from the scientific community to
identify unique and selective pathways that can be targeted for
development of new classes of antibiotics.
The aspartate pathway, absent in mammals but present in
microbes and plants, is a critical and essential pathway that meets
these criteria. We have targeted aspartate semialdehyde dehydro-
genase (ASADH), the enzyme which controls the first branch point
in this pathway, for the design of new, potent, and selective inhibi-
tors. Inhibition of ASADH will provide the basis for development of
Some parent aryl dicarboxylate structures have now been
designed with the inclusion of a secondary amine side chain that
can be further elaborated to test this approach (Scheme 1). Initial
attempts were made to directly mimic the 3-(3-carboxypropyl)ph-
thalate structure that showed good binding orientation at the active
1
9
site of spASADH through replacement of the carboxypropyl group
with an aminoethylcarboxylate side chain. This synthetic approach
failed because of rapid cyclization between the introduced sec-
ondary amine and the carboxyl group at the 2-position. Protection
of this amine through the presence of a methyl group did allow syn-
thesis of the desired structure (Scheme 2) which, unfortunately,
was no more potent than the original compound.

6
626
B. Thangavelu et al. / Bioorg. Med. Chem. 23 (2015) 6622–6631
Moving the aminoalkylcarboxyl side chain from the 3- to the 4-
presents a significant challenge in designing substrate analog inhi-
bitors that can demonstrate species-selectivity against this target
enzyme. Fortunately, the original compound in this phthalate ser-
ies, 3-(3-carboxypropyl)phthalate, binds in a unique position in the
active site of ASADH, base-stacking with the nicotinamide ring of
the NADP cofactor while also making electrostatic interactions
between the inhibitor carboxylate groups and the side chains of
Ser96, Arg99 and Arg245. However, minor structural alterations
leads to substantial changes in the bound orientation of these
structurally-related inhibitors, with the one carbon shorter homo-
log, 3-(3-carboxyethyl)phthalate, using the same electrostatic
binding partners but flipped by 180° in this binding pocket. This
orientational and conformational flexibility is the apparent cause
for the much lower measured affinity than would be predicted
based on the number of favorable binding interactions observed
in the optimally-oriented enzyme–inhibitor structure. In an
attempt to constrain this orientational flexibility an amine was
introduced into the carboxypropyl side chain (compound 3) that
can serve as a site for further structural elaborations. Derivatiza-
tion of this introduced secondary amine through the introduction
of para-hydrophobic benzyl substituents appears to make addi-
tional productive binding interactions that constrain these multi-
ple orientations, leading to the realization of potent and selective
inhibition of ASADH.
Docking of the most potent inhibitors into the active site of spA-
SADH offers a possible explanation both for the improved affinity
of these derivatives and for the observed ortholog selectivity. Using
the structure of 3-(3-carboxypropyl)phthalate in complex with
spASADH (PDB ID 4r4j) as a guide, the various para-substituted
benzylamine derivatives were modeled into the active site. This
enzyme structure contains an adjacent pocket near the active site,
surrounded by several hydrophobic amino acids (Leu326, Ala330,
Ala331 and Val14) that could make favorable interactions with
the para-hydrophobic substituents of the most potent inhibitors.
The shape of this hydrophobic pocket could also potentially accom-
modate a para-substituent on the benzyl ring as large as phenyl
(17), but interactions with hydrophobic substituents at either the
ortho- or meta-position would be less favorable. Further develop-
ment of this series of inhibitors would benefit from structures
bound to spASADH to identify the actual binding interactions that
have been incorporated and to gain a better sense of the directions
that these p-substituted benzyl derivatives can be elaborated.
The Val14 in this putative binding pocket is fully conserved
throughout the bacterial ASADH family. However, in the corre-
sponding region in the structure of vcASADH (PDB ID 2qz9) several
substitutions with polar amino acids in the 325–331 loop, includ-
ing Leu326 replaced by Asn and Asn325 replaced by a Gln that
would now extend into this site, would make this pocket much less
hydrophobic. These substitutions could explain the failure of these
inhibitors to show improved affinity with this Gram-negative
ortholog of ASADH. These same replacements are found at these
positions in other Gram-negative orthologs of ASADH, supporting
the planned development of this series of inhibitors as Gram-pos-
itive selective antimicrobials.
position on the benzyl ring avoided this cyclization reaction, and
has led to a much more productive series of ASADH inhibitors.
The parent compound 3 is already a high micromolar inhibitor of
the ASADHs with a 2-fold preference for the Gram-positive spA-
SADH. Exploring the reactivity of the introduced secondary amine
through the incorporation of a set of simple substituents did not
alter either the affinity or the selectivity of these derivatives relative
to the parent compound (Table 1). However, changes in affinities
were observed when substituted aryl substituents were introduced
at this amine position. Simple o- or m-substituted benzyl groups
were slightly poorer inhibitors of spASADH when compared to the
parent benzyl substituted analog (13), but the corresponding p-
substituted benzyl derivatives were substantially more potent spA-
SADH inhibitors and also showed 10-fold selectivity for this
enzyme form relative to vcASADH (Table 2). The more hydrophobic
fluorinated p-methylbenzyl derivatives gave a 2-fold additional
improvement in affinity, with slight differences observed between
di- and trifluoromethyl and between methyl and methoxy.
1
9
3
.3. Role of hydrophobic functional groups
Placing a hydrophobic group at the para-position is likely
accessing a hydrophobic pocket adjacent to the active site of spA-
SADH. Consistent with this hypothesis is the observation that the
p-tert-butylbenzyl derivative (33) is a much more potent inhibitor
in this series and also has over 40-fold selectivity for spASADH
(
Table 2). The most hydrophobic para-substituent tested, perfluo-
roisopropyl, is, as expected, the most potent inhibitor of spASADH,
with a low micromolar K value and 70-fold selectivity over the
i
Gram-negative vcASADH. Further elaboration at this position
through the introduction of either a p-styrene or a naphthyl group
in place of the benzyl group leads to a loss of activity, presumably
due to unfavorable interactions with the larger steric bulk. The
only additional aromatic hydrophobic group that allowed
improved binding affinity was a p-biphenyl group (17), suggesting
the presence of a deep but narrow hydrophobic pocket near the
active site that is being accessed by this side chain. Modifications
in the N-carboxymethyl side chain through the introduction of a-
substituents (5 and 6) does not lead to enhanced affinity, and car-
bon chain homologation (4) causes a substantial affinity loss, even
in the presence of the more optimal benzyl derivatives (35 and 36).
3
.4. Inhibitor selectivity
A major cause of ‘off-target’ effects with drug candidates is not
the lack of affinity for the target of interest, but a lack of specificity
that allows additional interactions with unintended targets. There
are no close mammalian orthologs of ASADH that would provide
obvious competing target proteins. While it is always difficult to
predict possible interactions between new chemical entities and
the wide range of possible in vivo binding partners, the most
potent of these inhibitors now displays enhanced selectivity
against the identical enzyme from another bacterial species. This
increases the confidence in the exquisite selectivity of this devel-
oped class of ASADH inhibitors.
4
. Conclusions
3
.5. Potential active site interactions
A secondary amine was introduced into the carboxypropyl side
There have been numerous structures of substrate analog
chain of a previously characterized modest and non-selective inhi-
bitor of ASADH to allow further structural elaborations. Significant
improvements in binding affinities, approaching three-orders of
magnitude, and dramatically enhanced ortholog selectivity have
now been achieved for certain substituted N-benzyl derivatives,
with the highest affinities driven by the incorporation of
hydrophobic para-substituents on the introduced benzyl ring.
1
5
11,13
inhibitors and reaction intermediates
bound at the active site
of different ASADHs. Modeling studies of substrate binding to a
wide range of ASADHs from different species concluded that the
substrate utilizes the same set of active site binding interactions
and binds in the same orientation despite the sequence diversity
2
1
in this enzyme family. This nearly identical substrate binding

B. Thangavelu et al. / Bioorg. Med. Chem. 23 (2015) 6622–6631
6627
5
5
. Experimental
DMF was air evaporated and the reaction mixture was purified
by flash column chromatography using 10–60% ethyl acetate:hex-
1
.1. General chemistry
anes to obtain compound 4Me. H NMR (600 MHz, CDCl
3
): d = 3.38
(
s, 2H), 3.70 (s, 3H), 3.84 (s, 2H), 3.86 (s, 3H), 3.87 (s, 3H), 7.48 (d,
All compounds and reagents purchased from commercial
sources were used without further purification. Reaction progress
was monitored by TLC carried out using silica coated glass plates
J = 7.8 Hz, 1H), 7.64 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H). MS (ESI) m/z:
(obs) [M+Na] 332.4, (calcd) 332.3. A solution of compound 4Me
+
(0.071 g, 0.229 mmol) was base-hydrolyzed as described above to
1
(
Analtech) and was visualized by 254 nm UV light. Flash column
chromatography was conducted in a 20 mm ꢀ 250 mm column
with 40–63 SiliaFlash P60 silica gel (Silicycle) using the specified
yield pure compound 4. H NMR (600 MHz, D
2
O): d = 2.47 (t,
J = 7.2 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 3.59 (s, 2H), 7.18 (d,
J = 7.8 Hz, 1H), 7.21 (s, 1H), 7.30 9 (d, J = 7.8 Hz, 1H).
l
1
ethyl acetate:hexanes gradient for elution. H NMR spectra were
recorded on either a Varian VXRS 400 MHz or a Varian INOVA
5.2.5. N-((2-Methyl)carboxymethyl)-3,4-dicarboxybenzylamine
6
00 MHz spectrometer, and were calibrated using residual non-
deuterated solvent as internal reference (CDCl , d = 7.26, for the
esters; D O, d = 4.80, for the sodium salts).
(5)
To a solution of compound 2Me (1.36 g, 4.7 mmol) in DMF was
3
added NaHCO
3.3 g, 23.7 mmol). The reaction mixture was stirred at rt for 8 h.
3
(2.0 g, 23.7 mmol) and
L
-alanine methyl esterꢁHCl
2
(
DMF was air evaporated and the reaction mixture was purified
5
5
.2. Parent compound synthesis
by flash column chromatography using 10–60% ethyl acetate:hex-
1
anes to obtain compound 5Me. H NMR (600 MHz, CDCl
3
): d = 1.32
.2.1. 4-Methylphthalate dimethyl ester (1Me)
To a solution of compound 1, 4-methyl-1,2-dicarboxylic acid
5 g, 27.7 mmol) in 12 ml of methanol, 5 ml of sulfuric acid was
added at rt, the reaction mixture was allowed to stir in a micro-
wave reactor at 80 °C for 8 h. Saturated NaHCO solution was
(
d, J = 6.6 Hz, 3H), 3.49 (m, J = 6.6 Hz, 1H), 3.71 (s, 4H), 3.84 (s, 2H),
3
.87–3.90 (m, 8H), 7.51 (d, J = 7.8 Hz, 1H), 7.66 (s, 1H), 7.69 (d,
(
+
J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na] 332.4, (calcd) 332.3.
A solution of compound 5Me (0.071 g, 0.229 mmol) was base-hy-
drolyzed as described above to yield pure compound 5. 1H NMR
3
added to the reaction mixture until slightly basic, with the product
precipitating as an oily liquid. The product was extracted with sev-
eral portions of dichloromethane (DCM), dried over anhydrous
sodium sulfate and concentrated in vacuum. The compound was
purified by column chromatography using 0–10% ethyl acetate:
(
600 MHz, D
2
O): d = 1.16 (d, J = 6.6 Hz, 3H), 3.11 (m, J = 7.2 Hz,
1
H), 3.52 (d, J = 12.6 Hz, 1H), 3.68 (d, J = 12.6 Hz, 1H), 7.27 (d,
J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.40 (d, J = 7.8 Hz, 1H).
hexanes to yield ester 1Me. 1H NMR (600 MHz, CDCl
5.2.6. N-((2-Hydroxymethyl)carboxymethyl)-3,4-dicarboxyben-
zylamine (6)
3
): d = 2.39
s, 3H), 3.87 (s, 3H), 3.88 (s, 3H), 7.30 (d, J = 7.8 Hz, 1H), 7.45 (s,
(
+
To a solution of compound 2Me (1.15 g, 4.0 mmol) in DMF was
1
H), 7.65 (d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na] 231.1,
added NaHCO
3
(1.7 g, 20.1 mmol) and
L
-serine methyl esterꢁHCl
(
calcd) 231.2.
(
3.1 g, 20.1 mmol). The reaction mixture was stirred at rt for 8 h.
DMF was air evaporated and the reaction mixture was purified
5
.2.2. 4-Bromomethylphthalate dimethyl ester (2Me)
To a solution of compound 2 (5 g, 24.8 mmol) in 25 ml dichlor-
by flash column chromatography using 10–60% ethyl acetate:hex-
1
anes to obtain compound 6Me. H NMR (600 MHz, CDCl
3
): d = 2.43
omethane was added NBS (4.9 g, 27.3 mmol). The reaction mixture
was stirred in the presence of light for 3 h, concentrated under vac-
uum and purified by flash column chromatography using 0–20%
(
s, 2H), 3.39 (m, 1H), 3.64 (m, 1H), 3.74 (s, 3H), 3.80 (m, 2H), 3.81–
3
.89 (m, 6H), 3.95 (d, J = 14.4 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.66
+
1
(s, 1H), 7.70 (d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na] 348.4,
calcd) 348.3. A solution of compound 6Me (0.071 g, 0.229 mmol)
was base-hydrolyzed as described above to yield pure compound
ethyl acetate:hexanes to obtain compound 2Me.
600 MHz, CDCl
H NMR
(
(
3
): d = 3.89 (s, 6H), 4.46 (s, 2H), 7.53 (d, J = 6.6 Hz,
+
1
3
H), 7.68–7.71 (m, 2H). MS (ESI) m/z: (obs) [M+Na] (isotopic)
09.3, 311.1 (calcd) (isotopic) 309.0, 311.0.
1
6.
2
H NMR (600 MHz, D O): d = 3.17 (t, J = 5.2 Hz, 1H), 3.61 (d,
1
3.2 Hz, 1H), 3.68–3.70 (m, 2H), 3.78 (d, J = 12.8 Hz, 1H), 7.31 (d,
J = 8 Hz, 1H), 7.35 (s, 1H), 7.42 (d, J = 7.8 Hz, 1H).
5
.2.3. N-Carboxymethyl-3,4-dicarboxybenzylamine (3)
To a solution of compound 2Me (3.1 g, 10.7 mmol) in DMF was
5
.2.7. 3-Methylphthalate dimethyl ester (7Me)
added NaHCO
3
(4.6 g, 54 mmol) and glycine methyl esterꢁHCl
To a solution of compound 7, 3-methylphthalic acid (5 g,
4 mmol) in 12 ml of methanol at rt was added 5 ml of concd sul-
(
6.8 g, 54 mmol). The reaction mixture was stirred at rt for 8 h.
2
DMF was air evaporated and the reaction mixture was purified
furic acid, with the reaction mixture stirred in a microwave reactor
at 80 °C for 8 h. Saturated NaHCO solution was added to the reac-
tion mixture until slightly basic, and the product was extracted
with DCM and concentrated in vacuum. The compound was puri-
fied by column chromatography using 0–10% ethyl acetate:hex-
by flash column chromatography using 10–50% ethyl acetate:hex-
3
1
anes to obtain compound 3Me. H NMR (600 MHz, CDCl
3
): d = 3.38
(
s, 2H), 3.70 (s, 3H) 3.84 (s, 2H), 3.86–3.87 (s, 6H), 7.48 (d,
J = 7.8 Hz, 1H), 7.64 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H). MS (ESI) m/z:
obs) [M+Na]+ 318.4, (calcd) 318.3. This ester was hydrolyzed by
adding H O (0.1 ml) to a solution of compound 3Me (0.04 g,
.14 mmol) in THF (0.4 ml). Then 1 N NaOH (0.4 ml, 0.42 mmol)
(
1
anes to furnish ester 7Me. H NMR (600 MHz, CDCl
3
): d = 2.33(s,
2
3
H), 3.87 (s, 3H), 3.93 (s, 3H), 7.35 (t, J = 7.8 Hz, 1H), 7.39 (d,
0
J = 7.8 Hz, 1H), 7.80 (d, J = 7.2 Hz, 1H). MS (ESI) m/z: (obs) [M
was added to the reaction mixture and stirred at rt for 3 h. The
+
+Na] 231.1, (calcd) 231.2.
reaction mixture was concentrated and dried for several hours
1
under high vacuum to yield pure compound 3.
400 MHz, D
H NMR
5
.2.8. 3-Bromomethylphthalate dimethyl ester (8Me)
To a solution of compound 7Me (1.8 g, 8.8 mmol) in dichloro-
(
1
2
O): d = 3.12 (s, 2H), 3.66 (s, 2H), 7.48 (d, J = 7.8 Hz,
H), 7.27–7.32 (m, 2H), 7.40 (s, 1H).
methane was added NBS (1.72 g, 9.7 mmol). The reaction mixture
was stirred in presence of light for 3 h, concentrated in vacuum
and purified by flash column chromatography using 0–15% ethyl
acetate:hexanes to obtain compound 8Me. H NMR (600 MHz,
CDCl ): d = 3.88 (s, 3H), 3.95 (s, 3H), 4.52 (s, 2H), 7.46 (t,
5
.2.4. N-Carboxyethyl-3,4-dicarboxybenzylamine (4)
To a solution of compound 2Me (0.52 g, 1.8 mmol) in DMF was
added NaHCO
(0.8 g, 9 mmol) and b-alanine methyl esterꢁHCl
1.3 g, 9 mmol). The reaction mixture was stirred at rt for 8 h.
1
3
3
(
J = 7.8 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H). MS

6
628
B. Thangavelu et al. / Bioorg. Med. Chem. 23 (2015) 6622–6631
(
3
ESI) m/z: (obs) [M+Na]+ (isotopic) 309.2, 311.1 (calcd) (isotopic)
09.0, 311.0.
5H), 7.59 (d, J = 8 Hz, 1H), 7.72 (m, J = 8 Hz, 2H). MS (ESI) m/z:
+
(obs) [M+Na] 408.3, (calcd) 408.4.
1
Sodium salt: H NMR (600 MHz, D
2
O): d = 2.88 (s, 2H), 3.62 (s,
5
.2.9. N-Methyl, N-carboxymethyl-2,3-dicarboxybenzylamine (9)
To a solution of compound 8Me (0.42 g, 1.46 mmol) in DMF was
4H), 7.20 (m, 7H), 7.29 (m, 1H).
added NaHCO
3
(0.61 g, 7.32 mmol) and N-methyl glycine methyl
5.3.5. N-(1-Naphthyl)-N-carboxymethyl-3,4-dicarboxybenzyl-
esterꢁHCl (1.1 g, 7.35 mmol). The reaction mixture was stirred at
rt for 8 h. DMF was air evaporated and the reaction mixture was
purified by flash column chromatography on silica gel using 10–
amine (14)
1
Triester: H NMR (600 MHz, CDCl
3
): d = 3.28 (s, 2H), 3.67 (s, 3H),
3.87 (s, 2H), 3.89 (s, 3H), 3.91 (s, 3H), 4.26 (s, 2H), 7.38 (s, J = 7.8 Hz,
1H), 7.44–7.53 (m, 4H), 7.65 (m, J = 7.8 Hz, 2H), 7.75 (d, J = 8.4 Hz,
1H), 7.81 (d, J = 8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+ 458.2,
(calcd) 458.5.
4
0% ethyl acetate:hexanes to obtain compound 9Me. 1H NMR
(
3
600 MHz, CDCl ): d = 2.23 (s, 3H), 3.15 (s, 2H), 3.57 (s, 3H), 3.65
(
s, 2H), 3.76 (s, 3H), 3.78 (s, 3H), 7.31 (t, J = 7.8 Hz, 1H), 7.54 (d,
J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M
Sodium salt: 1H NMR (600 MHz, D
2
O): d = 3.05 (s, 2H), 3.88 (s,
+
+
Na] 332.4, (calcd) 332.3. This ester was hydrolyzed as described
2H), 4.18 (s, 2H), 7.32(d, 1H), 7.38 (s, 1H), 7.41–7.49 (m, 5H),
7.83 (d, J = 7.8 Hz, 1H), 7.88–7.90 (m, J = 7.8 Hz, 1H), 7.93 (d,
J = 8.4 Hz, 1H).
1
above to yield pure compound 9. H NMR (600 MHz, D
s, 3H), 2.98 (s, 2H), 3.55 (s, 2H), 7.29 (t, J = 7.2 Hz, 1H), 7.50 (d,
J = 7.2 Hz, 1H), 7.53(d, J = 7.2 Hz, 1H).
2
O): d = 2.10
(
5
.3.6. N-(2-Naphthyl)-N-carboxymethyl-3,4-dicarboxybenzyl-
5
.3. General protocol for N-derivatized dicarboxybenzylamines
amine (15)
1
(
10–34)
Triester: H NMR (600 MHz, CDCl
3
): d = 3.32 (s, 2H), 3.67 (s, 3H),
3
.87 (s, 2H), 3.88–3.90 (m, 8H), 3.91 (s, 3H), 3.94 (s, 2H), 7.43–7.45
To a solution of compound 3Me (1 equiv) in acetonitrile was
added 1.1 equiv of various alkyl halides (Table 1) or various benzyl
halides (Table 2) along with 1.1 equiv of NaHCO . The reaction
mixture was stirred at rt for 3 h. CH CN was evaporated in vacuum
and purified by flash column chromatography on silica gel using 0–
5% ethyl acetate:hexanes to obtain corresponding N-derivatized
benzylamine esters. Ester hydrolysis was carried out by adding
.1 ml of H O to a solution of N-derivatized benzylamine esters
1 equiv) in THF (0.4 ml) with stirring. Then, 1 N NaOH (3.3 equiv)
(m, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 7.2 Hz, 1H), 7.70–7.74
(m, 3H), 7.79 (d, J = 8.4 Hz, 3H). MS (ESI) m/z: (obs) [M+Na]+
458.2, (calcd) 458.5.
3
Sodium salt: 1H NMR (600 MHz, D
O): d = 3.06 (s, 2H), 3.78 (s,
3
2
2H), 3.89 (s, 2H), 7.30 (d, J = 7.8 Hz, 1H), 7.34 (s, 1H), 7.41 (d,
J = 7.8 Hz, 1H), 7.47–7.51 (m, 3H), 7.80 (s, 1H), 7.86–7.89 (m,
J = 7.8 Hz, 1H).
2
0
(
2
5.3.7. N-(Ethylmorpholino)-N-carboxymethyl-3,4-dicarboxy-
was added to the reaction mixture and stirred at rt for 3 h. The
reaction mixture was concentrated and dried for several hours
under high vacuum to yield the corresponding sodium salts of
the N-derivatized benzylamines, compounds 10–34.
benzylamine (16)
Triester: 1H NMR (600 MHz, CDCl
): d = 2.38 (s, 3H), 2.44 (t,
3
J = 6.6 Hz, 2H), 2.77 (t, J = 6.6 Hz, 2H), 3.39 (s, 2H), 3.64 (s, 6H),
3.85–3.86 (m, 8H), 7.49 (d, J = 7.8 Hz, 2H), 7.64–7.66 (m, 3H). MS
+
(
ESI) m/z: (obs) [M+H] 408.3, (calcd) 408.4.
Sodium salt: H NMR (600 MHz, D O): d = 2.40 (s, 4H), 2.46 (t,
2
1
5
(
.3.1. N-Methyl, N-carboxymethyl-3,4-dicarboxybenzylamine
10)
Triester: 1H NMR (600 MHz, CDCl
H), 3.69 (s, 3H) 3.73 (s, 2H), 3.88 (s, 6H), 7.52 (d, J = 7.8 Hz, 1H),
J = 8.4 Hz, 2H), 2.67 (t, J = 7.2 Hz, 2H), 3.09 (s, 2H), 3.64 (s, 4H),
3.69 (s, 2H), 7.32 (d, J = 6.6 Hz, 2H), 7.40 (d, J = 8.4 Hz, 1H).
3
): d = 2.36 (9s, 3H), 3.28 (s,
2
7
3
+
.66 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]
32.4, (calcd) 332.3.
5.3.8. N-(4-Biphenyl)-N-carboxymethyl-3,4-dicarboxybenzyl-
amine (17)
Sodium salt: 1H NMR (600 MHz, D
Triester: H NMR (600 MHz, CDCl
1
2
O): d = 2.04 (s, 3H), 2.85 (s,
3
): d = 3.33 (s, 2H), 3.68 (s, 3H),
2
1
H), 3.46 (s, 2H), 7.15–7.17 (m, J = 7.2 Hz, 2H), 7.25 (d, J = 7.2 Hz,
H).
3.83 (s, 2H), 3.88–3.91 (m, 8H), 7.31–7.33 (m, J = 7.2 Hz, 1H), 7.40–
7.43 (m, 4H), 7.54, (d, J = 7.2 Hz, 2H), 7.58 (d, J = 7.2 Hz, 2H), 7.61
(
d, J = 7.8 Hz, 1H), 7.71–7.73 (m, 2H). MS (ESI) m/z: (obs) [M+Na]+
5
.3.2. N-Allyl, N-carboxymethyl-3,4-dicarboxybenzylamine (11)
484.2, (calcd) 484.5.
1
Sodium salt: 1H NMR (600 MHz, D
3
Triester: H NMR (600 MHz, CDCl ): d = 3.24 (s, 2H), 3.31 (s, 2H),
2
O): d = 3.03 (s, 2H), 3.74 (d,
3
5
.66 (s, 3H) 3.82 (s, 2H), 3.88–3.89 (s, 6H), 5.13–5.21 (m, 2H),
J = 14.4 Hz, 4H), 7.31–7.33 (m, J = 7.2 Hz, 1H), 7.29 (d, 1H), 7.33–
7.37 (m, J = 7.2 Hz, 2H), 7.39–7.42 (t, J = 8.4 Hz, 3H), 7.44–7.46 (t,
J = 7.8 Hz, 2H), 7.60 (d, J = 7.8 Hz, 2H), 7.73 (d, J = 7.8 Hz, 2H).
.78–5.84 (m, 1H) 7.54 (d, J = 7.8 Hz, 1H), 7.68 (m, J = 7.8 Hz, 2H).
+
MS (ESI) m/z: (obs) [M+Na] 358.4, (calcd) 358.3.
1
Sodium salt: H NMR (600 MHz, D
2
O): d = 2.9 (s, 2H), 3.04
(
s, 2H), 3.58 (s, 2H), 5.02–5.07 (m, 2H), 5.69–5.73 (m, 1H),
5.3.9. N-(2-Bromobenzyl)-N-carboxymethyl-3,4-dicarboxy-
7
.15–7.17 (m, 2H), 7.25 (d, 1H).
benzylamine (18)
1
Triester: H NMR (600 MHz, CDCl
3
): d = 3.31 (s, 2H), 3.67(s, 3H),
5
.3.3. N-Acetonitrile, N-carboxymethyl-3,4-dicarboxybenzylamine
3.86–3.88 (s, 6H), 3.92 (s, 4H), 7.08 (t, Hz, 1H), 7.25 (t, J = 7.6 Hz,
1H), 7.49 (t, J = 8.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 1H), 7.68 (d,
J = 7.6 Hz, 2H). MS (ESI) m/z: (obs) [M+Na] (isotopic) 486.2,
(12)
1
+
Triester: H NMR (400 MHz, CDCl
3
): d = 3.42 (s, 2H), 3.68 (s, 2H),
3
.72 (s, 3H) 3.84 (s, 2H), 3.89 (s, 6H), 7.55 (d, J = 7.6 Hz, 1H), 7.7 (m,
488.0 (calcd) (isotopic) 486.1, 488.1.
+
1
J = 7.6 Hz, 2H). MS (ESI) m/z: (obs) [M+Na] 357.4, (calcd) 357.3.
Sodium salt: H NMR (600 MHz, D
2H), 3.90 (s, 2H), 7.13–7.17 (t, 1H), 7.31–7.36 (m, J = 7.2 Hz, 3H),
7.39 (d, J = 8 Hz, 2H), 7.56 (d, J = 8 Hz, 2H).
2
O): d = 3.07 (s, 2H), 3.89 (s,
1
Sodium salt: H NMR (400 MHz, D
2
O): d = 2.97 (s, 2H), 2.99
(
s, 2H), 3.57 (s, 2H), 7.17–7.20 (m, 2H), 7.27 (d, 1H).
5
(
.3.4. N-Benzyl, N-carboxymethyl-3,4-dicarboxybenzylamine
13)
Triester: H NMR (400 MHz, CDCl
.79 (s, 2H), 3.87 (s, 2H), 3.89 (s, 3H), 3.91 (s, 3H), 7.23–7.34 (m,
5.3.10. N-(3-Bromobenzyl)-N-carboxymethyl-3,4-dicarboxy-
benzylamine (19)
1
1
3
): d = 3.30 (s, 2H), 3.68 (s, 3H),
Triester: H NMR (600 MHz, CDCl
3
): d = 3.26 (s, 2H), 3.65 (s, 3H),
3
3.73 (s, 2H), 3.83 (s, 2H), 3.86–3.88 (s, 6H), 7.14 (d, J = 7.8 Hz, 1H),

B. Thangavelu et al. / Bioorg. Med. Chem. 23 (2015) 6622–6631
6629
7
.24 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.48 (s, 1H), 7.54 (d,
7.49 (d, J = 7.8 Hz, 1H), 7.53 (t, J = 7.8 Hz, 2H) 7.57 (s, J = 7.8 Hz,
1H), 7.68 (d, J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 476.1,
(calcd) 476.4.
+
J = 7.8 Hz, 1H), 7.68 (d, J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]
(
isotopic) 486.2, 488.0 (calcd) (isotopic) 486.1, 488.1.
1
Sodium salt: 1H NMR (600 MHz, D
Sodium salt: H NMR (600 MHz, D
2
O): d = 3.01 (s, 2H), 3.70 (s,
2
O): d = 3.03 (s, 2H), 3.75 (s,
2
H), 3.74 (s, 2H), 7.22 (t, J = 7.2 Hz, 1H), 7.27–7.31 (m, 3H), 7.41
2H), 3.80 (s, 2H), 7.29–7.32 (m, J = 6 Hz, 2H), 7.16 (s, 1H), 7.41 (d,
J = 7.8 Hz, 1H), 7.48–7.51 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 7.8 Hz,
(
d, J = 7.8 Hz, 1H), 7.43–7.45 (d, J = 7.8 Hz, 1H), 7.51 (d, 1H).
1
H), 7.59 (d, J = 7.8 Hz, 1H), 7.65 (s, 1H).
5
.3.11. N-(4-Bromobenzyl)-N-carboxymethyl-3,4-dicarboxy-
benzylamine (20)
5.3.17. N-(4-Trifluoromethylbenzyl)-N-carboxymethyl-3,4-dicar-
1
Triester: H NMR (600 MHz, CDCl
3
): d = 3.25 (s, 2H), 3.65 (s, 3H),
boxybenzylamine (26)
1
3
7
.71 (s, 2H), 3.82 (s, 2H), 3.87–3.89 (s, 6H), 7.20 (d, J = 7.8 Hz, 2H),
.42 (d, J = 7.8 Hz, 2H), 7.54 (d, J = 7.8 Hz, 1H), 7.66–7.69 (m,
Triester: H NMR (600 MHz, CDCl
3
): d = 3.28 (s, 2H), 3.66 (s, 3H),
3.83 (s, 2H), 3.87 (s, 3H), 3.89 (s, 3H), 7.45 (d, J = 7.8 Hz, 2H), 7.54–
7.56 (m, J = 7.8 Hz, 3H), 7.68–7.70 (m, J = 7.8 Hz, 2H). MS (ESI) m/z:
+
J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na] (isotopic) 486.2,
+
4
88.0 (calcd) (isotopic) 486.1, 488.1.
Sodium salt: H NMR (600 MHz, D
(obs) [M+Na] 476.1, (calcd) 476.4.
1
1
2
O): d = 2.78 (s, 2H), 3.45 (s,
Sodium salt: H NMR (600 MHz, D
2
O): d = 2.89 (s, 2H), 3.61 (s,
2
1
H), 3.46 (s, 2H), 6.97 (d, J = 8.4 Hz, 2H), 7.05–7.07 (d, J = 7.8 Hz,
H), 7.12 (s, 1H), 7.21–7.24 (m, J = 7.8 Hz, 3H).
2H), 3.66 (s, 2H), 7.16 (m, J = 8.4 Hz, 2H), 7.27 (d, J = 7.2 Hz, 1H),
7.33 (d, J = 6 Hz, 2H), 7.50 (d, J = 6.6 Hz, 2H).
5
.3.12. N-(2-Methylbenzyl)-N-carboxymethyl-3,4-dicarboxy-
5
.3.18. N-(2-Trifluoromethoxybenzyl)-N-carboxymethyl-3,4-
benzylamine (21)
dicarboxybenzylamine (27)
1
Triester: H NMR (600 MHz, CDCl
3
): d = 2.31 (s, 3H), 3.24 (s, 2H),
1
Triester: H NMR (600 MHz, CDCl
3
): d = 3.27 (s, 2H), 3.65 (s, 3H),
3
3
7
4
.65 (s, 3H), 3.80 (s, 2H), 3.87 (s, 2H), 3.88–3.89 (s, 6H), 7.13 (m,
H), 7.28 (d, J = 6.6 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H) 7.64 (s, 1H),
.68 (d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na] 422.2, (calcd)
22.4.
3
.86 (s, 2H), 3.87 (s, 2H), 3.88 (s, 6H), 7.19 (d, J = 7.8 Hz, 1H), 7.25
(
m, J = 7.8 Hz, 2H), 7.55(m, J = 7.8 Hz, 2H) 7.68 (d, J = 7.8 Hz, 2H).
+
+
MS (ESI) m/z: (obs) [M+Na] 492.1, (calcd) 492.4.
1
Sodium salt: H NMR (600 MHz, D
H), 3.87 (s, 2H), 7.30–7.36 (m, 5H), 7.40 (d, J = 7.2 Hz, 1H), 7.45
d, J = 7.8 Hz, 1H).
2
O): d = 3.03 (s, 2H), 3.82 (s,
Sodium salt: 1H NMR (600 MHz, D
2
O): d = 2.13 (s, 3H), 3.04 (s,
2
(
2
7
H), 3.73 (s, 2H), 3.82 (s, 2H), 7.17 (m, 3H), 7.31–7.33 (m, 3H),
.41 (d, J = 7.8 Hz, 1H).
5
.3.19. N-(3-Trifluoromethoxybenzyl)-N-carboxymethyl-3,4-
5
.3.13. N-(3-Methylbenzyl)-N-carboxymethyl-3,4-dicarboxy-
dicarboxybenzylamine (28)
benzylamine (22)
1
1
Triester: H NMR (600 MHz, CDCl
3
): d = 3.28 (s, 2H), 3.65 (s, 3H),
Triester: H NMR (600 MHz, CDCl
3
): d = 2.31 (s, 3H), 3.28 (s, 2H),
3
7
7
.79 (s, 2H), 3.84 (s, 2H), 3.86–3.88 (s, 6H), 7.06 (d, J = 7.8 Hz, 1H),
.24 (s, J = 7.8 Hz, 1H), 7.30 (t, J = 7.8 Hz, 2H) 7.57 (d, J = 7.8 Hz, 1H),
.68 (d, J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na] 492.1, (calcd)
3
.66 (s, 3H), 3.73 (s, 2H), 3.84 (s, 2H), 3.87–3.88 (s, 6H), 7.03 (d,
J = 7.8 Hz, 1H), 7.12 (d, J = 7.8 Hz, 3H), 7.18 (m, J = 7.8 Hz, 1H)
.57 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 7.8 Hz, 2H). MS (ESI) m/z: (obs)
+
7
+
492.4.
Sodium salt: 1H NMR (600 MHz, D
H), 3.77 (s, 2H), 7.21 (d, J = 7.8 Hz, 1H), 7.28–7.32 (m, 4H), 7.39–
.42 (m, J = 7.8 Hz, 2H).
[
M+Na] 422.2, (calcd) 422.4.
1
2
O): d = 3.03 (s, 2H), 3.75 (s,
Sodium salt: H NMR (600 MHz, D
2
O): d = 2.28 (s, 3H), 3.01 (s,
2
7
2
1
H), 3.70 (s, 2H), 3.74 (s, 2H), 7.12–7.16 (m, J = 6 Hz, 2H), 7.16 (s,
H), 7.24–7.26 (t, J = 7.2 Hz, 1H), 7.29–7.31 (m, 2H), 7.41–7.42 (d,
J = 7.8 Hz, 1H).
5
.3.20. N-(4-Trifluoromethoxybenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (29)
5
.3.14. N-(4-Methylbenzyl)-N-carboxymethyl-3,4-dicarboxy-
1
Triester: H NMR (600 MHz, CDCl
3
): d = 3.27 (s, 2H), 3.66 (s, 3H),
benzylamine (23)
1
3.76 (s, 2H), 3.84 (s, 2H), 3.87 (s, 3H), 3.89 (s, 3H), 7.13 (d, J = 8.4 Hz,
H), 7.35 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 1H), 7.67–7.69 (m,
Triester: H NMR (600 MHz, CDCl
3
): d = 2.31 (s, 3H), 3.27 (s, 2H),
2
3
7
.66 (s, 3H), 3.73 (s, 2H), 3.87–3.89 (s, 6H), 7.11 (d, J = 7.8 Hz, 2H),
.22 (d, J = 7.8 Hz, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.69–7.70 (m,
+
J = 8.4 Hz, 2H). MS (ESI) m/z: (obs) [M+Na] 492.1, (calcd) 492.4.
1
+
Sodium salt: H NMR (600 MHz, D
2
O): d = 2.87 (s, 2H), 3.60 (s,
J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na] 422.2, (calcd) 422.4.
1
4H), 6.48–6.73 (m, 1H), 7.11 (d, J = 6 Hz, 2H), 7.16 (m, J = 6 Hz,
2
Sodium salt: H NMR (600 MHz, D O): d = 2.27 (s, 3H), 2.96 (s,
2
H), 7.22 (m, J = 6 Hz, 2H), 7.26 (m, J = 6 Hz, 1H).
2
7
H), 3.70 (s, 2H), 3.72 (s, 2H), 7.18–7.22 (m, J = 7.8 Hz, 4H), 7.28–
.30 (m, 2H), 7.40 (d, J = 7.8 Hz, 1H).
5
.3.21. N-(4-Difluoromethoxybenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (30)
5
.3.15. N-(2-Trifluoromethylbenzyl)-N-carboxymethyl-3,4-dicar-
1
Triester: H NMR (600 MHz, CDCl
3
): d = 3.26 (s, 2H), 3.65 (s, 3H),
boxybenzylamine (24)
1
3
(
7
.74 (s, 2H), 3.83 (s, 2H), 3.86–3.88 (s, 6H), 6.34–6.59 (m, 1H), 7.02
d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 1H),
.66–7.69 (m, J = 8.4 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 474.2,
Triester: H NMR (600 MHz, CDCl
3
): d = 3.28 (s, 2H), 3.66 (s, 3H),
3
7
2
.86 (s, 2H), 3.86–3.88 (s, 6H), 3.95 (s, 2H), 7.31 (t, J = 7.8 Hz, 1H),
.51 (t, J = 7.8 Hz, 1H), 7.57 (t, J = 7.8 Hz, 2H) 7.68 (m, J = 7.8 Hz,
H), 7.85 (d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na] 476.1,
+
(
calcd) 474.4.
Sodium salt: 1H NMR (600 MHz, D
2
O): d = 2.86 (s, 2H), 3.57 (s,
(
calcd) 476.4.
Sodium salt: 1H NMR (600 MHz, D
2
7
H), 3.60 (s, 2H), 6.48–6.73 (m, 1H), 6.95 (d, J = 8.4 Hz, 2H), 7.13–
.18 (m, J = 8.4 Hz, 4H), 7.24 (m, J = 7.8 Hz, 1H).
2
O): d = 3.05 (s, 2H), 3.79 (s,
2
H), 3.91 (s, 2H), 7.33 (m, 2H), 7.36–7.40 (m, 2H), 7.56 (t,
J = 7.8 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H).
5
.3.22. N-(4-Carboxybenzyl)-N-carboxymethyl-3,4-dicarboxy-
benzylamine (31)
5
.3.16. N-(3-Trifluoromethylbenzyl)-N-carboxymethyl-3,4-dicar-
1
Triester: H NMR (600 MHz, CDCl
3
): d = 3.26 (s, 2H), 3.64 (s, 3H),
boxybenzylamine (25)
1
3
.81 (s, 2H), 3.83 (s, 2H), 3.85 (s, 3H), 3.86 (s, 3H), 3.87 (s, 3H), 7.39
Triester: H NMR (600 MHz, CDCl
3
): d = 3.29 (s, 2H), 3.66 (s, 3H),
(
d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 1H), 7.66–7.68 (m, J = 8.4 Hz,
3
.83 (s, 2H), 3.85 (s, 2H), 3.86–3.89 (s, 6H), 7.40 (t, J = 7.8 Hz, 1H),

6
630
B. Thangavelu et al. / Bioorg. Med. Chem. 23 (2015) 6622–6631
2
(
H), 7.94 (d, J = 8.4 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 466.2,
calcd) 466.4.
Sodium salt: 1H NMR (600 MHz, D
5.3.28. N-Acetal-N-carboxymethyl-3,4-dicarboxybenzylamine
(37)
To a 0.8 ml solution of compound 3Me (0.15 g, 0.5 mmol) in for-
2
O): d = 2.83 (s, 2H), 3.54 (s,
2
7
H), 3.58 (s, 2H), 7.11 (d, 2H), 7.17–7.19 (m, 3H), 7.25 (d, 1H),
.61 (d, 2H).
mic acid 0.8 ml of acetic anhydride was added dropwise and the
reaction mixture was refluxed at 100 °C for 1 h. The reaction mix-
ture was cooled to rt and then concentrated by vacuum and diluted
5
.3.23. N-(4-Carboxamidebenzyl)-N-carboxymethyl-3,4-dicarb-
with DCM. The DCM portion was washed 4 times with 1 M NaHCO
and twice with water and dried over anhydrous Na SO . The con-
centrated product was dissolved in 0.4 ml of THF and the esters
3
oxybenzylamine (32)
2
4
1
Triester: H NMR (600 MHz, CDCl
3
): d = 3.27 (s, 2H), 3.65 (s, 3H),
d = 4.07 (m, 2H), 3.79–3.82 (m, 4H), 3.85–3.87 (s, 6H), 6.19–6.36
were hydrolyzed as described above to yield compound 37.
1
(
(
m, 2H), 7.37 (d, J = 7.8 Hz, 2H), 7.53 (d, J = 7.2 Hz, 1H), 7.64–7.66
m, 2H), 7.72 (d, J = 8.4 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]
3
Triester: H NMR (600 MHz, CDCl ): d = 3.64 (s, 3H), 3.83 (s, 3H),
+
3.84 (s, 3H), 3.88 (s, 2H), 4.53–4.59 (m, 2H), 7.34 (d, J = 6.6 Hz, 1H),
7.49 (d, J = 8.4 Hz, 1H), 7.63–7.69 (m, 1H), 8.12–8.29 (m, 1H). MS
4
51.3, (calcd) 451.4.
Sodium salt: H NMR (600 MHz, D
1
+
2
O): d = 3.02 (s, 2H), 3.77–3.79
(ESI) m/z: (obs) [M+Na] 364.4, (calcd) 364.3.
1
(
m, 4H), 7.31–7.34 (M, 2H), 7.37 (d, J = 7.8 Hz, 2H), 7.41 (d,
Sodium salt: H NMR (400 MHz, D
2
O): d = 3.0 (s, 2H), 3.55 (s,
J = 7.8 Hz, 1H), 7.78 (d, J = 8.4 Hz, 2H).
2H), 7.15–7.19 (m, 2H), 7.27–7.29 (d, J = 8 Hz, 1H), 8.26 (s, 1H).
5
.3.24. N-(4-t-Butylbenzyl)-N-carboxymethyl-3,4-dicarboxy-
5.3.29. N-((4-(2-Benzyl)vinyl)benzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (38)
benzylamine (33)
1
Triester: H NMR (400 MHz, CDCl
3
): d = 1.30 (s, 9H), 3.31 (s, 2H),
Compound 20 (0.48 g, 1.03 mmol) was weighed in a vial and bis
2
2,23
3
.67 (s, 3H), 3.77 (s, 2H), 3.87 (s, 2H), 3.88–3.91 (s, 6H), 7.27 (d,
(tri-tert-butylphosphinepalladium(0)
(0.1 g, 0.19 mmol) was
J = 8 Hz, 2H), 7.34 (d, J = 8 Hz, 2H), 7.59 (d, J = 8 Hz, 1H), 7.61 (d,
added under inert conditions in a nitrogen bag to the sealed vial.
Then, at rt and under inert conditions, was sequentially added
3 ml of anhydrous toluene, N-cyclohexyl-N-methyl cyclohexy-
lamine (0.27 ml, 1.24 mmol) and styrene (0.17 ml, 1.44 mmol).
The reaction mixture was allowed for stirring in a microwave reac-
tor at 110 °C for 16–18 h. Water was added to the reaction mixture,
followed by extraction with several portions of diethyl ether, dried
over anhydrous sodium sulfate and concentrated in vacuum. The
product was purified by column chromatography using 0–15%
ethyl acetate:hexanes. The resulting ester product was hydrolyzed
+
J = 7.6 Hz, 2H). MS (ESI) m/z: (obs) [M+Na] 464.3, (calcd) 464.5.
1
Sodium salt: H NMR (600 MHz, D
2
O): d = 1.26 (s, 9H), 3.01 (s,
2
H), 3.73 (s, 4H), 3.66 (s, 2H), 7.16 (m, J = 8.4 Hz, 2H), 7.28–7.31
m, 3H), 7.34 (s, 1H), 7.41–7.43 (d, J = 7.8 Hz, 1H), 7.44–7.46 (d,
J = 7.8 Hz, 2H).
(
5
.3.25. N-(4-(2-Perfluoropropyl))-N-carboxymethyl-3,4-dicar-
boxybenzylamine (34)
1
Triester: H NMR (600 MHz, CDCl
3
): d = 3.30 (s, 2H), 3.66 (s, 3H),
3
.84 (s, 2H), 3.87–3.88 (m, 8H), 7.47 (m, J = 7.8 Hz, 2H), 7.52 (m,
as described above (Section 5.3) to yield compound 38.
1
J = 8.4 Hz, 2H), 7.56 (d, J = 7.8 Hz, 1H), 7.69 (m, 2H). MS (ESI) m/z:
Triester: H NMR (600 MHz, CDCl
3
): d = 3.3 (s, 2H), 3.67 (s, 3H),
+
(
obs) [M+Na] 575.9, (calcd) 576.4.
3.78 (s, 2H), 3.86 (s, 2H), 3.88–3.90 (s, 6H), 7.08 (m, 2H), 7.25 (m,
1H), 7.32–7.35 (m, 4H), 7.45 (d, J = 7.8 Hz, 2H), 7.50 (d, J = 7.8 Hz,
2H), 7.60 (d, J = 7.8 Hz, 2H), 7.71(d, J = 8.4 Hz, 2H). MS (ESI) m/z:
1
Sodium salt: H NMR (600 MHz, D
2
O): d = 3.07 (s, 2H), 3.77 (s,
2
H), 3.83 (s, 2H), 7.33–7.34 (m, 1H), 7.39 (s, 1H), 7.45 (d,
+
J = 7.8 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 7.8 Hz, 2H).
(obs) [M+Na] 510.2, (calcd) 510.5.
1
Sodium salt: H NMR (400 MHz, D
2
O): d = 3.03 (s, 2H), 3.75 (s,
5
.3.26. N-(4-Bromobenzyl)-N-carboxyethyl-3,4-dicarboxybenzyl-
2H), 3.76 (s, 2H), 7.22 (m, 2H), 7.28–7.34 (m, J = 7.2 Hz, 5H),
7.38–7.43 (m, J = 7.8 Hz, 3H), 7.55–7.59 (d, J = 7.8 Hz, 4H).
amine (35)
4
-Bromo-benzyl bromide was added to a solution of compound
4
Me, followed by hydrolysis of the ester product as described
5.4. Enzyme purification and kinetic assay
above to yield compound 35.
1
Triester: H NMR (600 MHz, CDCl
3
): d = 2.46 (t, 2H), 2.74 (t, 2H),
ASADHs from S. pneumoniae and V. cholerae were cloned,
expressed and purified following our published procedure.24 The
concentrated enzymes were stored at ꢂ20 °C in a storage buffer
containing 50 mM HEPES (pH 7), 1 mM EDTA and 1 mM DTT.
Because of the instability of aspartyl phosphate the reaction was
followed in the reverse direction, with the increase in the absor-
bance of NADPH monitored at 340 nm. Initial velocity kinetics of
ASADH was carried out at rt in a reaction buffer composed of
3
(
7
(
.47 (s, 3H), 3.56 (s, 2H), 3.59 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 7.13
d, J = 7.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 2H), 7.46 (d, J = 7.8 Hz, 1H),
.59 (s, 1H), 7.66 (d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]
isotopic) 500.1, 501.9 (calcd) (isotopic) 500.1, 502.1.
3
Sodium salt: H NMR (600 MHz, CDCl ): d = 2.43 (t, 2H), 2.73 (t,
+
1
2
2
H), 3.54 (s, 2H), 3.59 (s, 2H), 7.19 (d, J = 7.8 Hz, 2H), 7.27–7.31 (m,
H), 7.41 (d, J = 7.2 Hz, 1H), 7.48 (d, J = 7.8 Hz, 2H).
1
20 mM CHES, pH 8.6 and 200 mM KCl, with ASA at either
5
.3.27. N-(4-Trifluoromethylbenzyl)-N-carboxymethyl-3,4-
0.15 mM (spASADH) or 0.3 mM (vcASADH), 1.5 mM NADP and
ꢂ1
dicarboxybenzylamine (36)
-Trifluroromethylbenzyl bromide was added to a solution of
20 mM phosphate in a final volume of 200
ll. Enzyme (30
l
g ml
)
4
was added to each well to initiate the reaction. To determine the
compound 4Me, followed by hydrolysis of ester product as
inhibition constant (K ) of each compound, inhibitor was added
i
described above to yield compound 36.
to each well by serial dilution to cover a suitable concentration
range. The measured initial velocities were then fitted to a Dixon
1
Triester: H NMR (600 MHz, CDCl
3
): d = 2.48 (t, 2H), 2.77 (t, 2H),
2
5
3
.58 (s, 2H), 3.59 (s, 2H), 3.60 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 7.40
plot that assumes competitive inhibition against ASA to deter-
mine the K value for each inhibitor.
(
d, J = 7.8 Hz, 2H), 7.49 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 7.8 Hz, 1H),
i
+
7
4
.61 (s, 1H), 7.66 (d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]
90.2, (calcd) 490.4.
Acknowledgements
1
Sodium salt: H NMR (600 MHz, CDCl
3
): d = 2.44 (t, 2H), 2.75 (t,
2
3
H), 3.61 (s, 2H), 3.64 (s, 2H), 7.33 (d, J = 7.8 Hz, 2H), 7.41–7.45 (m,
H), 7.64 (d, J = 7.8 Hz, 2H).
The authors thank Paul Erhardt (University of Toledo, Center for
Drug Design and Development) for helpful structure–activity rela-

B. Thangavelu et al. / Bioorg. Med. Chem. 23 (2015) 6622–6631
6631
tionship discussions. This work was supported by a grant to R.E.V.
from the National Institutes of Health (AI077720).
8. Hanzelka, B. L.; Greenberg, E. P. J. Bacteriol. 1996, 178, 5291.
Dickschat, J. S. Nat. Prod. Rep. 2010, 27, 343.
9.
10. Blanco, J.; Moore, R. A.; Kalabeeswaran, V.; Viola, R. E. Protein Sci. 2003, 12, 27.
1. Blanco, J.; Moore, R. A.; Viola, R. E. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12613.
1
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