Design and optimization of aspartate N-acetyltransferase inhibitors for the potential treatment of Canavan disease

Article abstract of DOI:10.1016/j.bmc.2016.11.060

Canavan disease is a fatal neurological disorder caused by defects in the metabolism of N-acetyl-L-aspartate (NAA). Recent work has shown that the devastating symptoms of this disorder are correlated with the elevated levels of NAA observed in these patients, caused as a consequence of the inability of mutated forms of aspartoacylase to adequately catalyze its breakdown. The membrane-associated enzyme responsible for the synthesis of NAA, aspartate N-acetyltransferase (ANAT), has recently been purified and examined (Wang et al., Prot Expr Purif. 2016;119:11). With the availability, for the first time, of a stable and soluble form of ANAT we can now report the identification of initial inhibitors against this biosynthetic enzyme, obtained from the screening of several focused compound libraries. Two core structures of these moderate binding compounds have subsequently been optimized, with the most potent inhibitors in these series possessing sub-micromolar inhibition constants (K_ivalues) against ANAT. Slowing the production of NAA via the inhibition of ANAT will lower the elevated levels of this metabolite and can potentially serve as a treatment option to moderate the symptoms of Canavan disease.

Full text of DOI:10.1016/j.bmc.2016.11.060

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and optimization of aspartate N-acetyltransferase inhibitors for
the potential treatment of Canavan disease
a
Bharani Thangavelu a,b, Vinay Mutthamsetty , Qinzhe Wang, Ronald E. Viola
⇑
Department of Chemistry and Biochemistry, University of Toledo, Toledo, OH 43606, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Canavan disease is a fatal neurological disorder caused by defects in the metabolism of N-acetyl-L-aspar-
Received 29 October 2016
Revised 27 November 2016
Accepted 29 November 2016
Available online xxxx
tate (NAA). Recent work has shown that the devastating symptoms of this disorder are correlated with
the elevated levels of NAA observed in these patients, caused as a consequence of the inability of mutated
forms of aspartoacylase to adequately catalyze its breakdown. The membrane-associated enzyme
responsible for the synthesis of NAA, aspartate N-acetyltransferase (ANAT), has recently been purified
and examined (Wang et al., Prot Expr Purif. 2016;119:11). With the availability, for the first time, of a
stable and soluble form of ANAT we can now report the identification of initial inhibitors against this
biosynthetic enzyme, obtained from the screening of several focused compound libraries. Two core struc-
tures of these moderate binding compounds have subsequently been optimized, with the most potent
inhibitors in these series possessing sub-micromolar inhibition constants (Ki values) against ANAT.
Slowing the production of NAA via the inhibition of ANAT will lower the elevated levels of this metabolite
and can potentially serve as a treatment option to moderate the symptoms of Canavan disease.
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Aspartate N-acetyltransferase
Canavan disease
Enzyme inhibitors
Fragment based drug discovery
Structure-activity relationships
1. Introduction
clinical mutants of this enzyme3 have led to the exploration of
enzyme replacement as a possible treatment therapy for Canavan
Defects in the metabolism of N-acetyl-
L
-aspartate (NAA) (Fig. 1)
disease.8 However, more recent work has shown that a knockout
of the Nat8l gene that codes for aspartate N-acetyltransferase
(ANAT), the enzyme which catalyzes NAA synthesis, leads to a
reversal of the CNS demyelination in an animal model of CD.9 On
the basis of these results an approach in which selective ANAT
inhibitors are designed and synthesized can be used to adjust the
elevated brain NAA levels back into the physiological range. Such
an approach has the potential to serve as a treatment for the symp-
toms of CD.
The connection between elevated brain NAA levels and CD has
been well established.1 In addition, recent work has identified
some intriguing links between NAA levels and several different
cancers. Overexpression of ANAT and the subsequent elevation of
NAA has been detected in a large percentage of adenocarcinoma
and squamous cell carcinoma cells isolated from patients with
late-stage lung cancer.10 Elevated NAA and high expression of
ANAT was also identified in high-grade ovarian cancer tissue sam-
ples,11 and has been correlated with worse overall patient survival
in this and in several other forms of cancers.
have been shown to be the underlying cause of a fatal neurological
disorder called Canavan disease (CD).1,2 Multiple mutations in the
aspA gene that codes for aspartoacylase (www.hgmd.org), the
enzyme responsible for the deacetylation of NAA in the brain, lead
to mutant enzyme forms3 with inadequate catalytic potential to
produce sufficient levels of acetate or to lower the accumulation
of NAA that is the diagnostic hallmark of CD.4
For many years the prevailing opinion among researchers in
this field had linked this acetate deficiency to the brain develop-
mental disorders that are observed in CD patients.5,6 Structural
and mechanistic characterization of aspartoacylase7 and several
Abbreviations: ANAT, aspartate N-acetyltransferase; CD, Canavan disease; DCM,
dichloromethane; DTNB, dithionitrobenzoate; EDCl, 1-ethyl-3-(3-(dimethylamino)
propyl)carbodiimide; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid;
IPTG, isopropyl-b-thiogalactoside; LB, Luria broth; LE, ligand efficiency; MBP,
maltose binding protein; NAA, N-acetyl-L-aspartate; NBS, N-bromosuccinimide;
TLC, thin layer chromatography; THF, tetrahydrofuran.
⇑
Corresponding author.
E-mail address: ron.viola@utoledo.edu (R.E. Viola).
B.T. and V.M. contributed equally to this work.
Present address: Department of Molecular Cardiology, Lerner Research Institute,
Identifying and developing effective enzyme inhibitors requires
the availability of a selective enzyme activity assay and the isola-
tion and purification of the target enzyme. A fixed-time ANAT
a
b
Cleveland Clinic Foundation, Cleveland, OH, United States.
http://dx.doi.org/10.1016/j.bmc.2016.11.060
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Fig. 1. Metabolism of N-acetyl-L-aspartate (NAA) in the brain. NAA is produced from L-aspartate and acetyl-coenzyme A catalyzed by aspartate N-acetyltransferase (ANAT).
After transport from neurons to oligodendrocytes NAA is hydrolyzed to release acetate catalyzed by aspartoacylase (ASPA), with acetate serving as the building block for the
synthesis of the fatty acid component of the myelin sheath.
assay that relies on the detection of radiolabeled NAA had previ-
ously been developed,12 but the membrane-associated nature of
ANAT had made enzyme purification a significant challenge.13,14
Several acyl-coenzyme A derivatives had been found to inhibit
the activity of ANAT in crude brain extracts,15 but without access
to purified enzyme there has been no progress to identify selective
and potent ANAT inhibitors. We recently developed the application
of fusion enzyme constructs to successfully solubilize and purify
ANAT as a maltose binding protein (MBP) fusion,16 and have used
an optimized, continuous thiol detection assay17 to kinetically
characterize this newly produced enzyme form. We now report
the results from a focused library screening effort to identify initial
ANAT inhibitors, followed by the systematic optimization of sev-
eral hits identified from these screens to yield selective, sub-micro-
molar inhibitors of this key regulator of brain NAA metabolism.
modified to determine the geometric properties that would lead to
optimal inhibitor binding. For this process of optimization a 2-
amino dioic acid was utilized as the parent structure, with the chain
length starting from that of the substrate,
L-aspartic acid (n = 1),
increasing to -glutamic acid (n = 2) and finally to 2-aminoadipic
L
acid (n = 3) (Scheme 1).
The length of the N-acylated side chain (indicated in red)
attached to these dioic acid core structures was also varied from
m = 0–3, as was the nature of the linker between the amino group
and the acyl side chain (Table 2). Each of these compounds was
synthesized as described in the Section 5, and were then kinetically
evaluated as potential inhibitors of ANAT.
The inhibition constants (Ki) determined from these studies
identified a systematic variation in binding affinity that is associ-
ated with changes in the values of n and m (Table 2). The kinetic
evaluation of this series of N-acyl dioic acid compounds against
ANAT suggests that a value for n of 1–2 (aspartate or glutamate)
and a value of m = 2, with either an acyl or an ester linkage, pro-
vides the optimal inhibition among this series of compounds. A
shorter dioic acid, aminomalonate, containing the reasonably
potent phenylpropanoyl side chain was also examined, but was
found to be a much less effective inhibitor than the longer dioic
acids (Table 2).
2. Results
2.1. Initial library screening
ANAT, the membrane-associated enzyme which catalyzes the
synthesis of NAA, has been found to be highly selective for L-aspar-
tate as the acetyl group acceptor. The introduction of a methyl
group at carbon-3 or the incorporation of a second amino group
are each somewhat tolerated, but the extension of the dioic acid
structure by one carbon (glutamate vs. aspartate) causes a drop
in catalytic efficiency to less than 1% that of the physiological sub-
strate.16 While the prevention of catalytic turnover is the goal of
inhibitor design, this inhibition must also possess improved affin-
ity that will enable an inhibitor to successfully compete with the
substrate for active site binding.
Our search for an initial set of inhibitors of ANAT started with
the screening of several focused compound libraries. The initial
libraries were composed of amino acids, amino acid derivatives,
various metabolites and metabolite analogs. Examination of a
library of 256 compounds in these structural groups yielded a total
of 17 weak inhibitors that showed at least 50% inhibition of ANAT
when examined at a concentration of 2 mM (Table 1). The most
potent initial inhibitor identified from these libraries, an N-chlor-
2.3. Optimization of dioic acid inhibitor design
Now that the geometric constraints of the ANAT inhibitor bind-
ing site has been systematically explored by varying the length of
the dioic acid structure and the length of the N-linked side chain
(Scheme 1), next the nature of the N-linkage, the requirement for
an aryl vs. alkyl side chain, and the effect of substituents on the aryl
side chain were explored.
2.3.1. Evaluation of the importance of the amide linkage in the
optimized structure
To determine if the amide linkage plays an important role in the
binding of these dioic acid inhibitors a series of structurally related
N-aryl derivatives of DL-aspartate were prepared and tested as
potential inhibitors of ANAT. While each of the N-aryl derivatives
were found to be inhibitors of ANAT (Table S1), these compounds
have only moderate affinities, even when adjusted for the correct
oacetyl derivative of the substrate, has a Ki value of 200 lM. Next,
a library of constrained amino acid analogs was examined, produc-
ing an additional set of five weak ANAT inhibitors. However, one of
L-isomer concentration, and the best inhibitors of this series (com-
these compounds, the N-carbobenzyloxy derivative of
L-aspartate,
pounds D19 and D20) were found to be significantly weaker inhi-
bitors than the corresponding N-acyl and N-ester linked
compounds (compounds D4 and D5) (Table 2).
gave an unexpectedly potent Ki value of 17 M (Table 1) and pro-
l
vided an excellent starting point for further inhibitor optimization.
2.2. Production of dioic acid derivatives (the D series)
2.3.2. Evaluation of the importance of an aromatic ring in the side
chain
Based on these initial screening results a core dioic acid struc-
ture that produced the best ANAT inhibitor was then systematically
To determine if the presence of an aryl side chain plays a
significant role in the binding affinity of these dioic acid inhibitors
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B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
3
Table 1
Summary of compound library screening and hit optimization against ANAT.
Library
Number of compounds
Weak inhibitorsa
Strong inhibitorsb
Most potent (Ki value)
N-Chloroacetyl- -aspartic acid (Ki = 200
2-Bromofumarate (Ki = 367 M)
N-Alanyl- -aspartic acid (Ki = 1.6 mM)
Amino acids
Metabolites
Amino acids II
Constrained analogs
96
96
64
77
3
13
1
0
0
0
1
L
lM)
l
L
5
N-Carbobenzyloxy-
L-aspartic acid (Ki = 17 lM)
Screened Compounds
(hit rate)
333
22 (6.6%)
1 (0.3%)
Dioic acids (D series)
Phthalates (P series)
41
60
4
11
N-Carbobenzyloxy-
4-Aminomethyl(N-carboethyl,N-4-carboxy-2,6-dichlorobenzyl) phthalate
L
-glutamic acid (Ki = 12
l
M)
(Ki = 0.6 M)
l
Total number
434
16
a
At least 50% inhibition when tested at 2 mM concentration.
Compounds with Ki values less than 100 lM.
b
derivatives achieved Ki values in the several hundred micromolar
range, significantly weaker than the N-acyl side chains on the same
dioic acid structures.
O
OH
HO
O
n
NH
O
2.3.3. Evaluation of the effect of aryl substitutions on inhibitor binding
Once the important of the N-aryl side chain had been con-
firmed, a series of substituents were introduced into the aromatic
ring to determine if either electronic or steric effects play an
important role in the binding of the aryl side chain to ANAT. The
addition of hydrophobic substituents on the N-benzyl ring does
lead to some alterations in the inhibition of ANAT relative to the
m
Scheme 1. Optimization of the dioic acid core structure.
a series of N-alkyl derivatives of
L-aspartate, L-glutamate, and
parent N-aryl-L-aspartate structure, with the ortho-CF3 substituent
L
-aminoadipate were prepared and tested. Several of these N-alkyl
(D33) showing the best inhibition among this group of derivatives
(Table S3). However, these changes are relatively small and did not
identify a significant trend for these modified derivatives in their
interactions with ANAT.
compounds did not show any inhibition against ANAT when tested
at 2 mM, while some were found to be only weak inhibitors
(Table S2). However, the best inhibitors among these N-alkyl
Table 2
N-Acylation derivatives of aminomalonic, aspartic, glutamic, and aminoadipic acids.
Cpd.
No.
m
2
Linker
Acyl
R group
Ki (mM)
1.4 0.2
Aminomalonate
derivaꢀve
(n=0)
D1
3-Phenylpropanoyl
D2
D3
0
1
2
2
3
0
1
2
2
3
0
1
2
2
3
Acyl
Acyl
Acyl
Ester
Acyl
Acyl
Acyl
Acyl
Ester
Acyl
Acyl
Acyl
Acyl
Ester
Acyl
Benzoyl
>2
Aspartate
derivaꢀves
(n = 1)
2-Phenylacetyl
3-Phenylpropanoyl a
Benzylcarbanoyl a
4-Phenylbutanoyl
Benzoyl
0.46 0.08
0.031 0.002
0.017 0.002
0.20 0.02
0.13 0.01
0.25 0.03
0.038 0.002
0.012 0.001
0.61 0.12
1.60 0.34
1.16 0.24
2.90 0.30
2.35 0.29
0.74 0.17
D4
D5
D6
D7
Glutamate
derivaꢀves
(n = 2)
D8
2-Phenylacetyl
3-Phenylpropanoyl a
Benzylcarbanoyl a
4-Phenylbutanoyl
Benzoyl
D9
D10
D11
D12
D13
D14
D15
D16
2-Aminoadipate
derivaꢀves
(n = 3)
2-Phenylacetyl
3-Phenylpropanoyl
Benzylcarbanoyl
4-Phenylbutanoyl
aSubstitutions shown in red indicate the best inhibitors in this compound class.
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4
B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Table 3
Synthesis and kinetic evaluation of 4-aminomethylphthalate and 4-aminoethylphthalate derivatives.
O
O
R1
O
O
O
O
R1
R1
NaHCO3
R2-X
CH3CN:H2O
NaO
O
O
N
R2
ONa
ONa
1N NaOH
THF:H2O
N
H
O
O
N
R2
O
O
O
O
O
Cpd. No.
R1
R2
Ki (l
M)
P3
H
H
H
H
H
H
n.i.b
P4a
P5
1710 333
n.i.b
(S)-CH3
(S)-CH2OH
P6
n.i.b
P10
P11
P12
P13
P14
P30
P31
P32
P33
P48a
P49a
H
H
H
H
H
H
H
H
H
H
H
CH3
n.i.b
Propionitrile
Acetaldehyde
Allyl
Benzyl
4-Biphenyl
N-Ethylmorpholino
2-Naphthyl
1-Naphthyl
4-Biphenyl
1-Naphthyl
n.i.b
1290 240
719 143
420 86
953 173
1080 380
538 75
115 12
550 103
34
5
a
N-Carboxymethyl group replaced with N-carboxyethyl.
No inhibition observed when examined at concentrations up to 2 mM.
b
2.4. Production of phthalate derivatives (the P series)
Ki of 1.7 mM (Table 3). To improve the affinity of the parent com-
pound P3 as a potential inhibitor of ANAT several additional
derivatives were synthesized through coupling with various
halides at the secondary amine position. The introduction of simple
alkyl or nitrile groups (P10 & P11) did not confer any inhibitory
properties to the parent compound, while the introduction of
either aldehyde or allyl groups (P12 & P13) produced only weak
inhibitors of ANAT with Ki values of 1.2 and 0.71 mM, respectively
(Table 3).
A related series of dioic acid structures, built on a conformation-
ally-constrained phthalic acid core, had previously been produced
and optimized as selective inhibitors of a Gram-positive form of
aspartate b-semialdehyde dehydrogenase obtained from
Streptococcus pneumonia.18 Given the similarity between the struc-
tures of the amino acid substrate of this enzyme to that of ANAT,
several members of this class of compounds were examined as
potential inhibitors of this enzyme. Compounds P3, with an
N-carboxymethyl side chain, as well as compounds P5 and P6, with
substitutions in the carboxymethyl branch, did not show any
measureable inhibition of ANAT. Compound P4 with a longer
N-carboxyethyl side chain does inhibit ANAT, but with a very weak
2.4.1. Introduction of aromatic functional groups
Based on the importance of N-aryl groups in conferring improved
binding affinity among the dioic acid inhibitors (the D series),
several N-aryl substituents were incorporated into the
Table 4
Synthesis and kinetic evaluation of 4-(N-substituted)benzylaminoalkylphthalates.
O
O
O
O
O
NaHCO3
NaO
O
O
1N NaOH
THF:H2O
N
Bn-R
ONa
ONa
N
Bn-R
O
O
R-Bn-X
N
H
O
O
n
n
n
CH3CN:H2O
O
O
O
O
R group
Aminomethylphthalates (n = 1)
Cpd. No.
Aminoethylphthalates (n = 2)
Cpd. No.
Ki (l
M)
Ki (lM)
2-Methyl
3-Methyl
4-Methyl
2-Bromo
3-Bromo
4-Bromo
2-Trifluroromethyl
3-Trifluoromethyl
4-Trifluoromethyl
4-tert-Butyl
P15
P16
P17
P18
P19
P20
P21
P22
P23
P28
P29
P34
1400 260
1700 220
4230 770
114 15
269 31
1200 50
390 50
P43
P44
P45
P37
P38
P39
P40
P41
P42
P46
P47
P50
724 91
563 89
2750 450
173 21
101
4
370 60
88
37
6
1
104
9
791 51
687 66
426 68
186 16
1660 330
587 72
4-(2-Perfluoropropyl)
4-Carboxy
104
9
29
2
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B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
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4-aminomethylphthalate core structure. Each of these aryl deriva-
tives were observed to be moderate inhibitors of ANAT, with the
benzyl (P14) and naphthyl (P32 & P33) derivatives leading to the
greatest improvement in affinity (Table 3). Introducing aryl
substituents into the homologous 4-aminoethylphthalate core
tuted series, it seems reasonable to suggest that this improvement
is likely due to an electrostatic interaction with a positively-
charged amino acid side chain within the binding pocket for this
aromatic side chain. To test this hypothesis the negatively-charged
p-carboxyl group was replaced with two different polar but
uncharged substituents, a carboxamide group (P35) and also a
ketone (P36). As predicted, these derivatives were found to be
much less effective inhibitors (Table S4), with Ki values around
2 mM that are significantly worse than even the unsubstituted
benzyl derivative.
structure led to
the 1-naphthyl substituent (P49) showing
inhibition (Ki = 34
a
further increase in binding affinity, with
50-fold greater
lM) when compared to the parent compound
a
(P4) (Table 3).
2.4.2. Incorporation of benzyl substituents
2.5. Development of the longer aminoethylphthalate structure
To further explore the binding affinity of the N-aryl derivatives
a series of substituted benzyl groups were introduced at this amine
position in both the 4-aminomethylphthalate and the 4-
aminoethylphthalate core structures. Coupling with benzyl halides
substituted with methyl groups at either the ortho-, meta- or para-
positions (P15–P17) actually led to weaker binding affinity of the
4-aminoalkylphthalates (Table 4) relative to the unmodified benzyl
derivative. The effect of changes in the nature of the methyl sub-
stituent was also examined. However, replacement of the methyl
group with either –OCF3 or –OCHF2 groups (P24–P27) did not sub-
stantially increase the affinity of these derivatives regardless of
their position on the benzyl ring (Table S4). Substitutions with
either a bromo or a trifluoromethyl group at the para-position of
the benzyl ring (P20 and P23) also resulted in a weaker inhibitor.
Changing the para-group to a bulkier substituent, such as p-tert-
butyl (P28) or p-perfluoropropyl (P29), also lead to only weak inhi-
bitors with no enhancement in their Ki values (Table 4). However,
substitution with either bromo or trifluoromethyl groups at the
ortho- or meta-positions (P18, P19, P21 and P22) lead to inhibitors
with some improvement in their Ki values against ANAT (Table 4)
relative to the unsubstituted benzyl derivative.
The parent aminomethylphthalate compound itself (P3) had
no inhibitory properties against ANAT until the introduction of
an N-aryl side chain. Now that the N-benzyl derivatives with
either bromo or trifluoromethyl group at the ortho- and meta-
position or with a p-carboxyl group, have been found to be good
inhibitors, several homologs of these derivatives were synthe-
sized starting from the parent aminoethylphthalate compound
P4 which had already been shown to be a moderate ANAT inhi-
bitor. The o- and m-methyl substituted derivatives (P43 and
P44) of this series also showed improved inhibition relative to
their shorter homologs (P15 and P16), however these derivatives
were still weaker inhibitors than the unsubstituted benzyl com-
pound (P14).
2.6. Substituent effects on aminoethylphthalate binding
Both the nature and the position of different benzyl sub-
stituents were then varied in an attempt to improve the affinity
of 4-(N-benzyl)aminoethylphthalates as ANAT inhibitors.
The o- and m-derivatives of either bromo (P37 and P38) or trifluo-
romethyl (P40 and P41) not only lead to better inhibitors than the
parent compound (Table 4), but these derivatives also have better
Ki values when compared to the same derivatives of the shorter
chain parent compound. For substituents at the para-position,
more hydrophobic alkyl groups such as t-butyl (P46) or perfluoro-
propyl (P47) were substantially weaker inhibitors than the parent
compound (Table 4). Further increases in the bulk of this benzyl
side chain led to mixed results. Replacing benzyl with biphenyl
(P48) had no significant effect on binding affinity, but replacement
with a 1-naphthyl ring (P49) resulted in a 10-fold improvement in
binding (Table 3). The p-bromobenzyl derivative (P39) gave
comparable inhibition as the parent compound, while the
p-trifluoromethyl substituent (P42) lead to a slight improvement
in affinity (Table 4). Based on the improvement observed with a
p-carboxyl group for the aminomethylphthalates, a similar deriva-
tive was synthesized for aminoethylphthalate. This compound
(P50) gave the lowest inhibition constant that had been observed
at this stage among the phthalate derivatives (Table 4).
While the incorporation of hydrophobic substituents in the
ortho- and meta-positions of the N-benzyl derivative resulted in
up to a 4-fold improvement in binding affinity, the presence of
hydrophobic substituents in the para-position proved to have a
deleterious effect on inhibitor binding. To examine the effect of a
hydrophilic substituent at this position, the p-carboxybenzyl
derivative (P34) was synthesized and tested against ANAT. Unlike
any of the other para-substituted derivatives this compound shows
enhanced inhibition, with a Ki value of 104 lM (Table 4). Because
this compound is the only improved inhibitor in the para-substi-
Table 5
Optimization of benzylaminoethylphthalates.
2.7. Optimization of phthalic acid inhibitor design
Cpd. No.
R1
R2
R3
Ki (μM)
After completion of the initial derivatization studies the two
best inhibitors obtained from the phthalate series of compounds
were the 1-napthyl derivative (P49) and the 4-carboxybenzyl
P52
P53
P54
P51
P57
P56
P55
P59
P58
-H
-CF3
-Phenyl
-Naphthyl
-F
-H
-H
-H
-H
-H
-H
-H
-Cl
-Br
-CF3
-H
-H
-H
-H
-H
-H
-H
-H
930 151
17.0 0.4
47.4 1.4
187 22
derivative (P50) with Ki values of 34 and 29 lM, respectively. This
observation led to the idea of combining the features of both
derivatives into a single molecule, by retaining the charged p-car-
boxyl group and then introducing hydrophobic groups at either the
ortho- or meta-positions. Unfortunately, the incorporation of a CF3
group at the meta-position (P52) resulted in a significant loss of
potency. However, placing this same group in the ortho-position
(P53) lead to a 2-fold improvement in binding relative to the start-
ing p-carboxyl derivative (Table 5). Expanding the size of the ortho-
4.06 0.08
1.70 0.04
2.01 0.07
0.61 0.01
0.77 0.01
-Cl
-Br
-Cl
-Br
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B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
hydrophobic group (P51 and P54) caused a decrease in affinity, but
replacement with an ortho-bromo substituent (P55) led to an addi-
tional 7-fold enhancement in affinity, to a Ki value of 2.4 lM
inhibitors. Unlike the acyclic dioic acids, many of which were
already weak inhibitors even without extensive derivatization
(Table 1), the parent N-carboxyalkylphthalates were either
non-inhibitors or extremely weak inhibitors of ANAT (Table 3).
However, the introduction of an N-aryl side chain into these core
structures conferred increasing affinity for our target enzyme.
The synthesis of a series of substituted benzyl derivatives clearly
defined the N-carboxyethyl side chain as the optimal length to best
position this acyl group into a binding pocket on ANAT, with most
derivatives showing a 2- to 4-fold enhancement in binding when
attached to the aminoethylphthalate (n = 2) structure compared
to the same derivatives of the aminomethylphthalates (n = 1)
(Table 4). This series of compounds also allowed the systematic
exploration of the role of the aryl side chain in binding. The intro-
duction of p-hydrophobic substituents on the benzyl ring were
detrimental to binding, but o- or m-hydrophobic substituents led
to as much as a 10-fold improvement in binding affinity (Table 4).
In contrast, the presence of a charged p-carboxyl group caused a
14-fold increased affinity relative to the parent N-benzyl deriva-
tive. The additional combination of o-dichloro groups with the p-
carboxyl substituent resulted in a nearly 700-fold improvement
(Table 5). Decreasing the size of the ortho-halo substituent from
bromo to chloro (P56) resulted in a further improvement in bind-
ing, while the further decrease to fluoro (P57) was less effective.
The dihalogenated derivatives showed the same trend, with the
dichloro substituent (P59) giving a slight improvement over the
dibromo derivative (P58). Each of these compounds now represent
the first sub-micromolar inhibitors of ANAT that have been pro-
duced (Table 5).
3. Discussion
3.1. Importance of ANAT as a drug development target
The overproduction of NAA has been shown to not only be a
consequence of the disruption of its subsequent metabolism, but
the underlying basis for the symptoms of Canavan disease.9,19
Because even the most impaired clinical mutants of aspartoacylase
have been demonstrated to still possess some residual catalytic
activity,4 slowing the production of NAA through the design of
selective ANAT inhibitors can potentially restore the balance in
NAA metabolism in the brain of CD patients (Fig. 1). With this goal
as a proposed therapeutic approach, we have screened some
focused compound libraries to identify inhibitors of the NAA
biosynthetic enzyme (ANAT), and have then proceeded to optimize
the structures of these initial inhibitors.
in affinity to a Ki value of 0.6 lM (Table 5). This best ANAT inhibitor
(P59) also has a very good ligand efficiency (LE) value of 0.27 kcal/-
mol/heavy atom,20 allowing the possibility of some additional opti-
mization that can lead to further enhancements in affinity, as well
as structural modifications to improve the bioavailability of this
compound series.
The amino acid binding site of ANAT has evolved to be specific
4. Conclusions
for L-aspartate as the acceptor of an acetyl group from acetyl-CoA,
and this site is highly selective for the binding of dicarboxylic acids.
Screening of an extensive group of amino acids and derivatives
found that only structures with an additional side chain carboxyl
group (or carboxylate mimic) showed any reasonable affinity for
binding to ANAT. The additional screening of a metabolite library
found that a wide variety of dicarboxylate structures were capable
of binding to the enzyme with moderate affinities, despite varia-
tions in the length of the core structure or the presence of addi-
tional functional groups. While the requirement of unmodified
carboxylate-type groups appears to be essential for binding to
ANAT, the amino group of aspartic acid is the acceptor for the
acetyl group in this reaction and must therefore remain accessible
to serve as the nucleophile for this group transfer reaction. We
have taken advantage of the required accessibility of this func-
tional group by designing and then elaborating a series of N-
derivatized dicarboxylic acids as more potent ANAT inhibitors.
A knock-out of the Nat8l gene completely blocked the synthesis
of NAA, and resulted in substantial recovery from the brain devel-
opmental defects that are the hallmark of CD.9 However, this
approach does come with some enhanced risks. The survival times
of the mice with this gene knock-out are significantly reduced,
even when the aspA gene that codes for aspartoacylase is not elim-
inated. When only one copy of the Nat8l gene is removed then the
survival times improve significantly.19 These results suggest that
extremely potent inhibitors of ANAT may not be desirable or even
advantageous as a drug. Slowing the production of NAA through
the interactions of moderately potent inhibitors with ANAT may
be sufficient to lower the abnormally high levels of this metabolite
and restore the balance to this pathway. Through systematic elab-
orations two different core structures have been converted into
reasonably potent inhibitors of the human ANAT enzyme that is
responsible for the overproduction of N-acetylaspartate in the
brain. With the overexpression of ANAT and elevated NAA levels
recently identified as correlating with poor patient survival in both
advanced stage lung cancer10 and in ovarian cancer,11 the effec-
tiveness of our ANAT inhibitors can also be tested in models of
these forms of cancer. The next step will be to convert these potent
enzyme inhibitors into more drug-like molecules, either by
replacement of the carboxylate functional groups with uncharged
isosteres or through the modification of these carboxylates with
hydrolysable derivatives.
3.2. Synthesis and optimization of dioic acid inhibitors of ANAT
Geometric optimization of a 2-amino dioic acid core structure
has led to either L-aspartate or L-glutamate derivatives as the best
dioic acid inhibitors, with either an acyl or an ester linkage to an
aromatic side chain. The most potent inhibitors in this series have
Ki values in the low micromolar range (Table 2), and have ligand
efficiency (LE) values between 0.3 and 0.4 kcal/mol/heavy atom.
When compared to typical drugs these are still relatively small
molecules. Further structural elaborations through the incorpora-
tion of new functional groups would make these inhibitors more
drug-like, and would likely lead to additional enhancements in
binding affinity.
5. Experimental
5.1. General chemistry
Solvents and reagents were used as purchased without further
purification. The fragment compound libraries were assembled
from commercially available compounds and prepared as
200 mM stock solutions with the pH adjusted as necessary using
either HCl or NaOH. For the newly synthesized enzyme inhibitors
3.3. Synthesis and optimization of phthalic acid inhibitors of ANAT
A series of conformationally-constrained dioic acids built on a
phthalic acid core structure were also examined as possible ANAT
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B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
7
the reaction time courses were monitored by TLC using an ethyl
acetate/hexane solvent system on Silica gel HLF plates (Analtech,
Inc., Newark, DE). The final products were purified by silica gel
(230–400 mesh) flash column chromatography. 1H and 13C NMR
spectra were recorded on either a Varian VXRS 400 MHz or a Var-
ian INOVA 600 MHz spectrometer. Mass spectra were measured on
a Bruker Esquire Liquid Chromatograph – Ion Trap Mass Spectrom-
eter by using electrospray ionization and detection in positive ion
mode. Compound purity was assessed by HPLC, using an Alltech
house library was kept at 4 °C. Acetyl-CoA was prepared as
10 mM stock in water and stored at À20 °C to minimize degrada-
tion. Before each set of assays, acetyl-CoA was diluted into a
2 mM working solution using HEPES buffer. 2 mM DTNB was
freshly prepared and kept on ice during the kinetic assay. For com-
pound library screening, 2
tor, and 116 l of buffer were added into each reaction well. 80
of freshly prepared pre-mix, which includes 4 l of acetyl-CoA, 4
of DTNB, 5 l of enzyme and 67
reaction. The reaction time course was monitored at 412 nm
= 14.15 mMÀ1 cmÀ1) for at least 15 min. For Ki determinations
ll of 200 mM L-aspartate, 2 ll of inhibi-
l
l
l
l
l
l
l
ll of buffer, was added to start the
alltima HP C18 amide 5
l
150 Â 4.6 mm column; with a linear gra-
dient from 90% water:10% methanol to 5% water:95% methanol in
20 min at a flow rate of 1.0 mL/min. For the compounds found to be
<95% pure, in most cases the primary impurity was the non-hydro-
lyzed ester precursor. The esters of this series of compounds were
found not to be inhibitors of ANAT.
(e
a twofold serial dilution of each inhibitor was performed starting
from either a 2 mM, 0.2 mM, or 0.02 mM stock solution, with the
starting concentration determined according to the potency of
the inhibitors. A negative control, in which no inhibitor was added,
and a background signal control, in which neither L-aspartate nor
inhibitor were added, were included in each set of assays. The
pH of the reaction wells with highest concentration of inhibitor
was tested to rule out any possible false positive due to pH
variations.
5.2. Enzyme production and activity assay
The recombinant maltose-binding protein-human aspartate N-
acetyltransferase (MBP-ANAT) fusion enzyme was expressed and
purified as reported previously.16 Briefly, NiCo21 (DE3) competent
E. coli cells (New England Biolabs, Ipswich, MA) were transformed
with an MBP-ANAT-his construct and selected on LB plates with
5.3. Compound library screening
30 lg/ml kanamycin. Colonies from these plates were used to inoc-
Four different compound libraries were used for the initial
screening of ANAT inhibitors. These fragment compound libraries
were assembled in-house: an amino acids library contained 96
compounds; a metabolite library contained 96 compounds and a
second amino acids library contained 64 compounds. An additional
constrained analog library contained 77 compounds that were also
purchased from commercial sources. Once the dioic acid core
structure was identified as a viable inhibitor of ANAT a library of
dioic acid compounds was synthesized for testing and
optimization.
ulate starter cultures in LB media. After diluting each starter cul-
ture by 100-fold in 1 L of LB media, cell growth was continued
for about 2 h at 37 °C until A600 reached 0.6. IPTG was then added
to a final concentration of 0.5 mM and protein expression was
induced at 16 °C for 20 h. To purify MBP-ANAT, a Ni Sepharose 6
Fast Flow column (GE Healthcare, Pittsburgh, PA) was equilibrated
with Buffer A (20 mM potassium phosphate, pH 7.4, 300 mM
sodium chloride, 10% glycerol and 20 mM imidazole). Cell lysate
was loaded on to the column and partially purified ANAT was then
eluted with a linear gradient of Buffer B (buffer A containing
400 mM imidazole). The active fractions were pooled and loaded
onto an amylose column and highly purified ANAT fusion was then
obtained by elution with a 0–10 mM linear maltose gradient. ANAT
activity was measured by an established DTNB-based assay17 using
a SpectraMax 190 spectrophotometer plate reader (Molecular
Devices, CA). A typical activity assay mixture contains 20 mM
5.4. General procedure for the preparation of N-acylated amino acids
The general synthetic scheme for the preparation of N-acylated
derivatives of aminomalonate (n = 0), aspartate (n = 1), glutamate
(n = 2) and 2-aminoadipate (n = 3) involves esterification to protect
the carboxylic acid groups, followed by coupling to different acyl
halides and then hydrolysis to yield the final products. The produc-
tion of specific dioic acid derivatives is illustrated for the synthesis
HEPES, pH 7.4, 150 mM NaCl, 5% glycerol, 40
acetyl-CoA, 2 mM -aspartate, 25 of enzyme and various
amount of inhibitors in a total volume of 200 l. Each compound
was prepared as 200 mM stock in water and compounds from in-
lM DTNB, 40 lM
L
lg
l
of two different N-acyl derivatives of L-aspartic acid (Scheme 2).
O
O
O
OH
OCH3
OCH3
b
a
HO
H3CO
H3CO
NH2
1
O
Cl NH3 O
NH
O
O
1Me
c
2Me
d
O
O
ONa
OCH3
O
NaO
O
H3CO
ONa
NaO
NH
O
O
NH O
NH
O
d
O
D4
3Me
D2
Scheme 2. Synthetic scheme for representative N-acyl-L-aspartate derivatives. Reagents and conditions: (a) SOCl2 in MeOH at 0 °C to RT 14 h; (b) benzoyl chloride, NaHCO3 in
DCM and H2O, RT, 8 h; (c) 3-phenylpropanoic acid, EDCl, Et3N in DCM, 0 °C to RT, 3 h; (d) NaOH in THF and H2O, RT, 5 h.
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8
B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
5.4.1. Synthesis of dimethyl
L-aspartate hydrochloride (1Me)
13C NMR (600 MHz, D2O): d = 24.97, 31.14, 31.90, 37.35, 39.19,
To an oven-dried round bottom flask,
L
-aspartic acid (1; 1 eq)
43.15, 67.79, 126.04, 126.37, 128.26, 140.71, 142.00, 168.27,
175.55, 176.67, 182.61.
was added and placed under a dry nitrogen gas atmosphere. The
material is then dissolved in anhydrous methanol and stirred at
0 °C for a few minutes until the desired temperature is attained.
Next, thionyl chloride (3 eq) was added dropwise until the reaction
mixture became homogeneous, then the reaction mixture was
warmed slowly to room temperature and left stirring for 14 h.
The reaction mixture was then concentrated under reduced pres-
sure to obtain compound 2 in the form of a pure white solid with
no addition purification required
Purity: HPLC – (>91%).
5.5.2. Benzoyl aspartic acid (D2)
Dimethylester: 1H NMR (600 MHz, CDCl3): d = 2.97–3.01 (dd,
J = 4.77, 17.66 Hz, 1H), 3.14–3.17 (dd, J = 4.77, 17.66 Hz, 1H),
d = 3.71 (s, 1H), 3.80 (s, 1H), 5.07 (m, 1H), 7.22 (d, 1H), 7.46 (t,
1H), 7.53 (t, 2H), 7.81 (d, 2H).
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.63 (m, 1H), 2.78 (m,
1H), 4.59 (q, J = 9.9 Hz, 1H), 7.51 (t, 1H), 7.60 (t, 2H), 7.78 (d, 2H).
13C NMR (600 MHz, D2O): d = 39.55, 53.70, 127.17, 128.65,
132.0, 133.59, 170.27, 178.63, 179.01.
5.4.2. Synthesis of dimethyl benzoyl-
L
-aspartate (2Me)
Dimethyl -aspartate hydrochloride (1Me; 1 eq) was added to a
L
round bottom flask, to which 5 ml of dichloromethane (DCM) and
2 ml of water was added. Sodium bicarbonate (1.5 eq) was added
to the resulting biphasic solution and the reaction mixture was
stirred for 2 min. Benzoyl chloride (1.4 eq) was added dropwise
to this solution and the reaction mixture was stirred at room tem-
perature overnight. After confirming completion of the reaction by
TLC, the organic layer was separated and the aqueous layer was
extracted three times with 50 ml of DCM. The organic extracts
were combined, washed with 75 ml of brine solution (aq), dried
over solid sodium sulfate, and then concentrated under reduced
pressure to obtain the crude product. Silica gel column chromatog-
raphy, with elution using 20% ethyl acetate in hexanes, afforded
pure compound 2Me as a white solid.
Purity: HPLC – (>99%).
5.5.3. (2-Phenylacetyl) aspartate (D3)
Dimethylester: 1H NMR (600 MHz, CDCl3): d = 2.82 (dd, J = 4.65,
16.96 Hz, 1H), 2.98 (dd, J = 4.65, 16.96 Hz, 1H), 3.60 (s, 2H), 3.61
(s, 3H), 3.72 (s, 3H), 4.83 (p, 1H), 6.45 (d, J = 7.57 Hz, 1H), 7.26–
7.31 (m, 3H), 7.36 (t, J = 7.08 Hz, 2H).
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.48 (dd, J = 9.54,
15.41 Hz, 1H), 2.66 (dd, J = 9.54, 15.41 Hz, 1H), 3.64 (q, 2H), 4.39
(q, J = 3.67, 9.54 Hz, 1H), 7.31–7.334 (m, 3H), 7.39 (t, J = 7.7 Hz, 2H).
13C NMR (600 MHz, D2O): d = 39.58, 42.13, 53.28, 127.11,
128.81, 129.25, 135.00, 171.01, 173.87, 178.55, 178.74.
Purity: HPLC – (>99%).
5.4.3. Synthesis of dimethyl (3-phenyl)propanoyl)-
L
-aspartate (3Me)
5.5.4. Dimethyl (3-phenylpropanoyl)aspartate (D4)
Dimethylester: 1H NMR (400 MHz, CDCl3): d = 2.55 (m, 2H), 2.75
(dd, J = 4.60 Hz, 17.65 Hz, 1H) 2.98 (t, J = 7.81 Hz, 2H), 3.01 (dd,
J = 4.15, 17.58 Hz, 1H), 3.67 (s, 3H), 3.75 (s, 1H), 4.86 (m, 1H),
6.40 (d, J = 8.06 Hz, 1H), 7.20 (m, 3H), 7.29 (t, J = 7.57 Hz, 2H).
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.38–2.43 (m, 2H),
2.49–2.52 (m, 2H), 2.85 (dt, J = 2.93, 7.69 Hz, 2H), 4.25 (q,
J = 4.03 Hz, 1H), 7.16–7.21 (m, 3H), 7.27 (t, J = 7.69 Hz, 2H).
13C NMR (600 MHz, D2O): d = 25.29, 31.21, 37.44, 39.52, 52.75,
58.29, 126.34, 128.38, 128.67, 140.76, 175.00, 178.58, 178.64
Purity: HPLC – (98.5%).
To a mixture of dimethyl -aspartate hydrochloride (1Me; 1 eq),
L
EDCl (1.5 eq) and trimethylamine (1.4 eq), DCM was added and the
solution stirred for 5 min. 3-phenylpropanoic acid (1 eq) was then
added to this solution with stirring for 3 h. After quenching the
reaction with saturated ammonium chloride, the reaction mixture
was extracted thrice with ethyl acetate. The organic extracts were
combined, washed with brine solution (aq), dried over solid
sodium sulfate, and then concentrated under reduced pressure to
obtain the crude product in the form of reddish oil. Purification
was performed by silica gel column chromatography with elution
using 50% ethyl acetate in hexanes to afford pure amide 3Me as
a yellow viscous liquid.
5.5.5. (4-Phenylbutanoyl)- -aspartic acid (D6)
L
Dimethylester: 1H NMR (600 MHz, CDCl3): d = 1.95 (m, 2H), 2.21
(t, 2H), 2.43 (t, 2H), 2.81 (m, 2H), 3.03 (m, 2H), 3.65 (s, 3H), 3.74 (s,
3H), 4.85 (t, 1H), 6.43 (d, 1H), 7.15–7.18 (m, J = 7.2 Hz, 3H), 7.23–
7.27 (m, J = 7.2 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 330.1, (calc)
330.1.
5.4.4. Hydrolysis of dimethyl-2-amino dioates (D2 or D4)
To the mixture of dimethyl-2-amino dioate (2Me or 3Me) (1 eq)
in THF and water (9:1 ratio), sodium hydroxide (2 eq) was added
and stirred for 5–8 h. After confirming the completion of reaction
by TLC, the reaction solution was concentrated for several hours
under reduced pressure to obtain the corresponding N-derivatized
sodium dioates (D2 or D4) as white/light yellow solids.
Sodium salt: 1H NMR (600 MHz, D2O): d = 1.85 (m, 3H), 2.22 (t,
2H), 2.43 (m, 1H), 2.60 (m, 3H), 4.34 (t, 1H), 7.21 (t, J = 7.2 Hz,
2H), 7.25 (m, J = 7.2 Hz, 2H), 7.31 (m, J = 7.8 Hz, 2H).
13C NMR (600 MHz, D2O): d = 26.94, 34.19, 35.03, 39.67, 53.03,
126.02, 128.54, 128.57, 128.65, 142.12, 175.92, 178.72, 178.77.
Purity: HPLC – (>99%).
5.5. General protocol for the synthesis of N-acyl dioic acid derivatives
(D1–D16)
A similar protocol to that described in Scheme 2 was used for
the synthesis of a series of N-acyl dioic acid derivatives, starting
with aspartic, glutamic or 2-aminoadipic acid and coupling with
different phenyl halides.
5.5.6. (2-Phenylacetyl)- -glutamic acid (D8)
L
Dimethylester: 1H NMR (600 MHz, CDCl3): d = 1.89 (m, 1H), 2.13
(m, 1H), 2.22–2.33 (m, 2H), 3.57 (s, 2H), 3.61 (s, 3H), 3.68 (s, 3H),
4.58 (m, 1H), 6.09 (d, 1H), 7.23–7.28 (m, J = 7.8 Hz, 3H), 7.32–
7.35 (m, J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 316.1, (calc)
316.0.
5.5.1. Diethyl 2-(3-phenylpropanamido)malonate (D1)
Diethylester: 1H NMR (400 MHz, CDCl3): d = 1.32 (t, J = 7.16 Hz,
6H), 2.60 (t, J = 7.64, 2H), 3.02 (m, 2H), 4.06 (d, J = 4.90 Hz, 1H)
4.24 (q, J = 7.13, 14.27 Hz, 4H), 6.06 (s, 1H), 7.24–7.35 (m, 5H).
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.45 (dt, J = 2.41, 7.93,
1H), 2.59 (dt, J = 2.41, 7.93 Hz, 1H), 2.90 (m, 2H), 3.72 (m, 1H),
7.23–7.28 (m, 3H), 7.32 (m, 2H).
Sodium salt: 1H NMR (600 MHz, D2O): d = 1.85 (m, 2H), 1.99 (d,
1H), 2.13 (m, 2H), 3.61 (m, 1H), 4.34 (d, 1H), 7.28 (m, 3H), 7.34 (m,
2H).
13C NMR (600 MHz, D2O): d = 28.43, 34.01, 42.19, 55.30, 127.16,
128.84, 129.12, 129.14, 135.06, 135.09, 174.10, 178.76, 182.13.
Purity: HPLC – (>99%).
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B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
9
5.5.7. (3-Phenylpropanoyl)-
L
-glutamic acid (D9)
(q, J = 7.34, 2H), 3.73 (s, 3H), 3.82 (s, 3H), 4.67–4.72 (m, 1H), 6.09
(d, J = 6.83 Hz, 1H), 7.25 (m, 3H), 7.36 (m, 2H). MS (ESI) m/z:
(obs) [M+Na]+ 358.4, (calc) 358.4.
Dimethylester: 1H NMR (600 MHz, CDCl3): d = 1.90 (m, 1H), 2.13
(m, 3H), 2.47–2.53 (m, 2H), 2.93 (m, 2H), 3.64 (s, 3H), 3.71 (s, 3H),
4.58 (m, 1H), 6.06 (d, 1H), 7.18 (d, J = 7.8 Hz, 3H), 7.23–7.27 (m,
J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 330.1, (calc) 330.1.
Sodium salt: 1H NMR (600 MHz, D2O): d = 1.52–1.69 (m, 4H),
2.42 (t, 2H), 2.71–2.20 (m, 2H), 3.84 (m, 1H), 4.34 (t, 1H), 7.06–
7.09 (m, J = 7.2 Hz, 3H), 7.17 (m, J = 7.2 Hz, 2H).
Sodium salt: 1H NMR (600 MHz, D2O): d = 1.5–1.75 (m, 4H), 1.89
(p, J = 7.48, 15.04 Hz, 2H), 2.11–2.19 (m, 2H), 2.27 (t, J = 7.46 Hz,
2H), 2.62 (t, J = 7.67 Hz, 2H), 4.04 (q, J = 4.54, 8.80 Hz, 2H), 7.23
(t, J = 7.31 Hz, 1H), 7.26 (d, J = 7.17 Hz, 2H), 7.33 (t, J = 7.74 Hz, 2H).
13C NMR (600 MHz, D2O): d = 22.54, 26.90, 31.40, 34.29, 35.01,
37.13, 39.93, 55.15, 126.05, 128.62, 142.02, 171.01, 176.10,
179.31, 183.12.
13C NMR (600 MHz, D2O): d = 28.42, 31.28, 33.77, 37.40, 54.86,
126.49, 128.42, 128.68, 140.36, 175.07, 178.73, 182.11.
Purity: HPLC – (98.0%).
Purity: HPLC – (94.4%).
5.5.8. (4-Phenylbutanoyl)-
L-glutamic acid (D11)
5.6. General phthalate derivative procedures
Dimethylester: 1H NMR (600 MHz, CDCl3): d = 1.94 (m, 3H), 2.18
(m, 3H), 2.33–2.41 (m, 2H), 2.62 (t, 2H), 3.63 (s, 3H), 3.72 (s, 3H),
4.60 (m, 1H), 6.06 (d, 1H), 7.16 (d, J = 8.4 Hz, 3H), 7.23–7.27 (m,
J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 344.1, (calc) 344.1.
Sodium salt: 1H NMR (600 MHz, D2O): d = 1.79 (m, 1H), 1.86 (m,
2H), 1.95 (m, 1H), 2.14 (t, 2H), 2.24 (t, 2H), 2.59 (t, 2H), 4.34 (m,
1H), 7.20–7.24 (m, J = 7.8 Hz, 3H), 7.30 (t, J = 7.2 Hz, 2H).
13C NMR (600 MHz, D2O): d = 26.87, 28.44, 34.07, 0.4.32, 35.06,
55.12, 126.06, 128.57, 128.61, 142.01, 176.11, 178.89, 182.15.
Purity: HPLC – (>99%).
Based on the observation of the dioic acid series and their
potency to bind with the ANAT, phthalate analogs were introduced
into the inhibitor design. This core structure allowed additional
structural elaborations and added complexity to the inhibitor
structure. To produce the parent compounds we followed our
previously published protocol,18 for the production of the amine-
containing 4-(carboxymethylaminomethyl) and 4-(2-carboxyethy-
laminomethyl)phthalate analogs P3 and P4, respectively, and also
for the amine-containing alanine methyl ester or serine methyl
ester analogs P5 and P6, respectively (Scheme 3).
5.5.9. 2-Benzamidohexanedioic acid (D12)
A similar protocol was used to produce compound P19 which is
analogous to compound P3 except for moving the position of the
side chain to carbon-3. Unfortunately, these 3-aminoalkylphtha-
late derivatives rapidly cyclized through attack by the adjacent car-
boxyl group. Protecting the amino group by N-methylation did
prevent cyclization, but this parent compound was not an effective
ANAT inhibitor. The affinities of the parent 4-aminoalkylphthalate
compounds were examined against ANAT and then improved
through the production of an extensive series of derivatives at
the introduced secondary amine.
Dimethylester: 1H NMR (600 MHz, CDCl3): d = 1.38–1.82 (m, 3H),
1.98 (m, 1H), 2.36 (m, 2H), 3.64 (s, 3H), 3.77 (s, 3H), 4.81 (m, 1H),
6.81 (d, 1H), 7.41–7.51 (m, J = 8.4 Hz, 5H). MS (ESI) m/z: (obs) [M
+Na]+ 316.1, (calc) 316.1.
Sodium salt: 1H NMR (600 MHz, D2O): d = 1.62–1.84 (m, 4H),
2.17 (m, 2H), 4.34 (m, 1H), 7.47 (m, 2H), 7.55 (m, 1H), 7.74 (m,
J = 6.6 Hz, 2H).
13C NMR (600 MHz, D2O): d = 22.67, 31.40, 37.14, 55.91, 127.16,
128.63, 131.99, 133.63, 170.61, 179.28, 183.20.
Purity: HPLC – (>99%).
5.6.1. 4-Methylphthalate dimethyl ester (4Me)
5.5.10. 2-(2-Phenylacetamido)hexanedioic acid (D13)
Dimethylester: 1H NMR (600 MHz, CDCl3): d = 1.50–1.61 (m, 3H),
1.84 (m, 1H), 2.26 (m, 2H), 3.58 (s, 2H), 3.62 (s, 3H), 3.68 (s, 3H),
4.56 (m, 1H), 6.81 (d, 1H), 7.26 (m, J = 6.6 Hz, 3H), 7.34 (m,
J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 330.1, (calc) 330.1.
Sodium salt: 1H NMR (600 MHz, D2O): d = 1.48–1.70 (m, 4H),
2.09 (s, 2H), 3.55 (m, 2H), 4.05 (m, 1H), 7.27 (m, 3H), 7.33 (m, 2H).
13C NMR (600 MHz, D2O): d = 22.43, 31.35, 37.07, 42.22, 44.42,
55.31, 127.18, 128.86, 129.07, 129.08, 135.09, 174.05, 179.18,
183.10.
To a solution of compound 4, 4-methyl-1,2-dicarboxylic acid (5 g,
27.7 mmol) in 12 mL of methanol, 5 mL of sulfuric acid was added at
r. t., the reaction mixture was allowed to stir in a microwave reactor
at 80 °C for 8 h. Saturated NaHCO3 solution was added to the reac-
tion mixture until slightly basic, with the product precipitating as
an oily liquid. The product was extracted with several portions of
dichloromethane (DCM), was dried over anhydrous sodium sulfate
and concentrated in vacuum. The compound was purified by column
chromatography using 0–10% ethyl acetate: hexanes to yield ester
4Me, yield: 90%. 1H NMR (600 MHz, CDCl3): d = 2.39 (s, 3H), 3.87
(s, 3H), 3.88 (s, 3H), 7.30 (d, J = 7.8 Hz, 1H), 7.45 (s, 1H), 7.65 (d,
J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+ 231.1, (calc) 231.2.
Purity: HPLC – (99.4%).
5.5.11. 2-(3-Phenylpropanamido)hexanedioic acid (D14)
Dimethylester: 1H NMR (600 MHz, CDCl3): d = 1.48–1.66 (m, 4H),
1.78–1.84 (m, 1H), 2.28 (dt, J = 2.69, 7.32 Hz, 2H), 2.49–2.59 (m,
2H), 2.96–2.99 (m, 2H), 3.67 (s, 3H), 3.73 (s, 3H), 4.58–4.61 (m,
1H), 5.96 (d, J = 7.57 Hz, 1H), 7.18–7.22 (m, 3H), 7.28 (t,
J = 7.32 Hz, 2H).
5.6.2. 4-Bromomethylphthalate dimethyl ester (5Me)
To a solution of compound 4Me (5 g, 24.8 mmol) in 25 mL
dichloromethane was added NBS (4.9 g, 27.3 mmol). The reaction
mixture was stirred in the presence of light for 3 h, concentrated
under vacuum and purified by flash column chromatography using
0–20% ethyl acetate: hexanes to obtain compound 5Me, yield: 78%.
1H NMR (600 MHz, CDCl3): d = 3.89 (s, 6H), 4.46 (s, 2H), 7.53 (d,
J = 6.6 Hz, 1H), 7.68–7.71 (m, 2H). MS (ESI) m/z: (obs) [M+Na]+ (iso-
topic) 309.3, 311.1 (calc) (isotopic) 309.0, 311.0.
Sodium salt: 1H NMR (600 MHz, D2O): d = 1.01 (m, 2H), 1.23–
1.40 (m, 2H), 1.81 (dt, J = 1.69, 5.81HZ, 2H), 2.40 (m, 2H), 2.72
(m, 2H), 3.77 (q, J = 4.17, 8.67 Hz, 1H), 7.03 (m, 3H), 7.12 (t,
J = 7.48 Hz, 2H).
13C NMR (600 MHz, D2O): d = 22.08, 31.31, 31.35, 37.06, 37.36,
54.95, 126.49, 128.40, 128.65, 140.36, 171.01, 175.08, 179.23,
182.95.
5.6.3. N-Carboxymethyl-3,4-dicarboxybenzylamine (P3)
To a solution of compound 5Me (3.1 g, 10.7 mmol) in DMF was
added NaHCO3 (4.6 g, 54 mmol) and glycine methyl ester.HCl
(6.8 g, 54 mmol). The reaction mixture was stirred at r. t. for 8 h.
DMF was air evaporated and the reaction mixture was purified
by flash column chromatography using 10–50% ethyl acetate: hex-
anes to obtain compound P3Me, yield: 64% 1H NMR (600 MHz,
Purity: HPLC – (98.5%).
5.5.12. 2-(4-Phenylbutanamido)hexanedioic acid (D16)
Dimethylester: 1H NMR (400 MHz, CDCl3): d = 1.66–1.82 (m, 2H),
1.92–2.09 (m, 4H), 2.32 (t, J = 7.46 Hz, 2H), 2.40–2.47 (m, 2H), 2.74
Please cite this article in press as: Thangavelu B., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.060

10
B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
O
OH
OH
O
NaO
N
O
4
ONa
ONa
H
O
NH2
MeOOC
O
a
O
c,d
P3
O
O
O
MeOOC
NH2
Br
O
O
O
O
NaO
N
H
ONa
ONa
c,d
b
O
O
4Me
O
NH2
5Me
MeOOC
c,d
P4
OH
O
O
NaO
c,d
NH2
MeOOC
N
H
ONa
ONa
O
OH
O
NaO
P5
N
H
ONa
ONa
O
O
P6
Scheme 3. Synthetic route to 4-aminomethylphthalate derivatives. Reagents and conditions: (a) H2SO4/MeOH, microwave, 80 °C, 8 h; (b) NBS/DCM, light, 3 h; (c) NaHCO3/
DMF, r.t., 8 h; (d) 1 N NaOH, r.t., 3 h.
CDCl3): 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, (calc) 318.3. This ester was hydrolyzed
by adding H2O (0.1 mL) to a solution of compound P3Me (0.04 g,
0.14 mmol) in THF (0.4 mL). Then 1 N NaOH (0.4 mL, 0.42 mmol)
was added to the reaction mixture and stirred at r. t. for 3 h. The
reaction mixture was concentrated and dried for several hours
under high vacuum to yield pure compound P3, yield: 100% 1H
NMR (400 MHz, D2O): d = 3.12 (s, 2H), 3.66 (s, 2H), 7.48 (d,
J = 7.8 Hz, 1H), 7.27–7.32 (m, 2H), 7.40 (s, 1H). 13C NMR
(600 MHz, D2O): d = 51.44, 51.62, 126.91, 127.54, 128.46, 136.26,
138.23, 139.58, 177.20, 177.58, 179.46.
(3.3 g, 23.7 mmol). The reaction mixture was stirred at r. t. for
8 h. DMF was air evaporated and the reaction mixture was purified
by flash column chromatography using 10–60% ethyl acetate: hex-
anes to obtain compound P5Me, yield: 58% 1H NMR (600 MHz,
CDCl3): d = 1.32 (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, (calc) 332.3. A solution of compound P5Me (0.071 g,
0.229 mmol) was base-hydrolyzed as described above to yield pure
compound P5, yield: 100% 1H NMR (600 MHz, D2O): d = 1.16 (d,
J = 6.6 Hz 3H), 3.11 (m, J = 7.2 Hz 1H), 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). 13C NMR (600 MHz, D2O): d = 18.27, 50.65,
58.27, 126.95, 127.54, 128.54, 138.22, 139.57, 177.22, 177.58,
183.34.
Purity: HPLC – (89.9%).
5.6.4. N-Carboxyethyl-3,4-dicarboxybenzylamine (P4)
Purity: HPLC – (98.2%).
To a solution of compound 5Me (0.52 g, 1.8 mmol) in DMF was
added NaHCO3 (0.8 g, 9 mmol) and b-alanine methyl ester.HCl
(1.3 g, 9 mmol). The reaction mixture was stirred at r. t. for 8 h.
DMF was air evaporated and the reaction mixture was purified
by flash column chromatography using 10–60% ethyl acetate: hex-
anes to obtain compound P4Me, yield: 66% 1H NMR (600 MHz,
CDCl3): d = 3.38 (s, 2H), 3.70 (s, 3H), 3.84 (s, 2H), 3.86 (s, 3H),
3.87 (s, 3H), 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]+ 332.4, (calc) 332.3. A
solution of compound P4Me (0.071 g, 0.229 mmol) was base-
hydrolyzed as described above to yield compound P4, yield:
100% 1H NMR (600 MHz, D2O): 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). 13C NMR (600 MHz, D2O): d = 37.03,
45.09, 51.91, 126.83, 127.51, 128.35, 136.14, 138.22, 139.79,
177.20, 177.61, 181.36.
5.6.6. N-((2-Hydroxymethyl)carboxymethyl)-3,4-
dicarboxybenzylamine (P6)
To a solution of compound 5Me (1.15 g, 4.0 mmol) in DMF was
added NaHCO3 (1.7 g, 20.1 mmol) and L-serine methyl ester.HCl
(3.1 g, 20.1 mmol). The reaction mixture was stirred at r. t. for
8 h. DMF was air evaporated and the reaction mixture was purified
by flash column chromatography using 10–60% ethyl acetate: hex-
anes to obtain compound P6Me, yield: 52% 1H NMR (600 MHz,
CDCl3): d = 2.43 (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 (s, 1H), 7.70 (d, J = 7.8 Hz, 1H). MS (ESI) m/z:
(obs) [M+Na]+ 348.4, (calc) 348.3. A solution of compound P6Me
(0.071 g, 0.229 mmol) was base-hydrolyzed as described above to
yield pure compound P6, yield: 100% 1H NMR (600 MHz, D2O):
d = 3.17 (t, J = 5.2 Hz 1H), 3.61 (d, 13.2 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). 13C NMR (600 MHz, D2O): d = 50.71, 62.96,
64.28, 126.91, 127.54, 128.54, 136.26, 138.20, 139.54, 177.21,
177.57, 179.75.
Purity: HPLC – (>99%).
5.6.5. N-((2-Methyl)carboxymethyl)-3,4-dicarboxybenzylamine (P5)
To a solution of compound 5Me (1.36 g, 4.7 mmol) in DMF was
added NaHCO3 (2.0 g, 23.7 mmol) and
L-alanine methyl ester.HCl
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B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
11
5.7. General protocol for N-derivatized dicarboxybenzylamines (P10-
P59)
5.7.5. N-Benzyl, N-carboxymethyl-3,4-dicarboxybenzylamine (P14)
Triester: 1H NMR (400 MHz, CDCl3): d = 3.30 (s, 2H), 3.68 (s, 3H),
3.79 (s, 2H), 3.87 (s, 2H), 3.89 (s, 3H), 3.91 (s, 3H), 7.23–7.34 (m,
5H), 7.59 (d, J = 8 Hz, 1H), 7.72 (m, J = 8 Hz, 2H). MS (ESI) m/z:
(obs) [M+Na]+ 408.3, (calc) 408.4.
To a solution of compound P3Me or P4Me (1 eq.) in acetonitrile
was added 1.1 eq of various alkyl halides or various benzyl halides
(Tables 3–5) along with 1.1 eq of NaHCO3. The reaction mixture
was stirred at r. t. for 3 h. CH3CN was evaporated in vacuum and
purified by flash column chromatography on silica gel using 0–
25% ethyl acetate: hexanes to obtain corresponding N-derivatized
benzylamine esters. Ester hydrolysis was carried out by adding
0.1 mL of H2O to a solution of N-derivatized benzylamine esters
(1 eq) in THF (0.4 mL) with stirring. Then, 1 N NaOH (3.3 eq) was
added to the reaction mixture and stirred at r. t. for 3 h. The reac-
tion mixture was concentrated and dried for several hours under
high vacuum to yield the corresponding sodium salts of the
N-derivatized benzylamines, compounds P10–P59. The percent
yield for the corresponding esters was typically about 80–90%
and the hydrolysis to the corresponding acids was essentially
quantitative.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.88 (s, 2H), 3.62 (s,
4H), 7.20 (m, 7H), 7.29 (m, 1H).
13C NMR (600 MHz, D2O): d = 55.76, 56.74, 56.91, 127.35,
127.47, 128.38, 128.41, 129.97, 130.10, 136.40, 137.42, 138.05,
138.15, 177.22, 177.61, 178.79.
Purity: HPLC – (>99%).
5.7.6. N-(2-Methylbenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P15)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.31 (s, 3H), 3.24 (s, 2H),
3.65 (s, 3H), 3.80 (s, 2H), 3.87 (s, 2H), 3.88–3.89 (s, 6H), 7.13 (m,
3H), 7.28 (d, J = 6.6 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H) 7.64 (s, 1H), 7.68
(d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+ 422.2, (calc) 422.4.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.13 (s, 3H), 3.04 (s,
2H), 3.73 (s, 2H), 3.82 (s, 2H), 7.17 (m, 3H), 7.31–7.33 (m, 3H),
7.41 (d, J = 7.8 Hz, 1H).
5.7.1. N-Methyl, N-carboxymethyl-3,4-dicarboxybenzylamine (P10)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.36 9 s, 3H), 3.28 (s,
2H), 3.69 (s, 3H) 3.73 (s, 2H), 3.88 (s, 6H), 7.52 (d, J = 7.8 Hz, 1H),
7.66 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+
332.4, (calc) 332.3.
13C NMR (600 MHz, D2O): d = 18.37, 53.86, 55.85, 57.38, 125.72,
127.30, 127.43, 128.44, 130.30, 130.31, 130.51, 136.38, 136.41,
138.78, 177.31, 177.62, 179.28.
Purity: HPLC – (99.0%).
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.04 (s, 3H), 2.85 (s,
2H), 3.46 (s, 2H), 7.15–7.17 (m, J = 7.2 Hz, 2H), 7.25 (d, J = 7.2 Hz,
1H).
5.7.7. N-(3-Methylbenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P16)
13C NMR (600 MHz, D2O): d = 41.85, 58.14, 61.44, 126.86,
127.17, 130.98, 132.84, 134.45, 140.35, 175.89, 177.76, 178.80.
Purity: HPLC – (92.6%).
Triester: 1H NMR (600 MHz, CDCl3): d = 2.31 (s, 3H), 3.28 (s, 2H),
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)
7.57 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 7.8 Hz, 2H). MS (ESI) m/z: (obs)
[M+Na]+ 422.2, (calc) 422.4.
5.7.2. N-Propionitrile, N-carboxymethyl-3,4-dicarboxybenzylamine
(P11)
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.28 (s, 3H), 3.01 (s,
2H), 3.70 (s, 2H), 3.74 (s, 2H), 7.12–7.16 (m, J = 6 Hz, 2H), 7.16 (s,
1H), 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).
Triester: 1H NMR (400 MHz, CDCl3): 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,
J = 7.6 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 357.4, (calc) 357.3.
Sodium salt: 1H NMR (400 MHz, D2O): d = 2.97 (s, 2H), 2.99 (s,
2H), 3.57 (s, 2H), 7.17–7.20 (m, 2H), 7.27 (d, 1H).
13C NMR (600 MHz, D2O): d = 57.17, 57.63, 127.33, 128.39,
130.16, 136.55, 137.53, 137.93, 177.30, 177.61, 178.92.
Purity: HPLC – (99.2%).
13C NMR (600 MHz, D2O): d = 20.36, 55.86, 56.65, 56.91, 127.06,
127.33, 127.99, 128.36, 128.37, 129.93, 130.70, 136.32, 137.61,
138.04, 138.39, 138.47, 177.25, 177.65, 178.97.
Purity: HPLC – (96.6%).
5.7.8. N-(4-Methylbenzyl)-N-carboxymethyl-3,4-
5.7.3. N-Acetaldehyde, N-carboxymethyl-3,4-dicarboxybenzylamine
(P12)
dicarboxybenzylamine (P17)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.31 (s, 3H), 3.27 (s, 2H),
3.66 (s, 3H), 3.73 (s, 2H), 3.87–3.89 (s, 6H), 7.11 (d, J = 7.8 Hz, 2H),
7.22 (d, J = 7.8 Hz, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.69–7.70 (m,
J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 422.2, (calc) 422.4.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.27 (s, 3H), 2.96 (s,
2H), 3.70 (s, 2H), 3.72 (s, 2H), 7.18–7.22 (m, J = 7.8 Hz, 4H), 7.28–
7.30 (m, 2H), 7.40 (d, J = 7.8 Hz, 1H).
Triester: 1H NMR (400 MHz, CDCl3): 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,
J = 7.6 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 357.4, (calc) 357.3.
Sodium salt: 1H NMR (400 MHz, D2O): d = 2.97 (s, 2H), 2.99 (s,
2H), 3.57 (s, 2H), 7.17–7.20 (m, 2H), 7.27 (d, 1H).
13C NMR (600 MHz, D2O): d = 51.44, 51.62, 126.92, 127.54,
128.48, 136.25, 138.23, 139.60, 171.03, 177.21, 177.59, 179.47.
Purity: HPLC – (96.8%).
13C NMR (600 MHz, D2O): d = 20.09, 55.73, 56.54, 127.33,
128.32, 128.93, 129.93, 130.14, 134.39, 136.30, 137.54, 138.04,
138.37, 177.23, 177.64, 178.97.
5.7.4. N-Allyl, N-carboxymethyl-3,4-dicarboxybenzylamine (P13)
Triester: 1H NMR (600 MHz, CDCl3): d = 3.24 (s, 2H), 3.31 (s, 2H),
3.66 (s, 3H) 3.82 (s, 2H), 3.88–3.89 (s, 6H), 5.13–5.21 (m, 2H), 5.78–
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, (calc) 358.3.
Purity: HPLC – (96.9%).
5.7.9. N-(2-Bromobenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P18)
Triester: 1H NMR (600 MHz, CDCl3): d = 3.31 (s, 2H), 3.67 (s, 3H),
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,
488.0 (calc) (isotopic) 486.1, 488.1.
Sodium salt: 1H NMR (600 MHz, D2O): 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), 7.15–7.17
(m, 2H), 7.25 (d, 1H).
13C NMR (600 MHz, D2O): d = 55.91, 56.28, 56.69, 119.17,
127.30, 128.47, 130.10, 134.30, 136.39, 137.93, 137.98, 177.23,
177.61, 178.84.
Sodium salt: 1H NMR (600 MHz, D2O): d = 3.07 (s, 2H), 3.89 (s,
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).
Purity: HPLC – (>99%).
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B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
13C NMR (600 MHz, D2O): d = 55.47, 56.29, 57.51, 124.74,
5.7.14. N-(4-Trifluoromethylbenzyl)-N-carboxymethyl-3,4-
127.28, 127.49, 128.49, 129.09, 130.06, 131.96, 132.80, 136.43,
137.30, 137.96, 138.63, 177.32, 177.60, 179.10.
Purity: HPLC – (99.3%).
dicarboxybenzylamine (P23)
Triester: 1H NMR (600 MHz, CDCl3): 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:
(obs) [M+Na]+ 476.1, (calc) 476.4.
5.7.10. N-(3-Bromobenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P19)
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.89 (s, 2H), 3.61 (s,
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).
Triester: 1H NMR (600 MHz, CDCl3): d = 3.26 (s, 2H), 3.65 (s, 3H),
3.73 (s, 2H), 3.83 (s, 2H), 3.86–3.88 (s, 6H), 7.14 (d, J = 7.8 Hz, 1H),
7.24 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.48 (s, 1H), 7.54 (d,
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 (calc) (isotopic) 486.1, 488.1.
Sodium salt: 1H NMR (600 MHz, D2O): d = 3.01 (s, 2H), 3.70 (s,
2H), 3.74 (s, 2H), 7.22 (t, J = 7.2 Hz, 1H), 7.27–7.31 (m, 3H), 7.41
(d, J = 7.8 Hz, 1H), 7.43–7.45 (d, J = 7.8 Hz, 1H), 7.51 (d, 1H).
13C NMR (600 MHz, D2O): d = 56.83, 121.71, 127.36, 128.32,
128.70, 129.88, 130.07, 130.25, 132.58, 136.39, 138.04, 138.25,
140.20, 177.24, 177.60, 178.86.
13C NMR (600 MHz, D2O): d = 56.10, 56.53, 56.89, 125.13,
125.16, 127.36, 128.29, 128.58, 128.79, 129.88, 130.18, 136.40,
138.06, 138.19, 142.09, 177.21, 177.60, 178.86.
Purity: HPLC – (85.3%).
5.7.15. N-(4-t-Butylbenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P28)
Triester: 1H NMR (400 MHz, CDCl3): d = 1.30 (s, 9H), 3.31 (s, 2H),
3.67 (s, 3H), 3.77 (s, 2H), 3.87 (s, 2H), 3.88–3.91 (s, 6H), 7.27 (d,
J = 8 Hz, 2H), 7.34 (d, J = 8 Hz, 2H), 7.59 (d, J = 8 Hz, 1H), 7.61 (d,
J = 7.6 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 464.3, (calc) 464.5.
Sodium salt: 1H NMR (600 MHz, D2O): d = 1.26 (s, 9H), 3.01 (s,
2H), 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).
Purity: HPLC – (98.3%).
5.7.11. N-(4-Bromobenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P20)
Triester: 1H NMR (600 MHz, CDCl3): d = 3.25 (s, 2H), 3.65 (s, 3H),
3.71 (s, 2H), 3.82 (s, 2H), 3.87–3.89 (s, 6H), 7.20 (d, J = 7.8 Hz, 2H),
7.42 (d, J = 7.8 Hz, 2H), 7.54 (d, J = 7.8 Hz, 1H), 7.66–7.69 (m,
J = 7.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ (isotopic) 486.2,
488.0 (calc) (isotopic) 486.1, 488.1.
13C NMR (600 MHz, D2O): d = 30.49, 33.84, 55.79, 56.41, 56.48,
125.33, 127.35, 128.31, 129.93, 130.02, 134.57, 136.30, 138.06,
138.34, 150.92, 177.23, 177.67, 178.97.
Purity: HPLC – (85.1%).
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.78 (s, 2H), 3.45 (s,
2H), 3.46 (s, 2H), 6.97 (d, J = 8.4 Hz, 2H), 7.05–7.07 (d, J = 7.8 Hz,
1H), 7.12 (s, 1H), 7.21–7.24 (m, J = 7.8 Hz, 3H).
5.7.16. N-(4-(2-Perfluoropropyl))-N-carboxymethyl-3,4-
dicarboxybenzylamine (P29)
13C NMR (600 MHz, D2O): d = 55.92, 56.28, 56.77, 120.60,
127.35, 128.31, 129.90, 131.23, 131.78, 136.36, 136.77, 138.05,
138.24, 177.22, 177.61, 178.87.
Triester: 1H NMR (600 MHz, CDCl3): 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,
J = 8.4 Hz, 2H), 7.56 (d, J = 7.8 Hz, 1H), 7.69 (m, 2H). MS (ESI) m/z:
(obs) [M+Na]+ 575.9, (calc) 576.4.
Purity: HPLC – (>99%).
Sodium salt: 1H NMR (600 MHz, D2O): d = 3.07 (s, 2H), 3.77 (s,
2H), 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).
13C NMR (600 MHz, D2O): d = 56.13, 56.39, 56.81, 119.36,
119.52, 121.26, 121.45, 124.91, 125.05, 125.61, 125.68, 127.37,
128.27, 129.87, 130.30, 136.39, 138.08, 138.22, 141.33, 177.22,
177.64, 178.88.
5.7.12. N-(2-Trifluoromethylbenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P21)
Triester: 1H NMR (600 MHz, CDCl3): d = 3.28 (s, 2H), 3.66 (s, 3H),
3.86 (s, 2H), 3.86–3.88 (s, 6H), 3.95 (s, 2H), 7.31 (t, J = 7.8 Hz, 1H),
7.51 (t, J = 7.8 Hz, 1H), 7.57 (t, J = 7.8 Hz, 2H) 7.68 (m, J = 7.8 Hz,
2H), 7.85 (d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+ 476.1,
(calc) 476.4.
Purity: HPLC – (95.4%).
Sodium salt: 1H NMR (600 MHz, D2O): d = 3.05 (s, 2H), 3.79 (s,
2H), 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).
13C NMR (600 MHz, D2O): d = 53.23, 56.29, 57.43, 125.69,
125.73, 127.17, 127.31, 128.12, 129.67, 131.02, 132.12, 136.33,
137.61, 137.98, 138.92, 177.31, 177.61, 179.32.
5.7.17. N-(4-Biphenyl)-N-carboxymethyl-3,4-dicarboxybenzylamine
(P30)
Triester: 1H NMR (600 MHz, CDCl3): d = 3.33 (s, 2H), 3.68 (s, 3H),
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]+
484.2, (calc) 484.5.
Purity: HPLC – (>99%).
5.7.13. N-(3-Trifluoromethylbenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P22)
Sodium salt: 1H NMR (600 MHz, D2O): d = 3.03 (s, 2H), 3.74 (d,
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).
13C NMR (600 MHz, D2O): d = 55.91, 56.58, 56.64, 126.77,
129.84, 127.36, 127.62, 128.34, 129.08, 129.93, 130.66, 136.34,
136.93, 138.07, 138.34, 139.50, 140.22, 177.23, 177.64, 178.95.
Purity: HPLC – (>99%).
Triester: 1H NMR (600 MHz, CDCl3): d = 3.29 (s, 2H), 3.66 (s, 3H),
3.83 (s, 2H), 3.85 (s, 2H), 3.86–3.89 (s, 6H), 7.40 (t, J = 7.8 Hz, 1H),
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,
(calc) 476.4.
Sodium salt: 1H NMR (600 MHz, D2O): d = 3.03 (s, 2H), 3.75 (s,
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,
1H), 7.59 (d, J = 7.8 Hz, 1H), 7.65 (s, 1H).
5.7.18. N-(Ethylmorpholino)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P31)
13C NMR (600 MHz, D2O): d = 56.09, 56.68, 56.88, 124.10,
124.13, 126.39, 126.41, 127.36, 128.33, 128.93, 129.90, 133.53,
138.05, 138.19, 138.72, 177.25, 177.59, 178.83.
Triester: 1H NMR (600 MHz, CDCl3): d = 2.38 (s, 3H), 2.44 (t,
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, (calc) 408.4.
Purity: HPLC – (98.6%).
Please cite this article in press as: Thangavelu B., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.060

B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
13
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.40 (s, 4H), 2.46 (t,
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).
13C NMR (600 MHz, D2O): d = 49.07, 25.60, 54.86, 57.41, 57.69,
65.98, 127.30, 128.29, 129.95, 136.50, 138.00, 138.21, 177.20,
177.48, 178.91.
13C NMR (600 MHz, D2O): d = 33.09, 49.73, 55.88, 57.04, 124.77,
127.29, 127.48, 128.64, 129.16, 130.12, 132.06, 132.85, 137.95,
138.15, 177.27, 177.49, 181.63.
Purity: HPLC – (95.0%).
5.7.23. N-(3-Bromobenzyl)-N-carboxyethyl-3,4-
Purity: HPLC – (90.6%).
dicarboxybenzylamine (P38)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.46 (t, 2H), 2.76 (t, 2H),
3.48 (s, 2H), 3.57 (s, 2H), 3.61 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 7.15
(m, J = 12 Hz, 2H), 7.34 (d, J = 7.8 Hz, 2H), 7.42 (s, 1H), 7.48 (d,
J = 7.8 Hz, 2H), 7.61 (s, 1H), 7.66 (d, J = 7.8 Hz, 2H) MS (ESI) m/z:
(obs) [M+Na]+ (isotopic) 500.4, 502.4 (calc) (isotopic) 500.1, 502.1.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.43 (t, 2H), 2.73 (t,
2H), 3.54 (s, 2H), 3.58 (s, 2 H), 7.23–7.288 (m, J = 7.8 Hz, 3H),
7.32 (s, J = 8.4 Hz, 1H), 7.41–7.47 (m, J = 7.2 Hz, 3 H).
13C NMR (600 MHz, D2O): d = 33.60, 49.65, 56.28, 56.63, 121.72,
127.40, 128.44, 128.75, 129.94, 130.11, 130.33, 132.63, 136.48,
137.97, 138.07, 139.86, 177.21, 177.52, 181.42.
5.7.19. N-(2-Naphthyl)-N-carboxymethyl-3,4-dicarboxybenzylamine
(P32)
Triester: 1H NMR (600 MHz, CDCl3): 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
(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, (calc) 458.5.
Sodium salt: 1H NMR (600 MHz, D2O): d = 3.06 (s, 2H), 3.78 (s,
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).
Purity: HPLC – (97.3%).
13C NMR (600 MHz, D2O): d = 56.01, 56.73, 57.04, 126.09,
126.33, 127.36, 127.54, 127.72, 127.81, 128.18, 128.38, 128.59,
129.94, 132.34, 132.87, 135.47, 136.35, 138.07, 138.35, 177.23,
177.63, 178.99.
5.7.24. N-(4-Bromobenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P39)
Triester: 1H NMR (600 MHz, CDCl3):d = 2.46 (t, 2H), 2.74 (t, 2H),
3.47 (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),
7.59 (s, 1H), 7.66 (d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+
(isotopic) 500.1, 501.9 (calc) (isotopic) 500.1, 502.1.
Sodium salt: 1H NMR (600 MHz, CDCl3):d = 2.43 (t, 2H), 2.73 (t,
2H), 3.54 (s, 2H), 3.59 (s, 2H), 7.19 (d, J = 7.8 Hz, 2H), 7.27–7.31
(m, 2H), 7.41 (d, J = 7.2 Hz, 1H), 7.48 (d, J = 7.8 Hz 2H).
13C NMR (600 MHz, D2O): d = 33.55, 49.63, 56.00, 56.60, 120.67,
127.38, 129.92, 131.26, 131.80, 136.45, 136.53, 137.96, 138.07,
177.16, 177.51, 181.45.
Purity: HPLC – (93.7%).
5.7.20. N-(1-Naphthyl)-N-carboxymethyl-3,4-dicarboxybenzylamine
(P33)
Triester: 1H NMR (600 MHz, CDCl3): 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, (calc)
458.5.
Sodium salt: 1H NMR (600 MHz, D2O): d = 3.05 (s, 2H), 3.88 (s,
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).
Purity: HPLC – (95.8%).
5.7.25. N-(2-Trifluoromethylbenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P40)
13C NMR (600 MHz, D2O): d = 53.94, 56.19, 57.73, 124.36, 125.46,
125.96, 126.30, 127.35, 128.04, 128.41, 128.54, 128.73, 130.08,
133.49, 134.02, 136.50, 138.05, 138.92, 177.28, 177.54, 179.34.
Purity: HPLC – (93.3%).
Triester: 1H NMR (600 MHz, CDCl3): d = 2.48 (t, 2H), 2.77 (t, 2H),
3.58 (s, 2H), 3.59 (s, 3H), 3.71 (s, 2H), 3.85 (s, 3H), 3.85 (s, 3H), 7.30
(d, J = 7.8 Hz, 1H), 7.50 (m, J = 7.2 Hz, 2H), 7.57 (d, J = 7.8 Hz, 1H),
7.62 (s, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 7.2 Hz, 1H). MS
(ESI) m/z: (obs) [M+Na]+ 490.2, (calc) 490.2.
5.7.21. N-(4-Carboxybenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P34)
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.44 (t, 2H), 2.77 (t,
2H), 3.63 (s, 2H), 3.78 (s, 2H), 7.34 (m, 2H), 7.39 (m, J = 8.4 Hz,
2H), 7.58 (t, J = 7.2 Hz, 1H), 7.66 (d, J = 7.8 Hz 1H), 7.73 (d,
J = 7.8 Hz 1H).
Triester: 1H NMR (600 MHz, CDCl3): d = 3.26 (s, 2H), 3.64 (s, 3H),
3.81 (s, 2H), 3.83 (s, 2H), 3.85 (s, 3H), 3.86 (s, 3H), 3.87 (s, 3H), 7.39
(d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 1H), 7.66–7.68 (m, J = 8.4 Hz,
2H), 7.94 (d, J = 8.4 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 466.2,
(calc) 466.4.
13C NMR (600 MHz, D2O): d = 33.90, 50.27, 53.10, 57.04, 125.74,
125.78, 127.17, 127.31, 128.11, 129.59, 130.94, 132.10, 136.36,
137.45, 137.98, 198.77, 177.27, 177.53, 181.64.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.83 (s, 2H), 3.54 (s,
2H), 3.58 (s, 2H), 7.11 (d, 2H), 7.17–7.19 (m, 3H), 7.25 (d, 1H),
7.61 (d, 2H).
Purity: HPLC – (98.8%).
13C NMR (600 MHz, D2O): d = 55.82, 56.64, 56.78, 127.35,
128.36, 128.81, 129.74, 129.94, 135.11, 136.37, 138.04, 138.19,
140.87, 175.54, 177.23, 177.63, 178.89.
5.7.26. N-(3-Trifluoromethylbenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P41)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.47 (t, 2H), 2.77 (t, 2H),
3.55 (s, 2H), 3.58 (s, 2H), 3.60 (s, 3H), 3.85 (s, 3H), 3.87 (s, 3H), 7.38
(t, J = 7.8 Hz, 1H), 7.45 (m, J = 7.2 Hz, 3H), 7.54 (s, 1H), 7.61 (s, 1H),
7.66 (d, J = 8.4 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+ 490.2, (calc)
490.2.
Purity: HPLC – (96.5%).
5.7.22. N-(2-Bromobenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P37)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.49 (t, 2H), 2.80 (t, 2H),
3.58 (s, 3H), 3.63 (s, 2H), 3.65 (s, 2 H), 3.85 (s, 3H), 3.87 (s, 3H), 7.05
(t, J = 12 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 7.44 (m, J = 7.8 Hz, 3H),
7.61 (s, 1H), 7.65 (d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+
(isotopic) 500.4, 502.4 (calc) (isotopic) 500.1, 502.1.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.29 (t, 2H), 2.60 (t,
2H), 3.46 (s, 2H), 3.49 (s, 2H), 7.14 (d, J = 7.8 Hz, 2H), 7.17 (s, 1H),
7.27 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 7.8 Hz 3H), 7.46 (d, J = 7.8 Hz
1H).
13C NMR (600 MHz, D2O): d = 33.55, 49.72, 56.37, 56.72, 124.18,
124.20, 126.44, 126.46, 127.40, 128.46, 128.97, 129.93, 133.59,
136.51, 137.88, 138.08, 138.42, 177.19, 177.48, 181.40.
Purity: HPLC – (>99%).
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.50 (t, 2H), 2.81 (t,
2H), 3.69 (s, 2H), 3.71 (s, 2 H), 7.16 (t, J = 7.8 Hz, 1H), 7.33 (m,
J = 8.4 Hz, 2H), 7.40 (m, J = 7.8 Hz, 3H), 7.58 (d, J = 7.8 Hz, 1H).
Please cite this article in press as: Thangavelu B., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.060

14
B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
5.7.27. N-(4-Trifluoromethylbenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P42)
(s, 3H), 7.20 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 7.50 (d,
J = 7.8 Hz, 1H), 7.62 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H). MS (ESI) m/z:
(obs) [M+Na]+ 478.3, (calc) 478.2.
Triester: 1H NMR (600 MHz, CDCl3):d = 2.48 (t, 2H), 2.77 (t, 2H),
3.58 (s, 2H), 3.59 (s, 2H), 3.60 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 7.40
(d, J = 7.8 Hz, 2H), 7.49 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 7.8 Hz, 1H),
7.61 (s, 1H), 7.66 (d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+
490.2, (calc) 490.4.
Sodium salt: 1H NMR (600 MHz, D2O): d = 1.26 (s, 9H), 2.45 (t,
2H), 2.73 (t, 2H), 3.57 (s, 2H), 3.59 (s, 2H), 7.27–7.301 (m,
J = 7.8 Hz, 3H), 7.34 (d, J = 7.8 Hz, 1H), 7.41–7.42 (m, J = 7.8 Hz,
1H), 7.44–7.46 (m, J = 7.8 Hz, 2H).
Sodium salt: 1H NMR (600 MHz, CDCl3):d = 2.44 (t, 2H), 2.75 (t,
2H), 3.61 (s, 2H), 3.64 (s, 2H), 7.33 (d, J = 7.8 Hz, 2H), 7.41–7.45
(m, 3H), 7.64 (d, J = 7.8 Hz 2H).
13C NMR (600 MHz, D2O): d = 30.47, 33.59, 33.83, 49.40, 56.18,
56.39, 125.36, 127.37, 128.40, 129.96, 130.02, 134.28, 136.37,
138.04, 138.08, 150.99, 177.18, 177.57, 181.52.
13C NMR (600 MHz, D2O): d = 36.63, 49.78, 56.27, 56.71, 123.36,
125.19, 127.39, 128.39, 128.63, 128.84, 129.92, 130.23, 136.48,
137.94, 138.08, 177.16, 177.52, 181.42.
Purity: HPLC – (84.0%).
5.7.32. N-(4-(2-Perfluoropropyl))-N-carboxyethyl-3,4-
dicarboxybenzylamine (P47)
Purity: HPLC – (99.5%).
Triester: 1H NMR (600 MHz, CDCl3): d = 2.48 (t, 2H), 2.78 (t, 3H),
3.57 (s, 2H), 3.58 (s, 3H), 3.60 (s, 2H), 3.85 (s, 3H), 3.87 (s, 3H), 7.40
(d, J = 7.8 Hz, 2H), 7.49 (m, J = 8.4 Hz, 3H), 7.61 (s, 1H), 7.67 (m,
J = 8.4 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+ 590.2, (calc) 590.1.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.46 (t, 2H), 2.76 (t,
2H), 3.63 (s, 2H), 3.65 (s, 2H), 7.29 (d, J = 7.8 Hz, 1H), 7.36 (s, 1H),
7.41 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.4 Hz,
2H).
5.7.28. N-(2-Methylbenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P43)
Triester:1H NMR (600 MHz, CDCl3): d = 2.26 (s, 3H), 2.46 (s, 2H),
2.76 (s, 3H), 3.53 (s, 2H), 3.56 (s, 2H), 3.58 (s, 3H), 3.86 (s, 3H), 3.88
(s, 3H), 7.10 (m, J = 7.2 Hz, 3H), 7.23 (m, J = 12 Hz, 1H), 7.45 (d,
J = 7.8 Hz, 1H) 7.56 (s, 1H), 7.64 (m, J = 7.8 Hz, 1H) MS (ESI) m/z:
(obs) [M+Na]+ 436.2, (calc) 436.2.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.09 (s, 3H), 2.47 (s,
2H), 2.78 (s, 2H), 3.56 (s, 2H), 3.63 (s, 2H), 7.16–7.19 (m, 3H),
7.16 (s, 1H), 7.30–7.33 (t, J = 7.8 Hz, 2H), 7.34 (s, 1H), 7.41 (d,
J = 7.8 Hz, 1H).
13C NMR (600 MHz, D2O): d = 33.64, 49.79, 55.98, 56.71, 124.99,
125.12, 125.64, 125.71, 127.41, 128.39, 129.89, 136.51, 137.86,
138.12, 141.01, 177.13, 177.50, 181.41.
Purity: HPLC – (95.7%).
13C NMR (600 MHz, D2O): d = 18.44, 33.19, 49.81, 53.55, 56.97,
125.71, 127.32, 127.47, 128.59, 130.08, 130.36, 130.56, 136.13,
136.49, 137.95, 137.97, 138.40, 177.26, 177.51, 181.75.
Purity: HPLC – (96.0%).
5.7.33. N-(4-Biphenyl)-N-carboxyethyl-3,4-dicarboxybenzylamine
(P48)
Triester: 1H NMR (400 MHz, CDCl3): d = 2.53 (s, 2H), 2.83 (s, 2H),
3.60 (s, 2H), 3.61 (s, 2H), 3.64 (s, 3H), 3.89 (s, 3H), 3.91 (s, 3H), 7.36
(m, J = 7.2 Hz, 3H), 7.43 (t, J = 7.2 Hz, 2H), 7.57, (m, J = 7.6 Hz 5H),
7.66 (s, 1H), 7.72 (d, J = 8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+
498.2, (calc) 498.2.
5.7.29. N-(3-Methylbenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P44)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.31 (s, 3H), 2.47 (s, 2H),
2.76 (s, 3H), 3.50 (s, 2H), 3.57 (s, 2H), 3.60 (s, 3H), 3.86 (s, 3H), 3.88
(s, 3H), 7.03 (d, J = 12 Hz, 1H), 7.07 (d, J = 7.8 Hz, 2H), 7.16 (m,
J = 7.8 Hz, 1H) 7.49 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.62
(d, J = 8.4 Hz, 1H) MS (ESI) m/z: (obs) [M+Na]+ 436.2, (calc) 436.2.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.28 (s, 3H), 2.44 (s, 2H),
2.74 (s, 2H), 3.55 (s, 2H), 3.59 (s, 2H), 7.11–7.15 (m, J = 7.2 Hz, 3H),
7.24–7.318 (m, J = 7.8 Hz, 3H), 7.42 (d, J = 7.8 Hz, 1H).
13C NMR (600 MHz, D2O): d = 20.31, 33.49, 49.59, 56.58, 56.62,
127.08, 127.35, 128.03, 128.38, 129.95, 130.73, 136.42, 137.36,
138.02, 138.06, 138.50, 177.19, 177.53, 181.53.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.46 (t, 2H), 2.76 (t,
2H), 3.61 (s, 1H), 3.62 (s, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.34–7.39
(m, J = 7.8 Hz, 4H), 7.43, (d, J = 7.8 Hz, 1H), 7.47 (t, J = 7.8 Hz, 2H),
7.62 (d, J = 7.8 Hz, 2H), 7.66 (d, J = 7.2 Hz, 2H).
13C NMR (600 MHz, D2O): d = 33.59, 49.62, 56.30, 56.56, 126.80,
126.86, 127.39, 127.63, 128.45, 129.08, 129.96, 130.69, 136.44,
136.67, 138.01, 138.11, 136.58, 140.20, 177.18, 177.54, 181.50.
Purity: HPLC – (95.2%).
5.7.34. N-(1-Naphthyl)-N-carboxyethyl-3,4-dicarboxybenzylamine
(P49)
Purity: HPLC – (97.3%).
Triester: 1H NMR (400 MHz, CDCl3): d = 2.52 (s, 2H), 2.86 (s, 2H),
3.49 (s, 3H), 3.63 (s, 2H), 3.87 (s, 3H), 3.89 (s, 3H), 4.10 (s, 2H), 7.46
(m, J = 7.8 Hz, 5H), 7.59 (m, J = 7.8 Hz, 2H), 7.74 (m, J = 7.6 Hz, 4H),
8.14 (d, J = 8.8 Hz, 2H). MS (ESI) m/z: (obs) [M+Na]+ 472.3, (calc)
472.2.
5.7.30. N-(4-Methylbenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P45)
Triester:1H NMR (600 MHz, CDCl3): d = 2.29 (s, 3H), 2.46 (s, 2H),
2.75 (s, 3H), 3.50 (s, 2H), 3.56 (s, 2H), 3.59 (s, 3H), 3.86 (s, 3H), 3.88
(s, 3H), 7.09 (t, J = 7.2 Hz, 2H), 7.15 (t, J = 7.8 Hz, 2H), 7.49 (d,
J = 7.8 Hz, 1H) 7.60 (s, J = 7.8 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), MS
(ESI) m/z: (obs) [M+Na]+ 436.2, (calc) 436.2.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.51 (t, 2H), 2.82 (t,
2H), 3.70 (s, 2H), 3.99 (s, 2H), 7.34 (d, J = 6.6 Hz, 1H), 7.41–7.53
(m, 6H), 7.75 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.90 (d,
J = 7.2 Hz, 1H).
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.12 (s, 3H), 2.28 (s,
2H), 2.57 (s, 2H), 3.39 (s, 2H), 3.43 (s, 2H), 7.03–7.06 (m,
J = 8.4 Hz, 4H), 7.12–7.13 (m, J = 8.4 Hz, 1H), 7.15 (s, 1H), 7.25 (d,
J = 7.8 Hz, 1H).
13C NMR (600 MHz, D2O): d = 32.87, 50.03, 53.48, 57.33, 124.15,
125.42, 125.93, 126.27, 127.40, 128.09, 128.43, 128.70, 128.78,
130.17, 131.87, 133.48, 133.66, 136.61, 138.07, 138.50, 177.24,
177.47, 181.80.
13C NMR (600 MHz, D2O): d = 20.08, 33.51, 49.47, 56.30, 56.45,
127.36, 128.43, 128.96, 129.95, 130.15, 134.14, 136.39, 137.61,
138.04, 138.07, 177.18, 177.54, 181.54.
Purity: HPLC – (97.3%).
Purity: HPLC – (96.8%).
5.7.35. N-(4-Carboxybenzyl)-N-carboxymethyl-3,4-
dicarboxybenzylamine (P50)
5.7.31. N-(4-t-Butylbenzyl)-N-carboxyethyl-3,4-
Triester: 1H NMR (600 MHz, CDCl3): d = 2.46 (s, 2H), 2.75 (s, 2H),
3.56–3.58 (m, 7H), 3.85–3.87 (m, 9H), 7.34 (d, J = 8.4 Hz, 2H), 7.48
(d, J = 7.8 Hz, 1H), 7.59 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.95 (d,
J = 8.4 Hz, 2H) MS (ESI) m/z: (obs) [M+Na]+ 480.2, (calc) 480.2.
dicarboxybenzylamine (P46)
Triester: 1H NMR (400 MHz, CDCl3): d = 1.27 (s, 9H), 2.48 (t, 2H),
2.77 (t, 2H), 3.52 (s, 2H), 3.58 (s, 2H), 3.59 (s, 3H), 3.86 (s, 3H), 3.86
Please cite this article in press as: Thangavelu B., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.060

B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
15
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.46 (t, 2H), 2.74 (t,
2H), 3.62 (s, 2H), 3.58 (s, 2H), 7.31 (t, J = 7.2 Hz, 1H), 7.35–7.38
(m, J = 7.8 Hz, 3H), 7.43 (m, J = 7.8 Hz, 1H), 7.80 (m, J = 7.8 Hz, 2H).
13C NMR (600 MHz, D2O): d = 33.54, 49.56, 56.43, 56.61, 127.39,
128.46, 128.84, 129.78, 129.99, 135.17, 136.43, 137.96, 138.07,
140.63, 175.49, 177.20, 177.56, 181.49.
13C NMR (600 MHz, D2O): d = 32.83, 49.43, 52.71, 56.88, 127.35,
127.40, 127.75, 128.19, 128.40, 129.54, 129.59, 130.35, 130.37,
134.85, 136.32, 138.01, 138.32, 138.63, 140.58, 142.46, 175.22,
177.20, 177.47, 181.34.
Purity: HPLC – (98.9%).
Purity: HPLC – (98.9%).
5.7.40. N-(2-Bromo-4-carboxybenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P55)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.50 (t, 2H), 2.82 (t, 2H),
3.61 (s, 3H), 3.65 (s, 2H), 3.69 (s, 2H), 3.86–3.88 (m, 10H), 7.50 (d,
J = 7.2 Hz, 2H), 7.56 (m, J = 7.2 Hz, 4H), 7.62 (s, 1H), 7.67 (d,
J = 7.8 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 8.16 (s, 1H). MS (ESI) m/z:
(obs) [M+Na]+ (isotopic) 558.1, 559.4 (calc) (isotopic) 558.6, 559.7.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.48 (t, 2H), 2.80 (t,
2H), 3.68 (s, 2H), 3.73 (s, 2H), 7.32 (d, J = 8 Hz, 1H), 7.37–7.45 (m,
3H), 7.72 (d, J = 8 Hz, 1H), 7.98 (d, 1H).
5.7.36. N-(1-Naphthyl-4-carboxybenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P51)
Triester: 1H NMR (400 MHz, CDCl3): d = 2.49 (s, 2H), 2.83 (s, 2H),
3.45 (s, 3H), 3.62 (s, 2H), 3.84 (s, 3H), 3.86 (s, 3H), 3.93 (s, 3H), 3.99
(s, 2H), 7.41 (d, J = 7.8 Hz, 2H), 7.49 (m, J = 7.2 Hz, 2H), 7.55 (m,
J = 7.2 Hz, 2H), 7.61 (d, J = 7.8 Hz, 2H), 8.04 (d, J = 7.2 Hz, 1H),
8.12 (d, J = 8.4 Hz, 1H), 8.86 (d, J = 8.8 Hz, 1H) MS (ESI) m/z: (obs)
[M+Na]+ 530.3, (calc) 530.2.
13C NMR (600 MHz, D2O): d = 33.23, 49.68, 55.77, 57.01, 124.45,
127.33, 127.64, 128.61, 130.09, 131.56, 133.15, 137.97, 138.13,
140.00, 173.87, 177.26, 177.52, 181.61.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.51 (t, 2H), 2.82 (t,
2H), 3.71 (s, 2H), 4.01 (s, 2H), 7.34–7.36 (s, J = 6.6 Hz, 1H), 7.44–
7.47 (m, J = 7.2 Hz, 3H), 7.49–7.53 (m, J = 7.2 Hz, 3H), 7.77 (d,
J = 8.4 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H).
Purity: HPLC – (99.5%).
13C NMR (600 MHz, D2O): d = 32.84, 49.93, 53.59, 57.31, 123.17,
124.42, 126.05, 126.21, 126.36, 127.44, 127.98, 128.74, 129.43,
130.24, 131.96, 134.47, 136.64, 137.81, 138.08, 138.45, 177.25,
177.50, 178.25, 181.80. Purity: HPLC – (94.5%).
5.7.41. N-(2-Chloro-4-carboxybenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P56)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.52 (t, 2H), 2.83 (t, 2H),
3.63 (s, 3H), 3.67 (s, 2H), 3.72 (s, 2H), 3.89 (s, 3H), 3.90–3.91 (s, 6H),
7.50 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.64 (s, J = 8.4 Hz,
1H), 7.68 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.99 (d,
J = 1.2 Hz 1H). MS (ESI) m/z: (obs) [M+Na]+ 514.3 (calc) 514.1.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.48 (t, 2H), 2.79 (t,
2H), 3.68 (s, 2H), 3.72 (s, 2H), 7.32 (d, J = 7.8 Hz, 1H), 7.37 (s, 1H),
7.39 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 7.8 Hz,
1H), 7.79 (s, 1H).
5.7.37. N-(2-Trifluromethyl-4-carboxybenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P52)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.47 (s, 2H), 2.77 (s, 2H),
3.59 (s, 3H), 3.60 (s, 2H), 3.73 (s, 3H), 3.83 (s, 3H), 3.86–3.88 (m,
5H), 7.48 (d, J = 7.8 Hz, 1H), 7.60 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H),
7.88 (d, J = 7.8 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.2 (s, 1H). MS
(ESI) m/z: (obs) [M+Na+ 548.2, (calc) 548.2.
13C NMR (600 MHz, D2O): d = 33.22, 49.73, 53.28, 57.06, 127.02,
127.33, 128.59, 129.78, 130.06, 131.72, 134.25, 136.53, 136.97,
137.97, 138.05, 138.19, 174.03, 177.24, 177.52, 181.58.
Purity: HPLC – (>99%).
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.43 (t, 2H), 2.77 (t,
2H), 3.64 (s, 2H), 3.81 (s, 2H), 7.35–7.41 (d, J = 7.8 Hz, 3H), 7.83
(d, J = 7.8 Hz, 1H), 8.01 (d, J = 7.2 Hz, 1H), 8.11 (d, 2H).
13C NMR (600 MHz, D2O): d = 33.55, 49.51, 56.11, 56.61, 126.78,
127.20, 127.23, 127.42, 128.49, 129.97, 133.57, 136.52, 137.67,
137.79, 138.08, 176.72, 177.19, 177.51, 181.40.
5.7.42. N-(2-Fluoro-4-carboxybenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P57)
Purity: HPLC – (98.8%).
Triester: 1H NMR (600 MHz, CDCl3): d = 2.52 (t, 2H), 2.81 (t, 2H),
3.63 (s, 3H), 3.65 (s, 2H), 3.67 (s, 2H), 3.89 (s, 3H), 3.91 (s, 6H), 7.45
(t, J = 7.8 Hz, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.63–7.703 (m, 3H), 7.78
(d, J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+ 498.1 (calc) (iso-
topic) 498.1.
5.7.38. N-(3-Trifluromethyl-4-carboxybenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P53)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.48 (s, 2H), 2.78 (s, 2H),
3.59–3.61 (m, 7H), 3.86–3.88 (m, 9H), 7.45 (d, J = 7.8 Hz, 1H), 7.50
(d, J = 7.8 Hz, 1H), 7.61 (s, 1H), 7.67 (m, J = 7.8 Hz, 2H), 7.7 (m,
J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+ 548.2, (calc) 548.2.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.43 (t, 2H), 2.77 (t,
2H), 3.64 (s, 2H), 3.81 (s, 2H), 7.35–7.41 (d, J = 7.8 Hz, 3H), 7.83
(d, J = 7.8 Hz, 1H), 8.01 (d, J = 7.2 Hz, 1H), 8.11 (d, 2H).
13C NMR (600 MHz, D2O): d = 34.05, 50.21, 53.09, 57.02, 126.29,
126.33, 127.36, 128.07, 129.55, 130.86, 132.16, 135.08, 136.35,
137.99, 138.78, 140.73, 174.03, 177.28, 177.59, 181.63.
Purity: HPLC – (98.7%).
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.45 (t, 2H), 2.74 (t,
2H), 3.64 (s, 2H), 3.65 (s, 2H), 7.29 (d, J = 7.8 Hz, 1H), 7.34–7.37
(m, J = 7.2 Hz, 2H), 7.40 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 10.8 Hz,
1H), 7.57 (d, J = 7.8 Hz, 1H).
13C NMR (600 MHz, D2O): d = 33.43, 49.54, 49.56, 56.93, 115.49,
115.64, 124.31, 127.05, 127.16, 128.47, 129.94, 132.19, 132.22,
136.49, 137.90, 137.96, 138.05, 160.23, 161.85, 174.13, 177.20,
177.53, 181.45.
Purity: HPLC – (97.5%).
5.7.43. N-(2,6-Dibromo-4-carboxybenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P58)
5.7.39. N-(2-Phenyl-4-carboxybenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P54)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.54 (t, 2H), 2.89 (t, 2H),
3.61 (s, 3H), 3.69 (s, 2H), 3.87 (s, 2H), 3.87 (s, 3H), 3.89 (s, 3H), 3.91
(s, 3H), 3.99 (s, 2H), 7.50 (d, J = 7.8 Hz, 1H), 7.57 (m, 2H), 8.13 (s,
2H). MS (ESI) m/z: (obs) [M+Na]+ (isotopic) 636.4, 638.0 (calc) (iso-
topic) 635.9, 637.9.
Triester: 1H NMR (600 MHz, CDCl3): d = 2.29 (t, 2H), 2.61 (t, 2H),
3.45 (s, 2H), 3.54 (s, 2H), 3.58 (s, 3H), 3.86 (s, 3H), 3.87 (s, 3H), 3.88
(s, 3H), 7.22 (d, J = 7.8 Hz, 2H), 7.39 (m, J = 6.6 Hz, 4H), 7.51 (s, 1H),
7.63 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.86 (s, 1H), 7.98 (d,
J = 7.8 Hz, 1H). MS (ESI) m/z: (obs) [M+Na]+ 556.2, (calc) 556.3.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.08 (t, 2H), 2.52 (t,
2H), 3.42 (s, 2H), 3.65 (s, 2H), 7.16 (d, J = 8 Hz, 1H), 7.21 (s, 1H),
7.34 (m, J = 7.2 Hz, 3H), 7.43 (m, J = 7.2 Hz, 3H), 7.54 (s, J = 8 Hz,
1H), 7.70 (s, 1H) 7.81 (d, J = 8 Hz, 1H).
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.55 (t, 2H), 2.77 (t, 2H),
3.72 (s, 2H), 3.96 (s, 2H), 7.32–7.39 (m, J = 7.2 Hz, 3H), 7.94 (s, 2H).
13C NMR (600 MHz, D2O): d = 32.57, 49.10, 56.32, 57.13, 126.31,
127.15, 128.79, 130.33, 132.75, 136.54, 137.79, 138.11, 138.41,
139.15, 172.17, 177.34, 177.52, 181.80.
Please cite this article in press as: Thangavelu B., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.060

16
B. Thangavelu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
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Purity: HPLC – (>99%).
the cause of Canavan disease:
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system. Molec. Brain Res. 1995;31:151–157.
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catabolism reduces brain acetate levels and myelin lipid synthesis in
Canavan’s disease. Proc Natl Acad Sci USA. 2005;102:5221–5226.
7. Le Coq J, Pavlovsky A, Malik R, Sanishvili R, Xu C, Viola RE. Examination of the
mechanism of human brain aspartoacylase through the binding of an
intermediate analogue. Biochemistry. 2008;47:3484–3492.
8. Zano S, Malik R, Szucs S, Matalon R, Viola RE. Modification of aspartoacylase for
potential use in enzyme replacement therapy for the treatment of Canavan
disease. Molec Genet Metab. 2011;102:176–180.
9. Guo F, Bannerman P, Ko EM, et al. Ablating N-acetylaspartate prevents
leukodystrophy in a Canavan disease model. Ann Neurol. 2015;77:884–888.
10. Lou T-F, Sethuraman D, Dospoy P, et al. Cancer-specific production of N-
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5.7.44. N-(2,6-Dichloro-4-carboxybenzyl)-N-carboxyethyl-3,4-
dicarboxybenzylamine (P59)
Triester: 1H NMR (600 MHz, CDCl3): d = 2.52 (t, 2H), 2.85 (t, 2H),
3.60 (s, 3H), 3.67 (s, 2H), 3.87 (s, 3H), 3.89–3.91 (m, 8H), 7.44 (d,
J = 7.8 Hz, 1H), 7.58 (s, 2H), 7.61 (d, J = 7.8 Hz, 1H), 7.90 (s, 2H).
MS (ESI) m/z: (obs) [M+Na]+ (isotopic) 548.2, 549.9 (calc) (isotopic)
548.1, 550.0.
Sodium salt: 1H NMR (600 MHz, D2O): d = 2.55 (t, 2H), 2.78 (t,
2H), 3.70 (s, 2H), 3.88 (s, 2H), 7.31 (d, J = 7.8 Hz, 1H), 7.37 (d,
J = 8.4 Hz, 2H), 7.71 (s, 2H).
13C NMR (600 MHz, D2O): d = 32.43, 49.52, 50.89, 57.39, 127.15,
128.59, 128.92, 130.46, 136.16, 136.44, 136.62, 137.75, 137.77,
138.09, 172.50, 177.32, 177.48, 181.73.
Purity: HPLC – (>99%).
Acknowledgements
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This work was partially supported by a grant from Turing
Pharmaceuticals LLC. The authors thank Sandeep Thanna
(University of Toledo) for assistance with the HPLC characteriza-
tion experiments.
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A. Supplementary data
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Please cite this article in press as: Thangavelu B., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.060