Exploration of inhibitors for diaminopimelate aminotransferase

Article abstract of DOI:10.1016/j.bmc.2010.02.001

Bacteria and higher plants make l-lysine from diaminopimelic acid (DAP). In mammals l-lysine is an essential amino acid that must be acquired from the diet as the biosynthetic pathway is absent for this key constituent of proteins. Recently, ll-diaminopim

Full text of DOI:10.1016/j.bmc.2010.02.001

Bioorganic & Medicinal Chemistry 18 (2010) 2141–2151
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploration of inhibitors for diaminopimelate aminotransferase
Chenguang Fan a, Matthew D. Clay b, Michael K. Deyholos c, John C. Vederas a, ,
*
a Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
b St. Mary’s University College, 14500 Bannister Road SE, Calgary, Alberta, Canada T2X 1Z4
c Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9
a r t i c l e i n f o
a b s t r a c t
Article history:
Bacteria and higher plants make L-lysine from diaminopimelic acid (DAP). In mammals L-lysine is an
Received 30 November 2009
Revised 31 January 2010
Accepted 2 February 2010
Available online 6 February 2010
essential amino acid that must be acquired from the diet as the biosynthetic pathway is absent for this
key constituent of proteins. Recently, LL-diaminopimelate aminotransferase (LL-DAP-AT), a pyridoxal-50-
phosphate (PLP)-dependent enzyme, was reported to catalyze a key step in the route to L-lysine in plants
and Chlamydia. Specific inhibitors of this enzyme could thus potentially serve as herbicides or antibiotics
that are non-toxic to mammals. In this work, 29,201 inhibitors were screened against LL-DAP-AT and the
IC50 values were determined for the top 46 compounds. An aryl hydrazide and rhodanine derivatives
were further modified to generate 20 analogues that were also tested against LL-DAP-AT. These analogues
provide additional structure–activity relationships (SAR) that are useful in guiding further design of
inhibitors.
Keywords:
Lysine biosynthesis
Aminotransferase
Diaminopimelate (DAP)
Arabidopsis thaliana
Pyridoxal-50-phosphate (PLP)
Chlamydia
Ó 2010 Elsevier Ltd. All rights reserved.
Enzyme inhibition
1. Introduction
transferase, and N-acyl-DAP deacylase, respectively.4 LL-DAP is
epimerized to meso-DAP by DAP epimerase18,19 and finally trans-
Lysine biosynthesis occurs via the diaminopimelate (DAP) path-
way in both plants and bacteria, but is absent in mammals, which
acquire this essential amino acid in their diet. It is believed that
effective inhibitors of enzymes in the DAP pathway would be
non-toxic to humans but could act as potent herbicides or antibiot-
ics. This has prompted numerous reviews1–4 and continuing stud-
ies on both the structures of the protein catalysts5–11 as well as
design of inhibitors12–15 since the mid 1980s.16 Lysine is often
the limiting amino acid in the human diet, and thus a complete
understanding of its biosynthetic route would also assist engineer-
ing of plants with increased lysine content for better nutrition.17
In most bacteria, the DAP pathway (Fig. 1a) begins with the con-
formed into L-lysine by DAP decarboxylase. This pathway was also
thought to be present in higher plants until 2006 when Gilvarg,
Leustek and co-workers found that an alternative shorter route is
used in higher plants and cyanobacteria for
The new route is similar to the bacterial pathway; however,
L
-lysine biosynthesis.20
L-
THDP is converted to LL-DAP by a single enzyme, LL-DAP amino-
transferase (LL-DAP-AT) (Fig. 1b), bypassing three enzymatic steps
present in bacteria. As in the bacterial path, LL-DAP is then con-
verted to meso-DAP by DAP epimerase6 and decarboxylated21 to
yield L-lysine. Recently, the crystal structure of LL-DAP-AT from
Arabidopsis thaliana was obtained by our group, and the mecha-
nism of substrate recognition and pyridoxal-50-phosphate (PLP)-in-
duced conformational changes in LL-DAP-AT were examined.7,22 In
densation of
ence of DHDP synthase to form
which is then reduced to -tetrahydrodipicolinate (THDP) by DHDP
reductase. THDP is then converted to LL-diaminopimelic acid
LL-DAP) through N-succinylation, transamination, and desucciny-
L-aspartate semialdehyde with pyruvate in the pres-
L-dihydrodipicolinate (DHDP),
this transamination, L-glutamate is used as the amino donor and
L
the reaction occurs via the typical PLP mechanism. The LL-DAP-AT
pathway is also shared by Chlamydia trachomatis and Protochlamy-
dia amoebophila.23 Chlamydiae are known to cause many diseases
in both humans and animals, including sexually transmitted infec-
tions, infectious blindness (C. trachomatis), coronary heart disease,
and atherosclerosis (C. pneumoniae), along with a variety of dis-
eases in poultry and livestock (Chlamydophila).23 Recent genetic
analysis has shown that LL-DAP-AT occurs in Cyanobacteria, Desulf-
uromonadales, Firmicutes, Bacteroidetes, Chlamydiae, Spirochaeta,
and Chloroflexi and two archaeal groups, Methanobacteriaceae and
Archaeoglobaceae.24,25
(
lation by the actions of THDP acyltransferase, N-acyl-DAP amino-
Abbreviations: DAP, 2,6-diaminopimelic acid; DAP-AT, diaminopimelate amino-
transferase; DHDP, dihydrodipicolinic acid; 2-OG, 2-oxoglutarate; OAB, 2-amino-
benzaldehyde; PLP, pyridoxal phosphate; SAR, structure–activity relationship;
THDP, tetrahydrodipicolinic acid.
* Corresponding author. Tel.: +1 780 492 5475; fax: +1 780 492 2134.
E-mail address: john.vederas@ualberta.ca (J.C. Vederas).
Canada Research Chair in Bioorganic and Medicinal Chemistry.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.001

2142
C. Fan et al. / Bioorg. Med. Chem. 18 (2010) 2141–2151
a
HO2C
O
Pyruvate
HO2C
CO2H
O
ii)
i)
iii)
+
O
HN
OHC
H2N
HO2C
N
CO2H
HO2C
N
CO2H
R
L-Dihydrodipicolinate
(L-DHDP)
Tetrahydrodipicolinate
(L-THDP)
CO2H
L-α-keto-ε-(N-succinylamino)pimelate R=CH2CH2CO2H
L-α-keto-ε-(N-acetylamino)pimelate
L-Aspartate
R=CH3
Semialdehyde
iv)
vii)
vi)
v)
HO2C
CO2H
HO2C
CO2H
NH2
HO2C
CO2H
O
CO2H
NH2
L-Lysine
NH2
NH2
NH2
LL-DAP
NH2
HN
NH2
meso-DAP
R
b
HO2C
CO2H
HO2C
CO2H
NH2
α-keto acid of THDPA
LL-DAP-AT
H2O
HO2C
N
CO2H
NH2
LL-DAP
NH2
O
Tetrahydrodipicolinate
(THDP)
HO2C
CO2H
NH2
L-Glutamate
HO2C
CO2H
O
α-ketoglutarate
Figure 1. (a) L-Lysine biosynthesis via DAP pathway in bacteria. Reagents: (i) DHDP synthase; (ii) DHDP reductase; (iii) THDP acyltransferase; (iv) N-acyl-DAP
aminotransferase; (v) N-acyl-DAP deacylase; (vi) DAP epimerase; (vii) DAP decarboxylase. (b) The shortcut of DAP pathway in plants and Chlamydiae.
In certain cases, compounds that inhibit DAP enzymes have al-
ready been shown to have antimicrobial activity.3,12,26 It thus
seemed that inhibitors that directly target LL-DAP-AT could be ap-
plied as herbicides or new antibiotics specific for Chlamydiae. Ra-
tional design of inhibitors of enzymes in the DAP pathway that
are based on corresponding substrates can be very effective, but
their antibiotic activity is often compromised by poor uptake by
bacterial cells.3 Hence, in this study a library of 29,201 accessible
drug-like compounds were robotically screened for inhibition of
LL-DAP-AT. The top 46 candidates were subjected to IC50 determi-
nation. Finally, preliminary structure–activity relationship (SAR)
studies were done based on modification of two lead structures,
a hydrazide and a rhodanine.
sured using an automated spectrophotometer, and the difference
was used as an indicator of enzyme activity. Activity was deter-
mined relative to the average of column 1 (positive control). The
assay procedure consisted of four steps and was done using an
automated workstation as described in the experimental section.
The percentage inhibition was determined at a concentration of
10 lM for all of the inhibitors (an estimated molecular weight of
500 g/mol was assumed for each inhibitor candidate when calcu-
lating the concentration in this robotic screening).
After robotic screening of the 29,201 compounds, the top 46
compounds were selected for further manual testing and analysis.
These substances displayed inhibition greater than 13% at 10 lM
concentration in both robotic screening and manual testing, and
thus were considered to be potentially good inhibitors. The IC50 va-
lue was measured manually using a UV–vis spectrophotometer.
Both robotic and manual testing results are listed in Table 1. An
interesting feature is that a high percentage of the compounds
with good inhibitory activity contain a ‘barbiturate’ or ‘thiobarbitu-
rate’ ring (compounds B-1 to B-16). Within the 29,201 compounds,
approximately 1000 compounds (ꢀ3%) possessed a moiety of this
type, whereas 16 of the top 46 compounds (ꢀ35%) contained one
of these groups as a potential pharmacophore.
2. Results and discussion
The initial 29,201 compounds were obtained from ChemBridge
Corporation. LL-DAP-AT from A. thaliana was cloned and overex-
pressed in Escherichia coli with a C-terminal hexahistidine tag to
aid in protein purification, as previously reported.7,22 A known 2-
aminobenzaldehyde (OAB)-based assay20 (Fig. 2) was adapted to
function reliably in a 96-well plate. The eight wells of column 1
of the 96-well plate served as a positive control (no inhibitor),
whereas column 12 served as a negative control (no enzyme).
The initial and final absorbance at 440 nm of each well was mea-
The best inhibitor was found to be C-1, with an IC50 of 5 lM.
However, this compound contains a hydrazide functionality having
a free NH2, a group that is well known to react readily with PLP.
NH2
HO2C
CHO
LL-DAP-AT
HO2C
CO2H
NH2
HN
N
CO2H
OH
HO2C
N
CO2H
NH2
LL-DAP
Tetrahydrodipicolinate
(THDP)
Monitor absorbance
increase at 440nm
HO2C
CO2H
NH2
L-Glutamate
HO2C
CO2H
O
α-ketoglutarate
Figure 2. Testing method for inhibitor against LL-DAP-AT.

C. Fan et al. / Bioorg. Med. Chem. 18 (2010) 2141–2151
2143
Table 1
Testing results
Entry
Structure
%Inhibitiona/IC50
(l
M)b
Entry
Structure
%Inhibitiona/IC50 M)b
(l
O
NH
O
S
N
S
N
O
O
S
S
S
O
25
70
18
194
A-1
A-2
N
O
O
S
O
O
O
O
O
NH
O
O
N
S
NH
52
78
19
141
S
O
O
A-3
A-5
A-7
A-4
A-6
A-8
O2N
N
NO2
O
NH
O
O
S
S
NH
22
>200
29
180
S
I
O
N
O
O
O
NH
N
NH
S
S
S
OH
13
85
13
46
O
Cl
Br
O2N
O
O
S
H
O
N
N
O
O
N
S
S
S
S
O
13
13
117
S
A-9
A-10
41c
Br
N
N
O
O
N
O
N
13
127
14
S
A-11
A-13
A-12
S
S
S
>200
S
S
O
Cl
N
S
Br
13
130
20
42
O
B-1
O
O
O
O
N
O
N
H
O
N
O
N
N
20
17
37
O
O
N
HO
B-2
B-3
45c
HO
O
N
N
O
O
N
H
O
(continued on next page)

2144
C. Fan et al. / Bioorg. Med. Chem. 18 (2010) 2141–2151
Table 1 (continued)
Entry
Structure
%Inhibitiona/IC50
(l
M)b
Entry
Structure
%Inhibitiona/IC50 M)b
(l
H
N
O
O
O
H
N
N
N
Cl
14
65
18
52
O
O
B-4
B-5
HO
N
O
N
F
O
O
N
H
O
O
N
N
O
N
NH
N
O
O
O2N
16
>200
13
>200
O
O
B-6
B-7
O
O
N
N
O
O
28
99
45
B-8
B-9
O
33d
HN
O
NH
N
O
N
H
O
O
N
H
S
N
N
O
O
35
75
33
69
B-10
B-12
B-14
B-11
B-13
B-15
N
N
O
N
H
S
O
N
H
S
O2N
Cl
O
O
O
O
N
O
O
48
121
20
62
N
NH
O
N
H
S
N
H
S
N
O
HN
15
16
164
O
NH
25c
N
O
N
H
S
O
N
H
S
Br
H2N
NH
O
O
O
S
17
>200
33
5c
O
N
O
B-16
C-2
C-1
C-3
N
H
O
N
H
S
O
O
O
HN
N
N
HN
NH
O
13
153
14
>200
NO2
N
N
O
S
O
H
O
N
HO
O
15
45
14
161
HN
N
C-4
C-6
O2N
C-5
C-7
N
OH
N
H
HN
N
F
O
O
O
N
HN
N
14
>200
16
N
HN
N
39c
H
HO
O

C. Fan et al. / Bioorg. Med. Chem. 18 (2010) 2141–2151
2145
Table 1 (continued)
Entry
Structure
%Inhibitiona/IC50
(l
M)b
Entry
Structure
%Inhibitiona/IC50 M)b
(l
O2N
H2N
O
N
N
NO2
O
HN
NH
N
13
>200
19
83
Cl
C-8
C-9
N
S
O
H
O
H
H
N
N
O
O
O
O
15
31
18
56
C-10
C-12
C-11
C-13
S
S
N
H
N
N
O
Cl
O
O
O
O
30
81
14
159
O
Cl
O
O
O
N
O
O
N
N
O
O
O
16
>200
25
>200
S
C-14
C-16
C-15
C-17
O
HN
O
N
F
N
N
O
SH
S
N
N
O
O
N
18
>200
14
>200
O
O
O
O
a
b
c
Robotic screening at inhibitor concentration of 10
Manually tested for IC50 value.
Time-independent type inhibitor (competitive, non-competitive, or uncompetitive).
lM.
d
Time-dependent type inhibitor (affinity label, mechanism-based irreversible inactivation, or slow-binding).
Thus, even though the compound C-1 shows high potency and
time-independent inhibition, this may be due to reaction of the
compound with PLP, rather than with the LL-DAP-AT enzyme.
Based on preliminary kinetic studies, compound C-1 appears to
be an uncompetitive inhibitor of LL-DAP-AT. Two analogues of C-
1 were also synthesized in which the hydrazine moiety was
masked (Scheme 1). In compound 2, the free hydrazide terminus
was masked by a six-membered ring, which would decrease the
nucleophilicity but retain the basicity of the nitrogen atom. In
compound 3, the amino group was acetylated to decrease both
the nucleophilicity and basicity of the nitrogen. Upon testing, it
was shown that neither analogue was an inhibitor of LL-DAP-AT,
suggesting that the inhibitory effect of compound C-1 may be at
least partly due to the reaction of the hydrazide amino group with
PLP. We therefore focused our initial efforts on other good inhibi-
tors identified by screening. However, it should be noted that
O
O
O
I)
Cl
S
Cl
N
O
O
Cl
HN
O
THF
O
OH
OH
NH2
NH
S
NH
O
O
II) DIPEA
O
S
O
NH2
N
1
2
No activity
O
I)
H
N
N
N
O
N
N
HN
O
DMF
O
NH
S O
II)
O
H2N
N
H
3
No activity
Scheme 1. Preparation and testing results of the analogues for compound C-1.

2146
C. Fan et al. / Bioorg. Med. Chem. 18 (2010) 2141–2151
certain useful drugs, for example, the antituberculosis agent isoni-
azid,27 possess such an unsubstituted hydrazide on an aromatic
ring.
The initial objective was to find inhibitors that displayed time-
independent behavior and interacted reversibly only with the en-
while the substituents on the aromatic ring were varied reveals that
an electron-donating group (e.g., –OCH3) on the aromatic ring in-
creases the activity of the inhibitor (e.g., [4a, 4d], [5a, 5c], [6a, 6b],
[7a, 7b]). This effect is most pronounced for those analogues in
which the nitrogen substituent is either a hydrogen or an amino
group. Replacement of the –OCH3 group with a slightly electron-
withdrawing chlorine atom (e.g., 4b, 5b, 6c, 7c) results in a drop in
activity in three of the four examples. The location of the substituent
also appears to play a role, as an electron-donating substituent
(–OH) added to the ortho position (4g) decreases the inhibition com-
pared to 4d, although it is still better than the unsubstituted ring.
Interestingly, the most potent analogue (7d) is substituted with a
furan ring instead of a benzene ring, as in the case of lead compound
A-8. Similar to A-9, compound 7d also possesses an electron-with-
drawing substituent in the 5-position of the furan ring, albeit a sub-
stantially larger one.
zyme. A number of inhibitors exhibited IC50 values of 20–60 lM,
and as mentioned above, a significant percentage of these contained
barbiturate and thiobarbiturate moieties with a conjugated exocy-
clic double bond. Similar barbiturate-based compounds have been
studied extensively in the past, and have been shown to be good
methionine aminopeptidase-1 inhibitors,28 as well as potent inhib-
itors of metalloenzymes.29 However, such unsaturated barbiturate
derivatives can be expected to be good Michael acceptors and may
react with nucleophilic groups on the enzyme to form covalent
bonds. Hence they would not be time-independent reversible inhib-
itors and would have the potential to react with other biomolecules,
which could lead to mammalian toxicity. Preliminary testing of
these compoundsas inhibitorsof LL-DAP-ATrevealedthatmost, such
as B-9, were time-dependent inhibitors, suggesting a non-reversible
reaction of the inhibitor with the enzyme or a slow-binding inhibi-
tion. Although a few barbiturate-based candidates, such as com-
pounds B-2 and B-14, did show time-independence, and
preliminary kinetic studies suggest that B-14 shows uncompetitive
inhibition, we chose to initially exclude barbiturate-based com-
pounds from the current work to avoid complications arising from
their ability to behave as Michael type acceptors.
Three of the best inhibitors were chosen for preliminary general
antimicrobial activity testing in vitro. The inhibitors examined were
B-14, C-1, and C-7, which had IC50 valueswith LL-DAP-ATof 25, 5, and
39
antimicrobial activity at concentrations lower than 200
preliminary tests againstE. coli DH5 , Pseudomonas aeruginosa ATCC
lM, respectively. As expected, none of these inhibitors showed
lg/mL in
a
15442, Salmonella typhimurium ATCC 23564, Staphylococcus aureus
ATCC 25923, and Carnobacterium divergens LV13. However, these
organisms do not use LL-DAP-AT in their biosynthetic pathway to L-
lysine. Studies targeted against plants and Chlamydia, both of which
In this study, the rhodanine-based inhibitors (A-1 to A-13) were
selected for further investigation because there were no obvious
functional groups reactive toward PLP or the enzyme, and thus
these compounds were more likely to be time-independent type
inhibitors. However, within this group of inhibitors, the inhibitory
activity was not particularly high and trends in SAR studies were
unclear. Fortunately, the synthesis of these types of compounds
was straightforward and allowed for facile preparation of ana-
logues. The top two inhibitors, A-8 and A-9, with IC50 values of
do use LL-DAP-AT, will be reported in the future.
3. Conclusions
Inthisstudy,46compoundsdisplayinggreaterthan13%inhibition
(at 10
29,201, using robotic screening. Fifteen of these 46 compounds had
IC50 values of 60 M or lower. Of the 46 compounds, 13 possessed a
lM) of LL-DAP-AT, were identified from a compound library of
l
rhodanine moiety, whereas 16 had a barbiturate or thiobarbiturate
moiety, suggesting potential pharmacophores. The best inhibitor is
46 and 41 lM, respectively, were shown to be time-independent
type inhibitors, and were chosen to be the two lead compounds.
The substituents on both of the nitrogens of the rhodanine ring
and on the aromatic ring were varied in this preliminary study.
The route used to synthesize the analogues is shown in Scheme 2,
and theresultsfor theinhibitiontestingare listedin Table 2. The data
show that no analogue exhibited better inhibition than the lead
compounds. However, several conclusions can be drawn that are
useful in guiding further study. Substitution on the nitrogen of the
rhodanine ring does not appear to have a significant effect on the
inhibitory potency of the compound as two of the best analogue
inhibitors (4c and 4d) possessed only a hydrogen atom on the nitro-
gen. Furthermore, comparisonof analogues in whichonly thesubsti-
tuent on nitrogen was varied (e.g., [4a, 5a, 6a, 7a], [4b, 5d, 6c, 7c], or
[4d, 5c, 6b, 7b]) does not reveal any significant differences. Although
the tested substituents are of somewhat different size, it is perhaps
more surprising to note the absence of any electronic effect despite
thedramaticallydifferentelectronicpropertiesof aminoand N-acet-
yl substituents. These results imply that the substituent on the rho-
danine does not dramatically affect activity. Comparison of
analogues in which the substituent on nitrogen was kept the same
hydrazide C-1, with an IC50 value of 5 lM. However, it is likely that
thisisatleastpartlyduetoreactionwith the PLPco-factor, ratherthan
direct inhibition of the target enzyme. The rhodanine-based inhibi-
tors were chosen as lead compounds for SAR studies. Upon analysis
of the results, it appears that the substituent on the nitrogen has little
effect on the activity of these inhibitors, whereas the presence of an
electron-donating group on the aromatic ring enhances activity. Sev-
eral compounds were also found to be good inhibitors aside from the
compounds mentioned above. Compounds C-4, C-7, C-10, and C-11
had IC50 values of 45, 39, 31, and 56 lM respectively. In the future,
additional analogues can be synthesized based on these compounds,
and also on aryl hydrazide C-1, as possible inhibitors of LL-DAP-AT
with useful antimicrobial or herbicidal activity.
4. Experimental
4.1. Inhibitor screening
4.1.1. Aliquot buffer solution to 30 plates
420 mg of 2-oxoglutarate (2-OG), 280 mg 2-aminobenzalde-
hyde (OAB), and 480 mg of racemic DAP were dissolved in
X
N
X
N
O
280 mL of 100 mM HEPES-KOH, pH 7.6, and 85 lL of this stock
O
O
Acetic acid
S
+
was aliquoted to empty 96-well assay plates using an automated
protocol on a Beckman Coulter Biomek 2000 Laboratory Automa-
tion Workstation. The final concentration of each component in
the stock solution is: 2-OG (10 mM); OAB (8.3 mM); LL-DAP
(assuming 25% of total DAP: 2.3 mM). Upon dilution to 100 lL in
the well plate, the final concentrations are: 2-OG (8.5 mM); OAB
S
S
NaOAc
reflux
R
H
S
R
4a - 4g: X=H
5a - 5f: X=Et
6a - 6c: X=NH2
7a - 7d: X=NHAc
(7 mM); LL-DAP (2.0 mM).
Scheme 2. General reaction for the synthesis of rhodanine-based inhibitors.

C. Fan et al. / Bioorg. Med. Chem. 18 (2010) 2141–2151
2147
Table 2
Testing results for rhodanine-based analogues
Entry
X
R
IC50
(
lM)
Entry
X
R
IC50 (lM)
4a
H
No activity
127
4b
H
>200
142
Cl
O
4c
H
4d
4f
H
H
N
NO2
4e
4g
5a
H
H
Et
No activity
>200
No activity
OH
OH
>200
5b
5d
Et
Et
>200
5c
5e
Et
Et
>200
ꢀ200
Cl
O
ꢀ200
5f
Et
190
155
N
6a
6c
7a
7c
NH2
No activity
No activity
No activity
No activity
6b
NH2
O
O
NH2
Cl
Cl
NHAc
NHAc
7b
7d
NHAc
NHAc
>200
73
O
NO2
4.1.2. Preparation of inhibitor working stock
The inhibitor source plates contained 0.25 mg of inhibitor in
L of DMSO. Inhibitor was present in columns 2–11 of 96-well
plates; columns 1 and 12 contained pure DMSO. Using an auto-
mated protocol on the Biomek 2000, 99 L of DMSO was added
to each well of a 96-well plate, followed by 1.0 L of inhibitor to
(with the exception of column 12, which served as a negative con-
trol) using an automated protocol on the Biomek 2000. This re-
sulted in 0.43 lg of enzyme in each well, or a concentration of
0.0043 mg/mL of LL-DAP-AT. The assay plates were then allowed
to sit at room temperature for 3 h, at which time a second absor-
bance reading at 440 nm was taken in triplicate.
25
l
l
l
create the inhibitor working stock. Assuming a molecular weight
of 500 g/mol, the final concentration of inhibitor in each well is
4.2. Determination of IC50
200
l
M. The working plates were stored at ꢁ20 °C.
4.2.1. Aliquot buffer solution
4.1.3. Addition of inhibitor
420 mg of 2-oxoglutarate and 480 mg of racemic DAP (commer-
cial) were dissolved in 280 mL of 100 mM HEPES-KOH, pH 7.6.
Seven milliliters of this solution were used to dissolve 7 mg of
A 5 L solution of inhibitor working stock was then added to the
l
assay plates containing buffer using an automated protocol on the
Biomek 2000. Columns 1 and 12 of the assay plates received pure
DMSO as no inhibitor was present in these columns in the inhibitor
source plates. Assuming a molecular weight of 500 g/mol, the final
2-aminobenzaldehyde (OAB) and 850 lL of this stock was added
into eight assay cells (1 mL). The final concentration of each com-
ponent in the stock solution is: 2-OG (10 mM); OAB (8.3 mM);
LL-DAP (assuming 25% of total DAP: 2.3 mM). Upon dilution to
1 mL in the assay cells, the final concentrations are: 2-OG
(8.5 mM); OAB (7 mM); LL-DAP (2.0 mM).
concentration of inhibitor in each well is 10 lM. In a few cases,
there were inhibitor solubility problems, but these tests were dis-
carded in the interest of high throughput. To remove air bubbles,
the assay plates were then centrifuged at 2400 rpm for 3 min.
The absorbance was then recorded at 440 nm in triplicate on a
Bio-TEK PowerWave XS Universal Microplate Spectrophotometer.
4.2.2. Preparation of inhibitor working stock
Eight micromoles of inhibitor were dissolved in 2 mL of DMSO,
which resulted 4
lmol/mL in concentration. The serial dilution
4.1.4. Addition of enzyme
gave 2, 1, 0.5, 0.2, and 0.1
l
mol/mL solutions. In the final assay cell,
A 0.043 mg/mL solution of LL-DAP-AT in 100 mM HEPES-KOH
the inhibitor concentrations were 200, 100, 50, 25, 10, and 5
lM,
pH 7.6 was prepared and 10 lL of this was added to each well
respectively.

2148
C. Fan et al. / Bioorg. Med. Chem. 18 (2010) 2141–2151
4.2.3. Addition of inhibitor
Fifty microliters of each concentration of inhibitor solutions
were added into cell 2–7. And 50 L of pure DMSO was added into
cell 1 and 8, which are control and negative control. The Varian
Cary 100 Bio UV–Visible spectrophotometer was used to measure
the absorbance at 440 nm. Under the program of Enzyme Kinetics
the absorbance for all the cells were then multi-zeroed.
Nicolet Magna 750 FTIR Spectrometer and Nic-Plan FTIR Micro-
scope. Varian Inova 300 MHz, 400 MHz, and Varian Mercury
400 MHz two-channel spectrometers were used to acquire 1H
and 13C NMR spectra. Chloroform-D and DMSO-d6 were used as
NMR solvents. Spin multiples are listed as s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet), and br (broad) and coupling
constant (J) values were estimated in hertz (Hz). High-resolution
mass spectra were obtained using Applied BioSystems Mariner
BioSpectrometry Workstation (orthogonal acceleration Time-of-
Flight, positive and negative ion ESI and APCI, Foster City, CA)
and Kratos MS50G (positive ion EI).
l
4.2.4. Addition of enzyme
A 0.043 mg/mL solution of LL-DAP-AT in 100 mM HEPES-KOH
pH 7.6 was prepared and 100 lL of this was added to each cell, ex-
cept the negative control (cell 8). The final concentration of the en-
zyme was 0.0043 mg/mL. The absorbance was recorded for
200 min with 2 min/cycle. The initial rate of increase of the absor-
bance is proportional to the rate of reaction. By comparing the ini-
tial slope of each absorbance curve of the inhibitor assay cell to the
control cell, the percentage inhibition was determined and the IC50
value was calculated based on this data.
4.6.1. 3-(Phenylsulfonamido)-2-naphthoic acid (1)
3-Amino-2-naphthoic acid (80%, 0.468 g, 2.00 mmol) and so-
dium carbonate (0.515 g, 4.80 mmol) were mixed with distilled
water (5 mL). The mixture was heated in an oil-bath to ꢀ60 °C.
Benzenesulfonyl chloride (0.31 mL, 2.4 mmol) was slowly added
into the mixture over the course of 15 min. The mixture was then
heated to 85 °C for a further 2 h. Norite (20 mg) was added into the
dark red mixture. The hot mixture was filtered through a pre-
heated funnel. The filtrate was slowly added with vigorous swirling
into a 50 mL flask, which contained of 6 N hydrochloric acid (1 mL).
A red solid formed. The solid was filtered and washed with of 1 N
hydrochloric acid (2 mL). The product (0.606 g, 93%) was a light red
solid and was used directly. IR (Microscope) 3221, 3070, 2861 (br),
4.3. Time dependent/independent testing
The concentration of inhibitor stock was chosen, based on the
IC50 value determined. The substrate buffer solution and the en-
zyme were prepared, as described above. Six portions of 100
of enzyme and 50 L of inhibitor solution were pre-incubated for
different lengths of time before addition into the cell, containing
850 L of substrate buffer solution. Cells 1 and 8 served as positive
lL
l
1676, 1448, 1160 cmꢁ1 1H NMR (400 MHz, DMSO-d6): d = 8.59 (s,
;
l
1H), 7.97 (d, J = 7.5, 1H), 7.91 (s, 1H), 7.89 (d, J = 7.5, 1H), 7.82 (m,
2H), 7.59 (m, 2H), 7.48 (m, 3H); 13C NMR (100 MHz, DMSO-d6):
d = 170.34, 139.26, 135.90, 135.70, 134.33, 134.13, 130.24,
130.08, 129.82, 129.22, 127.68, 127.62, 126.62, 118.25, 116.60;
ESI-HRMS m/z calcd for C17H13NO4S: 326.04925 [M+H+], found:
326.04881.
and negative control, respectively. The absorbance was recorded as
described above. For the time-independent inhibitors, the percent-
age inhibition did not change with different incubation times. For
the time-dependent inhibitors, the percentage inhibition increased
with longer pre-incubation times.
4.4. Type of inhibition testing
4.6.2. 3-(Phenylsulfonamido)-N-(piperidin-1-yl)-2-naphthamide
(2)
Preliminary testing was carried out according to the description
by Segel.30 The concentration of inhibitor stock was chosen, based
on the IC50 value determined. The concentration of LL-DAP was var-
3-(Phenylsulfonamido)-2-naphthoic acid (0.0818 g, 0.250 mmol)
was dissolved in dry THF (5 mL). The solution was cooled in an
ice-bath, after 10 min, oxalyl chloride (28 lL, 0.32 mmol) was
ied from 125
l
M to 5 mM. The UV absorbance was monitored at
added. Then two drops of DMF were added into the mixture. The
mixture was stirred for 30 min before being allowed to warm to
20 °C. The mixture was stirred for a further one hour. The solvent
and excess oxalyl chloride was removed in vacuo. Dry THF
(5 mL) was then added to the residue. N,N-Diisopropylethylamine
(0.3 mL, 1.7 mmol) was added, followed by slow addition of 1-
aminopiperidine (0.054 mL, 0.50 mmol). The mixture was stirred
for 16 h, and then the solvent was removed in vacuo. The residue
was dissolved in ethyl acetate (10 mL), and washed with saturated
sodium bicarbonate solution (2 ꢂ 15 mL), water (15 mL), and brine
(15 mL). The organic layer was dried over sodium sulfate followed
by solvent removal in vacuo. The product was purified by column
chromatography and yielded a white solid (0.073 g, 68%); Rf = 0.45
(2:1 ethyl acetate/hexanes); IR (Microscope) 3221, 3070, 2861 (br),
30 °C using a Spectra Max 340PC microplate reader controlled with
SOFTmaxÒ PRO software (Molecular Devices, Sunnyvale, CA) in the
kinetics read mode. The wavelength was set at 440 nm and each
well was monitored at 30 s intervals for up to 2 h. Data were then
graphed and analyzed using GraphPad PrismÒ Version 4. For Mich-
elis–Menten plots, a non-linear regression fit was employed. Data
were transformed as a Lineweaver–Burk (double reciprocal) plot.
4.5. Preliminary antimicrobial testing
Ten milliliters of sterile, soft agar (0.75% w/v agar) containing
the appropriate growth media (Luria Bertani for Gram-negative
bacteria and M17 for Gram-positive bacteria) were heated in boil-
ing water, until molten. After cooling to touch, the soft agar was
inoculated with an overnight culture of the indicator organism
(1%) and overlaid on a bed of solid agar (1.5% w/v agar). Solutions
to be tested, including appropriate controls (acetonitrile for C-1,
1676, 1448, 1160 cmꢁ1 1H NMR (400 MHz, CDCl3): d = 10.42 (br,
;
1H), 8.04 (s, 1H), 7.94 (br, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.72 (t,
J = 8.0 Hz, 2H), 7.50 (t, J = 8.0 Hz, 2H), 7.36 (m, 3H), 2.84 (br, 4H),
1.76 (br, 4H), 1.47 (br, 2H); 13C NMR (100 MHz, CDCl3): d =
166.87, 139.90, 135.10, 134.41, 132.91, 129.36, 129.13, 128.82,
128.59, 128.48, 127.70, 127.41, 126.25, 122.13, 119.06, 57.04,
25.29, 23.23; ESI-HRMS m/z calcd for C22H23N3O3S: 432.13523
[M+Na+], found: 432.13481.
methanol for B-14 and C-7), were spotted (10 lL) onto the agar
and allowed to air dry. Plates were incubated overnight at the
appropriate temperatures (37 °C for Gram-negative bacteria and
30 °C for Gram-positive bacteria) and examined for halos of growth
inhibition.
4.6.3. N-(3-(2-Acetylhydrazinecarbonyl)naphthalen-2-yl)benzene-
sulfonamide (3)
4.6. Chemical preparation of analogues
3-(Phenylsulfonamido)-2-naphthoic acid (0.0818 g, 0.250 mmol)
and carbonyldiimidazole (0.041 g, 0.25 mmol) were dissolved in
dry DMF (5 mL). The mixture was stirred for one hour.
All chemicals and solvents used in this study were purchased
from Sigma–Aldrich and TCI. Infrared spectra were obtained using

C. Fan et al. / Bioorg. Med. Chem. 18 (2010) 2141–2151
2149
Acetohydrazide (0.025 g, 0.30 mmol) was dissolved in DMF (1 mL)
and was added to the mixture. The reaction was allowed to stir for
16 h at 20 °C. The solvent was then removed in vacuo, and the
residue was dissolved in ethyl acetate (20 mL). The solution was
washed with 10% citric acid (2 ꢂ 20 mL), saturated sodium bicar-
bonate solution (2 ꢂ 20 mL), water (20 mL), and brine (20 mL).
The organic layer was dried over sodium sulfate, and solvent was
removed in vacuo. The product was purified by column chroma-
tography to yield a white solid (0.070 g, 73%); Rf = 0.28 (100% ethyl
acetate); IR (Microscope) 3267, 3225, 3020, 1698, 1412,
4.6.4.5. (Z)-5-(Naphthalen-1-ylmethylene)-2-thioxothiazolidin-
4-one (4e). Light yellow solid; 82% yield: IR (Microscope) 3152,
3056, 3017, 2857, 1691, 1437, 1170 cmꢁ1 1H NMR (400 MHz,
;
DMSO-d6): d = 8.27 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H), 8.07 (dd,
J = 6.4 Hz, 3.2 Hz, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.65 (m, 4H); 13C
NMR (100 MHz, DMSO-d6): d = 196.82, 169.48, 133.96, 131.84,
131.72, 130.75, 129.58, 129.45, 128.80, 128.24, 127.56, 127.53,
126.37, 123.99; EI-HRMS m/z calcd for C14H9NOS2: 271. 01254
[M+], found: 271.01231.
1156 cmꢁ1
;
1H NMR (400 MHz, DMSO-d6): d = 10.73 (s, 2H),
4.6.4.6. (Z)-5-(2-Nitrobenzylidene)-2-thioxothiazolidin-4-one
(4f). Light green solid; 98% yield: IR (Microscope) 3099, 3033,
10.08 (s, 1H), 8.28 (s, 1H), 7.84 (m, 5H), 7.53 (m, 5H), 1.99 (s,
3H); 13C NMR (100 MHz, DMSO-d6): d = 169.42, 167.97, 139.17,
134.85, 134.34, 134.04, 130.52, 130.14, 129.69, 129.21, 129.14,
127.84, 127.67, 126.88, 121.08, 117.01, 21.22; ESI-HRMS m/z calcd
for C19H17N3O4S: 406.08320 [M+Na+], found: 406.08265.
2853, 1732, 1533, 1455, 1302, 1200 cmꢁ1 1H NMR (400 MHz,
;
DMSO-d6): d = 8.16 (dd, J = 10.8 Hz, 1.6 Hz, 1H), 7.84 (td,
J = 10 Hz, 1.6 Hz, 1H), 7.82 (s, 1H), 7.68 (m, 2H); 13C NMR
(100 MHz, DMSO-d6): d = 196.46, 169.30, 148.65, 135.28,
131.93, 131.03, 130.08, 129.44, 128.53, 126.20; ESI-HRMS m/z
calcd for C10H6N2O3S2: 288.97121 [M+Na+], found: 288.97169.
4.6.4. General procedure for synthesis of rhodanine derivatives
Acetic acid (10 mL) was heated to ꢀ80 °C in an oil-bath. The
rhodanine derivative (2.5 mmol) and 10 mmol sodium acetate
were then added to the hot acid. The mixture was stirred for
5 min before adding 2.5 mmol of the aldehyde. The mixture was
then heated to reflux for 16 h, and cooled to room temperature
to give in a crystalline product. The mixture was poured into ice-
cold water (30 mL) and chilled in an ice-bath for an additional
30 min. The solid was filtered under suction. The crude compounds
were then purified by recrystallization with ethanol or a multi-sol-
vent method. The yields ranged from 37% to quantitative. Com-
pounds were characterized by IR, 1H NMR, 13C NMR, and high
resolution ESI-MS.
4.6.4.7. (Z)-5-(2-Hydroxybenzylidene)-2-thioxothiazolidin-4-
one (4g). Orange solid; 76% yield: IR (Microscope) 3496, 3437,
3096, 2846, 1699, 1450, 1163 cmꢁ1 1H NMR (400 MHz, DMSO-
;
d6): d = 13.66 (br, 1H), 10.62 (s, 1H), 7.83 (s, 1H), 7.28 (m, 2H),
6.94 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d = 196.68, 170.19,
158.22, 133.47, 129.94, 127.97, 124.52, 120.63, 120.59, 116.93;
ESI-HRMS m/z calcd for C10H7NO2S2: 259.98104 [M+Na+], found:
259.98150.
4.6.4.8. (Z)-5-Benzylidene-3-ethyl-2-thioxothiazolidin-4-one
(5a). Bright yellow solid; 82% yield: IR (dichloromethane cast)
3015, 2982, 2934, 2873, 1705, 1445, 1132 cmꢁ1
;
1H NMR
(400 MHz, DMSO-d6): d = 7.79 (s, 1H), 7.61 (dt, J = 8 Hz, 1.6 Hz,
2H), 7.52 (m, 3H), 4.04 (q, J = 7.2 Hz, 2H), 1.17 (t, J = 7.2 Hz, 3H);
13C NMR (100 MHz, DMSO-d6): d = 193.91, 167.39, 133.69,
133.55, 131.64, 131.32, 130.20, 123.25, 40.17, 12.58; ESI-HRMS
m/z calcd for C12H11NOS2: 250.03548 [M+H+], found: 250.03581.
4.6.4.1. (Z)-5-Benzylidene-2-thioxothiazolidin-4-one (4a). Yel-
low solid; 94% yield: IR (Microscope) 3157, 3064, 2852, 1701,
1445, 1195 cmꢁ1 1H NMR (400 MHz, DMSO-d6): d = 13.81 (br,
;
1H), 7.63 (s, 1H), 7.52 (m, 5H); 13C NMR (100 MHz, DMSO-d6):
d = 196.41, 170.07, 133.66, 132.32, 131.43, 131.16, 130.14, 126.23;
EI-HRMS m/z calcd for C10H7NOS2: 220.99690 [M+], found:
220.99690.
4.6.4.9. (Z)-3-Ethyl-5-(2-hydroxybenzylidene)-2-thioxothiazoli-
din-4-one (5b). Yellow solid; 56% yield: IR (dichloromethane
cast) 3256, 3052, 2980, 2916, 2849, 1678, 1456, 1131 cmꢁ1 1H
;
4.6.4.2. (Z )-5-(4-Chlorobenzylidene)-2-thioxothiazolidin-4-one
(4b). Light yellow solid; 56% yield: IR (Microscope) 3086, 3015,
NMR (400 MHz, DMSO-d6): d = 10.71 (br, 1H), 7.99 (s, 1H), 7.34
(m, 2H), 6.96 (m, 2H), 4.06 (q, J = 7.2 Hz, 2H), 1.18 (t, J = 7.2 Hz,
3H); 13C NMR (100 MHz, DMSO-d6): d = 193.53, 166.84, 157.57,
133.04, 129.57, 128.58, 120.82, 119.97, 119.92, 116.27, 40.17,
11.90; ESI-HRMS m/z calcd for C12H11NO2S2: 288.01234 [M+Na+],
found: 288.01190.
2852, 1718, 1488, 1188 cmꢁ1
;
1H NMR (400 MHz, DMSO-d6):
d = 7.61 (s, 1H), 7.58 (d, J = 2 Hz, 4H); 13C NMR (100 MHz, DMSO-
d6): d = 196.11, 170.02, 136.03, 132.74, 132.57, 130.86, 130.20,
127.00; EI-HRMS m/z calcd for C10H6ClNOS2: 254.95793 [M+],
found: 254.95776.
4.6.4.10. (Z )-3-Ethyl-5-(4-methoxybenzylidene)-2-thioxothiaz-
olidin-4-one (5c). Bright yellow solid; 53% yield: IR (dichloro-
4.6.4.3. (Z)-5-(4-(Dimethylamino)benzylidene)-2-thioxothiaz-
olidin-4-one (4c). Red solid; 61% yield: IR (Microscope) 3139,
methane cast) 3004, 2983, 2961, 2833, 1703, 1457, 1127 cmꢁ1
;
3042, 2909, 2853, 1682, 1436, 1180 cmꢁ1 1H NMR (400 MHz,
;
1H NMR (400 MHz, DMSO-d6): d = 7.75 (s, 1H), 7.58 (d, J = 8.8 Hz,
2H), 7.09 (d, J = 8.8 Hz, 2H), 4.03 (q, J = 7.2 Hz, 2H), 3.82 (s, 3H),
1.16 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): d = 193.65,
167.45, 162.22, 133.83, 133.61, 126.24, 119.86, 115.86, 56.28,
40.17, 12.60; ESI-HRMS m/z calcd for C13H13NO2S2: 280.04605
[M+H+], found: 280.04675.
DMSO-d6): d = 13.50 (br, 1H), 7.47 (s, 1H), 7.37 (d, J = 8.8 Hz, 2H),
6.77 (d, J = 8.8 Hz, 2H), 2.99 (s, 6H); 13C NMR (100 MHz, DMSO-
d6): d = 195.69, 170.12, 152.44, 133.92, 133.56, 120.45, 118.04,
112.85, 40.26; ESI-HRMS m/z calcd for C12H12N2OS2: 287.02833
[M+Na+], found: 287.02829.
4.6.4.4. (Z)-5-(4-Methoxybenzylidene)-2-thioxothiazolidin-4-
one (4d). Yellow solid; 85% yield: IR (Microscope) 3134, 3017,
4.6.4.11. (Z )-5-(4-Chlorobenzylidene)-3-ethyl-2-thioxothiazoli-
din-4-one (5d). Yellow solid; 79% yield: IR (dichloromethane
2934, 2853, 1687, 1446, 1170 cmꢁ1 1H NMR (400 MHz, DMSO-
;
cast) 3004, 2983, 2971, 2872, 1705, 1433, 1137 cmꢁ1 1H NMR
;
d6): d = 7.59 (s, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.8 Hz,
2H), 3.82 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d = 195.46,
169.39, 161.31, 132.65, 131.85, 125.45, 122.20, 115.06, 55.53;
ESI-HRMS m/z calcd for C11H9NO2S2: 273.99669 [M+Na+], found:
273.99682.
(400 MHz, DMSO-d6): d = 7.78 (s, 1H), 7.63 (d, J = 8.8 Hz, 2H),
7.58 (d, J = 8.8 Hz, 2H), 4.03 (q, J = 7.2 Hz, 2H), 1.16 (t, J = 7.2 Hz,
3H); 13C NMR (100 MHz, DMSO-d6): d = 193.61, 167.33, 133.26,
132.90, 132.59, 132.08, 130.26, 124.01, 40.17, 12.56; EI-HRMS m/
z calcd for C12H10ClNOS2: 282.98923 [M+], found: 282.98949.

2150
C. Fan et al. / Bioorg. Med. Chem. 18 (2010) 2141–2151
4.6.4.12. (Z)-3-Ethyl-5-(naphthalen-1-ylmethylene)-2-thioxo-
thiazolidin-4-one (5e). Yellow-brown solid; 31% yield: IR
(dichloromethane cast) 3075, 3055, 2974, 2931, 2873, 1704,
56.32, 20.99; ESI-HRMS m/z calcd for C13H12N2O3S2: 331.01815
[M+Na+], found: 331.01818.
1431, 1133 cmꢁ1 1H NMR (400 MHz, CDCl3): d = 8.49 (s, 1H),
;
4.6.4.19. (Z)-N-(5-(4-Chlorobenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl)acetamide (7c). Yellow solid; 80% yield: IR (dichloro-
8.16 (d, J = 8.8 Hz, 1H), 7.93 (m, 2H), 7.57 (m, 4H), 4.24 (q,
J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d = 194.03, 167.38, 133.99, 132.11, 131.66, 130.83, 130.01,
129.26, 127.71, 127.32, 127.05, 126.30, 125.64, 123.62, 40.04,
12.56; EI-HRMS m/z calcd for C16H13NOS2: 299.04385 [M+], found:
299.04404.
methane cast) 3199, 3013, 1732, 1680, 1405, 1128 cmꢁ1
;
1H
NMR (400 MHz, DMSO-d6): d = 7.92 (s, 1H), 7.72 (d, J = 8.5 Hz,
2H), 7.63 (d, J = 8.5 Hz, 2H), 2.07 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): d = 190.14, 167.56, 163.06, 135.83, 133.11, 132.31,
131.51, 129.50, 120.06, 20.15; ESI-HRMS m/z calcd for
C12H9ClN2O2S2: 334.96862 [M+Na+], found: 334.96818.
4.6.4.13. (Z)-3-Ethyl-5-(quinolin-5-ylmethylene)-2-thioxothiaz-
olidin-4-one (5f). Light yellow solid; 40% yield: IR (dichloro-
methane cast) 3063, 3042, 2978, 2935, 2876, 1712, 1439,
4.6.4.20. (Z)-N -(5-((5-(4-Nitrophenyl)furan-2-yl)methylene)-4-
oxo-2-thioxothiazolidin-3-yl) acetamide (7d). Orange solid; IR
(dichloromethane cast) 3239, 3038, 1729, 1678, 1511,
1134 cmꢁ1 1H NMR (400 MHz, CDCl3): d = 9.04 (d, J = 4.4 Hz, 1H),
;
1139 cmꢁ1 1H NMR (400 MHz, DMSO-d6): d = 11.14 (s, 1H), 8.37
;
8.38 (s, 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.84
(m, 1H), 7.71 (m, 1H), 7.47 (d, J = 4.8 Hz, 1H), 4.26 (q, J = 7.2 Hz,
2H), 1.36 (t, J = 7.2, 3H); 13C NMR (100 MHz, CDCl3): d = 192.63,
167.00, 150.03, 148.81, 138.99, 130.67, 130.63, 130.58, 128.12,
126.40, 126.25, 123.52, 119.72, 40.25, 12.52; ESI-HRMS m/z calcd
for C15H12N2OS2: 301.04638[M+H+], found: 301.04641.
(d, J = 9.2 Hz, 2H), 8.05 (d, J = 9.2 Hz, 2H), 7.79 (s, 1H), 7.61 (d,
J = 4.0 Hz, 1H), 7.44 (d, J = 4.0 Hz, 1H), 2.06 (s, 3H); 13C NMR
(100 MHz, DMSO-d6): d = 191.77, 168.41, 163.70, 156.51, 151.51,
147.68, 134.81, 126.00, 125.49, 124.28, 120.07, 117.96, 114.55,
20.99; ESI-HRMS m/z calcd for C16H11N3O5S2: 412.00323
[M+Na+], found: 412.00278.
4.6.4.14. (Z)-3-Amino-5-benzylidene-2-thioxothiazolidin-4-one
(6a). Bright yellow solid; 53% yield: IR (dichloromethane cast)
Acknowledgments
3299, 3225, 3031, 1706, 1448, 1125 cmꢁ1 1H NMR (400 MHz,
;
DMSO-d6): d = 7.85 (s, 1H), 7.64 (m, 2H), 7.53 (m, 3H), 5.93 (s,
2H); 13C NMR (100 MHz, DMSO-d6): d = 187.73, 163.69, 133.31,
132.95, 130.99, 130.70, 129.49, 120.26; ESI-HRMS m/z calcd for
C10H8N2OS2: 258.99703 [M+Na+], found: 258.99749.
Financial support was provided by the Natural Sciences and
Engineering Research Council of Canada (NSERC), the Alberta Her-
itage Foundation for Medical Research (AHFMR), and the Canada
Research Chair in Bioorganic and Medicinal Chemistry.
4.6.4.15. (Z)-3-Amino-5-(4-methoxybenzylidene)-2-thioxothiaz-
olidin-4-one (6b). Yellow solid; 26% yield: IR (dichloromethane
References and notes
cast) 3288, 3195, 2942, 2842, 1702, 1509, 1131 cmꢁ1 1H NMR
;
1. Hutton, C. A.; Perugini, M. A.; Gerrard, J. A. Mol. BioSyst. 2007, 3, 458–465.
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(400 MHz, DMSO-d6): d = 7.76 (s, 1H), 7.57 (d, J = 8.8 Hz, 2H),
7.04 (d, J = 8.8 Hz, 2H), 5.90 (s, 2H), 3.80 (s, 3H); 13C NMR
(100 MHz, DMSO-d6): d = 187.86, 164.42, 162.27, 134.27, 133.71,
126.23, 117.56, 115.84, 56.28; ESI-HRMS m/z calcd for
C11H10N2O2S2: 289.00759 [M+Na+], found: 289.00698.
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4.6.4.16. (Z)-3-Amino-5-(4-chlorobenzylidene)-2-thioxothiaz-
olidin-4-one (6c). Bright yellow solid; 73% yield: IR (dichloro-
methane cast) 3299, 3234, 3169, 3071, 1727, 1709, 1493,
1128 cmꢁ1 1H NMR (400 MHz, DMSO-d6): d = 7.83 (s, 1H), 7.65
;
(d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 5.92 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): d = 188.18, 164.36, 136.34, 133.01, 132.59,
132.56, 130.27, 121.75; ESI-HRMS m/z calcd for C10H7ClN2OS2:
292.95805 [M+Na+], found: 292.95793.
4.6.4.17. (Z)-N -(5-Benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-
acetamide (7a). Bright yellow solid; 88% yield: IR (dichlorometh-
ane cast) 3192, 3017, 1737, 1679, 1447, 1125 cmꢁ1 1H NMR
;
(400 MHz, DMSO-d6): d = 7.92 (s, 1H), 7.68 (m, 2H), 7.56 (m, 3H),
2.07 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d = 190.54, 167.70,
163.25, 134.58, 132.73, 131.27, 130.80, 129.53, 119.44, 20.28;
ESI-HRMS m/z calcd for C12H10N2O2S2: 301.00759 [M+Na+], found:
301.00714.
4.6.4.18. (Z)-N-(5-(4-Methoxybenzylidene)-4-oxo-2-thioxo-
thiazolidin-3-yl)acetamide (7b). Bright yellow solid; 30% yield:
IR (dichloromethane cast) 3194, 3013, 2963, 1729, 1679, 1421,
1134 cmꢁ1 1H NMR (400 MHz, DMSO-d6): d = 11.12 (s, 1H), 7.85
;
(s, 1H), 7.63 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H), 3.83 (s,
3H), 2.05 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d = 191.16,
168.36, 164.06, 162.52, 135.48, 133.91, 126.02, 116.67, 115.93,
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