5-Arylidenethioxothiazolidinones as Inhibitors of Tyrosyl-DNA Phosphodiesterase i

Article abstract of DOI:10.1021/jm3008773

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a cellular enzyme that repairs the irreversible topoisomerase I (Top1)-DNA complexes and confers chemotherapeutic resistance to Top1 inhibitors. Inhibiting Tdp1 provides an attractive approach to potentiating clinically used Top1 inhibitors. However, despite recent efforts in studying Tdp1 as a therapeutic target, its inhibition remains poorly understood and largely underexplored. We describe herein the discovery of arylidene thioxothiazolidinone as a scaffold for potent Tdp1 inhibitors based on an initial tyrphostin lead compound 8. Through structure-activity relationship (SAR) studies we demonstrated that arylidene thioxothiazolidinones inhibit Tdp1 and identified compound 50 as a submicromolar inhibitor of Tdp1 (IC₅₀ = 0.87 μM). Molecular modeling provided insight into key interactions essential for observed activities. Some derivatives were also active against endogenous Tdp1 in whole cell extracts. These findings contribute to advancing the understanding on Tdp1 inhibition.

Full text of DOI:10.1021/jm3008773

Article
pubs.acs.org/jmc
5‑Arylidenethioxothiazolidinones as Inhibitors of Tyrosyl−DNA
Phosphodiesterase I
Venkata Ramana Sirivolu,∥,† Sanjeev Kumar V. Vernekar,∥,† Christophe Marchand,∥,‡ Alena Naumova,‡
Adel Chergui,‡ Amelie Renaud,‡ Andrew G. Stephen,§ Feng Chen,† Yuk Y. Sham,† Yves Pommier,*,‡
and Zhengqiang Wang*,†
†Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, Minnesota 55455, United States
‡Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United
States
§Protein Chemistry Laboratory, Advanced Technology Program, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer
Research, Frederick, Maryland 21702, United States
S
* Supporting Information
ABSTRACT: Tyrosyl−DNA phosphodiesterase I (Tdp1) is a cellular enzyme
that repairs the irreversible topoisomerase I (Top1)−DNA complexes and
confers chemotherapeutic resistance to Top1 inhibitors. Inhibiting Tdp1
provides an attractive approach to potentiating clinically used Top1 inhibitors.
However, despite recent efforts in studying Tdp1 as a therapeutic target, its
inhibition remains poorly understood and largely underexplored. We describe
herein the discovery of arylidene thioxothiazolidinone as a scaffold for potent
Tdp1 inhibitors based on an initial tyrphostin lead compound 8. Through
structure−activity relationship (SAR) studies we demonstrated that arylidene thioxothiazolidinones inhibit Tdp1 and identified
compound 50 as a submicromolar inhibitor of Tdp1 (IC50 = 0.87 μM). Molecular modeling provided insight into key
interactions essential for observed activities. Some derivatives were also active against endogenous Tdp1 in whole cell extracts.
These findings contribute to advancing the understanding on Tdp1 inhibition.
a Tdp1−DNA covalent intermediate (Figure 1 C) and release
Top1.12 Hydrolysis of this intermediate turns over Tdp1 and
generates a DNA product with a 3′-phosphate (Figure 1 D and
E). Further repair presumably involves the polynucleotide
kinase phosphatase (PNKP), a bifunctional enzyme with 5′-
kinase and 3′-phosphatase activities that catalyze both the
hydrolysis of the 3′-phosphate and the phosphorylation of the
5′-DNA end to enable their rejoining.8 The critical role of
Tdp1 in the cellular repair of Top1-mediated DNA damage has
been established through three lines of evidence. First,
knocking out Tdp1 in yeast13 and in vertebrate cells14,15
renders such cells hypersensitive to camptothecin. Second,
Tdp1-defective SCAN1 (spinocerebellar ataxia with axonal
neuropathy-1) cells, which have a Tdp1 mutation N493R and
accumulate the normally transient Tdp1−DNA repair complex
(Figure 1C), are highly sensitive to CPT and accumulate DNA
strand breaks upon treatment with CPT.14,16 Third, over-
expression of wild-type Tdp1 protects cells against CPT-
induced cell death, whereas the H263A mutant does not.17
These observations provide the rationale for developing Tdp1
inhibitors for combination therapy with Top1 inhibitors.18,19
Tdp1 belongs to the phospholipase D (PLD) superfamily,20
which is defined by a conserved HKD catalytic motif21 and a
INTRODUCTION
■
Topoisomerase I (Top1) is an essential enzyme that catalyzes
the relaxation of supercoiling and the release of torsional strains
during DNA replication, transcription, recombination, and
chromatin remodeling.1,2 This enzymatic function involves a
dynamic equilibrium between the cleavage of the DNA
substrate and the religation of the resulting transient Top1−
DNA cleavage complex, which features a 3′-phosphotyrosyl
bond (Figure 1). Inhibitors of Top1 stall this equilibrium by
binding to and stabilizing an irreversible ternary Top1−DNA−
inhibitor complex, preventing the religation of the 5′-hydroxyl
group.3,4
This inhibition creates a DNA lesion and underlies the use of
camptothecin (CPT)-based Top1 inhibitors, such as topotecan
and irinotecan, for treating ovarian, lung, and colon cancers5,6
and the development of the indenoisoquinolines as non-
camptothecin Top1 inhibitors.7 Stalled Top1−DNA complexes
are repaired by Tdp1, a cellular enzyme conserved from yeast
to humans and which hydrolyzes the unique 3′-phosphotyrosyl
linkage of the Top1−DNA cleavage complex.8 Other than
Top1 inhibitors, cleavage complexes can also be generated by
endogenous DNA lesions such as strand breaks, abasic sites,
base mismatches, and specific oxidized or modified bases and
carcinogenic DNA adducts.9−11 The repair of the Top1−DNA
cleavage complex begins with a nucleophilic attack of the
histidine 263 of Tdp1 onto the 3′-phosphotyrosyl bond to form
Received: June 22, 2012
© XXXX American Chemical Society
A
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Figure 1. Tdp1 in the repair of the stalled Top1−DNA complex: (A) noncovalent Top1−DNA complex; (B) transient Top1−DNA complex, which
is trapped in the presence of a Top1 inhibitor; (C) the stalled Top1−DNA complex (cleavage complex) is repaired by Tdp1 via a transient Tdp1−
DNA covalent intermediate; (D) Top1 is released and the Tdp1−DNA covalent intermediate resolved by hydrolysis; (E) 3′-phospho−DNA−end
product.
signature catalytic mechanism12 featuring a covalent phospho-
histidine intermediate (Figure 1C). Two such motifs are
present in the N- and C-terminal domains of human Tdp1,
both containing the highly conserved histidine and lysine
residues (H263/K265 and H493/K495). The importance of
these residues for Tdp1 catalytic activity is manifested through
the observations that the mutation of H263 generates a
catalytically inactive enzyme and mutating H493, K265, or
K495 results in compromised enzymatic activity.20,22 Thus,
Tdp1 represents a distinct member of the PLD superfamily
with unique HKN motifs. Although the mechanism of Tdp1
catalysis is well established, its inhibition remains poorly
understood and largely unexplored. Early studies on Tdp1
discovered molecular probes that inhibit Tdp1 at millimolar
range, including aminoglycoside antibiotics and the ribosome
inhibitors,23 as well as tungstates and vanadates which served as
transition state mimics for crystallographic studies.24,25 Later
Figure 2. Known inhibitors of Tdp1.
on, a few micromolar inhibitors (Figure 2) were identified
through a high-throughput screening (HTS) assay,26 among
further advance the understanding of Tdp1 functions and
which 3 was confirmed to specifically inhibit Tdp1 through
SAR.11 Recently, Cushman and co-workers reported that a
subset of indenoisoquinoline derivatives potently inhibit both
Top1 and Tdp1, making them the first Top1/Tdp1 dual
inhibitors.27 Nevertheless, though inhibiting Tdp1 at micro-
molar concentrations, these compounds remain far from
therapeutic development. Therefore, there is a need to continue
to identify potent and specific small molecule inhibitors to
develop therapeutic candidates. Herein we report the design,
synthesis, molecular modeling, and biological evaluations of
thioxothiazolidinones as novel and potent Tdp1 inhibitors.
RESULTS AND DISCUSSION
■
Exploring Tdp1 as a therapeutic target was made possible by
the development of a biochemical screening assay using
recombinant human Tdp1.11,28 This gel-based assay is based
B
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Figure 3. Inhibition of Tdp1 by tyrphostin derivatives. (A) Schematic representation of the Tdp1 biochemical gel-based assay. Tdp1 hydrolyzes the
3′-phosphotyrosine bond and converts the N14Y DNA substrate to a 3′-phosphate oligonucleotide product (N14P); (B) representative gel
demonstrating concentration-dependent inhibition of Tdp1 by compounds 8 and 9; (C) representative compounds. The pharmacophore of lead
compound 8 comprises two domains: the functional head domain and the aromatic moiety.
on the cleavage by Tdp1 of the 14-mer 5′-32P-labeled 3′-
a
Table 1. Aromatic Domain SAR of Lead Compound 8
phosphotyrosyl DNA substrate N14Y (5′-GATCTAAAA-
GACTT-pY-3′) to a 14-mer 5′-32P-labeled 3′-phosphate DNA
product N14P (Figure 3, A). This assay was applied to a small
in-house chemical library constituted of benzylidenemaloni-
triles, a class of tyrphostins. This screening led to the
identification of compounds 8 and 9 as Tdp1 inhibitors with
IC50 values of 12 and 18 μM, respectively (Figure 3, B and C).
Notably, it was observed from this screening that only
tyrphostins with a thioamide functionality (compounds 8 and
9) in the head domain inhibited Tdp1. The carboxylic acid (4),
carboxamides (5 and 6), and cyano (7) analogues were
completely inactive at the highest tested concentration (333
μM) (Figure 3, C).
compound
Tdp1 IC50 (μM)
On the basis of these initial screening results, derivatives 10−
16 of lead compound 8 were prepared to explore the impact of
the aromatic domain (Table 1). While carboxylic acid analogue
13 demonstrated potent inhibitory activity against Tdp1 (IC50
= 12 μM, Table 1), all other analogues were inactive up to 333
μM. The phenolic hydroxyl group (compound 8) or the
carboxylic acid functionality (compound 13) appears essential
for the inhibition of Tdp1.
At this point, we shifted our focus onto constructing
heterocyclic scaffolds mimicking the head domain of lead
compound 8. Notably, rhodanine benzylidene derivatives such
as compound 18 have been reported as inhibitors of
phosphodiesterase,29 PTP,30 and recently as broad spectrum
antiviral.31 To mimic the head domain of compound 8, we
chose a few rhodanine-like five-membered heterocyclic
scaffolds with wide applications in medicinal chemistry (Figure
4): thiohydantoins 1932−34 and 20,35 thiazolidinedione 21,36−38
10
11
12
13
14
15
16
1
>333
>333
>333
12
>333
>333
>333
31 5.0
Furamidine 1 was used as a positive control,26 and its IC50 value is
expressed as mean SD from at least three independent experiments.
a
and thioxothiazolidinone 22.39,40 It must be pointed out that
the medicinal chemistry value of these scaffolds is a subject of
recent controversies. Many characterize these compounds as
promiscuous frequent hitters that tend to produce pan-assay
interference in a HTS setting.41 Others consider them
C
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a
Table 2. Impact of the Head Domain on Tdp1 Inhibition
compound
Tdp1 IC50 (μM)
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
74
>333
>333
111−333
13 3.0
>333
111−333
111−333
111−333
>111
Figure 4. Design of heterocyclic arylidene scaffolds 19−22 based on
lead compound 8 and PTP inhibitors 18.
>333
privileged scaffolds42 with an extraordinary ability to engage
with important biological targets as evidenced by the clinical
thiazolidinedione (21, aka glitazones) antidiabetic drugs.43−47
Recent systematic studies by Klein and co-workers found no
aggregation-based promiscuity or unspecific reactivity toward
protein,48 arguing against the exclusion of these scaffolds in
drug discovery efforts.
Synthetically, these scaffolds can be easily accessed through
an aldol condensation between a heterocyclic ring (27−30) and
an aromatic aldehyde (Scheme 1). The preparation of
>111
2.6 0.4
16 8.8
27 3.0
12 2.2
a
IC50 values are expressed as mean
independent experiments.
SD from at least three
thioxothiazolidinone scaffold (compounds 43−46) consistently
demonstrated significant potency. Of particular interest are the
3,4-dihydroxyl analogue 43 and the carboxylate analogue 44,
which inhibited Tdp1 at low micromolar concentrations (Table
2).
a
Scheme 1. Synthesis of Scaffolds 19−22
Having identified thioxothiazolidinone as the optimal
substructure for the head domain, we then conducted a SAR
on the aromatic domain. Toward this end, analogues with
variable aromatic ring or substitution patterns were prepared
based on the chemistry described in Scheme 1 (Table 3).
Analogues with two or three phenolic hydroxyl groups (43,
47−50, Table 3) were by far the best inhibitors of the series,
with 50 being a submicromolar Tdp1 inhibitor (Figure 5A and
B). When only one free hydroxyl group was present
(compounds 51−55, Table 3, Figure 5A and B), a substantial
drop in inhibitory activity was observed, suggesting that these
free hydroxyl groups may provide key interactions with the
enzyme possibly through hydrogen bonding.
The polyhydroxylated phenyl moiety, particularly the
pyrogallol (1,2,3-trihydroxyphenyl) functionality in compound
50, may potentially engage in excessive nonspecific protein
binding. To assess the protein binding property of this
functionality, 50 was tested against recombinant Tdp1 in
WCE gel-based assay buffer containing bovine serum albumin
(BSA, see Experimental Section) and exhibited an IC50 of 2.8
μM. Knowing that BSA does not impact Tdp1 activity (Huang,
S.H. and Pommier, Y., unpublished data), the 3-fold loss of
potency may reflect nonspecific BSA binding, albeit to the
extent typical of small molecule inhibitors. We have also tested
50 against recombinant Tdp2 in in a gel-based assay and
observed an IC50 of 17 μM, a 20-fold increase when compared
to that against Tdp1 (IC50 = 0.87 μM). These studies strongly
suggest that 50 is indeed a selective inhibitor of Tdp1. Efforts in
bioisosteric replacement51 of catechol and pyrogallol moieties
are currently underway, which could lead to more potent
inhibitors with improved physicochemical properties.
a
Reagents and conditions: (a) EtOAc, reflux, 48 h, 92%; (b) EtOH,
piperidine, reflux, 16 h, 41−78%; (c) 140 °C, 4 h, 72%; (d) Lawesson’s
reagent, toluene, reflux, 2 h, 98%; (e) NaOAc, AcOH, reflux, 2 h, 58−
90%.
thiohydantoin intermediates 27 and 28 featured a reaction
between a glycine derivative (23 and 25) and a thioisocyanate
24 or thiocyanate 26. The thioxothiazolidinone intermediate 30
was synthesized from commercial thiazolidine-2,4-dione 29
using Lawesson’s reagent. The key aldo condensation was
performed under thermodynamic conditions to yield the more
stable Z-isomers.49 The assignment of this stereochemistry is
consistent with literature reports.49−52
Four analogues of each scaffold were prepared and tested
against Tdp1. The assay results are summarized in Table 2.
Notably, with the exception of 35, which inhibited Tdp1 with
an IC50 value of 13 μM, analogues of the first three heterocyclic
scaffolds were essentially inactive against Tdp1. By contrast, the
The carboxylic acid analogues also demonstrated good
potency, which was not affected by the position of the
substitution (44, 56−57, Table 3). However, derivatization of
the acid appeared to improve the activity, as the two sodium
salts (58 and 59) and methyl ester 60 were all found to be
D
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a
Table 3. Aromatic Domain SAR of Thioxothiazolidinone Scaffold 22
a
IC50 values are expressed as mean SD from at least three independent experiments.
approximately twice as potent as the acid analogues. Other
substitutions, including halogens (45 and 62), nitro (63), and
cyano (64), all led to decreased inhibitory activity. In addition,
replacing the benzene ring with other heterocycles, such as
pyridines (65 and 66), thiophene (67), benzothiophene (68),
and indoles (46 and 69) also yielded less potent compounds.
Finally, bioisosteric replacement53 of the carboxylic acid was
explored, and analogues with tetrazole (70 and 71), sulfonic
acid (72), phosphonate (73), and phosphonic acid (74) were
prepared. The biochemical assay identified compound 74 as a
E
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Figure 5. (A) Representative gel demonstrating concentration-dependent inhibition of Tdp1 by compounds 48, 50, and 51. (C) Concentration−
response curves. Each point represents the mean value standard deviation of three independent experiments.
potent inhibitor (IC50 = 2.2 μM) with a 7.3-fold improvement
in potency over 44, demonstrating that the phosphonic acid
functional group is a viable bioisostere of the carboxylic acid.
To further assess the ability of our compounds to inhibit
human Tdp1 in a more complex pseudocellular environment,
we performed secondary gel-based assays using the same N14Y
DNA substrate while replacing recombinant Tdp1 by whole cell
extracts (WCEs) as a source of endogenous Tdp1. For this
purpose, the chicken Tdp1 gene in DT40 cells was knocked out
and recomplemented with the human Tdp1 gene.15 Lysates
were collected and used in place of recombinant Tdp1 in the
gel assays.15 Compounds with a single digit micromolar IC50
from the recombinant Tdp1 assay were tested against WCE,
and the results are shown in Table 4. Given the increased
stringency of this assay, the observation that these compounds
are generally active at micromolar concentrations in the WCE
assay strongly corroborates the earlier biochemical results and
confirms their inhibitory activities against endogenous Tdp1 in
a pseudocellular environment. The confirmed inhibitory activity
of this series of compounds, the SAR pattern and the
concentration-dependent inhibition fashion, suggest that these
controversial scaffolds can be valuable leads for inhibiting Tdp1.
Binding Analysis. The binding of our inhibitors to Tdp1
was directly measured using the Surface Plasmon Resonance
(SPR) technology. In this testing, Tdp1 was amine coupled to a
CM5 sensor chip, and a dilution series of the representative
inhibitor 50 was run through the surface. A surface without
coupled Tdp1 was used as a reference. As shown in Figure 6, a
concentration-dependent binding of 50 on Tdp1 was observed
(A). Similarly, a 14-base oligonucleotide modified with a biotin
at the 5′ end and a tyrosine at the 3′ end (biotin-
GATCTAAAAGACTT-Tyr) was immobilized onto the chip
surface and tested against the same dilution series of 50 (Figure
6, B). No binding was observed between 50 and the N14Y
oligomer, confirming the selective binding of our inhibitors to
Tdp1 under assay conditions.
Modeling and Docking. Close examination of the active
site identified three specific regions important for substrate
binding (Figure 7A)the hydrophobic (Y204, P461, W590),
the catalytic (K265, H263, N283, H493, K495, N516), and an
adjacent hydrophilic region (S400, S518, S536, E538). Both the
catalytic and hydrophilic regions consist of positively charged
and hydrophilic residues for the stabilization of the negatively
charged phosphate backbone of the DNA (Figure 7A). The
hydrophobic region is located at the top of the substrate
channel that provides essential interactions to both the tyrosyl
and the nucleoside group of the substrate. A docking study of
the previously identified Tdp1 inhibitors (1−3) showed
interactions to at least two of the three regions are required
for binding (Figure 1S, Supporting Information). Docking of
our first identified compounds 8 and 9 showed both inhibitors
bind to the “floor” of the substrate channel at the catalytic and
F
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Table 4. Assay Results with the DT40 WCE
Figure 6. Binding of 50 to Tdp1 was examined using SPR spectroscopy. A concentration series of 50 (50, 25, 6.25, 3.12, 1.56. 0.78, and 0.39 μM)
were injected over immobilized Tdp1 (A) or a tyrosyl-labeled oligonucleotide (GATCTAAAAGACTT-Tyr) (B). The theoretical maximal binding
response to Tdp1 was 45 RU and to the oligonucleotide was 140 RU.
the hydrophilic regions (see 9 in Figure 7B). The essential
interactions involved significant hydrogen bonding between the
thiocarbonyl and the catalytic histidine and lysine residues as
well as hydrogen bonding between the catechol and the
hydrophilic residues E538 and S536 (Figure 7B). The benzyl
group of 9 was easily accommodated within the tyrosyl binding
G
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Figure 7. Modeled structure of Human Tdp1 (PDB: 1NOP)25 with (A) Top1−DNA substrate, (B) 9, (C) 31, and (D) 50 docked within the active
site. The model shows three specific regions important for substrate bindingthe hydrophobic (Y204, P461, W590, labeled in white), the catalytic
(K265, H263, N283, H493, K495, N516, labeled in blue), and an adjacent hydrophilic region (S400, S518, S536, E538, labeled in red). Effective
binding of the hydrolytic tyrosyl−nucleosidic group of the Top1−DNA substrate involves the hydrophobic and the catalytic regions (boxed in
yellow).
site, which supports the observed similar activities between 8
and 9. Replacement of the catechol group with halobenzene or
a large aromatic group significantly disrupts this observed mode
of binding as the hydrophilic region energetically disfavors such
substitution. For the heterocyclic head domain, placement of
the catechol group directly adjacent to the thiocarbonyl is
essential for maintaining the observed mode of binding.
Placement of the thiocarbonyl at the 1,3 position to the
catechol results in the thiocarbonyl group pointing slightly away
from the catalytic lysine and histidine residues toward N283,
which results in a weaker activity observed with 31 (Figure 7C).
Addition of a benzyl group at the NH position of the
heterocyclic ring (35−38) is prohibited as it leads to steric
clashes with the interfacial region between the catalytic and
hydrophilic sites. One advantage observed for the heterocyclic
head is the ability to interact with P461 and Y204 within the
hydrophobic region as observed in the increase in activity from
12 μM of 8 to 2.6 μM of 43. The ability to interact with all
three regions enhances affinity to retain the observed mode of
binding and enables SAR modification of the catechol group
which would otherwise lead to significant loss of activities (10−
12, 14−16). As observed in Table 3, replacement of the
catechol by heteroaromatic groups and halobenzene after
heterocyclization is tolerable within the hydrophilic region with
only a 10-fold loss of activity. The replacement of the hydroxyl
groups of catechol by negatively charged groups such as
carboxylate, tetrazole, sulfonate, and phosphonate within the
hydrophilic site leads to a slight shift in mode of binding,
similar to that observed in 31 (Figure 7C). The observed effect
depends significantly on the size, the electronegativity of the
group, and the position of the aromatic substitution. Finally,
addition of a third hydroxyl group to catechol (50) enables the
formation of an additional hydrogen bond with E538 within the
hydrophilic site that correlates directly with the improved
activity of 0.87 μM (Figure 7D).
CONCLUSIONS
■
On the basis of the initial lead compound 8 identified from
screening a small focused library against Tdp1, we have
discovered 5-arylidene thioxothiazolidinone as a novel scaffold
for low micromolar Tdp1 inhibitors. SAR studies revealed that
analogues with multiple hydroxyl groups or a carboxylic acid
derivative on the terminal phenyl ring consistently inhibited
Tdp1 in the biochemical assay. Among these, compound 50
was identified as a submicromolar inhibitor. A modeling study
identified the most likely mode of inhibition that corroborates
the observed SAR. In addition, bioisosteric replacement of the
carboxylic acid also led to the discovery of phosphonic acid 74
as a potent Tdp1 inhibitor. Results from the more stringent
secondary WCE gel-based assay confirm the observed
inhibitory activities of these compounds.
EXPERIMENTAL SECTION
■
Chemistry. General Procedures. All commercial chemicals were
used as supplied unless otherwise indicated. Dry solvents (THF, Et2O,
CH2Cl2, and DMF) were dispensed under argon from an anhydrous
solvent system with two packed columns of neutral alumina or
molecular sieves. Flash chromatography was performed on a Teledyne
Combiflash RF-200 with RediSep columns (silica) and indicated
mobile phase. All reactions were performed under an inert atmosphere
of ultrapure argon with oven-dried glassware. 1H and 13C NMR
spectra were recorded on a Varian 600 MHz or a Varian 400 MHz
spectrometer. Mass data were acquired on an Agilent TOF II TOS/
MS spectrometer capable of ESI and APCI ion sources. Analysis of
sample purity was performed on a Varian Prepstar SD-1 HPLC system
with a Varian Microsorb-MW 100-5 C18 column (250 mm × 4.6
mm). HPLC conditions: solvent A = H2O, solvent B = MeCN; flow
rate =1.0 mL/min; compounds were eluted with a gradient of 5%
H
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MeCN/H2O to 80% MeCN for 40 min. Purity was determined by
total absorbance at 254 nm. All tested compounds have a purity ≥98%.
Compounds 4−9 were purchased from Axxora along with other
tyrphostins as a small focused library.
General Procedure a for the Aldol Condensation. To a mixture of
an aromatic aldehyde (2.0/mmol, 1 equiv) and cyanothioacetamide
(2.0 mmol, 1 equiv) in ethanol (5 mL) was added one drop of N-
methylmorpholine, and the resulting mixture was stirred at 35 °C for
30−60 min. The reaction mixture was evaporated to give the crude
product. The obtained solid was further purified by column
chromatography (SiO2, EtOAc/hexane).
(E)-2-Cyano-3-(4-fluorophenyl)prop-2-enethioamide (10). Pre-
pared from 4-fluorobenzaldehyde following the general procedure A.
Yield 62%; mp 155−158 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.10
(s, 1H), 9.61 (s, 1H), 8.06 (s, 1H), 8.06−7.98 (m, 2H), 7.43−7.41 (m,
2H); 13C NMR (150 MHz, DMSO-d6) δ 192.9, 165.4, 146.2, 133.5,
129.2, 117.3, 112.8, 101.2; HRMS-ESI(−) m/z calcd for C10H6FN2S
205.0236 [M − H]−, found 205.0214.
(E)-2-Cyano-3-(4-nitrophenyl)prop-2-enethioamide (11). Pre-
pared from 4-nitrobenzaldehyde following the general procedure A.
Yield 58%; mp 171−173 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.24−
8.26 (m, 3H), 7.81 (d, J = 7.2 Hz, 2H), 7.63 (br, 2H); 13C NMR (150
MHz, DMSO-d6) δ 194.6, 148.4, 148.0, 132.3, 131.9, 124.3, 123.7,
118.9; HRMS-ESI(−) m/z calcd for C10H6N3O2S 232.0259 [M −
H]−, found 232.0181.
crystallization afforded 27 (3.5 g, 18 mmol, 92%) as a brown solid. 1H
NMR (600 MHz, CDCl3) δ 7.53−7.46 (m, 3H), 7.33−7.32 (m, 2H),
4.23 (s, 2H).
Synthesis of 1-Benzyl-2-thiohydantoin (28). A suspension of N-
benzyl glycinethylester (3.86 g, 20 mmol) and ammonium thiocyanate
(4.56 g, 60 mmol) was heated to 40 °C. A clear orange solution was
observed which turned into dark red. After refluxing the reaction
mixture for 4 h, the solution was immediately poured into 40 mL of
(EtOH:H2O). The crystalline white solid was collected by suction
filtration, washed with 100 mL of (EtOH:H2O), followed by diethyl
ether (20 mL), and dried under vacuum to afford 28 (2.8 g, 13.7
mmol, 70%) as a pale yellow solid. 1H NMR (600 MHz, DMSO-d6) δ
11.84 (s, 1H), 7.27−7.36 (m, 5H), 4.85 (s, 2H), 4.07 (s, 2H).
4-Thioxo-thiazolidin-2-one (30).55 To a solution of compound 29
(0.61 g, 5.21 mmol) in dry toluene (12 mL) was added Lawesson’s
reagent (2.2 g, 5.2 mmol). The reaction mixture was refluxed for 2 h
and cooled to room temperature. The solid formed was filtered off and
crystallized from acetone to afford 0.64 g (98%) of 30 as yellow
1
crystals. H (600 MHz, DMSO-d6) δ 13.58 (s, 1H), 4.67 (s, 2H).
General Procedure B for Aldol Condensation.56 An aromatic
aldehyde (0.55 mmol, 1 equiv) and appropriate heterocyclic ring (27−
29) (0.73 mmol, 1.3 equiv) were dissolved in ethanol (14 mL) which
was followed by the addition of piperidine (90 μL). The reaction
mixture was refluxed for 6−14 h (monitored by TLC). The ethanol
was evaporated, and the residue was dissolved in ethylacetate (100
mL) and washed with water (50 mL) and brine (50 mL). The organic
layer was dried over Na2SO4, filtered, and concentrated to give the
crude product. The obtained solid was further purified by
crystallization from ethanol or by column chromatography to yield
the desired compound.
(E)-3-(4-Bromophenyl)-2-cyanoprop-2-enethioamide (12). Pre-
pared from 4-bromobenzaldehyde following the general procedure
1
A. Yield 59%; mp 176−179 °C; H NMR (600 MHz, DMSO-d6) δ
10.13 (s, 1H), 9.64 (s, 1H), 8.01 (s, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.77
(d, J = 8.4 Hz, 2H); 13C NMR (150 MHz, DMSO-d6) δ 192.7, 146.0,
133.0, 132.5, 131.8, 126.4, 116.7, 113.8; HRMS-ESI(−) m/z calcd for
C10H6BrN2S 264.9435 [M − H]−, found 264.9444.
(Z)-5-(3,4-Dihydroxybenzylidene)-3-phenyl-2-thioxoimidazolidin-
4-one (31). Prepared from 3,4-dihydroxybenzaldehyde and compound
27 (0.14 g, 0.73 mmol, 1.3 equiv) following the general procedure B.
Yield 51%; mp 214−220 °C; 1H NMR (600 MHz, DMSO-d6) δ 7.48−
7.45 (m, 2H), 7.41−7.37 (m, 2H), 7.32 (d, J = 7.8 Hz, 2H), 7.14−7.13
(m, 1H), 6.76 (d, J = 8.4 Hz, 1H), 6.54 (s, 1H); 13C NMR (150 MHz,
DMSO-d6) δ 178.7, 169.8, 146.0, 145.4, 139.9, 137.1, 129.0, 128.2,
126.7, 123.3, 117.4, 111.6; HRMS-ESI(−) m/z calcd for C16H11N2O3S
311.0490 [M − H]−, found 311.0505.
(Z)-4-((5-Oxo-1-phenyl-2-thioxoimidazolidin-4-ylidene)methyl)-
benzoic Acid (32). Prepared from 4-formylbenzoic acid and
compound 27 (0.14 g, 0.73 mmol, 1.3 equiv) following the general
procedure B. Yield 78%; mp > 300 °C; 1H NMR (600 MHz, DMSO-
d6) δ 13.07 (s, 1H), 12.70 (s, 1H), 7.96 (d, J = 8.4 Hz, 2H), 7.89 (d, J
= 8.4 Hz, 2H), 7.52−7.46 (m, 3H), 7.38 (d, J = 8.4 Hz, 2H), 6.70 (s,
1H); 13C NMR (150 MHz, DMSO-d6) δ 179.6, 167.3, 164.4, 137.2,
133.7, 131.1, 130.6, 129.9, 129.3, 129.2, 111.3; HRMS-ESI(−) m/z
calcd for C16H11N2O3S 323.0490 [M − H]−, found 323.0490.
(Z)-5-(4-Fluorobenzylidene)-3-phenyl-2-thioxoimidazolidin-4-
one (33). Prepared from 4-fluorobenzaldehyde and compound 27
(0.14 g, 0.73 mmol, 1.3 equiv) following the general procedure B.
Yield: 74%. 1H NMR (600 MHz, DMSO-d6) δ 12.69 (s, 1H), 7.98 (m,
2H), 7.61 (m, 2H), 7.37−7.56 (m, 5H), 6.78 (s, 1H).
(Z)-5-((1H-Indol-5-yl)methylene)-3-phenyl-2-thioxoimidazolidin-
4-one (34). Prepared from 1H-indole-5-carbaldehyde and compound
27 (0.14 g, 0.73 mmol, 1.3 equiv) following the general procedure B.
Yield 58%; mp 254−259 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.25
(s, 1H), 11.33 (s, 1H), 8.14 (s, 1H), 7.54−7.36 (m, 8 H), 6.80 (s, 1H),
6.50 (s, 1H); 13C NMR (150 MHz, DMSO-d6) δ 178.1, 164.4, 137.0,
133.9, 129.3,129.2, 129.0, 128.6, 127.3, 124.7, 124.1, 123.9, 123.7,
116.8, 112.3, 102.5; HRMS-ESI(−) m/z calcd for C18H12N3OS
318.0701 [M − H]−, found 318.0708.
(Z)-1-Benzyl-5-(3,4-dihydroxybenzylidene)-2-thioxoimidazolidin-
4-one (35). Prepared from 3,4-dihydroxybenzaldehyde and compound
28 (0.15 g, 0.73 mmol, 1.3 equiv) following the general procedure B.
Yield 63%; mp 252−255 °C; 1H NMR (600 MHz, CD3OD) δ 7.64 (s,
1H), 7.26−7.22 (m, 4H), 7.18−7.13 (m, 2H), 6.60 (d, J = 8.4 Hz,
1H), 6.27 (s, 1H), 5.28 (s, 2H); 13C NMR (150 MHz, CD3OD) δ
175.9, 162.8, 147.9, 144.4, 135.6, 128.4, 127.2, 126.7, 125.0, 124.3,
(E)-4-(3-Amino-2-cyano-3-thioxoprop-1-en-1-yl)benzoic Acid
(13). Prepared from 4-formylbenzoic acid following the general
1
procedure A. Yield 63%; mp >300 °C; H NMR (600 MHz, DMSO-
d6) δ 13.12 (s, 1H), 10.06 (s, 1H), 9.51 (s, 1H), 8.18 (s, 1H), 7.98 (d, J
= 8.4 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H); 13C NMR (150 MHz,
DMSO-d6) δ 192.2, 167.3, 161.9, 151.8, 140.1, 138.0, 132.6, 130.2,
128.5, 115.3; HRMS-ESI(−) m/z calcd for C11H7N2O2S 231.0228 [M
− H]−, found 231.0257.
(E)-N-(4-(3-Amino-2-cyano-3-thioxoprop-1-en-1-yl)phenyl)-
acetamide (14). Prepared from N-(4-formylphenyl)acetamide follow-
1
ing the general procedure A. Yield 74%; mp 231−236 °C; H NMR
(600 MHz, DMSO-d6) δ 10.36 (s, 1H), 9.98 (s, 1H), 9.48 (s, 1H),
8.01 (s, 1H), 7.92 (d, J = 8.9 Hz, 2H), 7.73 (d, J = 8.9 Hz, 2H), 2.07
(s, 3H); 13C NMR (150 MHz, DMSO-d6) δ 193.2, 169.7, 147.4, 143.7,
132.4, 126.8, 119.5, 117.4, 110.4, 24.8; HRMS-ESI(+) m/z calcd for
C12H12N3SO 246.0701 [M + H]+, found 246.0705.
(E)-2-Cyano-3-(1H-indol-5-yl)prop-2-enethioamide (15). Prepared
from 1H-indole-5-carbaldehyde following the general procedure A.
Yield 70%; mp 189−191 °C; 1H NMR (600 MHz, DMSO-d6) δ 11.58
(s, 1H), 9.90 (s, 1H), 9.41 (s, 1H), 8.24 (d, J = 10.2 Hz, 2H), 7.81 (d, J
= 8.4 Hz 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.48−7.47 (m, 1H), 6.59 (s,
1H); 13C NMR (150 MHz, DMSO-d6) δ 193.7, 150.6, 138.8, 128.6,
128.3, 125.8, 123.9, 123.4, 118.1, 113.1, 108.3, 103.5; HRMS-ESI(+)
m/z calcd for C12H10N3S 228.0595 [M + H]+, found 228.0581.
(E)-2-Cyano-3-(naphthalen-2-yl)prop-2-enethioamide (16). Pre-
pared from 2-naphthaldehyde following the general procedure A. Yield
1
67%; mp 141−144 °C; H NMR (600 MHz, DMSO-d6) δ 10.13 (s,
1H), 9.66 (s, 1H), 8.44 (s, 1H), 8.23 (s, 1H), 8.09−8.05 (m, 2H), 8.02
(d, J = 7.8 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.66 (t, J = 7.2 Hz, 1H),
7.61 (t, J = 7.2 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 193.1,
147.6, 134.9, 133.1, 130.2, 129.7, 129.5, 129.3, 128.5, 127.9, 125.5,
117.1, 113.1; HRMS-ESI(+) m/z calcd for C14H11N2S 239.0643 [M +
H]+, found 239.0650.
3-Phenyl-2-thioxo-imidazolidin-4-one (27).54 A suspension of
methylglycinate hydrochloride (2.5 g, 20 mmol), phenylisothiocyanate
(2.7 g, 20 mmol), and triethylamine (2.02 g, 2.8 mL, 20 mmol) in dry
ethylacetate was refluxed (80 °C) for 2 days. Solvent was evaporated
under reduced pressure. The residue was dissolved in EtOH, and
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123.3, 117.4, 114.5, 45.7; HRMS-ESI(−) m/z calcd for C17H13N2O3S
325.0647 [M − H]−, found 325.0637.
procedure C. Yield 63%; mp 210−214 °C; 1H NMR (600 MHz,
DMSO-d6) δ 13.62 (s, 1H), 7.92 (s, 1H), 7.05−7.03 (m, 2H), 6.86 (d,
J = 8.4 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 195.2, 171.1,
150.1, 146.5, 137.9, 125.9, 125.3, 117.0, 116.9; HRMS-ESI(−) m/z
calcd for C10H6NO3S2 251.9789 [M − H]−, found 251.9803.
(Z)-4-((2-Oxo-4-thioxothiazolidin-5-ylidene)methyl)benzoic Acid
(44). Prepared from 4-formylbenzoic acid following the general
procedure C. Yield 67%; mp > 300 °C; 1H NMR (600 MHz, DMSO-
d6) δ 13.22 (s, 1H), 8.08 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.75 (d, J =
8.4 Hz, 2H); 13C NMR (150 MHz, DMSO-d6) δ 195.8, 170.8, 166.9,
137.6, 134.6, 132.4, 132.1, 130.8, 130.5; HRMS-ESI(−) m/z calcd for
C11H6NO3S2 263.9789 [M − H]−, found 263.9808.
(Z)-5-(4-Fluorobenzylidene)-4-thioxothiazolidin-2-one (45). Pre-
pared from 4-fluorobenzaldehyde following the general procedure C.
Yield 53%; mp 192−194 °C; 1H NMR (600 MHz, CD3OD) δ 8.01 (s,
1H), 7.57−7.55 (m, 2H), 7.16−7.13 (m, 2H); 13C NMR (150 MHz,
CD3OD) δ 195.5, 170.2, 164.5, 162.8, 134.3, 132.5, 132.4, 130.4,
116.1, 115.9; HRMS-ESI(−) m/z calcd for C12H7N2OS2 237.9797 [M
− H]−, found 237.9827.
(Z)-5-((1H-Indol-5-yl)methylene)-4-thioxothiazolidin-2-one (46).
Prepared from 1H-indole-5-carbaldehyde following the general
procedure C. Yield 49%; mp 192−194 °C; 1H NMR (600 MHz,
CD3OD) δ 8.24 (s, 1H), 7.81 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.41
(d, J = 8.4 Hz, 1H), 7.24 (d, J = 3.1 Hz, 1H), 6.49 (d, J = 3.1 Hz, 1H);
13C NMR (150 MHz, CD3OD) δ 196.2, 172.8, 139.2, 137.6, 128.7,
126.5, 125.8, 125.2, 124.5, 123.7, 111.8, 102.4; HRMS-ESI(−) m/z
calcd for C12H7N2OS2 259.0000 [M − H]−, found 259.0007.
(Z)-5-(2,3-Dihydroxybenzylidene)-4-thioxothiazolidin-2-one (47).
Prepared from 2,3-dihydroxybenzaldehyde following the general
procedure C. Yield 69%; mp 188−192 °C; 1H NMR (600 MHz,
DMSO-d6) δ 12.89 (s, 1H), 9.99 (s, 1H), 9.76 (s, 1H), 8.49 (s, 1H),
6.92 (d, J = 6.6 Hz, 1H), 6.88 (d, J = 6.6 Hz, 1H), 6.76 (t, J = 8.4 Hz,
1H); 13C NMR (150 MHz, DMSO-d6) δ 195.8, 171.7, 161.6, 147.6,
146.3, 132.7, 128.5, 121.7, 120.1, 118.6; HRMS-ESI(−) m/z calcd for
C10H6NO3S2 251.9789 [M − H]−, found 251.9763.
(Z)-4-((3-Benzyl-5-oxo-2-thioxoimidazolidin-4-ylidene)methyl)-
benzoic acid (36). Prepared from 4-formylbenzoic acid and
compound 28 (0.15 g, 0.73 mmol, 1.3 equiv) following the general
procedure B. Yield 64%; mp 209−211 °C; 1H NMR (600 MHz,
DMSO-d6) δ 7.91 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.33−
7.32 (m, 5H), 6.49 (s, 1H), 5.35 (s, 2H); 13C NMR (150 MHz,
DMSO-d6) δ 169.6, 165.9, 137.7, 136.4, 134.7, 130.4, 130.2, 129.0,
127.7, 127.4, 118.6, 43.7; HRMS-ESI(−) m/z calcd for C18H13N2O3S
337.0647 [M − H]−, found 337.0624.
(Z)-1-Benzyl-5-(4-fluorobenzylidene)-2-thioxoimidazolidin-4-one
(37). Prepared from 4-fluorobenzaldehyde and compound 28 (0.15 g,
0.73 mmol, 1.3 equiv) following the general procedure B. Yield 47%;
1
mp 135−138 °C; H NMR (600 MHz, DMSO-d6) δ 12.62 (s, 1H),
8.02 (m, 2H), 7.24−7.34 (m, 5H), 7.20 (m, 2H), 6.64 (s, 1H), 5.32 (s,
2H); 13C NMR (150 MHz, DMSO-d6) δ 177.1, 163.2, 162.1, 135.8,
133.6, 133.5, 129.1, 128.8, 128.5, 127.9, 127.4, 119.8, 115.6, 45.7;
HRMS-ESI(−) m/z calcd for C16H11N2O3S 311.0654 [M − H]−,
found 311.0664.
(Z)-5-((1H-Indol-5-yl)methylene)-1-benzyl-2-thioxoimidazolidin-
4-one (38). Prepared from 1H-indole-5-carbaldehyde and compound
28 (0.15 g, 0.73 mmol, 1.3 equiv) following the general procedure B.
Yield 41%; 1H NMR (600 MHz, DMSO-d6) δ 12.49 (s, 1H), 12.29 (s,
1H), 8.32 (s, 1H), 7.83 (d, 1H), 7.23−7.36 (m, 7H), 6.75 (s, 1H), 6.44
(s, 1H), 5.40 (s, 2H).
(Z)-5-(3,4-Dihydroxybenzylidene)thiazolidine-2,4-dione (39). Pre-
pared from 3,4-dihydroxybenzaldehyde and commercially available
2,4-thiazolidinedione 29 (0.15 g, 1.3 mmol, 1.3 equiv) following the
1
general procedure B. Yield 65%; mp > 300 °C; H NMR (600 MHz,
DMSO-d6) δ 9.47 (br, 2H), 7.37 (s, 1H), 6.97 (s, 1H), 6.87 (d, J = 7.8
Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ
174.6, 171.9, 148.0, 146.1, 129.3, 125.8, 124.5, 123.4, 116.7, 116.5;
HRMS-ESI(−) m/z calcd for C10H6NO4S 236.0018 [M − H]−, found
236.0016.
(Z)-4-((2,4-Dioxothiazolidin-5-ylidene)methyl)benzoic Acid (40).
Prepared from 4-formylbenzoic acid and commercially available 2,4-
thiazolidinedione 29 (0.15 g, 1.3 mmol, 1.3 equiv) following the
(Z)-5-(2,4-Dihydroxybenzylidene)-4-thioxothiazolidin-2-one (48).
Prepared from 2,4-dihydroxybenzaldehyde following the general
procedure C. Yield 62%; mp 238−241 °C; 1H NMR (600 MHz,
CD3OD) δ 8.53 (s, 1H), 7.24 (d, J = 8.4 Hz, 1H), 6.31 (d, J = 8.4 Hz,
1H), 6.26 (s, 1H); 13C NMR (150 MHz, CD3OD) δ 194.3, 170.4,
161.7, 159.9, 132.1, 129.1, 112.8, 107.1, 101.2; HRMS-ESI(−) m/z
calcd for C10H6NO3S2 251.9789 [M − H]−, found 251.9776.
(Z)-5-(2,5-Dihydroxybenzylidene)-4-thioxothiazolidin-2-one (49).
Prepared from 2,5-dihydroxybenzaldehyde following the general
procedure C. Yield 62%; mp 226−229 °C; 1H NMR (600 MHz,
DMSO-d6) δ 13.7 (s, 1H), 9.49 (s, 1H), 9.18 (s, 1H), 8.41 (s, 1H),
6.82−6.80 (m, 3H); 13C NMR (150 MHz, DMSO-d6) δ 195.7, 175.1,
152.2, 150.6, 132.3, 128.0, 121.4, 121.0, 117.6, 113.2; HRMS-ESI(−)
m/z calcd for C10H6NO3S2 251.9789 [M − H]−, found 251.9781.
(Z)-4-Thioxo-5-(2,3,4-trihydroxybenzylidene)thiazolidin-2-one
(50). Prepared from 2,3,4-trihydroxybenzaldehyde following the
1
general procedure B. Yield 63%; mp > 300 °C; H NMR (600 MHz,
DMSO-d6) δ 13.17 (s, 1H), 12.68 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H),
7.80 (s, 1H), 7.67 (d, J = 8.4 Hz, 2H); 13C NMR (150 MHz, DMSO-
d6) δ 168.1, 167.6, 167.0, 137.4, 132.1, 130.9, 130.8, 130.7, 130.3,
126.4; HRMS-ESI(−) m/z calcd for C11H6NO4S 248.0018 [M − H]−,
found 247.9998.
(Z)-5-(4-Fluorobenzylidene)thiazolidine-2,4-dione (41). Prepared
from 4-fluorobenzaldehyde and commercially available 2,4-thiazolidi-
nedione 29 (0.15 g, 1.3 mmol, 1.3 equiv) following the general
1
procedure B. Yield 78%; mp > 300 °C; H (600 MHz, DMSO-d6) δ
7.55−7.54 (m, 2H), 7.28 (s, 1H), 7.23−7.26 (m, 2H); 13C NMR (150
MHz, DMSO-d6) δ 181.8, 175.2, 162.7, 161.1, 135.2, 132.6, 131.5,
122.4, 116.3, 116.1; HRMS-ESI(−) m/z calcd for C10H5NO2SF
222.0025 [M − H]−, found 222.0026.
1
general procedure C. Yield 60%; mp 227−231 °C; H NMR (600
(Z)-5-((1H-Indol-5-yl)methylene)thiazolidine-2,4-dione (42). Pre-
pared from 1H-indole-5-carbaldehyde and commercially available 2,4-
thiazolidinedione 29 (0.15 g, 1.3 mmol, 1.3 equiv) following the
general procedure B. Yield 67%; mp > 300 °C; 1H (600 MHz, DMSO-
d6) δ 12.42 (s, 1H), 11.41 (s, 1H), 7.87 (s, 1H), 7.82 (s, 1H), 7.51 (d, J
= 8.4 Hz, 1H), 7.42 (s, 1H), 7.31 (d, J = 8.4 Hz, 1H); 13C NMR (150
MHz, DMSO-d6) δ 168.7, 167.9, 137.3, 134.8, 134.7, 128.6, 127.9,
127.7, 124.4, 124.2, 123.4, 119.2; HRMS-ESI(−) m/z calcd for
C12H7N2O2S 243.0228 [M − H]−, found 243.0232.
MHz, CD3OD) δ 8.54 (s, 1H), 6.81 (d, J = 8.4 Hz, 1H), 6.36 (d, J =
8.4 Hz, 1H); 13C NMR (150 MHz, CD3OD) δ 195.1, 171.4, 149.8,
148.6, 133.2, 132.9, 124.9, 119.9, 114.3, 107.5; HRMS-ESI(−) m/z
calcd for C10H6NO4S2 267.9738 [M − H]−, found 267.9759.
(Z)-5-(4-Hydroxybenzylidene)-4-thioxothiazolidin-2-one (51).
Prepared from 4-hydroxybenzaldehyde following the general proce-
dure C. Yield 69%; mp 241−244 °C; 1H NMR (600 MHz, DMSO-d6)
δ 13.66 (s, 1H), 10.50 (s, 1H), 8.03 (s, 1H), 7.54 (d, J = 8.9 Hz, 2H),
6.92 (d, J = 8.9 Hz, 2H); 13C NMR (150 MHz, DMSO-d6) δ 195.3,
171.1, 161.2, 137.5, 137.4, 133.8, 133.7, 126.2, 124.9, 117.2; HRMS-
ESI(−) m/z calcd for C10H6NO2S2 235.9840 [M − H]−, found
235.9858.
(Z)-5-(3-Hydroxybenzylidene)-4-thioxothiazolidin-2-one (52).
Prepared from 3-hydroxybenzaldehyde following the general proce-
dure C. Yield 67%; mp 222−225 °C; 1H NMR (600 MHz, DMSO-d6)
δ 13.86 (s, 1H), 9.86 (s, 1H), 7.97 (s, 1H), 7.32−7.31 (m, 1H), 7.10
(d, J = 6.0 Hz, 1H), 7.02 (s, 1H), 6.91 (d, J = 7.8 Hz, 1H); 13C NMR
(150 MHz, DMSO-d6) δ 195.8, 175.0, 171.0, 158.4, 136.5, 134.9,
General Procedure C for Aldol Condensation.55 To a mixture of
aromatic aldehyde (1 equiv), 30 (1 equiv), and sodium acetate (1
equiv) was added glacial acetic acid (3 mL). The mixture was heated
under reflux for 2 h and then cooled to room temperature and poured
into cold water. The yellow solid precipitate was collected by filtration.
The obtained solid was further purified by crystallization from ethanol
or by column chromatography to yield the desired compound.
(Z)-5-(3,4-Dihydroxybenzylidene)-4-thioxothiazolidin-2-one (43).
Prepared from 3,4-dihydroxybenzaldehyde following the general
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130.9, 122.4, 118.8, 116.5; HRMS-ESI(−) m/z calcd for C10H6NO2S2
235.9840 [M − H]−, found 235.9860.
13C NMR (150 MHz, DMSO-d6) δ 195.7, 170.6, 165.8, 135.2, 134.7,
134.3, 131.3, 131.0, 130.7, 130.4, 52.5; HRMS-ESI(−) m/z calcd for
C12H8NO3S2 277.9946 [M − H]−, found 277.9951.
(Z)-5-(2-Bromobenzylidene)-4-thioxothiazolidin-2-one (62). Pre-
pared from 4-bromobenzaldehyde following the general procedure C.
Yield 40%; mp 165−168 °C; 1H NMR (600 MHz, CD3OD) δ 8.28 (s,
1H), 7.64 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 7.8
Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H); 13C NMR (150 MHz, CD3OD) δ
195.2, 179.7, 141.0, 134.2, 133.8, 133.4, 131.4, 128.7, 127.8, 125.9;
HRMS-ESI(−) m/z calcd for C10H5NBrOS2 297.8996 [M − H]−,
found 297.8992.
(Z)-5-(2-Hydroxybenzylidene)-4-thioxothiazolidin-2-one (53).
Prepared from 2-hydroxybenzaldehyde following the general proce-
dure C. Yield 63%; mp 210−212 °C; 1H NMR (600 MHz, DMSO-d6)
δ 13.73 (s, 1H), 10.62 (s, 1H), 8.45 (s, 1H), 7.40 (d, J = 7.2 Hz, 1H),
7.35−7.32 (m, 1H), 6.97−6.92 (m, 2H); 13C NMR (150 MHz,
DMSO-d6) δ 195.8, 186.0, 175.0, 171.2, 158.7, 132.2, 128.6, 126.0,
120.4; HRMS-ESI(−) m/z calcd for C10H6NO2S2 235.9840 [M −
H]−, found 235.9855.
(Z)-5-(4-Hydroxy-3-methoxybenzylidene)-4-thioxothiazolidin-2-
one (54). Prepared from 4-hydroxy-3-methoxybenzaldehyde following
the general procedure C. Yield 83%; mp 195−198 °C; 1H NMR (600
MHz, DMSO-d6) δ 13.66 (s, 1H), 10.1 (s, 1H), 8.04 (s, 1H), 7.22 (s,
1H), 7.16 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 2.47 (s, 3H);
13C NMR (150 MHz, DMSO-d6) δ 195.2, 171.0, 150.8, 148.6, 137.8,
(Z)-5-(4-Nitrobenzylidene)-4-thioxothiazolidin-2-one (63). Pre-
pared from 4-nitrobenzaldehyde following the general procedure C.
1
Yield 58%; mp 192−194 °C; H NMR (600 MHz, DMSO-d6) δ 8.32
(d, J = 8.4 Hz, 2H), 8.11 (s, 1H), 7.91 (d, J = 8.7 Hz, 2H); 13C NMR
(150 MHz, DMSO-d6) δ 195.3, 172.9, 147.9, 139.9, 131.9, 130.6,
123.8; HRMS-ESI(−) m/z calcd for C10H5N2O3S2 264.9742 [M −
H]−, found 264.9757.
126.4, 125.6, 123.3, 116.8, 115.2, 56.5; HRMS-ESI(−) m/z calcd for
C11H8NO3S2 265.9946 [M − H]−, found 265.9937.
(Z)-5-(4-Hydroxy-3,5-dimethoxybenzylidene)-4-thioxothiazoli-
din-2-one (55). Prepared from 4-hydroxy-3,5-dimethoxybenzaldehyde
(Z)-4-((2-Oxo-4-thioxothiazolidin-5-ylidene)methyl)benzonitrile
(64). Prepared from 4-formylbenzonitrile following the general
procedure C. Yield 61%; mp 178−181 °C; 1H NMR (600 MHz,
DMSO-d6) δ 8.03 (s, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.68 (d, 1H, J =
8.4 Hz, 2H); 13C NMR (150 MHz, DMSO-d6) δ 195.7, 170.6, 138.1,
133.5, 133.2, 131.2, 118.8, 112.7; HRMS-ESI(−) m/z calcd for
C11H5N2OS2 244.9843 [M − H]−, found 244.9861.
1
following the general procedure C. Yield 72%; mp 222−226 °C; H
NMR (600 MHz, DMSO-d6) δ 13.63 (s, 1H), 8.01 (s, 1H), 6.93 (s,
1H), 3.81 (s, 6H); 13C NMR (150 MHz, DMSO-d6) δ 195.0, 175.0,
171.0, 148.7, 140.1, 138.1, 126.6, 124.2, 109.3, 108.9, 56.6, 56.3;
HRMS-ESI(−) m/z calcd for C12H10NO4S2 296.0051 [M − H]−,
found 296.0069.
(Z)-5-(Pyridin-3-ylmethylene)-4-thioxothiazolidin-2-one (65).
Prepared from nicotinaldehyde following the general procedure C.
(Z)-2-((2-Oxo-4-thioxothiazolidin-5-ylidene)methyl)benzoic Acid
(56). Prepared from 2-formylbenzoic acid following the general
procedure C. Yield 58%; mp 206−209 °C; 1H NMR (600 MHz,
CD3OD) δ 8.71 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.67−7.65 (m, 1H),
7.62 (d, J = 7.2 Hz, 1H), 7.55−7.53 (m, 1H); 13C NMR (150 MHz,
CD3OD) δ 195.6, 171.2, 167.9, 137.0, 135.3, 133.1, 132.0, 131.9,
131.5, 130.7, 128.6; HRMS-ESI(−) m/z calcd for C11H6NO3S2
263.9789 [M − H]−, found 263.9808.
(Z)-3-((2-Oxo-4-thioxothiazolidin-5-ylidene)methyl)benzoic Acid
(57). Prepared from 3-formylbenzoic acid following the general
procedure C. Yield 58%; mp 242−244 °C; 1H NMR (600 MHz,
DMSO-d6) δ 8.15 (s, 1H), 8.07 (s, 1H), 8.00 (d, J = 7.2 Hz, 1H), 7.74
(d, J = 7.2 Hz, 1H), 7.52 (t, J = 8.4 Hz, 1H); 13C NMR (150 MHz,
DMSO-d6) δ 195.7, 170.7, 166.9, 135.0, 134.2, 132.3, 131.5, 131.2,
130.3, 126.7; HRMS-ESI(−) m/z calcd for C11H6NO3S2 263.9789 [M
− H]−, found 263.9807.
1
Yield 84%; mp 255−258 °C; H NMR (600 MHz, DMSO-d6) δ 8.80
(s, 1H), 8.64−8.63 (m, 1H), 8.07 (s, 1H), 8.03 (d, J = 8.4 Hz, 1H),
7.56 (dd, J = 6.0 Hz, J = 7.8 Hz, 1H); 13C NMR (150 MHz, DMSO-
d6) δ 195.7, 170.7, 152.1, 151.1, 136.7, 132.6, 132.2, 130.1, 124.7;
HRMS-ESI(−) m/z calcd for C9H5N2OS2 220.9843 [M − H]−, found
220.9863.
(Z)-5-(Pyridin-4-ylmethylene)-4-thioxothiazolidin-2-one (66).
Prepared from isonicotinaldehyde following the general procedure
1
C. Yield 67%; mp 252−254 °C; H NMR (600 MHz, DMSO-d6) δ
8.71 (d, J = 5.1 Hz, 2H), 7.97 (s, 1H), 7.59 (d, J = 5.4 Hz, 2H); 13C
NMR (150 MHz, DMSO-d6) δ 198.2, 172.4, 153.8, 153.1, 126.1,
124.4, 111.8; HRMS-ESI(−) m/z calcd for C9H5N2OS2 220.9843 [M
− H]−, found 220.9860.
(Z)-5-((5-Phenylthiophen-2-yl)methylene)-4-thioxothiazolidin-2-
one (67). Prepared from 5-phenylthiophene-2-carbaldehyde following
the general procedure C. Yield 65%; mp 250−255 °C; 1H NMR (600
MHz, DMSO-d6) δ 13.76 (s, 1H), 8.27 (s, 1H), 7.86 (d, J = 3.6 Hz,
1H), 7.78 (d, J = 7.8 Hz, 2H), 7.75 (d, J = 4.2 Hz, 1H), 7.47 (t, J = 7.8
Hz, 2H), 7.41 (d, J = 7.2 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ
194.3, 170.0, 151.9, 138.9, 137.6, 132.9, 129.7, 129.2, 127.5, 126.4,
126.5; HRMS-ESI(−) m/z calcd for C14H8NOS3 301.9768 [M − H]−,
found 301.9779.
(Z)-5-(Benzo[b]thiophen-3-ylmethylene)-4-thioxothiazolidin-2-
one (68). Prepared from benzo[b]thiophene-3-carbaldehyde following
the general procedure C. Yield 73%; mp 218−221 °C; 1H NMR (600
MHz, DMSO-d6) δ 13.83 (s, 1H), 8.35 (s, 1H), 8.24 (s, 1H), 8.10 (d, J
= 7.8 Hz, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.55 (t, J = 7.2 Hz, 1H), 7.50
(t, J = 7.8 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 195.2, 170.7,
139.5, 138.4, 132.8, 132.4, 131.1, 130.0, 125.4, 125.6, 123.8, 121.8;
HRMS-ESI(−) m/z calcd for C12H6NOS3 275.9612 [M − H]−, found
275.9628.
Sodium (Z)-4-((2-Oxo-4-thioxothiazolidin-5-ylidene)methyl)-
benzoate (58). Compound 44 (43 mg, 0.162 mmol) and NaOH
(6.5 mg, 0.162 mmol) were dissolved in 0.7 mL (H2O:MeOH/3:2)
and stirred at room temperature for 2 h. The solution was evaporated
and dried to afford 58 (43.8 mg, 94%) as a yellow colored compound;
1
mp > 300 °C; H NMR (600 MHz, DMSO-d6) δ 7.99 (d, 2H), 7.97
(s, 1H), 7.55 (d, J = 7.8 Hz, 2H); 13C NMR (150 MHz, DMSO-d6) δ
210.5, 180.2, 170.1, 140.6, 140.5, 136.2, 131.4, 129.9, 128.8, 128.6,
117.1; HRMS-ESI(−) m/z calcd for C11H6NO3S2 263.9789 [M −
Na]−, found 263.9838.
Sodium (Z)-2-((2-Oxo-4-thioxothiazolidin-5-ylidene)methyl)-
benzoate (59). Prepared from 56 following the procedure preparation
of 58. Yield 92%; mp 252−256 °C; 1H NMR (600 MHz, DMSO-d6) δ
8.68 (s, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.32 (t,
1H), 7.26 (t, 1H); 13C NMR (150 MHz, DMSO-d6) δ 210.5, 181.1,
172.5, 144.6, 140.4, 134.7, 131.4, 128.9, 127.3, 117.2; HRMS-ESI(−)
m/z calcd for C11H6NO3S2 263.9789 [M − Na]−, found 263.9829.
(Z)-Methyl 4-((2-oxo-4-thioxothiazolidin-5-ylidene)methyl)-
benzoate (60). Prepared from methyl 4-formylbenzoate following
the general procedure C. Yield 90%; mp 187−191 °C; 1H NMR (600
MHz, DMSO-d6) δ 8.08 (s, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.79 (d, J =
8.4 Hz, 2H), 3.86 (s, 3 H); 13C NMR (150 MHz, DMSO-d6) δ 195.8,
170.8, 165.9, 138.1, 134.3, 130.9, 130.4, 52.8; HRMS-ESI(−) m/z
calcd for C12H8NO3S2 277.9946 [M − H]−, found 277.9965.
(Z)-Methyl 3-((2-oxo-4-thioxothiazolidin-5-ylidene)methyl)-
benzoate (61). Prepared from methyl 3-formylbenzoate following
the general procedure C. Yield 79%; mp 200−203 °C; 1H NMR (600
MHz, DMSO-d6) δ 8.19 (s, 1H), 8.12 (s, 1H), 8.04 (d, J = 7.2 Hz,
1H), 7.93 (d, J = 7.2 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 3.88 (s, 3H);
(Z)-5-((1-Methyl-1H-indol-3-yl)methylene)-4-thioxothiazolidin-2-
one (69). Prepared from 1-methyl-1H-indole-3-carbaldehyde following
the general procedure C. Yield 59%; mp 224−228 °C; 1H NMR (600
MHz, DMSO-d6) δ 13.38 (s, 1H), 8.39 (s, 1H), 7.97 (s, 1H), 7.84 (d, J
= 8.4 Hz, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.2 Hz, 1H), 7.28
(t, J = 7.8 Hz, 1H), 3.93 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ
193.3, 175.0, 170.5, 137.5, 134.2, 128.9, 128.1, 124.1, 123.5, 122.5,
118.7, 111.6, 34.0; HRMS-ESI(−) m/z calcd for C13H9N2OS2
273.0156 [M − H]−, found 273.0171.
(Z)-5-(4-(1H-Tetrazol-5-yl)benzylidene)-4-thioxothiazolidin-2-one
(70). Prepared from 4-(1H-tetrazol-5-yl)benzaldehyde31 following the
1
general procedure C. Yield 84%; H NMR (600 MHz, DMSO-d6) δ
K
dx.doi.org/10.1021/jm3008773 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
13.9 (s, 1H), 8.16 (d, J = 8.4 Hz, 2H), 8.10 (s, 1H), 7.88 (d, J = 8.4
Hz, 2H); 13C NMR (150 MHz, DMSO-d6) δ 195.7, 170.8, 134.1,
131.7, 128.2, 117.4.
using a Typhoon 8600 (GE Healthcare), and densitometric analyses
were performed using the ImageQuant software (GE Healthcare).
Surface Plasmon Resonance Analysis. Materials. . Coupling
reagents N-ethyl-N′-(3dimethylaminopropyl)carbodiimide (EDC), N-
hydroxysuccinimide (NHS), and ethanolamine were purchased from
GE Heathcare (Piscatawy, NJ). Neutravidin was obtained from Pierce.
Binding experiments were performed on a Biacore T200 instrument
(GE, Piscatawy, NJ). Tdp1 was amine coupled to a CM5 sensor chip
(GE Healthcare, Piscatawy, NJ). The amine groups in the active site of
Tdp1 were protected from modification by binding a 14 base
oligonucleotide to Tdp1 during the coupling step. Specifically 1 μM
Tdp1 was incubated with 2 μM of a 14 base oligonucleotide
containing a phosphate group at the 3′ end (GATCTAAAAGACTT)
in 10 mM sodium acetate pH 4.5 for 20 min. The CM5 chip surface
was activated for 7 min with 0.1 M NHS and 0.4 M EDC at a flow rate
of 20 μL/min, and the Tdp1−oligonucleotide mixture was injected
until approximately 6000 RUs were attached. Activated amine groups
were quenched with an injection of 1 M solution of ethanolamine pH
8.0 for 7 min. Any bound oligonucleotide was removed by washing the
surface with 1 M NaCl. A reference surface was prepared in the same
manner without coupling of Tdp1. Using the same approach, 8000
RUs of Neutravidin were amine coupled to flow cells 3 and 4. A 14
base oligonucleotide modified with a biotin at the 5′ end and a
tyrosine at the 3′ end (biotin-GATCTAAAAGACTT-Tyr) was
captured in the flow cell 4. A dilution series of 50 (50, 25, 6.25,
3.12, 1.56. 0.78, and 0.39 μM) was prepared in running buffer [50 mM
Tris/HCl, 80 mM KCl, 0.01% tween 20 (v/v), 5% DMSO (v/v), 2
mM EDTA, 1 mM DTT, 40 μg/mL of BSA pH 7.5] and injected over
all flow cells at 30 μL/min at 25 °C. Following compound injections,
the surface was regenerated with a 30 s injection of 1 M NaCl, a 30 s
injection of 50% DMSO (v/v), and a 30 s running buffer injection.
Each cycle of compound injection was followed by a buffer cycle for
referencing purposes. A DMSO calibration curve was included to
correct for refractive index mismatches between the running buffer and
compound dilution series.
(Z)-5-(2-(1H-Tetrazol-5-yl)benzylidene)-4-thioxothiazolidin-2-one
(71). Prepared from 2-(1H-tetrazol-5-yl)benzaldehyde57 following the
1
general procedure C. Yield 41%; mp 206−210 °C; H NMR (600
MHz, CD3OD) δ 8.26 (s, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.70 (d, J =
7.8 Hz, 1H), 7.62 (t, J = 7.2 Hz, 1H), 7.56 (t, J = 7.2 Hz, 1H); 13C
NMR (150 MHz, CD3OD) δ 195.1, 170.9, 133.7, 133.2, 131.0, 130.1,
130.0, 128.2, 112.2, 109.9; HRMS-ESI(−) m/z calcd for C11H6N5OS2
288.0014 [M − H]−, found 288.0026.
(Z)-2-((2-Oxo-4-thioxothiazolidin-5-ylidene)methyl)-
benzenesulfonic Acid (72). Prepared from 2-formylbenzenesulfonic
acid sodium salt following the general procedure C. Yield 27%; mp
218−221 °C; 1H NMR (600 MHz, CD3OD) δ 9.02 (s, 1H), 8.04 (d, J
= 7.8 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.25
(t, J = 7.8 Hz, 1H); 13C NMR (150 MHz, CD3OD) δ 192.5, 176.7,
144.8, 134.6, 132.8, 130.2, 129.0, 128.1, 127.5, 127.0; HRMS-ESI(−)
m/z calcd for C10H6NO4S3 299.9459 [M − H]−, found 299.9445.
(Z)-Diethyl (4-((2-Oxo-4-thioxothiazolidin-5-ylidene)methyl)-
phenyl)phosphonate (73). Prepared from diethyl (4-formylphenyl)-
phosphonate58 following the general procedure C. Yield 51%; mp
1
151−154 °C; H NMR (600 MHz, CD3OD) δ 8.05 (s, 1H), 7.81−
7.77 (m, 2H), 7.68−7.66 (m, 2H), 4.04 (q, J = 6.6 Hz, J = 14.4 Hz,
4H), 1.24 (t, J = 7.2 Hz, 1H); 13C NMR (150 MHz, CD3OD) δ 195.3,
138.1, 133.3, 132.3, 132.0, 131.9, 129.9, 129.8, 62.6, 15.2; HRMS-
ESI(−) m/z calcd for C14H15NO4PS2 356.0180 [M − H]−, found
356.0213.
(Z)-(4-((2-Oxo-4-thioxothiazolidin-5-ylidene)methyl)phenyl)-
phosphonic Acid (74).59 To a solution of compound 73 (0.05 g, 0.14
mmol) in dry DCM (3 mL) was added TMSBr (0.3 g, 1.9 mmol) and
stirred at room temperature for 16 h. The solvent was evaporated
under vacuum and coevaporated with methanol. The reaction mixture
was poured into water, and the solid separated was filtered off and
1
washed with water to afford 74 (0.035 g, 84%). Mp 225−228 °C; H
Modeling and Docking. The study was carried out using the
Schrodinger modeling suite.60 The X-ray crystallographic structure of
human Tdp1 in complex with vanadate, DNA, and human Top1
derived peptide (PDB: 1NOP)25 was used as the starting point of the
study. The crystallographic vanadate ion was replaced with a
pyrophosphate linker to generate the complete Top1−DNA covalent
intermediate. All missing hydrogen atoms were added based on
ionization states determined by PROPKA.61 Restraint energy was
minimized with the implicit solvent model using the OPLS 2005 force
field.62 All Tdp1 inhibitors were built and prepared with Ligprep to
enumerate all possible ionization states, tautomers, and low ring
energy conformers. The docking studies were carried out using Glide
at standard precision without any constraint within the catalytic
binding site of Tdp1. The van der Waals radii of nonpolar atoms for
each of the ligands were scaled by a factor of 0.8 to account for
structure variability to specific ligand binding. Visual inspection was
employed to identify the potential mode of binding that best
corroborates with our observed SAR study.
NMR (600 MHz, CD3OD) δ 8.14 (s, 1H), 7.91−7.88 (m, 2H), 7.71−
7.61 (m, 2H); 13C NMR (150 MHz, CD3OD) δ 195.4, 170.2, 136.8,
133.9, 133.9, 131.6, 131.3, 131.2, 129.6, 129.5; HRMS-ESI(−) m/z
calcd for C10H7NO4PS2 299.9554 [M − H]−, found 299.9580.
Biology. Tdp1 Gel-Based Assay.11 5′-32P-labeled DNA substrate
(1 nM; N14Y; 5′-GATCTAAAAGACTT-pY-3′) was incubated with
0.02 nM Tdp1 in the absence or presence of inhibitor for 15 min at
room temperature in an assay buffer containing PBS 1X, pH 7.4, 80
mM KCl, and 0.01% Tween-20. Reactions were terminated by the
addition of 1 volume of gel loading buffer [99.5% (v/v) formamide, 5
mM EDTA, 0.01% (w/v) xylene cyanol, and 0.01% (w/v)
bromophenol blue]. Samples were subjected to a 16% denaturing
PAGE, and gels were exposed after drying to a PhosphorImager screen
(GE Healthcare). Gel images were scanned using a Typhoon 8600
(GE Healthcare), and densitometric analyses were performed using
the ImageQuant software (GE Healthcare).
WCE Tdp1 Gel-Based Assay. An amount of 1 ×107 DT40 knockout
cells for chicken Tdp1 and complemented with human Tdp1 were
collected and washed once with PBS. The supernatant was removed,
and the pellet was resuspended with 50 μL of CellLytic M Cell Lysis
Reagent (SIGMA C2978), pipetting several times over ice. After 15
min, the lysate was centrifuged at 12 000g for 10 min, and the protein
concentration of the supernatant was determined using the Protein
Assay Dye Reagent Concentrate (Bio-Rad #500−0006). 5′-32P-labeled
DNA substrate (1 nM; N14Y; 5′-GATCTAAAAGACTT-pY-3′) was
incubated with 1 μg/mL of whole cell extract in the absence or
presence of inhibitor for 15 min at room temperature in an assay buffer
containing 25 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM EDTA, 1
mM DTT, 40 μg/mL of BSA, and 0.01% Tween-20. Reactions were
terminated by the addition of 1 volume of gel loading buffer [99.5%
(v/v) formamide, 5 mM EDTA, 0.01% (w/v) xylene cyanol, and
0.01% (w/v) bromophenol blue]. Samples were subjected to a 16%
denaturing PAGE, and gels were exposed after drying to a
PhosphorImager screen (GE Healthcare). Gel images were scanned
ASSOCIATED CONTENT
■
S
* Supporting Information
Docking of previously reported inhibitors (1−3). This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: pommier@nih.gov. Phone: +1 (301) 496-5944. Fax:
+1 (301) 402-0752 (YP). E-mail: wangx472@umn.edu. Phone:
+1 (612) 626-7025. Fax: +1 (612) 625-8154 (ZW).
■
Author Contributions
∥These authors contributed equally.
Notes
The authors declare no competing financial interest.
L
dx.doi.org/10.1021/jm3008773 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(16) Miao, Z.-H.; Agama, K.; Sordet, O.; Povirk, L.; Kohn, K. W.;
Pommier, Y. Hereditary ataxia SCAN1 cells are defective for the repair
of transcription-dependent topoisomerase I cleavage complexes. DNA
Repair 2006, 5, 1489−1494.
(17) Barthelmes, H. U.; Habermeyer, M.; Christensen, M. O.;
Mielke, C.; Interthal, H.; Pouliot, J. J.; Boege, F.; Marko, D. TDP1
Overexpression in Human Cells Counteracts DNA Damage Mediated
by Topoisomerases I and II. J. Biol. Chem. 2004, 279, 55618−55625.
(18) Beretta, G. L.; Cossa, G.; Gatti, L.; Zunino, F.; Perego, P.
Tyrosyl-DNA phosphodiesterase 1 targeting for modulation of
camptothecin-based treatment. Curr. Med. Chem. 2010, 17, 1500−
1508.
ACKNOWLEDGMENTS
■
This research was supported by the Center for Drug Design at
the University of Minnesota and by the Center for Cancer
Research, Intramural Program of the National Cancer Institute,
National Institutes of Health. We thank the University of
Minnesota Supercomputing Institute for providing the
computational resources.
ABBREVIATIONS USED
■
Tdp1, tyrosyl−DNA phosphodiesterase; Top1, topoisomerase
I; SAR, structure−activity relationship; CPT, camptothecin;
PLD, phospholipase D; HTS, high-throughput screening; PTK,
protein tyrosine kinase; PTP, protein tyrosine phosphatase;
WCE, whole cell extracts; SPR, Surface Plasmon Resonance
(19) Dexheimer, T. S.; Antony, S.; Marchand, C.; Pommier, Y.
Tyrosyl-DNA phosphodiesterase as a target for anticancer therapy.
Anti-Cancer Agents Med. Chem. 2008, 8, 381−389.
(20) Interthal, H.; Pouliot, J. J.; Champoux, J. J. The tyrosyl-DNA
phosphodiesterase Tdp1 is a member of the phospholipase D
superfamily. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12009−12014.
(21) Waite, M. The PLD superfamily: insights into catalysis. Biochim.
Biophys. Acta, Mol. Cell Biol. Lipids 1999, 1439, 187−197.
(22) Raymond, A. C.; Rideout, M. C.; Staker, B.; Hjerrild, K.; Burgin,
A. B., Jr. Analysis of Human Tyrosyl-DNA Phosphodiesterase I
Catalytic Residues. J. Mol. Biol. 2004, 338, 895−906.
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