Design, synthesis, and cytoprotective effect of 2-aminothiazole analogues as potent poly(ADP-Ribose) polymerase-1 inhibitors

Article abstract of DOI:10.1021/jm800902t

A series of novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors were designed within 2-aminothiazole analogues (4-10) based on a constructed three-dimensional pharmacophore model. After synthesis, the inhibitory effect on PARP-1 activity and the cytoprotective action of these compounds were tested and evaluated. Among them, compounds 4-6 and 10 appeared to be potent PARP-1 inhibitors with IC₅₀ values less than 1 μM, which had been perfectly predicted by pharmacophore model. These compounds proved to be highly potent against cell injury induced by H₂O₂ and oxygen-glucose deprivation (OGD) in PC12 cells. These novel 2-aminothiazole analogues are potentially applicable as neuroprotective agents for the treatment of neurological diseases.

Full text of DOI:10.1021/jm800902t

718
J. Med. Chem. 2009, 52, 718–725
Design, Synthesis, and Cytoprotective Effect of 2-Aminothiazole Analogues as Potent
Poly(ADP-Ribose) Polymerase-1 Inhibitors
Wen-Ting Zhang,† Jin-Lan Ruan,†,§ Peng-Fei Wu,‡ Feng-Chao Jiang,*,†,§ Li-Na Zhang,† Wei Fang,† Xiang-Long Chen,‡
Yue Wang,† Bao-Shuai Cao,† Gang-Ying Chen,† Yi-Jing Zhu,† Jun Gu,‡ and Jian-Guo Chen*,‡,§
Department of Medicinal Chemistry and Department of Pharmacology, Tongji Medical College, Huazhong UniVersity of Science and
Technology, The Key Laboratory of Natural Medicinal Chemistry and Resource EValuation of Hubei ProVince, 13 Hangkong Road,
Wuhan 430030, China
ReceiVed July 21, 2008
A series of novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors were designed within 2-aminothiazole
analogues (4-10) based on a constructed three-dimensional pharmacophore model. After synthesis, the
inhibitory effect on PARP-1 activity and the cytoprotective action of these compounds were tested and
evaluated. Among them, compounds 4-6 and 10 appeared to be potent PARP-1 inhibitors with IC50 values
less than 1 µM, which had been perfectly predicted by pharmacophore model. These compounds proved to
be highly potent against cell injury induced by H2O2 and oxygen-glucose deprivation (OGD) in PC12 cells.
These novel 2-aminothiazole analogues are potentially applicable as neuroprotective agents for the treatment
of neurological diseases.
Introduction
also result in caspase-independent programmed cell death,
mediated by the translocation of apoptosis-inducing factor (AIF)
from the mitochondria to the nucleus.8
Poly(ADP-ribose) polymerase (PARPa) is known as a nuclear
enzyme that catalyzes poly(ADP-ribosyl)ation of DNA-binding
proteins, regulates the immediate cellular response to DNA
damage, and facilitates DNA repair.1 Eighteen different putative
PARP homologues were found, and at least seven members of
the PARP family were characterized to various degrees so far
(PARP-1-3, PARP-4 (VPARP), PARP-5a (tankyrase 1), PARP-
5b (tankyrase 2), PARP-7 (TiPARP)).2 PARP-1 is regarded as
the best characterized member of the PARP family, which is a
116 kDa nuclear protein composed of four main regions: an
N-terminal DNA binding domain with two zinc finger motifs,
a nuclear location signal containing a caspase-3 cleavage site,
an automodification domain, and a C-terminal catalytic domain.3
It is activated in response to DNA damage such as that induced
by DNA-binding agents, radiation, or oxidative stress. When
the DNA damage is moderate, PARP-1 participates in the DNA
repair process. Conversely, in the case of massive and unre-
pairable DNA injury, overactivation of PARP-1 can lead to
necrotic cell death caused by the depletion of NAD+ and ATP.4
Besides that, PARP-1 is involved in the regulation of inflam-
matory mediators and in the development of LPS-induced
endotoxic shock.5 PARP-1 is also associated with NMDA-
mediated excitotoxicity and myocardial postichemic injury.6,7
Recent findings suggest that extensive PARP-1 activation may
Because of its role in many common pathologies of various
central nervous system (CNS) diseases such as cell death,
inflammatory responses, excitotoxicity, and mitochondria func-
tional disorder, PARP-1 has attracted more and more attention
as a suitable target for neuroprotective agents.9 Extensive
investigations have been conducted in the identification of novel
PARP-1 inhibitors. Most of the PARP-1 inhibitors so far
developed are structural analogues of NAD+ and compete with
NAD+ at the level of the catalytic domain. Early PARP-1
inhibitors were benzamide derivatives, which characterized by
low potency and poor selectivity. Subsequently, there are various
compounds reported as PARP-1 inhibitors, including imida-
zobenzodiazepines,10 quinazolinone and quinoxaline deriva-
tives,11 substituted uracil derivatives,12 adenosine substituted
isoindolones,13 phthalazinones,14 phenanthridin-6-ones,15 and
naphthyridin-6-ones.16 Although some of these PARP-1inhibi-
tors displayed potent in vitro activity, they lacked specificity
and had poor physical properties, complicated synthesis, or in
vivo side effects. In this study, we devote ourselves to explore
novel PARP-1 inhibitors. Herein, a series of compounds with
2-aminothiazole framework have been schemed out via computer-
aided drug design.
Design
* To whom correspondence should be addressed. For F.-C.J.: phone,
(86)-027-83692749; fax, (86)-027-83692749; E-mail, fengchao@
mails.tjmu.edu.cn. For J.-G.C.: phone, (86)-027-83692628; fax, (86)-027-
83692847; E-mail, chenj@mails.tjmu.edu.cn.
The hypothesis generation methods (HipHop and HypoGen)
of the Catalyst software have been successfully used in drug
discovery research. The Catalyst generated pharmacophores can
be considered as the ensemble of steric and electrostatic features
of different compounds and effectively used for rational drug
design.17-20 In our previous research, the pharmacophore model
of PARP-1 inhibitors had been developed by HypoGen process
in Catalyst system, which can correlate the observed biological
activities of a series of compounds with their corresponding
structures. A training set consisting of 38 compounds with seven
different classes of diverse structures and high activity was
selected in order to improve the applicability and universality
of the pharmacophore model.10-16 The selected range of IC50
†
Department of Medicinal Chemistry, Tongji Medical College, Hua-
zhong University of Science and Technology.
‡
Department of Pharmacology, Tongji Medical College, Huazhong
University of Science and Technology.
§
The Key Laboratory of Natural Medicinal Chemistry and Resource
Evaluation of Hubei Province.
a Abbreviations: PARP, poly(ADP-ribose) polymerase; OGD, oxygen-
glucose deprivation; AIF, apoptosis-inducing factor; CNS, central nervous
system; HA, hydrogen bond acceptor; HPAR, hydrophobic aromatic; HD,
hydrogen bond donor; TPSA, total polar surface area; DCC, N,N-
dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine; 3-AB, 3-ami-
nobenzaminde; FITC, fluorescein isothiocyanate; PI, propidium iodide; HRP,
horseradish-peroxidase.
10.1021/jm800902t CCC: $40.75
2009 American Chemical Society
Published on Web 01/06/2009

2-Aminothiazole Analogues as PARP-1 Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 719
Table 1. The Matching with Pharmacophore Model and Calculated
Physicochemical Properties for Designed Compounds and 3-AB
values varied from 3.8 to 770 nM. The training set of
compounds was dealt with by generating molecular model,
making conformational analysis and generating hypothesis. Ten
models were obtained after automatic construction, and the best
pharmacophore in terms of statistics and predictive value
consisted of four features: two hydrogen bond acceptors (HA
units) and two hydrophobic aromatic cores (HPAR units). It
was selected with the lowest error cost, lowest rms difference,
and the best correlation coefficient (Weight ) 2.1, rms ) 0.46,
Correl ) 0.91, Config ) 15.97) from mapping of the training
set. Each unit met the special limitation in distance. As can be
seen in Figure 1, the activities of training set compounds was
perfectly predicted by the model (r ) 0.91). The pharmacophore
model had also been validated on 12 highly active test inhibitors
such as imidazobenzodiazepines, quinazolinone and quinoxaline
derivatives, phthalazinones, phenanthridin-6-ones, and naph-
thyridin-6-ones.21 Besides that, it is well-known that most
PARP-1 inhibitors have been designed to bind at the position
of the nicotiamide subsite of the NAD+ binding pocket.
Generally, inhibitors interact with Gly863 and Ser904 by the
hydrogen bonds, with Tyr896 and Tyr907 forming the walls of
the nicotinamide-binding pocket by hydrophobic interaction.22
The docking and QSAR analysis have demonstrated that the
amide moiety of the inhibitor invariably interacted with Gly863,
while the aromatic portion interacted possibly through π-π
interactions, and the extension of the contact surfaces between
inhibitors and enzyme was in favor of high inhibitory potency.23
Similarly, the pharmacophore model built in our experiment
also indicated that HA and HPAR interactions were the most
important elements that affected the inhibitory action of
compounds. Accordingly, the model which has good applicabil-
ity and forecast ability can be a guidance for discovering novel
PARP-1inhibitors.
On the basis of the constructed pharmacophore model, we
screened the library to find structures fitting this model. We
found that aminothiazoles have almost ubiquitous presence in
pharmaceutical test samples and have been used as attractive
candidates for treatment of CNS disorders. They have extensive
biological activity and possess fine stability and little side effects.
For example, 2-aminothiazole schiff base compounds have been
designed for cognition enhancers,24 and 2,6-diaminotetrahy-
drobenzothiazole analogue has shown a pronounced selectivity
for DA autoreceptors.25 The potential radical-scavenging ability
of 2-aminothiazole amide compounds was also known,26 and
2-aminothiazole analogue has been used as the lead compound
to explore novel p53 inactivators with neuroprotective effect.27
As a result, molecules with 2-aminothiazole framework were
chosen as a scaffold for further chemical modification to explore
potent PARP-1 inhibitors with neuroprotection (1-3). The
a The inosculated degree of pharmacophore model with the specific
conformation of the related compounds. b Physicochemical properties were
calculated using Pallas 3.3 software.
such as size, lipophilicity, hydrongen-bonding potential, charge,
and conformation. Herein, our approach to design novel PARP-1
inhibitors was based on the premise that they could match the
pharmacophore model and possess appropriate physical proper-
ties. The following criteria used for design:
(1) Compound was selected if the value of fit was more than
5.
(2) The estimated value of bioavailability for selected
compound accorded with the rule of 5.
(3) If estimated value of total polar surface area (TPSA) for
a compound was more than 90 Å, then it was deselected.
Only simple structures and several representatives of a certain
chemical series were selected. Finally, seven 2-aminothiazole
analogues (4-10) were selected and their activities of PARP-1
inhibition were predicted by pharmacophore. However, so far
as we are aware, there are no publications concerning the
PARP-1 inhibition in compounds from these chemical series.
As can be seen in Figure 2, the designed compounds successfully
mapped on all or at least three features of the selected
pharmacophore developed against PARP-1, whereas 3-ami-
nobenzamide (3-AB) only could map two features of it. And
the predicted physicochemical properties of these compounds
indicated that they possess good lipophilicity and CNS-penetrant
effect. The structures, values of fit, and calculated physico-
chemical properties of these compounds were shown in Table
1. Then they were synthesized and experimentally tested as
cytoprotective agents.
Chemistry
In this study, we prepared 2-aminothiazole analogues in which
the application of microwave technology was used for cycliza-
tion reaction. As shown in Scheme 1, the synthesis of 4,5-
substituted-2- aminothiazole (1-3) was according to a published
procedure.28,29 The cyclization step, carried by microwave, was
performed in only a few minutes compared to the conventional
thermal heating for 12 h. The crude product from the reaction
of commercially available acetophenone or cyclohexanone with
thiourea in the presence of iodine was extracted with ether,
dissolved in boiling water, and made basic with solid Na2CO3
to give compound 1-3 as crystal.
candidates with this framework were designed and screened out.
A 3D conformation with minimum energy was generated and
displayed based on the current 3D structure for each compound.
After conformational analysis, each molecule got a group of
conformations reasonable in energy using the “best quality”
process. Then View Hypothesis Workbench helped to map each
molecule onto the pharmacophoric units and compare the fit in
order to test the inosculated degree of pharmacophore model
with the specific conformation of the related compound. Besides,
CNS-penetrant compounds should possess appropriate properties

720 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Zhang et al.
Table 2. Inhibitory Effect on PARP-1 Activity (IC50) and
Cytoprotective Activity (EC50) of 2-Aminothiazole Analogues
responsible for the difference between estimated activity and
actual activity. Thus, the reason why the error values of
compounds 7-9 were greater is not the inaccuracy of the
pharmacophore model but the greater Econform values of them to
a
b
c
d
e
f
compd
IC50
IC50
error
Econform
EC50
EC50
4
5
6
7
8
9
140
260
150
92
580
88
240
753
224
4533
12399
9332
682
1.7
2.9
1.5
49.3
21.4
106.0
3.1
38.47
25.45
13.86
65.49
59.72
67.62
22.94
0
537
1548
129
1122
>3000
2089
324
143
191
559
527
961
1036
361
a great extent, although the values were within 84 kJ ·mol-1
.
Obviously, the inhibitory activity of 3-AB was accurately
predicted in despite of a low potency because its active
conformer was just the minimum energy conformer (Econform
)
0). So the pharmacophore model is creditable and we should
pay more attention to Econform values of compounds in further
design study.
10
3-AB
220
4000
9005
2.2
a Estimated IC50 values (nM) were predicted by Catalyst software.
b Experimental IC50 values (nM) were determined from logit plots of
inhibition% vs compound concentration and were calculated from mean
values at 3-5 concentrations (in each, correlation coefficients r2 >0.9).
c error ) IC50 (experimental)/IC50 (estimated). d Econform (kJ·mol-1) represents
the potential energy of the structure bearing the alignment with the
pharmacophore model. e Concentration of compounds required to induce
50% protection of PC12 cells from H2O2-induced cell death (EC50, nM).
f Concentration of compounds required to induce 50% protection of PC12
cells from OGD-induced cell death (EC50, nM).
Assays for Cytoprotective Action in PC12 Cells. The
oxidative stress may induce DNA damage and PARP-1 activa-
tion. First, the cytoprotective effects of these novel PARP-1
inhibitors against oxidant-induced cell death were tested with
an H2O2-induced PC12 cell injury model. In MTT assay,
compounds 6 and 10 were highly protective (EC50 129 and 324
nM, respectively, Table 2) and compounds 4, 5, and 7 were
moderately protective. Because inhibition of PARP-1 activity
have been shown to help prevent ischemic injury in the brain,30
we also generated an oxygen-glucose deprivation (OGD)
induced PC12 cell death model to evaluate the efficacy of these
compounds in the treatment of acute ischemic insults. As can
be seen in Figure 5A, compounds 4, 5, and 10 were more
protective (EC50 143, 191, and 361 nM, respectively, Table 2)
in MTT assay. Anyway, within the same cell, the common
biochemical pathway leading to cell death via PARP-1 can be
differentially stimulated by different DNA damage insults with
divergent pharmacological targets. In addition, when the IC50
values observed in the PARP-1 activity assay for these
compounds were compared with their EC50 values for the
reduction of H2O2-induced or OGD-induced cell death, a
significant correlation was found (r ) 0.90, P < 0.0053, Figure
4B; r ) 0.89, P < 0.0072, Figure 5B). Apparently, the
compounds possessing better PARP-1 inhibition showed better
cytoprotective activity in protecting PC12 cells exposed to H2O2
or OGD treatment. To a certain extent, a 50% effective
concentration for cytoprotection was commensurate with the
inhibition of PARP-1, although some extracellular cytoprotective
effects may act at the same time.
To confirm the cytoprotective effect of 2-aminothiazole
analogues, another experiment of measuring cell viability with
LDH release assay was performed after OGD treatment in PC12
cells. It has been previously established that LDH release
correlates linearly with the number of damaged cells after toxic
insult. In LDH assay, compounds 4-7 and 10 (3 µM)
significantly reduced the OGD-induced LDH release by 34.6%,
20.1%, 33.1%, 24.0%, and 28.4%, respectively (Figure 6). We
also performed a biparametric cytofluorimetric analysis using
fluorescein isothiocyanate (FITC)-conjugated annexin V and
propidium iodide (PI) double staining. Annexin V has been
utilized to detect the externalization of phosphatidylserine that
occurs at an early stage of apoptosis and PI was used as a marker
of necrosis due to cell membrane destruction. The distribution
of stained cells was shown in Figure 7. A concentration of 3
µM of compounds 4-6 significantly reduced OGD-induced total
apoptotic cells (early and late apoptotic cells) by 70.7%, 45.1%,
and 64.4%, respectively and exhibited a considerable protective
effect on OGD-induced PC12 cell damage, which indicated the
cytoprotective activity of potent PARP-1 inhibitors were relevant
to their antiapoptotic effect to some extent.
Compound 1 or 2 was stirred with acid or acyl chloride,
respectively. They were shaken in acetone at room temperature
for 24 h, extracted with acetone, and volatilized to give the crude
compound 4-9, which were recrystallized from alcohol to afford
compound 4-9 as crystal. Compound 3 reacted with aldehyde
in ethanol at 80-90 °C for two days and extracted with ether
to give the crude compound, which were recrystallized from
EtOH/Et2O to afford compound 10 as crystal, and the reaction
time was shortened from two days to 1 h by microwave.
Biological Results and Discussion
Assay for PARP-1 Inhibition in Vitro. The inhibitory effect
of these compounds on PARP-1 activity was summarized in
Table 2. The prototypical PARP-1 inhibitor, 3-AB, inhibited
the activity of PARP at a wide range of concentrations from 2
µM to 10 mM. The IC50 value of 3-AB was 9005 nM under the
same experimental conditions. As shown in Figure 3, com-
pounds 4-6 and 10 almost showed more than 50% PARP-1
inhibition at a concentration of 1 µM. Parenthetically, the
compounds 4-6 and 10 possess a 4-phenyl moiety and were
more potent (IC50 240, 753, 224, and 682 nM, respectively).
Analogue 4 has a 4-phenyl moiety and was more potent than
the analogous compound 8, which possesses a 4-(4′, 5′, 6′, 7′-
tetrahydrobenzo) moiety (IC50 > 10 µM). As exemplified above,
compounds with 4-phenyl group showed a large increase in
inhibitory activity.
As shown in Table 2, the experimental IC50 values of
compounds 4-6, 10, and 3-AB were closed to their estimated
values, while compounds 7-9 were on the contrary. And the
corresponding potential energies of them bearing the alignment
with pharmacophore model were lower than those of compounds
7-9. It is well-known that when a receptor recognizes and acts
with a ligand, the conformation of this ligand will change to
some extent according to the induced fit theory and the energy
of this conformer may be even higher than that of the minimum
energy conformer. An allowable energy interval between an
active conformer (Econform g 0) and the minimum energy
conformer (Econform ) 0) is maintained within a certain limit.
The system default value of the limit is 84 kJ · mol-1 in the
process of conformational analysis by Catalyst. It is obvious
that molecules with lower active conformation energy are prone
to approach the enzyme and bind with it. In actual environment,
when a compound acts with PARP-1 with the theoretically active
conformer calculated by pharmacophore model, it must conquer
the energy interval and leads to system instability, which is
Conclusion
We described herein a novel series of potent PARP-1
inhibitors with cytoprotective activity designed by pharmacor-

2-Aminothiazole Analogues as PARP-1 Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 721
Scheme 1. Synthesis of Compounds 1-10a
a Reagents and conditions: (a) acetophenone, iodine, microwave (130W-65W), Na2CO3; (b) cyclohexanone, iodine, microwave (130W-65W), Na2CO3;
(c) 1-(2,4-diethoxyphenyl)ethanone, iodine, microwave (130W-65W), Na2CO3; (d) aromatic acid or acyl chloride, DMAP or DCC, acetone, rt; (e) 1H-
indole-3-carbaldehyde, dehydrated alcohol, piperidine, microwave (65W-130W).
Figure 1. Correlation line of estimated activity vs actual activity for
the 38 training compounds from refs 10-16 including imidazobenzo-
diazepines, quinazolinone and quinoxaline derivatives, substituted uracil
derivatives, phenanthridin-6-ones, adenosine substituted 2,3-dihydro-
1H-isoindol-1-ones, and phthalazinones (r ) 0.91).21
Figure 2. Mapping of designed compounds 4-10 and 3-AB on the
optimal pharmacophore developed against PARP-1. The green and blue
contours represent the hydrogen bond accepting feature (HA) and
hydrophobic aromatic feature (HPAR) respectively.
phore model. To provide a better understanding of the involve-
ment of PARP-1 in neuronal cell death associated with CNS
diseases, further work to study the neuroprotective mechanisms
and optimize the structures of these compounds is necessary.
In addition, other possible protective mechanisms of these
2-aminothiazole analogues remain to be clarified, and this study
provided confidence for the usefulness of the selected pharma-
cophore model to identify and design more potent PARP-1
inhibitors with diverse structures.
Experimental Section
Chemistry. Melting points were obtained on Taike X-5 mi-
croautomatic melting point detector and were uncorrected. Infrared
absorption was detected on an impact 420 by FT-IR Spectrometer
one scanning between 400 and 4000 cm-1. 1H NMR was recorded
at 300 MHz on a PARIAN MERCURY VX-300 NMR. 13C NMR
was recorded at 100 MHZ on a Bruker AV400. And chemical shifts
are reported in parts per million (ppm, δ) relative to tetramethyl-
silane as an internal standard. Mass spectra were recorded on an
Agilent 7890A15975C. Microwave reactor refitted from SanluoWP-
650 household microwave oven (output power 650 W). The
structures of all compounds were consistent with their spectral data.
Unless otherwise noted, all materials were obtained from com-
mercial suppliers and used without further purification, and the
reported yields were not optimized.
Figure 3. 2-Aminothiazole analogues inhibit PARP-1 activity at a
concentration of 1 µM (n ) 3 cultures) and the mean is presented.
Signification difference from control group: “*”, P < 0.05.
General Procedure 1. Preparation for 4,5-substituted-2-ami-
nothiazole analogues. A mixture of acetophenone, thiourea, and
iodine was stirred in microwave for a few minutes. The reaction
mixture then was cooled, extracted with ether to remove ketone
and iodine, and then dissolved in hot water. The solution was
neutralized with Na2CO3, and recrystallized from alcohol.

722 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Zhang et al.
Figure 5. Cytoprotection by 2-aminothiazole analogues against OGD
toxicity in PC12 cells and correlation with their PARP-1inhibitory
potency. (A) Cell viability in each culture was quantified (n ) 10
cultures), and the mean is presented. No toxicity was associated with
the use of the designed compounds alone as assessed by MTT (data
not shown). Signification difference from control group: “#”, P < 0.005.
Significant difference from OGD treated group: “*”, P < 0.05 and “**”,
P < 0.01. (B) Positive correlation (r ) 0.89) between the IC50 values
of selected compounds for the inhibition of PARP-1 activity and their
EC50 values for reduction of OGD -induced cell death, P (that r is
zero) < 0.0072.
Figure 4. Cytoprotection by 2-aminothiazole analogues against H2O2-
induced damage in PC12 cells and correlation with their PARP-
1inhibitory potency. (A) Cell viability in each culture was quantified
(n ) 10 cultures), and the mean is presented. No toxicity was associated
with the use of the designed compounds alone as assessed by MTT
(data not shown). Signification difference from control group: “#”, P
< 0.001. Significant difference from H2O2 treated group: “*”, P < 0.05
and “**”, P < 0.01. (B) Positive correlation (r ) 0.90) between the
IC50 values of selected compounds for the inhibition of PARP-1 activity
and their EC50 values for reduction of H2O2-induced cell death, P (that
r is zero) < 0.0053.
4-Phenylthiazol-2-amine (1). Yield (63%); mp 149-151 °C
(lit.28 146-148 °C). IR (KBr) σ/cm-1 3446, 3437, 3255. 1H NMR
(CDCl3) δ 5.11 (s,2H), 6.73 (s,1H), 7.28 (t, J ) 7.2 Hz, 1H), 7.47
(t, J ) 7.2 Hz, 2H), 7.78 (d, J ) 7.2 Hz, 2H).
4,5,6,7-Tetrahydrobenzo[d]thiazol-2-amine (2). Yield (75%);
mp 87-88 °C (lit.29 87-88 °C). IR (KBr) σ/cm-1 3264, 3133,
1524. 1H NMR (CDCl3) δ 1.80 (s, 4H), 2.55 (s, 4H), 4.89 (s, 2H).
4-(2,4-Diethoxyphenyl)thiazol-2-amine (3). Yield (16%); mp
1
140-142 °C. H NMR (CDCl3) δ 1.32 (t, J ) 6.6 Hz, 3H), 1.46
(t, J ) 6.6 Hz, 3H), 4.11 (q, J ) 6.6 Hz, 2H), 4.13 (q, J ) 6.6 Hz,
2H), 5.94 (s, 2H), 6.45 (d, J ) 2.0 Hz, 1H), 6.51 (dd, J ) 7.2 Hz
and J ) 2.0 Hz, 1H), 6.73 (s, 1H), 7.63 (d, J ) 7.2 Hz, 1H).
Figure 6. OGD-induced LDH release was attenuated by 3 µM
2-aminothiazole analogues in PC12 cells. Data are expressed as mean
( SD (n ) 4 cultures). Signification difference from control group:
“#”, P < 0.001. Significant difference from OGD treated group: “*”,
P < 0.05; “**”, P < 0.001.
General Procedure 2. Synthesis of 4,5-substituted-2-aminothia-
zoles amide derivatives. 4,5-Substituted-2- aminothiazole (1 g) and
acid or acyl chloride (1:1.2) were dissolved in 15 mL of acetone
and then DMAP and DCC were added. The mixture was shaked
continually at 25 °C for 24 h. The mixture was filtered, washed
with 9 mL of acetone, and then the acetone solution was put
together. Volatilized acetone and yielded the yellow solid. The solid
was washed the by slightly distilled water and recrystallized with
75% alcohol.
δ 172.41, 169.37, 157.84, 149.47, 134.01, 128.82, 128.23, 126.13,
107.68, 61.10, 31.04, 29.03, 14.16. MS (ESI positive ion) m/z: 305.1
(M + 1).
N-(4-Phenylthiazol-2-yl)isonicotinamide (5). Yield (60%); mp
1
213-216 °C (lit. 206-208 °C). IR (KBr) σ/cm-1 3400, 1602. H
NMR (CDCl3) δ 7.23 (s, 1H), 7.25 (d, J ) 7.2 Hz, 1H), 7.32 (t, J
) 7.2 Hz, 2H), 7.58 (s, 2H), 7.67 (d, J ) 7.2 Hz, 2H), 8.64 (s,
2H), 11.49 (s, 1H). 13C NMR (CDCl3) δ 163.52, 158.59, 150.72,
150.27, 139.07, 133.74, 128.85, 128.37, 126.10, 120.91, 108.61.
MS (ESI positive ion) m/z: 282.2 (M + 1).
Compounds 4-9 were prepared according to general procedure
2.
Ethyl 4-oxo-4-(4-phenylthiazol-2-ylamino) butanoate (4). Yield
(44%); mp 168-169 °C. IR (KBr) σ/cm-1 3431, 3273, 1720, 1689.
1H NMR (CDCl3) δ 1.25 (t, J ) 7.2 Hz, 3H), 2.58 (t, J ) 6.6 Hz,
2H), 2.68 (t, J ) 6.6 Hz, 2H), 4.15 (q, J ) 7.2 Hz, 2H), 7.14 (s,
1H), 7.33 (d, J ) 7.2 Hz, 1H), 7.41 (dd, J ) 7.2 Hz and J ) 7.5
Hz, 2H), 7.82 (d, J ) 7.2 Hz, 2H), 10.00 (s, 1H). 13C NMR (CDCl3)
(E)-N-(4-Phenylthiazol-2-yl) cinnamamide (6). Yield (59%); mp
238-239 °C (lit. 225 °C). IR (KBr) σ/cm-1 3393, 2929, 1630, 1533,
1
696. H NMR (CDCl3) δ 6.15 (d, J ) 15.0 Hz, 1H), 7.26 (d, J )
7.2 Hz, 1H), 7.28 (s, 1H), 7.36-7.81 (m, 5H), 7.44 (t, J ) 7.2 Hz,

2-Aminothiazole Analogues as PARP-1 Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 723
Figure 7. Compounds 4-6 significantly reduced apoptotic cells (early and late apoptotic cells) induced by OGD treatment (n ) 3 cultures). (a-e)
Dot plot for PC12 cells with Annexin/PI double stain. (a) control; (b) OGD (4 h and 18 h reoxygeneration); (c-e) OGD (4 h and 18 h reoxygeneration)
+ 3 µM compounds 4-6, respectively. Shown are representative data from one of three independent experiments. (f) Data are expressed as the
mean ( SD of results in three independent experiments. Significant difference from untreated control: “#”, P < 0.005. Significant difference from
OGD-treated model group: “*”, P < 0.05; “**”, P < 0.005.
2H), 7.88 (d, J ) 15.0 Hz, 1H), 7.92 (d, J ) 7.2 Hz, 2H), 11.63 (s,
1H). 13C NMR (CDCl3) δ 163.69, 159.79, 149.71, 144.53, 134.12,
133.93,130.37,128.99,128.73,128.50,128.26,126.18,118.39,108.42.
MS (ESI positive ion) m/z: 307.1 (M + 1).
d6) δ 171.98, 169.52, 154.60, 143.84, 120.97, 59.91, 29.73, 28.35,
25.97, 22.92, 22.60, 22.18, 14.02. MS (ESI positive ion) m/z: 283.2
(M + 1).
(E)-4-(3,5-Dimethoxyphenyl)-1-(4,5,6,7-tetrahydrobenzo[d]thia-
zol-2-ylamino)but-3-en-2-one (9). Yield (40%); mp 207-209 °C.
IR (KBr) σ/cm-1 3445, 2847, 1620, 1542. 1H NMR (CDCl3) δ 1.81
(s, 4H), 2.73 (s, 4H), 3.80 (s, 6H), 6.49 (s, 1H), 6.50 (d, J ) 15.0
(E)-3-(3,5-Dimethoxyphenyl)-N-(4-phenylthiazol-2-yl)acryla-
mide (7). Yield (65%); mp 95-96 °C. IR (KBr) σ/cm-1 3466, 3051,
1
1590, 1543. H NMR (CDCl3) δ 3.77 (s, 6H), 6.16 (d, J ) 15.0
Hz, 1H), 7.62 (s, 2H), 7.68 (d, J ) 15.0 Hz, 1H), 11.4 (s, 1H,). 13
C
Hz, 1H), 6.35 (s, 2H), 6.45 (s, 1H), 7.22 (d, J ) 7.2 Hz, 1H), 7.26
(s, 1H), 7.34 (t, J ) 7.2 Hz, 2H), 7.61 (d, J ) 15.0 Hz, 1H), 7.85
(d, J ) 7.2 Hz, 2H), 11.63 (s, 1H). 13C NMR (DMSO-d6) δ 163.28,
160.73, 157.86, 149.05, 142.21, 136.20, 134.21, 128.64, 127.71,
125.60, 120.20, 108.41, 105.86, 102.15, 79.08, 55.94, 55.27. MS
(ESI positive ion) m/z: 367.1 (M + 1).
NMR (CDCl3) δ 162.71, 160.55, 156.23, 143.34, 143.01, 135.61,
122.97, 118.88, 105.62, 102.03, 54.88, 25.78, 22.67, 22.36. MS
(ESI positive ion) m/z: 345.1 (M + 1).
(E)-N-((1H-Indol-3-yl)methylene)-4-(2,4-diethoxyphenyl)thiazol-
2-amine (10). Compound 3 (1.6 g, 6 nmol) and 1H-indole-3-
carbaldehyde (0.9 g, 6 nmol) were dissolved in dehydrated alcohol
(25 mL). Piperidine was added dropwise to the stirring solution,
and the suspension was refluxed for 1 min by microwave at radiation
power of 65 W and then for 1 h at radiation power of 130 W.
Thereafter, the crude product was filtered off from the reaction
mixture, washed with a small amount of distilled water, and
Ethyl-4-oxo-5(4,5,6,7-tetrahydrobenzo[d]thiazol-2-ylamino)pen-
tanoate (8). Yield (55%); mp 125-126 °C. IR (KBr) σ/cm-1 3240,
1
3156, 2941, 1723, 1696, 1549, 1537. H NMR (CDCl3) δ 1.25 (t,
J ) 6.9 Hz, 3H), 1.84 (s, 4H), 2.65 (m, 2H), 2.69 (m, 2H), 2.73 (s,
4H), 4.15 (q, J ) 6.8 Hz, 2H), 10.88 (s, 1H). 13C NMR (DMSO-

724 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Zhang et al.
recrystallized from EtOH/Et2O to give compound 10. Yield (94%);
mp 218-220 °C. IR (KBr) σ/cm-1 3224, 3113, 1523.72, 1486.64.
1H NMR (DMSO-d6) δ 1.34 (t, J ) 6.6 Hz, 3H), 1.48 (t, J ) 6.6
Hz, 3H), 4.01 (q, J ) 6.6 Hz, 2H), 4.10 (q, J ) 6.6 Hz, 2H), 6.47
(d, J ) 2.4 Hz, 1H), 6.48 (dd, J ) 7.2 Hz and J ) 2.4 Hz, 1H),
7.16-7.21 (m, 2H), 7.58 (d, J ) 7.2 Hz, 1H), 7.68 (d, J ) 3.6 Hz,
1H), 7.85 (d, J ) 7.2 Hz, 1H), 8.15 (d, J ) 7.2 Hz, 1H), 8.38 (d,
J ) 3.6 Hz, 1H), 9.10 (s, 1H), 11.64 (s, 1H). 13C NMR (DMSO-
d6) δ 171.27, 164.70, 158.31, 156.20, 147.64, 136.52, 134.14,
129.73, 123.84, 122.37, 121.13, 120.62, 115.17, 113.67, 110.96,
104.21, 98.40, 62.74, 62.27, 13.77, 13.69. MS (ESI positive ion)
m/z: 392.2 (M + 1).
Biology. PARP Universal Colorimetric Assay. PARP-1 inhibi-
tion was determined in a functional in vitro test using a PARP
Universal Colorimetric Assay Kit (R&D catalogue no. 4677-096-
K). This kit measures the incorporation of biotinylated poly(ADP-
ribose) onto histone proteins in a 96-well histone-coated plate and
tests the inhibition of PARP-1 activity. The test to screen PARP-1
inhibitors consists of the following steps: 15 µL/well human rPARP-
HSA enzemy (0.5 unit/well, R&D 4668-050-01), 10 µL/well
dilutions of PARP inhibitor (s), 2.5 µL/well 10 × activated DNA
(R&D 4671-096-06), 2.5 µL/well 10 × PARP cocktail (contains
biotinylated NAD+, R&D 4671-096-03) and 20 µL/well 1 × PARP
buffer (R&D 4671-096-02) were incubated on a 96-well histone-
coated plate at room temperature for 1 h. After four washings with
ice-cold PBS-T (PBS containing 0.05% Tween 20), 50 µL of diluted
Strep-HRP (R&D 4800-30-06) was added to each well and the plate
was incubated at room temperature for 1 h. After four washings
with PBS-T, 50 µL of TACS-Sapphire (R&D 4822-96-08) was
added to each well and the plate was incubated for 15-30 min in
the dark. TACS-Sapphire is a horseradish-peroxidase (HRP)
substrate generating a soluble blue color. Then 50 µL of 0.2 N
HCl per well was added to stop this reaction. Then the plate can
be read at 450 nm with a microplate reader. The positive control,
3-aminobenzamide, was provided by this kit (4667-50-03). All of
the compounds were freshly prepared as stock solution with
dimethyl sulfoxide (DMSO) and diluted with the PARP buffer. The
final concentration of DMSO in the assay was less than 0.5%, and
the same concentration of DMSO in PARP buffer was used as a
control in this experiment.
Cell Culture. Rat PC12 cells were obtained from Chinese Type
Culture Collection and have been differentiated to be neuron-like
by treating with nerve growth factor (NGF). We have used these
PC12 cells as a cell culture model for screening novel neuropro-
tective agents. Some nature polyphenolic compounds in our
previous study, which have showed protective effects in oxidant
stress induced PC12 cell injury, such as breviscapine and tetrahy-
droxystilbene glucoside, also exhibited neuroprotection in MCAO
or 2VO animal models. Cultures of PC12 cells were maintained in
DMEM containing 5% heat-inactivated fetal calf serum, 10% heat-
inactivated horse serum, 100 U/mL penicillin, and 100 µg/mL
streptomycin in a humidified atmosphere of 95% air and 5% CO2
at 37 °C. The culture medium was changed and the cells were
passaged by trypsinization every 3 to 4 days.
OGD-Induced Cell Death in PC12 Cell Cultures. Cells (4 ×
104 cells/200 µL) were seeded into 96- or 6-well plates and then
incubated in a humidified atmosphere of 95% air and 5% CO2 at
37 °C for 2 days. To induce OGD stress, the cell culture medium
was removed and the cells were washed twice with a glucose-free
isotonic salt solution (OGD buffer pH 7.4) consisting of the
following in mM: 20 NaHCO3, 120 NaCl, 5.36 KCl, 0.33 Na2HPO4,
0.44 KH2PO4, 1.27 CaCl2, and 0.81 MgSO4 and incubated in the
same volume OGD buffer.31 Cells were then incubated at 37 °C in
an oxygen-free incubator (95% N2 and 5% CO2) for 4 h (OGD).
As a control for OGD, PC12 cells were incubated in OGD buffer
supplemented with 5% bovine calf serum, 10% horse serum, and
4.5 mg/mL glucose in normoxic conditions for the indicated length
of time. At the end of the OGD period, the medium was replaced
with culture medium for the indicated time, and the cultures were
put back in the incubator for an additional 18 h at the regular
atmospheric oxygen level (reoxygenation). When required com-
pounds (3, 0.3 and 0.03 µM) were added to the OGD buffer and
the reoxygenation culture, all of the compounds were freshly
prepared as stock solution with dimethyl sulfoxide (DMSO) and
diluted with OGD buffer or DMEM. Less than 0.5% DMSO has
no protective or toxic effects when used alone.
H2O2-Induced Cell Death in PC12 Cell Cultures. Cells (4 ×
104 cells/200 µL) were seeded into 96- well plates and then
incubated in a humidified atmosphere of 95% air and 5% CO2 at
37 °C for 2 days. In the experiment, the cells in DMEM were
exposed to different compounds (3, 0.3 and 0.03 µM) for 2 h at 37
°C. Then H2O2 was added to the cells at a final concentration of
550 µM. The plates were further incubated at 37 °C for 6 h.
MTT Assay. After various treatments, cell viability was
measured by using the MTT assay, which is based on the conversion
of MTT to formazan crystals by mitochondrial dehydrogenases. In
live cells, mitochondrial enzymes have the capacity to transform
MTT into insoluble formazan. Cell cultures were incubated with
MTT solution (5 mg/mL) for 4 h at 37 °C. Following this, the
medium was discarded and DMSO was added to solubilize the
reaction product formazan by shaking for 15 min. Absorbance at
492 nm was measured with a microplate reader (ELx800, Bio-Tek,
Winooski, VT). Cell viability of vehicle-treated control group not
exposed to either H2O2 or OGD was defined as 100%. Cell viability
was expressed as a percentage of the value in control cultures.
LDH assay. The LDH activity assay was performed according
to the protocols of a LDH kit. To confirm the cell death, the amount
of LDH released to the medium and total LDH were determined
18 h after OGD. Briefly, an aliquot of the culture supernatants
(extracellular) or cell dissociation solution (intracellular) was mixed
with nicotinamide adenine dinucleotide (NAD) and lactate solution.
Colorimetric absorbance was measured at 490 nm with a microplate
reader, respectively. Total LDH activity was calculated by adding
the values for extracellular and intracellular LDH that were
measured in live cells treated with 1% Triton X-100. The ratio of
released LDH (extracellular) versus total LDH (extracellular +
intracellular) was calculated and expressed as a percentage of total
LDH.
Assessment of Apoptosis. Flow cytometry was used to assess
the membrane and nuclear events during apoptosis. The assay was
performed with a two color analysis of FITC-labeled Annexin V
binding and PI uptake using the Annexin V-FITC apoptosis
detection kit. Briefly, after reoxygenation, apoptotic cells were
harvested by centrifugation of culture medium at 1000 rpm for 10
min, whereas adherent cells were trypsinized and subsequently
collected by centrifugation. The cells were washed with PBS and
resuspended in 1 mL of binding buffer. An aliquot of 100 µL was
incubated with 5 µL of Annexin V-FITC and 10 µL of PI for 15
min in dark at room temperature and 400 µL of PBS was added to
each sample. The FITC and PI fluorescence were measured through
a FL-1 filter (530 nm) and a FL-2 filter (585 nm), respectively, on
BD-LSR flow cytometer using Cell Quest software. Positioning of
quadrants on Annexin V/PI dot plots was performed and live cells
(Annexin V-/PI-, represent in the lower left quadrants of the
cytograms), early/primary apoptotic cells (Annexin V+/PI-,
represent in the lower right quadrants), and late/secondary apoptotic
cells (Annexin V+/PI+, represent in the upper right quadrants)
were distinguished.32 The upper left quadrants represent cells
damaged during the procedure.
Statistical Analysis. Data were expressed as means ( SD.
Comparison between two groups were evaluated by the unpaired
Student’s t test. Differences were considered significant at P < 0.05.
Acknowledgment. This work was supported by Graduates’
Innovation Fund of Huazhong University of Science and
Technology (no. HF0503707510) and grants from the National
Science Foundation for the Distinguished Young Scientists in
China (no. 30425024) and the National Basic Research Program
of China (973 Program, no. 2007CB507404).
Supporting Information Available: Details on pharmacophore
modeling and chromatographic analytical data for determining

2-Aminothiazole Analogues as PARP-1 Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 725
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