SAR, cardiac myocytes protection activity and 3D-QSAR studies of salubrinal and its potent derivatives

Article abstract of DOI:10.2174/092986712804485971

Salubrinal is a selective inhibitor of endoplasmic reticulum (ER) stress and affords remarkable protection to cardiomyocytes. By studying the structure-activity relationship (SAR) of salubrinal, it was found that modification of the quinoline ring terminus and thiourea unit could confer the compound PP1-24 with markedly enhanced cardioprotective activity (EC ₅₀ < 0.3 μM) that is 50-fold more potent than salubrinal. Comparative molecular field analysis (CoMFA) was performed using the obtained biological data and resulted in a statistically significant CoMFA model with high predictive power (q² = 0.741, r² = 0.991).

Full text of DOI:10.2174/092986712804485971

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6072
Current Medicinal Chemistry, 2012, 19, 6072-6079
SAR, Cardiac Myocytes Protection Activity and 3D-QSAR Studies of Salubrinal and
its Potent Derivatives
J. Liu1,2, K.-L. He3, X. Li3, R.-J. Li2, C.-L. Liu2, W. Zhong2,* and S. Li2,*
1Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering,
Shenyang Pharmaceutical University, Shenyang 110016, China; 2Laboratory of Computer-Aided Drug Design & Discovery, Beijing
3
Institute of Pharmacology & Toxicology, Beijing 100850, China; Department of Cardiology, General Hospital of Chinese PLA,
Beijing, China
Abstract: Salubrinal is a selective inhibitor of endoplasmic reticulum (ER) stress and affords remarkable protection to cardiomyocytes.
By studying the structure–activity relationship (SAR) of salubrinal, it was found that modification of the quinoline ring terminus and
thiourea unit could confer the compound PP1-24 with markedly enhanced cardioprotective activity (EC50 ꢀ 0.3 ꢁM) that is 50-fold more
potent than salubrinal. Comparative molecular field analysis (CoMFA) was performed using the obtained biological data and resulted in a
statistically significant CoMFA model with high predictive power (q2 = 0.741, r2 = 0.991).
Keywords: Cardiomyocytes protection, UPR, endoplasmic reticulum (ER) stress, eukaryotic translation initiation factor 2 subunit ꢂ (eIF2ꢂ)
phosphorylation, PP1/GADD34 complex inhibitor, salubrinal, structure–activity relationships (SAR), MTT, CoMFA model, predicted abili-
ties.
interested in investigating how cardiomyocytes would respond to
salubrinal treatment.
INTRODUCTION
Despite the extensive progress in the prevention and clinical
management of heart failure, cardiovascular diseases still remain
the leading cause of human mortality. Therefore, the development
of new drugs and the discovery of novel strategies for treating car-
diovascular diseases are needed.
In a previous study by our group, salubrinal was assayed for its
ability to protect cardiac myocytes from apoptosis induced by ER
stress. In this assay, neonate cardiac myocytes of 1-day-old Wistar
rats were treated with tunicamycin (Tm) and different doses of
salubrinal for 37 h, after which cell viability was assessed by meas-
urement of cellular ATP content. The data showed that salubrinal
protected cardiac myocytes with an EC50 of about 15 ꢁM, and res-
cued about 40% of cells when employed at a concentration of 40
ꢁM. From these studies, the ability of salubrinal to act as a cardio-
protective agent was quite apparent. However, salubrinal is still far
from being the ideal cardioprotective therapeutic agent due to its
side effects, poor solubility and high effective concentration.
Evidence obtained in recent years has demonstrated that ER-
mediated cell death plays an important role in many cardiac dis-
eases, including hypertrophic and failing hearts, cardiac arrest and
resuscitation, myocardial ischemia and reperfusion, and myocarditis
[1]. Various stimuli, such as ischemia, hypoxia, heat shock, genetic
mutation, and elevated protein synthesis, which are collectively
known as ER stressors [2], activate the unfolded protein response
(UPR) that bidirectionally regulates abnormal cardiomyocytes [3,
4]. Initially, the adaptive pathway of UPR deals with unfolded and
misfolded proteins to restore proteostasis. The apoptotic pathway of
UPR induced by severe and prolonged ER stress reconfigures cells
from survival to apoptosis. Thus, therapeutic interventions that
target the molecular components of the UPR and reduce ER stress
represent promising strategies to treat cardiovascular diseases.
The molecular mechanism of salubrinal action is still under in-
vestigation, so molecular design based directly on its protein struc-
ture is impossible. Also, a SAR study of salubrinal was carried out
by Long et al. with less than 30 compounds [12]. We describe
herein our efforts to further investigate its SAR and to find more
potent cardioprotective molecules. The primary modification dem-
onstrated that the trichloromethyl and cinnamide groups were in-
dispensable for the activity of this molecule, whereas the quinoline
ring terminus turned out to be a key site for modification. The re-
placement of the quinolyl group with a pyridyl group slightly im-
proved the activity, while by 4-chlorophenyl, termed as sal003 Fig.
(1), enhanced the potency at a lower concentration (25 ꢁM) with
EC50 unpublished [13]. Since modification of the quinoline ring
terminus resulted in dramatic changes in the molecule’s antiapop-
totic activity, our goal was to further explore the quinoline ring
terminus of salubrinal, as well as the untested thiourea group. In
this study, we perform the synthesis and in vitro evaluation of se-
lective inhibitors of eIF2ꢂ dephosphorylation.
In 2005, Boyce and colleagues reported that a small molecule
termed salubrinal Fig. (1) selectively inhibited the PP1/GADD34-
mediated dephosphorylation of eukaryotic translation initiation
factor 2 subunit ꢂ (eIF2ꢂ), thereby protecting 36% of PC12 cells
from ER stress-induced apoptosis, with cell viability increasing
from approximately 40% to 76% [5]. Due to its selectivity and non-
toxicity, salubrinal has drawn a great deal of attention as a protec-
tive agent, and indeed has shown remarkable cytoprotective activity
in many different kinds of cells. For example, salubrinal rescued
neuronal cells from ꢃ-amyloid-induced apoptosis [6], prevented
cadmium-mediated apoptosis in human renal proximal tubular cells
[7] and abolished homocysteine-induced apoptosis in endothelial
progenitor cells derived from patients with coronary heart disease
[8]. Nevertheless, different cell types have been shown to respond
differently to salubrinal treatment. It has been reported that salubri-
nal treatment caused pancreatic ꢃ-cell apoptosis [9], enhanced cis-
platin-induced nephrotoxicity [10] and induced the apoptosis of
EBV-transformed B cells [11]. Thus, our research group was
MATERIALS AND METHODS
Chemistry
1H-NMR spectra were recorded in DMSO-d6 on a Bruker-
ARX400 spectrometer with TMS as an internal standard. All
chemical shifts were quoted in ꢄ values (ppm). MS spectra were
recorded using a Micromass ZabSpect. Elemental analyses were
performed with an Elementar Vario MICRO CUBE (Germany).
Reagents and solvents were purchased from common commercial
suppliers. Intermediates were synthesized using previously reported
procedures.
*Address correspondence to these authors at the Laboratory of Computer-Aided Drug
Design
& Discovery, Beijing Institute of Pharmacology & Toxicology, Beijing
100850, China; Tel: (8610)66932624/(8610)66931250; Fax: (8610)68211656;
E-mails: zhongwu@bmi.ac.cn; lis@bmi.ac.cn
1875-533X/12 $58.00+.00
© 2012 Bentham Science Publishers

SAR, Cardiac Myocytes Protection Activity and 3D-QSAR Studies
Current Medicinal Chemistry, 2012 Vol. 19, No. 35
6073
Fig. (1). Chemical structures of salubrinal and sal003.
DMSO-d6) ꢀ2.28 (s, 3H); 6.49 (d, 1H); 7.13-7.18 (m, 3H); 7.36-
7.45(m, 4H); 7.66-7.72 (m, 2H); 8.03 (s, 1H); 8.97 (d, 1H); 10.21
(s, 1H). MS (ESI) 450.0 (M+).
COMPOUNDS
Synthesis of N, N-Asymmetrically Substituted Thiourea Deriva-
tives PP1-(1-16)
(E)-3-(Thiophen-2-yl)-N-(2,2,2-trichloro-1-(3-cyclohexylthi
1
oureido)ethyl)acrylamide (PP1-7): H-NMR (400 MHz, DMSO-
General procedures Aromatic formaldehyde (10 mmol) and
malonic acid (30 mmol) were stirred in dry pyridine (20 mL) for 8 h
with one drop of piperidine as catalyst. The mixture was poured
into 20 mL of water and 10% NaOH (aq) was used to adjust its pH
to 12. After three extractions by acetic ether, the water layer was
added with 2 N HCl (aq) until white precipitate appeared. The fil-
trate was dried and then resolved in 20 mL of dry THF. SOCl2 (4
mL) was added then refluxed for 2 h. The solvent was then evapo-
rated and the residue was added dropwise to cooled ammonia water
to give acrylamide. Toluene (100 mL) was added to the mixture of
acrylamide (5 mmol) and chloride hydrate (5 mmol). With 4 h re-
flux, a white solid was precipitated, filtrated and dried. The solid
was resolved in dry THF (20 mL) and chlorinated by reflux with
SOCl2 (2 mL) for 2 h. The yellow solid N-1, 2, 2, 2-tetrachloro
ethylacryl- amide, an important synthon, was yielded by evaporat-
ing the solvent and washing with cold petroleum. By reaction with
potassium thiocyanate (7.5 mmol), it was transformed to isocy-
anate. The mixture of isocyanate and various amines were refluxed
in THF (10 mL) for 1.5 h to obtain compounds PP1-(1-16).
d6) ꢀ1.14-1.31 (m, 5H); 1.51-1.64 (m, 3H); 1.88 (m, 2H); 3.97-3.99
(m, 1H); 6.53 (d, 1H); 7.13 (dd, 1H); 7.30-7.43 (m, 2H); 7.65-7.83
(m, 3H); 8.18 (d, 1H); 8.92 (d, 1H). MS (ESI) 440.0 (M+).
(E)-3-(Thiophen-2-yl)-N-(2,2,2-trichloro-1-(3-(3-isopropoxyl
propyl)thioureido)ethyl)acrylamide (PP1-8): 1H-NMR (400
MHz, DMSO-d6) ꢀ1.07 (d, 6H); 1.69 (t, 2H); 3.31-3.51 (m, 4H);
6.53 (d, 1H); 7.12 (dd, 1H); 7.31-7.43 (m, 2H); 7.64-7.70 (m, 2H);
7.87 (d, 1H); 8.21 (t, 1H); 8.89 (d, 1H). MS (ESI) 458.0 (M+).
(E)-3-(Thiophen-2-yl)-N-(2,2,2-trichloro-1-(3-(2-methoxycar
bonylphenyl)thioureido)ethyl)acryla-mide(PP1-9):
1H-NMR
(400 MHz, DMSO-d6) ꢀ3.79-3.80 (s, 3H); 6.60 (d, 1H); 7.13 (dd,
1H); 7.28-7.32 (m, 1H); 7.42-7.46 (m, 2H); 7.55-7.59 (m, 1H);
7.65-7.72 (m, 2H); 7.78 (d, 1H); 7.85 (dd, 1H); 8.88-9.01 (m, 2H).
MS (ESI) 492.0 (M+). Calcd for C18H17Cl3N3O3S2 (M+): 491.9771.
Found 491.9763.
(E)-3-(Thiophen-2-yl)-N-(2,2,2-trichloro-1-(3-(2-fluorophe
nyl)thioureido)ethyl)acrylamide (PP1-10): 1H-NMR (400 MHz,D
MSO-d6) ꢀ6.54 (d, 1H); 7.12-7.29 (m, 4H); 7.39-7.45 (m, 2H);
7.66-7.73 (m, 2H); 7.88 (t, 1H); 8.53 (d, 1H); 9.05 (d, 1H). MS
(ESI) 453.0(M+).
N-(2,2,2-Trichloro-1-(3-quinolin-8-ylthioureido)ethyl)cinna
mamide (salubrinal): 1H-NMR (400MHz, DMSO-d6) ꢀ 6.90 (d,
J=16Hz, 1H), 7.42-7.44 (m, 3H), 7.55-7.74 (m, 7H), 8.44 (dd, J=8,
1.2Hz, 1H), 8.95-9.06 (m, 3H), 9.58 (d, J=9.2Hz, 1H), 11.06 (s,
1H). FAB-MS m/z: 479.0[M+1]+.
(E)-3-(Thiophen-2-yl)-N-(2,2,2-trichloro-1-(3-cycloheptyl
thioureido)ethyl)acrylamide (PP1-11): 1H-NMR( 400 MHz,
DMSO-d6) ꢀ1.43-1.53 (m, 11H); 1.87-1.90 (m, 2H); 4.19 (s, 1H);
6.53 (d, 1H); 7.12 (dd, 1H); 7.33 (t, 1H); 7.42 (d, 1H); 7.64-7.78
(m, 3H); 8.21 (d, 1H); 8.88 (d, 2H). MS (ESI) 453.0 (M+).
N-(2,2,2-Trichloro-1-(3-(2-chlorophenyl)thioureido)ethyl)
1
cinnamamide (PP1-1): H-NMR (400MHz, DMSO-d6) ꢀ6.82 (d,
J=16Hz, 1H), 7.24-7.28 (m, 1H), 7.32-7.48 (m, 5H), 7.52-7.63 (m,
4H), 7.73 (d, J=8Hz, 1H), 8.60 (d, J=8Hz, 1H), 9.09 (d, J=8Hz,
1H), 9.96 (s, 1H). MS (ESI) 463.9(M+).
(E)-3-(Thiophen-2-yl)-N-(2,2,2-trichloro-1-(3-(2-methoxy
phenyl)thioureido)ethyl)acrylamide(PP1-12):1H-NMR
(400
MHz, DMSO-d6) ꢀ3.83 (s, 3H); 6.54 (d, 1H); 6.90-7.14 (m, 4H);
7.41-7.45 (m, 2H); 7.65-7.72 (m, 2H); 7.91 (d, 1H); 8.48 (s, 1H);
9.01 (d, 1H); 9.80 (s, 1H). MS (ESI) 464.0 (M+). HRMS (ESI):
Calcd for C17H17Cl3N3O2S2 (M+): 463.9822. Found 463.9829.
Anal. Calcd for C17H16Cl3N3O2S2: C 43.93, H 3.47, N 9.04. Found:
C 44.09, H 3.58, N 8.93.
N-(2,2,2-Trichloro-1-(3-(4-methylphenyl)thioureido)ethyl)
cinnamamide (PP1-2): H-NMR (400 MHz, DMSO-d6) ꢀ2.22 (s,
1H); 6.63-6.67 (t, 1H); 6.93 (d, 1H); 7.05 (d,2H); 7.32 (d, 2H);
7.40-7.44 (m, 2H); 7.48-7.61 (m, 7H); 9.20 (d, 1H); 9.60 (s, 1H).
MS (ESI) 448.0(M+).
1
N-(2,2,2-Trichloro-1-(3-(2,3-bimethyl)phenylthioureido)
ethyl)cinnamamide (PP1-3): 1H-NMR (400 MHz, DMSO-d6)
ꢀ2.08 (s, 3H); 2.26 (s, 3H); 6.73 (d, 1H); 7.05-7.13 (m, 3H); 7.41-
7.46 (m, 4H); 7.55 (d, 1H); 7.61-7.13 (m, 2H); 8.99 (d, 1H); 9.90 (s,
1H). MS (ESI) 456.0(M+).
(E)-3-(Thiophen-2-yl)-N-(2,2,2-trichloro-1-(3-benzylthiou
reido)ethyl)acrylamide (PP1-13): 1H-NMR (400 MHz, DMSO-
d6) ꢀ4.68 (d, 2H); 6.54 (d, 1H); 7.13 (dd, 1H); 7.31-7.43 (m, 7H);
7.64-7.71 (m, 2H); 8.01 (d, 1H); 8.62 (t, 1H); 8.95 (d, 1H). MS
(ESI) 447.9 (M+).
N-(2,2,2-Trichloro-1-(3-(2-methoxy)phenylthioureido)ethyl)
1
N-(2,2,2-trichloro-1-(3-(4-methoxylbenzyl)thioureido)ethyl)
cinnamamide (PP1-14): 1H-NMR (400 MHz, DMSO-d6) ꢀ3.72 (s,
3H); 4.59 (d, 2H); 6.79 (d, 1H); 6.91 (d, 2H); 7.25 (d, 2H); 7.36-
7.46 (m, 4H); 7.52-7.60 (m, 3H); 7.97 (d, 1H); 8.55 (t, 1H); 8.98 (d,
1H). MS (ESI) 472.8 (M+). Calcd for C20H20Cl3N3O2S1 (M+):
472.0300. Found 472.0412.
cinnamamide (PP1-4): H-NMR (400 MHz, DMSO-d6) ꢀ3.83 (s,
3H); 6.54 (d, 1H); 6.90-7.14 (m, 4H); 7.41-7.45 (m, 2H); 7.65-7.72
(m, 2H); 7.91 (d, 1H); 8.48 (s, 1H); 9.01 (d, 1H); 9.80 (s, 1H). MS
(ESI) 456.0(M+).
(E)-3-(Thiophen-2-yl)-N-(2,2,2-trichloro-1-(3-quinolin-8-ylt
hioureido)ethyl)acrylamide(PP1-5):1H-NMR(400 MHz, DMSO-
d6) ꢀ 6.65 (d, 1H); 7.13 (q, 1H); 7.43 (d, 1H); 7.53-7.73 (m, 6H);
8.43 (dd, 1H); 8.95-9.03 (m, 3H); 9.55 (d, 1H); 11.03 (s, 1H). MS
(ESI) 487.0 (M+).
N-(2,2,2-Trichloro-1-(3-morpholin-1-ylthioureido)ethyl)cin
1
namamide (PP1-15): H-NMR (400 MHz, DMSO-d6) ꢀ3.61-3.64
(m, 4H); 3.83 (m, 4H); 6.80 (d, 1H); 7.40-7.45 (m, 3H); 7.52-7.45
(m, 4H); 7.90 (d, 1H); 8.35(d, 1H). MS (ESI) 421.9(M+). Calcd for
C16H19Cl3N3O2S1 (M+): 422.0258. Found 422.0265.
(E)-3-(Thiophen-2-yl)-N-(2,2,2-trichloro-1-(3-(4-methylphe
nyl)thioureido)ethyl)acrylamide (PP1-6): 1H-NMR (400 MHz,

6074 Current Medicinal Chemistry, 2012 Vol. 19, No. 35
Liu et al.
N-(2,2,2-Trichloro-1-(3-(4-methylpiperazin-1-yl)thioureido)
ethyl)cinnamamide (PP1-16): 1H-NMR (400 MHz, DMSO-d6)
ꢀ2.18 (s, 3H); 2.31-2.40 (m, 4H); 3.78-3.83 (m, 4H); 6.78 (d, 1H);
7.41-7.45 (m, 3H); 7.52-7.60 (m, 2H); 7.65-7.67 (m, 2H); 7.84 (d,
1H); 8.33 (d, 1H). MS (ESI) 435.3 (M+).
the mixture was stirred at r.t. for 3 h. The organic layer was
then washed by brine and evaporated to give a brown oil, which
was transformed into ꢁ-bromoketone by reflux with CuBr2 (20
mmol) in acetic ether (10 mL) and CHCl3 (10 mL). The mixture
was filtrated and the filtrate was evaporated. The obtained oil was
added to thiourea (10 mmol) and refluxed in isopropanol (10 mL)
for 1.5 h until a pale yellow solid precipitated. After being filtrated,
Synthesis of Substituted Acrylamide Derivatives (PP1-17, 18)
the solid was added into
a solution of N-1, 2, 2, 2-
General procedures N-2, 2, 2-tetrachloro-1-amino ethylacryla-
mide (10 mmol) was added with various substituted acrylamides
(10 mmol) and following catalysts: HoBT (15 mmol), DIEA (20
mmol), EDCI (15 mmol) and DMAP (10 mmol). After stirring in
dry DMF for 24 h, the mixture was poured into 50 mL water, lead-
ing to the precipitation of a white solid, yielding compounds PP1-
17, 18.
tetrachloroethylacrylamide (10 mmol) in dry DMF and stirred for
24 h. The mixture was poured into 50 mL water and extracted by
acetic ether. The combined organic layer was evaporated and the
residue obtained was purified by chromatography over silica gel
using petroleum ether-acetic ether (4:1) to obtain compounds PP1-
(23-27).
N-(2,2,2-Trichloro-1-(4-phenylthiazol-2-ylamino)ethyl)cinna
mamide (PP1-23): 1H-NMR (400 MHz, DMSO-d6) ꢀ6.93 (d, 1H);
6.98 (t, 1H); 7.22 (s, 1H); 7.29 (t, 1H); 7.38-7.43 (m, 5H); 7.55-7.59
(m, 3H); 7.86 (dd, 2H); 8.68 (d, 1H); 9.02 (d, 1H). MS (ESI) 453.6
(M+).
(E)-3-(Furan-2-yl)-N-(2,2,2-trichloro-1-cinnamamidoethyl)
1
acrylamide (PP1-17): H-NMR (400 MHz, DMSO-d6) ꢀ6.61-6.62
(m, 1H); 6.72 (d, 1H); 6.83-6.88 (m, 2H); 6.96 (d, 1H); 7.36 (d,
1H); 7.40-7.46 (m, 3H); 7.55 (d, 1H); 7.59-7.61 (m, 2H); 7.82 (d,
1H); 8.93 (d, 1H); 9.00 (d, 1H). MS (ESI) 413.0 (M+).
N-(2,2,2-Trichloro-1-(4-(4-chlorophenyl)thiazol-2-ylamino)
ethyl)cinnamamide(PP1-24):1H-NMR(400MHz, DMSO- d6)
ꢀ6.93 (d, 1H); 6.98 (d, 1H); 7.30 (s, 1H); 7.40-7.48 (m, 5H); 7.55-
7.60 (m, 3H); 7.87-7.89 (m, 2H); 8.74 (d, 1H); 9.04 (d, 1H). HRMS
(ESI): Calcd for C20H16Cl4N3OS (M+): 485.9763. Found 485.9754.
Anal. Calcd for C20H15Cl4N3OS: C 49.30, H 3.10, N 8.62; Found: C
49.30, H 3.15, N 8.56.
(E)-3-(Pyridin-4-yl)-N-(2,2,2-trichloro-1-cinnamamidoethyl)
1
acrylamide (PP1-18): H-NMR (400 MHz, DMSO-d6) ꢀ6.84-6.89
(m, 1H); 6.95 (d, 1H); 7.40-7.46 (m, 3H); 7.53-7.61 (m, 6H); 8.64
(d, 1H); 8.98 (d, 1H); 9.12 (d, 1H). MS (ESI) 424.0 (M+).
Synthesis of N,ꢀN-Asymmetrically Substituted Urea Derivatives
PP1-(19-22)
N-(2,2,2-Trichloro-1-(4-(2,4-bifluorophenyl)thiazol-2-ylami
no)ethyl)cinnamamide (PP1-25): 1H-NMR (400 MHz, DMSO-d6)
ꢀ6.92 (d, 1H); 6.97 (t, 1H); 7.11 (d, 1H); 7.20-7.24 (m, 1H); 7.30-
7.34 (m, 1H); 7.39-7.45 (m, 3H); 7.56-7.60 (m, 3H); 8.08-8.12 (m,
1H); 8.77 (d, 1H); 9.05 (d, 1H). HRMS (ESI): Calcd for
C20H15Cl3F2N3OS (M+): 487.9964. Found 487.9956. Anal. Calcd
for C20H14Cl3F2N3OS: C 49.15, H 2.89, N 8.60. Found: C 49.11, H
2.93, N 8.61.
General procedures The synthon N-1, 2, 2, 2-tetra chloroethy-
lacrylamide (10 mmol) was converted to N-2, 2, 2-tetrachloro-1-
aminoethylacrylamide by resolving in THF (20 mL) and added to
cooled ammonia water. After stirring for 0.5 h, acetic ether was
used for extraction, and the organic layer was evaporated to give a
yellow solid. The solid was then resolved in dioxane and a solution
of triphosgene (10 mmol) in dioxane was added dropwise to it.
After refluxing overnight, various amines were added to the mix-
ture and refluxed for 2 h to yield compounds PP1-(19-22).
N-(2,2,2-Trichloro-1-(4-(4-methoxyphenyl)thiazol-2-ylami
no)ethyl)cinnamamide (PP1-26): 1H-NMR (400 MHz, DMSO-d6)
ꢀ3.76 (s, 3H); 6.93 (d, 1H); 6.98 (t, 1H); 7.20 (s, 1H); 7.34-7.39 (m,
5H); 7.53-7.57 (m, 3H); 7.84 (dd, 2H); 8.66 (d, 1H); 9.00 (d, 1H).
MS (ESI) 483.4 (M+).
N-(2,2,2-Trichloro-1-(3-quinolin-8-ylureido)ethyl)cinnama
mide (PP1-19): 1H-NMR (400 MHz, DMSO-d6) ꢀ6.77 (t, 1H);
6.93 (d, 1H); 7.40-7.44 (m, 3H); 7.53-7.55 (m, 2H); 7.59-7.64 (m,
4H); 8.38 (dd, 1H); 8.51 (dd, 1H); 8.57 (d, 1H); 8.91 (dd, 1H); 9.08
(d, 1H); 9.98 (s, 1H). MS (ESI) 441.0 (M+).
N-(2,2,2-Trichloro-1-(4-(4-methoxyphenyl)-5-phenylthiazol-
2-ylamino)ethyl)cinnamamide (PP1-27):1H-NMR (400MHz,
DMSO-d6) ꢀ3.74 (s, 3H); 6.85 (dd, 2H); 6.94-6.98 (m, 1H); 7.26-
7.46 (m, 6H); 7.58 (t, 1H); 8.72 (d, 1H); 9.04 (d, 1H). HRMS (ESI):
Calcd for C27H23Cl3N3O2S (M+): 558.0571. Found 558.0563.
N-(2,2,2-Trichloro-1-(3-p-tolylureido)ethyl)cinnamamide
1
(PP1-20): H-NMR (400 MHz, DMSO-d6) ꢀ2.22 (s, 1H); 6.65 (t,
1H); 6.93 (d, 1H); 7.05 (d, 2H); 7.32 (d, 2H); 7.42 (d, 2H); 7.40-
7.61 (m, 7H); 9.19 (d, 1H); 9.59 (s, 1H). MS (ESI) 426.0 (M+).
Biological Evaluation
N-(2,2,2-Trichloro-1-(3-(2-methoxyphenyl)ureido)ethyl)cin
namamide (PP1-21): 1H-NMR (400 MHz, DMSO-d6) ꢀ3.85 (s,
3H); 6.66 (t, 1H); 6.85-7.01 (m, 4H); 7.40-7.46 (m, 3H); 7.53-7.62
(m, 3H); 7.92 (d, 1H); 8.05 (dd, 1H); 8.58 (s, 1H); 9.02 (d, 1H). MS
(ESI) 441.0 (M+). HRMS (ESI): Calcd for C19H19Cl3N3O3 (M+):
442.0486. Found 442.0485.
Cell Culture and Treatments
Primary cultures of neonatal rat cardiomyocytes were isolated
from the ventricles of 1-day-old Wistar rats (Academy of Military
Medical Sciences, China). Briefly, rats were euthanized and their
hearts were excised. After scalpel homogenization, hearts were
digested with 0.125% trypsin and 0.1% collagenase II (4:1) at 37
°C for 10 min (repeated 4-5 times). Isolated cells were then col-
lected by centrifugation and resuspended in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal bovine serum
(FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. Cul-
tures were enriched for myocytes by preplating for 90 min to de-
plete the population of non-myocytes. Nonattached cells were
plated onto plastic culture dishes at an appropriate cell density. The
cells were cultured at 37 °C in a 95% air/5% CO2 atmosphere for
24 h, and then half of the liquid was changed to DMEM containing
10% FBS with 0.1% BrdU. Every 48 h the culture medium was
changed. All animal experiments were performed in accordance
with the ethical regulations of NUI. All reagents were obtained
from Gibco, unless otherwise stated.
N-(2,2,2-Trichloro-1-(3-(2-chlorophenyl)ureido)ethyl)cinna
1
mamide (PP1-22): H-NMR (400 MHz, DMSO-d6) ꢀ6.64 (t, 1H);
6.87 (d, 1H); 7.01-7.04 (m, 1H); 7.26-7.30 (m, 1H); 7.41-7.45 (m,
4H); 7.57 (d, 1H); 7.60-7.66 (m, 2H); 8.08-8.13 (m, 2H); 8.65 (s,
1H); 9.08 (d, 1H). MS (ESI) 445.0 (M+).
Synthesis of Thiazole Derivatives PP1-(23-27)
General procedures Substituted acetic acid (10 mmol) was re-
fluxed in SOCl2 (10 mL) for 2 h and the solvent was then evapo-
rated to give the corresponding acyl chloride, which was then added
dropwise to a cooled mixture of substituted benzene (10 mmol) and
AlCl3 (20 mmol) resolving in dry CHCl3 (20 mL). After addition,

SAR, Cardiac Myocytes Protection Activity and 3D-QSAR Studies
Current Medicinal Chemistry, 2012 Vol. 19, No. 35
6075
MTT Assay
Partial Least Squares (PLS) Analysis and Models Validation of
3D-QSAR Model
Cell viability was assessed by the methylthiazoletetrazolium
(MTT) (Amresco, USA) assay. Ten microliters of 5 mg/mL MTT
was added to each well for 4 hours, and then supernatants were
discarded and 100 μL of DMSO (Amresco, USA) was added. After
a 10-minute oscillation, the absorbance (A value) of each well at
550 nm was detected with a Universal Microplate Reader (Bio-Tek
instrument, Inc., USA). The relative number of viable cells was
determined by setting the value for untreated cells as 100%.
Partial Least Squares (PLS) analysis was used to derive a linear
correlation between the 3D-fields and their predicted ability to pro-
tect neonate cardiac myocytes from Tm-induced apoptosis. To se-
lect the best model, cross-validation analysis was performed using
the leave-One-Out (LOO) method, in which one compound was
removed from the data set and its activity was predicted using the
model built from rest of the data set. This analysis resulted in the
cross-validation correlation coefficient (q2) and the optimum num-
ber of components (N). The non-cross-validation was performed
with a column filter value of 2.0 to speed up the analysis and reduce
the noise.
Cell Viability Assay. A luminescent signal indicating ATP lev-
els was used as a marker of cell proliferation and viability. The
amount of ATP present was estimated with the use of a CellTiter-
Glo Luminescent Cell Viability Assay kit (Promega, USA) accord-
ing to the manufacturer’s protocol. Briefly, the media was equili-
brated to room temperature for approximately 30 minutes. A vol-
ume of CellTiter-Glo Reagent equal to the volume of cell culture
medium present in each well was added, and the mixture was mixed
for 2 minutes on an orbital shaker to induce cell lysis. The plate was
allowed to incubate at room temperature for 10 minutes to stabilize
the luminescent signal. ATP levels were measured and data were
normalized to the control group. The cytotoxicity of lectin was
determined as the percentage decrease in luminescence relative to
the control.
RESULTS
Salubrinal, as well as compounds PP1-(1-16), which were
modified on the quinoline ring terminus, were initially synthesized
as shown in Scheme 1 [12]. Reaction of commercially available
benzaldehyde and malonic acid yielded cinnamic acid 1, which was
then treated with thionyl dichloride and subsequently transformed
into the corresponding amine 2. Addition of 2 to chloral hydrate
resulted in the synthesis of chloralamide 3, which was then chlori-
zated by thionyl dichloride to afford the (2E)-N-2, 2, 2-trichloro-1-
chloroethyl-cinnamine 4, a key synthon for further elaboration.
Substitution reaction of 4 with potassium thiocyanate gave rise to
isothiocyanate 5, which, by reaction with various amines, was con-
verted to the desired compound salubrinal as well as PP1-(1-16).
Next, to investigate the thiourea group, we synthesized acrylamide
derivatives PP1-17 and PP1-18c as well as urea derivatives PP1-
(19- 22), as shown in Scheme 2. Via transforming 4 to 6, followed
by reactions with triphosgene to give isocyanate 7, PP1-(19-22)
were prepared. Condensation of 6 and acrylic acids yielded PP1-17
and 18.
Western Blot Analysis
Cells were lysed in whole cell lysis buffer (62.5 mM Tris-HCl
[pH 6.8 at 25 °C], 2% w/v SDS, 10% glycerol, 50 mM DTT) and
the homogenates were heated in 100 °C water for 10 min, followed
by centrifugation at 12000 g for 10 min at 4 °C. The supernatants
were used as protein samples. We used BCA to determine the pro-
tein concentration of each sample. Cellular proteins were separated
by electrophoresis on 10% SDS-polyacrylamide gels and trans-
ferred onto PVDF membranes. After blocking (1x TBS, 0.1%
Tween-20, 5% w/v nonfat dry milk), membranes were incubated
with antibodies against phospho-eIF2ꢀ, eIF-2ꢀ and ꢁ-actin (all ob-
tained from Cell Signaling Technology, 1:1000, rabbit polyclonal)
with gentle agitation overnight at 4 °C. Membranes were then incu-
bated with the appropriate horseradish peroxidase-conjugated sec-
ondary antibodies (Zhongshan Golden Bridge biotechnology CO.)
at a 1:5000 dilution, for 1 h at room temperature. The protein bands
were visualized by enhanced chemiluminescence detection reagents
(Applygen Technologies Inc., Beijing, China) and scanned with an
imaging densitometer (Alphamager5500, Alpha Innotech, USA).
The optical density was quantified using Multi-Analyst software.
All compounds were evaluated for their cardioprotective activ-
ity (ability to protect neonate cardiomyocytes from Tm-induced
apoptosis) via MTT assay [14, 15]. With salubrinal serving as a
positive control, the results are expressed as EC50 values (defined as
the concentration at which the compound produces a half-maximal
rescue from cell death versus the Tm-only control) in (Table 1).
As shown in (Table 1), compound PP1-5 had an EC50 close to
that of salubrinal, which means that as R1 substituents, the thienyl
and phenyl groups yield the same potency. Compounds PP1-4 and
PP1-12 showed best efficacy, with EC50 values of 6 and 4 μM,
respectively. Compounds PP1-7, 9, 13 had EC50 values close to that
of salubrinal. On the other hand, PP1-1, 8, 10, 11 were much less
potent, and PP1-2, 3, 6 were found to be cardiotoxic. Thus, as R2
substituents, 2-methoxyphenyl and morpholinyl groups improved
the activity, though the methylphenyl substitution resulted in toxic-
ity; other substituents, such as fluorophenyl, cycloalkyl, alkyl or
carbonyl, were much less potent. PP1-15, with a morpholinyl
group, showed higher efficacy than salubrinal; however, PP1-16,
with a 4-methylpiperazinyl group, was much less active. Based on
our observations of PP1-(19-22), we found that the replacement of
thiourea unit by urea was merely tolerated, while the data on PP1-
18 implied that the nitrogen linking the R2 terminus was not indis-
pensable.
D-QSAR Analysis
Data Set and Alignment
3D-QSAR studies were performed using SYBYL 6.9 molecular
modeling software. All parameters used in CoMFA, with the excep-
tion of those explained, were default. Active conformation selection
is a key step for 3D-QSAR analyses. Since the crystal structure of
the PP1/GADD34 complex with one of these compounds is not
available, a conformational search was performed with Chem3D
software to predict the global energy-minimum conformation for
salubrinal by rotating rotatable bonds. The rest of the molecules
were built by changing the substitutions of salubrinal which were
performed with the same way. Finally, Gasteiger-Hückel charges
were assigned to all molecules. Structural alignment is considered
to be one of the most sensitive parameters in CoMFA analysis. The
predictive accuracy of the CoMFA model and reliability of the
contour maps are directly dependent on the structural alignment
rule. Salubrinal was used as a template for superimposition, the
common fragment with (2E)-N-2,2,2-trichloro-1-ethylacrylamide
scaffold was selected for alignment, and all the molecules were
aligned on it.
We confirmed that compound PP1-12 indeed acts as a potent
inhibitor of eIF2ꢀ phosphorylation. As shown in Figs. (2 and 3),
respectively, eIF2ꢀ expression levels and its phosphorylation status
were monitored by immunoblotting, while CHOP expression was
examined by fluorescence microscopy [16].
Based on the above SAR, we designed and synthesized a series
of novel thiazole derivatives, PP1-(23-27), which are shown in
Scheme 3. The biological activities of these compounds are shown
in (Table 2). Starting from substituted acetic acid, via Friedel-Crafts

6076 Current Medicinal Chemistry, 2012 Vol. 19, No. 35
Liu et al.
O
1
O
2
O
3
CCl3
OH
CHO
R1
R1
a
OH
R1
NH2
R1
N
c
d
b
H
O
5
CCl3
NCS
O
CCl3
S
O
CCl3
Cl
R1
N
H
R1
N
N
R2
R1
N
H
H
e
f
H
PP1-(1-16), salubrinal
4
Scheme 1. Synthetic pathway of salubrinal and derivatives PP1-(1-16).
Reagents and conditions: a: malonic acid, pyridine, piperidine, reflux, 6 h; b: (1) SOCl2, THF; (2) NH3·H2O; c: CH(OH)2CCl3, toluene, reflux, 4 h; d: (1)
SOCl2, THF, 2 h; e: KSCN, acetone, reflux, 2 h; (f) R2NH2, THF, reflux, 2 h.
Table 1.
Structures, Experimental EC50 Values of Salubrinal and Derivatives PP1-(1-22)
CCl3
O
X
R1
N
H
N
R2
H
Compounds
R1
R2
X
EC50(μM)
Salubrinal
PP1-1
Phenyl
Phenyl
Quinolin-8-amino
2-chlorophenylamino
4-methylphenylamino
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
O
O
O
O
O
O
15
31
PP1-2
Phenyl
Toxica
Toxica
6
PP1-3
Phenyl
2,3-bimethylphenylamino
2-methoxyphenylamino
Quinolin-8-amino
PP1-4
Phenyl
PP1-5
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Thien-2-yl
Phenyl
16
PP1-6
4-methylphenylamino
Cyclohexylamino
Toxica
PP1-7
21
PP1-8
3-isopropoxypropylamino
43
PP1-9
2-methoxycarbonylphenylamino
2-fluorophenylamino
Cycloheptylamino
19
PP1-10
PP1-11
PP1-12
PP1-13
PP1-14
PP1-15
PP1-16
PP1-17
PP1-18
PP1-19
PP1-20
PP1-21
PP1-22
40
31
2-methoxyphenylamino
Benzylphenylamino
4-methoxybenzylamino
Morpholin-1-yl
4
25
30
Phenyl
10
Phenyl
4-methylpiperazin-yl
3-(2-furyl)propen-1-yl
3-(4-pyridyl)propen-1-yl
Quinolin-8-amino
56
Phenyl
Toxica
Phenyl
44
Phenyl
23
Phenyl
4-methylphenylamino
2-methoxyphenylamino
2-chlorophenylamino
Toxica
Phenyl
20
Phenyl
57
a: Compd PP-2, Compd PP-3 and Compd PP-6 90% dead cells at 50 ꢀM, Compd PP-6 and Compd PP-17 90% dead cells at 30 ꢀM.
O
4
CCl3
Cl
O
CCl3
NH2
R1
N
R1
N
H
b
d
H
a
O
CCl3
O
6
c
R1
N
N
R2
O
CCl3
NCO
H
H
R1
N
H
PP1-17,18
PP1-(19-22)
7
Scheme 2. Synthetic pathway of derivatives PP1-17, 18 and PP1-(19-22).
Reagents and conditions: a: NH3·H2O, 30 min; b: EDCI, HOBt, DMAP, DIEA, R2COOH, 24 h; c: triphosgene, CH2Cl2, 12 h; d: R2NH2, CH2Cl2, 2 h.

SAR, Cardiac Myocytes Protection Activity and 3D-QSAR Studies
Current Medicinal Chemistry, 2012 Vol. 19, No. 35
6077
Fig. (2). Western blot assessed ER stress related protein: eIF2ꢀ and p-eIF2ꢀ of PP1-12.
Fig. (3). CHOP expression in neonatal rat cardiomyocytes of PP1-12.
O
O
R2
OH
Br
R3
a
b
c
O
R3
R3
R4
R4
9
8
R3
R3
S
O
CCl3
S
R4
NH2
4
N
N
N
N
d
H
H
R4
10
PP1-(23-27)
Scheme 3. Synthetic pathway of compounds PP1-(23-27).
Reagents and conditions: a: AlCl3, CHCl3, 3 h; b: CuBr2, CHCl3, ethyl acetate, 2 h; c: thiourea, isopropanol, 1 h; d: DMF, K2CO3, 24 h.
Table 2.
Structures and EC50 Values of Compounds PP1-(23-27)
Compounds
R3
R4
EC50 (μM)
PP1-23
PP1-24
PP1-25
PP1-26
PP1-27
H
H
phenyl
4-chloro
30
ꢁ0.3
ꢁ0.8
61
H
2,4-bifluoro
4-methoxy
4-methoxy
H
phenyl
Toxica
a: Compd PP-27 90% dead cells at 30 ꢂM,
acylation, bromination, and cyclization reaction with thiourea, 10
was obtained. Reaction of 10 and 4 yielded PP1-(23-27).
PP1-23, which has an unsubstituted phenyl, compound PP1-25,
which has a bifluorophenyl group, provided a much better im-
provement, though compound PP1-26, with 4-methoxyphenyl, had
the opposite effect. 4,5-bisubstituted thiazole derivative PP1-27
was found to be cardiotoxic.
Compound PP1-24 was 50-fold more potent than salubrinal,
with significantly enhanced solubility. Compared with compound

6078 Current Medicinal Chemistry, 2012 Vol. 19, No. 35
Table 3. Cross-Validated Analyses of the CoMFA Models Using Salubrinal As the Structural Template
Liu et al.
Field Contribution
Steric Electrostatic
Method
SE
Compounds.
q2
r2
F
CoMFA
0.081
n1 = 6, n2 = 12
0.741
0.991
215.45
0.670
0.330
Table 4.
Experimental and CoMFA-Predicted pEC50 Values of Salubrinal and PP1-(1-26), in Both Training Set and Test Set
EC50
(μM)
Predict
Value
EC50
(μM)
Predict
Value
Compounds
pEC50
ꢀ
Compound
pEC50
ꢀ
Salubrinal
PP1-1
15
31
4.82
4.51
4.00
4.00
5.22
4.80
4.00
4.68
4.37
5.05
4.40
4.51
5.52
4.81
4.50
3.97
3.98
5.25
4.90
4.09
4.64
4.43
5.07
4.65
4.87
5.37
0.01
0.01
PP1-13
PP1-14
PP1-15*
PP1-16*
PP1-17*
PP1-18
PP1-19
PP1-20
PP1-21
PP1-22*
PP1-23*
PP1-24
PP1-25
25
30
4.60
4.52
5.00
4.25
4.00
4.35
4.64
4.00
4.79
4.24
4.52
6.52
6.09
4.51
4.56
4.92
4.56
5.36
4.34
4.66
3.91
4.87
4.43
6.28
6.47
6.14
0.09
-0.04
0.08
PP1-2
Toxic
Toxic
6
0.03
10
PP1-3
0.02
56
-0.31
-1.36
0.01
PP1-4
-0.03
-0.10
-0.09
0.04
Toxic
44
PP1-5
16
PP1-6
Toxic
21
23
-0.02
0.09
PP1-7
Toxic
20
PP1-8
43
-0.05
-0.02
-0.25
-0.36
0.15
-0.06
-0.19
-1.76
0.05
PP1-9
19
57
PP1-10*
PP1-11*
PP1-12
40
30
31
0.3
0.8
4
-0.05
Fig. (4). A Alignment of all compounds used in the training set; B CoMFA contour maps for the steric field: Green/yellow contours indicate regions where
steric bulky groups increase/decrease activity. Favored and disfavored levels of these displayed fields are fixed at 80% and 20%, respectively; C Electrostatic
field: Red/blue contours indicate regions where negative charge increase/decrease activity. Favored and disfavored levels of these displayed fields are fixed at
85% and 15%, respectively.

SAR, Cardiac Myocytes Protection Activity and 3D-QSAR Studies
Current Medicinal Chemistry, 2012 Vol. 19, No. 35
6079
To guide further modification, 3D-QSAR studies of all evalu-
ated compounds were subsequently performed using SYBYL 6.9
molecular modeling software. Molecules that were toxic were se-
lected for this study to identify the structural features responsible
for their toxicity. The EC50 value for all these molecules was set to
100 μM [17, 18]. The data set was randomly divided into test and
training sets that covered the activity range. For structural align-
ment, salubrinal was used as a template for superimposition, the
common fragment with (2E)-N-2,2,2-trichloro-1-ethylacrylamide
scaffold was selected for alignment, and all the molecules were
aligned on it. Twenty-six compounds (including salubrinal) with
available EC50 values were employed for 3D-QSAR analysis. Nine-
teen compounds (molecules listed in (Table 4) without an asterisk)
were selected as the training set for model construction, and the
remaining 7 compounds (asterisked molecules listed in Table 4)
were employed as the testing set for model validation. The align-
ment of all compounds used in the training set is shown in Fig.
(4A).
ABBREVIATIONS
SAR
=
=
Structure-Activity Relationship
3D-QSAR
Three Dimensional Quantitative Structure Ac-
tivity Relationships
ER
=
=
Endoplasmic Reticulum
UPR
Unfolded Protein Response
PP1/GADD34 = Protein Phosphatase 1/Growth Arrest and DNA
Damage-Inducible Gene34
EC50
MTT
=
=
Concentration for 50% of Maximal Effect
Methylthiazoletetrazolium
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CONFLICT OF INTEREST
The author(s) confirm that this article content has no conflicts
of interest.
ACKNOWLEDGEMENTS
Deswal, S.; Roy, N. A novel range based QSAR study of human neuropep-
tide Y (NPY) Y5 receptor inhibitors. Eur. J. Med. Chem., 2007, 42, 463–470.
We are grateful for the financial supports of the National Key
Technology R&D Program (2011BAI18B01) of China.
Received: May 15, 2012
Revised: September 20, 2012
Accepted: September 26, 2012