Synthesis and anti-endoplasmic reticulum stress activity of N-substituted-2-arylcarbonylhydrazinecarbothioamides

Article abstract of DOI:10.1007/s00044-019-02442-1

Misfolded or unfolded proteins are accumulated in lumen of endoplasmic reticulum (ER) in ER stress condition. It has been implicated in many pathological conditions such as Alzheimer’s disease, diabetic retinopathy, atherosclerosis, β-cell apoptosis and lung inflammation. We found a series of N-substituted-2-arylcarbonylhydrazinecarbothioamides to potently decrease ER stress signal, showing up to almost 300-fold better activity than 1-hydroxynaphthoic acid and tauro-ursodesoxycholic acid, positive controls, respectively. Structure?activity relationship (SAR) study showed that 2-arylcarbonyl moiety is critical for the activity of the hydrazinecarbothioamide analogues and side chains tethering on thioamide moiety were relatively insensitive to the activity. Some analogues were found to consistently exert the potency under more physiologically relevant condition where ER stress was induced by palmitic acid. ER stress markers such as CHOP and phosphorylated eIF2α and PERK were accordingly decreased in western blotting upon treatment of compound 4h. Potential ER stress inhibitory activity and novel structures could provide a novel platform for new chemical chaperone and therapy for protein misfolding diseases.

Full text of DOI:10.1007/s00044-019-02442-1

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02442-1
ORIGINAL RESEARCH
Synthesis and anti-endoplasmic reticulum stress activity of N-
substituted-2-arylcarbonylhydrazinecarbothioamides
Hoon Choi1 Wheesahng Yun1 Jung-hun Lee2 Seoul Jang1 Sang Won Park1 Dong Hwan Kim1
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Kyoung Pyo Seon1 Jung-mi Hyun2 Kwiwan Jeong2 Jin-mo Ku2 Tae-gyu Nam
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Received: 21 May 2019 / Accepted: 5 September 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Misfolded or unfolded proteins are accumulated in lumen of endoplasmic reticulum (ER) in ER stress condition. It has been
implicated in many pathological conditions such as Alzheimer’s disease, diabetic retinopathy, atherosclerosis, β-cell
apoptosis and lung inflammation. We found a series of N-substituted-2-arylcarbonylhydrazinecarbothioamides to potently
decrease ER stress signal, showing up to almost 300-fold better activity than 1-hydroxynaphthoic acid and tauro-
ursodesoxycholic acid, positive controls, respectively. Structure−activity relationship (SAR) study showed that 2-
arylcarbonyl moiety is critical for the activity of the hydrazinecarbothioamide analogues and side chains tethering on
thioamide moiety were relatively insensitive to the activity. Some analogues were found to consistently exert the potency
under more physiologically relevant condition where ER stress was induced by palmitic acid. ER stress markers such as
CHOP and phosphorylated eIF2α and PERK were accordingly decreased in western blotting upon treatment of compound
4h. Potential ER stress inhibitory activity and novel structures could provide a novel platform for new chemical chaperone
and therapy for protein misfolding diseases.
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Keywords Endoplasmic reticulum stress 2-Arylcarbonylhydrazinecarbothioamides Chemical chaperone Misfolded
protein
Introduction
Folding of newly synthesized proteins primarily takes place
in endoplasmic reticulum (ER). In a condition where
unfolded or misfolded peptides are accumulated inside ER
(ER stress), a series of cellular events, known as unfolded
protein responses (UPRs) or ER stress responses, are trig-
hun Lee
These authors contributed equally: Hoon Choi, Wheesahng Yun, Jung-
gered in order to adapt the cellular damages caused by ER
stress condition (Walter and Ron 2011). They consist of the
following canonical cellular processes: (i) translational
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02442-1) contains supplementary
material, which is available to authorized users.
attenuation, (ii) transcriptional activation of chaperones and
degradation factors, (iii) ER-associated degradation
(ERAD), and (iv) apoptosis. For example, expression of
chaperone molecules, such as GRP78, is upregulated to
* Kwiwan Jeong
assylum@gbsa.or.kr
* Jin-mo Ku
medichem@gbsa.or.kr
assist the protein folding process and to reduce the load of
* Tae-gyu Nam
the defect peptides.
tnam@hanyang.ac.kr
ER stress can be monitored by three representative and
evolutionarily conserved ER-resident sensor molecules:
1
Department of Pharmacy and Institute of Pharmaceutical Science
protein kinase RNA-activated (PKR)-like ER kinase
(PERK), activating transcription factor 6 (ATF6), and
inositol-requiring enzyme 1α (IRE1α), which relay complex
and interrelated downstream signal cascade deciding
and Technology, Hanyang University, 55 Hanyangdaehak-ro,
Ansan, Gyeonggi-do 15588, Republic of Korea
2
Bio-Center, Gyeonggido Business & Science Accelerater, 147
Gwanggyo-ro, Suwon, Gyeonggi-do 16229, Republic of Korea

Medicinal Chemistry Research
‘survival’ (adaptive signalling) or ‘suicide’ (apoptotic sig-
nalling) (Xu et al. 2005). The activated PERK phosphor-
ylates eukaryotic translation initiation factor 2α (eIF2α)
which attenuates protein synthesis to reduce ER protein
overloading (Harding et al. 1999) and also increases the
expression of activating transcription factor 4 (ATF4) to
augment the expression of many genes including apoptosis
inducing factors i.e. C/EBP homology protein (CHOP)
(Harding et al. 2003). ATF6 is also involved in activating
the transcription of many UPR mediators including an ER
chaperone protein, glucose-regulated protein (GRP78)
(Haze et al. 1999). IRE1α, a serine/threonine kinase, pro-
cesses X-box binding protein 1 (XBP1) mRNA through its
intrinsic endoribonuclease activity to produce active spliced
form which becomes a competent transcription factor for
UPR-related genes.
ER stress condition has been implicated in various dis-
eases and pathologies such as Alzheimer’s disease, diabetic
retinopathy, atherosclerosis, type 2 diabetes, β-cell apopto-
sis, and lung inflammation (Hetz et al. 2013). Recent studies
suggested intriguing role of ER stress in the initiation and
maintenance of diabetes to induce β-cell apoptosis (Papa
2012). Several chemical inducers of ER stress, such as
tunicamycin, thapsigargin, and dithiothreotol, are known.
Reversely, chemical version of chaperone molecules,
referred to as chemical chaperones (Fig. 1), are also iden-
tified and implicated for the potential treatment against the
ER stress-related pathologies. For example, 4-phenyburytic
acid (4-PBA) can exert therapeutic values against several
ER stress-related conditions (Iannitti and Palmieri 2011).
Salubrinal was also found to protect cells from ER stress by
hampering eIF2α from dephosphorylation which is one of
the hallmark events of UPR (Boyce et al. 2005). tUDCA
(tauro-ursodeoxycholic acid) is considered as one of the
best examples of chemical chaperones (Ozcan et al. 2006).
In search of novel chemical chaperones, we have
developed a cell-based assay where renilla luciferase
activity is driven by GRP78 promoter, which is one of the
most important chaperone molecules and plays a crucial
role in various UPR mechanisms such as PERK, ATF6,
and IRE1 pathways. We have reported that hydro-
xynaphthoic acids (HNAs) have EC50 ≅ 0.2 mM, about an
order of magnitude lower EC50 than salicylate and
tDUCA. They downregulate UPR markers such as
GRP78, CHOP and phosphorylated PERK as a potent
chemical chaperone (Jeong et al. 2013; Park et al. 2016).
For the discovery of new chemical scaffolds with more
drug-likeness than HNAs, we in this study identified N-
substituted-2-arylcarbonylhydrazinecarbothioamides (4)
as potent ER stress-reducing compounds. The new scaf-
fold is much more potent than HNA in reducing ER stress
signal and is much more diverse in heteroatom composi-
tions by which drug-likeness might be improved. Also, it
is possible that the open chain compound (4) can be
cyclized to afford 1,3,4-thiadiazoles (5) and 1,2,4-tria-
zoles (6). The cyclic structure can provide some rigidity
necessary for better binding.
We report here the synthesis and structure−activity
relationship of various N-substituted-2-arylcarbonylhy-
drazine-carbothioamides and their cyclic analogues
based on tunicamycin- and palmitate (PA)-induced
ER stress conditions. N-substituted-2-arylcarbonylhy-
drazinecarbothioamides
(4),
highly
heteroatom-
containing scaffold, were identified as strong hits with
EC50 < 10 μM, which is about three orders of magnitude
better EC50 than tUDCA and salicylate (Jeong et al.
2013), known chemical chaperones. It is noteworthy that
EC50 of 1-hydroxy-2-naphthoic acid (1-HNA), our initial
hit series, was 183 μM (Table 1) while best examples
shown here have EC50 in μM range.
Fig. 1 Examples of chemical
chaperones

Medicinal Chemistry Research
Table 1 Activity of (4), (5) and (6) against tunicamycin-induced ER
stress (data represent mean of three experiments in duplicate)
Chemical shifts (δ) were expressed in ppm using solvent as
an internal standard and coupling constant (J) in hertz. Low-
resolution mass spectra (LRMS) were obtained using an
Advion Expression CMS, and recorded in a positive ion
mode with an electrospray (ESI) source. High-resolution
mass spectra (HRMS) were obtained using a Thermo Sci-
entific LTQ Orbitrap XL mass spectrometer, and recorded
in positive ion mode with an electrospray (ESI) source.
Compounds
EC50 (μM)
CC50 (μM)
SIa
4a
1.52 0.09
2.77 0.24
1.13 0.12
4.53 0.37
1.61 0.24
6.96 1.39
3.19 0.25
0.62 0.11
0.87 0.11
>100
>100
>100
>100
>100
>100
>100
>100
>100
—
83.3
53.0
88.5
24.0
62.1
14.4
31.4
160
—
4b
4c
4d
4e
4f
General procedure for the synthesis of 4a–4t
4g
4h
4i
To a solution of hydrazide (2) (1.0 mmol) in EtOH was
added isothiocyanate (1.1 mmol). The reaction mixture was
refluxed overnight and cooled to room temperature. Half of
the solvent was removed under reduced pressure and the
residue was poured into ice. The precipitate was filtered and
washed with ice-cold EtOH to give a product.
4j
>100
>100
>100
—
1.0
4k
4l
94.9 9.49
47.6 9.50
31.0 7.47
80.0 12.0
82.1 9.03
>100
1.05
2.10
—
4m
4n
4o
>100
>100
>100
—
1.25
1.22
1.0
N-(3,4-Dichlorophenyl)-2-
picolinoylhydrazinecarbothioamide (4a)
4p
4q
4r
3.90 0.78
>100
—
Yield: 91%; m.p.: 208–210 °C; 1H-NMR (700 MHz,
DMSO-d6) δ 10.80 (s, 1H, C(=S)NH), 10.01 (s, 1H, NH
(C=S)), 9.84 (s, 1H, (C=O)NH), 8.70 (d, J = 4.1 Hz, 1H,
PyrC6H), 8.08 (d, J = 7.7 Hz, 1H, PyrC3H), 8.04 (t, J =
7.1 Hz, 1H, PyrC5H), 7.85 (s, 1H, Ph), 7.66 (t, J = 7.1 Hz,
1H, PyrC4H), 7.57–7.55 (m, 2H, Ph); 13C-NMR (175 MHz,
DMSO-d6) δ 181.11 (C=S), 164.30 (C=O), 149.76 (CPyr),
148.99 (CPyr), 139.97 (CPyr), 138.19 (CPyr), 130.46 (CPyr),
130.21 (CPh), 127.49 (CPh), 127.01 (CPh), 125.78 (CPh),
125.66 (CPh), 123.00 (CPh); MS (ESI) m/z: 341.1 [M+H]+;
HRMS (ESI) m/z calcd for C13H10Cl2N4OS [M+H]+
341.0025, found 341.0022.
>100
—
1.0
4s
3.38 0.37
16.9 0.21
19.1 3.05
>100
—
4t
—
—
5b
5e
>100
>100
>100
>100
>100
>100
>100
>100
5.23
1.0
5h
5i
>100
1.0
41.8 6.27
>100
2.39
1.0
6b
6e
>100
1.0
6h
6i
>100
1.0
>100
1.0
1-HNA
tUDCA
183
5.2 mM
2-Picolinoyl-N-(2-(trifluoromethyl)phenyl)
aSafety index is expressed as a ratio of CC50/EC50
Materials and methods
Chemistry
hydrazinecarbothioamide (4b)
1
White solid. Yield: 92%; m.p.: 192.9–195.0 °C; H-NMR
(700 MHz, DMSO-d6) δ 10.79 (s, 1H, C(=S)NH), 9.90 (s,
1H, NH(C=S)), 9.49 (s, 1H, (C=O)NH), 8.70 (d, J =
4.3 Hz, 1H, PyrC6H), 8.06–8.00 (m, 3H, PyrH), 7.68–7.65
(m, 3H), 7.47 (t, J = 7.4 Hz, 1H); 13C-NMR (175 MHz,
DMSO-d6) δ 183.17 (C=S), 164.31 (C=O), 149.86 (CPyr),
149.01 (CPyr), 138.19 (CPyr), 132.89 (CPh), 127.56 (CPh),
127.47 (CPh), 126.54 (CPyr), 126.51 (CF3), 124.74 (CPh),
123.18 (CPh), 122.95 (CPyr), 121.63 (CPh); MS (ESI) m/z:
341.1 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+
C14H11F3N4OS: 341.0678; found: 341.0676.
Unless noted otherwise, materials were purchased from
commercial suppliers and used without further purification.
Air- or moisture-sensitive reactions were carried out under
an inert gas atmosphere. Progress of reaction was monitored
by thin layer chromatography (TLC) using silica gel F254
plates. Purification of the products was performed by flash
column chromatography using silica gel 60 (70–230 mesh)
or by Biotage ‘Isolera One’ system with indicated solvents.
Melting points were determined using a Kruss melting
pointer metre and were not corrected. NMR spectra were
obtained using a Bruker spectrometer 400 and 700 MHz for
1H-NMR, 100 and 175 MHz for 13C-NMR, respectively.
N-Phenyl-2-picolinoylhydrazinecarbothioamide (4c)
White solid. Yield: 85%; 1H-NMR (400 MHz, DMSO-d6) δ
10.75 (s, 1H), 9.74 (br s, 2H), 8.69 (ddd, J = 4.7, 1.5,

Medicinal Chemistry Research
1.0 Hz, 1H), 8.11–7.95 (m, 2H), 7.64 (ddd, J = 7.3, 4.8,
1.5 Hz, 1H), 7.47 (d, J = 6.2 Hz, 2H), 7.32 (t, J = 7.8 Hz,
2H), 7.17–7.10 (m, 1H); MS (ESI) m/z 273.1 [M+H]+.
1H), 7.30 (d, J = 8.3 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 3.74
(s, 3H); MS (ESI) m/z 303.1 [M+H]+.
N-(4-Chlorophenyl)-2-picolinoylhydrazinecarbothioamide
N-(4-Chlorobenzyl)-2-picolinoylhydrazinecarbothioamide
(4h)
(4d)
White solid. Yield: 91%; 1H-NMR (400 MHz, DMSO-d6) δ
10.77 (s, 1H), 9.86 (br s, 1H), 9.78 (br s, 1H), 8.69 (d, J =
4.3 Hz, 1H), 8.10–7.97 (m, 2H), 7.65 (ddd, J = 7.2, 4.8,
1.5 Hz, 1H), 7.50 (d, J = 6.6 Hz, 2H), 7.37 (d, J = 8.8 Hz,
2H); 13C-NMR (100 MHz, DMSO) δ 180.72 (C=S), 163.79
(C=O), 149.39 (CPyr), 148.50 (CPyr), 138.28 (CPyr), 137.71
(CPh), 128.82 (CPh), 127.87 (CPh), 127.32(CPh), 126.98
(CPyr), 122.52(CPyr).; MS (ESI) m/z: 329.0 [M+Na]+.
Yield: 93%; m.p.: 192.2–194.1 °C; 1H-NMR (700 MHz,
DMSO-d6) δ 10.68 (s, 1H, HNC(=S)), 9.53 (s, 1H, C(=O)
NH), 8.68 (d, J = 5.2 Hz, 1H, PyrC6H)), 8.58 (s, 1H, C(=S)
NH), 8.06 (d, J = 7.8 Hz, 1H, PyrC3H), 8.02 (td, J = 7.7,
1.7 Hz, 1H, PyrC5H)), 7.64 (ddd, J = 7.5, 4.7, 1.2 Hz, 1H,
PyrC4H)), 7.35 (q, J = 8.6 Hz, 4H, Ph), 4.70 (d, J = 6.0 Hz,
2H, CH2); 13C-NMR (175 MHz, DMSO-d6) δ 182.48
(C=S), 164.14 (C=O), 149.86 (CPyr), 148.92 (CPyr), 138.97
(CPyr), 138.12 (CPh), 131.55 (CPh), 129.44 (CPh), 128.36
(CPh), 127.39 (CPyr), 123.00 (CPyr), 46.58 (CH2); MS (ESI)
m/z: 321.1 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+
C14H13ClN4OS: 321.0571; found: 321.0575.
N-Cyclohexyl-2-picolinoylhydrazinecarbothioamide (4i)
White solid. Yield: 82%; 1H-NMR (400 MHz, DMSO-d6) δ
10.49 (s, 1H), 9.31 (s, 1H), 8.70–8.63 (m, 1H), 8.07–7.96
(m, 2H), 7.85–7.47 (m, 2H), 4.30–4.02 (m, 1H), 1.80–1.78
(m, 2H), 1.67–1.66 (m, 2H), 1.57–1.53 (m, 1H), 1.32–1.15
(m, 4H), 1.08–1.03 (m, 1H); MS (ESI) m/z: 279.3 [M+H]+.
N-Butyl-2-picolinoylhydrazinecarbothioamide (4e)
1
White solid. Yield: 76%; m.p.: 141.5–143.2 °C; H-NMR
(400 MHz, DMSO-d6) δ 10.51 (s, 1H), 9.28 (s, 1H), 8.69 (d,
J = 4.7 Hz, 1H), 8.08–7.98 (m, 2H), 7.97 (t, J = 5.5 Hz,
1H), 7.64 (ddd, J = 6.8, 4.8, 2.2 Hz, 1H), 3.41 (dd, J =
13.3, 6.7 Hz, 2H), 1.52–1.39 (m, 2H), 1.32–1.19 (m, 2H),
0.86 (t, J = 7.3 Hz, 3H); 13C-NMR(100 MHz, DMSO-d6) δ
181.2 (C=S), 163.4 (C=O), 149.4 (CPyr), 148.5 (CPyr),
137.7 (CPyr), 126.9 (CPyr), 122.5 (CPyr), 43.4 (C1butyl), 30.9
(C2butyl), 19.4 (C3butyl), 13.8 (CH2); MS (ESI) m/z 253 [M
+H]+.
N-(4-Chlorophenyl)-2-nicotinoylhydrazinecarbothioamide
(4j)
White solid. Yield: 85%; 1H-NMR (400 MHz, DMSO-d6) δ
10.79 (s, 1H), 9.91 (s, 2H), 9.10 (d, J = 1.7 Hz, 1H), 8.76
(dd, J = 4.8, 1.7 Hz, 1H), 8.27 (dt, J = 8.0, 1.9 Hz, 1H),
7.56 (ddd, J = 8.0, 4.8, 0.8 Hz, 1H), 7.46 (s, 2H), 7.42–7.34
(m, 2H); MS (ESI) m/z: 307.7 [M+H]+.
N-(4-Chlorophenyl)-2-
N-Benzyl-2-picolinoylhydrazinecarbothioamide (4f)
isonicotinoylhydrazinecarbothioamide (4k)
White solid. Yield: 42 %; m.p.: 175–178 °C; 1H-NMR
(400 MHz, DMSO-d6) δ 10.64 (s, 1H, HNC(=S)), 9.48 (s,
1H, C(=O)NH)), 8.66 (ddd, J = 4.7, 1.4, 0.9 Hz, 1H,
PyrC6H), 8.54 (s, 1H, C(=S)NH)), 8.07–8.03 (m, 1H,
PyrC3H), 8.01 (td, J = 7.6, 1.7 Hz, 1H, PyrC5H), 7.63 (ddd,
J = 7.3, 4.8, 1.5 Hz, 1H, PyrC4H), 7.33–7.26 (m, 4H, Ph),
7.24–7.18 (m, 1H, Ph), 4.71 (d, J = 6.0 Hz, 2H, CH2); 13C-
NMR(100 MHz, DMSO-d6) δ 181.88, 163.64, 149.39,
148.42, 139.37, 137.62, 127.95, 127.05, 126.88, 126.50,
122.50, 46.72; MS (ESI) m/z 287.1 [M+H]+; CAS registry
211572-58-6.
White solid. Yield: 90%; 1H-NMR (400 MHz, DMSO-d6) δ
10.88 (s, 1H), 9.93 (s, 2H), 8.78 (dd, J = 4.4, 1.6 Hz, 2H),
7.85 (d, J = 6.0 Hz, 2H), 7.46 (s, 2H), 7.43–7.31 (m, 2H); 13
C-NMR (100 MHz, DMSO-d6) δ 181.10 (C=S), 164.48
(C=O), 150.25 (CPyr), 139.52 (CPyr), 138.14 (CPh), 129.27
(CPh), 127.99 (CPh), 127.68 (CPh), 121.69 (CPyr); MS (ESI)
m/z: 307.7 [M+H]+.
2-Benzoyl-N-(4-chlorophenyl)hydrazine-1-carbothioamide
(4l)
1
White solid. Yield: 95%; H-NMR (400 MHz, DMSO-d6) δ
N-(4-Methoxyphenyl)-2-picolinoylhydrazinecarbothioamide
(4g)
10.57 (s, 1H), 9.84 (s, 2H), 7.97 (d, J = 7.3 Hz, 2H),
7.61–7.57 (m, 1H), 7.52–7.49 (m, 4H), 7.42–7.33 (m, 2H); 13
C-NMR (100 MHz, DMSO-d6) δ 181.09 (C=S), 165.93
(C=O), 138.27 (CPh), 132.43 (CPh), 131.91 (CBenzoyl), 128.94
(CBenzoyl), 128.45 (CPh), 128.27 (CPh), 127.90 (CBenzoyl),
127.66 (CBenzoyl); MS (ESI) m/z: 328.0 [M+Na]+.
White solid. Yield: 81%; 1H-NMR (400 MHz, DMSO-d6) δ
10.71 (s, 1H), 9.63 (s, 2H), 8.68 (ddd, J = 4.7, 1.5, 1.0 Hz,
1H), 8.10–7.95 (m, 2H), 7.64 (ddd, J = 7.3, 4.8, 1.5 Hz,

Medicinal Chemistry Research
2-(2-chlorobenzoyl)-N-(5-methoxy-2-methylphenyl)
hydrazine-1-carbothioamide (4m)
ThiopheneC3H), 7.86 (d, J = 4.9 Hz, 1H, ThiopheneC5H),
7.35 (s, 2H, PhH), 7.20−7.21 (m, 3H, PhH + Thiophe-
neC4H), 2.88 (dt, J = 13.8, 6.9 Hz, 1H, CH), 1.21 (d, J =
6.9 Hz, 6H, CH3); 13C-NMR (175 MHz, DMSO-d6) δ
181.75 (C=S), 161.54 (C=O), 145.62 (Cph), 137.97 (Cph),
137.37 (Cthiophene), 132.25 (Cthiophene), 132.09 (Cthiophene),
130.26 (Cthiophene), 130.04 (Cph), 128.45 (Cph), 126.63 (Cph),
126.25 (Cph), 33.43 (CH), 24.40 (CH3), 24.29 (CH3); MS
(ESI) m/z: 320.1 [M+H]+; HRMS (ESI) m/z: Calcd. for [M
+H]+ C15H17N3OS2: 320.0886; found: 320.0889.
1
Yield: 82%; m.p.: 155.5–157.8 °C; H-NMR (700 MHz,
CDCl3) δ 10.10 (br s, 2H, NHC(=S)NH), 8.73 (s, 1H, C
(=O)NH), 7.90–7.80 (m, 1H, Cl-PhH), 7.81 (d, J =
7.5 Hz, 1H, Cl-PhH), 7.46–7.43 (m, 2H, Cl-PhH), 7.35
(t, J = 7.3 Hz, 1H, Ph), 6.98 (dd, J = 8.3, 1.1 Hz, 1H,
Ph), 6.79 (d, J = 8.4 Hz, 1H, Ph), 3.70 (s, 3H, OCH3),
2.31 (s, 3H, CH3); 13C-NMR (175 MHz, CDCl3) δ
181.51 (C=S), 161.43 (C=O), 149.31 (CPh), 132.39
(CPh), 131.67 (CPh), 131.22 (CPh), 130.72 (CPh), 130.61
(CPh), 129.54 (CPh), 128.53 (CPh), 127.19 (CPh), 125.85
(CPh), 124.89 (CPh), 111.14 (CPh), 55.76 (OCH3), 20.71
(CH3); MS (ESI) m/z: 350.1 [M+H]+; HRMS (ESI) m/z:
Calcd. for [M+H]+ C16H16ClN3O2S: 350.0725; found:
350.0724.
N-Cyclohexyl-2-(thiophene-2-carbonyl)
hydrazinecarbothioamide (4r)
White solid. Yield: 81%; 1H-NMR (400 MHz, DMSO-d6) δ
10.29 (s, 1H), 9.23 (s, 1H), 7.84 (d, J = 4.6 Hz, 2H), 7.80
(d, J = 8.2 Hz, 1H), 7.18 (t, J = 4.4 Hz, 1H), 4.16–4.13 (m,
1H), 1.78–1.76 (m, 2H), 1.69–1.68 (m, 2H), 1.58–1.57 (m,
1H), 1.35–1.13 (m, 4H), 1.06–1.05 (m, 1H); MS (ESI) m/z:
284.2 [M+H]+.
N-(4-Chlorophenyl)-2-(thiophene-2-carbonyl)hydrazine-1-
carbothioamide (4n)
1
White solid. Yield: 94%; m.p.: 193.3–193.6 °C; H-NMR
2-(5-Chlorothiophene-2-carbonyl)-N-
(400 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.93 (s, 1H), 9.86 (s,
1H), 7.86 (d, J = 4.9 Hz, 2H), 7.48 (s, 2H), 7.38 (d, J =
8.7 Hz, 2H), 7.21 (dd, J = 4.8, 4.0 Hz, 1H); 13C-NMR
(100 MHz, DMSO-d6) δ 181.2 (C=S), 161.1 (C=O), 138.2
(Cthiophene), 137.4 (Cph), 131.8 (Cph), 129.6 (Cthiophene), 129.1
(Cthiophene), 128.1 (Cph), 127.9 (Cph), 127.6 (Cthiophene); MS
(ESI) m/z: 312.8 [M+H]+.
cyclohexylhydrazinecarbothioamide (4s)
Yield: 92%; m.p.: 182.2–184.8 °C; 1H-NMR (700 MHz,
DMSO-d6) δ 10.37 (s, 1H, NHC(=S)), 9.23 (s, 1H, C(=O)
NH), 7.82 (d, J = 7.2 Hz, 1H, thiopheneC3H), 7.70 (s, 1H,
C(=S)NH), 7.24 (d, J = 4.0 Hz, 1H, thiopheneC4H),
4.30–4.10 (m, 1H), 1.79–1.78 (m, 2H), 1.71–1.70 (m, 2H),
1.59–1.58 (m, 1H), 1.34–1.21 (m, 4H), 1.07–1.06 (m, 1H);
13C-NMR (175 MHz, DMSO-d6) δ 181.14 (C=S), 160.47
(C=O), 137.26 (Cthiophene), 134.20 (Cthiophene), 129.83
(Cthiophene), 128.60 (Cthiophene), 53.59 (CH), 32.29 (CH2),
25.64 (CH2), 25.42 (CH2); MS (ESI) m/z: 318.1 [M+H]+;
HRMS (ESI) m/z: Calcd. for [M+H]+ C12H16ClN3OS2:
318.0496; found: 318.0499.
N-Phenyl-2-(thiophene-2-carbonyl)
hydrazinecarbothioamide (4o)
White solid. Yield: 79%; 1H-NMR (400 MHz, DMSO-d6) δ
10.56 (s, 1H), 9.88 (s, 1H), 9.74 (s, 1H), 7.86 (d, J = 4.8 Hz,
2H), 7.43 (s, 2H), 7.32 (t, J = 7.8 Hz, 2H), 7.25–7.07 (m,
2H); MS (ESI) m/z 278.4 [M+H]+.
N-Cyclohexyl-2-(4-ethyl-5-methylthiophene-3-carbonyl)
N-(4-Methoxyphenyl)-2-(thiophene-2-carbonyl)
hydrazinecarbothioamide (4t)
hydrazinecarbothioamide (4p)
Yield: 89%; m.p.: 177.3–179.3 °C; 1H-NMR (700 MHz,
DMSO-d6) δ 9.90 (s, 1H, NHC(=S)), 9.15 (s, 1H, thio-
pheneC2H), 7.84 (s, 1H, C(=O)NH), 7.55 (s, 1H, C(=S)
NH), 4.11 (s, 1H, CH), 2.71 (q, J = 7.4 Hz, 2H, CH2CH3),
2.35 (s, 3H, CH3), 1.80–1.79 (m, 2H), 1.73–1.64 (m, 2H),
1.57 (d, J = 12.5 Hz, 1H), 1.34–1.19 (m, 4H), 1.03 (t, J =
7.4 Hz, 3H, CH3+CH2); 13C-NMR (175 MHz, DMSO-d6) δ
184.22 (C=S), 164.03 (C=O), 140.07 (Cthiophene), 137.87
(Cthiophene), 133.87 (Cthiophene), 126.10 (Cthiophene), 53.20
(CH), 32.28 (CH2), 25.62 (CH2), 25.26 (CH2), 20.16 (CH2),
15.42 (CH3), 12.89 (CH3); MS (ESI) m/z: 326.1 [M+H]+;
HRMS (ESI) m/z: Calcd. for [M+H]+ C15H24N3OS2:
326.1355; found: 326.1358.
White solid. Yield: 87%; 1H-NMR (400 MHz, DMSO-d6) δ
10.53 (s, 1H), 9.76 (s, 1H), 9.64 (s, 1H), 7.89–7.82 (m, 2H),
7.27 (d, J = 8.2 Hz, 2H), 7.20 (dd, J = 4.9, 3.8 Hz, 1H),
6.89 (d, J = 8.9 Hz, 2H), 3.74 (s, 3H); MS (ESI) m/z 308.1
[M+H]+.
N-(4-Isopropylphenyl)-2-(thiophene-2-carbonyl)hydrazine-
1-carbothioamide (4q)
Yield: 89%; m.p.: 192.5–194.1 °C; 1H-NMR (700 MHz,
DMSO-d6) δ 10.54 (s, 1H, C=(S)NH)), 9.80 (s, 1H, NHC
(=S)), 9.68 (s, 1H, C(=O)NH), 7.88 (s, 1H,

Medicinal Chemistry Research
General procedure for the synthesis of 2a–2i, 2j–2l,
2n–2r
133.70 (C2-Thiophene), 123.76 (C2-Thiophene), 20.10 (CH2),
15.50 (CH3), 12.94 (CH3); MS (ESI) m/z: 185.1 [M+H]+.
To a solution of 100 mmol of ethyl picolinate (for 2a–2i),
ethyl nicotinate (for 2j), ethyl isonicotinate (for 2k), ethyl
benzoate (for 2l) or thiophene-2-carboxylate (for 2n–2r) in
EtOH was added hydrazine monohydrate (400 mmol). The
reaction mixture was refluxed overnight and cooled to room
temperature. Half of the solvent was removed under
reduced pressure and the residue was poured into ice. The
precipitate was filtered and washed with ice-cold EtOH to
give a product.
General procedure for the synthesis of 3d (y. 71%),
3m (y.75%) and 3q (74%), which are all known
compounds
To a solution of aniline analogues (2.0 mmol) in EtOH
(10 mL) were added CS2 (2.4 mmol), Boc2O (2.4 mmol),
Et3N (2.4 mmol) and 4-dimethylaminopyridine (0.2 mmol)
at 0 °C. The reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was concentrated under
reduced pressure, then diluted with CH2Cl2. Organic layer
was washed with water and brine, dried over anhydrous
MgSO4, and concentrated. The residue was purified on
silica gel column chromatography to give isothiocyanates.
Picolinohydrazide (2a–2i)
Yield: 79%; 1H-NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H),
8.64–8.57 (m, 1H), 8.03–7.94 (m, 2H), 7.56 (ddd, J = 6.1,
4.8, 2.9 Hz, 1H), 4.56 (s, 2H); MS (ESI) m/z 138.1
[M+H]+.
5-(Pyridin-2-yl)-N-(2-(trifluoromethyl)phenyl)-1,3,4-
thiadiazol-2-amine (5b)
Nicotinohydrazide (2j)
To a solution of hydrazinecarbothioamide (1 mmol) in
EtOH (3.5 mL) was added H2SO4 (553 μL, 0.01 mmol). The
reaction mixture was refluxed for 1 h and poured to ice-
water. The resulting slurry was filtered and the filter cake
was recrystallized with 33% AcOH in D.W. to give 5b as
white solid (y. 60%); m.p.: 249.6–250.6 °C; 1H-NMR
(400 MHz, DMSO-d6) δ 10.98 (s, 1H), 8.66 (ddd, J = 4.9,
1.6, 1.0 Hz, 1H), 8.28 (s, 1H), 8.17 (dt, J = 8.0, 1.0 Hz, 1H),
8.00 (td, J = 7.7, 1.7 Hz, 1H), 7.80 (dd, J = 7.9, 1.5 Hz,
1H), 7.61 (t, J = 8.0 Hz, 1H), 7.52 (ddd, J = 7.5, 4.9,
1.2 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H); MS (ESI) m/z 323 [M
+H]+.
Yield: 81%; 1H-NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H),
8.96 (dd, J = 2.3, 0.9 Hz, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H),
8.15 (ddd, J = 7.9, 2.3, 1.7 Hz, 1H), 7.49 (ddd, J = 7.9, 4.8,
0.9 Hz, 1H), 4.58 (s, 2H); MS (ESI) m/z 138.1 [M+H]+.
Isonicotinohydrazide (2k)
Yield: 88%; 1H-NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H),
8.64–8.57 (m, 1H), 8.01–7.93 (m, 2H), 7.56 (ddd, J = 6.1,
4.8, 2.9 Hz, 1H), 4.56 (s, 2H); MS (ESI) m/z 138.1 [M+H]+.
Benzohydrazide (2l)
General procedure for the synthesis of 5e, 5h and 5i
Yield: 94%; 1H-NMR (400 MHz, DMSO-d6) δ 9.77 (s, 1H),
7.86–7.78 (m, 2H), 7.54–7.40 (m, 3H), 4.49 (s, 2H); MS
(ESI) m/z 137.2 [M+H]+.
Hydrazinecarbothioamide (0.3 mmol) was added to H2SO4
(10 mL) at 0 °C and stirred for 3 h at room temperature. The
reaction mixture was neutralized with 2 N NaOH and fil-
tered. Filter cake was recrystallized with EtOH to give
thiadiazoles.
Thiophene-2-carbohydrazide (2n–2r)
Yield: 81%; 1H-NMR (400 MHz, DMSO-d6) δ 9.75 (s, 1H),
7.74 (dd, J = 5.0, 1.1 Hz, 1H), 7.70 (dd, J = 3.7, 1.1 Hz,
1H), 7.12 (dd, J = 5.0, 3.7 Hz, 1H), 4.45 (s, 2H); MS (ESI)
m/z 143.2 [M+H]+.
N-Butyl-5-(pyridin-2-yl)-1,3,4-thiadiazol-2-amine (5e)
1
White solid. Yield: 29%; m.p.: 110.9–112.8 °C; H-NMR
(400 MHz, DMSO-d6) δ 8.58 (ddd, J = 4.9, 1.5, 1.0 Hz,
1H), 8.08–8.04 (m, 1H, NH), 8.03 (t, J = 1.0 Hz, 1H,
PyrC3H), 7.91 (td, J = 7.7, 1.7 Hz, 1H, PyrC4H), 7.41 (ddd,
J = 7.5, 4.9, 1.1 Hz, 1H, PyrC5H), 3.33–3.29 (m, 2H,
NHCH2), 1.62–1.53 (m, 2H, CH2), 1.36 (dq, J = 14.4,
7.3 Hz, 2H, CH2), 0.91 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR
4-Ethyl-5-methylthiophene-3-carbohydrazide (2t)
1H-NMR (700 MHz, DMSO-d6) δ 9.38 (s, 1H), 7.54 (s,
1H), 4.46 (s, 2H), 2.70 (q, J = 7.5 Hz, 2H), 2.33 (s, 3H),
1.02 (t, J = 7.5 Hz, 3H); 13C-NMR (175 MHz, DMSO) δ
164.81 (C=O), 139.58 (C4-Thiophene), 135.90 (C5-Thiophene),
(100 MHz, DMSO-d6)
δ
169.9 (CThiadiazole), 157.7
(CThiadiazole), 149.6 (CPyr), 149.5 (CPyr), 137.3 (CPyr), 124.2

Medicinal Chemistry Research
(CPyr), 119 (CPyr), 44.5 (CH2), 30.6 (CH2), 19.5 (CH2), 13.6
4-(4-Chlorophenyl)-5-(pyridin-2-yl)-4H-1,2,4-triazole-3-thiol
(6h)
(CH3); MS (ESI) m/z 235 [M+H]+.
N-(4-Chlorophenyl)-5-(pyridin-2-yl)-1,3,4-thiadiazol-2-
amine (5h)
White solid. Yield: 96%; 1H-NMR (400 MHz, DMSO-d6) δ
14.29 (s, 1H), 8.35 (ddd, J = 4.8, 1.7, 1.0 Hz, 1H), 7.96
−7.85 (m, 2H), 7.56–7.47 (m, 2H), 7.41 (ddd, J = 7.4, 4.8,
1.4 Hz, 1H), 7.38−7.29 (m, 2H); MS (ESI) m/z 289
[M+H]+.
1
Yield: 41%; H-NMR (400 MHz, DMSO-d6) δ 10.75 (s,
1H), 8.64 (d, J = 4.7 Hz, 1H), 8.14 (d, J = 7.9 Hz, 1H), 7.97
(td, J = 7.8, 1.6 Hz, 1H), 7.76–7.66 (m, 2H), 7.49 (ddd, J =
7.4, 4.9, 0.9 Hz, 1H), 7.47–7.37 (m, 2H); MS (ESI) m/z 289
[M+H]+.
4-Cyclohexyl-5-(pyridin-2-yl)-4H-1,2,4-triazole-3-thiol (6i)
White solid. Yield: 81%; 1H-NMR (400 MHz, DMSO-d6) δ
14.02 (s, 1H), 8.76 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H), 8.01 (td,
J = 7.8, 1.8 Hz, 1H), 7.83 (dt, J = 7.9, 1.0 Hz, 1H), 7.60
(ddd, J = 7.7, 4.8, 1.2 Hz, 1H), 4.80 (tt, J = 12.3, 3.8 Hz,
1H), 2.29 (s, 2H), 1.70 (dd, J = 25.5, 12.6 Hz, 4H), 1.57 (d,
J = 12.6 Hz, 1H), 1.31–1.15 (m, 2H), 1.12–0.95 (m, 1H);
MS (ESI) m/z 261 [M+H]+.
N-Cyclohexyl-5-(pyridin-2-yl)-1,3,4-thiadiazol-2-amine (5i)
White solid. Yield: 71%; 1H-NMR (400 MHz, DMSO-d6) δ
8.57 (ddd, J = 4.9, 1.6, 1.0 Hz, 1H), 8.07–7.98 (m, 2H),
7.90 (td, J = 7.7, 1.7 Hz, 1H), 7.41 (ddd, J = 7.5, 4.9,
1.2 Hz, 1H), 3.58 (tt, J = 13.3, 6.8 Hz, 1H), 2.06 – 1.93 (m,
2H), 1.78 – 1.66 (m, 2H), 1.62 – 1.52 (m, 1H), 1.39 – 1.12
(m, 5H); 13C-NMR (100 MHz, DMSO-d6) δ 168.94 (C4′-
thiophene), 157.55 (C2′-thiophene), 149.55 (C2′-pyridine),
137.29 (C6′-pyridine), 124.15 (C4′-pyridiine), 118.94
(C3′- and C5′-pyridine), 53.74 (CH-cyclohexyl), 32.02
(C2′-cyclohexyl), 25.20 (C4′-cyclohexyl), 24.26 (C3′-
cyclohexyl); MS (ESI) m/z 261 [M+H]+.
Cell culture
HEK 293 cells (human embryonic kidney cell) and HepG2
cells (human hepatocarcinoma cell) were purchased from
ATCC. The cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum and penicillin and streptomycin in a humi-
dified incubator with 5% CO2 atmosphere at 37 °C.
General procedure for the synthesis of 6b, 6e, 6h
and 6i
Palmitate preparation
Hydrazinecarbothioamide (1 mmol) was added to 2 N
NaOH (6.6 mL) and refluxed for 3 h. The reaction mixture
was cooled to room temperature and acidified with 2 N HCl
to pH = 3. Resulting solid was filtered and recrystallized
with EtOH to give triazoles.
A 20 mM solution of palmitate in 0.01 N NaOH was
incubated at 80 °C for 30 min and 1 N NaOH was added up
to 0.2 N final concentration for clearing up the solution. The
fatty acid soaps were then combined with 5% fatty acid-free
BSA in phosphate-buffered saline (PBS) in 1:3 volume
ratio. PA/BSA conjugates consisted of 5 mM PA and 3.75%
BSA. PA/BSA conjugates were diluted in DMEM (4.5 g/L
glucose) supplemented with 10% FBS. Concentration of
BSA in 0.3 mM PA containing DMEM was approximately
0.5% and the molar ratio of PA to BSA in this solution was
approximately 5:1.
5-(Pyridin-2-yl)-4-(2-(trifluoromethyl)phenyl)-4H-1,2,4-
triazole-3-thiol (6b)
1
White solid. Yield: 71%; m.p.: 223.2–224.3 °C; H-NMR
(400 MHz, DMSO-d6) δ 14.33 (s, 1H), 8.26 (dt, J = 4.8,
1.4 Hz, 1H), 7.97–7.89 (m, 2H), 7.84–7.76 (m, 2H),
7.74–7.63 (m, 2H), 7.43–7.37 (m, 1H); MS (ESI) m/z 323
[M+H]+.
Multiplex luciferase assay
4-Butyl-5-(pyridin-2-yl)-4H-1,2,4-triazole-3-thiol (6e)
The stable human embryonic kidney (HEK) 293 cell line
harbouring a reporter gene expressed by human grp78
promoter was used as previously described. Synthesized
human grp78 promoter (−137 ~ +25) containing three ER
stress response elements (ERSE) in tandem was cloned into
pGL4.79 vector (Promega, Madison, USA). A stable human
embryonic kidney (HEK) 293 cell line harbouring the
White solid. Yield: 72%; 1H-NMR (400 MHz, DMSO-d6) δ
14.08 (s, 1H), 8.73 (dt, J = 4.8, 1.4 Hz, 1H), 8.04−7.98 (m,
2H), 7.60–7.53 (m, 1H), 4.49 (t, J = 7.6 Hz, 2H), 1.67−1.58
(m, 2H), 1.30−1.20 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H); MS
(ESI) m/z 235 [M+H]+.

Medicinal Chemistry Research
reporter gene was selected under 400 μg/mL of
G418 selection pressure. Five thousand selected stable cells
per well were seeded in white 384-well plate (Greiner,
Solingen, Germany). After 24 h, media were changed with
BSA or PA-conjugated BSA media, and serially diluted
compounds were added. Following 22 h incubation, 60 μM
of EnduRenTM live cell substrate (Promega, Madison, USA)
was added to medium and, after 2 h incubation, Renilla
luciferase (rLuc) activity was measured using EnVision®
(Perkin-Elmer). To normalize the rLuc activity, total viable
cells were counted using Celltiter-gloTM reagent (Promega).
The 50% inhibitory concentration (IC50) values for tested
compounds were determined by non-linear regression ana-
lysis of log-dose/response curves using Prism® 5 software
(Graphpad software Inc., CA, USA). Data from three
independent experiments were expressed as the geometric
mean IC50 and 95% confidence intervals (95% CI) were
calculated.
thiophenes, was screened in 384-well format at 20 mM
concentration to identify ER stress-reducing compounds.
Tunicamycin (1.2 μM final concentration) was treated to
induce ER stress and test compounds were treated 24 h after
Tm-induction. Compounds which decreased the luciferase
signal that was induced by Tm by more than two-fold of the
plate mean were selected as primary hits. Hydro-
xynaphthoic acids (HNAs) were identified as the initial hit
showing about tenfold better activity than salicylate, a
positive control. HNAs significantly decreased ER stress
markers such as CHOP and phosphorylated PERK and
eIF2α in western blot. Although HNAs are highly potent in
modulating ER stress markers, they still need to be
improved in drug-likeness and activity. N-Substituted-2-
arylcarbonylhydrazinecarbothioamides (4) were identified
in screening to show much better activity and to be more
drug-like due to higher heteroatom content than HNA.
Scheme 1 shows the structures and synthesis of N-sub-
stituted-2-arylcarbonylhydrazinecarbothioamides (4a–4t).
Except for the synthesis of 4s, various aryl ethyl esters (1)
were treated with hydrazine in refluxing ethanol to give the
corresponding acylhydrazides (2) in 76–94% yields. For the
synthesis of 4s, methyl 5-chlorothiophene-2-carboxylate
was prepared in refluxing MeOH under Fisher esterification
condition, then it was subsequently reacted with hydrazine
to afford the acylhydrazides (2s). All the prepared acylhy-
drazides (2a – 2t) were reacted with various isothiocyanates
(3) to afford hydrazinecarbothioamides (4a – 4t) in 79–94%
yields. Commercially unavailable isothiocyanates (3d, 3m
and 3q) were prepared through a reaction of the corre-
sponding amines with CS2 and Boc2O (Munch et al. 2008).
Ar group includes phenyl, substituted phenyl, 2- and 3-
thiophenyl, and 2-, 3-, and 4-pyridyl while R group ranges
from alkyl, cycloalkyl and substituted phenyl.
Immunoblot analysis
HepG2 cells in a six-well plate at a density of 500,000 cells/
well were treated with tunicamycin (Tm) and test compound
and incubated for 22 h. Cells were lysed in buffer A (50 mM
Hepes pH 7.4, 100 mM NaF, 50 mM NaCl, 10 mM sodium
pyrophosphate, 10 mM glycerol-2-phosphate, 5 mM EDTA,
5 mM EGTA and 1% TritonX-100) supplemented with
protease inhibitor cocktail (Roche, Germany) and phos-
phatase inhibitor cocktail (Sigma-Aldrich). Following
incubation on ice for 20 min, cleared lysates were boiled
with 6× SDS sample buffer for 5 min and subjected to SDS-
PAGE. Proteins were transferred to nitrocellulose mem-
brane (Bio-Rad, CA, USA) and analysed by immunoblot.
Anti-phospho PERK (Thr980), anti-phospho eIF2α (S51),
anti-CHOP were purchased from Santa Cruz Biotech (Santa
Cruz, CA, USA) and anti-beta-action was from Cell Sig-
naling (Beverly, MA, USA). AlexaFluor 488-conjugated
goat anti-rabbit secondary antibody was from Thermo
Fisher Scientific (MA, USA).
Next, we investigated the feasibility of the cyclization of
hydrazinecarbothioamides 4. 4b, 4e, 4h and 4i were chosen
as representative examples with high activities and under-
went further cyclization (Scheme 2). They were refluxed in
EtOH in the presence of 2 N NaOH to give corresponding
1,2,4-triazoles 6b, 6e, 6h, and 6i, respectively, in 71–96%
yields (Ainsworth 1960). On the other hand, 1,3,4-thiadia-
zoles 5 (Oruç et al. 2004) were generally obtained through a
Results and discussion
Previously, we have reported a cell-based reporter assay
(Jeong et al. 2013) in which renilla luciferase (rLuc)
activity is driven by human GRP78 promoter to search ER
stress-reducing compounds. Since GRP78 is one of the
most important chaperones and plays a pivotal role in var-
ious UPR mechanisms, monitoring the GRP78 signal in the
presence of test compounds can be a useful way to represent
ER stress-reducing activity of the compounds. Briefly, a
library of ~20,000 heteroatom-containing compounds,
including pyridines, pyrimidines, pyrroles, imidazoles,
Scheme 1 Synthetic scheme for compounds 4. Reagents and condi-
tions: (a) MeOH, H2SO4, reflux, 4 h, 94%; (b) H2NNH2·H2O (4 eq.),
EtOH, reflux, 4 h, 76–94%; (c) R-NCS (3), EtOH, reflux, 5 h, 79–94%;
(d) CS2, Boc2O, DMAP, Et3N, EtOH, 0 °C → r.t., 1 h, 71–75%

Medicinal Chemistry Research
cyclohexyl R group (4i) showed almost 100-fold less
activity than 2-pyridyl analogues with identical R groups
(4p, 4n, and 4r, respectively). Albeit not significant, elec-
tron withdrawing R groups and/or substitution on thiophene
ring are somewhat favoured for the activity. With the same
cyclohexyl R group attached, 4s which has a chloro sub-
stituent on thiophene ring showed the best activity among
4r – 4t.
Scheme 2 Synthetic scheme for 1,3,4-thiadiazoles 5 and 1,2,4-tria-
zoles 6. Reagents and conditions: (a) (i) H2SO4 or HCl, EtOH, 0 °C →
r.t., 2 h, 60% for 5b, 29% for 5e, 41% for 5h, 71% for 5i; (b) 2 N
NaOH, reflux, 2 h, 71% for 6b, 72% for 6e, 96% for 6h, 81% for 6i
Transformation of 4 to the more rigid structures (5 and 6)
generally resulted in detrimental effects to the activity.
Although they are all derived from active hydrazine-
carbothioamide compounds, cyclized analogues 5 and 6 did
not show comparable activity to their acyclic analogues (4b,
4e, 4h, and 4i). Interestingly, most compounds in this study
showed decent cytotoxicity (CC50) profile (Table 1),
thereby rendering maximized safety windows (SI).
reaction with hydrazinecarbothioamides 4 with 1 mol%
H2SO4 in refluxing EtOH. 5b was obtained in 60% yield
accordingly. However, other hydrazinecarbothioamides (4e,
4h and 4i) afforded complex mixture of products under the
same condition. The corresponding 1,3,4-thiadiazoles (5e,
5h and 5i) were obtained by addition of 4e, 4h, and 4i into
ice-cold H2SO4, followed by stirring at room temperature.
Compounds 4, 5, and 6 were evaluated using our cell-
based reporter assay where ER stress is induced by Tm. As
shown in Table 1, a number of compounds showed very
potent activities to reduce Tm-induced ER stress signal.
There are several features in the SAR. First of all, analogues
with pyridine and thiophene moiety on Ar position were
more active than those with phenyl and halogenated phenyl
groups (4l, 4m) and, 2-pyridyl analogues (4a–4i) showed
much superior activities over the other regioisomers (4j and
4k). Secondly, all the 2-pyridyl analogues with either alkyl
or aryl groups on R position showed significant activities.
Especially, when R = alkyl (4d–4f and 4i), cyclohexyl
analogue (4i) is the most active showing more than 200-fold
better than our initial hit compound, 1-HNA. Of aromatic R
groups, 4-chlorophenyl group showed the best activity (4h)
that is almost 300-fold better than 1-HNA. Moreover, 4h
showed considerably low cytotoxicity (CC50) suggesting a
significant safety profile (SI) of the compound. Lastly,
aromatic R groups should be attached directly to thiourea
moiety. A methylene spacer between aromatic R group and
thiourea moiety decreases the activity (4c vs. 4f; 4d vs. 4h)
by at least several folds.
With the identical R group of 4-chlorophenyl, 4j and 4k
showed much less activity than their 2-pyridyl analogue,
4h, demonstrating a high level of regioselectivity in pyridyl
substitution on Ar position. 2-Pyridyl analogues con-
sistently showed strong activities regardless of R group. It
does not seem that R group is a strong determinant for the
activity and shows significant electronic or steric effect on
the activity. For example, although 4-chlorophenyl R group
is commonly found in 2-pyridyl (4h), 3-pyridyl (4j), 4-
pyridyl (4k), and phenyl (4l) analogues, only 2-pyridyl
analogue (4e) showed the high level of activity while the
others showed much weaker activities. Dependency of
activity on Ar group was reproduced in thiophene series
(Table 1, entries 4n–4t) as well. Thiophene analogues with
R group of 4-methoxyphenyl (4g), 4-chlorophenyl, (4h) and
Tunicamycin (Tm) induced ER stress in our cell-based
assay for the sake of facile HTS campaign. It causes ER
stress by perturbing N-linked glycosylation which is
important in folding events of eukaryotic proteins (Heifetz
et al. 1979). It is well established that high level of free fatty
acids such as palmitic acid alone (lipotoxicity) or in com-
bination with high level of glucose is one of the most
important factors to trigger ER stress. The activity of the
analogues were tested in PA-induced ER stress assay
because high level of free fatty acids such as palmitic acid
(lipotoxicity) is considered to be more physiologically
important factors to trigger ER stress. At the same time, it
would be interesting to investigate the correlation of the
activity in between Tm- and PA-induced assays. As shown
in Table 2, some pyridinyl compounds slightly lost their
activities (4a, 4b, 4g–4i) while others gained activity (4c,
4d and 4f). However, level of changes remained within only
a few folds (1.1–3.9-fold decrease and 1.2–2.6-fold
increase) except for 4h and 4f which showed 6-fold
decrease and 5.4-fold increase, respectively, suggesting a
rough correlation between Tm-induced assay and PA-
induced assay. Thiophenyl analogues, 1,3,4-Thiadiazoles
(5) and 1,2,4-triazoles (6) which showed weak activity
under Tm-induced condition still showed trivial activities
(some data not shown).
Next, we investigated the effects of the active com-
pounds identified from our reporter assay on cellular ER
stress markers. Compound 4h was treated at various con-
centrations along with Tm to HepG2 cells and the changes
of some UPR markers were analysed by western blot (Fig.
2). When sensing ER stress, GRP78 which normally binds
to PERK is released from the complex, which is followed
by the oligomerization and transphosphorylation of PERK.
Compound 4h successfully diminished the phosphorylated
PERK and phosphorylated eIF2α both of which were
induced by Tm treatment. Level of CHOP were also atte-
nuated in line with well-documented PERK-eIF2α-AFT4

Medicinal Chemistry Research
Table 2 Activity of (4) against palmitate-induced ER stress
compounds, MOA by which our compounds decrease ER
stress or misfolding load is still unclear and requires a lots
of investigations. Nevertheless, we can speculate the MOA
based on literature. Chemical chaperones can usually be
classified into the following groups: (Papp and Csermely
2006) osmolytes, hydrophobic compounds or detergents,
and pharmacological chaperones. The most common types
are osmolytes which include glycerol, trimethylamine N-
oxide (TMAO) and proline and so on. MOA of osmolytes is
believed to limit free movement of proteins by elevating the
density of the solvent, thus preventing aggregation of
unfolded proteins. Therefore, this type of chemical cha-
perones typically require high enough concentration to exert
the activity and are usually charged or uncharged polar
compounds to be perfectly soluble in aqueous environment.
Based on polarity and water solubility of our compounds, it
is hard to suppose our compounds fit into the category. The
second group consists of hydrophobic compounds, e.g.,
sodium salt of PBA, lipids, and detergents. MOA was
proposed that hydrophobic moiety of these compounds
binds to the hydrophobic surface of unfolded proteins to
protect them from aggregation. Detergent-like structural
feature of this category could make our compounds distinct
from them. The last category is so-called pharmacological
chaperones such as enzyme inhibitors and receptor ligands.
Detailed MOA of this type of chemical chaperones is not
clearly understood. However, it is proposed that binding of
selective compound to a target protein can promote proper
folding and transport of the mutant or misfolded protein.
Pharmacological chaperones are to bind the native con-
formation of the target protein, stabilizing its conformation
and pushing the balance toward the native state. In this
regard, our compounds should bind to a target protein in a
specific manner to be pharmacological chaperones. Unfor-
tunately, we lack experimental data to identify their binding
partner, or, to the best of our knowledge, any binding
partners of similar compounds are not known at this point.
Investigation of MOA will be conducted as a future
direction.
Compounds
EC50 (μM)a
4a
4b
4c
4d
4f
3.13 0.68
4.58 0.73
0.96 0.19
4.20 1.26
1.29 0.16
3.39 0.51
3.72 0.30
3.41 0.46
>100
4g
4h
4i
4j
4k
4l
>100
25.6 2.8
61.6
4m
4n
4o
4p
4q
4r
4s
4t
24.1 3.6
>100
>100
45.5 5.9
>100
37.6
16.6 3.3
aData represent mean of three experiments
Mock Tm
Compound 4h
1
3
5
10 (μM)
p-PERK
PERK
p-eIF2
α
eIF2
α
In summary, we have identified and synthesized hydra-
zinecarbothioamides and their cyclized forms, thiadiazoles
and triazoles which reduce ER stress induced by tunica-
mycin. Open chain forms (4) showed up to about 300-fold
better activity than 1-hydroxynaphthoic acid (1-HNA)
which is, in turn, 10-fold better than salicylate, a positive
control. Some hydrazinecarbothioamides (4) also showed
strong activity under more physiologically relevant PA-
induced ER stress model. The cyclized analogues, 1,3,4-
thiadiazoles and 1,2,4-triazoles are in general less active
than 4. Indeed, a selected compound (4h) reduced repre-
sentative ER stress markers in western blot and we believe
this scaffold could be a platform for further investigation to
identify novel chemical chaperones.
CHOP
β-actin
Fig. 2 Western blot of ER stress markers upon treatment of 4h
pathway which is usually implicated in apoptosis. The
degree of those changes was in concert with the results of
our reporter assay, which demonstrated the robustness of
our reporter assay.
Although we have shown here that representative ER
stress markers are modulated upon treatment of our

Medicinal Chemistry Research
Acknowledgements This research was supported by Research Fund of
Hanyang University (HY-2013-P).
Hetz C, Chevet E, Harding HP (2013) Targeting the unfolded protein
response in disease. Nat Rev Drug Disco 12:703–719
Iannitti T, Palmieri B (2011) Clinical and experimental applications of
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Jeong KW, Ku JM, Park MW, Park SM, Yang JE, Nam TG (2013)
Hydroxynaphthoic acids identified in a high throughput screening
potently ameliorate endoplasmic reticulum stress as novel che-
mical chaperones. Chem Pharm Bull 61:740–746
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Munch H, Hansen JS, Pittelkow M, Christensen JB, Boas U (2008) A
new efficient synthesis of isothiocyanates from amines using di-
tert-butyl dicarbonate. Tetrahedron Lett 49:3117–3119
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Görgün CZ, Hotamisligil GS (2006) Chemical chaperones reduce
ER stress and restore glucose homeostasis in a mouse model of
type 2 diabetes. Science 313:1137–1140
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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