Design, synthesis, and structure-activity relationship of substrate competitive, selective, and in vivo active triazole and thiadiazole inhibitors of the c-jun n-terminal kinase

Article abstract of DOI:10.1021/jm801503n

We report comprehensive structure-activity relationship studies on a novel series of c-Jun N-terminal kinase (JNK) inhibitors. The compounds are substrate competitive inhibitors that bind to the docking site of the kinase. The reported medicinal chemistry

Full text of DOI:10.1021/jm801503n

J. Med. Chem. 2009, 52, 1943–1952
1943
Design, Synthesis, and Structure-Activity Relationship of Substrate Competitive, Selective, and
in Vivo Active Triazole and Thiadiazole Inhibitors of the c-Jun N-Terminal Kinase
Surya K. De,† John L. Stebbins,† Li-Hsing Chen,† Megan Riel-Mehan,† Thomas Machleidt,‡ Russell Dahl,† Hongbin Yuan,†
Aras Emdadi,† Elisa Barile,† Vida Chen,† Ria Murphy,† and Maurizio Pellecchia*,†
Infectious and Inflammatory Disease Center and Cancer Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road,
La Jolla, California 92037, and InVitrogen DiscoVery SerVices, InVitrogen Corporation, 501 Charmany DriVe, Madison, Wisconsin 53719
ReceiVed NoVember 28, 2008
We report comprehensive structure-activity relationship studies on a novel series of c-Jun N-terminal kinase
(JNK) inhibitors. The compounds are substrate competitive inhibitors that bind to the docking site of the
kinase. The reported medicinal chemistry and structure-based optimizations studies resulted in the discovery
of selective and potent thiadiazole JNK inhibitors that display promising in vivo activity in mouse models
of insulin insensitivity.
Introduction
several recent studies report preliminary success on the use of
D-JNK1 in early preclinical and clinical studies.9-13
Protein kinases comprise 2% of the mammalian genome and
catalyze the transfer of γ-phosphoryl group of ATP to specific
protein substrates.1 The c-Jun N-terminal kinases (JNKsa) are
a series of serine/threonine protein kinases of the mitogen
activated protein kinase (MAPK) family. Three distinct genes
encoding JNKs have been identified, JNK-1, JNK-2, and JNK-
3, and at least 10 different splicing isoforms exist in mammalian
cells.2-4 The three JNK isoforms share more than 90% amino
acid sequence identity, and the ATP pocket is >98% homolo-
gous. These proteins are activated in response to cellular stresses
such as heat shock, irradiation, hypoxia, chemotoxins, and
peroxides. They are also activated in response to various
cytokines and participate in the onset of apoptosis.5,6 It is
reported that up-regulation of JNK activity is associated with a
number of disease states such as type-2 diabetes, obesity, cancer,
inflammation, and stroke.1-3 Therefore, JNK inhibitors are
expected to be effective therapeutic agents against a variety of
diseases.
JNKs bind to substrates and scaffold proteins, such as JIP-1,
that contain a D-domain, as defined by the consensus sequence
R/KXXXXLXL.7,8 A peptide corresponding to the D-domain
of JIP-1 (aa 153-163; pep-JIP1) inhibits JNK activity in vitro
and displays remarkable selectivity with little inhibition of the
closely related Erk and p38 MAPKs.9-12 Recent in vivo data,
generated for studies focusing on pep-JIP1 fused to the cell
permeable HIV-TAT peptide, show that its administration in
various mouse models of insulin resistance and type-2 diabetes
restores normoglycemia without causing hypoglycemia in lean
mice.13 The peptide was further improved by the synthesis of
an all-D retroinverso peptide, D-JNK1, containing a cell-
penetrating sequence. However, the peptide’s instability in vivo,
its short half-life, and the costly and inefficient large-scale
manufacturing and purification processes all hamper the devel-
opment of novel therapies for diabetes based on D-JNK1 alone.
Nevertheless, despite some skepticism surrounding this peptide,
the efficacy of such substrate competitive peptides is such that
There has been considerable effort to identify JNK inhibitors
over the past several years.14-20 A drug discovery program in
our laboratory was initiated with the aim of identifying and
characterizing small-molecule JNK inhibitors as novel chemical
entities targeting the JIP docking site.21,22 As a result of these
efforts, we have reported on the identification of compound 12
(BI-78D3) (Figure 1A) (4-(2,3-dihydrobenzo[b][1,4]dioxin-6-
yl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-3-ol) from our
HTS libraries as the first potent and selective JNK inhibitor of
this class with demonstrated in vivo activity in mice model of
insulin resistance.21
As a continuation of our work21,22 we now report a
comprehensive structure-activity relationship studies describing
the discovery of novel JNK inhibitors that target the JIP-JNK
interaction site. We developed a triazole series followed by a
thiadiazole series based on structure-activity relationship (SAR)
studies carried out on the initial hit compound 12 (Figure 1A)21
which ultimately led to the discovery of compound 9 (Figure
1B). We describe here the pharmacological properties, design,
and SAR studies that have led to its identification.
Results and Discussion
Screening of our internal compound collection for JNK
inhibitors resulted in the identification of compounds belonging
to the triazole series.21 The 4-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-3-ol (compound
12, Figure 1A)21 was qualified as a hit and became the starting
point of our medicinal chemistry efforts.
To investigate the effects on potency induced by small
changes in the structure of 12, we developed the general
synthetic route for the preparation of this series. Compound 1,
thiobiurea, was synthesized according to the reported procedure
(Scheme 1).23 A variety of commercially available aryl isothio-
cyanates were treated with semicarbazide in the presence of
sodium acetate in acetonitrile at room temperature to give 1a-q
and 4a-e in good yields (Scheme 1). The thiobiureas (1a-e)
and 4a-e were heated at 100 °C in the presence of 2 M NaOH
aqueous solution for 5-8 h to afford 3-hydroxy-5-thiol deriva-
tives of triazoles in moderate to good yields. Compounds 3a-q
were synthesized by nucleophilic substitution of 2-bromo-5-
nitrothiazole with thiol of triazoles in NaOMe/MeOH solution.
* To whom correspondence should be addressed. Phone: 858-646-3159.
Fax: 858-713-9925. E-mail: mpellecchia@burnham.org.
†
Burnham Institute for Medical Research.
Invitrogen Corporation.
‡
a Abbreviations: JNK, c-Jun N-terminal protein kianse; MAPK, mitogen-
activated protein kinase; JIP1, JNK-interacting protein 1; EDC, N-ethyl-
N′-(3-dimethylaminopropyl)corbodiimide; DMF, N,N-dimethylformamide.
10.1021/jm801503n CCC: $40.75
2009 American Chemical Society
Published on Web 03/09/2009

1944 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
De et al.
Figure 1. Chemical structures and docked geometry: (A) chemical structure of the previously reported compound 12;21 (B) chemical structure of
compound 9; (C, D) docked structure of compound 9 in the JIP site of JNK1.
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) sodium acetate, CH3CN, room temp; (b)
2 M NaOH solution, 100 °C; (c) sodium methoxide, MeOH, 2-bromo-5-
nitrothiazole, room temp. 5-7: (a) R ) 3,4-benzodioxine, R1 ) phenyl; (b)
R ) 2,4-dichlorophenyl, R1) 1-methylpyrole; (c) R ) 3-methoxyphenyl,
R1 ) phenyl; (d) R ) furfuryl; R1 ) naphthyl; (e) R ) phenyl, R1
thiophene; (f) R ) phenyl, R1 ) 4-pyridyl; (g) R ) methyl, R1
pyrazinyloxadiazole.
)
)
a Reagents and conditions: (a) sodium acetate, CH3CN, room temp; (b)
2 M NaOH solution, 100 °C; (c) sodium methoxide, MeOH, 2-bromo-5-
nitrothiazole, room temp; (d) aryl or alkyl bromide, sodium methoxide,
MeOH, room temp. 1-3: (a) R ) 4-tert-butylphenyl; (b) R ) 3,4-
methylenedioxyphenyl; (c) R ) 4-fluoropheny; (d) R ) 2,4-dimethoxyphe-
nyl; (e) R ) 3,5-dimethoxyphenyl; (f) R) 3,4-dimethoxyphenyl; (g) R )
naphthyl; (h) R ) 2,5-dimethoxyphenyl; (i) R ) 4-trifluromethoxyphenyl;
(j) R ) 4-nitrophenyl; (k) R ) 3,4-difluorophenyl; (l) R ) 3-fluorophenyl;
(m) R ) 3-methoxyphenyl,; (n) R ) 2-methoxyphenyl; (o) R ) 3-trifluo-
romethylphenyl; (p) R ) 4-trifluromethylphenyl; (q) R ) cyclohexyl; (r)
R ) 4-methoxyphenyl. 4a: R ) phenyl, R1 ) n-butyl. 4b: R ) phenyl, R1
) ethyl. 4c: R ) 3,4-benzodioxine, R1 ) benzyl. 4d: R) phenyl, R1 )
5-nitrothiophene.
Exploration of triazole C-3 position (replacing hydroxyl to
alky or aryl) required a different synthesis of the core scaffold.
Arylbithioureas (5a-g) (Scheme 2) were prepared according
to published procedures.24 Compounds 6a-g were synthesized
following the similar conditions mentioned before in Scheme
1, and a few of them were commercially available (6b,d-g).
The final compounds (7a-g) were successfully obtained by
displacing bromide with thiols as previously described. The
dithioether of thiadiazoles (8a-g) were synthesized from
commercially available 1,3,4-thiadiazole-2,5-dithiol (Scheme 3).
Similarly, we prepared compounds 9 and 10 from 5-amino-
1,3,4-thiadiazole-2-thiol to introduce further diversity into the
series (Scheme 4).
We found that the thiol is consistently more reactive than
hydroxyl; we never isolated any hydroxyl derivative compounds,
even using excess amounts of 2-bromo-5-nitrothiazole. Simi-
larly, the compounds 4a-e were prepared using different alkyl
and aryl bromides.
In our work, the basis for the biochemical assay was JNK-1
mediated phosphorylation of ATF-2. High-throughput screening
identified compound 12, with an IC50 of 280 nM.21 Competition
experiments confirmed that this compound is a competitive
inhibitor of the interactions between JNK and pepJIP1.21

Triazole and Thiadiazole Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1945
Scheme 3a
Table 2. Modification of C-3 Position (Replacing Hydroxyl with Alkyl
or Aryl) on Triazoles
LanthaScreen kinase
activity assay,
IC50 (µM)
pepJIP1 DELFIA
displacement assay,
IC50 (nM)
compd
a Reagents and conditions: (a) alkyl or aryl bromide, sodium methoxide,
MeOH, room temp. 8a: R ) benzyl. 8b: R ) 4-fluorobenznyl. 8c: R )
4-methoxybenzyl. 8d: R ) 3-nitrobenzyl. 8e: R ) 4-nitrobenzyl. 8f: R )
5-nitrothiazole. 8g: R ) 5-carboxylthiazole.
7a
7b
7c
7d
7e
7f
27
2
108
83
168
57
107
122
131
7.9
13.6
>50
3.1
100
Scheme 4a
7g
Table 3. Dithioether of 1,3,4-Thiadiazoles
LanthaScreen kinase
activity assay,
pepJIP1 DELFIA
displacement assay,
IC50 (nM)
compd
IC50 (µM)
8a
8b
8c
8d
8e
8f
>100
>100
>100
>100
>100
0.8
NDa
NDa
NDa
NDa
NDa
187
8g
84
100
a Reagents and conditions: (a) 2-bromo-5-nitrothiazole, sodium meth-
oxide, MeOH, room temp; (b) 4-fluorobenzoic acid, EDC, HOBt, DIEA,
DMF, 50 °C; (c) 4-fluorobenzyl bromide, sodium methoxide, MeOH, room
temp.
a ND: no displacement at 100 µM.
Modifications investigated in the 4-phenyl moiety of the
triazole series are detrimental to JNK potency as shown in Table
1. The 3- and 4-positions of the phenyl group were more active.
Compound 3f has a 3,4-methylenedioxyphenyl group in place
of the 3,4-benzodioxin ring in the parent compound and has
comparable activity. However, a 4-tert-butylphenyl group was
not tolerated in this position likely because of its bulky size
(3a). The most active compound contains 4-nitrophenyl on the
4-position of the triazole, resulting in an IC50 of 0.4 µM (3j);
likewise, groups such as fluoro (3c), methoxy (3d, 3e, 3f, 3h,
3m, 3n, and 3r), trifluoromethyl (3o and 3p), trifluoromethoxy
(3i) show similar activities. However, a 3-fluoro on phenyl group
(3l) does not show any good activity and 3,4-difluorophenyl
(3k) has a moderate activity.
We further modified the 4- and 5-positions on the triazole
ring while maintaining the 5-nitrothiazole in the 3-position
(Table 2). In this series, phenyl group in the 5-position reduces
the activity in the kinase assay compared to the parent compound
12 but has a lower IC50 (108 nM) in the displacement assay
against pepJIP1, whereas replacing the dioxin ring with a
3-methoxypenyl group (7c) results in a 3-fold increase in
activity, indicating that two large ring structures in both the 4-
and 5-positions may constitute too much bulk. We subsequently
synthesized compounds containing smaller aromatic structures
in both positions (7b and 7d) and found that 1-methylpyrrole
is tolerated while both naphthyl and pyrazinyloxadiazole in the
5-position reduce the activity. We next focused on a 1,3,4-
thiadiazole-2,5-dithiol as the scaffold ring to further explore the
effect of the substituents in the 5-position (Table 3). We had
very little success with benzyl groups, but smaller moieties such
as a nitrothiazole and a carboxylthiazole were very promising,
which led to compound 9 (Table 4). The use of 5-amino-1,3,4-
thiadiazole-2-thiol as a scaffold allowed for nonsymmetrical
modifications; however, further modifications of the amine group
on compound 9 did not increase potency (10). Addition of a
methylphenyl group on the thiol resulted in a complete loss of
activity (11).
Table 1. Triazole Series
LanthaScreen kinase activity pepJIP1 DELFIA displacement
assay, IC50 (µM) or %
inhibition at 100 µM
assay, IC50 (nM) or %
inhibition at 100 µM
compd
4a
4b
4c
4d
3a
3b
3c
3d
3e
3f
25%
16%
2%
22%
39
3.3
1.8
1.1
4.3
1.0
2.7
2.4
2
NDa
NDa
NDa
55%
95
102
225
66
105
43
48
85
130
86
73%
626
118
53
160
75
3g
3h
3i
3j
3k
3l
0.4
6.6
84
3m
3n
3o
3p
3q
3r
16
2.2
2.9
3
25
17
56
45
a ND: no displacement at 100 µM.
We began our investigation of this initial scaffold by
synthesizing compounds with variations in the 3-, 4-, and
5-positions of the triazol. As shown in Table 1, among the
combinations tested, changes in the 4- and 5-positions are well
tolerated. We first synthesized and tested a series of alkyl thio
ethers, but none of these showed any remarkable JNK inhibition
(4a, 4b in Table 1). Aryl and benzyl thio ethers lead to a drastic
drop in activity. Replacing the thiazole with a thiophene also
results in a decrease in activity. We found that the only suitable
moiety in the 3-position was a 5-nitrothiazole, and changing
from nitro to carboxyl group on the thiazole unit was also not
tolerated. On the basis of our results, we retained the 5-position
as a 5-nitrothiazole and varied the other positions of the parent
compound 12 (Figure 1A).
Modeling studies suggest that compound 9 may bind at the
JIP site with the nitrothiazol group crossing the ridge close to
residues Arg127 and Cys163 (Figure 1C and Figure 1D). Its
thiazole group can form a hydrogen bond with Arg127 and can

1946 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Table 4. 2-Amino-1,3,4-thiadiazole Series
De et al.
effect of insulin on blood glucose levels was then measured
(Figure 3B). Compound 9 resulted in a statistically significant
reduction in blood glucose levels compared to the vehicle control
(Figure 3B). Hence, the ability of compound 9 to restore insulin
sensitivity is consistent with its proposed function as an effective
JNK inhibitor.21 Liquid chromatography/mass spectrometry
bioavailability analysis demonstrates that compound 9 has
favorable microsomal and plasma stability (T1/2 ) 27 min; see
Supporting Information) which support its use in further in vivo
experiments.
LanthaScreen kinase
activity assay,
IC50 (µM)
pepJIP1 DELFIA
displacement assay,
IC50 (nM)
compd
9
10
11
0.7
84
>100
239
NDa
>1000
a ND: no displacement at 100 µM.
Conclusion
We successfully developed a new series of JNK inhibitors,
many of which are very potent in vitro. Our initial in vivo
screens indicate that compound 9 possesses the ability to restore
insulin sensitivity in mice models of diabetes. Our results
indicate that targeting the protein-protein interaction between
JNK and JIP with a small molecule is a new and promising
avenue for the development of novel pharmacological tools that
inactivate the JNK pathway.
Figure 2. In vitro assays: (A) kinase inhibition assay for 9; (B) dose
dependent displacement of biotinylated pepJIP1 from GST-JNK1.
Table 5. Selectivity Profile
p38R, IC50
(µM)
furin, IC50
(µM)
LF, IC50
(µM)
compd
Akt1, IC50
Experimental Section
3j
7b
8f
9
>100
>100
>100
>100
20% at 100 µM
14% at 100 µM
27% at 100 µM
11% at 100 µM
>50
>50
>50
>50
>100
>100
>100
>100
General. Unless otherwise indicated, all anhydrous solvents were
commercially obtained and stored in SureSeal bottles under
nitrogen. All other reagents and solvents were purchased as the
highest grade available and used without further purification. Thin-
layer chromatography (TLC) analysis of reaction mixtures was
performed using Merck silica gel 60 F254 TLC plates and visualized
using ultraviolet light. NMR spectra were recorded on a Varian
300 or 500 MHz instrument. Chemical shifts (δ) are reported in
parts per million (ppm) referenced to 1H (Me4Si at 0.00). Coupling
constants (J) are reported in Hz throughout. Mass spectral data were
acquired on Shimadzu LCMS-2010EV for low resolution and on
an Agilent ESI-TOF for either high or low resolution. Purity of all
compounds was obtained in a HPLC Breeze from Waters Co. using
an Atlantis T3 3 µm, 4.6 mm × 150 mm reverse phase column.
The eluant was a linear gradient with a flow rate of 1 mL/min from
95% A and 5% B to 5% A and 95% B in 15 min followed by 5
min at 100% B (solvent A, H2O with 0.1% TFA; solvent B, ACN
with 0.1% TFA). The compounds were detected at λ ) 254 nm.
Purity of key compounds was established by elemental analysis as
performed on a Perkin-Elmer series II-2400 (see Supporting
Information).
bind into a subpocket nearby, which on the basis of this
hypothesis could tolerate groups with a similar size to substitute
the thiadiazole group of compound 9.
Compound 9 showed an IC50 of 0.7 µM in the kinase assay
(Figure 2A) in a substrate competitive manner. In addition,
compound 9 also showed an IC50 of 239 nM in displacing
pepJIP1, as measured by a DELFIA assay (Figure 2B).21
Compound 9 was found to be 142-fold less active against p38R,
a member of the MAPK family with high structural similarity
to JNK and completely inactive against Akt kinase (Akt
inhibitions at 100 µM for compounds 3j, 7b, 8f, and 9 were
20%, 14%, 27%, and 11%, respectively). These compounds
were also very selective against other protein targets under
investigation in our laboratory including the metalloprotease LF
(lethal factor) and the proprotein convertase furin (Table 5).
This selectivity is in agreement with our previous findings with
compound 12 and with the reported data on pepJIP1.10-13,21
In an attempt to profile the properties of compound 9 in the
context of a complex cellular milieu, we employed the cell-
based LanthaScreen kinase assay. In this assay compound 9 is
able to inhibit TNF-R stimulated phosphorylation of c-Jun in
cell (EC50 ) 6.23 µM; Figure 3A). It is noted that the cell-
based system employed makes use of a GFP-c-Jun stable
expression system. As a result, the levels of GFP-c-Jun in these
cells are higher than endogenous levels. This could have an
inflationary effect on the EC50 values obtained with this assay
when testing substrate competitive compounds. Nonetheless, this
finding establishes that compound 9 is able to function in a
cellular context.
2-(4-tert-Butylphenylcarbamothioyl)hydrazinecarboxamide (1a).
To a suspension of sodium acetate (216 mg, 2.63 mmol) and
semicarbazide (293 mg, 2.63 mmol) in acetonitrile (10 mL) was
added 4-tert-butylphenyl isothiocyanate (506 mg, 2.63 mmol), and
the reaction mixture was stirred for 24 h. The solvent was removed
by rotary evaporation, and the resulting residue was dissolved in 2
M NaOH aqueous solution (30 mL) and filtered through activated
charcoal to remove any unreacted isothiocyanate. The filtrate was
acidified with 1 N HCl (40 mL). The precipitate was collected and
washed with water (3 × 20 mL) and hexanes (2 × 20 mL) to afford
1
1a as a white solid (601 mg, 86%). H NMR (300 MHz, DMSO-
d6) δ 1.26 (s, 9 H), 6.03 (s, 2 H, NH2), 7.31 (d, J ) 8.4 Hz, 2 H),
7.39 (d, J ) 8.1 Hz, 2 H), 7.99 (s, 1 H, NH), 9.32 (s, 1 H), NH),
9.55 (s, 1 H, NH); MS m/z 555(2M + Na)+, 533 (2M + H)+, 289
(M + Na)+, 267 (M + H)+, 250, 224, 158, 88, 74; HRMS calcd
for C12H19N4OS (M + H) 267.1274, found 267.1279.
Following the above-mentioned procedure and with the appropri-
ate starting materials and reagents used, compounds 1b-q were
synthesized.
2-(Benzo[d][1,3]dioxol-5-ylcarbamothioyl)hydrazinecar-
boxamide (1b). Yield, 88%; 1H NMR (300 MHz, DMSO-d6) δ 5.99
(s, 2 H), 6.03 (s, 2 H, NH2), 6.80-6.86 (m, 2 H), 7.09 (s, 1 H),
7.97 (s, 1 H, NH), 9.32 (s, 1 H, NH), 9.54 (s, 1 H, NH); MS m/z
531 (2M + Na)+, 277 (M + Na)+, 255 (M + H)+, 238, 212, 151,
The link between the JNK pathway and type-2 diabetes has
been established previously.10-13 Thus, in an attempt to further
our bioanalysis of the JNK-inhibitory properties of compound
9, we monitored the ability of compound 9 to restore insulin
sensitivity in a mouse model of type-2 diabetes. For this analysis,
insulin insensitive mice from Harlan (Harlan Sprague Dawley,
Inc.; Indianapolis, IN) were injected once with 25 mg/kg of
compounds 9, 7b, and 8f 30 min prior to insulin injection. The

Triazole and Thiadiazole Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1947
Figure 3. Cell based and in vivo efficacy studies with compounds 9 and 7b: (A) TR-FRET analysis of c-Jun phosphorylation upon TNF-R stimulation
of HeLa cells in the presence of increasing concentrations 9; (B) effects on insulin resistance in 11-week-old BKS.Cg-+Leprdb/+Leprdb/OlaHsd
db/db mice (Harlan Sprague Dawley, Inc.; Indianapolis, IN); (diamonds) vehicle control; (triangles) 25 mg/kg 9; (circles) 25 mg/kg 7b; (squares)
25 mg/kg 8f. Data shown are the mean values ( SD (n ) 6): (*) P ) 0.0022; (**) P ) 0.0001.
147, 129, 106, 102, 97; HRMS calcd for C9H11N4O3S (M + H)
2-(3,4-Difluorophenylcarbamothioyl)hydrazinecarboxamide (1k).
Yield, 72%; 1H NMR (300 MHz, DMSO-d6) δ 6.08 (s, 2 H, NH2),
7.30-7.39 (m, 2 H), 7.70-7.724 (m, 1 H), 8.03 (s, 1 H, NH), 9.55
(s, 1 H, NH), 9.81 (s, 1 H, NH); MS m/z 515 (2M + Na)+, 269 (M
+ Na)+, 247 (M + H)+, 230, 204, 192, 147, 121, 102, 97; HRMS
calcd for C8H9F2N4OS (M + H) 247.0460, found 247.0455.
2-(3-Fluorophenylcarbamothioyl)hydrazinecarboxamide (1l).
Yield, 81%; 1H NMR (300 MHz, DMSO-d6) δ 6.08 (s, 2 H, NH2),
6.94 (t, J ) 8.4 Hz, 1 H), 7.31-7.36 (m, 2 H), 7.58-7.63 (m, 1
H), 8.03 (s, 1 H, NH), 9.53 (s, 1 H, NH), 9.78 (s, 1 H, NH); MS
m/z 251 (M + Na)+, 229 (M + H)+, 212, 186, 147, 118, 106, 85;
HRMS calcd for C8H10FN4OS (M + H) 229.0554, found 229.0553.
2-(3-Methoxyphenylcarbamothioyl)hydrazinecarboxamide (1m).
Yield, 84%; 1H NMR (300 MHz, DMSO-d6) δ 3.73 (s, 3 H, OMe),
6.05 (s, 2 H, NH2), 6.69 (d, J ) 7.8 Hz, 1 H), 7.10 (d, J ) 7.8 Hz,
1 H), 7.20 (t, J ) 7.8 Hz, 1 H), 7.26 (s, 1 H), 8.01 (s, 1 H, NH),
9.40 (s, 1 H, NH), 9.61 (s, 1 H, NH); MS m/z 503 (2M + Na)+,
481 (2M + H)+, 263 (M + Na)+, 241 (M + H)+, 224, 198, 166,
147, 129, 118, 107, 97; HRMS calcd for C9H12N4O2S (M + H)
241.0754, found 241.0755.
255.0546, found 255.0546.
2-(4-Fluorophenylcarbamothioyl)hydrazinecarboxamide (1c).
Yield, 76%; 1H NMR (300 MHz, DMSO-d6) δ 6.05 (s, 2 H, NH2),
7.10-7.18 (m, 2 H), 7.44-7.50 (m, 2 H), 8.02 (s, 1 H, NH), 9.41
(s, 1 H, NH), 9.69 (s, 1 H, NH); MS m/z 251 (M + Na)+, 229 (M
+ H)+, 212, 192, 186, 141, 129, 106, 102, 97, 85; HRMS calcd
for C8H10FN4OS (M + H) 229.0554, found 229.0560.
2-(2,4-Dimethoxyphenylcarbamothioyl)hydrazinecarboxamide
1
(1d). Yield, 81%; H NMR (300 MHz, DMSO-d6) δ 3.74 (s, 3 H,
OMe), 3.77 (s, 3 H, OMe), 6.07(s, 2 H, NH2), 6.48 (d, J ) 8.1 Hz,
1 H), 6.59 (s, 1 H), 7.84 (d, J ) 8.1 Hz, 1 H), 8.08 (s, 1 H, NH),
8.93 (s, 1 H, NH), 9.36 (s, 1 H, NH); HRMS calcd for C10H15N4O3S
(M + H) 271.0865, found 271.0866.
2-(3,5-Dimethoxyphenylcarbamothioyl)hydrazinecarboxamide
1
(1e). Yield, 85%; H NMR (300 MHz, DMSO-d6) δ 6.06 (s, 2 H,
NH2), 6.26 (s, 1 H), 6.87 (s, 2 H), 7.99 (s, 1 H, NH), 9.40 (s, 1 H,
NH), 9.56 (s, 1 H, NH); HRMS calcd for C10H15N4O3S (M + H)
271.0865, found 271.0868.
2-(3,4-Dimethoxyphenylcarbamothioyl)hydrazinecarboxamide
(1f). Yield, 82%; H NMR (300 MHz, DMSO-d6) δ 6.04 (s, 2 H,
2-(2-Methoxyphenylcarbamothioyl)hydrazinecarboxamide (1n).
Yield, 86%; 1H NMR (300 MHz, DMSO-d6) δ 3.81 (s, 3 H, OMe),
6.15 (s, 2 H, NH2), 6.94 (t, J ) 6.9 Hz, 1 H), 7.04 (d, J ) 7.2 Hz,
1 H), 7.09-7.14 (m, 2 H), 8.33 (s, 1 H, NH), 9.11 (1 H, NH), 9.56
(s, 1 H, NH); HRMS calcd for C9H12N4O2S (M + H) 241.0754,
found 241.0757.
2-(3-(Trifluoromethyl)phenylcarbamothioyl)hydrazinecarbo-
xamide (1o). Yield, 69%; 1H NMR (300 MHz, DMSO-d6) δ 6.10
(s, 2 H), 7.45-7.54 (m, 2 H), 7.87 (d, J ) 6.6 Hz, 1 H), 8.00 (s,
1 H), 8.07 (s, 1 H), 9.61 (s, 1 H), 9.96 (s, 1 H); MS m/z 579 (2M
+ Na)+, 557 (2M + H)+, 301 (M + Na)+, 279 (M + H)+, 262,
236, 169, 126, 88, 74; HRMS calcd for C9H9F3N4OS 279.0522 (M
+ H), found 279.0526.
1
NH2), 6.86 (d, J ) 8.7 Hz, 1 H), 6.99 (d, J ) 8.7 Hz, 1 H), 7.25
(s, 1 H), 7.99 (s, 1 H, NH), 9.28 (s, 1 H, NH), 9.51 (s, 1 H, NH);
HRMS calcd for C10H15N4O3S (M + H) 271.0865, found 271.0864.
2-(Naphthalen-1-ylcarbamothioyl)hydrazinecarboxamide (1g).
Yield, 79%; 1H NMR (300 MHz, DMSO-d6) δ 6.10 (s, 2 H, NH2),
7.38-7.55 (m, 4 H), 7.84 (d, J ) 7.8 Hz, 1 H), 7.91-7.97 (m, 2
H), 8.21 (s, 1 H, NH), 9.46 (s, 1 H, NH), 9.86 (s, 1 H, NH); MS
m/z 543 (2M + Na)+, 521 (2M + H)+, 283 (M + Na)+, 261 (M +
H)+, 244, 218, 159, 144, 85, 74, 56; HRMS calcd C12H13N4OS (M
+ H) 261.0805, found 261.0809.
2-(2,5-Dimethoxyphenylcarbamothioyl)hydrazinecarboxamide
2-(4-(Trifluoromethyl)phenylcarbamothioyl)hydrazinecarbo-
xamide (1p). Yield, 56%; 1H NMR (300 MHz, DMSO-d6) δ 6.10
(s, 2 H), 7.66 (d, J ) 8.7 Hz, 2 H), 7.82 (d, J ) 8.4 Hz, 2 H), 8.07
(s, 1 H), 9.63 (s, 1 H), 9.56 (s, 1 H); HRMS calcd for C9H9F3N4OS
279.0522 (M + H), found 279.0524.
1
(1h). Yield, 84%; H NMR (300 MHz, DMSO-d6) δ 3.68 (s, 3 H,
OMe), 3.77 (s, 3 H, OMe), 6.19 (s, 2 H, NH2), 6.65 (dd, J ) 9
and 2.7 Hz, 1 H), 6.95 (d, J ) 9 Hz, 1 H), 8.20 (s, 1 H), 8.28 (s,
1 H, NH), 9.14 (s, 1 H, NH), 9.62 (s, 1 H, NH); HRMS calcd for
C10H15N4O3S (M + H) 271.0865, found 271.0862.
2-(4-Methoxyphenylcarbamothioyl)hydrazinecarboxamide (1r).
Yield, 89%; 1H NMR (300 MHz, DMSO-d6) δ 3.77 (s, 3 H, OMe),
6.02 (s, 2 H, NH2), 6.87 (d, J ) 9 Hz, 2 H), 7.33 (d, J ) 9 Hz, 2
H), 7.98 (s, 1 H, NH), 9.28 (s, 1 H, NH), 9.53 (s, 1 H, NH); HRMS
calcd for C9H12N4O2S (M + H) 241.0754, found 241.0756.
4-(4-tert-Butylphenyl)-5-mercapto-4H-1,2,4-triazol-3-ol(2a).2-Thio-
biurea compound 1a (502 mg, 1.88 mmol) in 2 M NaOH aqueous
solution (20 mL) was stirred at reflux for 6 h. The reaction mixture
was allowed to cool to room temperature and then acidified with 1
N HCl. The resulting precipitate was collected by filtration and
washed with water (3 × 20 mL), hexanes (2 × 20 mL), 1:1 hexanes/
diethyl ether (20 mL) successively and dried in vacuo to afford 2a
2-(4-(Trifluoromethoxy)phenylcarbamothioyl)hydrazinecarbo-
xamide (1i). Yield, 82%; H NMR (300 MHz, DMSO-d6) δ 6.07
1
(s, 2 H, NH2), 7.29 (d, J ) 8.4 Hz, 2 H), 7.62 (d, J ) 9 Hz, 2 H),
8.07 (s, 1 H, NH), 9.51 (s, 1 H, NH), 9.83 (s, 1 H, NH); HRMS
calcd for C9H10F3N4O2S (M + H) 295.0477, found 295.047 78.
2-(4-Nitrophenylcarbamothioyl)hydrazinecarboxamide (1j).
Yield, 88%; 1H NMR (300 MHz, DMSO-d6) δ 6.13 (s, 2 H, NH2),
7.96 (d, J ) 9.0 Hz, 2 H), 8.13 (s, 1 H, NH), 8.18 (d, J ) 9.3 Hz,
2 H), 9.80 (s, 1 H, NH), 10.13 (s, 1 H, NH); MS m/z 278 (M +
Na)+, 256 (M + H)+, 253, 239, 169, 146, 107, 104, 87, 76; HRMS
calcd for C8H9N5O3S 256.0499 (M + H), found 256.0500.

1948 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
De et al.
1
as a white solid (355 mg, 76%). H NMR (300 MHz, DMSO-d6)
)5-Mercapto-4-(2-methoxyphenyl)-4H-1,2,4-triazol-3-ol (2n).
Yield, 65%; 1H NMR (300 MHz, DMSO-d6) δ 3.74 (s, 3 H, OMe),
7.04 (t, J ) 7.2 Hz, 1 H), 7.15-7.24 (m, 2 H), 7.4 (t, J ) 7.2 Hz,
1 H); HRMS calcd for C9H10N3O2S (M + H) 224.0488, found
224.0486.
δ 1.31 (s, 9 H), 7.28 (d, J ) 8.4 Hz, 2 H), 7.51 (d, J ) 8.4 Hz, 2
H); MS m/z 521 (2M + H)+, 497 (2M - H)-, 272 (M + Na)+,
250 (M + H)+, 151, 147, 134, 106, 97, HRMS calcd for
C12H16N3OS (M + H) 250.1009, found 250.1005.
5-Mercapto-4-(3-(trifluoromethyl)phenyl)-4H-1,2,4-triazol-
3-ol (2o). Yield, 68%; 1H NMR (300 MHz, DMSO-d6) δ 7.75-7.77
(m, 2 H), 7.82 (d, J ) 3.9 Hz, 1 H), 7.86 (s, 1 H); MS m/z 543
(2M + Na)+, 284 (M + Na)+, 262 (M + H)+, 226, 159, 141, 102,
74; HRMS calcd for C9H9F3N4OS 279.0522 (M + H), found
279.0526.
Following the above-mentioned procedure and with the appropri-
ate starting materials and reagents used, compounds 2b-q were
prepared.
4-(Benzo[d][1,3]dioxol-5-yl)-5-mercapto-4H-1,2,4-triazol-3-ol (2b).
Yield, 71%; 1H NMR (300 MHz, DMSO-d6) δ 6.10 (s, 2 H),
6.80-7.01 (m, 3 H); MS m/z 497 (2M + Na)+, 475 (2M + H)+,
260 (M + Na)+, 238 (M + H)+, 158, 120, 88, 56; HRMS calcd
for C9H8N3O3S (M + H) 238.0281, found 238.0278.
4-(4-Fluorophenyl)-5-mercapto-4H-1,2,4-triazol-3-ol (2c). Yield,
68%; 1H NMR (300 MHz, DMSO-d6) δ 7.28-7.37 (m, 2 H),
7.40-7.46 (m, 2 H); MS m/z 445 (2M + Na)+, 423 (2M + H)+,
234 (M + Na)+, 212 (M + H)+, 158, 88, 56; HRMS calcd for
C8H7FN3OS (M + H) 212.0288, found 212.0289.
4-(2,4-Dimethoxyphenyl)-5-mercapto-4H-1,2,4-triazol-3-ol (2d).
Yield, 65%; 1H NMR (300 MHz, DMSO-d6) δ 3.72 (s, 3 H, OMe),
3.81 (s, 3 H, OMe), 6.58 (d, J ) 8.7 Hz, 1 H), 6.69 (s, 1 H), 7.08
(d, J ) 8.7 Hz, 1 H); MS m/z 529 (2M + Na)+, 507 (2M + H)+,
276 (M + Na)+, 254 (M + H)+, 212, 158, 146, 141, 97, 88, 74,
56; HRMS calcd for C10H12N3O3S (M + H) 254.0595, found
254.0586.
5-Mercapto-4-(4-(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-ol
1
(2p). Yield, 45%; H NMR (300 MHz, DMSO-d6) δ 7.58 (d, J )
9.0 Hz, 2H), 8.06 (d, J ) 8.4 Hz, 2H); MS m/z 523 (2M + H)+,
284 (M + Na)+, 262 (M + H)+, 260 (M - H)-, 238, 102, 91, 74;
HRMS calcd for C9H6F3N3OS 262.0256 (M + H), found 262.0253.
4-Cyclohexyl-5-mercapto-4H-1,2,4-triazol-3-ol (2q). 2q was com-
mercially available.
5-Mercapto-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-ol (2r).
Yield, 69%; 1H NMR (300 MHz, DMSO-d6) δ 3.79 (s, 3 H, OMe),
7.02 (d, J ) 7.5 Hz, 2 H), 7.25 (d, J ) 7.5 Hz, 2 H); HRMS calcd
for C9H10N3O2S (M + H) 224.0488, found 224.0489.
4-(4-tert-Butylphenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-
3-ol (3a). To a solution of 2a (94 mg, 0.377 mmol) in MeOH (2
mL) was added MeONa (0.75 mL, 0.5 M solution in MeOH), and
the mixture was stirred. After 5 min, 2-bromo-5-nitrothiazole (78
mg, 0.377 mmol) was added to the reaction mixture, which was
stirred until the reaction was deemed complete by TLC (6 h). The
reaction mixture was acidified with 1 N HCl, and the resulting
precipitate was collected by filtration and washed with water (2 ×
30 mL), hexanes (2 × 30 mL), and 10% ethyl acetate in hexanes
(2 × 30 mL) to give a white solid. The residue was chromato-
graphed over silica gel (40% ethyl acetate in hexane) to afford 3a
4-(3,5-Dimethoxyphenyl)-5-mercapto-4H-1,2,4-triazol-3-ol (2e).
1
Yield, 68%; H NMR (300 MHz, DMSO-d6) δ 3.75 (s, 6 H, 2 ×
OMe), 6.54 (d, J ) 2.1 Hz, 2 H), 6.57 (d, J ) 2.4 Hz, 1 H); HRMS
calcd for C10H12N3O3S (M + H) 254.0595, found 254.0588.
4-(3,4-Dimethoxyphenyl)-5-mercapto-4H-1,2,4-triazol-3-ol (2f).
Yield, 71%; 1H NMR (300 MHz, DMSO-d6) δ 3.72 (s, 3 H, OMe),
3.79 (s, 3 H, OMe), 6.87 (d, J ) 8.4 Hz, 1 H), 6.94 (s, 1 H), 7.02
(d, J ) 8.4 Hz, 1 H); HRMS calcd for C10H12N3O3S (M + H)
254.0595, found 254.0588.
5-Mercapto-4-(naphthalen-1-yl)-4H-1,2,4-triazol-3-ol (2g). Yield,
74%; 1H NMR (300 MHz, DMSO-d6) δ 7.48-768 (m, 5 H),
8.01-8.112 (m, 2 H); MS m/z 509 (2M + Na)+, 487 (2M + H)+,
266 (M + Na)+, 244 (M + H)+, 186, 158, 144, 116, 85, 74, 67,
56; HRMS C12H10N3OS (M + H) 244.0539, found 244.0530.
4-(2,5-Dimethoxyphenyl)-5-mercapto-4H-1,2,4-triazol-3-ol (2h).
Yield, 68%; 1H NMR (300 MHz, DMSO-d6) δ 3.68 (s, 3 H, OMe),
3.71 (s, 3 H, OMe), 6.83 (d, J ) 2.7 Hz, 1 H), 7.03 (dd, J ) 8.4
and 2.7 Hz, 1 H), 7.08 (d, J ) 8.4 Hz, 1 H); HRMS calcd for
C10H12N3O3S (M + H) 254.0595, found 254.0589.
5-Mercapto-4-(4-(trifluoromethoxy)phenyl)-4H-1,2,4-triazol-
3-ol (2i). Yield, 72%; 1H NMR (300 MHz, DMSO-d6) δ 7.45-7.58
(m, 4 H); MS m/z 577 (2M + Na)+, 300 (M + Na)+, 278 (M +
H)+, 201, 178, 141, 134, 129, 106, 97, 85; HRMS calcd for
C9H7F3N3O2S (M + H) 278.0206, found 278.0211.
5-Mercapto-4-(4-nitrophenyl)-4H-1,2,4-triazol-3-ol (2j). Yield,
25%; 1H NMR (300 MHz, DMSO-d6) δ 7.79 (d, J ) 9.3 Hz, 2 H),
8.35 (d, J ) 9.0 Hz, 2 H); MS m/z 261 (M + Na)+, 242, 239 (M
+ H)+, 229, 130, 88, 85; HRMS calcd for C8H6N4O3S 239.0233
(M + H), found 239.0229.
4-(3,4-Difluorophenyl)-5-mercapto-4H-1,2,4-triazol-3-ol (2k).
Yield, 57%; 1H NMR (300 MHz, CD3OD) δ 7.02-7.08 (m, 1 H),
7.14-7.27 (m, 2 H); MS m/z 252 (M + Na)+, 230 (M + H)+, 184,
169, 107, 95, 85, 56; HRMS calcd for C8H6F2N3OS (M + H)
230.0194, found 230.0191.
1
(102 mg, 71%). H NMR (300 MHz, DMSO-d6) δ 1.27 (s, 9 H),
7.27 (d, J ) 8.4 Hz, 2 H), 7.47 (d, J ) 8.4 Hz, 2 H), 8.68 (s, 1 H);
MS m/z 777 (2M + Na)+, 755 (2M + H)+, 400 (M + Na)+, 378
(M + H)+, 354, 298, 250, 258, 207, 85, 74; HRMS calcd for
C15H16N5O3S2 (M + H) 378.0689, found 378.0690.
Following the above-mentioned procedure for 3a and with the
appropriate starting materials and reagents used, compounds 3b-q
were synthesized.
4-(Benzo[d][1,3]dioxol-5-yl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-
1
triazol-3-ol (3b). Yield, 60%; H NMR (300 MHz, DMSO-d6) δ
6.08 (s, 2 H), 6.84 (d, J ) 8.1 Hz, 1 H), 6.96 (s, 1 H), 7.01 (d, J
) 6 Hz, 1 H), 8.68 (s, 1 H); MS m/z 387 (M + Na)+, 365 (M +
H)+, 354, 349, 190, 158, 125, 102, 97; HRMS calcd for
C12H8N5O5S2 (M + H) 365.9961, found 365.9964.
4-(4-Fluorophenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-
1
3-ol (3c). Yield, 62%; H NMR (300 DMSO-d6) δ 7.28-7.35 (m,
2 H), 7.43-7.50 (m, 2 H), 8.65 (s, 1 H), 361 (M + Na)+, 339 (M
+ H)+, 294, 158, 85,74, 67, 60; HRMS calcd for C11H7FN5O3S2
(M + H) 339.9969, found 339.9964.
4-(2,4-Dimethoxyphenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-
1
triazol-3-ol (3d). Yield, 62%; H NMR (300 MHz, DMSO-d6) δ
3.66 (s, 3 H), 3.79 (s, 3 H), 6.65 (d, J ) 8.4 Hz, 2 H), 7.18 (s, 1
H), 8.69 (s, 1 H); MS m/z 785 (2M + Na)+, 763 (2M + H)+, 404
(M + Na)+, 382 (M + H)+, 359, 345, 158, 102, 85, 74, 55; HRMS
calcd for C13H12N5O5S2 (M + H) 382.0274, found 382.0281.
4-(3,5-Dimethoxyphenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-
1
triazol-3-ol (3e). Yield, 67%; H NMR (300 MHz, DMSO-d6) δ
4-(3-Fluorophenyl)-5-mercapto-4H-1,2,4-triazol-3-ol (2l). Yield,
56%; 1H NMR (300 MHz, DMSO-d6) δ 7.24-7.35 (m, 3 H),
7.52-7.56 (m, 1 H); HRMS calcd for C8H7FN3OS (M + H)
212.0288, found 212.0286.
3.70 (s, 6 H, 2 × OMe), 6.57 (s, 3 H), 8.69 (s, 1 H); HRMS calcd
for C13H12N5O5S2 (M + H) 382.0274, found 382.0280.
4-(3,4-Dimethoxyphenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-
1
triazol-3-ol (3f). Yield, 64%; H NMR (300 MHz, DMSO-d6) δ
3.67 (s, 3 H, OMe), 3.76 (s, 3 H, OMe), 6.88 (dd, J ) 8.4 and 1.8
Hz, 1 H), 6.98 (s, 1 H), 7.01 (d, J ) 8.4 Hz, 1 H), 8.68 (s, 1 H);
HRMS calcd for C13H12N5O5S2 (M + H) 382.0274, found 382.0282.
4-(Naphthalen-1-yl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-
3-ol (3g). Yield, 61%; 1H NMR (300 MHz, DMSO-d6) δ 7.38-7.76
(m, 5 H), 8.01-8.10 (m, 2 H), 8.53 (s, 1 H); MS m/z 394 (M +
5-Mercapto-4-(3-methoxyphenyl)-4H-1,2,4-triazol-3-ol (2m).
Yield, 74%; H NMR (300 MHz, DMSO-d6) δ 6.94 (d, J ) 8.7
Hz, 1 H), 6.96 (s, 1 H), 7.02 (d, J ) 8.4 Hz, 1 H), 7.40 (t, J ) 7.5
Hz, 1 H); MS m/z 246 (M + Na)+, 224 (M + H)+, 192, 147, 134,
106, 97; HRMS calcd for C9H10N3O2S (M + H) 224.0488, found
224.0488.
1

Triazole and Thiadiazole Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1949
Na)+, 372 (M + H)+, 217, 169, 130, 107,85, 74, 67, 45; HRMS
calcd for C15H10N5O3S2 (M + H) 372.0220, found 372.0224.
4-(2,5-Dimethoxyphenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-
5-(Ethylthio)-4-phenyl-4H-1,2,4-triazol-3-ol (4b). Yield, 72%; 1H
NMR (300 MHz, DMSO-d6) δ 1.35 (t, J ) 7.2 Hz, 3 H), 3.02 (t,
q, J ) 7.2 Hz, 2 H), 7.35-7.56 (m, 5 H), 10.51 (br s, 1 H, OH);
MS m/z 244 (M + Na)+, 222 (M + H)+, 200, 149, 121, 85; HRMS
calcd for C10H12N3OS (M + H) 222.0696, found 222.0697.
5-(Benzylthio)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4H-1,2,4-
1
triazol-3-ol (3h). Yield, 65%; H NMR (300 MHz, DMSO-d6) δ
3.62 (s, 3 H, OMe), 3.67 (s, 3 H, OMe), 6.93 (s, 1 H), 7.06 (dd, J
) 8.4 and 1.8 Hz, 1 H), 7.11 (d, J ) 8.4 Hz, 1 H), 8.69 (s, 1 H);
HRMS calcd for C13H12N5O5S2 (M + H) 382.0274, found 382.0282.
5-(5-Nitrothiazol-2-ylthio)-4-(4-(trifluoromethoxy)phenyl)-4H-
1
triazol-3-ol (4c). Yield, 75%; H NMR (300 MHz, DMSO-d6) δ
4.16 (s, 2 H), 4.26 (s, 4 H), 6.75 (d, J ) 8.4 Hz, 1 H), 6.82 (s, 1
H), 6.94 (d, J ) 8.7 Hz, 1 H), 7.22-7.31 (m, 5 H); MS m/z 705
((2M + Na)+, 683 (2M + H)+, 364 (M + Na)+, 342 (M + H)+,
326, 233, 158, 107, 87; HRMS calcd for C17H16N3O3S (M + H)
342.0907, found 342.0912.
1
1,2,4-triazol-3-ol (3i). Yield, 67%; H NMR (300 MHz, DMSO-
d6) δ 7.39-7.57 (m, 4 H), 8.63 (s,1 H); MS m/z 427 (M + Na)+,
405 (M + H)+, 364, 338, 306, 284, 192, 147, 106, 102, 97; HRMS
calcd for C12H7F3N5O4S2 (M + H) 405.9886, found 405.9891.
4-(4-Nitrophenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-3-
ol (3j). Yield, 60%; 1H NMR (300 MHz, DMSO-d6) δ 7.74 (d, J )
8.7 Hz, 2 H), 8.33 (d, J ) 8.7 Hz, 2 H), 8.62 (s, 1 H); MS m/z 367
(M + H)+, 360, 355, 85; HRMS calcd for C11H6N6O5S2 366.9914
(M + H), found 366.9900.
5-(5-Nitrothiophen-2-ylthio)-4-phenyl-4H-1,2,4-triazol-3-ol (4d).
1
Yield, 71%; H NMR (300 MHz, DMSO-d6) δ 7.06 (d, J ) 4.2
Hz, 1 H), 7.31-7.38 (m, 2 H), 7.41-7.49 (m, 3 H), 7.94 (d, J )
4.5 Hz, 1 H); MS m/z 321 (M + H)+, 277, 233, 200, 149, 97, 85;
HRMS calcd for C12H9N4O3S2 (M + H) 321.0111, found 321.0116.
2-Benzoyl-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)hydrazinecar-
bothioamide (5a). To a suspension of sodium acetate (226 mg, 2.76
mmol) and benzoylhydrazine (532 mg, 2.76 mmol) in acetonitrile
(5 mL) was added 2,3-dihydro-1,4-benzodioxin-6-yl isothiocyanate
(532 mg, 2.76 mmol), and the reaction mixture was stirred for 24 h
at room temperature. The solvent was removed by rotary evapora-
tion, and the resulting crude was dissolved in 2 M NaOH solution.
The filtrate was acidified with 1 N HCl. The precipitate was
collected and washed with water (3 × 20 mL) and hexanes (2 ×
4-(3,4-Difluorophenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-tria-
zol-3-ol (3k). Yield, 58%; 1H NMR (300 MHz, DMSO-d6) δ
7.28-7.59 (m, 3 H), 8.64 (s, 1 H); MS m/z 357 (M + H)+, 356,
355, 288, 226, 174, 130, 125, 102, 84, 56; HRMS calcd for
C11H6F2N5O3S2 (M + H) 357.9875, found 357.9866.
4-(3-Fluorophenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-
3-ol (3l). Yield, 68%; 1H NMR (300 MHz, DMSO-d6) δ 7.25-7.45
(m, 3 H), 7.49-7.56 (m, 1 H), 8.66 (s, 1 H); MS m/z 361 (M +
Na)+, 339 (M + H)+, 324, 229, 173, 158, 125, 116, 84, 74; HRMS
calcd for C11H7FN5O3S2 (M + H) 339.9969, found 339.9970.
4-(3-Methoxyphenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-
3-ol (3m). Yield, 61%; 1H NMR (300 MHz, DMSO-d6) δ 3.72 (s,
3 H, OMe), 6.95 (d, J ) 8.1 Hz, 1 H), 7.01 (s, 1 H), 7.02 (d, J )
8.1 Hz, 1 H), 7.37 (t, J ) 7.8 Hz, 1 H), 8.68 (s, 1 H); MS m/z 725
(2M + Na)+, 373 (M + Na)+, 352 (M + H)+, 338, 306, 284, 209,
201, 147, 106, 102, 97; HRMS calcd for C12H10N5O4S2 (M + H)
352.0169, found 352.0177.
1
20 mL) to afford 5a as a white solid (873 mg, 96%). H NMR
(300 MHz, DMSO-d6) δ 4.23 (s, 4 H), 6.81 (m, 2 H), 6.99 (s, 1 H,
NH), 7.49 (d, J ) 6.9 Hz, 1 H), 7.52 (d, J ) 7.8 Hz, 1 H), 7.58 (d,
J ) 7.2 Hz, 1 H), 7.96 (d, J ) 6.9 Hz, 2 H), 9.62 (s, 1 H), 9.68 (s,
1 H, NH), 10.50 (s, 1H, NH); MS m/z 352 (M + Na)+, 330 (M +
H)+, 306, 296, 284, 179, 138, 100, 83; HRMS calcd for
C16H15N3O3S 330.0907 (M + H), found 330.0902.
2-Benzoyl-N-(3-methoxyphenyl)hydrazinecarbothioamide (5c).
Yield, 78%; 1H NMR (300 MHz, DMSO-d6) δ 3.73 (s, 3 H, OMe),
6.72 (d, J ) 7.8 Hz, 1 H), 7.04 (d, J ) 7.8 Hz, 1 H), 7.22 (t, J )
8.4 Hz, 1 H), 7.45-7.59 (m, 3 H), 7.94 (d, J ) 7.5 Hz, 2 H), 9.72
(s, 1 H, NH), 9.73 (s, 1 H, NH), 10.53 (s, 1 H, NH).
4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-phenyl-4H-1,2,4-tria-
zole-3-thiol (6a). Compound 5a (1.15 g, 3.50 mmol) in 2 M NaOH
aqueous solution (35 mL) was stirred at reflux for 6 h. The reaction
mixture was allowed to cool to room temperature and then acidified
with 1 N HCl. The resulting precipitate was collected by filtration
and washed with water (3 × 20 mL), hexanes (2 × 20 mL), and
1:1 hexanes/diethyl ether (20 mL) successively and dried in vacuo
to afford 6a as a white solid (655 mg, 60%). 1H NMR (300 MHz,
DMSO-d6) δ 4.29 (s, 4 H), 6.75 (dd, J ) 2.4, 8.1 Hz, 1 H), 7.93
(d, J ) 8.7 Hz, 1 H), 7.94 (s, 1 H), 7.34-7.45 (m, 5 H); MS m/z
334 (M + Na)+, 312 (M + H)+, 280, 209, 179, 136, 100, 83; HRMS
calcd for C16H13N3O2S 312.0801 (M + H), found 312.0803.
4-(3-Methoxyphenyl)-5-phenyl-4H-1,2,4-triazole-3-thiol (6c).
Yield, 71%; 1H NMR (300 MHz, DMSO-d6) δ 3.72 (s, 3 H, OMe),
6.87 (d, J ) 7.8 Hz, 1 H), 7.02-7.08 (m, 2 H), 7.32-7.42 (m, 6
H); MS m/z 306 (M + Na)+, 284 (M + H)+, 252, 149, 129, 106,
97, 85; HRMS calcd for C15H14N3OS (M + H) 284.0852, found
284.0854.
4-(2-Methoxyphenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-
1
3-ol (3n). Yield, 69%; H NMR (300 MHz, DMSO-d6) δ 3.69 (s,
3 H, OMe), 7.03 (t, J ) 7.5 Hz, 1 H), 7.18 (d, J ) 8.4 Hz, 1 H),
7.30 (d, J ) 7.8 Hz, 1 H), 7.47 (t, J ) 7.8 Hz, 1 H), 8.69 (s, 1 H);
HRMS calcd for C12H10N5O4S2 (M + H) 352.0169, found 352.0172.
5-(5-Nitrothiazol-2-ylthio)-4-(3-(trifluoromethyl)phenyl)-4H-
1
1,2,4-triazol-3-ol (3o). Yield, 95%; H NMR (300 MHz, DMSO-
d6) δ 7.73-7.75 (m, 2 H), 7.83 (d, J ) 3.0 Hz, 1 H), 7.86 (s, 1 H),
8.66 (s, 1 H); MS m/z 412 (M + Na)+, 390 (M + H)+, 159, 141,
129, 102, 98, 90, 74; HRMS calcd for C12H6F3N5O3S2 389.9937
(M + H), found 389.9943.
5-(5-Nitrothiazol-2-ylthio)-4-(4-(trifluoromethyl)phenyl)-4H-
1
1,2,4-triazol-3-ol (3p). Yield, 94%; H NMR (300 MHz, DMSO-
d6) δ 7.55 (d, J ) 9.0 Hz, 2 H), 8.02 (d, J ) 9 Hz, 2 H), 8.65 (s,
1 H); MS m/z 412 (M + Na)+, 390 (M + H)+, 366, 221, 138, 74;
HRMS calcd for C12H6F3N5O3S3 389.9936 (M + H), found
389.9936.
4-Cyclohexyl-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-3-ol (3q).
1
Yield, 72%; H NMR (300 MHz, DMSO-d6) δ 1.01-1.26 (m, 3
H), 1.56-1.79 (m, 5 H), 2.01-2.10 (m, 2 H), 3.82-3.87 (m, 1 H),
8.75 (s, 1 H); MS m/z 677 (2M + Na)+, 350 (M + Na)+, 328 (M
+ H)+, 310, 306, 284, 281, 245, 204, 179, 138, 134, 129, 106,
100, 88, 60; HRMS calcd for C11H14N5O3S2 (M + H) 328.0533,
found 328.0548.
Compounds 6b,d-g were commercially available.
2-(4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-phenyl-4H-1,2,4-
triazol-3-ylthio)-5-nitrothiazole (7a). To a solution of 6a (173 mg,
0.556 mmol) in MeOH (1.4 mL) was added MeONa (1.3 mL, 0.5
M solution in MeOH) and stirred. After 5 min, 2-bromo-5-
nitrothiazole (127 mg, 0.608 mmol) was added to the reaction
mixture and stirred until the reaction was deemed complete by TLC
(18 h). The reaction mixture was acidified with 1 N HCl, and the
resulting precipitate was collected by filtration and washed with
water (2 × 30 mL), haxanes (2 × 30 mL), and 10% ethyl acetate
in hexanes (2 × 30 mL) to give a solid. The residue was
chromatographed over silica gel (50% ethyl acetate in hexane) to
4-(4-Methoxyphenyl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-
3-ol (3r). Yield, 71%; 1H NMR (300 MHz, DMSO-d6) δ 3.76 (s, 3
H, OMe), 7.01 (d, J ) 8.7 Hz, 2 H), 7.30 (d, J ) 8.7 Hz, 2 H),
8.68 (s, 1 H); HRMS calcd for C12H10N5O4S2 (M + H) 352.0169,
found 352.0174.
Similarly, compounds 4a-d were prepared using different aryl
and alkyl bromides.
5-(Butylthio)-4-phenyl-4H-1,2,4-triazol-3-ol (4a). Yield, 68%; 1H
NMR (300 MHz, CDCl3) δ 0.90 (t, J ) 7.2 Hz, 3 H), 1.39 (sextet,
J ) 7.8 Hz, 2 H), 1.66 (quintet, J ) 7.5 Hz, 2 H), 3.01 (t, J ) 7.5
Hz, 2 H), 7.35-7.57 (m, 5 H), 10.42 (br s, 1 H, OH); MS m/z 272
(M + Na)+, 250 (M + H)+, 233, 190, 149, 102, 85; HRMS calcd
for C12H16N3OS (M + H) 250.1009, found 250.1007.
1
afford the 7a (167 mg, 68%). H NMR (300 MHz, DMSO-d6) δ
4.29 (s, 4 H), 6.91 (dd, J ) 2.4, 7.8 Hz, 1 H), 6.89 (d, J ) 2.4 Hz,
1 H), 7.44-7.52 (m, 5 H), 8.73 (s, 1 H); MS m/z 462 (M + Na)+,

1950 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
De et al.
440 (M + H)+, 160, 150, 142, 138, 125, 102, 98, 60; HRMS calcd
for C19H13N5O4S2 440.0482 (M + H), found 440.0478.
Following the procedure as mentioned for 7a, compounds 7b-g
were synthesized.
2 H), 7.86 (d, J ) 7.8 Hz, 2 H), 8.12 (d, J ) 8.4 Hz, 2 H), 8.29 (s,
2H); HRMS calcd for C16H13N4O4S3 (M + H) 421.0093, found
421.0095.
2,5-Bis(4-nitrobenzylthio)-1,3,4-thiadiazole (8e). Yield, 72%; 1H
NMR (300 MHz, DMSO-d6) δ 4.63 (s, 4 H), 7.68 (d, J ) 8.7 Hz,
4 H), 8.17 (d, J ) 8.7 Hz, 4 H); MS m/z 442 (M + Na)+, 421 (M
+ H)+, 397, 364, 338, 306, 277, 201, 192, 147, 129, 118, 106,
102, 97; HRMS calcd for C16H13N4O4S3 (M + H) 421.0093, found
421.0097.
2-(4-(2,4-Dichlorophenyl)-5-(1-methyl-1H-pyrrol-2-yl)-4H-1,2,4-
1
triazol-3-ylthio)-5-nitrothiazole (7b). Yield, 91%; H NMR (300
MHz, DMSO-d6) δ 3.96 (s, 3 H), 5.59 (dd, J ) 1.5, 3.9 Hz, 1 H),
6.00 (dd, J ) 1.5, 3.6 Hz, 1 H) 7.06 (t, J ) 1.8 Hz, 1 H); MS m/z
475 (M + Na)+, 452 (M + H)+, 397, 369, 338, 159, 110, 98, 79;
HRMS calcd for C1cH10Cl2N6O2S2 452.9756 (M + H), found
452.9759.
2,5-Bis(5-nitrothiazol-2-ylthio)-1,3,4-thiadiazole (8f). Yield, 90%;
1H NMR (300 MHz, DMSO-d6) δ 8.86 (s, 2H); MS m/z 429 (M +
Na)+, 406 (M + H)+, 227, 159, 102, 56; HRMS calcd for
C8H2N6O4S5 406.8814 (M + H), found 406.8814.
2-(4-(3-Methoxyphenyl)-5-phenyl-4H-1,2,4-triazol-3-ylthio)-5-ni-
1
trothiazole (7c). Yield, 72%; H NMR (300 MHz, DMSO-d6) δ
2,2′-(1,3,4-Thiadiazole-2,5-diyl)bis(sulfanediyl)dithiazole-5-car-
3.71 (s, 3 H, OMe), 6.97 (d, J ) 7.8 Hz, 1 H), 7.10-7.16 (m, 2
H), 7.33-7.50 (m, 6 H), 8.70 (s, 1 H); MS m/z 434 (M + Na)+,
412 (M + H)+, 364, 354, 298, 252, 141, 138, 129, 102, 60; HRMS
calcd for C18H14N5O3S2 (M + H) 412.0533, found 412.0541.
1
boxylic Acid (8g). Yield, 78%; H NMR (300 MHz, DMSO-d6) δ
8.36 (s, 2 H); MS m/z 404 (M + H)+, 306, 253, 233, 169, 146,
102, 95, 87, 85; HRMS calcd for C10H5N4O4S5 (M + H) 404.8909,
found 404.8908.
2-(4-(Furan-2-ylmethyl)-5-(naphthalen-1-yl)-4H-1,2,4-triazol-3-
ylthio)-5-nitrothiazole (7d). Yield, 13%; 1H NMR (300 MHz,
DMSO-d6) δ 5.16 (s, 2 H), 6.00 (d, J ) 3.3 Hz, 1 H), 6.14 (d, J )
2.4 Hz, 1 H), 7.38 (s, 1 H), 7.62-7.78 (m, 5 H), 8.10 (d, J ) 6.9
Hz, 1 H), 8.23 (d, J ) 6.9 Hz, 1 H), 8.73 (s, 1 H); MS m/z 871
(2M + H)+, 458 (M + Na)+, 436 (M + H)+, 326, 179, 102, 88,
84; HRMS calcd for C20H13N5O3S2 436.0533 (M + H), found
436.0543.
5-Nitro-2-(4-phenyl-5-(thiophen-2-yl)-4H-1,2,4-triazol-3-ylthio)-
thiazole (7e). Yield, 72%; 1H NMR (300 MHz, DMSO-d6)
6.86-7.06 (m, 2 H), 7.53-7.74 (m, 6 H), 8.68 (s, 1 H); MS m/z
409 (M + Na)+, 387 (M + H)+, 354, 338, 326, 310, 284, 179,
147, 138, 121, 106, 97, 60; HRMS calcd C15H10N5O2S3 (M + H)
387.991, found 387.9996.
5-Nitro-2-(4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-ylthio)thia-
zole (7f). Yield, 62%; 1H NMR (300 MHz, DMSO-d6) δ 7.36 (d, J
) 6 Hz, 2 H), 7.50-7.61 (m, 5 H), 8.62 (d, J ) 5.4 Hz, 2 H), 8.69
(s, 1 H); MS m/z 405 (M + Na)+, 383 (M + H)+, 359, 310, 226,
158, 141, 138, 129, 121, 109, 97, 90, 60; HRMS calcd for
C16H11N6O2S2 (M + H) 383.0379, found 383.0381.
5-(5-Nitrothiazol-2-ylthio)-1,3,4-thiadiazol-2-amine (9). To a
solution of 5-amino-1,3,4-thiadiazole-2-thiol (211 mg, 1.586 mmol)
in MeOH (3 mL) was added MeONa (3.8 mL, 0.5 M solution in
MeOH) and stirred. After 5 min, 2-bromo-5-nitrothiazole (331 mg,
1.586 mmol) was added to the reaction mixture and stirred until
the reaction was deemed complete by TLC (12 h). The reaction
mixture was acidified with 1 N HCl, and the resulting precipitate
was collected by filtration and washed with water (2 × 30 mL),
hexanes (2 × 30 mL), and 10% ethyl acetate in hexanes (2 × 30
mL) to give a white solid. The residue was chromatographed over
silica gel (70% ethyl acetate in hexane) to afford the 9 (281 mg,
1
68%). H NMR (300 MHz, DMSO-d6) δ 7.94 (br s, 2 H, NH2),
8.74 (s, 1 H); MS m/z 283 (M + Na)+, 261 (M + H)+, 229, 193,
158, 126, 97, 84, 47; HRMS calcd C5H4N5O2S3 (M + H) 261.9522,
found 261.9515.
4-Fluoro-N-(5-(5-nitrothiazol-2-ylthio)-1,3,4-thiadiazol-2-yl)ben-
zamide (10). A solution of 9 (75 mg, 0.288 mmol), 4-fluorobenzoic
acid (40 mg, 0.288 mmol), EDC (66 mg, 0.34 mmol), HOBt (47
mg, 0.34 mmol), DIEA (0.15 mL, 0.86 mmol) in DMF (2 mL)
was stirred at 50 °C for 16 h. The reaction mixture was extracted
with ethyl acetate (50 mL), washed with 10% NaHCO3 solution
(30 mL), brine (2 × 30 mL), and water (2 × 30 mL), dried
(MgSO4), and concentrated in vacuo. The residue was chromato-
graphed over silica gel (30% ethyl acetate in hexane) to afford the
5-((4-Methyl-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-triazol-3-yl)methyl)-
1
3-(pyrazin-2-yl)-1,2,4-oxadiazole (7g). Yield, 79%; H NMR (300
MHz, DMSO-d6) δ 1.24 (s, 2 H), 3.50 (s, 3 H), 8.78 (s, 1 H), 8.80
(s, 1 H), 8.87 (s, 2 H), 9.31 (s, 1 H); MS m/z 871 (2M + H)+, 458
(M + Na)+, 436 (M + H)+, 326, 179, 102, 88, 84; HRMS calcd
for C20H13N5O3S2 436.0533 (M + H), found 436.0543.
1
10 (82 mg, 75%). H NMR (300 MHz, DMSO-d6) δ 7.42-7.46
(m, 2 H), 7.67 (s, 1 H, NH), 8.20-8.22 (m, 2 H), 8.87 (s, 1 H);
MS m/z 381 (M-H)+, 306, 248, 204, 180, 174, 154, 138, 96; HRMS
calcd for C12H5FN5O3S3 (M - H) 381.9544, found 381.9535.
5-(Benzylthio)-1,3,4-thiadiazol-2-amine (11). Yield, 69%; 1H
NMR (300 MHz, DMSO-d6) δ 4.28 (s, 2 H), 7.11-7.19 (m, 2 H),
7.34-7.42 (m, 5 H, Ar-H, NH2); MS m/z 264 (M + Na)+, 242
(M + H)+, 192, 147, 102, 97, 85; HRMS calcd C9H9H9FN3S2 (M
+ H) 242.0216, found 242.0215.
2,5-Bis(benzylthio)-1,3,4-thiadiazole (8a). To a solution of 1,3,4-
thiadiazole-2,5-dithiol (93 mg, 0.62 mmol) in MeOH (3 mL) was
added MeONa (2.8 mL, 0.5 M solution in MeOH) and stirred. After
5 min, benzyl bromide (233 mg, 1.364 mmol) was added to the
reaction mixture and stirred until the reaction was deemed complete
by TLC (6 h). The reaction mixture was acidified with 1 N HCl,
and the resulting precipitate was collected by filtration and washed
with water (2 × 30 mL), haxanes (2 × 30 mL), and 10% ethyl
acetate in hexanes (2 × 30 mL) to give a white solid. The residue
was chromatographed over silica gel (30% ethyl acetate in hexane)
DELFIA Assay (Dissociation Enhanced Lanthanide Fluoroim-
muno Assay). To each well of 96-well streptavidin-coated plates
(Perkin-Elmer) 100 µL of a 100 ng/mL solution of biotin-labeled
pep-JIP11 (biotin-lc-KRPKRPTTLNLF, where lc indicates a hy-
drocarbon chain of six methylene groups) was added. After 1 h of
incubation and elimination of unbound biotin-pep-JIP11 by three
washing steps, 87 µL of Eu-labeled anti-GST antibody solution
(300 ng/mL, 1.9 nM), 2.5 µL of DMSO solution containing test
compound, and 10 µL solution of GST-JNK for a final protein
concentration of 10 nM were added. After 1 h of incubation at 0
°C, each well was washed five times to eliminate unbound protein
and the Eu-antibody if displaced by a test compound. Subsequently,
200 µL of enhancement solution (Perkin-Elmer) was added to each
well and fluorescence measured after 10 min of incubation
(excitation wavelength, 340 nm; emission wavelength, 615 nm).
Controls include unlabeled peptide and blanks receiving no
compounds. Protein and peptide solutions were prepared in DELFIA
buffer (Perkin-Elmer).
1
to afford the 8a (204 mg, 80%). H NMR (300 MHz, DMSO-d6)
δ 4.49 (s, 4 H), 7.25-7.45 (m, 10 H); MS m/z 353 (M + Na)+,
331 (M + H)+, 192, 124, 102, 85; HRMS calcd for C16H15N2S2
(M + H) 331.0392, found 331.0398.
2,5-Bis(4-fluorobenzylthio)-1,3,4-thiadiazole (8b). Yield, 75%; 1H
NMR (300 MHz, DMSO-d6) δ 4.49 (s, 4 H), 7.11-7.19 (m, 4 H),
7.42-7.46 (m, 4 H); MS m/z 389 (M + Na)+, 367 (M + H)+, 338,
284, 192, 147, 106, 102, 97, 85; HRMS calcd for C16H13F2N2S3
(M + H) 367.0203, found 367.0208.
2,5-Bis(4-methoxybenzylthio)-1,3,4-thiadiazole (8c). Yield, 76%;
1H NMR (300 MHz, DMSO-d6) δ 3.72 (s, 6 H), 4.43 (s, 4 H),
6.87 (d, J ) 8.4 Hz, 4 H), 7.32 (d, J ) 8.4 Hz, 4 H); MS m/z 413
(M + Na)+, 391 (M + H)+, 305, 261, 226, 158, 149, 138, 106,
102, 97, 84; HRMS calcd for C18H19N2O2S3 (M + H) 391.0603,
found 391.0606.
2,5-Bis(3-nitrobenzylthio)-1,3,4-thiadiazole (8d). Yield, 73%; 1H
NMR (300 MHz, DMSO-d6) δ 4.64 (s, 4 H), 7.61(t, J ) 8.1 Hz,
In Vitro Kinase Assay. The LanthaScreen assay platform from
Invitrogen was utilized. The time-resolved fluorescence resonance

Triazole and Thiadiazole Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1951
energy transfer assay (TR-FRET) was performed in 384-well plates.
Each well received JNK (35 ng/mL), ATF2 (400 nM), and ATP
(200 µM) in 50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, and
0.01% Brij-35, pH 7.5, and test compounds. The kinase reaction
was performed at room temperature for 1 h, after which the terbium
labeled antibody and EDTA were added into each well. After an
additional hour of incubation, the signal was measured at 520/495
nm emission ratio on a fluorescence plate reader (Victor 2, Perkin-
Elmer).
of key compounds, and supporting figures. This material is available
free of charge via the Internet at http://pubs.acs.org.
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In Vivo Experiments. Insulin Tolerance Test. Eleven-week-
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Harlan (Harlan Sprague Dawley, Inc.; Indianapolis, IN) were
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Acknowledgment. In vivo experiments related to the insulin
tolerance were performed by Explora Biolabs. Elemental
analysis was performed by NuMega Laboratory, San Diego, CA.
Financial support was obtained from NIH Grants DK073274
and DK080263 and by a CSRA by Syndexa Pharmaceuticals
(to M.P.).
Supporting Information Available: Proton NMR spectra,
HPLC traces of key compounds (purity), elemental analysis data

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