Rationally Designed Small-Molecule Inhibitors Targeting an Unconventional Pocket on the TLR8 Protein-Protein Interface

Article abstract of DOI:10.1021/acs.jmedchem.9b02128

Rational designs of small-molecule inhibitors targeting protein-protein interfaces have met little success. Herein, we have designed a series of triazole derivatives with a novel scaffold to specifically intervene with the interaction of TLR8 homomerization. In multiple assays, TH1027 was identified as a highly potent and specific inhibitor of TLR8. A successful solution of the X-ray crystal structure of TLR8 in complex with TH1027 provided an in-depth mechanistic insight into its binding mode, validating that TH1027 was located between two TLR8 monomers and recognized as an unconventional pocket, thereby preventing TLR8 from activation. Further biological evaluations showed that TH1027 dose-dependently suppressed the TLR8-mediated inflammatory responses in both human monocyte cell lines, peripheral blood mononuclear cells, and rheumatoid arthritis patient specimens, suggesting a strong therapeutic potential against autoimmune diseases.

Full text of DOI:10.1021/acs.jmedchem.9b02128

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Article
Rationally designed small-molecule inhibitors targeting an
unconventional pocket on the TLR8 protein-protein interface
Shuangshuang Jiang, Hiromi Tanji, Kejun Yin, Shuting Zhang, Kentaro Sakaniwa,
Jian Huang, Yi Yang, Jing Li, Umeharu Ohto, Toshiyuki Shimizu, and Hang Hubert Yin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02128 • Publication Date (Web): 31 Mar 2020
Downloaded from pubs.acs.org on April 8, 2020
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Journal of Medicinal Chemistry
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Rationally designed small-molecule inhibitors targeting an uncon-
ventional pocket on the TLR8 protein-protein interface
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Shuangshuang Jiang1, Hiromi Tanji2, Kejun Yin1, Shuting Zhang1, Kentaro Sakaniwa2, Jian Huang1,
Yi Yang1, Jing Li3, Umeharu Ohto2, Toshiyuki Shimizu2 and Hang Yin1*
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1 Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education),
Department of Chemistry, School of Pharmaceutical Sciences, Tsinghua University-Peking University
Joint Center for Life Sciences, Tsinghua University, Beijing 100082, China
2 Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
3 Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital and
Chinese Academy of Medical Sciences, Key Laboratory of Rheumatology and Clinical Immunology,
Ministry of Education, Beijing 100032, China
Keywords: drug discovery, inhibitors, innate immunity, Toll-like receptors
ABSTRACT: Rational designs of small-molecule inhibitors targeting protein-protein interfaces have met little success. Herein, we
have designed a series of triazole derivatives with a novel scaffold to specifically intervene with the interaction of TLR8 homomer-
ization. In multiple assays, TH1027 was identified as a highly potent and specific inhibitor of TLR8. Successful solution of the X-
ray crystal structure of TLR8 in complex with TH1027 shed in-depth mechanistic insight of its binding mode, validating that
TH1027 located between two TLR8 monomers and recognized an unconventional pocket thereby preventing TLR8 from activation.
Further biological evaluations showed that TH1027 dose-dependently suppressed the TLR8-mediated inflammation responses in
both human monocyte cell lines, peripheral blood mononuclear cells (PBMCs), and rheumatoid arthritis patient specimens, suggest-
ing strong therapeutic potential against autoimmune diseases.
INTRODUCTION
es.12,13 Therefore, the development of small-molecule inhibitors
selectively targeting TLR8 may provide useful tools to further
understand the regulation mechanism and provide a new per-
spective for discovering new therapies for autoimmune diseas-
es. However, due to the flexible interface of the TLR8 dimeric
complex and the high similarity of TLR7 and TLR8 in se-
quence, it is a great challenge to develop small-molecule
TLR8-selective modulators. To our knowledge, resiquimod
(R848),14 the synthetic dual hTLR7/8 agonist, has been con-
firmed effective for the treatment of genital herpes in clinical.
A series of TLR8 specific agonists have been discovered, in-
cluding furo[2,3-c]pyridines,15 furo[2,3-c]quinolones,16 thia-
Toll-like receptor (TLR) family are a group of highly con-
served transmembrane proteins which form the first barrier of
the immune defense in higher animals.1 Through the recogni-
tion of pathogen-associated molecular patterns (PAMPs) and
danger-associated molecular patterns (DAMPs), TLRs trigger
inflammatory and antiviral responses.2 Ten different TLRs
(TLR 1 through 10) have been identified in human to date
which respond to a variety of PAMPs, including lipopeptides
(TLR1/2 or TLR2/6),3,4 viral double-stranded RNA (TLR3 and
10),5,6 lipopolysaccharide (TLR4),7 bacterial flagellin (TLR5),8
viral or bacterial single-stranded RNA (TLR7 and TLR8),9,10
and cytidine–phosphate guanosine (CpG)-rich unmethylated
DNA (TLR9).11 All these TLRs play a key role in the innate
and adaptive immune responses during pathogen invasion and
the repair of damaged tissues. However, the persistent activa-
tion of TLRs, for instance, TLR8 has also been suggested to
contribute to the pathogenesis of various autoimmune diseas-
zolo[4,5-c]quinolones,17
benzimidazol-2-amines,18
2-
aminoimidazoles,19 pyrimidine-2,4-diamines.20 There are also
TLR7 and TLR8 dual agonists including imidazo-quinolines,21
pyrimidine derivatives,22 pyrimidine-2,4-diamines.23 And
TLR7/8 dual antagonists under development are mainly 3H
imidazo-quinolines24 and benzanilide scaffolds.25 Despite that
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TLR8 was first discovered more than 20 years ago26 and re- aReagents and conditions: (A) EtOH, reflux, 10 h; (B) 2M
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markable clinical potential has been implied, the development
of TLR8 specific inhibitors lagged far behind. Most recently,
our group has reported the first series of inhibitors of TLR8,
designated as CU-CPTs,27,28 bearing a pyrazolo[1,5-a] pyrim-
idine scaffold and a quinoline scaffold, which stabilized the
preformed TLR8 dimer in its resting state (Figure S1). None-
theless, new chemical scaffolds are still much needed to target
TLR8 due to its biological relevance. Herein, we reported a
new series of small-molecule inhibitors of TLR8 with a 1,2,4-
triazole scaffold targeting the unliganded TLR8 dimeric com-
plex, laying the groundwork for future therapeutic develop-
ment.
NaOH (aq), reflux, 10 h, then 2M HCl (aq), stirred for 10 mins;
(C) CH3CN, K2CO3, rt, 12 h.
Scheme 2. Synthesis of compounds 1g and 1ha.
N
N
N
N
HO
O
CH3
CH3
R1'
S
S
N
N
A
Cl
Cl
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Cl
Cl
R1'= -CH3 (1g); CH3CO- (1h).
1a
aReagents and conditions: (A) NaH, anhydrous THF, CH3I, rt,
12 h (for 1g); acetic anhydride, 4-dimethylaminopyridine,
anhydrous dichloromethane, rt, 12 h (for 1h).
RESULTS and DISCUSSION
Structure-activity relationship studies
Rational design of small-molecule inhibitors of TLR8
With the scaffold identified, we developed a synthetic route
(Scheme 1-4 ) for 1,2,4-triazole derivatives and carried out
structure and activity relationship (SAR) studies (Table 1).
The SAR results indicated that the length of linker at the R1
position had no significant influence as long as the chain is not
too short (1a versus 56-G5, 1b, 1c, 1d and 1e). Comparison of
different compounds demonstrated that a three-carbon chain at
R1 position displayed relatively better inhibitory activity. Next,
removal of the hydroxyl group at R1 position (1f) rendered a
modest effect on its activity, while methylation (1g) or acety-
lation (1h) of hydroxyl group led to slight decrease of the po-
tency. These results implied that the proper length of the car-
bon chain and the hydroxyl group may be both important for
the formation of hydrogen bonds with surrounding amino acid
residues. Therefore, in the following SAR studies, the hydrox-
yl group linked with a three-carbon chain was fixed at the R1
position. Furthermore, when the methyl group was replaced
with hydrogen (2a) or more hydrophilic groups (2d, 2e) at the
R2 position, the inhibitory activity was completely compro-
mised. Introducing the ethyl (2b) or isopropyl group (2c) at
the R2 position sharply reduced the potency, suggesting a
small and hydrophobic substituent was favored here.
In order to identify inhibitors with a novel scaffold that can
specifically target the newly discovered binding site on the
interface of TLR8 dimer (Figure 1A),27 we carefully analyze
the crystal structure of apo TLR8 dimer (PDB: 3W3G) and
come up with following solutions: First of all, aiming to utilize
the hydrogen bonding potential of Y348 within the binding
pocket, we introduced different heterocycles especially with
more hydrogen bond acceptors which may increase the possi-
bility of forming hydrogen bonds and the water solubility.
Then we introduced aromatic rings which might have hydro-
phobic interactions with the surrounding hydrophobic amino
acid residues like F405, F261, F494*, F495* and F346
(throughout this paper, asterisks are used to indicate the resi-
dues of the second copy of TLR8 monomer). Aiming to these
molecular traits, we screened a Maybridge library containing
14,400 drug-like molecules. Using the SEAP reporter assay,
we identified 72 compounds as hits, which inhibited TLR8-
mediated signaling more than 85% at 4 µM. Within these
compounds, 13 hits were selected due to their negligible cyto-
toxicity (Listed in SMILES file). Eventually, three compounds,
56-G5, 169-B11 and 169-E2 were selected (Figure S2), as
they all shared a 1,2,4-triazole scaffold and met the demanding
of the above rationale. Then we chose the most active com-
pound 56-G5 as a starting compound to carry out further stud-
ies.
Scheme 3. Synthesis of compounds 2a-2ea.
N
N
N
N
HO
HO
R2
SH
Cl
S
N
N
A
Cl
Scheme 1. Synthesis of compounds 56-G5, 1a-1fa.
NCS
Cl
2a
Cl
Cl
Cl
Cl
H
N
S
A
R1
H
N
+
R1
R1= -CH2CH3 (2b); -CH(CH3)2 (2c);
-CONH2 (2d); -CH2CONH2(2e).
H2N
N
H
N
H
O
O
Cl
N
N
N
N
CH3
N
aReagents and conditions: (A) ethyl iodide, K2CO3, acetone, rt,
12 h (for 2b); 2-iodopropane, K2CO3, acetone, rt, 12 h (for 2c);
trichloroacetyl chloride, acetonitrile, DMF, rt, 1 h, then 7M
ammonia methanol solution, reflux for 3h (for 2d); 2-
iodoacetamide, K2CO3 and acetonitrile, rt, 12 h (for 2e).
R1
R1
SH
S
N
C
B
CH3I
+
Cl
Cl
Cl
Cl
R1= -(CH2)3OH (56-G5); -OH (1a); -CH2OH (1b); -(CH2)2OH (1c);
-(CH2)4OH (1d); -(CH2)5OH (1e); -(CH2)3 (1f).
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Journal of Medicinal Chemistry
Scheme 4. Synthesis of compounds 3a-3pa
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Table 1. Structure-activity relationship analyses of the TLR8
inhibitors in HEK-Blue hTLR8 cells
S
H
N
H
N
R3
A
N
N
+
N
H
N
H
OH
R2
H2N
OH
R3 NCS
R1
S
O
O
N
R3
N
N
N
N
HO
HO
CH3
C
B
SH
S
CH3I
+
N
N
No.
56-G5
1a
R1
R2
R3
IC50[μM] [a]
0.29 ± 0.01
9.00 ± 0.41
0.55 ± 0.07
0.59 ± 0.03
1.06 ± 0.10
0.52 ± 0.05
0.63 ± 0.02
1.18 ± 0.14
R3
________________________
R3
Cmpd
R3
Cmpd
R3
-(CH2)3OH
-OH
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
2,4-di-Cl-C6H3-
2,4-di-Cl-C6H3-
2,4-di-Cl-C6H3-
2,4-di-Cl-C6H3-
2,4-di-Cl-C6H3-
2,4-di-Cl-C6H3-
2,4-di-Cl-C6H3-
2,4-di-Cl-C6H3-
_____________________
3a
3b
3c
3d
3e
3f
4-Cl-C6H4-
3i
3j
3k
3l
2,3-di-Cl-C6H3-
2,4,6-tri-Cl-C6H2-
2,3,4-tri-Cl-C6H2-
3,4,5-tri-Cl-C6H2-
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4-OCH3-C6H4-
4-CF3-C6H4-
2-Cl-C6H4-
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1b
-CH2OH
-(CH2)2OH
-(CH2)4OH
-(CH2)5OH
-(CH2)2CH3
-(CH2)3OCH3
3-Cl-C6H4-
3m(TH1027) 3,4,5-tri-CH3-C6H2-
1c
3-CF3-C6H4-
3-CH3-C6H4-
3,4-di-Cl-C6H3-
3n
3o
3p
3-pyridyl-
3g
3h
cyclohexyl-
1-naphthyl-
1d
1e
aReagents and conditions: (A) EtOH, reflux, 10 h; (B) 2M
NaOH (aq), reflux, 10 h, then 2M HCl (aq), stirred for 10 mins;
(C) CH3CN, K2CO3, rt, 12 h.
1f
1g
To further investigate whether different substituents and
positions on the benzene ring would influence the activities,
we synthesized 4-Cl (3a), 4-OCH3 (3b), 4-CF3 (3c), 2-Cl (3d),
3-Cl (3e), 3-CF3 (3f) and 3-CH3 (3g) substituted compounds.
Results indicated that the activity of 3-substituted compounds
was significantly higher than those with 4-substitutions or 2-
substitutions. All 3–Cl (3e), 3-CF3 (3f) and 3-CH3 (3g) substi-
tuted compounds showed similar activities which indicated
that electronic effects are not obvious here. Next, we explored
the multi-substitutions on benzene ring (3h-3l) and results
showed that 3,4,5-tri-chlorophenyl group (3l) at R3 position
showed the most potent inhibitory activity, suggesting that this
substitutions fitted better to the hydrophobic pocket. Then we
tried to replace the 3,4,5-tri-Cl substitution with 3,4,5-tri-
methyl substitution and the inhibitory activity was increased
about 4-fold (3l versus TH1027), suggesting TH1027 may
match better to the target hydrophobic pocket. Also, cyclohex-
yl (3o) and 3-pyridyl (3n) substitution at the R3 position
showed no activities which may indicate the existence of a
hydrophobic pocket around R3 position. In summary, with
extensive and systematic SAR exploration, we have identified
TH1027 as the lead compound. Both synthetic small molecule
R848 and single stranded RNA are previously established as
nonselective TLR8 activators.10,14 In the cellular assay,
TH1027 showed dose-dependent inhibitory effects in blocking
R848- or ssRNA06-induced TLR8 activation with an IC50 of
30.0 ± 2.0 nM (Figure 1B). Importantly, TH1027 was found to
have no significant cytotoxicity at concentrations up to 100
μM in HEK-Blue TLR8 cells (Figure S3).
-
1h
-CH3
-H
2,4-di-Cl-C6H3-
2.08 ± 0.10
(CH2)3OCOCH3
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
3f
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
2,4-di-Cl-C6H3-
2,4-di-Cl-C6H3-
2,4-di-Cl-C6H3-
2,4-di-Cl-C6H3-
2,4-di-Cl-C6H3-
4-Cl-C6H4-
＞20
0.39 ± 0.04
10.12±0.81
＞20
-CH2CH3
-CH(CH3)2
-CONH2
-CH2CONH2
-CH3
＞20
1.61 ± 0.12
3.86 ± 0.31
4.84 ± 0.82
0.98 ± 0.14
0.35 ± 0.02
0.21 ± 0.02
-CH3
4-OCH3-C6H4-
4-CF3-C6H4-
2-Cl-C6H4-
-CH3
-CH3
-CH3
3-Cl-C6H4-
-CH3
3-CF3-C6H4-
3g
3h
3i
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
3-CH3-C6H4-
0.29 ± 0.03
0.19 ± 0.01
0.80 ± 0.07
2.81 ± 0.44
0.60 ± 0.10
0.11 ± 0.01
3,4-di-Cl-C6H3-
2,3-di-Cl-C6H3-
2,4,6-tri-Cl-C6H2-
2,3,4-tri-Cl-C6H2-
3,4,5-tri-Cl-C6H2-
3j
3k
3l
3m
-(CH2)3OH
-CH3
3,4,5-tri-CH3-C6H2-
0.030± 0.002
（
3n
3o
3p
TH1027
）
-(CH2)3OH
-(CH2)3OH
-(CH2)3OH
-CH3
-CH3
-CH3
3-pyridyl-
＞20
＞20
cyclohexyl-
1-naphthyl-
0.39 ± 0.06
Kd determination and on-target validation in cells
[a]
SEAP reporter assay was used to measure the inhibitory activities of
compounds. IC50 values and corresponding standard deviations were de-
termined from at least three biological replicates.
Next, isothermal titration calorimetry (ITC) confirmed that
TH1027 directly bound to the ectodomain of human TLR8
proteins with the dissociation constant (Kd) of 225 ± 38 nM
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(Figure 1C). The heat change induced by its synthetic ligand TH1027 on the mRNA level of pro-inflammatory cytokines
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R848 was markedly suppressed in the presence of TH1027,
indicating that TH1027 prevented R848 from binding to TLR8
(Figure S4).
by quantitative real-time PCR (RT-PCR) in HEK-Blue hTLR8
cells. The results showed that TH1027 completely abolished
the elevation of TNF-α and IL-8 mRNA levels induced by
R848. By contrast, the negative control, compound 2a, showed
negligible inhibition (Figure 1E).
A major challenge of developing TLR inhibitors is to
achieve high specificity. There are at least 13 homologous
TLRs present in murine macrophages and 10 homologous
TLRs in human, all sharing common structural motifs includ-
ing leucine-rich repeats.2 To evaluate its specificity, we tested
TH1027 against a panel of homologous TLRs, including
TLR1/2, TLR2/6, TLR3, TLR4, TLR5, TLR7 and TLR9, us-
ing TLR-specific ligands to selectively activate a particular
TLR signaling pathway. Result showed that TH1027 inhibited
TLR8-mediated signaling without affecting other TLRs, prov-
ing its highly selectivity in a complex, whole cell environment
(Figure 1D). We also investigated the inhibitory effects of
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Figure 1. Rational design of small-molecule TLR8 inhibitors. (A) X-ray crystal structure of the TLR8 protein (PDB: 3W3G).
The side view of unliganded conformation of the TLR8 ectodomain and the close-up view of the antagonist binding site of un-
liganded TLR8. (B) TH1027 dose-dependently inhibited R848- or ssRNA06-induced TLR8 activation with an IC50 of 30 nM. (C)
ITC thermogram of TH1027 titrated into human TLR8 (hTLR8) to determine its binding affinity and stoichiometry (Kd = 225 nM).
The raw data are presented on top and the integrated peak areas are shown and fitted below. Red line represents baseline for peak
integration. (D) Specificity test for TH1027 using TLR-specific ligands to selectively activate the respective TLRs: 100 ng/mL of
Pam3CSK4, 100 ng/mL of Pam2CSK4, 5 µg/mL of polyriboinosinic : polyribocytidylic acid [poly(I:C)], 20 ng/mL of LPS (lipopoly-
saccharide), 50 ng/mL of Flagellin, 1 µg/mL of R848, 2 µg/mL of R848 and 0.5 µM of ODN2006 were used to selectively activate
hTLR1/2, hTLR2/6, hTLR3, hTLR4, hTLR5, hTLR7, hTLR8 and hTLR9 cells, respectively (Data are mean s.d.). (E) TNF-α and
IL-8 mRNA levels in R848-treated HEK-Blue TLR8 cells in the presence or absence of 1 µM TH1027 or the negative control 2a (1
µM). Data are the average quantification of three independent biological replicates (Data are mean s.d.).
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Figure 2. Crystal structure of the TLR8/TH1027 complex (PDB ID: 6KYA, resolution: 2.89 Å). (A) Front view of the
TLR8/TH1027 complex. Two TLR8 monomers, TLR8 and TLR8*, are colored green and cyan, respectively. The ligand molecules
are illustrated by space-filling representations. The C, N, O and S atoms of the ligands are colored pink, blue, red and yellow, re-
spectively. (B) Side view of TLR8/TH1027 complex. (C) Close-up view of the antagonist binding site with TH1027. (D) Schemat-
ic representation of interactions between TH1027 and the TLR8 proteins. The hydrophobic pocket and hydrogen bonds are shown
as dashed gray arcs and a dashed red line, respectively.
X-ray crystal structure of the TLR8/TH1027 complex
(Figure 2C and 2D). In addition, hydroxyl group at the R1 po-
sition of TH1027 made another hydrogen bond with Y348
only in one protomer of the dimer, and its density was rather
poor, suggesting the hydrogen bond is weak. Moreover, no
hydrogen bond was observed in another protomer due to the
different orientation of hydroxyl group at the R1(Figure S5).
Considering these structural features, it was difficult to esti-
mate the significance of this interaction for the affinity from
the structure. Therefore we did not mark a dotted line to repre-
In order to elucidate the molecular mechanism of inhibition,
we successfully obtained the crystal structure of the
TLR8/TH1027 complex (PDB: 6KYA). Clearly, TH1027
bound to the target pocket and located between two copies of
TLR8 monomers, validating our rational design (Figure 2A
and 2B). TH1027 formed a hydrogen bond with the polypep-
tide backbone of the G351 residue and was sandwiched by
Y348 and F495*, contributing π-π interactions at this pocket
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sented a hydrogen bond between TH1027 and Y348. Interest- could also be inhibited by TH1027 (Figure 3A). The blots
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ingly, these structural results also were in good agreement
with the results of SAR studies discussed above. The proper
length of linker and hydroxyl group at R1 position was essen-
tial for the formation of hydrogen bonds. For example, the
hydrophobic pocket around R2 position is relatively small.
Therefore, TH1027 with a methyl substitution showed better
activity compared with compounds with larger or more polar
groups at R2 (2a - 2c). Further, hydrophobic residues including
V520*, A518*, F261, F346, F405, F494* and F495*, formed
a hydrophobic pocket around R3 position which fitted well
with 3,4,5-trimethyl benzene substitution. With 3-pyridyl- (3n)
and cyclohexyl- (3o) substitutions at R3 position, the inhibitory
activities of TH1027 totally disappeared, indicating the π-π
interaction is vital for the activities. Furthermore, our experi-
mental results were in good agreement with Lu lab.29 Interest-
ingly, superimposition of the structure of TLR8 derived from
the TLR8/TH1027 complex onto the unliganded TLR8 pro-
duced a root mean square deviation (RMSD) value of 2.2 Å.
By comparison, its superimposition with the agonist-bound,
active form of TLR8 produced a RMSD value of 7.5 Å, indi-
cating that the conformation of TLR8/TH1027 complex was
closer to the resting state. Also, its superimposition with the
structure of TLR8/CU-CPT8m complex, produced a RMSD
value of 0.39 Å, indicating that the conformation of
TLR8/TH1027 complex was very similar to TLR8/CU-
CPT8m. So we developed a series of TLR8 inhibitor with a
triazole scaffold showing almost the same binding mode, but
the interactions with surrounding amino acids were slightly
different. These results further confirmed the previous strategy
we had proposed,27 locking the resting state of TLR8 by target-
ing a feasible pocket on the TLR8-TLR8* interface.
were further analyzed with image J software for quantification
(Figure S6).
Finally, we employed more physiologically- and pathologi-
cally-relevant assays to confirm the activities of TH1027.
Results showed that TH1027 dose-dependently inhibited the
TLR8-mediated production of pro-inflammatory cytokines
TNF-α (Figure 3B), IL-6 (Figure 3C) and IL-1β (Figure 3D)
in human monocyte cell lines. Besides cultured cell lines, we
also investigated whether TH1027 could inhibit TLR8 signal-
ing pathway in primary cells. Human PBMCs (include lym-
phocytes, monocytes, and dendritic cells expressing various
TLRs) were isolated from the fresh blood of healthy donators
using established methods of density gradient centrifugation.30
The R848 treatment of PBMCs induced TNF-α secretion,
which could be inhibited by TH1027 in a dose-dependent
manner (Figure 3E), but not by the negative control, com-
pound 2a. It was worth noting that TNF-α secretion was not
inhibited to the baseline by TH1027 even at the concentration
up to 20 μM, most likely because both TLR7 and TLR8 were
activated by R848 but TH1027 only reversed the TLR8 acti-
vation. This result further proved that TH1027 was a TLR8-
specific inhibitor and did not affect TLR7-mediated signaling.
Previous studies showed that TLR8 deficiency can lead to
rheumatoid arthritis (RA).31 Murine TLR8 had mutants at the
ligand binding site and therefore could not be activated by
either ssRNA or R848, making it impossible to use wild type
mice to test the efficacy of these TLR8 inhibitors.32 As an al-
ternative to the mouse animal models, we tested TH1027 by
monitoring its ability to reduce TNF-α production in patients’
primary cells. Fresh blood from six RA patients were collected
and their PBMCs were isolated from these blood samples by
density gradient centrifugation30. Immediately after separation,
cells were co-cultured with various concentrations of TH1027
or inactive compound 2a for 24 hours. Then the culture media
were collected to measure the concentration of TNF-α. The
results indicated that TH1027 significantly inhibited sponta-
neous TNF-α production from these cultures compared to
untreated cells, pointing to remarkable therapeutic potential of
TH1027 and its derivatives (Figure 3F). Moreover, the cell
viability experiment showed that TH1027 had no toxicity in
PBMCs from RA patients (Figure S7).
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TH1027 Inhibited TLR8 downstream signaling in cultured
cells or patient specimens
Next, we carried out on-target validation for TH1027, in an
attempt to confirm that TH1027 indeed functioned by selec-
tively targeting TLR8 in the complex, cellular environment.
The phosphorylated IRAK4 (p-IRAK4), TRAF3 and p65
(component of NF-κB) are all signature downstream proteins
for the TLR8 signaling transduction (Figure 3A). In a good
agreement that TH1027 targeted TLR8, the expression of p-
IRAK4 and TRAF3 was elevated upon R848 treatment in hu-
man monocyte cell lines and this elevation can be suppressed
by TH1027 in a dose-dependent manner. Furthermore, the
translocation of p65 into the nucleus induced by the R848
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Figure 3. Inhibition of downstream signaling in cultured cells or human specimens. (A) The brief diagram of the TLR8 signal-
ing pathway. TH1027 downregulated the expression levels of proteins in the TLR8 signaling pathway. (B-D) ELISA assay results
showed that TH1027 dose-dependently inhibited the TNF-α, IL-6 and IL-1β production activated by 1 µg/ml R848 in human mon-
ocyte cell lines (20 µM 2a as a negative control). (E) Dose-dependent inhibitory effects of R848-induced TLR8 activation by
TH1027 (2a as a negative control) in human PBMCs. (F) TH1027 dose-dependently inhibited the production of TNF-α in PBMCs
harvested from six rheumatoid arthritis patients (2a as a negative control). Each data point represents an independent sample read.
Center lines indicate means, and whiskers indicate s.e.m. (P values were determined using one-way ANOVA; **P < 0.01; ****P <
0.0001).
CONCLUSIONS
in cultured cell lines, human PBMCs from healthy donators
and RA patients further demonstrated high efficacy and speci-
ficity of TH1027, suggesting strong therapeutic potential
against autoimmune diseases such as RA. Overall, the identifi-
cation of novel small-molecules as TLR8 inhibitors not only
further confirmed the feasibility of targeting interface of pro-
tein-protein with small molecules, but also provided more
In conclusion, our rational designs targeting an unconventional
pocket on the dimeric interface of TLR8 led to the discovery
of a next generation of highly potent and specific TLR8 inhibi-
tor, TH1027, with a novel scaffold. The X-ray crystallograph-
ic structure of TLR8/TH1027 complex validated the contacts
within the binding pocket. A variety of biological evaluations
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Page 8 of 20
candidate compounds for drug development in autoimmune
diseases.
added and the mixture was extracted with ethyl acetate (3 x 5
1
2
3
4
5
6
7
8
mL). The combined organic layers were washed with saturated
aqueous sodium chloride (10 mL), dried over anhydrous sodi-
um sulfate, filtered and concentrated to dryness. The resulting
residue was purified via flash SiO2 chromatography (eluted
with dichloromethane/methanol 20:1) to give 3-(4-(2,4-
EXPERIMENTAL SECTION
dichlorophenyl)-5-(methylthio)-4H-1,2,4-
Triazol-3-
Chemistry Method. All solvents and chemicals were used as
purchased without further purification. NMR spectra were
recorded on a Bruker AVANCE III HD 400 (1H at 400 MHz,
1
yl)propan-1-ol (79 mg, 75%) as a white solid. H NMR (400
MHz, DMSO-d6) δ 7.98 (d, J = 2.1 Hz, 1H), 7.72 – 7.63 (m,
2H), 4.42 (t, J = 5.2 Hz, 1H), 3.36 – 3.32 (m, 2H), 2.50 (s, 3H),
2.45 – 2.38 (m, 2H), 1.65 (dq, J = 8.2, 6.4 Hz, 2H).13C NMR
(101 MHz, DMSO-d6) δ 155.9, 151.0, 136.6, 133.3, 132.1,
130.8, 130.1, 129.7, 60.2, 29.8, 21.8, 15.3. HPLC purity anal-
ysis: 95.1% (229 nm). HRMS (ESI) calcd. for
C12H13Cl2N3OS[M+H]+ : 318.0235; found: 318.0230.
1
13C at 101 MHz) nuclear magnetic resonance spectrometer. H
9
NMR spectra were recorded at 400 MHz in CDCl3 or
(CD3)2SO using residual CHCl3 (7.26 ppm) and (CH3)2SO
(2.50 ppm) as the internal standard. 13C NMR spectra were
recorded at 101 MHz in CDCl3 or (CD3)2SO using residual
CHCl3 (77.16 ppm) and (CH3)2SO (39.6 ppm) as internal ref-
erence. High-resolution mass spectra were acquired on a
Thermo Scientific QExactive mass spectrometer (ESI). The
purity was determined by high performance liquid chromatog-
raphy (HPLC) on SHIMAZU (LC-20AP) except for 3o. Purity
of all final compounds was more than 95%. The column was a
Inertsil C8 (Lot: SB5-1449), 5 µm particle size (150 mm * 4.6
mm). The binary solvent system (A/B) was as follows: water
(A) and acetonitrile (B), A/B = 5/95 or 100% (B). The flow
rate was 0.15 - 0.5 mL/min depending on the pressure of
pumps. Of note, compounds 2a, 2d and 2e required MeOH to
ensure proper dissolution. All compounds were named using
ChemDraw 15.0.
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4-(2,4-dichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-ol
(1a) A solution of 2,4-dichloro-1-isothiocyanobenzene (400
mg, 1.95 mmol) in anhydrous ethanol (8 mL) was added me-
thyl carbazate (223 mg, 2.48 mmol) and then the mixture was
reflux overnight. After the reaction was completely converted,
the ethanol was evaporated, then 2 M NaOH (aq) (8 mL) was
added and refluxed overnight. Then 2 M HCl (aq) (9 mL) was
slowly added until a large amount of white solid was precipi-
tated. After filtration, the precipitation was washed with water
and then dried to obtain 4-(2,4-dichlorophenyl)-5-mercapto-
4H-1,2,4-triazole-3-ol (200 mg, 39%) as white solid without
further purification. Next,
a
solution of 4-(2,4-
dichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-ol (200 mg,
0.76 mmol) in acetonitrile (8 mL) was added potassium car-
bonate (276 mg , 2.00 mmol) and then methyl iodide (57 μL,
0.92 mmol) was added dropwise. The mixture was reacted at
room temperature overnight. The resulting residue was puri-
fied via flash SiO2 chromatography (eluted with dichloro-
methane/methanol 30:1 ) to give 4-(2,4-dichlorophenyl)-5-
(methylthio)-4H-1,2,4-triazole- 3-alcohol (79 mg, 85%) as a
white solid powder. 1H NMR (400 MHz, DMSO-d6) δ 12.09 (s,
1H), 8.03 – 7.86 (m, 1H), 7.64 (d, J = 1.7 Hz, 2H), 2.43 (s,
3H).13C NMR (101 MHz, DMSO-d6) δ 154.2, 144.2, 136.1,
134.2, 133.0, 130.4, 129.5, 129.2, 13.8. HPLC purity analysis:
3-(4-(2,4-dichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-
3-yl)propan-1-ol(56-G5). A solution of 2,4-dichloroaniline
(1.52 g, 9.39 mmol) in anhydrous chloroform (10 mL) was
added sodium carbonate (1.27 g, 11.98 mmol) and pre-cooled
in an ice bath. Then thiophosgene (660 μL, 8.66 mmol) was
slowly added and the solution was allowed to stir in an ice
bath for 2 hours. After this time, 2M sodium hydroxide solu-
tion (10 mL) was added to quench the reaction and the mixture
was extracted with dichloromethane (3 x 10 mL). The com-
bined organic layers were washed with saturated aqueous so-
dium chloride (10 mL), dried over anhydrous sodium sulfate,
filtered and concentrated to dryness. The residue obtained was
purified via flash SiO2 chromatography (eluted with petroleum
95.6%
(229
nm).
HRMS
(ESI)
calcd.
for
C9H7Cl2N3OS[M+H]+ : 275.9765; found: 275.9751.
ether/ethyl
acetate
20:1)
to
give
2,4-dichloro-1-
isothiocyanatobenzene (17.61 g, 99%) as pale yellow liquid.1H
NMR (400 MHz, Chloroform-d) δ 7.39 (d, J = 2.2 Hz, 1H),
7.20 (dd, J = 8.6, 2.2 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H). A
solution of 2,4-dichloro-1-isothiocyanobenzene (251 mg, 1.23
mmol) in anhydrous ethanol (8 mL) was added 4-
hydroxybutanehydrazide (160 mg, 1.35 mmol) and reflux
overnight. After this time, the ethanol was evaporated, then 2
M NaOH (aq) (8 mL) was added and refluxed overnight. Then
2 M HCl solution (9 mL)was slowly added until a large
amount of white solid was precipitated. After filtration, the
precipitation was washed with water and then dried to obtain
Synthesis of (4-(2,4-dichlorophenyl)-5-(methylthio)-4H-
1,2,4-triazol-3-yl)methanol (1b). A solution of methyl 2-
hydroxyacetate (200 mg, 2.22 mmol) in anhydrous ethanol (10
mL) was added hydrazine hydrate (166.7 mg, 2.66 mmol, 80%
in purity) and reflux for 3h. A large amount of white solid was
precipitated when the mixture cooled in room temperature.
After filtration, the precipitation was washed with water and
then dried to obtain 2-hydroxyacetohydrazide (160 mg, 80%)
as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.83 (s, 1H),
4.55 (s, 2H), 4.34 – 4.00 (m, 1H), 3.90 – 3.78 (m, 2H). A solu-
tion of 2,4-dichloro-1-isothiocyanobenzene (350 mg, 1.71
mmol) in anhydrous ethanol (8 mL) was added 2-
hydroxyacetohydrazide (200 mg, 2.22 mmol) and reflux over-
night. Then ethanol was evaporated, 2 M NaOH (aq) (8 mL)
was added and refluxed overnight. Then 2 M HCl (aq) (9 mL)
was slowly added until a large amount of white solid was pre-
cipitated. After filtration, the precipitation was washed with
water and then dried to obtain (4-(2,4-dichlorophenyl)-5-
mercapto-4H-1,2,4-triazol-3-yl)methanol (235 mg, 50%) as a
white solid. A solution of (4-(2,4-dichlorophenyl)-5-mercapto-
3-(4-(2,4-
dichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-
yl)propan-1-ol (110 mg, 76%) as a white solid.1H NMR (400
MHz, DMSO-d6) δ 13.80 (s, 1H), 7.98 (d, J = 2.2 Hz, 1H),
7.72 – 7.63 (m, 2H), 3.36 (s, 2H), 2.36 (dd, J = 8.6, 6.8 Hz,
2H), 1.66 (p, J = 6.7 Hz, 2H). A solution of 3-(4-(2,4-
dichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)propan-1-ol
(100 mg, 0.33 mmol) in acetonitrile (8 mL) was added potas-
sium carbonate (138 mg, 1.00 mmol) and methyl iodide (31
μL, 0.5 mmol) and the mixture was allowed to reacted at room
temperature overnight. After this time, water (10 mL) was
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Journal of Medicinal Chemistry
isothiocyanobenzene (200 mg, 0.977 mmol) in anhydrous
4H-1,2,4-triazol-3-yl)methanol (235 mg, 0.86 mmol) in ace-
1
2
3
4
5
6
7
8
tonitrile (8 mL) was added potassium carbonate (276 mg, 1.00
mmol) and methyl iodide (80 μL, 1.28 mmol) and the mixture
was allowed to reacted at room temperature overnight. After
this time, water (10 mL) was added and the mixture was ex-
tracted with ethyl acetate (3x10 mL). The combined organic
layers were washed with saturated aqueous sodium chloride
(10 mL), dried over anhydrous sodium sulfate, filtered and
concentrated to dryness. The resulting residue was purified via
flash SiO2 chromatography (eluted with dichloro-
methane/methanol 20:1) to give (4-(2,4-dichlorophenyl)-5-
(methylthio)-4H-1,2,4-triazol-3-yl)methanol (198mg, 81%) as
ethanol (8 mL) and 5-hydroxypentanehydrazide (181 mg, 1.37
mmol) was added. The mixture was refluxed overnight. Then
mixture was concentrated to dryness and 2 M NaOH (aq) (8
mL) was added and reflux overnight. After the mixture was
cooled, 2 M HCl solution was slowly added until a large
amount of white was appeared. The solid is filtered, washed
with water, and then dried to give 4-(4-(2,4-dichlorophenyl)-5-
mercapto-4H-1,2,4-triazol-3-yl)butan-1-ol (108 mg, 33%) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 13.81 (s, 1H),
7.98 (d, J = 2.2 Hz, 1H), 7.73 – 7.66 (m, 2H), 3.31 (s, 2H),
2.35 (td, J = 7.4, 3.2 Hz, 2H), 1.61 – 1.48 (m, 2H), 1.40 (dt, J
= 8.6, 6.3 Hz, 2H). A solution of 4-(4-(2,4-dichlorophenyl)-5-
mercapto-4H-1,2,4-triazol-3-yl)butan-1-ol (100 mg, 0.30
mmol) in acetonitrile (8 mL) was added potassium carbonate
(138 mg, 1.00 mmol) and methyl iodide (28 μL, 0.45 mmol)
and the mixture was allowed to reacted at room temperature
overnight. Then water (10 mL) was added and the mixture
was extracted with ethyl acetate (3x10 mL). The combined
organic layers were washed with saturated aqueous sodium
chloride (10 mL), dried over anhydrous sodium sulfate, fil-
tered and concentrated to dryness. The resulting residue was
purified via flash SiO2 chromatography (eluted with dichloro-
methane/methanol 20:1) to give 4-(4-(2,4-dichlorophenyl)-5-
(methylthio)-4H-1,2,4-triazol-3-yl)butan-1-ol (85mg, 85%) as
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a white solid. H NMR (400 MHz, DMSO-d6) δ 7.97 (t, J =
1.3 Hz, 1H), 7.66 (d, J = 1.3 Hz, 2H), 5.36 (t, J = 5.7 Hz, 1H),
4.38 (ddd, J = 47.9, 13.5, 5.8 Hz, 2H), 2.57 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 155.7, 152.4, 136.4, 133.2, 132.1,
130.5, 130.3, 129.3, 54.1, 15.2. HPLC purity analysis: 98.2%
(229 nm). HRMS (ESI) calcd. for C10H9Cl2N3OS [M+H]+:
289.9922; found: 289.9909.
2-(4-(2,4-dichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-
3-yl)ethan-1-ol (1c). A solution of oxetan-2-one (300 mg,
4.16 mmol) in diethyl ether (10 mL) was added sodium meth-
oxide (337.1 mg, 6.24 mmol) and reflux for 2 h, followed by
addition of hydrazine monohydrate (395 mg, 6.24 mmol, 80%
in purity), anhydrous ethanol (10mL) and the mixture was
refluxed overnight. Then 2,4-dichloro-1-isothiocyanobenzene
(204 mg, 1.00 mmol) was added and refluxed overnight. After
the reaction was completely converted, the ethanol solution
was spun dry, and then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. After the reaction was completely convert-
ed, 2 M HCl solution was slowly added until a large amount of
white solid was appeared. After suction filtration, it was
washed with water, and then dried to obtain 2-(4-(2,4-
dichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)ethan-1-ol
(147 mg, 48%) as a white solid.1H NMR (400 MHz, DMSO-
d6) δ 13.84 (s, 1H), 7.98 (d, J = 2.3 Hz, 1H), 7.69 (dd, J = 8.5,
2.3 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 4.78 (t, J = 5.6 Hz, 1H),
3.53 (dq, J = 8.5, 2.9 Hz, 2H), 2.58 (dd, J = 15.3, 6.6 Hz, 1H),
2.45 (dt, J = 15.3, 6.9 Hz, 1H). 13C NMR (101 MHz, DMSO-d6)
δ 168.2, 150.5, 136.1, 133.7, 133.3, 130.8, 130.5, 129.2, 58.2,
29.6. A solution of 2-(4-(2,4-dichlorophenyl)-5-mercapto-4H-
1,2,4-triazol-3-yl)ethan-1-ol (116mg, 0.38 mmol) in acetoni-
trile (8 mL) was added potassium carbonate (138 mg, 1.00
mmol) and methyl iodide (40 μL, 0.64 mmol) and the mixture
was allowed to reacted at room temperature overnight. Then
water (10 mL) was added and the mixture was extracted with
ethyl acetate (3x10 mL). The combined organic layers were
washed with saturated aqueous sodium chloride (10 mL),
dried over anhydrous sodium sulfate, filtered and concentrated
to dryness. The resulting residue was purified via flash SiO2
chromatography (eluted with dichloromethane/methanol 20:1)
to give 2-(4-(2,4-dichlorophenyl)-5-(methylthio)-4H-1,2,4-
triazol-3-yl)ethan-1-ol (198mg, 81%) as a white solid.1H NMR
(400 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.70 (s, 2H), 4.72 (t, J =
5.5 Hz, 1H), 3.64 – 3.50 (m, 2H), 2.66 (dt, J = 13.9, 6.8 Hz,
2H), 2.54 (s, 3H).13C NMR (101 MHz, DMSO-d6) δ 154.1,
151.2, 136.6, 133.2, 132.3, 130.7, 130.1, 129.6, 59.0, 28.9,
15.3. HPLC purity analysis: 98.3% (229 nm). HRMS (ESI)
calcd. for C11H11Cl2N3OS [M+H]+ : 304.0078; found: 304.0070.
1
a white solid. H NMR (400 MHz, DMSO-d6) δ 8.01 (d, J =
1.9 Hz, 1H), 7.75 – 7.67 (m, 2H), 4.32 (t, J = 5.1 Hz, 1H), 3.33
(m, 2H), 2.54 (s, 3H), 2.44 (t, J = 7.5 Hz, 2H), 1.61 – 1.50 (m,
2H), 1.44 – 1.33 (m, 2H).13C NMR (101 MHz, DMSO-d6) δ
155.4, 150.5, 136.0, 132.6, 131.5, 130.2, 129.6, 129.1, 60.1,
31.3, 24.0, 22.8, 14.8. HPLC purity analysis: 95.2% (229 nm).
HRMS (ESI) calcd. for C13H15Cl2N3OS [M+H]+ : 332.0391;
found: 332.0389.
5-(4-(2,4-dichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-
3-yl)pentan-1-ol (1e). A solution of tetrahydro-2H-pyran-2-
one (500 mg, 4.38 mmol) in ethanol(8 mL) was added hydra-
zine monohydrate (329 mg, 5.26 mmol, 80% in purity) and
reflux overnight, and spin dry to give crude product. Then
crude product (320 mg, 1.23 mmol) was added into a new 25
mL round bottom flask, 2,4-dichloro-1-isothiocyanobenzene
(324 mg, 1.58 mmol) and ethanol (8 mL) was added and re-
fluxed overnight. The ethanol was spun dry, then 2 M NaOH
(aq) (8 mL) was added and refluxed overnight. After the reac-
tion was completely converted, 2 M HCl (aq) was slowly add-
ed until the solution became weakly acidic, and a large amount
of white solid appeared. After suction filtration, it was washed
with water and then dried to give a crude 5-(4-(2,4-dichloro)
Phenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)pentan-1-ol (white
solid, 147 mg). Next, 5-(4-(2,4-dichlorophenyl)-5-mercapto-
4H-1,2,4-triazol-3-yl)pentan-1-ol (314 mg, 0.95 mmol) and
potassium carbonate solid (276 mg, 2 mmol) were added in 8
mL of acetone, and methyl iodide (89 μL, 1.42 mmol) was
added dropwise with stirring under a rotor, and allowed to
react at room temperature overnight. After rotary evaporation,
a
silica gel column was eluted using dichloro-
methane/methanol 20:1 to 10:1 to give 5-(4-(2,4-
dichlorophenyl)-5-(methylthio)-4H-1. 2,4-Triazol-3-yl)pentan-
1-ol as yellow oily liquid (280 mg, 85%). 1H NMR (400 MHz,
DMSO-d6) δ 8.01 (d, J = 2.1 Hz, 1H), 7.76 – 7.67 (m, 2H),
4.29 (t, J = 5.2 Hz, 1H), 3.33 (d, J = 5.5 Hz, 2H), 2.54 (s, 3H),
2.43 (t, J = 7.5 Hz, 2H), 1.50 (p, J = 7.2 Hz, 2H), 1.37 – 1.21
(m, 4H).13C NMR (101 MHz, DMSO-d6) δ 155.9, 151.1, 136.6,
4-(4-(2,4-dichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-
3-yl)butan-1-ol (1d).
A
solution of 2,4-dichloro-1-
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133.2, 132.1, 130.8, 130.2, 129.6, 60.9, 32.4, 26.5, 25.3, 24.9, dimethylaminopyridine (4.61 mg, 0.038 mmol) were added
1
2
3
4
15.3. HPLC purity analysis: 95.6% (229 nm). HRMS (ESI)
calcd. for C14H17Cl2N3OS [M+H]+ : 346.0548; found: 346.0532.
and allowed to react at room temperature overnight. Then add-
ed 10 mL of water and dichloromethane (10 mL×3), the or-
ganic phase was combined, washed with saturated brine, dried
over anhydrous sodium sulfate and then filtered over silica gel
4-(2,4-dichlorophenyl)-3-(methylthio)-5-propyl-4H-1,2,4-
triazole
(1f).
A
solution
of
2,4-dichloro-1-
to
abtain
(2,4-Dichlorophenyl)-5-(methylthio)-4H-1,2,4-
5
isothiocyanobenzene (200 mg, 0.98 mmol) in anhydrous etha-
nol (8 mL) and butyrohydrazide (102 mg, 1.00 mmol) was
added. The mixture was refluxed overnight. Then mixture was
concentrated to dryness and 2 M NaOH (aq) (8 mL) was add-
ed and reflux overnight. After the mixture was cooled, 2 M
HCl solution was slowly added until a large amount of white
was appeared. The solid is filtered, washed with water, and
then dried to give 4-(2,4-dichlorophenyl)-5-propyl-4H-1,2,4-
triazole-3-thiol (108 mg, 37%) as a white solid. Next 4-(2,4-
dichlorophenyl)-5-propyl-4H-1,2,4-triazole-3-thiol (100 mg,
0.30 mmol) in acetonitrile (8 mL) was added potassium car-
bonate (138 mg, 1.00 mmol) and methyl iodide (28 μL, 0.45
mmol) and the mixture was allowed to reacted at room tem-
perature overnight. After this time, water (10 mL) was added
and the mixture was extracted with ethyl acetate (3 x 10 mL).
The combined organic layers were washed with saturated
aqueous sodium chloride (10 mL), dried over anhydrous sodi-
um sulfate, filtered and concentrated to dryness. The resulting
residue was purified via flash SiO2 chromatography (eluted
with dichloromethane/methanol 20:1) to give 4-(2,4-
dichlorophenyl)-3-(methylthio)-5-propyl-4H-1,2,4-triazole as
triazol-3-yl)propyl acetate as a white solid (58mg, yield:
88%).1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 2.2 Hz, 1H),
7.48 (dd, J = 8.4, 2.2 Hz, 1H), 7.28 (d, J = 1.9 Hz, 1H), 4.10 (t,
J = 6.1 Hz, 2H), 2.68 (s, 3H), 2.59 (m, 2H), 2.10 – 2.02 (m,
2H), 2.00 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 170.9, 155.0,
152.3, 137.4, 133.7, 131.0, 130.5, 129.5, 128.8, 63.1, 25.8,
21.8, 20.9, 14.9. HPLC purity analysis: 99.7% (229 nm).
HRMS (ESI) calcd. for C14H15Cl2N3O2S [M+H]+ : 360.0340;
found: 360.0327.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3-(4-(2,4-dichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-
yl)propan-1-ol (2a). 2,4-dichloroaniline (1.52 g, 9.39 mmol)
was added into a 25 mL bottle, 10 mL anhydrous chloroform
and sodium carbonate (1.27 g, 11.98 mmol) were added and
stirred in an ice bath. Sulfur phosgene (660 μL, 8.66 mmol)
was slowly added and stirred for 2 h in an ice bath. When the
reactant was completely transformed, 2M sodium hydroxide
aqueous solution was slowly added to quench the reaction, and
then dichloromethane and saturated carbonic acid were added.
The organic phase was extracted with saturated aqueous sodi-
um chloride, dried over anhydrous sodium sulfate and evapo-
rated. The residue obtained was purified via flash SiO2 chro-
matography (10 g silica gel, gradient of hexanes to 5% ethyl
acetate/95% hexanes) to give isothiocyanobenzene was an oily
1
a white solid (50mg, 48%). H NMR (400 MHz, DMSO-d6) δ
8.03 (d, J=2.2 Hz, 1H), 7.75 (d, J=8.5 Hz, 1H), 7.71 (dd, J=8.5,
2.2 Hz, 1H), 2.55 (s, 3H), 2.42 (t, J=7.5 Hz, 2H), 1.54 (h,
J=7.2 Hz, 2H), 0.86 (t, J=7.4 Hz, 3H); 13C NMR(101MHz,
DMSO-d6) δ 155.2, 150.6, 136.1, 132.6, 131.6, 130.3, 129.6,
129.2, 26.3, 19.6, 14.8, 13.4. HPLC purity analysis: 99.9%
(229 nm). HRMS (ESI) calcd. for C12H14Cl2N3S [M+H]+ :
302.0286; found: 302.0271.
1
pale yellow liquid (1.91 g, 99%). H NMR (400 MHz, chloro-
form-d) δ 7.39 (d, J = 2.2 Hz, 1H), 7.20 (dd, J = 8.6, 2.2 Hz,
1H), 7.14 (d, J = 8.6 Hz, 1H). Next 2,4-dichloro-1-
isothiocyanobenzene (251 mg, 1.23 mmol) and 4-
hydroxybutanehydrazide (160 mg, 1.35 mmol) were added to
8 mL of anhydrous ethanol and refluxed overnight. After the
reaction was completely converted, the ethanol solution was
spun dry, and then 8 mL 2M aqueous sodium hydroxide solu-
tion was added and refluxed overnight. After the reaction was
completely converted, 2M aqueous HCl solution was slowly
added until the solution became acidic, and a large amount of
white solid was appeared. After suction filtration, washed with
water and then dried to give 3-(4-(2,4-dichlorophenyl) 5-5-
Mercapto-4H-1,2,4-triazol-3-yl)propan-1-ol as a white solid
4-(2,4-dichlorophenyl)-3-(3-methoxypropyl)-5(methylthio)-
4H-1,2,4-triazole
(1g).
3-(4-(2,4-Dichlorophenyl)-5-
(methylthio)-4H-1,2,4-triazol-3-yl)propan-1-ol (60 mg, 0.19
mmol) was added to a 25 mL round bottom flask, and 5 mL of
anhydrous tetrahydrofuran was added, followed by addition of
sodium hydride (60% dispersion in mineral oil, 15 mg, 0.38
mmol) at room temperature, then methyl iodide (19 μL, 0.30
mmol) was added, the mixture was stirred overnight. Then add
10 mL of water and dichloromethane (10 mL×3), the organic
phase is combined, washed with saturated brine, dried over
anhydrous sodium sulfate, and then filtered over silica gel 4-
Dichlorophenyl)-3-(3-methoxypropyl)-5-(methylthio)-4H-
1
(110 mg, 76%). H NMR (400 MHz, DMSO-d6) δ 13.80 (s,
1H), 7.98 (d, J = 2.2 Hz, 1H), 7.72 - 7.63 (m, 2H), 3.36 (s, 2H),
2.36 (dd, J = 8.6, 6.8 Hz, 2H), 1.66 (p, J = 6.7 Hz, 2H). HPLC
purity analysis: 99.7% (229 nm). 13C NMR (101 MHz,
DMSO-d6) δ 168.1, 152.5, 136.1, 133.7, 133.1, 130.8, 130.5,
129.3, 59.9, 40.2, 28.9, 22.4. HRMS (ESI) calculated for
C11H11CI2N3OS [M+H]+: 304.0078; found: 304.0068.
1
1,2,4-triazole as a white solid (58 mg, 88%). H NMR (400
MHz, CDCl3) δ 7.60 (d, J = 2.3 Hz, 1H), 7.42 (dd, J = 8.4, 2.3
Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 3.38 (t, J = 6.1 Hz, 2H),
3.22 (s, 3H), 2.63 (s, 3H), 2.52 (tt, J = 15.6, 7.6 Hz, 2H), 1.94
(p, J = 6.7 Hz, 2H).13C NMR (101 MHz, CDCl3) δ 155.8,
152.7, 137.4, 133.8, 131.0, 130.6, 129.8, 128.8, 71.3, 58.5,
26.7, 22.0, 15.0. HPLC purity analysis: 98.3% (229 nm).
HRMS (ESI) calcd. for C13H15Cl2N3OS[M+H]+ : 332.0391;
found: 322.0387.
3-(4-(2,4-dichlorophenyl)-5-(ethylthio)-4H-1,2,4-triazol-3-
yl)propan-1-ol (2b). 2a (50 mg, 0.16 mmol) was added in 25
mL flask, 5 mL of acetone, ethyl iodide (54 mg, 0.32 mmol)
and potassium carbonate (36 mg, 0.26 mmol) were added and
stirred at room temperature overnight. Then add 10 mL of
distilled water and dichloromethane (10 mL × 3), the organic
phase was combined, washed with a saturated sodium chloride
solution, dried over anhydrous sodium sulfate, and then passed
through a silica gel column, eluting with dichloromethane:
methanol 20:1. 3-(4-(2,4-dichlorophenyl)-5-(ethylthio)-4H-
1,2,4-triazol-3-yl)propan-1-ol was obtained as a white solid
3-(4-(2,4-dichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-
3-yl)propyl acetate (1h). 3-(4-(2,4-Dichlorophenyl)-5-
(methylthio)-4H-1,2,4-triazol-3-yl)propan-1-ol (60 mg, 0.19
mmol) into a 25 mL round bottom flask, 5 mL of anhydrous
dichloromethane, acetic anhydride (19 mg, 0.19 mmol) and 4-
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Journal of Medicinal Chemistry
1
(37 mg, 68%). H NMR (400 MHz, DMSO-d6) δ 8.03 (d, J =
2.1 Hz, 1H), 7.77 – 7.69 (m, 2H), 4.49 (t, J = 5.2 Hz, 1H), 3.38
(q, J = 6.1 Hz, 2H), 3.04 (qd, J = 7.3, 2.8 Hz, 2H), 2.49 – 2.44
(m, 2H), 1.69 (dt, J = 13.4, 6.5 Hz, 2H), 1.26 (t, J = 7.3 Hz,
3H). 13C NMR (101 MHz, DMSO-d6) δ 155.5, 149.6, 136.1,
132.7, 131.8, 130.3, 129.9, 129.2, 59.7, 29.5, 27.1, 21.5, 15.0.
HPLC purity analysis: 95.7% (229 nm). HRMS (ESI) HRMS
(ESI) calculated for C13H15CI2N3SO[M+H]+: 332.0391; found:
332.0389.
hydroxypropyl)-4H-1,2,4-triazol-3-yl)thio)acetamide (110mg,
yield: 90%) as white solid. H NMR (400 MHz, DMSO-d6) δ
1
1
2
3
4
5
6
7
8
8.02 (s, 1H), 7.71 (d, J = 1.8 Hz, 2H), 7.63 (s, 1H), 7.22 (s,
1H), 4.48 (td, J = 5.1, 1.7 Hz, 1H), 3.90 – 3.78 (m, 2H), 3.38
(s, 2H), 2.46 (d, J = 8.4 Hz, 2H), 1.75 – 1.62 (m, 2H).13C
NMR (101 MHz, DMSO-d6) δ 168.5, 155.6, 149.6, 136.2,
132.7, 131.7, 130.4, 129.5, 129.2, 59.7, 36.5, 29.5, 21.3.
HPLC purity analysis: 96.2% (229 nm). HRMS (ESI) calcd.
for C13H14Cl2N4O2S [M+H]+: 361.0293; found: 361.0275.
9
3-(4-(2,4-dichlorophenyl)-5-(isopropylthio)-4H-1,2,4-
3-(4-(4-chlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
triazol-3-yl)propan-1-ol (2c). 2a (80 mg, 0.26 mmol) was
added in 25 mL flask, 5 mL of acetone was added, then 2-
iodopropane (54 mg, 0.32 mmol) and potassium carbonate (72
mg, 0.52 mmol) were added and the mixture was stirred at
room temperature overnight. After this time, 10 mL water was
added and the mixture was extracted with dichloromethane (3
x 5 mL). The combined organic layers were washed with satu-
rated aqueous sodium chloride (10 mL), dried over magnesi-
um sulfate, filtered and concentrated to dryness. The residue
obtained was purified via flash SiO2 chromatography (10 g
silica gel, gradient of hexanes to 50% ethyl acetate/50% hex-
anes) to give 3-(4-(2,4-dichlorophenyl)-5-(isopropylthio)-4H-
1,2,4-triazol-3-yl)propan-1-ol as a pale yellow oily liquid (45
mg, 49%).1H NMR (400 MHz, DMSO-d6) δ 8.00 (dd, J = 2.1,
1.1 Hz, 1H), 7.72 – 7.67 (m, 2H), 4.46 (td, J = 5.2, 1.1 Hz, 1H),
3.38 (t, J = 5.7 Hz, 2H), 2.49 – 2.44 (m, 2H), 1.75 – 1.64 (m,
2H), 1.26 (d, J = 6.7 Hz, 6H), 1.14 (d, J = 1.1 Hz, 1H).13C
NMR (101 MHz, DMSO-d6) δ 155.4, 148.9, 135.9, 132.6,
131.6, 130.2, 130.1, 129.1, 59.7, 29.4, 23.1, 23.1, 21.4. HPLC
purity analysis: 99.2% (229 nm). HRMS (ESI) calcd. for
C14H17Cl2N3OS[M+H]+ : 346.0548; found: 346.0525.
yl)propan-1-ol
(3a).
A
solution
of
4-chloro-1-
isothiocyanobenzene (300 mg, 1.77 mmol) in anhydrous etha-
nol (6 mL) was added 4-hydroxybutanehydrazide (219 mg,
1.86 mmol) and reflux overnight. After this time, the ethanol
was evaporated, then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. Then 2 M HCl (aq) (9 mL)was slowly
added until a large amount of white solid was precipitated.
After filtration, the precipitation was washed with water and
then dried to obtain 3-(4-(2,4- dichlorophenyl)-5-mercapto-
4H-1,2,4-triazol-3-yl)propan-1-ol (350 mg, 73%) as a white
solid without further purification. Next a solution of 3-(4-(2,4-
dichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)propan-1-ol
(150 mg, 0.56 mmol) in acetonitrile (8 mL) was added potas-
sium carbonate (138 mg, 1.00 mmol) and methyl iodide (38
μL, 0.61 mmol) and the mixture was allowed to reacted at
room temperature overnight. After this time, water (10 mL)
was added and the mixture was extracted with ethyl acetate (3
x 10 mL). The combined organic layers were washed with
saturated aqueous sodium chloride (20 mL), dried over anhy-
drous sodium sulfate, filtered and concentrated to dryness. The
resulting residue was purified via flash SiO2 chromatography
(eluted with dichloromethane/methanol 20:1) to give 3-(4-(4-
chlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-yl)propan-1-
ol (87 mg, 55%) as a white solid. 1H NMR (400 MHz, DMSO-
d6) δ 7.68 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H), 4.47 (t,
J = 5.2 Hz, 1H), 3.37 (q, J = 6.1 Hz, 2H), 2.54 (m, 2H), 2.54 (s,
3H), 1.74 – 1.64 (m, 2H).13C NMR (101 MHz, DMSO-d6) δ
156.0, 151.0, 135.1, 132.5, 130.5, 129.8, 60.2, 29.9, 21. 9,
15.1. HPLC purity analysis: 95.4% (229 nm). HRMS (ESI)
calcd. for C12H14ClN3OS [M+H]+ : 284.0624 ; found: 284.0612.
S-(4-(2,4-dichlorophenyl)-5-(3-hydroxypropyl)-4H-1,2,4-
triazol-3-yl) carbamothioate (2d). 2a (100 mg , 0.33 mmol),
trichloroacetyl chloride (120 mg, 0.66mmol), 5mL acetonitrile
and 1mL DMF were added, then stirred for one hour. When
the reactants were completely converted, the solvent was re-
moved by rotary evaporation, then 7M ammonia methanol
solution(10 mL) was added and reflux for 3h. After the reac-
tion was completed, the reaction was concentrated to dryness.
After this time, the residue obtained was purified via flash
SiO2 chromatography (10 g silica gel, gradient of hexanes to
25% ethyl acetate/70% hexanes) to give S-(4-(2,4-
dichlorophenyl)-5-(3-hydroxypropyl)-4H-1,2,4-triazol-3-yl)
carbamothioate as white solid (77mg, 67%).1H NMR (400
MHz, DMSO-d6) δ 7.98 (d, J = 2.2 Hz, 1H), 7.71-7.62 (m, 2H),
4.51 (t, J = 5.1 Hz, 1H), 2.51 (m, 2H) , 2.36 (dd, J = 8.5, 6.8
Hz, 2H), 1.65 (dq, J = 8.1, 6.3 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 168.1, 152.6, 136.2, 133.7, 133.1, 132.5, 130.8,
130.5, 129.3, 59.9, 28.9, 22.4. HPLC purity analysis: 99.7%
(229 nm). HRMS (ESI) calcd. for C13H12Cl2N4O2S [M+H]+:
347.0134; found: 347.0541.
3-(4-(4-methoxyphenyl)-5-(methylthio)-4H-1,2,4-triazol-3-
yl)propan-1-ol (3b).
A solution of 1-isothiocyanato-4-
methoxybenzene (227 mg, 1.37 mmol) in anhydrous ethanol
(8 mL) was added 4-hydroxybutanehydrazide (186 mg, 1.00
mmol) and reflux overnight. After this time, the ethanol was
evaporated, then 2 M NaOH (aq) (8 mL) was added and re-
fluxed overnight. Then 2 M HCl (aq) was slowly added until
a large amount of white solid was precipitated. After filtration,
the precipitation was washed with water and then dried to ob-
tain 3-(5-mercapto-4-(4-methoxyphenyl)-4H-1,2,4-triazol-3-
yl)propan-1-ol as a white solid without further purification
(150 mg, 38%).1H NMR (400 MHz, DMSO-d6) δ 13.63 (s,
1H), 7.35 – 7.28 (m, 2H), 7.12 – 7.06 (m, 2H), 3.83 (s, 3H),
3.35 (t, J = 6.2 Hz, 2H), 2.42 (t, J = 7.6 Hz, 2H), 1.65 (dt, J =
8.3, 6.5 Hz, 2H). Next, a solution of 3-(5-mercapto-4-(4-
methoxyphenyl)-4H-1,2,4-triazol-3-yl)propan-1-ol (100 mg,
0.39 mmol) in acetone (8 mL) was added potassium carbonate
(138 mg, 1.00 mmol) and methyl iodide (64 μL, 1.02 mmol)
and the mixture was allowed to reacted at room temperature
overnight. After this time, water (10 mL) was added and the
mixture was extracted with ethyl acetate (3 x 10 mL). The
2-((4-(2,4-dichlorophenyl)-5-(3-hydroxypropyl)-4H-1,2,4-
triazol-3-yl)thio)acetamide (2e). 2a (80 mg , 0.26 mmol), 2-
iodoacetamide (59 mg, 0.66 mmol) and potassium carbonate
(138 mg, 1.00 mmol) were added in 10 mL acetonitrile, then
stirred overnight. After the reaction was completed, the reac-
tion was concentrated to dryness. After this time, the residue
obtained was purified via flash SiO2 chromatography (10 g
silica gel, gradient of hexanes to 10% methanol in dichloro-
methane)
to
give
2-((4-(2,4-dichlorophenyl)-5-(3-
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combined organic layers were washed with saturated aqueous triazol-3-yl)propan-1-ol (140 mg, 39%) as a pale yellow solid
1
2
3
4
5
6
7
8
sodium chloride (20 mL), dried over anhydrous sodium sulfate,
filtered and concentrated to dryness. The resulting residue was
purified via flash SiO2 chromatography (eluted with dichloro-
methane/methanol 20:1) to give 3-(4-(4-methoxyphenyl)-5-
(methylthio)-4H-1,2,4-triazol-3-yl)propan-1-ol (87 mg, yiled:
45%) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 7.36
(d, J = 8.8 Hz, 2H), 7.12 (d, J = 8.8 Hz, 2H), 4.49 (t, J = 5.1
Hz, 1H), 3.84 (s, 3H), 2.54 (m, 4H), 1.68 (d, J = 7.1 Hz, 2H).
13C NMR (101 MHz, DMSO-d6) δ 160.4, 156.3, 151.5, 129.1,
126.0, 115.5, 60.2, 56.0, 30.0, 21.9, 14.9. HPLC purity analy-
sis: 97.5% (229 nm). HRMS (ESI) calcd. for C13H17N3O2S
without further purification. 1H NMR (400 MHz, DMSO-d6) δ
13.79 (s, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.59 (dt, J = 15.2, 4.5
Hz, 3H), 3.35 (t, J = 6.2 Hz, 2H), 2.34 (t, J = 7.7 Hz, 2H), 1.65
(p, J = 6.7 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 156.2,
152. 5, 132.8, 132.0, 131.1, 131.0, 129.8, 128.5, 61.8, 29.3,
22.4. Next, A solution of 3-(4-(2-chlorophenyl)-5-mercapto-
4H-1,2,4-triazol-3-yl)propan-1-ol (80 mg, 0.30 mmol) in ace-
tonitrile (8 mL) was added potassium carbonate (138 mg, 1.00
mmol) and methyl iodide (28 μL, 0.45 mmol) and the mixture
was allowed to reacted at room temperature overnight. After
this time, water (10 mL) was added and the mixture was ex-
tracted with ethyl acetate (3x10 mL). The combined organic
layers were washed with saturated aqueous sodium chloride
(20 mL), dried over anhydrous sodium sulfate, filtered and
concentrated to dryness. The resulting residue was purified via
flash SiO2 chromatography (eluted with dichloro-
methane/methanol 20:1) to give 3-(4-(2-chlorophenyl)-5-
(methylthio)-4H-1,2,4-triazol-3-yl)propan-1-ol (52 mg, 61%)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[M+H]+:
280.1120,
[M+Na]+ :302.0930;
found:
[M+H]+:280.1107, [M+Na]+ : 302.0924.
3-(5-(methylthio)-4-(4-(trifluoromethyl)phenyl)-4H-1,2,4-
triazol-3-yl)propan-1-ol (3c). A solution of 1-isothiocyanato-
4-(trifluoromethyl)benzene (305 mg, 1.00 mmol) in anhydrous
ethanol (8 mL) was added 4-hydroxybutanehydrazide (186 mg,
1.00 mmol) and reflux overnight. After this time, the ethanol
was evaporated, then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. Then 2 M HCl (aq) was slowly added
until a large amount of white solid was precipitated. After
filtration, the precipitation was washed with water and then
dried to obtain 3-(5-mercapto-4-(4-(trifluoromethyl)phenyl)-
4H-1,2,4-triazol-3-yl)propan-1-ol as orange solid without fur-
ther purification (128 mg, 40%). 1H NMR (400 MHz, DMSO-
d6) δ 13.81 (s, 1H), 7.98 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 8.2
Hz, 2H), 3.35 (d, J = 6.1 Hz, 2H), 2.47 (t, J = 7.6 Hz, 2H),
1.66 (p, J = 6.7 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ
167.9, 152.5, 137.9, 130.0, 127.0, 125.6, 122.9, 59.9, 28.8,
1
as brown oil. H NMR (400 MHz, CDCl3) δ 7.61 (dd, J = 8.0,
1.5 Hz, 1H), 7.52 (td, J = 7.8, 1.7 Hz, 1H), 7.45 (td, J = 7.6,
1.5 Hz, 1H), 7.30 (dd, J = 7.7, 1.7 Hz, 1H), 3.69 (dq, J = 8.2,
5.1 Hz, 2H), 2.86 (s, 1H), 2.64 – 2.43 (m, 2H), 1.92 (qd , J =
6.9, 5.0 Hz, 2H).13C NMR (101 MHz, CDCl3) δ 156.2, 152. 5,
132.8, 132.0, 131.1, 131.0, 129.8, 128.5, 61.8, 29.3, 22.4, 15.0.
HPLC purity analysis: 98.9% (229 nm). HRMS (ESI) Calcu-
lated as C12H14ClN3OS [M+H]+: 284.0624; found: 284.0601.
3-(4-(3-chlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-
yl)propan-1-ol
(3e).
A
solution
of
3-chloro-1-
isothiocyanobenzene (339 mg, 2.00 mmol) in anhydrous etha-
nol (8 mL) was added 4-hydroxybutanehydrazide (248 mg,
2.10 mmol) and reflux overnight. After this time, the ethanol
was evaporated, then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. Then 2 M HCl (aq) was slowly added
until a large amount of white solid was precipitated. After
filtration, the precipitation was washed with water and then
dried to obtain 3-(4-(3-chlorophenyl)-5-mercapto-4H-1,2,4-
triazol-3-yl)propan-1-ol (166mg, 31%) as a white solid with-
22.6.
Finally,
a
solution
of
3-(5-mercapto-4-(4-
(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)propan-1-ol
(120 mg, 0.38 mmol) in acetone (8 mL) was added potassium
carbonate (138 mg, 1.00 mmol) and methyl iodide (35 μL,
0.56 mmol) and the mixture was allowed to reacted at room
temperature overnight. After this time, water (10 mL) was
added and the mixture was extracted with ethyl acetate (3 x 10
mL). The combined organic layers were washed with saturated
aqueous sodium chloride (20 mL), dried over anhydrous sodi-
um sulfate, filtered and concentrated to dryness. The resulting
residue was purified via flash SiO2 chromatography (eluted
with dichloromethane/methanol 20:1) to give 3-(5-
(methylthio)-4-(4-(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-
out further purification. Next,
a
solution of 3-(4-(2-
chlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)propan-1-ol
(100 mg, 0.35 mmol) in acetonitrile (8 mL) was added potas-
sium carbonate (138 mg, 1.00 mmol) and methyl iodide (33
μL, 0.53 mmol) and the mixture was allowed to reacted at
room temperature overnight. After this time, water (10 mL)
was added and the mixture was extracted with ethyl acetate
(3x10 mL). The combined organic layers were washed with
saturated aqueous sodium chloride (20 mL), dried over anhy-
drous sodium sulfate, filtered and concentrated to dryness. The
resulting residue was purified via flash SiO2 chromatography
(eluted with dichloromethane/methanol 20:1) to give 3-(4-(3-
chlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-yl)propan-1-
ol (56 mg, 56%) as a white solid.1H NMR (400 MHz, DMSO-
d6) δ 7.70 (d, J = 2.0 Hz, 1H), 7.69 – 7.66 (m, 1H), 7.63 (t, J =
7.8 Hz, 1H), 7.47 (dt, J = 7.6, 1.6 Hz, 1H), 4.50 (t, J = 5.2 Hz,
1H), 3.39 (d, J = 6.1 Hz, 2H), 2.57 (m, J = 4.8 Hz, 2H), 2.55 (s,
3H), 1.70 (m, J = 8.1, 6.4 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 155.9, 151.0, 134.9, 134.4, 132.0, 130.6, 127.9,
126.8, 60.2, 29.9, 21.9, 15.1. HPLC purity analysis: 97.9%
(229 nm). HRMS (ESI) calcd. for C12H14ClN3OS [M+H]+ :
284.0624; found: 284.0614.
1
yl)propan-1-ol as white solid (90 mg, 75%). H NMR (400
MHz, DMSO-d6) δ 7.95 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.3
Hz, 2H), 4.42 (t, J = 5.2 Hz, 1H), 3.32 (m, 2H), 2.48 (m, 2H),
2.46 (s, 3H), 1.66 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ
155.9, 150.8, 137.2, 130.6 (J = 32.32), 128.9, 127.6, 124.2 (J
= 273.7 Hz), 60.1, 29.9, 21.9, 15.2. HPLC purity analysis:
97.4% (229 nm). HRMS (ESI) calcd. for C13H14F3N3OS
[M+H]+ :318.0888; found: 318.0866.
3-(4-(2-chlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-
yl)propan-1-ol
(3d).
A
solution
of
2-chloro-1-
isothiocyanobenzene (200 mg, 1.18 mmol) in anhydrous etha-
nol (6 mL) was added 4-hydroxybutanehydrazide (139 mg,
1.18 mmol) and reflux overnight. After this time, the ethanol
was evaporated, then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. Then 2 M HCl (aq) was slowly added
until a large amount of white solid was precipitated. After
filtration, the precipitation was washed with water and then
dried to obtain 3-(4-(2-chlorophenyl)-5-mercapto-4H-1,2,4-
12
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Journal of Medicinal Chemistry
um sulfate, filtered and concentrated to dryness. The resulting
3-(5-(methylthio)-4-(3-(trifluoromethyl)phenyl)-4H-1,2,4-
1
2
3
4
5
6
7
8
triazol-3-yl)propan-1-ol (3f). A solution of 1-isothiocyanato-
3-(trifluoromethyl)benzene (305 mg, 1.00 mmol) in anhydrous
ethanol (8 mL) was added 4-hydroxybutanehydrazide (186 mg,
1.00 mmol) and reflux overnight. After this time, the ethanol
was evaporated, then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. Then 2 M HCl (aq) was slowly added
until a large amount of white solid was precipitated. After
filtration, the precipitation was washed with water and then
dried to obtain 3-(5-mercapto-4-(3-(trifluoromethyl)phenyl)-
4H-1,2,4-triazol-3-yl)propan-1-ol as white solid without fur-
ther purification (260 mg, 82%). 1H NMR (400 MHz, DMSO-
d6) δ 13.78 (s, 1H), 7.94 (d, J = 11.8 Hz, 2H), 7.87 – 7.76 (m,
2H), 2.46 (t, J = 7.6 Hz, 2H), 1.66 (p, J = 6.7 Hz, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 168.0, 152.6, 135.1, 133.3,
131.2, 130.7, 130.4, 126.8, 126.1, 59.9, 28.7, 22.6. Then a
solution of 3-(5-mercapto-4-(3-(trifluoromethyl)phenyl)-4H-
1,2,4-triazol-3-yl)propan-1-ol (120 mg, 0.38 mmol) in ace-
tone(8 mL) was added potassium carbonate (138 mg, 1.00
mmol) and methyl iodide (35 μL, 0.56 mmol) and the mixture
was allowed to reacted at room temperature overnight. After
this time, water (10 mL) was added and the mixture was ex-
tracted with ethyl acetate (3 x 10 mL). The combined organic
layers were washed with saturated aqueous sodium chloride
(20 mL), dried over anhydrous sodium sulfate, filtered and
concentrated to dryness. The resulting residue was purified via
flash SiO2 chromatography (eluted with dichloro-
methane/methanol 15:1) to give 3-(5-(methylthio)-4-(3-
(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)propan-1-ol as
residue was purified via flash SiO2 chromatography (eluted
with dichloromethane/methanol 15:1) to give 3-(5-
(methylthio)-4-(m-tolyl)-4H-1,2,4-triazol-3-yl)propan-1-ol as
1
white solid (231 mg, 68%). H NMR (400 MHz, CDCl3) δ
7.41 (t, J = 8.1 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.11 – 6.97
(m, 2H), 3.70 (q, J = 5.4 Hz, 2H), 3.27 (t, J = 5.4 Hz, 1H),
2.69 (t, J = 7.0 Hz, 2H), 2.64 (s, 3H), 2.43 (s, 3H), 1.98 – 1.87
(m, 2H). 13C NMR (101 MHz, CDCl3) δ 156.1, 152.4, 140.4,
133.1, 130.9, 129.8, 127.5, 124.0, 61.8, 29.4, 22.6, 21.3, 14.6.
HPLC purity analysis: 97.8% (229 nm). HRMS (ESI) calcd.
for C13H17N3OS [M+H]+ : 264.1171; found: 264.1173.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3-(4-(3,4-dichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-
3-yl)propan-1-ol (3h).
A
solution of 3,4-dichloro-1-
isothiocyanobenzene (306 mg, 1.50 mmol) in anhydrous etha-
nol (6 mL) was added 4-hydroxybutanehydrazide (186 mg,
1.57 mmol) and reflux overnight. After this time, the ethanol
was evaporated, then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. Then 2 M HCl (aq) was slowly added
until a large amount of white solid was precipitated. After
filtration, the precipitation was washed with water and then
dried to obtain 3-(4-(3,4-dichlorophenyl)-5-mercapto-4H-
1,2,4-triazol-3-yl)propan-1-ol (166mg, 31%) as a white solid
without further purification. Next, a solution of 3-(4-(3,4-
dichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)propan-1-ol
(100 mg, 0.33 mmol) in acetonitrile (8 mL) was added potas-
sium carbonate (138 mg, 1.00 mmol) and methyl iodide (33
μL, 0.53 mmol) and the mixture was allowed to reacted at
room temperature overnight. After this time, water (10 mL)
was added and the mixture was extracted with ethyl acetate
(3x10 mL). The combined organic layers were washed with
saturated aqueous sodium chloride (20 mL), dried over anhy-
drous sodium sulfate, filtered and concentrated to dryness. The
resulting residue was purified via flash SiO2 chromatography
(eluted with dichloromethane/methanol 20:1) to give 3-(4-
(3,4-dichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-
1
a colorless liquid (92 mg, 77%). H NMR (400 MHz, DMSO-
d6) δ 8.02 – 7.96 (m, 2H), 7.89 – 7.80 (m, 2H), 4.49 (t, J = 5.2
Hz, 1H), 2.55 (m, 4H), 1.75 – 1.66 (m, 2H).13C NMR (101
MHz, DMSO-d6) δ 156.0, 151.0, 134.5, 132.3, 131.8, 131.0 (J
= 32.3 Hz), 127.3, 123.9 (J = 273.7 Hz), 125.1, 60.1, 29.8,
21.9, 15.2. HPLC purity analysis: 99.9% (229 nm). HRMS
(ESI) calcd. for C13H14F3N3OS [M+H]+ : 318.0888, [M+Na]+:
340.0707; found: [M+H]+:318.0869, [M+Na]+: 340.0687.
1
yl)propan-1-ol (55 mg, 52%) as a white solid. H NMR (400
MHz, CDCl3) δ 7.63 (d, J = 8.5 Hz, 1H), 7.41 (d, J = 2.4 Hz,
1H), 7.14 (dd, J = 8.5, 2.4 Hz, 1H), 3.72 (t, J = 5.7 Hz, 2H),
2.92 – 2.74 (m, 1H), 2.70 (d, J = 7.0 Hz, 1H), 2.67 (s, 3H),
2.00 – 1.92 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 155.7,
152.2, 134.9, 134.3, 132.4, 131.8, 129.1, 126.5, 61.6, 29.2,
22.4, 14.7. HPLC purity analysis: 97.6% (229 nm). HRMS
3-(5-(methylthio)-4-(m-tolyl)-4H-1,2,4-triazol-3-yl)propan-
1-ol (3g). A solution of 1-isothiocyanato-3-methylbenzene
(298 mg, 2.00 mmol) in anhydrous ethanol (8 mL) was added
4-hydroxybutanehydrazide (284 mg, 2.10 mmol) and reflux
overnight. After this time, the ethanol was evaporated, then 2
M NaOH (aq) (8 mL) was added and refluxed overnight. Then
2 M HCl (aq) was slowly added until a large amount of white
solid was precipitated. After filtration, the precipitation was
washed with water and then dried to obtain 3-(5-mercapto-4-
(m-tolyl)-4H-1,2,4-triazol-3-yl)propan-1-ol as white solid
(ESI) calcd. for C12H13Cl2N3OS [M+H]+
found:318.0222.
:
318.0235;
3-(4-(2,3-dichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-
3-yl)propan-1-ol (3i). solution of 2,3-dichloro-1-
1
A
without further purification (260 mg, 32%). H NMR (400
isothiocyanobenzene (314 mg, 1.54 mmol) in anhydrous etha-
nol (6 mL) was added 4-hydroxybutanehydrazide (190 mg,
1.61 mmol) and reflux overnight. After this time, the ethanol
was evaporated, then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. Then 2 M HCl (aq) was slowly added
until a large amount of white solid was precipitated. After
filtration, the precipitation was washed with water and then
dried to obtain 3-(4-(2,3-dichlorophenyl)-5-mercapto-4H-
1,2,4-triazol-3-yl)propan-1-ol (400mg, 80%) as a white solid
without further purification. Next, a solution of 3-(4-(2,3-
dichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)propan-1-ol
(100 mg, 0.33 mmol) in acetonitrile (8 mL) was added potas-
sium carbonate (138 mg, 1.00 mmol) and methyl iodide (31
μL, 0.49 mmol) and the mixture was allowed to reacted at
MHz, DMSO-d6) δ 13.65 (s, 1H), 7.46 (t, J = 7.7 Hz, 1H),
7.35 (d, J = 7.7 Hz, 1H), 7.25 – 7.12 (m, 2H), 4.47 (t, J = 5.2
Hz, 1H), 3.36 (t, J = 5.8 Hz, 2H), 2.43 (t, J = 7.6 Hz, 2H), 2.38
(s, 3H), 1.65 (p, J = 6.7 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 168.1, 152.7, 139.5, 134.2, 130.6, 129.7, 129.1,
125.8, 60.0, 28.9, 22.6, 21.2. Next, a solution of 3-(5-
mercapto-4-(m-tolyl)-4H-1,2,4-triazol-3-yl)propan-1-ol (150
mg, 0.60 mmol) in acetone (8 mL) was added potassium car-
bonate (138 mg, 1.00 mmol) and methyl iodide (56 µL, 0.90
mmol) and the mixture was allowed to reacted at room tem-
perature overnight. After this time, water (10 mL) was added
and the mixture was extracted with ethyl acetate (3x10 mL).
The combined organic layers were washed with saturated
aqueous sodium chloride (20 mL), dried over anhydrous sodi-
13
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Journal of Medicinal Chemistry
Page 14 of 20
room temperature overnight. After this time, water (10 mL) carbonate (276 mg, 2.00 mmol) and methyl iodide (30 μL,
1
2
3
4
5
6
7
8
was added and the mixture was extracted with ethyl acetate
(3x10 mL). The combined organic layers were washed with
saturated aqueous sodium chloride (20 mL), dried over anhy-
drous sodium sulfate, filtered and concentrated to dryness. The
resulting residue was purified via flash SiO2 chromatography
(eluted with dichloromethane/methanol 20:1) to give 3-(4-
(3,4-dichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-
0.48 mmol) and the mixture was allowed to reacted at room
temperature overnight. After this time, water (10 mL) was
added and the mixture was extracted with ethyl acetate (3 x 10
mL). The combined organic layers were washed with saturated
aqueous sodium chloride (20 mL), dried over anhydrous sodi-
um sulfate, filtered and concentrated to dryness. The resulting
residue was purified via flash SiO2 chromatography (eluted
with dichloromethane/methanol 20:1) to give 3-(4-(2,3,4-
trichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-yl)propan-
1
yl)propan-1-ol (64 mg, 61%) as a white solid. H NMR (400
MHz, CDCl3) δ 7.68 (dd, J = 8.0, 1.5 Hz, 1H), 7.41 (t, J = 8.0
Hz, 1H), 7.24 (d, J = 1.5 Hz, 1H), 3.71 (dq, J = 10.1, 5.3 Hz,
2H), 2.94 (s, 1H), 2.66 (s, 3H), 2.64 – 2.52 (m, 2H), 1.95 (m,
2H). 13C NMR (101 MHz, CDCl3) δ 155.8, 152.1, 147.2, 135.1,
132.6, 131.9, 128.2, 127.9, 61.7, 29.1, 22.3, 14.9. HPLC purity
analysis: 99.6% (229 nm). HRMS (ESI) calcd. for
C12H13Cl2N3OS [M+H]+ : 318.0235; found: 318.0222.
1
9
1-ol as a white solid (35 mg, 32%). H NMR (400 MHz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
DMSO-d6) δ 7.96 (d, J = 8.6 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H),
4.48 (t, J = 5.2 Hz, 1H), 3.41 – 3.37 (m, 2H), 2.55 (s, 3H),
2.47 (t, J = 7.8 Hz, 2H), 1.77 – 1.65 (m, 2H).13C NMR (101
MHz, DMSO-d6) δ 155.9, 151.0, 135.8, 132.8, 132.4, 131.5,
130.6, 130.1, 60.1, 30.0, 21.7, 15.4. HPLC purity analysis:
97.8% (229 nm). HRMS (ESI) calcd. for C12H12Cl3N3OS
[M+H]+ : 351.9845; found: 351.9828.
3-(5-(methylthio)-4-(2,4,6-trichlorophenyl)-4H-1,2,4-
triazol-3-yl)propan-1-ol (3j). A solution of 2,4,6-trichloro-1-
isothiocyanobenzene (314 mg, 1.32 mmol) in anhydrous etha-
nol (6 mL) was added 4-hydroxybutanehydrazide (248 mg,
2.10 mmol) and reflux overnight. After this time, the ethanol
was evaporated, then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. Then 2 M HCl (aq) was slowly added
until a large amount of white solid was precipitated. After
filtration, the precipitation was washed with water and then
dried to obtain 3-(4-(2,4,6-trichlorophenyl)-5-mercapto-4H-
1,2,4-triazol-3-yl)propan-1-ol (330mg, 49%) as a white solid
without further purification. Next, a solution of 3-(4-(2,4,6-
trichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)propan-1-ol
(186 mg, 0.82 mmol) in acetonitrile (8 mL) was added potas-
sium carbonate (276 mg, 2.00 mmol) and methyl iodide (51
μL, 0.82 mmol) and the mixture was allowed to reacted at
room temperature overnight. After this time, water (10 mL)
was added and the mixture was extracted with ethyl acetate (3
x 10 mL). The combined organic layers were washed with
saturated aqueous sodium chloride (20 mL), dried over anhy-
drous sodium sulfate, filtered and concentrated to dryness. The
resulting residue was purified via flash SiO2 chromatography
(eluted with dichloromethane/methanol 20:1) to give 3-(4-
(2,4,6-trichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-
3-(5-(methylthio)-4-(3,4,5-trichlorophenyl)-4H-1,2,4-
triazol-3-yl)propan-1-ol (3l).
A
solution of 3,4,5-
trichloroaniline (786 mg, 4.00 mmol) in anhydrous chloroform
(10 mL) was added sodium carbonate (1.27 g, 11.98 mmol)
and pre-cooled in an ice bath. Then thiophosgene (335.5 μL,
5.37 mmol) was slowly added and the solution was allowed to
stir in an ice bath for 2 h. After this time, 2M sodium hydrox-
ide solution (10 mL) was added to quench the reaction and the
mixture was extracted with dichloromethane (3 x 10 mL) . The
combined organic layers were washed with saturated aqueous
sodium chloride (10 mL), dried over anhydrous sodium sulfate,
filtered and concentrated to dryness. The residue obtained was
purified via flash SiO2 chromatography (eluted with petroleum
ether/ethyl acetate 20:1) to give 3,4,5-trichloro-1-
isothiocyanatobenzene as a white solid (400 mg, 42%). 1H
NMR (400 MHz, DMSO-d6) δ 7.96 (s, 2H). Then a solution of
3,4,5-trichloro-1-isothiocyanobenzene (350 mg, 1.47 mmol) in
anhydrous
ethanol
(8
mL)
was
added
4-
hydroxybutanehydrazide (209 mg, 1.76 mmol) and reflux
overnight. After this time, the ethanol was evaporated, then 2
M NaOH (aq) (8 mL) was added and refluxed overnight. Then
2 M HCl (aq) was slowly added until a large amount of white
solid was precipitated. After filtration, the precipitation was
washed with water and then dried to obtain 3-(4-(3,4,5-
trichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)propan-1-ol
as a white solid without further purification (198 mg, 39%). 1H
NMR (400 MHz, DMSO-d6) δ 13.81 (s, 1H), 7.94 (d, J = 1.7
Hz, 2H), 4.52 (dt, J = 5.3, 2.5 Hz, 1H), 3.41 (s, 2H), 2.50 –
2.45 (m, 2H), 1.68 (dq, J = 8.1, 6.3 Hz, 2H). 13C NMR (101
MHz, DMSO-d6) δ 168.0, 152.5, 134.1, 133.9, 132.0, 130.2,
1
yl)propan-1-ol as a white solid (87 mg, 45%). H NMR (400
MHz, DMSO-d6) δ 8.12 (d, J = 11.4 Hz, 2H), 4.48 (p, J = 5.1
Hz, 1H), 3.41 (m, 2H), 2.59 (s, 3H), 2.45 (m, 2H), 1.72 (qd, J
= 7.9, 3.4 Hz, 2H).13C NMR (101 MHz, DMSO-d6) δ 155.6,
150.9, 137.3, 134.9, 130.6, 128.0, 60.1, 30.0, 21.5, 15.3.
HPLC purity analysis: 99.9% (229 nm). HRMS (ESI) calcd.
for C12H12Cl3N3OS [M+H]+ : 351.9845; found: 351.9830.
3-(5-(methylthio)-4-(2,3,4-trichlorophenyl)-4H-1,2,4-
60.0, 28.7, 22.5. Then
a
solution of 3-(4-(3,4,5-
triazol-3-yl)propan-1-ol (3k). A solution of 2,3,4-trichloro-1-
isothiocyanobenzene (358 mg, 1.50 mmol) in anhydrous etha-
nol (6 mL) was added 4-hydroxybutanehydrazide (186 mg,
1.58 mmol) and reflux overnight. After this time, the ethanol
was evaporated, then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. Then 2 M HCl (aq) was slowly added
until a large amount of white solid was precipitated. After
filtration, the precipitation was washed with water and then
dried to obtain 3-(4-(2,3,4-trichlorophenyl)-5-mercapto-4H-
1,2,4-triazol-3-yl)propan-1-ol (170mg, 33%) as a white solid
without further purification. Next, a solution of 3-(4-(2,3,4-
trichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)propan-1-ol
(107 mg, 0.32 mmol) in acetone (8 mL) was added potassium
trichlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)propan-1-ol
(190 mg, 0.56 mmol) in acetone (8 mL) was added potassium
carbonate (258 mg, 1.86 mmol) and methyl iodide (38 μL,
0.61 mmol) and the mixture was allowed to reacted at room
temperature overnight. After this time, water (10 mL) was
added and the mixture was extracted with ethyl acetate (3 x 10
mL). The combined organic layers were washed with saturated
aqueous sodium chloride (20 mL), dried over anhydrous sodi-
um sulfate, filtered and concentrated to dryness. The resulting
residue was purified via flash SiO2 chromatography (eluted
with dichloromethane/methanol 30:1) to give 3-(4-(3,4,5-
trichlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-yl)propan-
1
1-ol as a white solid (120 mg, 61%). H NMR (400 MHz,
14
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Page 15 of 20
Journal of Medicinal Chemistry
overnight. After this time, the ethanol was evaporated, then 2
DMSO-d6) δ 8.03 (s, 2H), 4.49 (t, J = 5.2 Hz, 1H), 3.42 – 3.37
1
2
3
4
5
6
7
8
(m, 2H), 2.63 – 2.56 (m, 2H), 2.55 (s, 3H), 1.73 (p, J = 6.6 Hz,
2H).13C NMR (101 MHz, DMSO-d6) δ 155.9, 150. 9, 134.4,
133.5, 132.4, 129.1, 60.2, 29.8, 21.8, 15.4. HPLC purity anal-
ysis: 97.2% (229 nm). HRMS (ESI) calcd. for
C12H12Cl3N3OS[M+H]+ : 351.9845; found: 351.9825.
M NaOH (aq)(8 mL) was added and refluxed overnight. Then
2 M HCl (aq) was slowly added until a large amount of white
solid was precipitated. After filtration, the precipitation was
washed with water and then dried to obtain 3-(5-mercapto-4-
(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)propan-1-ol as white solid
without further purification (323 mg, 62%). Then a solution of
3-(5-mercapto-4-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)propan-
1-ol (100 mg, 0.42 mmol) in acetone (8 mL) was added potas-
sium carbonate (138 mg, 1.00 mmol) and methyl iodide (40
µL, 0.64 mmol) and the mixture was allowed to reacted at
room temperature overnight. After this time, water (10 mL)
was added and the mixture was extracted with ethyl acetate
(3x10 mL). The combined organic layers were washed with
saturated aqueous sodium chloride (20 mL), dried over anhy-
drous sodium sulfate, filtered and concentrated to dryness. The
resulting residue was purified via flash SiO2 chromatography
(eluted with dichloromethane/methanol 15:1) to give 3-(4-(4-
chlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-yl)propan-1-
ol as white solid (231 mg, 68%). 1H NMR (400 MHz, DMSO-
d6) δ 8.74 (d, J = 4.8 Hz, 1H), 8.66 (d, J = 2.2 Hz, 1H), 7.97 (d,
J = 8.0 Hz, 1H), 7.62 (dd, J = 8.0, 4.8 Hz, 1H), 4.42 (t, J = 5.1
Hz, 1H), 3.33 (q, J = 6.0 Hz, 2H), 2.53 (m, 2H), 2.50 (s, 3H),
1.66 (p, J = 6.5 Hz, 2H).13C NMR (101 MHz, DMSO-d6) δ
156.2, 151.4, 151.2, 148.5, 135.9, 130.7, 125.1, 60.1, 29.9,
21.9, 15.3. HPLC purity analysis: 96.3% (229 nm). HRMS
(ESI) calcd. for C11H14N4OS[M+H]+ : 251.0967; found:
251.0968.
3-(5-(methylthio)-4-(3,4,5-trimethylphenyl)-4H-1,2,4-
triazol-3-yl)propan-1-ol (3m, TH1027). A solution of 3,4,5-
trimethyaniline (541 mg, 4.00 mmol) in anhydrous chloroform
(10 mL) was added sodium carbonate (1.27 g, 11.98 mmol)
and pre-cooled in an ice bath. Then thiophosgene (335.5 μL,
5.37mmol) was slowly added and the solution was allowed to
stir in an ice bath for 2 h. After this time, 2M sodium hydrox-
ide solution (10 mL) was added to quench the reaction and the
mixture was extracted with dichloromethane (3 x 10 mL). The
combined organic layers were washed with saturated aqueous
sodium chloride (10 mL), dried over anhydrous sodium sulfate,
filtered and concentrated to dryness. The residue obtained was
purified via flash SiO2 chromatography (eluted with petroleum
ether/ethyl acetate 20:1) to give 3,4,5-trimethy-1-
isothiocyanatobenzene as a white solid (443mg, 63%). 1H
NMR (400 MHz, chloroform-d) δ 6.85 (s, 2H), 2.23 (s, 6H),
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2.13 (s, 3H). Then
a
solution of 3,4,5-trimethy-1-
isothiocyanobenzene (430 mg, 2.43 mmol) in anhydrous etha-
nol (8 mL) was added 4-hydroxybutanehydrazide (295mg,
2.50 mmol) and reflux overnight. After this time, the ethanol
was evaporated, then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. Then 2 M HCl (aq) was slowly added
until a large amount of white solid was precipitated. After
filtration, the precipitation was washed with water and then
dried to obtain 3-(4-(3,4,5-trichlorophenyl)-5-mercapto-4H-
1,2,4-triazol-3-yl)propan-1-ol as a white solid without further
purification (500 mg, 45%). 1H NMR (400 MHz, DMSO-d6) δ
13.60 (s, 1H), 7.00 (s, 2H), 3.35 (t, J = 6.2 Hz, 2H), 2.43 –
2.35 (m, 2H), 2.29 (s, 6H), 2.18 (s, 3H), 1.66 (dq, J = 8.0, 6.3
Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 167.8, 152.5,
137.5, 136.6, 130.8, 126.9, 59.7, 28.6, 22.4, 20.3, 15.2. Then a
solution of 3-(4-(3,4,5- trimethyphenyl)-5-mercapto-4H-1,2,4-
triazol-3-yl)propan-1-ol (300 mg, 1.32 mmol) in acetone (8
mL) was added potassium carbonate (300 mg, 1.32 mmol)
and methyl iodide (93 μL, 1.49 mmol) and the mixture was
allowed to reacted at room temperature overnight. After this
time, water (10 mL) was added and the mixture was extracted
with ethyl acetate (3 x 10 mL). The combined organic layers
were washed with saturated aqueous sodium chloride (20 mL),
dried over anhydrous sodium sulfate, filtered and concentrated
to dryness. The resulting residue was purified via flash SiO2
chromatography (eluted with dichloromethane/methanol 30:1)
to give 3-(4-(3,4,5-trimethyphenyl)-5-(methylthio)-4H-1,2,4-
triazol-3-yl)propan-1-ol as a white solid (200 mg, 64%). 1H
NMR (400 MHz, DMSO-d6) δ 8.03 (s, 2H), 4.49 (t, J = 5.2 Hz,
1H), 3.42–3.37 (m, 2H), 2.63–2.56 (m, 2H), 2.55 (s, 3H),
2.27(s, 6H), 2.16(s, 3H), 1.73 (p, J = 6.6 Hz, 2H). 13C NMR
(101 MHz, DMSO-d6) δ 155.8, 151.0, 138.2, 137.2, 130.2,
125.8, 60.0, 29.8, 21.7, 15.3, 14.6. HPLC purity analysis: 98.5%
(229 nm). HRMS (ESI) calcd. for C15H21N3OS[M+H]+ :
292.1484; found: 292.1480.
3-(4-cyclohexyl-5-(methylthio)-4H-1,2,4-triazol-3-
yl)propan-1-ol (3o). A solution of isothiocyanatocyclohexane
(283 mg, 2.00 mmol) in anhydrous ethanol (6 mL) was added
4-hydroxybutanehydrazide (248 mg, 2.10 mmol) and reflux
overnight. After this time, the ethanol was evaporated, then 2
M NaOH (aq) (8 mL) was added and refluxed overnight. Then
2 M HCl (aq) was slowly added until a large amount of white
solid was precipitated. After filtration, the precipitation was
washed with water and then dried to obtain 3-(4-cyclohexyl-5-
mercapto-4H-1,2,4-triazol-3-yl)propan-1-ol as white solid
without further purification (230 mg , 48%). Then a solution
of 3-(4-cyclohexyl-5-mercapto-4H-1,2,4-triazol-3-yl)propan-
1-ol (100 mg, 0.41 mmol) in acetone (8 mL) was added potas-
sium carbonate (138 mg, 1.00 mmol) and methyl iodide (39
µL, 0.62 mmol) and the mixture was allowed to reacted at
room temperature overnight. After this time, water (10 mL)
was added and the mixture was extracted with ethyl acetate (3
x 10 mL). The combined organic layers were washed with
saturated aqueous sodium chloride (20 mL), dried over anhy-
drous sodium sulfate, filtered and concentrated to dryness. The
resulting residue was purified via flash SiO2 chromatography
(eluted with dichloromethane/methanol 20:1) to give 3-(4-
cyclohexyl-5-(methylthio)-4H-1,2,4-triazol-3-yl)propan-1-ol
1
as white solid (40 mg, 38%). H NMR (400 MHz, DMSO-d6)
δ 4.60 (t, J = 5.2 Hz, 1H), 4.02 (ddt, J = 12.4, 8.5, 3.8 Hz, 1H),
3.48 (q, J = 5.9 Hz, 2H), 2.78 (t, J = 7.6 Hz, 2H), 2.51 (s, 3H),
2.01 (qd, J = 12.4, 3.4 Hz, 2H), 1.79 (m, J = 14.4, 9.3, 3.8 Hz,
6H), 1.67 (d, J = 13.2 Hz, 1H), 1.39 (qt, J = 12.9, 3.4 Hz, 2H),
1.18 (ttd, J = 12.9, 9.6, 4.9 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 155.8, 149.1, 60.3, 55.5, 31.1, 30.8, 25.8, 25.1,
22.2, 15.9. Purity＞95% (by 1H-NMR). HRMS (ESI) calcd.
for C12H21 N3OS [M+H]+ : 256.1484, [M+Na]+ : 278.1303;
found [M+H]+ : 256.1471, [M+Na]+ : 278.1289.
3-(4-(4-chlorophenyl)-5-(methylthio)-4H-1,2,4-triazol-3-
yl)propan-1-ol (3n). A solution of 3-isothiocyanatopyridine
(300 mg, 2.20 mmol) in anhydrous ethanol (6 mL) was added
4-hydroxybutanehydrazide (260 mg, 2.20 mmol) and reflux
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3-(5-(methylthio)-4-(naphthalen-1-yl)-4H-1,2,4-triazol-3-
yl)propan-1-ol (3p). solution of
HEK-Blue TLR8 cells (4 × 104 cells/well) or human PBMCs
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(3 × l06 cells/mL) were placed in a 96-well plate and co-
incubated with indicated compounds at 37℃for 24 h. Next
Cell Counting Kit-8 (Bimake) was added to each well (1:10
dilution). Then cells were incubated at 37 °C until a color
change was observed (within 2 h) and absorbance was read at
450 nm. Data was normalized with the untreated cells control
as 100% survival.
isothiocyanatonaphthalene (370 mg, 2.00 mmol) in anhydrous
ethanol (6 mL) was added 4-hydroxybutanehydrazide (248 mg,
2.10 mmol) and reflux overnight. After this time, the ethanol
was evaporated, then 2 M NaOH (aq) (8 mL) was added and
refluxed overnight. Then 2 M HCl (aq) was slowly added
until a large amount of white solid was precipitated. After
filtration, the precipitation was washed with water and then
dried to obtain 3-(5-mercapto-4-(naphthalen-1-yl)-4H-1,2,4-
triazol-3-yl)propan-1-ol as white solid without further purifi-
cation (352 mg, 62%). Then a solution of 3-(5-mercapto-4-
(naphthalen-1-yl)-4H-1,2,4-triazol-3-yl)propan-1-ol (180 mg,
0.60 mmol) in acetone (8 mL) was added potassium carbonate
(276 mg, 2.00 mmol) and methyl iodide (55 µL, 0.87 mmol)
and the mixture was allowed to reacted at room temperature
overnight. After this time, water (10 mL) was added and the
mixture was extracted with ethyl acetate (3 x 10 mL). The
combined organic layers were washed with saturated aqueous
sodium chloride (20 mL), dried over anhydrous sodium sulfate,
filtered and concentrated to dryness. The resulting residue was
purified via flash SiO2 chromatography (eluted with dichloro-
methane/methanol 20:1) to give 3-(5-(methylthio)-4-
(naphthalen-1-yl)-4H-1,2,4-triazol-3-yl)propan-1-ol as white
TLR Selectivity assay
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The selectivity of TH1027 against the TLR family was exam-
ined in HEK-Blue cells overexpressing TLR2, TLR3, TLR4,
TLR5, TLR6, TLR7, TLR8 and TLR9. The assay was per-
formed in the same manner as “SEAP reporter assay”, using
their selective ligand:
100 ng/mL of Pam3CSK4 (N-
palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-
cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-
lysine3HCl), 5 µg/mL of polyriboinosinic : polyribocytidylic
acid (poly(I:C)), 20 ng/mL of LPS (lipopolysaccharide), 50
ng/mL of Flagellin, 100 ng/mL of Pam2CSK4 (S-[2,3-
bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-
lysyl-[S]-lysyl-[S]-lysyl-[S] lysine3CF3COOH), 0.5 µg/mL of
R848, 1 µg/mL of R848 and 0.5 µM ODN2006.
1
solid (97 mg, 54%). H NMR (400 MHz, DMSO-d6) δ 8.17
RT-qPCR analysis
(ddd, J = 28.4, 8.1, 3.6 Hz, 2H), 7.75 – 7.57 (m, 4H), 7.11 (dd,
J = 8.6, 3.1 Hz, 1H), 4.41 (q, J = 4.7 Hz, 1H), 3.36 (d, J = 3.5
Hz, 2H), 2.52 (s, 3H), 2.48 – 2.29 (m, 2H), 1.65 (ddt, J = 15.0,
7.7, 3.6 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 156.8,
152.1, 134.3, 131.2, 129.5, 129.2, 128.8, 127.7, 127.1, 126.3,
121.7, 60.1, 30.1, 21.9, 15.0. HPLC purity analysis: 98.6%
(229 nm). HRMS (ESI) calcd. for C16H17N3OS [M+H]+ :
300.1171; found: 300.1155.
HEK-Blue TLR8 cells were seeded at a density of 1 × 106
cells per well in a 12-well plate and the plate was incubated at
37°C in 5% CO2. After incubation for 24 h, the medium was
replaced by serum-free medium, and then the cells were left
untreated, treated with R848 (4 µg/mL), R848 (4 µg/mL) with
TH1027 (1 µM) or compound 2a (1 µM), respectively. After
incubation for 8 h, cells were scraped and re-suspended in
PBS and then centrifuged at 2000 rpm, 5 min to remove PBS.
Total RNA was extracted with the TRIZOL reagent (Invitro-
gen, Lot number: 15596018) using standard protocols. Re-
verse transcription was performed using iScriptTM cDNA
Synthesis Kit (Bio-Rad, Lot number: 1708890) according to
manufacturer’s instructions using an Applied Biosystems.
qPCR was performed using iTaq Universal SYBR Green Su-
permix (Bio-Rad, Lot number: 1725124) following the manu-
facturer’s protocol. TNF-α, IL-8 and GAPDH primers were
obtained from Ruibiotech company. Data was analyzed using
the ΔΔCt method using GAPDH gene as a housekeeping gene
and normalized to cell control.
Cell culture
HEK-Blue TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 and
TLR9 cells were purchased from InvivoGen and cultured in
complete culture medium: Dulbecco’s modified Eagle’s medi-
um (DMEM), 10% (v/v) FBS, 100 U/mL penicillin, 100
µg/mL streptomycin, 100 mg/mL normocin, and 2 mM L-
glutamine. Human monocyte cell lines were purchased from
ATCC and cultured in Roswell Park Memorial Institute (RPMI)
1640 medium supplemented with 10% (v/v) FBS, 2 mM L-
glutamine, 100 µg/mL streptomycin and 100 U/mL penicillin
and 0.05 mM 2-mercaptoethanol. All cultured cells were
grown at 37 °C in a humidified incubator containing 5% CO2.
The cultures were checked periodically and found to be free of
mycoplasma contamination.
Protein expression, purification and crystallization
The extracellular domain of human Toll-like receptor 8
(hTLR8, residues 27–827) was prepared as described previ-
ously33 and was concentrated to 8.2 mg/mL in 10 mM Tris-
HCl pH 8.0 and 150 mM NaCl. The protein solutions for the
co-crystallization of hTLR8 and TH1027 contained hTLR8
(7.0 mg/mL) and a ten-fold excess of TH1027 in a crystalliza-
tion buffer containing 10 mM Tris-HCl pH 8.0, 150 mM NaCl,
and 5% dimethyl sulfoxide (DMSO). Crystallization experi-
ments were performed with sitting-drop vapor-diffusion meth-
ods at 293 K. Crystals of hTLR8/TH1027 were obtained with
reservoir solutions containing 12-20% PEG 4000, 0.2 M calci-
um chloride, 0.1 M Tris-HCl pH 8.0, and 25% ethylene glycol.
SEAP reporter assay
HEK-Blue TLR8 cells were plated at the density of 4 × 104
cells per well in a 96-well plate in DMEM and treated with
1µg/mL R848 (Invivogen) and varying concentrations of ap-
propriate compounds. After incubation at 37 °C for 24 h, 50
µL of supernatant was collected and placed in a new 96-well
plate and then 50 µL of Quanti-Blue (Invivogen) was added to
the media, and incubated at 37 °C until color change was ob-
served (within 1 h). The plate were then measuring absorbance
at 620 nm. Data was normalized as readout of ligand-treated
cells are 100% activation and untreated cells are 0% activation.
Data collection and structure determination
Cell viability assay
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Journal of Medicinal Chemistry
Enzyme-Linked Immunosorbent Assay (ELISA) in human
Diffraction dataset for hTLR8/TH1027 crystals was collected
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on beamline PF-AR NE12A (Ibaraki, Japan) under cryogenic
condition at 100 K. The wavelength was set to 1.0000 Å. The
dataset was processed with the HKL2000 package34.
hTLR8/TH1027 structure was determined by the molecular
replacement method using the Molrep program35 with the
hTLR8/CUCUT8m structure (PDB ID: 5WYX) as a search
model. The model was further refined with stepwise cycles of
manual model building with the COOT program36 and re-
strained refinement with REFMAC37 until the R factor was
converged. TH1027 molecules, N-glycans were modeled into
the electron density map at the latter cycles of the refinement.
The quality of the final structure was validated with the PDB
validation server (http://wwpdb-validation.wwpdb.org/). The
favored and the allowed regions in the Ramachandran plot
contain 93% and 6% residues for TLR8/TH1027, respectively.
The statistics of the data collection and refinement are summa-
rized in Supplementary Table 1. The figures representing
structures were prepared with PyMOL (http://www.pymol.org).
Coordinate and structure factor of TLR8/TH1027 have been
deposited in the Protein Data Bank with PDB ID: 6KYA.
monocyte cell lines
Human monocyte cell lines were seeded at 1 × 106 per well in
1 mL RPMI medium supplemented with (10% (v/v) FBS, 2
mM L-glutamine, 100 µg/mL streptomycin, 100 U/mL penicil-
lin and 0.05 mM 2-mercaptoethanol in a 12-well plate and
incubated at 37 °C in a humidified 5% CO2 atmosphere. After
24 h, the medium was removed and cells were washed three
times using PBS buffer. Then 1 mL of RPMI medium was
added to each well and cells were treated with R848 (1 µg/mL)
and various concentrations of compounds TH1027 or left un-
treated. After incubation for 24 h, supernatants of the culture
medium were collected, and the levels of TNF-α, IL-6 and IL-
1β were determined using BD OptEIATM human TNF-ELISA
kit (BD Biosciences) , BD OptEIATM human IL-6-ELISA kit
(BD Biosciences) and BD OptEIATM human IL-1β-ELISA kit
(BD Biosciences) respectively, according to the manufactur-
er’s instructions.
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ELISA in human PBMCs from healthy donors and rheumatoid
arthritis patients
Isothermal Titration Calorimetry (ITC) assay
Human whole blood was collected by venipuncture from
healthy human volunteers and rheumatoid arthritis patients.
All experiments performed on human PBMCs have been de-
scribed and approved by the IRB of PUMCH (No. S-478) and
are consistent with Institutional Guidelines. Diagnosis of
rheumatoid arthritis (RA) was confirmed by senior consultant
rheumatologists according to 2010 American College of
Rheumatology (ACR) criteria for RA. Human PBMCs from
healthy donors and six RA patients were isolated using Densi-
ty Gradient Centrifugation. Immediately after separation, cells
were cultured at a density of 3 × l06 cells/mL in 0.2 mL of
RPMI 1640 in 96-well round-bottom plates (Thermo Scien-
tific). Cells were then treated with indicated TH1027 or 2a as a
negative control. After incubating for 24 h, the supernatants
were collected after centrifuged for 10 min at 4,000 r.p.m. at
4 °C and frozen at -80 °C until ready for TNF-α measurement.
The levels of TNF-α were determined using BD OptEIATM
human TNF-ELISA kit (BD Biosciences) according to the
manufacturer’s instructions.
ITC experiments were done in a buffer composed of 25 mM
MES pH 5.5, 0.20 M NaCl, and 5% DMSO at 298 K using a
MicroCal iTC200 (GE Healthcare). The titration sequence
included a single 0.4 µL injection followed by 18 injections, 2
µL each, with a spacing of 120 seconds between the injections.
The titration conditions were as follows: 100 µM TH1027 into
10 µM hTLR8; 100 µM R848 into 10 µM hTLR8; 100 µM
R848 into 10 µM hTLR8 in the presence of 50 µM TH1027.
OriginLab software (GE Healthcare) was used to analyze the
raw ITC data.
Western blot
Human monocyte cell lines were added in 6-well plates with a
density of 2x106 cells/well in RPMI 1640 medium supple-
mented with 10% FBS, 100 U/mL penicillin, 100 µg/mL strep-
tomycin, 2 mM L-glutamine, and 0.05 mM 2-mercaptoethanol.
Then cells were treated with phorbol-12-myristate-13-acetate
(PMA) (100 ng/mL) and incubated in humidified incubators
containing 5% CO2 at 37 C for 24 h. After differentiation, the
supernatant was removed and cells were washed three times
by PBS and then replaced with unsupplemented RPMI, these
cells were then treated with R848 (1 µg/mL) along with vari-
ous concentrations of TH1027. After incubation for 2 h, cells
were collected, nuclear protein fraction was extracted using
NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo
Fisher Scientific, Massachusetts, USA). BCA assay was then
used to determine protein concentration. Protein samples were
loaded and run in 10% Tris-glycine SDS-PAGE, and then
transferred onto a nitro-cellulose membrane (Bio-Rad, Cali-
fornia, USA) using electroblotting. P65 (CST, 1:1000),
TRAF3 (CST, 1:1000) and p-IRAK4 (CST, 1:1000) was then
used as primary antibody, and peroxidase-conjugated Affini-
Pure Goat Anti-Rabbit IgG (H+L) antibody (CST, 1:10000) as
secondary antibody, then blots were visualized using Thermo
SuperSignal West Pico Plus kit (Thermo Fisher Scientific,
Massachusetts, USA). Lamin A/C (transgen, 1:1000) was used
as internal controls for nuclear fractions and β-actin (CST,
1:1000) was used as internal controls for cytoplasmic fractions.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Figure S1. Crystal structure of inhibition and activation state of
TLR8 protein
Figure S2. The activity, toxicity and structure of three compounds
screened from a 14,400-membered commercial library
Figure S3. Toxicity of TH1027 in HEK-Blue hTLR8 cells
Figure S4. ITC experiments
Figure S5. Close-up view of each antagonist binding site
Figure S6. Quantitative data analyses of one representative West-
ern Blot experiment
Figure S7. Toxicity of TH1027 in PBMCs from RA patients
Table S1. Data collection and refinement statistic for
hTLR8/TH1027
NMR Spectra of compounds
HPLC traces of representative compound(TH1027)
Molecular formula strings (CSV)
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Accession Codes
kinase 4; TRAF3, tumor necrosis factor-alpha receptor-associated
1
2
3
factor 3; NF-κB, nuclear factor kappa-light-chain enhancer of
activated B cells; IL-6, interleukin-6; IL-1β, interleukin-1 beta;
RA, rheumatoid arthritis; ssRNA, single stranded-RNA.
PDB code for TLR8/TH1027 complex is 6KYA. Authors will
release the atomic coordinates upon article publication.
4
5
6
AUTHOR INFORMATION
Corresponding Author
REFERENCES
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*E-mail: yin_hang@tsinghua.edu.cn
ORCID
(1) Kawai, T.; Akira, S., The role of pattern-recognition recep-
tors in innate immunity: update on Toll-like receptors. Nat.
Immunol. 2010, 11 (5), 373-384.
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Hang Yin: https://orcid.org/0000-0002-9762-4818
(2) Takeuchi, O.; Akira, S., Pattern recognition receptors and
inflammation. Cell 2010, 140 (6), 805-820.
Author Contributions
(3) Yoshimura, A.; Lien, E.; Ingalls, R. R.; Tuomanen, E.;
Dziarski, R.; Golenbock, D., Cutting edge: recognition of
gram-positive bacterial cell wall components by the innate
immune system occurs via Toll-like receptor 2. J. Immunol.
1999, 163 (1), 1-5.
H.Y. supervised the overall project. S.J. designed the experi-
ments in consultation with H.Y. S.Z. performed the high
throughput screening. S.J., K.Y., and Y.Y. performed chemi-
cal synthesis of compounds. S.J. performed SEAP reporter
assay, cell viability assay, selectivity assay, RT-qPCR, ELISA,
Western Blot. H.T., K.S., U.O., and T.S. expressed protein,
solved the crystal structure, performed isothermal titration
calorimetry experiments. J.L. contributed to provide the pa-
tient specimens. S.J. performed PBMC isolation and patient
specimens studies. H.Y. and S.J. wrote the manuscript with
input from U.O., K.S., J.H., Y.Y. and K.Y.
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Notes
H.Y. and S.J. have filed a patent application based on the technol-
ogy reported in this manuscript.
.
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ACKNOWLEDGMENT
This work was funded by the National Natural Science Founda-
tion of China Grand No.20181371453 (H.Y.) and No.21807026
(S.Z.), Beijing Outstanding Young Scientist program Grand No.
BJJWZYJH01201910003013 (H.Y.), International Research Pro-
ject of Independent Research of Ministry of Education grand
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X.; Birdwell, D.; Alejos, E.; Silva, M.; Galanos, C.; Freuden-
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(UTokyo-Tsinghua
Collaborative
Research
Fund)
No.2019080048 (H.Y.) the Grant-in-Aid from the Japanese Min-
istry of Education, Culture, Sports, Science, and Technology
Grant Nos. 19H00976 (T.S.), 18H04669, 19H03164, 19H04950
(U.O.), 19J21830 (K.S.) and by Core Research for Evolutional
Science and Technology (T.S.). We thank the Beamline staff
members at Photon Factory for their assistance with data collec-
tion. This research was partially supported by Platform Project for
Supporting Drug Discovery and Life Science Research (Basis for
Supporting Innovative Drug Discovery and Life Science Research,
BINDS) from AMED under Grant Number JP19am0101071
(support number 0030). We thank Yifu Chen from Tianjin Uni-
versity for drawing the table of content and also thank Ying
Zhang for checking the manuscript.
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ABBREVIATIONS
TLRs, toll-like receptors; PBMCs, peripheral blood mononuclear
cells; PAMPs, pathogen-associated molecular patterns; DAMPs,
danger-associated molecular patterns; SEAP, secreted alkaline
phosphatase; THF, tetrahydrofuran; DMF, dimethyl formamide;
SAR, structure−activity relationship; RT-qPCR, real-time quanti-
tative polymerase chain reaction; ITC, isothermal titration calo-
rimetry; Kd, dissociation constant; LPS, lipopolysaccharide; TNF-
α, tumor necrosis factor-alpha; RMSD, root mean square devia-
tion; p-IRAK4, phosphorylated interleukin-1 receptor-associated
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