Discovery of a novel series of hDHODH inhibitors with anti-pulmonary fibrotic activities

Article abstract of DOI:10.1016/j.bioorg.2019.01.011

Human dihydroorotate dehydrogenase (hDHODH) is a flavin-dependent enzyme essential to pyrimidine de novo biosynthesis, which serves as an attractive therapeutic target for the treatment of autoimmune disorders. A novel series of hDHODH inhibitors was deve

Full text of DOI:10.1016/j.bioorg.2019.01.011

Accepted Manuscript
Discovery of a novel series of hDHODH inhibitors with anti-pulmonary fibrotic
activities
Kuan Lu, Yanfang Zhao, Guodong Wu, Hao Hu, Mingzhong Wang, Guowei
Gong, Yuyang Jiang
PII:
S0045-2068(18)31236-7
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.011
YBIOO 2715
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 October 2018
4 January 2019
6 January 2019
Please cite this article as: K. Lu, Y. Zhao, G. Wu, H. Hu, M. Wang, G. Gong, Y. Jiang, Discovery of a novel series
of hDHODH inhibitors with anti-pulmonary fibrotic activities, Bioorganic Chemistry (2019), doi: https://doi.org/
10.1016/j.bioorg.2019.01.011
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Discovery of a novel series of hDHODH inhibitors with
anti-pulmonary fibrotic activities
Kuan Lua,b, Yanfang Zhaoa, Guodong Wua, Hao Hua, Mingzhong Wangb, Guowei
Gonge* and Yuyang Jiang*a,c,d
a Department of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang,
Liaoning, 110016, PR China
b Shenzhen Kivita Innovative Drug Discovery Institute, Shenzhen, 518057, PR China
c
National & Local United Engineering Lab for Personalized Anti-tumor Drugs, The Graduate
School at Shenzhen, Tsinghua University, Shenzhen, 518055, PR China
d
Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua
University, Beijing, 100084, PR China
e
Department of bioengineering, Zunyi Medical University, Zhuhai Campus, Zhuhai 519041,
Guangdong, China
*Corresponding author.
E-mail address: jiangyy@sz.tsinghua.edu.cn (Yuyang Jiang);
amygong90@hotmail.com (Guowei Gong).
Abstract
Human dihydroorotate dehydrogenase (hDHODH) is a flavin-dependent enzyme
essential to pyrimidine de novo biosynthesis, which serves as an attractive therapeutic
target for the treatment of autoimmune disorders. A novel series of hDHODH
inhibitors was developed based on a lead which was obtained by a medicinal
chemistry exploration. Most compounds showed moderate to significant potency
against hDHODH, compounds 5d, 5e, and 6a effectively inhibited the activities of
hDHODH with IC50 values from 0.9 to 2.8 μM. Further studies showed that
compound 5e also effectively suppressed proliferation of the activated PBMCs (IC50 =
20.35 μM). Surprisingly, compound 5e also showed anti-pulmonary fibrotic activity
similar to that of pirfenidone in vitro assay. Therefore, compound 5e might have
potential to be developed as a novel hDHODH inhibitors for autoimmune diseases
therapy.
Keywords: Human dihydroorotate dehydrogenase (hDHODH) inhibitors;
Autoimmune diseases; Drug design; Anti-pulmonary fibrotic activity

1. Introduction
Human dihydroorotate dehydrogenase (hDHODH) is a flavin-dependent enzyme
essential to pyrimidine de novo biosynthesis. It has been identified as a therapeutic
target for the treatment of autoimmune disorders such as rheumatoid arthritis and
multiple sclerosis [1-3]. Leflunomide (Fig.1a) and Brequinar (Fig.1c) are two
representative hDHODH inhibitors. The former was approved more than 20 years ago
for the treatment of rheumatoid arthritis [1]. leflunomide acts as a prodrug and is
rapidly converted into its active metabolite teriflunomide (Fig.1b) which was
approved for treatment of multiple sclerosis. While, brequinar was initially evaluated
in clinical trials as a potential anticancer agent. However, it was discarded due to
severe side effects [4]. Recently, Johann Leban [5] and Honglin Li [6] respectively
reported several hDHODH inhibitors which bore thiophene carboxylic acid (Fig.1d)
and thiazole-scaffold (Fig.1e) with IC50 1 nM and 18 nM. However, despite recent
efforts, the quest to explore the therapeutic potential of hDHODH inhibitors remains
an urgent area of research.
Fig. 1. Selected Structures of the Reported DHODH Inhibitors
In our previous studies, compound f (Fig.2) has been identified as a potential
hDHODH inhibitor (IC50 = 1.5 μM). Docking analysis showed that compound f
adopted a binding mode similar with brequinar: The amide group forms two hydrogen
bonds with residues Gln47 and Arg136 of the enzyme and the terminal phenyl ring
extends into the hydrophobic pocket.
In order to improve the potency, we described a novel series of hDHODH
inhibitors designed by replacing the phenyl moiety (Fig.2 blue part) with thiophene
which exists in the effective hDHODH inhibitor compound d. The most promising
compound 5e (IC50 = 0.9 μM) exhibited 1.6-fold more potent than compound f and
displayed efficient anti-inflammation effect. Unexpectedly, some of the target
compounds also showed anti-pulmonary fibrotic activities. It suggested that they
might be developed as novel immune-suppressants without drug-induced pulmonary
fibrotic.

Fig. 2. Design strategy of the target compounds.
2. Results and discussion
2.1 Chemistry
The synthetic procedure for the target compounds is described in Scheme 1
3-amino-2-thiophenecarboxylate methyl ester was treated with NaNO2 to yield
the diazonium salt, which were not isolated but directly treated with NaN3 to
.
1
obtain azido derivative
hydrolysis. Intermediate
2
3
[7] which was converted to intermediate
was treated with SOCl2 under reflux to obtain
3 by
2-theonyl chloride derivative which reacted with ammonia or cyclopropylamine
to give the intermediates 4a-4b [8]. Intermediates 7a-n were synthesized by
reacting suitable aniline or phenol with propargyl bromide [9-11]. The target
compounds were yielded by click chemistry with copper-catalyzed alkyne-azide
1,3-dipolar cycloaddition (CuAAC) [12] between intermediates 2, 3, 4a-4b and
intermediates7a-o at room temperature. The target compounds were purified by
column chromatography on silica gel. The chemical structures of the target
compounds were confirmed by 1H NMR, 13C NMR and ESI-MS.
Scheme 1. Reagents and conditions: (i) NaNO2, HCl; NaN3, 1 h; (ii) NaOH, EtOH/H2O reflux;
(iii) a: SOCl2, reflux, 5 h; b: NH3.H2O/ cyclopropylamine, rt, 2h; (iv) alkyne, Sodium ascorbate,
CuSO4, t-BuOH/H2O (1:1), rt, 3-4h. (v) a: X=NH, 3-bromopropyne, K2CO3, DMF, rt, overnight;
b: X=O, 3-bromopropyne, K2CO3, MeCN, reflux.

In order to determine the stereochemistry of the target compounds, the
NOESY experiment of compound 5e was carried out, and an evident NOE
signal was observed between the proton (d, 7.42 ppm) on the 4-postion of the
thiophene amide moiety and the proton (s, 8.70 ppm) on the 1,2,3-triazole,
which existed only in the A-5e form due to the appropriate intramolecular H–H
distance (Fig. 3 and see Supporting Information).
Fig. 3. NOESY effect of the representative compound 5e
2.2. Biological evaluation
2.2.1Inhibition of hDHODH and structure-activity relationships
.
To get a preliminary insight into SARs, the thiophene derivatives with different R
group were synthesized and evaluated for enzyme inhibition assay. Human DHODH
inhibition assay was conducted using chromogen reduction method with
2,6-dichlorophenolindophenol (DCIP). Dihydroorotate (DHO) oxidation and
coenzyme Q (CoQ) reduction was coupled with chromogen reduction. Enzyme
inhibition was resulted in loss of chromogen absorbance at 600 nm. The preliminary
activities were measured at 10 μM concentration of synthesized compounds. The
results (Table 1). showed that compound 4c (R=NH2) exhibited higher potency than
2a and 3a.
Table 1 Inhibitory activities of the compounds against hDHODH under 10 μM concentration.
Compound
R
% Inhibition ±
SDa at 10 μM.
21 ± 0
OMe
OH
2a
3a
4c
12 ± 0
NH2
52 ± 0
a SD: standard deviation.
Due to the relatively weak activity of 4c, further modification of the target
compounds with different groups was undertaken. The results (Table 2) showed that
the position and property of R2 groups had a dramatic impact on activities against

hDHODH. When X is oxygen atom, the incorporation of a polar group such as CN or
CF3 group to 4-position of the terminal phenyl group led to a significant improvement
in potency (5d, 5e vs 4c). However, compounds 5a and 5c which possessed 4-position
halogen were inactive. Shifting fluorine from 4-position (5a) to 3-position (5b)
increased the activity, while introduction CF3 group on the 3-position showed a
significant decreased potency (5g vs 5e). Besides, conversion of the R1 substituted
phenyl ring to naphthalene (5j) also obtained low potency. When X is nitrogen atom,
compound 6a and 6c showed high potency (6a, 6c vs 4c), compounds 6e and 6f with
3,4-disubstituted halogen were inactive. Moreover, the introduction of cyclopropyl at
R1 result in the decline of activities against hDHODH (5f vs 5e, 6d vs 4c) The results
indicated that amide without substitution and a suitable substitution on the terminal
phenyl moiety were crucial for high potency.
Table 2 Inhibitory activities of the compounds against hDHODH under 10 μM concentration
Compound
X
R1
R2
% Inhibition ±
SDa at 10 μM.
NA
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H
4-F
5a
5b
5c
5d
5e
5f
3-F
49 ± 0
4-Br
4-CN
4-CF3
NA
86 ± 4
89 ± 0
cyclopropyl 4-CF3
49 ± 4
H
H
H
H
3-CF3
20 ± 11
NA
5g
5h
5i
3,4-2F
2-Br-4-F
49 ± 0
25 ± 11
5j
N
N
N
N
N
N
H
H
H
4-F
83 ± 4
57 ± 4
72 ± 0
12 ± 7
NA
6a
4-Br
6b
4-OMe
6c
cyclopropyl 4-CF3
6d
H
H
3,4-2F
6e
3-Cl-4-F
NA
6f
a SD: standard deviation.
Based on the preliminary assays, four compounds which showed significant
potency were selected for further test IC50 values against hDHODH in vitro. The
results were summarized in Table 3. As predicted, all of the selected compounds

displayed good to significant potency against hDHODH with IC50 values ranging from
0.9 to 4.6 μM and compound 5e (IC50 = 0.9 μM) exhibited relatively higher potency
than teriflunomide.
Table 3 IC50 values of synthesized derivatives against hDHODH
a
a
Compound
Compound
IC50 (μM)
IC50 (μM)
2.8
0.9
1.1
4.6
2.7
5d
6a
6c
5e
Teriflunomideb
a Values are the means of two independent experiments.
b Used as positive control.
2.2.2 Immunosuppression assay and metabolic stability
Based on the enzymatic assays, compound 5e was selected for further study the
immunosuppressive effect on the proliferation of stimulated peripheral blood
mononuclear cells (PBMCs) compared with teriflunomide. As shown in Table 4,
compound 5e was found to act similarly to teriflunomide, inhibiting activated PBMCs
proliferation (IC50 = 20.35 μM). Moreover, we performed the study of 5e in incubation
with human liver microsomes in vitro. The studies revealed that 5e possessed high
T1/2 >120 min and exhibited moderate CL. It demonstrated that 5e was stable in
human liver microsomes.
Table 4 Inhibition of proliferation of stimulated PBMCs and metabolic stability
a
Compound
T1/2 c(min)
IC50 (μM)
20.35
CL(μL/min/mg)
198.2
7.0
5e
teriflunomideb
14.17
a Values are the means of two independent experiments.
b Used as positive control.
c Conducted in human liver microsomes with final liver microsomal protein (0.5mg/mL).
2.2.3 anti-pulmonary fibrotic assay
Drug-induced interstitial lung disease (ILD), particularly pulmonary fibrosis, is a
serious clinical concern. For example, hDHODH inhibitor leflunomide, imatinib and
gefitinib, are known to induce ILD (pulmonary fibrosis). it can lead to patient death,
thereby restricting the therapeutic use of these drugs [13-15]. Recently, a rapid
method for anti-fibrotic drugs screening based on cellular stiffness was developed
[16]. A549 cells in vitro were stimulated with transforming growth factor-β1
(TGF-β1).It led the increase of the cellular stiffness which may be reversed by the
compounds with anti-pulmonary fibrotic activities. The alteration of cellular stiffness
was measured utilizing atomic force microscopy (AFM). The anti-pulmonary fibrotic
activities of compound 5d, 5e and 6c were evaluated by the method above and
anti-pulmonary fibrotic drug pirfenidone was used as positive control. The results
are summarized in Table 5. Compared with pirfenidone, compound 5e exhibited best

potency which could reduce the cellular stiffness of TGF-β1 stimulated A549 cells
from 9.25 Kpa to 6.23 Kpa.
Table 5 Alteration of cellular stiffness of TGF-β1 stimulated A549 cells
Compound
Stiffnessa (Kpa)
Compound
Stiffnessa (Kpa)
6.40±1.79
7.29±1.51
5d
6c
Controlb
pirfenidoned
6.23±1.58
9.25±1.02
4.48±1.96
6.09±0.30
5e
Modelc
a Values are the means of at least two independent experiments
b A549 cells without any stimulation
c TGF-β1 stimulated A549 cells without any compound
d Used as positive control.
2.2.4 Binding mode analysis
To further elucidate the binding mode, molecular docking of the potent compound
5e was performed in the binding site cavity of hDHODH. Crystal structure of
hDHODH (PDB ID: 1D3G) [17] and the target compound was pretreated by
Discovery Studio 3.0. All docking runs were utilizing CDOCKER protocol of
Discovery Studio 3.0 systems. The image files were generated by the PyMOL (The
PyMOL Molecular Graphics System, Version 1.4.1. Schrödinger, LLC).
Fig. 4. Docking mode of compound 5e with hDHODH (PDB ID:1D3G) a representation of the
overlap of docking model of 5e (blue) with brequinar (brown) in the binding site cavity of
hDHODH. For clarity, only interacting residues are displayed. The H-bond (Green arrows) are
displayed as dotted arrows.
Fig.4 shows the binding model overlay of the binding model of compound 5e
with brequinar. It can be observed that compound 5e adopted a brequinar-like binding
mode with matching occupancy of the hydrophobic pocket. The carboxyl group on
the thiophene amide moiety formed two hydrogen bonds with residues Gln47 and
Arg136. However, unlike brequinar, sulfur atom of thiophene moiety was founded to
reach the

small hydrophobic pocket and the hydrogen on the amide nitrogen atom formed
additional hydrogen bond with residues Thr360 which would be beneficial to increase
the binding affinity.
3. Conclusions
A previously discovered hDHODH inhibitor was further improved by
replacing the phenyl moiety by thiophene, a series of thiophene derivatives were
designed, synthesized and evaluated as hDHODH inhibitors. It led us to
discover compound 5e which displayed higher potency than teriflunomide.
Compound 5e also showed high antiproliferative activity against stimulated
PBMCs and possessed acceptable metabolic stability. Besides, compound 5e
reduced the stiffness of TGF-β1 stimulated A549 cells similar with
anti-pulmonary fibrotic drug pirfenidone. Taken together, compound 5e may
serve as a lead compound for the development of novel immunosuppressant
without inducing pulmonary fibrotic
.
Experimental section
4.1. Chemistry
Reagents and solvents were obtained from commercial sources and used without
further purification. Flash chromatography was performed using silica gel (300–400
mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). All reactions
were monitored by TLC on silica gel plates. Melting points were determined on a
Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland). The
1H and 13C NMR were recorded on Bruker ARX-400 or ARX-600 spectrometer
(Bruker Bioscience, Billerica, MA, USA) with TMS as an internal standard. Mass
spectra (MS) were measured in ESI mode on an Agilent 1100 LC-MS (Agilent, Palo
Alto, CA, USA). Elemental analysis was determined on a Carlo-Erba 1106 Elemental
analysis instrument (Carlo Erba, Milan, Italy). Propargyl intermediate (7a-7o) were
prepared following already described procedures [9-11].
4.1.1. Preparation of 3-azidothiophene-2-carboxylate methyl ester (2)
3-amino-2-thiophenecarboxylate methyl ester 1 (0.06 mol) was dissolved in
concentrated hydrochloric acid (15 mL), and ice (45 g) was added. When the mixture
was cooled to 0 °C, saturated sodium nitrite (5.2 g, 0.075 mol) aqueous solution was
added keeping the temperature below 5 °C. After 10 min, resinous sediment (if it was
formed) should be filtered. To the filtrated solution of the diazonium salt, sodium
azide (3.9 g; 0.06 mol) in 5 mL of water was added dropwise. The solution was left
for 30 min at room temperature and the azide was filtered as a white solid which was
1
used without subsequent cleaning. Yield: 80% (8.7g) m.p.:170.6-172.3°C. H NMR
(400 MHz, DMSO-d6) δH 7.95 (d, J = 5.4 Hz, 1H), 7.17 (d, J = 5.4 Hz, 1H), 3.79 (s,
3H).13C NMR (100 MHz, DMSO-d6) δC 161.15, 142.21, 133.45, 123.74, 116.78,
52.51. ESI-MS m/z: 206.3 [M+Na]+.
4.1.2. Preparation of 3-azidothiophene-2-carboxylic acid (
3)
Intermediate 2 (8.0 g, 0.043 mol) and NaOH (5.1 g, 0.13 mol) were added to a

solution of EtOH and H2O (80 mL, EtOH:H2O = 5:1). The mixture was stirred at 90°
C for 2h. Upon cooling to room temperature, ethanol was removed under vacuum, and
then H2O (40mL) was added to the mixture. The solution was adjusted to pH 2-3 with
3N HCl, and then the resulting precipitate was filtered and dried under reduced
1
pressure to afford a brown solid. Yield: 82% (5.9g) m.p.:137.2-139.1°C. H NMR
(400 MHz, DMSO-d6) δH 13.16 (s, 1H), 7.88 (d, J = 5.4 Hz, 1H), 7.10 (d, J = 5.4 Hz,
1H).13C NMR (100 MHz, DMSO-d6) δC 162.25, 141.34, 132.50, 123.87,112.64.
ESI-MS m/z: 168.2 [M-H]-.
4.1.3. General procedure for preparation of 3-azidothiophene-2-carboxamide
(4a-4b)
At room temperature, SOCl2 (20 mL) and 2 drops of DMF were added to
intermediate 3 (4g, 30.6 mmol) and the mixture was refluxed for 4 h. The excess
SOCl2 was evaporated. Then, 25% NH3.H2O (20 mL) was added to the residue at 0 ℃
and the mixture was stirred for 2-3h. A white precipitate was isolated by filtration.
The precipitate was washed with water and dried in vacuo to afford
2-azidobenzamide.
4.1.3.1. 3-azidothiophene-2-carboxamide (4a)
Yield: 72% (2.8 g), pale yellow solid, m.p.:151.3-152.4°C. H NMR (400 MHz,
1
DMSO-d6) δH: 7.83 (d, J = 5.4 Hz, 1H), 7.70 (s, 1H), 7.24 (d, J = 5.4 Hz, 1H), 7.15 (s,
1H); 13C NMR (100 MHz, DMSO-d6) δc 162.01, 136.56, 131.29, 123.80, 122.13;
ESI-MS m/z: 169.2 [M+H]+.
4.1.3.2. 3-azido-N-cyclopropylthiophene-2-carboxamide (4b)
Yield: 76% (3.7 g), yellow solid, m.p.:137.5-139.3°C. 1H NMR (600 MHz,
DMSO-d6) δH: 7.82 (d, J = 5.4 Hz, 1H), 7.66 (s, 1H), 7.21 (d, J = 5.4 Hz, 1H), 2.78
13
(m, 1H), 0.73 – 0.69 (m, 2H), 0.61 – 0.58 (m, 2H); C NMR (100 MHz, DMSO-d6)
δc 161.62, 136.06, 130.82, 123.61, 122.20, 23.34, 6.58; ESI-MS m/z: 209.3 [M+H]+ .
4.1.4. General procedure for preparation of the target compounds. (2a, 3a,4c,
5a-5i, 6a-6f)
A mixture of the appropriate azido derivative (0.2g, 1.2 mmol), the appropriate
alkyne derivative (1.4 mmol), copper sulphate (0.1 mmol) and sodium ascorbate (0.5
mmol) in 4 mL tBuOH/H2O (1:1) were stirred at room temperature for 3-4h. The
reaction mixture was diluted with water and the precipitate was collected by filtration.
After being washed with cold water (2 × 5 mL), the precipitate was dried under
vacuum. when necessary, they were purified by chromatograpy.
4.1.4.1.3-(4-(((4-(trifluoromethyl)phenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)
thiophene-2-carboxylate methyl ester (2a)
Yield: 77% (0.32g), White solid, M.p.: 140.2-141.8°C. H NMR (600 MHz,
1
DMSO-d6) δH: 8.51 (s, 1H), 8.10 (d, J = 5.3 Hz, 1H), 7.48 (d, J = 5.3 Hz, 1H), 7.38 (d,
J = 8.7 Hz, 2H), 6.96 (t, J = 5.9 Hz, 1H), 6.79 (d, J = 8.7 Hz, 2H), 4.46 (d, J = 5.9 Hz,
2H), 3.68 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δc: 156.74, 147.91, 141.19,
134.39, 129.46, 123.43, 122.85, 122.81, 122.12, 119.59, 119.57, 108.39, 49.02, 34.41;

ESI-MS m/z: 383.3 [M+H]+. Anal. calcd. For C16H13F3N4O2S (%):C, 50.26; H, 3.43;
N, 14.65. Found (%): C, 50.23; H, 3.47; N, 14.59.
4.1.4.2.3-(4-(((4-(trifluoromethyl)phenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)
thiophene-2-carboxylic acid (3a)
Yield: 68% (0.29 g), White solid, M.p.: 189.1-191.0°C. H NMR (600 MHz,
1
DMSO-d6) δH: 13.54 (s, 1H), 8.53 (s, 1H), 8.02 (d, J = 5.3 Hz, 1H), 7.44 (d, J = 5.3
Hz, 1H), 7.38 (d, J = 8.5 Hz, 2H), 6.94 (t, J = 5.4 Hz, 1H), 6.78 (d, J = 8.5 Hz, 2H),
4.44 (d, J = 5.4 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δc: 161.54, 151.75, 144.74,
137.65, 132.39, 127.22, 126.65, 126.61, 126.05, 124.87, 116.10, 112.15, 38.25;
ESI-MS m/z: 367.0 [M-H]-. Anal. calcd. For C15H11F3N4O2S (%):C, 48.91; H, 3.01; N,
15.21. Found (%): C, 48.95; H, 2.97; N, 15.23.
4.1.4.3.3-(4-(((4-(trifluoromethyl)phenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)
thiophene-2-carboxamide (4c)
Yield: 75% (0.33 g), White solid, M.p.: 182.7-184.1°C. H NMR (600 MHz,
1
DMSO-d6) δH: 8.49 (s, 1H), 7.88 (d, J = 5.3 Hz, 1H), 7.84 (s, 1H), 7.69 (s, 1H), 7.41
(d, J = 5.3 Hz, 1H), 7.39 (d, J = 8.6 Hz, 2H), 6.93 (t, J = 5.7 Hz, 1H), 6.79 (d, J = 8.6
Hz, 2H), 4.44 (d, J = 5.7 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δc: 161.97,
151.79, 145.33, 133.99, 129.12, 129.05, 126.67, 126.64, 125.71, 125.16, 112.16,
99.99, 38.26; ESI-MS m/z: 390.3 [M+Na]+. Anal. calcd. For C15H12F3N5OS (%):C,
49.04; H, 3.29; N, 19.06. Found (%): C, 49.01; H,3.27; N, 19.09.
4.1.4.4.3-(4-((4-fluorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxami
de (5a)
1
Yield: 81% (0.30 g), White solid, M.p.: 136.5-137.9°C. H NMR (600 MHz,
DMSO-d6) δH: 8.68 (s, 1H), 7.90 (d, J = 5.3 Hz, 1H), 7.86 (s, 1H), 7.68 (s, 1H), 7.44
(d, J = 5.3 Hz, 1H), 7.18 – 7.14 (m, 2H), 7.13 – 7.09 (m, 2H), 5.21 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δc: 161.92, 158.37, 154.85, 142.93, 134.05, 129.46, 128.96,
126.83, 125.87, 116.58, 116.35, 61.91; ESI-MS m/z: 341.5 [M+Na]+. Anal. calcd. For
C14H11FN4O2S (%):C, 52.82; H, 3.48; N, 17.60. Found (%): C, 52.85; H, 3.46; N,
17.63.
4.1.4.5.3-(4-((3-fluorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxami
de (5b)
1
Yield: 84% (0.32 g), Brown solid, M.p.: 168.1-169.2°C. H NMR (600 MHz,
DMSO-d6) δH: 8.69 (s, 1H), 7.89 (d, J = 5.3 Hz, 1H), 7.86 (s, 1H), 7.67 (s, 1H), 7.44
(d, J = 5.3 Hz, 1H), 7.34 (dd, J = 15.5, 8.1 Hz, 1H), 6.99 (m, 1H), 6.92 (m, 1H), 6.80
13
(m, 1H), 5.24 (s, 2H); C NMR (100 MHz, DMSO-d6) δc: 161.90, 159.97, 142.59,
134.07, 131.25, 131.15, 129.44, 128.93, 126.98, 125.88, 111.62, 108.04, 102.78,
61.69; ESI-MS m/z: 341.3 [M+Na]+. Anal. calcd. For C14H11FN4O2S (%):C, 52.82; H,
3.48; N, 17.60. Found (%): C,52.83; H, 3.50; N, 17.58.
4.1.4.6.3-(4-((4-bromophenoxy)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxami
de (5c)

1
Yield: 85% (0.38 g), White solid, M.p.: 134.2-134.9°C. H NMR (600 MHz,
DMSO-d6) δH: 8.68 (s, 1H), 7.89 (d, J = 5.3 Hz, 1H), 7.85 (s, 1H), 7.66 (s, 1H), 7.48
(d, J = 9.0 Hz, 2H), 7.43 (d, J = 5.3 Hz, 1H), 7.06 (d, J = 9.0 Hz, 2H), 5.22 (s, 2H);
13C NMR (100 MHz, DMSO-d6) δc: 161.90, 157.81, 142.69, 134.06, 132.65, 129.46,
128.95, 126.94, 125.87, 117.57, 112.86, 61.59; ESI-MS m/z: 401.4 [M+Na]+. Anal.
calcd. For C14H11BrN4O2S (%):C, 44.34; H, 2.92; N, 14.77. Found (%): C, 44.31; H,
2.93; N, 14.79.
4.1.4.7.
3-(4-((4-cyanophenoxy)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxamid
e
(5d)
1
Yield: 91% (0.35 g), Brown solid, M.p.: 190.3-192.1 °C. H NMR (600 MHz,
DMSO-d6) δH: 8.71 (s, 1H), 7.89 (d, J = 5.1 Hz, 1H), 7.85 (s, 1H), 7.81 (d, J =
8.5 Hz, 2H), 7.66 (s, 1H), 7.43 (d, J = 5.1 Hz, 1H), 7.27 (d, J = 8.5 Hz, 2H),
13
5.34 (s, 2H); C NMR (100 MHz, DMSO-d6) δc: 161.93, 161.89, 142.20, 134.70,
134.08, 129.47, 128.95, 127.20, 125.89, 119.55, 116.34, 103.73, 61.71; ESI-MS m/z:
326.4 [M+H]+. Anal. calcd. For C15H11N5O2S (%):C, 55.38; H, 3.41; N, 21.53. Found
(%): C, 55.41; H, 3.38; N, 21.55.
4.1.4.8.
3-(4-((4-(trifluoromethyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxa
mide (5e)
1
Yield: 86% (0.38 g), Yellow solid, M.p.: 147.9-149.2 °C. H NMR (600 MHz,
DMSO-d6) δH: 8.71 (s, 1H), 7.90 (d, J = 5.2 Hz, 1H), 7.86 (s, 1H), 7.69 (d, J = 8.4 Hz,
3H), 7.44 (d, J = 5.2 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 5.33 (s, 2H);13C NMR (100
MHz, DMSO-d6) δc: 161.34, 160.77, 141.89, 133.51, 128.89, 128.38, 126.91, 126.87,
126.52, 125.32, 121.56, 115.19, 61.08; ESI-MS m/z: 369.5 [M+H]+. Anal. calcd. For
C15H11F3N4O2S (%):C, 48.91; H, 3.01; N, 15.21. Found (%): C, 48.87; H, 3.04; N,
15.19.
4.1.4.9.
N-cyclopropyl-3-(4-((4-(trifluoromethyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)thiop
hene-2-carboxamide (5f)
1
Yield: 87% (0.42 g), Yellow solid, M.p.: 146.3-148.5 °C. H NMR (600 MHz,
DMSO-d6) δH: 8.65 (s, 1H), 8.50 (d, J = 3.1 Hz, 1H), 7.88 (d, J = 5.3 Hz, 1H), 7.69 (d,
J = 8.7 Hz, 2H), 7.45 (d, J = 5.3 Hz, 1H), 7.28 (d, J = 8.7 Hz, 2H), 5.33 (s, 2H), 2.71
13
(m, 1H), 0.65 – 0.61 (m, 2H), 0.46 – 0.42 (m, 2H); C NMR (100 MHz, DMSO-d6)
δc: 161.60, 161.31, 142.45, 133.82, 129.05, 128.70, 127.47, 127.44, 126.70, 125.25,
121.96, 115.75, 61.61, 23.37, 6.10; ESI-MS m/z: 409.5[M+H]+. Anal. calcd. For
C18H15F3N4O2S (%):C, 52.94; H, 3.70; N, 13.72. Found (%): C, 52.95; H, 3.72; N,
13.69.
4.1.4.10.
3-(4-((3-(trifluoromethyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxa
mide (5g)

1
Yield: 78% (0.34 g), White solid, M.p.: 142.7-144.3°C. H NMR (600 MHz,
DMSO-d6) δH: 8.71 (s, 1H), 7.91 (d, J = 5.3 Hz, 1H), 7.87 (s, 1H), 7.68 (s, 1H), 7.57
(t, J = 7.9 Hz, 1H), 7.45 (d, J = 5.3 Hz, 1H), 7.42 (m, 2H), 7.34 (d, J = 7.9 Hz, 1H),
5.34 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δc: 161.91, 158.81, 142.56, 134.07,
131.24, 130.67, 129.43, 128.95, 127.02, 125.87, 123.11, 119.53, 118.00, 111.92,
61.76; ESI-MS m/z: 369.5[M+H]+. Anal. calcd. For C15H11F3N4O2S (%):C, 48.91;
H,3.01; N, 15.21. Found (%): C, 48.93; H, 3.02; N,15.24.
4.1.4.11.
3-(4-((3,4-difluorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxamide
(5h)
1
Yield: 82% (0.33 g), White solid, M.p.: 145.8-147.6°C. H NMR (600 MHz,
DMSO-d6) δH: 8.68 (s, 1H), 7.89 (d, J = 5.3 Hz, 1H), 7.85 (s, 1H), 7.66 (s, 1H), 7.43
(d, J = 5.3 Hz, 1H), 7.38 (dd, J = 19.8, 9.4 Hz, 1H), 7.25 (m, 1H), 6.91 (m, 1H), 5.22
(s, 2H).; 13C NMR (100 MHz, DMSO-d6) δc: 161.33, 154.59, 154.51, 141.89, 133.51,
128.88, 128.37, 126.45, 125.32, 117.51, 111.07, 111.00, 104.40, 61.60; ESI-MS m/z:
337.4 [M+H]+. Anal. calcd. For C14H10F2N4O2S (%):C, 50.00; H, 3.00; N, 16.66.
Found (%): C,50.03; H, 3.01; N, 16.64.
4.1.4.12.
3-(4-((2-bromo-4-fluorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxa
mide (5i)
1
Yield: 73% (0.34g), White solid, M.p.: 147.9-149.5°C. H NMR (600 MHz,
DMSO-d6) δH: 8.69 (s, 1H), 7.89 (d, J = 5.3 Hz, 1H), 7.86 (s, 1H), 7.68 (s, 1H), 7.57
(dd, J = 8.1, 3.1 Hz, 1H), 7.45 (d, J = 5.3 Hz, 1H), 7.40 (dd, J = 9.1, 4.9 Hz, 1H), 7.26
13
(m, 1H), 5.30 (s, 2H); C NMR (100 MHz, DMSO-d6) δc: 161.37, 156.11, 151.07,
141.98, 133.46, 128.74, 128.37, 126.41, 125.26, 119.88, 115.29, 115.09, 111.36,
62.51; ESI-MS m/z: 397.4 [M+H]+. Anal. calcd. For C14H10BrFN4O2S (%):C, 42.33;
H, 2.54; N, 14.10. Found (%): C,42.31; H, 2.55; N, 14.12
4.1.4.13.
3-(4-((naphthalen-2-yloxy)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxamide
(5j)
1
Yield: 76% (0.32g), White solid, M.p.: 161.1-162.8°C. H NMR (600 MHz,
DMSO-d6) δH: 8.74 (s, 1H), 7.90 (d, J = 5.2 Hz, 1H), 7.85 (m, 4H), 7.68 (s, 1H), 7.56
(d, J = 1.5 Hz, 1H), 7.50 – 7.46 (m, 1H), 7.45 (d, J = 5.2 Hz, 1H), 7.37 (t, J = 7.1 Hz,
1H), 7.23 (m, 1H), 5.34 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δc: 161.93, 156.42,
142.93, 134.68, 134.08, 129.87, 129.47, 129.12, 128.96, 128.00, 127.26, 126.92,
126.92,125.89, 124.23, 119.12, 107.75, 61.45; ESI-MS m/z: 373.5 [M+Na]+. Anal.
calcd. For C18H14N4O2S (%):C, 61.70; H, 4.03; N, 15.99. Found (%): C,61.73; H,4.02;
N, 15.97

4.1.4.14.
3-(4-(((4-fluorophenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxamide
(6a)
1
Yield: 89% (0.33g), Yellow solid, M.p.: 154.1-155.3°C. H NMR (400 MHz,
DMSO-d6) δH: 8.46 (s, 1H), 7.88 (d, J = 5.3 Hz, 1H), 7.84 (s, 1H), 7.69 (s, 1H), 7.41
(d, J = 5.3 Hz, 1H), 6.93 (t, J = 8.9 Hz, 2H), 6.67 (dd, J = 8.8, 4.5 Hz, 2H), 6.05 (brs,
13
1H), 4.35 (d, J = 4.4 Hz, 2H); C NMR (100 MHz, DMSO-d6) δc: 161.97, 156.17,
153.87, 145.80, 133.96, 129.14, 129.07, 125.66, 125.07, 115.67, 113.68, 39.39;
ESI-MS m/z: 340.4 [M+Na]+. Anal. calcd. For C14H12FN5OS (%):C, 52.99; H, 3.81;
N, 22.07. Found (%): C,52.31; H,3.78; N, 22.08
4.1.4.15.
3-(4-(((4-bromophenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxamid
e (6b)
1
Yield: 85% (0.38g), Brown solid, M.p.: 157.7-158.5°C. H NMR (600 MHz,
DMSO-d6) δH: 8.46 (s, 1H), 7.88 (d, J = 5.2 Hz, 1H), 7.84 (s, 1H), 7.69 (s, 1H), 7.41
(d, J = 5.2 Hz, 1H), 7.21 (d, J = 8.5 Hz, 2H), 6.65 (d, J = 8.5 Hz, 2H), 6.38 (brs, 1H),
4.36 (d, J = 5.1 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δc: 161.98, 148.03, 145.74,
133.97, 131.82, 129.14, 129.07, 125.69, 125.10, 114.81, 107.28, 38.70; ESI-MS m/z:
400.3[M+Na]+. Anal. calcd. For C14H12BrN5OS (%):C, 44.46; H, 3.20; N, 18.52.
Found (%): C,44.45; H, 3.23; N, 18.54.
4.1.4.16.
3-(4-(((4-methoxyphenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxam
ide (6c)
1
Yield: 78% (0.30g), Dark yellow solid, M.p.: 185.1-186.7°C. H NMR (600 MHz,
DMSO-d6) δH: 8.44 (s, 1H), 7.88 (d, J = 5.3 Hz, 1H), 7.85 (s, 1H), 7.69 (s, 1H), 7.41
(d, J = 5.3 Hz, 1H), 6.73 (d, J = 8.8 Hz, 2H), 6.64 (d, J = 8.8Hz, 2H), 5.64 (brs, 1H),
4.33 (s, 2H), 3.64 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δc: 161.98, 151.57,
151.56, 133.98, 129.13, 129.09, 125.65, 124.98, 115.01, 114.10, 114.08, 55.76, 40.65;
ESI-MS m/z: 330.3 [M+H]+. Anal. calcd. For C15H15N5O2S (%):C, 54.70; H, 4.59; N,
21.26. Found (%): C,54.73; H, 4.61; N, 21.23.
4.1.4.17.N-cyclopropyl-3-(4-(((4-(trifluoromethyl)phenyl)amino)methyl)-1H-1,2,3-tri
azol-1-yl)thiophene-2-carboxamide (6d)
1
Yield: 83% (0.40g), Yellow solid, M.p.: 160.1-162.3°C. H NMR (600 MHz,
DMSO-d6) δH: 8.51 (d, J = 2.0 Hz, 1H), 8.40 (s, 1H), 7.85 (d, J = 5.2 Hz, 1H), 7.41 (d,
J = 5.2 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 6.93 (t, J = 5.7 Hz, 1H), 6.78 (d, J = 8.4 Hz,
13
2H), 4.44 (d, J = 5.7 Hz, 2H), 2.69 (m, 1H), 0.61 (m, 2H), 0.41 (m, 2H); C NMR
(100 MHz, DMSO-d6) δc: 161.12, 151.15, 144.85, 133.19, 128.08, 127.94, 126.09,
126.06, 124.44, 124.07, 115.60, 111.62, 37.66, 22.80, 5.53; ESI-MS m/z:
430.5[M+Na]+. Anal. calcd. For C18H16F3N5OS (%):C, 53.07; H, 3.96; N, 17.19.
Found (%): C,53.06; H, 3.98; N, 17.21.

4.1.4.18.
3-(4-(((3,4-difluorophenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-carboxa
mide (6e)
1
Yield: 81% (0.32 g), Pale yellow solid, M.p.: 174.5-175.6°C. H NMR (600 MHz,
DMSO-d6) δH: 8.53 (s, 1H), 7.94 (d, J = 5.3 Hz, 1H), 7.90 (s, 1H), 7.74 (s, 1H), 7.46
(d, J = 5.3 Hz, 1H), 7.17 (dd, J = 19.9, 9.0 Hz, 1H), 6.72 (m, 1H), 6.51 (d, J = 9.0 Hz,
1H), 6.42 (t, J = 5.8 Hz, 1H), 4.40 (d, J = 5.8 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6) δc: 161.97, 150.41, 146.45, 145.52, 141.75, 133.99, 129.13, 129.04,
125.69, 125.11, 117.82, 108.46, 100.87, 39.03; ESI-MS m/z: 334.5 [M-H]- Anal.
calcd. For C14H11F2N5OS (%):C, 50.15; H, 3.31; N, 20.89. Found (%): C, 50.12; H,
3.34; N, 20.92
4.1.4.19.
3-(4-(((3-chloro-4-fluorophenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)thiophene-2-car
boxamide (6f)
1
Yield: 71% (0.29 g), Yellow solid, M.p.: 151.5-152.5°C. H NMR (600 MHz,
DMSO-d6) δH: 8.47 (s, 1H), 7.89 (d, J = 5.3 Hz, 1H), 7.85 (s, 1H), 7.68 (s, 1H), 7.41
(d, J = 5.3 Hz, 1H), 7.12 (t, J = 9.1 Hz, 1H), 6.81 (m, 1H), 6.66 – 6.63 (m, 1H), 6.36 (t,
J = 5.8 Hz, 1H), 4.36 (d, J = 5.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δc: 161.97,
149.73, 146.39, 145.53, 133.98, 129.11, 129.04, 125.68, 125.10, 119.83, 117.33,
113.16, 112.51, 38.99; ESI-MS m/z:: 352.4 [M+H]+. Anal. calcd. For C14H11ClFN5OS
(%):C,47.80; H,3.15; N, 19.91 Found (%): C,47.78; H, 3.16 N, 19.93.
4.2. hDHODH inhibition assay
Coenzyme Q10 (CoQ10), dichlorophenolindophenol (DCIP) and L-dihydro
orotate (L-DHO) were obtained from Sigma Aldrich. teriflunomide were used as
reference compound, which was obtained from Sigma Aldrich. Stock solution of
synthesized compounds was retained in DMSO. Recombinant human DHODH was
purchased from Prospec Bio (ENZ-642). Inhibitory activity was assessed by
monitoring the reduction of 2,6-dichloroindophenol (DCIP), which is associated with
oxidation of dihydroorotate catalyzed by the DHODH enzyme. The enzyme was
pre-incubated for five minutes at 37 ℃ in Tris-buffer solution (pH 8.0) with coenzyme
Q10 (100 μM), tested compounds at different concentrations (0.04-10 μM), and DCIP
(50 μM). The reaction was initiated by addition of DHO (500 μM) and absorbance of
each
well
was
measured
at
600
nm.
Percent
inhibition
=
(max-conversion)/(max-min)*100.“max” stands for high control; “min” stands for
low control. an IC50 value was calculated using GraphPad Prism software. Values are
means ± SD of two independent experiments.
4.3. Immunosuppression assay
PBMCs were isolated by Ficoll/Isopaque (Lymphoprep) density gradient
centrifugation of buffy coat leukopheresis residues from fresh blood samples from
healthy donors. Purified cells were grown and maintained in culture medium at 37 ℃
in 5% CO2 humidified atmosphere. Cell proliferation was assessed by CellTiter-Glo®

assay. Cells were seeded in a white-opaque 96-well plate and exposed to increasing
concentrations (0.8-200 μM) of each compound or vehicle (DMSO) for 2 h and then
stimulated with 1 μg/mL anti-CD3 and 2 μg/mL anti-CD28 for 72 h. IC50 values were
determined using nonlinear regression plots with GraphPad Prism software. Values
are means ± SD of two independent experiments.
4.4 Microsomal metabolic stability assay
4.4.1 Solutions preparation
The stock solutions of test article and positive control were prepared at a
concentration of 10 mM using DMSO as diluents. All stock solutions were then
diluted to working concentrations at 0.25 mM with 70% acetonitrile. The cofactor
used in this study was NADPH regenerating system, that was composed of 6.5 mM
NADP, 16.5 mM G-6-P, 3 U/mL G-6-PD.The quench reagent was consisted of
acetonitrile containing tolbutamide and propanolol (serve as internal standard).The
buffer used in this study was 100 mM, phosphate buffer with 3.3 mM MgCl2.
Incubation mixtures containing 0.5 mg/mL liver microsomal protein and 1 μM test
article/positive control in 100 mM potassium phosphate buffer.
4.4.2 Assay procedure
The 0-minute samples were prepared by addition of an 80 µL aliquot of each
incubation mixture to 300 µL quench reagent to precipitate proteins. The samples
were vortexed, and then a 20 µL aliquot of the NADPH regenerating system was
added in. The reaction was initiated by addition of 80 µL of the NADPH regenerating
system to 320 µL of each incubation mixture. The final incubation conditions
achieved in 400 µL are: 0.5 mg/mL microsomal protein, 1 µM test article/positive
control, 1.3 mM NADP, 3.3 mM glucose 6 phosphate, 0.6 U/mL glucose 6 phosphate
dehydrogenase. The mixtures were incubated in a 37°C water bath with gentle
shaking. A 100 µL aliquot of each mixture was removed at 10, 30, 90 minutes to a
clean 96-well plate which contains 300 µL quench reagent to precipitate proteins, and
centrifuged (4000×g, 15 min).80 µL of supernatant are taken into 96-well assay plates
pre-added with 160 µL ultrapure water, and then analyzed by LC-MS/MS.
4.5 Anti-pulmonary fibrotic assay
4.5.1 Cell culture and treatment
The human alveolar epithelial cell line A549 was purchased from the Type
Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells
were cultured in RPMI-1640 (Hyclone, Logan, UT) containing 10% fetal bovine
serum (Hyclone), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma,St. Louis,
MO) in an incubator at 37 ℃ with 5% CO2. Cells were rendered quiescent by serum
free medium for 24 h, and then pretreated with for 3 h before simulation with 5 ng/ml
TGF-β1 for another 72 h. A549 cells simulated with PBS only were taken as the
control.

4.5.2 AFM imaging
Morphological and ultrastructural assays were performed using a Nanoscope VI
Dimension 3100 AFM (Veeco Company, Santa Barbara, CA) and oxide-sharpened
Si3N4 tips (MLCT, radius 10-40 nm; Bruker Company, Santa Barbara, CA). Cells
were seeded in 70 mm culture disk. For living cell imaging, the triangular cantilever
with a normal spring constant 0.01 N/m was employed. The spring constant was
calibrated with a Thermal Tune Adapter (Veeco Company). The probe was localized
onto the cell surface with the assistance of a CCD camera. The experiments were
performed at contact mode. The scan force was 50 pN and the scan rate was 0.3 Hz.
4.5.3. Measurement of cellular stiffness by AFM
The stiffness of cells was also detected at contact mode. Measurements were
conducted above the nucleus region of the cell to avoid the influence of the
underlying substrate. 100 Force-distance curves were obtained from 10 different cells
in each group at the ramp rate of 0.5 Hz. Force curves were obtained at the same
loading rate and were analyzed by Matlab 7.6.0.
4.6. Molecular Docking
The molecular modelling studies were performed with Accelrys Discovery
Studio 3.0. The crystal structure of hDHODH complexed with brequinar (PDB code:
1D3G) was obtained from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/).
Compound 5e was drawn with Chemdraw and fully minimized using the CHARMm
force field. Before docking, the crystal structure of the protein underwent an
optimization process using the Protein Preparation Wizard tool, implemented in
Discovery Studio 3.0. Missing hydrogen atoms were added and bond orders were
assigned while missing loops and residues not belonging to the binding site were
capped by adding ACE (N-acetyl) and NMA (N-methylamide) groups. Then, ORO,
nonstructural water molecules and impurities (such as solvent molecules) were
removed and the co-crystalized ligand brequinar was maintained. In the docking
process, the protein protocol was prepared by several operations, including
standardization of atom names and insertion of missing atoms in residues. Then, the
receptor model was typed with the CHARMm force field and a binding sphere with
radius of 9.0 Å was defined through the original ligand (hydroxylated azole) as the
binding site. Finally, they were docked into the binding site using the CDOCKER
with the default settings. And figures were prepared using PyMOL (The PyMOL
Molecular Graphics System, Version 1.4.1. Schrödinger, LLC).
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgements
We thank the Progarm for Innovation Research Team of the Ministry of
Education and Program for Liaoning Innovative Research Team in University

(IR1073) and Shenzhen Science and Technology innovation project
(JCYJ20170413113448742) for financial support.
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
A novel series of thiophene derivatives were designed, synthesized and
evaluated as hDHODH inhibitors
 Compared with teriflunomide, compound 5e displayed better potency against
hDHODH with IC50 0.9 μM.

Compound 5e also showed high antiproliferative activity against stimulated
PBMCs and possessed acceptable metabolic stability

Compound 5e reduced the stiffness of TGF-β1 stimulated A549 cells similar
with anti-pulmonary fibrotic drug pirfenidone.