Discovery of triazolo [1,5-a] pyridine derivatives as novel JAK1/2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127225

Small molecule JAK inhibitors have been demonstrated efficacy in rheumatoid arthritis, inflammatory bowel disease, and psoriasis with the approval of several drugs. Aiming to develop potent JAK1/2 inhibitors, two series of triazolo [1,5-a] pyridine derivatives were designed and synthesized by various strategies. The pharmacological results identified the optimized compounds J-4 and J-6, which exerted high potency against JAK1/2, and selectivity over JAK3 in enzyme assays. Furthermore, J-4 and J-6 effectively suppressed proliferation of JAK1/2 high-expression BaF3 cells accompanied with acceptable metabolic stability in liver microsomes. Therefore, J-4 and J-6 might serve as promising JAK1/2 inhibitors for further investigation.

Full text of DOI:10.1016/j.bmcl.2020.127225

Journal Pre-proofs
Discovery of triazolo [1,5-a] pyridine derivatives as novel JAK1/2 inhibitors
Kuan Lu, Weibin Wu, Cunlong Zhang, Zijian Liu, Boren Xiao, Zigao Yuan,
Anqi Li, Dawei Chen, Xin Zhai, Yuyang Jiang
PII:
S0960-894X(20)30325-5
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127225
BMCL 127225
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Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
31 March 2020
25 April 2020
27 April 2020
Please cite this article as: Lu, K., Wu, W., Zhang, C., Liu, Z., Xiao, B., Yuan, Z., Li, A., Chen, D., Zhai, X.,
Jiang, Y., Discovery of triazolo [1,5-a] pyridine derivatives as novel JAK1/2 inhibitors, Bioorganic & Medicinal
Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127225
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Discovery of triazolo [1,5-a] pyridine derivatives as novel
JAK1/2 inhibitors
a,b,1
b,c,1
b,c
b
f
b
Kuan Lu , Weibin Wu , Cunlong Zhang , Zijian Liu , Boren Xiao , Zigao Yuan ,
Anqi Li , Dawei Chen , Xin Zhai *, Yuyang Jianga,d,e,*
f
b
a,
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 Joint Key State Laboratory of Tumor Chemogenomics, Tsinghua Shenzhen International
Graduate School, Tsinghua University, Shenzhen 518055, PR China
e School of pharmaceutical sciences, Tsinghua University, Beijing 100084, PR China
f Department of Chemistry, Tsinghua University, Beijing 100084, PR China
*
Corresponding author.
E-mail address: jiangyy@sz.tsinghua.edu.cn (Yuyang Jiang);
zhaixin_syphu@126.com (Xin Zhai).
Abstract
Small molecule JAK inhibitors have been demonstrated efficacy in rheumatoid
arthritis, inflammatory bowel disease, and psoriasis with the approval of several drugs.
Aiming to develop potent JAK1/2 inhibitors, two series of triazolo [1,5-a] pyridine
derivatives were designed and synthesized by various strategies. The pharmacological
results identified the optimized compounds J-4 and J-6, which exerted high potency
against JAK1/2, and selectivity over JAK3 in enzyme assays. Furthermore, J-4 and J-
6
effectively suppressed proliferation of JAK1/2 high-expression BaF3 cells
accompanied with acceptable metabolic stability in liver microsomes. Therefore, J-4
and J-6 might serve as promising JAK1/2 inhibitors for further investigation.
Keywords: JAK1/2 inhibitors; Anti-inflammatory; Triazolo [1,5-a] pyridine
derivatives; Drug design
Autoimmune diseases are a pathophysiological state wherein immune responses are
directed against and damage the body's own tissues or organs including rheumatoid
arthritis (RA), inflammatory bowel disease etc. Dysregulation of cytokine signal
pathway plays a critical role in the occurrence and development of these diseases. It is
well documented that janus kinase-signal transducer and activator of transcription
(JAK-STAT) pathway mediates the signal of numerous cytokines and growth factors
involved in the regulation of immunity, inflammation, and hematopoiesis [1]. JAKs are
a family of cytoplasmic protein tyrosine kinases including JAK1, JAK2, JAK3, and
TYK2. Upon binding to the cytokine, its receptor units dimerize or polymerize which
leads to the phosphorylation and activation of JAKs. Subsequently, the corresponding

receptor is phosphorylated and activated JAKs recruit and phosphorylate STAT
proteins which translocate to the nucleus and modulate gene transcription [2]. The
overexpression and mutation of JAKs in this pathway will initiate severe symptoms
including autoimmune diseases and hematological disorders [3]. To date, diverse JAK
inhibitors have been developed for the treatment of immune-related disorders (Figure
1
). Tofacitinib 1 is the first approved JAK inhibitor for treating rheumatoid arthritis.
However, tofacitinib is a pan-JAK inhibitor, systemic administration of tofacitinib may
cause serious adverse events such as infection and thrombocytopenia [4]. Based on
significant therapeutic benefit, ruxolitinib 2 and barictinib 3, potent JAK1/2 inhibitors
have been approved for myelofibrosis and rheumatoid arthritis respectively with low
therapeutic dose and slight side effects [5]. Recently, selective JAK1 inhibitor
upadacitinib 4 has been approved by FDA for the treatment of medium or severe RA.
Notably, filgotinib 5, selective JAK1 inhibitor, is reported to be successful in Phase III
studies for RA and atopic dermatitis respectively. In addition, in the Phase II clinical
treatment of crohn's disease, filgotinib showed significant efficacy and acceptable
safety profile [6].
O
O
S
NC
N
N
NC
N
N
N
NC
N
N
O
N
N
N
N
N
N
N
N
H
H
H
1
Tofactinib
Approved
2 Ruxolitinib
Approved
3 Barictinib
Approved
O
S
O
N
N
O
N
N
F
F
H
O
F
N
N
N
N
N
N
N
H
H
4
Upadactinib
Approved
5 Filgotinib
Phase III
Figure 1. Chemical structures of representative JAK inhibitors
However, adverse effects were reported in filgotinib’s toxicological tests in rat and
dog models [7]. Due to the moderate activity of filgotinib in vivo, it is necessary to
increase the drug dose (100-200 mg) to achieve the expected potency [6]. By analysing
the SAR of filgotinib, it was found that its triazolopyridine and exocyclic NH were
crucial for maintain the activity. The substitution of cyclopropanecarbonyl group with
other acyl or alkyl group led to the decline of the activities. Besides, the phenyl moiety
has little effect on the activity, while the property and size of the terminal polar groups
have significant effect on the activity and selectivity. As show in Figure 2, the terminal
thiomorpholine dioxide group was modified by diverse polar motifs with CN or F atom
(series I) to further increase the potency and selectivity of the compounds. On the other
hand, series II were designed by replacing the phenyl moiety of filgotinib with
unsaturated piperidine to investigate the effect of flexibility on the activities and
selectivity.

Figure 2. Design strategy for the target compounds
Herein, we describe the identification and characterization of a novel
triazolopyridine series as JAK1/2 inhibitors. Synthetic method and detailed enzymatic
and cell-based studies of the target compounds were presented and discussed. The
results showed that compounds J-4 and J-6 exhibited high potency against JAK1/2 in
the enzymatic and cell-based assay accompanied with acceptable metabolic stability.
The synthetic procedure for the target compounds is described in Scheme 1 and
Scheme 2. Commercially available 2-amino-hydroxylaino-6-bromo-pyridine was
condensed with ethoxycarbonyl isothiocyanate and then reacted with hydroxylamine to
afford j-2. Next, intermediate j-2 was acylated with cyclopropanecarboxyl chloride to
yield j-3, followed by a Suzuki reaction to yield j-4 and j-5 [8]. The target compounds
J-1-J-5 were yielded by condensation reaction between j-4 and corresponding amines.
J-6 was synthesised by Baylis-Hillman reaction between j-5 and acrylonitrile at room
temperature. Furthermore, N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol
ester was coupled via a Suzuki reaction with j-3 to provide j-6 followed by removing
the Boc protecting group to yield j-7 (Scheme 2). Compounds J-7 and J-10-J-13 were
accessible using different functionalization of carboxylic acids by condensation
reaction with j-7. Using preformed imidazole-1-carboxamides under weak alkaline
conditions with j-7 provided compounds J-8 and J-9 [9].

Br
H
N
Br
N
NH2
Br
N
O
NH
O
N
N
b
N
a
NH2
S
O
j-2
j-1
R
Br
c
N
d
O
N
N
N
N
H
N
N
N
H
j-3
j-4 (R= CO2H)
j-5 (R= CHO)
R1
O
O
O
R1
=
e or f
N
CN
N
CF3
N
F
F
O
H
H
N
N
J-1
J-2
J-3
OH
N
N
H
O
O
CN
N
N
Series I J-1-J-6
F
F
CN
J-4
J-5
J-6
Scheme 1. Reagents and conditions: (a) ethoxycarbonyl isothiocyanate, DCM, rt; (b) hydroxylamine
hydrochloride, DIPEA, EtOH/MeOH, reflux; (c) Cyclopropanecarbonyl chloride, Et N, MeCN; (d) Ar-
, 1,4-dioxane/water, 90°C; (e) HNR R , HATU, DIPEA, DMF, rt; (f)
3
2
3
2 2 3
BPin, Pd(dppf)Cl , K CO
2
Acrylonitrile, DABCO, MeCN/H O, rt.
R'
N
R''
N
Br
O
O
O
N
N
N
N
N
N
N
N
i or j
N
g
N
N
H
H
N
H
j-3
j-6 R' = Boc
j-7 R' = H
Series II J-7-J-13
h
O
O
O
R'' =
CN
O
N
CN
CN
N
CF3
CF3
H
H
J-7
J-8
J-9
O
O
O
F
F
J-12
J-13
J-10
J-11
Scheme 2. Reagents and conditions: (g) N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester,
4
Pd(dppf)Cl
2
, K
2
CO
3
, 1,4-dioxane/water, 100°C; (h) 1,4-dioxane/HCl, rt; (i) R CO
2
H, HATU, DIPEA,
DMF, rt ; (j) N-substituted-1H-imidazole-1-carboxamide, TEA, EtOH, 60°C for J-8/9.
The enzymatic potency assay was conducted using a Caliper assay with the ATP
concentration at the Km for each of the JAK targets. As shown in Table 1, the steric
1
effect and property of R groups had an impact on activities and selectivity of series I
against JAKs. Compared to filgotinib, J-1 and J-2 showed similar selectivity with
relatively low potency against JAK-1/2. Through further modification, J-3-J-5 were

obtained by the incorporation of cyclic amine with CN or di-F group. Interestingly, the
potency of the compounds with substituted azetidine (J-3, J-4) were improved 2-5 folds
(
J-3, J-4 vs J-1, J-2). J-4 exhibited high potency against JAK1/2 (IC = 61 nM and
50
7
9nM) and showed good selectivity over JAK3 (JAK 3/1 ratio = 10.8). However, J-5
with 4,4-difluoropiperidine moiety was inactive (JAK1 IC = 393 nM) which may be
5
0
due to its steric effect. To further increase the inhibitory activity of the compound, J-6
with α-hydroxy acrylonitrile moiety was designed to increase polarity interaction with
the P-loop of JAKs. As we expected, J-6 had increased potency for all JAK isoforms.
J-6 effectively inhibited the activities of JAK1 and JAK2 with the value of IC 95 nM
5
0
and 55 nM respectively and 3-5 folds selectivity against JAK3.
Table 1. Enzymatic Potencies for series I compounds against JAK1, JAK2, and JAK3
R1
O
N
N
NH
N
Series I
compound
R1
JAK1 IC50
JAK2 IC50
(nM)a
JAK3 IC50
(nM)a
(
nM)a
H
N
J-1
J-2
J-3
286
299
1120
CN
O
O
H
N
230
124
214
139
1015
941
CF3
F
F
N
N
O
O
CN
J-4
J-5
61
79
659
923
F
F
393
401
N
O
J-6
95
55
74
285
623
CN
OH
Filgotinibb
115
a Experiments were performed in duplicate
b Used as positive control
Then, we investigated the conversion of phenyl moiety of filgotinib to
tetrahydropyridine and the effect of R’’ group on the activities of the compounds. As
shown in Table 2, Compared with J-1 and J-2, compounds J-7 and J-8 with similar
terminal cyano group displayed similar potency against JAK1/2. Subsequently, the
introduction of CF or difluorocyclopropyl at R’’ group showed a decreased potency
3
against all the three isoforms (J-9, J-10 vs J-7, J-8). The substitution of R’’ group by
acryloyl group led to a significant decline of activities against JAKs (JAK1, JAK2 and
JAK3 IC = 877, 424 and 2767 nM, respectively). Besides, compounds with CN or
5
0

CF substituted cyclopropyl group obtained low potency (J-12 and J-13). The results
3
indicated that the introduction of tetrahydropyridine could maintain the activities of the
compounds (J-7, J-8 vs J-1, J-9 vs J-2) and suitable polar functional group was crucial
for high potency and selectivity.
Table 2. Enzymatic Potencies for series II compounds against JAK1, JAK2, and JAK3
R''
N
O
N
N
NH
N
Series II
，
，
compound
J-7
R
JAK1 IC50
JAK2 IC50
(nM)a
JAK3 IC50
(nM)a
(
nM)a
193
157
1640
CN
O
H
N
J-8
170
234
283
877
175
347
192
424
441
317
74
983
1740
2409
2767
1547
5649
623
CN
O
O
H
N
J-9
CF3
F
F
J-10
O
J-11
O
J-12
1123
861
CN
O
J-13
CF3
O
Filgotinibb
115
a Experiments were performed in duplicate
b Used as positive control
Based on the enzymatic assays, compounds J-4 and J-6 were selected for further
study the anti-proliferation activities on BaF3-JAKs cells. As shown in Table 3,
similarly to filgotinib, J-4 exhibited high potency against BaF3-JAK1 cells (IC = 2.59
5
0
μM) and selectivity over BaF3-JAK2 cells (JAK 2/1 ratio = 2.9) which was inactive
against JAK3. Interestingly, J-6 effectively inhibited the proliferation of BaF3-JAK1
cells and BaF3-JAK2 cells with 7 folds selectivity against BaF3-JAK3 cells.

Table 3. Antiproliferation Activities of compounds against BaF3-JAKs cells
compound BaF3-JAK1 IC50a (μM) BaF3-JAK2 IC50a (μM) BaF3-JAK3 IC50a (μM)
J-4
2.59
1.18
1.43
7.43
1.03
4.45
＞30
7.05
J-6
Filgotinibb
13.26
a Experiments were performed in duplicate
b Used as positive control
Moreover, we performed the study of J-4 and J-6 in incubation with human and rat
liver microsomes in vitro (Table 4). The studies revealed that J-4 and J-6 possessed
high T1/2 and low CL in both HLM and RLM. It demonstrated that J-4 and J-6 were
stable in human and rat liver microsomes.
Table 4. In Vitro Liver Microsome Stability
compound
HLMa
HLMa
RLMb
T1/2 (min)
550
RLMb
CL ( μL/min/mg)
6.3
T1/2 (min)
CL ( μL/min/mg)
J-4
J-6
＞1000
＜2
381
9.1
129
26.9
a human liver microsomes
b rat liver microsomes
To further elucidate the binding mode, molecular docking analysis of compound
J-4 and J-6 was performed using Molegro Virtual Docker 2010 (v4.1) (Molegro ApS,
Aarhus, Denmark). As shown in Figure 3A and 3B, J-4 and J-6 showed similar binding
mode with the ATP binding pocket of JAK1. Exocyclic NH and the triazolo nitrogen
N3 of the triazolo[1,5-a]pyridine formed two critical hydrogen bonds with Leu959
residue and phenyl ring was located in the hydrophobic pocket. azetidine-3-carbonitrile
(J-4) and hydroxymethyl-acrylonitrile (J-6) are oriented to the glycine rich loop
respectively. Figure 3C and 3D expounded the binding mode of J-4 and J-6 with
JAK2. triazolo[1,5-a]pyridine skeleton of both compounds formed two hydrogen bonds
interactions with Leu932. The hydrophobic interactions were formed between 11e and
Ala880, Leu983, Val863 and Val911. The docking analysis indicated that J-4 and J-6
possess high potency against JAK1/2 in activity assays.

Figure 3. Docking mode of compound J-4 and J-6 with JAK1 (PDB code 4P7E) and JAK2 (PDB
code 6DBN). (A) binding conformation for J-4 in the binding site of JAK1, (B) binding
conformation for J-6 in the binding site of JAK1. (C) binding conformation for J-4 in the binding
site of JAK2, (D) binding conformation for J-6 in the binding site of JAK2, The H-bond (yellow or
green) are displayed as dotted arrows.
In summary, two series of triazolo[1,5-a]pyridine derivatives were designed,
synthesized as JAK1/2 inhibitors. Most compounds exhibited moderate to high potency
against JAK1/2. In particular, compounds J-4 and J-6 showed high potency against
JAK1/2 and selectivity over JAK3. Anti-proliferation assays showed that J-4 and J-6
could effectively inhibit the proliferation of JAK1 and JAK2 high-expression BaF3
cells. Furthermore, J-4 and J-6 exhibited preferable liver microsome stability. Overall,
J-4 and J-6 were identified as promising lead for JAK inhibitors for the treatment of
inflammatory diseases.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.
Acknowledgements
The work was financially supported by Shenzhen Sci & Tech Bureau
(JCYJ20170413113448742, JCYJ20170816170342439).
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Conflict of interest

The authors declared that they have no conflicts of interest to this work. Neither the entire paper nor
any part of its content has been published or has been accepted elsewhere.




A novel series of triazolo [1,5-a] pyridine derivatives were designed,
synthesized and evaluated as JAK1/2 inhibitors.
Compounds J-4 and J-6 displayed high potency against JAK1/2 and selectivity
over JAK3.
Compounds J-4 and J-6 effectively suppressed proliferation of BaF3-
JAK1/2 cells with selectivity over BaF3-JAK3 cells.
Compounds J-4 and J-6 possessed acceptable metabolic stability in liver
microsomes.