Design, synthesis and biological activity evaluation of novel 4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl) oxy) pyridine-2-yl) amino derivatives as potent transforming growth factor-β (TGF-β) type I receptor inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127339

TGF-β type I receptor (also known as activin-like kinase 5 or ALK5) plays a critical role in the progression of fibrotic diseases and tumor invasiveness and metastasis, as well. The development of small inhibitors targeting ALK5 has been validated as a potential therapeutic strategy for fibrotic diseases and cancer. Here, we developed various 4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl) oxy) pyridine-2-yl) amino derivatives as ALK5 inhibitors. The optimization led to identification of potent and selective ALK5 inhibitors 12r. The compound 12r exhibited strong inhibitory activity both in vitro and in vivo, and pharmacokinetics study showed an oral bioavailability of 57.6percent. Thus, compound 12r may provide as new therapeutic option as ALK5 TGF-βR1 inhibitor.

Full text of DOI:10.1016/j.bmcl.2020.127339

Journal Pre-proofs
Design, synthesis and biological activity evaluation of novel 4-((1-cycloprop‐
yl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl) oxy) pyridine-2-yl) amino
derivatives as potent transforming growth factor-β (TGF-β) type I receptor in‐
hibitors
Bin Tan, Xiaomeng Zhang, Xu Quan, Guochuang Zheng, Xue Li, Liwen
Zhao, Wei Li, Bo Li
PII:
S0960-894X(20)30450-9
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127339
BMCL 127339
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
24 March 2020
18 May 2020
6 June 2020
Please cite this article as: Tan, B., Zhang, X., Quan, X., Zheng, G., Li, X., Zhao, L., Li, W., Li, B., Design,
synthesis and biological activity evaluation of novel 4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-
pyrazol-4-yl) oxy) pyridine-2-yl) amino derivatives as potent transforming growth factor-β (TGF-β) type I
receptor inhibitors, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2020.127339
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Design, synthesis and biological activity evaluation of novel 4-((1-cyclopropyl-3-
(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl) oxy) pyridine-2-yl) amino
derivatives as potent transforming growth factor-β (TGF-β) type I receptor
inhibitors
Bin Tana,b,d, Xiaomeng Zhangc,d, Xu Quana, Guochuang Zhenga, Xue Lia, Liwen Zhaoa,*,
Wei Li c,*, Bo Li b,*
a R &D Center, Nanjing Sanhome Pharmaceutical Co. Ltd. , Nanjing, Jiangsu, 211135,
China
b Department of Pharmaceutical Analysis, School of Pharmacy, China Pharmaceutical
University, Nanjing, Jiangsu, 210009, China
c
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China; ;
Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources
Industrialization, Nanjing, Jiangsu, China; Jiangsu Key Laboratory for Functional
Substances of Chinese Medicine Stake Key Laboratory Cultivation Base for TCM
Quality and Efficacy School of Pharmacy, Nanjing University of Chinese Medicine,
Nanjing, Jiangsu, 210000, China
d These authors contributed to this work equally
* Corresponding authors
E-mail addresses: zhaolw@sanhome.com (Liwen Zhao), liwaii@126.com (Wei Li),
libo@cpu.edu.cn (Bo Li)
Abstract:
TGF-β type I receptor (also known as activin-like kinase 5 or ALK5) plays a
critical role in the progression of fibrotic diseases and tumor invasiveness and
metastasis, as well. The development of small inhibitors targeting ALK5 has been
validated as a potential therapeutic strategy for fibrotic diseases and cancer. Here, we
1

developed various 4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)
oxy) pyridine-2-yl) amino derivatives as ALK5 inhibitors. The optimization led to
identification of potent and selective ALK5 inhibitors 12r. The compound 12r
exhibited strong inhibitory activity both in vitro and in vivo, and pharmacokinetics
study showed an oral bioavailability of 57.6%. Thus, compound 12r may provide as
new therapeutic option as ALK5 TGF-βR1 inhibitor.
Keywords: TGF-β type I receptor; ALK5 inhibitors; inhibitory activity
TGF-β as a multifunctional growth factor, plays important role in regulating a
wide range of cellular processes, including cell growth, differentiation, migration,
adhesion, apoptosis, epithelial-mesenchymal transition (EMT), extracellular matrix
[3]
(ECM) remodeling [1, 2]. TGF-β belongs to the TGF-β superfamily , and the TGF-β
family members initiate intracellular downstream signaling by binding with cellular
surface serine/threonine kinase receptors. It is categorized into three subfamilies: TGF-
βRI, which is also known as activin receptor-like kinase 5 (ALK5), TGF-βRIIand TGF-
βRIII [4]. The TGF-βRIII is an auxiliary co-receptor and not directly involved in signal
transduction. Its main function is to increase the binding of TGF-β and TGF-βR on the
cell surface and deliver it to type I and type II receptors. The TGF-β/Smad signaling
pathway is the main pathway for TGF-β to exert biological effects. The signal
transduction of TGF-β pathway is rely on several signaling factors including
microenvironment, microRNAs (miRNAs), TGF-β family ligands, TGF-β
serine/threonine kinase receptors (ALK5 and TGFβRII), phosphorylation and
dephosphorylation kinases, ubiquitin ligase and deubiquitinase (Smads) [6]. TGF-β
signals through the binding of the TGF-β ligand to TGF-βRII receptor, which then
phosphorylates the GS domain (a highly conserved glycine and serine residue domain)
of ALK5. The activated ALK5 can trigger several signaling pathways by
[7]
phosphorylating downstream signaling molecules such as the Smad2 and Smad3
,
which form Smad2/3/4 heteromeric complex with another cytoplasmic transducer,
Smad4. The complex is translocated to nucleus and binds to a region called Smad-
binding element of TGF-β target genes through specific DNA-binding cofactors to
2

regulate the expression of TGFβ-downstream target genes, thereby regulating cell
proliferation, differentiation, migration, apoptosis, and extracellular matrix production
[8]. The overactivation of TGF-β/Smad signaling has been involved in various cancer
such as breast cancer [9] and Hepatocellular Carcinoma (HCC) [10]. Therefore, the TGF-
β signaling pathway seems to be a potential therapeutic target, especially the direct
inhibition of ALK5 could be a good approach for the treatment of cancer.
Recently, a variety of small molecule ALK5 inhibitors has been developed such
as LY-2157299 [11], EW-7197 [12], LY-3200882 [13], SD-208 [14], SB-525334 [15], A83-
01 [16] and GW788388 [17] (Figure 1) , which are able to specifically inhibit the TGF-
β/Smad signaling pathway by occupying the bind site of TGF-βRI to intervene the
binding of TGF-β and TGF-βRI. Among these inhibitors, LY-2157299, EW-7197 and
LY-3200882 are currently under clinical evaluation (Figure 1). LY-2157299 is a potent,
selective TGF-βRI inhibitor, and granted the orphan drug qualification of HCC in 2013.
LY-3200882, as a follow-up inhibitor of TGF-βRI, has improved inhibitory activity
and selectivity than LY-2157299, and it is initiated Phase 1 clinical investigation.
Figure 1 Example of ALK5 inhibitors under development
N
O
N
F
N
N
NH2
N
NH
OH
NH
O
O
N
N
NH
N
N
N
N
N
N
LY-2157299
EW-7197
LY-3200882
N
F
NH
N
N
N
NH
N
Cl
N
O
N
N
O
N
N
NH
N
N
NH
NH
N
N
N
S
N
SD-208
SB-525334
A83-01
GW788388
In order to rationalize the design idea (Figure 2), the binding mode of LY-
3200882 with TGF-βRI was studied. It was found that N-cyclopropyl-1H-pyrazole
showed a strong hydrogen bond interaction through a water bridge, which was critical
to maintain the binding efficacy. In addition, a hydrogen bond formed by hydroxyl
moiety with Asp-290. The structural modification in this study was to replace the
3

hydroxyl substituted pyridine of LY-3200882 with various substituted aryl, aromatic
and aliphatic heterocyclic derivatives, and the structure activity relationships of these
compounds was discussed, as well. We evaluated the novel TGF-βRI inhibitors for their
in vitro and in vivo activity, pharmacokinetic profile and preliminary toxicity. We
provided the novel TGF-βRI inhibitor for further investigation of study and a promising
clinical candidate.
Figure 2 Design of ALK5 inhibitors
water bridge
H-bond interactions
O
O
N
N
N
N
O
N
SAR analysis
O
N
N
activity improvement
solvent
channel
R1
N
N
HO
H
H
LY-3200882
H-bond interactions
O
O
O
OH
b, c
a
O
N
Br
d
+
O
O
Cl
O
N
HN
N
Cl
N
Cl
2
1
3
4
O
O
N
O
N
f
e
Cl
N
O
N
O
NH2
O
N
N
N
N
N
N
7
5
6
Scheme 1 Synthesis of intermediates 5 and 7. Reagents and conditions: (a) K2CO3,
DMF, 90 °C; (b) N, N-dimethylformamide dimethyl acetal, 100 °C; (c) AcOH,
hydrazine hydrate, r.t.; (d) 2,2'-bipyridine, Cu(OAc)2, Na2CO3, cyclopropylboronic
acid, 1,2-dichloroethane, 80 °C; (e) diphenylmethanimine, Pd(OAc)2, BINAP, Cs2CO3,
toluene, 95 °C; (f) HCl/1,4-dioxane, r.t..
The intermediates 5 and 7 were synthesized as shown in scheme 1. The
substitution reaction of pyridine 1 with brominated ketone 2 gave compound 3, which
4

was cyclized to obtain pyrazole derivative 4. Compound 4 was reacted with
cyclopropylboronic acid catalyzed by Cu(OAc)2 to afford intermediate 5, which was
further transformed to 7 via the palladium catalyzed coupling, followed by the acidic
deprotection.
O
N
N
R1
R1
12a
,
=
N
H2N
OH
O
8a c
-
N
O
N
R1
=
12b
,
a or b
N
N
R1
R1
=
12c
,
N
H
O
12a c
-
O
Boc
O
O
O
N
R2
=
12g
14
,
N
N
N
H2N
OH
N
9
N
N
O
N
CO2Et
O
N
O
N
R2
,
=
R3
O
N
Boc
R2
f
d or e
c
N
N
N
CO2H
R3
=
12h
,
N
Cl
N
N
O
H
H
H
13
12h
N
N
12g, 14
R4
O
O
O
O
5
H2N
, R4= -CN, R5
=
N
OH
N
N
N
N
N
12k
O
N
10a-b
O
N
O
N
O
i
h
g
R4
R4
O
R4
12l, R4= -F, R5
=
N
OH
R5
O
OH
N
N
, R4= -F, R5
=
N
N
H
12m
N
O
H
H
O
O
, R4= -CN
, R4= -F
, R4= -CN
, R4= -F
14a
14b
15a
15b
12k-m
R6
O
HN R7
H2N
S
O
O
N
11n-r
N
, R6= -H, R7= -H
, R6= -H, R7= -Me
, R6= -H, R7= -Et
, R6= -Me, R7= -H
12n
O
N
12o
12p
12q
12r
R6
HN R7
S
O
j
N
H
, R6= -F, R7= -H
O
12n-r
Scheme 2 Preparation of 12a-c, 12g-h and 12k-r. Reagents and conditions: (a)
Pd(OAc)2, XantPhos, Cs2CO3, 1,4-dioxane, 100 °C for 12a; (b) Pd2(dba)3, XantPhos,
NaOPh, 1, 4-dioxane, 100 °C for 12b and 12c; (c) Pd2(dba)3, XantPhos, t-BuONa, 1, 4-
dioxane, 100 °C; (d) HCl, 1,4-dioxane, r.t. then HATU, DIPEA, DCM, r.t. for 12g; (e)
HCl, 1,4-dioxane, r.t. then K2CO3, MeCN, reflux for 14; (f) NaOH, THF, H2O, r.t.; (g)
Pd2(dba)3, XantPhos, NaOPh, 1, 4-dioxane, 100 °C; (h) MeOH, H2O, LiOH, r.t.; (i)
HATU, DIPEA, DCM, r.t.; (j) Pd2(dba)3, XantPhos, NaOPh, 1, 4-dioxane, 100 °C.
Compound 5 was reacted with various amines via the palladium catalyzed
coupling reaction to give corresponding target compounds 12a-c and 12n-r, as well as
intermediates 13 and 14a-b. The Boc-group in compound 13 was removed under acidic
condition and then reacted with nucleophiles to obtain 12g and 14, and the 14 was
further hydrolyzed at LiOH/H2O/MeOH system to 12h. The cyanide and fluorine
derivatives 14a and 14b were processed with hydrolysis and coupling with
corresponding amines to obtain amide 12k-m.
5

O
N
R8=
R8=
R8
N
N
12d
Br
,
N
15d-f
a or b
N
O
N
H
N
12e
,
,
N
O
O
N
R8
H
R9=
N
CO2H
12f
12d-f
N
N
CN
O
O
R10
OH
Br
N
NH2
N
N
7
17i-j
N
R10= -H
12i
O
,
c
CN
R10= -Me
12j
,
R10
OH
N
N
H
N
12i-j
Scheme 3 Preparation of 12d-f and 12i-j. Reagents and conditions: (a) Pd2(dba)3,
XantPhos, Cs2CO3, 1, 4-dioxane, 100 °C for 12d and 12e; (b) Pd(OAc)2, Cs2CO3,
XantPhos, 1, 4-dioxane, 100 °C for 12f; (c) Pd2(dba)3, XantPhos, Cs2CO3, 1, 4-dioxane,
100 °C;
Compounds 15d-f were coupled with compound 7 via Buchwald-Hartwig reaction
to afford 12d-e and 16, and ester 16 was hydrolyzed under the basic condition to obtain
acid 12f. Compound 12i and 12j were synthesized from 7 with 17i-j via Buchwald-
Hartwig reaction, as well.
As shown in table 1, pyrazole derivative with propanol substitution 12a gave the
ALK5 inhibitory activity with IC50 = 77 nM, while compunds 12b and 12c substitution
resulted in a great increase of inhibitory activity. The pyridine derivatives 12d and 12e
displayed more potent ALK5 inhibitory activity which gave IC50 13 and 17 nM,
respectively. However, the carboxylic substitution (Table 1, 12f) lead to significant
decreased potency in the kinase assay. In addition, replacing the pyrazole and pyridine
ring with piperidine (Table 1, 12g and 12h) resulted in the complete loss of the activity.
Next, a series of phenyl substituted compounds were evaluated. Compound 12i and 12j
were able to maintain the high efficacy with the value of IC50 13 and 14 nM, while their
amide analogues 12k-12m displayed the reduction in potency. Phenylsulfonamide
6

derivatives exhibited highly inhibitory activity of ALK5. Phenyl sulfonamide 12n gave
the value of IC50 9.1 nM, and the substitution on either nitrogen or phenyl ring
possessed high potency as well (Table 1, 12o to 12r). The inhibitory activities of those
compounds were further verified in the cell-based luciferase inhibition assay using
NIH3T3 cell. In general, the ALK5 inhibitory activity of the compounds were well
represented in cellular assay with only several exceptions. Compound 12d possessed
good inhibitory activity in both ALK5 enzyme and NIH3T3 cell-based assays. The
methyl substituted phenlysulfonamides 12o and 12q exhibited 2-fold increase in
inhibitory activity over 12n, and fluorine-substituted compound 12r showed the
improved activity as well.
Table 1. Inhibitory activity of compounds 12 in vitro
IC50 (nM)a
Entry Compound
NIH3T3 cells
Luciferase
ALK5
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
77(22)
21(35)
32(21)
13(35)
17(21)
234(26)
>10000
>10000
13(16)
14(50)
211(41)
122(46)
114(36)
9.1(46)
15(37)
12(37)
16(38)
28(45)
215.2 (89.3)
128.3 (80.0)
338.0 (135.9)
54.9 (135.9)
133.4 (135.9)
N.D
N.D
N.D
N.D
85.9 (76.3)
N.D
N.D
N.D
167.7 (100.6)
92.7 (126.8)
N.D
69.5 (126.8)
126.4 (118.0)
a Data in parentheses were IC50 of the LY-3200882 tested in parallel
N.D: Not detected
7

To investigate the pharmacokinetic profiles, the sulfonamide compounds 12n, 12o,
12q and 12r were administered to BALB/C mice (three male and three female mice for
each compound) both oral gavage and intravenous injection in dosing of 10 mg/kg and
1 mg/kg, respectively. Compound 12n was favorable with oral dosing and had a half-
life of about 1.4 hours in vivo. The maximum concentration was 1508 ng/ml with 68.9%
of bioavailability. The compound 12o which showed good inhibitory activity in vitro,
gave only 359 ng/ml of the Cmax and short half-life (T1/2 = 0.3 h). Although compound
12q had similar value of Cmax and bioavailability compared with 12n, its relatively short
T1/2 was inappropriate for the further investigation of inhibitory activity in vivo.
Compound 12r had a superior PK profile, which exhibited over 3-fold improvement in
both Cmax and AUC with good bioavailability (F% = 74.3), and the half-life of the 12r
in plasma was 1.2 h.
Table 2. The results of PK for 12n,12o, 12q and 12r.
12n
1.4
12o
0.3
12q
0.5
12r
1.2
T1/2 (h)
Cmax (ng/ml)
1508
359
1127
5134
PO
(10 mg/kg)
AUC (0-∞)
(ng × h /ml)
2189
229
1244
7703
F (%)
T1/2 (h)
68.9
0.5
14.3
0.2
51.3
0.2
74.3
0.4
C0 (ng/ml)
549
562
797
3355
IV
(1 mg/kg)
AUC (0-∞)
(ng × h /ml)
338
3.1
143
7.1
232
4.4
1061
1.0
CL (L/h/kg)
Considering the good inhibitory activity in vitro and pharmacokinetic profile,
compound 12r was evaluated in vivo activity with BALB/C mice bearing xenograft
tumors derived from H22 hepatocarcinoma cell. Compound 12r was administrated at
30 mg/kg and 60 mg/kg twice daily by oral gavage, and the LY-3200882 was tested at
60 mg/kg and 120 mg/kg twice daily as well. Treatment with both 12r and LY-3200882
8

significantly reduced the tumor growth in comparison with the control group (Figure
3), and no significant loss of weight was observed (Figure 4). The compound 12r with
30 mg/kg and 60 mg/kg cohorts gave the 30% and 27% T/C value, while LY-3200882
gave 36% T/C value with dosing 120 mg/kg. The PK-PD analysis in treatment day 1
and day 19 were performed (Table 3). Dose-dependent exposal of the compound 12r
was observed, which gave 8907 h × ng/ml and 21045 h × ng/ml in the first treatment.
However, after the 19 days of administration, the concentration of 12r was decreased
to 5933 and 8870 h × ng/ml.
Figure 3 Tumor volume change of the mice in LY-3200882 and 12r treatment of H22
s.c xenografts
2000
Vehicle Control
1500
1000
500
0
LY-3200882 60 mg/kg p.o BID
LY-3200882 120 mg/kg p.o BID
12r 30 mg/kg p.o BID
12r 60 mg/kg p.o BID
0
5
10
15
20
Treatment (Day)
Figure 4 Mice body weight change in LY-3200882 and 12r treatment of H22 s.c
xenografts
26
24
Vehicle Control
22
LY-3200882 60 mg/kg p.o BID
LY-3200882 120 mg/kg p.o BID
20
12r 30 mg/kg p.o BID
18
12r 60 mg/kg p.o BID
16
0
5
10
15
20
Treatment (Day)
9

Table 3 PK-PD analysis of the LY-3200882 and 12r
AUC（h × ng/ml）
Dosing
(mg/kg)
Compound
LY-3200882
12r
T/C (%)
DAY1
9949
DAY19
1658
60
120
30
57
36
30
27
20232
8907
4864
5933
60
21045
8870
Compound 12r showed no inhibitory activity to hERG channel (IC50 > 30μM) and
the mini-Ames experiment indicated that no potential mutagenicity was observed as
well. In order to confirm the selectivity of the 12r to ALK5 over other kinases, a broad
spectrum of kinases was tested against compound 12r. The compound was treated in
single dose duplicate model at a concentration 10 μM. Among the 374 tested kinases,
ALK1, 2 and 4 which were the isoforms of ALK5 remained 2-29% enzyme activity
after treatment with 12r, none of the other kinases showed obvious inhibition with less
than 30% enzyme activity remaining (see Supplementary Material for details).
In order to rationalize the observed activities of these compounds, binding mode
of compound 12r and LY-3200882 in the binding pocket of TGF-β RI (PDB code:
2WOU) were studied (Figure 5). As displayed by the docking model, the pyrazole of
12r formed strong hydrogen bond with Glu-245 and Asp-351 through a water bridge,
which was an essential interaction to maintain inhibitory activity. The replacement of
the tertiary alcohol in LY-3200882 with sulfonamide in 12r could keep the key
hydrogen bonding interaction through bind with Asp-290.
Figure 5 Docking mode of LY-3200882 (pink) and compound 12r (green).
10

We have synthesized a series of 4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-
1H-pyrazol-4-yl) oxy) pyridine-2-yl) amino derivatives, which were evaluated as TGF-
βR1 inhibitors. The structure-activity relationship in this series of compounds were
discussed. Optimization of the structure led to the eventual identification of a potent
and selective TGF-β R1 inhibitor 12r. The introduction of phenlysulfonamides not only
improved the inhibitory activity but also increased the oral bioavailability. Compound
12r exhibited good enzyme inhibitory activity (IC50 = 28 nM), and luciferase inhibition
assay of 12r in NIH3T3 cells demonstrated that 12r had high inhibition activity which
IC50 was 126.4 nM, as well. Pharmacokinetics study with 12r gave an oral
bioavailability of 74.3% with high AUC of 7703 h × ng/mL and Cmax of 5134 ng/mL.
The in vivo study of 12r with H22 xenograft model showed 27% of T/C in dosing of
60mg/kg B.I.D. Based on these results, compound 12r was currently undergoing with
toxicology study as a promising candidate for clinical investigation.
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Declaration of interests
☒ 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.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
12

O
N
N
ALK5 IC50=28 nM
O
N
T/C=30%(30 mg/kg), 27%(60 mg/kg)
F
S
%
F=74.3
O
hERG IC50 > 30 uM
N
NH2
H
O
12r
13