Design, synthesis, biological evaluation and pharmacophore model analysis of novel tetrahydropyrrolo[3,4-c]pyrazol derivatives as potential TRKs inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113627

The tropomyosin receptor kinases TRKs are responsible for different tumor types which caused by NTRK gene fusion, and have been identified as a successful target for anticancer therapeutics. Herein, we report a potent and selectivity TRKs inhibitor 19m through rational drug design strategy from a micromolar potency hit 17a. Compound 19m significantly inhibits the proliferation of TRK-dependent cell lines (Km-12), while it has no inhibitory effect on TRK-independent cell lines (A549 and THLE-2). Furthermore, kinases selectivity profiling showed that in addition to TRKs, compound 19m only displayed relatively strong inhibitory activity on ALK. These data may indicate that compound 19m has a good drug safety. Partial ADME properties were evaluated in vitro and in vivo. Compound 19m exhibited a good AUC values and volume of distribution and low clearance in the pharmacokinetics experiment of rats. Finally, a pharmacophore model guided by experimental results is proposed. We hope this theoretical model can help researchers find type I TRK inhibitors more efficiently.

Full text of DOI:10.1016/j.ejmech.2021.113627

European Journal of Medicinal Chemistry 223 (2021) 113627
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis, biological evaluation and pharmacophore model
analysis of novel tetrahydropyrrolo[3,4-c]pyrazol derivatives as
potential TRKs inhibitors
Tianxiao Wu, Chu Zhang, Ruicheng Lv, Qiaohua Qin, Nian Liu, Wenbo Yin, Ruifeng Wang,
Yin Sun, Xiaoyan Wang, Yixiang Sun, Dongmei Zhao*, Maosheng Cheng
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The tropomyosin receptor kinases TRKs are responsible for different tumor types which caused by NTRK
gene fusion, and have been identified as a successful target for anticancer therapeutics. Herein, we report
a potent and selectivity TRKs inhibitor 19m through rational drug design strategy from a micromolar
potency hit 17a. Compound 19m significantly inhibits the proliferation of TRK-dependent cell lines (Km-
12), while it has no inhibitory effect on TRK-independent cell lines (A549 and THLE-2). Furthermore,
kinases selectivity profiling showed that in addition to TRKs, compound 19m only displayed relatively
strong inhibitory activity on ALK. These data may indicate that compound 19m has a good drug safety.
Partial ADME properties were evaluated in vitro and in vivo. Compound 19m exhibited a good AUC values
and volume of distribution and low clearance in the pharmacokinetics experiment of rats. Finally, a
pharmacophore model guided by experimental results is proposed. We hope this theoretical model can
help researchers find type I TRK inhibitors more efficiently.
Received 28 April 2021
Received in revised form
2 June 2021
Accepted 3 June 2021
Available online 15 June 2021
Keywords:
TRK inhibitors
Anticancer
Selectivity
Pharmacophore model
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
TRKB binds to BDNF and NT-4/5 [11], and TRKC specifically binds to
NT-3 [12]. Fusion of an NTRK might cause the loss of form or
The tropomyosin receptor kinases (TRKs), a family of three re-
ceptor tyrosine kinases (RTKs), regulates cell differentiation, pro-
liferation, survival and pain [1e5]. The three subtypes of the TRKs
are TRKA, TRKB, and TRKC, which are encoded by NTRK1, NTRK2,
and NTRK3 gene [6]. As a transmenbrane receptor proteins, TRKs
contain an extracellular domain for binding ligand, a trans-
function of the extracellular domain of the TRK protein, and ulti-
mately lead to continuous activation and overexpression [13,14].
The activation of TRKs receptors will trigger downstream signal
transduction pathways involving phosphatidylinositol 3 kinase
(PI3K)/AKT and RAS/RAF, which are considered as a pan-cancer
carcinogenic factor [15e17]. Considering that the fusion of TRKs
usually results in the loss of the extracellular domain, monoclonal
antibodies will not be able to target the mutated TRK protein.
Therefore, small molecule inhibitors may be the only key to target
TRKs to treat tumors.
A few potent TRKs inhibitors have been reported, including type
I, type II, and type III inhibitors, based on their site of binding to
TRKs. Among them, AZ-23 (1) is a pan-TRKs inhibitor with 2,4-
diaminopyrimidine core, which was active against TRKA
(IC50 ¼ 2 nM) and TRKB (IC50 ¼ 8 nM) [18]. In addition, the re-
searchers also obtained the co-crystal of AZ-23 and TRKA (PDB ID
4AOJ, 2.75 Å), revealing the binding mode of this type of inhibitors
and TRKs, laying the foundation for the follow-up study of further
works [19]. Entrectinib (2) is a multi-target kinases inhibitor which
menbrane domain and
a small intercellular domain which
comprised of approximately 70 amino acids before and 15 amino
acids after the kinase domain [7,8]. The kinase domain of the TRKs
is structurally conserved. TRKA, TRKB, and TRKC share more than
95% sequence identity in the active site where the protein interact
directly with ATP [9]. The growth factors of neurotrophin family
such as nerve growth factor (NGF), brain-derived neurotrophic
factor (BDNF) and various neurotrotrophins (NTs) can activate
TRKs, and different isoform is activated by different ligands. For
instance, TRKA mainly binds to NGF as well as NT-7/6 [10], while
* Corresponding author.
E-mail address: medchemzhao@163.com (D. Zhao).
https://doi.org/10.1016/j.ejmech.2021.113627
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
was developed by Roche and approved by the FDA on August 15,
2019. It has presented robust suppressive activity on ALK
(IC50 ¼ 12 nM), ROS1 (IC50 ¼ 7 nM), and all subtypes of TRKs (IC50
values for TRKA/B/C were 1 nM, 3 nM, and 5 nM, respectively)
[20,21]. Most recently, Ding et al. identified a series compounds
with 1H-indazole core as type I TRKs inhibitors (3). Compound 3
had activity on TRKA/B/C with IC50 values of 1.6 nM, 2.9 nM, and
2.0 nM, respectively. Moreover, 3 also exhibited excellent selec-
tivity over a large kinases panel [22]. Larotrectinib (4) was another
approved by FDA for treatment cancers containing NTRKs fusion,
regardless of cancer types [23e25]. PF-06273340 (5) is a type II
inhibitor for TRKs, which was reported by Pfizer and in Phase I
clinical trials currently. Su and his colleagues discovered 9H-fluo-
rene derivatives (6) as a type III inhibitor that bond to the juxta-
membrane (JM) domain of TRKA. Compound 6 displayed TRKA
activity and >250-fold selectivity over TRKB and TRKC (TRKA
IC50 ¼ 99 nM, TRKB IC50 > 8100 nM, TRKC IC50 ¼ 2500 nM) (Fig. 1)
[26].
(shown as different colors in Fig. 2), and aligns well with Entrec-
tinib in docking studies. The in vitro kinase assays showed that
compound 17a was weak active against TRKA (IC50 ¼ 1.24
mM).
Then, compound 7 (17h, IC50 ¼ 0.47
m
M) with stronger inhibitory
activity than 17a was discovered in hit to lead stage. Meanwhile, the
importance of the fluorine atom in contribution to activity was
realized. Therefore, we carried out the next structural modification
around the fluorine atom. With the 17l (IC50 ¼ 0.19
mM) and 17m
M) were obtained, furthermore optimization and
(IC50 ¼ 0.20
m
structure-activity relationship (SAR) study were conducted, which
led to identification of a potent and selective TRKs inhibitor 8 (19m,
IC50 ¼ 0.017
m
M). Herein, based on the above strategy, a series of
tetrahydropyrrolo[3,4-c]pyrazol
derivatives were reported
including the design, synthesis and biological evaluation of them.
And finally, a more accurate pharmacophore model was given to try
to help researchers to find selective type I TRKs inhibitors more
efficiently.
To analyze the structure of several type I inhibitors against TRKs
(Compound 1 to 3), it is not difficult to find that they have similar
structural features and binding modes: An aminopyrazole fragment
binds to the hinge of the kinases, and an aryl fragment extends into
the hydrophobic pocket adjacent to the DFG motif (see Fig.1). Based
on above analysis and with the aid of computer-aided drug design
(CADD), we constructed a ligand-based pharmacophore model of
competitive inhibitors of TRKs. Common features of this pharma-
cophore model include: One hydrogen bond donor and two
hydrogen bond acceptors form interactions with the hinge (shown
as blue in Fig. 2); A benzene or pyridine ring which is substituted by
fluorine atoms linked by the flexible chain extends towards a
hydrophic pocket composed of Asn-655, Cys-656, Gly-667, and so
on (shown as red in Fig. 2); The other two hydrophic centers form
hydrophic interactions with Phe-589, the gatekeeper residue, and
Leu-516 (shown as red in Fig. 2). Then, for the development of novel
type I TRKs inhibitors, we employed scaffold hopping strategy to
design and synthesize a tetrahydropyrolo derivative 17a, which
possesses all the aforementioned pharmacophore characteristics
2. Results and discussion
2.1. Chemistry
Target compounds 17a - 17v and 19a - 19s were synthesized in a
few steps from a key intermediate 14, a tetrahydropyrrolo[3,4-c]
pyrazol core protected with Boc. The key intermediate 14 was
prepared by a previously reported synthetic route, which was
optimized moderately [27]. The amino acid 10 was prepared by
utilizing
a
Michael addition reaction of 2-amino-2-
methylpropanoic acid (9) and acrylonitrile. The amino group of
10 was then protected as a Boc carbamate (11). Intermediate 11 was
reacted with iodomethane to yield methyl ester 12. Compound 13
was synthesized by the cyclization of 12 under basic conditions.
Finally, treatment of ketonitrile 13 with hydrazine under acetic acid
conditions, afforded the key intermediate 14.
For the synthesis of target compounds 17a - 17v, N atom located
at pyrazol ring of the key intermediate 14 was selectivity protected
by ethylcarbamoyl chloride (15), followed by the condensation
Fig. 1. Structures of potent TRK inhibitors.
2

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
Fig. 2. Design and modification strategies of novel and selective TRKs inhibitors.
reaction with benzoyl chloride obtained intermediate 16, which
could be separated by recrystallization in a moderate quantity
(>10 g) due to its smaller polarity. Then, the Boc group of 16 was
cleaved to give an amine, which subsequently nucleophilic attacked
on various substituted benzyl bromide, and finally deprotection
with DIPEA to obtain the corresponding compounds 17a - 17v for
one-pot.
Compounds 19a - 19s were synthesized according to the pro-
tocol described in the lower part of Scheme 1. Treatment of the key
intermediate 14 with different chloride prepared by corresponding
benzoic acid gave a mixture of compounds with mono-acylation
(18a - 18t) and bis-acylation, then the acylation at the pyrazol
ring was removed by basic conditions to convert the bis-acylated
compounds to mono-acylated compounds (18a - 18t). Depro-
tection of 18a - 18t, followed by substitution reactions, which was
similar to the preparation of 17a - 17v, obtained target compounds
19a - 19s.
TRKC with IC50 values of 3.4 nM, 4.7 nM, and 0.9 nM, respectively,
which was similar to previously outlined data [22].
According to the constructed pharmacophore model, we first
designed and synthesized the compound 17a (IC50 ¼ 1.24
mM) as a
hit. The docking study indicated that aminopyrazol moiety in 17a
formed three key hydrogen bonds with Glu-590 and Met-592 in
hinge region of the TRKA. The tetrahydropyrrolo ring occupying the
adenine region, and two methyls in the ring formed hydrophobic
interactions with gatekeeper Phe-589 and neighbouring residues.
The benzyl moiety located downward in a hydrophobic pocket, and
generated anion-p interactions with Asp-668 from DFG motif
(Fig. 3A).
Guided by docking study, we found that the hydrophobic cavity
adjacent to the DFG motif was not occupied completely, suggested
that introducing a suitable substituent at the meta-position of the
benzyl moiety in 17a could further occupy the cavity and might
improve the inhibitory activity of compounds (Fig. 3B). Therefore,
we synthesized and tested the inhibitory activity of analogues 17b
to 17i, and the results were shown in Table 1. Compared to com-
pound 17a, introduction of a bulky substituent (17b to 17f) at the 3-
position of the benzyl moiety resulted no significant improve in
activity, indicating that the cavity is limited in size. While analogue
2.2. Target compounds design and in vitro activity against TRKs
kinase
The in vitro inhibitory activity against TRKs of the novel syn-
thesized compounds were evaluated by using the homogeneous
time-resolved fluorescence (HTRF) assays, and Larotrectinib (4)
was involved as a comparison. Under our experimental conditions,
Larotrectinib exhibited suppressive activity on TRKA, TRKB, and
17g (IC50 ¼ 0.67
more, switching chlorine atom to fluorine atom (17h) led to further
increase in activity (IC50 ¼ 0.47 M). The potency improve of 17h
might be explained by the fluorine atom formed halogen
mM) displayed better potency than 17a. Further-
m
3

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
Scheme 1. Synthesis of compounds 17a - 17v and 19a - 19s. Reagents and conditions: (a) acrylonitrile, NaOH (aq), r.t., 24 h; (b) (Boc)2O, TMAH, TEA, DMAP, CH3CN, 40 ꢀC, 10 h; (c)
MeI, K2CO3, DMF, 40 ꢀC, 2 h; (d) NaH, dry 1,4-dioxane, reflux, 3 h; (e) NH2NH2, AcOH, EtOH, reflux, 9 h; (f) Ethylcarbamoyl chloride, DIPEA, dry THF, ꢁ40 ꢀC, 2 h; (g) PhCOCl, DIPEA,
dry DCM, 30 ꢀC, overnight; (h) i. TFA, DCM, r.t., 1 h; ii. appropriate benzyl bromide, DIPEA, MeOH, 45 ꢀC, 3 h; (i) i. appropriate chloride, DIPEA, dry DCM, ꢁ20 ꢀC, 2 h; ii. TEA, MeOH,
reflux, 2 h; (j) 1-(bromomethyl)-3,5-difluorobenzene, DIPEA, MeOH, 45 ꢀC, 8 h.
Fig. 3. Molecular docking of 17a (A and B) and 17h (C) in the ATP-binding site of TRKA. The kinase is depicted in green ribbons, and interactions are illustrated with red dashed lines.
4

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
Table 1
activity to TRKA (IC50 ¼ 0.19
mM, and 0.20 mM, respectively).
In vitro TRKA kinase inhibitory activity of the 17a to 17ia.
For further exploring the structure-activity relationships (SAR),
we tried to replace fluorine atoms with a larger methyl group
provided 17r and 17s, with decreased enzyme activity
Entry
R3
TRKA IC50
(
m
M)
Entry
R3
TRKA IC50(mM)
17a
17b
17c
17d
17e
H
1.24
1.10
1.12
0.98
1.36
17f
17g
17h(7)
17i
Larotrectinib
Br
Cl
F
NO2
e
0.94
0.67
0.47
0.60
(IC50 ¼ 0.94
fluorine atom with one or two trifluoromethyls (17u,17t) also led to
M, and 16.36 M,
mM, and 0.88 mM, respectively). Replacement of the
Me
MeO
CN
I
obvious activity loss against TRKA (IC50 ¼ 1.00
m
m
0.0034
respectively). These results might confirm that the cavity is limited
in size. Analogue 17v also exhibited significant potency loss
a
The IC50 values are shown as the mean (mM) values from two separate
experiments.
(IC50 ¼ 6.96
mM), which might be explained by adverse effects of
para-position fluorine atom. In conclude, Compound 17l and 17m
with the best potency of them were chosen for further
optimization.
interactions with Cys-656, Arg-654, and Asn-644 (Fig. 3C). Com-
pound 17h (7) with moderate potency and minor molecular weight,
was an acceptable lead compound for further optimization.
Since the performance of the fluorine atom in hit to lead stage,
we focused our attention on introducing more fluorine atoms or
changing the position of fluorine atoms on the benzyl group of the
lead compound 17i. (Table 2). The in vitro kinase assays with
analogue 17j, 17k, and 17h was showing that the analogue 17j with
ortho-position fluorine and 17h with meta-position fluorine
exhibited approximately same potency, while the 4-position of
We next focused on the Ar1 moieties by fixing benzyl moieties
with 2,3-difluorophenyl and 3,5-difluorophenyl (Table 3). In vitro
kinase assays with analogues 19a to 19g indicated that hydrophonic
substituents in Ar1 were detrimental to activity. Based on the above
results, we then chose to introduce some hydrophilic fragments on
the benzene ring (19h to 19m). All of these compounds exhibited
stronger inhibitory activity than 17l and 17m, which were preferred
compounds in last round of structural modification.
Furthermore, using the 3,5-difluorobenzyl moiety to ionize
fluorine (17k) was not tolerated (IC50 ¼ 1.18
mM). Incorporation of
analogue 19m (IC50 ¼ 0.017
mM) gave quaternary ammonium salt
another fluorine atom in benzyl of 17k afforded analogue 17o and
17p, and their potency were weaker than the lead, yet
19n (IC50 ¼ 0.095 M), which displayed measurable decreases in
m
potency. Changing the position of substituent of 19h to 19m from
para to meta provided analogues 19o to 19t, without improving
enzyme activity, yet. Finally, we selected analogues 19h, 19i, 19l,
19m, 19p and 19t because of their stronger inhibitory activity, to
test activity on the enzyme level of TRKB and TRKC (Table 4). The
results showed that all the analogues were pan-TRK inhibitor, as
their significant inhibitory effect on each subtype of TRKs.
(IC50 ¼ 0.68
mM, and 2.32 mM, respectively). The other analogues
with difluorophenyl fragment (17l, 17m, 17n, and 17q) displayed
moderate inhibitory activity. In particular, the 3,5-difluorophenyl
and 2,3-difluorophenyl at Ar1 (17l and 17m) exhibited the best
Table 2
In vitro TRKA kinase inhibitory activity of the 17j to 17va.
Entry Ar1
TRKA
Entry
Ar1
TRKA
2.3. Antiproliferative activity
IC50 (uM)
IC50 (uM)
Above six analogues (19h, 19i, 19l, 19m, 19p and 19t) were
further evaluated for in vitro anticancer screening. Their IC50 values
against Km-12 cells line which harbors TRKA rearrangement was
tested and listed in Table 5. Meanwhile, the lung cancer cells line
A549 and the liver epithelial cells line THLE-2, neither of them have
TRKA gene fusion, had also been tested to evaluate potential off-
target effects of TRKs inhibitors. As shown in Table 5, compared
with A549 and THLE-2, analogues 19h, 19i, 19l and 19m exhibited
more than 200-fold selectivity to Km-12. This means that the risk of
off-target effects of these compounds may be low. In addition, these
compounds also showed potent antiproliferative effects on Km-
12 cell lines. Among them, analogue 19m has the strongest activity
17j
0.45
1.18
0.19
0.20
17q
0.52
0.94
0.88
16.36
17k
17l
17r
17s
17t
(IC50 ¼ 0.082
mM) and selectivity (Km-12 vs A549, 882-fold; Km-12
17m
vs THLE-2, >1220-fold). As a result, analogue 19m was selected for
further research.
2.4. Effects of analogue 19m on cell apoptosis
17n
17o
17p
0.48
0.68
2.32
17u
1.00
The ability of analogue 19m to induce apoptosis of Km-12 cells
was tested by flow cytometry. Analogue 19m strongly induced
apoptosis in a concentration-dependent manner with apoptotic
17v
6.96
rates of 29.44%, 36.38% and 69.79% at concentrations of 0.0625
0.125 M and 0.25 M, respectively. When the concentration was
greater than 0.25 M, with the further increase of the administra-
mM,
m
m
m
tion concentration, the compound had no significant effects on
apoptosis (Fig. 4).
17h(7)
Larotrectinib
0.47
2.5. Effects of analogue 19m on cell cycle
e
0.0034
a
We further examined the effects of analogue 19m on the cell
The IC50 values are shown as the mean (uM) values from two separate
experiments.
cycle. With the increase in the concentration of administration, the
5

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
Table 3
Table 3 (continued )
In vitro TRKA kinase inhibitory activity of the 19a to 19sa.
Entry
Ar2
R1
H
R2
F
TRKA
IC50 (uM)
19n
0.095
Entry
Ar2
R1
F
R2
H
TRKA
IC50 (uM)
19a
0.46
0.44
0.18
19o
F
H
0.27
19b
19c
H
H
F
F
19p
H
F
0.12
19d
19e
19f
F
H
F
0.21
0.35
0.95
0.71
0.029
H
F
19q
19r
19s
F
H
F
0.42
0.27
0.21
H
F
19g
19h
H
F
H
F
H
H
19i
H
F
0.014
19t
H
F
0.15
19j
19k
19l
F
H
F
0.069
0.096
0.017
Larotrectinib
e
e
e
0.0034
a
The IC50 values are shown as the mean (mM) values from two separate
H
F
experiments.
Table 4
In vitro TRKs kinase inhibitory activity of compounds.a
H
Entry
TRKA
TRKB
TRKC
IC50 (uM)
IC50 (uM)
IC50 (uM)
19h
19i
19l
19m
19p
19t
0.029
0.014
0.017
0.017
0.12
0.035
0.027
0.023
0.028
0.17
0.010
0.0090
0.013
0.011
0.075
0.10
19m
H
F
0.017
0.15
0.19
Larotrectinib
0.0013
0.0047
0.0010
a
The IC50 values are shown as the mean (mM) values from two separate
experiments.
G1 phase ratio of the Km-12 cells line gradually increased, the S
phase ratio gradually decreased, and finally leveled off at 0.25 M,
m
6

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
Table 5
Relative IC50 (uM) values and selectivity for selected compounds against Km-12, A549 and THLE-2 cells.
Km-12 select.indexb
a
Entry
IC50
Km-12 (
mM)
A549 (
m
M)
THLE-2 (
m
M)
Km-12 vs A549
Km-12 vs THLE-2
19h
19i
19l
19m
19p
19t
0.146 0.015
0.211 0.077
0.216 0.098
0.082 0.008
>1
88.53 1.57
>100
64.18 2.14
72.29 5.06
e
49.48 1.48
606x
>474x
297x
882x
e
339x
>474x
>463x
>1220x
e
>100
>100
>100
e
>1
e
e
e
e
a
IC50: concentration of the compound (uM) producing 50% cell growth inhibition after 72 h of drug exposure, as determined by the CCK-8 assay.
A549 IC50 or THLE-2 IC50/Km-12 IC50, x ¼ fold.
b
Fig. 4. Effects of analogue 19m on Km-12 cells apoptosis.
which was compatible with the results of the apoptosis test. These
data demonstrated that analogue 19m could effectively lead to G1/
S phase arrest in Km-12 cells (Fig. 5).
19m possessed favorable plasma stability with t1/2 > 4.8 h. Liver
microsomal experiments displayed that 19m had relatively
acceptable metabolic stability. Without NADPH, the recovery rate
was 90%, which indicated that analogue 19m was metabolized
through the NADPH-dependent oxidation pathway (Table 8).
Analogue 19m was then evaluated with a panel of cytochrome
P450 isoforms (1A2, 2C9, 2C19, 2D6 and 3A4). None of them were
obviously inhibited by 19m at a concentration of 10.0 mM, indi-
cating minimal drug-drug potential with compounds cleared pre-
dominantly via these metabolic pathways (Table 9).
2.6. Kinase selectivity profile
In order to further evaluate the selectivity of this series, the
potent analogue 19m was profiled against a panel of 28 kinases at a
concentration of 1.0 mM, and the percent inhibition values were
shown in Table 6 and Fig. 6. The analogue 19m had good kinase
selectivity, with only TRKs and ALK producing greater than 80%
inhibition (red columns in Fig. 6). Four kinases were inhibited by
19m to an extent 40%e80% (yellow columns in Fig. 6), and the other
kinases were inhibited less than 40% (green columns in Fig. 6).
These above experimental results were consistent with the cell
antiproliferative test results.
2.8. In vivo pharmacokinetics properties
Then, the pharmacokinetic profile in rat for analogue 19m was
evaluated and the corresponding data were showed on Table 10.
The rats were treated with 19m at the dose of 3 mg/kg or 1 mg/kg
which depended on the different way of administration (p.o. or i.v.).
Analogue 19m exhibited moderate half-time of 1.89 0.06 h for p.o.
and 1.51 0.36 h for i.v. A good area under the curve was detected,
whether the way of administration was p.o. or. i.v. Analogue 19m
also had a low plasma clearance of 8.08 1.00 mL/min/kg and a
2.7. In vitro metabolic stability and CYPs inhibition
Next, the in vitro plasma stability and liver microsomal stability
of analogue 19m were also evaluated. As shown in Table 7, analogue
7

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
Fig. 5. Effects of analogue 19m on Km-12 cells cycle.
Table 6
Kinase selectivity profile of analogue 19ma.
Kinases
%inhibition @1.0
m
M
Kinases
%inhibition @1.0
m
M
Kinases
%inhibition @1.0
m
M
Kinases
%inhibition @1.0 mM
TRKA
TRKB
TRKC
ALK
JAK2
FGFR1
AMPK
102
100
99
82
76
FAK
BTK
FLT3
MELK
RAF1
Aurora A
ROS1
51
35
31
22
11
11
10
MEK
TBK1
cMet
EGFR
PAK4
BRAF
PAK1
8
7
6
4
4
3
3
CDK2
PRKCA
AKT1
mTOR
PLK4
3
3
2
1
0
0
0
60
53
ERK1
PIM1
Profiling Service from Life Technologies. The results represent the mean of three indepedent experiments performed in triplicate.
a
Selectivity profile of analogue 19m measured at a concentration of 1.0 mM in a panel of 28 kinase generated with the SelectScreen®.
good volume of distribution (892 150 mL/kg). The oral bioavail-
ability was 44.3%, which was an acceptable result that will be
improved in further research.
tetrahydropyrrolo scaffold with the hydrophobic side chains of
gatekeeper Phe-589. Meanwhile, the fluorine atom on benzyl motif
served as a fluorine bond donor for Asn-655 and Gly-667. In
addition, the benzyl moiety also interacts with Asp-668 of the DFG
motif via anion-p interaction, and the N-methylpiperazine frag-
2.9. Binding mode and pharmacophore model analysis
ment towards the solvent region.
Through the above analysis of the binding mode and the test
results of in vitro enzyme activity, we abstracted the structural
characteristics of the inhibitor into four pharmacophore charac-
teristics. Different from the pharmacophore model that we relied
on calculation at the beginning, the new pharmacophore model has
been revised by experimental results and thus become more reli-
able. The new pharmacophore model includes a hydrogen bond
center (C, blue in Fig. 8), two hydrophobic centers (B and D, red in
To investigate the interaction mode of analogue 19m, a docking
study of this analogue in the ATP-binding site of TRKA (PDB entry: 5
kvt) was performed. The best predicted binding mode is shown in
Fig. 7, and has a calculated binding energy of ꢁ12.55 kcal/mol. As
shown in Fig. 7, the aminopyrazole fragment forms three hydrogen
bonds to the backbone carbonyl oxygen of Glu-590, the NH and
carbonyl oxygen of Met-592 in the hinge region. Further stabili-
zation may be achieved through the hydrophobic interaction of
8

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
Fig. 6. Selectivity profile of analogue 19m measured at a concentration of 1.0
and green columns denote <40%).
m
M in a pannel of 28 kinases (red columns denote >80%, yellow columns denote between 40% and 80%,
Table 9
Table 7
Cytochrome P450 inhibition of analogue 19ma.
Plasma stability of analogue 19m.
Isozyme
CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4
mM (%) 12.9 16.0 11.5 0.0 6.4
Time Point (min)
0
10
30
60
120
Inhibition at 10
Remaining (%)
T1/2 (min)
100.0
>289.1
96.8
95.7
99.2
100.7
a
Assay were performed in pooled human liver microsomes.
3. Conclusion
Fig. 8), and a hydrophilic center (E, green in Fig. 8), the four of which
are cross-linked to form a non-strict isosceles triangle. Among
them, the hydrogen bond center C used to form hydrogen bonds
with hinge region and the hydrophobic center D with fluorine
atoms are two most critical pharmacophores, which play a key role
in maintaining the potent activity of the inhibitor. In addition, in
the study of the structure-activity relationship, we found that
introduction of a hydrophilic center E can further increase the ac-
tivity of inhibitors, while the benzene ring connected to the center
E may only serve as a backbone support. Because of this, we
replaced the hydrophobic center A corresponding to the benzoyl
group in the original pharmacophore model with the hydrophilic
center E, which is also the core difference between the new and old
pharmacophore models. We hope that this renew and more accu-
rate pharmacophore model can help researchers more efficiently
discover selective TRKs inhibitors with a novel parent scaffold. The
new pharmacophore-based drug design of TRK inhibitors are
ongoing by us, and will be reported in due course.
By analyzing the structural characteristics and binding mode of
type I TRK inhibitors, we first constructed a rough pharmacophore
model. Under the guidance of the early pharmacophore, a series of
novel tetrahydropyrrolo [3,4-c] pyrazol derivatives were obtained
using scaffold hopping strategy. One of the best compounds, 19m
(TRKA IC50
¼
0.017
M), showed potent anti-proliferative activity on Km-
M) and remarkable selectivity
m
M, TRKB IC50
¼
0.028 mM, TRKC
IC50 ¼ 0.011
m
12 cell lines (IC50 ¼ 0.082 0.008
m
between TRK-dependent cell lines and TRK-independent cell lines
(>800-fold). Compound 19m strongly induced apoptosis and G1/S
phase arrest on Km-12 cell lines. The kinase selectivity evaluation
displayed that compound 19m has good kinome selectivity. In
addition, compound 19m possesses favorable plasma stability,
moderate liver microsomal stability, and low CYPs inhibition
in vitro, and good ADME properties in vivo. Taken together, com-
pound 19m may serve as a lead compound for drug discovery to
treat cancer caused by NTRK gene fusion. Moreover, during the
research process, we delved into the structure-activity relationship
Table 8
Liver microsomal stability of analogue 19m.
Parameters
R2
T1/2
CLint(mic)
L/min/mg)
CLint(liver)
L/min/mg)
Remaining (%)
(T ¼ 60 min)
Remaining (%) (NCFa, T ¼ 60min)
(
m
(
m
In human
0.99
29.6
46.8
42.1
22.4
90.9
a
NCF: abbreviation of no co-factor. No NADPH was added to NCF samples (replaced by buffer) during the 60 min incubation.
9

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
Table 10
In vivo ADME parameters of analogue 19m.
Dose (mg/kg) Adminstration Mean value & SD T1/2 (h) Tmax (h) Cmax (ng/mL) AUC last (h*ng/mL) AUC inf (h*ng/mL) CL (mL/min/kg) MRT (h) Vss (mL/kg) F (%)
3
1
p.o.
i.v.
Mean
SD
Mean
SD
1.89
0.06
1.51
0.36
1.67
0.58
e
603
50
e
2701
244
2034
224
2887
264
2084
240
e
3.49
0.16
1.85
0.37
e
44.3
e
e
8.08
1.00
892
150
e
e
and improved the previously immature pharmacophore model
based on the experimental results. The renew pharmacophore
model will guide our research on type I TRK inhibitors more
accurately, and related follow-up research will be published later.
fluorescence F-254 and visualized with UV light. Column chroma-
tography was performed on silica gel (200e300 mesh). 1H NMR, 13
C
NMR and DEPT spectral data were recorded in DMSO‑d6 or CDCl3 on
Bruker ARX-600 NMR or Bruker ARX-400 NMR spectrometers with
TMS as an internal standard. High-resolution accurate mass spec-
trometry (HRMS) determinations for all final target compounds
were obtained on a Bruker micromass time of flight mass spec-
trometer equipped with an electrospray ionization (ESI) detector.
4. Experimental section
4.1. Chemistry
Starting materials, reagents and solvents were obtained from
commercial supplier and used without further purification unless
otherwise indicated. Anhydrous solvents were dried and stored
according to standard procedures. All reactions were monitored by
thin-layer chromatography (TLC) on silica gel plates with
4.1.1. 2-[(2-cyanoethyl)amino]2-methylpropanoic acid (10)
Sodium hydroxide (19.39 g, 0.48 mol) was slowly added at 0 ꢀC
to a 2-amino-2-methylpropanoic solution in water (100 mL), and
then acrylonitrile (34.3 mL, 0.52 mol) was added. The mixture was
stirred at room temperature for 24 h. The pH of reaction solution
Fig. 7. Molecular docking of analogue 19m in the ATP-binding site of TRKA. (A) Detailed interactions with the protein rediues. The kinase is depicted in green ribbons, and in-
teractions are illustrated with red dashed lines. (B) The two-dimensional interaction diagram showing the interaction of analogue 19m with key amino acid residues in TRKA active
site. Each of the pink dashed arrows represent hydrogen bonds.
Fig. 8. Two pharmacophore models, old (A) and renew (B). Red, blue and green balls represent hydrophobic centers, hydrogen bond centers and hydrophilic centers, respectively.
The number on the dotted line indicate the ideal distance between the two pharmacophores, and the unit is Å. The red dotted line links the two most important pharmacophores.
10

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
was adjusted to 3 with concentrated hydrochloric acid under ice
bath, and then absolute ethanol (50 mL) was added. The suspension
was stirred for an additional 15 min, and filtered under reduced
pressure to obtain a white solid in 82% yield, which was used in the
next step without additional purification.
were cooled to ꢁ40 ꢀC and then a solution of ethyl carbon-
ochloridate (11.84 mL, 0.12 mol) in anhydrous tetrahydrofuran
(300 mL) was added drop wised. After the TLC showed that ma-
jority of the starting material was converted to the product, the
tetrahydrofuran was removed, and oil residue was dissolved in
dichloromethane (300 mL). The organic layer was washed with
water (150 mL ꢂ 3), and evaporated to dryness to provide a yellow.
Then ethyl ether (80 mL) was added to the residue and the resulting
slurry was stirred at room temperature for 2 h, filtered and the fiter
cake was the target product as a white solid. Yield, 42%.
4.1.2. 2-[(tert-butoxycarbonyl)(2-cyanoethyl)amino]-2-
methylpropanoic acid (11)
To solution of 10 (62.10 g, 0.40 mol) and tetramethylammonium
hydroxide (79.27 g, 0.44 mol) in acetonitrile (500 mL) were care-
fully added the mixture of di-tert-butyl dicarbonate (216.95 g,
0.99 mol), triethylamine (138.17 mL, 0.99 mol) and 4-
dimethylaminopyridine (12.14 g, 0.10 mol) in acetonitrile
(500 mL) at 0 ꢀC. The mixture was heated to 40 ꢀC for 10 h, and then
cooled to room temperature. The solvent was removed. The oil
residue was subsequently dissolved in water (500 mL), and washed
with ethyl acetate (250 mL ꢂ 3). The pH of aqueous layer was
adjusted to 4 with citric acid, extracted with ethyl acetate (250
mL ꢂ 3), and washed with brine (250 mL ꢂ 3). The organic layer
was dried over anhydrous sodium sulfate and concentrated to give
a orange oil in 53% yield, which was used in the next step without
additional purification.
4.1.7. 5-(tert-butyl)2-ethyl3-benzamido-6,6-dimethylpyrrolo[3,4-c]
pyrazole-2,5(4H,6H)-dicarboxylate (16)
A solution of 15 (18.82 g, 0.058 mol) in anhydrous dichloro-
methane (200 mL) was added N,N-disopropylethylamine
(20.22 mL, 0.12 mol). The solution was cooled to 0 ꢀC, and then a
solution of benzoyl chloride (7.36 mL, 0.064 mol) in anhydrous
dichloromethane (100 mL) was added dropwise. The reaction
mixture was heated to 30 ꢀC for overnight. The solvent was
removed, and then recrystallized with petroleum ether to give the
pure product as a white solid. Yield, 84%. 1H NMR (600 MHz,
DMSO‑d6)
d
10.85 (s, 1H), 7.92 (d, J ¼ 7.7 Hz, 2H), 7.69 (t, J ¼ 7.4 Hz,
1H), 7.61 (t, J ¼ 6.9 Hz, 2H), 4.55 (d, J ¼ 13.6 Hz, 2H), 4.46 (q,
J ¼ 7.1 Hz, 2H), 1.62 (2s, 6H, rotamers.), 1.47 (2s, 9H, rotamers.), 1.37
(t, J ¼ 7.1 Hz, 3H).
4.1.3. Methyl 2-[(tert-butoxycarbonyl)(2-cyanoethyl)amino]-2-
methylpropanoate (12)
A well stirred mixture of 11 (54.01 g, 0.21 mol), potassium car-
bonate (87.38 g, 0.63 mol), and iodomethane (26.24 mL, 0.42 mol)
in N,N-dimethylformamide (400 mL) was heated to 40 ꢀC for 2 h.
The suspension was poured into ice saturated ammonium chloride
solution (2000 mL), extracted with ethyl acetate (500 mL ꢂ 3) and
washed with brine (500 mL ꢂ 3). The combined organic fractions
were dried over anhydrous sodium sulfate, filtered, and evaporated
to dryness to obtain crude product as a yellow oil in 94% yield,
which was used in the next step without further purification.
4.1.8. General procedure for the synthesis of target compounds 17a
- 17v
A mixture of 16 (0.20 g, 0.47 mmol) and trifluoroacetic acid
(0.50 mL) in dichloromethane (1.00 mL) were stirred at room
temperature for 1 h. The reaction mixture was evaporated to dry-
ness, redissolved in methanol (2 mL), N,N-disopropylethylamine
was added to adjust the pH of mixture to 10, then treated with
corresponding benzyl bromide (2.35 mmol), and stirred at 45 ꢀC for
an additional 3 h. The reaction was subsequently evaporated to
dryness, redissolved in ethyl acetate (70 mL), and washed with
water (35 mL ꢂ 3). The crude organic was concentrated and pu-
rified with silica chromatography (1% Methanol/Dichloromethane)
gave the title compounds 17a - 17v in moderate yield.
4.1.4. Tert-butyl 4-cyano-2,2-dimethyl-3-oxopyrrolidine-1-
carboxylate (13)
A solution of 12 (53.55 g, 0.20 mol) in 1,4-dioxane (400 mL) was
cooled to 0 ꢀC, treated with 60% sodium hydride carefully, then
heated at reflux for 3 h. The reaction mixture was cooled to room
temperature and cautiously quenched with water (9 mL). The sol-
vent was removed, and the resulting oil was redissolved in water
(200 mL). The pH of the aqueous layer was adjusted to 4 with citric
acid, extracted with ethyl acetate (200 mL ꢂ 3), washed with brine
(200 mL ꢂ 3), and concentrated to give a yellow oil in 93% yield,
which was used in the next step without further purification.
4.1.8.1. N-(5-benzyl-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]
pyrazol-3-yl)benzamide (17a). White solid. Yield: 51%. m.p.
190.4e191.1 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d 12.24 (s, 1H), 10.66
(s, 1H), 7.91 (d, J ¼ 7.7 Hz, 2H), 7.62e7.40 (m, 3H), 7.38 (t, J ¼ 5.3 Hz,
2H), 7.33 (q, J ¼ 6.8 Hz, 2H), 7.24 (q, J ¼ 6.5 Hz, 1H), 3.78 (s, 2H),
3.64e3.46 (m, 2H), 1.37 (s, 6H).13C NMR (151 MHz, DMSO‑d6)
d
164.49, 140.76, 132.14,128.82, 128.79, 128.66, 128.16, 127.16, 59.78,
4.1.5. tert-butyl-3-amino-6,6-dimethyl-3a,4,6,6a-
tetrahydropyrrolo[3,4-c]pyrazole-5(1H)-carboxylate (14)
52.39, 40.54, 24.04. Rf ¼ 0.075 (4% Methanol/Dichloromethane).
HRMS (ESI, m/z) calcd for C21H22N4O [MþH]þ, 347.1872; found
347.1893.
To solution of 13 (43.90 g, 0.18 mol) and acetic acid (84.92 mL,
1.47 mol) in absolute ethanol (500 mL) were added 80% hydrazine
hydrate (57.63 mL, 0.92 mol), then heated at reflux for 9 h. The
reaction mixture was cooled to room temperature, concentrated,
and redissolved in water (300 mL). The pH of aqueous layer was
adjusted to 9 with saturated sodium carbonate solution, extracted
with ethyl acetate (150 mL ꢂ 3), and washed with brine (150 mL ꢂ
3). The combined organic layers were dried over anhydrous so-
dium sulfate, and filtered. The filtrate was concentrated to yield a
yellow oil in 75% yield, which was used in the next step without
further purification.
4.1. 8. 2. N-[6, 6-dimethyl-5-(3-methylbenzyl)-1, 4, 5, 6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17b). White solid.
Yield: 53%. m.p. 152.1e152.9 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d
10.68 (s, 1H), 7.92 (d, J ¼ 7.7 Hz, 2H), 7.54 (s, 1H), 7.47 (d, J ¼ 8.4 Hz,
2H), 7.25e7.12 (m, 3H), 7.05 (d, J ¼ 7.4 Hz, 1H), 3.74 (s, 2H), 3.56 (s,
2H), 2.30 (s, 3H),1.36 (s, 6H).13C NMR (151 MHz, DMSO‑d6)
164.49,
d
130.14, 129.39, 128.80, 128.54, 128.17, 127.83, 125.96, 59.74, 52.36,
40.54, 24.01, 21.53. Rf ¼ 0.075 (4% Methanol/Dichloromethane).
HRMS (ESI, m/z) calcd for C22H24N4O [MþH]þ, 361.2028; found
361.2043.
4.1.6. 5-(tert-butyl) 2-ethyl 3-amino-6,6-dimethylpyrrolo[3,4-c]
pyrazole-2,5(4H,6H)-dicarboxylate (15)
A solution of 14 (20.00 g, 0.14 mol) and N,N-disopropylethyl-
amine (48.13 mL, 0.28 mol) in anhydrous tetrahydrofuran (300 mL)
4.1.8.3. N-[5-(3-methoxybenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17c). White solid.
Yield: 35%. m.p. 124.8e125.7 ꢀC 1H NMR (600 MHz, DMSO‑d6)
11

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
d
10.69 (s, 1H), 7.92 (d, J ¼ 7.7 Hz, 2H), 7.54 (d, J ¼ 8.2 Hz,1H), 7.46 (s,
3.95 (s, 2H), 3.72e3.48 (m, 2H), 1.40 (s, 6H).13C NMR (151 MHz,
DMSO‑d6) d 164.53, 148.45, 143.55, 135.54, 130.25, 128.79, 128.16,
123.06, 122.32, 60.03, 51.59, 40.53, 24.19. Rf ¼ 0.88 (10% Methanol/
Dichloromethane). HRMS (ESI, m/z) calcd for C21H21N5O3 [MþH]þ,
392.1723; found 392.1741.
2H), 7.24 (t, J ¼ 7.8 Hz, 1H), 6.99e6.90 (m, 2H), 6.82e6.80 (m, 1H),
3.75 (s, 2H), 3.74 (s, 3H), 3.59 (s, 2H), 1.35 (s, 6H). Rf ¼ 0.075 (4%
Methanol/Dichloromethane). HRMS (ESI, m/z) calcd for C22H24N4O2
[MþH]þ, 377.1978; found 377.1983.
4 .1. 8 . 4 . N - [ 5 - ( 3 - c ya n o b e n z yl ) - 6 , 6 - d i m e t h yl - 1, 4 , 5 , 6 -
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17d). White solid.
Yield: 57%. m.p. 129.7e130.6 ꢀC 1H NMR (600 MHz, DMSO‑d6)
4.1. 8.10. N-[5-(2-fluorobenzyl)-6, 6-dimethyl-1, 4, 5, 6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17j). White solid.
Yield: 54%. m.p. 193.1e194.2 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d
12.27 (s, 1H), 10.70 (s, 1H), 7.92 (d, J ¼ 7.6 Hz, 2H), 7.81 (s, 1H), 7.74
(t, J ¼ 8.2 Hz, 2H), 7.61e7.37 (m, 4H), 3.85 (s, 2H), 3.68e3.47 (m, 2H),
1.37 (s, 6H).13C NMR (151 MHz, DMSO‑d6)
164.53, 142.77, 133.82,
d
12.25 (s, 1H), 10.67 (s, 1H), 7.93 (d, J ¼ 7.5 Hz, 2H), 7.63e7.37 (m,
4H), 7.31 (tt, J ¼ 7.3, 3.5 Hz, 1H), 7.22e7.12 (m, 2H), 3.83 (s, 2H),
d
3.72e3.49 (m, 2H), 1.36 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
132.17, 131.14, 130.00, 128.80, 128.17, 119.45, 111.70, 59.98, 51.58,
40.54, 24.14. Rf ¼ 0.20 (4% Methanol/Dichloromethane). HRMS (ESI,
m/z) calcd for C22H21N5O [MþH]þ, 372.1824; found 372.1829.
d
164.54, 161.23 (d, J ¼ 244.9 Hz), 131.48 (d, J ¼ 4.5 Hz), 129.13 (d,
J ¼ 7.8 Hz), 128.80, 128.18, 127.22 (d, J ¼ 13.9 Hz), 124.70, 124.68,
115.62 (d, J ¼ 21.7 Hz), 60.02, 45.26, 40.53, 23.87. Rf ¼ 0.75 (10%
Methanol/Dichloromethane). HRMS (ESI, m/z) calcd for C21H21FN4O
[MþH]þ, 365.1778; found 365.1796.
4.1.8.5. N-[5-(3-iodobenzyl)-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo
[3,4-c]pyrazol-3-yl]benzamide (17e). White solid. Yield: 55%. m.p.
212.8e213.9 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d 12.26 (s, 1H), 10.68
4.1. 8.11. N-[5 -(4 -flu orobenz yl) -6, 6 -dim ethyl-1, 4 , 5, 6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17k). White solid.
Yield: 56%. m.p. 211.5e212.5 ꢀC 1H NMR (600 MHz, DMSO‑d6)
(s, 1H), 7.93 (s, 2H), 7.75 (t, J ¼ 1.7 Hz, 1H), 7.61 (dt, J ¼ 7.8, 1.5 Hz,
1H), 7.57e7.42 (m, 3H), 7.40 (dt, J ¼ 7.7, 1.3 Hz, 1H), 7.15 (t, J ¼ 7.7 Hz,
1H), 3.76 (s, 2H), 3.65e3.47 (m, 2H), 1.36 (s, 6H).13C NMR (151 MHz,
d
12.24 (s, 1H), 10.68 (s, 1H), 7.92 (d, J ¼ 7.6 Hz, 2H), 7.61e7.43 (m,
DMSO‑d6)
d 164.52, 143.76, 137.22, 135.93, 130.94, 128.78, 128.30,
3H), 7.41 (dd, J ¼ 8.3, 5.6 Hz, 2H), 7.15 (t, J ¼ 8.7 Hz, 2H), 3.77 (s, 2H),
128.19, 95.31, 59.87, 51.70, 40.54, 24.10. Rf ¼ 0.19 (4% Methanol/
Dichloromethane). HRMS (ESI, m/z) calcd for C21H21IN4O [MþH]þ,
473.0838; found 473.0849.
3.70e3.42 (m, 2H), 1.36 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
d
163.39, 160.51 (d, J ¼ 241.9 Hz), 135.77, 129.43 (d, J ¼ 8.2 Hz),
127.64, 127.07, 114.25 (d, J ¼ 21.0 Hz), 58.72, 50.45, 39.44, 23.00.
Rf ¼ 0.23 (5% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C21H21FN4O [MþH]þ, 365.1778; found 365.1788.
4 .1. 8. 6. N -[ 5 -( 3 -bro m ob en zyl) - 6 , 6 -d im e thyl -1, 4, 5, 6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17f). White solid.
Yield: 51%. m.p. 201.1e201.9 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d
12.26 (s, 1H), 10.70 (s, 1H), 7.93 (d, J ¼ 7.7 Hz, 2H), 7.61e7.51 (m,
4.1.8.12. N-[5-(3,5-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17l). Yellow solid.
Yield: 58%. m.p. 170.0e171.5 ꢀC 1H NMR (600 MHz, DMSO‑d6)
2H), 7.44 (d, J ¼ 8.1 Hz, 2H), 7.39 (d, J ¼ 7.6 Hz, 1H), 7.31 (t, J ¼ 7.7 Hz,
1H), 3.79 (s, 2H), 3.62 (s, 2H), 1.36 (s, 6H).13C NMR (151 MHz,
DMSO‑d6)
d 164.52, 131.32, 130.90, 130.09, 128.79, 128.18, 127.89,
d
12.28 (s, 1H), 10.73 (s, 1H), 7.93 (d, J ¼ 7.7 Hz, 2H), 7.62e7.41 (m,
3H), 7.10 (d, J ¼ 8.2 Hz, 3H), 3.82 (s, 2H), 3.65 (s, 2H), 1.36 (s, 6H). 13
C
122.14, 59.93, 51.75, 40.54, 24.09. Rf ¼ 0.20 (4% Methanol/
Dichloromethane). HRMS (ESI, m/z) calcd for C21H21BrN4O [MþH]þ,
425.0977; found 425.0989.
NMR (151 MHz, DMSO‑d6)
d
164.54, 162.93 (dd, J ¼ 245.8, 13.1 Hz),
146.22 (t, J ¼ 8.7 Hz), 132.13, 130.13, 128.79, 128.17, 111.41 (d,
J ¼ 21.5 Hz), 109.86, 102.57 (t, J ¼ 25.9 Hz), 59.95, 51.65, 40.54, 24.17.
Rf ¼ 0.13 (5% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C21H20F2N4O [MþH]þ, 383.1683; found 383.1707.
4 .1. 8 . 7 . N - [ 5 - ( 3- chl o ro b e n z yl ) - 6, 6 - di m e t hyl- 1, 4 , 5 , 6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17g). White solid.
Yield: 53%. m.p. 193.7e195.0 ꢀC 1H NMR (400 MHz, DMSO‑d6)
d 7.93
(d, J ¼ 7.6 Hz, 2H), 7.61e7.44 (m, 2H), 7.43 (s, 1H), 7.40e7.32 (m, 3H),
4.1.8.13. N-[5-(2,3-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17m). White solid.
Yield: 59%. m.p. 184.1e185.0 ꢀC 1H NMR (600 MHz, DMSO‑d6)
3.80 (s, 2H), 3.60 (s, 2H), 1.36 (s, 6H).13C NMR (151 MHz, DMSO‑d6)
d
164.52, 133.46, 130.57, 128.79, 128.43, 128.18, 127.48, 127.18, 59.91,
51.79, 40.53, 24.09. Rf ¼ 0.21 (4% Methanol/Dichloromethane).
HRMS (ESI, m/z) calcd for C21H21ClN4O [MþH]þ, 381.1482; found
381.1497.
d
12.06 (s, 1H), 10.69 (s, 1H), 7.93 (d, J ¼ 7.8 Hz, 2H), 7.46e7.53 (m,
3H), 7.40e7.25 (m, 2H), 7.19 (td, J ¼ 7.9, 4.7 Hz, 1H), 3.88 (s, 2H),
3.75e3.49 (m, 1H), 1.36 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
d
164.55, 150.32 (dd, J ¼ 205.6, 12.5 Hz), 148.69 (dd, J ¼ 206.9,
4 .1. 8 . 8 . N - [ 5 - ( 3 - fl u o ro b e n z yl ) - 6 , 6 - d i m e t hyl - 1, 4 , 5 , 6 -
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17h). White solid.
Yield: 50%. m.p. 143.1e144.1 ꢀC 1H NMR (600 MHz, DMSO‑d6)
12.5 Hz),130.11 (d, J ¼ 10.1 Hz),134.16, 132.10,128.79, 128.18, 126.49,
124.97e124.85 (m), 116.20 (d, J ¼ 16.7 Hz), 109.83, 60.13, 45.25,
40.53, 23.89. Rf ¼ 0.25 (5% Methanol/Dichloromethane). HRMS (ESI,
m/z) calcd for C21H20F2N4O [MþH]þ, 383.1683; found 383.1707.
d
10.70 (s, 1H), 7.91 (d, J ¼ 7.7 Hz, 2H), 7.60e7.44 (m, 3H), 7.42 (d,
J ¼ 1.9 Hz, 1H), 7.38e7.32 (m, 2H), 7.30 (d, J ¼ 7.3 Hz, 1H), 3.79 (s,
2H), 3.59 (s, 2H), 1.35 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
d
162.22, 160.56 (d, J ¼ 243.0 Hz), 141.81, 128.25 (d, J ¼ 8.2 Hz),
4.1.8.14. N-[5-(2,5-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17n). Yellow solid.
Yield: 35%. m.p. 111.9e112.6 ꢀC 1H NMR (600 MHz, DMSO‑d6)
127.84, 126.49, 125.88, 122.42, 112.88 (d, J ¼ 21.1 Hz), 111.64 (d,
J ¼ 20.9 Hz), 57.58, 49.57, 38.24, 21.82. Rf ¼ 0.31 (5% Methanol/
Dichloromethane). HRMS (ESI, m/z) calcd for C21H21FN4O [MþH]þ,
365.1778; found 365.1798.
d
12.26 (s,1H),10.75 (s,1H), 7.94 (d, J ¼ 7.7 Hz, 2H), 7.55 (t, J ¼ 7.3 Hz,
1H), 7.47 (t, J ¼ 7.5 Hz, 2H), 7.30 (ddd, J ¼ 9.0, 5.5, 3.2 Hz, 1H), 7.24
(td, J ¼ 9.2, 4.4 Hz, 1H), 7.15 (td, J ¼ 8.2, 4.1 Hz, 1H), 3.83 (s, 2H), 3.67
4.1.8.9. N-[6,6-dimethyl-5-(3-nitrobenzyl)-1,4,5,6-tetrahydropyrrolo
(s, 2H), 1.36 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 155.28, 147.35,
[3,4-c]pyrazol-3-yl]benzamide (17i). Yellow solid. Yield: 51%. m.p.
144.37, 138.19, 137.22, 136.47, 132.31, 131.40, 130.26, 130.13, 129.27,
127.50, 60.84, 40.55, 35.16, 19.11.Rf ¼ 0.29 (5% Methanol/Dichloro-
methane). HRMS (ESI, m/z) calcd for C21H20F2N4NaO [MþNa]þ,
405.1503; found 405.1528.
178.1e178.8 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d 12.29 (s, 1H), 10.71
(s, 1H), 8.23 (t, J ¼ 2.1 Hz, 1H), 8.16e8.09 (m, 1H), 7.91 (d, J ¼ 7.7 Hz,
2H), 7.89e7.84 (m, 1H), 7.65 (t, J ¼ 7.9 Hz, 1H), 7.59e7.41 (m, 3H),
12

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
4.1.8.15. N-[5-(3,4-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17o). White solid.
Yield: 55%. m.p. 177.4e178.5 ꢀC 1H NMR (600 MHz, DMSO‑d6)
2H), 7.72e7.56 (m, 1H), 7.52 (t, J ¼ 7.7 Hz, 2H), 4.87 (d, J ¼ 10.8 Hz,
1H), 4.75 (dd, J ¼ 12.6, 7.2 Hz,1H), 4.66 (dd, J ¼ 12.7, 9.8 Hz,1H), 4.12
(dd, J ¼ 12.7, 5.0 Hz, 1H), 1.89 (s, 3H), 1.66 (s, 3H). Rf ¼ 0.35 (5%
Methanol/Dichloromethane). HRMS (ESI, m/z) calcd for
d
12.06 (s, 1H), 10.70 (s, 1H), 7.93 (d, J ¼ 7.6 Hz, 2H), 7.48e7.51 (m,
J ¼ 3H), 7.39 (ddt, J ¼ 13.7, 11.0, 5.2 Hz, 2H), 7.28e7.18 (m, 1H), 3.78
C
23H20F6N4O [MþH]þ, 483.1620; found 483.1637.
(s, 2H), 3.62 (s, 2H), 1.36 (s, 6H).13C NMR (151 MHz, DMSO‑d6)
d
164.52, 150.14 (dd, J ¼ 171.7, 12.6 Hz), 148.52 (dd, J ¼ 170.4,
4.1.8.21. N-{5-[3-fluoro-5-(trifluoromethyl)benzyl]-6,6-dimethyl-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzamide (17u).
White solid. Yield: 55%. m.p. 199.7e201.6 ꢀC 1H NMR (600 MHz,
DMSO‑d6)
10.72 (s, 1H), 7.92 (d, J ¼ 7.6 Hz, 2H), 7.60 (s, 1H),
7.57e7.51 (m, 3H), 7.46 (s, 2H), 3.92 (s, 2H), 3.70e3.52 (m, 2H), 1.37
(s, 6H). 13C NMR (151 MHz, DMSO‑d6)
146.30 (d, J ¼ 6.9 Hz), 134.06, 132.17, 131.32 (qd, J ¼ 32.6, 8.8 Hz),
128.79, 128.18, 123.95 (dd, J ¼ 271.6, 4.0 Hz), 121.21, 119.56 (d,
J ¼ 21.3 Hz), 111.57 (d, J ¼ 24.8 Hz), 60.04, 51.47, 40.53, 24.19.
Rf ¼ 0.35 (5% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C21H20F4N4O [MþH]þ, 433.1651; found 433.1674.
12.7 Hz), 138.80, 134.06, 132.17, 128.80, 128.17, 125.16 (d, J ¼ 5.9 Hz),
117.60 (d, J ¼ 17.1 Hz), 117.32 (d, J ¼ 16.5 Hz), 59.92, 51.29, 40.53,
24.11. Rf ¼ 0.26 (5% Methanol/Dichloromethane). HRMS (ESI, m/z)
calcd for C21H20F2N4O [MþH]þ, 383.1683; found 383.1698.
d
d
162.61 (d, J ¼ 245.6 Hz),
4.1.8.16. N-[5-(2,4-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17p). White solid.
Yield: 57%. m.p. 186.2e186.9 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d
10.70 (s, 1H), 7.92 (d, J ¼ 7.7 Hz, 2H), 7.57e7.41 (m, 4H), 7.18 (td,
J ¼ 9.9, 2.6 Hz, 1H), 7.06 (td, J ¼ 8.4, 2.6 Hz, 1H), 3.79 (s, 2H), 3.61 (s,
2H), 1.34 (s, 6H).13C NMR (151 MHz, DMSO‑d6) 13C NMR (151 MHz,
DMSO‑d6)
d
164.54, 161.82 (dd, J ¼ 244.7, 16.1 Hz), 161.06 (dd,
4.1.8.22. N-[6,6-dimethyl-5-(3,4,5-trifluorobenzyl)-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17v). White solid.
Yield: 58%. m.p. 179.1e180.3 ꢀC 1H NMR (600 MHz, DMSO‑d6)
J ¼ 244.7, 10.9 Hz), 134.06, 132.52 (t, J ¼ 7.7 Hz), 132.18, 128.80,
128.18, 123.62, 111.69 (d, J ¼ 22.0 Hz), 104.12 (t, J ¼ 25.8 Hz), 60.05,
44.89, 40.53, 23.84. Rf ¼ 0.26 (5% Methanol/Dichloromethane).
HRMS (ESI, m/z) calcd for C21H20F2N4O [MþH]þ, 383.1683; found
383.1681.
d
12.28 (s, 1H), 10.73 (s, 1H), 7.93 (d, J ¼ 7.6 Hz, 2H), 7.67e7.39 (m,
3H), 7.32 (dd, J ¼ 8.9, 6.7 Hz, 2H), 3.79 (s, 2H), 3.73e3.43 (m, 2H),
1.35 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
164.57, 150.70 (ddd,
d
J ¼ 246.9, 9.9, 3.7 Hz), 137.87 (dt, J ¼ 247.8, 11.3, 9.4 Hz), 134.04,
132.18, 130.13, 128.82, 128.15, 112.67 (d, J ¼ 16.8 Hz), 59.97, 51.21,
40.50, 24.16. Rf ¼ 0.34 (5% Methanol/Dichloromethane). HRMS (ESI,
m/z) calcd for C21H19F3N4O [MþH]þ, 401.1589; found 401.1607.
4.1.8.17. N-[5-(2,6-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17q). White solid.
Yield: 49%. m.p. 215.2e216.1 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d
12.26 (s, 1H), 10.69 (s, 1H), 7.94 (d, J ¼ 7.7 Hz, 2H), 7.64e7.44 (m,
3H), 7.39 (ddd, J ¼ 14.9, 8.4, 6.5 Hz, 1H), 7.09 (t, J ¼ 7.8 Hz, 2H), 3.83
4.1.9. General procedure for the synthesis of target compounds 18a
- 18t
(s, 2H), 3.75e3.55 (m, 2H), 1.37 (s, 6H). 13C NMR (151 MHz,
DMSO‑d6)
d
163.35, 160.58 (dd, J ¼ 247.2, 8.3 Hz), 132.88, 130.90,
A solution of various benzoic acid (2.34 mmol) in anhydrous
dichloromethane (20 mL) was added oxalyl chloride (0.40 mL,
4.67 mmol) and a catalytic amount of N,N-dimethylformamide. The
reaction was stirred at room temperature for 6 h. The solvent was
removed, and then redissolved in anhydrous tetrahydrofuran
128.73 (t, J ¼ 10.8 Hz), 127.56, 126.96, 114.50 (t, J ¼ 18.8 Hz), 110.73
(d, J ¼ 4.8 Hz), 110.59 (d, J ¼ 4.8 Hz), 58.96, 39.29, 37.78, 22.22.
Rf ¼ 0.31 (5% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C21H20F2N4O [MþH]þ, 383.1683; found 383.1676.
(20 mL). The solution was cooled to
0
ꢀC, and N,N-dis-
4.1.8.18. N-[5-(3-fluoro-4-methylbenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17r). White solid.
Yield: 51%. m.p. 189.1e190.5 ꢀC 1H NMR (600 MHz, DMSO‑d6)
opropylethylamine (0.58 mL, 3.27 mmol) was added. 14 (0.20 g,
0.47 mmol) was subsequently added dropwise. After addition, the
resulting solution was stirred at ꢁ20 ꢀC for 2 h. The tetrahydrofuran
was removed, and then dissolved in methanol. The solution was
stirred at reflux for another 2 h. After the reaction was cooled to
room temperature, removal of the solvent gave a residue that was
purified by silica chromatography (5%e10% methanol/dichloro-
methane) to obtain 18a - 18t in moderate yield.
d
12.23 (s, 1H), 10.69 (s, 1H), 7.91 (d, J ¼ 7.7 Hz, 2H), 7.60e7.36 (m,
3H), 7.22 (t, J ¼ 7.8 Hz, 1H), 7.12e7.10 (m, 1H), 7.09 (s, 1H), 3.74 (s,
2H), 3.57 (s, 2H), 2.20 (s, 3H), 1.34 (s, 6H). 13C NMR (151 MHz,
DMSO‑d6)
d
163.39, 160.11 (d, J ¼ 242.1 Hz), 139.96, 132.97, 131.06,
130.65 (d, J ¼ 5.3 Hz), 127.70, 127.07, 123.38, 121.68 (d, J ¼ 17.1 Hz),
113.87 (d, J ¼ 21.7 Hz), 50.55, 39.44, 22.96, 13.26 (d, J ¼ 3.3 Hz).
Rf ¼ 0.26 (5% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C22H23FN4O [MþH]þ, 379.1934; found 379.1946.
4.1.10. General procedure for the synthesis of target compounds 19a
- 19s
The method of synthesizing compounds 19a - 19s were similar
to the method of synthesizing compounds 17a - 17v except that the
starting materials were different.
4.1.8.19. N-[5-(2-fluoro-5-methylbenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (17s). White solid.
Yield: 53%. m.p. 184.4e185.3 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d
12.25 (s, 1H), 10.68 (s, 1H), 7.91 (d, J ¼ 7.7 Hz, 2H), 7.60e7.38 (m,
3H), 7.17 (dt, J ¼ 8.3, 4.6 Hz, 2H), 6.96 (td, J ¼ 8.5, 2.9 Hz, 1H), 3.76 (s,
4.1.10.1. N-[5-(2,3-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
2H), 3.68e3.44 (m, 2H), 2.32 (s, 3H),1.36 (s, 6H). 13C NMR (151 MHz,
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-2-naphthamide
White solid. Yield: 56%. m.p. 190.0e191.1 ꢀC 1H NMR (600 MHz,
DMSO‑d6)
(19a).
DMSO‑d6)
d
164.55, 161.02 (d, J ¼ 240.1 Hz), 140.75 (d, J ¼ 6.5 Hz),
134.07,133.20,132.16,131.94 (d, J ¼ 7.5 Hz),128.80,128.18,115.97 (d,
J ¼ 21.4 Hz), 113.50 (d, J ¼ 20.8 Hz), 60.08, 50.14, 40.53, 23.79, 18.44.
Rf ¼ 0.29 (5% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C22H23FN4O [MþH]þ, 379.1934; found 379.1928.
d
12.29 (s, 1H), 10.87 (s, 1H), 8.59 (s, 1H), 8.00 (q, J ¼ 12.0,
11.0 Hz, 4H), 7.67e7.56 (m, 2H), 7.37e7.28 (m, 2H), 7.24e7.16 (m,
1H), 3.89 (s, 2H), 3.71 (s, 2H), 1.38 (s, 6H).13C NMR (151 MHz,
DMSO‑d6) 13C NMR (151 MHz, DMSO‑d6)
d 164.56, 150.33 (dd,
J ¼ 202.7, 12.8 Hz), 148.71 (dd, J ¼ 204.0, 12.5 Hz), 134.80, 132.54,
131.33, 130.12, 129.46, 128.82, 128.37, 128.09, 127.27, 126.56, 124.79,
116.24 (d, J ¼ 16.9 Hz), 60.17, 45.26, 40.53, 23.87. Rf ¼ 0.31 (5%
Methanol/Dichloromethane). HRMS (ESI, m/z) calcd for
4.1.8.20. N-{5-[3,5-bis(trifluoromethyl)benzyl]-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzamide (17t). White solid.
Yield: 39%. m.p. 216.0e217.2 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d
11.17 (s, 1H), 10.69 (s, 1H), 8.44 (s, 2H), 8.31 (s, 1H), 7.95e7.91 (m,
C
25H22F2N4O [MþH]þ, 433.1840; found 433.1840.
13

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
4.1.10.2. N-[5-(3,5-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
Yield: 54%. m.p. 204.0e204.8 ꢀC 1H NMR (400 MHz, DMSO‑d6)
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-2-naphthamide
White solid. Yield: 58%. m.p. 206.9e207.8 ꢀC 1H NMR (600 MHz,
DMSO‑d6) 12.11 (s, 1H), 10.89 (s, 1H), 8.75e8.51 (m, 1H), 8.11e7.95
(19b).
d
12.11 (s, 1H), 10.84 (s, 1H), 7.87 (d, J ¼ 8.1 Hz, 2H), 7.67 (d,
J ¼ 7.0 Hz, 2H), 7.10 (dd, J ¼ 7.0, 3.7 Hz, 3H), 3.82 (s, 2H), 3.64 (s, 2H),
1.35 (s, 6H).13C NMR (151 MHz, DMSO‑d6)
162.69, 161.81 (dd,
d
d
(m, 4H), 7.62 (d, J ¼ 11.5 Hz, 2H), 7.10 (dd, J ¼ 14.4, 8.6 Hz, 3H), 3.84
J ¼ 236.0, 14.3 Hz), 145.10, 132.18, 130.72, 129.23, 124.86, 110.31 (d,
J ¼ 21.7 Hz), 101.49 (t, J ¼ 25.8 Hz), 58.88, 50.53, 39.44, 23.06.
Rf ¼ 0.42 (5% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C21H19BrF2N4O [MþH]þ, 461.0789; found 461.0772.
(s, 2H), 3.78e3.52 (m, 2H), 1.36 (s, 6H). 13C NMR (151 MHz,
DMSO‑d6)
d
163.50, 161.87 (dd, J ¼ 245.9, 13.1 Hz), 145.13, 133.74,
131.47, 130.28, 128.39, 127.74, 127.30, 127.03, 126.20, 123.72, 110.41
(d, J ¼ 21.1 Hz), 101.54 (t, J ¼ 25.8 Hz), 58.93, 50.59, 39.47, 23.08.
Rf ¼ 0.32 (5% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C25H22F2N4O [MþH]þ, 433.1840; found 433.1835.
4.1.10.8. N-[5-(2,3-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-4-morpholinobenzamide
(19h). White solid. Yield: 53%. m.p. 117.4e118.3 ꢀC 1H NMR
4.1.10.3. N-[5-(3,5-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]quinoline-7-carboxamide (19c).
White solid. Yield: 57%. m.p. 245.2e246.1 ꢀC 1H NMR (600 MHz,
(600 MHz, DMSO‑d6)
d
12.19 (s, 1H), 10.40 (s, 1H), 7.84 (d, J ¼ 8.1 Hz,
2H), 7.35e7.28 (m, 2H), 7.22e7.16 (m, 1H), 6.96 (s, 2H), 3.87 (s, 2H),
3.77e3.70 (m, 4H), 3.63 (s, 2H), 3.23 (t, J ¼ 5.0 Hz, 4H), 1.35 (s, 6H).
DMSO‑d6)
d
10.98 (s, 1H), 8.99 (dd, J ¼ 4.2, 1.8 Hz, 1H), 8.64 (s, 1H),
13C NMR (151 MHz, DMSO‑d6)
d 163.00, 152.59, 149.21 (dd,
8.46 (d, J ¼ 8.4 Hz, 1H), 8.23 (dd, J ¼ 8.8, 2.0 Hz, 1H), 8.07 (d,
J ¼ 8.8 Hz, 1H), 7.62 (dd, J ¼ 8.4, 4.2 Hz, 1H), 7.16e7.03 (m, 3H), 3.84
(s, 2H), 3.68 (s, 2H), 1.37 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
J ¼ 205.3, 12.8 Hz), 147.59 (dd, J ¼ 206.7, 12.7 Hz), 129.04 (d,
J ¼ 10.1 Hz), 128.47, 125.39, 123.81 (t, J ¼ 5.5 Hz), 115.08 (d,
J ¼ 17.0 Hz),112.62, 65.28, 58.98, 46.54, 44.16, 39.43, 22.77. Rf ¼ 0.75
(10% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd for
d
162.99, 161.83 (dd, J ¼ 241.6, 13.0 Hz), 151.66, 148.17, 145.01,
136.56, 130.77, 128.35, 128.19, 127.30, 126.40, 121.61, 110.39 (dd,
J ¼ 19.9, 4.0 Hz), 101.51 (t, J ¼ 25.8 Hz), 58.95, 50.53, 39.42, 23.04.
Rf ¼ 0.12 (5% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C24H22F2N5O [MþH]þ, 434.1792; found 434.1728.
C
25H27F2N5O2 [MþH]þ, 468.2211; found 468.2214.
4.1.10.9. N-[5-(3,5-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-4-morpholinobenzamide (19i).
White solid. Yield: 53%. m.p. 125.3e126.1 ꢀC 1H NMR (600 MHz,
4.1.10.4. N-[5-(2,3-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
DMSO‑d6)
d
12.20 (s, 1H), 10.64e10.27 (m, 1H), 7.84 (d, J ¼ 8.4 Hz,
tetrahydropyrrolo[3,4-c]pyrazol-3-yl])-4-fluorobenzamide
White solid. Yield: 47%. m.p. 203.2e204.2 ꢀC 1H NMR (600 MHz,
DMSO‑d6)
12.28 (s, 1H), 10.74 (s, 1H), 8.01 (d, J ¼ 10.0 Hz, 2H),
7.38e7.24 (m, 4H), 7.23e7.13 (m, 1H), 3.88 (s, 2H), 3.73e3.54 (m,
2H), 1.36 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
163.54 (d,
(19d).
2H), 7.13e7.05 (m, 3H), 6.96 (s, 2H), 3.82 (s, 2H), 3.76e3.69 (m, 4H),
3.61 (s, 3H), 3.23 (t, J ¼ 4.9 Hz, 4H), 1.34 (s, 6H). 13C NMR (151 MHz,
d
DMSO‑d6)
d
164.08, 162.93 (dd, J ¼ 245.6, 13.2 Hz), 153.69, 146.26 (t,
J ¼ 8.5 Hz),129.56,123.30,113.72,111.41 (dd, J ¼ 20.1, 4.5 Hz),102.55
(t, J ¼ 26.0 Hz), 66.38, 59.90, 51.68, 47.63, 40.53, 24.15. Rf ¼ 0.75
(10% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd for
d
J ¼ 249.8 Hz), 162.43, 154.16, 149.24 (dd, J ¼ 205.6, 12.5 Hz), 147.62
(dd, J ¼ 207.2, 12.7 Hz), 139.31, 129.87 (d, J ¼ 9.0 Hz), 129.61, 129.02
(dd, J ¼ 10.3, 4.2 Hz), 125.40, 123.83 (t, J ¼ 5.6 Hz), 115.13 (d,
J ¼ 16.9 Hz), 114.64 (d, J ¼ 21.8 Hz), 108.74, 59.06, 44.17, 39.45, 22.81.
Rf ¼ 0.24 (5% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C21H19F3N4O [MþH]þ, 401.1589; found 401.1581.
C
25H27F2N5O2 [MþH]þ, 468.2211; found 468.2213.
4.1.10.10. N-[5-(2,3-difluorobenzyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]
pyrazol-3-yl]-4-(dimethylamino)benzamide (19j). Yellow solid.
Yield: 51%. m.p. 110.0e111.3 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d
12.15 (s, 1H), 10.29 (s, 1H), 7.82 (d, J ¼ 8.6 Hz, 2H), 7.37e7.28 (m,
2H), 7.22e7.17 (m, 1H), 6.75e6.64 (m, 2H), 3.87 (s, 2H), 3.61 (s, 2H),
2.97 (s, 6H), 1.35 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
163.23,
4.1.10.5. N-[5-(3,5-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-4-fluorobenzamide
White solid. Yield: 40%. m.p. 213.7e214.8 ꢀC 1H NMR (600 MHz,
DMSO‑d6)
12.08 (s, 1H), 10.75 (s, 1H), 8.01 (t, J ¼ 6.5 Hz, 2H),
(19e).
d
151.82,149.22 (dd, J ¼ 205.5, 12.9 Hz),147.60 (dd, J ¼ 206.7, 12.6 Hz),
129.07 (d, J ¼ 10.1 Hz), 128.55, 125.41, 123.82 (t, J ¼ 5.7 Hz), 119.16,
115.09 (d, J ¼ 16.9 Hz), 110.11, 58.97, 44.18, 39.44, 22.78. Rf ¼ 0.66
(10% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd for
d
7.48e7.21 (m, 2H), 7.10 (d, J ¼ 8.3 Hz, 3H), 3.82 (s, 2H), 3.69e3.55
(m, 2H), 1.36 (s, 6H). Rf ¼ 0.25 (5% Methanol/Dichloromethane).13
C
NMR (151 MHz, DMSO‑d6)
d
165.31 (d, J ¼ 305.3 Hz), 162.34, 161.78
C
23H25F2N5O [MþH]þ, 426.2105; found 426.2109.
(dd, J ¼ 246.1, 13.3 Hz), 145.02, 130.99 (d, J ¼ 19.4 Hz), 129.78 (d,
J ¼ 9.0 Hz), 129.43, 128.00, 114.59 (dd, J ¼ 21.8, 4.7 Hz), 110.26 (dd,
J ¼ 21.8, 4.8), 101.43 (t, J ¼ 26.0 Hz), 58.85, 50.48, 39.38, 23.00.
HRMS (ESI, m/z) calcd for C21H19F3N4O [MþH]þ, 401.1589; found
401.1584.
4.1.10.11. N-[5-(3,5-difluorobenzyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]
pyrazol-3-yl]-4-(dimethylamino)benzamide (19k). Yellow solid.
Yield: 55%. m.p. 179.1e180.3 ꢀC 1H NMR (600 MHz, DMSO‑d6)
d
10.30 (s, 1H), 7.82 (d, J ¼ 8.4 Hz, 2H), 7.13e7.06 (m, 3H), 6.69 (d,
J ¼ 8.1 Hz, 2H), 3.81 (s, 2H), 3.60 (s, 2H), 2.97 (s, 6H), 1.34 (s, 6H). 13
C
4.1.10.6. 4-Bromo-N-[5-(2,3-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (19f). White solid.
Yield: 52%. m.p. 173.3e174.3 ꢀC 1H NMR (600 MHz, DMSO‑d6)
NMR (151 MHz, DMSO‑d6)
d
163.22, 161.84 (dd, J ¼ 245.8, 13.0 Hz),
151.82, 145.19 (t, J ¼ 8.4 Hz), 128.54, 119.20, 110.32 (dd, J ¼ 20.6,
4.5 Hz), 110.11, 101.46 (t, J ¼ 26.0 Hz), 58.80, 50.60, 39.44, 23.06.
Rf ¼ 0.66 (10% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C23H25F2N5O [MþH]þ, 426.2105; found 426.2098.
d
12.29 (s, 1H), 10.80 (s, 1H), 7.87 (d, J ¼ 8.3 Hz, 2H), 7.76e7.61 (m,
2H), 7.31 (dt, J ¼ 12.1, 7.5 Hz, 2H), 7.19 (td, J ¼ 8.0, 4.9 Hz,1H), 3.87 (s,
2H), 3.76e3.51 (m, 2H), 1.37 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
d
162.57, 149.22 (dd, J ¼ 205.7, 12.6 Hz), 147.59 (dd, J ¼ 206.9,
4.1.10.12. N-[5-(2,3-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-4-(4-methylpiperazin-1-yl)
benzamide (19l). White solid. Yield: 49%. m.p. 192.1e193.5 ꢀC 1H
12.4 Hz), 132.16, 130.73, 129.24, 128.99 (d, J ¼ 10.5 Hz), 125.39,
124.88, 123.83 (t, J ¼ 6.3, 4.3 Hz), 115.12 (d, J ¼ 16.8 Hz), 59.05, 44.14,
39.44, 22.78. Rf ¼ 0.26 (5% Methanol/Dichloromethane). HRMS (ESI,
m/z) calcd for C21H19BrF2N4O [MþH]þ, 461.0789; found 461.0771.
NMR (600 MHz, DMSO‑d6)
d 12.16 (s, 1H), 10.38 (s, 1H), 7.82 (d,
J ¼ 7.4 Hz, 2H), 7.31 (td, J ¼ 10.5, 9.3, 4.3 Hz, 2H), 7.23e7.17 (m, 1H),
6.94 (s,1H), 3.87 (s, 2H), 3.62 (s, 2H), 3.26 (s, 4H), 2.47e2.40 (m, 4H),
4.1.10.7. 4-Bromo-N-[5-(3,5-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]benzamide (19g). White solid.
2.21 (s, 3H), 1.35 (s, 6H).13C NMR (151 MHz, DMSO‑d6)
d
164.09,
153.54, 150.31 (dd, J ¼ 205.8, 12.4 Hz), 148.68 (dd, J ¼ 206.9,
14

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
12.5 Hz), 130.15 (d, J ¼ 9.7 Hz), 129.58, 126.50, 124.91 (t, J ¼ 5.7 Hz),
116.18 (d, J ¼ 16.9 Hz), 113.84, 60.07, 54.83, 47.28, 46.19, 45.26,
40.54, 23.87. Rf ¼ 0.24 (10% Methanol/Dichloromethane). HRMS
6.94e6.84 (m, 1H), 3.88 (s, 2H), 3.65 (s, 2H), 2.93 (s, 6H), 1.36 (s, 6H).
13C NMR (151 MHz, DMSO‑d6)
d 164.00, 149.64, 149.21 (dd,
J ¼ 206.2, 12.7 Hz), 147.58 (dd, J ¼ 207.4, 12.7 Hz), 133.59, 129.03 (d,
J ¼ 9.9 Hz), 128.20, 125.36, 123.82 (t, J ¼ 5.9, 5.3 Hz), 115.08 (d,
J ¼ 16.5 Hz), 114.72, 110.66, 58.99, 44.14, 39.48, 39.43, 22.78.
Rf ¼ 0.67 (10% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd
for C23H25F2N5O [MþH]þ, 426.2105; found 426.2108.
(ESI, m/z) calcd for
C
26H30F2N6O [MþH]þ, 481.2527; found
481.2531.
4.1.10.13. N-[5-(3,5-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-4-(4-methylpiperazin-1-yl)
benzamide (19m). White solid. Yield: 58%. m.p. 200.9e202.0 ꢀC 1H
4.1.10.18. N-[5-(3,5-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-3-(dimethylamino)benzamide
(19r). White solid. Yield: 45%. m.p. 110.3e111.5 ꢀC 1H NMR
NMR (600 MHz, DMSO‑d6)
d 12.18 (s, 1H), 10.39 (s, 1H), 7.82 (d,
J ¼ 8.4 Hz, 2H), 7.13e7.06 (m, 3H), 6.99e6.85 (m, 2H), 3.81 (s, 2H),
3.59 (s, 2H), 3.26 (t, J ¼ 5.1 Hz, 4H), 2.43 (t, J ¼ 5.0 Hz, 4H), 2.21 (s,
(600 MHz, DMSO‑d6)
(m, 3H), 6.88 (d, J ¼ 7.9 Hz,1H), 3.82 (s, 2H), 3.62 (s, 2H), 2.93 (s, 6H),
1.35 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
165.09, 162.94 (dd,
d 10.60 (s, 1H), 7.28e7.14 (m, 3H), 7.13e7.04
3H), 1.34 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 164.09, 162.93 (dd,
J ¼ 245.7, 13.2 Hz), 153.48, 146.28 (d, J ¼ 8.8 Hz), 130.13, 129.65,
129.58, 122.80, 114.10, 113.88, 111.41 (dd, J ¼ 19.9, 4.6 Hz), 102.56 (t,
J ¼ 25.9 Hz), 59.91, 54.71, 51.67, 47.16, 46.02, 40.51, 24.14. Rf ¼ 0.23
(10% Methanol/Dichloromethane). HRMS (ESI, m/z) calcd for
d
J ¼ 245.7, 13.1 Hz), 150.75, 146.26 (t, J ¼ 8.5 Hz), 134.68, 130.14,
115.81 (d, J ¼ 12.7 Hz), 111.77, 111.40 (dd, J ¼ 17.7, 4.7 Hz), 102.58 (t,
J ¼ 25.9 Hz), 59.93, 51.66, 40.59, 40.54, 24.17. Rf ¼ 0.68 (10%
Methanol/Dichloromethane). HRMS (ESI, m/z) calcd for
C
26H30F2N6O [MþH]þ, 481.2527; found 481.2526.
C
23H25F2N5O [MþH]þ, 426.2105; found 426.2115.
4.1.10.14. 1-(3,5-difluorobenzyl)-4-(4-{[5-(3,5-difluorobenzyl)-6,6-
dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl]carbamoyl}
phenyl)-1-methylpiperazin-1-ium (19n). White solid. Yield: 37%.
Decompose without heating to the m.p. 1H NMR (600 MHz,
4.1.10.19. N-[5-(2,3-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-3-(4-methylpiperazin-1-yl)
benzamide (19s). White solid. Yield: 48%. m.p. 113.6e114.5 ꢀC 1H
DMSO‑d6)
d
10.51 (s, 1H), 7.91 (d, J ¼ 8.5 Hz, 2H), 7.52 (tt, J ¼ 9.3,
NMR (600 MHz, DMSO‑d6) d 12.23 (s, 1H), 10.64 (s, 1H), 7.47 (s, 1H),
2.4 Hz, 1H), 7.41e7.36 (m, 2H), 7.13e7.03 (m, 5H), 4.72 (s, 2H),
3.96e3.87 (m, 2H), 3.82 (s, 2H), 3.62 (dd, J ¼ 13.3, 9.9 Hz, 4H), 3.51
(td, J ¼ 12.8, 10.9, 3.2 Hz, 4H), 3.11 (s, 3H), 1.35 (s, 6H). 13C NMR
7.35e7.26 (m, 4H), 7.22e7.17 (m, 1H), 7.10 (d, J ¼ 8.1 Hz, 1H), 3.88 (s,
2H), 3.64 (s, 2H), 3.19 (d, J ¼ 5.2 Hz, 4H), 2.47 (d, J ¼ 5.2 Hz, 4H), 2.23
(s, 3H), 1.36 (s, 6H). Rf ¼ 0.11 (10% Methanol/Dichloromethane).
HRMS (ESI, m/z) calcd for C26H30F2N6O [MþH]þ, 481.2527; found
481.2527.
(151 MHz, DMSO‑d6)
d
163.89, 162.93 (dd, J ¼ 245.8, 13.1 Hz), 162.76
(dd, J ¼ 247.5, 13.4 Hz), 158.30 (d, J ¼ 30.4 Hz), 152.03, 146.27, 131.47
(t, J ¼ 10.0 Hz), 130.13, 129.66, 118.85, 117.26e116.91 (m), 116.86 (dd,
J ¼ 18.9, 5.4 Hz),114.45,111.41 (d, J ¼ 23.3 Hz),106.66 (t, J ¼ 25.6 Hz),
102.56 (t, J ¼ 25.9 Hz), 66.09, 59.92, 59.05, 51.67, 45.34, 41.33, 40.53,
24.15. Rf ¼ 0.66 (50% Methanol/Dichloromethane). HRMS (ESI, m/z)
calcd for C33H37F2N6O [MþH]þ, 608.2887; found 608.2848.
4.1.10.20. N-[5-(3,5-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-3-(4-methylpiperazin-1-yl)
benzamide (19t). White solid. Yield: 55%. m.p. 114.7e115.4 ꢀC 1H
NMR (600 MHz, DMSO‑d6) d 12.26 (s, 1H), 10.66 (s, 1H), 7.48 (s, 1H),
7.33 (d, J ¼ 6.8 Hz, 1H), 7.28 (s, 1H), 7.10 (d, J ¼ 7.9 Hz, 4H), 3.82 (s,
4.1.10.15. N-[5-(2,3-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-3-morpholinobenzamide
(19o). White solid. Yield: 44%. m.p. 182.1e183.6 ꢀC 1H NMR
2H), 3.63 (s, 2H), 3.25e3.11 (m, 4H), 2.46 (s, 4H), 2.23 (s, 3H), 1.35 (s,
6H). 13C NMR (151 MHz, DMSO‑d6)
d
164.71, 162.93 (dd, J ¼ 245.4,
13.4 Hz),151.32,146.25,134.71,130.13,129.39,118.82,118.55,114.72,
111.78e110.98 (m), 102.58 (dd, J ¼ 20.8, 4.1 Hz), 59.94, 54.93, 51.66,
48.28, 46.12 (d, J ¼ 6.2 Hz), 40.54, 24.18. Rf ¼ 0.11 (10% Methanol/
Dichloromethane). HRMS (ESI, m/z) calcd for C26H30F2N6O [MþH]þ,
481.2527; found 481.2522.
(600 MHz, DMSO‑d6)
d 12.25 (s, 1H), 10.66 (s, 1H), 7.48 (s, 1H),
7.39e7.27 (m, 4H), 7.23e7.16 (m, 1H), 7.11 (d, J ¼ 8.1 Hz, 1H), 3.88 (s,
2H), 3.79e3.63 (m, 4H), 3.65 (s, 2H), 3.16 (dd, J ¼ 5.8, 3.9 Hz, 4H),
1.37 (s, 6H).13C NMR (151 MHz, DMSO‑d6)
d 164.69, 151.46, 150.32
(dd, J ¼ 205.6, 12.6 Hz), 148.69 (dd, J ¼ 207.1, 12.8 Hz), 134.73, 130.12
(d, J ¼ 10.0 Hz), 129.44, 126.48, 124.93 (t, J ¼ 5.9 Hz), 118.91, 118.67,
116.20 (d, J ¼ 16.6 Hz), 114.48, 66.51, 60.11, 48.76, 45.24, 40.54,
23.89. Rf ¼ 0.79 (10% Methanol/Dichloromethane). HRMS (ESI, m/z)
calcd for C25H27F2N5O2 [MþH]þ, 468.2211; found 468.2208.
4.2. Pharmacological assay
4.2.1. TRKs HTRF assay
The purified kinases were purchased from Carna Biosciences
(Japan) and the TK kinEASE kit was commercially purchased from
Cisbio Bioassays (France). The HTRF assays were performed ac-
cording to the instructions of manufactures. A few optimization
experiments were implemented to find the optimal concentration
of kinases. The concentrations of TRKA, TRKB and TRKC used were
0.111 ng/uL, 0.037 ng/uL and 0.037 ng/uL, respectively. The con-
centrations of ATP were determined by similar experiments, and
4.1.10.16. N-[5-(3,5-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-3-morpholinobenzamide
(19p). White solid. Yield: 41%. m.p. 192.5e193.6 ꢀC 1H NMR
(600 MHz, DMSO‑d6)
d 12.27 (s, 1H), 10.67 (s, 1H), 7.50 (s, 1H), 7.36
(s, 1H), 7.30 (s, 1H), 7.14e7.06 (m, 4H), 3.82 (s, 2H), 3.79e3.69 (m,
4H), 3.64 (s, 2H), 3.24e3.10 (m, 4H), 1.35 (s, 6H). 13C NMR (151 MHz,
DMSO‑d6)
d
164.67, 162.94 (dd, J ¼ 245.3, 13.0 Hz), 155.12, 151.46,
the concentrations of ATP were 14.68
and 25.64 M (TRKC).
First, compounds were diluted from a concentrated stock of
8 mM in 100% DMSO and with serial kinase reaction buffer di-
mM (TRKA), 4.77 mM (TRKB)
146.24 (t, J ¼ 8.4 Hz), 140.61, 134.75, 129.42, 118.94, 114.49, 111.40
(dd, J ¼ 20.2, 4.6 Hz), 102.58 (t, J ¼ 26.1 Hz), 66.51, 59.94, 51.66,
48.76, 40.54, 24.18. Rf ¼ 0.67 (10% Methanol/Dichloromethane).
HRMS (ESI, m/z) calcd for C25H27F2N5O2 [MþH]þ, 468.2211; found
468.2214.
m
lutions. Second, a 5
solutions and ATP solutions whose concentrations were displayed
in the previous were prepared. For each assays, 4 L of dispensed
compounds, 2 L of ATP, 2 L of substrate TK and 2 L of kinase were
mM TK solution, 0.5 mM XL-665 solution, kinases
m
m
4.1.10.17. N-[5-(2,3-difluorobenzyl)-6,6-dimethyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-3-(dimethylamino)benzamide
(19q). White solid. Yield: 47%. m.p. 99.3e101.2 ꢀC 1H NMR
m
m
added to the assay wells. The assay wells were incubated at 25 ꢀC
for 30e40 min. The enzymatic reaction time was related to the type
of kinase. (The reaction time of TRKA was 30 min, however the
(600 MHz, DMSO‑d6) d 12.24 (s,1H),10.60 (s,1H), 7.36e7.15 (m, 6H),
15

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
reaction time of TRKB and TRKC were 40 min) Finally, a mixture of
quenching solution which containing 200 ng/mL tolbutamide and
5
mL of XL-665 and 5
mL of TK-Antibody-Cryptate was added to
200 ng/mL labetalol in acetonitrile were added sequentially. 50 mL
terminate reactions. The assay well were incubated at 25 ꢀC for
another 60 min, and then HTRF signals were obtained by reading
plates using as Infinite® F500 microplate reader (Tecan,
Switzerland). The ratio of fluorescence at 665 nm to fluorescence at
620 nm was calculated for each well. The data were analysed using
Graphpad prism 8. Every experiment was repeated at least two
times.
of buffer was added to the NCF60 plate, mixed three times thor-
oughly, and then the plate will be incubated at 37 ꢀC for 60 min
while shaking. 44
and then the plate will be incubated at 37 ꢀC for 60 min as well. At 5,
15, 30, 45, and 60 min, add 180 L quenching solution to
‘Quenching’ plates, mix once, and serially transfer 60 L sample
mL of NADPH cofactor was added to the T60 plate,
m
m
from T60 plate per time point to ‘Quenching’ plates. All sampling
plates are shaken for 10 min, then centrifuged at 4000 rpm for
4.2.2. Cell proliferation assay
20 min at 4 ꢀC, and 80
mL of supernatant was removed into 240 mL
The antiproliferative activities of the compounds were evalu-
ated against Km-12, A549 and THLE-2 cell lines by the standard Cell
Counts Kit-8 (CCK-8) assay in vitro. Cells were seeded in 96 well
plates. After incubation 24 h, the cells were exposed to various
concentrations of compounds for addition 72 h. The IC50 values
were calculated by concentration-response curve fitting using
prism 8.
pure water and mixed by plate shaker for 10 min. Each bioanalysis
plate was sealed and shaken for 10 min prior to LC-MS/MS analysis.
4.2.6. Cytochrome P450 inhibition assay
Cytochrome P450 inhibition was evaluated in human liver mi-
crosomes (0.253 mg/mL) using five probe substrates (CYP1A2,
phenacetin; CYP2C9, diclofenac; CYP2C19, S-mephenytoin;
CYP2D6, dextromethorphan; and CYP3A4, midazolam.) in the
4.2.3. Flow cytometry
presence of compound 19m (10 mM). After preincubation for 10 min
The FITC-Annexin V and PI apoptosis kit was purchased from US
Everbright® Inc. (item: F6012), and the experimental operation was
carried out according to the protocol provided by the manufacturer.
Briefly, Km-12 cells were incubated with compound 19m (0.0625,
at 37 ꢀC, an NADPH-regenerating system was added. After the
mixed system was incubated for 15 min at 37 ꢀC, the reaction was
stopped by adding 400 mL of cold stop solution (200 ng/mL tolbu-
tamide and 200 ng/mL labetalol in acetonitrile). The incubation
mixtures were then centrifuged, and supernatants were analyzed
by LC-MS/MS.
0.125, 0.25, 0.5, or 1
trypsin, harvested, and washed twice with PBS. The cells were
counted, and 105 cells were dispersed in 100 L of Annexin V
binding buffer solution, followed by the addition of 4 L of FITC-
Annexin V working fluid and 2 L of PI working fluid to the solu-
tion. After incubation at room temperature in the dark for 10 min,
400 L of PBS were added, and the fluorescence intensity was
mM) for 48 h; then, the cells were digested by
m
m
4.2.7. Pharmacokinetic study in SD rats
m
Pharmacokinetic properties were investigated in SD rats (n ¼ 3,
pear group) after single intravenous (1 mg/kg) and oral adminis-
tration (3 mg/kg). The blood samples were collected in heparin-
containing tubes at specified time points and centrifuged imme-
diately at 4 ꢀC and 4000 rpm for 20 min. Noncompartmental
pharmacokinetic parameters were fitted using phoenix WinNonlin
6.3 based on the LC-MS/MS quantitation data.
m
detected by flow cytometry (FITC-Annexin V: Abs/Em ¼ 494/
518 nm, PI: Abs/Em ¼ 535/617 nm).
4.2.4. Plasma stability assay
The pooled frozen human plasma (BioreclamationIVT, batch No.
BRH1569252) was thawed in a water bath at 37 ꢀC prior to exper-
iment. Plasma was centrifuged at 4000 rpm for 5 min and the clots
were removed if any. The pH will be adjusted to 7.4 0.1 if required.
4.2.8. Molecular docking study
The TRKA crystal structure (PDB code: 5KVT), which was
downloaded from protein data bank (https://www.rcsb.org/), was
processed with the Protein Preparation Wizard in the Schrçdinger
suite. The protein structure was adjusted and modified, followed by
adding hydrogen atoms, deleting solvent water molecules, and
defining right bonds orders using Prime. The protonation and
tautomeric states of Asp, Lys, and His were assigned at pH 7.4 state.
Afterward, all hydrogen atoms of TRKA complexes were optimized
with OPLS_2005 force field, which minimized and converged heavy
atoms to a RMSD of 0.3. The selected inhibitors were prepared by
using LigPrep from the Schrçdinger suite with the OPLS_2005 force
field. The structure of inhibitors was also adjusted and modified,
followed by adding all hydrogen atoms, checking the bond order
and atom types.
Receptor grids were generated before docking with allosteric
site determined by the literatures. The prepared proteineligand
complex was imported into Glide 9.7, which defined it as the re-
ceptor structure with size box (15 Å ꢂ 15 Å ꢂ 15 Å). Based on the
OPLS_2005 force field, the grid of TRKA crystal structure was
generated. The extra precision (XP) mode was set for docking
studies with two crucial residues Glu-590 and Met-592 in con-
strained binding to get accurate results.
100
L of 10 mM stock solution with 495
The other detected compound solution was prepared by the same
method. 98 L of blank plasma was spiked with 2 L of dosing
solution (100 M) to achieve 2 M of the final concentration in
duplicate and samples were Incubated at 37 ꢀC in a water bath. At
each time point (0, 10, 30, 60 and 120min), 400 L of stop solution
mM propantheline bromide solution was prepared by diluting
5
m
mL H2O as a positive control.
m
m
m
m
m
(200 ng/mL tolbutamide and 200 ng/mL Labetalol in ACN) was
added to precipitate protein and mixed thoroughly. Centrifuged
sample plates at 4000 rpm for 15 min. An aliquot of supernatant
(50 mL) was transferred from each well and mixed with 100 mL ultra
pure water. The samples were shaked at 800 rpm for about 10 min
before submitting to LC-MS/MS analysis.
4.2.5. Liver microsomal stability assay
The empty ‘Incubation’ plates T60 and NCF60 were pre-warmed
for 10 min. The liver microsomes (Human, Corning, Lot No.38295)
was diluted to 0.59 mg/mL as working solutions in 100 mM po-
tassium phosphate buffer. 445
transferred into pre-warmed ‘Incubation’ plates T60 and NCF60.
Then, both of plates were shook at 37 ꢀC for 10 min, 5
L of com-
pound working solution (100 M) was added and mixed three
times thoroughly after 54 L of liver microsomes was transferred to
another black plate. To the black plate, 6 L of NADPH solution
(NADPH powder which purchased from Chem-Impex International,
Cat No.00616 diluted into 10 mM MgCl2 solution) and 180
mL of working solutions were
m
m
Declaration of competing interest
m
m
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.
mL
16

T. Wu, C. Zhang, R. Lv et al.
European Journal of Medicinal Chemistry 223 (2021) 113627
activity of the thyroid TRK-T3 oncogene requires signalling through Shc, Br. J.
Canc. 87 (2002) 645e653.
Acknowledgment
[18] T. Wang, M.L. Lamb, D.A. Scott, H. Wang, M.H. Block, P.D. Lyne, J.W. Lee,
A.M. Davies, H.J. Zhang, Y. Zhu, F. Gu, Y. Han, B. Wang, P.J. Mohr, R.J. Kaus,
J.A. Josey, E. Hoffmann, K. Thress, T. Macintyre, H. Wang, C.A. Omer, D. Yu,
Identification of 4-aminopyrazolylpyrimidines as potent inhibitors of Trk ki-
nases, J. Med. Chem. 51 (2008) 4672e4684.
We gratefully acknowledge the Program for Innovative Research
Team of the Ministry of Education and the Program for Liaoning
Innovative Research Team in University.
[19] T. Wang, M.L. Lamb, M.H. Block, A.M. Davies, Y.X. Han, E. Hoffmann,
S. Ioannidis, J.A. Josey, Z.Y. Liu, P.D. Lyne, T. MacIntyre, P.J. Mohr, C.A. Omer,
T. Sjogren, K. Thress, B. Wang, H.Y. Wang, D.W. Yu, H.J. Zhang, Discovery of
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G. Canevari, L. Ceriani, M. Colombo, L. Corti, D. Donati, M. Fasolini, E. Felder,
C. Fiorelli, F. Fiorentini, A. Galvani, A. Isacchi, A.L. Borgia, C. Marchionni,
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113627.
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