N-(Pyrimidin-2-yl)-1,2,3,4-tetrahydroisoquinolin-6-amine Derivatives as Selective Janus Kinase 2 Inhibitors for the Treatment of Myeloproliferative Neoplasms

Article abstract of DOI:10.1021/acs.jmedchem.0c01488

In this study, we described a series of N-(pyrimidin-2-yl)-1,2,3,4-tetrahydroisoquinolin-6-amine derivatives as selective JAK2 (Janus kinase 2) inhibitors. Systematic exploration of the structure-activity relationship though cyclization modification based on previously reported compound 18e led to the discovery of the superior derivative 13ac. Compound 13ac showed excellent potency on JAK2 kinase, SET-2, and Ba/F3V617F cells (high expression of JAK2V617F mutation) with IC50 values of 3, 11.7, and 41 nM, respectively. Further mechanistic studies demonstrated that compound 13ac could downregulate the phosphorylation of downstream proteins of JAK2 kinase in cells. Compound 13ac also showed good selectivity in kinase scanning and potent in vivo antitumor efficacy with 82.3% tumor growth inhibition in the SET-2 xenograft model. Moreover, 13ac significantly ameliorated the disease symptoms in a Ba/F3-JAK2V617F allograft model, with 77.1% normalization of spleen weight, which was more potent than Ruxolitinib.

Full text of DOI:10.1021/acs.jmedchem.0c01488

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Article
N‑(Pyrimidin-2-yl)-1,2,3,4-tetrahydroisoquinolin-6-amine
Derivatives as Selective Janus Kinase 2 Inhibitors for the Treatment
of Myeloproliferative Neoplasms
Tao Yang,§ Mengshi Hu,§ Yong Chen,§ Mingli Xiang, Minghai Tang, Wenyan Qi, Mingsong Shi, Jun He,
Xue Yuan, Chufeng Zhang, Kongjun Liu, Jiewen Li, Zhuang Yang,* and Lijuan Chen*
Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c01488
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ABSTRACT: In this study, we described a series of N-(pyrimidin-2-yl)-1,2,3,4-tetrahydroisoquinolin-6-amine derivatives as
selective JAK2 (Janus kinase 2) inhibitors. Systematic exploration of the structure−activity relationship though cyclization
modification based on previously reported compound 18e led to the discovery of the superior derivative 13ac. Compound 13ac
showed excellent potency on JAK2 kinase, SET-2, and Ba/F3V617F cells (high expression of JAK2V617F mutation) with IC50 values of
3, 11.7, and 41 nM, respectively. Further mechanistic studies demonstrated that compound 13ac could downregulate the
phosphorylation of downstream proteins of JAK2 kinase in cells. Compound 13ac also showed good selectivity in kinase scanning
and potent in vivo antitumor efficacy with 82.3% tumor growth inhibition in the SET-2 xenograft model. Moreover, 13ac
significantly ameliorated the disease symptoms in a Ba/F3-JAK2V617F allograft model, with 77.1% normalization of spleen weight,
which was more potent than Ruxolitinib.
JAK2 signal are present in many MPN patients. This continuous
activation leads to malignant cell proliferation and inhibits
normal cell apoptosis, thereby causing the occurrence of
MPNs.10−14 Because of the vital function of JAK2 in MPNs,
another JAK2 inhibitor Fedratinib was approved to treat
myelofibrosis (MF) in 2019.15,16 Several other JAK2 inhibitors
with varying degrees of selectivity are in mid to late-stage clinical
trials for MF such as Lestaurtinib (CEP701), Momelotinib
(CYT-387), Gandotinib (LY2784544), and Pacritinib.17−22
Although these drugs have shown excellent disease modification,
they all exhibited more or less side effects especially Fedratinib,
which was given a “black box warning” on the risk of serious and
INTRODUCTION
■
Myeloproliferative neoplasms (MPNs) are a group of malignant
bone marrow proliferative diseases originating from hemato-
poietic stem cells, which manifest as excessive proliferation of
one or more lines of myeloid cells.1,2 Such diseases include
polycythemia vera (PV), essential thrombocythemia (ET), and
primary myelofibrosis (PMF), and these diseases can be
transformed into each other.3 In myelofibrosis, stem cell
transplantation and small molecule therapy are preferred
methods of treatment for genetic or clinical high-risk diseases.4
The study of MPNs has also facilitated the development of Janus
kinase (JAK) inhibitors, such as JAK1/JAK2 inhibitor
Ruxolitinib, which has shown promising activity in controlling
constitutional symptoms and splenomegaly in MF and PV.5,6
In the JAK kinase family, JAK2 plays a key role in MPNs,
especially the JAK2V617F mutation, which is up to 95% in PV and
50% in ET and PMF.7−9 The JAK2V617F mutation function has
been fully verified, and studies of gene expression profiles have
clearly shown that this mutation and the continuously activated
Received: August 26, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01488
© XXXX American Chemical Society
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A

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Figure 1. Representative drugs in clinical trials or launched JAK2 inhibitors.
Figure 2. (A) Docking results of 18e in the JAK2 protein (PDB code: 2XA4); (B) Docking results of 18e in the FLT3 protein (PDB code: 4RT7); (C)
Modifying plans for newly designed compounds.
fatal encephalopathy by the FDA.23 The side effects may be
caused by multitarget activity. Therefore, the development of
novel selective JAK2 inhibitors was in need, such as the
prospective candidate BMS-911543 (Figure 1).24
In our previous studies, we reported a dual JAK2/FLT3 kinase
inhibitor 18e. The 2-aminopyrimidine scaffold provided a novel
hinge binding motif for the design of small-molecular ATP-
competitive JAK2/FLT3 inhibitors.25 We analyzed the crystal
structure and docking data of 18e and found that the
aminopiperidine tail formed hydrogen bonds with receptor
residues such as Met665 and Asp829, which played a vital role in
interaction with FLT3 protein while the tail was exposed to the
solvent region in the JAK2-binding pocket (Figure 2A,B). We
hypothesized that the modification of the tail might provide a
solution to reduce the binding activity for FLT3 but retain the
activity for JAK2. Cyclization is a commonly used method in
drug design to improve selectivity, such as the broad-spectrum
anticancer drug Repotrectinib, Larotrectinib, and the fourth
generation EGFR inhibitor BI-4020.26−28 Therefore, our group
considered cyclizing the tail and modifying the newly formed
B
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ring with various substituents to improve the JAK2 selectivity
and activity (Figure 2C). In this article, we described our efforts
toward identifying potent, selective JAK2 inhibitors for the
treatment of MPNs.
As shown in Table 2, when R1 was substituted by F, the
activity of compounds 13f, 13i, and 13l−13n against JAK2
slightly decreased while the FLT3 potency was increased
apparently. We thought that the electron-withdrawing effect of F
might enhance the interaction between the amino acid residue
and pyrimidine amine, which resulted in an increase in the
activity for JAK2 and FLT3. However, the effect of F went
against selectivity of JAK2 versus FLT3. Furthermore, the F
atom introduced into the benzene at R2 position (13k) caused
apparent loss of activity both on JAK2 and FLT3. Compounds
with a rigid urea bond (13g, 13h, and 13j) reduced the activity
toward JAK2 and FLT3 sharply, which indicated the R3
substituent played a key role in activity and selectivity. Among
these compounds, 13d and 13e showed the most potent cellular
activity and acceptable selectivity, but the cellular activity on the
JAK2 related cell lines was still unsatisfactory. Accordingly,
based on 1,2,3,4-tetrahydro-isoquinolin, a series of substituents
at the R3 position were modified for exploring a more effective
and selective compound.
RESULTS AND DISCUSSION
■
In Vitro Structure−Activity Analysis. Our initial SAR
efforts were directed toward cyclizing the tail to retain JAK2
potency and reduce FLT3 potency. SET-2 and Ba/F3 JAK2V617F
cells, which overexpressed JAK2V617F, were chosen to determine
the JAK2 potency. For FLT3, the activity against MOLM-13,
which represents a human acute myeloid leukemia (AML) cell
line harboring FLT3 internal tandem duplication (FLT3-ITD),
was detected.29 At first, analogs containing four kinds of rings
with active substituents hydroxyethyl and hydroxypropyl were
synthesized.25 As illustrated in Table1, 5,6,7,8-tetrahydro-1,6-
Table 1. Structures and In Vitro Biological Activities of
Compounds 13a−e
In the previous research, compounds 13d and 13e showed
selectivity for FLT3, therefore, hydrophilic saturated alkane
substituents with different chain length were introduced to the
R3 position (13o−13p). As shown in Table3, the selectivity and
cellular activity of these two compounds did not improve with
the increase of the chain length. Comparison of the structures of
13q and 13r suggested that the terminal heteroatom played a
certain role in maintaining the activity. Inspired by this result,
the heteroatom substituent cyano was introduced to the same
position and resulted in a significant improvement in cellular
activity and an 8.8-fold increase in selectivity (13t). Sub-
sequently, we extended the cyano chain to obtain compound
13u, and the activity against JAK-relative cells decreased.
Considering that the cyano group is an unsaturated bond, some
other unsaturated groups were also introduced to the R3
position. Obviously, rigid unsaturated substituents had no
positive effect on activity enhancement (13s, 13v, and 13w). We
further analyzed the cyano group and realized the cyano group
possessed potential of hydrolyzing to carboxyl, thus, the carboxyl
derivatives were synthesized. Compared to 13y, 13x showed
more excellent activity and about 35-fold selectivity for FLT3,
which indicated the importance of the double bond. This
conclusion was also confirmed by the two groups of compounds
13aa and 13ab and 13ac and 13ad. As compound 13x possessed
good activity, we further investigated the conjugated structure
by replacing with a larger conjugate system (13z, 13aa).
Obviously, the large conjugated system contributed to
selectivity and activity. Both carbonyl and amino groups
contributed electron-donating effects in the formation of
hydrogen bonds, therefore, the benzoyl was replaced by
dimethylaminoethyl (13ac). Not surprisingly, compound 13ac
also showed potent activity and 75.2-fold selectivity for FLT3. In
view of the metabolic instability of the ester bond of compound
13x, the ester bond was replaced with a more stable amide bond
(13ae−13ah). However, the activities of these compounds were
reduced to certain degrees. So far, we had obtained a number of
compounds with excellent cellular activity and acceptable
selectivity, occasionally, during the synthetic preparation
process, we found that the 6- and 7-amino groups of
tetrahydroisoquinoline were similar in space. Hence, the 7-
amino tetrahydroisoquinoline analogues were also synthesized
on the basis of the earlier active substituent.
a
IC50 = compound concentration required to inhibit tumor cell
proliferation by 50%; data are expressed as the mean SEM from the
dose−response curves of at least three independent experiments. SI
= selectivity index (IC50 (MOLM-13)/IC50 (SET-2)), which
represents the selectivity for JAK2 on cells. % control = kinase
activity remained.
b
c
phthyridine (13a), isoindoline (13b), indoline (13c), and
1,2,3,4-tetrahydro-isoquinolin (13d−13e) all showed obviously
weak potency on FLT3, however, the JAK2 potency also
decreased apparently except for compounds 13d and 13e.
Compared to compound 18e, 13d−13e demonstrated a similar
inhibition for JAK2 and poorer activity for FLT3. We speculated
that the small ring of 13b and 13c limited the conformation of
the substituent, which resulted in poor interactions with the
amino acid residue. Because 1,2,3,4-tetrahydro-isoquinolin
exhibited desirable selectivity, it was selected as a skeleton.
Next, we further examined the effects of substituents around the
skeleton.
As shown in Table 4, most of the newly synthesized
compounds (13ai−13ak) exhibited excellent cellular activity
C
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Table 2. Structures and In Vitro Biological Activities of Compounds 13f−n
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%; data are expressed as the mean SEM from the dose−
b
response curves of at least three independent experiments. SI = selectivity index (IC50 (MOLM-13)/IC50 (SET-2)), which represents the
selectivity for JAK2 on cells. % control = kinase activity remained. = no detected.
c
d
for JAK2 but demonstrated decreased selectivity for FLT3.
These results also indicated the spatial position of the
substituent had a certain influence on the selectivity.
Consideration of the structure novelty, compounds (13t, 13x,
13z, 13aa, 13ac, 13aj, 13al, and 13am) with excellent cellular
activity and selectivity were selected for the next kinase
selectivity screening.
both proved that 13ac was relatively safe. Based on the above
experimental results, 13ac was chosen for further study.
Kinase Selectivity Profiling of 13ac. To further
investigate the kinase selectivity of 13ac, its kinome selectivity
profile was then examined with Kinase Profile technology
developed by Eurofins Discovery. A panel of 361 kinases was
tested at a 0.1 μM concentration of 13ac (Table S1). The
kinome screening revealed that 13ac exhibited expected
selectivity for JAK2 within the human kinome. In addition to
high activity on JAK2, 13ac also demonstrated activity against
TrkA (Figure 3). TrkA belongs to a subfamily of receptor
tyrosine kinases, which is activated by phosphorylation of
specific tyrosine sites through dimerization, subsequently, in
turn activates downstream signaling pathways, including Ras/
MAPK, PI3K/AKT, and PLCγ pathways.32,33 These signaling
pathways are consistent with JAK2’s downstream signaling
pathways, besides, patients with MPNs can secondary activate
downstream pathways such as PI3K and MAPK via the JAK-
STAT signaling pathway, there may be a certain synergy
between JAK2 and TrkA for treating MPNs, and the follow-up
process was still being conducted.34 Overall, 13ac was relatively
selective for JAK2.
To test the potency of these compounds for JAK2 and FLT3,
kinase assays were conducted by Eurofins Discovery. Because of
the high homology of the JAK family, it was thought to be
selective when the selectivity was greater than 10-fold. As shown
in Table 5, compounds 13x, 13aa, 13ac, and 13al exhibited
relative selectivity for JAK2. Ruxolitinib, as the first marketed
drug for the treatment of myelofibrosis, has varying degrees of
damage to the blood cell reduction.30 Another marketed drug
Fedratinib showed obvious damage on liver and spleen.31
Therefore, safety is an important factor in the development of
myelofibrosis drugs. The above-mentioned four compounds all
had good performance on cellular and enzymatic selectivity, so
we tested their toxicity on a series of normal cells. As shown in
Table 6, in comparison with other compounds, 13ac was
relatively safe on a panel of normal cells especially normal liver
cell. At the same time, after continuous oral administration of 30
and 60 mg/kg of 13ac for 14 days, the routine blood test was
conducted (Figure S5), compound 13ac only showed a slight
effect on the hematocrit (HCT %) compared to normal mice.
This result was consistent with in vitro cell safety screening, and
Pharmacokinetics of 13ac in Rats. In vitro screening
indicated that compound 13ac was a potential JAK2 inhibitor,
subsequently, the pharmacokinetic study was conducted. In
vitro liver microsomal metabolism showed that T1/2 of 13ac was
54.6 min, and the in vitro clearance rate was 25.4 L/h/kg, which
D
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Table 3. continued
Table 3. Structures and In Vitro Biological Activities of
Compounds 13o−13h
a
IC50 = compound concentration required to inhibit tumor cell
proliferation by 50%; data are expressed as the mean SEM from the
b
dose−response curves of at least three independent experiments. SI
= selectivity index (IC50 (MOLM-13)/IC50 (SET-2)), which
represents the selectivity for JAK2 on cells. % control = kinase
activity remained. = no detected.
c
d
Table 4. Structures and In Vitro Biological Activities of
Compounds 13ai−am
a
IC50 = compound concentration required to inhibit tumor cell
proliferation by 50%; data are expressed as the mean SEM from the
b
dose−response curves of at least three independent experiments. SI
= selectivity index (IC50 (MOLM-13)/IC50 (SET-2)), which
c
represents the selectivity for JAK2 on cells. % control = kinase
activity remained.
indicated the potential druggability of 13ac. Therefore, in vivo
studies were also performed. The PK properties of 13ac are
summarized in Table 7. In rats, 13ac showed fast absorption
(Tmax = 0.9 h), with a peak concentration of 51.1 ng/mL, an
AUC of 253.6 ng h/mL, and a terminal half-life of ∼1.8 h
following a single oral dose of 5 mg/kg. The oral bioavailability
of 13ac in rat was 25.7%.
In Vivo Xenograft and Allograft Model Experiments.
As compound 13ac showed acceptable pharmacokinetic
properties and excellent activity and kinase selectivity in vitro,
the efficacy of 13ac in vivo was tested in the SET-2 cell-
inoculated xenograft NOD/SCID mouse models. The average
tumor volume at the start was 163.8 mm3, mice were dosed
orally daily for 16 days with three doses of 13ac. In comparison
with Ruxolitinib, 13ac exhibited a significant tumor growth
inhibition of 82.3% without obvious weight change (Table 8,
Figures 4A and S6A). After termination of the treatment, tumor
growth was measured up to day 36. All mice in the control group
died at the 20th day, while only one mouse died in the treatment
group after 36 days (Figure 4C). The survival time of mice was
improved, which indicated the relative safety and effectiveness of
13ac.
In order to further verify the antitumor effect of 13ac in vivo, a
Ba/F3-JAK2V617F allograft model by tail vein injection was
established (detailed experiment method shown in Supporting
Information). The Ba/F3-JAK2V617F mouse allograft represents
a model for a JAK2-driven disease exhibiting hallmark symptoms
E
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Table 5. Kinome Activity and Selectivity of the Preferred Compounds
a
kinase IC50 (nM)
compd
13t
13x
13z
13aa
13ac
13aj
13al
13am
kinase subtype
JAK1
JAK2
JAK3
TYK2
43
7
54
56
108
6.1
7.7
8.0
26
2
77
88
23
13
38.5
44
11.5
47
10
120
79
60
3
50
100
30
20
16.6
33.3
10
42
3
30
2
24
0.9
28
30
18
26.6
31
33
91
9
147
45
23
10
16
5.0
2.5
94
75
62
14
31
25
20.7
89
52
16
15
44.5
26
8
FLT3
34
selectivity (folds)
JAK2/JAK1
JAK2/JAK3
JAK2/TYK2
JAK2/FLT3
4.7
12
7.9
3.4
15.4
20
a
The biochemical tests were provided by Eurofins Discovery. All data were obtained by double testing.
Table 6. Inhibition of Cellular Proliferation in Normal Cell Lines by Preferred Compounds
a
growth inhibition, IC50 (μM)
cell line
Vero
H9c2 (2-1)
LO2
cell type
13x
13aa
13ac
13al
African green monkey kidney cell
rat cardiomyocyte
human normal liver cell
2.23 0.45
2.89 0.13
1.53 0.22
1.05 0.52
1.43 0.32
2.66 0.11
6.75 0.28
6.93 0.18
7.22 0.41
2.47 0.48
1.69 0.16
2.03 0.15
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%; data are expressed as the mean SEM from the dose−
response curves of at least three independent experiments.
of myeloproliferative diseases such as splenomegaly and
hepatomegaly. In this model, murine Ba/F3-JAK2V617F cells
were engrafted by tail vein injection. Treatment with 13ac at
doses of 30 and 60 mg/kg p.o. q.d. was started 4 days after cell
inoculation for 20 consecutive days. Ruxolitinib was chosen as a
positive control. At the study end, model mice exhibited
splenomegaly and hepatomegaly (∼5.9-fold and 1.5-fold,
respectively). Compound 13ac treatment at 30 mg/kg p.o.
q.d. significantly ameliorated the disease symptoms. Compared
to Ruxolitinib, at a dose of 60 mg/kg, 13ac showed a superior
treatment effect with 77.1% normalization of spleen weight
(Figures 4D,E and S6D).
Molecular Docking Study of 13ac. The screening results
at the cell and kinase levels indicated our previous hypothesis,
modification of the tail of lead compound 18e retained the
activity for JAK2 but reduced the potency for FLT3. Further in
vivo study indicated the effectiveness in mice. In order to explore
the protein-binding mode of 13ac, the crystal structure of the
protein kinase domain of JAK2 from the Protein Data Bank
(PDB code: 2XA4) was selected as the docking model.35,36 The
docking results are presented in Figure 5 (see docking methods
in Supporting Information).37−42 As demonstrated, the newly
synthesized cycle was still toward outside the binding pocket, the
reasonable conformation of the 13ac−JAK2 complex suggested
that the pyrimidine amino and one nitrogen atom pyrimidine
formed two strong hydrogen bonds with the Leu932 in the
distance of acceptor−donor of 2.98 and 3.06 Å. The pyrazole
region also connected with Val863, Lys882, Val911, Met929,
Leu983, Gly993, and Asp994 with hydrophobic interaction.
Meanwhile, the pyrimidine ring formed arene-H interaction
with Leu983 and Gly935 at the same time. Simultaneously, the
terminal amine of the isoquinoline exposed in the solvent
formed unexpected electrostatic interaction with Asp939, which
might increase the binding activity. Those interactions
suggested that 13ac possessed a good affinity for JAK2 protein.
Cell Cycle Analysis and Apoptosis Assays in SET-2
Cells. JAK2 plays a vital role in cell proliferation and survival;
therefore, we examined the effect of 13ac on the cell cycle. The
experimental results showed that 13ac induced cell arrest in the
G0/G1 phase in a concentration-dependent manner (Figure
6A). A flow cytometry assay was also performed to examine cell
apoptosis upon treatment with compound 13ac. Compound
13ac induced apoptosis in SET-2 cells in a similar pattern
(Figure 6B). At a concentration of 0.80 μM, an apoptosis rate of
49.3% was observed.
Signaling Inhibition of 13ac in JAK2V617F-Expressing
Cell Lines and Spleen Lysates. In order to investigate
whether the excellent enzymatic activity of compound 13ac
regulates the JAK2 signaling pathway, the phosphorylation
status of JAK2 and downstream substrates was detected by
Western blot analysis of JAK2V617F-expressing cell lines. After a 2
h treatment with increasing concentrations of compound 13ac,
SET-2 and Ba/F3-JAK2V617F cells were harvested and lysed for
an IP/wt assay. As shown in Figure 7A, compound 13ac
inhibited JAK2 and STAT5 phosphorylation in a dose-
dependent manner. To explore the selectivity of 13ac in the
signaling pathway, FLT3 and downstream signaling protein
AKT were detected.29 Compared to JAK2, poorer inhibition of
phosphorylated FLT3 and AKT indicated the selectivity of 13ac.
Selectivity is reflected not only in the kinase level for different
subtypes, but also in the downstream effector molecules in the
signaling pathway. The analysis for STAT of different subtypes
was conducted on the Ba/F3 JAK2V617F cell. As illustrated in
Figure 7B, compound 13ac reduced STAT5 phosphorylation at
a concentration of 50 nM while barely showed obvious
inhibition for other STAT subtypes even at a concentration of
1000 nM. Furthermore, considering the excellent efficacy of
13ac in vivo, we also investigated the influence of 13ac on the
JAK2 signaling pathway on the spleen tissue. On day 25, mice
were killed and tumor lysates were analyzed for JAK2 signaling
by Western blot. As shown in Figure 7C, 13ac demonstrated a
full inhibition of STAT5 phosphorylation.
F
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Figure 3. Kinome-wide selectivity profiling of compound 13ac with the Kinase Profile assay by Eurofins Discovery. Measurements were performed at a
concentration of 0.1 μM of the inhibitor in duplicate. The % control means remaining active kinase percentage. The affinity was defined with respect to
a dimethyl sulfoxide (DMSO) control. The TREEspot image was mapped with the KinMap software tool provided by Cell Signaling Technology, Inc.
(www.cellsignal.com).
G
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Table 7. In Vivo Profiling of Compound 13ac
a
ADME parameter
compd
administration (5 mg/kg)
Cmax (ng/mL)
Tmax (h)
T
1/2 (h)
Clz (L/h/kg)
AUC0-t (ng·h/mL)
F %
13ac
oral
intravenous
51.1 26.5
479.3 28.3
0.9 0.4
0.08
1.8 0.5
4.0 1.1
28.8 9.5
5.01 0.71
253.6 24.1
987.9 110.2
25.7
a
Sprague-Dawley rats (n = 5) were treated with solution (2% ethanol and 1% tween 80, pH5) at a dose of 5 mg/kg.
Table 8. Summary of Tumor Growth Inhibition of Compound 13ac
administration
a
compd
13ac
tumor model
SET-2
schedule
route
P.O
dose (mg/kg)
survivors
5/5
inhibition rate (%)
QD × 16
15
30
60
60
51.4
67.8
82.3
52.8
Ruxolitinib
a
QD, every day.
Figure 4. (A) NOD/SCID mice bearing SET-2 tumors (n = 5) were treated for 16 consecutive days with 15, 30, and 60 mg/kg 13ac p.o. q.d. Tumor
volumes were measured every two days. ***, P < 0.01 ANOVA, Dunnett’s post test; (B) body weight change in SET-2 cell-inoculated xenograft NOD/
SCID mouse models; (C) NOD/SCID mice bearing SET-2 tumors (n = 5) were treated for 36 consecutive days with 15, 30, and 60 mg/kg 13ac p.o.
q.d. the survival curve was analyzed; (D) Ba/F-JAK2V617F-bearing BALB/c-nude mice (n = 6) were treated for 20 consecutive days with 30 and 60 mg/
kg 13ac p.o. q.d. On day 25, mice were sacrificed and spleen and liver weights determined. ***, P < 0.01 ANOVA, Dunnett’s post test; (E) inhibition of
the liver growth in the Ba/F-JAK2V617F allograft model. ***, P < 0.01; **, 0.01 < P < 0.05 ANOVA, Dunnett’s post test; and (F) spleen weight to body
weight ratio in the Ba/F-JAK2V617F allograft model.
tolerated doses. To summarize, 13ac is a promising inhibitor of
JAK2 for the treatment of MPNs.
CONCLUSIONS
■
Based on the progress made in JAK2/FLT3 dual target inhibitor
18e, we designed selective 1,2,3,4-tetrahydro-isoquinolin series
of JAK2 inhibitors. SAR developed around the designed cycle
moieties combined with structure-based design led to the
discovery of 13ac, which possessed >14-fold selectivity over
JAK1,3, FLT3 in enzyme assays. Full kinase spectrum screening
indicated 13ac possesses a relatively high selectivity. Further-
more, the western blot assay confirmed that the antitumor
activities were indeed on-target. Compound 13ac could also
stimulate cell cycle arrest in the G0/G1 phase and induce tumor
cell apoptosis. An efficacy study in a model of JAK2V617F-driven
disease demonstrated dose-dependent tumor growth inhibition
and normalization of splenomegaly and hepatomegaly at well-
CHEMISTRY
■
The syntheses of the 4-(1H-pyrazol-4-yl)-pyrimidin-2-amine
derivatives require the preparation of two pairs of building
blocks, that is, 4-(1H-pyrazol-4-yl)-pyrimidine and anilines. The
synthesis of amines is described in Scheme 1. The intermediate 2
was synthesized from commercially available phenethylamine by
an amidation. Subsequently, the intermediate 2 was cyclized
under concentrated sulfuric acid and paraformaldehyde to
obtain intermediate 3. Treatment of 3 with concentrated sulfuric
acid and potassium nitrate at 0 °C provided 4a−b, from which
analogues without protection could be accessed. Compounds
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Figure 5. (A) 3D Model of 13ac bound to the kinase catalytic domain of JAK2; (B) 2D Model of 13ac bound to the kinase catalytic domain of JAK2
(PDB: 2XA4).
Figure 6. (A) Cell cycle effects of 13ac on SET-2 cells. Cells were treated with increasing concentrations of 13ac for 24 h, harvested, fixed, and stained
with propidium iodide prior to flow cytometric analysis. (B) Effects of 13ac on the induction of apoptosis. SET-2 cells were treated with 13ac for 2 h at
0.05, 0.2, 0.4, 0.8, and 1.6 μM, respectively.
solid loaders packed with Scharlau silica gel (0.04−0.06 mm) on
Biotage FlashMaster Personal + flash chromatography. Counter-
current Chromatography separations was finished with a SCU-Prep
instrument manufactured by our team. The volume of the counter-
current chromatography column is 1500 mL and the rotation speed is
1200 rpm. 1H NMR and 13C NMR spectra were recorded on a Bruker
Avance 400 spectrometer (Bruker Company, Germany) or a Varian
spectrometer (Varian, Palo Alto, CA), using TMS as an internal
standard. Chemical shifts were given in ppm (parts per million). Mass
spectra were recorded on a Q-TOF Premier mass spectrometer
(Micromass, Manchester, UK). The purity of each compound (>95%)
was determined on a Waters e2695 series LC system (column, Xtimate
C18, 4.6 mm × 150 mm, 5 μm; mobile phase, methanol (60%)/H2O
(40%); low rate, 1.0 mL/min; UV wavelength, 254−400 nM;
temperature, 25 °C; and injection volume, 10 μL).
5a−b were further amidated under (BOC)2O. Primary amines
(7a−b) were synthesized by hydrogen reduction.
The primary intermediates 10a−b were synthesized via
alkylation of 8 with 2-iodopropane to provide 9, followed by the
Suzuki reaction with 2, 4-dichloropyrimidine derivatives. The
anilines (7a−b) and pyrimidine intermediates (10a−b) were
coupled via a Buchwald−Hartwig reaction to get 11a−f. The
Boc groups were removed from 11a−f with hydrochloric acid to
produce the corresponding piperazines. The final products
13a−am were obtained by alkylation or amidation (Scheme 2).
EXPERIMENTAL SECTION
■
Synthesis. All the chemical solvents and reagents were purchased
from commercial sources and were used as received. Thin-layer
chromatography was performed on 0.20 mm Silica Gel 60 F254 plates
(Qingdao Haiyang Chemical, China) and visualized under UV light
(254 nm). Flash column chromatography was carried out using fritted
2,2,2-Trifluoro-N-phenethylacetamide (2). To a solution of 2-
phenylethan-1-amine (12.1 g, 0.1 mol) in acetonitrile (200 mL),
K2CO3 (34.5 g, 0.25 mmol) and trifluoroacetic anhydride (17.0 mL,
0.12 mol) were added, and the mixture was stirred at room temperature
I
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Figure 7. Inhibition of the JAK2 signaling pathway by 13ac. (A) SET-2, (B) Ba/F3 JAK2V617Fcells, and (C) spleen lysates of the Ba/F3 JAK2V617F
allograft model were treated for 3 h with 13ac. After lysis, the proteins were detected by immunoblotting with antibodies.
a
Scheme 1. Synthesis of Amino Derivatives 7a−b
a
Reagents and conditions: (a) trifluoroacetic anhydride, K2CO3, acetonitrile, rt; (b) paraformaldehyde, concentrated sulfuric acid, rt; (c)
concentrated sulfuric acid, potassium nitrate, 0 °C; (d) hydrochloric acid, methanol, reflux; (e) (BOC)2O, 60 °C; and (f) Pd/C, H2, methanol, rt.
a
Scheme 2. Synthesis of 13a−am
a
Reagents and conditions: (g) 2-iodopropane, K2CO3, acetonitrile, reflux; (h) 2,4-dichloropyrimidine derivatives, dioxane/EtOH/water (v/v/v, 7/
3/4), 80 °C, 2 h; (i) amines, dioxane, reflux, 4 h, N2 atmosphere; (j) hydrochloric acid, EA, rt; (k) appropriate halide, K2CO3, acetonitrile, rt. And
(l) appropriate acid, HATU, DIEA, DCM, rt.
(rt) for 1 h. The completion of the reaction was checked with TLC on
silica (silica gel: DCM/MeOH = 20:1). The mixture was dried under
reduced pressure. 50 mL of water was added to the concentrated
solution, the solid was precipitated, filtered with suction, and dried to
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obtain the title compound. Yield: 88%. 1H NMR (400 MHz,
Chloroform-d): δ 7.67 (t, J = 6.3 Hz, 1H), 7.33−7.24 (m, 2H),
7.25−7.16 (m, 3H), 3.46 (dt, J = 6.4, 5.6 Hz, 2H), 2.76 (tt, J = 5.6, 1.0
Hz, 2H). HRMS (ESI) m/z: 217.0774 [M + H+].
(13a). 1H NMR (400 MHz, DMSO-d6): δ 9.19 (s, 1H), 8.38−8.30 (m,
2H), 8.11 (t, J = 4.2 Hz, 2H), 7.47 (d, J = 8.5 Hz, 1H), 4.63 (h, J = 6.6
Hz, 1H), 4.44 (d, J = 38.4 Hz, 1H), 3.55−3.44 (m, 4H), 2.80 (t, J = 5.9
Hz, 2H), 2.73 (t, J = 6.1 Hz, 2H), 2.54 (d, J = 7.1 Hz, 2H), 2.33 (s, 3H),
1.72−1.64 (m, 2H), 1.48 (d, J = 6.7 Hz, 6H). 13C NMR (101 MHz,
DMSO): δ 159.93, 158.01, 157.82, 152.89, 151.72, 139.55, 136.47,
129.69, 123.47, 120.45, 117.59, 110.21, 59.81, 55.19, 54.71, 53.83,
50.98, 32.20, 30.45, 23.03, 17.19. HRMS (ESI) m/z: 408.5245 [M +
H+].
2-(5-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
yl)amino)indolin-1-yl)ethan-1-ol (13b). 1H NMR (400 MHz,
DMSO-d6): δ 8.90 (s, 1H), 8.24 (d, J = 41.8 Hz, 2H), 8.05 (s, 1H), 7.49
(s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 6.45 (d, J = 8.4 Hz, 1H), 4.63 (dq, J =
20.3, 6.8, 6.1 Hz, 2H), 3.61 (q, J = 6.1 Hz, 2H), 3.31 (d, J = 8.9 Hz, 1H),
3.05 (t, J = 6.3 Hz, 2H), 2.87 (t, J = 8.2 Hz, 2H), 2.28 (s, 3H), 1.47 (d, J
= 6.7 Hz, 6H). 13C NMR (101 MHz, DMSO): δ 159.79, 159.38,
157.61, 148.07, 139.29, 132.27, 129.86, 129.27, 120.84, 118.60, 117.13,
115.43, 107.01, 59.57, 54.30, 53.78, 52.86, 29.01, 23.04, 17.11. HRMS
(ESI) m/z: 379.4856 [M + H+].
1-(3,4-Dihydroisoquinolin-2(1H)-yl)-2,2,2-trifluoroethan-1-
one (3). To a solution of 2,2,2-trifluoro-N-phenethylacetamide (18.5 g,
0.085 mol) in concentrated sulfuric acid (100 mL) was slowly added
paraformaldehyde (3.0 g, 0.1 mol), and the mixture was stirred at room
temperature (rt) for 2 h. After the reaction completed, the mixture was
very slowly added to ice water and extracted with ethyl acetate (200
mL) twice. The organic phases were collected together and
concentrated under reduced pressure. The solid was washed with
ether (50 mL) to obtain the title compound. Yield: 60%. 1H NMR (400
MHz, Chloroform-d): δ 7.23 (ddt, J = 6.1, 3.9, 0.9 Hz, 1H), 7.20−7.11
(m, 3H), 4.55 (d, J = 1.0 Hz, 2H), 3.77 (dt, J = 8.6, 5.6 Hz, 2H), 2.90
(ddt, J = 8.7, 5.8, 0.7 Hz, 2H). HRMS (ESI) m/z: 229.0768 [M + H+].
2,2,2-Trifluoro-1-(6-nitro-3,4-dihydroisoquinolin-2(1H)-yl)-
ethan-1-one (4a). A solution of 1-(3,4-dihydroisoquinolin-2(1H)-
yl)-2,2,2-trifluoroethan-1-one (11.0 g, 0.048 mol) in concentrated
sulfuric acid (100 mL) was cooled to 0 °C, and potassium nitrate (12.1
g, 0.12 mol) was slowly added to the mixture while keeping the
temperature below 0 °C. After 2 h, the reaction solution was very slowly
added to ice water and extracted with ethyl acetate (150 mL) twice. The
organic phases were collected together and concentrated under reduced
pressure. The residue was pretreated by counter-current chromatog-
raphy (solvent system: n-hexane/ethyle acetate/methanol:/water
5:1:5:1; mobile phase: upper phase; rotation speed: 1200 rpm; and
flow rate: 50 mL/min) and then purified by FlashMaster Personal +
chromatography (silica gel: PE/EA = 90:10) to give the desired product
4a and 4b. 1H NMR (400 MHz, Chloroform-d): δ 7.99 (dd, J = 8.2, 2.3
Hz, 1H), 7.78 (dt, J = 2.2, 1.0 Hz, 1H), 7.40 (dt, J = 8.2, 1.0 Hz, 1H),
4.59 (d, J = 1.0 Hz, 2H), 3.76 (ddd, J = 8.8, 6.0, 1.8 Hz, 2H), 2.94 (ddt, J
= 8.4, 5.8, 1.1 Hz, 2H). HRMS (ESI) m/z: 274.0628 [M + H+].
6-Nitro-1,2,3,4-tetrahydroisoquinoline (5a). To a solution of
2,2,2-trifluoro-1-(6-nitro-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-
one (7.8 g, 0.029 mol) in MeOH (60 mL) was added hydrochloric acid
(10 mL) and refluxed for 2 h. The mixture was concentrated under
reduced pressure and the desired product was obtained without
purification. Yield: 94%. 1H NMR (400 MHz, Chloroform-d): δ 7.96
(dd, J = 8.4, 2.2 Hz, 1H), 7.76 (dt, J = 2.1, 1.0 Hz, 1H), 7.32 (dt, J = 8.5,
1.0 Hz, 1H), 4.06 (dd, J = 7.5, 1.0 Hz, 2H), 3.00 (dddd, J = 7.8, 6.6, 5.9,
4.0 Hz, 2H), 2.86 (dddd, J = 11.6, 6.6, 3.8, 0.9 Hz, 2H), 2.66 (tt, J = 7.5,
5.9 Hz, 1H). HRMS (ESI) m/z: 179.0756 [M + H+].
2-(5-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
1
yl)amino)isoindolin-2-yl)ethan-1-ol (13c). H NMR (400 MHz,
DMSO-d6): δ 9.29 (s, 1H), 8.30 (d, J = 21.5 Hz, 2H), 8.08 (s, 1H), 7.76
(s, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 4.63 (p, J =
6.7 Hz, 1H), 4.51 (s, 1H), 3.85 (d, J = 20.6 Hz, 4H), 3.64−3.47 (m,
2H), 2.76 (t, J = 6.3 Hz, 2H), 2.31 (s, 3H), 1.48 (d, J = 6.7 Hz, 6H). 13C
NMR (101 MHz, DMSO): δ 159.84, 159.00, 157.71, 140.81, 140.30,
139.34, 132.79, 129.43, 122.29, 120.66, 117.42, 116.47, 112.67, 60.46,
59.70, 59.04, 58.44, 53.82, 23.03, 17.15. HRMS (ESI) m/z: 379.4866
[M + H+].
2-(6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-ol (13d).
1H NMR (400 MHz, DMSO-d6): δ 9.20 (s, 1H), 8.29 (d, J = 22.7
Hz, 2H), 8.08 (s, 1H), 7.65 (d, J = 2.3 Hz, 1H), 7.46 (dd, J = 8.3, 2.3 Hz,
1H), 6.94 (d, J = 8.4 Hz, 1H), 4.62 (h, J = 6.6 Hz, 1H), 4.46 (s, 1H),
3.67−3.46 (m, 4H), 2.80 (d, J = 5.8 Hz, 2H), 2.71 (t, J = 5.8 Hz, 2H),
2.57 (t, J = 6.2 Hz, 2H), 2.31 (s, 3H), 1.48 (d, J = 6.7 Hz, 6H). 13C NMR
(101 MHz, DMSO): δ 159.88, 159.00, 157.63, 139.42, 139.31, 134.44,
129.38, 127.92, 126.71, 120.74, 118.31, 116.72, 116.35, 60.74, 59.30,
56.07, 53.83, 51.64, 29.63, 23.03, 17.15. HRMS (ESI) m/z: 393.5070
[M + H+].
3-(6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)propan-1-ol (13e).
1H NMR (400 MHz, DMSO-d6): δ 9.20 (s, 1H), 8.32 (s, 1H), 8.26
tert-Butyl 6-Nitro-3,4-dihydroisoquinoline-2(1H)-carboxy-
late (6a). A solution of 6-nitro-1,2,3,4-tetrahydroisoquinoline (4.8 g,
0.027 mol) in (BOC)2O (20 mL) was heated at 60 °C for 2 h, filtered,
and washed with ether once to get the target compound. Yield: 90%. 1H
NMR (400 MHz, Chloroform-d): δ 7.99 (dd, J = 8.1, 2.3 Hz, 1H), 7.78
(dt, J = 2.1, 1.0 Hz, 1H), 7.28 (dt, J = 8.2, 1.0 Hz, 1H), 4.61 (d, J = 1.1
Hz, 2H), 3.73 (ddd, J = 9.1, 6.4, 4.2 Hz, 2H), 2.97 (ddd, J = 9.1, 6.4, 1.1
Hz, 2H), 1.47 (s, 7H). HRMS (ESI) m/z: 279.1356 [M + H+].
tert-Butyl 6-Amino-3,4-dihydroisoquinoline-2(1H)-carboxy-
late (7a). A solution of tert-butyl 6-nitro-3,4-dihydroisoquinoline-
2(1H)-carboxylate (5.2 g, 0.024 mol) and Pd/C (0.52 g) in MeOH (50
mL) was reduced under the atmosphere of H2. The mixture was filtered
and concentrated to obtained the intermediate. Yield: 98%. 1H NMR
(400 MHz, Chloroform-d): δ 7.19 (dt, J = 8.3, 1.0 Hz, 1H), 6.60 (dd, J =
8.1, 2.3 Hz, 1H), 6.54 (dt, J = 2.1, 1.0 Hz, 1H), 4.75 (s, 1H), 4.59 (d, J =
0.9 Hz, 2H), 3.73 (dd, J = 9.1, 6.4 Hz, 2H), 2.82 (dddd, J = 9.2, 7.4, 6.4,
1.0 Hz, 2H), 1.47 (s, 6H). HRMS (ESI) m/z: 271.1567 [M + Na +].
The synthesis method of intermediates 9−12 was described in the
previous literature, herein, we just described the synthesis of amide.
General Synthetic Procedure 1. Amidation. To a solution of
secondary amine(0.01 mmol, 1.0 equiv), HATU (0.01 mmol, 1.0
equiv), and N,N-diisopropylethylamine (0.025 mmol, 2.5 equiv) in
DCM (20 mL) was added acid (0.01 mmol, 1.0 equiv). The reaction
mixture was stirred at room temperature for 0.5 h and filtered, and the
solid was washed with EA to give the desired product.
(s, 1H), 8.08 (s, 1H), 7.65 (d, J = 2.2 Hz, 1H), 7.47 (dd, J = 8.3, 2.3 Hz,
1H), 6.95 (d, J = 8.3 Hz, 1H), 4.75−4.39 (m, 2H), 3.57−3.42 (m, 4H),
2.80 (t, J = 5.8 Hz, 2H), 2.64 (t, J = 5.9 Hz, 2H), 2.50 (s, 2H), 2.31 (s,
3H), 1.68 (p, J = 6.5 Hz, 2H), 1.48 (d, J = 6.7 Hz, 6H). 13C NMR (101
MHz, DMSO): δ 159.89, 159.00, 157.64, 139.46, 139.31, 134.46,
129.39, 127.83, 126.75, 120.74, 118.30, 116.75, 116.36, 59.96, 55.86,
55.56, 53.83, 51.25, 30.34, 29.66, 23.04, 17.15. HRMS (ESI) m/z:
407.5340 [M + H+].
1-(6-((5-Fluoro-4-(1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxyethan-
1-one (13f). 1H NMR (400 MHz, DMSO-d6): δ 9.55 (d, J = 4.7 Hz,
1H), 8.44 (d, J = 45.8 Hz, 2H), 8.09 (s, 1H), 7.68 (s, 1H), 7.55 (t, J = 6.6
Hz, 1H), 7.11 (dd, J = 13.3, 8.1 Hz, 1H), 4.76−4.42 (m, 4H), 4.19 (d, J
= 5.2 Hz, 2H), 3.64 (dt, J = 51.4, 5.8 Hz, 2H), 2.83 (dt, J = 28.2, 5.8 Hz,
2H), 1.48 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, DMSO): δ 170.87,
156.92, 150.02, 147.54, 146.90, 146.25, 139.54, 138.88, 134.92, 129.87,
126.97, 126.44, 118.39, 117.31, 115.71, 60.74, 54.01, 43.89, 41.70,
29.42, 22.96. HRMS (ESI) m/z: 411.4546 [M + H+].
N-(3-Chlorophenyl)-6-((4-(1-isopropyl-1H-pyrazol-4-yl)-5-
methylpyrimidin-2-yl)amino)-3,4-dihydroisoquinoline-2(1H)-
carboxamide (13g). 1H NMR (400 MHz, DMSO-d6): δ 9.30 (s, 1H),
8.73 (s, 1H), 8.31 (d, J = 21.9 Hz, 2H), 8.09 (s, 1H), 7.72 (d, J = 26.0
Hz, 2H), 7.57 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.26 (t, J =
8.1 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 7.9 Hz, 1H), 4.61 (d, J
= 16.3 Hz, 3H), 3.71 (t, J = 5.8 Hz, 2H), 2.85 (t, J = 5.9 Hz, 2H), 2.32 (s,
3H), 1.49 (d, J = 6.7 Hz, 6H). 13C NMR (101 MHz, DMSO): δ 159.90,
158.95, 157.69, 155.14, 142.73, 139.93, 139.34, 135.07, 133.20, 130.35,
3-(2-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
yl)amino)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)propan-1-ol
K
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129.44, 126.68, 126.38, 121.69, 120.70, 119.38, 118.38, 118.27, 117.16,
116.57, 53.84, 45.80, 41.95, 29.27, 23.04, 17.16. HRMS (ESI) m/z:
503.0190 [M + H+].
8.4, 1.9 Hz, 2H), 6.98 (d, J = 8.3 Hz, 1H), 4.67 (dt, J = 13.3, 6.6 Hz, 2H),
4.35 (t, J = 4.9 Hz, 2H), 3.50 (s, 1H), 3.44−3.37 (m, 5H), 2.86−2.75
(m, 4H), 2.66 (s, 0H), 2.44 (d, J = 5.0 Hz, 1H), 1.58−1.40 (m, 8H),
1.33 (dt, J = 15.3, 7.1 Hz, 3H). HRMS (ESI) m/z: 439.5514 [M + H+].
4-(6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)butan-1-ol (13o).
1H NMR (400 MHz, DMSO-d6): δ 9.19 (s, 1H), 8.29 (d, J = 23.0
Hz, 2H), 8.08 (s, 1H), 7.64 (d, J = 2.3 Hz, 1H), 7.46 (dd, J = 8.3, 2.3 Hz,
1H), 6.95 (d, J = 8.4 Hz, 1H), 4.63 (q, J = 6.6 Hz, 1H), 4.56 (d, J = 26.0
Hz, 1H), 3.56−3.35 (m, 4H), 2.80 (t, J = 5.9 Hz, 2H), 2.63 (t, J = 5.8
Hz, 2H), 2.43 (t, J = 7.0 Hz, 2H), 2.31 (s, 3H), 1.55 (q, J = 7.2 Hz, 2H),
1.48 (d, J = 6.7 Hz, 6H). 13C NMR (101 MHz, DMSO): δ 159.03,
158.27, 158.19, 140.86, 139.50, 131.80, 129.78, 127.32, 121.05, 120.48,
118.02, 117.76, 116.99, 60.49, 60.22, 55.58, 53.89, 52.27, 49.47, 29.86,
29.68, 25.74, 23.02, 22.91, 21.01, 17.16. HRMS (ESI) m/z: 421.5610
[M + H+].
N-Ethyl-6-((4-(1-isopropyl-1H-pyrazol-4-yl)-5-methylpyrimi-
din-2-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxamide
(13h). 1H NMR (400 MHz, DMSO-d6): δ 9.26 (s, 1H), 8.30 (d, J =
22.7 Hz, 2H), 8.08 (s, 1H), 7.70 (d, J = 2.2 Hz, 1H), 7.53 (dd, J = 8.3,
2.3 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.49 (t, J = 5.4 Hz, 1H), 4.62 (h, J
= 6.5 Hz, 1H), 4.41 (s, 2H), 3.54 (t, J = 5.8 Hz, 2H), 3.08 (dt, J = 12.5,
6.3 Hz, 2H), 2.75 (t, J = 5.9 Hz, 2H), 2.31 (s, 3H), 1.48 (d, J = 6.6 Hz,
6H), 1.03 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO): δ 159.90,
158.95, 157.80, 157.67, 139.71, 139.32, 135.21, 129.43, 126.95, 126.59,
120.70, 118.43, 117.08, 116.51, 53.83, 45.46, 41.38, 35.38, 29.23, 23.04,
17.15, 16.15. HRMS (ESI) m/z: 320.5330 [M + H+].
3-(6-((5-Fluoro-4-(1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)propan-1-ol (13i).
1H NMR (400 MHz, DMSO-d6): δ 10.24 (s, 1H), 9.68 (s, 1H), 8.53
5-(6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)pentan-1-ol (13p).
1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.32 (s, 1H), 8.27
(d, J = 3.0 Hz, 1H), 8.40 (d, J = 2.0 Hz, 1H), 8.10 (d, J = 1.1 Hz, 1H),
7.76 (d, J = 2.2 Hz, 1H), 7.60 (dd, J = 8.4, 2.2 Hz, 1H), 7.14 (d, J = 8.5
Hz, 1H), 4.67 (p, J = 6.6 Hz, 1H), 4.25 (d, J = 36.4 Hz, 2H), 3.37 (s,
4H), 3.24 (t, J = 8.1 Hz, 2H), 3.12 (s, 2H), 1.94 (dq, J = 11.9, 6.0 Hz,
2H), 1.48 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, DMSO): δ 156.98,
149.96, 147.48, 146.97, 146.18, 139.09, 138.86, 134.51, 128.24, 126.82,
118.38, 116.85, 115.73, 59.94, 55.80, 55.52, 54.00, 51.18, 30.28, 29.56,
22.94. HRMS (ESI) m/z: 411.4974 [M + H+].
6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
yl)amino)-N-propyl-3,4-dihydroisoquinoline-2(1H)-carboxa-
mide (13j). 1H NMR (400 MHz, DMSO-d6): δ 9.26 (s, 1H), 8.30 (d, J
= 22.9 Hz, 2H), 8.09 (s, 1H), 7.71 (d, J = 2.2 Hz, 1H), 7.54 (dd, J = 8.4,
2.2 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.50 (t, J = 5.5 Hz, 1H), 4.63
(hept, J = 6.7 Hz, 1H), 4.43 (s, 2H), 3.55 (t, J = 5.8 Hz, 2H), 3.02 (q, J =
7.8, 7.0 Hz, 2H), 2.76 (t, J = 5.8 Hz, 2H), 2.32 (s, 3H), 1.46 (dd, J =
18.8, 6.9 Hz, 8H), 0.84 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz,
DMSO): δ 159.90, 158.95, 157.88, 157.67, 139.70, 139.32, 135.21,
129.43, 126.99, 126.60, 120.70, 118.43, 117.08, 116.51, 53.83, 45.50,
42.47, 41.44, 29.22, 23.56, 23.04, 17.15, 11.87. HRMS (ESI) m/z:
434.5600 [M + H+].
2-(7-Fluoro-6-((4-(1-isopropyl-1H-pyrazol-4-yl)-5-methyl-
pyrimidin-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)ethan-
1-ol (13k). 1H NMR (400 MHz, DMSO-d6): δ 8.56 (s, 1H), 8.26 (d, J
= 25.6 Hz, 2H), 8.01 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 6.92 (d, J = 11.5
Hz, 1H), 4.61 (p, J = 6.7 Hz, 1H), 4.54 (s, 1H), 3.82−3.42 (m, 4H),
2.96−2.63 (m, 4H), 2.55 (t, J = 6.5 Hz, 2H), 2.30 (s, 3H), 1.46 (d, J =
6.6 Hz, 6H). 13C NMR (101 MHz, DMSO): δ 159.91, 159.09, 157.81,
154.07, 151.66, 139.34, 130.75, 129.94, 129.42, 123.69, 120.56, 116.87,
113.15, 60.46, 59.27, 55.69, 53.82, 51.37, 28.78, 23.02, 17.08. HRMS
(ESI) m/z: 411.4974 [M + H+].
(s, 1H), 8.08 (s, 1H), 7.65 (d, J = 2.2 Hz, 1H), 7.47 (dd, J = 8.3, 2.3 Hz,
1H), 6.95 (d, J = 8.3 Hz, 1H), 4.63 (p, J = 6.7 Hz, 1H), 4.34 (s, 1H),
3.40 (t, J = 5.7 Hz, 2H), 2.80 (t, J = 5.9 Hz, 2H), 2.63 (t, J = 5.9 Hz, 2H),
2.43 (t, J = 7.3 Hz, 2H), 2.31 (s, 3H), 1.58−1.41 (m, 10H), 1.35 (qd, J =
9.8, 8.4, 4.5 Hz, 2H). 13C NMR (101 MHz, DMSO): δ 158.46, 157.32,
157.25, 152.31, 138.89, 133.23, 128.99, 125.84, 125.17, 122.18, 119.75,
119.28, 117.87, 62.50, 55.23, 54.16, 51.07, 31.69, 28.71, 27.04, 24.94,
23.19, 15.49. HRMS (ESI) m/z: 435.5880 [M + H+].
2-(2-Ethoxyethyl)-N-(4-(1-isopropyl-1H-pyrazol-4-yl)-5-
methylpyrimidin-2-yl)-1,2,3,4-tetrahydroisoquinolin-6-amine
(13q). 1H NMR (400 MHz, DMSO-d6): δ 9.20 (s, 1H), 8.30 (d, J =
22.7 Hz, 2H), 8.08 (s, 1H), 7.65 (d, J = 2.2 Hz, 1H), 7.47 (dd, J = 8.3,
2.2 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 4.63 (hept, J = 6.7 Hz, 1H), 3.61−
3.51 (m, 4H), 3.46 (q, J = 7.0 Hz, 2H), 2.79 (d, J = 5.8 Hz, 2H), 2.70 (t,
J = 5.8 Hz, 2H), 2.64 (t, J = 6.0 Hz, 2H), 2.31 (s, 3H), 1.49 (d, J = 6.7
Hz, 6H), 1.12 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO): δ
159.87, 159.01, 157.63, 139.46, 139.32, 134.35, 129.35, 127.83, 126.68,
120.76, 118.31, 116.74, 116.34, 68.48, 65.94, 57.73, 56.03, 53.83, 51.59,
29.65, 23.02, 17.15, 15.62. HRMS (ESI) m/z: 421.5610 [M + H+].
2-Butyl-N-(4-(1-isopropyl-1H-pyrazol-4-yl)-5-methylpyrimi-
din-2-yl)-1,2,3,4-tetrahydroisoquinolin-6-amine (13r). 1H NMR
(400 MHz, DMSO-d6): δ9.19 (s, 1H), 8.29 (d, J = 23.5 Hz, 2H), 8.09
(s, 1H), 7.65 (s, 1H), 7.47 (d, J = 7.7 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H),
4.70−4.56 (m, J = 6.8 Hz, 1H), 3.46 (s, 2H), 2.79 (s, 2H), 2.61 (s, 2H),
2.41 (s, 2H), 2.31 (s, 3H), 1.48 (d, J = 6.8 Hz, 8H), 1.31 (p, J = 7.3 Hz,
2H), 0.90 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6): δ
159.88, 158.98, 157.65, 139.71, 139.32, 134.06, 129.40, 126.80, 120.72,
118.22, 116.86, 116.43, 57.61, 55.28, 53.83, 50.97, 29.10, 28.73, 23.03,
20.51, 17.16, 14.34. HRMS (ESI) m/z: 405.5620 [M + H+].
2-(6-((5-Fluoro-4-(1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-
1
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-ol (13l). H
2-Allyl-N-(4-(1-isopropyl-1H-pyrazol-4-yl)-5-methylpyrimi-
din-2-yl)-1,2,3,4-tetrahydroisoquinolin-6-amine (13s). 1H NMR
(400 MHz, DMSO-d6): δ 9.21 (s, 1H), 8.33 (s, 1H), 8.27 (d, J = 0.8 Hz,
1H), 8.08 (s, 1H), 7.66 (d, J = 2.2 Hz, 1H), 7.47 (dd, J = 8.3, 2.2 Hz,
1H), 6.95 (d, J = 8.3 Hz, 1H), 5.91 (ddt, J = 16.6, 10.2, 6.3 Hz, 1H), 5.26
(dq, J = 17.2, 1.7 Hz, 1H), 5.18 (ddt, J = 10.1, 2.2, 1.2 Hz, 1H), 4.62 (h, J
= 6.7 Hz, 1H), 3.48 (s, 2H), 3.12 (dt, J = 6.4, 1.4 Hz, 2H), 2.81 (t, J = 5.9
Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H), 2.31 (s, 3H), 1.49 (d, J = 6.6 Hz, 6H).
13C NMR (101 MHz, DMSO): δ 159.87, 159.01, 157.64, 139.49,
139.32, 136.22, 134.40, 129.36, 127.75, 126.70, 120.75, 118.35, 117.80,
116.77, 116.35, 61.23, 55.59, 53.83, 50.75, 29.62, 23.03, 17.16. HRMS
(ESI) m/z: 389.5190 [M + H+].
2-(6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)acetonitrile (13t).
1H NMR (400 MHz, DMSO-d6): δ 9.24 (s, 1H), 8.30 (d, J = 21.8
Hz, 2H), 8.09 (s, 1H), 7.69 (d, J = 2.3 Hz, 1H), 7.51 (dd, J = 8.3, 2.2 Hz,
1H), 7.00 (d, J = 8.4 Hz, 1H), 4.63 (hept, J = 6.7 Hz, 1H), 3.91 (s, 2H),
3.62 (s, 2H), 2.86 (d, J = 6.0 Hz, 2H), 2.77 (d, J = 5.7 Hz, 2H), 2.32 (s,
3H), 1.49 (d, J = 6.7 Hz, 6H). 13C NMR (101 MHz, DMSO): δ 159.87,
158.97, 157.66, 139.77, 139.34, 133.54, 129.35, 126.77, 126.45, 120.74,
118.35, 116.96, 116.47, 116.34, 53.85, 53.79, 49.74, 45.59, 29.43, 23.01,
17.16. HRMS (ESI) m/z: 388.4910 [M + H+].
NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.49 (d, J = 2.9 Hz, 1H),
8.38 (s, 1H), 8.08 (s, 1H), 7.62 (s, 1H), 7.48 (d, J = 8.3 Hz, 1H), 6.99
(d, J = 8.4 Hz, 1H), 4.66 (td, J = 14.4, 13.9, 7.2 Hz, 2H), 3.83−3.50 (m,
4H), 2.78 (d, J = 66.0 Hz, 6H), 1.48 (d, J = 6.7 Hz, 6H). 13C NMR (101
MHz, DMSO): δ 53.94, 147.50, 143.35, 142.60, 139.98, 138.80, 132.96,
128.37, 127.01, 126.49, 120.09, 118.70, 117.83, 59.30, 56.76, 56.40,
54.17, 51.12, 28.89, 23.16. HRMS (ESI) m/z: 397.4704 [M + H+].
4-(6-((5-Fluoro-4-(1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)butan-1-ol (13m).
1H NMR (400 MHz, DMSO-d6): δ 9.48 (s, 1H), 8.49 (d, J = 3.0 Hz,
1H), 8.38 (d, J = 1.9 Hz, 1H), 8.08 (d, J = 1.3 Hz, 1H), 7.60 (d, J = 2.2
Hz, 1H), 7.46 (dd, J = 8.3, 2.2 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 4.68 (h,
J = 6.6 Hz, 1H), 4.56 (s, 1H), 3.42 (t, J = 6.3 Hz, 2H), 2.81 (t, J = 5.9 Hz,
2H), 2.64 (t, J = 5.8 Hz, 2H), 2.44 (t, J = 7.1 Hz, 2H), 1.60−1.40 (m,
10H). 13C NMR (101 MHz, DMSO): δ 157.01, 149.95, 147.47, 146.97,
145.96, 139.05, 138.85, 134.58, 129.78, 128.38, 126.82, 118.39, 116.83,
115.73, 61.22, 58.22, 55.74, 54.00, 51.11, 31.13, 29.61, 23.80, 22.95.
HRMS (ESI) m/z: 424.5244 [M + H+].
5-(6-((5-Fluoro-4-(1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)pentan-1-ol (13n).
1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 8.49 (d, J = 3.0 Hz,
1H), 8.38 (d, J = 1.6 Hz, 1H), 8.08 (s, 1H), 7.59 (s, 1H), 7.46 (dd, J =
L
https://dx.doi.org/10.1021/acs.jmedchem.0c01488
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
3-(6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)propanenitrile
(13u). 1H NMR (400 MHz, DMSO-d6): δ 9.21 (s, 1H), 8.29 (d, J =
22.7 Hz, 2H), 8.08 (s, 1H), 7.67 (d, J = 2.2 Hz, 1H), 7.48 (dd, J = 8.3,
2.2 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 4.63 (hept, J = 6.7 Hz, 1H), 2.76
(ddt, J = 20.6, 9.8, 5.7 Hz, 8H), 2.31 (s, 3H), 1.48 (d, J = 6.6 Hz, 6H).
13C NMR (101 MHz, DMSO-d6): δ 159.89, 158.98, 157.64, 139.57,
139.31, 134.24, 129.40, 127.33, 126.74, 120.73, 120.50, 118.32, 116.81,
116.41, 54.95, 53.83, 53.09, 50.51, 29.46, 23.03, 17.15, 15.80. HRMS
(ESI) m/z: 402.5180 [M + H+].
3.82 (dt, J = 10.1, 5.9 Hz, 2H), 2.88 (dt, J = 23.6, 6.0 Hz, 2H), 2.40 (s,
3H), 2.32 (s, 3H), 1.48 (dd, J = 6.7, 1.7 Hz, 6H). 13C NMR (101 MHz,
DMSO): δ 189.47, 164.43, 164.20, 159.91, 158.92, 157.70, 144.87,
140.16, 140.04, 139.33, 135.04, 134.85, 134.59, 134.11, 133.90, 133.72,
130.06, 129.46, 129.31, 126.99, 126.82, 125.73, 125.62, 120.66, 118.21,
117.29, 117.17, 116.62, 53.84, 47.03, 44.33, 43.85, 29.88, 28.79, 23.04,
21.70, 17.16. HRMS (ESI) m/z: 521.6370 [M + H+].
1-(6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)-4-(p-tolyl)butane-
1,4-dione (13ab). 1H NMR (400 MHz, DMSO-d6): δ 9.30 (d, J = 5.9
Hz, 1H), 8.31 (d, J = 20.9 Hz, 2H), 8.09 (d, J = 1.7 Hz, 1H), 7.88 (d, J =
8.0 Hz, 2H), 7.75 (dd, J = 11.7, 1.8 Hz, 1H), 7.61−7.51 (m, 1H), 7.33
(d, J = 7.8 Hz, 2H), 7.11 (dd, J = 25.0, 8.4 Hz, 1H), 4.71−4.49 (m, 3H),
3.71 (dt, J = 35.7, 5.9 Hz, 2H), 3.24 (t, J = 6.2 Hz, 2H), 2.95−2.71 (m,
4H), 2.35 (d, J = 24.3 Hz, 6H), 1.49 (d, J = 6.6 Hz, 6H). 13C NMR (101
MHz, DMSO-d6): δ 198.87, 170.54, 170.46, 159.90, 158.95, 157.69,
143.72, 140.03, 139.92, 139.34, 135.26, 135.03, 134.80, 129.62, 129.44,
128.41, 126.84, 126.47, 125.93, 120.70, 118.31, 118.24, 117.19, 116.59,
116.56, 53.84, 46.43, 43.82, 43.09, 33.38, 29.66, 29.02, 27.73, 27.30,
23.03, 21.59, 17.16. HRMS (ESI) m/z: 523.6530 [M + H+].
N-(4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-yl)-
2-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroisoquinolin-6-amine
1
(13v). H NMR (400 MHz, DMSO-d6): δ 9.22 (s, 1H), 8.30 (d, J =
22.4 Hz, 2H), 8.09 (s, 1H), 7.75−7.61 (m, 1H), 7.56−7.42 (m, 1H),
6.98 (d, J = 8.4 Hz, 1H), 4.63 (p, J = 6.7 Hz, 1H), 3.59 (s, 2H), 3.51−
3.40 (m, 2H), 3.23−3.13 (m, 1H), 2.84 (s, 2H), 2.72 (s, 2H), 2.32 (s,
3H), 1.49 (d, J = 6.7 Hz, 6H). 13C NMR (101 MHz, DMSO): δ 159.89,
158.99, 157.65, 139.51, 139.32, 133.99, 129.39, 127.46, 126.76, 120.73,
118.34, 116.82, 116.40, 79.84, 76.20, 53.90, 53.83, 49.71, 46.51, 29.64,
23.04, 17.15. HRMS (ESI) m/z: 387.5030 [M + H+].
2-(But-2-yn-1-yl)-N-(4-(1-isopropyl-1H-pyrazol-4-yl)-5-
methylpyrimidin-2-yl)-1,2,3,4-tetrahydroisoquinolin-6-amine
(13w). 1H NMR (400 MHz, DMSO-d6): δ 9.21 (s, 1H), 8.33 (s, 1H),
8.27 (s, 1H), 8.08 (s, 1H), 7.66 (d, J = 2.3 Hz, 1H), 7.48 (dd, J = 8.4, 2.2
Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 4.63 (hept, J = 6.7 Hz, 1H), 3.38 (q, J
= 2.4 Hz, 2H), 2.82 (t, J = 5.8 Hz, 2H), 2.70 (t, J = 5.9 Hz, 2H), 2.32 (s,
3H), 1.83 (t, J = 2.3 Hz, 3H), 1.49 (d, J = 6.6 Hz, 6H). 13C NMR (101
MHz, DMSO): δ 159.88, 159.00, 157.65, 139.47, 139.32, 134.07,
129.38, 127.60, 126.75, 120.74, 118.31, 116.78, 116.38, 81.12, 75.17,
54.12, 53.83, 49.85, 47.01, 29.68, 23.03, 17.15, 3.55. HRMS (ESI) m/z:
401.5300 [M + H+].
Methyl-(E)-4-(6-((4-(1-isopropyl-1H-pyrazol-4-yl)-5-methyl-
pyrimidin-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)but-2-
enoate (13x). 1H NMR (400 MHz, DMSO-d6): δ 9.22 (s, 1H), 8.33
(s, 1H), 8.29 (s, 1H), 8.08 (s, 1H), 7.67 (d, J = 2.2 Hz, 1H), 7.52−7.44
(m, 1H), 6.96 (d, J = 8.8 Hz, 2H), 6.10 (d, J = 15.7 Hz, 1H), 4.63 (hept,
J = 6.7 Hz, 1H), 3.68 (s, 3H), 3.52 (s, 2H), 2.83 (t, J = 5.8 Hz, 2H), 2.68
(t, J = 5.8 Hz, 2H), 2.32 (s, 3H), 1.49 (d, J = 6.6 Hz, 6H). 13C NMR
(101 MHz, DMSO): δ 166.38, 159.88, 158.99, 157.64, 146.64, 139.58,
139.31, 134.22, 129.37, 127.43, 126.73, 122.53, 120.74, 118.34, 116.78,
116.39, 58.56, 55.68, 53.83, 51.80, 50.99, 29.53, 23.02, 17.15. HRMS
(ESI) m/z: 447.5550 [M + H+].
Methyl-4-(6-((4-(1-isopropyl-1H-pyrazol-4-yl)-5-methylpyri-
midin-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)butanoate
(13y). 1H NMR (400 MHz, DMSO-d6): δ 9.18 (s, 1H), 8.32 (s, 1H),
8.26 (s, 1H), 8.07 (s, 1H), 7.64 (s, 1H), 7.46 (d, J = 8.4 Hz, 1H), 6.95
(d, J = 8.3 Hz, 1H), 4.62 (dt, J = 13.2, 6.6 Hz, 1H), 3.57 (d, J = 9.1 Hz,
3H), 3.46 (s, 2H), 2.78 (d, J = 5.3 Hz, 2H), 2.62 (t, J = 5.6 Hz, 2H), 2.44
(t, J = 6.9 Hz, 2H), 2.36 (t, J = 7.2 Hz, 2H), 2.31 (s, 3H), 1.83−1.74 (m,
2H), 1.48 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, DMSO): δ 173.86,
159.90, 158.99, 157.63, 139.44, 139.31, 134.48, 129.40, 127.83, 126.74,
120.73, 118.28, 116.72, 116.36, 57.30, 55.71, 53.83, 51.61, 51.04, 31.80,
29.63, 23.04, 22.51, 17.14. HRMS (ESI) m/z: 449.5710 [M + H+].
(E)-1-(6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimi-
din-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)-4-phenyl-
but-2-ene-1,4-dione (13z). 1H NMR (400 MHz, DMSO-d6): δ 9.31
(d, J = 2.2 Hz, 1H), 8.33 (s, 1H), 8.28 (s, 1H), 8.13−8.02 (m, 3H),
7.85−7.79 (m, 1H), 7.76 (d, J = 2.2 Hz, 1H), 7.74−7.68 (m, 1H),
7.64−7.50 (m, 4H), 7.15 (dd, J = 8.5, 4.3 Hz, 1H), 4.73 (d, J = 36.9 Hz,
2H), 4.63 (p, J = 6.7 Hz, 1H), 3.83 (dt, J = 11.2, 5.9 Hz, 2H), 2.88 (dt, J
= 23.7, 6.0 Hz, 2H), 2.32 (s, 3H), 1.49 (dd, J = 6.7, 1.9 Hz, 6H). 13C
NMR (101 MHz, DMSO): δ 188.40, 167.72, 157.94, 151.75, 138.93,
138.16, 132.71, 129.12, 125.20, 121.82, 120.43, 119.95, 117.79, 54.16,
47.73, 44.20, 29.77, 23.16, 15.47. HRMS (ESI) m/z: 507.6100 [M +
H+].
(E)-4-(Dimethylamino)-1-(6-((4-(1-isopropyl-1H-pyrazol-4-
yl)-5-methylpyrimidin-2-yl)amino)-3,4-dihydroisoquinolin-
2(1H)-yl)but-2-en-1-one (13ac). 1H NMR (400 MHz, DMSO-d6): δ
9.30 (d, J = 5.2 Hz, 1H), 8.30 (d, J = 20.8 Hz, 2H), 8.09 (s, 1H), 7.75 (d,
J = 6.4 Hz, 1H), 7.56 (t, J = 9.0 Hz, 1H), 7.10 (t, J = 8.8 Hz, 1H), 6.85
(d, J = 15.1 Hz, 1H), 6.70−6.56 (m, 1H), 4.71 (s, 1H), 4.65−4.57 (m,
2H), 3.77 (dt, J = 18.4, 5.8 Hz, 2H), 3.51 (d, J = 6.6 Hz, 2H), 2.84 (dt, J
= 28.8, 6.0 Hz, 2H), 2.32 (s, 3H), 1.49 (d, J = 6.6 Hz, 6H). 13C NMR
(101 MHz, DMSO): δ 164.68, 164.48, 159.88, 158.92, 157.67, 148.07,
140.10, 139.97, 139.46, 139.34, 139.22, 135.13, 134.82, 129.41, 127.95,
126.92, 126.65, 126.19, 125.84, 125.23, 120.69, 119.55, 118.33, 118.23,
117.26, 117.15, 116.59, 65.38, 59.54, 53.85, 44.57, 44.17, 43.46, 29.96,
23.01, 17.15. HRMS (ESI) m/z: 460.5980 [M + H+].
4-(Dimethylamino)-1-(6-((4-(1-isopropyl-1H-pyrazol-4-yl)-5-
methylpyrimidin-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-
yl)butan-1-one (13ad). 1H NMR (400 MHz, DMSO-d6): δ 9.30 (d, J
= 6.4 Hz, 1H), 8.33 (s, 1H), 8.28 (s, 1H), 8.09 (s, 1H), 7.75 (dd, J =
10.1, 2.2 Hz, 1H), 7.56 (ddd, J = 14.8, 8.4, 2.2 Hz, 1H), 7.10 (d, J = 8.4
Hz, 1H), 4.68−4.54 (m, 3H), 3.68 (dt, J = 12.1, 5.9 Hz, 2H), 3.09−2.99
(m, 2H), 2.88 (s, 8H), 2.56−2.51 (m, 2H), 2.32 (s, 3H), 1.87 (p, J = 7.0
Hz, 2H), 1.48 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, DMSO): δ
170.49, 170.41, 159.92, 158.92, 157.68, 140.07, 139.95, 139.34, 135.21,
134.95, 129.44, 126.88, 126.71, 126.30, 125.78, 120.66, 118.34, 118.21,
117.22, 116.66, 116.62, 57.26, 53.86, 46.45, 43.82, 43.07, 42.89, 30.17,
29.84, 29.58, 28.94, 23.02, 22.89, 20.15, 17.13. HRMS (ESI) m/z:
462.6140 [M + H+].
(E)-4-(6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimi-
din-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)but-2-enoic
Acid (13ae). 1H NMR (400 MHz, DMSO-d6): δ 9.21 (s, 1H), 8.30 (d,
J = 22.4 Hz, 2H), 8.08 (s, 1H), 7.67 (d, J = 2.3 Hz, 1H), 7.48 (dd, J = 8.3,
2.2 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.79 (dt, J = 15.6, 5.9 Hz, 1H),
6.02−5.94 (m, 1H), 4.62 (h, J = 6.6 Hz, 1H), 3.51 (s, 2H), 3.27 (d, J =
5.5 Hz, 2H), 3.18 (s, 1H), 2.83 (t, J = 5.8 Hz, 2H), 2.67 (t, J = 5.8 Hz,
2H), 2.31 (s, 3H), 1.49 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz,
DMSO-d6): δ 170.53, 158.50, 157.32, 152.38, 144.77, 138.89, 133.26,
129.05, 125.84, 125.17, 122.18, 120.54, 119.69, 119.22, 117.89, 56.52,
55.36, 54.11, 50.45, 28.84, 23.16, 15.48. HRMS (ESI) m/z: 433.5680
[M + H+].
(E)-4-(6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimi-
din-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)-N-(2-(piperi-
din-1-yl)ethyl)but-2-enamide (13af). 1H NMR (400 MHz,
DMSO-d6): δ 9.22 (s, 1H), 8.33 (s, 1H), 8.27 (s, 1H), 8.09 (s, 2H),
7.68 (d, J = 2.2 Hz, 1H), 7.48 (dd, J = 8.3, 2.2 Hz, 1H), 6.96 (d, J = 8.3
Hz, 1H), 6.67 (dt, J = 15.4, 5.9 Hz, 1H), 6.14 (d, J = 15.5 Hz, 1H), 4.62
(h, J = 6.6 Hz, 1H), 3.53 (s, 2H), 3.29−3.21 (m, 3H), 2.87−2.81 (m,
2H), 2.69 (d, J = 8.0 Hz, 2H), 2.32 (s, 3H), 1.49 (d, J = 6.6 Hz, 12H).
13C NMR (101 MHz, DMSO-d6): δ 167.03, 157.94, 157.26, 151.81,
(E)-1-(6-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimi-
din-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)-4-(p-tolyl)-
1
but-2-ene-1,4-dione (13aa). H NMR (400 MHz, DMSO-d6): δ
141.33, 138.80, 138.62, 134.04, 128.44, 125.75, 125.22, 122.48, 120.43,
120.19, 120.13, 117.84, 56.00, 55.63, 54.14, 53.65, 50.44, 38.81, 28.20,
26.43, 23.84, 23.16, 15.43. HRMS (ESI) m/z: 543.7320 [M + H+].
9.31 (d, J = 2.1 Hz, 1H), 8.33 (s, 1H), 8.28 (s, 1H), 8.09 (s, 1H), 7.96
(dd, J = 8.3, 2.8 Hz, 2H), 7.84−7.72 (m, 2H), 7.59−7.48 (m, 2H), 7.39
(d, J = 8.0 Hz, 2H), 7.14 (dd, J = 8.5, 3.8 Hz, 1H), 4.79−4.58 (m, 3H),
M
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Article
(E)-N-(tert-Butyl)-4-(6-((4-(1-isopropyl-1H-pyrazol-4-yl)-5-
methylpyrimidin-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-
yl)but-2-enamide (13ag). 1H NMR (400 MHz, DMSO-d6): δ 9.21
(s, 1H), 8.30 (d, J = 22.0 Hz, 2H), 8.09 (s, 1H), 7.71−7.56 (m, 2H),
7.48 (dd, J = 8.3, 2.2 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.58 (dt, J = 15.4,
5.9 Hz, 1H), 6.15 (dd, J = 15.4, 1.6 Hz, 1H), 4.63 (p, J = 6.6 Hz, 1H),
3.50 (s, 2H), 3.26−3.15 (m, 2H), 2.84 (t, J = 5.8 Hz, 2H), 2.66 (t, J = 5.8
Hz, 2H), 2.32 (s, 3H), 1.49 (d, J = 6.6 Hz, 6H), 1.29 (s, 9H). 13C NMR
(101 MHz, DMSO): δ 164.52, 159.90, 158.99, 157.65, 139.53, 139.31,
138.56, 134.34, 129.41, 127.68, 127.63, 126.77, 120.71, 118.32, 116.76,
116.39, 58.83, 55.77, 53.83, 51.06, 50.47, 29.59, 29.03, 23.05, 17.15.
HRMS (ESI) m/z: 488.6520 [M + H+].
(E)-N,N-Diethyl-4-(6-((4-(1-isopropyl-1H-pyrazol-4-yl)-5-
methylpyrimidin-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-
yl)but-2-enamide (13ah). 1H NMR (400 MHz, DMSO-d6): δ 9.21
(s, 1H), 8.30 (d, J = 22.5 Hz, 2H), 8.08 (s, 1H), 7.67 (d, J = 2.2 Hz, 1H),
7.48 (dd, J = 8.4, 2.2 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.71 (dt, J = 15.1,
6.0 Hz, 1H), 6.61−6.52 (m, 1H), 4.63 (hept, J = 6.7 Hz, 1H), 3.52 (s,
2H), 3.38 (s, 3H), 3.30−3.25 (m, 2H), 2.83 (t, J = 5.9 Hz, 2H), 2.68 (t, J
= 5.8 Hz, 2H), 2.32 (s, 3H), 1.49 (d, J = 6.7 Hz, 6H), 1.17−0.99 (m,
7H). 13C NMR (101 MHz, DMSO): δ 159.85, 158.70, 157.79, 154.11,
139.32, 137.19, 137.08, 129.48, 120.53, 120.22, 116.78, 114.55, 106.91,
63.14, 53.84, 48.24, 35.15, 33.95, 23.05, 18.71, 17.15, 11.35. HRMS
(ESI) m/z: 488.6542 [M + H+].
2-(7-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-
yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)acetonitrile (13ai).
1H NMR (400 MHz, DMSO-d6): δ 9.25 (s, 1H), 8.30 (d, J = 21.5
Hz, 2H), 8.09 (s, 1H), 7.65 (d, J = 2.3 Hz, 1H), 7.50 (dd, J = 8.3, 2.2 Hz,
1H), 7.04 (d, J = 8.4 Hz, 1H), 4.63 (hept, J = 6.7 Hz, 1H), 3.93 (s, 2H),
3.68 (s, 2H), 2.84−2.71 (m, 4H), 2.32 (s, 3H), 1.49 (d, J = 6.6 Hz, 6H).
13C NMR (101 MHz, DMSO): δ 159.90, 158.96, 157.66, 139.32,
139.29, 133.95, 133.95, 129.38, 128.93, 125.94, 120.72, 117.55, 116.47,
116.34, 116.20, 54.40, 53.85, 49.90, 45.51, 40.00, 28.39, 23.04, 17.16.
HRMS (ESI) m/z: 388.4910 [M + H+].
(E)-4-(Dimethylamino)-1-(7-((4-(1-isopropyl-1H-pyrazol-4-
yl)-5-methylpyrimidin-2-yl)amino)-3,4-dihydroisoquinolin-
2(1H)-yl)but-2-en-1-one (13am). 1H NMR (400 MHz, DMSO-d6):
δ 9.32 (d, J = 10.3 Hz, 1H), 8.34 (d, J = 5.4 Hz, 1H), 8.28 (s, 1H), 8.10
(s, 1H), 7.76 (d, J = 13.3 Hz, 1H), 7.54 (dd, J = 8.3, 2.3 Hz, 1H), 7.10
(d, J = 8.4 Hz, 1H), 6.95 (dd, J = 23.4, 15.0 Hz, 1H), 6.65 (dt, J = 14.7,
6.8 Hz, 1H), 4.79 (s, 1H), 4.69 (s, 1H), 4.63 (q, J = 6.7 Hz, 1H), 3.79
(dt, J = 15.3, 5.9 Hz, 2H), 3.68 (d, J = 6.9 Hz, 2H), 2.79 (dt, J = 30.7, 5.8
Hz, 2H), 2.63 (s, 6H), 2.32 (s, 3H), 1.50 (d, J = 6.7 Hz, 6H). 13C NMR
(101 MHz, DMSO): δ 166.48, 157.98, 157.32, 151.95, 139.51, 138.89,
138.61, 131.22, 130.08, 126.99, 125.17, 122.18, 119.89, 119.75, 118.46,
58.03, 54.16, 46.57, 45.19, 44.61, 29.28, 23.19, 15.48. HRMS (ESI) m/
z: 460.5988 [M + H+].
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01488.
■
sı
Binding affinities of 13ac with 365 protein kinases;1H
NMR, 13C NMR, MS/MS, and HPLC spectra of
compound 13ac; routine blood test in normal mice; and
effect of 13ac on mouse models (PDF)
PDB code 2XA4 used for modeling docking in JAK2 of
13ac (PDB)
PDB code: 4RT7 used for modeling docking in FLT3 of
18e (PDB)
(PDB)
Molecular formula strings of the prepared compounds
(CSV)
AUTHOR INFORMATION
Corresponding Authors
■
(E)-1-(7-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimi-
din-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)-4-phenyl-
Zhuang Yang − State Key Laboratory of Biotherapy and Cancer
Center, National Clinical Research Center for Geriatrics, West
China Hospital of Sichuan University, Chengdu 610041,
China; Chengdu Zenitar Biomedical Technology Co., Ltd.,
Chengdu 610041, China; Email: young9008@126.com
Lijuan Chen − State Key Laboratory of Biotherapy and Cancer
Center, National Clinical Research Center for Geriatrics, West
China Hospital of Sichuan University, Chengdu 610041,
China; Chengdu Zenitar Biomedical Technology Co., Ltd.,
Chengdu 610041, China; orcid.org/0000-0002-8076-
163X; Phone: +86-28-85164063; Email: chenlijuan125@
163.com
1
but-2-ene-1,4-dione (13aj). H NMR (400 MHz, DMSO-d6): δ
9.31 (d, J = 3.6 Hz, 1H), 8.34 (s, 1H), 8.28 (d, J = 7.3 Hz, 1H), 8.12−
8.01 (m, 3H), 7.85−7.75 (m, 2H), 7.73−7.67 (m, 1H), 7.62−7.50 (m,
4H), 7.10 (d, J = 8.3 Hz, 1H), 4.79 (d, J = 40.6 Hz, 2H), 4.61 (dq, J =
13.3, 6.7 Hz, 1H), 3.82 (dt, J = 10.9, 5.9 Hz, 2H), 2.82 (dt, J = 24.6, 5.9
Hz, 2H), 2.32 (d, J = 4.6 Hz, 3H), 1.48 (dd, J = 16.7, 6.6 Hz, 6H). 13C
NMR (101 MHz, DMSO): δ 188.48, 167.69, 157.94, 151.81, 138.93,
138.61, 138.16, 132.55, 132.01, 131.74, 130.78, 128.97, 127.02, 125.22,
121.82, 119.95, 118.78, 54.13, 46.66, 44.21, 29.07, 23.19, 15.44. HRMS
(ESI) m/z: 507.6100 [M + H+].
Methyl-(E)-4-(7-((4-(1-isopropyl-1H-pyrazol-4-yl)-5-methyl-
pyrimidin-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)but-2-
enoate (13ak). 1H NMR (400 MHz, DMSO-d6): δ 9.21 (s, 1H), 8.32
(s, 1H), 8.26 (d, J = 5.5 Hz, 1H), 8.07 (d, J = 6.0 Hz, 1H), 7.61 (s, 1H),
7.50−7.45 (m, 1H), 7.00 (t, J = 9.9 Hz, 1H), 6.93 (dt, J = 15.7, 5.8 Hz,
1H), 6.10 (d, J = 15.7 Hz, 1H), 4.62 (dt, J = 13.3, 6.6 Hz, 1H), 3.68 (s,
3H), 3.58 (s, 2H), 3.32−3.29 (m, 2H), 2.75 (d, J = 5.1 Hz, 2H), 2.67 (t,
J = 5.4 Hz, 2H), 2.30 (d, J = 9.3 Hz, 3H), 1.48 (d, J = 6.7 Hz, 6H). 13C
NMR (101 MHz, DMSO): δ 166.38, 159.89, 158.97, 157.64, 146.65,
139.26, 139.16, 134.82, 129.36, 128.84, 126.72, 122.54, 120.75, 117.36,
116.39, 116.26, 65.38, 58.42, 56.33, 53.83, 51.81, 51.08, 28.45, 23.02,
17.14, 15.62. HRMS (ESI) m/z: 447.5550 [M + H+].
(E)-1-(7-((4-(1-Isopropyl-1H-pyrazol-4-yl)-5-methylpyrimi-
din-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)-4-(p-tolyl)-
but-2-ene-1,4-dione (13al). 1H NMR (400 MHz, DMSO-d6): δ 9.31
(d, J = 3.4 Hz, 1H), 8.39−8.21 (m, 2H), 8.08 (d, J = 10.4 Hz, 1H), 7.96
(dd, J = 8.0, 4.3 Hz, 2H), 7.88−7.68 (m, 2H), 7.61−7.47 (m, 2H), 7.38
(dd, J = 8.1, 4.2 Hz, 2H), 7.10 (d, J = 8.3 Hz, 1H), 4.94−4.49 (m, 3H),
3.82 (dt, J = 9.7, 5.9 Hz, 2H), 2.81 (dt, J = 24.2, 5.9 Hz, 2H), 2.40 (d, J =
3.7 Hz, 3H), 2.32 (d, J = 4.5 Hz, 3H), 1.48 (dd, J = 16.3, 6.7 Hz, 6H).
13C NMR (101 MHz, DMSO): δ 188.51, 167.69, 157.92, 151.75,
Authors
Tao Yang − State Key Laboratory of Biotherapy and Cancer
Center, National Clinical Research Center for Geriatrics, West
China Hospital of Sichuan University, Chengdu 610041,
China
Mengshi Hu − State Key Laboratory of Biotherapy and Cancer
Center, National Clinical Research Center for Geriatrics, West
China Hospital of Sichuan University, Chengdu 610041,
China
Yong Chen − State Key Laboratory of Biotherapy and Cancer
Center, National Clinical Research Center for Geriatrics, West
China Hospital of Sichuan University, Chengdu 610041,
China
Mingli Xiang − State Key Laboratory of Biotherapy and Cancer
Center, National Clinical Research Center for Geriatrics, West
China Hospital of Sichuan University, Chengdu 610041,
China
142.36, 138.67, 136.61, 131.44, 130.18, 129.22, 128.97, 126.98, 125.22,
121.82, 119.95, 118.51, 54.14, 46.66, 44.23, 29.07, 23.16, 21.34, 15.44.
HRMS (ESI) m/z: 521.6370 [M + H+].
Minghai Tang − State Key Laboratory of Biotherapy and
Cancer Center, National Clinical Research Center for
N
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Geriatrics, West China Hospital of Sichuan University,
Chengdu 610041, China
Wenyan Qi − State Key Laboratory of Biotherapy and Cancer
Center, National Clinical Research Center for Geriatrics, West
China Hospital of Sichuan University, Chengdu 610041,
China
Mingsong Shi − State Key Laboratory of Biotherapy and
Cancer Center, National Clinical Research Center for
Geriatrics, West China Hospital of Sichuan University,
Chengdu 610041, China
Jun He − State Key Laboratory of Biotherapy and Cancer
Center, National Clinical Research Center for Geriatrics, West
China Hospital of Sichuan University, Chengdu 610041,
China
Xue Yuan − State Key Laboratory of Biotherapy and Cancer
Center, National Clinical Research Center for Geriatrics, West
China Hospital of Sichuan University, Chengdu 610041,
China
Chufeng Zhang − State Key Laboratory of Biotherapy and
Cancer Center, National Clinical Research Center for
Geriatrics, West China Hospital of Sichuan University,
Chengdu 610041, China
Kongjun Liu − State Key Laboratory of Biotherapy and Cancer
Center, National Clinical Research Center for Geriatrics, West
China Hospital of Sichuan University, Chengdu 610041,
China
Jiewen Li − State Key Laboratory of Biotherapy and Cancer
Center, National Clinical Research Center for Geriatrics, West
China Hospital of Sichuan University, Chengdu 610041,
China
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01488
Author Contributions
§T.Y, M.H., and Y.C. contributed equally and should be
considered as co-first authors
́
́
(12) Dupont, S.; Masse, A.; James, C.; Teyssandier, I.; Lecluse, Y.;
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Larbret, F.; Ugo, V.; Saulnier, P.; Koscielny, S.; Le Couedic, J. P.;
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(13) Jang, Y.-N.; Baik, E. J. JAK-STAT pathway and myogenic
differentiation. JAK-STAT 2013, 2, e23282.
The authors greatly appreciate the financial support from the
National Science Foundation of China (81527806, 82073693,
and 81673289) and Post-Doctor Research Project, West China
Hospital, Sichuan Univeristy (2018HXBH054). The authors
also greatly appreciate the financial support from 1.3.5 project
for disciplines of excellence, West China Hospital, Sichuan
University. Thanks to West China College of Pharmacy Sichuan
University for providing Schrodinger software and technical
support.
́
(14) James, C.; Ugo, V.; Le Couedic, J.-P.; Staerk, J.; Delhommeau, F.;
Lacout, C.; Garcon, L.; Raslova, H.; Berger, R.; Bennaceur-Griscelli, A.;
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ABBREVIATIONS
■
JAK1, Janus kinase 1; JAK2, Janus kinase 2; JAK3, Janus kinase
3; TFA, trifluoroacetic acid; TLC, thin layer chromatography;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; PE, petroleum ether; EA, ethyl acetate; DCM,
dichloromethane
(17) Hexner, E. O.; Serdikoff, C.; Jan, M.; Swider, C. R.; Robinson, C.;
Yang, S.; Angeles, T.; Emerson, S. G.; Carroll, M.; Ruggeri, B.;
Dobrzanski, P. Lestaurtinib (CEP701) is a JAK2 inhibitor that
suppresses JAK2/STAT5 signaling and the proliferation of primary
erythroid cells from patients with myeloproliferative disorders. Blood
2008, 111, 5663−5671.
(18) Tyner, J. W.; Bumm, T. G.; Deininger, J.; Wood, L.; Aichberger,
K. J.; Loriaux, M. M.; Druker, B. J.; Burns, C. J.; Fantino, E.; Deininger,
M. W. CYT387, a novel JAK2 inhibitor, induces hematologic responses
and normalizes inflammatory cytokines in murine myeloproliferative
neoplasms. Blood 2010, 115, 5232−5240.
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