Discovery of 4-piperazinyl-2-aminopyrimidine derivatives as dual inhibitors of JAK2 and FLT3

Article abstract of DOI:10.1016/j.ejmech.2019.111590

Hybridization strategy is an effective strategy to obtain multi-target inhibitors in drug design. In this study, we assembled the pharmacophores of momelotinib and tandutinib to get a series of 4-piperazinyl-2-aminopyrimidine derivatives. All compounds were tested for the inhibition of JAK2 and FLT3 enzymes, of which, compounds with potent enzyme activities were assayed for antiproliferative activities against three cancer cell lines (HEL, MV4-11, and HL60). The structure-activity relationship studies were conducted through variations in two regions, the “A” phenyl ring and “B” phenyl ring. Compound 14j showed the most balanced in vitro inhibitory activity against JAK2 and FLT3 (JAK2 IC₅₀ = 27 nM, FLT3 IC₅₀ = 30 nM), and it also showed potent inhibition against the above tested cell lines. In the cellular context, 14j strongly induced apoptosis by arresting cell cycle in the G₁/S phase, and was selected as a promising JAK2/FLT3 dual inhibitor.

Full text of DOI:10.1016/j.ejmech.2019.111590

Journal Pre-proof
Discovery of 4-piperazinyl-2-aminopyrimidine derivatives as dual inhibitors of JAK2
and FLT3
Yingxiu Li, Tianyu Ye, Le Xu, Yuhong Dong, Yong Luo, Chu Wang, Yufei Han, Ke
Chen, Mingze Qin, Yajing Liu, Yanfang Zhao
PII:
S0223-5234(19)30724-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111590
EJMECH 111590
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 May 2019
Revised Date: 18 July 2019
Accepted Date: 5 August 2019
Please cite this article as: Y. Li, T. Ye, L. Xu, Y. Dong, Y. Luo, C. Wang, Y. Han, K. Chen, M. Qin, Y. Liu,
Y. Zhao, Discovery of 4-piperazinyl-2-aminopyrimidine derivatives as dual inhibitors of JAK2 and FLT3,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111590.
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Graphical Abstract
A series of 4-piperazinyl-2-aminopyrimidine derivatives were designed, synthesized and evaluated
for their biological activities.

Discovery of 4-piperazinyl-2-aminopyrimidine derivatives as dual inhibitors of JAK2
and FLT3
a
a
a
a
a
a
a
Yingxiu Li , Tianyu Ye , Le Xu , Yuhong Dong , Yong Luo , Chu Wang , Yufei Han , Ke Chen
a
a, b
a*
a†
,
Mingze Qin , Yajing Liu , Yanfang Zhao
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, China
b
Chinese People’s Liberation Army Logistics Support Force No.967 Hospital, Dalian 116021, PR China
Abstract: Hybridization strategy is an effective strategy to obtain multi-target
inhibitors in drug design. In this study, we assembled the pharmacophores of
momelotinib and tandutinib to get a series of 4-piperazinyl-2-aminopyrimidine
derivatives. All compounds were tested for the inhibition of JAK2 and FLT3 enzymes,
of which, compounds with potent enzyme activities were assayed for antiproliferative
activities against three cancer cell lines (HEL, MV4-11, and HL60). The
structure-activity relationship studies were conducted through variations in two
regions, the “A” phenyl ring and “B” phenyl ring. Compound 14j showed the most
balanced in vitro inhibitory activity against JAK2 and FLT3 (JAK2 IC =27 nM,
50
FLT3 IC =30 nM), and it also showed potent inhibition against the above tested cell
5
0
lines. In the cellular context, 14j strongly induced apoptosis by arresting cell cycle in
the G /S phase, and was selected as a promising JAK2/FLT3 dual inhibitor.
1
Keywords: Hybridization strategy; 4-piperazinyl-2-aminopyrimidine derivatives; dual inhibitor.
*
Corresponding author
†
Corresponding author
E-mail address: lyjpharm@126.com (Yajing Liu), yanfangzhao@126.com (Yanfang Zhao)
1

1
. Introduction
Hematologic malignancies are a group of diseases caused by disorders of the
hematopoietic system, which mainly include leukemia, lymphoma, myelodysplastic
syndrome, and multiple myeloma diseases [1]. Current treatments of hematological
malignancies, such as bone marrow transplantation, hormones, and chemotherapy,
have known disadvantages such as serious side effects, low cure rate, and high
recurrence rate. Researchers have conducted an in-depth study of the pathogenesis of
hematologic malignancies and thus found that certain genetic mutations, including
mutations in the JAK2 and FLT3 genes, lead to signal transduction disorders in
different tissues and cells [2-3]. Therefore, based on the pathogenesis of such diseases,
a variety of single-target and multi-target inhibitors have been developed [4-7] such as
tofactinib, ruxolitinib, momelotinib, and pacritinib. Due to the unique mechanism of
JAK2 and FLT3 kinases in vivo and their close relationship with hematologic
malignancies, the development of JAK2/FLT3 dual inhibitors has brought new hopes
for patients to cure such diseases safely and effectively.
The Janus kinases (JAK1, JAK2, JAK3, and TYK2) belong to the family of
intracellular protein tyrosine kinases, which play an essential role in the signaling of a
variety of cytokines. Activation of JAKs by different cytokines results in
phosphorylation and dimerization of the STAT (signal transducers and activators of
transcription) proteins, which further translocate to the nucleus and activate gene
transcription [8]. JAK2-STATs pathway is one of the most important pathways of cell
signal transduction, and plays an important role in regulating the normal physiological
2

and pathological responses of the human body. The abnormal activity of JAK2 can
dysregulate the JAK2-STATs signaling pathway, which leads to various malignant
diseases [3, 9-10].
Fig. 1. Structures of representative JAK2 or FLT3 inhibitors
Currently, a number of pan-JAK and selective JAK inhibitors have been
discovered (Fig. 1). JAK1/2 inhibitor ruxolitinib (I) and pan-JAK inhibitor tofacitinib
(II) were successively approved by FDA for the treatment of myelofibrosis (MF),
polycythemia vera (PV), and rheumatoid arthritis (RA), respectively. [11]. Another
JAK1/2 inhibitor, momelotinib (III), is currently in phase III clinical tial [12].
Although JAK2 inhibitors provide a new option for the treatment of MF or other
blood diseases, studies have shown that toxic side effects such as myelosuppression
3

and cytopenia are the main factors limiting the use of these drugs [13].
FLT3 (FMS-like tyrosine kinase-3), a class III RTK, is the most frequently
mutated gene in acute myeloid leukemia (AML) and plays an important role in the
maintenance, growth, and development of hematopoietic and non-hematopoietic cells
[
14]. The first wave of FLT3 inhibitors belongs to tyrosine kinase inhibitor (TKIs),
which were initially developed for the treatment of solid tumors. These drugs were
originally designed to inhibit other kinases, but occasionally found to be active
against FLT3 (e.g., sorafenib, tandutinib, sunitinib) (V-VII, Fig. 1). Because these
TKIs can inhibit a variety of kinases and FLT3, off-target effects and significant
toxicity are inevitable [15]. In contrast to the numerous multi-kinase inhibitors that
have been adopted as FLT3 inhibitors, quizartinib (VIII, Fig. 1) was designed
specifically to target FLT3, which is in the New Drug Application (NDA) phase now.
[
16]
Studies have shown that JAK2/FLT3 dual inhibitors could regulate JAK2-STATs
signaling pathway and inhibit phosphorylated FLT3 kinase. In the SET2 cell AML
mouse models carrying JAK2-V617F and FLT3-ITD mutations, JAK2/FLT3
inhibitors could effectively inhibit the growth and transfer of tumor [18]. Pacritinib
(SB1518, IV, Fig. 1), a JAK2/FLT3 inhibitor in the New Drug Application (NDA)
phase, showed better therapeutic effects than ruxolitinib in patients with severe
thrombocytopenia in its clinical trials, while its bone marrow toxicity and clinical
response were comparable to ruxolitinib [19-20]. These results indicate that
JAK2/FLT3 combined inhibitors have great potential on the treatment of MF and
4

AML.
In this paper, we utilized the hybridization strategy to design and synthesize a
series of 4-piperazinyl-2-aminopyrimidine analogues as dual JAK2/FLT3 inhibitors.
All compounds were assayed for the in vitro enzymatic inhibitory activities against
JAK2 and FLT3. Based on the enzymatic inhibitory results, potent compounds were
assayed for antiproliferative activities against three cancer cell lines, namely, HEL,
MV4-11, and HL60. To further clarify the primary mechanism, cell apoptosis and cell
cycles on HEL cell line were examined for 14j by flow cytometry.
2
. Results and discussion
2
.1 JAK2/FLT3 dual inhibitors Designed by Hybridization strategy
Pharmacophore of JAK2 inhibitors is composed of two hydrophobic groups (two
phenyl rings), a hinge-region binder (usually an amino-pyrimidine), and a
solvent-exposed region [21]. FLT3 inhibitors (Fig. 1) such as quizartinib (V) and
tandutinib (VI), usually consist of a urea linker, two hydrophobic groups (two phenyl
rings), and a solvent-exposed tail. Given that the 2-aminopyrimidine derivatives have
been widely reported as JAK2 inhibitors, the piperazinyl-1-carboxamide (4, Fig. 2), a
bioisostere of diaryl urea based on tandutinib (2, Fig. 2) as a FLT3 inhibitor, was
introduced to 4-position of pyrimidine moiety in order to obtain dual inhibitors
against JAK2 and FLT3 (Fig. 2).
5

Fig. 2. Design strategy for the target compounds.
2
.2 Chemistry
The synthetic route of target compounds is shown in Scheme 1. Commercially
available starting material 2,4-dichloropyrimidine (5) reacted with N-Boc-piperazine
to yield 6 [22], which was then reacted with various anilines 9a-h in the catalysis of
trifluoroacetic acid in n-butyl alcohol to yield intermediates 10a-h [23]. After treating
1
0a-h with trifluoroacetic acid in dichloromethane, intermediates 11a-h were obtained.
Subsequently, target compounds 14a-x, 15a-c, 16a-c, 17-19a were available via
condensation of 11a-h with different phenyl carbamates, respectively.
Anilines 9a-h were prepared in two steps. Aromatic nucleophilic substitution of
fluorine in 4-fluoromitrobenzene by reacting alcohols or secondary amines was
o
processed to yield intermediates 8a-h in DMF at 25 C, which were subsequently
reduced to corresponding anilines 9a-h by using H -Pd/C in EtOH and generally used
2
without further purification [24]. An assortment of phenylcarbamates was synthesized
by treating different substituted anilines with phenyl chloroformate in the presence of
6

o
sodium carbonate at 25 C [25].
Scheme 1. General scheme for the synthesis of target compounds; Reagents and conditions: (a)
o
N-Boc-piperazine, TEA, DMF, rt, 4 h; (b) TFA, n-BuOH, 120 C, 1 h; (c) TFA, DCM, rt, 12 h; (d)
o
o
DIPEA, DMF, 40 C, 12 h; (e) TEA, acetonitrile, 80 C, 3 h; (f) Pd-C, H , EtOH; (g) phenyl
2
chloroformate, sodium carbonate, THF/EA/H O, rt, 12 h.
2
2
2
.3 Bioactivity and discussion
.3.1 Design Rationale and Structure−Activity Relationship (SAR) Exploration.
We first synthesized compound 14a, which retains the iso-propoxyl group at “A”
phenyl ring from tandutinib, and 14b-14h with other different substituents. The
results of the kinase-inhibition assays are listed in Table 1 with pacritinib,
momelotinib and tandutinib as the positive controls.
Table 1. SAR exploration of different substituents in “A” phenyl ring
a
a
Cpd
R1
JAK2 inhibition @ 100nM
FLT3 inhibition @ 100nM
1
4a
11.1%
22.9%
7

1
4b
4c
4d
4e
4f
4g
4h
53.7%
54.8%
53.3%
48.7%
41.2%
79.3%
78.0%
35.2%
86.7%
64.4%
86.3%
68.1%
40.6%
20.0%
75.8%
86.0%
94.7%
27.8%
27.8%
27.4%
21.7%
0%
1
1
1
1
1
84.4%
70.8%
36.5%
80.7%
62.8%
81.1%
35.8%
33.1%
17.2%
53.9%
79.7%
1
1
4i
1
4j
1
4k
4l
4m
4n
1
1
1
1
4o
4p
4q
1
1
b
momelotinib
tandutinib
pacritinib
-
-
-
ND
b
ND
46.1%
96.0%
92.0%
a
b
All compounds were assayed at least twice, and the inhibitory values were averaged.
Not determined.
As shown in Table 1, compounds with electron-withdrawing groups (EWGs),
such as 14g and 14h, showed more potent inhibitory activities against JAK2 and
8

FLT3 than those with electron-donating groups (EDGs, see 14a-f). Base on the results,
a hypothesis was proposed that EWGs confers greater potency against both JAK2 and
FLT3 than EDGs on the “A” phenyl ring. Therefore, some other EWGs were
introduced to the “A” phenyl ring and compounds 14i-q were obtained (Table 1).
When the carbonyl-like groups were introduced (14j, 14l, and 14q), both JAK2
and FLT3 inhibition rates increased up to 80%. The resulting increase in inhibition
rates might be attributed to the H-bond formed between the carbonyl and ARG980 in
JAK2 and ASP829 in FLT3 (Fig. 3A). However, when the “length” of amide in 14l
was enhanced, such as compounds 14m-o, a decrease in their inhibitory activities
against JAK2 and FLT3 was observed, especially against FLT3. The probable cause
was the tail group of compounds located in a quite narrow channel which was formed
by VAL675, PHE691, CYS828 and ASP829 in FLT3 (Fig. 3C). Therefore, the size of
the tail group cannot be too large.
Table 2. SAR exploration of EWG substituents in “A” phenyl ring
a
a
Cpd
R3
R4
JAK2 inhibition @ 100nM
FLT3 inhibition @ 100nM
1
4r
4s
8.1%
35.2%
21.8%
2.0%
9
15.9%
36.5%
15.0%
3.3%
1
1
4t
1
4u

1
4v
4w
4x
18.9%
74.0%
9.5%
13.8%
39.8%
12.5%
1
1
a
All compounds were assayed at least twice, and the inhibitory values were averaged.
To investigate the effects of the substituents and substituent positions at “A”
phenyl ring, compounds 14r-x were synthesized (Table 2). After a comparison among
4j, 14u, and 14v, it can be easily concluded that the compound with the
1
para-substituent, 14j, showed the greatest potency against JAK2 and FLT3, and a
similar trend can be found among other analogues with their parent compounds.
Therefore, para-substituted compounds 14j, 14l, and 14q were chosen for further
modifications. Although compound 14g showed similar activities to 14j, it was not
included because the nitro group is a well-known structural alert in drug design.
In the following modifications, we paid attention to the hydrophilic groups at
4
-position of “B” phenyl ring, and the HEL cell line harboring the JAK2-V617F
mutation was applied in further cellular profiling (Table 3). Replacement of the
morpholino with N-methylpiperazinyl group (15a-c) retained the activities against
JAK2 (inhibition rates at 100 nM range from 80.8% to 86.0%) and FLT3 (inhibition
rates at 100 nM range from 85.0% to 87.5%), but replacement of morpholino group
with 4-(2-pyrrolidinyl)ethoxy (16a-c) decreased activities against JAK2 (inhibition
rates at 100 nM range from 45.0% to 59.0%) and FLT3 (inhibition rates at 100 nM
range from 69.0% to 82.0%). It is noteworthy that compounds with amide moiety (14l,
1
5b, and 16b) or sulfonamide moiety (14q, 16c, and 15c) lose their antiproliferative
activities against HEL cell line (>50 µM). This situation might be caused by an
1
0

increase in the compounds’ t-PSA, which influences their permeability (Table 3).
Therefore, amide and sulfonamide moieties were not considered in the next
modification.
Compounds 17-21a were synthesized to investigate the effects of other
hydrophilic groups, such as thiomorpholinyl, 4-methylpiperidinyl, and
4
-(2-pyrrolidinyl)ethoxy. The results showed that they all have a decrease in
inhibitory activities against JAK2 and FLT3 at 100 nM; similar trend was observed in
cellular potency against HEL (GI values ranged from 3.3 µM to 26.4 µM). As a
5
0
result, the morpholinyl group was found to be the best hydrophilic substituent in the
designed compounds.
Table 3. SAR optimization of hydrophilic groups at “B” phenyl ring
JAK2
FLT3
HEL
c
Cpd
R1
R2
inhibition @ inhibition @
t-PSA
b
GI50
a
a
1
00nM
100nM
1
4j
4l
86.7%
86.3%
86.0%
80.8%
84.0%
76.0%
80.7%
81.1%
79.7%
87.5%
85.0%
86.0%
4.3µM
>50µM
>50µM
14.1µM
>50µM
>50µM
102.93
128.95
154.40
96.94
1
1
4q
5a
5b
5c
1
1
122.96
148.41
1
11

O
N
1
6a
45.0%
53.0%
59.0%
73.9%
53.0%
60.7%
61.7%
60.9%
69.0%
74.0%
82.0%
77.6%
54.0%
83.2%
70.6%
70.9%
30.3µM
>50µM
>50µM
4.7µM
26.4µM
3.3µM
8.9µM
10.7µM
102.93
128.95
154.40
93.70
1
6b
16c
17a
18a
19a
20a
21a
93.70
102.93
112.16
114.96
a
b
c
All compounds were assayed at least twice, and the inhibitory values were averaged.
All GI values are the average of three replicates.
5
0
The t-PSA values were calculated using Marvin Sketch, version 6.1.0, with standard settings.
Among the series of compounds mentioned above, compounds having more than
8
0% inhibition rates against JAK2 and FLT3 were selected for further in vitro kinase
and cellular profiling (Table 4). Compared to 14j, compounds 14l and 14q both had
higher t-PSA, and the aforementioned decrease in their permeability resulted in the
loss of antiproliferation against HEL, MV4-11, and HL60 cell lines; therefore, amide
and sulfonamide moieties at “B” phenyl ring were unfavorable. In summary,
compound 14j exhibited an overall balanced profile meeting all target criteria. It was
shown to be active against JAK2 with IC value of 27 nM and FLT3 with IC value
5
0
50
of 30 nM. Moreover, 14j showed good inhibition against HEL with IC value of 4.3
5
0
µM and moderate inhibition against MV4-11 and HL60 (11.0 µM and 16.5 µM,
respectively). Thus, 14j was selected as the candidate for further study.
1
2

Table 4. In vitro profiling leading to 14j as a candidate
JAK2
FLT3
HEL
MV4-11
HL60
Cpd
4j
4l
R1
R2
a
a
b
b
b
IC50
IC50
GI50
GI50
GI50
1
27nM
11nM
30nM
21nM
30nM
12nM
40nM
13nM
4.3µM
>50µM
>50µM
14.1µM
4.7µM
3.3µM
1.1µM
11.0µM
>50µM
>50µM
32.0µM
17.8µM
11.0µM
1.6µM
16.5µM
>50µM
>50µM
46.4µM
>50µM
29.4µM
1.4µM
1
14q
15a
17a
19a
9.3nM
13.8nM
29.8nM
65.0nM
c
c
Pacritinib
-
-
23.0nM
22nM
a
All compounds were assayed at least twice, and the inhibitory values were averaged.
All GI values are the average of three replicates.
b
c
5
0
Data from J. Med. Chem, 2012, 55, 2623−2640.
2
.3.2 Effect of compound 14j on cell cycle phases
The effect of compound 14j on various phases of cell cycle progression was
tested in HEL cells. After treatment of HEL cells with compound 14j for 72 h at
varied concentrations (0.33µM, 1.0µM, 3.0µM, 9.0 µM), the cells were fixed and
stained with propidium iodide (PI) and the DNA content was analyzed by flow
cytometry. The obtained results were compared with those of non-treated HEL cells as
control. As shown in Fig. 3, the percentage of cells in the G populations increased
1
from 43.12% (as control group) to 54.07%, 61.44%, 71.85%, and 78.05%,
respectively. These results confirmed that compound 14j arrested the cell-cycle
1
3

progression of the former cell line into the G /S phases in a dose-dependent manner.
1
Fig. 3. Effect of compound 14j on the cell cycle distribution of HEL cells. HEL cells treated with
1
4j at 0.33 µM – 9 µM for 72 h were assayed by flow cytometer after staining with PI.
2
.3.3 Cell apoptosis study of compound 14j
To characterize the mode of cell death induced by compound 14j,
biparametric-flow-cytometric analysis with Annexin-V and propidium iodide (PI) was
performed. Flow cytometry was used to investigate whether the antiproliferative
effect of 14j was caused by the activation of cellular apoptosis in HEL cells.
Quantitative analysis of early-apoptotic cells and advanced-apoptotic and necrotic
cells was determined via an Annexin V-FITC and PI assay. HEL cells were stimulated
with 0.33 µM−9 µM concentrations of compound 14j for 72 h. DMSO was used as
the negative control. As shown in Fig. 4, the apoptosis induced by compound 14j was
much greater than that by the control, and HEL cells underwent both early apoptosis
(Annexin V+ and PI−, lower right) and advanced apoptosis and necrosis (Annexin V+
and PI+, upper right). The results showed that compound 14j could effectively induce
cell apoptosis starting from 1 µM with enhanced effects at higher concentrations. The
apoptosis ratios of compound 14j measured at different concentrations were 8.37%
1
4

(
0.33 µM), 39.37% (1 µM), 65.47% (3 µM), and 65.93% (9 µM), and the apoptotic
effect increased in a dose-dependent manner. From these results, we can conclude that
4j caused effective apoptotic effects on the HEL cell line.
1
Fig. 4. Cell apoptosis induced by compound 14j. HEL cells were treated with 14j at 0.33 µM – 9
µM for 72 h. Apoptosis was examined by flow cytometer.
2
.3.4 Binding modes of compound 14j
Compound 14j shows optimal JAK2 and FLT3 inhibitory activities; hence, the
binding mode of 14j was elucidated using a docking model (Fig. 5). As shown in Fig.
A, 14j binds to the ATP-binding site of JAK2 in an orientation similar to that of
3
pacritinib (Fig. 5B). The 2-aminopyrimidine moiety of 14j forms two conserved
hydrogen bonds with LEU932 in the hinge region of JAK2 which guaranteed the
JAK2 potency; meanwhile the pyrimidinyl group together with adjacent ALA880,
LEU855, VAL863, and LEU983 formed a netlike alkyl-π interaction. In addition, the
morpholine moiety was exposed in the solvent area and formed a hydrogen bond with
LYS943, and another hydrogen bond was formed between NH of
1
5

N-phenylpiperazine-1-carboxamide and LEU 855.
Fig. 5. Binding-mode analysis of compound 14j to JAK2 kinase and FLT3 kinase, hydrogen bonds
were shown as red dashes. (A) Compound 14j docked into JAK2 kinase (PDB code 4AQC). (B)
Overlapping images of compound 14j (green) and pacritinib (violet) complexed with JAK2. (C)
Compound 14j docked into FLT3 kinase (PDB code 4XUF). (D) Overlapping images of
compound 14j (green) and quizartinib (yellow) complexed with FLT3.
In Fig. 3C, we found the configuration of 14j resembles that of quizartinib (Fig.
5
D). A hydrogen bond was observed between NH of 2-aminopyrimidine and CYS695,
and the morpholine tail was fully stretched into the solvent-exposed area. Furthermore,
a π-π stacked interaction was formed by “B” phenyl ring with TYR696. Another
hydrogen bond was formed between the oxygen atom of acetophenone moiety with
ASP829, and the “A” phenyl ring was strongly fixed at the narrow hydrophobic
channel by two edge-to-face π-π stacked interactions with PHE691 and PHE830. In
general, the docking results further confirm the rationality of our design strategy.
3
. Conclusion
A series of novel 4-piperazinyl-2-aminopyrimidine derivatives were initially
designed and synthesized as JAK2/FLT3 dual inhibitors. In particular, compound 14j
1
6

showed the most balanced antiproliferative activity against JAK2 and FLT3 with IC50
value of 27 nM and 30 nM, respectively. It also showed good inhibition against HEL
with GI value of 4.3 µM and moderate inhibition against MV4-11 and HL60 (GI
5
0
50
values of 11.0 µM and 16.5 µM, respectively). Furthermore, cell cycle analysis of 14j
on HEL cells by flow cytometry showed that cell cycle was arrested in G /S phase,
1
and thereby induced the apoptosis of HEL cells in a dose-dependent manner. All these
reported results suggested that compound 14j might be a “hit” compound for
JAK2-FLT3 dual inhibitor.
4
. Experimental procedures
4
.1. Chemistry
All melting points were acquired on a Mettler Melting Point MP70 apparatus
(Mettler, Toledo, Switzerland) without calibration. Mass spectra (MS) were taken in
ESI mode on Agilent 1100 LC-MS (Agilent, palo Alto, CA, USA). High-resolution
mass spectra (HRMS) were measured with an Agilent Accurate-Mass Q-TOF 6530 in
ESI mode (Agilent, Santa Clara, CA, USA). The reverse phase HPLC was conducted
on an Agilent 1260 Infinity chromatograph, which was equipped with ZORBAX
SB-C18 column (250 mm × 4.6 mm). The mobile phase A was methanol, and mobile
phase B was 30 mM NaH PO in water (pH 2.5). The gradient of 5−95% A was run at
2
4
a flow rate of 1.0 mL/min over 30 min. Reactions were monitored by thin layer
1
chromatography (TLC) on silica plates (F-254) and visualized under UV light. H
1
3
NMR and C NMR spectra were performed using Bruker spectrometers (Bruker
Bioscience, respectively, Billerica, MA, USA) with TMS as an internal standard.
1
7

Column chromatography was run on silica gel (200-300 mesh) from Qingdao Ocean
Chemicals (Qingdao, Shandong, China). Unless otherwise noted, all materials were
obtained from commercially available sources and used without further purification.
4
.1.1. General procedure for preparation of intermediate 6
A solution of 2, 4-dichloropyrimidine (5) (10.0 g, 67.1 mmol) and Et N (11.1 mL,
3
o
8
0.5 mmol) in DMF (100 mL) was added dropwise to a cooled solution (-10 C) of
N-Boc-piperazine (13.1 g, 70.5 mmol) in DMF (100 mL). The mixture was then
stirred for 4 h at room temperature. The resulting mixture was poured into stirring
ice-water (500 mL), and then the white solid was filtered and dried under reduced
pressure. The crude product was further purified by flash column chromatography
1
using PE/EtOAc (1:1) as eluent to afford 15.3 g of 6 as a white solid; yield: 76.2%; H
NMR (600 MHz, DMSO-d ) δ 8.10 (d, J = 6.2 Hz, 1H), 6.83 (d, J = 6.2 Hz, 1H), 3.62
6
1
3
(
m, 4H), 3.47 – 3.37 (m, 4H), 1.42 (s, 9H); C NMR (151 MHz, DMSO-d ) δ 162.76,
6
o
1
59.89, 157.86, 154.17, 102.85, 79.61, 43.57, 28.40; m.p.: 170.6 – 173.2 C.
4
4
1
.1.2 General procedure for preparation of intermediates 8a-h
.1.2.1. 4-(4-nitrophenyl)morpholine (8a). To
a
stirred solution of
-fluoro-4-nitrobenzene 7 (2.0 g, 14.2 mmol) in 20 mL acetonitrile was added
morpholine (1.9 g, 21.3 mmol) followed by triethylamine (4.3 g, 5.9 mL, 42.5 mmol).
The mixture was stirred at reflux for 3 h. After the reaction was cooled to room
temperature, it was poured into 80 mL water and extracted with ethyl acetate (2×80
mL). The combined organic layers were washed with brine (60 mL), dried over
1
8

sodium sulfate, concentrated in vacuo, then dried under vacuum to obtain 2.7 g of 8a
as yellow solid; yield: 92%; 8a was used without further purification.
4
.1.2.2. 1-methyl-4-(4-nitrophenyl)piperazine (8b). Synthesized using the procedure
for 8a using 2.0 g (14.2 mmol) of 7 and 2.1 g (21.3 mmol) of 1-methylpiperazine in
0 mL of acetonitrile and 5.9 mL of trimethylamine, and 2.8 g of 8b was obtained as
yellow solid; yield: 89%.
2
4
.1.2.3. 4-methyl-1-(4-nitrophenyl)piperidine (8c). Synthesized using the procedure
for 8a using 2.0 g (14.2 mmol) of 7 and 2.1 g (21.3 mmol) of 4-methylpiperidine in
0 mL of acetonitrile and 5.9 mL of trimethylamine, and 2.8 g of 8c was obtained as
yellow solid; yield: 91%.
2
4
.1.2.4. 4-(4-nitrophenyl)thiomorpholine (8d). Synthesized using the procedure for 8a
using 2.0 g (14.2 mmol) of 7 and 2.2 g (21.3 mmol) of thiomorpholine in 20 mL of
acetonitrile and 5.9 mL of trimethylamine, and 2.7 g of 8d was obtained as yellow
solid; yield: 87%.
4
.1.2.5. 1-(2-(4-nitrophenoxy)ethyl)pyrrolidine (8e). Synthesized using the procedure
for 8a using 2.0 g (14.2 mmol) of 7 and 2.5 g (21.3 mmol) of
-(pyrrolidin-1-yl)ethan-1-ol in 20 mL of acetonitrile and 5.9 mL of trimethylamine,
and 1.9 g of 8e was obtained as yellow solid; yield: 58%.
2
1
9

4
.1.2.6. 1-(3-(4-nitrophenoxy)propyl)pyrrolidine (8f). Synthesized using the procedure
for 8a using 2.0 g (14.2 mmol) of 7 and 2.8 g (21.3 mmol) of
-(pyrrolidin-1-yl)propan-1-ol in 20 mL of acetonitrile and 5.9 mL of trimethylamine,
and 2.2 g of 8f was obtained as yellow oil; yield: 62%.
3
4
.1.2.7. 4-(2-(4-nitrophenoxy)ethyl)morpholine (8g). Synthesized using the procedure
for 8a using 2.0 g (14.2 mmol) of 7 and 2.8 g (21.3 mmol) of 2-morpholinoethan-1-ol
in 20 mL of acetonitrile and 5.9 mL of trimethylamine, and 2.6 g of 8g was obtained
as yellow oil; yield: 73%.
4
.1.2.8. 4-(3-(4-nitrophenoxy)propyl)morpholine (8h). Synthesized using the
procedure for 8a using 2.0 g (14.2 mmol) of 7 and 3.1 g (21.3 mmol) of
3
2
-morpholinopropan-1-ol in 20 mL of acetonitrile and 5.9 mL of trimethylamine, and
.6 g of 8h was obtained as yellow oil; yield: 69%.
4
4
8
.1.3 General procedure for preparation of intermediates 9a-h
.1.3.1. 4-morpholinoaniline (9a). Pd-C (0.27g, 10% m/m) was added to a solution of
a (2.7g, 13.0 mmol) in ethanol (20ml) and hydrogenated for 12 h at room
temperature. The resultant was filtered, washed with ethanol and concentrated. 2.2 g
1
of 9a as purple solid was obtained; yield: 94%; H NMR (600 MHz, DMSO-d ) δ
6
6
.68 (d, J = 8.5 Hz, 2H), 6.50 (d, J = 8.5 Hz, 2H), 4.62 (s, 2H), 3.69 (t, J = 4.8Hz ,4H),
2
0

+
2
.87 (t, J = 4.8Hz ,4H). MS (ESI) m/z: 179.2 [M+H] .
4
.1.3.2. 4-(4-methylpiperazin-1-yl)aniline (9b). Synthesized using the procedure for
9
a using 0.28 g (10% m/m) of Pd-C and 2.8 g (12.7 mmol) of 8b in 20 mL of ethanol,
1
and 2.1 g of 9b was obtained as purple solid; yield: 88%; H NMR (600 MHz,
DMSO-d ) δ 6.67 (d, J = 8.8 Hz, 2H), 6.48 (d, J = 8.8 Hz, 2H), 4.57 (s, 2H), 2.89 (t, J
6
+
=
4.8 Hz, 4H), 2.41 (t, J = 4.8 Hz, 4H), 2.19 (s, 3H). MS (ESI) m/z: 192.3 [M+H] .
4
.1.3.3. 4-(4-methylpiperidin-1-yl)aniline (9c). Synthesized using the procedure for 9a
using 0.28 g (10% m/m) of Pd-C and 2.8 g (12.7 mmol) of 8c in 20 mL of ethanol,
1
and 2.3 g of 9c was obtained as violet solid; yield: 96%; H NMR (600 MHz,
DMSO-d ) δ 6.67 (d, J = 7.2 Hz, 2H), 6.47 (d, J = 7.2 Hz, 2H), 4.57 (s, 2H), 3.29 (d, J
6
=
10.2 Hz, 2H), 2.44 (t, J = 11.0 Hz, 2H), 1.65 (d, J = 11.7 Hz, 2H), 1.45 – 1.32 (m,
1
H), 1.23 (q, J = 11.1 Hz, 2H), 0.92 (d, J = 6.5 Hz, 3H). MS (ESI) m/z: 191.3
+
[
M+H] .
4
.1.3.4. 4-thiomorpholinoaniline (9d). Synthesized using the procedure for 9a using
0
.27 g (10% m/m) of Pd-C and 2.7 g (12.0 mmol) of 8d in 20 mL of ethanol, and 1.7
1
g of 9d was obtained as light yellow solid; yield: 72%; H NMR (600 MHz,
DMSO-d ) δ 6.68 (d, J = 8.6 Hz, 2H), 6.48 (d, J = 8.6 Hz, 2H), 4.64 (s, 2H), 3.15 (t, J
6
+
=
4.8 Hz, 4H), 2.68 (d, J = 4.8 Hz, 4H). MS (ESI) m/z: 195.2 [M+H] .
2
1

4
.1.3.5. 4-(2-(pyrrolidin-1-yl)ethoxy)aniline (9e). Synthesized using the procedure for
9
a using 0.19 g (10% m/m) of Pd-C and 1.9 g (8.0 mmol) of 8e in 20 mL of ethanol,
1
and 1.2 g of 9e was obtained as brown oil; yield: 73%; H NMR (600 MHz, DMSO-d )
6
δ 6.64 (d, J = 8.7 Hz, 2H), 6.50 (d, J = 8.7 Hz, 2H), 4.59 (s, 2H), 3.90 (t, J = 6.0 Hz,
+
2
H), 2.71 (t, J = 6.0 Hz, 2H), 2.48 (s, 4H), 1.67 (s, 4H). MS (ESI) m/z: 207.3 [M+H] .
4
.1.3.6. 4-(3-(pyrrolidin-1-yl)propoxy)aniline (9f). Synthesized using the procedure
for 9a using 0.22 g (10% m/m) of Pd-C and 2.2 g (8.8 mmol) of 8f in 20 mL of
1
ethanol, and 1.8 g of 9f was obtained as light yellow solid; yield: 92%; H NMR (600
MHz, DMSO-d ) δ 6.62 (d, J = 8.8 Hz, 2H), 6.49 (d, J = 8.8 Hz, 2H), 4.59 (s, 2H),
6
3
.84 (t, J = 6.4 Hz, 2H), 2.54 (t, J = 7.3 Hz, 2H), 2.47 (s, 4H), 1.82 (p, J = 6.6 Hz, 2H),
+
1
.68 (p, J = 3.2 Hz, 4H). MS (ESI) m/z: 221.3 [M+H] .
4
.1.3.7. 4-(2-morpholinoethoxy)aniline (9g). Synthesized using the procedure for 9a
using 0.26 g (10% m/m) of Pd-C and 2.6 g (10.3 mmol) of 8g in 20 mL of ethanol,
1
and 2.0 g of 9g was obtained as purple solid; yield: 89%; H NMR (600 MHz,
DMSO-d ) δ 6.64 (d, J = 8.8 Hz, 2H), 6.49 (d, J = 8.8 Hz, 2H), 4.64 (s, 2H), 3.92 (t, J
6
=
5.8 Hz, 2H), 3.57 (d, J = 4.8 Hz, 4H), 2.61 (t, J = 5.8 Hz, 2H), 2.44 (s, 4H). MS
+
(
ESI) m/z: 223.3 [M+H] .
4
.1.3.8. 4-(3-morpholinopropoxy)aniline (9h). Synthesized using the procedure for 9a
using 0.26 g (10% m/m) of Pd-C and 2.6 g (9.8 mmol) of 8h in 20 mL of ethanol, and
2
2

1
2
.1 g of 9h was obtained as brown solid; yield: 91%; H NMR (600 MHz, DMSO-d )
6
δ 6.63 (d, J = 8.8 Hz, 2H), 6.49 (d, J = 8.8 Hz, 2H), 4.60 (s, 2H), 3.84 (t, J = 6.6 Hz,
2
H), 3.56 (t, J = 4.5 Hz, 4H), 2.38 (t, J =6.6 Hz, 2H), 2.34 (s, 4H), 1.79 (p, J = 6.5 Hz,
+
2
H). MS (ESI) m/z: 237.3 [M+H] .
4
.1.4 General procedure for preparation of compounds 10a-h
4
.1.4.1.
tert-butyl-4-(2-((4-morpholinophenyl)amino)pyrimidin-4-yl)piperazine-1-carboxylate
10a). To a mixture of 6 (2.0 g, 6.7 mmol, 1 equiv.) and trifluoroacetate (2.3 g, 20.1
(
mmol, 3 equiv.) in 20 mL n-BuOH, 9a (1.4 g, 8.0 mmol, 1.2 equiv.) was added and
o
the resulted mixture was heated to 120 C and stirred for 1 h. When the mixture was
cooled to room temperature, it was poured into 40 mL water and the mixture was
neutralized with 10% sodium hydroxide. Blue-gray precipitate was filtered off and
washed with water; the crude product (10a) was dried in a vacuum desiccator and was
1
then used directly without further purification. H NMR (600 MHz, DMSO-d ) δ 9.60
6
(s, 1H), 7.92 (d, J = 6.7 Hz, 1H), 7.44 (d, J = 9.0 Hz, 2H), 6.93 (d, J = 9.0 Hz, 2H),
6
.39 (d, J = 6.7 Hz, 1H), 3.75 – 3.72 (m, 4H), 3.67 (t, J = 5.2 Hz, 4H), 3.43 (t, J = 5.2
1
3
Hz, 4H), 3.09 – 3.03 (m, 4H), 1.42 (s, 9H); C NMR (151 MHz, DMSO-d ) δ 161.83,
6
1
55.70, 154.18, 131.04, 122.17, 115.94, 95.43, 79.66, 66.51, 49.31, 44.10, 28.42; m.p.:
o
+
1
97.3 – 200.6 C. MS (ESI) m/z: 441.4 [M+H] .
4
.1.4.2.
2
3

tert-butyl-4-(2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)piperazine-1
carboxylate (10b). Synthesized using the procedure for 10a using 2.0 g (6.7 mmol) of
and 2.3 g (20.1 mmol) of trifluoroacetate and 1.5 g (8.0 mmol) of 9b in 20 mL of
-
6
n-BuOH, crude product of 10b was obtained and directly used without purification.
1
H NMR (600 MHz, DMSO-d ) δ 8.85 (s, 1H), 7.94 (d, J = 6.0 Hz, 1H), 7.54 (d, J =
6
9
.0 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 6.19 (d, J = 6.0 Hz, 1H), 3.57 (s, 4H), 3.40 (s,
o
4
H), 3.12 (s, 4H), 2.76 (s, 4H), 2.44 (s, 3H), 1.42 (s, 9H). m.p.: 175.2 – 177.5 C. MS
+
(
ESI) m/z: 454.5 [M+H] .
4
.1.4.3.
tert-butyl-4-(2-((4-(4-methylpiperidin-1-yl)phenyl)amino)pyrimidin-4-yl)piperazine-1-
carboxylate (10c). Synthesized using the procedure for 10a using 2.0 g (6.7 mmol) of
6
and 2.3 g (20.1 mmol) of trifluoroacetate and 1.5 g (8.0 mmol) of 9c in 20 mL of
1
n-BuOH, crude product of 10c was obtained and directly used without purification. H
NMR (600 MHz, DMSO-d ) δ 10.08 (s, 1H), 7.91 (d, J =4.2 Hz, 1H), 7.36 (d, J =1.2
6
Hz, 2H), 7.13(d, J =1.2 Hz, 2H), 6.52 (d, J = 7.2 Hz, 1H), 3.73 (s, 4H), 3.63 (d, J =
1
2.1 Hz, 2H), 3.45 (s, 4H), 2.72 (s, 2H), 1.71 (d, J = 11.0 Hz,2H), 1.53 (s, 1H), 1.42 (s,
o
9
H), 1.26 (d, J = 11.0 Hz, 2H), 0.94 (d, J = 6.5 Hz, 3H). m.p.: 221.8 – 224.8 C. MS
+
(
ESI) m/z: 453.5 [M+H] .
4
.1.4.4.
tert-butyl-4-(2-((4-thiomorpholinophenyl)amino)pyrimidin-4-yl)piperazine-1-carboxyl
2
4

ate (10d). Synthesized using the procedure for 10a using 2.0 g (6.7 mmol) of 6 and
2
.3 g (20.1 mmol) of trifluoroacetate and 1.6 g (8.0 mmol) of 9d in 20 mL of n-BuOH,
1
crude product of 10d was obtained and directly used without purification. H NMR
(
600 MHz, DMSO-d ) δ 10.23 (s, 1H), 7.92 (s, 1H), 7.34 (d, J = 7.8 Hz, 2H), 6.97 (d,
6
J =7.8Hz, 2H), 6.56 (d, J =7.4 Hz, 1H), 3.76 (s, 4H), 3.51 (t, J = 4.8 Hz 4H), 3.45 (s,
o
4
H), 2.67 (t, J = 4.8 Hz 4H), 1.42 (s, 9H). m.p.: 215.8 – 217.4 C. MS (ESI) m/z:
+
4
57.4 [M+H] .
4
.1.4.5.
tert-butyl-4-(2-((4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)amino)pyrimidin-4-yl)piperazine
1-carboxylate (10e). Synthesized using the procedure for 10a using 2.0 g (6.7 mmol)
-
of 6 and 2.3 g (20.1 mmol) of trifluoroacetate and 1.7 g (8.0 mmol) of 9e in 20 mL of
1
n-BuOH, crude product of 10e was obtained and directly used without purification. H
NMR (600 MHz, DMSO-d ) δ 8.89 (s, 1H), 7.94 (d, J = 6.0 Hz, 1H), 7.57 (d, J = 9.0
6
Hz, 2H), 6.84 (d, J = 9.0 Hz, 2H), 6.20 (d, J = 6.0 Hz, 1H), 4.02 (t, J = 5.9 Hz, 2H),
3
4
.57 (s, 4H), 3.40 (s, 4H), 2.83 (t, J = 5.9 Hz, 2H), 2.59 (s, 4H), 1.70 (p, J = 3.2 Hz,
o
+
H), 1.42 (s, 9H). m.p.: 141.0 – 143.1 C. MS (ESI) m/z: 469.5 [M+H] .
4
.1.4.6.
tert-butyl-4-(2-((4-(3-(pyrrolidin-1-yl)propoxy)phenyl)amino)pyrimidin-4-yl)piperazin
e-1-carboxylate (10f). Synthesized using the procedure for 10a using 2.0 g (6.7 mmol)
of 6 and 2.3 g (20.1 mmol) of trifluoroacetate and 1.8 g (8.0 mmol) of 9f in 20 mL of
2
5

1
n-BuOH, crude product of 10f was obtained and directly used without purification. H
NMR (600 MHz, DMSO-d ) δ 8.89 (s, 1H), 7.94 (d, J = 6.0 Hz, 1H), 7.57 (d, J = 9.0
6
Hz, 2H), 6.83 (d, J = 9.0 Hz, 2H), 6.20 (d, J = 6.0 Hz, 1H), 3.95 (t, J = 6.3 Hz, 2H),
3
.58 (s, 4H), 3.41 (s, 4H), 2.62 (t, J = 7.0 Hz, 2H), 2.54 (s, 4H), 1.92 – 1.84 (m, 2H),
o
+
1
.71 (s, 4H), 1.43 (s, 9H). m.p.: 150.9 – 153.1 C. MS (ESI) m/z: 483.4 [M+H] .
4
.1.4.7.
tert-butyl-4-(2-((4-(2-morpholinoethoxy)phenyl)amino)pyrimidin-4-yl)piperazine-1-ca
rboxylate (10g). Synthesized using the procedure for 10a using 2.0 g (6.7 mmol) of 6
and 2.3 g (20.1 mmol) of trifluoroacetate and 1.8 g (8.0 mmol) of 9g in 20 mL of
n-BuOH, crude product of 10g was obtained and directly used without purification.
1
H NMR (600 MHz, DMSO-d ) δ 8.88 (s, 1H), 7.94 (d, J = 6.0 Hz, 1H), 7.57 (d, J =
6
9
2
9
.0 Hz, 2H), 6.84 (d, J = 9.0 Hz, 2H), 6.20 (d, J = 6.0 Hz, 1H), 4.02 (t, J = 5.4 Hz,
H), 3.58 (t, J = 4.2Hz, 8H), 3.40 (s, 4H), 2.67 (t, J = 5.4Hz, 2H), 2.47 (s, 4H), 1.42 (s,
o
+
H). m.p.: 184.5 – 186.4 C. MS (ESI) m/z: 485.5 [M+H] .
4
.1.4.8.
tert-butyl-4-(2-((4-(3-morpholinopropoxy)phenyl)amino)pyrimidin-4-yl)piperazine-1-
carboxylate (10h). Synthesized using the procedure for 10a using 2.0 g (6.7 mmol) of
6
and 2.3 g (20.1 mmol) of trifluoroacetate and 1.9 g (8.0 mmol) of 9h in 20 mL of
n-BuOH, crude product of 10h was obtained and directly used without purification.
1
H NMR (600 MHz, DMSO-d ) δ 8.88 (s, 1H), 7.94 (d, J = 6.0 Hz, 1H), 7.56 (d, J =
6
2
6

9
2
1
.1 Hz, 2H), 6.83 (d, J = 9.1 Hz, 2H), 6.20 (d, J = 6.0 Hz, 1H), 3.94 (t, J = 6.4 Hz,
H), 3.57 (s, 8H), 3.40 (s, 4H), 2.41 (s, 2H), 2.36 (s, 4H), 1.84 (p, J = 6.6 Hz, 2H),
o
+
.42 (s, 9H). m.p.: 185.1 – 186.2 C. MS (ESI) m/z: 499.5 [M+H] .
4
.1.5 General procedure for preparation of compounds (11a-h)
4
.1.5.1. N-(4-morpholinophenyl)-4-(piperazin-1-yl)pyrimidin-2-amine (11a). The
crude product (10a) from the previous step was dissolved in 10 mL of
dichloromethane; 10 mL of trifluoroacetate was then slowly added. After stirring
overnight at room temperature, the mixture was evaporated under reduced pressure
and the resultant was added to 20mL water, adjusting pH to 8 with saturated sodium
hydrogen carbonate, the precipitate was filtered off and washed with water, then dried
under vacuum to obtain 1.8 g of 11a as blue-grey solid; yield: 77%; MS (ESI) m/z:
+
3
41.2 [M+H] .
4
.1.5.2.
N-(4-(4-methylpiperazin-1-yl)phenyl)-4-(piperazin-1-yl)pyrimidin-2-amine
(11b). Synthesized using the procedure for 11a, 10 mL of dichloromethane and 10 mL
of trifluoroacetate were used, 2.0 g of 11b were obtained; yield: 84%; MS (ESI) m/z:
+
3
54.2 [M+H] .
4
.1.5.3.
N-(4-(4-methylpiperidin-1-yl)phenyl)-4-(piperazin-1-yl)pyrimidin-2-amine
(11c). Synthesized using the procedure for 11a, 10 mL of dichloromethane and 10 mL
of trifluoroacetate were used, 1.8 g of 11c were obtained; yield: 78%; MS (ESI) m/z:
2
7

+
3
53.3 [M+H] .
4
.1.5.4. 4-(piperazin-1-yl)-N-(4-thiomorpholinophenyl)pyrimidin-2-amine (11d).
Synthesized using the procedure for 11a, 10 mL of dichloromethane and 10 mL of
trifluoroacetate were used, 1.9 g of 11d were obtained; yield: 81%; MS (ESI) m/z:
+
3
57.4 [M+H] .
4
.1.5.5. 4-(piperazin-1-yl)-N-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)pyrimidin-2-amine
(11e). Synthesized using the procedure for 11a, 10 mL of dichloromethane and 10 mL
of trifluoroacetate were used, 1.8 g of 11e were obtained; yield: 72%; MS (ESI) m/z:
+
3
69.3 [M+H] .
4
4
.1.5.6.
-(piperazin-1-yl)-N-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)pyrimidin-2-amine (11f).
Synthesized using the procedure for 11a, 10 mL of dichloromethane and 10 mL of
trifluoroacetate were used, 1.7 g of 11f were obtained; yield: 65%; MS (ESI) m/z:
+
3
83.3 [M+H] .
4
.1.5.7. N-(4-(2-morpholinoethoxy)phenyl)-4-(piperazin-1-yl)pyrimidin-2-amine (11g).
Synthesized using the procedure for 11a, 10 mL of dichloromethane and 10 mL of
trifluoroacetate were used, 1.7 g of 11g were obtained; yield: 68%; MS (ESI) m/z:
+
3
85.2 [M+H] ,
2
8