Discovery of (R)-1-(3-(4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-(dimethylamino)ethanone (CHMFL-FLT3-122) as a Potent and Orally Available FLT3 Kinase Inhibitor for FLT3-ITD Positive Acute Myeloid Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.5b01611

FLT3-ITD mutant has been observed in about 30% of AML patients and extensively studied as a drug discovery target. On the basis of the structure of PCI-32765 (ibrutinib), a BTK kinase inhibitor that was recently reported to bear FLT3 kinase activity through a structure-guided drug design approach, we have discovered compound 18 (CHMFL-FLT3-122), which displayed an IC₅₀ of 40 nM against FLT3 kinase and achieved selectivity over BTK kinase (over 10-fold). It significantly inhibited the proliferation of FLT3-ITD positive AML cancer cell lines MV4-11 (GI₅₀ = 22 nM), MOLM13/14 (GI₅₀ = 21 nM/42 nM). More importantly, 18 demonstrated 170-fold selectivity between FLT3 kinase and c-KIT kinase (GI₅₀ = 11 nM versus 1900 nM) in the TEL-fusion isogenic BaF3 cells indicating a potential to avoid the FLT3/c-KIT dual inhibition induced myelosuppression toxicity. In the cellular context it strongly affected FLT3-ITD mediated signaling pathways and induced apoptosis by arresting the cell cycle into the G0/G1 phase. In the in vivo studies 18 demonstrated a good bioavailability (30%) and significantly suppressed the tumor growth in MV4-11 cell inoculated xenograft model (50 mg/kg) without exhibiting obvious toxicity. Compound 18 might be a potential drug candidate for FLT3-ITD positive AML.

Full text of DOI:10.1021/acs.jmedchem.5b01611

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Article
Discovery of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-
d]pyrimidin-1-yl)piperidin-1-yl)-2-(dimethylamino)ethanone
(CHMFL-FLT3-122) as a Potent and Orally Available FLT3
Kinase Inhibitor for FLT3-ITD Positive Acute Myeloid Leukemia
Xixiang Li, Aoli Wang, Kailin Yu, Ziping Qi, Cheng Chen, Wenchao Wang,
Chen Hu, Hong Wu, Jiaxin Wu, Zheng Zhao, Juan Liu, Fengming Zou, Li
Wang, Beilei Wang, Wei Wang, Shanchun Zhang, Jing Liu, and Qingsong Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01611 • Publication Date (Web): 02 Dec 2015
Downloaded from http://pubs.acs.org on December 5, 2015
Just Accepted
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
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of their duties.

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Discovery of (R)ꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ3ꢀ(4ꢀ
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phenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀ
yl)piperidinꢀ1ꢀyl)ꢀ2ꢀ(dimethylamino)ethanone
(CHMFLꢀFLT3ꢀ122) as a Potent and Orally
Available FLT3 Kinase Inhibitor for FLT3ꢀITD
Positive Acute Myeloid Leukemia
Xixiang Li1,2,6, Aoli Wang1,3,6, Kailin Yu1,3,6, Ziping Qi1,2,6, Cheng Chen1,2, Wenchao Wang1,2,
Chen Hu1,2, Hong Wu1,3, Jiaxin Wu1,3, Zheng Zhao1,2, Juan Liu1,2, Fengming Zou1,2, Li Wang1,2,
Beilei Wang1,2, Wei Wang1,2, Shanchun Zhang2,4, Jing Liu1,2*, Qingsong Liu1,2,3,5*
1. High Magnetic Field Laboratory, Chinese Academy of Sciences, Mailbox 1110, 350
Shushanhu Road, Hefei 230031, Anhui, P. R. China
2. CHMFLꢀHCMTC Target Therapy Joint Laboratory, Shushanhu Road, Hefei 230031,
Anhui, P. R. China
3. University of Science and Technology of China, P. R. China, Anhui, Hefei, 230036
4. Hefei Cosource Medicine Technology Co. LTD., 358 Ganquan Road, Hefei, 230031,
Anhui, P. R. China
5. Hefei Science Center, Chinese Academy of Sciences, Shushanhu Road, Hefei 230031,
Anhui, P. R. China
6. These authors contribute equally
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ABSTRACT: FLT3ꢀITD mutant has been observed in about 30% AML patients and extensively
studied as a drug discovery target. Based on the structure of PCIꢀ32765 (Ibrutinib), a BTK kinase
inhibitor that was recently reported to bear FLT3 kinase activity, through a structureꢀguided drug
design approach, we have discovered compound 18 (CHMFLꢀFLT3ꢀ122), which displayed an
IC50 of 40 nM against FLT3 kinase and achieved selectivity over BTK kinase (over 10ꢀfold). It
significantly inhibited the proliferation of FLT3ꢀITD positive AML cancer cell lines MV4ꢀ11
(GI50: 22 nM), MOLM13/14 (GI50: 21 nM/42 nM). More importantly, 18 demonstrated 170ꢀfold
selectivity between FLT3 kinase and cꢀKIT kinase (GI50: 11 nM versus 1900 nM) in the TELꢀ
fusion isogenic BaF3 cells indicating a potential to avoid the FLT3/cꢀKIT dual inhibition
induced myelosuppression toxicity. In the cellular context it strongly affected FLT3ꢀITD
mediated signaling pathways and induced apoptosis by arresting the cell cycle into G0/G1 phase.
In the in vivo studies 18 demonstrated a good bioavailability (30%) and significantly suppressed
the tumor growth in MV4ꢀ11 cell inoculated xenograft model (50 mg/kg) without exhibiting
obvious toxicity. Compound 18 might be a potential drug candidate for FLT3ꢀITD positive
AML.
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INTRODUCTION
Acute Myeloid Leukemia (AML) accounts for approximately 80ꢀ90% of leukemia in adults.
Over ten thousand new AML patients are diagnosed and lead to similar number of deaths per
year in US alone.1, 2 The current standard chemotherapy for AML usually lacks the durable
efficacy and is also poorly tolerated especially in the elder patients. FMSꢀLike tyrosine kinase
(FLT3) is a type III receptor tyrosine kinase that plays important roles in the differentiation and
survival of hematopoietic stem cells in bone marrow and has been observed overꢀexpressed in
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the AML and ALL.3ꢀ5 A variety of gainꢀofꢀfunction mutations have been identified in the AML
patients such as FLT3ꢀITD (internal tandem duplication), FLT3ꢀD835Y/E/V/H, and FLT3ꢀ
K663Q etc, among which FLT3ꢀITD accounts for about 30% and is associated with poor
prognosis.6 The mutated FLT3ꢀITD kinase promotes the AML blast survival and proliferation
through the downstream signaling mediators including Stat5, ERK and AKT.7 Therefore, FLT3ꢀ
ITD kinase has been considered as a validated drug discovery target for FLT3ꢀITD positive
AML.
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A number of small molecule inhibitors have been reported to exhibit potent FLT3 kinase
inhibitory activities such as sunitinib8, sorafenib,9 PKC412,10, CEPꢀ701,11 UNC2025,12
MLN518,13 KWꢀ244914 and AMGꢀ92515 etc. In addition, the relatively more selective second
generation FLT3 inhibitors such as AC2206, crenolanib16 and PLX339717 are being tested in the
clinic now which have shown initial transient responses but usually followed by quick
development of resistance. Besides, the cꢀKIT kinase and FLT3 kinase dual inhibition induced
synthetic lethal myelosuppression toxicity observed from the most advanced clinical trial
compounds such as PKC412 and AC220 is also a big safety concern.18 Currently there is no
FLT3 targeted therapies get approved for clinical application yet.19 Hence, there is still a critical
unmet medical need remaining for FLT3ꢀITD positive AML.
Recently we discovered that PCIꢀ32765 (1), a clinically used BTK kinase inhibitor for Mantle
Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), also displayed subꢀ
micromolar GI50 against FLT3ꢀITD positive AML cancer cell lines.20, 21 In addition, compound 1
did not exhibit apparent cꢀKIT kinase inhibition which indicated a better safety profile for this
class of compounds. In order to further improve the FLT3 kinase potency and selectivity of this
new class of compounds, starting from 1, through a detailed SAR study, we discovered a potent,
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selective and orally available FLT3 kinase inhibitor 18 (CHMFLꢀFLT3ꢀ122) that displayed
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impressive in vitro and in vivo activities against FLT3ꢀITD positive AML (Figure 1).
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Figure 1. Chemcial structure of PCIꢀ32765 (1) and CHMFLꢀFLT3ꢀ122 (18).
RESULTS AND DISCUSSTION
We first examined the binding mode of PCIꢀ32765 to BTK and FLT3 kinase in order to direct
the chemical modification (Figure 2). In BTK kinase, PCIꢀ32765 forms three hydrogen bonds
with Met477, Glu475 and Thr474 respectively in the hinge binding area. The signature of this
binding is that the acrylamide of PCIꢀ32765 forms a covalent bond with Cys481 to achieve the
irreversible binding. The Oꢀbridged diphenyl moiety directs into the hydrophobic pocket formed
by DFG motif and αꢀcHelix (Figure 2A). In comparison, PCIꢀ32765 forms two similar hydrogen
bonds with Cys694 and Glu692 of FLT3 kinase in the hinge binding area. However, the Cys481
in BTK is replaced by Asp698 in FLT3 which makes the irreversible binding impossible (Figure
2B). Superimposition of BTK and FLT3 kinase reveals that the Phe830 in the DFG motif of
FLT3 kinase (white in Figure 2C) flips more flat than Phe540 in BTK kinase (purple in Figure
2C), which might provide more space for this hydrophobic pocket. However, the αꢀcHelix of
FLT3 kinase shifts closer to the hinge binding area than the αꢀcHelix of BTK kinase hence this
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will shrink the hydrophobic pocket (Figure 2C). Combining all of the analyses, we proposed that
there are two directions we can take to improve the binding affinity for new lead compounds
against FLT3 kinase. First, we can modify the Oꢀbridged diphenyl group (R2, Figure 2D) to
investigate if there is a larger pocket created by αꢀcHelixꢀin and Phe830ꢀout. Second, since in
FLT3 kinase there is no approachable cysteine residues required by irreversible binding, we can
modify the piperidine acrylamide moiety (R1 and R3, Figure 2D) to achieve the better selectivity
and stronger binding affinity against FLT3 kinase.
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Figure 2. Binding mode analysis of PCIꢀ32765 (1) to BTK kinase and FLT3 kinase. (A) PCIꢀ
32765 docked into BTK kinase (PDB ID: 3GEN). (B) PCIꢀ32765 docked into FLT3 kinase
(PDB ID: 1RJB). (C) Superimposition of BTK and FLT3 kinase in complex with PCIꢀ32765
(purple, BTK kinase; white, FLT3 kinase). (D) Illustration of the chemical modification strategy
of compound 1.
We first made compound 2 which bears a double bond linked diphenyl moiety to explore the
possibility of FLT3 kinase occupying a larger inner hydrophobic pocket formed by DFG motif
and αꢀcHelix. However, compared with 1, it significantly lost the activity against intact AML
cancer cell line MV4ꢀ11 (GI50: 10 ꢀM versus 0.33 ꢀM) (Table 1), which expresses FLT3ꢀITD
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mutant. In the isogenic BaF3 cells whose proliferation is dependent on FLT3ꢀITD, compound 2
also lost about 15ꢀfold activity (GI50: 1.8 ꢀM versus 0.12 ꢀM). This indicated that FLT3 might
not tolerate the larger hydrophobic moiety. We then turned our attention to the hinge binding
area and made a series of modifications at R1 position to explore the SAR. Since there is no
approachable cysteine residue which could be utilized to form the covalent bond as in BTK
kinase, we first saturated the double bond in 1 to make 3 and found that it exhibited better
inhibitory activities against MV4ꢀ11 (GI50: 0.13 ꢀM) and FLT3ꢀITDꢀBaF3 isogenic cell line
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(GI50: 0.028 ꢀM). But it also displayed a GI50 of 3.8 ꢀM against parental BaF3 cell line which
indicated there was still selectivity window could be explored (ideally, the compound that does
not show apparent inhibitory activity against the parental BaF3 cells i.e. >10 ꢀM is preferred).
Variation of terminal ethyl group, e.g. methyl group (4) or bulky ones such as propyl (5), tertꢀ
butyl (6), 2ꢀmethylꢀbutyl (7), dimethyl propyl (8) and cyclopropyl (9) all led to significant lose
of inhibitory activity. However, introduction of the methyl cyclopropyl group (10) started to gain
back the activity against MV4ꢀ11 (GI50: 0.41 ꢀM) and FLT3ꢀITDꢀBaF3 cells (GI50: 0.082 ꢀM),
though still weaker than 3. Methyl piperidine (11) and 1,4ꢀdimethyl piperazine (12) exhibited
comparably moderate activities to 3. Compounds that switching the linking carbonyl group into
sulfonamide and bearing different sizes of substitutes (13ꢀ15) did not improve the antiꢀ
proliferation efficacy either, despite that all of them still remained the moderate potencies.
Table 1. Compounds (1-15) with different substituents at R1/R2 position and their antiꢀ
proliferation efficacies against intact and isogenic cancer cell linesa
MV4ꢀ11
BaF3
BaF3ꢀFLT3ꢀITD
Compd.
R1
R2
(GI50: ꢀM)
(GI50: ꢀM)
(GI50: ꢀM)
0.33
0.12
>10
1
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>10
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0.13
0.028
0.35
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0.45
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>10
>10
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>10
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0.082
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0.093
0.048
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a All GI50s were obtained by triplet testing.
We then went back to analyze the binding mode again and envisioned that the Asp698 might
serve as a hydrogen bond acceptor. If the substitutes at proper R1 position could provide a
hydrogen bond donor then it might increase the potency. Therefore we replaced the acrylamide
group with a series of amino acids and derivatives for SAR study. The glycine moiety (16)
remarkably improved the antiꢀproliferation efficacy against MV4ꢀ11 (GI50: 0.022 ꢀM) and
BaF3ꢀFLT3ꢀITD (GI50: 0.007 ꢀM) meanwhile still remained proper selectivity against parental
BaF3 cell (GI50: 3.8 ꢀM) (Table 2), while the betaꢀalanine (17) lost over 30ꢀfold activity against
MV4ꢀ11. The N, Nꢀdimethyl glycine (18) kept the activity against MV4ꢀ11 (GI50: 0.022 ꢀM) and
BaF3ꢀFLT3ꢀITD (GI50: 0.011 ꢀM) and showed a better selectivity against parental BaF3 (GI50:
4.9 ꢀM). Introduction of other amino acids, e.g., alanine (19), serine (20), valine (21), threonine
(22) and leucine (24) led to similar activities except that isoleucine (23) lost around 6ꢀfold
activities. These results indicated that the hydrogen bond formed between Asp698 and the
nitrogen atom in R1 was critical for the binding (Figure 3).
Table 2. Compounds (16-24) with amino acids and derivatives at R1 position and their antiꢀ
proliferation efficaciesa
MV4ꢀ11
BaF3ꢀFLT3ꢀITD
BaF3
Compd.
R1
(GI50: ꢀM)
(GI50: ꢀM)
(GI50: ꢀM)
0.022
0.007
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a All GI50s were obtained by triplet testing.
Figure 3. Compound 18 docked into FLT3 kinase (PDB ID: 1RJB).
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Since compound 18 displayed the best activities so far, we then placed more efforts to investigate
the R3 part. Inversion of the R stereo center on the piperidine ring of 18 to S (25) resulted in over
10ꢀfold loss of activities indicating that the R conformation was preferred at this position (Table
3). Shifting the amide moiety from 3ꢀ position of piperidine to 4- position (26-28) or replacing
the piperidine ring by the pyrrolidine ring (29-32) all led to the activity loss.
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Table 3. Antiꢀproliferation efficacies of compounds (25-32) bearing different R1 and R3
moietiesa
MV4ꢀ11
BaF3ꢀFLT3ꢀITD
BaF3
Compd.
R1
R3
(GI50: ꢀM)
(GI50: ꢀM)
(GI50: ꢀM)
0.28
0.11
0.12
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>10
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2.15
>10
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32
a All GI50s were obtained by triplet testing.
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Considering the potent antiꢀproliferation activities against FLT3ꢀITD positive cell line and the
selectivity window between the FLT3ꢀITDꢀBaF3 cell and parental BaF3 cell, we decided to
choose 18 for further characterization. We first examined compound 18’s selectivity with
DiscoveRx’s KinomeScanTM technology. The results showed that compound 18 was very
selective (S score (1)=0.03 at 1ꢀM). Besides FLT3wt and FLT3/ITD, it also displayed strong
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binding against BLK, BTK, ERBB3, HCK, LCK, MEK5, PDGFRα, RET and SRMS kinases
(percent activity remaining less than 1% at 1ꢀM). (Fig. 4A and B and Supplemental Table 1)
Given the fact that KinomeScanTM is a binding assay and may not fully reflect the inhibitory
activity, we then used ADPꢀGlo based biochemical activity assay with the purified kinase
proteins to confirm the compound 18’s inhibition activity against FLT3, BTK and cꢀKIT. The
results demonstrated that compound 18 inhibited FLT3 kinase with an IC50 of 40 nM (Figure 4C).
It also demonstrated a good selectivity over BTK kinase (IC50: 421 nM) and cꢀKIT kinase (IC50:
559 nM). We also used TEL transformed BaF3 system to further confirm the onꢀtarget activity
and selectivity against other potential offꢀtargets revealed by KinomeScanTM binding assay
(Table 4). Interestingly, compound 18 was also very potent against FLT3ꢀD835Y/V/H mutants,
which are other important oncogenic mutations in the AML, in the BaF3 isogenic cell lines.
While for the drug resistance mutations such as FLT3ꢀITDꢀD835Y, FLT3ꢀITDꢀF691L, it lost 16ꢀ
20 fold activities comparing to FLT3ꢀITD itself. In addition, 18 also exhibited good selectivity
over cꢀKit kinase (GI50: 1.9 ꢀM versus 0.011 ꢀM for FLT3ꢀITD, 170 fold selectivity) and other
protein kinases such as MET, EGFR, JAK1/2/3, RET, PDGFRα and BMX (around 25ꢀ200 fold)
(Table 4). In the SRC kinase family, compound 18 did not potently inhibits HCK (GI50: 0.88
ꢀM), and LCK (GI50: 0.31 ꢀM) and BLK (GI50: 0.94 ꢀM). Combining the results from all of
these different assay formats, we concluded that compound 18 is a highly potent FLT3 kinase
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inhibitor. Compound 18 displayed similar potencies in the FLT3ꢀITD dependent AML cancer
cell line MOLM13 (GI50: 0.021 ꢀM) and MOLM14 (0.042 ꢀM) as in MV4ꢀ11 (Table 5 and 2).
As expected, it did not exhibit potent inhibitory activities against FLT3 wt or negative intact
cancer cell lines such as U937, SUꢀDHLꢀ2, U2932, JVMꢀ2, Namalwa and NB4 cell lines.
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Figure 4. Selectivity profiling of compound 18 (A) KinomeScanTM profiling of compound 18 at
a concentration of 1 ꢀM against 468 kinases. (B) Kinases showed strong binding to compound
18 (less than 1% activity remaining comparing to DMSO control in the assay format with 1 ꢀM
of 18). (C) ADPꢀGlo biochemical characterization of compound 18 against FLT3 kinase, BTK
kinase and cꢀKIT kinase.
Table 4. Antiꢀproliferation effects of compound 18 against a variety of isogenic BaF3 cell linesa
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Cell lines
Cell lines
GI50: ꢀM
0.016
0.17
GI50: ꢀM
1.9
BaF3ꢀTELꢀFLT3 wt
BaF3ꢀTELꢀcꢀKIT
BaF3ꢀTELꢀMET
BaF3ꢀTELꢀEGFR
BaF3ꢀTELꢀJAK1
BaF3ꢀTELꢀJAK2
BaF3ꢀTELꢀJAK3
BaF3ꢀTELꢀBMX
BaF3ꢀTELꢀHCK
BaF3ꢀTELꢀPDGFRb
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BaF3ꢀFLT3ꢀITDꢀ
D835Y
2.2
BaF3ꢀFLT3ꢀITDꢀ
F691L
0.22
1.8
BaF3ꢀFLT3ꢀD835Y
BaF3ꢀFLT3ꢀD835V
BaF3ꢀFLT3ꢀD835H
BaF3ꢀTELꢀBLK
0.033
0.003
0.004
0.94
3.8
0.3
2.5
0.28
0.88
0.68
BaF3ꢀTELꢀLCK
0.31
BaF3ꢀTELꢀRET
0.65
a All GI50s were obtained by triplet testing.
Table 5. Antiꢀproliferation effects of compound 18 against a variety of intact cancer cell lines a
Cell lines
Cell lines
GI50:
ꢀM
GI50: ꢀM
MOLM13
MOLM14
U937
0.021
0.042
9.7
U2932
JVMꢀ2
Namalwa
NB4
1.6
2.6
6.4
3.8
SUꢀDHLꢀ2
5.5
a All GI50s were obtained by triplet testing
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We then investigated the inhibitory effect of 18 on FLT3 mediated signaling pathway. It
significantly affected FLT3 autoꢀphosphorylation at Tyr589/591 site in MV4ꢀ11 and MOLM14
cell line (EC50 less than 30 nM) as well as MOLM13 cell line (EC50 about 100 nM) (Figure 5).
FLT3 kinase downstream mediator Stat5’s phosphorylation was also remarkably inhibited in
these cell lines (EC50 less than 30 nM). In addition, the phosphorylation of ERK, AKT and
expression of cMYC were also significantly inhibited, which exhibited a similar trend to the
wellꢀestablished FLT3 inhibitor AC220. Compound 18 could also arrest cell cycle progression
of these cell lines into the G0/G1 phase and induction of apoptosis (Figure 6).
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Figure 5. Compound 18 inhibited FLT3 kinase mediated signaling pathway in MV4ꢀ11,
MOLM13 and MOLM14 cancer cell lines.
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Figure 6. Compound 18 arrested cell cycle progression and induction of apoptosis in MV4ꢀ11,
MOML14 and MOLM13 cells.
We also evaluated the compound 18’s PK properties in rats following intravenous and oral
administration (Table 6). The halfꢀlife of the 18 was about 1.4 h with IV and 1.2 h with PO and
the bioavailability was about 30%. Overall it had a good volume distribution but relatively quick
clearance.
Table 6. PK properties of compound 18.
MRT
Compd.
T1/2 Tmax
Cmax
AUC(0ꢀt) AUC(0ꢀ∞)
Vz
CLz
(0ꢀ∞) F (%)
(hr)
(hr) (hr) (ng/mL) ng/mL*hr Ng/mL*hr
mL/kg
mL/hr/kg
18
IV
(1mg/kg)
Mean
1.38 0.02 1503.83
606.89
20.78
619.33
23.28
3233.38
779.55
1616.20
62.03
1.15
0.12
NA
NA
SD
0.31 0.00
121.81
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PO (10
mg/kg)
Mean
1.24 1.67
0.24 0.58
458.24
103.77
1834.84
360.99
1883.80
378.29
NA
NA
NA
NA
2.75
0.32
30.42
NA
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SD
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We next tested the antitumor efficacy of compound 18 in MV4ꢀ11cell inoculated xenograft
mouse model. With oral gavage, the 12.5, 25 and 50 mg/Kg/day administration did not
significantly affect the mice body weight (Figure 7A). However, the 25mg/kg/day and 50
mg/kg/day dosage could almost completely suppress the tumor progression (Figure 7 B, C, D).
It is worth to mention that the PK profile obtained from rat may not be directly transformed into
the mice due to the species difference.
Figure 7. Compound 18’s antiꢀtumor efficacy in MV4ꢀ11 xenograft model. Female nu/nu mice
bearing established MV4ꢀ11 tumor xenografts were treated with 18 at 12.5, 25.0, 50.0 mg/kg/d,
or vehicle. Daily oral administration was initiated when MV4ꢀ11 tumors had reached a size of
200 to 400 mm3. Each group contained 5 animals. Data, mean ± SEM. (A) Body weight and (B)
tumor size measurements from MV4ꢀ11 xenograft mice after 18 administration. Initial body
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weight and tumor size were set as 100%. (C) Representative photographs of tumors in each
group after 12.5, 25.0 or 50.0 mg/kg/d 18 or vehicle treatment. (D) Comparison of the final
tumor weight in each group after 22ꢀday treatment period. Numbers in columns indicate the
mean tumor weight in each group. ns, p>0.05, *p<0.05, **p<0.01.
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CONCLUSION
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Starting from irreversible BTK kinase inhibitor compound 1 (PCIꢀ32765), we used a focused
medicinal chemistry approach guided by structure based drug design to afford compound 18
(CHMFLꢀFLT3ꢀ122) which possessed highly potent inhibitory activities against FLT3ꢀITD
positive AML cancer cell lines meanwhile showed good selectivity against the nonꢀFLT3
dependent cancer cell lines. In addition, compound 18 has achieved better selectivity between the
BTK kinase and FLT3 kinase comparing to compound 1. What is worth to mention is that 18 has
also achieved good selectivity over cꢀKIT kinase, which potentially will reduce the
myelosuppression toxicity. Compound 18 exhibited suitable PK properties and strong in vivo
antiꢀtumor activity. Currently compound 18 is under extensive preclinical evaluation and we
believe it will be a useful pharmacological candidate for further exploration of the therapeutic
potential in the FLT3ꢀITD positive AML cancer.
EXPERIMENTAL SECTION
1
General Procedures. All solvents and reagents were used as obtained. H NMR spectra and
13C NMR spectra were recorded with a Bruker 400 NMR spectrometer and referenced to
deuterium dimethyl sulfoxide (DMSOꢀd6) or deuterium chloroform (CDCl3). Chemical shifts are
expressed in ppm. In the NMR tabulation, s indicates singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; and br, broad peak. LC/MS were performed on an Agilent 6224 TOF using an ESI
source coupled to an Agilent 1260 Infinity HPLC system operating in reverse mode with an
Agilent Eclipse Plus C18 1.8µm 3.0×50 mm column. Purification of final compounds were
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performed with Isco CombiFlash Rf + system (Silica Flash Column 4g) using a gradient of 0ꢀ
10% MeOH in DCM over 20 min at a flow rate of 18 mL/min. The purities of all compounds
were above 95%.
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Reagents and conditions: (a) DMF, NIS, 80 °C, 8 h; (b) DMF/H2O, Pd(dppf)Cl2, K3PO4, (4ꢀ
phenoxyphenyl)boronic acid, 120 °C, 12 h; (c) THF, triphenylphosphine, DIAD, (S)ꢀtertꢀbutyl 3ꢀ
hydroxypiperidineꢀ1ꢀcarboxylate, 0 °Cꢀrt, 12 h; (d) 4 N HCl in EtOAc, rt, 1 h; (e) THF,
triphenylphosphine, DIAD, (S)ꢀtertꢀbutyl 3ꢀhydroxypyrrolidineꢀ1ꢀcarboxylate, 0 °Cꢀrt, 12 h; (f)
THF, triphenylphosphine, DIAD, tertꢀbutyl 4ꢀhydroxypiperidineꢀ1ꢀcarboxylate, 0 °Cꢀrt, 12 h.
3ꢀiodoꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine (S1) The mixture of 1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ
4ꢀamine (10 g, 74 mmol) and Nꢀiodoꢀsuccinimide (25 g, 111 mmol) in DMF (100 mL) was
stirred at 80 °C for 8 h. The resulting mixture was allowed to cool to room temperature, and then
diluted with 300 mL of water. The precipitate was filtered and washed with 2×60 mL of
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saturated aqueous sodium sulfite, 2×100 mL of water, respectively and dried under vacuum to
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yield S1 as a yellow solid (14 g, 73%), which was used directly without further purification. H
NMR (400 MHz, DMSOꢀd6) δ 8.29 (br, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ 156.13, 154.69,
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153.35, 102.79, 91.06; LC/MS (ESI, m/z): 261.9591 [M+H]+ .
3ꢀ(4ꢀphenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine (S2) To a mixture of S1 (10 g, 38.3
mmol) and DMF/H2O (150 mL, v/v, 3/2) was added (4ꢀphenoxyphenyl)boronic acid (9.8 g, 45.9
mmol), K3PO4 (12.1 g, 57.4 mmol) and Pd(dppf)Cl2 (1.4 g, 1.9 mmol). The reaction mixture was
placed into an oil bath preheated to 120 °C, with stirring at this temperature for 12 h under argon.
The resulting mixture was allowed to cool to room temperature, then diluted with 150 mL of
water. The precipitate was filtered, and dried under the vacuum. The crude product was
1
recrystallized with MeOH to afford S2 as an offꢀwhite color solid (7.2 g, 62%). H NMR (400
MHz, DMSOꢀd6) δ 13.57 (s, 1H), 8.23 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.45 (t, J = 7.2 Hz, 2H),
7.26 – 7.08 (m, 5H); 13C NMR (100 MHz, DMSOꢀd6) δ 158.59, 157.53, 156.77, 156.57, 155.26,
144.41, 130.54, 130.51, 128.93, 124.24, 119.44, 119.38, 97.46; LC/MS (ESI, m/z): 304.1217
[M+H]+.
General Procedure for the Preparation of Compounds S3, S4 and S5
(R)ꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1ꢀ(piperidinꢀ3ꢀyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine (S3) To a
mixture of triphenyl phosphine (5.1 g, 19.8 mmol) in THF (300 mL) was added diisopropyl
azodicarboxylate (3.9 mL, 19.8 mmol) at 0 °C. The reaction mixture was stirred at this
temperature for 0.5 h under argon, then (S)ꢀtertꢀbutyl 3ꢀhydroxypiperidineꢀ1ꢀcarboxylate (3.9 g,
19.8 mmol) was added. The reaction mixture was stirred at 0 °C for 0.5 h, after that, S2 (3.0 g,
9.9 mmol) was added. The reaction mixture was allowed to warm to room temperature with
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stirring for 12 h. The resulting mixture was then concentrated to afford the crude product, which
was purified by silica gel column chromatography (eluting 0%ꢀ2% MeOH in DCM) to provide
the desired Bocꢀprotected monoamine as an offꢀwhite solid (3.3 g). Then the solid in EtOAc (5
mL) was added 4 N HCl in EtOAc (15 mL). The reaction mixture was stirred at room
temperature for 1 h. After complete conversion of the starting material, excess EtOAc was
removed under vacuum. The residue was diluted in EtOAc and water. The water layer was
basified with 2 N NaHCO3 solution and extracted with EtOAc (2×100 mL). The organic layers
were then washed with water followed by brine. The organic layers were dried over sodium
sulfate, filtered, and concentrated to provide S3 as an offꢀwhite solid (2.2 g, 84%) without further
purification. 1H NMR (400 MHz, DMSOꢀd6) δ 8.25 (s, 1H), 7.67 (d, J = 7.9 Hz, 2H), 7.43 (t, J =
7.2 Hz, 2H), 7.15 (dt, J = 12.0, 7.8 Hz, 5H), 4.70 (t, J = 10.3 Hz, 1H), 3.09 (d, J = 9.0 Hz, 1H),
3.19 (s, 1H), 2.95 (dd, J = 24.7, 12.8 Hz, 2H), 2.47 (d, J = 11.5 Hz, 1H), 2.22 – 2.07 (m, 1H),
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2.01 (d, J = 17.3 Hz, 1H), 1.75 (d, J = 12.3 Hz, 1H), 1.56 (d, J = 12.2 Hz, 1H); C NMR (100
MHz, DMSOꢀd6) δ 158.64, 157.52, 156.80, 155.93, 154.24, 143.26, 130.57, 130.52, 128.61,
124.22, 119.43, 97.83, 54.40, 51.29, 45.91, 30.75, 26.47; LC/MS (ESI, m/z): 387.1904 [M+H]+.
(R)ꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1ꢀ(pyrrolidinꢀ3ꢀyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine (S4) Overall
1
yield from S2=53%. H NMR (400 MHz, DMSOꢀd6) δ 8.26 (s, 1H), 7.68 (d, J = 8.2 Hz, 2H),
7.42 (t, J = 7.5 Hz, 2H), 7.15 (dt, J = 12.5, 7.9 Hz, 5H), 5.32 (s, 1H), 3.59 (s, 1H), 3.29 – 3.01
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(m, 3H), 2.93 (s, 1H), 2.28 – 2.06 (m, 2H); C NMR (100 MHz, DMSOꢀd6) δ 158.64, 157.55,
156.80, 156.01, 154.38, 143.44, 130.56, 128.59, 124.21, 119.41, 98.00, 57.27, 53.26, 46.94,
32.96; LC/MS (ESI, m/z): 373.1783 [M+H]+.
3ꢀ(4ꢀphenoxyphenyl)ꢀ1ꢀ(piperidinꢀ4ꢀyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine (S5) Overall yield
from S2=53%. 1H NMR (400 MHz, DMSOꢀd6) δ 8.25 (s, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.43 (t, J
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= 7.3 Hz, 2H), 7.25 – 7.07 (m, 5H), 4.74 (t, J = 11.3 Hz, 1H), 3.07 (d, J = 11.6 Hz, 2H), 2.63 (t, J
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= 11.7 Hz, 2H), 2.05 (d, J = 8.4 Hz, 3H), 1.83 (d, J = 10.6 Hz, 2H); C NMR (100 MHz,
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DMSOꢀd6) δ 158.64, 157.52, 156.79, 155.87, 153.90, 143.21, 130.57, 130.49, 128.68, 124.23,
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119.45, 119.41, 97.88, 54.92, 45.89, 33.05; LC/MS (ESI, m/z): 387.1911 [M+H]+.
o
Reagents and conditions: (a) bis(pinacolato)diboron, 1,4ꢀdioxane, KOAc, Pd(dppf)Cl2, 100 C,
12 h; (b) THF, triphenylphosphine, DIAD, (S)ꢀtertꢀbutyl 3ꢀhydroxypiperidineꢀ1ꢀcarboxylate, 0
°Cꢀrt, 12 h; (c) 1,4ꢀdioxane/H2O, K2CO3, Pd(PPh3)4, 100 °C, 12 h; (d) 4 N HCl in EtOAc, rt, 1 h.
(E)ꢀ4,4,5,5ꢀtetramethylꢀ2ꢀ(4ꢀstyrylphenyl)ꢀ1,3,2ꢀdioxaborolane (S6) To a solution of (E)ꢀ1ꢀ
bromoꢀ4ꢀstyrylbenzene (1.0 g, 3.8 mmol) in 1,4ꢀdioxane (10 mL) was added
bis(pinacolato)diboron (1.1 g, 4.2 mmol), KOAc (0.56 g, 5.7 mmol) and Pd(dppf)Cl2 (0.14 g,
0.19 mmol) at room temperature. The reaction mixture was stirred overnight at 100 °C under
argon protection. The resulting mixture was concentrated and purified by silica gel column
1
chromatography (dichloromethane) to afford S6 as a light yellow solid (1.0 g, 84%). H NMR
(400 MHz, CDCl3) δ 7.85 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 7.5 Hz, 4H), 7.51 – 7.36 (m, 2H), 7.36
13
– 7.26 (m, 1H), 7.19 (q, J = 16.3 Hz, 2H), 1.47 (d, J = 62.0 Hz, 12H); C NMR (100 MHz,
CDCl3) δ 140.09, 137.23, 135.25, 129.72, 128.71, 127.87, 126.69, 125.88, 83.82, 83.55, 25.03.
(R)ꢀtertꢀbutylꢀ3ꢀ(4ꢀaminoꢀ3ꢀiodoꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidineꢀ1ꢀcarboxylate
(S7) To a mixture of 3ꢀiodoꢀ1Hꢀpyrazoloꢀ[3,4ꢀd]pyrimidinꢀ4ꢀamine (1.0 g, 3.3 mmol) in THF
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(150 mL) was added triphenylphosphine (1.7 g, 6.6 mmol), diisopropyldiazeneꢀ1,2ꢀdicarboxylate
(1.3 mL, 6.6 mmol), and (S)ꢀtertꢀbutyl 3ꢀhydroxypiperidineꢀ1ꢀcarboxylate (1.3 g, 6.6 mmol) at
room temperature. The reaction mixture was stirred for 16 h and was judged complete by
LC/MS. The resulting mixture was concentrated to give the crude product, which was purified by
silica gel column chromatography (eluting 0%ꢀ2% MeOH in DCM) to provide S7 as a white
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solid (1.0 g, 69%). H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H), 6.41 (s, 2H), 4.76 (m, 1H), 4.12
(d, J = 6.7 Hz, 2H), 3.36 (s, 1H), 2.84 (t, J = 11.5 Hz, 1H), 2.30 – 2.06 (m, 2H), 1.88 (d, J = 11.4
Hz, 1H), 1.66 (d, J = 11.7 Hz, 1H), 1.45 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 157.77, 155.73,
154.58, 153.47, 103.92, 86.34, 79.96, 53.45, 30.19, 28.39; LC/MS (ESI, m/z): 445.0993 [M+H]+.
(R,E)ꢀtertꢀbutylꢀ3ꢀ(4ꢀaminoꢀ3ꢀ(4ꢀstyrylphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidineꢀ1ꢀ
carboxylate (S8) To a solution of S6 (0.07 g, 0.2 mmol), S7 (0.1 g, 0.2 mmol), K2CO3 (0.06 g,
0.4 mmol) in 1,4ꢀdioxane/H2O (12 mL, V/V=5/1) was added Pd(PPh3)4 (0.013g, 0.01mmol) at
room temperature. The reaction mixture was stirred overnight at 100 °C under argon protection.
The resulting mixture was concentrated and purified by silica gel column chromatography
(eluting 0%ꢀ2% MeOH in DCM) to afford S8 as a white solid (0.1 g, 90%). 1H NMR (400 MHz,
CDCl3) δ 8.35 (s, 1H), 7.69 (s, 4H), 7.55 (d, J = 7.3 Hz, 2H), 7.39 (t, J = 7.1 Hz, 2H), 7.30 (d, J
= 7.7 Hz, 1H), 7.19 (d, J = 5.7 Hz, 2H), 4.86 (s, 1H), 4.12 (d, J = 7.0 Hz, 2H), 2.87 (t, J = 12.2
Hz, 1H), 2.36 – 2.14 (m, 2H), 2.07 (d, J = 20.1 Hz, 1H), 1.91 (s, 1H), 1.71 (d, J = 11.9 Hz, 1H),
13
1.24 (s, 9H); C NMR (100 MHz, CDCl3) δ 158.13, 155.22, 154.69, 154.03, 144.15, 138.12,
136.91, 132.12, 129.93, 128.75, 127.99, 127.63, 127.33, 126.64, 98.51, 79.92, 74.99, 52.93,
30.11, 28.39, 24.85; LC/MS (ESI, m/z): 497.2689 [M+H]+.
(E)ꢀ1ꢀ(piperidinꢀ3ꢀyl)ꢀ3ꢀ(4ꢀstyrylphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine (S9) To
a
solution of S8 (0.1 g, 0.2 mmol) in EtOAc (2 mL) was added 4 N HCl in EtOAc (4 mL). The
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reaction mixture was stirred at room temperature for 1 h. After complete conversion of the
starting material, excess EtOAc was removed under vacuum. The residue was added EtOAc and
water. The water layer was basified with 2 N NaHCO3 solution and extracted with EtOAc (2×50
mL). The organic layers were then washed with water followed by brine. The organic layers
were dried over sodium sulfate, filtered, and concentrated to provide S9 as an offꢀwhite solid (65
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mg, 82%) without further purification. H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 7.70 (s, 4H),
7.57 (d, J = 7.0 Hz, 2H), 7.40 (d, J = 7.3 Hz, 2H), 7.36 – 7.26 (m, 1H), 7.21 (s, 2H), 5.94 (s, 2H),
4.88 (s, 1H), 3.34 (s, 1H), 3.10 (d, J = 11.3 Hz, 1H), 2.81 (d, J = 10.5 Hz, 1H), 2.34 (d, J = 9.4
13
Hz, 1H), 2.21 (m, 1H), 2.06 (s, 1H), 1.91 (m, 1H), 1.72 (m, 1H), 1.28 (m, 1H); C NMR (100
MHz, CDCl3) δ 158.19, 155.42, 153.96, 144.16, 138.09, 136.93, 132.26, 129.91, 128.78, 128.01,
127.66, 127.35, 126.66, 98.47, 53.92, 50.14, 45.41, 30.14, 25.53; LC/MS (ESI, m/z): 397.2135
[M+H]+.
Reagents and conditions: Method A: RC(O)Cl or RSO2Cl, DIEA, DCM, 10min; Method B:
RCOOH, HATU, DIEA, DMF, 30min; Method C: (i) NꢀBocꢀAAꢀOH, HATU, DIEA, DMF,
30min, (ii) 4 N HCl in EtOAc, 30min.
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Reagents and conditions: Method A: MeSO2Cl, DIEA, DCM, 10min; Method B: 2ꢀ
(dimethylamino)acetic acid, HATU, DIEA, DMF, 30 min; Method C: (i) NꢀBocꢀAAꢀOH,
HATU, DIEA, DMF, 30 min, (ii) 4 N HCl in EtOAc, 30 min.
Reagents and conditions: Method A: MeSO2Cl, DIEA, DCM, 10 min; Method B: 2ꢀ
(dimethylamino)acetic acid, HATU, DIEA, DMF, 30 min; Method C: (i) NꢀBocꢀ(D)LeuꢀOH,
HATU, DIEA, DMF, 30 min, (ii) 4 N HCl in EtOAc, 30 min.
Method A: General Procedure for the Preparation of Compounds 1-9, 13-15, 27 and 32.
(R)ꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidinꢀ1ꢀyl)propꢀ
2ꢀenꢀ1ꢀone (1, PCIꢀ32765). To a solution of S3 (20 mg, 0.05 mmol) in dichloromethane (3 mL)
o
was added DIEA (7.4 mg, 0.05 mmol) and acryloyl chloride (5.1 mg, 0.05 mmol) at 0 C. The
resulting mixture was stirred for 3 min. Then it was quenched by MeOH (1 mL), concentrated
and purified with CombiFlash system (0ꢀ10% MeOH in DCM) to afford the title compound 1
1
(18 mg, 81%) as a white solid. H NMR (400 MHz, DMSOꢀd6) δ 8.27 (s, 1H), 7.67 (d, J = 6.9
Hz, 2H), 7.45 (t, J = 7.3 Hz, 2H), 7.27 – 7.08 (m, 5H), 6.97 – 6.81 (m, 0.5H), 6.81 – 6.64 (m,
0.5H), 6.11 (dd, J = 26.1, 16.8 Hz, 1H), 5.66 (dd, J = 47.4, 9.8 Hz, 1H), 4.72 (s, 1H), 4.57 (d, J =
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10.8 Hz, 0.5H), 4.22 (s, 1H), 4.09 (d, J = 13.2 Hz, 0.5H), 3.73 (d, J = 10.9 Hz, 0.5H), 3.21 (d, J =
9.7 Hz, 1H), 3.02 (s, 0.5H), 2.28 (d, J = 11.2 Hz, 1H), 2.14 (s, 1H), 1.94 (d, J = 12.4 Hz, 1H),
1.59 (s, 1H); 13C NMR (100 MHz, DMSOꢀd6) δ 165.04, 158.68, 157.61, 156.79, 156.14, 154.46,
143.80, 130.58, 128.82, 128.43, 127.91, 127.69, 124.24, 119.44, 97.90, 53.23, 52.54, 49.78,
46.23, 30.12, 25.41; LC/MS (ESI, m/z): 441.2015 [M+H]+.
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(R,E)ꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ3ꢀ(4ꢀstyrylphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidinꢀ1ꢀyl)propꢀ2ꢀ
1
enꢀ1ꢀone (2) Yield=80%. H NMR (400 MHz, DMSOꢀd6) δ 8.29 (s, 1H), 7.79 (d, J = 7.2 Hz,
2H), 7.68 (dd, J = 16.2, 7.3 Hz, 4H), 7.40 (dd, J = 15.3, 6.8 Hz, 4H), 7.31 (d, J = 6.8 Hz, 1H),
6.85 (dd, J = 42.3, 27.4 Hz, 1H), 6.22 – 6.02 (m, 1H), 5.71 (dd, J = 33.8, 24.5 Hz, 1H), 4.74 (s,
1H), 4.57 (s, 0.5H), 4.24 (s, 1H), 4.11 (s, 0.5H), 3.74 (s, 0.5H), 3.31 – 3.14 (m, 1H), 3.04 (s,
0.5H), 2.30 (d, J = 10.9 Hz, 1H), 2.16 (s, 1H), 1.95 (d, J = 11.6 Hz, 1H), 1.60 (s, 1H); 13C NMR
(100 MHz, DMSOꢀd6) δ 165.06, 158.71, 156.15, 154.58, 144.04, 137.77, 137.40, 132.41, 129.68,
129.22, 129.06, 128.83, 128.31, 127.61, 127.07, 97.96, 55.38, 49.78, 46.23, 30.11, 25.40;
LC/MS (ESI, m/z): 451.2244 [M+H]+.
(R)ꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidinꢀ1ꢀ
yl)propanꢀ1ꢀone (3) Yield=81%. 1H NMR (400 MHz, DMSOꢀd6) δ 8.27 (d, J = 9.5 Hz, 1H), 7.67
(d, J = 7.4 Hz, 2H), 7.44 (t, J = 7.1 Hz, 2H), 7.25 – 7.08 (m, 5H), 4.75 (s, 0.5H), 4.64 (s, 0.5H),
4.55 (d, J = 11.4 Hz, 0.5H), 4.25 (d, J = 12.4 Hz, 0.5H), 4.03 (d, J = 11.6 Hz, 0.5H), 3.89 (d, J =
12.1 Hz, 0.5H), 3.66 – 3.49 (m, 0.5H), 3.11 (d, J = 8.6 Hz, 1H), 2.83 (d, J = 10.6 Hz, 0.5H), 2.35
(d, J = 26.7 Hz, 1H), 2.33 – 2.18 (m, 2H), 2.12 (s, 1H), 1.89 (s, 1H), 1.72 – 1.45 (m, 1H), 1.00
13
(dd, J = 15.6, 7.5 Hz, 3H); C NMR (100 MHz, DMSOꢀd6) δ 171.97, 158.68 , 157.61, 156.78,
156.15, 156.15, 154.51, 143.74, 130.56, 128.44, 124.23, 119.43, 97.96, 52.61, 49.62, 45.85,
30.12, 26.10, 23.98, 9.89; LC/MS (ESI, m/z): 443.2293.
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(R)ꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidinꢀ1ꢀ
yl)ethanone (4) Yield=81%. 1H NMR (400 MHz, DMSOꢀd6) δ 8.27 (d, J = 10.3 Hz, 1H), 7.67 (d,
J = 7.5 Hz, 2H), 7.61 – 7.39 (m, 2H), 7.28 – 7.07 (m, 5H), 4.77 (s, 0.5H), 4.63 (s, 05H), 4.52 (d,
J = 12.5 Hz, 0.5H), 4.19 (d, J = 11.8 Hz, 0.5H), 4.00 (d, J = 12.9 Hz, 0.5H), 3.85 (d, J = 12.6 Hz,
0.5H), 3.23 – 3.01 (m, 1H), 2.85 (t, J = 11.6 Hz, 1H), 2.23 (d, J = 11.5 Hz, 1H), 2.09 (d, J = 19.9
Hz, 3H), 2.00 – 1.83 (m, 2H), 1.75 – 1.44 (m, 1H); 13C NMR (100 MHz, DMSOꢀd6) δ 168.83,
158.68, 157.60, 156.79, 156.16, 154.46, 143.71, 130.58, 128.43, 124.25, 119.44, 97.90, 53.09,
50.45, 45.60, 29.79, 23.87, 21.74; LC/MS (ESI, m/z): 429.2036 [M+H]+.
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(R)ꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidinꢀ1ꢀyl)butanꢀ
1
1ꢀone (5) Yield=81%. H NMR (400 MHz, DMSOꢀd6) δ 8.27 (d, J = 9.3 Hz, 1H), 7.67 (d, J =
7.3 Hz, 2H), 7.44 (t, J = 7.1 Hz, 2H), 7.26 – 7.07 (m, 5H), 4.74 (s, 0.5H), 4.63 (s, 0.5H), 4.54 (d,
J = 11.0 Hz, 0.5H), 4.22 (d, J = 12.1 Hz, 0.5H), 4.04 (d, J = 11.6 Hz, 0.5H), 3.90 (d, J = 13.2 Hz,
0.5H), 3.60 (t, J = 11.2 Hz, 0.5H), 3.11 (s, 1H), 2.86 (t, J = 11.1 Hz, 0.5H), 2.34 (d, J = 6.2 Hz,
1H), 2.26 (d, J = 5.9 Hz, 1H), 2.14 (d, J = 18.7 Hz, 1H), 1.90 (s, 1H), 1.54 (dd, J = 14.0, 7.0 Hz,
13
3H), 1.35 – 1.19 (m, 1H), 0.87 (dt, J = 27.4, 6.7 Hz, 3H); C NMR (100 MHz, DMSOꢀd6) δ
171.18, 158.62, 157.61, 156.78, 156.08, 154.37, 143.75, 130.58, 128.41, 124.24, 119.43, 97.95,
52.63, 49.72, 45.77, 34.77, 30.10, 25.20, 18.70, 14.27; LC/MS (ESI, m/z): 457.2321 [M+H]+.
(R)ꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidinꢀ1ꢀyl)ꢀ2,2ꢀ
dimethylpropanꢀ1ꢀone (6) Yield=81%. 1H NMR (400 MHz, DMSOꢀd6) δ 8.27 (s, 1H), 7.68 (d, J
= 8.2 Hz, 2H), 7.44 (t, J = 7.5 Hz, 2H), 7.17 (dt, J = 12.8, 7.9 Hz, 5H), 4.70 (s, 1H), 4.45 (d, J =
12.0 Hz, 1H), 4.29 (d, J = 12.5 Hz, 1H), 3.27 (t, J = 11.6 Hz, 1H), 2.96 (d, J = 11.4 Hz, 1H), 2.38
– 2.22 (m, 1H), 2.13 (s, 1H), 1.92 (d, J = 12.7 Hz, 1H), 1.59 (d, J = 12.4 Hz, 1H), 1.29 – 1.16 (m,
9H); 13C NMR (100 MHz, DMSOꢀd6) δ 175.61, 158.63, 157.60, 156.78, 156.05, 154.41, 143.77,
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130.59, 130.56, 128.41), 124.25, 119.44, 97.89, 52.96, 49.15, 45.43, 38.74, 30.02, 28.58, 24.89;
LC/MS (ESI, m/z): 471.2512 [M+H]+.
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(R)ꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidinꢀ1ꢀyl)ꢀ2,2ꢀ
dimethylbutanꢀ1ꢀone (7) Yield=81%. 1H NMR (400 MHz, DMSOꢀd6) δ 8.27 (s, 1H), 7.68 (d, J =
7.8 Hz, 2H), 7.45 (t, J = 7.1 Hz, 2H), 7.25 – 7.07 (m, 5H), 4.68 (s, 1H), 4.47 (d, J = 12.2 Hz,
1H), 4.31 (d, J = 12.0 Hz, 1H), 2.97 (s, 1H), 2.29 (d, J = 11.6 Hz, 1H), 2.14 (s, 1H), 1.97 (dd, J =
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33.6, 10.6 Hz, 2H), 1.61 (s, 3H), 1.18 (d, J = 10.0 Hz, 6H), 0.83 (t, J = 6.9 Hz, 3H); C NMR
(100 MHz, DMSOꢀd6) δ 174.72, 158.67, 157.60, 156.78, 156.11, 154.44, 143.74, 130.57, 130.54,
128.43, 124.24, 119.43, 97.92, 53.02, 49.08, 45.27, 42.90, 33.06, 30.00, 26.74, 24.94, 9.91;
LC/MS (ESI, m/z): 485.2644 [M+H]+.
(R)ꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidinꢀ1ꢀyl)ꢀ3,3ꢀ
1
dimethylbutanꢀ1ꢀone (8) Yield=82%. H NMR (400 MHz, DMSOꢀd6) δ 8.28 (s, 1H), 7.68 (s,
2H), 7.45 (s, 2H), 7.14 (dd, J = 22.4, 12.5 Hz, 5H), 4.74 (s, 0.5H), 4.60 (s, 0.5H), 4.32 (s, 0.5H),
4.15 (d, J = 12.1 Hz, 0.5H), 4.00 (d, J = 13.1 Hz,0.5H), 3.64 (d, J = 10.0 Hz, 0.5H), 3.13 (d, J =
12.6 Hz, 1H), 2.83 (s, 1H), 2.27 (dd, J = 22.0, 9.2 Hz, 2H), 2.13 (s, 1H), 1.92 (s, 1H), 1.25 (s,
13
2H), 0.99 (d, J = 18.5 Hz, 9H). C NMR (100 MHz, DMSOꢀd6) δ 170.09, 158.67, 157.59,
156.80, 156.10, 154.38, 143.72, 130.58, 128.45, 124.24, 119.46, 119.42, 97.98, 52.71, 50.45,
45.60, 44.29, 41.39, 31.37, 30.20, 25.16; LC/MS (ESI, m/z): 485.2643 [M+H]+.
(R)ꢀ(3ꢀ(4ꢀaminoꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidinꢀ1ꢀ
yl)(cyclopropyl)methanone (9) Yield=82%. 1H NMR (400 MHz, DMSOꢀd6) δ 8.26 (s, 1H), 7.68
(d, J = 8.1 Hz, 2H), 7.44 (t, J = 7.4 Hz, 2H), 7.17 (dt, J = 12.9, 7.8 Hz, 5H), 4.79 (s, 0.5H), 4.65
(s, 0.5H), 4.50 (s, 0.5H), 4.37 (d, J = 11.5 Hz, 0.5H), 4.26 (s, 0.5H), 4.17 (s, 0.5H), 3.79 (s,
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0.5H), 3.29 – 3.08 (m, 0.5H), 2.96 (s, 1H), 2.28 (d, J = 10.2 Hz, 1H), 2.15 (s, 1H), 2.08 – 1.81
(m, 1H), 1.61 (d, J = 47.1 Hz, 1H), 1.28 (dt, J = 19.1, 9.6 Hz, 1H), 0.63 (d, J = 95.8 Hz, 4H); 13C
NMR (100 MHz, DMSOꢀd6) δ 171.70, 158.64, 157.59, 156.79, 156.09, 154.39, 143.72, 130.59,
130.54, 128.43, 124.24, 119.46, 119.43, 97.86, 53.92, 49.63, 46.43, 30.19, 25.41, 18.50, 10.93,
7.32; LC/MS (ESI, m/z): 455.2236 [M+H]+.
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(R)ꢀ1ꢀ(1ꢀ(methylsulfonyl)piperidinꢀ3ꢀyl)ꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀ
amine (13) Yield=85%. 1H NMR (400 MHz, DMSOꢀd6) δ 8.29 (s, 1H), 7.68 (d, J = 8.0 Hz, 2H),
7.45 (t, J = 7.5 Hz, 2H), 7.17 (td, J = 14.2, 7.7 Hz, 5H), 4.84 (s, 1H), 3.77 (d, J = 9.0 Hz, 1H),
3.62 (d, J = 11.8 Hz, 1H), 3.34 (s, 3H), 3.17 (t, J = 10.9 Hz, 1H), 2.83 (t, J = 11.6 Hz, 1H), 2.28
13
– 2.08 (m, 2H), 1.99 (d, J = 13.3 Hz, 1H), 1.75 (d, J = 12.4 Hz, 1H); C NMR (100 MHz,
DMSOꢀd6) δ 158.55, 157.72, 156.62, 156.21, 154.33, 144.01, 130.60, 128.11, 124.38, 119.43,
97.91, 52.94, 49.65, 45.72, 35.09, 29.00, 24.20; LC/MS (ESI, m/z): 304.1217 [M+H]+ TOF
LC/MS: (ESI) m/z: 465.1754 [M+H]+.
(R)ꢀ1ꢀ(1ꢀ(ethylsulfonyl)piperidinꢀ3ꢀyl)ꢀ3ꢀ(4ꢀphenoxyphenyl)ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀ
amine(14) Yield=83%. 1H NMR (400 MHz, DMSOꢀd6) δ 8.29 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H),
7.44 (d, J = 7.3 Hz, 2H), 7.17 (td, J = 14.2, 7.8 Hz, 5H), 4.81 (s, 1H), 3.81 (d, J = 11.4 Hz, 1H),
3.66 (d, J = 11.1 Hz, 1H), 3.28 (d, J = 10.6 Hz, 1H), 3.11 (d, J = 7.1 Hz, 2H), 2.94 (t, J = 11.7
Hz, 1H), 2.31 – 2.06 (m, 2H), 1.97 (d, J = 13.4 Hz, 1H), 1.72 (d, J = 11.0 Hz, 1H), 1.21 (t, J =
7.1 Hz, 3H). 13C NMR (100 MHz, DMSOꢀd6) δ 158.68, 157.63, 156.79, 156.23, 154.53, 143.89,
130.58, 130.56, 128.35, 124.24, 119.46, 119.42, 97.94, 53.10, 49.52, 45.62, 43.75, 29.24, 24.75,
8.02; LC/MS (ESI, m/z): 479.1836 [M+H]+.
28
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