Allosteric Inhibitors of SHP2 with Therapeutic Potential for Cancer Treatment

Article abstract of DOI:10.1021/acs.jmedchem.7b01520

SHP2, a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene, is involved in multiple cell signaling processes including Ras/MAPK and Hippo/YAP pathways. SHP2 has been shown to contribute to the progression of a number of cancer types inclu

Full text of DOI:10.1021/acs.jmedchem.7b01520

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Article
Allosteric inhibitors of SHP2 with therapeutic potential for cancer treatment
Jingjing Xie, Xiaojia Si, Shoulai Gu, Mingliang Wang, Jian Shen,
Haoyan Li, Jian Shen, Dan Li, Yanjia Fang, Cong Liu, and Jidong Zhu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01520 • Publication Date (Web): 20 Nov 2017
Downloaded from http://pubs.acs.org on November 20, 2017
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Allosteric inhibitors of SHP2 with therapeutic
potential for cancer treatment
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†
,
⊥
,#
∥,#
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‡
†,
⊥
†,
⊥
Jingjing Xie,
Xiaojia Si,
Shoulai Gu, Mingliang Wang, Jian Shen, Haoyan Li, Jian
§
†
†,*
†,*
∇
Shen, Dan Li, Yanjia Fang, Cong Liu, Jidong Zhu
†
Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, 26 Qiuyue Road, Shanghai 201203, China
⊥
University of the Chinese Academy of Sciences，19 A Yuquan Road, Shijingshan District ，
Beijing 100049, China
‡
Department of Natural Products Chemistry, Fudan University, 826 Zhangheng Road, Shanghai
2
01203, China
§
Viva Biotech Ltd. 334 Aidisheng Road, Shanghai 201203, China
∥
OrganischꢀChemisches Institut，RuprechtꢀKarlsꢀUniversität Heidelberg, Im Neuenheimer Feld
2
70, 69120, Heidelberg , Germany
∇Key laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of
Education), BioꢀX Institutes, Shanghai Jiao Tong University, Shanghai 200240, China
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KEYWORDS: SHP2, phosphatase, SH2 domain, allosteric inhibitor, cancer.
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ABSTRACT
SHP2, a cytoplasmic proteinꢀtyrosine phosphatase encoded by the PTPN11 gene, is involved in
multiple cell signaling processes including Ras/MAPK and Hippo/YAP pathways. SHP2 has
been shown to contribute to the progression of a number of cancer types including leukemia,
gastric and breast cancer. It also regulates Tꢀcell activation by interacting with inhibitory
immune checkpoint receptors such as the programmed cell death 1 (PDꢀ1) and Bꢀ and Tꢀ
lymphocyte attenuator (BTLA). Thus, SHP2 inhibitors have drawn great attention by both
inhibiting tumor cell proliferation and activating T cell immune responses toward cancer cells. In
this study, we report the identification of an allosteric SHP2 inhibitor 1ꢀ(4ꢀ(6ꢀbromonaphthalenꢀ
2ꢀyl)thiazolꢀ2ꢀyl)ꢀ4ꢀmethylpiperidinꢀ4ꢀamine（23） that locks SHP2 in a closed conformation
by binding to the interface of the Nꢀterminal SH2, Cꢀterminal SH2, and phosphatase domains. 23
suppresses MAPK signaling pathway and YAP transcriptional activity and shows antiꢀtumor
activity in vivo. The results indicate that allosteric inhibition of SHP2 could be a feasible
approach for cancer therapy.
INTRODUCTION
Protein tyrosine phosphorylation is a key modification controlling all aspects of crucial cellular
processes including proliferation, differentiation, growth and apoptosis. Dysregulation of
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tyrosine phosphorylation has been associated with the developmental pathologies of various
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, 2
human diseases such as cancer, diabetes, and autoimmune disorders. Over the past decades,
great success has been made to develop protein tyrosine kinase (PTK) inhibitors for clinical
application; however, the attempts to develop protein tyrosine phosphatase (PTP) drugs have
been hampered due to the less understanding of their biological functions and the poor
pharmaceutical properties of PTP inhibitors. Accumulating evidences suggest that some PTPs,
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such as SHP2, PTP1B, and CDC25, are potential therapeutic targets. Thus, developing
selective PTP inhibitors may open a new gate accessing more effective therapeutics for human
diseases.
SHP2 is a nonreceptor PTP that plays a positive role in cell signaling transduction by growth
6ꢀ8
factors and is involved in cell survival, proliferation and migration. Accumulated evidences
demonstrate that SHP2 is critical for Ras/MAPK signaling downstream of receptor tyrosine
9
ꢀ13
kinases activation.
SHP2 contains two tandem Src homology 2 (SH2) domains, a PTP domain
and a Cꢀterminal tail. At basal state, the NꢀSH2 domain of SHP2 protein binds to the PTP
domain and blocks its substrate access, therefore, resulting in suppressed PTP activity. When the
SH2 domains bind to specific phosphotyrosine motifs, the autoꢀinhibitory interactions are
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abolished. The phosphatase is then in an open conformation that allows SHP2 activation. Gainꢀ
ofꢀfunction mutations in SHP2 that cause hyperactivation of its catalytic activity have been
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5, 16
identified in the developmental disorder Noonan syndrome (～50%)
and various cancer
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types.
The mutated residues are located in the interface between NꢀSH2 and PTP domains.
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Hyperactivated SHP2 also contributes to tumorigenesis of gastric and breast cancer. SHP2 is
implicated in promoting YAP oncoprotein transcriptional activity and stimulating TEAD target
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genes. Moreover, SHP2 associates with PDꢀ1 following PDꢀL1 stimulation and inhibits T cell
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activation,
which makes it a promising target for cancer immunotherapy.
Development of small molecule inhibitors targeting PTPs has proven to be considerably difficult
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due to the highly conserved and positively charged nature of the PTP catalytic domain. Indeed,
the previously reported SHP2 inhibitors (1ꢀ7) have not achieved satisfactory selectivity over
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6ꢀ32
33, 34
other PTPs (Fig.1).
Until recently, an allosteric inhibitor, 8 (SHP099),
was reported to
selectively block SHP2 phosphatase activity by stabilizing SHP2 in an autoꢀinhibited
conformation. 8 inhibits cancer cell growth in vitro and in mouse tumor xenograft models,
demonstrating that inhibition of SHP2 is a valid therapeutic approach for the treatment of
cancers. We describe here an independent identification of SHP2 allosteric inhibitors with 2ꢀ
aminothiazole scaffold through a mutant E76A SHP2 biochemical assay. The inhibitor 23 has an
IC of 700 nM for SHP2 with more than 30ꢀfold selectivity over other PTPs. The coꢀcrystal
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structure reveals that the inhibitor binds to the same pocket formed by NꢀSH2, CꢀSH2 and PTP
33, 34
domains as that identified in 8 case
. We show that 23 not only inhibits RASꢀERK signaling,
but also suppresses YAP transcriptional activity. 23 inhibits the proliferation of multiple cancer
cell lines. In addition, it exhibits acceptable pharmacokinetic properties and inhibits tumor
growth in mouse xenograft models, corroborating the potential of SHP2 allosteric inhibitors for
the treatment of cancers.
RESULTS
Discovery of SHP2 allosteric inhibitors through a mutant E76A SHP2 biochemical screen.
The catalytic sites of protein tyrosine phosphatases are highly conserved and polar, which makes
it very challenging to develop selective PTP inhibitors. Recently, a few phosphatase allosteric
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inhibitors
have been reported to achieve great selectivity and cell permeability, which
enlightened us to develop allosteric inhibitors that can freeze SHP2 in its autoꢀinhibited state.
Glutamate 76 plays a key role in bridging NꢀSH2 and PTP domains, including a hydrogen bond
with S502 hydroxyl and a salt bridge interacting with R265. Substituting glutamate 76 leads to
weakened interactions between these two domains and thus arouses the destabilization of the
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autoꢀinhibited conformation. Mutations of E76 residue have been frequently identified in
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E76A
Noonan syndrome and leukemia. Indeed, mutant E76A full length SHP2 (SHP2
) exhibited
WT
much higher phosphatase activity than wild type SHP2 (SHP2 ) in an in vitro biochemical
C459S 38
assay using DIFMUP as a surrogate substrate. In contrast, C459S SHP2 (SHP2
) , which
replaces the catalytic center cysteine to serine, totally abolished its phosphatase activity (Fig.
E76A
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A). A biochemical screen against SHP2
was conducted with a library of about 20,000
compounds at 20 ꢁM. The hits came from the primary screen were further profiled in a
PTP 34
phosphatase assay using SHP2 PTP domain (SHP2 ) to filter out the catalytic inhibitors and
nonꢀspecific binders. One compound, 9 (Fig. 2B), was identified to show inhibition against
E76A
PTP
SHP2
with an IC of 19.1 ꢁM, but no effect on SHP2 (Fig. 2C). Thus, 9 could serve as a
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starting point to develop SHP2 allosteric inhibitors. We speculated that the amine part may gain
certain hydrogen bonds critical for the interaction. Therefore, we kept the amine part intact and
enriched the complexity of 9 by changing the phenyl with several common aryl groups. A brief
replacement of phenyl with biphenyl (10 and 11) or naphthyl (12) led to improved potency with
IC of 3.27, 5.32 and 2.55 ꢁM, respectively (Fig. 2D).
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The atomic structure of SHP2 in complex with 10. To elucidate the structural basis underlying
E76A
recognition of inhibitors to SHP2, we determined the crystal structure of SHP2
in complex
E76A
with 10 (Table 1). SHP2
adopted an autoꢀinhibited conformation as previously observed in
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E76Q
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wildꢀtype SHP2 and SHP2
structures (Fig. 3A)
. 10 is accommodated in the same
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pocket, as previously described for 8 but with distinct binding features (Fig. 3B)
. The amino
linker of 10 forms hydrogen bond with mainꢀchain carbonyl of Arg111 and the methyl groups of
tetramethylpiperidine make several van der Waals contacts with Thr108, Glu110, His114,
Glu249, and Thr253. The diphenyl moiety is sandwiched between sideꢀchains of Arg111 and
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Lys492 via cationꢀπ interaction , and the hydrophobic patch formed by the side chains of
Leu254, Gln257, Pro491 and Gln495 further stabilizes the diphenyl moiety (Fig. 3C). Compared
with the 6ꢀmember aminopyrazine core in 8, the 5ꢀmember thiazole core of 10 provides a
different trajectory for both the aryl and the piperidine regions, which may result in distinct
structureꢀactivity relationship (SAR). The hydrogen bond between the amino linker and Arg111
is unique to 10, although this interaction is relatively weak as indicated by the 3.5Å distance
between the donor and the acceptor, possibly due to the steric hindrance between the
tetramethylpiperidine group and sideꢀchain of His114. The cationꢀπ interaction by diphenyl
moiety is more toward the terminal phenyl ring and involves both Arg111 and Lys492,
consistent with the 6ꢀfold increase of potency from 9 to 10. The central binding pocket located in
the interface of the three domains (Fig.S1) is within the hinge region of SHP2 ，which is
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proposed to rearrange upon substrate binding. From SHP1 structures, the hinge region is
observed to undergo dramatic conformational changes to release PTP from NꢀSH2 domain for
substrate binding (Fig. S1). The binding of 10 in the central pocket of SHP2 locks the hinge
region and prevents the substrate binding and phosphatase activity of PTP domain by allosteric
regulation. Thus, the complex structure provided structural basis of allosteric inhibition of 10 to
SHP2 which served as a guideline for improvement of allosteric inhibitors with higher potency
by structureꢀbased design.
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E76A
SAR analysis of SHP2 inhibitors. Intrigued by the complex structure of 10 with SHP2
, we
turned our focus to improve its potency. Since the aminothiazole core of 10 provides a different
binding trajectory compared with 8, we decided to maintain the aminothiazole core and
independently explore the SAR of the aryl and the piperidine regions (Scheme 1 and Scheme 2).
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In our approach to build SAR on amine regions, we synthesized 2ꢀbromoꢀ4ꢀ(naphthaleneꢀ2ꢀ
yl)thiazole (38d, Scheme 1) as a key intermediate to enrich the diversity of amine by Buchwald
reaction. The commercial available amines were coupled with 38d followed by deprotection
when necessary as exampled in Scheme 1. Similarly, in order to explore SAR on the aryl
substitutions, a different synthetic approach (Scheme 2) was deployed. Dibromothiazole 43 was
substituted with amine (exampled as 44, Scheme 2) followed by Suzuki coupling with various
boronic acids, to afford compounds 20-22, 25, 28-35.
The brief SAR was summarized in Table 2 and Table 3. Removal of four methyl groups in
piperidine increased compound activity by 3.5 fold (Table 2, e.g., 12, 13), likely due to the
improved Hꢀbond interaction between the amino linker and Arg111 by removing the steric
hindrance to His114. Increasing the nitrogen substitution reduced inhibition (e.g., 13, 14),
suggesting the terminal NH group may be involved in important interactions such as hydrogen
bond with surrounding residues, which most likely involve water molecules since we did not
observe any direct interaction in the crystal structure. Replacement of piperidine with pyrrolidine
or adding one CH in N linker is tolerable for SHP2 inhibition (e.g., 15, 16). We then directly
2
linked thiazole with piperidine. Compound 17 compensates the loss of hydrogenꢀbond between
the amino linker and Arg111 with a potentially more favorable hydrogenꢀbonding interaction by
the terminal NH2 group, resulting in slightly improved activity as compared to 12. The amine
functional group of 17 is same as that of 8, suggesting the amino group is optimal to capture the
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Hꢀbond interactions with surrounding residues. Other substitutions on piperidine resulted in
compromised activity against SHP2 (e.g., 18, 19). Compound 12-17 showed comparable activity,
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however, some of the compounds (e.g. 13) were not stable in DMSO solution even at ꢀ20 C
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stock. 17 showed stable chemical stability and good bioactivity, therefore, we chose compound
1
7 as a template to further optimize the aryl region. In general, substitutions on naphthaline such
as F, Cl, Br, OMe, CN and CO Me were well accepted to inhibit SHP2, suggesting the
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hydrophobic cleft is tolerable to electron donating and withdrawing groups (Table 3, e.g., 20ꢀ25).
However, polar substitutions with acid or hydroxyl abolished the compound activity, consistent
to the nonꢀpolar environment of the hydrophobic cleft (e.g., 26, 27). The potency of 1, 3ꢀ
diphenyl substitutions was generally better than that of 1, 4ꢀdiphenyl, suggesting that the
terminal phenyl in 1, 3ꢀdiphenyl adopted a favorable position for piꢀcation interaction with the
side chain of Arg111 (e.g., 28ꢀ32). Other aryl groups we tested led to dramatic reduced inhibition
against SHP2 (33ꢀ35). After two rounds of optimization and SAR study, we identified
E76A
compound 23 as a SHP2 allosteric inhibitor with an IC50 of 0.7 ꢁM for SHP2
, but no
PTP
inhibition on SHP2 (Fig. S2).
WT
31,
To compare the reported biochemical assay using SHP2 stimulated with 2PꢀIRSꢀ1 peptide
3
2
E76A
with that using SHP2
in this study, we tested 23 and 8 in both assay formats (Fig. S3). 8
WT
E76A
exhibited an IC of 0.06 ꢁM for SHP2 (2PꢀIRSꢀ1) and 0.12 ꢁM for SHP2
. Similarly, 23
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showed comparable IC s in the two assays, suggesting that both assays are able to identify
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SHP2 allosteric inhibitors with similar sensitivity. Selectivity profiling revealed that 23 exhibited
more than 30ꢀfold selectivity over other PTPs, including LmwPTP, MKP3, PTP1B, TCꢀPTP, et
al. (Table 4). Of particular note, 23 displayed a 48ꢀfold preference for SHP2 over its closely
related homologue SHP1.
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3 inhibits MAPK signaling pathway and antagonizes YAP transcription activity. To
examine if 23 could interfere with ERK activation in cancer cells, we treated a largeꢀcell lung
carcinoma cell line NCIꢀH661 with 23 at indicated concentrations. NCIꢀH661 cells harbor SHP2
N58S gainꢀofꢀfunction mutation, whose activity was inhibited by 23 with an IC of 1.2 ꢁM (Fig.
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S4). As shown in Fig 4A, the levels of both phosphorylated ERK and AKT were downregulated
upon 23 treatment, whereas the inactive compound 34, had no effect even at 20 ꢁM (Fig. 4A).
The inhibition of ERK activation by 23 was timeꢀdependent, reaching max inhibition at 60min
(Fig. 4B). SHP2 plays a key role in RASꢀMAPK activation mediated by receptor tyrosine
9
ꢀ13
kinases . We then tested 23 in H1975 cells harboring the T790M mutation in EGFR, whose
4
1
proliferation was reported to be dependent on SHP2. Treatment of 23 diminished the
immunofluorescence of pꢀERK1/2, indicating that 23 effectively blocked MAPK signaling
pathway mediated by activated SHP2 (Fig. 4C).
SHP2 has been shown to physically associate with the oncoprotein YAP and potentiate its
2
1
transcriptional coactivator function.
Indeed, we found that FlagꢀYAP could be coꢀ
immunoprecipitated by HAꢀSHP2 and vice versa in HEK293T cells (Fig. 5A). To ask if SHP2
phosphatase activity is involved in regulation of Hippo/YAP signaling pathway, we then tested
2
3 in a YAPꢀdependent luciferase reporter assay. SF268, a neuroblastoma cell line harboring
YAP amplification, was stably expressed with a luciferase reporter driven by YAP responsive
promoter. The luciferase activity was decreased by 23 treatment in a doseꢀdependent manner, but
not by the negative compound 34 (Fig. 5B). Moreover, the mRNA levels of YAP target genes
42
CTGF, CYR61 and ANKRD1 , but not YAP itself, were downregulated by the treatment of 23
(Fig. 5C). Notably, 23 elevated the level of tyrosine phosphorylation on YAP (Fig. 5D). It has
been shown that cꢀAbl antagonizes the YAP oncogenic function by phosphorylating YAP at
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Y357. We then asked if SHP2 could regulate YAP Y357 phosphorylation. Indeed, treatment of
A549 cells harboring KRAS mutation with 23 increased Y357 phosphorylation (Fig. 5E), while
pꢀERK1/2 was not obviously affected. To further investigate whether YAP is a direct substrate
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of SHP2, we performed in vitro dephosphorylation assay using phosphorylated Y357 peptide.
E76A
SHP2
decreased the phosphorylation level of the peptide, which was reversed in the presence
of 23 (Fig. 5F).
2
3 inhibits cancer cell proliferation in vitro and tumor growth in vivo. Because 23 effectively
inhibited proꢀsurvival and proꢀgrowth signaling pathways such as MAPK and YAP, we then
examined its effect on cancer cell proliferation. We tested 23 activity in a panel of cancer cell
lines including lung, breast, esophageal and hematopoietic tumors. The SHP2 inhibitor exhibited
a broad antiꢀtumor activity with great potency in a subset of leukemia cell lines such as MV4;11
and MOLMꢀ13 which contain FLT3ꢀITD mutation (Fig. 6A). 23 also attenuated the colony
formation of H1975 cells, showing that 23 inhibited anchorageꢀindependent cancer cell growth
(
Fig. 6B).
Pharmacokinetics studies revealed that 23 had good oral exposure (10 mg/kg PO AUC: 9860 nM
•h) and bioavailability (67% F) with halfꢀlife of 13.3 h (Table S1). The promising PK properties
stimulated us to further evaluate the antiꢀtumor activity of 23 in vivo. We chose MV4;11 cells to
establish a xenograft model by subcutaneously implanting cells into immunocompromised mice.
A daily oral dose of 10 or 30 mg/kg of 23 led to 44.6% and 56.0% tumor growth inhibition,
respectively, while the body weight was not significantly affected (Fig. 6C). The tumor volumes
on the last day of treatment and the statistical analysis were presented in Fig 6D.
DISCUSSION AND CONCLUSION
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SHP2 has drawn great attention as an antiꢀtumor target due to its direct genetic link to cancer and
critical function in proꢀsurvival signaling pathways. In our study, we established a new screen
E76A
strategy for SHP2 allosteric inhibitors using SHP2
protein and successfully discovered 9 as a
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novel allosteric inhibitor of SHP2. Further optimization led to a more potent compound 23 with
E76A
WT
an IC of 0.7 ꢁM. The IC s of 23 or 8 in SHP2
and SHP2 (2PꢀIRSꢀ1) biochemical assays
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were comparable (Fig. S3), suggesting that either assay format can serve as a feasible approach
to identify SHP2 allosteric inhibitors. 23 effectively suppressed ERK1/2 and AKT activation in
cancer cells. SHP2 has been involved in regulation of YAP oncogene transcriptional activity;
however, it is not elucidated if the regulation is SHP2 phosphatase activity dependent. Our data
showed that 23 inhibited YAPꢀdependent reporter and the expression of YAP target genes,
suggesting that YAP activity is modulated by SHP2 PTP catalytic function. 23 inhibited the
proliferation of a variety of cancer cell lines and suppressed the colony formation of lung cancer
cell H1975. It exhibited good PK profiling and antiꢀtumor activity in a MV4;11 xenograft model.
Our study described herein corroborates that allosteric inhibition of SHP2 could be a feasible
approach to develop novel anticancer therapies.
EXPERIMENTAL SECTION
Materials. DIFMUP (6,8ꢀDifluoroꢀ4ꢀMethylumbelliferyl Phosphate) was purchased from
Thermo Fisher Scientific. Rabbit antiphosphoꢀAKT, antitotal AKT, antiphosphoꢀERK1/2,
antitotal ERK1/2 and mouse antiphosphotyrosine pYꢀ100 were purchased from Cell Signaling
Technology. Mouse antiꢀYAP was from Santa Cruze Biotechnology. And rabbit antiphosphoꢀ
YAP(Y357) was from Abcam.
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General Methods for Chemistry. All reagents and starting materials were obtained from
commercial suppliers and used without further purification unless otherwise stated. Reaction
progress was monitored by thin layer chromatography (TLC) on preloaded silica gel 60 F254
plates. Visualization was achieved with UV light and iodine vapor. All reactions involving
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oxygenꢀ or moistureꢀsensitive compounds were carried out under a dry N atmosphere. THF was
2
distilled from sodium/benzophenone immediately prior to use. Toluene was distilled from
1
sodium immediately prior to use. Yields were of purified product and were not optimized. H
13
NMR, C NMR were recorded on Bruker AMꢀ400, AgilentꢀNMRꢀvnmrs 400 spectrometers in
1
the corresponding solvent. H NMR spectra were referenced to the residual solvent peaks as
13
internal standards (7.26 ppm for CDCl , 2.50 ppm for DMSOꢀd , and 3.34 ppm for CD OD). C
3
6
3
NMR spectra were referenced to the residual solvent peaks as internal standards (39.52 ppm for
DMSOꢀd ), NMR data were recorded as follows: multiplicity (s = singlet, d = doublet, t =triplet,
6
m = multiplet or unresolved, coupling constant (solid) in Hz, integration). Mass spectra were
determined on an Agilent 5973N MSD (EI), Shimadzu LCMSꢀ2010EV (ESI) mass spectrometer
or Agilent G6100 LC/MSD (ESI) single Quand mass spectrometer and IonSpec HiResMALDI.
High resolution mass spectra were recorded on Waters Micromass GCT Premier (EI), Bruker
Daltonics, Inc. APEXIII 7.0 TESLA FTMS (ESI) mass spectrometers and IonSpec 4.7 Tesla
FTMS (MALDI). The purity was determined by high performance liquid chromatography
(
HPLC). Purity of all final compounds was 95% or higher. The instrument was an Agilent
Technologies 6120 LC/MS system. The column was a Phenomenex Luna C18, 100A, 2.0 50mm,
um.
5
General Procedure A: Buchwald−Hartwig Coupling. To an ovenꢀdried roundꢀbottomed flask
equipped with a stir bar was added Pd (dba) (23 mg, 0.025 mmol,5 mol %), Xantphos (29 mg,
2
3
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.05 mmol,10 mol %), sodium tertꢀbutoxide (96 mg, 1.0 mmol, 2.0 equiv), and the aryl bromide
(0.5 mmol, 1.0 equiv). Toluene (0.5M) was added followed by the amine (1.5 mmol, 3.0 equiv),
and the reaction mixture was degassed by sparging with N (g) for 10 min, and the resulting
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suspension was heated at 120 °C for 3ꢀ12 h, the reaction mixture was allowed to cool to r.t, then
diluted with ethyl acetate and subsequently filtered with celite. After the filtrate was concentrated
under reduced pressure, the resulting residue was purified by chromatography as specified.
General Procedure B: SuzukiꢀCoupling and Bocꢀdeprotection. The corresponding brominated
aromatic compound (0.5 mmol, 1.0 equiv) was dissolved in THF (10 mL/ mmol), the
corresponding boronic acid or boronic acid ester (0.6 mmol,1.2 equiv) and an aqueous 2.0 M
Na CO solution (0.75 mL, 3.0 equiv) were added. The mixture was deoxygenated under
2
3
reduced pressure and flushed with nitrogen. After having repeated this cycle several times,
Pd(PPh ) (29 mg, 0.025 mmol, 5 mol %) was added, and the resulting suspension was heated at
3
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80 °C for 12−16 h. After cooling, ethyl acetate and water were added and the organic phase was
separated. The water phase was extracted with ethyl acetate. The combined organic phases were
washed with brine, dried over Na SO , concentrated in vacuo, and purified by chromatography to
2
4
afford Bocꢀprotected product, which was treated with trifluoroacetic acid to get Bocꢀdeprotected
product.
4
ꢀPhenylꢀNꢀ(2, 2, 6, 6ꢀtetramethylpiperidinꢀ4ꢀyl)thiazolꢀ2ꢀamine (9). The title compound was
prepared from 38a and 2,2,6,6ꢀtetramethylpiperidinꢀ4ꢀamine following general procedure A.
1
Yellow solid (66 mg,42% yield ). H NMR (400 MHz, DMSOꢀd ) δ ppm 9.37 (s, 1H), 7.85 –
6
7
.80 (m, 3H), 7.37 (t, J = 7.6 Hz, 1H), 7.26 (t, J = 7.2 Hz, 1H), 7.10 (s, 1H), 4.18ꢀ4.11 (m, 1H),
1
3
2
.13 (dd, J = 3.2 Hz, J = 5.2 Hz,2H), 1.59ꢀ1.53 (m, 2H), 1.47 (d, J = 18.4 Hz, 12H); C NMR
(100 MHz, DMSOꢀd ) δ ppm 166.88, 149.77, 134.80, 128.47, 127.30, 125.59, 101.26, 56.68,
6
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+
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5.11, 40.54, 29.73, 24.26; HRMSꢀESI: calcd. for C H N S [M + H] : 316.1842, found:
18 26 3
3
16.1845.
4ꢀ([1,1'ꢀBiphenyl]ꢀ3ꢀyl)ꢀNꢀ(2,2,6,6ꢀtetramethylpiperidinꢀ4ꢀyl)thiazolꢀ2ꢀamine (10). The title
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compound was prepared from 38b and 2,2,6,6ꢀtetramethylpiperidinꢀ4ꢀamine following general
1
procedure A. Yellow solid (90 mg, 46% yield). H NMR (400 MHz, DMSOꢀd ) δ ppm 8.15 (s,
6
1
H), 7.82 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 7.2 Hz, 1H), 7.61ꢀ7.55 (m, 2H), 7.49ꢀ7.44 (m, 3H),
7
.40ꢀ7.34 (m, 1H), 7.19 (s, 1H), 4.03ꢀ3.90 (m, 1H), 1.97 (dd, J = 2.4 Hz, J = 12.0 Hz, 2H), 1.25
13
(
s, 6H), 1.09 (s, 6H), 1.05ꢀ0.98 (m, 2H); C NMR (100 MHz, DMSOꢀd ) δ ppm 167.36, 149.66,
6
1
40.29, 140.18, 135.52, 129.12, 128.91, 127.52, 126.63, 125.51, 124.54, 124.03, 101.04, 51.04,
+
47.71, 43.91, 34.04, 28.19; HRMSꢀESI: calcd. for C H N S [M + H] : 392.2155, found:
24
30
3
3
92.2153.
4
ꢀ([1,1'ꢀBiphenyl]ꢀ4ꢀyl)ꢀNꢀ(2,2,6,6ꢀtetramethylpiperidinꢀ4ꢀyl)thiazolꢀ2ꢀamine (11). The title
compound was prepared from 38c and 2,2,6,6ꢀtetramethylpiperidinꢀ4ꢀamine following general
1
procedure A. Yellow solid (100 mg, 51% yield). H NMR (400 MHz, DMSOꢀd ) δ ppm 9.38 (s,
6
1
1
2
1
H), 7.93 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 7.2 Hz, 1H), 7.71ꢀ7.68 (m, 4H), 7.47 (t, J = 7.6 Hz,
H), 7.36 (t, J = 7.6 Hz, 1H), 7.17 (s, 1H), 4.22ꢀ4.12 (m, 1H), 2.14 (dd, J = 2.4 Hz, J = 13.2 Hz,
1
3
H), 1.56ꢀ1.40 (m, 14H); C NMR (100 MHz, DMSOꢀd ) δ ppm166.93, 149.42, 139.70, 138.81,
6
33.93, 128.95, 127.74, 126.70, 126.16, 101.54, 56.63, 45.17, 40.60, 29.81, 24.33; HRMSꢀESI:
+
calcd. for C H N S [M + H] : 392.2155, found: 392.2157.
2
4
30
3
4ꢀ(Naphthalenꢀ2ꢀyl)ꢀNꢀ(2, 2, 6, 6ꢀtetramethylpiperidinꢀ4ꢀyl) thiazolꢀ2ꢀamine (12). The title
compound was prepared from 38d and 2,2,6,6ꢀtetramethylpiperidinꢀ4ꢀamine following general
1
procedure A. White solid (97 mg, 53% yield). H NMR (400 MHz, DMSOꢀd δ ppm 8.35 (s,
6
)
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H), 7.98 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H ), 7.90ꢀ7.83 (m, 3H), 7.65 (d, J = 8.0 Hz,1H), 3.98ꢀ3.94
1
3
(m, 1H), 1.95 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H ), 1.24 (s, 6H), 1.09ꢀ1.03 (m, 8H); C NMR (100
MHz, DMSOꢀd ) δ ppm 167.62, 149.87, 133.16, 132.43, 132.35, 128.07, 127.93, 127.58, 126.38,
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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4
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4
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5
5
5
5
5
5
5
5
6
0
1
2
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5
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7
8
9
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1
2
3
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7
8
9
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1
2
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7
8
9
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1
25.83, 124.08, 101.40, 50.81, 48.08, 44.24, 34.42, 28.52 . HRMSꢀESI: calcd. for C H N S [M
22 28 3
+
+
H] : 366.1998, found: 366.1995.
4
ꢀ(Naphthalenꢀ2ꢀyl)ꢀNꢀ(piperidinꢀ4ꢀyl)thiazolꢀ2ꢀamine (13). The title compound was prepared
from 38d and piperidinꢀ4ꢀamine following general procedure A. Yellow solid (56 mg, 36 %
1
yield). H NMR (400 MHz, CDCl ) δ ppm 8.32 (s, 1H), 7.98 – 7.72 (m, 4H), 7.46ꢀ7.44 (m, 2H),
3
6
.81 (s, 1H), 5.52 (s, 1H), 3.53 (s, 1H), 3.13 (d, J = 12.0 Hz, 2H), 2.74 (t, J = 11.3 Hz, 2H), 2.48
1
3
(s, 1H), 2.15 (d, J = 11.3 Hz, 2H), 1.52ꢀ1.41 (m, 2H); C NMR (101 MHz, DMSOꢀd ) δ ppm
6
1
4
67.06, 149.85, 133.15, 132.36, 132.29, 128.02, 127.89, 126.35, 125.83, 124.14, 124.06, 102.12,
+
8.73, 41.78, 21.13; HRMSꢀESI: calcd. for C H N S [M + H] : 310.1372, found: 310.1369.
1
8
20
3
Nꢀ(1ꢀmethylpiperidinꢀ4ꢀyl)ꢀ4ꢀ(naphthalenꢀ2ꢀyl)thiazolꢀ2ꢀamine (14). The title compound was
prepared from 38d and 1ꢀmethylpiperidinꢀ4ꢀamine following general procedure A. Yellow solid
1
(79 mg, 49 % yield). H NMR (400 MHz, CDCl ) δ ppm 8.29 (s, 1H), 7.88 – 7.79 (m, 4H), 7.49
3
–
2
1
5
7.40 (m, 2H), 6.80 (s, 1H), 5.88 (s, 1H), 3.71 (s, 1H), 3.13 – 3.09 (m, 2H), 2.58 – 2.43 (m, 5H),
1
3
.26 (d, J = 11.7 Hz, 2H), 1.96ꢀ1.85 (m, 2H); C NMR (101 MHz, DMSOꢀd ) δ ppm 167.31,
6
49.88, 133.15, 132.35, 132.34, 128.03, 127.89, 126.34, 125.81, 124.15, 124.01, 101.87, 54.92,
+
2.94, 44.40, 30.06; HRMSꢀESI: calcd. for C H N S [M + H] : 324.1529, found: 324.1531.
1
9
22
3
4ꢀ(Naphthalenꢀ2ꢀyl)ꢀNꢀ(pyrrolidinꢀ3ꢀyl)thiazolꢀ2ꢀamine (15). The title compound was prepared
from 38d and pyrrolidinꢀ3ꢀamine following general procedure A. Yellow solid (60 mg, 41 %
1
yield). H NMR (400 MHz, DMSOꢀd ) δ ppm 9.40 (s, 1H), 8.37 (s, 1H), 8.13 (d, J = 5.2 Hz,
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H), 7.99 (d, J = 1.2 Hz, J = 8.4 Hz, 1H), 7.94ꢀ7.88 (m, 3H), 7.53ꢀ7.46 (m, 2H), 7.30 (s, 1H),
.49ꢀ4.43 (m, 1H), 3.57ꢀ3.52 (m, 1H), 3.39ꢀ3.26 (m, 3H), 2.33ꢀ2.24 (m, 1H), 2.07ꢀ2.00 (m, 1H);
1
3
C NMR (100 MHz, DMSOꢀd ) δ ppm 167.59, 150.33, 133.63, 132.87, 132.61, 128.52, 128.45,
6
0
1
2
3
4
5
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8
9
0
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8
9
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1
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7
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9
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1
28.05, 126.87, 126.37, 124.59, 124.57, 103.38, 53.83, 49.85, 43.98, 30.55; HRMSꢀESI: calcd.
+
for C H N S [M + H] : 296.1216, found: 296.1213.
1
7
18
3
4
ꢀ(Naphthalenꢀ2ꢀyl)ꢀNꢀ(piperidinꢀ3ꢀylmethyl)thiazolꢀ2ꢀamine (16). Following the general
procedure A from 38d and tertꢀbutyl 3ꢀ(aminomethyl)piperidineꢀ1ꢀcarboxylate to afford tertꢀ
butyl 3ꢀ(((4ꢀ(naphthalenꢀ2ꢀyl)thiazolꢀ2ꢀyl)amino)methyl)piperidineꢀ1ꢀcarboxylate, which was
1
treated with trifluoroacetic acid to get compound 16. Yellow solid (58 mg, 36 %yield). H NMR
(400 MHz, DMSOꢀd ) δ ppm 8.92 (s, 1H), 8.36 (s, 1H), 7.99ꢀ7.87 (m, 4H), 7.53ꢀ7.45 (m, 2H),
6
7
1
.22 (s, 1H), 3.22ꢀ3.19 (m, 2H), 2.80ꢀ2.65 (m, 2H), 2.18ꢀ2.14 (m, 1H), 1.87ꢀ1.80 (m, 2H), 1.67ꢀ
13
.64 (m, 2H), 1.30ꢀ1.23 (m, 2H); C NMR (100 MHz, DMSOꢀd ) δ ppm 168.44, 149.85,
6
133.18, 132.37, 132.32, 128.09, 127.91, 127.57, 126.36, 125.85, 124.15, 124.12, 101.96, 47.33,
+
4
6.42, 43.36, 33.56, 26.11, 21.50; HRMSꢀESI: calcd. for C H N S [M + H] : 324.1529, found:
1
9
22
3
3
24.1527.
4
ꢀMethylꢀ1ꢀ(4ꢀ(naphthalenꢀ2ꢀyl)thiazolꢀ2ꢀyl)piperidinꢀ4ꢀamine (17). Following the general
procedure A from 38d and tertꢀbutyl (4ꢀmethylpiperidinꢀ4ꢀyl)carbamate to afford tertꢀbutyl (4ꢀ
methylꢀ1ꢀ(4ꢀ(naphthalenꢀ2ꢀyl)thiazolꢀ2ꢀyl)piperidinꢀ4ꢀyl)carbamate, which was treated with
1
trifluoroacetic acid to get compound 17. White solid (73 mg, 45% yield). H NMR (400 MHz,
DMSOꢀd ) δ ppm 8.44 (s, 1H), 8.40 (s, 1H), 8.32 (s, 2H), 8.01ꢀ7.89 (m, 4H), 7.53ꢀ7.47 (m, 2H),
6
7
.44 (s, 1H), 3.88ꢀ3.84 (m, 2H), 3.46ꢀ3.36 (m, 2H), 1.94ꢀ1.90 (m, 2H), 1.83ꢀ1.80 (m, 2H), 1.40
1
3
(s, 3H) ; C NMR (100 MHz, DMSOꢀd ) δ ppm 170.05, 150.56, 133.16, 132.48, 132.08, 128.10,
6
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28.02, 127.59, 126.45, 125.98, 124.24, 124.08, 103.60, 51.80, 44.13, 33.76, 22.06; HRMSꢀESI:
+
calcd. for C H N S [M + H] : 324.1529, found: 324.1525.
1
9
22
3
2ꢀ(3,5ꢀDimethylpiperazinꢀ1ꢀyl)ꢀ4ꢀ(naphthalenꢀ2ꢀyl)thiazole (18). Following the general
procedure A from 38d and tertꢀbutyl 2,6ꢀdimethylpiperazineꢀ1ꢀcarboxylate to afford tertꢀbutyl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
,6ꢀdimethylꢀ4ꢀ(4ꢀ(naphthalenꢀ2ꢀyl)thiazolꢀ2ꢀyl)piperazineꢀ1ꢀcarboxylate, which was treated
1
with trifluoroacetic acid to get compound 18. White solid (76 mg, 47% yield). H NMR (400
MHz, DMSOꢀd ) δ ppm 8.40 (s, 1H), 8.01ꢀ7.89 (m, 4H), 7.53ꢀ7.46 (m, 2H), 7.44 (s, 1H), 3.95
6
(dd, J = 2.4 Hz, J = 12.0 Hz,2H), 3.07 (s, 2H), 2.77 (t, J = 11.2 Hz, 2H), 1.17 (d, J = 6.0 Hz, 6H);
1
3
C NMR (100 MHz, DMSOꢀd ) δ ppm 170.08, 150.50, 133.14, 132.46, 132.05, 128.09, 127.98,
6
127.56, 126.40, 125.95, 124.23, 124.08, 103.48, 53.00, 50.02, 17.70; HRMSꢀESI: calcd. for
+
C H N S [M + H] : 324.1529, found: 324.1525.
19
22
3
(1ꢀ(4ꢀ(Naphthalenꢀ2ꢀyl)thiazolꢀ2ꢀyl)piperidinꢀ3ꢀyl)methanamine (19). Following the general
procedure A from 38d and tertꢀbutyl(piperidinꢀ3ꢀylmethyl)carbamate to afford tertꢀbutyl 2,6ꢀ
dimethylꢀ4ꢀ(4ꢀ(naphthalenꢀ2ꢀyl)thiazolꢀ2ꢀyl)piperazineꢀ1ꢀcarboxylate, which was treated with
1
trifluoroacetic acid to get compound 19. White solid (69mg, 43% yield). H NMR (400 MHz,
DMSOꢀd ) δ ppm 8.42 (s, 1H), 8.01 (dd, J = 2.0 Hz, J = 8.8 Hz, 1H), 7.95ꢀ7.89 (m, 3H), 7.54ꢀ
6
7
.47 (m, 2H), 7.44 (s, 1H), 4.10 (dd, J = 2.4 Hz, J = 12.8 Hz, 1H), 3.96ꢀ3.87 (m, 1H), 3.49ꢀ3.30
(
(
m, 2H), 3.23ꢀ3.05 (m, 1H), 2.92ꢀ2.72 (m, 3H), 1.92ꢀ1.79 (m, 3H), 1.62ꢀ1.53 (m, 1H), 1.27ꢀ1.23
1
3
m, 1H); C NMR (100 MHz, DMSOꢀd ) δ ppm 170.94, 150.93, 133.64, 132.93, 132.61, 128.53,
6
128.41, 128.04, 126.87, 126.39, 124.73, 124.57, 103.63, 52.13, 49.23, 40.63, 34.93, 27.94, 23.99;
+
HRMSꢀESI: calcd. for C H N S [M + H] : 324.1529, found: 324.1525.
1
9
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Journal of Medicinal Chemistry
Page 18 of 54
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ꢀ(4ꢀ(6ꢀChloronaphthalenꢀ2ꢀyl)thiazolꢀ2ꢀyl)ꢀ4ꢀmethylpiperidinꢀ4ꢀamine
(20).
The
title
compound was prepared from 45 and 2ꢀ(6ꢀchloronaphthalenꢀ2ꢀyl)ꢀ4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
1
dioxaborolane following general procedure B. White solid (111 mg, 62% yield). H NMR (400
0
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
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7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz, CD OD) δ ppm 8.29 (s, 1H), 7.91ꢀ 7.73 (m, 4H), 7.56 (s, 2H), 7.39 (d, J = 8.0 Hz, 1H),
3
13
7
.02 (s, 1H), 4.00ꢀ3.89 (m, 2H), 3.46 – 3.31 (m, 2H), 2.01 – 1.85 (m, 4H), 1.47 (s, 3H); C
NMR (100 MHz, DMSOꢀd ) δ ppm 170.12, 150.19, 133.08, 132.59, 131.56, 130.38, 130.26,
6
1
27.35, 126.89, 126.23, 125.27, 124.13, 103.92, 50.95, 44.28, 34.49, 23.44; HRMSꢀESI: calcd.
+
for C H N ClS [M + H] : 358.1139, found: 358.1137.
1
9
21
3
1
ꢀ(4ꢀ(6ꢀFluoronaphthalenꢀ2ꢀyl)thiazolꢀ2ꢀyl)ꢀ4ꢀmethylpiperidinꢀ4ꢀamine
(21).
The
title
compound was prepared from 45 and 2ꢀ(6ꢀfluoronaphthalenꢀ2ꢀyl)ꢀ4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
1
dioxaborolane following general procedure B. White solid (116 mg, 68% yield). H NMR (400
MHz, DMSOꢀd ) δ ppm 8.44 (s, 1H), 8.44 (s, 1H), 8.38 (s, 2H), 8.05ꢀ8.02 (m, 2H), 7.91 (d, J =
6
8.8 Hz, 1H), 7.70 (dd, J = 2.0 Hz, J = 10.0 Hz,1H), 7.43ꢀ7.40 (m, 2H), 3.89ꢀ3.84 (m, 2H), 3.45ꢀ
1
9
3
.40 (m, 2H), 1.95ꢀ1.90 (m, 2H), 1.83ꢀ1.80 (m, 2H), 1.40 (s, 3H) ; FꢀNMR: ꢀ114.43 to ꢀ114.49;
13
C NMR (100 MHz, DMSOꢀd ) δ ppm 170.07, 160.03 (d, J = 242.4 Hz), 150.31, 133.16 (d, J =
6
9
1
.4 Hz), 131.62 (d, J = 2.6 Hz), 130.98 (d, J = 9.0 Hz), 130.33, 127.51 (d, J = 5.3 Hz), 125.19,
24.29, 116.53 (d, J = 25.0 Hz), 110.72 (d, J = 20.6 Hz), 103.54, 51.83, 44.10, 33.71, 21.98;
+
HRMSꢀESI: calcd. for C H FN S [M + H] : 342.1435, found: 342.1437.
1
9
21
3
1
ꢀ(4ꢀ(6ꢀMethoxynaphthalenꢀ2ꢀyl)thiazolꢀ2ꢀyl)ꢀ4ꢀmethylpiperidinꢀ4ꢀamine (22). The title
compound was prepared from 45 and 2ꢀ(6ꢀMethoxynaphthalenꢀ2ꢀyl)ꢀ4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
1
dioxaborolane following general procedure B. White solid (104 mg, 59% yield). H NMR (400
MHz, DMSOꢀd ) δ ppm 8.31 (s, 1H), 7.94 (dd, J = 1.6 Hz, J = 8.8 Hz, 2H), 7.86ꢀ7.80 (m, 2H),
6
7.33 (s, 1H), 7.31 (d, J = 2.4 Hz, 1H), 7.16 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 3.88 (s, 3H), 3.84ꢀ
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.79 (m, 2H), 3.50 – 3.37 (m, 2H), 1.89 – 1.76 (m, 4H), 1.36 (s, 3H); C NMR (100 MHz,
DMSOꢀd ) δ ppm 170.00, 157.39, 150.77, 133.78, 130.01, 129.63, 128.48, 126.90, 124.55,
6
124.18, 118.89, 106.03, 102.40, 55.22, 51.28, 44.24, 34.22, 22.89; HRMSꢀESI: calcd. for
+
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C H N OS [M + H] : 354.1635, found: 354.1630.
20
24
3
1
ꢀ(4ꢀ(6ꢀBromonaphthalenꢀ2ꢀyl)thiazolꢀ2ꢀyl)ꢀ4ꢀmethylpiperidinꢀ4ꢀamine (23). Compound 42
was treated with trifluoroacetic acid and to get compound 23. White solid (174 mg, 87% yield).
1
H NMR (400 MHz, DMSOꢀd ) δ ppm 8.41 (s, 1H), 8.18 (s, 1H), 8.13 (s, 2H), 8.05 (d, J = 8.8
6
Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.47 (s, 1H), 3.90ꢀ3.80 (m, 2H), 3.44
1
3
–
3.36 (m, 2H), 1.90 – 1.80 (m, 4H), 1.39 (s, 3H); C NMR (100 MHz, DMSOꢀd ) δ ppm
6
170.07, 150.22, 133.61, 132.65, 131.71, 130.33, 129.51, 129.41, 127.33, 125.23, 124.20, 119.07,
+
1
04.22, 51.91, 44.19, 33.67, 21.71; HRMSꢀESI: calcd. for C H N BrS [M + H] : 402.0634,
1
9
21
3
found: 402.0635.
6ꢀ(2ꢀ(4ꢀAminoꢀ4ꢀmethylpiperidinꢀ1ꢀyl)thiazolꢀ4ꢀyl)ꢀ2ꢀnaphthonitrile (24). To an ovenꢀdried
roundꢀbottomed flask was added compound 42（50mg, 0.1 mmol）, Zn(CN) （14 mg, 0.15
2
mmol）and Pd(PPh ) (58 mg, 0.05 mmol), then DMF (5 mL) was added, and the reaction
3
4
mixture was degassed by sparging with N (g) for 10 min, at which time it was equipped with a
2
condenser and placed in a preheated 130 °C oilꢀbath. After 8 h, the reaction mixture was allowed
to cool to r.t, then diluted with ethyl acetate and the reaction mixture was partitioned between
ethyl acetate and water. The organic layer was washed with water and brine successively, dried
over anhydrous sodium sulfate, and concentrated in vacuum. The resulting residue was purified
by silica gel chromatography (dichloromethane/methanol, v/v, 99:1to 95:5) to give the desired
1
product 24. Yellow solid (10 mg, 29% yield). H NMR (400 MHz, DMSOꢀd ) δ ppm 8.54 (s,
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2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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H), 8.50 (s, 1H), 8.17 – 8.12 (m, 2H), 8.06 (d, J = 8.8 Hz, 1H), 7.77 (dd, J = 1.2 Hz, J = 8.4
13
Hz,1H), 7.55 (s, 1H), 3.70ꢀ3.65 (m, 2H), 3.56ꢀ3.39 (m, 2H), 1.66ꢀ1.64 (m, 4H), 1.22 (s, 3H) ; C
NMR (100 MHz, DMSOꢀd ) δ ppm 170.30, 149.81, 135.07, 134.73, 134.01, 131.28, 129.56,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
28.76, 126.83, 125.74, 124.11, 119.30, 107.99, 105.12, 48.90, 44.73, 36.34, 26.94; HRMSꢀESI:
+
calcd. for C H N S [M + H] : 349.1481, found: 349.1483.
2
0
21
4
Methyl 6ꢀ(2ꢀ(4ꢀaminoꢀ4ꢀmethylpiperidinꢀ1ꢀyl)thiazolꢀ4ꢀyl)ꢀ2ꢀnaphthoate (25). The title
compound was prepared from 45 and methyl 6ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ2ꢀ
1
naphthoate following general procedure B. White solid (124 mg, 65% yield). H NMR (400
MHz, DMSOꢀd ) δ ppm 8.62 (s, 1H), 8.47 (s, 1H), 8.15 (d, J = 8.8 Hz, 1H), 8.10 (dd, J = 1.2 Hz,
6
J = 8.4 Hz, 1H), 8.06ꢀ8.02 (m, 3H), 7.98 (dd, J = 1.6 Hz, J = 9.6 Hz,1H), 7.57 (s, 1H), 3.94 (s,
13
3
H), 3.92ꢀ3.85 (m, 2H), 3.50ꢀ3.40 (m, 2H), 1.90ꢀ1.80 (m, 4H), 1.40 (s, 3H) ; C NMR (100
MHz, DMSOꢀd ) δ ppm 170.08, 166.33, 150.11, 135.39, 134.42, 131.52, 130.29, 129.64, 128.64,
6
126.70, 125.29, 125.01, 123.95, 105.11, 52.24, 51.88, 44.18, 33.66, 21.70; HRMSꢀESI: calcd. for
+
C H N O S [M + H] : 382.1584, found: 382.1583.
21
24
3
2
6
ꢀ(2ꢀ(4ꢀAminoꢀ4ꢀmethylpiperidinꢀ1ꢀyl)thiazolꢀ4ꢀyl)ꢀ2ꢀnaphthoic acid (26). To an ovenꢀdried
roundꢀbottomed flask was added compound 25（76 mg, 0.2 mmol）, and KOH (34 mg, 0.6
mmol), then 1,4ꢀdioxane (3 mL) and MeOH (3 mL) was added, and the reaction mixture was
degassed by sparging with N (g) for 10 min, and stirred at 70 °C for 2 h, the reaction mixture
2
was allowed to cool to r.t, water (10 mL) was added and acified with 1N HCl to PH to 2, then
diluted with ethyl acetate and the reaction mixture was partitioned between ethyl acetate and
water. The organic layer was washed with water and brine successively, dried over anhydrous
Na SO , and concentrated in vacuum. The resulting residue was purified by silica gel
2
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chromatography (dichloromethane/methanol, v/v, 99:1to 95:5) to give the desired product 26.
1
Yellow solid (56 mg, 76%yield). H NMR (400 MHz, DMSOꢀd ) δ ppm 13.04 (s, 1H), 8.57 (s,
6
1H), 8.46 (s, 1H), 8.39 (s, 2H), 8.12ꢀ8.09 (m, 2H), 8.07ꢀ7.95 (m, 2H), 7.54 (s, 1H), 3.89ꢀ3.86 (m,
13
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
H), 3.46ꢀ3.37 (m, 2H), 1.97ꢀ1.93 (m, 2H), 1.90ꢀ1.81 (m, 2H), 1.41 (s, 3H) ; C NMR (100
MHz, DMSOꢀd ) δ ppm 170.08, 167.39, 150.16, 135.24, 134.17, 131.57, 130.25, 129.54, 128.41,
6
1
27.88, 125.66, 124.84, 123.95, 104.85, 51.85, 44.11, 33.86, 21.93; HRMSꢀESI: calcd. for
+
C H N O S [M + H] : 368.1427, found: 368.1423.
20
22
3
2
(6ꢀ(2ꢀ(4ꢀAminoꢀ4ꢀmethylpiperidinꢀ1ꢀyl)thiazolꢀ4ꢀyl)naphthalenꢀ2ꢀyl)methanol (27). To the
solution of compound 25（76 mg, 0.2 mmol）in THF (10 mL) was added LiAlH (1M/THF, 0.5
4
mL) at 0 °C, then stirred at 25 °C for 2 h, the reaction mixture was allowed to cool to r.t,
Na SO .10H O was added, then diluted with ethyl acetate and subsequently filtered with celite.
2
4
2
After the filtrate was concentrated under reduced pressure, the resulting residue was purified by
1
chromatography to give the desired product 27. Yellow solid (57 mg, 81% yield). H NMR (400
MHz, DMSOꢀd ) δ ppm 8.43 (s, 1H), 8.10 (s, 2H), 8.10 (dd, J = 1.6 Hz, J = 8.4 Hz, 1H), 8.04ꢀ
6
7
3
.95 (m, 3H), 8.10 (dd, J = 1.6 Hz, J = 8.4 Hz, 1H), 7.49 (s, 1H), 5.60 (s, 2H), 3.88ꢀ3.84 (m, 2H),
1
3
.50ꢀ3.40 (m, 2H), 1.90ꢀ1.79 (m, 4H), 1.90ꢀ1.81 (m, 2H), 1.40 (s, 3H) ; C NMR (100 MHz,
DMSOꢀd ) δ ppm 170.04, 150.33, 133.15, 132.81, 132.03, 131.18, 128.73, 128.29, 128.03,
6
1
26.70, 124.67, 124.06, 104.14, 69.94, 51.92, 44.19, 33.65, 22.06; HRMSꢀESI: calcd. for
+
C H N OS [M + H] : 354.1635, found: 354.1631.
20
24
3
1
ꢀ(4ꢀ([1,1'ꢀBiphenyl]ꢀ3ꢀyl)thiazolꢀ2ꢀyl)ꢀ4ꢀmethylpiperidinꢀ4ꢀamine (28). The title compound
was prepared from 45 and 2ꢀ([1,1'ꢀbiphenyl]ꢀ3ꢀyl)ꢀ4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolane
1
following general procedure B. White solid (129 mg, 74% yield). H NMR (400 MHz, DMSOꢀ
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
d6) δ ppm 8.11 (brs, 3H), 7.86 (d, J = 6.8 Hz, 1H), 7.73ꢀ7.67 (m, 2H), 7.60ꢀ7.55 (m, 1H), 7.51ꢀ
7
.46 (m, 3H), 7.44 (s, 1H), 7.42ꢀ7.36 (m, 1H), 3.85ꢀ3.79 (m, 2H), 3.42ꢀ3.35 (m, 2H), 1.89ꢀ1.78
13
(m, 4H), 1.38 (s, 3H) ; C NMR (100 MHz, DMSOꢀd ) δ ppm 169.98, 150.51, 140.56, 140.17,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
35.26, 129.20, 128.96, 127.57, 126.82, 126.01, 124.89, 123.95, 103.36, 51.90, 44.16, 33.65,
+
1.68; HRMSꢀESI: calcd. for C H N S [M + H] : 350.1685, found: 350.1679.
2
1
24
3
3
'ꢀ(2ꢀ(4ꢀAminoꢀ4ꢀmethylpiperidinꢀ1ꢀyl)thiazolꢀ4ꢀyl)ꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀcarbonitrile (29). The
title compound was prepared from 45 and 3'ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ[1,1'ꢀ
1
biphenyl]ꢀ4ꢀcarbonitrile following general procedure B. White solid (129 mg, 69% yield). H
NMR (400 MHz, DMSOꢀd ) δ ppm 8.25ꢀ8.11 (m, 3H), 8.01ꢀ7.89 (m, 5H), 7.66 (d, J = 8.8 Hz,
6
1H), 7.53 (t, J = 7.6 Hz, 1H), 7.48 (s, 1H), 3.83ꢀ3.80 (m, 2H), 3.53ꢀ3.39 (m, 2H), 1.89ꢀ1.78 (m,
13
4
1
H), 1.38 (s, 3H) ; C NMR (100 MHz, DMSOꢀd ) δ ppm 170.05, 150.19, 144.63, 138.67,
6
35.52, 132.91, 129.52, 127.75, 126.33, 126.17, 124.20, 118.93, 110.17, 103.76, 51.86, 44.17,
+
33.68, 21.68; HRMSꢀESI: calcd. for C H N S [M + H] : 375.1638, found: 375.1635.
2
2
23
4
Methyl 3'ꢀ(2ꢀ(4ꢀaminoꢀ4ꢀmethylpiperidinꢀ1ꢀyl) thiazolꢀ4ꢀyl)ꢀ[1, 1’ꢀbiphenyl]ꢀ4ꢀcarboxylate
30). The title compound was prepared from 45 and methyl 3'ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
(
dioxaborolanꢀ2ꢀyl)ꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀcarboxylate following general procedure B. White solid
1
(
132 mg, 65% yield). H NMR (400 MHz, DMSOꢀd ) δ ppm 8.22 (s, 1H), 8.06 (brs, 2H), 8.01ꢀ
6
7
.95 (m, 4H), 7.74 (d, J = 8.4 Hz, 2H), 7.64 (t, J = 8.0 Hz, 1H), 7.39 (s, 1H), 3.90 (s, 3H), 3.84ꢀ
1
3
3
.81 (m, 2H), 3.42ꢀ3.39 (m, 2H), 1.89ꢀ1.78 (m, 4H), 1.39 (s, 3H) ; C NMR (100 MHz, DMSOꢀ
d6) δ ppm 170.02, 166.19, 150.10, 140.28, 137.94, 134.24, 131.37, 130.39, 129.58, 128.12,
1
26.95, 126.89, 126.41, 103.46, 52.31, 51.94, 44.18, 33.63, 21.66; HRMSꢀESI: calcd. for
+
C H N O S [M + H] : 408.1740, found: 408.1745.
23
26
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1
ꢀ(4ꢀ(4'ꢀFluoroꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)thiazolꢀ2ꢀyl)ꢀ4ꢀmethylpiperidinꢀ4ꢀamine (31). The title
compound was prepared from 45 and methyl 2ꢀ(4'ꢀfluoroꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolane following general procedure B. White solid (127 mg, 69%
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
yield). H NMR (400 MHz, DMSOꢀd ) δ ppm 8.51 (s, 3H), 7.94 (d, J = 8.0 Hz, 2H), 7.74ꢀ7.66
6
(m, 4H), 7.35ꢀ7.27 (m, 3H), 3.84ꢀ3.81 (m, 2H), 3.42ꢀ3.37 (m, 2H), 1.97ꢀ1.92 (m, 2H), 1.80ꢀ1.79
19
13
(m, 2H), 1.40 (s, 3H) ; FꢀNMR: ꢀ115.42 to ꢀ115.50; C NMR (100 MHz, DMSOꢀd ) δ ppm
6
1
70.04, 161.87 (d, J = 242.9 Hz), 150.14, 138.00, 136.17 (d, J = 3.0 Hz), 133.74, 128.49 (d, J =
8
.1 Hz), 126.72, 126.29, 116.76 (d, J = 21.2 Hz), 102.96, 51.70, 44.04, 33.74, 22.10; HRMSꢀESI:
+
calcd. for C H FN S [M + H] : 368.1591, found: 368.1593.
2
1
23
3
4'ꢀ(2ꢀ(4ꢀAminoꢀ4ꢀmethylpiperidinꢀ1ꢀyl)thiazolꢀ4ꢀyl)ꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀol
(32).
The title
compound was prepared from 45 and 4'ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ[1,1'ꢀ
1
biphenyl]ꢀ4ꢀol following general procedure B. White solid (130 mg, 71% yield). H NMR (400
MHz, DMSOꢀd ) δ ppm 9.60 (s, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.52 (d,
6
J = 8.4 Hz, 2H), 7.21 (s, 1H), 6.85 (d, J = 8.8 Hz, 2H), 3.57ꢀ3.49 (m, 4H), 1.57ꢀ1.47 (m, 4H),
13
1
1
.10 (s, 3H) ; C NMR (100 MHz, DMSOꢀd ) δ ppm 170.24, 157.21, 150.41, 139.07, 132.89,
6
30.42, 127.56, 126.16, 125.88, 101.60, 46.85, 45.05, 38.16, 30.44; HRMSꢀESI: calcd. for
+
C H N OS [M + H] : 366.1635, found: 366.1631.
21
24
3
4
ꢀMethylꢀ1ꢀ(4ꢀ(quinolinꢀ3ꢀyl)thiazolꢀ2ꢀyl)piperidinꢀ4ꢀamine (33). The title compound was
prepared from 45 and quinolinꢀ3ꢀylboronic acid following general procedure B. Yellow solid
1
(115 mg, 71% yield). H NMR (400 MHz, DMSOꢀd ) δ ppm 9.41 (d, J = 1.6 Hz, 1H), 8.72 (s,
6
1
H), 8.02 (t, J = 6.8 Hz, 1H), 7.73 (t, J = 7.2 Hz, 1H), 7.63 – 7.48 (m, 4H), 3.84ꢀ3.81 (m, 2H),
1
3
3
.50ꢀ3.35 (m, 2H), 1.87ꢀ1.75 (m, 4H), 1.35 (s, 3H) ; C NMR (100 MHz, DMSOꢀd ) δ ppm
6
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1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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1
4
70.43, 148.86, 147.89, 146.84, 131.29, 129.32, 128.75, 128.37, 127.66, 127.09, 104.69, 50.95,
+
4.33, 34.49, 23.46; HRMSꢀESI: calcd. for C H N S [M + H] : 325.1481, found: 325.1479.
1
8
21
4
1ꢀ(4ꢀ(Benzo[b]thiophenꢀ2ꢀyl)thiazolꢀ2ꢀyl)ꢀ4ꢀmethylpiperidinꢀ4ꢀamine (34). The title compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was prepared from 45 and benzo[b]thiophenꢀ2ꢀylboronic acid following general procedure B.
1
Yellow solid (95 mg, 58% yield). H NMR (400 MHz, DMSOꢀd ) δ ppm 8.10 (s, 2H), 7.93ꢀ7.91
6
(m, 1H), 7.83ꢀ7.81 (m, 1H), 7.75 (s, 1H), 7.39 – 7.31 (m, 3H), 3.82ꢀ3.77 (m, 2H), 3.43ꢀ3.34 (m,
1
3
2
1
2
H), 1.89 – 1.78 (m, 4H), 1.39 (s, 3H); C NMR (100 MHz, DMSOꢀd ) δ ppm 169.98, 145.20,
6
40.02, 138.61, 138.57, 124.65, 124.48, 123.63, 122.43, 119.68, 103.93, 51.85, 39.94, 33.58,
+
1.61; HRMSꢀESI: calcd. for C H N S [M + H] : 330.1093, found: 330.1095.
1
7
20
3 2
1ꢀ(4ꢀ(Bibenzo[b,d]furanꢀ4ꢀyl)thiazolꢀ2ꢀyl)ꢀ4ꢀmethylpiperidinꢀ4ꢀamine (35). The title compound
was prepared from 45 and dibenzo[b,d]furanꢀ4ꢀylboronic acid following general procedure B.
1
Yellow solid (126 mg, 69% yield). H NMR (400 MHz, DMSOꢀd ) δ ppm 8.19ꢀ8.16 (m, 2H),
6
8.12ꢀ8.09 (m, 3H), 7.82ꢀ7.45 (m, 2H), 7.57 (t, J = 8.0 Hz, 1H), 7.49ꢀ7.43 (m, 2H), 3.89ꢀ3.85 (m,
1
3
2
H), 3.47ꢀ3.37 (m, 2H), 1.92 – 1.80 (m, 4H), 1.40 (s, 3H); C NMR (100 MHz, DMSOꢀd ) δ
6
ppm 169.43, 155.40, 152.03, 145.44, 127.72, 126.06, 124.12, 123.39, 123.30, 123.12, 121.21,
20.15, 119.27, 111.84, 107.49, 51.91, 44.21, 33.65, 21.67; HRMSꢀESI: calcd. for C H N OS
1
21 22
3
+
[
M + H] : 364.1478, found: 364.1473.
1
ꢀ(Naphthalenꢀ2ꢀyl)ꢀ2ꢀthiocyanatoethanꢀ1ꢀone (37d). A solution of 2ꢀbromoꢀ1ꢀ(naphthalenꢀ2ꢀ
yl) ethanꢀ1ꢀone (36d) (249 mg, 1.0 mmol) and potassium thiocyanate (194 mg, 2 mmol, ) in
ethanol (5 mL) was stirred at 85°C for 2h. The reaction was extracted with diethyl ether and
dried with MgSO , and then the solvent was removed at low pressure. The crude product was
4
1
purified by chromatography to get compound 37d (225mg, 99% yield) as white solid. H NMR
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(
300 MHz, CDCl ) δ ppm 8.45 (s, 1H), 7.95 (dd, J = 20.5, 11.0 Hz, 4H), 7.65 (dt, J = 15.0, 6.9
3
Hz, 2H), 4.89 (s, 2H).
2ꢀBromoꢀ4ꢀ(naphthalenꢀ2ꢀyl)thiazole (38d). A suspension of 37d (227 mg, 1.0 mmol) in 2 mL
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
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4
4
4
4
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5
6
0
1
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7
8
9
0
1
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8
9
0
1
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5
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7
8
9
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8
9
0
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8
9
0
3
0% hydrogen bromide in acetic acid and 3 mL acetic acid was stirred under nitrogen at room
temperature overnight. The mixture was quenched with Na CO aqueous solution, then extracted
2
3
with ethyl acetate and washed with water and brine, the organic was dried with MgSO and
4
solvent was removed at low pressure. The crude product was purified by chromatography to get
1
compound 38d (261 mg, 90% yield) as white solid. H NMR (400 MHz, CDCl ) δ ppm 8.43 (s,
3
1
H), 7.93 – 7.83 (m, 4H), 7.53 – 7.49 (m, 3H).
2ꢀBromoꢀ4ꢀ(6ꢀbromonaphthalenꢀ2ꢀyl)thiazole (41). The compound was prepared by following a
1
procedure similar to that of 38d. White solid (321 mg, 87%yield). H NMR (400 MHz, CDCl ) δ
3
ppm 8.51 (s, 1H), 8.32 (s, 1H), 8.23(d, J = 1.6 Hz , 1H), 8.09 (dd, J = 8.4 Hz, J =1.6 Hz, 1H),
1
3
8.01ꢀ7.97 (m, 2H); C NMR (100 MHz, DMSOꢀd ) δ ppm 154.01, 136.53, 133.94, 131.66,
6
1
30.87, 130.65, 129.69, 129.59, 127.85, 124.92, 124.89, 119.78, 119.73.
Tertꢀbutyl (1ꢀ(4ꢀ(6ꢀbromonaphthalenꢀ2ꢀyl)thiazolꢀ2ꢀyl)ꢀ4ꢀmethylpiperidinꢀ4ꢀyl)carbamate (42).
The title compound was prepared from 41 and tertꢀbutyl (4ꢀmethylpiperidinꢀ4ꢀyl)carbamate
1
following general procedure A. Yellow solid (245 mg, 56% yield). H NMR (400 MHz, DMSOꢀ
d6) δ ppm 8.41 (s, 1H), 8.18(d, J = 0.8 Hz , 1H), 8.04 (dd, J = 1.6 Hz, J = 8.8 Hz,1H), 7.94 – 7.89
(m, 2H), 7.62 (dd, J = 2.0 Hz, J = 8.8 Hz,1H), 7.43 (s, 1H), 6.68 (s, 1H), 3.68ꢀ3.64 (m, 2H),
1
3
3.34ꢀ3.28 (m, 2H), 2.18ꢀ2.15 (m, 2H), 1.59ꢀ1.52 (m, 2H), 1.39 (s, 9H), 1.27 (s, 3H) ; C NMR
100 MHz, DMSOꢀd ) δ ppm 170.38, 150.18, 133.58, 132.75, 131.75, 130.39, 129.50, 129.37,
(
6
1
27.28, 125.24, 124.11, 119.01, 103.65, 77.49, 49.73, 44.56, 34.26, 28.32, 25.98.
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Tertꢀbutyl (1ꢀ(4ꢀbromothiazolꢀ2ꢀyl)ꢀ4ꢀmethylpiperidinꢀ4ꢀyl)carbamate (45). A mixture of 2, 4ꢀ
dibromothiazole (43) (1.22 g, 5.0 mmol), tertꢀbutyl (4ꢀmethylpiperidinꢀ4ꢀyl)carbamate (44) (1.61
g, 7.5 mmol) in DMF ( 50 mL) and TEA ( 3.0 mL, 15 mmol) was heated to 90 °C for 8 h. The
reaction mixture was cooled to room temperature, diluted with water and extracted with ethyl
0
1
2
3
4
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9
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1
2
3
4
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8
9
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9
0
acetate and washed with water and brine, the organic was dried with MgSO and solvent was
4
removed at low pressure. The crude product was purified by chromatography to get compound 7
1
(
1.65 g, 87%) as light yellow solid. H NMR (400 MHz, DMSOꢀd ) δ ppm 6.84 (s, 1H), 6.66 (s,
6
1
1
H), 3.51ꢀ3.41 (m, 2H), 3.24ꢀ3.17 (m, 2H), 2.11ꢀ2.08 (m, 2H), 1.52ꢀ1.45 (m, 2H), 1.38 (s, 9H),
1
3
.24(s, 3H); C NMR (100 MHz, DMSOꢀd ) δ ppm 170.18, 154.51, 120.57, 104.23, 77.51,
6
56.06, 49.59, 44.15, 34.07, 28.31
WT
Biological Evaluation. SHP2 protein expression and purification. Genes encoding SHP2 ,
E76A
C459S
PTP
SHP2
, SHP2
and SHP2
were inserted into pET28a. These constructs were used to
o
transform BL21(DE3) competent cells and cultured at 37 C in Luria broth(LB). When OD600
o
reached 0.6, 0.5 mM IPTG was added to induce SHP2 protein expression for 5 hours at 30 C.
Cells were collected by centrifugation (4500rpm 15min) and resuspended in lysis buffer
containing 20 mM TrisꢀHCl (pH7.5), 500 mM NaCl and 0.1 mM PMSF. After the cell lysis by
o
ultrasonication, cell lysates were centrifuged at 4 C for 40min at 16000rpm. Then the
supernatants were loaded onto HisTrap HP column, and gradient eluted by 25, 50,100, 250 mM
imidazole. Eluents containing SHP2 protein were further loaded onto HiLoad Superdex 200PG
column. Fractions were collected according to the results of SDSꢀPAGE and then concentrated
the protein to 10 mg/ml or more.
In vitro phosphatase assay. Artificial substrate 6,8ꢀdifluoroꢀ4ꢀmethylumbelliferyl phosphate
(DIFMUP) (Invitrogen) was used to examine the catalytic activity of phosphatases. Reactions
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were performed at room temperature in 96ꢀwell black polystyrene plate with a final volume of
E76A
1
00ꢁL. The SHP2
enzyme (1.5nM) and various concentrations of compounds were preꢀ
incubated for 20min (buffer condition: 0.1M sodium acetate, pH 5.0), then added the substrate
DIFMUP, incubated another 20min, and measured EX/EM 358/455. For selectivity study,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
4
4
4
4
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4
4
4
4
4
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5
5
5
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5
5
5
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0
1
2
3
4
5
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7
8
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1
2
3
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5
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8
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1
2
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7
8
9
0
different phosphatases including LmwPTP, MKP3, PTP1B, TCꢀPTP, SHP1, VHR and CDC25A
E76A
were purified from E.coli and measured as the same condition as SHP2
, with the exception
of the DIFMUP concentration corresponding to their respective Km value. For IC50
determination, eight concentrations of compounds at 3ꢀfold dilution were tested. Each
experiment was performed in duplicate, and IC50 data were derived from at least three
independent experiments. The curve fitting program Prism 4 (GraphPad Software) was used to
calculate the IC value.
5
0
Immunoblot analysis. NCIꢀH661 cells were cultured in 6ꢀwell plate, and treated with SHP2
o
o
inhibitors for 2h at 37 C. Cells were lysed with RIPA buffer. After centrifugation at 4 C for
0min at 12000rpm, 3 volumes of protein extracts were mixed with 1 volume of 4× loading
buffer and loaded onto the SDSꢀPAGE gel. Proteins were then transferred to PVDF membranes
3
(Millipore), blocked with 5% nonꢀfat milk, and incubated with pERK1/2, ERK1/2, pAKT and
o
AKT (Cell Signaling Technology) and other antibodies overnight at 4 C. Corresponding
secondary antibodies were applied and further detected using Amersham Imager 600. The results
have been repeated for at least three times.
Immunofluorescence cell staining. NCIꢀH1975 cells were seeded in 24ꢀwell plate with a
coverslip in each well and grown overnight. After the treatment of 10 ꢁM SHP2 inhibitor for 2
hours, aspirated the medium and fixed the cells with 4% PFA. Treated the coverslip with 0.4%
Triton Xꢀ100 and blocked with 5% BSA. Diluted phosphoꢀERK1/2 antibody (Cell Signaling
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o
Technology) was applied to the coverslip and incubated overnight at 4 C. After PBS washing,
corresponding fluorochromeꢀlabeled secondary antibody was applied at room temperature for 1h
and further PBS washed. After mounting with DAPI, examined the slides under the fluorescence
microscope. The results have been repeated for at least three times.
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Luciferase reporter assay. SF268 cells stably expressing YapꢀLuc reporter were plated out onto
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6ꢀwell plate in RPMIꢀ1640. On day 1, SHP2 inhibitors at the concentrations of 3, 6 and 12ꢁM
were added. 12 hours later, cells in each well were lysed and used to measure Renilla luciferase
Promega) and CellTiterꢀGlo (Promega), respectively. Luciferase of the Yap reporter was
(
normalized by the luciferase of CellTiterꢀGlo.
RT-PCR experiments. RNA was extracted from SF268 cells treated with DMSO, 10ꢁM and 20
ꢁ
M SHP2 inhibitor for 2h and then performed cDNA synthesis. Quantstudio 6 Flex was used to
conduct quantitative PCR(qPCR) analysis and the primer pairs used were as follows:
Gene (human)
CTGF
Forward primer
Reverse primer
5'ꢀGAAGCTGACCTGGAAGAGAACA ꢀ3'
5’ꢀAAACATCCAGGTTTCCTCCAꢀ3’
5’ꢀGCAAATTCTCCAAAATGTCAGGꢀ3’
5'ꢀGCAAATTCCATGGCACCGTꢀ3'
5'ꢀCGTCGGTACATACTCCACAGAA ꢀ3'
5’ꢀTTTGGCAATTGTGGAGAAGTTAꢀ3’
5’ꢀCGGGAGAAGACACTGGATTTꢀ3’
5'ꢀTCGCCCCACTTGATTTTGGꢀ3'
ANKRD1
YAP1
GAPDH
Cell proliferation assay. Cells (3000 cells/well) of each cell line were seeded in 96ꢀwell plates
and grown overnight. The cells were treated with SHP2 inhibitors at indicated concentrations for
2
days. Cell viability was examined by CellTiterꢀGlo (Promega).
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Page 29 of 54
Journal of Medicinal Chemistry
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Soft agar assay. Soft agar assay was performed in 6ꢀwell plate and consisted of two layers. The
bottom layer was filled with 2 mL 1% low melting temperature agarose. In the top layer, cells
were reꢀsuspended in media containing 0.4% agarose and seeded at 10,000 cells per well in a
volume of 2mL. Varying concentrations of SHP2 inhibitors were applied and supplemented 3ꢀ5
drops twice a week. 2 weeks later, colonies were stained with iodonitrotetrazolium chloride
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(Sigma) and visualized with Gel Imager (BioꢀRad).
In vitro dephosphorylation. YAP Y357 peptide (SGLSMSSYSVPRTPD) and pY 357 peptide
(SGLSMSSpYSVPRTPD) were synthesized to perform in vitro dephosphorylation experiments
E76A
E76A
with purified SHP2
protein. The SHP2
enzyme (1.5nM) and 20ꢁM 23 were preꢀ
incubated for 15min (buffer condition: 20mM TrisꢀHCl pH 7.4, 100mM NaCl, 1mM EDTA,
2
mM DTT), then added the substrate 100ꢁM pY357 peptide, incubated another 15min. The
E76A
reactions were stopped by separating the SHP2
phosphatase from the mixture using 30KD
filters and then analyzed the results using pꢀYAP(Y357) antibody (Abcam).
Dot blot. 2ꢁL of the reaction products from in vitro dephosphorylation experiments were loaded
onto nitrocellulose membrane using narrowꢀmouth pipette tip and let the membrane dry. Block
nonꢀspecific sites by soaking in 5% BSA in TBST for 1h, then diluted (1:1000) pꢀYAP(Y357)
antibody (Abcam) can be applied for 1h at room temperature. After 3 times of TBST washing,
secondary antibody was incubated. Another 3 times of TBST washing, incubate ECL reagent for
1
minute, cover with Saranꢀwrap, and expose Xꢀray film in the dark room.
Crystallization and X-ray data collection. Hanging drop vapor diffusion was applied to
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crystalize SHP2
(1ꢀ534). The reservoir solution consisted of 0.2 M Sodium formate, 0.1 M
E76A
Bicine pH8.5 and 15% w/v PEG 5000MME. 1 ꢁL SHP2
(1ꢀ534) (8.8mg/ml) was mixed with
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