Discovery of Potent, Selective Stem Cell Factor Receptor/Platelet Derived Growth Factor Receptor Alpha (c-KIT/PDGFRα) Dual Inhibitor for the Treatment of Imatinib-Resistant Gastrointestinal Stromal Tumors (GISTs)

Article abstract of DOI:10.1021/acs.jmedchem.7b00468

Stem cell factor receptor (c-KIT) and platelet derived growth factor receptor alpha (PDGFRα) kinases play an important role in gastrointestinal stromal tumors (GISTs). Here, we have discovered an c-KIT/PDGFRα dual inhibitor, compound 31, with single-digit nanomolar potency against c-KIT and PDGFRα. Compared to Imatinib (1), 31 showed better antiproliferative efficacy against various TEL-c-KIT/PDGFRα-BaF3 isogenic cells, including three 1-resistant BaF3 cell lines, as well as against GIST-T1 and GIST-882 cell lines. Furthermore, compound 31 showed a good KinomeScan selectivity (468 kinases) (S score (1) = 0.01 at 1 μM concentration), good metabolic stability in liver microsomes, and no hERG inhibitory activity. It was worth noting that 31 inhibited GIST-T1 tumor growth (TGI = 81.5%) and even the BaF3-TEL-cKIT-T670I tumor progression (TGI = 41.9%, 1-resistant GISTs) at a dosage of 100 mg/kg/day without exhibiting apparent toxicity.

Full text of DOI:10.1021/acs.jmedchem.7b00468

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Article
Discovery of Potent, Selective Stem Cell Factor Receptor/Platelet Derived
Growth Factor Receptor Alpha (c-KIT/PDGFR#) Dual Inhibitor for the
Treatment of Imatinib-Resistant Gastrointestinal Stromal Tumors (GISTs)
Yanli Lu, Fei Mao, Xiaokang Li, Xinyu Zheng, Manjiong Wang, Qing Xu, Jin Zhu, and Jian Li
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 25 May 2017
Downloaded from http://pubs.acs.org on May 25, 2017
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Discovery of Potent, Selective Stem Cell Factor Receptor/Platelet
Derived Growth Factor Receptor Alpha (cꢀKIT/PDGFRα) Dual
Inhibitor for the Treatment of ImatinibꢀResistant Gastrointestinal
Stromal Tumors (GISTs)
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a,†
a,†
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Yanli Lu , Fei Mao , Xiaokang Li , Xinyu Zheng , Manjiong Wang , Qing Xu , Jin
a
a,
Zhu , Jian Li *
a
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China
University of Science and Technology, Shanghai 200237, China
†
These authors contributed equally to this work.
*
To whom correspondence should be addressed: jianli@ecust.edu.cn
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ABSTRACT
cꢀKIT and platelet derived growth factor receptor alpha (PDGFRα) kinase play
an important role in gastrointestinal stromal tumors (GISTs). Here, we have
discovered an cꢀKIT/PDGFRα dual inhibitor, compound 31, with singleꢀdigit
nanomolar potency against cꢀKIT and PDGFRα. Compared to Imatinib (1), 31
showed better antiproliferative efficacy against various TELꢀcꢀKIT/PDGFRαꢀBaF3
isogenic cells, including three 1ꢀresistant BaF3 cell lines, as well as against GISTꢀT1
and GISTꢀ882 cell lines. Furthermore, compound 31 showed a good KinomeScan
selectivity (468 kinases) (S score (1) = 0.01 at 1 µM concentration), good metabolic
stability in liver microsomes and no hERG inhibitory activity. It was worth noting that
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31 inhibited GISTꢀT1 tumor growth (TGI
=
81.5%) and even the
BaF3ꢀTELꢀcKITꢀT670I tumor progression (TGI = 41.9%, 1ꢀresistant GISTs) at a
dosage of 100 mg/kg/day without exhibiting apparent toxicity.
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INTRODUCTION
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal
neoplasms originating from the intestitial cells of Cajal, the pacemaker cells of the gut
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and pervading the entire area of the gastrointestinal tract. Epidemiological data shows
that the annual incidence of GISTs is about 1ꢀ2/100,000, and approximately 30% of
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GISTs are malignant. The treatment of GISTs faces great challenges due to its
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complex etiology. Prior to 2010, surgical resection appeared to be the only available
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treatment for GISTs, resulting in 5ꢀyear survival rates of 48ꢀ54% for resectable cases ,
while the median survival period was only 19 months, and just 5ꢀ10% of irresectable
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or metastasized GISTs patients have a 5ꢀyear survival rate. In addition, over oneꢀthird
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of patients will suffer tumor metastases after macroscopically complete surgery.
Unfortunately, GISTs respond poorly to conventional cytotoxic chemotherapy, and
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radiation therapy has not been shown to be effective.
The year 1998 witnessed a critical breakthrough for GISTs, which began with the
discovery of the protoꢀoncogene stem cell factor receptor (cꢀKIT). Approximately
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0% of GISTs are positive for cꢀKIT expression, which has facilitated the diagnosis
of GISTs. About 80%ꢀ85% of GISTs arise from gainꢀofꢀfunction mutations in cꢀKIT
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or the platelet derived growth factor receptorꢀα (PDGFRα) gene, both of which
encode type III receptor tyrosine kinases (RTK). The common sites of mutations are
usually located in cꢀKIT exon 11 (~70%), cꢀKIT exon 9 (~10%), or PDGFRα exons
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12 or 18 (~10%), which cause sustained activation of the tyrosine kinase resulting in
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uncontrolled cellular proliferation and resistance to apoptosis . Due to the critical
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role of cꢀKIT and PDGFRα kinases in cellular transformation and differentiation to
the tumorigenesis of GISTs, cꢀKIT and PDGFRα kinases have been extensively
evaluated as important drug discovery targets for antiꢀGIST therapy.
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Currently, there are three nonꢀselective cꢀKIT/PDGFRα kinase inhibitors
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approved for clinical use (Figure 1). Imatinib (1) , a small molecule tyrosine kinase
inhibitor (TKI) with activity against ABL/cꢀKIT/PDGFR kinases, was the first TKI
approved by the US Food and Drug Administration (FDA) as frontline therapy for
metastatic or unresectable GISTs. Compound 1 binds competitively to the adenosine
triphosphate (ATP) binding site of the target kinases, preventing substrate
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phosphorylation and signaling, thereby inhibiting tumor proliferation. However,
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approximately 14% patients are initially insensitive to 1. And oneꢀhalf of the
responding patients can acquire secondary cꢀKIT mutations within 2 year of 1
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treatment, which contains a T670I substitution within the ATPꢀbinding pocket. The
T670 residue is situated at the gatekeeper position of cꢀKIT kinase and provides one
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of the key hydrogen bonds for the binding to 1. This mutation prevents the full
access of 1 to the ATP binding pocket due to steric hindrance in the drug binding
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site. In addition, experimental evidence showed that cꢀKIT/T670I mutant will
contribute to an early, more aggressive metastasis and shorter progressionꢀfree
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survival. Additionally, nearly 5%ꢀ7% of patients with GISTs who have PDGFRα
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mutations are generally 1ꢀresistant. Sunitinib (2), a VEGFR/PDGFR/cꢀKIT/FLT3
TKI, is approved as secondꢀline therapy for patients with advanced GISTs who are 1
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resistant or intolerant. Regorafenib (3), a VEGFR/PDGFR/FGFR/cꢀKIT/RET/Rafꢀ1
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TKI, is a thirdꢀline therapy for GISTs that progressed on 1 and 2. Moreover, a number
of multitarget inhibitors (Figure 1) are undergoing clinical investigation, such as
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nilotinib (4), masitinib (5) and dovitinib (6).
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Figure 1
Although the above agents for the treatment of GISTs have achieved some
success stories, the clinical resistance still demand close attention for the
heterogeneity of patterns of mutations in the cꢀKIT and PDGFRα proteins. In addition,
nonꢀselective profiles targeting cꢀKIT and PDGFRα frequently induced different and
more side effects. The patients occurring serious adverse events (grade 3 or 4) with 1
treatment were 21.1% and suffering mild or moderate oedema, fatigue were
frequently seen with 1 treatment. While 61% patients suffered the drugꢀrelated
12, 21, 25
adverse events of grade 3 or higher with 3 treatment.
Hypertension, diarrhea,
palmar plantar erythrodysesthesia and fatigue were commonly observed in therapy
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with 2 or 3. Moreover, skin discoloration (up to 30%, yellow color) and hair
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discoloration were more representative with 2 treatment, while weight loss,
hypophosphatemia and hyperlipasemia were more often observed with 3 treatment.
In this text, we planned to design and synthesize a series of novel, potent, and
selective dualꢀtarget cꢀKIT/PDGFRα inhibitors, with powerful efficacy against
cꢀKIT/PDGFRa kinase and mutants, and anticipated finding a good lead compound
for the treatment of GISTs, especially 1ꢀresistant GISTs.
DESIGN OF cꢀKIT/PDGFRα DUAL INHIBITORS
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Linifanib (7) (Figure 2), a 3ꢀaminoindazoleꢀbased orally active drug containing
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an N, N’ꢀdiaryl urea moiety (Figure 2), is a novel available multiꢀtarget inhibitor of
the vascular endothelial growth factor receptor (VEGFR) and PDGFR family
members with minimal activity against unrelated RTKs. Inhibitor 7 has demonstrated
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potent antitumor efficacy in a broad range of clinical trials. At the beginning of our
efforts to discover new antitumor chemotypes targeting VEGFR2 and PDGFRα, 7
was selected as a hit compound (Table 1; PDGFRα: IC50=15 nM, VEGFR2: IC50=250
nM). With the aid of a wellꢀdocumented structureꢀactivity relationship (SAR) as well
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as the synthetic accessibility of 7, we modified three regions (R ꢀR , Figure 2) of this
molecule to obtain new SAR information and improve TKI activity.
To obtain novel analogs and clarify the SARs under the premise of minimizing
1
the structural changes, we designed and synthesized compounds 8ꢀ15 (R , Table 1) to
determine if the 3ꢀaminoindazole moiety is necessary for VEGFR2 and PDGFRα
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inhibitory activities, compounds 16ꢀ17 (R , Table 1) to demonstrate the significance
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of the oxygen atom in the urea group as a key linker, and compounds 18ꢀ22 (R , Table
3) to examine the influence of different substituents on the 3ꢀposition of
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pyrazolo[3,4ꢀb]pyridine scaffold. Furthermore, compounds 23ꢀ65 (R , Table 4) were
prepared to determine if the type of Nꢀsubstitutions would affect cꢀKIT/PDGFRα
inhibitory activities.
Figure 2
Tables 1ꢀ4
CHEMISTRY
As outlined in Scheme 1, ethyl trifluoroacetate was selected as the starting
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material and was reacted with acetonitrile by likeꢀnucleophilic substitution to produce
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,4,4ꢀtrifluoroꢀ3ꢀoxobutanenitrile (66), which was not further purified because of its
difficult detection by TLC and low boiling point. Then, 66 was cyclized in the
presence of hydrazine hydrate to yield the intermediate 67a.
Scheme 1
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Scheme 2 depicts the synthetic route for the preparation of target compounds
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ꢀ17 and 20ꢀ27. Intermediate 68aꢀb was synthesized by coupling 67a and 67b
(
commercially available) with 4ꢀnitro benzaldehyde and 2,2ꢀDimethylꢀ1,3ꢀdioxane
4,6ꢀdione through an effective oneꢀcontainer threeꢀcomponent approach. The
ꢀ
reduction of 68b by catalytic hydrogenation of Pd/C yielded the key intermediate 69,
followed by treatment with two substituted phenyl isocyanates to produce target
compounds 8ꢀ9. 68aꢀb was treated with 2,3ꢀdichloroꢀ5,6ꢀdicyanobenzoquinone (DDQ)
and N,Oꢀbis(trimethylsilyl) trifluoroacetamide (BSTFA) to provide aromatic
intermediates 70aꢀb via oxidative dehydrogenation. The nitro group of 70b was
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reduced using SnCl to form another key intermediate 71, followed by treatment with
isocyanates to produce target compounds 10ꢀ11. The chlorination of 70aꢀb with
phenylphosphonic dichloride (BPOD) gave intermediates 72aꢀb, and a reductive
elimination reaction with zinc and hydrazine hydrate then produced the last key
intermediates 73aꢀb, followed by coupling with various commercially available
substituted isocyanates and isothiocyanates to achieve target compounds 12ꢀ17 and
20ꢀ27.
Scheme 2
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The synthetic route for the preparation of target compounds 18ꢀ19 and 41ꢀ65 is
shown in Scheme 3. Boc protection of 73b gave triꢀsubstitued intermediate 74,
followed by bromination to provide 75, which was hydrolyzed to produce 76. The
nucleophilic addition of substituted isocyanates with 76 generated target compounds
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8ꢀ19. Different isocyanates were in situ synthesized using N, N'ꢀcarbonyl
diimidazole (CDI) from various commercially available aliphatic amines, aryl amines,
and aralkyl amines. Nucleophilic addition of these isocyanates with 73b gave target
compounds 41ꢀ65.
Scheme 3
Intermediates 77aꢀm were reduced by the catalytic hydrogenation of Pd/C to
provide the corresponding amines 78aꢀm, respectively, then reacted with phenyl
chloroformate and 73b to generated target compounds 28ꢀ40. The details of the
synthetic procedures and structural characterizations of target compounds 8ꢀ65 are
described in the Experimental Section. The purities of all targeted compounds were
determined by HPLC (Table S1, Supporting Information). The details of the synthetic
procedures and structural characterizations of intermediates 66ꢀ78 are shown in the
Supporting Information.
Scheme 4
RESULTS AND DISCUSSION
Kinase Enzymatic Assay
Based on our initial investigations of 7, the novel designed analogs were first
optimized according to the potencies (IC50) against PDGFRα, VEGFR2 and fibroblast
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growth factor receptor 1 (FGFR1). From Table 1, we could see that compounds 8ꢀ15
caused a distinct loss of activity against VEGFR2 (IC50 > 1000 nM) compared to 7
(IC50=250 nM) and weak inhibitory activity against FGFR1 (IC50 > 1000 nM), similar
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to 7 (IC =5479 nM). This finding indicated that the potencies against VEGFR2 and
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FGFR1 may not tolerate the change of the core scaffold. Furthermore, in the studied
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set of three new core scaffolds (R ), the potency (especially PDGFRα) was increased
in the order 3ꢀmethylꢀpyrazolo[3,4ꢀb]pyridine (12ꢀ13) > 3ꢀmethylꢀ6ꢀhydroxyꢀ
pyrazolo[3,4ꢀb]pyridine (10ꢀ11) > 3ꢀmethylꢀ6ꢀhydroxyꢀ4,5ꢀdihydroꢀpyrazolo[3,4ꢀb]
pyridine (8ꢀ9). A small set of analogs with various Nꢀaryl substituents (12ꢀ15, Table 1,
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R ) showed that R substituent enabled good variability for potent PDGFRα inhibitory
activity. With the novel good core scaffold retained, we turned our attention to the
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“
urea linker” (R ) and prepared thiourea compounds (16ꢀ17). Unfortunately, these two
compounds both obviously lost activity against all three kinases (IC50 > 1000 nM).
Considering the multiꢀtargeted profiles of 7, we decided to select compound 13, the
most potent PDGFRα inhibitor among compounds 8ꢀ17, as the lead compound
(Figure 2) to preliminarily explore its spectrum of activity against other RTKs (Table
2). Surprisingly, compound 13 showed very potent inhibitory activity against cꢀKIT at
the singleꢀdigit nanomolar concentration (IC50=2.1 nM). It is interesting that
compound 13 seemed to be a selective cꢀKIT/PDGFRα dual inhibitor. Based on this
result, the subsequent work was aimed to developing a dualꢀtargeted compound
against cꢀKIT and PDGFRα, and VEGFR2 was used as a typical kinase to monitor the
selective profile.
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To examine the influence of different substituents on the 3ꢀposition of
pyrazolo[3,4ꢀb]pyridine scoffold, we replaced the 3ꢀmethyl substituent with
hydroxymethyl (18ꢀ19) and trifluoromethyl (20ꢀ21). The results in Table 3 showed
that replacement of the 3ꢀmethyl group with a hydroxymethyl group (18 vs 13)
maintained fairly potent cꢀKIT inhibition (IC50= 2.4 vs 2.1 nM) but led to a 2ꢀfold loss
of PDGFRα activity (IC50=87 vs 40 nM). Changing the 3ꢀmethyl group to the
trifluoromethyl group (20 vs 13) could not be tolerated, leading to a complete loss of
all kinase activities (IC50 > 50,000 nM). Therefore, the 3ꢀmethylꢀpyrazolo[3,4ꢀb]
pyridine moiety represented seemingly the best pharmacophore for inhibiting both
cꢀKIT and PDGFRα.
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Considering the 3ꢀmethylꢀ1Hꢀpyrazolo[3,4ꢀb]pyridine scaffold and urea linker as
a promising novel template for a dualꢀtargeted cꢀKIT/PDGFRα inhibitor, we next
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focused SAR exploration on substituent (R , Figure 2) at the terminal nitrogen of urea.
Shifting the methyl group from 3ꢀposition (13) to 2ꢀposition (23) or 4ꢀposition (24)
both led to significant loss of activity against cꢀKIT/PDGFRα (Table 4). Among
various 3ꢀsubstituents (13ꢀ15 and 25ꢀ28), 3ꢀmethyl group was the best one for
cꢀKIT/PDGFRα inhibition. Surprisingly, a Nꢀmethyl piperazinyl group at 3ꢀposition
(28) only moderately decreased the cꢀKIT/PDGFRα inhibitory activities compared to
13 (Table 4). Inspired by this result, a combination of 3ꢀmethyl and Nꢀmethyl
piperazinyl or morpholinyl group on the terminal phenyl obtained compounds 29ꢀ31,
of which 31 displayed more potent inhibitory activities than lead compound 13
against cꢀKIT/PDGFRα kinases (IC50 = 2.4 and 7.2 nM, respectively), and also
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retained the selectivity over VEGFR2. However, when we changed the combined type
or position of methyl, Nꢀmethyl piperazinyl or morpholinyl group on the terminal
phenyl (32ꢀ35), it caused a significant loss of activities against all three kinases.
Furthermore, introducing diethylamino group (36ꢀ37) or dimethylamino group (38ꢀ39)
on the terminal phenyl started to gain back the cꢀKIT/PDGFRα activities but inferior
to 31. Replacement of 3ꢀdimethylamino group with 3ꢀdimethylaminomethyl group
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(40) also led to complete loss of activities to all three kinases.
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To explore more effective compounds, further screening of R group (41ꢀ65) was
implemented (Table 4). For the linear alkyl groups (41ꢀ45), the cꢀKIT/PDGFRα
inhibitory activity was substantially improved with an increasing length of alkyl
groups. Changing linear alkyl (43) into branched alkyl (46ꢀ47) or cycloalky (48ꢀ51)
groups could not enhance the cꢀKIT/PDGFRα inhibitory activity compared to 13. For
the aralkyl groups (52ꢀ59), the heteroarylmethyl groups (52ꢀ54) were favorable for
cꢀKIT/PDGFRα inhibition compared to the phenylmethyl group (55) but inferior to 13.
With the different length set of phenylalkyl groups (55ꢀ59), the potency against
cꢀKIT/PDGFRα was increased in the order phenylpropyl (57) > phenylethyl (56) >
2,3ꢀdihydroꢀindene (59) > phenylbutyl (58). Although the cꢀKIT inhibitory activity of
5
7 was slightly better than that of 13 and 31, the PDGFRα inhibitory activity of 57
was weaker than that of 31 (IC =24 nM vs 7.2 nM). Additionally, replacement of the
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aryl group (R ) with heteroaryl groups (60ꢀ62) all led to a reduction of kinase potency
greater than 6ꢀfold, but introduction of large fused aryl groups (63ꢀ65) seemingly was
beneficial, leading to a visible increase of cꢀKIT/PDGFRα inhibition compared to 13
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but still not improving PDGFRα inhibition at the singleꢀdigit nanomolar
concentration.
Collectively, the structural optimization and SAR studies led to the discovery of
a number of novel compounds, such as 31, 45, 57 and 63, that exhibited a higher
dualꢀtarget potency against cꢀKIT (IC50 < 3 nM) and PDGFRα (IC50 < 30 nM) as well
as maintained the appropriate selective window against VEGFR2 (IC50 > 1800 nM).
Especially, 31 had the most surprising singleꢀdigit nanomolar potency against
PDGFRα, similar to the launched compound 1 (IC50= 7.2 nM vs 4.6 nM, Table 4), and
had powerful cꢀKIT activity (IC50 = 2.4 nM), providing a 17ꢀfold improvement
compared to 1 (Table 4).
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Cellular Property and Biochemical Evaluation
In light of the previous research of kinase inhibitory activities, compound 31 was
the best cꢀKIT/PDGFRa dual inhibitor. To further evaluate whether 31 was the
optimal compound, we selected 11 compounds to examine the inhibitory activities
30
against cellular cꢀKIT and PDGFRα, including four BaF3 cells whose viability were
KITꢀdependent or PDGFRαꢀdependent. As shown in Table 5, the
3ꢀmethylꢀ1Hꢀpyrazolo[3,4ꢀb]pyridine scaffold was favorable for the antiproliferation
against cꢀKIT/PDGFRα cell lines. Moreover, the inhibitory activities of cellular level
were consistent with the kinase inhibitory activities in vitro. Excitingly, compound 31
showed the best antiproliferation activities against the four BaF3 cell lines, which was
superior to the hit compound 7 with a 5ꢀfold increase of TelꢀcꢀKITꢀBaF3 inhibitory
potency (GI50 = 0.0181 ꢁM vs 0.0887 ꢁM, Table 5). Therefore, 31 was chosen for
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further inꢀdepth studies of antitumor potencies.
Table 5
First, the inhibitory activities of optimal compound 31 against
cꢀKIT/PDGFRαꢀBaF3 cell lines were comprehensively evaluated. The results are
exhibited in Table 6 and Figure 3, including the four cell lines examined above.
According to the data, 31 was more potent against all of the mutations except
TelꢀcꢀKIT/V559DꢀBaF3 (GI50= 0.0965 vs 0.0310 ꢁM) mutation compared to 1. Based
on exhibiting a similar trend to 1 in the parental BaF3, 31 improved the
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antiꢀproliferative activity more than 21ꢀfold towards the native cꢀKIT (GI = 0.0181
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vs 0.3819 ꢁM). The inhibitory activity of TelꢀcꢀKIT/C674SꢀBaF3 mutation (GI50
=
=
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1
0.0105 ꢁM) was consistent with a point mutation TelꢀcꢀKIT/L576PꢀBaF3 (GI50
0
.0106 ꢁM) in exon 11, which showed poor sensitivity for 1 (Figure 3). In addition,
32
31 was an effective inhibitor of all four ATP pocket mutations (Figure 3) and
brought approximately more than 250ꢀfold activity improvement in the 1ꢀresistance
mutations of TelꢀcꢀKIT/T670IꢀBaF3 (GI50
=
0.0392 vs >10 ꢁM) and
TelꢀcꢀKIT/V559D/T670IꢀBaF3 (GI50 = 0.0364 vs >10 ꢁM) compared to 1. With four
Aꢀloop mutations (Figure 3), 31 exhibited a highly inhibitory activity of
TelꢀcꢀKIT/N822KꢀBaF3 mutation (GI50 = 0.0111 ꢁM) and TelꢀcꢀKIT/N829KꢀBaF3
mutation (GI = 0.0116 ꢁM) in contrast to TelꢀcꢀKIT/D816VꢀBaF3 mutation (GI >
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0 ꢁM), but showed a moderate activity (GI50 = 0.1341 ꢁM) against
TelꢀcꢀKIT/D816HꢀBaF3 mutation. More excitingly (Table 5), 31 improved the
antiproliferation activity of TelꢀPDGFRα/T674IꢀBaF3 mutation (GI50 = 0.1348 ꢁM),
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which was insensitive to 1 (GI50 > 10 ꢁM), while retaining activity against the native
PDGFRα (GI50 = 0.0104 ꢁM). Combining the results from all of these different BaF3
cell lines, we concluded that 31 is promising as a powerful dualꢀtargeted
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cꢀKIT/PDGFRα kinase inhibitor that is even superior to 1.
Table 6
Figure 3
Next, the inhibitory effects of compound 31 against a variety of intact cancer cell
lines were investigated (Table 7). The results showed that the inhibitory effect of 31
3
3
against proliferation was more potent than 1 against the GISTꢀT1 cell line; these
cells expressed the characteristics of GISTs that were strongly positive for CD34 and
cꢀKIT but not for desmin, Sꢀ100 protein, or αꢀsmooth muscle actin. Most interestingly,
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1 exhibited powerful antiproliferative activity against the GISTꢀ882 cell line with
single cꢀKIT mutation and gained significant improvement of antiꢀproliferation
35
activity compared to 1 (GI50 < 0.003 ꢁM vs 0.020 ꢁM). For the GIST 48B cell line
harboring the activating cꢀKIT mutation but expressing no detectable cꢀKIT transcript
or cꢀKIT protein, 31 showed the same drug resistance as 1. In addition, 31 completely
abolished the inhibitory activity against the BCRꢀABL driven CML cell lines, such as
K562 (GI50 > 10 ꢁM), KU812 (GI50 > 10 ꢁM) and MEGꢀ01 (GI50 = 9.092 ꢁM) cells in
contrast with 1. Moreover, 31 had no antiproliferative activity against normal Chinese
hamster ovary CHL and CHO cells, implying a good safety profile.
Table 7
To understand kinase spectrum selective of 31 comprehensively, we then
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examined its kinome wide selectivity profile with DiscoveRx’s KinomeScan
3
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technology. The results are shown in Figure 4 and Table S2 (Supporting
Information). We were delighted to find that 31 possessed a good selectivity (S score
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(
1) = 0.01) in a panel of 468 kinases and mutants at 1 µM concentration. Besides high
binding against cꢀKIT and PDGFRα kinases, it also exhibited strong binding affinity
to PDGFRβ, CSF1R kinases and mutant kinases including cꢀKIT/V559D/T670I,
cꢀKIT/L576P, cꢀKIT/V559D and FLT3/N841I (percent activity remaining <1% at 1
ꢁ
M) (Figure 4A and 4B). We further calculated the values of S score (10) and S score
(35), and the results also (S score (10) = 0.03 at 1 ꢁM, S score (35) = 0.04 at 1 ꢁM)
illustrated that 31 exhibited a good selectivity at 1 µM concentration (Supporting
Information, Table S3). Furthermore, considering the fact that KinomeScan is a
competitive binding assay and may not fully reflect the inhibitory activity, 31’s
inhibitory activities against six kinds of available kinases (CSF1R, PDGFRβ, FLT1,
FLT3, FLT4 and RET kinases) with POC < 10 at 1 ꢁM were further tested by using
TM
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Invitrogen’s Z’ꢀLYTE assay and enzymeꢀlinked immunosorbent assay (ELISA)
TM
and the results were shown in Table 8. The Z’ꢀLYTE assay demonstrated that 31
inhibited CSF1R kinase with an IC50 of 160 nM. The ELISA showed that 31 inhibited
PDGFRβ kinase with an IC50 below 1 nM, FLT1 with an IC50 of 159 nM, FLT3 with
an IC of 2.0 nM, FLT4 with an IC of 72 nM and RET with an IC above 1000 nM
5
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(Table 8). The good inhibitory activity against PDGFRβ and FLT3 kinases was
understandable since cꢀKIT, CSF1R, PDGFRα, PDGFRβ and FLT3 kinases are
members of the type III receptor tyrosine kinase family. In addition, compound 31
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showed high percent of control values (POC > 35%) against all kinds of ABL kinases
and mutants (Supporting Information, Table S4) indicating weak binding and
consisting with the results that compound 31 showed almost no inhibitory activity
against the BCRꢀABL driven CML cell lines which had been evaluated in Table 7.
Table 8
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Figure 4
Pharmacokinetic and in Vitro Safety Properties
Because of its potency and attractive selectivity profile, compound 31 was
evaluated in an in vivo rat pharmacokinetic (PK) model following intravenous (IV),
oral and intraperitoneal (IP) administration. As shown in Table 9, 31 demonstrated a
z
short halfꢀlife (T1/2 = 0.69 h), a moderate plasma clearance (CL = 0.95 L/h/kg), small
volume distribution (Vss = 0.97 L/kg) and a good plasma exposure in
SpragueꢀDawley rats (AUC(0ꢀt) = 2243.82 ng/mL·h) with intravenous (IV) injection at
a dose of 2 mg/kg. However, there was no absorption in the oral administration with a
dose of 10 mg/kg, which prevented 31 from the oral application in the animal model.
And then, pharmacokinetic characterization of 31 with IP injection was tested with
two dosages of 25 mg/kg and 100 mg/kg (Table 10). At the dosage of 100 mg/kg, 31
showed a good halfꢀlife (T1/2 =10.97 h), and a better plasma exposure in
SpragueꢀDawley rats (AUC(0ꢀt) = 4245.24 ng/mL·h) which was much better than the
plasma level at the dosage of 25 mg/kg (AUC(0ꢀt) = 691.08 ng/mL·h).
Moreover, the metabolic stability of compound 31 in rat liver microsomes was
further tested in vitro and the result showed that its halfꢀlife was 46.76 min, indicating
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1 was metabolically stable (Supporting Information). What's more, the safety
evaluation experiment (Supporting Information) displayed that 31 possessed almost
no hERG inhibitory activity (IC50 > 40 ꢁM).
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Table 9
Table 10
In Vivo Evaluation of 31 in inoculated xenograft mouse model with GISTꢀT1 Cell
To assess the potential of 31 for further medicinal chemistry development, we
investigated its antitumor efficacy using the GISTꢀT1 cell inoculated xenograft mouse
model. Intraperitoneal (IP) injection was selected based upon the PK data (Table 8). It
was noteworthy that the PK profile obtained from rats can’t be transformed into mice
due to the differences of species and administration. Figure 5A showed that no animal
lethality or obvious weight loss was observed by the administration of 25 mg/kg or
1
00 mg/kg of 31 during 28 consecutive treatment days. Furthermore, 31 showed a
high inhibition of tumor growth (TGI = 81.5%) which was slightly weaker than 1
TGI = 87.3%) at the dosage of 100 mg/kg. When the dosage of 31 was decreased to
5 mg/kg, the treatment also resulted in a modest suppression against tumor
(
2
progression (TGI = 20.2%) (Figures 5BꢀD). Immunohistochemistry staining
demonstrated that 31 could strongly suppress the cellular proliferation marker (Ki67
stain) and induce the apoptosis effect (TUNEL stain) in the tumor tissues (Figure 5E).
Figure 5
In Vivo Evaluation of 31 in inoculated xenograft mouse model with
BaF3ꢀTELꢀcKITꢀT670I Cell
Considering 31 with a strong suppression against GISTꢀT1 progression, we next
wished to determine the antiproliferation efficacy in BaF3ꢀTELꢀcKITꢀT670I cells
inoculated xenograft mouse model. We also chose the IP injection administration with
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the dosage of 25 and 100 mg/kg/day for 11 days of continuous treatment and the
results were shown in Figure 6. The changes of all mice body weights were in a
reasonable range as shown in Figure 6A. As expected, 1 could not suppress the cꢀKIT
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T670I tumor growth while compound 31 demonstrated moderate tumor growth
inhibition (TGI = 41.9%) at 100 mg/kg/day dosage (Figure 6BꢀC) without obvious
toxicity. In addition, The Figure 6D showed that 31 affected cꢀKIT pY719, pY703,
and pY823 autophosphorylation sites of cꢀKIT at the dosage of 100 mg/kg/day. The
results of immunohistochemistry staining also indicated the proliferation of tumor
cells was greatly inhibited and apoptosis was well induced (Figure 6E).
CONCLUSIONS
In conclusion, the application of lead optimization based on multiꢀtargeted RTK
inhibitor 7 contributed to the design and synthesis of 58 new compounds (8ꢀ65) and
the novel 3ꢀsubstitutedꢀpyrazolo[3,4ꢀb]pyridine scaffold shifted the kinase inhibitory
spectrum from VEGFR/PDGFR to cꢀKIT/PDGFRα. Using kinase inhibitory assays
provided an integrated assessment of kinase selectivity and potency of all compounds.
Preliminary SARs were obtained, which showed that the 3ꢀmethylꢀpyrazolo[3,4ꢀb]
pyridine core scaffold of new compounds was an important pharmacophore of the
cꢀKIT/PDGFRα dual inhibitor; the urea linker and terminal Nꢀsubstituents, e.g.,
3ꢀmethylꢀ4ꢀ(morpholinꢀ4ꢀyl)phenyl, long linear alkyl, aralkyl and large fused
heteroaryl, substantially improved the potency.
The preferred compound 31, harboring singleꢀdigit nanomolar potency against
cꢀKIT (IC = 2.4 nM) and PDGFRα (IC = 7.2 nM), also exhibited better inhibition
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compounds. So its further inꢀdepth antiꢀGISTs investigations in vitro and in vivo were
performed. In vitro, broad antiproliferative efficacy of 31 was found across a range of
TELꢀcꢀKIT/PDGFRα ꢀBaF3 isogenic cells, which were almost suppressed except for
the TELꢀcꢀKIT/D816VꢀBaF3 cells that were also insensitive to 1. In particular, 31
showed more potent inhibitory activities against the cꢀKIT/T670IꢀBaF3,
cꢀKIT/V559D/T670IꢀBaF3 and PDGFRα/T674IꢀBaF3 cell lines, which were all
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1
ꢀresistant. In parallel, the potency of 31 against a variety of intact cancer cell lines
was observed and improved almost 10 times or more against GISTꢀ882 cells and
completely abolished the inhibitory activity against BCRꢀABL driven CML cell lines
compared to 1. Moreover, 31 showed a good selectivity (S score (1) = 0.01 at 1 ꢁM)
among 468 kinases and mutants tested with DiscoveRx’s KinomeScan technology and
exhibited almost no effects on the growth of two normal cells (CHL: GI50 = 7.036 ꢁM,
CHO: GI50 > 10 ꢁM) as well as possessing almost no hERG inhibitory activity (IC50
0 ꢁM), indicating a good therapeutic window. 31 also displayed a high metabolic
stability in rat liver microsomes with good halfꢀlife (T1/2 = 46.76 min). Furthermore,
1 was assessed in a GISTꢀT1 xenograft mouse model and showed significant in vivo
>
4
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antitumor activities, suppressing tumor growth (TGI = 81.5%) and inducing apoptosis.
More excitingly, 31 demonstrated 41.9% tumor growth inhibition in the
BaF3ꢀTELꢀcKITꢀT670I isogenic cell inoculated xenograft mouse model (insensitive
to 1) and blocked cKIT pY703, pY719, pY823 autophosphorylation sites at the
dosage of 100 mg/kg/day.
In general, the favorable in vitro and in vivo potency of 31 makes it possible for
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serving as a dualꢀtargeted compound against cꢀKIT and PDGFRα for the treatment of
GISTs, especially 1ꢀresistant GISTs, as well as a useful prototype for medicinal
chemistry programs to further study the GISTs.
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EXPERIMENTAL SECTION
General Chemistry. Synthetic starting materials, reagents, and solvents were
purchased from Energy Chemical, J&K, Adamasꢀbeta, TCI, Shanghai Shaoyuan Co.
Ltd. and Alfa Aesar and were used without further purification. Analytical thinꢀlayer
chromatography (TLC) was performed on an HSGF 254 (150ꢀ200 ꢁm thickness;
Yantai Huiyou Co., China), and the peak purity was verified with UV spectroscopy
(254 nm and 365 nm). Melting points were measured in capillary tubes on an SGW
Xꢀ4 melting point apparatus without correction. Nuclear magnetic resonance (NMR)
spectroscopy was performed on a Bruker AMXꢀ400 NMR (IS as TMS). Chemical
shifts were reported in parts per million (ppm, δ) downfield from tetramethylsilane.
Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (br). Highꢀresolution mass spectra (LRMS and HRMS)
were obtained with electric ionization (EI) and electrospray ionization (ESI) produced
by a Waters GCT Premie and Waters LCT. HPLC data analysis of compounds 8ꢀ65
were performed on an Agilent 1100 with a quaternary pump and diodeꢀarray detector
(DAD). All compounds were confirmed to have a purity ≥ 95%.
General procedure for the preparation of compounds 8-27
3
To a solution of 69/71/73/76 (0.5 mmol) in DMSO (5 mL) was added Et N (50
ꢁ
L) and corresponding isocyanates or isothiocyanates (0.6 mmol). The reaction
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mixture was stirred at room temperature for 12 h. Water (15 mL) was added and the
mixture was extracted with EtOAc (3 × 50 mL). The organic layer was separated and
2 4
dried over Na SO . Removal of the solvents produced a residue, which was purified
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
by column chromatography eluted with a mixture of MeOH/DCM to yield
corresponding target compounds 8ꢀ27.
N-(2-Fluoro-5-methylphenyl)-N’-(4-(3-methyl-6-hydroxy-4,5-dihydro-2H-pyrazolo[
3
,4-b]pyridin-4-yl)phenyl)urea (8)
Yellow solid. Yield = 21%. Mp 163ꢀ166 °C; HꢀNMR (400 MHz, DMSOꢀd
1.81 (s, 1H), 10.29 (s, 1H), 9.04 (s, 1H), 8.45 (d, J = 2.5 Hz, 1H), 7.98 (dd, J = 8.0,
.0 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.13 – 7.05 (m, 3H), 6.81 – 6.76 (m, 1H), 4.49
1
6
): δ
1
2
– 4.43 (m, 1H), 3.39 (d, J = 5.2 Hz, 1H), 3.17 (d, J = 5.2 Hz, 1H), 2.26 (s, 3H), 1.84 (s,
+
3
H). HRMS (ESI) m/z calcd for C21
H20FN
5
O
2
[M+H] 394.1679, found 394.1679.
N-(3-Methylphenyl)-N’-(4-(3-methyl-6-hydroxy-4,5-dihydro-2H-pyrazolo[3,4-b]pyri
din-4-yl)phenyl)urea (9)
1
Yellow solid. Yield = 43%. Mp 231ꢀ233 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
1
1.81 (s, 1H), 10.29 (s, 1H), 8.62 (s, 1H), 8.56 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.29
(s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H),
.78 (d, J = 7.2 Hz, 1H), 4.08 (t, J = 6.4 Hz, 1H), 2.76 (dd, J = 15.6, 6.8 Hz, 1H), 2.54
6
(dd, J = 15.6, 6.4 Hz, 1H), 2.27 (s, 3H), 1.85 (s, 3H); HRMS (ESI) m/z calcd for
+
C
21
H
21
N
5
O
2
[M+H] 376.1772, found 376.1773.
N-(2-Fluoro-5-methylphenyl)-N’-(4-(3-methyl-6-hydroxy-2H-pyrazolo[3,4-b]pyridin
4-yl)phenyl)urea (10)
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5
5
5
5
5
5
5
5
5
6
1
Light yellow solid. Yield = 11%. Mp 131−134 °C; HꢀNMR (400 MHz,
DMSOꢀd ): δ 12.89 (s, 1H), 11.56 (s, 1H), 9.38 (s, 1H), 8.60 (s, 1H), 7.99 (d, J =7.6
Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H), 7.12 (dd, J = 11.2, 8.4 Hz,
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
H), 6.82 (m, 1H), 5.85 (s, 1H), 2.28 (s, 3H), 2.09 (s, 3H); HRMS (ESI) m/z calcd for
+
C
21
H
18FN
5
O
2
[M+H] 392.1524, found 392.1523.
N-(3-Methylphenyl)-N’-(4-(3-methyl-6-hydroxy-2H-pyrazolo[3,4-b]pyridin-4-yl)phe
nyl)urea (11)
1
Yellow solid. Yield = 18%. Mp 173−175 °C; HꢀNMR (400 MHz, DMSOꢀd
): δ
6
1
2.87 (s, 1H), 11.57 (s, 1H), 8.91 (s, 1H), 8.71 (s, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.41
(
d, J = 8.4 Hz, 2H), 7.31 (s, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H),
6.80 (d, J = 7.6 Hz, 1H), 5.87 (s, 1H), 2.29 (s, 3H), 2.10 (s, 3H); HRMS (ESI) m/z
+
calcd for C21
H
19
N
5
O
2
[M+H] 374.1618, found 374.1617.
N-(2-Fluoro-5-methylphenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phen
yl)urea (12)
1
White solid. Yield = 62%. Mp 215ꢀ222 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
1
3.34 (s, 1H), 9.41 (s, 1H), 8.64 (s, 1H), 8.47 (d, J = 4.7 Hz, 1H), 8.09 – 7.94 (m, 1H),
7.65 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.12 (dd, J = 11.3, 8.4 Hz, 1H),
7
.03 (d, J = 4.7 Hz, 1H), 6.82 (s, 1H), 2.27 (d, J = 13.8 Hz, 6H); HRMS (EI) m/z
+
calcd for C H FN O (M ) 375.1495, found 375.1499.
21
18
5
N-(3-Methylphenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea
(13)
1
White solid. Yield = 52%. Mp 225ꢀ229 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
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3.34 (s, 1H), 9.16 (s, 1H), 8.92 (s, 1H), 8.47 (d, J = 4.6 Hz, 1H), 7.65 (d, J = 8.5 Hz,
H), 7.49 (d, J = 8.4 Hz, 2H), 7.34 (s, 1H), 7.29 (d, J = 7.9 Hz, 1H), 7.18 (t, J = 7.7
2
Hz, 1H), 7.03 (d, J = 4.6 Hz, 1H), 6.81 (d, J = 7.2 Hz, 1H), 2.28 (d, J = 13.5 Hz, 6H);
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
HRMS (EI) m/z calcd for C H N O (M ) 357.1590, found 357.1593.
21
19
5
N-(3-Bromophenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea
14)
(
1
White solid. Yield = 86%. Mp 232ꢀ235 °C; HꢀNMR (400 MHz, DMSOꢀd
3.34 (s, 1H), 9.19 (s, 2H), 8.47 (d, J = 4.3 Hz, 1H), 7.89 (s, 1H), 7.65 (d, J = 8.0 Hz,
H), 7.51 (d, J = 7.9 Hz, 2H), 7.36 (d, J = 7.8 Hz, 1H), 7.26 (t, J = 7.9 Hz, 1H), 7.17
6
): δ
1
2
(
d, J = 7.6 Hz, 1H), 7.03 (d, J = 4.2 Hz, 1H), 2.25 (s, 3H). HRMS (EI) m/z calcd for
+
C
20
H
16BrN
5
O (M ) 423.0518, found 423.0523.
N-(3-(Trifluoromethyl)phenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridine-4-yl)ph
enyl)urea (15)
1
White solid. Yield = 82%. Mp 234ꢀ237 °C; HꢀNMR (400 MHz, DMSOꢀd ): δ
6
13.35 (s, 1H), 9.41 (d, J = 50.8 Hz, 2H), 8.47 (d, J = 4.7 Hz, 1H), 8.06 (s, 1H), 7.65
(
dd, J = 16.5, 8.5 Hz, 3H), 7.53 (dd, J = 13.4, 8.3 Hz, 3H), 7.33 (d, J = 7.6 Hz, 1H),
+
7.03 (d, J = 4.7 Hz, 1H), 2.25 (s, 3H); HRMS (EI) m/z calcd for C21
11.1307, found 411.1306.
H
16
F
3
N
5
O (M )
4
N-(3-Bromophenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)thioure
a (16)
1
Light yellow solid. Yield = 60%. Mp 93ꢀ101°C; HꢀNMR (400 MHz, DMSOꢀd
6
):
δ 13.36 (s, 1H), 10.17 (s, 1H), 10.06 (s, 1H), 8.49 (d, J = 4.7 Hz, 1H), 7.83 (s, 1H),
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1
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
2
.67 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 6.7 Hz, 1H), 7.32 (s,
5
H), 7.05 (d, J = 4.7 Hz, 1H), 2.23 (s, 3H); HRMS (ESI) m/z calcd for C20H17BrN S
+
[
M+H] 438.0368, found 438.0383.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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6
7
8
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1
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3
4
5
6
7
8
9
0
N-(3-(Trifluoromethyl)phenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridine-4-yl)ph
enyl)thiourea (17)
1
Light yellow solid. Yield = 64%. Mp 86ꢀ95 °C; HꢀNMR (400 MHz, DMSOꢀd
6
):
δ 13.37 (s, 1H), 10.25 (s, 1H), 10.18 (s, 1H), 8.50 (dd, J = 9.1, 4.7 Hz, 1H), 7.97 (s,
H), 7.78 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.63–7.52 (m, 3H), 7.48 (d, J
1
=
7.8 Hz, 1H), 7.05 (d, J = 4.7 Hz, 1H), 2.24 (s, 3H); HRMS (ESI) m/z calcd for
+
C
21
H
17
F
3
N
5
S [M+H] 428.1151, found 428.1151.
N-(3-Methylphenyl)-N’-(4-(3-hydroxymethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl
)urea (18)
1
White solid. Yield = 59%. Mp 211ꢀ214 °C; HꢀNMR (400 MHz, MeOD): δ 8.52
d, J = 4.7 Hz, 1H), 8.45 (d, J = 4.9 Hz, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.31 (s, 1H),
(
7.27 (d, J = 8.2 Hz, 1H), 7.19 (dd, J = 16.4, 6.2 Hz, 2H), 7.10 (d, J = 4.9 Hz, 1H),
6
.88 (t, J = 9.5 Hz, 3H), 4.70 (d, J = 8.4 Hz, 2H), 2.36 (s, 3H); HRMS (ESI) m/z calcd
+
for C21
H
19
N
5
NaO
2
[M+Na] 396.1434, found 396.1431.
N-(3-Ethylphenyl)-N’-(4-(3-hydroxymethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)
urea (19)
1
White solid. Yield = 34 %. Mp 209ꢀ213 °C; HꢀNMR (400 MHz, MeOD): δ 8.40
(d, J = 4.8 Hz, 1H), 8.33 (d, J = 4.8 Hz, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.22 (s, 1H),
7
.19 – 7.08 (m, 2H), 7.05 (d, J = 4.8 Hz, 1H), 6.98 (d, J = 4.8 Hz, 1H), 6.80 (d, J =
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.3 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 4.58 (d, J = 8.0 Hz, 2H), 2.54 (q, J = 7.6 Hz,
+
21 5 2
H), 1.15 (t, J = 7.6 Hz, 3H); HRMS (ESI) m/z calcd for C22H N NaO [M+Na]
10.1588, found 410.1588.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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55
56
57
58
59
60
N-(3-Methylphenyl)-N’-(4-(3-trifluoromethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)pheny
l)urea (20)
1
White solid. Yield = 46%. Mp 294ꢀ298 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
1
4.71 (s, 1H), 8.88 (s, 1H), 8.68 (d, J = 4.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.41 (d,
J = 8.4 Hz, 2H), 7.33 (s, 1H), 7.28 – 7.23 (m, 2H), 7.18 (t, J = 8.0 Hz, 1H), 6.81 (d, J
+
=
7.6 Hz, 1H), 2.29 (s, 3H). HRMS (EI) m/z calcd for C H F N O (M ) 411.1307,
21 16
3
5
found 411.1305.
N-(3-Ethylphenyl)-N’-(4-(3-trifluoromethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)
urea (21)
1
White solid. Yield = 65%. Mp 297ꢀ299 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
1
4.71 (s, 1H), 8.87 (s, 1H), 8.71 – 8.67 (m, 2H), 7.60 (d, J = 8.4Hz, 2H), 7.41 (d, J =
8.4 Hz, 2H), 7.35 (s, 1H), 7.30 – 7.24 (m, 2H), 7.20 (t, J = 7.6 Hz, 1H), 6.84 (d, J =
7
.6 Hz, 1H), 2.59 (q, J = 7.6 Hz, 1H), 1.19 (t, J = 7.6 Hz, 1H). HRMS (EI) m/z calcd
+
for C22
H
18
F
3
N
5
O (M ) 425.1463, found 425.1464.
N-(3-Ethylphenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea
(22)
1
White solid. Yield = 48%. Mp 165ꢀ170 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
13.34 (s, 1H), 9.07 (s, 1H), 8.86 (s, 1H), 8.47 (d, J = 4.6 Hz, 1H), 7.65 (d, J = 8.5 Hz,
2
H), 7.50 (d, J = 8.5 Hz, 2H), 7.36 (s, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 7.8
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2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
Hz, 1H), 7.03 (d, J = 4.6 Hz, 1H), 6.84 (d, J = 7.4 Hz, 1H), 2.59 (q, J = 7.5 Hz, 2H),
+
2
.26 (s, 3H), 1.19 (s, 3H); HRMS (EI) m/z calcd for C22
71.1744.
H
21
N
5
O (M ) 371.1746, found
3
0
1
2
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
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
N-(2-Methylphenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea
23)
(
1
White solid. Yield = 45%. Mp 222ꢀ226 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
1
1
7
3.34 (s, 1H), 9.54 (s, 1H), 8.47 (d, J = 4.7 Hz, 1H), 8.20 (s, 1H), 7.85 (d, J = 8.0 Hz,
H), 7.67 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 8.6 Hz, 2H), 7.18 (dd, J = 15.0, 7.5 Hz, 2H),
.03 (d, J = 4.7 Hz, 1H), 6.97 (dd, J = 10.6, 4.1 Hz, 1H), 2.27 (d, J = 11.0 Hz, 6H);
+
HRMS (EI) m/z calcd for C21
H
19
N
5
O (M ) 357.1590, found 357.1595.
N-(4-Methylphenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea
24)
(
1
White solid. Yield = 25%. Mp 234ꢀ238 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
1
3.33 (s, 1H), 9.30 (s, 1H), 9.05 (s, 1H), 8.47 (d, J = 4.7 Hz, 1H), 7.64 (d, J = 8.6 Hz,
2H), 7.49 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.2 Hz, 2H), 7.03
+
(d, J = 4.7 Hz, 1H), 2.26 (s, 6H); HRMS (EI) m/z calcd for C21
H
19
N
5
O (M ) 357.1590,
found 357.1595.
N-(3-Fluorophenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea
(25)
1
White solid. Yield = 65%. Mp 263ꢀ266 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
13.34 (s, 1H), 9.26 (d, J = 11.4 Hz, 2H), 8.47 (d, J = 4.7 Hz, 1H), 7.66 (d, J = 8.6 Hz,
2
H), 7.60 – 7.46 (m, 3H), 7.32 (dd, J = 15.2, 8.1 Hz, 1H), 7.22 – 7.11 (m, 1H), 7.03 (d,
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7
8
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J = 4.7 Hz, 1H), 6.80 (td, J = 8.4, 2.0 Hz, 1H), 2.26 (s, 3H); HRMS (EI) m/z calcd for
+
C
20
H
16FN
5
O (M ) 361.1339, found 361.1344.
N-(3-Chlorphenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea
26)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
1
White solid. Yield = 84%. Mp 238ꢀ245 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
1
1
–
3.34 (s, 1H), 9.56 (d, J = 7.9 Hz, 2H), 8.47 (d, J = 4.7 Hz, 1H), 7.75 (d, J = 0.9 Hz,
H), 7.66 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 1.0 Hz, 2H), 7.17
+
6.89 (m, 2H), 2.25 (s, 3H); HRMS (EI) m/z calcd for C20
H
16ClN
5
O (M ) 377.1043,
found 377.1042.
N-(3-Cyanophenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea
(27)
1
White solid. Yield = 53%. Mp 173ꢀ177 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
13.35 (s, 1H), 9.54 (d, J = 37.3 Hz, 2H), 8.47 (d, J = 4.7 Hz, 1H), 8.02 (s, 1H), 7.72 (d,
J = 8.1 Hz, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.58 – 7.48 (m, 3H), 7.44 (d, J = 7.6 Hz,
+
1H), 7.04 (d, J = 4.7 Hz, 1H), 2.25 (s, 3H); HRMS (EI) m/z calcd for C21
68.1386, found 368.1395.
General procedure for the preparation of compounds 28-40.
H
16
N
6
O (M )
3
Under ice cooling, after a solution of phenyl chloroformate (0.5 mL, 4 mmol) in
THF (0.5 mL) was added dropwise to a solution of the intermediate 78/80 (3.2 mmol)
and pyridine (1.2 mL) in DMF (5 mL), the mixture was stirred at room temperature
for 3 h. Ethyl acetate (40 mL) was added thereto, and the whole mixture was washed
with water (40 mL) twice and brine (40 mL) twice. The organic layer was dried over
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1
1
1
1
1
1
1
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
anhydrous Na
2
SO
4
, and the solvent was evaporated under reduced pressure. The
resulting solid was collected by filtration with diethyl ether and dried under reduced
pressure without further purification. This product was dissolved in 1,4ꢀdioxane (10
mL) with 73b (0.5 mmol) and 1ꢀmethyl pyrrolidine (20 ꢁL). The mixture was stirred
at 80 °C for 12 h and then cooled to room temperature and concentrated. The residue
was purified by column chromatography eluting with a mixture of MeOH/DCM to
afford corresponding target compounds 28ꢀ40.
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
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7
8
9
0
N-(3-(4-Methylpiperazin-1-yl)phenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-
yl)phenyl)urea (28)
1
Yellow solid. Yield = 27%. Mp 108ꢀ109 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
13.33 (s, 1H), 8.91 (s, 1H), 8.68 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.63 (d, J = 8.4 Hz,
H), 7.49 (d, J = 8.4 Hz, 2H), 7.20 (s, 1H), 7.12 (t, J = 8.0 Hz, 1H), 7.02 (d, J = 4.8
2
Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.59 (d, J = 8.0 Hz, 1H), 3.13 (brs, 4H), 2.50 (brs,
+
4
H), 2.25 (d, 6H); HRMS (ESI) m/z calcd for C H N O [M+H] 442.2350, found
2
5
28
7
442.2348.
N-(5-Methyl-2-(4-methylpiperazin-1-yl)phenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]
pyridin-4-yl)phenyl)urea (29)
1
White solid. Yield = 19%. Mp 247ꢀ250 °C; HꢀNMR (400 MHz, DMSOꢀd
6
): δ
13.33 (s, 1H), 9.81 (s, 1H), 8.47 (d, J = 4.8 Hz, 1H), 8.08 (s, 1H), 7.89 (s, 1H), 7.68 (d,
J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 4.8 Hz,
1H), 6.79 (d, J = 8.0 Hz, 1H), 2.79 (t, J = 4.0 Hz, 4H), 2.64 (brs, 4H), 2.31 (s, 3H),
+
2
.25 (s, 6H); HRMS (ESI) m/z calcd for C26
H
30
N
7
O [M+H] 456.2506, found
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4
56.2504.
N-(3-Methyl-4-(4-methylpiperazin-1-yl)phenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]
pyridin-4-yl)phenyl)urea (30)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
White solid. Yield = 20%. Mp 236ꢀ 238 °C; HꢀNMR (400 MHz, DMSOꢀd ): δ
6
1
3.34 (s, 1H), 8.87 (s, 1H), 8.58 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.62 (d, J = 8.6 Hz,
H), 7.48 (d, J = 8.6 Hz, 2H), 7.27 (s, 1H), 7.24 (d, J = 8.4 Hz ,1H), 7.02 (d, J = 4.8
2
Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 2.79 (t, J = 4.4 Hz, 4H), 2.45 (brs, 4H), 2.24 (t, 9H);
+
HRMS (ESI) m/z calcd for C26
H
30
N
7
O [M+H] 456.2506, found 456.2506.
N-(3-Methyl-4-(morpholin-4-yl)phenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-
4
-yl)phenyl)urea (31)
White solid. Yield = 18%. Mp 253ꢀ255 °C; HꢀNMR (400 MHz, DMSOꢀd
3.32 (s, 1H), 8.85 (s, 1H), 8.57 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.62 (d, J = 8.4 Hz,
1
6
): δ
1
2H), 7.48 (d, J = 8.4 Hz, 2H), 7.28 (s, 1H), 7.26 (d, J = 8.4 Hz,1H), 7.03 (d, J = 4.8
Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 3.73 (t, J = 4.4 Hz, 4H), 2.79 (t, J = 4.4 Hz, 4H),
+
2.25 (s, 6H); HRMS (ESI) m/z calcd for C25
H
27
N
6
O
2
[M+H] 443.2190, found
4
43.2188.
N-(2-Methyl-4-(4-methylpiperazin-1-yl)phenyl)-N’-(4-(3-methyl-1H-pyrazolo[3,4-b]
pyridin-4-yl)phenyl)urea (32)
1
Pink solid. Yield = 24%. Mp 263ꢀ264 °C; HꢀNMR (400 MHz, DMSOꢀd ): δ
6
1
3.33 (s, 1H), 9.09 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.86 (s, 1H), 7.62 (d, J = 8.4 Hz,
1H), 7.48 (d, J = 8.4 Hz, 3H), 7.02 (d, J = 4.8 Hz, 1H), 6.81 (s, 1H), 6.75 (d, J = 8.8
Hz, 1H), 3.07 (t, J = 4.4 Hz, 4H), 2.45 (t, J=4.4Hz, 4H), 2.25 (s, 3H), 2.21 (s, 6H);
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