Rapid Discovery and Structure-Activity Relationships of Pyrazolopyrimidines That Potently Suppress Breast Cancer Cell Growth via SRC Kinase Inhibition with Exceptional Selectivity over ABL Kinase

Article abstract of DOI:10.1021/acs.jmedchem.6b00065

Novel pyrazolopyrimidines displaying high potency and selectivity toward SRC family kinases have been developed by combining ligand-based design and phenotypic screening in an iterative manner. Compounds were derived from the promiscuous kinase inhibitor PP1 to search for analogs that could potentially target a broad spectrum of kinases involved in cancer. Phenotypic screening against MCF7 mammary adenocarcinoma cells generated target-agnostic structure-activity relationships that biased subsequent designs toward breast cancer treatment rather than to a particular target. This strategy led to the discovery of two potent antiproliferative leads with phenotypically distinct anticancer mode of actions. Kinase profiling and further optimization resulted in eCF506, the first small molecule with subnanomolar IC₅₀ for SRC that requires 3 orders of magnitude greater concentration to inhibit ABL. eCF506 exhibits excellent water solubility, an optimal DMPK profile and oral bioavailability, halts SRC-associated neuromast migration in zebrafish embryos without inducing life-threatening heart defects, and inhibits SRC phosphorylation in tumor xenografts in mice.

Full text of DOI:10.1021/acs.jmedchem.6b00065

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Article
Rapid Discovery and Structure-Activity Relationships of Pyrazolopyrimidines
that Potently Suppress Breast Cancer Cell Growth via SRC
Kinase Inhibition with Exceptional Selectivity over ABL Kinase
Craig Fraser, John C. Dawson, Reece Dowling, Douglas R. Houston, Jason T. Weiss,
Alison F. Munro, Morwenna Muir, Lea Harrington, Scott P Webster, Margaret C Frame,
Valerie G. Brunton, E Elizabeth Patton, Neil O. Carragher, and Asier Unciti-Broceta
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00065 • Publication Date (Web): 26 Apr 2016
Downloaded from http://pubs.acs.org on April 27, 2016
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Rapid Discovery and StructureꢀActivity Relationships of Pyrazolopyrimidines
that Potently Suppress Breast Cancer Cell Growth via SRC Kinase Inhibition
with Exceptional Selectivity over ABL Kinase
9
Craig Fraser,† John C. Dawson,† Reece Dowling,†,‡ Douglas R. Houston,§ Jason T. Weiss,† Alison F.
Munro,† Morwenna Muir,† Lea Harrington,¥ Scott P. Webster,¶ Margaret C. Frame,† Valerie G. Brunton,† E.
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,
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Elizabeth Patton,† ‡ Neil O. Carragher† * and Asier UncitiꢀBroceta† *
† Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe
Road South, Edinburgh EH4 2XR, UK.
‡ MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South,
Edinburgh EH4 2XR, UK.
§ Institute of Structural and Molecular Biology, University of Edinburgh, Edinburgh EH9 3BF, UK.
¥ Faculty of Medicine, University of Montreal, Institute for Research in Immunology and Cancer, Chemin de
Polytechnique, Montreal, Quebec H3T 1J4, Canada
¶ University/BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, University of Edinburgh,
Edinburgh EH16 4TJ, UK
* Correspondence to: Neil.Carragher@igmm.ed.ac.uk; Asier.UncitiꢀBroceta@igmm.ed.ac.uk
ABSTRACT
Novel pyrazolopyrimidines displaying high potency and selectivity towards SRC family kinases
have been developed by combining ligandꢀbased design and phenotypic screening in an iterative
manner. Compounds were derived from the promiscuous kinase inhibitor PP1 to search for analogs
that could potentially target a broad spectrum of kinases involved in cancer. Phenotypic screening
against MCF7 mammary adenocarcinoma cells generated targetꢀagnostic structureꢀactivity
relationships that biased subsequent designs towards breast cancer treatment rather than to a
particular target. This strategy led to the discovery of two potent antiproliferative leads with
phenotypicallyꢀdistinct anticancer mode of actions. Kinase profiling and further optimization
resulted in eCF506, the first small molecule with subnanomolar IC50 for SRC that requires 3 orders
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of magnitude greater concentration to inhibit ABL. eCF506 exhibits excellent water solubility, an
optimal DMPK profile and oral bioavailability, halts SRCꢀassociated neuromast migration in
zebrafish embryos without inducing lifeꢀthreatening heart defects and inhibits SRC phosphorylation
in tumor xenografts in mice.
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Introduction
Modern drug discovery programs typically begin with a screening campaign (e.g. biochemical,
virtual or biophysical) for agonists, antagonists or inhibitors of a nominated target associated to a
particular disease.1ꢀ4 After hit identification, subsequent chemical optimization is fundamentally
based upon "onꢀtarget" potency.1 The generation of highꢀaffinity ligands (soꢀcalled lead
compounds) is followed by chemical refinement into derivatives of superior potency, selectivity and
desirable pharmacokinetic properties.1,5 Selected drug candidates are then validated in vivo and,
upon verification of safety and efficacy, progressed to human trials.5 While the merits of this wellꢀ
defined process are undeniable, including several major breakthroughs in anticancer therapy,6 this
strategy is also associated with declining productivity in the pharmaceutical industry and limited
success to tackle the most aggressive cancers of unmet therapeutic need.7,8 High attrition rates at
late stages of drug development underlines that cancer heterogeneity across patients and adaptive
drug resistance mechanisms are major obstacles for the development of effective and longꢀlasting
anticancer targeted therapies.9ꢀ12 These challenges have stimulated outꢀofꢀtheꢀbox thinking in
pharmacotherapy research (e.g. targeted polypharmacology,10 antibodyꢀdrug conjugates,13
innovative prodrug approaches,14ꢀ17 etc.) and the reexamination of the core principles of drug
discovery in complex diseases.18ꢀ20 The rise of modern phenotypic drug discovery18,19 together with
the use of more clinicallyꢀrelevant disease models to guide early drug development20 are
representative examples of the paradigm shift initiated in the field to trigger a positive inflection
point.
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Protein kinases are integral components of signal transduction cascades. They govern a wide
range of basic intracellular functions and coordinate cellꢀtoꢀcell and extracellular matrixꢀtoꢀcell
communication to modulate cell and tissue physiology. Consequently, their malfunctioning is
directly linked to progressive diseases including cancer and inflammation.21 The success in the
clinic of several anticancer kinase inhibitors has validated a number of kinases as oncotargets,22
while the increasing understanding of cancer cell biology has demonstrated the essential role of
different kinases in tumor suppressor pathways (antitargets).23ꢀ26
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The vast majority of kinase inhibitors bind to the kinase adenosine triphosphate (ATP)
pocket. Since all kinases (>500) necessarily possess this relatively wellꢀconserved catalytic site,
there is a great potential for crossꢀreactivity.10 In fact, even though most kinase inhibitors are
developed from single target hypotheses, they typically display broad selectivity profiles which, in
some cases, have resulted in unanticipated clinical applications (e.g. sorafenib).26 Inhibitor
promiscuity may also be advantageous for anticancer therapy when offꢀtarget activities assist to
address bioactivity issues related to pathway redundancies, molecular heterogeneity or resistance
mechanisms.9,10,26 However, if these activities result in the inhibition of antiꢀoncogenic pathways or
lead to severe side effects, drug promiscuity becomes a major drawback.27,28 Paradoxically, some
kinases may behave as a target or an antiꢀtarget depending on the cancer context. The expression of
the activated fusion oncoprotein BCRꢀABL is a genetic abnormality associated with chronic
myeloid leukemia (CML) and ABL inhibitors (imatinib, dasatinib) are clinically used in chronic
phase CML treatment.29 Also, ABL family kinases are abnormally activated in various solid tumors,
supporting their involvement in oncogenesis.29 However, ABL (ABL1) and ARG (ABL2) have
been found to negatively modulate breast cancer progression in vivo,30ꢀ32 indicating that ABL
inhibition could be counterproductive for breast cancer treatment (= antitarget). This example
serves to delineate the complexity of cancer etiology and highlights the necessity of developing
kinase inhibitors with tailorꢀmade pharmacodynamic profiles for the effective targeting of each
cancer subtype.33 Unfortunately, despite significant investments in the development of kinase
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inhibitors and related biomedical knowledge built over several decades, our still limited
understanding of cancer biology prevents us from anticipating and optimally targeting the complex
orchestrated actions that generate, maintain and progress most neoplastic processes.
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Acknowledging these limitations, many research groups including ours are frontloading the
collection of robust empirical data to progress anticancer drug development programs away from
classical blackꢀandꢀwhite anticancer target hypotheses to more unbiased and evidenceꢀled strategies
for hit selection and lead generation. Following that principle, in this manuscript we show that
cooperative ligandꢀbased design and phenotypic screening, complemented with biochemical assays
and the use of published data (literature, patents, etc.), can be effectively applied to accelerate the
generation of preclinical drug candidates. Our strategy builds on three wideꢀranging hypotheses: (i)
targeting the kinase ATP pocket with compounds derived from promiscuous kinase inhibitors can
enable “rationallyꢀbiased” serendipitous discoveries; (ii) early optimization of drugꢀlikeness can be
concurrently applied to explore pharmacodynamic diversity; and (iii) phenotypic screening of
chemically related compounds in designated models of cancer can generate targetꢀagnostic
structureꢀbioactivity relationships and tailor ligand optimization to particular cancer types/subtypes.
By means of this pragmatic approach to anticancer kinase inhibitor discovery, target deconvolution
of identified hits and leads is largely simplified, thereby assisting the mechanistic elucidation of the
molecular targets and antitargets involved in the observed phenotype.
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In this work we describe how the implementation of such an approach led to the discovery
of a kinase inhibitor with potent activity against breast cancer cells and a unique selectivity profile:
the first small molecule able to inhibit SRC at subnanomolar levels with a thousandꢀfold selectivity
over ABL.
Results and discussion
Design, synthesis and screening of compounds 7aꢀl. PP1 is a promiscuous inhibitor that
indiscriminately targets protein tyrosine kinases, many of which are involved in oncogenesis such
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as the SRC family of kinases (SFK), RET, KIT and ABL.34ꢀ37 Moreover, related derivatives
developed thereafter38ꢀ40 have shown strong inhibition of a variety of kinases with relevance in
cancer including IGFꢀ1R, EGFR, BTK, VEGFR, PDGFR, PI3K and mTOR. According to the coꢀ
crystal structure of PP1 with HCK41 and RET42 kinases, this small molecule is an archetypical type
I kinase inhibitor, with its N5 and 4ꢀNH2 groups forming multiple Hꢀbonds with the hinge region of
the kinase ATP site (Fig 1). The C3 pꢀtolyl group is positioned towards a hydrophobic region wellꢀ
conserved across tyrosine kinases, thus being responsible for the partial selectivity of PP1 over
other kinase families. Although PP1’s potent inhibition of diseaseꢀassociated kinases make it a
valuable biological tool, its use is limited by low solubility in water and poor selectivity, major
limiting factors for the clinical translation of many drug candidates.
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Figure 1. Upper panel: ATP and PP1 (neutral forms). Lower
panel: general structure of the novel compounds and
retrosynthetic analysis.
We envisaged that the substitution of PP1’s tertꢀbutyl group at the N1 position with flexible
waterꢀsolubilizing groups could be used to improve drugꢀlike properties and, at the same time,
explore the accessible sugar/phosphate regions occupied by the natural ligand ATP in the search for
novel binding affinity profiles. As shown in Fig 1, compounds were designed to display a cyclic
tertiary amine connected to the N1 position of the pyrazolopyrimidine ring through an ethylene
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linker. Following the route described in Scheme 1 (see full details in the Supp. Inf.), a highlyꢀ
focused 12ꢀmember library was generated by coupling a selection of cyclic secondary amines to an
aldehydeꢀmodified derivative43 of PP1 (6) via reductive amination.
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Scheme 1. Fiveꢀstep synthesis of compounds 7a-l from commercially available 5ꢀaminoꢀ1Hꢀpyrazoleꢀ4ꢀ
carbonitrile 1. Compound 6 was employed as the common intermediate to generate compounds 7a-l by
reductive amination using 12 commerciallyꢀavailable cyclic secondary amines (piperidines, morpholine and
piperazines).
The 12ꢀmember library was then tested against human mammary adenocarcinoma MCF7
cells, using the inhibition of cell growth as the primary output of the screening. Liveꢀcell highꢀ
content imageꢀbased phenotypic assays were carried out to kinetically profile the antiproliferative
response of MCF7 breast cancer cells following treatment with a single dose (100 ꢁM) of
compounds 7aꢀl for 5 d. Experiments were performed in triplicate, using cells treated with PP1 (100
ꢁM) or DMSO as a positive and negative control, respectively. Cell proliferation was monitored by
timeꢀlapse imaging using an IncuCyteTM ZOOM microscope and analyzed by its software, enabling
determination of cell density (% confluence) over time after compound addition. As shown in Fig
2a, compounds 7d, 7i, 7j, 7k and 7l (colored lines) led to a strong inhibition of cell proliferation,
maintaining confluence levels equal or below those imaged at time zero (point of compound
addition). Interestingly, all these hits presented at least two tertiary amines in their N1 motifs,
providing the first insights into structure/antiproliferative activity relationships.
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Figure 2. (a) Timeꢀlapse imaging analysis of breast cancer MCF7 cell proliferation over 5 d. Cells were
imaged every 3 h using an IncuCyteTM ZOOM microscope and growth measured by its integrated software.
Curves represent % confluence of cells treated with compounds 7a-l (100 ꢁM, 0.1 % DMSO) over 5 d of
incubation. DMSO (0.1 %, v/v) was used as untreated cell control (in black). Error bars: ± SD from n = 3. (d)
Dose response curves and calculated EC50 values determined by PrestoBlue® cell viability assay after
incubation of MCF7 cells with compounds 7d, 7i, 7j, 7k, 7l and the positive control PP1 (dose range: 0.03ꢀ
100 ꢁM). Error bars: ± SD from n = 3.
Half maximal effective concentration (EC50) values were then calculated for the five hits
and PP1 in MCF7 cells using an 8ꢀpoint halfꢀlog dose response study (0.03 ꢁM to 100 ꢁM). Based
on the MCF7 growth kinetics data provided by the IncuCyte assay, cell viability was determined at
day 5 using the PrestoBlue® reagent. Fig 2b shows that derivatives 7j and 7k were the most potent
among the novel compounds, with EC50 values of 12 ꢁM. Due to its potency and lower molecular
weight, the dimethylaminoꢀcontaining piperidinyl group of compound 7j was chosen as the N1
motif for the preparation of a second library of pyrazolopyrimidines.
Design, synthesis and screening of compounds 8aꢀf and 9aꢀf. Several investigations have
reported that substitutions of the pꢀtolyl group at the C3 position of PP1 by different aryl moieties
(even closely related ones) have a major impact on proteinꢀligand binding. Medicinal chemistry at
that position has generated inhibitors for a variety of kinases, including receptor and nonꢀreceptor
tyrosine kinases (e.g. SRC, ABL, RET, EGFR, BTK, PDGFRs, VEGFRs, KIT)34ꢀ38,40 and nonꢀ
tyrosine kinases (e.g. PI3Ks, mTOR).39,44 To exploit this feature as an opportunity to expand the
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prospective pharmacodynamic scope of 7j, a selection of 5 arylboronic acids and phenylacetylene
were employed to functionalize the C3 position of the heterocyclic ring by palladiumꢀcatalyzed
crossꢀcoupling chemistry to synthesize derivatives 9aꢀf (Scheme 2). Together with the piperidineꢀ
containing compounds 9aꢀf, the corresponding acetal intermediates 8aꢀf were selected for testing
against breast cancer cells to increase the chemical diversity of the screening.
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Scheme 2. Second library of pyrazolopyrimidines:
compounds 8a-f and 9a-f.
Antiproliferative properties of compounds 8aꢀf and 9aꢀf were evaluated in MCF7 cells (dose
response studies), using compound 5 (acetal intermediate of library 1), 7j (most potent derivative of
library 1), and PP1 as controls. As shown in Fig 3, compounds 8d and 9d (both having a 7ꢀ
azaindolꢀ5ꢀyl group at C3) exhibited superior antiproliferative properties (EC50 < 2 ꢁM).
Remarkably, timeꢀlapse imaging of compounds 8d and 9d (1ꢀ100 ꢁM) revealed patentlyꢀdistinct
antiproliferative mode of actions (Fig 3b and Movies S1 and S2). Compound 9d —containing the
piperidinyl moiety of 7j— induced cell death, while its acetal precursor 8d halted cell division.
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Figure 3. (a) EC50 values calculated after treatment of MCF7 cells with acetalꢀfunctionalized compounds 5
and 8a-f (top panel) and piperidineꢀfunctionalized derivatives 7j and 9a-f (bottom panel). Dose range: 0.03ꢀ
300 ꢁM. DMSO and PP1 were used as negative and positive controls, respectively. Cell viability was
determined using the PrestoBlue® reagent. (b) IncuCyteTM ZOOM images of MCF7 cells treated with 8d, 9d
and DMSO at 0 h, 48 h and 120 h after compound addition. Inhibitor dose: 100 ꢁM.
To identify the target/s that could be responsible of the phenotype induced by hits 8d and 9d
in MCF7 cells and thereby shed some light over further optimization campaigns, inhibition
activities were tested for PP1, 7iꢀk, 8d and 9d against a selection of kinases involved in human
cancer. Proteins were chosen in accordance with the profile of related pyrazolopyrimidines.34ꢀ39
Kinase profiling (Reaction Biology Corp., USA) was performed by measuring 33P incorporation on
the substrate (poly [Glu, Tyr] 4:1) relative to DMSO. Doseꢀresponse curves and calculated half
maximal inhibitory concentration (IC50) values are shown in Fig S1 and Table 1, respectively.
Derivatives 7iꢀk and 9d strongly inhibited SFK members, whereas acetalꢀfunctionalized compound
8d preferentially inhibited mTOR (> 28ꢀfold more potent than PP1). The most potent inhibitor was
compound 9d, which displayed similar potency to PP1 against SRC but higher selectivity over
RET, PDGFRα and KIT. Interestingly, according to the inhibition profile of compounds 7iꢀk (only
varying on the spacer between the dimethylamino group and the piperidine), potency and selectivity
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for SRC was enhanced by the proximity of the dimethylamino to the ring (IC50(ABL)/IC50(SRC) for
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7i = 50).
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Using the information provided by the phenotypic screening and the kinase inhibition
profiling of leads 8d and 9d, two independent optimization campaigns were implemented towards
the generation of anticancer drug candidates inhibiting mTOR and SFK, respectively. Optimization
of lead 8d into the highlyꢀselective mTOR inhibitor eCF309 has been reported elsewhere.45 Herein
is described the generation and structureꢀactivity relationships (SAR) of pyrazolopyrimidine
derivatives with high potency and selectivity for SFK.
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Table 1. IC50 values (in nM) calculated for PP1, 7iꢀk, 8d, 9d, 11a and dasatinib in a selection of
recombinant kinases.
Kinase \ Hit
ABL
PP1
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7i
6323 7525 7249 1207 116
311 913 1964 1226 38
7j
7k
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11a
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Dasatinib
<0.5
FYNa
<0.5
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9,318 >104 >104 >104 8565 >104 >104
>104 >104 >104 >104 >104 >104 9.9
6584 >104 >104 598 >104 433
289
1040 2453 27
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PDGFRα
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657
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SRCa
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303
<0.5
<0.5
YESa
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344
696
566
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<0.5
<0.5
IC50(ABL)
/IC50(SRC)
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0.5
4
>950
1
aMember of SFK.
Design, synthesis and phenotypic screening of compounds 11a,b. The data collected from the
kinase profiling study indicated that the 2ꢀ[4ꢀ(dimethylamino)ꢀ1ꢀpiperidyl]ethyl group at the N1
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position was optimal to generate SFK inhibitors of high selectivity, while the superior potency of
9d against SRC suggested that onꢀtarget potency could be further enhanced by optimization of the
substituent at the C3 position. A literature survey on SRC inhibitors was thus performed to assist in
the design of derivatives with improved SRC binding. We focused our attention on the C3
substituent of PP20, a potent dual SRC/ABL inhibitor developed by Apsel et al. (Fig 4a).36 Novel
derivatives were designed by substituting the 7ꢀazaindolꢀ5ꢀyl group at C3 of 9d with the
functionalized phenyl ring found in PP20, whereas the piperidines used for 7i and 7j were
introduced at the N1 position. Acetal deprotection of compound 4 in TFA/water (1:1) followed by
reductive amination with either 4ꢀ(dimethylamino) or 4ꢀ(dimethylaminomethyl)piperidine
generated iodo intermediates 10a,b, respectively, which afforded derivatives 11a (also known as
eCF506) and 11b (Fig 4a) by Suzuki crossꢀcoupling with 4ꢀ(NꢀBocꢀamino)ꢀ3ꢀ
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methoxyphenylboronic acid (see synthesis in the Supp. Inf.).
Antiproliferative properties of compounds 11a,b were then tested in cells using PP20 and
dasatinib (a clinicallyꢀapproved dual SRC/ABL inhibitor)22 as positive controls. Along with MCF7
cells, breast adenocarcinoma MDAꢀMBꢀ231 cells were tested as a model of triple negative breast
cancer known to be particularly sensitive to SRC inhibitors.46 As shown in Fig 4b, compound 11a
induced a very potent antiproliferative effect in both MCF7 and MDAꢀMBꢀ231 cells, significantly
superior to the activity displayed by derivatives 9d, 11b and PP20. Notably, 11a also outperformed
the goldꢀstandard SRC inhibitor dasatinib.
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Figure 4. (a) Structures of PP20 and compounds 11a and 11b. (b) EC50 values calculated after treating
MCF7 and MDAꢀMBꢀ231 cells with compounds 11a, 11b, PP20 and dasatinib (dose range: 1ꢀ10,000 nM) for
5 d. Cell viability was determined using the PrestoBlue® reagent. Error bars: ± SD from n = 3. (c) Western
blot analysis of SRC and FAK activity in MDAꢀMBꢀ231 cells treated with 11a and dasatinib for 1.5 h. (d)
Scratchꢀwound migration assay. MDAꢀMBꢀ231 cells were treated with 11a or dasatinib (10 nM) and cell
migration compared with untreated cell control (DMSO, 0.1 %, v/v) at 6, 12 and 24 h. Cells were imaged and
analyzed using an IncuCyteꢀZOOMTM microscope with integrated scratchꢀwound migration software module.
Error bars: ± SD from n = 3; p<0.001, ***; p<0.01, **. (e) Snapshots of the scratch wound area of MDAꢀMBꢀ
231 cells at 0 and 24 h. Yellow lines highlight the gap formed by the scratch. Blue lines indicate the advance
of cells into the wound after 24 h incubation in the absence and presence of 11a (10 nM).
Compound 11a was then selected for further screening. Inhibition of SRC kinase activity in
cells was studied by western blot. Upon activation, SRC undergoes autophosphorylation of the
Y416 residue within its kinase domain.47 Study of phosphoꢀSRCY416 levels can then serve both as
evidence for the presence of active SRC and to evaluate direct inhibition of SRC kinase activity.
Phosphorylation of the Focal Adhesion Kinase (FAK), a downstream substrate of SRC with high
relevance in cancer progression,48 was also studied in both MDAꢀMBꢀ231 (Fig 4c) and MCF7 (Fig
S2) cells. Dasatinib was used as a positive control. Cell lysates were prepared following 24 h of
serum starvation and 1.5 h exposure to each inhibitor and serum stimulation for 1 h. Analyses
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demonstrated that 11a inhibits phosphorylation of SRC and FAK at low nanomolar levels, with
complete inhibition observed at 100 nM. Dasatinib activity was close to that of 11a, although it also
induced a doseꢀdependent increment of total SRC (Fig 4c).
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Having demonstrated that 11a targets SRC in cells, we investigated FAK activity and cell
viability in cells that lack expression of SRC. SYF murine embryonic fibroblasts (SRCꢀ/ꢀ, YESꢀ/ꢀ,
FYNꢀ/ꢀ) were treated with either 11a or dasatinib and levels of total / phosphoꢀFAK analyzed by
protein immunoblot. As expected (Fig S3), FAK activity was found to be unaffected by drug
treatments, indicating that the inhibition of phosphoꢀFAKY861 in the breast cancer cell lines was a
consequence of SRC inhibition. Compounds 11a and 11b displayed significantly less
antiproliferative activity in SYF cells than dasatinib suggesting increased selectivity against SRC
family kinases (Fig S3).
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An automated 96ꢀwell scratchꢀwound cell migration assay (analyzed in realꢀtime by the
IncuCyteTM ZOOM system) was then set up to determine whether compound 11a could halt
migration of MDAꢀMBꢀ231 cells, as would be expected for a SRC inhibitor.49 Cells treated with
11a were monitored for 24 h at a dose range (1 to 10,000 nM) and compared with untreated cells
(0.1 % v/v DMSO) and dasatinib treatment. Compound 11a significantly reduced cell motility at 10
nM as early as 6 h into the study, with equivalent efficacy to dasatinib (Fig 4d,e). 11a treatments at
higher concentrations induced greater inhibition of cell migration (Fig S4), although cell viability
was also significantly reduced. Imageꢀbased measurement of caspase 3/7 activity in MCF7 breast
cancer cells demonstrated significant levels of apoptotic cell death after 5 d treatment with 11a at
concentrations ≥100 nM (Fig S4), in accordance with the EC50 values observed for this cell line.
Kinome profiling of SRC inhibitor 11a. IC50 values were determined for 11a and dasatinib
against the panel of recombinant kinases used in previous screenings (see IC50 values in Table 1 and
dose response curves in Fig S5). As expected, dasatinib induced potent inhibition of nonꢀreceptor
tyrosine kinases, such as ABL and SRC,22 but also high potency against receptor tyrosine kinases
(e.g. PDGFRα, KIT). In contrast, 11a exclusively inhibited SFK, with subnanomolar IC50 values
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against SRC and YES (activities for the rest SFK members are shown in Table S1). It is important
to highlight that 11a displayed a vast difference in activity (>950ꢀfold difference) between ABL and
its primary target SRC. While small molecule inhibitors with reverse binding capabilities have been
developed (e.g. imatinib strongly target ABL without inhibiting SRC),49 this is —to the best of our
knowledge— the first case of a small molecule with subnanomolar IC50 for SRC that requires a
concentration 3 orders of magnitude greater to reach the same level of inhibition in ABL. Such
properties have only been previously reported in peptideꢀbased bisubstrate inhibitors of SRC.50
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Design and synthesis of compounds 12aꢀy and SAR analysis. To shed light over the structural
features responsible for the high antiproliferative properties and unique selectivity profile of 11a, a
library of 24 closelyꢀrelated analogs was developed (see syntheses in the Supp. Inf.) by introducing
small changes on key functional groups of 11a (see Figure 5). The activity of compounds 12aꢀy was
then tested in cells. MDAꢀMBꢀ231 was the cell line chosen for the screening due to its superior
sensitivity to 11a treatment. Cell viability assays were performed as previously described and EC50
values calculated for each compound. IC50 values were subsequently determined against SRC and
ABL for those compounds exhibiting high antiproliferative activity (12d, 12e, 12m, 12x and 12y)
and for selected compounds with low activity (12a, 12i, 12j, 12t and 12w).
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O
Removal / substitution / translocation of the carbamate group
O
HN
O
Removal / substitution / translocation of the methoxy group
NH2
N
Removal of the pyrimidine ring
N
N
N
6
N
7
8
Removal / substitution / translocation of the
diaminomethyl group
N
11a
9
EC50 (MDAꢀMBꢀ231) = 9 nM
IC50 (SRC) < 0.5 nM
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IC50 (ABL) = 479 nM
O
O
O
O
O
O
O
O
O
O
N
O
HN
HN
HN
O
HN
HN
H2
N
HN
O
O
O
O
O
O
O
O
NH2
N
NH2
N
NH2
N
NH2
N
NH2
N
NH2
N
NH2
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
N
N
N
N
N
12a
12b
12c
12d
12e
12g
12h
12f
EC50 (MDAꢀMBꢀ231) = 2,760 nM
IC50 (SRC) = 21 nM
EC50 (MDAꢀMBꢀ231) = 1,990 nM EC50 (MDAꢀMBꢀ231) = 1,960 nM EC50 (MDAꢀMBꢀ231) = 39 nM
EC50 (MDAꢀMBꢀ231) = 81 nM
IC50 (SRC) = 0.6 nM
EC50 (MDAꢀMBꢀ231) = 32,600 nM
EC50 (MDAꢀMBꢀ231)= 6,720 nM EC50 (MDAꢀMBꢀ231) >100,000 nM
IC50 (SRC) < 0.5 nM
IC50 (ABL) = 407 nM
IC50 (ABL) = 4,690 nM
IC50 (ABL) = 808 nM
O
N
O
O
O
O
N
N
N
O
N
HN
HN
HN
HN
HN
HN
H
O
O
O
O
O
O
O
NH2
NH2
NH2
NH2
N
NH2
N
NH2
N
NH2
N
NH2
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
12o
N
N
N
N
N
12i
12j
12k
12l
12p
12m
12n
EC50 (MDAꢀMBꢀ231)= 15,230 nM EC50 (MDAꢀMBꢀ231)= 13,590 nM EC50 (MDAꢀMBꢀ231)= 12,180 nM EC50 (MDAꢀMBꢀ231) >100,000 nM
EC50 (MDAꢀMBꢀ231) = 4,150 nM
EC50 (MDAꢀMBꢀ231) = 167 nM EC50 (MDAꢀMBꢀ231) = 40,200 nM EC50 (MDAꢀMBꢀ231) = 4,140 nM
IC50 (SRC) = 194 nM
IC50 (ABL) = 9,440 nM
IC50 (SRC) = 114 nM
IC50 (SRC) = 3.5 nM
IC50 (ABL) > 10,000 nM
IC50 (ABL) = 10,000 nM
O
O
O
O
O
O
O
O
O
O
OH
O
O
H
HN
HN
HN
H
N
N
O
HO
O
N
O
NH2
N
NH2
N
NH2
N
NH2
N
NH2
N
NH2
N
NH2
N
NH2
O
O
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
12q
12r
12s
12t
12u
12v
12w
12x
EC50 (MDAꢀMBꢀ231) = 26,770 nM EC50 (MDAꢀMBꢀ231) = 28,590 nM EC50 (MDAꢀMBꢀ231) = 9,710 nM EC50 (MDAꢀMBꢀ231) = 3,570 nM EC50 (MDAꢀMBꢀ231) = 17,740 nM EC50 (MDAꢀMBꢀ231) > 100,000 nM EC50 (MDAꢀMBꢀ231) = 73 nM
EC50 (MDAꢀMBꢀ231) = 31 nM
IC50 (SRC) = 1 nM
IC50 (SRC) = 71 nM
IC50 (SRC) > 10,000 nM
IC50 (ABL) > 10,000 nM
IC50 (SRC) = 1 nM
IC50 (ABL) > 10,000 nM
IC50 (ABL) = 2,950 nM
IC50 (ABL) = 370 nM
Figure 5. Compounds 12a-x and their biological activities. Structural motifs modified on 11a for the
generation of compounds 12a-x are highlighted as colored circles. EC50 values against MDAꢀMBꢀ231 breast
cancer cells are shown below each structure. SRC and ABL inhibition values (as IC50) are reported for
selected compounds.
Analysis of the properties of compounds 12aꢀe (Figure 5) indicates that the presence of a
tertiary amino group at the position 4 of the pyperidinyl ring is essential for the activity.
Substitution of the methyl groups by larger aliphatic rings was tolerated, however, with compounds
12d and 12e being among the most potent SRC inhibitors of the series. Removal of the pyrimidine
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ring (see compound 12f) resulted in >3,000ꢀfold decrease in antiproliferative activity. Screening of
compounds 12gꢀx evidenced the limited structural variations permitted at the top of the molecule to
achieve high bioactivity. Most modifications of the Boc and the methoxy groups of the phenyl
moiety at the C3 position led to a significant reduction in bioactivity. Even minor chemical
modifications such as the substitution of the carbamate group by urea (12i) or amide (12j), or the
change of the methoxy by OH (12s), resulted in >200ꢀfold decrease in both antiproliferative
properties and SRC inhibitory activity. Remarkably, introduction of a benzylamino group instead of
the Boc was tolerated well, as observed from the bioactivity exhibited by compound 12n. However,
introduction of endocyclic nitrogen atoms in the ring (see 12n and 12o) dramatically reduced
compounds’ activity, particularly in the ortho position. Interestingly, while the methoxy group is
required for maintaining high levels activity when the Boc group is present in the structure (see 12sꢀ
u), substitution of the Boc group by an ester tolerated the elimination of the methoxy group (see
12w and 12x).
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Biochemical and in silico studies of 11a binding to the SRC kinase. Most kinase inhibitors ꢀ
especially adenine analoguesꢀ bind to the catalytic domain of the enzyme in its active
conformation,22 thus competing with the natural substrate ATP. To determine whether this was the
mode of binding of 11a to its primary target SRC, competitive inhibition studies with ATP were
performed (Reaction Biology Corp., USA). Experiments were designed to obtain Km values for
ATP in the absence and presence of different concentrations of inhibitor 11a. The reactions were
monitored every 10 min to obtain progress curves with time course (Fig S6). These were found to
be linear regardless of the compound’s concentration, suggesting that SRC inhibition by 11a is not
timeꢀdependent (= reversible). The slopes (ꢁM/min) were represented against ATP concentration
for a MichaelisꢀMenten plot, and a subsequent LineweaverꢀBurk plot (doubleꢀreciprocal plot), using
GraphPad Prism software (Fig S6). Apparent Km increased when inhibitor concentration increased
and all lines converged on the Yꢀaxis in the doubleꢀreciprocal plot, thus suggesting that 11a is
competitive with respect to ATP against SRC. Dose response curves of SRC inhibition by 11a at
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different concentrations of ATP are plotted in Fig S7. Calculated IC50 values increased in direct
proportion to ATP concentration, further confirming that 11a is a type I kinase inhibitor and
therefore binds to the SRC ATP site in its active conformation. Global fit analysis using GraFit
software provided a Ki of 0.8 nM (Fig S7).
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To gain insight on the selective binding affinity of 11a for SRC over ABL, in silico docking
studies were performed with both the dual ABL/SRC inhibitor PP20 and the SRCꢀselective
inhibitor 11a. Docking of PP20 into SRC and ABL produced a plausible predicted binding mode in
both cases (Fig 6a,b), with the adenineꢀmimicking pyrazolopyrimidine making Hꢀbonds to the hinge
region of the proteins. The tertꢀbutyl group of the carbamate occupies a hydrophobic pocket formed
by several lipophilic residues in SRC and ABL, including the Phe of their
aspartate−phenylalanine−glycine (DFG) motif. Free energy of binding was predicted by Autodock
to be approximately the same for both proteins (ꢀ8.5 and ꢀ8.9 kcal/mol, respectively). Docking of
11a into SRC also produced a plausible predicted binding mode (Fig 6c) and a higher predicted
affinity than PP20 (calculated free energy of binding of ꢀ10.6 kcal/mol). In SRC, the dimethylamino
group of the ethylpiperidinyl moiety on N1 interacts with Asp404, one of the protein's phosphate
binding residues (see Fig 6c,e).21,22 In contrast, the large N1 group of 11a (compared to the
isopropyl on PP20) clashes with Tyr253 in the catalytic site of ABL. This resulted in Autodock
being unable to find a position in the ATP binding site of ABL where 11a could mimic the
interactions made by the adenine moiety of ATP (Fig 6d). This study indicates that the flexible
polyamine linker on N1 is responsible for the low affinity of compound 11a for ABL.
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Figure 6. (a-d) Predicted binding modes of PP20 and 11a in SRC and ABL kinases. (a) PP20 in SRC, (b)
PP20 in ABL, (c) 11a in SRC and (d) 11a in ABL. The Pꢀloop, αC helix, and DFG motif are in red, orange,
and magenta, respectively. PP20 and 11a are represented by sticks, where carbon atoms are in black,
nitrogen atoms are in blue, and oxygen atoms are in red. (e) 11a in SRC active site compared to adenosine
monophosphate (AMP) (from PDB 3DQX). AMP is represented by sticks, where carbon atoms are in green,
nitrogen atoms are in blue, oxygen atoms are in red and the phosphorus atom in orange. Predicted binding
mode indicates that the dimethylamino group at the piperidine ring is optimally placed to interact with the
carboxylic group of Asp404 (DFG motif, in pink).
Phenotypic screening of SRC inhibitor 11a in zebrafish. Developing zebrafish provides a rapid
phenotypic assay to simultaneously test safety and efficacy of novel compounds in a living
vertebrate.51 Small molecule phenotypic based screens in zebrafish have recently implicated SRC
kinase in the migration of the posterior lateral line primordium,52 a cohesive cluster of cells that
migrates horizontally under the skin along the myoseptum to the end of the tail, periodically
depositing neuromasts. To determine the effects of 11a on cell migration in vivo, we treated
Tg(brn3c:mGFP) transgenic zebrafish53 that express green fluorescent protein (GFP) in the
mechanosensory hair cells of the lateral line (which form part of the neuromasts) with 11a and
dasatinib for 2 d and measured the distance of the last neuromast to the tip of the tail (marked by the
end of the notochord and the presence of black melanocytes, Fig 7a, in red). 11a significantly
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reduced neuroblast migration (> 100 microns in average) with minimal effect on the development
of the embryos (Fig 7aꢀc). In contrast, dasatinib treatment at >10 µM resulted in severe
cardiotoxicity and death of most embryos. At concentrations that were compatible with embryo
survival (1ꢀ10 µM), dasatinib did not inhibit the migration of neuroblasts whereas it did still induce
a patent cardiotoxic phenotype (note heart enlargement in Fig 7c). Further safety studies (see Fig
S8) showed that dual ABL/SRC inhibitor PP20 also induces severe cardiotoxicity in zebrafish even
after short treatment. These results, which correlates with the essential role of ABL in heart
development and healing,54,55 suggests that the selectivity of 11a over ABL might be advantageous
for therapy when ABL inhibition is not required.
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Figure 7. (a,b) Neuromast migration assay. Fresh E3 media with DMSO or 11a (500 µM) was added to
zebrafish embryos at 20 hpf, 36 hpf and 48 hpf, and imaged at 72 hpf. (a) Representative images of the tail
of a 3 dpf zebrafish without (top) and with 11a treatment (bottom). Neuromasts are identified by GFP
expression (green) and the tip of the tail as a red line. Yellow arrow indicates shortest distance from tail tip to
a neuromast. (b) Imaging analysis of the distance between the last neuromast and the tip of the tail (n=10)
under treatment with DMSO (negative control) or 11a (500 µM). P value calculated from tꢀtest. (c) Study of
zebrafish heart development under short treatment with 11a (500 µM) and dasatinib (10 µM). Compounds
were added to 2 dpf zebrafish embryos and incubated for 4 h (n=5). Subsequently, fresh media was added
and fish imaged after 48 h incubation.
Physicochemical and drug metabolism and pharmacokinetics (DMPK) profiling of SRC
inhibitor 11a in vitro and in vivo. Compound 11a displays very high solubility in water (> 100
mg/mL, pH= 7.4), a significant advantage over dasatinib, whose solubility in water ranges from
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moderate (18 mg/mL at pH= 2.6) to extremely low (<0.001 mg/mL at pH= 7).56 Notably, analysis
of cLogP with the OSIRIS Property Explorer57 provided an estimated value of 2.2 for 11a, while
experimental LogD at pH= 7.4 was determined to be –0.04.
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Inhibition of hERG channel and cytochrome P450 (CYP) enzymes was determined for 11a
to assess offꢀtarget liabilities associated with cardiotoxicity (QT interval prolongation) and potential
drugꢀdrug metabolic interactions. Results demonstrated weak hERG inhibition (50.1 % inhibition at
25 ꢁM) and marginal inhibition of CYP enzymes at 10 ꢁM (Fig S9).
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A plasma protein binding assay was performed with compound 11a to test to which extent
the compound can bind to proteins in the blood, an important factor to predict free drug levels in the
body. Warfarin (positive control, 99ꢀ100 % bound) and 11a were incubated with human or rat
plasma in duplicates at 37 °C for 24 h and the free (unbound) fraction of compound determined by
LCꢀMS/MS. Percentages of unbound 11a were 9.4 and 19.1 % in rat and human plasma,
respectively.
In vitro metabolic stability of 11a was systematically assessed in human liver microsomes
(HLM), blood plasma and primary hepatocytes (Cyprotex, UK). LCꢀMS/MS analysis of 11a
showed excellent compound stability (94 %) after 30 min incubation with HLM at 37 °C. 11a was
subsequently incubated (1 µM, 2 h, 37 °C) with plasma from either human, mouse or rat, and
analysed by LCꢀMS/MS at different time points. Regardless of the species, analyses showed full
plasma stability (Fig S10). Lastly, compound 11a (3 µM) was incubated with mouse and human
primary hepatocytes for 60 min and samples analysed by LCꢀMS/MS at various time points.
Verapamil and umbelliferone were used as control compounds. In agreement with the HLM study,
compound 11a showed high stability in human hepatocytes with a halfꢀlife of 751 min (Fig S10).
As expected, faster clearance was observed in mice hepatocytes (7ꢀfold decrease in halfꢀlife).
Encouraged by the in vitro DMPK profile of 11a, an in vivo study was performed in mice to
determine oral bioavailability and halfꢀlife (Cyprotex, UK). Female CD1 mice were given a single
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dose of 10 mg/kg (0.25 mg / 25 g mouse) of compound 11a orally (as a solution in nanopure water)
or via IV injection (as a saline solution). Blood/plasma samples were taken at different time points
(final sample taken after 8 h) and compound levels measured by LCꢀMS/MS (Fig S11). The
calculated halfꢀlife value for 11a was 2.9 h, substantially higher than that reported for dasatinib in
mice (0.9 h).58 Compound 11a showed moderate oral bioavailability (25.3 %), although it is
important to note that no excipients (just pure water) were used for its oral formulation.
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In vivo SRC inhibition study in a tumor xenograft model. The presence of active
(phosphorylated) SRC in human colorectal cancer HCT116 cells and its inhibition under 11a
treatment was verified by western blot (Fig S12). Subsequently, an in vivo PD study was performed
in a xenograft model of HCT116 cells in mice.59 HCT116 cells were injected subcutaneously and
tumors allowed to grow up to 3ꢀ4 mm in diameter. Subsequently, mice were dosed daily for 3 d
with 11a (50 mg/kg, in nanopure water) or vehicle (nanopure water) by oral gavage and culled 3 h
after the last dose (n=4). Tumors were excised, fixed and sections labelled for phosphoꢀSRCY416 and
stained with hematoxylin. As shown in Fig 8, microscopy analysis demonstrated significant
reduction of phosphoꢀSRCY416 in the xenograft sections from mice treated with 11a relative to the
untreated animal controls.
Figure 8. Immunohistochemical analysis of phosphoꢀSRCY416 in human tumor xenografts. (a) Images of
representative sections (low and high resolution) of HCT116 xenografts from: (left) untreated mice and
(right) mice treated with 11a (n=4). (b) Histoscore analysis (6ꢀ7 sections analysed per experiment).
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Quantification of immunohistochemistry across tumor tissue sections from untreated animals (water) and 11a
treated groups performed in blinded fashion. P value calculated from tꢀtest.
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Conclusions
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To move away from orthodox targetꢀcentric strategies, a pragmatic approach for rapid discovery of
novel highꢀquality anticancer kinase inhibitors was implemented through the generation and
phenotypic screening of analogues of PP1, a pyrazolopyrimidine known to inhibit multiple
oncogenic pathways.32ꢀ34 Derivatives of this promiscuous kinase inhibitor were designed to improve
physicochemical properties and explore favorable pharmacological features through small chemical
modifications. To accelerate the advance from hits to leads to drug candidates, compounds were
screened by phenotypic assays in the search for derivatives with potent antiproliferative properties.
Chemical design was biased towards human breast cancer treatment (rather than to a particular
target) by using the breast adenocarcinoma cell line MCF7 as a discriminating cell model. Such a
pseudo targetꢀagnostic strategy identified compounds that inhibited pathways involved in breast
cancer survival and also disregarded compounds with low cell penetrability (= deficient drugꢀ
likeness). After two rounds of design, synthesis and screening of highlyꢀfocused libraries, novel
candidates with low micromolar activity in MCF7 cells and high solubility in water were identified
(compounds 8d and 9d). Elucidation of the potential target/s linked with their bioactivity was
followed by a literature survey to explore chemical motifs that could further improve these
phenotypic leads. From that point, two independent medicinal chemistry campaigns were initiated.
This manuscript has focused on the development of SRC inhibitor 11a, a novel pyrazolopyrimidine
analogue that exhibits potent antiproliferative properties against breast cancer cells, halts MDAꢀ
MBꢀ231 cell migration and inhibits intracellular SRC signaling at low nanomolar concentrations.
Remarkably, kinase profiling revealed that 11a is the first small molecule that inhibits SRC at
subnanomolar concentration (IC50 < 0.5 nM) with a thousandꢀfold selectivity over ABL. This is of
relevance because the manifestation of cardiac events ꢀespecially in elderly patientsꢀ is a wellꢀ
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established adverse effect of ABL inhibition.51,52 Synthesis and phenotypic screening of 24 closelyꢀ
related derivatives of 11a was carried out to gain further insight into the optimal chemical space for
the generation of potent, selective and cellꢀactive SRC inhibitors. Analysis of SAR provided
evidence of the strict structural requirements of the pyrazolopyrimidine scaffold required to create
such a class of antiproliferative SRC/noꢀABL inhibitors. The relatively low molecular weight of
11a (510 Da) and its good physicochemical properties, including excellent water solubility and
cLogP of 2.2, prompted us to investigate further the biological properties of this compound.
Biochemical and in silico studies confirm that 11a is a Type I kinase inhibitor of SRC and provided
rationale for its selective affinity for SRC over ABL. Screening of hERG channel and CYP
induction, together with in vitro and in vivo PK / PD assays (including zebrafish PD / safety studies)
support the nomination of 11a as a SRCꢀtargeting drug candidate that could potentially offer a
superior therapeutic window than dual SRC/ABL inhibitors.60,61 This novel SRCꢀtargeting inhibitor
belongs to a second generation of SRC inhibitors that do not target the ABL kinase,50,62 being the
first one to demonstrate suppression of SRC phosphorylation in vivo. Finally, in concordance with
the primary role of SRC in driving tumor invasion and drug resistance mechanisms,63,64 more inꢀ
depth preclinical proofꢀofꢀconcept studies using appropriate in vivo models of disease progression
and rational drug combination studies will be required to demonstrate the efficacy and therapeutic
value of SRC/noꢀABL inhibitors in cancer treatment.
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EXPERIMENTAL SECTION
1. Synthetic Procedures. General Methods. Microwaveꢀirradiated reactions were carried out in a
Biotage Initiator microwave synthesizer. Nonꢀmicrowave reactions were performed under an inert
atmosphere of nitrogen using anhydrous solvents. All commercially available chemicals were
obtained from either Fisher Scientific, Matrix Scientific, SigmaꢀAldrich or VWR International Ltd.
NMR spectra were recorded at ambient temperature on a 500 MHz Bruker Avance III spectrometer.
Samples were dissolved in deuterated solvents commercially available from SigmaꢀAldrich.
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Chemical shifts are reported in parts per million (ppm) relative to the peak of the solvent. The data
is presented as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet, m = multiplet), coupling constants (in Hertz, Hz) and interpretation. TLCs were ran on
Merck TLC Silica gel 60 F254 plates, typically 5 cm x 10 cm, and monitored using a 254 nm UV
source or permanganate staining. Purifications were carried out using flash column chromatography
with commercially available silica gel and solvents. All compounds used in the biological
screenings were determined to be >95% pure by analytical HPLC with evaporative light scattering
detection (Agilent).
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2. Synthesis of compound 11a. 1H-pyrazolo[3,4-d]pyrimidin-4-amine (2). 5ꢀaminoꢀ1Hꢀpyrazoleꢀ
4ꢀcarbonitrile, 1 (3 g, 27.77 mmol), and formamide (15 mL) were added to a 20 mL microwave vial
and the mixture heated at 180 °C for 2 h using microwave radiation. The precipitate formed on
cooling was filtered off and washed with water (50 mL) and allowed to dry giving the product as a
pale brown solid (3.5 g, 25.9 mmol, 93 %). The experiment was repeated to give a second batch of
product (3.44 g, 25.5 mmol, 92 %). 1H NMR (500 MHz, DMSO) δ 13.34 (s, 1H), 8.13 (s, 1H), 8.07
(s, 1H), 7.69 (br. m, 2H); 13C NMR (126 MHz, DMSO) δ 158.2, 156.0, 155.0, 132.8 (CH), 99.8;
MS (ES +ve) (M+H)+: 136.0, 157.9 (+Na), (ES ꢀve) (MꢀH)ꢀ: 133.9.
3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3). Compound 2 (1.5 g, 11.11 mmol) was suspended
in 15 mL of DMF and Nꢀiodosuccinimide (1.2 eq., 3.0 g, 13.3 mmol) added. The mixture was
heated at 180 °C in the microwave for 40 min. EtOH (80 mL) was added to the reaction and a
precipitate began to form, which was aided by sonication. The precipitate was filtered and washed
with EtOH (x3, 20 mL) and allowed to dry in an oven at 40 °C overnight to give a sand colored
solid (2.115 g, 8.1 mmol, 73 %). 1H NMR (500 MHz, DMSO) δ 13.80 (s, 1H), 8.16 (s, 1H), 7.79 ꢀ
6.44 (m, 2H); 13C NMR (126 MHz, DMSO) δ 157.6, 156.1, 155.0, 102.5, 89.8; MS (ES +ve)
(M+H)+: 283.9 (+Na), (ES ꢀve) (MꢀH)ꢀ: 259.9, 287.8 (Na+).
1-(2,2-diethoxyethyl)-3-iodo-pyrazolo[3,4-d]pyrimidin-4-amine (4). To a solution of 3 (500 mg, 1.9
mmol) in DMF (15 mL) was added sodium hydride (1.5 eq., 2.9 mmol, 60 % dispersion in mineral
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oil, 115.2 mg) and the solution allowed to stir for 30 min until the gas evolution stopped.
Bromoacetaldehyde diethyl acetal (1.5 eq. 2.9 mmol, 0.435 mL) was then added dropwise and the
mixture heated at 150 °C in the microwave for 40 min. EtOAc and water (50 mL) were added to the
mixture and the organics separated. The aqueous layer was washed with EtOAc (50 mL, x3) and the
organics combined and washed with water (x3, 30 mL), dried over anhydrous MgSO4 and
concentrated in vacuo. The crude product was purified by column chromatography MeOH/DCM (0ꢀ
5 %) to give a light orange solid (461 mg, 1.2 mmol, 64 %). 1H NMR (500 MHz, DMSO) δ 8.21 (s,
1H), 7.90 ꢀ 6.30 (m, 2H), 4.93 (t, J = 5.7, 1H), 4.33 (d, J = 5.8, 2H), 3.62 (dq, J = 9.4, 6.9, 2H), 3.40
(dq, J = 9.6, 7.0, 2H), 0.98 (t, J = 7.0, 6H); 13C NMR (126 MHz, DMSO) δ 157.9, 156.3 (CH),
154.0, 103.2, 99.5, 89.5, 61.4 (CH2), 48.8 (CH2), 15.39 (CH3); MS (ES +ve) (M+H)+: 377.8, 400.0
(Na+), (ES ꢀve) (MꢀH)ꢀ: 376.0.
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1-(2,2-diethoxyethyl)-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrazolo[3,4-d]pyrimidin-4-amine (8d). To
a solution of 4 (1.135 g, 3.0 mmol) in dioxane/water (10 mL/1 mL) was added 7ꢀazaindoleꢀ5ꢀ
boronic acid pinacol ester (1.5 eq., 614 mg, 4.5 mmol), potassium carbonate (1.5 eq., 624.7 mg, 4.5
mmol) and followed by palladium acetate (5 mol %, 33.8 mg) and the mixture heated in the
microwave at 120 °C for 1 h. EtOAc and water (50 mL) were added to the mixture and the organic
layer separated, dried over anhydrous MgSO4 and concentrated in vacuo. The crude products were
purified by column chromatography. Purified by column chromatography, MeOH/DCM (0ꢀ6 %), to
give a white solid (93 mg, 0.253 mmol, 96 %). 1H NMR (500 MHz, CDCl3) δ 9.53 (s, 1H), 8.62 (d,
J = 1.9, 1H), 8.41 (s, 1H), 8.24 (d, J = 2.0, 1H), 7.45 (d, J = 3.4, 1H), 6.62 (d, J = 3.5, 1H), 6.29 –
5.86 (br. s, 2H), 5.14 (t, J = 5.7, 1H), 4.62 (d, J = 5.7, 2H), 3.79 (dq, J = 9.4, 7.0, 2H), 3.55 (dq, J =
9.4, 7.0, 2H), 1.14 (t, J = 7.0, 6H); 13C NMR (126 MHz, CDCl3) δ 156.8, 154.6, 153.4 (CH), 148.9,
143.8, 142.8 (CH), 128.8 (CH), 126.8 (CH), 121.3, 120.5, 101.8 (CH), 99.9 (CH), 98.5, 62.2 (CH2),
49.3 (CH2), 15.3 (CH3); MS (ES +ve) [M+H]+: 368.2, 390.2 (Na+), (ES –ve) [MꢀH]ꢀ: 366.2; HRMS
(ES +ve), C18H22N7O2 (M+H)+: calculated 368.18295, found 368.18090.
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Synthesis of 1-[2-[4-(dimethylaminomethyl)-1-piperidyl]ethyl]-3-(1H-pyrrolo[2,3-b]pyridin-5-
yl)pyrazolo[3,4-d]pyrimidin-4-amine (9d). 60 mg of 8d was added to a 20 mL microwave vial. 5
mL of water was added followed by 5 mL of TFA and the mixture heated at 100 °C for 1 h. The
mixture was concentrated in vacuo to give a light brown oil which was used without further
purification. The crude compound was dissolved in 2 mL of DCM. N,Nꢀdimethylꢀ1ꢀ(4ꢀ
piperidyl)methanamine (1.5 eq., 0.245 mmol, 34.8 mg) was added followed by a drop of acetic acid.
The mixture was allowed to stir for 10 min then sodium triacetoxyborohydride (1.5 eq., 0.245
mmol, 51.9 mg) was added and the mixture allowed to stir for 2 h. The mixture was concentrated in
vacuo and the product purified by column chromatography, MeOH/DCM (5ꢀ10% then 10% with 5ꢀ
20 drops of NH3 aq. / 100 mL) to give a light orange solid (15.3 mg, 0.0365 mmol, 15 %). 1H NMR
(500 MHz, MeOD) δ 8.52 (s, 1H), 8.29 (d, J = 2.0, 1H), 8.28 (s, 1H), 7.52 (d, J = 3.5, 1H), 6.62 (d,
J = 3.5, 1H), 4.64 (t, J = 6.4, 2H), 3.25 (d, J = 11.7, 2H), 3.13 (t, J = 6.3, 2H), 2.96 (d, J = 7.2, 2H),
2.84 (s, 6H), 2.37 (t, J = 11.4, 2H), 1.88 (m, 1H), 1.80 (d, J = 13.1, 2H), 1.36 – 1.31 (m, 2H); 13C
NMR (126 MHz, MeOD) δ 161.8, 158.6, 155.5 (CH), 154.3, 148.2, 143.7, 141.8 (CH), 128.7 (CH),
127.1 (CH), 120.7, 100.6 (CH), 98.2, 62.7 (CH2), 56.3 (CH2), 52.2 (CH2), 43.4 (CH2), 42.7 (CH3),
30.9 (CH), 28.4 (CH2); MS (ES +ve) [M+H]+: 420.2; HRMS (ES +ve), C22H29N9 [M+H]+:
calculated 420.25404, found 420.254249.
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1-[2-[4-(dimethylamino)-1-piperidyl]ethyl]-3-iodo-pyrazolo[3,4-d]pyrimidin-4-amine (10a). 150
mg (0.398 mmol) of 4 was added to a 10 mL microwave tube. 2.5 mL of water and 2.5 mL of TFA
were added and the mixture heated to 100 °C for 1 h. The mixture was concentrated in vacuo to
give a white solid which was used without further purification. The aldehyde intermediate was
suspended in 3 mL of DCM. 4ꢀ(N,Nꢀdimethylamino)piperidine (1.5 eq., 0.598 mmol, 76.6 mg) was
added followed by a drop of acetic acid and the mixture allowed to stir for 10 min. Sodium
triacetoxyborohydride (1.5 eq., 0.598 mmol, 126.8 mg) was added and the mixture allowed to stir
for 17 h overnight. The mixture was concentrated in vacuo and the product purified by column
chromatography, MeOH/DCM (0ꢀ10 % then 5ꢀ20 drops of NH3 aq. per 100 mL) to give a light
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orange/brown solid (163.8 mg, 0.405 mmol, 99 %). 1H NMR (500 MHz, MeOD) δ 8.22 (s, 1H),
4.49 (t, J = 6.4, 2H), 3.32 (s, 3H), 3.15 (d, J = 12.1, 2H), 2.96 (ddd, J = 16.0, 8.0, 4.0, 1H), 2.91 (t, J
= 6.4, 2H), 2.72 (s, 6H), 2.15 (td, J = 12.0, 2.0, 2H), 2.04 – 1.96 (m, 2H), 1.54 (qd, J = 12.2, 3.9,
2H); 13C NMR (126 MHz, MeOD) δ 158.07 (C), 155.67 (CH), 153.70 (C), 103.59 (C), 86.97 (C),
63.18 (CH), 55.86 (CH2), 51.38 (CH2), 44.37 (CH2), 39.31 (CH3), 26.42 (CH2); MS (ES +ve)
[M+H]+: 416.2.
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Tert-butyl N-[4-[4-amino-1-[2-[4-(dimethylamino)-1-piperidyl]ethyl]pyrazolo[3,4-d]pyrimidin-3-
yl]-2-methoxy-phenyl]carbamate (11a, a.k.a. eCF509). To a solution of 10a (50 mg, 0.1205 mmol)
in dioxane/water (4.5 mL/0.5 mL) was added [4ꢀ(tertꢀbutoxycarbonylamino)ꢀ3ꢀmethoxyꢀ
phenyl]boronic acid (1.5 eq., 48.3 mg, 0.181 mmol), potassium carbonate (1.5 eq., 25.0 mg, 0.181
mmol) and triphenylphosphine (20 mol %, 9.5 mg) followed by palladium acetate (5 mol %) and
the mixture heated in the microwave at 120 °C for 1 h. EtOAc (50 mL) and water (50 mL) were
added to the mixture and the organic layer separated. The aqueous layer was washed with EtOAc
(20 mL, x2) and the organics combined dried over anhydrous MgSO4 and concentrated in vacuo.
The crude product was purified by column chromatography, MeOH/DCM (0ꢀ10 % then 5ꢀ20 drops
of NH3 aq. per 100 mL) to give a light brown solid (23.1 mg, 0.0453 mmol, 38 %). 1H NMR (500
MHz, MeOD) δ 8.27 (s, 1H), 8.08 (d, J = 8.2, 1H), 7.30 (d, J = 1.8, 1H), 7.26 (dd, J = 8.2, 1.9, 1H),
4.56 (t, J = 6.7, 2H), 3.98 (s, 3H), 3.14 (d, J = 11.9, 2H), 2.94 (t, J = 6.7, 2H), 2.39 (m, 7H), 2.14
(dd, J = 12.0, 10.0, 2H), 1.90 (d, J = 12.5, 2H), 1.57 (s, 9H), 1.49 (qd, J = 12.1, 3.6, 2H); 13C NMR
(126 MHz, MeOD) δ 158.53 (C), 155.40 (CH), 154.12 (C), 153.46 (C), 149.24 (C), 145.02 (C),
128.78 (C), 127.38 (C), 120.43 (CH), 119.56 (CH), 110.28 (CH), 97.73 (C), 80.18 (C), 62.29 (CH),
56.22 (CH2), 55.07 (CH3), 52.20 (CH2), 44.00 (CH2), 40.06 (CH3), 27.24 (CH2), 27.21 (CH3); MS
(ES +ve) [M+H]+: 511.3; HRMS (ES +ve), C26H38N8O3 [M+H]+: calculated 511.31396, found
511.3151.
3. Biological Studies.
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3.1. General Methods. MCF7, MDAꢀMBꢀ231 and SYF cells were grown in Dulbecco’s Modified
Eagle medium (DMEM) supplemented with serum (10% fetal bovine serum) and Lꢀglutamine (2
mM) and incubated in a Heracell 240i tissue culture incubator at 37 °C and 5 % CO2.
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Time-lapse cell growth assay. Cells were plated in 96ꢀwell Nunc™ black opticalꢀbottom plates
(Thermo Scientific) at 1,000 cells/well in 100 µl of DMEM medium containing 10% FBS and 2
mM Lꢀglutamine and incubated for 48 h in an incubator at 37 °C and 5% CO2. The media was
replaced with fresh media containing 100 µM concentration of 7aꢀl, PP1 or DMSO (0.1% v/v) and
the plates imaged in the IncuCyteTM ZOOM system. Cell growth was monitored at sequential timeꢀ
points over 5 d using the brightfield microscopic images acquired by the IncuCyteTM ZOOM
system. Cell confluence determination was performed by the IncuCyte software.
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3.2. Dose response viability assay. Cells were plated in 96ꢀwell plates at 2,000 cells/well in 100 µl
of DMEM medium containing 10% FBS and 2 mM Lꢀglutamine and incubated for 48 h in an
incubator at 37 °C and 5% CO2. After 48 hours, the media was aspirated from each well and
replaced with 95 µl of fresh medium. Compounds, including DMSO, were prepared at 20x in
DMEM medium in a separate 96ꢀwell intermediate plate. 5 µl from the intermediate plate was then
added to each well containing cells. Untreated cells were incubated with DMSO (0.1% v/v). After 5
d, PrestoBlue™ cell viability reagent (10 µl) was added to each well and the plates incubated for 60
ꢀ 90 min. Fluorescence emission was detected using a Envision® fluorescence plate reader
(excitation 540 nm, emission 590 nm). All conditions were normalised to the untreated cells (100%)
and curves were fitted using a four parameter logistic fit with minimum value constrained to zero
using GraphPad Prism software, to calculate EC50 values.
3.3. Apoptosis assay. Cells were plated in 96ꢀwell Nunc™ black opticalꢀbottom plates (Thermo
Scientific) at 3,000 cells/well in 100 µl of DMEM medium containing 10% FBS and 2 mM Lꢀ
glutamine and incubated for 48h in an incubator at 37 oC and 5% CO2. The media was replaced
with 95 µl of fresh media containing NucView™ 488 from Biotium, at 1 µM concentration and
drugs or DMSO added along a concentration gradient, as described in the cell viability assay, and
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the plates imaged in the IncuCyte. Cell growth was monitored over 5 days using brightfield and
NucView™ 488 fluorescence (excitation 460 nm, emission 524 nm) microscopy. Cell confluence
and apoptotic count (positive NucView™ 488 emission signal threshold above background signal)
was performed by the IncuCyte software. The numbers generated from the applied confluence and
NucViewTM 488 labelled cells masks were divided so as to create a ratio of number of apoptotic
cells to cell confluence. The data were then normalised to DMSO to account for the decreased
number of cells found in higher concentrations of drug treatment.
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3.4. Western blotting. Cells were plated at 1x106 cells/well in 2 mL of DMEM medium containing
10% FBS and 2 mM Lꢀglutamine in 6ꢀwell plates and incubated at 37 °C with 5% CO2. After 24 h,
the media was aspirated and replaced with 2 mL of DMEM medium containing 0.1 % FBS and 2
mM Lꢀglutamine and the cells incubated for a further 24 h. 2 µl of compounds dissolved in DMSO
at appropriate concentration was then added to each well and plates incubated for 1.5 h. 222 µl of
FBS was then added to each well (giving a final concentration of 10%) and cells incubated for 1 h.
Cell lysates were then prepared using 100 µl of cell lysis buffer (1% Triton Xꢀ100, 50 mM HEPES,
pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium
pyrophosphate, 1mM Na3VO4, 10% glycerol and protease and phosphatase inhibitors) per well. The
total cell protein concentration in each lysate was determined using Precision Red Advanced
Protein Reagent #2 from Cytoskeleton. Lysates were normalised to 2 mg/mL and were boiled at 100
°C for 3 min in SDSꢀPAGE sample buffer (40% glycerol, 8% SDS, 0.1M DTT, 0.25 M TrisꢀHCl,
pH 6.8). Samples were resolved by SDSꢀPAGE using BioRad 4ꢀ15% precast gels over 60 min at
140 V and transferred to PVDF membranes over 150 min at 210 mA. Nonꢀspecific antibody
binding was blocked by incubation of membranes for one hour at room temperature using western
blocking reagent solution (Roche) prior to adding primary antibodies in 0.5 % blocking buffer at 4
°C overnight. Membranes were washed with TBS/T (x3, 5 min) then secondary antibody linked to
horseradish peroxidase (HRP) added for one hour at room temperature. Following further washing
with TBS/T (x3, 5 min) and TBS (x2, 5 min) HRP was detected by peroxidase enhanced
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