Discovery and pharmacological characterization of a novel series of highly selective inhibitors of cyclin-dependent kinases 4 and 6 as anticancer agents

Article abstract of DOI:10.1111/bph.13974

Background and Purpose: Cyclin D-dependent kinases 4 and 6 (CDK4/6) are crucial regulators of the G1 to S phase transition of the cell cycle and are actively pursued as therapeutic targets in cancer. We sought to discover a novel series of orally bioavailable and highly selective small molecule inhibitors of CDK4/6. Experimental Approach: The discovery of pharmacological inhibitors and optimization for potency, selectivity and drug properties were achieved by iterative chemical synthesis, biochemical screening against a panel of kinases, cell-based assays measuring cellular viability, cell cycle distribution, induction of apoptosis and the level of retinoblastoma tumour suppressor protein (Rb) phosphorylation and E2 factor (E2F)-regulated gene expression and in vitro biopharmaceutical and in vivo pharmacokinetic profiling. Key Results: We discovered several lead compounds that displayed >1000-fold selectivity for CDK4/6 over other members of the CDK family. The lead compounds, 82, 91 and 95, potently inhibited the growth of cancer cells by inducing G1 arrest with a concomitant reduction in the phosphorylation of Rb at S780 and in E2F-regulated gene expression. With a remarkable selectivity for CDK4 over 369 human protein kinases, 91 was identified as a highly potent and orally bioavailable drug candidate. Conclusions and Implications: We have identified unique and new inhibitors of CDK4/6 as potential drug candidates. Compound 91 represents an ideal candidate for further development as targeted cancer therapy.

Full text of DOI:10.1111/bph.13974

Discovery and Pharmacological Characterisation of a Novel Series of Highly Selective
Inhibitors of Cyclin-Dependent Kinases 4 and 6 as Anticancer Agents
Running Title
Highly Selective CDK4/6 Inhibitors as Anticancer Agents
Solomon Tadesse1, Laychiluh Bantie1, Khamis Tomusange1, Mingfeng Yu1, Saiful Islam1,
Nataliya Bykovska1, Benjamin Noll1, Ge Zhu1, Peng Li1, Frankie Lam1, Malika Kumarasiri1,
Robert Milne1 and Shudong Wang1
1Centre for Drug Discovery and Development, Sansom Institute for Health Research, School
of Pharmacy and Medical Sciences, and Centre for Cancer Biology, University of South
Australia, Adelaide, South Australia 5001, Australia.
*Correspondence: Professor Shudong Wang, Centre for Drug Discovery and Development,
Sansom Institute for Health Research, School of Pharmacy and Medical Sciences, and Centre
for Cancer Biology, University of South Australia, Adelaide, South Australia 5001, Australia,
shudong.wang@unisa.edu.au
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/bph.13974
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BACKGROUND AND PURPOSE
Cyclin D dependent kinases 4 and 6 (CDK4/6) are crucial regulators of the G1 to S phase
transition of the cell cycle, and are actively pursued as therapeutic targets in cancer targets.
We sought to discover a novel series of orally bioavailable, and highly selective small
molecule inhibitors of CDK4/6.
EXPERIMENTAL APPROACH
The discovery of pharmacological inhibitors and optimisation for potency, selectivity and
drug properties were achieved by iterative chemical synthesis, biochemical screening against
a panel of kinases, cell-based assays measuring cellular viability, cell cycle distribution,
induction of apoptosis, and the level of retinoblastoma tumour suppressor protein (Rb)
phosphorylation and E2 factor (E2F) regulated gene expression, and in vitro
biopharmaceutical and in vivo pharmacokinetic profiling.
KEY RESULTS
We identified several lead compounds that displayed > 1,000-fold selectivity for CDK4/6
over other members of the CDK family. The lead compounds i.e. 82, 91 and 95, potently
inhibited the growth of cancer cells by inducing G1 arrest with a concomitant reduction in the
phosphorylation of Rb at S780 and in E2F-regulated gene expression. With a remarkable
selectivity for CDK4 over 369 human protein kinases, 91 was identified as a highly potent
and orally bioavailable drug candidate.
CONCLUSION AND IMPLICATIONS
We have provided unique and new members of CDK4/6 inhibitor drug candidates.
Compound 91 represents an ideal nominee for further development as targeted cancer
therapy.
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Abbreviations:
CDK, cyclin dependent kinase
CYP450, cytochrome P450
E2F, E2 factor
FOXM1, forkhead box M1
GI50, 50% cell growth inhibitory concentration
hERG, ether-a-go-go related gene
HRP, horseradish peroxidase
MEP50, methylosome protein 50
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
PK, pharmacokinetics
pKa, dissociation constant
PRMT5, Arg N-methyltransferase 5
Rb, retinoblastoma tumour suppressor protein
RPMI, roswell park memorial institute
SMAD2/3, mothers against decapentaplegic homolog 2/3
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Introduction
Cell cycle dysregulation is frequently mediated by alterations in the activity of CDKs. These
perturbations cause abnormal cell proliferation resulting in cancer (Malumbres & Barbacid,
2009). For instance, control of the CDK4/6→Rb→E2F signalling pathway, which is involved
in regulating progress of the cell from the G1 to S phases of its cycle, is disrupted in virtually
all cancers through a multitude of genomic alterations (Hamilton & Infante, 2016). Thus,
components of the CDK4/6→Rb→E2F pathway represent molecular targets for cancer
therapy.
CDK4/6 phosphorylate serine/threonine residues of Rb. Once phosphorylated, Rb loses its
E2F inhibitory function, allowing the transcription of E2F-regulated genes, and G1 to S phase
progression (Hamilton & Infante, 2016). CDK4/6 also phosphorylate other transcription
factors to suppress senescence and stimulate proliferation (Anders et al., 2011). In contrast to
cancer cells, most normal cells can proliferate in the absence of CDK4/6 and/or D-type
cyclins (Kozar & Sicinski, 2005). Therefore, inhibition of CDK4/6 in cancer cells is expected
to trigger accumulation of cancer cells at G1, cause senescence and facilitate the induction of
apoptosis, while sparing normal cells (Figure 5A) (Choi et al., 2012).
The earlier generation of CDK inhibitors were non-specific. Thus, toxicities have hampered
their utilization (O'Leary, Finn & Turner, 2016). More recently, a new generation of specific
CDK4/6 inhibitors, PD0332991 (palbociclib) and LEE011 (ribociclib), have been FDA-
approved for the treatment of breast cancer. (abemaciclib) has also received a breakthrough
therapy designation (Chen et al., 2016). CDK4/6 inhibitor pre-treatment has also been shown
to protect normal cells from the cytotoxic effects of chemotherapy by reversibly halting their
proliferation (Bisi, Sorrentino, Roberts, Tavares & Strum, 2016). However, while these
agents are relatively selective for CDK4/6, they also inhibit other kinases (Chen et al., 2016;
Sumi, Kuenzi, Knezevic, Remsing Rix & Rix, 2015), and these molecules have unique
impacts on patient physiology, due to their different selectivity and pharmacological profiles.
This, along with the need for better research tools to address the biological and toxicological
issues, has resulted in an increasing demand for new inhibitors with novel chemical structures
and enhanced selectivity for CDK4/6.
Here in, we describe the discovery and characterisation of a novel series of potent inhibitors,
which exhibit excellent selectivity for CDK4/6 over a large panel of protein kinases. These
molecules possess admirable anti-proliferative activity on cancer cell lines, which is
consistent with CDK4/6-targeted mechanism of action. Lead inhibitor 91 boasts an optimal
balance of in vitro activities, safety and pharmacokinetic properties.
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Methods
Chemical synthesis and characterisation of compounds
The chemical synthesis of 4-(thiazol-5-yl)-2-(phenylamino)pyrimidine derivatives (1 and 3)
and 4-(thiazol-5-yl)-N-(pyridin-2-yl)pyrimidin-2-amines (2 and 4-29) shown in Table 1 is
delineated in Figures 1-3. The synthesis involved the parallel preparation of enaminones
(Figure 1) and guanidines (Figures 2), and their subsequent coupling under microwave
irradiation (Figure 3). The preparation of enaminones and guanidines was carried out
according to the methods described previously (Shao et al., 2013; Tadesse et al., 2017). To
prepare compounds 72-101 (Figure 3), an appropriate crude guanidine trifluoroacetate (2.0
eq.) and NaOH (2.0 eq.) were added to a solution of an appropriate enaminone (1.0 eq.) in 2-
methoxy ethanol. The reaction mixture was heated at 180 °C under microwave irradiation for
1 h, cooled to room temperature and concentrated under reduced pressure. The residue was
purified by FlashMaster Personal+ chromatography (silica gel, solvent system I: PE ramping
to 100% EtOAc, solvent system II: DCM ramping to DCM:MeOH = 9:1, or solvent system
III: DCM ramping to DCM:MeOH:NH4OH = 9:1:0.1) to give the desired product.
Compounds 72-101 were characterised by proton-1 and carbon-13 nuclear magnetic
resonance spectroscopy (1H- and 13C-NMR), melting points, high resolution mass
1
spectrometry (HRMS) and high performance liquid chromatography (HPLC). H NMR and
13C NMR spectra were obtained using a Bruker Avance III HD spectrometer (Faellanden,
Switzerland) at 500 and 125 MHz respectively, and were analysed using a Bruker Topspin
3.2 program. Chemical shifts are reported in parts per million (ppm), and are referenced to 1H
signals of residual non deuterated solvents and to 13C signals of the deuterated solvents.
Multiplicity of 1H NMR signals is reported as: s = singlet, d = doublet, t = triplet, q = quartet,
dd = doublet of doublets, m = multiplet, and br = broad. Coupling constants J (Hz) are quoted
to the nearest 0.1 Hz. Melting points were determined using an open capillary on a Stuart
SMP10 melting point apparatus and are uncorrected. High-resolution mass spectra were
recorded using an AB SCIEX TripleTOF 5600 mass spectrometer (Concord, ON, Canada),
and ionisation of all samples was carried out using ESI. The purity of compounds used for
biological evaluation was determined to be greater than 95% using Shimadzu Prominence
UFLC system (UltraFast Liquid Chromatograph, Kyoto, Japan) equipped with a CBM-20A
communications bus module, a DGU-20A5R degassing unit, an LC-20AD liquid
chromatograph pump, an SIL-20AHT auto-sampler, an SPD-M20A photo diode array
detector, a CTO-20A column oven and a Phenomenex Kinetex 5 µ C18 100A 250 mm × 4.60
mm column. Method A (gradient 5% to 95% MeOH containing 0.1% formic acid (FA) over 7
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min at a flow rate of 1 mLmin-1, followed by 95% MeOH containing 0.1% FA over 13 min)
and method B (gradient 5% to 95% MeCN containing 0.1% FA over 7 min at a flow rate of 1
mLmin-1, followed by 95% MeCN containing 0.1% FA over 13 min) were used for analytical
RP-HPLC. Data acquired were processed using LabSolutions Analysis Data System. The
details of the characterisation of each of the 30 final compounds are provided in the
Supporting Information.
Kinase assays
The in vitro kinase assay was performed by Reaction Biology Corporation (Malvern, PA,
USA, www.reactionbiology.com). In brief, specific kinase-substrate pairs together with the
required cofactors were prepared in freshly made base reaction buffer (20 mM HEPES, pH
7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij 35, 0.02 mgmL-1 BSA, 0.1 mM Na3VO4, 2 mM
DTT, 1% DMSO). Compounds in DMSO were delivered into the reaction mixture by
Acoustic technology (Echo550; nanoliter range). The reaction mixtures were incubated for 20
33
min at rt and P-ATP (specific activity 10 µCiµL-1) added to initiate the reaction. Reactions
were carried out at rt for 2 h, followed by spotting of the reactions onto P81 ion exchange
filter papers. Phosphoric acid (0.75%) was used to wash unbound phosphate from the filters.
Enzyme activity was determined through measuring the percentage of remaining kinase
activity in test samples compared to vehicle (DMSO) reactions after subtraction of
background derived from control reactions containing inactive enzyme. IC50 values and curve
fits were obtained using Prism (GraphPad Software, La Jolla, CA, USA). Ki values were
calculated using the Cheng-Prusoff equation: Ki = IC50/(1 + ([ATP]/Km(ATP))), where [ATP]
is Km (ATP) according to the Reaction Biology Corporation binning structure. A kinome
activity
map
was
prepared
using
Kinome
Mapper
(http://www.reactionbiology.com/apps/kinome/mapper/LaunchKinome.htm).
Cell culture
All cell lines were obtained from the cell bank at the Centre for Drug Discovery and
Development, University of South Australia. The cell lines were cultured in Roswell Park
Memorial Institute (RPMI)-1640 Medium, Dulbecco’s Modified Eagles Medium or
Minimum Essential Medium supplied with 10% foetal bovine serum (FBS) (Sigma-Aldrich,
Castle Hill, NSW, Australia) within a humidified 5% CO2, 37 °C incubator.
Cell viability assays
3-(4,5-Dimethylthiazol-2-thiazolyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium bromide (MTT) (Life Technologies, Mulgrave, VIC, Australia) and resazurin
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(Sigma Aldrich, Castle Hill, NSW, ) assays were performed as previously reported (Diab et
al., 2016). Compound concentrations required to inhibit 50% of cell growth (GI50) were
calculated using non-linear regression analysis.
Cell cycle analysis
Cell cycle analysis was performed as described previously (Diab et al., 2016). Cells were
seeded at 8 × 104 cells per well using six-well plates and incubated overnight at 37 °C, 5%
CO2. After adding each compound, cells were incubated for 24 h. Cells were transferred to
FACS tubes and centrifuged at 300g for 5 min. Cell pellets were collected and re-suspended
in 1 mL of phosphate-buffered saline (PBS) and centrifuged at 300g for 5 min. The
supernatant PBS was removed and cell pellets were fixed by adding 500 µL of ice-cold 70%
EtOH dropwise on ice for 15 min and collected again after being centrifuged at 300g for 5
min. The supernatant was removed and pellets were incubated with propidium iodide (PI) cell
cycle solution in PBS (50 μgmL-1 propidium iodide, 0.1 mgmL-1 RNase A, 0.05% Triton X-
100) at RT for 1.5 h and analysed using a Gallios flow cytometer (Beckman Coulter, Brea,
CA, USA). Data were analysed using Kaluza v1.2 (Beckman Coulter, Brea, CA, USA).
Detection of apoptosis
The apoptosis analysis was performed as described previously (Diab et al., 2016). Cells were
seeded at 8 × 104 cells per well using a six-well plate and incubated overnight at 37 °C, 5%
CO2. After adding each compound, the cells were incubated for 24 h. Cells were transferred
to FACS tubes and centrifuged at 300g for 5 min. Cell pellets were collected and re-
suspended in 1 mL of warm PBS and centrifuged at 300g for 5 min. The supernatant PBS
was removed, and cell pellets were diluted to 1 × 105 cellsmL-1 with warm PBS and
centrifuged at 300g for 5 min. The supernatant was removed, cell pellets were re-suspended
with 1 mL of ice-cold PBS and centrifuged at 300g for 5 min. The supernatant was removed,
and cell pellets were re-suspended with 100 µL of binding buffer, whereupon 3 µL of
annexin V and 3 µL of PI were added to each sample with slight vortexing and cells were
incubated in the dark for 15 min. Afterwards, 200 µL of binding buffer was added to each
sample and analysed by the Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Data
were analysed using Kaluza v1.2 software (Beckman Coulter, Brea, CA, USA).
Western blot analysis
Western blotting was performed as described previously (Diab et al., 2016). The following
antibodies (Cell Signaling Technology, Danvers, MA, USA) were used for protein detection:
total Rb (Cat: 9309, Rb (4H1) Mouse mAb, Lot: 7), phosphorylated Rb S780 (Cat: 3590,
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Phospho-Rb (S780) (C84F6) Rabbit mAb, Lot:5) and β-actin (Cat:4 970, β-Actin (13E5)
Rabbit mAb, Lot: 14). Both anti-mouse and anti-rabbit immunoglobulin G (IgG) horseradish
peroxidase (HRP)-conjugated antibodies were obtained from Dako, Kingsgrove, NSW,
Australia.
RNA isolation and reverse transcription
Total RNA was extracted from 1 × 106 MDA-MB-453 cells in two independent repeats using
a High Pure RNA Isolation Kit (Roche Applied Science, Castle Hill, NSW, Australia). Total
RNA (1 μg) was reverse transcribed to synthesise the first-strand cDNA using a mixture of
anchored-oligo(dT)18 and random hexamer primer and a Transcriptor First Strand cDNA
Synthesis Kit (Roche Applied Science, Castle Hill, NSW, Australia).
Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
qRT-PCR was carried out in duplicate with the customised control primer (Table S1) (Sigma
Aldrich, CastleHill, NSW, Australia) and the synthesised cDNA template using 2 × FastStart
Essential DNA Green Master (Roche Applied Science, Castle Hill, NSW, Australia) in a
LightCycler® 480 Multiwell Plate 96 (Roche Applied Science, Castle Hill, NSW, Australia).
The plate was centrifuged at 1500g for 2 min and loaded into the LightCycler® 96 (Roche
Applied Science, Penzberg, Germany). The reaction conditions were as follows: a pre-
incubation step at 95 °C for 10 min, a three-step amplification of 45 PCR cycles at 95 °C for
10 sec; 60 °C for 10 sec; 72 °C for 10 sec, a melting step at 95 °C for 10 sec; 65°C for 60 sec;
97 °C for 1 sec, and followed by a cooling step at 40 °C for 10 sec. Results were normalised
to β-actin mRNA levels after examining primer efficiency using the ∆∆CT (threshold cycle)
quantification method (Livak and Schmittgen, 2001; Schmittgen and Livak, 2008).
Biopharmaceutical profiling
The physicochemical characterisation, cytochrome P450 (CYP450) inhibition assay and
ether-a-go-go related gene (hERG/ Kv11.1) safety experiments were carried out by Cyprotex
Discovery Limited (Macclesfield, UK). Partition coefficient was measured using a pH-metric
method. Dissociation constant was determined by Shake Flask method. Apparent
permeability coefficient was measured by Caco-2 assay. CYP450 inhibition assay was
performed using human liver microsomes and NADPH in the presence of a CYP450 isoform-
specific probe substrate: midazolam for CYP3A4, dextromethorphan for CYP2D6,
ethoxyresorufin for CYP1A2, tolbutamide for CYP2C9 and mephenytoin for CYP2C19.
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CHO-hERG cells were used for cardiac toxicity assay. Details are available at
http://www.cyprotex.com.
Pharmacokinetic determinations
Intravenous doses were prepared in a formulation of polyethylene glycol 400/N-methyl-2-
pyrrolidone, 9:1, vv-1 and oral doses were suspended in 1% carboxy methyl cellulose, wv-1.
For cassette dosing a total of four healthy male albino Wistar rats (250-350 g) were used. Up
to four compounds were concurrently administered intravenously or orally, with an internal
standard (IS) which has well-characterised PK. Intravenously, a mixture of 2 mgkg-1 of each
of inhibitors 77, 82, 85 and IS was administered to one rat, and a mixture of inhibitors 86, 91,
94, 95 and IS (2 mgkg-1 of each) to another. Orally, a mixture of compounds 86, 91, 94 and
95 (10 mgkg-1 of each) to one rat, and a mixture of compounds 77, 82 and 85 (4 mgkg-1 of
each) was administered to another rat. Compounds 82, 91 and 95 appeared to have
comparable oral absorption to the standard, and were further assessed individually in a single
rat at a dose of 10 mg·kg-1 (PO) and 5 mg·kg-1 (IV). For follow-up full PK studies of 82 and
91 (85 was excluded because of poor exposure), healthy male adult BALB/c mice (20-25 g)
or male albino Wistar rats (250-350 g) were split into weight matched groups of three and
two per group, respectively and fasted for 2 h, then administered a single oral or intravenous
dose of compound 82 or 91 (2 mgkg-1 for mice, 5 mgkg-1 for rats) via the tail vein or by oral
gavage (10 mgkg-1 for mice, 20 mgkg-1 for rats).
Blood samples were collected from animals by jugular vein cannula (rats) or under anesthesia
by cardiac puncture (mice) at time zero and at intervals up to 24 h. Harvested blood was
centrifuged at 7000g for 3 min, and the plasma aspirated and frozen at −20 °C until analysis.
Concentrations of the compounds in plasma were quantified using a validated LC/MS/MS
method with a triple TOF-MS 5600 (AB Sciex). Briefly, the samples were extracted with
ethyl acetate, and the organic phase was separated and dried under nitrogen. The extracts
were reconstituted in acetonitrile/water (50:50), injected into a Shimadzu Nexera HPLC
system, and resolved on a Kinetex C18 2.6 µ 50 mm × 3 mm column (Phenomenex) at a
mobile phase flow rate of 0.4 mLmin-1. Mobile phase A (MPA) was 5% acetonitrile and 0.1%
formic acid in water and mobile phase B (MPB) was 95% acetonitrile and 0.1% formic acid.
The mobile phase timetable was set as a gradient from 20% MPB initially to 95% MPB in 1
min, held at 95% MPB for 2 min, then 20% MPB for 30 sec in preparation for the next
sample. Peak areas were obtained from the compounds, and IS and known concentrations of
calibrators were used to construct a calibration curve from compounds/IS area ratios. The
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limit of quantification was 5 ngmL-1. The intraday and interday variability for each
compound was within 15%. Noncompartmental pharmacokinetics (PK) analysis was
performed with Phoenix WinNonlin (Pharsight, St. Louis, MO) for each concentration–time
profile. Following the final blood sample animals were killed via inhalation euthanasia using
carbon dioxide (CO2).
Compliance with requirements for studies using animals
Animal experiments were performed under the approval of the SA Pathology Animal Ethics
Committee (Animal Ethics Number: 91C-12). All procedures adopted for housing and
handling of animals were in strict compliance with Australian guidelines for laboratory
animal welfare. Animal studies are reported in compliance with the ARRIVE and BJP
guidelines (Kilkenny, et al., 2010; McGrath & Lilley, 2015).
Computational methods
Protein structures of CDK6 and CDK9 were based on in-house homology models generated
using X-ray crystallographic structures (PDB IDs: 4BCI and 2EUF, respectively).
PD0332991 and 91 were prepared for docking using the Ligand Preparation module of the
Schrödinger software suite. The protonation states of ligands were calculated based on a pH
value of 7.4 ± 1.0. Prepared ligands were docked using Schrödinger Glide XP protocol
(Friesner et al., 2004; Halgren et al., 2004). Docking protocols were initially verified by re-
docking crystallised ligands, to reproduce crystallised ligand poses. The best scoring of the
five binding poses for each ligand was then subjected to MM-GBSA binding energy
calculations, where the side chains of residues in a 4 Å sphere around the ligand were
allowed to be flexible.
Data and statistical analysis
In vitro experiments were performed in triplicate and repeated at least twice, and data are
given as mean values ± SEM; representative data were selected for generating figures.
Statistical comparisons for G1 arrest and gene expression were performed using one way
ANOVA and values of p ≤ 0.05 were considered significant. Data were blindly analysed
using GraphPad Prism version 6.03 for Windows (GraphPad Software, La Jolla, CA, USA).
In vivo pharmacokinetic parameters are given as mean values from three animals.
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Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in
http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS
Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the
Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).
Results
Discovery, screening and optimisation
In our pursuit to develop kinase targeted anticancer agents, we previously characterised a
series of 4-(thiazol-5-yl)-2-(phenylamino)pyrimidine derivative CDK inhibitors (Shao et al.,
2013), but high selectivity for individual CDKs was not achieved. In this study, we embarked
on a cascade approach to identify highly selective CDK4/6 inhibitors. Subsequent
optimisation of the scaffold culminated in the identification of 4-thiazol-N-(pyridin-2-
yl)pyrimidin-2-amine as novel and proprietary CDK4/6 inhibitor pharmacophore.
Using our screening cascade, compounds with GI50 < 1 µM were subjected to biochemical
assay against CDK4 at a concentration of 10 µM, and those causing > 80% inhibitory activity
were profiled against CDK1, 2, 4, 6, 7 and 9. Low-nanomolar inhibitors with  500-fold
selectivity for CDK4 over its isotypes were assessed for their cell cycle effects. Subsequently,
compounds with G1 cell cycle arrest profile were investigated for their influence on Rb
phosphorylation and apoptosis. This strategy led to the identification of 82, 91 and 95 as
potent and highly selective CDK4/6 inhibitors (Table 1).
The first two 4-(4-methylthiazol-5-yl)-N-(pyridin-2-yl)pyrimidin-2-amine derivatives
prepared were 73 and 75. Comparison with their corresponding anilino counterparts 72 and
74 pinpointed a distinct difference in selectivity, with the pyridinyl analogues favouring
inhibition of CDK4 over that of CDK1, 2, 7 and 9, at the expense of a modest loss in their
potency against CDK4. This might be due to the hydrogen bond interaction between the
pyridinyl nitrogen-which is part of the general N-NH-N sequence of the pyrimidine-amine-
pyridine system-common in all the clinical CDK4/6 inhibitors, and the histidine (rarely
conserved in the CDKs) of the hinge region of the kinases. Recently, LY2835219 has been
co-crystallised with CDK6 showing such type of interaction through a water bridge (Chen et
al., 2016).
In addition, a brief examination of the position of the nitrogen atom on the pyridine ring was
performed to determine the effect of transposition to meta position; the meta compound 101
was a pan-CDK inhibitor with 16-fold higher Ki value for CDK4 when compared with its
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ortho counterpart 77. Consequently, in all the remaining analogues the pyridine ring was
essentially unaltered. The structural optimisation was thus turned to the C5 position of the
pyridine ring. Although 73 and 75 conferred good potency and selectivity towards CDK4, the
bulkiness and lipophilicity of the benzyl ring suggested further attenuation would most likely
be required. Specifically, an improvement in overall physical properties was anticipated with
substituents that could lower the molecular weight and introduce greater polarity at the R3
position. Therefore, compounds incorporating piperazinyl moieties were synthesised. The
data in Table 1 identified most of these compounds as remarkably potent and selective
CDK4/6 inhibitors. While showing a varying degree of selectivity towards CDK2, the
majority of them did not inhibit CDK1, 7 and 9. Particularly, 82, 91 and 95 demonstrated
high level of selectivity and potency. In contrast, compounds lacking substituent on C5 of the
pyridine ring (93), those incorporating a morpholinyl (79, 83, 88 and 97) or piperidinyl (89)
substituents were either inactive against CDK4-cyclin D1 (89 and 93) or could not
significantly discriminate between CDK2 and CDK4 (79, 83, 88 and 97). We also explored
the effects of substitution at C5 position of the pyrimidine ring. The introduction of a fluorine
atom (84-88) or a carbonitrile group (80-83) considerably improved potency for CDK4
without significantly affecting selectivity. Similarly, a tertiary amino group in the place of the
methylamino group at C2 position of the thiazole ring preserved CDK4 inhibitory activity in
the fluorinated analogues (94-96). However, similar modifications on the non-fluorinated
analogues (76 and 78 vs 98 and 99, respectively) resulted in a modest reduction in potency
for CDK4.
Kinase inhibition
Several compounds from this series inhibited CDK4/6 potently, and exhibited selectivity over
closely related cell cycle and transcriptional CDK family members. Remarkably, 82, 91 and
95 were inhibitors of CDK4 with approximately 2-4 orders of magnitude greater potency than
of CDK1, 2, 7 and 9. The selectivity of the three compounds over CDK1, inhibition of which
is highly toxic to cells, was > 1,250-fold. Furthermore, these lead compounds exhibited far
less activity against CDK9 when compared with PD0332991. To better explore kinase
selectivity, the lead compounds were further evaluated in an expanded panel of 48
representative kinases at 10 µM, which represented profiling at > 2,500–fold of CDK4 Ki
value (Table S2). Compounds 82, 91 and 95 were highly selective against the majority of
these kinases, except DYRKs and HIPK2 to which > 90% inhibition was observed. In
addition, while 82 and 95 targeted FLTs 1 & 4 and Aurora kinase B respectively, 91 inhibited
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FLT3 and MYLK4 potently (> 90% inhibition at 10 µM). On the basis of its excellent drug
properties (vide infra), 91 was selected for a further investigation of its selectivity against a
panel of 369 human protein kinases. Compound 91 proved to be highly selective, only
effecting on CLK2, DYRKs, and MYLK4 with a residual kinase activity < 10% (Figure 4A,
Table S3). Consequently, Ki value for each of these kinases was determined (Figure 4B), and
the inhibitory activity was seen against DYRKs, and to lesser extent, CLK2, FLT3 and
MYLK4.
Compounds 82, 91 and 95 have a broad-spectrum of anticancer activity
In MV4-11 cell line, GI50 values were below 1 µM for 27 of the 30 compounds, indicating
potent activity on cancer cells of haematopoietic origin. In contrast, compounds were mostly
less active against MDA-MB-453 cells with GI50 values ranging from 0.237 to 5.84 µM
(Table 1). To determine the potential of 82, 91 and 95 as broad spectrum anticancer agents,
we further investigated their anti-proliferative potency against a panel of 13 human solid and
3 haematological cancer cell lines (Table 2). 82, 91 and 95 potently inhibited the growth of
cancer cell lines with GI50 values in a range of 0.028 to 10.5 μM. Particularly, in MOLM13
and A2780 cells, 91 and 95 suppressed tumour cell proliferation with similar potency to
PD0332991, and were more potent than PD0332991 in M249 and M249R melanoma cells
and PC3 prostate cancer cells. To investigate whether the anti-proliferative effects were
selective towards malignant over normally proliferating cells, 82, 91 and 95 were evaluated
in non-transformed WI-38 and MRC-5 fibroblast cells (Table 2). The data showed that they
were preferably cytotoxic to cancer cells. Taken together, these results suggested that the lead
compounds possessed a broad spectrum of anticancer activity without a significant effect on
the non-cancerous cells. Moreover, by comparing inhibition of the growth of Rb-positive
(MDA-MB-453) and Rb-negative (MDA-MB-468) breast cancer cells by 82, 91 and 95, their
cellular selectivity was found to be about 11-, 22- and 36-fold, respectively (Figure 5B).
Consistent with CDK4/6 targeted mechanism, these compounds exhibited far less anti-
proliferative activity against the Rb-negative MDA-MB-468 breast cancer and DU145 (Table
2) prostate carcinoma cells.
82, 91 and 95 arrest Rb-positive cancer cells exclusively at G1 phase of the cell cycle
The effectiveness of 82, 91 and 95 in causing G1 arrest was tested in Rb-positive MV4-11
cells. Each of the compounds caused a concentration-dependent accumulation of these cells
in the G1 phase with a simultaneous decrement in the S-phase (Figure 5C). For example,
treatment with 0.5 µM (5 × GI50) of 91 resulted in > 30% increase in G1 cells compared to
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untreated cells. This signifies that 91 caused a G1 arrest at > 300-fold the Ki for CDK4. A
similar increase in the G1 cell-cycle was also observed with the MDA-MB-453 cells 24 h
post-exposure to 91 (Figure 5D). In contrast, there was no apparent G1 cell-cycle
accumulation in the Rb-negative MDA-MB-468 cells (Figure 5D), evidencing highly specific
on-target effect of 91.
82, 91 and 95 trigger apoptosis in MV4-11 and MDA-MB-453 cells
To determine whether apoptosis contributes to the observed anti-proliferative effects of 82,
91 and 95, we used annexin V/ PI double staining of MV4-11 and MDA-MB-453 cells,
following 24, 48, 72 and 96 h treatment. 82, 91 and 95 triggered cancer cell apoptosis in
MV4-11 cells (Figure 5E). At the highest dose tested (10 × GI50), 82, 91 and 95 caused over
30% annexin V-positive cells post 72 h treatment. Impressively, about 70% of treated cells
also entered apoptosis after 96 h treatment with 95 at a concentration of 1 µM. In contrast,
similar treatment with PD0332991 caused little apoptotic MV4-11 cells. In MDA-MB-453
cells, both 91 and PD0332991 were capable of inducing apoptotic cells in a dose- and time-
dependent manner (Figure 5F), but the effects were less pronounced compared to those on
MV4-11 cells.
82, 91 and 95 inhibit phosphorylation of Rb
To directly investigate the impact of the lead compounds on the phosphorylation of Rb at
S780, time-course western blot experiments were performed on MV4-11 and MDA-MB-453
cells. The level of S780-phosphorylated Rb (pRb(S780)) declined in both cell lines exposed
to each of compounds 82, 91 and 95, with maximum decline at 24 h (Figures 6A and 6C).
The level of pRb(S780) decreased in a dose-dependent manner except for compounds 95 and
82 at 12 h on MDA-MB-453 cells. A decrease in the level of total Rb was also seen in both
MV4-11 and MDA-MB-453 cell lines, except for 91 at 24 h in MV4-11 cells. However, in
MV4-11 cells neither the total Rb nor pRb(S780) were affected at 4 h (Figure 6B).
Interestingly, the decrease in phosphorylated Rb after exposure to 91 was more pronounced
and sustained than 82 and 95. Due to its high potency, selectivity, and favourable
biopharmaceutical and pharmacokinetic properties (vide infra) 91 was further studied in two
Rb-positive breast cancer cell lines (MCF7 and T47D). Post 24 h treatment of MCF7 and
T47D with 91 resulted in blockage of the Rb phosphorylation in a dose-dependent manner
(Figure 6D). Collectively, the data were consistent with the cellular CDK4/6 targeted
mechanism of action.
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82, 91 and 95 decrease transcription of Rb1 and E2F-target genes in MDA-MB-453 cells
Based on the results obtained from the western blot, we wished to determine the effect of 82,
91 and 95 on the regulation of Rb1 and two E2F-target genes viz. CCNA2 and CCNE2.
Similar to PD0332991, treatment of MDA-MB-453 cells with each of 82, 91 and 95 resulted
in a repression of Rb1, CCNA2 and CCNE2 transcripts when compared with untreated cells
(Figures 7A-C). Consistent with the western blot data, MDA-MB-453 cells treated with 91
for 24 h exhibited a dramatic suppression in CCNA2 and CCNE2 gene transcription when
compared with cells treated with each of 82 and 95. Furthermore, at their respective GI50
concentration and post 24 h treatment, when compared with PD0332991, 91 elicited a better
response in reducing the levels of CCNA2 and CCNE2 (p < 0.05), and the suppression
appeared to be time-dependant (p < 0.05). Thus, these results demonstrated that the lead
compounds decreased the expression of cyclin A2 and cyclin E2 by a mechanism of
inhibiting CDK4/6 in cells.
91 exhibits excellent biopharmaceutical properties
Compound 91 was found to be soluble up to 100 µM in sodium phosphate buffer (10 µM, pH
7.4) and has low lipophilicity (Log D7.4 = 1.9). The dissociation constant (pKa) values of 91
were found to be 3.80 and 5.41. Compound 91 was highly permeable across the Caco-2
monolayer with Papp = 48.7 nm/s. An efflux ratio of 3.4 suggests that it is an unlikely
substrate for potential efflux transporters. Compound 91 possessed no inhibition against all
the CYP450 enzymes tested (IC50 > 25 μM), and showed no activity against hERG (IC50 > 25
μM), indicating very low potential for metabolic and cardiotoxicity.
91 possesses favourable pharmacokinetic properties
Poor pharmacokinetics (PK) is one of the primary causes of failures in drug development that
prevent new chemical entities from reaching the market. Hence, PK evaluation has moved to
the early stage of drug discovery (Smith, Raynaud & Workman, 2007). In an attempt to
accelerate the PK throughput and reduce animal usage, cassette dosing, which involves the
administration of a cocktail of compounds to an animal, was used for initial screening of
compounds with the desired potency and selectivity profiles. The compounds were selected
for, or eliminated from further PK analysis, based on their performance (Figures S1A-C). 82,
91 and 95 appeared to have comparable oral absorption to the standard, and were further
assessed individually in a single rat. 82 and 91 showed high exposure when orally
administered (Table S4). The follow-up full PK studies showed both 82 and 91 were
absorbed and reached their highest maximum concentrations within 2.5 to 5.7 h, except for
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82 in mice, which was absorbed rapidly (Table 3). The plasma clearance of 91 was rapid and
the compound appeared highly distributed in both animal species. This could be attributed in
part to the extensive red blood cell binding with mean blood to plasma concentration ratio of
4.3:1. Following oral administration, 91 exhibited the high plasma exposure with AUC = 4.4
and 4.3 M in rats and mice, respectively. Its half-life time (t1/2) in plasma was 20 h in rats
and 2.7 h in mice. The oral bioavailability of 91 was very high giving F values of 95% and
129% in rat and mice, respectively (Table 3).
Discussion and conclusion
In the present study, we report the discovery, optimisation and characterisation of a new
series of small-molecule inhibitors that are selective for CDK4/6. Importantly, we reveal the
biological impacts of these inhibitors and demonstrate the high potential for their application
in cancer therapy. The lead compound 91 has an advantage of an excellent kinase selectivity
profile. In addition to CDK4/6, PD0332991, LY2835219 and LEE011 target CDK9 among
other kinases. Our computational modelling of 91 bound to CDK6 (Figure 4C) and CDK9
provided a plausible rationale for its enhanced selectivity compared to PD0332991. Like
PD0332991, 91 has low binding energies to CDK6, suggesting strong binding affinity.
However, PD0332991 prefers binding to CDK9 substantially with a ΔΔG° value of -5.9
kcal/mol. These preliminary binding affinity calculations corroborate with the trends of Ki
values observed. Furthermore, unlike PD0332991 and LEE011, which show similar level of
potency towards CDK4 and CDK6, 91 is 93-fold more potent for CDK4D1 over CDK6D3.
Given the fact that LY2835219’s lower incidence of neutropenia in the clinic, which is a
dose-limiting toxicity for PD0332991 and LEE011, has been associated with its higher
potency and selectivity for CDK4 than for CDK6 compared to PD0332991 and LEE011
(Barroso-Sousa, Shapiro & Tolaney, 2016), 91 would provide an even better safety option for
the clinic due to its superior selectivity for CDK4 over CDK6D3 to LY2835219.
An investigation on the mechanism of the anti-proliferative action of our lead compounds 82,
91 and 95 revealed that they inhibit cellular CDK4/6, induce G1 cell cycle arrest by inhibiting
Rb phosphorylation, and suppress the expression of several Rb-E2F-regulated genes.
Importantly, the repression of the cell-cycle genes encoding cyclins A and E, both of which
bind to and facilitate to activate CDK2, leads to the suppression of various transcription
factors that contribute to cell division (Bertoli, Skotheim & de Bruin, 2013). It should also be
noted that the lead compounds reduced the transcription of Rb1 gene and caused a
corresponding decrease in the expression of Rb protein. Although the mechanism underlying
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this partial reduction is not clear, the possibility of proteasomal degradation of Rb cannot be
completely discounted. Sathe et al proposed that the loss of Rb transcription might be a
prerequisite for the therapeutic effects of CDK4/6 inhibitors (Sathe et al., 2016). We also note
that several publications showed the reduction in Rb protein level by CDK4/6 inhibitors but
did not discuss its implications (Zhang et al., 2014).
In cancer cells that lack Rb function, the E2Fs are active and the CDK4/6 pathway is
redundant (O'Leary et al., 2016). As expected, our lead compounds were significantly less
active against the Rb-negative MDA-MB-468 and DU145 cells. Nevertheless, Rb is intact in
the vast majority of cancers (Dick, 2007), substantiating the rationale behind the observed
activity by our inhibitors against a broad panel of human cancer cell lines (Table 2),
rendering them as potentially highly effective therapeutics for these cancers. Our CDK4/6
inhibitors were capable of inducing apoptosis in MV4-11 and MDA-MB-453 cells, and were
more potent than PD0332991. The apparent differential effects of our compounds and
PD0332991 on their ability to induce apoptosis may result from their different kinase
selectivity profiles. As such, the potential synergistic effects of the combined targeting of
CDK4 and DYRKs by 91 will be investigated. Indeed, recent studies have implicated that
DYRKs are overly activated in cancers, and cancer cells treated with a DYRK inhibitor
underwent apoptosis (Friedman, 2013).
Translation of in vitro CDK inhibitory and cancer cell growth inhibitory effects into in vivo
efficacy depends on the biopharmaceutical and PK properties of drug molecules, as drug
exposure profiles define the level and duration of target inhibition (Chen et al., 2016). 82, 91,
and 95 seem comparable in terms of their kinase inhibitory potency and selectivity as well as
anti-proliferative activity in cells; however, upon further evaluation, 91 outperformed 82 and
95 with overall superior profiles of potency, selectivity, biopharmaceutical characteristics and
PK. The excellent in vitro cellular potency of 91, along with its superior pharmaceutical
profile, warrants its advancement into in vivo efficacy studies.
In conclusion, using biochemical, cell-based, in vitro ADME and pharmacological screening
cascade, we have successfully identified a novel library of 4-(4-methylthiazol-5-yl)-N-
(pyridin-2-yl)pyrimidin-2-amine derivatives as single-digit nanomolar inhibitors of CDK4/6.
The lead i.e. 91 demonstrated not only high potency and selectivity, but also superior
pharmacokinetic properties.
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Acknowledgments
S.T. acknowledges the support from the Australian Government Research Training Program
Scholarship.
Author contributions
Conception, overall study design and supervision: S.W., chemistry experiments and data
analysis: S.T., N.B., and M.Y., biological experiments: L.B., F.L., S.I., G. Z., K.T., and P.L.
in vivo experiments: L.B. and B.N. computational experiments: M.K., wrote/revised/edited
the manuscript: S.T., S.W., M.Y., R.M., M.K., L.B., B.N., and K.T.
Conflict of interest statement
The authors declare no conflict of interest.
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List of the hyperlinks applied to the article
Targets
1. CDK
2. CDK1
3. CDK2
4. CDK4
5. CDK6
6. CDK7
7. CDK9
8. CLK2
9. CYP1A2
10.CYP2C19
11.CYP2C9
12.CYP2D6
13.CYP450
14.FLT3
15.HIPK2
16.Kv11.1
17.MYLK4
Ligands
1. Rb
2. Palbociclib
3. Ribociclib
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4. Abemaciclib
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Table 1. Structures, CDK inhibitory and in vitro anti-proliferative activities of 4-(4-methylthiazol-5-yl)-N-
(pyridin-2-yl)pyrimidin-2-amine derivatives
Anti-proliferative activity,
GI50b (μM)c
MV4-11 MDA-MB-453
0.001 0.036 0.089 0.017 0.101 0.034 0.037 ± 0.004 0.109 ± 0.024
0.016 0.036 > 5 > 5 > 5 0.999 0.084 ± 0.006 0.128 ± 0.015
0.006 0.225 0.133 0.037 0.067 0.117 0.038 ± 0.004 0.160 ± 0.001
CDK inhibition, Ki (μM)a
4D1d 6D3d 1Bd 2Ad 7Hd
Structure
X
9Td
Cmpd
R1
R2
R3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CN CH
NBn
NBn
NBn
NBn
NH
NCH3
NCOCH3
O
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
CN
H
H
H
H
H
H
CN
CN
CN
CN
F
F
F
F
F
F
F
F
F
N
CH
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
0.169 2.710
0.006 0.114
0.005 0.050 3.205 0.650
0.578 3.032 3.101 0.310
0.020 0.061
0.001 0.040 4.645 0.212
0.004 0.032
0.004 0.030
0.004 0.064
0.004 0.032 1.360 0.236
0.002 0.010
0.005 0.020
> 5
> 5
> 5
0.889
> 5
> 5
> 5
> 5
> 5
> 5
> 5
> 5
> 5
> 5
> 5
> 5
> 5
> 5
-
> 5
> 5
> 5
-
> 5
> 5
> 5
> 5
> 5
> 5
> 5
> 5
> 10
1.015 ± 0.238
2.850 0.259 ± 0.172 0.665 ± 0.213
1.820 0.297 ± 0.044 0.544 ± 0.039
> 5
> 5
0.338 0.014 ± 0.004 0.458 ± 0.277
0.485 0.056 ± 0.003 1.047 ± 0.374
1.678 0.170 ± 0.019 0.813 ± 0.016
0.154 ± 0.052 5.840 ± 0.141
0.643 ± 0.013 1.458 ± 0.279
> 5
0.459
NH
NCH3
NCOCH3
O
> 5
> 5
> 5
0.301
4.466
0.201
> 5
0.465 ± 0.091 0.090 ± 0.007
NH
0.784 0.012 ± 0.002 0.248 ± 0.031
1.905 0.011 ± 0.003 0.381 ± 0.068
2.925 0.065 ± 0.001 0.528 ± 0.033
4.070 0.314 ± 0.127 4.912 ± 0.305
> 5
-
NCH3
NCH2CH3
NCOCH3
O
> 5
> 5
0.365
0.665
0.017 0.046 1.820 0.178
0.005 0.030 3.315 0.100
0.405 ± 0.014 5.407 ± 0.567
0.606 ± 0.027 0.463 ± 0.053
0.425 ± 0.052 0.660 ± 0.065
0.107 ± 0.016 0.325 ± 0.045
H
H
H
H
CH2
> 5
0.037 0.297 0.580 0.076
> 5 3.335
0.003 0.032 2.370 0.206
-
-
-
NSO2CH3
> 5
> 5
CHN(CH3)2 0.003 0.279
CH2NH2
3.037 0.080 ± 0.009 0.362 ± 0.002
-
> 5
H
F
F
F
F
NSe
> 5
-
-
-
2.158 ± 0.305 2.941 ± 0.389
0.048 ± 0.002 0.237 ± 0.031
CH3
CH3
CH3
CH3
CH3
CH3
CH3
NH
0.004 0.040
0.002 0.055
0.024 0.366
> 5
> 5
> 5
> 5
> 5
> 5
> 5
ND
> 5
0.335
1.390
1.040
0.069
0.903
1.430
0.976
NCH3
NCOCH3
O
NH
NCH3
NCOCH3
4.360 0.073 ± 0.007 0.638 ± 0.030
> 5
> 5
> 5
> 5
> 5
0.208 ± 0.021 2.369 ± 0.018
4.675 ± 0.211 5.358 ± 0.354
0.011 ± 0.015 0.894 ± 0.064
0.400 ± 0.057 2.102 ± 0.556
0.537 ± 0.094 4.718 ± 0.506
F
0.044
ND
H
H
H
0.021 0.056
0.030 0.154
0.087 0.234
0.080 0.127
0.004 0.025
1.500 0.280 0.030 0.008 ± 0.001
> 5 > 5 0.386 0.050 ± 0.025 0.326 ± 0.047
-
PD0332991
aApparent inhibition constants (Ki) were calculated using IC50 values and the appropriate Km (ATP) values for
b
c
each kinase. GI50 is the compound concentration required to inhibit 50% of cell growth. Antiproliferative
activity was determined by 72 h resazurin and MTT assays using MV4-11 and MDA-MB-453 cell lines,
d
respectively. The data given are derived from at least two replicates, and are presented as mean ± SEM. 4D1,
6D3, 1B, 2A, 7H, and 9T1 represent CDK4-cyclin D1, CDK6-cyclin D3, CDK1-cyclin B, CDK2-cyclin A,
e
CDK7-cyclin H-MAT1 and CDK9-cyclinT1, respectively. NS indicates no substitution at C5 of the pyridine
ring.
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Table 2. Anti-proliferative activities of 82, 91 and 95 against a panel of human cancer cell lines
Anti-proliferative activity, GI50 (μM)a
Cancer type
Cell line
MCF7
T47D
82
91
95
PD0332991
0.557 ± 0.040
1.180 ± 0.071
1.170 ± 0.085
1.290 ± 0.262
1.400 ± 0.113
0.061 ± 0.003
0.033 ± 0.067
1.080 ± 0.085
1.220 ± 0.431
1.970 ± 0.184
1.620 ± 0.368
1.990 ± 0.368
2.960 ± 0.156
0.045 ± 0.013
0.740 ± 0.212
8.596 ± 0.106
Breast
0.660 ± 0.190
-
1.390 ± 0.190
0.839 ± 0.092
2.220 ± 0.247
3.610 ± 0.113
3.750 ± 0.410
0.076 ± 0.006
0.431 ± 0.096
2.860 ± 0.276
3.380 ± 0.573
2.750 ± 0.445
1.180 ± 0.028
0.550 ± 0.092
0.313 ± 0.049
0.028 ± 0.002
0.220 ± 0.049
5.657 ± 0.312
0.290 ± 0.092
-
Colorectal
Leukaemia
Melanoma
COLO205
HCT116
HT29
3.610 ± 0.311
7.470 ± 0.148
5.610 ± 0.877
0.408 ± 0.018
0.511 ± 0.100
4.920 ± 0.014
2.210 ± 0.290
2.420 ± 0.014
1.950 ± 0.092
5.570 ± 0.134
10.50 ± 0.919
0.552 ± 0.098
0.230 ± 0.106
> 10
2.130 ± 0.481
1.240 ± 0.007
1.390 ± 0.041
0.098 ± 0.008
0.120 ± 0.006
1.370 ± 0.849
2.830 ± 0.601
1.090 ± 0.007
4.690 ± 1.287
0.960 ± 0.071
1.100 ± 0.003
0.041 ± 0.004
1.240 ± 0.375
9.405 ± 0.421
MOLM13
NB4
U937
M229
M238
M238R1
M249
M249R
A2780
PC3
Ovarian
Prostate
DU145
Untransformed
Embryonic lung
fibroblasts
WI-38
> 10
> 10
> 10
> 10
> 10
> 10
MRC-5
8.320 ± 0.305
8.110 ± 0.028
aAnti-proliferative activity was determined by 72 h resazurin and MTT assays using leukaemia and solid cancer
cell lines, respectively. GI50 is the compound concentration required to inhibit 50% of cell growth. The data
given are derived from at least two replicates, and are presented as mean ± SEM.
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Table 3. Pharmacokinetic studies of compounds 82 and 91 in rodents
82
91
PK parametera
route
rat
IV
5
28
2.6
6.5
8.5
-
mouse
IV
2
66
2.2
1.1
4.2
-
rat
IV
5
155
27.5
1.2
1.4
-
mouse
IV
2
PO
20
-
PO
10
-
PO
20
-
PO
10
-
dose (mg·kg-1)
CL (mL·min-1·kg-1)
Vss (L·kg-1)
AUC (µM·h)
Cmax (µM)
90
-
-
-
15.7
0.7
1.3
-
2.9
-
-
10.1
0.6
5.7
4.6
39
0.8
0.4
0.5
2.7
14
4.4
0.3
4.3
20.3
95
4.3
0.6
2.5
2.7
129
Tmax (h)
t1/2 (h)
1.4
-
1.0
-
2.1
-
F (%)
a
Non-compartmental PK analysis was performed using WinNonlin (Pharsight, St.Louis, US) for each
concentration versus time profile and the mean value from three animals is presented.
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Figure 1. Synthetic scheme for enaminones 3-6 and 10. Reagents and conditions: (a) 3-chloro-2,4-pentadione,
C5H5N, MeOH, rt, o/n, 53%; (b) DMF-DMA, reflux, 8 h, 3: 21%, 10: 62%, or microwave, 150 °C, 1 h, 4: 38%;
(c) Selectfluor, ice bath, 1 h, 30%; (d) CH3I, NaH, THF, 40 °C, 3.5 h, 82%; (e) ethyl-2-chloroacetoacetate,
DMAP, CH2Cl2, rt, 4 h, 85%; (f) di-tert-butyl dicarbonate, DCM, Et3N, 8 h, rt, 93%; (g) LiNiPr2, CH3CN, -78
°C, 10 min; o/n, HCl, H2O, 48%.
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Figure 2. Synthetic scheme for guanidine derivatives 49 and 61-71. Reagents and conditions: (a) 1-fluoro-4-
nitrobenzene, K2CO3, DMSO, 90 °C, 6.5 h, 36: 100%, or 5-bromo-2-nitropyridine, Et3N, DMSO, 120 °C, 16 h,
24, 25, 27, 29-35: 37-92%; (b) 25 or 27, di-tert-butyl dicarbonate, DCM, Et3N, DMAP, 8 h, rt, 26: 73%, 28:
90%; (c) H2, 10%Pd/C, CH3OH, 6.5-12 h, rt, 37-47: 82-100%; (d) 47, cyanamide, HNO3, EtOH, reflux, 20 h,
49: 69%; (e) N,N′-bis-Boc-S-methylisothiourea, Et3N, HgCl2, DCM, 0 °C, 0.5 h, rt, 14 h, 50-60: 22-82%; (f)
a
TFA/DCM/H2O (18:9:1), rt, 16 h, 61-71: 90-100%. no substitution at carbon 5 of the pyridine ring. 48 was
purchased from Combi Blocks, In., US.
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Figure 3. Synthetic scheme for the 4-(4-methylthiazol-5-yl)-N-(pyridin-2-yl)pyrimidin-2-amine derivatives 72-
101 Reagents and conditions: (a) NaOH, 2-methoxyethanol, microwave, 180 °C, 1 h, final products 72-101: 14-
50%. See Table 1 for the individual structures of 72-101.
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Figure 4. Kinome selectivity of compound 91. (A) Kinome activity map of 91. Kinome binding specificity of 91
was measured by a radiometric kinase inhibition assay. Red, yellow and blue colours represent < 10%, 10 - 20%
and > 20% residual activity of the kinase at 1 µM inhibitor, respectively. The figure was generated using
Reaction Biology Corporation’s Kinome Mapper. See also Table S3 for detailed data. (B) Ki values for a subset
of kinases with residual activities of < 10% in the presence of 1 µM 91. (C) Binding mode of 91 to CDK6. The
protein backbone is in white and the hinge region is highlighted in yellow. 91 makes 4 hydrogen bonds with
CDK6 (2 × with Val101, 1 × with T107, 1 × with D163). There is also a weak hydrophobic interaction with the
gatekeeper, through the methyl group and the fluoride. The figure was prepared using PyMOL1.3 (Schrödinger
Inc., NY, USA).
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Figure 5. Cellular mechanisim of action of CDK4/6 inhibitors. (A) In cancer cells, activation of CDK4/6
accelerates G1-S transition, while suppressing senescence and apoptosis. CDK4/6 inhibitors, therefore, are
capable of causing G1 arrest, triggering senescence and inducing apoptosis. (B) 82, 91 and 95 inhibited the
proliferation of the Rb-positive MDA-MB-453 cancer cells, but not that of the Rb-deficient MDA-MB-468
cells. (C) MV4-11 cell lines displayed a clear G1 arrest and a reduction in fraction of S phase cells after
incubation with 82, 91, and 95 for 24 h, at concentrations of their respective GI50, 5 × GI50 and 10 × GI50. (D) 91
caused a significant G1 cell-cycle arrest in the Rb-positive MDA-MB-453 cells but not in the Rb-negative
MDA-MB-468 cells when compared with the untreated cells. Cell cycle progression was assessed by flow
cytometery. Data represent 2 independent experiments and are plotted as mean. One way ANOVA: p < 0.05
considered significant, for all statistics relative to untreated cells and for comparison of the effect of different
doses on G1 accumulation. For 82 and 91 the differences were not significant between the last 2 doses. (E)
Treatment with each of compounds 82, 91 and 95 induced apoptosis in MV4-11 cells and 91 induced apoptosis
in MDA-MB-453 cells (F). Cells were treated with each compound for 24, 48, 72 and 96 h, at concentrations of
their respective GI50, 5 × GI50 and 10 × GI50. Cells were stained with annexin V and PI. The percentage of cells
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undergoing apoptosis is defined as the sum of early apoptotic (annexin V+/PI-) cell and late apoptotic (annexin
V+/PI+) cell percentages. Anti-proliferative data given are the mean ± SEM from at least two replicates. Cell
cycle analysis and determination of apoptosis were performed in at least two replicates and the mean values
were used to generate graphs C-F.
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