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

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 Milne¹and Shudong Wang^1

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

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.

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

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.

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

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^-1BSA, 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. IC₅₀values and curve

fits were obtained using Prism (GraphPad Software, La Jolla, CA, USA). K_ivalues were

calculated using the Cheng-Prusoff equation: K_i= IC₅₀/(1 + ([ATP]/K_m(ATP))), where [ATP]

is K_m(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

(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 × 10⁴cells 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^-1propidium iodide, 0.1 mgmL^-1RNase 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 × 10⁴cells 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 × 10⁵cellsmL^-1with 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,

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 × 10⁶MDA-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)₁₈and 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 ∆∆C_T(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/ K_v11.1) 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.

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^-1and 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^-1of 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^-1of each) to another. Orally, a mixture of compounds 86, 91, 94 and

95 (10 mgkg^-1of each) to one rat, and a mixture of compounds 77, 82 and 85 (4 mgkg^-1of

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^-1for mice, 5 mgkg^-1for rats) via the tail vein or by oral

gavage (10 mgkg^-1for mice, 20 mgkg^-1for 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

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.

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 K_ivalue for CDK4 when compared with its

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 K_i

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

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, K_ivalue 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, GI₅₀values 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 GI₅₀values 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 GI₅₀values 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

untreated cells. This signifies that 91 caused a G1 arrest at > 300-fold the K_ifor 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.

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 D_7.4= 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 P_app= 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

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 (t_1/2) 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 K_i

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

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.

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.

References

Alexander SPH, Fabbro D , Kelly E, Marrion N , Peters JA, Benson HE et al. (2015). The

Concise Guide to PHARMACOLOGY 2015/16: Enzymes. Br J Pharmacol 172: 6024–6109.

Anders L, Ke N, Hydbring P, Choi YJ, Widlund HR, Chick JM et al. (2011). A systematic

screen for CDK4/6 substrates links FOXM1 phosphorylation to senescence suppression in

cancer cells. Cancer Cell 20: 620-634.

Barroso-Sousa R, Shapiro GI & Tolaney SM (2016). Clinical development of the CDK4/6

inhibitors Ribociclib and Abemaciclib in breast cancer. Breast Care 11: 167-173.

Bertoli C, Skotheim JM, & de Bruin RA (2013). Control of cell cycle transcription during G1

and S phases. Nat Rev Mol Cell Biol 14: 518-528.

Bisi JE, Sorrentino JA, Roberts PJ, Tavares FX, & Strum JC (2016). Preclinical

characterization of G1T28: A novel CDK4/6 inhibitor for reduction of chemotherapy-induced

myelosuppression. Mol Cancer Ther 15: 783-793.

Chen P, Lee N, Hu W, Xu M, Ferre RA, Lam H et al. (2016). Spectrum and degree of CDK

drug interactions predicts clinical performance. Mol Cancer Ther 15: 2273-2281.

Choi YJ, Li X, Hydbring P, Sanda T, Stefano J, Christie AL, et al. (2012). The requirement

for cyclin D function in tumor maintenance. Cancer Cell 22: 438-451.

Diab S, Li P, Basnet SK, Lu J, Yu M, Albrecht H et al. (2016). Unveiling new chemical

scaffolds as Mnk inhibitors. Future Med Chem 8: 271-285.

Dick FA (2007). Structure-function analysis of the retinoblastoma tumor suppressor protein -

is the whole a sum of its parts? Cell Div 2: 26.

Friedman E (2013). Mirk/dyrk1B kinase in ovarian cancer. Int J Mol Sci 14: 5560-5575.

Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT et al. (2004). Glide: A

New approach for rapid, accurate docking and scoring. 1. method and assessment of docking

accuracy. J Med Chem 47: 1739-1749.

Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT et al. (2004). Glide:

a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database

screening. J Med Chem 47: 1750-1759.

Hamilton E, & Infante JR (2016). Targeting CDK4/6 in patients with cancer. Cancer Treat

Rev 45: 129-138.

Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG, et al. (2010). Animal research:

reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160: 1577-1579.

Kozar K, & Sicinski P (2005). Cell cycle progression without cyclin D-CDK4 and cyclin D-

CDK6 complexes. Cell Cycle 4: 388-391.

Livak KJ, & Schmittgen TD (2001). Analysis of relative gene expression data using real-time

quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402-408.

Malumbres M, & Barbacid M (2009). Cell cycle, CDKs and cancer: a changing paradigm.

Nat Rev Cancer 9: 153-166.

McGrath JC, & Lilley E (2015). Implementing guidelines on reporting research using animals

(ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol 172: 3189-3193.

O'Leary B, Finn RS, & Turner NC (2016). Treating cancer with selective CDK4/6 inhibitors.

Nat Rev Clin Oncol 13: 417-430.

Sathe A, Koshy N, Schmid SC, Thalgott M, Schwarzenbock SM, Krause BJ et al. (2016).

CDK4/6 inhibition controls proliferation of bladder cancer and transcription of RB1. J Urol

195: 771-779.

Schmittgen TD, & Livak KJ (2008). Analyzing real-time PCR data by the comparative C(T)

method. Nat Protoc 3: 1101-1108.

Shao H, Shi S, Huang S, Hole AJ, Abbas AY, Baumli S et al. (2013). Substituted 4-(thiazol-

5-yl)-2-(phenylamino)pyrimidines are highly active CDK9 inhibitors: synthesis, X-ray crystal

structures, structure-activity relationship, and anticancer activities. J Med Chem 56: 640-659.

Smith NF, Raynaud FI, & Workman P (2007). The application of cassette dosing for

pharmacokinetic screening in small-molecule cancer drug discovery. Mol Cancer Ther 6:

428-440.

Southan C, Sharman JL, Benson HE, Faccenda E, Pawson AJ, Alexander SP et al. (2016).

The IUPHAR/BPS Guide to PHARMACOLOGY in 2016: towards curated quantitative

interactions between 1300 protein targets and 6000 ligands. Nucl Acids Res 44: D1054-1068.

Sumi NJ, Kuenzi BM, Knezevic CE, Remsing Rix LL, & Rix U (2015). Chemoproteomics

reveals novel protein and lipid kinase targets of clinical CDK4/6 inhibitors in lung cancer.

ACS Chem Biol 10: 2680-2686.

Tadesse S, Yu M, Mekonnen LB, Lam F, Islam S, Tomusange K et al. (2017). Highly potent,

selective and orally bioavailable 4-thiazol-N-(pyridin-2-yl)pyrimidin-2-amine cyclin-

dependent kinase 4 and 6 inhibitors as anticancer drug candidates: Design, synthesis and

evaluation. J Med Chem 60: 1892-1915

Zhang YX, Sicinska E, Czaplinski JT, Remillard SP, Moss S, Wang Y et al. (2014).

Antiproliferative effects of CDK4/6 inhibition in CDK4-amplified human liposarcoma in

vitro and in vivo. Mol Cancer Ther 13: 2184-2193.

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.K_v11.1

17.MYLK4

Ligands

1. Rb

2. Palbociclib

3. Ribociclib

4. Abemaciclib

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,

GI₅₀^b(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, K_i(μM)^a

4D1^d6D3^d1B^d2A^d7H^d

Structure

X

9T^d

Cmpd

R¹

R²

R³

H

CN CH

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

CN

F

N

CH

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

0.889

> 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

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

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

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

CH₂

> 5

0.037 0.297 0.580 0.076

> 5 3.335

0.003 0.032 2.370 0.206

-

NSO₂CH₃

> 5

CHN(CH₃)₂0.003 0.279

CH₂NH₂

3.037 0.080 ± 0.009 0.362 ± 0.002

-

> 5

H

F

NS^e

> 5

-

2.158 ± 0.305 2.941 ± 0.389

0.048 ± 0.002 0.237 ± 0.031

CH₃

NH

0.004 0.040

0.002 0.055

0.024 0.366

> 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

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

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 (K_i) were calculated using IC₅₀values and the appropriate K_m(ATP) values for

b

c

each kinase. GI₅₀is 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.

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

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. GI₅₀is 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.

Table 3. Pharmacokinetic studies of compounds 82 and 91 in rodents

82

91

PK parameter^a

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)

V_ss(L·kg^-1)

AUC (µM·h)

C_max(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

T_max(h)

t_1/2(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.

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%.

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.

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.

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) K_ivalues 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).

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 × GI₅₀and 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 × GI₅₀and 10 × GI₅₀. Cells were stained with annexin V and PI. The percentage of cells

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.

Article Doi

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

DOI: 10.1111/bph.13974

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Article abstract of DOI:10.1111/bph.13974

Full text of DOI:10.1111/bph.13974

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