Synthesis, structure-activity relationship and biological evaluation of 2,4,5-trisubstituted pyrimidine CDK inhibitors as potential anti-tumour agents

Article abstract of DOI:10.1016/j.ejmech.2013.08.052

A series of 2,4,5-trisubstituted pyrimidines have been synthesised and characterised, which exhibited potent CDK inhibition and anti-proliferative activities. The structure-activity relationship is analysed and a rational for CDK9 selectivity is discussed

Full text of DOI:10.1016/j.ejmech.2013.08.052

Accepted Manuscript
Synthesis, structure-activity relationship and biological evaluation of 2,4,5-
trisubstituted pyrimidine CDK inhibitors as potential anti-tumour agents
Hao Shao, Shenhua Shi, David W. Foley, Frankie Lam, Abdullah Y. Abbas, Xiangrui
Liu, Shiliang Huang, Xiangrui Jiang, Nadiah Baharin, Peter M. Fischer, Shudong
Wang
PII:
S0223-5234(13)00572-2
10.1016/j.ejmech.2013.08.052
EJMECH 6402
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 June 2013
Revised Date: 27 July 2013
Accepted Date: 21 August 2013
Please cite this article as: H. Shao, S. Shi, D.W. Foley, F. Lam, A.Y. Abbas, X. Liu, S. Huang, X. Jiang,
N. Baharin, P.M. Fischer, S. Wang, Synthesis, structure-activity relationship and biological evaluation
of 2,4,5-trisubstituted pyrimidine CDK inhibitors as potential anti-tumour agents, European Journal of
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.08.052.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Synthesis, structure-activity relationship and biological evaluation of
2,4,5-trisubstituted pyrimidine CDK inhibitors as potential anti-tumour
agents
Hao Shaoa, Shenhua Shia, David W. Foleya, Frankie Lama,b, Abdullah Y. Abbasa, Xiangrui
Liua, Shiliang Huanga, Xiangrui Jianga, Nadiah Baharina, Peter M. Fischera and Shudong
Wanga,b*
aSchool of Pharmacy and Centre for Biomolecular Sciences, University of Nottingham,
Nottingham NG7 2RD, UK
bSansom Institute for Health Research, School of Pharmacy and Medical Sciences, University
of South Australia, Adelaide SA 5001, Australia
*Corresponding author. Sansom Institute for Health Research, School of Pharmacy and
Medical Sciences, University of South Australia, Adelaide SA 5001, Australia.
Tel.: +61 883022372; fax: +61 883021087.
E-mail address: shudong.wang@unisa.edu.au (S. Wang).
Keywords: CDK9 inhibitors, inhibitor design, RNA polymerase II, Mcl-1 anti-apoptotic
proteins, anti-cancer drug discovery.
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ABSTRACT
A series of 2, 4, 5-trisubstituted pyrimidines have been synthesized and characterised, which
exhibited potent CDK inhibition and anti-proliferative activities. The structure-activity
relationship is analysed and a rational for CDK9 selectivity is discussed. Compound 9s,
possessing appreciable selectivity for CDK9 over other CDKs, is capable of activating
caspase 3, reducing the level of Mcl-1 anti-apoptotic protein, and inducing cancer cell
apoptosis.
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1. Introduction
Cyclin-dependent kinases (CDKs) are a family of serine threonine protein kinases
involved in cell cycle and transcription. The family includes two major groups: cell cycle
CDKs and transcriptional CDKs. Cyclin D-CDK4/6 and cyclin E-CDK2 promote the cell
cycle transition from G1 to S phase by sequentially phosphorylating the retinoblastoma
protein (Rb). Cyclin A-CDK1/2 and Cyclin B-CDK1 are crucial for progression of S-phase
and G2/M transition, respectively [1, 2]. Development of CDK inhibitors initially focussed
on cell-cycle CDKs, but this approach was challenged when functional redundancy amongst
these CDKs was demonstrated. Cancer cells depleted of CDK1, CDK2 or CDK4/6 continue
to proliferate and CDK2 or CDK4/6 knockout mice are viable [3-7]. The recent advancement
of the selective CDK4/6 inhibitor PD-0332991 into Phase III clinical trial for treatment of
oestrogen receptor (ER)-positive breast cancer however, has revived interest in cell cycle
inhibitors. These inhibitors may be useful therapeutically if administered to appropriate
patients [8, 9].
The second group of CDKs, mainly CDK7 and CDK9, are involved in transcription
regulation through the sequential phosphorylation of the carboxyl terminal domain (CTD)
hepta peptide repeat of the largest subunit of RNA polymerase II. The general transcription
factor IIH, a complex of cyclin H-CDK7 and the RING-finger protein MAT1, phosphorylates
Ser5 of the CTD to initiate transcription [10, 11]. After initiation of transcription, positive
transcription elongation factor b (P-TEFb), consisting of cyclin T-CDK9, first phosphorylates
the SpT5 (p160) subunit of DRB-sensitivity-inducing factor (DSIF) and the negative
elongation factor (NELF), followed by Ser2, and occasionally Ser5, on the CTD of RNAP II
to promote productive RNA elongation [12-14]. CDK9 regulates the transcription, while
CDK7 is also known as a CDK-activating kinase (CAK) and is involved in the cell cycle [10].
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CDKs have been pursued as anti-cancer drug targets for more than a decade and a number
of compounds have been subjected to clinical trials. The first generation of pan-CDKs
inhibitors, such as flavopiridol and R-Roscovitine (seliciclib), are also potent CDK9
inhibitors and their anti-tumour activity has been attributed to down-regulation of CDK9-
mediated anti-apoptotic proteins, especially Mcl-1 [15-17]. An analogue of R-Roscovitine,
CR-8, also reduces the level of Mcl-1 and induces apoptosis through inhibition of CDK9 in
neuroblastoma cells [18]. Dinaciclib, a selective inhibitor of CDK 1, 2 and 9, has been
recently advanced into Phase III clinical studies for the treatment of refractory chronic
lymphocytic leukaemia (CLL), all of which validates continued interest in CDK9 as a
therapeutic target in cancer [19]. Our research into CDK inhibitors has identified several
classes of chemical compounds [20-23]. Here, we report the design, synthesis and biological
evaluation of a novel class of 2,4,5-trisubstituted pyrimidine CDK inhibitors as potential anti-
cancer agents.
2. Chemistry
2.1. Synthesis
The general chemistry for the synthesis of 2,4,5-trisubstituted pyrimidine compounds was
adapted from the methods reported previously [20, 24] and is outlined in Scheme 1. Briefly,
treatment of appropriate thioureas 1 with 3-chloro-2,4-pentadione resulted in 5-
acetylthiazoles 2, followed by heating in N,N-dimethylformamide dimethyl acetal (DMF-
DMA) to get the corresponding enaminones 3. The fluorinated enaminones 4a and 4b were
obtained from 3 by treatment with selectfluor in methanol. The synthesis of tert-butyl 5-(2-
cyanoacetyl)-4-methylthiazol-2-yl(methyl)carbamate 4c started from ethyl-4-methyl-2-
(methylamino)thiazole-5-carboxylate 5, which can be easily formed through cyclization of
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ethyl 2-chloro-3-oxobutanoate and 1-methylthiourea. Following Boc protection and treatment
of 6 with acetonitrile in the presence of LDA afforded 5-(2-cyanoacetyl)-thiazole 7 in high
yield. Enaminone 4c was obtained by heating 7 in DMF-DMA. The guanidine 8 was prepared
by heating aniline and cyanamide in the presence of trimethylsilyl chloride. The final
compounds 9a-s were obtained by treating 4a-c and corresponding guanidines 8 under
microwave-aided reaction conditions.
2.2. Structure-activity relationship analysis
We observed previously that 2C-methylamino group of the thiazole formed hydrogen
bonds in the co-crystal structures of CDK9, which was not always the case in CDK2 [25]. We
therefore designed and synthesised several analogues to probe the effect on potency and
selectivity. All compounds were tested against CDK1, 2, 7 and 9, as well as two human
tumour cell lines (Table 1). The primary amino analogues, i.e. 9a, 9b and 9d, are more potent
in cells compared with the corresponding methylamino analogues, but no CDK9 selectivity
advantage is observed.
Compound 9a, containing a sulfonamide at the meta-position of aniline, inhibits CDK9,
CDK1 and CDK2 potently with single-digit Ki, ranging from 3-7 nM. Replacing the
sulfonamide with bulkier functional groups resulted in 9b (R3 = m-4-acetylpiperazin-1-yl)
and 9c (R3 = m-4-methylpiperazin-1-yl), showing 4-6 fold and 7-fold selectivity for CDK9
over CDK1 and CDK2 respectively. Compound 9b demonstrated the best selectivity over
CDK7, 145-fold, of this series of analogues.
Our previous studies has demonstrated that the CDK9 ATP binding pocket is more
malleable and can accommodate larger functional groups on the aniline ring when compared
with CDK2 [25]. A series of analogues with increased flexibility at the meta position of the
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aniline were thus designed and synthesized. Not only do the sulfonyl and carbonyl linkages
chosen allow for different conformations to be sampled, they may also have beneficial effects
on the drug-like properties of the compounds. Compound 9e (R1 = NHMe, R2 = CN and R3 =
SO2Me) is a potent CDK9 inhibitor, but shows only modest selectivity for CDK9.
Alkylsulfone analogue 9f (R2 = F, R3 = SO2NH(CH2)2OMe) and 9g (R2 = CN, R3 =
SO2NH(CH2)2OMe) did not offer improvement in selectivity when compared to 9e; a
considerable enhancement in CDK9 inhibitory potency was however observed with 9g,
indicating tolerance of methoxyethyl benzenesulfonamide, and that 5C-fluoro is superior to
5C-cyano for CDK9 inhibition. Compound 9h (R2 = CN, R3 = m-SO2-morpholin-4-yl), with a
bulkier morpholinosulfonamide at the meta-position of aniline, enhanced the selectivity for
CDK9 over other CDKs when compared to 9e-g. Introduction of a methyl group at the para-
position while keeping the meta-morpholinosulfonamide resulted in compounds 9i-j, which
showed a further improvement in selectivity for CDK9 over CDK1 and CDK2, however, the
selectivity over CDK7 decreased dramatically. This is consistent with the SAR reported for
another series of CDK9 inhibitors [26]. The C5-fluoro and C5-cyano substituted analogues
were more potent than the corresponding C5-unsubstituted analogues, as evidenced by
compounds 9j and 9i, 9m and 9l. This may be due to electronic effects of the C5-fluoro and
C5-cyano enhancing binding to the hinge region of CDK9. Compound 9k (R1 = NHMe, R2 =
CN, R3 = m-SO2-4-methylpiperazin-1-yl) was the most potent CDK7 inhibitor in this series
with a Ki value of 24 nM. Compounds 9l-o exhibited a similar CDK inhibitory profile, but
9m (R2 = CN, R3 = m-CO-morpholine) offered ~ 43-fold selectivity for CDK9 over CDK7,
albeit with slightly reduced potency. Analogue 9p (R2 = CN, R3 = m-CO-4-methylpiperazin-
1-yl, o-Cl) containing an additional chloro at the ortho-position of aniline showed
dramatically reduced kinase and cellular potency, although it showed a very interesting
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kinase selectivity profile for CDK9 over CDK1, CDK2 and CDK7. Modification of keto-N-
(1-methylpiperidin-4-yl) at the para or meta-position resulted in inhibitors 9q-s. 9s (R2 = CN,
R3 = m-CO-N-1-methylpiperidin-4-yl) exhibited a better selectivity profile for CDK9 versus
CDK1 and CDK2 compared to 9r (R2 = CN, R3 = p-CO-N-1-methylpiperidin-4-yl) and 9q
(R2 = F, R3 = m-CO-N-1-methylpiperidin-4-yl). Interestingly, 9s was also a more potent
CDK9 inhibitor than its corresponding C5-fluoro analogue 9q.
2.3. Molecular docking
Molecular docking experiements were carried out in order to rationalise the observed
selecitivty differences between 9q and 9s. We recently solved the crystal structures of several
members of this series bound to CDK9 and CDK2 [25]. We revealed that ligand binding
could induce significant CDK9 structural changes via a downward movement of the glycine
rich loop. This plasticity, absent in CDK2, may be critical for obtaining CDK9 specificity.
We therefore used crystal structures representative of the open [PDB ID: 4BCF] and closed
[PDB ID: 4BCG] forms of CDK9 as well as a comparable [PDB ID: 4BCP] CDK2 crystal
structure to investigate the binding models of 9q and 9s in an attempt to rationalise the
selectivity profiles observed.
Upon docking the compounds into the closed CDK9 structure [4BCG], both 9q and 9s
adopted an identical conformation (Figure 1A and 1B), giving Chemgauss 4 scores of -
13.2131 and -12.6736, respectively. When the open CDK9 structure [4BCF] was used for the
docking study, as shown in Figure 1C, 9s adopted a conformation similar to that of the
9s/4BCG, resulting in Chemgauss 4 score of -13.5206. In contrast, 9q exhibited a rather
different binding conformation than 9q/4BCG, which resulted in a lower Chemgauss 4 score
of -15.0470 (Figure 1D). This energy difference was driven by the formation of an internal
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hydrogen bond between the 4N-methylpiperdine and the 2C-NH-methylthiazole. We have
shown that the ability of CDK9 to accommodate a closed conformation around these ligands
is crucial for selectivity [23]. 9q appears to prefer the open conformation of CDK9 and this
preference may explain the reduced selectivity when compared to 9s.
In the structure of CDK2 (PDB ID: 4BCP) 9q adopted a conformation such that it became
involved in water-mediated hydrogen bonds connecting Asn132 and Asp145 (Figure 1E). No
such hydrogen-bonding network was observed when 9s was docked into 4BCP which may
also rationalise the increased affinity for CDK2 of 9q when compared to 9s.
3. Pharmacology
3.1. Cytotoxicity in human tumour cell lines
The anti-proliferative activity of each compound was assessed against human HCT-116
colorectal carcinoma and MCF-7 breast cancer cells. Determination of proliferation using the
MTT assay showed that growth was inhibited for the cell lines tested, with 50% growth
inhibition (GI50) values in a range of nM - low µM (Table 1). The pan-CDK inhibitors 9a (R1
= NH2, R2 = F, R3 = m-SO2NH2) and 9d (R1 = NH2, R2 = F, R3 = m-4-methylpiperazin-1-yl)
showed the most potent anti-proliferative properties with GI50 values of 0.05 - 0.09 µM.
However a considerable loss in cellular potency was observed with 9n (R1 = NHMe, R2 = CN,
R3 = m-CO-4-acetylpiperazin-1-yl) giving the GI50 values of 4.96 - 5.88 µM in the tumour
cell lines.
3.2. Cellular mechanism of action
Based on the observed CDK9 selectivity and cellular potency, 9s was selected for detailed
cellular mechanistic studies. A panel of cell lines representing a range of tumour types,
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including colorectal, breast, lung, ovarian, cervical and pancreatic cancers, were treated with
9s for 48 h. As shown in Table 2, 9s exhibited a broad spectrum of anti-proliferative activity
with GI50 ranging from 0.64 – 2.01 µM. HCT-116 and MCF-7 appeared to be the most
sensitive with GI50 = 0.79 and 0.64 µM respectively. We next investigated whether the anti-
proliferative effect of 9s was a consequence of activation of cellular apoptosis. Induction of
caspase 3 activity was measured in HCT-116 cancer cells after treatment with 9s for 24 h
(Figure 2), showing significant activation of caspase 3 at 5× and 10 × GI50 µM concentrations.
Annexin V/PI double staining was then performed to confirm the ability of 9s in induction of
apoptosis. As shown in Figure 3, 9s effectively induced cell apoptosis (Annexin V positive/PI
negative cells) starting from 5 × GI50 µM with an enhanced effect at higher concentrations.
We further examined the effects of a 24-hour exposure of 9s on cell cycle progression (Figure
4). Flow cytometric analysis of HCT-116 cells indicated a slight increase in G1-phase DNA
content at the GI50 concentration, but G2-M arrest predominates following drug exposure at
higher concentrations (5 × and 10 × GI50 µM), accompanied by substantial sub-G1 DNA at
10 × GI50, indicating cell death. The cell-cycle profile is consistent with that previously
observed for other CDK9 inhibitors [20].
Consistent with cellular inhibition of CDK9 by 9s, western blotting analysis of HCT-116
cells upon 24-hours drug treatment showed that phosphorylation at Ser2 of RNAPII CTD was
reduced at 1.0 µM and completely abolished at 5.0 µM (Figure 5). A decrease in phospho-
Ser5 as well as total RNA polymerase II levels at 10 µM was also observed. Anti-apoptotic
protein Mcl-1 expression was reduced at 1.0 µM, the concentration causing cellular CDK9
inhibition, and completely inhibited at 5.0 µM. This was accompanied by PARP cleavage
indicating cell apoptosis.
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4. Conclusion
In conclusion, a series of 2,4,5-trisubstituted pyrimidines were synthesized and their
structure activity relationship was investigated. Most compounds demonstrated low-nM
CDK9 inhibition and anti-proliferative activity in tumour cell lines. Lead compound 9s
showed >20-fold selectivity for CDK9 over CDK1 and CDK2, and 10-fold selectivity over
CDK7. The Cellular mode of action investigation confirmed that 9s is capable of inducing
apoptosis through CDK9 mediated down-regulation of anti-apoptotic protein Mcl-1 and
activation of caspase-3.
5. Experimental section
5.1. Chemistry
Chemical reagents and solvents were obtained from commercial sources. When necessary,
1
solvents were dried and/or purified by standard methods. H NMR and 13C NMR spectra
were obtained using a Bruker 400 Ultrashield spectrometer at 400 and 100 MHz, respectively.
These were analyzed using the Bruker TOPSPIN 2.1 program. Chemical shifts are reported in
parts per million relative to the residual solvent signal. Coupling constants (J) are quoted to
the nearest 0.1 Hz. The following abbreviations are used: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad. High resolution mass spectra were obtained using a Waters
2795 single quadrupole mass spectrometer/micromass LCT platform. Purity for final
compounds was greater than 95% and was measured using Waters high performance liquid
chromatography (Waters 2487 dual λ absorbance detector) with Phenomenex Gemini-NX 5u
C18 110A 250 mm × 4.60 mm column, UV detector at 254 nm, using system A (10% MeCN
containing 0.1% TFA for 2 min, followed by linear gradient 10−100% over 10 min at a flow
rate of 1 mL/min); system B (10-70 % MeCN over 20 min at a flow rate of 1 mL/min);
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system C (10% MeOH containing 0.1% TFA for 4 min, followed by linear gradient 10−100%
over 10 min at a flow rate of 1 mL/min); and system D (10% MeOH containing 0.1% TFA
for 4 min, followed by linear gradient 10−100% over 6 min at a flow rate of 1 mL/min).
Flash chromatography was performed using either a glass column packed with silica gel
(200−400 mesh, Aldrich Chemical) or prepacked silica gel cartridges (FlashMaster systems,
Biotage). Melting points were determined with an Electrothermal melting point apparatus and
are uncorrected.
5.1.1. N'-(5-3-(dimethylamino)-2-fluoroacryloyl)-4-methylthiazol-2-yl)-N,N-
dimethylformimidamide (4b).
To a well-stirred solution of N'-(5-(3-(dimethylamino)acryloyl)-4-methylthiazol-2-yl)-
N,N-dimethylformimidamide 3b (5.0 mmol) in MeOH under ice bath, selectfluor (7.5 mmol)
was added and the mixture was stirred for 1 hour. After completion of the reaction, the
mixture was concentrated and purified by column chromatography using EtoAc/MeOH to
1
yield the titled compound. Yellow solid (22 %). H-NMR (DMSO-d6): δ 2.41 (s, 3H, CH3),
2.98 (s, 3H, CH3), 3.06 (d, 6H, J = 1.6 Hz, 2×CH3), 3.18 (s, 3H, CH3), 6.95 (d, 1H, J = 29.6
Hz, CH), 8.38 (s, 1H, NH). HR-MS (ESI+): m/z [M + H]+ C12H17FN4OS calcd for 285.1185;
found 285.1206.
5.1.2. General method for the synthesis of compounds (9a-9s).
A mixture of the appropriate enaminones 4a-c, 1-phenylguanidine 8 (2 equivalent mmol)
in 2-methoxyethanol (0.2 mL/mmol) was heated in a Discovery Microwave at 100-140 °C for
30-45 minutes. Upon cooling, the residue was purified by flash chromatography using
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appropriate mixtures of EtOAc/PE or EtOAc/MeOH as the eluant. The products were further
purified by crystallization from EtOAc-MeOH mixtures.
5.1.3. 3-((4-(2-Amino-4-methylthiazol-5-yl)-5-fluoropyrimidin-2-yl)amino)benzenesulfon
amide (9a).
From N'-(5-(3-(dimethylamino)-2-fluoroacryloyl)-4-methylthiazol-2-yl)-N,N-
dimethylformimidamide and 3-guanidinobenzenesulfonamide. Light Yellow powder (10 %);
mp: 264 °C (dec). Anal. RP-HPLC: tR 9.00 min (method A), purity 95 %. 1H-NMR (DMSO-
d6): δ 2.48 (d, 3H, J = 2.0 Hz, CH3), 7.30 (s, 2H, NH2), 7.41 (dt, 1H, J = 8.0, 1.2 Hz, Ph-H),
7.47 (t, 1H, J = 8.0 Hz, Ph-H), 7.60 (s, 2H, NH2), 7.91-7.97 (m, 1H, Ph-H), 8.20 (t, 1H, J =
2.0 Hz, Ph-H), 8.46 (d, 1H, J = 3.6 Hz, Py-H), 9.82 (s, 1H, NH). 13C-NMR (DMSO-d6): δ
19.19, 109.90 (d, J = 9.0 Hz), 115.92, 118.79, 121.90, 129.52, 141.47, 144.91, 145.88 (d, J =
26 Hz), 147.82, 147.98 (d, J = 249 Hz), 155.47, 156.06, 170.74 (d, J = 5.0 Hz). HR-MS
(ESI+): m/z [M + H]+ calcd for C14H14FN6O2S2, 381.0604; found 381.0475.
5.1.4. 1-(4-(3-(4-(2-Amino-4-methylthiazol-5-yl)-5-fluoropyrimidin-2-ylamino)phenyl)
piperazin-1-yl)ethanone (9b).
From 1-(4-acetylpiperazin-1-yl)phenyl)guanidine and N'-(5-(3-(dimethylamino)-2-
fluoroacryloyl)-4-methyl thiazol-2-yl)-N,N-dimethyl formimidamide. Light Yellow powder
1
(10%); mp 235 °C (dec). Anal. RP-HPLC: tR 8.90 min (method A), purity 98 %. H-NMR
(DMSO-d6): δ 2.05 (s, 3H, CH3), 2.41 (d, 3H, J = 2.8 Hz, CH3), 3.08 (apparent t, 2H, J = 5.2
Hz, CH2), 3.15 (apparent t, 2H, J = 5.2 Hz, CH2), 3.53-3.63 (m, 4H, 2×CH2), 6.57 (dd, 1H, J
= 8.0, 2.0 Hz, Ph-H), 7.12 (t, 1H, J = 8.0 Hz, Ph-H), 7.22 (dd, 1H, J = 8.0, 1.2 Hz, Ph-H),
7.38 (t, 1H, J = 2.0 Hz, Ph-H), 7.56 (s, 2H, NH2), 8.42 (d, 1H, J = 3.6 Hz, Py-H), 9.36 (s, 1H,
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NH). 13C-NMR (DMSO-d6): δ 18.98 (d, J = 6 Hz), 21.68, 41.17, 45.94, 49.04, 49.47, 107.01,
109.87, 110.80, 110.91(d, J = 8 Hz), 129.31, 141.76, 146.23 (d, J = 25 Hz), 147.28 (d, J = 12
Hz), 147.71 (d, J = 248 Hz), 151.70, 154.26, 156.46 (d, J = 2 Hz), 168.78, 170.49 (d, J = 4
Hz). HR-MS (ESI+): m/z [M + H]+ calcd for C20H23FN7OS, 428.1669; found 428.1736.
5.1.5 5-(5-Fluoro-2-(3-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-yl)-N,4-
dimethylthiazol-2-amine (9c).
From 1-(3-(4-methylpiperazin-1-yl)phenyl)guanidine and 3-(dimethylamino)-2-fluoro-1-
(4-methyl-2-(methylamino)thiazol-5-yl)prop-2-en-1-one. Yellow solid (10 %); mp 179-180
°C. Anal. RP-HPLC: tR 8.73 min (method A), purity 99 %. 1H-NMR (DMSO-d6): δ 2.23 (s,
3H, CH3), 2.44 (s, 3H, CH3), 2.80-2.93 (m, 4H, 2×CH2), 3.12 (s, 4H, 2×CH2), 6.54 (d, 2H, J
= 6.8 Hz, Ph-H), 7.09 (t, 1H, J = 7.6 Hz, Ph-H), 7.18 (d, 1H, J = 7.6 Hz, Ph-H), 7.33 (s, 1H,
Ph-H), 8.10 (s, 1H, NH), 8.43 (s, 1H, Py-H), 9.32 (s, 1H, NH). HR-MS (ESI+): m/z [M + H]+
calcd for C20H25FN7S, 414.1876; found 414.1973.
5.1.6 5-(5-Fluoro-2-(3-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-yl)-4-
methylthiazol-2-amine (9d).
From 1-(3-(4-methylpiperazin-1-yl)phenyl)guanidine and N'-(5-(3-(dimethylamino)-2-
fluoroacryloyl)-4-methylthiazol-2-yl)-N,N-dimethylformimidamide. Light yellow powder (6
%); mp 146-148 °C. Anal. RP-HPLC: tR 11.10 min (method C), purity 98 %. 1H-NMR
(DMSO-d6): δ 2.23 (s, 3H, CH3), 2.41 (d, 3H, J = 2.8 Hz, CH3), 2.46 (apparent t, 4H, J = 4.8
Hz, 2×CH2), 3.12 (apparent t, 4H, J = 4.4 Hz, 2×CH2), 6.54 (dd, 2H, J = 8.4, 2.0 Hz, Ph-H),
7.09 (t, 1H, J = 8.0 Hz, Ph-H), 7.18 (d, 1H, J = 8.0 Hz, Ph-H), 7.32 (s, 1H, Ph-H), 7.55 (s, 2H,
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NH2), 8.42 (d, 1H, J = 3.6 Hz, Py-H), 9.31 (s, 1H, NH). HR-MS (ESI+): m/z [M + H]+ calcd
for C19H23FN7S, 400.1720; found 400.1459.
5.1.7
pyrimidine-5-carbonitrile (9e).
From tert-butyl
4-(4-Methyl-2-(methylamino)thiazol-5-yl)-2-((3-(methylsulfonyl)phenyl)amino)
(5-(2-cyano-3-(dimethylamino)acryloyl)-4-methylthiazol-2-
yl)(methyl)carbamate and 1-(3-(methylsulfonyl)phenyl)guanidine. Yellow solid (12 %); mp
1
277-278 °C. Anal. RP-HPLC: tR 5.47 min (method B), purity 97%. H-NMR (DMSO-d6): δ
2.44 (s, 3H, CH3), 2.90 (d, 3H, J = 4.8 Hz, CH3), 3.21 (s, 3H, CH3), 7.56-7.66 (m, 2H, 2×Ph-
H), 7.93-8.04 (m, 1H, Ph-H), 8.31 (q, 1H, J = 4.8 Hz, NH), 8.41 (s, 1H, Ph-H), 8.85 (s, 1H,
Py-H), 10.58 (s, 1H, NH). 13C-NMR (DMSO-d6): δ 20.08, 31.42, 44.15, 94.78, 118.08,
118.36, 121.59, 125.25, 130.28, 140.39, 141.76, 156.49, 159.20, 161.45, 164.19. HR-MS
(ESI+): m/z [M + H]+ calcd for C17H17N6O2S2, 401.0854; found 401.2841.
5.1.8
methoxyethyl)benzenesulfonamide (9f).
From tert-butyl (5-(2-cyano-3-(dimethylamino)acryloyl)-4-methylthiazol-2-
3-((5-Cyano-4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)-N-(2-
yl)(methyl)carbamate and 3-guanidino-N-(2-methoxyethyl)benzenesulfonamide. Yellow
1
solid (7 %); mp 202-203 °C. Anal. RP-HPLC: tR 4.87 min (method B), purity 97 %. H-
NMR (DMSO-d6): δ 2.44 (s, 3H, CH3), 2.89 (d, 3H, J = 4.8 Hz, CH3), 2.93 (q, 2H, J = 5.6
Hz, CH2), 3.16 (s, 3H, CH3), 3.29 (t, 2H, J = 5.6 Hz, CH2), 7.48 (dt, 1H, J = 8.0, 1.6 Hz, Ph-
H), 7.56 (t, 1H, J = 8.0 Hz, Ph-H), 7.71 (t, 1H, J = 5.6 Hz, NH), 7.93 (d, 1H, J = 8.4 Hz, Ph-
H), 8.24 (s, 1H, Ph-H), 8.29 (q, 1H, J = 4.8 Hz, NH), 8.83 (s, 1H, Py-H), 10.50 (br s, 1H,
NH). 13C-NMR (DMSO-d6): δ 20.03, 31.39, 42.67, 58.31, 70.97, 94.65, 118.12, 118.48,
14

ACCEPTED MANUSCRIPT
121.16, 124.20, 129.95, 140.06, 141.65, 159.27, 161.48, 164.16, 170.91. HR-MS (ESI+): m/z
[M + H]+ calcd for C19H22N7O3S2, 460.1226; found, 460.1713.
5.1.9
3-(5-Fluoro-4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-ylamino)-N-(2-
methoxyethyl)benzenesulfonamide (9g).
From 3-guanidino-N-(2-methoxyethyl)benzenesulfonamide and 1-(4-(4-(5-fluoro-4-(4-
methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-ylamino)phenyl)piperazin-1-yl)ethanone.
Yellow solid (20 %); mp: 193-195 °C. Anal. RP-HPLC: tR 13.97 min (method C), purity 100
1
%. H-NMR (DMSO-d6): δ 2.49 (s, 3H, CH3), 2.88 (d, 3H, J = 4.8 Hz, CH3), 2.93 (q, 2H, J =
6.0 Hz, CH2), 3.16 (s, 3H, CH3), 3.31 (t, 2H, J = 6.0 Hz, CH2), 7.36 (dt, 1H, J = 8.0, 1.6 Hz,
Ph-H), 7.49 (t, 1H, J = 8.0 Hz, Ph-H), 7.65 (t, 1H, J = 5.6 Hz, NH), 7.92 (dd, 1H, J = 8.4, 1.6
Hz, Ph-H), 8.14 (q, 1H, J = 4.8 Hz, NH),8.23 (t, 1H, J = 2.0 Hz, Ph-H), 8.47 (d, 1H, J = 3.6
Hz, Py-H), 9.85 (s, 1H, NH). 13C-NMR (DMSO-d6): δ 19.38 (d, J = 5 Hz), 31.34, 42.65,
58.32, 70.99, 110.05 (d, J = 8 Hz), 116.57, 119.29, 122.30, 129.75, 141.46, 141.67, 145.99 (d,
J = 25 Hz), 147.62 (d, J = 12 Hz), 147.98 (d, J = 249 Hz), 155.59, 155.99, 171.36. HR-MS
(ESI+): m/z [M + H]+ calcd for C18H22FN6O3S2, 453.1179; found 453.0728.
5.1.10
amino)pyrimidine-5-carbonitrile (9h).
From tert-butyl (5-(2-cyano-3-(dimethylamino)acryloyl)-4-methylthiazol-2-
4-(4-Methyl-2-(methylamino)thiazol-5-yl)-2-((3 (morpholinosulfonyl)phenyl)
yl)(methyl)carbamate and 1-(3-(morpholinosulfonyl)-phenyl)guanidine. Yellow solid (25 %);
1
mp: 240-241 °C. Anal. RP-HPLC: tR 9.8 min (method B), purity 95 %. H-NMR (DMSO-
d6): δ 2.43 (s, 3H, CH3), 2.89 (d, 7H, J = 4.8 Hz, CH3 & 2 × CH2), 3.63 (apprent t, 4H, J =
4.8 Hz, 2×CH2), 7.40 (d, 1H, J = 8.4 Hz, Ph-H), 7.63 (t, 1H, J = 8.0 Hz, Ph-H), 8.03 (d, 1H,
15

ACCEPTED MANUSCRIPT
J = 8.0 Hz, Ph-H), 8.22 (s, 1H, Ph-H), 8.29 (q, 1H, J = 4.8 Hz, NH), 8.85 (s, 1H, Py-H),
10.55 (br s, 1H, NH). 13C-NMR (DMSO-d6): δ 19.98, 31.38, 46.41, 65.74, 95.03, 118.03,
119.08, 122.21, 125.04, 130.19, 135.38, 140.48, 159.24, 161.57, 164.15, 170.93. HR-MS
(ESI+): m/z [M + H]+ calcd for C20H22N7O3S2, 472.1226; found, 472.1476.
5.1.11
5-(5-Fluoro-2-(4-methyl-3-(morpholinosulfonyl)phenylamino)pyrimidin-4-yl)-N,4-
dimethylthiazol-2-amine (9j).
From 1-(4-methyl-3-(morpholinosulfonyl) phenyl)guanidine and 1-(4-(4-(5-fluoro-4-(4-
methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-ylamino)phenyl)piperazin-1-yl)ethanone.
Yellow solid (19 %); mp: 232-234 °C. Anal. RP-HPLC: tR 14.75 min (method A), purity
100 %. 1H-NMR (DMSO-d6): δ 2.48 (d, 3H, J = 2.8 Hz, CH3), 2.88 (d, 3H, J = 4.8 Hz, CH3),
3.05 (apparent t, 4H, J = 4.8 Hz, 2×CH2), 3.63 (apparent t, 4H, J = 4.8 Hz, 2×CH2), 7.36 (d,
1H, J = 4.8 Hz, Ph-H), 7.95 (dd, 1H, J = 8.4, 2.4 Hz, Ph-H), 8.13 (q, 1H, J = 4.8 Hz, NH),
8.16 (d, 1H, J = 2.4 Hz, Ph-H), 8.46 (d, 1H, J = 3.2 Hz, Py-H), 9.78 (s, 1H, NH). 13C-NMR
(DMSO-d6): δ 19.34 (d, J = 5 Hz), 20.12, 31.32, 45.77, 60.22, 66.04, 110.06 (d, J = 8 Hz),
119.66, 123.17, 129.66, 133.51, 135.04, 139.44, 145.95 (d, J = 27 Hz), 147.70 (d, J = 13 Hz ),
147.26 (d, J = 248 Hz), 155.37, 156.04, 171.24. HR-MS (ESI+): m/z [M + H]+ calcd for
C20H24FN6O3S2, 479.1335; found 479.1472.
5.1.12 4-(4-Methyl-2-(methylamino)thiazol-5-yl)-2-((3-((4-methylpiperazin-1-yl)sulfonyl)
phenyl)amino)pyrimidine-5-carbonitrile (9k).
From
tert-butyl
(5-(2-cyano-3-(dimethylamino)acryloyl)-4-methylthiazol-2-
yl)(methyl)carbamate and 1-(3-((4-methylpiperazin-1-yl)sulfonyl)phenyl)guanidine. Brown
1
solid (17 %); mp: > 300 °C. Anal. RP-HPLC: tR 11.03 min (method D), purity 97 %. H-
16

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NMR (DMSO-d6): δ 2.12 (br s, 3H, CH3), 2.30-2.38 (m, 2H, CH2), 2.40-2.46 (m, 2H, CH2),
2.84-2.95 (m, 7H, CH3 & 2 × CH2), 3.33 (s, 3H, CH3), 7.39 (d, 1H, J = 7.9 Hz, Ph-H), 7.60 (t,
1H, J = 8.0 Hz, Ph-H ), 8.01 (d, 1H, J = 7.9 Hz, Ph-H), 8.21 (br s, 1H, Ph-H), 8.30 (q, 1H, J =
4.8 Hz, NH), 8.84 (s, 1H, Py-H), 10.54 (br s, 1H, NH). 13C-NMR (DMSO-d6): δ 19.5, 30.9,
45.3, 45.8, 53.5, 94.5, 114.1, 117.6, 118.5, 121.6, 124.4, 129.6, 135.4, 139.9, 155.8, 158.8,
161.1, 163.7, 170.6. HR-MS (ESI+): m/z [M + H]+ calcd for C21H25N8O2S2 485.1536; found,
485.1530.
5.1.13
(3-((4-(4-Methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)phenyl)
(morpholino)methanone (9l).
From 3-(dimethylamino)-1-(4-methyl-2-(methylamino)thiazol-5-yl)prop-2-en-1-one and
1-(3-(morpholine-4-carbonyl)phenyl)guanidine. Yellow solid (64 %); mp 262-264 °C. Anal.
1
RP-HPLC: tR 11.56 min (method B), purity 100 %. H-NMR (DMSO-d6): δ 2.48 (s, 3H,
CH3), 2.86 (d, 3H, J = 4.8Hz, CH3), 3.61 (s, 8H, 4×CH2), 6.90-7.00 (m, 2H, Py-H & Ph-H),
7.35 (t, 1H, J = 8.0 Hz, Ph-H), 7.81 (dd, 1H, J = 8.4, 1.2 Hz, Ph-H), 7.90 (s, 1H, CH), 8.08 (q,
1H, J = 4.8 Hz, NH), 8.35 (d, 1H, J = 5.2Hz, Py-H), 9.60 (s, 1H, NH). 13C-NMR (DMSO-d6):
δ 19.11, 31.33, 66.62, 107.64, 117.45, 118.17, 119.97, 120.04, 129.03, 136.25, 141.17,
153.06, 158.09, 159.11, 159.81, 169.73, 169.94. HR-MS (ESI+): m/z [M + H]+ calcd for
C20H23N6O2S, 411.1603; found, 411.1607.
5.1.14
thiazol-5-yl)pyrimidine-5-carbonitrile (9n).
From tert-butyl (5-(2-cyano-3-(dimethylamino)acryloyl)-4-methylthiazol-2-
yl)(methyl)carbamate and carbonyl)acrylonitrile
2-((3-(4-Acetylpiperazine-1-carbonyl)phenyl)amino)-4-(4-methyl-2-(methylamino)
and 1-(3-(4-acetylpipera-zine-1-
17

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carbonyl)phenyl)guanidine. Yellow solid (54 %); mp 200-201°C. Anal. RP-HPLC: tR 8.12
min (method B), purity 100 %. 1H-NMR (DMSO-d6): δ 2.02 (s, 3H, CH3), 2.42 (s, 3H, CH3),
2.88 (d, 3H, J = 4.8 Hz, CH3), 3.45 (br s, 8H, 4×CH2), 7.11 ( d, 1H, J = 7.6 Hz Ph-H), 7.42 ( t,
1H, J = 8.0 Hz Ph-H), 7.75 ( d, 1H, J = 8.0 Hz Ph-H), 7.90 ( s, 1H, Ph-H), 8.26 (q, 1H, J =
4.8 Hz, NH), 8.81 (s, 1H, Py-H), 10.39 (br s, 1H, NH). 13C-NMR (DMSO-d6): δ 19.89, 21.69,
31.31, 94.50, 118.15, 119.20, 121.88, 122.03, 129.29, 136.42, 139.51, 159.36, 161.51, 164.05,
169.00, 169.50, 170.65. HR-MS (ESI+): m/z [M + H]+ calcd for C23H25N8O2S, 477.1821;
found, 477.1925.
5.1.15 4-(4-Methyl-2-(methylamino)thiazol-5-yl)-2-((3-(4-methylpiperazine-1-carbonyl)
phenyl)amino)pyrimidine-5-carbonitrile (9o).
From
tert-butyl
(5-(2-cyano-3-(dimethylamino)acryloyl)-4-methylthiazol-2-
yl)(methyl)carbamate and 1-(3-(4-methylpiperazine-1-carbonyl)phenyl)guanidine. Yellow
1
solid (21 %); mp 139-140 °C. Anal. RP-HPLC: tR 3.04 min (method B), purity 100 %. H-
NMR (MEOD-d4): δ 0.80 (s, 3H, CH3), 0.89 (s, 2H, CH2), 0.97 (s, 3H, CH3), 1.03 (s, 2H,
CH2), 1.48 (s, 3H, CH3), 2.00 (s, 2H, CH2), 2.28 (s, 2H, CH2), 5.71 (d, 1H, J = 7.6 Hz, Ph-H),
5.92 ( t, 1H, J = 7.6 Hz, Ph-H), 6.17 (d, 1H, J = 7.6 Hz, Ph-H), 6.52 (s, 1H, Ph-H), 7.17 (s,
1H, Py-H). 13C-NMR (DMSO-d6): δ 19.92, 31.35, 45.99, 54.69, 94.47, 118.16, 118.98,
121.62, 121.89, 129.25, 136.74, 139.46, 159.34, 161.51, 164.02, 169.22. HR-MS (ESI+): m/z
[M + H]+ calcd for C22H25N8OS, 449.1827; found, 449.3006.
5.1.16
2-((2-Chloro-5-(4-methylpiperazine-1-carbonyl)phenyl)amino)-4-(4-methyl-2-
(methylamino)thiazol-5-yl)pyrimidine-5-carbonitrile (9p).
18

ACCEPTED MANUSCRIPT
From
tert-butyl
(5-(2-cyano-3-(dimethylamino)acryloyl)-4-methylthiazol-2-
yl)(methyl)carbamate and 1-(2-chloro-5-(4-methylpiperazine-1-carbonyl)phenyl)guanidine.
Yellow solid (9 %); mp: 162-163 °C. Anal. RP-HPLC: tR 3.04 min (method B), purity 100 %.
1H-NMR (MEOD-d4): δ 0.89 (s, 3H, CH3), 0.97 (s, 3H, CH3), 1.18 (br s, 4H, 2×CH2), 1.47
(s, 3H, CH3), 2.15 (br s, 4H, 2×CH2), 5.77 (dd, 1H, J = 8.0, 2.0 Hz, Ph-H), 6.11 (apparant d,
1H, J = 8.0 Hz, Ph-H), 6.60 (d, 1H, J = 1.6 Hz, Ph-H), 7.16 (s, 1H, Py-H). HR-MS (ESI+):
m/z [M + H]+ calcd for C22H24ClN8OS, 483.1482; find 483.1646.
5.1.17
3-(5-Fluoro-4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-ylamino)-N-(1-
methylpiperidin-4-yl)benzamide (9q).
From 3-guanidino-N-(1-methylpiperidin-4-yl)benzamide and (5-(3-(dimethylamino)-2-
fluoroacryloyl)-4-methyl thiazol-2-yl)-N,N-dimethyl formimidamide. Yellow solid (10 %);
mp: 235-237 °C. Anal. RP-HPLC: tR 8.60 min (method A), purity 100 %. 1H-NMR (DMSO-
d6): δ 1.51-1.66 (m, 2H, CH2), 1.76 (apparent d, 2H, J = 10.4 Hz, CH2), 1.99 (t, 2H, J = 12.0
Hz, CH2), 2.19 (s, 3H, CH3), 2.48 (d, 3H, J =1.6 Hz, CH3), 2.79 (d, 2H, J = 11.2 Hz, CH2),
2.88 (d, 3H, J = 4.8 Hz, CH3), 3.67-3.80 (m, 1H, CH), 7.30-7.34 (m, 2H, 2×Ph-H), 7.80 (d,
1H, J = 8.0 Hz, Ph-H), 8.07-8.21 (m, 3H, Ph-H & 2×NH), 8.44 (d, 1H, J = 3.2 Hz, Py-H),
9.62 (s, 1H, NH). 13C-NMR (DMSO-d6): δ 19.41, 31.29, 31.72, 46.24, 46.80, 54.85, 109.99,
118.72, 120.47, 121.72, 128.57, 135.92, 140.98, 146.06 (d, J = 26 Hz), 147.41 (d, J = 12 Hz),
147.78 (d, J = 248 Hz), 155.42, 156.29, 166.58, 171.13. HR-MS (ESI+): m/z [M + H]+ calcd
for C22H27FN7OS, 456.1982; found 456.1998.
5.1.18
4-(5-Cyano-4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-ylamino)-N-(1-
methylpiperidin-4-yl)benzamide (9r).
19

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From 4-guanidino-N-(1-methylpiperidin-4-yl)benzamide and 3-(dimethylamino)-2-(4-
methyl-2-(methylamino)thiazole-5-carbonyl)acrylonitrile. Yellow solid (13 %); mp: 230-232
1
°C. Anal. RP-HPLC: tR 8.40 min (method A), purity 100 %. H-NMR (DMSO-d6): δ 1.52-
1.66 (m, 2H, CH2), 1.76 (apparent d, 2H, J = 9.6 Hz, CH2), 2.00 (t, 2H, J = 10.8 Hz, CH2),
2.20 (s, 3H, CH3), 2.44 (s, 3H, CH3), 2.81 (d, 2H, J = 11.6 Hz, CH2), 2.90, (d, 3H, J = 4.4 Hz,
CH3), 3.68-3.80 (m, 1H, CH), 7.78-7.89 (m, 4H, 4×Ph-H), 8.13 (d, 1H, J = 7.6 Hz, NH), 8.28
(q, 1H, J = 4.4 Hz, NH), 8.83 (s, 1H, Py-H), 10.45 (s, 1H, NH). 13C-NMR (DMSO-d6): δ
20.02, 31.32, 31.74, 46.19, 46.79, 54.85, 94.66, 114.35, 118.12, 119.66, 128.42, 129.23,
141.99, 155.95, 159.20, 161.47, 164.06, 165.68, 170.75. HR-MS (ESI+): m/z [M + H]+
calcd for C23H27N8OS, 463.2029; found 463.1533.
5.1.19
3-(5-Cyano-4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-ylamino)-N-(1-
methylpiperidin-4-yl)benzamide (9s).
From 3-guanidino-N-(1-methylpiperidin-4-yl)benzamide and 3-(dimethylamino)-2-(4-
methyl-2-(methylamino)thiazole-5-carbonyl)acrylonitrile. Yellow solid (18 %); mp: 168-170
°C. Anal. RP-HPLC: tR 8.39 min (method A), purity 99 %. 1H-NMR (DMSO-d6): δ 1.51-1.63
(m, 2H, CH2), 1.76 (apparent d, 2H, J = 10.0 Hz, CH2), 1.99 (t, 2H, J = 11.2 Hz, CH2), 2.19
(s, 3H, CH3), 2.42 (s, 3H, CH3), 2.79 (d, 2H, J = 11.2 Hz, CH2), 2.89 (d, 3H, J = 4.8 Hz, CH3),
3.17 (s, 3H, CH3), 3.68-3.80 (m, 1H, CH), 7.40 (t, 1H, J = 8.0 Hz, Ph-H), 7.51 (d, 1H, J = 8.0
Hz, Ph-H), 7.81 (d, 1H, J = 7.6 Hz, Ph-H), 8.17 (s, 1H, Ph-H), 8.21 (d, 1H, J = 7.6 Hz, NH),
8.28 (q, 1H, J = 4.8 Hz, NH), 8.79 (s, 1H, Py-H), 10.32 (s, 1H, NH). 13C-NMR (DMSO-d6): δ
20.03, 31.31, 31.70, 46.24, 46.86, 54.83, 93.99, 118.29, 120.53, 122.33, 123.60, 128.77,
135.95, 139.32, 156.49, 159.42, 161.40, 164.11, 166.21, 170.64. HR-MS (ESI+): m/z [M
+ H]+ C23H27N8OS, 463.2027; found 463.2188.
20

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5.2. Modelling Methods
Up to 2000 conformations of the individual compounds to be docked were generated using
OpenEye OMEGA software (version 2.4.6), with all other settings on default. Receptors of
PDB structures 4BCF, 4BCG and 4BCP were generated using the OpenEye MAKE
RECEPTOR program, using default settings. Hydrogen bond acceptor constraints between
the ligand and the amide nitrogen of Cys106 (CDK9) or Leu83 (CDK2) and hydrogen bond
donor constraints between the ligand and the carbonyl of Cys106 (CDK9) or Leu83 (CDK2)
were enforced. Docking was performed using OpenEye OEDOCK software (version 3.0.0).
The number of alternate poses retuned was set to 10 and all other settings were on default.
FRED Chemgauss 4 was used as the scoring function. Results were visualised using
OpenEye VIDA software (version 4.1.2) and images generated using DeLano Scientific LLC
PyMOL freeware (version 0.99).
5.3. Biological assays
5.3.1 Kinase assay
Inhibition of kinases was measured by radiometric assay using the Millipore
KinaseProfiler services. Half-maximal inhibition (IC50) values were calculated from 10-point
dose–response curves and apparent inhibition constants (Ki) were calculated from the IC50
values and appropriate KM (ATP) values for the kinases in question.
5.3.2 MTT toxicity assays
All cancer cell lines were obtained from the cell bank at the Centre for Biomolecular
Sciences, University of Nottingham, UK, and were maintained in RPMI-1640 with 10% FBS.
21

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MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) assays were
performed as reported previously [27]. The concentrations required to inhibit 50% of cell
growth (GI50) were calculated using non-linear regression analysis.
5.3.3 Caspase-3 assay
Caspase-3 activity was determined following the manufacturer’s instructions (CASP3F-
1KT, Sigma). Fluorescence was measured at 460 nm at room temperature using an EnVision
multilabel plate reader (PerkinElmer).
5.3.4 Annexin V/PI staining and Cell cycle analysis
Cells (2×105) were seeded in 6-well plate and were treated with varying concentrations of
inhibitor for 24 hours. Cell cycle status was analyzed using a Beckman Coulter EPICS-XL
MCL flow cytometer, and data were analyzed using EXPO32 software. Apoptosis was also
confirmed using FITC annexin V/PI (propidium idodide) staining according to the protocols
(BD Bioscience). The annexin V/PIpositive apoptotic cells were enumerated using flow
cytometry. The percentage of cells undergoing apoptosis was defined as the sum of early
apoptosis (annexin V-positive and PI-negative cells) and late apoptosis (annexin V-positive
and PI-positive cells).
5.3.5 Western blots
Western blotting was performed either with Simple Western assay by Simon (Protein
Simple) according to manufacturer instruction or as previously described. Antibodies used
were as follows: total RNAPII, phosphorylated RNAPII Ser2 and Ser5 (Covance), β-Actin,
Mcl-1, and PARP (Cell Signalling Technologies). Both anti-mouse and anti-rabbit
22

ACCEPTED MANUSCRIPT
immunoglobulin G (IgG) horseradish peroxidase conjugated antibodies were obtained from
Dako.
ACKNOWLEDGMENTS
This study was supported by Cancer Research UK Grants C21568/A8988 and
C21568/A12474.
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Table 1 Structure and biological activity summary
Cytotoxicity
Structure
Kinase inhibition Ki (nM)a
(GI50, µM)b
Comp
R1
R2 R3
CDK9T1 CDK1B CDK2A CDK7H HCT-116 MCF-7
m-SO2NH2
NH2
F
F
9a
9b
3
7
3
252
0.05
0.17
0.41
0.42
m-4-acetylpiperazin-1-
yl
NH2
11
41
77
1,457
m-4-methylpiperazin-1-
yl
m-4-methylpiperazin-1-
yl
NHMe
F
F
9c
7
32
65
41
68
541
476
0.51
0.09
0.62
0.09
NH2
9d
11
m-SO2Me
NHMe CN
NHMe CN
9e
9f
5
19
34
10
73
43
22
6
110
180
30
0.20
0.19
0.30
0.30
0.43
0.26
0.72
0.82
m-SO2NH(CH2)2OCH3
16
3
NHMe
NHMe
F
m-SO2NH(CH2)2OCH3
m-SO2-morpholine
9g
9h
CN
14
55
893
m-SO2-morpholine, p-
NHMe
NHMe
H
F
9i
27
11
35
357
101
576
294
130
584
150
133
24
0.94
0.70
0.61
1.61
1.50
1.15
Me
m-SO2-morpholine, p-
9j
9k
Me
m-SO2-4-methyl
NHMe CN
piperazin-1-yl
NHMe
NHMe
H
m-CO-morpholine
9l
95
43
261
205
485
199
393
0.52
0.59
0.55
0.82
CN m-CO-morpholine
m-CO-4-
9m
1,870
NHMe CN
NHMe CN
NHMe CN
9n
90
9p
9q
9r
9s
17
19
715
36
8
166
313
134
164
552
840
5.88
0.72
4.96
1.38
1.93
0.94
0.51
0.64
acetylpiperazin-1-yl
m-CO-4-methyl
piperazin-1-yl
m-CO-4-methyl
piperazin-1-yl,o-Cl
m-CO-N-(1-
methylpiperidin-4-yl)
p-CO-N-(1-
methylpiperidin-4-yl)
m-CO-N-(1-
>5,000 >5,000 >5,000 1.47
NHMe
F
64
231
32
542
304
163
0.47
0.18
0.79
NHMe CN
NHMe CN
43
14
262
316
methylpiperidin-4-yl)
aApparent inhibition constants (Ki) were calculated from IC50 values and the appropriate Km
b
(ATP) values for each kinase. Anti-proliferative activity by MTT-48 h assay; the data given
are mean values derived from at least three replicates.

ACCEPTED MANUSCRIPT
Table 2 Anti-proliferative activity of 9s in human cancer cell lines
Human Cell line
Origin
48h-MTT
Designation
HCT-116
MCF-7
GI50 (µM) + S.D.
0.79 ± 0.08
0.64 ± 0.08
Colon Carcinoma
Breast Carcinoma
MDA-MB468 1.51 ± 0.34
Lung Carcinoma
A549
A2780
HeLa
2.01 ± 0.55
1.00 ± 0.11
0.90 ± 0.07
1.25 ± 0.26
Ovarian Carcinoma
Cervical Carcinoma
Pancreatic carcinoma Miacapa-2