Discovery of 5-(2-(phenylamino)pyrimidin-4-yl)thiazol-2(3H)-one derivatives as potent Mnk2 inhibitors: Synthesis, SAR analysis and biological evaluation

Article abstract of DOI:10.1002/cmdc.201300552

Phosphorylation of eIF4E by human mitogen-activated protein kinase (MAPK)-interacting kinases (Mnks) is crucial for human tumourigenesis and development. Targeting Mnks may provide a novel anticancer therapeutic strategy. However, the lack of selective Mnk inhibitors has so far hampered pharmacological target validation and clinical drug development. Herein, we report, for the first time, the discovery of a series of 5-(2-(phenylamino) pyrimidin-4-yl)thiazole-2(3H)-one derivatives as Mnk inhibitors. Several derivatives demonstrate very potent Mnk2 inhibitory activity. The most active and selective compounds were tested against a panel of cancer cell lines, and the results confirm the cell-type-specific effect of these Mnk inhibitors. Detailed cellular mechanistic studies reveal that Mnk inhibitors are capable of reducing the expression level of anti-apoptotic protein Mcl-1, and of promoting apoptosis in MV4-11 acute myeloid leukaemia cells. Highly active Mnk2 inhibitors: Mnk-related cancer biology is an area of intensive research, but its inhibitor discovery has lagged behind due to a lack of understanding of the protein structure. Herein we report the discovery of Mnk2 inhibitors (e.g. 8 e). These potent and selective inhibitors are extremely valuable for target validation and drug discovery.

Full text of DOI:10.1002/cmdc.201300552

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DOI: 10.1002/cmdc.201300552
Discovery of 5-(2-(Phenylamino)pyrimidin-4-yl)thiazol-
2(3H)-one Derivatives as Potent Mnk2 Inhibitors:
Synthesis, SAR Analysis and Biological Evaluation
Sarah Diab, Theodosia Teo, Malika Kumarasiri, Peng Li, Mingfeng Yu, Frankie Lam,
Sunita K. C. Basnet, Matthew J. Sykes, Hugo Albrecht, Robert Milne, and Shudong Wang*[a]
Phosphorylation of eIF4E by human mitogen-activated protein
kinase (MAPK)-interacting kinases (Mnks) is crucial for human
tumourigenesis and development. Targeting Mnks may provide
a novel anticancer therapeutic strategy. However, the lack of
selective Mnk inhibitors has so far hampered pharmacological
target validation and clinical drug development. Herein, we
report, for the first time, the discovery of a series of 5-(2-(phe-
nylamino)pyrimidin-4-yl)thiazole-2(3H)-one derivatives as Mnk
inhibitors. Several derivatives demonstrate very potent Mnk2
inhibitory activity. The most active and selective compounds
were tested against a panel of cancer cell lines, and the results
confirm the cell-type-specific effect of these Mnk inhibitors.
Detailed cellular mechanistic studies reveal that Mnk inhibitors
are capable of reducing the expression level of anti-apoptotic
protein Mcl-1, and of promoting apoptosis in MV4-11 acute
myeloid leukaemia cells.
Introduction
Mitogen-activated protein (MAP) kinase-interacting kinases
(Mnk1/2) are members of a family of serine-threonine kinases,
and are emerging as potential anticancer targets.[1] Identified
as one of the key effectors for MAPK pathways, Mnks are acti-
vated by Erk or p38 kinases in response to various extracellular
factors. Subsequently, Mnks phosphorylate Ser209 of the eu-
karyotic initiation factor 4E (eIF4E), a key player in translational
control.[2] Recent findings suggest that eIF4E activity is a key
determinant of PI3K/Akt/mTOR- and Ras/Raf/MEK/Erk- mediat-
ed tumourigenic activity, and that targeting eIF4E should have
a major impact on all of these pathways in human cancer.[2]
Phosphorylation of eIF4E by Mnks is considered to be critical
for its oncogenic role. It was previously demonstrated that
mice carrying eIF4E mutated at Ser209 were incapable of de-
veloping cancer.[3] Furthermore, a constitutively active, mutated
Mnk1 was able to promote tumourigenesis in a similar way to
eIF4E.[4] Conversely, silencing Mnks through the infection of
human glioma cell line U87MG with virus expressing Mnk1
small hairpin RNA (shRNA) attenuates the level of phosphory-
lated eIF4E and tumour formation.[2,5] Mnk1/2-double knock-
out tPTENꢀ/ꢀ mice showed an attenuated tumour growth com-
pared to the parental tPTENꢀ/ꢀmice.[5] Most importantly, Mnks
seem to be not required for normal growth.[6] Collectively,
these results provide strong biological evidence that Mnks are
oncoproteins and their pharmacological inhibition provides
a safe and effective anticancer strategy. Thus, development of
selective pharmacological inhibitors of Mnks is highly desira-
ble.
Only a few Mnk inhibitors have so far been identified, but
they lack selectivity. CGP57380 has a low-nanomolar inhibitory
effect against Mnks, but was shown to inhibit other kinases.[7]
It has been shown to impair mRNA recruitment to the poly-
some by inhibiting the phosphorylation of various substrates
of the major signalling pathways that regulate cap-dependent
translation, including rpS6.[7a] Likewise, cercosporamide, an an-
tifungal agent, was identified as a potent Mnk inhibitor
through high-throughput screening, and was found to inhibit
Jak3 and Mnks with similar potency.[8] More recently, a class of
Mnk inhibitors was designed based on hypothemycin, a cova-
lent Erk2 inhibitor.[9] However, the selectivity of these com-
pounds remains unknown.
Development of selective kinase inhibitors has been a formi-
dable challenge, primarily due to the high similarity of the ATP
binding sites among kinases. The X-ray crystal structures of
Mnks revealed two distinct structural features.[10] Firstly, Mnks
contain a DFD motif instead of the common DFG motif typical-
ly found in other kinases. Secondly, the catalytic domain con-
tains three Mnk-specific inserts (i.e., insertion I1, I2 and I3) not
observed in other kinases. In the ATP-free structure of Mnks,
the DFD motif predominantly adopts a DFG/D-out conforma-
tion, whereby residue DFD/Phe points into the ATP binding
pocket, prevents accessing of ATP and exposes an additional
hydrophobic/allosteric site.[11] Tailoring existing kinase inhibitor
scaffolds to exploit the new binding environment might offer
Mnk inhibitors with high selectivity. Nevertheless, the lack of
structural information available for the ATP-bound Mnk has
hindered inhibitor design and pharmacological drug develop-
ment.
[a] S. Diab, T. Teo, Dr. M. Kumarasiri, Dr. P. Li, Dr. M. Yu, Dr. F. Lam,
S. K. C. Basnet, Dr. M. J. Sykes, Dr. H. Albrecht, Dr. R. Milne, Prof. S. Wang
Centre for Drug Discovery and Development
Sansom Institute for Health Research
School of Pharmacy and Medical Sciences
University of South Australia, Adelaide, SA 5001 (Australia)
E-mail: shudong.wang@unisa.edu.au
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Results and Discussion
Previously, we have explored the 2-anilino-pyrimidine scaf-
fold with various C4-heterocyclic substituents as cyclin-depen-
dent kinase (CDK) inhibitors.[12] Many compounds were inhibi-
tors of CDK2 and CDK9 with low-nanomolar potency, which
led to identification of several preclinical drug candidates.[12b,c]
Co-crystallisation of 2-anilino-4-(2-methylaminothiazol-5-yl)pyri-
midine analogues with CDK2
Chemistry
Synthetic chemistry of 3,4-dimethyl-5-(2-(phenylamino)pyrimi-
din-4-yl)thiazol-2(3H)-one derivatives is outlined in Scheme 1.
and CDK9 showed similar bind-
ing modes.[13] The pyrimidine
ring occupies the adenine site of
the ATP binding pocket where
its N1 and C2-NH form hydrogen
bonds with the hinge region.
The aniline moiety is positioned
in front of the ATP binding site,
forming van der Waals interac-
tions with Ile10 in CDK2 (Ile25 in
CDK9). The thiazole moiety
adopts two distinct conforma-
tions in CDK2, whereas only one
is seen in CDK9. In both cases,
the thiazol-2-amine site forms
a hydrogen bond with DFG/Asp
to achieve high potency against
CDKs.
Scheme 1. Synthesis of 3,4-dimethyl-5-(2-(phenylamino)pyrimidin-4-yl)thiazol-2(3H)-one derivatives. Reagents and
conditions: a) (i) KSCN, Me2CO, RT, 6 h; (ii) EtOH, HCl, reflux, overnight, 93%; b) MeI, K2CO3, DMF, RT, overnight,
48%; c) DMF-DMA, reflux, 3.5 h, 40%; d) Fe, MeOH/MeCOOH, reflux, 2 h, 37–98%; e) CN2H2, HNO3, EtOH/H2O,
reflux, overnight, 36–98%; f) NaOH, CH3CN, microwave 200–300 W, 1608C, 30 min, 7–41%.
In a program to identify Mnk
inhibitors, we screened our in-
house kinase inhibitor library, and several 2-anilino-4-(2-amino-
thiazol-5-yl)pyrimidines were found to inhibit Mnk2 activity.
Using analogue design and optimisation approaches, we de-
signed and synthesised a series of 3,4-dimethyl-5-(2-(phenyla-
mino)pyrimidin-4-yl)thiazol-2(3H)-one derivatives. Replacement
of the thiazol-2-amine moiety of 2-anilino-4-(2-aminothiazol-5-
yl)pyrimidine with a thiazol-2(3H)-one system would interrupt
the hydrogen-bonding interactions between the C2-NH of thia-
zole and Gln131 and Asp86 of CDK2, but this alteration might
have a minimal effect on Mnk activity. The structure–activity re-
lationship (SAR) analysis of the pyrimidinyl C2-aniline moiety
was previously described.[12] Many meta- and para-substituted
anilines, in the context of 4-thiazol-pyrimidine, gave rise to
CDK inhibition, whereas substituents at the ortho-position
were detrimental to CDK inhibitory activity. As expected,
a number of 5-(2-(phenylamino)pyrimidin-4-yl)thiazol-2(3H)-one
analogues were found to be potent Mnk inhibitors with appre-
ciable selectivity against CDK2 and CDK9. A lead compound
8e exhibited an excellent selectivity profile with 34-fold and
fourfold greater potency towards Mnk2 relative to the respec-
tive CDK2 and CDK9. Cellular mechanistic investigations con-
firmed that the inhibitors were capable of reducing the level
of eIF4E phosphorylation and of inducing apoptosis in MV4-11
and A2780 tumour cell lines.
5-Acetyl-4-methylthiazol-2(3H)-one (2) was obtained by treat-
ing 3-chloropentane-2,4-dione (1) with potassium thiocyanate.
Alkylation of 2 with iodomethane gave 5-acetyl-3,4-dimethyl-
thiazol-2(3H)-one (3). Condensation of 3 with N,N-dimethylfor-
mamide dimethylacetal (DMF-DMA) at 1108C afforded 5-(3-(di-
methylamino)acryloyl)-3,4-dimethylthiazol-2(3H)-one (4). In par-
allel, nitrophenyl derivatives were reduced to anilines (6), fol-
lowed by conversion to the corresponding guanidines (7) in
the presence of aqueous cyanamide.[12a] A series of 3,4-dimeth-
yl-5-(2-(phenylamino)pyrimidin-4-yl)thiazol-2(3H)-ones
(8a–r)
was obtained by the condensation reaction of enaminone (4)
with guanidines (7) using microwave-assisted chemistry.
SAR analysis
Biological activities of compounds 8a–r were evaluated using
Mnk2, CDK2 and CDK9 biochemical assays and cellular prolifer-
ation assays in A2780 ovarian cancer cell line. The results are
summarised in Table 1. Compound 8a, where R1, R2 and R3 are
hydrogen atoms, inhibits Mnk2 with Ki =0.25 mm, but it is sig-
nificantly more potent for CDK2 and CDK9 (Ki =0.04 and
0.01 mm respectively). This compound is also a very effective
cytotoxic agent with GI50 =0.05 mm. Introduction of a methyl
or ethyl group at its anilino para position leading to respective
compounds 8b or 8c, abolishes Mnk2 inhibition (Ki >10 mm),
while maintaining a similar potency for CDK2 and CDK9 as 8a.
The loss in Mnk2 activity seems to cause a minimal effect on
cellular cytotoxicity; compounds 8b and 8c are able to inhibit
A2780 cell proliferation at a GI50 value of 0.06 and 0.35 mm, re-
spectively. Replacement of the methyl or ethyl groups with
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effect on the A2780 cancer cell line (GI50 >10 mm)
when compared to 8d. Replacement of the methoxy
with a bulkier ethoxy group in 8h (R1 =OEt, R2 =R3 =
H) is not tolerated for all three kinases. Introduction
of a slightly less bulky difluoromethoxy or trifluoro-
methyl group at the ortho position of aniline afforded
the respective compounds 8i (R1 =OCHF2, R2 =R3 =
H) and 8j (R1 =CF3, R2 =R3 =H). Compound 8i is the
most potent Mnk2 inhibitor (Ki =0.12 mm) identified
in this chemical series. Both 8i and 8j display a >45-
fold selectivity for Mnk2 over CDK2. Compounds 8k–
m, bearing an ethyl, methyl or fluoro group at the
ortho or para position of aniline, also exhibit favoura-
ble Mnk2 inhibition with Ki values in a range of 0.26–
0.55 mm; however, no appreciable selectivity profile is
observed. All these compounds show modest cellular
potency (GI50 =6.6–9.24 mm). Keeping the o-methyl
group, and introducing a methanesulfonamide at the
meta position of 8n (R1 =Me, R2 =NHSO2Me, R3 =H)
resulted in a slight decrease of Mnk2 potency com-
pared to 8l (R1 =Me, R2 =R3 =H), but enhanced
CDK2 and CDK9 inhibition by >twofold. Compounds
8o–r, where R1 =R3 =H, and R2 =NHSO2Me (8o), Me
(8p), OMe (8q), or COMe (8r), are able to inhibit
Mnk2 within the potency of Ki <1 mm. However, all
these compounds are highly potent CDK2 and CDK9
inhibitors, and as expected, they are also highly toxic
to A2780 cancer cells.
Table 1. Structure and biological activity of 3,4-dimethyl-5-(2-(phenylamino)pyrimidin-
4-yl)thiazol-2(3H)-one derivatives 8a–r.
Compd R1
R2
R3
Ki [mm][a]
GI50 [mm][b]
Mnk2
0.25
>10
>10
CDK2A CDK9T1
8a
8b
8c
8d
8e
8 f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.04
0.04
0.05
0.70
0.01
0.02
0.03
0.03
1.66
0.05ꢁ0.01
0.06ꢁ0.01
0.35ꢁ0.06
0.23ꢁ0.01
8.26ꢁ0.89
5.70ꢁ2.83
Me
Et
OMe
OMe
Cl
H
H
H
H
H
H
F
H
H
H
H
H
0.32
OMe
OMe
OMe
OEt
OCHF2
CF3
Et
Me
Me
Me
H
0.37 >10
>10 2.23 >10
0.20 2.47 0.97 > 10
>10 >10 >10
0.12 5.36
0.20 >10
4.93ꢁ1.22
0.13
0.21
0.09
0.11
0.17
0.06
0.01
0.01
0.02
<0.01
7.49ꢁ0.45
9.24ꢁ4.78
8.54ꢁ2.00
6.94ꢁ0.01
6.63ꢁ0.29
1.84ꢁ0.14
0.67ꢁ0.14
0.11ꢁ0.07
0.63ꢁ0.02
0.03ꢁ0.00
0.33
0.26
0.55
0.33
0.17
0.48
0.27
0.92
1.23
0.46
0.49
0.19
0.03
0.02
0.07
0.02
H
NHSO2Me
NHSO2Me
Me
OMe
COMe
H
H
H
[a] Kinase inhibition: ATP concentrations were within 15 mm of KM values. Apparent in-
hibition constants (Ki) were calculated using IC50 values and the appropriate KM (ATP)
values for each kinase. [b] Cytotoxicity: antiproliferative activity was determined by
72 h MTT assay using A2780. The data given are meanꢁSD derived from at least two
replicates.
Taken together, 3,4-dimethyl-5-(2-(phenylamino)-
pyrimidin-4-yl)thiazol-2(3H)-one is a valuable chemi-
cal scaffold for Mnk2 inhibitors. Selectively targeting
a methoxy, as shown in 8d (R1 =R2 =H and R3 =OMe), gives
a >31-fold increase in Mnk2 inhibition, indicating a favourable
electron-donating effect on Mnk2. Interestingly, 8d is >14-fold
less potent for CDK2 in comparison with 8b and 8c, but re-
tains similar CDK9 potency. The most selective Mnk2 inhibition
is achieved by 8e, where both R1 and R3 are methoxy groups
(R2 =H). Compound 8e displays not only potent Mnk2 inhibi-
tion (Ki =0.37 mm), but also excellent selectivity towards Mnk2
by >34-fold and fourfold compared to CDK2 (Ki >10 mm) and
CDK9 (Ki =1.66 mm), respectively. While 8e is >14-fold and 55-
fold less potent for the respective CDK2 and CDK9 compared
to 8d, it shows a fourfold reduced cellular potency (GI50 =
8.26 mm) against the A2780 tumour cell line. This result sug-
gests that CDK2 and CDK9 inhibition may be the main contrib-
utor to the cytotoxicity in A2780 cells observed with this com-
pound. Replacing the methoxy with chlorine at the para posi-
tion of aniline gave rise to compound 8 f (R1 =OMe, R2 =H and
R3 =Cl) that abolishes both Mnk2 and CDK9 activities, and
gives a poor antiproliferative activity. To investigate the effects
of monosubstitution at the ortho position of aniline, analogues
8g–l were synthesised and evaluated. Compound 8g (R1 =
OMe, R2 =R3 =H), one of the most potent Mnk2 inhibitors (Ki =
0.20 mm), is more potent as well as more selective towards
Mnk2 than its para analogue 8d, confirming the unfavourable
interactions between ortho substitution and CDK2 and CDK9
proteins. Compound 8g has a significantly lower cytotoxic
Mnk2 can be achieved by manipulation of the ortho and para
substitutions of the aniline moiety in the context of 4-thiazo-
lone-pyrimidine-2-aniline system. Being the most active and se-
lective Mnk inhibitors of this series, compounds 8i, 8e and 8j
were subjected to further structural analysis and cellular mech-
anistic investigation.
Molecular basis for selectivity
Given that no ATP-bound Mnk structure is available, we used
CDK2 and CDK9 to rationalise the observed selectivity of the
compounds. Compounds 8a–r were docked to multiple CDK2
and CDK9 X-ray crystallographic structures, and the results
were evaluated based on the extra-precision docking scores
and binding poses (Schrçdinger Inc., 2013). The binding
modes obtained for these 3,4-dimethyl-5-(2-(phenylamino)pyri-
midin-4-yl)thiazol-2(3H)-ones were consistent with previously
reported co-crystal structures of 2-anilino-4-(2-methylamino-
thiazol-5-yl)pyrimidine analogues bound to CDK2 and CDK9.[12c]
The docking pose of 8a in CDK2 depicts hinge region interac-
tions with the backbone of Leu83, through the N1 and C1-NH
of the pyrimidine moiety (Figure 1A). A similar binding mode
is observed in CDK9, in which the corresponding position is
Cys106 (Figure 1B). Furthermore, 8a is seen interacting with
Lys48 of CDK9 (Lys33 in CDK2) through the carbonyl of the
thiazolone ring. In CDK9, the thiazolone ring adopts only one
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more easily. Evidently, the loss of
o-methoxy (8d) or p-methoxy
(8g) is favourable for binding to
CDK2 and CDK9, thus reducing
the selectivity for Mnk2 when
compared to 8e.
Interestingly, compound 8j,
which lacks a para substituent
but contains an o-trifluoromethyl
group, demonstrates a similar
loss of CDK2 activity compared
to 8e. Comparing the best-scor-
ing docking poses of 8j with 8e
reveals that the binding confor-
mations to CDK2 are similar (Fig-
ure 1C). Thus, in CDK2, the com-
bined steric and electronic ef-
fects introduced by the o-tri-
fluoromethyl group may be simi-
lar to that of the o- and p-
methoxy groups on the aniline
ring. In contrast, the Ki value of
8j reflects a slightly higher affini-
ty for CDK9 compared to 8e.
Binding modes of 8j and 8e at
the hinge region are nearly iden-
tical with the o-trifluoromethyl
group positioned at the location
of the oxygen atom of o-me-
thoxy (Figure 1D). This is expect-
ed as the differences in Ki values
of 8e and 8j are small enough
Figure 1. Superposition of computationally generated modes of binding of 8a, 8e and 8j to CDK2 and CDK9.
A) In CDK2, the aniline ring of 8e (grey capped sticks) is displaced away from the hinge region compared to 8a
(white capped sticks) due to the addition of the ortho- and para -substituents. B) In CDK9, this movement is not
pronounced as there is more space available for 8e (grey capped sticks) at the site of the aniline ring. C) Binding
modes of 8e (grey capped sticks) and 8j (white capped sticks) are similar in CDK2, resulting in similar lowering of
binding affinities. D) In CDK9, the positionings of the ligand atoms at the hinge region are nearly identical. The
binding modes were generated by docking the compounds to multiple X-ray structures of CDK2 and CDK9 using
Glide (Schrçdinger Inc., 2013). The best scoring poses are illustrated here. The figures were generated using
Pymol v1.6.
conformation, in which it interacts with Lys48 through hydro-
gen bonding In contrast, in CDK2, it flips occasionally by
ꢂ1808 to break the Lys hydrogen bond so that the hydropho-
bic interaction between the C4-methyl group of the thiazolone
and the Phe80 gatekeeper residue of CDK2 can be established.
A similar effect was observed in previously reported X-ray
structures in complex with its 2-thiazole-pyrimidinyl ana-
logues,[13] where the thiazole ring was confined to a single con-
formation in CDK9 while it alternated between two in CDK2.
Compound 8e, containing simultaneous ortho and para sub-
stituents on the aniline ring, demonstrates marked reduction
of CDK2 affinity. In CDK2, the aniline ring of 8e is tilted away
from the protein by ꢂ1.5 ꢁ at the point of maximum displace-
ment, compared to 8a (Figure 1A). This movement appears to
be caused by the steric hindrance between the p-methoxy and
the side chain of Lys89. Additionally, compared to 8a, the ani-
line ring of 8e has also moved slightly away from the hinge
region. Thus, the net effect of the presence of o- and p-me-
thoxy groups in 8e appears to destabilise the CDK2 interac-
tions, as evident by the higher Ki value in biochemical assays
as well as lower docking score compared to 8a. This effect is
not pronounced in CDK9 due to the presence of Gly112 in the
corresponding position of Lys89 of CDK2 (Figure 1B). Due to
the lack of a side chain, Gly112 creates more space for the
ligand in CDK9, accommodating ortho and para substituents
that the computational predictions of the correct trends are
beyond the scope of the current docking methods. Tracing the
origins of such subtle effects would require highly accurate cal-
culations, which necessitate significant computational resour-
ces.
Cellular mode of action
Potent antiproliferative activity
To investigate cell-type sensitivity, compounds 8e and 8i were
tested against a panel of six tumour cell lines, including acute
myelocytic leukaemia (MV4-11), breast carcinoma (MCF-7),
colon carcinoma (HCT-116), ovarian carcinoma (A2780), prostat-
ic carcinoma (LNCaP) and pancreatic carcinoma (PANC-1),
using cytotoxic assays. The results are summarised in Table 2.
CGP57380 was included as a comparator in the assays, as it is
a potent Mnk2 inhibitor (Ki =0.34 mm) and has no inhibitory ac-
tivity for CDK2 and CDK9 at concentrations >10 mm.
CGP57380 exhibits modest antiproliferative activity in all five
tumour cell lines with GI50 values in the range of 4.2–9.1 mm,
except MCF-7, which has shown a little effect (GI50 >10 mm).
Among the cell lines tested, CGP57380 exhibited its greatest
cytotoxicity to MV4-11 cells. The same trend is observed with
compounds 8e and 8i. MV4-11cells are most sensitive in re-
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ment of MV4-11 cells with 20 mm of 8e or 8i gave
rise to the significant alteration of cell distributions,
that is, resulting in 78.6% and 61.2% cells, respec-
tively, with the G1-phase DNA content compared to
58% with DMSO or CGP57380 treatment. Decrease in
S- and G2/M-phase cells, accompanied by substantial
sub-G1 cell accumulations were observed for all
three compounds. The results are in agreement with
the findings that inhibiting phosphorylated eIF4E re-
sults in a G0/G1 cell cycle arrest in nasopharyngeal
carcinoma cell lines,[15] and that treatment of Ba/F3
cells with CGP57380 for 48 h induced G0/G1 arrest.[7a]
It was suggested that the cell cycle effects might be
caused by targeting c-Myc and MMP9 in nasophar-
yngeal cells, as well as cyclin-D in Ba/F3 cells.
Table 2. Antiproliferative activity of CGP57380, 8e and 8i against a panel of human
cancer cell lines.
Cell Line
GI50 [mm][a]
8e
8i
CGP57380
Acute myelocytic leukaemia (MV4-11)[b]
Breast carcinoma (MCF-7)
Colon carcinoma (HCT-116)
Ovarian carcinoma (A2780)
Prostatic carcinoma (LNCaP)
Pancreatic carcinoma (PANC-1)
0.78ꢁ0.07
0.88ꢁ0.29
4.19ꢁ0.15
>10
>10
>10
>10
8.20ꢁ0.90
6.36ꢁ1.60
9.14ꢁ1.17
5.72ꢁ0.68
8.54ꢁ0.10
8.80ꢁ0.24
9.62ꢁ0.57
>10
>10
>10
>10
[a] Determined using 96 h MTT assay; data given are the meanꢁSD derived from at
least two replicates. [b] Determined using 96 h resazurin assay.
sponse to treatment with 8e or 8i, resulting in their respective
To confirm cellular Mnk inhibition and selectivity, we next
GI50 values of 0.78 and 0.88 mm, respectively, being over five-
fold more potent than CGP57380. Both 8e and 8i exhibit mod-
erate antiproliferative activity against A2780 cells, but fail to
show growth inhibition in other cell types at concentrations
>10 mm. The different sensitivities towards the Mnk inhibitors
may be attributed to the different expression levels of phos-
phorylated eIF4E in these cell lines. It has been shown, for ex-
ample, that MCF-7 breast cancer cells are resistant to
CGP57380 because of the low level of Mnk-mediated eIF4E
phosphorylation.[14]
performed western blot analysis on A2780 cells following treat-
ment with 8e, 8i or CGP57380. The cells were incubated with
20 mm of each compound for a period of 24 h or 72 h (Fig-
ure 2D). Antibodies against phosphorylated eIF4E show that
the level of the phosphorylated eIF4E was reduced by 24 h
post-treatment, indicating cellular Mnk inhibitory activity of
8e, 8i or CGP57380. The same treatment showed little effect
on the expression of other proteins, including phosphorylated
RNAP II serine 2 (p-Ser2 RNAPII) and anti-apoptotic proteins
myeloid cell leukaemia sequence 1 (Mcl-1) and B-cell lympho-
ma 2 (Bcl-2). After 72 h treatment with 8e, 8i or CGP57380,
the eIF4E phosphorylation in the A2780 cells was completely
abolished. Like CGP57380,[7b] 8e had no effect on the p-Ser2
RNAPII, confirming its lack of CDK9 inhibitory activity.[12c] eIF4E
has been found to promote tumourigenesis through its anti-
apoptotic effect,[16] and inhibition of eIF4E resulted in a reduc-
tion of the anti-apoptotic protein Mcl-1.[8] In agreement with
these findings, both CGP57380 and 8e downregulated Mcl-
1 and caused poly(adenosine diphosphate [ADP]-ribose) poly-
merase (PARP) cleavage, indicating apoptosis. Compound 8i
caused a significant loss in p-Ser2 RNAPII, which is consistent
with its potent CDK9 inhibition in biochemical assay. The inhib-
ition of both Mnk2 and CDK9 may be responsible for the
greater activity of 8i in abrogation of Mcl-1 and suppression of
Bcl-2 in A2780 cells when compared to CGP57380 and 8e. We
carried out further studies in MV4-11 leukaemia cells by treat-
ment with 20 mm of CGP57380, 8e and 8i for a period of 24 h.
As shown in Figure 2D, the treatment resulted in complete in-
hibition of eIF4E phosphorylation, which is consistent with
their greater antiproliferative potencies in MV4-11 cells when
compared to that in A2780 cells. In addition, 8e is more effec-
tive in blockage of Mcl-1 expression when compared to
CGP57380 and 8i in the MV4-11 cells. All three compounds are
capable of cleaving PARP, but cause the minimal alterations to
Bcl-2 protein expression.
Mnk inhibition and induction of apoptosis in cancer cells
In order to assess whether the antiproliferative activity ob-
served with the Mnk inhibitors is the consequence of apopto-
sis, we used annexin V/propidium iodide (PI) staining of A2780
or MV4-11 cells following treatment with 8e, 8i or CGP57380
for a period of 48 h. A concentration of 20 mm of inhibitor
compounds was chosen as CGP57380 has been shown to
block eIF4E phosphorylation at this concentration.[14] As shown
in Figure 2A, 8e and 8i induce A2780 cell death with around
5% annexin V+/PI- and 15% annexin V+/PI+ cells when
compared to DMSO control. Similarly, CGP57380 causes A2780
cell death, but being less effective; it only results in 3% annex-
in V+/PIꢀ and 8% annexin V+/PI+ cells. Experiments were
also performed in MV4-11 leukaemia cells and confirmed that
these cells were significantly more sensitive than A2780 cells
towards the inhibitor compounds. The same treatment with
CGP57380, 8e and 8i resulted in24%, 27% and 16% increase,
respectively, in apoptotic (annexin V+/PIꢀ) cells when com-
pared to DMSO control.
Cell death induced by inhibitor compounds can occur
through caspase-dependent or caspase-independent apopto-
sis. We next measured caspase 3/7 activity in MV4-11 cells after
treatment with 20 mm of CGP57380, 8e or 8i for a period of
48 h (Figure 2B). No significant activation of caspase 3/7 was
observed. We further examined the effects of a 48 h exposure
of 20 mm of 8e, 8i or CGP57380 on cell cycle progression (Fig-
ure 2C). Flow cytometric analysis of the treated A2780 cells in-
dicated a slight loss in the G2/M phase DNA content but a sub-
stantial increase in sub-G1 cells, indicating cell death. Treat-
Conclusions
In this study, we described the discovery of a novel series of
3,4-dimethyl-5-(2-(phenylamino)pyrimidin-4-yl)thiazol-2(3H)-
ones Mnk inhibitors. Most of these compounds showed potent
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anistic investigations of lead
compounds 8e and 8i con-
firmed their Mnk2 inhibitory po-
tency and selectivity. Important-
ly, the antiproliferative activity of
8e, the most selective Mnk in-
hibitor in the series, was found
to be cell-type specific with
marked tumour cell growth in-
hibition against MV4-11 cells.
Compound 8e blocked the
Mnk2-mediated phosphorylated
eIF4E and abrogated the expres-
sion of anti-apoptotic protein
Mcl-1 in a time- and cell-depen-
dent manner. Consistent with
these findings, 8e has been
shown to arrest cells at the G1
phase and to induce significant
cell apoptosis in MV4-11 cells.
However, this compound had
a limited effect on the cell cycle
distribution of A2780 ovarian
cancer cells. Our study provided
a rationale for development of
Mnk2 inhibitors for the targeted
treatment of certain human ma-
lignancies.
Experimental Section
Chemistry
Chemical reagents and solvents,
including anhydrous solvents, were
purchased from commercial sour-
ces, and were used as received
unless otherwise specified. Micro-
wave-assisted synthesis was per-
formed in an Asynt DrySyn insert
(10 mL, Isleham, UK) using a CEM
Discover SP and Explorer 48/72/96
microwave system (Matthews, NC,
USA) controlled by Synergy soft-
ware
(Firmware
version
DSCA02.17). Thin-layer chromatog-
raphy was performed on Merck
silica gel 60 F254 precoated alumini-
um plates (0.2 mm). Flash column
chromatography was carried out
using either prepacked silica gel
cartridges KP-SIL (ca. 0.05 mm) on
Biotage Isolera Four automated
flash chromatography (Uppsala,
Sweden) or fritted solid loaders
packed with Scharlau silica gel
Figure 2. Cellular mechanism of action of 8e and 8i. A) Induction of apoptosis by CGP57380, 8e or 8i in A2780
and MV4-11 cells. Cells were exposed to 20 mm each compound for 48 h, and analysed by Annexin V/PI staining.
The percentage of cells undergoing apoptosis was defined as the sum of early apoptosis (annexin V-positive cells)
and late apoptosis (annexin V-positive and PI-positive cells). B) Caspase 3/7 activity in MV4-11 cells (in relative fluo-
rescence units, RFU) after treatment with 8e, 8i or CGP57380 for a period of 48 h at concentrations shown.
C) Cell cycle analysis of A2780 and MV4-11 cells after treatment with 20 mm of CGP57380, 8e or 8i for 48 h.
D) Western blot analysis of A2780 cells treated with 20 mm of 8e and 8i respectively for a period of 24 h (left
panel) and 72 h (middle panel), and of MV4-11 cells treated for 24 h (right panel). b-Actin antibody was used as
an internal control. DMSO diluent was used as control in each experiment.
Mnk2 inhibition with the various selectivity profiles against
(0.04–0.06 mm) on Biotage FlashMaster Personal+ flash chromatog-
raphy. 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. 1H NMR
CDK2 and CDK9. The computational docking studies of 8e and
8j bound in CDK9/cyclin T and CDK2/cyclin A provided the ra-
tionale for the observed potency and selectivity. Cellular mech-
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and 13C NMR spectra were obtained using a Bruker Avance III HD
spectrometer (Faellanden, Switzerland) at 500.16 and 125.76 MHz
respectively, and were analysed using a Bruker Topspin 3.2 pro-
gram. Chemical shifts are reported in parts per million (ppm), and
are referenced to 1H signals of residual nondeuterated solvents
and to 13C signals of the deuterated solvents. Multiplicity of
1H NMR signals is reported as follows: s=singlet, d=doublet, t=
triplet, q=quartet, dd=doublet of doublets, dt=doublet of trip-
lets, m=multiplet, and br=broad. Coupling constants J (Hz) are
quoted to the nearest 0.1 Hz. The purity of compounds used for
biological evaluation was determined to be greater than 95%
using Shimadzu Prominence UFLC system (UltraFast Liquid Chro-
matograph, Kyoto, Japan) equipped with a CBM-20A communica-
tions 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 Phe-
nomenex Kinetex 5u C18 100A 250 mmꢂ4.60 mm column. Meth-
od A (gradient 5% to 95% MeOH containing 0.1% FA over 7 min
at a flow rate of 1 mLminꢀ1, followed by 95% MeOH containing
0.1% FA over 13 min) and method B (gradient 5% to 95% CH3CN
containing 0.1% FA over 7 min at a flow rate of 1 mLminꢀ1, fol-
lowed by 95% CH3CN containing 0.1% FA over 13 min) were used
for analytic HPLC. Melting points were determined in an open ca-
pillary using a Stuart SMP10 melting point apparatus (Stone, UK),
and are uncorrected.
16 h and concentrated in vacuo. The residue was triturated with
2m HCl (10 mL), and the resulting precipitate was filtered, and
washed with 2m HCl (20 mL) to give 5n as a beige powder (6.78 g,
1
90%): H NMR ([D6]DMSO): d=2.37 (s, 3H, CH3), 3.06 (s, 3H, CH3),
7.46 (apparent t, 1H, J=7.0 Hz, Ph-H), 7.62 (d, 1H, J=7.0 Hz, Ph-H),
7.78 (d, 1H, J=7.5 Hz, Ph-H), 9.55 ppm (s, 1H, NH); HRMS (ESI): m/z
[M+H]+ calcd for C8H11N2O4S: 231.0434, found: 231.0441.
N-(3-Nitrophenyl)methanesulfonamide (5o): Methanesulfonyl
chloride (2.95 mL, 38.1 mmol) was added dropwise to a solution of
3-nitroaniline (4.36 g, 38.1 mmol) in pyridine (20 mL) on an ice
bath. The reaction mixture was stirred at RT overnight and concen-
trated in vacuo. The residue was triturated with H2O (20 mL), and
the precipitate was filtered, and washed with 2m HCl (20 mL) to
1
give 5o as a white powder (7.81 g, 95%): H NMR ([D6]DMSO): d=
3.10 (s, 3H, CH3), 7.58–7.67 (m, 2H, Ph-H), 7.92–7.96 (m, 1H, Ph-H),
8.04 (s, 1H, Ph-H), 10.37 ppm (s, 1H, NH); HRMS (ESI): m/z [M+H]+
calcd for C7H9N2O4S: 217.0278, found: 217.0400.
General procedure for preparation of 6: Iron powder (5 equiv)
was added to a solution of nitro compound 5 (1 equiv) in MeOH/
MeCOOH (1:1). The reaction mixture was heated at 508C for 3 h
under N2 and concentrated in vacuo. EtOAc (100 mL) and 1m
NaOH (50 mL) were added to the residue, and the precipitate was
removed by centrifugation. The aqueous layer was separated and
extracted with EtOAc (3ꢂ50 mL). The organic layers and extracts
were combined, dried over MgSO4, filtered and concentrated in
vacuo to give the desired amine.
5-Acetyl-4-methylthiazol-2(3H)-one (2): 3-Chloropentane-2,4-
dione (7.31 mL, 61.7 mmol) was added dropwise to a solution of
KSCN (6.00 g, 61.7 mmol) in Me2CO (50 mL) on an ice bath. The re-
action mixture was warmed to RT, stirred for 6 h, and concentrated
in vacuo. EtOH (50 mL) and 2m HCl (10 mL) were added to the res-
idue. The reaction mixture was heated at reflux for 14 h, cooled to
RT, and concentrated in vacuo. The residue was washed with cold
MeOH (50 mL) and Et2O (50 mL) to give 2 as a beige solid (9.00 g,
1
4-Methoxyaniline (6d): Brown powder (98%); H NMR ([D6]DMSO):
d=3.61 (s, 3H, OCH3), 6.48–6.52 (m, 2H, Ph-H), 6.61–6.65 ppm (m,
2H, Ph-H); HRMS (ESI): m/z [M+H]+ calcd for C7H10NO: 124.0757,
found: 124.0680.
4-Chloro-2-methoxyaniline (6 f): Black oil (94%); 1H NMR
([D6]DMSO): d=3.77 (s, 3H, OCH3), 4.83 (s, 2H, NH2), 6.60 (d, 1H,
J=8.4 Hz, Ph-H), 6.70 (dd, 1H, J=8.3, 2.3 Hz, Ph-H), 6.81 ppm (d,
1H, J=2.3 Hz, Ph-H); HRMS (ESI): m/z [M+H]+ calcd for C7H9ClNO:
158.0367, found: 158.0504.
1
93%): H NMR ([D6]DMSO): d=2.35 (s, 3H, CH3), 2.40 (s, 3H, CH3),
11.93 ppm (s, 1H, NH); HRMS (ESI): m/z [M+H]+ calcd for
C6H8NO2S: 158.0270, found: 157.9903.
5-Acetyl-3,4-dimethylthiazol-2(3H)-one
(3):
Iodomethane
o-Toluidine (6l): Pale brown oil (65%); 1H NMR ([D6]DMSO): d=
2.04 (s, 3H, CH3), 4.76 (s, 2H, NH2), 6.45 (dt, 1H, J=7.4, 1.2 Hz, Ph-
H), 6.59 (dd, 1H, J=7.9, 1.1 Hz, Ph-H), 6.86 (dt, 1H, J=7.8, 1.5 Hz,
Ph-H), 6.90 ppm (d, 1H, J=8.3 Hz, Ph-H); HRMS (ESI): m/z [M+H]+
calcd for C7H10N: 108.0808, found: 108.0557.
(16.0 mL, 255 mmol) was added dropwise to a solution of 2
(10.0 g, 63.7 mmol) and K2CO3 (17.6 g, 127 mmol) in N,N-dimethyl-
formamide (DMF, 30 mL). The reaction mixture was stirred at RT
overnight and concentrated in vacuo. H2O (20.0 mL) was added,
and the mixture was extracted with EtOAc (4ꢂ100 mL). The organ-
ic layers were combined, washed with brine (20 mL), dried over
MgSO4, filtered, and concentrated in vacuo to give 3 as a yellow
powder (5.20 g, 48%): H NMR (CDCl3): d=2.34 (s, 3H, CH3), 2.58 (s,
3H, CH3), 3.33 ppm (s, 3H, NCH3); HRMS (ESI): m/z [M+H]+ calcd
for C7H10NO2S: 172.0427, found: 172.0586.
N-(3-Amino-2-methylphenyl)methanesulfonamide (6n): Orange
powder (73%); 1H NMR ([D6]DMSO): d=2.01 (s, 3H, CH3), 2.89 (s,
3H, CH3), 4.93 (s, 2H, NH2), 6.49 (d, 1H, J=7.8 Hz, Ph-H), 6.54 (d,
1H, J=8.0 Hz, Ph-H), 6.86 (t, 1H, J=7.9 Hz, Ph-H), 8.80 ppm (s, 1H,
NHSO2); HRMS (ESI): m/z [M+H]+ calcd for C8H13N2O2S: 201.0692,
found: 201.0721.
1
5-(3-(Dimethylamino)acryloyl)-3,4-dimethylthiazol-2(3H)-one (4):
Dimethylformamide dimethyl acetal (DMF-DMA, 25.0 mL,
188 mmol) was added to 3 (7.88 g, 46.1 mmol). The reaction mix-
ture was heated at 1008C for 3.5 h, cooled to RT, and stirred for
1 h. The precipitate was filtered, and washed with Et2O (30 mL) to
N-(3-Aminophenyl)methanesulfonamide (6o): Yellow oil (37%);
1H NMR ([D6]DMSO): d=2.92 (s, 3H, CH3), 5.15 (s, 2H, NH2), 6.29
(dd, 1H, J=8.0, 1.3 Hz, Ph-H), 6.34 (dd, 1H, J=7.9, 1.2 Hz, Ph-H),
6.45 (t, 1H, J=2.0 Hz, Ph-H), 6.92 (t, 1H, J=7.5 Hz, Ph-H), 9.37 ppm
(s, 1H, NHSO2); HRMS (ESI): m/z [M+H]+ calcd for C7H11N2O2S:
187.0536, found: 187.0645.
1
give 4 as an orange powder (4.18 g, 40%): H NMR ([D6]DMSO): d=
2.52 (s, 3H, CH3), 2.83 (s, 3H, CH3), 3.12 (s, 3H, CH3), 3.32 (s, 3H,
CH3), 5.10 (d, 1H, J=12.0 Hz, CH), 7.61 ppm (d, 1H, J=12.5 Hz,
CH); HRMS (ESI): m/z [M+H]+ calcd for C10H15N2O2S: 227.0849,
found: 227.0882.
General procedure for preparation of 7: Nitric acid (69%) was
added dropwise to a solution of the aniline derivative 6 (1 equiv)
in EtOH (50 mL), followed by addition of a solution of cyanamide
(5 equiv) in a minimal amount of H2O. The reaction mixture was
heated at reflux for 18–36 h, and concentrated in vacuo. The resi-
due was covered with Et2O (50–100 mL) and left at 48C overnight
N-(2-Methyl-3-nitrophenyl)methanesulfonamide (5n): Methane-
sulfonyl chloride (2.70 mL, 34.9 mmol) was added dropwise to a so-
lution of 2-methyl-3-nitroaniline (5.00 g, 32.9 mmol) in pyridine
(20 mL) on an ice bath. The reaction mixture was stirred at RT for
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to be solidified. The solid was filtered, and washed with Et2O to
give the corresponding guanidine.
1H, J=7.0, 1.9 Hz, Ph-H), 7.35 (dt, 1H, J=7.7, 1.2 Hz, Ph-H), 7.37
(dd, 1H, J=7.8, 2.2 Hz, Ph-H), 9.11 ppm (brs, 1H, NH); HRMS (ESI):
m/z [M+H]+ calcd for C9H14N3: 164.1182, found: 164.1350.
1
Phenylguanidine (7a): Yellow powder (30%); H NMR ([D6]DMSO):
1-(o-Tolyl)guanidine (7l): Beige powder (92%); 1H NMR
([D6]DMSO): d=2.19 (s, 3H, CH3), 6.57 (brs, 3H, NH2, NH), 7.41 (ap-
parent s, 2H, Ph-H), 7.46–7.49 ppm (m, 2H, Ph-H), one secondary
amine proton (NH) signal not observed; HRMS (ESI): m/z [M+H]+
calcd for C8H12N3: 150.1026, found: 150.1056.
d=6.59 (s, 2H, NH2), 7.24 (dd, 2H, J=8.6 1.2 Hz, Ph-H), 7.29 (t, 1H,
J=7.5 Hz, Ph-H), 7.39 (brs, 1H, NH), 7.44 (dt, 2H, J=8.0, 0.7 Hz, Ph-
H), 9.58 ppm (brs, 1H, NH); HRMS (ESI): m/z [M+H]+ calcd for
C7H10N3: 136.0869, found: 136.0893.
1-(p-Tolyl)guanidine (7b): Beige powder (92%); 1H NMR
([D6]DMSO): d=2.32 (s, 3H, CH3), 7.13 (d, 2H, J=8.3 Hz, Ph-H), 7.24
(brs, 3H, NH2, NH), 7.25 (d, 2H, J=8.1 Hz, Ph-H), 9.30 ppm (brs,
1H, NH); HRMS (ESI): m/z [M+H]+ calcd for C8H12N3: 150.1026,
found: 150.1018.
1-(4-Fluoro-2-methylphenyl)guanidine (7m): Purple powder
1
(96%); H NMR ([D6]DMSO): d=2.20 (s, 3H, CH3), 7.11 (dt, 1H, J=
8.5, 2.9 Hz, Ph-H), 7.21 (brs, 3H, NH2, NH), 7.22–7.28 (m, 2H, Ph-H),
8.98 ppm (brs, 1H, NH); HRMS (ESI): m/z [M+H]+ calcd for
C8H11FN3: 168.0932, found: 168.1104.
1-(4-Ethylphenyl)guanidine (7c): Beige powder (66%); 1H NMR
([D6]DMSO): d=1.19 (t, 3H, J=7.6 Hz, CH3), 2.62 (q, 2H, J=7.6 Hz,
CH2), 7.15 (d, 2H, J=8.3 Hz, Ph-H), 7.25 (brs, 3H, NH2, NH), 7.29 (d,
2H, J=8.6 Hz, Ph-H), 9.40 ppm (brs, 1H, NH); HRMS (ESI): m/z [M+
H]+ calcd for C9H14N3: 164.1182, found: 164.1181.
1-(m-Tolyl)guanidine (7p): White powder (90%); 1H NMR
([D6]DMSO): d=3.77 (s, 3H, CH3), 7.03 (d, 1H, J=8.0 Hz, Ph-H), 7.05
(s, 1H, Ph-H), 7.12 (d, 1H, J=7.6 Hz, Ph-H), 7.32 (apparent t, 1H,
J=7.7 Hz, Ph-H), 7.33 (brs, 3H, NH2, NH), 9.56 ppm (brs, 1H, NH);
HRMS (ESI): m/z [M+H]+ calcd for C8H12N3: 150.1026, found:
150.1019.
1-(4-Methoxyphenyl)guanidine (7d): Brown powder (78%);
1H NMR ([D6]DMSO): d=3.77 (s, 3H, CH3), 6.98–7.02 (m, 2H, Ph-H),
7.15–7.19 (m, 2H, Ph-H), 7.30 ppm (brs, 4H, NH2, 2ꢂNH); HRMS
(ESI): m/z [M+H]+ calcd for C8H12N3O: 166.0975, found: 166.1102.
1-(3-Methoxyphenyl)guanidine (7q): Beige powder (98%);
1H NMR ([D6]DMSO): d=3.77 (s, 3H, CH3), 6.78–6.82 (m, 2H, Ph-H),
6.87 (ddd, 1H, J=8.4, 2.4, 0.9 Hz, Ph-H), 7.20–7.39 (m, 4H, Ph-H,
NH2, NH), 9.53 ppm (brs, 1H, NH); HRMS (ESI): m/z [M+H]+ calcd
for C8H12N3O: 166.0975, found: 166.1026.
1-(2,4-Dimethoxyphenyl)guanidine (7e): Purple powder (64%);
1H NMR ([D6]DMSO): d=3.79 (s, 3H, CH3), 3.81 (s, 3H, CH3), 6.57
(dd, 1H, J=8.6, 2.4 Hz, Ph-H), 6.70 (d, 1H, J=2.3 Hz, Ph-H), 7.05
(brs, 3H, NH2, NH), 7.13 (d, 1H, J=8.6 Hz, Ph-H), 8.79 ppm (s, 1H,
NH); HRMS (ESI): m/z [M+H]+ calcd for C9H14N3O2: 196.1081,
found: 196.0785.
1-(3-Acetylphenyl)guanidine (7r): Beige powder (79%); 1H NMR
([D6]DMSO): d=2.60 (s, 3H, CH3), 7.49 (brs, 3H, NH2, NH), 7.60 (ap-
parent s, 2H, Ph-H), 7.78 (apparent s, 1H, Ph-H), 7.88 (apparent s,
1H, Ph-H), 9.67 ppm (brs, 1H, NH); HRMS (ESI): m/z [M+H]+ calcd
for C9H12N3O: 178.0975, found: 178.1009.
1-(4-Chloro-2-methoxyphenyl)guanidine (7 f): Pink powder
1
(62%); H NMR ([D6]DMSO): d=3.85 (s, 3H, CH3), 7.07 (dd, 1H, J=
8.4, 2.3 Hz, Ph-H), 7.22 (brs, 3H, NH2, NH), 7.24 (d, 1H, J=8.4 Hz,
Ph-H), 7.25 (d, 1H, J=2.2 Hz, Ph-H), 9.12 ppm (brs, 1H, NH); HRMS
(ESI): m/z [M+H]+ calcd for C8H11ClN3O: 200.0585, found:
200.0109.
N-(3-Guanidino-2-methylphenyl)methanesulfonamide (7n): Tri-
methylsilylchloride (2.50 mL) was added dropwise to solution of
6n (2.00 g, 10.0 mmol) and cyanamide (840 mg, 20.0 mmol) in ace-
tonitrile (40 mL) on an ice bath. The reaction mixture was allowed
to warm up to RT and then heated at reflux for 15 h. After cooling
down to RT, the precipitate was filtered and washed with EtOAc to
1-(2-Methoxyphenyl)guanidine (7g): Purple powder (90%);
1H NMR ([D6]DMSO): d=3.82 (s, 3H, CH3), 7.00 (t, 1H, J=7.5 Hz, Ph-
H), 7.15 (d, 1H, J=8.2 Hz, Ph-H), 7.20 (brs, 3H, NH2, NH), 7.22 (d,
1H, J=7.7, Hz, Ph-H), 7.35 (t, 1H, J=7.5 Hz, Ph-H), 8.51 ppm (brs,
1H, NH); HRMS (ESI): m/z [M+H]+ calcd for C8H12N3O: 166.0975,
found: 166.0982.
1
give 7n as a pale pink powder (2.30 g, 95%): H NMR ([D6]DMSO):
d=2.19 (s, 3H, CH3), 3.00 (s, 3H, CH3), 7.14 (d, 1H, J=7.2, Ph-H),
7.29 (t, 1H, J=7.9 Hz, Ph-H), 7.29–7.39 (m, 4H, Ph-H, NH2, NH), 9.28
(s, 1H, NH), 9.59 ppm (s, 1H, NH); HRMS (ESI): m/z [M+H]+ calcd
for C9H15N4O2S: 243.0910, found: 243.0945.
1-(2-Ethoxyphenyl)guanidine (7h): Beige powder (36%); 1H NMR
([D6]DMSO): d=1.33 (t, 3H, J=7.0 Hz, CH3), 4.10 (q, 2H, J=7.0 Hz,
CH2), 6.99 (t, 1H, J=7.5 Hz, Ph-H), 7.14 (d, 1H, J=8.2 Hz, Ph-H),
7.17 (brs, 3H, NH2, NH), 7.23 (dd, 1H, J=7.8, 1.4 Hz, Ph-H), 7.32 (dt,
1H, J=7.3, 1.4 Hz, Ph-H), 8.96 ppm (brs, 1H, NH); HRMS (ESI): m/z
[M+H]+ calcd for C9H14N3O: 180.1131, found: 180.0705.
N-(3-Guanidinophenyl)methanesulfonamide (7o): Trimethylsilyl-
chloride (6.80 mL) was added dropwise to solution of 6o (3.13 g,
16.9 mmol) and cyanamide (1.15 g, 27.4 mmol) in acetonitrile
(60 mL) on an ice bath. The reaction mixture was allowed to warm
up to RT and then heated at reflux overnight. After cooling down
to RT, the mixture was filtered, and the filtrate was concentrated in
1-(2-(Difluoromethoxy)phenyl)guanidine (7i): White powder
(31%);1H NMR ([D6]DMSO): d=7.22 (t, 1H, J=73.4 Hz, CHF2), 7.39
(d, 1H, J=7.2 Hz, Ph-H), 7.44 (t, 1H, J=7.3 Hz, Ph-H), 7.32–7.36 (m,
5H, 2ꢂPh-H, NH2, NH), 8.91 ppm (brs, 1H, NH); HRMS (ESI): m/z
[M+H]+ calcd for C8H9F2N3O: 202.0786, found: 202.0839.
1
vacuo to give 7o as a yellow oil (2.54 g, 66%): H NMR ([D6]DMSO):
d=3.03 (s, 3H, CH3), 6.94 (d, 1H, J=7.4 Hz, Ph-H), 7.09 (s, 1H, Ph-
H), 7.11 (d, 1H, J=8.1 Hz, Ph-H), 7.37 (t, 1H, J=7.7 Hz, Ph-H), 7.61
(s, 3H, NH2, NH), 10.00 (s, 1H, NH), 10.14 ppm (s, 1H, NH); HRMS
(ESI): m/z [M+H]+ calcd for C8H13N4O2S: 229.0754, found:
229.0833.
1-(2-(Trifluoromethyl)phenyl)guanidine (7j): Beige powder (60%);
1H NMR ([D6]DMSO): d=7.32 (brs, 3H, NH2, NH), 7.55 (d, 1H, J=
7.7 Hz, Ph-H), 7.63 (t, 1H, J=7.4 Hz, Ph-H), 7.79 (t, 1H, J=7.5 Hz,
Ph-H), 7.85 (d, 1H, J=7.7 Hz, Ph-H), 8.98 ppm (brs, 1H, NH); HRMS
(ESI): m/z [M+H]+ calcd for C8H9F3N3: 204.0743, found: 204.0473.
General procedure for preparation of 8: The corresponding gua-
nidine (2 equiv) and NaOH (2 equiv) were added to a suspension
of 4 (1 equiv) in CH3CN. The reaction mixture was heated under
microwave at 1608C for 30 min, concentrated in vacuo and puri-
fied by flash column chromatography (silica gel, petroleum
ether!petroleum ether/EtOAc 1:4) to give the desired product.
1-(2-Ethylphenyl)guanidine (7k): Beige powder (57%); 1H NMR
([D6]DMSO): d=1.14 (t, 3H, J=7.6 Hz, CH3), 2.56 (q, 2H, J=7.6 Hz,
CH2), 7.17 (brs, 3H, NH2, NH), 7.21 (d, 1H, J=7.1 Hz, Ph-H), 7.30 (dt,
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3,4-Dimethyl-5-(2-(phenylamino)pyrimidin-4-yl)thiazol-2(3H)-one
(8a): Yellow powder (39%); mp: 192–1948C; 1H NMR ([D6]DMSO):
d=2.56 (s, 3H, CH3), 3.30 (s. 3H, CH3), 6.95 (d, 1H, J=5.5 Hz, pyri-
midinyl-H), 6.96 (t, 1H, J=8.0 Hz, Ph-H), 7.29 (t, 2H, J=8.5 Hz, Ph-
H), 7.73 (d, 2H, J=7.5 Hz, Ph-H), 8.43 (d, 1H, J=5.3 Hz, pyrimidinyl-
H), 9.60 ppm (s, 1H, NH); 13C NMR ([D6]DMSO): d=14.3, 29.6, 108.3,
109.8, 119.1, 121.6, 128.5, 137.9, 140.3, 157.8, 158.6, 159.5,
169.8 ppm, two carbon signals overlapping or obscured; HRMS
(ESI): m/z [M+H]+ calcd for C15H15N4OS: 299.0961, found:
299.1525; Anal. RP-HPLC: method A: tR =11.58 min, purity>96%,
method B: tR =10.36 min, purity>96%.
1H, J=8.6, 2.2 Hz, Ph-H), 7.12 (d, 1H, J=2.2 Hz, Ph-H), 8.00 (d, 1H,
J=8.6 Hz, Ph-H), 8.21 (s, 1H, NH), 8.41 ppm (d, 1H, J=5.3 Hz, pyri-
midinyl-H); 13C NMR ([D6]DMSO): d=14.2, 29.6, 56.2 108.7, 109.4,
111.5, 119.9, 122.2, 126.9, 127.4, 138.2, 150.7, 158.0, 158.7, 159.4,
169.7 ppm; HRMS (ESI): m/z [M+H]+ calcd for C16H16ClN4O2S:
363.0677, found: 363.0549; Anal. RP-HPLC: method A: tR =
12.74 min, purity >97%, method B: tR =11.63 min, purity>95%.
5-(2-((2-Methoxyphenyl)amino)pyrimidin-4-yl)-3,4-dimethylthia-
1
zol-2(3H)-one (8g): White powder (12%); mp: 165–1668C; H NMR
([D6]DMSO): d=2.53 (s, 3H, CH3), 3.29 (s. 3H, CH3), 3.85 (s, 3H,
CH3), 6.94 (d, 1H, J=5.4 Hz, pyrimidinyl-H), 6.96–6.70 (m, 1H, Ph-
H), 7.02–7.08 (m, 2H, Ph-H), 8.04 (d, 1H, J=7.8 Hz, Ph-H) 8.12 (s,
1H, NH), 8.40 ppm (d, 1H, J=5.3 Hz, pyrimidinyl-H); 13C NMR
([D6]DMSO): d=14.2, 29.6, 55.7, 108.4, 109.5, 110.9, 120.3, 121.0,
123.2, 128.3, 138.0, 149.6, 157.9, 158.7, 159.5, 169.7 ppm; HRMS
(ESI): m/z [M+H]+ calcd for C16H17N4O2S: 329.1067; found:
329.1035. Anal. RP-HPLC: method A: tR =12.04 min, purity>99%,
method B: tR =10.85 min, purity>99%.
3,4-Dimethyl-5-(2-(p-tolylamino)pyrimidin-4-yl)thiazol-2(3H)-one
1
(8b): Yellow powder (23%); mp: 240–2418C; H NMR ([D6]DMSO):
d=2.25 (s, 3H, CH3), 2.56 (s. 3H, CH3), 3.30 (s, 3H, CH3), 6.91 (d, 1H,
J=5.3 Hz, pyrimidinyl-H), 7.10 (d, 2H, J=8.3 Hz, Ph-H), 7.62 (d, 2H,
J=1.7 Hz, Ph-H), 8.41 (d, 1H, J=5.3 Hz, pyrimidinyl-H), 9.49 ppm (s,
1H, NH); 13C NMR ([D6]DMSO): d=14.2, 20.4, 29.6, 108.0, 109.9,
119.2, 128.9, 130.4, 137.7, 137.8, 157.8, 158.5, 159.6, 169.7 ppm,
two carbon signals overlapping or obscured; HRMS (ESI): m/z [M+
H]+ calcd for C16H17N4OS: 313.1118, found: 313.1052; Anal. RP-
HPLC: method A: tR =11.98 min, purity>96%, method B: tR =
10.76 min, purity>96%.
5-(2-(2-Ethoxyphenylamino)pyrimidin-4-yl)-3,4-dimethylthiazol-
2(3H)-one (8h): yellow powder (32%); mp: 177–1788C; 1H NMR
([D6]DMSO): d=1.35 (t, 3H, J=7.0 Hz, CH3), 2.54 (s. 3H, CH3), 3.29
(s, 3H, CH3), 4.11 (q, 2H, J=7.0 Hz, CH2), 6.95 (d, 1H, J=5.2 Hz, pyr-
imidinyl-H), 6.97 (dt, 1H, J=7.8, 1.8 Hz, Ph-H), 7.01 (dt, 1H, J=8.0,
1.7 Hz, Ph-H), 7.04 (dd, 1H, J=8.1, 1.8 Hz, Ph-H), 8.06 (s, 1H, NH),
8.09 (dd, 1H, J=7.9, 1.6 Hz, Ph-H), 8.41 ppm (d, 1H, J=5.3 Hz, pyri-
midinyl-H); 13C NMR ([D6]DMSO): d=14.2, 14.7, 29.6, 63.9, 108.5,
109.6, 111.8, 120.3, 120.5, 123.0, 128.4, 138.1, 148.5, 157.9, 158.7,
159.4, 169.7 ppm; HRMS (ESI): m/z [M+H]+ calcd for C17H19N4O2S:
343.1223, found: 343.1671; Anal. RP-HPLC: method A: tR =
12.44 min, purity>97%, method B: tR =11.29 min, purity>97%.
5-(2-(4-Ethylphenylamino)pyrimidin-4-yl)-3,4-dimethylthiazol-
2(3H)-one (8c): Yellow powder (10%); mp: 204–2058C; 1H NMR
([D6]DMSO): d=1.17 (t, 3H, J=7.6 Hz, CH3), 2.55 (q, 2H, J=7.7 Hz,
CH2), 2.56 (s. 3H, CH3), 3.30 (s, 3H, CH3), 6.92 (d, 1H, J=5.4 Hz, pyri-
midinyl-H), 7.13 (d, 2H, J=8.5 Hz, Ph-H), 7.63 (d, 2H, J=8.5 Hz, Ph-
H), 8.41 (d, 1H, J=5.3 Hz, pyrimidinyl-H), 9.50 ppm (s, 1H, NH);
13C NMR ([D6]DMSO): d=14.2, 15.8, 27.6, 29.6, 108.0, 109.9, 119.3,
127.7, 137.0, 137.7, 137.9, 157.8, 158.5, 159.6, 169.8 ppm, two
carbon signals overlapping or obscured; HRMS (ESI): m/z [M+H]+
calcd for C17H19N4OS: 327.1274, found: 327.1524; Anal. RP-HPLC:
method A: tR =12.38 min, purity>96%, method B: tR =11.17 min,
purity>99%.
5-(2-(2-(Difluoromethoxy)phenylamino)pyrimidin-4-yl)-3,4-dime-
thylthiazol-2(3H)-one (8i): White powder (7%); mp: 126–1278C;
1H NMR (CD3OD): d=2.59 (s, 3H, CH3), 3.38 (s. 3H, CH3), 6.84 (t, 1H,
J=73.4 Hz, CHF2), 6.96 (d, 1H, J=5.4 Hz, pyrimidinyl-H), 7.10 (dt,
1H„ J=8.2, 1.6 Hz, Ph-H), 7.21–7.28 (m, 2H, Ph-H), 8.24 (dd, 1H„
J=8.2, 1.6 Hz, Ph-H), 8.38 ppm (d, 1H, J=5.3 Hz, pyrimidinyl-H),
one secondary amine proton signal (NH) not observed; 13C NMR
(CD3OD): d=14.5, 30.1, 110.2, 112.3, 118.2 (t, JCꢀF =257.3 Hz), 120.3,
123.7, 124.6, 126.7, 132.6, 139.1, 143.2, 159.5, 160.3, 161.0,
173.1 ppm; HRMS (ESI): m/z [M+H]+ calcd For C16H15F2N4O2S:
365.0878, found: 365.0864; Anal. RP-HPLC: method A: tR =
11.89 min, purity>99%, method B: tR =10.83 min, purity>99%.
5-(2-((4-Methoxyphenyl)amino)pyrimidin-4-yl)-3,4-dimethylthia-
zol-2(3H)-one (8d): Yellow powder (21%); mp: 217–2198C;
1H NMR ([D6]DMSO): d=2.55 (s, 3H, CH3), 3.29 (s, 3H, CH3), 3.73 (s.
3H, CH3), 6.88–6.90 (m, 3H, Ph-H, pyrimidinyl-H), 7.60–7.62 (m, 2H,
Ph-H), 8.39 (d, 1H, J=5.3 Hz, pyrimidinyl-H), 9.45 ppm (s, 1H, NH);
13C NMR ([D6]DMSO): d=14.2, 29.6, 55.2, 107.8, 109.9, 113.7, 120.9,
133.3, 137.6, 154.3, 157.7, 158.5, 159.6, 169.7 ppm (two carbon sig-
nals overlapping or obscured); HRMS (ESI): m/z [M+H]+ calcd for
C16H17N4O2S: 329.1067, found: 329.0706; Anal. RP-HPLC: method A:
tR =12.30 min, purity>98%, method B: tR =10.10 min, purity>
97%.
3,4-Dimethyl-5-(2-((2-(trifluoromethyl)phenyl)amino)pyrimidin-4-
yl)thiazol-2(3H)-one (8j): White powder (10%); mp: 135–1378C;
1H NMR ([D6]DMSO): d=2.42 (s, 3H, CH3), 3.24 (s. 3H, CH3), 6.82 (d,
1H, J=5.4 Hz, pyrimidinyl-H), 7.43 (t, 1H, J=7.5 Hz, Ph-H), 7.64 (d,
1H, J=7.9 Hz, Ph-H), 7.69 (t, 1H, J=7.9 Hz, Ph-H), 7.74 (d, 1H, J=
7.8 Hz, Ph-H), 8.33 (d, 1H, J=5.3 Hz, pyrimidinyl-H), 8.86 ppm (s,
1H, NH); 13C NMR ([D6]DMSO): d=13.9, 29.5, 108.3, 109.0, 123.9 (q,
5-(2-((2,4-Dimethoxyphenyl)amino)pyrimidin-4-yl)-3,4-dime-
thylthiazol-2(3H)-one (8e): Yellow powder (29%); mp: 154–1568C;
1H NMR ([D6]DMSO): d=3.27 (s, 3H, CH3), 3.32 (s, 3H, CH3), 3.76 (s.
3H, CH3), 3.79 (s, 3H, CH3), 6.53 (dd, 1H, J=8.7, 2.7 Hz, Ph-H), 6.63
(d, 1H, J=2.7 Hz, Ph-H), 6.81 (d, 1H, J=5.4 Hz, pyrimidinyl-H), 7.66
(d, 1H, J=8.8 Hz Ph-H), 8.10 (s, 1H, NH), 8.32 ppm (d, 1H, J=
5.3 Hz, pyrimidinyl-H); 13C NMR ([D6]DMSO): d=14.0, 29.5, 55.3,
55.7, 98.9, 103.9, 107.6, 109.5, 121.3, 124.0, 137.7, 152.2, 156.5,
157.9, 158.6, 160.2, 169.6 ppm; HRMS (ESI): m/z [M+H]+ calcd for
C17H19N4O3S: 359.1172, found: 359.1518; Anal. RP-HPLC: method A:
tR =11.64 min, purity >95%, method B: tR =10.31 min, purity>
95%.
J
CꢀF =271.7 Hz), 124.9 (q, JCꢀF =28.8 Hz), 125.9, 126.4 (q, JCꢀF =
5.0 Hz), 130.0, 133.0, 137.4, 138.1, 158.1, 158.8, 161.1, 169.6 ppm;
HRMS (ESI): m/z [M+H]+ calcd for C16H14F3N4OS: 367.0835, found:
367.0783; Anal. RP-HPLC: method A: tR =12.09 min, purity>96%,
method B: tR =11.17 min, purity>95%.
5-(2-((2-Ethylphenyl)amino)pyrimidin-4-yl)-3,4-dimethylthiazol-
2(3H)-one (8k): Yellow powder (25%); mp: 118–1208C; 1H NMR
([D6]DMSO): d=1.10 (t, 3H, J=7.6 Hz, CH3), 2.45 (s, 3H, CH3), 2.61
(q, 2H, J=7.6 Hz, CH2), 3.25 (s, 3H, CH3), 6.75 (d, 1H, J=5.3 Hz, pyr-
imidinyl-H), 7.13 (dt, 1H, J=7.4, 1.3 Hz, Ph-H), 7.18 (dt, 1H, J=7.6,
1.6 Hz, Ph-H), 7.24 (dd, 1H, J=7.4, 1.4 Hz, Ph-H), 7.36 (dd, 1H, J=
7.9, 1.1 Hz, Ph-H), 8.30 (d, 1H, J=5.3 Hz, pyrimidinyl-H), 8.80 ppm
5-(2-(4-Chloro-2-methoxyphenylamino)pyrimidin-4-yl)-3,4-dime-
thylthiazol-2(3H)-one (8 f): Orange powder (18%); mp: 236–
1
2378C; H NMR ([D6]DMSO): d=2.53 (s, 3H, CH3), 3.29 (s. 3H, CH3),
3.86 (s, 3H, CH3), 6.94 (d, 1H, J=5.3 Hz, pyrimidinyl-H), 7.02 (dd,
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(s, 1H, NH); 13C NMR ([D6]DMSO): d=14.0, 14.1, 23.9, 29.5, 107.5,
109.3, 125.2, 125.8, 126.6, 128.3, 137.1, 137.7, 138.9, 158.0, 158.7,
161.0, 169.6 ppm; HRMS (ESI): m/z [M+H]+ calcd for C17H19N4OS:
327.1274, found: 327.0047; Anal. RP-HPLC: method A: tR =
11.92 min, purity>98%, method B: tR =10.75 min, purity>99%.
8.43 (d, 1H, J=5.3 Hz, pyrimidinyl-H), 9.53 ppm (s, 1H, NH);
13C NMR ([D6]DMSO): d=14.2, 21.4, 29.6, 108.2, 109.9, 116.3, 119.6,
122.3, 128.3, 137.5, 137.9, 140.2, 157.7, 158.6, 159.6, 169.8 ppm;
HRMS (ESI): m/z [M+H]+ calcd for C16H17N4O2S: 313.1118, found:
313.1270; Anal. RP-HPLC: method A: tR =11.99 min, purity>99%;
method B: tR =10.76 min, purity>99%.
3,4-Dimethyl-5-(2-(o-tolylamino)pyrimidin-4-yl)thiazol-2(3H)-one
(8l): White powder (20%); mp: 138–1408C. 1H NMR ([D6]DMSO):
d=2.21 (s, 3H, CH3), 2.47 (s. 3H, CH3), 3.26 (s, 3H, CH3), 6.79 (d, 1H,
J=5.3 Hz, pyrimidinyl-H), 7.06 (dt, 1H, J=8.5, 1.1 Hz, Ph-H), 7.17
(dt, 1H, J=7.5, 1.0 Hz, Ph-H), 7.22 (d, 1H, J=7.4 Hz, Ph-H), 7.43 (d,
1H, J=7.4 Hz, Ph-H), 8.32 (d, 1H, J=5.3 Hz, pyrimidinyl-H),
8.81 ppm (s, 1H, NH); 13C NMR ([D6]DMSO): d=14.0, 18.1, 29.5,
107.6, 109.4, 124.5, 125.4, 125.8, 130.2, 132.5, 137.7, 158.0, 158.7,
160.6, 169.6 ppm, one carbon signal overlapping or obscured;
HRMS (ESI): m/z [M+H]+ calcd for C16H17N4OS: 313.1118, found:
313.0653; Anal. RP-HPLC: method A: tR =11.61 min, purity>96%,
method B: tR =10.34 min, purity>97%.
5-(2-(3-Methoxyphenylamino)pyrimidin-4-yl)-3,4-dimethylthia-
zol-2(3H)-one (8q): Yellow powder (33%); mp: 180–1818C;
1H NMR ([D6]DMSO): d=2.56 (s, 3H, CH3), 3.30 (s. 3H, CH3), 3.75 (s,
3H, CH3), 6.54 (dd, 1H, J=8.1, 2.1 Hz, Ph-H), 6.97 (d, 1H, J=5.3 Hz,
pyrimidinyl-H), 7.18 (t, 1H, J=8.2 Hz, Ph-H), 7.28 (dd, 1H, J=8.1,
1.0 Hz, Ph-H), 7.48 (t, 1H, J=2.1 Hz, Ph-H), 8.44 (d, 1H, J=5.3 Hz,
pyrimidinyl-H), 9.59 ppm (s, 1H, NH); 13C NMR ([D6]DMSO): d=14.3,
29.6, 55.0, 104.9, 106.9, 108.4, 109.8, 111.5, 129.2, 137.9, 141.5,
158.5, 159.5, 159.6, 169.8 ppm, one carbon signal overlapping or
obscured; HRMS (ESI): m/z [M+H]+ calcd for C16H17N4O2S:
329.1067, found: 329.1153; Anal. RP-HPLC: method A: tR =
11.58 min, purity>99%, method B: tR =10.35 min, purity>99%.
5-(2-(4-Fluoro-2-methylphenylamino)pyrimidin-4-yl)-3,4-dime-
5-(2-(3-Acetylphenylamino)pyrimidin-4-yl)-3,4-dimethylthiazol-
2(3H)-one (8r): Yellow powder (30%); mp: 225–2268C; 1H NMR
([D6]DMSO): d=2.57 (s, 3H, CH3), 2.58 (s. 3H, CH3), 3.30 (s, 3H,
CH3), 6.99 (d, 1H, J=5.3 Hz, pyrimidinyl-H), 7.45 (t, 1H, J=7.9 Hz,
Ph-H), 7.57 (d, 1H, J=7.7 Hz, Ph-H), 7.99 (dd, 1H, J=8.1, 1.3 Hz,
Ph-H), 8.34 (s, 1H, Ph-H), 8.47 (d, 1H, J=5.3 Hz, pyrimidinyl-H),
9.81 ppm (s, 1H, NH); 13C NMR ([D6]DMSO): d=14.3, 26.9, 29.6,
108.8, 109.6, 118.3, 121.6, 123.6, 128.8, 137.3, 138.1, 140.7, 157.9,
158.6, 159.4, 169.8, 197.8 ppm; HRMS (ESI): m/z [M+H]+ calcd for
C17H17N4O2S: 341.1067, found: 341.1006; Anal. RP-HPLC: method A:
tR =11.34 min, purity>95%, method B: tR =9.99 min, purity>96%.
thylthiazol-2(3H)-one (8m): Yellow powder (41%); mp: 175–
1768C; H NMR ([D6]DMSO): d=2.20 (s, 3H, CH3), 2.46 (s. 3H, CH3),
1
3.25 (s, 3H, CH3), 6.78 (d, 1H, J=5.3 Hz, pyrimidinyl-H), 6.98 (dt,
1H, J=8.6, 3.0 Hz, Ph-H), 7.09 (dd, 1H, J=9.7, 2.9 Hz, Ph-H), 7.36
(dd, 1H, J=8.7, 5.7 Hz, Ph-H), 8.31 (d, 1H, J=5.3 Hz, pyrimidinyl-H),
8.83 ppm (s, 1H, NH); 13C NMR ([D6]DMSO): d=14.0, 18.1, 29.5,
107.6, 109.3, 112.3 (d, JCꢀF =21.7 Hz), 116.4 (d, JCꢀF =22.0 Hz), 127.7
(d, JCꢀF =8.6 Hz), 134.1 (d, JCꢀF =2.8 Hz), 135.9 (d, JCꢀF =8.1 Hz),
137.8, 158.0, 158.7, 159.5 (d, JCꢀF =239.1 Hz), 160.7, 169.6 ppm;
HRMS (ESI): m/z [M+H]+ calcd for C16H16FN4OS: 331.1023, found:
331.1652; Anal. RP-HPLC: method A: tR =11.66 min, purity>97%,
method B: tR =10.39 min, purity>97%.
N-(3-((4-(3,4-Dimethyl-2-oxo-2,3-dihydrothiazol-5-yl)pyrimidin-2-
yl)amino)-2-methylphenyl)methanesulfonamide
(8n):
White
Computational methods
powder (31%); mp: 244–2458C; 1H NMR ([D6]DMSO): d=2.18 (s,
3H, CH3), 2.47 (s, 3H, CH3), 2.98 (s, 3H, CH3), 3.25 (s, 3H, CH3), 6.79
(d, 1H, J=5.3 Hz, pyrimidinyl-H), 7.13 (d, 1H, J=7.7 Hz, Ph-H), 7.18
(t, 1H, J=7.9 Hz, Ph-H), 7.34 (d, 1H, J=7.6 Hz, Ph-H), 8.32 (d, 1H,
J=5.3 Hz, pyrimidinyl-H), 8.91 (s, 1H, NH), 9.10 ppm (s, 1H, NH);
13C NMR ([D6]DMSO): d=13.5, 14.0, 29.5, 107.8, 109.3, 123.2, 124.2,
125.5, 130.5, 136.0, 137.8, 138.8, 158.0, 158.7, 160.7, 169.6 ppm,
one carbon signal overlapping or obscured; HRMS (ESI): m/z [M+
H]+ calcd for C17H20N5O3S2: 406.1002, found: 406.0977; Anal. RP-
HPLC: method A: tR =10.18 min, purity >99%, method B: tR =
8.98 min, purity>99%.
The required atomic coordinates of CDK2 (PDB IDs: 2XMY, 2XNB,
3QTR, 3QTU, 3QUO, 3QXP, 3RAL, 3RJC, 3RKB, 3TNW, 4BCK, 4BCM,
4BCO, 4BCP) and CDK9 (PDB IDs: 3MYI, 3BLQ, 3BLR, 3 MIA, 4BCF,
4BCG, 4BCH, 4BCI, 4BCJ, 4IMY) were obtained from the Protein
Data Bank. These were prepared for docking using the Protein
Preparation Wizard of Schrçdinger Maestro (Schrçdinger Inc.,
2013). Any missing atoms in the receptor structures were added
and mutations were reverted during this process. Docking grids for
each receptor were generated with the centre at the adenine bind-
ing pocket of the ATP binding site. Compounds 8a–r were sepa-
rately prepared using the Ligprep utility of Schrçdinger. Subse-
quently, Glide was used to dock all the compounds to the different
receptor conformations.[17] During docking, the extra-precision
mode was used while the maximum number of energy minimiza-
tion steps was raised to 400 to ensure convergence. The results
were sorted according to the Glide-XP docking score and the
poses were visualized in Maestro. The best scoring pose of each
ligand–receptor complex was used for analysis.
N-(3-((4-(3,4-Dimethyl-2-oxo-2,3-dihydrothiazol-5-yl)pyrimidin-2-
yl)amino)phenyl)methanesulfonamide (8o): Orange powder
1
(13%); mp: 287–2888C; H NMR ([D6]DMSO): d=2.57 (s, 3H, CH3),
2.99 (s. 3H, CH3), 3.30 (s, 3H, CH3), 6.82 (d, 1H, J=7.8 Hz, Ph-H),
6.95 (d, 1H, J=5.3 Hz, pyrimidinyl-H), 7.24 (t, 1H, J=8.1 Hz, Ph-H),
7.55 (s, 1H, Ph-H), 7.58 (d, 1H, J=8.3 Hz, Ph-H), 8.43 (d, 1H, J=
5.3 Hz, pyrimidinyl-H), 9.65 ppm (s, 2H, 2ꢂNH); 13C NMR
([D6]DMSO): d=14.3, 29.6, 108.6, 109.6, 111.1, 113.5, 115.0, 129.1,
138.0, 138.5, 141.1, 158.0, 158.5, 159.5, 169.8 ppm, one carbon
signal overlapping or obscured; HRMS (ESI): m/z [M+H]+ calcd for
C16H18N5O3S2: 392.0846, found: 392.1049; Anal. RP-HPLC: meth-
od A: tR =10.47 min, purity>95%, method B: tR =9.27 min,
purity>95%.
Biology
Kinase assays: Inhibition of Mnk, CDK2 and CDK9 were measured
by radiometric assay using the Millipore KinaseProfiler services
using ATP KM(app) concentration of ATP of each kinase. Half-maximal
inhibition (IC50) values were calculated from 10-point dose–re-
sponse curves and apparent inhibition constants (Ki) were calculat-
ed from the IC50 values and appropriate KM (ATP) values for the kin-
ases in question.
3,4-Dimethyl-5-(2-(m-tolylamino)pyrimidin-4-yl)thiazol-2(3H)-one
(8p): White powder (21%); mp: 196–1978C; 1H NMR ([D6]DMSO):
d=2.30 (s, 3H, CH3), 2.57 (s. 3H, CH3), 3.30 (s, 3H, CH3), 6.78 (d, 1H,
J=7.5 Hz, Ph-H), 6.94 (d, 1H, J=5.3 Hz, pyrimidinyl-H), 7.17 (t, 1H,
J=7.8 Hz, Ph-H), 7.48 (d, 1H, J=8.2 Hz, Ph-H), 7.64 (s, 1H, Ph-H),
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 962 – 972 971

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Cell culture: A2780 cells were purchased from the European Collec-
tion of Cell Culture (ECACC). MV4-11 cells were kindly provided by
Prof. R. D’Andrea (University of South Australia). All other cell lines
were obtained from the cell bank at the Centre for Drug Discovery
and Development (University of South Australia). All cell lines were
maintained in RPMI-1640 with 10% fetal bovine serum (FBS).
Keywords: anticancer drug discovery · apoptosis · CGP57380 ·
eIF4E phosphorylation · Mnk inhibitors
Proliferation assays: Resazurin (Sigma) assays were performed in
MV4–11 cell lines according to the manufacturer instructions. Cells
were seeded at 5ꢂ103 cells/well into 96-well plates and incubated
overnight at 378C, 5% CO2. Tested compounds were diluted from
a 10 mm stock solution to prepare a fivefold dilution series in
100 mL of cell medium, added to cells (in triplicates), and incubated
at the corresponding time point at 378C, 5% CO2. Resazurin was
made up as a stock of 0.1 mgmLꢀ1 in cell medium, and the solu-
tion was filter-sterilized. The resazurin solution was then added at
20 mL/well and incubated in the dark at 378C, 5% CO2 for 4 h. The
plate was left at RT for 10–15 min, and absorbance was measured
at 585 nm using an EnVision multi-label plate reader (PerkinElmer,
Beaconsfield, Buckinghamshire, UK). MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide, Sigma) assays were performed
as reported previously.[12a] Compound concentrations required to
inhibit 50% of cell growth (GI50) were calculated using non-linear
regression analysis.
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Caspase-3/7 assay: Activity of caspase 3/7 was measured using the
Apo-ONE Homogeneous Caspase-3/7 kit (G7790 Promega, Madi-
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Cell cycle analysis and detection of apoptosis: A2780 (5ꢂ104) and
MV4-11 (1ꢂ105) cells were seeded and incubated overnight at
378C, 5% CO2. Followed by the treatment with inhibitors, cells
were trypsinised (not applied to suspension cell line MV4-11) and
centrifuged (300 g, 5 min). Cell pellets were collected and fixed
with 70% EtOH on ice for 15 min, followed by centrifugation
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solution (50 mgmLꢀ1 propidium iodide (PI), 0.1 mgmLꢀ1 RNase A,
0.05% Triton X-100) at 378C for 1 h and analysed with Gallios flow
cytometer (Beckman Coulter, Brea, CA, USA). 1ꢂ105 cells were pro-
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commercial kit (Becton Dickenson, Franklin Lakes, NJ, USA). Sam-
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Acknowledgements
This study is supported by Australia Government National Health
and Medical Research Council (research grant 1050825).
Received: December 23, 2013
Published online on February 12, 2014
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 962 – 972 972