Synthesis and evaluation of the NSCLC anti-cancer activity and physical properties of 4-aryl-N-phenylpyrimidin-2-amines

Article abstract of DOI:10.1016/j.bmcl.2017.08.063

Reported herein are efforts to profile 4-aryl-N-phenylpyrimidin-2-amines in terms of their anti-cancer activity towards non small-cell lung carcinoma (NSCLC) cells. We have synthesized new 4-aryl-N-phenylpyrimidin-2-amines and assessed them in terms of their cytotoxicity (A549, NCI-H187, MCF7, Vero & KB) and physicochemical properties (logD_7.4 and solubility). 13f and 13c demonstrated potent anti-cancer activity in A549 cells (0.2 μM), compared to 0.4 μM for the NSCLC drug Doxorubicin. 13f also displayed low experimental logD_7.4 (2.9) and the best solubility (～40 μM). Compounds 13b and 13d showed the best balance of A549 anti-cancer activity and selectivity. 13g showed good activity and selectivity comparable with the anti-cancer drug Doxorubicin.

Full text of DOI:10.1016/j.bmcl.2017.08.063

Accepted Manuscript
Synthesis and Evaluation of the NSCLC Anti-cancer Activity and Physical
Properties of 4-aryl-N-phenylpyrimidin-2-amines
Borvornwat Toviwek, Praphasri Suphakun, Kiattawee Choowongkomon, Supa
Hannongbua, M. Paul Gleeson
PII:
S0960-894X(17)30855-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.063
BMCL 25257
Reference:
Bioorganic & Medicinal Chemistry Letters
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19 August 2017
25 August 2017
Please cite this article as: Toviwek, B., Suphakun, P., Choowongkomon, K., Hannongbua, S., Gleeson, M.P.,
Synthesis and Evaluation of the NSCLC Anti-cancer Activity and Physical Properties of 4-aryl-N-
phenylpyrimidin-2-amines, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/
j.bmcl.2017.08.063
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Synthesis and Evaluation of the NSCLC anti-
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aryl-N-phenylpyrimidin-2-amines
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Borvornwat Toviweka, Praphasri Suphakunb, Kiattawee Choowongkomonb Supa Hannongbuaa and M. Paul
Gleesona,c*
a Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
b Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
c Department of Biomedical Engineering, Faculty of Engineering, King Mongkut’s Institute of Technology
Ladkrabang, Bangkok 10520, Thailand.

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Synthesis and Evaluation of the NSCLC Anti-cancer Activity and Physical
Properties of 4-aryl-N-phenylpyrimidin-2-amines
Borvornwat Toviweka, Praphasri Suphakunb, Kiattawee Choowongkomonb Supa Hannongbuaa and M. Paul
Gleesona,c∗
a Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
b Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
c Department of Biomedical Engineering, Faculty of Engineering, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand.
ARTICLE INFO
ABSTRACT
Article history:
Received
Reported herein are efforts to profile 4-aryl-N-phenylpyrimidin-2-amines in terms of their anti-
cancer activity towards non small-cell lung carcinoma (NSCLC) cells. We have synthesized new
4-aryl-N-phenylpyrimidin-2-amines and assessed them in terms of their cytotoxicity (A549,
NCI-H187, MCF7, Vero & KB) and physicochemical properties (logD7.4 and solubility). 13f and
9c demonstrated potent anti-cancer activity in A549 cells (0.2 µM), compared to 0.4 µM for the
NSCLC drug Doxorubicin. 13f also displayed low experimental logD7.4 (2.9) and the best
solubility (~40 µM). Compounds 13b and 13d showed the best balance of A549 anti-cancer
activity and selectivity. 13g showed good activity and selectivity comparable with the anti-
cancer drug Doxorubicin.
Received in revised form
Accepted
Available online
Keywords:
Computational design
Lipophilicity
A549
2017 Elsevier Ltd. All rights reserved.
Solubility
Cytotoxicity
Lung cancer is one of the largest contributors to cancer
fatalities in the world according to the World Health
Organization (WHO).1 The incidence rates are highest in less
developed regions, and generally higher in men compared to
women. It is found that the incidence rates in different regions of
the world reflect different historical exposures to tobacco
smoking.1, 2 There are two main types of lung cancer: Non small-
cell lung carcinoma (NSCLC) and small-cell lung carcinoma
(SCLC), the former accounting for approximately 80% of the
total.3-5 Recent estimates put its five-year survival rate at ~15%
confirming the need for improved therapeutic treatments.6
1
2
NHSO2Me
Me
S
O
O
NH2
NH2
S
S
N
N
O
O
N
NH
N
NH
µ
JNK1 IC50 = 0.019 M,
β
GSK3 IC50 = 0.02
µ
M
µ
JNK3 IC50 = 0.007
M
4
3
OMe
OMe
A number of targeted treatments exist for NSCLC. Target
based approaches have dominated drug discovery due to benefits
associated with selectively modulating a particular target relevant
to the disease pathway; ease of automation as well as its high
throughput nature.7 Protein kinases offer great potential due to
their role in regulating the majority of cellular pathways,
including cellular growth, differentiation and apoptosis.8 These
inhibitors work on certain patient sub-populations and are
sensitive to gene mutations,9, 10 meaning new of treatments are
constantly sought. Current kinase inhibitors used to treat NSCLC
include Erlotinib, Lapatinib (EGFR) and Crizotinib and Ceritinib
(ALK).8
NH2
O
NH2
S
N
N
O
N
NH
N
NH
µ
β
IKK IC50 = 40
µ
M
CDK5 IC50 = 81
M
Figure 1. A selection of 2-Phenylamino-pyrimidine compounds targeting
JNK211, IKK212, GSK313 and CDK5. 14
In this work we investigate the anti-cancer activity of 4-aryl-
N-phenylpyrimidin-2-amines. These compounds are known to
act at a range of targets (Fig. S1), including exemplars in Fig. 1
that target protein kinases.11-14
———
∗ Corresponding authors. Tel.: +66-86-9779678; fax: +66-2-329-8346; e-mail: paul.gl@kmitl.ac.th

R1
Cl
N
R1
R1
R2
(a)
(b)
N
N
N
+
+
NH2
B(OH)2
R2
Cl
N
7-12
Cl
N
13a-q
NH
Scheme 1. General reagents and conditions used to produce 1. (a) Pd(PPh3)4, 2M Na2CO3, dioxane, 100oC, 6-15 h; (b) TFA, isopropanol, 90oC, overnight.
Substructure searches of the ChEMBL database using the N-
phenyl-4-(aryl)pyrimidin-2-amine core reveals 2599 unique
target-based activities (532 unique compounds) and 476 unique
cell based cytotoxicities (184 unique compounds). Cytotoxicity
measurements represent 34% of total measurements but were
collected onjust 18% of the total compounds. The advantage of
taking a cell based approach is the perceived increased disease
relevance15-19 and the possibility of identifying compounds with
desirable polypharmacology becomes more likely.20, 21
SCLC cell line (H187); HeLa contaminated cervical
adenocarcinoma cells cell line (KB); breast cancer cell (MCF7),
as are the experimental logD and phosphate buffer solubility at
pH7.4
.
A selection of new compounds were preparedModification to the
2-position and the 4-position of the pyrimidine scaffold were
undertaken as shown in Table 1. Intermediates 7-12 were
prepared as detailed in Scheme 1 using established synthetic
methods.11,
37, 38
. This intermediates were produced by the
coupling of 2,4 dichloropyrimidine with a range of arylboronic
acids or esters using Suzuki–Miyaura reaction in dioxane or
Less decorated 4-aryl-N-phenylpyrimidin-2-amines, as
exemplified by the structures in Fig. 1, act on targets previously
reported to have implications for NSCLC. JNK plays a role in
mediating cell growth and self renewal in A549 cells.22-27 It was
also reported CDK5 regulates the proliferation, motility and
invasiveness of lung cancer in A549 cells28 and that it was a
potential prognostic biomarker for NSCLC.29 IKKβ depletion in
tumor cells significantly attenuated tumor proliferation and
significantly prolonged mouse survival30 and its selective
targeting was reported to potentiate the efficacy of Taxol in
NSCL cancer.31 It was also reported that maintaining GSK3 cross
activity was needed to maintain the effectiveness of mTOR
inhibitors.32 Preparation of new compounds 5 and 6 (Fig. 2) was
therefore undertaken and these were subsequently shown to be
micro-molar inhibitors of the human adenocarcinomic alveolar
basal epithelial cells (A549) prompting a more extensive
investigation.
DMSO.11
The
reactions
utilized
tetrakis(triphenylphosphine)palladium(0) and 2M Na2CO3 giving
intermediates 7-12 in yields ranging from 30-80% Compounds
13a-q were produced by the acid catalyzed substituted of the 2-
chloropyrimidine of each intermediate with a range of anilines.
o
The typical procedure involved reactions at 90 C in isopropanol
with either TFA or 1M HCl for up to 10-24 hrs.
The cytotoxicity of compounds were evaluated in A549 cells
(ATCC CCL-185) using an MTT based assay. The compounds
displayed IC50 values between 0.2 and >100 µM (Table 1).
Compounds 13c and 13f showed the most potent activity, with
IC50 of 0.2 µM. This compares to 0.3 µM and 22 µM for the
standards Doxorubicin and Gefitinib, respectively. Six different
aryl boronic acids groups have been incorporated at the R1
position. Conisidering compounds with 3-sulfonamido aniline at
the R2, phenyl at the R1 (13f) gave rise to the best activity, 8 fold
greater than the initial hit 5, which possesses a 4-methoxy-
phenyl. The equivalent 3-methoxy-phenyl analog (13g) and N-4-
acetamido phenyl (13j) were over 10 fold less potent than 13f,
while all 4-methylsulphonyl phenyl compounds (13h and 13i)
were inactive. Compounds with 2-thiophene-5-Me at R1 showed
comparable or slightly weaker activity than the comparable 4-
methoxyphenyl compounds (6 vs 13n and 13b vs 13o). The data
collectively points to hydrophobicity being preferred at the R1
for strong A549 activity.
OMe
OMe
O
S
O
H2N
O
OH
N
N
N
NH
N
NH
The preference for hydrophobicity at the R1 necessitated
greater effort to incorporate polar modifications at R2 in order to
maintain a balance in the overall molecular properties (Fig. 3).
Compounds with 3-sulfonamido aniline (5, 13f, 13g, 13j)
6
A549 IC50= 20.3 µM
5
A549 IC50= 1.5 µM
Figure. 2. Initial compounds prepared showing good activity following
cytotoxicity assessment in the NSCLC based A549 cell line.
displayed
the good to reasonable
activity.
3-N-
methylsulphonamido aniline also led to very good activity (13c).
4-sulfonamido aniline was over 2 fold less active than the 3-
isomer (5 vs 13e). 3 and 4-amidobenzamide and amino-benzoic
acid were less active than the comparable sulphonamides as were
bicyclic amines represented by 13l and 13m. The SAR is broadly
in-line with that expected for certain MAPK kinases, which
possess relatively small hydrophobic back pocket and allow for
polar interactions towards the solvent accessible pocket (Fig. 4).
Herein, we report the design, synthesis and biological
new
evaluation
of
4-aryl-N-phenylpyrimidin-2-amine
compounds. Our goal is to improve our SAR understanding of
this series by screening additional analogs in the A549 cell line
making subtle modifications to scaffold and evaluating their
cytotoxicity, and the overall physical properties.33-35. We also
assess the cytotoxicity of the series using monkey kidney
epithelial cell line (Vero).36 For the most effective compounds,
the activity at additional human cell lines is also evaluated;

Table 1Activities and yields of compounds 1, 2, 13a-q. The selectivity of compound for A549 over Vero cells is also reported (SI).
Vero
IC50/uM
ID
R1
R2
clogPa
MWTa
Yield %
A549 IC50/µM
SI
0
OMe
5
2.65
356.4
45
1.6 0.5
1.1 0.6
SO2NH2
COOH
6
OMe
OMe
OMe
OMe
3.57
2.89
2.89
2.42
321.3
320.4
320.4
370.4
34
57
34
59
20.3 13.7
6.7 1.4
5.3 0.9
0.2 0.1
>100
>4.9
0
13a
13b
13c
2.7 0.4
5.4 1.2
0.9 0.14
CONH2
CONH2
0
4.5
NHSO2CH3
CO2CH3
SO2NH2
OMe
OMe
13d
13e
4.05
2.65
335.4
356.4
31
48
6.0 3.0
4.4 8.5
4.5 0.1
2.4 1.1
0
0
13f
2.81
2.65
326.4
356.4
39
38
0.2 0.2
2.6 1.4
0.8 0.4
1.1 0.4
4
0
SO2NH2
SO2NH2
13g
OMe
13h
13i
13j
SO2Me
SO2Me
1.89
1.65
2.05
368.4
404.5
383.4
20
47
42
>100
>100
37.9 17.1
>100
0
0
CONH2
SO2NH2
SO2NH2
NHCOMe
6.6 1.8
8.7 2.2
1.3
NHCOMe
OMe
13k
13l
2.29
3.33
3.37
347.4
317.4
317.4
51
50
37
33.0 11.8
65.1 21.3
13.9 4.1
>100
>3
0
CONH2
H
N
N
7.9 3.7
14.8 2.8
H
N
OMe
13m
1.1
N
S
S
13n
13o
COOH
4.15
3.34
311.4
310.4
62
40
18.2 9.6
30.0 0.1
80.8 1.2
6.2 0.1
4.4
0
Me
CONH2
NO2
Me
Me
S
S
13p
4.13
328.4
55
>100
>100
0
HO
13q
3.34
3.75
310.4
34
-
>100
>100
>100
0
CONH2
Me
Gefitinib
-
-
446.91
21.9 4.8
>4.5

Doxorubicin
0.53
543.5
-
0.4 0.1
1.4 0.1
4.6
-
-
a MWT and clogP was calculated using JChem Version 14.9.100.
resazurin based cytotoxicity assays (Table 2).42 Compounds 5
and 13g, showed good potency at the 3 additional cell lines,
similar in overall profile to Doxorubicin. 13f showed good
activity at 2 of the 3 additional cell lines in line. Compounds 6,
13a, 13b, 13c and 13d were more selective, showing either weak
to negligible inhibition of the additional cell lines.
All compounds lie within druglike property space and it
appears a combination of moderate calculated lipophilicity
(clogP) and size showed the best A549 activity (Figure 3). We
determined the experimental solubility and the partition
coefficient at pH7.4 (logD7.4), using shake flask based
methodologies, to more thoroughly assess their potential as leads
(Table 2). Lead-like physicochemical properties are considered
logD7.4<4, while acceptable solubility is considered >10 µM.43
The compounds showed favorable logD7.4 values in the range of
1.2-3.0, with an average of 2.7. 5 was the most soluble in
phosphate buffer pH7.4 (38.4 µM). Compound 6 had ~3 fold
lower solubility and remainder had solubility <10 µM.
As noted previously, the role of JNK in mediating cell growth
and self renewal in A549 cells has been reported on extensively22-
26
and this coupled with the similarity to known JNK inhibitors
suggests inhibition of protein kinome is a probable contributor to
the mode of action (Fig. 1). However, minimally decorated 4-
aryl-N-phenylpyrimidin-2-amines are active against a number
targets, particularly within the CMGC group.44 To more clearly
illustrate the origin of this SAR finding we undertook
computational docking to JNK (3E7O)45 and GSK3β (1UV5)46
kinases. Docking was performed using GOLD5.147 using the
methodology previously described by us elsewhere.48 Briefly,
solvent and co-factors were also removed from the crystal
structures, and docking was undertaken using default setting with
an additional condition that a H-bond between the hinge H-bond
donor and a substrate acceptor was a requirement for a valid
solution. The top scored poses obtained in both JNK2 and
GSK3β (Fig. 4) were consistent with classic aminopyridine-hinge
interactions found in experimental structures including those in
JNK,49, 50 IKK2,51 JAK2,52 and VEGFR253.
Figure 3. Plot of the molecular weight vs clogP for the compounds
synthesized in this study. Data colored and labelled by A549 cytotoxicity.
Exemplar structures are illustrated. Data visualization performed in
Dotmatics Vortex.
Additional selectivity studies were undertaken using monkey
kidney epithelial cells (Vero, ATCC CCL-81)39, 40 using an MTT
based approach.41. Doxorubicin is found to have approximately 5
fold selectivity over A549 cells, and Gefitinib approximately 4.5
fold. The 4-aryl-N-phenylpyrimidin-2-amines are found to
display low levels of selectivity overall. Compounds 6, 13c, 13f,
13k and 13n display comparable levels of selectivity to
Doxorubicin (~4 fold). Eight of the most potent and/or selective
compounds were further evaluated KB (ATCC CCL-17), MCF7
(ATCC HTB-22) and NCI-H186 (ATCC CRL-5804) using a
Table 2
Activity of compounds selected for kinase selectivity and further cytotoxicity assessment
IC50/µM [%10 µM or 10 µg/ml for standards]
Solubility pH7.4
µg/ml (µM)
/
Expt.
logD7.4
Calc
logD7.4
ID
KB
1.29 ( 0.31) [76.3%]
- [0%]
H186
MCF7
5.95 ( 1.98) [63.3%]
- [0%]
5
6
2.08 ( 1.22) [82.3%]
- [23.5%]
13.7 (38.4)
3.9 (12.1)
1.3 (4.1)
< 0.2 <0.6)
< 0.2 (<0.5)
2.3 (6.9)
1.1 (3.4)
< 0.2 (<0.6)
-
2.7
1.2
3.1
2.8
3.2
2.9
2.9
3.0
-
2.7
0.4
3.0
2.9
2.2
4.1
2.8
2.7
-
13a
- [0%]
- [18.6%]
- [0%]
13b
- [0%]
- [28.1%]
- [1.9%]
13c
- [43.7%]
5.35 ( 0.94) [84.8%]
- [23.8%]
- [41.0%]
13d
- [0%]
- [0%]
13f
2.44 ( 0.61) [93.6%]
3.56 ( 0.88) [94.9%]
1.35 ( 0.34) [99.7%]
1.18 ( 0.11) [99.8%]
1.34 ( 0.28) [96.6%]
0.06 ( 0.01) [96.98%]
- [0%]
13g
1.50 ( 0.36) [63.8%]
8.98 ( 1.98) [93.1%]
Doxorubicin

Ellipticine
Tamoxifen
2.81 ( 1.39) [96.3%]
-
0.95 ( 0.14) [99.24%]
-
-
-
-
-
-
-
-
7.94 ( 1.98) [97.6%]
Figure 4. Compound 13f docked to JNK2 (green) and GSK3β (blue).
Compounds of this series can bind to the ATP binding site of
a number of kinases as confirmed from their known SAR (Table
S1) and the results from the initial docking. The aminopyrimidine
core can make 2 H-bond interactions to Met149. The phenyl and
4-methoxy phenyl substituent of the most potent compounds bind
well within the relatively small hydrophobic back pocket of the
two proteins. These compounds can also form two additional H-
bond interactions, between their 3-sulphonamide substituents and
the carbonyl of Asp150 and the amide side chain of Gln155, or
Pro136 and Arg141 in the case of GSK3β.
The activity and promiscuity profile of the latter compounds are
comparable to the anti-cancer drug Doxorubicin. Compounds
13b and 13d showed the best balance of A549 anti-cancer
activity and selectivity towards other cells tested. Further efforts
are now needed to gain better understanding of the precise kinase
inhibition profile of the compounds and to improve their overall
solubility.
Acknowledgments
The authors are grateful for financial support provided by the
Kasetsart University Research and Development Institute
(KURDI) and the Thailand Research Fund (TRF, DBG5980001).
BT would like to acknowledge support from the National
Research University (NRU) and the Royal Golden Jubilee (RGJ)
Scholarship (PHD57K0003).
The potential targets(s) of the top 8 molecules was probed
further using computational methods. The possible targets were
identified using cheminformatics and bioinformatics similarity
methods whereby the chemical similarity to known compounds
were used followed by sequence similarity of the identified
targets to suggest further homologs, paralogs and orthologs.54, 55
Targets including IKK2, GSK3, P38 JNK and CDK were
identified (Table S1). Compounds were subsequently docked to
these targets and their overall fitness score assessed (Table S2).
IKK2 and CDK2 targets showed the highest overall scores, with
P38 being the lowest. It was observed that the CDK2 fitness
score also showed the highest correlation with the A549 IC50
(r2=0.44). The value was close to zero for targets except for IKK2
(0.13) and P38 (0.27). While not conclusive, the strong
correlation is consistent with the SAR associated with the CDK
pocket (relatively small, non-polar). Additional experimental
kinase profiling is needed at these targets to conclusively
determine the precise targets involved and the level of inhibition.
Supplementary Material
Experimental details and spectra of compounds (NMR, MS,
HRMS, IR, and HPLC-UV) are provided as supplementary data.
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OMe
OMe
O
S
O
H2N
O
9c
9g
N
OH
N
N
NH
N
NH
1
2
A549 EC50= 20.3
M
1
A549 EC50= 1.5 M
SAR
9n
9l
expansion
R1
R1
Cl
N
R1
R2
NH2
(a)
(b)
N
N
N
+
Cl
+
Cl
B(OH)2
R2
9f
N
9a-q
NH
N
3-8

 Nineteen 4-aryl-N-phenylpyrimidin-2-amines were designed, synthesized.
 Cytotoxicities in A549, NCI-H187, MCF7, Vero and KB cell assessed.
 Physicochemical properties (logD7.4 & phosphate buffer solubility pH7.4) of key
compounds were assessed.
 9f and 9c display potent anti-cancer activity in A549 cells compared to Doxorubicin.
 1 displayed the lowest experimental logD7.4 (2.9) and the best solubility (40 M).