Design, synthesis, and in vitro antiproliferative and kinase inhibitory effects of pyrimidinylpyrazole derivatives terminating with arylsulfonamido or cyclic sulfamide substituents

Article abstract of DOI:10.1080/14756366.2016.1190715

A novel series of substituted pyrimidine compounds bearing N-phenylpyrazole and terminating with aryl and cyclic sulfonamido moiety were designed, synthesized, and evaluated in vitro as antiproliferative agents against a panel of 53 cell lines of differen

Full text of DOI:10.1080/14756366.2016.1190715

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis, and in vitro antiproliferative and
kinase inhibitory effects of pyrimidinylpyrazole
derivatives terminating with arylsulfonamido or
cyclic sulfamide substituents
Mahmoud M. Gamal El-Din, Mohammed I. El-Gamal, Mohammed S. Abdel-
Maksoud, Kyung Ho Yoo, Daejin Baek, Jungseung Choi, Huiseong Lee &
Chang-Hyun Oh
To cite this article: Mahmoud M. Gamal El-Din, Mohammed I. El-Gamal, Mohammed S.
Abdel-Maksoud, Kyung Ho Yoo, Daejin Baek, Jungseung Choi, Huiseong Lee & Chang-
Hyun Oh (2016): Design, synthesis, and in vitro antiproliferative and kinase inhibitory
effects of pyrimidinylpyrazole derivatives terminating with arylsulfonamido or cyclic
sulfamide substituents, Journal of Enzyme Inhibition and Medicinal Chemistry, DOI:
10.1080/14756366.2016.1190715
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1190715
Published online: 02 Jun 2016.
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Date: 09 June 2016, At: 02:38

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–12
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1190715
!
RESEARCH ARTICLE
Design, synthesis, and in vitro antiproliferative and kinase inhibitory
effects of pyrimidinylpyrazole derivatives terminating with
arylsulfonamido or cyclic sulfamide substituents
1
,2,3
4,5,6
1,2,3
7
Mahmoud M. Gamal El-Din , Mohammed I. El-Gamal , Mohammed S. Abdel-Maksoud , Kyung Ho Yoo ,
8
Daejin Baek , Jungseung Choi , Huiseong Lee , and Chang-Hyun Oh
8
8
1,2
1
2
Center for Biomaterials, Korea Institute of Science and Technology, Seoul, Republic of Korea, Department of Biomolecular Science, University of
3
Science and Technology, Daejeon, Republic of Korea, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki-Giza,
4
Egypt, Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah, United Arab Emirates, Sharjah Institute for
5
6
Medical Research, University of Sharjah, Sharjah, United Arab Emirates, Department of Medicinal Chemistry, Faculty of Pharmacy, University of
7
Mansoura, Mansoura, Egypt, Chemical Kinomics Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea, and
8
Department of Chemistry, Hanseo University, Seosan, Republic of Korea
Abstract
Keywords
A novel series of substituted pyrimidine compounds bearing N-phenylpyrazole and terminating Antiproliferative activity, arylsulfonamide,
with aryl and cyclic sulfonamido moiety were designed, synthesized, and evaluated in vitro as
antiproliferative agents against a panel of 53 cell lines of different tissues at the NCI. Among
them, compound 1d with p-chlorobenzenesulfonamido terminal moiety, ethylene spacer, and 4-
chloro-3-methoxyphenyl ring at position 3 of the pyrazole nucleus showed the highest mean
percentage inhibition value over the whole cancer cell line panel at 10 mM concentration.
It showed broad-spectrum antiproliferative activity over many cell lines of different cancer types.
For instance, compound 1d inhibited the growth of HL-60 (TB), SR leukemia, and T-47D and MCF-
1
,3,4-triarylpyrazole, RAF kinases
History
Received 29 February 2016
Revised 4 May 2016
Accepted 5 May 2016
Published online 1 June 2016
7
breast cancer cell line by 135.92%, 119.44%, 95.32%, and 82.03% at 10 mM, respectively. And it
inhibited the growth of COLO 205 colon, HT29 colon, BT-549 breast, and ACHN renal cancer cell
lines by more than 80% at the same test concentration. However, testing compound 1d upon
determining its IC50 against the most sensitive cell lines showed to good extent selectivity
against HT29 colon cancer cell line than HL-60 leukemia and MRC-5 lung fibroblasts (normal
cells). Compound 1d was further tested against 12 kinases of different kinase families, and the
highest inhibitory effect was exerted against RAF1, V600E-B-RAF, and V600K-B-RAF kinases.
Introduction
In addition, much attention has been paid to the chemistry and
1
1–15
biological activities of 1,3,4-triarylpyrazole scaffold
. In the
Cancer is considered the second leading cause of death after
1
,2
present study, a new series of 1,3,4-triarylpyrazole derivatives
containing terminal arylsulfonamido or cyclic sulfamide moieties
were designed with similarity to SB203580 and Encorafenib
cardiovascular diseases all over the world . It is a collection of
serious diseases caused by uncontrolled growth of cells invading
surrounding tissues and spread to other parts of the body. In the
statistics according to new report from the World Health
Organization’s International Agency for Cancer Research
(IARC) suggests that the incidence of cancer worldwide will
grow by 75%, nearly doubling in some of the developing
3
countries . Those increases will add much more burden on the
poorly developed health-care systems in such countries. That is
why intensive research should be done in this field for getting
more potent drugs with less toxic side effects.
Many research articles have recently reported the potential
antiproliferative activity of sulfonamide and cyclic sulfonamide
(
LGX818) (Figure 1). SB203580 was reported to bind to P38/
MAP kinase via hydrophobic and hydrogen bonding interactions
of the fluorophenyl ring with a hydrophobic region, hydrogen
bonding of the pyridyl and imidazole rings with the kinase hinge
region, and the methylsulfinylphenyl ring with the phosphate
16,17
binding region
. There is another hydrophobic region below
16
the pyridyl ring which was not occupied by SB203580 . That’s
why the terminal chain on the pyridyl ring was important. Our
target compounds were also designed similar to Encorafenib
through ligand-based design approach. The N-phenyl ring on the
pyrazole ring mimics the N-isopropyl group of Encorafenib and
estimated to undergo similar interaction. The two aryl rings at
positions 3 and 4 of the pyrazole ring mimic the two aryl rings at
the same positions on the Encorafenib structure. And the side
chain on the pyridyl ring mimics that of Encorafenib with
replacement of the terminal methoxycarbonyl moiety of
Encorafenib with arylsulfonyl or cyclic sulfamide moiety.
4
–10
moieties against a variety of cancer cell lines
.
Address for correspondence: Chang-Hyun Oh, Center for Biomaterials,
Korea Institute of Science and Technology, PO Box 131, Cheongryang,
Seoul 130-650, Republic of Korea. Tel: +82 2 958 5160. Fax: +82 2 958
5
308. E-mail: choh@kist.re.kr

2
M. M. Gamal El-Din et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Figure 1. Structures of SB203580,
Encorafenib (LGX818), and rationale design
to the target compounds 1a–l and 2a–l.
O
N
S
N
N
F
NH
N
NH
O
N
S
NH
HN
O
Cl
N
O
O
F
SB203580
Encorafenib (LGX818)
N
N
R1
O
N
NH
N
n
R2
Cl
1
2
R
= H, CH3
R = ArSO2NH, cyclic sulfamide
n = 1, 2
In the present study, we report synthesis of a new series of ACQUITY UPLC BEH C18 (1.6 mm, 2.1 ꢀ 100 mm) (Waters,
ꢁ
1,3,4-triarylpyrazole scaffold having amino pyrimidine moiety Milford, MI) at 25 C using acetonitrile and water (containing
similar to that part present in compound B which have been 0.1% formic acid as mobile phase in gradient elution mode at a
reported as an important fragment presented in many antiproli- flow rate of 0.4 mL/min, and injection volume: 5 mL. The data
1
8–20
ferative agents
instead of pyridine and the aminopyrimidine acquisition was under the control of Mass Hunter workstation
was substituted with ethylene or propylenediamino aryl or cyclic software. Melting points were obtained on a Walden Precision
sulfonamide derivatives to test their anticancer activities as Apparatus Electrothermal 9300 apparatus (Barnstead, UK) and are
illustrated in (Figure 1). Their in vitro antiproliferative activities uncorrected. Solvents and liquid reagents were transferred using
against NCI-60 cancer cell line panel are reported.
hypodermic syringes. All solvents and reagents were commercially
available and used without further purification.
Synthesis of the ethylenediamine and propylenediamine sul-
fonamides side chain compounds 3a–l was carried out through a
Experimental
Chemistry
General
1
pathway as previously reported .
9
Chloro-2-methoxy-4-methylbenzene (5)
The target compounds were purified by column chromatography
using silica gel (0.040–0.063 mm, 230–400 mesh) and technical
To a solution of 2-chloro-5-methylphenol (4, 21.5 g, 150 mmol),
dimethyl sulfate (20.8 g, 165 mmol) in acetone (250 mL),
anhydrous K CO (51.8 g, 375 mmol) was added. The reaction
1
13
grade solvents. H NMR and C NMR spectra were recorded on a
Bruker Avance 400 or 300 spectrometer (Bruker Bioscience,
Billerica, MA) using tetramethylsilane as an internal standard. IR
analysis (KBr discs, New Jersey, NJ) was carried out using a
Bruker FT-IR instrument (Bruker Bioscience, Billerica, MA).
Mass spectra (MS) were determined by LC-MS analysis using the
following system: Waters 2998 photodiode array detector, Waters
2
3
mixture was heated under reflux for 3 h. The mixture was
filtered, and the filtrate was evaporated under reduced pressure.
Water (150 mL) was added to the residue, and the resulting
mixture was carefully extracted with ethyl acetate. The organic
layer was separated and the aqueous layer was extracted with
ethyl acetate (3 ꢀ 50 mL). The combined organic extracts were
washed with brine, dried over anhydrous Na SO , and filtered.
3
Waters 2545 binary gradient module, Waters reagent manager,
100 mass detector, Waters SFO system fluidics organizer,
2
4
The organic solvent was evaporated under reduced pressure, and
the residue was used in the next step without further purifica-
+
tion. MS m/z: 157.5 [M + H].
TM
Waters 2767 sample manager, Sunfire
C18 column
(
4.6 ꢀ 50 mm, 5 mm particle size) (Waters, UK); solvent gradi-
ent ¼ 95% A at 0 min, 1% A at 5 min; solvent A: 0.035%
trifluoroacetic acid (TFA) in water; solvent B: 0.035% TFA in
CH OH; flow rate ¼ 3.0 mL/min. Solvents and liquid reagents
Chloro-3-methoxybenzoic acid (6)
3
were transferred using hypodermic syringes. Purity percentage of A stirred mixture of compound 5 (11.17 g, 71 mmol), potassium
all the target compounds were determined by LC-MS and found to permanganate (35.0 g, 221 mmol), pyridine (36 mL), and water
ꢁ
be 495%. The high-resolution mass spectrometry was done on (107 mL) was heated at 50 C for 24 h. The mixture was then
positive mode with capillary: 3.1 KV, desolvation gas: 800 L/h, stirred at room temperature for 13 h. The mixture was filtered and
and extraction cone 4.0. Chromatography was performed on MnO was suspended in hot water and again filtered off. The
2

DOI: 10.1080/14756366.2016.1190715
Antiproliferative and kinase inhibitory effects of pyrimidinylpyrazole derivatives
3
combined aqueous filtrates were washed with ethyl acetate was added. The mixture was stirred at room temperature for 16 h.
(
3 ꢀ 75 mL), and then acidified with 2 N H SO . The precipitate The organic solvent was evaporated under reduced pressure and
2
4
was filtered off, washed with water, and dried to give the title the remaining aqueous solution was extracted with CH Cl2
2
compound (11.98 g, 90%). mp: 215–216 C (lit. mp: 217– (50 mL) and the organic layer was separated. The aqueous layer
2
ꢁ
ꢁ
21
19 C ).
was extracted with CH Cl (3 ꢀ 25 mL) and the combined organic
2
2
layer extracts were washed with brine, dried over anhydrous
MgSO , and filtered. The organic solvent was evaporated under
Methyl 4-chloro-3-methoxybenzoate (7)
4
reduced pressure and the residue was purified by flash column
chromatography. 2.5 g of the title compound was obtained, yield
To a solution of the acid 6 (1.0 g, 5.4 mmol) in MeOH (40 mL) at
ꢁ
0
C, acetyl chloride (1.9 mL, 28.1 mmol) was added dropwise at
1
8%. H NMR (400 MHz, CDCl ) ꢀ 8.49 (s, 1H), 8.39 (d, 1H,
9
3
this temperature and the reaction mixture was then stirred at room
temperature for 15 h. After evaporation of the organic solvent, the
residue was purified by flash column chromatography (silica gel,
hexane–ethyl acetate 5:1 v/v) to give 4 (0.91 g, 85%) as a
J ¼ 5.2 Hz), 7.46–7.29 (m, 5H), 6.92–6.85 (m, 2H), 6.79 (d, 1H,
1
3
J ¼ 6.0 Hz), 3.71 (s, 3H), 2.89 (s, 3H); C NMR (100 MHz,
CDCl ) ꢀ 173.5, 159.1, 157.1, 156.1, 141.4, 140.8, 139.1, 130.7,
3
ꢁ
1
129.6, 128.9, 128.1, 125.3, 123.7, 123.3, 120.0, 114.1, 112.5,
5
crystalline solid. mp: 49–50 C; H NMR (400 MHz, DMSO-d ) d
6
6.5, 43.1.
7
.62–7.55 (m, 3H), 3.94 (s, 3H), 3.88 (s, 3H).
1-(4-Chloro-3-methoxyphenyl)-2-(2-(methylthio)pyrimidin-4-
yl)ethan-1-one (8)
General procedure for preparation of the target
compounds 1a–l
To a solution of compound 7 (1.0 g, 5.0 mmol) and, 4-methyl-2-
(methylthio)pyrimidine (0.78 g, 5.6 mmol) in THF (5 mL) in a
A mixture of mesyl compound 10 (0.2 g, 0.5 mmol), amino side
chain compound 3a–l (1.2 mmol), and diisopropylethylamine
ꢁ
cooled bath at ꢂ78 C, LHMDS (3.7 mL, 1.0 M solution in
ꢁ
(0.29 mL, 1.7 mmol) in DMSO (5 mL) was stirred at 80 C for
THF,19.9 mmol) was slowly added to maintain the temperature at
ꢂ78 C. The resulting mixture was stirred overnight at room
1
6 h. The mixture was cooled to room temperature, quenched
with water (20 mL), then extracted with ethyl acetate
3 ꢀ 20 mL). The combined organic layer extracts were
washed with brine, dried over anhydrous Na SO , filtered,
ꢁ
temperature. After disappearance of the starting material, the
mixture was quenched with saturated aqueous NH Cl. Ethyl
(
4
2
4
acetate (20 mL) was added and the organic layer was separated.
The aqueous layer was extracted with ethyl acetate (3 ꢀ 10 mL).
The combined organic layer extracts were washed with brine
and dried over anhydrous Na SO . The organic solvent was
and concentrated under reduced pressure. The residue was
purified by flash column chromatography (silica gel, appropri-
ate ratio of hexane–ethyl acetate) to obtain the pure target
compounds.
2
4
evaporated under reduced pressure and the residue was purified
by flash column chromatography (silica gel, hexane ethyl acetate
1
:1 v/v then switching to hexane–ethyl acetate 1:5 v/v) to yield N-(2-((4-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
ꢁ
1
compound 6 (0.58 g, 45%). mp: 85–88 C; H NMR (400 MHz, yl)pyrimidin-2-yl)amino)-ethyl)benzenesulfonamide (1a)
CDCl ) ꢀ 8.24–8.23 (m, 1H), 7.65–7.60 (m, 1H), 7.36–7.21
3
1
H NMR (400 MHz, CDCl ) ꢀ 8.21 (s, 1H), 8.02 (d, 1H,
J ¼ 5.2 Hz), 7.76 (d, 2H, J ¼ 8.0 Hz), 7.51–7.21 (m, 9H), 6.81 (d,
H, J ¼ 8.0 Hz), 6.76 (s, 1H), 6.35 (brs, 1H), 5.85 (t, 1H,
3
(
(
m, 4H), 6.95–6.93 (m, 1H), 6.84–6.82 (m, 1H), 3.95 (s, 5H), 2.65
1
s, 3H); C NMR (100 MHz, CDCl ) ꢀ 194.4, 157.2, 156.4,
3
3
1
1
4
55.4, 155.1, 135.8, 134.9, 130.3, 118.8, 112.8, 109.5, 56.3,
7.9, 14.1.
1
3
J ¼ 6.4 Hz), 3.66 (s, 3H), 3.32 (brs, 2H), 3.05 (brs, 2H); C NMR
(100 MHz, CDCl ) ꢀ 162.3, 159.7, 157.4, 155.0, 141.0, 140.5,
3
1
1
40.0, 139.2, 132.4, 129.0, 128.9, 128.0, 126.9, 125.3, 120.5,
14.2, 107.7, 56.3, 43.6, 40.9.
4
(
-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-2-
methylthio)-pyrimidine (9)
Compound 8 (1.1 g, 3.8 mmol) was added to DMF-DMA
ꢁ
(
3
5.14 mL, 38.2 mmol) and the mixture was stirred at 80 C for N-(2-((4-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
h. The resulting solution was concentrated to dryness to furnish yl)pyrimidin-2-yl)amino)ethyl)-4-methylbenzenesulfonamide (1b)
oil which was used in the next step without purification. To a
portion of the oil from the previous step (0.137 g, 0.457 mmol) in
EtOH (3 mL) was added phenylhydrazine (98 mg, 0.9 mmol). The
reaction mixture was stirred overnight at room temperature. Water
1
H NMR (400 MHz, CDCl ) ꢀ 8.21 (s, 1H), 8.03 (d, 1H,
3
J ¼5.2 Hz), 7.65 (d, 2H, J ¼ 8.0 Hz), 7.33–7.20 (m, 8H), 6.81 (dd,
1
H, J ¼ 8.0, 1.2 Hz) 6.76 (d, 1H, J ¼ 1.2 Hz), 6.35 (brs, 1H), 5.77
13
(
t, 1H, J ¼ 6.4 Hz), 3.67 (s, 3H), 3.31 (brs, 2H), 3.05 (brs, 2H),
(5 mL) was added to the reaction mixture and the organics were
2
1
1
2
.37 (s, 3H); C NMR (100 MHz, CDCl ) ꢀ 162.3, 159.7, 157.8,
55.0, 143.2, 141.0, 140.5, 139.2, 130.5, 129.8, 129.6, 128.9,
28.0, 126.9, 125.3, 123.4, 120.5, 114.2, 107.7, 56.2, 43.6, 40.8,
3
extracted with ethyl acetate (3 ꢀ 5 mL). The combined organic
layer extracts were washed with brine and dried over anhydrous
Na SO . After evaporation of the organic solvent, the residue was
+
1.5; LC-MS (m/z): 574.8 (M ).
2
4
purified by column chromatography (silica gel, hexane–ethyl
acetate 1:1 v/v then switching to hexane–ethyl acetate 1:5 v/v) to
1
yield compound 9 (1.18 g, 81%). H NMR (400 MHz, CDCl ) ꢀ
N-(2-((4-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)ethyl)-4-methoxybenzenesulfonamide
3
8
.30 (s, 1H), 8.26 (d, 1H, J ¼ 5.2 Hz), 7.36–7.21 (m, 5H), 6.82–
.80 (m, 2H), 6.71 (d, 1H, J ¼ 6.4 Hz), 3.68 (s, 3H), 2.34 (s, 3H);
(1c)
6
1
3
1
C NMR (100 MHz, CDCl ) ꢀ 172.5, 158.9, 156.9, 155.1, 141.2,
3
H NMR (400 MHz, CDCl ) ꢀ 8.21 (s, 1H), 8.03 (d, 1H,
3
1
1
40.6, 139.1, 130.6, 129.4, 128.9, 128.0, 125.3, 123.7, 123.3,
20.0, 114.1, 112.5, 56.2, 13.7.
J ¼ 5.2 Hz), 7.70 (dd, 2H, J ¼ 9.2, 2.0 Hz), 7.34–7.21 (m, 6H),
6
5
3
1
.90–6.87 (m, 2H), 6.82 (dd, 1H, J ¼ 8.4, 2.0 Hz), 6.35 (brs, 1H),
.77 (t, 1H, J ¼ 6.4 Hz), 3.81 (s, 3H), 3.67 (s, 3H), 3.33 (brs, 2H),
4-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-2-
methylsulfonyl)pyrimidine (10)
13
.05 (brs, 2H); C NMR (100 MHz, CDCl ) ꢀ 162.7, 162.3,
59.7, 157.8, 155.0, 141.0, 140.5, 139.3, 130.5, 129.8, 129.1,
3
(
To a solution of compound 9 (2.4 g, 6 mmol) in methanol 128.9, 128.0, 125.3, 123.4, 120.5, 114.2, 114.1, 107.7, 56.2, 55.6,
250 mL), a solution of oxone (12.3 g, 18 mmol) in water (50 mL) 43.6, 40.8.
(

4
M. M. Gamal El-Din et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
4
pyrazol-4-yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (1d) 1H), 5.76 (s, 1H), 3.66 (s, 3H), 2.88 (brs, 2H), 2.72 (brs, 2H), 1.45
-Chloro-N-(2-((4-(3-(4-chloro-3-methoxyphenyl)-1-phenyl-1H-
7.41–7.28 (m, 6H), 6.82 (s, 1H), 6.92–6.86 (m, 2H), 6.55 (brs,
1
(brs, 2H); C NMR (100 MHz, CDCl
157.8, 154.7, 141.3, 141.1, 140.5, 139.8, 139.6, 137.7, 130.4,
3
ꢂ1
) ꢀ 162.6, 161.1, 159.7,
3
IR (KBr disc, cm ): 3378, 3264, 3088, 2931, 2855, 1577, 1499;
1
H NMR (400 MHz, CDCl ) ꢀ 8.36 (brs, 1H), 8.09 (d, 1H,
3
1
5
29.7, 129.4, 128.9, 128.5, 126.1, 124.0, 120.8, 115.5, 107.9,
6.7, 55.2, 42.0, 29.5.
J ¼ 5.2 Hz), 7.76 (d, 3H, J ¼ 8.0 Hz), 7.62 (d, 2H, J ¼ 8.4 Hz),
7
6
2
1
1
4
.41–7.28 (m, 6H), 6.82 (d, 1H, J ¼ 1.2 Hz), 6.92–6.86 (m, 2H),
.47 (brs, 1H), 5.76 (s, 1H), 3.67 (s, 3H), 3.07 (brs, 2H), 2.70 (brs,
1
H); C NMR (100 MHz, CDCl ) ꢀ 162.3, 161.1, 159.7, 157.8,
3
N-(3-((4-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)propyl)-3-fluorobenzenesulfonamide (1j)
3
54.7, 141.3, 141.1, 140.5, 139.7, 139.6, 137.7, 130.4, 129.7,
29.4, 128.9, 128.5, 126.1, 124.0, 120.8, 115.5, 107.9, 56.7, 55.4,
+
2.2; LC-MS (m/z): 594.8 (M ).
1
H NMR (400 MHz, CDCl ) ꢀ 8.21 (s, 1H), 8.08 (d, 1H,
3
J ¼ 5.2 Hz), 7.45–7.16 (m, 9H), 6.82–6.80 (m, 2H), 6.40 (brs, 1H),
5
2H), 1.59 (brs, 2H); C NMR (100 MHz, CDCl ) ꢀ 163.8, 162.4,
.47 (t, 1H, J ¼ 6.4 Hz), 3.68 (s, 3H), 3.20 (brs, 2H), 2.89 (brs,
1
3
N-(2-((4-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)ethyl)-3-fluorobenzenesulfonamide (1e)
3
161.0, 159.8, 157.4, 155.0, 142.1, 141.1, 140.5, 139.2, 130.8,
130.4, 129.7, 128.9, 128.0, 126.9, 125.3, 123.4, 122.0, 120.3,
1
H NMR (400 MHz, CDCl ) ꢀ 8.22 (s, 1H), 8.02 (d, 1H,
3
1
19.7, 119.5, 114.3, 114.1, 107.8, 56.2, 39.8, 37.4, 30.3.
J ¼ 5.2 Hz), 7.54 (d, 2H, J ¼ 8.0 Hz), 7.43–7.18 (m, 9H), 6.82 (d,
1
H, J ¼ 8.0 Hz), 6.77 (s, 1H), 6.38 (brs, 1H), 5.87 (t, 1H,
1
3
2-(2-((4-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)ethyl)-5-ethyl-1,2,5-thiadiazolidine 1,1-
dioxide (1k)
J ¼ 6.4 Hz), 3.67 (s, 3H), 3.31 (brs, 2H), 3.05 (brs, 2H); C NMR
(
100 MHz, CDCl ) ꢀ 163.6, 162.3, 161.1, 159.7, 157.8, 155.0,
3
1
1
1
42.1, 141.1, 140.5, 139.2, 130.8, 130.4, 129.7, 128.9, 128.0,
26.9, 125.3, 123.4, 122.0, 120.3, 119.7, 119.5, 114.3, 114.1,
07.8, 56.2, 43.8, 40.8.
1
H NMR (400 MHz, CDCl ) ꢀ 8.18 (s, 1H), 8.06 (brs, 1H), 7.28–
3
7.14 (m, 6H), 6.76 (dd, 1H, J ¼ 8.0, 2.0 Hz), 6.68 (d, 1H,
J ¼ 2.0 Hz), 6.38 (brs, 1H), 5.62 (t, 1H, J ¼ 6.0 Hz), 3.61 (s, 3H),
3.46 (brs, 2H), 3.38–3.21 (m, 4H), 3.12 (brs, 2H), 3.00 (m, 2H),
N-(3-((4-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)propyl)benzenesulfonamide (1f)
1
3
1.18 (t, 3H, J ¼ 7.2 Hz); C NMR (100 MHz, CDCl ) ꢀ 161.3,
3
1
159.9, 157.4, 155.0, 141.0, 140.6, 130.5, 129.8, 128.9, 128.0,
H NMR (400 MHz, CDCl ) ꢀ 8.19 (s, 1H), 8.06 (d, 1H,
3
1
3
25.3, 123.5, 120.7, 114.1, 107.7, 56.2, 47.1, 46.1, 45.1, 42.6,
9.3, 13.0.
J ¼ 5.2 Hz), 7.68 (brs, 2H), 7.51–7.24 (m, 9H), 6.85–6.80 (m, 2H),
6
2
ꢀ
.40 (brs, 1H), 5.85 (t, 1H, J ¼ 6.4 Hz), 3.67 (s, 3H), 3.20 (brs,
1
3
H), 2.89 (brs, 2H), 1.58 (brs, 2H); C NMR (100 MHz, CDCl )
3
2-(3-((4-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)propyl)-5-ethyl-1,2,5-thiadiazolidine
162.5, 159.8, 157.9, 154.9, 141.3, 140.3, 140.2, 139.3, 132.3,
30.3, 129.6, 128.9, 128.1, 126.8, 125.6, 123.3, 120.5, 114.6,
07.6, 56.3, 39.9, 37.3, 30.1.
1
1
1
,1-dioxide (1l)
1
H NMR (400 MHz, CDCl ) ꢀ 8.24 (s, 1H), 8.09 (d, 1H,
3
N-(3-((4-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)propyl)-4-methylbenzenesulfonamide
J ¼5.2 Hz), 7.36–7.21 (m, 6H), 6.81(dd, 1H, J ¼ 8.4, 2.0 Hz), 6.76
(d, 1H, J ¼ 2.0 Hz), 6.33 (brs, 1H), 5.31 (t, 1H, J ¼ 6.0 Hz), 3.68
(s, 3H), 3.40 (brs, 2H), 3.30 (brs, 4H), 3.14–3.08 (m, 4H), 1.85
(1g)
1
3
1
(brs, 2H), 1.26 (t, 3H, J ¼ 7.6 Hz); C NMR (100 MHz, CDCl
3
) ꢀ
62.4, 159.6, 157.9, 154.9, 140.8, 140.6, 139.4, 130.4, 129.9,
H NMR (400 MHz, CDCl ) ꢀ 8.19 (s, 1H), 8.06 (d, 1H,
3
1
1
4
J ¼ 5.2 Hz), 7.71 (d, 2H, J ¼ 8.0 Hz), 7.37–7.25 (m, 8H), 6.85 (dd,
28.9, 128.0, 127.9, 125.3, 123.5, 120.9, 114.2, 107.5, 56.2, 45.5,
5.0, 42.5, 38.7, 27.5, 12.9.
1
H, J ¼ 8.0, 1.2 Hz), 6.72 (d, 1H, J ¼ 1.2 Hz), 6.40 (brs, 1H), 5.67
(
t, 1H, J ¼ 6.4 Hz), 3.66 (s, 3H), 3.08 (brs, 2H), 2.89 (brs, 2H),
1
.34 (s, 3H), 1.58 (brs, 2H); C NMR (100 MHz, CDCl ) ꢀ 162.2,
3
2
1
1
5
3
59.8, 157.9, 154.9, 143.1, 141.2, 140.4, 139.1, 130.4, 129.8, General procedure for preparation of the target
29.6, 128.9, 128.0, 126.9, 125.3, 123.4, 120.5, 114.2, 107.7, compounds 2a–l
6.2, 43.6, 40.8, 30.3, 21.5.
A solution of the appropriate methoxy compound 1a–l (0.2 mmol)
ꢁ
in anhydrous dichloromethane (3 mL) was cooled to ꢂ78 C, and
N-(3-((4-(3-(4-Chloro-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)propyl)-4-methoxybenzenesulfonamide
BBr (1M in DCM solution, 3 mL, 2.1 mmol) was added dropwise
3
thereto under nitrogen over a period of 10 min. The reaction
mixture was stirred at the same temperature for 1 h, then the
IR (KBr disc, cm ): 3380, 3275, 3091, 2928, 1580, 1500; H temperature was allowed to reach room temperature and kept
(1h)
ꢂ1
1
NMR (400 MHz, CDCl ) ꢀ 8.20 (s, 1H), 8.08 (d, 1H, J ¼ 5.2 Hz), overnight at same temperature. 10% Na CO aqueous solution
3
2
3
7
3
3
.72 (dd, 2H, J ¼ 9.2, 2.0 Hz), 7.33–7.19 (m, 6H), 6.92–6.82 (m, (15 mL) and ethyl acetate (15 mL) were added, and the alkaline
H), 6.41 (brs, 1H), 5.67 (t, 1H, J ¼ 6.4 Hz), 3.80 (s, 3H), 3.65 (s, aqueous layer was extracted with ethyl acetate (3 ꢀ 20 mL). The
1
H), 3.08 (brs, 2H), 2.88 (brs, 2H), 1.58 (brs, 2H); C NMR combined organic layer extracts were washed with brine,
3
(100 MHz, CDCl ) ꢀ 162.5, 162.1, 159.8, 157.7, 155.0, 141.1, separated, and dried over anhydrous Na SO . The organic solvent
3 2 4
1
1
40.4, 139.2, 130.5, 129.8, 129.1, 128.9, 128.0, 125.3, 123.4, was evaporated under reduced pressure, and the residue was
20.5, 114.2, 114.1, 107.7, 56.3, 55.7, 39.6, 37.3, 29.2; LC-MS purified by column chromatography to obtain the purified title
+
(m/z): 604.9 (M ).
compounds.
4
-Chloro-N-(3-((4-(3-(4-chloro-3-methoxyphenyl)-1-phenyl-1H-
N-(2-((4-(3-(4-Chloro-3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (2a)
pyrazol-4-yl)pyrimidin-2-yl)amino)propyl)benzenesulfonamide
1i)
(
1
H NMR (400 MHz, CDCl ) ꢀ 8.15 (s, 1H), 7.85 (d, 1H,
3
1
H NMR (400 MHz, CDCl ) ꢀ 8.34 (brs, 1H), 8.11 (d, 1H, J ¼ 5.2 Hz), 7.75 (d, 2H, J ¼ 7.2 Hz), 7.47–7.15 (m, 9H), 6.85 (d,
3
J ¼ 4.8 Hz), 7.76 (d, 3H, J ¼ 8.4 Hz), 7.66 (d, 1H, J ¼ 8.4 Hz), 1H, J ¼ 1.6 Hz), 6.59 (dd, 1H, J ¼ 8.4, 5.0 Hz), 6.35 (brs, 1H),

DOI: 10.1080/14756366.2016.1190715
Antiproliferative and kinase inhibitory effects of pyrimidinylpyrazole derivatives
5
1
3
5
(
.74 (t, 1H, J ¼ 6 Hz), 3.26 (brs, 2H), 2.99 (brs, 2H); C NMR (s, 1H), 6.73 (d, 2H, J ¼ 8.0 Hz), 6.38 (brs, 1H), 3.28 (brs, 2H),
1
100 MHz, CDCl ) ꢀ 161.8, 159.9, 157.2, 152.4, 141.1, 140.4, 2.92 (brs, 2H), 2.38 (s, 3H), 1.62 (brs, 2H); C NMR (100 MHz,
3
3
1
1
39.8, 139.0, 132.5, 130.0, 129.8, 128.9, 128.0, 126.9, 125.2, CDCl ) ꢀ 162.6, 159.7, 158.9, 153.4, 142.9, 141.2, 140.5, 139.5,
3
22.8, 121.7, 120.3, 118.8, 107.7, 43.2, 40.9.
138.1, 130.6, 129.4, 128.4, 128.0, 125.7, 122.5, 120.9, 118.8,
06.5, 43.3, 41.0, 29.5, 21.4.
1
N-(2-((4-(3-(4-Chloro-3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)ethyl)-4-methylbenzenesulfonamide (2b)
N-(3-((4-(3-(4-Chloro-3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)propyl)-4-hydroxybenzenesulfonamide
1
H NMR (400 MHz, CDCl ) ꢀ 8.20 (s, 1H), 8.04 (d, 1H,
3
J ¼ 5.2 Hz), 7.71 (d, 2H, J ¼ 8.0 Hz), 7.29–7.19 (m, 8H), 7.08 (s, (2h)
1
3
1
1
2
H), 6.56 (dd, 2H, J ¼ 8.0, 2.0 Hz), 6.26 (brs, 2H), 3.34 (brs, 2H),
1
H NMR (400 MHz, CDCl ) ꢀ 8.22 (s, 1H), 8.11 (d, 1H,
3
1
.01 (brs, 2H), 2.32 (s, 3H); C NMR (100 MHz, CDCl ) ꢀ 162.6,
3
3
J ¼ 5.2 Hz), 7.82 (dd, 2H, J ¼ 9.2, 2.0 Hz), 7.36–7.21 (m, 6H),
.96–6.88 (m, 3H), 6.41 (brs, 1H), 5.67 (t, 1H, J ¼ 6.4 Hz), 3.12
59.7, 158.9, 152.2, 143.3, 140.9, 138.8, 136.7, 129.9, 129.7,
28.3, 127.1, 125.3, 122.7, 121.9, 119.6, 118.9, 107.5, 41.1, 30.9,
1.5.
6
13
brs, 2H), 2.92 (brs, 2H), 1.68 (brs, 2H); C NMR (100 MHz,
(
CDCl ) ꢀ 162.5, 162.1, 159.8, 157.7, 155.0, 141.1, 140.4, 139.2,
3
1
1
30.5, 129.8, 129.1, 128.9, 128.0, 125.3, 123.4, 120.5, 114.2,
14.1, 107.7, 37.4, 35.3, 29.2.
N-(2-((4-(3-(4-Chloro-3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)ethyl)-4-hydroxybenzenesulfonamide
(
2c)
4
-Chloro-N-(3-((4-(3-(4-chloro-3-hydroxyphenyl)-1-phenyl-1H-
1
H NMR (400 MHz, CDCl ) ꢀ 8.21 (s, 1H), 8.03 (d, 1H,
3
pyrazol-4-yl)pyrimidin-2-yl)amino)propyl)benzenesulfonamide
2i)
J ¼ 5.2 Hz), 7.70 (dd, 2H, J ¼ 9.2, 2.0 Hz), 7.34–7.21 (m, 6H),
(
6
5
(
1
1
.90–6.87 (m, 2H), 6.82 (dd, 1H, J ¼ 8.4, 2.0 Hz), 6.35 (brs, 1H),
1
1
3
H NMR (400 MHz, DMSO-d ) ꢀ 8.36 (brs, 1H), 8.14 (d, 1H,
6
.77 (t, 1H, J ¼ 6.4 Hz), 3.35 (brs, 2H), 3.09 (brs, 2H); C NMR
J ¼ 5.2 Hz), 7.79 (d, 3H, J ¼ 8.0 Hz), 7.70 (d, 1H, J ¼ 8.4 Hz),
100 MHz, CDCl ) ꢀ 162.9, 162.4, 159.8, 157.9, 154.9, 141.4,
3
7
1
.47–7.32 (m, 6H), 6.89 (s, 1H), 6.98–6.89 (m, 2H), 6.65 (brs,
H), 5.86 (s, 1H), 2.95 (brs, 2H), 2.80 (brs, 2H), 1.61 (brs, 2H);
40.7, 139.8, 130.5, 129.8, 129.4, 128.9, 128.0, 125.3, 123.4,
20.5, 114.2, 114.2, 107.7, 41.6, 30.9.
1
3
C NMR (100 MHz, DMSO-d ) ꢀ 162.6, 161.1, 159.9, 157.1,
6
1
1
2
53.4, 141.2, 140.6, 139.9, 139.6, 137.6, 129.8, 129.4, 128.9,
28.4, 128.1, 125.7, 122.5, 121.8, 120.9, 118.8, 107.6, 41.1, 31.2,
9.5.
4-Chloro-N-(2-((4-(3-(4-chloro-3-hydroxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (2d)
1
H NMR (400 MHz, CDCl ) ꢀ 8.15 (brs, 1H), 7.85 (d, 1H,
3
J ¼ 5.2 Hz), 7.65 (d, 2H, J ¼ 8.8 Hz), 7.32–7.15 (m, 8H), 6.85 (d,
N-(3-((4-(3-(4-Chloro-3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)propyl)-3-fluorobenzenesulfonamide (2j)
1
H, J ¼ 1.6 Hz), 6.60 (d, 1H, J ¼ 8.0 Hz), 6.36 (brs, 1H), 5.79 (t,
1
3
1
H, J ¼ 6.0 Hz), 3.23 (brs, 2H), 2.97 (brs, 2H); C NMR
1
(100 MHz, CDCl ) ꢀ 161.1, 159.9, 157.1, 152.5, 141.2, 140.4,
3
H NMR (400 MHz, DMSO-d ) ꢀ 8.22 (s, 1H), 8.13 (d, 1H,
6
1
1
38.9, 138.8, 138.3, 130.0, 129.8, 129.3, 128.9, 128.4, 125.5, J ¼ 5.2 Hz), 7.75–7.29 (m, 11H), 7.00–6.72 (m, 3H), 6.51 (brs,
1
3
22.8, 121.8, 120.2, 118.8, 107.6, 43.1, 40.8.
1H), 3.21 (brs, 2H), 2.81 (brs, 2H), 1.55 (brs, 2H); C NMR
100 MHz, DMSO-d ) ꢀ 163.5, 162.6, 161.0, 159.2, 158.5, 153.4,
(
6
N-(2-((4-(3-(4-Chloro-3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)ethyl)-3-fluorobenzenesulfonamide (2e)
143.1, 141.2, 140.6, 139.6, 132.1, 130.5, 129.4, 128.4, 125.7,
123.2, 122.5, 120.9, 120.0, 119.8, 118.8, 114.1, 113.8, 106.6,
41.1, 38.3, 29.6.
1
H NMR (400 MHz, CDCl ) ꢀ 8.25 (s, 1H), 8.09 (d, 1H,
3
J ¼ 5.2 Hz), 7.58 (d, 2H, J ¼ 8.0 Hz), 7.73–7.28 (m, 9H), 6.92 (d,
1
H, J ¼ 8.0 Hz), 6.83 (s, 1H), 6.57 (brs, 1H), 5.96 (t, 1H, 2-(2-((4-(3-(4-Chloro-3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-
1
3
J ¼ 6.4 Hz), 3.35 (brs, 2H), 3.09 (brs, 2H); C NMR (100 MHz, yl)pyrimidin-2-yl)amino)ethyl)-5-ethyl-1,2,5-thiadiazolidine 1,1-
CDCl ) ꢀ 163.9, 162.8, 161.5, 159.9, 157.7, 155.6, 142.4, 141.3, dioxide (2k)
3
1
1
40.7, 139.7, 131.3, 130.0, 129.8, 128.8, 128.3, 126.9, 125.3,
23.4, 122.0, 120.3, 119.7, 119.5, 114.3, 114.2, 107.5, 41.2, 30.8.
1
H NMR (400 MHz, CDCl ) ꢀ 8.21 (s, 1H), 8.06 (d, 1H,
3
J ¼ 5.2 Hz), 7.32–7.18 (m, 6H), 6.77 (dd, 1H, J ¼ 8.0, 2.0 Hz),
6
3
1
.72 (d, 1H, J ¼ 2.0 Hz), 6.38 (brs, 1H), 5.35 (t, 1H, J ¼ 6.0 Hz),
13
N-(3-((4-(3-(4-Chloro-3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)propyl)benzenesulfonamide (2f)
.48 (brs, 2H), 3.34 (brs, 2H), 3.13–3.05 (m, 4H), 1.87 (brs, 2H),
.31 (t, 3H, J ¼ 7.2 Hz); C NMR (100 MHz, CDCl ) ꢀ 162.5,
3
ꢂ1
IR (KBr disc, cm ): 3366, 3269, 3071, 2955, 2853, 1577, 1499; 159.8, 157.7, 155.1, 141.1, 140.5, 139.5, 130.5, 129.8, 128.9,
1
H NMR (400 MHz, CDCl ) ꢀ 8.17 (s, 1H), 7.89 (d, 1H, 128.0, 127.9, 125.3, 123.6, 120.9, 114.2, 107.7, 42.5, 41.9, 41.3,
3
J ¼ 5.2 Hz), 7.81 (d, 2H, J ¼ 7.2 Hz), 7.51–7.18 (m, 9H), 6.87 (d, 36.5, 12.4.
1
5
2
1
1
H, J ¼ 1.6 Hz), 6.47 (dd, 1H, J ¼ 8.4, 5.0 Hz), 6.35 (brs, 1H),
.74 (t, 1H, J ¼ 6.0 Hz), 3.26 (brs, 2H), 2.99 (brs, 2H), 1.84 (brs, 2-(3-((4-(3-(4-Chloro-3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-
1
3
H); C NMR (100 MHz, CDCl ) ꢀ 161.9, 159.8, 157.4, 152.6, yl)pyrimidin-2-yl)amino)propyl)-5-ethyl-1,2,5-thiadiazolidine
3
41.4, 140.5, 139.7, 138.8, 132.6, 130.0, 129.8, 128.9, 128.0, 1,1-dioxide (2l)
26.9, 125.2, 122.8, 121.7, 120.3, 118.8, 107.7, 43.2, 40.9, 29.5;
1
H NMR (400 MHz, CDCl ) ꢀ 8.25 (s, 1H), 8.11 (d, 1H,
3
+
LC-MS (m/z): 559.9 (M ).
J ¼ 5.2 Hz), 7.46–7.23 (m, 6H), 6.84 (dd, 1H, J ¼ 8.0, 2.0 Hz),
6
3
1
1
.86 (d, 1H, J ¼ 2.0 Hz), 6.36 (brs, 1H), 5.47 (t, 1H, J ¼ 6.0 Hz),
13
N-(3-((4-(3-(4-Chloro-3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)pyrimidin-2-yl)amino)propyl)-4-methylbenzenesulfonamide
.43 (brs, 2H), 3.35 (brs, 4H), 3.18–3.12 (m, 4H), 1.89 (brs, 2H),
.37 (t, 3H, J ¼ 7.6 Hz); C NMR (100 MHz, CDCl ) ꢀ 162.6,
3
(2g)
59.8, 157.7, 155.0, 141.2, 140.2, 139.5, 130.5, 129.8, 128.9,
1
H NMR (400 MHz, CDCl ) ꢀ 8.17 (s, 1H), 8.06 (d, 1H, 128.0, 127.9, 125.3, 123.7, 120.9, 114.2, 107.5, 42.5, 42.0, 41.5,
3
J ¼ 5.2 Hz), 7.61 (d, 2H, J ¼ 7.2 Hz), 7.31–7.21 (m, 10H), 6.97 36.6, 25.4, 12.5.

6
M. M. Gamal El-Din et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Õ
Cancer cell line screening at the NCI
CellTiter 96 Non-Radioactive Cell Proliferation Assay (MTT)
reagent was purchased from Promega (Madison, WI). HL-60,
HT29 and MRC-5 cell lines were purchased from American Type
Culture Collection (Manassas, VA). HL-60 cells were grown in
IMDM medium, HT-29 in McCoy’s 5a medium, and MRC-5 cells
in EMEM medium. 100 mg/mL penicillin, 100 mg/mL strepto-
ꢂ5 mycin, and 10% fetal bovine serum were added to all of the
Screening against the cancer cell lines was carried out at the
National Cancer Institute (NCI), Bethesda, MD, applying the
standard protocol of the NCI (DTP Human Tumor Cell Line
Screen
Process:
http://www.dtp.nci.nih.gov/branches/btb/
ivclsp.html). Single-dose protocol: compounds submitted to the
NCI 60 Cell screen are tested initially at a single high dose (10
M) in the full panel of 53 cancer cell line. The single-dose data
will be reported as a mean graph of the percentage growth of
treated cells and will be similar in appearance to mean graphs
from the 5-dose assay. The number reported for the single-dose
assay is growth relative to the no-drug control and to the time zero
number of cells. This allows detection of both growth inhibition
ꢁ
culture media. Cultures were maintained at 37 C in a humidified
atmosphere of 5% CO2 and 95% air. Compound 1d and the
reference compound staurosporine were diluted with DMSO in
1
0-dose and 3-fold serial dilutions. 225 nL of 1d or 25 nL of
staurosporine was delivered from the source plate to the wells of a
84-well cell culture plate by Echo 550 Liquid Handler. 25 mL of
3
culture medium containing 2000 of cells was added to the wells of
ꢁ
(
values between 0 and 100) and lethality (values less than 0). The
the 384-well culture plate. The plate was incubated at 37 C in a
humidified atmosphere of 5% CO and 95% air for 72 h. 5 mL of
percentage growth is calculated at each of the drug concentrations
levels. Percentage growth inhibition is calculated as:
ꢃ [(Ti ꢂ Tz)/(C ꢂ Tz)] ꢀ 100 for concentrations for which
Ti ꢄ Tz
2
MTT Dye Solution was added to each well. The plate was
ꢁ
incubated at 37 C in a CO incubator for 6 h. After incubation,
2
the formazan was produced in the cells. 25 mL of Solubilization/
Stop Solution was added to each well and the plate was incubated
ꢃ [(Ti ꢂ Tz)/Tz] ꢀ 100 for concentrations for which Ti5Tz.
ꢁ
for another 1 h at 37 C in a CO incubator to dissolve the
2
MTT cell viability assay
formazan crystals. The absorbance of each sample was measured
at 590 nm using Envision 2104 Multilabel Reader (PerkinElmer,
Santa Clara, CA). The cell viability was determined based on the
Materials and methods
Compound 1d was submitted to Reaction Biology Corporation quantification of the color intensity in each culture well. The IC50
http://www.reactionbiology.com) for MTT assay. The targeted curves were plotted and the IC50 values were calculated using the
(
compound was dissolved in DMSO at 10 mM stock. Control GraphPad Prism 4 program based on a sigmoidal dose–response
compound was purchased from Sigma-Aldrich (Saint Louis, MO). equation.
H O
N
O
S
i
H N
ArSO Cl
H N
2
+
ii
2
2
n
NH2
n
IIa-e
I(a, b)
R
3a-j
O
O
N
O
O
S
S
HN
H2N
NH2
III
IV
Cbz
Cbz
HN
O
S
O
S
O
O
H N
iii
iv
vi
2
H N
OH
n
Cbz
OMs
n
v
N
N
2
n
OH
N
N
N
N
n
n
H
H
V(a, b)
VI(a,b)
VII(a,b)
VIII (a,b)
3k,l
O
Cl
OH
O
O
O
Cl
Cl
Cl
Cl
O
b
d
e
c
O
O
O
H
O
N
N
4
5
6
7
S
8
O
OH
O
O
Cl
Cl
Cl
Cl
N
N
g
N
i
N
f
N
N
N
h
N
N
N
N
N
N
N
N
O
N
HN
HN
S
S
10
9
O
n
n
R
1
R
2
a-l
a-l
ꢁ
Scheme 1. Reagents and conditions: (a) (i) triethylamine, CH
2
Cl
2
, 0 C, RT, overnight; (ii) N-ethyl ethylenediamine, C
ꢁ
5
H
5
N, reflux, 3 h; (iii)
ꢁ
benzyloxycarbonyl chloride, TEA, CH
DMF; (vi) H
chloride, CH
2
Cl
2
, 0 C; (iv) methanesulfonyl chloride, TEA, CH
2
Cl
2
, 0 C; (v) IV, NaH (60% dispersion in mineral oil),
ꢁ
2
/Pd-C, MeOH, RT, 1 h; (b) (CH
3 2
) SO
4
, K
2
CO
3
, acetone, reflux, 3 h; (c) KMnO
ꢁ
4
, C
H N, H O, 50 C, 24 h, then RT, 13 h; (d) acetyl
5 5 2
ꢁ
3
OH, RT, 15 h; (e) 4-methyl-2-(methylthio)pyrimidine, LiHMDS, THF, ꢂ78 C, 2 h then RT, overnight; (f) (i) DMF-DMA, 80 C, 3 h; (ii)
ꢁ
phenylhydrazine, C
ꢁ
2
H
5
OH, RT, overnight; (g) oxone, MeOH, H O, RT, 16 h; (h) 3a–l, diisopropylethylamine, DMSO, 80 C, 16 h; (i) BBr
2
3
, CH
2
Cl
2
,
ꢂ78 C, 1 h; RT, overnight.

DOI: 10.1080/14756366.2016.1190715
Antiproliferative and kinase inhibitory effects of pyrimidinylpyrazole derivatives
7
Table 1. Structures of the target compounds 1a–l and 2a–l, their yield percentage, and melting points.
(
continued )

8
M. M. Gamal El-Din et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 1. Continued
*
Means the rest of the attached compound.

DOI: 10.1080/14756366.2016.1190715
Antiproliferative and kinase inhibitory effects of pyrimidinylpyrazole derivatives
9
Table 2. Mean percentage growth of the 53 cancer cell line
panel after treatment with the tested target compounds
Table 3. IC50 (mM) results exerted by the compound 1d and staurosporine
against HL-60 leukemia cell line, HT29 colon cancer cell line, and MRC-
5 lung fibroblasts.
(10 mM concentration).
a
Compound No.
Mean percentage growth
IC50 (mM)
compound 1d
IC50 (nM)
Staurosporine
Cell line
1
1
1
1
1
1
1
2
2
2
2
a
d
h
i
j
k
l
f
i
k
l
90.00
33.29
68.88
88.81
84.29
94.93
84.76
67.91
74.12
99.38
96.07
HL-60 leukemia cell line
HT29 colon cancer cell line
MRC-5 lung fibroblasts
(normal cells)
28.87
13.37 (2.77)
37.02
81.90
186.0 (0.015)
2.8
b
b
a
IC50 value means the concentration required to inhibit 50% of the cell
growth.
Selectivity index calculated by dividing the IC50 values against
fibroblasts by IC50 values against HT29 colon cancer cell line.
b
Bold figures indicate the most active compounds.
derivatives shown in Table 2 were selected on the basis of degree
of structural variation technique for testing their antiproliferative
service (http:// activity. The selected compounds were subjected to an in vitro
In vitro kinase screening
SM
Reaction Biology Corp. Kinase HotSpot
www.reactionbiology.com) was used for screening of compound anticancer assay against a full panel of 53 cancer cell lines of nine
1
1
0
d. Assay protocol: in a Enal reaction volume of 25 mL, kinase (5– different cancer types; leukemia, non-small cell lung, colon, CNS,
0 mU) is incubated with 25 mM Tris pH 7.5, 0.02 mM EGTA, melanoma, ovarian, renal, prostate, and breast cancers. The
.66 mg/mL relevant substrate, and 10 mM magnesium acetate compounds were tested at a single-dose concentration of 10 mM,
and [g P-ATP] (speciEc activity approx. 500 cpm/pmol, con- and the percentages of growth inhibition over the 53 tested cell
centration 1 mM). The reaction is initiated by the addition of the lines were determined. The mean percentage growth of the NCI-
Mg-ATP mix. After incubation for 40 min at room temperature, 60 cancer cell line panel after treatment with each of the tested
the reaction is stopped by the addition of 5 mL of a 3% phosphoric compound is illustrated in Table 2.
3
3
acid solution. 10 mL of the reaction is then spotted onto a P30
Upon investigating the effect of linker length on antiprolifera-
Eltermat and washed thrice for 5 min in 75 mM phosphoric acid tive activity, it was found that compound 1d with ethylene linker
and once in methanol prior to drying and scintillation counting.
was more active than the corresponding propylene derivative 1i.
The ethylene spacer might be optimal for appropriate fitting at the
receptor site. Regarding the terminal moieties (arylsulfonamido
and the cyclic sulfamide present in the terminal moieties), it was
found that compounds 1a, d, h–j, 2f, and 2i possessing
Results and discussion
Chemistry
Synthesis of the target designed compounds 1a–l and 2a–l was arylsulfonamido showed higher antiproliferative activity than
achieved through the pathway shown in Scheme 1. Methylation of cyclic ones 1k, 1l, 2k, and 2l. This may be attributed to that
the phenolic hydroxyl group of 2-chloro-5-methylphenol (4) using increased hydrophobicity by the aromatic moieties might enhance
2
2
dimethyl sulfate gave 1-chloro-2-methoxy-4-methylbenzene (5) . the compound penetration through the cell membrane to inside
Oxidation of the methyl group of 5 using KMnO produced 4- the cell and/or stronger affinity at the receptor site due to stronger
4
2
1
chloro-3-methoxybenzoic acid (6) . Esterification of the car- hydrophobic interactions. Any or both of these effects might lead
boxylic acid 6 using methanol in the presence of acetyl chloride to stronger antiproliferative activity. This assumption could be
produced the corresponding methyl ester 7. 4-Methyl-2- supported by the slightly higher antiproliferative activity of the
(
methylthio)pyrimidine was prepared by methylation of 4- methoxy compounds 1k and 1l than the corresponding hydroxyl
methylpyrimidine-2-thiol with iodomethane in the presence of analogs 2k and 2l. The results also showed that the compounds
aqueous NaOH. Upon reacting the ester with the pyrimidine with substituted arylsulfonamide moieties 1d and 1h–j were more
methyl thioether in the presence of lithium bis(trimethylsilyl)a- active than those with unsubstituted phenyl ring 1a. The increased
mide (LiHMDS), the ketide intermediate 8 was formed. Reaction bulkiness and lipophilicity induced by those substituents might
of compound 8 with dimethylformamide dimethyl acetal (DMF- lead to enhanced activity due to more appropriate fitting at the
DMA) followed by subsequent treatment with phenylhydrazine receptor site and/or more penetration to inside the cancer cells.
2
3
yielded the N-phenylpyrazole intermediate 9 . Oxidation of the
The percentage inhibition values of the most active compounds
1
9
methylsulfide moiety of compound 9 using oxone produced the over the most sensitive cell lines are summarized in Table 3. SR
corresponding mesyl compound 10. Heating compound 10 with leukemia, MCF-7, and T-47D breast cancer cell lines were the
the appropriate amino reagents 3a–l in the presence of most sensitive cell lines to this series of compounds. The most
diisopropylethylamine led to displacement of the mesyl group active compounds against SR leukemia were 1d, 2f, and 1h with
of compound 10 by the amino group and formation of the target percentage inhibitions of 119.44%, 58.02, and 56.01%, respect-
compounds 1a–l. Demethylation of the methoxy group of 1a–l ively. MCF-7 and T-47D breast cancer cell line were also sensitive
using BBr afforded the corresponding hydroxyl derivatives 2a–l. to the most potent compounds. Figure 2 illustrates the percentage
3
Structures of the target compounds, their yield percentages, and inhibition values of the tested target compounds against SR
melting points are illustrated in Table 1.
leukemia, MCF-7, and T-47D breast cancer cell line. It is obvious
from the figure that compounds 1d then 2f are the most active
among the 10 tested compounds against those three cell lines.
Among all the tested derivatives, compound 1d was the most
active. It exerted broad-spectrum antiproliferative activity against
In vitro antiproliferative activity against NCI-60 cancer cell
line panel (single dose)
Structures of the target synthesized compounds were submitted to different cell lines of different cancer types. It exerted the highest
National Cancer Institute (NCI, Bethesda, MD), and the 11
activities against HL-60 (TB) and SR leukemia cell lines.

1
0
M. M. Gamal El-Din et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
(
a)
% Inhibition 1d
1
1
1
1
60
40
20
00
80
60
40
20
0
Leukemia
Non-Small Cell Lung Cancer
Colon Cancer
CNS Cancer
Melanoma
Ovarian Cancer
Renal Cancer
Prostate
Cancer
Breast Cancer
(
b)
% Inhibiꢀon 1h
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
−10
Leukemia
Non-Small Cell Lung
Cancer
Colon Cancer
CNS Cancer
Melanoma
Ovarian Cancer
Renal Cancer
Prostate
Cancer
Breast Cancer
%
Inhibiꢀon 2f
(
c)
80
70
60
50
40
30
20
10
0
Leukemia
Non-Small Cell Lung
Cancer
Colon Cancer
CNS Cancer
Melanoma
Ovarian Cancer
Renal Cancer
Prostate
Cancer
Breast Cancer
Figure 2. Percentage inhibition expressed by compounds 1d (a), 1h (b), and 2f (c) at 10 mM concentration over all cell lines of the NCI cancer cell line
panel of nine different cancer types. The inhibition percentages are calculated by subtracting the growth percentages from 100.
Compounds 1d, 1h, and 2f showed the highest mean inhib- was significantly more active than compounds 1h and 2f. The
itions. The percentages of inhibition of these three compounds p-chlorophenylsulfonamido terminal moiety of compound 1d was
over each tested cell line of the NCI-60 panel at 10 mM more optimal than p-methoxyphenylsulfonamido and phenylsul-
concentration are depicted in Figure 2. The three compounds fonamido moieties of compounds 1h and 2f, respectively. As
exerted broad-spectrum antiproliferative activities against differ- mentioned previously, it might enhance the penetration into inside
ent cell lines of different cancer types. In general, compound 1d the cells and/or increase the affinity at the receptor site. The

DOI: 10.1080/14756366.2016.1190715
Antiproliferative and kinase inhibitory effects of pyrimidinylpyrazole derivatives 11
Table 4. Percentage inhibition and IC50 (mM) results exerted by the most Conclusion
potent compounds over the selected kinases.
In this study, a novel series of pyrimidinylpyrazoles possessing
terminal arylsulfonamido or cyclic sulfamide moiety separated by
IC50 (mM) ethyl or propyl spacer was synthesized. The target compounds
were tested for in vitro antiproliferative activities over NCI-60
cancer cell line panel of nine different cancer types. Among them,
Percentage inhibition
at 10 mM concentration
Kinase
ABL1
ABL1 (T315I)
FLT3
V600E-B-RAF
RAF1
ALK5
B-RAF (wild type)
V600K-B-RAF
EGFR
ERK2/MAPK1
JNK3
7.31% ± 0.38%
ꢂ2.96% ± 0.60%
17.01% ± 0.42%
74.47% ± 0.28%
78.04% ± 0.32%
7.90% ± 0.63%
6.26% ± 0.23%
72.46% ± 0.37%
ꢂ3.57% ± 0.25%
ꢂ7.68% ± 1.96%
ꢂ3.07% ± 0.25%
ꢂ26.57% ± 5.96%
ND
ND
compound 1d possessing p-chlorobenzenesulfonamido terminal
moiety, ethylene linker, and 4-chloro-3-methoxyphenyl ring at
position 3 of the pyrazole nucleus showed the highest activity. So
these moieties together may comprise potential pharmacophoric
hits for this series of compounds. Compound 1d exerted broad-
spectrum antiproliferative activities over different cell lines of
different cancer types. The inhibition percentage values of
compound 1d over HL-60 (TB) and SR leukemia, HT29 colon,
and T-47D and MCF-7 breast cancer cell lines were 135.92%,
ND
3.57
2.47
ND
ND
ND
ND
ND
ND
ND
p38a/MAPK14
1
19.44%, 88.66%, 95.32%, and 82.03%, respectively. SR leuke-
mia, MCF-7, and T-47D breast cancer cell lines were found to be
highly sensitive to the most active compounds. Compound 1d
gave 13.37 mM IC50 value upon testing against HT29 colon cancer
cell line, but it showed lower potency against HL-60 leukemia and
MRC-5 lung fibroblasts. It showed higher selectivity toward
HT29 colon cancer cell line than the normal fibroblasts, compared
to staurosporine. So despite of inferior potency than staurosporine
against HT29 colon cancer cell line, it is much safer for the
normal cells. Upon testing the compound against kinases, it
showed selectivity against RAF1 and V600E-B-RAF kinases
which are over-expressed in HT29 colon cancer cell line from
which we can deduce that this compound might express its
antiproliferative effect, at least in part, through inhibition of RAF
kinases. Therefore, compound 1d could be utilized as a lead
compound for future development of active anticancer agents
inhibiting RAF kinases. Further modifications of this series of
compounds in order to improve their anticancer activity are
currently in progress.
ethylene spacer of compound 1d seems to be more optimal for
appropriate fitting at the receptor site than the propylene linker of
compounds 1h and 2f. And the methoxy group on the aryl ring at
position 3 of the pyrazole ring could be more favorable than
hydroxyl due to hydrophobicity, steric, and electronic differences
between them.
MTT assay
Compound 1d was further tested at Reaction Biology Corporation
to identify its IC50 against HL-60 (TB) leukemia and HT-29 colon
cancer against which our compound showed the most promising
growth inhibition percentage and also were available in reaction
biology cell line screening panels. It was also tested against MRC-
5
lung fibroblasts normal cell line in order to investigate the
selectivity index. The IC50 values are illustrated in Table 3. The
compound showed higher potency against HT29 colon cancer cell
line than HL-60 leukemia cell line, with good selectivity index
toward cancer cells than normal cells. On the other hand,
staurosporine, the reference standard drug, was much more potent
against normal fibroblasts than cancer cells. So, compound 1d has
the merit of higher safety than staurosporine.
Declaration of interest
The authors have declared no conflict of interest.
This work was supported by Korea Institute of Science and
Technology (KIST), KIST Project (2E25260). We would like to
thank the National Cancer Institute (NCI), Bethesda, Maryland,
USA, for performing the in vitro anticancer testing over the cell
lines.
In vitro kinase screening
In order to investigate the probable mechanism of action of
compound 1d at molecular level, it was selected to be tested at a
single-dose concentration of 10 mM over 12 kinases; from which
V600E-B-RAF and RAF1 which are over-expressed in HT29
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