Design and synthesis of new potent anticancer pyrazoles with high FLT3 kinase inhibitory selectivity

Article abstract of DOI:10.1016/j.bmc.2010.04.029

A new series of 1H- and 2H-pyrazole derivatives (35 final compounds) has been designed and synthesized in this study. A selected group (13 compounds) was then tested over a panel of 60 cancer cell lines at a single dose concentration of 10 μM. At this concentration, six compounds have showed moderate to strong mean inhibitions, and were further tested at five-dose testing mode to determine their IC₅₀ over the 60 cell lines. The IC₅₀ values of the tested compounds indicated high potency (as for compound 10f) as well as high efficacy (as for compound 11e). Accordingly, compound 10f was then tested at a single dose concentration of 10 μM over a panel of 54 kinases to determine its kinase inhibitory profile. The compound has showed good selectivity towards FLT3 kinase, associated with a moderate potency, with an IC₅₀ value of 1.74 μM.

Full text of DOI:10.1016/j.bmc.2010.04.029

Bioorganic & Medicinal Chemistry 18 (2010) 3961–3973
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of new potent anticancer pyrazoles with high FLT3
kinase inhibitory selectivity
*
Ibrahim Mustafa El-Deeb, So Ha Lee
Life/Health Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
Department of Biomolecular Science, University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 1H- and 2H-pyrazole derivatives (35 final compounds) has been designed and synthesized
Received 16 March 2010
Revised 8 April 2010
Accepted 9 April 2010
Available online 14 May 2010
in this study. A selected group (13 compounds) was then tested over a panel of 60 cancer cell lines at a
single dose concentration of 10 lM. At this concentration, six compounds have showed moderate to
strong mean inhibitions, and were further tested at five-dose testing mode to determine their IC50 over
the 60 cell lines. The IC50 values of the tested compounds indicated high potency (as for compound 10f) as
well as high efficacy (as for compound 11e). Accordingly, compound 10f was then tested at a single dose
Keywords:
Anticancer
Pyrazoles
FLT3
concentration of 10
pound has showed good selectivity towards FLT3 kinase, associated with a moderate potency, with an
IC50 value of 1.74 M.
lM over a panel of 54 kinases to determine its kinase inhibitory profile. The com-
l
Ó 2010 Elsevier Ltd. All rights reserved.
Receptor tyrosine kinase
1. Introduction
nases.5,6 Hence, the targeting of oncogenic kinases has recently be-
come one of the new effective strategies for the development of
selective therapies for specific cancers.7
Cancer has been the leading cause of death worldwide, and it
accounted for 7.4 million deaths (around 13% of all deaths) in
2004 according to WHO reports.1 The conventional cancer treat-
ments such as surgery, radiation and cytotoxic chemotherapy seem
to be no longer effective neither for the complete eradication of
disease nor for the improvement of cancer patients’ life. This has
prompted the development of a variety of new therapies that are
capable of selectively targeting the substantial cause of cancer,
resulting in a better and more complete response and at the same
time, minimal side effects, and more improvement of patient’s life.
The deregulated cell growth in cancer occurs as a result of per-
turbed signal transduction defined, in its broadest sense, as all cel-
lular signals that modulate or alter cellular behavior or function.2,3
Consequently, cancers do not necessarily arise as a result of an in-
creased rate of cell proliferation. Rather, it is the critical balance
between the rate of cell-cycle progression (cell division) and cell
growth (cell mass) on one hand, and programmed cell death (apop-
tosis) on the other.4 During normal embryonic development and in
adult life, signaling needs to be precisely coordinated and inte-
grated at all times, and properly regulated differentiation signals
are critical for preventing oncogenesis. Protein kinases are impor-
tant players in the process of cell signaling and signal transduction,
and the dysregulation of such signals is most probably caused by
mutations or abnormalities in the substantial controlling ki-
The hematopoietic class III receptor tyrosine kinase (RTK) FLT3
(FLK2, STK1) has recently received much attention as a potential
drug target in cancer treatment.8–10 The activation of FLT3 by dif-
ferent types of mutations plays an important role in proliferation,
resistance to apoptosis, and prevention of differentiation of leuke-
mia blasts in acute myeloid leukemia (AML). The signal transduc-
tion of FLT3 involves activation of several conserved pathways,
including the RAS/MAP-kinase and the phosphoinositied-3-ki-
nase/Akt signaling cascades. A number of potent FLT3 inhibitors,
such as Lestaurtinib (CEP-701),11 ABT-869,12 MLN-518,13 and
Fostamatinib,14 which differ in their selectivity profiles, both with
respect to other kinases and among wild-type FLT3, are currently
under preclinical and clinical investigations as potential drug can-
didates for AML.
In this study, a number of 1H- and 2H-pyrazole derivatives (35
final compounds) have been designed in rationale to the potent
B-Raf inhibitor ‘GDC-0879’.15 As showed in Figure 1, the com-
pounds share a general structure at which, the N-(dihydroinden-
3-ylidene)hydroxylamine group of GDC-0879 has been replaced
with a 3-(methoxy/hydroxyl)-5-methylphenyl group attached to
position 3 of the pyrazole nucleus (instead of position 4 in GDC-
0879), and an acetonitrile group (instead of the 2-hydroxyethyl
group in GDC-0879) is attached either to position 1 (in 1H-pyra-
oles) or position 2 (in 2H-pyrazoles). In position 4 of the pyrazole
ring, a pyridin-4-yl group (similar to that in GDC-0879) or a pyri-
din-3-yl group was attached, to which, a series of variable substi-
* Corresponding author. Tel.: +82 2 958 6834; fax: +82 2 958 5189.
E-mail address: LSH6211@kist.re.kr (S.H. Lee).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.029

3962
I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3961–3973
OR
good selectivity towards FLT3 kinase, associated with a moderate
OR
potency, with an IC50 value of 1.74 lM.
CN
N
CN
N
N
N
2. Results and discussion
2.1. Chemistry
N
HO
N
N
Ar
Ar
OH
9a-g
11a-e
10a-g
12a-e
For the preparation of the target compounds, the key ester,
methyl 3-methoxy-5-methylbenzoate (4), was first prepared as
illustrated in Scheme 1. The synthesis started with the preparation
of the sodium salt of ethyl 2-hydroxy-4-oxopent-2-enoate (1)
according to the literature procedure, through the condensation
of diethyl oxalate with acetone in the presence of sodium ethoxide
in absolute ethanol.16 The resulted salt 1 was then cyclized into
Claisen furan derivative 2 by heating in 50% acetic acid followed
by acidification with sulfuric acid.17 The resulted Claisen com-
pound underwent rearrangement and aromatization into 3-hydro-
xy-5-methylbenzoic acid (3) within less than one hour by heating
with magnesium oxide in boiling water, followed by acidification
with hydrochloric acid to precipitate the product.17 Methyl esteri-
fication and O-methylation of the resulted phenolic acid 3 were
achieved in a single step and in high yield (94%) to give compound
4 through a little modification of the literature procedure,18 where
the acid 3 was refluxed with excess potassium carbonate and iodo-
methane in acetone, and in the presence of a catalytic amount of
DMAP.
N
N
N
OR
OR
GDC-0879
CN
(AR00341677)
CN
N
N
N
N
Ar
N
Ar
N
18a-c
20a,b
17a-c
19a-c
Figure 1. Structure of GDC-0879 and the new rationally designed compounds.
tuted aryl moieties were linked through a group of Suzuki cou-
plings. A selected group (13 compounds) from the target com-
pounds was then tested over a panel of 60 cancer cell lines at a
single dose concentration of 10 lM. At this concentration, six com-
pounds have showed very good activity and were accordingly fur-
ther tested at five-dose testing mode to determine their IC50 over
the 60 cell lines. The IC50 values of the tested compounds indicated
high potency (as for compound 10f) as well as high efficacy (as for
compound 11e). Accordingly, compound 10f was then tested at a
In Scheme 2, the benzoate ester 4 underwent a nucleophilic at-
tack at its carboxylic carbon by the activated methylene group of
2-chloro-4-methylpyrimidine. The activation of this methyl group
into an active methylene was achieved by dropwise addition of
lithium bis(trimethylsilyl)amide (LHMDS) in dry THF at room tem-
single dose concentration of 10 lM over a panel of 54 kinases to
determine its kinase inhibitory profile. The compound has showed
perature. The resulted tautomeric
a,b-unsaturated ketone 5 was
O
OH
OCH3
O
O
O
O
OEt
OEt
O
ii
i
iv
iii
O
+
O
OCH3
O
H3C
CH3
O
H3C
COOH
ONa O
H3C
O
O
O
2
4
1
3
Scheme 1. Reagents and conditions: (i) NaOEt, abs EtOH, rt, 4 h, 87%; (ii) acetic acid/H2O (1:1), rt, 2 h, 50%; (iii) MgO, H2O, reflux, 45 min, 42%; (iv) K2CO3, CH3I, DMAP,
acetone, 65 °C, 12 h, 94%.
OCH3
OCH3
OCH3
OCH3
OCH3
CN
OCH3
N
O
OH
CN
N
N
N
H3C
H3C
H3C
H3C
H3C
ii , iii
iv
NH
N
Cl
i
N
+
OCH3
H3C
N
N
N
N
N
O
Cl
Cl
Cl
Cl
Cl
5
4
8
6
7
OH
OCH3
OH
OCH3
CN
CN
CN
N
N
N
CN
H3C
N
H3C
H3C
v
H3C
vi
N
N
N
N
+
+
N
N
N
N
R
R
R
R
10a-g
11a-e
12a-e
9a-g
Scheme 2. Reagents and conditions: (i) LHMDS, THF, N2, rt, 18 h, 72%; (ii) DMF–DMA, reflux, 12 h; (iii) hydrazine hydrate, abs EtOH, rt, 2 h, 81%; (iv) K2CO3, iodoacetonitrile,
acetone, reflux, 4 h, 92%; (v) arylboronic acid, Pd(PPh3)2Cl2, Na2CO3, N2, THF/H2O; (4:1), 70 °C, 12 h; (vi) BF3ÁS(CH3)2, dichloromethane, N2, rt, 24 h.

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3961–3973
3963
Section 4 and in the 1H NMR spectra in Supplementary data. In
the 2H-isomers, such protons appeared at a d value of around
4.95–4.97, while in the 1H-isomers, these protons were more
downfield shifted and appeared in the range of 5.10–5.20 ppm.
The final hydroxyl derivatives 11a–e and 12a–e were obtained
by demethylation of the methoxy group of the corresponding
methoxy compound using 10 equiv of borontrifluoride–dimethyl-
sulfide complex in dichloromethane.
NOE
OH
OH
H
H
H
H
2
6
CN
2
6
CN
H
N
H3C
N
H3C
N
N
H
H
H
a
b
a: The distance between acetonitrile -CH2- and the aromatic protons
doesn't allow for NOE interaction in compound 9e; b: NOE effect between
acetonitrile -CH2- and the aromatic protons in compound 10e.
Typical procedures to those used in Scheme 2 have been applied
in Scheme 3. The only difference was in the starting amine, where
in Scheme 3, 2-chloro-5-methylpyridine was used instead of 2-
chloro-4-methylpyridine in Scheme 2. All compounds synthesized
were listed in Table 1.
Figure 2. NOE interactions in compounds 9e and 10e.
then converted to the required pyrazole derivative 6 through two
successive steps. In the first step, compound 5 was heated with
excess N,N-dimethylformamide dimethylacetal for 12 h, and the
resulted product was taken to the next step without further
purification, where it was cyclized with hydrazine monohydrate
in absolute ethanol into the pyrazole derivative 6. The reaction of
the resulted pyrazole 6 with iodoacetonitrile in the presence of
excess potassium carbonate produced two different regioisomers;
a major isomer 7 with Rf value of 0.74 (EtOAc–hexane, 1:1, v/v),
and a minor isomer 8 with Rf value of 0.84 (EtOAc–hexane, 1:1,
v/v). A mixture of these two isomers was taken to the next step
without separation, where it underwent a series of Suzuki coupling
reactions with a group of arylboronic acids, in the presence of di-
chloro bis(triphenylphosphine)Pd(II) and potassium carbonate, in
a mixed solvent of THF and water in a (4:1) ratio. These series of
Suzuki coupling reactions produced two isomers in every reaction
9a–g and 10a–g, which were separated in a pure form by prepara-
tive TLC. In all of these reactions, the 1H-pyrazole isomer (isomer
9) was the product with the lower Rf while the 2H-pyrazole isomer
(isomer 10) was the product with the higher Rf. This was proved by
the 2D-NOESY NMR spectrum of compounds 9e and 10e as a model
for the whole series. The absence of any cross peak between the
acetonitrile –CH2– and any of the aromatic protons of 3-meth-
oxy-5-methylphenyl group in the 2D-NOESY NMR spectrum of
compound 9e while the presence of such peaks in the 2D-NOESY
NMR spectrum of compound 10e has confirmed the assigned struc-
tures (Fig. 2). Furthermore, the expected higher shielding of the
two protons of the acetonitrile –CH2– in all of the 2H-pyrazole iso-
mers caused by the anisotropic effect of the nearby aromatic elec-
tron cloud, was proved in the assigned structures as showed in
2.2. Biological Screening
2.2.1. In vitro anticancer screening
The structures of the final products were submitted to National
Cancer Institute (NCI),19 Bethesda, Maryland, USA, and the 13 com-
pounds showed in Table 2 were selected on the basis of degree of
structural variation and computer modeling techniques for evalu-
ation of their antineoplastic activity. The selected compounds were
subjected to in vitro anticancer assay against tumor cells in a full
panel of 60 cell lines taken from nine different tissues (blood, lung,
colon, CNS, skin, ovary, kidney, prostate and breast). The com-
pounds were tested at a single dose concentration of 10 lM, and
the percentages of growth inhibition over the 60 tested cell lines
were determined. The mean inhibition percentages of all of the
tested compounds over the full panel of cell lines are illustrated
in Table 2. As showed in Table 2, the mean inhibition was strong
in compounds 9f, 10f, 11d and 17c, moderate in compounds 10c
and 11e, while weak in compounds 9c, 9d, 9e, 9g, 10d, 11a and
11c. The figures showing the multiple inhibitions exerted by the
tested compounds are illustrated in Supplementary data.
By referring to the inhibitory effects of the tested compounds
summarized in Table 2, and correlating between the structural
variations and the resulted anticancer activities, we can conclude
that the best aryl substitution at the terminal pyridyl moiety in
Scheme 2 compounds is that using 4-(dimethylamino)phenyl group.
The derivatives containing such group were found to possess the
highest anticancer activity over the 60 tested cell lines. It was found
that both the 1H (9f) and 2H (10f) isomers containing such group
have comparable activity, which means that the relative position
of the acetonitrile group has a little effect on activity, relative to
OCH3
OCH3
OCH3
OCH3
OCH3
CN
OCH3
N
O
OH
CN
N
N
N
H3C
Cl
H3C
Cl
H3C
Cl
H3C
Cl
H3C
Cl
Cl
ii , iii
NH
N
iv
N
+
OCH3
i
H3C
N
N
O
N
N
N
14
16
13
15
4
OCH3
OCH3
OH
OH
CN
CN
CN
CN
N
N
N
N
H3C
H3C
H3C
H3C
v
vi
N
N
N
N
+
+
N
N
N
N
R
R
R
R
X
X
X
X
18a-c
19a-c
20a,b
17a-c
Scheme 3. Reagents and conditions: (i) LHMDS, THF, N2, rt, 18 h, 55%; (ii) DMF–DMA, reflux, 12 h; (iii) hydrazine hydrate, abs EtOH, rt, 4 h, 62%; (iv) K2CO3, iodoacetonitrile,
acetone, reflux, 4 h, 80%; (v) arylboronic acid, Pd(PPh3)2Cl2, Na2CO3, N2, THF/H2O (4:1), 70 °C, 12 h; (vi) BF3ÁS(CH3)2, dichloromethane, N2, rt, 24 h.

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I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3961–3973
Table 1
Table 2 (continued)
Synthesized derivatives from Schemes 2 and 3 and their corresponding yields
Compd
Structure
Inhibition %a
Compd
X
R
Yield%
O
9a
9b
9c
9d
9e
9f
9g
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
N
H
H
N
H
H
N
H
H
H
H
H
3-Ac
4-Ac
3-NH–Ac
4-CN
4-N(CH3)2
4-OPh
H
74
79
68
75
82
79
72
62
77
75
63
77
50
69
70
61
81
75
62
65
42
55
72
61
55
48
76
61
77
80
61
77
75
48
72
CN
N
N
9e
14.38
N
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
11e
12a
12b
12c
12d
12e
17a
17b
17c
18a
18b
18c
19a
19b
19c
20a
20b
3-Ac
4-Ac
CN
O
3-NH–Ac
4-CN
4-N(CH3)2
4-OPh
H
CN
N
N
3-Ac
4-CN
4-N(CH3)2
4-OPh
3-Ac
9f
92.30
N
4-Ac
3-NH-Ac
4-CN
4-OPh
H
2-Ac
3-Ac
H
2-Ac
3-Ac
H
N
O
CN
N
N
N
9g
13.48
2-Ac
3-Ac
2-Ac
3-Ac
O
O
CN
N
N
10c
33.36
N
Table 2
Compounds selected for single dose cancer cell-line screening and their mean
inhibitory percentages
Compd
Structure
Inhibition %a
O
O
O
CN
CN
N
N
N
N
9c
18.23
10d
9.40
N
N
O
N
H
O
O
O
CN
N
CN
N
O
N
N
9d
1.18
10f
88.36
N
N
N
H
N

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3961–3973
3965
11e. In this case, the activity of the hydroxyl derivative 11e was
found to be higher than that of the methoxy derivative 9g. A possible
reason for this inversion in activity between the methoxy and hydro-
xyl analogs could be owed to the possible variation in binding modes
betweenthe4-phenoxyphenylderivativesandtherelativelysmaller
4-(dimethylamino)phenyl derivatives to their target site, since the
relatively larger size of the 4-phenoxyphenyl derivatives may force
the compounds to bind differently to their target site. By comparing
the activities of compounds 9c and 10c, both with a 4-acetylphenyl
group at the terminal pyridine, it was found that the activity of 10c
(the 2H-isomer) is higher than that of 9c (the 1H-isomer), and this
may suggest (along with the IC50 data of compounds 9f and 10f) that
the presence of the acetonitrile group at position 2 in the 2H-isomer
is more favored for activity. The only screened compound from
Scheme 3 (compound 17c) has showed also good activity, but unfor-
tunately, no other compounds were selected for screening from the
same scheme, so that a reliable SAR could be made to this group of
compounds.
Table 2 (continued)
Compd
Structure
Inhibition %a
OH
CN
N
N
11a
16.41
N
OH
CN
N
N
11c
20.75
N
After this initial single dose screening of the 13 selected com-
pounds, the compounds showing the highest activity at the single
dose were further tested in a five-dose testing mode, in order to
determine their IC50 values over the 60 tumor cell lines. These
compounds are 9f, 10c, 10f, 11d, 11e and 17c. For each of these
compounds, the IC50 (the concentration producing 50% inhibition),
TGI (the concentration producing 100% inhibition) and LC50 (the
concentration causing 50% lethality or 50% tumor regression) were
recorded. The five-dose testing results of these six compounds are
showed in Table 3.
CN
OH
CN
N
N
11d
65.35
N
As showed in Table 3, the six tested compounds have showed
good potency over almost all of the 60 tested cell lines. However,
the five-dose testing data were so much interesting in compounds
9f, 10f and 11e. In compounds 9f and 10f, a very high potency was
observed over almost the whole cell-lines panel, with IC50 values in
the nM scale. The potency was however higher in compound 10f,
with IC50 values as small as of 23 nM at the melanoma cell-line
MDA-MB-435. The two compounds (9f and 10f) have showed also
high efficacies, but only at a limited range of cell lines. For exam-
ples, the TGI and LC50 values of compound 9f over the NSCLC cell
N
OH
CN
N
N
N
11e
46.59
line NCI-H522 were 0.492
for compound 10f, the values were 0.085
l
M and 3.24
l
M, respectively, while
O
l
M and 3.22 M, respec-
l
tively, at the same cell line. Among the six tested compounds, com-
pound 11e has showed the greatest efficacies over a very broad
range of cell lines. The compound was capable of inducing total
growth inhibition (TGI) and 50% lethality (LC50) at almost all of
O
N
CN
N
N
the 60 cell lines with TGI and LC50 values as small as of 2.9
lM
and 5.38 M, respectively, at the colon cancer cell line HCT-116.
l
17c
78.65
2.2.2. In vitro kinase screening
In order to investigate the mechanism of action and the kinase
inhibitory profile of this new class of compounds, compound 10f
with the highest potency at the cancer cell lines was tested at a sin-
O
a
% Inhibition represents the mean inhibition percentages over the 60 cell lines.
The inhibition percentages are calculated by subtracting the growth percentages
from 100.
gle dose concentration of 10 lM over a panel of 54 kinases at Reac-
tion Biology Corporation.20 It worth also to mention that a selected
group from these compounds was previously screened against ROS
receptor tyrosine kinase (RTK), where the discovery of a potent
ROS RTK inhibitor has been reported.21 As showed in Figure 3,
compound 10f has showed very good selectivity for FLT3 kinase
with an inhibition percentage of 82.3% at the testing dose. Accord-
ingly, the compound was further tested against FLT3 kinase in a
10-dose IC50 testing mode with threefold serial dilutions starting
the effect exerted by the aromatic ring substituted at the terminal
pyridyl group. Furthermore, the hydroxy-isomer of compound 9f
(compound 11d) which contains the same 4-(dimethyl-
amino)phenyl group was found also to have good activity, but
slightly lower than that of compound 9f. This means that the meth-
oxy group is more favored than the hydroxy for good anticancer
activity within this group of compounds. This was however con-
tradicted by the relative activities of compounds 9g and 11e. Both
of these two compounds are substituted with 4-phenoxyphenyl
moiety at the terminal pyridine group, and the only difference be-
tween them lies in the methoxy group of compound 9g which was
demethylated to the corresponding hydroxy group in compound
at 20
l
M concentration using hot-spot technique. The compound
M. This
has showed a moderate potency with an IC50 of 1.74
l
means that this compound can be used as a good new lead for
the development of selective FLT3 kinase inhibitors. However,
the moderate potency of the compound over FLT3 kinase cannot
justify for its strong and broad spectrum anticancer activity, and

Table 3
IC50, TGI and LC50 values in lM of compounds 9f, 10c, 10f, 11d, 11e, and 17c over 60 cancer cell lines
Cell line
9f
10c
10f
11d
11e
17c
TGIb
LC50
IC50
TGIb
LC50
IC50
TGIb
LC50
IC50
TGIb
LC50
IC50
TGIb
LC50
IC50
TGIb
LC50
a
c
a
c
a
c
a
c
a
c
a
c
IC50
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
0.454
0.293
0.161
0.626
1.36
>100
0.923
>100
>100
10.7
>100
>100
>100
>100
>100
>100
2.97
2.26
0.795
2.63
2.59
2.87
>100
6.50
>100
>100
9.02
>100
>100
>100
>100
>100
>100
0.237
0.105
0.058
0.271
0.266
NA
>100
0.465
3.82
NA
1.96
NA
>100
>100
>100
>100
>100
NA
3.59
3.14
2.55
3.17
3.71
2.64
>100
12.9
32.8
46.0
22.2
>100
>100
>100
>100
>100
>100
>100
2.85
2.25
0.049
2.03
2.25
3.77
>100
5.94
6.02
6.82
6.52
37.2
>100
>100
>100
>100
>100
>100
2.61
1.78
0.365
3.35
3.30
1.62
>100
15.1
>100
>100
18.6
>100
>100
>100
>100
>100
>100
>100
0.461
>100
>100
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
0.583
0.696
0.390
0.557
0.968
0.514
NA
48.3
>100
18.7
26.6
12.7
>100
>100
1.68
0.492
>100
>100
>100
>100
>100
>100
>100
94.7
3.68
5.80
3.35
1.46
5.47
3.90
>100
2.41
1.93
>100
>100
>100
7.56
>100
>100
>100
NA
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.155
0.290
0.263
<0.010
NA
0.276
0.331
0.058
0.027
>100
>100
>100
0.055
NA
>100
>100
0.282
0.085
>100
>100
>100
>100
NA
>100
>100
4.99
3.22
4.18
3.92
2.82
3.03
4.18
4.48
6.08
2.79
1.93
54.5
92.8
23.4
46.8
21.8
24.8
59.8
10.2
7.14
>100
>100
>100
>100
>100
>100
>100
70.5
2.00
2.16
11.7
1.59
3.22
1.94
5.68
1.87
2.72
5.07
8.95
24.7
5.24
12.2
3.97
19.2
3.58
10.4
24.7
>100
52.3
39.5
64.5
8.13
48.6
6.85
35.1
3.87
3.98
3.07
2.63
4.29
3.45
4.36
2.04
1.64
30.8
50.7
36.9
9.09
82.3
84.0
50.8
5.72
6.72
>100
>100
>100
>100
>100
>100
>100
26.6
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
0.371
0.196
3.24
6.96
68.1
>100
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
0.320
0.455
0.353
0.350
0.481
0.310
0.332
0.769
1.93
1.64
8.79
2.88
1.62
>100
3.91
9.69
>100
>100
37.2
25.5
>100
1.95
3.41
1.70
3.14
3.21
2.87
2.27
3.70
>100
5.40
>100
>100
>100
>100
7.02
0.220
NA
>100
NA
1.03
>100
0.211
0.264
>100
>100
NA
>100
>100
NA
1.95
3.19
2.71
3.36
4.65
1.63
2.83
4.13
13.2
10.4
18.2
20.8
7.66
17.5
8.78
37.2
41.9
>100
93.9
29.2
>100
1.80
1.91
1.56
2.14
1.83
1.92
1.94
3.26
3.39
2.90
5.38
3.84
4.87
4.33
5.90
6.02
5.38
26.1
8.06
15.3
9.63
1.85
2.08
2.33
2.14
3.28
2.23
1.30
4.13
6.77
10.6
>100
18.7
13.9
19.3
9.20
54.4
87.4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.050
0.077
0.038
0.048
0.039
KM12
SW-620
>100
>100
CNS cancer
Melanoma
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
0.478
0.304
0.259
0.567
0.286
0.415
8.90
1.91
0.596
>100
0.848
5.86
>100
>100
>100
>100
>100
>100
3.14
5.84
2.02
>100
2.08
2.89
>100
>100
4.66
>100
NA
>100
>100
>100
>100
>100
>100
0.302
0.053
0.127
0.362
0.069
0.178
>100
0.315
NA
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.55
2.18
2.12
4.54
1.86
3.14
16.9
8.71
5.33
43.9
5.94
12.9
77.9
84.1
27.3
>100
>100
61.3
3.96
4.42
3.38
10.7
3.82
1.61
15.5
21.2
13.5
23.4
17.1
2.99
41.4
75.2
44.0
51.1
42.0
5.56
4.07
2.63
1.73
6.62
1.96
3.17
81.4
>100
4.65
>100
6.24
13.5
>100
>100
>100
>100
>100
>100
>100
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
0.681
NA
0.258
0.235
1.01
0.776
0.269
0.448
0.337
14.8
>100
>100
>100
>100
>100
>100
24.0
3.39
5.82
1.97
1.93
5.88
7.66
1.94
5.24
4.05
>100
>100
NA
>100
>100
>100
4.96
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.067
0.060
0.054
0.023
0.204
NA
0.039
0.036
0.062
>100
>100
0.483
0.067
>100
NA
0.309
>100
>100
>100
>100
>100
>100
>100
NA
>100
>100
>100
5.13
4.56
1.83
1.01
4.15
5.22
1.99
4.35
3.05
17.8
39.2
4.76
3.21
15.8
32.4
5.75
35.6
14.0
47.7
>100
20.4
>100
>100
>100
22.9
>100
72.1
1.87
1.81
1.70
2.32
3.44
4.92
2.93
2.38
4.67
3.39
3.58
3.12
6.84
11.7
17.0
9.90
8.38
18.1
6.13
7.12
5.73
60.6
44.2
41.4
31.5
31.7
44.4
3.52
3.76
1.17
0.341
3.73
3.63
1.54
3.61
4.36
>100
96.4
3.30
1.60
41.3
93.6
6.79
>100
>100
>100
>100
NA
>100
>100
>100
39.8
>100
>100
>100
0.769
0.613
45.5
>100
0.672
>100
40.7
>100
>100
Ovarian cancer
IGROV1
0.402
0.204
0.549
3.98
0.522
0.253
0.304
30.1
>100
NA
3.57
2.61
5.13
>100
2.86
2.87
4.22
>100
8.05
>100
>100
>100
>100
>100
>100
>100
0.061
0.030
0.270
>100
0.236
0.048
0.504
40.9
NA
>100
>100
>100
>100
>100
>100
>100
3.91
1.91
4.85
14.9
4.20
2.54
2.85
24.8
4.78
>100
60.4
69.4
7.93
8.03
>100
21.2
1.74
1.88
4.56
4.84
2.08
2.06
12.0
3.75
3.95
16.0
19.2
5.54
4.56
24.4
8.07
8.29
48.6
48.3
21.9
15.8
49.6
2.73
1.34
3.76
2.43
3.16
1.31
3.13
10.4
3.30
>100
33.9
35.2
6.06
9.10
>100
8.13
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
0.448
>100
>100
41.6
0.655
0.882
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Renal cancer
786-0
A498
0.420
0.246
0.499
0.345
0.258
0.730
1.01
5.43
0.818
12.4
18.2
0.633
33.1
78.9
>100
>100
>100
>100
>100
>100
>100
3.11
2.03
3.76
4.24
2.32
4.49
NA
>100
>100
>100
>100
6.02
>100
>100
>100
>100
NA
0.085
0.132
0.068
0.037
NA
>100
>100
>100
0.089
NA
>100
>100
>100
>100
NA
3.41
3.03
3.73
2.89
2.87
4.28
8.40
16.6
13.4
23.7
16.4
6.14
30.0
57.8
>100
>100
>100
>100
>100
>100
>100
2.14
3.87
2.84
1.80
2.08
1.80
4.25
5.38
16.7
9.14
5.78
4.31
4.02
15.1
18.9
44.1
33.8
41.0
8.92
9.01
43.6
3.52
1.08
4.19
1.91
1.49
6.08
3.28
93.0
5.14
>100
10.5
3.59
>100
11.5
>100
72.5
>100
>100
8.62
ACHN
CAKI-1
RXF 393
SN12C
TK-10
>100
>100
>100
>100
0.398
0.636
>100
>100
>100
>100
>100
>100

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3961–3973
3967
this suggests the presence of other underlying mechanisms that (in
addition to the moderate FLT3 kinase inhibition) controls the activ-
ity of these new compounds against cancer.
3. Conclusions
A new series of highly potent and efficient anticancer pyrazoles
has been designed and synthesized in this study. A structure–activ-
ity relationship (SAR) has been made to correlate between com-
pounds structures and activity. The SAR made has revealed that
for best activity the compounds should be substituted with a 4-
(dimethylamino)phenyl moiety at the 2-position of the terminal
pyridyl group. In the second place for good activity comes the 4-
phenoxyphenyl group, and then the 4-acetyl group. The presence
of the acetonitrile group at the position 2 of the pyrazole ring
(2H-isomers) was found also to be favored for activity rather than
in position 1. The effect of demethylation of the methoxy group to
produce the hydroxyl analog was found not to be constant within
the derivatives, which suggests the possibility of variable binding
modes for such derivatives at their target’s binding site, or the
presence of different targets, the binding mode at each of which
would likely be different. The screening of compound 10f over 54
kinases showed high selectivity of the compound over FLT3 kinase,
but unfortunately with moderate potency, which means that the
compounds may have also another target that may contribute, to-
gether with the FLT3 inhibition to the anticancer effect of these
new compounds. Currently compounds 9f, 10f and 11e are being
further tested in vivo in NCI, USA. The compounds would be also
screened by our group over a large number of potential molecular
targets (such as tubulin, phosphatases and other cell-cycle specific
targets) in order to further investigate their possible mechanism of
action.
4. Experimental
4.1. General
1H NMR (300 MHz) and 13C NMR (75 MHz) were recorded on a
Bruker Avance 300 spectrometer with TMS as an internal standard.
The IR spectra were recorded on Perkin Elmer Spectrum GX spec-
trometer. Melting points were taken on a Thomas-Hoover capillary
melting apparatus and were uncorrected. Column chromatography
was performed on Merck Silica Gel 60 (230–400 mesh). TLC was
carried out using glass sheets precoated with Silica Gel 60 F254
prepared by E. Merck. All the commercially available reagents were
obtained from Aldrich and Tokyo Kasei Chemicals and generally
used without further purification.
4.2. Synthesis
4.2.1. 2-(2-Chloropyridin-4-yl)-1-(3-methoxy-5-methylphenyl)
ethanone (5)
To a solution of the ester 4 (9.0 g, 50 mmol) and 2-chloro-4-
methyllpyridine (7.0 g, 55 mmol) in THF (90 mL) was added drop-
wise lithium bis(trimethylsilyl)amide 1 M/THF (75 mL, 75 mmol)
at 0 °C under N2 atmosphere. The mixture was warmed to room
temperature and stirred for 18 h. Saturated aqueous ammonium
chloride solution (150 mL) was added to the mixture, followed
by extraction with ethyl acetate (300 mL Â 2). The combined or-
ganic layers were washed with saturated NaCl solution, dried over
anhydrous MgSO4 and evaporated under vacuum to yield crude 5
(9.92 g, 72%) which was used for the next step without further
purification; mp 88–89 °C; IR
m
/cmÀ1: 3432, 3055, 2954, 2919,
2834, 1683, 1598, 1466, 1346, 1323, 1288, 1181, 1155, 1041,
847, 679, 579; 1H NMR (CDCl3): d 2.41 (s, 3H), 3.84 (s, 3H), 4.26

3968
I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3961–3973
100
80
60
40
20
0
Kinase
Figure 3. Inhibition percentages of compound 10f at a single dose concentration of 10 lM over 54 kinases.
(s, 2H), 6.98 (s, 1H), 7.13 (d, J = 4.5 Hz, 1H), 7.25 (s, 1H), 7.30 (s, 1H),
7.38 (s, 1H), 8.34 (d, J = 4.6 Hz, 1H); 13C NMR (CDCl3): 21.36, 21.52,
44.14, 55.41, 55.47, 110.12, 120.79, 121.82, 123.82, 125.45, 137.23,
140.16, 146.86, 149.57, 151.69, 159.97, 195.14.
4.2.3.1. 2-(4-(2-Chloropyridin-4-yl)-3-(3-methoxy-5-methylphe
nyl)-1H-pyrazol-1-yl)acetonitrile (7). IR
/cmÀ1: 3432, 2938,
m
1596, 1532, 1379, 1168, 1130, 1063, 850, 778, 731; 1H NMR
(CDCl3): d 2.29 (s, 3H), 3.71 (s, 3H), 5.19 (s, 2H), 6.73 (s, 1H), 6.75
(s, 1H), 6.85 (s, 1H), 7.06 (d, J = 4.4 Hz, 1H), 7.26 (s, 1H), 7.79 (s,
1H), 8.24 (d, J = 4.8 Hz, 1H); 13C NMR (CDCl3): 21.53, 39.97,
55.29, 110.91, 111.12, 113.65, 115.67, 118.75, 121.59, 121.65,
122.78, 130.58, 132.37, 140.16, 143.28, 149.68, 151.45, 151.79,
159.71, 162.33.
4.2.2. 2-Chloro-4-(3-(3-methoxy-5-methylphenyl)-1H-pyrazol-
4-yl)pyridine (6)
A mixture of compound 5 (9.1 g, 33 mmol) and N,N-dimethyl-
formamide dimethylacetal (30 mL, 255 mmol) was heated under
reflux in an oil bath for 12 h. Excess N,N-dimethylformamide
dimethylacetal was removed under vacuum, and then the crude
product was dissolved in absolute ethanol (150 mL). Hydrazine
monohydrate (3.3 g, 66 mmol) was added and the mixture was
stirred at room temperature for 2 h. The solvent was then removed
under vacuum and the crude product was crystallized from ethyl
acetate/hexane to yield pure 6 as buff to brown powder (8.0 g,
4.2.3.2. 2-(4-(2-Chloropyridin-4-yl)-5-(3-methoxy-5-methylphe
nyl)-1H-pyrazol-1-yl)acetonitrile (8). IR
m
/cmÀ1: 3429, 3937,
1596, 1464, 1372, 1156, 844, 803; 1H NMR (CDCl3): d 2.39 (s,
3H), 3.83 (s, 3H), 4.94 (s, 2H), 6.63 (s, 1H), 6.74 (s, 1H), 6.92–6.96
(m, 2H), 7.19 (s, 1H), 7.93 (s, 1H), 8.17 (d, J = 5.2 Hz, 1H); 13C
NMR (CDCl3): 21.56, 37.78, 55.48, 112.15, 113.91, 117.08, 118.16,
119.88, 121.36, 122.48, 128.46, 139.57, 141.60, 142.19, 142.85,
149.78, 151.95, 160.48.
81%); mp 131–132; IR
m
/cmÀ1: 3442, 3112, 2915, 2848, 1596,
1064, 866, 832, 769; 1H NMR (CDCl3): d 2.33 (s, 3H), 3.75 (s, 3H),
6.78 (s, 1H), 6.80 (s, 1H), 6.85 (s, 1H), 7.13 (dd, J = 1.1, 4.2 Hz,
1H), 7.32 (s, 1H), 7.76 (s, 1H), 8.25 (d, J = 5.3 Hz, 1H); 13C NMR
(CDCl3): 21.54, 55.30, 111.11, 115.63, 115.89, 121.03, 121.49,
122.27, 140.41, 144.28, 149.61, 151.84, 159.91.
4.2.4. General procedure for the synthesis of compounds 9a–g
and 10a–g
A mixture of starting material 7 and 8 mixture (300 mg,
0.886 mmol), the appropriate aryl boronic acid (0.974 mmol),
dichlorobis(triphenylphosphine)Pd(II) (31 mg, 0.0443 mmol) and
K2CO3 (130 mg, 0.886 mmol) was placed in a mixed solvent of THF
and water (4:1, 10 mL). N2 gas was bubbled into this mixture for
10 min, and then the mixture was heated at 70 °C while stirring un-
der N2 atmosphere for 12 h. The reaction mixture was left to cool at
room temperature, and then poured into ice water (100 mL) and ex-
tracted with ethyl acetate (100 mL Â 3). The combined organic ex-
tracts were dried over anhydrous MgSO4 and evaporated under
vacuum. The target products were separated in pure form by pre-
parative TLC on silica plates using the proper ratio of ethyl acetate/
hexane. After four runs using the specified mobile phase; the prod-
ucts were extracted from silica using ethyl acetate (100 mL Â 2) fol-
lowedbymethanol(100 mL). Forcalculation of yields, it wasroughly
assumed that the amount of compounds 7 and 8 in every starting
300 mg of starting mixture are 200 and 100 mg, respectively.
4.2.3. General procedure for the synthesis of compounds 7 and 8
A mixture of compound 6 (4.6 g, 15.35 mmol) and ground
K2CO3 (10.6 g, 76.73 mmol) in acetone (100 mL) was stirred under
reflux for 2 h. To the reaction mixture was added iodoacetonitrile
(1.34 mL, 18.42 mmol) dropwise. Heating and stirring were main-
tained for two more hours, after which acetone was removed un-
der vacuum, and the residue was partitioned between water
(200 mL) and ethyl acetate (200 mL). The aqueous layer was fur-
ther extracted with ethyl acetate (100 mL), and then the combined
organics were dried over anhydrous MgSO4 and evaporated under
vacuum. The crude product was purified by column chromatogra-
phy (silica gel, ethyl acetate–hexane 2:3 v/v) to give a pale yellow
oily mixture of compounds 7 and 8 in an approximate ration of (2:
1), respectively, that has been used in the next step without sepa-
ration of the two isomers (4.78 g, 92%).

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3961–3973
3969
4.2.4.1. 2-(3-(3-Methoxy-5-methylphenyl)-4-(2-phenylpyridin-
4-yl)-1H-pyrazol-1-yl)acetonitrile (9a). Preparative TLC separa-
tion was carried out using (ethyl acetate–hexane 1:2 v/v). Yield:
4.2.4.6. 2-(4-(2-(4-(Dimethylamino)phenyl)pyridin-4-yl)-3-(3-
methoxy-5-methylphenyl)-1H-pyrazol-1-yl)acetonitrile (9f). Pre-
parative TLC separation was carried out using (ethyl acetate–
(166 mg, 74%); mp 55–56 °C; IR
m
/cmÀ1: 3428, 2924, 1596, 1447,
hexane 1:3 v/v). Yield: (197 mg, 79%); mp 98–99 °C; IR m :
/cmÀ1
1418, 1166, 1154, 1060, 850, 777, 697; 1H NMR (CDCl3): d 2.32
(s, 3H), 3.71 (s, 3H), 5.14 (s, 2H), 6.79 (s, 1H), 6.84 (s, 1H), 6.96
(s, 1H), 7.13 (d, J = 4.9 Hz, 1H), 7.41–7.48 (m, 3H), 7.65 (s, 1H),
7.79 (s, 1H), 7.88 (d, J = 6.9 Hz, 2H), 8.61 (d, J = 5.1 Hz, 1H); 13C
NMR (CDCl3): 21.51, 39.86, 55.25, 110.90, 113.61, 115.61, 119.78,
120.42, 121.24, 121.76, 126.89, 128.78, 129.11, 130.05, 132.90,
139.19, 139.98, 140.77, 149.89, 151.46, 157.75, 159.75.
3409, 2934, 1606, 1531, 1441, 1194, 1167, 1060, 820, 462; 1H
NMR (CDCl3): d 2.31 (s, 3H), 3.01 (s, 6H), 3.70 (s, 3H), 5.10 (s,
2H), 6.75–6.78 (m, 3H), 6.84 (s, 1H), 6.97–7.00 (m, 2H), 7.56 (s,
1H), 7.72 (s, 1H), 7.80 (d, J = 8.8 Hz, 2H), 8.52 (d, J = 5.0 Hz, 1H);
13C NMR (CDCl3): 21.53, 39.79, 40.34, 55.25, 110.68, 112.16,
113.70, 115.67, 118.33, 119.88, 120.69, 121.74, 126.77, 127.72,
130.10, 132.99, 139.89, 140.46, 149.55, 151.13, 151.35, 157.78,
159.68.
4.2.4.2. 2-(4-(2-(3-Acetylphenyl)pyridin-4-yl)-3-(3-methoxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (9b). Preparative
TLC separation was carried out using (ethyl acetate–hexane 1:1
4.2.4.7. 2-(3-(3-Methoxy-5-methylphenyl)-4-(2-(4-phenoxyphe-
nyl)pyridin-4-yl)-1H-pyrazol-1-yl)acetonitrile (9g). Preparative
TLC separation was carried out using (ethyl acetate–hexane 1:3
v/v). Yield: (197 mg, 79%); mp 73–74 °C; IR m
/cmÀ1: 3436, 2937,
1683, 1607, 1423, 1247, 1167, 1061, 804, 695, 590; 1H NMR
(CDCl3): d 2.27 (s, 3H), 2.60 (s, 3H), 3.67 (s, 3H), 5.19 (s, 2H), 6.76
(s, 1H), 6.80 (s, 1H), 6.91 (s, 1H), 7.13 (s, 1H), 7.49 (br s, 1H), 7.64
(s, 1H), 7.85 (s, 1H), 7.95 (d, J = 5.4 Hz, 1H), 8.04 (d, J = 5.7 Hz,
1H), 8.39 (s, 1H), 8.56 (s, 1H); 13C NMR (CDCl3): 21.51, 26.78,
39.89, 55.23, 111.03, 113.93, 115.51, 119.76, 119.90, 121.63,
121.77, 126.71, 128.60, 128.76, 129.11, 130.54, 131.45, 132.06,
132.92, 137.52, 139.61, 140.00, 141.09, 149.91, 151.38, 156.47,
159.69, 198.16.
v/v). Yield: (201 mg, 72%); mp 80–81 °C; IR m
/cmÀ1: 3419, 3064,
2937, 2835, 1596, 1510, 1490, 1238, 1167, 1059, 871, 840, 692;
1H NMR (CDCl3): d 2.31 (s, 3H), 3.72 (s, 3H), 5.18 (s, 2H), 6.79 (s,
1H), 6.83 (s, 1H), 6.94 (s, 1H), 7.04 (d, 8.0 Hz, 4H), 7.13–7.22 (m,
2H), 7.37 (t, J = 7.3 Hz, 2H), 7.64 (s, 1H), 7.79 (d, J = 8.3 Hz, 2H),
7.94 (s, 1H), 8.57 (d, J = 5.0 Hz, 1H); 13C NMR (CDCl3): 21.53,
39.89, 55.28, 111.05, 113.70, 115.65, 118.72, 119.42, 119.67,
119.74, 121.03, 121.80, 123.92, 128.57, 129.81, 129.94, 130.86,
132.40, 132.82, 140.09, 142.48, 148.36, 151.65, 156.10, 156.43,
158.98, 159.81.
4.2.4.3. 2-(4-(2-(4-Acetylphenyl)pyridin-4-yl)-3-(3-methoxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (9c). Preparative
TLC separation was carried out using (ethyl acetate–hexane 1:2
4.2.4.8. 2-(5-(3-Methoxy-5-methylphenyl)-4-(2-phenylpyridin-
4-yl)-1H-pyrazol-1-yl)acetonitrile (10a). Preparative TLC sepa-
ration was carried out using (ethyl acetate–hexane 1:2 v/v). Yield:
v/v). Yield: (170 mg, 68%); mp 99–100 °C; IR m
/cmÀ1: 3463, 2924,
(70 mg, 62%); mp 51–52 °C; IR m
/cmÀ1: 3428, 2964, 1596, 1445,
1678, 1667, 1603, 1270, 1166, 1070, 849, 467; 1H NMR (CDCl3): d
2.32 (s, 3H), 2.65 (s, 3H), 3.72 (s, 3H), 5.20 (s, 2H), 6.79 (s, 1H),
6.82 (s, 1H), 6.94 (s, 1H), 7.20 (d, J = 5.1 Hz, 1H), 7.69 (s, 1H), 7.88
(s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 8.63 (d,
J = 5.1 Hz, 1H); 13C NMR (CDCl3): 21.54, 26.80, 39.95, 55.27,
110.97, 113.53, 115.58, 120.20, 121.74, 121.88, 127.00, 128.82,
130.02, 132.77, 137.19, 140.06, 140.95, 143.39, 150.15, 151.54,
156.35, 159.77, 197.91.
1414, 1386, 1263, 1155, 1072, 1037, 843, 801, 777, 694; 1H NMR
(CDCl3): d 2.41 (s, 3H), 3.81 (s, 3H), 4.95 (s, 2H), 6.76 (s, 1H), 6.82
(s, 1H), 6.95 (s, 1H), 7.06 (d, J = 4.5 Hz, 1H), 7.41–7.44 (m, 3H),
7.58 (s, 1H), 7.80 (d, J = 6.3 Hz, 2H), 8.01 (s, 1H), 8.53 (d,
J = 4.8 Hz, 1H); 13C NMR (CDCl3): 21.57, 37.76, 55.49, 112.31,
114.07, 116.90, 118.29, 119.60, 119.70, 122.75, 126.83, 128.71,
129.03, 129.11, 139.21, 139.61, 140.45, 141.47, 141.89, 149.88,
157.70, 160.51.
4.2.4.4. N-(3-(4-(1-(Cyanomethyl)-3-(3-methoxy-5-methylphe
nyl)-1H-pyrazol-4-yl)pyridin-2-yl)phenyl)acetamide (9d). Pre-
parative TLC separation was carried out using (ethyl acetate–hex-
4.2.4.9. 2-(4-(2-(3-Acetylphenyl)pyridin-4-yl)-5-(3-methoxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (10b). Preparative
TLC separation was carried out using (ethyl acetate–hexane 1:2
m :
/cmÀ1
v/v). Yield: (96 mg, 77%); mp 65–66 °C; IR m
/cmÀ1: 3440, 2938,
ane 1:2 v/v). Yield: (194 mg, 75%); mp 103–105 °C; IR
1684, 1606, 1420, 1358, 1248, 1230, 1156, 846, 804, 689, 589; 1H
NMR (CDCl3): d 2.39 (s, 3H), 2.62 (s, 3H), 3.79 (s, 3H), 4.96 (s,
2H), 6.74 (s, 1H), 6.80 (s, 1H), 6.95 (s, 1H), 7.10 (br s, 1H), 7.50 (t,
J = 7.5 Hz, 1H), 7.56 (s, 1H), 7.94–8.00 (m, 3H), 8.29 (s, 1H), 8.52
(d, J = 4.2 Hz, 1H); 13C NMR (CDCl3): 21.59, 26.78, 37.77, 55.78,
112.33, 114.14, 116.93, 118.18, 119.32, 120.14, 122.64, 126.60,
128.68, 129.01, 131.39, 137.53, 139.59, 139.72, 140.66, 141.54,
141.93, 150.05, 156.54, 160.45, 197.89.
3281, 3083, 2937, 1674, 1595, 1549, 1453, 1321, 1167, 1062,
848, 797, 699; 1H NMR (CDCl3): d 2.13 (s, 3H), 2.47 (s, 3H), 3.68
(s, 3H), 5.14 (s, 2H), 6.74 (s, 1H), 6.78 (s, 1H), 6.89 (s, 1H), 7.03
(d, J = 4.7 Hz, 1H), 7.28–7.34 (m, 1H), 7.51–7.57 (m, 2H), 7.62 (d,
J = 7.8 Hz, 1H), 7.71 (s, 1H), 8.09 (s, 1H), 8.27 (s, 1H), 8.47 (d,
J = 4.8 Hz, 1H); 13C NMR (CDCl3): 21.48, 24.44, 39.80, 55.27,
110.92, 113.82, 115.56, 118.46, 119.72, 120.02, 120.70, 121.51,
121.82, 122.60, 129.42, 130.37, 132.93, 138.68, 139.83, 139.99,
140.96, 149.59, 151.33, 157.11, 159.65, 169.06.
4.2.4.10. 2-(4-(2-(4-Acetylphenyl)pyridin-4-yl)-5-(3-methoxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (10c). Preparative
TLC separation was carried out using (ethyl acetate–hexane 1:2 v/
4.2.4.5. 4-(4-(1-(Cyanomethyl)-3-(3-methoxy-5-methylphenyl)-
1H-pyrazol-4-yl)pyridin-2-yl)benzonitrile (9e). Preparative TLC
separation was carried out using (ethyl acetate–hexane 1:2 v/v).
v). Yield: (93 mg, 75%); mp 156–157 °C; IR m
/cmÀ1: 3473, 2939,
Yield: (196 mg, 82%); mp 83–84 °C; IR m
/cmÀ1: 3421, 2938, 2227,
1676, 1604, 1594, 1423, 1267, 1172, 839, 471; 1H NMR (CDCl3): d
2.42 (s, 3H), 2.65 (s, 3H), 3.82 (s, 3H), 4.97 (s, 2H), 6.75 (s, 1H),
6.82 (s, 1H), 6.96 (s, 1H), 7.12 (d, J = 4.9 Hz, 1H), 7.60 (s, 1H), 7.87
(d, J = 8.1 Hz, 2H), 7.99–8.02 (m, 3H), 8.56 (d, J = 5.1 Hz, 1H); 13C
NMR (CDCl3): 21.60, 26.78, 37.78, 55.52, 112.34, 114.02, 116.86,
118.62, 119.33, 120.38, 122.67, 126.90, 128.76, 129.01, 137.12,
139.56, 140.61, 141.58, 141.96, 143.49, 150.20, 156.29, 160.54,
197.88.
1604, 1546, 1451, 1167, 1059, 842; 1H NMR (CDCl3): d 2.31 (s,
3H), 3.71 (s, 3H), 5.19 (s, 2H), 6.79 (s, 1H), 6.82 (s, 1H), 6.93 (s,
1H), 7.22 (d, J = 4.6 Hz, 1H), 7.66 (s, 1H), 7.71 (d, J = 8.3 Hz, 2H),
7.87 (s, 1H), 7.97 (d, J = 8.3 Hz, 2H), 8.63 (d, J = 5.1 Hz, 1H); 13C
NMR (CDCl3): 21.50, 39.96, 55.27, 111.11, 112.47, 113.57, 115.51,
118.78, 119.97, 120.10, 121.75, 122.22, 127.38, 130.09, 132.53,
132.80, 140.06, 141.17, 143.30, 150.26, 151.51, 155.44, 159.81.

3970
I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3961–3973
4.2.4.11. N-(3-(4-(1-(Cyanomethyl)-5-(3-methoxy-5-methylphenyl)-
1H-pyrazol-4-yl)pyridin-2-yl)phenyl)acetamide (10d). Pre-
parative TLC separation was carried out using (ethyl acetate–
4.2.5.1. 2-(3-(3-Hydroxy-5-methylphenyl)-4-(2-phenylpyridin-
4-yl)-1H-pyrazol-1-yl)acetonitrile (11a). Column chromatogra-
phy (silica gel, ethyl acetate–hexane 1:1 v/v). Yield: (31 mg,
hexane 1:1 v/v). Yield: (81 mg, 63%); mp 97–98 °C; IR
m
/cmÀ1
:
70%); mp 110–111 °C; IR m
/cmÀ1: 3433, 2922, 1609, 1547, 1448,
3302, 3080, 2936, 1674, 1595, 1551, 1465, 1374, 1322, 846,
794, 698; 1H NMR (CDCl3): d 2.15 (s, 3H), 2.39 (s, 3H), 3.81 (s,
3H), 4.95 (s, 2H), 6.74 (s, 1H), 6.79 (s, 1H), 6.93 (s, 1H), 7.01 (d,
J = 4.7 Hz, 1H), 7.32–7.37 (m, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.57
(s, 1H), 7.75–7.77 (m, 2H), 7.98 (s, 1H), 7.99 (s, 1H), 8.46 (d,
J = 4.8 Hz, 1H); 13C NMR (CDCl3): 21.56, 24.51, 37.76, 55.52,
112.24, 114.07, 117.00, 118.36, 119.50, 119.89, 120.67, 122.39,
122.78, 129.08, 129.39, 138.57, 139.60, 139.92, 140.49, 141.52,
141.88, 149.81, 157.12, 160.45, 168.55.
1168, 852, 777, 699; 1H NMR (CDCl3): d 2.31 (s, 3H), 5.09 (s, 2H),
6.54 (s, 1H), 6.73 (s, 1H), 6.98–7.03 (m, 2H), 7.28–7.32 (m, 3H),
7.51 (s, 1H), 7.62–7.65 (m, 3H), 8.30 (s, 1H); 13C NMR (CDCl3):
21.51, 39.82, 112.71, 113.60, 117.21, 119.73, 120.33, 120.77,
121.35, 127.14, 128.79, 129.29, 130.37, 132.21, 138.30, 140.62,
141.52, 148.83, 149.41, 150.24, 151.30, 156.81, 157.68.
4.2.5.2. 2-(4-(2-(3-Acetylphenyl)pyridin-4-yl)-3-(3-hydroxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (11b). Column chro-
matography (silica gel, ethyl acetate–hexane 1:1 v/v). Yield: (30 mg,
61%); mp 84–85 °C; IR m
/cmÀ1: 3408, 3059, 2924, 1683, 1607, 1425,
4.2.4.12. 4-(4-(1-(Cyanomethyl)-5-(3-methoxy-5-methylphenyl)
-1H-pyrazol-4-yl)pyridin-2-yl)benzonitrile (10e). Preparative
TLC separation was carried out using (ethyl acetate–hexane 1:2
1358, 1299, 1250, 1166, 804, 725, 694, 542; 1H NMR (CDCl3): d 2.29
(s, 3H), 2.49 (s, 3H), 5.16 (s, 2H), 6.59 (s, 1H), 6.73 (s, 1H), 6.97 (s,
1H), 7.11 (d, J = 4.8 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.53 (s, 1H),
7.77 (s, 1H), 7.83 (t, J = 7.2 Hz, 2H), 8.05 (s, 1H), 8.35 (d, J = 5.1 Hz,
1H).
v/v). Yield: (92 mg, 77%); mp 161–162 °C; IR
m
/cmÀ1: 3420,
2941, 2218, 1605, 1467, 1386, 1155, 855, 834; 1H NMR (CDCl3):
d 2.42 (s, 3H), 3.85 (s, 3H), 4.96 (s, 2H), 6.75 (s, 1H), 6.82 (s, 1H),
6.96 (s, 1H), 7.13 (d, J = 4.9 Hz, 1H), 7.58 (s, 1H), 7.71 (d,
J = 8.1 Hz, 2H), 7.91 (d, J = 8.1 Hz, 2H), 8.02 (s, 1H), 8.56 (d,
J = 5.1 Hz, 1H); 13C NMR (CDCl3): 21.58, 37.80, 55.51, 112.40,
112.47, 113.95, 116.85, 118.53, 118.73, 119.20, 120.71, 122.65,
127.30, 129.01, 132.48, 139.51, 140.82, 141.60, 142.00, 143.39,
150.32, 155.46, 160.59.
4.2.5.3. 4-(4-(1-(Cyanomethyl)-3-(3-hydroxy-5-methylphenyl)-
1H-pyrazol-4-yl)pyridin-2-yl)benzonitrile (11c). Column chro-
matography (silica gel, ethyl acetate–hexane 1:1 v/v). Yield:
(38 mg, 81%); mp 147–149 °C; IR
m
/cmÀ1: 3401, 2229, 1605,
1547, 1449, 1300, 1167, 843; 1H NMR (CDCl3): d 2.27 (s, 3H),
5.18 (s, 2H), 6.66 (s, 1H), 6.71 (s, 1H), 6.92 (s, 1H), 7.19 (d,
J = 4.9 Hz, 1H), 7.59–7.64 (m, 3H), 7.80–7.84 (m, 3H), 8.51 (d,
J = 5.1 Hz, 1H); 13C NMR (CDCl3): 21.37, 39.94, 112.47, 112.71,
113.50, 117.05, 118.58, 119.64, 120.60, 121.23, 122.22, 127.53,
130.27, 132.51, 132.59, 140.57, 141.52, 142.90, 149.56, 151.42,
155.48, 156.43.
4.2.4.13. 2-(4-(2-(4-(Dimethylamino)phenyl)pyridin-4-yl)-5-(3-
methoxy-5-methylphenyl)-1H-pyrazol-1-yl)aceto-nitrile
(10f). Preparative TLC separation was carried out using (ethyl
acetate–hexane 1:3 v/v). Yield: (62 mg, 50%); mp 150–151 °C; IR
m
/cmÀ1: 3443, 2920, 1600, 1531, 1417, 1371, 1200, 1172, 820,
805; 1H NMR (CDCl3): d 2.41 (s, 3H), 3.02 (s, 6H), 3.81 (s, 3H),
4.95 (s, 2H), 6.76–6.81 (m, 4H), 6.91–6.93 (m, 2H), 7.50 (s, 1H),
7.73 (d, J = 8.6 Hz, 2H), 8.00 (s, 1H), 8.46 (d, J = 5.1 Hz, 1H); 13C
NMR (CDCl3): 21.58, 37.74, 40.38, 55.48, 112.12, 114.12, 116.82,
116.95, 118.33, 119.98, 122.82, 127.04, 127.63, 129.23, 139.68,
139.94, 141.33, 141.70, 149.68, 151.05, 157.84, 160.42.
4.2.5.4. 2-(4-(2-(4-(Dimethylamino)phenyl)pyridin-4-yl)-3-(3-
hydroxy-5-methylphenyl)-1H-pyrazol-1-yl)acetonitrile
(11d). Column chromatography (silica gel, ethyl acetate–hexane
1:2 v/v). Yield: (37 mg, 75%); mp 138–139 °C; IR m
/cmÀ1: 3420,
2922, 1607, 1531, 1444, 1167, 819; 1H NMR (CDCl3): d 2.29 (s,
3H), 2.92 (s, 6H), 4.99 (s, 2H), 6.60–6.62 (m, 3H), 6.73 (s, 1H),
6.83 (d, J = 4.3 Hz, 1H), 6.96 (s, 1H), 7.41 (s, 1H), 7.54–7.57 (m,
3H), 8.11 (d, J = 4.8 Hz, 1H); 13C NMR (CDCl3): 21.45, 39.58,
40.22, 112.14, 112.87, 113.69, 117.15, 119.17, 119.94, 120.39,
125.73, 128.03, 130.51, 132.53, 140.40, 141.31, 148.21, 151.12,
151.27, 156.95, 157.46.
4.2.4.14.
2-(5-(3-Methoxy-5-methylphenyl)-4-(2-(4-phenoxy-
phenyl)pyridin-4-yl)-1H-pyrazol-1-yl)acetonitrile (10g). Pre-
parative TLC separation was carried out using (ethyl acetate–
hexane 1:3 v/v). Yield: (96 mg, 69%); mp 69–70 °C; IR
m :
/cmÀ1
3037, 2937, 2835, 1595, 1489, 1423, 1237, 1167, 838, 691; 1H NMR
(CDCl3): d 2.41 (s, 3H), 3.81 (s, 3H), 4.96 (s, 2H), 6.76 (s, 1H), 6.82
(s, 1H), 6.95 (s, 1H), 7.05 (d, 7.3 Hz, 5H), 7.14 (t, J = 7.1 Hz, 1H), 7.36
(t, J = 7.1 Hz, 2H), 7.56 (s, 1H), 7.78 (d, J = 8.0 Hz, 2H), 8.01 (s, 1H),
8.51 (d, J = 3.8 Hz, 1H); 13C NMR (CDCl3): 21.59, 37.78, 55.50,
112.34, 114.14, 116.90, 117.87, 118.75, 119.23, 119.46, 122.73,
123.69, 128.38, 129.07, 129.88, 133.77, 139.63, 140.88, 141.50,
141.99, 149.50, 156.74, 156.77, 158.46, 160.43.
4.2.5.5. 2-(3-(3-Hydroxy-5-methylphenyl)-4-(2-(4-phenoxyphe-
nyl)pyridin-4-yl)-1H-pyrazol-1-yl)acetonitrile (11e). Column
chromatography (silica gel, ethyl acetate–hexane 1:2 v/v). Yield:
(34 mg, 62%); mp 104–105 °C; IR
m
/cmÀ1: 3041, 2919, 1608,
1490, 1448, 1237, 1167, 845, 762, 691; 1H NMR (CDCl3): d 2.29
(s, 3H), 5.09 (s, 2H), 6.62 (s, 1H), 6.71 (s, 1H), 6.90–7.01 (m, 6H),
7.13 (t, 7.2 Hz, 1H), 7.34 (t, J = 7.7 Hz, 2H), 7.49 (s, 1H), 7.58 (d,
J = 8.4 Hz, 2H), 7.68 (s, 1H), 8.28 (d, J = 4.8 Hz, 1H); 13C NMR
(CDCl3): 21.47, 39.81, 112.66, 117.23, 118.49, 119.43, 119.83,
120.32, 120.52, 121.02, 123.84, 128.67, 129.90, 130.37, 132.36,
133.15, 140.57, 141.54, 148.89, 151.23, 156.42, 156.72, 157.08,
158.56.
4.2.5. General procedure for the synthesis of compounds 11a–e
and 12a–e
To a solution of the starting methoxy compound 9a–g or 10a–g
(0.12 mmol) in dichloromethane (4 mL) was added borontrifluo-
ride–methyl sulfide complex (0.126 mL, 1.2 mmol) dropwise at
room temperature and under N2 atmosphere. The resulting sus-
pension was stirred for 24 h, and then the mixture was concen-
trated under vacuum. The residue was partitioned between ethyl
acetate (100 mL) and brine (50 mL). The organic layer was sepa-
rated and dried over anhydrous MgSO4, then evaporated under
vacuum. The residue was then purified by column chromatography
to yield the pure hydroxyl product.
4.2.5.6.
2-(4-(2-(3-Acetylphenyl)pyridin-4-yl)-5-(3-hydroxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (12a). Column chro-
matography (silica gel, ethyl acetate–hexane 1:1 v/v). Yield: (32 mg,
65%); mp 123–125 °C; IR m
/cmÀ1: 3429, 2923, 1684, 1608, 1422,
1360, 1307, 1234, 851, 692, 594; 1H NMR (CDCl3): d 2.34 (s, 3H),
2.58 (s, 3H), 4.95 (s, 2H), 6.37 (s, 1H), 6.69 (s, 1H), 6.87 (s, 1H),
7.00 (d, J = 5.0 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.53 (s, 1H), 7.89–
7.95 (m, 3H), 8.19 (s, 1H), 8.37 (d, J = 5.1 Hz, 1H), 9.51 (s, 1H); 13C

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3961–3973
3971
NMR (CDCl3): 21.51, 26.80, 37.66, 114.02, 114.09, 118.73, 118.95,
119.21, 120.55, 121.27, 126.96, 128.32, 129.03, 129.23, 131.81,
137.26, 139.15, 139.69, 141.50, 141.72, 142.24, 149.21, 156.63,
157.89, 198.40.
55.26, 111.11, 120.06, 122.73, 123.65, 131.09, 132.09, 139.48,
139.62, 148.43, 149.70, 159.49, 167.06.
4.2.7. 2-Chloro-5-(3-(3-methoxy-5-methylphenyl)-1H-pyrazol-
4-yl)pyridine (14)
A mixture of compound 13 (7.28 g, 26.5 mmol) and N,N-
dimethylformamide dimethylacetal (24 mL, 204 mmol) was
heated under reflux in an oil bath for 12 h. The excess unreacted
N,N-dimethylformamide dimethylacetal was removed under vac-
uum, and the residue was dissolved in absolute ethanol
(120 mL). Hydrazine monohydrate (2.65 g, 53 mmol) was added
to the ethanolic solution, and the mixture was stirred at room tem-
perature for 4 h, then the solvent was removed under vacuum. The
crude product was purified by column chromatography (silica gel,
ethyl acetate–hexane 1:2 v/v) to yield pure 14 (4.92 g, 62%) as red
4.2.5.7.
2-(4-(2-(4-Acetylphenyl)pyridin-4-yl)-5-(3-hydroxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (12b). Column chro-
matography (silica gel, ethyl acetate–hexane 1:1 v/v). Yield: (21 mg,
42%); mp 215–216 °C; IR m
/cmÀ1: 3107, 2993, 2926, 1672, 1604,
1404, 1302, 1268, 1166, 836; 1H NMR (CDCl3): d 2.37 (s, 3H), 2.61
(s, 3H), 4.95 (s, 2H), 6.56 (s, 1H), 6.73 (s, 1H), 6.91 (s, 1H), 7.11 (s,
1H), 7.51 (s, 1H), 7.75 (d, J = 7.6 Hz, 2H), 7.92–7.98 (m, 3H), 8.46
(s, 1H).
4.2.5.8. N-(3-(4-(1-(Cyanomethyl)-5-(3-hydroxy-5-methylphen
yl)-1H-pyrazol-4-yl)pyridin-2-yl)phenyl)acetamide (12c). Col-
umn chromatography (silica gel, ethyl acetate). Yield: (28 mg,
sticky oil; IR m
/cmÀ1: 3161, 2938, 1595, 1454, 1285, 1155, 1113,
1062, 837; 1H NMR (CDCl3): d 2.29 (s, 3H), 3.71 (s, 3H), 6.74 (s,
2H), 6.82 (s, 1H), 7.25 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H),
7.68 (s, 1H), 8.38 (s, 1H), 12.25 (s, 1H); 13C NMR (CDCl3): 21.53,
55.50, 110.99, 114.96, 115.19, 121.30, 123.93, 128.18, 131.31,
138.10, 140.29, 148.56, 149.33, 159.90.
55%); mp 142–143 °C; IR m
/cmÀ1: 3380, 2927, 2857, 1671, 1605,
1550, 1452, 1311, 1164, 850, 795, 699; 1H NMR (CD3OD): d 2.16
(s, 3H), 2.36 (s, 3H), 5.14 (s, 2H), 6.66 (s, 1H), 6.75 (s, 1H), 6.89
(s, 1H), 7.23 (d, J = 5.1 Hz, 1H), 7.36 (d, J = 3.9 Hz, 2H), 7.65 (br s,
2H), 7.97 (s, 1H), 8.17 (s, 1H), 8.41 (d, J = 5.2 Hz, 1H); 13C NMR
(CD3OD): 20.07, 22.46, 37.03, 113.48, 114.37, 117.72, 118.52,
118.69, 118.76, 119.85, 120.61, 121.12, 122.30, 128.80, 129.11,
139.00, 139.26, 139.62, 141.44, 141.61, 142.38, 149.04, 157.39,
158.40, 170.35.
4.2.8. General procedure for the synthesis of compounds 15 and
16
A mixture of compound 14 (4.6 g, 15.35 mmol) and ground
K2CO3 (10.6 g, 76.73 mmol) in acetone (100 mL) was stirred under
reflux for 2 h. To the reaction mixture was added iodoacetonitrile
(1.34 mL, 18.42 mmol) dropwise. Heating and stirring were main-
tained for two more hours, after which acetone was removed un-
der vacuum, and the residue was partitioned between water
(200 mL) and ethyl acetate (200 mL). The aqueous layer was fur-
ther extracted with ethyl acetate (100 mL), and then the combined
organics were dried over anhydrous MgSO4 and evaporated under
vacuum. The crude product was purified by column chromatogra-
phy (silica gel, ethyl acetate–hexane 2:3 v/v) to give an oily mix-
ture of compounds 15 and 16 in an approximate ration of (2:1),
respectively, that has been used in the next step without separa-
tion of the two isomers (Yield: 4.16 g, 80%).
4.2.5.9. 4-(4-(1-(Cyanomethyl)-5-(3-hydroxy-5-methylphenyl)-
1H-pyrazol-4-yl)pyridin-2-yl)benzonitrile (12d). Column chro-
matography (silica gel, ethyl acetate–hexane 1:1 v/v). Yield:
(34 mg, 72%); mp 225–227 °C; IR
m
/cmÀ1: 3065, 2993, 2219,
1607, 1460, 1390, 1305, 1166, 1105, 839; 1H NMR (CDCl3): d 2.37
(s, 3H), 4.96 (s, 2H), 6.61 (s, 1H), 6.75 (s, 1H), 6.90 (s, 1H), 7.14
(d, J = 5.1 Hz, 1H), 7.54 (s, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.83 (d,
J = 8.4 Hz, 2H), 8.00 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H).
4.2.5.10.
2-(5-(3-Hydroxy-5-methylphenyl)-4-(2-(4-phenoxy-
phenyl)pyridin-4-yl)-1H-pyrazol-1-yl)acetonitrile (12e). Col-
umn chromatography (silica gel, ethyl acetate–hexane 1:2 v/v).
Yield: (34 mg, 61%); mp 101–102 °C; IR
m
/cmÀ1: 3041, 2923,
1607, 1490, 1425, 1238, 1167, 870, 836, 691, 461; 1H NMR (CDCl3):
d 2.36 (s, 3H), 4.91 (s, 2H), 6.45 (s, 1H), 6.72 (s, 1H), 6.86 (s, 1H),
6.94–7.03 (m, 5H), 7.15 (t, 7.4 Hz, 1H), 7.36 (t, J = 8.0 Hz, 2H),
7.49 (s, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.95 (s, 1H), 8.32 (d,
J = 5.3 Hz, 1H), 9.11 (s, 1H); 13C NMR (CDCl3): 21.47, 37.67,
113.97, 114.03, 118.68, 118.88, 118.98, 119.28, 119.58, 121.62,
123.80, 128.62, 129.90, 133.57, 139.59, 141.19, 141.71, 142.15,
149.05, 156.53, 157.20, 157.79, 158.47.
4.2.8.1. 2-(4-(6-Chloropyridin-3-yl)-3-(3-methoxy-5-methylphe-
nyl)-1H-pyrazol-1-yl)acetonitrile (15). IR
m
/cmÀ1: 2964, 2835,
1595, 1544, 1464, 1434, 1262, 1166, 1100, 1061, 1031, 803; 1H
NMR (CDCl3): d 2.28 (s, 3H), 3.66 (s, 3H), 5.16 (s, 2H), 6.71 (s,
2H), 6.84 (s, 1H), 7.24 (d, J = 8.3 Hz, 1H), 7.49 (dd, J = 2.3, 5.9 Hz,
1H), 7.70 (s, 1H), 8.32 (s, 1H); 13C NMR (CDCl3): 21.57, 39.89,
55.23, 110.80, 113.76, 115.28, 117.76, 121.47, 124.02, 127.24,
129.92, 132.57, 138.58, 140.12, 148.71, 149.92, 151.17, 159.69,
162.33.
4.2.6. 2-(2-Chloropyridin-4-yl)-1-(3-methoxy-5-methylphenyl)
ethanone (13)
4.2.9. General procedure for the synthesis of compounds 17a–c
and 18a–c
The same procedure used for the preparation of compounds 9a–
g and 10a–g was applied here starting with an un-isolated mixture
of compounds 15 and 16.
To a solution of the ester 4 (9 g, 50 mmol) and 2-chloro-5-meth-
ylpyridine (7.0 g, 55 mmol) in THF (90 mL) was added dropwise
lithium bis(trimethylsilyl)amide 1 M/THF (75 mL, 75 mmol) at
0 °C under N2 atmosphere. The mixture was warmed to room tem-
perature and stirred for 18 h. Saturated aqueous ammonium chlo-
ride solution (150 mL) was added to the mixture, followed by
extraction with ethyl acetate (300 mL Â 2). The combined organic
layers were washed with saturated NaCl, dried over anhydrous
MgSO4 and evaporated under vacuum to yield crude 13 (7.58 g,
55%) as a sticky solid which was used for the next step without fur-
4.2.9.1. 2-(3-(3-Methoxy-5-methylphenyl)-4-(6-(pyridin-3-yl)pyri-
din-3-yl)-1H-pyrazol-1-yl)acetonitrile (17a). Preparative TLC sep-
aration was carried out using (ethyl acetate–hexane 3:1 v/v). Yield:
(124 mg, 55%); mp 66–67 °C; IR m
/cmÀ1: 2936, 2835, 1594, 1562,
1464, 1440 1416, 1292, 1166, 1062, 848, 808, 707; 1H NMR (CDCl3):
d 2.27 (s, 3H), 3.67 (s, 3H), 5.16 (s, 2H), 6.71 (s, 1H), 6.76 (s, 1H), 6.90
(s, 1H), 7.36–7.40 (m, 1H), 7.62–7.69 (m, 2H), 7.73 (s, 1H), 8.29 (d,
J = 7.7 Hz, 1H), 8.62–8.65 (m, 2H), 9.19 (s, 1H); 13C NMR (CDCl3):
21.51, 39.85, 55.18, 110.91, 113.73, 115.30, 118.78, 119.96, 121.57,
ther purification; mp <40 °C; IR m
/cmÀ1: 3390, 2964, 1660, 1596,
1263, 1103, 1064, 802; 1H NMR (CDCl3): d 2.29 (s, 3H), 3.74 (s,
3H), 3.83 (s, 2H), 6.84 (s, 1H), 7.14 (s, 1H), 7.29 (s, 1H), 7.35–7.39
(m, 2H), 8.12 (s, 1H); 13C NMR (CDCl3): 17.55, 21.23, 52.00,

3972
I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3961–3973
123.62, 127.41, 129.76, 132.92, 134.10, 134.35, 136.58, 139.97,
148.09, 149.28, 149.93, 151.30, 153.13, 159.71.
4.2.10. General procedure for the synthesis of compounds 19a–c
and 20a,b
The same procedure used for the preparation of compounds
11a–e and 11a–e was applied here starting with the appropriate
methoxy compounds 17a–c and 18a–c.
4.2.9.2. 2-(4-(6-(2-Acetylphenyl)pyridin-3-yl)-3-(3-methoxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (17b). Preparative
TLC separation was carried out using (ethyl acetate–hexane 1:2 v/
v). Yield: (120 mg, 48%); mp 176–177 °C; IR
m
/cmÀ1: 3128, 2977,
4.2.10.1. 2-(3-(3-Hydroxy-5-methylphenyl)-4-(6-(pyridin-3-yl)pyri-
din-3-yl)-1H-pyrazol-1-yl)acetonitrile (19a). Column chromatogra-
phy (silica, acetone–ethyl acetate 1:50 v/v). Yield: (27 mg, 61%); mp
2922, 1681, 1594, 1551, 1436, 1418, 1170, 1154, 1066, 843, 762,
594; 1H NMR (CDCl3): d 2.25 (s, 3H), 2.28 (s, 3H), 3.70 (s, 3H), 5.14
(s, 2H), 6.72 (s, 1H), 6.76 (s, 1H), 6.89 (s, 1H), 7.44–7.65 (m, 6H),
7.70 (s, 1H), 8.54 (s, 1H); 13C NMR (CDCl3): 21.51, 30.58, 39.79,
55.19, 110.80, 113.82, 115.30, 118.66, 121.54, 121.89, 127.06,
127.63, 128.80, 128.96, 130.02, 130.36, 132.92, 136.56, 138.15,
139.93, 141.68, 148.55, 151.23, 155.76, 159.71, 204.39.
258–259 °C; IR m
/cmÀ1: 3429, 3121, 2993, 2948, 2646, 1596, 1567,
1419, 1178, 859, 806, 698; 1H NMR (DMSO-d6): d 2.31 (s, 3H), 5.60
(s, 2H), 6.59 (s, 2H), 6.73 (s, 1H), 7.49–7.54 (m, 1H), 7.74 (d,
J = 7.5 Hz, 1H), 8.06 (d, J = 8.2 Hz, 1H), 8.25 (s, 1H), 8.45 (d, J = 7.8 Hz,
1H), 8.61 (s, 2H), 9.29 (s, 1H), 9.39 (s, 1H); 13C NMR (DMSO-d6):
21.53, 112.66, 116.34, 116.37, 117.27, 120.01, 120.71, 124.29, 128.23,
132.36, 133.80, 134.03, 134.16, 136.89, 139.61, 148.07, 149.12,
150.28, 150.32, 152.24, 157.74.
4.2.9.3. 2-(4-(6-(3-Acetylphenyl)pyridin-3-yl)-3-(3-methoxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (17c). Preparative
TLC separation was carried out using (ethyl acetate–hexane 1:2
4.2.10.2. 2-(4-(6-(2-Acetylphenyl)pyridin-3-yl)-3-(3-hydroxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (19b). Column chro-
matography (silica, ethyl acetate–hexane 1:1 v/v). Yield: (38 mg,
v/v). Yield: (190 mg, 76%); mp 73–74 °C; IR m
/cmÀ1: 2937, 1683,
1595, 1564, 1423, 1293, 1242, 1166, 1065, 847, 802, 692; 1H
NMR (CDCl3): d 2.27 (s, 3H), 2.65 (s, 3H), 3.66 (s, 3H), 5.16 (s,
2H), 6.71 (s, 1H), 6.77 (s, 1H), 6.91 (s, 1H), 7.54 (t, J = 7.7 Hz, 1H),
7.60–7.72 (m, 3H), 8.97 (d, J = 7.5 Hz, 1H), 8.19 (d, J = 7.5 Hz, 1H),
8.58 (s, 1H), 8.63 (s, 1H); 13C NMR (CDCl3): 21.55, 26.83, 39.84,
55.19, 110.91, 113.92, 115.23, 118.77, 120.03, 121.58, 126.53,
127.18, 128.77, 129.14, 129.93, 131.22, 133.02, 136.57, 137.63,
139.31, 139.97, 149.01, 151.22, 154.56, 159.68, 198.12.
77%); mp 108–109 °C; IR m
/cmÀ1: 3116, 3059, 2924, 1684, 1596,
1436, 1298, 1165, 849, 761, 735; 1H NMR (CDCl3): d 2.24 (s, 3H),
2.39 (s, 3H), 5.13 (s, 2H), 6.39 (s, 1H), 6.58 (s, 1H), 6.99 (s, 1H),
7.46–7.66 (m, 6H), 7.70 (s, 1H), 8.46 (s, 1H); 13C NMR (CDCl3):
21.38, 29.89, 39.77, 112.53, 113.80, 116.70, 118.53, 120.26, 122.31,
127.24, 128.01, 128.85, 129.05, 129.83, 130.97, 132.38, 137.25,
138.70, 140.20, 140.65, 148.32, 151.07, 156.37.
4.2.9.4.
pyridin-3-yl)-1H-pyrazol-1-yl)acetonitrile
TLC separation was carried out using (ethyl acetate–hexane 3:1
v/v). Yield: (69 mg, 61%); mp 64–65 °C; IR
/cmÀ1: 2923, 2835,
2-(5-(3-Methoxy-5-methylphenyl)-4-(6-(pyridin-3-yl)
4.2.10.3. 2-(4-(6-(3-Acetylphenyl)pyridin-3-yl)-5-(3-hydroxy-5-
(18a). Preparative
methylphenyl)-1H-pyrazol-1-yl)acetonitrile
chromatography (silica, ethyl acetate–hexane 1:1 v/v). Yield:
(37 mg, 75%); mp 100–101 °C; IR
/cmÀ1: 3418, 3121, 1682,
(19c). Column
m
1594, 1465, 1418, 1196, 1168, 1156, 849, 811, 704; 1H NMR
(CDCl3): d 2.37 (s, 3H), 3.79 (s, 3H), 4.94 (s, 2H), 6.71 (s, 1H), 6.77
(s, 1H), 6.88 (s, 1H), 7.36–7.40 (m, 1H), 7.60–7.67 (m, 2H), 7.92
(s, 1H), 8.27 (d, J = 7.7 Hz, 1H), 8.60–8.62 (m, 2H), 9.15 (s, 1H);
13C NMR (CDCl3): 21.60, 37.81, 55.41, 112.42, 114.14, 116.71,
118.30, 120.26, 122.69, 123.62, 127.25, 128.99, 134.09, 134.43,
135.01, 139.48, 141.21, 141.39, 147.98, 148.25, 149.80, 152.64,
160.40.
m
1598, 1425, 1299, 1244, 1166, 850, 803, 693; 1H NMR (CDCl3): d
2.31 (s, 3H), 2.65 (s, 3H), 4.96 (s, 2H), 6.55 (s, 1H), 6.67 (s, 1H),
6.79 (s, 1H), 7.55–7.74 (m, 3H), 7.90 (s, 1H), 7.98 (d, J = 7.5 Hz,
1H), 8.10 (d, J = 7.3 Hz, 1H), 8.49 (br s, 2H); 13C NMR (CDCl3):
21.45, 26.81, 37.75, 113.82, 114.03, 117.46, 118.45, 121.49,
121.74, 126.91, 127.88, 128.48, 129.38, 131.48, 136.59, 137.63,
139.40, 141.53, 141.68, 146.30, 153.28, 157.69, 198.13.
4.2.9.5. 2-(4-(6-(2-Acetylphenyl)pyridin-3-yl)-5-(3-methoxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (18b). Preparative
TLC separation was carried out using (ethyl acetate–hexane 1:2
4.2.10.4. 2-(4-(6-(2-Acetylphenyl)pyridin-3-yl)-5-(3-hydroxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (20a). Column chro-
matography (silica, ethyl acetate–hexane 1:1 v/v). Yield: (21 mg,
v/v). Yield: (96 mg, 77%); mp 72–73 °C; IR m
/cmÀ1: 2924, 1689,
48%); mp 142–143 °C; IR m
/cmÀ1: 3099, 3064, 2925, 2853, 1684,
1594, 1355, 1243, 1168, 1155, 847, 760, 700, 593; 1H NMR (CDCl3):
d 2.18 (s, 3H), 2.36 (s, 3H), 3.78 (s, 3H), 4.95 (s, 2H), 6.68 (s, 1H),
6.75 (s, 1H), 6.86 (s, 1H), 7.43–7.59 (m, 6H), 7.91 (s, 1H), 8.49 (s,
1H); 13C NMR (CDCl3): 21.55, 30.49, 37.81, 55.38, 112.37, 114.78,
116.72, 118.33, 122.13, 122.67, 126.78, 127.62, 128.64, 128.92,
128.99, 130.26, 134.89, 138.22, 139.49, 141.23, 141.33, 141.58,
147.50, 155.33, 160.40, 204.23.
1597, 1356, 1303, 1250, 1160, 860, 765, 732; 1H NMR (CDCl3): d
2.23 (s, 3H), 2.27 (s, 3H), 4.92 (s, 2H), 6.45 (s, 1H), 6.62 (s, 1H),
6.71 (s, 1H), 7.46–7.63 (m, 6H), 7.88 (s, 1H), 8.41 (s, 1H); 13C NMR
(CDCl3): 21.37, 29.95, 37.78, 113.96, 114.07, 117.74, 118.48,
121.59, 122.25, 127.46, 127.52, 128.32, 128.62, 129.22, 130.69,
136.18, 137.35, 139.38, 141.22, 141.39, 141.60, 146.14, 154.62,
157.66.
4.2.10.5. 2-(4-(6-(3-Acetylphenyl)pyridin-3-yl)-3-(3-hydroxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (20b). Column chro-
matography (silica, ethyl acetate–hexane 1:1 v/v). Yield: (35 mg,
4.2.9.6. 2-(4-(6-(3-Acetylphenyl)pyridin-3-yl)-5-(3-methoxy-5-
methylphenyl)-1H-pyrazol-1-yl)acetonitrile (18c). Preparative
TLC separation was carried out using (ethyl acetate–hexane 1:2
72%); mp 103–105 °C; IR m
/cmÀ1: 3429, 2958, 2926, 1683, 1596,
v/v). Yield: (100 mg, 80%); mp 149–150 °C; IR
m
/cmÀ1: 3436,
1426, 1359, 1301, 1244, 850, 804, 700; 1H NMR (CDCl3): d 2.30 (s,
3H), 2.60 (s, 3H), 5.12 (s, 2H), 6.60 (s, 1H), 6.69 (s, 1H), 7.03 (s,
1H), 7.39–7.48 (m, 2H), 7.61–7.64 (m, 2H), 7.79 (d, J = 7.6 Hz, 1H),
7.95 (d, J = 7.5 Hz, 1H), 8.30 (s, 1H), 8.50 (s, 1H), 8.84 (s, 1H); 13C
NMR (CDCl3): 21.48, 26.78, 39.82, 112.17, 113.66, 117.03, 118.13,
120.49, 120.77, 126.74, 127.72, 129.11, 130.06, 131.26, 132.48,
137.49, 137.58, 138.19, 140.74, 147.90, 151.07, 154.30, 156.70,
198.22.
2943, 1686, 1595, 1463, 1355, 1245, 1173, 1028, 852, 810, 701;
1H NMR (CDCl3): d 2.37 (s, 3H), 2.67 (s, 3H), 3.78 (s, 3H), 4.95 (s,
2H), 6.71 (s, 1H), 6.77 (s, 1H), 6.87 (s, 1H), 7.51–7.70 (m, 3H),
7.92 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.53
(s, 1H), 8.57 (d, J = 1.2 Hz, 1H); 13C NMR (CDCl3): 21.61, 26.82,
37.80, 55.41, 112.41, 114.22, 116.66, 118.39, 120.33, 122.71,
126.53, 126.96, 128.67, 129.04, 129.09, 131.18, 135.01, 137.58,
139.36, 139.48, 141.15, 141.36, 147.96, 154.21, 160.37, 198.06.

I. M. El-Deeb, S. H. Lee / Bioorg. Med. Chem. 18 (2010) 3961–3973
3973
4.3. Cell-line screening
4.4. Enzyme screening
Cell-line screening was applied at the National Cancer Institute
(NCI), Bethesda, Maryland, USA,19 applying the following procedure.
The human tumor cell lines of the cancer screening panel are grown
in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM
Kinase assays were performed at Reaction Biology Corporation
using the ‘HotSpot’ assay platform.20,21 Kinase Assay Protocol. Reac-
tion Buffer: base Reaction buffer; 20 mM Hepes (pH 7.5), 10 mM
MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4,
2 mM DTT, 1% DMSO. Reaction procedure: To a freshly prepared buf-
fer solution was added any required cofactor for the enzymatic reac-
L-glutamine. For a typical screening experiment, cells are inoculated
into 96-well microtiter plates in 100 L at plating densities ranging
l
from 5000 to 40,000 cells/well depending on the doubling time of
individual cell lines. After cell inoculation, the microtiter plates are
incubated at 37 °C, 5% CO2, 95% air and 100% relative humidity for
24 h prior to addition of experimental drugs. After 24 h, two plates
of each cell line are fixed in situ with TCA, to represent a measure-
ment of the cell population for each cell line at the time of drug addi-
tion (Tz). Experimentaldrugs are solubilized in dimethylsulfoxideat
400-fold the desired final maximum test concentration and stored
frozen prior to use. At the time of drug addition, an aliquot of frozen
concentrate is thawed and diluted to twice the desired final
maximum test concentration with complete medium containing
tion, followed by the addition of the selected kinase at
concentration of 20 M. The contents were mixed gently, and then
the compound under test (compound 10f) dissolved in DMSO was
added to the reaction mixture in the 10 M concentration. 339-
ATP (specific activity 500 Ci/ L) was added to the mixture in order
to initiate the reaction, and the mixture was incubated at room tem-
perature for 2 h. Staurosporine was used as a control compound in a
five-dose IC50 mode with 10-fold serial dilutions starting at 20 lM,
and the reaction was carried out at 10 lM ATP concentration.
a
l
l
l
l
Acknowledgments
50
tions are made to provide a total of five drug concentrations plus
control. Aliquotsof100 Lofthesedifferentdrugdilutionsareadded
to the appropriate microtiter wells already containing 100 L of
medium, resulting in the required final drug concentrations. Follow-
ing drug addition, the plates are incubated for an additional 48 h at
37 °C, 5% CO2, 95%air, and100% relativehumidity. For adherent cells,
the assay is terminated by the addition of cold TCA. Cells are fixed
lg/mL gentamicin. Additional four, 10-fold or 1/2 log serial dilu-
This research was supported by Korea Institute of Science and
Technology. We would like to express our gratitude and thanks
to the National Cancer Institute (NCI), Bethesda Maryland, USA
for performing the anticancer testing of the new compounds. Our
appreciations also for Dr. Sean W. Deacon and Dr. Haiching Ma
from Reaction Biology Corporation for carrying out the kinase
screening.
l
l
in situ by the gentle addition of 50 lL of cold 50% (w/v) TCA (final
concentration, 10% TCA) and incubated for 60 min at 4 °C. The super-
natant is discarded, and the plates are washed five times with tap
Supplementary data
water and air dried. Sulforhodamine B (SRB) solution (100 lL) at
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.029.
0.4% (w/v) in 1% acetic acid is added to each well, and plates are incu-
bated for 10 min at room temperature. After staining, unbound dye
is removed by washing five times with 1% acetic acid and the plates
are air dried. Bound stain is subsequently solubilized with 10 mM
trizma base, and the absorbance is read on an automated plate read-
er at a wavelength of 515 nm. For suspension cells, the methodology
is the same except that the assay is terminated by fixing settled cells
References and notes
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at the bottom of the wells by gently adding 50 lL of 80% TCA (final
concentration, 16% TCA). Using the seven absorbance measurements
[time zero, (Tz), control growth, (C), and test growth in the presence
of drug at the fiveconcentrationlevels (Ti)], the percentagegrowthis
calculated at each of the drug concentrations levels. Percentage
growth inhibition is calculated as:
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[(Ti À Tz)/(C À Tz)] Â 100 for concentrations for which Ti P Tz
[(Ti À Tz)/Tz] Â 100 for concentrations for which Ti <Tz.
Three dose–response parameters are calculated for each exper-
imental agent. Growth inhibition of 50% (IC50) is calculated from
[(Ti À Tz)/(C À Tz)] Â 100 = 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as mea-
sured by SRB staining) in control cells during the drug incubation.
The drug concentration resulting in total growth inhibition (TGI) is
calculated from Ti = Tz. The LC50 (concentration of drug resulting in
a 50% reduction in the measured protein at the end of the drug
treatment as compared to that at the beginning) indicating a net
loss of cells following treatment is calculated from [(Ti À Tz)/
Tz] Â 100 = À50. Values are calculated for each of these three
parameters if the level of activity is reached; however, if the effect
is not reached or is exceeded, the value for that parameter is ex-
pressed as greater or less than the maximum or minimum concen-
tration tested.
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