Synthesis of novel 3,5-diaryl pyrazole derivatives using combinatorial chemistry as inhibitors of tyrosinase as well as potent anticancer, anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2010.06.046

In the present article, we have synthesized a combinatorial library of 3,5-diaryl pyrazole derivatives using 8-(2-(hydroxymethyl)-1-methylpyrrolidin-3- yl)-5,7-dimethoxy-2-phenyl-4H-chromen-4-one (1) and hydrazine hydrate in absolute ethyl alcohol under the refluxed conditions. The structures of the compounds were established by IR, ¹H NMR and mass spectral analysis. All the synthesized compounds were evaluated for their anticancer activity against five cell lines (breast cancer cell line, prostate cancer cell line, promyelocytic leukemia cell line, lung cancer cell line, colon cancer cell line) and anti-inflammatory activity against TNF-α and IL-6. Out of 15 compounds screened, 2a and 2d exhibited promising anticancer activity (61-73% at 10 μM concentration) against all selected cell lines and IL-6 inhibition (47% and 42% at 10 μM concentration) as in comparison to standard flavopiridol (72-87% inhibition at 0.5 μM) and dexamethasone (85% inhibition at 1 μM concentration), respectively. Cytotoxicity of the compounds checked using CCK-8 cell lines and found to be nontoxic to slightly toxic. Out of 15, four 3,5-diaryl pyrazole derivatives exhibiting potent inhibitory activities against both the monophenolase and diphenolase actions of tyrosinase. The IC₅₀ values of compounds (2a, 2d, 2h and 2l) for monophenolase inhibition were determined to range between 1.5 and 30 μM. Compounds 2a, 2d, 2h and 2l also inhibited diphenolase significantly with IC₅₀ values of 29.4, 21.5, 2.84 and 19.6 μM, respectively. All four 3,5-diaryl pyrazole derivatives were active as tyrosinase inhibitors (2a, 2d, 2h and 2l), and belonging to competitive inhibitors. Interestingly, they all manifested simple reversible slow-binding inhibition against diphenolase.

Full text of DOI:10.1016/j.bmc.2010.06.046

Bioorganic & Medicinal Chemistry 18 (2010) 6149–6155
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel 3,5-diaryl pyrazole derivatives using combinatorial
chemistry as inhibitors of tyrosinase as well as potent anticancer,
anti-inflammatory agents
c
Babasaheb P. Bandgar a,b, , Jalinder V. Totre , Shrikant S. Gawande b,c, C. N. Khobragade d,
*
Suchita C. Warangkar d, Prasad D. Kadam a
a Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur 413 255, India
b Organic Chemistry Research Laboratory, School of Chemical Sciences, Swami Ramanand Teerth Marathawada University, Nanded 431 606, India
c Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem 91120, Israel
d Biochemistry Research Laboratory, School of Life Sciences, Swami Ramanand Teerth Marathawada University, Nanded 431 606, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present article, we have synthesized a combinatorial library of 3,5-diaryl pyrazole derivatives
using 8-(2-(hydroxymethyl)-1-methylpyrrolidin-3-yl)-5,7-dimethoxy-2-phenyl-4H-chromen-4-one (1)
and hydrazine hydrate in absolute ethyl alcohol under the refluxed conditions. The structures of
the compounds were established by IR, 1H NMR and mass spectral analysis. All the synthesized com-
pounds were evaluated for their anticancer activity against five cell lines (breast cancer cell line, pros-
tate cancer cell line, promyelocytic leukemia cell line, lung cancer cell line, colon cancer cell line) and
Received 12 May 2010
Revised 13 June 2010
Accepted 15 June 2010
Available online 19 June 2010
Keywords:
3,5-Diaryl pyrazole derivatives
Rohitukine
anti-inflammatory activity against TNF-
ited promising anticancer activity (61–73% at 10
IL-6 inhibition (47% and 42% at 10 M concentration) as in comparison to standard flavopiridol (72–
87% inhibition at 0.5 M) and dexamethasone (85% inhibition at 1 M concentration), respectively.
a
and IL-6. Out of 15 compounds screened, 2a and 2d exhib-
l
M concentration) against all selected cell lines and
l
Tyrosinase
l
l
Polyphenol oxidase
Diphenolase inhibitors
Anticancer activity
Anti-inflammatory activity
Cytotoxicity of the compounds checked using CCK-8 cell lines and found to be nontoxic to slightly
toxic. Out of 15, four 3,5-diaryl pyrazole derivatives exhibiting potent inhibitory activities against
both the monophenolase and diphenolase actions of tyrosinase. The IC50 values of compounds (2a,
2d, 2h and 2l) for monophenolase inhibition were determined to range between 1.5 and 30
pounds 2a, 2d, 2h and 2l also inhibited diphenolase significantly with IC50 values of 29.4, 21.5, 2.84
and 19.6 M, respectively. All four 3,5-diaryl pyrazole derivatives were active as tyrosinase inhibitors
lM. Com-
l
(2a, 2d, 2h and 2l), and belonging to competitive inhibitors. Interestingly, they all manifested simple
reversible slow-binding inhibition against diphenolase.
Ó 2010 Published by Elsevier Ltd.
1. Introduction
sets of structurally related compounds, allowing determination
of the chemical structure that is the strongest possible lead
With the advent of combinatorial chemistry, the number of
new chemical entities (NCEs) can be produced in a short space
of time by drug discovery teams. Although this has produced a
wealth of possible new therapeutic compounds, it has also raised
an important question about screening of most therapeutically
active ones, from thousands of compounds. One approach to
solve this problem has been the use of in vitro screens to iden-
tify the characteristics of an NCE, particularly with respect to its
drug metabolism. Such information is crucial to the decision-
making process of which compounds to progress with and which
to discard. Such an approach can also be used to screen smaller
candidate.1,2
Increased generation of reactive oxygen species (ROS) has been
observed in cancer, degenerative diseases and other pathological
conditions. ROS can stimulate cell proliferation, promote genetic
instability and induce adaptive responses that enable cancer cells
to maintain their malignant phenotypes. However, when cellular
redox balance is severely disturbed, high levels of ROS might cause
various damages leading to cell death. Cancer is the second leading
cause of death in the present society after cardiovascular diseases.
A great deal of efforts have been underway to treat various forms of
cancer for decades; and until recently, chemoprevention of cancer
is receiving its due to share of attention. Combinatorial chemistry
and high-throughput screening against pure molecular targets and
cancer cells are established methods for primary anticancer drug
discovery.3–5
* Corresponding author. Tel./fax: +91 217 2351300.
E-mail address: bandgar_bp@yahoo.com (B.P. Bandgar).
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.06.046

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B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 6149–6155
Inflammation is the body’s way of dealing with infections and
2. Results and discussion
2.1. Chemistry
tissue damage, but there is a fine balance between the beneficial
effects of inflammation cascades and their potential for long-term
tissue destruction. If they are not controlled or resolved, inflamma-
tion cascades can lead to the development of diseases such as
chronic asthma, rheumatoid arthritis, multiple sclerosis, inflamma-
tory bowel disease and psoriasis. Within many inflammation cas-
cades or pathways, there are often pivotal molecular targets that,
when antagonized or neutralized, block the output of the pathway.
A relatively small number of pivotal targets has been identified
that have yielded many successful anti-inflammatory drugs. These
targets include the enzymes (COX 1 and COX 2), cytokines (tumor
Natural products have played an important role in drug discov-
ery and are source of scaffolds for the development of new entities.
Rohitukine is one of such compound isolated, characterized and
synthesized at Hoechst Research Centre, Bombay India. Rohitukine
is a novel 4H-1-benzopyrane-4-one with combined anti-inflamma-
tory and immunomodulatory properties in acute and chronic mod-
el of inflammation. Further from Rohitukine a synthetic compound
was synthesized and tasted for anticancer activity was Flavopir-
idol. Flavopiridol is a novel semi synthetic flavone. Flavopiridol is
known to inhibit potently the activity of multiple cyclin-dependent
kinases (IC50 40 nm).
necrosis factor-a, interleukin-6 and interleukin-2) and the receptor
for the Cysteinyl leukotrienes C4 and D4 and nuclear membrane
receptors (corticosteroids).6,7 Therefore, inhibition of these targets
has become a major focus of current drug discovery and develop-
ment, and an important in vitro method for evaluating the bioac-
tivity of drugs.8,9
While synthesizing Rohitukine, five membered ring contracted
2-hydroxymethyl pyrrolidine moiety was observed.32. Which was
used to synthesize flavones having anticancer activity, we have
decided to derivatize these flavones to pyrazoles.
Tyrosinase (EC 1.14.18.1), which is also referred to as poly-
phenoloxidase (PPO), is most widely associated with the produc-
tion of melanin for the protection of skin from solar radiation.
However, this beneficial trait comes in hand with some severe
vices and human maladies since the overproduction of melanin
results in skin hypigmentation, characterized by age spots, mel-
asma and chloasama.10–12 It has also been suggested that tyros-
inase may contribute to the neurodegeneration associated with
Parkinson’s disease.13 Thus, the unregulated action of tyrosinase
is factor in a number of human disease etiologies. Tyrosinase
inhibition has thus been avidly explored as an avenue for thera-
pies to these diseases. The inhibition of melanin formation by
tyrosinase is also applicable to fruit preservation by the allevia-
tion of browning, rendering inhibitors of even broader impor-
tance. In addition, tyrosinase is known to be involved in the
molting process of insect and adhesion of marine organisms.14
In the last decades, a large number of naturally occurring and
synthetic compounds acting as tyrosinase inhibitors were re-
ported15–23 and the most representative tyrosinase inhibitor so
far is tropolone. However, only few of them are put into practi-
cal use due to their weak individual activities or safety concerns.
Undoubtedly, more efforts are still needed to search and develop
more effective and safe tyrosinase inhibitors.
In the present investigation 2-(2-hydroxymethyl-1-methyl-
pyrrolidin-3-yl)-3,5-dimethoxy-6-(5-phenyl-1H-pyrazol-3-yl)-
phenol (2) has been synthesized from 8-(2-(hydroxymethyl)-1-
methylpyrrolidin-3-yl)-5,7-dimethoxy-2-phenyl-4H-chromen-
4-one (1) and hydrazine hydrate in absolute ethyl alcohol under
the refluxed conditions for appropriate time (Scheme 1). Substi-
tuted
8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-dime-
thoxy-2-phenyl-chromen-4-one (1) were prepared according to
known literature method.32 The residue was purified on column
chromatography (silica gel with 1% MeOH and 1% aq ammonia in
CHCl3). The compound’s structure was confirmed by spectral data
(IR, 1H NMR and MS). The chemical profile of the compounds is
as shown in Table 1.
2.2. Biological evaluation
In vitro assays are increasingly being used in drug metabolism
studies to screen novel chemicals. Their advantages are twofold:
first, they allow testing early in the drug discovery phase, provid-
ing important information on chemical characteristics; second, hu-
man cells or cell constituents can be utilized, increasing the
relevance to man.1
Tyrosinase itself is an enzyme containing a binuclear copper
active site. It catalyzes two steps in the conversion of tyrosine
to melanin. This process proceeds via 3,4-dihydroxy phenylala-
nine (DOPA), which is formed by tyrosinase monophenolase
activity on tyrosine. The next step is the oxidation of DOPA into
DOPA quinine, which is a process catalysed by active dipheno-
lase.24–26 These quinines spontaneously polymerize to high
molecular weight brown pigmented species, known as melanin.
Tyrosinase inhibitors normally either render the copper within
the active site inactive by chelation, obviating the substrate–en-
zyme interaction, or inhibit oxidation via an electrochemical pro-
cess.27,28 Since, it has been observed that flavonoids, stilbenes,
and tropolones manifest tyrosinase inhibitory activities, we have
honed our research goals both to evaluate other phenolic skele-
tons for their tyrosinase inhibition properties and also to eluci-
date their kinetic modes.
The 3,5-diaryl-4,5-dihydropyrazoles has been reported as po-
tent and selective inhibitors of Kinesin spindle protein (KSP).29
Dihydropyrazoles are potent, cell active KSP that induces apoptosis
and generates aberrant mitotic spindles in human ovarian carci-
noma cells. Based on literature search it has been found that pyra-
zole derivatives could have potential biological activity, and
therefore, we had decided to work on the pyrazole derivatives,
their synthesis, characterization and evaluation for their biological
activity.30,31
Anticancer activity of the synthesized compounds was evalu-
ated by using cell line inhibition viz. MCF-7 (breast cancer cell
line), PC-3 (prostate cancer cell line), HL-60 (promyelocytic leuke-
mia cell line), H-460 (lung cancer cell line) and HCT-116 (colon
cancer cell line) (Table 2). The compound 2a and 2d revealed
promising anticancer activity (61–73%), followed by 2i, 2n, 2b, 2c
and 2o compounds (51–67%). While compounds 2e, 2f, 2g, 2h,
2k, 2l and 2m (11–29%) revealed slight to moderate activity. Struc-
ture–activity relationship (SAR) study observed that only halo sub-
stituents (Cl and Br) are involved in improving the anticancer
activity except fluoro substituent. However, the substituent CH3
H
N
O
O
O
O
N
R
a
O
O
OH
R
OH
OH
N
N
1
2
Scheme 1. Reagents and conditions: (a) hydrazine hydrate, ethanol, reflux, 6–8 h.

B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 6149–6155
6151
Table 1
Preparation of 3,5-diaryl pyrazole derivatives
Compound
Substituents (R)
Molecular formula
Molecular weight
Yield (%)
Reaction time (h)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2-Cl
2-Br
C
C
C
23H26ClN3O4
23H26BrN3O4
23H25Cl2N3O4
443
488
478
488
427
423
439
469
443
488
423
427
439
443
488
52
51
46
48
41
57
55
59
54
63
59
46
63
46
50
6
6
6
6
6
6
6
6.5
6
6
6
6
6
6
6
2,4-Cl
2-Cl - 4-NO2
2-F
2- CH3
2-OCH3
2,4-OCH3
3-Cl
3-Br
4-CH3
4-F
C23H25ClN4O6
C23H26FN3O4
C24H29N3O4
C24H29N3O5
C25H31N3O6
C
C
23H26ClN3O4
23H26BrN3O4
C24H29N3O4
23H26FN3O4
C24H29N3O5
C
4-OCH3
4-Cl
4-Br
C
C
23H26ClN3O4
23H26BrN3O4
Table 2
Anticancer activity of 3,5-diaryl pyrazole derivatives
Table 3
Anti-inflammatory activity of 3,5-diaryl pyrazole derivatives
Compound
Anticancer activity at 10
l
M concn
Compound
% Inhibition at 10
lM
Toxicity
MCF-7
PC-3
HL-60
H-460
HCT-116
TNF-a
IL-6
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
63
55
58
61
15
11
13
21
59
65
17
19
15
61
63
74
69
51
63
65
21
17
29
16
67
58
11
15
22
65
59
77
67
62
59
63
19
14
17
24
61
52
21
26
19
63
57
72
69
53
61
65
23
18
15
19
55
61
13
16
13
58
61
78
73
61
59
67
19
16
21
17
63
59
19
21
16
55
49
87
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
24
19
18
21
0
0
0
0
11
13
0
11
0
16
17
71
47
39
41
42
11
13
10
11
35
43
10
28
12
35
31
85
0
0
0
23
7
18
0
0
0
0
14
11
0
0
0
Flavopiridol 0.5
lM
Dexamethasone (1
lM)
0
MCF-7—breast cancer cell line.
PC-3—prostate cancer cell line.
HL-60—promyelocytic leukemia cell line.
H-460—lung cancer cell line.
More detailed bioassay for some compounds was subsequently con-
ducted. Data shown in Table 4, all 3,5-diaryl pyrazole derivatives
examined for tyrosinase inhibition, apart from all 3,5-diaryl pyra-
zole derivatives showed dose dependent inhibition against both
monophenolase and diphenolase activity. As the concentration of
inhibitors raised, the residual enzyme activity drastically decreased
(Fig. 1). All inhibitors manifested a similar relationship between the
enzyme activity and enzyme concentration. From the progress
curve obtained, compounds (2a, 2d, 2h and 2l) showing solid lines
below the line of enzyme activity has indicative of enzyme inhibi-
tion and vice versa.
Taken as an ensemble, the following general features of SAR can
be deduced from compounds (2a–2o). The presence of the meth-
oxy groups at 2,4-position is important for inhibition (2h,
IC50 = 1.75). While, compounds lacking this substitution showed
10 fold higher IC50 values from other inhibitors (2a, IC50 = 22.8,
2d, IC50 = 17.8 and 2l, IC50 = 15.5). This effect of great magnitude
obtained in IC50 values was due to the presence of halo substitu-
ents (–Cl, –Br and –F).
HCT-116—colon cancer cell line.
and OCH3 imparted low activity. In general all the cells lines MCF-
7, PC-3, HL-60, H-460 are equally inhibited by the synthesized
compounds except HCT-116.
Ant-inflammatory activity of the compound was determined for
TNF-
to moderate cytokine inhibition in the range of 10–47% for IL-6 and
only 11–21% for TNF- . Compounds 2a, 2c, 2d and 2i give IL-6
inhibitory activity while 2a and 2d inhibit TNF- actively. However
a and IL-6 inhibition (Table 3). The compounds showed good
a
a
rest of the compounds has shown slight to moderate TNF-
inhibition.
a
The rule of three, relating to activity–exposure–toxicity, presents
the single most difficult challenge in the design and advancement of
drug candidates to the development stage. Absorption, distribution,
metabolism and excretion (ADME) studies are widely used in drug
discovery to optimize this balance of properties necessary to convert
lead compounds into drugs that are both safe and effective for hu-
man patients.33 Therefore, in vitro bioavailability of the synthesized
compoundswasdeterminedusingCCK-8cells. Thecompoundswere
found to be nontoxic except 2d, 2f, 2k and 2l but at an acceptable
range. Therefore, the compounds are biologically safe and used as
the therapeutic agent for future drug discovery study.
The kinetic behavior of the L-tyrosine on L-DOPA oxidation cata-
lyzed by the mushroom tyrosinase at different concentrations of
compounds (2h, 2l, 2d and 2a) were studied. In this experiment,
the initial velocity of the enzyme was monitored via dopachrome
formation at 475 nm. As shown in Figure 2, the Lineweaver–Burk
plots of 1/v versus 1/[S] result in a family of straight lines with the
same y-axis intercept, as illustrated, respectively for the four tyros-
In a preliminary screening, using mushroom tyrosinase as a rep-
resentative enzyme, we observed that 3,5-diaryl pyrazole deriva-
tives showed significant inhibition of tyrosinase L-DOPA oxidation.
inase inhibitors. In Figure 2 the reciprocal of concentration of
L-tyro-
sine is the abscissa 1/[ -DOPA] and the ordinate 1/v is the reciprocal
L

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B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 6149–6155
Table 4
Inhibitory effects of 3,5-diaryl pyrazole derivatives on mushroom tyrosinase activities
Entry
L
-Tyrosine
L-DOPA
IC50
(
lM)
Type of inhibition (Ki,
lM)
IC50
(
lM)
Type of inhibition (Ki, lM)
2a
2d
2h
2l
22.8
17.8
1.75
15.5
Competitive (10.6)
Competitive (9.85)
Competitive (0.84)
Competitive (11.2)
29.4
21.5
2.84
19.6
Competitive (11.8)
Competitive (14.6)
Competitive (0.96)
Competitive (12.6)
pyrazole derivatives were assayed for mushroom tyrosinase inhibi-
tion. Five of these compounds, emerged to be the potent inhibitors
of Mushroom tyrosinase. We progressed to clearly demonstrate
that all of these derivatives were competitive inhibitors. This study
explored novel compounds that can fight against tyrosinase born
illness as well as food depreciation.
7.0
control
2g
2a
2d
2l
6.0
5.0
4.0
2h
4. Experimental
4.1. General
3.0
2.0
1.0
Melting points were recorded in open capillaries with an elec-
trical melting point apparatus and were uncorrected. An IR spec-
trum (KBr disks) was recorded using
a Perkin–Elmer 237
spectrophotometer. 1H NMR spectra was recorded on a Bruker
Avance (300 MHz) spectrometer in CDCl3 solutions, with TMS as
an internal reference. A mass spectrum was recorded on a Shima-
dzu GCMS QP 1000 EX. All the reagents and solvents used were of
analytical grade, and were used as supplied, unless otherwise sta-
ted. TLC was performed on aluminum plates coated with silica gel
(Merck) for monitoring the reactions.
0
2
4
6
8
10
Time (S)
Figure 1. Progress curve for oxidation of L-DOPA catalyzed by mushroom tyrosi-
nase in the presence of of 3,5-diaryl pyrazole derivatives (2h > 2l > 2d > 2a > 2g).
Data shown in the figure is mean of three different experiments.
4.2. Synthesis of 3,5-diaryl pyrazole derivatives
of the change of velocity, which is reciprocal of tyrosinase activity.
The pattern of lines and their arrangement in presence of each inhib-
itor for each graph indicated that they are all competitive inhibitors
To a solution of 8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-
yl)-5,7-dimethoxy-2-phenyl-chromen-4-one (1) (1.16 mmol) in
absolute ethanol (20 mL) was added hydrazine hydrate (87 ll,
1.74 mmol) and refluxed for 6 h (Scheme 1). Reaction mixture
was cooled to 25 °C diluted with chloroform adsorbed on silica
and passed through a silica gel column eluting with 1% MeOH/
CHCl3 and 1% aq ammonia to get required pyrazole derivatives.
The physical and spectral data of pyrazole derivatives are given
below.
[2a, Ki = 10.6
lM, 2d, Ki = 9.85 lM, 2h, Ki = 0.84 lM and 2l,
Ki = 11.2 M] with their inhibitory activity toward tyrosinase
l
decreasing with the increasing concentrations of the substrate. Most
competitive inhibitors of tyrosinase share some structural common-
ality with that of product. As reported from earlier studies, for suc-
cessful diphenolase inhibition, a 3,4-dihydroxy group in the B-ring
of required in order to make the structure of the inhibitor molecule
resemble L-DOPA. This leads to the competitive displacement of L-
DOPA from the active site of the cofactor in a lock-and key model.
This is also seen in molecules such as quercetin and chalcones.34,35
Tepper et al. have reported that halides ions such as Iꢀ, Fꢀ, Clꢀ, and
Brꢀ directlyinteractedwiththecopper ionsintheactivesiteoftyros-
inase from Streptomyces antibioticus and this resulted in the inhibi-
4.2.1. 2-[5-(2-Chloro-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2a)
:
Mp: 210–212 °C; IR cmꢀ1 3422, 1648, 1598; 1H NMR
(300 MHz, CDCl3): d 7.74 (m, 1H), 7.4 (m, 1H), 7.32 (m, 2H), 7.31
(s, 1H), 6.1 (s, 1H), 4.06 (m, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.69
(m, 1H), 3.49 (m, 1H), 3.26 (m, 1H), 2.92 (m, 1H), 2.5 (s, 3H), 2.43
(m, 1H), 2.08 (m, 2H); MS: m/z 444 (M+).
tion of L
-DOPA conversion.36 These results implied that different
several manners of inhibition mechanism by halide ions on tyrosi-
nase from various sources could exist. Likewise, this is consistent
to the general recognition that mushroom tyrosinase activity was
inhibited by those 3,5-diaryl pyrazolederivatives whichposes either
nucleophilic methoxy group or halides ions such as Fꢀ, Clꢀ, and Brꢀ
along with the phenolic skeleton.
4.2.2. 2-[5-(2-Bromo-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy–
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2b)
:
Mp: 188–190 °C; IR cmꢀ1 3423, 1649, 1596; 1H NMR
(300 MHz, CDCl3): d 7.71 (m, 1H), 7.46 (m, 1H), 7.34 (m, 2H),
7.30(s, 1H), 6.1 (s, 1H), 4.02 (m, 1H), 3.97 (s, 3H), 3.95 (s, 3H),
3.68 (m, 1H), 3.48 (m, 1H), 3.26 (m, 1H), 2.92 (m, 1H), 2.51 (s,
3H), 2.42 (m, 1H), 2.08 (m, 2H); MS: m/z 488 (M+).
3. Conclusion
In an attempt to generate broad spectrum 3,5-diaryl pyrazole
derivatives, we have synthesized total 15 compounds with signifi-
cant anticancer, anti-inflammatory and Tyrosinase inhibitory
activity. Compounds 2a and 2d were identified as the potent anti-
cancer and inflammatory agent against all selected cell lines and
IL-6 (an anti-inflammatory target), respectively. The 3,5-diaryl
4.2.3. 2-[5-(2,4-Dichloro-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2c)
:
Mp: 128–130 °C; IR cmꢀ1 3418, 1644, 1596; 1H NMR
(300 MHz, CDCl3): d 7.78 (d, 1H, J = 8.5), 7.58 (d, 1H, J = 8.6),

B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 6149–6155
6153
0 μM
5 μM
10 μM
15 μM
A
B
0 μM
10 μM
20 μM
30 μM
12
10
15
8
10
5
6
4
2
-0.004
-0.004
-0.002
-0.002
0
0.002 0.004 0.006 0.008 0.01 0.012
0
0.002 0.004 0.006 0.008 0.01 0.012
1/[L-DOPA], (μM)
1/[L-DOPA], (μM)
C
D
0 μM
0. 5 μM
0 μM
1.0 μM
2.0 μM
3.0 μM
1.0 μM
1.5 μM
15
30
20
10
10
5
-
-0.004
- 0.002
0.004 - 0.002
0
0.002 0.004 0.006 0.008 0.01 0.012
0
0.002 0.004 0.006 0.008 0.01 0.012
1/[L-DOPA], (μM)
1/[L-DOPA], (μM)
Figure 2. Lineweaver–Burk plots for the inhibition of diphenolase activity of Mushroom tyrosinase by 3,5-diaryl pyrazole derivatives (2a, 2d, 2h and 2l). (A) Concentrations of
derivative 2a for curves from top to bottom were 0, 10, 20 and 30 M. (B) Derivative 2d (0, 5, 10 and 15 M). (C) Derivative 2h (0, 1.0, 2.0 and 3.0 M) and derivative 2l (0, 0.5,
1.0 and 1.5 M), respectively.
l
l
l
l
7.46 (s, 1H), 7.3 (s, 1H), 5.59 (s, 1H), 4.02 (m, 1H), 3.96 (s,
3H), 3.94 (s, 3H), 3.67 (m, 1H), 3.47 (m, 1H), 3.25 (m, 1H),
2.91 (m, 1H), 2.5 (s, 3H), 2.44 (m, 1H), 2.06 (m, 2H); MS:
m/z 479 (M+).
(m, 1H), 3.47 (m, 1H), 3.24 (m, 1H), 2.91 (m, 1H), 2.48 (s, 3H), 2.4
(m, 1H), 2.04 (m, 2H). MS: m/z 428 (M+).
4.2.6. 2-(2-Hydroxymethyl-1-methyl-pyrrolidin-3-yl)-3,5-dimeth-
oxy-6-(5-o-tolyl-1H-pyrazol-3-yl)-phenol (2f)
4.2.4. 2-[5-(2-Chloro-4-nitro-phenyl)-1H-pyrazol-3-yl]-6-(2-
hydroxymethyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-
phenol (2d)
Mp: 152–156 °C; IR cmꢀ1
:
3426, 1653, 1596; 1H NMR
(300 MHz, CDCl3): d 7.6–7.32 (m, 4H), 7.3 (s, 1H), 6.0 (s, 1H), 4.04
(m, 1H), 3.92 (s, 3H), 3.90 (s, 3H), 3.67 (m, 1H), 3.44 (m, 1H),
3.21 (m, 1H), 2.9 (m, 1H), 2.48 (s, 3H), 2.43 (m, 1H), 2.32 (s, 3H),
2.08 (m, 2H); MS: m/z 424 (M+).
Mp: 197–199 °C; IR cmꢀ1
:
3426, 1646, 1592; 1H NMR
(300 MHz, CDCl3): d 8.6 (s, 1H), 8.2 (d, 1H, J = 8.6), 7.82 (d, 1H,
J = 8.4), 7.4 (s, 1H), 6.1 (s, 1H), 4.01 (m, 1H), 3.94 (s, 3H), 3.90 (s,
3H), 3.6 (m, 1H), 3.4 (m, 1H), 3.24 (m, 1H), 2.91 (m, 1H), 2.48 (s,
3H), 2.42 (m, 1H), 2.07 (m, 2H). MS: m/z 489 (M+).
4.2.7. 2-(2-Hydroxymethyl-1-methyl-pyrrolidin-3-yl)-3,5-dimeth-
oxy-6-[5-(2-methoxy-phenyl)-1H-pyrazol-3-yl]-phenol (2g)
Mp: 137–141 °C; IR cmꢀ1
:
3424, 1650, 1598; 1H NMR
4.2.5. 2-[5-(2-Fluoro-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2e)
(300 MHz, CDCl3): d 7.64–7-4 (m, 4H), 7.31 (s, 1H), 5.59 (s, 1H),
4.06 (m, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 3.86 (s, 3H), 3.69 (m, 1H),
3.46 (m, 1H), 3.24 (m, 1H), 2.91 (m, 1H), 2.5 (s, 3H), 2.41 (m, 1H),
2.06 (m, 2H); MS: m/z 440 (M+).
Mp: 115–121 °C; IR cmꢀ1
:
3421, 1646, 1592; 1H NMR
(300 MHz, CDCl3): d 7.78 (m, 1H), 7.4 (m, 1H), 7.36 (m, 2H), 7.3
(s, 1H), 5.58 (s, 1H), 4.04 (m, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.67

6154
B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 6149–6155
4.2.8. 2-[5-(2,4-Dimethoxy-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2h)
Mp: 88–90 °C; IR cmꢀ1: 3428, 1656, 1600; 1H NMR (300 MHz,
CDCl3): d 7.7 (d, 1H, J = 8.7), 7.62 (d, 1H, J = 8.4), 7.42 (s, 1H), 7.30
(s, 1H), 6.1 (s, 1H), 4.04 (m, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.9 (s,
3H), 3.86 (s, 3H), 3.66 (m, 1H), 3.46 (m, 1H), 3.24 (m, 1H), 2.90
(m, 1H), 2.5 (s, 3H), 2.46 (m, 1H), 2.04 (m, 2H); MS: m/z 470 (M+).
4.3. Anticancer activity
Cytotoxic assay is performed on MCF-7 (breast cancer cell line),
PC-3 (prostate cancer cell line), HL-60 (promyelocytic leukemia
cell line), H-460 (lung cancer cell line) and HCT-116 (colon cancer
cell line) cell lines using propidium iodide fluorescence assay.37
Dyes such as propidium iodide (PI), which bind DNA, provide a ra-
pid and accurate means for quantitating total nuclear DNA. The
fluorescence signal intensity of the PI is directly proportional to
the amount of DNA in each cell, PI is not able to penetrate an intact
membrane, and so cells must first be permeabilized. Seed cells of
4.2.9. 2-[5-(3-Chloro-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2i)
:
Mp: 143–148 °C; IR cmꢀ1 3420, 1646, 1597; 1H NMR
(300 MHz, CDCl3): d 7.78 (s, 1H), 7.66 (d, 1H, J = 7.8), 7.44 (d, 1H,
J = 7.8), 7.32 (s, 1H), 6.0 (s, 1H), 4.06 (m, 1H), 3.97 (s, 3H), 3.94 (s,
3H), 3.67 (m, 1H), 3.47 (m, 1H), 3.24 (m, 1H), 2.91 (m, 1H), 2.48
(s, 3H), 2.41 (m, 1H), 2.07 (m, 2H); MS: m/z 444 (M+).
3000–7500 cells/well were placed in 2000 ll of tissue culture
grade 96 well plates and allowed them to recover for 24 h in
humidified 5% CO2 incubator at 37 °C. After culturing for 24 h com-
pounds (in 0.1% DMSO) were added onto triplicate wells with
10
48 h in humidified 5% CO2 incubator at 37 °C condition, the med-
ium was removed and treated with 25 l of propidium iodide
(50 g/mL in water/medium) per well. The plates were frozen at
lM concentrations. 0.1% DMSO alone was used as control. After
4.2.10. 2-[5-(3-Bromo-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2j)
l
Mp: 157–163 °C; IR cmꢀ1
:
3421, 1646, 1598; 1H NMR
l
(300 MHz, CDCl3): d 7.79 (s, 1H), 7.6 (d, 1H, J = 7.6), 7.48 (d, 1H,
J = 7.8), 7.30 (s, 1H), 6.0 (s, 1H), 4.04 (m, 1H), 3.96 (s, 3H), 3.92 (s,
3H), 3.66 (m, 1H), 3.42 (m, 1H), 3.24 (m, 1H), 2.90 (m, 1H), 2.48
(s, 3H), 2.41 (m, 1H), 2.06 (m, 2H); MS: m/z 488/490 (M+).
ꢀ80 °C for 24 h then thawed and allowed it to come at room tem-
perature and the plate absorbance was read on a fluorometer (Po-
lar-Star BMG Tech), using 530 nm excitation and 620 nm emission
wavelength. Lastly, percent cytotoxicity of the compounds was cal-
culated by using following formula:
4.2.11. 2-(2-Hydroxymethyl-1-methyl-pyrrolidin-3-yl)-3,5-di-
methoxy-6-(5-p-tolyl-1H-pyrazol-3-yl)-phenol (2k)
Reading of control ꢀ Reading of treated cells
% Cytotoxicity ¼
Reading of control
Mp: 89–94 °C; IR cmꢀ1: 3420, 1646, 1592; 1H NMR (300 MHz,
CDCl3): d 7.58 (d, 2H, J = 8), 7.24 (d, 2H, J = 7.8), 7.3 (s, 1H), 6.04
(s, 1H), 4.06 (m, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 3.65 (m, 1H), 3.47
(m, 1H), 3.24 (m, 1H), 2.91 (m, 1H), 2.42 (s, 3H), 2.43 (m, 1H), 2.3
(s, 3H), 2.08 (m, 2H); MS: m/z 424 (M+).
ꢁ 100
The results were compared with the standard drug inhibitors
flavopiridol (0.5 lM).
4.4. Anti-inflammatory and cytotoxicity assay
4.2.12. 2-[5-(4-Fluoro-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy–
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2l)
Proinflammatory cytokine production by lipopolysaccharide
(LPS) in THP-1 cells was measured according to the method de-
scribed by Hwang et al.38 During the assay, THP-1 cells were cul-
tured in the RPMI 1640 culture media (Gibco BRL, Pasley, UK)
containing 100 U/mL penicillin and 100 mg/mL streptomycin con-
taining 10% fetal bovine serum (FBS, JRH). Cells were differentiated
with phorbol myristate acetate (PMA, Sigma). Following cell plat-
ing, the test compounds in 0.5% DMSO was added to each well
and the plate was incubated for 30 min at 37 °C. Finally, LPS (Esch-
erichia coli 0127: B8, Sigma Chemical Co., St. Louis, MO) was added,
Mp: 146–150 °C; IR cmꢀ1
:
3423, 1649, 1596; 1H NMR
(300 MHz, CDCl3): d 7.68 (t, 2H, J = 6.6), 7.42 (t, 2H, J = 8.7), 7.3
(s, 1H), 6.06 (s, 1H), 4.04 (m, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.66
(m, 1H), 3.48 (m, 1H), 3.25 (m, 1H), 2.91 (m, 1H), 2.49 (s, 3H),
2.42 (m, 1H), 2.06 (m, 2H). MS: m/z 428 (M+).
4.2.13. 2-(2-Hydroxymethyl-1-methyl-pyrrolidin-3-yl)-3,5-di-
methoxy-6-[5-(4-methoxy-phenyl)-1H-pyrazol-3-yl]-phenol
(2m)
Mp: 108–114 °C; IR cmꢀ1
:
3421, 1651, 1594; 1H NMR
at a final concentration of 1
incubated at 37 °C for 24 h in 5% CO2. After incubation, superna-
tants were harvested, and assayed for TNF- and IL-6 by ELISA as
lg/mL in each well. Plates were further
(300 MHz, CDCl3): d 7.61 (d, 2H, J = 8.2), 7.30 (d, 2H, J = 8.2), 7.30
(s, 1H), 6 (s, 1H), 4.06 (m, 1H), 3.92 (s, 3H), 3.90 (s, 3H), 3.86 (s,
3H), 3.66 (m, 1H), 3.48 (m, 1H), 3.25 (m, 1H), 2.92 (m, 1H), 2.46
(s, 3H), 2.41 (m, 1H), 2.04 (m, 2H); MS: m/z 440 (M+).
a
described by the manufacturer (BD Biosciences). The cells were
simultaneously evaluated for cytotoxicity using CCK-8 from Dojin-
do Laboratories. Percent inhibition of cytokine release in compari-
son to the control was calculated. The 50% inhibitory concentration
(IC50) values were calculated by a nonlinear regression method.
4.2.14. 2-[5-(4-Chloro-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2n)
:
Mp: 112–117 °C; IR cmꢀ1 3420, 1646, 1595; 1H NMR
4.5. Assay of tyrosinase activity
(300 MHz, CDCl3): d 7.78 (d, 2H), 7.64 (d, 2H), 7.32(s, 1H), 6.1 (s,
1H), 4.08 (m, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.66 (m, 1H), 3.48
(m, 1H), 3.24 (m, 1H), 2.91 (m, 1H), 2.48 (s, 3H), 2.41 (m, 1H),
2.06 (m, 2H); MS: m/z 444 (M+).
Mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was
used as described previously with some modifications, using
either, L-DOPA (diphenolase) or L-tyrosine (monophenolase) as
substrate.39 In spectrophotometric experiments, enzyme activity
was monitored by observing dopachrome formation at 475 nm
with a UV–vis spectrophotometer (Spectro UV–vis Double beam;
Shimadzu, Inc.) at 30 °C. All samples were first dissolved in 0.1%
4.2.15. 2-[5-(4-Bromo-phenyl)-1H-pyrazol-3-yl]-6-(2-hydroxy-
methyl-1-methyl-pyrrolidin-3-yl)-3,5-dimethoxy-phenol (2o)
Mp: 165–171 °C; IR cmꢀ1
:
3424, 1651, 1596; 1H NMR
DMSO at 10
(Km = 180 M) or 5.4 mM
was mixed with 2687 L of 0.25 M phosphate buffer (pH 6.8).
Then, 100 L of the sample solution and 13 L of the same
l
M–50
l
M. First, 200
l
L of a 2.7 mM
L-tyrosine
(300 MHz, CDCl3): d 7.8 (d, 2H), 7.66 (d, 2H), 7.31 (s, 1H), 6.1 (s,
1H), 4.06 (m, 1H), 3.95 (s, 3H), 3.92 (s, 3H), 3.68 (m, 1H), 3.46
(m, 1H), 3.26 (m, 1H), 2.90 (m, 1H), 2.47 (s, 3H), 2.4 (m, 1H), 2.07
(m, 2H); MS: m/z 488/490 (M+).
l
L
-DOPA (Km = 360
lM) aqueous solution
l
l
l

B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 6149–6155
14. Shiino, M.; Watanabe, Y.; Umezawa, K. Bioorg. Med. Chem. 2001, 9, 1233.
6155
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ducted as three separate replicates. The inhibitor concentration
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The authors are thankful to Mr. Mahesh Nambiar and Mrs. Asha
Almeida, Piramal Life Sciences Ltd, Mumbai for anti-inflammatory
and anticancer activities and Director School of Life Sciences, for
tyrosinase inhibitory activity and also to Ms. Diana Ickowicz, The
Hebrew University of Jerusalem.
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