Design and synthesis of 3,5-diaryl-4,5-dihydro-1H-pyrazoles as new tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.12.054

In this study, twenty 3,5-diaryl-4,5-dihydro-1H-pyrazole derivatives with hydroxyl(s) (1a-1p, 2a-2d) were synthesized and their inhibitory activity on mushroom tyrosinase was examined. The results showed that among these compounds, 1-(5-(3,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl) ethanone 1d was found to be the most potent tyrosinase inhibitor with IC ₅₀ value of 0.301 μM. Kinetic study revealed that these compounds were competitive inhibitors of tyrosinase and their structure-activity relationships were investigated in this article.

Full text of DOI:10.1016/j.bmc.2012.12.054

Bioorganic & Medicinal Chemistry 21 (2013) 2156–2162
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of 3,5-diaryl-4,5-dihydro-1H-pyrazoles as new
tyrosinase inhibitors
⇑
Zhixuan Zhou, Jiaru Zhuo, Sujun Yan, Lin Ma
School of Chemistry and Chemical Engineering, Sun Yat-sen University, 135 Xin Gang Xi Road, Guangzhou 510275, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, twenty 3,5-diaryl-4,5-dihydro-1H-pyrazole derivatives with hydroxyl(s) (1a–1p, 2a–2d)
were synthesized and their inhibitory activity on mushroom tyrosinase was examined. The results
showed that among these compounds, 1-(5-(3,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)ethanone 1d was found to be the most potent tyrosinase inhibitor with IC50 value of
Received 6 November 2012
Revised 13 December 2012
Accepted 15 December 2012
Available online 11 January 2013
0.301
lM. Kinetic study revealed that these compounds were competitive inhibitors of tyrosinase and
their structure–activity relationships were investigated in this article.
Keywords:
Ó 2013 Published by Elsevier Ltd.
4,5-Dihydro-1H-pyrazole
Mushroom tyrosinase
Inhibit
1. Introduction
zylbenzamides,9 and benzoate ester derivatives10 are a class of
tyrosinase inhibitors that had been investigated intensively. They
often contain two hydroxylated benzene rings separated by three
atoms and often constructed with one of two distinct substruc-
tures: a 4-substituted resorcinol or catechol moiety. Introduction
of a heterocycle in the compounds, including oxazolones,11 pyra-
zoles,12 oxadiazoles,13 thiazoles,14 and carbazoles,15 the com-
pounds show more potent tyrosinase inhibition.
Based on these studies, we designed new 3,5-diaryl-4,5-dihy-
dro-1H-pyrazole with hydroxyls in order to discover effective
tyrosinase inhibitors (Fig. 1). At first, 3,5-diaryl-4,5-dihydro-1H-
pyrazole ethanones were synthesized by our designed route. We
found that these ethanones derivatives are also potent tyrosinase
inhibitors. Based on the results of ethanones, we modified the reac-
tion conditions and successfully synthesized four compounds of
polyhydroxyl 3,5-diaryl-4,5-dihydro-1H-pyrazoles. We carried
out on their inhibitory effect (inhibitory activity and inhibitory
kinetics), and investigated the structure–activity relationship on
the enzyme inhibitory activity. Mushroom tyrosinase was used
throughout our studies. This research aimed at discovering and fil-
tering effective compounds as tyrosinase inhibitors, which can
have potential application in cosmetic careers and other fields to
inhibit enzymatic browning.
Tyrosinase (EC 1.14.18.1), which is also referred to as polyphe-
noloxidase, is a multifunctional copper-containing enzyme widely
exists in plants and animals. It catalyzes two steps in the conver-
sion of tyrosine to melanin. This process proceeds via 3,4-dihy-
droxy phenylalanine (DOPA), which is formed by tyrosinase
monophenolase activity on tyrosine. Then DOPA was oxidized into
DOPA quinine, which is a process catalyzed by active diphenolase.
These quinines can polymerize spontaneously to form melanins,
which are brown pigments of high molecular weight and deter-
mine the color of mammalian skin and hair.1 However, the accu-
mulation of an excessive level of epidermal pigmentation causes
melasama, age spots and other dermatological disorders.2 There-
fore, tyrosinase inhibitors have become increasingly important
for medicinal and cosmetic products that may be used to prevent
or treat pigmentation disorders.3 Tyrosinase also play a vital role
in undesirable enzymatic browning reactions of some vegetables
and fruits.4 Moreover, tyrosinase is one of the most important
key enzymes in the insect molting process, investigation of its
inhibitors may be important in finding alternative insect control
agents.5 These phenomena have encouraged researchers to seek
new potent tyrosinase inhibitors for use in foods, cosmetics and in-
sect control.
Many efforts have been addressed to the search and synthesis
for effective and safe tyrosinase inhibitors. Polyphenol derivatives,
such as flavanones,6 chalcones,7 resveratrol and it analogs,8 N-ben-
2. Results and discussion
2.1. Chemistry
⇑
Polyhydroxyl 3,5-diaryl-4,5-dihydro-1H-pyrazole derivatives
were synthesized according to the routes in Scheme 1. Chalcones
Corresponding author. Tel.: +86 20 8411 3690, +86 136 408 02515.
E-mail address: cesmal@mail.sysu.edu.cn (L. Ma).
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.12.054

Z. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 2156–2162
2157
HO
OH
O
HO
OH
HO
OH
N
OH
Chalcone, (E)-3-(2,4-dihydroxyphenyl)-1-(2,4,6-
trihydroxyphenyl)prop-2-en-1-one as example.
IC50=1 µM
OH
NH
3,5-diary-4,5-dihydro-1H-pyrazole derivative, 4-(5-(3-hydroxyphenyl)-4,5-
dihydro-1H-pyrazol-3-yl)benzene-1,3-diol as example
IC50=0.837 µM
OH
O
OH
OCH3
HO
O
Design
HO
OH
N
OH
OH
Synthesis
Flavanone, 2-(2,4-dihydroxyphenyl)-5,7-
dihydroxychroman-4-one as example.
IC50=26.5 µM
N
O
OCH3
3,5-diary-4,5-dihydro-1H-pyrazole ethanone derivative, 1-(3-(2,4-
dihydroxyphenyl)-5-(4-hydroxy-3-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)ethanone as example
S
IC50=0.301 µM
N
OCH3
HO
OH
Thiazole,4-(2-(3,5-dimethoxyphenyl)thiazol-4-
yl)benzene-1,3-diol as example
µ
M
IC50=2
Figure 1. Design and synthesis of compounds in this article.
were synthesized through the Claisen–Schmidt condensation of
the corresponding acetophenones and benzaldehydes under alka-
line condition, as described in synthesis of isoliquiritigenin
(2,4,40-trihydroxychalcone).16 Polyhydroxylchalcones can also be
synthesized under acidic condition,17 or using protection and de-
protection on hydroxyl group,5 but these methods suffered from
more reaction steps, prolonged reaction time, and toxic reagents.
In terms of economization and time-saving, we conducted the
reactions under alkaline condition. The reactions were processed
in presence of solid phase (neutral Al2O3) and KOH catalyst under
microwave irradiation. After irradiation, the mixture was treated
with water, then the resulting solution was acidified and the pre-
cipitate was filtered to afford chalcone. Attempts to synthesis
2,4,20,40-tetrahydroxychalcone by this method resulted in the for-
mation of polymeric products.
4,5-dihydro-1H-pyrazole was acetylated by the solution, and
yielded 3,5-diaryl-4,5-dihydro-1H-pyrazole ethanones.
2.2. 3,5-Diaryl-4,5-dihydro-1H-pyrazole derivatives on the
diphenolase activity of mushroom tyrosinase
Kojic acid, a widely used skin-whitening material for its high
inhibitory activity against tyrosinase18 and synthesized com-
pounds 1a–1p, 2a–2d were tested for their enzymatic inhibitory
activities against tyrosinase. The two hydroxylated benzene rings
in the compounds were asymmetric; therefore, different position
of hydroxyl group(s) on ring A and B may result in different inhib-
itory effect on tyrosinase. The results were shown in Table 1. Ele-
ven of the synthesized compounds show IC50 lower than kojic
acid, indicating these compounds were more effective than kojic
acid when used as tyrosinase inhibitor.
Chalcones were purified and dissolved in ethanol, then hydra-
zine hydrate was added and the mixture was heated under reflux
to afford polyhydroxyl 3,5-diaryl-4,5-dihydro-1H-pyrazoles. When
the reaction was processed in acetic acid, the active hydrogen of
The position and number of hydroxyl substituted on ring A had
a great influence on the inhibitory activity against tyrosinase. Com-
pounds 1a–1d and 2a–2c with a 2,4-dihydroxyphenyl moiety on
R2
R1
(ii)
N
N
O
(i)
R1
R2
B
R2
A
R1
+
(iii)
O
O
O
R2
R1
N
NH
Scheme 1. Reagents and conditions: (i) KOH, neutral Al2O3, microwave irradiation, 30–70 s; (ii) acetic acid, hydrazine hydrate, reflux, 50–90 min; (iii) ethanol, hydrazine
hydrate, reflux, 5 h.

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Z. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 2156–2162
Table 1
with 4-dihydroxyphenyl moiety on ring A. Compounds 1o, 1p with
a 3,5-dihydroxyphenyl moiety demonstrated practically low activ-
ity. The results were in agreement with previous studies on chal-
cones, suggesting that compounds possessing 2, 4-
dihydroxyphenyl moiety on ring A exhibited better inhibitory
activity against tyrosinase than other compounds. These results
were in good agreement with previous studies, which showed that
4-resorcinol moiety is the key substituted group in exerting potent
inhibitory activity.19
In contrast, the effects caused by different position of substi-
tuted groups attached on ring B were not as conspicuous as on ring
A. According to earlier studies, a 30,40-dihydroxyphenyl moiety was
required on ring B for successful diphenolase inhibition in order to
Inhibition effect of 3,5-diaryl-4,5-dihydro-1H-pyrazole derivatives on mushroom
tyrosinase activities
A
B
R2
R1
A
B
R2
R1
N
N
N
NH
O
1a-1p
2a-2d
Compounds
R1
R2
IC50 (lM)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
2a
2b
2c
2d
Kojic acid
2,4-OH
2,4-OH
2,4-OH
2,4-OH
2,4-OH
4-OH
4-OH
4-OH
4-OH
2-OH
2-OH
2-OH
2-OH
2-OH
3,5-OH
3,5-OH
2,4-OH
2,4-OH
2,4-OH
2-OH
30,40-OH
40-OH
30-OH
2.74 0.21
1.69 0.03
0.863 0.102
0.301 0.090
>200
1.07 0.14
3.51 0.04
147
>200
>200
13.1 0.8
>200
13.4 0.3
>200
161 16
>200
30-OMe, 40-OH
30-NO2, 40-OH
30,40-OH
make the structure of the inhibitor molecule similar to
L-DOPA.
This leads to the competitive displacement of -DOPA from the ac-
L
40-OH
30-OH
tive site of the cofactor in a lock and key model. 3,5-Diaryl pyrazole
derivatives with nucleophilic methoxy groups or halogen groups
such as fluoro, chloro, and bromo along with the phenolic skeleton
also recognized as tyrosinase inhibitor.12,20 In the present study,
addition of methoxy greatly influenced the inhibitory activity of
the compounds, when compared the inhibitory activity of com-
pound 1d and 1k with methoxy at C-30 and hydroxy at C-40 and
compound 1a and 1j with a 30,40-dihydroxyphenyl moiety on ring
B, compound 1d and 1m shows more potent inhibitory activity.
However, compound 1i exhibited practically no activity and com-
pound 1f was a very active inhibitor against tyrosinase with IC50
5
30-OMe, 40-OH
30,40-OH
40-OH
30-OH
30-OMe, 40-OH
20-OMe, 40-OH
40-OH
30-OH
40-OH
0.882 0.057
0.837 0.148
2.43 0.41
11.8 1.5
18.3 0.7
30-OH
30-OMe, 40-OH
40-OH
at 1.07 lM. The substitution position of methoxy was also of
importance, as compound 1n with a methoxy at C-20 exhibits no
inhibitory activity, while compound 1m was active. And, addition
of a –NO2 at C-30 (compound 1e) diminished the inhibitory activity.
These results indicated that different several manners of inhibition
mechanism on tyrosinase from various sources could exist.
ring A were all very active members of the synthesized com-
pounds, even better than the standard inhibitor, kojic acid. Com-
pound 1d showed most potent tyrosinase inhibition and the IC50
The chemical groups between the two hydroxylated benzene
rings were another major factor that influenced the inhibition
activity. As shown in Figure 2 and Table 2, where compounds with
similar 4-substituted resorcinol and same number of atoms be-
tween two hydroxylated benzene rings were taken into compari-
son, when a ring such as thizole and 4,5-dihydro-1H-pyrazole
was introduced between the the two hydroxylated benzene rings,
the compounds are more effective inhibitor of tyrosinase than
those with a linear chain that separates the two hydroxylated ben-
zene rings like chalcones and benzyl benzoates.21 These results
is 0.301 l
M. When there was only hydroxy substituted at C-30
and C-40, compounds exhibited slightly lower inhibition. For com-
pounds 1f–1i with a p-hydroxy on ring A, most compounds exhib-
ited lower inhibitory activity against tyrosinase compared to those
with 2,4-dihydroxy substituent. Only compound 1f with a 3,4-
dihydroxyphenyl moiety on ring B showed higher potency than
corresponding 1a with 2,4-dihydroxyphenyl moiety. Similarly,
compounds 1j–1m possessing an 2-dihydroxyphenyl on ring A also
exhibited lower inhibition potency, and even lower than those
HO
OH
O
OH
HO
OH
O
HO
OH
HO
OH H3CO
N
OCH3
O
S
(a)
(b)
(c)
OCH3
HO
OH
OH
HO
OH
HO
OH
HO
OH
OH
O
O
N
NH
O
O
N
N
O
OH
OH
(f)
(g)
OH
OH
(d)
(e)
Figure 2. Comparison between tyrosinase inhibitors with different chemical groups (red) which separated two hydroxylated benzene rings. (a) Benzyl benzoate; (b)
chalcone; (c) bis-resorcinol-substituted thiazole; (d) flavanone; (e) flavone; (f) 3,5-diaryl-4,5-dihydro-1H-pyrazole ethanone; (g) 3,5-diaryl-4,5-dihydro-1H-pyrazole.

Z. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 2156–2162
2159
Table 2
and 2b as an example). The plots of 1/V versus 1/[S] were charac-
terized by a family of straight lines with different slopes, which
presented different fixed substrate concentrations. All these lines
were intersected each other at vertical axis, indicating that the
Comparison between compounds different chemical groups which separated two
hydroxylated benzene rings
Compound structure
IC50
(
lM)
Inhibitory type
inhibitory type of 1d was competitive manner with L-DOPA as a
Benzyl benzoate
Chalcone
Bis-resorcinol-substituted thiazole
Flavanone
Flavone
4.69
5
2
26.5
>200
Competitive
Competitive
—
substrate. This data suggested that compound 1d to be an effective
inhibitor by binding to active site of the enzyme. Identical research
was carried out on 1a, 1b, 1c, 2a and 2b, and same conclusions
were drawn that the five compounds were competitive inhibitors.
a
Competitive
a
—
3,5-Diaryl-4,5-dihydro-1H-pyrazole ethanone
3,5-Diaryl-4,5-dihydro-1H-pyrazole
0.301
0.837
Competitive
Competitive
a
3. Conclusions
No data afforded from the article.
In summary, series of polyhydroxyl 3,5-diaryl-4,5-dihydro-1H-
pyrazole derivatives were synthesized and tested. The results
showed that polyhydroxyl 3,5-diaryl pyrazole represent a new
class of tyrosinase inhibitors. Judging from the results, position of
hydroxyl moiety remarkably influenced their inhibitory activity
on tyrosinase. When compared the synthesized compounds to
compounds with similar structure, it can be concluded that chem-
ical groups between the two hydroxylated benzene rings of the
compounds also influenced the inhibitory activity on tyrosinase,
addition of a heterocycle could increase inhibitory activity. Among
the synthesized compounds, compound 1d presented the most po-
tent inhibition against the enzyme tyrosinase. These compounds
will be of potential use for cosmetic, clinical applications, and other
fields which need to inhibit over activity of tyrosinase. The results
also provide guidance for further studies on tyrosinase and design
of its effective inhibitors.
indicated that a hydrophobic site exists in tyrosinase and a hydro-
phobic ring of proper size allowed compounds possess a stronger
interaction with tyrosinase, thus enhance their inhibitory activity.
A different type of ring closure, flavanone for example, resulted in
decrease in inhibitory activity.
In order to get a better understanding of influence of pyrazole
ring and acetyl group, based on the results of compounds with
an acetyl group (1a–1p), and previous studies on tyrosinase inhibi-
tion, compounds without an acetyl group (2a–2d) were synthe-
sized and evaluated. Compounds 2a, 2b and 2d showed a similar
structure–activity relationship with their corresponding acetylated
compounds while exerting more potent inhibitory activity. How-
ever, to our surprise, 2c exhibited diminish in inhibitory activity
when compared to corresponding 1d. This result indicated that
the influence of acetyl group on the pyrazole ring and methoxy
group on ring B might be complicate. Generally the acetyl group
on the pyrazole ring diminish the inhibitory activity, possible
due to its steric hindrance. Further studies are needed to get a bet-
ter understanding about the influence of the chemical groups be-
tween the two hydroxylated benzene rings, as well as the role of
acetyl group and methoxy group.
4. Experimental
Tyrosinase was purchased from Sigma–Aldrich Chemical Co. (St.
Louis, MO). 3,4-Dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxy-
benzaldehyde, 3-hydroxybenzaldehyde, 2,4-dihydroxyactetophe-
none were purchased from Alfa Aesar. 4-Hydroxybenzaldehyde,
4-hydroxyactetophenone, 2-hydroxyactetophenone, hydrazine hy-
drate were purchased from Aladdin reagent Co., Shanghai, China.
Other reagents were purchased from commercial suppliers and
were dried and purified when necessary. Water used was re-dis-
tilled and ion-free.
All UV–vis absorbance were measured on a SHIMADZU UV-
2501PC spectrometer without correction. IR spectra were recorded
as KBr flakes on a Thermo Nicolet 330FT-IR spectrometer. 1H NMR
2.3. Inhibitory types of 3,5-diaryl-4,5-dihydro-1H-pyrazole
derivatives on mushroom tyrosinase
Kinetic behavior of the oxidation of L-DOPA catalyzed by tyros-
inase of compounds 1a, 1b, 1c, 1d, 2a and 2b, which showed excep-
tional tyrosinase inhibition, were further studied by method
steady-state kinetic analysis. Inhibition data were then analyzed
using Lineweaver–Burk plot shown in Figure 3 (compounds 1d
Figure 3. (a) Lineweaver–Burk plot of mushroom tyrosinase inhibition by compound 1d. Enzyme activity was measured at 475 nm in the presence of 0.2
while the substrate. -DOPA, varied from 20 to 80 M. (b) Lineweaver–Burk plot of mushroom tyrosinase inhibition by compound 2b. Enzyme activity was measured at
475 nm in the presence of 0.5 M compound 2b while the substrate. -DOPA, varied from 20 to 80 M.
lM compound 1d
L
l
l
L
l

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Z. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 2156–2162
and 13C NMR spectra were recorded with a Varian Mercury-Plus
300 instrument (1H, 300 MHz), (13C, 75 MHz) in DMSO-d6. Melting
points were determined on a SGW X-4 melting point apparatus
and were uncorrected. Reactions were monitored by TLC on silica
gel plates in either iodine chamber or in UV chamber.
4.3.3. 1-(3-(2,4-Dihydroxyphenyl)-5-(3-hydroxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (1c)
Yield 49%, light brown powder, mp 193–194 °C, IR(KBr):
m
(cmꢀ1): 3211(
m
O–H), 1670(
mC@O), 1618(mC@N), 1462, 1420 (mas),
1301(
m
C–N). 1H NMR (DMSO-d6): d (ppm): 10.84(s, 2H, OH),
9.31(s, 1H, OH), 7.58–6.17(m, 7H, ArH), 5.31(dd, 1H, CH, J = 11.57,
3.28 Hz), 3.83(dd, 1H, HM, J = 10.34, 17.73 Hz), 3.16(dd, 1H, HA,
J = 17.73, 3.67 Hz), 1.77(s, 3H, COCH3).
4.1. Assay of the diphenolase activity
All the synthesized compounds were screened for diphenolase
inhibitory activity of tyrosinase, using
inhibitors were dissolved in DMSO and prepared at concentrations
of 10 mmol/L. 20 units of mushroom tyrosinase (2000 U/mL), 10
of DMSO and 900 L of phosphate buffer (pH 6.8) were mixed and
pre-incubated for 20 min at 30 °C. Then, the -DOPA (2 mg/mL) was
L
-DOPA as substrate. The
4.3.4. 1-(3-(2,4-Dihydroxyphenyl)-5-(4-hydroxy-3-
methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (1d)
lL
Yield 45%, light brown powder, mp 228–230 °C, IR(KBr):
(cmꢀ1): 3437(
O–H), 1674( C@O), 1624( C@N), 1597, 1577, 1512,
1428( as), 1315(
C–N). 1H NMR (DMSO-d6): d (ppm): 9.73(s, 2H,
m
l
m
m
m
L
m
m
added into this blending and the reaction was monitored for 1 min
by measuring the change in absorbance at 475 nm, due to the for-
mation of DOPAchrome. In subsequent experiments, DMSO was re-
placed by equivalent inhibitors, whose concentration would be
OH), 9.03(s, 1H, OH), 7.33–6.23(m, 6H, ArH), 5.37(dd, 1H, CH,
J = 11.47, 3.90 Hz), 3.65(s, 3H, OCH3), 3.89(dd, 1H, HM, J = 11.50,
17.88 Hz), 3.16(dd, 1H, HA, J = 18.38, 4.04 Hz), 1.75(s, 3H, COCH3).
13C NMR (DMSO-d6): d (ppm): 174.01, 160.31, 159.53, 156.87,
150.01, 148.02, 131.57, 129.89, 121.34, 117.98, 117.31, 114.61,
113.82, 110.54, 70.07, 57.75, 41.29, 25.82.
decreased from 200 lM until the inhibition was less than 50%.
The extent of inhibition by the addition of the sample was ex-
pressed as the percentage necessary for 50% inhibition (IC50). As
a control, the IC50 of kojic acid was also measured.
4.3.5. 1-(3-(2,4-Dihydroxyphenyl)-5-(4-hydroxy-3-nitrophenyl)-
4,5-dihydro-1H-pyrazol-1-yl) (1e)
4.2. General procedure for the preparation of
polyhydroxychalcones
Yield 68%, yellow powder, mp 275–276 °C, IR(KBr):
3227( O–H), 1673( C@O), 1629( C@N), 1597, 1535, 1488, 1421
as), 1317(
C–N). 1H NMR (DMSO-d6): d (ppm): 11.35(s, 1H, OH),
m
(cmꢀ1):
m
m
m
(m
m
Polyhydroxyactetophenone (2 mmol) and polyhydroxybenzal-
dehyde (2 mmol) was mixed with neutral Al2O3 (1.6 g) and grinded
in mortar, then KOH (0.8 g) was added and mixed. The obtained
mixture was irradiated at 400 W under domestic microwave oven.
After 30–70 s, the reaction mixture had become reddish orange.
Upon completion, monitored by TLC, the reaction mixture was
cooled to room temperature and then treated with water. The mix-
ture was filtered and the resulting solution was acidified by 1 N
HCl. The resultant precipitate was filtered off, washed with water,
and purified by recrystallization or column chromatography.
11.21(s, 2H, OH), 7.41–6.19(m, 6H, ArH), 5.37(dd, 1H, CH,
J = 11.68, 4.13 Hz), 3.80(dd, 1H, HM, J = 11.55, 18.20 Hz), 3.11(HA,
d, merged & appeared as doublet), 1.83(s, 3H, COCH3).
4.3.6. 1-(5-(3,4-Dihydroxyphenyl)-3-(4-hydroxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (1f)
Yield 40%, light yellow powder, mp 216–217 °C, IR(KBr):
(cmꢀ1): 3247(
O–H), 1670( C@O), 1611( C@N), 1574, 1521, 1485,
1441( as), 1364(
C–N). 1H NMR (DMSO-d6): d (ppm): 10.03(s, 1H,
m
m
m
m
m
m
OH), 9.43(s, 2H, OH), 8.22–6.77(m, 7H, ArH), 5.29(dd, 1H, CH,
J = 11.39, 3.75 Hz), 3.82(dd, 1H, HM, J = 11.55, 17.85 Hz), 3.03(HA,
d, merged & appeared as doublet), 1.93(s, 3H, COCH3).
4.3. General procedure for the preparation of compounds 1a–1p
To a stirred solution of polyhydrochalcone (1 mmol) in acetic
acid, hydrazine hydrate (80% aqueous solution, 5 mmol) was
added. The mixture was then refluxed for a period of 50–90 min,
monitored by TLC. Upon completion, the solution was cooled to
room temperature. Saturated Na2CO3 solution was added to neu-
tralize the solution. The resultant precipitate was filtered and
recrystallized from methanol to afford compound 1a–1p.
4.3.7. 1-(3,5-Bis(4-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone (1g)
Yield 53%, orange powder, mp 171–173 °C, IR(KBr):
m
(cmꢀ1):
C@N), 1572, 1516, 1441( as),
C–N). 1H NMR (DMSO-d6): d (ppm): 9.63(s, 2H, OH), 7.59(d,
3319(
m
O–H), 1655(
m
C@O), 1609(
m
m
1362(
m
2H, ArH), 7.00–6.40(m, 4H, ArH), 5.37(dd, 1H, CH, J = 11.32,
3.73 Hz), 3.83(dd, 1H, HM, J = 11.56, 17.54 Hz), 3.13(HA, d, merged
& appeared as doublet), 1.81(s, 3H, COCH3).
4.3.1. 1-(3-(2,4-Dihydroxyphenyl)-5-(3,4-dihydroxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (1a)
4.3.8. 1-(5-(3-Hydroxyphenyl)-3-(4-hydroxyphenyl)-4,5-
Yield 26%, light brown powder, mp 259–261 °C, IR(KBr):
(cmꢀ1): 3256(
O–H), 1672( C@O), 1614( C@N), 1565, 1514,
1480( as), 1363(
C–N). 1H NMR (DMSO-d6): d (ppm): 11.06(s, 1H,
OH), 10.94(s, 1H, OH), 9.30(s, 1H, OH), 9.13(s, 1H, OH), 7.84(d,
2H, ArH), 6.96(m, 4H, ArH), 5.37(dd, 1H, CH, J = 11.49, 3.43 Hz),
3.80(dd, 1H, HM, J = 11.67, 17.90 Hz), 3.19(1H, HA, d, merged & ap-
peared as doublet), 1.90(s, 3H, COCH3).
m
dihydro-1H-pyrazol-1-yl)ethanone (1h)
m
m
m
m
Yield 57%, light yellow powder, mp 134–135 °C, IR(KBr):
(cmꢀ1): 3241(
O–H), 1653( C@O), 1604( C@N), 1518, 1458( as),
1365(
C–N). 1H NMR (DMSO-d6): d (ppm): 10.02(s, 1H, OH),
m
m
m
m
m
m
m
9.55(s, 1H, OH), 7.80–6.35(m, 8H, ArH), 5.43(dd, 1H, CH, J = 11.49,
3.93 Hz), 3.12(dd, 1H, HM, J = 10.23, 17.90 Hz), 2.91(dd, 1H, HA,
J = 17.89, 4.19 Hz), 1.85(s, 3H, COCH3).
4.3.2. 1-(3-(2,4-Dihydroxyphenyl)-5-(4-hydroxyphenyl)-4,5-
4.3.9. 1-(5-(4-Hydroxy-3-methoxyphenyl)-3-(4-
dihydro-1H-pyrazol-1-yl)ethanone (1b)
hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (1i)
Yield 42%, yellow brown powder, mp 233–235 °C, IR(KBr):
(cmꢀ1): 3276(
O–H), 1672( C@O), 1626( C@N), 1515, 1475,
1419( as), 1248(
C–N). 1H NMR (DMSO-d6): d (ppm): 10.40(s, 1H,
m
Yield 51%, light yellow powder, mp 216–217 °C, IR(KBr):
(cmꢀ1): 3438(
O–H), 1665( C@O), 1610( C@N), 1596, 1576, 1514,
1433( as), 1364(
C–N). 1H NMR (DMSO-d6): d (ppm): 9.98(s, 1H,
m
m
m
m
m
m
m
m
m
m
m
OH), 9.90(s, 1H, OH), 9.14(s, 1H, OH), 6.89(m, 7H, ArH), 5.34(dd,
1H, CH, J = 11.41, 3.54 Hz), 3.80(dd, 1H, HM, J = 11.43, 17.76 Hz),
3.14(dd, 1H, HA, J = 18.16, 3.78 Hz), 1.78(s, 3H, COCH3).
OH), 9.64(s, 1H, OH), 7.85–6.41(m, 7H, ArH), 5.37(dd, 1H, CH,
J = 11.50, 3.81 Hz), 3.71(s, 3H, OCH3), 3.82(dd, 1H, HM, J = 11.58,
18.01 Hz), 3.03(dd, 1H, HA, J = 17.86, 4.28 Hz), 1.83(s, 3H, COCH3).

Z. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 2156–2162
2161
4.3.10. 1-(5-(3,4-Dihydroxyphenyl)-3-(2-hydroxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (1j)
4.4. General procedure for the preparation of compounds 2a–2d
Yield 45%, light yellow powder, mp 119–120 °C, IR(KBr):
m
To a stirred solution of polyhydrochalcone (1 mmol) in ethanol,
hydrazine hydrate (80% aqueous solution, 10 mmol) was added.
The mixture was then refluxed for a period of 6 h, monitored by
TLC. Upon completion, the solution was cooled to room tempera-
ture. Water was added to the reaction mixture. The products were
extracted from the one-third concentrated solution of reaction
mixture using chloroform. The organic layer was then evaporated
to yield the required crude 3,5-diaryl pyrazole derivatives. Resi-
dues obtained were purified by column chromatography to afford
pure 3,5-diaryl pyrazole derivatives.
(cmꢀ1): 3233(
m
O–H), 1659(
mC@O), 1612(mC@N), 1563, 1494,
1447(mas), 1287(m
C–N). 1H NMR (DMSO-d6): d (ppm): 10.53(s, 1H,
OH), 9.05(s, 2H, OH), 7.87–6.27(m, 7H, ArH), 5.30(dd, 1H, CH,
J = 11.45, 3.80 Hz), 3.38(dd, 1H, HM, J = 11.61, 17.59 Hz), 3.18(dd,
1H, HA, J = 18.39, 4.29 Hz), 1.74(s, 3H, COCH3).
4.3.11. 1-(3-(2-Hydroxyphenyl)-5-(4-hydroxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (1k)
Yield 50%, yellow powder, mp 242–243 °C, IR(KBr):
m
(cmꢀ1):
as),
C–N). 1H NMR (DMSO-d6): d (ppm): 11.31(s, 1H, OH),
3346(
m
m
O–H), 1655(mC@O), 1608(mC@N), 1592, 1564, 1513, 1448(m
1263(
4.4.1. 4-(5-(4-Hydroxyphenyl)-4,5-dihydro-1H-pyrazol-3-
9.14(s, 1H, OH) 8.85–6.25(m, 8H, ArH), 5.30(dd, 1H, CH, J = 11.32,
3.62 Hz), 3.88(dd, 1H, HM, J = 11.44, 17.92 Hz), 3.15(HA, d, merged
& appeared as doublet), 1.90(s, 3H, COCH3).
yl)benzene-1,3-diol (2a)
Yield 19%, orange powder, mp 225–226°C, IR(KBr):
m
(cmꢀ1):
as), 1314( C–N).
3323(
m
O–H), 1657(
m
C@N), 1573, 1559, 1509, 1436(
m
m
1H NMR (DMSO-d6): d (ppm): 11.43(s, 1H, NH), 10.06(s, 1H, OH),
9.92(s, 1H, OH), 9.68(s, 1H, OH), 7.70–6.40(m, 7H, ArH), 4.92(dd,
1H, CH, J = 11.82, 11.02 Hz), 3.56(dd, 1H, HM, J = 16.45, 10.87 Hz),
3.18(dd, 1H, HA, J = 13.91, 13.70 Hz).
4.3.12. 1-(3-(2-Hydroxyphenyl)-5-(3-hydroxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (1l)
Yield 55%, light brown powder, mp 177–178 °C, IR(KBr):
(cmꢀ1): 3173(
O–H), 1681( C@O), 1636( C@N), 1589, 1461( as),
1311(
C–N). 1H NMR (DMSO-d6): d (ppm): 10.33(s, 1H, OH),
m
m
m
m
m
m
4.4.2. 4-(5-(3-Hydroxyphenyl)-4,5-dihydro-1H-pyrazol-3-
9.49(s, 1H, OH), 7.60–6.66(m, 8H, ArH), 5.38(dd, 1H, CH, J = 11.61,
3.74 Hz), 3.89(dd, 1H, HM, J = 11.80, 17.25 Hz), 3.18(dd, 1H, HA,
J = 18.36, 4.23 Hz) 1.78(s, 3H, COCH3).
yl)benzene-1,3-diol (2b)
Yield 26%, orange powder, mp 179–181 °C, IR(KBr):
m
(cmꢀ1):
3287( O–H), 1650( C@N), 1579, 1511, 1452( as), 1330(m
m
m
m
C–N). 1H
NMR (DMSO-d6): d (ppm): 10.92(s, 1H, NH), 10.38(s, 1H, OH),
10.01(s, 1H, OH), 9.41(s, 1H, OH), 7.80–6.30(m, 7H, ArH), 5.12(dd,
1H, CH, J = 10.12, 9.43 Hz), 3.33(dd, 1H, HM, J = 14.92, 10.23 Hz),
3.06(dd, 1H, HA, J = 14.72, 12.00 Hz). 13C NMR (DMSO-d6): d
(ppm): 160.01, 159.42, 156.96, 144.19, 131.47, 130.02, 122.17,
122.48, 118.32, 117.77, 110.65, 109.12, 100.66, 69.89, 41.50.
4.3.13. 1-(5-(4-Hydroxy-3-methoxyphenyl)-3-(2-
hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (1m)
Yield 49%, light brown powder, mp 211–213 °C, IR(KBr):
(cmꢀ1): 3430(
O–H), 1677( C@O), 1630( C@N), 1569, 1507,
1440( as), 1345(
C–N). 1H NMR (DMSO-d6): d (ppm): 11.30(s, 1H,
m
m
m
m
m
m
OH), 9.89(s, 1H, OH), 7.54–6.14(m, 7H, ArH), 5.35(dd, 1H, CH,
11.63, 3.90 Hz), 3.76(s, 3H, OCH3), 3.84(dd, 1H, HM, J = 11.91,
17.60 Hz), 3.09(HA, d, merged & appeared as doublet), 1.82(s, 3H,
COCH3).
4.4.3. 4-(5-(4-Hydroxy-3-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-3-yl)benzene-1,3-diol (2c)
Yield 22%, brown powder, mp 126–127°C, IR(KBr):
m
(cmꢀ1):
3309( O–H), 1661( C@N), 1591, 1556, 1434( as), 1351(m
m
m
m
C–N). 1H
4.3.14. 1-(5-(4-Hydroxy-2-methoxyphenyl)-3-(2-
hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (1n)
NMR (DMSO-d6): d (ppm): 11.23(s, 1H, NH), 10.45(s, 1H, OH),
9.82(s, 1H, OH), 9.32(s, 1H, OH), 7.40–6.20(m, 6H, ArH), 4.91(dd,
1H, CH, J = 9.62, 8.49 Hz), 3.72(s, 3H, OCH3), 3.62(dd, 1H, HM,
J = 16.15, 12.09 Hz), 3.15(dd, 1H, HA, J = 16.16, 10.09 Hz). 13C
Yield 42%, light brown powder, mp 175–176 °C, IR(KBr):
(cmꢀ1): 3263(
O–H), 1694( C@O), 1657( C@N), 1598, 1560, 1519,
1428( as), 1309(
C–N). 1H NMR (DMSO-d6): d (ppm): 9.64(s, 1H,
m
m
m
m
m
m
NMR (DMSO-d6): d (ppm): 160.92, 160.18, 149.41, 146.23,
OH) 8.54(s, 1H, OH), 7.45–6.50(m, 7H, ArH), 5.33(dd, 1H, CH,
J = 11.21, 3.45 Hz), 3.63(s, 3H, OCH3), 3.88(dd, 1H, HM, J = 11.41,
17.32 Hz), 3.14(HA, d, merged & appeared as doublet), 1.79(s, 3H,
COCH3).
145.88, 128.69, 126.34, 122.48, 118.74, 116.33, 115.51, 114.70,
112.37, 70.23, 58.19, 41.52
4.4.4. 2-(5-(4-Hydroxyphenyl)-4,5-dihydro-1H-pyrazol-3-
yl)phenol (2d)
4.3.15. 1-(3-(3,5-Dihydroxyphenyl)-5-(4-hydroxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (1o)
Yield 33%, brown powder, mp 232–234 °C, IR(KBr):
m
(cmꢀ1):
3271( O–H), 1663( C@N), 1569, 1506, 1452( as), 1387(m
m
m
m
C–N). 1H
Yield 59%, orange powder, mp 264–265 °C, IR(KBr):
m
(cmꢀ1):
C@N), 1591, 1565, 1448( as),
C–N). 1H NMR (DMSO-d6): d (ppm): 9.55(s, 3H, OH), 6.93(d,
NMR (DMSO-d6): d (ppm): 12.10(s, 1H, NH), 11.13(s, 1H, OH),
10.32(s, 1H, OH), 7.30–6.50(m, 8H, ArH), 5.03(dd, 1H, CH, J = 9.83,
8.62 Hz), 3.62(dd, 1H, HM, J = 16.31, 10.45 Hz), 3.15(dd, 1H, HA,
J = 16.34, 8.31 Hz).
3346(
1303(
m
m
O–H), 1661(
m
C@O), 1610(
m
m
2H, ArH), 6.66(d, 2H, ArH), 6.60(d, 2H, ArH), 6.28(t, 1H, ArH),
5.36(dd, 1H, CH, J = 11.57, 4.12 Hz), 3.83(dd, 1H, HM, J = 11.65,
17.13 Hz), 3.14(dd, HA, J = 18.13, 3.31 Hz), 1.74(s, 3H, COCH3).
Acknowledgments
4.3.16. 1-(3-(3,5-Dihydroxyphenyl)-5-(3-hydroxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (1p)
The project was supported by the National Natural Science
Foundation of China (J1103305).
Yield 63%, light brown powder, mp 165–167 °C, IR(KBr):
m
The authors thank Yurong Liu for technical assistance.
(cmꢀ1): 3288( C–N). 1H
m
O–H), 1662( C@N), 1588, 1463( as), 1288(
m
m
m
NMR (DMSO-d6): d (ppm): 9.38(s, 2H, OH), 9.20(s, 1H, OH), 7.30–
6.30(m, 7H, ArH), 5.37(dd, 1H, CH, J = 11.33, 4.23 Hz), 3.82(dd,
1H, HM, J = 11.54, 17.45 Hz), 2.96(dd, 1H, HA, J = 17.79, 3.90 Hz),
1.75(s, 3H, COCH3).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.054.

2162
Z. Zhou et al. / Bioorg. Med. Chem. 21 (2013) 2156–2162
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