Synthesis, biological evaluation, and molecular docking studies of new pyrazol-3-one derivatives with aromatase inhibition activities

Article abstract of DOI:10.1111/cbdd.12812

A new series derived from 4-(2-chloroacetyl)-1,2-dihydro-1,5-dimethyl-2-phenyl-3H-pyrazol-3-one was synthesized, characterized and its pharmacological activity toward aromatase enzyme inhibition was screened and compared to the reference native ligand letrozole. The most active compound of the series was 16, showing IC₅₀ value of 0.0023?±?0.0002?μm compared to letrozole with IC₅₀ of 0.0028?±?0.0006?μm. In addition, compounds 26 and 36 exhibit good inhibition activities close to letrozole with IC₅₀ values 0.0033?±?0.0001 and 0.0032?±?0.0003?μm, respectively. Moreover, molecular docking studies were conducted to support the findings.

Full text of DOI:10.1111/cbdd.12812

Received: 6 April 2016
Revised: 5 June 2016
Accepted: 11 June 2016
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DOI: 10.1111/cbdd.12812
R E S E A R C H A R T I C L E
Synthesis, biological evaluation, and molecular docking studies of
new pyrazol-3-one derivatives with aromatase inhibition activities
Xue-Jing Yi1
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Tamer T. El-Idreesy2,3
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Taha M. A. Eldebss2
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Ahmad M. Farag2
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Mohamed M. Abdulla4
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Shaimaa A. Hassan2 Yahia N. Mabkhot5
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1Yueyang Vocational Technical College,
Yueyang, China
2Department of Chemistry, Faculty of
Science, Cairo University, Giza, Egypt
3Department of Chemical Engineering, Faculty
of Engineering, The British University in Egypt,
El-Shorouk City, Egypt
4Research Unit, Saco Pharm. Co., 6th October
City, Egypt
A new series derived from 4-(2-chloroacetyl)-1,2-dihydro-1,5-dimethyl-2-phenyl-
3H-pyrazol-3-one was synthesized, characterized and its pharmacological activity
toward aromatase enzyme inhibition was screened and compared to the reference
native ligand letrozole. The most active compound of the series was 16, showing IC50
value of 0.0023 0.0002 μm compared to letrozole with IC50 of 0.0028 0.0006 μm.
In addition, compounds 26 and 36 exhibit good inhibition activities close to letrozole
with IC50 values 0.0033 0.0001 and 0.0032 0.0003 μm, respectively. Moreover,
molecular docking studies were conducted to support the findings.
5Department of Chemistry, College of
Science, King Saud University, Riyadh, Saudi
Arabia
K E Y W O R D S
Correspondence
Tamer T. El-Idreesy, tamertawhid@yahoo.com;
Taha M. A. Eldebss, taha_eldebss@yahoo.com
aromatase activity inhibitors, IC50, letrozole, molecular docking studies, pyrazol-3-one
The majority of hormone-dependent cancers such as breast
cancer are related to estrogen hormone. Estrogen was found
to play a key role in the progression and metastasis of such
tumors.[1,2] Tamoxifen represents one of the treatments for
hormone-dependent cancers by blocking the binding of
estrogen to the estrogen receptor.[3–5] However, resistance
to tamoxifen therapy was developed and consequently, the
search for an alternative treatment protocol has become a
crucial matter.[6] Inhibition of estrogen synthesis represents
another promising therapeutic approach causing the body to
produce less estrogen through the inhibition of aromatase
enzyme.[7–9] Aromatase is an enzyme found in the liver and
is responsible for the conversion of the androgens, andro-
stenedione, and testosterone into the estrogens, estrone,
and estradiol.[10,11] Several generations of aromatase
inhibitors were developed, and letrozole was found to be a
highly potent competitor of tamoxifen as estrogen blocker
(Figure 1).[12–16]
several applications in the field of medicinal chemistry and
pharmaceuticals. Compounds with pyrazole subunit show
potent anticancer,[17–22] antibacterial,[23] and antifungal[24]
activities. Phenazone and ampyrone are two potent drugs
showing a 3-pyrazolone ring in their structure that is believed
to contribute to their effective pharmacological behavior
(Figure 1). Phenazone drug is used as analgesic, non-steroidal
anti-inflammatory drug,[25] whereas ampyrone shows antiox-
idant and anti-inflammatory activity.[26] Other 3-pyrazolone
drugs were reported to reduce fever, treat arthritis,[27,28]
be used in veterinary medicine,[29] and also to show anti-
estrogenic activity,[21,22] This anti-estrogenic pharmacologi-
cal activity stimulated our attention to synthesize a new series
of heterocyclic systems containing the 3-pyrazolone core
and examine their biological activity as aromatase enzyme
inhibitors. For this purpose, we synthesized 4-(2-chloroace-
tyl)-1,2-dihydro-1,5-dimethyl-2-phenyl-3H-pyrazol-3-one[30]
which was then converted to 1,2-dihydro-1,5-dimethyl-2-phe
nyl-4-thiocyanatoacetyl-3H-pyrazol-3-one (2), the main pre-
cursor of this series of functionalized 3-pyrazolones.
Diverse pharmacological activities have been associat-
ed with pyrazole-containing heterocyclic compounds with
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Chem Biol Drug Des 2016; 1–12
wileyonlinelibrary.com/journal/cbdd
© 2016 John Wiley & Sons A/S.
1

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FIGURE 1 The structure of
letrozole, ampyrone and phenazone.
washed by water, and dried. Recrystallization from ethanol
afforded oxathiine-2-imine derivative 4 in 60% yield, m.p.:
208–210 °C, IR (KBr) ν (cm−1) = 3449 (NH), 1663 (C=O),
1597 (C=N). 1HNMR (DMSO-d6) δ (ppm) = 2.49 (s, 3H),
3.36 (s, 3H), 7.1 (s, 1H, NH/D2O-exchangeable), 7.36–7.59
(m, 5H, ArH), 8.1 (s, 1H, CH-oxathiole). MS, m/z = 288
(M+ + 1), 287 (M+), 215, 187, 77, 56. For C14H13N3SO2,
(287.34) Calcd.: C: 58.52; H: 4.56; N: 14.62; S: 11.16%.
Found: C: 58.45; H: 4.65; N: 14.62; S: 11.18%.
1
EXPERIMENTAL PROTOCOL
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All melting points were measured on a Gallenkamp melt-
ing point apparatus. The infrared spectra were recorded
in potassium bromide disks on a Pye Unicam SP 3300
(Pye Unicam Ltd., Cambridge, UK) and Shimadzu FT IR
8101 PC (Schimadzu, Tokyo, Japan) infrared spectropho-
tometers. The NMR spectra were recorded on a Varian
1
Mercury VX-300 NMR spectrometer. H (300 MHz) and
13C NMR (75 MHz) were run in deuterated chloroform
(CDCl3) or dimethylsulfoxide (DMSO-d6). Chemical
shifts were related to that of the solvent. Mass spectra
were recorded on a Shimadzu GCMS-QP 1000 EX mass
spectrometer (Schimadzu) at 70 eV. Elemental analyses
were carried out at the Microanalytical Center of Cairo
University, Giza, Egypt.
1.3
4-[4-(1,2-Dihydro-1,5-dimethyl-2-
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phenyl-3-oxo-3H-pyrazol-4-ylcarbonyl)]-5-
phenylamino-1,3-dithiole-2-imine (7)
To a stirred solution of potassium hydroxide (0.56 g,
0.01 mmol) in dimethylformamide (20 mL) was added 1,2
-dihydro-1,5-dimethyl-2-phenyl-4-thiocyanatoacetyl-3H-py
razol-3-one (2) (2.87 g, 0.01 mmol). Phenyl isothiocyanate
(1.35 g, 0.01 mmol) was added to the produced mixture
after stirring for 30 min. Stirring was continued for 6 h and
then poured over crushed ice containing hydrochloric acid.
The formed product was filtered off, washed with water,
dried, and finally recrystallized from DMF/water to give
1,3-dithiole-2-imine derivative 7 in 70% yield, m.p.: 197–
199 °C, IR (KBr) ν (cm−1) = 3449, 3419 (2NH), 1643, 1634
(2C=O), MS, m/z; 442(M+), 388, 256, 215, 177, 77, 56,
1HNMR (DMSO-d6) δ (ppm) = 2.57 (s, 3H), 3.33 (s, 3H),
7.1 (s, 1H, NH/D2O-exchangeable), 7.3–7.49 (m, 10H, ArH),
7.8 (s, 1H, NH/D2O-exchangeable). For C21H18N4O2S2
(422.53), Calcd.: C: 59.70; H: 4.29; N: 13.26; S: 15.18%.
Found: C: 59.88; H: 4.34; N: 13.28; S: 15.38%.
1.1
1,2-Dihydro-1,5-dimethyl-2-phenyl-4-
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thiocyanatoacetyl-3H-pyrazol-3-one (2)
To a solution of 4-(2-chloroacetyl)-1,2-dihydro-2,3-dime
thyl-1-phenylpyrazol-5-one (1) (26.45 g, 0.1 mol) in etha-
nol was added potassium thiocyanate (97 g, 0.1 mol). The
reaction mixture was refluxed for 1 h then allowed to cool.
The reaction mixture poured onto cold water with continu-
ous stirring. The solid product was collected by filtration,
washed with water, dried, and finally recrystallized from
ethanol to afford the corresponding product in 90% yield,
m.p.: 145–147 °C, IR (KBr) ν (cm−1) = 2152 (SCN), 1643,
1633 (2C=O), 1589 (C=N), MS, m/z; 287 (M+, 80%), 215
1
(90%), 187 (18%), 100 (10%), 77 (20%), HNMR (DMSO-
d6) δ (ppm) = 2.49 (s, 3H), 3.36 (s, 3H), 4.5 (s, 2H), 7.36–
7.59 (m, 5H). For C14H13N3SO2 (287.34), Calcd.: C: 58.52;
H: 4.56; N: 14.62; S: 11.16%. Found: C: 58.45; H: 4.65; N:
14.62; S: 11.18%.
1.4
2-[4-(1,2-Dihydro-1,5-dimethyl-
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2-phenyl-3-oxo-3H-pyrazol-4-yl)]-1,3,4-
thidiazole[3,2-a]-1,3-quinazoline-2-imine (10a)
To a cold solution of compound 2 (2.87 g, 0.01 mmol) in
ethanol (50 mL) and sodium acetate trihydrate (5 g) was
added the diazonium salt solution of anthranilonitrile (1.18 g,
0.01 mmol), while stirring over a period of 30 min. After
the addition was complete, the reaction was stirred for fur-
ther 3 h at 0–5 °C and left to stand in an ice box for 12 h
then diluted with water. The solid so formed was filtered
1.2
6-[4-(1,2-Dihydro-1,5-dimethyl-
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2-phenyl-3-oxo-3H-pyrazol-4-yl)-1,2,3,4-
tetrahydro-1,3-oxathiine-2-imine (4)
A solution of compound 2 in pyridine was refluxed for 1 h
then allowed to cool. The reaction mixture was diluted
with water. The solid product was collected by filtration,

Yi et al.
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off, washed with water, and dried. Recrystallization from
DMF afforded thidiazole[3,2-a]-1,3-quinazoline-2-imine
derivative 10a in 70% yield, m.p.: 262–264 °C, IR (KBr) ν
(cm−1) = 3425 (NH), 1643, 1633 (2C=O), 1HNMR (DMSO-
d6) δ (ppm) = 2.61 (s, 3H), 3.2 (s, 3H), 4.5 (s, 1H), 7.35–7.56
(m, 9H, ArH). For C21H16N6O2S, (416.46) Calcd.: C: 60.57;
H: 3.87; N: 20.18; S: 7.70%. Found: C: 60.57; H: 3.99; N:
19.99; S: 7.55%.
of 1 h and heating was continued for an additional 1 h.
The reaction flask was removed from the oil bath, left
to cool, and triturated with water. The solid product was
filtered off, washed with water several times, and dried.
Recrystallization from ethanol afforded the corresponding
product thiocyanato-3-ethoxypropenone derivative 11 in
80% yield, m.p.: 286–287 °C, IR (KBr) ν (cm−1) = 2152
(SCN), 1643, 1633 (2C=O), MS, m/z; 343 (M+), 328, 287,
1
215, 187, 77, 56. HNMR (DMSO-d6) δ (ppm) = 1.36 (t,
3H). 2.49 (s, 3H), 3.3 (s, 3H), 3.9 (q, 2H), 6.9 (s, 1H), 7.35–
7.59 (m, 5H, ArH). 13C NMR (DMSO-d6) δ (ppm) = 13.8,
15.2, 33.4 (3CH3), 56.3 (CH2),117.8, 122.5, 125.9, 130.7,
145.3, 152.5 (Ar-C), 125.1 (CH=O), 126.3 (C=), 136.8
(SCN). 164.8 (C=O). For C17H17N3SO3 (343.41), Calcd.:
C: 59.46; H: 4.99; N: 12.24; S: 9.34%. Found: C: 59.51; H:
5.01; N: 12.34; S: 9.36%.
1.5
2-[4-(1,2-Dihydro-1,5-dimethyl-
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2-phenyl-3-oxo-3H-pyrazol-4-yl]-1,3,4
-thiazolo[3,2-a]-1,3-quinazolin-2-one (10b)
1.5.1
Method A
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To a cold solution of compound 2 (2.87 g, 0.01 mmol) in
ethanol (50 mL) and sodium acetate trihydrate (5 g) was
added the diazonium salt solution of methyl anthranilate
(1.51 g, 0.01 mmol), while stirring over a period of 30 min.
After the addition was complete, the reaction mixture was
stirred for further 3 h at 0–5 °C and left to stand in an ice box
for 12 h then diluted with water. The solid that formed was
filtered off, washed with water, and dried. Recrystallization
from DMF afforded thidiazole [3,2-a]-1,3-quinazoline-2-one
derivative 10b in 70% yield, m.p.: 270–272 °C, IR (KBr) ν
(cm−1) = 1710, 1690, 1639 (3C=O), 1598, 1590 (2C=N);
1HNMR (DMSO-d6) δ (ppm) = 2.7 (s, 3H), 3.3 (s, 3H),
7.39–8.2 (m, 9H, ArH). For C21H15N5O3S (417.45), 13C
NMR (DMSO-d6) δ (ppm) = 13.8 (CH3), 30.4 (CH3), 114.8,
122.5, 122.7, 125.9, 126.5, 126.8, 128.0, 128.2, 128.6, 128.9,
129.7, 130.7, 135.3, 142.5 (Ar-C), 162.8, 163.5. 164.8
(3C=O) ppm. Calcd.: C: 60.42; H: 3.62; N: 16.78; S: 7.68%.
Found: C: 60.44; H: 3.42; N: 16.87; S: 7.68%.
1.7
1-Phenyl-3-[4-(1,2-Dihydro-1,5-
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dimethyl-2-phenyl-3-oxo-3H-pyrazol-4-
ylcarbonyl]-4-thiocyanatopyrazole (13)
To a solution of thiocyanato-3-ethoxypropenone deriva-
tive 11 (3.43 g, 0.01 mmol) in ethanol (20 mL) was added
phenylhydrazine hydrate (1.26 g, 0.01 mmol). The reac-
tion mixture was refluxed for 2 h then cooled, and the solid
precipitate was filtered off, washed with water, and dried.
Recrystallization from ethanol afforded the corresponding
product derivative thiocyanatopyrazole 13 in 70% yield,
m.p.: 256–257 °C, IR (KBr) ν (cm−1) = 2052 (SCN), 1627
1
(C=O), 1597 (C=N). HNMR (DMSO-d6) δ (ppm) = 2.49
(s, 3H), 3.36 (s, 3H), 5.8 (s, 1H, CH-pyrazole), 7.36–7.59
(m, 10H, ArH). 13C NMR (DMSO-d6) δ (ppm) = 14.8
(CH3), 34.4 (CH3), 117.8, 122.5, 124.7, 125.9, 126.8,
128.0, 128.2, 128.6, 128.9, 129.7, 130.7, 135.3, 142.5 (Ar-
C), 136.8 (SCN). 164.8 (C=O). MS; m/z: 417.13 (100.0%),
418.13 (24.9%), 419.12 (4.5%), For C22H19N5O2S (417.13),
Calcd.: C: 63.29; H: 4.59; N: 16.78; S: 7.68%. Found: C:
63.31; H: 4.58; N: 16.78; S: 7.70%.
1.5.2
Method B
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To a cold solution of compound 10a (12.48 g, 30 mmol) in
ethanol (20 mL) was added hydrochloric acid (37%, 5 mL).
The reaction mixture was refluxed for 4 h then left to cool, and
the precipitate so formed was collected by filtration, washed
with water, and dried. Recrystallization from dioxane afford-
ed compound which is identical in all respects (m.p., mixed
m.p., and spectra) with that obtained by method A above.
1.8
3-[4-(1,2-Dihydro-1,5-dimethyl-
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2-phenyl-3-oxo-3H-pyrazol-4-ylcarbonyl]
isoxazole (16)
To a solution of thiocyanato-3-ethoxypropenone deriva-
tive 11 (3.43 g, 0.01 mmol) in ethanol (20 mL) was added
hydroxylamine hydrochloride (0.69 g, 0.01 mmol) in pres-
ence of equivalent amount of potassium carbonate. The
reaction mixture was heated under reflux for 2 h then left
to cool. The precipitate was filtered off, washed with water,
and dried. Recrystallization from ethanol afforded the cor-
responding product isoxazole derivative 16 in 65% yield,
m.p.: 223–225 °C, IR (KBr) ν (cm−1) = 20 525 (SCN), 1641
(C=O). 1HNMR (DMSO-d6) δ (ppm) = 2.49 (s, 3H), 3.36 (s,
1.6
1-[4-(1,2-Dihydro-1,5-dimethyl-2-
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phenyl-3-oxo-3H-pyrazol-4-yl)-3-ethoxy-2-
thiocyanatopropenone (11)
In a 250 mL three-necked round-bottomed flask, fitted
with air condenser and thermometer were placed triethyl
orthoformate (29.6 g, 200 mmol). The flask was immersed
in an oil bath heated to 145–150 °C and then compound 2
(5.74 g, 200 mmol) was added portion wise over a period

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Yi et al.
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3H), 6.3 (s, 1H, CH-isooxazole), 7.36–7.59 (m, 5H, ArH).
MS; m/z: 314.08 (80.0%), 315.09 (16.5%), 316.08 (14.8%),
315.08 (2.3%), For C15H14N4O2S (314.08), Calcd.: C: 57.31;
H: 4.49; N: 17.82; O: 10.18; S: 10.20%. Found: C: 57.31; H:
4.49; N: 17.82; O: 10.18; S: 10.20.
260–262 °C, IR (KBr) ν (cm−1) = 1643, 1633 (2C=O) 1587
(C=N). MS; m/z: 388.10 (60.0%), 389.10 (25.1%), 390.10
(7.5%). 1HNMR (DMSO-d6) δ (ppm) = 2.49 (s, 3H), 3.36 (s,
3H), 6.2 (s, 1H, CH-thiazole), 7.26–7.79 (m, 9H). 13C NMR
(DMSO-d6) δ (ppm) = 14.8 (CH3), 34.8 (CH3), 117.8, 122.5,
124.7, 125.9, 126.8, 128.0, 128.2, 128.6, 128.9, 129.7, 130.7,
135.3, 137.3, 139.7 142.5 (Ar-C), 162.8, 163.5 (2C=O) For
C21H16N4O2S (388.45), Calcd.: C: 64.93; H: 4.15; N: 14.42;
S: 8.25%. Found: C: 65.01; H: 4.15; N: 14.34; S: 8.25%.
1.9
2-Hydroxy-4-([4-(1,2-dihydro-1,5-
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dimethyl-2-phenyl-3-oxo-3H-pyrazol-4-yl)
thiazole (25)
A mixture of compound 2 (2.87 g, 10 mmol), sulfuric
acid (5 mL), and acetic acid (5 mL) was refluxed for
30 min then cooled. The solid product was filtered off,
washed with water, and dried. Recrystallization from
ethanol afforded thiazole derivative 25 in 70% yield,
m.p.: 222–224 °C, IR (KBr) ν (cm−1) = 3418 (OH), 1643
(C=O). 1HNMR (DMSO-d6) δ (ppm) = 2.49 (s, 3H),
3.36 (s, 3H), 6.1 (s, 1H, CH-thiazole), 9.1 (s, 1H, OH/
D2O-exchangeable), 7.36–7.59 (m, 5H, ArH). MS; m/z:
287.07 (70.0%), 288.08 (15.4%), 289.08 (11.6%). For
C14H13N3O2S (287.07), Calcd.: C: 58.52; H: 4.56; N:
14.62; S: 11.16%. Found: C: 58.44; H: 4.66; N: 14.62;
S: 11.17%.
1.11.2
Method B
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A mixture of 2-chloro-4-[4-(1,2-dihydro-1,5-dimethyl-2-phe
nyl-3-oxo-3H-pyrazol-4-yl)thiazole (26) (3.88 g, 0.01 mmol)
and methyl anthranilate hydrobromide (3.32 g, 0.01 mmol)
in presence of phenol (4 mL) was heated into an oil bath at
150–180 °C for 7 h then left to cool. The precipitated solid
was filtered off, washed with water, dried, and recrystallized
from acetic acid to afford a product identical in all respects
(m.p., mixed m.p., and spectra) with that obtained from reac-
tion of compound 2 with methyl anthranilate hydrobromide
as in method A above.
1.12
1-[4-(1,2-Dihydro-1,5-dimethyl-
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2-phenyl-3-oxo-3H-pyrazol-4-yl)]-3-(4-
1.10
2-Chloro-4-[4-(1,2-dihydro-1,5-
methoxyphenyl)-2-thiocyanatopropenone (30)
|
dimethyl-2-phenyl-3-oxo-3H-pyrazol-4-yl)
thiazole (26)
To a solution of compound 2 (2.87 g, 0.01 mmol)in etha-
nol (20 mL) was added p-methoxy benzaldehyde (1.36 g,
0.01 mmol) in presence of few drops of piperidine as cata-
lyst. The reaction mixture was refluxed for 3 h then cooled.
The solid product so formed was collected by filtration,
washed with ethanol, and dried. Recrystallization from eth-
anol afforded compound 30 in 90% yield, m.p.: 255 °C, IR
(KBr) ν (cm−1) = 2152 (SCN), 1643, 1633 (2C=O), 1589
(C=N), 1HNMR (DMSO-d6) δ (ppm) = 2.49 (s, 3H), 2.6 (s,
3H), 3.2 (s, 3H), 3.4 (s, 1H), 4.5 (s, 1H), 7.3–7.56 (m, 10H,
ArH). MS; m/z: 387.16 (100.0%), 388.16 (26.3%), 389.17
(3.0) %. For C23H21N3O3 (387.16), Calcd.: C: 71.30; H:
5.46; N: 10.85%. Found: C: 71.30; H: 5.46; N: 10.85%.
A solution of compound 25 (10 mmol) in ether (30 mL)
was cooled to 10–15 °C and saturated with dry hydrochloric
acid gas during 1.5–2 h. After the reaction was completed,
the solid product was filtered off, washed with ether, and
recrystallized from dioxane to give compound 26 in 63%
yield, m.p.: 216–217 °C, IR (KBr) ν (cm−1) = 1643 (C=O),
1587 (C=N). MS; m/z: 305.04 (60.0%), 306.04 (17.1%).
For C14H12ClN3OS (305.04), Calcd.: C: 54.99; H: 3.96; Cl:
11.59; N: 13.74; S: 10.49%. Found: C: 54.99; H: 3.98; Cl:
11.69; N: 13.70; S: 10.50%.
1.11
3-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-
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dihydro-1H-pyrazol-4-yl)-5H-thiazolo[2,3-b]
quinazolin-5-one (21)
1.13
5-( p-Methoxyphenylmethylene)-6-[4-
|
(1,2-dihydro-1,5-dimethyl-2-phenyl-3-oxo-3H-
pyrazol-4-yl)]-1,2,4-triazine (34)
1.11.1
Method A
|
To a solution of compound 30 (3.87 g, 0.01 mmol) in etha-
nol (20 mL) was added hydrazine hydrate (1 g, 20 mmol) in
presence of a catalytic amount of piperidine. The mixture was
refluxed for 4 h; the solid product so formed was collected
by filtration, washed with ethanol, dried, and recrystallized
from ethanol to afford 60% yield of 34, m.p.: 252–254 °C,
IR (KBr) ν (cm−1) = 1643 (C=O), 1587 (C=N). MS; m/z:
To a solution of 4-thiocyanatopyrazolone 2 (2.87 g, 10 mmol)
in ethanol (20 mL) was added methyl anthranilate hydrobro-
mide (3.32 g, 10 mmol). The mixture was refluxed for 6–8 h.
The solid that formed was collected by filtration, washed
with water, and dried. Recrystallization from DMF afforded
9-(4-(1,2-dihydro-1,5-dimethyl-2-phenyl-3-oxo-3H-pyraz
ol-4-yl)thiazolo[1,2-a]quinazolin-3-one in 66% yield, m.p.:
1
387.17 (100.0%), 388.17 (25.7%), 389.18 (2.8%). HNMR

Yi et al.
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(DMSO-d6) δ (ppm) = 2.49 (s, 3H), 2.6 (s, 3H), 3.8 (s, 3H),
4.5 (s, 1H), 7.3–7.56 (m, 10H, ArH). 13C NMR (DMSO-
d6): δ (ppm) = 13.8, 33.4. 52.8 (3CH3), 117.8, 122.5, 124.7,
125.9, 126.8, 128.0, 128.2, 128.9, 129.7, 130.7, 135.3, 137.3,
142.5 (Ar-C), 126.8 (CH=), 164.8 (C=O). For C22H21N5O2
(387.17), Calcd.: C: 68.20; H: 5.46; N: 18.08%. Found: C:
68.21; H: 5.47; N: 18.10%.
2-[4-(1,2-Dihydro-1,5-dimethyl-2-phenyl-3-oxo-3H-pyr
azol-4-yl]-1,3,4-thiadiazolo[3,2-a]-1,3-quinazoline-2-imine
10a was obtained via treatment of compound 2 with diazo-
tized anthranilonitrile in ethanol, in the presence of sodium
acetate at 0–5 °C (Scheme 2). The structure of the latter prod-
uct was supported by elemental analysis, IR, and NMR data.
For example, the IR spectrum showed a stretching NH band
at 3425 cm−1 and two carbonyl absorption bands at 1643 and
1633 cm−1. Its 1HNMR spectrum exhibited a singlet signal at
4.5 ppm due to NH proton.
In a similar manner, when compound 2 was treated
with diazotized anthranilic acid or methyl anthranilate, it
afforded one and the same product identified as 2-[4-(1,
2-dihydro-1,5-dimethyl-2-phenyl-3-oxo-3H-pyrazol-4-yl]
-2,3,4-thiadiazolo[3,2-a]-1,3-quinazoline-2-one 10b. The
IR spectrum of 10b revealed three carbonyl absorption
bands at 1710, 1690, and 1639 cm−1, and its mass spec-
trum showed a peak corresponding to its molecular ion.
Chemical confirmation of the structure of 10b was also
established on basis of an alternative synthetic pathway
from 10a by refluxing in ethanolic hydrochloric acid solu-
tion (Scheme 2).
2
RESULTS AND DISCUSSION
Chemistry
|
2.1
|
Reaction of 4-(2-chloroacetyl)-1,2-dihydro-2,3-dimethyl-
1-phenylpyrazol-5-one (1) with potassium thiocyanate, in
refluxing ethanol, afforded a single product in good yield
which was identified as 1,2-dihydro-1,5-dimethyl-2-phen
yl-4-thiocyanatoacetyl-3H-pyrazol-3-one (2) (Scheme 1).
The structure of compound 2 was inferred from its elemental
analysis and spectral data. Thus, the IR spectrum of com-
pound 2 revealed a band at 2152 cm−1 assignable to SCN
group, in addition to two carbonyl absorption bands at 1643
1
and 1633 cm−1. Its HNMR spectrum displayed two singlet
signals at 2.49 and 3.36 ppm due to two methyl protons and
another singlet signal at 4.2 ppm due to methylene protons,
whereas its mass spectrum exhibited a peak at m/z 287 cor-
responding to its molecular ion.
Fusion of compound 2 with triethyl orthoformate afforded
a single product identified as 1-[4-(1,2-dihydro-1,5-dimethyl-
2-phenyl-3-oxo-3H-pyrazol-4-yl)]-2-thiocyanato-3-ethoxyprop
enone (11) on the basis of its elemental analysis and spectral
data. For example, its IR spectrum showed absorption band
at 2152 cm−1 due to SCN group, in addition to two carbonyl
absorption bands at 1643 and 1633 cm−1. The 1HNMR showed
a triplet signal at 1.21 ppm due to the methyl protons and a
quartet signal at δ 4.49 ppm assigned to the methylene protons
and a singlet signal at 7.91 ppm due to the methine proton. In
addition, the mass spectrum showed a peak at m/z 343 corre-
sponding to its molecular ion.
Treatment of 11 with phenylhydrazine in refluxing etha-
nol, in the presence of catalytic amount of piperidine, afford-
ed a single product identified as 3-[4-(1,2-dihydro-1,5-dimet
hyl-2-phenyl-3-oxo-3H-pyrazol-4-ylcarbonyl]-4-thiocyanato
pyrazole (13). The structure of the isolated product was con-
firmed on the basis of elemental analysis and spectral data.
For example, its IR spectrum showed a strong absorption
band at 2052 cm−1 due to SCN group and a carbonyl absorp-
tion band at 1627 cm−1. Also, the absence of IR bands corre-
sponding to NH2, NH, and a second carbonyl group excluded
the possible structures 12 and 14.
Heating of compound 2 in ethanol at reflux temperature
afforded a single product which was analyzed correctly for
C14H13N3SO2 and identified as 2-imino-5-[4-(1,2-dihydro-1,
5-dimethyl-2-phenyl-3-oxo-3H-pyrazol-4-yl)-4H-1,3-oxathiin
e (4). The structure of the isolated product was established on
the basis of its elemental analysis and spectral data. For exam-
ple, the IR spectrum of compound 3 revealed the presence of
NH absorption band at 3095 cm−1 and the presence of only one
carbonyl bands at 1663 cm−1 in addition to the absence of SCN
band in its corresponding region. Its mass spectrum displayed a
peak at m/z 287 corresponding to its molecular ion (Scheme 1).
Treatment of compound 2 with phenyl isothiocyanate, in
dimethylformamide in the presence of potassium hydroxide,
at room temperature, afforded the corresponding potassium
salt 5 that was converted into the non-isolable intermediate 6
upon treatment with dilute hydrochloric acid. The latter com-
pound 6 underwent intramolecular cyclization to afford the
corresponding imino-1,3-dithiole derivative 7. The structure
of the isolated product was confirmed on the basis of its ele-
mental analysis and spectral data. For example, its IR spec-
trum revealed two absorption bands at 3449 and 3419 cm−1
due to two NH groups and two carbonyl absorption bands
Reaction of compound 11 with hydroxylamine hydrochlo-
ride, in refluxing ethanol in presence of equivalent amount of
potassium carbonate, afforded the corresponding isoxazole
derivative 16 in moderate yield. Confirmation of the structure
of the product was based on elemental analysis and spectral
data. For example, its IR spectrum revealed an absorption band
at 2052 cm−1 assigned to SCN group, in addition to a carbonyl
absorption band at 1641 cm−1.
1
at 1643 and 1634 cm−1. Its HNMR exhibited two singlet
signals at 2.57 and 3.33 ppm due to two methyl protons and
another two singlet signals at 7.1 and 9.78 ppm assigned to
two NH protons, whereas its mass spectrum displayed a peak
at m/z 442 corresponding to its molecular ion.

6
Yi et al.
|
SCHEME 1
When compound 2 was treated with the hydrobromide salt
all aspects (m.p., mixed m.p., spectra, and TLC) to that of 21
(Scheme 6).
of methyl anthranilate in refluxing ethanol, it afforded a com-
pound identified as 2-[4-(1,2-dihydro-1,5-dimethyl-2-phenyl-3-
oxo-3H-pyrazol-4-yl]-1,3,4-thiazolo[3,2-a]-1,3-quinazoline-2-o
ne (21) (Scheme 5). The structure of the latter product was con-
firmed on the basis of its elemental analysis and spectral data.
The IR spectrum showed two carbonyl absorption bands at 1643
and 1633 cm−1, whereas, its mass spectrum showed a molecular
ion peak at m/z at 388 corresponding to its molecular ion. These
data provided an additional support for the assigned structure.
The structure of the isolated product was proven to be a lin-
ear 21 rather than the angular isomer 23; this was supported
by an alternative chemical synthesis of compound 21 via the
reaction of the methyl anthranilate hydrobromide with 2-chlo
ro-4-[4-(1,2-dihydro-1,5-dimethyl-2-phenyl-3-oxo-3H-pyrazol
-4-yl]-1,3-thiazole (26) that afforded a compound identical in
1-[4-(1,2-dihydro-1,5-dimethyl-2-phenyl-3-oxo-3H-
pyrazol-4-yl)]-3-(4-methoxyphenyl)-2-thiocyanatopro-
penone (30) was prepared via condensation reaction of
compound 2 with p-anisaldehyde, in refluxing ethanol,
in presence of catalytic amount of piperidine to afford a
single product (Scheme 7). The structure of the isolated
product was supported on the basis of its elemental analy-
sis and spectral data. For example, its IR spectrum showed
two carbonyl absorption bands at 1643 and 1633 and SCN
1
absorption at 2152 cm−1; its HNMR revealed three sin-
glet signals at 2.49, 2.6, and 3.2 ppm due to three meth-
yl protons, in addition to singlet signal at 4.5 ppm due to
methine proton, whereas its mass spectrum showed a peak
at m/z 387 corresponding to its molecular ion. Compound

Yi et al.
7
|
SCHEME 2
SCHEME 3
This fluorescence-based assay measures the rate at which
recombinant human aromatase (baculovirus/insect cell-
expressed) converts the substrate 7-methoxy-trifluoromethy
lcoumarin (MFC) into a fluorescent product 7-ethynyl-triflu
oromethylcoumarin (HFC; λex = 409 nm, λem = 530 nm) in
a NADPH regenerating system. Briefly, concentrated stock
solutions of test compounds were prepared in acetonitrile.
Hundred microliters of samples containing serial dilutions of
test compounds (dilution factor of 3 between samples) and
cofactor mixture (0.4 U/mL glucose-6-phosphate dehydro-
genase; 16.2 μm NADP+; 825 μm MgCl2; 825 μm glucose-6-
phosphate; 50 μm citrate buffer, pH 7.5) was prepared in a
96-well plate. After incubating the plate for 10 min at 37 °C,
30 reacted with hydrazine hydrate in refluxing ethanol to
afford the corresponding 1,2,4-triazine derivative 34 as
confirmed by its elemental analysis and spectral data (see
Section 1).
3
PHARMACOLOGICAL
|
EVALUATION
3.1
Aromatase inhibition activity assay
|
Inhibitory potencies of compounds toward aromatase enzyme
were determined according to an established procedure using
a commercially available aromatase test kit from BD Gentest.

8
Yi et al.
|
SCHEME 4
SCHEME 5

Yi et al.
9
|
SCHEME 6
SCHEME 7

10
Yi et al.
|
TABLE 1 Aromatase inhibition activity of the synthesized compounds
100 μL of an aromatase/P450 reductase/substrate solution
(105 μg protein/mL enzyme; 50 μm MFC; 20 mm phosphate
buffer, pH 7.4) was added to each well. The plate was covered
and incubated for 30 min at 37 °C. Seventy-five microliters
of 0.5 m Tris base was then added to stop the reaction, and the
fluorescence of the formed de-methylated MFC was measured
with a plate reader (Spectra Max Gemini; Molecular Devices,
Sunnyvale, CA, USA). Fluorescence intensities, which were
proportional to the amount of reaction product generated by
aromatase, were graphed as a function of inhibitor concentra-
tion and then fit to a 3-parameter logistic function. Inhibitory
potencies were expressed in terms of the IC50 value, the
inhibitor concentration necessary to reduce the enzyme activ-
ity by half. Each experiment was performed at least in trip-
licate. The aromatase inhibition activity of the synthesized
compounds was tested and summarized (Table 1). As seen
in Table 1, the most active compound of the series was 16,
showing IC50 value of 0.0023 0.0002 μm compared to the
native ligand letrozole with IC50 of 0.0028 0.0006 μm. In
addition, compounds 26 and 36 exhibit good inhibition activ-
ities close to letrozole with IC50 values 0.0033 0.0001 and
0.0032 0.0003 μm, respectively.
and the native ligand letrozole
Compound
IC50 (μm)
2
4
7
10a
10b
13
16
21
0.0067 0.0002
0.0054 0.0002
0.0046 0.0001
0.0041 0.0001
0.0039 0.0002
0.0037 0.0002
0.0023 0.0002
0.0034 0.0001
0.0035 0.0002
0.0033 0.0001
0.0032 0.0003
0.0028 0.0006
25
26
36
Letrozole
were removed. The hydrogen atoms were added to make the
system closer to reality, Kollman united atom charges and
salvation parameters were added, and the energy of the pro-
tein was minimized using the Swiss pdb-viewer 4.1.0.
It was found that bulky compounds are difficult to enter
the binding site as it needed to rotate its side chains to fit the
cavity entrance, as by analyzing the binding site of aromatase
by PDBsum (31) explained that aromatase binding site vol-
umes occupy 1525.92 Å and the binding site diameter when
measured by PYMOL was 3.24 Å. Traditional approaches to
Pyrazolone development focus on inhibiting aromatase by
binding to its active site. It may be worthwhile to consider
the development of drugs that may sterically block access of
natural substrate to the entrance of the binding site. From oth-
er previous studies, it was observed that the catalytic binding
residue is M374 (methionine 374). The structures of newly
synthesized pyrazolone compound 16 exhibited the lowest
IC50 value (0.0023 0.0002 μM) compared to the native
3.2
Molecular modeling and molecular
|
dynamics studies
Protein structure of aromatase with its equilibrium molecu-
lar dynamic simulation is obtained directly from protein data
bank, as the molecular dynamics simulation of the protein
was performed to relax to its equilibrium conformation. The
protein co-ordinates have been downloaded from Protein
Data Bank website (PDB ID: 4GL7) with 2 ligands (PDB
ID: OXJ) (6α,8α)-6-(pent-2-yn-1-yloxy)androsta-1,4-diene-
3,17-dione C24H30O3, (PDB ID: HEM) protoporphyrin IX
containing Fe. The water molecules and all other substruc-
tures including HEAM (protoporphyrin IX containing Fe)
FIGURE 2 MOE docking
showing the interaction between
Methionine 374 with the nitrogen atom
of the thiocyanate group in the ligand
16 which is 2.71 Å and 36%..

Yi et al.
11
|
FIGURE 3 Autodock and
visualized by UCSF Chimera
showing the Hydrogen bond between
Methionine and the ligand 16.
TABLE 2 The approximate free energy of binding (ΔGb) and
inhibition constants (Ki) calculated by autodock
ligand letrozole IC50 (0.0028 0.0006 μM). The structures
were built with chemsketch software (Advanced Chemistry
Development, Inc., Toronto, ON, Canada) and minimized
by the moe 2008.10. (Macrovision corporation 2830 De la
cruz Blvd Santa Clara, CA, USA) Next, the ligand structures
were prepared for docking by merging non-polar hydrogen
atoms, adding Gasteiger partial charges, and defining rotat-
able bonds. The energy-optimized ligand (16) was docked
into the binding site in the protein using moe 2008.10 and
autodock 4 (Molecular Graphics Laboratory, The Scripps
Research Institute, La Jolla, CA, USA) to elucidate the bind-
ing mode with aromatase. The maximum distance between
hydrogen bond donor and acceptor was set to 3.5 Å. And all
the parameters were set as default values. By this tool, we
can determine all type of interactions as hydrogen bond, π–π
interaction, and cation–π interaction. Finally docking algo-
rithm files were run on CYGWIN-I and CYGWIN-II for anal-
ysis and visualization of result by ucsf chimera, Regents of
the University of California, USA (Figures 2 and 3).
Lowest binding energy
−7.95 kcal/mol
−7.91 kcal/mol
−7.95 kcal/mol
1.48 μm
Mean binding energy
Estimated free energy of binding
Estimated inhibition constant, Ki
[temperature = 298.15 K]
Final intermolecular energy
vdW + H bond + desolvation energy
Electrostatic energy
−8.25 kcal/mol
−8.19 kcal/mol
−0.06 kcal/mol
−0.55 kcal/mol
+0.30 kcal/mol
−0.55 kcal/mol
106.718 Å
Final total internal energy
Torsional free energy
Unbound system’s energy
RMSD from reference structure
Statistical mechanical analysis calculated by autodock.
Partition function, Q = 10.13 at temperature, T = 298.15 K.
Free energy, A ~ −1372.15 kcal/mol at temperature, T = 298.15 K.
Internal energy, U = −7.91 kcal/mol at temperature, T = 298.15 K.
Entropy, S = 4.58 kcal/mol/K at temperature, T = 298.15 K.
Furthermore, the free energies of binding (ΔGb) and
inhibition constants (Ki) as calculated by autodock are sum-
marized (Table 2). Docking calculations were performed
with autodock, version 4.2 using the Lamarckian genetic
algorithm. A grid box size of 50 × 64 × 78 Å points with a
grid spacing of 0.375 Å was generated using AutoGrid. The
grid was centered at x, y, z co-ordinates of 85.363, 61.426,
and 42.824, which was reported as the binding site residues
autodock parameter set- and distance-dependent dielectric
functions were used for calculating the van der Waals and the
electrostatic terms, respectively. The initial position, orienta-
tion, and torsions of the ligand molecules were set random-
ly. The docked compound was derived from 10 independent
docking runs that were set to terminate after a maximum of
2.5 × 106 energy evaluations with mutation rate of 0.02 and
crossover rate of 0.8. The search for low-energy binding ori-
entations was performed by Lamarckian genetic algorithm
using a translational step of 0.2 Å, a quaternion step of 5 Å,
and a torsion step of 5 Å. To validate the accuracy of the
docking system, the nature substrate androstenedione was re-
docked to aromatase and its orientation with respect to the
crystal structure was determined.
4
CONCLUSION
|
In conclusion, a new series derived from 4-(2-chloroacety
l)-1,2-dihydro-2,3-dimethyl-1-phenylpyrazol-5-one
was
synthesized and characterized, and its pharmacological

12
Yi et al.
|
[11] C. J. Fabian, Int. J. Clin. Pract. 2007, 61, 2051.
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and compared to the reference native ligand letrozole (IC50 of
0.0028 0.0006μM). Moreover, molecular docking studies
were conducted to support the findings. One of the synthe-
sized compounds (16) was found to be more potent inhibitor
(IC50 value of 0.0023 0.0002μM) than the reference letro-
zole. In addition, two other compounds (26 and 36) exhibited
good inhibition activities close to letrozole with IC50 values
0.0033 0.0001 and 0.0032 0.0003μM, respectively.
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ACKNOWLEDGMENTS
[18] R. Dayam, F. Aiello, J. X. Deng, Y. Wu, A. Garofalo, X. Y. Chen, N. Nea-
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The authors extend their sincere appreciation to the Deanship
of Scientific Research at King Saud University for its funding
this Prolific Research group (PRG-1437-29).
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