Synthesis, anti-inflammatory and neuroprotective activity of pyrazole and pyrazolo[3,4-d]pyridazine bearing 3,4,5-trimethoxyphenyl

Article abstract of DOI:10.1007/s00044-017-1870-5

A new series of 3,4,5-trimethoxyphenyl bearing pyrazole (4a–g) and pyrazolo[3,4-d]pyridazine (5a–g) scaffolds were synthesized in good yield. The newly synthesized compounds were characterized on the basis of elemental and spectroscopic analyses. Their inhibitory activity against the pro-inflammatory inducible nitric oxide synthase and cyclooxygenase-2 proteins expression in lipopolysaccharide-stimulated murine RAW 264.7 macrophages were assessed and showed various potencies. All pyrazolo[3,4-d]pyridazine compounds (5a–g) strongly down regulated lipopolysaccharide inducible nitric oxide synthase expression to the range of 20.3 ± 0.6–51.3 ± 3.5% relative to the bioactive pyrazole derivatives 4b, 4c, 4e and 4g. With the exception of inactive compounds 4c and 4d, all other synthesized compounds inhibited cyclooxygenase-2 expression below 100% in the lipopolysaccharide-stimulated cells, which being declined maximally to 42.8 ± 1.4% by one of the pyrazolo[3,4-d]pyridazine compounds (5d). Moreover, the neuroprotective activity of the less cytotoxic compounds 4b, (4e–g) and (5a–g) were evaluated against 6-hydroxydopamine (6-OHDA)-induced neuroblastoma SH-SY5Y cell death and exhibited significant (p < 0.05) cell protection. The pyrazolo[3,4-d]pyridazine compound (5e) exhibited more than 100% of relative neuroprotection (110.7 ± 4.3%) with an additional advantage of having the highest cell viability index (107.2 ± 2.9%).

Full text of DOI:10.1007/s00044-017-1870-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1870-5
ORIGINAL RESEARCH
Synthesis, anti-inflammatory and neuroprotective activity of
pyrazole and pyrazolo[3,4-d]pyridazine bearing 3,4,5-
trimethoxyphenyl
1 ●
2,3 ●
4 ●
Mashooq A. Bhat
Atallah F. Ahmed
Zhi-Hong Wen
1 ●
5
Mohamed A. Al-Omar Hatem A. Abdel-Aziz
Received: 10 October 2016 / Accepted: 8 March 2017
©
Springer Science+Business Media New York 2017
Abstract A new series of 3,4,5-trimethoxyphenyl bearing
pyrazole (4a–g) and pyrazolo[3,4-d]pyridazine (5a–g)
scaffolds were synthesized in good yield. The newly syn-
thesized compounds were characterized on the basis of
elemental and spectroscopic analyses. Their inhibitory
activity against the pro-inflammatory inducible nitric oxide
synthase and cyclooxygenase-2 proteins expression in
lipopolysaccharide-stimulated murine RAW 264.7 macro-
phages were assessed and showed various potencies. All
pyrazolo[3,4-d]pyridazine compounds (5a–g) strongly
down regulated lipopolysaccharide inducible nitric oxide
synthase expression to the range of 20.3 ± 0.6–51.3 ± 3.5%
relative to the bioactive pyrazole derivatives 4b, 4c, 4e and
by one of the pyrazolo[3,4-d]pyridazine compounds (5d).
Moreover, the neuroprotective activity of the less cytotoxic
compounds 4b, (4e–g) and (5a–g) were evaluated against 6-
hydroxydopamine (6-OHDA)-induced neuroblastoma SH-
SY5Y cell death and exhibited significant (p < 0.05) cell
protection. The pyrazolo[3,4-d]pyridazine compound (5e)
exhibited more than 100% of relative neuroprotection
(110.7 ± 4.3%) with an additional advantage of having the
highest cell viability index (107.2 ± 2.9%).
●
●
●
Keywords iNOS COX-2 Anti-inflammatory
●
●
Neuroprotective Pyrazole derivatives Pyrazolo[3,4-d]
pyridazine derivatives
4
g. With the exception of inactive compounds 4c and 4d, all
other synthesized compounds inhibited cyclooxygenase-2
expression below 100% in the lipopolysaccharide-stimu-
lated cells, which being declined maximally to 42.8 ± 1.4%
Introduction
*
*
*
Mashooq A. Bhat
mashooqbhat@rediffmail.com
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely
used to alleviate inflammation and pain associated with
many pathological conditions. Traditional NSAIDs act by
inhibiting the cyclooxygenase isoforms (COXs) and hence
inhibiting the biosynthesis of prostaglandins from arachi-
donic acid (Jouzeau et al. 1997; Cairns 2007). COX-2 is
inducible in inflammation and carcinogenesis whereas the
isoform (COX-1) is constitutively expressed under all
physiological conditions. Selective COX-2 inhibitors such
as the diaryl-substituted pyrazole derivative have less side
effects as compared to traditional NSAIDs (Michaux and
Charlier 2004). However, long term use of both traditional
NSAIDs and COXIBS has been reported to cause sig-
nificant cardiovascular side effects (Patel et al. 2004), and
thus discovery of new, safe and effective drugs is still a
necessity. Pyrazole scaffold possess a broad spectrum of
Atallah F. Ahmed
afahmed@ksu.edu.sa
Hatem A. Abdel-Aziz
hatem_741@yahoo.com
1
2
3
4
5
Department of Pharmaceutical Chemistry, College of Pharmacy,
King Saud University, Riyadh 11451, Saudi Arabia
Department of Pharmacognosy, College of Pharmacy, King Saud
University, Riyadh 11451, Saudi Arabia
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura
University, Mansoura 35516, Egypt
Department of Marine Biotechnology and Resources, National
Sun Yat-sen University, Kaohsiung 80424, Taiwan
Applied Organic Chemistry Department, National Research
Centre, Dokki, Cairo, Egypt

Med Chem Res
applications including anti-inflammatory (Sharma et al.
010; Tewari et al. 2010; el-Hawash and el-Mallah 1998;
Gokhan-Kelekci et al. 2007; Domiati et al. 2016), analgesic
Hall et al. 2008), neuroprotective (Jayaraj et al. 2013),
anticonvulsant, and antidepressant activity (Abdel-Aziz
et al. 2009; Özdemir et al. 2007). Pyrazolopyridazine deri-
vatives are also reported as good anti-inflammatory agents
then left to cool at room temperature. The precipitate
obtained was filtered off, washed with diethyl ether, dried
under vacuum and recrystallized from absolute ethanol to
give enaminone 2 as orange crystals. Yield: 90%; M.p.:
2
(
−
1
128–130 °C; IR (KBr): νmax/cm : 1638 (C=O), 1543
1
(C=C), 1118 (C–O); H NMR (DMSO–d ) δ ppm: 7.71
6
(1H, d, J = 12 Hz, -CH), 7.1 (2H, m, Ar–H), 5.8 (1H, d, J
= 12 Hz, –CH), 3.8 (6H, s, 2 × OCH ), 3.7 (3H, s, OCH ),
(
Tewari and Mishra 2001; Tewari et al. 2011) and as
3
3
1
3
analgesic in neuropathic pain (Myatt et al. 2010). The lit-
erature survey reveals that many pyrazoline derivatives
have been used for clinical applications as NSAIDs. The
pyrazoline NSAIDs like phenylbutazone, celecoxib, and
deracoxib are potent anti-inflammatory and analgesic
agents. This motivated us to design and synthesize novel
series of 3,4,5-trimethoxyphenyl bearing pyrazole (4a–g)
and pyrazolo[3,4-d]pyridazine (5a–g), which have been
found to possess an interesting profile for anti-COX-2/iNOS
expression-related anti-inflammatory and neuroprotective
activity.
3.1 (3H, s, N–CH ), 2.9 (3H, s, N–CH ); C NMR
3 3
(DMSO–d ) δ ppm: 184.4 (C=O), 154.1 (Ar–C), 152.4
6
(Ar–C), 139.8 (Ar–CH), 135.8 (Ar–CH), 104.6 (C=C),
90.7 (C=C), 60.0 (OCH ), 55.9 (OCH ), 44.4 (N–CH ),
3
3
3
+
37.1 (N–CH ); MS (ESI) m/z: 265.0 [M] ; Analysis: for
3
C H NO , calcd. C 63.38, H 7.22, N 5.28%; found C
1
4
19
4
63.14, H 7.20, N 5.30%.
General synthesis of 1-[1-(4-substituted phenyl)-4-(3,4,5-
trimethoxybenzoyl)-1H-pyrazol-3-yl]ethanones (4a–g)
To an equimolar mixture of enaminone 2 (5.0 mmol) and
appropriate hydrazonoyl chloride (3a–g) (5.0 mmol) in
absolute ethanol was added triethylamine (10.0 mmol) and
the mixture was refluxed for 4 h. The precipitated solid was
filtered off, washed with absolute ethanol, dried under
vacuum and recrystallized from ethanol to give corre-
sponding pyrazole derivative (4a–g).
Materials and methods
Chemistry
Experimental
All the solvents were procured from Merck. The purity of
the synthesized compounds was checked by thin layer
chromatography (TLC) performed on Silica gel 60 F254
coated plates (Merck). UV light was used for the visuali-
zation of TLC spots. The Fourier transform infrared spec-
troscopy (FT-IR) spectra were performed in KBr pellets on
a (Spectrum BX) Perkin Elmer FT-IR spectrophotometer.
Melting points were checked on a Gallenkamp melting
point apparatus, and the thermometer was uncorrected.
1-[1-(4-Methoxyphenyl-4-(3,4,5-trimethoxybenzoyl)-1H-
pyrazol-3-yl]ethanone (4a)
Yield: 85%; IR (KBr): νmax/cm− : 1684 (C=O), 1651
1
1
(C=O), 1519 (C=N), 1231 (C-N), 1130 (C–O); H NMR
(DMSO-d ) δ ppm: 8.9 (1H, s, pyrazole), 7.9 (2H, d, J =
6
8.5 Hz, Ar–H), 7.1 (4H, m, Ar–H), 3.6 (12H, s, 4 × OCH ),
3
1
3
2.59 (3H, s, COCH3); C NMR (DMSO-d ) δ ppm: 192.7
6
(C=O), 187.8 (C=O), 158.7 (Ar–C), 152.6 (Ar–C), 149.8
(Ar–C), 142.0 (Ar–C), 132.1 (Ar–C), 122.2 (Ar–C), 121.0
NMR Spectra were processsed in DMSO-d on a Bruker
6
nuclear magnetic resonance NMR spectrophotometer
(Ar–C), 114.7 (Ar–C), 106.7 (Ar–C), 60.1 (OCH ), 56.0
3
+
1
13
operating at 500 MHz for H and 125 MHz for C at the
Research Center, College of Pharmacy, King Saud Uni-
versity, Saudi Arabia. Triple quadruple mass spectroscopy
was used for the measurement of molecular masses of
compounds. The elemental analysis of the compounds was
performed on the CHN Elementar (Analysensysteme
GmbH, Germany). The elemental analysis for C, H and N
were within the limit of ±0.4% of the theoretical values.
(OCH ), 27.3 (COCH ); MS (ESI) m/z: 411.2 [M] , 243.0
3
3
[100 %]; Analysis: for C H N O , calcd. C 64.38, H 5.40,
2
2 22 2 6
N 6.83% found C 64.13, H 5.41, N 6.84%.
1-[1-(4-Flourophenyl-4-(3,4,5-trimethoxybenzoyl)-1H-
pyrazol-3-yl]ethanone (4b)
Yield: 90%; IR (KBr): νmax/cm− : 1684 (C=O), 1635
1
1
(
C=O), 1506 (C=N), 1233 (C–N), 1132 (C–O); H NMR
Synthesis of (2E)-3-(dimethylamino)-1-(3,4,5-
(DMSO-d ) δ ppm: 9.0 (1H, s, pyrazole), 8.0 (2H, d, J =
6
trimethoxyphenyl)prop-2-en-1-one (2)
8.5 Hz, Ar–H), 7.4 (2H, d, J = 8.5 Hz, Ar–H), 7.1 (2H, s,
1
3
Ar–H), 3.8 (9H, s, 3 × OCH ), 2.6 (3H, s, COCH );
C
3
3
A mixture of 3,4,5-trimethoxy acetophenone 1 (0.02 mol)
and dimethylformamide-dimethylacetal (DMF–DMA)
NMR (DMSO-d ) δ ppm: 192.7 (C=O), 187.8 (C=O),
152.7 (Ar–C), 131.8 (Ar–C), 121.7(Ar–C), 116.6 (Ar–C),
6
(
0.023 mol) was refluxed in solvent free condition for 10 h,
106.7 (Ar–C), 60.1 (OCH ), 56.0 (OCH ), 27.3 (COCH );
3
3
3

Med Chem Res
+
MS (ESI) m/z: 399.2 [M] ; Analysis: for C H N O F,
1-[1-(4-Bromophenyl-4-(3,4,5-trimethoxybenzoyl)-1H-
2
1 19 2 5
calcd. C 63.31, H 4.81, N 7.03% found C 63.55, H 4.82, N
pyrazol-3-yl]ethanone (4f)
7
.01%.
Yield: 85%; IR (KBr): νmax/cm− : 1686 (C=O), 1635
(
1
1
C=O), 1506 (C=N), 1233 (C–N), 11231 (C–O); H NMR
DMSO-d ) δ ppm: 9.1 (1H, s, pyrazole), 8.0 (2H, d, J =
.5 Hz Ar–H), 7.8 (2H, d, J = 8.5 Hz, Ar–H), 7.1 (2H, s,
Ar–H), 3.8 (9H, s, 3 × OCH ), 2.6 (3H, s, COCH );
NMR (DMSO-d ) δ ppm: 192.7 (C=O), 187.5 (C=O),
53.3 (Ar–C), 132.5 (Ar–C), 121.4 (Ar–C), 106.8 (Ar–C),
0.1 (OCH ), 56.0 (OCH ), 27.3 (COCH ); MS (ESI) m/z:
1
-[1-(4-Methylyphenyl-4-(3,4,5-trimethoxybenzoyl)-1H-
(
6
pyrazol-3-yl]ethanone (4c)
8
1
3
Yield: 80%; IR (KBr): νmax/cm−1: 1684 (C=O), 1636
C
3
3
1
6
(
C=O), 1506 (C=N), 1232 (C–N), 1131 (C–O); H NMR
1
6
4
(
DMSO-d ) δ ppm: 8.9 (1H, s, pyrazole), 7.9 (2H, d, J =
6
3
+
3
3
8
.5 Hz, Ar–H), 7.4 (2H, d, J = 8.5 Hz, Ar–H), 7.1 (2H, s,
60.2 [M] ; Analysis: for C H N O Br, calcd. C 54.92,
2
1 19 2 5
Ar–H), 3.7 (9H, s, 3 × OCH ), 3.6 (3H, s, CH ), 2.6 (3H, s,
COCH3); C NMR (DMSO-d ) δ ppm: 192.7 (C=O),
3
3
1
3
H 4.17, N 6.10%; found C 55.12, H 4.16, N 6.12%.
6
1
1
1
2
87.6 (C=O), 156.7 (Ar–C), 152.4 (Ar–C), 149.5 (Ar–C),
41.0 (Ar–C), 132.1 (Ar–C), 122.8 (Ar–C), 121.5 (Ar–C),
14.2 (Ar–C), 106.8 (Ar–C), 60.1 (OCH ), 56.0 (OCH ),
1-[1-(4-Chlorohenyl-4-(3,4,5-trimethoxybenzoyl)-1H-
pyrazol-3-yl]ethanone (4g)
3
+
3
7.3 (COCH ) MS (ESI) m/z: 395.2 [M] , 227.0 [100 %].
3
Yield: 70%; IR (KBr): νmax/cm− : 1688 (C=O), 1635
1
Analysis: for C H N O , calcd. C 66.99, H 5.62, N
2
2 22 2 5
1
7
.10%; found C 66.73, H 5.63, N 7.12%.
(C=O), 1505 (C=N), 1232 (C–N), 11231 (C–O); H NMR
(
DMSO-d ) δ ppm: 9.0 (1H, s, pyrazole), 8.0 (2H, d, J =
6
8
.5 Hz Ar–H), 7.6 (2H, d, J = 8.5 Hz, Ar–H), 7.1 (2H, s,
1
-[1-Phenyl-4-(3,4,5-trimethoxybenzoyl)-1H-pyrazol-3-yl]
1
3
Ar–H), 3.8 (9H, s, 3 × OCH ), 2.6 (3H, s, COCH );
NMR (DMSO-d ) δ ppm: 192.7 (C=O), 187.5 (C=O),
C
3
3
ethanone (4d)
6
Yield: 70%; IR (KBr): νmax/cm−1: 1685 (C=O), 1664
153.0 (Ar–C), 129.6 (Ar–C), 121.12 (Ar–C), 106.8 (Ar–C),
1
60.0 (OCH ), 56.0 (OCH ), 25.0 (COCH ); MS (ESI) m/z:
3
3
3
(
(
C=O), 1506 (C=N), 1233 (C–-N), 1127 (C–O); H NMR
DMSO-d6) δ ppm: 9.0 (1H, s, pyrazole), 8.0 (1H, t, Ar–H),
+
4
16.0 [M] ; Analysis: for C H N O Cl, calcd. C 60.80,
21 19 2 5
H 4.62, N 6.75 %; found C 60.65, H 4.61, N 6.73 %.
7
7
.6 (2H, d, J = 7 Hz, Ar–H), 7.4 (2H, d, J = 7 Hz, Ar–H),
.1 (2H, s, Ar–H), 3.8 (9H, s, 3 × OCH ), 2.6 (3H, s,
3
1
3
COCH3); C NMR (DMSO-d ) δ ppm: 192.7 (C=O),
General synthesis of pyrazolo[3,4-d]pyridazine derivatives
6
1
1
1
5
2
87.8 (C=O), 152.7 (Ar–C), 150.1 (Ar–C), 142.0 (Ar–C),
38.6 (Ar–C), 132.4 (Ar–C), 129.7 (Ar–C), 127.9(Ar–C),
(5a–g)
22.4(Ar–C), 119.4 (Ar–C), 106.7 (Ar–C), 60.1 (OCH ),
A mixture of pyrazole derivative (4a–g) (3.0 mmol) and
hydrazine hydrate 99% (3.0 mmol) in absolute ethanol (30
mL) was refluxed for 1 h and then cooled at room tem-
perature. The product that was separated out was filtered,
dried under vacuum and recrystallized from ethanol.
3
+
6.0 (OCH ), 27.2 (COCH ); MS (ESI) m/z: 381.1 [M] ,
3
3
13.0 [100 %]; Analysis: for C H N O , calcd. C 66.31,
2
1 20 2 5
H 5.30, N 7.36%; found C 66.56, H 5.32, N 7.34%.
1
1
-[1-(4-Sulfonamidophenyl-4-(3,4,5-trimethoxybenzoyl)-
H-pyrazol-3-yl]ethanone (4e)
7-Methyl-2-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-
2
H-pyrazolo[3,4-d]pyridazine (5a)
Yield: 75%; IR (KBr): νmax/cm− : 3410 (NH ), 1690
1
2
Yield: 80%; IR (KBr): ν /cm− : 1507 (C=N), 1264
(C–N), 1126 (C–O); H NMR (DMSO-d ) δ ppm: 9.5 (1H,
1
(
(
C=O), 1652 (C=O), 1506 (C=N), 1334 (S=O), 1232
C–N), 1131 (C-O); H NMR (DMSO-d ) δ ppm: 9.1 (1H,
max
1
1
6
6
s, pyrazole), 8.2 (2H, d, J = 8.5 Hz, Ar–H), 8.0 (2H, d, J =
.5 Hz, Ar–H), 7.5 (2H, s, –NH , D O exchang.), 7.1 (2H,
s, pyrazole), 8.1 (2H, d, J = 9 Hz, Ar–H), 7.3 (2H, s, Ar–H),
8
7.2 (2H, d, J = 9 Hz, Ar–H), 3.8 (9H, s, 3 × OCH ), 3.6
2
2
3
1
3
13
m, Ar–H), 3.8 (9H, s, 3 × OCH ), 2.6 (3H, s, COCH );
C
(3H, s, OCH ), 2.8 (3H, s, COCH ); C NMR (DMSO-d6)
3
3
3
3
NMR (DMSO-d ) δ ppm: 192.7 (C=O), 187.6 (C=O),
δ ppm: 159.7 (Ar–C), 153.4 (Ar–C), 150.8 (Ar–C), 143.6
(Ar–C), 139.0 (Ar–C), 132.5 (Ar–C), 132.0 (Ar–C), 124.5
(Ar–C), 122.9 (Ar–C), 115.2 (Ar–C), 114.7 (Ar–C), 105.5
(Ar–C), 60.0 (OCH ), 56.0 (OCH ), 55.6 (OCH ), 18.0
6
1
1
1
2
52.7 (Ar–C), 150.6 (Ar–C), 143.0 (Ar–C), 142.1 (Ar–C),
40.6 (Ar–C), 132.3 (Ar–C), 127.3 (Ar–C), 122.8 (Ar–C),
19. (Ar–C)6, 106.8 (Ar–C), 60.1 (OCH ), 56.0 (OCH ),
3
+
3
3
3
3
+
7.3 (COCH ); MS (ESI) m/z: 460.2 [M] ; Analysis: for
(CH3); MS (ESI) m/z: 407.2 [M] ; Analysis: for
3
C H N O S, calcd. C 54.89, H 4.61, N 9.15S 6.98%;
C H N O , calcd. C 65.01, H 5.46, N 13.78%; found C
22 22 4 4
2
1
21
3
7
found C 54.67, H 4.62, N 9.14S 6.97%.
65.26, H 5.45, N 13.74%.

Med Chem Res
7
2
-Methyl-2-(4-flourophenyl)-4-(3,4,5-trimethoxyphenyl)-
H-pyrazolo[3,4-d]pyridazine (5b)
–NH , D O exchang.), 7.3 (2H, m, Ar–H), 3.9 (6H, s, 2 ×
2
2
1
3
OCH ), 3.7 (3H, s, OCH ), 2.9 (3H, s, –CH ); C NMR
3
3
3
(
DMSO-d ) δ ppm: 153.7 (Ar–C), 153.2 (Ar–C), 151.2
6
Yield: 90%; IR (KBr): ν /cm− : 1507 (C=N), 1220
1
(Ar–C), 144.3 (Ar–C), 141.2 (Ar–C), 139.2 (Ar–C), 131.8
(Ar–C), 127.2 (Ar–C), 126.0 (Ar–C), 121.9 (Ar–C), 115.4
(Ar–C), 105.6 (Ar–C), 60.1 (OCH ), 56.1 (OCH ), 18.0
(CH3); MS (ESI) m/z: 456.2 [M] ; Analysis: for
C H N O S, calcd. C 55.37, H 4.65, N 15.38S 7.04%;
max
1
(
C–N), 1127 (C–O); H NMR (DMSO-d ) δ ppm: 9.6 (1H,
s, pyrazole), 8.2 (2H, d, J = 9 Hz, Ar–H), 7.5 (2H, d, J = 9
Hz, Ar–H), 7.3 (2H, s, Ar–H), 3.7 (9H, s, 3 × OCH ), 2.8
3H, s, COCH3); C NMR (DMSO-d ) δ ppm: 153.2
6
3
3
+
3
1
3
(
6
21 21 5 5
(
(
(
[
Ar–C), 139.1 (Ar–C), 131.9 (Ar–C), 125.4 (Ar–C), 123.8
Ar–C), 116.7 (Ar–C), 115.2 (Ar–C), 105.5 (Ar–C), 60.0
OCH ), 56.0 (OCH ), 18.0 (CH ); MS (ESI) m/z: 395.2
found C 55.58, H 4.66, N 15.42S 7.05%.
7-Methyl-2-(4-bromophenyl)-4-(3,4,5-trimethoxyphenyl)-
2H-pyrazolo[3,4-d]pyridazine (5f)
3
3
3
+
M] , 102.3 [100%]; Analysis: for C H N O F, calcd. C
2
1 19 4 3
6
1
3.95, H 4.86, N 14.21%; found C 63.75, H 4.87, N
3.25%.
Yield: 82%; IR (KBr): ν /cm− : 1497 (C=N), 1233
1
max
1
(
C–N), 1128 (C–O); H NMR (DMSO-d ) δ ppm: 9.7 (1H,
6
7
2
-Methyl-2-(4-methylphenyl)-4-(3,4,5-trimethoxyphenyl)-
H-pyrazolo[3,4-d]pyridazine (5c)
s, pyrazole), 8.2 (2H, d, J = 8.5 Hz, Ar–H), 7.9 (2H, d, J =
8.5 Hz, Ar–H), 7.4 (2H, s, Ar–H), 4.0 (9H, s, 3 × OCH ),
3
1
3
2
.9 (3H, s, CH3); C NMR (DMSO-d ) δ ppm: 153.1
6
Yield: 85%; IR (KBr): ν /cm− : 1507 (C=N), 1229
1
(Ar–C), 153.0 (Ar–C), 151.5 (Ar–C), 144.6 (Ar–C), 141.0
(Ar–C), 139.1 (Ar–C), 131.6 (Ar–C), 127.2 (Ar–C), 126.2
(Ar–C), 121.0 (Ar–C), 115.3 (Ar–C), 105.1 (Ar–C), 60.0
(OCH ), 56.0 (OCH ), 18.0 (CH ); MS (ESI) m/z: 456.2
max
1
(
C–N), 1127 (C–O); H NMR (DMSO-d ) δ ppm: 9.0 (1H,
6
s, pyrazole), 8.1 (2H, d, J = 9 Hz, Ar–H), 7.5 (2H, d, J = 9
Hz, Ar–H), 7.3 (2H, s, Ar–H), 3.6 (9H, s, 3 × OCH ), 2.6
3
3
3
3
1
3
+
(
3H, s, COCH3); C NMR (DMSO-d ) δ ppm: 153.1
[M] ; Analysis: for C H N O Br, calcd. C 55.40, H 4.21,
21 19 4 3
6
(
(
(
[
Ar–C), 139.0 (Ar–C), 131.7 (Ar–C), 125.2 (Ar–C), 123.5
Ar–C), 116.5 (Ar–C), 115.0 (Ar–C), 105.1 (Ar–C), 60.1
OCH ), 56.0 (OCH ), 18.0 (CH ); MS (ESI) m/z: 391.3
N 12.31%; found C 55.62, H 4.20, N 12.35%.
7-Methyl-2-(4-chlorophenyl)-4-(3,4,5-trimethoxyphenyl)-
2H-pyrazolo[3,4-d]pyridazine (5g)
3
3
3
+
M] , 254.9 [100%]; Analysis: for C H N O , calcd. C
2
2 22 4 3
6
1
7.68, H 5.68, N 14.35%; found C 67.87, H 5.67, N
4.30%.
Yield: 85%; IR (KBr): ν /cm− : 1505 (C=N), 1233
1
max
1
(
C–N), 1127 (C–O); H NMR (DMSO-d ) δ ppm: 9.7 (1H,
6
7
-Methyl-2-phenyl-4-(3,4,5-trimethoxyphenyl)-2H-pyrazolo
s, pyrazole), 8.3 (2H, d, J = 9 Hz, Ar–H), 7.8 (2H, d, J = 9
[
3,4-d]pyridazine (5d)
Hz, Ar–H), 7.4 (2H, s, Ar–H), 4.0 (9H, s, 3 × OCH ), 2.9
3
1
3
(
3H, s, CH3); C NMR (DMSO-d ) δ ppm: 153.2 (Ar–C),
6
Yield: 80%; IR (KBr): ν /cm− : 1506 (C=N), 1231
1
138.0 (Ar–C), 129.7 (Ar–C), 123.2 (Ar–C), 105.6 (Ar–C),
60.0 (OCH ), 56.1 (OCH ), 18.0 (CH ); MS (ESI) m/z:
max
1
(
C–N), 1128 (C–O); H NMR (DMSO-d ) δ ppm: 9.6 (1H,
6
3
3
3
+
s, pyrazole), 8.2 (2H, d, J = 7 Hz, Ar–H), 7.7 (1H, t, J =
.5 Hz, Ar–H), 7.6 (2H, d, J = 7 Hz, Ar–H), 7.4 (2H, s,
Ar–H) 4.0 (6H, s, 2 × OCH ), 3.8 (3H, s, 3 × OCH ), 2.9
412.0 [M] ; Analysis: for C H N O Cl, calcd. C 61.39,
21 19 4 3
7
H 4.66, N 13.64%; found C 61.50, H 4.65, N 13.60%.
3
3
1
3
(
(
(
(
(
3H, s, COCH3); C NMR (DMSO-d ) δ ppm: 153.6
Ar–C), 153.2 (Ar–C), 151.0 (Ar–C), 143.8 (Ar–C), 139.2
Ar–C), 131.9 (Ar–C), 129.7 (Ar–C), 125.4 (Ar–C), 121.5
Ar–C), 115.2 (Ar–C), 105.5 (Ar–C), 60.0 (OCH ), 56.0
OCH ), 18.0 (CH ); MS (ESI) m/z: 377.2 [M] ; Analysis:
Biological assays
6
Cell cultures
3
+
The murine RAW 264.7 macrophage and SH-SY5Y neu-
roblastoma cells were obtained from the American Type
Culture Collection (ATCC, MD, USA). The macrophage
cells were cultured in Dulbecco’s modified essential med-
ium (DMEM, Invitrogen-Gibco, CA, USA) containing 10%
inactivated fetal bovine serum (FBS, Sigma-Aldrich, MO,
USA). The neuroblastoma cells were grown in a 1:1
DMEM and Hams F12 nutrient medium (Invitrogen-Gibco)
containing 10% heat-inactivated FBS. Each cell line was
3
3
for C H N O , calcd. C 67.01, H 5.36, N 14.88%; found
C 67.27, H 5.37, N 14.92%.
2
1 20 4 3
7
-Methyl-2-(4-sulfonamidophenyl)-4-(3,4,5-
trimethoxyphenyl)-2H-pyrazolo[3,4-d]pyridazine (5e)
Yield: 75%; IR (KBr): νmax/cm− : 3354 (NH ), 1506
1
2
1
(
C=N), 1334 (S=O), 1239 (C–N), 1134 (C–O); H NMR
(
DMSO-d ) δ ppm: 9.7 (1H, s, pyrazole), 8.4 (2H, d, J =
incubated at 37 °C in a humidified 5% CO , 95% air
6
2
8
.5 Hz, Ar–H), 8.0 (2H, d, J = 8.5 Hz, Ar–H), 7.5 (2H, s,
incubator.

Med Chem Res
Cell viability assay
of the product was identified by elemental analysis and
spectral data. H NMR spectrum displayed two singles at δH
1
Human neuroblastoma SH-SY5Y or murine RAW 264.7
macrophage cells were seeded in 96-well culture plates at an
initial density of 3 × 10 cells/well (Corning, NY, USA).
The testing compound at 1 and 10 µM was incorporated into
the cell culture in DMEM medium in humidified incubator
2.93, 3.14 ppm due to the N,N-dimethyl protons and two
doublets at δ 5.83 and 7.71 ppm (d, J = 12 Hz) due to the
H
4
ethylenic protons, in addition to the multiplet at the region
δH 7.18 ppm (2H, m, aromatic). The value of coupling
constant (J = 12 Hz) for the ethylenic protons indicate that
the enaminone 2 exist in the E-configuration, which is in
complete agreement with the results recently reported
(Shaaban et al. 2007). The reaction of enaminone 2 with the
nitrilamine, generated in situ by the reaction of triethyla-
mine on the (1E)-N-(4-methoxyphenyl)-2-oxopropanehy-
drazonoyl chloride 3a in refluxing absolute ethanol afforded
within a 5% CO atmosphere at 37 °C for 24 h. The survival
2
of the cells was determined using Alamar blue dye (Invi-
trogen, CA, USA) according to the manufacturer instruc-
tion. The absorbance (A) was measured at 570 nm using a
microplate reader. This cell viability assay is similar in
principle to that of 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MMT) which has been previously
validated by Bohlken et al. 2009.
a
single product (1-[1-(4-methoxyphenyl-4-(3,4,5-tri-
1
methoxybenzoyl)-1H-pyrazol-3-yl]ethanone 4a. H NMR
spectrum of the isolated product 4a revealed three singlets
at δ 2.59, 3.34 and 8.94 ppm due to acetyl-CH , methoxy-
In vitro anti-inflammatory assay
H
3
CH and pyrazole-5-CH protons, respectively, in addition to
3
By using murine RAW 264.7 macrophages and a bacterial
lipopolysaccharide (LPS), the in vitro anti-inflammatory
activity of compounds (10 µM) was performed according to
the previously described assay (Ahmed et al. 2006) mod-
ified from the method of Park et al. 2005. The expression of
iNOS, COX-2 proinflammatory proteins in the LPS-
stimulated cells was determined by western-immunoblot
analysis after being separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and transferred to a
polyvinylidene difluoride membrane. β-actin was used as an
internal loading control to normalize the blot data.
aromatic protons (6H, m) in the region of δ 7.12–7.93 ppm.
The structure of 4a was further confirmed by its reaction
with hydrazine hydrate 99% to afford a white crystalline
product, which was identified as 7-methyl-2-(4-methox-
yphenyl)-4-(3,4,5-trimethoxyphenyl)-2H-pyrazolo[3,4-d]
pyridazine (5a) (Scheme 1). IR spectrum of 4a showed
−
1
two carbonyl absorption bands at λmax 1684 and 1651 cm
1
which disappeared in IR spectrum of 5a. The H NMR
spectrum of the isolated product revealed a singlet at δH
9.52 ppm, which indicates the presence of pyrazole 5-CH
1
3
proton (Hamdy and El-Senousy 2013). Also the C NMR
of compound 5a is in agreement with its chemical structure.
Under the same experimental conditions, the enaminone 2
was allowed to react with hydrazonoyl chlorides (3b–g) to
afford the corresponding pyrazole derivatives (4b–g). The
latter product underwent cyclocondensation upon treatment
with hydrazine hydrate 99% in refluxing ethanol to yield
pyrazolo[3,4-d]pyridazine derivatives (5b–g). All the
structures were confirmed on the basis of spectral data and
elemental analysis. The data of the physiochemical prop-
erties of all compounds is given in (Table 1).
In vitro neuroprotective assay
Human neuroblastoma SH-SY5Y cells were seeded in 96-
well culture (vide supra), treated with the tested compounds
at 1 µM for 1 h and then exposed to the neurotoxin 6-
hydroxydopamine (6-OHDA, Sigma-Aldrich, MO, USA) at
2
0 μM. Following a further 18 h incubation period, SH-
SY5Y cells survival was determined using Alamar blue
assay by measuring the produced pink dye at 595 nm.
Relative protection (%) was calculated as [(optical density
(
OD) of 6-OHDA/compounds-treated cells −OD of 6-
The anti-inflammatory activity
OHDA-treated cells) / (OD of control cells − OD of 6-
OHDA-treated cells)].
Gram-negative bacterial LPS is a key component linked to
septic shock which is associated by a buildup of inflam-
matory mediators such as prostaglandins and NO (Hewett
and Roth 1993) due to overexpression of the proin-
flammatory COX-2 and iNOS proteins, respectively.
Therefore, management of the inflammatory responses is
achievable by compounds which can down regulate the
LPS-induced expression of COX-2 or iNOS or both pro-
teins in the affected tissues. In this work, LPS-stimulated
RAW264.7 macrophages and immunoblot analysis were
employed to recognize which compounds possess an
Results
Chemistry
Treatment of 3,4,5-trimethoxy acetophenone 1 with (DMF-
DMA) under solvent free condition, afforded a orange
crystalline product identified as (2E)-3-(dimethylamino)-1-
(
3,4,5-trimethoxyphenyl)prop-2-en-1-one 2. The structure

Med Chem Res
Scheme 1 Synthetic route of
compounds (4a–g) and (5a–g)
in vitro inhibitory effect on the expression of proin-
flammatory proteins. The well-known anti-inflammatory
drug dexamethasone was used as a positive control since it
inhibits iNOS and COX-2 expression (Korhonen et al.
Table 1 Physico-chemical properties of compounds (4a–g) and
(5a–g)
Compound
R
MP (°C) M. formula
M. weight
2
002; Koistinaho et al. 1999; Oh et al. 2015). Before the
4a
4b
4c
4d
4e
4f
OCH
F
3
205–207
235–237
225–227
C
C
C
22
21
22
H
H
H
22
N
2
O
6
410.4
398.3
394.4
380.3
459.4
459.2
414.8
406.4
394.3
390.4
376.4
455.4
455.3
410.8
anti-inflammatory assay, compounds (4a–g) and (5a–g)
were proved to be non-cytotoxic based on cell viability
19FN
2
O
5
CH
H
3
22
N
O
2 5
(
>90%) when assessed against the growth of RAW264.7
21 20 2
198–200 C H N O
5
macrophage cells via alamar blue assay at the same con-
centration (10 μM).
SO NH
21 21 3 7
237–239 C H N O S
2
2
Br
240–242 C H BrN O
2
1
19
2
5
4
5
5
5
5
5
5
5
g
a
b
c
d
e
f
Cl
245–247
230–232
255–257
250–252
203–205
315–317
260–262
265–267
C
C
C
C
C
C
C
C
21
H
22
H
21
H
22
H
21
H
21
H
21
H
21
H
2
19ClN O
5
The effect of pyrazoline and pyrazolo[3,4-d]pyridazine
derivatives on LPS-induced iNOS expression
OCH
F
3
22 4 4
N O
19FN
4
O
3
CH
H
3
22
20
21
N
N
N
O
4 3
O
4 3
O
5 5
In this experiment, the synthetic compounds exerted sig-
nificant (p < 0.05) down regulation on the LPS-induced
iNOS expression, except compounds 4a, 4d and 4f which
showed no activity. Based on whether the compounds
belong to pyrazole or pyrazolo[3,4-d]pyridazine derivatives,
two levels of iNOS expression inhibitory activity have been
observed. All pyrazolo[3,4-d]pyridazine derivatives (5a–g)
strongly inhibited the iNOS expression in the LPS-
stimulated cells to less than 51.3% while the active pyr-
azole derivatives showed moderate anti-iNOS-based anti-
inflammatory effect in the range of 58.1 ± 3.7–73.9 ± 6.3%
at the same concentration (10 μM) (Fig. 1). In contrast to the
inactive compounds 4a, 4d, and 4f regarding the effect on
the LPS-induced iNOS expression, their correspondent
pyrazolo[3,4-d]pyridazine derivatives 5a, 5d, and 5f dis-
closed strong inhibition (41.8 ± 1.4, 28.2 ± 8.0, and 20.3 ±
SO
Br
Cl
2
NH
2
S
19BrN
19ClN
4 3
O
g
4 3
O
0.6%, respectively). The pyrazolo[3,4-d]pyridazine deriva-
tives having 4-fluorophenyl and 4-chlorophenyl moieties 5b
and 5g reduced the anti-iNOS expression by 2.5-fold and by
more than 1/3 potency of dexamethasone in comparison to
their corresponding pyrazole derivatives 4b and 4g (Fig. 1).
The reaction of hydrazine hydrate with the pyrazole deri-
vatives 4c and 4e could escalate the iNOS inhibitory
activity by 14.0 and 12.6% as demonstrated by the effect of
compounds 5c and 5e, respectively.

Med Chem Res
Fig. 2 Effect of 3,4,5-trimethoxybenzene bearing pyrazole a and
pyrazolo[3,4-d]pyridazine b scaffolds on COX-2 protein expression in
LPS-stimulated RAW264.7 macrophage cells by immunoblot analysis.
The values are mean ± S.E.M. (n = 6). Equal loading of protiens was
verified by β-actin immunoblot. Relative intensity of the LPS-
stimulated control group was taken as 100%. Significantly different
from the untreated control group (*p < 0.05) and from LPS-stimulated
group (#p < 0.05)
Fig. 1 Effect of 3,4,5-trimethoxybenzene bearing pyrazole a and
pyrazolo [3,4-d]pyridazine b scaffolds on iNOS protein expression in
LPS-stimulated RAW264.7 macrophage cells by immunoblot analysis.
The values are mean ± S.E.M. (n = 6). Equal loading of proteins was
verified by β-actin immunonblot. Relative intensity of the LPS-
stimulated control group was taken as 100%. Significantly different
from untreated control group (*p < 0.05) and from LPS-stimulated
group (#p < 0.05)
The effect of pyrazoline and pyrazolo[3,4-d]pyridazine
derivatives on LPS-induced COX-2 expression
sulfonamidophenyl 4e moieties together with their corre-
sponding pyrazolo[3,4-d]pyridazine compounds 5a, 5b, 5f,
5g, and 5e exerted moderate activity by inhibiting the LPS-
The 3,4,5-trimethoxybenzene bearing pyrazole (4a–g) and
pyrazolo[3,4-d]pyridazine (5a–g) derivatives had been
assessed for their inhibitory effect on COX-2 expression in
LPS-stimulated cells. Among the fourteen tested com-
pounds, only two pyrazole derivatives 4c and 4d were
devoid of anti-COX-2 expression activity. However, the
corresponding pyrazolo[3,4-d]pyridazine derivatives 5c and
induced COX-2 expression to the range of 66.3 ± 8.2 to
81.1 ± 1.9% (p < 0.05) (Fig. 2).
The neuroprotective activity
A neuroprotective assay based on the protection potential of
the tested compound against 6-OHDA (20 µM)-induced
toxicity in a human dopaminergic neuroblastoma SHSY5Y
cells was performed as reported previously (Lee et al. 2006;
Chen et al. 2012). Prior to carrying out this assay, the
cytotoxic activity of the synthetic pyrazole, pyrazolo[3,4-d]
pyridazine derivatives, and 6-OHDA was measured at the
same concentration (1.0 μM) against SH-SY5Y cells, via
alamar blue assay. The compounds which showed cell
5
d, produced upon condensation with hydrazinehydrate
with pyrazole derivatives (Scheme 1) weakly and strongly
down regulate the COX-2 expression to the level of 93.3 ±
4
.7% and 42.8 ± 1.4% (p < 0.05), respectively, at 10 μM
relative to that expressed (100%) in the LPS-stimulated
control cells. Other pyrazole derivatives having 4-
methoxyphenyl 4a, 4-halophenyl 4b, 4f, and 4g, and 4-

Med Chem Res
Fig. 3 Effect of selected 3,4,5-trimethoxybenzene bearing pyrazole
4b and 4e–4g) and pyrazolo[3,4-d] pyriadazine (5a–5g) scaffolds on
neuroblastoma SH-SY5Y cells at 1.0 μM and on 6-OHDA (20 μM)
treated neuroblastoma SH-SY5Y cells. Cell viabiliaty was determined
for 6-OHDA and tested compounds at 1.0 μM. The values are mean ±
S.E.M. (n = 6). Significantly different from untreated control group
(*p < 0.05) and from 6-OHDA (20 μM)-treated control (#p < 0.05)
(
viability >50% at 1.0 μM were selected and then evaluated
for their protection potential against 6-OHDA-induced
toxicity in SH-SY5Y cells (Fig. 3). Compounds 4a, 4c,
and 4d were thus excluded from the neuroprotective assay
as they resulted in very low cell viability <40% at 1.0 μM.
degree (27.1 ± 12.4 to 53.1 ± 2.4%) by pretreatment 4b,
(4e–g), and (5a–c) at 1.0 µM (Fig. 3). However, the higher
cell viability index achieved by 5e, 5d, 5f, and 5g (107.2 ±
2.9, 87.8 ± 1.6, 80.8 ± 3.8, 76.8 ± 1.9%, respectively) rela-
tive to 4b, (4e–g) and (5a–c) represent an advantageous
merit added to their neuroprotective effect.
The neuroprotective activity of pyrazole and pyrazolo
[
3,4-d]pyridazine derivatives against 6-OHDA-induced
SH-SY5Y cell death
Discussion
From the results shown in Fig. 3, the pryrazole 4b, (4e–g)
and pyrazolo[3,4-d]pyridazine (5a–g) derivatives demon-
strated significant (p < 0.05) neuroprotective activity against
Recent research shows that chronic inflammatory diseases,
share common pathways of cellular and molecular dysre-
gulation. Neuroinflammation is also known to have a major
role in neuronal damage in neurological and neurodegen-
erative disorders such as Alzheimer’s disease (AD), Par-
kinson’s disease (PD), and multiple sclerosis (Skaper 2007).
Altered gene expression induced by epigenetic mechanisms
contributes further to the development of chronic inflam-
mation (Garn et al. 2016). These facts encourage scientists
to develop new preventive and therapeutic strategies to
effectively control inflammation. In this work, we have
constructed novel series of pyrazole (4a–g) and pyrazolo
[3,4-d]pyridazine (5a–g) scaffolds bearing trimethox-
6
-OHDA (20 µM)-induced SH-SY5Y cell death with vari-
able potencies (>25% protection) relative to the 6-OHDA
20 µM)-intoxicated control (0.0% protection). Compound
e: 7-methyl-2-(4-sulfonamidophenyl)-4-(3,4,5-trimethox-
(
5
yphenyl)-2H-pyrazolo[3,4-d]pyridazine exhibited more
than 100% relative neuroprotection (110.7 ± 4.3%) against
6
-OHDA toxicity in human neuroblastoma cells, followed
by the related pyrazolo[3,4-d]pyridazine derivatives 5d, 5f,
and 5g in decreasing order (82.0 ± 2.4, 71.6 ± 5.7, 65.6 ±
2
1
.8%, respectively) in the same experimental condition at
.0 mM. It was also observed that the cytotoxicity of 6-
yphenyl through condensation of
a
trimethoxyar-
OHDA on SH-SY5Y cells could be reduced in a moderate
ylenaminone 2 with seven N-substituted arylhydrazonoyl

Med Chem Res
chlorides (3a–g) in presence of triethyl amine (TEA) and
Conclusion
then hydrazine, respectively (Scheme 1). Compounds
(
4a–g) and (5a–g) thus possessed 1,3,4-trisubstituted pyr-
In conclusion, novel series of pyrazole and pyrazolo[3,4-d]
pyridazine derivatives of 3,4,5-trimethoxyphenyl were
synthesized. The reactions were clean and products were
obtained as single spot. All the compounds gave satisfactory
spectral data. The compounds were evaluated for anti-
inflammatory and neuroprotective activities. The pyrazolo
[3,4-d]pyridazine derivative 5d strongly down regulated the
LPS-induced COX-2 expression. Furthermore, the pyrazolo
[3,4-d]pyridazine derivatives 5a, 5d, and 5f disclosed strong
inhibition in the LPS-induced iNOS expression. Compound
5e exhibited potent neuroprotection against 6-OHDA toxi-
city in human neuroblastoma cells. From the results of
neuroprotectivity, we suggest that further investigation of
(5a–g) for their therapeutic potential against neurodegen-
erative diseases is worthy.
azole nucleus. Since celecoxib (1,3,5-trisubstituted pyr-
azole), its analogous and related compounds (including
1
effects by different mechanisms and indices (Alegaon
et al. 2014; Ragab et al. 2013; Villa et al. 2016), the anti-
inflammatory activity of compounds (4a–g) and (5a–g)
were tested in vitro. The inhibitory potential of the com-
pounds against the expression of iNOS and COX-2 protein
in the LPS-stimulated RAW264.7 macrophages and against
,3,4-trisubstituted pyrazoles) showed anti-inflammatory
6
-OHDA-induced cytotoxicity in neuroblastoma SH-SY5Y
cells were measured and considered as indices for the anti-
inflammatory activity. The tested pyrazolo[3,4-d]pyridazine
compounds (5a–g) exerted much stronger down regulation
effect against the proinflammatory iNOS protein expression
in the LPS-stimulated macrophages to the range of
2
0.3–51.3% relative to the correspondent pyrazole deriva-
Acknowledgements The authors would like to extend their sincere
appreciation to the Deanship of Scientific Research at King Saud
University for funding this research group no. (RG 1435–006).
tives (4a–g), which showed moderate activity (58.1–73.9%)
while compounds 4a, 4d, and 4f were found to be inactive
(
Fig. 1). Moreover, except of the inactive compounds 4c
Conflict of interest The authors declare that they have no competing
and 4d, the tested compound showed anti-COX-2-based
activity (Fig. 2) but with variable potencies. Among them,
the pyrazolo[3,4-d]pyridazine derivative 5d exhibited the
strongest potential to suppress the COX-2 expression to the
level of 42.8% relative to that expressed (100%) in the LPS-
stimulated control cells. The anti-COX-2 and/or iNOS-
based anti-inflammatory activity shown by the synthetic
compounds has encouraged us to measure their neuropro-
tective activity against 6-OHDA-induced toxicity in a
human dopaminergic neuroblastoma SHSY5Y cells. Only
compounds which showed good cell viability 4b, (4e–g)
and (5a–g) were assessed and fortunately showed positive
activity with variable potencies. The pyrazolo[3,4-d]pyr-
idazine derivative 7-Methyl-2-(4-sulfonamidophenyl)-4-
interests.
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