Non-ulcerogenic pyrazolyl 2-hydroxychalcones and pyrazolylpyrazolines derived from naturally existing furochromone (khellin): semi-synthesis, docking study and anti-inflammatory activity

Article abstract of DOI:10.1080/14786419.2021.1907383

Novel pyrazolyl 2-hydroxychalcone derivatives 3a–e and pyrazolylpyrazoline derivatives 4a–e and 5a–j derived from the naturally existing furochromone (Khellin) were synthesized and evaluated for their in?vivo anti-inflammatory activity. Most of the synthesized compounds showed better or comparable activity to that of Diclofenac as reference drug. Twelve compounds were evaluated for their ulcerogenic potential and exhibited no ulcerogenic effect. In addition compounds 3c, 5c and 5h as examples showed PGE₂ inhibition % 88.86, 65.87 and 44.06, respectively and TNFα inhibition % 48.62, 31.11 and 16.02, respectively in rat serum samples. Compounds 3c, 5c, 5h and Celecoxib were subjected to in?vitro COX-1 and COX-2 inhibition assay, showed selectivity index 45.04, 102.04, 131.58 and 185.18, respectively. The computational finding supported those of in?vitro, where the pyrazolylpyrazolines interacted with the COX-2 enzyme in a similar orientation to that of Celecoxib, while chlacones were found to exhibit similar orientation to that of Diclofenac.

Full text of DOI:10.1080/14786419.2021.1907383

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Non-ulcerogenic pyrazolyl 2-hydroxychalcones
and pyrazolylpyrazolines derived from naturally
existing furochromone (khellin): semi-synthesis,
docking study and anti-inflammatory activity
Fatma Abd El-Fattah Ragab, Yassin Mohammed Nissan, M. Alaraby Salem,
Mamdouh Moawad Ali & Ahmed Abbass Mohamed Abdelrahman Selim
To cite this article: Fatma Abd El-Fattah Ragab, Yassin Mohammed Nissan, M. Alaraby
Salem, Mamdouh Moawad Ali & Ahmed Abbass Mohamed Abdelrahman Selim (2021): Non-
ulcerogenic pyrazolyl 2-hydroxychalcones and pyrazolylpyrazolines derived from naturally existing
furochromone (khellin): semi-synthesis, docking study and anti-inflammatory activity, Natural
Product Research, DOI: 10.1080/14786419.2021.1907383
To link to this article: https://doi.org/10.1080/14786419.2021.1907383
Published online: 05 Apr 2021.
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2021.1907383
Non-ulcerogenic pyrazolyl 2-hydroxychalcones and
pyrazolylpyrazolines derived from naturally existing
furochromone (khellin): semi-synthesis, docking study
and anti-inflammatory activity
Fatma Abd El-Fattah Ragaba, Yassin Mohammed Nissana,b, M. Alaraby Salemb,c
Mamdouh Moawad Alid and Ahmed Abbass Mohamed Abdelrahman Selime
,
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt;
bPharmaceutical Chemistry Department, Faculty of Pharmacy, October University for Modern
c
Sciences and Arts (MSA), Giza, Egypt; School of Life and Medical Sciences, University of
Hertfordshire hosted by Global Academic Foundation, New Administrative Capital, Cairo, Egypt;
dBiochemistry Department, Genetic Engineering and Biotechnology Research Division, National
Research Centre, Giza, Egypt; eMedicinal and Aromatic Plants Department, Desert Research Centre,
Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Received 23 January 2021
Accepted 19 March 2021
Novel pyrazolyl 2-hydroxychalcone derivatives 3a–e and pyrazolyl-
pyrazoline derivatives 4a–e and 5a–j derived from the naturally
existing furochromone (Khellin) were synthesized and evaluated
for their in vivo anti-inflammatory activity. Most of the synthesized
compounds showed better or comparable activity to that of
Diclofenac as reference drug. Twelve compounds were evaluated
for their ulcerogenic potential and exhibited no ulcerogenic
effect. In addition compounds 3c, 5c and 5h as examples showed
PGE2 inhibition % 88.86, 65.87 and 44.06, respectively and TNFa
inhibition % 48.62, 31.11 and 16.02, respectively in rat serum sam-
ples. Compounds 3c, 5c, 5h and Celecoxib were subjected to
in vitro COX-1 and COX-2 inhibition assay, showed selectivity
index 45.04, 102.04, 131.58 and 185.18, respectively. The compu-
tational finding supported those of in vitro, where the pyrazolyl-
pyrazolines interacted with the COX-2 enzyme in a similar
orientation to that of Celecoxib, while chlacones were found to
exhibit similar orientation to that of Diclofenac.
KEYWORDS
Pyrazole; pyrazolylpyrazo-
line; khellin; anti-
inflammatory
CONTACT Fatma Abd El-Fattah Ragab
fatmarag@hotmail.com; Ahmed Abbass Mohamed Abdelrahman Selim
ahmed-abbass-10@hotmail.com
Supplemental data for this article can be accessed online at https://doi.org/10.1080/14786419.2021.1907383.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
F. A. RAGAB ET AL.
1. Introduction
Despite their side effects non-steroidal anti-inflammatory drugs have been extensively
used to treat inflammation and increase pain threshold (Day and Graham 2013). These
side effects (renal malfunction, delayed labour and mainly gastric irritation that may
lead to peptic or duodenal ulcer) were mainly attributed to the inhibition of cyclooxy-
genase-1 enzyme (COX-1), a constitutive isoform with no definite role in inflammation
process (Matsui et al. 2011). On the contrary, the second isoform of cyclooxygenase
enzyme (COX-2), an inducible isoform, was recognized as the key enzyme in synthesis
of prostaglandins (PGs) inflammatory mediators with a major role in inflammation pro-
cess (Forey and Yonida 2002). Discovering selective COX-2 inhibitors was considered a
breakthrough to control inflammation without suffering from these side effects (Forey
and Yonida 2002). However, two selective COX-2 inhibitors (Rofecoxib and Valdecoxib)
were withdrawn due to their serious cardiotoxicity (Back et al. 2012). Explanation of
these side effects depends on the fact that selective COX-2 inhibitors may disturb the
balance between prostacyclin (PGI2) and thromboxane (TxA2) towards TxA2 which may
lead to coronary artery disease, heart attack and stroke (Back et al. 2012). Therefore,
seeking for mildly selective COX-2 inhibitors is still a goal for medicinal chemists in
order to control inflammation without suffering from COX-1 inhibition side effect (pep-
tic ulcer) or selective COX-2 inhibition side effect (cardiotoxicity).
In the other hand, for millennia medicinal plants have been a valuable source of
therapeutic agents and still many of today’s drugs are plant-derived natural products
or their derivatives (Kinghorn et al. 2011). It has been estimated that approximately
over half of the pharmaceuticals in clinical use today are derived from natural prod-
ucts (Newman and Cragg 2012). In the past decades, pharmaceutical industry focused
mainly on libraries of synthetic compounds as drug discovery source, they are compar-
ably easy to produce and resupply and demonstrate good compatibility with estab-
lished high throughput screening (HTS) platforms (David et al. 2015). However, at the
same time there has been a declining in the number of new drugs reaching the mar-
ket (Scannell et al. 2012), which rising and renewed scientific interest in drug discovery
from natural sources despite of its known challenges (Kingston 2011).
Ammi visnaga (khella) is a widely distributed ancient egyption medicinal plant, the
furochromone khellin I (major constituent of ammi visnaga seeds) has been used to
treat a variety of diseases including renal colic, kidney stones, coronary diseases, bron-
€
chial asthma, vitilligo and psoriasis (Gunaydin and Beyazit 2004; Badr et al. 2015; Khalil
et al. 2020). The existence of benzofuran moiety in khellin encourages the medicinal
chemist to use it as synthone in the search for new anti-inflammatory agents, as ben-
zofuran containing structures were reported to possess anti-inflammatory activity
(Deshpande et al. 2012; Ragab et al. 2020) and exhibited promising gastro-protective
effect (Ragab et al. 2007; Hassan and Soliman 2010; Ragab et al. 2020). Furthermore,
the existence of pyrazole moiety in several anti-inflammatory agents especially, the
well-known selective COX-2 inhibitor (Celecoxib) (Penning et al. 1997) encouraged
many researchers to develop anti-inflammatory agents with pyrazole scaffold carrying
a vicinal diaryl substitution pattern which thought to be responsible for the selectivity
of Celecoxib (Ren et al. 2018). Several semisynthetic derivatives of khellin containing
pyrazole scaffold have been semi-synthesized and showed good anti-inflammatory

NATURAL PRODUCT RESEARCH
3
N
O
OCH3
OCH3
OCH3
O
O
OCH3
N
R4
R3
NH
N
N
OH
R1
O
NNH2
O
CH3
O
OH
HC
OH
R2
OCH3
OCH3
SO2NH2
IV
III
I
II
Figure 1. Khellin, pyrazolyl khellin derivatives and 2-hydroxychalcone as anti-inflammatory agents.
properties II and III (Figure 1) (Ragab et al. 1992; Hassan et al. 2014). Chalcones are a
group of plant-derived polyphenolic compounds belonging to the flavonoids family,
chalcones either natural or synthetic have been reported to exhibit diverse biological
activity including anti-inflammatory activity (Singh et al. 2014). 2-hydroxychalcones IV
have demonstrated anti-inflammatory activity with strong inhibition of prostagalndins
(PGs) the main inflammatory mediators, presenting promising candidates in the search
for potent anti-inflammatory agents (Figure 1) (Dao-Tran et al. 2009).
Motivated by the above information the present investigation deals with the semi-
synthesis of pyrazolyl 2-hydroxychalcones 3a–e derived from naturally occurring furo-
chromone khellin to be tested as anti-inflammatory agents. As pyrazoline nucleus is
present as a core in a variety of leading drugs as phenazone (antipyrine) and metami-
zole, which possess anti-inflammatory, antipyretic and analgesic activity (Elattar and
Fadda 2016). The synthesized chalcones 3a–e are used to construct the corresponding
pyrazolylpyrazoline derivatives 4a–e and 5a–j hoping that these compounds contain-
ing the three active anti-inflammatory motifs pyrazole, pyrazoline and hydroxybenzo-
furanyl may exhibit enhanced anti-inflammatory activity, also in 5a–j the pyrazoline
nucleus is disubstituted with vicinal aryl rings (substituted pyrazole and phenyl) hop-
ing to increase COX-2 selectivity. The most active compounds were evaluated for their
ulcerogenic effect. Compounds 3c, 5c and 5h as examples were subjected to in vitro
COX-1 and COX-2 inhibition activity as well as their ability to inhibit PGE2 and TNFa in
rat serum samples. Furthermore, docking study on the active site of COX-2 enzyme
was conducted for all compounds.
2. Results and discussion
2.1. Chemistry
The pyran ring of khellin I was subjected to alkaline hydrolysis with KOH to reveal kel-
linone 1 which was confirmed by its melting point 95 ꢀC (Spath and Gruber 1938). On
the other hand, 1,3-diarylpyrazol-4-carboxaldehydes 2a–e were prepared through
Vilsemier-Haack reaction, the structures of the formed products were confirmed on
the basis of spectral analyses as well as their melting points (Yogi et al. 2015).
Khellinone 1 was further subjected to Claisen-Schmidt condensation with pyrazol-4-
carboxaldehyde derivatives 2a–e to give the pyrazolyl 2-hydroxychalcones 3a–e,
respectively (Figure 2). The structures of compounds 3a–e were confirmed on the

4
F. A. RAGAB ET AL.
R
O
O
O
O
OH
O
O
O
O
a, R= H
a
N
b, R= Br
c, R= Cl
d, R= F
N
O
O
e, R= OCH3
Khellinone
1
Khellin
I
2 a-e
b
R
R
O
O
O
O
N
HN
c
O
N
N
N
N
O
HO
HO
3 a-e
d
O
a, R= H
4 a-e
b, R= Br
c, R= Cl
d, R= F
e, R= OCH3
O
R
O
OH
N
O
N
N
N
5 a-j
R1
a, R=H, R1=H
f, R=H, R1= Cl
b, R= Br, R1=H
c, R= Cl, R1=H
d, R= F, R1=H
g, R=Br, R1= Cl
h, R= Cl, R1= Cl
i, R= F, R1= Cl
e, R= OCH3, R1=H j, R=OCH3, R1= Cl
Solvents and Reagents: a: H2O, 5%KOH; b: ethanol, NaOH; c: ethanol, hydrazine hydrate
d: ethanol, phenylhydrazine hydrochloride or 4-chlorophenylhydrazine hydrochloride
Figure 2. Scheme for semi-synthesis of compounds 3a–e, 4a–e and 5a–j.
1
basis of spectral and elemental analyses. H NMR spectra of compounds 3a–e revealed
beside other peaks two new doublets assigned to the two olefinic protons of a and b
1
unsaturated ketones confirming chalcones formation (Figures S1–S13). For example H
NMR spectra of compound 3c showed the presence of two doublet at 7.06 and
7.29 ppm corresponding to (-CO-CH ¼ CH) J ¼ 15.89, 15.90 Hz indicating the trans con-
figuration (Figures S7–S9). The pyrazolylpyrazoline derivatives 4a–e and 5a–j were
obtained by reacting chalcones 3a–e with hydrazine hydrate, phenylhydrazine

NATURAL PRODUCT RESEARCH
5
hydrochloride or 4-chlorophenylhydrazine hydrochloride, respectively (Figure 2). The
structures of pyazolylpyrazoline derivatives were confirmed on the basis of spectral
1
and elemental analyses. H NMR spectra of compounds 4a–e and 5a–j revealed two
doublet of doublet assigned to two protons of C4 pyrazoline ring and a triple doublet
or doublet of doublet assigned to one proton of C5 pyrazoline ring (Figures S14–S59).
For example 1H NMR spectra of compounds 5c revealed beside other peaks three
doublet of doublet at 3.47 ppm J ¼ 7.76, 18.03 Hz, 4.22 ppm J ¼ 11.99, 18.04 Hz and
5.51 ppm J ¼ 7.78, 11.95 Hz corresponding to two protons of C4 pyrazoline and one
proton of C5 pyrazoline, respectively and the disappearance of the two doublet corre-
sponding to (-CO-CH ¼ CH) (Figures S36–S38), while 13C NMR revealed beside other
peaks the appearance of two aliphatic carbon peak at 46.75 ppm and 55.03 ppm corre-
sponding to C4 and C5 of pyrazoline, respectively (Figure S39), the mechanism of for-
mation of 4a–e and 5a–j illustrated in Figure S60.
2.2. Biological screening
2.2.1. In vivo anti-inflammatory activity
All the newly synthesized compounds 3a–e, 4a–e and 5a–j have been evaluated for
their in vivo anti-inflammatory activity using carrageenan-induced paw edema in rats
model (50 mg/kg interperitoneal dose) (Winter et al. 1962). The protocol of animal
experiments was approved by ethical committee. The results of the anti-inflammatory
activity revealed that all the tested compounds showed good activity with a percent-
age inhibition of rat paw edema after four hours ranged from 66.02–95.24% (Table
S1). It was also noticed that all of the tested compounds showed higher inhibition
than Diclofenac (21.25%) after one hour interval except compounds 4d and 5f which
showed nearly equal percentage inhibition, while after four hours interval twelve com-
pounds were still more active than Diclofenac (84.20%). Concerning chalcone deriva-
tives 3a–e this series showed excellent anti-inflammatory activity with percentage
inhibition ranged from 73.67–92.72% after four hours, four derivatives were superior in
activity than Diclofenac. It was obvious that the nature of substituent at the 3rd pos-
ition of the pyrazole nucleus influenced the activity. The 4-chlorophenyl derivative 3c
exhibited the greatest activity. Conversion of 3a–e to the corresponding pyrazolylpyra-
zolines 4a–e with unsubstituted N1 pyrazoline also showed good anti-inflammatory
activity although less than the chalcone series with percentage inhibition after four
hours ranged from 66.02 to 87.75% but still two compounds 4c and 4e possessed
superior activity than Diclofenac. Again the nature of substituent at the 3rd position of
the pyrazole nucleus affected the activity. The most active compound was 4c with its
4-chlorophenyl substituent. While conversion of 3a–e to the corresponding pyrazolyl-
pyrazolines 5a–e with N1 pyrazoline substituted with phenyl moiety showed better
activity than the previous series 4a–e and equal or better activity than its chalcones
3a–e. The percentage of inhibition after four hours ranged from 80.29–95.24%. Four
compounds revealed improved activity than Diclofenac. The derivative 5c with 4-chlor-
ophenyl moiety at the 3rd position of the pyrazole nucleus showed the most superior
activity. Pyrazolylpyrazolines 5f–j carrying 4-chlorophenyl substituent at N1 pyrazoline
exhibited decreased activity than those with N1 phenyl substituent 5a–e with

6
F. A. RAGAB ET AL.
percentage inhibition after four hours ranged from 77.31–94.92%. Two compounds 5h
and 5j were more active than Diclofenac. Again the most active compound was the 4-
chlorophenyl substituted derivative 5h.
From this study we concluded that in the pyrazolylpyrazoline derivatives both sub-
stitutions at 3rd position of the pyrazole and N1 position of the pyrazoline affected
the anti-inflammatory activity, the best substituents were 4-chlorophenyl in the pyra-
zole and phenyl in the pyrazoline moieties. The best member was 5c, with percentage
inhibition 95.24%. While in chalcones substitution at 3rd position of the pyrazole
affected the anti-inflammatory activity, again the best substituents were 4-chloro-
phenyl in the pyrazole nucleus. The best member was 3c with percentage inhibition
92.72%. Replacement of the 4-chlorophenyl moiety substitutions at 3rd position of the
pyrazole with its isostere 4-bromophenyl moiety decreased the activity, while the 4-
fluorophenyl derivatives showed marked decrease in the anti-inflammatory activity
than the 4-chlorophenyl or 4-bromophenyl derivatives.
2.2.2. Ulcerogenic effect
The ulcerogenic potential of the 3a–c, 3e, 4c, 4e, 5a–c, 5e, 5h and 5j with reference
to Diclofenac (in an oral dose 100 mg/kg/day) was evaluated (Hassan et al. 2014). The
results revealed that the twelve tested compounds exhibited no ulcerogenic effect. In
the contrary, Diclofenac showed marked ulcerogenic effect (ulcer index 40 6 2.21).
These results greatly supported our main objective to avoid gastric insult caused by
COX-1 inhibition.
2.2.3. Evaluation of PGE2 inhibition in rat serum samples
The PGE2 concentration in rat serum samples for the three most active compounds
3c, 5c, 5h and Diclofenac were assayed and the percentages of PGE2 inhibition were
calculated. Two compounds 3c and 5c were superior in activity than Diclofenac
(45.49%) with percentage inhibition 88.86 and 65.87%, respectively, while the deriva-
tive 5h was nearly equal to Diclofenac with percentage inhibition 44.06% (Table S2).
2.2.4. Evaluation of TNFa inhibition in rat serum samples
The Tumor necrosis factor alpha (TNFa) concentration for compounds 3c, 5c, 5h and
Diclofenac were assayed and the percentages of TNFa inhibition were calculated.
Again two compounds 3c and 5c were more active than Diclofenac (19.43%) with per-
centage inhibition of 48.62 and 31.11%, respectively, while the derivative 5h were
slightly less than Diclofenac with 16.02% (Table S2).
2.2.5. In vitro COX-1 and COX-2 inhibition assay
Compounds 3c, 5c, 5h and Celecoxib have been screened for their inhibitory activity
of COX-1 and COX-2 isozymes using an ovine-COX-1/COX-2 assay kit (Hassan et al.
2014). All the tested compounds showed no inhibitory effect on COX-1 up to 50 mM.
Moreover, compounds 3c, 5c, 5h and Celecoxib showed in vitro COX-2 inhibitory
activity with IC50 1.11, 0.49, 0.38 and 0.27 mM, respectively. Pyrazolyl 2-hydroxychalcone
3c (SI 45.04) and pyrazolylpyrazoline derivatives 5c and 5h (SI 102.04 and 131.58,
respectively) showed less selectivity against COX-2 than Celecoxib (SI 185.18) (Table

NATURAL PRODUCT RESEARCH
7
S3). These results mean that our compounds may have lower or absent cardiovascular
side effects due to its mild selectivity on COX-2.
2.3. Docking study
The structure of the COX-2 enzyme differs from that of COX-1 enzyme in the volume
of the active site. The active site of COX-2 possesses an additional binding pocket,
which is thought to be responsible for the selectivity of selective COX-2 inhibitors
(Blobaum and Marnett 2007). In order to elucidate the mechanism of selectivity shown
by compounds 3c, 5c and 5h, compounds 3a–e, 4a–e, 5a–j, Diclofenac and Celecoxib
were docked into the active site of COX-2 enzyme PDB code: 3LN1 (Wang et al. 2010)
€
€
using Maestro 11.4 (Schrodinger Release 2017-4: Maestro; Schrodinger, LLC: New York,
NY, USA, 2017) (Table S4 and Figures S61-S71). The computational finding supported
those of in vitro, where the pyrazolylpyrazoline derivatives were found to exhibit high
probability (high number of pose for the same compound) to interact with the active
site in a similar orientation to that of Celecoxib by fitting one of its aromatic rings in
the same additional pocket the Celecoxib fit its N1-phenyl pyrazole and forming bonds
with some of the amino acids Celecoxib binds to (Arg 106, Arg 499, Phe 504, Gln 178
and Ser 339) (Figures S64, S66 and S68). While chlacone derivatives were found to
exhibit high probability to orientate similarly to the non-selective COX inhibitor
Diclofenac (Figures S70 and S71).
3. Conclusion
New pyrazolyl 2-hydroxychalcones 3a–e, and pyrazolylpyrazolines 4a–e and 5a–j
were semi-synthesized and showed good in vivo anti-inflammatory activity with a
percentage inhibition of rat paw edema after four hours ranged from 66.02–
95.24%. Twelve compounds were still more active than Diclofenac (84.20%) after
four hour interval. Concerning chalcone derivatives 3a–e the best compound was
3c with percentage inhibition 92.72%, while for pyrazolylpyrazoline derivatives 5a–j
the best compounds were 5c and 5h 95.24 and 94.92%, respectively. Twelve com-
pounds were evaluated for their ulcerogenic potential and exhibited no ulcerogenic
effect. In addition compounds 3c, 5c and 5h as examples showed PGE2 inhibition
% 88.86, 65.87 and 44.06, respectively and TNFa inhibition % 48.62, 31.11 and
16.02, respectively in rat serum samples. Moreover compounds 3c, 5c, 5h and
Celecoxib were subjected to in vitro COX-1 and COX-2 inhibition assay and showed
selectivity index 45.04, 102.04, 131.58 and 185.18, respectively. The docking study
supported those of in vitro, where the pyrazolylpyrazolines interacted with the COX-
2 enzyme in a similar orientation to that of Celecoxib, while chlacones were found
to exhibit similar orientation to that of Diclofenac. So our study suggest that these
classes of compounds may be able to control inflammation without suffering from
COX-1 inhibition side effect (peptic ulcer) or selective COX-2 inhibition side effect
(cardiotoxicity) due to its safety on stomach mucosa and its moderate selectivity
against COX-2 enzyme.

8
F. A. RAGAB ET AL.
Disclosure statement
No potential conflict of interest was reported by the author(s).
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