Synthesis, characterization and molecular docking studies of substituted 4-coumarinylpyrano[2,3-c]pyrazole derivatives as potent antibacterial and anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2016.09.021

A green, eco-friendly and efficient protocol has been developed and synthesized a series of coumarin based pyrano[2,3-c]pyrazole derivatives (3) by multi-component reaction (MCR). Unexpected 3-coumarinyl-3-pyrazolylpropanoic acids (4) have been isolated by the reaction of compound (3) in acidic conditions. Further, intramolecular cyclization of compounds (4) leads to C₄[sbnd]C₄chromons (9) and these compounds were screened for their biological activities using array of techniques. Most of the compounds exhibited promising antibacterial activity, in particular Gram-positive bacteria. The anti-inflammatory assay was evaluated against protein denaturation as well as HRBC membrane stabilization methods and compounds exhibit excellent anti-inflammatory activity in both methods. Molecular docking study has been performed for all the synthesized compounds with S.?aureus dihydropteroate synthetase (DHPS) and results obtained are quite promising.

Full text of DOI:10.1016/j.ejmech.2016.09.021

Accepted Manuscript
Synthesis, characterization and molecular docking studies of substituted 4-
coumarinylpyrano[2,3-c]pyrazole derivatives as potent antibacterial and anti-
inflammatory agents
Bahubali M. Chougala, S. Samundeeswari, Megharaja Holiyachi, Lokesh A. Shastri,
Suneel Dodamani, Sunil Jalalpure, Sheshagiri R. Dixit, Shrinivas D. Joshi, Vinay A.
Sunagar
PII:
S0223-5234(16)30752-8
10.1016/j.ejmech.2016.09.021
EJMECH 8887
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 April 2016
Revised Date: 22 August 2016
Accepted Date: 7 September 2016
Please cite this article as: B.M. Chougala, S. Samundeeswari, M. Holiyachi, L.A. Shastri, S. Dodamani,
S. Jalalpure, S.R. Dixit, S.D. Joshi, V.A. Sunagar, Synthesis, characterization and molecular
docking studies of substituted 4-coumarinylpyrano[2,3-c]pyrazole derivatives as potent antibacterial
and anti-inflammatory agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.09.021.
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ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis, characterization and molecular docking studies of substituted 4-
coumarinylpyrano[2,3-c]pyrazole derivatives as potent antibacterial and anti-
inflammatory agents
a
a
a
a,
Bahubali M. Chougala, Samundeeswari S, Megharaja Holiyachi, Lokesh A. Shastri, * Suneel
b
b,c
d
d
e
Dodamani, Sunil Jalalpure, Sheshagiri R Dixit, Shrinivas D. Joshi, Vinay A Sunagar
a
Department of Chemistry, Karnatak University, Dharwad, 580 003, Karnataka, India.
b
Dr. Prabhakar Kore Basic Science Research Center, KLE University, Belgaum 590010,
Karnataka, India.
c
KLE University’s College of Pharmacy, Nehru Nagar, Belgaum 590010, Karnataka, India.
d
Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry,
S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-580002, Karnataka, India.
e
Department of Chemistry, G.S.S. College, Belagavi, Karnataka, India.
OC H5
2
O
NH
2
NH
2
.H
2
O
O
O
O
NH2
N
CH
CN
R'
3
HN
R'
CHO
O
R
R
O
O
2
3
OH
O
O
O
O
N
N
HN
HN
OH
O
O
R
R
O
4
9
*
Corresponding author: e-mail: drlashastri@kud.ac.in (Lokesh A. Shastri)

ACCEPTED MANUSCRIPT
Synthesis, characterization and molecular docking studies of substituted 4-
coumarinylpyrano[2,3-c]pyrazole derivatives as potent antibacterial and anti-
inflammatory agents
a
a
a
a,
Bahubali M. Chougala, Samundeeswari S, Megharaja Holiyachi, Lokesh A. Shastri, * Suneel
b
b,c
d
d
e
Dodamani, Sunil Jalalpure, Sheshagiri R Dixit, Shrinivas D. Joshi, Vinay A Sunagar
a
Department of Chemistry, Karnatak University, Dharwad, 580 003, Karnataka, India.
b
Dr. Prabhakar Kore Basic Science Research Center, KLE University, Belgaum 590010,
Karnataka, India.
c
KLE University’s College of Pharmacy, Nehru Nagar, Belgaum 590010, Karnataka, India.
d
Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry,
S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-580002, Karnataka, India.
e
Department of Chemistry, G.S.S. College, Belagavi, Karnataka, India.
*
Corresponding author: e-mail: drlashastri@kud.ac.in (Lokesh A. Shastri)
Abstract:
A green, eco-friendly and efficient protocol has been developed and synthesized a series of
coumarin based pyrano[2,3-c]pyrazole derivatives (3) by multi-component reaction (MCR).
Unexpected 3-coumarinyl-3-pyrazolylpropanoic acids (4) have been isolated by the reaction of
compound (3) in acidic conditions. Further, intramolecular cyclization of compounds (4) leads to
C -C chromons (9) and these compounds were screened for their biological activities using
4
4
array of techniques. Most of the compounds exhibited promising antibacterial activity, in
particular Gram-positive bacteria. The anti-inflammatory assay was evaluated against protein
denaturation as well as HRBC membrane stabilization methods and compounds exhibit excellent
anti-inflammatory activity in both methods. Molecular docking study has been performed for all
the synthesized compounds with S. aureus dihydropteroate synthetase (DHPS) and results
obtained are quite promising.
Key words: DMAP, Pyrano[2,3-c]pyrazole, Pyranopyrazolo-pyrimidine, 3-Coumarinyl-3-
pyrazolylpropanoic acids, C -C pyranone, Antibacterial, Anti-inflammatory, Molecular docking.
4
4

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1
. Introduction
Coumarin analogues are a group of bioactive molecules, found substantially in nature
with a wide range of structural modifications [1]. Coumarins are a privileged oxygen
heterocycles widely distributed in various species of plants as well as animals and microbial
metabolite, playing an important role in the agricultural and pharmaceutical industries [2]. They
exhibit antiviral [3], anti-cancer [4], anti-fungal [5], anti-inflammatory [6], anti-HIV [7]
properties. They have been known to be particularly effective against Gram-positive bacterial
species [8]. Incorporation of bio-compatible fragments like vanillin and paracetamol onto
coumarin nucleus has resulted 4-aryloxymethyl coumarins which are exhibiting anti-
inflammatory activity [9]. Coumarin based anti-biotics viz. novobiocin and clorobiocin affect the
functioning of DNA gyrase, which is the basis for their broad spectrum of antibacterial activity
[10]. The naturally occurring bromotyrosine derivatives have exhibited anti-microbial effect on
the methicillin resistant S. aureus (MRSA) [11].
Pyrazolones fused pyran rings compose a very important class of compounds in the
heterocyclic area [12], and are extensively used as important precursors in the field of medicinal
chemistry due to their potential biological and pharmacological properties such as antimicrobial
[13], anti-inflammatory [14], anticancer [15] insecticidal [16], and inhibitors of human Chk1
kinase [17]. Medicinally important pyrano[2,3-c]pyrazole derivatives have been synthesized via
two component [18], three component [19], and more importantly four component reactions by
using different homogeneous and heterogeneous catalysts [20].
Computational biology and bioinformatics play a major role in drug designing and
accelerating the drug discovery process. Molecular docking of the drug molecule with the
receptor (target) provides important information about drug-receptor interactions and is
commonly used to identify the binding orientation of drug candidates to their protein targets in
order to predict the affinity and activity [21]. DNA gyrase is an enzyme that relieves strain, while
double-strand DNA is being unwound by helicase, this causes negative supercoiling of the DNA.
DNA gyrase is the target of many antibiotics, including nalidixic acid, novobiocin and
ciprofloxacin. The antibiotics such as ciprofloxacin prevents the two enzymatic steps from
replicating of DNA [22].
Knowing the therapeutic importance of coumarin, pyrazole and pyran scaffolds, the
present investigation pertains to the hybridization of these three pharmacophoric motifs in a

ACCEPTED MANUSCRIPT
single molecule. We hereby report the synthesis, characterization and biological studies of some
novel coumarin based pyrano[2,3-c]pyrazoles and pyrazolylpropanoic acid using conventional
method. The newly synthesized compounds were screened for their antibacterial and anti-
inflammatory activities, further molecular docking studies were carried out to understand the
drug–receptor interactions.
2
. Chemistry
Base catalyzed one-pot four component approach has been used for the synthesis of
substituted 4-coumarinyl-6-amino-1,4-dihydro-3-methylpyrano[2,3-c]pyrazole-5-carbonitrile 3
under optimized conditions (Scheme 1).
Insert Scheme 1
Encouraged by gaining acceptable results in our first attempt, initial optimization studies
were performed by employing 6-methyl-4-formylcoumarin 2 [23] as a precursor. Table 1 shows
the optimizing effect for the synthesis of pyrano[2,3-c]pyrazoles 3 using different ratios of water
and ethanol (0 : 100, 50 : 50, 80 : 20 and 100 : 0). The best results were obtained in terms of
yield (> 90%) by using DMAP in water-ethanol medium (Table 1, entry 8) at ambient
temperature.
Insert Table 1
The mechanism of reaction is believed to involve the Knoevenagel condensation [24]
with compound 2 and malononitrile to form an alkene intermediate followed by Michael type
addition with pyrazolin-5-one (formed from ethylacetoacetate and hydrazine hydrate) and intra-
molecular cyclization to give polyfunctionalized pyrano[2,3-c]pyrazoles (Scheme 2). The
structures for the newly synthesized pyrano[2,3-c]pyrazoles were assigned on the basis of their
spectral data and X-ray single crystal studies.
Insert Scheme 2
Insert Table 2
The literature revels that pyrimidinone derivatives have wide range of applications in
medicinal and pharmaceutical area; therefore we thought of synthesizing coumarin based
pyranopyrazole pyrimidines 5 by utilizing polyfunctional compound 3. The sequence of reaction
illustrates (Scheme 3) the construction of desired compound 5 starting from compound 3 with

ACCEPTED MANUSCRIPT
urea and thiourea in presence of acetic acid and HCl (3:1) by using literature methods [25].
Unfortunately, we noticed that the isolated expected product 5 was not supporting with spectral
data, whereas spectral data agrees well with the unexpected compound 4. Further, this
observation inspired us to study the earlier reported reaction for the synthesis of various
substituted pyranopyrazolo-pyrimidine derivatives using variety of reagents at different
experimental conditions [26]. Most of the reported methods afforded desired product pyrimidine
skeleton starting from 6-amino-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile [26]. The model
reaction was initiated to construct pyrimidine skeleton starting from compound 3 with various
reagents such as acetic anhydride in acetic acid, alone acetic anhydride, formic acid in polar
solvents, alone formic acid, water-HCl with acetamide and formamide in polar solvents etc.
Interestingly, we observed the formation of compound 4 almost >80% instead of pyrimidine
structures 5, 6, 7 and 8 (Scheme 3).
Insert Scheme 3
There are very few references in the literature for the conversion of compound 3 into 3-
aryl-3-(5-hydroxy-3-methyl-1H-pyrazol-4-yl)propanoic acid derivatives 4 using formic acid
[27]. Finally, we noticed that reaction of compound 3 with various reagents in acidic condition
resulted in the formation of compound 4 in high yield (Table 3). Mechanism of the reaction
involves opening of the pyran ring, under acidic condition with subsequent hydrolysis of nitrile
or ester group, accompanied by decarboxylation of one of the carboxylic group giving the
compound 4, the general plausible mechanism is presented in Scheme 4.
Insert Scheme 4
Insert Table 3
In order to confirm the formation of compound 4, the study was extended to
intramolecular cyclization to generate new class of compounds i.e. tail-tail pyranone (pyranone-
4
-pyranone or C -C pyranone 9) structure starting from compound 4 with thionyl chloride
4 4
(Scheme 5). Successfully we achieved the synthesis of desired product 4,5-dihydropyrano[2,3-
c]pyrazol-6(1H)-one 9 from compound 4 with excellent yield and purity (Table 4).
Insert Scheme 5
Insert Table 4

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3
. Results and Discussion
All new synthesized compounds were characterized and confirmed by spectral analysis.
Formation of product 3 was supported by spectral data. In case of compound 3a, IR spectrum
-1
-1
shows intense bands at 1707 cm and 2201 cm due to lactone carbonyl and nitrile groups
respectively. Whereas, pyrazole and pyran rings NH and NH stretching bands were observed at
2
-1
-1
1
3
339 cm and 3311 cm . It was further confirmed by H-NMR, wherein the NH proton of
pyrazole ring appeared as a singlet at 12.25 ppm. The C H of coumarin was resonated as singlet
5
8 7
at 7.55 ppm and coumarin C H was observed as a doublet at 7.41 ppm (J = 8.8 Hz). The C H of
coumarin was appeared as a doublet at 7.32 ppm (J = 8.8 Hz). The NH proton of pyran ring was
2
observed as a singlet at 7.15 ppm. Two singlet at 6.34 ppm and 5.13 ppm due to C H of
3
coumarin and C H of pyran respectively. Whereas, singlet at 2.28 ppm due to C -CH of
4
6
3
coumarin and one more singlet at 1.87 ppm due to C -CH of pyrazole. The protons correlations
3
3
have been confirmed by using 2D NMR (Fig. 1).
Insert Figure 1
Formation of product 4 is well supported by spectral data. In case of compound 4a, IR
-1
-1
spectrum shows two intense bands at 1712 cm and 1727 cm , due to carboxylic acid and
-1
lactone carbonyl. The band at 3327 cm is due to NH and OH groups. It was further confirmed
1
by H-NMR, wherein the NH of pyrazole ring and OH of propanoic acid appeared as a broad
singlet at 11.26 ppm. The C H of coumarin was resonated as a doublet at 7.69 ppm (J = 1.2 Hz).
5
The C
8 7
H of coumarin was observed as a doublet at 7.38 ppm (J = 8.4 Hz) and C H of coumarin
appeared as a doublet at 7.27 ppm (J = 8.4 Hz). The C H of coumarin was observed as a singlet
3
3
at 6.41 ppm and C H of propanoic acid was observed as a triplet at 4.57 ppm (J= 7.6 Hz) and
phenolic OH of pyrazole ring has appeared as broad singlet at 3.3 ppm. The two protons on C of
2
propanoic acid was resonated as two doublets of doublet at 3.08 ppm (J = 16.4 Hz & 8.0 Hz) and
2
6 3
.89 ppm (J = 16.0 Hz & 7.2 Hz). The methyl groups on C of coumarin and C of pyrazole
moiety were observed as two singlets at 2.34 ppm and 2.07 ppm respectively. Further this was
confirmed by DEPT-135, in this two methyl group carbon containing odd number of protons
were observed at 10.26 ppm and 20.22 ppm with +ve mode. The C of propanoic acid containing
3
odd number of proton has appeared at 30.66 ppm with +ve mode and remaining aromatic
carbons containing odd number of protons was resonated in expected region with +ve mode.

ACCEPTED MANUSCRIPT
Whereas, C carbon of propanoic acid containing even number of proton were observed at 36.46
2
ppm with –ve mode. Further, proton correlations have been confirmed by 2D NMR (Fig.2).
Insert Figure 2
-1
In case of compound 9a, IR spectrum shows two intense bands at 1722 cm and 1748
-1
-1
cm , due to lactone carbonyls of coumarin and pyran nucleus and band at 3327 cm due to -NH
1
of pyrazole ring. It was further confirmed by H-NMR, wherein the NH proton of pyrazole ring
appeared as a singlet at 11.23 ppm. The C H of coumarin was resonated as singlet at 7.69 ppm
5
and C H of coumarin was observed as a doublet at 7.40 ppm (J = 8.4 Hz). The C H of coumarin
8
7
was appeared as a doublet at 7.28 ppm (J = 8.4 Hz) and C
3
H of coumarin was observed as a
singlet at 6.43 ppm. The two protons on C of pyran ring have appeared as two doublets of
3
doublet at 3.28 ppm (J = 14.8 Hz and 6.8 Hz) and 2.88 ppm (14.2 Hz and 6.7 Hz) respectively.
Whereas, C H of pyran was resonated as a triplet at 4.60 ppm (J= 7.6 Hz). The C and C methyl
4
3
6
groups of pyrazole and coumarin moiety were resonated as two singlets at 2.06 ppm and 2.48
ppm respectively. Further, the protons correlations have been confirmed by 2D NMR (Fig. 3).
Insert Figure 3
4
. X-Ray Diffraction Studies
The single crystals of compound 3b were developed by slow evaporation of methanol as
a solvent. Suitable crystal was selected using polarizing microscope, then mounted on a Mitegen
Micromount and refined using the Bruker SAINT Software package using a narrow-frame
algorithm. The structure was solved and refined using Bruker SHELXTL Software Package.
Crystal data was refined [28,29] and their properties were studied. Crystal information files
(CIF) have been deposited at Cambridge Crystallographic Data Centre, the CCDC number for 3b
is 1463750. The crystal and structure refinement data of compound 3b is given in Table 5.
X-ray studies and molecular structure of pyranopyrazoles revealed that it exists in the
crystal phase as 2H instead of 1H tautomer as shown in Fig. 4, which is in congruence with the
literature reports of pyrano[2,3-c]pyrazole [30]. In contrast to this, N1-substituted pyrano[2,3-
c]pyrazole derivatives exist as ‘immobilized’ 1H-tautomers [31]. The crystal structure of
compound 3b is non-planar in nature with a significant dihedral angle 88.18° with coumarin and
pyranopyrazole moiety (Fig. 5 and 6).
Insert Figure 4

ACCEPTED MANUSCRIPT
Insert Figure 5
Insert Figure 6
Insert Table 5
5
. Pharmacological Screening
5
.1. Antibacterial Studies
All the synthesized compounds 3a-j, 4a-e and 9a-e were evaluated for their antibacterial
activity against two Gram-positive (S. aureus and E. faecalis) and two Gram-negative (E. coli
and P. aeruginosa) bacterial strains with ciprofloxacin as a standard by using Broth
microdilution method [32]. The minimum inhibitory concentration (MIC) of the synthesized
compounds were compared with ciprofloxacin, it revealed that almost all the newly synthesized
compounds showed excellent antibacterial activity against Gram-positive S. aureus and E.
faecalis bacterial strain. But in case of Gram-negative E. coli and P. aeruginosa bacterial strains
some of the synthesized compounds have showed selective antibacterial activity.
The antibacterial screening of compounds 3a-j reveals that almost all the coumarin
substituted pyrano[2,3-c]pyrazoles are highly active against S. aureus at concentration of 1.56 to
6
.25 µg/mL. Compounds 3b (OCH substitution at C of coumarin and CN, NH substations on
3
6
2
pyran), 3c (Cl substitution at C
6
of coumarin and CN, NH
2
substations on pyran), 3f (CH
3
substitution at C of coumarin and COOEt, NH substations on pyran), 3g (OCH substitution at
6
2
3
C of coumarin and COOEt, NH substations on pyran), 3h (Cl substitution at C of coumarin
6
2
6
and COOEt, NH substations on pyran), 3i (CH substitution at C of coumarin and COOEt, NH
2
2
3
7
substations on pyran) and 3j (benzo substitution at C -C of coumarin and COOEt, NH
2
7
8
substations on pyran) have shown significant activity at lower concentration than standard drug,
but compounds 3a (CH substitution at C of coumarin and CN, NH substations on pyran), 3d
3
6
2
(
CH substitution at C of coumarin and CN, NH substations on pyran) and 3e (benzo
3 7 2
substitution at C -C of coumarin and CN, NH substations on pyran) have exhibited good
7
8
2
activity at 6.25µg/mL, which is same as standard drug concentration used (Table 6). Among 3a-
j, compound 3g is more effective against S. aureus, showing the least MIC value of 1.56 µg/mL.
The efficiency of the compounds (3a-j) were screened against E. faecalis, it was observed that,
among these only compound 3g exhibited excellent activity at lower concentration (3.125
µg/mL). Whereas, other compounds such as 3c, 3f and 3h were found to be equally active

ACCEPTED MANUSCRIPT
compared to standard. However, compounds 3a, 3b, 3d, 3e, 3i and 3j were found to be less
3 6 2
active. It has been observed that compound 3g with OCH at C of coumarin and COOEt, NH
on pyran nucleus is highly active against both Gram +ve bacterial strains. Similarly, activity
against E. coli, compound 3d is found to be highly active at 0.78µg/mL compared to standard.
Compound 3c and 3i are equally active, however other synthesized compounds were less active.
In case of P. aeruginosa, compound 3b (OCH substitution at C of coumarin and CN, NH
3
6
2
substations on pyran) and 3d (CH
3
substitution at C
7
of coumarin and CN, NH
2
substations on
pyran) are highly active. Whereas, other compounds are less active and results obtained are
summarized in Table 6.
The antibacterial activity of substituted pyrazolylpropanoic acids (4a-e) revealed that
compounds 4a (CH substitution at C of coumarin), 4b (OCH substitution at C of coumarin),
3
6
3
6
4
c (Cl substitution at C
6
of coumarin) and 4d (CH
3
substitution at C
7
of coumarin) are highly
active against S. aureus. Whereas, compound 4e (benzo substitution at C -C of coumarin) shows
7
8
good activity, the results obtained are summarized in Table 6. Activity against E. faecalis,
compound 4b was found to be highly active at lower concentration (1.56µg/mL). However,
compounds 4a, 4c and 4d showed good activity (6.25µg/mL) and compound 4e shows less
activity against E. faecalis. Whereas, all the compounds 4a-e were less active against E. coli and
P. aeruginosa bacterial strains.
The antibacterial activity of coumarin substituted 4,5-dihydropyrano[2,3-c]pyrazol-
6
(1H)-ones 9 were highly active against S. aureus bacterial strain. Among these compound 9b
(
OCH substitution at C of coumarin) shows activity at lower concentration (3.125 µg/mL) than
3
6
standard, but compounds 9a (CH
3 6 6
substitution at C of coumarin), 9c (Cl substitution at C of
coumarin) and 9d (CH substitution at C of coumarin) showed moderate activity compared to
3
7
standard drug. However compound 9a-e were less active against E. faecalis, E. coli and P.
aeruginosa antibacterial strains, the results obtained are discussed in Table 6.
Insert Table 6
5
.2. Anti-inflammatory Studies
5
.2.1. Egg albumin denaturation method

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All the synthesized compounds were subjected to anti-inflammatory effect against
denaturation of hen’s egg albumin method [33] at the concentration (31.25-1000 µg/mL) with
standard aceclofenac drug (31.25-1000 µg/mL).
The outcome of anti-inflammatory screening of compounds (3a-j, 4a-e and 9a-e) by
using egg albumin denaturation method are summarized in Table 7. The percentage inhibition of
all the synthesized compounds showed very high active against the denaturation of protein.
Among these compounds 3g, 4b and 4c exhibited an excellent inhibition of heat induced protein
denaturation 51.43%, 63.11% and 42.64% respectively and these compounds are almost ten
times more active compared to standard aceclofenac drug (5.50%). Whereas, compounds 9c and
9
e shows less activity and the remaining compounds showed good inhibitory anti-inflammatory
activity against the denaturation of protein method.
Insert Table 7
5
.2.2. Human Red Blood Cell (HRBC) membrane stabilization method
The anti-inflammatory activity of newly synthesized compounds was also screened by
using Human Red Blood Cell (HRBC) membrane stabilization technique [34]. This technique is
conventional, sensitive and well accepted for screening of newer anti-inflammatory agents. The
activity of compounds was screened at the concentration of 100 µg/mL oral dose and the same
dose of standard drug acetyl salicylic acid is used. The results are presented in Table 8.
The bio-screening results disclosed that all the compounds exhibited inhibitions of HRBC
membrane stabilization at the concentration of 100 µg/mL and most of the compounds showed
more than half maximal (50%) inhibition except compounds 3a, 3d, 3e, 9c and 9e. Compounds
3
g, 3h, 3j, 4a, 4b, 4c, 4d, 4e and 9b exhibited excellent activity with inhibitions 54.06%,
3
9.86%, 38.56%, 37.26%, 55.26%, 50.66%, 42.56%, 49.56% and 36.33% respectively in
comparison with the standard acetyl salicylic acid inhibition 36.16%. Whereas, compounds 3b,
c, 3f, 3i, 9a and 9d showed potent inhibition erythrocyte hemolysis membrane stabilization
close to the standard drug.
The synthesized compounds have shown significant anti-inflammatory activity in protein
3
denaturation as well as HRBC membrane stabilization methods. Among all the synthesized
compounds, 3g and 4a-e exhibit very good inhibitions in both the methods.
Insert Table 8

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6
. Computational studies
The newly synthesized compounds have exhibited excellent antibacterial activity, in
particular against Gram +ve bacteria S. aureus. The S. aureus can cause a range of illnesses from
minor skin infections to life-threatening diseases. Today, S. aureus has become resistant to many
commonly used antibiotics and only 2% of all S. aureus isolates are found to be sensitive to
penicillin. The β-lactamase resistant penicillins (methicillin, oxacillin, cloxacillin and
flucloxacillin) were developed to treat penicillin resistant S. aureus and are still used as first-line
treatment. S. aureus seems to employ specific resistance mechanisms, which include the
modification of the antibiotic structure, mutagenesis of key amino acids in the protein targets of
specific chemotherapeutics or drug efflux from the cell. In this line Ciprofloxacin is a synthetic
chemotherapeutic antibiotic of the fluoroquinolone drug class and is a second-generation
antibacterial agent, which kills bacteria by inhibiting the enzyme DNA-gyrase.
To understand the mechanism of antibacterial activity of newly synthesized compounds,
molecular modeling and docking studies were performed on X-ray crystal structure of the
dihydropteroate synthetase (DHPS) complexed with OH-CH -pterin-pyrophosphate from S.
2
aureus (PDB ID:1AD4) [35]. The docking study was obtained from the Protein Data Bank by
using Surflex-Dock programme of Sybyl-X software. All the twenty compounds were docked
into the active site of the DHPS (Fig. 7), the predicted binding energies and the observed C-score
values of all the compounds are ranging from 2.49-5.57, the score values are listed in Table 9.
Insert Figure 4
In order to know, the high activity of synthesized compounds against S. aureus,
molecular docking study was performed to support the interaction and preferred binding mode of
Ciprofloxacin with S. aureus DHPS (PDB ID:1AD4). The binding interaction of standard
Ciprofloxacin with dihydropteroate synthetase active sites shows five bonding interactions and
the docked view of the same has been depicted in Fig. 8.
Insert Figure 5
Insert Table 9
As shown in Fig. 9, compound 3g, forms eight hydrogen bonding interaction at the active
site of the enzyme (PDB ID:1AD4). Oxygen atoms of ester forms three hydrogen bonding
2 2
interaction with hydrogen of ASN11 & ARG239 (-CH COO---H-ASN11, -CH COO---H-

ACCEPTED MANUSCRIPT
nd
ASN11 & -CH COO---H-ARG239) and hydrogen atom of amine group present at 2 position of
2
2
pyran ring forms hydrogen bonding interaction with oxygen atom of ASN11 (-NH ---O-ASN11).
One hydrogen bonding interaction raised from methoxy group oxygen present at C of coumarin
6
ring ASN103 (-CH O---H-ASN103). The remaining three interaction are raised from the
3
pyrazole ring, two hydrogen bonding interaction raised from nitrogen atom of pyrazole ring (-N--
-H-ARG52 & -N---H-SER50) and another from amine of pyrazole ring with oxygen atom of
SER50 (-NH---O-SER50).
Insert Figure 6
Compounds 4b forms six hydrogen bonding interaction with amino acid residues (Fig
rd
1
0), among them two bonding interactions are due to hydroxyl group present at 3 position of
pyrazole ring with hydrogen of ARG239 & oxygen of ASN11 (-HO---H-ARG239, 2.21 Å; -OH-
-O-ASN11, 2.10 Å) respectively. Nitrogen of pyrazole ring forms hydrogen bonding interaction
-
with hydrogen of ASN11 (-N---H-ASN11, 2.14 Å) and hydrogen of -NH of pyrazole ring forms
a bonding interaction with nitrogen of HIS241 (-NH---N-HIS241, 2.46 Å). Carboxyl group
oxygen forms hydrogen bonding interaction with hydrogen of LYS203 (-COOH---H-LYS203,
th
2
.30 Å), and methoxy group oxygen present at 6 position of coumarin ring with hydrogen atom
of SER50 (-CH O---H-SER50, 2.54 Å).
3
Insert Figure 7
The comparative molecular docking study of synthesized compounds and standard
Ciprofloxacin drug highlighted that the synthesized compounds exhibited high C-score value.
Ciprofloxacin C-score value 3.58 whereas the compounds 3b, 3c, 3f, 3g, 3h, 3i, 3j, 4a, 4b, 4c,
4
d, 4e and 9b have higher C-score values than the Ciprofloxacin. The docking study reveals that,
the compound 3g and 4b are most potent among all the synthesized compounds.
Moreover docking study in Sybyl-X-2.0 software, has been applied for the synthesized
compounds to determine the Lipinski’s parameters (Table 10). All the synthesized compounds
did not indicate any violation against Lipinski rule of five. It was observed that the newly
synthesized compounds followed Lipinski’s rule of five, indicating more ‘drug-like’ nature.
Insert Table 10.
7
. Experimental protocol

ACCEPTED MANUSCRIPT
7
.1. Materials and methods
All the chemicals were purchased by commercial source and were used without further
purification unless otherwise stated. The reactions monitored by thin-layer chromatography
(
TLC) was performed with Merck Kieselgel 60 F254 plates and visualized using UV light at 254
nm and KMnO staining or iodine vapour, the purity of the compound was also checked by TLC.
Column chromatography was generally performed on silica gel (60-120 mesh size). The melting
4
1
13
points were determined by open capillary method and are uncorrected. H NMR and C NMR
spectra were measured on a Bruker 400 MHz and Jeol solution for innovation 500 MHz
spectrometer (400 MHz or 100 MHz, respectively) using DMSO-d as solvent and TMS as an
6
internal standard. Chemical shifts are reported in δ ppm relative to internal tetramethylsilane
standard (TMS, δ 0.00). The peak patterns are indicated as follows: s, singlet; d, doublet; t,
triplet; m, multiplet; q, quartet; dd, doublet of doublets; br, broad. The coupling constants, J, is
reported in Hertz (Hz). The X-ray diffraction study were performed on a BRUKER AXS
SMART APEX CCD diffractometer. The IR spectra were recorded on a Nicolet 5700 FT-IR
spectrometer in KBr disc method and mass spectra were recorded using Shimadzu
GCMSQP2010S. The CHN elemental analyses of all the compounds were recorded by LECO
TRUSPEC CHN analyzer.
7
.2. General Procedure and Spectral Characterization of Compounds 3a-j:
The mixture of hydrazine hydrate 96% (1 mmol) and ethyl acetoacetate (1 mmol) in aqueous
ethanol (20%) was stirred for 5-10 min. Aldehyde (1 mmol), malononitrile or ethylcyanoacetate
(1 mmol) and base (5 mmol %) were added to it successively at room temperature under open
atmosphere with vigorous stirring for 2 h. The progress of the reaction was monitored by TLC.
The precipitated solid was filtered and washed with water, to afforded pure product 3 in good
yield and no further purification is required
7
.2.1.
c]pyrazole-5-carbonitrile (3a):
White; Yield 94%; m.p: 236-238 C; IR (KBr) cm 3339, 3311, 2201, 1707; H NMR (400
MHz, DMSO-d ) δ (ppm): 12.25 (s, 1H, NH of Pyrazole), 7.55 (s, 1H, C H of Coumarin), 7.41
d, 1H, J= 8.8 Hz, C H of Coumarin), 7.32 (d, 1H, J= 8.8 Hz, C H of Coumarin), 7.15 (s, 2H,
6-Amino-1,4-dihydro-3-methyl-4-(6-methyl-2-oxo-2H-chromen-4-yl)pyrano[2,3-
o
-1
1
6
5
(
8
7
2 3 4 3
NH of Pyran), 6.34 (s, 1H, C H of Coumarin), 5.13 (s, 1H, C H of Pyran), 2.28 (s, 3H, CH of

ACCEPTED MANUSCRIPT
1
3
Pyrazole), 1.87 (s, 3H, CH of Coumarin); C NMR (100 MHz, DMSO-d ) δ (ppm): 161.62,
3
6
1
60.07, 154.71, 151.89, 136.19, 133.17, 132.98, 124.71, 120.01, 116.96, 114.96, 112.98, 108.16,
1
01.92, 94.69, 53.95, 20.67, 9.90; MS (m/z): 334. Elemental Analysis for C H N O (%),
1
8
14
4
3
Calcd: C, 64.66; H, 4.22; N, 16.76; found: C, 64.59; H, 4.18; N, 16.71.
7
.2.2.
6-Amino-1,4-dihydro-4-(6-methoxy-2-oxo-2H-chromen-4-yl)-3-methylpyrano[2,3-
c]pyrazole-5-carbonitrile (3b):
o
-1
1
Light green; Yield 91%; m.p: 204-206 C; IR (KBr) cm 3378, 3308, 2186, 1708; H NMR (400
MHz, DMSO-d ) δ (ppm): 12.28 (s, 1H, NH of Pyrazole), 7.39 (d, 1H, J= 8.8 Hz, C H of
6
8
Coumarin), 7.20 (d, 1H, J= 8.4 Hz, C
7
H of Coumarin), 7.13 (s, 1H, C
5
H of Coumarin), 7.06 (s,
2
H, NH of Pyran), 6.47 (s, 1H, C H of Coumarin), 5.09 (s, 1H, C H of Pyran), 3.68 (s, 3H,
2 3 4
1
3
3 3 6
OCH of Coumarin), 1.90 (s, 3H, CH of Pyrazole); C NMR (100 MHz, DMSO-d ) δ (ppm):
1
1
61.59, 160.05, 159.99, 154.88, 154.66, 154.61, 148.17, 136.50, 136.43, 119.93, 119.17, 119.11,
18.28, 107.56, 107.50, 55.33, 53.79, 9.84; MS (m/z): 350. Elemental Analysis for C H N O
4
1
8
14
4
(%), Calcd: C, 61.71; H, 4.03; N, 15.99; found: C, 61.67; H, 3.98; N, 15.92.
7
.2.3.
6-Amino-4-(6-chloro-2-oxo-2H-chromen-4-yl)-1,4-dihydro-3-methylpyrano[2,3-
c]pyrazole-5-carbonitrile (3c):
White; Yield 89%; m.p: 240-242 C; IR (KBr) cm 3433, 3315, 2197, 1719; H NMR (400
MHz, DMSO-d ) δ (ppm): 12.30 (s, 1H, NH of Pyrazole), 7.45 (d, 1H, J= 2.4 Hz, C H of
o
-1
1
6
5
Coumarin), 7.34 (d, 1H, J= 8.8 Hz, C H of Coumarin), 7.26 (d, 1H, J= 8.8 Hz, C H of
8
7
Coumarin), 7.21 (s, 2H, NH
2
3 4
of Pyran), 6.37 (s, 1H, C H of Coumarin), 5.17 (s, 1H, C H of
1
3
Pyran), 2.30 (s, 3H, CH of Pyrazole); C NMR (100 MHz, DMSO-d ) δ (ppm): 160.11, 159.32,
3
6
1
9
55.38, 151.92, 134.26, 126.72, 119.89, 119.13, 118.70, 116.17, 111.71, 108.16, 107.73, 106.92,
4.90, 53.87, 9.96; MS (m/z): 354. Elemental Analysis for C17 (%), Calcd: C, 57.56;
4 3
H11ClN O
H, 3.13; N, 15.79; found: C, 57.51; H, 3.09; N, 15.74.
7
.2.4.
6-Amino-1,4-dihydro-3-methyl-4-(7-methyl-2-oxo-2H-chromen-4-yl)pyrano[2,3-
c]pyrazole-5-carbonitrile (3d):
o
-1
1
White; Yield 92%; m.p: 230-232 C; IR (KBr) cm 3343, 3305, 2190, 1719; H NMR (400
MHz, DMSO-d ) δ (ppm): 12.04 (s, 1H, NH of Pyrazole), 7.31 (d, 1H, J= 8.8 Hz, C H of
6
5
8 7
Coumarin), 7.23 (d, 1H, J= 2.4 Hz, C H of Coumarin), 7.17 (d, 1H, J= 8.8 Hz, C H of

ACCEPTED MANUSCRIPT
Coumarin), 6.90 (s, 2H, NH of Pyran), 6.41 (s, 1H, C H of Coumarin), 5.12 (s, 1H, C H of
2
3
4
1
3
Pyran), 2.32 (s, 3H, CH
3 3
of Pyrazole), 1.93 (s, 3H, CH of Coumarin); C NMR (100 MHz,
DMSO-d ) δ (ppm): 161.73, 160.11, 155.22, 151.87, 136.20, 133.15, 132.74, 125.01, 120.85,
6
1
17.26, 115.09, 113.13, 108.19, 102.31, 94.57, 54.65, 21.07, 9.94; MS (m/z): 334. Elemental
Analysis for C18 (%), Calcd: C, 64.66; H, 4.22; N, 16.76; found: C, 64.60; H, 4.18; N,
14 4 3
H N O
1
6.71.
7
.2.5.
6-Amino-1,4-dihydro-3-methyl-4-(2-oxo-2H-benzo[h]chromen-4-yl)pyrano[2,3-
c]pyrazole-5-carbonitrile (3e):
Light brown; Yield 90%; m.p: 270-272 C; IR (KBr) cm 3432, 3314, 2196, 1712; H NMR
o
-1
1
(
400 MHz, DMSO-d ) δ (ppm): 12.30 (s, 1H, NH of Pyrazole), 8.40 (d, 1H, J= 8.8 Hz, C H of
6
5
Coumarin), 8.07 (d, 1H, J= 8.6 Hz, C10H of Coumarin), 7.70-7.81 (m, 4H, C
6
H, C
7
H, C
8
H and
C H of Coumarin), 7.26 (s, 2H, NH of Pyran), 6.55 (s, 1H, C H of Coumarin), 5.21 (s, 1H, C H
9
2
3
4
1
3
of Pyran), 1.89 (s, 3H, CH of Pyrazole); C NMR (100 MHz, DMSO-d ) δ (ppm): 161.50,
3
6
1
1
59.75, 154.58, 150.89, 136.38, 134.08, 129.06, 127.86, 127.55, 123.73, 122.58, 121.75, 120.32,
19.93, 114.73, 112.98, 108.16, 101.92, 94.67, 53.83, 9.86; MS (m/z): 370. Elemental Analysis
14 4 3
for C21H N O (%), Calcd: C, 68.10; H, 3.81; N, 15.13; found: C, 68.06; H, 3.76; N, 15.10.
7
.2.6.
c]pyrazole-5-carboxylate (3f):
White; Yield 92%; m.p: 206-208 C; IR (KBr) cm 3337, 3313, 1727, 1711; H NMR (400
Ethyl-6-amino-1,4-dihydro-3-methyl-4-(6-methyl-2-oxo-2H-chromen-4-yl)pyrano[2,3-
o
-1
1
MHz, DMSO-d
6
) δ (ppm): 11.40 (s, 1H, NH of Pyrazole), 7.72 (s, 1H, C
5
H of Coumarin), 7.64
(
s, 2H, NH of Pyran), 7.43 (d, 1H, J= 8.4 Hz, C H of Coumarin), 7.31 (d, 1H, J= 8.8 Hz, C H of
2
8
7
Coumarin), 6.64 (s, 1H, C H of Coumarin), 5.16 (s, 1H, C H of Pyran), 4.09 (m, 2H, CH of
3
4
2
3 3 3
Ethyl group), 2.33 (s, 3H, CH of Pyrazole), 2.09 (s, 3H, CH of Coumarin), 1.08 (m, 3H, CH of
1
3
Ethyl group); C NMR (100 MHz, DMSO-d ) δ (ppm): 164.90, 159.75, 153.11, 151.43, 138.15,
6
1
2
5
33.73, 133.00, 124.55, 117.57, 116.76, 116.13, 113.15, 112.44, 96.11, 94.95, 62.60, 34.63,
0.50, 13.44, 10.53; MS (m/z): 381. Elemental Analysis for C H N O (%), Calcd: C, 62.99; H,
2
0
19
3
5
.02; N, 11.02; found: C, 62.95; H, 4.97; N, 10.98.
7
.2.7. Ethyl-6-amino-1,4-dihydro-4-(6-methoxy-2-oxo-2H-chromen-4-yl)-3-methylpyrano[2,3-c]
pyrazole-5-carboxylate (3g):

ACCEPTED MANUSCRIPT
o
-1
1
White; Yield 89%; m.p: 210-212 C; IR (KBr) cm 3387, 3278, 1720, 1711; H NMR (400
MHz, DMSO-d ) δ (ppm): 11.75 (s, 1H, NH of Pyrazole), 7.40 (s, 1H, C H of Coumarin), 7.24
d, 1H, J= 8.4 Hz, C H of Coumarin), 7.18 (d, 1H, J= 8.8 Hz, C H of Coumarin), 7.15 (s, 2H,
6
5
(
8
7
NH of Pyran), 6.44 (s, 1H, C H of Coumarin), 5.10 (s, 1H, C H of Pyran), 4.06 (m, 2H, CH of
2
3
4
2
Ethyl group), 3.67 (s, 3H, OCH
3
of Coumarin), 2.37 (s, 3H, CH
3
of Pyrazole), 1.07 (m, 3H, CH
3
1
3
of Ethyl group); C NMR (100 MHz, DMSO-d ) δ (ppm): 164.67, 160.03, 153.14, 151.49,
6
1
5
6
38.17, 133.77, 132.98, 124.57, 120.18, 117.60, 116.81, 114.89, 112.58, 97.16, 95.93, 64.67,
5.67, 53.33, 13.68, 10.60; MS (m/z): 397. Elemental Analysis for C H N O (%), Calcd: C,
2
0
19
3
6
0.45; H, 4.82; N, 10.57; found: C, 60.39; H, 4.77; N, 10.50.
7
.2.8.
Ethyl-6-amino-4-(6-chloro-2-oxo-2H-chromen-4-yl)-1,4-dihydro-3-methylpyrano[2,3-
c]pyrazole-5-carboxylate (3h):
o
-1
1
White; Yield 86%; m.p: 238-240 C; IR (KBr) cm 3407, 3368, 1723, 1712; H NMR (400
MHz, DMSO-d ) δ (ppm): 11.85 (s, 1H, NH of Pyrazole), 7.45 (s, 1H, C H of Coumarin), 7.34
6
5
(d, 1H, J= 8.8 Hz, C
8
H of Coumarin), 7.27 (d, 1H, J= 8.4 Hz, C
7
H of Coumarin), 7.17 (s, 2H,
NH of Pyran), 6.32 (s, 1H, C H of Coumarin), 5.11 (s, 1H, C H of Pyran), 4.10 (m, 2H, CH of
2
3
4
2
1
3
Ethyl group), 2.30 (s, 3H, CH
3 3
of Pyrazole), 1.17 (m, 3H, CH of Ethyl group); C NMR (100
MHz, DMSO-d ) δ (ppm): 163.61, 159.87, 154.67, 150.84, 136.19, 133.23, 132.86, 123.17,
6
1
20.09, 116.71, 114.92, 112.96, 108.20, 102.02, 94.71, 54.63, 53.95, 20.67, 9.90; MS (m/z): 401.
Elemental Analysis for C19
H, 3.96; N, 10.38.
3 5
H16ClN O (%), Calcd: C, 56.80; H, 4.01; N, 10.46; found: C, 56.76;
7
.2.9.
Ethyl-6-amino-1,4-dihydro-3-methyl-4-(7-methyl-2-oxo-2H-chromen-4-yl)pyrano[2,3-
c]pyrazole-5-carboxylate (3i):
o
-1
1
White; Yield 91%; m.p: 212-214 C; IR (KBr) cm 3350, 3321, 1722, 1709; H NMR (400
MHz, DMSO-d ) δ (ppm): 12.05 (s, 1H, NH of Pyrazole), 7.42 (d, 1H, J= 8.4 Hz, C H of
6
5
8 6
Coumarin), 7.35 (d, 1H, J= 2.4 Hz, C H of Coumarin), 7.31 (d, 1H, J= 8.4 Hz, C H of
Coumarin), 7.12 (s, 2H, NH of Pyran), 6.30 (s, 1H, C H of Coumarin), 5.13 (s, 1H, C H of
2
3
4
Pyran), 3.84 (m, 2H, CH of Ethyl group), 2.23 (s, 3H, CH of Pyrazole), 1.45 (s, 3H, CH of
2
3
3
1
3
Coumarin), 1.02 (m, 3H, CH
3
of Ethyl group); C NMR (100 MHz, DMSO-d
6
) δ (ppm): 162.52,
1
60.17, 155.73, 151.91, 136.23, 133.13, 132.78, 124.87, 120.71, 116.98, 113.16, 112.98, 107.15,

ACCEPTED MANUSCRIPT
1
01.72, 94.69, 54.95, 34.86, 20.67, 13.64, 10.13; MS (m/z): 381. Elemental Analysis for
20 19 3 5
C H N O (%), Calcd: C, 62.99; H, 5.02; N, 11.02; found: C, 62.93; H, 4.96; N, 10.94.
7
.2.10.
c]pyrazole-5-carboxylate (3j):
White; Yield 88%; m.p: 207-209 C; IR (KBr) cm 3367, 3288, 1720, 1712; H NMR (400
MHz, DMSO-d ) δ (ppm): 12.28 (s, 1H, NH of Pyrazole), 8.37 (d, 1H, J= 8.4 Hz, C H of
Coumarin), 8.34 (d, 1H, J= 8.8 Hz, C H of Coumarin), 8.23 (m, 2H, C H and C H of
Ethyl-6-amino-1,4-dihydro-3-methyl-4-(2-oxo-2H-benzo[h]chromen-4-yl)pyrano[2,3-
o
-1
1
6
5
1
0
8
9
Coumarin), 7.85 (d, 1H, J= 8.4 Hz, C H of Coumarin), 7.68 (d, 1H, J= 8.8 Hz, C H of
7
6
Coumarin), 7.28 (s, 2H, NH
2
3 4
of Pyran), 6.34 (s, 1H, C H of Coumarin), 5.18 (s, 1H, C H of
Pyran), 4.08 (m, 2H, CH of Ethyl group), 2.28 (s, 3H, CH of Pyrazole), 1.87 (s, 3H, CH of
2
3
3
1
3
Coumarin), 1.58 (m, 3H, CH
3
of Ethyl group); C NMR (100 MHz, DMSO-d
6
) δ (ppm): 161.92,
1
1
60.21, 157.41, 149.67, 134.21, 133.29, 133.00, 128.65, 127.81, 124.04, 122.82, 121.53, 120.29,
17.07, 114.86, 113.08, 108.16, 101.90, 94.67, 54.72, 34.78, 20.71, 10.11; MS (m/z): 417.
(%), Calcd: C, 66.18; H, 4.59; N, 10.07; found: C, 66.14; H,
19 3 5
Elemental Analysis for C23H N O
4
.53; N, 10.02.
7
.3. General Procedure and Spectral Characterization of compounds 4a-e:
A mixture of compound 3 (1 mmol) and formic acid (10 mL) was prepared and refluxed on oil
bath for about 4-5 h. The reaction completion was confirmed by TLC, after that reaction mixture
was allowed to cool and poured into ice cold water with stirring. Solid separated was filtered,
repeatedly washed with water and dried to obtain the pure white product 4 in excellent yield and
no purification required further.
7
.3.1. 3-(5-hydroxy-3-methyl-1H-pyrazol-4-yl)-3-(6-methyl-2-oxo-2H-chromen-4-yl)propanoic
acid (4a):
o
-1
1
White; Yield 83%; m.p: 268-270 C; IR (KBr) cm 3327, 1727, 1712; H NMR (400 MHz,
DMSO-d ) δ (ppm): 11.26 (s, 2H, NH of Pyrazole and OH of Propanoic acid), 7.69 (d, 1H, ,
6
J=1.2 Hz C H of Coumarin), 7.38 (d, 1H, J= 8.4 Hz, C H of Coumarin), 7.27 (d, 1H, J= 8.4 Hz,
5
8
C
7
H of Coumarin), 6.41 (s, 1H, C
3
3
H of Coumarin), 4.57 (t, 1H, J= 7.6 Hz, C H of Propanoic
acid), 3.30 (s, 1H, OH of Pyrazole), 3.08 (dd, 1H, J= 16.4 & 8.0 Hz, C H of Propanoic acid),
2
2
2 3
.89 (dd, 1H, J= 16.0 & 7.2 Hz, C H of Propanoic acid), 2.34 (s, 3H, CH of Pyrazole), 2.07 (s,

ACCEPTED MANUSCRIPT
1
3
3
H, CH of Coumarin); C NMR (100 MHz, DMSO-d ) δ (ppm): 163.65, 161.88, 154.73,
3 6
1
51.86, 136.22, 133.17, 132.59, 124.39, 120.01, 116.37, 111.96, 108.16, 101.92, 36.46, 30.36,
2
0.22, 10.26; MS (m/z): 328. Elemental Analysis for C H N O (%), Calcd: C, 62.19; H, 4.91;
1
7
16
2
5
N, 8.53; found: C, 62.14; H, 4.85; N, 8.48.
7
.3.2. 3-(5-hydroxy-3-methyl-1H-pyrazol-4-yl)-3-(6-methoxy-2-oxo-2H-chromen-4-yl)propanoic
acid (4b):
o
-1
1
Light green; Yield 80%; m.p: 256-258 C; IR (KBr) cm 3328, 1725, 1705; H NMR (400 MHz,
DMSO-d ) δ (ppm): 12.21 (s, 1H, NH of Pyrazole), 11.44 (s, 1H, OH of Propanoic acid), 7.42 (d,
6
1
H, J= 8.4 Hz, C
8 7 5
H of Coumarin), 7.26 (d, 1H, J= 8.8 Hz, C H of Coumarin), 7.17 (s, 1H, C H
of Coumarin), 6.48 (s, 1H, C H of Coumarin), 4.59 (t, 1H, J= 7.4 Hz, C H of Propanoic acid),
3
3
3
.72 (s, 3H, OCH
3
of Coumarin), 3.31 (s, 1H, OH of Pyrazole), 3.04 (dd, 1H, J= 16.0 & 8.4 Hz,
C H of Propanoic acid), 2.90 (dd, 1H, J= 16.4 & 7.6 Hz, C H of Propanoic acid), 2.04 (s, 3H,
2
2
1
3
CH of Pyrazole); C NMR (100 MHz, DMSO-d ) δ (ppm): 162.89, 161.27, 155.09, 151.88,
3
6
1
1
8
36.26, 133.16, 132.56, 125.10, 120.03, 117.39, 111.97, 109.04, 101.95, 53.79, 36.51, 30.39,
0.14; MS (m/z): 344. Elemental Analysis for C H N O (%), Calcd: C, 59.30; H, 4.68; N,
17
16
2
6
.14; found: C, 59.24; H, 4.62; N, 8.08.
7
.3.3. 3-(6-chloro-2-oxo-2H-chromen-4-yl)-3-(5-hydroxy-3-methyl-1H-pyrazol-4-yl)propanoic
acid (4c):
White; Yield 77%; m.p: 266-268 C; IR (KBr) cm 3338, 1727, 1711; H NMR (400 MHz,
DMSO-d ) δ (ppm): 11.81 (s, 1H, NH of Pyrazole), 11. 37 (s, 1H, OH of Propanoic acid), 7.44
d, 1H, J= 2.4 Hz, C H of Coumarin), 7.35 (d, 1H, J= 8.4 Hz, C H of Coumarin), 7.28 (d, 1H, J=
o
-1
1
6
(
5
8
8
.8 Hz, C H of Coumarin), 6.36 (s, 1H, C H of Coumarin), 4.53 (t, 1H, J= 7.4 Hz, C H of
7
3
3
Propanoic acid), 3.36 (s, 1H, OH of Pyrazole), 3.07 (dd, 1H, J= 16.4 & 8.4 Hz, C
2
H of Propanoic
acid), 2.97 (dd, 1H, J= 16.4 & 7.8 Hz, C H of Propanoic acid), 2.19 (s, 3H, CH of Pyrazole);
2
3
1
3
C NMR (100 MHz, DMSO-d
32.58, 124.96, 120.07, 117.26, 112.05, 108.78, 101.92, 35.55, 30.25, 10.31; MS (m/z): 348.
Elemental Analysis for C H ClN O (%), Calcd: C, 55.10; H, 3.76; N, 8.03; found: C, 55.06;
6
) δ (ppm): 163.12, 161.34, 154.81, 151.92, 136.23, 133.19,
1
16
13
2
5
H, 3.73; N, 7. 98.

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7
.3.4. 3-(5-hydroxy-3-methyl-1H-pyrazol-4-yl)-3-(7-methyl-2-oxo-2H-chromen-4-yl)propanoic
acid (4d):
o
-1
1
White; Yield 82%; m.p: 260-262 C; IR (KBr) cm 3375, 1719, 1710; H NMR (400 MHz,
DMSO-d ) δ (ppm): 12.05 (s, 1H, NH of Pyrazole), 11.39 (s, 1H, OH of Propanoic acid), 7.46 (d,
6
1
5 8
H, J=8.4 Hz C H of Coumarin), 7.32 (d, 1H, J= 2.4 Hz, C H of Coumarin), 7.23 (d, 1H, J= 8.4
Hz, C H of Coumarin), 6.31 (s, 1H, C H of Coumarin), 4.56 (t, 1H, J= 7.6 Hz, C H of Propanoic
6
3
3
acid), 3.37 (s, 1H, OH of Pyrazole), 3.11 (dd, 1H, J= 16.0 & 7.6 Hz, C
2
H of Propanoic acid),
2
.93 (dd, 1H, J= 16.0 & 8.4 Hz, C H of Propanoic acid), 2.24 (s, 3H, CH of Pyrazole), 2.06 (s,
2 3
1
3
3
H, CH of Coumarin); C NMR (100 MHz, DMSO-d ) δ (ppm): 164.22, 160.17, 155.11,
3 6
1
52.09, 136.21, 133.20, 132.73, 124.91, 120.81, 117.30, 112.06, 108.86, 102.12, 36.49, 30.95,
2
0.65, 10.36; MS (m/z): 328. Elemental Analysis for C H N O (%), Calcd: C, 62.19; H, 4.91;
1
7
16
2
5
N, 8.53; found: C, 62.13; H, 4.86; N, 8.46.
7
.3.5. 3-(5-hydroxy-3-methyl-1H-pyrazol-4-yl)-3-(2-oxo-2H-benzo[h]chromen-4-yl)propanoic
acid (4e):
Light brown; Yield 79%; m.p: 233-235 C; IR (KBr) cm 3364, 1727, 1712; H NMR (400
MHz, DMSO-d ) δ (ppm): 11.79 (s, 1H, NH of Pyrazole), 11.27 (s, 1H, OH of Propanoic acid),
.21 (d, 1H, J= 8.8 Hz, C H of Coumarin), 8.15 (d, 1H, J= 8.8 Hz, C H of Coumarin), 8.07 (m,
o
-1
1
6
8
2
5
10
H, C H and C H of Coumarin), 7.89 (d, 1H, J= 8.6 Hz, C H of Coumarin), 7.83 (d, 1H, J= 8.8
8
9
6
Hz, C
7
H of Coumarin), 6.43 (s, 1H, C
3
3
H of Coumarin), 4.50 (t, 1H, , J= 7.6 Hz, C H of
Propanoic acid), 3.28 (s, 1H, OH of Pyrazole), 3.10 (dd, 1H, J= 16.4 & 8.4 Hz, C H of Propanoic
2
2 3
acid), 2.98 (dd, 1H, J= 16.0 & 7.6 Hz, C H of Propanoic acid), 2.31 (s, 3H, CH of Pyrazole);
1
3
C NMR (100 MHz, DMSO-d ) δ (ppm): 163.90, 161.17, 158.23, 153.19, 149.67, 134.24,
6
1
3
33.19, 132.87, 128.71, 127.89, 123.97, 121.96, 120.21, 116.92, 113.72, 108.20, 100.97, 35.79,
16 2 5
0.47, 10.43; MS (m/z): 364. Elemental Analysis for C20H N O (%), Calcd: C, 65.93; H, 4.43;
N, 7.69; found: C, 65.88; H, 4.39; N, 7.61.
7
.4. General Procedure and Spectral Characterization of compounds 9a-e:
Compound 4 (1 mmol) in acetonitrile was charged into round bottomed flask to this SOCl (5
2
mmol) was added in cold condition and the reaction mixture was refluxed on oil bath for about 3

ACCEPTED MANUSCRIPT
h. The progress of the reaction was monitored by TLC, then poured into ice cold water and
3
neutralized by NaHCO . The separated solid was filtered, washed with water and dried. The
crude product obtained was purified by column chromatography on silica 60–120, eluent:
EtOAc-hexane, 4:6.
7
.4.1. 4,5-dihydro-3-methyl-4-(6-methyl-2-oxo-2H-chromen-4-yl)pyrano[2,3-c]pyrazol-6(1H)-
one (9a):
o
-1
1
White; Yield 85%; m.p: 215-218 C; IR (KBr) cm 3327, 1748, 1722; H NMR (400 MHz,
DMSO-d ) δ (ppm): 11.23 (s, 1H, NH of Pyrazole), 7.67 (s, 1H, C H of Coumarin), 7.40 (d, 1H,
6
5
J= 8.4 Hz, C
8
H of Coumarin), 7.28 (d, 1H, J= 8.4 Hz, C
7
3
H of Coumarin), 6. 43 (s, 1H, C H of
Coumarin), 4.60 (t, 1H, J= 7.6 Hz, C H of Pyran), 3.16 (dd, 1H, J= 14.8 & 6.8 Hz, C H of
4
3
Pyran), 3.01 (dd, 1H, J= 14.2 & 6.7 Hz, C
3
H of Pyran), 2.34 (s, 3H, CH
3
of Pyrazole), 2.06 (s,
1
3
3
1
2
H, CH of Coumarin); C NMR (100 MHz, DMSO-d ) δ (ppm): 162.72, 160.91, 158.71,
3 6
50.24, 138.17, 134.57, 128.91, 125.73, 121.01, 120.46, 118.96, 115.98, 112.16, 34.36, 30.05,
0.37, 10.31; MS (m/z): 310. Elemental Analysis for C17 (%), Calcd: C, 65.80; H, 4.55;
14 2 4
H N O
N, 9.03; found: C, 65.75; H, 4.49; N, 8.93.
7
.4.2. 4,5-dihydro-4-(6-methoxy-2-oxo-2H-chromen-4-yl)-3-methylpyrano[2,3-c]pyrazol-6(1H)-
one (9b):
o
-1
1
Light green; Yield 82%; m.p: 221-224 C; IR (KBr) cm 3379, 1720, 1707; H NMR (400 MHz,
DMSO-d ) δ (ppm): 11.27 (s, 1H, NH of Pyrazole), 7.59 (d, 1H, J= 8.4 Hz, C H of Coumarin),
6
8
7
.43 (d, 1H, J= 8.8 Hz, C
7
H of Coumarin), 7.31 (s, 1H, C
5
3
H of Coumarin), 6.48 (s, 1H, C H of
Coumarin), 4.68 (t, 1H, J= 7.6 Hz, C H of Pyran), 3.67 (s, 3H, OCH of Coumarin), 3.13 (dd,
4
3
1
3
1
1
8
H, J= 16.8 & 7.8 Hz, C H of Pyran), 2.99 (dd, 1H, J= 16.4 & 7.6 Hz, C H of Pyran), 2.38 (s,
3
3
1
3
H, CH
3
of Pyrazole); C NMR (100 MHz, DMSO-d
6
) δ (ppm): 163.11, 161.03, 159.69, 154.81,
45.62, 140.23, 138.17, 123.54, 122.46, 116.83, 114.15, 111.71, 110.08, 53.79, 34.45, 29.89,
14 2 5
0.24; MS (m/z): 326. Elemental Analysis for C17H N O (%), Calcd: C, 62.57; H, 4.32; N,
.59; found: C, 62.54; H, 4.29; N, 8.52.
7
.4.3. 4-(6-chloro-2-oxo-2H-chromen-4-yl)-4,5-dihydro-3-methylpyrano[2,3-c]pyrazol-6(1H)-
one (9c):

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o
-1
1
White; Yield 81%; m.p: 228-230 C; IR (KBr) cm 3403, 1722, 1711; H NMR (400 MHz,
DMSO-d ) δ (ppm): 11.33 (s, 1H, NH of Pyrazole), 7.55 (s, 1H, J= 2.4 Hz, C H of Coumarin),
.38 (d, 1H, J= 8.4 Hz, C H of Coumarin), 7.30 (d, 1H, J= 8.8 Hz, C H of Coumarin), 6.40 (s,
6
5
7
8
7
1
H, C H of Coumarin), 4.65 (t, 1H, J= 7.6 Hz, C H of Pyran), 3.11 (dd, 1H, J= 16.0 & 7.6 Hz,
3 4
C
3 3 3
H of Pyran), 3.00 (dd, 1H, J= 16.8 & 8.0 Hz, C H of Pyran), 2.38 (s, 3H, CH of Pyrazole);
1
3
C NMR (100 MHz, DMSO-d ) δ (ppm): 163.63, 161.47, 159.71, 147.80, 139.19, 135.06,
6
1
32.08, 128.71, 125.01, 122.96, 122.17, 112.88, 109.14, 35.05, 28.64, 10.37; MS (m/z): 330.
Elemental Analysis for C H ClN O (%), Calcd: C, 58.11; H, 3.35; N, 8.47; found: C, 58.08;
16
11
2
4
H, 3.31; N, 8.40.
7
.4.4. 4,5-dihydro-3-methyl-4-(7-methyl-2-oxo-2H-chromen-4-yl)pyrano[2,3-c]pyrazol-6(1H)-
one (9d):
o
-1
1
White; Yield 84%; m.p: 198-200 C; IR (KBr) cm 3393, 1719, 1705; H NMR (400 MHz,
DMSO-d ) δ (ppm): 11.25 (s, 1H, NH of Pyrazole), 7.49 (d, 1H, J=8.4 Hz, C H of Coumarin),
6
5
7
.38 (d, 1H, J= 2.4 Hz, C
8
H of Coumarin), 7.25 (d, 1H, J= 8.4 Hz, C
7
H of Coumarin), 6. 34 (s,
1
H, C H of Coumarin), 4.62 (t, 1H, J= 7.6 Hz, C H of Pyran), 3.13 (dd, 1H, J= 16.4 & 8.0 Hz,
3 4
C
3 3 3
H of Pyran), 2.97 (dd, 1H, J= 16.0 & 7.6 Hz, C H of Pyran), 2.27 (s, 3H, CH of Pyrazole),
1
3
2
1
3
4
.03 (s, 3H, CH of Coumarin); C NMR (100 MHz, DMSO-d ) δ (ppm): 163.07, 161.31,
3 6
58.96, 148.14, 138.87, 134.83, 132.11, 128.73, 123.01, 122.46, 118.73, 115.89, 112.13, 34.26,
14 2 4
0.25, 20.42, 10.21; MS (m/z): 310. Elemental Analysis for C17H N O (%), Calcd: C, 65.80; H,
.55; N, 9.03; found: C, 65.77; H, 4.51; N, 8.96.
7
.4.5.
4,5-dihydro-3-methyl-4-(2-oxo-2H-benzo[h]chromen-4-yl)pyrano[2,3-c]pyrazol-6(1H)-
one (9e):
o
-1
1
Light brown; Yield 83%; m.p: 207-210 C; IR (KBr) cm 3412, 1723, 1712; H NMR (400
MHz, DMSO-d ) δ (ppm): 11.29 (s, 1H, NH of Pyrazole), 8.31 (d, 1H, J= 8.4 Hz, C H of
6
5
8 9
Coumarin), 8.23 (d, 1H, J= 8.8 Hz, C10H of Coumarin), 8.16 (m, 2H, C H and C H of
Coumarin), 8.02 (d, 1H, J= 8.6 Hz, C H of Coumarin), 7.89 (d, 1H, J= 8.4 Hz, C H of
6
7
Coumarin), 6.53 (s, 1H, C H of Coumarin), 4.66 (t, 1H, , J= 7.8 Hz, C H of Pyran), 3.09 (dd, 1H,
3
4
J= 16.0 & 7.8 Hz, C
3
H of Pyran), 2.95 (dd, 1H, J= 16.4 & 7.6 Hz, C
3
H of Pyran), 2.30 (s, 3H,
1
3
CH of Pyrazole); C NMR (100 MHz, DMSO-d ) δ (ppm): 164.09, 161.13, 159.21, 154.24,
3
6
1
39.67, 135.04, 133.96, 127.47, 126.81, 126.59, 126.04, 124.27, 121.97, 120.71, 120.12, 115.72,

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1
6
09.20, 34.12, 29.47, 10.33; MS (m/z): 346. Elemental Analysis for C H N O (%), Calcd: C,
2
0
14
2
4
9.36; H, 4.07; N, 8.09; found: C, 69.32; H, 4.03; N, 8.02.
7
.5. Biological evaluation
7
.5.1 Antibacterial screening
The susceptibility of the test organisms to synthetic compounds were assessed using broth
dilution assay, as minimum inhibitory concentration (MIC) Triplicates were performed for each
of the standard strains.
Culture media: Brain Heart Infusion (BHI) broth Test organisms: Four microorganisms were
selected for the study: S. aureus microbial type culture collection MTCC 12598, E. faecalis
MTCC 35550, E. coli MTCC 443 and P. aeruginosa MTCC 25668. All microorganisms were
previously sub cultured in appropriate media and under gaseous conditions to confirm their
purity at 35 °C for 48 h prior to testing of the vehicles.
Inoculum preparation: The growth method or the log phase method was performed as follows.
At least three to five well isolated colonies of the same morphological type were selected from
an agar culture plate. Top of each colony was scooped with a loop, and the growth was
transferred into a tube containing 4–5 mL of BHI broth. The broth culture was incubated at 35°C
for 2–6 h until it achieved the turbidity of the 0.5 McFarland standard. The turbidity of actively
growing broth culture was adjusted with broth to obtain a final turbidity optically comparable to
that of the 0.5 McFarland standard, done visually by comparing the inoculum tube and the
standard against a white card with contrasting black lines.
Broth dilution method: A total of 10 tubes were taken and nine dilutions of the vehicle were done
with BHI for MIC. In the initial tube, only 200 ꢀL of vehicle was added. For further dilutions,
2
00 ꢀL of BHI broth was added to the next nine tubes separately. In the second tube, 200 ꢀL of
vehicle was added which already contained 200 ꢀL of BHI broth. This was considered as 10
dilution. From the 10 diluted tube, 200 ꢀL was transferred to the second tube to make 10
dilution. The serial dilution was repeated up to 10 dilution for each vehicle. From the maintained
stock cultures of the required microorganisms, 5 ꢀL was taken and added to 2 mL of BHI broth.
In each serially diluted tube, 200 ꢀL of the above culture suspension was added. The last tube
contained only the media and the culture suspension, i.e. the negative control. The tubes were
kept for incubation for 24 h at 37 °C in bacteriological incubator and observed for turbidity.

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7
7
.5.2. Anti-inflammatory activity
.5.2.1. Egg albumin denaturation method
The mixture (5 mL) consisted of 0.2 mL of egg albumin (from fresh hen’s egg), 2.8 mL of
phosphate buffered saline (PBS, pH 6.4) and 2 mL of varying concentrations of coumarin based
pyrano[2,3-c]pyrazole compounds so that final concentrations become 31.25, 62.5, 125, 250,
5
00, 1000 µg/mL and similar volume of double-distilled water served as control. Then the
mixtures were incubated at (37±2) °C in an incubator (Bio-technics, India) for 15 min and then
heated at 70 °C for 5 min. After cooling, their absorbance was measured at 660 nm
(SHIMADZU, UV-1800 Spectrophotometer) by using vehicle as blank. Aceclofenac sodium at
the final concentration of (31.25, 62.5, 125, 250, 500, 1000 µg/mL) was used as reference drug
and treated similarly for determination of absorbance. The percentage inhibition of protein
denaturation was calculated by using the following formula
%
Inhibition = 100 x (Abs of control -Abs of sample) /Abs of control.
7
.5.2.2. Human Red Blood Cell (HRBC) membrane stabilization method
Collection of blood samples: Human RBCs were collected for the study. 7 ml of blood was
collected from healthy male human volunteers (aged 24-26 years) without a history of oral
contraceptive or anticoagulant therapy and free from diseases using protocol issued by
Institutional Ethics Committee. The collected RBCs were kept in a test tube with an
anticoagulant EDTA under standard conditions of temperature 23±2 °C and relative humidity
5
5±10%.
Assay of membrane stabilization
Erythrocyte suspension: The blood was washed three times using isotonic solution (0.9% saline).
The volume of saline was measured and reconstituted as a 40% (v/v) suspension with isotonic
.
buffer solution (pH 7.4) which contained in 1 L of distilled water: NaH PO 2H O, 0.26 g;
2
4
2
Na HPO , 1.15 g; NaCl, 9 g (10 mM sodium phosphate buffer). Thus the suspension finally
2
4
collected was the stock erythrocyte (RBC) suspension.
Heat-induced hemolysis: Aliquots (5 ml) of the isotonic buffer, containing 0.1 mg/mL of
synthesized compounds were put into two duplicate sets of centrifuge tubes [36].The vehicle, in

ACCEPTED MANUSCRIPT
the same amount, was added to another tube as control. Erythrocyte suspension (30 µL) was
added to each tube and mixed gently by inversion. One pair of the tubes was incubated at 54 °C
for 20 min in a water bath. The other pair was maintained at 0-5 °C in an ice bath. The reaction
mixture was centrifuged for 3 min at 1300 rpm and the absorbance of the supernatant was
measured at 540 nm using UV-VIS spectrometer (SHIMADZU, UV-1800). The percentage
inhibition or acceleration of hemolysis in tests and was calculated using the following equation.
%
Inhibition of Hemolysis=100 x 1- [OD -OD /OD -OD ]
2 1 3 1
Where, OD =Test sample unheated,
1
OD
2
=Test sample heated and
OD =Control sample heated.
3
7
.5.3. Computational Studies
For the docking of ligands to protein active sites and for estimating the binding affinities of
docked compounds, Surflex-Dock module, a fully automatic docking tool available on Sybyl X-
2
.0 version was used in this study.
Docking simulations: The X-ray Crystal Structure of S. aureus dihydropteroate synthetase (PDB
ID:1AD4) enzyme [35] was obtained from protein data bank in PDB format as starting point.
The synthesized compounds and the standard compounds tested in this study were docked to S.
aureus dihydropteroate synthetase (PDB ID:1AD4) enzyme using Surflex-Dock programme in
Sybyl software by incremental construction approach of building the structure in the active site
so as to favour the binding affinity [37,38]. Finally, the docked ligands were ranked based on a
variety of scoring functions that have been compiled into the single consensus score (C-score)
[39].
8
. Conclusions
To explore different scaffold structures, we have designed and synthesized a series of coumarin
based pyrano[2,3-c]pyrazole derivatives with yields ranging from 77-94%. Most of them
exhibited excellent activity toward antibacterial against gram +ve bacterial strains and also anti-
inflammatory activity. Among all these synthesized scaffolds, compounds 3g and 4b are highly
active and more potent in both biological as well as molecular docking simulation studies. These

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results also suggested a new and potential route in the discovery of drug against antibacterial and
anti-inflammatory activities.
Acknowledgement
The authors acknowledge the University Scientific Instrumentation Centre (USIC), Karnatak
University, Dharwad for IR, Mass and SC-XRD data. We are also grateful the Department of
Science and Technology (DST) and also UGC-UPE and UGC-DSA New Delhi, for the financial
support. Authors also thank to NMR Research Centre, Indian Institute of Science (IISc),
Bengaluru, India for carrying out the spectral analyses.
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O
O
NH NH .H O
H C
OC H5
2
2
2
H
3
2
O
O
NH2
N
N
Base, Aq. C H OH (20%)
2
5
R'
CHO
O
CN
2
-3 h Stirring
R'
R
R
O
O
2
3
R= 6-Me, 6-OMe, 6-Cl, 7-Me, 7,8-Benzo; R'= CN, COOEt
Scheme 1: Construction of various coumarin substituted pyrano[2,3-c]pyrazoles 3a-j

ACCEPTED MANUSCRIPT
H
O
N
N
N
N
N
NC
R'
H
H
C
H
C
H
R
C
C
R'
R'
O
O
1
N
H
N
C
N
C
N
C
OH
H
O
H
R'
O
R'
R'
H
H
R
R
R
O
O
O
O
O
O
O
H
H C
H C
3
3
OEt
O
OH
N
N
N
N
H
H
NH -NH2
2
N
N
H
N
OH
C
C
N
H
R'
O
R'
O
H
H C
H C
3
3
OH
R
R
N
N
H
O
O
H
N
H
O
NH2
O
NH2
OH
NH
R'
N
N
C
HN
N
N
R'
O
R'
H C
H C
H C
3
3
3
R
R
R
O
O
O
O
O
3
(2H)
3 (1H)
Scheme 2: Mechanism for the synthesis of 4-coumarinyl-6-amino-1,4-dihydro-3-
methylpyrano[2,3-c]pyrazole-5-carbonitriles.

ACCEPTED MANUSCRIPT
H
H
OH
O
N
N
O
O
N
NH
2
N
N
OH
N
O
O
NH2
O
CO2
NH3
R
R
4
5
x
H
O
O
N
R1
H
N
N
OH
N
Urea/Thiourea,
N
N
OH
R = H, CH3
CH COOH:HCl (3:1)
3
1
NH2
x
O
O
R
H
O
O
NH2
R
CO2
NH3
N
O
N
O
O
8
R'
4
R CONH
1
2
HCOOH
H
OH
CH
3
COOH:HCl (3:1)
H
N
N
N
N
R
N
O
OH
NH
3
NH3
CO2
O
O
O
O
x
R
R
AcOH/ Ac O
O
2
O
4
6
x
H
H
N
N
OH
O
O
O
N
N
N
NH
OH
O
O
O
O
R
R
CO2 NH3
7
4
R= 6-Me, 6-OMe, 6-Cl, 7-Me, 7,8-Benzo; R'= CN, COOEt
Scheme 3: Synthesis of substituted 3-coumarinyl-3-(5-hydroxy-3-methyl-1H-pyrazole-4-yl)
propanoic acid derivatives 4a-e.