Study of antimicrobial and antioxidant activities of pyrrole-chalcones

Article abstract of DOI:10.1016/j.molstruc.2020.129483

The resistance of pathogenic microorganisms to accessible antibiotics, anxiolytics, sedatives, hypnotics and anti-convulsants are rapidly forming a foremost problem worldwide. The detailed spectroscopic, DFT, antimicrobial, and antioxidant analysis of synthesized pyrrole chalcone derivatives (3a, 3c, 3h, 3i, and 5a) have been performed and interpreted. Occurring of doublet in ¹H NMR spectra of (3a, 3c, 3h, 3i, and 5a) compounds in the range of δ 7.656-6.960 confirms the presence of β-vinyl (=C-H) proton and another doublet at δ 7.206-6.707 confirms the presence of α-vinyl (=C-H) proton, respectively, confirming the formation of reported products. The global electrophilicity index (ω = 5.26 eV) shows that the (3g) molecule is a strong electrophile among all the studied compound. All compounds tends to the formation of pyrazoline, oxazoline heterocyclic compounds which may have considerable pharmacological activities and material applications. The solvent-induced effects on the non-linear optical properties (NLO) were studied by using self-consistent reaction field (SCRF) method. As the solvent polarity increases, the β value have been found to increases monotonically. The compound (3a, 3c, 3g, 3h, 3i, and 5a) displayed better non-linear optical (NLO) responses than the standard p-Nitro aniline (pNA) in solvent and as well as in gas phase. 3g and 3i have high NLO values even in gas phase which can be attributed to their unsymmetrical and high non-planar structure. All studied compounds (3a, 3c, 3g, 3h, 3i, and 5a) show good antifungal and antibacterial activity against A. Niger and gram-positive bacteria B. subtilis but 3g, 3h, and 3i showed more prominent activity which can be attributed to their either substituents. Compounds 3h, 3c, 3a, and 3g showed better free radical scavenging than standard BHT, whereas 3h, 3c, 3a showed better Fe²⁺ ion chelating activity than standard EDTA, and better Total reductive capability than standard BHA. Thus they are suitable for both material and biological applications.

Full text of DOI:10.1016/j.molstruc.2020.129483

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Study of antimicrobial and antioxidant activities of pyrrole-chalcones
Poonam Rawat , R.N. Singh , Alok Ranjan , Anshu Gautam ,
Sweta Trivedi , Mukesh Kumar
PII:
S0022-2860(20)31798-1
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.129483
MOLSTR 129483
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Journal of Molecular Structure
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Revised date:
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29 March 2020
20 September 2020
17 October 2020
Please cite this article as: Poonam Rawat , R.N. Singh , Alok Ranjan , Anshu Gautam ,
Sweta Trivedi , Mukesh Kumar , Study of antimicrobial and antioxidant activities of pyrrole-chalcones,
Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.129483
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Highlights




All pyrrole-chalcones showed π→π* electronic excitations.
Compound (3a, 3c, 3g, 3h, 3i and 5a) displayed better NLO responses than standard p-NA.
Studied compounds (3a, 3c, 3g, 3h, 3i and 5a) have good antifungal and antibacterial activity.
Studied compounds showed promising free radical scavenging and Fe+2 ion chelating activity.
1

Study of antimicrobial and antioxidant activities of
pyrrole-chalcones
Poonam Rawat, R. N. Singh*, Alok Ranjan, Anshu Gautam, Sweta Trivedi, Mukesh Kumar
Department of Chemistry, University of Lucknow, Lucknow–226007, U.P., India
Abstract
The resistance of pathogenic microorganisms to accessible antibiotics, anxiolytics,
sedatives, hypnotics and anti-convalsunts are rapidly forming a foremost problem worldwide.
The detailed spectroscopic, DFT, antimicrobial, and antioxidant analysis of synthesized pyrrole
chalcone derivatives (3a, 3c, 3h, 3i, and 5a) have been performed and interpreted. Occurring of
doublet in 1H NMR spectra of (3a, 3c, 3h, 3i, and 5a) compounds in the range of δ 7.656-6.960
confirms the presence of β-vinyl (=C-H) proton and another doublet at δ 7.206-6.707 confirms
the presence of α-vinyl (=C-H) proton, respectively, confirming the formation of reported
products. The global electrophilicity index (ω = 5.26 eV) shows that the (3g) molecule is a
strong electrophile among all the studied compound. All compounds tend the formation of
pyrazoline, oxazoline heterocyclic compounds which may have considerable pharmacological
activities and material applications. The solvent-induced effects on the non-linear optical
properties (NLO) were studied by using self-consistent reaction field (SCRF) method. As the
solvent polarity increases, the β value have been found to increases monotonically. The
compound (3a, 3c, 3g, 3h, 3i, and 5a) displayed better non-linear optical (NLO) responses than
the standard p-Nitro aniline (pNA) in solvent and as well as in gas phase. 3g and 3i have high
NLO values even in gas phase which can be attributed to their unsymmetrical and high non-
planar structure. All studied compounds (3a, 3c, 3g, 3h, 3i, and 5a) show good antifungal and
2

antibacterial activity against A. Niger and gram-positive bacteria B. subtilis but 3g, 3h, and 3i
showed more prominent activity which can be attributed to their either substituents. Compounds
3h, 3c, 3a, and 3g showed better free radical scavenging than standard BHT, whereas 3h, 3c, 3a
showed better Fe2+ ion chelating activity than standard EDTA, and better Total reductive
capability than standard BHA. Thus they are suitable for both material and biological
applications.
Keywords: NMR, TD–DFT, NBO analysis, reactivity descriptor, NLO
* Corresponding author. e-mail: rnsvk.chemistry@gmail.com; Tel.: +919451308205
3

1. Introduction
Heterocyclic chemistry encompasses one of the largest divisions of chemistry, with
important applications in biological [1], pharmaceutical [2], therapeutic [3], medicinal [4],
catalytic [5], advanced materials [1-3] chemistry and the production of (non)-natural
compounds [6]. The pyrrole moiety is one of the ubiquitous heterocyclic structures throughout
both the plant and animal kingdoms [7]. The growing abundance of pyrrolic components in
natural products, pharmaceuticals, and new materials lead the chemistry of pyrrole and its
derivatives towards the center of interest. From the point of view of its intense utilization, the
synthetic pyrrole chemistry has dominated. The chemistry of pyrroles is attracting steady
attention because these heterocycles play an important role in nature and, at the same time,
possess rich synthetic potential making them valuable synthons for the design of novel
organometallic magnetic compounds or materials for optoelectronics [8], light-harvesting
systems and photosensitizers for photodynamic cancer diagnostics and therapy [9], conducting
organic polymers, pesticides. Pyrrole is also widely known as a biologically active scaffold
which possesses a diverse nature of activities.
Scientific investigations on the bioavailability of chalcones from food sources are
limited, but varieties of synthetic chalcones have been reported to possess a wide range of
pharmaceutically important biological activities [10]. The chalcones have shown a wide range
of biological activities [11] depending on the substitution pattern on the two aromatic rings
around enone moiety. Several synthetic chalcones have been designed, synthesized, and have
found numerous applications as pesticides; photo-protectors in plastics; solar creams, and food
4

additives [12]. In plants, chalcones are important intermediates in the biosynthesis of flavonoids
and isoflavonoids [13]. Subsequently, they are precursors in the biosynthesis of a large number
of flavonoid groups, including flavones, flavonols, dihydroflavonols, aurones, and isoflavones
[14]. Chalcones constitute an important group of natural products that serve as precursors for
the synthesis of various heterocyclic compounds like furans, pyrroles, pyrimidines, imidazoles,
pyrazoles, 2-pyrazolines, and flavonoids [15-19]. Numerous studies dealing with chalcones have
been carried out in the past few years [20-24]. Among many organic NLO materials, chalcone
derivatives are noticeable materials for their excellent blue light transmittance [25] much better
than those observed in inorganic crystals and show preference to crystallize as
noncentrosymmetric structures. For this reason, they have been the objective of several
experimental and theoretical studies, aimed mainly at the determination of their crystal
structures [26]
On taking into account, the enormous areas of applicability of both types of moieties pyrrole and
chalcone, it is a matter of great interest to have combined both. One of the rings of 1, 3-diaryl-2-
propen-1-one skeleton is occupied by pyrrole derivatives, it generates Pyrrole-chalcone. In α, β-
unsaturated carbonyl frame pyrrole moiety may have a position on β carbon or carbonyl carbon
depends on reactants' functional during synthesis. Based on pyrrole position in α, β-unsaturated
carbonyl frame two categories of chalcones are formed: (i) Pyrrole moiety attached to β carbon
of α, β-unsaturated carbonyl frame of chalcone and (ii) Pyrrole moiety attached to the carbonyl
carbon of α, β-unsaturated carbonyl frame of chalcone.
The combination of different pharmacophores in a pyrrole-chalcones has led to the formation of
more active compounds. Pyrrole containing analogs are considered as a potential source of
5

biologically active compounds that contains a significant set of advantageous properties and can
be found in many natural products. The marketed drugs containing a pyrrole ring system are
known to have many biological properties such as antipsychotic, β-adrenergic antagonist,
anxiolytic, anticancer (leukemia, lymphoma, and myelofibrosis, etc.), antibacterial, antifungal,
antiprotozoal, antimalarial and many more. Due to the diversity of these analogs in the
therapeutic response profile, many researchers have been working to explore this skeleton to its
maximum potential against several diseases or disorders.
Ethyl 3,5–dimethyl–4-acetyl–1H–pyrrole–2–carboxylate (EDAPC) and Ethyl 3,5–
dimetyl–4–formyl–1H–pyrrole–2–carboxylate (EDFPC) are found to be a suitable precursor for
the synthesis of the pyrrole containing chalcones, however, biological and antioxidant activities
of pyrrole-chalcones derived from these pyrrole precursors have not been yet studied and
reported. Therefore, in present work biological and antioxidant activities of pyrrole-chalcones
having different carbonyl positions with respect to pyrrole have been studied and the effect of
others substituents have also been studied. Among the various properties of chemical
compounds, biological activity plays a crucial role suggesting uses of the compounds in the
medical applications.
2.
Experimental Methodology
All the chemicals were used of an analytical grade. Ethyl 3,5–dimetyl–4–formyl–1H–pyrrole–
2–carboxylate (EDFPC) (1a) and Ethyl 3,5–dimethyl-4-acetyl–1H–pyrrole–2–carboxylate
1
(EDAPC) (1b) were prepared by an earlier reported method [27]. The H NMR spectra of
EDFPC and EDAPC were recorded in DMSO–d6 on Bruker DRX–300 spectrometer using TMS
6

as an internal reference. The FT–IR spectra were recorded in KBr medium on a Bruker
spectrometer. The UV–Visible absorption spectra of EDFPC and EDAPC, (1×10–5 M in
DMSO) were recorded on V–670 JASCO spectrophotometer. The synthesis of all the
synthesized compounds has been given in the supplementary material.
2.1 Antimicrobial Screening
The synthesized compounds were evaluated for their in vitro antifungal and antibacterial
activity using A. Niger, B. subtilis strain, respectively, in potato dextrose agar media (PDA).
The composition for this Nutrient Agar Media (per liter) is as Potato (200), Agar-agar (20), and
Dextrose (20) gm. The solution (2.5 ml) of a synthesized compound (different concentrations)
and 50 ml of molten sterile nutrient PDA media (5%) was poured into sterilized glass
petridishes. A control PDA plate was also prepared for the test to compare the effect of test
samples and to nullify the effect of solvent. All these plates were kept for 24 hours in UV
chamber so that the samples had sufficient time to diffuse over a considerable area of the plate.
After solidification of the medium, these plates were freshly seeded with a small portion of the
mycelium of fungus in the form of 0.5 mm discs, carefully over the center of each PDA plate
with the help of a sterilized needle. The plates were kept in incubation at 25±1°C for 96 hours.
Ethanol was used as a solvent to prepare a desired solution of the synthesized compounds
initially and also to maintain proper control.
The antibacterial activity was performed by the agar diffusion method at the concentration level
of 200μg/ml and 100μg/ml. The agar media were inoculated with 1.0 ml of liquid cultures
containing 105 microorganisms/ml. Diffusion time was 24 hours at 5oC for all bacteria, and
7

incubation time was 12 hours at 30oC. DMSO was used as solvent control. Inhibitory activity
was measured (in mm) as the diameter of the observed inhibition zones.
2.2 Antioxidant activity
2.2.1 Free radical scavenging activity by DPPH method: Free radical scavenging capacities of
synthesized compounds were determined according to the reported procedure [28]. The different
concentrations (25-100 µg/mL) of newly synthesized compounds were added to each test tube
and volume was made up to 4 ml using methanol and 3 ml of 0.004% DPPH in methanol was
added. The mixtures were kept for incubated at room temperature under the dark condition for 30
min. The absorbance was recorded at 517 nm using UV-Visible spectrophotometer (Shimadzu
UV-1800, Japan) and butylated hydroxyl toluene (BHT), was used as a reference. A control
sample was prepared using the same volume without any compound and BHT, 95% methanol
served as blank. The test was performed in triplicate and the results were averaged. Radical
scavenging activity was calculated using the formula:
% of radical scavenging activity=[(Acontrol–Atest)/A] × 100
Where Acontrol is the absorbance of the control sample (DPPH solution without test sample) and
Atest is the absorbance of the test sample (DPPH solution+test compound).
2.2.2 Iron chelating ability: The chelating effect was determined according to the literature
method [29]. The test solution (2 ml) of different concentrations (25-100 µg/mL) in methanol
was added to a solution of 2mM FeCl2 (0.05 ml), the reaction was initiated by adding 5 mM
ferrozine (0.2 ml) and total volume was adjusted to 5 ml with methanol. Then, the mixture was
shaken vigorously and left at room temperature for 10 min. Absorbance of the solution was
8

measured spectrophotometrically at 562 nm. EDTA was used as a standard. The inhibition
percentage of ferrozine-Fe complex formations was calculated using the formula:
Metal chelating effect (%)=[(Acontrol–Asample)/Acontrol] × 100
Where Acontrol is the absorbance of control and Asample is the absorbance of test compounds.
Ascorbic acid is used as a control. The test was performed in triplicate and the results were
averaged.
2.2.3 Reducing power assay: Reducing power of the test samples was evaluated by following
the literature method [30]. Various concentrations of test compounds were mixed thoroughly
with a mixture of 2.5 ml of 0.2 mM phosphate buffer (pH 7.4) and 2.5 ml of potassium
ferricyanide. The resulting mixture was incubated at 50°C for 20 min, followed by the addition
of 2.5 ml of trichloroacetic acid (10% w/v) and the mixture was centrifuged at 3000 rpm for 10
min. The upper layer of the solution was collected and mixed with 2.5 ml distilled water and
later with 0.5 ml of ferrous chloride (0.1% w/v). The absorbance was measured at 700 nm
against a blank sample. An increase in the absorbance of the reaction mixture indicated a higher
reducing power of the test compounds.
3. Computational methodology
After the establishment of the structure of the molecule with the help of FT-IR, 1H and 13C
NMR, UV-Visible, and elemental analysis, the initial geometries were generated with the help
of Chem-Draw and Chem-3D Ultra program. All the quantum chemical calculations have been
carried out with Gaussian 09 program package [31] using DFT method, B3LYP functional, and
6–311+G(d,p) basis set. Frequency calculation was conducted at the same level to test the
stability of molecular structures. DFT frequencies are known to be overestimated than the
experimental frequencies; these discrepancies were corrected directly scaling the calculated
9

wavenumbers with proper scaling factor [32]. The electronic transitions of the product were
calculated using time dependent density functional (TD–DFT) theory which has emerged as a
reliable standard tool for the theoretical treatment of electronic excitation spectra and recent
works demonstrate the good accuracy for a wide range of systems [33]. Visualization of
molecule, molecular orbital, and molecular electrostatic potential surfaces (MEPS) was
performed with Gauss View program package [34]. QTAIM analysis has been found to be
successful in the evaluation of associations, the existence of the hydrogen bonding and other
weaker interactions, change of electronic properties in the formation of hydrogen bonds, red or
blue shift in FT-IR spectrum, molecular geometry-distant consequences of H-bonding, π-
electron delocalization shaping the H-bonding feature. The advantage of the QTAIM theory is
that one can obtain information on changes in the electron density distribution as a result of
either bond formation or complexes formation [35]. All QTAIM calculations were performed
with the help of AIMALL program package [36]. The chemical reactivity and site selectivity of
the molecular systems have been determined on the basis of Koopman’s theorem [37]. Basis set
superposition error (BSSE) was corrected by the counterpoise procedure of Boys and Bernardi
[38], as ∆E (BSSE) = E (complex) – E (monomer Aunit) – E (Monomer Bunit). DFT derived
global and local reactivity descriptors have been calculated using standard formula [39-45].
Global reactivity descriptors as electronegativity (χ) = –1/2(εLUMO + εHOMO), chemical
potential (μ) = 1/2 (εLUMO + εHOMO), global hardness (η) = 1/2 (εLUMO – εHOMO), global
softness (S) = 1/2η and electrophilicity index (ω) = μ2/2η are highly successful in predicting
global reactivity trends.
10

4. Result and Discussion
4.1 Structure, conformations, QTAIM and Reactivity
The Scheme 1 shows ten synthesized pyrrole-chalcones (3a-i, and 5b). The spectroscopic, DFT,
QTAIM and Chemical reactivity of 3b, 3d, 3e, and 3f compounds have been already published
[41, 44, 45]. This manuscript present antimicrobial and antioxidants activity of 3b, 3d, 3e, and
3f compounds and compared with the studied molecules (3a, 3c, 3g, 3h, 3i) and (5b). The
product (3a, 3c, 3g, 3h, 3i) and (5b) is depicted in Scheme 1.
11

Scheme 1. The route for the formation of compound (3a-i) and (5a)
The product Ethyl 3, 5-Dimethyl-4-(3-oxo-3-phenyl-propenyl)-1H-pyrrole-2-carboxylate (3a),
(E)–ethyl 4–(3–(3–chlorophenyl)acryloyl)–3,5–dimethyl–1H–pyrrole–2–carboxylate(3c), (E)–
ethyl 4–(3–(2,6–dimethoxyphenyl)acryloyl)–3,5–dimethyl–1H–pyrrole–2–carboxylate (3g),
(E)–ethyl 4–(3–(3–methoxyphenyl)acryloyl)–3,5–dimethyl–1H–pyrrole–2–carboxylate (3h) and
(E)–ethyl4–(3–(4–(benzyloxy)phenyl)acryloyl)–3,5–dimethyl–1H–pyrrole–2–carboxylate (3i)
differ from Ethyl 3, 5-dimethyl-4-(3-phenyl-acryloyl)-1H-pyrrole-2-carboxylate (5a) in the
position of pyrrole substitution around enone –C(=O)CH=CH- (α, β-unsaturated carbonyl)
framework. All products have been obtained by the Claisen-Schmidt condensation of
substituted pyrrole methyl ketone with benzaldehyde derivatives and substituted pyrrole
aldehyde with benzene methyl ketones or acetophenone. The isomers of (3a, 3c, 3g, 3h, 3i) and
(5b) show double bond exist in the trans configuration since the cis configuration is unstable
due to the strong steric effects between the pyrrole ring and the carbonyl group. According to
our calculation, the trans configuration is indeed more stable than the cis one. Thus, only the
trans configuration was considered in this study. There are different conformers for the trans
configuration of the studied molecules (3a, 3c, 3g, 3h, 3i) and (5b) as shown in Figure 1. The
Potential energy surface scans, studies have been performed to understand the conformers’
stability in (3a, 3c, 3g, 3h, 3i) and (5b). The potential energy curve is obtained as a function of
12

the dihedral angle around the C5-C4-C6-C15, C5-C4-C7-C15, C5-C4-C7-C15, C5-C4-C7-C15,
C5-C4-C7-C15 bond by scanning it in steps of 10º, starting from the 0º value in (3a, 3c, 3g, 3h,
3i) and (5b), respectively. The 3aI, 3cI, 3gI, 3hI, 3iI, and 5aI conformers are of lower energy.
3cII
3aII
3aI
3cI
3hII
3gII
3gI
3hI
3III
5aII
5aI
3II
Figure 1. The conformers of (3a-i) and (5a) compound.
13

The enthalpy difference between 3aI / 3aII, 3cI / 3cII, 3gI / 3gII, 3hI / 3hII, 3iI / 3iII and 5aI / 5aII
conformers calculated as -0.5251, -0.6152, -0.9465, -0.8703, -0.5183 and 0.2887 kcal /mol,
respectively. The geometrical parameters of 3a, 3c, 3g, 3h, 3i, and 5b are listed in
supplementary material Table S1, S2, S3, S4, S5, and S6, respectively. From torsion angle data,
it can be seen that the enone unit in (3a, 3c, 3g, 3h, 3i) and (5b) are essentially planar, with a
C=C-C=O torsion angle of average -174.69-176.77º, respectively.
It has been seen in the literature that those compounds having methyl and ethyl ester
group on α and α’ position of pyrrole derivative have the tendency to form dimers [27, 40-45].
Both the pyrrolic N-H and ester C=O group, the role lies in molecular association for dimer
formation. The conformer 3aI, 3cI, 3gI, 3hI, 3iI, and 5aI of (3a, 3c, 3g, 3h, 3i) and (5b),
respectively, have found suitable geometry for dimer formation through intermolecular
hydrogen bonding by pyrrolic N-H and ester C=O. The calculated molecular geometry of
dimers (3a, 3c, 3g, 3h, 3i) and (5b) are shown in supplementary material Figure S1. The
binding energy of dimers is found to be 13.89, 13.98, 26.07, 27.42, 13.81, and 14.68 kcal/mol of
(3a, 3c, 3g, 3h, 3i) and (5b), respectively, as per DFT calculation. The binding energy of dimer
formation after basis set superposition error (BSSE) correction via the standard counterpoise
method [38] are found to be 9.09, 10.4, 10.99, 11.23, 10.89, 9.06 kcal/mol in case of (3a, 3c, 3g,
3h, 3i) and (5b), respectively.
The calculated molecular graphs of (3a, 3c, 3g, 3h, 3i) and (5b) dimers are shown in
Supplementary material Figure S2. The calculated binding energy of (3a) and (3b) found to be
16.62, 16.84, 24.77, 15.12, 16.70, and 14.71 kcal/mol. The binding energy of (3g) dimer is
higher than (3a, 3c, 3h, 3i, and 5a) due to the presence of weaker interactions. In (3a, 3c, 3g,
3h, 3i, and 5a) is higher than (5a) due to the presence of the carbonyl group (interaction C6-O7-
14

--C9/ C60-O67-C59, C6 ---O14-C7 / C47 ---O55-C48, C8--- H35-C15 / C49 ---H76-C56, C6---
O14-C7 / C51---O59-C52, C6--- O14-C7 / C61---O69-C62) near to the pyrrole ring provides
extra stability to the dimer as compared to (5a).
The calculated global parameters are given in Table 1. The calculated value of
electrophilic charge transfer (ECT = -0.3485 (3a), -0.2518 (3b) eV) indicates that, charge flow
from EDAPC to benzaldehyde and EDFPC to acetophenone, respectively. Therefore, EDAPC
and EDFPC as electron donor and benzaldehyde and acetophenone as the electron acceptor. The
increased value of global electrophilicity ω reveals that (3a) molecule is more electrophilic.
Table 1. Calculated εHOMO, εLUMO, energy band gap (εL – εH), chemical potential (μ),
electronegativity (χ), global hardness (η), global softness (S), global electrophilicity
index (ω) for (1a, 1b), (2a, 2c, 2g, 2h, 2i and 4a), (3a, 3c, 3g, 3h, 3i and 5a) and
Elecrophilicity based charge transfer (ECT) for reactant system [(1a, 1b)↔(2a, 2b)].
εH
εL
εL– εH
μ = - χ
η
S
ω
ECT
-6.1609
-6.9446
-7.129
-1.0163 5.1446 3.5886 2.5720 0.1943 2.5032
(1a)
(2b)
(2c)
(2g)
(2h)
(2i)
-1.7257 5.2189 4.3352 2.6094 0.1916 3.6010 -0.3485
-2.0433 5.0857 4.5861 2.5428 0.1966 4.1356 -0.4084
-1.4011 4.9772 3.8897 2.4886 0.2009 3.0398 -0.1679
-1.4944 4.8154 3.9021 2.4077 0.2076 3.1620 -0.2255
-1.3714 4.9580 3.8504 2.4790 0.2016 2.9902 -0.1581
-1.8373 4.0959 3.8849 2.0479 0.2441 3.6848
-2.0460 3.9810 4.0365 1.9905 0.2511 4.0927
-2.5780 3.8760 4.5160 1.9380 0.2579 5.2616
-1.4887 4.0896 3.5335 2.0448 0.2445 3.0530
-1.6631 4.0404 3.6833 2.0202 0.2475 3.3577
-0.8158 5.2156 3.4236 2.6078 0.1917 2.2473 -0.2518
-1.4808 5.2450 4.1033 2.6225 0.1906 3.2102
-1.7220 4.0207 3.7328 2.0103 0.2487 3.4656
-6.3783
-6.3098
-6.3294
-5.9329
-6.0270
-6.4540
-5.5783
-5.7035
-6.0314
-6.7258
-5.7432
(3a)
(3c)
(3g)
(3h)
(3i)
(4a)
(5b)
(6c)
εH, εL, εL– εH, χ, μ, η, ω (in eV) and S (in eV–1)
15

Electrostatic potential surfaces of (3a, 3c, 3g, 3h, 3i, and 5a) are shown in Figure 2 to
understand the reactivity of the molecules and correlate with dipole moment, electronegativity,
partial charges, and site of chemical reactivity of the molecule. In (3a, 3c, 3g, 3h, 3i, and 5a),
the nucleophilic region present is around ester carbonyl group and the electrophilic region
around the hydrogen atom of pyrrole N-H. In (3a, 3c, 3g, 3h, 3i) dimer formation due to the
charge transfer, the equalization of chemical potential and electronegativity take place from the
most positive electrostatic potential region of hydrogen of pyrrole N-H and α-methyl to the
most electronegative electrostatic potential region of ester carbonyl group C=O. Similar
observations have been seen in the case of (5a).
(3g)
(3a)
(3c)
16

(3h)
(5a)
(3i)
Figure 2. The molecular electrostatic potential map of (3a, 3c, 3g, 3h, 3i and 5a) of lower energy conformer.
4.2 Spectroscopic analysis
The structures of compounds have been established on the basis of spectral data. Detailed
discussions of the spectral outcome for both compounds are described as follows. The simulated
spectrum of FT-IR of (3a, 3c, 3g, 3h, 3i) and (5a) compounds are given in Figure 3. The
calculated and experimental vibrational wavenumbers with assignment are given in Table 2.
17

Figure 3. The FT-IR spectrum of (3a, 3c, 3g, 3h, 3i and 5a) compounds of lower energy
conformer
The N-H stretching generally seems in the region of 3500-3220 cm-1 and its accurate position
generally depends upon the degree of hydrogen bonding as well as upon the degree physical
state of the sample for frequency record. The experimental and calculated IR spectrum of (3a,
3c, 3g, 3h, 3i, and 5a) shows N-H stretching vibration at 3340, (3355 (dimer), 3446
(monomer)), 3305, (3471 (dimer), 3639 (monomer)), 3290 (3474 (dimer), 3642 (monomer)),
3301 (3474 (dimer), 3641 (monomer)), 3306 (3473 (dimer), 3641 (monomer)), and 3312 (3292
(dimer), 3430 (monomer)), cm-1, due to intermolecular hydrogen bonding, respectively. The
calculated values of dimer corroborate well with the experimental values as compared to
monomer. This observation correlates well with the reported literature involving intermolecular
hydrogen bonded N–H system at 3358 cm-1 [46] and also with other pyrrole-chalcones and
hydrazones derivatives [27, 33, 40-45]. The free νNH band reported in the literature (pyrrole-2-
18

carboxylic acid) at wavenumber 3465 cm-1 recorded in CCl4 solution [46]. Therefore, we
concluded that the solid-state IR spectrum of (3a, 3c, 3g, 3h, 3i) and (5a) indicates that all
studied molecules exist in dimer form and the optimized geometry of dimer is shown in
Supplementary Figure S1. The observed and calculated wagging mode of the N–H group
appears at 789, 620, 630, 630, 628, 731 and 790, 694, 650, 745, 676, 742 cm-1 in (3a, 3c, 3g,
3h, 3i) and (5a), respectively, confirms the involvement of the pyrrolic N–H group in
intermolecular attraction. The appearances of C=O stretching vibrations in (3a, 3c, 3g, 3h, 3i)
and (5a) again indicate that studied molecules exist in dimer form in the solid-state. In (3a, 3c,
3g, 3h, 3i) and (5a) molecules ester carbonyl group stretching vibrations (νC=O) are observed at
1676, 1687, 1670, 1686, 1684, 1653 cm-1, and calculated as 1722, 1733, 1732, 1730, 1735 and
1688 cm-1, respectively in dimer, and 1752, 1758, 1776, 1756, 1757 and 1691 cm-1 in monomer.
This observed νC=O absorption band at 1676, 1687, 1670, 1686, 1684, 1653 cm-1 agrees well
with the calculated wavenumber of the dimer as compared to the monomer in (3a, 3c, 3g, 3h,
3i) and (5a), respectively. This finding again closely correlates with the reported hydrogen
bonded νC=O vibration at 1665 cm–1 for dimer of syn–pyrrole–2–carboxylic acid [46]. The
vibrational frequencies of this mode again confirm carbonyl group (C=O) involvement in
intermolecular hydrogen bonding. In the case of α-β unsaturated compounds the carbonyl
stretching frequency shift away from the normal position due to the presence of strong
electronegative substituent. The stretching vibration of the keto vinylic group (νC=O) -CH=CH-
CO- in (3a, 3c, 3g, 3h, 3i) calculated at 1649, 1657, 1714, 1720, 1716 cm-1 lower wavenumber
than (5a) at 1760 cm-1. The appended carbonyl group in α-β unsaturated framework of (3a, 3c,
3g, 3h, 3i) indicated that this forms a >C=O---C< interaction with that of methyl group present
at α- position of pyrrole ring.
19

Table 2. Theoretical and Experimental (selected) vibrational wavenumbers of (3a, 3c, 3g, 3h, 3i, and 5b), and their approximate assignments
Cal. (3a) Exp. Cal.(3c) Exp. Cal.(3g) Exp. Cal.(3h) Exp. Cal.(3i) Exp. Cal.(5b) Exp. Rep.
Assignments
Dimer
3355
Dimer
3340 3471
Dimer
3305 3474
Dimer
3290 3474
Dimer
3301 3473
Dimer
3306 3312
3292 3358
υ(N-H)-pyrrole
3094
3069
2940
3229
3303 3225
2990 3141,
3133
2936 3234
2858 3329
2734 3110
2994 3225
2948 3195
2839 3132
2957 3224
2919 3328
2884 3195
2926 3111
2853 3061
2853 2937
2744
3020 - 2900 υ(C-H)-Vinylic
3043 3100-2900 υ(C-H)-benzene
2927 2990-2850 υ(C-H)-ester Me +CH2
1722
1649
1676 1733
1656 1657
1687 1732
1644 1714
1670 1730
1640 1720
1686 1735
1650 1716
1684 1688
1646 1760
1653 1665
1641 1633-1668 -CH=CH-CO-
Keto vinylic group
υ(C=O)ester
1514
-
1552 1516
1608 1648
1578 1670
1599 1644
1601 1614
1565 1600
- 1400-1600 υ(C=C)-Benzoxyl ring
υ(C=C)-Benzene ring
-
-
-
-
-
-
-
1614
1574
-
1501
1445 1512
1297 1392
1011
1418 1599
1505 1645
1429 1697
1198 1643
1180 1228
1510 1598
1427 1202
1192 1143
1103 873
1510 1416
1439 1400-1540 υ(C=C)-keto vinylic
group
1349 1420-1360 υ(C=C)-pyrrole
1303
1220
1295 1332
1429 1349
948
1145
1166
1279
1279 1300-1000 δip -(C-H) benzene
1215 1300-1000 υ(C-O-C) ester
1097
1025
924
860
790
1066 906
1011 885
825
837
778
795
690
620
883
876
831
789
650
1033 1205
934
830
773
630
1045
844
777
745
981
770
682
630
843
832
777
676
990
771
695
628
1101
1093 1000-800
δoop(C-H)- Benzene
891
857
742
876
832
731
800-700
690
602
δoop(C-H)- Me
δoop(C=C)- Benzene ring
ω(N-H)-pyrrole
697
789
694
20

The experimental and calculated HC=CH stretching vibration in (3a, 3c, 3g, 3h, 3i) and (5a)
appears at 1445, 1518, 1505, 1510, 1510, 1439 and 1501, 1512, 1599, 1645, 1598, 1416 cm-1
respectively, and assigned to the HC=CH stretching vibration of chalcone framework.
1
The H NMR chemical shift values of (3a, 3c, 3g, 3h, 3i) and (5a) are given in
Supplementary Table S7. The presence of a broad singlet at δ 9.033 (3a), 11.806 (3c), 11.575
(3g), 11.661 (3h), 11.871 (3i) 9.165 (5a) ppm corresponds to pyrrolic N-H. A multiplet of five
protons of phenyl ring appears in the range δ 7.414-7.7.061 ppm (3a), 7.132-7.679 ppm (3c),
δ7.531-7.559, 6.898-6.699, 6.844-6.860 ppm (3g), δ 7.426-7.482, 6.720-6.748 ppm (3h), δ.
7.681-7.709, 7.078-7.147, 6.939-7.049, 7.376-7.461 ppm (3i) and δ 7.792-7.192 ppm (5a)
experimental spectrum. A detection of doublet in the experimental spectrum of (3a) at δ 7.656-
7.602 (J=16.2 Hz), 7.735-7.762 (J=15.3 Hz) (3c), δ 7.211-7.263 (J=15.6 Hz) (3g), δ 7.862-
7.915 (J=15.9 Hz) (3h), 7.835-7.888 (J=15.9 Hz) (3i), δ 6.998- 6.960 (J = 11.4 Hz) (5a)
confirms the presence of β-vinyl (=C-H) proton and another doublet at δ 7.206-7.154 (J=16.2
Hz), (3a), δ 6.687-6.738 (J=15.3 Hz) (3c), δ 7.146-7.198 (J=15.6 Hz) (3g), δ 7.639-7.692
(J=15.9 Hz) (3h), δ 7.172-7.225 (J=15.9 Hz) (3i) δ 6.747-6.707 (J=12 Hz) (5a) confirms the
presence of α-vinyl (=C-H) proton, respectively. The quartet observed at δ 4.367-4.344 (J = 6.9
Hz), 4.217-4.287, (J = 7.2 Hz), δ 4.258-4.325, (J = 6.6 Hz), 4.151-4.271, (J = 6.6 Hz), 4.255-
4.321, (J = 6.6 Hz), 4.345-4.321 (J = 8 Hz), triplet at δ 1.414-1.367 (J = 7.1 Hz), δ 1.289-1.333
(J = 6.6 Hz), δ 1.233-1.280, (J = 6.3 Hz), δ 1.292-334 (J = 6.3 Hz), δ 1.446-1.490 (J = 6.3 Hz),
δ 1.404-1.356 (J = 7.1 Hz) confirm the presence of methylene and methyl of ester group in the
(3a, 3c, 3g, 3h, 3i) and (5a) molecule, respectively. The presence of singlets at δ 2.591, 2.523, δ
2.234, 2.434, δ 2.271, 2.390, δ 2.271, 2.353, δ 2.271, 2.351 ppm and 2.575, 2.527, confirm the
presence of methyl group at α and β positions of (3a, 3c, 3g, 3h, 3i) and (5a).
21

4.3 Linear and Nonlinear optical property
Electronic absorption spectra of (3a, 3c, 3g, 3h, 3i) and (5a) show an intense lowest
energy charge-transfer absorption band in the UV-visible region. The calculated electronic
excitations of high oscillatory strength and experimental are listed in Table 3. The theoretical
UV–Visible spectrum for (3a, 3c, 3g, 3h, 3i) and (5a) is shown in Figure 4.
Figure 4. The Theoretical UV-Vis spectrum of (3a, 3c, 3g, 3h, 3i, and 5a) compounds
The UV–Visible spectrum study reveals that the compound absorption is observed at
309, 249 (3a), 310, 245 (3c), 352, 242 (3g), 349, 244 (3h), 353, 249 (3i), 350, 240 (5a) nm in
UV range as a result of π–π* transition.
22

Table 3. Calculated and experimental electronic excitations for (3a, 3c, 3g, 3h, 3i and 5a): E
/ eV, oscillatory strength (f), (λmax / nm) at TD–DFT/B3LYP/6–311+G(d,p) level.
S. No
Excitations
E (eV)
(f)
λ
λ
Assignment
calcd.
Exp.
3a
1
2
78 -> 80
79 -> 81
3.9777
5.0420
0.6979
0.3853
311
253
309
249
π→π*
n→π*
3c
1
2
86 -> 88
87 -> 89
3.9670
4.9063
0.7273 312.54
0.3863 252.70
310
245
π→π*
n→π*
3g
1
2
87 -> 88
86 -> 89
3.3916
4.7776
0.8811 365.89
0.4757 259.51
352
242
π→π*
n→π*
3h
1
2
95 -> 96
94 -> 97
3.3921
4.7691
0.6496 365.51
0.4488 259.97
349
244
π→π*
n→π*
3i
1
2
105 ->108
106 ->109
3.3897
4.7785
0.9197 365.77
0.4739 259.46
353
249
π→π*
n→π*
5a
1
2
79 -> 80
79 -> 81
3.5067
4.5694
0.6832 353.57
0.1812 271.34
350
240
π→π*
n→π*
Total static dipole moment (μ0), mean polarizability (|α0|), anisotropy of polarizability
(∆α) and first hyperpolarizability (β0), using x,y,z components are listed in Table 4. We have
studied the solvent-induced effects on the dipole moment, polarizability, and non-linear optical
properties (NLO) by using self-consistent reaction field (SCRF) method. Today computational
calculation as an alternate choice provides extensive properties of materials, eg.
hyperpolarizability which is a difficult task to measure directly [47]. Therefore, nowadays
theoretical methods have been considered as useful techniques for prediction of polarizabilities
and hyperpolarizabilities avoiding an expensive large amount of experimental synthetic work
that precedes the measuring of NLO properties, which may not lead to the desired compound
for practical applications. Total static dipole moment (μ0), mean polarizability (|α0|), anisotropy
23

of polarizability (∆α) and first hyperpolarizability (β0), have been calculated by reporting
literature methods [48].
The dipole moment in a molecule is an important property, which is mainly used to
study the intermolecular interactions involving the nonbonded type dipole–dipole interactions,
because the higher the dipole moment, the stronger the intermolecular interactions will be. The
calculated dipole moment for (3c) (1.9385, 2.4776, 2.7575) molecule is lower than (3a) (2.146,
2.252, 2.446 D), (3g) (4.0842, 4.9932, 5.3373), (3h) (5.5814, 7.0277, 7.6641), (3i) (4.2309,
5.0652, 5.3872), (5a) (2.776, 3.336, 3.573), in vacuum, chloroform and methanol.
Polarizability is an important electronic property, the calculated polarizability α0, and the
anisotropy of the polarizability Δαfor (3a, 3c, 3g, 3h, 3i) and (5a) forms of still using
B3LYP/6-311+G(d,p) in vacuum and solvent are listed in Table 4. The softness and
polarizability are related to each other as ''a soft species is also more polarizable.'' Thus, a hard
(soft) species are known to correspond to a low (high) value of the polarizability as well as a
small (large) size. The polarizability value of (3a, 3c, 3g, 3h, 3i) and (5a) increases
monotonically as the solvent polarity increases. The compound (3a) is a hard species in
comparison to (3b). Therefore, the polarizability of (3b) is higher. The first static
hyperpolarizability (β0) for compounds (3a, 3c, 3g, 3h, 3i) and (5a) increases with the polarity
of the solvent and is better than standard para Nitro aniline (p–NA). Hence, the investigated
molecule will show non–linear optical response and might be used as non–linear optical (NLO)
material.
24

Table 4. Calculated Dipole moment (µ0), Polarizability (|α0|),
anisotropy of Polarizability (Δα), First Hyperpolarizability (β0)
and their components for (3a, 3c, 3g, 3h, 3i and 5a) compounds.
B3LYP/6-311+G(d,p)
Dipole
moment
Polarizability
First
hyperpolarizability
β0
µ0
|α0|
Δα
2.1466 28.8620
1.9385 33.8419
4.0842 35.1639
5.5814 36.7125
4.2309 30.7277
2.7765 30.9897
105.9135
115.9501
132.2266
127.8132
169.3611
105.4309
8.0984
5.2470
33.6791
7.5079
41.5952
14.5947
3a
3c
3g
3h
3i
5a
B3LYP/6-311+G(d,p) CHCl3
2.2527 26.3461
125.6804
141.4556
160.8954
156.9115
194.0433
128.2914
16.9891
13.3619
70.5265
21.4372
76.8431
32.4755
3a
3c
3g
3h
3i
2.4776 41.5621
4.9932 42.9021
7.0277 44.9638
54.6171
5.0652
3.3361 38.0834
5a
B3LYP/6-311+G(d,p) MeOH
2.4466 28.2548
133.7775
21.1303
3a
2.7575 44.7474
5.3373 46.1228
7.6641 48.3512
5.3872 58.4812
3.5735 40.9316
151.4366
171.9840
168.5086
205.4506
137.1315
18.0572
73.0719
22.3735
94.8966
42.1254
3c
3g
3h
3i
5a
µ0 in Debye; |α0| and Δα in 10-24 esu; β0 in 10-30 esu,
4.4 Evaluation of Biological Activity
4.4.1 Evaluation of Antimicrobial activity
Living organisms provide material structures and environment for complicated
chemistry of living. Chemical and physical reactions provide energy to maintain living
functions and to renew structural material. Thus, consideration of biological properties is a
natural extension of physical and chemical properties. Biological activity describes the
beneficial or adverse effects of compounds on living matter. When a drug is a complex
25

chemical, this activity is exerted by the substance's pharmacophore but can be modified by the
other constituents. Antifungal and antibacterial activity of study compounds (3a-3i) and (5a)
were studied against A. Niger and human pathogenic gram-positive bacteria B. subtilis. All the
synthesized compounds (3a-3i) and (5a) screened, exhibited remarkable in vitro activity against
test organism strain. The bar diagram of inhibition of the zone for fungal and bacterial strain is
shown in Figure 5 and 6, respectively and antifungal and antibacterial activity data reported in
Table 5. The systematic perusal of antifungal activity with A. Niger found to be in order of 3h
>3i > 3g>3f >3e > 3c >3b > 3d > 3a >5a. Antibacterial activity against human pathogenic
gram-positive bacteria B. subtilis found to be in order of 3h > 3g > 3i ≈ 3a > 3f > 3e >3b > 3c >
3d > 5a. A close inspection of screening data reveals that the directional position of the α, β-
unsaturated group (-CH=CH-C=O) in chalcone and the nature of substitution at aromatic rings
are highly influencing the antifungal activity of these compounds.
The pyrrole chalcones in which pyrrole is attached to carbonyl group have high
antifungal activity than pyrrole is attached to unsaturated β carbon of group (-CH=CH-C=O).
The ether substituents (3h, 3g and 3i) show good activity for both antifungal activity and
antibacterial study relative to other substituents. Both donating and withdrawing groups are
found to enhance the antifungal activity. Another observation noticed significantly from Table 5
is that degree of inhibition increases with the increasing concentration of testing these pyrrole
chalcones.
26

Figure 5. The bar diagram of antifungal activity of (3a, 3c, 3g, 3h, 3i and 5a) compounds
27

Figure 6. The bar diagram of antibacterial activity of (3a, 3c, 3g, 3h, 3i and 5a) compounds
Table 5. Antimicrobial screening data against for study compounds
Comp
Aspergillus niger
Bacillus subtilis
Conc. of
comp. in
µg/ml
Zone of
Conc. of
Zone of
inhibition in
mm
inhibition
in mm
20
comp. in
µg/ml
50
50
28
3a
25
10
25
13
50
25
50
25
22
10
22
14
50
25
50
25
25
11
22
14
3b
3c
50
25
50
25
50
25
50
21
14
23
14
23
14
24
50
25
50
25
50
25
50
21
10
25
12
27
14
30
3d
3e
3f
3g
28

25
16
25
18
50
25
26
18
50
25
32
19
3h
3i
50
25
25
18
50
25
28
18
50
20
50
25
19
11
32
20
50
25
50
25
19
12
36
22
5a
Control
Nystatin /
Tetracycline
4.4.2 Free radical scavenging activity by DPPH method: The graphical representation of free
radical scavenging activity is given in Figure 7. All the synthesized compounds were screened
for their free radical scavenging activity by DPPH method. The Freshly prepared solution
exhibits a deep blue color with the absorption maximum at 517 mm. This deep blue color
generally fades when the antioxidant is present in the solution. All compounds have exhibited
varied free radical scavenging capacity by comparison with the standard Butylated hydroxy
toluene (BHT) [49]. DPPH assay (IC50 µg/mL) value of DPPH radical scavenging of tested
compounds (3a-i and 5a) is reported in Table 6. The systematic of free radical scavenging
activity with respect to standard BHT (48.63) for synthesized chalcones found to be in order of
3h (38.12) > 3c (43.38) > 3a (44.58) > 3g (45.58) > 3i (50.03) > 5a (52.55) >3e (62.63) >3b
(75.58) > 3d (82.56) > 3f (87.41). Further, it was observed that the compound 3h having orth-
methoxy substituent has excellent activity with minimum IC50 value. The other compounds
showed moderate to good activity. From the activity results it is revealed that among the tested
compounds, compounds (3h, 3c, 3a and 3g) displayed better free radical scavenging activity
with least IC50 value than standard BHT.
29