Highly functionalized pyranopyrazoles: synthesis, antimicrobial activity, simulation studies and their structure activity relationships (SARs)

Article abstract of DOI:10.1007/s11164-018-3569-8

Abstract: Development of potent antibacterial and antifungal agents is a permanently new and unremitting investigation in the therapeutic field. Still, medicinal research wants to find the best antimicrobial agent. To attain this goal , here, the present work synthesis, simulation, and antimicrobial studies of pyranopyrazole derivatives were discussed. Among the reported compounds, 4b has dominant antimicrobial activity that was due to its higher dipole moment. Also, this compound has the most hydrophilic nature and low E_g value. Besides, compound 4e has the next higher dipole moment, hydrophilic property and efficient biological activity. The rationale of these results explained that the comparison of electronic results with biological data is the better way to find the potent pharmaceutical drug compounds. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11164-018-3569-8

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-018-3569-8(0123456789(). -, vo Vl
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Highly functionalized pyranopyrazoles: synthesis,
antimicrobial activity, simulation studies and their
structure activity relationships (SARs)
Guda Mallikarjuna Reddy1,2 Gundala Sravya2 Gutha Yuvaraja3
•
•
•
Alexandre Camilo Jr.4 Grigory V. Zyryanov2,5 Jarem Raul Garcia1
•
•
Received: 6 June 2018 / Accepted: 16 August 2018
Ó Springer Nature B.V. 2018
Abstract
Development of potent antibacterial and antifungal agents is a permanently new and
unremitting investigation in the therapeutic field. Still, medicinal research wants to
find the best antimicrobial agent. To attain this goal , here, the present work syn-
thesis, simulation, and antimicrobial studies of pyranopyrazole derivatives were
discussed. Among the reported compounds, 4b has dominant antimicrobial activity
that was due to its higher dipole moment. Also, this compound has the most
hydrophilic nature and low Eg value. Besides, compound 4e has the next higher
dipole moment, hydrophilic property and efficient biological activity. The rationale
of these results explained that the comparison of electronic results with biological
data is the better way to find the potent pharmaceutical drug compounds.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11164-
018-3569-8) contains supplementary material, which is available to authorized users.
&
Jarem Raul Garcia
nagareddy.organic@gmail.com
Extended author information available on the last page of the article
123

G. M. Reddy et al.
Graphical abstract
Keywords Microwave synthesis Á Antimicrobial activity Á SARs Á HOMO–LUMO Á
Energy gap Á Electronic parameters
Introduction
In and around 1980 there was a slogan, ‘‘now the time to close the volume on
bacterial sickness as the main health risk’’ by US doctors. At that time antibiotic
drugs were considered to create a sensation. Later, the resistance to antibiotics is the
major problem facing us now everywhere. Novel therapy agents are needed to fight
these microbial pathogens, for this, the detection of new platforms should be an
important phenomenon [1, 2]. Countless heterocycles were shown to be in
biological and pharmacological actions. In that, 2-pyrones and their fused
derivatives have fascinated us due to their biotic activities [3–5]. In addition,
pyrazoles act as a core nucleus in various drugs due to their actions such as
antimicrobials [6], antioxidants [7, 8] cytotoxicity [9], and other properties [10]. The
combination of two heterocycles into one molecule could afford a novel entity with
increase bioactivities [11–18]. On the way, dihydro-1H-pyrano[2,3-c]pyrazoles
signify a motivating template in therapeutic chemistry and play an essential role in
the pharmaceutical field. Such compounds displayed biological activities like
antifungal, antibacterial, anticancer and antiinflammatory [19, 20]. Recently, Yang
et al, stated some pyranopyrazoles have good antioxidant and cytotoxicity
properties [21]. Additionally, the most vital aspect is SARs, and it has become
helpful in understanding numerous aspects of chemical–biological interactions in
drug, pesticide research and other areas. In a way, an electronic structure of a
compound displays a fundamental role in a mechanism associated with biological
and pharmacological activities [22]. Yan et al., explained the theoretical calcula-
tions, biological assay and their relationships of pyrazole derivatives [23]. Thus,
electronic properties such as the molecular electrostatic profile (MESP), the lowest
123

Highly functionalized pyranopyrazoles: synthesis…
unoccupied molecular orbital (LUMO), the highest occupied molecular orbital
(HOMO), energy gap, and dipole moment are useful parameters for an accurate
understanding of the chemical reactivity of molecules [24–30]. Peculiarly, MESP
has proven a tool for exploring molecular electronic structure, reactivity patterns,
and structure–activity relationship studies [31–33].
Herein, we report a microwave synthetic method, theoretical calculations and
antimicrobial activity of the pyranopyrazole derivatives. To identify the potent
antimicrobial agent, structure–activity relationships allowed a correct correlation of
biological activity with some appropriate quantum descriptors such as HOMO,
LUMO, energy gap and dipole moment.
Experimental data
General
Computational methodology and chemistry
By using the Density Functional Theory (DFT) method, electronic parameters such
as Molecular Electrostatic Potential (MEP), dipole moment, HOMO (Highest
Occupied Molecular Orbital), LUMO (Lowest Unoccupied Molecular Orbitals) and
heat of formation (HF) were calculated for all the reported compounds. These
calculations were calculated from all electronic energies of the cationic (E?),
anionic (E-) and neutral (E0) representations. For DFT calculations executed on the
GAMESS-US [34] line up package, for Iodine atom, SPK-DZP (Sapporo Double
Zeta Potential) [35] basis set was applied, and 6-31?G(p) basis set, and B3LYP
hybrid functional were applied for atoms like Cl, C, S, O, H, N, and F. In relation of
entire system energy, the compound structure was completely relaxed. Proton NMR
and carbon NMR data were noted at 500 MHz and 75 MHz. Internal standards were
tetramethylsilane. Melting point statistics were pointed out with a micromelting
point device and were uncorrected. Synthetic methods were performed in a
microwave oven, Catalyst-4R. The mass spectra were recorded on Jeol JMS-D 300
and Finnigan Mat 1210 B mass spectrometers at 70 eV with an emission current of
100 lA. The elemental analysis was determined by using Perkin-Elmer 240C
elemental analyzer. HRMS data and was obtained using Electro spray ionization
(ESI).
Protocol for the preparation of pyranopyrazole derivatives (4a–m)
Substituted aromatic aldehyde (1a) (1.0 mmol) and malononitrile (2) (1.0 mmol)
were added to the ethanol solution (5 mL) consisting of methyl-1H-pyrazol-5(4H)-
one (1.0 mmol) (3). The total reaction blend was irradiated with microwaves for
10 min at 130 °C (250 W). The reaction monitored, and product confirmation was
observed by TLC. Then, the crud was poured into chilled ice, extracted with
dichloromethane, and concentrated by rotary evaporation. The residue was purified
by silica gel column chromatography with hexane–EtOAc (4:1) as eluent to afford
123

G. M. Reddy et al.
final compound 4a. All the other products were followed by the same procedure
with their respective starting material.
4-(p-Tolyl)-3-methyl-6-amino-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
(4a)
Off white solid in (0.26 g, 85%); M.p. 212–214 °C; 1H NMR (500 MHz,
CDCl3 ? DMSO-d6: d 2.04 (s, 3H, CH3), 2.49 (s, 3H, Ar-CH3), 4.71 (s, 1H, 4H),
5.53 (s, 2H, NH2), 7.22–7.61 (m, 4H, Ar–H), 11.59 (s, 1H, NH) ppm;13C NMR
(75 MHz, DMSO-d6): d 9.6, 20.5, 35.7, 57.3, 97.6, 117.1, 120.6, 127.2, 128.8,
135.4, 141.3, 154.6, 160.6 ppm; MS m/z: 266.11 (M ?). Anal. Calcd for
C15H14N4O: C, 67.65; H, 5.30; N, 21.04; Found: C, 67.59; H, 5.24; N, 21.01;
HRMS (ES?) Calcd for (M ? H)? 267.12404 found m/z 267.1235.
4-(4-Nitrophenyl)-6-amino-3-methyl-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
(4b) White solid in (0.29 g, 91%); M.p. 263–265 °C; 1H NMR (500 MHz,
DMSO-d6: d 1.40 (s, 3H, CH3), 4.31 (s, 1H, 4H), 5.50 (s, 2H, NH2), 7.02 (d, 2H, Ar–
H, J = 12.5 HZ), 7.71 (d, 2H, Ar–H, J = 12.5 HZ), 11.43 (s, 1H, NH) ppm; 13C NMR
(75 MHz, DMSO-d6): d 9.6, 35.7, 55.8, 96.4, 120.3, 123.7, 128.7, 135.7, 146.2,
151.9, 154.5, 161.0 ppm; MS m/z: 297.08 (M ?). Anal. Calcd for C14H11N5O3: C,
56.56; H, 3.73; N, 23.56; Found: C, 56.62; H, 3.85; N, 23.61; HRMS (ES?) Calcd
for (M ? H)? 298.09347 found m/z 298.0929.
4-Phenyl-6-amino-3-methy-l-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (4c) White
solid in (0.25 g, 88%); M.p. 174–177 °C; 1H NMR (500 MHz, DMSO-d6: d 1.78 (s,
3H, CH3), 3.99 (s, 1H, NH), 4.96 (s, 1H, 4H), 6.98–7.06 (m, 2H, Ar–H), 7.15–7.25
(m, 3H, Ar–H) ppm; 13C NMR (75 MHz, DMSO ? CDCl3-d6): d 9.5, 26.8, 42.4,
48.6, 97.9, 112.7, 127.2, 127.4, 128.3, 138.5, 159.4, 174.0 ppm; MS m/z: 252.10
(M ?). Anal. Calcd for C14H12N4O: C, 66.65; H, 4.79; N, 22.21; Found: C, 66.48;
H, 4.62; N, 22.14; HRMS (ES?) Calcd for (M ? H)? 253.10839 found m/z
253.1079.
(4-Fluorophenyl)-6-amino-4-3-methyl-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
1
(4d) Light yellow solid in (0.27 g, 90%); M.p. 242–244 °C; H NMR (500 MHz,
DMSO-d6: d 1.42 (s, 3H, CH3), 4.21 (s, 1H, 4H), 5.30 (s, 2H, NH2), 6.50–6.79 (m,
4H, Ar–H) ppm; 13C NMR (75 MHz, DMSO ? CDCl3-d6): d 9.4, 35.5, 59.0, 96.7,
114.4, 114.6, 119.9, 128.5, 135.5, 138.8, 160.0 ppm; MS m/z: 270.09 (M ?). Anal.
Calcd for C14H11FN4O: C, 62.22; H, 4.10; N, 20.73; Found: C, 62.18; H, 3.98; N,
20.71; HRMS (ES?) Calcd for (M ? H)? 271.09897 found m/z 271.0984.
3-Methyl-6-amino-4-((4-trifluromethyl)phenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-car-
bonitrile (4e) Yellow solid in (0.32 g, 84%); M.p. 191–193 °C; 1H NMR
(500 MHz, DMSO-d6: d 2.11 (s, 3H, CH3), 4.16 (s, 1H, 4H), 5.17 (b, 1H, NH),
6.70–6.75 (d, 2H, Ar–H, J = 12.3 HZ), 7.34–7.36 (d, 2H, Ar–H, J = 13.0 HZ) ppm;
13C NMR (75 MHz, DMSO ? CDCl3-d6): d 9.6, 27.4, 42.1, 55.5, 98.0, 113.8,
114.5, 122.1, 128.9, 131.8, 133.1, 138.5, 159.3, 165.1 ppm; MS m/z: 320.08 (M ?).
Anal. Calcd for C15H11F3N4O: C, 56.25; H, 3.46; N, 17.49; Found: C, 56.31; H,
3.56; N, 17.60; HRMS (ES?) Calcd for (M ? H)? 321.2692 found m/z 321.2698.
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Highly functionalized pyranopyrazoles: synthesis…
3-Methyl-6-amino-4-(4-chlorophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
1
(4f) Light yellow solid in (0.28 g, 89%); M.p. 160–162 °C; H NMR (500 MHz,
DMSO-d6: d 1.90 (s, 3H, CH3), 3.88 (s, 1H, 4H), 4.65 (s, 1H, NH), 6.62–6.72 (m,
4H, Ar–H) ppm; 13C NMR (75 MHz, DMSO-d6): d 9.5, 16.9, 48.7, 97.4, 112.5,
128.3, 128.9, 133.0, 137.1, 138.7, 159.4 ppm; MS m/z: 286.06 (M ?). Anal. Calcd
for C14H11ClN4O: C, 58.65; H, 3.87; N, 19.54; Found: C, 58.51; H, 3.76; N, 19.36;
HRMS (ES?) Calcd for (M ? H)? 287.06942 found m/z 287.0688.
3-Methyl-6-amino-4-(4-bromophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
(4g) Yellow solid in (0.33 g, 92%); M.p. 172–174 °C; 1H NMR (500 MHz,
DMSO-d6: d 1.83 (3H, CH3, S,), 3.91 (d, 1H, 4H), 4.72 (d, 1H, NH), 6.61–6.69 (m,
4H, Ar–H) ppm;13C NMR (75 MHz, DMSO-d6): d 9.5, 16.4, 47.9, 97.0, 113.4,
129.3, 129.5, 132.5, 135.4, 138.5, 155.4 ppm; MS m/z: 330.01 (M ?). Anal. Calcd
for C14H11BrN4O: C, 50.77; H, 3.35; N, 16.92; Found: C, 50.81; H, 3.49; N, 17.03;
HRMS (ES?) Calcd for (M ? H)? 332.1673 found m/z 332.1370.
3-Methyl-6-amino-4-(2-bromophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
1
(4h) Light yellow solid in (0.33 g, 81%); M.p. 232–235 °C; H NMR (500 MHz,
DMSO-d6: d 1.85 (s, 3H, CH3), 3.87 (d, 1H, 4H), 4.69 (d, 1H, NH), 6.61–6.74 (m,
4H, Ar–H), ppm; 13C NMR (75 MHz, DMSO-d6): d 9.4, 16.5, 47.1, 95.9, 115.1,
126.0, 129.8, 134.5, 138.4, 139.4, 158.5 ppm; MS m/z: 330.01 (M ?). Anal. Calcd
for C14H11BrN4O: C, 50.77; H, 3.35; Br, 24.13; N, 16.92; Found: C, 50.68; H, 3.32;
N, 16.88; HRMS (ES?) Calcd for (M ? H)? 332.1673 found m/z 332.1652.
3-Methyl-6-amino-4-(2-fluorophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
1
(4i) Light yellow solid in (0.27 g, 83%); M.p. 214–216 °C; H NMR (500 MHz,
DMSO-d6: d 1.63 (s, 3H, CH3), 4.20 (s, 1H, 4H), 5.28 (s, 2H, NH2), 6.59 (q, 2H, Ar–
H, J = 12.3 HZ), 6.90 (t, 2H, Ar–H, J = 12.3 HZ) ppm; 13C NMR (75 MHz,
DMSO ? CDCl3-d6): d 9.6, 27.0, 34.5, 61.0, 94.1, 115.4, 117.2, 121.6, 127.9,
135.0, 139.2, 158.9 ppm; MS m/z: 270.09 (M ?). Anal. Calcd for C14H11FN4O: C,
62.22; H, 4.10; N, 20.73; Found: C, 62.19; H, 4.15; N, 20.65; HRMS (ES?) Calcd
for (M ? H)? 271.09897 found m/z 271.0984.
3-Methyl-6-amino-4-(3-Chlorophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
1
(4j) Light yellow solid in (0.28 g, 92%); M.p. 181–183 °C; H NMR (300 MHz,
DMSO-d6: d 1.29 (s, 3H, CH3), 4.11 (s, 1H, 4H), 5.46 (s, 2H, NH2), 6.76–6.99 (m,
2H, Ar–H), 7.69 (t, 2H, Ar–H, J = 13.5 HZ), ppm; 13C NMR (75 MHz,
DMSO ? CDCl3-d6): d 9.5, 35.4, 58.6, 96.2, 120.4, 122.3, 125.8, 129.1, 132.5,
137.5, 143.2, 145.7, 156.5, 162.3 ppm; MS m/z: 286.06 (M ?). Anal. Calcd for
C14H11ClN4O: C, 58.65; H, 3.87; N, 19.54; Found: C, 58.69; H, 4.01; N, 19.58;
HRMS (ES?) Calcd for (M ? H)? 287.7163 found m/z 286.7058.
3-Methyl-6-amino-4-(2-aminophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
(4k) Yellow solid in (0.26 g, 89%); M.p. 201–203 °C; 1H NMR (500 MHz,
CDCl3 ? DMSO-d6: d 1.80 (s, 3H, CH3), 5.20 (t, 3H, 4H ? NH2, J = 12.5 HZ),
6.06 (bs, 2H, NH2), 7.19–7.35 (m, 1H, Ar–H), 7.47–7.88 (m, 3H, Ar–H) ppm; 13C
NMR (75 MHz, DMSO-d6): d 9.5, 27.4, 48.9, 97.2, 114.5, 125.1, 128.1, 129.2,
123

G. M. Reddy et al.
132.0, 133.2, 138.9, 149.3, 159.4, 172.6 ppm; MS m/z: 267.11 (M ?). Anal. Calcd
for C14H13N5O: C, 62.91; H, 4.90; N, 26.20; Found: C, 62.84; H, 4.79; N, 26.16;
HRMS (ES?) Calcd for (M ? H)? 268.2859 found m/z 268.2796.
3-Methyl-6-amino-4-(2-iodophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (4l)
Light yellow solid in (0.37 g, 90%); M.p. 212–214 °C; 1H NMR (500 MHz,
CD3OD-d4: d 1.83 (s, 3H, CH3), 4.56 (s, 1H, 4H), 6.41–6.70 (m, 3H, Ar–H), 7.17 (t,
1H, Ar–H, J = 12.5 HZ) ppm; 13C NMR (75 MHz, CD3OD-d4): d 9.7, 35.2, 82.8,
115.8, 116.5 117.5, 122.4, 127.9, 131.5, 1357.3, 145.2, 153.4, 157.2, 160.5 ppm;
MS m/z: 377.99 (M ?). Anal. Calcd for C14H11IN4O: C, 44.46; H, 2.93; N, 14.82;
Found: C, 44.39; H, 2.85; N, 14.75; HRMS (ES?) Calcd for (M ? H)? 378.1678
found m/z 378.1596.
3-Methyl-6-amino-4-(4-iodophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (4m)
Light yellow solid in (0.37 g, 86%); M.p. 250–252 °C; 1H NMR (500 MHz,
CD3OD-d4: d 1.69 (s, 3H, CH3), 4.52 (s, 1H, 4H), 6.55–6.71 (m, 3H, Ar–H), 7.14 (t,
1H, Ar–H, J = 11.5 HZ) ppm; 13C NMR (75 MHz, CD3OD-d4): d 9.7, 37.4, 82.2,
114.8, 115.5 117.2, 120.8, 125.9, 131.3, 138.9, 146.0, 151.4, 158.2, 162.3 ppm; MS
m/z: 377.99 (M ?). Anal. Calcd for C14H11IN4O: C, 44.46; H, 2.93; N, 14.82;
Found: C, 44.37; H, 2.90; N, 14.79; HRMS (ES?) Calcd for (M ? H)? 379.1678
found m/z 379.1650.
Biological experiment
By the agar well diffusion method [36, 37] against Bacillus subtilis (ATCC 6633),
Staphylococcus aureus (ATCC 19433) (Gram-positive (G(?))), Escherichia coli
(ATCC 8739), Proteus vulgaris (ATCC 29213) (Gram-negative(G(-))) bacterial
stains, Aspergillus niger (MTCC 1881) and Aspergillus flavus (MTCC 1884) fungi
were used to determine in vitro antibacterial and antifungal activities of reported
compounds. In this, dimethyl sulfoxide was used as control and no visible inhibition
zone was observed in control groups. Distilled water was used to prepare the test
solutions. Two references, namely Chloramphenicol and Ciprofloxacin were used
for antibacterial standard drugs and Ketoconazole and Fluconazole were used for
antifungal standard drugs. A bacterial suspension was added to the sterile nutrient
broth, and the mixture was poured into a Petri dish on a horizontal levelled surface.
After the medium was solidified, wells of the dish were made in the agar medium.
Subsequently, 25, 50, and 100 lL of the reported compound’s suspensions were
loaded into the wells separately. The Petri dishes were incubated at 37 °C for one
day for bacteria, and 28 °C for 48 h for fungi in an oven. After, appropriate
incubation,the inhibition zone diameter of the well was measured Experiments were
carried out in triplicate and average zone diameters were measured.
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Highly functionalized pyranopyrazoles: synthesis…
Results and discussion
Synthetic results
All the targeted motifs 4(a–m) were synthesized by a one-pot synthesis method
using microwave irradiation to reduce the reaction time. Our successful efforts gave
the products in quantitative yield within a short time of 10 min (Scheme 1). To a
solution of methyl-1H-pyrazole-5(4H)-one (3) in ethanol were added arylaldehyde 1
and malononitrile (2), followed by irradiation to afford the pyranopyrazole
derivatives 4(a–m). The proton NMR value at d 4.71 ppm belongs to 4H hydrogen
of the pyranopyrazole ring, and the peak appeared at d 5.53 ppm due to the NH2
group attached to the pyranopyrazole ring indicating the formation of product 4a.
Scheme 1 Microwave synthesis of pyranopyrazole and their analogs
123

G. M. Reddy et al.
The other prepared compounds 4H proton range of d 3.78–4.96 ppm and amino
group protons range from d 50.2 to 6.14 ppm revealed the formation of products
4(b–m). Total yields of the products were in the range of 92–81%. Moreover, bromo
and chloro substituted compounds 4g and 4j were formed in the highest yields in
comparison with those of other products. Thus, the present synthetic method may be
used to prepare pyranopyrazole derivatives within a short time. All the compounds
were characterized by proton NMR, carbon NMR, and mass elemental analysis.
Biological results
The compounds (Scheme 1) were screened for their in vitro antimicrobial activity
against two Gram-positive, two Gram-negative bacteria, and two fungal strains.
Most of the compounds showed excellent inhibitory antimicrobial activity against
four bacterial and two fungal strains, few compounds displayed good antibacterial
and antifungal activity, while some compounds were inactive against all strains. In
this study, for comparison purpose, we were used two types of standard drugs for
bacterial and for fungal uses. Biological results tabulated in Tables 1 and 2. From
the data in Table 1, compound 4b displayed higher antibacterial activity than other
Table 1 In vitro antibacterial activity of compounds 4(a–m)
Compound no.
MIC in lg/mL (Zone of inhibition in mm)
G (?)
G (-)
B. subtilis (ATCC- S. aureus (ATCC-
6633)
E. coli (ATCC- P. vulgaris (ATCC-
8739)
19433)
29213)
4a
4b
4c
4d
4e
4f
–
–
–
–
50 (15)
–
100 (16)
–
100 (15)
–
100 (15)
100 (3)
50 (8)
100 (14)
50 (12)
100 (10)
50 (8)
100 (5)
100 (3)
–
50 (7)
100 (13)
50 (10)
50 (7)
100 (7)
100 (4)
–
100 (5)
25 (15)
25 (11)
100 (6)
100 (7)
50 (3)
–
50 (6)
100 (10)
50 (10)
100 (8)
100 (6)
25 (4)
50 (3)
–
4g
4h
4i
4j
4k
4l
–
–
100 (8)
50 (10)
100 (8)
100 (9)
100 (18)
100 (20)
–
100 (9)
100 (8)
100 (19)
100 (17)
–
50 (10)
100 (12)
100 (14)
100 (13)
–
4m
Chloramphenicol 100 (22)
Ciprofloxacin
100 (21)
Control (DMSO)
–
MIC = minimum inhibitory concentration; G (?) = gram-positive; G (-) = gram-negative
‘–’ no activity
123

Highly functionalized pyranopyrazoles: synthesis…
Table 2 In vitro antifungal activity of compounds 4(a–m)
Compound no.
MIC in lg/mL (zone of inhibition in mm)
Antifungal
Aspergillus niger (MTCC 1881)
Aspergillus flavus (MTCC 1884)
4a
–
50 (6)
100 (30)
–
4b
100 (26)
–
4c
4d
–
100 (10)
50 (20)
100 (17)
100 (16)
25 (9)
100 (7)
–
4e
100 (22)
50 (16)
100 (15)
100 (9)
25 (8)
–
4f
4g
4h
4i
4j
4k
–
–
4l
50 (11)
100 (13)
100 (29)
100 (28)
–
100 (9)
100 (12)
100 (31)
100 (34)
–
4m
Ketoconazole
Fluconazole
Control (DMSO)
active compounds. It exhibited higher antibacterial activity towards Gram-positive
bacteria, against Staphylococcus aureus and its inhibition zone value of 16 mm at
100 lg/mL. In bacterial strains, an interesting point was noted that this compound
displayed a greater inhibition zone than the standard drugs, namely, Chloram-
phenicol and Ciprofloxacin against Proteus vulgaris (ATCC 29213) Gram-negative
bacteria strain and its zone of inhibition value of 15 mm at 100 lg/mL
concentration. The next lead antibacterial compound was 4e, and it showed a
second higher antibacterial inhibitory zone against all four bacterial strains. Besides,
the best outcome of this motif was shown by an inhibition zone equal to
Chloramphenicol bacterial standard, inhibition zone value 14 mm at 100 lg/mL
concentration and greater than Ciprofloxacin standard inhibition zone value 13 mm
at 100 lg/mL. Meanwhile, compounds 4a, and 4k were inactive against all the
bacterial strains. While the compound 4j showed inhibition zone against Gram-
negative bacteria, and the compound 4c displayed antibacterial activity against
Proteus vulgaris organism, in other organisms it was inactive. The compounds 4f,
4l, and 4m were exposed for good antibacterial activity towards Gram-negative
Proteus vulgaris organism with their inhibition zone values 12, 12 and 10 mm at a
concentration of 50, 100, and 50 lg/mL. Attractively, the other compounds showed
moderate to low antibacterial activities. In addition, compounds 4b, and 4e
displayed higher antibacterial activity towards a gram-positive Staphylococcus
aureus strain. But, the other active compounds showed a higher zone of inhibition
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G. M. Reddy et al.
against Proteus vulgaris bacteria that did Gram-negative bacteria. Finally, the above
results indicated that, compounds 4b, and 4e showed higher antibacterial activity
than the other active compounds towards Staphylococcus aureus strain.
As presented in Table 2, the reported compounds screened for their antifungal
minimal inhibitory concentrations (MIC lg/mL) and zone of inhibition (mm)
against Aspergillus niger (MTCC 1881) and Aspergillus flavus (MTCC 1884)
fungi’s. The results revealed that some active compounds displayed higher
antifungal activity towards Aspergillus niger, while, few other active compounds
showed a higher inhibition zone against Aspergillus flavus. Among the tested
compounds, compound 4b showed a higher inhibition zone than the other active
compounds. Its value was 30 mm at 100 lg/mL towards Aspergillus flavus strain
and in Aspergillus niger, its inhibition zone value was 26 mm at the same
concentration. In addition, compound 4e exhibited next the most inhibition zones
than did the other active compounds of its value 22 mm towards Aspergillus niger at
100 lg/mL, and 20 mm against Aspergillus flavus. In this, the compound 4b has the
most active use against Aspergillus flavus, while, compound 4e was a higher
inhibition zone towards Aspergillus niger at the same concentration of 100 lg/mL.
On the other hand, compounds 4f, and 4g displayed excellent antifungal activity and
their inhibition zone values of 16, 17 mm at 50, 100 lg/mL against Aspergillus
niger fungi, and 15, 16 mm at 100 lg/mL towards Aspergillus flavus strain. Besides
compounds 4c, 4j, and 4k were inactive against two fungal strains. Interestingly,
compounds 4a, and 4d displayed an inhibition zone towards Aspergillus flavus only
and those were inactive against another strain. Also, activity values of compounds
4a, and 4d were 6 and 10 mm at a minimal inhibitory concentration of 50 and
100 lg/mL. Compound 4m exhibited an identifiable antifungal inhibition zone at
100 lg/mL against all fungi for its values 13, and 12 mm. In addition, the other
compounds displayed moderate antifungal activity against both strains.
Simulation results
In simulation studies, the electronic structural orientation, HOMO and LUMO
orbital location of synthesized compounds were observed, along with these, were
calculated their energies, HOMO–LUMO energy gap (Eg), dipole moment, and heat
of formation (HF).
The most important parameters like electron densities and energies of frontier
molecular orbitals are used for unsaturated structure to determine the reactive sites
[38]. HOMO energy, and LUMO energy are related to ionization potential and
electron affinity. While, the former energy is exemplifying the vulnerability of the
molecule towards an attack by electrophiles whereas, the second one is towards
attack by nucleophiles. According to the HOMO and LUMO data presented in
Table 3, HOMO energy of compounds 4(a–m) were ranged from - 8.73 to
- 9.35 eV. In this, the high negative HOMO value - 9.35 eV belonged to
compound 4b. Moreover, the compound 4e has - 9.24 eV HOMO energy, and the
compound 4f has - 9.10 eV. The average * - 9.06 eV of HOMO value
corresponded to compounds 4d, 4g, 4h, 4i, and 4j. Meanwhile, rest of the
compounds have low negative HOMO energy. Further, compound 4b showed the
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Highly functionalized pyranopyrazoles: synthesis…
Table 3 Simulation results of compounds 4(a–m)
Compound
HF (kcal/mol)
HOMO (eV)
LUMO (eV)
Eg (eV)
Dipole moment
4a
4b
4c
4d
4e
4f
62.738
66.626
72.480
24.860
- 88.826
62.463
75.520
75.938
26.039
62.262
62.842
89.398
88.154
- 8.906
- 9.358
- 8.957
- 9.090
- 9.246
- 9.107
- 9.098
- 9.039
- 9.044
- 9.082
- 8.916
- 8.791
- 8.753
0.003
- 1.183
- 0.040
- 0.184
- 0.610
- 0.247
- 0.247
- 0.201
- 0.144
- 0.220
- 0.014
- 0.206
- 0.255
8.909
8.175
8.917
8.906
8.636
8.860
8.851
8.838
8.900
8.862
8.902
8.585
8.498
4.251
7.563
4.371
4.931
6.146
5.195
5.111
4.779
4.584
4.227
4.130
5.023
5.309
4g
4h
4i
4j
4k
4l
4m
most negative LUMO energy value - 1.18 eV, and - 0.61 eV was the next lead
LUMO energy corresponded to the compound 4e. In the case of 4f, 4g, 4h, 4j, 4l,
and 4m compounds, their LUMO energy was in the order 4m [ 4f [ 4g [ 4j [
4l [ 4h. Interestingly, compound 4a has 0.003 eV of LUMO energy. At the same
time, other compounds illustrated least negative LUMO energy value.
Accurately, the other important parameter is an energy gap (Eg) of HOMO and
LUMO energies [39]. The Eg of all the reported molecules ranges from 8.17 to
8.91 eV, and, if the blend has a high reactivity, that means compound has a low
energy gap. Similarly, the compound having a high energy gap leads to stumpy
reactive nature or in another sense; a high strength of compounds has a low Eg vice
versa.
Furthermore, compounds dipole moment was calculated, and the dipole moment
values range from 4.25 to 7.56 Debye. In addition, there was calculated HF of all the
reported compounds (4(a–m)). Herein, compounds with varying activities have a
very great dissimilarity in their HF. So, there was no clear relationship of HF and
biological activity.
SARs discussion
To gain a better understanding of the antimicrobial capacity of pyranopyrazole
derivatives, and search for potent derivatives, SARs is a key point. In all synthesized
compounds 4(a–m), except 4l and 4m, the HOMO orbitals were situated on the
fused ring (Structures 1 and 2), whereas, the same orbitals of later compounds were
located on an aryl group (Structures 3 and 4). But, in case of LUMO orbitals, those
were positioned differently with respect to the compounds, and; hence, there was no
proper relation between the orbital orientation and biological results.
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G. M. Reddy et al.
Structure 1 Electronic HOMO orbital location of compounds 4b, 4c, 4d, 4j and 4k
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Highly functionalized pyranopyrazoles: synthesis…
Structure 2 HOMO orbitals of compounds 4a, 4e, 4f, 4g, 4h and 4i
Dipole moment, lipophilic nature and their relationship with biological activity
For understanding the SARs of synthesized compounds and biological assay, dipole
moment was the main parameter (Table 3) and this gave some acumen information
about the compounds lipophilicity and hydrophilicity. Generally, lipophilic
compounds are low dipole moment or a non-polar motif, while the hydrophilic
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G. M. Reddy et al.
Structure 3 Electronic and HOMO orbital location of compound 4l
Structure 4 Electronic and HOMO orbital location of compound 4m
compounds are polar, and they will tend to have a polar bond with polar solvents
like water, and; hence, makes it have a dipole moment. Moreover, hydrophilic
compounds are of a water-soluble nature and if these compounds possess potent
biological activity then, those are the most useful in the medicinal chemistry region
for production of drugs. From the reported data, the lipophilic nature of the
compound increases when the dipole moment decreases land eads to polarity
decreases [40]. That means, as the high dipole moment leads to high polarity, for
which in turn the lipophilic nature of the compound decreases, concludes the
increases of hydrophilicity nature. Among the calculated compounds, compound 4b
has the highest dipole moment 7.56 Debye revealed that, the highest hydrophilic
nature and dominant antimicrobial active compound. But, the compound 4k was
inactive, and it has a lipophilic nature that was due to its low dipole moment of 4.13
Debye. Besides, 4e and 4m compounds have apparent biological activity due to
their excellent dipole moment of 6.14 and 5.30 Debye value and have the next
higher hydrophilic nature. In fact, compounds 4f, 4g and 4l showed dipole moment
5.19, 5.11 and 5.02 Debye, its hydrophilic order was 4f [ 4g [ 4l, while the other
compounds consisted of their dipole moments range of 4.93–4.22 Debye to show
different antimicrobial activity.
HOMO–LUMO energy gap (vs) biological activity
The eventual charge transfer interaction in the molecule is responsible for
bioactivity, which is explained by the decrease in HOMO and LUMO energy gap
(Eg) [41, 42]. In reported compounds, compound 4b has low Eg value 8.17 eV
(Fig. 1) and was the highest potent antimicrobial agent. In fact, compounds 4e, 4l,
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Highly functionalized pyranopyrazoles: synthesis…
Fig. 1 Compound 4b
and 4m have the next lowest Eg values of 8.63, 8.58 and 8.49 eV and excellent
antibacterial and antifungal activity. Further, the better Eg values showed
compounds 4f, 4g, 4h and 4j, and their values of 8.86, 8.85, 8.83 and 8.86 eV, in
decreased order of 4j [ 4f [ 4g [ 4h. Not much difference in their Eg, exhibited
different antimicrobial activity. On the other hand, compounds 4a, 4c, 4d, 4i, and 4k
have the Eg value range of 8.90–8.91 eV and exhibited moderate to low biological
activity. Thus, compound 4b has the low Eg value and higher antimicrobial activity
and the compounds 4e, 4l, and 4m displayed excellent biological activity due to
their next low Eg values.
The justification of above results and their comparison with respect to each other
explained that the correlation of simulation parameters with the biological data is a
better way to classify the potent therapeutic compounds.
Conclusions
In this work, synthetic technique, simulation studies, antimicrobial activity and their
connection to pyranopyrazoles were discussed. Screening of antimicrobial activity
of targeted amalgams revealed that the compound 4b produced higher antibacterial
and antifungal activity than other active compounds. The electronic parameters
supported this result. Dipole moment and the HOMO–LUMO energy gap revealed
that 4b has a dominant high dipole moment, hydrophilic nature and low energy gap
(Eg). In addition, compound 4 k was inactive, and it has a lipophilic nature that was
due to its low dipole moment. The rationale of these consequences clarified that
123

G. M. Reddy et al.
correlation of electronic results with biological data is a key technique to categorize
the potent pharmaceutical drug compounds.
Acknowledgements Guda Mallikarjuna Reddy is grateful to the Brazilian Higher Education Personnel
´
Training Coordination (CAPES: Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nıvel Superior) under
the PNPD program and authors GMR and GS give thanks to Ural Federal University, Russia.
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Affiliations
Guda Mallikarjuna Reddy1,2 Gundala Sravya2 Gutha Yuvaraja3
•
•
•
Alexandre Camilo Jr.4 Grigory V. Zyryanov2,5 Jarem Raul Garcia1
•
•
1
Department of Chemistry, State University of Ponta Grossa, Ponta Grossa, Parana 84030-900,
Brazil
2
Ural Federal University, Chemical Engineering Institute, Yekaterinburg, Russian Federation
620002
3
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s
Republic of China
4
Department of Physics, State University of Ponta Grossa, Ponta Grossa, Parana 84030-900,
Brazil
5
I. Ya. Postovskiy Institute of Organic Synthesis, Ural Division of the Russian Academy of
Sciences, 22 S. Kovalevskoy Street, Yekaterinburg, Russian Federation 620219
123