Synthesis of new bis-pyrazole linked hydrazides and their in vitro evaluation as antimicrobial and anti-biofilm agents: A mechanistic role on ergosterol biosynthesis inhibition in Candida albicans

Article abstract of DOI:10.1111/cbdd.13509

Literature reports suggest that pyrazoles and hydrazides are potential antimicrobial pharmocophores. Considering this fact, a series of nineteen conjugates containing hybrids of bis-pyrazole scaffolds joined through a hydrazide linker were synthesized and

Full text of DOI:10.1111/cbdd.13509

DR CHITYAL GANESH KUMAR (Orcid ID : 0000-0003-4954-5258)
Article type : Research Article
Corresponding author mail id: cgkumar5@gmail.com
Synthesis of new bis-pyrazole linked hydrazides and their in vitro
evaluation as antimicrobial and anti-biofilm agents: A mechanistic role on
ergosterol biosynthesis inhibition in Candida albicans
Irfan Khan,a,b K. Sirishaa,b, Mohd Adil Shareef,a,b Thipparapu Ganapathi,c Anver Basha Shaik,a Kunta
Chandra Shekar, a Ahmed Kamal,d and C. Ganesh Kumara,b*
aOrganic Synthesis and Process Chemistry Division, CSIR-Indian Institute of Chemical Technology, Tarnaka,
Hyderabad 500007, India
bAcademy of Scientific and Innovative Research, Ghaziabad 201002, India
cStem Cell Research Division, Department of Biochemistry, ICMR-National Institute of Nutrition, Tarnaka,
Hyderabad 500007, India
dSchool of Pharmaceutical Education and Research, Jamia Hamdard University, New Delhi 110062, India
Abstract
Literature reports suggest that pyrazoles and hydrazides are potential antimicrobial pharmocophores.
Considering this fact, a series of nineteen conjugates containing hybrids of bis-pyrazole scaffolds joined through
a hydrazide linker were synthesized and further evaluated for their antimicrobial activity against a panel of
Gram-positive and Gram negative bacteria along with Candida albicans MTCC 3017 strain. Although the
derivatives exhibited good antibacterial activity, some of the derivatives (13d, 13j, 13l, 13p and 13r) showed
excellent anti-Candida activity with MICs values of 3.9 µg/mL, which was equipotent to that of the standard
Miconazole (3.9 µg/mL), which has inspired us to further explore their anti-Candida activity. The same
compounds were also tested for anti-biofilm studies against various Candida strains and among them,
compounds 13l and 13r efficiently inhibited the formation of fungal biofilms. Field emission scanning electron
micrographs revealed that one of the promising compound 13r showed cell damage and in turn cell death of the
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Candida strain. These potential conjugates (13l and 13r) also demonstrated promising ergosterol biosynthesis
inhibition against some of the strains Candida albicans, which were further validated through molecular
docking studies. In silico computational studies were carried out to predict the binding modes and
pharmacokinetic parameters of these conjugates.
KEYWORDS: Bis-pyrazole; hydrazides; antifungal;
antibiofilm
1 | INTRODUCTION
Microbial infections in humans have breached the alarming levels across the globe especially with the
rise of multidrug resistant pathogens which has also reduced the effectiveness of the available arsenal of
antibiotics.[1,2] This has warranted the search for novel structures showing broad spectrum antibacterial and
antifungal properties. In particular, fungal infections caused by the most common fungal pathogens, Candida
albicans, Cryptococcus neoformans and Aspergillus fumigatus, pose a serious threat to immunocompromised
patients.[3] Amongst all these fungal pathogens, Candida albicans is one of the widespread agent causing
nosocomial infections which are linked with biofilm growth on indwelling medical devices (e.g. ocular lenses,
catheters, heart valves, dental implants, vascular bypass grafts, artificial joints and central nervous system
shunts)[4,5] Candida albicans exhibits dimorphic growth pattern in the nature (i.e., planktonic and biofilm).[6]
Biofilm cell communities of different Candida spp. such as Candida albicans, Candida glabrata, Candida
krusei and Candida parapsilosis are relatively resistant to various antifungal agents such as amphotericin B,
nystatin, clotrimazole, fluconazole, voriconazole, miconazole, etc. Similarly, Aspergillus fumigatus biofilms are
relatively resistant to itraconazole and to some extent caspofungin.[8,9]
The mortality rate due to fungal infections is over 1.35 million per annum, despite the availability of
four different kinds of chemotherapeutic antifungal drugs and amongst them azole containing drugs [fluconazole
(Fig.1,(i)), itraconazole, voriconazole, miconazole and posaconazole] is widely applied as first-line antifungal
therapy that functions by inhibiting the fungal lanosterol 14α-demethylase (CYP51) via binding between
nitrogen of azole to the heme iron atom in the binding site of enzyme,[10,11] whereas the inhibition of lanosterol
results in the interference in the function of ergosterol as a membrane component and plasma membrane
disruption that alter the activities of several membrane bound enzymes.[12] On the other hand, most of the azole
drugs have shown limitation in terms of their drug resistance, narrow antifungal spectrum, and low
bioavailability in practical applications.[13] Considering these facts, there is an urgent emphasis to identify novel
azole antifungal agents exhibiting potent broad spectrum activity, low toxicity and low resistance.
Pyrazole is one of the privileged structure from azole family and is currently gaining renewed attention
as a potential biomolecule in view of its interesting pharmacological properties including antimicrobial,[14] (Fig.
1,vi), anticancer,[15] analgesic,[16] antihyperglycemic,[17] anti-inflammatory,[18] antihypertensive,[19] CNS-like
activity, antidepressant,[20] etc.; however, the potential of pyrazoles as antifungal agents is still not explored to a
greater extent. Hydrazide is obtained from oxoacids, in which –OH is replaced by–NRNR2 (R groups are
commonly H), as in carbohydrazides, RC(=O)NHNH2; sulfonohydrazides, RS(=O)2NHNH2; and phosphonic
dihydrazides, RP(=O)(NHNH2). Hydrazide has attracted attention of medicinal chemists in view of their
diverse array of biological activities like antibacterial, antifungal (Fig. 1,v), anticonvulsant, anti-inflammatory,
antimalarial and anti-tuberculosis, etc.[21-28] Prompted by the aforementioned findings and in continuation of our
ongoing research on the synthesis of bioactive conjugates,[29,30] we prepared hybrid derivatives comprising of
two substituted pyrazole moieties joined through a hydrazide linker and later evaluated their antibacterial, anti-
Candida and anti-biofilm activities. Further, mechanistic aspects of the promising leads on ergosterol
biosynthesis inhibition in different Candida albicans strains were demonstrated, which were further validated
through molecular docking studies.
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Fig. 1. Structures of some antimicrobial agents
2 | METHODS AND MATERIALS
2.1 | Chemistry
All chemicals and reagents were obtained from Aldrich (Sigma-Aldrich, St. Louis, MO, USA),
Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill, MA, USA), or Spectrochem Pvt. Ltd. (Mumbai,
India) and were used as such without any further purification. Reactions were monitored by TLC performed on
silica gel 60 GF-254 glass plates and visualization was achieved by UV light or iodine indicator. Column
1
chromatography was performed with Merck 60-120 mesh silica gel. H NMR spectra were recorded on Bruker
UXNMR/XWIN-NMR (300 MHz) or Innova Varian-VXR-Unity (400, 500 MHz) instruments. 13C NMR
spectra were recorded on Bruker UXNMR/XWIN-NMR (75 MHz) instrument. Chemical shifts (δ) are reported
in ppm downfield from an internal TMS standard. Purity was evaluated by analytical HPLC (LC-20AD,
Shimadzu) interfaced with a PDA detector. Mobile phase: Methanol-water (80:20, v/v) (buffer: ammonium
acetate, pH 4.2). The column used was Phenomenex Luna (C-18) 250 × 4.6 mm particle size. Flow rate: 0.9 mL
min-1; injection volume: 5 mL; retention times were given in minutes. ESI spectra were recorded on a Micro
mass Quattro LC using ESI software with capillary voltage 3.98 kV and ESI mode positive ion trap detector.
High-resolution mass spectra (HRMS) were recorded on a QSTAR XL Hybrid MS-MS mass spectrometer.
Melting points were determined with an Electrothermal melting point apparatus and are uncorrected.
2.1.1 Synthesis of (E)-3-phenyl-N'-((3-phenyl-1H-pyrazol-5-yl)methylene)-1H-pyrazole-5
carbohydrazides 13(a-s)
The different substituted pyrazole carbaldehydes (12a-d) and substituted pyrazole hydrazides (10a-e)
used in this study were adopted from our previous reports[31,32]. To the substituted pyrazole-5-carbaldehyde
(12a) prepared in the previous step was added to the corresponding substituted pyrazole hydrazide in ethanol in
presence of catalytic amount of glacial acetic acid. The reaction mixture was stirred at room temperature for 5-6
h and progress of the reaction was monitored by TLC. After completion, ethanol was evaporated under vacuum
than filtered to obtain the residue which was washed 3-4 times with methanol and vacuum-dried to remove
solvent traces.
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(E)-N'-((3-(3,4-dimethoxyphenyl)-1H-pyrazol-5
yl)methylene)-3-phenyl-1H-pyrazole-5-carbohydrazide (13a)
1
Yellow colored solid, Yield: 76%; mp: 280-285 °C; H NMR (400 MHz, DMSO-d6) δ 3.89 (d, 6H), 6.97 (m, 2H), 7.35 (m, 3H), 7.57 (bs,
1H), 7.74 (s, 1H), 7.98 (m, 3H), 8.57 (s, 1H), 11.82 (bs, 1H), 13.79 (bs, 1H), 13.96 (bs, 1H); 13C NMR (125 MHz, DMSO-d6) δ 55.4, 98.9,
103.5, 109.0, 111.9, 117.6, 125.3, 128.5, 129.0, 143.6, 146.7, 148.9, 158.0. MS (ESI) m/z 417 [M+H]; HR-MS (ESI) m/z for C22H20N6O3
calculated m/z: 417.16696, found m/z: 417.16697.
(E)-3-phenyl-N'-((3-(3,4,5-trimethoxyphenyl)-1H-pyr
azol-5-yl)methylene)-1H-pyrazole-5-carbohydrazide (13b)
Yellow colored solid, Yield: 72%; mp: 270-275 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.68 (s, 3H), 3.86 (s, 6H), 7.17 (s, 2H), 7.23 (s, 1H),
7.41 (d, 1H), 7.49 (t, 2H), 7.84 (d, 2H), 8.55 (s, 1H), 11.79 (bs,1H), 13.52 (bs, 1H), 13.84 (bs, 1H); 13C NMR (125 MHz, CDCl3); δ 55.4,
98.9, 103.5, 109.0, 111.9, 117.6, 125.3, 128.5, 129.0, 143.6, 146.7, 148.9, 158.0. MS (ESI) m/z 447 [M+H]; HR-MS (ESI) m/z for
C23H22N6O4 calculated m/z: 447.17753, found m/z: 447.17968.
(E)-3-phenyl-N'-((3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-5-yl)methylene)-1H-pyrazole-5-
carbohydrazide (13c)
White colored solid, Yield: 65%; mp: 240-245 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.25 (d, 2H), 7.40 (d, 1H), 7.49 (t, 2H), 7.70 (s, 2H),
7.84 (d, 2H), 8.16 (s, 1H), 8.21 (s, 1H), 8.55 (s, 1H), 11.92 (bs, 1H), 13.45 (bs, 1H), 13.65 (bs, 1h); 13C NMR (125 MHz, CDCl3) 100.7,
103.5, 121.2, 121.6, 125.3, 128.5, 129.0, 129.8, 130.1, 136.9, 142.2, 143.7, 146.5, 149.5, 158.0, 206.4 MS (ESI) m/z 445 [M+Na]; HR-MS
(ESI) m/z for C21H15N6OF3Na calculated m/z: 445.0523, found m/z: 445.05446
(E)-N'-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)-3-phenyl-1H-pyrazole-5-
carbohydrazide (13d)
Pale yellow colored solid, Yield: 70%; mp: 255-260 °C; 1H NMR (400 MHz, DMSO-d6) δ 6.04 (s, 2H), 6.88 (s, 2H), 7.14 (s, 1H), 7.36 (m,
3H), 7.84 (bs, 4H), 8.53 (s, 1H), 13.20 (bs, 1H), 13.56 (bs ,1H), 13.89 (bs, 1H); 13C NMR (125 MHz, DMSO-d6) δ 99.0, 101.2, 102.3, 103.4,
105.7, 108.7, 119.1, 122.8, 125.3, 128.5, 128.9, 132.9, 142.5, 143.6, 146.9, 147.2, 147.8, 157.9. MS (ESI) m/z 401 [M+H]; HR-MS (ESI)
m/z for C21H16N6O3 calculated m/z: 401.13566, found m/z: 401.40545
(E)-N'-((3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)methylene)-3-(4-methoxyphenyl)-1H-pyrazole-5-
carbohydrazide (13e)
Red colored solid, Yield: 64%; mp: 276-280 °C; 1H NMR (400 MHz, CDCl3) δ 3.84 (s, 6H), 3.90 (s, 3H), 7.37 (d, 2H), 7.60 (s, 3H), 7.73 (d,
2H), 8.09 (d, 2H), 8.55 (s, 1H), 11.6 (bs, 1H), 13.53 (bs, 1H), 13.87 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ 56.0, 60.1, 102.8, 102.9, 108.3,
110.7, 120.1, 124.4, 125.2, 125.8, 126.9, 129.2, 137.7, 141.2, 153.3, 169.2. MS (ESI) m/z 447 [M+H]; HR-MS (ESI) m/z for C23H22N6O4
calculated m/z: 447.17753, found m/z: 447.17968.
(E)-3-(4-methoxyphenyl)-N'-((3-(3,4,5-trimethoxy phen yl)-1H-pyrazol-5-yl)methylene)-1H-pyrazole-
5-carbohy- drazide (13f)
Red colored solid, Yield: 64%; mp: 268-270 °C; 1H NMR (400 MHz, CDCl3) δ 3.84 (s, 6H), 3.92 (s, 6H), 6.99 (d, 2H), 7.08 (s, 3H), 7.71 (d,
2H), 7.77 (s, 1H), 8.49 (s, 2H), 11.64 (bs, 1H), 13.62 (bs, 1H), 13.71 (bs, 1H); 13C NMR (125 MHz, DMSO-d6) δ 55.1, 56.0, 60.0, 99.7,
102.7, 102.5, 114.3, 121.1, 137.4, 142.4, 143.5, 146.6, 149.0, 153.2, 158.0, 159.4. MS (ESI) m/z 477 [M+H]; HR-MS (ESI) m/z for
C23H22N6O4 calculated m/z: 477.18792, found m/z: 477.18809.
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(E)-3-(4-methoxyphenyl)-N'-((3-(4-(trifluoromethyl)
pyrazole-5-carbohydrazide (13g)
phenyl)-1H-pyrazol-5-yl)methylene)-1H-
Light brown colored solid, Yield: 60%; mp: 250-245 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.83 (s, 3H), 6.76 (s, 1H), 7.03 (d, 2H), 7.55 (d,
2H), 7.57 (d, 2H), 7.66 (d, 2H), 8.69 (s, 1H), 10.87 (bs,1H), 13.53 (bs, 1H), 13.75 (bs, 1H); 13C NMR (125 MHz, CDCl3) δ 55.1, 100.6
102.6, 103.1, 113.6, 114.3, 121.1, 126.8, 127.9, 129.7, 130.1, 130.5, 136.8, 142.1, 143.6, 159.4, 160.4. MS (ESI) m/z 455 [M+H]; HR-MS
(ESI) m/z for C22H18N6OF3 calculated m/z: 455.14378, found m/z: 455.42060
(E)-N'-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)
carbohydrazide (13h)
methylene)-3-phenyl-1H-pyrazole-5-
White colored solid, Yield: 78%; mp: 265-270 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.83 (s, 3H), 6.07 (s, 2H), 6.96 (s, 2H), 7.02 ( d, 1H),
7.05 (d, 2H), 7.23 (d, 1H), 7.28 (d, 1H), 7.57 (d, 2H), 8.56 (s, 1H), 11.86 (bs, 1H), 13.77 (bs, 1H), 13.93 (bs, 1H); 13C NMR (125 MHz,
DMSO-d6) δ 55.6, 101.2, 102.3, 108.5, 109.1, 114.4, 119.9, 124.8, 128.3, 133.5, 140.0, 146.9, 148.5, 150.6, 156.9, 160.9. MS (ESI) m/z 431
[M+H]; HR-MS (ESI) m/z for C21H16N6O3 calculated m/z: 431.14623, found m/z: 431.43145.
(E)-3-(3,4,5-trimethoxyphenyl)-N'-((3-(3,4,5-trimethoxy
pyrazole-5-carb ohydrazide (13i)
phenyl)-1H-pyrazol-5-yl)methylene)-1H-
White colored solid, Yield: 80%; mp: 295-300 °C; 1H NMR (400 MHz, CDCl3+ DMSO-d6) δ 3.81 (s, 6H), 3.88 (s, 12H), 6.86 (s, 2H), 7.03
(s, 2H), 7.05 (s, 2H), 8.63 (s, 1H), 11.86 (bs, 1H), 13.53 (bs, 1H); 13C NMR (125 MHz, CDCl3+DMSO-d6) δ 53.9, 54.1, 54.3, 54.5, 58.4,
102.5, 103.0, 103.6, 107.9, 109.8, 116.1, 116.9, 119.2, 126.7, 133.0, 139.2, 142.0, 145.3, 147.4, 147.8, 151.3, 161.3. MS (ESI) m/z 537
[M+H]; HR-MS (ESI) m/z for C26H28N6O7 calculated m/z: 537.13902, found m/z: 537.15010
(E)-N'-((3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-5-yl)methylene)-3-(3,4,5-trimethoxyphenyl)-1H-
pyrazole-5-carbohydrazide (13j)
Yellow colored solid, Yield: 63%; mp: 282-285 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.67 (s, 3H), 3.88 (s, 6H), 7.17 (s, 2H), 7.47 (d, 1H),
7.74 (d, 2H), 8.15 (d, 1H), 8.27 (s, 1H), 8.56 (s, 1H), 11.86 (bs, 1H), 13.53 (bs, 1H), 13.99 (bs ,1H); 13C NMR (125 MHz, CDCl3) δ 54.3,
58.5, 99.8, 101.3, 104.3, 106.0, 106.7, 122.2, 136.2, 136.8, 147.3, 146.5, 151.6, 161.9. MS (ESI) m/z 515 [M+H]; HR-MS (ESI) m/z for
C24H22N6O4F3 calculated m/z: 515.10302, found m/z: 515.10556
(E)-N'-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)
pyrazole-5-carbohydrazide (13k)
methylene)-3-(3,4,5-trimethoxyphenyl)-1H-
White colored solid, Yield: 65%; mp: 263-265 °C; 1H NMR (400 MHz, DMSO-d6); δ 3.70 (s, 3H), 3.87 (s, 6H), 6.08 (s, 2H), 7.01 (s, 1H),
7.04 (d, 1H), 7.14 (s, 1H), 7.35 (d, 1H), 7.43 (s, 2H), 8.54 (s, 1H), 8.91 (bs, 1H), 12.93 (bs,1H), 13.11 (bs,1H); 13C NMR (125 MHz, DMSO-
d6) δ 56.0, 60.0, 101.1, 101.3, 102.8, 105.7, 105.8, 108.7, 119.0, 119.3, 137.5, 147.1, 147.7, 147.8, 147.5, 153.2. MS (ESI) m/z 491 [M+H];
HR-MS (ESI) m/z for C24H23N6O6 calculated m/z: 491.16736, found m/z: 491.16992
(E)-N'-((3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)methylene)-3-(4(trifluoromethyl)phenyl)-1H-
pyrazole-5-carbohydrazide (13l)
1
White colored solid, Yield: 80%; mp: 270-275 °C; H NMR (400 MHz, DMSO-d6) δ 3.89 (d, 6H), 6.99 (m, 1H), 7.31 (m, 2H), 7.61 (bs,
1H), 8.07 (s, 4H), 8.57 (s, 1H), 11.75 (bs, 1H), 13.98 (bs, 1H); 13C NMR (125 MHz, CDCl3+DMSO-d6) δ 55.1, 100.9, 105.5, 108.3, 113.3,
113.7, 118.9, 119.4, 125.1, 129.2, 129.3, 147.0, 147.6, 161.8. MS (ESI) m/z 485 [M+H]; HR-MS (ESI) m/z for C23H20N6O3F3 calculated m/z:
485.08756, found m/z: 485.08904
(E)-3-(4-(trifluoromethyl)phenyl)-N'-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)methylene)-1H-
pyrazole-5-carbohydrazide (13m)
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White brown colored solid, Yield: 60%; mp: 296-300 °C; 1H NMR (500 MHz, DMSO-d6); δ 3.67 (s, 3H), 3.88 (s, 6H), 7.17 (s, 2H), 7.47 (d,
1H), 7.74 (d, 2H), 8.15 (d, 1H), 8.27 (s, 1H), 8.56 (s, 1H), 11.86 (bs, 1H), 13.53 (bs, 1H), 13.99 (bs, 1H); 13C NMR (125 MHz, CDCl3); δ
56.1, 60.8, 100.7, 102.3, 124.1, 125.6, 126.1, 127.3, 131.0, 136.3, 139.2, 147.6, 148.3, 153.1, 157.6. MS (ESI) m/z 515 [M+H]; HR-MS
(ESI) m/z for C24H22N6O4F3 calculated m/z: 515.10302, found m/z: 515.10556.
(E)-3-(4-(trifluoromethyl)phenyl)-N'-((3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-5-yl)methylene)-
1H-pyrazole-5-carbohydrazide (13n)
Yellow colored solid, Yield: 63%; mp: 235-240 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.31 (s, 1H), 7.46 (s, 1H), 7.74 (m, 4H), 8.20 (m, 4H),
8.56 (s, 1H), 11.99 (bs,1H), 14.44 (bs ,1H); 13C NMR (125 MHz, DMSO-d6) δ 101.3, 124.1, 125.6, 126.1, 131.0, 133.6, 136.3, 139.2, 147.3,
147.6, 148.6, 157.6. MS (ESI) m/z 493 [M+H]; HR-MS (ESI) m/z for C22H14N6OF6 calculated m/z: 493.11902, found m/z: 493.12346.
(E)-N'-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)-3-(4-(trifluoromethyl)phenyl)-1H-
pyrazole-5-carbohydrazide (13o)
Yellow colored solid, Yield: 70%; mp: 258-260 °C; 1H NMR (400 MHz, DMSO-d6); δ 6.07 (s, 2H), 7.01 (s, 2H), 7.34 (s, 1H), 7.40 (s, 2H),
7.73 (s, 2H), 7.77 (d, 1H), 8.16 (Bs, 1H), 8.24 (s, 1H), 11.91 (bs, 1H), 11.98 (bs, 1H); 13C NMR (125 MHz, DMSO-d6) δ 101.2, 102.3,
108.4, 108.5, 120.8, 124.1, 125.6, 126.1, 128.3, 131.0, 133.6, 139.2, 147.6, 148.8, 149.3, 157.6. MS (ESI) m/z 469 [M+H]; HR-MS (ESI)
m/z for C22H15N6O3F3 calculated m/z: 469.12305, found m/z: 469.40366
(E)-3-(3,4-dichlorophenyl)-N'-((3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)methylene)-1H-pyrazole-5-
carbohydrazide (13p)
White colored solid, Yield: 65%; mp: 274-277 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.90 (d, 6H), 6.98 (s, 2H), 7.36 (m, 2H), 7.57 (Bs, 1H),
7.74 (d, 1H), 7.98 (m, 2H), 8.54 (s, 1H), 11.80 (bs, 1H), 13.77 (bs, 1H), 13.97 (bs ,1H); 13C NMR (125 MHz, DMSO-d6) δ 55.50, 55.58,
107.7, 108.9, 110.6, 112.0, 117.9, 121.1, 123.4, 124.2, 125.1, 125.8, 127.0, 129.1, 141.2, 143.3, 147.5, 147.3, 149.0, 169.2. MS (ESI) m/z
485 [M+H]; HR-MS (ESI) m/z for C22H19N6O3Cl2 calculated m/z: 485.06902, found m/z: 485.08904
(E)-3-(3,4-dichlorophenyl)-N'-((3-(3,4,5-
trimethoxyphenyl)-1H-pyrazol-5-yl)methylene)-1H-pyrazole-5-carbohydrazide (13q)
Pale yellow colored solid, Yield: 60%; mp: 272-275 °C; 1H NMR (500 MHz, CDCl3) δ 3.83 (s, 3H), 3.88 (s, 6H), 6.98 (s, 1H), 7.02 (s, 1H),
7.23 (s, 2H), 7.46 (d, 1H), 7.56 (d, 1H), 7.91 (s, 1H), 8.45 (s, 1H), 11.86 (bs, 1H), 13.99 (bs, 1H); 13C NMR (125 MHz, CDCl3) 56.0, 60.0
101.1, 101.3, 102.8, 105.7, 105.8, 108.7, 119.0, 119.3, 137.5, 147.1, 147.5, 147.7, 153.4, MS (ESI) m/z 515 [M+H]; HR-MS (ESI) m/z for
C23H21N6O4Cl2 calculated m/z: 515.10325, found m/z: 515.10556
(E)-3-(3,4-dichlorophenyl)-N'-((3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-5-yl)methylene)-1H-
pyrazole-5-carbohydrazide (13r)
Yellow colored solid, Yield: 63%; mp: 255-260 °C; 1H NMR (500 MHz, DMSO-d6) δ 7.02 (s, 2H), 7.46 (d, 2H), 7.49 (d, 1H), 7.59 (d, 1H),
7.76 ( d, 1H), 7.86 (d, 1H), 8.56 (s, 1H), 12.86 (bs, 1H), 13.99 (bs, 1H); 13C NMR (125 MHz, DMSO-d6) 101.5 102.8, 106.2, 107.7, 108.4,
124.3, 125.4, 128.7, 129.0, 138.6, 148.0, 148.8, 163.7. MS (ESI) m/z 493 [M+H]; HR-MS (ESI) m/z for C21H13N6OF3Cl2 calculated m/z:
493.11245, found m/z: 493.12346
(E)-N'-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)-3-(3,4-dichlorophenyl)-1H-
pyrazole-5-carbohydrazide (13s)
1
Pale yellow colored solid, Yield: 75%; mp: 240-245 °C; H NMR (500 MHz, DMSO-d6); δ 6.08 (s, 2H), 7.01 (m, 1H), 7.39 (m, 3H), 7.73
(d, 1H), 7.77 (d, 1H), 7.87 (d, 1H), 8.19 (s, 1H), 8.54 (s,1H), 9.91 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 101.2, 102.3, 108.4, 108.5,
120.8, 127.0, 128.8, 130.7, 132.5, 132.7, 133.4, 139.2 147.6, 148.8, 149.3, 157.6. MS (ESI) m/z 469 [M+H]; HR-MS (ESI) m/z for
C21H15N6O3Cl2 calculated m/z: 469.05772, found m/z: 469.06031
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3 | RESULTS AND DISCUSSION
3.1 | Chemistry
Synthesis of bis-pyrazole linked through a hydrazide moiety 13(a-s) were outlined in Scheme 1(A-B),
wherein the final step was carried out by condensation reaction using equimolar mixtures of substituted phenyl-
1H-pyrazole-5-carbaldehydes 12(a-d) and substituted pyrazole hydrazide 10(a-e) in the presence of glacial
acetic acid in ethanol. The key intermediates, 1H-pyrazole-5-carbohydrazides 10(a–e) were prepared
conveniently in three sequential steps: Initially, the substituted acetophenones 7(a-e) were oxylated in presence
of sodium ethanolate in ethanol which yielded ethyl 3-oxo-4- (substituted phenyl) butanoates 8(a-e). These
diketoesters were further cyclized with NH2-NH2.2HCl in refluxing ethanol resulting in substituted pyrazole
carboxylates 9(a–e), which were further reacted with NH2–NH2·H2O in methanol to furnish 3-phenyl-1H-
pyrazole-5-carbohydrazides 10(a-e), while substituted phenyl-1H-pyrazole-5-carbaldehydes 12(a-d) were
synthesized by reducing pyrazole carboxylates 9(a-e) to corresponding pyrazole alcohols 11(a-d) by using
LiAlH4. These were selectively oxidized to 3-substituted phenyl-1H-pyrazole-5-carbaldehydes 12(a-d) by 2-
iodoxybenzoic acid (IBX) in DMSO. The final target conjugates 13(a-s) obtained were further characterized by
1H NMR, 13C NMR and mass spectral data.
Reagents and conditions: (i) Diethyloxalate, NaOEt/ Ethanol, 0 °C-rt, 4 h; (ii) N2H4.2HCl/ ethanol, reflux, 3-4
h; (iii) N2H4.H2O/ethanol, reflux, 3-4 h; (iv) LiAlH4/THF, 2 h, 0 °C-rt; (v) IBX/dry DMSO, 1 h, rt; (vi) EtOH,
glacial acetic acid.
Scheme 1. Synthesis of bis-pyrazole hydrazide derivatives 13(a-s)
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3.2 | Antimicrobial activity
The synthesized hybrids, bis-pyrazole hydrazides 13(a-s), were evaluated for their in vitro antimicrobial assay
by employing agar well diffusion method.[33] The bacterial strains were procured from Microbial Type Culture
Collection and Gene Bank (MTCC), CSIR-Institute of Microbial Technology, Chandigarh, India. Bacterial
strains used in the study included Gram-positive strains such as Micrococcus luteus MTCC 2470,
Staphylococcus aureus MTCC 96, Staphylococcus aureus MLS-16 MTCC 2940, Bacillus subtilis MTCC 121
and Gram-negative strains such as Escherichia coli MTCC 739, Pseudomonas aeruginosa MTCC 2453 and
Klebsiella planticola MTCC 530. Among all the screened derivatives, compounds 13d, 13j, 13l, 13p and 13r
exhibited moderate antimicrobial activity towards the entire spectrum of tested Gram-positive and Gram-
negative bacterial strains and also against Candida albicans MTCC 3017 with MIC values ranging between 3.9
to 15.6 µg mL-1.While derivatives, 13l and 13r exhibited moderate antibacterial activity with MIC values of 3.9-
7.8 µg/ mL specifically towards all the tested bacterial strains; however both the derivatives, 13l and 13r
exhibited excellent MIC value of 3.9 µg/ mL against Candida albicans MTCC 3017 which were equipotent to
miconazole and fluconazole with MIC value of 3.9 µg/ mL.
Table 1. Antimicrobial activity of various bis-pyrazole linked hydrazides 13(a-s)
Test
cmd.
13a
13b
13c
Minimum Inhibitory Concentration (MIC, µg/mL)
ML
3.9
SA
3.9
3.9
3.9
7.8
SL
7.8
BS
7.8
EC
PA
KP
CA
>125
>125
>125
7.8
>125
>125
>125
15.6
>125
>125
>125
15’6
>125
>125
>125
>125
15.6
3.9
7.8
7.8
3.9
7.8
7.8
13d
13e
7.8
7.8
7.8
>125
>1
25
>125
>125
>125
>125
>125
3.9
3.9
7.8
7.8
>125
>125
>125
>125
>125
>125
>125
>125
13f
>125
>1
25
>125
>125
13g
>125
>1
25
>125
>125
>125
>125
>125
>125
13h
7.8
7.8
7.8
7.8
7.8
7.8
7.8
7.8
7.8
>125
7.8
>125
15.6
>125
15.6
13i
13j
13k
7.8
>125
>1
25
>125
>125
>125
>125
>125
>125
13l
7.8
3.9
3.9
3.9
7.8
15.6
3.9
3.9
>125
>1
25
>125
>125
>125
>125
>125
>125
13m
>125
3.9
>1
25
>125
7.8
7.8
7.8
>125
>125
>125
>125
>125
15.6
>125
>125
13n
13o
3.9
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13p
13q
7.8
7.8
7.8
7.8
7.8
7.8
15.6
15.6
>125
>1
25
>125
>125
>125
>125
>125
>125
13r
13s
3.9
3.9
15.6
7.8
3.9
7.8
3.9
3.9
>125
>1
25
>125
>125
>125
>125
>125
>125
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
MCZ
FCZ
CLZ
CPF
3.9
3.9
7.8
-
0.9
0.9
0.9
0.9
0.9
0.9
0.9
ML = Micrococcus luteus MTCC 2470, SA = Staphylococcus aureus MTCC 96, SL = Staphylococcus aureus MLS-16 MTCC 2940, BS =
Bacillus subtilis MTCC 121, EC = Escherichia coli MTCC739, PA = Pseudomonas aeruginosa MTCC 2453, KP = Klebsiella planticola
MTCC 530, CA = Candida albicans MTCC 3017, CPF = ciprofloxacin, MCZ = Miconazole, FCZ = Fluconazole; CLZ = Clotrimazole
From a structure–activity relationship perspective, it was observed that 13l was more potent when R1 was
substituted
by electronegative CF3 and R2 was substituted with electron
donating group like dimethoxy group, while in case of 13r both R1 and R2 were substituted by electronegative
groups like dichloro and CF3, respectively, which exhibited promising antibacterial as well as antifungal
activities as compared to other screened molecules in the series. The MIC values to this regard are tabulated in
Table 1.
3.3 | Antifungal activity
Candidiasis is the most prevalent fungal infection in humans caused by various Candida members, of
which Candida albicans is the most common causative agent. Candida infections are prevalent in patients
showing immune deficient complications, under medications for diabetes, chemotherapy, HIV-AIDS and also in
children. It is reported that three out of every four women exhibit Candida infections during their life time.[34]
Further, different Candida spp. form biofilms over medical prosthetics.[35] Considering the above pathogenic
effects of different Candida spp., the potential derivatives 13d, 13j, 13l, 13p and 13s were screened for their
anti-Candida activity against various Candida strains namely, Candida albicans MTCC 183, C. albicans MTCC
227, C. albicans MTCC 1637, C. albicans MTCC 3017, C. albicans MTCC 3018, C. albicans MTCC 4748, C.
albicans MTCC 7315, Candida parapsilosis MTCC 1744, Candida glabrata MTCC 3019, Candida krusei
MTCC 3020, Issatchenkia hanoiensis MTCC 4755. All the screened derivatives exhibited anti-Candida activity
with MIC values ranging from 3.9-15.6 µg mL-1 against various Candida strains. The anti-Candida activity
results to this regard are tabulated in Table 2.
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3.4 | Minimum fungicidal concentration (MFC)
The antifungal properties of bis-pyrazole hydrazide conjugates were further evaluated and the most
promising hybrids 13d, 13j, 13l, 13p and 13s were screened for minimum fungicidal concentration and the
results were found to be almost double to the MIC values . The MFC results are shown in Table S1
(Supplementary Information).
3.5 | Inhibition of Candida biofilms
The most promising conjugates 13l and 13r showing potential MIC values against various Candida
strains were also tested for their biofilm inhibition activity employing crystal violet staining assay[36] using
standard drug, Miconazole as control. The results of anti-Candida biofilm activity are shown in Table 2.
Wherein, both the derivatives 13l and 13r, exhibited significant biofilm inhibition at doses ranging between 25-
52 µM against various Candida biofilms. The derivative 13l showed biofilm inhibition activity formed by
Candida albicans MTCC 227 at a dose of 27.32 µM, and 13r showed promising biofilm inhibition activity
against Candida albicans MTCC 3017 at a dose of 25.14 µM, while Miconazole showed biofilm inhibitory
activity of 26.18 µM against the same strain. Similarly, derivative 13r also inhibited biofilms formed by
Candida albicans MTCC 227 at a concentration of 25.23 µM, which was close to that of Miconazole (24.02
µM). These results recommended that the synthesized derivatives effectively inhibited in vitro biofilms formed
by all the tested Candida strains.
3.6 | Field emission scanning electron microscopic (FE-SEM) studies
The FE-SEM micrographs[37] (Fig. 2) clearly indicated that the control group or untreated Candida
albicans MTCC 3017 hyphal stage cells were enveloped by the secreted biofilm. The micrograph also showed
that the architecture of each Candida cell was intact and protected by the secreted biofilm. In contrast, the
Candida group treated with the compound 13r at its MFC value, showed a clear disruption of biofilm formed by
hyphal stage cells of Candida albicans MTCC 3017, which showed good correlation with the results of MFC
and in vitro biofilm enveloped in the secreted biofilm and individual hyphal stage cells intact in a microcolony,
adhered to each other and showing normal morphology, while Figs. 2c and 2d
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Table 2. Anti-fungal activity of the promising derivatives (13d, 13j, 13l, 13p, 13s) as well as anti-biofilm activity of derivatives, 13l and 13r against various Candida strains
represent Candida biofilms treated with derivative 13r, which showed disruption of Candida biofilm and majority of the hyphal stage Candida cells lost adherence which
either showed disrupted morphology or cell damage with release of intracellular contents or were dead. Thus it can be considered that derivative 13r is a promising inhibitor
of Candida albicans biofilm formation.
Tested fungal strin
Minimum inhibitory concentration (µg/ml)
Biofilm inhibitory concentration (µM)
13d
13j
13p
13l
13r
MCZ
(Std.)
3.9
CLZ
FCZ
13l
13r
MCZ
(Std.)
15.6
7.8
31.2
7.8
15.6
15.6
3.9
7.8
3.9
7.8
15.6
3.9
7.8
7.8
3.9
3.9
3.9
3.9
3.9
47.88 ± 0.15
27.32 ± 0.06
42.36 ± 0.08
42.36 ± 0.08
45.18 ± 0.20
39.91 ± 0.08
23.08 ± 0.22
24.02 ± 0.05
28.06 ± 0.03
26.18 ± 0.08
29.12 ± 0.07
C.albicans MTCC 183
C. albicans MTCC 227
C. albicans MTCC 1637
C. albicans MTCC 3017
C. albicans MTCC 3018
7.8
7.8
3.9
25.23 ± 0.1
38.91 ± 0.17
25.14 ± 0.17
46.15 ± 0.23
7.8
7.8
7.8
3.9
7.8
15.6
15.6
15.6
15.6
3.9
7.8
3.9
15.6
15.6
7.8
15.6
15.6
15.6
15.6
15.6
15.6
15.6
15.6
15.6
15.6
7.8
15.6
15.6
7.8
7.8
7.8
7.8
7.8
7.8
15.6
7.8
7.8
7.8
7.8
7.8
15.6
3.9
3.9
3.9
7.8
3.9
3.9
7.8
7.8
7.8
15.6
7.8
7.8
3.9
3.9
3.9
7.8
15.6
7.8
44.82 ± 0.18
42.18 ± 0.08
43.24 ± 0.18
45.16 ± 0.17
48.19 ± 0.09
47.11 ± 0.17
39.65 ± 0.18
37.15 ± 0.06
39.18 ± 0.02
38.16 ± 0.17
38.17 ± 0.09
39.78 ± 0.07
23.07 ± 0.08
26.18 ± 0.13
23.12 ± 0.17
24.06 ± 0.20
28.01 ± 0.09
22.10 ± 0.11
C. albicans MTCC 4748
C. albicans MTCC 7315
C. parapsilosis MTCC1744
C. glabrata MTCC 3019
C. krusei MTCC 3020
Issatchenkia hanoiensis
MTCC 4755
15.6
15.6
15.6
15.6
15.6
15.6
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3.7 | Inhibition of ergosterol biosynthesis
Most of the antifungals are fungistatic that inhibit the fungal growth for a limited time period. An ideal
antifungal should be able to kill various Candida strains by inhibiting the crucial target site. Ergosterol plays an
important role in maintaining the structural integrity of cell wall of different Candida strains. Since ergosterol is
present only in fungal or Candida cell walls and is absent in the animal cells, it is an attractive target for most of
the antifungal agents like Miconazole, Clotrimazole, Fluconazole, etc. The most important feature of ergosterol
as a target is,= its ability to screen for antifungals that can be both fungicidal and/or fungistatic.[38] The
promising anti-Candida activity exhibited by the synthesized bis-pyrazole hydrazide derivatives led us to further
investigate the effect of these conjugates on the inhibition of ergosterol biosynthesis. Modified sterol
quantification assay[39] was employed to quantify the steady state amounts of ergosterol formed in various
Candida strains. The ergosterol detected was 100% in the control group Candida members. While there was a
decrease in the ergosterol content among the Candida strains treated with the derivatives 13l and 13r and
standard Miconazole drug. As the dose of the synthesized derivatives increased from 0 to 16 µg mL-1, the
concentration of ergosterol and dehydroergosterol lowered.
The UV-visible scans showed a flat line at the effective doses of compounds 13l and 13r, indicating no
detectable levels of ergosterol formed. These results support that the newly synthesized bis-pyrazole linked
hydrazide derivatives inhibited the biosynthesis of ergosterol which showed a perfect correlation with the anti-
biofilm and antifungal activity results. The results to this regard are represented in Fig. 3
a
b
Cell damage and release of
intracellular contents
c
d
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Fig. 2. FE-SEM micrographs of treated and untreated (Control) Candida biofilms.
Fig. 3 Ergosterol inhibition assay for the most promising bis-pyrazole linked hydrazide derivatives
3.8 | Cytotoxicity assay
The cytotoxic evaluation of the promising derivatives 13d, 13j, 13l, 13p and 13r was checked against
normal human lung fibroblast cell line (MRC5, ATCC No. CCL171) and the results to this regard are shown in
Table 3. Based on the results, it was evident that majority of the compounds were moderate to less cytotoxic
with values ranging from 33 µg/ml to 51 µg/ml. However, the conjugates 13d and 13l were found to be more
toxic as compare to the antifungal drugs like miconazole (71.13 µg/ml) and fluconazole (42.84 µg/ml), while
conjugate 13j, 13p and 13r were found to be less toxic than fluconazole but more toxic than miconazole.
Whereas the antifungal activity of many of these conjugates is below 10 μg/mL. These results suggest that the
compounds exhibited 3-5 times lower cytotoxicity to normal cells as compared to the anti-Candida activity and
can be of immense therapeutic potential.
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Table 3. Cytotoxicity assay of most promising bis-pyrazole hydrazide derivatives
Test compounds
Human lung fibroblast cell line
(MRC-5; IC50, µg/ml)
33.28 ± 0.1
50.47 ± 0.5
34.92 ± 0.2
43.65 ± 0.9
51.27 ± 1.2
71.13 ± 1.3
42.84 ± 1.2
13d
13j
13l
13p
13r
Miconazole
Fluconazole
3.9 | IN SILICO PREDICTION
3.9.1 | Toxicity prediction
The web server Protox[40] estimates rodent oral toxicity derived from compounds of known drug
candidates and their toxicity by their toxic fragments or chemical structure and compares the similarity of
structures of synthesized molecules which will be encumbered in the server with database of molecules
consisting previously known toxicity data and identifies the toxic fragments of loaded molecules and their
possible toxicity targets. Toxic doses are calculated as LD50 values in mg per kg body weight. The LD50 is the
median lethal dose defined as the dose at which 50% of test subjects are killed upon exposure to the chemical.
The predicted LD50 (lethal dose) for all the compounds (13d, 13j, 13l, 13p and 13r) were about 1000 mg kg-1
and it falls within the class IV compounds. While the reference antifungal agents, miconazole and fluconazole,
showed LD50 (lethal dose) in the range of 463 mg kg-1 and 1273 mg kg-1, respectively. This toxicity prediction
study demonstrated that the synthesized derivatives were less toxic as compared to miconazole and slightly
more toxic as compared to fluconazole, which suggests that these derivatives can be promising leads for further
investigation.
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Table 4. In silico toxicity prediction studies for most promising derivatives
Entry Predictd Predictd Average
Predictn Toxic
LD50
(mg/kg)
1000
1000
1000
1000
1000
463
Toxicity
class
IV
Similarit
y(%)
accuray
fragments
44.0
44.8
46.4
43.5
42.7
100
54.26%
54.26%
54.26%
54.26%
54.26%
100
Nil
Nil
Nil
Nil
Nil
Nil
Nil
13d
13j
IV
IV
13l
IV
13p
IV
13r
IV
MCZ
FCZ
1271
IV
100
100
3.9.2 | Molecular docking studies
It was observed that the amino acid residues present in the active binding site of the protein 3GW9 can
frequently interact with compounds. The docked poses of the compounds with the protein 3GW9 are shown in
Fig. 4, which clearly demonstrates the binding positions of ligands with the protein. Analysis of the receptor
ligand complex models generated after successful molecular docking of the synthesized compounds with the
3GW9 was done based on the parameters such as H-Bond Energy, Protein-Ligand Interactions, MolDock[41]
Score and Rerank Score of the docked compound within the active site. Protein-ligand interactions like
hydrogen bonds, steric and electrostatic interactions between the ligand and the active site of the receptor were
found to be responsible for mediating the biological activity. The results on H-Bond Energy, MolDock Score,
Rerank Score and Protein-Ligand Interactions are shown in Table 5 and the ligand interactions with the specific
amino acid residues were represented in Fig. 5.
Among all the potent molecules subjected for docking studies, 13j showed highest MolDock Score
followed by 13r, 13l, 13p, 13d and standard drug (MCZ) whereas, 13l showed highest hydrogen bond energy
followed by Miconazole (MCZ), 13d, 13r, 13j and 13p. Meanwhile, by considering the standard drug as
control, the binding affinities were calculated from the docking results and the samse are represented in the
Table S3 (Supplementary Information). When compared to the standard 13j, 13r, 13l, 13p and 13d showed
32%, 22%, 17%, 13% and 12% more binding affinity, respectively. Fig. 6 illustrates the protein-ligand
interaction stability in terms of their binding affinity of the potent molecules in comparison with Miconazole.
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Table 5. Protein-ligand interactions of the most promising bis-pyrazole hydrazides with the target 3GW9
Liga
nd
Mol
Rerank
Score
H-Bond Protein-Ligand Interactions
Energy
Dock
Score
-167.
938
-129.
191
-5.000
Tyr116, Leu130, Ala291,
Ala288, Gly424, Gly428,
Ser296
13d
13j
Ala288,
Ser296
-196.
4
-160
.953
-4.054
His420, Leu356, Gly414,
Pro355, Thr299, Gly428,
Ser296, Leu134, Ala291,
Ile423, HOH526[A]
Thr299,
Ser296,
HOH52
6[A]
-175.
08
-134
.017
-5.844
His420, Cys422, Ser296,
Thr299, Gly424, Gly428,
Ile350, Phe415, Thr295
13l
Cys422,
Ser296,
Thr299
-169.
192
-112.
64
-3.255
Ala115, Phe110, Met284,
Ala288, Ser296, Gly424,
Gly428, Phe415, Thr295
13p
13r
Ala288,
Ser296
-182
.85
-154.
025
-4.662
His420, Cys422, Gly414,
Ile350, Phe415, Gly292,
Thr299, Ser296, Thr295,
HOH526[A]
Thr299,
Ser296,
Thr295,
HOH52
6[A]
-148.
67
-122.
40
-5.573
Ser296, Gly424, Ala288,
Leu134, Ile423, Cys422
MCZ
Ile423,
Cys422
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Fig. 6. Protein-ligand interaction stability/binding affinity of
the promising bis-pyrazole hydrazides as compared to the standard Miconazole
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Fig. 4. All the promising bis-pyrazole hydrazides docked ligand poses in the target active site
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Fig. 5. Protein-ligand interactions of the promising bis-pyrazole hydrazides with 3GW9
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4 | CONCLUSIONS
In summary, a series of bis-pyrazole hydrazide conjugates 13(a-s) were synthesized and evaluated for
their antimicrobial activity which exhibited very promising anti-Candida and antibacterial activities. Most
promising compounds like 13d, 13j, 13l, 13p and 13r from the series were tested for their biofilm inhibition
assay, among which 13l and 13r inhibited Candida biofilms efficiently in the range of 27.32 µM and 25.14 µM
which was almost similar to the Miconazole (standard) having value of 24.02 µM and 26.18 µM with the same
Candida strain, respectively. In addition, the biofilm inhibition profile was further supported by FE-SEM
images. The inhibition of ergosterol biosynthesis in Candida strains elucidated the mode of their action which
was further validated with the help of docking studies. Furthermore, in silico studies revealed a good
pharmacokinetic profile, binding affinity and drug likeliness properties for the synthesized bis-pyrazole
hydrazide hybrids
Supporting Information Summary
The detailed biological experimental section, 1H and 13C NMR,HRMS spectra and ADME pharmacoprediction
studies are provided in the supporting information
Acknowledgments
The authors would like to acknowledge UGC, New Delhi for the award of research fellowship to Mr.
Irfan Khan. Authors would also like to thank Council of Scientific
and Industrial Research (CSIR), Government of India for financial support under the 12th Five Year plan (2012-
17) project “Affordable Cancer Therapeutics” (CSC0301).
Conflicts of interest
There are no conflicts of interest to declare.
Figure Legends
Fig. 1. Structures of some antimicrobial agents
Fig. 2. FE-SEM micrographs of treated and untreated (Control) Candida biofilms.
Fig. 3 Ergosterol inhibition assay for the most promising bis-pyrazole linked hydrazide derivatives
Fig. 4. All the promising bis-pyrazole hydrazides docked ligand poses in the target active site
Fig. 5. Protein-ligand interactions of the promising bis-pyrazole hydrazides with 3GW9
Fig. 6. Protein-ligand interaction stability/binding affinity of the promising bis-pyrazole hydrazides as compared
to the standard Miconazole
Scheme 1. Synthesis of bis-pyrazole hydrazide derivatives 13(a-s)
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