Synthesis, molecular docking and in vitro antimicrobial studies of new hexahydroindazole derivatives of curcumin

Article abstract of DOI:10.2174/157018013804725161

A series of hexahydroindazole analogues of curcumin were synthesized and investigated for in vitro and in silico antimicrobial activity. The structures of synthesized compounds were identified on the basis of satisfactory analytical and spectral data (¹H NMR, ¹³C NMR, EI-MASS techniques and elemental analysis). Synthesized compounds showed moderate to high activity against both bacterial and fungal strains. All compounds were docked computationally to the active site of enzyme L-glutamine: D-fructose-6-phosphate amido-transferase [GlcN-6-P] (EC 2.6.1.16). The autodock programme 4.0 was employed to perform automated molecular docking. (E)-1-(7-(3-methoxybenzylidene) -3-(3- methoxyphenyl)-3,3a,4,5,6,7-hexahydro-2H-indazol-2-yl)ethanone (A7) turned out to be the most potent analogue of the series, showing best activity against bacterial and fungal strains. Compound A7 showed minimum binding and docking energy and may be considered as good inhibitor of GlcN-6-P synthase. Further investigation and optimization of this lead could provide new antimicrobial molecules.

Full text of DOI:10.2174/157018013804725161

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Letters in Drug Design & Discovery, 2013, 10, 119-128
119
Synthesis, Molecular Docking and In Vitro Antimicrobial Studies of New
Hexahydroindazole Derivatives of Curcumin
Dileep Kumar1, Harish B.G2, Mayank Gangwar3, Manish Kumar1, Dharmendra Kumar4,
RaginiTilak4, Gopal Nath4, Ashok kumar*,1 and Sushil Kumar Singh*,1
1Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutics, Indian Institute of Technology,
2
(BHU), Varanasi- 221005, India; Department of Biotechnology, M.S. Ramaiah Institute of Technology, MSR Nagar,
Bengaluru, Karnataka, India; 3Department of Pharmacology, Institute of Medical Sciences, BHU, Varanasi,
India;4Department of Microbiology, Institute of Medical Sciences, BHU, Varanasi, India
Abstract: A series of hexahydroindazole analogues of curcumin were synthesized and investigated for in vitro and in
silico antimicrobial activity. The structures of synthesized compounds were identified on the basis of satisfactory
analytical and spectral data (1H NMR, 13C NMR, EI-MASS techniques and elemental analysis). Synthesized compounds
showed moderate to high activity against both bacterial and fungal strains. All compounds were docked computationally
to the active site of enzyme L-glutamine: D-fructose-6-phosphate amido-transferase [GlcN-6-P] (EC 2.6.1.16). The
autodock programme 4.0 was employed to perform automated molecular docking. (E)-1-(7-(3-methoxybenzylidene)-3-(3-
methoxyphenyl)-3,3a,4,5,6,7-hexahydro-2H-indazol-2-yl)ethanone (A7) turned out to be the most potent analogue of the
series, showing best activity against bacterial and fungal strains. Compound A7 showed minimum binding and docking
energy and may be considered as good inhibitor of GlcN-6-P synthase. Further investigation and optimization of this lead
could provide new antimicrobial molecules.
Keywords: Autodock 4.0; Bacterial strain; Fungal strains; GlcN-6-P synthase; Hexahydroindazole; In silico.
INTRODUCTION
antimicrobial studies. The enzyme catalyses uridine 5’di-
phospho-N-acetyl-D-glucosamine, the first and practically
irreversible step in the pathway of hexosamine metabolism
end-product, and serves as an activated form of N-acetyl-
glucosamine. This amino sugar is incorporated into several
macromolecules viz. peptidoglycan, lipopolysacc-harides
and teichoic acids in bacteria, chitin in fungi, insects and
crustaceans, and glycoproteins, glycosaminoglycans and
mucopolysaccharides in mammals. All these molecules are
essential for the assembly of the cell wall [5].ꢀSeveral GlcN-
6-P inhibitors, exhibiting antimicrobial activity have been
reported, from both natural and synthetic origin.
In recent years, a number of antibiotics have lost their
effectiveness due to the development of resistant strains.
Extremely resistant bacteria such as methicillin-resistant
Styphlococoous aureus (MRSA) and vancomycin-resistant
Enterococci accounts for a soaring percentage of hospital
acquired infections [1]. Multidrug resistance (MDR) to
antibiotics presents a serious therapeutic problem in the
treatment of bacterial infections. The importance of
mechanism of resistance in clinical settings is reflected in
increasing number of reports of multidrug resistant isolates
[2]. In addition to this problem, antibiotics are sometimes
associated with adverse effects including hypersensitivity,
immune-suppression allergic reactions etc. Therefore, there
is a need to develop novel antimicrobial drugs for the
treatment of infectious diseases. The search for not only the
improved versions of existing drugs but also new drug
targets have become an urgent need. Large number of
marketed drugs have enzymes or membrane receptors as
molecular targets [3]. In most cases, elucidating the
intermolecular interactions in these drug-target complexes
was a pre-requisite of the success [4].
Curcumin, a naturally occurring polyphenol is extracted
from the rhizomes of Curcuma longa. It has been used as an
important dietary component for long time. It exhibits
various biological activities including antiproliferative
activity against various cancer cells, antioxidant activity,
wound healing ability and antimicrobial activity. Curcumin
possesses antibacterial property against a number of Gram-
positive and Gram-negative bacteria. It has been shown to
kill several pathogenic Gram-positive bacteria such as
Staphylococcus aureus, Staphylococcus epidermidis and
Enterococcus that cause skin infections, pneumonia,
meningitis and urinary tract infections in human being.
Understanding the mechanism of the antibacterial activity of
curcumin will greatly assist in designing potent analogues of
curcumin, a natural remedy for several bacterial diseases [6-
10].
The enzyme, L-glutamine:D-fructose-6-phosphateamido-
transferase, known under the trivial name of glucosamine-6-
phosphatesynthase (EC 2.6.1.16) is a new target for
*Address correspondence to these authors at the Pharmaceutical Chemistry
Research Laboratory, Department of Pharmaceutics, Indian Institute of
Technology, (BHU), Varanasi- 221005, India; Tel.: +91-542-6702736;
Fax: +91-542-368428; E-mail: sksingh.phe@iitbhu.ac.in
Department of Pharmaceutics, Indian Institute of Technology, (BHU),
Varanasi-221005, India; Tel: 0542-6702815, 09336915232;
Compounds containing hexahydroindazole are biolo-
gically active and have been reported as potent antimicrobial,
anti-inflammatory, depressant of central nervous system and
additionally some are also found to be monoamine oxidase
inhibitors [11-12].
Fax No. 0542-2316428; E-mail: akmourya.rs.phe@itbhu.ac.in
1570-1808/13 $58.00+.00
© 2013 Bentham Science Publishers

120 Letters in Drug Design & Discovery, 2013, Vol. 10, No. 2
Kumar et al.
O
O
(1)
CHO
R
R
R
+
(3)
(2)
O
N
N
N
N
R
R
R
R
Scheme 1. Synthesis of hexahydroindazole derivatives of Curcumin (A1-A9) and (B1-B5). Reagents and conditions: (1) 10 % NaOH, EtOH,
H2O; (2) Hydrazine hydrate, Glacial acetic acid, 12 h reflux; (3) Phenyl hydrazine, Glacial acetic acid, 12 h reflux.
Minu et al. reported synthesis and antimicrobial activity
LC-MS-2010A spectrometer and mass values are reported in
m/z. Elemental analysis of synthesized compounds were
recorded on EXETER-CE-440 elemental analyzer.
of
2,3-disubstituted-3,3a,4,5,6,7-hexahydro-2H-indazole
derivatives. They observed that the compounds with electron
withdrawing groups were generally more active than other
derivatives. Among the electron withdrawing halogen
groups, presence of p-flurophenyl group at third position of
hexahydroindazole improved the antibacterial activity.
Whereas, the presence of p-chlorophenyl group at third
position of hexahydroindazole improved the antifungal
activity [13].
Molecular Docking Studies
Automated docking was used to determine the orientation
of inhibitors bound to the active site of GlcN-6-P synthase.
A genetic algorithm method, implemented in the program
AutoDock 4.0, was employed [15]. The 3D structure file of
all the curcumin analogues and fluconazole molecule were
loaded on to PRODRG server [16] and PreADMET server
for energy minimization and drug likeliness prediction
respectively. The protein structure file 1Jxa was downloaded
from Protein Data Bank (www.rcsb.org/pdb) was edited by
removing the hetero atoms, adding C-terminal oxygen [17].
For docking calculations, Gasteigere-Marsili partial charges
[18] were assigned to the ligands and non-polar hydrogen
atoms were merged. All torsions were allowed to rotate
during docking. The grid map was centered at the residues of
the protein predicted from the CASTp server [19]. The
Lamarckian genetic algorithm and the pseudo-Solis and
Wets methods were applied for minimization using default
parameters. The number of docking runs was 50, the
population in the genetic algorithm was 250, the number of
energy evaluations was 100,000, and the maximum number
of iterations was 10,000. The docking results for ligand
molecules against glucosamine-6-phosphate synthase [PDB
Id: 1jka], showed minimum docking energy, binding energy,
inhibition constant, intermolecular energy with 0.0 RMS as
documented. Computer with Microsoft Windows XP
operating system, Intel Pentium 3.40 GHz processor, 1 GB
RAM and Hard disk: 500 GB, Python: 2.4 was used.
Synthesis of furfurylidene containing hexahydroin-
dazoles with different pharmacophore fragments (furan and
pyrazoline cycles, nitro-, azomethine, and other groups) was
reported by Golikov et al. They observed that nitrofuran
cycle linked to the pyrazoline ring containing azomethine
group via avinilydene unit was most potent against gram
positive bacteria. While, other substitutions (replacement of
nitro group in furan by a methyl group or hydrogen) to
hexahydroindazole resulted in diminished activity against all
the tested organisms [14].
Present study includes the synthesis, molecular docking
and antimicrobial studies of some novel hexahydroindazole
derivatives of curcumin and attempt has been made to
ascertain the role of various substituents on aryl ring with
respect to the antibacterial activity.
MATERIALS AND METHODS
All reagents were obtained from commercial suppliers
and were used without further purification. Reaction
progress was monitored by thin layer chromatography (TLC)
on pre-coated Merck alufoil plates (silica gel 60F-254, 0.25
mm thickness). Melting points were determined on a Veego
1
capillary melting point apparatus and are uncorrected. H
Synthesis
NMR spectra were recorded on a Bruker 300 MHz
spectrophotometer. All NMR spectra were obtained in
deuterated chloroform (CDCl3); chemical shifts are reported
in parts per million, and coupling constants in hertz (Hz).
Multiplicities are reported as follows: s (singlet), d (doublet),
t (triplet), m (multiplet). Mass spectra were recorded on a
The compounds were prepared by coupling substituted
benzaldehydes with cyclohexanone (2:1) in a base catalyzed
Claisen-Schmidt condensation followed by reflux with
hydrazines (hydrazine hydrate, phenyl-hydrazine) and acetic
acid (scheme 1, Table 1).

Synthesis, Molecular Docking and Antimicrobial Studies
Letters in Drug Design & Discovery, 2013, Vol. 10, No. 2 121
Table 1. Different substitutions on aryl ring of synthesized compounds.
O
O
H
A1
A8
2,3,4
triOCH3
N
N
OCH3
OCH3
N
N
H3CO
H3CO
OCH3
OCH3
O
O
2-Cl
A2
4-N(CH3)2
A9
N
N
Cl
N
N
Cl
N
N
O
3-OH
4-Cl
A3
A4
3-Cl
4-Cl
B1
B2
N
N
N
N
HO
OH
Cl
Cl
O
N
N
N
N
Cl
Cl
Cl
Cl
O
2,4-diCl
A5
A6
2,4-diCl
B3
Cl
N
N
Cl
Cl
Cl
N
N
Cl
Cl
Cl
Cl
O
3-NO2
4-OCH3
B4
B5
N
N
N
N
O2N
NO2
H3CO
OCH3
O
3-OCH3
A7
4-N(CH3)2
N
N
N
N
H3CO
OCH3
N
N
Synthesis of E-1-(7-(substituted bezylidene)-3-(substitu-
ted phenyl)-3,3a,4,5,6,7-hexahydro 2 H indazole-2yl)
Ethanones (A1-A9)
reaction mixture was further stirred for 45 minutes. The solid
separated was filtered off, washed with distilled water, and
subjected to further purification by recrystallization with
ethyl alcohol.
These were prepared in two steps. In First step,
respective dibenzylidiene cyclohexanones were synthesized.
A mixture of ethanol (20ml) and sodium hydroxide solution
(10%, 10 ml) was taken in a beaker, maintained at
temperature between 15 to 25ºC. The solution was
vigorously stirred and one half of previously prepared
mixture of appropriate aromatic aldehyde and cyclohexanone
(2:1) was added to it. After 20 min, the remaining of the
aromatic aldehyde-cyclohexanone mixture was added. The
In second step, synthesized substituted dibenzylidene
cycloheaxanone (1.2 mmol) from first step was dissolved in
glacial acetic acid (5 mL), and hydrazine hydrate (1.5 mmol)
was added to the solution. The solution was refluxed for 12
hours and reaction was monitored by TLC .The solvent was
removed in vacuum and the residue was recrystallized with
ethyl alchol. All the derivatives were also prepared by the
same procedure.

122 Letters in Drug Design & Discovery, 2013, Vol. 10, No. 2
Kumar et al.
(E)-1-(7-benzylidene-3-phenyl-3,3a,4,5,6,7-hexahydro-2H-
(E)-1-(7-(3-nitrobenzylidene)-3-(3-nitrophenyl)-3,3a,4,5,6-
indazol-2-yl)ethanone (A1):
,7-hexahydro-2H-indazol-2-yl) ethanone (A6):
1
Yellow powder, mp 130-1320 C. H NMR (CDCl3, 300
Yellow powder, mp 90-920C.1H NMR (CDCl3, 300
MHz, 25 °C): 7.61-7.25 (m, 10H), 6.31 (s, 1H, =CH), 4.72
(d, 1H), 2.67 (m, 1H), 2.12 (s, 3H, CH3), 1.81-1.42 (m, 6H).
13C NMR (CDCl3, 75 MHz, 25 °C): 166.9, 154.4, 141.7,
136.0, 134.9, 131.9, 128.5, 127.6, 127.1, 127.9, 126.3, 70.2,
41.8, 27.5, 24.8, 23.7. ESI-MS: m/z: (M+, %) 330.14 (M+
6%), 329(97%), 200(100%). Elemental analysis found
(Calc.) for C22H22N2O: C, 79.97 (79.65); H, 6.71 (6.73); N,
8.48(8.45).
MHz, 25 °C): 8.29-7.54 (m, 8H), 6.42 (s, 1H, =CH), 4.4 (d,
1H), 2.63 (m, 1H), 2.02 (s, 3H, CH3), 1.81-1.43 (m, 6H). 13
C
NMR (CDCl3, 75 MHz, 25 °C): 167.5, 154.6, 146.8, 146.2,
140.4, 136.1, 135.3, 134.6, 134.2, 129.5, 129.3, 128.7, 123.8,
122.7, 121.7, 120.7, 69.7, 42.8, 27.8, 24.5, 23.4. ESI-MS:
m/z: 420(M+ 12%),.418(100%). Elemental analysis found
(Calc.) for C22H20 N4O5 : C, 62.85(62.60); H,4.79 (4.80); N,
13.33(13.28) .
(E)-1-(7-(3-methoxybenzylidene)-3-(3-methoxyphenyl)-3,3-
a,4,5,6,7-hexahydro-2H-indazol-2-yl)ethanone (A7):
(E)-1-(7-(2-chlorobenzylidene)-3-(2-chlorophenyl)-3,3a,4,-
5,6,7-hexahydro-2H-indazol-2-yl)ethanone (A2):
Yellow powder, mp 110-1120 C.1H NMR (CDCl3, 300
MHz, 25 °C): 7.49-6.21 (m,8 H), 6.11 (s, 1H, =CH), 4.1 (d,
1H), 3.75 (s, 6H), 2.68 (m, 1H), 2.04 (s, 3H, CH3), 1.78 -1.32
(m, 6H). 13C NMR (CDCl3, 75 MHz, 25 °C): 169.5, 161.5,
160.4, 155.6, 142.5, 136.0, 134.8, 128.6, 126.5, 120.8, 120.4,
112.5, 112.0, 111.5, 71.2, 55.8, 42.8, 27.1, 24.5, 23.4. ESI-
MS: m/z: 390((M+ 8%), 389(26%), 212(100%). Elemental
analysis found (Calc.) for C24H26N2O3 : C, 73.82(73.63); H,
6.71(6.72); N, 7.17(7.16).
Yellow powder, mp 102-1040C.1H NMR (CDCl3, 300
MHz, 25 °C): 7.72-7.20 (m, 8H), 6.69 (s, 1H, =CH), 4.5 (d,
1H), 2.67 (m, 1H), 2.02 (s, 3H, CH3), 1.85-1.43 (m, 6H). 13
C
NMR (CDCl3, 75 MHz, 25 °C): 166.5, 154.6, 141.5, 136.5
134.7, 133.5, 133.1, 130.7, 129.5, 129.3, 129.3, 128.2, 127.1,
127.0, 126.6, 126.4, 65.9, 42.4, 23.3. ESI-MS: m/z: (M+, %)
398.07. (M+ 9%), 397(22%), 220(100%). Elemental analysis
found (Calc.) for C22H20Cl2N2O : C, 66.17(66.43); H,
5.05(5.03); N, 7.02(7.30).
(E)-1-(7-(2,3,4-trimethoxybenzylidene)-3-(2,3,4-trimethoxy-
phenyl)-3,3a,4,5,6,7-hexahydro-2H-indazol-2-yl)ethanone
(A8):
(E)-1-(7-(3-hydroxybenzylidene)-3-(3-hydroxyphenyl)-3,-
3a,4,5,6,7-hexahydro-2H-indazol-2-yl)ethanone (A3):
Yellow powder, mp 166-1680 C.1H NMR (CDCl3, 300
MHz, 25 °C): 7.52-6.51 (m, 8 H), 6.31 (s, 1H, =CH), 5.30 (s,
2H), 4.6 (d, 1H), 2.55 (m, 1H), 2.32 (s, 3H, CH3), 1.75-1.37
(m, 6H). 13C NMR (CDCl3, 75 MHz, 25 °C): 167.2, 158.9,
156.6, 155.5, 140.7, 135.6, 135.1, 131.7, 130.4, 129.7, 121.8,
120.4, 115.6, 113.4, 113.2, 22.2. ESI-MS: m/z: (M+, %) 362
(M+ 7%),361(96%), 221( 100%). Elemental analysis found
(Calc.) for C22H22N2O3 : C, 72.91(72.71); H,6.12 (6.09); N,
7.73(7.76).
Yellow powder, mp 210-2120 C.1H NMR ( CDCl3 , 300
MHz, 25 °C): 7.47 -6.25 (m,4 H), 6.59 (s, 1H, =CH), 4.4 (d,
1H), 3.75 (s, 9H), 2.68 (m, 1H), 2.04 (s, 3H, CH3), 1.80-1.35
(m, 6H). 13C NMR (CDCl3, 75 MHz, 25 °C): 168.5, 156.8,
155.9, 152.9, 151.1, 149.1, 142.3, 141.6, 136.0, 135.7, 132.7,
122.7, 121.4, 119.6, 108.2, 106.8, 104.7, 65.3, 61.7, 60.8,
60.3, 56.1, 43.1, 27.8, 24.5, 23.4. ESI-MS: m/z: 510(M+
12%), 507(20%), 215(100%). Elemental analysis found
(Calc.) for C28H34N2O7: C, 65.87(65.92); H, 6.71(6.70); N,
5.49(5.48).
(E)-1-(7-(4-chlorobenzylidene)-3-(4-chlorophenyl)-3,3a,4,-
5,6,7-hexahydro-2H-indazol-2-yl)ethanone (A4):
(E)-1-(7-(4-(dimethylamino)benzylidene)-3-(4-(dimethyla-
mino)phenyl)-3,3a,4,5,6,7-hexahydro-2H-indazol-2-
yl)ethanone (A9) :
Yellow powder, mp 126-1280 C.1H NMR (CDCl3, 300
MHz, 25 °C): 7.66-7.58 (m,8 H), 6.27 (s, 1H, =CH), 4.2 (d,
Yellow powder, mp 150-1520 C.1H NMR (CDCl3, 300
MHz, 25 °C): 7.64 -6.56 (m,10 H), 6.25 (s, 1H, =CH), 4.7 (d,
1H), 3.78 (s, 12 H), 2.68 (m, 1H), 2.54 (s, 3H, CH3), 1.81-
1.36 (m, 6H). 13C NMR (CDCl3, 75 MHz, 25 °C): 167.5,
154.6, 151.3, 149.3, 133.7, 132.1, 131.4, 128.7, 124.7, 112.7,
111.7, 72.9, 41.3, 26.8, 24.5, 23.4. ESI-MS: m/z: 416(M+ 9
%), 415(21%), 221(100%). Elemental analysis found (Calc.)
for C26H32N4O: C, 74.97(74.96); H, 7.74(7.72); N,
13.45(13.43).
1H), 2.65 (m, 1H), 2.01 (s, 3H, CH3), 1.77-1.45 (m, 6H). 13
C
NMR (CDCl3, 75 MHz, 25 °C): 168.5, 155.6, 139.7, 136.0,
133.3, 131.5, 129.0, 128.6, 128.4, 70.9, 42.8, 27.8, 24.5,
21.4. ESI-MS: m/z: (M+,%) 398 (M+ 12%),
397(47%),229(100%). Elemental analysis found (Calc.) for
C22H20 Cl2N2O2 : C, 63.62(63.36); H,4.85 (4.86); N,6.75
(6.77).
(E)-1-(7-(2,4-dichlorobenzylidene)-3-(2,4-dichlorophenyl)-
3,3a,4,5,6,7-hexahydro-2H-indazol-2-yl)ethanone (A5):
Synthesis of E-7-(substituted benzylidene)-2-phenyl-3-
(substituted phenyl)-3, 3a, 4,5,6,7 hexahydro 2H inda-
zoles (B1-B5)
Yellow powder, mp 152-1540 C.1H NMR (CDCl3 , 300
MHz, 25 °C): 7.71-7.02 (m, 6H), 6.56 (s, 1H, =CH), 4.7 (d,
1H), 2.66 (m, 1H), 2.02 (s, 3H, CH3), 1.85-1.23 (m, 6H). 13
C
In the first step, the dibenzylidene cycloheaxanones were
synthesized as reported. The synthesized dibenzylidene
cycloheaxanon(1.2 mmol) from first step was dissolved in
glacial acetic acid (5 mL) and phenyl hydrazine (1.5mml)
was added to the solution. The solution was refluxed for 12
hours and monitored by TLC. Solvent was removed in
vacuum and recrystallized with ethlyl alcohol. The
NMR (CDCl3, 75 MHz, 25 °C): 163.5, 152.6, 150.1, 137.6,
136.8, 132.4, 132.1, 131.4, 131.0, 130.3, 127.9, 126.8, 126.5,
125.2, 120.8, 75.8, 42.1, 27.9, 24.3, 23.1. ESI-MS: m/z: (M+,
%) 468(M+ 12%), 467(22%), 221(100%). Elemental analysis
found (Calc.) for C22H18 Cl4N2O : C, 56.44(56.66); H,3.88
(3.83); N, 5.98 (5.96).

Synthesis, Molecular Docking and Antimicrobial Studies
Letters in Drug Design & Discovery, 2013, Vol. 10, No. 2 123
Table 2. Antimicrobial activity of new hexahydroindazole derivatives of curcumin.
Bacterial Strains
Fungal Strains
Zone of inhibition (in mm)
C. tropicalis ATCC
Comp
no.
Zone of inhibition (in mm)
E.coli
S. typhi
K.
S. aureus
E. faecalis
C. krusie
C. albicans
ATCC 90028
750
ATCC 35218
MTCC 3216
pneumonia
ATCC 25323
A1
A2
A3
A4
A5
A6
A7
A8
A9
B1
B2
B3
B4
B5
Cip
Fluc
NA
NA
14.37±0.12
14.93±0.18
NA
NA
14.87±0.15
17.63±0.09
NA
10.37±0.38
13.20±0.17
16.77±0.03
11.73±0.38
11.70±0.12
10.23±0.15
22.53±0.09
17.0±0.16
10.03±0.18
7.10±0.17
6.77±0.15
NA
9.70±0.36
15.83±0.19
14.57±0.15
10.50±0.21
NA
NA
NA
12.53±0.18
11.30±0.15
NA
NA
12.83±0.15
11.20±0.15
NA
15.60±0.26
13.30±0.25
9.40±0.12
8.93±0.09
8.07±0.09
21.43±0.18
19.67±0.11
10.90±0.06
13.93±0.09
8.07±0.06
7.87±0.10
9.33±0.07
8.90±0.08
26.23±0.16
NA
14.80±0.25
16.27±0.23
13.37±0.09
12.67±0.09
NA
NA
10.27±0.09
NA
NA
NA
NA
9.03±0.12
20.33±0.15
15.23±0.20
12.10±0.20
6.63±0.09
4.07±0.18
NA
NA
NA
17.20±0.17
15.10±0.15
NA
20.40±0.11
16.80±0.17
NA
23.0±0.12
20.33±0.12
11.93±0.09
7.63±0.05
NA
21.50±0.12
18.20±0.17
NA
19.77±0.15
17.87±0.19
12.47±0.07
NA
NA
8.87±0.13
NA
5.73±0.12
NA
NA
NA
NA
NA
6.70±0.13
NA
NA
5.93±0.09
NA
8.33±0.09
NA
NA
5.57±0.12
NA
4.87±0.09
4.17±0.15
22.50±0.09
NA
NA
NA
NA
NA
3.30±0.17
NA
19.98±0.06
NA
26.71±0.08
NA
27.44±0.15
NA
NA
NA
25.33±0.05
24.65±0.05
24.06±0.04
The value of each compound consisted of Mean ± SE of 03 replicates.
Level of significance p < 0.05
remaining derivatives were also prepared by the same
procedure.
(E)-7-(2,4-dichlorobenzylidene)-3-(2,4-dichlorophenyl)-2-
phenyl-3,3a,4,5,6,7-hexahydro-2H-indazole (B3):
(E)-7-(3-chlorobenzylidene)-3-(3-chlorophenyl)-2-phenyl-
3,3a,4,5,6,7-hexahydro-2H-indazole (B1):
Yellow powder, mp 70-720 C.1H NMR (CDCl3, 300
MHz, 25 °C): 7.58-6.75 (m, 13H), 6.59 (s, 1H, =CH), 3.8 (d,
1H), 2.27 (m, 1H), 1.87-1.30 (m, 6H). 13C NMR (CDCl3, 75
MHz, 25 °C): 154.6, 150.5, 141.8, 138.6, 136.0, 133.8,
133.1, 131.7, 131.1, 130.3, 129.4, 128.9, 128.1, 126.7, 120.8,
116.7, 58.0, 41.4, 28.7, 24.5. ESI-MS: m/z: 502(M+ 3%),
501(15%), 225(100%). Elemental analysis found (Calc.) for
C26H20Cl4N2 : C, 62.17(62.31); H, 4.01(4.02); N, 5.58(5.56).
Yellow powder, mp 58-600 C.1H NMR (CDCl3, 300
MHz, 25 °C): 7.34-6.75 (m, 13 H), 6.24 (s, 1H, =CH), 3.7 (d,
1H), 2.25 (m, 1H), 1.78-1.43 (m, 6H). 13C NMR (CDCl3, 75
MHz, 25 °C): 145.6, 143.8, 140.9, 136.0, 134.9, 134.2,
134.1, 130.0, 129.9, 129.5, 128.0, 126.9, 126.2, 119.7, 62.6,
40.5, 26.8, 24.8, 23.5. ESI-MS: m/z: 432(M+ 7%),
430(100%). Elemental analysis found (Calc.) for
C26H22Cl2N2: C, 72.06(71.97); H, 5.12(5.11); N, 6.46(6.52).
(E)-7-(4-methoxybenzylidene)-3-(4-methoxyphenyl)-2-phe-
nyl-3,3a,4,5,6,7-hexahydro-2H-indazole (B4):
(E)-7-(4-chlorobenzylidene)-3-(4-chlorophenyl)-2-phenyl-
Yellow powder, mp 80-820 C.1H NMR (CDCl3, 300
MHz, 25 °C): 7.52-6.84 (m,8 H), 6.32 (s, 1H, =CH), 3.5 (d,
1H), 3.73 (S, 6H), 2.25 (m, 1H), 1.84-1.42 (m, 6H). 13C
NMR (CDCl3, 75 MHz, 25 °C): 158.8, 156.8, 144.8, 134.0,
131.8, 131.2, 130.2, 129.5, 127.8, 127.5, 121.8, 116.7, 113.2,
112.1, 63.1, 55.8, 42.9, 27.8, 24.8, 24.5. ESI-MS: m/z:
424(M+ 4%), 215(100%). Elemental analysis found (Calc.)
for C28H28N2O2: C, 79.22(79.43); H, 6.65(6.66); N,
6.60(6.62).
3,3a,4,5,6,7-hexahydro-2H-indazole (B2):
Yellow powder, mp 78-800 C.1H NMR (CDCl3, 300
MHz, 25 °C): 7.78-6.73 (m, 13 H), 6.23 (s, 1H, =CH), 3.5 (d,
1H), 2.26 (m, 1H), 1.82-1.41 (m, 6H). 13C NMR (CDCl3, 75
MHz, 25 °C): 154.6, 143.8, 138.6, 136.0, 133.5, 133.3,
131.7, 131.5, 129.0, 129.5, 128.6, 128.4, 116.7, 63.1, 42.9,
28.6, 27.8, 24.8, 24.5. ESI-MS: m/z: 432(M+ 6%),
430(100%). Elemental analysis found (Calc.) for
C28H24Cl2N2: C, 73.20(73.37); H, 5.27(5.28); N, 6.10(6.11).

124 Letters in Drug Design & Discovery, 2013, Vol. 10, No. 2
Kumar et al.
Fig. (1). Antimicrobial activity of hexahydroindazole derivatives of Curcumin (a) antifungal activity against candida albicans with refrence
to standard drug Fluconazole, A7 showing the strongest inhibition compare to A3 and A8 (b) antifungal activity against C.tropicalis, A7
showing clear zone of inhibition as compare to A5, B5 and A2 (c) Antibacterial against S.aureus with reference to standard drug
Ciprofloxacin, A3, A8 and A7 showing inhibition much better than B3, B2,and B4 (d) antibacterial against S.typhi, A3, A5, A8, A7, A2, A5
and B4 showing inhibition zone (e) antibacterial against E.faecalis with reference to Ciprofloxacin as standard, A5, B4, and B1 showing less
inhition compare to standard (f) antibacterial activity against E-coli, A7 and A3 showing good inhibition as compared to standard
Ciprofloxacin.
(E)-4-(7-(4-(dimethylamino) benzylidene)-2-phenyl-3, 3a,
4, 5, 6, 7-hexahydro-2H-indazol-3-yl)-N,N-dimethylaniline
(B5):
fungal strains of Candida according to the guidelines of
National Committee for Clinical Laboratory Standards
(NCCLS, 1997).
Yellow powder, mp 178- 1790 C.1H NMR (CDCl3, 300
MHz, 25 °C): 7.62-7.01 (m, 13 H), 6.24 (s, 1H, =CH), 3.1 (d,
1H), 3.07 (s, 12 H) 2.31 (m, 1H), 1.78 -1.20 (m, 6H). 13C
NMR (CDCl3, 75 MHz, 25 °C): 154.2, 150.1, 146.3, 143.8,
136.7, 130.7, 130.4, 130.0, 129.7, 129.5, 128.9, 128.4, 127.1,
126.9, 116.7, 112.7, 111.7, 63.1, 42.9, 40.3, 27.8, 24.8, 24.5.
ESI-MS: m/z: 450(M+ 3%), 135(100%). Elemental analysis
found (Calc.) for C30H34N4 : C, 79.96(80.02); H, 7.61(7.62);
N, 12.43(12.41).
Freshly grown microbial strains were mixed in sterile
saline (0.85%) and the turbidity was matched with
McFarland No. 2 system to achieve concentration of 107
CFU/ml. Sterile petri plates containing 20 mL of Mueller
Hinton agar (MHA, Hi-Media) were used for all bacterial
culture and Sabouraud’s dextrose agar (SDA)/Potato
dextrose agar (PDA) (Hi-Media) were used for all fungal
culture. The bacterial inoculums’ suspension was spread on
the surface of agar plates. Sterile disc (5mm) of Whatman
paper no. 1 was then placed on the surface of the media and
the test compounds (25ꢀl/ml) were put and allowed to
diffuse and plates were incubated for 24 h at 37ºC for
bacterial cultures and 72 h at 25 ºC for fungal culture.
Ciprofloxacin (5ꢀg/disc, Hi-Media) was used as positive
control for bacteria and Fluconazole (10ꢀg/disc, Hi-
Media), was used as a positive control for fungi. Zone of
Antimicrobial Studies
Antimicrobial activities of newly synthesized compounds
were first screened by disc diffusion method [20] against
various Gram positive and Gram negative human pathogenic
bacteria viz. Escherichia coli ATCC 25922, Pseudomonas
aeruginosa ATCC 27893, S. typhi MTCC 3216, E. faecalis
and Staphylococcus aureus ATCC 25323 and different

Synthesis, Molecular Docking and Antimicrobial Studies
Letters in Drug Design & Discovery, 2013, Vol. 10, No. 2 125
Table 3. Theoretical prediction of different properties of new hexahydroindazole derivatives of curcumin using PreADMET
Server.
Water solubility
in buffer system
Human intestinal
Absorption
(%)
In vitro skin
permeability
(logKp, cm/hour)
In vitro plasma
protein binding
(%)
Entry
Logp
Mg/L
A1
A2
A3
A4
A5
A6
A7
A8
A9
B1
B2
B3
B4
B5
4.62
5.70
3.61
5.98
7.01
3.69
4.69
4.10
4.82
8.23
8.27
8.85
7.07
7.10
99.54
99.68
94.30
99.68
99.59
93.90
97.37
98.91
98.31
100
-2.45
-2.42
-2.95
-2.45
-2.27
-2.42
-2.82
-3.40
-2.52
-1.77
-1.76
-1.69
-2.05
-1.78
93.96
94.26
90.87
100
403.4
45.21
207.1
144.0
100
5.57
93.48
89.91
87.51
92.61
100
5.53
128.1
15.57
55.88
5.37x10-6
1.50x10-5
5.77x10-7
3.77x10-5
5.83x10-6
100
100
100
100
99.32
100
96.34
93.07
Table 4. Molecular docking of new hexahydroindazole derivatives of curcumin with glucosamine-6-phosphate synthase.
Docking Energy
(Kcal/mol)
Inhibition
constant
Bond
Length
Binding Energy
(Kcal/mol)
Amino acid residue involved in H-
bond
Entry
RMSD
(M)
( Å)
Arg 73:HH1::Dp1:NA
His 77:HE2::Dp1:NA
Arg 73:HE::Dp2:OA
Arg 73:HH4::Dp2:NA
Gly 99:HN::Dp3:OA
No H- Bonds
1.67
2.18
2.05
2.08
1.90
--
A1
A2
-9.25
-9.99
-10.03
-10.78
1.67x10-7
0.81
0.92
4.79x10-8
A3
A4
A5
A6
A7
-9.57
-8.19
-9.56
--
-10.28
-8.54
-9.64
--
9.63x10-8
9.97x10-7
9.80x10-8
--
0.99
1.12
1.20
No H- Bonds
--
--
--
-10.02
-10.96
4.50x10-8
Gly 99:HN::Dp7:OA
Arg 73:HH21::Dp8:OA
His 77:HE2::Dp8:OA
His 71:HE2::Dp9:NB
His 77:HE2::Dp10:NA
No H- Bonds
2.00
1.68
2.13
1.88
2.01
--
0.56
1.02
A8
-6.93
-8.48
8.57x10-6
A9
B1
B2
B3
B4
B5
-5.40
-7.64
-7.51
-8.69
-8.53
-7.12
-4.86
-8.35
-8.40
-9.07
-8.40
-7.55
1.10x10-4
2.49x10-6
3.14x10-6
4.29x10-7
5.59x10-7
6.05x10-6
1.11
0.96
1.23
1.20
0.95
1.23
No H- Bonds
--
Cys 1:SG::Dp13:OA
No H- Bonds
2.93
--
Arg 73:HH21::DF:OA
Arg 73:HE::DF:OA
His 77:HE2::Dp10:NA
2.20
2.09
1.87
Fluconazole
-5.81
-5.36
5.53x10-5
1.97

126 Letters in Drug Design & Discovery, 2013, Vol. 10, No. 2
Kumar et al.
Fig. (2). Interaction of compound A7 with GlcN-6-P.
inhibition was measured in millimeters after 24 h. All tests
were performed in triplicate.
compound A3 also showed moderate activity against three
fungal strains viz. C. albicans, C. tropicalis, C. krusie.
Among the halogenated derivatives chloro substitution at
ortho or para position increased antifungal potency
appreciably (compound A2 zone of inhibition = 14.80mm),
(compound A4 zone of inhibition = 13.37mm) Fig. (1).
RESULT AND DISCUSSION
The compounds were prepared by coupling substituted
benzaldehydes with cyclohexanone in (ratio of 2:1) in a base
catalyzed Claisen-Schmidt condensation followed by reflux
with hydrazines (hydrazine hydrate, phenyl-hydrazine) and
acetic acid. The results of antimicrobial screening of
synthesized hexahydroindazole derivatives of curcumin are
presented in table (Table 2).
Chloro substitution at both ortho and para positions
resulted in marginal decrease in potency (compound A5 zone
of inhibition = 12.67mm). This may be attributed to
successful interaction of the compound with target protein(s)
when chloro substitution occurred either at ortho or para
position. Out of 14 compounds synthesized, majority of them
showed considerable antimicrobial activity against tested
strains.
The results of antibacterial screening revealed that among
the compounds screened compounds A2, A3, B1 and B2
showed moderate antibacterial activity while compound A7
& A8 displayed good antibacterial activity when compared
with ciprofloxacin used as standard. Particularly, compound
A7 which is carrying methoxy group on aryl ring appears to
exhibit maximum antibacterial activity (zone of inhibition up
to 21 mm) against E. coli and S. aureus. Interestingly, triple
substitution of methoxy group at ortho, meta and para
positions leads to a slight decrease in the potency (compound
A8 zone of inhibition = 19.67mm), this may be due to the
steric hindrance by these groups. Electron donating group
(methoxy) significantly increases antibacterial activity.
The combinatorial chemistry and virtual screening have
rapidly increased in drug discovery. Need for minimizing
extremely time-consuming steps of synthesis, biological
screening and good tools for predicting drug-likeness is very
much desired. The molecular descriptors have been
employed to predict various human ADMET processes and
other pharmacokinetic parameters such as oral absorption,
bioavailability, skin penetration, clearance, volume of
distribution, and metabolism [21-22]. Predicting ADME
properties at an early stage of drug discovery and
development process is very important to remove
compounds with poor pharmacokinetic properties and
minimize extremely expensive and time-consuming steps.
Different properties viz. drug-likeliness, Human intestinal
absorption, In vitro skin permeability, in vitro plasma protein
binding and water solubility in buffer system of all the 14
synthesized hexahydroindazole analogues of curcumin were
determined using PreADMET server. The results are
summarized in table (Table 3).
Derivatization of B1 with chloro substitution on phenyl
ring enhanced the potency (Compound B1 zone of inhibition
= 13.93mm) indicating that substitutions may result in
restoration of potency. Whereas, methoxy (Compound B4
zone of inhibition = 9.33mm) and N, N-dimethylamino (B5,
zone of inhibition = 8.90mm) substitutions exhibited
moderate potency. Phenyl pyrazoline analogues (B1-B5) did
not significantly increase the antibacterial potency.
The results of antifungal screening indicated that
compounds, A2, A3, A4, A7 and A8 showed moderate to
excellent antifungal activity. Compounds A2, A3 and A4
showed moderate activity against C. albicans. Interestingly,
Comparative docking of glucosamine-6-phosphate
synthase (GlcN-6-P synthase) protein with the curcumin
analogues and the standard drug fluconazole and

Synthesis, Molecular Docking and Antimicrobial Studies
Letters in Drug Design & Discovery, 2013, Vol. 10, No. 2 127
amphotericin B yielded best possible conformations with
parameters including the binding energy, docked energy,
inhibition constant and RMSD (Table 4). Theoretically,
molecules showed very good binding energy and docking
energy ranging from -5.40 Kcal/mol to -10.02 Kcal/mol and
-4.86 Kcal/mol to -10.96 Kcal/mol respectively Fig. (2). The
minimum docked energy was found in the analogue A7 (-
10.96 kcal/mol) with an estimated inhibition constant of
4.50x10-8 and RMSD 0.56. Whereas, docked energy of the
standard drugs fluconazole was -5.67 kcal/mol with an
inhibition constant of 5.53x10-5 and RMSD 1.97.
CONFLICT OF INTEREST
The authors confirm that this article content has no
conflicts of interest.
ACKNOWLEDGEMENTS
One of the authors, Dileep Kumar acknowledges the
nancial support from University Grant Commission, Govt.
of India in the form of fellowship.
SUPPLEMENTARY MATERIAL
The accuracy of dockings was evaluated by the root-
mean square deviation (RMSD) of docked ligand from
original crystal structure. RMSD less than 1.0 Å was
considered as excellent.
Supplementary material is available on the publishers
Web site along with the published article.
A significant number of drugs and drug candidates in
clinical development are halogenated structures. The
formation of halogen bonds in ligand-target complexes is
recognized as a kind of intermolecular interaction that
favorably contributes to the stability of protein-ligand
complexes. The insertion of halogen atoms has been used in
innumerous cases of hit-to-lead or lead-to-drug conversions
[23-25].
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