Effect of acetate and nitrate anions on the molecular structure of 3-(hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)hydrazone

Article abstract of DOI:10.1016/j.molstruc.2016.12.044

A novel Schiff base ligand 3-(hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)hydrazone has been synthesized by the condensation reaction of 2-Hydrazinobenzimidazole with diacetyl monoxime in presence of acetic acid catalyst. The ligand has crystallized as its acetate salt, due to the charge-assisted hydrogen bonding between protonated benzimidazole ring and acetate anion. Efforts to synthesize the zinc(II) complex of the title compound, has resulted in the formation of a nitrate salt of the ligand, instead of coordination complex of zinc(II). Acetate salt has crystallized in monoclinic P 2₁/n, while the nitrate salt has crystallized in a triclinic crystal system with P -1 space group. Hirshfeld surface analysis is presented for both of the crystal structures. Structures of synthesized molecules are even computationally optimized using DFT. A comparative structural approach between the synthesized molecules and DFT optimized structure of bare ligand without any counterions is analyzed in terms of bond parameters. Hydrogen bonding is explained keeping the anions as the central dogma. Mass fragmentation pattern of the organic molecule and comparative account of IR, ¹H and ¹³C NMR chemical shifts are also presented. Compounds are screened for their antibacterial and antifungal potencies against few pathogenic microorganisms. The organic motif is found be an excellent antifungal agent.

Full text of DOI:10.1016/j.molstruc.2016.12.044

Accepted Manuscript
Effect of acetate and nitrate anions on the molecular structure of 3-(hydroxyimino)-2-
butanone-2-(1H-benzimidazol-2-yl)hydrazone
Vinayak Kamat, Krishna Naik, Vidyanand K. Revankar
PII:
S0022-2860(16)31361-8
10.1016/j.molstruc.2016.12.044
MOLSTR 23242
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 November 2016
Revised Date: 14 December 2016
Accepted Date: 15 December 2016
Please cite this article as: V. Kamat, K. Naik, V.K. Revankar, Effect of acetate and nitrate anions on
the molecular structure of 3-(hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)hydrazone, Journal of
Molecular Structure (2017), doi: 10.1016/j.molstruc.2016.12.044.
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ACCEPTED MANUSCRIPT
Ortep projections of 3-(Hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)hydrazone in its two different salt
forms, viz., acetate and nitrate.

ACCEPTED MANUSCRIPT
Effect of acetate and nitrate anions on the molecular structure
of 3-(hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)hydrazone
Vinayak Kamata, Krishna Naika and Vidyanand K. Revankara*
a Department of Chemistry, Karnatak University, Dharwad-580003, India.
* Corresponding author
Abstract
A novel Schiff base ligand 3-(hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)
hydrazone has been synthesized by the condensation reaction of 2-Hydrazinobenzimidazole
with diacetyl monoxime in presence of acetic acid catalyst. The ligand has crystallized as its
acetate salt, due to the charge-assisted hydrogen bonding between protonated benzimidazole
ring and acetate anion. Efforts to synthesize the zinc(II) complex of the title compound, has
resulted in the formation of a nitrate salt of the ligand, instead of coordination complex of
zinc(II). Acetate salt has crystallized in monoclinic P 21/n, while the nitrate salt has
crystallized in a triclinic crystal system with P -1 space group. Hirshfeld surface analysis is
presented for both of the crystal structures. Structures of synthesized molecules are even
computationally optimized using DFT. A comparative structural approach between the
synthesized molecules and DFT optimized structure of bare ligand without any counterions is
analyzed in terms of bond parameters. Hydrogen bonding is explained keeping the anions as
the central dogma. Mass fragmentation pattern of the organic molecule and comparative
account of IR, 1H and 13C NMR chemical shifts are also presented. Compounds are screened
for their antibacterial and antifungal potencies against few pathogenic microorganisms. The
organic motif is found be an excellent antifungal agent.
Keywords
benzimidazole-based Schiff base ligand, 2-Hydrazinobenzimidazole, charge-assisted
hydrogen bonding, antifungal agent.
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1. Introduction
The benzimidazole scaffold is a very useful structural motif in developing molecules
of pharmaceutical or biological interest [1]. Substituted benzimidazole derivatives have found
a variety of therapeutic applications as in antiulcers, anticancers, antihypertensives, antivirals,
antifungals and antihistaminics [2]. This has resulted in benzimidazole-based structures in
various drugs that are serving as life-saving agents such as anthelmintics; oxfendazole,
albendazole, fenbendazole, thiabendazole, mebendazole; inhibitors of proton pump
lansoprazole, pantoprazole, omeprazole [3].
One of the often ways to manipulate the chemical, biological, physical, and economic
characteristics of medicinal agents, is to convert them to a salt form [4]. One among these
characteristics is solubility, which is a key contributor to the developmental challenges [5].
Formation of salt is the most common and effective method of increasing aqueous solubility
and dissolution rates of acidic and basic drugs. Hence it is the most preferred approach for the
development of liquid formulations for parenteral administration [6]. In last few years, this
has become the focus of many high-throughput screening methods developed. The root cause
of solubility of this kind of molecules can be well understood in terms cation-anion
interactions in the solid state, that operates in them. And here crystal engineering plays a
substantial role [7].
The driving force to facilitate the formation of salts in hydrazone compounds is the
bifurcated N+−H···O− hydrogen bond synthon [8]. Hydrogen donor belonging to a cation and
the acceptor belonging to an anion is one of the most employed devices in the construction of
crystalline architectures. In these, charge-assisted bonds combine the intrinsic strength and
directionality of the neutral hydrogen bond with a favorable location of the ionic charges and
they are easily obtained in crystal synthesis via acid-base reactions [9]. These sort of
intermolecular interactions in organic molecules, play important role in crystal packing and
the fields like supramolecular chemistry give special attention to these [10]. Cooperativity
effect of combined weak interactions contributes a lot to the stability of crystal lattice [11].
Quantitative measurements of weak interactions in elucidating the molecular structures can
be effectively carried out by Hirshfeld surface analysis [12] and other computational
methods. Considering these facts, herein we report the synthesis, spectroscopic
characterization, crystallographic studies, Hirshfeld surface analysis and antimicrobial
evaluation of acetate and nitrate salts of a benzimidazole-based Schiff base ligand, 3-
(hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)hydrazone.
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2. Experimental
2.1. General considerations
All reagents were purchased commercially and were used as supplied. Solvents were
purified and dried according to the standard procedures. 2-Hydrazinobenzimidazole was
prepared by a new synthetic route, which used slightly altered procedure than previously
reported. The 1H and 13C NMR spectra were recorded in Bruker 400 MHz spectrometer, in a
DMSO-d6 solvent. Infrared spectra were recorded in KBr discs in the region 4000–400 cm−1
on a Nicolet-6700 FT-IR spectrometer. The mass spectrum was obtained on a Shimadzu
GCMS-QP 2010S spectrometer. Thermal behavior was analyzed on a Universal V2.4F TA
instrument.
2.2. Synthesis and Crystallization
2.2.1. 3-(Hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)hydrazonium acetate (LHAc)
The schematic representation of the synthesis of LHAc is given in Scheme 1. First, 2-
bromobenzimidazole was obtained from 2-mercaptobenzimidazole by the reported method
[13]. In the next step, 2-bromobenzimidazole (1.97 g, 0.01 mol) was refluxed in 20 mL of
80% hydrazine hydrate solution, to obtain 2-Hydrazinobenzimidazole (1.35 g, Yield : 91%).
Finally, diacetyl monoxime (1.01g, 0.01 mol) was added to the 20 ml methanolic solution of
2-hydrazinobenzimidazole (1.48 g, 0.01 mol) in presence of 3 drops of glacial acetic acid.
The resulting mixture was refluxed for 4 hours. On cooling, the white crystals separated were
filtered and washed with cold methanol to get the desired product (1.6 g, yield : 55%, M.P
2400C ).
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H
N
H
N
Br2, HBr
SH
Br
N
N
CH3COOH, RT, Stir 4 h
Yield : 80%
MP : 190-192 0
C
Reflux 4 h
NH2-NH2.H2O (80%)
CH3COO
12
4
13
H
H
N
N
3
N
2
H
N
5
10
11
7
N
OH
Diacetyl monoxime
NH
9
NH
1
6
N
NH2
Methanol, Reflux 4h
8
Yield : 91%
MP : 224-2250C
Yield : 55%
MP : 2400C
Scheme 1. Synthesis of LHAc.
2.2.2. 3-(Hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)hydrazonium nitrate (LHNO3)
The schematic representation of the synthesis of LHNO3 is given in scheme 2. LHAc
(0.15 g, 0.5 mmol) was refluxed with Zn(NO3)2.6H2O (0.15 g, 0.5 mmol) in 10 ml of ethanol
for 4 hours. The greenish clear solution obtained was slowly evaporated to get greenish white
coloured crystals (0.11 g, yield : 78%, M.P 2320C).
CH3COO
4
NO3
5
H
H
N
N
3
NH
NH
N
N
10
2
11
N
N
OH
OH
Zn(NO3)2.6H2O
NH
9
NH
1
6
Ethanol, Reflux 4h
8
7
Yield : 78%
MP : 2320C
Scheme 2. Synthesis of LHNO3.
2.3. Spectroscopic data
1
LHAc : H NMR (400 MHz, DMSO- d6): δ (ppm) 11.36 (s, 1H), 7.26 (dd, J = 5.6 Hz, J =
3.2Hz, 2H), 6.99 (dd, J = 5.6 Hz, J = 3.2Hz, 2H), 3.15 (s, 2H), 2.16 (s, 3H), 2.12 (s, 3H),
13
1.88 (s, 3H); C NMR (100 MHz, DMSO-d6): δ 172.85, 155.45, 154.30, 146.53, 120.64,
21.57, 11.99, 9.97; EI-MS (m/z): 231 (M+) IR (KBr, v, cm−1): 3516, 3168, 1673, 1016, 748.
LHNO3 : 1H NMR (400 MHz, DMSO- d6): δ (ppm) 11.8 (s, 1H), 7.50 (dd, J = 5.6 Hz, J =
3.2Hz, 2H), 7.29 (dd, J = 5.6 Hz, J = 3.2Hz, 2H), 3.45 (s, 2H), 2.20 (s, 3H), 2.16 (s, 3H); 13C
4

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NMR (100 MHz, DMSO-d6): δ 154.70, 152.54, 149.7, 131.17, 123.81, 112.77, 11.25, 9.91;
EI-MS (m/z): 231 (M+) IR (KBr, v, cm−1): 3308, 1671, 1384, 1016, 754.
2.4. Structure Determination and Refinement
A summary of crystallographic data along with refinement parameters for both the
structures is tabulated in Table 1. The X-ray diffraction data were obtained at 296.15 K on a
Bruker SMART APEX2 CCD area detector diffractometer using a graphite monochromated
Mo-Kα (λ = 0.71073 Å) radiation source. The frames were integrated with the Bruker SAINT
Software package [14] using a narrow-frame algorithm. Using Olex2 [15], the structure was
solved with the ShelXT [16] structure solution program using Direct Methods and refined
with the XL [17] refinement package using CGLS minimisation. All non-hydrogen atoms
were refined anisotropically. Molecular graphics were generated using Mercury-3.8 package
[18]. The corresponding crystallographic information files can be downloaded from the
website http://www.ccdc.cam.ac.uk. (CCDC 1480955 and CCDC 1480896).
Table 1. Crystal data and structure refinement parameters.
LHAc
LHNO3
Chemical formula
C11H14N5O·C2H3O2·CH4O
323.36
2(C11H14N5O)·2(NO3)
588.56
Mr
Crystal system, space Monoclinic, P21/n
Triclinic, P-1
group
Temperature (K)
296
296
a, b, c (Å)
11.9358 (7), 7.2800 (5), 7.3242 (2), 8.8206 (2),
19.7823 (12)
11.5090 (3)
90, 103.213 (4), 90
98.293 (2), 100.400 (2),
α, β, γ (°)
108.232 (2)
678.38 (3)
1
V (Å3)
1673.43 (18)
Z
4
Radiation type
Mo Kα
Mo Kα
µ (mm-1)
0.10
0.11
No.
of
measured, 12374, 3389, 1397
and
19704, 5421, 3444
independent
observed [I
>
2σ(I)]
5

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reflections
Rint
0.090
0.039
(sin θ/λ)max (Å-1)
R[F2 > 2σ(F2)], wR(F2), S
No. of reflections
No. of parameters
∆〉max, ∆〉min (e Å-3)
0.624
0.787
0.051, 0.127, 0.79
3389
0.055, 0.175, 1.06
5421
222
220
0.23, -0.22
0.40, -0.30
2.5. Hirshfeld surface analysis
Crystal Explorer 3.1 [19] was used to generate Hirshfeld surfaces (HSs) and 2D
fingerprint plots (FPs) from corresponding crystallographic information files [20]. The
function dnorm is a ratio encompassing the distances of any surface point to the nearest interior
(di) and exterior (de) atom and the van der Waals radii of the atoms. The red color in HSs is a
visualization of the negative value of dnorm indicating the sum of di and de to be shorter than
the sum of the relevant van der Waals radii. Intermolecular distances close to van der Waals
contacts with dnorm equal to zero are indicated in white color, whereas contacts longer than
the sum of van der Waals radii with positive dnorm values are colored with blue. 2D
fingerprint plots are obtained by the plotting of di versus de to show the different types of
intermolecular interactions.
2.6. Computational methods
The geometry of the molecules with and without counter anions was optimized using
density functional theory. The crystal structures obtained from CIF were used as an initial
molecular geometry for LHAc and LHNO3. ORCA program package (version 3.0.3) was
used for DFT calculations. Tight convergence criteria were chosen for all calculations. All
calculations were performed using the hybrid functional BP in combination with Ahlrichs
split-valence double-ξ basis set def2-SVP [21] for all atoms. ORCA calculation times were
minimized using the RI approximation [22] [23] using SVP/J auxiliary basis sets [22] from
the TurboMole basis set library II. The outputs were visualized using molecular visualizer
tool Avogadro 1.1.1 [24] and Mercury-3.8 package [18].
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2.7. Growth inhibitory activity of microbial strains[25]
The MIC values of all synthesized compounds for the growth inhibition of six
different pathogenic microbial genomes S. epidermidis ,E. faecalis, K. pneumoniae, E. coli,
A. fumigatus and C. albicans were determined in HIMEDIA M210 Brain heart infusion broth
(BHI), which is used for the propagation of pathogenic cocci and other fastidious organisms
associated with allied pathological investigations.
Each sample was diluted 9 times with BHI for MIC determination. 20 ꢀL of the drug
was added to the 380 ꢀL of BHI broth in the initial tube. 200 ꢀL of BHI broth was added into
the next 9 tubes separately, for dilutions. Then from the initial tube 200ꢀL was transferred to
the first tube containing 200 ꢀL of BHI broth. This was considered as 10-1 dilution. From 10-1
diluted tube, 200 ꢀL was transferred to the second tube to make 10-2 dilution. The serial
dilution was repeated up to 10-9 dilution for each drug. From the maintained stock cultures of
required organisms, 5 ꢀL was taken and added into 2 mL of BHI broth. In each serially
diluted tube, 200 ꢀL of above culture suspension was added. For facultative anaerobes, tubes
were incubated at 37°C for 48-72 hrs in CO2 jar while fort strict anaerobes, tubes were
incubated in anaerobic jars for 48-72 hrs. After the incubation period the tubes were observed
for turbidity.
3. Results and Discussion
3.1. Synthesis
The primary aim of the work was to condense, 2-hydrazinobenzimidazole with
diacetyl monoxime to obtain a novel Schiff base ligand, which can act as trinitrogen chelator.
However, the reaction did not proceed directly. When few drops of acetic acid were added to
facilitate the reaction by adjusting the pH to acidic [26], product was formed with ease. And
the desired condensation product had crystallized as its acetate salt.
The ligand in its acetate form was used to prepare the coordination complex with
Zn(NO3)2. The isolated product was greenish white crystals. The mere observation of IR, 1H
and 13C NMR peak shifts of this, lead us to a wrong conclusion. Initially, the formed product
was thought to be a coordination complex of zinc(II). The NMR shifts were thought to be
due to the coordination of the nitrogen atoms of the ligand to zinc(II) ion [27], while the
nitrate peak in the IR spectra was ascribed to counter nitrate ions in the complex [28].
However, the single crystal structure of the compound clearly ruled out the possibility of
coordination complex and answered the observed shifts in IR and NMR spectral studies.
7

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3.2. Mass fragmentation
LHAc as well as LHNO3 have given similar mass fragmentation pattern for the
organic motif. Fig.1. depicts the mass spectrum of LHAc. The plausible fragmentation pattern
for the same is presented in scheme 3. The molecular ion is observed at m/z 231. This has lost
the hydroxyl group, triggered by the homolytic fission of N-O bond to give rise to a species
with m/z 214. The subsequent loss of two acetonitrile fragments in two distinct steps is
responsible for the ions observed at m/z 173 and the base peak at m/z 132.
H
H
N
H
N
H
N
N
-
CH3CN
NH
N
NH
N
- CH3CN
NH
N
NH
N
N
N
C
N
N
N
OH
m/z = 132
m/z = 173
m/z =231
m/z = 214
Scheme 3. Fragmentation pattern.
Fig.1. Mass spectrum of LHAc.
3.3. IR Spectral analysis
The υ(O-H) of oxime functionality, has given stretching vibrations at 3516 cm-1 and
3308 cm-1 in LHAc and LHNO3 respectively [29]. In addition, LHAc has shown a broad peak
at 3168 cm-1 which is possibly alluded to the still intact carboxylic acid group, and the peak
shift indicating possible hydrogen bonding [30]. The sharp intense band observed at 1673 and
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1671 in both the structures are assigned to the υ(C=N) vibrations, while the bands at 1016 to
υ(N-O) [31]. Further, the presence of ionic nitrate in LHNO3 is evidenced in a sharp peak
observed at 1384 cm-1 [32].
3.4. 1H and 13C NMR studies
1
The H NMR signal for hydrazone NH is observed downfield at 11.36 ppm [33],
while the benzimidazole NH signals are observed upfield at 3.2 ppm [34] in LHAc. In
LHNO3, Both of these signals have shifted to 11.8 and 3.5 ppm respectively. Similarly, the
slight shifts in δ values of all other peaks, are attributed to the replacement of anion. A sharp
singlet observed at 1.9 ppm in the case of LHAc, evidences the acetate ion. The OH and
benzimidazole NH protons being labile, have shown rapidity of exchange with the solvent.
In the 13C NMR spectrum of LHAc, two distinct resonances due to acetate anion have
occurred at 21.57 and 172.85 ppm. The disappearance of these peaks and significant shifts in
13
the δ values of all other C resonances in LHNO3, assures the replacement of acetate anion
by nitrate in the bulk of the matter under investigation.
3.5. Single crystal X-ray diffraction Structures and DFT optimized structures
Single crystals suitable for X-ray diffraction of LHAc were grown directly from the
methanolic reaction mixture, while LHNO3 from the ethanolic reaction mixture. Ortep
representations of both the structures with 50% displacement ellipsoids along with unit cell
diagrams are presented Fig.2 and Fig.3. The organic part in the synthesized molecules LHAc
and LHNO3 are cationic, due to the protonation of the imidazole ring. Hence charge on the
organic part is stabilized by the presence of an anion in these molecules. The neutral organic
molecule without any anion (L) is not synthesized. Hence the structure of L is obtained from
DFT calculations. The DFT optimized structures of L, LHAc and LHNO3 are given in Fig.4.
These structures are quite close to the structures obtained from single crystals. Comparison of
bond lengths of all the structures is summarized in Table 2, while bond angles are given in
Table 3. The calculated bond lengths and bond angles are in reasonable agreement with the
corresponding experimental values for the both compounds. However, the small deviations
observed in the case of LHNO3 may be attributed to the solid state intermolecular interactions
related to crystal packing effects [35].
9

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Fig.2.a) Ortep representation of LHAc with 50% displacement ellipsoids b) Unit cell.
10

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Fig.3. a) Ortep representation of LHNO3 with 50% displacement ellipsoids b) Unit cell.
a) L
b) LHAc
11

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c) LHNO3
Fig.4. DFT optimized structures
Table 2. Comparison of bond lengths for L, LHAc and LHNO3 [Å].
Atom
Atom
L
LHAc
(Expt)
LHAc
(DFT)
LHNO3
(Expt)
LHNO3
(DFT)
1.345
1.259
1.355
1.260
1.439
1.343
1.399
1.307
1.396
1.496
1.502
1.509
1.385
1.387
1.387
1.398
1.387
1.386
-
(DFT)
1.322
1.298
1.379
1.298
1.414
1.354
1.340
1.358
1.345
1.494
1.518
1.519
1.393
1.367
1.408
1.415
1.393
1.408
-
O1
N1
N2
N2
N3
N5
N5
N4
N4
C2
N1
C2
N3
C3
C5
C5
C11
C5
C6
C3
C1
C4
C10
C6
C9
C8
C7
C7
C12
1.401(3) 1.402 1.3919(15)
1.292(3) 1.297 1.2822(17)
1.386(3) 1.387 1.3828(15)
1.290(3) 1.289 1.2897(16)
1.330(3) 1.331 1.3369(15)
1.321(3) 1.320 1.3392(16)
1.398(3) 1.398 1.3937(16)
1.341(3) 1.340 1.3311(16)
1.405(3) 1.404 1.3930(16)
1.478(3) 1.478 1.4799(18)
C2
1.483(3) 1.483
1.489(2)
C3
1.492(3) 1.491 1.4892(18)
1.372(3) 1.371 1.3860(17)
1.379(3) 1.379 1.3911(17)
C11
C11
C10
C9
1.385(3) 1.386
1.375(4) 1.375
1.382(2)
1.386(2)
C6
1.382(3) 1.387 1.3853(18)
C8
1.386(4) 1.387
1.259(3) 1.258
1.390(2)
-
O3
12

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O2
C12
O4
N6
N6
N6
C12
C13
C14
O2
-
-
-
-
-
-
1.253(3) 1.253
1.493(3) 1.493
1.402(3) 1.402
-
-
-
-
-
-
-
-
-
-
-
-
1.2308(17)
1.2435(17)
1.2428(16)
1.248
1.210
1.189
O3
O4
Table 3. Comparison of bond angles for LHAc and LHNO3 [°].
Atom Atom Atom
L
LHAc LHAc
LHNO3
(Expt)
LHNO3
(DFT)
(DFT) (Expt) (DFT)
C2
C3
N1
N2
N3
N5
N4
C2
C2
C2
C3
C3
C3
C5
C5
C5
C11
C11
C11
C10
C9
C6
C6
C6
C8
O1 126.04 111.4(2) 111.39 112.87(12) 115.21
N3 126.26 116.3(2) 116.32 115.36(11) 119.42
N2 119.50 115.6(2) 115.61 115.44(10) 119.02
C11 104.19 108.5(2) 108.51 108.25(10) 108.16
C5
C5
C5
C6
C3 118.17 114.2(2) 114.30 113.08(11) 114.53
C1 123.6 124.7(2) 124.65 125.74(13) 126.21
103.3 108.0(2) 108.04 108.40(10) 108.15
N1
N1
C3
C1 118.14 121.1(2) 121.04 121.13(12) 119.28
C2 117.92 113.7(2) 113.75 115.14(11) 115.65
C4 124.04 125.8(2) 125.80 124.26(11) 123.96
C4 118.04 120.5(2) 120.44 120.54(11) 120.40
N4 123.42 126.7(2) 126.63 125.60(11) 126.91
N3 123.23 123.2(2) 123.19 124.30(11) 122.37
N2
N2
C2
N3
N5
N5
C10
C10
C6
N4
113.3 110.1(2) 110.17 110.09(11) 110.71
N5 128.70 130.4(2) 130.45 132.16(12) 132.17
C6 122.16 122.5(2) 122.56 121.30(12) 122.32
N5 109.14 107.0(2) 106.98 106.53(10) 105.51
C9 117.44 116.2(3) 116.19 116.61(13) 116.42
C10 120.47 121.6(3) 121.58 122.06(13) 121.42
N4 110.01 106.3(2) 106.30 106.73(10) 107.47
C7 121.87 121.5(2) 121.49 122.22(12) 121.50
N4 110.01 132.2(2) 132.21 131.05(12) 131.01
C7 120.48 122.2(3) 122.20 121.68(14) 121.78
C11
C8
C11
C11
C7
C9
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C6
O3
O2
O2
O2
O2
O3
C7
C12
C12
C12
N6
C8 117.57 116.0(3) 115.98 116.11(14) 116.57
C13
O3
-
-
-
-
-
-
117.4(2) 117.40
122.9(2) 122.87
119.8(2) 119.73
-
-
-
-
C13
O4
-
-
-
-
-
-
-
-
118.68
121.51
119.77
119.36
118.03
122.61
N6
O3
N6
O4
LHAc has crystallized in monoclinic crystal system with P 21/n space group.
Replacement of acetate counter anion with the nitrate anion has resulted in triclinic crystal
system with P -1 space group in LHNO3. Asymmetric units contain protonated organic
-
molecule (LH+) along with acetate (Ac-) anion in LHAc and nitrate (NO3 ) anion in LHNO3.
In addition, LHAc contains a methanol molecule as a solvent of crystallization. Diazine and
azomethine modules of the imine functionality of LH+, have maintained coplanarity in both
the structures. This coplanarity is even maintained in the DFT optimized structure of L.
Further in LHAc and L, hydroxylimine group is also in the mean plane of the molecule. But
in LHNO3, hydroxylimine group is out of the mean plane of the molecule, with a dihedral
angle of 20.360. The imine linkages in diacetyl fragment of the ligand (C2-N1and C3-N2) in
both the cases averages to 1.29 Å as expected for C=N bond length, confirming the double
bond character [36]. The bond lengths of 3 C-N bonds arising from C5 of the benzimidazole
ring (C5-N3, C5-N4 and C5-N5) in LHAc and LHNO3 averages to 1.33 Å. This is in
accordance with previous reports for benzimidazolium salts[37]. The C-N bond lengths in the
imidazole ring are in the range 1.32–1.35Å, indicating partial double-bond character [38].
The torsional angle of -178.90 is shown by C1-C2-C3-C4 bonds in the case of LHAc and -
179.99 in the case of L, conveys the S-trans configuration of methyl groups in the diacetyl
fragment. Same torsional angle in the case of LHNO3 has deviated to 167.460. This is a
consequence of the free rotation of C2-C3 bond, which takes hydroxylimine group out of the
mean plane of the molecule. Hence the molecule L is found to be planar, resembling LHAc
than LHNO3. Previously reported computational chemistry calculations on these type of
molecules have proved that the charge assisted hydrogen-bonded salts are more stable than
the individual molecules or the hydrogen bonded neutral molecules [30]. Accordingly,
charge-assisted hydrogen bonding has stabilized both the structures in the present work. This
has also reflected in the calculated single point energies (SPE). SPE calculates the
wavefunction and charge density, and hence the energy, of a particular arrangement of nuclei.
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The calculated SPE for L, LHAc and LHNO3 are found to be -770.5, -1049.8 and -1112.83
eV respectively.
The optimized structures were used to calculate the energies of the molecular orbitals
(MOs). Besides the HOMO and LUMO, HOMO-1 and LUMO+1 are also considered for all
the three structures. The contributions of each atom in the molecular orbital are reflected in
the molecular density distribution through the MO. The localization of the density for the
orbitals and energies of L, LHAc and LHNO3 are depicted in Fig.5. The highest contribution
for the HOMO for all the three structures is attributed to the delocalized π electrons of the
benzene ring. In the LUMO orbital, in addition to the localized density from the benzene ring,
there is some contribution from other atoms as shown in Fig. 5.
Fig.5. Density distribution and energies of the molecular orbitals for L, LHAc and
LHNO3.
3.6. Hydrogen bonding
Hydrogen bonding interactions in LHAc are depicted in Fig.6 and Table 4
summarizes the same. Each acetate ion in the extended structure of LHAc has accepted four
15

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hydrogen bonds with its two oxygen atoms (O2 and O3). All these four bonds are assisted by
the charge delocalized over oxygen atoms of acetate anion. O2 has accepted a hydrogen bond
from hydrazide of NH (N3H....O2;2.84 Å) of one molecule while the other hydrogen bond is
with the hydroxyl group of the next molecule (O1H....O2;2.67 Å). O3 has made a hydrogen
bond with NH of benzimidazole (N5H....O3;2.60 Å) while the other is with the hydroxyl
group of methanol (O4H....O3;2.67 Å). In addition to these, the hydroxyl oxygen of methanol
has accepted a hydrogen bond from the other NH of benzimidazole (N4H....O4;2.82 Å).
Fig.6. Hydrogen bonding interactions in LHAc.
Table 4. Hydrogen bond parameters in LHAc [Å and °].
D-H...A
d(D-H)
d(H...A)
d(D...A)
<(DHA)
O(1)-H(1)...O(2)#1
N(3)-H(3)...O(2)
N(5)-H(5)...O(3)
N(4)-H(4)...O(4)
O(4)-H(4D)...O(3)#2
1.01(3)
1.00(2)
0.86
1.67(3)
1.91(3)
1.76
2.672(2)
2.840(3)
2.605(3)
2.822(3)
2.673(3)
173(3)
153(2)
168.8
0.90(3)
0.82
1.99(3)
1.86
154(3)
168.7
Symmetry transformations used to generate equivalent atoms:
#1 -x+3/2,y-1/2,-z+1/2 #2 -x+2,-y+1,-z
Hydrogen bonding interactions in LHNO3 are depicted in Fig.7 and Table 5
summarizes the same. Each nitrate ion in the extended structure of LHNO3 has accepted four
hydrogen bonds. Even these bonds are assisted by charge, delocalized over oxygen atoms. O4
has accepted a hydrogen bond from hydrazide NH (N3H....O4;2.87 Å) of first LH+ unit and
also with hydroxyl hydrogen of the second LH+ unit (O1H....O4;2.90 Å). O3 has made a
16

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hydrogen bond with benzimidazole-NH of first LH+(N5H....O3;2.85 Å). Nitrate is linked to a
third LH+ with O2 by making a hydrogen bond with benzimidazole-NH (NH....O2;2.82 Å).
Fig.7. Hydrogen bonding interactions in LHNO3.
Table 5. Hydrogen bond parameters in LHNO3 [Å and °].
D-H...A
d(D-H)
0.88(2)
0.94(2)
0.86
d(H...A)
2.03(2)
1.94(2)
2.10
d(D...A)
2.8245(17)
2.8493(17)
2.8741(15)
2.9014(17)
<(DHA)
150.0(18)
162.8(17)
149.0
N(4)-H(4)...O(2)
N(5)-H(5)...O(3)#1
N(3)-H(3)...O(4)#1
O(1)-H(1)...O(4)#2
0.89(3)
2.03(3)
169(2)
Symmetry transformations used to generate equivalent atoms:
#1 x,y-1,z #2 -x+1,-y+1,-z+2
3.7. Hirshfeld surface analysis
Hirshfeld surface is a unique surface for a given crystal structure, which suggests the
possibility of obtaining additional insights into the crystal structure [39]. The molecular HSs
generated for LHAc and LHNO3 are given in Fig.8. The similarity in shapes of HSs
indicates similar type of crystal packing and intermolecular interactions. The quantitative
measures like volume, area, globularity and asphericity calculated using HSs are found to be
286.64 Å3, 281.09 Å2, 0.748, 0.343 and 278.51 Å3, 280.14 Å2, 0.736, 0.339 for LHAc and
LHNO3 respectively. The value of the term globularity is found to be <1 in both the cases
indicating the molecular surface to be more structured rather than a sphere [40].
17

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a) LHAc
b) LHNO3
Fig.8. Hirshfeld surfaces mapped with dnorm of LHAc and LHNO3.
Two intense red spots near to hydrazide and ring-N hydrogens on the surface are in
consistence with the strong hydrogen bonding interactions. The crystal packing is mainly
controlled by these dominant interactions. Relatively intense red spots near the other ring-N
and hydroxyl hydrogen is due to the hydrogen bonded contact of the surface atoms. Besides
these, weak red spot observed near methyl hydrogens in LHAc indicates the H-C short
contacts with the crystallization solvent methanol. In LHNO3, the hydrogen atom of a methyl
group is in close proximity with the oxime nitrogen. This mutual intermolecular interaction
has resulted in weak red spots on the HS of LHNO3. An additional moderately intense red
spot near the benzene ring on the HS of LHNO3 is due to the close proximity of aromatic
hydrogen with the oxime oxygen of the next molecule in the crystal lattice. In conclusion, the
HS analysis indicates more intermolecular interactions in the case of LHNO3 than in the case
of LHAc.
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Medicinal molecules can be authorized for some rare disease only if they are found to
be dissimilar to authorized orphan drugs. 2D fingerprints can be used to show the extent of
the relationship between computed levels of structural similarity for pairs of molecules [41].
Intermolecular interactions in LHAc and LHNO3 are depicted in the form of di versus de 2D
fingerprint plots in Fig.9. This gives the distance (di) from the nearest atom inside the surface
against the distance (de) to the nearest atom external to the surface. Individual contributions
of the intermolecular contacts to the total Hirshfeld surface are graphed in Fig. 10.
Fig.9. 2D fingerprint plots of LHAc and LHNO3 depicting all intermolecular contributions.
19

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LHAc LHNO3
LHNO3
H-H
49.6%
17.9%
11.6%
11.0%
4.9%
38.3%
26.9%
14.3%
9.7%
5.5%
3.0%
1.6%
0.3%
H-O
H-C
H-N
C-N
LHAc
C-C 3.8%
0.7%
N-N
0.6%
C-O
Fig.10. Individual contributions of the intermolecular contacts to the total Hirshfeld surface.
The comparison in the shapes of FP plots of LHAc and LHNO3 is indicating the
similarity in interionic interactions. This is due to the isostructural cation. A major
contribution to HS is coming from interactions in which H atoms are involved. i.e., H⋯H,
H⋯O, H⋯C and H⋯N. C⋯N and C⋯C are contributing significantly while N⋯N and C⋯O
interactions are negligibly contributing to the total. The significantly high value of H⋯O
contributions is due to the charge-assisted hydrogen bonding that occurs via O atoms. This
contribution is 26.9% in LHNO3 and 17.9% in LHAc, is in accordance with the more number
of short contacts in LHNO3 which are evidenced on the HS as red spots. Contributions from
H⋯O contacts to the total HS is depicted in Fig.11. in the form of 2D FP plots.
20

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Fig.11. 2D fingerprint plots of LHAc and LHNO3 depicting H⋯O contributions
3.8. Thermal analysis
LHAc and LHNO3 were studied for TG and DT analysis over the temperature range
of 25-6000C at a heating rate of 100C/min under nitrogen atmosphere. LHAc has remained
thermally stable up to a temperature of 960C without any significant weight loss. Between 96-
1100, the weight loss of about 9.3 % (calc. 9.9%) corresponds to loss of lattice held methanol
molecule. This is evident on the DTA curve in the form of an endothermic peak at 1120C.
Further, both the compounds have lost major fractions of their weights above 2400C. This is
due to the loss of respective counter anions accompanied by the oxidative decomposition of
the organic part.
3.9. Antibacterial and antifungal assay
Benzimidazole derivatives are well known antimicrobial agents and hence they find
chemotherapeutic importance. To evaluate the antimicrobial potency of newly synthesized
organic molecule 3-(hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)hydrazone, LHAc
and LHNO3 were screened against gram-positive bacteria (S. epidermidis ,E. faecalis, gram-
negative bacteria (K. pneumoniae, E. coli) and fungi (A. fumigatus and C. albicans.
Ciprofloxacin and flucanazole were used as standards for antibacterial and antifungal assays
respectively. The results are tabulated in table 6. Synthesized compounds have shown
excellent growth inhibition activity against S. epidermidis and K. pneumoniae with a MIC
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value of 0.8 and 0.4 ꢀg/mL respectively. This is almost twice the activity of standard
ciprofloxacin in both the cases. The antifungal activity of organic motif of the compounds
which is responsible for the growth inhibition is found to be far better than that of standard
flucanazole. Compounds are inhibiting the growth of A. fumigatus and C. albicans even at a
low concentration of 0.4 ꢀg/mL. The comparison of antifungal potencies of LHAc and
LHNO3 with the standard flucanazole is presented in fig. 12.
Table 6. In vitro antimicrobial activity (MIC)
Microbial
Strains
Gram-positive
bacteria
Gram-negative
bacteria
Fungi
S.
E.
K.
E.
A.
C.
epidermidis faecalis pneumoniae coli fumigatus albicans
MIC
LHAc
0.8
0.8
1.6
-
12.5
6.25
1.6
-
0.4
0.4
0.8
-
6.25
12.5
1.6
-
0.4
0.4
-
0.4
0.4
-
ꢀg/mL
LHNO3
Ciprofloxacin
Flucanazole
16
8
3
2.5
2
LHAc
1.5
1
LHNO3
Flucanazole
0.5
0
A. fumigatus
C. albicans
Fig.12. Comparision of antifungal potencies.
22

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4. Conclusions
A novel Schiff base is synthesized by the condensation of 2-Hydrazinobenzimidazole
with diacetyl monoxime. The compound is successfully characterized by IR, Mass and NMR
spectral techniques. The new compound has crystallized in two forms viz. acetate and nitrate
salts. Acetate salt has crystallized in monoclinic P 21/n, while the nitrate salt has crystallized
in a triclinic crystal system with P -1 space group. Charge assisted hydrogen bonding
between anions and the organic motif has stabilized both the structures. DFT optimized
structures of salts as well as the bare organic motif, 3-(hydroxyimino)-2-butanone-2-(1H-
benzimidazol-2-yl)hydrazone, are close to the experimental crystal structures. Hirshfeld
surface analysis of the salts has indicated more intermolecular interactions in the case of
nitrate salt than in the case of acetate salt. Synthesized compounds have shown very
promising antifungal activity with the MIC value of 0.4 ꢀg/mL, proving the new compounds
to be excellent antifungal agents than the standard tested.
Acknowledgements
The authors thank USIC, Karnatak University, Dharwad, for providing spectral and
XRD facilities. One of the authors (Vinayak Kamat) is thankful to UGC-New Delhi for JRF
(101864/June 2013).
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Highlights
•
A novel Schiff base ligand 3-(hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)
hydrazone has been synthesized.
•
Crystal structure and Hirshfeld analysis is carried out for acetate and nitrate salts of
the title compound.
•
•
Structures of synthesized molecules are computationally optimized using DFT.
Antibacterial and antifungal activity of the synthesized compounds is presented.