Evaluation of some classical hydrazones of ketones and 1,2-diketones as antileishmanial, antibacterial and antifungal agents

Article abstract of DOI:10.1007/s12272-012-0608-7

The paper describes the synthesis and antimicrobial (antileishmanial, antibacterial and antifungal) activity of some classical hydrazones of benzophenones and of 1,2-diketones. N-(Diaryl) acyl derivatives of these hydrazones have also been synthesized and evaluated. 4,4,- Demthoxybenzil monohydrazone and 4,4'-dimethoxybenzophenone hydrazone showed significant antileishmanial activity. The effect of substituents on the bioactivity is discussed.

Full text of DOI:10.1007/s12272-012-0608-7

Arch Pharm Res Vol 35, No 6, 1009-1013, 2012
DOI 10.1007/s12272-012-0608-7
Evaluation of Some Classical Hydrazones of Ketones and 1,2-Diketones
as Antileishmanial, Antibacterial and Antifungal Agents
Yasser M. S. A. Al-kahraman1, Masoom Yasinzai2, and Girija S. Singh3
2
1Institute of Biochemistry, University of Baluchistan, Quetta, Pakistan, Quaid-I-Azam University, Islamabad, Pakistan, and
3Chemistry Department, University of Botswana, P/Bag: 0022, Gaborone, Botswana
(Received October 16, 2011/Revised December 8, 2011/Accepted December 30, 2011)
The paper describes the synthesis and antimicrobial (antileishmanial, antibacterial and anti-
fungal) activity of some classical hydrazones of benzophenones and of 1,2-diketones.
N-(Dia-
ryl)acyl derivatives of these hydrazones have also been synthesized and evaluated. 4,4,-
Demthoxybenzil monohydrazone and 4,4’-dimethoxybenzophenone hydrazone showed signifi-
cant antileishmanial activity. The effect of substituents on the bioactivity is discussed.
Key words: Hydrazones,
N-(diaryl)acyl hydrazones, Antileishmanial, Antibacterial, Antifungal
for several weeks. Recent years have witnessed consider-
able interest in design and development of antileishmanial
INTRODUCTION
The infectious diseases are the second major cause compounds. Many compounds containing carbon-ni-
of death worldwide and third leading cause of death in trogen double bond in different structural environments
economically advanced countries (Nathan, 2004). Bac- are reported to have promising antileishmanial activ-
terial strains are getting resistant towards antibiotics ity (Croft et al., 2006; Khan et al., 2008). We recently
in clinical use. The ability of bacteria to deceive any launched a program to synthesize diverse types of
kind of conventional therapy has become apparent nitrogen and sulfur containing compounds and to
and pathogens resistant to one or more antibiotics are evaluate them as antileishmanial agents. We observ-
emerging and spreading worldwide (Clark et al., 2003). ed significant antileishmanial activity in some
N-sub-
Unnecessary use of antibiotics has further fuelled this stitutes imines from benzaldehydes, cinnamaldehyde,
problem. The discovery of vancomycin resistant Sta- furan-2-carbaldehyde; thiophene-2-carbaldehyde, sali-
phylococcus aureus (VRSA) and multiresistant S. aureus cylaldehyde and isatin (Al-Kahraman et al., 2010, 2011).
has generated worldwide concern. It has thus become It was therefore considered pertinent to evaluate hydra-
evident that there is urgent need for novel antibacterial zones for their antileishmanial, and also antibacterial
drugs with broader spectrum, less side effects, and and antifungal activities. In the present paper, we wish
without cross-resistance to antibiotics in use.
to report the results of antileishmanial, antibacterial
Leishmaniasis is a tropical disease caused by infec- and antifungal screening of some hydrazones and
N-
tion of protozoa, Leishmania. It has been recognized by (diaryl)acyl hydrazones derived from benzophenones
the World Health Organization (WHO) as an increas- and benzils that have never been evaluated for their
ing health problem (Modabber, 1991). Many parts of bioactivity.
Asia and Africa are vulnerable to leishmaniasis. Most
of the medicines available currently in the market for
treatment of leishmaniasis are costly, have side-effects,
and are getting resistant to pathogen after treatment
MATERIALS AND METHODS
Materials
Melting points (m.p.) have been recorded on a Stuart
Scientific melting point apparatus and are uncorrect-
ed. The IR spectra were recorded on a Perkin-Elmer-
781 IR spectrophotometer using KBr disc of the sample.
The NMR spectra were recorded on a Jeol FX 90Q
Correspondence to: Girija S. Singh, Chemistry Department,
University of Botswana, P/Bag: 0022, Gaborone, Botswana
Tel: 267-3552501, Fax: 267-3554578
E-mail: singhgs@mopipi.ub.bw
1009

1010
Y. M. S. A. Al-kahraman et al.
spectrometer using TMS as an internal standard.
The ketones and hydrazine hydrate were Aldrich Parasite culture (100 µL) was added in all wells. In
(20
µ
L) was added in medium and serially diluted.
products. The 2-diazo-1,2-diarylethanones were syn- negative controls, DMSO was serially diluted in medium
thesized according to the reported method (Nenitzescu while the positive control contained varying concen-
and Solomonica, 1950).
trations of standard antileishmanial compound i.e.
Leishmania major was cultured from the blood amphotericin B. The plates were incubated for 72 h at
taken directly from the infected patients at the Bollan 24oC. The culture was examined microscopically on an
Medical College, Quetta, Pakistan, in artificial medium improved Neubauer counting chamber and IC50 values
199. The bacterial and fungal cultures, identified pre- of compounds possessing antileishmanial activity
viously by 16S and 18S rRNA, were obtained from the were calculated. All the assays were run in duplicate.
H. E. J. Karachi University, Pakistan.
IC50 of samples was determined by using the Prism
software.
Synthesis of hydrazones 1-3
To a warmed solution containing appropriate 1,2-
diketone (10 mmol) in 20 mL of methanol was added
Antibacterial activity
In vitro evaluation of antibacterial activity was carried
slowly with stirring 15 mmol of hydrazine hydrate out by disk diffusion method (Kirby-bauer method)
(80%, aq.). The solution was heated under reflux for 15 (Cappuccino and Sherman, 1996) against Staphylococcus
min and then allowed to attain the room temperature. aureus, Bacillus subtilis, Klebsiella pneumonia and
The white solid appeared in the flask that was filtered Escherichia coli. The tests were repeated three times
and washed with cold methanol (Nenitzescu and and the results are reported as means of least three
Solomonica, 1950).
times. The minimum inhibitory concentrations (MICs)
of the compounds were recorded as the lowest concen-
tration of each of the compounds in Petri plates with
Synthesis of hydrazones 4-6
The solution of appropriate ketone (10 mmol), hydra- no turbidity (i.e. no growth) of inoculated bacteria.
zine hydrate (15 mmol, 80%, aq.) in 1 mL of n-butanol
was heated under reflux for 2-4 h. The hot solution was
poured into 5 mL of methanol. The white solid obtained
Antifungal activity
The antifungal bioassay was performed by the Agar
was filtered and recrystallized from methanol (Baltzly Tube Dilution Method (Choudhary et al., 1995), in
et al., 1961).
which the test Compounds were screened for activity
against the following organisms: Candida albicans,
Microsportum canis and Fusarium solani and the per-
Synthesis of
N-(diaryl)acyl hydrazones 7-11
A solution of appropriate hydrazone and 2-diazo-1,2- centage inhibition is reported. In all tests, the linear
diarylethanone (10 mmol of each) in 60 mL of dry growth in control was 100 mm.
benzene was refluxed under nitrogen atmosphere for
8 h. The solvent was removed under reduced pressure
and the residue obtained was triturated with ethanol
to get white crystalline product (Singh et al., 1984;
Singh, 2004).
RESULTS
The hydrazones 1-3 were synthesized by refluxing
the appropriate 1,2-diketone with hydrazine hydrate
in methanol (Scheme 1). The hydrazones were pre-
pared by refluxing appropriate ketones with hydrazones
4-6
Antileishmanial activity
The title compounds were screened for their antilei- in minimum amount of tert-butanol (Scheme 2). The
shmanial activity against the pre-established culture -(diaryl)acyl hydrazones were prepared by refluxing
N
of L. major. Parasites were cultured in Medium M199 equimolar solution of appropriate ketohydrazone and
with 10% foetal bovine serum; 25 mmol HEPES, and 2-diazo-1,2-diarylethanones 12 (acylating agent) in
0.22 mg of penicillin and streptomycin, respectively at dry benzene (Scheme 3). The physical data of all the
24oC in a shaking incubator (Ali et al., 1997).
Each compound to be tested and amphotericin B (as
a positive control) were dissolved in DMSO to a con-
centration of 1 mg/mL. Parasites at log phase were
centrifuged at 3000 rpm for 3 min. Parasites were
diluted in fresh culture medium to a final density of 2
× 106 cells/mL. In 96-well plates, 180
was added in different wells. Experimental compound
µ
L of medium
Scheme 1. Preparation of benzil monohydrazones 1-3.

Evaluation of Some Classical Hydrazones
1011
Table I. Physical data and antileishmanial activity of
hydrazones 1-11
Yield m. p.
(%)
Compds. No.
Mol Formula* L. major*
(oC)
1
2
3
94 149-151 C14H12N2O 0.73 ± 0.03
92 141-142 C16H16N2O 0.62 ± 0.08
88 145-147 C14H16N2O3 0.58 ± 0.05
Scheme 2. Preparation of benzophenone hydrazones 4-6.
4
5
6
7
8
9
10
11
85
90
80
97-98
85-86 C15H16N2O2 0.59 ± 0.01
103 C13H11N2Cl 0.60 ± 0.02
C13H12N2 0.62 ± 0.01
compounds are shown in Table I.
The results of in vitro screening against L. major
are shown in Table I. All the compounds have been
screened for antibacterial and antifungal acitivites
and the results are described in Tables II and III,
respectively.
80 147-149 C27H22N2O 0.89 ± 0.04
56 119-120 C29H26N2O3 0.70 ± 0.09
52
80 147-148 C29H26N2O 0.59 ± 0.04
72 144-146 C29H25N2OCl 0.67 ± 0.02
97-99 C27H21N2OCl 0.90 ± 0.01
DISCUSSION
DMSO as
−ve
0.99 ± 0.00
Control
Chemistry
Standard Drug IC50
(1 g/mL ± S.D.)
(Amphotericin B)
All the hydrazones were characterized on the basis
of comparison of m.p. and spectral (IR, and NMR) data
with the literature value (Nenitzescu and Solomonica,
1950; Baltzly et al., 1961; Singh et al., 1984; Singh,
µ
0.56 + 0.06
*Percentage inhibition activity: 0.99 = insignificant; 0.95-0.80
= low; 0.79-0.70 = moderate; 0.69-0.60 = good; < 0.59-0.56
= significant activity.
2004). Briefly, the IR spectra of hydrazones
1-3 ex-
hibited four absorption bands at 3400-3430 cm−1,
3280-3305 cm−1, 1620-1630 cm−1, and 1605-1610 cm−1. 1605 cm−1, respectively. The IR spectra of hydrazones
The first two bands correspond to the symmetric and
7-
11 showed absorptions at 3280 cm−1, 1660 cm−1 and
asymmetric stretching vibrations of the amino group 1600 cm−1 due to NH, amido carbonyl and azomethine
1
whereas the latter two bands correspond to the car- linkage, respectively. The H-NMR spectra of hydra-
bonyl group and azomethine linkage, respectively. The zones 7-11 displayed the typical singlet signal at around
IR spectra of hydrazones 4-6 showed two high frequency 6.1 ppm corresponding to the methine proton present
1
absorption bands - one in the range of 3380-3410 cm−
in the (diaryacyl) group.
The formation of -(diaryl)acyl hydrazones
and been explained as shown in Scheme 3 by the reaction
showed absorptions at 1610 cm−1, 1635 cm−1 and of diarylketenes 14, generated in situ by thermal de-
and other in the range of 3280-3295 cm−1 due to amino
N
7-11 has
group. The azomethine linkage in hydrazones 4, 5
6
Scheme 3. Preparation of
N-(diaryl)acyl hydrazones 7-11 and mechanism of their formation.

1012
Y. M. S. A. Al-kahraman et al.
Table III. Antifungal activity of hydrazones
1-
11
composition of the diazoketones 12 via the Wolff-rear-
rangement of the initially formed aroyl aryl carbenes
13, with ketohydrazone leading to formation of the
intermediate 15 (Singh, 2004). A 1,3-proton transfer
Compds. No.
C. albicans M. canis
F. solani
1
2
3
4
5
6
7
8
9
*20*
30
41
−
52
−
38
43
62
31
55
−
24
16
20
29
38
−
22
27
28
24
20
27
32
21
33
30
63
−
34
21
60
in this intermediate leads to the formation of
acyl hydrazones 11
N-(diaryl)
7-
.
Antileishmanial activity of hydrazones 1-11
The -unsubstituted hydrazones and bearing
N
3
5
strong electron donating methoxy group showed signi-
ficant activity (0.58 ± 0.05 and 0.59 ± 0.01, respecti-
vely) against L. major which were almost parallel to
10
11
the standard drug amphotericin. The hydrazones
and 11 showed good activity. The hydrazone
showed moderate activity on L. major. Thus, the
unsubstituted hydrazones were observed more active
than the -substituted series. The high in vitro anti-
1, 2,
4,
6
8
DMSO as
−ve Control
0
0
0
N-
miconazole Standard
drug (2 mg/1 mL)
110.8
98.4
73.25
N
*Percentage inhibition activity: 0-39 = low (insignificant); 40-
59 = moderate; 60-69 = good; > 70 = significant activity.
leishmaniasis activity of these compounds makes them
promising leads for development of effective therapeutic
agents.
inhibition zone) and 11 (with 67% inhibition zone).
The MIC of compounds
3 and 11 for this strain was
Antibacterial activity of hydrazones 1-11
According to the data shown in Table II, all the observed as 125 and 500 g/mL, respectively. The hydra-
hydrazones exhibited good to moderate inhibitory zone with 48% inhibition zone (MIC = 500 g/mL)
activity against the two Gram (+) strains, S. aureus was the most active against S. aureus
9
.
and B. subtilis compared to the standard drug im-
ipenem at the tested concentration. Many of them were
moderately active against K. pneumoniae. Almost all
Antifungal activity of hydrazones 1-11
It is evident from the antifungal bioassay that the
of them were inactive against E. coli. The two most compound
active hydrazones against B. subtilis are (with 62% whereas compounds
activity against this fungus. Compounds
9
has good activity against C. albicans
and 11 exhibit modearte
and 11
3
3, 5, 8
7
have good activity against Microsportum canis. All the
compounds showed only poor or insignificant activity
on F. solani.
In conclusion, the study reports for the first time
observation of significant antileishmanial activity in
some simple hydrazones and good to moderate anti-
bacterial and antifungal activities. The high in vitro
Table II. Antibacterial activity of hydrazones
1
-
11
) strains
E.
Gram (+) strains
S. B.
aureus subtilis pneuminiae coli
Gram (−
Compds.
No.
K.
1
2
−
6
*5*
8
6
−
−
−
−
−
−
−
−
−
−
3
4
2
−
11
−
7
2
antileishmaniasis activity of compounds
3 and 5 makes
them promising leads for development of effective
antileishmanial agents.
5
8
4
5
6
7
8
9
10
11
3
6
1
10
6
6
8
4
7
8
12
−
2
3
4
2
4
ACKNOWLEDGEMENTS
The authors are grateful the Chemistry Department,
University of Botswana, and the Institute of Biochem-
istry, University of Baluchistan, for providing the
facilities.
1
−
9
DMSO as
−ve
0
0
0
0
Control
Imipenem Standard
drug (2 mg/1 mL)
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