A green approach to synthesize and in vitro antimicrobial activity of indeno-imidazole derivatives and ninhydrin-nucleophile adducts

Article abstract of DOI:10.14233/ajchem.2016.19775

Ninhydrin (1) reacted with weak nitrogen nucleophiles (2a-2f) in the presence of H2O/EtOH medium to give the corresponding indenoimidazole derivatives (3a-3d) and ninhydrin-nucleophile adducts (3e-3f). The structures of the compounds (3a-3f) were confirme

Full text of DOI:10.14233/ajchem.2016.19775

Asian Journal of Chemistry; Vol. 28, No. 7 (2016), 1611-1616
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2016.19775
A Green Approach to Synthesize and in vitro Antimicrobial Activity of
Indeno-Imidazole Derivatives and Ninhydrin-Nucleophile Adducts
*
*
SAMIA RIFAT , KHORSHADA JAHAN, U.K.R. ROMMAN , KAWSARI AKHTER and MD. ERSHAD HALIM
Department of Chemistry, University of Dhaka, Dhaka-1000, Bangladesh
Corresponding authors: Fax: +880 2 8615583; Tel: +880 01711549709; E-mail: dr.romman@gmail.com; samia2690chem@gmail.com
Received: 14 December 2015; Accepted: 17 February 2016; Published online: 31 March 2016; AJC-17859
*
Ninhydrin (1) reacted with weak nitrogen nucleophiles (2a-2f) in the presence of H
2
O/EtOH medium to give the corresponding indeno-
imidazole derivatives (3a-3d) and ninhydrin-nucleophile adducts (3e-3f). The structures of the compounds (3a-3f) were confirmed by
1
13
their UV, IR, H NMR, C NMR spectra and elemental analyses. The synthesized compounds were evaluated for their antimicrobial
activity by Kirby-Bauer disk diffusion method. Most of the compounds showed good to moderate antimicrobial activity.
Keywords: Ninhydrin, Weak nitrogen nucleophiles, Antimicrobial activity, Green chemistry.
(
Gram-negative) using Kirby-Bauer method. The primary
INTRODUCTION
purpose of the study was to evaluate antimicrobial potency of
synthetic products against the particular bacteria.
Imidazole nucleus is a constituent of many bioactive
heterocyclic compounds that are of wide interest because of
their diverse biological and clinical applications. This created
interest in researcher’s to synthesize variety of imidazole deri-
vatives and screen them for their various biological activities
i.e. antifungal drugs and antibiotics and the sedative midazolam
1-4]. Imidazole drug has become an important part in pharma-
ceuticals. Synthetic imidazoles are present in many fungicides
5], antifungal, antiprotozoal, antiviral, anti HIV, anti-infla-
EXPERIMENTAL
Purity of the newly synthesized compounds was checked
by TLC on silica gel plates (Merck, Silica gel G). Melting points
were determined on an electro thermal micro melting point
apparatus and are uncorrected. The UV spectra were measured
by a SHIMADZU-UV-166V ultraviolet Spectrophotometer by
using Ethanol as a solvent. The IR spectra were recorded on a
SHIMADZU-IR-470 infrared spectrophotometer by direct
[
[
mmatory, anticancer and hypertensive medications and drug
targets in inflammation, neurodegenerative disease and tumor
present in nervous system [6,7]. Several derivatives of indeno-
imidazole showed good antibacterial, antimicrobial and
Cholinesterase Enzymes inhibitory activities [8,9]. Thus
several methods [8-11] were reported for the synthesis of these
derivatives. Though these entire reactions produced somewhat
higher yield but they also suffer from the drawbacks with the
use of expensive and hazardous solvents. With this background
the objective of our research is to perform the reaction in a
neutral medium and accessible solvent which makes the
reaction framework more environmentally benign as well as
economic. Here we tried to develop a simple reaction method
for the synthesis of indeno-imidazole derivatives with higher
yield (Fig. 1) and their structures were confirmed by UV,
1
13
transmittance using KBr pellet techniques. H NMR and C
NMR spectra of the samples were recorded in DMSO on a
BRUKER 400 MHz NMR spectrophotometer.
General procedure of reactions: A reaction mixture of
ninhydrin (1), (10 mmol) and weak nitrogen nucleophiles [urea
(2a), thiourea (2b), diphenyl thiourea (2c), guanidine (2d),
cyanoacetamide (2e) and diphenyl amine (2f)] (10 mmol) in
1:1 molar ratio were well dissolved in water (2a-2c) and ethanol
(2d-2f). Then the mixture was stirred over water bath at room
temperature for 40-75 min. The solvent was removed by filtra-
tion to give the solid product which was then recrystallized
from water (3a-3c) and ethanol (3d-3f), respectively. The
physico-chemical data of the synthesized compounds are given
in Table-1.
1
13
IR, H NMR, C NMR spectra and elemental analyses. The
synthesized compounds (3a-3f) were evaluated for in vitro
antimicrobial activity against Staphylococcus aureus, Bacillus
cereus (Gram-positive); Escherichia coli, Salmonella typhimerium
in vitro Antimicrobial activity: The test organisms used
in this study were as follows: Bacillus cereus (ATCC 19637),
Staphylococcus aureus (Coagulate), Escherichia coli (ATCC

1
612 Rifat et al.
Asian J. Chem.
O
O
O
C
4
3
1
NH2
CH2
CN
8
9
OH
5
(
2e)
O
2
6
NH
C
CH CN
2
7
1'
2'
3'
4'
(
3e)
R
3
X
R
X
HO
N
2
O
4
7
9
OH
11
5
RHN
C
NHR
1
Water/EtOH
r. t.
N
1
0
6
4
0-75 min
(2a-2d)
2a, X=O, R=H
b, X=S, R=H
2c, X=S, R=-C H
12
8
OH
OH
2
O
O
6
5
(3a-3d)
(1)
2
d, X=NH, R=H
3
3
3
3
a, X=O, R =H
b, X=S, R=H
c, X=S, R =-C6H5
d, X=NH, R =H
O
4
8
9
3
OH
5
2
"
3
"
C H
NH
2f)
C H
5
2
6
5
6
(
6
1"
4"
1
N
7
6
'
1
'
O
6"
5
"
2
'
5'
(
3f)
3'
4'
Fig. 1. Synthetic route of indeno-imidazole derivatives (3a-3d) and ninhydrin-nucleophile adducts (3e-3f)
TABLE-1
PHYSICO-CHEMICAL DATA OF INDENO-IMIDAZOLE DERIVATIVES (3a-3d) AND NINHYDRIN-NUCLEOPHILE ADDUCTS (3e-3f)
Time
Solvent of TLC
(EtOAc: CHCl3)
Elemental analysis (%): Found (calcd.)
Yield
(%)
Ref. m.p.
(°C)
Compd.
R value
f
m.p. (°C)
(min)
C
H
N
3
a
50
45
45
75
40
60
4:1
3:2
3:2
3:2
2:3
53.09 (54.55)
50.18 (50.85)
68.10 (68.04)
53.01 (54.79)
55.75 (59.01)
74.26 (76.59)
3.22 (3.64)
3.27 (3.39)
6.58 (7.22)
3.79 (4.11)
2.55 (3.27)
3.83 (4.55)
12.01 (12.73)
11.09 (11.86)
3.99 (4.12)
19.11 (19.18)
12.53 (11.48)
4.15 (4.26)
0.48
0.57
0.68
0.51
0.49
0.56
98
98
95
92
91
96
216-218
220-224
210-212
212-214
172-176
152-154
–
3b
220-222 [5]
208-212 [6]
3
c
3d
–
–
–
3
3
e
f
5 (CHCl )
3
20120829) and Salmonella typhimerium (JCM-1692). Routine
RESULTS AND DISCUSSION
culture was performed on Tryptone Soya Broth (pH 7.3 ± 0.2
at 25 °C) in a required number inoculation tube. 5 falcon tube
containing 5 mL nutrient rich media each was inoculated
through sterilized loop tip with each isolated bacteria strain.
The incubation of the strain for the antimicrobial test was
executed in an aerobic chamber at 37 °C in an incubator for
The one-pot synthesis 3a,8a-dihydroxy-tetrahydro indeno-
imidazole derivatives and ninhydrin-nucleophile adducts
(Fig. 1) were obtained by the two component condensation of
ninhydrin (1) and weak nitrogen nucleophiles (2a-2f). All of
the reaction was performed at room temperature condition in
water (3a-3c) and ethanol (3d-3f) media. The structures of
2
4 h. After that the growth was checked by a cloudy haze in
1
the media. Test sample were prepared in DMSO with concen-
tration 1 mg/mL.
(3a-3f) was confirmed with the help of their UV, IR, H NMR
13
and C NMR spectra and elemental analyses.

Vol. 28, No. 7 (2016)
Synthesis of Indeno-Imidazole Derivatives and Ninhydrin-Nucleophile Adducts 1613
The absorption bands in the range 246-267 nm may be
assigned to the π → π* transition of C=O in these compounds.
The n → π* transition of these compounds due to C=O group
were assigned in the range of 332-355 nm. The observed λmax
values in the UV spectrum (Table-2) of compounds (3a-f) agree
well to the expected values [12].
The N-H protons at positions 1 and 3 in compounds (3a-
3d) were appeared as singlet in their H NMR spectra [Table-
1
3(a)] and at a range of δ 6.92-6.46. The N-H protons at position
1 in these compounds were found comparatively more
deshielded than protons at position 3. In compound (3b) more
deshielding of the N-H protons were observed due to presence
of thiocarbonyl group. This may be attributed to the greater
polarizability of sulfur in comparison to oxygen (3a)and nitrogen
(3d). The hydroxyl proton at position 9 and 10 in (3a-3d)
appeared as a singlet in a deshielded region. The chemical
shifts were observed at δ 7.88-10.71. The highly deshielded
region is attributed in compound 3b and 3c due to the presence
of hydrogen bonding [8,9]. Compounds 3a and 3d also follow
the same pattern. All aromatic protons of structures (3a-3d)
were observed at δ 7.97-7.13.
In Table-3(b), the hydroxyl proton of compounds (3e-3f)
at position-2 was observed as singlet at a range δ 6.78-6.81.
All aromatic protons of compounds (3e-3f) were assigned in
the range δ8.05-7.00. For compound 3e two singlet’s for 1′-H
and 3′-H were observed at δ 6.97 and δ 3.35, respectively.
This presence of -CO- group adjacent to the –NH group might
account for the highly deshielded region.
Infrared of the compounds (3a-f) (Table-2) showed sharp
-1
bands in the range of (νmax) 3569-3409 cm , indicating the
presence of O-H group. Compound 3b and 3c showed –OH
-1
stretching bands at 3428 and 3409 cm , respectively indicating
the presence of intermolecular hydrogen bonding [8,9]. The
-1
compounds 3a and 3d showed bands at 3428 and 3450 cm ,
respectively due to the same reason. The compounds 3e showed
-1
-1
a band at 3569 cm and 3f at 3650 cm indicate a ‘free’ sharp
O-H stretching for both of them. The absorption bands at 3317-
-1
3
307 and 1552-1511 cm indicate the presence of N-H stretching
and bending modes, respectively for compounds 3a 3b and 3d.
-1
The bands at 3133-3031 and 958-693 cm were assigned to
aromatic -CH stretching and bending, respectively. The absorp-
-1
tion band at 1741-1704 cm indicate the presence of C=O group.
-1
The absorption bands at 1610-1597 and 1494-1432 cm indicate
the presence of C=C of aromatic rings. Additional bands were
-1
observed at 1365-1269 and 1293-1055 cm due to the presence
The structure of the compounds (3a-3f) was further confir-
13
of C-N and C-O bonds, respectively. For compound (3e), 3268,
med by their C NMR spectral data (Table-4a). The chemical
shifts of carbonyl carbon at 8-C for compounds (3a-3d) were
found to be deshielded in the range of δ 198.01-194.10.
2
787, 2252 and 1631 were assigned for –NH, –CH (aliphatic
2
stretching), C≡N and -CONH bond, respectively.
2
TABLE-2
IR AND UV SPECTRAL DATA OF THE COMPOUNDS (3a-3f)
-1
UV, λmax
IR (cm )
(
nm)
Compd.
*
Aromatic
Aromatic
Aromatic
(–CH bend.)
π–
π*
ν(–OH)
ν
(C=O)
ν
(C–N)
ν
(C–O)
Other functional group
ν(–CH str.)
ν(C=C str.)
ν
n–
π
1
1
234,
107
958, 894,
773, 706
3310 –NH (s), 1511 –NH (b),
3
a
3428
3428
3409
3450
3128
3128
3133
3060
1733
1725
1710
1736
1602, 1446
1305
1269
1365
1355
267,341
267,339
343
1682 (CO-NH )
2
1
1
1
234,
1
957, 893,
773, 706
3b
1602, 1450
1610, 1494
1605, 1490
3307 –NH (s), 1511 –NH (b)
–
107
292,
1
946, 896,
776, 737, 693
3
c
055
293,
3d
902, 840, 698 3317 –NH (s), 1552 –NH (b)
246,342
1
220
3
268 –NH, 2787 (-CH2),
1
1
205,
087
950, 902,
874, 774
3
e
3569
3560
3039
3031
1741
1704
1602, 1432
1597, 1453
1296
1319
2252 (C≡N),
631 (-CO-NH )
253,355
332
1
2
1
1
258,
186
878, 814,
749, 695
3
f
–
TABLE-3a
H NMR (DMSO) SPECTRAL DATA OF COMPOUNDS (3a-3d) (δ ppm)
1
Compd.
3-H
2-H
1-H
Aromatic
9-H
10-H
3
a
6.46 (s, 1H, NH)
6.84 (s, 1H, NH)
6.57 (s, 1H, NH)
6.92 (s, 1H, NH)
7.85-7.57 (m, 4H, 4, 5, 6, 7)
7.97-7.61 (m, 4H, 4, 5, 6, 7)
7.97-7.77 (m, 4H, Ar-H);
7.88 (s, 1H, OH)
9.49 (s, 1H, OH)
10.09 (s, 1H, OH)
8.04 (s, 1H, OH)
9.77 (s, 1H, OH)
10.34 (s, 1H, OH)
3b
3
c
7.57-7.13 (m, 10H, Ar-H)
3d
6.85 (s, 1H, NH) 6.89 (s, 1H, NH) 6.87 (s, 1H, NH)
7.96-7.57 (m, 4H, 4, 5, 6, 7)
10.64 (s, 1H, OH)
10.71 (s, 1H, OH)
TABLE-3b
H NMR (DMSO) SPECTRAL DATA OF COMPOUNDS (3e-3f) (δ ppm)
1
Compound
2-H
Aromatic
1'- H
3'-H
3.35 (s, 2H, -CH )
2
3
e
6.78 (s, 1H, OH)
6.81 (s, 1H, OH)
7.98-7.63 (m, 4H, 4, 5, 6, 7)
8.05-7.00 (m, 14H, Ar-H)
6.97 (s, 1H, NH)
–
3
f
–

1
614 Rifat et al.
Asian J. Chem.
TABLE-4a
1
3
C NMR (DMSO) SPECTRAL DATA OF COMPOUNDS (3a-3d) (δ ppm)
Compound
8-C (C=O)
198.01
196.33
2-C
Aromatic carbons (6C)
152.05-123.55
10-C
86.77
90.12
88.73
9-C
3
3
a
b
156.85
178.26
178.62
86.77
89.61
88.24
150.83-123.58
150.62-127.88
3
c
194.43
136.29-125.19 (1'-6')
1
36.17-125.40 (1"-6")
3
d
194.10
153.39
149.03-126.06
92.58
89.51
In the compound 3a the chemical shifts of carbonyl
at 2-C were found to be at δ 178.62-178.26. This explains that
the replacement of a carbonyl group by a thiocarbonyl group
results in a downfield shift [13]. In compound 3d, the 2-C
carbon is appeared in the most shielded region (153.39)
carbons at 2-C was found to be at δ 156.85 which is relatively
less deshielded due to the resonance of amide functional group.
In the compounds 3b, 3c the chemical shifts of thioxo carbon
X
RHN
C
NHR
O
O
(2a-2d)
O
H
O
OH
H O
2
H
O
O
OH
H
O
O
O
(
1)
(3)
(
2)
Proton from
solvent
R
H
N
X
R
X
R
HO
N
O
C
NHR
OH
OH
N
H
OH
OH2
(
4)
(
5)
O
O
R
X
HO
N
N
R
OH
O
(
3a-3d)
Fig 2. Plausible reaction pathway for compounds (3a-3d)

Vol. 28, No. 7 (2016)
Compound
Synthesis of Indeno-Imidazole Derivatives and Ninhydrin-Nucleophile Adducts 1615
TABLE-4b
13
C NMR (DMSO) SPECTRAL DATA OF COMPOUNDS (3e-3f) (δ ppm)
1-C
2-C
3-C
Aromatic carbons (6C)
148.58 - 130.63
2'-C
161.94
–
3'-C
58.01
–
4'-C
122.81
–
3e
195.86
199.71
85.54
78.83
195.76
199.82
3f
152.81-127.71
1
42.80-116.65(1′-6′)
142.43-116.47(1″-6″)
compared to 2-C of (3a-3c). This might account for the lower
polarizability of nitrogen in comparison to oxygen (3a) and
sulfur (3b-3c). The chemical shift values for 10-C and 9-C in
these compounds were observed at δ 92.58-86.77 and 89.61-
measured on the underside of the plates with a metric ruler.
Five zone diameters for particular compounds were recorded
on different plates. The disk potency and their zone of inhibition
are summarized in Table-5.
86.77, respectively. The 10-C of the compounds showed chemical
shift values in slightly deshielded region due to the presence
of electron withdrawing group adjacent to it. All aromatic
carbons showed chemical shift value at δ 152.05-123.55 which
had a good correlation with literature value [12].
TABLE-5
ANTIMICROBIAL TEST DATA OF COMPOUNDS (3a-3f)
Disk
potency
(µg/mL)
Zone of inhibition (mm)
Compd.
S.
E.
coli
S.
B.
cereus
In compounds (3e-3f), the chemical shifts of carbon at
position 1-C and 3-C were observed in a range of δ 195.76-
aureus
typhimerium
3a
3b
30
6
19
13
6
6
6
6
20
19
6
6
6
6
6
6
6
6
6
19
21
21
19
20
17
30
1
99.82 (Table-4b). All aromatic carbons of these compounds
3c
30
30
30
30
were observed at δ 148.58-116.47. The carbon at 2′-C, 3′-C
and 4′-C were assigned at δ 161.94, 58.01 and 122.81,
respectively (Table-4b).
According to Fig. 2, the general reaction mechanism of
the compounds (3a-3d) is proceed by the plausible pathway.
While, the general reaction mechanism [14] of compounds
3d
3e
3f
Standard
control
30
19
19
20
15
(
3e-3f) follow the plausible pathway given in Fig. 3.
Compounds (3a-3e) and 3f showed strong and intermediate
(
17 mm Z.D.) inhibitory activity, respectively for Bacillus cereus
O
O
compared with nalidixic acid (15 mm). Compounds 3b and
3
c showed strong and intermediate inhibitory activity for
OH
Staphylococcus aureus, respectively. Compound 3b and 3c
both had strong activity against Escherichia coli. Compound
O
+
Ph
N
Ph
OH
H
3a, 3eand 3f are found to be resistant for strain of Staphylococcus
(2f)
O
O
aureus, Escherichia coli and Salmonella typhimerium.
(1)
(2)
Conclusion
After investigating literature no earlier reports were found
for the synthesis of products (Fig. 1) using water (3a-3d). The
reaction time is very short and a high yield of product is
obtained. The method neither involved the use of any high
boiling solvents nor the use of costly and environmentally toxic
catalyst. Therefore the performances of reaction are green in
nature and hundred percent atoms economic. Most of the syn-
thesized compounds showed good to moderate antimicrobial
activity. Kirby Bauer disc-diffusion test demonstrated the result
for the 30 µg concentration of particular compound (3a-3f)
are summarized in Table-6.
O
O
O
OH
H
N
Ph
N
Ph
Ph
O
O
Ph
(
3)
(3f)
Fig. 3. Plausible reaction pathway for compounds (3e-3f)
Microbial test for compounds (3a-3f): Susceptibility
tests were performed by disk diffusion method of Bauer et al.
15]. Zone of inhibition were measured after 24 h of incubation.
Commercial antibiotic (nalidixic acid) was used as controls.
Bacteria were transferred directly to the prepared petri plate
TABLE-6
[
ANTIMICROBIAL DATA OF COMPOUNDS (3a-3f)
S.
Compd.
S. aureus
E. coli
B. cereus
typhimerium
(
containing 20 mL Tryptone Soya Agar, pH 7.4 in each plate)
3
a
Resistant
Susceptible
Intermediate Susceptible Resistant
Resistant
Resistant
Resistant
Resistant
Susceptible Resistant
Resistant
Susceptible
Susceptible
Susceptible
Susceptible
Susceptible
Intermediate
with a wet swab containing the bacterial broth culture. The
synthetic compounds impregnated disks were placed on the
surface of the agar, using forceps. In each 100 mm plate 5
disks were placed for individual kind of bacteria. All plates
then incubated at 37 °C overnight. Zone of inhibition were
3b
3
c
3d
3e
3f
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant

1
616 Rifat et al.
Asian J. Chem.
8
.
R.M. Ghalib, R. Hashim, S.F. Alshahateet, S.H. Mehdi, O. Sulaiman,
K.-L. Chan, V. Murugaiyah and A. Jawad, J. Chem. Crystallogr., 42,
783 (2012).
REFERENCES
1
.
J.D. Hepworth, C.D. Gabbutt and B.M. Heron, in eds.: A.R. Katritzky,
C.W. Rees and E.F. Scriven, In Comprehensive Heterocyclic Chemistry
II: Pyrans and their Benzo Derivatives: Synthesis, Pergamon Press,
Oxford, vol. 5 pp. 351-468 (1984).
9. R.M. Ghalib, R. Hashim, S.F. Alshahateet, S.H. Mehdi, O. Sulaiman,
V. Murugaiyah and C.A. Aruldass, J. Mol. Struct., 1005, 152 (2011).
10. M. Chakrabarty, A. Mukherji, S. Arima, Y. Harigaya and G. Pilet,
Monatsh. Chem., 140, 189 (2009).
11. R.M. Shaker, A.F. Mahmoud and F.F. Abdel-Latif, J. Chil. Chem. Soc.,
52, 563 (2005).
12. D.L. Pavia, G.M. Lampman and G.S. Kriz, Introduction to Spectroscopy,
Thomson Brooks/Cole, edn 3 (2001).
13. H.H. Otto and J. Triepel, Liebigs Ann. Chem., 1982 (1976).
14. M.G. Ahmed, S.A. Ahmed, U.Q.R. Romman and M.K. Alam, J.
Bangladesh Acad. Sci., 5, 13 (1981).
2
3
4
5
6
7
.
.
.
.
.
.
M.R. Grimmett, Imidazole and Benzimidazole Synthesis,Academic Press
(
1997).
E.G. Brown, Ring Nitrogen and Key Biomolecules, Kluwer Academic
Press (1998).
A.F. Pozharskii, A.T. Soldatenkov and A.R. Katritzky, Heterocycles in
Life and Society, John Wiley & Sons, New York (1997).
L. Shargel, A.H. Mutnick, P.F. Souney and L.N. Swanson, Comprehensive
Pharmacy Review, Lippincott Williams &Wilkins, edn 6, p. 930 (2006).
T. Castaño, A. Encinas, C. Pérez, A. Castro, N.E. Campillo and C. Gil,
Bioorg. Med. Chem., 16, 6193 (2008).
15. A.W. Bauer, W.M. Kirby, J.C. Sherris and M. Turck, Am. J. Clin. Pathol.,
45, 493 (1966).
R.G. Bogle, G.S. Whitley, S.C. Soo, A.P. Johnstone and P. Vallance,
Br. J. Pharmacol., 111, 1257 (1994).