Synthesis, characterization and antimicrobial evaluation of novel derivatives of isoniazid

Article abstract of DOI:10.1007/s00044-011-9634-0

In the present investigation, a series of new Mannich bases were prepared by the reaction of 2-ethoxybenzaldehyde with isoniazid to form acid hydrazone (3a). Further, C-Mannich bases of the above acid hydrazone were prepared by aminomethylation with formaldehyde and substituted secondary amines (3b-3k). The structures of newly synthesized compounds were evaluated by elemental analyses and spectral (IR, ¹H NMR, ¹³C NMR) studies. All the synthesized compounds were evaluated for their antimicrobial activity. Amoxicillin was used as a standard drug for antibacterial activity while Nystatin was used as a standard drug for antifungal activity. Preliminary pharmacological evaluation revealed that the compound (3f, 3i, 3j, 3k) showed better performance against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans and Candida gabrata. The result demonstrates the potential and importance of developing new mannich bases which would be effective against resistant bacterial and fungal strain. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9634-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1237–1244
DOI 10.1007/s00044-011-9634-0
ORIGINAL RESEARCH
Synthesis, characterization and antimicrobial evaluation of novel
derivatives of isoniazid
•
Manav Malhotra Sagun Sharma Aakash Deep
•
Received: 16 September 2010 / Accepted: 24 March 2011 / Published online: 10 April 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In the present investigation, a series of new
Mannich bases were prepared by the reaction of 2-eth-
oxybenzaldehyde with isoniazid to form acid hydrazone
(3a). Further, C-Mannich bases of the above acid hydra-
zone were prepared by aminomethylation with formalde-
hyde and substituted secondary amines (3b–3k). The
structures of newly synthesized compounds were evaluated
by elemental analyses and spectral (IR, 1H NMR, 13C
NMR) studies. All the synthesized compounds were eval-
uated for their antimicrobial activity. Amoxicillin was used
as a standard drug for antibacterial activity while Nystatin
was used as a standard drug for antifungal activity. Pre-
liminary pharmacological evaluation revealed that the
compound (3f, 3i, 3j, 3k) showed better performance
against Bacillus subtilis, Staphylococcus aureus, Pseudo-
monas aeruginosa, Escherichia coli, Candida albicans and
Candida gabrata. The result demonstrates the potential and
importance of developing new mannich bases which would
be effective against resistant bacterial and fungal strain.
Introduction
Morbidity and mortality caused due to bacterial and fungal
infections are the major cause of health problem in
developing countries (Qadri et al., 2005; Devasia et al.,
2006). Millions of people were infected and around 20,000
deaths were reported in the tropical regions every year
because of bacterial infections (Datta et al., 1974). So,
there is an urgent need for identification of novel lead
structure for designing of new, potent and less toxic agents
which ideally shorten the duration of therapy and are
effective against the resistant strain (Murphy et al., 2007).
Hydrazones belong to the Schiff base family containing
azomethine –NHN=CH– protons and they are considered
as the important class of compounds for the development
of new drugs (Rollas and Kucukguzel, 2007). Hydrazones
have been reported to posses antimicrobial (Rollas et al.,
2002), antitubercular (Imramovsky et al., 2007; Janin,
2007), antileprotic (Buuhoi et al., 1956), anticonvulsant
(Dimmock et al., 2000), analgesic (Lima et al., 2000), anti-
inflammatory (Salgin-Goksen et al., 2007; Kalsi et al.,
1990), antiplatelet (Silva et al., 2004), anticancer (Savini
et al., 2004; Bijev, 2006), antifungal (Lonce et al., 2004),
antiviral (Abdel-Aal et al., 2006), antitumor (El-Hawash
et al., 2006; Cocco et al., 2006), antibacterial (Capilla
et al., 2003), and antimalarial (Walcourt et al., 2004)
activities. The first hydrazine derivative is characterized by
Fischer and Deutsch (1875). Hydrazones have been
received much attention and many studies have been
reported due to their chemotherapeutic value in the
development of novel antimicrobial agents (Papakonstan-
tinou et al., 2002; Vacini et al., 2002; Masunari and Tav-
ares, 2007; Joshi et al., 2008; Kumar et al., 2009; Ozdemir
et al., 2009). Hydrazones are used as plasticizers, poly-
merization initiators, antioxidants, and in the determination
Keywords Mannich bases ꢀ Hydrazones ꢀ Isoniazid ꢀ
Antibacterial & antifungal activity
M. Malhotra ꢀ S. Sharma
Research Scholar, Department of Pharmaceutical Chemistry,
ISF College of Pharmacy, Ferozpur Road, Moga 142001,
Punjab, India
A. Deep (&)
Department of Pharmaceutical Sciences,
G.V.M. College of Pharmacy, Sonepat 131001, India
e-mail: aakashdeep82@gmail.com
123

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Med Chem Res (2012) 21:1237–1244
of carbonyl containing compound (Singh et al., 1982).
Antimicrobial resistance to a drug can be overcome by
designing the new derivatives (Wechter et al., 1975).
Further, pharmacokinetic and cellular permeability of the
drug can be increased by derivatization to bioreversible
form of this drug, namely hydrazones (Maccari et al.,
2002). The Mannich base of isonicotinoyl hydrazone has
improved lipid solubility (Joshi et al., 2004). Mannich
reaction is a three-component condensation reaction
involving active hydrazone containing compound, formal-
dehyde, and a secondary amine (Sujith et al., 2009). It is
believed that the Mannich base functional group increases
the lipophilicity of parent amines and amides and results
into enhancement of absorption through biomembranes
(Gamal El-Din et al., 2009). The lipophilicity of mannich
bases enables them to cross bacterial and fungal mem-
branes. So, looking at the antimicrobial importance of
hydrazone and its mannich bases, prompted us to synthe-
size some new derivatives of Mannich bases. So, consid-
ering this entire concept, we report the synthesis,
characterization, and antimicrobial study of hydrazone and
its mannich bases.
characteristic C=O signals appeared at around d 163.59–
163.18 ppm, pyridine d 149.81–122.12 ppm, –N=C–H d
143.66–143.13 ppm, benzylidene d 158.13–114.75 ppm, d
O–CH2 65.57–64.15 ppm, d Ar–CH2–N 55.91–45.13 ppm.
Materials and methods
Melting points of the synthesized compounds were deter-
mined in open-glass capillaries on Stuart SMP10 melting
point apparatus and were uncorrected. The purity of the
compounds was checked by thin layer chromatography
(TLC). Silica gel plates kiesel gel 0.25 mm, 60G F254,
precoated sheets obtained from Merck, Darmstadt (Ger-
many) were used for TLC and the spots were visualized by
iodine vapors/ultraviolet light as visualizing agent. The IR
spectra (t, cm-1) were obtained with a Perkin-Elmer 1600
FTIR spectrometer in KBr pellets. 1H-NMR spectra (d,
ppm) were recorded in DMSO-d6 solutions on a Varian-
Mercury 300 MHz spectrometer using tetramethylsilane as
the internal reference. 13C-NMR spectra were recorded on
in DMSO-d6 solutions on a Bruker Avance II 400 spec-
trometer at 400 MHz using tetramethylsilane as the inter-
nal reference. Elemental analyses were performed on an
ECS 4010 Elemental Combustion System. The necessary
chemicals were purchased from Loba Chemie.
Chemistry
The synthesis of target compounds were carried outline in
synthetic Scheme 1. Compounds 3a–3k wwere readily
prepared in good yields and purity. Equimolar quantity of
2-ethoxybenzaldehyde (1.50 g, 0.01 mol) and Isoniazid
(1.37 g, 0.01 mol) in 15 ml of absolute ethanol was refluxed
for 7 h to form 2-ethoxybenzylidene isonicotinohydrazide.
The completion of reaction was confirmed by thin layer
chromatography (TLC). Then 2-ethoxybenzylidene ison-
icotinohydrazide (646 mg, 0.0024 mol) along with (0.1 ml,
0.0036 mol) of formaldehyde and (0.0024 mol) of substi-
tuted secondary amines were refluxed in the presence of
50 ml of super dry ethanol and the pH was adjusted to four
with hydrochloric acid. The purity of the compounds was
checked by TLC, elemental analyses and characterized by
spectral data. In general, IR spectra of all compounds 3a–3k
showed absorption band at around 3284–3257, 2869–2838,
2929–2926, 1676–1665, 1669–1651, 1582–1539, 1188–
1118, 1088–1049 cm-1 regions, conforming the presence of
NH, CH2, CH, C=N, C=O, C=C, C–N, and C–O, respec-
Synthesis of 2-ethoxybenzylidene Isonicotinohydrazide
A mixture of 2-ethoxybenzaldehyde (1.50 g, 0.01 mol) and
isoniazid (1.37 g, 0.01 mol) in 15 ml of super dry etha-
nol(method of preparation of dry ethanol: Take 1 l of
ethanol and add 25 g of magnesium metals. Reflux until the
metal is consumed (add a few drops of chloroform if it
doesn’t start to get cloudy).It will take a good 24 h to
convert the metal to magnesium ethoxide. Then just distill
the ethanol off. It will be very dry) was refluxed for 7 h.
The completion of reaction was confirmed by TLC. The
reaction mixture was then poured onto ice cold water and
the precipitate obtained was filtered and dried in oven at
low temperature. The product was recrystallised from
absolute ethanol.
N-(2-ethoxybenzylidine)Isonicotinohydrazide (3a)
1
tively. The H NMR spectra, the signals of the respective
Yield 68%; m.p. 192–195°C; IR (KBr; cm-1): 3261, 2934,
2865, 2838, 1674, 1652, 1561, 1116, 1064. 1H-NMR
(300 MHz, DMSO-d6, d ppm): 12.05 (s, 1H, –NH–N=),
8.82 (d, 2H, pyridine, J = 4.5 Hz), 8.74 (s, 1H, –N = C–
H), 7.88 (d, 2H, pyridine, J = 4.2 Hz), 7.82 (d, 2H, ben-
zylidene, J = 7.8 Hz), 7.40 (d, 2H, benzylidene, J = 7.5),
3.86 (m, 2H, OCH2), 1.28 (t, 3H, CH3); 13C-NMR
(400 MHz, DMSO d6, d ppm): 163.45, 157.32, 149.81,
prepared derivatives were confirmed on the basis of their
chemical shifts, multiplicities, and coupling constants. The
spectra of most compounds showed the characteristic NH
proton d 12.05–11.74 ppm, 1H proton of –N=C–H at d
8.54–8.31 ppm, 4H proton of pyridine were at around d
8.89–7.32 ppm, and characteristic protons of benzylidene at
d 7.82–6.94 ppm. 13C-NMR spectra of compounds 3a–3k
123

Med Chem Res (2012) 21:1237–1244
1239
H5C2O
Scheme 1 Synthetic pathway
for the formation of the title
compounds
H
5C 2O
O
C
O
C
O
C 2H 5OH
NH
N
C
H
N
N
NH
NH
H
C
2
+
Isoniazid
2-Ethoxybenzaldehyde
2-Ethoxybenzylidene isonicotinohydrazide
(3a)
R1
HCHO
R=NH
R2
H5C2
O
CH2 R
O
N
C
NH
N
C
H
Substituted Mannich bases (3b-3k)
R= 3b N(CH3)2, 3c N(C2H5)2, 3d N(C3H7)2, 3e N(C4H9)2, 3f N(C6H5)2,
N
NCH3
N
NH
N
N
N
O
3g
, 3h
, 3i
, 3j
, 3k
143.17, 139.87, 131.73, 129.88, 122.69, 120.51, 117.33,
114.75, 65.32, 14.92. Anal.: Calcd. for C15H15N3O2
(269.30): C 66.90, H 5.61, N 15.60. Found: C 65.95, H
5.63, N 15.53.
44.74, 13.87. Anal.: Calcd. for C18H24N4O2. (326.39) C
66.24, H 6.79, N 17.17. Found: C 66.18, H 6.75, N 17.27.
N-3-((diethyamino)methyl)-2-
ethoxybenzylidene)isonicotinohydrazide (3c)
Synthesis of substituted mannich bases (3b–3k)
Yield 55%; m.p. 220–222°C; IR (KBr; cm-1): 3284, 2958,
2863, 2842, 1671, 1658, 1555, 1124, 1067. 1H-NMR
(300 MHz, DMSO-d6, d ppm): 11.74 (s, 1H, –NH–N=), 8.71
(d, 2H, pyridine, J = 4.2 Hz), 8.39 (s, 1H, –N=C–H), 7.69
(d, 2H, pyridine, J = 3.9 Hz), 7.64 (d, 2H, benzylidene,
J = 3.5 Hz), 7.12 (t, 1H, benzylidene), 3.91 (m, 2H, OCH2),
3.62 (s, 2H, Ar–CH2–N), 2.87 (m, 4H, N–2CH2), 1.14 (m,
9H, 3CH3); 13C-NMR (400 MHz, DMSO d6, d ppm):
163.34, 157.64, 149.39, 143.39, 139.74, 131.98, 127.71,
122.82, 121.69, 119.74, 115.19, 64.84, 52.17, 48.63, 14.81,
13.39. Anal.: Calcd. for C20H26N4O2. (354.45) C 67.77, H
7.39, N 15.81. Found: C 67.82, H 7.38, N 15.77.
The 2-ethoxy-benzylidene isonicotinohydrazide (646 mg,
0.0024 mol) along with (0.1 ml, 0.0036 mol) of formal-
dehyde and (0.0024 mol) of substituted secondary amines
was placed in 100 ml round bottom flask to which 50 ml of
super dry ethanol was added and the pH was adjusted to
four with hydrochloric acid and refluxed for 38–43 h. The
completion of reaction was confirmed by TLC. The reac-
tion mixture was then concentrated on water bath and
allowed it to cool at room temp for half an hour to which
diethyl ether was added. The reaction mixture was kept for
3–5 h in refrigerator and filtered and washed with n-hex-
ane. The products were recrystallised from absolute
ethanol.
N-3-((dipropylamino)methyl)-2-
ethoxybenzylidene)isonicotinohydrazide (3d)
N-3-((dimethyamino)methyl)-2-
ethoxybenzylidene)isonicotinohydrazide (3b)
Yield 55%; m.p. 215–217°C; IR (KBr; cm-1): 3265, 2954,
2853, 2841, 1671, 1658, 1541, 1118, 1052. 1H-NMR
(300 MHz, DMSO-d6, d ppm): 11.86 (s, 1H, –NH–N=), 8.64
(d, 2H, pyridine, J = 4.2 Hz), 8.39 (s,1H,–N=C–H), 7.93 (d,
2H, pyridine, J = 3.9 Hz), 7.34 (d, 2H, benzylidene,
J = 3.4 Hz), 7.11 (t, 1H, benzylidene), 3.78 (m, 2H, OCH2),
3.69 (s, 2H, Ar–CH2–N), 2.18 (m, 8H, N–4CH2), 1.12 (m,
9H, 3CH3); 13C-NMR (400 MHz, DMSO d6, d ppm):
163.18, 157.59, 149.18, 143.23, 139.93, 131.81, 128.12,
122.57, 121.59, 120.27, 116.61, 64.86, 56.18, 51.17, 22.54,
14.88, 11.52. Anal.: Calcd. for C22H30N4O2. (382.50) C
69.08, H 7.91, N 14.65. Found: C 69.15, H 7.85, N 14.64.
Yield 52%; m.p. 225–227°C; IR (KBr; cm-1): 3267, 2924,
2869, 2845, 1676, 1654, 1562, 1118, 1058. 1H-NMR
(300 MHz, DMSO-d6, d ppm): 11.82 (s, 1H, –NH–N=),
8.69 (d, 2H, pyridine, J = 4.2 Hz), 8.44 (s, 1H, –N=C–H),
7.74 (d, 2H, pyridine, J = 3.8 Hz), 7.58 (d, 2H, benzyli-
dene, J = 3.2 Hz), 7.28 (t, 1H, benzylidene), 3.82 (m, 2H,
OCH2), 3.57 (s, 2H, Ar–CH2–N), 3.32 (s, 6H, N–2CH3),
1.12 (t, 3H, OCH3); 13C-NMR (400 MHz, DMSO d6, d
ppm): 163.59, 158.13, 149.37, 143.41, 139.79, 132.74,
127.39, 122.38, 121.52, 119.87, 115.18, 64.57, 54.91,
123

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Med Chem Res (2012) 21:1237–1244
N-3-((dibutylamino)methyl-2-
ethoxybenzylidene)isonicotinohydrazide (3e)
(300 MHz, DMSO-d6, d ppm): 11.88 (s, 1H, –NH–N=),
8.79 (d, 2H, pyridine, J = 4.5 Hz), 8.42 (s, 1H, –N=C–H),
7.78 (d, 2H, pyridine, J = 4.3 Hz), 7.59 (d, 2H, benzyli-
dene, J = 3.2 Hz), 7.19 (t, 1H, benzylidene), 3.75 (m, 2H,
OCH2), 3.55 (s, 2H, Ar–CH2–N), 2.37 (d, 4H, N–2CH2,
pyrrolidine), 1.37 (m, 4H, 2CH2, pyrrolidine), 1.25 (t, 3H,
CH3); 13C-NMR (400 MHz, DMSO d6, d ppm): 163.44,
157.28, 149.53, 143.18, 139.47, 131.39, 128.74, 122.91,
121.67, 120.19, 116.76, 64.79, 56.89, 51.27, 26.73, 14.82.
Anal.: Calcd. for C20H24N4O2. (352.19) C 68.16, H 6.86, N
15.90. Found: C 68.15, H 6.85, N 15.92.
Yield 52%; m.p. 208–210°C; IR (KBr; cm-1): 3266, 2958,
2851, 2843, 1674, 1655, 1539, 1124, 1062. 1H-NMR
(300 MHz, DMSO-d6, d ppm): 11.75 (s, 1H, –NH–N=), 8.69
(d, 2H, pyridine, J = 4.1 Hz), 8.39 (s, 1H, –N=C–H), 7.89
(d, 2H, pyridine, J = 3.8 Hz), 7.58 (d, 2H, benzylidene,
J = 3.2 Hz), 7.34 (t, 1H, benzylidene), 3.72 (m, 2H, OCH2),
3.66 (s, 2H, Ar–CH2–N), 2.26 (m, 4H, N–2CH2), 1.75 (m,
8H, 4CH2), 1.14 (t, 9H, 3CH3); 13C-NMR (400 MHz, DMSO
d6, d ppm): 163.27, 157.19, 149.37, 143.13, 139.86, 131.77,
128.57, 122.12, 120.18, 116.88, 64.91, 55.26, 51.28, 32.54,
21.19, 14.91, 13.55. Anal.: Calcd. for C24H34N4O2. (410.55)
C 70.21, H 8.35, N 13.65. Found: C 70.28, H 8.38, N 13.55.
N-(2-ethoxy-3-((morpholinomethyl)benzylidene)
isonicotinohydrazide (3i)
Yield 45%; m.p. 219–221°C; IR (KBr; cm-1): 3264, 2989,
2863, 2845, 1668, 1655, 1582, 1184, 1079. 1H-NMR
(300 MHz, DMSO-d6, d ppm): 11.88 (s, 1H, –NH–N=),
8.89 (d, 2H, pyridine, J = 4.3 Hz), 8.46 (s, 1H, –N=C–H),
7.79 (d, 2H, pyridine, J = 3.8 Hz), 7.38 (d, 2H, benzyli-
dene, J = 3.1 Hz), 7.19 (t, 1H, benzylidene), 3.92 (m, 2H,
OCH2), 3.67 (s, 2H, Ar–CH2–N), 3.57 (t, 4H, O–2CH2,
morpholine), 2.42 (t, 4H, 2CH2, morpholine), 1.25 (t, 3H,
CH3); 13C-NMR (400 MHz, DMSO d6, d ppm): 163.19,
157.34, 149.53, 143.66, 139.48, 131.67, 128.53, 122.53,
121.91, 120.49, 116.13, 67.51, 54.19, 51.29, 15.22. Anal.:
Calcd. for C20H24N4O3. (368.43) C 65.20, H 6.57, N 15.21.
Found: C 65.23, H 6.47, N 15.28.
N-(3((diphenylamino)-methyl)-2-
ethoxybenzylidene)isonicotinohydrazide (3f)
Yield 53%; m.p. 199–201°C; IR (KBr; cm-1): 3269, 2989,
2865, 2841, 1669, 1655, 1558, 1139, 1075. 1H-NMR
(300 MHz, DMSO-d6, d ppm): 11.92 (s, 1H, –NH–N=),
8.74 (d, 2H, pyridine, J = 4.5 Hz), 8.56 (s, 1H, –N=C–H),
7.39 (d, 2H, pyridine, J = 4.2 Hz), 7.24–6.94 (m, 13 Ar–
H, benzylidene), 3.98 (m, 2H, OCH2), 3.62 (s, 2H, Ar–
CH2–N), 1.27 (s, 3H, CH3); 13C-NMR (400 MHz, DMSO
d6, d ppm): 163.19, 156.27, 149.83, 143.34, 139.87,
129.72, 127.81, 122.59, 119.45, 118.64, 116.77, 65.15,
45.13, 15.24. Anal.: Calcd. for C28H26N4O2. (450.21) C
74.65, H 5.82, N 12.44. Found: C 74.59, H 5.88, N 12.44.
N-(2-ethoxy-3-((piperazin-1-
yl)methyl)benzylidene)isonicotinohydrazide (3j)
N-(2-ethoxy-3-((piperidine-1-
yl)methyl)benzylidene)isonicotinohydrazide (3g)
Yield 49%; m.p. 205–207°C; IR (KBr; cm-1): 3259, 2984,
2865, 2841, 1673, 1658, 1567, 1188, 1049. 1H-NMR
(300 MHz, DMSO-d6, d ppm): 11.81 (s, 1H, –NH–N=),
8.69 (d, 2H, pyridine, J = 4.1 Hz), 8.69 (s, 1H, –N=C–H),
7.59 (d, 2H, pyridine, J = 3.7 Hz), 7.38 (d, 2H, benzyli-
dene, J = 3.2 Hz), 7.19 (t, 1H, benzylidene), 3.78 (m, 2H,
OCH2), 3.65 (s, 2H, Ar–CH2–N), 2.62 (m, 8H, N–4CH2,
piperazine), 2.37 (m, 1H, NH, piperazine), 1.28 (t, 3H,
CH3); 13C-NMR (400 MHz, DMSO d6, d ppm): 163.25,
157.55, 149.67, 143.18, 139.67, 131.54, 128.18, 122.86,
121.51, 120.55, 116.77, 64.59, 55.86, 51.67, 46.35, 15.23.
Anal.: Calcd. for C20H25N5O2. (367.44) C 65.37, H 6.86, N
19.06. Found: C 65.56, H 6.72, N 19.01.
Yield 58%; m.p. 191–193°C; IR (KBr; cm-1): 3265, 2963,
2864, 2842, 1674, 1649, 1561, 1131, 1055. 1H-NMR
(300 MHz, DMSO-d6, d ppm): 11.92 (s, 1H, –NH–N=),
8.74 (d, 2H, pyridine, J = 4.2 Hz), 8.56 (s, 1H, –N=C–H),
7.94 (d, 2H, pyridine, J = 3.8 Hz), 7.28 (d, 2H, benzyli-
dene, J = 3.2 Hz), 7.13 (t, 1H, benzylidene), 3.84 (m, 2H,
OCH2), 3.47 (s, 2H, Ar–CH2–N), 2.65 (t, 4H, N–2CH2,
piperidine), 1.24 (m, 6H, 3CH2, piperidine), 1.15 (t, 3H,
3CH3); 13C-NMR (400 MHz, DMSO d6, d ppm): 163.34,
157.18, 149.38, 143.27, 139.84, 131.22, 128.37, 122.74,
121.18, 120.74, 116.93, 64.94, 55.16, 51.77, 25.17, 14.81.
Anal.: Calcd. for C21H26N4O2. (366.46) C 68.83, H 7.15, N
15.29. Found: C 68.58, H 7.18, N 15.24.
N-(2-ethoxy-3-((4-methylpiperazin-1-
yl)methyl)benzylidene)isonicotinohydrazide (3k)
N-(2-ethoxy-3-((pyrrolidin-1-
yl)methyl)benzylidene)isonicotinohydrazide (3h)
Yield 51%; m.p. 210–212°C; IR (KBr; cm-1): 3264, 2977,
2862, 2842, 1665, 1651, 1555, 1158, 1078. 1H-NMR
(300 MHz, DMSO-d6, d ppm): 11.95 (s, 1H, –NH–N=),
8.77 (d, 2H, pyridine, J = 4.2 Hz), 8.39 (s,1H, –N=C–H),
Yield 57%; m.p. 196–198°C; IR (KBr; cm-1): 3257, 2959,
2862, 2842, 1669, 1655, 1561, 1187, 1088. 1H-NMR
123

Med Chem Res (2012) 21:1237–1244
1241
7.69 (d, 2H, pyridine, J = 3.9 Hz), 7.34 (d, 2H, benzyli-
dene, J = 3.3 Hz), 7.13 (t, 1H, benzylidene), 3.89 (m, 2H,
OCH2), 3.66 (s, 2H, Ar–CH2–N), 2.47 (t, 8H, N–4CH2,
piperazine), 2.12 (s, 3H, NCH3, piperazine), 1.22 (t, 3H,
CH3); 13C-NMR (400 MHz, DMSO d6, d ppm): 163.28,
157.51, 149.34, 143.27, 139.44, 131.84, 128.53, 122.92,
121.47, 120.23, 116.75, 64.86, 56.13, 52.17, 51.25, 43.71,
15.18. Anal.: Calcd. for C21H27N5O2. (381.47) C 66.12, H
7.13, N 18.36. Found: C 66.25, H 7.18, N 18.18.
Hot-plate diffusion method was used; six equidistant (1 cm
diameter) holes were made using sterile cork borer in malt
extract agar and nutrient agar sterile plates (10 9 10 cm),
which had previously been seeded with tested bacterial and
fungal isolates. Holes are filled with 100 lg/ml concen-
tration of each of the synthesized compounds after com-
pletely dissolving in DMSO. Controlled holes were filled
with DMSO solvent. Plates were left in a cooled incubator
at 37 ( 2)°C for bacterial isolates and incubation at 28
(
2)°C for fungal isolates used. Inhibition zones devel-
Antimicrobial activity
oped due to active ingredients were measured after
24–48 h of incubation time. Amoxicillin was used as
standard antibacterial agent while Nystatin was used as a
standard antifungal agent.
Culture media
Two specific media were used for detecting the antimi-
crobial activity, nutrient agar medium was used for bac-
terial growth [beef extract, 3 g; bacteriological peptones,
5 g; agar, 20 g, the pH was adjusted to 6.2 0.2 at 25
( 2)°C], while malt extract agar (MEA) for fungal isolates
[malt extract, 20 g; bacteriological peptone, 5 g; agar,
20 g, the pH was adjusted to 5.4 0.2 at 25 ( 2)°C]. Each
medium was prepared by dissolving the solid ingredient in
1 l of cold distilled water and then heated to 60–70°C with
stirring. Media were sterilized by autoclaving at 121°C
(1.5 atm) for 15–20 min (Atlas 1993).
Minimum inhibitory concentration (MIC) was deter-
mined by broth dilution technique. The Nutrient Broth
contained logarithmic serially two fold diluted amount of
test compound and controls. The inoculum size was
approximately 106 colony forming units (CFU/ml). The
tubes were incubated at 37 1°C for 24 h (bacteria) and
25°C for 7 days (A. Niger) and the growth was monitored
visually and spectrophotometrically. The lowest concen-
tration (highest dilution) required to arrest the growth of
microbes was regarded as minimum inhibitory concentra-
tions (MIC). To obtain the minimum fungal concentration
(MFC), 0.1 ml volume was taken from each tube and
spread on agar plates. The number of cfu was counted after
3 days of incubation at 30°C. MFC was defined as the
lowest drug concentration at which 99.9% of the inoculums
were killed. MIC50 were calculated mathematically
depending on the number of inhibited colonies of the
Antimicrobial assays
By diffusion agar technique, the antibacterial and anti-
fungal potentialities against several species are expressed
as the measurement of diameter of their inhibition zone.
Table 1 Zone of inhibition of the tested compounds
Compound
Concentration
(lg/ml)
Zone of inhibition (mm)
Gram positive bacteria
Gram negative bacteria
Fungal strain
B. subtilis
(ATCC 25923)
S. aureus
(ATCC 29213)
P. aeruginosa
(ATCC 27863)
E. coli
(ATCC 25922)
C. albicans
(ATCC 10231)
C. gabrata
(ATCC 10233
3a
100
100
100
100
100
100
100
100
100
100
100
50
15
17
19
21
21
26
23
22
27
27
26
25
–
19
21
21
22
23
31
25
26
32
34
33
30
–
14
15
16
18
19
22
20
20
22
23
22
21
–
18
20
20
22
24
32
25
27
31
33
32
30
–
13
13
15
15
15
21
16
18
21
24
22
–
11
14
14
15
15
22
16
19
21
23
20
–
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
Amoxicillin
Nystatin
50
–
–
123

1242
Med Chem Res (2012) 21:1237–1244
medium with the respective Mannich base compounds or
standard drug dilution compared to the control medium
colonies without drugs.
nutrient agar at 100 lg/ml concentration was shown in
Table 1. Amoxicillin standard was active at 50 lg/ml on all
the Gram (?ve) and Gram (-ve) bacteria. From the anti-
bacterial screening, it was concluded that compounds 3f, 3i,
3j, and 3k show larger zone of inhibition as compared to
standard drug amoxicillin against Bacillus subtilis (26, 27,
27, 26 mm), Staphylococcus aureus (31, 32, 34, 33 mm),
and Gram (-ve) bacteria Pseudomonas aeruginosa (22, 22,
23, 22 mm) and Escherichia coli (32, 31, 33, 32 mm).
Result and discussion
The antimicrobial sensitivity testing of the synthesized
compounds assayed using cup plate technique in the
Table 2 MIC50 of tested compounds
Compound
MIC50
Gram positive bacteria
Gram negative bacteria
Fungal strain
B. subtilis
S. aureus
P. aeruginosa
E. coli
C. albicans
C. gabrata
3a
25.6
12.8
12.8
12.8
12.8
6.4
12.8
12.8
12.8
6.4
12.8
12.8
12.8
6.4
6.4
6.4
6.4
6.4
6.4
6.4
6.4
3.2
–
25.6
12.8
12.8
6.4
6.4
3.2
6.4
3.2
3.2
3.2
3.2
1.6
–
25.6
12.8
12.8
12.8
12.8
6.4
25.6
25.6
25.6
12.8
12.8
12.8
12.8
12.8
12.8
12.8
12.8
–
3b
3c
3d
3e
6.4
3f
3.2
3g
12.8
12.8
6.4
6.4
12.8
6.4
3h
6.4
3i
3.2
6.4
3j
6.4
3.2
6.4
3k
6.4
3.2
6.4
Amoxicillin
Nystatin
3.2
1.62
–
–
–
3.2
6.4
Table 3 MIC and MFC of tested compounds
Compound
MIC and MFC
Gram positive bacteria
Gram negative bacteria
Fungal strain
B. subtilis
S. aureus
P. aeruginosa
E. coli
C. albicans
C. gabrata
MFC
MFC
3a
50
25.0
25.0
25.0
25.0
12.5
6.25
12.5
12.5
6.25
3.12
6.25
3.12
–
25.0
25.0
25.0
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
6.25
–
50.0
25.0
25.0
12.5
12.5
50.0
25.0
25.0
25.0
25.0
12.5
12.5
12.5
12.5
12.5
12.5
–
50
100
25.0
50
25.0
25.0
25.0
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
–
100
100
100
100
100
50
3b
50
3c
25.0
25.0
25.0
12.5
25.0
25.0
12.5
12.5
12.5
6.25
–
3d
3e
100
25
3f
6.25
12.5
6.25
3g
100
25
100
100
50
3h
3i
6.25
6.25
6.25
3.12
–
25
3j
25
50
3k
25
50
Amoxicillin
Nystatin
–
–
6.25
12.5
12.5
25.0
MIC Minimum inhibitory concentration
MFC Minimum fungicidal concentration
123

Med Chem Res (2012) 21:1237–1244
1243
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Compounds 3g and 3h showed nearly moderate zone of
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A series of mannich bases were synthesized for their
antimicrobial activity. The highest antibacterial activity
against Gram positive species Bacillus subtilis, Staphylo-
coccus aeuras and Gram negative species P. aeruginosa,
E. coli were shown by compounds 3f, 3i, 3j, and 3k. The
compounds 3f, 3i, 3j, and 3k also exhibited high antifungal
activity against C. albicans and C. gabrata species. So, it
was concluded that antimicrobial activity increases with
increase in chain length from dimethyl amine to dibutyl
amine. So, the significant antimicrobial activity of com-
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morpholine, piperazine, and N-methyl piperazine moiety in
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