Synthesis and biological evaluation of 4-(4-chlorophenyl)cyclohexane carbohydrazide derivatives as anti-bacterial agents

Article abstract of DOI:10.14233/ajchem.2015.18731

The present paper describes the synthesis and antibacterial activity of novel hydrazone derivatives 4a-s derived from 4-(4-chlorophenyl)-cyclohexane carboxylic acid. All the ninteen newly synthesized novel hydrazone derivatives 4a-s were evaluated for their in vitro antibacterial activity against Staphylococcus aureus and S. pyogenes (Gram-positive bacteria) and Escherichia coli and Pseudomonas aeruginosa (Gram-negative bacteria). Antibacterial activity data revealed that the basic scaffold with R = nitrogen heterocyclic ring such as pyridine, quinoline, imidazole and indole showed significant antibacterial activity (excellent activity), whereas the hetrocyclic ring like benzo[b]furan, furan, thiophene moiety showed good antibacterial activity.

Full text of DOI:10.14233/ajchem.2015.18731

Asian Journal of Chemistry; Vol. 27, No. 6 (2015), 2267-2272
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2015.18731
Synthesis and Biological Evaluation of 4-(4-Chlorophenyl)cyclohexane
Carbohydrazide Derivatives as Anti-Bacterial Agents
1
2
3
3
3
G. VENKATASATYANARAYANA1,2,3,*, V. LAKSHMANA RAO , M. THIRUMALA CHARY , B. RAM , B. BALRAM and E. LAXMINARAYANA
1Mylan Laboratories Limited, Hyderabad-500 034, India
2JNTUH College of Engineering, Nachupally (Kondagattu), Kodimyal mandal, Karimnagar-505 327, India
3Green Evolution Laboratories, Wangapally Village, Nalgonda-500 085, India
*Corresponding author: E-mail: gvenkatasatyanarayana2014@gmail.com
Received: 10 September 2014;
Accepted: 24 November 2014;
Published online: 17 March 2015;
AJC-17008
The present paper describes the synthesis and antibacterial activity of novel hydrazone derivatives 4a-s derived from 4-(4-chlorophenyl)-
cyclohexane carboxylic acid.All the ninteen newly synthesized novel hydrazone derivatives 4a-s were evaluated for their in vitro antibacterial
activity against Staphylococcus aureus and S. pyogenes (Gram-positive bacteria) and Escherichia coli and Pseudomonas aeruginosa
(Gram-negative bacteria). Antibacterial activity data revealed that the basic scaffold with R = nitrogen heterocyclic ring such as pyridine,
quinoline, imidazole and indole showed significant antibacterial activity (excellent activity), whereas the hetrocyclic ring like benzo[b]furan,
furan, thiophene moiety showed good antibacterial activity.
Keywords: Atovaquone, Cyclohexanecarbohydrazide, Hydrazones, Antibacterial Activity.
treatment of toxoplasmosis and babesiosis22. Due to the broad
INTRODUCTION
range of applications and encouraged by these reported biolo-
The emergence of new infectious diseases, the resurgence
gical activities the present research work is aspired to describe
of several infections that appeared to have been controlled and
the synthesis and antibacterial activity of novel hydrazone
the increase in bacterial resistance have created the necessity
derivatives (4a-s) derived from 4-(4-chlorophenyl)cyclohexane
for studies directed towards the development of new antimicro-
carboxylic acid.
bials agents1. The chemical properties of hydrazones have been
intensively studied in several research fields because of their
EXPERIMENTAL
high physiological activity and chelating capability2. Extensive
investigations have revealed that the lone pair on trigonally
hybridized nitrogen atom of the azomethine group is respon-
sible for the chemical and biological activity3-7. Several hydrazide-
hydrazone derivatives exhibited broad spectrum of biological
activities such as antimicrobial8,9, antitubercular10-12, anti-
candida, anticancer, antiviral, antioxidant and antiinflamma-
Chemical and solvents used were purchased either from
Fluka or Merck. All the reagents were of analytical grade.
Thin-layer chromatography (TLC) was performed on E. Merck
AL silica gel 60 F254 plates and visualized under UV light.
IR spectra were recorded as KBr pellet with a perkin-elmer
spectrum gx FTIR instrument and only diagnostic and/or
intense peaks are reported. 1H NMR spectra were recorded in
DMSO-d6 with a Varian Mercury plus 400 MHz instrument.
13C NMR spectra were recorded in DMSO-d6 with a Varian
Gemini 100 MHz instrument. Signals due to the residual
protonated solvent (1H NMR) served as the internal standard.
All the chemical shifts were reported in δ (ppm) using TMS as
an internal standard. The 1H NMR chemical shifts and coupling
constants were determined assuming first-order behaviour.
Mass spectra were recorded with a PE Sciex model API 3000
instrument. All the reactions were carried out under argon
atmosphere. 4-(4-Chlorophenyl)cyclohexane carboxylic acid
tory13-17
.
The starting material 4-(4-chlorophenyl)cyclohexane carbo-
xylic acid is an important precursor used for the synthesis of
atovaquone18.Atovaquone is the hydroxyl-1,4-naphthoquinone
analogue of coenzyme Q10 and has an antipneumocystic
activity19 and it is approved and marketed as a prescription
drug for the treatment of Pneumocystis carinii pneumonia
(PCP), a common parasitic lung infection of immune com-
promized patients20,21. It is not only used for the treatment of
Pneumocystis carinii pneumonia, but also displays potent
activity as an antimalarial agent and has been used in the

2268 Venkatasatyanarayana et al.
Asian J. Chem.
(1) and all the benzaldehydes used for the preparation of 4a-s
were purchased from commercial sources.
7.40 (m, 5H), 6.99-6.96 (m, 2H), 3.79 (s, 6H), 3.19 (*2.58,
m, 1H), 2.58-2.22 (m, 1H), 1.84-1.91 (m, 4H), 1.66-1.46 (m,
4H); ESI-MS: m/z, 400.90.
(E)-N'-(3,4,5-Trimethoxybenzylidene)-4-(4-chloro-
phenyl)cyclohexane carbohydrazide (4d): White solid;Yield:
82 %; m.p.: 115-116 °C; 1H NMR (400 MHz, DMSO-d6): δ
11.42 (*11.2, s, 1H), 8.12 (*7.9, s, 1H), 7.34-7.30 (m, 4H),
6.99 (s, 2H), 3.83 (s, 6H), 3.86 (s, 3H), 2.54-2.52 (m, 1H),
2.23-2.20 (m, 1H), 1.70-1.40 (m, 4H), 1.90-1.80 (m, 4H); ESI-
MS: m/z, 431.1.
(E)-N'-(4-Ethoxy-3-methoxybenzylidene)-4-(4-chloro-
phenyl)cyclohexane carbohydrazide (4e): Yellow solid;Yield:
75 %; m.p.: 120-121 °C; IR (KBr, νmax, cm-1): 3207.0, 2933.2,
1655.5, 1598.7, 1556.3, 1511.9, 1270.9; 1H NMR (400 MHz,
DMSO-d6): δ 11.26 (*11.08, s, 1H), 8.10 (*7.91, s, 1H), 7.28-
7.36 (m, 5H), 7.12 ( d, 1H, J = 9.0 Hz), 6.98 (d, 1H, J = 6.0
Hz), 4.04 (q, 2H, J = 6.0 Hz), 3.80 (s, 3H), 3.42 (* 2.56, m,
1H), 2.25 ( m, 1H), 1.88-1.85 (m, 4H), 1.66-1.46 (m, 4H),
1.32 (t, 2H, J = 6.4 Hz); ESI-MS: m/z, 415.1.
Synthesis of methyl 4-(4-chlorophenyl)cyclohexane
carboxylate (2): To a solution of compound 1 (2 g, 8.4 mmol)
in methanol (20 mL ) was added sulphuric acid (0.1 mL) and
refluxed for 10 h. After completion of the reaction, methanol
was evaporated under reduced pressure and the obtained
residue was taken in ethyl acetate (30 mL,), washed with 10 %
aq. NaHCO3 solution (2 × 10 mL) followed by water and brine
solution. The organic layer was separated, dried over Na2SO4,
filtered and evaporated to afford compound 2. Yellow oily
1
liquid, Yield: 2 g, 94 %; H NMR (400 MHz, DMSO-d6): δ
7.32 (d, J = 12.0 Hz, 2H), 7.24 (d, J = 12.0 Hz, 2H), 3.61 (s,
3H), 2.51-2,42 (m, 1H), 2.39-2.35 (m, 1H), 1.97 (d, J = 12
Hz, 2H), 1.82 (d, J = 12 Hz, 2H), 1.82 (d, J = 12 Hz, 2H),
1.54-1.40 (m, 4H).
Synthesis of 4-(4-chlorophenyl)cyclohexane carbohy-
drazide (3): To a solution of compound 2 (1.5 g, 5.95 mmol)
in ethanol (15 mL) was added hydrazine hydrate (24 m mol)
and heated to reflux for 3 h. After completion of the reaction,
ethanol was concentrated under reduced pressure to obtain
crude compound 3. The crude compound was slurred in n-
hexane, filtered at the high vaccum pump and dried to obtain
compound 3. White solid, Yield: 1.35 g, 86 %; m.p.: 121-122
(E)-N'-(3-Methoxy-4-propoxybenzylidene)-4-(4-chloro-
phenyl)cyclohexane carbohydrazide (4f): Brown solid;Yield:
78 %; m.p.: 122-123 °C; 1H NMR (400 MHz, DMSO-d6): δ
11.26 (*11.09, s, 1H), 8.10 (*7.9, s, 1H), 7.36-7.25 (m, 5H),
7.12 ( d, 1H, J = 9.0 Hz), 6.98 (d, 1H, J = 9.0 Hz), 3.93 (q, 2H,
J = 3.0 Hz), 3.83 (s, 3H), 2.58-2.56 ( m, 1H), 2.2 ( m, 1H),1.88-
1.85 (m, 4H), 1.78-1.71 (m, 4H), 1.63-152 (q, 2H, J = 9.0
Hz), 0.95-1.0 (t, 3H, J = 9.0 Hz); ESI-MS: m/z, 429.2.
(E)-N'-(4-(Dimethylamino)benzylidene)-4-(4-chloro-
phenyl)cyclohexane carbohydrazide (4g): Pale brown solid;
Yield: 71 %; m.p.: 102-103 °C; IR (KBr, νmax, cm-1): 3287.1,
1
°C; H NMR (400 MHz, DMSO-d6): δ 8.97 (br.s, 1H), 7.32
(d, J = 12.0 Hz, 2H), 7.24 (d, J = 12.0 Hz, 2H), 4.16 (br.s, 2H),
2.55-2,47 (m, 1H), 2.16-2.08 (m, 1H), 1.82-1.60 (m, 4H), 1.32-
1.56 (m, 4H).
General experimental procedure for the synthesis of
hydrazone derivatives (4a-s): To a stirred solution of com-
pound 3 (100 mg, 0.40 mmol) in ethanol was added corres-
ponding benzaldehydes (1 mmol) and refluxed for 1 h. The
reaction medium was poured into water and extracted with
ethyl acetate. The organic layer was washed with water follo-
wed by brine solution, dried over anhydrous sodium sulphate,
filtered and concentrated under reduced pressure, to obtain
the pure compounds. Yields of the products varied between
80 and 94 %.
(E)-N'-(2,4-Dimethoxybenzylidene)-4-(4-chlorophenyl)-
cyclohexane carbohydrazide (4a): White solid;Yield: 82 %;
m.p.: 102-103 °C; IR (KBr, νmax, cm-1): 3225.4, 2933.2, 1655.6,
1608.3, 1595.8, 1554.3, 1503.2, 1209.1, 1159.0; 1H NMR (400
MHz, DMSO-d6): δ 11.23 (*11.00, s, 1H), 8.44 (*8.23, s, 1H),
7.70-7.75 (m, 1H), 7.27-7.42 (m, 4H), 6.90-6.60 (m, 2H), 3.81
(s, 3H), 3.91 (s, 3H), 3.35-3.18 (*2.57-2.50, m, 1H), 2.27-
2.19 (m, 1H), 1.84-1.87 (m, 4H), 1.65-1.38 (m, 4H); ESI-MS:
m/z, 400.90.
1
2934.2, 1692.2, 1539.9, 1492.6, 1310.4, 1300.8, 1144.5; H
NMR (400 MHz, DMSO-d6): δ 11.06 (*10.9, s, 1H), 8.03
(*7.86, s, 1H), 7.49-7.46 (d, 1H, J = 9.0 Hz), 7.29 (m, 4H),
6.75-6.72 (d, 1H, J = 9.0 Hz), 2.95 (s, 6H), 3.34-3.20 (m, 1H),
2.27-2.25 ( m, 1H), 1.85-1.84 (m, 4H), 1.58-1.55 (m. 4H);
ESI-MS: m/z, 384.1.
(E)-N'-(4-(Methanesulfonyl)benzylidene)-4-(4-chloro-
phenyl)cyclohexane carbohydrazide (4h): Yellow solid;
Yield: 68 %; m.p.: 131-132 °C; 1H NMR (400 MHz, DMSO-d6):
δ 11.65 (*11.44, s,1H), 8.28 (*8.16, s, 1H), 7.92-8.08 (m, 4H),
7.27-7.37 (m, 4H), 3.26 (s, 3H), 3.46 (*2.57, m, 1H), 2.36-
2.23 ( m, 1H), 1.85-1.94 (m, 4H), 1.4-1.67 (m. 4H); ESI-MS:
m/z, 419.1.
(E)-N'-(4-(Methyl)benzylidene)-4-(4-chlorophenyl)-
cyclohexane carbohydrazide (4i): Brown solid;Yield: 68 %;
m.p.: 125-126 °C; IR (KBr, νmax, cm-1): 3209.9, 2926.4, 1660.4,
1
1604.5, 1563.0, 1491.7; H NMR (400 MHz, DMSO-d6): δ
(E)-N'-(2,5-Dimethoxybenzylidene)-4-(4-chlorophenyl)-
cyclohexane carbohydrazide (4b): Yellow solid; Yield: 80
%; m.p.: 112-113 °C; 1H NMR (400 MHz, DMSO-d6): δ 11.41
(*11.17, s, 1H), 8.52 (*8.29, s, 1H), 7.40-7.24 (m, 5H), 6.99-
6.96 (m, 2H), 3.79 (s, 6H), 3.19 (*2.58, m, 1H), 2.58-2.22
(m, 1H), 1.91-1.84 (m, 4H), 1.66-1.46 (m, 4H); ESI-MS: m/z,
400.90.
(E)-N'-(3,4-Dimethoxybenzylidene)-4-(4-chloro-phenyl)-
cyclohexane carbohydrazide (4c): Yellow solid; Yield: 82
%; m.p.: 118-119 °C; IR (KBr, νmax, cm-1): 3233.1, 2935.1,
1659.4, 1600.6, 1556.3, 1512.90, 1268.0; 1H NMR (400 MHz,
DMSO-d6): δ 11.41 (*11.17, s, 1H), 8.52 (*8.29, s, 1H), 7.27-
11.38 (*11.12, s, 1H), 8.47 (*8.27, s, 1H), 7.78-7.70 (m, 1H),
7.37-7.27 (m, 7H), 3.18 (*2.6, m, 1H), 2.50 (s, 3H), 2.20 ( m,
1H), 1.94-1.85 (m, 4H), 1.63-1.44 (m. 4H); ESI-MS: m/z,
355.2.
(E)-4-(4-Chlorophenyl)-N'-((quinoline-4-yl)methy-
lene)cyclohexane carbohydrazide (4j): Brown solid; Yield:
72 %; m.p.: 134-135 °C; IR (KBr, νmax, cm-1): 3210.9, 2927.4,
1
1668.1, 1558.2; H NMR (400 MHz, DMSO-d6): δ 11.80
(*11.55, s, 1H), 8.99 ( d, J = 8.0 Hz, 1H), 8.81-8.75 (*8.09, s,
1H), 8.64 (d, J = 8.0 Hz, 1H), 7.86-7.71 (m, 3H), 7.34-7.29
(m, 5H), 3.36 (*2.59, m, 1H), 2.36-2.34 (m, 1H), 1.94-1.91
(m, 4H), 1.65-1.43 (m. 4H); ESI-MS: m/z, 392.4.

Vol. 27, No. 6 (2015)
Synthesis of 4-(4-Chlorophenyl)cyclohexane Carbohydrazide Derivatives 2269
(E)-4-(4-Chlorophenyl)-N'-((pyridine-2-yl)methylene)-
cyclohexane carbohydrazide (4k): Brown solid; Yield: 67
%; m.p.: 127-128 °C; 1H NMR (400 MHz, DMSO-d6): δ 11.50
(*11.28, s,1H), 8.69 (d, J = 8.0 Hz, 1H), 8.16 (*7.96, m, 1H),
7.82-7.77 (m, 2H), 7.42-7.28 (m, 5H), 3.34 (*2.59, m, 1H),
2.38- 2.36 (m, 1H), 1.92-1.90 (m, 4H), 1.64-1.44 (m. 4H);
ESI-MS: m/z, 342.40.
Yield: 76 %; m.p.: 144-145 °C; 1H NMR (400 MHz, DMSO-d6):
δ 11.60 (s, 1H), 11.47 (*11.27, s, 1H), 8.35 (*8.24, s, 1H),
8.04 (s, 1H), 7.14-7.18 (m, 9H), 3.60 (*2.31, m, 1H), 2.51 (m,
1H), 1.89 (m, 4H), 1.44 (m. 4H); ESI-MS: m/z, 407.20
in vitro Antibacterial assay: All the microbial cultures
were adjusted to 0.5 McFarland standard, which is visually
comparable to a microbial suspension of approximately 1.5 ×
108 cfu/mL23. The antibacterial activity of newly synthesized
4-(4-chlorophenyl)cyclohexane carbohydrazide derivatives
was evaluated by agar well diffusion method24,25. Into the each
petri plate, 20 mL of Mueller Hinton agar medium was poured
and the agar plates were swabbed with 100 µL inocula of each
test bacterium and kept for 15-20 min for adsorption. Using
sterile cork borer of 8 mm diameter, wells were bored into
seeded agar plates and these were loaded with a 100 µL volume
with concentration of 4 mg/mL of each compound reconsti-
tuted in dimethyl sulphoxide (DMSO). All the plates were
incubated at 37 °C for 24 h.
(E)-4-(4-Chlorophenyl)-N'-((pyridin-4-yl)methylene)-
cyclohexane carbohydrazide (4l): Yellow solid;Yield: 66 %;
m.p.: 122-123 °C; IR (KBr, νmax, cm-1): 3241.8, 2929.3, 1668.1,
1
1550.5; H NMR (400 MHz, DMSO-d6): δ 11.50 (*11.6, s,
1H), 8.64 (s, 2H), 8.30 (*8.17, s, 1H), 7.67 (d, J = 8.0 Hz,
2H), 7.30-7.28 (m, 4H), 3.42-3.40 (m, 1H), 2.54-2.51 (m, 1H),
1.27-1.25 (m, 4H), 1.82-1.80 (m. 4H); ESI-MS: m/z, 342.40.
(E)-N'-(Benzofuran-2-yl)methylene)-4-(4-chloro-phenyl)-
cyclohexane carbohydrazide (4m): Dark yellow solid;Yield:
95 %; m.p.: 136-137 °C; 1H NMR (400 MHz, DMSO-d6): δ
11.3 (*11.6, s, 1H), 8.10 (*8.30, s, 1H), 7.64-7.60 (m, 2H),
7.40-7.20 (m, 7H), 3.62-3.58 (m, 1H), 2.54-2.52 (m, 1H), 1.82-
1.80 (m, 4H), 1.62-1.60 (m. 4H); ESI-MS: m/z, 381.50.
(E)-4-(4-Chlorophenyl)-N'-((5-methylfuran-2-yl)-
methylene)cyclohexane carbohydrazide (4n): Brown solid;
Yield: 86 %; m.p.: 130-132 °C; IR (KBr, νmax, cm-1): 3233.1,
Antibacterial activity of the newly synthesized 4-(4-
chlorophenyl)cyclohexanecarbohydrazide derivatives was
evaluated by measuring the zone of growth inhibition against
the test bacteria. DMSO was used as a negative control whereas
ciprofloxacin was used as a positive control. The experiments
were performed in triplicates. The antibacterial activity of the
compounds was compared with ciprofloxacin as standard.
1
2934.2, 1655.6, 1624.0, 1561.1, 1450.2, 1260.3, 1241.0; H
NMR (400 MHz, DMSO-d6): δ 11.25 (*11.03, s,1H), 7.97
(*7.81, s, 1H), 7.36-7.23 (m, 5H), 6.76-6.73 (m, 1H), 6.24
(s, 1H), 3.66 (*3.01, m, 1H), 2.82-2.80 (m, 1H), 1.87-1.85
(m, 4H), 1.67-1.65 (m. 4H); ESI-MS: m/z, 345.4.
(E)-4-(4-Chlorophenyl)-N'-((5-nitrothiophen-3-yl)-
methylene)cyclohexane carbohydrazide (4o): Yellow solid;
Yield: 82 %; m.p.: 140-141 °C; 1H NMR (400 MHz, DMSO-
d6): δ 11.80 (*11.63, s,1H), 8.47 (*8.17, s, 1H), 8.14-8.12 (m,
1H), 7.55-7.52 (m, 1H), 7.36-7.27 (m, 4H), 3.31-3.29 (m, 1H),
2.52-2.50 (m, 1H), 1.86-1.85 (m, 4H), 1.55-1.52 (m. 4H); ESI-
MS: m/z, 392.3.
(E)-4-(4-Chlorophenyl)-N'-((5-nitrothiophen-2-yl)-
methylene)cyclohexane carbohydrazide (4p):Yellow solid;
Yield: 85 %; m.p.: 145-146 °C; 1H NMR (400 MHz, DMSO-
d6): δ 11.53 (*11.30, s, 1H), 8.25 (d, J = 12.0 Hz, 2H), 8.21
(*7.99, s, 1H), 8.12 (m, 1H), 7.36-7.27 (m, 4H), 3.31-3.29 (m,
1H), 2.52-2.50 (m, 1H), 1.88-1.85 (m, 4H), 1.54-1.52 (m. 4H);
ESI-MS: m/z, 392.3.
(E)-N'-((1H-Indole-2-yl)methylene)-4-(4-chlorophenyl)-
cyclohexane carbohydrazide (4q): White solid;Yield: 79 %;
m.p.: 138-139 °C; 1H NMR (400 MHz, DMSO-d6): δ 11.54 (s,
1H), 11.06 (*10.89, s,1H), 8.37 (*8.25, s, 1H), 8.23 (s, 1H),
7.77 (s, 1H), 7.12-7.10 ( m, 7H), 3.32-3.29 (*2.3, m, 1H),
2.60-2.57 (m, 1H), 1.92-1.89 (m, 4H), 1.46-1.44 (m. 4H); ESI-
MS: m/z, 380.4.
RESULTS AND DISCUSSION
A series of 4-(4-chlorophenyl)cyclohexane carboxylic
acid related hydrazones 4a-s are synthesized (Scheme-I) and
the structures of the target compounds (4a-s) were confirmed
by 1H NMR, Mass and IR spectral data. The target compounds
(4a-s) was obtained in three steps, the methyl ester 2 was
prepared from corresponding carboxylic acid 1 in presence of
catalytic quantity of concentrated sulphuric acid in methanol
at reflux for 10 h and the reaction of methyl 4-(4-chlorophenyl)-
cyclohexane carboxylate (2) with hydrazine hydrate in ethanol
at reflux for 3 h afforded 4-(4-chlorophenyl)cyclohexane carbo-
hydrazide (3). The target compounds 4a-s were synthesized
via the nucleophilic addition-elimination reaction of 4-(4-
chlorophenyl)cyclohexane carbohydrazide (3) with various
aryl and heteroaryl aldehydes.
The FT-IR spectra of target compounds (4a-s) showed
absorption bands at 1692-1655 cm-1 due to the presence of
C=O functional group, while the bands observed at 1563-1550
cm-1 corresponded to C=N linkage and the band appearing at
1
3245-3197 cm-1 represented -NH group. From the H NMR
spectra, the structures of the synthesized compounds (4a-s)
were confirmed on the basis of the fact that the aldehydic
proton (which was visible at δ 10.55) present in the corres-
ponding aldehydes disappeared and a new singlet due to the
azomethine (CH=N) group appeared at δ values between 7.86-
8.52 ppm in all the compounds. Also, the protons of CONH
and CH=N exhibited two separate signals in 1H NMR spectra
at 11, 11.80 and 7.86, 8.52 ppm, respectively due to the nitrogen
inversion. Furthermore, all the synthesized hydrazones deriva-
tives 4a-4s were found to exist as a mixture of two rotameric
forms in solution26 e.g. anti-periplanar (ap) and syn-periplanar
(sp) as indicated by their 1H NMR spectra. Two sets of signals
(E)-N'-(4-Pyridin-2-yl)benzylidene)-4-(4-chlorophenyl)-
cyclohexane carbohydrazide (4r): White solid;Yield: 75 %;
m.p.: 128-129 °C; IR (KBr, νmax, cm-1): 3197.4, 2934.2, 2853.2,
1655.6, 1555.3, 1491.7, 1463.7; 1H NMR (400 MHz, DMSO-
d6): δ 11.48 (*11.28, s,1H), 8.36 (s, 2H), 8.24 (*8.04, s, 1H),
7.83-7.77 (m, 5H), 7.34-7.24 (m, 4H), 7.14 (s, 1H), 3.24
(*2.31, m, 1H), 2.53-2.51 (m, 1H), 1.88-1.85 (m, 4H), 1.46-
1.44 (m. 4H); ESI-MS: m/z, 417.93.
(E)-4-(4-Chlorophenyl)-N'-((5-phenyl-1H-imidazol-2-
yl)methylene)cyclohexane carbohydrazide (4s): White solid;
1
were observed for all groups in H NMR spectra of each

2270 Venkatasatyanarayana et al.
Asian J. Chem.
Cl
Cl
Cl
Cl
a
b
c
N
R
NH2
O
N
O
N
O
OH
O
O
H
H
2
3
4a - 4s
1
OMe
OMe
OMe
OMe
OMe
OMe
O
MeO
OMe
MeO
c
a
OMe
d
b
e
CHO
OMe
O
O
S
N
N
O
f
g
h
i
j
OHC
CHO
R =
OHC
O2N
CHO
NO2
O
S
o
OHC
S
p
O
m
N
k
CHO
N
l
n
CHO
HN
CHO
N
CHO
N
H
N
q
r
s
Scheme-I:Synthesis of hydrazone derivatives of 4-(4-chlorophenyl)cyclohexane carboxylic acid (4a-s); Experimental conditions: (a) Conc; H2SO4, methanol,
reflux, 10 h; (b) NH2NH2·H2O, ethanol, reflux, 3 h; (c) Benzaldehydes (a-s), ethanol, reflux, 1 h
compound indicating the possibility of equilibrium and
interconversion between rotamers (and/or configurationally
isomers) in solution26.
mass spectra of compounds showed (M+1) peaks and are in
agreement with their molecular formula.
in vitro Antibacterial activity: All the newly synthesized
4-(4-chlorophenyl)cyclohexane carbohydrazide derivatives
(4a-4s) were evaluated for their in vitro antibacterial activity
against Staphylococcus aureus and S. pyogenes (Gram-positive
bacteria) and Escherichia coli and Pseudomonas aeruginosa
(Gram-negative bacteria). The results were recorded for each
tested compound as the average diameter of inhibition zones
of bacterial growth surrounding the well in mm. The minimum
inhibitory concentration (MIC) measurements were performed
using a macrodilution tube method27. On the basis of MIC,
As a representative example, the 1H NMR spectra of the
compound 4a is as follows, the broad singlets at 11.23 (*11.00
ppm) and 8.44 ppm (*8.23 ppm) corresponds to the protons
representing to -CONH-N=C- -N=CH- groups, respectively.
The phenyl ring protons appears at 6.6-7.75 ppm and the
cyclohexy protons were observed at 1.38-1.84 ppm and all
the other aliphatic protons were observed at expected regions.
The 1H NMR data for the remaining hydrazone derivatives in
the series are in agreement with the assigned structures. The

Vol. 27, No. 6 (2015)
Compound no.
Synthesis of 4-(4-Chlorophenyl)cyclohexane Carbohydrazide Derivatives 2271
TABLE-1
ANTIBACTERIAL ACTIVITY OF INTERMEDIATES AND COMPOUNDS 4a-s
Zone of inhibition in mmb
Gram-negative bacteria
E. coli P. aeruginosa
Gram-positive bacteria
S. aureus
S. pyogenes
MTCC 443
13
MTCC 424
MTCC 96
15
MTCC 442
13
13
13
22
15
14
23
23
16
28
26
28
22
22
24
23
27
28
25
14
15
13
21
14
16
21
23
15
27
28
27
24
25
24
24
27
28
26
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
14
15
21
17
16
22
22
17
28
26
26
23
24
23
23
26
13
16
23
14
17
23
22
17
30
29
29
25
24
23
24
28
4r
4s
29
25
30
27
Ciprofloxacina
aConcentration: 4 mg/mL-1 of DMSO; bValues, including diameter of the well (8 mm), are means of three replicates; cNo activity
none of the compounds was found to have substantial anti-
bacterial activity28 .
which can be further transformed to a promising antibacterial
agent.
The antibacterial activity data of the tested compounds
4a-s is presented in Table-1. Interpretation of the results of
the antibacterial activity data was done on the basis of zone of
inhibition (measured in mm). Compounds 4j (R = quinoline
ring), 4k (R = pyridine ring), 4l (R = pyridine ring), 4q (R =
indole ring), 4r (R = pyridine ring with phenyl substitution at
4th position), 4s (R = imidazole ring) with zone of inhibition
ranging between 26-30 mm, exhibited excellent antibacterial
activity against all the tested bacterial strains when compared
to the standard drug ciprofloxacin.
Compounds 4d (R = -3,4,5-trimethoxy phenyl), 4g (R =
4-N,N-dimethyl phenyl), 4h (R = 4-methane sulphonyl phenyl),
4m (R = benzo[b]furan ring), 4n (2-methyl-furan ring), 4o (R
= 5-nitro-thiophene ring) and 4p (2-nitro-thiophene ring)
displayed good antibacterial activity (with zone of inhibition
21-25 mm), while the compounds 4a (R = -2,4-dimethoxy
phenyl), 4b (R = -2,5-dimethoxy phenyl), 4c (R = -3,4-dime-
thoxy phenyl), 4e (R = -3-methoxy-4-ethoxy phenyl), 4f (R =
2,4-dimethoxy phenyl) and 4i (R = 4-methyl-phenyl) showed
moderate antibacterial activity.
A careful observation from the above data reveals that
the scaffold with R = nitrogen heterocyclic ring such as
pyridine, quinoline, imidazole and indole showed significant
antibacterial activity (excellent activity), whereas the hetrocyclic
ring like benzo[b]furan, furan, thiophene moiety showed good
antibacterial activity.Among the aromatic moieties, the phenyl
ring with substitutions such as 3,4,5-trimethoxy, -4-N,N-
dimethyl and 4-SO2Me also showed good antibacterial activity.
Therefore it may be suggested that a suitable structural
modification of the basic scaffold with an appropriate R
substitution may pave path to the discovery of a lead compound
Conclusion
The present paper describes the synthesis and antibacterial
activity of novel hydrazones derivatives 4a-s derived from 4-(4-
chlorophenyl)cyclohexane carboxylic acid (1) as starting
material. The structures of the synthesized compounds were
confirmed by 1H NMR, Mass and IR spectral data. The anti-
bacterial activity of newly synthesized 4-(4-chlorophenyl)-
cyclohexane carbohydrazide derivatives (4a-s) was evaluated
by agar well diffusion method by measuring zone of inhibition.
Compounds 4j (R = quinoline ring), 4k (R = pyridine ring), 4l
(R = pyridine ring), 4q (R = indole ring), 4r (R = pyridine
ring with phenyl substitution at 4th position), 4s (R = imidazole
ring) with zone of inhibition ranging between 26-30 mm,
exhibited excellent antibacterial activity against all the tested
bacterial strains when compared to standard drug ciproflo-
xacin.
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