JOURNAL OF THE CHINESE

Article CHEMICAL SOCIETY

Synthesis of Hydrazones from Amino Acids and their Antimicrobial and Cytotoxic
Activities

Obaid-ur-Rahman Abid ,a* Ghamama Khatoon,a Muhammad Arfan,b Imran Sajid,c

Peter Langer,d Wajid Rehman,a,e Fazal Rahim,a Muhammad Yasir,a Muhammad Waqara
and Kashif Syed Haleemf
a
Department of Chemistry, Hazara University, Mansehra, Pakistan
b
Department of Chemistry, School of Natural Sciences, National University of Sciences and Technology, Islamabad,
Pakistan
c
Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan
d
Institut für Chemie, Universität Rostock, Rostock, Germany
e
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190 China
f
Department of Microbiology, Hazara University, Mansehra, Pakistan

(Received: December 10, 2016; Accepted: June 7, 2017; DOI: 10.1002/jccs.201600859)

Hydrazones 6a–6n were synthesized from different amino acids with various aldehydes under
reflux in methanol/ethanol. The structures of synthesized compounds were ascertained by elemen-
tal analysis and spectroscopic techniques. A comparative study of the antimicrobial activity and
cytotoxicity was carried out of the N-protected amino acids, their esters, hydrazides, and the
respective hydrazones, providing good results in cytotoxicity studies.

Keywords: Hydrazones; Cytotoxicity; Amino acids; Antimicrobial.

INTRODUCTION synthesis is the best strategy for this purpose. Generally,

Hydrazones are potential candidates for drug
development because of their reactive azomethine
group (−CO–NH–N CH–).1 Many researchers in the
organic chemistry and medicinal fields have focused on
the study of such molecules for years because of their
important applications. Hyrdrazones have been found
to exhibit diverse biological properties including
antiviral,2 antifungal,3 antibacterial,4 antimalarial,5
antitubercular,6,7 inflammatory,8 antiplatelet,9
anticonvulsant,10 antitumor,11 and anticancer activ-
ities.12,13 Among the significant pharmacophores in case
of antiinflammatory and antiplatelet drug structures are
hydrazine and acyl hydrazine moieties.9,14 Hydrazones
with the cyanoacetyl group have been reported for their
antitumor activities.15–17
Amino acids have been used in the synthesis of Schiff
bases and their complexes. Such moieties have shown
promising results in their application studies such as fluo-
rescent properties18 and biological activities over a wide
range.19–23 Hydrazones from N-protected amino acids are
less reported. Protection via sulfonamide or benzamide
benzamide derivatives are known for their medicinal
importance such as a muscle relaxant as well as antemetic,
antipsychotic, and antiarrahythmic agents.24,25 Recently,
hydrazone derivatives of benzoyl-protected glycine have
been reported for their anticonvulsant activities.26
Determination of the biological activity of synthetic
and biological compounds has played a great role in drug
design during the last few years. Antimicrobial activity
screening is important in drug development, which in turn
is helpful in curing human and animal diseases. Keeping
in mind the significance of hydrazones and their amino
acid derivatives in relation to their biological activities,
this paper reports the synthesis of hydrazones from
N-benzoylated amino acids and different substituted alde-
hydes and their biological evaluation.
RESULTS AND DISCUSSION
Chemistry
Amino acids (1a–1c) were converted to N-
benzoylated amino acids by stirring their solutions in
NaOH (10%) with benzoyl chloride for 3–4 h. A solid

*Corresponding author. Email: obaid_chem@yahoo.com

J. Chin. Chem. Soc. 2017 © 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1
Article
material appeared in solution, to which ice and hydro-
chloric acid (dil + cold) were added till the pH reached
3. After filtration and washing with cold distilled water,
the benzoyl-protected amino acid was dried. IR spectro-
scopic data indicated the completion of protection of
the amino group by the disappearance of two separate
bands for the NH2 asymmetric and symmetric stretch-
ing vibrations, whereas only one band for NH stretch-
ing appeared in the range 3365–3296 cm−1. Appearance
of amidic carbonyl bands in the range 1692–1646 cm−1
and aromatic C C bands in the range 1578–1542 cm−1
also indicated the successful protection.
Each protected amino acid (2a–2c) was dissolved
separately in dry methanol, a few drops of sulfuric acid
were added, and reaction was refluxed for 10–19 h. The
solvent was removed using a rotary evaporator. The
remaining material was then worked up. IR spectro-
scopic data indicated the formation of the ester by the
disappearance of broad band due to the OH group cen-
tered near 3000 cm−1. The appearance of ester carbonyl
band in the range 1742–1718 cm−1 indicated the suc-
cessful esterification.
Each ester (3a–3c) was separately dissolved in eth-
anol, and then hydrazine hydrate was added and
refluxed for 3–11 h. The solvent was removed, and the
product was filtered, washed with hexane, and dried. IR
spectroscopic data indicated the formation of hydra-
zides by appearance of two separate bands for NH2
asymmetric (3485–3458 cm−1) and symmetric
(3298–3269 cm−1) stretching vibrations in addition to
the NH band. Disappearance of the ester carbonyl
band and the appearance of a broad band for two the
amidic carbonyls in the range 1678–1649 cm−1 also
O H R
H R
(a) (b)
H2N COOH N COOH
H H
(1a-c) (2a-c)
R=H, CH3, CH2CH(CH3)2
O H R R1
NHN (d)
N C
H H 1
O R CHO
(6a-n) (5a-h)
Scheme 1. Synthesis of Schiff bases (a) PhCOCl,
MeOH (b) dry MeOH, conc. H2SO4,
reflux (c) MeOH, hydrazine hydrate
80%, reflux (d) MeOH, R1CHO, reflux.
2 www.jccs.wiley-vch.de © 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Abid et al.
indicated the synthesis of the hydrazide. Each hydrazide
(4a–4c) was dissolved in 5 mL of ethanol by stirring,
and an equivalent mmol of the aldehyde and few drops
of acetic acid were added. The solution was refluxed for
3–9 h, and then the product formed was filtered by add-
ing cold distilled water and dried. Products (6a–6n)
were characterized by elemental analysis and spectro-
scopic data. The results of elemental analysis were in
good agreement with the proposed stoichiometry. IR
spectroscopic data indicated the formation of imines by
the disappearance of two separate bands for the NH2
asymmetric and symmetric stretching vibrations. The
appearance of the imine (C N) band in the range
1651–1589 cm−1 also indicated the synthesis of imines.
In the 1H NMR spectra, the presence of NH and imine
CH protons in the range 12.20–7.93 ppm and the rele-
vant aromatic protons of the protecting group and the
aldehyde group confirmed the synthesis of the desired
products. Electrospray ionization mass spectrometry
(EI-MS) results showed the molecular ion peaks in only
two cases, whereas the fragment with m/z 105 (benzoyl
cation) mostly appeared as a prominent fragment and
as base peak in six cases. The confirmation of target
molecules was finally achieved through high-resolution
mass spectrometry (HRMS) (Scheme 1 and Table 1).
Biological activity
Antimicrobial activity The newly synthesized com-
pounds were investigated for their antimicrobial activity
against a panel of Gram-positive and Gram-negative
pathogenic bacteria and fungi. The test strains included
Staphylococcus aureus, MRSA (methicillin-resistant
S. aureus), Escherichia coli, Klebsiella sp., Actinobacter
sp., and Candida albicans. In this analysis, the protected
O H R
amino acids, esters, hydrazides, and hydrazones with
N COOMe
diverse aldehydes were studied comparatively. Each of
(3a-c) the compounds exhibited a different of antimicrobial
(c) response against the different tested strains. The com-
pound 6k exhibited good antimicrobial activity against
O H R
Gram-negative bacteria such as E. coli and against
NHNH2
N C
H C. albicans with inhibitory zones of 20 and 19 mm,
O
respectively. Compounds 2a, 2c, 3a, 4b, 4c, 6a, 6b, and
(4a-c)
6c showed moderate to low antimicrobial activity
against Gram-positive bacteria, and inhibitory zones in
the range of 7 to 16 mm were observed against
S. aureus and MRSA. Compounds 3b, 3c, 4a, and 6i,
J. Chin. Chem. Soc. 2017
 JOURNAL OF THE CHINESE
Hydrazones from Amino Acids CHEMICAL SOCIETY

Table 1. Structures of the synthesized compounds Table 1. Continued

Sample code R R1 Sample code R R1

6a H 6n CH3

6b H

however, were found to be inactive against all the tested

6c H N strains (Table 2).The results of this antimicrobial
screening indicate that N-protected amino acids 2a, 2c,
the ester 3a, the hydrazide 4c, and the hydrazones 6a,
6d H
6b are promising candidates against Gram-positive bac-
teria. The synthesized hydrazones such as 6f, 6h, 6j, 6k,
and 6m exhibit a broad spectrum of activities against
MeO OMe
both Gram-positive and Gram-negative bacteria and
6e H
Me2N also against the fungal test strain, so these compounds
can be the lead candidates for further biological investi-
6f H gation and structural modifications. It is to be noted
H3CO that the hydrazones were found to be more active than
6g CH2CH(CH3)2 the protected amino acids as well as their esters and
hydrazides. The active compounds were also tested for
their minimum inhibitory concentrations (MICs)
against S. aureus and E. coli. In the case of Gram-
6h CH2CH(CH3)2 positive S. aureus, most of the compounds exhibited
OH MIC in the range 32–128 μg/mL, while in case of
Gram-negative E. coli, MIC in the range 16–256 μg/mL
was exhibited by compounds 6f and 6k, respectively
OH (Table 2).
6i CH2CH(CH3)2 Cytotoxicity (% larval mortality) The synthesized com-
pounds were subjected to cytotoxicity screening by the
6j CH2CH(CH3)2 N microwell cytotoxicity assay against the brine shrimp
Artimia salina. Most of the compounds exhibited mod-
erate to high cytotoxicity. The compounds 6a and 6b
6k CH2CH(CH3)2 were found to be less cytotoxic, with larval mortality in
the range 20–30%; however, most of the compounds
showed 50–70% larval mortality. Compounds 4b, 6f, 6j,
MeO OMe and 6k showed the highest percentage of larval mortal-
6l CH2CH(CH3)2 ity (70–85%) and hence were the most cytotoxic com-
Me2N pounds. Owing to the antimicrobial response of these
compounds, the broad spectrum activity of the com-
6m CH2CH(CH3)2 OH pounds like 6f, 6h, 6j, 6k, and 6m can be due to their
high nonspecific cytotoxicity. The high cytotoxicity of
the compound 6k might be due to the presence of two
methoxy groups, which are the activating groups.

J. Chin. Chem. Soc. 2017 © 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.jccs.wiley-vch.de 3
Article Abid et al.

Table 2. Antimicrobial activity of the synthesized compounds

Antimicrobial activity against MDR bacteria and fungi Minimum inhibitory concentrations
(zone of inhibition in mm) (MICs) μg/mL

Sample codes S. aureus MRSA E. coli Kleb Acineto Candida albicans S. aureus E. coli
2a 14 18 — — — — 32 —
2b — 10 — — — — — —
2c 12 12 — 10 — — 64 —
3a 10 11 — 09 — — 64 —
3b — — — — — — — —
3c — — — — — — — —
4a — — — — — — — —
4b 09 13 — 10 — — 128 —
4c 12 16 — 10 — — 64 —
6a 10 14 — 07 — — 128 —
6b 10 11 — — — — — —
6c 07 12 — — — — 128 —
6d — — — 09 08 — — —
6e 10 — — — — — 128 —
6f 10 — 10 — 10 12 128 256
6g — — — — — 10 — —
6h — — 19 08 11 13 — 32
6i — — — — — — — —
6j 16 — 16 12 10 18 32 32
6k — — 20 10 09 18 — 16
6l — — 10 — — — — —
6m 12 — 10 — 14 07 64 64
6n — — 14 — — 10 — 64
Kleb, Klebsella sp.; Acineto, Acinetobacter sp.

However, these compounds can be the promising candi- CONCLUSIONS

dates for antitumor or anticancer activity and should be The hydrazones 6a–6n synthesized from three
investigated further for these kinds of biological activ- amino acids were characterized and evaluated for their
ities (Table 3). antimicrobial activity and cytotoxicity. Compounds 6f,
6h, 6j, 6k, and 6m exhibited a broad spectrum of antibac-
Table 3. Cytotoxicity results of synthesized compounds
terial activity and can be the lead candidates for further
Cytotoxicity Cytotoxicity biological investigations and structural modifications.
Samples (% mortality) Samples (n% mortality) Cytotoxicity studies revealed that compounds 4b, 6f, 6j,
2a 50.0 6d — and 6k were the most cytotoxic. And these compounds
2b — 6e 63.8 can be promising candidates for antitumor or anticancer
2c 58.9 6f 81.0 activity and should be investigated further for such bio-
3a 64.1 6g — logical activities. In general, hydrazones were found to be
3b — 6h 38.6 more active than their precursors.
3c — 6i —
4a — 6j 76.1
4b 71.2 6k 85.0 EXPERIMENTAL
4c 64.1 6l 65.2 Different chemicals/reagents were used for the syn-
6a 26.3 6m 55.0 thesis of the desired products, which included amino
6b 21.6 6n 65.8 acids, benzoyl chloride, hydrochloric acid, sulfuric acid,
6c — — —
methanol (dry), magnesium sulfate(anhydrous), ethanol,

4 www.jccs.wiley-vch.de © 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim J. Chin. Chem. Soc. 2017

Hydrazones from Amino Acids
acetic acid, hydrazine hydrate (80%), distilled water, hex-
ane, dichloromethane, ethyl acetate, acetone, chloroform,
and different aldehydes. All the chemicals used were of
analytical grade and were purchased from Merck, Riedel-
deHaen, Scharlau, Aldrich, or Fluka.
All synthesized compounds were characterized by
their physical constants as well as spectroscopic data
such as IR, 1H NMR, GC-MS, HRMS, and elemental
analysis. Thin-layer chromatography (TLC) was per-
formed on precoated silica gel GF-254 aluminum plates
(Kieselgel 60, 20 × 20, 0.2–0.5 mm thick; E. Merck,
Germany). Detection under UV light illumination was
done with 254 and/or 366 nm. Melting points were
determined on a Sanyo Gallenkamp digital melting
point apparatus (MPD 350) in open capillaries. 1H
NMR was carried out on a Bruker ARX 300 spectrome-
ter. Mass Spectroscopy was carried out on AMD
MS40, AMD 402 (AMD Intectra), Varian MAT CH
7, MAT 731, and HRMS on Finnigan MAT 95 or Var-
ian MAT 311, Bruker FTCIR, AMD 402 (AMD Intec-
tra) instruments. IRs spectra were recorded on a Bruker
IFS 66 (FT IR), Nicolet 205 FT IR, Nicolet Protege
460, or Nicolet 360 Smart Orbit (ATR) instrument
using KBr and ATR. The elemental analyzer used was
LECO CHNS-932 or Thermoquest Flash EA 1112.
Procedure for the N-protection of amino acids
Ten millimoles of the amino acid was dissolved
2.5 mL of NaOH (10%). Then, 1.5 mL of benzoyl chlo-
ride was added, and the solution was stirred for 3–4
h. A solid material was obtained, which was further
acidified with hydrochloric acid (dilute + cold) to pH 3.
After filtration and washing by cold distilled water, the
benzoyl-protected amino acid was dried and then
weighed.
Procedure for the esterification of N-protected amino
acids
The protected amino acid (8.0 mmol) was dis-
solved in 5 mL of dry methanol in a round-bottom
flask, and a few drops of sulfuric acid were added. The
solution was stirred and refluxed for 10–19 h. After
completion of the reaction, the solvent was evaporated
and ethyl acetate was added, and the solution was
washed with NaHCO3 solution and distilled water.
Then MgSO4 was added and the organic layer was
J. Chin. Chem. Soc. 2017 © 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
filtered. The solvent was evaporated to obtain the
product.
Procedure for the synthesis of hydrazides
Six millimoles of the ester was dissolved in 4 mL
methanol by stirring in a round-bottom flask, and then
1.5 mL of hydrazine hydrate was added and refluxed
for 3–11 h in a reflux condenser. The solvent was
removed, and the product was filtered, washed with
n-hexane, and dried.
Procedure for the synthesis of hydrazones
One millimole of hydrazide was dissolved in 5 mL
of ethanol by stirring in a round-bottom flask, and an
equivalent mmol of aldehydes and few drops of acetic
acid were added. The solution was refluxed for 3–9 h,
and the product was filtered, washed by cold distilled
water, and dried. Characteristic data for all the synthe-
sized compounds 6a–6n are given in detail below.
N0 -(Anthracene-10-ylmethylene)-2-benzamidoacetohydra-
zide 6a
Yellow powder; Yield: 89%; m.p: 202–205 C; IR
−1
(cm ): 3352 (NH), 1680 (C O), 1643(C N), 1537
(C C); Elemental analysis: calcd. for C24H19N3O2: C,
75.57; H, 5.02; N, 11.02, Found: C, 75.69; H, 4.91; N,
11.18; 1H NMR (DMSO-d6, 300 MHz); δ 11.72 (s, 1H,
NH), 8.77–8.74 (m, 2H, NH/CH), 8.63(d, 2H,
J = 6.6 Hz, Ar–H), 8.17 (d, 2H, J = 8.4 Hz, Ar–H),
7.98–7.92 (m, 2H, Ar–H), 7.69–7.48 (m, 8H, Ar–H),
4.49 (d, 2H, J = 6.0 Hz, CH2); MS (GC, 70 eV): m/z
(%): 204(16), 203(100), 202(10), 176(9), 175(8), 150(5),
101(7), 88(11), 75(6); HRMS Pos (ESI) calcd. for
C24H19N3O2 [M + H,]+ 382.155, Found 382.15492 and
[M + Na]+ calcd. 404.13695, Found 404.1369 and the
exact ion mass is 381.14773.
N0 -(p-Biphenylmethylene)-2-benzamidoacetohydrazide 6b
Light brown powder; Yield: 86%; m.p:
238–239 C; IR (cm−1): 3315 (NH), 1687 (C O),1634
(C N), 1547 (C C); Elemental analysis: calcd. for
C22H19N3O2: C, 73.93; H, 5.36; N, 11.76. Found: C,
73.87; H, 5.19; N, 11.94. 1H NMR (DMSO-d6,
300 MHz); δ 11.58 (s, 1H, NH), 8.71 (t, 1H,
J = 6.6 Hz, NH), 8.06 (s, 1H, CH), 7.92(d, 2H,
J = 6.3 Hz, Ar–H), 7.82–7.77 (m, 4H, Ar–H), 7.73 (d,
2H, J = 8.7 Hz, Ar–H), 7.57–7.47 (m, 5H, Ar–H), 7.41
(d, 1H, J = 6.3 Hz, Ar–H) 4.46 (d, 2H, J = 6.0 Hz,
www.jccs.wiley-vch.de 5
Article
CH2); MS (GC, 70 eV): m/z (%): 358 (M + 1+,1),
357 (M+, 5), 223(10), 197(15), 196(83), 195(13), 179
(13), 167(12), 165(27), 152(16), 135(13), 134(27), 105
(100), 77(52), 51(11); HRMS (EI) calcd. for
C22H19N3O2 [M+]: 357.14718, Found: 357.1483.
2-Benzamido-N0 -(quinolin-2-ylmethylene)acetohydrazide 6c
Light brown powder; Yield: 93%; m.p:
226–229 C; IR (cm−1): 3391 (NH), 1697 (C O),1641
(C N), 1538 (C C); Elemental analysis: calcd. for
C19H16N4O2: C, 68.66; H, 4.85; N, 16.86. Found: C,
68.83; H, 4.77; N, 16.72; 1H NMR (DMSO-d6,
300 MHz); δ 11.90 (s, 1H, NH), 8.78 (t, 1H,
J = 6.6 Hz, NH), 8.42 (d, 1H, J = 8.7 Hz, Ar–H), 8.21
(s, 1H, CH), 8.14 (d, 1H, J = 8.7 Hz, Ar–H), 8.05–8.0
(m, 2H, Ar), 7.93 (d, 2H, J = 6.6 Hz, Ar–H), 7.80 (dt,
1H, J = 6.6, 2.7 Hz, Ar–H), 7.64 (dt, 1H, J = 6.6,
2.7 Hz, Ar–H), 7.57–7.50 (m, 3H, Ar–H), 4.53 (d, 2H,
J = 6.0 Hz, CH2); MS (GC, 70 eV): m/z (%): 290(5),
198(54), 170(62), 142(68), 115(82), 105(100), 77(71),
51(18); HRMS Pos (ESI) calcd. for C19H16N4O2
[M + H]+ calcd. 333.1346, Found: 333.13527 and
[M + Na]+ calcd. 355.11655, Found 355.11638; exact
ion mass 332.12733.
N0 -(3,5-Dimethoxybenzylidene)-2-benzamidoacetohydraz-
ide 6d
Floral white powder; Yield: 91%; m.p: 174–175 C;
IR (cm−1): 3255 (NH), 1687 (C O),1631 (C N), 1573
(C C); Elemental analysis: calcd. for C18H19N3O4: C,
63.33; H, 5.61; N, 12.31, Found: C, 63.47; H, 5.53; N,
12.44; 1H NMR (DMSO-d6, 300 MHz); δ 11.51 (s, 1H,
NH), 8.70 (t, 1H, J = 6.6 Hz, NH), 7.93 (s, 1H, CH),
7.90 (d, 2H, J = 6.6 Hz, Ar–H), 7.56–7.49 (m, 3H, Ar),
6.86 (t, 2H, J = 3.0 Hz, Ar–H), 6.55(t, 1H, J = 3.0 Hz,
Ar–H), 4.42 (d, 2H, J = 6.3 Hz, CH2), 3.78 (s, 6H,
2OCH3); MS (GC, 70 eV): m/z (%): 341 (M+, 1), 207
(26),180(18), 178(11), 153(17), 135(11), 134(24),
105 (100), 77 (67), 51 (14); HRMS Pos (ESI) calcd. for
C18H19N3O4 [M + H]+; calcd. 342.14483, Found
342.14496, and [M + Na]+ calcd. 364.12678, Found
364.12699; exact ion mass 341.13756.
N0 -(anthracene-10-ylmethylene)-2-benzamido-4-methylpe-
ntanehydrazide 6g
Dark orange powder; Yield: 79%; m.p:
225–230 C; IR (cm−1): 3298 (NH), 1663 (C O), 1624
(C N), 1549 (C C); Elemental analysis: calcd. for
6 www.jccs.wiley-vch.de © 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Abid et al.
C28H27N3O2 : C, 76.86; H, 6.22; N, 9.60, Found: C,
76.69; H, 6.17; N, 9.81; 1H NMR (DMSO-d6,
300 MHz); δ 12.11 (s, 1H, NH), 9.69 (s, 1H, NH), 8.79
(s, 1H, CH), 8.75 (d, 2H, J = 6.6 Hz, Ar–H), 8.18 (d,
2H, J = 6.3 Hz, Ar–H), 8.05 (d, 2H, J = 6.3 Hz, Ar–
H), 7.70–7.58 (m, 8H, Ar–H), 4.44 (dd, 1H,
J = 8.0,7.5 Hz, CH), 1.88–1.79 (m, 3H, CH/CH2), 0.92
(d, 3H, J = 6.3 Hz, CH3), 0.85 (d, 3H, J = 6.3 Hz,
CH3); MS (GC, 70 eV): m/z (%): 204(19), 203(100), 202
(12), 176(13), 174(8), 88(10), 75(8), 61(6); HRMS Pos
(ESI) calcd. for C28H27N3O2, [M + H]+ calcd.
438.13354, found 438.13364 and [M + Na]+ calcd.
460.11548, found 460.11583; exact ion mass 437.12626.
N0 -(2,4-Dihydroxybenzylidene)-2-benzamido-4-methylpe-
ntanehydrazide 6h
Dark brown powder; Yield: 72%; m.p:
204–209 C; IR (cm−1): 3318 (NH), 1688 (C O), 1651
(C N), 1561 (C C); Elemental analysis: calcd. for
C20H23N3O4: C, 65.03; H, 6.28; N, 11.37, Found: C,
65.16; H, 6.45; N, 11.20; 1H NMR (DMSO-d6,
300 MHz); δ 11.60 (s, 1H, NH), 9.33 (bs, 2H, OH),
8.54 (d, 1H, J = 8.7 Hz, NH), 8.27 (s, 1H, CH), 7.90
(d, 2H, J = 6.6 Hz, Ar–H), 7.57 (d, 1H, J = 6.6 Hz,
Ar–H), 7.53 (d, 2H, J = 6.3 Hz, Ar–H), 7.26 (d, 1H,
J = 3.0 Hz, Ar–H), 6.94 (dd, 1H, J = 6.3,3.0 Hz, Ar–
H), 6.79 (d, 1H, J = 8.7 Hz, Ar–H), 4.51 (dd, 1H,
J = 8.0, 7.5 Hz, CH), 1.94–1.88 (m, 3H, CH/CH2),
0.91 (d, 3H, J = 6.3 Hz, CH3), 0.87 (d, 3H, J = 6.3 Hz,
CH3); MS (GC, 70 eV): m/z (%): 256(22), 135(24), 122
(32), 105(100), 77(47), 51(14), 29(11); HRMS Pos (ESI)
calcd. for C20H23N3O4 [M + H]+ calcd. 370.17613,
Found 370.17713; [M + Na]+ calcd. 392.15808, Found
392.15769, exact ion mass 369.16886.
N0 -(p-Biphenyl)-2-benzamido-4-methylpentanehydrazide 6i
Floral white powder, Yield: 89%; m.p: 234–238 C;
IR (cm−1): 3345 (NH), 1672 (C O), 1619 (C N), 1558
(C C); Elemental analysis: calcd. for C26H27N3O2: C,
75.52; H, 6.58; N, 10.16, Found: C, 75.82; H, 6.73; N,
10.03; 1H NMR (DMSO-d6, 300 MHz);δ 11.89 (s, 1H,
NH), 8.52 (s, 1H, CH), 7.94 (d, 2H, J = 6.60 Hz, Ar–
H), 7.82–7.77 (m, 4H, Ar–H), 7.74 (d, 2H, J = 6.3 Hz,
Ar–H), 7.61–7.47(m, 5H, Ar–H), 7.42 (d, 1H,
J = 8.7 Hz, Ar–H), 4.55 (dd, 1H, J = 8.0,7.5 Hz, CH),
1.79–1.73 (m, 3H, CH/CH2), 0.92 (d, 3H, J = 6.3 Hz,
CH3), 0.85 (d, 3H, J = 6.3 Hz, CH3); MS (GC, 70 eV):
J. Chin. Chem. Soc. 2017

Hydrazones from Amino Acids
m/z (%): 300(5), 180(20), 179(100), 165(11), 152(7), 105
(80), 77(40), 51(8); HRMS Pos (ESI) calcd. for
C26H27N3O2 [M + H]+ calcd. 414.13354, Found
414.13404 and [M + Na]+ calcd. 436.11548, Found
436.11598; exact ion mass 413.12626.
2-Benzamido-4-methyl-N0 -(quinolin-2-ylmethylene)penta-
nehydrazide 6j
Peach powder; Yield: 66%; m.p: 110–113 C; IR
−1
(cm ): 3312 (NH), 1637 (C O), 1589 (C N), 1565
(C C); Elemental analysis: calcd. for C23H24N4O2: C,
71.11; H, 6.23; N, 14.42, Found: C, 71.29; H, 6.09; N,
14.49; 1H NMR (DMSO-d6, 300 MHz); δ 12.20 (s, 1H,
NH), 8.69 (d, 1H, J = 8.7 Hz, NH), 8.63 (s, 1H, CH),
8.44 (d, 1H, J = 8.7 Hz, Ar–H), 8.14 (d, 1H,
J = 8.7 Hz, Ar–H), 8.07–8.01 (m, 2H, Ar–H), 7.96 (d,
2H, J = 6.3 Hz, Ar–H), 7.81 (dt, 1H, J = 8.7, 3.0 Hz,
Ar–H), 7.68–7.55 (m, 4H, Ar–H), 4.59 (dd, 1H,
J = 8.0,7.5 Hz, CH), 1.90–1.84 (m, 3H, CH/CH2), 0.98
(d, 3H, J = 6.3 Hz, CH3), 0.94 (d, 3H, J = 6.3 Hz,
CH3); MS (GC, 70 eV): m/z (%): 275(2), 171(12), 170
(84), 142(59), 140(15), 115(100), 105(63), 77(97), 51(37),
50(18); HRMS Pos (ESI) calcd. for C23H24N4O2
[M + H,]+ calcd. 389.1972, Found 389.19777;
[M + Na]+ calcd. 411.17915, found 411.1787; exact ion
mass 388.188993.
N0 -(3,5-Dimethoxybenzylidene)-2-benzamido-4-methylpe-
ntanehydrazide 6k
White powder; Yield: 83%; m.p: 190–193 C; IR
−1
(cm ): 3346 (NH), 1681 (C O), 1645 (C N), 1547
(C C); Elemental analysis: calcd. for C22H27N3O4:C,
66.48; H, 6.85; N, 10.57, Found: C, 66.58; H, 6.71; N,
10.76; 1H NMR (DMSO-d6, 300 MHz); δ 11.87 (s, 1H,
NH), 8.52 (d, 1H, J = 8.7 Hz, NH), 8.39 (s, 1H, CH),
7.92 (d, 2H, J = 6.3 Hz, Ar–H), 7.61–7.53 (m, 3H, Ar–
H), 6.88 (d, 2H, J = 3.0 Hz, Ar–H), 6.58 (t, 1H,
J = 3.0 Hz, Ar–H), 4.56 (dd, 1H, J = 8.0, 7.5 Hz, CH),
3.80 (s, 6H, 2OCH3),1.84–1.76 (m, 3H, CH/CH2), 1.02
(d, 3H, J = 6.3 Hz, CH3), 0.94 (d, 3H, J = 6.3 Hz,
CH3); MS (GC, 70 eV): m/z (%): 284(9), 164(12), 163
(75), 106(9), 105(100), 77(77), 76(10), 51(25), 50(9);
HRMS Pos (ESI) calcd. for C22H27N3O4 [M + H]+
calcd. 398.20743; Found 398.2069 and [M + Na]+
calcd. 420.18938, Found 420.18889; exact ion mass
397.20016.
J. Chin. Chem. Soc. 2017 © 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
N0 -(4-(Dimethylamino)benzylidene)-2-benzamido-4-meth-
ylpentanehydrazide 6l
Orange powder; Yield: 96%;m.p: 130–135 C; IR
−1
(cm ): 3315 (NH), 1648 (C O), 1593(C N), 1524
(C C); Elemental analysis: calcd. for C22H28N4O2: C,
69.45; H, 7.42; N, 14.73, Found: C, 69.61; H, 7.33; N,
14.51; 1H NMR (DMSO-d6, 300 MHz); δ 11.54 (s, 1H,
NH), 8.52 (d, 1H, J = 8.7 Hz, NH), 8.32 (s, 1H, CH),
7.90 (d, 2H, J = 6.6 Hz, Ar–H), 7.58–7.49 (m, 5H, Ar–
H), 6.77 (d, 2H, J = 8.7 Hz, Ar–H), 4.56 (dd, 1H,
J = 8.0, 7.5 Hz, CH), 2.98 (s, 6H, NMe2), 1.81–1.74
(m, 3H, CH/CH2), 1.01 (d, 3H, J = 6.0 Hz, CH3), 0.93
(d, 3H, J = 6.3 Hz, CH3); MS (GC, 70 eV): m/z (%):
267(44), 162(17), 148(29), 146(100), 132(8), 118(14), 105
(34), 91(8), 77(31), 51(9); HRMS Pos (ESI) calcd. for
C22H28N4O2 [M + H]+ calcd. 381.11314, Found
381.11369 and [M + Na]+ calcd. 413.09508, Found
413.09564; and exact ion mass 380.10586.
2-Benzamido-N0 -((1-hydroxynaphthalen-2-yl)methylene)-
4-methylpentanehydrazide 6m
Lemon yellow powder; Yield: 95%; m.p:
217–220 C; IR (cm−1): 3265 (NH), 1644 (C O), 1621
(C N), 1579 (C C); Elemental analysis: calcd. for
C24H25N3O3:C, 71.44; H, 6.25; N, 10.41, Found: C,
71.33; H, 6.19; N, 10.55; 1H NMR (DMSO-d6,
300 MHz); δ 12.78 (s, 1H, OH), 12.20 (s, 1H, NH),
9.50 (s, 1H, CH), 8.23 (d, 1H, J = 8.7 Hz, Ar–H),
8.02–7.90 (m, 4H, Ar–H), 7.66–7.57 (m, 4H, Ar–H),
7.42 (t, 1H, J = 6.3 Hz, Ar–H), 7.25 (d, 1H,
J = 6.3 Hz, Ar–H), 4.64 (dd, 1H, J = 8.0, 7.5 Hz, CH),
1.93–1.85 (m, 3H, CH/CH2), 0.98 (d, 3H, J = 6.0 Hz,
CH3), 0.94 (d, 3H, J = 6.0 Hz, CH3); MS (GC, 70 eV):
m/z (%): 217(18), 174(22),161(26), 147(30), 130(16), 105
(100), 77(37), 51(9); HRMS Pos (ESI) calcd. for
C24H25N3O3 [M + H]+ calcd. 404.10582, Found
404.10614 and [M + Na]+ calcd. 426.15637, found
426.15685; and exact ion mass 403.06308.
N0 -(anthracen-10-ylmethylene)-2-benzamidopropanehydr-
azide 6n
Orange powder; Yield: 54%; m.p: 219–221 C; IR
−1
(cm ): 3254 (NH), 1670 (C O), 1634 (C N), 1532
(C C); Elemental analysis: calcd. for C25H21N3O2:C,
75.93; H, 5.35; N, 10.63, Found: C, 75.69; H, 5.47; N,
10.79; 1H NMR (DMSO-d6, 300 MHz); δ 11.81 (s, 1H,
NH), 8.78 (m, 1H, NH), 8.73 (s, 1H, CH), 8.67 (d, 2H,
www.jccs.wiley-vch.de 7
Article
J = 6.3 Hz, Ar–H), 8.16 (d, 1H, J = 8.7 Hz, Ar–H),
8.00–7.93 (m, 2H, Ar–H), 7.67–7.48 (m, 8H, Ar–H),
4.66 (q, 1H, J = 7.8 Hz, CH), 1.52 (d, 3H, J = 8.1 Hz,
CH3): MS (GC, 70 eV): m/z (%): 204(14), 203(100), 201
(9), 176(8), 175(10), 150(6), 88(8), 87(7), 75(7), 63(4),
50(4); HRMS Pos (ESI) calcd. for C25H21N3O2
[M + H]+ calcd. 396.17065, found 396.17106 and
[M + Na]+ calcd. 418.1526, found 418.15308; exact ion
mass 395.16338.
DETERMINATION OF ANTIMICROBIAL
ACTIVITY, MINIMUM INHIBITORY
CONCENTRATIONS (MICS), AND
CYTOTOXICITY
The antimicrobial activity of the synthesized com-
pounds was determined by the agar diffusion method
against the following test strains; S. aureus, MRSA,
E. coli, Klebsiella sp., Acintobacter sp., and C. albicans.
Luria–Bertani (LB) agar plates were prepared by pour-
ing 16 mL of molten agar in to Petri plates. After solid-
ification, the plates were overlayed with 4 mL of seed
agar having a standardized inoculum of the test strains.
The plates were left for 15 min at room temperature,
and the agar wells were made with the help of a sterile
cork borer. Then 50 μL of the samples was loaded in
each well, which were labeled accordingly. The plates
were left at room temperature for 2 h and incubated at
37 C for 18–24 h. The plates were observed for clear
zones around the wells, which indicated growth inhibi-
tion of the test organism by the compounds. The zones
of inhibition were measured in millimeters.
The minimum inhibitory concentrations (MICs) of
the compounds were determined by the broth dilution
method against the two representative Gram-positive
and Gram-negative test organisms including S. aureus
and E. coli, respectively. The pure cultures of the test
organisms were grown in the LB broth and the inocu-
lum was standardized by comparing with 0.5 McFar-
land standard. Twofold dilutions of each of the
compounds were prepared in sterile LB broth in the fol-
lowing range: 1024, 512, 256, 128, 64, 32, 16, 8, 4,
2, and 1 μg/mL. Each test tube contained 5 mL of LB
broth, which was inoculated with 50 μL of the cell sus-
pension. The tubes were incubated at 37 C for 18–24 h
and were observed for the growth of the test organism,
which was indicated by turbidity. The lowest dilution
that did not exhibit any turbidity or growth of the
8 www.jccs.wiley-vch.de © 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Abid et al.
organism was considered as the MIC of the respective
sample.
The cytotoxicity of the compounds was deter-
mined by microwell cytotoxicity assay against the brine
shrimp (Artimia salina). The eggs powder of the
A. salina (0.5 g) was added to a 1-L separating funnel
filled with 500 mL of artificial seawater. The suspension
was aerated by bubbling air into the funnel and kept
for 24–48 h at room temperature. Later, the aeration
was stopped and the suspension was kept undisturbed
for 1 h to let the remaining eggs settle down. In order
to use only the active larvae, one side of the separating
funnel was covered with an aluminum foil and the other
side illuminated with a lamp to gather the phototropic
larvae at the illuminated side. Then with the help of a
micropipette, 30–40 shrimp larvae were transferred to a
deep-well microtiter plate, with each well filled with
0.2 mL of sea water. Initially, the dead larvae were
counted (value N), then 20 μg of the compounds in
DMSO was added to the designated wells, and the plate
was kept at room temperature in the dark. After 24 h,
the number A of the dead larvae in each well was
counted again under the microscope. The surviving lar-
vae were killed by the addition of 0.5 mL methanol to
each well, and subsequently the total number G of the
larvae was determined. Each test row was accompanied
by a blind sample containing pure DMSO instead of
the test compounds. As a positive control with 100%
mortality, namely actinomycin D (20 μg/mL), was used.
The percentage mortality M was calculated using the
following formula:
M = [(A – B – N)/(G – N)] × 100
where
M = percent of the dead larvae after 24 h.
A = number of the dead larvae after 24 h.
B = average number of the dead larvae in the
blind samples after 24 h.
N = number of the dead larvae before starting of
the test.
G = total number of larvae.
ACKNOWLEDGMENTS
The authors acknowledge the support provided by
HEC, Pakistan, under its ASIP program for the analy-
sis of the products synthesized.
J. Chin. Chem. Soc. 2017

Hydrazones from Amino Acids
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