Synthesis and biological evaluation of 4-(2-(dimethylamino)ethoxy)benzohydrazide derivatives as inhibitors of Entamoeba histolyica

Article abstract of DOI:10.1016/j.ejmech.2016.08.022

In the quest for potent antiamoebic agents, a series of hydrazone hybrids (H1[sbnd]H30) have been designed and sequentially synthesized. The dimethylaminoethoxy and hydrazone entities incorporated into one molecule proved to be more persuasive and selecti

Full text of DOI:10.1016/j.ejmech.2016.08.022

Accepted Manuscript
Synthesis and biological evaluation of 4-(2-(dimethylamino)ethoxy) benzohydrazide
derivatives as inhibitors of Entamoeba histolyica
Afreen Inam, Sonam Mittal, Maitreyi S. Rajala, Fernando Avecilla, Amir Azam
PII:
S0223-5234(16)30665-1
10.1016/j.ejmech.2016.08.022
EJMECH 8818
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 April 2016
Revised Date: 20 July 2016
Accepted Date: 11 August 2016
Please cite this article as: A. Inam, S. Mittal, M.S. Rajala, F. Avecilla, A. Azam, Synthesis and biological
evaluation of 4-(2-(dimethylamino)ethoxy) benzohydrazide derivatives as inhibitors of Entamoeba
histolyica, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.022.
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Graphical Abstract
Synthesis and biological evaluation of 4-(2-(dimethylamino)ethoxy) Leave this area Blank for abstract info
benzohydrazide derivatives as inhibitors of Entamoeba histolyica
Afreen Inama, Sonam Mittalb, Maitreyi S. Rajalab, Fernando Avecillac, Amir Azama*
aDepartment of Chemistry, Jamia Millia Islamia, Jamia Nagar, 110 025, New Delhi, India
bSchool of Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India
cDepartamento de Química Fundamental, Universidade da Coruña, Campus da Zapateira, 15071 A Coruña,
Spain
Hydrazones formed by the combination of two pharmacophores, the N-dimethylaminoethoxy tail and different
substituted aldehydes. The hybrids (H1-H30) have been evaluated against the HM1: ISS strain of E.histolytica and
cell viability on lung cancer cell line (A549 cells) by MTT assay.
O
N
NH
N
N
O
Compound (H23)

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Synthesis and biological evaluation of 4-(2-(dimethylamino)ethoxy)benzohydrazide
derivatives as inhibitors of Entamoeba histolyica
Afreen Inama, Sonam Mittalb, Maitreyi S. Rajalab, Fernando Avecillac, Amir Azama*
aDepartment of Chemistry, Jamia Millia Islamia, Jamia Nagar, 110 025, New Delhi, India
bSchool of Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India
cDepartamento de Química Fundamental, Universidade da Coruña, Campus da Zapateira,
15071 A Coruña, Spain
*To whom correspondence should be addressed: tel.: +91 11 2698 1717/3254; fax: +91 11
26980229; e-mail address: amir_sumbul@yahoo.co.in
Abstract
In the quest for potent antiamoebic agents, a series of hydrazone hybrids (H1-H30) have
been designed and sequentially synthesized. The dimethylaminoethoxy and hydrazone
entities incorporated into one molecule proved to be more persuasive and selective approach
towards designing of antiamoebic agent. The synthesized compounds exhibited promising
results against E.histolytica. The compound N’-(2-chlorobenzylidene)-4-(2-(dimethylamino)
ethoxy)benzohydrazide was most impending among the series. Cytotoxicity profile showed
better cell viability on lung cancer cell line (A549 cells) by MTT assay.
Key word: Hydrazones; antiamoebic activity; Cytotoxicity; MTT assay; X-ray
crystallography

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1. Introduction
Amoebiasis is an acute infection caused by the protozoan Entamoeba histolytica, which
causes intestinal disease (e.g. colitis) and extra intestinal symptoms, including liver abscess
and pleuropulmonary, cardiac, and cerebral dissemination [1]. E histolytica is transmitted
primarily through the fecal-oral route. Infective cysts can be found in fecal contaminated food
and water supplies [2]. According to reports approximately 50 million cases of invasive E
histolytica disease occur worldwide and 40,000 to 100,000 people die annually due to
amoebiasis each year [3]. The prevalence of amoebiasis is elevated in developing countries
[4]. Several studies have evaluated that amebic liver abscess is an emerging parasite infection
in individuals with HIV infection in disease-endemic areas, as well as in non–disease-
endemic areas [5]. Treatment of amoebiasis includes pharmacologic therapy, surgical
intervention, and preventive measures. The various detrimental aspects of the currently used
drugs include numbness of hands or feet, blurred vision, burning in urination etc. Numerous
severe side effects associated with the use of metronidazole are: seizures, ataxia, peripheral
neuropathy, transient myopia, gastric mucus irritation, spermatozoid damage and haematuria.
Hence, there is an intense need for the development of potential molecules acting against this
dreadful infection [6]. Hydrazones constitute a resourceful class of compounds in organic
chemistry. These compounds possess extensive range of biological properties, such as anti-
inflammatory, analgesic, anticonvulsant, antitubercular, antitumor, anti-HIV and
antimicrobial activity [7-12]. Due to its positive traits, hydrazones have been under study for
a long time, but much of their basic chemistry remains unexplored. Hydrazone is a well-
known group of antiparasitic agents, which includes antiamoebic properties [13, 14]. A
number of hydrazone derivatives were synthesized in our laboratory and found to be
endowed with antiamoebic activity (Figure 1) [15-17]. It was observed that hydrazones
showed better antiplasmodial and antiamoebic activity as compared to its hydrazide [15]. The

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derivatives bearing quinoline nucleus with a hydrazone linkage [-NH-N- C(CH3)-R] having a
free N-H group exhibited higher antiamoebic activity as compared with their cyclised
products [16].
Another entity which has gained importance in the drug designing is the dimethylamino
ethoxy moiety which is present in the number of currently used cancer drugs such as
tamoxifin, toremefin, 4-hydroxytamoxifin, ferrocifen (Figure 2) [18-21]. Moreover,
compounds bearing dimethylamino ethoxy entity have also been reported recently against
amoebiasis (Figure3) [22, 23]. An effort has been made towards designing and synthesis of a
series of hybrid molecules that have dimethylaminoethoxy and hydrazone entities
incorporated into one molecule which were expected to show remarkable antiamoebic profile
(Figure 4).
2.
Result and Discussion
Chemistry
2.1
The synthetic route employed to obtain the title compounds is outlined in Scheme 1. The first
step involves acid catalysed esterification of p-hydroxybenzoic acid with methanol. Further it
was followed by protection of hydroxyl group by condensation reaction of p-
hydroxymethylbenzoate with 2-Dimethylaminoethylchloride to give the compound (2). The
conversion of ester (2) to hydrazide was done by nucleophilic substitution reaction with
hydrazine hydrate. Finally, the condensation of the hydrazide with different aromatic
aldehydes gave the final products (H1-H30) in a good yield (80-90%). All the compounds
were characterized by spectroscopic techniques and their data is present in experimental
section. The molecular composition of the compounds was established using the CHNS
1
elemental analysis. The evidence for the formation of the compounds was obtained by H
NMR spectroscopy. The methyl-4-(2-(dimethylamino)ethoxy)benzoate (2) showed a sharp

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singlet at δ3.88 ppm for –OCH3 protons. In N’-Benzylidene-4- (2-(dimethylamino)ethoxy)
benzohydrazide (3) the peak for COOCH3 protons disappeared and a broad singlet can be
seen at δ7.27 ppm for (NH) proton indicating formation of the hydrazide (3). A sharp singlet
was observed at δ2.34ppm for the six protons of the two methyl groups , a triplet appears for
(-OCH2) is slightly downfield as compared to the triplet of CH2-N which appeared at δ4.11-
4.10 ppm and δ2.74-2.75 ppm respectively. The NH2 peak was not observed in the PMR of
N’-Benzylidene-4-(2-(dimethylamino)ethoxy)benzohydrazide (3) may be due to long range
H-bonding but it appeared 3310 cm-1 in IR spectra. In all the compounds (H1-H30) the -NH
protons appeared as broad singlet in the range δ9.86-9.21ppm. A singlet is observed δ8.46-
8.26ppm for –N=CH group in (H1-H30) which was absent in compounds (2) and (3). In IR
spectra the band for the formation of Methyl-4-(2-(dimethylamino)ethoxy) benzoate (2) was
confirmed by the presence of the bands for ester and the ether tail at 1712cm-1(COO- ester
group) and 1247cm-1 (Ar-O-C,C-O-C asym.str) respectively. In p-hydroxymethylbenzoate (1)
the peaks for ester and hydroxyl groups were present. Further the formation of N’-
Benzylidene-4-(2-(dimethylamino)ethoxy) benzohydrazide (3) can be confirmed by the
appearance of the peak for NH2 group at 3317cm-1 (N-H str) and for carbonyl group at
1605cm-1 (C=O str). In Methyl-4-(2-(dimethylamino)ethoxy)benzoate (2) C=O stretching
band for ester group was found at 1712cm-1 which was absent in (3) suggesting conversion of
ester group to hydrazide group. The hydrazone derivatives (H1-H30) show all the
characteristic bands such as the band at 3392-3061 cm-1 was assigned to N-H stretching and
1712-1643 cm-1 to C=O stretching which suggested the condensation of different aromatic
aldehyde with hydrazide. The mass spectra of the compounds showed the expected
fragmentation pattern m/z corresponding to [M+1] which confirmed their respective
molecular weights.
2.3.Single crystal structure

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Single Crystal Structure of compound H23
ORTEP diagram for the compound 4-(2-(dimethylamino)ethoxy)-N-(pyridine-2-
ylmethylene)benzohydrazide (H23) is showed in Figure 5. The compound H23 crystallizes
in the orthorhombic system, with Pbca space group. In the asymmetric unit, there are one
molecule of H23 and one water molecule. Selected bond distances and angles are given in
Table 2.
In H23 compound, atoms, C(11), C(12), O(2), N(2) and N(3), of hydrazide group are
coplanar with benzyl group, C(5), C(6), C(7), C(8), C(9) and C(10) (mean deviation from the
least-squares plane being 0.0762(16) Å). Torsion angle for this part of the molecule with
pyridine group, C(13), C(14), C(15), C(16), C(17) and N(4), was found to be 24.06(8)º ,
which indicate a light torsion due to intermolecular forces.
Intermolecular hydrogen bonds between water molecule and different groups of the
hydrazide derivative determine the crystal packing of the compound (Table 3). π-π lateral
stacking interactions between the bonds of the benzyl rings establish the linked between
frameworks and determine the structure (Figure 6) [24]. The distances between π clouds of
the centroids in the bonds of benzyl groups are: dc1-c2 = 3.577 Å [c1: C(5B)-C(6B) and c2:
C(5K)-C(6K)] and repeated for other centroids]. Table 2 contains selected bond lengths and
angles for compound H23.
2.4.
Biology
2.4.1. Antiamoebic activity
Preliminary experiments were carried out to determine the in vitro antiamoebic activity of all
the compounds (H1-H30) by microdilution method using HM1: IMSS strain of E.histolytica.
The results are summarized in Table 4. The data are present in terms of percent growth
inhibition relative to untreated controls, and plotted as probit values as a function of drug

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concentration. The antiamoebic effect was compared with the most widely used antiamoebic
medication metronidazole (Mtz) which had a 50% inhibitory concentration (IC50) of 1.89µM
in our experiments. The structure activity relationships (SAR) can be explained on the basis
of the position, number and nature of substituent present in the phenyl ring. However, it is
important to mention that the strength of the group can also be considered as an interesting
factor for the activity of the compounds. As the alkyl group attached to the phenyl ring
increases the electron density which makes azomethine group electron rich correspondingly
antiamoebic activity decreases. The same was observed in few compounds whilst from
phenyl group (H1, IC50: 1.43 ± 0.012 µM) to 4-isopropylphenyl group (H10, IC50: 3.45 ±
0.015 µM) and 4-dimethylamino group (H21, IC50: 1.89 ± 0.008 µM) where IC50 values
increases and thereby slightly decreasing the antiamoebic activity. All the halogen substituted
compounds showed an interesting inhibitory behaviour. The compounds H2, H3, H4 having
chloro group at ortho, meta and para positions respectively, showed activity higher than the
compound H1 containing unsubstituted phenyl ring. The ortho-Cl (H2, IC50: 0.23 ± 0.015
µM) exhibited the least IC50 value than meta-Cl (H3, IC50: 1.01 ± 0.014µM) and para-Cl (H4
IC50: 0.79 ± 0.011µM). The nitro group containing compounds showed slightly higher IC50
values (H5, IC50: 2.80 ± 0.021 µM & H6, IC50: 2.40 ± 0.029 µM), though para-nitro showed
lower IC50 value than meta- nitro but both can be considered as moderately active.
Compound H7 with –OH at ortho position was endowed with activity which can be observed
from its percentage inhibition value (H7, IC50: 1.76 ± 0.017 µM), while the compounds H8
para –OH and H28 with dihydoxy substitution were moderately active with higher inhibitory
concentration values (H8, IC50: 2.21 ± 0.030 µM & H28, IC50: 6.40 ± 0.022 µM)).The results
obtained for the alkyl and alkoxy groups were quite similar, wherein the activity reduces
from methyl (H15, IC50: 1.86 ± 0.010 µM) to ethyl (H9, IC50: 5.65 ± 0.002 µM) and from
methoxy (H22, IC50: 1.53 ± 0.019 µM) to ethoxy (H11, IC50: 3.77 ± 0.011 µM) and

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isopropoxy (H12, IC50: 7.653 ± 0.003 µM) substituted compounds. The presence of the
heterocyclic rings like pyridine (H23) and thiophene (H24) reduced the activity by showing
higher IC50 values. From the above discussion, it can be concluded that compounds with –Cl
substitution H2, H3, H4 seems to be the most potent of all the compounds screened.
3.3.1. Cytotoxicity Profile
Cytotoxic effects of the compounds (H1-H30) were studied in A549cells (Non small cell lung
carcinoma cells) by MTT assay. Metronidazole, known anti amoebic drug was used as a
reference drug. Sub confluent monolayers of A549 cells were treated with different
concentration of each compound. Each concentration was taken in triplicates. Toxicity
creasing concentration of these compounds and percentage of viable cells were measured at
48 hours post drug treatment (Figure 7a). IC50 value of each compound was determined.
Two compounds, H4 and H19 had relatively low IC50 values of 69.379 µm and 81.719 µm
respectively while for rest of the compound including MNZ, IC50 was found be very high that
is >100 µm. Corresponding IC50 values of each compound was given in Table 4 and
graphical representation was also shown besides the graph showing percentage of viability
(Figure 7 & Figure 8).
4. Conclusion
In the present study hydrazones derivatives (H1-H30) were designed and sequentially
synthesized in four steps. The combinational chemistry was used in the designing of the
final compounds. The compounds proved to be more potent and selective antiamoebic
agent. Out of thirty synthesized compounds fourteen exhibited promising results against
E.histolytica. Cytotoxicity effects of the compounds found active against E.histolytica were
studied in A549cells (Non small cell lung carcinoma cells) by MTT assay which showed
good cell viability results.

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5. Experimental Protocol
Materials and Methods
All the required chemicals were purchased from Merck and Aldrich Chemical Company
(USA). Precoated aluminum sheets (silica gel 60 F254, Merck Germany) were used for thin-
layer chromatography (TLC) and spots were visualized under UV light. IR spectra were
1
recorded on Bruker FT-IR spectrophotometer under neat condition on a Zn Se crystal. H
NMR was recorded on Bruker Spectrospin DPX 300 MHz using CDCl3 as a solvent and
tetramethylsilane (TMS) as an internal standard. Splitting patterns are designated as follows;
s, singlet; d, doublet; m, multiplet. Chemical shift values are given in ppm. Electrospray (ES)
mass spectra were carried out on Microtof-Q II 10262. Melting points were recorded on a
Veego melting point apparatus (model REC-2203882) and were uncorrected. Elemental
analyses were performed in an Elementar Vario analyzer and the results lay within ± 0.3% of
the theoretical values.
6.3.2 Synthesis
General procedure for the synthesis of ester (1): Acidic esterification of p-hydroxybenzoic
acid (5g) was done in methanol (50mL) and catalytic amount of conc. H2SO4 was added to
the reaction mixture which was refluxed overnight. White solid product was obtained after
removing methanol in vacuo. The solid obtained was washed with chilled water, dried and
crystallized from methanol.
p-hydroxymethylbenzoate (1): White solid, Yield 85%, IR νmax (cm-1): 3506(O-H str),
3030(ArC-H, C-H str), 1676 (COO ester group), 1273 (Ar-O-C,C-O-C asym. str), 846(p-
disubstituted benzene,C-H out of plane bending). Elemental analysis calcd (%) for C8H8O3:
C 63.15, H 5.30, N 0.00; found: C 63.40, H 5.54, N 0.00.

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Synthesis of Methyl-4-(2-(dimethylamino)ethoxy)benzoate (2): To the stirring solution of
THF and potassium tert-butoxide, the ester p-hydroxymethylbenzoate (1), (32.89mmol) was
added which was refluxed for 30 minutes followed by addition of 2-
dimethylaminoethylchloride (4.7384g, 32.89mmol) and was further refluxed for 50 h. the
reaction mixture was poured into water and extraction by ethyl acetate gave a viscous mass.
1
Brown viscous solid, Yield 65%, H NMR (CDCl3, 300MHz, δppm): 7.99(d, 2H, J=8.4,Ar-
H), 6.94(d, 2H, J=8.7, Ar-H), 4.11(t, 2H, J=5.7Hz, -OCH2), 3.88(ss, 3H, COOCH3), 2.75(t,
2H, J=5.4Hz, -CH2), 2.34 (ss, 6H, N(CH3)2); IR νmax (cm-1): 2948(ArC-H, C-H str),
1712(COO ester group), 1604(C=O str), 1247(Ar-O-C,C-O-C asym. str), 846(p-disubstituted
benzene,C-H out of plane bending). Elemental analysis calcd (%) for C12H17NO3: C 64.55, H
7.67, N 6.27; found: C 64.40, H 7.54, N 6.53.
Synthesis of N- substituted-4-(2-(dimethylamino)ethoxy)benzohydrazide (H1-H30): The
Methyl-4-(2-(dimethylamino)ethoxy)benzoate (2) was refluxed with hydrazine hydrate in
ethanol, for 6h gave 4-(2-(dimethylamino)ethoxy) benzohydrazide (3). A mixture of
hydrazide (3) and appropriate aromatic aldehydes in absolute ethanol (50ml) was refluxed for
4hrs with continuous stirring. On reaction completion (TLC) the reaction mixture was cooled
and the solid product was filtered and recrystallized in ethanol yielded the final product (H1-
H30).
N’-Benzylidene-4-(2-(dimethylamino)ethoxy)benzohydrazide (3): Cream coloured solid;
1H NMR (CDCl3, 300MHz, δppm): 7.72-7.69(m, 2H, Ar-H), 7.27(bs, 1H, NH), 6.96-6.93(m,
2H, Ar-H), 4.10 (t, 2H, J=5.7Hz, OCH2), 2.74(t, 2H, J=5.4Hz,CH2), 2.34 (ss, 6H, N(CH3)2).
IR νmax (cm-1): 3317(NH2, N-H str) , 2973(ArC-H, C-H str), 1605(C=O str), 1250(Ar-O-C,C-
O-C asym. str), 1172(N-N), 846(p-disubstituted benzene,C-H out of plane bending).
Elemental analysis calcd (%) for C11H17N3O2: C 59.17, H 7.67, N 18.82; found: C 59.10, H
7.54, N 18.93.

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N’-Benzylidene-4-(2-(dimethylamino)ethoxy)benzohydrazide (H1): White solid, Yield:
1
85%, m.pt. 172oC ; HNMR (CDCl3, 300MHz, δppm): 9.43 (bs,1H, -NH), 8.07(d, 2H,
J=6.9Hz, Ar-H), 8.31 (s, 1H, -CHN),7.88-7.72 (m, 2H, Ar-H), 7.48-7.37 (m, 3H, Ar-H), 6.98
(d, 2H, J=8.7Hz, Ar-H), 4.12(t, 2H, J=5.7Hz, OCH2), 2.77 (t, 2H, J=5.7Hz, CH2), 2.36 (ss,
6H, N(CH3)2), 1.91 (s, 5H, Ar-H). IR νmax (cm-1): 3363 (NH2, N-H str) , 3022 (ArC-H, C-H
str), 1699 (C=O str), 1250 (Ar-O-C, C-O-C asym. str), 1171 (N-N); Elemental analysis calcd
(%) for C18H21N3O2: C 69.43, H 6.80, N 13.49; found: C 69.14, H 6.54, N 13.33; ES-MS:
m/z 312.52 (M+1).
N’-(2-chlorobenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H2): Colourless
1
solid, Yield: 85%, m.pt. 100oC; H NMR (CDCl3, 300MHz, δppm): 9.36 (bs,1H, -NH), 8.19
(s, 1H, CHN), 7.89(bs, 2H, Ar-H), 7.38-7.29(m, 4H, Ar-H), 7.01 (d, 2H, J=8.7, Ar-H), 4.13
(t, 2H, J=6Hz, OCH2), 2.76 (t, 2H, J=5.7Hz, CH2), 2.35 (ss, 6H, N(CH3)2). IRνmax (cm-1):
3184 (N-H str) , 1649 (C=O str), 1554 (N=CH), 1256 (Ar-O-C, C-O-C asym. str), 1177(N-
N); Elemental analysis calcd (%) for C18H20N3O2Cl: C 62.52, H 5.83, N 12.15; found: C
62.34, H 5.54, N 12.33; ES-MS: m/z 346.16 (M+1).
N’-(3-chlorobenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H3): Colourless
solid, Yield: 82%, m.pt. 99oC; 1H NMR (CDCl3, 300MHz, δppm): 9.21 (bs,1H, -NH), 8.26(s,
1H, CHN), 7.87 (bs, 2H, Ar-H), 7.73 (bs, 1H, Ar-H),7.58 (bs, 1H, Ar-H), 7.37-7.29 (m, 2H,
Ar-H), 7.00 (bs, 1H, CHN), 4.13 (t, 2H, J=5.7Hz, OCH2), 2.76 (t, 2H, J=5.4Hz, CH2),
2.35(ss, 6H, N(CH3)2). IR νmax (cm-1): 3061(N-H str) , 3061(ArC-H, C-H str), 1643(C=O str),
1248(Ar-O-C, C-O-C asym. str), 1552(-N=CH), 1182(N-N); Elemental analysis calcd (%) for
C18H20N3O2Cl: C 62.52, H 5.83, N 12.15; found: C 62.34, H 5.54, N 12.33; ES-MS: m/z
346.15 (M+1).
N’-(4-chlorobenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H4): Colourless
1
solid, Yield: 84%, m.pt. 188oC; H NMR (CDCl3, 300MHz, δppm): 9.37(bs,1H, -NH),

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8.28(s, 1H, CHN), 7.86(bs, 2H, Ar-H), 7.65 (bs, 2H, Ar-H),7.37 (d, 2H, J=8.1, Ar-H), 6.99
(d, 2H, J=8.4, Ar-H), 4.12 (t, 2HJ=5.1Hz, OCH2), 2.75(t, 2H, J=5.7Hz, CH2), 2.35(ss, 6H,
N(CH3)2). IR νmax (cm-1): 3392 (N-H str) , 1695 (C=O str), 1565 (N=CH), 1257 (Ar-O-C,
C-O-C asym. str), 1178 (N-N); Elemental analysis calcd (%) for C18H20N3O2Cl: C 62.52, H
5.83, N 12.15; found: C 62.34, H 5.54, N 12.33; ES-MS: m/z 346.28 (M+1).
N’-(3-Nitrobenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H5): Colourless
1
solid, Yield: 82%, m.pt.130oC ; H NMR (CDCl3, 300MHz, δppm): 9.43(bs,1H, -NH),
8.46(s, 1H, CHN), 8.24(d, 2H,J=8.7, Ar-H), 8.13(bs, 1H, Ar-H) 7.89(d, 2H,J=7.5, Ar-H),
7.58(t, 1H, J=8.1, Ar-H), 7.01 (d, 2H, J=8.7, Ar-H), 4.13(t, 2H, J=5.7Hz, OCH2), 2.78 (t, 2H,
J=5.7Hz,-CH2), 2.35 (ss, 6H, N(CH3)2). IR νmax (cm-1): 3204(N-H str) , 1684(C=O str),
1562(N=CH), 1259(Ar-O-C, C-O-C asym. str), 1177(N-N); Elemental analysis calcd (%) for
C18H20N4O4: C 60.66, H 5.66, N 15.72; found: C 60.34, H 5.54, N 15.43; ES-MS: m/z 357.11
(M+1).
N’-(4-Nitrobenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H6): Yellow
solid, Yield: 82%, m.pt.99oC ; 1H NMR (CDCl3, 300MHz, δppm): 9.84(bs,1H, -NH), 8.46(s,
1H, CHN), 8.25(d, 2H,J=8.7, Ar-H), 7.87 (d, 4H,J=6.9, Ar-H) 6.98(d, 2H,J=8.7, Ar-H),
4.19(t, 2H, J=5.4Hz, OCH2), 2.87(t,2H,J=5.7Hz, CH2), 2.42 (ss, 6H, N(CH3)2). IR νmax (cm-
1): 3367 (N-H str) , 1653(C=O str), 1565(N=CH), 1247 (Ar-O-C, C-O-C asym. str), 1168(N-
N); Elemental analysis calcd (%) for C18H20N4O4: C 60.66, H 5.66, N 15.72; found: C 60.34,
H 5.54, N 15.43; ES-MS: m/z 357.32 (M+1).
N’-(2-hydroxybenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H7): Yellow
1
solid, Yield: 70%, m.pt. 150oC ; H NMR (CDCl3, 300MHz, δppm): 11.06 (bs, 1H), 9.12
(s,1H), 8.46 (s, 1H), 7.82 (d, 2H,J=8.7), 7.33-7.33 (m, 2H), 7.23-7.20 (m, 1H), 7.00-6.97 (m,
3H), 4.13 (t, 2H, J=5.4Hz), 2.77(t,2H,J=5.7Hz), 2.35 (ss, 6H). IR νmax (cm-1): 3355 (N-H
str) , 1646(C=O str), 1561(N=CH), 1252 (Ar-O-C, C-O-C asym. str), 1150(N-N); Elemental

ACCEPTED MANUSCRIPT
analysis calcd (%) for C18H21N3O3: C 66.04, H 6.47, N 12.84; found: C 66.14, H 6.47, N
12.80 ES-MS: m/z 328.00 (M+1).
N’-(4-hydroxybenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H8): Yellow
1
solid, Yield: 62%, m.pt.111oC ; H NMR (CDCl3, 300MHz, δppm): 11.51 (s,1H, -NH), 9.92
(s, 1H, OH), 8.33(s, 1H, CHN), 7.88(d, 2H, J=6.3Hz, Ar-H), 7.55(d, 2H,J=8.7Hz, Ar-H),
7.06 (d, 2H, J=6.3Hz, Ar-H), 6.84 (d, 2H, J=6.0 Hz, Ar-H), 4.13 (t, 2H, J=5.4Hz, OCH2),
2.81 (t, 2H, J=5.7Hz, CH2), 2.22 (ss, 6H, N(CH3)2); IRνmax (cm-1): 3590 (-OH), 3301 (N-H
str) , 1644 (C=O str), 1557 (N=CH), 1283 (Ar-O-C, C-O-C asym. str), 1166 (N-N);.
Elemental analysis calcd (%) for C18H21N3O3: C 66.04, H 6.47, N 12.84; found: C 66.34, H
6.45, N 12.83; ES-MS: m/z 328.55 (M+1).
N’-(4-ethylbenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H9): solid, Yield:
1
60%, m.pt.155oC ; H NMR (CDCl3, 300MHz, δppm): 9.11 (bs,1H), 8.24 (s, 1H), 7.85 (bs,
2H), 7.65 (bs, 2H),7.26-7.21 (m, 2H), 6.99 (d, 2H, J=7.2Hz), 4.14(t, 2H, J=5.7Hz), 2.77 (t,
2H, J=5.4Hz), 2.71 (m, 2H), 2.35 (ss, 6H), 1.27 (t, 3H, J=7.5Hz). IRνmax (cm-1): 3357 (N-H
str) , 1648 (C=O str), 1560 (N=CH), 1270 (Ar-O-C, C-O-C asym. str), 1173 (N-N);
Elemental analysis calcd (%) for C20H25N3O2: C 70.77, H 7.42, N 12.38; found: C 70.64, H
7.54, N 12.43; ES-MS: m/z 340.10 (M+1).
N’-(4-isopropylbenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H10): solid,
Yield: 60%, m.pt.150oC; 1H NMR (CDCl3, 300MHz, δppm): 9.41 (bs,1H), 8.24 (s, 1H), 7.96-
7.84 (m, 2H), 7.68-7.61 (m, 2H), 7.25-7.24 (m, 2H), 6.99 (d, 2H, J=8.7Hz), 4.14 (t, 2H,
J=5.4Hz), 2.97-2.91 (m, 1H), 2.78 (t, 2H, J=5.7Hz), 2.35 (s, 6H), 1.27 (d, 6H, J=6.9Hz);
IRνmax (cm-1): 3384 (N-H str) , 1647 (C=O str), 1557 (N=CH), 1250 (Ar-O-C, C-O-C asym.
str), 1176 (N-N); Elemental analysis calcd (%) for C21H27N3O2: C 71.36, H 7.70, N 11.89;
found: C 71.35, H 7.70, N 11.88; ES-MS: m/z 354.25 (M+1).

ACCEPTED MANUSCRIPT
N’-(4-ethoxybenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H11): solid,
Yield: 65%, m.pt.162oC ; 1H NMR (CDCl3, 300MHz, δppm): 9.24 (bs,1H), 8.21 (s, 1H), 7.84
(bs, 2H), 7.67 (bs, 2H), 6.98 (d, 2H, J=8.7Hz), 6.90 (d, 2H, J=8.7Hz), 4.13 (t, 2H, J=5.4Hz),
4.07-4.02 (m, 2H), 2.77 (t, 2H, J=5.7Hz), 2.34 (s, 6H), 1.44(t, 3H, J=6.9Hz); IRνmax (cm-1):
3301 (N-H str) , 1644 (C=O str), 1557 (N=CH), 1250 (Ar-O-C, C-O-C asym. str), 1167 (N-
N); Elemental analysis calcd (%) for C20H25N3O3: C 67.58, H 7.09, N 11.82; found: C 67.58,
H 7.09, N 11.85; ES-MS: m/z 356.10 (M+1).
N’-(4-propoxybenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H12): solid,
Yield: 72%, m.pt.190oC; 1H NMR (CDCl3, 300MHz, δppm): 9.08 (bs,1H), 8.19 (s, 1H), 7.96-
7.83 (m, 2H), 7.65-7.60 (m, 2H), 6.99 (d, 2H, J=8.7Hz), 6.90 (d, 2H, J=9.0Hz), 4.65 (m, 1H),
4.14 (t, 2H, J=5.4Hz), 2.77 (t, 2H, J=5.7Hz), 2.34 (s, 6H), 1.36(d, 6H, J=6.0Hz); IRνmax
(cm-1): 3361 (N-H str) , 1644 (C=O str), 1563 (N=CH), 1240 (Ar-O-C, C-O-C asym. str),
1142 (N-N); Elemental analysis calcd (%) for C21H27N3O3: C 68.27, H 7.37, N 11.37;
found: C 68.27, H 7.39, N 11.34; ES-MS: m/z 370.22 (M+1).
N’-(4-hydroxy-3-methoxybenzylidene)-4-(2-(dimethylamino)ethoxy)benzo- hydrazide
1
(H13): solid, Yield: 76%, m.pt.147oC; H NMR (CDCl3, 300MHz, δppm): 11.02 (s, 1H),
8.24 (s, 2H), 7.91-7.49 (m, 3H), 7.02-6.87 (m, 4H), 4.13 (t, 2H, J=5.4Hz), 3.94 (s, 3H) 2.78-
2.74 (m, 2H), 2.33 (s, 6H); IRνmax (cm-1): 3358 (N-H str) , 1640 (C=O str), 1504 (N=CH),
1241 (Ar-O-C, C-O-C asym. str), 1173 (N-N); Elemental analysis calcd (%) for C19H23N3O4:
C 63.85, H 6.49, N 11.76; found: C 63.89, H 6.52, N 11.75; ES-MS: m/z 358.11(M+1).
N’-(2-bromo-4-hydroxy-3-methoxybenzylidene)-4-(2-(dimethylamino)ethoxy)
1
benzohydrazide (H14): solid, Yield: 68%, m.pt.197oC; H NMR (CDCl3, 300MHz, δppm):
11.68 (s,1H), 10.12 (bs, 1H), 8.29 (s, 1H), 7.89 (d, 2H, J=5.7Hz), 7.40-7.30 (m, 2H), 7.10-
7.07 (m, 2H), 4.14 (t, 2H, J=5.4Hz), 3.89 (s, 3H), 2.64 (t, 2H, J=5.7Hz), 2.22 (s, 6H);
IRνmax (cm-1): 3356 (N-H str) , 1640 (C=O str), 1502 (N=CH), 1250 (Ar-O-C, C-O-C asym.

ACCEPTED MANUSCRIPT
str), 1169 (N-N); Elemental analysis calcd (%) for C19H22BrN3O4: C 52.30, H 5.08, N 9.63;
found: C 52.33, H 5.18, N 9.66; ES-MS: m/z 437.36 (M+1).
N’-(4-methylbenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H15): solid,
Yield: 55%, m.pt.164oC; 1H NMR (CDCl3, 300MHz, δppm): 9.11 (bs,1H), 8.22 (s, 1H), 7.83
(bs, 2H), 7.65 (bs, 2H), 7.22 (d, 2H, J=8.1Hz), 7.00(d, 2H, J=8.1Hz), 4.14(t, 2H, J=5.4Hz),
2.78 (t, 2H, J=5.7Hz), 2.38 (ss, 9H); IRνmax (cm-1): 3353 (N-H str) , 1647 (C=O str),
1562(N=CH), 1276 (Ar-O-C, C-O-C asym. str), 1176 (N-N); Elemental analysis calcd (%)
for C19H23N3O2: C 70.13, H 7.12, N 12.91; found: C 70.15, H 7.16, N 12.89; ES-MS: m/z
326.41 (M+1).
N’-(3,4-dimethoxybenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide
(H16):
1
solid, Yield: 64%, m.pt.125oC; H NMR (CDCl3, 300MHz, δppm): 9.43 (bs,1H), 8.22 (s,
1H), 7.85 (bs, 2H), 7.48 (bs, 1H), 7.10 (d, 1H, J=8.4Hz), 6.97(d, 2H, J=8.4Hz), 6.85 (d, 1H,
J=8.1Hz), 4.12 (t, 2H, J=5.4Hz), 3.91 (ss, 6H), 2.77 (t, 2H, J=5.4Hz), 2.34 (s, 6H); IRνmax
(cm-1): 3354 (N-H str), 1641 (C=O str), 1540 (N=CH), 1240 (Ar-O-C, C-O-C asym. str),
1165 (N-N); Elemental analysis calcd (%) for C20H25N3O4: C 64.67, H 6.78, N 11.31; found:
C 64.65, H 6.79, N 11.32; ES-MS: m/z 372.10 (M+1).
N’-(benzo[1,3]dioxol-5-ylmehylene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H17):
solid, Yield: 69%, m.pt.149oC; 1H NMR (CDCl3, 300MHz, δppm): 9.21(bs,1H), 8.20 (s, 1H),
7.84 (bs, 2H), 7.39 (bs, 1H), 7.06 (d, 1H, J=8.1Hz), 6.98 (d, 2H, J=8.7Hz), 6.81 (d 1H,
J=8.1Hz), 6.00 (ss, 2H), 4.13(t, 2H, J=5.4Hz), 2.77 (t, 2H, J=5.7Hz), 2.34 (s, 6H); IRνmax
(cm-1): 3299 (N-H str) , 1641 (C=O str), 1561 (N=CH), 1250 (Ar-O-C, C-O-C asym. str),
1171 (N-N); Elemental analysis calcd (%) for C19H21N3O4: C 64.21, H 5.96, N 11.82; found:
C 64.20, H 5.97, N 11.80; ES-MS: m/z 356.10 (M+1).
N’-(3,5-dibromo-4-hydroxybenzylidene)-4-(2-(dimethylamino)ethoxy) benzohydrazide
1
(H18): solid, Yield: 56%, m.pt.152oC; H NMR (CDCl3, 300MHz, δppm): 11.63 (s,1H),

ACCEPTED MANUSCRIPT
8.19(s, 1H), 7.89-7.87 (m, 2H), 7.77-7.61(m, 2H), 7.06-7.04 (m, 2H), 4.27 (t, 2H, J=5.4Hz),
2.84 (t, 2H, J=5.7Hz), 2.43 (s, 6H); IRνmax (cm-1): 3356 (N-H str) , 1612 (C=O str),
1571(N=CH), 1252 (Ar-O-C, C-O-C asym. str), 1175 (N-N); Elemental analysis calcd (%)
for C18H19Br2N3O3: C 44.56, H 3.95, N 8.66; found: C 44.56, H 3.95, N 8.66; ES-MS: m/z
486.05 (M+1).
N’-(2,4,6-trimethylbenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide
(H19):
1
Solid, Yield: 60%, m.pt.196oC; H NMR (CDCl3, 300MHz, δppm): 11.57 (s,1H), 8.73 (s,
1H), 7.90-7.89 (m, 2H), 7.07-7.05 (m, 2H), 6.91 (s, 2H), 4.12 (t, 2H, J=5.4Hz), 2.65(t, 2H,
J=5.7Hz), 2.42 (s, 6H), 2.24(s, 3H), 2.21 (s, 6H); IRνmax (cm-1): 3227 (N-H str) , 1643 (C=O
str), 1542 (N=CH), 1255 (Ar-O-C, C-O-C asym. str), 1179 (N-N); Elemental analysis calcd
(%) for C21H27N3O2: C 71.36, H 7.70, N 11.89; found: C 71.35, H 7.73, N 11.86; ES-MS:
m/z 354.16 (M+1).
N’-(3-methoxy-4-propoxybenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide
(H20): Solid, Yield: 55%, m.pt.126oC; 1H NMR (CDCl3, 300MHz, δppm): 9.24 (bs,1H), 8.20
(s, 1H), 7.85 (bs, 2H), 7.48 (bs, 1H), 7.09 (d, 1H, J=6.3Hz), 6.98 (d, 2H, J=9.0Hz), 6.87 (d,
1H, J=8.4Hz), 4.63 (m, 1H), 4.13 (t, 2H, J=5.4Hz), 3.89-3.84 (m, 3H), 2.77 (t, 2H, J=5.7Hz),
2.37 (s, 6H), 1.40 (d, 6H, J=6.3Hz); IRνmax (cm-1): 3386 (N-H str) , 1642 (C=O str),
1536(N=CH), 1238 (Ar-O-C, C-O-C asym. str), 1150 (N-N); Elemental analysis calcd (%)
for C22H29N3O4: C 66.14, H 7.32, N 10.52; found: C 66.12, H 7.30, N 10.51; ES-MS: m/z
400.16 (M+1).
N’-(4-(dimethylamino)benzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H21):
solid, Yield: 56%, m.pt.216oC; 1H NMR (CDCl3, 300MHz, δppm): 8.94(bs,1H), 8.10 (s, 1H),
7.91 (bs, 2H), 7.63 (bs, 2H), 6.98 (d, 2H, J=8.7Hz), 6.70 (d, 2H, J=8.7Hz), 4.31 (t, 2H,
J=5.4Hz), 3.02 (ss, 6H), 2.79 (t, 2H, J=5.7Hz), 2.37 (s, 6H); IRνmax (cm-1): 3352 (N-H str) ,
1644 (C=O str), 1517 (N=CH), 1253 (Ar-O-C, C-O-C asym. str), 1177 (N-N); Elemental

ACCEPTED MANUSCRIPT
analysis calcd (%) for C20H26N4O2: C 67.77, H 7.39, N 15.81; found: C 67.75, H 7.40, N
15.81; ES-MS: m/z 355.16 (M+1).
N’-(4-methoxybenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H22): solid,
1
Yield: 69%, m.pt.120oC; H NMR (CDCl3, 300MHz, δppm): 9.24 (bs,1H), 8.22 (s, 1H),
7.84(bs, 2H), 7.68 (bs, 2H), 6.97-6.89 (m, 4H), 4.13 (t, 2H, J=5.4Hz), 3.83(ss, 3H), 2.77 (t,
2H, J=5.7Hz), 2.34 (s, 6H); IRνmax (cm-1): 3390 (N-H str), 1644 (C=O str), 1559 (N=CH),
1241 (Ar-O-C, C-O-C asym. str), 1164 (N-N); Elemental analysis calcd (%) for C19H23N3O4:
C 66.84, H 6.79, N 12.31; found: C 66.84, H 6.79, N 12.31; ES-MS: m/z 342.66 (M+1).
4-(2-(dimethylamino)ethoxy)-N-(pyridine-2-ylmethylene)benzohydrazide (H23): solid,
Yield: 52%, m.pt.102oC; 1H NMR (CDCl3, 300MHz, δppm): 9.38(bs,1H), 8.61 (s, 1H), 8.23-
8.05 (m, 2H), 8.00-7.90 (m, 2H), 7.76 (t, 1H, J=7.8Hz), 7.31-7.28 (m, 1H), 7.013 (d, 2H,
J=8.7Hz), 4.152 (t, 2H, J=5.4Hz), 2.781 (t, 2H, J=5.7Hz), 2.35 (s, 6H); IRνmax (cm-1): 3379
(N-H str) , 1649 (C=O str), 1560 (N=CH), 1272 (Ar-O-C, C-O-C asym. str), 1179 (N-N);
Elemental analysis calcd (%) for C17H20N4O2: C 65.37, H 6.45, N 17.94; found: C 65.36, H
6.46, N 17.96; ES-MS: m/z 313.23(M+1).
4-(2-(dimethylamino)ethoxy)-N-((3-methylthiophene-2-yl)methylenen) benzohydrazide
(H24): solid, Yield: 61%, m.pt.141oC; 1H NMR (CDCl3, 300MHz, δppm): 9.31 (bs,1H), 9.20
(s, 1H), 7.83 (bs, 1H), 7.27 (bs, 1H), 6.97 (d, 2H, J=8.7Hz), 6.84 (d, 2H, J=5.1Hz), 4.13 (t,
2H, J=5.4Hz), 2.77 (t, 2H, J=5.7Hz), 2.34 (s, 9H); IRνmax (cm-1): 3324 (N-H str) , 1642
(C=O str), 1558 (N=CH), 1244 (Ar-O-C, C-O-C asym. str), 1177 (N-N); Elemental analysis
calcd (%) for C17H21N3O2S: C 61.61, H 6.39, N 12.68; found: C 61.61, H 6.39, N 12.65; ES-
MS: m/z 332.49 (M+1).
Ferrocenyl-4-(2-(dimethylamino)ethoxy)benzohydrazide (H25): solid, Yield: 60%,
1
m.pt.192oC; H NMR (CDCl3, 300MHz, δppm): 8.84 (bs,1H), 8.22 (bs, 1H), 7.81 (bs, 2H),
6.99 (d, 2H, J=8.7Hz),4.69 (s, 2H), 4.41 (s, 2H), 4.221 (s, 4H), 4.15 (t, 2H, J=5.4Hz), 2.78(t,

ACCEPTED MANUSCRIPT
2H, J=5.4Hz), 2.36 (s, 6H); IRνmax (cm-1): 3397 (N-H str) , 1641 (C=O str), 1558 (N=CH),
1256 (Ar-O-C, C-O-C asym. str), 1188 (N-N); Elemental analysis calcd (%) for
C22H25FeN3O2: C 62.96, H 5.39, N 10.01; found: C 62.95, H 5.39, N 10.04; ES-MS: m/z
421.50 (M+1).
N’-(3,4-dichlorobenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide (H26): solid,
1
Yield: 69%, m.pt. 96oC; H NMR (CDCl3, 300MHz, δppm): 8.34 (bs,1H), 7.84(s, 1H), 7.81
(bs, 3H), 7.54 (bs, 1H), 7.47 (d, 1H, J=8.1Hz), 6.98 (d, 2H, J=8.4Hz), 4.14(t, 2H, J=5.4Hz),
2.74 (t, 2H, J=5.7Hz), 2.35 (s, 6H); IRνmax (cm-1): 3260 (N-H str) , 1649 (C=O str), 1554
(N=CH), 1247 (Ar-O-C, C-O-C asym. str), 1173 (N-N); Elemental analysis calcd (%) for
C18H19Cl2N3O2: C 56.85, H 5.04, N 11.05; found: C 56.85, H 5.02, N 11.02; ES-MS: m/z
381.28 (M+1).
2-methoxy-4-[2-{4-(dimethylamino)ethoxybenzoyl)hydrazinylidene}methyl]phenyl
1
acetate (H27): solid, Yield: 56%, m.pt. 83oC; H NMR (CDCl3, 300MHz, δppm): 9.66
(bs,1H), 8.10 (bs, 1H), 7.87(bs, 2H), 7.47 (s, 1H), 6.99-6.93 (m, 4H), 4.13 (t, 2H, J=6.0Hz),
3.82 (s, 3H), 2.78-2.75 (m, 2H), 2.35 (s, 6H), 2.31 (s, 3H); IRνmax (cm-1): 3329 (N-H str), 1646
(C=O str), 1554 (N=CH), 1284 (Ar-O-C, C-O-C asym. str), 1171 (N-N); Elemental analysis
calcd (%) for C21H25N3O5: C 63.14, H 6.31, N 10.52; found: C 63.14, H 6.33, N 10.55; ES-MS:
m/z 400.07(M+1).
N’-(3,4-dihydroxybenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide
(H28):
1
solid, Yield: 64%, m.pt. 195oC; H NMR (CDCl3, 300MHz, δppm): 11.47 (s,1H), 9.73-9.29
(bs, 2H, OH), 8.24 (s, 1H), 7.78-7.66 (m, 2H), 7.30 (s, 1H), 7.05-7.03 (m, 2H), 6.78-6.76 (m,
1H), 6.60-6.56 (m, 1H), 4.13 (t, 2H, J=5.4Hz), 2.63 (t, 2H, J=5.7Hz), 2.22(s, 6H) IRνmax
(cm-1): 3314 (N-H str) , 1645 (C=O str), 1505 (N=CH), 1248 (Ar-O-C, C-O-C asym. str),
1176 (N-N); Elemental analysis calcd (%) for C18H21N3O4: C 62.96, H 6.16, N 12.24; found:
C 62.93, H 6.17, N 12.25; ES-MS: m/z 344.64 (M+1).

ACCEPTED MANUSCRIPT
N’-(2,5-dimethoxybenzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide
(H29):
1
solid, Yield: 59%, m.pt. 124oC; H NMR (CDCl3, 300MHz, δppm): 9.22 (bs,1H), 8.57 (bs,
1H), 7.95 (bs, 2H), 7.64 (bs, 1H), 6.99-6.93 (m, 3H), 6.86-6.83 (m, 1H), 4.14- (t, 2H,
J=4.8Hz), 3.82 (ss, 6H), 2.78 (t, 2H, J=4.5Hz), 2.36 (s, 6H); IRνmax (cm-1): 3457 (-OH),
3336 (N-H str) , 1642 (C=O str), 1563 (N=CH), 1246 (Ar-O-C, C-O-C asym. str), 1170 (N-
N); Elemental analysis calcd (%) for C20H25N3O4: C 64.67, H 6.78, N 11.31; found: C 64.67,
H 6.78, N 11.31; ES-MS: m/z 372.18 (M+1).
N’-(4-(diethoxymethyl)benzylidene)-4-(2-(dimethylamino)ethoxy)benzohydrazide(H30):
solid, Yield: 69%, m.pt.75oC; 1H NMR (CDCl3, 300MHz, δppm): 9.05 (bs,1H), 7.86 (s, 1H),
7.72-7.52 (m, 4H), 7.50 (d, 2H, J=8.1Hz), 7.00(d, 2H, J=8.7Hz), 5.52 (s, 1H), 4.15 (t, 2H,
J=5.7Hz), 3.62-3.49 (m, 4H), 2.79 (t, 2H, J=5.7Hz), 2.36 (s, 6H), 1.26 (m, 6H); IRνmax (cm-
1): 3389 (N-H str) , 1649 (C=O str), 1558 (N=CH), 1259 (Ar-O-C, C-O-C asym. str), 1176
(N-N); Elemental analysis calcd (%) for C23H31N3O4: C 66.81, H 7.56, N 10.16; found C
66.85, H 7.55, N 10.14; ES-MS: m/z 414.11 (M+1).
5. Pharmacological Evaluation
5.1. Antiamoebic activity
All the compounds were screened in vitro for antiamoebic activity against HM1:IMSS strain
of E. histolytica by micro dilution method [25]. E. histolytica trophozoites were cultured in
wells of 96-well microtiter plate by using Diamond TYIS-33 growth medium [26]. The test
compounds (1 mg) were dissolved in ethanol (50 mL, level at which no inhibition of amoeba
occurs) [27]. The stock solutions of the compounds were prepared freshly before use at a
concentration of 1 mg/mL. Each plate included metronidazole as standard amoebicidal drug,
negative control (culture medium plus amoeba) and a blank (culture medium only). All the
compounds were used in triplicate concentrations. The parasite suspension was adjusted to
105 cells per mL by adding fresh medium and then distributed to a 96-wells microtiter plate

ACCEPTED MANUSCRIPT
(Corning). Compounds were added to the respective wells and the plate was sealed, gassed
for 10 min with nitrogen and then incubated at 37 °C for 72 h. After incubation, the amoeba
growth was checked under microscope. The culture medium was removed by inverting the
plate and shaking gently. Plate was then immediately washed with sodium chloride (0.9%) at
37 °C, dried at room temperature and the cells fixed with methanol and once dried, stained
with aqueous eosin (0.5%) for 15 min. The optical density of the resulting solution in each
well was determined at 490 nm in a micro plate reader. The percentage inhibition of parasite
growth was calculated from the optical densities of the control and test compounds. A non-
linear regression analysis was used to determine the best fitting line from which the IC50
values were estimated. Each compound was tested at least in two independent experiments.
5.2 Cytotoxicity
Cytotoxic effects of the series (H1-H30) were determined in A549 cells (lung epithelial
cells). The concentration range used for all the compounds were between 5-640µm.
Compounds were reconstituted initially in ethanol, but further dilutions were made in culture
medium. A549 cells were cultured in 96 well plates at a density of 8000 cells/well in Ham’s
F-12 medium (Hi Media) supplemented with 10% FBS. Once the cells attached to 80 to 90%
confluence, medium was aspirated off and replaced with medium containing desired
concentration of each drug and 1%FBS. Each drug concentration was taken in triplicates.
Cells treated only with medium were used as negative controls. Plates were incubated at 37oC
with 5% CO2. At 48hrs post drug treatment, cell viability was measured by MTT assay. In
brief, cells in each well were incubated with 20µl of MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyl-2H-tetrazolium bromide) reagent at a concentration of 5mg/ml in 5% methanol for
4hrs. Then, MTT reagent was removed and the resulting formazan crystals were solubilized
in 200µl of DMSO. The quantity of formazan which is directly proportional to the number of
cells viable was measured by recording absorbance at wavelength of 560nm with the

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reference wavelength of 670nm [28]. The percentage of viability was calculated taking
negative controls into account and plotted against the concentration of the drug. IC50 value
for each compound was determined by using graph pad prism∞5.0 software.
5.3 X-Ray crystal structure determination
Three-dimensional X-ray data were collected on a Bruker Kappa Apex CCD diffractometer at
low temperature for H23 by the
φ
-
ω
scan method. Reflections were measured from a
. A total of 73793
hemisphere of data collected from frames, each of them covering 0.3º in
ω
reflections measured were corrected for Lorentz and polarization effects and for absorption
by multi-scan methods based on symmetry-equivalent and repeated reflections. Of the total,
2381 independent reflections exceeded the significance level ( F /
σ F ) > 4.0. After data
collection, in each case an multi-scan absorption correction (SADABS) [29] was applied, and
the structure was solved by direct methods and refined by full matrix least-squares on F2 data
using SHELX suite of programs [30]. Hydrogen atoms were located in difference Fourier
map and left to refine freely, except for C(1) and C(2), which were included in calculation
position and refined in the riding mode. Refinements were done with allowance for thermal
anisotropy of all non-hydrogen atoms. A final difference Fourier map showed no residual
2
density in the crystal 0.337 and -0.290 e.Å–3. A weighting scheme w = 1/[σ2(Fo ) + (0.091800
P)2 + 0.000000 P] was used in the latter stages of refinement. Further details of the crystal
structure determination are given in Table 1. CCDC 1427081 contains the supplementary
crystallographic data for the structure reported in this paper. These data can be obtained free
of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223
336 033; or e-mail: deposit@ccdc.cam.ac.uk.

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Acknowledgement
One of the authors (AI) is thankful to University Grants Commission, New-Delhi, India for
BSR (SRF) Fellowship.
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Captions and headings
Figures
Figure1. Promising antiamoebic compounds containing hydrazone linkage
Figure2. Structures of currently used drugs containing dimethylaminoethoxy tail
Figure3. : Promising antiamoebic compounds bearing N-substituted aminoethoxy tail
Figure4. Rational designing of the compounds
Figure5. ORTEP plot for the compound 4-(2-(dimethylamino)ethoxy)-N-(pyridine-2-
ylmethylene)benzohydrazide (H23). All the non-hygrogen atoms are presented by their
30% probability ellipsoids. Hydrogen atoms are omitted for clarity.
Figure6. Intermolecular π-π interactions in H23. Dashed lines link the centres of the π
clouds involving in each interaction
Figure7a Measurement of cell viability following treatment with H1, H2, H3 and H4
compounds by MTT assay
Figure7b. Measurement of cell viability following treatment with H7, H15, H17 and H18
compounds by MTT assay
Figure7c. Measurement of cell viability following treatment with H19, H21, H22, H27,
H29 and H30 compounds by MTT assay
Figure8. IC50 Value of compounds (H1-H30) for MTT assay

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Tables
Table 1. Crystal data and structure refinement for H23.
4
Table 2. Atomic coordinates ( x 10 ) and equivalent isotropic displacement parameters
2
3
ij
(Å x 10 ) for H2303. U(eq) is defined as one third of the trace of the orthogonalized U
tensor.
Table 3. Bond lengths [Å] and angles [°] fossr H23
Table 4. In vitro antiamoebic activity of (H1-H30) against HM1:IMSS strain of E. histolytica
and cytotoxicity profile of active compounds.
Scheme
Scheme 1: Synthesis of 4-(2-(dimethylamino)ethoxy)benzohydrazide derivatives (H1-
H30)

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2
Cl
N
NH O
O
R
N
N
O
N
N
N
NH
R
Quinoline based Hydrazones
O
1
N
R
NH
N
Hydrazone bearing pyridyl moiety
Figure 1: Promising antiamoebic compounds containing hydrazone linkage
O
N
O
N
Cl
Toremifene
Tamoxifen
Fe
O
N
O
N
OH
OH
4-Hydroxytamoxifen
Ferrocifen
Figure 2: Structures of currently used drugs containing dimethylaminoethoxy entity

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O
N
N
N
N
N
O
N
O
IC50 1.40uM
IC50 0.50uM
O
O
N
N
O
O
OCH3
IC50 0.12uM
IC50 0.10uM
Figure 3: Promising antiamoebic compounds bearing N-substituted aminoethoxy
entity
Antiamoebic agents
Combination of two
-
O
pharmacophore
Ar
N
NH
N
Hydrazone
linkage
O
Dimethylamino
ethoxy entity
(H1-H30)
Figure 4: Designing of the hybrid molecules

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Figure5. ORTEP plot for the compound 4-(2-(dimethylamino)ethoxy)-N-(pyridine-2-
ylmethylene)benzohydrazide (H23). All the non-hygrogen atoms are presented by their 30%
probability ellipsoids. Hydrogen atoms are omitted for clarity.
Figure6. Intermolecular π-π interactions in H23. Dashed lines link the centres of the π clouds
involving in each interaction