European Journal of Medicinal Chemistry 124 (2016) 445e455

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Short communication

Synthesis and biological evaluation of 4-(2-(dimethylamino)ethoxy)

benzohydrazide derivatives as inhibitors of Entamoeba histolyica
Afreen Inam a, Sonam Mittal b, Maitreyi S. Rajala b, Fernando Avecilla c, Amir Azam a, *
a
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, 110 025, New Delhi, India
b
School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India
c
Departamento de Química Fundamental, Universidade da Corun ~ a, Campus da Zapateira, 15071 A Corun
~ a, Spain

a r t i c l e i n f o a b s t r a c t

Article history: In the quest for potent antiamoebic agents, a series of hydrazone hybrids (H1eH30) have been designed
Received 1 April 2016 and sequentially synthesized. The dimethylaminoethoxy and hydrazone entities incorporated into one
Received in revised form molecule proved to be more persuasive and selective approach towards designing of antiamoebic agent.
20 July 2016
The synthesized compounds exhibited promising results against E. histolytica. The compound N0 -(2-
Accepted 11 August 2016
Available online 13 August 2016
chlorobenzylidene)-4-(2-(dimethylamino) ethoxy)benzohydrazide was most impending among the se-
ries. Cytotoxicity profile showed better cell viability on lung cancer cell line (A549 cells) by MTT assay.
© 2016 Published by Elsevier Masson SAS.
Keywords:
Hydrazones
Antiamoebic activity
Cytotoxicity
MTT assay
X-ray crystallography

1. Introduction spermatozoid damage and haematuria. Hence, there is an intense

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 pleu-
ropulmonary, 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 nonedisease-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, pe-
ripheral neuropathy, transient myopia, gastric mucus irritation,
* Corresponding author.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
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 [7e12]. 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
(Fig. 1) [15e17]. It was observed that hydrazones showed better
antiplasmodial and antiamoebic activity as compared to its hy-
drazide [15]. The derivatives bearing quinoline nucleus with a
hydrazone linkage [eNHeNeC(CH3)eR] having a free NeH 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, tor-
emefin, 4-hydroxytamoxifin, ferrocifen (Fig. 2) [18e21]. Moreover,
compounds bearing dimethylamino ethoxy entity have also been
reported recently against amoebiasis (Fig. 3) [22,23]. An effort has

http://dx.doi.org/10.1016/j.ejmech.2016.08.022
0223-5234/© 2016 Published by Elsevier Masson SAS.
446 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455
Fig. 1. Promising antiamoebic compounds containing hydrazone linkage.
Fig. 2. Structures of currently used drugs containing dimethylaminoethoxy tail.
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 (Fig. 4).
2. Result and discussion
2.1. Chemistry
The synthetic route employed to obtain the title compounds is
Fig. 3. Promising antiamoebic compounds bearing N-substituted aminoethoxy tail.
Fig. 4. Rational designing of the compounds.
outlined in Scheme 1. The first step involves acid catalysed esteri-
fication 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 hydra-
zide was done by nucleophilic substitution reaction with hydrazine
hydrate. Finally, the condensation of the hydrazide with different
aromatic aldehydes gave the final products (H1eH30) in a good
yield (80e90%). All the compounds were characterized by spec-
troscopic techniques and their data is present in experimental
section. The molecular composition of the compounds was estab-
lished using the CHNS elemental analysis. The evidence for the
formation of the compounds was obtained by 1H NMR spectros-
copy. The methyl-4-(2-(dimethylamino)ethoxy)benzoate (2)
showed a sharp singlet at d3.88 ppm for eOCH3 protons. In N0 -
Benzylidene-4- (2-(dimethylamino)ethoxy) benzohydrazide (3)
the peak for COOCH3 protons disappeared and a broad singlet can
be seen at d7.27 ppm for (NH) proton indicating formation of the
hydrazide (3). A sharp singlet was observed at d2.34 ppm for the six
protons of the two methyl groups, a triplet appears for (eOCH2) is
slightly downfield as compared to the triplet of CH2eN which
appeared at d4.11e4.10 ppm and d2.74e2.75 ppm respectively. The
NH2 peak was not observed in the PMR of N0 -Benzylidene-4-(2-
(dimethylamino)ethoxy)benzohydrazide (3) may be due to long
range H-bonding but it appeared 3310 cm1 in IR spectra. In all the
compounds (H1eH30) the eNH protons appeared as broad singlet
in the range d9.86e9.21 ppm. A singlet is observed d8.46e8.26 ppm
for eN]CH group in (H1eH30) 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 pres-
ence of the bands for ester and the ether tail at 1712 cm1(COO-
 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455
Scheme 1. Synthesis of 4-(2-(dimethylamino)ethoxy)benzohydrazide derivatives (H1eH30).
ester group) and 1247 cm1 (AreOeC,CeOeC asym.str) respec-
tively. In p-hydroxymethylbenzoate (1) the peaks for ester and
hydroxyl groups were present. Further the formation of N0 -Benzy-
lidene-4-(2-(dimethylamino)ethoxy) benzohydrazide (3) can be
confirmed by the appearance of the peak for NH2 group at
3317 cm1 (NeH str) and for carbonyl group at 1605 cm1 (C]O
str). In Methyl-4-(2-(dimethylamino)ethoxy)benzoate (2) C]O
stretching band for ester group was found at 1712 cm1 which was
absent in (3) suggesting conversion of ester group to hydrazide
group. The hydrazone derivatives (H1eH30) show all the charac-
teristic bands such as the band at 3392e3061 cm1 was assigned to
NeH stretching and 1712e1643 cm1 to C]O stretching which
suggested the condensation of different aromatic aldehyde with
hydrazide. The mass spectra of the compounds showed the ex-
pected fragmentation pattern m/z corresponding to [Mþ1] which
confirmed their respective molecular weights.
2.2. Single crystal structure
2.2.1. Single crystal structure of compound H23
ORTEP diagram for the compound 4-(2-(dimethylamino)
ethoxy)-N-(pyridine-2-ylmethylene)benzohydrazide (H23) is
showed in Fig. 5. The compound H23 crystallizes in the ortho-
rhombic 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) , 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). p-p lateral stacking in-
teractions between the bonds of the benzyl rings establish the
linked between frameworks and determine the structure (Fig. 6)
Fig. 5. 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.
447
[24]. The distances between p clouds of the centroids in the bonds
of benzyl groups are: dc1-c2 ¼ 3.577 Å [c1: C(5B)eC(6B) and c2:
C(5K)eC(6K)] and repeated for other centroids]. Table 2 contains
selected bond lengths and angles for compound H23.
2.3. Biology
2.3.1. Antiamoebic activity
Preliminary experiments were carried out to determine the
in vitro antiamoebic activity of all the compounds (H1eH30) 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 concentration. The
antiamoebic effect was compared with the most widely used
antiamoebic medication metronidazole (Mtz) which had a 50%
inhibitory concentration (IC50) of 1.89 mM 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 azome-
thine group electron rich correspondingly antiamoebic activity
decreases. The same was observed in few compounds whilst from
phenyl group (H1, IC50: 1.43 ± 0.012 mM) to 4-isopropylphenyl
group (H10, IC50: 3.45 ± 0.015 mM) and 4-dimethylamino group
(H21, IC50: 1.89 ± 0.008 mM) 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 mM) exhibited the least IC50 value
than meta-Cl (H3, IC50: 1.01 ± 0.014 mM) and para-Cl (H4 IC50:
0.79 ± 0.011 mM). The nitro group containing compounds showed
slightly higher IC50 values (H5, IC50: 2.80 ± 0.021 mM & H6, IC50:
2.40 ± 0.029 mM), though para-nitro showed lower IC50 value than
448 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455
Fig. 6. Intermolecular p-p interactions in H23. Dashed lines link the centres of the p clouds involving in each interaction.
meta-nitro but both can be considered as moderately active.
Compound H7 with eOH at ortho position was endowed with ac-
tivity which can be observed from its percentage inhibition value
(H7, IC50: 1.76 ± 0.017 mM), while the compounds H8 para eOH and
H28 with dihydoxy substitution were moderately active with
higher inhibitory concentration values (H8, IC50: 2.21 ± 0.030 mM &
H28, IC50: 6.40 ± 0.022 mM)). The results obtained for the alkyl and
alkoxy groups were quite similar, wherein the activity reduces from
methyl (H15, IC50: 1.86 ± 0.010 mM) to ethyl (H9, IC50:
5.65 ± 0.002 mM) and from methoxy (H22, IC50: 1.53 ± 0.019 mM) to
ethoxy (H11, IC50: 3.77 ± 0.011 mM) and isopropoxy (H12, IC50:
7.653 ± 0.003 mM) 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 dis-
cussion, it can be concluded that compounds with eCl substitution
H2, H3, H4 seems to be the most potent of all the compounds
screened.
2.3.2. Cytotoxicity profile
Cytotoxic effects of the compounds (H1eH30) were studied in
A549 cells (Non small cell lung carcinoma cells) by MTT assay.
Metronidazole, known anti amebic 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 com-
pounds and percentage of viable cells were measured at 48 h post
drug treatment (Fig. 7a). IC50 value of each compound was deter-
mined. Two compounds, H4 and H19 had relatively low IC50 values
of 69.379 mm and 81.719 mm respectively while for rest of the
compound including MNZ, IC50 was found be very high that
is > 100 mm. Corresponding IC50 values of each compound was
given in Table 4 and graphical representation was also shown be-
sides the graph showing percentage of viability (Figs. 7 and 8).
3. Conclusion
In the present study hydrazones derivatives (H1eH30) were
designed and sequentially synthesized in four steps. The combi-
national chemistry was used in the designing of the final com-
pounds. The compounds proved to be more potent and selective
antiamoebic agent. Out of thirty synthesized compounds fourteen
exhibited promising results against E. histolytica. Cytotoxicity ef-
fects of the compounds found active against E. histolytica were
studied in A549 cells (Non small cell lung carcinoma cells) by MTT
assay which showed good cell viability results.
4. Experimental protocol
4.1. 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 recorded on Bruker FT-IR spectrophotometer under
neat condition on a Zn Se crystal. 1H NMR was recorded on Bruker
Spectrospin DPX 300 MHz using CDCl3 as a solvent and tetrame-
thylsilane (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 Ele-
mentar Vario analyzer and the results lay within ±0.3% of the
theoretical values.
4.2. Synthesis
General procedure for the synthesis of ester (1): Acidic
esterification of p-hydroxybenzoic acid (5g) was done in methanol
(50 mL) and catalytic amount of conc. H2SO4 was added to the re-
action 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 nmax
(cm1): 3506(OeH str), 3030(ArC-H, CeH str), 1676 (COO ester
group), 1273 (AreOeC,CeOeC asym. str), 846(p-disubstituted
benzene,CeH 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.
Synthesis of Methyl-4-(2-(dimethylamino)ethoxy)benzoate
(2): To the stirring solution of THF and potassium tert-butoxide,
the ester p-hydroxymethylbenzoate (1), (32.89 mmol) was added
which was refluxed for 30 min followed by addition of 2-
dimethylaminoethylchloride (4.7384g, 32.89 mmol) and was
further refluxed for 50 h the reaction mixture was poured into
water and extraction by ethyl acetate gave a viscous mass. Brown
viscous solid, Yield 65%, 1H NMR (CDCl3, 300 MHz, dppm): 7.99 (d,
2H, J ¼ 8.4,AreH), 6.94 (d, 2H, J ¼ 8.7, AreH), 4.11 (t, 2H, J ¼ 5.7 Hz,
eOCH2), 3.88(ss, 3H, COOCH3), 2.75 (t, 2H, J ¼ 5.4 Hz, eCH2), 2.34
(ss, 6H, N(CH3)2); IR nmax (cm1): 2948 (ArCeH, CeH str), 1712
(COO ester group), 1604(C]O str), 1247(AreOeC,CeOeC asym.
str), 846(p-disubstituted benzene, CeH 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 (H1eH30): The Methyl-4-(2-(dimethylamino)
ethoxy)benzoate (2) was refluxed with hydrazine hydrate in
ethanol, for 6 h gave 4-(2-(dimethylamino)ethoxy) benzohydrazide
(3). A mixture of hydrazide (3) and appropriate aromatic aldehydes
in absolute ethanol (50 ml) was refluxed for 4 h with continuous
stirring. On reaction completion (TLC) the reaction mixture was
 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455 449

Fig. 7. a. Measurement of cell viability following treatment with H1, H2, H3 and H4 compounds by MTT assay. b. Measurement of cell viability following treatment with H7, H15,
H17 and H18 compounds by MTT assay. c. Measurement of cell viability following treatment with H19, H21, H22, H27, H29 and H30 compounds by MTT assay.

cooled and the solid product was filtered and recrystallized in
ethanol yielded the final product (H1eH30).
N0 -Benzylidene-4-(2-(dimethylamino)ethoxy)benzohy-
drazide (3): Cream coloured solid; 1H NMR (CDCl3, 300 MHz,
dppm): 7.72e7.69 (m, 2H, AreH), 7.27(bs, 1H, NH), 6.96e6.93 (m,
2H, AreH), 4.10 (t, 2H, J ¼ 5.7 Hz, OCH2), 2.74 (t, 2H, J ¼ 5.4 Hz,CH2),
2.34 (ss, 6H, N(CH3)2). IR nmax (cm1): 3317(NH2, NeH str),
2973(ArCeH, CeH str), 1605(C]O str), 1250(AreOeC,CeOeC
asym. str), 1172(NeN), 846(p-disubstituted benzene,CeH 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.
N0 -Benzylidene-4-(2-(dimethylamino)ethoxy)benzohy-
drazide (H1): White solid, Yield: 85%, m.pt. 172  C; 1HNMR (CDCl3,
300 MHz, dppm): 9.43 (bs, 1H, eNH), 8.07 (d, 2H, J ¼ 6.9 Hz, AreH),
8.31 (s, 1H, eCHN),7.88e7.72 (m, 2H, AreH), 7.48e7.37 (m, 3H,
AreH), 6.98 (d, 2H, J ¼ 8.7 Hz, AreH), 4.12 (t, 2H, J ¼ 5.7 Hz, OCH2),
2.77 (t, 2H, J ¼ 5.7 Hz, CH2), 2.36 (ss, 6H, N(CH3)2), 1.91 (s, 5H,
AreH). IR nmax (cm1): 3363 (NH2, NeH str), 3022 (ArCeH, CeH str),
450 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455
Fig. 8. IC50 Value of compounds (H1eH30) for MTT assay.
1699 (C]O str), 1250 (AreOeC, CeOeC asym. str), 1171 (NeN);
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 (Mþ1).
N0 -(2-chlorobenzylidene)-4-(2-(dimethylamino)ethoxy)ben-
zohydrazide (H2): Colourless solid, Yield: 85%, m.pt. 100  C; 1H
NMR (CDCl3, 300 MHz, dppm): 9.36 (bs, 1H, eNH), 8.19 (s, 1H, CHN),
7.89(bs, 2H, AreH), 7.38e7.29 (m, 4H, AreH), 7.01 (d, 2H, J ¼ 8.7,
AreH), 4.13 (t, 2H, J ¼ 6 Hz, OCH2), 2.76 (t, 2H, J ¼ 5.7 Hz, CH2), 2.35
(ss, 6H, N(CH3)2). IRnmax (cm1): 3184 (NeH str), 1649 (C]O str),
1554 (N]CH), 1256 (AreOeC, CeOeC asym. str), 1177(NeN);
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 (Mþ1).
N0 -(3-chlorobenzylidene)-4-(2-(dimethylamino)ethoxy)ben-
zohydrazide (H3): Colourless solid, Yield: 82%, m.pt. 99  C; 1H NMR
(CDCl3, 300 MHz, dppm): 9.21 (bs, 1H, eNH), 8.26 (s, 1H, CHN), 7.87
(bs, 2H, AreH), 7.73 (bs, 1H, AreH),7.58 (bs, 1H, AreH), 7.37e7.29
(m, 2H, AreH), 7.00 (bs, 1H, CHN), 4.13 (t, 2H, J ¼ 5.7 Hz, OCH2), 2.76
(t, 2H, J ¼ 5.4 Hz, CH2), 2.35(ss, 6H, N(CH3)2). IR nmax (cm1):
3061(NeH str), 3061(ArCeH, CeH str), 1643(C]O str),
1248(AreOeC, CeOeC asym. str), 1552(eN]CH), 1182(NeN);
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 (Mþ1).
N0 -(4-chlorobenzylidene)-4-(2-(dimethylamino)ethoxy)ben-
zohydrazide (H4): Colourless solid, Yield: 84%, m.pt. 188  C; 1H
NMR (CDCl3, 300 MHz, dppm): 9.37(bs, 1H, eNH), 8.28 (s, 1H, CHN),
7.86(bs, 2H, AreH), 7.65 (bs, 2H, AreH),7.37 (d, 2H, J ¼ 8.1, AreH),
6.99 (d, 2H, J ¼ 8.4, AreH), 4.12 (t, 2HJ ¼ 5.1 Hz, OCH2), 2.75 (t, 2H,
J ¼ 5.7 Hz, CH2), 2.35(ss, 6H, N(CH3)2). IR nmax (cm1): 3392 (NeH
str), 1695 (C]O str), 1565 (N]CH), 1257 (AreOeC, CeOeC asym.
str), 1178 (NeN); 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 (Mþ1).
N0 -(3-Nitrobenzylidene)-4-(2-(dimethylamino)ethoxy)ben-
zohydrazide (H5): Colourless solid, Yield: 82%, m.pt.130  C; 1H
NMR (CDCl3, 300 MHz, dppm): 9.43(bs, 1H, eNH), 8.46 (s, 1H, CHN),
8.24 (d, 2H,J ¼ 8.7, AreH), 8.13(bs, 1H, AreH) 7.89 (d, 2H,J ¼ 7.5,
AreH), 7.58 (t, 1H, J ¼ 8.1, AreH), 7.01 (d, 2H, J ¼ 8.7, AreH), 4.13 (t,
2H, J ¼ 5.7 Hz, OCH2), 2.78 (t, 2H, J ¼ 5.7 Hz,eCH2), 2.35 (ss, 6H,
N(CH3)2). IR nmax (cm1): 3204(NeH str), 1684(C]O str), 1562(N]
CH), 1259(AreOeC, CeOeC asym. str), 1177(NeN); 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 (Mþ1).
N0 -(4-Nitrobenzylidene)-4-(2-(dimethylamino)ethoxy)ben-
zohydrazide (H6): Yellow solid, Yield: 82%, m.pt.99  C; 1H NMR
(CDCl3, 300 MHz, dppm): 9.84(bs, 1H, eNH), 8.46 (s, 1H, CHN), 8.25
(d, 2H,J ¼ 8.7, AreH), 7.87 (d, 4H,J ¼ 6.9, AreH) 6.98 (d, 2H,J ¼ 8.7,
AreH), 4.19 (t, 2H, J ¼ 5.4 Hz, OCH2), 2.87 (t, 2H,J ¼ 5.7 Hz, CH2), 2.42
(ss, 6H, N(CH3)2). IR nmax (cm1): 3367 (NeH str), 1653(C]O str),
1565(N]CH), 1247 (AreOeC, CeOeC asym. str), 1168(NeN);
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 (Mþ1).
N0 -(2-hydroxybenzylidene)-4-(2-(dimethylamino)ethoxy)
benzohydrazide (H7): Yellow solid, Yield: 70%, m.pt. 150  C; 1H
NMR (CDCl3, 300 MHz, dppm): 11.06 (bs, 1H), 9.12 (s, 1H), 8.46 (s,
1H), 7.82 (d, 2H,J ¼ 8.7), 7.33e7.33 (m, 2H), 7.23e7.20 (m, 1H),
7.00e6.97 (m, 3H), 4.13 (t, 2H, J ¼ 5.4 Hz), 2.77 (t, 2H,J ¼ 5.7 Hz),
2.35 (ss, 6H). IR nmax (cm1): 3355 (NeH str), 1646(C]O str),
1561(N]CH), 1252 (AreOeC, CeOeC asym. str), 1150(NeN);
Elemental 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 (Mþ1).
N0 -(4-hydroxybenzylidene)-4-(2-(dimethylamino)ethoxy)
benzohydrazide (H8): Yellow solid, Yield: 62%, m.pt.111  C; 1H
NMR (CDCl3, 300 MHz, dppm): 11.51 (s, 1H, eNH), 9.92 (s, 1H, OH),
8.33 (s, 1H, CHN), 7.88 (d, 2H, J ¼ 6.3 Hz, AreH), 7.55 (d,
2H,J ¼ 8.7 Hz, AreH), 7.06 (d, 2H, J ¼ 6.3 Hz, AreH), 6.84 (d, 2H,
J ¼ 6.0 Hz, AreH), 4.13 (t, 2H, J ¼ 5.4 Hz, OCH2), 2.81 (t, 2H,
J ¼ 5.7 Hz, CH2), 2.22 (ss, 6H, N(CH3)2); IRnmax (cm1): 3590 (eOH),
3301 (NeH str), 1644 (C]O str), 1557 (N]CH), 1283 (AreOeC,
CeOeC asym. str), 1166 (NeN); . 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 (Mþ1).
N0 -(4-ethylbenzylidene)-4-(2-(dimethylamino)ethoxy)ben-
zohydrazide (H9): solid, Yield: 60%, m.pt.155  C; 1H NMR (CDCl3,
300 MHz, dppm): 9.11 (bs, 1H), 8.24 (s, 1H), 7.85 (bs, 2H), 7.65 (bs,
2H),7.26e7.21 (m, 2H), 6.99 (d, 2H, J ¼ 7.2 Hz), 4.14 (t, 2H,
J ¼ 5.7 Hz), 2.77 (t, 2H, J ¼ 5.4 Hz), 2.71 (m, 2H), 2.35 (ss, 6H), 1.27 (t,
3H, J ¼ 7.5 Hz). IRnmax (cm1): 3357 (NeH str), 1648 (C]O str),
1560 (N]CH), 1270 (AreOeC, CeOeC asym. str), 1173 (NeN);
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 (Mþ1).
N0 -(4-isopropylbenzylidene)-4-(2-(dimethylamino)ethoxy)
benzohydrazide (H10): solid, Yield: 60%, m.pt.150  C; 1H NMR
(CDCl3, 300 MHz, dppm): 9.41 (bs, 1H), 8.24 (s, 1H), 7.96e7.84 (m,
2H), 7.68e7.61 (m, 2H), 7.25e7.24 (m, 2H), 6.99 (d, 2H, J ¼ 8.7 Hz),
4.14 (t, 2H, J ¼ 5.4 Hz), 2.97e2.91 (m, 1H), 2.78 (t, 2H, J ¼ 5.7 Hz),
2.35 (s, 6H), 1.27 (d, 6H, J ¼ 6.9 Hz); IRnmax (cm1): 3384 (NeH str),
1647 (C]O str), 1557 (N]CH), 1250 (AreOeC, CeOeC asym. str),
1176 (NeN); 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
(Mþ1).
N0 -(4-ethoxybenzylidene)-4-(2-(dimethylamino)ethoxy)ben-
zohydrazide (H11): solid, Yield: 65%, m.pt.162  C; 1H NMR (CDCl3,
300 MHz, dppm): 9.24 (bs, 1H), 8.21 (s, 1H), 7.84 (bs, 2H), 7.67 (bs,
2H), 6.98 (d, 2H, J ¼ 8.7 Hz), 6.90 (d, 2H, J ¼ 8.7 Hz), 4.13 (t, 2H,
J ¼ 5.4 Hz), 4.07e4.02 (m, 2H), 2.77 (t, 2H, J ¼ 5.7 Hz), 2.34 (s, 6H),
1.44 (t, 3H, J ¼ 6.9 Hz); IRnmax (cm1): 3301 (NeH str), 1644 (C]O
str), 1557 (N]CH), 1250 (AreOeC, CeOeC asym. str), 1167 (NeN);
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 (Mþ1).
N0 -(4-propoxybenzylidene)-4-(2-(dimethylamino)ethoxy)
benzohydrazide (H12): solid, Yield: 72%, m.pt.190  C; 1H NMR
(CDCl3, 300 MHz, dppm): 9.08 (bs, 1H), 8.19 (s, 1H), 7.96e7.83 (m,
2H), 7.65e7.60 (m, 2H), 6.99 (d, 2H, J ¼ 8.7 Hz), 6.90 (d, 2H,
J ¼ 9.0 Hz), 4.65 (m, 1H), 4.14 (t, 2H, J ¼ 5.4 Hz), 2.77 (t, 2H,
J ¼ 5.7 Hz), 2.34 (s, 6H), 1.36 (d, 6H, J ¼ 6.0 Hz); IRnmax (cm1): 3361
(NeH str), 1644 (C]O str), 1563 (N]CH), 1240 (AreOeC, CeOeC
asym. str), 1142 (NeN); 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 (Mþ1).
N0 -(4-hydroxy-3-methoxybenzylidene)-4-(2-(dimethyla-
mino)ethoxy)benzo-hydrazide (H13): solid, Yield: 76%,
m.pt.147  C; 1H NMR (CDCl3, 300 MHz, dppm): 11.02 (s, 1H), 8.24 (s,
 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455
2H), 7.91e7.49 (m, 3H), 7.02e6.87 (m, 4H), 4.13 (t, 2H, J ¼ 5.4 Hz),
3.94 (s, 3H) 2.78e2.74 (m, 2H), 2.33 (s, 6H); IRnmax (cm1): 3358
(NeH str), 1640 (C]O str), 1504 (N]CH), 1241 (AreOeC, CeOeC
asym. str), 1173 (NeN); 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 (Mþ1).
N0 -(2-bromo-4-hydroxy-3-methoxybenzylidene)-4-(2-
(dimethylamino)ethoxy) benzohydrazide (H14): solid, Yield: 68%,
m.pt.197  C; 1H NMR (CDCl3, 300 MHz, dppm): 11.68 (s, 1H), 10.12
(bs, 1H), 8.29 (s, 1H), 7.89 (d, 2H, J ¼ 5.7 Hz), 7.40e7.30 (m, 2H),
7.10e7.07 (m, 2H), 4.14 (t, 2H, J ¼ 5.4 Hz), 3.89 (s, 3H), 2.64 (t, 2H,
J ¼ 5.7 Hz), 2.22 (s, 6H); IRnmax (cm1): 3356 (NeH str), 1640 (C]O
str), 1502 (N]CH), 1250 (AreOeC, CeOeC asym. str), 1169 (NeN);
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 (Mþ1).
N0 -(4-methylbenzylidene)-4-(2-(dimethylamino)ethoxy)
benzohydrazide (H15): solid, Yield: 55%, m.pt.164  C; 1H NMR
(CDCl3, 300 MHz, dppm): 9.11 (bs, 1H), 8.22 (s, 1H), 7.83 (bs, 2H),
7.65 (bs, 2H), 7.22 (d, 2H, J ¼ 8.1 Hz), 7.00 (d, 2H, J ¼ 8.1 Hz), 4.14 (t,
2H, J ¼ 5.4 Hz), 2.78 (t, 2H, J ¼ 5.7 Hz), 2.38 (ss, 9H); IRnmax (cm1):
3353 (NeH str), 1647 (C]O str), 1562(N]CH), 1276 (AreOeC,
CeOeC asym. str), 1176 (NeN); 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 (Mþ1).
N0 -(3,4-dimethoxybenzylidene)-4-(2-(dimethylamino)
ethoxy)benzohydrazide (H16): solid, Yield: 64%, m.pt.125  C; 1H
NMR (CDCl3, 300 MHz, dppm): 9.43 (bs, 1H), 8.22 (s, 1H), 7.85 (bs,
2H), 7.48 (bs, 1H), 7.10 (d, 1H, J ¼ 8.4 Hz), 6.97 (d, 2H, J ¼ 8.4 Hz),
6.85 (d, 1H, J ¼ 8.1 Hz), 4.12 (t, 2H, J ¼ 5.4 Hz), 3.91 (ss, 6H), 2.77 (t,
2H, J ¼ 5.4 Hz), 2.34 (s, 6H); IRnmax (cm1): 3354 (NeH str), 1641
(C]O str), 1540 (N]CH), 1240 (AreOeC, CeOeC asym. str), 1165
(NeN); 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 (Mþ1).
N0 -(benzo[1,3]dioxol-5-ylmehylene)-4-(2-(dimethylamino)
ethoxy)benzohydrazide (H17): solid, Yield: 69%, m.pt.149  C; 1H
NMR (CDCl3, 300 MHz, dppm): 9.21(bs, 1H), 8.20 (s, 1H), 7.84 (bs,
2H), 7.39 (bs, 1H), 7.06 (d, 1H, J ¼ 8.1 Hz), 6.98 (d, 2H, J ¼ 8.7 Hz),
6.81 (d 1H, J ¼ 8.1 Hz), 6.00 (ss, 2H), 4.13 (t, 2H, J ¼ 5.4 Hz), 2.77 (t,
2H, J ¼ 5.7 Hz), 2.34 (s, 6H); IRnmax (cm1): 3299 (NeH str), 1641
(C]O str), 1561 (N]CH), 1250 (AreOeC, CeOeC asym. str), 1171
(NeN); 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 (Mþ1).
N0 -(3,5-dibromo-4-hydroxybenzylidene)-4-(2-(dimethyla-
mino)ethoxy) benzohydrazide (H18): solid, Yield: 56%,
m.pt.152  C; 1H NMR (CDCl3, 300 MHz, dppm): 11.63 (s, 1H), 8.19 (s,
1H), 7.89e7.87 (m, 2H), 7.77e7.61 (m, 2H), 7.06e7.04 (m, 2H), 4.27
(t, 2H, J ¼ 5.4 Hz), 2.84 (t, 2H, J ¼ 5.7 Hz), 2.43 (s, 6H); IRnmax
(cm1): 3356 (NeH str), 1612 (C]O str), 1571(N]CH), 1252
(AreOeC, CeOeC asym. str), 1175 (NeN); 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 (Mþ1).
N0 -(2,4,6-trimethylbenzylidene)-4-(2-(dimethylamino)
ethoxy)benzohydrazide (H19): Solid, Yield: 60%, m.pt.196  C; 1H
NMR (CDCl3, 300 MHz, dppm): 11.57 (s, 1H), 8.73 (s, 1H), 7.90e7.89
(m, 2H), 7.07e7.05 (m, 2H), 6.91 (s, 2H), 4.12 (t, 2H, J ¼ 5.4 Hz), 2.65
(t, 2H, J ¼ 5.7 Hz), 2.42 (s, 6H), 2.24 (s, 3H), 2.21 (s, 6H); IRnmax
(cm1): 3227 (NeH str), 1643 (C]O str), 1542 (N]CH), 1255
(AreOeC, CeOeC asym. str), 1179 (NeN); 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 (Mþ1).
N0 -(3-methoxy-4-propoxybenzylidene)-4-(2-(dimethyla-
mino)ethoxy)benzohydrazide (H20): Solid, Yield: 55%,
m.pt.126  C; 1H NMR (CDCl3, 300 MHz, dppm): 9.24 (bs, 1H), 8.20 (s,
1H), 7.85 (bs, 2H), 7.48 (bs, 1H), 7.09 (d, 1H, J ¼ 6.3 Hz), 6.98 (d, 2H,
J ¼ 9.0 Hz), 6.87 (d, 1H, J ¼ 8.4 Hz), 4.63 (m, 1H), 4.13 (t, 2H,
451
J ¼ 5.4 Hz), 3.89e3.84 (m, 3H), 2.77 (t, 2H, J ¼ 5.7 Hz), 2.37 (s, 6H),
1.40 (d, 6H, J ¼ 6.3 Hz); IRnmax (cm1): 3386 (NeH str), 1642 (C]O
str), 1536(N]CH), 1238 (AreOeC, CeOeC asym. str), 1150 (NeN);
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 (Mþ1).
N0 -(4-(dimethylamino)benzylidene)-4-(2-(dimethylamino)
ethoxy)benzohydrazide (H21): solid, Yield: 56%, m.pt.216  C; 1H
NMR (CDCl3, 300 MHz, dppm): 8.94(bs, 1H), 8.10 (s, 1H), 7.91 (bs,
2H), 7.63 (bs, 2H), 6.98 (d, 2H, J ¼ 8.7 Hz), 6.70 (d, 2H, J ¼ 8.7 Hz),
4.31 (t, 2H, J ¼ 5.4 Hz), 3.02 (ss, 6H), 2.79 (t, 2H, J ¼ 5.7 Hz), 2.37 (s,
6H); IRnmax (cm1): 3352 (NeH str), 1644 (C]O str), 1517 (N]CH),
1253 (AreOeC, CeOeC asym. str), 1177 (NeN); Elemental 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 (Mþ1).
N0 -(4-methoxybenzylidene)-4-(2-(dimethylamino)ethoxy)
benzohydrazide (H22): solid, Yield: 69%, m.pt.120  C; 1H NMR
(CDCl3, 300 MHz, dppm): 9.24 (bs, 1H), 8.22 (s, 1H), 7.84(bs, 2H),
7.68 (bs, 2H), 6.97e6.89 (m, 4H), 4.13 (t, 2H, J ¼ 5.4 Hz), 3.83(ss, 3H),
2.77 (t, 2H, J ¼ 5.7 Hz), 2.34 (s, 6H); IRnmax (cm1): 3390 (NeH str),
1644 (C]O str), 1559 (N]CH), 1241 (AreOeC, CeOeC asym. str),
1164 (NeN); 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
(Mþ1).
4-(2-(dimethylamino)ethoxy)-N-(pyridine-2-ylmethylene)
benzohydrazide (H23): solid, Yield: 52%, m.pt.102  C; 1H NMR
(CDCl3, 300 MHz, dppm): 9.38(bs, 1H), 8.61 (s, 1H), 8.23e8.05 (m,
2H), 8.00e7.90 (m, 2H), 7.76 (t, 1H, J ¼ 7.8 Hz), 7.31e7.28 (m, 1H),
7.013 (d, 2H, J ¼ 8.7 Hz), 4.152 (t, 2H, J ¼ 5.4 Hz), 2.781 (t, 2H,
J ¼ 5.7 Hz), 2.35 (s, 6H); IRnmax (cm1): 3379 (NeH str), 1649 (C]O
str), 1560 (N]CH), 1272 (AreOeC, CeOeC asym. str), 1179 (NeN);
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 (Mþ1).
4-(2-(dimethylamino)ethoxy)-N-((3-methylthiophene-2-yl)
methylenen) benzohydrazide (H24): solid, Yield: 61%, m.pt.141  C;
1
H NMR (CDCl3, 300 MHz, dppm): 9.31 (bs, 1H), 9.20 (s, 1H), 7.83 (bs,
1H), 7.27 (bs, 1H), 6.97 (d, 2H, J ¼ 8.7 Hz), 6.84 (d, 2H, J ¼ 5.1 Hz),
4.13 (t, 2H, J ¼ 5.4 Hz), 2.77 (t, 2H, J ¼ 5.7 Hz), 2.34 (s, 9H); IRnmax
(cm1): 3324 (NeH str), 1642 (C]O str), 1558 (N]CH), 1244
(AreOeC, CeOeC asym. str), 1177 (NeN); 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 (Mþ1).
Ferrocenyl-4-(2-(dimethylamino)ethoxy)benzohydrazide
(H25): solid, Yield: 60%, m.pt.192  C; 1H NMR (CDCl3, 300 MHz,
dppm): 8.84 (bs, 1H), 8.22 (bs, 1H), 7.81 (bs, 2H), 6.99 (d, 2H,
J ¼ 8.7 Hz),4.69 (s, 2H), 4.41 (s, 2H), 4.221 (s, 4H), 4.15 (t, 2H,
J ¼ 5.4 Hz), 2.78 (t, 2H, J ¼ 5.4 Hz), 2.36 (s, 6H); IRnmax (cm1): 3397
(NeH str), 1641 (C]O str), 1558 (N]CH), 1256 (AreOeC, CeOeC
asym. str), 1188 (NeN); 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 (Mþ1).
N0 -(3,4-dichlorobenzylidene)-4-(2-(dimethylamino)ethoxy)
benzohydrazide (H26): solid, Yield: 69%, m.pt. 96  C; 1H NMR
(CDCl3, 300 MHz, dppm): 8.34 (bs, 1H), 7.84 (s, 1H), 7.81 (bs, 3H),
7.54 (bs, 1H), 7.47 (d, 1H, J ¼ 8.1 Hz), 6.98 (d, 2H, J ¼ 8.4 Hz), 4.14 (t,
2H, J ¼ 5.4 Hz), 2.74 (t, 2H, J ¼ 5.7 Hz), 2.35 (s, 6H); IRnmax (cm1):
3260 (NeH str), 1649 (C]O str), 1554 (N]CH), 1247 (AreOeC,
CeOeC asym. str), 1173 (NeN); 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 (Mþ1).
2-methoxy-4-[2-{4-(dimethylamino)ethoxybenzoyl)hydrazi-
nylidene}methyl]phenyl acetate (H27): solid, Yield: 56%, m.pt.
83  C; 1H NMR (CDCl3, 300 MHz, dppm): 9.66 (bs, 1H), 8.10 (bs, 1H),
7.87(bs, 2H), 7.47 (s, 1H), 6.99e6.93 (m, 4H), 4.13 (t, 2H, J ¼ 6.0 Hz),
3.82 (s, 3H), 2.78e2.75 (m, 2H), 2.35 (s, 6H), 2.31 (s, 3H); IRnmax
(cm1): 3329 (NeH str), 1646 (C]O str), 1554 (N]CH), 1284
452 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455

(AreOeC, CeOeC asym. str), 1171 (NeN); Elemental analysis calcd Table 1
(%) for C21H25N3O5: C 63.14, H 6.31, N 10.52; found: C 63.14, H 6.33, Crystal Data and Structure Refinement for the compound 4-(2-(dimethylamino)
ethoxy)-N-(pyridine-2-ylmethylene)benzohydrazide (H23$H2O).
N 10.55; ES-MS: m/z 400.07 (Mþ1).
N0 -(3,4-dihydroxybenzylidene)-4-(2-(dimethylamino) Formula C17 H22 N4 O3 (H23$H2O)
ethoxy)benzohydrazide (H28): solid, Yield: 64%, m.pt. 195  C; 1H Formula weight 330.39
T, K 100 (2)
NMR (CDCl3, 300 MHz, dppm): 11.47 (s, 1H), 9.73e9.29 (bs, 2H, OH), Wavelength, Å 0.71073
8.24 (s, 1H), 7.78e7.66 (m, 2H), 7.30 (s, 1H), 7.05e7.03 (m, 2H), Crystal system Orthorhombic
6.78e6.76 (m, 1H), 6.60e6.56 (m, 1H), 4.13 (t, 2H, J ¼ 5.4 Hz), 2.63 (t, Space group Pbca
2H, J ¼ 5.7 Hz), 2.22 (s, 6H) IRnmax (cm1): 3314 (NeH str), 1645 a/Å 8.4474 (10)
b/Å 11.8401 (16)
(C]O str), 1505 (N]CH), 1248 (AreOeC, CeOeC asym. str), 1176
c/Å 34.235 (4)
(NeN); Elemental analysis calcd (%) for C18H21N3O4: C 62.96, H 6.16, V/Å3 3424.2 (7)
N 12.24; found: C 62.93, H 6.17, N 12.25; ES-MS: m/z 344.64 (Mþ1). Z 8
N0 -(2,5-dimethoxybenzylidene)-4-(2-(dimethylamino) F000 1408
ethoxy)benzohydrazide (H29): solid, Yield: 59%, m.pt. 124  C; 1H Dcalc/g cm3 1.282
m/mm1 0.090
NMR (CDCl3, 300 MHz, dppm): 9.22 (bs, 1H), 8.57 (bs, 1H), 7.95 (bs, q/( ) 1.19e26.83
2H), 7.64 (bs, 1H), 6.99e6.93 (m, 3H), 6.86e6.83 (m, 1H), 4.14- (t, Rint 0.1243
2H, J ¼ 4.8 Hz), 3.82 (ss, 6H), 2.78 (t, 2H, J ¼ 4.5 Hz), 2.36 (s, 6H); Crystal size/mm3 0.27  0.25  0.17
IRnmax (cm1): 3457 (eOH), 3336 (NeH str), 1642 (C]O str), 1563 Goodness-of-fit on F2 1.036
R1 [I > 2s(I)]a 0.0542
(N]CH), 1246 (AreOeC, CeOeC asym. str), 1170 (NeN); Elemental
wR2 (all data)b 0.1582
analysis calcd (%) for C20H25N3O4: C 64.67, H 6.78, N 11.31; found: C Largest differences peak and hole (eÅ3) 0.337 and 0.290
64.67, H 6.78, N 11.31; ES-MS: m/z 372.18 (Mþ1). a
R1 ¼ SrrForrFcrr/SrFor.
N0 -(4-(diethoxymethyl)benzylidene)-4-(2-(dimethylamino) b
wR2 ¼ {S[w (rrFor2rFcr2r)2]r/S[w (F2o)2]}1/2.
ethoxy)benzohydrazide(H30): solid, Yield: 69%, m.pt.75  C; 1H
NMR (CDCl3, 300 MHz, dppm): 9.05 (bs, 1H), 7.86 (s, 1H), 7.72e7.52
(m, 4H), 7.50 (d, 2H, J ¼ 8.1 Hz), 7.00 (d, 2H, J ¼ 8.7 Hz), 5.52 (s, 1H), Table 2
4.15 (t, 2H, J ¼ 5.7 Hz), 3.62e3.49 (m, 4H), 2.79 (t, 2H, J ¼ 5.7 Hz), Bond lengths [Å] and angles [ ] for the compound 4-(2-
(dimethylamino)ethoxy)-N-(pyridine-2-ylmethylene)benzo-
2.36 (s, 6H), 1.26 (m, 6H); IRnmax (cm1): 3389 (NeH str), 1649 (C]
hydrazide (H23·H2O).
O str), 1558 (N]CH), 1259 (AreOeC, CeOeC asym. str), 1176
(NeN); Elemental analysis calcd (%) for C23H31N3O4: C 66.81, H 7.56, Bond lengths H23$H2O

N 10.16; found C 66.85, H 7.55, N 10.14; ES-MS: m/z 414.11 (Mþ1). O(1)eC(5) 1.357 (2)
O(1)eC(4) 1.432 (3)
N(1)eC(1) 1.457 (3)
4.3. Pharmacological evaluation
N(1)eC(2) 1.462 (3)
N(1)eC(3) 1.463 (3)
4.3.1. Antiamoebic activity O(2)eC(11) 1.224 (2)
All the compounds were screened in vitro for antiamoebic ac- N(2)eC(11) 1.364 (3)
tivity against HM1:IMSS strain of E. histolytica by micro dilution N(2)eN(3) 1.379 (2)
N(3)eC(12) 1.281 (3)
method [25]. E. histolytica trophozoites were cultured in wells of
96-well microtiter plate by using Diamond TYIS-33 growth me- Angles H23·H2O
dium [26]. The test compounds (1 mg) were dissolved in ethanol C(5)eO(1)eC(4) 117.90 (15)
(50 mL, level at which no inhibition of amoeba occurs) [27]. The C(3)eN(1)eC(1) 108.62 (17)
stock solutions of the compounds were prepared freshly before use C(3)eN(1)eC(2) 111.50 (17)
C(1)eN(1)eC(2) 109.78 (17)
at a concentration of 1 mg/mL. Each plate included metronidazole C(11)eN(2)eN(3) 117.98 (17)
as standard amoebicidal drug, negative control (culture medium C(12)eN(3)eN(2) 115.31 (18)
plus amoeba) and a blank (culture medium only). All the com- N(1)eC(3)eC(4) 115.99 (19)
pounds were used in triplicate concentrations. The parasite sus- O(1)eC(4)eC(3) 108.45 (18)
pension was adjusted to 105 cells per mL by adding fresh medium
and then distributed to a 96-wells microtiter plate (Corning).
Compounds were added to the respective wells and the plate was Table 3
sealed, gassed for 10 min with nitrogen and then incubated at 37  C Hydrogen bonds in the compound 4-(2-(dimethylamino)ethoxy)-N-(pyridine-2-
ylmethylene)benzohydrazide (H23·H2O).
for 72 h. After incubation, the amoeba growth was checked under
microscope. The culture medium was removed by inverting the DeH…A d(DeH) d(H … A) d(D … A) <(DHA)
plate and shaking gently. Plate was then immediately washed with N(2)eH(2N)…O(1W)#1 0.94 (2) 1.91 (2) 2.828 (2) 162 (2)
sodium chloride (0.9%) at 37  C, dried at room temperature and the O(1W)eH(1WB)…O(2)#2 0.92 (4) 2.00 (3) 2.849 (2) 152 (3)
cells fixed with methanol and once dried, stained with aqueous O(1W)eH(1WB)…N(3)#2 0.92 (4) 2.46 (4) 3.171 (2) 134 (3)
O(1W)eH(1WA)…N(1)#3 1.04 (4) 1.74 (4) 2.775 (2) 174 (3)
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 Symmetry transformations used to generate equivalent atoms:
#1 xþ1/2,yþ1/2,z #2 x,y1,z #3 x1/2, yþ1/2, z.
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
all the compounds were between 5 and 640 mm. Compounds were
tested at least in two independent experiments.
reconstituted initially in ethanol, but further dilutions were made
in culture medium. A549 cells were cultured in 96 well plates at a
4.3.2. Cytotoxicity
density of 8000 cells/well in Ham's F-12 medium (Hi Media) sup-
Cytotoxic effects of the series (H1eH30) were determined in
plemented with 10% FBS. Once the cells attached to 80e90%
A549 cells (lung epithelial cells). The concentration range used for
 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455 453

Table 4
In vitro antiamoebic activity of (H1eH30) against HM1:IMSS strain of E. histolytica and cytotoxicity profile of active compounds.

Compound R Antiamoebic activity IC50 ± S.D. (mM) Cytotoxicity IC50 ± S.D. (mM)

H1 1.43 ± 0.012 267.79 ± 24.65

H2 0.23 ± 0.015 134.00 ± 11.90

H3 1.01 ± 0.014 139.59 ± 25.00

H4 0.79 ± 0.011 69.37 ± 5.87

H5 2.80 ± 0.021 N.D.

H6 2.40 ± 0.029 N.D.

H7 1.76 ± 0.017 404.47 ± 9.39

H8 2.21 ± 0.030 N.D.

H9 5.65 ± 0.002 N.D.

H10 3.45 ± 0.015 N.D.

H11 3.77 ± 0.011 N.D.

H12 7.65 ± 0.003 N.D.

H13 2.41 ± 0.009 N.D.

H14 3.61 ± 0.020 N.D.

H15 1.86 ± 0.010 475.03 ± 3.32

H16 2.69 ± 0.005 N.D.

(continued on next page)

454 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455

Table 4 (continued )

Compound R Antiamoebic activity IC50 ± S.D. (mM) Cytotoxicity IC50 ± S.D. (mM)

H17 1.06 ± 0.009 548.04 ± 45.66

H18 1.17 ± 0.010 281.41 ± 20.9

H19 1.89 ± 0.004 81.71 ± 4.77

H20 4.13 ± 0.001 N.D.

H21 1.89 ± 0.008 481.48 ± 57.5

H22 1.53 ± 0.019 126.01 ± 16.70

H23 2.37 ± 0.021 N.D.

H24 4.98 ± 0.020 N.D.

H25 5.04 ± 0.029 N.D.

H26 2.81 ± 0.031 N.D.

H27 0.79 ± 0.036 185.66 ± 45.34

H28 6.40 ± 0.022 N.D.

H29 1.81 ± 0.006 110.35 ± 3.30

H30 1.14 ± 0.005 171.78 ± 17.70

 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455
Table 4 (continued )
Compound R Antiamoebic activity IC50 ± S.D. (mM)
Mtz
* N.D. Not Determined, S.D. Standard Deviation.
The values in bold shows that the compounds are active towards E. histolytica.
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
37  C with 5% CO2. At 48 h post drug treatment, cell viability was
measured by MTT assay. In brief, cells in each well were incubated
with 20 ml of MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-
tetrazolium bromide) reagent at a concentration of 5 mg/ml in 5%
methanol for 4 h. Then, MTT reagent was removed and the resulting
formazan crystals were solubilized in 200 ml of DMSO. The quantity
of formazan which is directly proportional to the number of cells
viable was measured by recording absorbance at wavelength of
560 nm with the reference wavelength of 670 nm [28]. The per-
centage 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.
4.4. 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 f-u
scan method. Reflections were measured from a hemisphere of
data collected from frames, each of them covering 0.3 in u. A total
of 73793 reflections measured were corrected for Lorentz and po-
larization 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 (rFr/
srFr) > 4.0. After data collection, in each case an multi-scan ab-
sorption 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 density in the
crystal 0.337 and 0.290 e.Å3. A weighting scheme w ¼ 1/
[s2(Fo2) þ (0.091800 P)2 þ 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: (þ44) 1223 336
033; or e-mail: deposit@ccdc.cam.ac.uk.
Acknowledgement
One of the authors (AI) is thankful to University Grants
455
Cytotoxicity IC50 ± S.D. (mM)
1.89 ± 0.002 >100
Commission, New-Delhi, India for BSR (SRF) Fellowship.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.08.022.
References
[1] B.S. Pritt, C.G. Clark, Mayo Clin. Proc. 83 (2008) 1154.
[2] H.P. Verkerke, W.A. Petri Jr., C.S. Marie, Semin. Immunopathol. 34 (2012) 771.
[3] (a) Heiman F.L. Wertheim, Peter Horby, John P. Woodall, Atlas of Human In-
fectious Diseases, first ed., Blackwell Publishing Ltd, 2012;
(b) G. Choudhuri, M. Rangan, Indian J. Gastroenterol. 4 (2012) 153.
[4] W. Stauffer, M. Abd-Alla, J.I. Ravdin, Arch. Med. Res. 37 (2006) 266.
[5] C.C. Hung, D.D. Ji, H.Y. Sun, Y.T. Lee, S.Y. Hsu, S.Y. Chang, PLoS Negl. Trop. Dis. 2
(2008) 175.
[6] (a) J. Evans, D. Levesque, K. Knowles, R. Longshore, S. Plummer, J. Vet. Intern.
Med. 17 (2003) 304;
(b) A. Azam, S.M. Agarwal, Curr. Bioact. Compd. 3 (2007) 121;
[7] S. Rollas, S.G. Kucukguzel, Molecules 12 (2007) 1910.
[8] F. Chimenti, E. Maccioni, D. Secci, A. Bolasco, P. Chimenti, A. Granese, O. Befani,
P. Turini, S. Alcaro, F. Ortuso, M.C. Cardia, S.J. Distinto, Med. Chem. 50 (2007)
707.
[9] J. Mao, Y. Wang, B. Wan, A.P. Kozikowski, S.G. Franzblau, Chem. Med. Chem. 2
(2007) 1624.
[10] A. Andreani, S. Burnelli, M. Granaiola, A. Leoni, A. Locatelli, R. Morigi,
M. Rambaldi, L. Varoli, N. Calonghi, C. Cappadone, G. Farruggia, M. Zini,
C. Stefanelli, L. Masotti, N.S. Radin, R.H. Shoemaker, J. Med. Chem. 51 (2008)
809.
[11] E. Noulsri, R. Richardson, S. Lerdwana, S. Fucharoen, T. Yamagishi,
D.S. Kalinowski, K. Pattanapanyasat, Am. J. Hematol. 84 (2009) 170.
[12] P. Vicini, M. Incerti, I.A. Doytchinova, P. Colla, B. Busonera, R. Loddo, Eur. J.
Med. Chem. 41 (2006) 624.
[13] L. Vehmanen, I. Elomaa, C. Blomqvist, T. Saarto, J. Clin. Oncol. 24 (2006) 675.
[14] N. Price, O. Sartor, T. Hutson, S. Mariani, Clin. Prostate Cancer 3 (2005) 211.
[15] A. Inam, S.M. Siddiqui, T.S. Macedo, D.R.M. Moreira, A.C.L. Leite, M.B.P. Soares,
A. Azam, Eur. J. Med. Chem. 75 (2014) 67.
[16] F. Hayat, A. Salahuddin, A. Umar, A. Azam, Eur. J. Med. Chem. 45 (2010) 4669.
[17] S.M. Siddiqui, A. Salahuddin, A. Azam, Eur. J. Med. Chem. 49 (2012) 411.
[18] S. Specht, S.R. Sarite, I. Hauber, J. Hauber, U.F. Go €rbig, C. Meier, D. Bevec,
A. Hoerauf, A. Kaiser, Parasitol. Res. 102 (2008) 1177e1184.
[19] P. Melnyk, V. Leroux, C. Sergheraert, P. Grellier, Bioorg. Med. Chem. 16 (2006)
31e35.
[20] S. Top, J. Tang, A. Vessie res, D. Carrez, C. Provot, G. Jaouen, Chem Commun. 8
(1996) 955.
[21] S. Top, A. Vessie res, G. Leclercq, J. Quivy, J. Tang, J. Vaissermann, M. Huche ,
G. Jaouen, Chem. Eur. J. 9 (2003) 5223.
[22] M. Abid, K. Husain, A. Azam, Bioorg. Med. Chem. Lett. 15 (2005) 4375.
[23] S.L. Zaidi, S. Mittal, M.S. Rajala, F. Avecilla, M. Husain, A. Azam, Eur. J. Med.
Chem. 98 (2015) 179.
[24] M.R. Maurya, S. Dhaka, F. Avecilla, Polyhedron 81 (2014) 154.
[25] C.W. Wright, M.J. O'Neill, J.D. Phillipson, D.C. Warhurst, Antimicrob. Agents
Chemother. 32 (1988) 1725.
[26] L.S. Diamond, D.R. Harlow, C. Cunnick, Trans. Soc. Trop. Med. Hyg. 72 (1978)
431.
[27] A.T. Keene, A. Harris, J.D. Phillipson, D.C. Warhurst, Planta Med. 52 (1986) 278.
[28] M.V. Berridge, P.M. Herst, A.S. Tan, Biotechnol. Annu. Rev. 11 (2005) 127.
[29] G.M. Sheldrick, SADABS, Version 2.10, University of Go €ttingen, Germany,
2004.
[30] SHELX, G.M. Sheldrick, Acta Crystallogr. Sect. A 64 (2008) 112.