Synthesis, characterization and anti-cancer activity of hydrazide derivatives incorporating a quinoline moiety

Article abstract of DOI:10.3390/molecules21070916

Identification of the novel (E)-N1-((2-chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio) propanehydrazide scaffold 18 has led to the development of a new series of biologically active hydrazide compounds. The parent compound 18 and new quinoline derivatives 19-26 were prepared from the corresponding quinoline hydrazones and substituted carboxylic acids using EDC-mediated peptide coupling reactions. Further modification of the parent compound 18 was achieved by replacement of the quinoline moiety with other aromatic systems. All the newly synthesized compounds were evaluated for their anti-cancer activity against the SH-SY5Y and Kelly neuroblastoma cell lines, as well as the MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines. Analogues 19 and 22 significantly reduced the cell viability of neuroblastoma cancer cells with micromolar potency and significant selectivity over normal cells. The quinoline hydrazide 22 also induced G₁ cell cycle arrest, as well as upregulation of the p27^kip1 cell cycle regulating protein.

Full text of DOI:10.3390/molecules21070916

molecules
Article
Synthesis, Characterization and Anti-Cancer Activity
of Hydrazide Derivatives Incorporating a
Quinoline Moiety
Murat Bingul 1 , Owen Tan , Christopher R. Gardner , Selina K. Sutton , Greg M. Arndt 2,3,
,2
2
1,2
2
Glenn M. Marshall 2 , Belamy B. Cheung *, Naresh Kumar * and David StC. Black *
,4
2,
1,
1,
1
School of Chemistry, The University of New South Wales Australia, Sydney, NSW 2052, Australia;
muratbingul1983@gmail.com (M.B.); z3217087@gmail.com (C.R.G.)
Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre,
2
The University of New South Wales Australia, Sydney, NSW 2031, Australia; Otan@ccia.unsw.edu.au (O.T.);
ssutton@ccia.unsw.edu.au (S.K.S.); garndt@ccia.unsw.edu.au (G.M.A.);
gmarshall@ccia.unsw.edu.au (G.M.M.)
3
ACRF Drug Discovery Centre for Childhood Cancer, Children’s Cancer Institute Australia for Medical
Research, Lowy Cancer Research Centre, The University of New South Wales Australia, Sydney,
NSW 2052, Australia
Kids Cancer Centre, Sydney Children’s Hospital, Randwick, NSW 2031, Australia
4
*
Correspondence: bcheung@ccia.unsw.edu.au (B.B.C.); n.kumar@unsw.edu.au (N.K.);
d.black@unsw.edu.au (D.StC.B.); Tel.: +61-2-9385-2450 (B.B.C.); +61-2-9385-4698 (N.K.);
+
61-2-9385-4657 (D.StC.B.)
Academic Editor: Jean Jacques Vanden Eynde
Received: 24 May 2016; Accepted: 8 July 2016; Published: 14 July 2016
1
Abstract: Identification of the novel (E)-N -((2-chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio)
propanehydrazide scaffold 18 has led to the development of a new series of biologically active
hydrazide compounds. The parent compound 18 and new quinoline derivatives 19–26 were
prepared from the corresponding quinoline hydrazones and substituted carboxylic acids using
EDC-mediated peptide coupling reactions. Further modification of the parent compound 18 was
achieved by replacement of the quinoline moiety with other aromatic systems. All the newly
synthesized compounds were evaluated for their anti-cancer activity against the SH-SY5Y and Kelly
neuroblastoma cell lines, as well as the MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines.
Analogues 19 and 22 significantly reduced the cell viability of neuroblastoma cancer cells with
micromolar potency and significant selectivity over normal cells. The quinoline hydrazide 22 also
kip1
induced G cell cycle arrest, as well as upregulation of the p27
cell cycle regulating protein.
1
Keywords: quinoline; hydrazide-hydrazone; anticancer; neuroblastoma; breast cancer
1
. Introduction
Cancer is a heterogeneous type of genetic diseases, resulting from the alteration of
many key genetic and molecular pathways [
conventional chemotherapies often not being effective, also inducing side-effects and encountering
drug-resistance [ ]. An important class of chemotherapeutics that effectively induce cell death, but
can become ineffective due to drug-resistance, are the histone deacetylase inhibitors (HDACi) [ ].
These molecules exert efficacy by regulating transcription of genes that suppress cell proliferation
and angiogenesis, induce cell differentiation and promote apoptosis [ ]. In particular, the HDACi
suberoylanilide hydroxamic acid (SAHA, Figure 1) has shown potent cytotoxic effects towards a
1]. Treatment regimens are often harsh, with
2
3
4,5
number of tumour types with low toxicity towards normal cells [
6,
7]. As SAHA targets only a few
Molecules 2016, 21, 916; doi:10.3390/molecules21070916
www.mdpi.com/journal/molecules

Molecules 2016, 21, 916
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signalling pathways, single agent treatment with SAHA has been found to be ineffective against
several cancers [ ]. Cytotoxic drugs are found to be most effective when given in combination to
8,9
achieve additive or synergistic effects. The combination of SAHA with other chemotherapeutic agents
that possess different mechanisms of action has been considered to be a more promising approach to
reduce drug resistance and minimize overlapping toxicities [8,10].
Figure 1. Structure of the HDACi suberoylanilide hydroxamic acid (SAHA).
In order to provide a basis for the discovery of novel SAHA enhancers, a subset (10,560
compounds) of the Walter & Eliza Hall Institute (WEHI) compound library was screened to identify
molecules that can act synergistically with a clinical dose of SAHA to overcome resistance in
SAHA-resistant MDA-MB-231 breast cancer cells, as previously reported [11]. From this screening,
1
we identified the quinoline-hydrazide scaffold (E)-N -((2-chloro-7-methoxyquinolin-3-yl)methylene)-
-(phenylthio)propane hydrazide (15, Figure 2) as a novel anti-cancer lead compound, which reduced
3
the MDA-MB-231 cell viability by <30% in the absence of SAHA, >60% in the presence of SAHA and
also displayed a differential of >55% between these conditions.
Figure 2. The structure of parent compound 15 from the WEHI screening.
Quinoline is a pharmacologically valuable scaffold that is prevalent in a variety of biologically
active synthetic and natural compounds [12]. Throughout the 20th century, the chemistry of quinolines
has been the subject of intense study and different interesting bioactivity such as antibacterial,
antifungal, anti-inflammatory, antimalarial and anticancer activities [13
quite broad, with quinoline derivatives having been used against many cancers, such as those of the
breast, prostate, gastrointestinal tract, colon and liver [19 22]. Importantly, a number of quinoline-based
–18]. This anticancer activity is
–
anticancer drugs have also been used clinically, including camptothecin and its analogues (irinotecan
and topotecan) [23,24] and bosutinib (Figure 3) [25,26].
Figure 3. Structures of quinoline-containing anticancer drugs.
The hydrazide-hydrazone moiety (–(C=O)NHN=CH) has been identified as an important fragment
for various biological activities including antibacterial, antifungal, analgesic, antiinflammatory,
antidepressant and anticancer activities [27–29]. When used in conjunction with a quinoline

Molecules 2016, 21, 916
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system, this combination has afforded compounds with antimicrobial [30], antimycobacterial [31],
anti-tubercular [32 33], anticonvulsant [34] and cytotoxic activity [35]. These biological activities are
,
related to the many interesting and important properties of the hydrazidehydrazone moiety, such as
their relatively higher metabolic stability towards proteases than amides and their tuneable, labile
nature in acidic pH. The anticancer activity of quinoline-based compounds is exerted through many
mechanisms, such as apoptosis [36], inhibition of angiogenesis [37], receptor inhibition and DNA
intercalation [38
–41]. Many compounds also exert their activity through cell cycle arrest, particularly
at the G /M phase [42
cell cycle arrest occurs at the G /G phase [49
–
45] or the S phase [46
–
,
48]. However, a few examples have been reported where
2
kip1
50]. p27
is involved in the regulation of G to S phase
0
0
1
transitions by regulating the activity of cyclin-dependent kinases [51,52]. While p27 is rarely mutated
or deleted in human cancers, it is frequently deregulated through reduced expression or mislocalization
to the cytoplasm [53]. Currently, p27 expression levels in cancer serve prognostic purposes [54], as
they can correlate to treatment response in either a positive or inverse fashion depending upon the
disease model [55
–
60]. However, therapeutics targeting p27 have also been investigated in numerous
62]. In this paper, we report the quinoline-hydrazide
E)-N -((2-chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio)propane hydrazide (15, Figure 2)
cancers to enforce apoptotic consequences [61
,
1
(
as a novel anticancer lead compound. We further report the synthesis, characterization and in vitro
biological evaluation of a series of quinoline-based and non-quinoline-based analogues, including
kip1
their effects on cell cycle distribution and the expression of the cell cycle-related p27
protein.
2
. Results and Discussion
2
.1. Synthesis of Quinoline-Based Hydrazide-Hydrazones
In order to generate a diverse array of analogues, a convergent synthesis was envisaged, whereby
the targeted hydrazide-hydrazone structure could be formed by an amide-coupling reaction between
an alkyl carboxylic acid and aryl hydrazone (Scheme 1).
Scheme 1. Synthesis of quinoline-based hydrazide-hydrazones. Reagents and conditions: i. DMF, POCl3,
˝
9
0 C, 12 h; ii. NH NH ¨ H O, EtOH, rt, 18 h. iii. EDC, DMF, Et N, rt, 18 h.
2
2
2
3
The alkyl acids
1
–
4
were prepared following literature methods [63]. Similarly, the substituted
2-chloro- quinoline-3-carbaldehydes 5–7 were isolated in yields of 47%–60% following Vilsmeier

Molecules 2016, 21, 916
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cyclisation of the corresponding acetanilides
Following aqueous work-up, carbaldehydes
8
5
–
–
10, according to literature methods (Scheme 1) [64
were then treated with neat hydrazine hydrate at
13 in 74%–87% yields (Scheme 1).
19 were obtained by EDC-coupling of the hydrazones
using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC), which gave hydrazide-hydrazones 14–19 in yields of 62%–68% (Scheme 1).
,65].
7
room temperature in ethanol to generate the quinoline hydrazones 11
Finally, the desired hydrazide-hydrazones 14
11 13) to the appropriate carboxylic acids
–
–
(
–
1–4
2
.2. Synthesis of Non-Quinoline Hydrazide-Hydrazones
In order to elaborate on the structure-activity relationship and identify other heterocyclic motifs
with potential cytotoxic activity, the quinoline motif was replaced with naphthalene, indole and
benzaldehyde. Naphthalene was selected in order to investigate the necessity of the quinoline nitrogen,
which might participate in target interaction. Indole was selected not only for its known biological
activities, but also to determine if the ring size is essential for activity. Benzene was selected in order
to gain insight into the necessity for a system containing a pendant ring. The aldehydes 20
subjected to the same synthetic protocol described above, firstly being converted to the corresponding
hydrazones 23 25, followed by EDC-coupling to acid 11 to give the targeted hydrazide-hydrazones
6–28 (Scheme 2).
–22 were
–
2
Scheme 2. Synthesis of non-quinoline hydrazide-hydrazones. Reagents and conditions: i. NH NH2¨ H O,
2
2
EtOH, rt, 18 h; ii. 11, EDC, DMF, Et N, rt, 18 h.
3
The new hydrazide-hydrazones 14
IR, H-NMR, C-NMR and EIMS). The IR spectra of the derivatives showed the azomethine C–H
stretching doublet absorption band at 2850–2950 cm , the C=N absorption band at 1550–1600 cm ,
a carbonyl stretch at 1620–1680 cm–1 and a strong N–H stretch at 3180–3220 cm . In the H-NMR
spectra of the hydrazides, the azomethine proton appeared at 8.22–8.90 ppm as a sharp singlet, whereas
the corresponding amide N–H proton appeared as a broad singlet at 9.18–9.37 ppm. The protons of
the heterocyclic systems and carboxylic acid nucleus showed resonances at the expected chemical
shift values.
– , 26–28 were characterized by physical and spectral data
19
1
13
(
–1
–1
–1
1
As stereochemistry is an essential factor for biological activity, single crystals of 28 were examined
by X-ray diffraction in order to determine the relative stereochemistry of the C=N bond (Figure 4).
The X-ray diffraction study clearly indicated an E conformation for the C=N bond, as well as showing
the alkyl chain in a bent conformation. Theoretically, this orientation might impact on the potential
binding of the hydrazide-hydrazone motif to transition metals, which is a mechanism seen in multiple
quinoline-hydrazone compounds [66–69]. Furthermore, it could also affect any selectivity between
larger binding pockets, as opposed to shallower grooves.

Molecules 2016, 21, 916
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Figure 4. ORTEP diagram of X-ray crystal structure of 15 grown from chloroform.
.3. Biological Results
2
2
.3.1. Cell Viability Assays
The newly synthesized compounds were evaluated for their ability to inhibit the growth of
SH-SY5Y and Kelly neuroblastoma, and MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines
using the Alamar blue (Resazurin) assay [70]. Briefly, the cells were allowed to attach for 24 h in a
9
6-well culture plate before being exposed to the ligands at a concentration of 10 µM in DMSO for 72 h,
either in the presence or absence of SAHA. Comparative values for cell viability in each well were
determined by a Wallac 1420 Victor III spectrophotometer, which measured light absorbance in each
well at 570 nm. The mean and standard deviation (SD) values for each compound were calculated
from at least three replicate experiments. The anticancer activity of the compounds was evaluated by
comparison to a negative (DMSO) control (Table 1 and Figure 5).
MCF-7
MDA-MB-231
1
1
20
00
120
100
80
60
40
20
0
-
SAHA
+SAHA
-SAHA
+SAHA
8
6
4
2
0
0
0
0
0
1
4
15 16 17 18 19 26 27 28
Compounds (10µM)
14 15 16 17 18 19 26 27 28
Compounds (10µM)
SH-SY5Y
KELLY
1
1
20
00
120
100
80
60
40
20
0
-SAHA
+SAHA
-SAHA
+SAHA
8
6
4
2
0
0
0
0
0
1
4
15 16 17 18 19 26 27 28
Compounds (10µM)
14 15 16 17 18 19 26 27 28
Compounds (10µM)
Figure 5. Cell viability of cancer cell lines after treatment at 10
the negative control (DMSO). Red line represents the cell viability of cell lines after treatment with
M SAHA (0.5 M SAHA for KELLY cells). Data represents the mean values ( S.D.) for three
independent determinations.
µM for 72 h, given as a percentage of
1
µ
µ
˘

Molecules 2016, 21, 916
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Table 1. Cell viability of cancer cell lines after treatment at 10 µM for 72 h, given as a percentage of the
negative control (DMSO). Data represents the mean values (˘S.D.) for three independent determinations.
Cell Viability (% of Negative Control)
Compound
MCF-7
MDA-MB-231
SH-SY5Y
Kelly
14
15
16
17
18
19
26
27
28
98.5 ˘ 1.0
94.8 ˘ 3.8
88.4 ˘ 3.8
66.8 ˘ 1.2
93.1 ˘ 0.6
85.6 ˘ 4.9
93.1 ˘ 1.0
95.1 ˘ 1.7
93.1 ˘ 3.8
93.8 ˘ 3.6
86.9 ˘ 0.5
72.0 ˘ 2.6
61.8 ˘ 8.4
100.2 ˘ 2.0
78.0 ˘ 4.1
71.5 ˘ 1.5
76.4 ˘ 8.0
87.9˘ 1.5
104.2 ˘ 6.2
74.3 ˘ 1.0
32.4 ˘ 6.4
18.4 ˘ 7.0
92.8 ˘ 6.6
85.8 ˘ 5.5
71.0 ˘ 5.0
78.8 ˘ 2.5
83.6 ˘ 4.9
80.0 ˘ 13.7
52.7 ˘ 8.7
12.4 ˘ 3.2
3.7 ˘ 1.0
89.7 ˘ 13.7
65.2 ˘ 10.5
47.6 ˘ 9.9
70.2 ˘ 13.7
70.6 ˘ 2.4
In general, it was found that these compounds displayed similar levels of activity whether in
the presence or absence of SAHA. It was also observed that the hydrazide-hydrazone compounds
had a greater cytotoxic effect on the neuroblastoma cells (SH-SY5Y and Kelly) compared to the breast
cancer cell lines (MCF-7 and MDA-MB-231). MCF-7 cells were found to be the most resistant towards
treatment with the hydrazide compounds, with reductions in cell viability ranging from 5%–33%.
The compounds were generally more effective in the MDA-MB-231 breast adenocarcinoma cell line,
with cell viability values being up to 20% lower than those in the MCF-7 cell line. Similarly, these
compounds were also observed to have a greater effect on the cell viability of the Kelly neuroblastoma
cell line than the SH-SY5Y cell line, again with up to 20% lower cell viability.
Notably, compound 17, bearing an additional methoxy group, was found to be the most active
agent against both breast adenocarcinoma cell lines, with cell reductions of 33% and 38% in MCF-7
and MDA-MB-231 cells, respectively. Further, this compound was also the most active agent against
the neuroblastoma cell lines, with reductions in cell viability of 82% and 96% in the SH-SY5Y and
Kelly cell lines, respectively. The importance of the methoxy groups was further demonstrated by the
observation that compound 14, which bears no methoxy substituents, had lower activity than the lead
compound 15. While compound 14 showed no activity towards breast adenocarcinoma cell lines, the
highest activity was observed with the 20% reduction in cell viability of Kelly cells.
It was also found that replacing the sulfane linkage with an ether linkage increased the potency of
the synthesized compounds. The ether 16, which was found to be the second most active compound
of those screened, displayed an enhanced potency of 42% and 40% against the SH-SY5Y and Kelly cell
lines, respectively, compared to the corresponding sulfane lead compound 15. However, only 15% and
6% of additional reductions were observed for the MDA-MB-231 and MCF-7 cell lines respectively.
The ether 19 showed 7% and 25% greater reductions in cell viability towards the SH-SY5Y and Kelly
cell lines, respectively, than was observed for the corresponding sulfane 18. Similarly, 7% and 22% of
additional reductions were observed for the MCF-7 and MDA-MB-231 cell lines respectively.
Hydrazides with shorter chains were also found to be more potent than their longer chain
counterparts. With respect to compounds 15 (n = 1) and 18 (n = 4), bearing sulfane linkages, it was
observed that compound 15 reduced viability of Kelly cells by up to 37% more than compound 18
In the case of SH-SY5Y cell, 18% of additional reduction was observed. Similarly, the shorter chain
analogue 16 reduced cell viability by up to 53% more than its longer chain analogue 19 for both Kelly
and SH-SY5Y neuroblastoma cell lines.
.
Additionally, the replacement of the quinoline system with other aromatic groups generally led to
a reduction in activity. Replacement of the quinoline (compound 15) with naphthalene (compound 26
)
resulted in similar levels of cytotoxicity across all cell lines and exhibited up to a 54% of reduction in
the case of Kelly cells. MCF-7 was found to be the most resistant cell with only 7% reduction. However,
the indole (compound 27) and phenyl (compound 28) derivatives showed significant decrease in

Molecules 2016, 21, 916
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activity compared to 15, with 28 typically having lower activity than 27. This suggests that the size of
this heterocycle is important for its cytotoxic activity, with bicyclic systems more favourable than the
tested monocyclic counterpart.
Dose response experiments were also conducted for the most potent compounds 16 and 17
.
These compounds were tested against the neuroblastoma and breast adenocarcinoma cell lines at
concentrations ranging from 0.1–25.0
µM, with their IC50 values determined (Table 2). Compound 16
showed IC50 values of 5.7 and 2.4 M against SH-SY5Y and Kelly neuroblastoma cells, respectively,
µ
with the IC50 value against the MCF-7 breast adenocarcinoma cell line greater than the maximum
tested dosage. On the other hand, the most active compound 17, showed IC50 values of 2.9 and
1
.3
µM against the SH-SY5Y and Kelly cells, respectively, and values of 14.1 and 18.8 µM for MCF-7
and MDA-MB-231 breast cancer cells, respectively.
Table 2. IC50 values of compounds 16 and 17 against four different cancer cell lines.
IC50 (µM) a
Compound
MCF-7
MDA-MB-231
SH-SY5Y
Kelly
1
1
6
7
>25.0
14.1
22.1
18.8
5.7
2.9
2.4
1.3
a
Compound concentration required to reduce cell viability by 50%, as determined by the Alamar Blue assay,
under conditions allowing untreated controls to undergo at least five consecutive rounds of multiplication.
2
.3.2. Toxicity Study on Normal Human Cells
Compounds 16 and 17 were then further tested against the MRC-5 and WI-38 normal human
lung fibroblast cell lines (Figure 6). At a concentration of 10 µM, 16 reduced MRC-5 and WI-38 cell
viability by only 10% and 5% respectively, while 17 reduced cell viability by about 30% in the two
normal lung fibroblast cell lines. Notably, these two compounds were significantly less toxic towards
the normal lung fibroblast cells compared to neuroblastoma cells. Taken together, the data suggests
that these novel quinoline hydrazides warrant further investigation as potential anti-cancer agents,
particularly for the treatment of neuroblastoma.
Figure 6. Comparative toxicity of compounds 29 and 30 against the tested cell lines at 10 µM after 72 h
exposure. Cell viability values are quoted as a percentage of the negative control.
2
.3.3. Effects on Cell Cycle and the Expression of Related Proteins
In order to determine if the synthesized compounds 16 and 17 play an active role in cell cycle
progression, we analysed their effect on the cell cycle progression of SH-SY5Y cells after 72 h treatment.
It was found that cells treated with 10 M of compound 16 showed no significant difference in the
proportions of cells across the cell cycle, compared to the DMSO control (Figure 7). Interestingly,
cells treated with 10 M of compound 17 showed an increased proportion of cells in G /G , with
9% of cells in G /G , compared with the solvent control treated cells with 61% of cells in G /G
µ
µ
0
1
6
0
1
0
1

Molecules 2016, 21, 916
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(
Figure 7). Furthermore, treatment with compound 17 also reduced the number of cells in the S phase
and G /M phase.
2
8
6
4
2
0
0
0
0
0
DMSO
*
1
1
6
7
*
*
*
Sub
/G
G
0
/G
1
S
G2/M
G0
1
Figure 7. Comparative cell cycle distribution of SH-SY5Y cells after 72 h exposure to 10
compounds 16 and 17. * p < 0.05.
µM of
To further investigate their mechanism of action on the cell cycle, immunoblot analysis was
performed on proteins harvested from SH-SY5Y cells treated with compounds 16 and 17. No changes
were observed in the levels E2F1, pRb and p27kip1 protein expression, in comparison to the negative
control, following 72 h treatment with 10 µM of 16 (Figure 8).
DMSO
16
DMSO
17
p27kip1
pRb
E2F1
α-tubulin
Figure 8. Immunoblot analysis of p27kip1 and E2F1 proteins isolated from SH-SY5Y cells treated with
0 µM of compounds 16 and 17 over 72 h. Anti-α-tubulin antibody was used for the loading control.
1
On the other hand, p27kip1 was highly up-regulated in SH-SY5Y cells treated with 10
µM of
compound 17, in comparison with the solvent control (Figure 8). However, two other cell cycle
kip1
regulatory proteins, E2F1 and pRb, were unchanged by treatment with compound 17. Since p27
is
a protein that prevents cell progression from the G to S phase, the results suggest that compound 17
1
kip1
induces cell cycle arrest by up-regulating p27
.
3
. Materials and Methods
3
.1. General Information
Commercially available reagents were purchased from Fluka (Sydney, NSW, Australia), Aldrich
Sydney, NSW, Australia), Acros Organics (Morris Plains, NJ, USA), Alfa Aesar (Lancashire, UK) and
(
Lancaster (Lancashire, UK) and purified if necessary. The synthetic procedures have been reported for
all compounds as general methods and appropriate references have been given for known compounds.

Molecules 2016, 21, 916
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1H (300 MHz) and 13C-NMR (75 MHz) spectra were obtained in the designated solvents on a DPX
00 spectrometer (Bruker, Sydney, NSW, Australia). Melting points were measured using a Mel-Temp
3
melting point apparatus and are uncorrected. Infrared spectra were recorded on a Avatar Series
FTIR spectrophotometer as KBr disks (Thermo Nicolet, Waltham, MA, USA). Ultraviolet spectra were
measured using a Cary 100 spectrophotometer (Varian, Santa Clara, CA, USA) in the designated
´
1
´1
solvents and data reported as wavelength (
λ
) in nm and adsorption coefficient (
ε
) in cm
M .
High-resolution [ESI] mass spectra were recorded by the UNSW Bioanalytical Mass Spectrometry
Facility, on an Orbitrap LTQ XL (Thermo Scientific, Waltham, MA, USA) ion trap mass spectrometer
using a nanospray (nano-electrospray) ionization source.
3
.1.1. General Procedure for the Synthesis of Substituted Carboxylic Acids 1–4
To a solution of thiophenol or phenol (10 mmol) in aqueous 10% KOH (25 mL) and EtOH
(30 mL) was added 3-bromopropionic acid or 6-bromohexanoic acid (11 mmol) in a saturated solution
of K CO (10 mL, 5.5 mmol). The resulting mixture was heated at reflux for 5 h. The reaction mixture
2
3
was neutralized with 18% HCl solution and the resulting precipitate was collected via filtration, washed
with water and crystallized from petroleum ether to give the product as a white solid.
˝
˝
3
-(Phenylthio)propanoic acid (
1
) [63]. White solid, yield: 88%; m.p. 59–60 C (lit. [63] m.p. 58–59 C);
1
H-NMR (CDCl3):
δ 7.41–7.33 (4H, m, ArH), 7.27–7.22 (1H, m, ArH), 3.22 (2H, t, J = 7.2 Hz, CH ), 2.65
2
(2H, t, J = 7.2 Hz, CH2).
˝
˝
3
-Phenoxypropanoic acid (
2
) [71]. White solid, yield: 85%; m.p. 92–94 C (lit. [71] m.p. 95–96 C);
1
H-NMR (CDCl3):
δ
7.34–7.29 (4H, m, ArH), 7.01–6.92 (1H, m, ArH), 4.29 (2H, t, J = 6.3 Hz, CH ), 2.88
2
(2H, t, J = 6.3 Hz, CH2).
˝
-(Phenylthio)hexanoic acid (3) [72]. White crystals, yield: 95%; m.p. 62–63 C (lit. [72] m.p. not given);
6
1
H-NMR (CDCl3):
δ 7.31–7.26 (2H, m, ArH), 6.96–6.90 (3H, m, ArH), 4.02 (2H, t, J = 7.2 Hz, CH ), 2.36
2
(
2H, t, J = 7.5 Hz, CH ), 1.87–1.65 (4H, m, CH -CH ), 1.61–1.50 (2H, m, CH ).
2 2 2 2
˝
6
-Phenoxyhexanoic acid (
4
) [72]. White crystals, yield: 89%; m.p. 66–68 C (lit. [72] m.p. not given);
1
H-NMR (CDCl3):
δ 7.38–7.35 (3H, m, ArH), 7.30–717 (2H, m, ArH), 3.00 (2H, t, J = 6.4 Hz, CH ), 2.32
2
(
2H, t, J = 6.2 Hz, CH ), 1.69–1.60 (4H, m, CH -CH ), 1.57–1.46 (2H, m, CH ).
2 2 2 2
3
.1.2. General Procedure for the Synthesis of Acetylated Anilines 8–10
A mixture of the appropriate aniline (50 mmol) and dimethoxyaminopyridine (DMAP, 50 mmol)
in dichloromethane (100 mL) was stirred for 10 min at room temperature. Acetic anhydride (50 mmol)
was added dropwise and the resulting mixture was stirred for a further 4 h at room temperature. Water
(
25 mL) was added and the resulting precipitate was thoroughly washed with ethanol followed by
˝
water to remove unreacted acetic anhydride and acetic acid. The desired product was dried at 50 C
under vacuum to yield the acetyl derivatives as a white solid.
˝
˝
N-Phenylacetamide (
8
) [73]. White solid, yield: 94%; m.p. 112–114 C (lit. [73] m.p. 113–115 C);
1
H-NMR (CDCl3):
δ
7.49 (2H, t, J = 4.4 Hz, H2 and H6), 7.37 (1H, bs NH), 7.34 (2H, t, J = 8.0 Hz, H3
and H5), 7.13 (1H, t, J = 7.4 Hz, H4), 2.20 (3H, s, CH3).
˝
˝
N-(3-Methoxyphenyl)acetamide (
H-NMR (CDCl3):
9
) [74]. White solid, yield: 88%; m.p. 80–82 C (lit. [74] m.p. 81–83 C);
1
δ 7.45 (1H, bs, NH), 7.30–7.23 (2H, m, H4–H5), 6.99 (1H, d, J = 8.0 Hz, H2), 6.69 (1 H,
dd, J = 2.1, 8.1 Hz, H6), 3.82 (3H, s, OMe), 2.20 (3H, s, CH3).
˝
N-(3,5-Dimethoxyphenyl)acetamide (10) [75]. White solid, yield: 90%; m.p. 152–154 C (lit. [75] m.p.
1
not given); H-NMR (CDCl ):
δ 7.53 (1H, bs, NH), 6.78 (2H, d, J = 2.34 Hz, H2 and H4), 6.27 (1H, t,
3
J = 2.34 Hz, H6), 3.80 (6H, s, 2 ˆ OMe), 2.20 (3H, s, CH3).

Molecules 2016, 21, 916
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3
.1.3. General Procedure for the Synthesis of 2-Chloro-3-Formylquinolines (5–7).
N,N-Dimethylformamide (0.025 mol) was cooled in an ice-salt bath and treated with phosphoryl
chloride (0.070 mol) for 20 min. The resulting mixture was added dropwise to a cooled solution of
the appropriate acetylated aniline ( 10) (0.010 mol) in N,N-dimethylformamide (10 mL) with stirring.
8
–
˝
The mixture was stirred at 90 C for 12 h. A small amount of crushed ice was added and the mixture
was basified to pH 14 with 5 M NaOH. After stirring at ambient temperature for 1 h, the precipitate
was filtered, washed with water, and dried to give the title compound.
˝
2
1
-Chloroquinoline-3-carbaldehyde (
5
) [76]. Yellow solid, yield: 60%; m.p. 150–152 C (lit. [76] m.p.
˝
46–149 C); H-NMR (CDCl ): δ 10.59 (1H, s, CHO), 8.79 (1H, s, H4), 8.11–7.65 (4H, m, H5, H6,
3
1
H7, H8).
˝
2
-Chloro-7-methoxyquinoline-3-carbaldehyde (
6
) [64]. Yellow solid, yield: 55%; m.p. 195–197 C (lit. [64
]
˝
1
m.p. 196–197 C); H-NMR (CDCl ):
δ
10.54 (1H, s, CHO), 8.70 (1H, s, H4), 7.89 (1H, d, J = 9.0 Hz, H5),
3
7
.42 (1H, d, J = 2.5 Hz, H8), 7.32 (1H, dd, J = 2.5, 9.0 Hz, H6) 4.00 (3H, s, OMe).
˝
-Chloro-5,7-dimethoxyquinoline-3-carbaldehyde (7). Yellow solid, yield: 47%; m.p. 160–162 C; UV-vis
2
´
1
´1
1
(
(
(
1
[
MeOH): λmax 314 nm (
1H, d, J = 8.7 Hz, H6), 7.32 (1H, d, J = 8.7 Hz, H8), 6.66 (1H, s, H4), 4.13 (6H, s, 2
CDCl3): 190.18 (C=O), 164.21 (C5), 160.97 (C7), 152.89 (C2), 150.39 (aryl C), 133.91 (C4), 119.99 (C3),
13.75 (aryl C), 111.16 (C6), 92.73 (C8), 106.87 (C8), 56.66 and 56.23 (OMe) HRMS (ESI): C H ClNO
ε
7,100 cm
M
), 347 (9,500); H-NMR (CDCl ):
δ
11.18 (1H, s, CHO), 8.43
3
ˆ
OMe); 13C-NMR
δ
12
10
3
+
M] requires 274.0241, found 274.0241.
3
.1.4. General Procedure for the Preparation of Hydrazones 11–13, 23–25
A mixture of the appropriate aldehyde 20 22 (5 mmol) and excess hydrazine hydrate was
5
–
7,
–
stirred overnight in ethanol. After completion of the reaction, the solvent was evaporated. Water was
added to the residue and the resulting mixture was extracted with ethyl acetate. The organic extract
was dried with anhydrous sodium sulfate and the solvent was evaporated under reduced pressure to
give the title compound.
˝
(
E)-2-Chloro-3-(hydrazonomethyl)quinolone (11) [77]. Yellow solid, yield: 87%; m.p. 140–142 C (lit. [77
]
˝
1
m.p. 135 C); H-NMR (CDCl ):
δ
8.63 (1H, s, H4), 8.09–8.06 (1H, m, H5), 8.06 (1H, s, CH), 7.93–7.91
3
(1H, m, H8), 7.79–7.73 (1H, m, H6) 7.66–7.60 (1H, m, H7), 7.58 (2H, bs, NH2).
˝
(
(
E)-2-Chloro-3-(hydrazonomethyl)-7-methoxyquinoline (12) [77]. Yellow solid, yield: 80%; m.p. 166–168 C
˝
lit. [77] m.p. 165 C); H-NMR (CDCl ): δ 8.57 (1H, s, H4), 8.20 (1H, s, CH), 7.74 (1H, d, J = 9.0 Hz,
3
1
H5), 7.34 (1H, d, J = 2.5 Hz, H8), 7.22 (1H, dd, J = 2.5, 9.0 Hz, H6) 5.86 (2H, s, NH ), 3.97 (3H, s, OMe).
2
(
1
(
E)-2-Chloro-3-(hydrazonomethyl)-5,7-dimethoxyquinoline (13).
Yellow solid, yield: 74%; m.p.
˝ ´1
98–200 C; IR (KBr): νmax 3403, 2942, 1610, 1499, 1466, 1349, 1263, 1220, 1175, 1132 cm ; UV-vis
´
1
´1
), 362 (4,900); H-NMR (CDCl ): δ 8.44 (1H, d, J = 8.7 Hz, H6),
3
1
MeOH): λmax 315 nm (
ε
2,800 cm
M
8
3
.40 (1H, s, CH), 7.38 (1H, d, J = 8.7 Hz, H8), 7.02 (1H, s, H4), 6.63 (2H, bs, NH ), 4.08 and 3.98 (each
2
H, s, OMe). 13C-NMR (CDCl3):
δ 159.73 (C5), 156.24 (C7), 151.29 (C2), 147.74 (aryl C), 139.93 (CH),
1
33.86 (C4), 119.17 (C3), 114.03 (aryl C), 102.36 (C6), 94.04 (C8), 106.87 (C8), 56.85 and 55.86 (OMe);
+
C H ClN NaO [M + Na] requires 288.0516, found 288.0503.
12
12
3
2
˝
(
E)-(Naphthalen-2-ylmethylene)hydrazine (23) [78]. Yellow solid, yield: 83%; m.p. 152–154 C (lit. [78
]
˝
1
m.p. 148–151 C); H-NMR (CDCl ):
δ
7.94 (1H, s, CH), 7.91–7.83 (5H, m, ArH), 7.50–7.46 (2H, m, H3
3
and H4), 5.63 (2H, s, NH2).
˝
E)-3-(Hydrazonomethyl)-1H-indole (24) [79]. Yellow solid, yield: 76%; m.p. 158–160 C (lit. [79] not
(
1
given); H-NMR (CDCl ):
δ
11.69 (1H, bs, NH), 8.92 (1H, s, CH), 8.35 (1H, dd, J = 0.9, 1.2 Hz, H4), 7.93
3
(1H, s, H2), 7.49 (1H, dd J = 0.9,1 .2 Hz, H7), 7.27–7.17 (2H, m, H5 and H6) 6.62 (2H, bs, NH2).

Molecules 2016, 21, 916
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˝ ˝
E)-Benzylidenehydrazine (25) [80]. Yellow crystal, yield: 88%; m.p. 92–94 C (lit. [80] m.p. 89–90 C);
(
1
H-NMR (CDCl3):
δ 8.72 (1H, s, CH), 7.91–7.88 (2H, m ArH), 7.51–7.49 (3H, m, ArH), 5.63 (2H, bs, NH2).
3
.1.5. General Procedure for EDC Coupling (compounds 14–19, 26–28)
The appropriate hydrazone 11 13 23 25 (4 mmol) and substituted carboxylic acid 1–4 (4 mmol)
–
,
–
and EDC (4.4 mmol) were dissolved in dry N-N-dimethylformamide. Triethylamine (4.4 mmol) was
added and the solution was stirred overnight at room temperature. The reaction mixture was diluted
with ethyl acetate (300 mL) and washed with 0.5 M hydrochloric acid solution (100 mL) and saturated
NaHCO solution (100 mL). The organic phase was dried over MgSO , filtered and concentrated under
3
4
reduced pressure to give the title compound.
1
E)-N -((2-Chloroquinolin-3-yl)methylene)-3-(phenylthio)propanehydrazide (14). Yellow solid, yield: 65%;
log P: 4.79; m.p. 172–174 C; IR (KBr): νmax 3181, 3070, 2960, 1668, 1616, 1598, 1571, 1486,1436, 1409,
(
˝
1
(
397, 1328, 1275, 1212 cm 1; UV-vis (MeOH): λmax 323 nm (
CDCl3): 9.28 (1H, bs, NH), 8.61 (1H, s, H4), 8.27 (1H, s, CH), 8.06 (1H, d, J = 8.4 Hz, H5), 7.92 (1H,
d, J = 8.4 Hz H8), 7.84–7.79 (1H, m, H6) 7.68–7.62 (1H, m, H7), 7.46–7.31 (5H, m, ArH ) 3.38 (2H, t,
´
ε
17,300 cm
´1
M
´1
), 352 (7,900); H-NMR
1
δ
13
J = 7.3 Hz, CH ), 3.20 (2H, t, J = 7.3 Hz„ CH ); C-NMR (CDCl3):
δ
173.43 (C=O), 148.94 (C2), 148.54
2
2
(
aryl C), 138.96 (CH), 135.67 (C4), 131.64 (aryl C), 129.60 and 129.04 (aryl CH), 128. 50 (C7), 128.37 (aryl
C), 127.77 (C5 and C8), 127.00 (C6), 126.33 (C3), 125.72 (2
ˆ
aryl CH), 32.70 (CH ) and 28.61 (CH )
2 2
+
HRMS (ESI): C H N O S [M] requires 285.1062, found 285.1053.
16
17
2
3
1
E)-N -((2-Chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio)propanehydrazide (15). Yellow solid,
(
˝
yield: 65%; log P: 4.66; m.p. 180–182 C; IR (KBr): νmax 3171, 3071, 2961, 1675, 1616, 1599, 1488, 1446,
1
3
407, 1365, 1331, 1274, 1232, 1163, 1126 cm 1; UV-vis (MeOH): λmax 324 nm (
´
ε
15,800 cm
´1
M ),
´1
1
52 (14,600); H-NMR (CDCl ):
δ
9.37 (1H, bs, NH), 8.51 (1H, s, H4), 8.24 (1H, s, CH), 7.79 (1H, d,
3
J = 9.0 Hz, H5), 7.44 (1H, d, J = 2.4 Hz, H8), 7.32–7.26 (5H, m, ArH), 7.23 (1H, dd, J = 2.4, 9.0 Hz, H6),
13
3
.99 (3H, s, OMe), 3.37 (1H, t, J = 7.2 Hz, CH ), 3.19 (1H, t, J = 7.2 Hz, CH ); C-NMR (CDCl3): δ
2
2
171.21 (C=O), 162.57 (C2), 150.02 (aryl C), 149.46 (C7), 139.45 (CH), 135.78 (C4), 135.22 (aryl C), 129.54
(
(
C5), 129.47, 129.41, 129.20, 129.02, and 126.28 (aryl CH), 123.02 (aryl C), 122.26 (C3), 121.14 (C6), 106.61
+
C8), 55.76 (OMe), 32.69 (CH ) and 28.62 (CH ) HRMS (ESI): C H ClN O SNa [M + Na] requires
2
2
21 20
2
2
4
22.0706, found 422.0697.
1
E)-N -((2-Chloro-7-methoxyquinolin-3-yl)methylene)-3-phenoxypropanehydrazide (16). Yellow solid, yield:
(
˝
7%; log P: 4.1; m.p. 206–208 C; IR (KBr): νmax 3186, 3065, 2932, 1674, 1619, 1600, 1489, 1458, 1400, 1365,
6
1
334, 1292, 1237, 1163, 1127 cm 1; UV-vis (MeOH): λmax 322 nm (
´
ε
17,500 cm
9.30 (1H, bs, NH), 8.63 (1H, s, H4), 8.26 (1H, s, CH), 7.81 (1H, d, J = 9.0 Hz, H5),
.40–7.20 (5H, m, ArH), 7.00 (1H, d, J = 2.4 Hz, H8), 6.98 (1H, dd, J = 2.4, 9.0 Hz, H6), 4.47 (1H, t,
´1
M
´1
), 350 (16,800);
1
H-NMR (CDCl3):
δ
7
13
J = 7.2 Hz, CH ), 3,99 (3H, s, OMe), 3.37 (1H, t, J = 7.2 Hz, CH ); C-NMR (CDCl3):
δ 172.65 (C=O),
2
2
1
1
62.59 (C2), 158.59 (aryl C), 150.04 (aryl C), 149.47 (C7), 139.40 (CH), 135.26 (C4), 129.75 (C5), 129.52,
29.44 (aryl CH) and 123.02 (aryl C), 122.31 (C3), 121.15 (aryl CH), 121.00 (C6), 114.64 (aryl CH), 106.61
+
(C8), 63.22 (CH2), 55.76 (OMe), 32.87 (CH ) HRMS (ESI): C H ClN O [M + H] requires 384.1115,
2
21 21
2
3
found 384.1105.
1
E)-N -((2-chloro-5,7-dimethoxyquinolin-3-yl)methylene)-3-(phenylthio)propanehydrazide (17). Yellow solid,
(
˝
yield: 63%; log P: 4.54; m.p. 168–170 C; IR (KBr): νmax 3174, 3074, 2940, 1666, 1612, 1585, 1566, 1497,
1
479, 1448, 1398, 1367, 1346, 1275, 1239 cm 1; UV-vis (MeOH): λmax 313 nm (
´
ε
8,700 cm
´1
M ), 356
´1
1
(8,000); H-NMR (CDCl ):
δ
8.80 (1H, s, CH), 8.58 (1H, bs, NH) 8.44 (1H, d, J = 8.1 Hz, H6), 8.41 (1H, d,
3
J = 8.1 Hz, H8), 7.45–7.42 (2H, m, ArH), 7.32–7.26 (3H, m, ArH), 6.70 (1H, s, H4), 4.09 and 4.00 (each 3H,
13
s, OMe), 3.41 (2H, t, J = 7.2 Hz, CH ), 3.17 (2H, t, J = 7.2 Hz„ CH ); C-NMR (CDCl3):
δ 172.89 (C=O),
2
2
1
1
60.85 (C7), 157.56 (C5), 152.15 (C2), 148.65 (aryl C), 139.93 (CH), 135.92 (aryl C) 134.01 (C4), 129.47,
29.21, 128.28 (aryl CH), 125.59 (aryl CH), 119.56 (C3), 114.36 (aryl C), 108.54 (C6), 93.74 (C8), 56.65

Molecules 2016, 21, 916
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+
(
OMe), 56.01 (OMe), 33.65 (CH ) and 28.44(CH )HRMS (ESI): C H ClN O SNa [M + Na] requires
2 2 21 20 3 3
4
52.0812, found 452.0806.
1
E)-N -((2-Chloro-7-methoxyquinolin-3-yl)methylene)-6-(phenylthio)hexanehydrazide (18). Yellow solid,
(
˝
yield: 68%; log P: 5.78; m.p. 138–140 C; IR (KBr): νmax 3202, 3052, 2928, 1669, 1622, 1597, 1566,
1
3
550, 1491, 1479, 1465, 1436, 1363, 1334, 1260 cm 1; UV-vis (MeOH): λmax 322 nm (
´
ε
18,100 cm
´1
M ),
´1
1
52 (16,900); H-NMR (CDCl ):
δ
9.18 (1H, bs, NH), 8.63 (1H, s, H4), 8.22 (1H, s, CH), 7.81 (1H, d,
3
J = 9.0 Hz, H5), 7.40–7.20 (5H, m, ArH), 7.19 (1H, d, J = 2.4 Hz, H8), 7.18 (1H, dd, J = 2.4, 9.0 Hz, H6),
,99 (3H, s, OMe), 2.99 (2H, t, J = 7.6 Hz, CH ), 2.84 (2H, t, J = 7.6 Hz, CH ), 1.85–1.73 (4H, m, CH -CH ),
3
1
1
2
2
2
2
.67–1.60 (4H, m, CH2); 13C-NMR (CDCl3):
38.79 (CH), 136.76 (C4), 135.10 (aryl C), 129.42 (C5), 129.06, 128.94, 128.86, 125.77 (aryl CH), 123.23
δ 175.42 (C=O), 162.52 (C2), 149.96 (aryl C), 149.46 (C7),
(
2
aryl C), 122.33 (C3), 121.10 (C6), 106.60 (C8), 55.76 (OMe), 33.46 (CH2), 32.50 (CH ), 28.90 (CH ) 28.48,
2 2
8.28 and 24.08(CH ) HRMS (ESI): C H ClN O SNa [M + Na] requires 464.1163, found 464.1170.
2 23 24 3 2
+
1
(
6
1
3
E)-N -((2-Chloro-7-methoxyquinolin-3-yl)methylene)-6-phenoxyhexanehydrazide (19). Yellow solid, yield:
˝
2%; log P: 5.21; m.p. 134–136 C; IR (KBr): νmax 3191, 3082, 2932, 1667, 1618, 1490, 1467, 1399,
337, 1286, 1232, 1164, 1127, 1105, 1081 cm 1; UV-vis (MeOH): λmax 323 nm (
50 (17,200); H-NMR (CDCl ):
´
ε
18,200 cm
´1
M ),
´1
1
δ
9.32 (1H, bs, NH), 8.63 (1H, s, H4), 8.24 (1H, s, CH), 7.79 (1H, d,
3
J = 9.0 Hz, H5), 7.35 (1H, d, J = 2.4 Hz, H8), 7.31–7.23 (5H, m, ArH), 6.92 (1H, dd, J = 2.4, 9.0 Hz,
H6), 4.02 (2H, t, J = 7.6 Hz, CH ), 3,99 (3H, s, OMe), 2.88 (2H, t, J = 7.6 Hz, CH ), 1.94–1.87 (4H, m,
2
2
13
CH -CH ), 1.67–1.65 (4H, m, CH ); C-NMR (CDCl3):
δ
175.54 (C=O), 162.50 (C2), 159.03 (aryl C),
2
2
2
1
49.95 (aryl C), 149.46 (C7), 138.79 (CH), 135.09 (C4), 129.43 ( C5) and 123.26 (aryl C), 122.33 (C3), 121.09
(
2
2
ˆ
aryl CH), 120.56 (C6), 114.47 (2
ˆ
aryl CH), 106.59 (C8), 67.61 (CH2), 55.75 (OMe), 32.87 (CH ),
2
+
9.15, 25.91 and 24.33 (CH ) HRMS (ESI): C H ClN O [M + H] requires 426.1575, found 426.1579.
2
23 25
3
3
(
4
7
E)-N'-(Naphthalen-2-ylmethylene)-3-(phenylthio)propanehydrazide (26). Yellow solid, yield: 72%; log P:
˝
.8; m.p. 166–168 C; IR (KBr): νmax 3325, 3276, 1686, 1550, 1501, 1441, 1284, 1220, 1237, 1002, 832, 719,
´
1
´1
´1
), 309 (36,100); H-NMR (CDCl ): δ 9.40
3
1
60 cm ; UV-vis (MeOH): λmax 300 nm (
ε
30,300 cm
M
(1H, s, CH), 7.94–7.87 (5H, m, ArH,), 7.58–7.55 (2H, m, H6 and H7 npthl.), 7.49–7.46 (2H, m, ArH),
7
.37–7.32 (2H, m, ArH). 7.28–7.25 (1H, m, ArH), 3.38 (2H, t, J = 7.2 Hz, CH ), 3.21 (2H, t, J = 7.2 Hz,
2
13
CH2); C-NMR (CDCl3):
δ 173.73 (C=O), 143.98 (CH), 135.84 (aryl C), 134.32 (aryl C), 133.12 (aryl C),
1
29.79 and 129.04 (2
ˆ
aryl CH), 128.70 (C2), 128.37 (C5), 127.93 (C8), 127.21 (C4), 126.75 (C1 and C3),
+
126.33 (C6 and C7), 122.71 (aryl CH), 32.97 (CH ) and 28.90 (CH ) HRMS (ESI): C H N OS [M + H]
2 2 20 19 2
requires 335.1218, found 335.1213.
(
3
1
9
E)-N
'-((1H-Indol-3-yl)methylene)-3-(phenylthio)propanehydrazide (27). Yellow solid, yield: 58%; log P:
˝
.35; m.p. 226–228 C; IR (KBr): νmax 3385, 3134, 3060, 2917, 1721, 1624, 1521, 1490, 1448,1438, 1390,
364, 1330, 1299, 1231 cm 1; UV-vis (MeOH): λmax 344 nm (
.04 (1H, bs, NH), 8.91 (1H, bs, NH), 8.88 (1H, s, CH), 8.48 (1H, s, H2), 8.45 (1H, dd, J = 0.9, 1.2 Hz,
´
´1
´1
), H-NMR (CDCl ): δ
3
1
ε
38,600 cm
M
H4), 8.29 (1H, dd J = 0.9, 1.2 Hz, H7), 7.51–7.41 (5H, m, ArH) 7.36–7.23 (2H, m, H5 and H6), 3.48 (2H, t,
1
3
J = 7.2 Hz, CH ), 3.32 (2H, t, J = 7.2 Hz, CH ); C-NMR (CDCl3):
δ
170.93 (C=O), 153.95 (CH), 137.74
aryl CH), 125.45 (aryl C), 126.34
CH), 121.78 (C4 and C6), 117.98 (C5), 112.57 (C7), 112.20 (C3) 36.80 (CH ) and 27.51 (CH ) HRMS
2
2
(
(
(
aryl C), 136.52 (aryl C), 131.06 (C2), 129.67 (2
ˆ
aryl CH), 128.59 (2
ˆ
2
2
+
ESI): C H N OS [M + H] requires 324.1171, found 324.1163.
18
19
3
(
1
1
E)-N'-Benzylidene-3-(phenylthio)propanehydrazide (28). Yellow solid, yield: 68%; log P: 3.81; m.p.
˝
04–106 C; IR (KBr): νmax 3176, 3089, 2965, 1667, 1607, 1583, 1571, 1481, 1438, 1423, 1356, 1315,
298, 1263 cm 1; UV-vis (MeOH): λmax 301 nm (
´
ε
11,000 cm
´1
M
´1
); H-NMR (CDCl ): δ 10.21
3
1
(
1H, bs, NH), 7.87 (1H, s, CH), 7.65–7.62 (2H, m ArH), 7.47 (1H, m, ArH), 7.45–7.42 (4H, m, ArH),
.36–7.24 (3H, m, ArH), 3.35 (2H, t, J = 7.2 Hz, CH ), 3.16 (2H, t, J = 7.2 Hz, CH ); C-NMR (CDCl3):
2 2
13
7
δ
174.37 (C=O), 144.40 (CH), 135.85 (aryl C), 133.70 (C1), 130.23 (C5), 129.78 (C2 and C3), 129.02
aryl CH), 126.31 (2 aryl CH), 32.97 and 28.97 (CH2)
HRMS (ESI): C H N OSNa [M + Na] requires 307.0881, found 307.0872.
(aryl C), 128.75 (C4 and C6), 127.26 (2
ˆ
ˆ
+
1
6
16
2

Molecules 2016, 21, 916
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3
.2. Cell Biology Techniques
The SH-SY5Y and Kelly human neuroblastoma cell lines were generously donated by
Dr. J. Biedler (Memorial Sloan-Kettering Cancer Center, New York, NY, USA). The MDA-MB-231
and MCF-7 breast cancer cell lines were purchased from the American Type Culture Collection. All
˝
cell lines were cultured under standard conditions at 37 C in 5% CO as an adherent monolayer in
2
Dulbecco’s modified Eagle’s medium supplemented with L-glutamine (DMEM) (Invitrogen, Waltham,
MA, USA) and 10% fetal calf serum (FCS) (Thermo Fisher Scientific, Waltham, MA, USA).
3
.2.1. Method for Cell Viability Assays
Cell viability was measured by the standard Alamar blue assay, as previously described [34].
Briefly, cells were allowed to attach for 24 h in 96-well culture plates. The cells were then continuously
exposed to serial dilutions of the hydrazide-hydrazone derivatives for 72 h, either in the presence
or absence of SAHA (0.5 or 1
µM), with five replicate wells for each determination. Cell viability
was determined by the addition of 22
µL of Alamar blue reagent, recorded at comparative 0 h
and 5 h values, using a Wallac 1420 Victor III spectrophotometer (GMI, Ramsey, MN, USA), which
measured light absorbance in each well at 570 nm. The cell viability of each plate was calculated as
a percentage compared to matched DMSO controls (0.5%). The mean (+/
independent experiments.
´SEM) is shown for three
3
.2.2. Cell Cycle Study by Propidium Iodide Staining
The cell cycle phase distribution of a cell population was determined by measuring cell DNA
content using flow cytometry with propidium iodide (PI) (Sigma Aldrich) staining. The cells were
6
trypsinized and resuspended at a concentration of 1
ˆ
10 in a complete medium, then pelleted by
cold centrifugation at 1200 r.p.m., rinsed with PBS and recollected as a cell pellet. The cells were then
resuspended in 0.1 mL cold PBS and were fixed in 80% cold ethanol by adding 1 mL on top of the
cell suspension and incubated at 4 C for 30 min. Prior to flow cytometric analysis, the cells were
˝
pelleted and the entire supernatant carefully discarded. The samples were treated with RNase A
(
Sigma, Sydney, NSW, Australia) for 30 min at room temperature and then stained with propidium
iodide (50 g/mL, final concentration) for 45 min at room temperature. The samples were analysed
using a BD Caliber Flow cytometer (BD, San Jose, CA, USA). The cell cycle analysis was performed
in FlowJo. Cell populations were divided into three subsets of cells that represented the sub G /G ,
µ
0
1
G /G phase, S phase and G /M (as determined from comparison with untreated cell control gating).
0
1
2
3
.2.3. Immunoblotting of Cell Cycle Regulatory Protein Expression
Whole-cell lysates were prepared in RIPA buffer as described [81]. 20
µg of lysates were
loaded onto Criterion Gels TRIS-HCl 10%–14% Polyacrylamide gels (Bio-Rad, NSW, Australia).
kip1
Antibodies used were p27
(
(1/500, BD), pRb (1/500, Cell Signaling, San Jose, CA, USA), E2F1
-tubulin (1/2000, Sigma-Aldrich, Sydney, NSW, Australia)
1/500, Zymed, Waltham, MA, USA), and
α
as loading control.
3
.3. Statistical Analysis
Results of the cell viability studies were statistically analyzed using the two-tailed, unpaired
Student’s t-test. Results are expressed as mean values with 95% confidence intervals.
4
. Conclusions
The quinoline-hydrazide-hydrazone motif was identified in a library screen as a promising lead
scaffold for anti-cancer activity against neuroblastoma cells. This work describes the synthesis of a
series of related quinoline hydrazide-hydrazones as well as three derivatives containing different
aromatic systems, via an EDC-mediated coupling. The methodology involved the simple reaction

Molecules 2016, 21, 916
14 of 19
of hydrazones with substituted carboxylic acids under EDC-mediated conditions. Preliminary SAR
investigations revealed that the presence of a bicyclic system comprised of 6-membered rings is
favoured for activity. Furthermore, the presence of methoxy groups was found to increase activity, as
was exchange of the use of an ether linkage, rather than a sulfane. The two most potent analogues
16 and 17 showed low micromolar potency against neuroblastoma cells. Furthermore, these two
compounds displayed significant selectivity for neuroblastoma cancer cells over normal human lung
fibroblast cells. It was also found that compound 17 induced upregulation of the cell cycle mediator
kip1
p27
, with a corresponding cell cycle arrest at G in SH-SY5Y neuroblastoma cells.
1
Acknowledgments: We thank the University of New South Wales, and the Australian Research Council (ARC)
for funding to N.K and D.StC.B. Mass spectrometric analysis for this work was carried out at the Bioanalytical
Mass Spectrometry Facility, UNSW and was supported in part by infrastructure funding from the New South
Wales Government as part of its co-investment in the National Collaborative Research Infrastructure Strategy.
This research was supported by Australian Postgraduate Research Scholarships, University of NSW, Program
Grants from the NHMRC Australia, Cancer Institute NSW Australia and Cancer Council NSW Australia. The
Children’s Cancer Institute Australia for Medical Research is affiliated with the University of NSW and Sydney
Children's Hospital.
Author Contributions: N.K., D.StC.B. and B.C. conceived and designed the experiments, G.M.A. provided the
data from library screening, M.B., S.S., O.T. and C.R.G. performed the experiments, analyzed and interpreted the
data, M.B. and C.R.G. wrote the paper, N.K. and G.M. supported financially.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
EDC
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
Histone deacetylase inhibitors
Suberoylanilide hydroxamic acid
Walter & Eliza Hall Institute
Dimethylformamide
HDACi
SAHA
WEHI
DMF
SD
Standart Deviation
DMSO
DMAP
KBr
Dimethylsulfoxide
Dimethoxyaminopyridine
Potassium Bromide
Et N
3
Triethylamine
PI
Propidium iodide
SEM
ORTEP
Standart Error of Mean
Oak Ridge thermal ellipsoid
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Sample Availability: Samples of the compounds 14, 15, 16, 17, 18, 19, 26, 27 and 28 are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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