Identification of N-acylhydrazone derivatives as novel lactate dehydrogenase A inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.06.028

Abstract Glycolysis is drastically increased in tumors and it is the main route to energy production with a minor use of oxidative phosphorylation. Among the key enzymes in the glycolytic process, LDH is emerging as one of the most interesting targets for the development of new inhibitors. In this context, in the present work, we carried out a virtual screening procedure followed by chemical modifications of the identified structures according to a "hit-to-lead" process. The effects of the new molecules were preliminary probed against purified human LDH-A. The compounds active at low micromolar level were additionally characterized for their activity on some cellular metabolic processes by using Raji human cell line. Within the series, 1 was considered the best candidate, and a more detailed characterization of its biological properties was performed. In Raji cells exposed to compound 1 we evidenced the occurrence of effects usually observed in cancer cells after LDH-A inhibition: reduced lactate production and NAD/NADH ratio, apoptosis. The flow cytometry analysis of treated cells also showed cell cycle changes compatible with effects exerted at the glycolytic level. Finally, in agreement with the data obtained with other inhibitors or by silencing LDH-A expression, compound 1 was found to increase Raji cells response to some commonly used chemotherapeutic agents. Taken together, all these finding are in support of the LDH-A inhibiting activity of compound 1.

Full text of DOI:10.1016/j.ejmech.2015.06.028

European Journal of Medicinal Chemistry 101 (2015) 63e70
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Identification of N-acylhydrazone derivatives as novel lactate
dehydrogenase A inhibitors
a
a
,
1
b
b
Sebastiano Rupiani , Rosa Buonfiglio , Marcella Manerba , Lorenza Di Ianni ,
b
c
a
c
Marina Vettraino , Elisa Giacomini , Matteo Masetti , Federico Falchi ,
Giuseppina Di Stefano , Marinella Roberti , Maurizio Recanatini a
b
a, *
a
Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via S. Giacomo 14, 40126 Bologna, Italy
D3 Computation, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova, Italy
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 January 2015
Received in revised form
Glycolysis is drastically increased in tumors and it is the main route to energy production with a minor
use of oxidative phosphorylation. Among the key enzymes in the glycolytic process, LDH is emerging as
one of the most interesting targets for the development of new inhibitors. In this context, in the present
work, we carried out a virtual screening procedure followed by chemical modifications of the identified
structures according to a “hit-to-lead” process. The effects of the new molecules were preliminary
probed against purified human LDH-A. The compounds active at low micromolar level were additionally
characterized for their activity on some cellular metabolic processes by using Raji human cell line. Within
the series, 1 was considered the best candidate, and a more detailed characterization of its biological
properties was performed. In Raji cells exposed to compound 1 we evidenced the occurrence of effects
usually observed in cancer cells after LDH-A inhibition: reduced lactate production and NAD/NADH ratio,
apoptosis. The flow cytometry analysis of treated cells also showed cell cycle changes compatible with
effects exerted at the glycolytic level. Finally, in agreement with the data obtained with other inhibitors
or by silencing LDH-A expression, compound 1 was found to increase Raji cells response to some
commonly used chemotherapeutic agents. Taken together, all these finding are in support of the LDH-A
inhibiting activity of compound 1.
27 May 2015
Accepted 10 June 2015
Available online 18 June 2015
Keywords:
Tumor metabolism
Glycolysis
LDH-A inhibitors
Virtual screening
N-acylhydrazone derivatives
Anticancer drug
©
2015 Elsevier Masson SAS. All rights reserved.
1
. Introduction
Lactate dehydrogenase (LDH) constitutes a key enzyme in
glycolysis, catalyzing the inter-conversion of pyruvate to lactate
with simultaneous oxidation of NADH to NAD , which is essential
þ
Reprogramming of cells energy metabolism is an emerging
hallmark of cancer [1]. Cancer cells, compared to their normal
counterparts, increase the rate of glucose uptake and mainly
metabolize it to lactate, as opposed to the more energy-efficient but
oxygen-dependent mitochondrial oxidative phosphorylation pro-
cess. This metabolic shift termed Warburg effect occurs even in the
presence of abundant oxygen levels, and provides ATP and sub-
strates for cell growth and division. Given the dependence of cancer
cells on glycolytic ATP, this peculiar metabolic feature could be
exploited for selective anticancer therapies [2,3].
to maintain the glycolytic flow. LDH is a homo- or hetero-
tetrameric enzyme consisting of two different subunits (A and B)
encoded by the highly related genes, ldh-a and ldh-b [4,5]. Actually,
the importance of the A isoform of the enzyme as a critical factor in
tumorigenesis is supported by a plethora of data, whereas con-
flicting results have been reported concerning the implications of
LDH-B [6,7], and its function in cancer cells has not yet been fully
elucidated. LDH-A expression is constantly up-regulated in tumors
and was found to correlate with tumor size and poor prognosis
[8e10]. In several tumor models, silencing LDH-A expression by
siRNA or shRNA was found to inhibit cell growth, migration and
in vivo tumorigenesis [11,12]. Inhibition of LDH-A was also found a
way to overcome the resistance of breast cancer cells to taxol and
trastuzumab [13,14]. A further interesting observation is that when
*
Corresponding author.
E-mail address: marinella.roberti@unibo.it (M. Roberti).
Present address: Chemistry Innovation Centre, Discovery Sciences, AstraZeneca
1
R&D M o€ lndal. SE-43183 M o€ lndal, Sweden.
http://dx.doi.org/10.1016/j.ejmech.2015.06.028
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

64
S. Rupiani et al. / European Journal of Medicinal Chemistry 101 (2015) 63e70
LDH-A expression was silenced in noncancerous cultured cells,
proliferation and protein synthesis were not impaired [15]. There-
fore, compounds which inhibit LDH-A enzymatic activity may
constitute safe agents able to hinder cancer cell metabolism and
growth without causing damage to normal tissues.
Several LDH-A inhibitors have been reported so far including the
natural product gossypol [16], its derivative FX-11 [17], the pyruvate
mimetic oxamate [18], the N-hydroxindole-derivatives [19,20].
More recently, novel inhibitors were developed through either
a fragment based approach by AstraZeneca [21] and Ariad Phar-
maceuticals [22], or screening by Genentech [23] and GSK [24].
Even though some of the reported compounds showed inhibitory
activity at low micromolar and nanomolar range, the authors
reported limited cellular activity or not suitable pharmacokinetic
properties. In this context, the identification of a new structure
showing both inhibitory activity and potential chemical manage-
ability can offer a further chance to be exploited.
We recently reported the identification of galloflavin (GF), a
novel LDH-A inhibitor selected by a Virtual Screening (VS)
campaign [25e27]. From preliminary tests, GF was found to inhibit,
at micromolar range, both the enzymatic activity of the purified
LDH and lactate production in cultured cells, proving good cell
permeability. Moreover, it was shown to be not harmful for mito-
chondrial respiration, suggesting tolerability for normal cell
metabolism. Disappointingly, both the poor solubility and chemical
tractability of GF did not allow the consequent optimization.
Herein, we describe the identification of novel LDH-A inhibitors,
which were selected by VS and chemically manipulated for
structure-activity relationship (SAR) studies.
deeper cavity where hydrophobic residues (e.g. Ile251 and Leu164)
are located. Similarly, the furan ring of 1e3 occupies the inner
lipophilic domain corresponding to the nicotinamide and ribose
binding site. Finally, the remaining portion of the molecules over-
laps with the ribose and phosphate NADH domain without making
interactions with the distal adenine pocket. In particular, the
hydrazone groups are involved in hydrogen bonds with Ala29,
Val30 and Gly96 and the terminal phenyl ring, nitrile group and
pyridine ring of 1, 2 and 3 respectively are located in a solvent
exposed pocket (see Fig. 1 for the detailed interactions).
Compounds 1e3 were then re-synthesized by us (Scheme 1) to
be tested on cultured cancer cells. As a model for these experi-
ments, we chose the Raji cell line, derived from a Burkitt's lym-
phoma and characterized by overexpression of the MYC protein.
This alteration, which drives the neoplastic change leading to
Burkitt's lymphoma, directly alters cell metabolism and causes
increased LDH-A levels, rendering cells very responsive to LDH-A
inhibition [26]. The results obtained with compounds 1e3 are re-
ported in Table 1. Compound 1 [32]exerted a marked effect on
lactate production in cells at the same concentration inhibiting
purified LDH-A (IC50, 42
penetration. Moreover, 1 inhibited cell growth with an IC50 of
38 M, whereas compound 2 [32,33] and 3 did not affect lactate
mM), thus showing a good capacity of cell
m
production and cell growth.
To explore the SAR of this class of compounds, a series of
N-acylhydrazone analogues was designed (4e16, Scheme 1,
Table 1). The docking study suggested that portion C engaged
similar interactions as the endogenous substrate and therefore the
carboxylic group was kept unchanged in most variants, while we
focused on modifications concerning portions A and B (addressing
the cofactor binding site).
2
. Virtual screening and hit identification
An Ensemble-Based Virtual Screening was performed, aimed at
3
. Chemistry
including LDH-A flexibility in the screening campaign. In particular,
we used a conformational ensemble consisting of three confor-
mations, which were sampled, validated and selected through a
computational protocol previously developed [28]. Three parallel
ligand docking runs were carried out with Schr o€ dinger suite as
The common synthetic strategy to achieve the desired com-
pounds 1e16 (Scheme 1), consisted in two main step: the Suzuki
reaction to couple the appropriate bromo-substituted heterocyclic
aldehydes 17a-e (portion B) and the suitable boronic acids 18a-
d (portion C) to obtain the bicyclic aldehydes 19a-i, that in the
second step underwent a condensation with the appropriate hy-
drazides 20a-f (portions A) affording the final N-acylhydrazones
described in the Supplementary data [29e31]. In particular, Glide
standard precision (SP) and extra precision (XP) were used to dock
appropriately filtered Asinex database. The first 10% of the docking
poses scored according to Glide SP were selected for a further study
with Glide XP. Subsequently, 1000 Glide XP best-ranked com-
pounds per conformation were clustered and visually inspected.
The most representative ligands for each cluster were taken into
account, and the conserved interactions with the catalytic residues,
along with the binding pockets (substrate and/or cofactor binding
sites as shown in Fig. S1) occupied by ligands were the main criteria
to guide the hits selection. Finally, based also on the commercial
availability, 67 molecules were chosen for biological tests. The most
frequent feature of these molecules was a carboxylic function,
mimicking the same functional group of the endogenous substrate.
In details, it interacted with the catalytic amino acids of the sub-
strate binding site, such as the Arg168, protonated His192 and
Arg105.
The selected compounds were purchased and tested on purified
human LDH-A as described in Supplementary data. Three of them
1e3, Table 1), showing a common N-acylhydrazone scaffold, were
found to cause enzyme inhibition at micromolar level. Fig. 1 shows
the binding mode of these three compounds as resulted from the
Glide XP docking run. As before mentioned, they enter the posi-
tively charged substrate binding pocket through the negative
carboxylate. Although 2 is characterized by the carboxylic group in
the para-position, this moiety is involved in electrostatic in-
teractions similarly to 1 and 3, whereas its aromatic ring reaches a
1
e16. Both reactions were carried out using sealed vessel micro-
wave heating. Not commercially available hydrazides 20c-f were
obtained by reaction of dihydrated NH NH with the corresponding
2
2
ethyl or methyl esters 21a-d, using sealed vessel microwave heat-
ing (Scheme 2). The synthesis of aldehyde 17c and esters 21b-c are
described in the Supplementary data.
4. Biology
Initially all the compounds were evaluated for their inhibitory
activity on purified human LDH-A. The compounds able to cause
enzyme inhibition at micromolar level (1e3, 6e12) and 4 that was
too fluorescent to be analyzed by fluorimetric method, were also
investigated for their activity on lactic acid production and cell
proliferation on Raji cell line. Further studies were performed on 1,
the most active compound of the series. LDH-A enzymatic assays
(see Supplementary data) allowed to calculate the inhibition con-
stants vs pyruvate and NADH. Moreover, 1 was evaluated on Raji
cells for its effect on NAD/NADH balance, apoptotic activity, cell
cycle progression and cell viability of Raji cultures and normal
lymphocytes. Finally compound 1 was evaluated for its potential in
combination tests with commonly used chemotherapeutic agents.
The detail biological protocols are reported in Supplementary data.
(

S. Rupiani et al. / European Journal of Medicinal Chemistry 101 (2015) 63e70
65
Table 1
Activity of N-acylhydrazone analogues 1e13, 15 on purified human LDH-A and on lactic acid production and cell proliferation on Raji cells.
Compound
hLDH-A
IC50
M)a
Lactate production
IC50
M)a
Cell growth
IC50
M)a
(
m
(
m
(m
1
2
37 ± 5
37 ± 6
42 ± 3
>200
38 ± 7
n.d.b
3
4
5
6
7
8
9
43 ± 5
n.d.b
>200
>200
n.d.b
>200
80 ± 9
n.d.b
>200
125 ± 7
32 ± 6
41 ± 11
46 ± 6
41 ± 11
38 ± 10
48 ± 3
>200
134 ± 27
52 ± 9
105 ± 17
>200
100 ± 10
48 ± 14
95 ± 27
>200
10
11
12
13
15
>200
45 ± 15
115 ± 14
64 ± 9
n.d.b
100 ± 30
115 ± 3
n.d.b
>200
n.d.b
n.d.b
a
All points were tested in triplicate with error bars indicating the standard deviations.
Not determined.
b
5
. Results and discussion
fluorimetric method. The dropped potency after introduction of 2,6
pyridine ring in compounds 5 and 13 suggested that 6-atom rings
may cause a different arrangement within the binding site. Consis-
tently with this hypothesis, the docking results showed that 5 and
13 were oriented in a different way compared to the active com-
pounds and fully occupied the cofactor binding site without reaching
the catalytic residues. The modification of portion C gave conflicting
results. Considering compound 1, the shift of the carboxylic function
from the m-to the p-position, as in derivative 6, induced a decrease of
activity. Differently, no relevant change in the activity was observed
between compound 2 showing a p-benzoic acid and the analogue
12 bearing a m-benzoic acid. Finally the esterification of compounds
1e2 afforded insoluble derivatives 14 and 16, while 15, the methyl
ester of compound 3, was inactive. Despite some evident effects on
the inhibitory activity, no clear SAR pattern could be identified
through the latter series. Considering the ability of 6e12 to inhibit
human LDH-A, we investigated their activity on lactic acid produc-
tion and cell proliferation on Raji cells (Table 1). Compound 7
showed the same cellular activity as the parent 1. On the contrary,
the other derivatives exhibited reduced lactate production and cell
growth activity.
Through a Virtual Screening procedure 67 molecules were
selected, purchased and tested on purified human LDH-A as
described in Supplementary data. Three of them (1e3, Table 1),
showing a common N-acylhydrazone scaffold, were found to cause
enzyme inhibition at micromolar level and were then re-
synthesized by us (Scheme 1) to be tested on cultured cancer
cells. To explore the SAR of this class of compounds, a series of N-
acylhydrazone analogues was designed (4e16, Scheme 1, Table 1).
Initially, we investigated the inhibitoryactivity of new derivatives
4e16 on purified human LDH-A (Table 1). Compounds 9e11, in
which the portion A is represented respectively by 3-hydroxymethyl
benzoyl, 5-1H indazolyl and 5-indolyl moieties, maintained a com-
parable activity to the parent compound 1. In terms of binding mode,
they preserved the interactions showed by compound 1 in the
substrate binding pocket, and reached the Asp51 in the cofactor
cavity. When portion B of 1 is replaced by 2,3 furan or 2,5 thiophene
rings as in compounds 7e8, respectively, once again no change in the
activity was observed, whereas the substitutionwith 2,4 pyrrole ring
present in 4, made this compound too fluorescent to be analyzed by

66
S. Rupiani et al. / European Journal of Medicinal Chemistry 101 (2015) 63e70
Fig. 1. Binding mode of compounds 1, 2 and 3 shown in the panel A, B and C respectively. Dotted lines represent hydrogen bonds.
On the basis of these results, further studies were only per-
compound 1 caused a small but statistically significant decrease of
formed on compound 1. LDH-A enzymatic assays (see
Supplementary data) allowed to calculate the inhibition constants
the cell fraction in S phase; on the contrary, cells in G2/M phase
resulted significantly increased. This latter result is in agreement
with published data obtained with oxamate [35] (a well studied
inhibitor of LDH) and dichloroacetate [36], a compound inhibiting
glycolysis and lactate production. A recent investigation concerning
the relationships between cell cycle progression and energy
metabolism in cancer cells showed that the ATP requirement for G1
and S phases is largely met by accelerated glycolysis, while the
energetic needs for G2/M phase are mainly derived from mito-
chondrial oxidative phosphorylation. On the basis of the published
reports [37], our flow cytometry data, indicating a reduction of the
cell population entering S phase and an increased fraction of G2/M
cells, are compatible with effects exerted at the glycolytic level and
can be a further evidence of the LDH-A inhibiting activity of com-
pound 1. To assess whether the effect of compound 1 was not only
limited to cell growth arrest, we measured by Western blot the
expression of apoptosis markers (Fig. 2C). The evaluated proteins
(BAX and BCL2) are well studied regulators of the mitochondrial
apoptosis pathway [38]. Moreover, increased BAX levels were
already found in cancer cells treated with oxamate [35] and other
LDH inhibitors [27]. As shown in Fig. 2C, an 18 h treatment with
(
Ki) vs pyruvate (39 mM) and NADH (47 mM) (Fig. S2). Further ex-
periments were addressed at verifying the occurrence of biological
effects usually observed in cancer cells after LDH-A inhibition.
These experiments, which are summarized in Fig. 2, were per-
formed on Raji cells after 18 h exposure to compound 1 at 40
This dose was chosen on the basis of the data reported on Table 1
50% inhibition on both lactate production and cell growth). The
mM.
(
major function of LDH-A is to assure the rapid reoxidation of NADH,
needed to sustain the glycolytic flux [34]. Fig. 2A shows that, in
agreement with previous results obtained with small molecules
LDH-A inhibitors [17,26], treatment with compound 1 reduced
þ
NAD regeneration and caused a statistically significant shifting of
the redox balance in favor of the reduced form of the dinucleotide.
The extent of the observed NADH increase (þ50%) fits well with the
effect caused by 40 mM compound 1 on lactate production in Raji
cells (50% inhibition). The effects caused by compound 1 on cell
cycle phases distribution were studied by propidium iodide stain-
ing of the treated cells and subsequent flow cytometry analysis
(Fig. 2B). This experiment showed that after an 18 h treatment,
ꢀ
Scheme 1. Synthesis of N-Acylhydrazone derivatives. Reagents and conditions: (a) compounds 19a-g: Pd(N,N-Dimethyl-alaninate)
2
, Na
2
CO
3
, EtOH/H
2
O, MW, 100 W, 120 C, 5 min,
ꢀ
ꢀ
yield 50e70%; compounds 19h-i: Pd(N,N-Dimethyl-alaninate)
yield 80e90%.
2 2 3
, Na CO , EtOH/H
2
O, MW, 50 W, 120 C, 10 min, yield 40e60%; (b) NH
2
NH
2
$2H
2
O, EtOH, MW, 130 W, 120 C, 45 min,

S. Rupiani et al. / European Journal of Medicinal Chemistry 101 (2015) 63e70
67
Table 2
Association compound 1 with chemotherapeutic agents.
Chemotherapeutic agent
Combination indexa
Cisplatin
1.00 ± 0.02
0.88 ± 0.15
0.86 ± 0.01
1.08 ± 0.01
Daunomycin
Etoposide
Sunitinib
a
Association experiments were repeated twice. A result ranging from 0.8
to 1.2 is indicative of additive effects.
Scheme 2. Synthesis of hydrazides. Reagents and conditions: (a) MeOH, MW, 100 W,
ꢀ
140 C, 60 min, yield 40e50%.
Supplementary data. The results are shown in Fig. 2D. No statisti-
cally significant (ANOVA) effect was found on normal cells viability
even at the dose of 200 mM.
Although preliminary, all the obtained results were in support of
the LDH inhibitory effect of compound 1; they also suggested a
good tolerability of the molecule on normal lymphocytes and its
capacity of improving the effects of commonly administered
chemotherapy.
compound 1 did not substantially alter the level of BCL2 protein,
but caused a 6.7-fold increase in BAX expression, denoting the in-
duction of cell death signaling. Interestingly, contrary to oxamate,
which was observed to trigger the mitochondrial apoptosis
pathway in cells after a prolonged period of exposition (48 h) [35],
the effects caused by compound 1 on Raji cultures appeared to
occur at earlier time. A further experiment with compound 1 was
aimed at evaluating its potential in combination tests with
commonly used chemotherapeutic agents. Compound 1 (40 mM)
6. Conclusion
was tested on Raji cells in combination with four anticancer drugs
usually employed in the therapy of hematological neoplasms. For
each drug, we previously determined the lowest dose level causing
statistically significant effects on cell viability at 24 h (see
Supplementary data). This dose was subsequently tested in asso-
ciation with compound 1 to calculate the combination index, ac-
cording to the procedure described in Supporting data. A result
ranging from 0.8 to 1.2 is indicative of additive effects (Table 2). As
already observed after LDH inhibition by oxamate or after LDH-A
silencing, compound 1 showed the potential of increasing the
therapeutic efficacy of commonly used chemotherapeutic agents.
Finally, we compared the effects on cells viability caused by com-
pound 1 on Raji cells and on normal lymphocytes, one of the cell
populations more susceptible to the adverse effects of anticancer
chemotherapy. The detailed experimental procedure is reported in
In continuation of our research for innovative antitumor lead
candidates, through a VS campaign followed by SAR studies, we
identified a new class of LDH-A inhibitors in a series of N-acylhy-
drazone derivatives. The new molecules were active at the micro-
molar range on purified LDH-A; notably 1 showed a marked effect
on lactate production in cells at the same concentration inhibiting
purified LDH-A. A more detailed characterization of its biological
properties confirmed 1 to be a suitable lead structure in the field of
LDH-A inhibitors. Noteworthy, from a medicinal chemistry point of
view, the N-acylhydrazone scaffold is a privileged structure, in
which the biological relevance meets the synthetic accessibility,
allowing to rapidly obtain variously substituted analogues, making
the follow-up studies of the identified hits more efficient.
7. Experimental section
7
7
.1. Chemistry
.1.1. General chemical methods
Reaction progress was monitored by TLC on pre-coated silica gel
plates (Kieselgel 60 F254, Merck) and visualized by UV254 light.
Flash column chromatography was performed on silica gel (particle
size 40e63 mM, Merck). If required, solvents were distilled prior to
use. All reagents were obtained from commercial sources and used
without further purification. When stated, reactions were carried
out under an inert atmosphere. Reactions involving microwave
irradiation were performed using a microwave synthesis system
(
CEM Discover® SP, 2.45 GHz, maximum power 300 W), equipped
with infrared temperature measurement. Compounds were named
relying on the naming algorithm developed by CambridgeSoft
1
13
Corporation and used in Chem-BioDraw Ultra 12.0. H NMR and
NMR spectra were recorded on Varian Gemini at 400 MHz and
00 MHz respectively. Chemical shifts ( ) are reported relative to
C
1
d
H
TMS as internal standard. Purity of compounds was determined by
elemental analyses; purity for all the tested compounds was ꢁ95%
(see Supplementary data).
7
.1.2. General procedure for the Suzuki coupling reaction to obtain
9a-g
To a solution of the appropriate bromo-substituted five or six
membered heterocyclic aldehydes 17a-e (1.0 mmol) in EtOH/H
:3 (tot 12 mL) in a 35 mL CEM microwave vessel, the correspon-
dent carboxyphenyl boronic acids 18a-b (1.2 mmol), Na CO 2M
1
Fig. 2. Experiments performed on Raji cell cultures treated with compound 1: A:
Evaluation of NAD/NADH balance; B: Analysis of cell cycle phases by flow cytometry; C:
Apoptosis evaluation; D: effect of compound 1 on cell viability of Raji cultures and
normal lymphocytes.
2
O
5
2
3

68
S. Rupiani et al. / European Journal of Medicinal Chemistry 101 (2015) 63e70
(
2.0 mmol) and Pd(N,N-Dimethyl
b
-alaninate)
2
[39] (5 mol%) were
product as a colored solid ranging from yellow to red color (yield
added. The vessel was capped and placed in a microwave reactor
and the reaction carried out with the following method in dynamic
mode: 120 C, 5 min, 100 W, with high stirring. After completion
40e60%) (Scheme 1, Table 1).
ꢀ
7.1.5.1. 3-(5-((2-(3,4,5-trimethoxybenzoyl)
hydrazono)methyl)
the vessel was allowed to cool to room temperature, HCl 2M was
added until pH turned acidic, and the mixture was extracted with
EtOAc (3 X 10 mL). The organic phase was collected, dried over
furan-2-yl) benzoic acid (1). The product was prepared using the
general procedure for the N-acylhydrazone formation starting from
1
19a and 20a (pale yellow solid, yield 73%). H NMR (DMSO-d6)
anhydrous Na
crude product was then purified via silica gel column chromatog-
raphy (CH Cl /MeOH elution gradient from a 100/0 ratio to a 90/10
ratio) to obtain the pure compounds (yield 50e70%) (Scheme 1).
2
SO
4
, and the solvent evaporated under vacuum. The
d 13.18 (bs, 1H), 11.74 (s, 1H), 8.45 (s, 1H), 8.33 (s, 1H), 8.01 (d,
J ¼ 7.6 Hz, 1H), 7.91 (d, J ¼ 7.6 Hz, 1H), 7.61 (t, J ¼ 7.6 Hz, 1H), 7.27 (d,
2
2
J ¼ 2.6 Hz, 1H), 7.24 (s, 2H), 7.09 (d, J ¼ 2.6 Hz, 1H), 3.87 (s, 6H), 3.77
13
(s, 3H) ppm; C NMR (400 MHz, DMSO-d6)
d 166.92, 162.57,
153.75, 152.73, 149.43, 140.50, 137.30, 131.66, 129.85, 129.49, 128.88,
7
.1.2.1. 3-(5-formylfuran-2-yl)benzoic acid 19a. The product was
128.44, 128.15, 124.36, 116.34, 109.21, 10.521, 60.15, 56.12 ppm; MS
þ
þ
þ
-
prepared using the general procedure starting from 17a and 18a
(ES): m/z 425 [Mþ H] , 447 [Mþ Na] , 463 [Mþ K] , 423 [M- H] .
Anal. Calcd. for C22 (%): C, 62.26; H, 4.75; N, 6.60. Found (%):
C, 62.16; H, 4.68; N, 6.62.
1
(
yield 70%). H NMR (DMSO-d6)
H), 8.06 (d, J ¼ 8.4 Hz, 1H), 8.00 (d, J ¼ 8.4 Hz, 1H), 7.88 (t,
J ¼ 8.4 Hz, 1H), 7.69 (d, J ¼ 3.7 Hz, 1H), 7.45 (d, J ¼ 3.7 Hz, 1H) ppm;
d
13.10 (bs,1H), 9.66 (s, 1H), 8.24 (s,
20 2 7
H N O
1
13
C NMR (DMSO-d6)
d
178.49, 167.11, 157.50, 152.30, 132.41, 130.61,
7.1.5.2. 4-(5-((2-(2-cyanoacetyl)hydrazono)methyl)furan-2-yl)ben-
zoic acid (2). The product was prepared using the general proce-
dure for the N-acylhydrazone formation starting from 19c and 20b
129.91, 129.55, 129.45, 125.75, 125.49, 109.87 ppm.
1
7
.1.3. General procedure for the Suzuki coupling reaction to obtain
9h-i
To a solution of the appropriate bromo-substituted heterocyclic
aldehydes 17a (1.0 mmol) in EtOH/H 0 5:3 (tot 12 mL) in a 35 mL
CEM microwave vessel, the correspondent boronic acids 18c-
d (1.2 mmol), Na CO 2M (2.0 mmol) and Pd(N,N-Dimethyl -ala-
ninate) (5 mol%) were added. The vessel was capped and placed in
(red solid, yield 55%). H NMR (DMSO-d6, mixture of two cisetrans
1
amide bond rotamers) d 13.04 (bs, 1H), 11.84 (s, 0.69H), 11.75 (s,
0.31H), 8.04-7.97 (m, 2H), 7.94-7.86 (m, 2H), 7.40 (d, J ¼ 3.6 Hz,
2
0.31H), 7.30 (d, J ¼ 3.6 Hz, 0.69H), 7.12 (d, J ¼ 3.6 Hz, 0.31H), 7.09 (d,
13
J ¼ 3.6 Hz, 0.69H), 4.22 (s, 1.38H), 3.83 (s, 0.62H) ppm; C NMR
2
3
b
(DMSO-d6) d 166.81, 164.75, 158.88, 153.89, 153.53, 149.43, 149.36,
2
137.14, 133.92, 133.06, 130.08, 130.03, 129.93, 124.18, 123.90, 123.81,
116.23, 115.99, 110.37, 40.14, 39.93, 39.72, 39.51, 39.31, 39.10, 38.89,
a microwave reactor and the reaction carried out with the following
method in dynamic mode: 120 C, 10 min, 50 W, with high stirring.
After completion the vessel was allowed to cool to room temper-
ature and the mixture was extracted with EtOAc (3 X 10 mL). The
ꢀ
þ
þ
24.88, 24.27 ppm; MS (ES): m/z 298 [Mþ H] , 320 [Mþ Na] , 336
þ
- -
[Mþ K] , 296 [M- H] , 332 [Mþ Cl] . Anal. Calcd. for C15
11 3 4
H N O (%):
C, 60.61; H, 3.73; N, 14.14. Found (%): C, 60.52; H, 3.60; N, 14.23.
2 4
organic phase was collected, dried over anhydrous Na SO , and the
solvent evaporated under vacuum. The crude product (containing a
small portion of the ethyl ester as a transesterification product) was
then purified via silica gel column chromatography (petroleum
ether/EtOAc elution gradient from a 90/10 ratio to a 80/20 ratio) to
obtain the pure compounds (yield 40e60%) (Scheme 1).
7.1.5.3. 3-(5-((2-nicotinoylhydrazono)methyl)furan-2-yl)benzoic
acid (3). The product was prepared using the general procedure for
the N-acylhydrazone formation starting from 19a and 20c (pale
1
yellow solid, yield 60%). H NMR (DMSO-d6)
d 13.22 (bs, 1H), 12.03
(s, 1H), 9.07 (s, 1H), 8.78 (d, J ¼ 4.2 Hz, 1H), 8.39 (s, 1H), 8.34 (s, 1H),
.26 (d, J ¼ 7.7 Hz,1H), 8.05 (d, J ¼ 7.7 Hz,1H), 7.92 (d, J ¼ 7.7 Hz,1H),
7.66e7.54 (m, 2H), 7.28 (d, J ¼ 3.3 Hz, 1H), 7.13 (d, J ¼ 3.3 Hz, 1H)
8
7.1.4. General procedure for the synthesis of hydrazides 20c-f
13
A solution of the appropriate ethyl or methyl esters 21a-
ppm; C NMR (DMSO-d6)
148.54, 137.84, 135.45, 131.68, 129.81, 129.52, 129.13, 128.95, 128.23,
124.40, 123.68, 116.84, 109.25 ppm. Anal. Calcd. for C18 (%):
d 166.92, 161.67, 153.94, 152.36, 149.25,
d (1.0 mmol) in methanol (5 mL) was prepared in a 10 mL CEM
microwave vessel. Hydrazine hydrate 50% (5.0 mmol) was added,
the vessel was capped and placed in a microwave reactor and the
13 3 4
H N O
C, 64.48; H, 3.91; N, 12.53. Found (%): C, 64.55; H, 3.84; N, 12.67.
reaction carried out with the following method in dynamic mode:
ꢀ
1
40 C, 60 min, 100 W, with high stirring. After completion the
7.1.5.4. 3-(4-((2-(3,4,5-trimethoxybenzoyl)hydrazono)methyl)-1H-
reaction mixture was transferred to a round bottom flask and the
solvent evaporated under reduced pressure. The crude product was
transferred to an Erlenmeyer flask and suspended in dichloro-
pyrrol-2-yl)benzoic acid (4). The product was prepared using the
general procedure for the N-acylhydrazone formation starting from
1
19b and 20a (gray solid, yield 72%). H NMR (DMSO-d6)
d
13.03 (bs,
ꢀ
methane, heated at 50 C for 5 min, rapidly vacuum filtered and
1H), 11.89 (s, 1H), 11.33 (s, 1H), 8.37 (s, 1H), 8.26 (s, 1H), 7.95 (d,
washed with the same solvent, to obtain the pure product (yield
J ¼ 7.8 Hz, 1H), 7.78 (d, J ¼ 7.8 Hz, 1H), 7.51 (t, J ¼ 7.8 Hz, 1H), 7.35 (s,
13
8
0e90%). When the product was found to be still not pure, the
1H), 7.22 (s, 2H), 6.94 (s, 1H), 3.86 (s, 6H), 3.72 (s, 3H) ppm; C NMR
purification procedure was repeated.
(DMSO-d6)
31.90, 129.56, 129.48, 128.58, 127.42, 124.67, 123.85, 121.30, 110.01,
105.51, 103.96, 60.54, 56.53 ppm. Anal. Calcd. for C22 (%): C,
62.41; H, 5.00; N, 9.92. Found (%): C, 62.29; H, 5.11; N, 9.80.
d 167.71, 162.38, 153.07, 144.76, 140.57, 132.86, 132.66,
1
7.1.5. General procedure for the synthesis of N-acylhydrazones
21 3 6
H N O
1
e16
A solution of the appropriate aldehydes 19a-i (1.0 mmol) in
ethanol (20 mL) was prepared in a 35 mL CEM microwave vessel.
The correspondent hydrazides 20a-f (1.0 mmol) were added, the
vessel was capped and placed in a microwave reactor and the re-
7.1.5.5. 3-(6-((2-(3,4,5-trimethoxybenzoyl)hydrazono)methyl)pyr-
idin-2-yl)benzoic acid (5). The product was prepared using the
general procedure for the N-acylhydrazone formation starting from
1
action carried out with the following method in dynamic mode:
19f and 20a (white solid, yield 66%). H NMR (DMSO-d6)
d
13.12 (bs,
ꢀ
1
20 C, 45 min, 130 W. After completion the vessel was allowed to
1H), 11.90 (s, 1H), 8.75 (s, 1H), 8.66 (s, 1H), 8.35 (d, J ¼ 7.9 Hz, 1H),
cool to room temperature and then placed in a refrigerator for 1 h.
The product precipitated from the cold reaction mixture was
collected by vacuum filtration and dried on filter. Purification was
achieved by recrystallization with methanol, yielding the pure
8.09 (d, J ¼ 7.9 Hz, 1H), 8.06e7.95 (m, 3H), 7.65 (t, J ¼ 7.9 Hz, 1H),
13
7.28 (s, 2H), 3.88 (s, 6H), 3.74 (s, 3H) ppm; C NMR (DMSO-d6)
d
167.18, 154.92, 153.25, 152.76, 148.22, 139.38, 138.36, 138.31,
130.64, 130.00, 129.23, 128.20, 127.41, 120.88, 119.03, 110.70, 109.61,

S. Rupiani et al. / European Journal of Medicinal Chemistry 101 (2015) 63e70
69
105.32, 60.17, 56.16, 30.69 ppm. Anal. Calcd. for C23
H
21
N
3
O
6
(%): C,
7.1.5.11. 3-(5-((2-(1H-indole-5-carbonyl)hydrazono)methyl)furan-2-
6
3.44; H, 4.86; N, 9.65. Found (%): C, 63.32; H, 4.76; N, 9.60.
yl)benzoic acid (11). The product was prepared using the general
procedure for the N-acylhydrazone formation starting from 19a
1
7
2
.1.5.6. 4-(5-((2-(3,4,5-trimethoxybenzoyl)hydrazono)methyl)furan-
-yl)benzoic acid (6). The product was prepared using the general
and 20f (orange solid, yield 62%). H NMR (DMSO-d6)
d
13.19 (bs,
1H), 11.79 (s, 1H), 11.41 (s, 1H), 8.44 (s, 1H), 8.34 (s, 1H), 8.22 (s, 1H),
8.05 (d, J ¼ 7.7 Hz,1H), 7.91 (d, J ¼ 7.7 Hz,1H), 7.70 (d, J ¼ 8.1 Hz,1H),
7.61 (t, J ¼ 8.1 Hz, 1H), 7.51 (s, 1H), 7.49e7.46 (m, 1H), 7.26 (d,
procedure for the N-acylhydrazone formation starting from 19c and
1
2
1
2
0a (orange solid, yield 66%). H NMR (DMSO-d6)
1.74 (s, 1H), 8.45 (s, 1H), 8.03 (d, J ¼ 8.1 Hz, 2H), 7.91 (d, J ¼ 8.1 Hz,
H), 7.33 (d, J ¼ 2.3 Hz, 1H), 7.24 (s, 2H), 7.12 (d, J ¼ 2.3 Hz, 1H), 3.87
d 12.94 (bs, 1H),
13
J ¼ 4.1 Hz, 1H), 7.05 (d, J ¼ 4.1 Hz, 1H), 6.59 (s, 1H) ppm; C NMR
(DMSO-d6) d 166.99, 164.16, 153.47, 149.79, 137.71, 136.16, 131.75,
13
(
s, 6H), 3.74 (s, 3H) ppm; C NMR (DMSO-d6)
54.12, 153.15, 150.41, 140.99, 137.69, 133.59, 130.54, 130.33, 128.84,
24.27, 116.65, 110.95, 110.02, 105.66, 60.58, 56.56 ppm. Anal. Calcd.
for C22 (%): C, 62.26; H, 4.75; N, 6.60. Found (%): C, 62.31; H,
d
167.26, 162.99,
129.92,128.72,128.19,128.00,127.03,124.55,124.29,120.49,120.33,
115.78, 110.95, 109.61, 109.06, 102.36, 102.14 ppm. Anal. Calcd. for
1
1
C H
21 15
N
3
O
4
(%): C, 67.56; H, 4.05; N, 11.25. Found (%): C, 67.61; H,
20 2 7
H N O
4.13; N, 11.16.
4
.67; N, 6.73.
7
.1.5.12. 3-(5-((2-(2-cyanoacetyl)hydrazono)methyl)furan-2-yl)ben-
7
3
.1.5.7. 3-(5-((2-(3,4,5-trimethoxybenzoyl)hydrazono)methyl)furan-
-yl)benzoic acid (7). The product was prepared using the general
zoic acid (12). The product was prepared using the general proce-
dure for the N-acylhydrazone formation starting from 19a and 20b
procedure for the N-acylhydrazone formation starting from 19d
and 20a (yellow solid, yield 43%). H NMR (DMSO-d6)
1
1
(orange solid, yield 74%). H NMR (DMSO-d6, mixture of two
1
d 13.98 (bs,
cisetrans amide bond rotamers)
d 13.12 (bs, 1H), 11.81 (s, 0.63H),
H), 11.75 (s, 1H), 8.49 (s, 1H), 8.39 (s, 1H), 8.19 (s, 1H), 7.92 (d,
1
1.72 (s, 0.37H), 8.27 (s, 1H), 8.01 (d, J ¼ 7.9 Hz, 1H), 7.91 (s, 1H), 7.89
J ¼ 8.1 Hz, 1H), 7.87 (d, J ¼ 8.1 Hz, 1H), 7.54 (t, J ¼ 8.1 Hz, 1H), 7.49 (s,
(
(
(
d, J ¼ 7.9 Hz, 1H), 7.58 (t, J ¼ 7.9 Hz, 1H), 7.24 (d, J ¼ 3.3 Hz, 1H), 7.08
13
1
d
H), 7.24 (s, 2H), 3.87 (s, 6H), 3.73 (s, 3H) ppm; C NMR (DMSO-d6)
167.34, 162.55, 152.76, 150.56, 141.96, 140.54, 137.19, 131.50,
d, J ¼ 3.3 Hz, 0.37H), 7.05 (d, J ¼ 3.3 Hz, 0.63H), 4.20 (s, 1.26H), 3.81
13
s, 0.74H) ppm; C NMR (DMSO-d6)
d
166.92,164.71,153.71,148.91,
1
5
6
29.79, 129.26, 128.38, 128.21, 127.15, 126.16, 111.84, 105.27, 60.19,
6.17 ppm. Anal. Calcd. for C22 (%): C, 62.26; H, 4.75; N,
.60. Found (%): C, 62.29; H, 4.80; N, 6.67.
1
1
6
37.29, 134.22, 131.67, 129.79, 129.53, 128.95, 128.12, 124.40, 116.23,
16.00, 109.18, 24.89, 24.28 ppm. Anal. Calcd. for C15 (%): C,
0.61; H, 3.73; N, 14.14. Found (%): C, 60.49; H, 3.82; N, 14.03.
20 2 7
H N O
11 3 4
H N O
7
.1.5.8. 3-(5-((2-(3,4,5-trimethoxybenzoyl)hydrazono)methyl)thio-
7
.1.5.13. 4-(6-((2-(2-cyanoacetyl)hydrazono)methyl)pyridin-2-yl)
phen-2-yl)benzoic acid (8). The product was prepared using the
general procedure for the N-acylhydrazone formation starting from
benzoic acid (13). The product was prepared using the general
procedure for the N-acylhydrazone formation starting from 19g
and 20b (white solid, yield 83%). H NMR (DMSO-d6, mixture of
two cisetrans amide bond rotamers)
0
3
1
19e and 20a (pale yellow solid, yield 68%). H NMR (DMSO-d6)
1
d
12.90 (bs, 1H), 11.74 (s, 1H), 8.68 (s, 1H), 8.22 (s, 1H), 7.99 (d,
d
13.08 (bs, 1H), 12.10 (s,
J ¼ 7.5 Hz, 1H), 7.91 (d, J ¼ 7.5 Hz, 1H), 7.66 (d, J ¼ 3.2 Hz, 1H), 7.59 (t,
.77H), 11.99 (s, 0.23H), 8.26 (s, 1H), 8.23 (d, J ¼ 7.9 Hz, 2H), 8.09 (m,
J ¼ 7.5 Hz, 1H), 7.51 (d, J ¼ 3.2 Hz, 1H), 7.23 (s, 2H), 3.87 (s, 3H), 3.73
1
3
H), 8.01 (d, J ¼ 7.9 Hz, 2H), 4.30 (s, 1.54H), 3.89 (s, 0.46H) ppm;
C
(
s, 2H) ppm; 13C NMR (DMSO-d6)
d 166.89, 162.48, 152.74, 144.25,
NMR (DMSO-d6)
d
167.49, 165.64, 155.24, 153.22, 145.09, 142.30,
1
1
C
4
42.68, 140.61, 138.97, 133.53, 132.16, 131.99, 129.72, 129.70, 128.96,
28.47, 125.96, 125.10, 105.34, 60.19, 56.19 ppm. Anal. Calcd. for
S (%): C, 59.99; H, 4.58; N, 6.36. Found (%): C, 60.04; H,
138.60, 131.72, 130.25, 127.14, 121.92, 119.72, 116.37, 109.99,
2
12 4 3
4.75 ppm. Anal. Calcd. for C16H N O (%): C, 62.33; H, 3.92; N,
22 20 2 6
H N O
.71; N, 6.26.
18.17. Found (%): C, 62.44; H, 3.87; N, 18.20.
7
.1.5.14. Methyl 3-(5-((2-(3,4,5-trimethoxybenzoyl)hydrazono)
7
.1.5.9. 3-(5-((2-(3-(hydroxymethyl)benzoyl)hydrazono)methyl)
methyl)furan-2-yl)benzoate (14). The product was prepared using
the general procedure for the N-acylhydrazone formation starting
from 19h and 20a (pale yellow solid, yield 68%). H NMR (DMSO-
furan-2-yl)benzoic acid (9). The product was prepared using the
general procedure for the N-acylhydrazone formation starting from
1
1
19a and 20d (yellow solid, yield 75%). H NMR (DMSO-d6)
d
13.26
d6)
.94 (d, J ¼ 7.6 Hz, 1H), 7.64 (t, J ¼ 7.8 Hz, 1H), 7.30 (d, J ¼ 3.3 Hz, 1H),
.24 (s, 2H), 7.10 (d, J ¼ 2.4 Hz, 1H), 3.92 (s, 3H), 3.87 (s, 6H), 3.74 (s,
d
11.72 (s, 1H), 8.46 (s, 1H), 8.33 (s, 1H), 8.08 (d, J ¼ 7.6 Hz, 1H),
(
2
bs, 1H), 11.91 (s, 1H), 8.67 (s, 1H), 8.42 (s, 1H), 8.35 (d, J ¼ 11.8 Hz,
7
7
H), 8.07 (dd, J ¼ 15.0, 8.0 Hz, 2H), 7.62 (dd, J ¼ 15.0, 8.0 Hz, 2H),
7.36 (d, J ¼ 2.9 Hz,1H), 7.27 (t, J ¼ 4.0 Hz,1H), 7.09 (d, J ¼ 2.9 Hz,1H),
5
d
13
13
3H) ppm; C NMR (DMSO-d6)
37.32, 131.28, 130.48, 129.97, 129.66, 128.69, 127.61, 123.96, 120.24,
16.34, 110.98, 109.41, 106.37, 105.21, 60.12, 56.90, 56.11, 55.61,
d 165.81, 159.99, 152.70, 149.49,
.36 (t, J ¼ 3.6 Hz, 1H), 4.59 (d, J ¼ 3.6 Hz, 2H); C NMR (DMSO-d6)
1
1
5
6
172.56, 168.30, 163.63, 154.80, 149.65, 143.43, 137.73, 133.58,
1
30.18, 129.72, 129.37, 129.30, 128.68, 126.31, 126.04, 124.88, 116.66,
09.06, 62.98, 21.60 ppm. Anal. Calcd. for C20 (%): C, 65.93;
H, 4.43; N, 7.69. Found (%): C, 65.84; H, 4.34; N, 7.80.
2.34 ppm. Anal. Calcd. for C23
H
22
N
2
O
7
(%): C, 63.01; H, 5.06; N,
1
16 2 5
H N O
.39. Found (%): C, 63.12; H, 5.00; N, 6.42.
7
2
.1.5.10. 3-(5-((2-(1H-indazole-5-carbonyl)hydrazono)methyl)furan-
-yl)benzoic acid (10). The product was prepared using the general
7.1.5.15. Methyl
3-(5-((2-nicotinoylhydrazono)methyl)furan-2-yl)
benzoate (15). The product was prepared using the general pro-
procedure for the N-acylhydrazone formation starting from 19a
cedure for the N-acylhydrazone formation starting from 19h and
1
1
and 20e (dark yellow solid, yield 67%). H NMR (DMSO-d6)
d
13.36
20c (white solid, yield 48%). H NMR (DMSO-d6)
d
12.03 (s, 1H),
(
1
bs, 1H), 13.26 (bs, 1H), 11.93 (s, 1H), 8.43 (s, 2H), 8.34 (s, 1H), 8.28 (s,
H), 8.05 (d, J ¼ 8.2 Hz, 1H), 7.91 (d, J ¼ 8.0 Hz, 2H), 7.66 (d,
9.07 (s, 1H), 8.78 (d, J ¼ 6.0 Hz, 1H), 8.40 (s, 1H), 8.33 (s, 1H), 8.26 (d,
J ¼ 7.8 Hz, 1H), 8.09 (d, J ¼ 7.8 Hz, 1H), 7.94 (d, J ¼ 8.2 Hz,1H), 7.65 (t,
J ¼ 8.2 Hz,1H), 7.61 (t, J ¼ 8.2 Hz,1H), 7.28 (d, J ¼ 2.8 Hz,1H), 7.08 (d,
J ¼ 8.2 Hz, 1H), 7.61e7.55 (m, 1H), 7.31 (d, J ¼ 3.5 Hz, 1H), 7.14 (d,
13
13
J ¼ 2.8 Hz, 1H) ppm; C NMR (DMSO-d6)
49.61, 141.10, 136.72, 134.89, 131.68, 129.89, 129.49, 128.83, 128.13,
25.64, 125.53, 124.32, 122.31, 121.22, 116.02, 110.20, 109.19 ppm.
Anal. Calcd. for C20 (%): C, 64.17; H, 3.77; N, 14.97. Found
%): C, 64.24; H, 3.72; N, 14.90.
d
166.94, 163.43, 153.61,
J ¼ 3.5 Hz, 1H), 3.92 (s, 3H) ppm; C NMR (DMSO-d6)
d 165.55,
1
1
164.23, 161.56, 153.82, 152.26, 149.29, 148.50, 137.89, 135.45, 130.50,
129.82, 129.04, 128.55, 128.48, 124.02, 123.72, 117.08, 109.57,
H
14
N
4
O
4
30.66 ppm. Anal. Calcd. for C19
15 3 4
H N O (%): C, 65.32; H, 4.33; N,
(
12.03. Found (%): C, 65.38; H, 4.45; N, 11.97.

70
S. Rupiani et al. / European Journal of Medicinal Chemistry 101 (2015) 63e70
7.1.5.16. Methyl 4-(5-((2-(2-cyanoacetyl)hydrazono)methyl)furan-2-
breast cancer, Breast Cancer Res. 10 (2008) R84.
[
[
19] C. Granchi, S. Roy, C. Giacomelli, M. Macchia, T. Tuccinardi, A. Martinelli,
M. Lanza, L. Betti, G. Giannaccini, A. Lucacchini, N. Funel, L.G. Leon,
E. Giovannetti, G.J. Peters, R. Palchaudhuri, E.C. Calvaresi, P.J. Hergenrother,
F. Minutolo, Discovery of N-hydroxyindole-based inhibitors of human lactate
dehydrogenase isoform A (LDH-A) as starvation agents against cancer cells,
J. Med. Chem. 54 (2011) 1599e1612.
20] C. Granchi, S. Roy, A. De Simone, I. Salvetti, T. Tuccinardi, A. Martinelli,
M. Macchia, M. Lanza, L. Betti, G. Giannaccini, A. Lucacchini, E. Giovannetti,
R. Sciarrillo, G.J. Peters, F. Minutolo, N-Hydroxyindole-based inhibitors of
lactate dehydrogenase against cancer cell proliferation, Eur. J. Med. Chem. 46
yl)benzoate (16). The product was prepared using the general
procedure for the N-acylhydrazone formation starting from 19i and
1
2
0b (orange solid, yield 53%). H NMR (DMSO-d6)
d
11.89 (s, 1H),
8
.42 (s, 1H), 8.05 (d, J ¼ 7.7 Hz, 2H), 7.95 (d, J ¼ 7.7 Hz, 2H), 7.35 (d,
J ¼ 3.8 Hz, 1H), 7.15 (d, J ¼ 3.8 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 2H) ppm;
1
3
C NMR (DMSO-d6)
32.65, 129.98, 125.11, 124.83, 124.03, 111.93, 110.75, 109.54, 52.27,
0.63 ppm. Anal. Calcd. for C16 (%): C, 61.73; H, 4.21; N,
3.50. Found (%): C, 61.65; H, 4.26; N, 13.56.
d 178.28, 165.73, 165.67, 156.58, 149.85, 133.43,
1
3
1
13 3 4
H N O
(2011) 5398e5407.
[21] R.A. Ward, C. Brassington, A.L. Breeze, A. Caputo, S. Critchlow, G. Davies,
L. Goodwin, G. Hassall, R. Greenwood, G.A. Holdgate, M. Mrosek, R.A.
Norman, S. Pearson, J. Tart, J.A. Tucker, M. Vogtherr, D. Whittaker, J.
Wingfield, J. Winter, K. Hudson, Design and synthesis of novel lactate
dehydrogenase A inhibitors by fragment-based lead generation, J. Med.
Chem. 55 3285e3306.
Acknowledgments
This work was supported by a PRIN2012 (prot. 2012ZN9YH)
Project Grant from MIUR, Italy.
[
22] A. Kohlmann, S.G. Zech, F. Li, T. Zhou, R.M. Squillace, L. Commodore,
M.T. Greenfield, X. Lu, D.P. Miller, W.S. Huang, J. Qi, R.M. Thomas, Y. Wang,
S. Zhang, R. Dodd, S. Liu, R. Xu, Y. Xu, J.J. Miret, V. Rivera, T. Clackson,
W.C. Shakespeare, X. Zhu, D.C. Dalgarno, Fragment growing and linking lead
to novel nanomolar lactate dehydrogenase inhibitors, J. Med. Chem. 56 (2013)
Appendix A. Supplementary data
1
023e1040.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.06.028.
[23] S. Labadie, P.S. Dragovich, J. Chen, B.P. Fauber, J. Boggs, L.B. Corson, C.Z. Ding,
C. Eigenbrot, H. Ge, Q. Ho, K.W. Lai, S. Ma, S. Malek, D. Peterson, H.E. Purkey,
K. Robarge, L. Salphati, S. Sideris, M. Ultsch, E. VanderPorten, B. Wei, Q. Xu,
I. Yen, Q. Yue, H. Zhang, X. Zhang, A. Zhou, Optimization of 3-Hydroxy-5-(2,6-
dichlorophenyl)-2-mercaptocyclohex-2-enones as potent inhibitors of
HumanLactate Dehydrogenase, Bioorg. Med. Chem. Lett. 25 (2015) 75e82
(and references therein).
[24] J. Billiard, J.B. Dennison, J. Briand, R.S. Annan, D. Chai, M. Colon, C.S. Dodson,
S.A. Gilbert, J. Greshock, J. Jing, H. Lu, J.E. McSurdy-Freed, L.A. Orband-Miller,
G.B. Mills, C.J. Quinn, J.L. Schneck, G.F. Scott, A.N. Shaw, G.M. Waitt, R.F. Wooster,
K.J. Duffy, Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and
reverse aerobic glycolysis in cancer cells, Cancer Metab. 1 (2013) 19.
[25] M. Manerba, M. Vettraino, L. Fiume, G. Di Stefano, A. Sartini, E. Giacomini,
R. Buonfiglio, M. Roberti, M. Recanatini, Galloflavin (CAS 568-80-9): a novel
inhibitor of lactate dehydrogenase, ChemMedChem 7 (2012) 311e317.
[26] M. Vettraino, M. Manerba, M. Govoni, G. Di Stefano, Galloflavin suppresses
lactate dehydrogenase activity and causes MYC downregulation in Burkitt
lymphoma cells through NAD/NADH-dependent inhibition of sirtuin-1,
Anticancer Drugs 24 (2013) 862e870.
[27] F. Farabegoli, M. Vettraino, M. Manerba, L. Fiume, M. Roberti, G. Di Ste-
fano, Galloflavin, a new lactate dehydrogenase inhibitor, induces the death
of human breast cancer cells with different glycolytic attitude by affecting
distinct signaling pathways, Eur. J. Pharm. Sci. 47 (2012) 729e738.
[28] R. Buonfiglio, M. Ferraro, F. Falchi, A. Cavalli, M. Masetti, M. Recanatini,
Collecting and assessing human lactate dehydrogenase-A conformations for
structure-based virtual screening, J. Chem. Inf. Model 53 (2013)
2792e2797.
[29] T.A. Halgren, R.B. Murphy, R.A. Friesner, H.S. Beard, L.L. Frye, W.T. Pollard,
J.L. Banks, Glide: a new approach for rapid, accurate docking and scoring. 2.
Enrichment factors in database screening, J. Med. Chem. 47 (2004) 1750e1759.
[30] R.A. Friesner, J.L. Banks, R.B. Murphy, T.A. Halgren, J.J. Klicic, D.T. Mainz,
M.P. Repasky, E.H. Knoll, M. Shelley, J.K. Perry, D.E. Shaw, P. Francis, P.S. Shenkin,
Glide: a new approach for rapid, accurate docking and scoring. 1. Method and
assessment of docking accuracy, J. Med. Chem. 47 (2004) 1739e1749.
[31] R.A. Friesner, R.B. Murphy, M.P. Repasky, L.L. Frye, J.R. Greenwood,
T.A. Halgren, P.C. Sanschagrin, D.T. Mainz, Extra precision glide: docking and
scoring incorporating a model of hydrophobic enclosure for protein-ligand
complexes, J. Med. Chem. 49 (2006) 6177e6196.
[32] N.R. Nouri, S.D. Compounds with Hiv-iintegrase inhibitory activity and
use thereof as anti-Hiv/Aids Therapeutics, U.S. Pat. Appl. 20090088420,
2009.
[33] D.S. Goldfarb, Method for altering The Lifespan Of Eukaryotic Organisms: U.S.
Pat. Appl. 20090163545, 2009.
[34] L. Fiume, M. Manerba, M. Vettraino, G. Di Stefano, Inhibition of lactate de-
hydrogenase activity as an approach to cancer therapy, Future Med. Chem. 6
(2014) 429e445.
[35] X. Zhai, Y. Yang, J. Wan, R. Zhu, Y. Wu, Inhibition of LDH-A by oxamate induces
G2/M arrest, apoptosis and increases radiosensitivity in nasopharyngeal car-
cinoma cells, Oncol. Rep. 30 (2013) 2983e2991.
[36] B.M. Madhok, S. Yeluri, S.L. Perry, T.A. Hughes, D.G. Jayne, Dichloroacetate
induces apoptosis and cell-cycle arrest in colorectal cancer cells, Br. J. Cancer
102 (2010) 1746e1752.
[37] Y. Bao, K. Mukai, T. Hishiki, A. Kubo, M. Ohmura, Y. Sugiura, T. Matsuura,
Y. Nagahata, N. Hayakawa, T. Yamamoto, R. Fukuda, H. Saya, M. Suematsu,
Y.A. Minamishima, Energy management by enhanced glycolysis in G1-phase in
humancoloncancercells invitroand invivo, Mol. CancerRes.11 (2013) 973e985.
[38] A. Chalah, R. Khosravi-Far, The mitochondrial death pathway, Adv. Exp. Med.
Biol. 615 (2008) 25e45.
References
[
[
[
1] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144
(
2011) 646e674.
2] G. Kroemer, J. Pouyssegur, Tumor cell metabolism: cancer's Achilles' heel,
Cancer Cell 13 (2008) 472e482.
3] R. Scatena, P. Bottoni, A. Pontoglio, L. Mastrototaro, B. Giardina, Glycolytic
enzyme inhibitors in cancer treatment, Expert Opin. Investig. Drugs 17 (2008)
1533e1545.
[
4] J.A. Read, V.J. Winter, C.M. Eszes, R.B. Sessions, R.L. Brady, Structural basis for
altered activity of M- and H-isozyme forms of human lactate dehydrogenase,
Proteins 43 (2001) 175e185.
[
[
5] J. Everse, N.O. Kaplan, Lactate dehydrogenases: structure and function, Adv.
Enzymol. Relat. Areas Mol. Biol. 37 (1973) 61e133.
6] M.L. McCleland, A.S. Adler, Y. Shang, T. Hunsaker, T. Truong, D. Peterson,
E. Torres, L. Li, B. Haley, J.P. Stephan, M. Belvin, G. Hatzivassiliou,
E.M. Blackwood, L. Corson, M. Evangelista, J. Zha, R. Firestein, An integrated
genomic screen identifies LDHB as an essential gene for triple-negative breast
cancer, Cancer Res. 72 (2012) 5812e5823.
[
7] N.J. Brown, S.E. Higham, B. Perunovic, M. Arafa, S. Balasubramanian, I. Rehman,
Lactate dehydrogenase-B is silenced by promoter methylation in a high fre-
quency of human breast cancers, PLoS One 8 (2013) e57697.
[
8] A.J. Levine, A.M. Puzio-Kuter, The control of the metabolic switch in cancers by
oncogenes and tumor suppressor genes, Science 330 (2010) 1340e1344.
9] R.J. DeBerardinis, J.J. Lum, G. Hatzivassiliou, C.B. Thompson, The biology of
cancer: metabolic reprogramming fuels cell growth and proliferation, Cell
Metab. 7 (2008) 11e20.
[
[
10] M.R. Buchakjian, S. Kornbluth, The engine driving the ship: metabolic steering
of cell proliferation and death, Nat. Rev. Mol. Cell. Biol. 11 (2010) 715e727.
11] Z.Y. Wang, T.Y. Loo, J.G. Shen, N. Wang, D.M. Wang, D.P. Yang, S.L. Mo,
X.Y. Guan, J.P. Chen, LDH-A silencing suppresses breast cancer tumorigenicity
through induction of oxidative stress mediated mitochondrial pathway
apoptosis, Breast Cancer Res. Treat. 131 (2012) 791e800.
[
[
[
12] Y. Rong, W. Wu, X. Ni, T. Kuang, D. Jin, D. Wang, W. Lou, Lactate dehydro-
genase A is overexpressed in pancreatic cancer and promotes the growth of
pancreatic cancer cells, Tumour Biol. 34 (2013) 1523e1530.
13] M. Zhou, Y. Zhao, Y. Ding, H. Liu, Z. Liu, O. Fodstad, A.I. Riker,
S. Kamarajugadda, J. Lu, L.B. Owen, S.P. Ledoux, M. Tan, Warburg effect in
chemosensitivity: targeting lactate dehydrogenase-A re-sensitizes taxol-
resistant cancer cells to taxol, Mol. Cancer 9 (2010) 33.
[
14] Y. Zhao, H. Liu, Z. Liu, Y. Ding, S.P. Ledoux, G.L. Wilson, R. Voellmy, Y. Lin,
W. Lin, R. Nahta, B. Liu, O. Fodstad, J. Chen, Y. Wu, J.E. Price, M. Tan, Over-
coming trastuzumab resistance in breast cancer by targeting dysregulated
glucose metabolism, Cancer Res. 71 (2011) 4585e4597.
[
15] D.W. Jeong, I.T. Cho, T.S. Kim, G.W. Bae, I.H. Kim, I.Y. Kim, Effects of lactate
dehydrogenase suppression and glycerol-3-phosphate dehydrogenase over-
expression on cellular metabolism, Mol. Cell. Biochem. 284 (2006) 1e8.
16] M.S. Gomez, R.C. Piper, L.A. Hunsaker, R.E. Royer, L.M. Deck, M.T. Makler,
D.L. Vander Jagt, Substrate and cofactor specificity and selective inhibition of
lactate dehydrogenase from the malarial parasite P. falciparum, Mol. Biochem.
Parasitol. 90 (1997) 235e246.
[
[
17] A. Le, C.R. Cooper, A.M. Gouw, R. Dinavahi, A. Maitra, L.M. Deck, R.E. Royer,
D.L. Vander Jagt, G.L. Semenza, C.V. Dang, Inhibition of lactate dehydrogenase
A induces oxidative stress and inhibits tumor progression, Proc. Natl. Acad.
Sci. U. S. A. 107 (2010) 2037e2042.
[39] X. Cui, T. Qin, J.-R. Wang, L. Liu, Q.-X. Guo, Pd(N,N-Dimethyl beta-alaninate)
2
[18] J.M. Thornburg, K.K. Nelson, B.F. Clem, A.N. Lane, S. Arumugam, A. Simmons,
J.W. Eaton, S. Telang, J. Chesney, Targeting aspartate aminotransferase in
as a high-turnover-number, phosphine-free catalyst for the suzuki reaction,
Synthesis 3 (2007) 393e399.