N-(1'-naphthyl)-3,4,5-trimethoxybenzohydrazide as microtubule destabilizer: Synthesis, cytotoxicity, inhibition of cell migration and in vivo activity against acute lymphoblastic leukemia

Article abstract of DOI:10.1016/j.ejmech.2015.02.041

Tubulin-interacting agents, like vinca alkaloid and taxanes, play a fundamental role in cancer chemotherapy, making cellular microtubules (MT), one of the few validated anticancer targets. Cancer resistance to classical MT inhibitors has motivated the development of novel molecules with increased efficacy and lower toxicity. Aiming at designing structurally-simple inhibitors of MT assembly, we synthesized a series of thirty-one 3,4,5-trimethoxy-hydrazones and twenty-five derivatives or analogs. Docking simulations suggested that a representative N-acylhydrazone could adopt an appropriate stereochemistry inside the colchicine-binding domain of tubulin. Several of these compounds showed anti-leukemia effects in the nanomolar concentration range. Interference with MT polymerization was validated by the compounds' ability to inhibit MT assembly at the biochemical and cellular level. Selective toxicity investigations done with the most potent compound, a 3,4,5-trimethoxy-hydrazone with a 1-naphthyl group, showed remarkably selective toxicity against leukemia cells in comparison with stimulated normal lymphocytes, and no acute toxicity in vivo. Finally, this molecule was as active as vincristine in a murine model of human acute lymphoblastic leukemia at a weekly dose of 1 mg/kg.

Full text of DOI:10.1016/j.ejmech.2015.02.041

Accepted Manuscript
N-(1'-naphthyl)-3,4,5-trimethoxybenzohydrazide as microtubule destabilizer:
synthesis, cytotoxicity, inhibition of cell migration and in vivo activity against acute
lymphoblastic leukemia
Lívia B. Salum, Alessandra Mascarello, Rafael R. Canevarolo, Wanessa F. Altei,
Angelo B.A. Laranjeira, Patrícia D. Neuenfeldt, Taisa R. Stumpf, Louise D. Chiaradia-
Delatorre, Laura L. Vollmer, Hikmat N. Daghestani, Carolina P. de Souza Melo, André
B. Silveira, Paulo C. Leal, Marisa J.S. Frederico, Leandro F. do Nascimento, Adair
R.S. Santos, Adriano D. Andricopulo, Billy W. Day, Rosendo A. Yunes, Andreas Vogt,
José A. Yunes, Ricardo J. Nunes
PII:
S0223-5234(15)00137-3
10.1016/j.ejmech.2015.02.041
EJMECH 7723
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 November 2014
Revised Date: 20 February 2015
Accepted Date: 21 February 2015
Please cite this article as: L.B. Salum, A. Mascarello, R.R. Canevarolo, W.F. Altei, A.B.A. Laranjeira,
P.D. Neuenfeldt, T.R. Stumpf, L.D. Chiaradia-Delatorre, L.L. Vollmer, H.N. Daghestani, C.P. de Souza
Melo, A.B. Silveira, P.C. Leal, M.J.S. Frederico, L.F. do Nascimento, A.R.S. Santos, A.D. Andricopulo,
B.W. Day, R.A. Yunes, A. Vogt, J.A. Yunes, R.J. Nunes, N-(1'-naphthyl)-3,4,5-trimethoxybenzohydrazide
as microtubule destabilizer: synthesis, cytotoxicity, inhibition of cell migration and in vivo activity
against acute lymphoblastic leukemia, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.02.041.
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Colchicine
N-acylhydrazone
6)
(
Control
N-acylhydrazone
12)
(
G /G1
0
G /M
2
S

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N-(1'-naphthyl)-3,4,5-trimethoxybenzohydrazide as microtubule
destabilizer: synthesis, cytotoxicity, inhibition of cell migration and in vivo activity
against acute lymphoblastic leukemia
1
#
2#
3
1
Lívia B. Salum , Alessandra Mascarello , Rafael R. Canevarolo , Wanessa F. Altei , Angelo B. A.
3
2
2
2
Laranjeira , Patrícia D. Neuenfeldt , Taisa R. Stumpf , Louise D. Chiaradia-Delatorre , Laura L.
4
5
3
3
2
Vollmer , Hikmat N. Daghestani , Carolina P. de Souza Melo , André B. Silveira , Paulo C. Leal ,
6
6
6
1
Marisa J. S. Frederico , Leandro F. do Nascimento , Adair R. S. Santos , Adriano D. Andricopulo ,
7
2
4,8
3,
2,
Billy W. Day , Rosendo A. Yunes , Andreas Vogt , José A. Yunes *, Ricardo J. Nunes *
1
2
3
4
5
6
Laboratório de Química Medicinal e Computacional, Universidade de São Paulo, São Carlos, SP 13560-590, Brasil;
Laboratório Estrutura e Atividade, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brasil;
Centro Infantil Boldrini, Campinas, SP 13083-210, Brasil;
Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA;
Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA;
Laboratório de Neurobiologia da Dor e Inflamação, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-
9
00, Brasil;
7
Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261, USA;
8
Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
#
These authors contributed equally to this work
Corresponding authors:
*
Ricardo José Nunes: nunesricardoj@gmail.com, phone number: +55 48 3721-9853.
José Andres Yunes: andres@boldrini.org.br, phone number: +55 19 3787-5070.
Keywords: acylhydrazones; colchicine; tubulin; microtubule assembly; cell migration; leukemia;
cancer.

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Abbreviations:
ABT-751, N-(2-(4-hydroxyphenylamino)pyridin-3-yl)-4-methoxybenzenesulfonamide;
CHNS,
elemental analysis; DAMA-colchicine, N-deacetyl-N-(2-mercaptoacetyl)-colchicine; DMEM,
Dulbecco's Modified Eagle Medium; DMSO, dimethylsulfoxide; DNA, deoxyribonucleic acid; FBS,
Fetal Bovine Serum; FITC, Fluorescein Isothiocyanate; HeLa, human cervical carcinoma cell line;
HL60, human promyelocytic leukemia cell line; IC , half maximal inhibitory concentration; i.p.,
5
0
intraperitoneal injection; IR, Infrared; Jurkat, human T-cell acute lymphoblastic leukemia cell line;
MDA-MB-231, breast cancer cell line; MDEC, Minimum Detectable Effective Concentration;
mRNA, messenger RNA; MT, Microtubules; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; NMR, Nuclear Magnetic Resonance; PDB, Protein Data Bank; PBMC,
peripheral blood mononuclear cells; REH, human precursor B-cell acute lymphoblastic leukemia cell
line; RPMI, Roswell Park Memorial Institute Medium; SD, Standard Deviation; TBAI;
tetrabutylammonium iodide.

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ABSTRACT
Tubulin-interacting agents, like vinca alkaloid and taxanes, play a fundamental role in cancer
chemotherapy, making cellular microtubules (MT), one of the few validated anticancer targets. Cancer
resistance to classical MT inhibitors has motivated the development of novel molecules with increased
efficacy and lower toxicity. Aiming at designing structurally-simple inhibitors of MT assembly, we
synthesized a series of thirty-one 3,4,5-trimethoxy-hydrazones and twenty-five derivatives or analogs.
Docking simulations suggested that a representative N-acylhydrazone could adopt an appropriate
stereochemistry inside the colchicine-binding domain of tubulin. Several of these compounds showed
anti-leukemia effects in the nanomolar concentration range. Interference with MT polymerization was
validated by the compounds’ ability to inhibit MT assembly at the biochemical and cellular level.
Selective toxicity investigations done with the most potent compound, a 3,4,5-trimethoxy-hydrazone
with a 1-naphthyl group, showed remarkably selective toxicity against leukemia cells in comparison
with stimulated normal lymphocytes, and no acute toxicity in vivo. Finally, this molecule was as
active as vincristine in a murine model of human acute lymphoblastic leukemia at a weekly dose of 1
mg/kg.

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1
. INTRODUCTION
Tubulin-interacting agents have played a fundamental role in cancer chemotherapy over the
past decades, making cellular microtubules (MT) a well validated anticancer target [1, 2]. Despite the
clinical success with taxoids (paclitaxel and docetaxel) and vinca alkaloids (vinblastine and
vincristine), the search for novel and structurally more simple modulators of the microtubule/tubulin
system has been strongly encouraged through the last several years due to drug resistance, adverse
side effects, synthetic complexity, scarceness and limited bioavailability [3, 4].
Colchicine (here compound 1) (Figure 1A) is a naturally occurring MT depolymerizing and
cytotoxic agent that, in spite of its central role in elucidating physical properties and biological
functions of microtubules, has not been used as a therapeutic alternative for the treatment of cancer
due to its high toxicity [5]. Recently, however, compounds targeting the colchicine-binding site have
attracted particular attention as anticancer agents [6]. The enormous interest in such molecules is
reflected in the fact that combretastatin A-4 (compound 2) analogs and N-(2-(4-
hydroxyphenylamino)pyridin-3-yl)-4-methoxybenzenesulfonamide (ABT-751, compound 3), both
structurally simple tubulin inhibitors with potent cytotoxic and antimitotic activities, are currently
under clinical investigation as potential new chemotherapeutic drugs [3, 4, 7, 8, 9]. In this context, our
group has previously synthesized a series of cytotoxic 3,4,5-trimethoxychalcones (e.g., compounds 4
and 5) capable of modulating MT assembly (Figure 1A) [10]. The significance of the 3,4,5-
trimethoxyphenyl motif for inhibition of tubulin assembly and antimitotic activity has been discussed
for this series of chalcone analogs [3, 10, 11, 12].
In this work, as a continuation of our research focused on finding novel structurally-simple
mitotic arresters that inhibit MT assembly, we have designed and synthesized a series of thirty-one
3
,4,5-trimethoxy-hydrazones (compounds 6-36) (Figure 1B) and assessed their cytotoxic properties
against leukemia cells. Like chalcones, N-acylhydrazones (6-36) and their derivatives/analogs
compounds 37-61) (Figure 2) proved to be attractive chemical scaffolds for the design of novel MT
inhibitors due to their chemical versatility, simplicity and efficient synthesis.
(

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2
2
. RESULTS AND DISCUSSION
.1. Synthesis
Thirty-one 3,4,5-trimethoxy-acylhydrazones (6-36) were prepared as shown in Figure 1B,
with yields varying between 25 and 95%. The N-acylhydrazones were obtained by condensation of the
suitable benzohydrazide and the appropriate aldehyde under reflux [11].
Subsequently, twenty five novel analogs or derivatives of compound 12 were synthesized
(Figure 2). The six acylhydrazones (37-42) and the analog 48 were obtained as described above [11].
Some derivatives of compound 12 were synthesized by direct cyclization of the 3,4,5-
trimethoxyhydrazones. The three 1,3,4-oxadiazoles (43-45) were prepared by cyclization of
hydrazones with acetic anhydride under reflux [14], with yields varying between 25-35%. The
thiazolidinones (53, 54 and 56-61) were obtained with yields from 60 to 92%, using mercaptoacetic
acid in toluene and the appropriate 3,4,5-trimethoxyphenyl-substrate, under reflux in Dean-Stark
apparatus [15].
Pyrazoline 55 was synthesized by the cyclization of chalcone A23 (previously prepared
according procedure described [16]) and phenylhydrazine in acetic acid [17], with yield of 83%.
Hydrazone 49 was synthesized using 3,4,5-trimethoxyhydrazide and naphthoyl chloride in the
presence of pyridine in toluene, under reflux [11], yielding 91%. Imides 46 and 47 were obtained from
3
,4,5-trimethoxyhydrazide and the corresponding anhydride in toluene, under reflux [18], with yields
of 76 and 78%, respectively. Urea 50 was obtained from 3,4,5-trimethoxyhydrazide and 1-isocyanato-
naphthalene in acetone, under magnetic stirring, at room temperature [19], with a yield of 84%.
Sulfonamides 51 was synthesized by the reaction of 3,4,5-trimethoxyaniline with N-
acetylbezenesulfonyl chloride [20] with a yield of 95% . The N-acetyl group in compound 51 was
hydrolyzed to give sulfonamide 52, in 81% yield.
Reagents used were commercially available (Sigma-Aldrich), except the 3,4,5-
trimethoxybenzohydrazide, which was prepared as previously described [21], with yield of 80%.
Among the synthesized compounds, twenty-six (6, 8, 13-19, 21, 23-25, 27-33 and 37-42) have been

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previously published by other authors [22, 23] and five (7, 9, 11, 12 and 20) by us [24]. Seven novel
compounds (10, 22, 26, 34 and 43-45) were recently patented by us [25] and thirteen are unpublished
until now (35, 36, 46-61).
1
13
All compounds were characterized by melting points, H and C NMR, including the already
1
13
published compounds (Supporting Information). Detailed spectral characterization ( H NMR,
C
NMR, IR and elemental analysis or mass spectrometry) for novel compounds is presented in the
Material and Methods section.
2
.2. Molecular modeling
Docking simulations were performed to explore possible binding modes of the proposed N-
acylhydrazones in the colchicine-binding domain. Previous molecular dynamic simulations on N-
acylhydrazone derivatives suggested that different conformational states might coexist in solution
[
13]. For this reason, four N-acylhydrazone conformers were modeled and investigated (Figure 3A).
The results suggested that one of the probable conformational states (Figure 3A, conformation “b”)
could adopt an appropriate stereochemistry inside the α/β intradimer interface, resembling the
conformation previously proposed for chalcone 4 [10].
As shown in Figure 3B, both N-acylhydrazone 6 and chalcone 4 overlapped well with
DAMA-colchicine in the tubulin cavity, with their 3,4,5-trimethoxyphenyl groups sharing the same
hydrophobic sub pocket. In addition, the carbonyl oxygen of compounds 4 and 6 similarly established
hydrogen bonds with Asp β251 and Leu β255 whereas van der Waals interactions were important for
the interaction of the aromatic B-rings, positioned at the opposite side of the binding cavity, in the
same region as the tropone ring of colchicine.
Compounds were synthesized to explore the relation with the activity of: i) the presence of the
3
,4,5-trimethoxyphenyl substituent on the A-ring, ii) the influence of the number and position of
substituents on the B-ring, and the replacement of the B-phenyl ring by different fused or heterocyclic
rings (compounds 6-36), iii) the importance of 3,4,5-trimethoxy moiety compared with
acylhydrazones non-substituted on A ring (compounds 37-42), iv) the modifications in the skeleton of

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,4,5-trimethoxyacylhydrazones (compounds 46-52), v) the effect of the direct cyclization of the
3
studied acylhydrazones, forming 1,3,4-oxadiazoles 43-45 and thiazolidinones 53-54, and vi) the
influence of other cyclic conformationally-restricted analogs on the biological activity (pyrazoline 55
and thiazolidinones 56-61).
2
2
.3. Biological Evaluation
.3.1. In vitro cell growth inhibitory activity
The 3,4,5-trimethoxyacylhydrazones synthesized were initially evaluated at three
concentrations (1, 0.1, and 0.01 µM) for their cytotoxic effects against REH and Jurkat human acute
lymphoblastic leukemia cell lines of B- and T-cell origins, respectively. As shown in Table 1 and
Supplementary Table 1, in this 3,4,5-trimethoxybenzohydrazone series (6-36), replacement of the
substituent on the B-ring significantly affected anti leukemic activity. Compound 6, which represents
a general backbone for this series of compounds (except for compounds 22 and 27, in which the
phenyl B-rings were replaced by the aromatic bioisosteres imidazole and thiophene), did not show
significant cytotoxic activity. In general, compounds having tri-substituted B-rings were not active
(compounds 9, 10, 18 and 19). Interestingly, several 3,4,5-trimethoxyhydrazones were more potent
than the best 3,4,5-trimethoxychalcones previously developed by our group [10].
Compounds presenting activity at the nanomolar concentration range (<40% survival at 0.1
µM) were selected for the determination of their half maximal inhibitory concentration (IC ) against
5
0
leukemia cells (Table 2). The 1-naphthalene derivative 12 was as potent as colchicine, showing IC50
values around 15 nM. Except for compounds 5, 35 and 36, all other derivatives also inhibited cell
proliferation in both cell lines at nanomolar concentrations, especially compounds 7 and 21, which
showed IC around 40 nM and 70 nM, respectively.
5
0
Based on the most active compound 12, we designed and synthesized twenty five additional
derivatives and analogs (37-61, Figure 2), which were assayed under the same conditions (Table 3).
While compounds containing the general backbone (E)-N’-benzylidene-benzohydrazide (phenyl group

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as A-ring) substituted at B-ring (37-42) did not show activity, the presence of the 3,4,5-trimethoxy
group as A-ring in the (E)-N’-benzylidene-benzohydrazide scaffold led to some very active
compounds (7, 12, 13-16 and 21), confirming the importance of the 3,4,5-trimethoxyphenyl motif for
the cytotoxic activity of the proposed analogs [10, 26, 27, 28]. The 1,3,4-oxadiazole derivatives (43-
4
5) were also not active against leukemia cells, presumably (by docking analysis) because they were
locked in a conformation that is not conducive for the interaction within the colchicine-binding site.
With respect to compounds resulting from direct modifications in the prototype 12, structures
4
6, 47, 48, 49 and 50 did not show antiproliferative activity. Only compound 51 was active against
REH and Jurkat cell lines, with IC values around 7 and 2 µM, respectively, being the best non-
5
0
hydrazide analog obtained in this work. Compound 51 differs from analogue 52 (non active) by the
presence of an acetyl group, which seems to contribute to the activity of this molecule.
Regarding analogues obtained by direct cyclization of acyl-hydrazones 12 and 13, both
thiazolidinones (compounds 54 and 53, respectively) showed activity against leukemia cells. The IC50
values obtained for 54 and 53, although ~500-fold higher than their synthetic precursors (12 and 13),
follow the same pattern, wherein the 1-naphthyl-substituted derivative (12 and 54) are more active
than the 2-naphthyl substituted (13 and 53).
Analogs obtained by cyclization of 3,4,5-trimethoxy-derivatives (compounds 55-61) showed
variable anti-leukemia activities, but at concentrations significantly higher (~1,000-fold) than
prototype 12. Among thiazolidinones with a methylene group between the 3,4,5-trimethoxyphenyl
and thiazo rings, compound 56 (which has a 1-naphthyl substituent) showed higher activity than 57
(which has one hydrogen from the 1-naphthyl group of 56 substituted by the 4-dimethylamino group).
When the methylene group was deleted from 56 and 57, a reverse pattern was observed, wherein
compound 59 (correspondent to 57) had higher activity than 58 (correspondent to 56). The same
profile was observed for compounds 60 and 61. The pyrazoline 55 was not active in the tests.
The analysis of the relation structure activity showed that the 3,4,5-trimethoxyphenyl group,
which is present also in colchicine and was found important for docking in a hydrophobic sub pocket
of the colchicine-cavity in tubulin (see above). The relationship between the other part of the molecule

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and its biological activity seems more complex and may depend on the polarity, distance and
conformation of the groups as exemplified by differences observed with the naphtyl group and same
substituent, as 4-dimethylamino and acetyl.
2
.3.2. Inhibition of tubulin assembly
To address whether N-acylhydrazones directly interact with tubulin, representative N-
acylhydrazones bearing the 3,4,5-trimethoxyphenyl substituent with high, low or no activity against
leukemia cell lines were tested for inhibition of MT assembly. As shown in Table 4, in agreement
with their nanomolar cytotoxic potencies, compounds 7, 12, 13, and 21 were also nanomolar
inhibitors of MT polymerization in vitro, exceeding the activity of colchicine 1 and of the most potent
previously described chalcone 4. In agreement with their lack of cytotoxic effects, compounds 18 (a
3
,4,5-trisubstitued analog), 20 (an extended non-branched side chain analog), and 22 (phenyl B-ring
replaced by a heterocyclic aromatic ring) did not inhibit MT polymerization at concentrations up to 10
µM. In general, in agreement with our docking studies, some B-mono-substituted analogs presented
increased activity against tubulin assembly (as for compounds 7, 8, 16 and 21), while tri-substituted
analogs had reduced potency (as for compounds 10 and 18), probably due to steric hindrance [14].
Steric factors may also explain the somewhat decreased activity of the binuclear compounds 13 and
1
5 (both with beta orientation) when compared to 12 (alpha orientation).
2
.3.3. Cellular microtubule perturbation, cell cycle arrest, and apoptosis induction
Microtubule-interacting agents characteristically provoke cell cycle arrest and cellular
microtubule perturbation [1, 10, 33]. To examine the phenotypic alterations in cells caused by the
selected N-acylhydrazones, multi-parameter high-content analyses were performed in HeLa cells. We
evaluated the effects of the 12 representative derivatives (6-8, 10, 12, 13, 15, 16, 18, 20-22) and
colchicine 1 on microtubule perturbation, apoptotic morphology, cell cycle arrest and histone H3
phosphorylation. Fluorescence micrographs of representative images of nuclei (blue), tubulin (green),
and phosphohistone H3 (red) staining are shown in Figure 4. In comparison to control (vehicle),

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colchicine treatment resulted in microtubule disorganization and increased numbers of phosphohistone
H3 positive cells. Similar results were obtained with equivalent concentrations of the most potent
derivative 12 and with high-nanomolar concentrations of the general backbone of this series
(compound 6). The mitotic arrest phenotype was then quantified by high-content analysis as
previously described [10] (Table 4). A representative experiment showing quantitative analysis of
IC values for cell loss and minimum detectable effective concentrations (MDEC) for mitotic arrest
5
0
from ten-point, two-fold concentration gradients is presented in Supplementary Figure 1.
Cellular toxicity, mitotic arrest, nuclear morphology and microtubule perturbation results were
all in accordance with the ability of these compounds to inhibit MT assembly in vitro (Table 4). Most
of the compounds promoted cell loss in the mid- to high-nanomolar range, either through detachment
or lysis. In agreement with the preliminary assessment of cytotoxicity against leukemia cells, (E)-N’-
benzylidene-3,4,5-trimethoxybenzohydrazides 7, 12, 13, 15, 16 and 21 were the most potent ones in
affecting cell density, and to a lesser extent compound 8 and the B-non-substituted analog 6.
Compounds 18, 20 and 22, which did not inhibit MT assembly, were not cytotoxic at concentrations
as high as 50 µM. Compounds having trisubstituted B-rings (10 and 18) were among the least active.
All tested agents, except for the inactive compounds 18, 20 and 22, showed chromatin condensation,
decreases in cellular tubulin staining, and increased numbers of phospho-histone H3 positive cells at
mid- to high nanomolar concentrations, consistent with mitotic arrest (Table 4). The most potent
agent (12) was equipotent to colchicine (1) and represented a substantial improvement over the
general backbone of this series (6) (Table 4).
Measurements of nuclear morphology and DNA content (not shown) after Hoechst 33342
counterstaining revealed the presence of condensed and fragmented nuclei, suggesting that the
cytotoxic effect promoted by compound 12 occurs by cell cycle arrest in G2/M and apoptosis (Table
4
and Supplementary Figure 1). This was confirmed by flow cytometry of DNA content and
annexin V staining in Jurkat cells (Figure 5), which documented that compound 12 is a strong inducer
of apoptosis.

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2
.3.4. Effects on cell migration
The identification of compounds with nanomolar cytotoxic potencies motivated further
evaluations of their ability to inhibit metastatic breast cancer cell migration. Colchicine (1) and
compounds 12 (the most potent among the evaluated compounds), 6 (backbone similar to 12, but
activity against leukemia at micromolar concentrations, Supplementary Table 1) and 37 (the general
backbone that did not show significant activity against leukemia cell), were selected as representative
compounds for such evaluation.
The breast cancer cell line MDA-MB-231, an estrogen-independent aggressive breast cancer
model, was used for the assays due to its high metastatic potential. Initially, the selected compounds
were evaluated in a wound healing assay [29, 30], which involves a migratory response of confluent
monolayer cells after mechanical injury. As expected, compound 37 demonstrated no ability to inhibit
the migration of MDA-MB-231 cells even at high concentrations (data not shown). In contrast,
representative N-acylhydrazones bearing the 3,4,5-trimethoxyphenyl group (6 and 12), efficiently
inhibited MDA-MB-231 migration at 1 µM (Table 5) and even lower concentrations (Figure 6).
Compound 12 showed inhibitory activity as strong as colchicine (1) in the scratch-wound assay.
The antimigratory activities of compounds 6 and 12 were confirmed in transwell cell culture
chambers [31, 32]. Again, data obtained from this assay indicated that compound 12 represented an
improvement over compound 6, with potency similar to colchicine (Table 5 and Supplementary
Figure 2).
2
.3.5. Selective cytotoxicity and in vivo acute toxicity
Selective targeting and low toxicity for normal host tissues are important requisites for novel
anticancer agents. Peripheral blood mononuclear cells from healthy donors and the leukemia cell lines
Jurkat and REH were treated with increasing doses of compound 12 in order to investigate the
selective cytotoxicity of compound 12 against cancer cells. As shown in Figure 7, compound 12 was
at least three orders of magnitude more toxic to leukemia cells than to normal lymphocytes,
documenting exquisite selectivity for malignant over nonmalignant cells.

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We have previously shown that acylhydrazones (compounds 7, 12, 13 and 24) are rapidly
absorbed after oral administration, as judged by their remarkable effect on glucose tolerance tests in
Wistar rats, at 10 mg/kg. Importantly, no tissue damage was recorded after 180 min of acylhydrazone
administration, as evaluated by serum lactate dehydrogenase levels (Frederico et al., 2012). In this
work, we extended the in vivo toxicity evaluation of acylhydrazones by performing acute toxicity tests
on mice. Groups of twelve mice (six of each gender) received a single oral dose of compound 12 (0.1
to 1,000 mg/kg). Mortality, behavioral changes, clinical signs, body weight (Supplementary Figure
3
), intake of food and drink (Supplementary Table 2) and macroscopic organ appearance at sacrifice
were measured for 14 days. No obvious acute toxic symptoms were observed, suggesting that the
estimated p.o. LD50 for compound 12 is higher than 1,000 mg/kg. Moreover, macroscopic
examination (general appearance of the organ, consistency, shape, color and weight) of heart, lung,
liver, kidney, spleen, stomach, salivary gland, adrenal gland, thymus, mesenteric lymph nodes and
brain revealed no pathological changes after compound 12 treatment (Supplementary Table 3 and 4).
2
.3.6. In vivo sensitivity of ALL xenografts to compound 12
To test the in vivo efficacy of compound 12 against leukemia, NOD/SCID mice were
transplanted with a patient-derived xenograft B-cell precursor ALL cells. Animals were weekly
monitored for the presence of ALL cells. Mice were randomized and treatment began when the ALL
percentage in the peripheral blood of half of the animals reached ≥0.5%. Compound 12 was well
tolerated by mice during the whole experiment. As shown in Figure 8A, weekly i.p. doses of 1 mg/kg
of compound 12 were able to significantly slow the progression of primary human ALL in
NOD/SCID mice, the effect being comparable to that obtained with vincristine (0.15 mg/kg/1x week).
At the end of four weeks, animals were sacrificed and the infiltration of ALL in different organs was
evaluated. Animals treated with compound 12 had lower infiltration of leukemic cells in both bone
marrow, spleen and liver compared to control. In general the results obtained with compound 12 were
similar to those with vincristine (Figure 8B).

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3
. CONCLUSIONS
We have designed and synthesized a series of N-acylhydrazones and analogs, and assessed
their potential as anti-leukemia agents. Some compounds showed activity at nanomolar concentrations
against acute lymphoblastic leukemia cell lines, a human cervical cancer cell line (HeLa), and
inhibited mobility of a breast cancer cell line (MDA-MB-231). The compounds induced mitotic arrest
in G2/M and cell death through apoptosis. The most potent agent (12) displayed only modest toxicity
toward normal T lymphocytes and in vivo model of acute toxicity. Importantly, compound 12 was as
active as vincristine in a murine model of human acute lymphoblastic leukemia. The proposed
mechanism of action of these compounds is interference with MT polymerization, as demonstrated by
their ability to inhibit in vitro MT assembly at concentrations similar to colchicine. An attractive
feature of these molecules is that they bind to tubulin at the colchicine binding site, distinct from the
binding sites of the vinca alkaloids and taxanes, but the same site as an antitumor molecule
(combretastatin A4 phosphate) in Phase 2 clinical studies for solid tumors. In sum, some of the N-
acylhydrazones described here are attractive lead compound for the design of novel microtubule
modulators that complement existing MT-targeted therapies.
4
4
4
. EXPERIMENTAL SECTION
.1. Synthesis
.1.1. General procedure for the synthesis and purification of 3,4,5-trimethoxybenzohydrazide [21]
The 3,4,5-trimethoxybenzohydrazide was obtained from a mixture of gallic acid (6 mmol),
dimethyl sulphate (28 mmol), anhydrous K CO (26 mmol) and tetrabutylammonium iodide (TBAI)
2
3
(0.1 g) in acetone (30 mL) [21]. The mixture was refluxed for 12 h, after the precipitate was filtered
and washed with acetone. The ester obtained (6 mmol) was treated with a solution of 99% N H (4.4
2
4
mmol) in methanol (20 mL) and refluxed for 5 h, and then kept at room temperature overnight. The

ACCEPTED MANUSCRIPT
solid obtained was filtered and recrystallized from hot methanol to afford the 3,4,5-
trimethoxybenzohydrazide (80%).
4
.1.2. General procedure for the synthesis and purification of (E)-N’-benzylidene-benzohydrazide (6-
4
2)
The hydrazones were synthesized from the 3,4,5-trimethoxybenzohydrazide (2 mmol) or
benzohydrazide (2 mmol) and the appropriate aldehydes (2 mmol) in methanol (15 mL) and refluxed
for 2 h. After cooling, the crude product was collected by filtration, washed and recrystallized from
hot ethanol to give white solids [11]. Hydrazones 10, 22, 26 and 34 are novel compounds, which were
recently patented by our group [25].
4
.1.3. General procedure for the synthesis and purification of oxadizoles derivatives (43-45)
The 1,3,4-oxadiazoles were prepared by cyclization of the previously obtained (E)-N’-
benzylidene-benzohydrazide with acetic anhydride under reflux for 3 h. After cooling, the crude
product was collected by filtration, washed and recrystallized from acetone/water to give white solids
[
14]. All 1,3,4-oxadiazoles synthesized in this work (43, 44 and 45) are novel compounds, which were
recently patented by our group [25].
4
.1.4. General procedure for the synthesis and purification of thiazolidinones (53, 54, 56-61)
The thiazolidinones 53 and 54 were synthesized by adding of 3,4,5-trimethoxyhydrazone (1
mmol) and mercaptoacetic acid (0.5 mL) in toluene (35 mL), under reflux with a Dean-Stark
apparatus for 16 h until complete removal of water from the system. The mixture was washed with
saturated solution of NaHCO (3 x 10 mL), the organic phase was dried with MgSO and the solvent
3
4
evaporated. The product was recrystallized from hexane [15]. The methods to obtain the compounds
6-61 were by one pot cycloaddition with respective 3,4,5-trimethoxyamine (1 mmol), 1-
naphtaldehyde or 4-dimethylamine-1-naphtaldehyde (1 mmol) and mercaptoacetic acid (3 mmol) in
5

ACCEPTED MANUSCRIPT
toluene (35 mL), following the same methods described above. These thiazolidinones were then
recrystallized from ethanol. All of them are novel compounds.
4
.1.5. General procedure for the synthesis and purification of pyrazoline (55)
The 1,3,5-triaryl-2-pyrazoline 55 were prepared by the reaction of the chalcone (E)-3-
(naphthalen-1-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (1 mmol) previously synthesized [16]
with phenylhydrazine (3 mmol), under reflux in acetic acid (10 mL) for 3 hours [17]. The crude
product was poured in an ice bath, being the precipitate filtered and recrystallized from ethanol to
form pyrazoline 55, a novel compound.
4
.1.6. General procedure for the synthesis and purification of imides (46-47)
The imides 46 and 47 were synthesized using the methodology proposed by Brosse et al. [18].
The reaction from 3,4,5-trimethoxyhydrazide (1 mmol) and the corresponding anhydride (1 mmol) in
0 mL of toluene was kept under reflux for 3 h. Further, the solution was cooled to 0 °C to precipitate
5
the product, which was recrystallized from hexane/ethyl acetate. The imides 46 and 47 are novel
compounds.
4
.1.7. General procedure for the synthesis and purification of compounds 48 and 49
The compound 48 was synthesized with the same methodology described for the N’-
benzylidene-benzohydrazide [11]. Compound 49 was synthesized using 3,4,5-trimethoxyhydrazide (1
mmol) and naphthoyl chloride (1 mmol) in the presence of pyridine (1 mmol) in toluene (50 mL),
under reflux for 2.5 h. The solution was cooled to 0 °C to precipitate the product, which was
recrystallized from hexane. Both compounds are original.
4
.1.8. General procedure for the synthesis and purification of a urea derivative (50)
The urea 50 was synthesized using 3,4,5-trimethoxyhydrazide (1 mmol) and 1-
isocyanatonaphthalene (1 mmol) in acetone (10 mL) at room temperature for 3 hours [19]. The crude

ACCEPTED MANUSCRIPT
product was poured in an ice bath, being the precipitate filtered and recrystallized in ethanol. The urea
derivative 50 is a novel compound.
4
.1.9. General procedure for the synthesis and purification of sulfonamides (51-52)
The sulfonamide 51 was synthesized by reaction of the 3,4,5-trimethoxyaniline (1 mmol) with
N-acetylbezenesulfonyl chloride (1 mmol) in dichlorometane (10 mL) and presence of pyridine
1mmol) [20]. Subsequently, the solvent was evaporated and the crude product was washed with water
(
to give N-(4-(N-(3,4,5-trimethoxyphenyl)sulfamoyl)phenyl)acetamide 51. The compound 52 was
obtained by controlled hydrolysis of acetyl group with hydrochloric acid 6M. Thereafter, the solution
was treated with charcoal to remove colored impurities. The last step was the reaction neutralization
with sodium bicarbonate solution, resulting in the precipitation of compound 52. The sulfonamides 51
and 52 are novel compounds.
4
.1.10. Physicochemical data on synthesized compounds
The purified compounds were obtained with yields ranging from 25% to 95%. Melting points
were determined with a Microquímica MGAPF-301 apparatus and are uncorrected. IR spectra were
1
13
recorded with an AbbBomen FTLA 2000 spectrometer on KBr disks. NMR ( H and C NMR) were
recorded on a Varian Oxford AS-400 (400 MHz) spectrometer, using tetramethylsilane as an internal
standard. Elemental analysis was carried out with a CHNS EA 1110. Percentages of C and H were in
agreement with the product formula (within + 0.4% of theoretical values to C). High resolution mass
data was collected on a micrOTOF-QII (Bruker Daltonics) electrospray/ion trap instrument in positive
and negative ion modes.
(E)-N'-(4-hydroxy-3-iodo-5-methoxybenzylidene)-3,4,5-trimethoxybenzohydrazide (10). White solid,
1
mp 247-248 ºC; yield 69%; H NMR (DMSO-d ) δ 3.71 (s, 3H, p-OCH ), 3.85 (s, 6H, m-OCH ), 3.86
6
3
3
(
s, 3H, m-OCH ), 7.20 (s, 2H, H2, H6), 7.32 (s, 1H, H6’), 7.59 (s, 1H, H2’), 8.29 (s, 1H, HC=N),
3
13
1
0.07 (1H, OH), 11.65 (s, 1H, NH). C NMR (DMSO-d ) δ 56.78 (m-OCH ), 60.81 (p-OCH ), 85.17
6 3 3

ACCEPTED MANUSCRIPT
(C3’), 105.84 (C2, C6), 109.72 (C6’), 128.28 (C1), 129.31 (C1’), 130.75 (C2’), 141.03 (C4), 147.35
-
1
(
C=N), 147.97 (C5’), 149.00 (C4’), 153.36 (C3, C5), 163.16 (C=O). IR λmax/cm 3382 (N-H), 1636,
228 (C=O), 1565 (C=N), 1290, 1045 (C-O), 2999, 2839, 1585, 1490, 1334, 1137, 997 (Ar) (KBr).
Anal. Calcd for C H IN O : C 44.46, H 3.94, N 5.76. Found: C 44.64, H 3.99, N 5.88.
1
1
8
19
2
6
(E)-N'-((1H-imidazol-5-yl)methylene)-3,4,5-trimethoxybenzohydrazide (22). White solid, mp 230-231
1
ºC; yield 75%; H NMR (DMSO-d ) δ 3.74 (s, 3H, p-OCH ), 3.88 (s, 6H, m-OCH ), 7.23 (s, 2H, H2,
6
3
3
H6), 7.53 (s, 1H, H2’), 7.66 (m, 1H, H4’), 8.02 (s, 1H, NH5’), 8.43 (s, 1H, HC=N), 11.37 (s, 1H, NH).
1
3
C NMR (DMSO-d ) δ 56.76 (m-OCH ), 60.81 (p-OCH ), 105.71 (C2, C6), 129.51 (C1), 131.94
6
3
3
-1
(
C1’), 132.16 (C2’), 135.37 (C4’), 145.45 (C=N), 153.35 (C3, C5), 162.29 (C=O). IR λmax/cm 3212
(N-H), 1623, 1234 (C=O), 1580 (C=N), 1280, 1054 (C-O), 2994, 2941, 2838, 1503, 1456, 1411, 1344,
1
125, 1006, 844 (Ar) (KBr). Anal. Calcd for C H N O : C 55.26, H 5.33, N 18.41. Found: C 55.35,
14 16 4 4
H 5.53, N 18.23.
(E)-N'-(2,6-dimethoxybenzylidene)-3,4,5-trimethoxybenzohydrazide (26). White solid, mp 245-246 ºC;
1
yield 93%; H NMR (DMSO-d ) δ 3.71 (s, 3H, p-OCH ), 3.79 (s, 6H, o-OCH ), 3.85 (s, 6H, m-
6
3
3
OCH ), 6.72 (d, J = 8.0 Hz, 2H, H3’, H5’), 7.23 (s, 2H, H2, H6), 7.34 (t, J = 8.0 Hz, 1H, H4’), 8.60 (s,
3
1
3
1
H, HC=N), 11.52 (s, 1H, NH). C NMR (DMSO-d ) δ 56.72 (m-OCH ), 56.78 (o-OCH ), 60.76 (p-
6 3 3
OCH ), 105.08 (C3’, C5’), 105.76 (C2, C6), 111.75 (C1’), 129.37 (C1), 131.87 (C4’), 140.88 (C4),
3
-
1
1
43.92 (C=N), 153.31 (C3, C5), 159.38 (C2’, C6’), 162.81 (C=O). IR λmax/cm 3186 (N-H), 1644,
240 (C=O), 1586 (C=N), 1258, 1068 (C-O), 3002, 2928, 2838, 1502, 1473, 1417, 1378, 1342, 1121,
007, 783 (Ar) (KBr). Anal. Calcd for C H N O : C 60.95, H 5.92, N 7.48. Found: C 60.56, H 6.10,
1
1
1
9
22
2
6
N 7.55.
(E)-N'-(4-chloro-3-(trifluoromethyl)benzylidene)-3,4,5-trimethoxybenzohydrazide (34). White solid,
1
mp 203-204 ºC; yield 83%; H NMR (DMSO-d ) δ 3.72 (s, 3H, p-OCH ), 3.85 (s, 6H, m-OCH ), 7.23
6
3
3
(s, 2H, H2, H6), 7.82 (d, J = 8.6 Hz, 1H, H5’), 8.02 (d, J = 8.6 Hz, 1H, H6’), 8.15 (s, 1H, H2’), 8.52

1
3
ACCEPTED MANUSCRIPT
s, 1H, HC=N), 11.96 (s, 1H, NH). C NMR (DMSO-d ) δ 56.65 (m-OCH ), 60.73 (p-OCH ), 105.96
6 3 3
(
(
(
C2, C6), 126.29 (C2’), 127.98 (C1’), 128.88 (C1), 132.31 (CF ), 132.91 (C4’), 133.01 (C5’), 134.82
3
-1
C6’), 141.27 (C4), 145.61 (C=N), 153.40 (C3, C5), 163.50 (C=O). IR λmax/cm 3182 (N-H), 1655,
1
242 (C=O), 1587 (C=N), 1269, 1039 (C-O), 3008, 2938, 2838, 1506, 1480, 1417, 1336, 1316, 1173,
121, 1006, 958, 666 (Ar) (KBr). Anal. Calcd for C H ClF N O : C 51.87, H 3.87, N 6.72. Found:
1
18
16
3
2
4
C 51.70, H 4.05, N 6.74.
(E)-3,4,5-trimethoxy-N’-(quinoxalin-6-yl-methylene)benzohydrazide (35). Yellow solid, m.p. 207-
1
2
08°C; yield 83%; H NMR (DMSO-d ) δ 3.75 (s, 3H, OCH ); 3.90 (s, 6H, OCH ); 7.29 (s, 2H, H2,
6 3 3
H6); 8.17 (d, J=8,0Hz, 1H, H2’); 8.34 (m, 2H, H9’, H10’); 8.70 (s, 1H, HC=N); 8.99 (d, J=8,0Hz, 2H,
1
3
H5’, H6’); 12.02 (s, 1H, NH). C NMR (DMSO-d ) δ 56.56 (2C, OCH ); 60.59 (1C, OCH ); 105.73
6
3
3
(C2, C6); 127.39 (C1); 128.70 (C10’); 129.44 (C1’); 130.29 (C2’, C9’); 136.62 (C4); 142.91 (C5’,
-1
C6’); 143.61 (C3’); 146.55 (C8’); 146.86 (C3, C5); 153.16 (CH); 163.22 (C=O). IR λmax/cm 3446
NH), 1663 (C=O), 1576 (C=N), 1233 (C-O), 2941, 2832, 1008, 913, 862, 767 (Ar) (KBr). Anal.
Calcd for C H N O : C 62.26; H 4.95; N 15.29. Exp.: C 62.80; H 5.16; N 15.21.
(
1
9
18
4
4
(E)-N’-[(4-(dimethylamino)naphtalen-1-yl)methylene]-3,4,5-trimethoxybenzohydrazide (36). Yellow
1
solid, m.p. 214-215°C; yield 89%; H NMR (DMSO-d ) δ 2.90 (s, 6H, CH ); 3.75 (s, 3H, OCH ); 3.90
6
3
3
(
s, 6H, OCH ); 7.17 (d, J=8Hz, 1H, H3’); 7.30 (s, 2H, H2, H6); 7.58-7.67 (m, 2H, H7’, H8’); 7.83 (d,
3
1
3
J=8,0Hz, H9’); 8.22 (d, J=8,0Hz, H6’); 8.97-9.00 (m, 2H, HC=N, H2’); 11.70 (s, 1H, NH). C NMR
(
DMSO-d ) δ 45.03 (2C, CH ); 56.56 (2C, OCH ); 60.58 (1C, OCH ); 105.59 (C2, C6); 113.78 (C3’);
6 3 3 3
1
23.91 (C1’); 125.23 (C6’); 125.42 (C7’); 125.77 (C8’, C2’); 127.51 (C1); 128.26 (C9’); 129.17
(C5’); 129.44 (C10’); 132.09 (C4); 140.80 (CH); 148.66 (C4’); 153.17 (C3, C5); 163.22 (C=O). IR
-1
λmax/cm 3454 (NH), 1663 (C=O), 1576 (C=N), 1233 (C-O), 2941, 2833, 1008, 862, 767 (Ar) (KBr).
Anal. Calcd for C H N O : C 67.80; H 6.18; N 10.31. Exp.: C 67.46; H 6.11; N 10.42.
2
3
25
3
4

ACCEPTED MANUSCRIPT
-(2-(3-bromo-4-acethyl-5-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-3(2H)-
1
1
yl)ethanone (43). White solid, mp 177-179 ºC; yield 30%; H NMR (DMSO-d ) δ 2.36 (s, 3H, CH ),
6
3
2
.38 (s, 3H, CH ), 3.84 (s, 3H, p-OCH ), 3.91 (s, 3H, m-OCH ), 3.92 (s, 6H, m-OCH ), 6.99 (s, 1H,
3 3 3 3
1
3
H2’), 7.06 (s, 1H, H6’), 7.11 (s, 2H, H2, H6), 7.17 (s, 1H, HC-N). C NMR (DMSO-d ) δ 20.68
6
(
CH ), 21.75 (CH ), 56.56 (m-OCH ), 61.25 (p-OCH ), 91.52 (C-N), 104.44 (C2, C6), 117.93 (C5’),
3 3 3 3
1
19.37 (C2’), 122.52 (C6’), 136.02 (C1’, C1), 139.24 (C4’), 141.49 (C4), 152.90 (C3’), 153.64 (C3,
-1
C5), 155.84 (C=N), 168.00 (C=O), 168.32 (C=O). IR λmax/cm 1766, 1238 (C=O), 1667, 1582 (C=N),
1
1
254, 1047 (C-O), 1177 (C-N), 3445 (OH), 1130, 621 (C-Br), 3004, 2941, 2838, 1466, 1416, 1366,
+
306, 1190, 998, 858 (Ar) (KBr). MS (ESI) m/z 523.07 (M + H) .
1
-(2-(4-acethyl-3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone
1
(
44). White solid, mp 170-172 ºC; yield 25%; H NMR (DMSO-d ) δ 2.37 (s, 3H, CH ), 2.38 (s, 3H,
6
3
CH ), 3.83 (s, 3H, p-OCH ), 3.91 (s, 3H, m-OCH ), 3.93 (s, 6H, m-OCH ), 6.98-7.07 (m, 3H, H2’,
3
3
3
3
1
3
H6’), 7.11 (s, 2H, H2, H6), 7.18 (s, 1H, HC-N), 7.45 (m, 1H, H5’); C NMR (DMSO-d ) δ 20.82
6
(
CH ), 21.54 (CH ), 56.27 (m-OCH ), 56.33 (m-OCH ), 61.02 (p-OCH ), 91.14 (C-N), 92.06 (C2’),
3 3 3 3 3
1
04.22 (C2, C6), 119.16 (C5’), 128.09 (C6’), 136.402 (C1’, C1), 141.24 (C4’), 141.96 (C4), 151.78
-1
(
C3’), 153.42 (C3, C5), 155.60 (C=N), 167.75 (C=O), 168.10 (C=O). IR λmax/cm 1767, 1243 (C=O),
1
1
4
665, 1581 (C=N), 1250, 1043 (C-O), 1177 (C-N), 3445 (OH), 1129, 644 (C-Br), 2967, 2945, 2838,
507, 1466, 1417, 1365, 1036, 1287, 1197, 1083, 997, 958, 861, 699 (Ar) (KBr). MS (ESI) m/z
+
45.14 (M + H) .
1
-(2-(naphthalen-1-yl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (45). White
1
solid, mp 189-192 ºC; yield 35%; H NMR (DMSO-d ) δ 2.47 (s, 3H, CH ), 3.87 (s, 9H, OCH ), 7.08
6
3
3
(s, 2H, H2, H6), 7.48 (t, J = 8.0 Hz, 1H, H3’), 7.55 (t, J = 8.0 Hz, 1H, H7’, H8’), 7.62 (t, J = 8.0 Hz,
1
3
1
H, H7’), 7.76 (m, 1H, H4’), 7.92 (d, J = 8.0 Hz, 1H, H6’), 8.22 (d, J = 8.0 Hz, 1H, H9’). C NMR
(DMSO-d ) δ 21.57 (CH ), 56.28 (m-OCH ), 60.98 (p-OCH ), 91.17 (C-N), 104.27 (C2, C6), 119.61
6 3 3 3
(C1), 123.04 (C2’), 125.07 (C9’), 125.20 (C3’), 126.05 (C7’), 126.95 (C8’), 128.95 (C4’), 130.51

ACCEPTED MANUSCRIPT
(C6’), 130.61 (C10’), 130.79 (C5’), 134.04 (C1’), 141.11 (C4), 153.29 (C3, C5), 155.83 (C=N),
-
1
1
2
68.26 (C=O). IR λmax/cm 1731, 1243 (C=O), 1669, 1587 (C=N), 1254, 1039 (C-O), 1124 (C-N),
997, 2941, 2827, 1509, 1465, 1416, 1369, 1332, 1191, 1006, 980, 847, 784, 699 (Ar) (KBr). MS
+
(
ESI) m/z 429.14 (M + Na) .
N-(1,3-dioxoisoindolin-2-yl)-3,4,5-trimethoxybenzamide (46). White solid, m.p. 198-199°C; yield
1
7
3%; H NMR (DMSO-d ) δ 3.74 (s, 3H, OCH ); 3.85 (s, 6H, OCH ); 7.30 (s, 2H, H2, H6); 7.95-8.02
6
3
3
1
3
(
m, 4H, H4’, H5’, H6’, H7’); 11.25 (s, 1H, NH). C NMR (DMSO-d ) δ 56.52 (2C, OCH ); 60.64
6 3
(
1C, OCH ); 105.72 (C2, C6); 124.34 (C4’, C7’); 126.02 (C1); 129.93 (C2’, C3’); 135.88 (C5’, C6’);
3
-
1
1
1
6
41.59 (C4); 153.28 (C3, C5); 165.15 (C=O); 165.89 (C=O). IR λmax/cm 3441 (NH), 1796 (C=O),
746 (C=O), 1657 (C=O), 1234 (C-O), 1002, 882, 707 (Ar) (KBr). Anal. Calcd for C H N O : C
1
8
16
2
6
0.67; H 4.53; N 7.86. Exp.: C 60.83; H 4.79; N 7.87.
N-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,4,5-trimethoxybenzamide (47). White solid, m.p. 146-
1
1
2
6
47°C; yield 78%; H NMR (DMSO-d ) δ 3.71 (s, 3H, OCH ); 3.83 (s, 6H, OCH ); 6.43-6.32 (dd,
6
3
3
1
3
H, H3’, H4’); 7.23 (s, 2H, H2, H6); 10.57 (s, 1H, NH). C NMR (DMSO-d ) δ 56.45 (2C, OCH );
6
3
0.56 (1C, OCH ); 105.48 (C2, C6); 127.43 (C4’); 127.66 (C3’); 133.38 (C1); 140.91 (C4); 153.10
3
-1
(
C3, C5); 163.69 (C=O); 165.06 (C=O); 167.39 (C=O). IR λmax/cm 3437 (NH), 1704 (C=O), 1663
(
C=O), 1590 (C=C), 1126 (C-O), 991, 851, 773 (Ar) (KBr). Anal. Calcd for C H N O : C 54.90; H
1
4
14
2
6
4
.61; N 9.15. Exp.: C 54.32; H 4.22; N 9.06.
1
N-benzoyl-3,4,5-trimethoxybenzohydrazide (48). White solid, m.p. 188-189°C; yield 88%; H NMR
(
DMSO-d ) δ 3.71 (s, 3H, OCH ); 3.83 (s, 6H, OCH ); 7.25 (s, 2H, H2, H6); 7.50-7.61 (m, 3H, H3’,
6
3
3
1
3
H4’, H5’); 7.91 (d, J=8,0Hz, H2’, H6’); 10.45 (s, 1H, NH); 10.49 (s, 1H, NH). C NMR (DMSO-d )
6
δ 56.45 (2C, OCH ); 60.57 (1C, OCH ); 105.39 (C2, C6); 127.88 (C2’, C6’); 128.92 (C3’, C5’);
3
3
1
32.66 (C1); 140.75 (C4’); 148.92 (C1’); 149.80 (C4); 152.84 (C3); 152.84 (C3); 164.96 (C=O);

-1
ACCEPTED MANUSCRIPT
1
65.58 (C=O). IR λmax/cm 3450 (NH), 1651 (C=O), 1588 (C=O), 1239 (C-O), 1002, 863, 708 (Ar)
(
KBr). Anal. Calcd for C H N O : C 61.81; H 5.49; N 8.48. Found: C 61.27; H 5.78; N 8.19.
1
7
18
2
5
1
N-(3,4,5-trimethoxybenzoil)-2-naphtohydrazide (49). White solid, m.p. 210-211°C; yield 91%; H
NMR (DMSO-d ) δ 3.75 (s, 3H, OCH ); 3.88 (s, 6H, OCH ); 7.34 (s, 2H, H2, H6); 7.63 (m, 2H, H5’,
6
3
3
H6’); 7.69 (d, J=8,0Hz, H10’); 8.01-8.10 (m, 3H, H4’, H7’, H9’); 8.43 (d, J=8,0Hz, H2’); 10.49 (s,
1
3
1
H, NH); 10.61 (s, 1H, NH). C NMR (DMSO-d ) δ 56.47 (2C, OCH ); 60.87 (1C, OCH ); 105.40
6 3 3
(C2, C6); 125.47 (C10’); 125.94 (C6’, C1); 126.90 (C2’); 127.33 (C7’); 127.85 (C5’); 128.66 (C9’);
1
1
28.69 (C4’); 130.38 (C8’); 130.73 (C1’); 133.31 (C3’); 133.54 (C4); 153.23 (C3, C5); 165.59 (C=O);
-1
68.58 (C=O). IR λmax/cm 3445 (NH), 1688 (C=O), 1644 (C=O), 1238 (C-O), 1008, 852, 776 (Ar)
(
KBr). Anal. Calcd for C H N O : C 66.31; H 5.30; N 7.36. Exp.: C 66.49; H 4.98; N 6.90.
21 20 2 5
N-(naphthalen-1-yl)-2-(3,4,5-trimethoxybenzoyl)hydrazinecarboxamide (50). White solid, m.p 225°C.
1
yield 84%; H NMR (DMSO-d ) δ 3.73 (s, 3H, 4-OCH ); 3.86 (s, 6H, 3-OCH , 5-OCH ); 7.30 (s, 2H,
6
3
3
3
13
H2, H6); 8.13-7.44 (m, 7H, naphthyl ring); 8.49 (s, 1H, NH); 8.95 (s, 1H, NH); 10.39 (s, 1H, NH). C
NMR (DMSO-d ) δ 56.4 (3-OCH3, 5-OCH ); 60.5 (4-OCH3); 105.5 (C2, C6); 122.4-134.7 (naphthyl
6
3
ring and C1); 140.8 (C4); 153.0 (C3, C5); 156.8 (C=O); 166,2 (C=O). HRMS (ESI+) m/z: calculated
+
for C H N O [M ]: 396.1554; found, 396.1554.
2
1
21
3
5
N-(4-(N-(3,4,5-trimethoxyphenyl)sulfamoyl)phenyl)acetamide (51). Gray solid, m.p. 191°C; yield
1
9
5%; H NMR (DMSO-d ) δ 2.06 (s, 3H, COCH ); 3.55 (s, 3H, 4-OCH ); 3.64 (s, 6H, 3-OCH , 5-
6
3
3
3
OCH ); 6,37 (s, 2H, H2, H6); 7.71 (s, 4H, H2’, H3’, H5’, H6’); 10.01 (s, 1H, NH); 10.32 (s, 1H, NH).
3
1
3
C NMR (DMSO-d ) δ 24.5 (COCH ); 56.1 (3-OCH , 5-OCH ); 60.4 (4-OCH ); 98.2 (C2, C6);
6
3
3
3
3
1
1
18.9 (C2’, C6’); 128.5 (C3’, C5’); 133.3 (C1); 134.2 (C4’); 134.4 (C1’); 143.4 (C4); 153.3 (C3, C5);
+
69.4 (C=O). HRMS (ESI+) m/z: calculated for C H N O S [M ]: 381.1115; found, 381.1114
1
7
20
2
6

1
ACCEPTED MANUSCRIPT
4
-amino-N-(3,4,5-trimethoxyphenyl)benzenesulfonamide (52). White solid, m.p. 173°C; yield 81%; H
NMR (DMSO-d ) δ 3.55 (s, 3H, 4-OCH ); 3.65 (s, 6H, 3-OCH , 5-OCH ); 6.37 (s, 2H, H2, H6); 6.56
6
3
3
3
1
3
(
d, 2H, H3’, H5’, J= 8.7 Hz); 7.43 (d, 2H, H2’, H6’, J= 8.7 Hz); 9.70 (s, 2H, NH). C NMR (CDCl )
3
δ 56.0 (3-OCH3, 5-OCH3); 60.4 (4-OCH3); 97.6 (C2, C6), 113.0 (C2’, C6’); 124.7 (C1); 129.3 (C3’,
+
C5’); 134.0 (C4’); 134.9 (C1’); 153.2 (C3, C5). HRMS (ESI+) m/z: calculated for C H N O S [M ]:
1
5
18
2
5
3
39.1009; found, 339.1015.
3
,4,5-trimethoxy-N-(2-(naphtalen-2-yl)-4-oxotiazolidin-3-yl)benzamide (53). White solid, m.p. 107-
1
1
09°C; yield 89%; H NMR (CDCl ) δ 3.67 (s, 6H, OCH ); 3.76 (s, 3H, OCH ); 3.74-3.80 (m, 1H,
3
3
3
CH ); 3.97-3.93 (m, 1H, CH ); 6.23 (s, 1H, CH); 6.81 (s, 2H, H2, H6); 7.95-7.18 (m, 7H, naphthyl);
2
2
1
3
9
.02 (br, 1H, NH). C NMR (CDCl ) δ 30.60 (CH ); 55.97 (OCH ); 60.69 (OCH ); 63.58 (CH);
3 2 3 3
1
04.55 (C2, C6); 124.25-133.94 (naphthyl); 141.30 (C4); 152.68 (C3, C5); 164.79 (C=O); 171.86
-1
(
C=O). IR λmax/cm 3252 (NH); 1706 (C=O tiazol.); 1670 (C=O); 1240 (C-O); 1127 (C-N); 1415,
1
337, 1003, 852, 806, 747 (Ar). Anal. Calcd for C H N O S: C 63.00; H 5.06; N 6.39; S 7.31. Exp.:
23 22 2 5
C 62.97; H 5.41; N 6.24; S 7.58.
3
,4,5-trimethoxy-N-(2-(naphtalen-1-yl)-4-oxotiazolidin-3-yl)benzamide (54). Yellow solid, m.p. 114-
1
1
16°C; Yield 92%; H NMR (CDCl ) δ 3.67 (s, 6H, OCH ); 3.76 (s, 3H, OCH ); 3.76-3.80 (m, 1H,
3 3 3
1
3
CH ); 3.93-3.97 (m, 1H, CH ); 6.80 (s, 2H, H2, H6); 7.91-7.16 (m, 7H, naftil); 9.45 (br, 1H, NH). C
2
2
NMR (CDCl ) δ 30.47 (CH ); 56.02 (OCH ); 60.69 (OCH ); 104.79 (C2, C6); 122.42 (C2’); 125.28
3
2
3
3
(
(
(
C8’); 125.38 (C7’); 125.45 (C9’); 125.47 (C4’); 126.15 (C3’); 128.20 (C1); 129.01 (C6’); 129.14
-1
C10’); 129.17 (C5’); 130.73 (C1’); 141.25 (C4); 152.62 (C3, C5); 165.08 (C=O). IR λmax/cm 3247
NH); 1704 (C=O thiazol.); 1668 (C=O); 1235 (C-O); 1126 (C-N); 1414, 1336, 1004, 852, 776 (Ar).
Anal. Calcd for C H N O S: C 63.00; H 5.06; N 6.39; S 7.31. Exp.: C 62.86; H 5.37; N 6.25; S 7.37.
2
3
22
2
5
5
-(naphthalen-1-yl)-3-(3,4,5-trimethoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (55).
Yellow
solid, m.p: 207-208°C, yield 83%; ¹H NMR (acetone-d ) δ 3.18 (dd, 1H, Ha, J= 17.1 Hz, 6.2 Hz); 3.74
6

ACCEPTED MANUSCRIPT
(s, 3H, p-OCH ); 3.87 (s, 6H, m-OCH ); 4.24 (dd, 1H, Hb, J = 17.5 Hz, 12.5 Hz); 6.18 (dd, 1H, Hc, J=
3 3
1
2.1 Hz, 7.0 Hz); 6.72 (m, 1H, H4’’); 7.02 (d, 2H, H2’’, H6’’, J = 7.8 Hz); 7.14 (m, 4H, H2’, H6’,
H3’’, H5’’); 7.33 (m, 1H, H2); 7.42 (dd, 1H, H3, J = 7.8 Hz, 7.4 Hz); 7.62 (dd, 1H, H7, J = 7.4 Hz,
.0 Hz); 7.69 (dd, 1H, H6, J = 7.8 Hz, 7.4 Hz); 7.87 (d, 1H, H4, J = 8.2 Hz); 8.03 (d, 1H, H5, J = 8.2
7
1
3
Hz); 8.36 (d, 1H, H8, J = 7.8 Hz). C NMR (acetone-d ) δ 43.2 (C ); 56.3 (m-OCH ); 60.4 (p-
6
ab
3
OCH ); 62.8 (Cc); 103.3 (C2’, C6’); 113.6 (C2’’, C6’’); 119.2 (C4’’); 124.1 (C2, C8); 124.9 (C6);
3
1
26.3 (C7); 126.6 (C4); 127.1 (C3); 128.5 (C1’); 128.9 (C4’); 129.5 (C3’’,C5’’); 129.6 (C5); 129.9
(
C4 ); 130.7 (C8 ); 137.9 (C1); 146.5 (C1’’); 148.4 (C=N); 154.1 (C3’, C5’). HRMS (ESI+) m/z:
a
a
+
calculated for C H N O [M ]: 438.1937; found, 438.1940.
2
8
26
2
3
2
-(naphthalen-1-yl)-3-(3,4,5-trimethoxybenzyl)thiazolidin-4-one (56). Beige solid, m.p. 142-143°C;
1
yield 80%; H NMR (CDCl ) δ 3.58 (d, 1H, CH , J= 14.4 Hz); 3.72 (s, 6H, 3-OCH , 5-OCH ); 3.81
3
2
3
3
(
d, 1H, CH -S, J= 15.6 Hz); 3.83 (s, 3H, 4-OCH ); 3.96 (dd, 1H, CH -S, J= 15.6 Hz, J= 1.56 Hz);
2 3 2
5
.06 (d, 1H, CH , J= 14.4 Hz); 5.60 (d, 1H, CH, J= 1.56 Hz); 6.26 (s, 2H, H2, H6); 7.40-7.90 (m, 7H,
2
1
3
naphthyl ring). C NMR (CDCl ) δ 33.2 (CH -S); 43.3 (CH ); 56.0 (3-OCH , 5-OCH ); 60.8 (4-
3
2
2
3
3
OCH ); 63.2 (CH); 105.5 (C2, C6); 124.0-139.2 (naphthyl ring and C1); 146.8 (C4); 153.5 (C3, C5);
3
+
1
71.2 (C=O). HRMS (ESI+) m/z: calculated for C H NO S [M ]: 410.1421; found, 410.1423
2
3
23
4
2
-(4-(dimethylamino)naphthalen-1-yl)-3-(3,4,5-trimethoxybenzyl)thiazolidin-4-one (57). White solid,
1
m.p. 161-162°C; yield 71%; H NMR (CDCl ) δ 2.92 (s, 6H, N(CH ) ); 3.64-3.93 (m, 12H, OCH ,
3
3 2
3
1
3
CH , CH -S); 5.19 (m, 1H, CH ); 6.22 (s, 2H, H2, H6); 7.46-8.34 (m, 6H, naphthyl ring). C NMR
2
2
2
(
(
(
CDCl ) δ 32.9 (CH -S); 45.0 (N(CH ) ); 47.0 (CH ); 55.9 (3-OCH , 5-OCH ); 60.7 (4-OCH3); 105.6
3 2 3 2 2 3 3
C2, C5); 125.2-137.6 (naphthyl ring and C1); 151.7 (C4) 153.3 (C2, C6); 171.8 (C=O). HRMS
+
ESI+) m/z: calculated for C H N O S [M ]: 453.1843; found, 453.1837
2
5
28
2
4
2
-(naphthalen-1-yl)-3-(3,4,5-trimethoxyphenyl)thiazolidin-4-one (58). Yellow solid, m.p. 145-146°C;
1
yield 72%; H NMR (CDCl ) δ 3.62 (s, 6H, 3-OCH , 5-OCH ); 3.73 (s, 3H, 4-OCH ); 3.93 (d, 1H,
3
3
3
3

ACCEPTED MANUSCRIPT
CH , J= 15.6 Hz); 4.07 (dd, 1H, CH , J= 15.6Hz J= 1.95 Hz); 6.11 (s, 1H,CH); 6.33 (s, 2H, H2, H6);
2
2
1
3
7
.48-7.88 (m, 7H, naphtyl ring). C NMR (CDCl ) δ 33.4 (CH ); 55.9 (3-OCH , 5-OCH ); 60.7 (4-
3 2 3 3
OCH ); 66.4 (CH); 103.9 (C2, C6); 124.0-136.0 (naphtyl ring and C1); 153.2 (C3, C5); 164.2 (C4);
3
+
1
71.1 (C=O). HRMS (ESI+) m/z: calculated for C H NO S [M ]: 396.1264; found, 396.1264
2
2
21
4
2
-(4-(dimethylamino)naphthalen-1-yl)-3-(3,4,5-trimethoxyphenyl)thiazolidin-4-one (59). Yellow
1
solid, m.p. 156-157°C; yield 73%; H NMR (DMSO-d ) δ/ppm 2.78 (s, 6H, N-(CH ) ); 3.55 (s, 3H, 4-
6
3 2
OCH ); 3.26 (s, 6H, 3-OCH , 5-OCH ); 3.73 (m,2H, CH ); 6.85 (s, 2H, H2, H6); 7.48-8.19 (m, 6H,
3
3
3
2
1
3
naphthyl ring). C NMR (DMSO-d ) δ/ppm 30.0 (CH ); 45.1 (N-(CH ) ); 56.4 (3-OCH , 5-OCH );
6
2
3 2
3
3
6
0.3 (4-OCH ); 124.6-136.2 (naphthyl and phenyl rings); 146.4 (C4); 152.9 (C3, C5); 171.1 (C=O).
3
+
HRMS (ESI+) m/z: calculated for C H N O S [M ]: 439.1686; found, 439.1685
2
4
26
2
4
4
-(2-(naphthalen-1-yl)-4-oxothiazolidin-3-yl)-N-(3,4,5-trimethoxyphenyl)benzenesulfonamide
(60).
1
White solid, m.p. 256°C; yield 74%; H NMR (DMSO-d ) δ/ppm 3.45 (s, 3H, 4-OCH ); 3.49 (s, 6H,
6
3
3
-OCH , 5-OCH ); 4.00 (m, 2H, CH ); 6.20 (s, 2H, H2, H6); 6.77 (s, 1H, CH); 7.48-7.85 (m, 11H,
3 3 2
1
3
naphthyl and phenyl rings); 9.98 (s, 1H, NH). C NMR (DMSO-d ) δ/ppm 33.3 (CH ); 55.9 (3-OCH ,
6
2
3
5
-OCH ); 60.4 (4-OCH ); 63.5 (CH); 98.9 (C2, C6); 124.5-137.1 (naphthyl and phenyl rings); 141.8
3 3
+
(
C4); 153.2 (C3, C5); 171.3 (C=O). HRMS (ESI+) m/z: calculated for C H N O S [M ]: 551.1305;
28 26 2 6 2
found, 551.1303.
4
-(2-(4-(dimethylamino)naphthalen-1-yl)-4-oxothiazolidin-3-yl)-N-(3,4,5-trimethoxyphenyl)benzene
1
sulfonamide (61). Yellow solid, m.p. 234°C; yield 60%; H NMR (DMSO-d ) δ/ppm 2.77 (s, 6H, N-
6
(
CH ) ); 3.73 (m, 11H, CH and 3,4,5-tri-OCH ); 6.26 (s, 2H, H2, H6); 6.38 (s, 1H, CH); 7.39-8.16
3 2 2 3
1
3
(
m, 10H, naphthyl and phenyl rings); 10.03 (s, 1H, NH). C NMR (DMSO-d ) δ/ppm 33.0 (CH );
6 2
4
5.0 (N-(CH ) ) 56.1 (3-OCH , 5-OCH ); 60.4 (4-OCH ); 98.7 (C2, C6); 119.3-134.6 (naphthyl and
3 2 3 3 3
+
phenyl rings); 153.3 (C3, C5); 167.8 (C=O). HRMS (ESI+) m/z: calculated for C H N O S [M ]:
3
0
31
3
6 2
5
94.1727; found, 594.1722.

ACCEPTED MANUSCRIPT
4
.2. Molecular modeling
The structures of the N-acylhydrazone derivatives were constructed using the standard tools
available in SYBYL 8.0. Four different conformers were modeled for each derivative [13] for which
the energy values were minimized employing the Tripos force field and Gasteiger-Huckel charges.
Docking protocols were used as implemented in GOLD 3.1 [34]. The X-ray crystal structures of the
tubulin-colchicine, tubulin-ABT751 and tubulin-T138067 complexes (PDB IDs 1SA0, 3HKC and
3
HKE, respectively) were retrieved from the Protein Data Bank [35, 36]. For the docking calculations,
the ligands were removed and hydrogen atoms were added using the Biopolymer module (SYBYL
.0). Residues in the binding site were manually checked for possible flipped orientation, protonation
8
and tautomeric states. The binding cavity was centered on the C22 atom of DAMA-colchicine and a
radius sphere of 10 Å was considered for the docking procedures, which were repeated 10 times for
each selected conformation of the N-acylhydrazone derivatives.
4
.3. Antiproliferative assay
REH and Jurkat human acute lymphoblastic leukemia cell lines were maintained in RPMI-
640, supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin, at 37 °C and 5%
1
CO . For the cytotoxic assay, compounds were dissolved in DMSO to obtain a stock solution of 20
2
mM. Further dilutions were made in culture medium immediately before use. Eighty microliters of a
5
REH or Jurkat suspension (3.75 x 10 cells/mL) were seeded in a 96-well plate. Immediately, 20 µL of
compound dilutions were added to each well, in triplicate, in final concentrations ranging from 0.01 to
1
µM. Culture plates were then kept at 37 °C and 5% CO for 48 h, when 20 µL of MTT reagent (5
2
mg/mL) was added. After 4.5 h, the precipitated formazan crystals were dissolved by the addition of
00 µL of an acid sodium dodecyl sufate solution (10% SDS, 0.01 mol/L HCl). Following overnight
1
incubation, absorbance was measured at 570 nm. Percentage of cell survival was calculated in relation
to controls, and IC values were determined using GraphPad Prism 5 software.
5
0

ACCEPTED MANUSCRIPT
4
.4. Cell migration assays
4
.4.1. Cell culture. Breast cancer cell line MDA-MB-231 was kindly provided by Prof. Heloisa
Sobreiro Selistre de Araújo (Departamento de Ciências Fisiológicas, Universidade Federal de São
2
Carlos, Brazil). The stock cells were maintained in a 75-cm flask in DMEM (Dulbecco’s Modified
Eagle’s Medium) supplemented with 10% FBS. Cells were incubated at 37 °C in 5% CO humidified
2
atmosphere. The Trypan Blue exclusion test was used to evaluate cell viability before experiments.
4
.4.2. Wound healing assay. For cell motility determination, cells were seeded in 24-well tissue
culture plate (Becton Dickinson) and grown to 80-90% confluence. After aspirating the medium, the
center of the cell monolayers were scraped with a sterile micropipette tip to create a denuded zone.
Subsequently, cellular debris was washed away with DMEM (without FBS supplementation) and cells
were incubated at 37 °C for 22 h, in growth medium containing different concentrations of
compounds. Photographs of the wounded monolayers were taken at time points 0 and 22 h, and the
percentage of wound closure was calculated by Java’s Image J software.
4
.4.3. Boyden chamber migration assay. Cell migration was assayed with Boyden chambers [8.0 ꢀm
pore size, polyethylene terephthalate membrane, FALCON cell culture insert (Becton-Dickinson)].
MDA-MB-231 cells were cultured in DMEM until 80-90% confluency. For the migration assay, cells
4
were detached with trypsin and resuspended in serum-free medium. After cell counting, 4.0 x 10 cells
(in 350 µL of serum free medium) were added in the upper chamber. In the lower chamber, 700 µL of
culture medium containing 10% FBS (a chemoattractant) was added. The transwells were incubated
for 6 h at 37 °C with different concentrations of the compounds in both upper and lower chambers.
Controls were given the vehicle in the same way. After incubation, cells inside the inserts were
removed with a cotton swab and cells on the underside of the insert were fixed with methanol and
stained with toluidine blue stain (Sigma). Transmigrated cells were counted in a light microscope,
using six random regions of the membrane. The NIS Elements software (NIKON) was used to count
the cells. Two independent experiments were carried out in duplicate for each compound.

ACCEPTED MANUSCRIPT
4
.5 Inhibition of MT Assembly
Electrophoretically homogeneous bovine brain tubulin (final concentration 10 µM; 1mgꢁ mL)
was pre-incubated with test agents dissolved in DMSO (1% v/v final concentration) and monosodium
glutamate (0.8 M final concentration) at 30 °C in 96-well plates. The reaction mixtures were cooled
on ice for 10 min and GTP (0.4 mM final concentration) was added. The absorbance at 350 nm was
immediately followed in a spectrophotometer. Baselines were established and temperature was
quickly raised to 37 °C. The turbidity value after 20 min at 30 °C for DMSO 1% was assigned as
1
00% assembly and for colchicine 10 µM, as 0% assembly. The IC were calculated by nonlinear
50
regression from the percent assembly values at the same time-point obtained with 0.625, 1.25, 2.5, 5
and 10 µM of the tested agents.
4
.6. Automated High-Content Cellular Analyses
The effects of 12 representative N-acylhydrazones on mitotic arrest, nuclear morphology and
cellular microtubules were studied as previously described using a multiparameter, high-content
imaging assay on an ArrayScan II (Thermo Fisher Cellomics, Pittsburgh, PA) [10]. Briefly,
asynchronously growing HeLa cells were treated for 18 h with each compound or vehicle (DMSO
0
.1% final concentration) in collagen-coated 384-well microplates, fixed, and incubated with primary
antibodies for tubulin and the mitotic marker protein phosphohistone H3, followed by addition of
fluorescein isothiocyanate (FITC) and Cy3-conjugated secondary antibodies, respectively. Cells were
detected by nuclear counterstaining with Hoechst 33342, which also provided information about
chromatin condensation and cell density as markers of cell death. Analysis of cell cycle arrest, mitotic
index, nuclear morphology, and cellular toxicity was performed exactly as described [10].
4
.7. Flow cytometry for Cell Cycle and Apoptosis Analysis
Jurkat cells were maintained in RPMI-1640, supplemented with 10% FBS and
penicillin/streptomycin, 37 °C and 5% CO . Cells were incubated for 12 h in fresh medium before
2

6
ACCEPTED MANUSCRIPT
experiments. 2 x 10 cells were treated with 125 nM of compound 12 or DMSO for 18 h. For cell
cycle analysis, cells were fixed in 70% ethanol for 2 h, washed in PBS and incubated for 30 min in
PBS 20 µg/mL propidium iodide, 80 µg/mL RNase A and 0.1% Triton X-100. For apoptosis analysis,
cells were incubated with Alexa Fluor® 488 Annexin V conjugate according to manufacturer
protocol. Stained cells were read in a BD Biosciences FACSCalibur flow cytometer.
4
.8. Selective cytotoxicity
Peripheral blood mononuclear cells (PBMC) were isolated from the blood of two healthy
6
individuals using Ficoll-Paque gradient. PMBC (2.5 x 10 cells/mL), REH and Jurkat cell (both at
5
3
.75 x 10 cells/mL) were resuspended in RPMI-1640 supplemented with 10% FBS and
penicillin/streptomycin. PBMC´s culture medium was supplemented with phytohemagglutinin (25
µL/mL, Cultilab) and Interleukin 2 (50U/mL) to induce proliferation of T lymphocytes. Eighty
microliters of cell suspensions were seeded in a 96-well plate and incubated for 24 h at 37 °C and 5%
CO for cell acclimation. Next, 20 µL of culture medium with increasing concentrations of compound
2
1
2 or vehicle (DMSO) were added to each well and citotoxicity was evaluated by the MTT reduction
assay after 48 h of treatment. Survival was calculated in relation to controls.
4
.9. Toxicity assays in vivo
To investigate the safety profiles of compound 12, we performed acute toxicity tests on mice,
according to guidelines of the Organization for Economic Co-operation and Development (OECD,
001). All experimental protocols were carried out following recommendations of the National
2
Institutes of Health Animal Care Guidelines (NIH publications No. 80–23), and conducted in
accordance to the protocol approved by the Committee of the Ethical Use of Animals of the Federal
University of Santa Catarina (CEUA/UFSC). Briefly, 7- to 8-week-old Swiss mice (25 – 35 g) were
acquired from the animal facility of the Federal University of Santa Catarina (UFSC, Florianópolis,
SC, Brazil). Animals were housed in cages (six animals per cage), at 22 ± 1°C, under a 12-hour
light/12-h dark cycle (lights on at 06:00 hours), 60–80% humidity, with free access to food (Nuvital,