Allylic isothiouronium salts: The discovery of a novel class of thiourea analogues with antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2017.02.013

A series of 28 aryl- and alkyl-substituted isothiouronium salts were readily synthesized in high yields through the reaction of allylic bromides with thiourea, N-monosubstituted thioureas or thiosemicarbazide. The S-allylic isothiouronium salts substitute

Full text of DOI:10.1016/j.ejmech.2017.02.013

Accepted Manuscript
Allylic isothiouronium salts: The discovery of a novel class of thiourea analogues with
antitumor activity
Misael Ferreira, Laura Sartori Assunção, Adny Henrique Silva, Fabíola Branco
Filippin-Monteiro, Tânia Beatriz Creczynski-Pasa, Marcus Mandolesi Sá
PII:
S0223-5234(17)30078-8
10.1016/j.ejmech.2017.02.013
EJMECH 9209
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 November 2016
Revised Date: 19 January 2017
Accepted Date: 6 February 2017
Please cite this article as: M. Ferreira, L.S. Assunção, A.H. Silva, F.B. Filippin-Monteiro, T.B.
Creczynski-Pasa, M.M. Sá, Allylic isothiouronium salts: The discovery of a novel class of thiourea
analogues with antitumor activity, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.02.013.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Allylic isothiouronium salts: the discovery of a novel class of
thiourea analogues with antitumor activity
Misael Ferreiraa,+, Laura Sartori Assunçãob,+, Adny Henrique Silvac, Fabíola Branco
Filippin-Monteirod, Tânia Beatriz Creczynski-Pasab, Marcus Mandolesi Sáa,*
a Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis,
SC 88040-900, Brazil
b Departamento de Ciências Farmacêuticas, Universidade Federal de Santa
Catarina, Florianópolis, SC 88040-900, Brazil
c Departamento de Bioquímica, Universidade Federal de Santa Catarina,
Florianópolis, SC 88040-900, Brazil
d Departamento de Análises Clínicas, Universidade Federal de Santa Catarina,
Florianópolis, SC 88040-900, Brazil
* Corresponding Author: Prof. Dr. M. M. Sá
MESOLab (Laboratory of Methodology and Organic Synthesis)
Departamento de Química, Universidade Federal de Santa Catarina
Florianópolis, SC 88040-900, Brazil
Tel.: +55-48-37213642; fax: +55-48-37216850
E-mail: marcus.sa@ufsc.br
+ As first authors, M. Ferreira and L. S. Assunção contributed equally in the studies related to
chemistry and biology, respectively.

ACCEPTED MANUSCRIPT
Allylic isothiouronium salts: the discovery of a novel class of thiourea
analogues with antitumor activity
Abstract
A series of 28 aryl- and alkyl-substituted isothiouronium salts were readily
synthesized in high yields through the reaction of allylic bromides with thiourea, N-
monosubstituted thioureas or thiosemicarbazide. The S-allylic isothiouronium salts
substituted with aliphatic groups were found to be the most effective against
leukemia cells. These compounds combine high antitumor activity and low toxicity
toward non-tumoral cells, with selectivity index higher than 20 in some cases.
Furthermore, the selected isothiouronium salts induced G2/M cell cycle arrest and
cell death, possibly by apoptosis. Therefore, these compounds can be considered as
a promising class of antitumor agents due to the potent cytostatic activity associated
with high selectivity.
Keywords
isothiouronium salts; allylic bromides; antitumor activity; DNA fragmentation;
leukemia
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1. Introduction
Cancer is the leading cause of death worldwide and the development and
discovery of new anticancer agents are the key focus of several research groups.1
Leukemia, a type of cancer characterized by the clonal proliferation of malignant
white blood cell precursors, can be subdivided based on the predominant cell of
origin (lymphoid or myeloid) and the rate of progression (acute or chronic) [2-4].
Different forms of therapy have been used to treat leukemia, such as radiotherapy,
chemotherapy, immunotherapy, and bone marrow transplant, with chemotherapy
being the most frequently used [5-7]. The existing chemotherapeutic treatments are
known to display a wide range of side effects, caused by the low selectivity and high
toxicity of the drugs employed. Furthermore, a large number of anticancer agents
promote the death of cancer cells by cell cycle arrest, induction of apoptosis and
differentiation, as well as through inhibition of cell growth and proliferation, or a
combination of two or more of these mechanisms [8-10].
The treatment selected for a leukemia patient is dependent on many factors,
such as prognostic features and the stage and type of leukemia [11]. Despite the
remarkable progress achieved in the treatment of acute and chronic leukemia, truly
effective therapies are absent for the majority of the patients affected by this disease.
The most common reasons for failure include drug resistance and treatment-related
toxicity associated with intensive therapies [12]. A common example is the
anthracycline antibiotic doxorubicin, a widely used anticancer agent that, due to its
low bioavailability, requires high doses for therapeutic response, which increases the
cumulative risk of cardiotoxicity, myelosuppression and nephrotoxicity [13].
Therefore, the search for more efficient chemotherapies for leukemia, which
minimize the toxicity to normal tissues, is of great importance. One of the ways to
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achieve this goal is the development of new chemical entities that are more active,
more selective, less toxic, and readily available through resourceful methods.
One of the classes of interest in the search for new chemotherapeutic agents
is the group of isothiouronium salts. These compounds can exert biological effects
and have attracted the attention of researchers due to their diverse activities, such as
inhibitors of protein kinase C [14] as well as agonists of the GABA-type [15, 16] and
histamine-H3 receptors [17]. Furthermore, they also exhibit a variety of
pharmacological properties, such as local anesthetic [18] and antimicrobial [19-22]
activity. Recently, we reported the moderate activity of selected S-allylic
isothiouronium salts against Mycobacterium tuberculosis [23]. However, despite
displaying these biological properties, the antitumor activity of isothiouronium salts
has been scarcely explored [24, 25].
Therefore, the aim of this study was to evaluate whether this class of
compounds could also exhibit antitumor activity. Specifically, this Communication
reports the chemical synthesis of twenty-eight S-allylic isothiouronium salts and the
assessment of their cytotoxicity profile, including an evaluation of possible cell cycle
alterations as well as the potential selectivity against leukemia cells.
2. Results and discussion
2.1. Chemistry
A variety of isothiouronium salts 1, 2, and 3 were readily synthesized in two
steps from α-methylene-β-hydroxy esters 4 (Morita-Baylis-Hillman adducts) [26]. The
synthetic strategy involves the preparation of the corresponding allylic bromide 5, a
key precursor obtained through the reaction of α-methylene-β-hydroxyesters 4 with
LiBr in acidic medium at 25 °C [27] (Scheme 1). This two-step protocol to obtain
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allylic bromides 5 was adapted for the multigram scale, with overall yields of 65-85%,
thus constituting a practical route to access these structurally versatile starting
materials.
Insert Scheme 1.
The subsequent treatment of the allylic bromides 5 with thiourea (6a) as well
as N-monosubstituted thioureas (6b-e) led to the formation of the corresponding
isothiouronium salts [28] 1a-k, 2a-g, and 3a-j in nearly quantitative yields (Scheme
1). The choice of the solvent was shown to be crucial to achieving successful
transformations. While the N-unsubstituted isothiouronium salts 1, originating from
thiourea (6a), could be formed smoothly in aqueous acetone as an insoluble
crystalline solid, the preparation of N-substituted isothiouronium salts 2a,b and 3a-j
could not be achieved under similar conditions. However, running the reaction in
acetonitrile at 25 °C furnished the expected N-substituted isothiouronium salts 2 and
3 as crystalline products that precipitated out and were readily collected by filtration,
thus facilitating the work-up and isolation steps. After purification, the isothiouronium
salts were analyzed by IR, 1H/13C NMR, and HRMS. Stretching bands related to C=O
and C=C groups can be observed on the IR spectrum at 1704-1730 cm-1 and 1634-
1658 cm-1, respectively. Furthermore, 1H NMR analysis revealed the presence of the
expected peak for the olefinic hydrogen (=CH) at 7.5-8.0 ppm, in the case of aryl-
substituted derivatives (1a-h, 2a-e, 3a-i), or at 6.9-7.1 ppm, in the alkyl-derived salts
(1i-k, 2f,g, 3j). This observation unequivocally supports the stereochemistry of the
double bond being Z for all products, which is also in line with the analysis of the
decoupled 13C NMR spectra [28] (see the Experimental section in the Supplementary
Material).
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For the synthesis of the N-amino substituted salts 2c-e derived from
thiosemicarbazide (6d), the conditions using acetonitrile as the solvent were also
successfully employed, but in these particular cases the products were characterized
by NMR as mixtures of isomers in solution, possibly due to the fast equilibrium
between tautomeric forms of the allylic isothioureido N-C=N moiety [29].
2.2. Biological activity
2.2.1. Cytotoxicity and selectivity index of S-allylic isothiouronium salts related to
leukemia and non-tumoral cell lines
To evaluate the cytotoxicity potential of the S-allylic isothiouronium salts, the
MTT assay was performed with a murine leukemia cell line (L1210) and a murine
non-tumoral cell line (NIH-3T3). The IC50 concentration (concentration required to
reduce cell viability by 50%) [30] was determined by non-linear regression and the
selectivity index (SI) was calculated. A selectivity index higher than 1 indicates that
cytotoxicity on tumoral cells surpassed healthy non-tumoral ones [31]. To compare
the selectivity of the compounds studied here, we classified them as remarkable (SI
above 12), moderate (from 6 to 12) and weak (from 1 to 5) selectivity. The
antineoplasic agent mitoxantrone was used as positive control. Mitoxantrone is a
synthetic molecule structurally similar to the anthracyclines. This drug was developed
to reduce the cardiototoxicity presented by the naturally occurred anthracycline
doxorubicin. It is used in breast and prostate cancer, acute myeloid leukemia, and
lymphoma treatment [32, 33], also presenting improvements in the survival rate of
children suffering from acute lymphoblastic leukemia relapse [34].
The results for the cytotoxicity are summarized in Tables 1 and 2. The effect of
substitution on both the allylic chain (R = aryl [Table 1] or alkyl [Table 2] groups) and
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the isothiourea moiety (N-unsubstituted or substituted by a variety of R1 groups) was
evaluated and both were significant to the activity. Table 1 shows the results for a
diversity of aromatic-derived allylic salts (R = aryl) 1a-h, 2a-e, and 3a-i with different
substitution patterns at R1 originating from the thiourea portion. A relatively low IC50
(4-18 µM) was observed for the N-unsubstituted analogues 1a-h (Table 1, entries 1-
8). Likewise, mitoxantrone presented cytotoxicity toward leukemia cell line, with an
IC50 of 15 µM, however with low selectivity (SI = 2.8). As previously mentioned, the
low selectivity of an anticancer agent might represent a risk of toxicity to patients in
cumulative doses. Conversely, three of the N-unsubstituted analogues (1a, 1e, 1h)
showed remarkable selectivity (SI = 17.5, > 6.3, and 10.2, respectively), which in
turns could present less risk of toxic effects in cancer treatment eventually
Insert Table 1.
Insert Table 2.
On the other hand, substitution of a hydrogen at one of the nitrogen atoms of
the isothioureido portion by hydrophobic methyl or allyl groups (2a,b, where R1 ≠ H)
was deleterious for both the activity and selectivity (Table 1, entries 9 and 10).
Conversely, the introduction of the more polar and hydrophilic amino group, as in the
thiosemicarbazide derivatives 2c-e (logP ~ 1 [35]), caused an increment in the
activity (entries 11-13).
Remarkably, N-substitution by a more lipophilic phenyl group as in N-phenyl
isothioureas 3a-i (logP ~ 4-5 [35]; see Table 1, entries 14-22) was found to improve
both the antitumor activity as well as the selectivity. Besides the high SI found for the
phenyl-substituted salt 3a (SI = 9.0, entry 14), the analogues 3c-g containing
electron-withdrawing groups at the phenyl ring showed SI values ranging from c.a. 3
to 13 (Table 1, entries 16-20). In the particular case of the nitro-substituted
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derivatives 3e and 3g (Table 1, entries 18 and 20), the very low cytotoxicity toward
non-tumoral cells is worth noting, as nitro groups are usually related to a variety of
biological effects [36, 37]. Thus, the N-phenyl-substituted isothioureas 3a,c-g appear
as promising leads for the development of selective antitumor agents, not only
because they show good SI values but also due to their relatively high lipophilicity,
which may improve the penetration of these compounds into cells.
Another attractive class of potential antileukemic leads was found to be
derived from the aliphatic allylic salts 1i-k, 2f,g, and 3j (R = alkyl, Table 2), wherein
high antitumor activity (IC50 = 3-14 µM) as well as selectivity (SI = 5.3-23.7) were
observed for the N-unsubstituted analogues 1i-k (Table 2, entries 1-3). When the
selectivity index was analyzed, the allylic salts substituted with alkyl groups, such as
ethyl (R = CH3CH2; SI(1i) = 23.7) and methyl (R = CH3; SI(1j) > 20), exerted a greater
toxic effect against leukemia (L1210) cells with a lower concentration of each salt (3
and 5 µM, respectively). The relative low lipophilicity can be modulated by adding or
removing carbon atoms from the R chain (logP [35] varying from 0.61 for 1j to 1.91
for 1k), and the optimum chain size was determined as being the two-carbon moiety
as in 1i (Table 2, entry 1).
As expected, based on the results for the aromatic series (see Table 1, entries
9 and 10), the N-methyl- and N-allyl-substituted salts 2f,g of the alkyl series (Table 2,
entries 4 and 5) are comparatively less cytotoxic and less selective than their N-
unsubstituted analogues 1i-k (entries 1-3). A different trend was observed for 3j
(Table 2, entry 6), the least lipophilic N-phenyl-substituted salt tested (logP = 3.18
[35]), where its high antitumor activity was partially obfuscated by the pronounced
cytotoxicity toward non-tumoral cells, thus giving a low SI value of 2.8. Therefore, the
alkyl-substituted isothiouronium salts 1i and 1j were also considered as promising
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leads for further development as selective antileukemic agents.
2.2.2. Effect of S-allylic isothiouronium salts on DNA content of leukemia cells in cell
cycle distribution
Cell division is controlled by checkpoints that avoid genomic instability and
ensure the proper cell cycle progression. The checkpoints act during the transition
from the G1 to the S phase (DNA replication) and from the G2 to the M phase
(mitosis). Chemicals and other agents can either delay replication or cause
irreparable DNA damage that is recognized by the checkpoints, leading to a cell cycle
arrest and/or death [38, 39].
In order to investigate whether the effects of the S-allylic isothiouronium salts
are associated with cell cycle alterations, the leukemia cells were 24-h exposed with
the corresponding IC50 of each tested compound. Flow cytometry analysis of cell
cycle, using propidium iodide (PI) as DNA label, was performed to observe the
distribution of cells in the specific phases of the cycle for replication. It was observed
that the IC50 of the N-unsubstituted salts 1a-h (data not shown for 1g) induced DNA
fragmentation, represented by an increase of approximately 20% in the number of
events in the sub-G1 compared to the control group (Figure 1C). Our previous study
demonstrated similar results for melanoma cells exposed to compounds 1a and 1e
[29]. In that case, compound 1c showed greater activity against melanoma cells,
leading to DNA fragmentation of more than 50% of cell population under evaluation
[29]. However, DNA fragmentation in leukemia cells induced by 1c was observed in
about 20% of cell population. This difference observed in both studies might be
explained by the biological distinctions between the two cancer cell types, which
reflect in specific responses to the stimulus [9].
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Insert Figure 1.
The N-methyl salt 2a induced cell cycle arrest in the G2/M phase with an
increase of 14% in the number of events, while N-phenyl salt 3b induced a significant
DNA fragmentation in the sub-G1 when compared to cells without treatment (Figures
1D and 1E, respectively). The aliphatic allylic salts 1i (data not shown), 1k and 2f
induced cell cycle arrest in the G2/M phase leading to cell death, represented by an
increase in the DNA fragmentation in the sub-G1 phase (Figure 1F). This G2/M arrest
might be explained by a phenomenon in the cell cycle progression due to an
extensive damage in the DNA content or chromosome segregation caused by these
compounds, which make it impossible for the cell to continue its replication and, as a
result, it enters into the death state [40]. Furthermore, compounds 1j and 2g also
induced DNA fragmentation, increasing by 27% the number of events in sub-G1
without affecting the G2/M phase significantly. In terms of the mechanism of action,
this observation suggests that compounds 1i, 1k, and 2f differ from the others in the
group.
Other isothiouronium salts showed no significant alteration in the cell cycle
distribution. This might be due to the fact that the IC50 of the compounds was
determined by a mitochondrial activity assay, mainly using dehydrogenases. Since
changes in mitochondrial metabolism and mitochondrial damages are earlier noticed
in cell death process, changes in DNA content might not be observed in flow
cytometry due to a time-dependent response [41-44].
The G2/M cell cycle arrest observed in L1210 cells caused by the exposure to
compounds 1i, 1k and 2f might be an interesting approach in cancer therapy, since
they increase the sensitivity when combined with other anticancer agents and
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eventually augment the cell death rates [45, 46]. Also, effective treatment of cancer
with agents that induce G2/M arrest can be carried out with lower drug
concentrations, thus reducing the side effects [47, 48].
In order to investigate the cell death caused by the S-allylic isothiouronium
salts, leukemia cells were exposed for 24 h to compounds that presented SI higher
than 6 (1a, 1e, 1h, 3a, 3g, and 1j) and were subsequently stained with Annexin-V-
FITC and PI for further evaluation through flow cytometry (Figure 2). It was observed
that compound 3g presented an increase of events stained with annexin-V-FITC,
which is suggestive of an apoptotic process, since annexin conjugates to
phosphatidylserine once it is externalized in cell membrane during apoptotic events.
Moreover, cells exposed to salts 1a and 1e presented a portion of its population
double stained, suggesting late apoptotic or necrotic process. Furthermore, another
fraction of cell population exposed to these same compounds also presented PI
labeling, suggestive of a necrotic process characterized by loss of extracellular
membrane integrity, allowing PI penetration [49-51].
Insert Figure 2.
3. Conclusions
In summary, a diverse set of S-allylic isothiouronium salts were readily
prepared in high yields and screened against leukemia cells. A representative group
of N-unsubstituted isothiouronium salts not only showed high potent activity against
tumoral cells but also displayed remarkable selectivity. Similarly, the externalization of
phosphatidylserine caused by the nitro-substituted derivative 3g suggests an
apoptotic process, being an interesting approach to cancer treatment, once this
mechanism of death does not induce an inflammatory process, unlike necrosis.
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Furthermore, the alkyl-substituted derivatives are more potent than the aryl-
substituted analogues, inducing G2/M cell cycle arrest and, possibly, apoptosis.
However, further studies should be done to characterize better the cell death in all
cases.
4. Experimental section
4.1. Chemistry
4.1.1. General considerations
All chemicals were of reagent grade and were used as received. Melting
points were determined using a Microquímica MQPF301 hot-plate apparatus and are
uncorrected. Infrared spectra were acquired with a Perkin-Elmer FT-IR 1600
spectrometer (range 4000-400 cm-1) using KBr for all samples. 1H NMR spectra were
recorded at 400 MHz or 200 MHz and 13C NMR spectra (fully decoupled) were
recorded at 100 MHz or 50 MHz. Samples were prepared in an appropriate
deuterated solvent (CDCl3, D2O, DMSO-d6). Chemical shifts are reported in parts per
million (ppm, δ) relative to TMS at 0.00 ppm or solvent (CDCl3 at 7.26 ppm or D2O at
4.64 ppm or DMSO-d6 at 2.50 ppm for 1H NMR, and CDCl3 at 77.16 ppm or DMSO-
13
d6 at 39.52 ppm for C NMR) as the internal standard. Coupling constants (J) are
measured in Hertz (Hz) and coupling patterns are designated as s (singlet); d
(doublet); dd (doublet of doublets); t (triplet); appt (apparent triplet); dq (doublet of
quartet); m (multiplet); brs (broad signal). Elemental analyses were conducted in a
CHNS-O Carlo Erba EA-1110 analyzer. The ESI-QTOF mass spectrometer was
operated in the positive ion mode at 4.5 kV and at a desolvation temperature of 180
°C. The standard electrospray ion (ESI) source was used to generate the ions. The
instrument was calibrated in the range m/z 50-3000 using a calibration standard (low
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concentration tuning mix solution) and data were processed with the aid of computer
software. The isothiouronium bromides 1a,c,e-j, isothiosemicarbazide bromides 2d,e
and N-phenylisothiouronium bromides 3a,i were prepared and purified according to
the previously described methods and showed physical and spectral data in
accordance with their expected structure and by comparison with spectral data in
literature [23, 28, 29].
4.1.2. Typical procedure for the synthesis of isothiouronium salts 1
To a stirred solution of allylic bromide 5 (1.0 mmol) in 5.0 mL of acetone:H2O
(4:1 v/v) at 25 °C was added 0.95 mmol of thiourea (6a). After stirring for 1 h, the
organic salt was concentrated under reduced pressure and the resulting solid was
crushed with ethyl ether/CH2Cl2 (4:1) and filtered to give pure isothiouronium salts
1b,d. For 1k, the resulting product was washed with ethyl ether to give pure
isothiouronium salt as oil.
4.1.2.1.
Methyl
(Z)-2-(isothioureidomethyl)-3-(4-methylphenyl)-2-propenoate
hydrobromide (1b)
Yield 96% (348 mg); white solid, mp 172.0-174.0 °C; 1H NMR (400 MHz, D2O):
13
δ
2.17 (s, 3H), 3.66 (s, 3H), 4.01 (s, 2H), 7.10 (s, 4H), 7.61 (s, 1H); C NMR (100
MHz, D2O, DMSO-d6 as internal standard):
δ 22.2 (CH3), 30.6 (CH2), 54.5 (OCH3),
124.4 (C), 131.3 (2 × CH), 131.4 (2 × CH), 131.9 (C), 142.8 (=CH), 146.9 (C), 170.1
(C), 172.1 (C); IR (KBr) νmax/cm-1: 3292, 3253, 3081, 2949, 1709, 1636, 1437, 1322,
1271; Anal. Calcd for C13H17BrN2O2S (%): C, 45.22; H, 4.96; N, 8.11; found: C, 45.38;
H, 5.35; N, 8.19.
4.1.2.2.
Methyl
(Z)-3-(4-fluorophenyl)-2-(isothioureidomethyl)-2-propenoate
hydrobromide (1d)
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Yield 94% (602 mg); white solid, mp 177.0-179.0 °C; 1H NMR (200 MHz, D2O):
δ
3.70 (s, 3H), 4.08 (s, 2H), 7.07 (t, J = 8.5 Hz, 2H), 7.31 (dd, J = 5.6, 8.5 Hz, 2H),
7.78 (s, 1H); 13C NMR (100 MHz, D2O, DMSO-d6 as internal standard):
δ 30.4 (CH2),
54.5 (OCH3), 117.7 (d, J = 24.9 Hz, 2 × CH), 125.6 (C), 131.2 (C), 133.4 (d, J = 7.3
Hz, 2 × CH), 145.8 (=CH), 164.8 (d, J = 247.7 Hz, C), 169.9 (C), 171.9 (C); IR (KBr)
νmax/cm-1: 3368, 3213, 3106, 3037, 2954, 1704, 1650, 1597, 1510, 1438, 1233, 1204,
1162; Anal. Calcd for C12H14BrFN2O2S (%): C, 41.27; H, 4.04; N, 8.02; found: C,
41.16; H, 3.52; N, 8.08.
4.1.2.3. Methyl (Z)-2-isothioureidomethyl-2-hexenoate hydrobromide (1k)
Yield 98% (293 mg); clear yellow oil; 1H NMR (400 MHz, CDCl3):
δ 0.90 (t, J =
7.4 Hz, 3H), 1.37-1.56 (m, 2H), 2.19-2.30 (m, 2H), 3.60 (brs, 2H), 3.75 (s, 3H), 4.07
13
(s, 2H), 7.02 (t, J = 7.6 Hz, 1H), 8.70-8.90 (m, 4H); C NMR (100 MHz, CDCl3):
δ
14.0 (CH3), 21.8 (CH2), 28.0 (CH2), 31.5 (CH2), 53.2 (OCH3), 126.1 (C), 151.1 (=CH),
168.5 (C), 172.0 (C); IR (neat) νmax/cm-1: 3289, 3189, 3064, 2961, 2874, 2735, 1712,
1646, 1440, 1298, 1198, 1071; HRMS (ESI+): m/z calcd for C9H17N2O2S [M]+:
217.1005; found: 217.1002.
4.1.3. Typical procedure for the synthesis of N-substituted isothiouronium salts 2 and
3
To a stirred solution of allylic bromide 5 (1.0 mmol) in 5.0 mL of acetonitrile at
25 °C was added 0.95 mmol of N-substituted thiourea (5b or 5c or 5e) or
thiosemicarbazide (5d). After stirring for 1 h, the organic salt was concentrated under
reduced pressure and the resulting solid was crushed with acetonitrile and filtered to
give pure isothiouronium salts 2a-c. For 2f,g and 3j, the resulting crude product was
triturated with ethyl ether to give pure isothiouronium salt as oil.
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4.1.3.1.
Methyl
(Z)-2-[(N-methylisothioureido)methyl]-3-(4-methoxyphenyl)-2-
propenoate hydrobromide (2a)
Yield 89% (232 mg); white solid, mp 126.0-128.0 °C; 1H NMR (400 MHz, D2O):
δ
2.69 (s, 3H), 3.64 (s, 6H), 4.01 (s, 2H), 6.81 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz,
13
2H), 7.49 (s, 1H); C NMR (50 MHz, D2O, DMSO-d6 as internal standard):
δ 31.2
(CH2), 31.7 (CH3), 54.4 (OCH3), 57.0 (OCH3), 116.1 (2 × CH), 123.7 (C), 127.5 (C),
133.4 (2 × CH), 145.8 (=CH), 162.0 (C), 168.1 (C), 170.1 (C); IR (KBr) νmax/cm-1:
3168, 3072, 2991, 2947, 2908, 1717, 1702, 1646, 1601, 1511, 1439, 1259, 1181;
Anal. Calcd for C14H19BrN2O3S (%): C, 44.81; H, 5.10; N, 7.46; found: C, 45.16; H,
5.53; N, 7.53.
4.1.3.2. Methyl (Z)-2-[(N-allylisothioureido)methyl]-3-(4-methoxyphenyl)-2-propenoate
hydrobromide (2b)
1
Yield 98% (242 mg); white solid, mp 162.0-163.5 °C; H NMR (400 MHz,
DMSO-d6):
δ 3.75 (s, 3H), 3.78 (s, 3H), 4.02 (s, 2H), 4.38 (s, 2H), 5.19-5.26 (m, 2H),
5.76-5.85 (m, 1H), 7.04 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.79 (s, 1H),
9.33 (brs, 1H), 9.52 (brs, 1H), 9.95 (brs, 1H); 13C NMR (100 MHz, DMSO-d6):
δ
30.0
(CH2), 45.7 (CH2), 52.6 (OCH3), 55.5 (OCH3), 114.7 (2 × CH), 117.5 (=CH2), 121.3
(C), 125.7 (C), 131.4 (=CH), 132.1 (2 × CH), 143.5 (=CH), 160.8 (C), 165.7 (C), 166.6
(C); IR (KBr) νmax/cm-1: 3294, 3189, 3026, 2839, 1714, 1638, 1598, 1512, 1440,
1281, 1261, 1175; Anal. Calcd for C16H21BrN2O3S (%): C, 47.88; H, 5.27; N, 6.98;
found: C, 47.98; H, 5.03; N, 7.06.
4.1.3.3.
Methyl
(Z)-2-[(N-aminoisothioureido)methyl]-3-phenyl-2-propenoate
hydrobromide (2c)
1
Yield 92% (856 mg); white solid, mp 157.0-159.0 °C; H NMR (400 MHz,
DMSO-d6): mixture of isomers (92:8) coexisting in solution (data for the major isomer)
15

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δ
3.77 (s, 3H), 4.20 (s, 2H), 7.43-7.53 (m, 5H), 7.84 (s, 1H); 13C NMR (100 MHz,
DMSO-d6):
δ 28.8 (CH2), 52.6 (OCH3), 125.0 (C), 129.1 (2 × CH), 129.6 (2 × CH),
130.0 (CH), 133.6 (C), 143.4 (=CH), 166.3 (C), 166.4 (C); IR (KBr) νmax/cm-1: 3345,
3311, 3103, 2950, 2779, 1721, 1654, 1626, 1545, 1436, 1284, 1205, 1166, 949; Anal.
Calcd. for C12H16BrN3O2S (%): C, 41.63; H, 4.66; N, 12.14; found: C, 41.89; H, 4.69;
N, 12.22.
4.1.3.4. Methyl (Z)-2-[(N-methylisothioureido)methyl]-2-pentenoate hydrobromide (2f)
Yield 98% (341 mg); clear yellow oil; 1H NMR (400 MHz, DMSO-d6):
δ 1.00 (t,
J = 7.6 Hz, 3H), 2.27 (dq, J = 7.6, 7.8 Hz, 2H), 2.88 (d, J = 4.0 Hz, 3H), 3.69 (s, 3H),
4.12 (s, 2H), 6.92 (t, J = 7.8 Hz, 1H), 9.12 (brs, 1H), 9.42 (brs, 1H), 9.71 (brs, 1H);
13C NMR (100 MHz, DMSO-d6):
δ 13.1 (CH3), 22.0 (CH2), 28.1 (CH2), 30.8 (CH3),
52.3 (OCH3), 124.8 (C), 150.2 (=CH), 165.8 (C), 165.9 (C); IR (neat) νmax/cm-1: 3155,
3060, 2972, 1714, 1653, 1604, 1438, 1315, 1287, 1199; HRMS (ESI+): m/z calcd for
C9H17N2O2S [M]+: 217.1005; found: 217.1006.
4.1.3.5. Methyl (Z)-2-[(N-allylisothioureido)methyl]-2-pentenoate hydrobromide (2g)
Yield 96% (373 mg); clear yellow oil; 1H NMR (400 MHz, DMSO-d6):
δ 1.00 (t,
J = 7.4 Hz, 3H), 2.28 (dq, J = 7.4, 7.6 Hz, 2H), 3.69 (s, 3H), 3.94-3.98 (m, 2H), 4.15
(s, 2H), 5.20-5.24 (m, 2H), 5.76-5.85 (m, 1H), 6.92 (t, J = 7.6 Hz, 1H), 9.21 (brs, 1H),
9.48 (brs, 1H), 9.92 (brs, 1H); 13C NMR (100 MHz, DMSO-d6):
δ 13.1 (CH3), 22.1
(CH2), 28.2 (CH2), 45.7 (CH2), 52.3 (OCH3), 117.5 (=CH2), 124.9 (C), 131.4 (=CH),
150.1 (=CH), 165.4 (C), 165.9 (C); IR (neat) νmax/cm-1: 3143, 3070, 2975, 1717,
1644, 1594, 1438, 1315, 1287, 1251, 1199; HRMS (ESI+): m/z calcd for C11H19N2O2S
[M]+: 243.1162; found: 243.1165.
4.1.3.6. Methyl (Z)-2-[(N-phenylisothioureido)methyl]-2-pentenoate hydrobromide (3j)
Yield 98% (391 mg); clear yellow oil; 1H NMR (400 MHz, DMSO-d6):
δ
1.00 (t,
16

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J = 7.6 Hz, 3H), 2.31 (dq, J = 7.6, 7.8 Hz, 2H), 3.71 (s, 3H), 4.24 (s, 2H), 6.97 (t, J =
7.8 Hz, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.40 (t, J = 7.4 Hz, 1H), 7.51 (appt, J = 7.6 Hz,
13
2H), 11.42 (brs, 1H); C NMR (50 MHz, DMSO-d6):δ 13.1 (CH3), 22.1 (CH3), 28.4
(CH2), 52.4 (OCH3), 124.7 (C), 125.2 (2 × CH), 128.3 (CH), 130.0 (2 × CH), 135.0
(=CH), 150.7 (C), 166.0 (C), 167.3 (C); IR (neat) νmax/cm-1: 3266, 3063, 2971, 2877,
2801, 1713, 1638, 1578, 1494, 1439, 1286, 1199, 1146; HRMS (ESI+): m/z calcd for
C14H19N2O2S [M]+: 279.1162; found: 279.1167.
4.2. Biological activity
4.2.1. Materials
The cell culture media, fetal bovine serum and antibiotics
(penicillin/streptomycin) were purchased from GIBCO (Grand Island, NY, USA).
Dimethyl sulfoxide (DMSO) and ethylene diaminetetraacetic acid disodium salt
(EDTA) were purchased from Merck (Darmstadt, Germany). Binding buffer was
purchased from Millipore (Billerica, MA, USA) and other reagents were supplied by
Sigma-Aldrich (St. Louis, MO, USA).
4.2.2. Cell lines and culture conditions
The cell lines L1210 (murine lymphocytic leukemia) and NIH-3T3 (murine
fibroblast) were purchased from the Rio de Janeiro Cell Bank. The cells were
cultured in RPMI-1640 (L1210) or DEMEM (NIH-3T3) medium, supplemented with
1.5 g/L sodium bicarbonate, 10 mM HEPES, pH 7.4, 100 U/mL penicillin G, 100
µg/mL streptomycin and 10% fetal calf serum at 37 °C in a humidified atmosphere
consisting of 95% air and 5% CO2. Cells were passaged 3 times a week and cultures
with greater than 95% of viable cells in trypan-blue exclusion tests were used for the
17

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experiments.
4.2.3. Viability assay
The MTT method was used to determine the cell viability [52]. NIH-3T3 cells (1
× 104 cells/well) and L1210 cells (1 × 105 cells/well) were seeded in 96-well plates
and incubated for 24 h with increasing concentrations of the compounds, ranging
from 1 to 100 µM. For the control group, cells were incubated without treatment. After
incubation, the old culture medium was replaced by fresh culture medium with 5
mg/mL of MTT, followed by incubation for 1-4 h at 37 °C. MTT-formazan crystals
were dissolved in 100 µL of DMSO and the absorbance was measured at 540 nm
using a micro-well system reader. The IC50 values were calculated using the software
Prism 5.0 (GraphPad Software) through non-linear regression analysis of the
logarithm concentration as a function of the normalized response (percentage of
viable cells after 24 h incubation).
The activity of the compounds in tumoral cells was classified according to IC50 as:
High: < 5 µM; Good: 5 to 10 µM; Moderate: 11 to 25 µM; Low: > 26 µM
4.2.4. Selectivity Index
The selectivity index was calculated as the ratio of the IC50 of the non-tumoral
cell line to the IC50 of the tumoral cell line, as presented in the following formula. An
SI value higher than 1 indicates that the cytotoxicity against tumoral cells surpassed
that toward non-tumoral cells [31].
18

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4.2.5. Flow cytometry analysis
To analyze the cell cycle of cells treated with isothiouronium salts, flow
cytometry was used following a previously described method [53]. Leukemia cells (1
× 106/well) were incubated with the IC50 concentrations of each compound for 24 h, in
a 12-well plate. After incubation, the cells were centrifuged at 400 × g at room
temperature for 10 min. The cells were then washed with 1 mL of PBS and
centrifuged again. The supernatant was discarded and cells were fixed with 200 µL of
70% ethanol for 30 min at 4 °C. PBS (1 mL) with 2% of BSA was then added and the
cells were centrifuged for 10 min at 400 × g. The supernatants were removed and the
cells were permeabilized with lysis buffer (0.1% Triton-X in PBS) and 0.5 µL of
RNase (100 µg/mL). The DNA content was stained with propidium iodide (PI) (20
µg/mL) and analyzed using a FACSCanto flow cytometer (Becton Dickinson). The
DNA content of the cell population in each phase of the cell cycle was determined
using Flowing 2.5.1 software (University of Turku, Turku, Finland).
To evaluate cell death a flow cytometric assay using Annexin-V-FITC and
propidium iodide was performed as previously described, with minor modifications
[51]. Briefly, 1 × 106 cells/well were exposed to the IC50 of each compound for 24 h, in
a 12-well plate, centrifuged for 10 minutes at 400 g and 4 °C , washed twice with cold
PBS (1 mL), resuspended in binding buffer 1× and incubated with 2.5 µL Annexin-V-
FITC for 15 min. After, 5 µL of PI solution (20 µg/mL) was added in the moment of
cells acquisition. Cell death was analyzed using FACSCanto flow cytometry
equipment (Becton Dickinson) and Flowing 2.5.1 software (University of Turku, Turku,
Finland). The percentage of apoptotic and/or necrotic cells induced by the
compounds exposure was compared to cells without treatment (control group).
19

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Acknowledgements
The authors wish to thank the Central de Análises (Departamento de Química,
UFSC, Florianópolis) for spectroscopic analysis. Special thanks to CEBIME
(Laboratório Central de Biologia Molecular e Estrutural), for obtaining the mass
spectra, and LAMEB I (Laboratório Multiusuário de Estudos em Biologia I) for the
analysis carried out on the Flow Cythometer BD FACSCanto II. M.F. and L.S.A. thank
CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for fellowships.
M.M.S. is grateful to CNPq for a research fellowship. Financial support from INCT-
Catalysis/CNPq is also gratefully acknowledged.
Conflict of interest
The authors confirm that this article content has no conflict of interest.
Appendix A. Supplementary data
Supplementary data associated with this article (selected NMR spectra for novel
compounds) can be found, in the online version, at http://
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S
O
S
6a
NH2
O
HO
O
H2N
LiBr, H2SO4
R
OCH3
R
OCH3
R
OCH3
Acetonitrile
Ref. 26
Acetone:H2O
1 h, 25 oC
Br
Br
4a-k
5
H2N
O
NH2
1a-k
S
S
6b-d
6e
H2N
R1
2a-g
O
S
3a-j
H2N
N
N
H
H
R
OCH3
R
OCH3
Acetonitrile
1 h, 25 oC
Acetonitrile
1 h, 25 oC
S
Br
Br
R1
N
R: Aryl, Alkyl
R1: CH3 (6b), CH2=CHCH2 (6c), NH2 (6d)
H2N
H2N
N
H
H
Scheme 1. Synthesis of S-allylic isothiouronium salts 1-3 from allylic bromides 5 and
thioureas 6.
25

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Figure 1. Flow cytometry analysis of cell cycle after 24-h exposure with IC50 of the
tested compounds. (A) Representative histogram of undamaged DNA content
26

ACCEPTED MANUSCRIPT
distribution in the cell cycle. (B) Representative histogram of DNA fragmentation in
sub-G1 and G2/M arrest induced by treatment with compound 1k. (C) Cell cycle
distribution of L1210 cells exposed to N-unsubstituted salts, (D and E) to N-
substituted salts, and (F) to aliphatic allylic salts. G0/G1, S, and G2/M represent cell
cycle phases and sub-G1 indicates DNA fragmentation. Each phase was calculated
using the Flowing 2.5.1 software and compared to the control group (without
treatment). Data represent mean ± SD of three independent experiments.
***p<0.0001, **p<0.001, *p<0.005.
27

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Figure 2. Cell death after 24-h exposure with IC50 of the tested compounds. Flow
cytometry with double staining annexin V-FITC and propidium iodide. (A)
Representative dotplot of L1210 cells labeled with Annexin V/FITC and PI. Alive cells
are double negative for Annexin V/PI, Annexin
V
positive indicates
phosphatidylserine externalization, PI positive indicates cell membrane disruption
and double positive indicates both events at the same cell. (B) Quantification of
L1210 cells labeled with Annevin V/FITC and PI after compounds exposure for 24 h.
Percentage of cell death was calculated using the Flowing 2.5.1 program followed by
GraphPad Prism 5 through one-way ANOVA analysis followed by Dunnet post hoc
test. Data represent mean ± SD of four independent experiments. ***p < 0.0001, **p
< 0.001.
28