11a-N-Tosyl-5-deoxi-pterocarpan (LQB-223), a promising prototype for targeting MDR leukemia cell lines

Article abstract of DOI:10.1016/j.ejmech.2014.03.039

Aza-deoxi-pterocarpans (1) were synthesized through palladium-catalyzed aza-arylation of dihydronaphtalen, and showed antineoplastic effect on MDR leukemic cell lines (K562, Lucena-1 and FEPS). Compounds 1c-d were prepared to identify the pharmacophoric g

Full text of DOI:10.1016/j.ejmech.2014.03.039

European Journal of Medicinal Chemistry 78 (2014) 190e197
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
11a-N-Tosyl-5-deoxi-pterocarpan (LQB-223), a promising prototype
for targeting MDR leukemia cell lines
, ,
,c, ,
Camilla D. Buarque a 1, Eduardo J. Salustiano b 1, Kevin C. Fraga a, Bruna R.M. Alves a,
*
*
,
Paulo R.R. Costa c
*
a Departamento de Química, Pontifícia Universidade Católica do Rio de Janeiro, Rua Marquês de São Vicente 225, Gávea-Rio de Janeiro, RJ 22435-900, Brazil
b Laboratório de Imunologia Tumoral, Instituto de Bioquímica Médica Leopoldo de Meis, Centro de Ciências da Saúde, Bloco H, Universidade Federal do Rio
de Janeiro, RJ 21941-590, Brazil
c Laboratório de Química Bioorgânica, Núcleo de Pesquisas de Produtos Naturais, Centro de Ciências da Saúde, Bloco H, Universidade Federal do Rio de
Janeiro, RJ 21941-590, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Aza-deoxi-pterocarpans (1) were synthesized through palladium-catalyzed aza-arylation of dihy-
dronaphtalen, and showed antineoplastic effect on MDR leukemic cell lines (K562, Lucena-1 and FEPS).
Compounds 1ced were prepared to identify the pharmacophoric group responsible for the activity as
well as compounds 2aec were prepared to evaluate the structural requirements in the D-ring. LQB-223
(1b) is the most promising antileukemic agent since it was the most active on MDR cells without
detectable toxicity to normal immune system cells.
Received 21 December 2013
Received in revised form
13 March 2014
Accepted 13 March 2014
Available online 15 March 2014
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Aza-heck
Aza-arylation
Antileukemic activity
Sulfonamide
1. Introduction
the nitrogen atom in the C-ring (compounds 1c,d) to see if this
moiety is part of the pharmacophore. In analogues 2a,b, halogen
During the course of our studies aiming for the discovery of new
pterocarpan-based antineoplastic compounds [1e6], we reported
the synthesis of aza-pterocarpan 1a and its deoxi-analogue 1b
(Fig. 1) [7].
These compounds presented antineoplastic activity on leukemic
cell lines (K562 and HL-60) as well as colon cancer (HCT-8), glio-
blastoma (SF-295) and melanoma (MDA-MB435) and 1b was
shown to be the most potent [7].
atoms at D-ring were introduced in order to modify the partition
coefficient. In addition, halogen bonds occur in many biological
systems, being employed by nature for the molecular recognition.
These bonds may target diverse set of relevant proteins due to the
short halogen$$$N/O/S contacts in the binding pocket [14]. Com-
pound 2c was planned to increase the cytotoxicity, since com-
pounds containing the nitro group may generate reactive oxygen
species through the action of nitroreductases [15,16].
As the development of multidrug resistance (MDR) is a major
problem in cancer chemotherapy, we decided to extend the study
of 1b to human leukemia cell lines with MDR phenotype and
evaluate its mechanism of action. We also present some structuree
activity relationships (SAR) studies aiming to identify the phar-
macophoric groups in this new class of compounds. Since sulfon-
amide moiety is present in the structure of several bioactive
compounds [8e13], our first goal was to modify the substituent at
2. Results and discussion
2.1. Chemistry
2-Iodoanilines 5a,b were prepared by the reaction of 2-
iodoaniline 3a with tosyl chloride 4a or mesyl chloride 4b,
respectively, in pyridine (condition i) while the reaction of 3a with
benzylchloroformate (4c) in basic media (condition ii) or acetic
anhydride (4d) in acid media (condition iii) led to 2-iodoanilines
5c,d (Table 1). Finally, 5e was prepared by reaction between 3b
and 4a. All these products were obtained in good to excellent yields.
* Corresponding authors.
E-mail addresses: camilla-buarque@puc-rio.br (C.D. Buarque), salustiano@
bioqmed.ufrj.br (E.J. Salustiano).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.03.039
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

C.D. Buarque et al. / European Journal of Medicinal Chemistry 78 (2014) 190e197
191
Scheme 1. Halogenation and nitration of 1b.
the results obtained with the parental K562. The latter is derived
from a patient with chronic myeloid leukemia and known to be
resistant against oxidative stress damage due to its high expression
of glutathione and catalase [19,4]. Lucena-1 and FEPS, both derived
from K562 as described [17,18], among other mechanisms stand out
for their higher expression and activity of ABCB1 and ABCC1 pro-
teins, responsible for the MDR phenotype [20]. The cytotoxic effect
of the six compounds is summarized on Table 2. The results ob-
tained showed that for K562 and Lucena-1 the presence of the tosyl
group at the nitrogen atom is essential for the antineoplastic effect
while the substitution at the D-ring led to a decrease in potency,
being 1b 4e25-fold more potent than the other compounds.
Despite the fact that FEPS is described as more resistant to
chemotherapeutic agents such as vincristine (VCR), daunorubicin
(DNR), cisplatin and even imatinib mesylate [18], the IC50 obtained
for all compounds was consistently lower in this cell line when
compared with K562 and Lucena-1. Compound 1b was also in this
case 2e10-fold more potent than the other compounds. These re-
sults suggest that the new synthetic aza-deoxi-pterocarpans exert
cytotoxic activity independent of the presence of the MDR proteins
ABCB1 and ABCC1.
Fig. 1. Aza-deoxi-pterocarpans 1 and 2.
As previously described [7], compound 1b was prepared in good
yield by a palladium-catalyzed aza-arylation of dihydronaphthalen
(6) with N-tosyl-o-iodoanilin (5a), as shown in Table 1. Under
thermal conditions the reaction took 4 h to completion leading to
1b in 85% yield. It was also possible to accomplish the aza-arylation
of 6 with aniline derivatives 5b,c and 5e, leading to aza-deoxi-
pterocarpans 1ced and 2a in reasonable to good yields. In
contrast, a low yield of 1e was obtained when 6 was allowed to
react with N-acetyl orto-iodoanilin (5d).
To prepare compounds 2b and 2c we took advantage of the great
reactivity of the D-ring in 1b over the A-ring for electrophilic
substitution reaction. These compounds were obtained from 1b by
halogenation with NCS in chloroform solution, or by nitration with
fuming nitric acid, respectively. As expected, these reactions were
chemoselective for D-ring (Scheme 1).
2.2.2. DNA incorporation
Since 1b was the most active compound evaluated, it was
selected for further tests. In order to evaluate if this compound
could lead to inhibition of cellular proliferation, we incubated K562,
2.2. Evaluation of biological activity
Lucena-1 and FEPS with 1.25, 2.5 and 5.0 mM of this compound for
24e72 h and measured DNA duplication by incorporation of [3H]-
thymidine after 6 h. As seen in Fig. 2, both K562 and Lucena-1
showed a dose and time-dependent inhibition in [3H]-thymidine
2.2.1. Anticancer activity
The antineoplastic effect of the new aza-deoxi-pterocarpans
was evaluated on two human erythroleukemic cell lines with
MDR phenotype, Lucena-1 [17] and FEPS [18] and compared with
incorporation. After 24 h of exposure to 2.5 mM of 1b proliferation
of all three cell lines was inhibited in nearly 25%, and the sub-toxic
Table 1
Synthesis of compounds 1b-d and 2a.
I
I
H
H
6
4a-e
R2
H2N
R2
N
R2
H
iv
i, ii or iii
R1
N
1 (+/-)
R
3a: R2 = H
3b: R2 = Br
5a-e
1b-e, 2a
i. TsCl(4a) ou MsCl (4b), pyridine, 800C, 12h
ii. ClCbZ (4c), NaOH, t.a, 1h
iv. acetone, 10 mol% Pd(OAc)2, 3 equiv. Ag2CO3, 700C, 4 hours
iii. Ac2O (4d), H2SO4, t.a, 5 min.
R1
R2
Compound
Yield (%)
R1
R2
Compound
Yield (%)
Ts
H
H
H
H
Br
5a
5b
5c
5d
5e
75
70
89
85
67
Ts
H
H
H
H
Br
1b
1c
1d
1e
2a
85
45
67
10
75
Ms
Cbz
Ac
Ts
Ms
CbZ
Ac
Ts

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C.D. Buarque et al. / European Journal of Medicinal Chemistry 78 (2014) 190e197
Table 2
2.2.4. Toxicity against normal lymphocytes
Effect of the aza-deoxi-pterocarpans on the growth of human leukemic cells.
For a drug to be successfully used in the clinic toxicity to normal
cells should be minimal; hence, selectivity of a newly synthesized
compound should be assessed with care. In order to investigate if
1b could sensitize normal cells, we collected a population of sple-
nocytes from healthy Swiss mice and cultured these cells in pres-
Compound
K562
Lucena-1
FEPS
2.12 ꢀ 0.73
1b
1c
1d
2a
2b
2c
2.90 ꢀ 0.65
40.64 ꢀ 16.12
>50
16.60 ꢀ 1.79
28.65 ꢀ 6.79
12.91 ꢀ 1.15
2.49 ꢀ 0.14
27.03 ꢀ 10.90
>50
14.33 ꢀ 9.93
34.77 ꢀ 5.50
23.23 ꢀ 3.68
18.07 ꢀ 11.98
20.65 ꢀ 5.05
6.76 ꢀ 3.36
15.42 ꢀ 6.13
4.20 ꢀ 1.21
ence or absence of 5
induce proliferation in immune system cells [22]. As shown in
Fig. 4, viability of normal lymphocytes incubated with 10 M 1b
mg/mL of ConA, a lectin known for its ability to
m
Results are reported as IC50 values ꢀ SD (concentration required to inhibit cell
was higher than 90%, regardless of their activation by ConA
(PIþ ¼ 3.67%; PI þ ConA ¼ 7.69%). Different types of immune sys-
tem cells can be obtained from the spleen; mainly T and B lym-
phocytes, but also macrophages and dendritic cells [23,24]. Thus,
the low toxicity against ex vivo cultured lymphocytes is indicative
of a lower toxicity against the immune system.
growth by 50%) in M. Data represent means of three independent experiments,
m
with each concentration tested in triplicate. Assays were performed as described in
the Experimental Section.
concentration of 1.25 mM was not able to induce the same effect
even after 72 h. When cells were incubated with a greater con-
centration than the IC50 inhibition was as high as 50% after 24 h.
FEPS cells presented a different pattern, as inhibition after 48 h
seems higher than in 72 h. Since the doubling time of these cells is
roughly 24 h [18], viable cells at 48 h could still be incorporating
[3H]-thymidine after 72 h, leading to this discrepancy. Taken
together, data suggests that the mechanism of action exerted by 1b
involves impairment of DNA duplication, at least in K562 and
Lucena-1 cells.
3. Conclusions
In conclusion, the newly synthesized aza-deoxi-pterocarpans
exhibited antineoplastic effect depending on the pattern of sub-
stitution at the nitrogen in C-ring and at the D-ring. Our data
suggests the importance of the tosyl group (a sulfonamide), since
the cytotoxic effect is dramatically reduced upon removal of tosyl
group from the structure (compounds 1c and 1d). In other N-tosyl
derivatives of 1b substituted at the D-ring (2aec), IC50 values were
lower when electron withdrawing groups such as the halogens
chlorine and bromine and a nitro group presented at D ring. The
most active compound, 1b, exerts its cytotoxicity by inhibition of
DNA duplication independent of MDR phenotype. Furthermore, 1b
seems to lead the three cell lines to accumulate at the G2 phase of
the cycle. Interestingly, FEPS cells are only affected later than K562
and Lucena-1. According to data obtained this arrest precedes the
apoptotic cell death, since DNA fragmentation was observed after
72 h. Finally, 1b demonstrated a selective antitumoral activity,
without reducing the viability of immune system cells. Since
multidrug resistance is the most important cause of therapeutic
failure, our data suggest that 1b could be a candidate for treatment
of unresponsive leukemias with a lower chance of side effects.
2.2.3. Cell cycle analysis
Furthermore, our next step was to find out if this inhibition of
nucleotide incorporation would lead to disruption of the cell cycle.
To test this hypothesis, we cultured K562, Lucena-1 and FEPS
within the same conditions of the previous experiment, and
analyzed the percentage of cells in each phase of the cell cycle by
flow cytometry. In Fig. 3A, it can be observed that K562 and its MDR
counterpart Lucena-1 presented similar behavior after 24 h of in-
cubation with 2.5 mM of 1b, consistent with the inhibition of cell
proliferation previously observed. Both cells were arrested in the
G2 phase of the cycle (G2/M K562 ¼ 31.24%; G2/M Lucena-
1 ¼44.02%) and it was maintained up to 48 h in Lucena-1. As seen in
Fig. 3B, FEPS cells exhibited a similar pattern, but in a timely
fashion; only after 48 h of incubation cells accumulated in G2 phase
(G2/M FEPS ¼ 33.89%). This could be explained by the lengthier cell
cycle demonstrated by FEPS when compared to K562 and Lucena-1
[18]. After 72 h massive DNA fragmentation was observed (Fig. 3Ce
H K562 ¼ 64.73%; H Lucena-1 ¼ 34.79%; H FEPS ¼ 55.69%), indic-
ative of apoptotic cell death. These results corroborate previous
findings of our group, when three hybrid pterocarpanquinones
bearing similarities with 1b on the C and D-rings, were described as
capable of inducing apoptosis in both K562 and Lucena-1 cells after
72 h [3,21].
4. Experimental section
4.1. Chemistry
Melting points were determined with
a ThomaseHoover
apparatus and are uncorrected. Column chromatography was per-
formed on silica gel 230e400 mesh (Aldrich). 1H NMR spectrum
was recorded on a Bruker Avance 400 (400.013 MHz) spectrometer
at room temperature. All J values are given in Hz. Chemical shifts
are expressed in parts per million downfield shift from
Fig. 2. Inhibitory effect of 1b on cellular proliferation. Proliferation was measured by incorporation of [3H]-thymidine into the DNA. After 24e72 h cultured with 1.25, 2.5 and 5.0
mM
of 1b, 0.5
m
Ci of [3H]-thymidine was added to each cell line. Proliferation of control cells was considered as 100%. Bars represent means þ SD of three independent experiments, with
each concentration tested in triplicate.

C.D. Buarque et al. / European Journal of Medicinal Chemistry 78 (2014) 190e197
193
Fig. 3. Effect of compound 1b on the cell cycle distribution of human leukemic cell lines. Cells were treated with 2.5 mM of 1b for 24, 48 or 72 h, then stained with PI to analyze DNA
content by flow cytometry. (A), cells incubated for 24 h; (B), incubated for 48 h; (C), incubated for 72 h. Histograms representative of three independent experiments; H, hypodiploid
cells (apoptotic); G0/G1, S, G2/M, phases of the cell cycle; >4n, cell doublets. Numbers represent the percentage of cellular population in each phase of the cycle.
tetramethylsilane as an internal standard, and reported as position
a Bruker Avance 400 (100.003 MHz) spectrometer at room tem-
perature with complete proton decoupling. Data are expressed in
parts per million downfield shift from tetramethylsilane as an in-
ternal standard and reported as position (dC).
(
dH) (relative integral, multiplicity (s ¼ singlet, d ¼ doublet,
dd ¼ double doublet, dt ¼ double triplet, m ¼ multiplet)), coupling
constant (J Hz) and assignment. 13C NMR spectrum was recorded on

194
C.D. Buarque et al. / European Journal of Medicinal Chemistry 78 (2014) 190e197
Fig. 3. (continued).
4.2. Preparation of aryl-sulfonamides
7.66e7.62 (4H, m); 7.32e7.26 (1H, m); 7.21 (1H, d, J ¼ 8.1 Hz); 6.84e
6.81 (2H, m) LRMS (EI) m/z 218, 91.
A solution of aryl-sulphonyl chloride (6.5 mmol) and 2-iodo-
aniline (5 mmol) in 1 mL of pyridine was stirred overnight at 80 ꢁC.
The mixture was concentrated under reduced pressure and the
products were purified either by recrystallization from ethanol
followed hexane or by column chromatography (hexane/ethyl
acetate).
4.2.2. Substance 5b
After column chromatography using n-hexane/ethyl acetate
(90:10) as eluent, this compound was obtained as a white solid in
70%, mp 90e92 ꢁC 1H (400 MHz, CDCl3)
d (ppm): 7.82 (1H, d,
J ¼ 9.3 Hz); 7.63 (2H, d, J ¼ 6.7 Hz); 7.38 (1H, d, J ¼ 7.3 Hz); 6.93 (1H,
t, J ¼ 7.7 Hz); 3.02 (3H, m); LRMS (EI) m/z 297, 218.
4.2.1. Substance 5a
After recrystallization from ethanol, followed by washing
several times with hexane, the compound was obtained as a white
4.2.3. Substance 5e
After column chromatography using n hexane/ethyl acetate
(95:5) as eluant, this compound was obtained as a white solid in
solid in 70% yield, mp 88e90 ꢁC 1H NMR (400 Mz, CDCl3)
d (ppm):
Fig. 4. Effect of compound 1b on the viability of murine lymphocytes. Splenocytes were exposed to 10 mM of 1b for 72 h, in presence or absence of 5 mg/mL of ConA. Cells were
collected on a flow cytometer, and the region corresponding to the lymphocytes was gated. Fluorescence of PI was measured, and PI-positive lymphocytes were considered non-
viable. Histograms representative of three independent experiments. Numbers represent the percentage of viable and non-viable lymphocytes.

C.D. Buarque et al. / European Journal of Medicinal Chemistry 78 (2014) 190e197
195
67%, mp 92e94 ꢁC 1H NMR (400 MHz, CDCl3)
J ¼ 2.0 Hz); 7.62 (2H, d, J ¼ 8.2 Hz); 7.53 (1H, d, J ¼ 8.7 Hz); 7.43 (1H,
dd, J ¼ 8.7 and 2.0 Hz); 7.24 (2H, d, J ¼ 8.2 Hz); 2.40 (3H, s) LRMS (EI)
m/z 451, 296.
d
(ppm): 7.78 (1H, d,
40%, mp 136e138 ꢁC; IR (neat) 2953, 2928, 1341, 1155 cmꢃ1 1H NMR
(400 MHz, CDCl3)
(ppm): 7.84 (1H, d, J ¼ 7.7 Hz); 7.41 (1H, d,
d
J ¼ 7.2 Hz); 7.25e7.13 (5H, m), 6.98 (1H, d, J ¼ 7.4 Hz), 5.55 (1H, d,
J ¼ 8.9 Hz), 4.09e4.05 (1H, m), 2.83 (3H, s), 2.66e2.53 (2H, m),
2.34e2.30 (1H, m), 2.25e2.19 (1H, m) 13C NMR (100 MHz, CDCl3):
141.98 (C); 138.07 (C); 135.03 (C); 134.33 (C); 130.50 (CH); 128.24
(CH); 128.11 (CH); 127.54 (CH); 126.87 (CH); 125.47 (CH); 123.96
(CH); 118.21 (CH); 64.36 (CH); 40.32 (CH); 37.36 (CH3); 25.24 (CH2);
24.69 (CH2) HRMS calc for C17H17NSO2 [MþNa]þ: 322.0872 Found:
322.0876.
4.2.4. Substance 5c
To 2-iodoaniline (756 mg, 7.46 mmol) was added an aqueous
sodium hydroxide solution (3.5 mL, 186 mmol). The resulting sus-
pension was stirred vigorously as benzylchloroformate (0.75 mL,
3.47 mmol) was added slowly from an addition funnel at room
temperature. The progress of the reaction was followed by loss of
starring by TLC. When 2-iodoaniline was completely consumed
(30 min), the reaction mixture was poured into ethyl acetate. The
ethyl acetate layer was separated and the aqueous layers were
extracted two times with ethyl acetate (50 mL). The organic layer
was combined, dried over Na2SO4, filtered, and concentrated under
pressure. After column chromatography using n-hexane/ethyl ac-
etate (95:5) as eluent, this compound was obtained as a white solid
4.3.3. Substance 1d
After column chromatography using n-hexane/ethyl acetate
(95:5) as eluant, the compound was obtained as a white solid in
63%, mp 112e115 ꢁC; IR (neat) 3038, 1709 cmꢃ1 1H NMR (400 MHz,
CDCl3)
d (ppm): 7.45e7.43 (2H, m); 7.39e7.33 (5H, m); 7.17e7.13
(5H, m); 7.02e6.97 (2H, m); 5.73 (1H, s broad); 5.34 (2H, s); 3.96e
3.94 (1H, m); 2.69e2.61 (1H, m); 2.54 (1H, t, J ¼ 4.3 Hz); 2.50 (1H, t,
in 92%, mp 58e60 ꢁC. 1H NMR (400 MHz, CDCl3)
d (ppm): 8.07 (1H,
J ¼ 4.3 Hz); 2.20e2.15 (2H, m); 13C NMR (100 MHz, CDCl3)
d (ppm):
d, J ¼ 8.2 Hz); 7.75 (1H, dd, J ¼ 7.9 and 1.5 Hz); 7.44e7.31 (6H, m);
154.0 (C); 138.8 (C); 138.6 (C); 136.1 (C); 135.6 (C); 134.0 (C); 128.6
(CH); 128.6 (CH); 128.6 (CH); 128.4 (CH); 128.2 (CH); 128.1 (CH);
127.6 (CH); 127.2 (CH); 126.5 (CH); 123.5 (CH); 123.4 (CH); 67.7
(CH2); 61.3 (CH); 39.7 (CH); 38.5 (CH); 25.9 (CH2); 24.8 (CH2) HRMS
calc for C24H21NO2: 378.1465 [MþNa]þ Found: 378.1450.
6.80 (1H, dt, J ¼ 7.9 and 1.5 Hz) LRMS (EI) m/z 353, 91.
4.2.5. Substance 5d
One drop of concentrated H2SO4 was added to a stirred solution
of 2-iodoaniline (100 mg; 0.46 mmol) in acetic anhydride (100 mL).
The resulting mixture was stirred at room temperature for 5 min
and then quenched with water and extracted with ethyl acetate
(3 ꢂ 5 mL). The combined organic layers were washed with water,
brine and dried with anhydrous sodium sulfate. The solvent was
removed and the crude product was crystallized from ethanol to
give the N-acetyl derivative as a crystalline solid in 85% yield, mp:
4.3.4. Substance 2a
After column chromatography using n-hexane/ethyl acetate
(98:2) as eluant, the compound was obtained as a white solid in
79%, mp 186e188 ꢁC 1H NMR (400 MHz, CDCl3)
d (ppm): 7.95 (1H, d,
J ¼ 7.8 Hz); 7.52 (2H, d, J ¼ 8.0 Hz); 7.48 (1H, d, J ¼ 8.5 Hz); 7.32e7.27
(2H, m); 7.20 (2H, d, J ¼ 7.9 Hz); 7.17e7.13 (2H, m); 6.94 (1H, d,
J ¼ 7.5 Hz); 5.41 (1H, d, J ¼ 8.6 Hz); 3.11e3.07 (1H, m); 2.57e2.46
(2H, m); 2.39 (3H, s); 2.09e1.97 (2H, m) 13C NMR (100 MHz, CDCl3):
144.3(C), 141.5(C), 138.7(C), 137.3(C), 135.5(C), 133.9 (C), 130.9 (CH);
130.4 (CH), 129.8 (CH), 128.1 (CH), 127.6 (CH), 127.1 (CH), 126.9 (CH),
126.6 (CH), 64.3 (CH), 39.7 (CH), 24.8 (CH2), 23.7 (CH2), 21.7 (CH3)
HRMS calc for C23H20BrNO2S: 476.0290 [MþNa]þ Found:476.0275.
102e104 ꢁC. 1H RMN (CDCl3, 400 MHz):
d (ppm) 8.21 (1H, d,
J ¼ 8.2 Hz); 7.78 (1H, d, J ¼ 7.9); 7.34 (1H, t, J ¼ 7.9 Hz); 6.84 (1H, t,
J ¼ 8.2 Hz); 2.23 (3H,s) LRMS (EI) m/z 219, 134.
4.3. Aza-arylation reactions: synthesis of 1bed and 2a
To a stirred solution of 6 (0.5 mmol), tosyl-2-iodoaniline 5a or 5e
(0.75 mmol), mesyl-2-iodo-aniline 5b (0.75 mmol), Cbz-2-
iodoaniline 5c (0.75 mmol) or Acetyl-2iodoaniline 5d
(0.75 mmol), in acetone (5 mL), silver-carbonate (1.5 mmol) and
Pd(OAc)2 (0.005 mmol) were added. The reaction mixture was
refluxed for 4 h and filtered in celite with ethyl acetate. The organic
layer was washed with brine, dried over anhydrous Na2SO4 and
concentrated. The crude product was washed in n-hexane and
purified by flash chromatography on silica.
4.3.5. Substance 2b
After column chromatography using n-hexane/ethyl acetate
(99:1) as eluent, the compound was obtained as a white solid in
35%, mp 176e180 ꢁC 1H NMR (400 MHz, CDCl3)
d (ppm): 7.96 (1H, d,
J ¼ 7.8 Hz); 7.54e7.51 (3H, m); 7.29e7.27 (1H, m); 7.20e7.13 (5H,
m); 6.94 (1H, d, J ¼ 7.5 Hz); 5.41 (1H, d, J ¼ 8.6 Hz); 3.11e3.07 (1H,
m); 2.56e2.46 (2H, m); 2.38 (3H, s); 2.09e1.96 (2H, m) 13C NMR
(100 MHz, CDCl3): 144.2(C), 140.9(C), 138.3(C), 137.3(C), 135.3(C),
133.9 (C), 131.2 (C); 130.4 (CH), 129.7 (CH), 128.1 (CH), 128.0(CH);
127.6 (CH), 127.1 (CH), 126.9 (CH), 123.7 (CH), 120.9 (CH); 64.3 (CH),
39.5 (CH), 24.9 (CH2), 23.2 (CH2), 21.7 (CH3) HRMS calc for C23 H20
ClN O2S: [MþNa]þ 432.0795 Found: 432.0788.
4.3.1. Substance 1b
After column chromatography using n-hexane/ethyl acetate
(1:99) as eluant, the compound was obtained as a yellow solid in
85% yield; mp 160e165 ꢁC; IR (neat) 3060, 2923,1349,1165 cmꢃ1 1H
NMR (CDCl3, 400 MHz):
d
(ppm): 8.0 (1H, d, J ¼ 7.8 Hz); 7.61 (1H, d,
J ¼ 7.9 Hz); 7.51 (1H, d, J ¼ 8.1 Hz); 7.29e7.09 (7H, m); 7.04 (1H, d,
J ¼ 7.0 Hz); 6.93 (1H, d, J ¼ 7.5 Hz); 5.42 (1H, d, J ¼ 8.5 Hz); 3.13e3.11
(1H, m); 2.54e2.45 (2H, m); 2.37 (3H, m); 2.13e2.09 (1H, m); 2.06e
4.3.6. Substance 2c
After filtration in celite with ethyl acetate, the product was ob-
tained in 50% yield as a yellow solid, mp 190e192 ꢁC 1H NMR
1.98(1H,m); 13C NMR (CDCl3, 100 MHz):
d
(ppm): 143.8 (C);
(400 MHz, CDCl3)
d
(ppm): 8.12 (1H, dd, J ¼ 8.8 and 2.3 Hz); 7.96
142.1(C); 137.6 (C); 136.2 (C); 135.6 (C); 134.4 (C); 30.5 (CH); 129.6
(CH); 129.6 (CH); 127.9 (CH); 127.8 (CH); 127.3(CH); 127.1(CH);
127.1 (CH); 126.8 (CH); 125.7 (CH); 123.4 (CH); 119.9 (CH); 64.0
(CH); 39.5 (CH); 24.8 (CH2); 23.6 (CH2); 21.5 (CH3). HRMS calc for
(1H, d, J ¼ 7.8 Hz); 7.92 (1H, s); 7.70 (1H, d, J ¼ 8.8 Hz); 7.55 (1H, d,
J ¼ 8.2 Hz); 7.31e7.16 (5H, m); 6.97 (1H, d, J ¼ 7.6 Hz); 5.56 (1H, d,
J ¼ 8.9 Hz); 3.34e3.28 (1H, m); 2.55e2.53 (2H, m); 2.39 (3H, s);
2.13e2.09 (1H, m); 13C NMR (100 MHz, CDCl3): 147.9(C); 145.5(C);
144,8(C); 137.6(C), 137.3(C); 135.5(C); 133.2(C); 130.4(CH);
129.9(CH); 128.3(CH); 128.0(CH); 127.2(CH); 126.8(CH); 124.6(CH);
119.5(CH); 118.8(CH); 65.2(CH); 39.6(CH); 24.9(CH2); 23.8(CH2);
21.5(CH3) HRMS calc for C22H19N2NaSO4: 443.1036 [MþNaþ]
Found: 443.1034.
C
23H21NO2S [MþNa]þ: 398.1185 Found 396.1191.
4.3.2. Substance 1c
After column chromatography using n-hexane/ethyl acetate
(95:5) as eluent, the compound was obtained as a white solid in

196
C.D. Buarque et al. / European Journal of Medicinal Chemistry 78 (2014) 190e197
4.4. Cell lines
4.8. Cell cycle assay
The human erythroleukemic cell lines K562 and its multidrug
resistant (MDR) variants Lucena-1 and FEPS were all maintained in
RPMI-1640 medium, supplemented with 50 mM b-mercaptoetha-
The cell cycle assay was performed by quantifying cells in
each phase of the cycle after 24, 48 and 72 h in presence or
absence of 1b. Cells were maintained in culture at a concentra-
tion of 2 ꢂ 104 cells/mL in 24-well plates with RPMI-1640 sup-
plemented with 10% FCS and kept in 5% CO2 atmosphere at 37 ꢁC,
for periods aforementioned. Cell concentrations were adjusted to
105 cells per well, washed with Hank’s balanced salt solution
nol, 25 mM HEPES, pH adjusted to 7.4 with NaOH, 60 mg/L peni-
cillin, 100 mg/L streptomycin (all obtained from Sigma Chemical
Co., Saint Louis, USA) and 10% fetal bovine serum (FBS) (Cultilab,
São Paulo, Brazil), inactivated at 56 ꢁC for 1 h.
b-Mercaptoethanol
was added due to its capacity of reducing reactive oxygen species
and protective effect against toxic metabolites produced by
cultured cells, thus improving the microenvironment and pro-
moting in vitro cell growth [28]. Lucena-1 and FEPS cells were
developed in our laboratory by continuous exposure of K562 cells
to increasing concentrations of the cytotoxic drugs vincristine
sulfate (VCR) (Sigma) and daunorubicin hydrochloride (DNR)
(Sigma), as described before [17,18]. 60 nM VCR and 500 nM DNR
were added to Lucena-1 and FEPS cultures respectively in order to
maintain the MDR phenotype. All cells were passaged at a con-
centration of 2 ꢂ 104 cells/mL every three days and kept at 37 ꢁC in a
5% CO2 humidified environment. Before the experiments, vincris-
tine and daunorubicin were removed from the cultures and cell
lines were resuspended in medium with fetal bovine serum. The
cell lines Lucena-1 and FEPS were kindly donated by Dr. Vivian M.
Rumjanek.
(HBSS) (Sigma), and resuspended in 250
a 5ꢂ concentrated cell cycle solution containing 50
m
L of HBSS and 50
mL of
m
g/mL pro-
pidium iodide (PI), 1 mg/mL RNAse and 0.2% Triton X-100 (all
obtained from Sigma). Subsequently, cells were incubated at
room temperature for 15 min and analyzed by flow cytometry.
This assay was performed to analyze cell cycle profile and assess
DNA fragmentation, which was quantified by the percentage of
cells in sub-G0/G1 phase.
4.9. Animals
Swiss mice were bred in the animal facilities at the Instituto de
Bioquímica Médica Leopoldo de Meis from UFRJ (IBqM-UFRJ), and
housed under standard laboratory conditions (20e25 ꢁC, 12-h light
regimen) with free access to water and standard chow (ad libitum).
Two-month female mice were used in experiments. Procedures
were approved by the Centro de Ciências da Saúde Ethics Comitee
for Animal Use (CEUA-CCS, UFRJ) under the protocol number
IBQM082.
4.5. Cell treatment
Leukemic cell lines were exposed to the different aza-
pterocarpans in culture for 72 h. Stock solutions of 25 mM dis-
solved in DMSO were prepared, and further dilutions were made in
4.10. Isolation of splenocytes
Mice were anaesthetized with ethyl ether (Reagen, Rio de
Janeiro, Brazil) and euthanized by cervical dislocation. Spleens were
surgically removed and macerated with the help of a sterile rubber
pressed through a nylon mesh, and fresh cell suspension was ho-
mogenized in 3 mL cold RPMI-1640 medium supplemented with
5% FCS. Afterwards, cell suspension was washed and further diluted
with cold PBS, incubated with 0.08% Trypan Blue dye (Sigma) and
counted in an optical microscope.
RPMI-1640 medium. Briefly, 2 ꢂ 104 cells/mL in 200
mL were seeded
in 96-well microtiter plates in drug-free medium or in medium
containing different concentrations of each compound and main-
tained for 72 h at 37 ꢁC in an atmosphere of 5% CO2, and cell
viability was then measured.
4.6. Cell viability
4.11. Ex vivo viability assessment of splenocytes
Cell viability was determined by the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay
[25e27]. MTT can be reduced by dehydrogenases present in active
mitochondria of living cells. After incubation in presence or
Viability of the normal splenocytes was measured by quanti-
fying the percentage of PI-positive cells after 72 h exposure to 1b.
Cells were maintained in culture at
a
concentration of
absence of the compounds being tested, 20
(Sigma) was added to each well. Plates were then kept at 37 ꢁC in
L of DMSO
mL of MTT (5 mg/mL)
5 ꢂ 105 viable cells/mL in 24-well plates with RPMI-1640 sup-
plemented with 10% FCS and kept in 5% CO2 atmosphere at 37 ꢁC.
5% CO2 for 3 h. After centrifugation at 200 ꢂ g, 200
m
5 mg/mL concanavalin A (ConA) (Sigma) was added to cultures in
(Sigma) was added to all wells in order to dissolve the dark blue
crystals formed by MTT reduction. Absorbance was measured with
order to stimulate cell proliferation. Cell concentration was
adjusted to 105 cells per well, washed with PBS supplemented
a
Tecan Sunrise ELISA reader at 490 nm (Tecan Group,
with 5% FCS (Sigma), and resuspended in 300
mL of PBS containing
Switzerland). Absorbance was directly proportional to the amount
of formazan (reduction product) present, indicating the percent-
age of living cells. The IC50 values were obtained by nonlinear
regression on the GraphPad Prism v4.0 program (GraphPad Soft-
ware, San Diego, USA).
50 g/mL propidium iodide (PI). Subsequently, cells were incu-
m
bated at room temperature for 15 min and analyzed by flow
cytometry.
4.12. Flow cytometry analysis
4.7. Cell proliferation assay
Ten thousand cells were examined under each condition using a
FACSCalibur flow cytometer (Becton, Dickinson and Company, San
Jose, USA). Cells were acquired based on forward and side scatter
parameters, representative of cell size and granulosity. The region
corresponding to the lymphocytes was gated, and fluorescence of PI
was measured. PI-positive lymphocytes were considered non-
viable. All analysis was performed using the Summit v4.3 soft-
ware (Dako Colorado, Inc., Fort Collins, USA).
Cell proliferation was assessed by incorporation of [3H]-thymi-
dine into the DNA of cells incubated with 1b. After different incu-
bation periods (24e72 h) with 1b, 0.5 m
Ci of [3H]-thymidine diluted
in RPMI-1640 was added to the cell cultures and the radioactivity
was measured in a PerkineElmer Tri-Carb 2810TR liquid scintilla-
tion counter (PerkineElmer Inc., Waltham, USA) after 6 h.

C.D. Buarque et al. / European Journal of Medicinal Chemistry 78 (2014) 190e197
197
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We would like to thank Prof. Vivian M. Rumjanek for the use of
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financial support from Fundação do Câncer (FAF), Conselho
Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico
(CNPq), Financiadora de Estudos e Projetos (FINEP), Instituto
Nacional de Ciência e Tecnologia (INCT) para o Controle do Câncer
and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado
do Rio de Janeiro (FAPERJ).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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files and InChiKeys of the most important compounds described in
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