Synthesis of hybrids thiazole-quinoline, thiazole-indole and their analogs:in vitroanti-proliferative effects on cancer cell lines, DNA binding properties and molecular modeling

Article abstract of DOI:10.1039/d1nj02105b

A convenient synthesis under ultrasound (US) irradiation of 4-thiazolidinone, thiazole, dihydrothiazole, and thiazine hybrid compounds containing quinoline and indole nucleus is described. All the title compounds were characterized by NMR and HRMS. The synthetic protocol affords highly selective conversions, short reaction times, simple work-up procedures, and good yields compared with conventional methods. All the synthesized compounds were tested forin vitrocytotoxic activity against glioblastoma (SF-295), leukemia (HL-60), and prostate cancer (PC-3) cell lines. Three compounds (4c-e) presented moderate to high activity against all cancer cell lines evaluated. Compound4cstood out with its promising cytotoxicity activity against the HL-60 cell line with an IC₅₀value of 2.41 μM and an SI of 10.5. The electrochemical behavior of4cwas studied using differential pulse voltammetry (DPV) on a glassy carbon electrode modified with dsDNA and with ssDNA in the solution. As a result, the pre-concentration of the compound on the dsDNA biosensor surface and modification of the oxidation currents of guanosine and adenosine bases in ssDNA experiments demonstrated an interaction between4cand DNA. The affinity of4cwas evaluated against ctDNA by exploring spectroscopic techniques, showing that this compound acts preferentially as a groove binder. Molecular docking and dynamics simulations proposed that4cinteractsviagroove binding and intercalation, corroborating the experimental results. The dominating interactions were conventional hydrogen bonds and van der Waals forces. Finally, our findings suggest the4cderivative to be a potential anticancer prototype against HL-60.

Full text of DOI:10.1039/d1nj02105b

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Synthesis of hybrids thiazole–quinoline,
thiazole–indole and their analogs: in vitro
anti-proliferative effects on cancer cell lines,
DNA binding properties and molecular modeling†
Cite this: New J. Chem., 2021,
45, 13847
a
a
Paulo Fernando da S. Santos-Junior,‡ Igor Jose dos S. Nascimento,‡
´
a
a
b
Edjan Carlos D. da Silva, Kadja Luana C. Monteiro, Johnnatan D. de Freitas,
Samaysa de Lima Lins, Thamilla Maria S. Maciel, Bruno C. Cavalcanti,
Jose de Brito V. Neto, Fabiane C. de Abreu, Isis M. Figueiredo,
Claudia do O´ Pessoa, Edeildo F. da Silva-Junior,
Joao X. de Araujo-Junior and Thiago M. de Aquino
c
d
e
e
c
d
´
d
e
a
Josue Carinhanha C. Santos,
´
´
a
a
*
˜
´
´
A convenient synthesis under ultrasound (US) irradiation of 4-thiazolidinone, thiazole, dihydrothiazole,
and thiazine hybrid compounds containing quinoline and indole nucleus is described. All the title
compounds were characterized by NMR and HRMS. The synthetic protocol affords highly selective
conversions, short reaction times, simple work-up procedures, and good yields compared with
conventional methods. All the synthesized compounds were tested for in vitro cytotoxic activity against
glioblastoma (SF-295), leukemia (HL-60), and prostate cancer (PC-3) cell lines. Three compounds (4c–e)
presented moderate to high activity against all cancer cell lines evaluated. Compound 4c stood out with
its promising cytotoxicity activity against the HL-60 cell line with an IC50 value of 2.41 mM and an SI of
10.5. The electrochemical behavior of 4c was studied using differential pulse voltammetry (DPV) on a
glassy carbon electrode modified with dsDNA and with ssDNA in the solution. As a result, the pre-
concentration of the compound on the dsDNA biosensor surface and modification of the oxidation
currents of guanosine and adenosine bases in ssDNA experiments demonstrated an interaction between
4c and DNA. The affinity of 4c was evaluated against ctDNA by exploring spectroscopic techniques,
showing that this compound acts preferentially as a groove binder. Molecular docking and dynamics
simulations proposed that 4c interacts via groove binding and intercalation, corroborating the experimental
results. The dominating interactions were conventional hydrogen bonds and van der Waals forces. Finally,
our findings suggest the 4c derivative to be a potential anticancer prototype against HL-60.
Received 29th April 2021,
Accepted 9th June 2021
DOI: 10.1039/d1nj02105b
rsc.li/njc
Introduction
Cancer goes back to ancient history, being found in Egyptian
a
Laboratory of Medicinal Chemistry, Institute of Pharmaceutical Sciences, Federal
University of Alagoas-UFAL, Campus A. C. Sim o˜ es, 57072-900, Macei o´ , Alagoas,
Brazil. E-mail: thiago.aquino@iqb.ufal.br
1
,2
mummies dating back to more than 3000 years B. C. with
global economic impact measured in 2010 at approximately
b
c
3
Instrumental Analysis Laboratory, Federal Institute of Alagoas-IFAL, 57020-600,
Macei o´ , Alagoas, Brazil
US$1.16 trillion. Currently, it is the second leading cause of
death in the world (1 in 6 deaths), with about 18.1 million cases
and 9.6 million deaths reported in 2018, including lung cancer
Laboratory of Electrochemistry and Microsystems for Analysis, Institute of
Chemistry and Biotechnology, Federal University of Alagoas-UFAL,
Campus A. C. Sim o˜ es, 57072-900, Macei o´ , Alagoas, Brazil
Laboratory of Instrumentation and Development in Analytical Chemistry, Institute
of Chemistry and Biotechnology, Federal University of Alagoas-UFAL, Campus A.
C. Sim o˜ es, 57072-900, Macei o´ , Alagoas, Brazil
(
2.09 million), breast cancer (2.09 million), and colorectal
4
–6
d
e
cancer (1.8 million).
incidence to 29.5 million cases of cancer in 2040.
Estimates have shown an increase in
7
In medicinal chemistry, indole and quinoline are important
Drug Research and Development Center-NPDM, Department of Physiology and
Pharmacology, Faculty of Medicine, Federal University of Cear a´ -UFC, Fortaleza,
Cear a´ , Brazil
8–13
moieties due to their versatile activities against various diseases,
including cancer.
14–19
These heterocycles are widely found in many
natural sources and have been exhaustively explored by pharma-
†
‡
Electronic supplementary information (ESI) available. See DOI: 10.1039/d1nj02105b
These authors contributed equally to this work.
20–24
ceutical companies.
Also, thiazole is an essential 5-membered
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Fig. 1 Chemical structures and anticancer activities of intercalators and groove binders of DNA, and general chemical structures of designed quinoline
and indole hybrid compounds.
heterocyclic compound that has increased the interest of the most critical aspects of biological and toxicological studies of
3
,19,20
scientific community due to its remarkable biological activities pharmaceutical development processes.
2
5–27
28,29
toward several diseases
such as Toxoplasmosis
and
In this study, to explore the anticancer potential of thiazole,
as well as due to its antidiabetic, anti- dihydrothiazole, 4-thiazolidinone, and thiazine analogs, we report
3
0,31
32
Chagas Disease,
2
6,33
34
35–41
HIV,
antimicrobial, and anticancer activities.
Fig. 1 the design and efficient conditions for US-assisted synthesis of
shows many anticancer compounds containing these hetero- hybrid compounds, including quinoline and indole moieties
cycles, especially intercalators or groove binders of DNA, which (Fig. 1). All designed compounds were screened for their ability
were used as starting materials for the design of the title to inhibit the in vitro growth of glioblastoma (SF-295), leukemia
compounds.
(HL-60), and prostate cancer (PC-3) cell lines. For the most
Some methods used for the synthesis of organic compounds active derivative (4c), we studied its mechanism of interaction
feature severe reaction conditions such as high temperature with DNA using voltammetry, spectroscopic techniques, and in
and long reaction periods, which in many cases produce low silico approaches.
yields. Given these factors, there is a need for an efficient and
versatile reaction method to improve the reaction process. In
this sense, the ultrasound (US) reactions are increasingly used
because they are clean and environmentally sustainable, providing
an alternative for preparing organic compounds of synthetic
Experimental
Chemistry
origin. Compared to conventional heating, which provides thermal
energy in the macro-system, US reduces the reaction time, purchased from Sigma-Aldrich and were used without further
Apparatus and analysis. The chemicals and solvents were
s
1
13
improves yields, and minimizes side products by providing purification. H and C NMR spectra were recorded on a
4
2–44
s
the activation energy into the microenvironment.
Bruker Avance Spectrometer operating at 400 MHz and 100
Synthetic and natural drugs exhibit high toxicity and mutation MHz, respectively. The chemical shifts were reported in d units,
characteristics in cells, enzymes, and DNA (humans and micro- and coupling constants (J) were measured in hertz. The peaks
10,18
organisms).
This effect on DNA can be measured using are presented as s (singlet), d (doublet), t (triplet), q (quartet),
dsDNA biosensors and spectroscopic methodologies. These quint (quintet), br s (broad singlet), dd (double doublet), td
analyses are essential for development of new hits and lead (triplet of doublets), and m (multiplet). The ultrasonic assisted
compounds, since drug interactions with DNA are among the reactions were carried out in a Spectralab model UMC 20 Ultrasonic
1
3848
|
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13
cleaner with a frequency of 40 kHz and a nominal power 250 W. 11.86 (1H, br s, NH); C NMR (100 MHz, DMSO-d
6
) d 14.15;
Melting points were detected with open capillaries using an 15.94; 60.38; 98.59; 114.24; 119.06; 125.52; 127.19; 138.09; 138.64;
+
+
MSTecnopon PFMII digital melting point apparatus, and are 143.34; 161.29. HRMS (ESI ) [M + H] : calcd For C H ClN OS:
1
4
10
4
uncorrected. All the reactions were monitored using thin layer 318.0264, found: 318.3047.
chromatography (TLC) on aluminum-backed plates coated with 2-(2-(7-Chloroquinolin-4-yl)hydrazinyl)-4-phenylthiazole (4d).
Merck Kieselgel 60 F254 silica gel, and were visualized by exposure Brown amorphous solid; yield: 92%; m.p. 295 1C; HPLC-UV
1
to ultraviolet light (257 nm). The purity of all compounds was (methanol/formic acid 0.1%): 2.05 min/99%; H NMR (400 MHz,
6
determined by HPLC (Shimadzu SIL-20AHT) on a Discovery C-18 DMSO-d ) d 7.14 (1H, d, J = 6.8 Hz, ArH), 7.29 (1H, t, J = 7.3 Hz,
Supelco column, and methanol, methanol/formic acid, methanol/ ArH), 7.36–7.40 (2H, m, ArH), 7.81 (2H, m, ArH), 7.89 (1H, dd,
water or water was used as the mobile phase. HRMS were J = 1.8 and 9.0 Hz, ArH), 8.15 (1H, d, J = 1.5 Hz, ArH), 8.65–8.67
s
13
measured on a Bruker microQTOF mass spectrometer.
(2H, m, ArH), 10.55 (1H, br s, NH), 11.79 (1H, br s, NH);
C
Synthesis of 2-(7-chloroquinolin-4-yl)hydrazinecarbothioamide NMR (100 MHz, DMSO-d ): d 99.08, 104.38, 113.78, 115.00,
6
(
2) and (E)-2-((1H-indol-3-yl)methylene)hydrazinecarbothioamide 119.41, 125.26, 125.58, 127.78, 128.61, 134.01, 138.55, 138.63,
45
+
+
(
3). See Hammoud et al.
General procedure for the synthesis of 4a–f. To a glass tube
containing a solution of 2 (1 mmol) in 5 mL of appropriate
144.01, 150.05, 15659, 169.22; HRMS (ESI ) [M ꢀ H] : calcd for
11ClN S: 351.0393, found: 351.0440.
2-(2-(7-Chloroquinolin-4-yl)hydrazinyl)-4,5-dihydrothiazole (4e).
18
C H
4
solvent (4 : 1 MeOH/DMF for 4a, c–f; 4 : 1 toluene/DMF for 4b), Pale yellow amorphous solid; yield: 90%; m.p. 230 1C; HPLC-UV
1
the required dielectrophile (1.5 eq. for 4a, c–f; 4.5 eq. for 4b) (methanol/formic acid 0.1%): 2.15 min/99%; H NMR (400 MHz,
and sodium acetate (2 eq.) were added. The tube was sealed and DMSO-d ): d 3.64 (2H, t, J = 7.1 Hz, CH ), 3.99 (2H, t, J = 7.4 Hz,
6
2
left under ultrasonic irradiation at 85 1C until complete con- CH ), 6.20 (1H, d, J = 6.3 Hz, ArH), 7.40 (1H, d, J = 8.2 Hz, ArH), 7.60
2
version of the starting material (20 min for 4c; 25 min for 4d; (1H, s, ArH), 7.78 (1H, s, ArH), 8.30 (1H, d, J = 8.7 Hz, ArH), 8.59
1
3
3
5 min for 4e, f; 40 min for 4b; 55 min for 4a). Thereafter, the (1H, s, ArH) 9.45 (1H, br s, NH); C NMR (100 MHz, DMSO-d
6
):
reaction mixture was left to stand at room temperature, and the d 25.79, 53.15, 96.93, 117.02, 117.98, 124.05, 126.86, 136.12,
+
+
separated solid product was filtered off, and washed with ethyl 138.44, 138.63, 161.74, 165.04; HRMS (ESI ) [M + H] : calcd for
ether and water. Finally, the final compounds were purified
12 4
C H11ClN S: 279.0393, found: 279.0480.
by recrystallization in methanol/water (4a, c–f) and column
2-(2-(7-Chloroquinolin-4-yl)hydrazinyl)-5,6-dihydro-4H-1,3-thi-
chromatography on silica gel using 9.5 : 0.5 dichloromethane/ azine (4f). Pale yellow amorphous solid; yield: 95%; m.p. 245 1C;
1
methanol (4b), and then dried under vacuum.
HPLC-UV (methanol/formic acid 0.1%): 2.04 min/99%; H NMR
2
-(2-(7-Chloroquinolin-4-yl)hydrazinyl)thiazol-4(5H)-one (4a). (400 MHz, DMSO-d ); d 2.35 (2H, s, CH ), 3.32 (2H, s, CH ), 3.59
6
2
2
Pale yellow amorphous solid; yield: 87%; m.p. 210 1C; HPLC-UV (2H, s, CH
(
2
), 6.00 (1H, d, J = 5.4 Hz, ArH), 7.39 (1H, d, J = 8.5 Hz,
methanol/formic acid 0.1%): 4.72 min/99%; H NMR (400 MHz, ArH), 7.62 (1H, s, ArH), 7.77 (1H, s, ArH), 8.04 (1H, br s, NH), 8.29
DMSO-d ) d 3.78 (2H, s, CH ), 6.36 (1H, d, J = 7.3 Hz, ArH), (1H, d, J = 8.8 Hz, ArH), 8.79 (1H, br s, NH from tautomer); 12.01
.18 (1H, d, J = 8.2 Hz, ArH), 7.24 (2H, s, ArH), 8.09 (1H, d, (1H, br s, NH); C NMR (100 MHz, DMSO-d
J = 8.5 Hz, ArH), 10.67 (1H, br s, NH), 11.57 (1H, br s, NH); 48.46, 96.25, 116.93, 118.21, 123.97, 127.02, 136.03, 138.60,
1
6
2
1
3
7
6
): d 23.17, 25.79,
1
3
+
+
C NMR (100 MHz, DMSO-d ) d 32.55, 98.40, 116.18, 119.85, 157.34, 161.11; HRMS (ESI ) [M + H] : calcd for C H ClN S:
6
13 13
4
1
22.80, 125.58, 134.32, 134.46, 139.34, 151.54, 155.85, 173.61; 293.0549, found: 293.0638.
+
+
HRMS (ESI ) [M + H] : calcd for C H ClN OS: 292.0186, found:
General procedure for the synthesis of 5a–f. A sealed glass
tube containing a solution of 3 (1 mmol), the required dielectro-
phile (4.5 eq. for 5a–c, f; 1.2 eq. for 5d; 3 eq. for 5e) and sodium
1
2
9
4
293.0250.
2
-(2-(2-(7-Chloroquinolin-4-yl)hydrazinyl)-4-oxo-4,5-dihydro-
thiazol-5-yl)acetic acid (4b). Yellow crystalline solid; yield: 87%; acetate (3 eq. for 5a, d, f; 2 eq. for 5c, e) in 6 mL of appropriate
1
m.p. 285 1C; HPLC-UV (methanol): 2.15 min/99%; H NMR solvent (MeOH for 5a, c, d; toluene and drops of DMF for 5b;
6
(400 MHz, DMSO-d ) d 2.54 (1H, br s, NH); 2.72 (1H, s, NH from isopropanol for 5e, f) was left under ultrasonic irradiation at 70 1C
tautomer); 2.80 (1H, dd, J = 9.3 and 17.4 Hz, CH ); 2.99 (1H, dd, (85 1C for 5b) until complete conversion of the starting material
2
a
J = 3.7 and 17.4 Hz, CH ); 4.24 (1H, dd, J = 3.7 and 9.3 Hz, CH); (20 min for 5c, d; 40 min for 5a, b, e, f). After this, the reaction
2
b
6.37 (1H, d, J = 7.6 Hz, ArH); 6.62 (1H, br s, NH); 7.17 (1H, dd, mixture was left to stand at room temperature, and the separated
J = 2.1 and 8.6 Hz, ArH); 7.24 (2H, s, ArH); 8.01 (1H, d, J = 8.7 Hz, solid product was filtered off, washed with methanol and water,
1
3
ArH); 10.66 (1H, br s, COOH). C NMR (100 MHz, DMSO-d
6
) d and dried under vacuum. Only compound 5b was purified by
3
1
7.25; 43.03; 98.36; 116.21; 119.84; 122.84; 125.64; 133.92; recrystallization in methanol/water.
+
34.46; 139.36; 151.53; 165.91; 171.74; 174.88. HRMS (ESI )
(E)-2-(2-((1H-indol-3-yl)methylene)hydrazinyl)thiazol-4(5H)-
+
[M + H] : calcd for C14
H10ClN OS: 318.0264. Found: 318.3044. one (5a). White solid crystals; yield: 89%; m.p. 257–258 1C;
4
1
Ethyl 2-(2-(7-chloroquinolin-4-yl)hydrazinyl)-4-methylthiazole- HPLC (9 : 1 methanol/water): 3.21 min/95%; H NMR: see Makam
46 13
46
+
+
5-carboxylate (4c). Pale yellow amorphous solid; yield: 90%; m.p. et al.;
C NMR: see Makam et al.; HRMS (ESI ) [M + H] : calcd
2
82 1C; HPLC-UV (methanol/formic acid 0.1%): 1.86 min/99%; for C H N OS: 259.0575, found: 259.0634.
1
2 10 4
1
H NMR (400 MHz, DMSO-d
.45 (3H, s, CH ), 4.15 (2H, q, J = 7.0 Hz, CH
ArH), 7.80 (1H, s, ArH), 8.02 (1H, s, ArH), 8.57 (2H, m, ArH), 80%; m.p. 230–232 1C; HPLC (methanol/formic acid 0.1%):
6
) d 1.20 (3H, t, J = 7.0 Hz, CH
3
),
(E)-2-(2-(2-((1H-indol-3-yl)methylene)hydrazinyl)-4-oxo-4,5-dihydro-
2
3
2
), 6.90 (1H, s, thiazol-5-yl)acetic acid (5b). Yellow amorphous solid; yield:
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.12 min/98%; H NMR (400 MHz, DMSO-d
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1
3
6
): d 2.88 (1H, dd,
An activity scale was utilized to appraise the cytotoxic
J = 17.3 and 8.5 Hz, CH), 3.01 (1H, dd, J = 17.5 and 3.9 Hz, potential of the tested samples: (i) inactive samples; (ii) samples
CH ), 4.33 (1H, dd, J = 8.5 and 3.9 Hz, CH ), 7.13–7.21 (2H, m, with low activity (LA, cytotoxic activity between 1 and 50%);
2
a
2b
ArH), 7.43 (1H, d, J = 7.3 Hz, ArH), 7.83 (1H, d, J = 2.8 Hz, ArH), (iii) moderate activity (MA, cytotoxic activity between 50 and
.15 (1H, d, J = 7.0 Hz, ArH), 8.51 (1H, s, CHQN), 11.65 (1H, br 75%); and (iv) high activity (HA, cytotoxic activity between
8
1
3
s, NH), 12.26 (1H, br s, OH); C NMR (100 MHz, DMSO-d
6
): d 75 and 100%). Only compounds that showed moderate or high
3
1
6.83, 43.48, 111.96, 112.02, 120.80, 122.01, 122.77, 124.52, activity in at least one cell line were studied further in an
+
+
31.89, 137.16, 152.52, 171.85, 175.52; HRMS (ESI ) [M + H] : expanded panel of human cell lines besides the lineages used
S: 317.0630, found: 317.0684. previously: HCT-116 human colon cancer cells at a density of
12 4 3
calcd for C14H N O
5
ꢀ1
(
E)-ethyl 2-(2-((1H-indol-3-yl)methylene)hydrazinyl)-4-methyl- 0.7 ꢁ 10 cells mL . The L929 murine fibroblast cell line (0.1 ꢁ
6
ꢀ1
thiazole-5-carboxylate (5c). Yellow crystalline solid; yield: 94%; 10 cells mL ; Rio de Janeiro Cell Bank, Brazil) was used as a
m.p. 255–257 1C; HPLC (water): 13.37 min/97%; H NMR: see model of non-cancer cells to calculate the selectivity index. After
1
4
6
13
46
+
Makam et al.;
C NMR: see Makam et al.; HRMS (ESI ) 72 h exposure to the selected compounds at a concentration
M + H] : calcd for C16H16N4O2S: 329.0994, found: 329.1046. ranging from 0.31 to 10 mg in prescreening, the plates were
E)-2-(2-((1H-indol-3-yl)methylene)hydrazinyl)-4-phenylthiazole centrifuged. The medium was replaced by fresh medium
+
[
(
ꢀ
1
(5d). Red crystalline solid; yield: 80%; m.p. 140–142 1C; HPLC (150 mL) containing 0.5 mg mL MTT. Three hours later, the
1
47 13
(water): 3.58 min/98%; H NMR: see Mahmoodi et al.;
C
MTT formazan product was dissolved in 150 mL of DMSO, and
4
7
+
+
NMR: see Mahmoodi et al.; HRMS (ESI ) [M + H] : calcd for the absorbance was measured using a multiplate reader (Spectra
C18H14N4S: 319.0939, found: 319.0994. Count, Packard, Ontario, Canada). The drug effect was quanti-
E)-2-(2-((1H-indol-3-yl)methylene)hydrazinyl)-4,5-dihydro- fied as the percentage of control absorbance of the reduced dye
thiazole (5e). Orange crystalline solid; yield: 90%; m.p. 180– at 595 nm. Control groups received the same amount of vehicle
(
1
s
1
(
82 1C; HPLC (9 : 1 methanol/water): 6.35 min/98%; H NMR and doxorubicin (Doxolem , Zodiac Produtos Farmac ˆe uticos S/
400 MHz, DMSO-d ): d 3.66 (2H, t, J = 7.4 Hz, CH ), 4.44 (2H, t, A, Brazil), the latter of which was used as a positive control. Each
), 7.18 (1H, t, J = 7.6 Hz, ArH), 7.24 (1H, t, sample was tested in two independent experiments performed
6
2
J = 7.26 Hz, CH
2
J= 7.6 Hz, ArH), 7.49 (1H, d, J = 7.6 Hz, ArH), 8.04 (1H, d, J = in triplicate. The results consisted of the average value for each
1
1
.9 Hz, ArH), 8.38 (1H, d, J = 7.8 Hz, ArH), 8.48 (1H, s, CHQN), experimental unit. The IC50 (cytotoxic concentration at 50%
0.08 (1H, br s, NH), 11.94 (1H, br s, NH); C NMR (100 MHz, level) values and their 95% confidence intervals were obtained
1
3
DMSO-d ): d 25.4, 49.68, 110.24, 112.09, 121.23, 122.50, 123.17, by nonlinear regression using the GraphPad Prism program 6
6
+
+
1
23.75, 134.12, 137.15, 148.41, 166.21; HRMS (ESI ) [M + H] : (Intuitive Software for Science, San Diego, CA).
calcd for C12 S: 245.0783, found: 245.0888.
E)-2-(2-((1H-indol-3-yl)methylene)hydrazinyl)-5,6-dihydro-4H-
12 4
H N
Electrochemical studies
(
1
,3-thiazine (5f). Yellow crystalline solid; yield: 95%; m.p. 189– Cyclic voltammetry (CV) and differential pulse voltammetry
1
190 1C; HPLC (9 : 1 methanol/water): 3.00 min/99%; H NMR (DPV) experiments were performed using a three-electrode cell,
(
(
(
400 MHz, DMSO-d
6 2
): d 2.40 (2H, quint, J = 6.0 Hz, CH ), 3.27 and an AutolabPGSTAT-30 potentiostat (Eco Chemie, Utrecht,
2H, t, J = 5.5 Hz, CH ), 4.00 (2H, t, J = 6.0 Hz, CH ), 7.16–7.26 Netherlands) coupled to a microcomputer, interfaced by GPES
2H, m, ArH), 7.50 (1H, d, J = 7.8 Hz, ArH) 8.15 (1H, d, J = 2.9 Hz, 4.9 software. A glassy carbon electrode (GC, diameter = 3 mm)
2
2
ArH), 8.21 (1H, d, J = 7.6 Hz, ArH), 8.59 (1H, br s, NH), 8.76 (1H, s, was used as the working electrode, a Pt wire as the counter
ꢀ
CHQN), 9.58 (1H, br s, NH from tautomer), 12.00 (1H, br s, NH); electrode, and an Ag/AgCl, Cl system (saturated) as the refer-
1
3
C NMR (100 MHz, DMSO-d): d 23.28, 25.10, 46.02, 110.10, ence electrode. The GC was cleaned by polishing with alumina
1
1
12.17, 121.21, 121.90, 123.04, 124.11, 134.09, 137.08, 148.97, on polishing felt. An inert gas was used for degassing the
+
+
61.23; HRMS (ESI ) [M + H] : calcd for C13
H
14
N
4
S: 259.0939, solution, and the solution was covered with an argon blanket
during the experiments. The pH was measured (QUIMIS). All
experiments were conducted at room temperature (25 1C). The
solvent used in aprotic media studies was extra dry dimethyl-
found: 259.1030.
Inhibition of cell proliferation
The MTT assay was used to evaluate the cytotoxic potential of all formamide (DMF) purchased from Acros Organics. Electrochemical
compounds. The derivatives were diluted in DMSO and evaluated studies in protic media were performed using a phosphate buffer
ꢀ1
at a single concentration (10 mg mL ) in three human cancer cell pH 7.0 (ionic strength 0.2) or ethanol 5% and acetate buffer pH 4.5
48
lines during 72 h exposure. The cell lines were SF295 (glio- (ionic strength 0.2 M).
blastoma), HL-60 (promyelocytic leukemia), and PC3 (prostate),
The DNA biosensor (dsDNA) used was a Calf Thymus DNA
(Sodium salt, Type I) obtained from Sigma Chemical Co, which
6
and were plated in 96-well microplates at a density of 0.1 ꢁ 10
6
(
SF295 and PC3 cells) and 0.3 ꢁ 10 cells per mL (HL-60 cells). was prepared by covering the glassy carbon electrode with
Human tumor cell lines were obtained from National Cancer 12 mg of dsDNA dissolved in 1 mL of pH 4.5 acetate buffer,
Institute (Bethesda, MD, USA) and were cultured in RPMI1640 which was then left in a refrigerator for 24 h for homogenization
medium, supplemented with 10 or 20% fetal bovine serum and and to prevent heat degradation. Later, 10 mL was applied to the
1% antibiotics (penicillin and streptomycin). Cultures were main- surface of the CV electrode. This DNA-modified glassy carbon
tained in a humidified incubator at 37 1C in a 5% CO atmosphere. electrode was then left to dry by inert gas flow and placed in the
2
1
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s 52
electrochemical cell containing acetate buffer (pH 4.5). The DPV and a blind docking was performed using Autodock Vina . The
technique was used with a potential range of 0 to +1.4 V, with lowest energy conformation was chosen as the initial conformation
amplitude 0.05 V. To produce ssDNA by acid–base treatment, for molecular dynamics (MD) simulations. MD was performed using
s
dsDNA (3 mg) was dissolved in 1 mL of HCl (1.0 M) by heating at the GROMACS MD package. The interaction poses were visualized
s
9
1
5 1C in a sealed glass tube immersed in a boiling water bath for using UCSF Chimera and VMD software. The RMSD values during
s
h, followed by neutralization with 1 mL of NaOH (1.0 M). A the simulation were calculated using GROMACS , and the RMSD
s
52
freshly prepared solution, consisting of 2 mL of ssDNA and chart was generated using Xmgrace software.
mL of acetate buffer pH 4.5, was added to the electrochemical
cell. Single-scan DPV experiments were conducted between 0
8
ꢀ
1
Results and discussion
and +1.4 V versus AgCl, KCl (0.1 M) (n = 10 mV s , pulse
amplitude = 50 mV and pulse width = 70 ms). Peaks corresponding Synthesis
to the oxidation of guanine and adenine appeared at +0.815 V and
In the synthesis of the title compounds, initially, thiosemicarbazide
1) undergoes a nucleophilic substitution with 4,7-dichloroquinoline
or a condensation reaction with indole-3-carboxaldehyde, yielding
+1.164 V. After rinsing the surface, the GCE was immersed in a
(
solution containing 4c (1 to 100 mM), and the DPV experiment was
repeated to observe possible interference of oxidation waves of DNA.
thiosemicarbazone intermediates 2 and 3, respectively, according
4
8
to conventional methods described in the literature. Then,
-thiazolidinone (4a–b and 5a–b), thiazole (4c–d and 5c–d),
ctDNA spectroscopic interaction studies
4
Reagents and solutions. The ctDNA solution (Calf thymus,
Sigma) was prepared by weighing 10 mg of the nucleic acid,
which was solubilized in 20 mL of Tris–HCl buffer (10 mM,
pH = 7.40 ꢂ 0.10), after constant mechanical agitation for 24 h.
The stock solution was stored at 4 1C. The concentration of the
ctDNA solution was determined by using the absorption at
dihydrothiazole (4e and 5e), and thiazine (4f and 5f) derivatives
were prepared via thia-Michael cyclization, or substitution
followed by intramolecular cyclization between 2 or 3 with
dielectrophiles (ethyl chloroacetate, maleic anhydride, ethyl
2
-chloroacetoacetate, bromo-acetophenone, dibromoethane,
30
and dibromopropane) using US irradiation (Scheme 1).
To determine the most efficient reaction conditions for the
synthesis of title compounds, we compared the effects of
ꢀ1
2
60 nm, using the molar extinction coefficient e260 = 6600 L mol at
4
9
25 1C. To evaluate the purity of DNA against protein contam-
ination, we used the ratio of absorbance at 260 and 280 nm
A260/A280); if the value obtained is between 1.8 and 1.9 the DNA
(
5
0
is protein-free. The stock solution (1.40 mM) of 4c was
prepared by weighing and solubilizing in dimethylsulfoxide
(DMSO, Sigma), and the subsequent dilutions were prepared
in Tris–HCl buffer. The maximum percentage of DMSO in the
4
9
samples was 4% (v/v). In the UV-vis assays, the spectra of
the free 4c (2.5 to 20 mM), 4c bound to the ctDNA (10 mM), and
the free ctDNA were measured. In the molecular fluorescence
experiments, the final concentration of the fluorescent probes
and the ctDNA was fixed ([EB] = [AO] = 1.5 mM, [Ho] = 0.5 mM
and [ctDNA] = 6.0 mM, while [DAPI] = 1.0 mM and [ctDNA] =
10 mM) and increasing amounts of 4c (2.5 to 120 mM, depending
on the probe) were added, using a stock solution of 50 mM
for all probes. In molecular fluorescence measurements, each
system was excited at a specific wavelength: Ho-DNA (lex
=
350 nm), DAPI-DNA (lex = 496 nm), EB-DNA (lex = 525 nm) and
AO-DNA (lex = 490 nm).
Apparatus
The spectrofluorimetric titrations were carried out in an
s
RF-5301 spectrofluorimeter (Shimadzu , Japan) equipped with
a xenon lamp (150 W) using a quartz cuvette with 10 mm
optical path. UV-vis spectra were recorded in an AJX-6100PC
double beam spectrophotometer (Micronal S.A., Brazil).
Docking and molecular dynamics studies
For the in silico study, the most stable conformation of the ctDNA
(
PDB entry: 1G3X) was selected after molecular dynamics (MD)
51
simulations, as previously described by our research group.
Scheme 1 Synthetic route for the thiazole, 4-thiazolidinone, dihydrothia-
Subsequently, the structure of 4c was energetically minimized, zole and thiazine derivatives under ultrasound (US) irradiation.
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Table 1 Scope of the thia-Michael cyclization or substitution followed by
intramolecular cyclization involving compound 2 or 3 with dielectrophiles
under ultrasound irradiation
Table
2
Antiproliferative activity of 4-thiazolidinone, thiazole, dihy-
drothiazole and thiazine coupled with quinoline and indole nucleus against
three different tumor cell lines
a
Dielectrophile
(equiv.)
Time
(min)
Yield
(%)
Cell line
Code
Solvent
T (1C)
SF-295
HL-60
PC-3
4
4
4
4
4
4
5
a
b
c
d
e
f
MeOH/DMF
Toluene/DMF
MeOH/DMF
MeOH/DMF
MeOH/DMF
MeOH/DMF
MeOH
EtOH
Toluene/DMF
MeOH
EtOH
MeOH
EtOH
Isopropanol
Isopropanol
1.5
4.5
1.5
1.5
1.5
1.5
4.5
85
85
85
85
85
85
70
55
40
20
25
35
35
40
87
87
90
92
90
95
89
90
80
94
86
90
89
90
95
Code
RVC (%)
ꢂSD
RVC (%)
ꢂSD
RVC (%)
ꢂSD
2
3
20.7
27.2
2.5
18.2
97.3
79.3
40.0
25.8
0.5
19.2
40.9
25.6
20.9
17.8
99.5
3.8
4.0
2.5
0.5
0.7
0.9
1.7
0.9
2.8
2.5
2.2
3.1
4.0
1.9
0.6
30.9
2.4
0.8
2.5
1.2
3.0
0.5
0.6
1.3
5.9
5.5
5.4
5.9
1.5
3.6
2.9
0.3
23.5
9.4
2.7
3.7
3.2
2.9
0.2
1.9
2.4
4.6
3.2
2.7
1.4
1.3
0.8
5.0
0.5
4
4
4
4
4
4
5
5
5
5
5
a
b
c
d
e
f
a
b
c
d
e
34.5
40.8
93.2
91.3
70.4
19.4
27.3
35.9
16.1
59.8
34.3
34.5
100
12.3
17.2
98.8
79.8
32.2
10.1
8.1
a
a
a
a
a
1.0
rt
120
5
5
b
c
4.5
4.5
1.0
85
70
78
40
20
60
a
a
a
a
5
d
1.2
1.0
70
70
20
120
3.4
a
a
a
a
16.8
55.9
17.0
16.0
98.0
5
5
e
f
3.0
4.5
70
70
40
40
a
46,47,53
5f
DOX
Conventional synthesis for 5a, 5c, and 5d.
b
a
All data are presented as reduction of cell viability (RCV in %) ꢂ
ultrasound application and changing parameters such as solvent, standard deviation (SD), obtained from two independent experiments
b
performed in quadruplicate, after 72 hours post-incubation. Doxor-
dielectrophile equivalents, reaction time, and temperature. In
this context, it was found that a mixture of toluene/DMF (ratio
ubicin (DOX) was employed as a positive control.
4
: 1) was the appropriate solvent for the synthesis of 5b. An
increase of maleic anhydride equivalents (1.0 to 4.5 equiv.) at
5 1C accelerates the reaction time (40 min), and the desired
tumor lines. It exhibited cytotoxicity comparable to that of
doxorubicin, especially against SF295 and PC3 cells displaying
RCV values higher than 97%. Furthermore, the structure–
activity relationship (SAR) for all derivatives evaluated in this
study will be discussed in detail.
8
product was isolated in 80% yield. When the small screening was
extended to the synthesis of different analogs, a similar result was
observed for 4b. On the other hand, we found diverse appropriate
solvents (MeOH/DMF for 4a,c–f; MeOH for 5a,c,d; isopropanol
for 5e,f), temperature (85 1C for 4a, c–f; 70 1C for 5a, c–f), and
dielectrophile stoichiometry (1.5 equiv. for 4a, c–f; 4.5 equiv. for
For SAR discussion of thiazole–quinoline derivatives and
analogs, the quinolone-thiosemicarbazide intermediate (2) was
initially considered the starting point by comparison with its
derivatives. It was observed that this compound presents more
antiproliferative effects against HL-60 cells, showing an RCV
value of 30.9%. Also, it exhibits similar effects against SF-295
and PC-3 cells, with RCV values of 20.7 and 23.5%, respectively.
The cyclization of compound 2 by reaction with ethyl-2-
chloroacetate provides the most poorly active derivative (4a).
However, it presents increased activity against HL-60 cells (RCV
of 34.5%) compared to its precursor. Additionally, it is possible
to verify that the 4-thiazolidinone ring drastically reduces 8- and
2-fold the antiproliferative activity against SF-295 and PC-3,
respectively, compared with 2. Posteriorly, the reaction with maleic
anhydride yields a derivative containing a 4-thiazolidinone sub-
stituted at position 5 by a carboxylic acid function (4b). It presents
higher antiproliferative effects than non-substituted at 4 position
5a,c,f; 1.2 equiv. for 5d; 3.0 equiv. for 5e) as the best synthetic
conditions (Table 1). For all reactions performed without sodium
acetate, the limiting reactants 2 and 3 were not completely
consumed after 3 hours. In general, the US-assisted protocol
described afforded highly selective conversions and minimum
side product formation, short reaction times (20–55 min), simple
work-up procedures, and high yields (80–96%). When compared
with studies found in the literature, our synthesis methods
increased the reaction velocity three times for indole derivatives
4
6,47,53
5a and 5c (Table 1).
The structures of the synthesized
1
13
compounds were confirmed by H NMR, C NMR, and HRMS
(see ESI†). For compounds 5a, 5c, 5e 5d, spectroscopic data were
46,47,53
identical to that reported in the literature.
(
4
4a), with an RCV value of 40.8% towards HL-60 cells. Similarly to
a, this compound exhibits weak activity against SF-295 and PC-3
Biological evaluation
The synthesized hybrid compounds 4a-f and 5a-f were initially cell lines, with RCV values of 18.2 and 17.2%, respectively. The
ꢀ1
screened at a single dose of 10 mg mL (Table 2). Based on the quinoline moiety, coupled to the thiazole ring substituted with
results, in general, the compounds containing the quinoline methyl and ethyl ester groups at 4 and 5 positions, respectively,
moiety were more active than indole analogs. Derivative 4e represents the most active derivative (4c) found in this study. In
showed a moderate effect on the HL-60 cell line (RCV values general, it is possible to observe that this compound exhibits a
higher than 70%), but low activity against SF295 and PC3 cells. similar biological profile, showing a non-specific behavior with
Compound 4d exerted a moderate cytotoxic effect on SF295 and antiproliferative activity ranging from 93.2 to 98.8%, with a better
PC3 cells but was extremely cytotoxic (greater than 90%) to affinity for PC-3 (RCV of 98.3%) and SF-295 (RCV of 97.3%) tumor
HL-60. Finally, compound 4c was very cytotoxic against all cell lines.
1
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The introduction of a 4-phenyl ring at the thiazole ring Table 3 IC50 values for the most promising thiazole–quinoline and
dihydrothiazole–quinoline hybrid compounds against different tumor
provides a compound (4d) with similar antiproliferative activity
and murine (L929) cell lines
(RCV of 91.3%) against HL-60 to 4c. In contrast, this substitution
a
approximately reduces 20% of activity against SF-295 and PC-3
cell lines. In a sense, it can be suggested that this phenyl ring
increases the affinity for HL-60 cells, generating a more selective
analog. The quinoline hybrid containing a dihydrothiazole (non-
IC50 (mM)
Code PC-3
c
HL-60
SF-295
L929
SI
4
4
c
d
20.7 ꢂ 4.9
428.4
45.97
0.76 ꢂ 0.34 0.02 ꢂ 0.01
2.41 ꢂ 0.85
47.62
25.32 ꢂ 9.0 10.5
14.72 ꢂ 4.32 428.4
48.40
1.9
1.4
substituted thiazole) ring (4e) presents increased activity against 4e
25.5 ꢂ 10.8
45.97
435.97
b
DOX
0.24 ꢂ 0.07 0.66 ꢂ 0.34 33
all three cell lines compared to its precursor 4a. It is also verified
that 4e shows selectivity for HL-60 cells, demonstrating an RCV
a
IC50 ꢂ SD values were obtained from two independent experiments in
b
value of 70.4%. This compound displays low antiproliferative duplicate, after 72 h. Doxorubicin (DOX) was employed as a positive
c
control. SI was calculated as IC50 L929/IC50 HL-60.
effects than its more substituted analogs, such as 4c and 4d. The
replacement of the thiazole by a thiazine ring, generating
compound 4f, resulted in substantial activity reduction. This (2.41 mM) against HL-60 leukemic cells and the best selectivity
compound exhibits more affinity for SF-295 cells with a low RCV index among the compounds tested. This compound has similar
value of 25.8%.
cytotoxic effects against HL-60 cells as thiazoloindolo[3,2-c]quino-
Similarly, for SAR discussion of thiazole–indole hybrids and line and thiazole derivatives, exhibiting a moderate-to-strong
54,55
analogs, indole-3-thiosemicarbazide (3) will be used as a starting activity.
Finally, the analogs 4d and 4e exhibited low SI values
point, considering that it gives rise to the 5a–f series of com- (1.9 and 1.4, respectively), revealing that these compounds are not
pounds. This compound presents poor activity against all cell selected due to their high cytotoxicity.
lines evaluated, but has more affinity for SF-295 cells (RCV value
of 27.2%). The introduction of a 4-thiazolidinone nucleus com-
pletely inactivates the molecule (5a) towards SF-295 tumor cells
compared to its precursor 3. In contrast, an increase in HL-60
antiproliferative effects is observed, showing an RCV value of
Spectroscopic interaction studies with ctDNA
Considering the promising antitumor potential of 4c against the
HL-60 line cell and selectivity, a ctDNA interaction study was
performed to understand its possible action mechanism. Prelimin-
ary studies showed that 4c did not exhibit intrinsic fluorescence
27.3%. Additionally, this compound is considered as the most
inactive in this series of indole derivatives.
(
l
ex = 278 and 432 nm) at concentrations up to 120 mM. Therefore,
the evaluation of the interaction process directly between ctDNA and
c could not be performed by molecular fluorescence studies. In
The insertion of a carboxylic acid function at 5 position in
the thiazolidinone ring of 5a generates a new analog (5b),
which was inactive against HL-60 and showed modest activity
against SF-295 cells (RCV of 19.2%), in comparison with its
precursor 5a. The indole moiety coupled to the thiazole ring
substituted with methyl and ethyl ester groups at 4 and 5 positions
4
this way, a competition study exploring classic fluorescent probes
selective to DNA was used to evaluate the binding mode of 4c and
obtain information about ctDNA binding parameters.
(
4
5c) presents more affinity for SF-295 cells, with an RCV value of
0.9%. Also, it presents increased activity against PC-3 cells (RCV
Molecular fluorescence studies
of 16.8%) and decreased activity against HL-60 cells (RCV of Several compounds cause variation in the fluorescence intensity
6.1%) compared with its precursor 5a. In this series of indole of the probe–DNA complex, thus providing information about
1
derivatives, the derivative containing the thiazole ring substituted the mode of binding. In this sense, probes having a well-
with a phenyl group at 4 position was found to be the most active established binding mode such as Hoechst 33258 (Ho), DAPI,
compound (5d) against both HL-60 and PC-3 cell lines, with RCV ethidium bromide (EB), and acridine orange (AO) have been
of 59.8 and 55.9%, respectively. Besides, it presents low activity for used to evaluate the binding mode of 4c to ctDNA. EB and AO
SF-295 tumor cells, showing an RCV value of 25.6%. Finally, it is are widely used as fluorescent probes that bind to DNA by
observed that the non-substituted hybrid compounds containing intercalation, and in the free form, they present low emission
dihydrothiazole (5e) and thiazine rings (5f) present a very similar of fluorescence. However, a considerable increase in fluores-
antiproliferative profile, showing RCV values of B20, 34, and cence intensity occurs (Fig. 2) when the probe intercalates
B17% against SF-295, HL-60, and PC-3 tumor cells, respectively. between the DNA base pairs in the presence of a hydrophobic
Furthermore, it is verified that the sizes of these rings are not medium. The presence of a ligand with the same DNA binding
significantly essential for antiproliferative activity.
mode in the system can lead to displacement of the intercalated
The most active compounds were chosen to estimate IC50 EB or AO and, consequently, decrease fluorescence intensity.
values against cancer cell lines and murine L929 fibroblasts (as The competition assay principle with Ho and DAPI is similar.
a non-cancer cell model). The cell lines were treated with However, they bind to DNA via the minor groove and have high
5
6
different concentrations of thiazole–quinoline and dihydrothia- selectivity for DNA with AT-rich base sequences.
zole–quinoline hybrids 4c–e for 72 h, and doxorubicin was used Thus, to evaluate the preferential binding mode between 4c
as a positive control. All compounds were tested in duplicate and ctDNA, increasing amounts of the compound were added
in two independent experiments, and the IC50 values are shown to the system containing Ho-ctDNA, DAPI-ctDNA, EB-ctDNA,
in Table 3. The derivative 4c exhibited the lower IC50 value and AO-ctDNA. As shown in Fig. 2A–E, a significant change in
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Fig. 2 Evaluation of 4c–DNA interaction mode, (A) competition of 4c with the Ho–ctDNA complex; (B) competition of 4c with the DAPI–ctDNA complex;
C) competition of 4c with the EB–ctDNA complex; (D) competition of 4c with the AO–ctDNA complex; (E) Stern–Volmer plot for 4c (quenching process); (F)
double logarithmic curve for calculation of the binding constant of 4c with ctDNA. Condition: pH = 7.4 (10 mM Tris–HCl) at 25 1C.
(
6
0
fluorescence intensity was observed with the addition of an binding sites (n) were calculated according to eqn (2), where
increasing concentration of 4c, indicating that this compound Kb is the binding constant, n is the stoichiometric ratio, and [L]
displaces the probes from the double helix and binds to the is the concentration of 4c. The values of K and n are obtained
b
5
7
DNA via groove binding and intercalation. This result demon- from the intercept and slope, respectively, of the graph
strates that the probes were displaced from DNA, suggesting log[(F
that the binding mode of 4c is groove binding and intercala-
tion. This behavior is possibly associated with the structure of
0
ꢀ F)/F] vs. log[4c], as shown in Fig. 2F.
ꢀ
ꢁ
F0 ꢀ F
log
¼ log Kb þ n log ½Lꢃ
(2)
F
4c, which presents groups capable of interacting with DNA via
both modes simultaneously.
Table 4 shows the values obtained for KSV and K
b
. The obtained
Due to the displacement of all probes from the probe–ctDNA
complex by 4c, the information obtained by spectrofluorometric
titration of this system was used to calculate the binding para-
meters of 4c to ctDNA (or probe–ctDNA) and establish which mode
of interaction is preferred. As previously described, when 4c was
added, there was a gradual reduction in the analytical signal. This
reduction is called fluorescence quenching and is described by the
4
ꢀ1
K
SV value (0.56 to 3.12 ꢁ 10
M
) indicates the interaction
b
between 4c and the macromolecule (ctDNA), while the K value
5
ꢀ1
(
0.023 to 4.30 ꢁ 10 M ) demonstrates their degree of affinity.
Additionally, the value of n (0.91 to 1.18) suggests a stoichiometric
ratio of 1 : 1 (4c : ctDNA). The results indicate that 4c has a greater
capacity to displace the Ho and DAPI probes, indicating that the
preferred mode of interaction occurs via groove binding. However,
the compounds are also intercalated with DNA.
58,59
Stern–Volmer equation (eqn (1)).
0
F and F are the fluorescence
intensities in the absence and presence of 4c, respectively; [Q] is
the concentration of 4c that acts as a quencher; and KSV is the
quenching constant, which can be obtained by the slope of the
To confirm the relative affinity and the preferred binding
mode, the probabilities of intercalative DNA binding mode of
c were determined. The result suggests that the intercalative
4
0
graph F /F vs. [Q], as shown in Fig. 2E.
binding mode (%I) of 4c constitutes 32% of the overall binding
mechanism. Hence this compound is predominantly a groove
binder.
F 0
¼
1 þ KSV½Qꢃ
(1)
F
Moreover, to evaluate the magnitude of the probe–ctDNA
The binding parameters obtained agree with works that
binding to 4c, the binding constant (K
b
) and the number of evaluated the interaction process of similar synthetic compounds
1
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Table 4 Values of the Stern–Volmer (KSV) constants, binding constant (K
mode of the complex between 4c and ctDNA (or probe–ctDNA) at 25 1C
b
), number of binding sites (n) and probabilities of intercalative DNA binding
a
Stern–Volmer constant
Binding parameters
Probabilities of intercalative DNA by 4c
4
ꢀ1
5
ꢀ1
Probe
K
SV (10 M
)
r
K
b
(10 M
)
r
n
IEB/GDAPI
%I
Ho
DAPI
BE
3.12 ꢂ 0.03
2.99 ꢂ 0.04
0.59 ꢂ 0.01
0.56 ꢂ 0.01
0.9963
0.9981
0.9955
0.9875
4.30 ꢂ 0.06
1.02 ꢂ 0.10
0.9954
0.9942
0.9796
0.9834
1.18 ꢂ 0.04
1.10 ꢂ 0.05
0.91 ꢂ 0.01
0.97 ꢂ 0.03
0.47
32
0.023 ꢂ 0.003
AO
0.038 ꢂ 0.002
a
Calculation criteria: I50/G50 = IEB/IDAPI, where Iprobe = log[Kb(probe)]/C50. The parameter C50 is the concentration (M) of 4c at 50% fluorescence
ꢀ
1 ꢀ1
56
6
quenching of EB or DAPI under experimental conditions. The %I was determined as %I = [1 + (I50/G50
)
]
ꢁ 100%.
K
b
(DAPI) = 0.93 ꢁ 10 and
6
K
b(EB) = 7.75 ꢁ 10 .
16,61–63
64
65
66
with DNA.
Additionally, Phadte et al. and Williams et al.
may be an indicator of intercalation in UV-vis studies. Fig. 3
have reported several bioactive compounds (dibenzodioxins, shows that by adding an amount of ctDNA, a decrease in the
phenazines, amaranth, berenil, and imipramine, among others) absorbance signal is observed, characterizing the hypochromic
that interact with DNA by intercalation and groove binding effect, demonstrating that 4c is bound on the outside of the helix.
simultaneously, but with one of these modes being the pre- However, with the addition of ctDNA at concentrations 1.5 and
ferred. Thus, the biological activity of 4c against human tumor 2 times higher than that of 4c, there was an increase in the
cells may be associated with its capacity to interact with DNA.
absorbance signal (hyperchromic effect), and a redshift, which
characterizes the bathochromic effect (based on complex spectra).
Indeed, it is possible to infer that 4c may change its non-
covalent binding mode to the ctDNA with different biomolecule
concentrations. The result suggests that 4c interacts with ctDNA
via the minor groove (lower concentrations of DNA) and inter-
calation (higher concentrations of DNA), confirming the results
of molecular fluorescence. Finally, Chi et al. and Silva et al.
have shown similar profiles by UV-vis in the studies of toluidine
blue and acridine derivatives with calf thymus DNA.
UV-VIS interaction studies
In this assay, the absorption spectra of the ctDNA, free 4c, and
the respective 4c–ctDNA complex formed are analyzed (Fig. 3).
Compound 4c exhibits maximum absorption in the visible
region, in which the ctDNA does not absorb. The maximum
absorption values of 4c and ctDNA are located at 432 and
67
68
260 nm (not shown), respectively. When ctDNA was added in
the solution, spectral changes occurred immediately, indicating
the interaction of the compounds with ctDNA and corroborating
the results of molecular fluorescence.
Electrochemical studies
In the UV-vis spectra, a hypochromic effect and blue shift
indicate external non-covalent binding (groove), while a redshift
Derivative 4c was used as a model for electrochemistry experi-
ments and interaction with DNA. In these steps, studies using
cyclic voltammetry in a protic medium were performed in a pH
7.03, 5% ethanol phosphate buffer, to promote solubility in
water. Fig. 4 shows the voltammetric profile (100 mM) for this
medium. The cyclic voltammogram (CV) showed a reversible
system represented by Epc = ꢀ0.085 V and Epa = ꢀ0.056 V to
ꢀ
1
1
00 mV s , which shows the oxidation of the hydrazine group
(R–NH–NH–R), involving two electrons/two protons generating
the Azo (R–N = N–R) derivative. These results are compatible
6
9
with the studies carried out by Patai et al. in their mechanistic
studies of the oxireduction of the azo group.
Experiments of interaction with DNA demonstrate that
derivative 4c reacts with nucleic acids in vitro. In cases where DNA
becomes the main target in vivo, DNA damage may also depend on
the stability of the intermediate product formed by the reduction/
oxidation of the bioactive compound. The electrochemical behavior
of 4c was also studied using differential pulse voltammetry (DPV) on
a glassy carbon electrode modified with dsDNA. One of the dsDNA
biosensor applications is the investigation of the interactions
between macromolecules and biologically active compounds. The
DNA biosensor is characterized by not presenting a redox response
in the studied environment, and peaks of oxidation occur when the
substance interacts with dsDNA, breaking hydrogen bonds and
breaking its complementary base, leaving both exposed to oxidation.
Fig. 3 Absorption spectrum of 4c (10 mM) with increasing additions of
ctDNA (0, 2.5, 10, 15 and 20 mM, curves a–e, respectively). Condition: pH =
7
.4 (Tris–HCl buffer) at 25 1C.
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In studies on the dsDNA biosensor, it can be seen that there
are no evident changes in the voltammetric behavior in the
presence of 4c. On the other hand, its oxidation wave at
Epa = +0.08 V is present in the voltammogram. It is possible
to observe an increase in current intensity due to the preconcen-
tration of 4c as a function of the contact time, proving the
bioactive interaction with the double-stranded DNA (Fig. 5).
When purine bases are exposed, waves of oxidation are observed
around + 0.85 V, indicating the oxidation of guanine and, with a
7
0
more positive potential, the oxidation of adenine.
Derivative 4c interacts with the ssDNA, a behavior that was
confirmed by modifying the oxidation currents of the guanosine
and adenosine bases in the ssDNA (Fig. 6A and B). In the analysis
as a function of concentration, a high potential deviation from
the guanine oxidation peak and disappearance of the adenine
oxidation peak can be observed for high concentrations of 4c, but
at lower concentrations, the base peaks undergo potential
deviation, and the peak decreases over a contact time of 60 min.
Fig. 4 Cyclic voltammogram of the electrochemical behavior of 4c
ꢀ5
ꢀ1
ꢀ1
(c = 3.0 ꢁ 10 mol L ); phosphate buffer 0.2 mol L , pH 7.03; 5% PA
ethanol; CV electrode; dE: 0.3 to ꢀ0.3V.
Molecular modeling
To understand the mode of interaction of 4c with DNA, molecular
docking simulation was performed. The binding pose exhibiting the
ꢀ1
lowest binding energy (ꢀ7.9 kcal mol ) was selected as the initial
coordinates for the MD simulations of the DNA–ligand complex
within 100 ns. The initial conformation for 4c in the molecular
dynamic simulations (from the molecular docking) is represented in
Fig. 7A, where derivative 4c binds to the DNA groove. The most
prevalent conformations during MD are illustrated in Fig. 7B and C.
It was observed that the compound initially remains as a groove
binder, where the complex was stabilized by intermolecular van der
Waals interactions (Fig. 7B). During the simulation, 4c intercalates
with DNA bases. The new pose was stabilized by conventional
hydrogen bonds with residues DT20 and DA06, as well as van der
Waals interactions between the quinoline moiety and the base pairs
DT19 and DA06 (Fig. 7C).
Fig. 5 Differential pulse voltammograms of dsDNA-4c as a function of
time variation. Acetate buffer pH = 4.5; glassy carbon electrode; pulse
The Root Mean Square Deviation (RMSD) of the DNA back-
bone during the simulation is presented in Fig. 7D, indicating
ꢀ1
amplitude 50 mV; pulse width 70 ms; n = 50 mV s
.
Fig. 6 (A) Differential pulse voltammograms of ssDNA in the presence of different concentrations of 4c. Acetate buffer pH = 4.5; glassy carbon
ꢀ1
electrode; pulse amplitude 50 mV; pulse width 70 ms; n = 50 mV s . (B) Differential pulse voltammograms of ssDNA in the presence of 4c as a function
ꢀ6
ꢀ1
ꢀ1
.
of time. Acetate buffer pH = 4.5. 4c: 4.0 ꢁ 10 mol L ; glassy carbon electrode; pulse amplitude 50 mV; pulse width 70 ms; n = 50 mV s
1
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Fig. 7 (A) Best docking conformations for 4c. (B and C) The best conformations of 4c with DNA as a groove binder and intercalator after MD simulation,
respectively. (D) RMSD plot for the DNA backbone during the molecular dynamics simulation.
that the DNA–ligand complex remains stable until the end of Conflicts of interest
simulations without causing DNA denaturation. The more
pronounced structural changes in the oligonucleotide occur
when derivative 4c changes from groove binding to intercalation
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
(identified by the arrows in Fig. 7D), justified by the opening
between the base pairs DT19, DT20, DA5, and DA6, as observed
7
1
in Fig. 7C. Finally, Murugavel et al. have shown a similar
intercalant binding mode of some chloroquinoline derivatives
Acknowledgements
with ctDNA by docking studies, due to p–p interactions invol- We are grateful to the National Council for Scientific and Techno-
ving the planar quinoline moiety and the DNA bases.
logical Development – CNPq (Grant numbers 308431/2017-0-
CNPq-BR, 409146/2018-8-CNPq-BR, and 465536/2014-0-CNPq-BR)
and Fundaç ˜a o de Amparo `a Pesquisa do Estado de Alagoas –
FAPEAL (Grant numbers: EFP_00007992-PPSUS/FAPEAL-BR) for
the financial support of this work. This study was partially
supported by the Coordenaç ˜a o de Aperfeiçoamento de Pessoal
de N ´ı vel Superior – Brasil/CAPES (Finance Code 001).
Conclusions
A series of thiazole–quinoline hybrids, thiazole–indole derivatives,
and analogs have been synthesized with good yields and shorter
reaction time under ultrasound irradiation. All intermediates and
title compounds are characterized by NMR and HRMS. Among all
derivatives screened against three cancer cell lines, some of them
showed moderate to high activity, and compound 4c displayed the
lowest value of IC50 against HL-60 leukemic cells. The electroche-
mical and spectroscopic experiments established that 4c interacts
with DNA preferentially by the groove mode. The binding interac-
tions were identified as conventional hydrogen bonds and van der
Waals forces through MD simulation. Modifications to improve
the potency and selectivity of this active derivative are currently in
progress in our laboratory.
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