Med Chem Res (2016) 26:27–43 MEDICINAL

DOI 10.1007/s00044-016-1705-9
CHEMISTRY
RESEARCH
ORIGINAL RESEARCH

Synthesis and in vitro evaluation of novel triazole/azide chalcones

Fernanda C. G. Evangelista1 Maralice O. Bandeira2 Graziele D. Silva3
● ● ●

Marina G. Silva3 Silmara N. Andrade2 Deisielly R. Marques2 Luciana M. Silva4

● ● ● ●

Whocely V. Castro2 Fabio V. Santos2 Gustavo H. R. Viana2 José A. F. P. Villar3

● ● ● ●

Adriano P. Sabino1 Fernando P. Varotti 2

●

Received: 10 November 2015 / Accepted: 26 July 2016 / Published online: 23 September 2016
© Springer Science+Business Media New York 2016

Abstract A series of 30 novel triazole/azide chalcone Introduction

derivatives were synthesized by Claisen-Schmidt and Cu(I)-
catalyzed cycloaddition reactions. The antiproliferative Cancer is a generic term designating a group of diseases that
activity of each compound was evaluated against HeLa, can affect different tissues in an organism and can be
RKO-AS45-1 and Wi-26VA4 cell lines. Terminal deox- characterized by disordered cell growth (Zhang et al.,
ynucleotidyl transferase dUTP nick end labeling assays 2013b). This disorder occurs because the cancer cells lose
indicated that compounds 4j and 5j significantly reduced the ability to limit and regulate their own growth (Tanaka
the HeLa and RKO-AS45-1cell populations compared to et al., 2009).
the controls. The relative expression of the TP53 gene This group of diseases has an important impact on
revealed changes in both cell lines after exposure to com- human health, causing millions of deaths every year.
pounds 5j and 4j. The increased expression of the TP53 According to the World Health Organization, until 2030,
gene suggests a cellular attempt to repair DNA damage and more than 12 million people across the world are expected
indicates these triazole/azide chalcone derivatives as pro- to die from some type of cancer.
mising anticancer agents. One of the therapeutic alternatives against cancer is the
use of chemical agents, alone or in combination (Abu et al.,
Keywords Chalcones Cytotoxicity TP53 gene
● ●
2013). The aim of chemotherapy is to destroy the malignant
cells without affecting the normal ones. However, most
chemotherapeutical agents act non-selectively, damaging
both cancer and normal cells (Shin et al., 2013; Singh et al.,
2013; Bracci et al., 2014). The innate characteristics of the
cancerous cells, such as uncontrolled proliferation and the
* Adriano P. Sabino
adriansabin@farmacia.ufmg.br ability to metastasize and to avoid apoptosis, render therapy
* Fernando P. Varotti
a continuous challenge. Despite advances in chemotherapy,
varotti@ufsj.edu.br problems like side effects and development of resistance are
the main causes of treatment failure (Abbas et al., 2014). In
1
Departamento de Análises Clínicas e Toxicológicas, Faculdade de this scenario, the search for new compounds with antitumor
Farmácia, UFMG, Av. Antonio Carlos 6627, Pampulha, Belo
activity is necessary.
Horizonte, MG 31270901, Brazil
2
Chalcones are an important class of natural products,
Núcleo de Pesquisa em Química Biológica (NQBio), UFSJ, Av.
precursors of flavonoids. These compounds are present in
Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501296,
Brazil most plants, concentrating in the seeds and peel of fruits,
3 and especially in the flowers (Zhang et al., 2013b). The term
Laboratório de Síntese Orgânica e NanoEstruturas, UFSJ, Av.
Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501296, chalcone is used to designate a family of compounds
Brazil characterized by the presence of the 1,3-diphenyl-2-propen-
4
Fundação Ezequiel Dias (FUNED), Rua Conde Pereira Carneiro, 1-one moiety as the fundamental nucleus of the structure
80, Belo Horizonte, MG 30510010, Brazil (Singh et al., 2012). Chemically, chalcones consist of
28 Med Chem Res (2016) 26:27–43

open-chain flavonoids, wherein two aromatic rings are resonance spectra (1H NMR) were obtained using a Bruker
joined by a three-carbon α, β-unsaturated ketone. They are AVANCE 400 MHz or Bruker AC 200 MHz. Spectra
classified according to their chemical structures. Chalcones were recorded in CDCl3 or dimethylsulfoxide (DMSO)-d6
and their derivatives hold many biological activities solutions. Chemical shifts are reported as δ (ppm)
including anticancer, anti-inflammatory, antioxidant, cyto- downfield from tetramethylsilane, and the J values are
toxic, antimicrobial, analgesic and antipyretic, anti- reported in Hz. Preparative chromatography was performed
hepatotoxic, antiallergic, and antimalarial (Kim et al., 2001; using silica gel (230–400 mesh) following the methods
Achanta et al., 2006; Lim et al., 2007; Bandgar et al., 2010; described by Still et al. (1978). Thin layer chromatography
Hans et al., 2010; Moon et al., 2010; Gutteridge et al., 2011; (TLC) was performed using silica gel GF254, 0.25 mm
Mielcke et al., 2012; Sharma et al., 2013; Kumar et al., thickness. For visualization, TLC plates were either placed
2014; Sharma et al., 2014). under ultraviolet light or stained with iodine vapor or acidic
Several chalcones have been reported to have cytotoxic vanillin.
activity due to their potential ability to inhibit various High performance liquid chromatography (HPLC) ana-
molecular targets/pathways involved in carcinogenesis lysis was performed in a Shimadzu Prominence liquid
(Mahapatra et al., 2015), acting in different points of the chromatograph, LC-20AT pump, SPD-M20A photodiode
cells, including inhibition of tubulin assembly, angiogen- array detector, SIL-20A automatic injector, and C-18 Shim-
esis, kinase inhibition, and induction of cell death by pack VP-ODS reverse phase column (4.6 × 250 mm, i.d.
apoptosis (Champelovier et al., 2013). According to Melo 5 μm). High-resolution mass spectrometry (HRMS) analysis
et al. (2006), Mourad et al. (2012), and Zhang et al. (2013a), was recorded in a Bruker micrOTOF-QII. Infrared (IR)
chalcones have proved to be a promising class against a analysis was recorded in a Shimadzu IRAffinity-1.
range of tumor cells, both in vitro and in vivo. Their bio-
logical activity and solubility depend on the presence of
different groups on the aromatic rings. The presence of General procedure of O-alkylation of 1-(2,4-
hydroxy substituents on both rings causes enhanced activity dihydroxyphenyl)ethan-1-one (resacetophenone 1)
and solubility (Tomar et al., 2010).
Previous structure–activity studies allowed inference that Resacetophenone 1 (2.0 g, 13.1 mmol), 5.4 g (39.1 mmol)
the addition of substituents on chalcone structures is an of anhydrous K2CO3, 5.18 g (24.2 mmol) of 1,4-dibromo-
important strategy to reduce their toxicity and increase their butane and 15 mL of anhydrous acetonitrile were added to a
potential biological activity (Ethiraj et al., 2013). According round-bottom flask. After stirring for 5 h at 40 °C, 50 mL of
to Abonia et al. (2012) and Zhang et al. (2013b), the bio- water was added and extracted with ethyl acetate (3 × 50
logical activity of these compounds increased by inserting mL). The organic layer was washed with brine, dried over
heterocyclic rings into their structures. Some authors syn- anhydrous Na2SO4 and concentrated in a vacuum. The
thesized chalcones with a 1,2,3-triazole ring, due to their crude product was purified by silica column chromato-
low toxicity (Kamal et al., 2011), high stability, high dipole graphy (EtOAc/Hex 3:1).
moment and ability to form hydrogen bonds with potential
molecular targets (Chinthala et al., 2015). Furthermore,
these heterocyclic moieties have been suggested to have a 1-[4-(4-Bromobutoxy)-2-hydroxyphenyl]ethan-1-one 2
cytotoxic profile against many cancer lines (Yin et al.,
2014). However, there is little information regarding the White solid 2.26 g (7.9 mmol, 72 %); 1H NMR (400 MHz,
cytotoxic activity of triazole chalcones. Linking triazole CDCl3) δ (ppm): 1.93–2.00 (m, 2H, BrCH2CH2); 2.02–2.09
rings to chalcone structures seems to be an interesting way (m, 2H, OCH2CH2); 2.56 (s, 3H, CH3); 3.48 (t, 2H, J =
of producing new derivatives (Guantai et al., 2010). 6.40 Hz, BrCH2CH2); 4.04 (t, 2H, J = 6.05 Hz, OCH2CH2);
Therefore, the objective of this study was to evaluate the 6.40 (d, 1H, J = 2.47 Hz, ArH); 6.43 (dd, 1H, J = 8.85 Hz,
in vitro antitumor activity of synthetic triazole/azide chal- J = 2.47 Hz, ArH); 7.63 (d, 1H, J = 8.85 Hz, ArH); 12.74 (s,
cone derivatives. 1H, ArOH); 13C NMR (100 MHz, CDCl3) δ (ppm): 202.51
(C, COCH3); 165.31 (C, C-2); 165.16 (C, C-4); 132.29
(CH, C-6); 113.88 (C, C-1); 107.83 (CH, C-5); 101.27 (CH,
Materials and methods C-3); 67.20 (CH2, OCH2); 33.19 (CH2, BrCH2); 29.28
(CH2, BrCH2CH2); 27.60 (CH2, OCH2CH2); 26.21 (CH3);
Chemistry IR (KBr) νmax/cm−1: 1631.78; HRMS calculated for
C12H15BrO3 [M+H+] 287.0277; found 287.0270; HPLC
Reagents and solvents were purchased as reagent grade and RT: 8.50 min, solvent: MeOH 30 %, THF 30 %, H2O 40 %;
used without further purification. Proton nuclear magnetic C18 250 mm × 4.6 mm.
Med Chem Res (2016) 26:27–43 29

Preparation of 1-[4-(4-azidobutoxy)-2-hydroxyphenyl] (C, C-1″); 128.60 (CH, C-4″); 128.43 (CH, C-3″; C-5″);
ethan-1-one 3 120.20 (CH, Cα); 114.07 (CH, C-1′); 108.02 (CH, C-5′);
101.49 (CH, C-3′); 67.56 (CH2, OCH2); 51.09 (CH2,
NaN3 (0.13 g, 2 mmol) and 4 mL of DMSO were added to a N3CH2); 26.24 (CH2, OCH2CH2); 25.66 (CH2,
round-bottom flask; after total dissolution of NaN3, 0.29 g N3CH2CH2); IR (KBr) νmax/cm−1: 1635; 2089; HRMS (m/
(1 mmol) of bromide (3) was added. After stirring for 3 h at z): [M+Na]+ 360.1312 (C19H19N3NaO3+ = 360.1319).
room temperature, 20 mL of water was added and extracted
with ethyl acetate (3 × 30 mL). The organic layer was (2E)-1-[4-(4-Azidobutoxy)-2-hydroxyphenyl]-3-(4-
washed with brine, dried over anhydrous Na2SO4 and methoxyphenyl)prop-2-en-1-one 4b
concentrated in a vacuum. The crude product was filtered in
a little silica column to afford a low melting oil. Recrystallization over MeOH, yellow solid, yield 0.19 g
Yellow oil, 0.22 g (0.88 mmol, 86 %); 1H NMR (400 (0.52 mmol, 52 %); 1H NMR (400 MHz, CDCl3) δ (ppm):
MHz, CDCl3) δ (ppm): 1.75–1.83 (m, 2H, N3CH2CH2); 1.75–1.84 (m, 2H, N3CH2CH2); 1.85–1.94 (m, 2H,
1.85–1.92 (m, 2H, OCH2CH2); 2.56 (s, 3H, CH3); 3.36 (t, OCH2CH2); 3.39 (t, 2H, J = 6.73 Hz, N3CH2CH2); 3.86 (s,
2H, J = 6.73 Hz, N3CH2CH2); 4.03 (t, 2H, J = 5.98 Hz, 3H, CH3); 4.05 (t, 5.95 Hz, 2H, OCH2CH2); 6.44 (d, 1H, J
OCH2CH2); 6.40 (d, 1H, J = 2.47 Hz, ArH); 6.44 (dd, 1H, = 2.46 Hz, ArH); 6.48 (dd, 1H, J = 8.89 Hz, J = 2.46 Hz,
J = 8.85 Hz, J = 2.47 Hz, ArH); 7.63 (d, 1H, J = 8.85 Hz, ArH); 6.94 (d, 2H, J = 8.72 Hz, ArH); 7.46 (d, 1H, J =
ArH); 12.74 (s, 1H, ArOH); 13C NMR (100 MHz, CDCl3) δ 15.39 Hz, Hα); 7.62 (d, 2H, J = 8.72 Hz, ArH); 7.83 (d, 1H,
(ppm): 202.51(C, COCH3); 165.33 (C, C-2); 165.20 (C, C- J = 8.89 Hz, ArH); 7.86 (d, 1H, J = 15.39 Hz, Hβ); 13.56 (s,
4); 132.29 (CH, C-6); 113.92 (C, C-1); 107.86 (CH, C-5); 1H, ArOH); 13C NMR (100 MHz, CDCl3) δ (ppm): 191.81
101.30 (CH, C-3); 67.52 (CH2, OCH2); 51.09 (CH2, (C, COCH=CH); 166.54 (C, C-4′); 165.24 (C, C-2′);
N3CH2); 26.24 (CH2, OCH2CH2); 26.19 (CH2, 161.78 (C, C-4″); 144.26 (CH, Cβ); 131.12 (CH, C-6′);
N3CH2CH2); 25.64 (CH3); IR (KBr) νmax/cm−1: 1651.07; 130.34 (CH, C-2″;C-6″) 127.49 (C, C-1″); 117.76 (CH,
2096.62; HPLC RT: 8.98 min, solvent MeOH 70 %:H2O Cα); 114.43 (CH, C-3″; C5″); 114.14 (C, C-1′); 107.83
30 %; C18 250 mm × 4.6 mm. (CH, C-5′); 101.50 (CH, C-3′); 67.51 (CH2, OCH2); 55.41
(OCH3,); 51.09 (CH2, N3CH2); 26.25 (CH2, OCH2CH2);
General procedure for the synthesis and purification of 25.65 (CH2, N3CH2CH2); IR (KBr) νmax/cm−1: 1637; 2089;
chalcones HRMS (m/z): [M+Na]+ 390.1420 (C20H21N3NaO4+ =
390.1424).
Compound 3 (0.25 g, 1 mmol), 1.2 mmol of the appropriate
benzaldehyde and 2 mL of methanol were added to a round- (2E)-1-[4-(4-Azidobutoxy)-2-hydroxyphenyl]-3-(4-
bottom flask. To this solution, 2 mL of a solution of KOH in ethoxyphenyl)prop-2-en-1-one 4c
methanol (5 M) was added dropwise. After stirring for 48 h
at room temperature, 20 mL of water was added and Recrystallization over MeOH, yellow solid, yield 0.17 g
extracted with ethyl acetate (3 × 30 mL). The organic layer (0.45 mmol, 45 %); 1H NMR (400 MHz, CDCl3) δ (ppm):
was washed with brine, dried over anhydrous Na2SO4 and 1.43 (t, 1H, J = 7.00 Hz, OCH2CH3); 1.76–1.84 (m, 2H,
concentrated in a vacuum. N3CH2CH2); 1.86–1.94 (m, 2H, OCH2CH2); 3.38 (t, 2H, J
= 6.57 Hz, N3CH2CH2); 4.04 (t, 2H, J = 6.04 Hz,
(2E)-1-[4-(4-Azidobutoxy)-2-hydroxyphenyl]-3-phenylprop- OCH2CH2); 4.08 (q, 2H, J = 7.00 Hz, OCH2CH3); 6.44 (d,
2-en-1-one 4a 1H, J = 2.46 Hz, ArH); 6.47 (dd, 1H, J = 8.91 Hz, J = 2.46
Hz, ArH); 6.93 (d, 2H, J = 8.75 Hz, ArH); 7.45 (d, 1H, J =
Recrystallization over MeOH, yellow solid, yield 0.17 g 15.42 Hz, Hα); 7.60 (d, 2H, J = 8.75 Hz, ArH); 7.86 (d, 1H,
(0.50 mmol, 50 %); 1H NMR (400 MHz, CDCl3) δ (ppm): J = 8.91 Hz, ArH); 7.86 (d, 1H, J = 15.42 Hz, Hβ); 13.57 (s,
1.75–1.85 (m, 2H, N3CH2CH2); 1.86–1.92 (m, 2H, 1H, ArOH); 13C NMR (100 MHz, CDCl3) δ (ppm): 191.82
OCH2CH2); 3.39 (t, 2H, J = 6.73 Hz, N3CH2CH2); 4.06 (t, (C, COCH=CH); 166.53 (C, C-4′); 165.22 (C, C-2′);
2H, J = 5.95 Hz, OCH2CH2); 6.46 (d, 1H, J = 2.47 Hz, 161.21 (C, C-4″); 144.34 (CH, Cβ); 131.11 (CH, C-6′);
ArH-3′); 6.49 (dd, 1H, J = 8.90 Hz, J = 2.47 Hz, ArH-5′); 130.35 (CH, C-2″;C-6″); 127.29 (C, C-1″); 117.59 (CH,
7.40–7.47 (m, 3H, ArH); 7.59 (d, 1H, J = 15.47 Hz, Hα); Cα); 114.89 (CH, C-3″; C5″); 114.14 (C, C-1′); 107.81
7.62–7.70 (m, 2H, ArH); 7.84 (d, 1H, J = 8.90 Hz, ArH); (CH, C-5′); 101.49 (CH, C-3′); 67.50 (CH2, OCH2); 63.66
7.90 (d, 1H, J = 15.47 Hz, Hβ); 13.44 (s, 1H, ArOH); 13C (OCH3,); 51.08 (CH2, N3CH2); 26.24 (CH2, OCH2CH2);
NMR (100 MHz, CDCl3) δ (ppm): 191.78 (C, COCH=CH); 25.64 (CH2, N3CH2CH2); 14.70 (OCH3); IR (KBr) νmax/
166.65 (C, C-4′); 165.45 (C, C-2′); 144.47 (CH, Cβ); cm−1: 1633.71; 2090.84; HRMS (m/z): [M+Na]+ 404.1586
134.74 (CH, C-6′); 131.33 (CH, C-2″;C-6″); 128.96 (C21H23N3NaO4+ = 404.1581).
30 Med Chem Res (2016) 26:27–43

(2E)-1-[4-(4-Azidobutoxy)-2-hydroxyphenyl]-3-(4- 0.98 (t, 3H, J = 7.42 Hz, CH2CH3); 1.46–1.57 (m, 2H,
butoxyphenyl)prop-2-en-1-one 4d CH2CH3); 1.76–1.94 (m, 6H, OCH2CH2; N3CH2CH2;
CH2CH2CH3); 3.37 (t, 2H, J = 6.67 Hz, N3CH2CH2); 3.34
Recrystallization over MeOH, yellow solid, yield 0.28 g (s, 3H, OCH3); 3.99–4.15 (m, 4H, OCH2; OCH2); 6.44 (d,
(0.69 mmol, 68 %); 1H NMR (400 MHz, CDCl3) δ (ppm): 1H, J = 2.48 Hz, ArH); 6.47 (dd, 1H, J = 8.98 Hz, J = 2.48
0.97 (t, 3H, J = 7.30 Hz, CH2CH3); 1.38–1.62 (m, 2H, Hz, ArH); 6.90 (d, 1H, J = 8.35 Hz, ArH); 7.16 (d, 1H, J =
CH2CH3); 1.70–1.97 (m, 6H, OCH2CH2; N3CH2CH2; 1.91 Hz, ArH); 7.23 (dd, 1H, J = 8.35 Hz, J = 1.91 Hz,
CH2CH2CH3); 3.36 (t, 2H, J = 6.28 Hz, N3CH2CH2); 3.96– ArH); 7.43 (d, 1H, J = 15.25 Hz, Hα); 7.84 (d, 1H, J = 8.98
4.10 (m, 4H, OCH2; OCH2); 6.47 (m, 2H, ArH); 6.92 (d, 2H, Hz, ArH); 7.85 (d, 1H, J = 15.25 Hz, Hβ); 13.56 (s, 1H,
J = 8.74 Hz, ArH); 7.48 (d, 1H, J = 15.38 Hz, Hα); 7.60 (d, ArOH); 13C NMR (100 MHz, CDCl3) δ (ppm): 191.80 (C,
2H, J = 8.74 Hz, ArH); 7.82 (d, 1H, J = 8.68 Hz, ArH); 7.86 COCH=CH); 166.62 (C, C-4′); 165.30 (C, C-2′); 151.36
(d, 1H, J = 15.38 Hz, Hβ); 13.58 (s, 1H, ArOH); 13C NMR (C, C-3″); 149.58 (CH, C-4″); 144.77 (CH, Cβ); 131.18
(100 MHz, CDCl3) δ (ppm): 191.84 (C, COCH=CH); 166.53 (CH, C-6′); 127.55 (C, C-1″); 123.43(CH, C-6″); 117.79
(C, C-4′); 165.22 (C, C-2′); 161.45 (C, C-4″); 144.37 (CH, (CH, Cα); 114.19(C, C-1′); 112.37(CH, C-5″); 110.73(CH,
Cβ); 131.11 (CH, C-6′); 130.34 (CH, C-2″;C-6″); 127.25 (C, C-2″); 107.91(CH, C-5′); 101.54 (CH, C-3′); 68.76 (CH2,
C-1″); 117.57 (CH, Cα); 114.93 (CH, C-3″; C5″); 114.17 (C, Ar″OCH2); 67.58(CH2, Ar′OCH2); 56.18 (OCH3); 51.15
C-1′); 107.80 (CH, C-5′); 101.52 (CH, C-3′); 67.88 (CH2, Ar (CH2, N3CH2); 31.11 (CH2, Ar″OCH2CH2); 26.31 (CH2,
″OCH2); 67.51 (CH2, Ar′OCH2); 51.10 (CH2, N3CH2); 31.17 Ar′OCH2CH2); 25.71(CH2, N3CH2CH2); 19.23 (CH2,
(CH2, Ar″OCH2CH2); 26.26 (CH2, Ar′OCH2CH2); 25.66 CH2CH3); 13.90 (CH3); IR (KBr) νmax/cm−1: 1641; 2087;
(CH2, N3CH2CH2); 19.19 (CH2, CH2CH3); 13.80 (CH3); IR HRMS (m/z): [M+Na]+ 462.1998 (C24H29N3NaO5+ =
(KBr) νmax/cm−1: 1635; 2094; HRMS (m/z): [M+Na]+ 462.1999).
432.1899 (C23H27N3NaO4+ = 432.1894).
(2E)-1-[4-(4-Azidobutoxy)-2-hydroxyphenyl]-3-(4-
(2E)-1-[4-(4-Azidobutoxy)-2-hydroxyphenyl]-3-(3- chlorophenyl)prop-2-en-1-one 4g
butoxyphenyl)prop-2-en-1-one 4e
Purification over hexane/ethyl acetate (7:1) column, yellow
Recrystallization over MeOH, yellow solid, yield 0.22 g solid, 0.30 g (0.81 mmol, 81 %); 1H NMR (400 MHz,
(0.53 mmol, 53 %); 1H NMR (400 MHz, CDCl3) δ (ppm): DMSO-d6) δ (ppm): 1.65–1.73 (m, 2H, N3CH2CH2); 1.76–
0.98 (t, 3H, J = 7.30 Hz, CH2CH3); 1.2–1.61 (m, 2H, 1.83 (m, 2H, OCH2CH2); 3.43 (t, 2H, J = 6.81 Hz,
CH2CH3); 1.71–1.94 (m, 6H, OCH2CH2; N3CH2CH2; N3CH2CH2); 4.12 (t, 2H, J = 6.17 Hz, OCH2); 6.52 (d, 1H,
CH2CH2CH3); 3.73 (t, 2H, J = 6.31 Hz, N3CH2CH2); 3.96– J = 2.47 Hz, ArH); 6.57 (dd, 1H, J = 9.06 Hz, J = 2.47 Hz,
4.10 (m, 4H, OCH2; OCH2); 6.41–6.54 (m, 2H, ArH); ArH); 7.54 (d, 2H, J = 8.53 Hz, ArH); 7.81 (d, 1H, J =
6.91–7.02 (m, 1H, ArH); 7.13–7.28 (m, 2H, ArH); 7.31 (t, 15.49 Hz, Hα); 7.96 (d, 2H, J = 8.53 Hz, ArH); 8.04 (d, 1H,
1H, J = 7.87 Hz, ArH); 7.55 (d, 1H, J = 15.50 Hz, Hα); 7.83 J = 15.49 Hz, Hβ); 8.28 (d, 1H, J = 9.06 Hz, ArH), 13.38 (s,
(d, 1H, J = 8.41 Hz, ArH); 7.85 (d, 1H, J = 15.50 Hz, Hβ); 1H, ArOH); 13C NMR (100 MHz, DMSO-d6) δ (ppm):
13.43 (s, 1H, ArOH); 13C NMR (100 MHz, CDCl3) δ 191.58 (C, COCH=CH); 165.64 (C, C-4′); 165.31 (C, C-
(ppm): 191.81(C, COCH=CH); 166.67 (C, C-4′); 165.48 2′); 142.47(CH, Cβ); 135.17(C, C-1″); 133.42(CH, C-4″);
(C, C-2′); 159.56 (C, C-3″); 144.45 (CH, Cβ); 136.12 (C, 132.67(CH, C-6′); 130.66 (CH, C-3″; C-5″); 128.84 (CH,
C-1″); 131.30 (CH, C-6′); 129.94(CH, C-5″); 121.08 (CH, C-2″; C-6″); 121.90 (CH, Cα); 113.72 (C, C-1′); 107.68
Cα); 120.52 (CH, C-6″); 116.84 (CH, C-4″); 114.21 (CH, (CH, C-5′); 101.25 (CH, C-3′); 67.51 (CH2, ArOCH2);
C-2″); 114.14 (C, C-1′); 107.99 (CH, C-5′); 101.57 (CH, C- 50.22 (CH2, N3CH2); 25.59(CH2, ArOCH2CH2); 24.88
3′); 67.86 (CH2, Ar″OCH2); 67.59 (CH2, Ar′OCH2); 51.12 (CH2, N3CH2CH2); IR (KBr) νmax/cm−1: 1641; 2087;
(CH2, N3CH2); 31.31 (CH2, Ar″OCH2CH2); 26.28 (CH2, HRMS (m/z): [M+H]+ 372.135 (C19H19ClN3O3+ =
Ar′OCH2CH2); 25.67(CH2, N3CH2CH2); 19.26 (CH2, 372.111).
CH2CH3); 13.85 (CH3); IR (KBr) νmax/cm−1: 1636; 2093;
HRMS (m/z): [M+Na]+ 432.1893 (C23H27N3NaO4+ = (2E)-1-[4-(4-Azidobutoxy)-2-hydroxyphenyl]-3-(4-
432.1894). bromophenyl)prop-2-en-1-one 4h

(2E)-1-[4-(4-Azidobutoxy)-2-hydroxyphenyl]-3-(4-butoxy- Recrystallization over MeOH, yellow solid, yield 0.25 g

3-methoxyphenyl)prop-2-en-1-one 4f (0.61 mmol, 61 %); 1H NMR (400 MHz, CDCl3; CDCl3) δ
(ppm): 1.76–1.84 (m, 2H, N3CH2CH2); 1.86–1.95 (m, 2H,
Recrystallization over MeOH, yellow solid, yield 0.30 g OCH2CH2); 3.37 (t, 2H, J = 6.56 Hz, N3CH2CH2); 4.04 (t,
(0.68 mmol, 68 %); 1H NMR (400 MHz, CDCl3) δ (ppm): 2H, J = 6.10 Hz, OCH2); 6.45 (d, 1H, J = 2.45 Hz, ArH);
Med Chem Res (2016) 26:27–43 31

6.48 (dd, 1H, J = 8.93 Hz, J = 2.45 Hz, ArH); 7.51 (d, 2H, 131.69(CH, C-6′); 131.32(CH, C-4″); 127.36 (CH, C-5″);
J = 8.61 Hz, ArH); 7.56 (d, 2H, J = 8.81 Hz, ArH); 7.56 (d, 125.96(CH, C-6″); 124.22(CH, Cα); 113.95(C, C-1′);
1H, J = 15.45 Hz, Hα); 7.81 (d, 1H, J = 15.45 Hz, Hβ); 7.81 108.23(CH, C-5′); 101.57 (CH, C-3′); 67.65(CH2,
(d, 1H, J = 8.93 Hz, ArH); 13.37 (s, 1H, ArOH); 13C NMR ArOCH2); 51.09(CH2, N3CH2); 26.25 (CH2, ArOCH2CH2);
(100 MHz, CDCl3) δ (ppm): 191.44 (C, COCH=CH); 25.65 (CH2, N3CH2CH2); IR (KBr) νmax/cm−1: 1637; 2090;
166.70 (C, C-4′); 165.58 (C, C-2′); 142.92 (CH, Cβ); HRMS (m/z): [M+Na]+ 428.0538 (C19H17Cl2N3NaO3+ =
133.67 (C, C-1″); 132.22 (C, C-3″; C-5″); 131.21(CH, C- 428.0539).
6′); 129.83 (CH, C-2″; C-6″); 124.93 (CH, C-4″); 120.84
(CH, Cα); 114.00(C, C-1′); 108.10 (CH, C-5′); 101.52 (CH, General procedure to cycloaddition
C-3′); 67.59 (CH2, ArOCH2); 51.08 (CH2, N3CH2); 26.23
(CH2, ArOCH2CH2); 25.64 (CH2, N3CH2CH2); IR (KBr) The appropriate azide chalcone (1 mmol), 0.23 mL
νmax/cm−1: 1635; 2104; HRMS (m/z): [M+Na]+ 438.0429 (4 mmol) of propargyl alcohol or 0.44 mL (4 mmol) phe-
(C19H18BrN3NaO3+ = 438.0424). nylacetylene, 4 mL of DMSO and a catalytic amount of CuI
were added to a round-bottom flask. After stirring for 2 h at
(2E)-1-[4-(4-Azidobutoxy)-2-hydroxyphenyl]-3-(3- room temperature, 10 mL of water was added and acidified
nitrophenyl)prop-2-en-1-one 4i with HCl (1.2 mol). The crude reside was extracted with
ethyl acetate (3 × 30 mL) and the organic layer was washed
Recrystallization over MeOH, yellow solid, yield 0.25 g with brine, dried over anhydrous Na2SO4 and concentrated
(0.66 mmol, 66 %); 1H NMR (400 MHz, CDCl3) δ (ppm): in a vacuum. The residue was filtered in a little silica col-
1.77–1.85 (m, 2H, N3CH2CH2); 1.87–1.96 (m, 2H, umn and recrystallized over MeOH.
OCH2CH2); 3,39 (t, 2H, J = 6.56 Hz, N3CH2CH2); 4.06 (t,
2H, J = 6.10 Hz, OCH2); 6.47 (d, 1H, J = 2.46 Hz, ArH); (2E)-1-(2-Hydroxy-4-{4-[4-(hydroxymethyl)-1H-1,2,3-
6.52 (dd, 1H, J = 9.01 Hz, J = 2.46 Hz, ArH); 7.62 (t, 1H, J triazol-1-yl]butoxy}phenyl)-3-phenylprop-2-en-1-one 5a
= 7.98 Hz, ArH); 7.69 (d, 1H, J = 15.42 Hz, Hα); 7.85 (d,
1H, J = 9.01 Hz, ArH); 7.89 (d, 1H, J = 15.42 Hz, Hβ); Recrystallization over MeOH, yellow solid, 0.35 g
7.94–7.91 (m, 1H, ArH); 8.24–8.29 (m, 1H, ArH); 8.52 (t, (0.88 mmol, 88 %); 1H NMR (400 MHz, DMSO-d6) δ
J = 1.82 Hz, 1H, ArH); 13.24 (s, 1H, ArOH); 13C NMR (ppm): 1.66–1.76 (m, 2H, OCH2CH2); 1.93–2.02 (m, 2H,
(100 MHz, CDCl3) δ (ppm): 190.93 (C, COCH=CH); N3CH2CH2); 4.10 (t, 2H, J = 5.97 Hz, OCH2CH2); 4.44 (t,
166.88 (C, C-4′); 165.93 (C, C-2′); 148.77(C, C-3″); 141.24 2H, J = 7.0 Hz, NCH2CH2); 4.54 (d, 2H, J = 5.35 Hz,
(CH, Cβ); 136.58 (C, C-1″); 134.50(CH, C-6″); 131.39(CH, HOCH2); 5.20 (t, 1H, J = 5.61 Hz, HOCH2); 6.52 (d, 1H, J
C-6′); 130.11(CH, C-5″); 124.74 (CH, C-4″); 123.25 (CH, = 2.46 Hz, ArH); 6.57 (dd, 1H, J = 9.10 Hz, J = 2.46 Hz,
C-2″); 122.31 (CH, Cα); 113.95(C, C-1′); 108.39 (CH, C- ArH); 7.44–7.52 (m, 3H, ArH); 7.84 (d, 1H, J = 15.51 Hz,
5′); 101.63(CH, C-3′); 67.73 (CH2, ArOCH2); 51.13(CH2, Hα); 7.89–7.96 (m, 2H, ArH); 8.00 (s, 1H, CHN); 8.03 (d,
N3CH2); 26.29 (CH2, ArOCH2CH2); 25.69 (CH2, 1H, J = 15.51 Hz, Hβ); 8.29 (d, 1H, J = 9.10 Hz, ArH);
N3CH2CH2); IR (KBr) νmax/cm−1: 1645; 2095; HRMS (m/ 13.45 (s, 1H, ArOH); 13C NMR (100 MHz, DMSO-d6) δ
z): [M+Na]+ 405.1168 (C19H18N4NaO5+ = 405.1169). (ppm): 191.77 (C, COCH=CH); 165.64 (C, C-4′); 165.25
(C, C-2′); 147.87(C, HOCH2C=CH); 144.06(CH, Cβ);
(2E)-1-[4-(4-Azidobutoxy)-2-hydroxyphenyl]-3-(2,3- 134.45 (C, C-1″); 132.67 (CH, C-6′); 130.73 (CH, CHN);
dichlorophenyl)prop-2-en-1-one 4j 129.03 (CH, C-3″; C-5″); 128.85 (CH, C-2″; C-6″); 122.59
(CH, C-4″); 121.12 (CH, Cα); 113.75 (C, C-1′); 107.70
Purification over hexane/ethyl acetate (3:1) column, yellow (CH, C-5′); 101.29 (CH, C-3′); 67.38 (CH2, ArOCH2);
solid, yield 0.28 g (0.69 mmol, 68 %); 1H NMR (400 MHz, 55.00 (CH2, NCH2); 48.75 (CH2, HOCH2); 26.40 (CH2,
CDCl3) δ (ppm): 1.76–1.84 (m, 2H, N3CH2CH2); 1.87–1.95 ArOCH2CH2); 25.40 (CH2, NCH2CH2); IR (KBr)
(m, 2H, OCH2CH2); 3.37 (t, 2H, J = 6.56 Hz, N3CH2CH2); νmax/cm−1: 1631.78; 1568.13; HRMS (m/z): [M+H]+
4.05 (t, 2H, J = 6.11 Hz, OCH2); 6.46 (d, 1H, J = 2.45 Hz, 394.1758 (C22H24N3O4+ = 394.1761).
ArH); 6.48 (dd, 1H, J = 8.85 Hz, J = 2.45 Hz, ArH); 7.27 (t,
1H, J = 7.51 Hz, ArH); 7.52 (d, 1H, J = 15.44 Hz, Hα); 7.52 (2E)-1-(2-Hydroxy-4-{4-[4-(hydroxymethyl)-1H-1,2,3-
(dd, 1H, J = 7.93 Hz, J = 1.32 Hz, ArH); 7.65 (dd, 1H, J = triazol-1-yl]butoxy}phenyl)-3-(4-methoxyphenyl)prop-2-en-
7.93 Hz, J = 1.32 Hz, ArH), 7.79 (d, 1H, J = 8.85 Hz, ArH); 1-one 5b
8.24 (d, 1H, J = 15.44 Hz, Hβ); 13.27 (s, 1H, ArOH); 13C
NMR (100 MHz, CDCl3) δ (ppm): 191.19 (C, COCH=CH); Recrystallization over MeOH, yellow solid, 0.35 g (0.81
166.81 (C, C-4′); 165.74 (C, C-2′); 140.03 (CH, Cβ); mmol, 81 %); 1H NMR (400 MHz, DMSO-d6; CDCl3) δ
135.56 (C, C-3″); 134.17 (C, C-1″); 133.54 (CH, C-2″); (ppm): 1.76–1.85 (m, 2H, OCH2CH2); 2.03–2.13 (m, 2H,
32 Med Chem Res (2016) 26:27–43

NCH2CH2); 3.86 (s, 3H, CH3); 4.06 (t, 2H, J = 6.18 Hz, (t, 3H, J = 7.40 Hz, OCH3); 1.48–1.55 (m, 2H, CH2CH3);
OCH2CH2); 4.44 (t, 2H, J = 7.00 Hz, NCH2CH2); 4.63 (d, 1.75–1.87 (m, 4H, CH2CH2CH3; OCH2CH2); 2.09–2.16
2H, J = 3.27 Hz, HOCH2); 5.10 (t, 1H, J = 3.27 Hz, 1H); (m, 2H, NCH2CH2); 3.97–4.06 (m, 4H, OCH2CH2;
6.42 (d, 1H, J = 2.45 Hz, HOCH2); 6.49 (dd, 1H, J = 9.03 OCH2CH2); 4.34 (s, 1H, HOCH2); 4.44 (t, 2H, J = 7.04 Hz,
Hz, J = 2.45 Hz, ArH); 6.97 (d, 2H, J = 8.79 Hz, ArH); 7.65 NCH2CH2); 4.80 (s, 2H, HOCH2); 6.42 (d, 1H, J = 2.34 Hz,
(d, 1H, J = 15.35 Hz, Hα); 7.73 (d, 2H, J = 8.79 Hz, ArH); ArH); 6.45 (dd, 1H, J = 8.94 Hz, J = 2.34 Hz, ArH); 6.92
7.81 (s, 1H, CHN); 7.81 (d, 1H, J = 15.35 Hz, Hβ); 8.04 (d, (d, 2H, J = 8.70 Hz, ArH); 7.44 (d, 1H, J = 15.60 Hz, Hα);
J = 9.03 Hz, ArH); 13.54 (s, 1H, ArOH); 13C NMR (100 7.57 (d, 1H, J = 8.94 Hz, ArH); 7.60 (d, 2H, J = 8.70 Hz,
MHz, DMSO-d6; CDCl3) δ (ppm): 191.24(C, COCH=CH); ArH); 7.81 (s, 1H, CHN); 7.86 (d, 1H, J = 15.60 Hz, Hβ);
165.66 (C, C-4′); 164.64 (C, C-2′); 161.19 (CH, C-4″) ; 13.57 (s, 1H, ArOH); 13C NMR (100 MHz, CDCl3) δ
147.90 (C, HOCH2C=CH); 143.67 (CH, Cβ); 131.36 (CH, (ppm): 191.85 (C, COCH=CH); 166.44 (C, C-4′); 164.99
C-6′); 130.17 (CH, C-2″; C-6″); 126.78 (C, C-1″); 121.71 (C, C-2′); 161.46 (CH, C-4″); 147.73 (C, HOCH2C=CH);
(CH, CHN); 117.54 (CH, Cα); 113.90 (CH, C-3″; C-5″); 144.48 (CH, Cβ); 131.17 (CH, C-6′); 130.37 (CH, C-2″;
113.48 (C, C-1′); 107.04(CH, C-5′); 100.96(CH, C-3′); C-6″); 127.19 (C, C-1″); 121.60 (CH, CHN); 117.48
66.83 (CH2, ArOCH2); 55.18 (CH2, NCH2); 54.87 (OCH3); (CH, Cα); 114.92 (CH, C-3″; C-5″); 114.25 (C, C-1′);
48.85 (CH2, HOCH2); 26.35 (CH2, ArOCH2CH2); 25.27 107.66 (CH, C-5′); 101.55 (CH, C-3′); 67.88 (CH2,
(CH2, NCH2CH2); IR (KBr) νmax/cm−1: 1635.64; 1577.77; Ar’OCH2); 67.19 (CH2, Ar″OCH2); 56.51 (CH2, HOCH2);
HRMS (m/z): [M+H]+ 424.1869 (C23H26N3O5+ = 49.93 (CH2, NCH2); 31.16 (CH2, CH2CH2CH3); 27.08
424.1867). (CH2, ArOCH2CH2); 25.95 (CH2, NCH2CH2); 19.18
(CH2, CH2CH3); 13.81 (OCH3); IR (KBr) νmax/cm−1:
(2E)-3-(4-Ethoxyphenyl)-1-(2-hydroxy-4-{4-[4- 1631.78; 1566.2; HRMS (m/z): [M+Na]+ 488.2153
(hydroxymethyl)-1H-1,2,3-triazol-1-yl]butoxy}phenyl)prop- (C26H31N3NaO5+ = 488.2156).
2-en-1-one 5c

Recrystallization over MeOH, yellow solid, 0.39 g (0.89 (2E)-3-(3-Butoxyphenyl)-1-(2-hydroxy-4-{4-[4-

mmol, 89 %); 1H NMR (400 MHz, CDCl3) δ (ppm): 1.43 (hydroxymethyl)-1H-1,2,3-triazol-1-yl]butoxy}phenyl)prop-
(t, 3H, J = 6.93 Hz, OCH2CH3); 1.79–1.97 (m, 2H, 2-en-1-one 5e
OCH2CH2); 2.08–2.16 (m, 2H, NCH2CH2); 4.01 (t, 2H, J
= 5.96 Hz, OCH2CH2); 4.08 (q, 2H, J = 7.00 Hz, Recrystallization over MeOH, yellow solid, 0.39 g (0.83
OCH2CH3); 4.43 (t, 2H, J = 7.02 Hz, NCH2CH2); 4.79 mmol, 83 %); 1H NMR (400 MHz, CDCl3) δ (ppm): 0.99
(s, 2H, HOCH2); 6.41 (d, 1H, J = 2.18 Hz, ArH); 6.44 (dd, (t, 3H, J = 7.41 Hz, OCH3); 1.48–1.57 (m, 2H, CH2CH3);
1H, J = 9.12 Hz, J = 2.18 Hz, ArH); 6.93 (d, 2H, J = 8.68 1.75–1.88 (m, 4H, CH2CH2CH3; OCH2CH2); 2.08–2.18
Hz, ArH); 7.44 (d, 1H, J = 15.64 Hz, Hα); 7.58 (s, 1H, (m, 2H, NCH2CH2); 3.99–4.06 (m, 4H, OCH2CH2;
CHN); 7.60 (d, 2H, J = 8.68 Hz, ArH); 7.82 (d, 1H, J = OCH2CH2); 4.44 (t, 2H, J = 7.03 Hz, NCH2CH2); 4.80 (s,
9.12 Hz, ArH); 7.85 (d, 1H, J = 15.64 Hz, Hβ); 13.56 (s, 2H, HOCH2); 6.42 (d, 1H, J = 2.47 Hz, ArH); 6.46 (dd,
1H, ArOH); 13C NMR (100 MHz, CDCl3) δ (ppm): 191.84 1H, J = 8.97 Hz, J = 2.47 Hz, ArH); 6.94–6.99 (m, 1H,
(C, COCH=CH); 166.43 (C, C-4′); 165.00 (C, C-2′); ArH); 7.15–7.17 (m, 1H, ArH); 7.20–7.24 (m, 1H, ArH);
161.23 (CH, C-4″); 147.78 (C, HOCH2C=CH); 144.44 7.32 (t, 1H, J = 7.96 Hz, ArH); 7.54 (d, 1H, J = 15.47 Hz,
(CH, Cβ); 131.17 (CH, C-6′); 130.37 (CH, C-2″; C-6″); Hα); 7.58 (s, 1H, CHN); 7.82 (d, 1H, J = 8.97 Hz, ArH);
127.24 (C, C-1″); 121.61 (CH, CHN); 117.53 (CH, Cα); 7.84 (d, 1H, J = 15.47 Hz, Hβ); 13.42 (s, 1H, ArOH);
114.89 (CH, C-3″; C-5″); 114.23(C, C-1′); 107.64 (CH, C- 13
C NMR (100 MHz, CDCl3) δ (ppm): 191.81(C,
5′); 101.55 (CH, C-3′); 67.18(CH2, Ar′OCH2); 63.66 (CH2, COCH=CH); 166.56 (C, C-4′); 165.23 (C, C-2′); 159.52
Ar″OCH2CH3); 56.42 (CH2, NCH2); 49.89 (CH2, HOCH2); (CH, C-3″); 147.77 (C, HOCH2C=CH); 144.52 (CH, Cβ);
27.05 (CH2, ArOCH2CH2); 25.93 (CH2, NCH2CH2); 14.68 136.04 (C, C-1″); 131.34 (CH, C-6′) 129.92 (CH, C-5″);
(OCH3); IR (KBr) νmax/cm−1: 1639.49; 1568.13; HRMS (m/ 121.56 (CH, CHN); 121.06 (CH, Cα); 120.41 (CH, C-6″);
z): [M+Na]+ 460.1838 (C24H27N3NaO5+ = 460.1843). 116.84 (CH, C-4″); 114.19 (CH, C-2″ ;C, C-1′); 107.81
(CH, C-5′); 101.58 (CH, C-3′); 67.83 (CH2, Ar′OCH2);
(2E)-3-(4-Butoxyphenyl)-1-(2-hydroxy-4-{4-[4- 67.23 (CH2, Ar″OCH2); 56.52 (CH2, HOCH2); 49.90
(hydroxymethyl)-1H-1,2,3-triazol-1-yl]butoxy}phenyl)prop- (CH2, NCH2); 31.27 (CH2, CH2CH2CH3); 27.06 (CH2,
2-en-1-one 5d ArOCH2CH2); 25.94 (CH2, NCH2CH2); 19.22 (CH2,
CH2CH3); 13.82 (OCH3); IR (KBr) νmax/cm−1: 1635.64;
Recrystallization over MeOH, yellow solid, 0.37 g (0.80 1570.06; HRMS (m/z): [M+H]+ 466.2336 (C26H32N3O5+
mmol, 80 %); 1H NMR (400 MHz, CDCl3) δ (ppm): 0.97 = 466.2336).
Med Chem Res (2016) 26:27–43 33

(2E)-3-(4-Butoxy-3-methoxyphenyl)-1-(2-hydroxy-4-{4-[4- νmax/cm−1: 1643.35; 1577.77; HRMS (m/z): [M+Na]+

(hydroxymethyl)-1H-1,2,3-triazol-1-yl]butoxy}phenyl)prop- 450.1195 (C22H22ClN3NaO4+ = 450.1191).
2-en-1-one 5f
(2E)-3-(4-Bromophenyl)-1-(2-hydroxy-4-{4-[4-
Recrystallization over MeOH, yellow solid, 0.45 g (0.90 (hydroxymethyl)-1H-1,2,3-triazol-1-yl]butoxy}phenyl)prop-
mmol, 90 %); 1H NMR (400 MHz) δ (ppm): 0.98 (t, 3H, J 2-en-1-one 5h
= 7.39 Hz, CH2CH3); 1.46–1.56 (m, 2H, CH2CH3); 1.79–
1.90 (m, 4H, CH2CH2CH3; OCH2CH2); 2.08–2.18 (m, 2H, Recrystallization over MeOH, yellow solid, 0.43 g (0.92
NCH2CH2); 3.95 (s, 3H, OCH3); 4.02 (t, 2H, J = 5.97 Hz, mmol, 92 %); 1H NMR (400 MHz, acetone-d6, DMSO-d6) δ
OCH2CH2); 4.07 (t, 2H, J = 6.73 Hz, OCH2CH2); 4.44 (t, (ppm): 1.66–1.76 (m, 2H, OCH2CH2); 1.92–2.02 (m, 2H,
2H, J = 7.00 Hz, NCH2CH2); 4.80 (s, 2H, HOCH2); 6.42 (d, NCH2CH2); 4.10 (t, 2H, J = 6.40 Hz, OCH2CH2); 4.42 (t,
1H, J = 2.37 Hz, ArH); 6.45 (dd, 1H, J = 9.11 Hz, J = 2.37 2H, J = 6.98 Hz, NCH2CH2); 4.52 (brs, 2H, HOCH2); 5.22
Hz, ArH); 6.90 (d, 1H, J = 8.34 Hz, ArH); 7.15–7.18 (m, (t, 1H J = 5.0 Hz, HOCH2); 6.52 (d, 1H, J = 2.45 Hz, ArH);
1H, ArH); 7.21–7.25 (m, 1H, ArH); 7.42 (d, 1H, J = 15.14 6.56 (dd, 1H, J = 9.08 Hz, J = 2.45 Hz, ArH); 7.68 (d, 2H,
Hz, Hα); 7.58 (s, 1H, CHN); 7.83 (d, 1H, J = 9.11 Hz, J = 8.52 Hz, ArH); 7.79 (d, 1H J = 15.48 Hz, Hα); 7.98 (d,
ArH); 7.84 (d, 1H, J = 15.14 Hz, Hβ); 13.56 (s, 1H, ArOH); 2H, J = 8.52 Hz, ArH); 8.00 (s, 1H, CHN); 8.06 (d, 1H, J =
13
C NMR (100 MHz) δ (ppm): 191.77 (C, COCH=CH); 15.48 Hz, Hβ); 8.28 (d, 1H, J = 9.08 Hz, ArH); 13.39 (s,
166.45 (C, C-4′); 165.02(C, C-2′); 151.34(CH, C-4″); 1H, ArOH); 13C NMR (100 MHz, DMSO-d6) δ (ppm):
149.53 (CH, C-3″); 147.75(C, HOCH2C=CH); 144.80 191.62 (C, COCH=CH); 165.68 (C, C-4′); 165.35 (C, C-
(CH, Cβ); 131.19 (CH, C-6′); 127.46 (C, C-1″); 123.39 2′); 147.90 (C, HOCH2C=CH); 142.65 (CH, Cβ); 133.79
(CH, C-6″); 121.59 (CH, CHN); 117.68 (CH, Cα); 114.22 (C, C-1″); 132.74 (CH, C-6′); 131.84 (CH, C-3″; C-5″);
(C, C-1′); 112.36 (CH, C-5″); 110.78(CH, C-2″); 107.67 130.92 (CH, C-2″; C-6″); 124.15 (CH, C-4″); 122.63 (CH,
(CH, C-5′); 101.55 (CH, C-3′); 68.71 (CH2, Ar′OCH2); CHN); 122.01 (CH, Cα); 113.79(C, C-1′); 107.75 (CH, C-
67.19 (CH2, Ar″OCH2); 56.48 (CH2, Ar″OCH3); 56.13 5′); 101.31 (CH, C-3′); 67.43 (CH2, ArOCH2); 55.01 (CH2,
(CH2, HOCH2); 49.91 (CH2, NCH2); 31.04 (CH2, HOCH2); 48.79 (CH2, NCH2); 26.43(CH2, ArOCH2CH2);
CH2CH2CH3); 27.05 (CH2, ArOCH2CH2); 25.93 (CH2, 25.42 (CH2, NCH2CH2); IR (KBr) νmax/cm−1: 1633.71;
NCH2CH2); 19.15 (CH2, CH2CH3); 13.81 (OCH3); IR 1579.7; HRMS (m/z): [M+Na]+ 494.0715
(KBr) νmax/cm−1: 1639.49; 1568.13; HMRS (m/z): [M (C22H22BrN3NaO4 = 494.0686).
+

+Na]+ 518.2265 (C27H33N3NaO6+ = 518.2262).

(2E)-3-(3-Nitrophenyl)-1-(2-hydroxy-4-{4-[4-
(2E)-3-(4-Chlorophenyl)-1-(2-hydroxy-4-{4-[4- (hydroxymethyl)-1H-1,2,3-triazol-1-yl]butoxy}phenyl)prop-
(hydroxymethyl)-1H-1,2,3-triazol-1-yl]butoxy}phenyl)prop- 2-en-1-one 5i
2-en-1-one 5g
Recrystallization over MeOH, yellow solid, 0.34 g (0.79
Recrystallization over MeOH, yellow solid, 0.37 g (0.87 mmol, 79 %); 1H NMR (400 MHz, DMSO-d6) δ (ppm):
mmol, 87 %); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.67–1.75 (m, 2H, OCH2CH2); 1.93–2.02 (m, 2H,
1.60–1.83 (m, 2H, OCH2CH2); 1.84–2.08 (m, 2H, NCH2CH2); 4.11 (t, 2H, J = 6.39 Hz, OCH2CH2); 4.42 (t,
NCH2CH2); 4.11 (t, 2H, J = 6.36 Hz, OCH2CH2); 4.43 (t, 2H, J = 7.04 Hz, NCH2CH2); 4.52 (d, 2H, J = 5.59 Hz,
2H, J = 6.99 Hz, NCH2CH2); 4.52 (d, 2H, J = 4.7 Hz, HOCH2); 5.19 (t, 1H, J = 5.59 Hz, HOCH2); 6.53 (d, 1H, J
HOCH2); 5.16 (t, 1H, J = 4.7 Hz, HOCH2); 6.52 (d, 1H, J = 2.45 Hz, ArH); 6.58 (dd, 1H, J = 9.08 Hz, J = 2.45 Hz,
= 2.42 Hz, ArH); 6.57 (dd, 1H, J = 9.05 Hz, J = 2.42 Hz, ArH); 7.75 (t, 1H, J = 8.00 Hz, ArH); 7.92 (d, 1H, J =
ArH); 7.54 (d, 1H, J = 8.52 Hz, ArH); 7.81 (d, 1H, J = 15.55 Hz, Hα); 8.01 (s, 1H, CHN); 8.24 (d, 1H, J = 15.55
15.49 Hz, Hα); 7.96 (d, 2H, J = 8.52 Hz, ArH); 8.00 (s, 1H, Hz, Hβ); 8.26–8.30 (m, 1H, ArH); 8.34–8.37 (m, 1H, ArH);
CHN); 8.04 (d, 1H, J = 15.49 Hz, Hβ); 8.28 (d, 1H, J = 8.36 (d, 1H, J = 9.08 Hz, ArH); 8.81–8.83 (m, 1H, ArH);
9.05 Hz, ArH); 13.38 (s, 1H, ArOH); 13C NMR (100 MHz, 13.32 (s,1H, ArOH); 13C NMR (100 MHz, DMSO-d6) δ
DMSO-d6) δ (ppm): 191.60 (C, COCH=CH); 165.62 (C, C- (ppm): 191.50 (C, COCH=CH); 165.72 (C, C-4′); 165.49
4′); 165.29 (C, C-2′); 147.85 (C, HOCH2C=CH); 142.50 (C, C-2′); 148.36 (CH, C-3″); 147.89 (C, HOCH2C=CH);
(CH, Cβ); 135.18 (C, C-1″); 133.42 (CH, C-4″); 132.68 141.44 (CH, Cβ); 136.36 (C, C-1″); 135.26(CH, C-6″);
(CH, C-6′); 130.67 (CH, C-3″; C-5″); 128.85(CH, C-2″; C- 132.98 (CH, C-6′); 130.28 (CH, C-5″); 124.72 (CH, C-4″);
6″); 122.54(CH, CHN); 121.92 (CH, Cα); 113.73(C, C-1′); 124.05(CH, C-2″); 123.06(CH, CHN); 122.60 (CH, Cα);
107.69 (CH, C-5′); 101.27 (CH, C-3′); 67.37 (CH2, 113.76 (C, C-1′); 107.83 (CH, C-5′); 101.25 (CH, C-3′);
ArOCH2); 54.97(CH2, NCH2); 48.72 (CH2, HOCH2); 26.36 67.43 (CH2, ArOCH2); 55.00(CH2, HOCH2); 48.75
(CH2, ArOCH2CH2); 25.37 (CH2, NCH2CH2); IR (KBr) (CH2, NCH2); 26.40(CH2, ArOCH2CH2); 25.40 (CH2,
34 Med Chem Res (2016) 26:27–43

NCH2CH2); IR (KBr) νmax/cm−1: 1639.49; 1577.77; HRMS (2E)-1-{2-Hydroxy-4-[4-(4-phenyl-1H-1,2,3-triazol-1-yl)

(m/z): [M+Na]+ 461.1434 (C22H22N4NaO6+ = 461.1432). butoxy]phenyl}-3-(4-methoxyphenyl)prop-2-en-1-one 6b

(2E)-3-(2,3-Dichlorophenyl)-1-(2-hydroxy-4-{4-[4- Recrystallization over MeOH, yellow solid, 0.43 g (0.92

(hydroxymethyl)-1H-1,2,3-triazol-1-yl]butoxy}phenyl)prop- mmol, 92 %); 1H NMR (400 MHz, CDCl3) δ (ppm): 1.82–
2-en-1-one 5j 1.93 (m, 2H, OCH2CH2); 2.13–2.23 (m, 2H, NCH2CH2);
3.87 (s, 3H, OCH3); 4.06 (t, 2H, J = 5.95 Hz, OCH2CH2);
Recrystallization over MeOH, yellow solid, 0.43 g (0.93 4.51 (t, 2H, J = 7.04 Hz, NCH2CH2); 6.42–6.48 (m, 2H,
mmol, 93 %); 1H NMR (400 MHz, DMSO-d6) δ (ppm): ArH); 6.95 (d, 2H, J = 8.8 Hz, ArH); 7.3–7.36 (m, 1H,
1.65–1.77 (m, 2H, OCH2CH2); 1.91–2.02 (m, 2H, ArH); 7.39–7.45 (m, 2H, ArH); 7.45 (d, 1H, J = 15.4 Hz,
NCH2CH2); 4.12 (t, 1H, J = 6.35 Hz, OCH2CH2); 4.43 (t, Hα); 7.6 (d, 2H, J = 8.8 Hz, ArH); 7.78 (s, 1H, CHN);
1H, J = 6.96 Hz, NCH2CH2); 4.52 (s, 2H, HOCH2); 5.17 (s, 7.79–7.84 (m, 3H, ArH); 7.86 (d, 1H, J = 15.4 Hz, Hβ);
1H, HOCH2); 6.53 (d, 1H, J = 2.40 Hz, ArH); 6.58 (dd, 1H, 13.52 (s, 1H, ArOH); 13C NMR (100 MHz, CDCl3) δ
J = 9.01 Hz, J = 2.40 Hz, ArH); 7.49 (t, 1H, J = 7.97 Hz, (ppm): 191.88(C, COCH=CH); 166.53 (C, C-4′); 165.07
ArH); 7.74 (dd, 1H, J = 8.00 Hz, J = 1.19 Hz, ArH); 8.00 (s, (C, C-2′); 161.85 (C, C-4″); 147.94 (C, Ar″′CH2C=CH);
1H, CHN); 8.09 (d, 1H, J = 15.53 Hz, Hα); 8.11 (d, 1H, J = 144.36 (CH, Cβ); 131.22 (CH, C-6′); 130.58 (C, C-1″′);
15.53 Hz, Hβ); 8.22 (dd, 1H, J = 8.00 Hz, J = 1.19 Hz, 130.37(CH, C-2″; C-6″); 128.83 (CH, C-3″′; C-5″′);
ArH); 8.26 (d, 1H, J = 9.01 Hz, ArH); 13.17 (s, 1H, ArOH); 128.17 (CH, C-4″′); 127.53 (C, C-1″); 125.71 (CH, C-2″′;
13
C NMR (100 MHz, DMSO-d6) δ (ppm): 191.01(C, C-6″′); 119.44 (CH, CHN); 117.81 (CH, Cα); 114.49
COCH=CH); 165.60 (C, C-4′); 165.50 (C, C-2′); 147.85(C, (CH, C-3″;C-5″); 114.32 (C, C-1′); 107.63 (CH, C-5′);
HOCH2C=CH); 138.09 (CH, Cβ); 134.62(CH, C-3″); 101.68 (CH, C-3′); 67.25 (CH2, Ar′OCH2); 55.45
132.82(C, C-1″); 132.45 (C, C-2″); 131.99 (CH, C-6′); (OCH3); 49.98 (CH2, NCH2); 27.16 (CH2, Ar
131.95 (CH, C-4″); 128.29 (CH, C-5″); 127.26 (CH, C-6″); ´OCH2CH2); 26.02 (CH2, NCH2CH2); IR (KBr) νmax/
125.45(CH, CHN); 122.54 (CH, Cα); 113.77 (C, C-1′); cm−1: 1633.71; 1570.06; HRMS (m/z): [M+Na]+
107.84 (CH, C-5′); 101.29 (CH, C-3′); 67.42 (CH2, 492.1893 (C28H27N3NaO4+ = 492.1894).
ArOCH2); 54.97 (CH2, HOCH2); 48.72 (CH2, NCH2);
26.36 (CH2, ArOCH2CH2); 25.36 (CH2, NCH2CH2); IR
(KBr) νmax/cm−1: 1633.71; 1571.99; HRMS (m/z): [M (2E)-3-(4-Ethoxyphenyl)-1-{2-hydroxy-4-[4-(4-phenyl-1H-
+Na]+ 484.0801 (C22H21Cl2N3NaO4+ = 484.0801). 1,2,3-triazol-1-yl)butoxy]phenyl}prop-2-en-1-one 6c

(2E)-1-{2-Hydroxy-4-[4-(4-phenyl-1H-1,2,3-triazol-1-yl) Recrystallization over MeOH, yellow solid, 0.40 g (0.83

butoxy]phenyl}-3-phenylprop-2-en-1-one 6a mmol, 83 %); 1H NMR (400 MHz, DMSO-d6) δ (ppm):
1.34 (t, 3H, J = 6.96 Hz, OCH2CH3); 1.69–1.8 (m, 2H,
Recrystallization over MeOH, yellow solid, 0.42 g (0.96 OCH2CH2); 1.98–2.09 (m, 2H, NCH2CH2); 4.05–4.16 (m,
mmol, 96 %); 1H NMR (400 MHz, CDCl3) δ (ppm): 1.83– 4H, OCH2; OCH2); 4.49 (t, 2H, J = 6.96 Hz, NCH2CH2);
1.94 (m, 2H, OCH2CH2); 2.13–2.25 (m, 2H, NCH2CH2); 6.5 (d, 1H, J = 2.43 Hz, ArH); 6.55 (dd, 1H, J = 9.0 Hz, J
4.06 (t, 2H, J = 5.97 Hz, OCH2CH2); 4.51 (t, 2H, J = 7.03 = 2.43 Hz, ArH); 7.00 (d, 2H, J = 8.8 Hz, ArH); 7.29–7.35
Hz, NCH2CH2); 6.4–6.49 (m, 2H, ArH); 7.3–7.36 (m, 1H, (m, 1H, ArH); 7.4–7.47 (m, 2H, ArH); 7.79 (d, 1H, J =
ArH) 7.39–7.46 (m, 5H, ArH); 7.57 (d, J = 15.5 Hz, 1H, 15.35 Hz, Hα); 7.82–7.9 (m, 5H, ArH); 8.26 (d, 1H, J =
Hα); 7.62–7.68 (m, 2H, Hβ); 7.78 (s, 1H, CHN); 7.8–7.85 9.0 Hz, ArH); 8.61 (s, 1H, CHN); 13.61 (s, 1H, ArOH); 13C
(m, 3H, ArH); 7.89 (d, 1H, J = 15.5 Hz, Hβ); 13.4 (s, 1H, NMR (100 MHz, DMSO-d6) δ (ppm): 191.75 (C,
ArOH); 13C NMR (100 MHz, CDCl3) δ (ppm): 191.85 (C, COCH=CH); 165.61 (C, C-4′); 165.01 (C, C-2′); 160.78
COCH=CH); 166.62 (C, C-4′); 165.27 (C, C-2′); 147.95 (C, C-4″); 146.21 (C, Ar″′CH2C=CH); 144.18 (CH, Cβ);
(C, Ar″′CH2C=CH); 144.49 (CH, Cβ); 134.79 (C, C-1″); 132.44 (CH, C-6′); 131.03 (CH, C-2″; C-6″); 130.73 (C, C-
131.35 (CH, C-6′); 130.69 (C, C-1″′); 130.57 (CH, C-3″′; 1″′); 128.76 (CH, C-3″′; C-5″′); 127.68 (CH, C-4″′);
C-5″′); 129.00 (CH, C-3″; C-5″); 128.84(CH, C-2″; C-6″); 126.95 (C, C-1″); 125.01 (CH, C-2″′; C-6″′); 121.23 (CH,
128.54(CH, C-4″′); 128.17(CH, C-2″; C-6″); 125.70 (CH, CHN); 118.26 (CH, Cα); 114.72 (CH, C-3″; C-5″); 113.71
CHN); 120.32 (CH, C-4″); 119.44 (CH, Cα); 114.26 (C, C- (C, C-1′); 107.51 (CH, C-5′); 101.27 (CH, C-3′); 67.31
1′); 107.78 (CH, C-5′); 101.70 (CH, C-3′); 67.28 (CH2, Ar′ (CH2, Ar′OCH2); 63.29 (CH2, Ar″OCH2); 49.08 (CH2,
OCH2); 49.97 (CH2, NCH2); 27.14(CH2, Ar′OCH2CH2); NCH2) ; 26.26 (CH2, Ar′OCH2CH2); 25.40 (CH2,
26.02 (CH2, NCH2CH2); IR (KBr) νmax/cm−1: 1639.49; NCH2CH2); 14.44 (CH3); IR (KBr) νmax/cm−1: 1635.64;
1573.91; HRMS (m/z): [M+H]+ 440.248 (C27H26N3O3+ = 1571.99; HRMS (m/z): [M+H]+ 484.237 (C29H29N3NaO4+
440.197). = 484.223).
Med Chem Res (2016) 26:27–43 35

(2E)-3-(4-Butoxyphenyl)-1-{2-hydroxy-4-[4-(4-phenyl-1H- ArOCH2); 67.27 (CH2, ArOCH2); 49.97(CH2, NCH2) ;

1,2,3-triazol-1-yl)butoxy]phenyl}prop-2-en-1-one 6d 31.31 (CH2, OCH2CH2CH2); 27.14 (CH2, NCH2CH2);
26.01 (CH2, OCH2CH2); 19.26 (CH2, CH2CH3); 13.85
Recrystallization over MeOH, yellow solid, 0.43 g (0.84 (CH3); IR (KBr) νmax/cm−1: 1643.35; 1577.77; HRMS (m/
mmol, 84 %); 1H NMR (400 MHz, DMSO-d6) δ (ppm): z): [M+Na]+ 534.2362 (C31H33N3NaO4+ = 534.2363).
0.94 (t, 3H, J = 7.41 Hz, CH3); 1.40–1.50 (m, 2H,
CH2CH3); 1.67–1.82 (m, 4H, CH2CH2CH3; OCH2CH2); (2E)-3-(4-Butoxy-3-methoxyphenyl)-1-{2-hydroxy-4-[4-(4-
2.00–2.10 (m, 2H, NCH2CH2); 4.05 (t, 2H, J = 6.4 Hz, phenyl-1H-1,2,3-triazol-1-yl)butoxy]phenyl}prop-2-en-1-
OCH2CH2); 4.13 (t, 2H, J = 6.2 Hz, OCH2CH2); 4.50 (t, one 6f
2H, J = 7.0 Hz, NCH2CH2); 6.5 (d, 1H, J = 2.3 Hz, ArH);
6.56 (dd, 1H, J = 9.0 Hz, J = 2.3 Hz, ArH); 7.02 (d, 1H, J = Recrystallization over MeOH, yellow solid, 0.44 g (0.81
8.7 Hz, ArH); 7.33 (t, 1H, J = 7.4 Hz, ArH); 7.45 (t, 2H, J mmol, 81 %); 1H NMR (400 MHz, DMSO-d6) δ (ppm):
= 7.6 Hz, ArH); 7.80–7.90 (m, 6H, ArH; Hα; Hβ); 8.25 (d, 0.93 (t, 3H, J = 7.44 Hz, CH2CH3); 1.38–1.50 (m, 2H,
1H, J = 9.1 Hz, ArH); 8.59 (s, 1H, CHN); 13.56 (s, 1H, CH2CH3); 1.68–1.81 (m, 4H, CH2CH2CH3; OCH2CH2);
ArOH).13C NMR (100 MHz, DMSO-d6) δ (ppm): 191.79 2.01–2.09 (m, 2H, NCH2CH2); 3.87 (s, 3H, OCH3); 4.02 (t,
(C, COCH=CH); 165.61 (C, C-4′); 165.04 (C, C-2′); 2H, J = 6.54 Hz, OCH2CH2); 4.12 (t, 2H, J = 6.36 Hz,
161.00 (C, C-4″); 146.27 (C, Ar″′CH2C=CH); 144.18 (CH, OCH2CH2); 4.49 (t, 2H, J = 7.00 Hz, NCH2CH2); 6.51 (d,
Cβ); 132.45 (CH, C-6′); 131.01(CH, C-2″; C-6″); 130.80 1H, J = 2.46 Hz, ArH); 6.56 (dd, 1H, J = 8.97 Hz, J = 2.46
(C, C-1″′); 128.77 (CH, C-3″′; C-5″′); 127.69 (CH, C-4″′); Hz, ArH); 7.03 (d, 1H, J = 8.40 Hz, ArH); 7.33 (tt, 1H, J =
127.01 (C, C-1″); 125.07 (CH, C-2″′; C-6″′); 121.23 (CH, 8.00 Hz, J = 1.58 Hz, ArH); 7.38 (dd, 1H, J = 8.36 Hz, J =
CHN); 118.34 (CH, Cα); 114.82 (CH, C-3″;C-5″); 113.80 1.86 Hz, ArH); 7.44 (d, 1H, J = 15.27 Hz, Hα); 7.42–7.47
(C, C-1′); 107.52 (CH, C-5′); 101.35 (CH, C-3′); 67.40 (m, 1H, ArH); 7.55 (d, 1H, J = 1.86 Hz, ArH); 7.78 (d, 1H,
(CH2, ArOCH2); 67.36 (CH2, ArOCH2); 49.14 (CH2, J = 15.27 Hz, Hβ); 7.82–7.88 (m, 3H, ArH); 8.27 (d, 1H, J
NCH2); 30.59 (CH2, OCH2CH2CH2); 26.28 (CH2, = 9.10 Hz, ArH); 8.58 (s, 1H, CHN); 13.59 (s, 1H, ArOH);
NCH2CH2); 25.46 (CH2, OCH2CH2); 18.62 (CH2, 13
C NMR (100MHz, DMSO-d6) δ (ppm): 191.79 (C,
CH2CH3); 13.58 (CH3); IR (KBr) νmax/cm−1: 1635.64; COCH=CH); 165.64 (C, C-4′); 165.03 (C, C-2′); 150.96
1575.84; HRMS (m/z): [M+H]+ 512.2546 (C31H34N3O4+ = (C, C-3″); 149.17 (C, C-4″); 146.25 (C, Ar″′CH2C=CH);
512.2544). 144.69 (CH, Cβ); 132.45 (CH, C-6′); 130.74 (C, C-1″′);
128.76 (CH, C-3″′;C-5″′); 127.69 (CH, C-4″′); 127.21 (C,
(2E)-3-(3-Butoxyphenyl)-1-{2-hydroxy-4-[4-(4-phenyl-1H- C-1″); 125.05 (CH, C-2″′; C-6″′); 124.29 (CH, C-6″);
1,2,3-triazol-1-yl)butoxy]phenyl}prop-2-en-1-one 6e 121.21 (CH, CHN); 118.35 (CH, Cα); 113.75 (C, C-1′);
112.51 (CH, C-5″); 111.23 (CH, C-2″); 107.52 (CH, C-5′);
Recrystallization over MeOH, yellow solid, 0.46 g (0.9 101.29 (CH, C-3′); 67.89 (CH2, ArOCH2); 67.35 (CH2,
mmol, 90 %); 1H NMR (400 MHz, CDCl3) δ (ppm): 0.99 (t, ArOCH2); 55.82 (CH2, ArOCH3); 49.13(CH2, NCH2);
3H, J = 7.41 Hz, CH2CH3); 1.48–1.57 (m, 2H, CH2CH3); 30.61(CH2, OCH2CH2CH2); 26.26 (CH2, NCH2CH2);
1.76–1.84 (m, 2H, CH2CH2CH3); 1.85–1.92 (m, 2H, 25.44 (CH2, OCH2CH2); 18.62 (CH2, CH2CH3); 13.56
OCH2CH2); 2.15–2.22 (m, 2H, NCH2CH2); 4.00 (t, 2H, J (CH3); IR (KBr) νmax/cm−1: 1635.64; 1573.91; MS (m/z):
= 6.49 Hz, OCH2CH2); 4.05 (t, 2H, J = 5.99 Hz, [M+H]− 541.80.
OCH2CH2); 4.50 (t, 2H, J = 7.02 Hz, NCH2CH2); 6.44 (d,
1H, J = 2.39 Hz, ArH); 6.47 (dd, 1H, J = 8.71 Hz, J = 2.39 (2E)-3-(4-Chlorophenyl)-1-{2-hydroxy-4-[4-(4-phenyl-1H-
Hz, ArH); 6.97 (ddd, 1H, J = 8.2, J = 2.5 Hz, J = 0.9 Hz, 1,2,3-triazol-1-yl)butoxy]phenyl}prop-2-en-1-one 6g
ArH); 7.15–7.18 (m, 1H, ArH); 7.2–7.25 (m, 1H, ArH);
7.30–7.36 (m, 2H, ArH); 7.40–7.45 (m, 2H, ArH); 7.54 (d, Recrystallization over MeOH, yellow solid, 0.38 g (0.80
1H, J = 15.5 Hz, Hα); 7.78 (s, 1H, ); 7.8–7.87 (m, 4H); mmol, 81 %); 1H NMR (400 MHz, DMSO-d6) δ (ppm):
13.31 (s, 1H, ArOH); 13C NMR (100 MHz, CDCl3) δ 1.7–1.82 (m, 2H); 1.98–2.11 (m, 2H); 4.13 (t, J = 6.1 Hz,
(ppm): 191.83(C, COCH=CH); 166.60 (C, C-4′); 165.26 2H); 4.5 (t, J = 6.9 Hz, 2H); 6.52 (d, J = 2.0 Hz, 1H); 6.57
(C, C-2′); 159.56 (C, C-3″); 147.94 (C, Ar″′CH2C=CH); (dd, J = 9.0 Hz, J = 2.0 Hz, 1H); 7.29–7.37 (m, 1H); 7.4–
144.51 (CH, Cβ); 136.09 (C, C-1″); 131.37 (CH, C-6′); 7.49 (m, 2H); 7.55 (d, J = 8.4 Hz, 2H); 7.77–7.87 (m, 3H);
130.56 (C, C-1″′); 129.94 (CH, C-5″); 128.83 (CH, C-3″′; 7.97 (d, J = 8.4 Hz, 2H); 8.05 (d, J = 15.48 Hz, 1H); 8.29
C-5″′); 128.17 (CH, C-4″′); 125.70 (CH, C-2″′; C-6″′); (d, J = 9.0 Hz, 1H); 8.62 (s, 1H); 13.39 (s, 1H); 13C NMR
121.08 (CH, CHN); 120.48 (CH, Cα); 119.44 (CH, C-6″); (100 MHz, DMSO-d6) δ (ppm): 191.60 (C, COCH=CH);
116.85 (CH, C-4″); 114.25 (C, C-1′); 114.22 (CH, C-2″); 165.62 (C, C-4′); 165.29 (C, C-2′); 146.20 (C, Ar″′
107.76 (CH, C-5′); 101.68 (CH, C-3′); 67.86 (CH2, CH2C=CH); 142.51 (CH, Cβ); 135.18 (C, C-1″); 133.42
36 Med Chem Res (2016) 26:27–43

(C, C-4″); 132.69 (CH, C-6′); 130.71 (C, C-1″′); 130.68 130.61 (C, C-1″′); 129.90 (CH, C-5″); 128.42 (CH, C-2″′;
(CH, C-3″; C-5″); 128.86 (CH, C-2″; C-6″); 128.75 C-6″′); 127.38 (CH, C-4″′); 124.89 (CH, C-3″′; C-5″′);
(CH, C-3″′; C-5″′); 127.67 (CH, C-4″′); 124.99 (CH, 124.28 (CH, C-4″); 123.83 (CH, C-2″); 122.73(CH, CHN);
C-2″′; C-6″′); 121.92 (CH, Cα); 121.22 (CH, CHN); 113.73 120.83 (CH, Cα); 113.61 (C, C-1′); 107.49 (CH, C-5′);
(C, C-1′); 107.69 (CH, C-5′); 101.28 (CH, C-3′); 67.37 101.03 (CH, C-3′); 67.14 (CH2, ArOCH2); 48.98 (CH2,
(CH2, ArOCH2); 49.07 (CH2, NCH2) ; 26.24 (CH2, NCH2); 26.21 (CH2, NCH2CH2); 25.31 (CH2, OCH2CH2);
NCH2CH2); 25.38 (CH2, OCH2CH2); IR (KBr) νmax/cm−1: IR (KBr) νmax/cm−1: 1631.78; 1585.49; HRMS (m/z): [M
1635.64; 1577.77; HRMS (m/z): [M+H]+ 474.1579 +Na]+ 507.1638 (C27H24N4NaO5+ = 507.1639).
(C27H24ClN3NaO3+ = 474.1579).
(2E)-3-(2,3-Dichlorophenyl)-1-{2-hydroxy-4-[4-(4-phenyl-
(2E)-3-(4-Bromophenyl)-1-{2-hydroxy-4-[4-(4-phenyl-1H- 1H-1,2,3-triazol-1-yl)butoxy]phenyl}prop-2-en-1-one 6j
1,2,3-triazol-1-yl)butoxy]phenyl}prop-2-en-1-one 6h
Recrystallization over MeOH, yellow solid, 0.45 g (0.89
Recrystallization over MeOH, yellow solid, 0.47 g (0.90 mmol, 89 %); 1H NMR (400 MHz, CDCl3) δ (ppm): 1.84–
mmol, 90 %). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.73– 1.93 (m, 2H, OCH2CH2); 2.13–2.24 (m, 2H, NCH2CH2);
1.80 (m, 2H, OCH2CH2); 2.00–2.09 (m, 2H, NCH2CH2); 4,06 (t, 2H, J = 5.98 Hz, OCH2CH2); 4.51 (t, 2H, J = 7.02
4.12 (t, 2H, J = 6.34 Hz, OCH2CH2); 4.48 (t, 2H, J = 7.00 Hz, NCH2CH2); 6.43–6.48 (m, 2H, ArH); 7.27 (t, 1H, J =
Hz, NCH2CH2); 6.51 (d, 1H, J = 2.45 Hz, ArH); 6.56 (dd, 7.9 Hz, ArH); 7.3–7.36 (m, 1H, ArH); 7.39–7.45 (m, 2H,
1H, J = 8.98 Hz, J = 2.47 Hz, ArH); 7.32 (tt, 1H, J = 7.97 ArH); 7.45 (d, 1H, J = 15.5 Hz, Hα); 7.52 (dd, 1H, J = 1.4
Hz, J = 1.55 Hz, ArH); 7.42–7.46 (m, 2H, ArH); 7.67 (d, Hz; J = 7.9 Hz, ArH); 7.6 (dd, 1H, J = 1.4 Hz; J = 7.9 Hz,
2H, J = 8.48 Hz, ArH); 7.78 (d, 1H, J = 15.51 Hz, Hα); ArH); 7.75–7.84 (m, 2H); 8.24 (d, 1H J = 15.5 Hz, Hβ);
7.82–7.89 (m, 4H, ArH); 8.03 (d, 1H, J = 15.51 Hz, Hβ); 13.24 (s, 1H, ArOH); 13C NMR (100 MHz, CDCl3) δ
8.25 (d, 1H, J = 9.14 Hz, ArH); 8.58 (s, 1H, CHN); 13.31 (ppm): 191.27 (C, COCH=CH); 166.78 (C, C-4′); 165.56
(s, 1H, ArOH); 13C NMR (100 MHz, CDCl3) δ (ppm): (C, C-2′); 147.97 (C, Ar″′CH2C=CH); 140.13 (CH, Cβ);
191.62 (C, COCH=CH); 165.59 (C, C-4′); 165.30 (C, C- 135.59 (C, C-3″); 134.22 (C, C-1″); 133.56 (C, C-2″);
2′); 142.55 (CH, Cβ); 133.79 (C, C-1″); 132.69 (CH, C-6′); 131.74 (CH, C-6′); 131.43 (CH-C-4″); 130.57 (C, C-1″′);
131.80 (CH, C-3″; C-5″); 130.85 (CH, C-3″′; C-5″′); 128.86 (CH, C-2″′; C-6″′); 128.21 (CH, C-4″′); 127.40
130.76 (C, C-1″′); 128.74 (CH, C-2″; C-6″); 127.66 (CH, (CH, C-5″); 126.00 (CH, C-6″); 125.72 (CH, C-3″′; C-5″′);
C-4″′); 125.03 (CH, C-2″′; C-6″′); 124.06 (C, C-4″); 122.10 124.24 (CH, Cα); 119.46 (CH, CHN); 114.11 (C, C-1′);
(CH, Cα); 121.20 (CH, CHN); 113.81 (C, C-1′); 107.69 108.02 (CH, C-5′); 101.74 (CH, C-3′); 67.35 (CH2,
(CH, C-5′); 101.35 (CH, C-3′); 67.40 (CH2, ArOCH2); ArOCH2); 49.98 (CH2, NCH2); 27.14(CH2, NCH2CH2);
49.10 (CH2, NCH2); 26.24 (CH2, NCH2CH2); 25.42 (CH2, 26.02 (CH2, OCH2CH2); IR (KBr) νmax/cm−1: 1635.64;
OCH2CH2); IR (KBr) νmax/cm−1: 1637,56; 1570,06; HRMS 1577.77; HRMS (m/z): [M+Na]+ 530.1006
(m/z): [M+Na]+ 540.0892 (C27H24BrN3NaO3+ = 540.0893). (C27H23Cl2N3NaO3 = 530.1009).
+

(2E)-3-(3-Nitrophenyl)-1-{2-hydroxy-4-[4-(4-phenyl-1H- Cytotoxicity assay

1,2,3-triazol-1-yl)butoxy]phenyl}prop-2-en-1-one 6i
The cytotoxic effects of the synthetic chalcones were
Recrystallization over MeOH, yellow solid, 0.31 g (0.64 assessed against tumoral human cell lines HeLa (cervical
mmol, 64 %); 1H NMR (400 MHz, DMSO-d6) δ (ppm): carcinoma ATCC CCL2), RKO-AS45-1 (colorectal carci-
1.77–1.85 (m, 2H, OCH2CH2); 2.06–2.14 (m, 2H, noma ATCC CRL-2579) and Wi-26VA4 (lung fibroblast
NCH2CH2); 4.12 (t, 2H, J = 6.27 Hz, OCH2CH2); 4.50 (t, ATCC CCL-95.1), respectively. The cell viability after each
2H, J = 6.96 Hz, NCH2CH2); 6.50 (d, 1HJ = 2.30 Hz, ArH); chemical treatment was determined by MTT colorimetric
6.55 (dd, 1H, J = 8.98 Hz, J = 2.30 Hz, ArH); 7.31 (t, 1H, J assay (Carmichael et al., 1987; Park et al., 1987). Briefly,
= 7.59 Hz, ArH); 7.41 (t, 2H, J = 7.59 Hz, ArH); 7.72 (t, the method consisted of plating 1 × 106 cells in 96-well
1H, J = 7.98 Hz, ArH); 7.84 (d, 2H, J = 7.48 Hz, ArH); 7.91 microplates, in which they were homogenized in RPMI
(d, 1H, J = 15.24 Hz, Hα); 8.19 (d, 1H, J = 15.24 Hz, Hβ); 1640 medium supplemented with fetal bovine serum (FBS)
8.24–8.29 (m, 2H, ArH); 8.31 (d, 1H, J = 8.95 Hz, ArH); and penicillin-streptomycin antibiotics. Then, microplates
8.51 (s, 1H, CHN); 8.79 (s, 1H, ArH); 13.27 (s, 1H, ArOH); were incubated overnight at 37 °C, 5 % CO2, followed by
13
C NMR (100 MHz, DMSO-d6) δ (ppm): 191.20 (C, the addition of each chalcone solubilized in DMSO 0.1 %
COCH=CH); 165.75 (C, C-4′); 165.26 (C, C-2′); 148.19 (v/v). Negative control groups were constituted with cells
(C, C-3″); 146.19 (C, Ar″′CH2C=CH); 141.07 (CH, Cβ); without treatment. Five serial dilutions (1:10) were made
136.27 (C, C-1″); 134.94 (CH, C-6″); 132.59 (CH, C-6′); from stock solution (10 mg mL−1) using RPMI
Med Chem Res (2016) 26:27–43 37

supplemented with 1 % FBS. Cell viability was evaluated was performed in triplicate using the ΔΔCT comparison. All
after incubation for 48 h, by removing and discarding the reactions were subjected to the same conditions of analysis
medium and adding 100 μL of MTT 5 %, followed by 3 h of and normalized by ROX passive reference dye signal to
incubation. After this time, supernatant was removed and correct fluctuations in readings due to changes in volume
discarded and the insoluble formazan product was dissolved and evaporation along the reaction.
in DMSO. The optical density (OD) of each well of the 96-
well plates was measured using a microplate spectro- Statistical analysis
photometer at 550 nm. The OD of formazan formed in
untreated control cells was defined as 100 % cell viability. The IC50 values were calculated using the OriginPro 8.0
All assays were performed in triplicate (Lee et al., 2003). program (OriginLab Corporation, Northampton, MA,
USA). The gene expression results were analyzed using
TUNEL (terminal deoxynucleotide transferase dUTP Sigma Stat version 2.03 (Systat Software, San Jose, CA,
nick end labeling) assay USA). For data with normal distribution, analysis of var-
iance (ANOVA) followed by Tukey’s multiple comparison
Apoptosis analysis was performed using an APO-BrdU test was used. For those without normal distribution, we
TUNEL kit (Invitrogen, CA, USA). A typical characteristic used ANOVA for comparison of more groups, the non-
of cell death by apoptosis is the activation of nucleases, parametric Kruskal–Wallis test followed by the Holm-Sidak
which eventually degrades nuclear DNA into fragments of multiple comparison test. A significance value of p < 0.05
about 200 base pairs. The TUNEL assay allows identifica- was used.
tion of apoptotic cells by detecting these DNA fragments
through fluorescence microscopy. The DNA fragments
resulting from the breakage of genomic DNA can be
identified by labeling the free 3′-OH ends. An enzymatic Results and discussion
reaction catalyzes the incorporation of nucleotides labeled
with fluorescein. This assay was performed in HeLa and Synthesis
RKO-AS45-1 cell lines by determining the IC50 values after
24 h of exposure to effective concentrations of each Compounds proposed in this work were synthesized
compound. employing a previously described procedure (da Silva et al.,
2012) (Scheme 1). O-Alkylation at position 4 of the resa-
TP53 gene expression cetophenone 1 in the first step following the reaction with
NaN3 afforded azide 3 at 62 % overall yield (Alvarez and
Total RNA extraction was performed using Trizol LS Alvarez, 1997). Chalcone-azide derivatives 4a–j were
reagent (Invitrogen), strictly following the manufacturer’s synthesized by Claisen-Schmidt condensation with different
recommendations. Only the compounds with a satisfactory aromatic aldehydes at moderate to good yield (Scheme 1)
cytotoxic effect were investigated. Extraction was per- after purification by recrystallization from MeOH or by
formed before drug exposure, and 6, 12, and 24 h after silica gel column chromatography. Triazole chalcones 5a–j
treatment. The concentration range of each compound was (CH2OH) and 6a–j (Ph) were synthesized by 1,3-dipolar
that corresponding to the IC50, 0.5 × IC50, and 0.25 × IC50, cycloaddition catalyzed by CuI in DMSO using chalcone-
respectively. Quantification of the total RNA obtained after azide derivatives 4a–j and either propargyl alcohol or
the extraction process was performed by spectrophotometry phenyl acetylene. The desired products were obtained at
(NanoVue, GE Healthcare Life Sciences, UK). Samples excellent yields after silica gel filtration or recrystallization
used in the study presented concentrations higher than from MeOH.
50 ng μL−1 and purity between 1.7 and 1.9, respectively. All compounds were characterized by 1H NMR, 13C
Extracted RNA was subjected to the reverse transcriptase NMR, HRMS, and IR spectroscopy.
reaction to produce cDNA (High Capacity cDNA Reverse
Transcription Kit®, Applied Biosystems, USA). Cytotoxicity assay
TP53 gene expression analysis was performed by real-
time PCR using TaqMan methodology (Applied Biosys- In order to assess the cytotoxic activity of the compounds,
tems, USA) following the recommendations of the supplier. MTT assays were performed on human HeLa, RKO-AS45-
PCR was performed on the StepOne Real Time PCR Sys- 1 (tumor cell lines) and WI-4-26VA (non-tumor cell line)
tem (Applied Biosystems). The PCR conditions were: 2 min cell lines using etoposide as a standard cytotoxic com-
at 50 °C, 10 min at 95 °C, followed by 40 cycles at 95 °C for pound. Table 1 shows the data from the cytotoxicity and
15 s and 1 min at 60 °C. The quantification of each sample cell viability tests.
38 Med Chem Res (2016) 26:27–43

1
1
2 3

3,

3
3
2

1
1

2 2

Scheme 1 Route for the synthesis of chalcone azides 4a–j and triazole chalcones 5a–j and 6a–j

It was observed that for the HeLa cell line, compounds 4j cells (Mojzis et al., 2008; Shi et al., 2010; Zhao et al.,
and 6f presented effects on cell viability comparable with 2014).
the standard compound etoposide, a chemical agent already Over recent years, many chalcone derivatives with anti-
used in chemotherapy (p > 0.05) (Sinkule, 1984). In the tumor activity have been developed by introducing a het-
RKO-AS45-1 cell line, the most effective compounds were erocyclic skeleton (Guantai et al., 2010; Kamal et al., 2011;
5i, 5f, 5j, and 5g (p > 0.05). Mesenzani et al., 2011; Yin et al., 2014).
In the non-tumor cell line (WI-26VA4), most of the Numerous substituents have been tested and more eva-
compounds evaluated had an IC50 higher than observed for luations are still needed in order to identify the mechanism
etoposide and a selectivity index > 100. of action of the chalcones, which could represent an
Although several studies have reported antiproliferative important therapeutic alternative for different tumor types.
and cytotoxic effects of chalcones in several cell lines, the In addition, synthetic chalcones have a high therapeutic
mechanisms of action described, including induction of index, are easy to prepare and present potential for oral
apoptosis, interference with the mitotic phase of the cell administration (Chauhan et al., 2014).
cycle, and modulation of P-glycoprotein and redox activity,
are still controversial (Hans et al., 2010; Kim et al., 2010; TUNEL assay
Pilatova et al., 2010; Firoozpour et al., 2012; Nagaraju et
al., 2012; Syam et al., 2012; Ethiraj et al., 2013; Jin et al., The TUNEL assay allows identification of fragmented
2013; Noushini et al., 2013; Chauhan et al., 2014). Further DNA, observed as a result of the final phase of cell death by
studies are required in order to clarify these mechanisms; apoptosis. In Fig. 1, it can be seen that the compounds 4j
although the presence of polar substituents is important for (Fig. 1c) and 5j (Fig. 1f) significantly reduced the cell
the cytotoxicity, it may reduce the lipophilicity of the rings population compared to the controls (Figs. 1a and d);
of the generic structure and therefore hinder the compound however, there was no detection of DNA fragmentation. It
in crossing the lipid membrane and penetrating the cancer can be seen that the tests with etoposide (Figs. 1b and e)
Med Chem Res (2016) 26:27–43 39

Table 1 IC50 values obtained in in vitro assays with HeLa, RKO- cycle and induces apoptosis of cells is still controversial
AS45-1, and WI-4-26VA cell lines
(Hans et al., 2010; Firoozpour et al., 2012; Mourad et al.,
Compound IC50 (µM) ± S.D.a 2012; Syam et al., 2012; Abu et al., 2013; Champelovier et
HeLa RKO-AS45-1 WI-26-VA4 al., 2013; de Vasconcelos et al., 2013; Abbas et al., 2014).
Nevertheless, the absence of apoptosis induction by certain
4a 69.84 ± 7.32 65.66 ± 1.88 >100 chalcone derivatives was also observed elsewhere (Ethiraj
4b >100 >100 >100 et al., 2013; Zhang et al., 2013a), suggesting that other
4c >100 >100 >100 possible mechanisms of action may take place depending on
4d >100 80.61 ± 4.24 >100 the substituents present in the structure.
4e >100 64.73 ± 11.02 76.03 ± 1.76 However, no apoptosis detection in this assay may be
4f >100 81.97 ± 3.20 >100 directly related to the fact that the kit employed detects late
4g 72.30 ± 2.99 77.95 ± 0.77 >100 apoptosis.
4h 51.23 ± 3.44 >100 >100
4i >100 61.24 ± 9.09 98.58 ± 1.85
4j <0.1 61.18 ± 7.42 >100 Analysis of TP53 gene expression
5a >100 60.92 ± 0.70 >100
5b 55.35 ± 1.21 81.41 ± 0.03 >100 Real-time qPCR reactions were performed to calculate the
5c >100 55.10 ± 3.50 71.77 ± 2.02 relative expression of the TP53 gene, using the comparative
5d 81.75 ± 0.49 78.20 ± 2.39 93.36 ± 1.80 ΔΔCT method. When the HeLa and RKO-AS45-1 cell lines
5e >100 82.10 ± 1.82 <0.1 were exposed to compounds 5j and 4j, it was observed that
5f 54.46 ± 4.54 <0.1 >100
the total number of transcripts reached approximately 2.2
times the value of the expression of the reference group (no
5g >100 <0.1 >100
treatment). The TP53 expression levels of compound 4j in
5h >100 76.92 ± 5.93 87.91 ± 1.65
the HeLa cell line is shown in Fig. 2 and compound 5j in
5i >100 <0.1 >100
the RKO-AS45-1 cell line in Fig. 3. All measurements were
5j 49.47 ± 3.28 <0.1 >100
normalized by endogenous control and the quantification of
6a 69.99 ± 9.71 56.80 ± 1.10 >100
the reference sample (no treatment, 0 h) was standardized as
6b 67.89 ± 4.55 >100 >100
the value 1.0.
6c 60.55 ± 2.48 >100 >100
For all tested concentrations, an increase of TP53
6d >100 >100 >100 expression was observed in the HeLa cell line after 12 h of
6e >100 64.47 ± 1.80 >100 exposure to compound 4j. Similar results were observed for
6f <0.1 51.81 ± 10.84 198.74 ± 0.74 the RKO-AS45-1 cell line; however, the augmentation of
6g >100 >100 >100 expression became more evident after 24 h of incubation
6h >100 >100 >100 with compound 5j. The increase in gene expression was
6i 65.84 ± 4.85 60.48 ± 1.77 >100 significant for the IC50 concentration (0.1 µM) for com-
6j 57.38 ± 3.87 258.07 ± 7.20 >100 pounds 4j and 5j in both cell lines.
Etoposide <0.1 <0.1 57.85 ± 1.09 The TP53 gene encodes a nuclear phosphoprotein (p53)
a
S.D Standard deviation that has an active role in cell cycle arrest and induction of
DNA repair gene expression (Rao et al., 2010; Ethiraj et al.,
2013; Khan et al., 2013). The increased expression of the
exhibit green fluorescence due to binding with DNA TP53 gene could indicate that compounds 4j and 5j induced
strands, and red fluorescence indicates that few of the DNA DNA damage at the conditions employed in the present
strains remained intact. study. As a consequence of TP53 induction, p53 protein
Since there was no evidence of apoptosis in the tests levels are augmented (Liu et al., 2014). This protein binds
carried out, it is possible to consider that the antitumor transcription factors, preventing cells from entering S phase.
potential of chalcone derivatives is related to their ability to A high level of p53 also promotes overexpression of the
reduce cell proliferation by blockade of the cell cycle at the protein p21, which inhibits the active molecular complex
G2/M phase, as well as in the G1 phase, depending on the cyclin-dependent kinase (Mahapatra et al., 2015). There-
chalcones and their chemical structure (Orlikova et al., fore, the cell cycle is interrupted as an attempt to repair
2011). Several studies have shown that certain chalcones damage to the DNA of cancer cells caused by the tested
may also block the cell cycle in the G0/G1 phase (Romag- compounds (Haupt et al., 2003; Guo et al., 2012). However,
noli et al., 2009). However, the effect of chalcones and their if the cells are not able to repair this damage, the apoptotic
derivatives in signal transduction, which controls the cell process can be triggered.
40 Med Chem Res (2016) 26:27–43

Fig. 1 Results from apoptosis

induction by TUNEL assay tests
with compounds 4j (HeLa) and
5j (RKO-AS45-1) (scale 20 μm)

Fig. 2 Relative quantification of TP53 gene in HeLa line at different Fig. 3 Relative quantification of TP53 in RKO-AS45-1 strain at dif-
concentrations of compound 4j (*p < 0.05) ferent concentrations of compound 5j (*p < 0.05)

The cytotoxic mechanism of action of chalcones is still up 2014). Nevertheless, Singh and colleagues (2014) evaluated
for debate (Champelovier et al., 2013; Zhang et al., 2013b; the action of chalcones on lung adenocarcinoma, associating
Abbas et al., 2014). Rao et al. (2010) showed that is not the increased p53 expression with DNA damage.
currently clear whether chalcone derivatives can cause DNA In vitro system models for genotoxicity studies are some-
damage and trigger p53 activation. Upregulation of p53 may times prone to false negative results, and some of this loss of
be one of the molecular mechanisms by which those com- predictive power can be caused by inactivation or low p53
pounds can inhibit the growth of cancer cells and induce cell expression in some cell models (Khoo et al., 2014). Within this
cycle arrest in G1 (Rao et al., 2010; Iqbal et al., 2014). context, when choosing cell lines for assays of in vitro geno-
However, other studies suggest that the cytotoxic effect is toxicity, it is important to be aware of the p53 status and thus
independent of the p53 status (Iqbal et al., 2014; Singh et al., how this state can influence responses in the trials. According
Med Chem Res (2016) 26:27–43 41

to Zhuang et al. (2012), p53 wild-type cells can clearly have cell lines. The TUNEL assay indicated a reduction in the
two main cellular responses to DNA damage: cell cycle arrest cell population compared to controls, but there was no
and apoptosis. According to the American Type Culture Col- detection of DNA fragmentation after 24 h of exposure.
lection (ATCC), the HeLa and RKO-AS45-1 cell lines used in Further, qPCR studies proved that compounds 4j and 5j
this study are able to express p53. Most malignant cervical induced the expression of TP53, which could be related to
cells, including human cells of cervical carcinoma (HeLa) cell cycle arrest. In summary, our results demonstrated that
contain wild-type p53 (Johnson et al., 2002). However, the E6 compounds analogous to triazole/azide chalcones are pro-
gene product of human papillomavirus (HPV) may interact mising anticancer agents.
with p53, which results in rapid degradation of p53 via the
ubiquitin-proteasome pathway (Johnson et al., 2002). There- Acknowledgments The authors thank Fundação de Amparo a Pes-
quisa do Estado de Minas Gerais (FAPEMIG) and Conselho Nacional
fore, although HeLa cells contain a wild type of TP53 gene,
de Desenvolvimento Científico e Tecnológico (CNPq) for financial
the expression of the E6 protein of HPV may have the same support.
functional effects as a mutant TP53 (Johnson et al., 2002).
Thus, the results of TP53 expression may be underestimated. Compliance with ethical standards
Associating the results obtained in qPCR studies for TP53
Conflict of interest The authors declare that they have no competing
gene expression with that observed in the TUNEL assay, it can interests.
be inferred that the absence of apoptosis detected at 24 h of
exposure to the compounds could be a consequence of G1/S
arrest of the cell cycle due to the damage induced by upre-
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