Synthesis and anticancer activities of novel guanylhydrazone and aminoguanidine tetrahydropyran derivatives

Article abstract of DOI:10.3390/molecules21060671

In this paper we present the convenient syntheses of six new guanylhydrazone and aminoguanidine tetrahydropyran derivatives 2-7. The guanylhydrazone 2, 3 and 4 were prepared in 100% yield, starting from corresponding aromatic ketones 8a-c and aminoguanidi

Full text of DOI:10.3390/molecules21060671

molecules
Article
Synthesis and Anticancer Activities of Novel
Guanylhydrazone and Aminoguanidine
Tetrahydropyran Derivatives
Fábio Pedrosa Lins Silva 1, Bruna Braga Dantas 2, Gláucia Veríssimo Faheina Martins 2,
Demétrius Antônio Machado de Araújo 2 and Mário Luiz Araújo de Almeida Vasconcellos 1,
*
1
Departamento de Química, Campus I, Laboratório de Síntese Orgânica Medicinal da Paraíba (LASOM-PB),
Universidade Federal da Paraíba, João Pessoa, CEP:58051-900, Paraíba, Brazil; pedrosalinssilva@gmail.com
Departamento de Biotecnologia, Campus I, Laboratório de Biotecnologia Celular e Molecular,
Universidade Federal da Paraíba, João Pessoa, CEP:58051-900, Paraíba, Brazil;
brunabdantas@gmail.com (B.B.D.); glauciafaheina@yahoo.com.br (G.V.F.M.);
demetrius@cbiotec.ufpb.br (D.A.M.A.)
2
*
Correspondence: mlaav@quimica.ufpb.br; Tel.: +55-83-3216-7589
Academic Editor: Derek J. McPhee
Received: 28 March 2016; Accepted: 11 May 2016; Published: 21 June 2016
Abstract: In this paper we present the convenient syntheses of six new guanylhydrazone and
aminoguanidine tetrahydropyran derivatives
100% yield, starting from corresponding aromatic ketones 8a
accessed by microwave irradiation. The aminoguanidine
guanylhydrazone with sodium cyanoborohydride (94% yield of
The aromatic ketones 8a were prepared from the Barbier reaction followed by the Prins cyclization
reaction (two steps, 63%–65% and 95%–98%). Cytotoxicity studies have demonstrated the effects of
compounds in various cancer and normal cell lines. That way, we showed that these compounds
2
–7
. The guanylhydrazone
and aminoguanidine hydrochloride
and were prepared by reduction of
, and 100% yield of and ).
2, 3 and 4 were prepared in
–
6
c
5
,
7
2
–
4
5
6
7
–c
2–7
decreased cell viabilities in a micromolar range, and from all the compounds tested we can state that,
at least, compound 3 can be considered a promising molecule for target-directed drug design.
Keywords: Prins cyclization reaction; tetraydropyran derivatives; anticancer; guanylhydrazone;
aminoguanidine
1. Introduction
Despite the availability of improved drugs, including targeted cancer therapies, cancer is still
one of the leading causes of mortality worldwide. It is estimated to have accounted for 8.2 million
deaths (around 13% of all deaths) in 2007, and ~1.4 million new cancer cases and ~566,000 deaths
from cancer occurred in the United States in 2008 [1–4]. Additionally, a 70% increase in new cases of
cancer is expected over the next two decades. Therefore, new, more effective treatment strategies are
required to address this issue. A substantial number of new antineoplastic agents have been discovered.
Considerable insight has been gained into the mechanisms by which many of these compounds affect
cellular growth and this knowledge has been used in the design of new chemotherapeutic drugs [5].
In recent years, several compounds based on the tetrahydropyran moiety have been described as
presenting high anticancer activities [6–11]. Substituted tetrahydropyranyl moieties are extensively
distributed in the natural products’ structures that present a large pharmacological profile [12].
There are many synthetic methodologies to prepare this moiety, i.e., the Prins cyclization reaction
and others. In 2014, Ahmed et al. described several 2,4,6-diaryltetraydropyran scaffolds that possess
immense importance with antiproliferative activity against human cancer cell lines (Figure 1) [13].
Molecules 2016, 21, 671; doi:10.3390/molecules21060671
www.mdpi.com/journal/molecules

Molecules 2016, 21, 671
2 of 11
Figure 1. The compound 2,4,6-Diaryltetraydropyran has antiproliferative activity against human
cancer cell lines.
In connection to our interest in the stereoselective synthesis of tetraydropyran derivatives using
the Prins cyclization reaction [14 18], and considering that guanylhydrazone [19] and aminoguanidine
groups [20 21] potentiate the anticancer activity of some drugs, and these nitrogenated bases
could be considered pharmacophoric points, we present in this article the syntheses of six novel
tetrahydropyranyl guanylhydrazone and aminoguanidine (Figure 2). That way, we performed
–
,
2–7
in vitro evaluation for anticancer activity against cancer cell lines, such as the chronic myeloid leukemia
(K562), human acute myeloid leukemia (HL-60), human breast adenocarcinoma (MCF-7), human colon
adenocarcinoma (HT-29), and L929 (murine fibroblast) and the human peripheral blood of patients
with chronic myeloid leukemia (PBMC/CML) cells. Normal cell lines, L929 (murine fibroblast) cells
and human peripheral blood cells (PBMC) were also evaluated.
Figure 2. Compounds synthesized and evaluated against tumor cells line in this work.
It is important to note that compounds 2–7 are Meso compounds which greatly simplifies these
syntheses, since only relative configuration (cisztrans) must be controlled [12].
2. Results and Discussion
2.1. Chemistry
Our experimental work starts with the preparation in good yield of homoallylic alcohols shown in
Scheme 1 (step i) from the Barbier reaction [22] between the corresponding aldehydes (benzaldehyde,
4-fluorobenzaldehyde and 2-naphthaldehyde) with allyl bromide in the presence of stannous chloride
(96%–98% yields). In step ii the corresponding tetrahydropyran rings of 1a–c are prepared with control
of relative configuration (2,4,6-cis) from the Prins cyclization reaction [12], followed by hydrolysis of the
acetyl intermediate with potassium carbonate in methanol (60%–63% yields). The cis stereoselectivity of
these reactions is anticipated based on the proposed mechanisms 4 for these reactions and determined
by bi-dimensional spectroscopic study (NOESY spectra, see in Supplementary Materials) and by X-ray
crystallography of
1 [23]. The oxidation of tetrahydropyran alcohols to the corresponding ketones
8a (step iii, Scheme 1) was performed in high yields by using PCC in dichloromethane as a solvent
–c

Molecules 2016, 21, 671
3 of 11
(95%–98% yields). The guanylhydrazone
2
–
4
(Figure 1) were prepared in quantitative yields (100%
yields) by reacting ketones 8a
catalysts were used) promoted by 5 min of microwave irradiation (250 W) at 100 ˝C (step iv). Finally,
the reductions of aminoguanidine were performed in very high yields (94%–100% yields) by
reacting with sodium cyanoborohydride in ethanol as a solvent at room temperature (step v,
Scheme 1). The relative configurations of C4 of aminoguanidine
were determined by 1H-NMR
spectroscopic studies (NOESY spectrum, see in Supplementary Materials).
–c with aminoguanidine hydrochloride and ethanol as a solvent (no
2–4
2–4
5–7
Scheme 1. Synthesis of guanylhydrazone
2
–
4
and aminoguanidine
5–7. (i) SnCl2, KI, H2O, NH4Cl,
0 ˝C, 2 h, r.t., 96%–98% yields; (ii) 1-PhH, AcOH, BF3
¨
Et2O, 0 ˝C, 3 h, r.t.; 2-K2CO3, MeOH, 30 min, r.t.,
60%–63% yields; (iii) PCC, CH2Cl2, r.t., 2.5 h , 95%–98% yields; (iv) aminoguanidine hydrochloride,
EtOH, microwave, 100 ˝C, 5 min, 100% yields; (v) NaCNBH3, 0˝C, 2 h, r.t., 94%–100% yields.
2.2. Biology
The anticancer drug discovery screening has been designed to distinguish between
broad-spectrum anticancer compounds and cancer-selective agents [24]. The anticancer activities
of
2
–
7
were investigated against different types of cancer cells and non-cancer cells using MTT assay.
mol
´1) for each compound
Each cell line was incubated with different concentrations (3–100
and this was used to create a concentration-response relationship curve. The response parameter
(IC50) was calculated for each cell line. The IC50 value corresponds to the compound’s concentration
µ
¨L
causing 50% of reduction viability at the end of the incubation period (24 or 72 h). Compounds 2–7
indicated potency anticancer activity against K562, HL-60, MCF-7, HT-29 and PBMC/CML cell lines.
The analogues showed a broad-spectrum anticancer activity independent of the incubation period
(Tables 1 and 2).
The effects of compounds 3 and 7, which were the most promissory substances, had similar action
on K562 and PBMC/CML cells. Moreover, those cells showed to be the most sensitive cells. It is
important because those types of chronic myeloid leukemia have expressed the BCR-ABL gene, which
stimulates cell growth, and it is highly resistant to anticancer therapy [25,26]. Therefore, our result can
address an association with a possible clinical application.
In this work, etoposide has been used as the reference substance, because this compound is
known is a standard drug used in the treatment of a wide spectrum of solid tumors, lymphomas and
leukemias. It is a topoactive drug which inhibits topoisomerase II–DNA cleavable complex, resulting
in DNA damage, and often results in cell death apoptosis [27,28].

Molecules 2016, 21, 671
4 of 11
The results have demonstrated that DNA is vulnerable to damage after treatment with
compounds and , similar to therapeutics such as etoposide. Many anticancer drugs exert their
effect on the cell cycle which is known as being cell-cycle-specific. The compounds and induced
3
7
3
7
cell-cycle arrest (Supplement Figures S1 and S2) in the G1 phase, corresponding to IC50 in 24 h, as
well as an increase of cells in the sub-G1 phase, representing sub diploid content that corresponds to
apoptotic cells with fragmented DNA or condensed chromatin [29]. This event demonstrates that the
addition of the compound inhibits the cell cycle and induces cell death by apoptosis, probably because
these cells were unable to repair the damage caused by molecules [30].
Our results also showed that the different substituents added around the tetrahydropyran ring
were critical to the motifs’ efficacy and our cytotoxic data showed that compounds
at least, two-fold more potent than compounds and (Tables 1 and 2). The analogs
become more powerful than and , probably due to the addition of the fluor atom at position
the aromatic ring. Compounds and
2, 3 and 4 are,
5,
6
7
3
and
6
2
5
4
in
4
7
were even more potent than the other analogs, probably due
to the presence of two naphthyl rings, which alter the molecular properties, i.e., lipophilicity, among
others. Other studies corroborate with our results, demonstrating increased activity of tetrahydropyran
derivates with a further lipophilicity group [31].
The six compounds 2–7 demonstrated potent anticancer activity. Moreover, compound 3 showed
the most promising effects due to both its powerful cytotoxic activity in K562 cells and being the most
selective compound tested [32].
Table 1. Inhibitory effect of compounds and etoposide on cell growth of human cancer and normal cell
lines after 24 h of incubation.
Compound
HL-60
K562
HT-29
MCF-7
LMC
L929
PBMC
2
3
4
5
6
7
16.0 ˘ 4.6
12.5 ˘ 4.5
9.2 ˘ 4.6
42.0 ˘ 4.4
85.4 ˘ 5.2
35.3 ˘ 5.2
5.8 ˘ 1.1
13.6 ˘ 4.5
8.9 ˘ 4.0
7.5 ˘ 4.9
44.7 ˘ 4.8
58.8 ˘ 4.1
15.5 ˘ 5.1
>50
15.9 ˘ 3.7
11.2 ˘ 4.0
7.3 ˘ 4.7
46.9 ˘ 4.2
59.5 ˘ 4.3
25.8 ˘ 4.8
>100
52.0 ˘ 4.6
31.7 ˘ 4.7
16.7 ˘ 5.2
>100
126.9 ˘ 4.3
43.3 ˘ 5.8
>100
nt
18.1 ˘ 5.0
20.0 ˘ 4.2
10.8 ˘ 5.5
65.8 ˘ 4.7
72.2 ˘ 3.9
39.7 ˘ 5.5
nt
19.4 ˘ 3.2
14.3 ˘ 3.2
5.9 ˘ 3.3
51.5 ˘ 3.0
72.7 ˘ 3.4
20.8 ˘ 3.5
>100
7.5 ˘ 2.4
nt
nt
nt
14.8 ˘ 2.4
Etoposide
nt
Note: nt (not tested); PBMC, peripheral blood mononuclear cell; SEM, standard error of the mean. HL-60:
leukemia myeloid acute, K562: leukemia myeloid chronic, MCF-7: breast adenocarcinoma cell line, HT-29: colon
adenocarcinoma cell line and PBMC were incubated with compounds for 24 h separately. The cell viability
was determined by MTT assay. Results are displayed as IC50 in micromolar values and expressed as the
mean ˘ SEM, obtained from at least three independent experiments in triplicate.
Table 2. Inhibitory effect of compounds and etoposide on cell growth of human cancer and normal cell
lines after 72 h of incubation.
Compound
HL-60
K562
HT-29
MCF-7
PBMCLMC
L929
PBMC
2
3
4
5
6
7
14.4 ˘ 5.2
14.9 ˘ 5.4
6.9 ˘ 4.9
31.0 ˘ 4.5
29.0 ˘ 4.9
19.2 ˘ 5.3
71.4 ˘ 5.0
>100
9.6 ˘ 4.7
6.0 ˘ 4.6
3.4 ˘ 4.7
19.9 ˘ 3.9
36.9 ˘ 3.3
11.8 ˘ 4.3
34.8 ˘ 3.8
20.8 ˘ 4.0
13.7 ˘ 5.0
47.2 ˘ 3.5
42.9 ˘ 4.2
24.14 ˘ 3.5
nt
23.9 ˘ 4.0
17.2 ˘ 4.7
3.9 ˘ 3.8
4.7 ˘ 2.9
6.7 ˘ 3.1
2.2 ˘ 2.9
11.4 ˘ 2.7
12.5 ˘ 2.9
7.5 ˘ 3.4
14.6 ˘ 3.4
nt
nt
41.4 ˘ 5.8
66.5 ˘ 5.3
25.4 ˘ 5.3
46.9 ˘ 4.4
79.4 ˘ 3.8
16.88 ˘ 4.1
nt
43.6 ˘ 5.6
18.7 ˘ 2.7
Note: nt (not tested); PBMC, peripheral blood mononuclear cell; SEM, standard error of the mean. HL-60:
leukemia myeloid acute, K562: leukemia myeloid chronic, MCF-7: breast adenocarcinoma cell line, HT-29: colon
adenocarcinoma cell line and PBMC were incubated with compounds for 24 h separately. The cell viability
was determined by MTT assay. Results are displayed as IC50 in micromolar values and expressed as the
mean ˘ SEM, obtained from at least three independent experiments in triplicate.

Molecules 2016, 21, 671
5 of 11
3. Materials and Methods
3.1. Chemistry
3.1.1. General Methods
All commercially available reagents were purchased from Aldrich and used without further
purification. Reactions were monitored by thin-layer chromatography (TLC) using Silica gel 60 UV254
Macherey-Nagel pre-coated silica gel plates; detection was by means of a UV lamp and revelation to
vanillin. Flash column chromatography was performed on 300–400 mesh silica gel. Organic layers were
dried over anhydrous MgSO4 or Na2SO4 prior to evaporation on a rotary evaporator. The reactions
performed under microwave irradiation were performed in a microwave reactor model CEM Discover
System-Benchmate (CEM Corporation, 3100 Smith Farm Road Matthews, NC. USA, 28104) quipped
with a system for continuous irradiation with programmable
µ
W power in the range from 0 to 300 W,
with temperature monitored by infrared sensor. H- and 13C-NMR spectra were obtained by using
a Varian Mercury Spectra AC 200 (200 MHz for 1H and 50 M for 13C) (Varian, Palo Alto, CA, USA)
or in CDCl3, DMSO or CD3OD. Spectral patterns are designated as s singlet; d doublet; dd doublet
of doublets; ddd double doublet of doublets; t triplet; dt double triplet; q quartet; m multiplet. The
1
chemical shifts (δ
, ppm) were reported in values relative to tetramethylsilane (0 ppm) for 1H-NMR and
were reported in the scale relative to CDCl3 (77.00 ppm), DMSO (39.52 ppm) or CD3OD (49.00 ppm) for
13C-NMR and coupling constants (J) were expressed in. The Fourier Transform Infrared Spectroscopy
spectra were obtained using a spectrophotometer IR-Prestige-21 (Shimadzu, Kyoto, Japan). MS data
were measured with a Shimadzu GCMS–QP2010 mass spectrometer (Shimadzu). The elementary
analyses of unpublished compounds were performed in an analyzer organic elemental CHNS-O,
FLASH 2000 of Perkin-Elmer, Waltham, MA, USA).
3.1.2. General Procedure for Homoallylic Alcohol Synthesis
The synthesis of homoallylic alcohol was performed through
a Barbier reaction.
The corresponding aldehyde (8 mmol) was added to water (40 mL) containing potassium
iodide (24 mmol), stannous chloride dehydrate (12 mmol) and allyl bromide (1 mL). The orange
solution turns white by addition of saturated ammonium chloride (20 mL). The stirring was
continued for 2 h at room temperature. After the end of reaction, the reaction mixture was extracted
with CH2Cl2 (2
ˆ
50 mL) gave an organic phase, washed with water, dried with sodium sulfate
anhydrous, concentrated under reduced pressure and purified by flash chromatography on silica
gel (EtOAc:Hexane as eluent). The products were concentrated under reduced pressure yielding the
homoallylic alcohol, respectively [23].
1-Phenylbut-3-en-1-ol. This product was obtained using benzaldehyde. The product was purified by
silica gel column chromatography using EtOAc/hexane (1:9) as eluent, resulting in 97% yield. IR (KBr)
´1
/cm 3371, 3070, 3028, 2904, 1639, 1492, 1049, 995, 914, 756, 702; 1H-NMR (200 MHz, CD3OD)
δ 7.33
ν
(m, 5H), 5.83 (m, 1H), 5.18 (m, 2H), 4.74 (t, 1H, J = 6.0 Hz), 2.53 (m, 3H); 13C-NMR (50 MHz, CDCl3)
δ 144.57, 135.18, 129.09, 128.22, 126.54, 119.02, 74.03, 44.49.
1-(4-Fluorophenyl)but-3-en-1-ol. This product was obtained using 4-fluorobenzaldehyde. The product
was purified by silica gel column chromatography using EtOAc/hexane (3:7) as eluent, resulting in
96% yield. IR (KBr) ν
/cm´1 3383, 3078, 2981, 2908, 1604, 11508, 1049, 995, 914, 837; 1H-NMR (200 MHz,
CDCl3)
δ 7.41 (m, 2H), 7.14 (m, 2H), 5.88 (m, 1H), 5.25 (m, 2H), 4.80 (t, 1H, J = 6.0 Hz), 2.57 (m, 3H);
13C-NMR (50 MHz, CDCl3)
115.71, 73.45, 73.33, 44.62.
δ 165.29, 160.42, 140.31, 140.25, 134.96, 134.88, 128.29, 128.13, 119.36, 116.13,
1-(Naphthalen-2-yl)but-3-en-1-ol. This product was obtained using 2-naphthaldehyde. The product was
purified by silica gel ´co1lumn chromatography using EtOAc/hexane (1:9) as eluent, resulting in 98%
yield. IR (KBr)
ν
/cm 3278, 3051, 2947, 2854, 1600, 1508, 1060, 991, 952, 821, 748; 1H-NMR (200 MHz,

Molecules 2016, 21, 671
6 of 11
CDCl3)
δ 7.67 (m, 7H), 5.84 (m, 1H), 5.18 (m, 2H), 4.90 (t, 1H, J = 6.0 Hz), 2.60 (m, 2H), 2.34 (s, 1H);
13C-NMR (50 MHz, CDCl3)
124.75, 119.23, 74.19, 44.45.
δ 141.98, 135.11, 133.98, 133.67, 128.94, 128.70, 128.42, 126.86, 126.55, 125.26,
3.1.3. General Procedure for Synthesis of 4-Hydroxy-2,6-diaryl-tetrahydropyran
A round bottom flask equipped with a magnetic stir bar was charged with 0.81 mL of acetic acid,
7 mL of benzene, 4.5 mmol of homoallylic alcohol and 9 mmol of aldehyde. The mixture was cooled to
0 ˝C and after slow addition of 1.2 mL of boron trifluoride etherate was stirred in this temperature
for 3 h. To the reaction media was added saturated NaHCO3 in water (10 mL) followed extraction
with EtOAc (3
ˆ
10 mL). The combined organic phase are washed with brine (3
ˆ
10 mL), dried over
Na2SO4, filtered and concentrated under vacuum. The acetylated thus obtained in this reaction was
dissolved in methanol (10 mL) and stirred over potassium carbonate (500 mg) for 0.5 h. Then methanol
was removed under reduced pressure and water (20 mL) was added. The mixture was extracted with
EtOAc (2
ˆ
20 mL) and the combined organic layers were dried over anhydrous Na2SO4 and the
solvent was removed under reduced pressure. The resulting crude product was purified by column
chromatography on silica gel to give compound 4-Hydroxy-2,6-disubstituted tetrahydropyran [23].
2,6-Diphenyl-tetrahydro-2H-pyran-4-ol (1a). This product was obtained using 4.5 mmol of 1-phenylbut-
3-en-1-ol and 9 mmol of benzaldehyde. The product was purified by silica gel column chromatography
using EtOAc/hexane (1:3) as eluent, resulting in 60% yield. IR (KBr) ν
/cm´1 3263, 3032, 2947, 2858,
1604, 1496, 1319, 1060, 752, 694; 1H-NMR (200 MHz, CDCl3)
δ 7.37 (m, 10H), 4.57 (d, 2H, J = 12.0 Hz),
4.10 (m, 1H), 2.27 (dd, 2H, J = 12.0, 4.0 Hz), 2.12 (m, 1H), 1.60 (q, 2H, J = 12.0 Hz); 13C-NMR (50 MHz,
CDCl3) δ 142.49, 128.85, 128.02, 126.39, 78.32, 69.03, 43.51.
2,6-Bis(4-fluorophenyl)-tetrahydro-2H-pyran-4-ol (1b). This product was obtained using 4.5 mmol of
1-(4-fluorophenyl) but-3-en-1-ol and 9 mmol of 4-Fluorobenzaldehyde. The product was purified by
silica gel column chromatography using EtOAc/hexane (3:7) as eluent, resulting in 62% yield. IR (KBr)
´1
/cm 3329, 3051, 2943, 2854, 1604, 1512, 1230, 1068, 821; 1H-NMR (200 MHz, CDCl3)
δ 7.33 (m, 4H),
ν
7.04 (m, 4H), 4.53 (d, 2H, J = 12.0 Hz), 4.09 (m, 1H), 2.23 (m, 2H), 2.02 (s, 1H), 1.56 (dd, 2H, J = 14.0,
12.0 Hz); 13C-NMR (50 MHz, CDCl3)
78.14, 78.06, 69.09, 43.70.
δ 165.36, 160.48, 138.39, 138.33, 128.41, 128.25, 116.18, 115.75,
2,6-Di(naphthalen-2-yl)-tetrahydro-2H-pyran-4-ol (1c). This product was obtained using 4.5mmol of
1-(naphthalen-2-yl)but-3-en-1-ol and 9 mmol of 2-naphthaldehyde. The product was purified by silica
gel co´lu1mn chromatography using EtOAc/hexane (1:1) as eluent, resulting in 63% yield. IR (KBr)
/cm 3278, 3020, 2947, 2854, 1600, 1508, 1357, 1060, 821, 748; 1H-NMR (500 MHz, DMSO)
δ 7.92 (m,
ν
8H), 7.63 (dd, 2H, J = 10.0, 5.0 Hz), 7.50 (m, 4H), 4.82 (d, 2H, J = 10.0 Hz), 4.11 (m, 1H), 3.82 (s, 1H), 2.29
(dd, 2H, J = 10.0, 5.0 Hz), 1.58 (dd, 2H, J = 25.0, 10.0 Hz); 13C-NMR (125 MHz, DMSO)
δ 140.76, 133.40,
132.94, 128.41, 128.30, 128.02, 126.59, 126.28, 125.08, 124.64, 77.81, 67.35, 43.63.
3.1.4. General Procedure for Oxidation of 4-Hydroxy-2,6-diaryl Tetrahydropyrans
In a typical reaction, 1 mmol of 4-hydroxy-2,6-disubstituted tetrahydropyran was dissolved in
6 mL of CH2Cl2 dry in one round bottom flask and treated with 1 mmol of PCC. The stirring was
continued for 2.5 h at room temperature where the completion of the reaction was monitored by
thin-layer chromatography (TLC). The mixture was extracted with CH2Cl2 and filtered through a short
pad of silica gel with hexane and the solvent evaporated to 2,6-disubstituted–tetrahydropyran -4-one.
No side products were observed to be formed [23].
2,6-Diphenyl-tetrahydropyran-4-one (8a). This product was obtai´n1ed using 1 mmol of 2,6-diphenyl-
tetrahydro-2H-pyran-4-ol, resulting in 98% yield. IR (KBr) ν/cm 3032, 2970, 2858, 1720, 1604, 1496,
1056, 1060, 752, 698; 1H-NMR (500 MHz, CDCl3)
δ
7.40 (m, 10H), 4.85 (dd, 2H, J = 10.0, 5.0 Hz), 2.73 (m,
4H); 13C-NMR (125 MHz, CDCl3) δ 206.39, 141.10, 128.99, 128.43, 126.01, 79.31, 50.06.

Molecules 2016, 21, 671
7 of 11
2,6-Bis(4-fluorophenyl)-tetrahydropyran-4-one (8b). This product was obtained using 1 mmol of
2,6-bis(4-fluorophenyl)-tetrahydro-2H-pyran-4-ol, resulting in 97% yield. IR (KBr)
/cm-1 3070, 2970,
7.41 (m, 4H), 7.08 (m, 4H), 4.82 (dd,
206.25, 165.65, 160.74, 137.14, 137.08,
ν
2893,1720, 1608, 1512, 1053, 1138,829; 1H-NMR (200 MHz, CDCl3)
δ
2H, J = 10.0, 4.0 Hz), 2.66 (m, 4H); 13C-NMR (50 MHz, CDCl3)
δ
128.28, 128.12, 116.54, 116.11, 79.12, 50.35.
2,6-Di(Naphthalen-2-yl)-tetrahydropyran-4-one (8c). Product 8c was obtained using 1 mmol of
2,6-di(naphthalen-2-yl)-tetrahydro-2H-pyran-4-ol, resulting in 95% yield. IR (KBr)
/cm´1 3051,
7.69 (m, 14H), 5.06 (m, 2H),
206.76, 138.76, 133.95, 133.85, 129.33, 128.83, 128.48, 127.10,
ν
2978, 2897, 1708, 1600, 1508, 1045, 825, 748; 1H-NMR (200 MHz, CDCl3)
δ
2.84 (m, 4H); 13C-NMR (50 MHz, CDCl3)
126.95, 125.38, 124.41, 79.97, 50.42.
δ
3.1.5. General Procedure for the Preparation of Guanylhydrazone (2–4)
The hydrazones were prepared by reaction of ketones with aminoguanidine hydrochloride with
the aid of a microwave device. A mixture of 2,6-di-substituted-tetrahydropyran-4-one (0.5 mmol)
and aminoguanidine hydrochloride (0.5 mmol) in 1 mL of ethanol were placed in a glass tube for
specific microwave reactor along with a magnetic. A shaker reaction was performed under microwave
˝
irradiation to 100 C (read monitored by infrared sensor) for 5 min (“Hold Time”) under conditions of
a closed vessel. After completion of the reaction, the solvent was evaporated under reduced pressure.
This crude product was then subjected to flash column chromatography to yield a solid.
2-(2,6-Diphenyl-2H-pyran-4(3H)-ylidene)hydrazinecarboximidamide (2). This product was obtained
using 0.5 mmol of 2,6-diphenyl-tetrahydropyran-4-one (8a). The product was purified by column
chromatography on silica gel using methanol/EtOAc (1:9) as eluent, resulting in quantitative yield
(100%). IR (KBr)
698; 1H-NMR (200 MHz, CD3OD)
(dt, 1H, J = 14.0, 2.0 Hz), 2.51 (dd, 1H, J = 14.0, 12.0 Hz), 2.27 (dd, J = 14.0, 12.0 Hz, 1H); 13C-NMR
(50 MHz, CD3OD) 159.01, 158.57, 144.10, 130.80, 130.24, 128.20, 82.20, 44.71, 38.37; Anal. Calcd for
ν
/cm´1 3348, 3309, 3155, 3062, 3035, 2862, 1674, 1597, 1627, 1492, 1091, 1064, 756,
δ
7.36 (m, 10H), 4.68 (m, 2H), 3.17 (dt, 1H, J = 16.0, 2.0 Hz), 2.76
δ
C18H21ClN4O C, 62.69; H, 6.14; N, 16.25; Found C, 62.27, H, 6.24, N, 16.15.
2-(2,6-Bis(4-fluorophenyl)-2H-pyran-4(3H)-ylidene)hydrazinecarboximidamida (3). This product was
obtained using 0.5 mmol of 2,6-bis(4-fluorophenyl)-tetrahydropyran-4-one (8b). The product was
purified by column chromatography on silica gel using methanol/EtOAc (1:9) as eluent, resulting in
quantitative yield (100%). IR (KBr)
1080, 1056, 825. H-NMR (200 MHz, CD3OD)
ν
/cm´1 3390, 3352, 3271, 3066, 2958, 2850, 1670, 1604, 1627, 1508,
1
δ 7.45 (dd, 4H, J 14.0, 8.0), 7.04 (t, 4H, J = 8.0 Hz), 4.63 (t,
2H, J = 12.0 Hz), 2.72 (d, 1H, J = 16.0 Hz), 2.48 (dd, 1H, J = 14.0, 12.0 Hz), 2.22 (dd, 1H, J = 14.0, 12.0
Hz), 2.08 (s, 1H); 13C-NMR (50 MHz, CD3OD)
δ
167.43, 162.56, 159.86, 157.84, 140.17, 130.29, 117.19,
81.64, 44.65, 38.24; Anal. Calcd for C18H19ClF2N4O C, 56.77; H, 5.03; N, 14.71; Found C, 56.63, H, 5.06,
N, 14.77.
2-(2,6-Di(naphthalen-2-yl)-2H-pyran-4(3H)-ylidene)hydrazinecarboximidamida (4). This product was
obtained using 0.5 mmol of 2,6-di(naphthalen-2-yl)-tetrahydropyran-4-one (8c). The product was
purified by column chromatography on silica gel using methanol/EtOAc (3:7) as eluent, resulting in
quantitative yield (100%). IR (KBr)
1H-NMR (200 MHz, CD3OD)
7.87 (m, 8H), 7.60 (m, 2H), 7.43 (m, 4H), 4.78 (dd, 2H, J = 12.0, 2.0 Hz),
2.69 (m, 4H); 13C-NMR (50 MHz, CD3OD)
158.98, 158.34, 141.44, 135.97, 135.83, 130.43, 130.04, 128.55,
ν
/cm´1 3423, 3340, 3155, 3055, 1672, 1600, 1625, 1508, 1078, 817, 748.
δ
δ
128.41, 127.13, 126.90, 126.35, 82.24, 44.49, 38.26; Anal. Calcd for C26H24N4O C, 76.45; H, 5.92; N, 13.72;
Found C, 76.55, H, 6.01, N, 13.64.
3.1.6. General Procedure for the Preparation of Aminoguanidine (5–7)
The aminoguanidine were prepared by reduction of guanylhydrazone with sodium
cyanoborohydride. The guanylhydrazone 0.5 mmol was added to a dry flask under magnetic stirring,

Molecules 2016, 21, 671
8 of 11
˝
followed by addition of 1 mL of ethanol at 0 C. Then, 48 mg of sodium cyanoborohydride was
slowly added to the flask, and then the mixture remained stirring at room temperature for two hours.
After completion of the reaction, the solvent was evaporated under reduced pressure. This crude
product was then subjected to flash column chromatography to yield a solid.
2-(2,6-Diphenyltetrahydro-2H-pyran-4-yl)hydrazinecarboximidamida (
0.5 mmol of 2-(2,6-diphenyl-2H-pyran-4-(3H)-ylidene) hydrazinecarboximidamida (
was purified by column chromatography on silica gel using EtOAc as the eluent, resulting in a yield
of 94%. IR (KBr)
/cm´1 3568, 3448, 3062, 3032, 2862, 1674, 1631, 1631, 1492, 1126, 1064, 756, 698;
1H-NMR (200 MHz, CD3OD)
7.33 (m, 10H), 5.40 (s, 1H), 4.60 (m, 2H), 3.10 (d, 1H, J = 14.6 Hz), 2.33
(m, 2H), 1.92 (m, 2H), 1.16 (t, 2H, J = 8.0 Hz); 13C-NMR (50 MHz, CD3OD)
161.81, 159.32, 158.24,
5). This product was obtained using
2). The product
ν
δ
δ
145.58, 145.09, 144.08, 143.32, 130.76, 130.54, 128.19, 82.02, 80.46, 44.71, 42.66, 40.69, 38.21; Anal. Calcd
for C18H23ClN4O C, 62.33; H, 6.68; N, 16.15; Found C, 62.38, H, 6.64, N, 16.19.
2-(2,6-Bis-(4-fluorophenyl)tetrahydro-2H-pyran-4-yl)hydrazinecarboximidamida (6). This product was obtained
using 0.5 mmol of 2-(2,6-bis(4-fluorophenyl)-2H-pyran-4(3H)-ylidene)-hydrazinecarboximidamida (
3).
The product was purified by column chromatography on silica gel using EtOAc as eluent, resulting
in quantitative yield (100%). IR (KBr)
1076, 833; 1H-NMR (200 MHz, CD3OD)
ν
δ
/cm´1 3568, 3452, 2924, 2870, 1666, 1639, 1639, 1512, 1126,
7.47 (m, 4H), 7.07 (m, 4H), 4.7 (m, 2H), 3.53 (m, 2H), 1.61 (m,
8H); 13C-NMR (50 MHz, CD3OD)
δ 168.48, 163.63, 159.25, 142.88, 142.82, 142.44, 142.38, 131.49, 118.63,
118.21, 81.27, 77.61, 41.92, 39.37. Anal. Calcd for C18H20F2N4O C, 62.42; H, 5.82; N, 16.18; Found C,
62.48, H, 5.79, N, 16.16.
2-(2,6-Di(naphthalen-2-yl)tetrahydro-2H-pyran-4-yl)hydrazinecarboximidamida (
using 0.5 mmol of 2-(2,6-di(naphthalen-2-yl)-2H-pyran-4(3H)-ylidene)hydrazinecarboximidamida (
The product was purified by column chromatography on silica gel using EtOAc as eluent, resulting
in quantitative yield (100%). IR (KBr)
/cm-1 3525, 3448, 3055, 2943, 1674, 1631, 1124, 1070, 819,
750; 1H-NMR (200 MHz, CD3OD) 7.69 (m, 14H), 4.79 (s, 2H), 2.44 (m, 5H), 1.22 (m, 2H); 13C-NMR
(50 MHz, CD3OD) 163.17, 160.56, 159.60, 144.35, 143.83, 142.76, 142.04, 137.29, 137.14, 136.96, 131.64,
7). This product was obtained
4).
ν
δ
δ
131.58, 131.23, 129.78, 129.66, 129.40, 128.47, 128.40, 128.20, 128.01, 127.93, 127.67, 83.64, 82.10, 78.42,
60.92, , 45.87, 41.97, 39.44; Anal. Calcd for C26H27ClN4O C, 69.87; H, 6.09; N, 12.53; Found C, 69.83, H,
6.12, N, 12.64.
3.2. Pharmacology
Fetal bovine serum (FBS) (Cripion, Brazil); Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden);
Trypan blue (Inlab, São Paulo, Brazil); trypsin–EDTA and MTT (3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide) (Amresco, Toronto, ON, Canada); RPMI1640 and DMEM medium
was purchased from HIMEDIA (Mumbai, Maharashtra, India) and Penicillin/streptomycin solution
was obtained by Sigma-Aldrich (St. Louis, MO, USA).
3.2.1. Preparation of Stock Solutions
Preparation of stock solutions of the test compounds were prepared in dimethyl sulfoxide (DMSO)
at 20 mmol L µm), and diluted for use in the nutrient medium
´1, filtered through adequate filters (0.22
¨
to the relevant working concentrations. For all of the cells used, the nutrient medium was RPMI 1640
or DMEM, according to the cell line tested. We did not verify any DMSO effect, incubating only this
dispersing at the highest concentration used in the experiments.
3.2.2. Cell Culture
K562, HL-60, MCF-7, HT-29 and L929 cell lines were acquired from Rio de Janeiro Cell Bank
(Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil). Leukemia cell lines and MCF-7
cells were maintained in the nutrient RPMI 1640 medium supplemented with 10% FBS, 2 mmol
L´1
¨

Molecules 2016, 21, 671
9 of 11
´1
´1
µg mL streptomycin. Others cell lines were maintained
glutamine, 100 U
¨
mL penicillin, and 100
¨
˝
in the nutrient DMEM similarly supplemented. All of these cells were grown at 37 C in 5% CO2 and
humidified air atmosphere. The PBMC cells were separated by Histopaque-1077 (GE Healthcare), from
whole blood of healthy, non-smoking donors who had not taken any drugs for at least 15 days prior
to sampling donors. Thus, using the gradient centrifugation, the interface cells were washed with
PBS (phosphate buffered saline), counted and resuspended in nutrient medium to the required cell
concentration. The blood used to obtain PBMC cells were collected from the blood bank of the João
Pessoa (Hemocentro), Paraíba, Brazil. Similar procedure was performed for obtaining PBMC/CML.
These cells were collected from blood of patients recently diagnosed with CML and without being
subject to treatment at this moment. The blood samples from patients PBMC/CML, were collected
in the Napoleão Laureano Hospital, João Pessoa, Paraíba, Brazil. This study was approved by the
Institutional Ethical Committee of Lauro Wanderley Hospital (CEP/HULW), from Federal University
of Paraíba, protocol numbers, 378119 and 05878712.7.0000.5183.
3.2.3. Cell Viability–MTT
The cytotoxicity of molecules to cells was evaluated using the original enzymatic reduction
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to produce formazan
crystals [33]. Cells were seeded at 3
Cells were exposed to different concentrations of tetrahydropyrans substituted (3–100
dissolved in the medium (three wells per concentration). After 24 h or 72 h of incubation, plates were
ˆ
104 or 5
ˆ
104 cells per well in 96-well tissue culture plates.
mol
L´1
µ
¨
)
centrifuged (500 g, 5 min) and the supernatant was removed, followed by the addition of MTT solution
˝
(0.5 mg
¨
mL´1 in PBS) and incub´at1ion for 4 h at 37 C. After 4 h, the MTT formazan product was
dissolved in SDS/HCl 0.01 mol
Winooski, VT, USA).
¨
L
and absorbance was measured at 570 nm in reader plate (Biotek,
4. Conclusions
In this paper we present efficient syntheses of six new guanylhydrazone and aminoguanidine
tetrahydropyran derivatives We studied the cytotoxic activity of studied tetraydropyran
compounds ( ) in various cancer and normal cell lines; in K562 cells the cytotoxicity is mediated by
2–7.
2–7
cell-cycle arrest in the G1 phase. These results can address a promising starting point for furthering
structural modifications in these compounds as a new class of anticancer compounds, leading to the
possibility of target-directed drug design for cancer treatment.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
6/671/s1.
Acknowledgments: This work has been supported by CNPq and CAPES. Chemistry Institute of Federal
University of Goiás (UFG) by CHNS analysis.
Author Contributions: Fábio Pedrosa Lins Silva was responsible for the chemical experiments and
analyzed the data; Bruna Braga Dantas and Gláucia Veríssimo Faheina Martins were responsible for
pharmacology experiments Demétrius Antônio Machado de Araújo was responsible for pharmacology
orientations; Mário Luiz Araújo de Almeida Vasconcellos was responsible for design of article, analyzed the
data, Fabio P.L Silva orientation and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
Varmus, H. The New Era in Cancer Research. Science 2006, 312, 1162. [CrossRef] [PubMed]
Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Thun, M.J. Cancer statistics, 2009. CA Cancer J. Clin. 2009, 59,
225–249. [PubMed]
3.
4.
WHO Websites. Available online: http://www.who.int/cancer/en (accessed on 23 August 2015).
Cancer Trends Progress Report. Available online: http://progressreport.cancer.gov (accessed on 22
June 2015).

Molecules 2016, 21, 671
10 of 11
5.
6.
7.
Verweij, J.; Jonge, M.J.A. Achievements and future of chemotherapy. Eur. J. Cancer 2000, 36, 1479–1487.
[CrossRef]
Carrillo, R.; Leon, L.G.; Martín, T.; Martín, V.S.; Padrón, J.M. Synthesis and antiproliferative activity of
(2R,3R)-disubstituted tethahydropyrans. Bioorg. Med. Chem. Lett. 2006, 16, 6135–6138. [CrossRef] [PubMed]
Carrillo, R.; Leon, L.G.; Martín, T.; Martín, V.S.; Padrón, J.M. Synthesis and antiproliferative activity of
(2R,3R)-disubstituted tetrahydropyrans. Part 2: Effect of side chain homologation. Bioorg. Med. Chem. Lett.
2007, 17, 780. [CrossRef] [PubMed]
8.
9.
Singh, P.; Bhardwaj, A. Mono-, Di-, and Triaryl Substituted Tetrahydropyrans as Cyclooxygenase-2 and
Tumor Growth Inhibitors. Synthesis and Biological Evaluation. J. Med. Chem. 2010, 53, 3707–3717. [PubMed]
Al-Tel, T.H. Design and synthesis of novel tetrahydro-2H-Pyrano[3,2-c]Pyridazin-3(6H)-one derivatives as
potential anticancer agents. Eur. J. Med. Chem. 2010, 45, 5724–5731. [CrossRef] [PubMed]
10. Al-Tel, T.H. Design, synthesis and qualitative structure–activity evaluations of novel hexahydropyrano
[3,2-c][1,2]diazepin-3(4H)-one and tetrahydropyrano[3,2-b]pyrrol-2(1H)-one derivatives as anticancer agents.
Eur. J. Med. Chem. 2010, 45, 4615–4621. [CrossRef] [PubMed]
11. Judd, W.R.; Slattum, P.M.; Hoang, K.C; Bhoite, L.; Valppu, L.; Alberts, G.; Brown, B.; Roth, B.;
Ostanin, K.; Huang, L. Discovery and SAR of Methylated Tetrahydropyranyl Derivatives as Inhibitors
of Isoprenylcysteine Carboxyl Methyltransferase (ICMT). J. Med. Chem. 2011, 54, 5031–5047. [CrossRef]
[PubMed]
12. Vasconcellos, M.L.A.A.; Miranda, L.S.M. A reação de ciclização de Prins: Uma estratégia eficiente para
síntese estereosseletiva de anéis tetraidropirânicos substituídos. Quím. Nova 2006, 29, 834–839. [CrossRef]
13. Ahmeda, N.; Konduru, K.; Ahmad, S.; Owais, M. Synthesis of flavonoids based novel tetrahydropyran
conjugates (Prins products) and their antiproliferative activity against human cancer cell lines. Eur. J.
Med. Chem. 2014, 75, 233–246. [CrossRef] [PubMed]
14. Miranda, L.S.M.; Marinho, B.G.; Leitão, S.G.; Matheus, M.E.; Fernandes, P.D.; Vasconcellos, M.L.A.A.
( )-cis-(6-Ethyl-tetrahydropyran-2-yl)-formic acid: A novel substance with antinociceptive properties.
˘
Bioorg. Med. Chem. Lett. 2004, 14, 1573–1575. [CrossRef] [PubMed]
15. Marinho, B.G.; Miranda, L.S.M.; Gomes, N.M.; Matheus, M.E.; Leitão, S.G.; Vasconcellos, M.L.A.A.;
Fernandes, P.D. Antinociceptive action of ( )-cis-(6-ethyl-tetrahydropyran-2-yl)-formic acid in mice.
˘
Eur. J. Pharmacol. 2006, 550, 47–53. [CrossRef] [PubMed]
16. Marinho, B.G.; Miranda, L.S.M.; Marinho, B.G.; Vasconcellos, M.L.A.A.; Matheus, M.E.; Pereira, V.L.P.;
Fernandes, P.D. Antinociceptive activity of (´)-(2S,6S)-(6-ethyl-tetrahydropyran-2-yl)-formic acid on acute
pain in mice. Behav. Pharmacol 2011, 22, 564–572. [CrossRef] [PubMed]
17. Capim, S.L.; Carneiro, P.H.P.; Castro, P.C.; Barros, M.R.M.; Marinho, B.G.; Vasconcellos, M.L.A.A. Design,
Prins-cyclization reaction promoting diastereoselective synthesis of 10 new tetrahydropyran derivatives and
in vivo antinociceptive evaluations. Eur. J. Med. Chem. 2012, 58, 1–11. [CrossRef] [PubMed]
18. Capim, S.L.; Gonçalves, G.M.; dos Santos, G.C.M.; Marinho, B.G.; Vasconcellos, M.L.A.A. High
analgesic and anti-inflammatory in vivo activities of six new hybrids NSAIAs tetrahydropyran derivatives.
Bioorg. Med. Chem. 2013, 21, 6003–6010. [CrossRef] [PubMed]
19. LaFrate, A.L.; Gunther, J.R.; Carlson, K.E.; Katzenellenbogen, J.A. Synthesis and biological evaluation of
guanylhydrazone coactivator binding inhibitors for the estrogen receptor. Bioorg. Med. Chem. 2008, 16,
10075–10084. [CrossRef] [PubMed]
20. Pignatellol, R.; Panicol, A.; Mazzonez, P.; Pinizzotto, M.R.; Garozzo, A.; Furneri, P.M. Schiff bases of
N-hydroxy-N1-aminoguanidines as antiviral, antibacterial and anticancer agents. Eur. J. Med. Chem. 1994, 29,
781–785.
21. Basua, A.; Sinha, B.N.; Saiko, P.; Graser, G.; Szekeres, T. N-Hydroxy-N1-aminoguanidine as anti-cancer lead
molecule: QSAR, synthesis and biological evaluation. Bioorg. Med. Chem. Lett. 2011, 21, 3324–3328.
22. Zhou, J.Y.; Jia, Y.; Sun, G.F.; Wu, S.H. Barbier-Type Allylation of Aldehydes and Ketones with Metallic Lead
in Aqueous Media. Synth. Commun. 1997, 27, 1899–1906. [CrossRef]
23. Silva, F.P.L.; Sabino, J.R.; Martins, F.T.; Vasconcellos, M.L.A.A. Diastereoselective syntheses via
Prins cyclization, crystal structures determination and theoretical studies of cis-2,6-diphenyl-
4-hydroxytetrahydropyran and analogues. J. Mol. Structure 2013, 1036, 478–487. [CrossRef]

Molecules 2016, 21, 671
11 of 11
24. El-Azab, A.; Al-Omar, M.A.; Abdzel-Aziz, A.A.M.; Abdzel-Aziz, N.I.; El-Sayed, M.A.A.; Aleisa, A.M.;
Sayed-Ahmed, M.M.; Abdel-Hamide, S.G. Design, synthesis and biological evaluation of novel quinazoline
derivatives as potential antitumor agents: Molecular docking study. Eur. J. Med. Chem. 2010, 45, 4188–4198.
[CrossRef] [PubMed]
25. Rowley, J.D. A New Consistent Chromosomal Abnormality in Chronic Myelogenous Leukaemia identified
by Quinacrine Fluorescence and Giemsa Staining. Nature 1973, 243, 290–293. [CrossRef] [PubMed]
26. Baccarani, M.; Cortes, J.; Pane, F.; Niederwieser, D.; Saglio, G.; Apperley, J.; Cervantes, F.; Deininger, M.;
Gratwohl, A.; Guilhot, F.; et al. Chronic Myeloid Leukemia: An Update of Concepts and Management
Recommendations of European LeukemiaNet. J. Clin. Oncol. 2009, 27, 6041–6051. [CrossRef] [PubMed]
27. Slevin, M.L. The clinical pharmacology of etoposide. Cancer 1991, 67, 319–329. [CrossRef]
28. Saleh, E.M. Inhibition of topoisomerase IIα sensitizes FaDu cells to ionizing radiation by diminishing DNA
repair. Tumour Biol. 2015, 10, 8985–8992. [CrossRef] [PubMed]
29. Torres, F.Q.J.; Díaz, J.G.; Carmona, A.J.; Estévez, F. Trifolin acetate-induced cell death in human leukemia
cells is dependent on caspase-6 and activates the MAPK pathway. Apoptosis 2008, 13, 716–728. [CrossRef]
[PubMed]
30. Roos, W.P.; Thomas, A.D.; Kaina, B. DNA damage and the balance between survival and death in cancer
biology. Nat. Rev. Cancer 2016, 16, 20–33. [CrossRef] [PubMed]
31. Szücová, L.; Lukáš, S.; Dolez, K.; Zatloukal, M.; Greplová, J.; Galuszka, P.; Kryštof, V.; Voller, J.; Popa, I.;
Massino, F.J.; et al. Synthesis, characterization and biological activity of ring-substituted 6-benzylamino-
9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine derivatives. Bioorg. Med. Chem. 2009, 17, 1938–1947.
[CrossRef] [PubMed]
32. Pedram, B.; van Oeveren, A.; Mais, D.E.; Marschke, K.B. A Tissue-Selective Nonsteroidal Progesterone
Receptor Modulator: 7,9-Difluoro-5-(3-methylcyclohex-2-enyl)-2,2,4-trimethyl-1,2-dihydrochromeno[3,4-f]
quinoline. J. Med. Chem. 2008, 51, 3696–3999. [CrossRef] [PubMed]
33. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and
cytotoxicity assays. J. Imunol. Methods 1983, 65, 55–63. [CrossRef]
Sample Availability: Samples of the compounds 2–7 are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).