Synthesis and Cytotoxicity of 1,4-Dihydropyridines and an Unexpected 1,3-Oxazin-6-one

Article abstract of DOI:10.1002/hlca.201500265

Eight heterocycles have been prepared in a one-pot reaction manner based on the Hantzsch dihydropyridine synthesis. The synthesis afforded seven dihydropyridines (DHP) and one unexpected 1,3-oxazin-6-one. Their structures were confirmed based on NMR spect

Full text of DOI:10.1002/hlca.201500265

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Helv. Chim. Acta 2016, 99, 310 – 314
FULL PAPER
Synthesis and Cytotoxicity of 1,4-Dihydropyridines and an Unexpected
1,3-Oxazin-6-one
by Louis Pergaud Sandjo*a), Victor Kueteb)c), Frederic Nanad), Gilbert Kirschd), and Thomas Efferth*c)
a) Department of Pharmaceutical Sciences, CCS – Universidade Federal de Santa Catarina, Trindade, 88040-900
ꢀ
Florianopolis, SC, Brazil (phone: +55-4837-213408; e-mail: p.l.sandjo@ufsc.br)
b) Department of Biochemistry, University of Dschang, P.O. Box 67, Dschang, Cameroon
c) Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5,
DE-55128 Mainz (phone: +49-6131-3925751; fax: +49-6131-39-23752; e-mail: efferth@uni-mainz.de)
d) UMR-SRSMC 7565, University of Lorraine, Molecular Engineering Laboratory and Formerly Pharmacological
^
Biochemistry, 1 Boulevard Arago, FR-57070 Metz Technopole
Eight heterocycles have been prepared in a one-pot reaction manner based on the Hantzsch dihydropyridine synthesis.
The synthesis afforded seven dihydropyridines (DHP) and one unexpected 1,3-oxazin-6-one. Their structures were confirmed
based on NMR spectroscopy and mass spectrometry. The obtained products have been evaluated for their cytotoxicity against
eight cancer cell lines and one normal cell line. Two halogenated DHPs (7 and 8) displayed cytotoxicity toward all the nine
tested cancer cell lines with IC50 values from 4.10 to 58.90 lM, while others showed selective activities. DHPs (7 and 8)
bearing a Me group at C(2) and C(6) as well as a halogenated substituent at C(40) were more antiproliferative than the
others.
Keywords: Halogenated dihydropyridines, 1,3-Oxazin-6-one, Hantzsch DHP synthesis, Cytotoxic activity.
products with moderate to no cytotoxicity against the
eight cancer cell lines studied.
Introduction
Cancer is a group of diseases that affects organ tissues
and is characterized by abnormal growth of cells with pos-
Results and Discussion
sibility of invading other healthy organs of the body
through metastasis [1]. By 2020, about 15 million of new
Chemistry
cancer cases will be diagnosed among the world popula-
tion and 80% of those patients may die from that disease
[2]. This dramatic increase is caused by the development
of multidrug resistance of some cancer cells leading to a and ethyl benzoylacetate), and one equivalent of
Different DHP analogs were prepared from an arylalde-
hyde, two equivalents of a b-ketoester (ethyl acetoacetate
high and continuous demand of new antiproliferative sub-
stances. Previous studies had reported 1,4-dihydropyri-
dines (DHP) as cytotoxic [3], reducer of DNA damage
and stimulator of DNA repair in human cells [4], anti-
microbial [5], antioxidant [6], hypotensive [7], and anti-
diabetic [8] agents.
AcONH4 as NH3 source. The reaction catalyzed by BiCl3
was carried out in THF under reflux and was stirred for
6 h (Scheme 1). Products (5 – 11) were obtained with
yields from 10% to 92% and their structures were con-
firmed by NMR and MS data in addition to those
reported.
1,4-Dihydropyridines, such as amlodipine, felodipine,
nitrendipine, and nifedipine (1 – 4, Fig. 1), known as cal-
cium channel blockers [9] used in cardiovascular and ang-
ina pectoris therapy, were reported as inhibitors of LDL
An unexpected 1,3-oxazin-6-one was obtained from
the similar reaction involving 3-nitrobenzaldehyde, ethyl
benzoylacetate (2 equiv.), and AcONH4 (1.5 equiv.) in
THF (Scheme 2). BiCl3 was also used as catalyst and the
oxidation and as antiproliferative agents against cholan- mixture was heated at 85° under stirring. After 6 h, the
giocarcinoma cells [10]. Therefore, the abovementioned TLC of the mixture revealed a new entity formed in small
observation aroused interest in synthesizing heterocycles, quantity. After 24 h, the product afforded, after the
including seven DHP and one unexpected 1,3-oxazin-6-
workup, was identified by NMR and MS data as the
one, and to assess their cytotoxicity. Different substituted oxazinone derivative 12. The 1,3-oxazin-6-one ring was
substrates used for the heterocycles preparation afforded
determined by comparing its C-shifts with those previously
€
© 2016 Verlag Helvetica Chimica Acta AG, Zurich
DOI: 10.1002/hlca.201500265

Helv. Chim. Acta 2016, 99, 310 – 314
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Fig. 1. Drugs with structures based on DHP scaffold.
Scheme 1. Synthesis of DHPs 5 – 11.
reported for similar heterocycles [11] and pyrimidin-6-ones lines CCRF-CEM and CEM/ADR5000, respectively.
(Fig. 2) [12]. The conditions used for this synthesis turns to Interestingly, both DHPs were more antiproliferative
be a new method for oxazinone preparation and the mech- against CEM/ADR5000 cell line than doxorubicin (refer-
anism is tentatively explained in Scheme 3. A preliminary ence drug). Halogenated DHPs bearing a Me group at
aldol condensation took place forming A which was con- C(2)/C(6) of the heterocyclic moiety (Scheme 1) turned
verted into the imine B by reacting with AcONH4. The to be more cytotoxic than those containing a phenyl
imine B undergoes a condensation with 3-nitrobenzaldehyde group on the same C-atoms. However, DHPs with phenyl
followed by a rearrangement of the H-atom through the
residues at C(2)/C(6), such as 5 and 6, showed selective
intermediates C and D to afford the 1,3-oxazin-6-one. antiproliferative activity (IC50 at 6.21 and 5.75 lM, respec-
Previously, 1,3-oxazin-6-ones analogs were prepared either tively) against CCRF-CEM when they contain a halogen
by converting N-thioacylisoxazolones with triphenylphos- atom. In general, the normal hepatocyte cell line AML12
phine [13] or by rearranging N-acyl-4-acyloxy-b-lactam was weakly sensitive to all the tested compounds. DHP
derivatives under basic conditions [14]. Moreover, isoxa- 10 displayed moderate cytotoxicity against all cancer lines
zolone and oxazoline derivatives also served as precursor except for HCT116 (p53+/+) with IC50 comprised between
for 1,3-oxazin-6-ones synthesis. Thus, after the treatment
with a 1,1-dichloro-containing compound and DBN, the
first intermediate led to expected product [15], whereas HT29 were previously reported on DHP 11 with IC50 val-
21.86 and 59.93 lM.
Weak antiproliferative effect against MDA-MB and
the same heterocycle was obtained by CO insertion into the
second precursor in the presence of a cobalt catalyst [16].
ues at 244.1 and 500 lM, respectively [20]. Nevertheless, it
was described as an inhibitor of capacitative calcium entry
The structures of 6 – 9 and 11 were previously in HL60 as its analogs nifedipine and nitrendipine [21].
reported [17 – 19], while 5, 10, and 12 are reported for Therefore, replacement of substituents in the DHP core
the first time.
by bioisosteres could be of interest in developing potent
cytotoxic compounds.
Antiproliferative Activity
Conclusion
The cytotoxicity of compounds (5 – 12) was evaluated
against nine cancer cell lines, including CCRF-CEM, CEM/ Seven 1,4-dihydropyridine derivatives and an unexpected
ADR5000, MDA-MB231, MDA-MB231/BCRP, HCT116 1,3-oxazin-6-one were prepared from an arylaldehyde,
(p53+/+), HCT116 (p53ꢀ/ꢀ), U87MG, U87MG.DEGFR, and two equivalents of a b-ketoester (ethyl acetoacetate and
HepG2 (Table S1). Compounds 7 and 8 displayed cyto-
toxicity toward all the tested cancer cell lines with the
ethyl benzoylacetate), and one equivalent of AcONH4 as
NH3 source. The same synthetic condition afforded a new
lowest IC50 values at 4.63 and 4.10 lM, respectively. These 1,3-oxazin-6-one derivative. The cytotoxicity of all
sensitivities were observed with the leukemia cancer cell
compounds was evaluated against seven cancer cell lines
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Helv. Chim. Acta 2016, 99, 310 – 314
Scheme 2. Synthesis of 1,3-oxazinone-6-one 12.
M.p.: MQAPF-301 apparatus; uncorrected. 1D- and 2D-
NMR: Bruker DRX-400 MHz instrument; d in ppm rel. to
Me4Si as internal standard, J in Hz. HR-ESI-MS: Waters
Xevo G2-S QToF mass spectrometer equipped with an
ESI probe; in m/z.
Synthesis of Products 5 – 11
Each aldehyde (400 mg) was treated with 2 equiv. of
ethyl benzoylacetate (or ethyl acetoacetate) and
1.5 equiv. of AcONH4. After adding BiCl3 (3 equiv.) and
THF (2 ml), the medium was stirred and heated at 85°
for 6 h. The halogenated products were recovered by add-
ing CH2Cl2 which caused the precipitation of BiCl3. Fur-
thermore, its separation was performed by filtration, and
the filtrate was evaporated in vacuo. The obtained residue
was recrystallized in EtOH and filtered to yield yellow
bright solid. Reactions from non-halogenated aldehydes
afforded a brown mixture, separated from BiCl3 by filtra-
tion after adding CH2Cl2. The solvent was evaporated by
rotary evaporation and purified by CC (SiO2) to give the
non-halogenated products.
Fig. 2. Comparison of oxazinone and pyrimidone C-atom shifts [11]
[12].
and one normal cell line. It turned out that the presence
of a bulky substituent, such as Ph, at C(2) and C(6) might
reduce the cytotoxicity, while the presence of a 4-halogen-
ophenyl group at C(4) improve the activity. Therefore,
modifying 7 and 8 structures could lead to more potent
cytotoxic derivatives.
Synthesis of Product 12
3-Nitrobenzaldehyde (400 mg) was mixed with 2 equiv. of
ethyl benzoylacetate, 1.5 equiv. of AcONH4, and BiCl3
(3 mol equiv.). THF (2 ml) was used as a solvent and the
mixture was heated at 85° under stirring for 24 h. Then,
CH2Cl2 was added to the mixture inducing the precipita-
tion of BiCl3 and the product. Therefore, the product was
poured onto H2O and extracted three times with CH2Cl2.
The oxazinone was obtained after evaporation of the
V. K. is grateful for an 18 months fellowship in Germany
through the ʻGeorg Foster Research Fellowship for Expe-
rienced Researcherʼ program.
Supporting Information
Additional Supporting Information may be found in the solvent.
online version of this article:
Diethyl 4-(4-Bromophenyl)-1,4-dihydro-2,6-diphenyl-
Cytotoxicity of compounds 5 – 12 toward sensitive and pyridine-3,5-dicarboxylate (5). Yield: 90% (1.044 g). Yel-
drug-resistant cancer cell lines and normal cells as deter-
mined by the resazurin assay.
low bright crystals. M.p. 204.8 – 205.1°. 1H-NMR
(400 MHz, CDCl3): 0.88 (t, J = 7.1, 2 Me); 3.88 (q,
J = 7.1, 2 CH2); 7.34 – 7.36 (m, 4 H, Ph); 7.38 – 7.40 (m,
4 H, Ph); 7.39 – 7.41 (m, 2 H, Ph); 7.43 (J = 8.75, 2 H,
4-BrC6H4); 7.45 (J = 8.75, 2 H, 4-BrC6H4); 5.18 (s,
H–C(4)); 5.99 (s, H–N(1), exchangeable). 13C-NMR
(100 MHz, CDCl3): 13.8 (2 MeCH2); 60.0 (2 MeCH2);
166.8 (2 C=O); 136.6 (2 C, Ph); 128.2 (4 C, Ph); 128.6 (4
Experimental Part
General
TLC: Silica gel GF254. Column chromatography (CC): sil-
ica gel 60H (particle size 90% < 45 mm, 200 – 300 mesh). C, Ph); 129.5 (2 C, Ph); 146.6 (C(10)); 129.9 (C(20,60));
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Helv. Chim. Acta 2016, 99, 310 – 314
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Scheme 3. Mechanism proposed for the oxazinone formation.
131.5 (C(30,50)); 120.5 (C(40)); 145.9 (C(2,6)); 104.1
Diethyl
1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)
(C(3,5)); 29.9 (C(4)). HR-ESI-MS: 532.1115 ([M + H]+, pyridine-3,5-dicarboxylate (11) [18]. Yield: 87% (0.862 g).
C29H2779BrNO+4; calc. 532.1123); 534.1100 ([M + H]+, Yellow bright crystals. M.p. 162.4 – 163.1°. HR-ESI-MS:
C29H2781BrNO+4; calc. 534.1103).
Diethyl 4-(4-Chlorophenyl)-1,4-dihydro-2,6-diphenyl-
pyridine-3,5-dicarboxylate (6) [17]. Yield: 91% (1.27 g).
375.1557 ([M + H]+, C19H23N2O+6; calc. 375.1551).
2-(3-Nitrophenyl)-5-[(3-nitrophenyl)methyl]-4-phenyl-
6H-1,3-oxazin-6-one (12). Yield: 50% (0.568 g). Brownish
Yellow bright crystals. M.p. 186.9 – 187.1°. HR-ESI-MS: solid. M.p. 247.7 – 247.9°. 1H-NMR (400 MHz, C5D5N):
488.1626 ([M + H]+, C29H2735ClNO+4; calc. 488.1629); 7.52 – 7.54 (m, 2 H, Ph); 7.56 – 7.58 (m, 2 H, Ph);
490.1601 ([M + H]+, C29H2737ClNO+4; calc. 490.1599).
Diethyl 4-(4-Chlorophenyl)-1,4-dihydro-2,6-dimethyl-
pyridine-3,5-dicarboxylate (7) [18]. Yield: 88% (0.913 g).
7.80 – 7.82 (m, 1 H, Ph); 4.30 (s, CH2 (10)); 8.31 (t,
J = 2.0, H–C(30)); 8.06 (ddd, J = 1.0, 2.4, 8.0, H–C(50));
7.39 (t, J = 8.0, H–C(60)); 7.65 (d, J = 8.0, H–C(70)); 9.31
Yellow bright crystals. M.p. 149.5 – 150.5°. HR-ESI-MS: (t, J = 2.0, H–C(200)); 8.37 (ddd, J = 1.2, 2.4, 8.2,
364.1309 ([M + H]+, C19H2335ClNO+4; calc. 364.1316); H–C(400)); 7.65 (d, J = 8.0, H–C(500)); 8.81 (dt, J = 1.2, 8.0,
366.1284 ([M + H]+, C19H2337ClNO+4; calc. 366.1286).
H–C(600)). 13C-NMR: 139.7 (C, Ph); 129.6 (2 CH, Ph);
Diethyl 4-(4-Bromophenyl)-1,4-dihydro-2,6-dimethyl- 129.3 (2 CH, Ph); 130.0 (CH, Ph); 32.7 (C(10)); 143.3
pyridine-3,5-dicarboxylate (8) [19]. Yield: 92% (0.818 g).
Yellow bright crystals. M.p. 163.4 – 164.5°. HR-ESI-MS: (C(6 )); 135.4 (C(70)); 138.2 (C(100)); 123.6 (C(200)); 149.3
(C(200)); 124.2 (C(30)); 149.1 (C(40)); 121.9 (C(50)); 130.2
408.0811 ([M + H]+, C19H2379BrNO+4; calc. 408.0810); (C(300)); 126.1 (C(400)); 130.5 (C(500)); 134.7 (C(600)); 157.6
410.0796 ([M + H]+, C19H2381BrNO+4; calc. 410.0790).
Diethyl 1,4-Dihydro-4-(4-methoxyphenyl)-2,6-diphe-
nylpyridine-3,5-dicarboxylate (9) [17]. Yield: 10%
(0.142 g). Brown gum. HR-ESI-MS: 484.2121 ([M + H]+,
C30H30NO+5; calc. 484.2118).
(C(2)); 164.9 (C(4)); 119.2 (C(5)); 168.2 (C(6)). HR-ESI-
MS: 429.0963 (M+, C23H15N3O+6; calc. 429.0961).
Cytotoxicity Assays
Diethyl 1,4-Dihydro-2,6-diphenyl-4-(3,4,5-trimethoxy- The resazurin reduction assay [22] was performed to assess
phenyl)pyridine-3,5-dicarboxylate (10). Yield: 88% the cytotoxicity of compounds 5 – 12 and doxorubicin as
(0.975 g). Yellowish solid. M.p. 189.7 – 190.1°. 1H-NMR
control drug toward various sensitive and drug-resistant
(400 MHz, CDCl3): 0.90 (t, J = 7.1, 2 Me); 3.91 (q, J = 7.1, cancer cell lines, including the CCRF-CEM and CEM/
2 CH2); 7.35 – 7.41 (m, 10 H, Ph); 6.82 (s, H–C(20,60)); 5.20 ADR5000 leukemia, MDA-MB231 breast cancer cells and
(s, H–C(4)); 3.86 (s, MeO–C(30,50)); 3.84 (s, MeO–C(40));
6.00 (s, H–N(1), exhangeable). 13C-NMR (100 MHz,
its resistant subline MDA-MB231/BCRP, HCT116p53+/+
colon cancer cells and its resistant subline HCT116p53ꢀ/ꢀ
,
CDCl3): 13.9 (2 MeCH2), 60.2 (2 MeCH2); 167.8 (2 C=O); U87MG glioblastoma cells and its resistant subline
136.9 (2 C, Ph); 128.3 (4 C, Ph); 128.6 (4 C, Ph); 129.4 (2 C,
U87MG.DEGFR and HepG2 hepatocarcinoma cells and
Ph); 143.1 (C(10)); 104.8 (C(20,60)); 153.2 (C(30,50)); 136.8 normal AML12 hepatocytes The assay is based on the
(C(40)); 145.6 (C(2,6)); 104.4 (C(3,5)); 40.3 (C(4); 56.2 reduction of the indicator dye, resazurin, to the highly fluo-
(MeO–C(30,50)); 61.0 (MeO–C(40)). HR-ESI-MS: 544.2335 rescent resorufin by viable cells. Non-viable cells rapidly
([M + H]+, C32H34NO+7; calc. 544.2330).
lose their metabolic capacity to reduce resazurin and, thus,
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ꢀ
ꢀ
ꢀ
[5] P. Olejnıkova, L. Svorc, D. Olsovska, A. Panakova, Z.
Vihonska, K. Kovaryova, S. Marchalın, Sci. Pharm. 2014, 82,
ꢁ
ꢀ
ꢀ
ꢀ
do not produce fluorescent signals anymore. Briefly, adher-
ent cells were detached by treatment with 0.25% trypsin/
EDTA (Invitrogen, Darmstadt, Germany) and an aliquot
of 1 9 104 cells was placed in each well of a 96-well cell cul-
ture plate (Thermo Scientific, Langenselbold, Germany) in
a total volume of 200 ll. Cells were allowed to attach over-
night and then were treated with different concentrations
of compounds. For suspension cells, aliquots of 2 9 104
cells per well were seeded in 96-well-plates in a total vol-
ume of 100 ll. The studied compound was immediately
added in varying concentrations in an additional 100 ll of
culture medium to obtain a total volume of 200 ll/well.
After 72 h, resazurin (Sigma-Aldrich, Schnelldorf, Ger-
many) (20 ll, 0.01% w/v) in distilled H2O was added to
each well and the plates were incubated at 37° for 4 h. Flu-
orescence was measured on an Infinite M2000 ProTM plate
reader (Tecan, Crailsheim, Germany) using an excitation
wavelength of 544 nm and an emission wavelength of
590 nm. Each assay was done at least twice with six repli-
cates each. The viability was evaluated based on a compar-
ison with untreated cells. IC50 values represent the
compound concentrations required to inhibit 50% of cell
proliferation and were calculated from a calibration curve
by linear regression using Microsoft Excel.
ꢀ
ꢀ
221.
[6] L. Cominacini, A. Fratta Pasini, U. Garbin, A. M. Pastorino, A.
Davoli, C. Nava, M. Campagnola, P. Rossato, V. Lo Cascio,
Biochem. Biophys. Res. Commun. 2003, 302, 679.
[7] A. Ashimori, T. Ono, T. Uchida, Y. Ohtaki, C. Fukaya,
M. Watanabe, K. Yokoyama, Chem. Pharm. Bull. 1990, 38,
2446.
[8] J. Briede, M. Stivrina, B. Vigante, D. Stoldere, G. Duburs, Cell
Biochem. Funct. 2008, 26, 238.
[9] R. A. Coburn, M. Wierzba, M. J. Suto, A. J. Solo, A. M. Trig-
gle, D. J. Triggle, J. Med. Chem. 1988, 31, 2103.
[10] M. Syamala, Org. Prep. Proced. Int. 2009, 41, 1.
[11] J. U. Jeong, X. Chen, A. Rahman, D. S. Yamashita, J. I.
Luengo, Org. Lett. 2004, 6, 1013.
[12] Y. S. Chun, J. H. Kim, S. Y. Choi, Y. O. Ko, S. G. Lee, Org.
Lett. 2012, 14, 6358.
[13] D. S. Millan, R. H. Prager, J. Chem. Soc., Perkin Trans. 1998,
1, 3245.
ꢀ
ꢀ
[14] M. Alajarın, A. Vidal, P. Sanchez-Andrada, F. Tovar, G.
Ochoa, Org. Lett. 2000, 2, 965.
[15] E. M. Beccalli, T. Benincori, A. Marchesini, Synthesis 1988, 8,
630.
[16] H. Xu, L. Jia, Org. Lett. 2003, 5, 1575.
[17] B.-J. Zhao, X.-Q. Zhu, J.-Q. He, C.-Z. Xia, J.-P. Cheng, Chem.
J. Chin. Univ. 1999, 20, 1733.
[18] H. Niaz, H. Kashtoh, J. A. J. Khan, A. Khan, A.-T. Wahab, M.
T. Alam, K. M. Khan, S. Perveen, M. I. Choudhary, Eur. J.
Med. Chem. 2015, 95, 199.
REFERENCES
[19] C. P. Sharma, M. Gupta, Green Chem. 2015, 17, 1100.
[20] S. N. Amgoth, M. Porika, S. Abbagani, A. Garlapati, M. R.
Vanga, Med. Chem. Res. 2013, 22, 147.
[1] T. Reya, S. J. Morrison, M. F. Clarke, I. L. Weissman, Nature
2001, 414, 105.
[21] J. L. Harper, C. S. Camerini-Otero, A.-H. Li, S.-A. Kim, K. A.
Jacobson, J. W. Daly, Biochem. Pharmacol. 2003, 65, 329.
[22] J. O’Brien, I. Wilson, T. Orton, F. Pognan, Eur. J. Biochem.
2000, 267, 5421.
[2] P. Anand, A. B. Kunnumakara, C. Sundaram, K. B. Harikumar,
S. T. Tharakan, O. S. Lai, B. Sung, B. B. Aggarwal, Pharm.
Res. 2008, 25, 2097.
[3] R. Miri, K. Javidnia, Z. Amirghofran, S. H. Salimi, Z. Sabetghadam,
S. Meili, A. R. Mehdipour, Iran. J. Pharm. Res. 2011, 10, 497.
[4] N. I. Ryabokon, R. I. Goncharova, G. Duburs, J. Rzeszowska-
Wolny, Mutat. Res. 2005, 587, 52.
Received August 31, 2015
Accepted January 15, 2016
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