β-Nitro substituted carboxylic acids and their cytotoxicity

Article abstract of DOI:10.1016/j.bmcl.2014.06.021

β-Nitro-substituted ethyl carboxylates are a new class of cytotoxic agents; they can be easily obtained in fair to good yields in a single-step reaction by a Pd-catalyzed asymmetric conjugate addition of aryl boronic acids to 2-nitro-acrylates. Of all the tested derivatives, 2-(4-chlorophenyl)-3- nitropropionic acid ethyl ester (6) is most cytotoxic especially against the human ovarian cancer cell line A2780 therefore making this compound an interesting candidate for further investigations.

Full text of DOI:10.1016/j.bmcl.2014.06.021

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4011–4013
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
b-Nitro substituted carboxylic acids and their cytotoxicity
a
a
b
b
b
René Csuk a, , Lucie Heller , Bianka Siewert , Andrey Gutnov , Oliver Seidelmann , Volkmar Wendisch
⇑
a Martin-Luther Universität Halle-Wittenberg, Bereich Organische Chemie, Kurt-Mothes-Str. 2, D-06120 Halle (Saale), Germany
b Chiroblock GmbH, Andresenstr. 1a, D-06766 Bitterfeld-Wolfen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
b-Nitro-substituted ethyl carboxylates are a new class of cytotoxic agents; they can be easily obtained in
fair to good yields in a single-step reaction by a Pd-catalyzed asymmetric conjugate addition of aryl boro-
nic acids to 2-nitro-acrylates. Of all the tested derivatives, 2-(4-chlorophenyl)-3-nitropropionic acid ethyl
ester (6) is most cytotoxic especially against the human ovarian cancer cell line A2780 therefore making
this compound an interesting candidate for further investigations.
Received 29 April 2014
Revised 5 June 2014
Accepted 8 June 2014
Available online 21 June 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
b-Nitro carboxylic acids
Cytotoxicity
SRB assay
Cancer
The first synthesis of 3-nitro-propanoic acid (3-NP, Fig. 1) from
3-iodopropionic acid was described 135 years ago by
Lewkowitsch,1 and in 1920 3-NP has been found2 as a ‘natural’
product in the glycoside hiptagin. But only 40 years ago, 3-NP
has been identified3 as the toxic substance of many plants. Since
then, this aliphatic nitro compound has been detected in
several—more or less toxic—different plants, and it was shown to
be toxic4 to animals causing acute as well as chronic syndromes.
3-NP crosses the blood–brain barrier; it is used as a synthetic tool
to study the mechanisms involved in Huntington’s disease5,6 for its
ability to mimic the hyperkinetic as well as the hypokinetic symp-
toms of this inheritable autosomal-dominant disorder. The causal
mechanisms of Huntington’s disease remain still unknown.
3-NP is a suicidal inhibitor7,8 of the enzyme succinate dehydro-
genase, and first studies performed in the 90s of the last century
showed9 this compound to inhibit the cell proliferation of cultured
murine embryonal carcinoma cells.
Figure 1. Structure of 3-nitro-propanoic acid (3-NP).
to various Michael acceptors are effective tools for the synthesis
of biologically relevant molecules. More recently, Pd-catalyzed
asymmetric conjugate additions of aryl boronic acids to
2-nitroacrylates in the presence of chiral catalysts enabled a quick
access13–15 to 3-nitro-2-arylpropanoates. Here, we apply a similar
strategy (Scheme 1) for the synthesis of compounds 1–15 using
the DIPHOS ligand, and the products were obtained in 25–90%
isolated yield.
Although first studies on the cytotoxicity of 3-NP have been
performed almost 20 years ago (vide supra), no systematic studies
have been made using
a-substituted b-nitro carboxylic acids or
esters. Cytotoxicity was determined as IC50 values using photomet-
ric sulforhodamin B assays (SRB)16,17 employing different human
tumor cell lines as well as non-malignant mouse fibroblasts (NiH
3T3). Our own preliminary studies showed a higher cytotoxicity
for compounds bearing an aromatic substituent in a-position than
for alkyl-substituted analogs. Therefore, we focused our investiga-
b-Nitro carboxylic acids are valuable precursors for the synthe-
sis of b-amino acids. The latter compounds have been used10,11 as
nonnatural chiral building blocks; they have been obtained in
excellent yields from the corresponding nitro compounds by
reduction. b-Nitro carboxylic acids bearing an extra substituent
in
a-position adjacent to the carboxyl moiety can be accessed in
tion on these aromatic compounds.
high yields (as well as in a stereoselective manner) and most con-
veniently from Michael addition reactions12 using nitro-acrylates
and organometallic compounds in the presence of a metal catalyst.
These Pd-catalyzed conjugate additions of aryl metal compounds
Whereas no significant activity (IC50 >30 lM as a cut-off) was
found for unsubstituted 1 (Fig. 2, Table 1) as well as for p-methyl
substituted 2, the trifluoromethyl derivative 3 showed significant
cytotoxic activity against six tumor cell lines. Interestingly enough,
analog 3 displayed good cytotoxic activity for the tumor cell lines
SW1736 (human thyroid carcinoma), MCF-7 (human breast adeno-
carcinoma) and A549 (human adenocarcinomic alveolar basal
⇑
Corresponding author. Tel.: +49 345 5525660; fax: +49 345 5527030.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
http://dx.doi.org/10.1016/j.bmcl.2014.06.021
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

4012
R. Csuk et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4011–4013
Scheme 1. General scheme for the synthesis of the alkyl b-nitro propanoates.
O
O
N
R'
O2N
R
H2N
OEt
R1
R2
R3
Cl
Cl
17
1-15
16
No
R
R1
R2
R3
H
R’
H
Yield [%]
56
1
2
OEt
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
OEt
H
CH3
CF3
Cl
F
H
42
3
OEt
H
H
25
4
OEt
H
CH3
H
32
5
OEt
H
27
6
OEt
H
Cl
OMe
H
H
26
7
OEt
H
H
33
8
OEt
Cl
H
H
27
9
OEt
H
H
26
10
11
12
13
14
15
NH2
NMe2
OEt
H
Cl
Cl
H
H
88
H
H
90
CF3
OCF3
H
H
31
OEt
H
H
30
OMe
CH2-Ph
Cl
Cl
H
29
H
H
44
Figure 2. Structures and yields of compounds 1–17.
epithelial cells). However, the selectivity in distinguishing between
tumor cells and non-malignant NiH 3T3 cells (expressed as the
quotient IC50 non-malignant/IC50 malignant), remained low for this
Almost no cytotoxicity was found for p-OMe substituted 7.
Shifting of the chlorine substituent from the para position (as in
6) into meta (as in 8) or ortho position (as in 9) also lowered the
cytotoxicity of the compounds. The same phenomenon was noted
for the carboxamide 10 or the dimethyl-carbamate 11.
compound. The presence of an
a-hydrogen substituent seems
important, since a decreased toxicity was found for methyl substi-
tuted compound 4.
Interestingly enough, whereas
a shifting of the chloro-
Cytotoxicity decreased for the 4-fluoro-substituted compound 5;
this is an interesting finding because the 4-chloro-analog 618 showed
moderate (against cell lines A549, DLD-1 (human colorectal adeno-
carcinoma cells), SW1736) to excellent (against cell line A2780,
substituent from the para into the meta position lowered cytotox-
icity, no such effect was observed for the meta trifluoromethyl
substituted compound 12 compared to its para substituted analog
3. Cytotoxicity decreased, however, for the meta trifluoromethoxy
substituted compound 13.
human epithelial ovarian cancer, IC50 = 1.61 lM) cytotoxicity.

R. Csuk et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4011–4013
4013
Table 1
Cytotoxicity of 1–17 (IC50 values in lM from SRB assays; independent experiments were at least performed in triplicate; standard errors are given) employing human tumor cell
lines (518A2, 8505C, A253, A2780, A549, DLD-1, Lipo, MCF7 and SW1736) and non-malignant mouse fibroblasts NiH 3T3
518A2
8505
A253
A549
A2780
DLD-1
Lipo
MCF-7
SW1736
NiH 3T3
1
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
2
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
3
4
15.2 0.3
>30
13.8 0.4
>30
9.8 0.2
>30
9.9 0.3
>30
6.6 0.2
>30
11.0 0.3
>30
15.2 0.3
>30
6.7 0.2
>30
6.3 1.4
>30
9.7 0.4
>30
5
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
6
7
17.3 0.2
>30
>30
>30
>30
>30
14.9 0.7
>30
1.6 0.1
>30
10.9 0.3
>30
19.2 0.2
>30
22.8 0.4
>30
>30
>30
7.7 0.2
>30
8
9
7.3 0.2
>30
>30
14.9 0.7
>30
>30
10.6 0.9
>30
>30
3.5 0.1
>30
>30
14.4 0.7
9.9 0.6
>30
14.5 0.4
>30
>30
>30
8.2 0.2
>30
5.2 0.2
22.5 0.3
>30
19.5 0.3
>30
>30
>30
10.9 0.3
>30
8.7 0.3
28.9 0.6
>30
17.9 0.6
>30
>30
>30
7.6 0.2
>30
3.4 0.1
18.2 0.8
>30
8.3 0.1
>30
>30
21.1 0.9
>30
>30
>30
10.2 0.3
>30
2.1 0.1
9.2 0.4
25.7 0.7
23.5 0.3
10
11
12
13
14
15
16
17
>30
>30
>30
>30
>30
>30
8.6 0.1
14.5 0.2
5.8 0.1
23.9 0.5
>30
20.7 0.3
22.1 0.2
11.6 0.5
>30
>30
>30
7.2 0.1
11.8 0.2
5.4 0.2
21.4 0.4
>30
13.6 0.2
19.9 0.1
10.5 0.7
>30
>30
>30
6.3 0.1
2.2 0.1
2.5 0.1
12.1 0.2
22.3 0.4
15.4 0.3
4.7 0.2
16.6 0.2
5.8 0.3
23.9 0.7
>30
>30
>30
>30
>30
14.2 0.2
>30
To evaluate the influence of the ester moiety, two analogs of the
most active compound 6 of this investigation were prepared, and
the cytotoxicity of the methyl ester 14 and of the benzyl derivative
15 were determined. This change led to some changes of the
cytotoxicity of the compounds: Whereas IC50 values improved for
the methyl ester 14, cytotoxicity decreased for 15. Reduction of
the nitro group in compound 6 gave the b-amino acid 16. This com-
pound exhibited almost no cytotoxicity in the SRB assays; weak
activity was determined for the mouse fibroblasts as well as for
human epithelial ovarian cancer cell line A2780. The presence of
References and notes
1. Leukowitsch, J. J. Prakt. Chem. 1879, 2, 159.
2. Gorter, K. Bull. Jard. bot. Buitenzorg 1920, 3, 187 [CAN: 1921, 15, 7141].
3. Majak, W.; Pass, M. A. Aliphatic Nitrotoxins In Toxicans of Plant Origin,
Glycosides; Cheeke, P. R., Ed.; CRC Press: Boca Raton, 1989; Vol. 2, pp 143–159.
4. Liu, X.; Luo, X.; Hu, W. Biomed. Environm. Sci. 1992, 5, 161.
5. Ramaswamy, S.; McBrid, J. L.; Kordower, J. H. ILAR J. 2007, 48, 356.
6. Tunez, I.; Tasset, I.; Perez-de la Cruz, V.; Santamaria, A. Molecules 2010, 15, 878.
7. Alston, T. A.; Mela, L.; Bright, H. J. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 3767.
8. Gould, D. H.; Wilson, M. P.; Hamar, D. W. Toxicol. Lett. 1985, 27, 83.
9. Pass, M. A.; Carlisle, C. H.; Reuhl, K. R. Nat. Toxins 1994, 2, 386.
10. Juaristi, E.; Soloshonok, V. Enantioselective Synthesis of b-Amino Acids, 2nd ed.;
Wiley-Interscience: N.Y., 2005.
a
b-nitro ester moiety seems to be essential for providing
11. Seebach, D.; Beck, A. K.; Capone, S.; Deniau, G.; Grosel, U.; Zass, E. Synthesis
2009, 1.
12. Kastl, R.; Wennemers, H. Angew. Chem. 2013, 52, 7228.
high cytotoxicity since almost no activity was found for the
2-(4-chlorophenyl)acrylonitrile (17).
Additional biological screening experiments were performed
using most active and previously unknown compound 6 using
the human melanoma cell line 518A2. Results from an annexin
V/propidium iodide staining experiment as well as the close
inspection of the cells by fluorescence microscopy revealed an
increased number of cells died by apoptosis as well as by second-
ary necrosis.
These results show b-nitro-substituted ethyl carboxylates as a
new class of cytotoxic agents; they can be easily obtained in fair
to good yields in a single-step reaction. Of all the tested derivatives,
novel compound 6 was cytotoxic especially against the human
ovarian cancer cell line A2780 therefore making this compound
an interesting candidate for further investigations.
13. Petri, A.; Seidelmann, O.; Eilitz, U.; Leßmann, F.; Reißman, S.; Wendisch, V.;
Gutnov, A. Tetrahedron Lett. 2014, 55, 267.
14. Gutnov, A. Eur. J. Org. Chem. 2008, 4547.
15. Miyaura, N. Synlett 2009, 2039.
16. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
17. Cell lines and culture conditions: The cell lines and their origin are specified in
the Supplementary material. SRB-Assays were prepared according to Ref. 16.
The cell lines were amplified with a starting cell number between 1000 and
2500 and counted with an automatic cell counter (Invitrogene™ countessÒ).
EC50 values were calculated from semi logarithmic dose response curves by
non-linear regression applying a two-parametrical Hill-slope equation.
18. Representative synthetic procedure for the synthesis of 2-(4-chlorophenyl)-3-
nitropropionic acid ethyl ester (6): DIPHOS (0.3 g, 0.75 mmol) and Pd(OAc)2
(0.17 g, 0.75 mmol) were suspended in acetone (60 mL). After 15 min stirring
at 20 °C, ethyl 2-nitroacrylate (2.18 g, 15 mmol), p-chlorophenyl-boronic acid
(3.52 g, 22.5 mmol), AgBF4 (0.29 g, 1.5 mmol), deionized H2O (30 mL), and
HBF4 (3 mL, 24 mmol, 50% in H2O) were added, and the turbid reaction was
stirred at 20 °C for 18 h. The reaction mixture was partitioned between ethyl
acetate (50 mL) and water (100 mL), the organic layer separated, dried
(Na2SO4), and the solvents were evaporated at diminished pressure. The
crude oily residue was purified by chromatography (silica gel, hexane/ethyl
acetate) to yield analytically pure 6 (1.06 g, 27% yield) as slightly yellowish
oil.1H NMR (500 MHz, CDCl3): d = 7.35 and 7.21 (each m, 2 H), 7.09 and 7.14
(AA0BB0, J = 2.36, 7.99, 0.39, 0.39, 7.99 Hz, 2H), 5.10 and 4.56 (AB of ABX, J = 5.7,
9.5, 14.2 Hz, CH2-NO2), 4.42 (X of ABX, J = 5.7, 9.5 Hz, CH), 4.15 and 4.22 (qxAB,
J = 7.3, 10.4 Hz, CH2 of Et), 1.22 (t, J = 7.3 Hz, CH3 of Et) ppm; 13C NMR (100 Hz,
CDCl3): d = 13.93 (CH3 of Et), 48.17 (CH), 62.09 (CH2 of Et), 75.58 (CH2-NO2),
129.25 (2 ꢀ CHarom.), 129.55 (2 ꢀ CHarom.), 131.87 (Ci, Cl), 134.78 (Ci), 170.12
(CO); ESI-MS (MeOH): m/z = 258.
Acknowledgments
This research was supported by the German Ministry of
Economy and Technology (BMBF). The cell lines were kindly pro-
vided by Dr. Thomas Müller (Dept. of Haematology/Oncology,
Martin-Luther Universität Halle-Wittenberg). Support by the
‘Gründerwerkstatt–Biowissenschaften’ is gratefully acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.06.
021.