R. Quesada et al.
Varian Mercury 300 MHz and Varian Unity Inova 400 MHz spectrome-
ters. Chemical shifts (d) are reported in ppm, referenced to the residual
solvent peak; coupling constants (J) are reported in Hz. HRMS were re-
corded on a Micromass Autospec S-2 spectrometer by using the electro-
spray ionization technique at 70 eV. Microwave reactions were performed
in a Biotage Initiator 2.0 microwave. Compound 5 was prepared as de-
scribed.[10]
Compound 6: Compound 5 (0.15 g, 0.44 mmol) was suspended in water
(18 mL). The mixture was heated at 1708C for 35 min under MW irradia-
tion. Once cold, the solid was filtered off and washed with water to
afford
6
(0.096 g, 91%) as a greenish powder. 1H NMR (300 MHz,
[D6]DMSO): d=11.86 (s, 1H; NH), 11.46 (s, 1H; NH), 9.42 (s, 1H;
CHO), 7.53 (d, J=6.9 Hz, 1H; ArH), 7.39 (d, J=6.9 Hz, 1H; ArH), 7.13
(t, J=6.9 Hz, 1H), 7.11 (s, 1H), 7.01 (t, J=6.9 Hz, 1H; ArH), 6.55 (s,
1H), 3.88 ppm (s, 3H; OCH3); 13C NMR (75 MHz, [D6]DMSO): d=
173.12, 158.14, 136.80, 131.84, 129.47, 128.12 (CH), 122.44 (CH), 120.44
(CH), 119.79 (CH), 118.63 (CH), 111.31 (CH), 100.69, 93.28, 57.97 ppm
(CH3).
Figure 8. Dose–response curves for compounds 1–4 on the viability of
GLC4 cell line. The results are the meanÆSEM for three independent
experiments and are expressed as % viability compared with control.
~
~
&
*
Error bars represent the standard deviation. ( 1a, 1b, 1c, &2, 3,
Compound 1a·HCl: Under an inert atmosphere, 6 (0.1 g, 0.42 mmol) was
dissolved in methanol (5 mL), then 2,4-dimethylpyrrole (86 mL,
0.83 mmol) was added, followed by dropwise addition of a solution of
HCl in methanol (1.25m, 0.66 mL). The color changed to dark purple and
the mixture was stirred overnight. The solid formed was filtered off and
washed with cold methanol (1 mL) to afford 1a·HCl (0.137 g, 93%) as a
dark-purple crystalline solid. 1H NMR (300 MHz, CDCl3): d=12.89 (s,
1H; NH), 12.82 (s, 1H; NH), 12.26 (s, 1H; NH), 7.57 (d, J=7.2 Hz, 1H;
ArH), 7.53 (d, J=7.2 Hz, 1H; ArH), 7.29 (t, J=7.2 Hz, 1H; ArH), 7.08
(t, J=7.2 Hz, 1H; ArH), 7.07 (s, 1H), 6.98 (s, 1H), 6.24 (s, 1H), 6.05 (s,
1H), 3.97 (s, 3H; OCH3), 2.60 (s, 3H; CH3), 2.28 ppm (s, 3H; CH3);
13C NMR (75 MHz, CDCl3): d=164.89, 150.39, 146.21, 142.50, 138.80,
127.81, 127.03, 125.54, 125.41 (CH), 121.31 (CH), 120.69 (CH), 119.72,
115.67 (CH), 114.67 (CH), 112.50 (CH), 108.77 (CH), 94.01 (CH), 58.64
(OCH3), 14.10 (CH3), 11.96 ppm (CH3); HRMS (EI): m/z calcd for
C19H20N3O: 317.15; found: 317.15.
*
4).
in the anion-transport assays. Compound 4 was found to be
inactive in these assays and displayed no cytotoxicity. The
compounds studied showed no marked toxicity on nonma-
lignant HaCaT cells at IC50 concentrations.
Conclusion
In this study we have investigated the anion-binding proper-
ties of obatoclax (1a) by using DFT calculations, as well as
solution and structural studies. This compound is well suited
to interact with anions through hydrogen bonds and prefer-
entially adopts a b conformation. Compound 1a and related
analogues 1b and 1c are highly active anionophores capable
of promoting chloride and bicarbonate (and nitrate) trans-
port in model phospholipid liposomes. Manipulation of
these compounds by variation of the number of hydrogen-
bond donors resulted in diminished anion-carrier ability.
The anionophoric activity correlated well with the results
observed in the in vitro studies. Active anion carriers are
able to discharge pH gradients in living cells and the most
active anionophores were found to be the most potent cyto-
toxic derivatives. Inactive, yet structurally very related mole-
cules (such as compound 4), did not show cytotoxicity
against the GLC4 cell line. These findings led us to suggest
that anion transport plays a relevant role in the mechanism
of action of these compounds. Thus, development of new de-
rivatives with improved anion-transport abilities could lead
to new drugs with improved pharmacological properties. In
this regard, the straightforward liposome assays could be
useful for the screening of new candidates. Efforts in these
directions are currently underway in our laboratories.
Compound 1a·MeSO3H: Compound 1a·MeSO3H was obtained as de-
scribed for 1a·HCl by using methanesulfonic acid as a catalyst to afford
1a·MeSO3H (0.142 g, 82%) as a dark-purple crystalline solid. 1H NMR
(300 MHz, CDCl3): d=12.23 (s, 1H; NH), 11.98 (s, 1H; NH), 11.43 (s,
1H; NH), 7.69 (d, J=8.3 Hz, 1H; ArH), 7.52 (d, J=8.0 Hz, 1H; ArH),
7.26 (t, J=5.7 Hz, 1H; ArH), 7.13 (s, 1H), 7.05 (t, J=7.2 Hz, 1H; ArH),
7.01 (s, 1H), 6.31 (d, J=1.9 Hz, 1H), 6.08 (s, 1H), 4.01 (s, 3H; OCH3),
2.97 (s, 3H; SCH3), 2.62 (s, 3H; CH3), 2.29 ppm (s, 3H, CH3).
Compound 1a: Compound 1a·HCl (0.040 g, 0.11 mmol) was dissolved in
CH2Cl2 (20 mL). The solution was treated with a 1% aqueous solution of
NaOH (30 mL) and the color changed from deep red to orange. The or-
ganic phase was dried over anhydrous Na2SO4 and the solvent was re-
moved under reduced pressure. Compound 1a (0.035 g, 98%) was ob-
tained as an orange solid. 1H NMR (300 MHz, CDCl3): d=12.05 (s, 1H;
NH), 7.49 (d, J=8.1 Hz, 1H; ArH), 7.11 (s, 1H), 7.02 (t, J=8.1 Hz, 1H;
ArH), 6.93 (t, J=8.1 Hz, 1H; ArH), 6.90 (s, 1H), 6.77 (d, J=8.1 Hz, 1H;
ArH), 6.34 (s, 1H), 5.67 (s, 1H), 4.08 (s, 3H; OCH3), 2.17 (s, 3H; CH3),
1.78 ppm (s, 3H; CH3); 13C NMR (75 MHz, CDCl3): d=168.87, 158.55,
140.97, 137.75, 136.30, 133.98, 133.82, 128.43, 126.71, 123.31 (CH), 120.76
(CH), 119.52 (CH), 115.67 (CH), 112.61 (CH), 111.49 (CH), 104.68 (CH),
96.01 (CH), 58.57 (OCH3), 12.15 (CH3), 11.44 ppm (CH3); HRMS (EI):
m/z calcd for C19H20N3O: 317.1528; found: 317.1539.
Compound 1b·HCl: Compound 1b·HCl was prepared as described for
1a·HCl, from 6 (0.1 g, 0.42 mmol), 2,4-dimethyl-3-ethylpyrrole (112 mL,
0.83 mmol), and a solution of HCl in methanol (1.25m, 0.66 mL). Com-
pound 1b·HCl (0.130 g, 82%) was obtained as a dark-purple solid.
1H NMR (300 MHz, CDCl3): d=12.93 (s, 1H; NH), 12.83 (s, 1H; NH),
12.26 (s, 1H; NH), 7.58 (d, J=7.5 Hz, 1H; ArH), 7.56 (d, J=7.5 Hz, 1H;
ArH), 7.28 (t, J=7.5 Hz, 1H; ArH), 7.09 (t, J=7.5 Hz, 1H; ArH), 7.08
(s, 1H), 7.06 (s, 1H), 6.28 (s, 1H), 4.00 (s, 3H; OCH3), 2.60 (s, 3H; CH3),
2.41 (q, J=7.8 Hz, 2H; CH2) 2.23 (s, 3H; CH3), 1.08 ppm (t, J=7.8 Hz,
3H; CH3); 13C NMR (75 MHz, CDCl3): d=164.39, 150.27, 145.18, 139.43,
138.74, 129.44, 127.86, 127.35, 125.29, 125.20 (CH), 121.19 (CH), 120.66
(CH), 119.29, 114.39 (CH), 112.60 (CH), 108.05 (CH), 93.88 (CH), 58.59
Experimental Section
General procedures and methods: Commercial reagents were used as re-
ceived without any further purification. NMR spectra were recorded on
14080
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 14074 – 14083