In vitro cytotoxic evaluation of a novel phosphinyl derivative of boldine

Article abstract of DOI:10.3390/molecules16032253

2,9-Dimethoxymethylboldine (2), 2,9-dimethoxymethyl-3-bromoboldine (3) and 2,9-dimethoxymethyl-3-diphenylphosphinylboldine (4) have been synthesized in an effort to find compounds with potential pharmacological applications. The cytotoxic activities of th

Full text of DOI:10.3390/molecules16032253

Molecules 2011, 16, 2253-2258; doi:10.3390/molecules16032253
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Communication
In Vitro Cytotoxic Evaluation of a Novel Phosphinyl Derivative
of Boldine
Franz A. Thomet 1, Pablo Piñol 2, Joan Villena 2 and Patricio G. Reveco 1,*
1
Department of Chemistry, Universidad Técnica Federico Santa María, Avenida España N° 1680,
Valparaíso, Chile; E-Mail: franz.thomet@usm.cl (F.A.T.)
2
Centro Regional de Estudios en Alimentos Saludables (CREAS), Department of Biomedical
Science, School of Medicine, Universidad de Valparaíso, Avenida Hontaneda N° 2664, Valparaíso,
Chile; E-Mails: pignol@yahoo.com (P.P.); juan.villena@uv.cl (J.V.)
* Author to whom correspondence should be addressed; E-Mail: patricio.reveco@usm.cl;
Tel.: +56-32-2654236; Fax: +56-32-2654782.
Received: 10 January 2011; in revised form: 23 February 2011 / Accepted: 4 March 2011 /
Published: 7 March 2011
Abstract: 2,9-Dimethoxymethylboldine (2), 2,9-dimethoxymethyl-3-bromoboldine (3) and
2,9-dimethoxymethyl-3-diphenylphosphinylboldine (4) have been synthesized in an effort
to find compounds with potential pharmacological applications. The cytotoxic activities of
the natural precursor 1 and these three derivatives have been measured as IC50 inhibitory
growth. The diphenylphosphinyl derivative 4 showed a significant cytotoxic activity on
two breast cancer cell lines, namely MCF-7 and MDA-MB-231, with IC50 values of 55.5
and 62.7 [µM], respectively. These results suggest that the kind of structural modifications
introduced to synthesize compound 4 represent a promising way to enhance the cytotoxic
activity of boldine derivatives.
Keywords: synthesis; phosphinyl derivative; boldine; cytotoxic activity
1. Introduction
Boldine (1), the major alkaloidal constituent of the Chilean boldo tree (Peumus boldus Molina,
Monimiaceae) has been extensively studied due to its anti-inflammatory, antipyretic and antioxidant
activity (in both in vitro as well as in vivo models) [1], and derivatives of boldine with significant

Molecules 2011, 16
2254
biological activity have already been reported in the literature [2]. A synthetic route that uses lithiation
to introduce a different functional group on the aporphinic skeleton has been previously published by
our research group [3].
The search for a potential biological application of the cytotoxicity of boldine has already yielded
encouraging results. A methanolic leaf extract of Peumus boldus exhibiting cytotoxic activity on two
types of human cancer cells [4], the demonstration of an intercalating agent behavior of boldine [5] and
its antiproliferant activity against a glioma cell line [6] are examples of promising findings that
motivate further research in this area.
In the search of coordination compounds involving the pharmacologically active metals Pt(II),
Ru(II) and Au(I) and boldine, various groups were attached to the natural product to make possible the
coordination. The present work focuses on the preparation of a new diphenylphosphinyl derivative 4 of
boldine and the assessment of its in vitro inhibitory effect on the growth of two breast cancer lines
(MDA-MB-231, MCF-7) and one dermal human fibroblast cell line (DHF). These inhibitory effects
have been compared to those obtained for compounds 1–3.
2. Results and Discussion
2.1. Chemistry
The new diphenylphosphinyl derivative 4 was prepared in good yield employing compound 3 as a
precursor for the lithium-bromine exchange reaction (Scheme 1).
Scheme 1. Boldine derivatives synthesized.
O
R2
P
3
4
R1O
O
O
2
3a
1b
5
1
1) n-BuLi / THF / -78°C
2) PPh2Cl
N
H
N
6a
H3CO
CH3
H3CO
H
CH3
1a
H
11a
7
H
7a
11
8
10
H
H3CO
H
O
H3CO
9
OR3
O
(1) R1 = R2 = R3 = H
(2) R1 = R3 = CH2OCH3 ; R2 = H
(3) R1 = R3 = CH2OCH3 ; R2 = Br
(4)
1
The appearance of ten additional aromatic protons in the H-NMR spectrum associated to the
diphenylphosphinyl group, and the strong upfield shift of the dioxymethylene protons on position C-2
from 5.24 and 5.33 ppm (J = 5.7 Hz) in 3 to 3.85 and 4.78 ppm (J = 4.9 Hz) in 4 indicated the
substitution on the C-3 position. 13C-NMR (including dept-135, together with 1D-TOCSY and 2D

Molecules 2011, 16
2255
31
HSQC/HMBC experiments) and P-NMR confirmed the structure proposed for 4. Oxidation of the
diphenylphosphine to the diphenylphosphinyl function was attributed to the chromatographic elution
conditions (CHCl3/MeOH = 99/1), given that in the halo-dehydroxylation procedures PPh3 is
employed [7]. This conclusion is supported by the MS parent ion at m/z 616.2481.
2.2. Cytotoxic Activity
To quantify the cytotoxic effect of boldine (1) and the synthetic derivatives 2–4, the IC50 value of
each compound was measured (the IC50 value is defined as the µM concentration of the compound that
achieves a 50% reduction in cellular growth after 72 hours of drug exposure). Table 1 shows the IC50
values for compounds 1 through 4 for two types of breast cancer cell (MDA-MB-231 and MCF-7) and
the DHF fibroblast cell line. The results clearly indicate that only the diphenylphosphinyl derivative 4
exhibits a significant cytotoxic activity, and that such activity emerges in the breast
cancer cells.
Table 1. Cytotoxic activity (IC50) of boldine (1) and its synthetic derivatives 2–4.
IC50 (µM)
MDA-MB-231
>100
Compound
MCF-7
>100
>100
>100
55.5 ± 6.7
DHF
>100
>100
>100
>100
1
2
3
4
>100
>100
62.7 ± 4.8
3. Experimental
3.1. General
NMR experiments were performed using an Avance 400 Digital Bruker NMR spectrometer,
operating at 400.13 MHz for 1H, 100.61 MHz for 13C and 161.97 MHz for 31P. Chemical shifts () are
1
given in ppm and coupling constant (J) in Hz. H-NMR chemical shifts are relative to the proton
13
resonance resulting from incomplete deuteration of CDCl3 ( 7.26), those of C-NMR are relative to
31
the carbon of the CDCl3 ( 77.0) and those of P-NMR are relative to 85% H3PO4 external standard.
High resolution mass spectrum was recorded by direct injection on a LTQ Orbitrap XL (Thermo
Fischer Scientific) with electrospray ionization (ESI/MS) in positive mode. The data were acquired an
analyzed using the software Xcalibur v. 2.0.7. Boldine (1) was extracted from Peumus boldus Molina
using a established procedure. 2,9-Dimethoxymethylboldine (2) and 2,9-dimethoxymethyl-3-bromo-
boldine (3) were prepared following a previously reported procedure [2,3]. Chlorodiphenylphosphine
was freshly distilled prior to use. All reagents were obtained from Aldrich Co. and were used without
further purification. Solvents were purchased from either J.T. Baker or Tedia Company Inc.

Molecules 2011, 16
2256
3.2. Chemistry: Synthesis of 2,9-Dimethoxymethyl-3-diphenylphosphinylboldine (4)
To a solution of 2,9-dimethoxymethyl-3-bromoboldine (3, 135 mg, 0.27 mmol) in dry THF (9 mL),
cooled at −78 °C, n-BuLi (0.50 mL, 1.6 M, 0.80 mmol) was added under an inert atmosphere. After
1 minute, the cooling bath was removed and neat chlorodiphenylphosphine (0.25 mL, 0.81 mmol) was
added dropwise. The mixture was further stirred at room temperature for 1 hour. A saturated NH4Cl
solution (10 mL) was then added for quenching. The organic phase was separated, dried over
anhydrous Na2SO4 and the solvent was removed under reduced pressure at 40 °C. The raw product
was chromatographed (silica gel 0.040–0.063 mm, Merck) employing a mixture of CHCl3/MeOH =
1
99/1 as eluting solvent. Yield: 100 mg (0.16 mmol, 60% yield). H-NMR (CDCl3) : 2.51 (3H, s, N-
CH3), 3.12 (3H, s, 2-OCH3), 3.50 (3H, s, 1-OCH3), 3.55 (3H, s, 9-OCH3), 3.85 (1H, d, J = 4.9 Hz,
2-OCH2O-), 3.88 (3H, s, 10-OCH3), 4.78 (1H, d, J = 4.9 Hz, 2-OCH2O-), 5.28 (2H, s, 9-OCH2O-),
7.06 (1H, s, H-8), 7.40–7.55 (6H, m, Ar-Hmeta + Ar-Hpara), 7.67 (1H, dd, J = 7.8, 1.4 Hz, Ar-Hortho),
7.70 (1H, dd, J = 7.8, 1.5 Hz, Ar-Hortho), 7.78 (1H, dd, J = 7.5, 1.5 Hz, Ar-Hortho), 7.81 (1H, dd,
13
J = 7.4, 1.6 Hz, Ar-Hortho), 7.97 (1H, s, H-11); C-NMR (CDCl3) : 27.8 (C-4), 33.6 (C-7), 43.6 (N-
CH3), 52.6 (C-5), 56.2 (10-OCH3), 56.3 (9-OCH3), 57.2 (2-OCH3), 60.2 (1-OCH3), 63.3 (C-6a), 95.2
(9-OCH2O-), 99.1 (2-OCH2O-), 112.6 (C-11), 115.0 (C-8), 121.3 (C-3), 124.7 (C-11a), 128.2 (Ar-
Cmeta), 128.3 (Ar-Cmeta), 128.4 (Ar-Cmeta), 128.5 (Ar-Cmeta), 130.1 (C-3a), 130.6 (Ar-Cortho), 130.7 (Ar-
Cortho), 130.8 (Ar-Cpara), 131.4 (2C, Ar-Cpara + Ar-Cortho), 131.5 (Ar-Cortho), 132.4 (C-1a), 133.9 (O=P-
31
C), 135.0 (O=P-C), 146.6 (C-9), 147.5 (C-1), 148.2 (C-10), 151.9 (C-2); P-NMR (CDCl3) : 29.1;
HRMS (CI) Found: m/z 616.2481 (M+H+), Calcd. for C35H39NO7P: m/z 616.2459.
3.3. Cell Lines
The experimental cell cultures were obtained from the American Type Culture Collection
(Rockville, MD, USA). MCF-7 and MDA-MB-231 cells were grown in DMEM-F12 medium
containing 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin and 1 mM glutamine. DHF
dermal human fibroblast cells were grown in DMEM-F12 medium containing 10% FCS, 100 U/mL
penicillin, 100 µg/mL streptomycin and 1 mM glutamine. Cells were seeded into 96 well microtiter
plates in 100 µL at plating density of 3 × 103 cells/well. After 24 h of incubation at 37 °C in a
humidified 5% CO2: 95% air mixture to allow cell attachment, the cells were treated with different
concentrations of drugs [boldine (1) and derivatives 2–4] and incubated for 72 h under the same
conditions. Stock solutions of compounds were prepared in ethanol and the final concentration of this
solvent was kept constant at 1%. Control cultures received 1% ethanol alone.
3.4. In vitro Growth Inhibition Assay
The sulforhodamine B assay was used according to the method of Skehan et al. [8] with some
modifications [9]. Briefly, the cells were set up 3 × 103 cells per well of a 96-well, flat-bottomed
200 μL microplate. Cells were incubated at 37 °C in a humidified 5% CO2 / 95% air mixture and
treated with the compounds at different concentrations for 72 hours. At the end of the drug exposure,
cells were fixed with 50% trichloroacetic acid (TCA) at 4 °C (TCA final concentration 10%). After
washing with distilled water, cells were stained with 0.1% sulforhodamine B, dissolved in 1% acetic

Molecules 2011, 16
2257
acid (50 µL/well) for 30 min, and subsequently washed with 1% acetic acid to remove unbound stain.
Protein-bound stain was solubilised with 100 µL of 10 mM unbuffered Tris base, and the cell density
was determined using a fluorescence plate reader (wavelength 540 nm). Values shown are the %
viability vs. ctrl + SD, with three independent experiments in triplicate.
4. Conclusions
The derivatization of boldine with a phosphinyl group, introduces a distinct capability as a potential
drug for breast cancer treatment which is not found in the boldine itself nor in 2,9-dimethoxy-
methylboldine or 2,9-dimethoxymethyl-3-bromoboldine. This capability, due to the phosphinyl group
in 4, may be attributed to the enhanced lipophilicity as well as an increasing intercalating behavior.
This in vitro inhibitory effect in the growth of two breast cancer lines cells opens a promising way to
introduce structural modifications in this natural product which enhances its biological activity.
Acknowledgements
This work was supported by Universidad Técnica Federico Santa María (DGIP 13.09.24) and
Universidad de Valparaiso (grant DIPUV 27/2006). We thank Hugo Peña-Cortés, Head of the
Biotechnology Centre at Universidad Técnica Federico Santa María for providing the LTQ Orbitrap
XL to perform the HRMS.
References and Notes
1. O´Brien, P.; Carrasco-Pozo, C.; Speisky, H. Boldine and its antioxidant or health-promoting
properties. Chem. Biol. Inter. 2006, 159, 1-17.
2. Reveco, P.G.; Asencio, M.; Sanguinetti, M.E.; Thomet, F.A. The synthesis of methoxymethyl
derivatives of boldine: Versatile protected precursors of substituted boldine derivatives. Synth.
Commun. 2005, 35, 341-347.
3. Reveco, P.G.; Thomet, F.A.; Asencio, M.; Sanguinetti, M.E. Novel methoxymethyl 3-
bromoboldine derivatives with improved capabilities as precursors for organic synthesis. Synth.
Commun. 2007, 37, 821-828.
4. Garbarino, J.; Troncoso, N.; Frasca, G.; Cardile, V.; Russo, A. Potential anticancer activity
against human epithelial cancer cells of Peumus boldus leaf extract. Nat. Prod. Commun. 2008, 3,
2095-2098.
5. Hoet, S.; Stevigny, C.; Block, S.; Opperdoes, F.; Colson, P.; Baldeyrou, B.; Lansiaux, A.; Bailly,
C.; Quetin-Leclercq, J. Alkaloids from Cassytha filiformis and related aporphines:
Antitrypanosomal activity, cytotoxicity, and interaction with DNA and topoisomerases. Planta
Med. 2004, 70, 407-413.
6. Gerhardt, D.; Horn, A.P.; Gaelzer, M.M.; Frozza, R.L.; Delgado-Cañedo, A.; Pelegrini, A.L.;
Henriques, A.T.; Lenz, G.; Salbego, Ch. Boldine: A potential new antiproliferative drug against
glioma cell lines. Invest. New Drug. 2009, 27, 517-525.
7. March, J. Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th ed.; John
Wiley & Sons Inc.: New York, NY, USA, 1992; pp. 431-433.

Molecules 2011, 16
2258
8. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.T.;
Bokesch, H.; Kenney, S.; Boyd, M.R. New colorimetric cytotoxicity assay for anticancer-drug
screening. J. Natl. Cancer Inst. 1990, 82, 1107-1112.
9. Vichai, V.; Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screenin. Nat.
Protoc. 2006, 1, 1112-1116.
Sample Availability: Samples of the compounds 2–3 are available from the authors.
© 2011 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 license
(http://creativecommons.org/licenses/by/3.0/).