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[email protected] Medicinal Chemistry, 2014, 10, 000-000
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Design, Synthesis and Cytotoxic Activity Evaluation of New Aminosubstituted Benzofurans Konstantinos Daniilidesa, Nikolaos Lougiakisa, Nicole Poulia, Panagiotis Marakosa*, Pinelopi Samarab and Ourania Tsitsilonisb a
University of Athens, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Panepistimiopolis-Zografou, Athens 15771, Greece; bUniversity of Athens, Department of Animal & Human Physiology, Faculty of Biology, Panepistimiopolis, Athens 15784, Greece Abstract: A number of new aminosubstituted benzofuran analogues have been prepared and their cytotoxic/cytostatic activity was investigated against five human tumor cell lines (MCF-7, SKBR3, SKOV3, HCT-116 and HeLa). Certain compounds showed noticeable tumor cell growth inhibition, indicative of possible structure-activity relationships.
Keywords: Benzofurans, Cytotoxicity, Heterocycles, Sonogashira cross-coupling, Wadsworth-Emmons reaction. 1. INTRODUCTION Benzofuran derivatives have drawn considerable attention over the last few years due to their widespread occurrence in nature [1], as well as their broad range of biological activities [2], while new targets of this kind of analogues have been recently reported. These include histamine H3 receptor antagonism [3], 2 adrenergic receptor antagonism [4] and inhibition of calcium-activated chloride channels, which are present in many tissues and are mediators in numerous physiological processes [5]. Moreover, benzofuran derivatives are involved in inhibition of protein tyrosine phosphatase-1B which holds a key role in the insulindependent signaling cascade and has attracted considerable attention as a potential target for the treatment of type-2 diabetes [6]. One of the most important characteristics of benzofuran derivatives synthesized over the last decade, is their remarkable cytotoxic activity, based on their ability to inhibit enzymes involved in cell proliferation and normal cellular functions, such as farnesyltransferase [7], mitogen-activated protein kinase phosphatase-1 [8] and glycogen synthase kinase 3 [9]. Furthermore, some 2-arylbenzofurans were reported to exhibit cytotoxicity against human cell lines, and interestingly, they inhibited mitosis and tubulin polymerization through an interaction with the colchicine binding site of tubulin [10]. Finally, most recent data showed that 5,6dihydroxybenzofuran can provide a scaffold for the design of estrogen receptor (ER) ligands, as it significantly inhibited the proliferation of ER+ breast cancer cells [11]. On the other hand, it is known that the incorporation of basic side chains in the molecule of several classes of cytotoxic agents is an important component for the expression of cytotoxicity, resulting in the introduction of cationic charges, which can *Address correspondence to this author at the University of Athens, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, PanepistimiopolisZografou, Athens 15771, Greece; Tel: +302107274830; Fax. +302107274747; E-mail:
[email protected] 1573-4064/14 $58.00+.00
improve solubility under physiological conditions and enable electrostatic binding to the phosphate moieties of DNA. [12] Prompted by the remarkable pharmacological profile of benzofuran derivatives, we considered interesting to prepare a number of 3-aminosubstituted 7-methoxy-2-phenylbenzo[b] furans, with the objective to study the effect of this substitution pattern on the potential cytotoxic activity of the compounds. 2. RESULTS AND DISCUSSION 2.1. Chemistry For the preparation of the target compounds, vanillin (1) was used as the starting material which was converted to the benzaldehyde 4, in 74% overall yield (Scheme 1). This conversion proceeds via a three-step route, involving iodination of 1, acetylation of the iodo derivative 2 with the use of acetic anhydride, and Sonogashira cross-coupling reaction of compound 3 and phenylacetylene [13], with the use of appropriate catalysts. Compound 4 underwent carbonylative cyclization in methanol saturated with CO [14], in the presence of (PPh3)2PdCl2 and CuCl2·2H2O, using potassium carbonate and sodium acetate as bases to provide the corresponding 2arylbenzo[b]furan methyl ester 5 in 89% yield (Scheme 2). From this reaction, a byproduct was also isolated in 10% yield. This compound was 7-methoxy-2-phenylbenzo[b]furan-5-carboxaldehyde (5a) [15], as was evident from the NMR data that indicated the absence of the methylester group and the appearance of the 3-aromatic proton. The formation of this byproduct was due to the attack of the hydroxyl anion, derived from partial deacetylation of 4 by the bases used, on the triple bond and subsequent cyclisation, prior the complexation of the carbonylated palladium catalyst with the triple bond. Following an investigation of the reaction conditions, it was observed that formation of 5a was minimized when potassium carbonate and sodium acetate were added, ten minutes after the addition of catalysts. © 2014 Bentham Science Publishers
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Medicinal Chemistry, 2014, Vol. 10, No. ??
OHC
Daniilides et al.
OHC
OHC
I
OH
c
OH
OCH3
OCH3
1
2
OHC
I
b
a
OAc
OAc OCH3
OCH3 3
4
Scheme 1. a) ICl, HCl 18%, 50°C, 1h then rt, 72h, 80%; b) Ac2O, rt, 20h, 98%; c) phenylacetylene, CuI, (PPh3)2PdCl2, Et3N, rt, 20h, 94%.
OEt
R OHC
OHC
a
CO2CH3
b (from 5) EtO O
OAc OCH3
O
OCH3
OCH3
5 R = CO2CH3 5a R = H
4
6 c
OH
OH
OEt
OH
OHC
EtO2C
d
e
EtO
O
O
O
OCH3
OCH3
OCH3
9
8
7
.
Scheme 2. a) CO, MeOH, (PPh3)2PdCl2, CuCl2·2H2O, K2CO3, CH3COONa, rt, 48h, 89%; b) CH(Ot)3, TsOH H2O, rt, 1.5h, 99%; c) LiAlH4, anh.THF, reflux, 1.5h, 94%; d) acetone, H2O, TsOH, rt, 20min, 97%; e) NaH, THF (dry), (EtO)2P(O)CH2COOEt, rt, 45min, 56%.
The formyl group of compound 5 was then protected and the resulting diethyl acetal 6 was reduced to the corresponding 3-hydroxymethyl analogue 7. The formyl group of compound 7 was deprotected under acidic conditions to result in aldehyde 8 (overall yield for three reactions 90%). Aldehyde 8 was then subjected to Wadsworth-Emmons reaction with the use of triethyl phosphonoacetate [16] and was converted to the corresponding ethyl acrylate 9. From this reaction, only the E-isomer was formed, as indicated by the NMR spectrum of ester 9, where the J constant of the olefinic protons was 15.9 Hz. Compound 9 was then converted to the corresponding chloride 10, which was submitted to nucleophilic substitution by secondary amines, without any further purification, to afford the target molecules 11a-c (Scheme 3). Reduction of the acrylates 11a-c with LiAlH4 provided the corresponding benzofuranepropenols 12a-c. It should be noted, that the reducing agent was used in a three equivalent excess and the reaction was carried out at 0 °C to avoid simultaneous reduction of the conjugated olefinic bond. Finally, the alcohols 12a-c were converted to the acetates 13ac with the use of acetic anhydride (Scheme 3). 2.2. Biological Evaluation The cytotoxic activity of the new derivatives was assessed against five human tumor cell lines, namely, breast
MCF-7 and SKBR3, ovarian SKOV3, colon HCT-116 and cervix HeLa adenocarcinomas. The compounds were evaluated as hydrochlorides (11a-c, 13a) or fumarates (12a-c, 13b), or as free bases (13c) in the case where a stable hydrochloride, fumarate or maleate salt could not be obtained. The anthracycline doxorubicin, the anthracenedione mitoxantrone and the synthetic analogue of genistein phenoxodiol, were used as reference compounds. The results are presented in (Table 1). Among the tested derivatives, the ethyl esters 11ac possessed weak cytotoxicity against the five cell lines tested, with a 50% inhibitory concentration (IC50) ranging from 38-118 M. On the contrary, the carbinols 12, as well as the corresponding acetates 13 showed improved cytotoxic activity. Among the latter two series, compounds 12 presented a more interesting cytotoxic profile, with IC50 values within the range of 9-37 M. It would be of interest to notice that derivatives bearing the pyrolidine-substituted side chain (12b, 13b) were more active in inhibiting cancer cell growth than their corresponding counterparts. The pyrolidine analogue 12b, which proved to be the most potent cytotoxic derivative of the series, showed favorable cytotoxic activity against the five cell lines, with IC50 values of 14.6, 15.7, 20.3, 10.5 and 9.3 M against MCF-7, SKBR3, SKOV3, HCT-116 and HeLa, respectively. The cytotoxicity of 12b was not associated with selectivity, as it showed similar activity against all cell lines. Nevertheless, it did not reach the
Aminobenzofurans
Medicinal Chemistry, 2014, Vol. 10, No. ??
OH EtO2C
Cl
NRR
EtO2C
a
b
O
EtO2C
O
OCH3
OCH3
9
10
3
O OCH3 11a-c c
NRR: a) N(CH3)2
b)
NRR
NRR
N
HO
AcO
d O
O c)
N
OCH3
OCH3
12a-c
13a-c
Scheme 3. a) SOCl2, toluene (dry), reflux, 1h; b) R2NH, THF (dry), reflux, 1.5h, 74-93%; c) LiAlH4, anh.THF, 0°C, 45min, 45-62%; d) Ac2O, Et3N, rt, 2h, 65-75%. Table 1. In vitro cytotoxicity of the 3-aminosubstituted 7-methoxy-2-phenylbenzo[b]furans. IC50 values in M a Compound NRR
MCF-7 (Breast)
SKBR3 (Breast)
SKOV3 (Ovarian)
HCT-116 (Colon)
HeLa (Cervix)
11a b
N(CH3)2
37.67 ± 1.89
41.83 ± 3.76
40.63 ± 4.02
49.00 ± 2.71
67.10 ± 5.77
11b b
N(CH2)4
42.77 ± 2.90
42.67 ± 2.16
40.93 ± 3.02
33.93 ± 3.43
70.27 ± 3.56
11c b
N(CH2)5
90.20 ± 3.74
89.50 ± 2.44
85.53 ± 1.60
94.60 ± 5.14
118.13 ± 4.89
12a c
N(CH3)2
22.83 ± 2.24
32.10 ± 3.59
31.03 ± 1.83
16.93 ± 3.19
14.40 ± 3.30
12b c
N(CH2)4
14.60 ± 3.32
15.73 ± 1.65
20.33 ± 2.04
10.53 ± 2.27
9.30 ± 2.56
12c c
N(CH2)5
36.80 ± 1.49
28.87 ± 1.35
31.40 ± 1.14
21.43 ± 2.50
23.10 ± 2.60
13a b
N(CH3)2
42.47 ± 2.56
44.23 ± 2.37
38.33 ± 1.34
52.20 ± 7.82
80.17 ± 5.32
c
N(CH2)4
45.47 ± 4.22
35.17 ± 2.93
41.73 ± 1.91
45.00 ± 4.65
59.63 ± 8.00
N(CH2)5
76.87 ± 5.37
72.90 ± 3.06
52.33 ± 4.14
90.77 ± 4.26
102.20 ± 7.43
Doxorubicin
0.092 ± 0.007
0.095 ± 0.008
0.114 ± 0.011
0.192 ± 0.029
0.329 ± 0.042
Mitoxantrone
0.006 ± 0.001
0.022 ± 0.003
0.019 ± 0.003
0.031 ± 0.007
0.039 ± 0.009
Phenoxodiol
19.30 ± 0.76
41.92 ± 3.50
25.52 ± 2.29
27.21 ± 1.42
13b 13c
a
b
20.95 ± 1.21 c
IC50 was determined after 72 h exposure to each compound. The results represent mean (±standard deviation) of 3–5 independent experiments. Hydrochloride. Fumarate.
high cytotoxic efficacy of either doxorubicin or mitoxantrone, being 2-3 orders of magnitude less potent than both drugs. However, the cytotoxic activity of compound 12b against MCF-7 and SKOV3 was comparable to that of the isoflavone phenoxodiol, previously shown to induce cell death in a wide range of cancer cells, including ovarian that are resistant to conventional chemotherapeutics like carboplatin and paclitaxel [17], melanoma [18] and promyelocytic leukemic cells [19].
3. CONCLUSION In conclusion, we have prepared a number of new 3dialkylaminomethyl-substituted benzofuran analogues and have evaluated in vitro their cytotoxic activity. The aminobenzofurans were prepared from vanillin that upon iodination, acetylation and cross-coupling with phenylacetylene was converted to the corresponding ethynylbenzaldehyde. The latter was subjected to carbonylative cyclization to pro-
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Medicinal Chemistry, 2014, Vol. 10, No. ??
vide a substituted 5-formylbenzofuran-3-carboxylate that was protected and reduced to the corresponding 3hydroxymethyl-analogue, which was then deprotected and subjected to Wadsworth-Emmons reaction to give the corresponding ethyl acrylate. This compound was converted to the corresponding chloride, which was submitted to nucleophilic substitution by secondary amines to afford the target amino-substituted carboxylates. Subsequent reduction of the carboxylates provided the corresponding benzofuranepropenols, which were finally converted to their acetates. The compounds were assessed for their anticancer effect in five human tumor cell lines of various origin and showed differential cytotoxic activities, with IC50 values ranging from 9118 M. Among the three classes of the synthesized derivatives, the most active analogues were found to be the alkylamino-substituted carbinols 12. In particular, the 3pyrrolidinemethyl derivative 12b exhibited the strongest cytotoxicity, indicating that this substitution pattern could be in favour of the cytotoxic activity concerning this class of compounds. In our study, in parallel to the standard drugs doxorubicin and mitoxantrone, we used as reference compound phenoxodiol, a synthetic derivative of the naturally occurring isoflavone genistein, with improved anticancer activity. Further to its chemo sensitizing potency, phenoxodiol was reportedly shown to directly promote cancer cell apoptosis through multiple mechanisms [20]. The rational for using phenoxodiol as a reference was dual. Firstly, because of its partial structural similarity with our derivatives, but also because, although its precise targets were yet to be identified, it was granted “fast track” approval for clinical trials by the US Food and Drug administration, which regrettably failed in phase III [21]. As shown herein, the in vitro cytotoxic activity of compound 12b was at least analogous (MCF-7, SKOV3) and in three out of the five cell lines even improved (SKBR3, HCT-116, HeLa), compared to phenoxodiol. Therefore, compound 12b might hold some promise and appropriate modifications of its structure may eventually lead to a more bioactive derivative. 4. EXPERIMENTAL SECTION All chemicals were purchased from Aldrich Chemical Co. Melting points were determined on a Büchi apparatus and are uncorrected. Flash chromatography was performed on Merck silica gel 60 (0.040-0.063 mm). Analytical thin layer chromatography (TLC) was carried out on precoated (0.25 mm) Merck silica gel F-254 plates. 1H-NMR spectra and 2D spectra were recorded on a Bruker Avanche 400 instrument, whereas 13C-NMR spectra were recorded on a Bruker AC 200 spectrometer in deuterated solvents and were referenced to TMS ( scale). The signals of 1H and 13C spectra were unambiguously assigned by using 2D NMR techniques: 1H–1H COSY, NOESY HMQC and HMBC. Elemental analyses were performed on a Perkin-Elmer PE 240C Elemental Analyzer (Norwalk, CT, U.S.A.) and were within ± 0.4% of the theoretical values. The oily analytical samples, upon the appropriate chromatographic purification were dried in vacuo (vacuum pump) at 90 °C in the presence of phosphorous pentoxide for 12 h. 4-Acetoxy-3-methoxy-5-phenylethynylbenzaldehyde (4) To a solution of the ester 3 (4.58 g, 14.31 mmol) in acetonitrile (40 mL) were added under Ar phenylacetylene (2.5
Daniilides et al.
mL, 22.76 mmol), CuI (272 mg, 1.43mmol), (PPh3)2PdCl2 (505 mg, 0.72 mmol) and Et3N (2 mL, 143.5 mmol) and the reaction mixture was stirred at room temperature for 20 hrs. Upon completion of reaction, the solvent was vacuum evaporated and the residue was purified by column chromatography (silica gel) using a mixture of cyclohexane/CH2 Cl2 (1/1, v/v) as the eluent to give pure 4 (3.97 g, 94%) as a solid. mp 107-108 °C (Et2O- n-hexane). 1H-NMR (400MHz, CDCl3): 2.44 (3H, s, COCH3), 3.93 (3H, s, OCH3), 7.31 (1H , m , H-4), 7.38 (2H, m, H-3, H-5), 7.46 (1H, d, J = 1.47 Hz, H-3), 7.51 (2H, m, H-2, H-6), 7.65 (1H, d, J = 1.47 Hz, H-5), 9.92 (1H, s, CH=O). 13C-NMR (50MHz, CDCl3): 19.95 (COCH3), 56.52 (OCH3), 82.68 (CCC6H5), 95.54 (CC-C6H5), 109.97 (C-3), 122.40 (C-1), 127.79 (C-5), 128.25 (C-3, C-5), 128.62 (C-4), 129.13 (C6), 131.54 (C-2, C-6), 134.25 (C-4), 145.50 (C-1), 152.06 (C-2), 167.83 (COCH3), 190.62 (CH=O). Anal. Calcd for C18H14O4: C, 73.46; H, 4.79. Found: C, 73.35; H, 4.73. Methyl 5-formyl-7-methoxy-2-phenylbenzofuran-3-carboxylate (5) Carbon monoxide was bubbled for 5 minutes into a refluxing solution of the carboxaldehyde 4 (500 mg, 1.70 mmol) in anhydrous methanol (40 mL), followed by addition of (PPh3)2PdCl2 (65 mg, 0.09 mmol) and CuCl2·2H2O (860 mg, 6.40 mmol). The mixture was stirred for 10 minutes, then K2CO3 (465 mg, 3.36 mmol) and CH3COONa (280 mg, 3.42 mmol) were added and the reaction was stirred at room temperature for 48 hrs under CO atmosphere. Insoluble materials were filtered off through a celite pad and the filtrate was evaporated under vacuum. A 9% aqueous HCl solution was added to the residue which was then extracted with CH2Cl2. The organic layer was dried (Na2SO4) and concentrated to dryness and the residue was purified by column chromatography (silica gel) using a mixture of cyclohexane/CH2 Cl2 (3/7, v/v) as the eluent to give pure 5 (470 mg, 89%) as a light orange solid. mp 144-145 °C (Et2O- nhexane). 1H-NMR (400MHz, CDCl3): 3.96 (3H, s, COOCH3), 4.05 (3H, s, OCH3), 7.39 (1H, d, J = 1.47 Hz, H6), 7.50 (3H, m, H-3, H-4, H-5), 8.04 (2H, m, H-2, H-6), 8.12 (1H, d, J = 1.47 Hz, H-4), 10.04 (1H, s, CH=O). 13CNMR (50MHz, CDCl3): 52.31 (COOCH3), 57.00 (OCH3), 104.82 (C-6), 121.70 (C-4), 129.20 (C-3, C-5), 129.67 (C1), 130.14 (C-2, C-6), 132.01 (C-4), 136.24 (C-3), 146.55 (C-7), 147.49 (C-7a), 163.43 (C-2), 165.30 (COOCH3), 192.96 (CH=O). Anal. Calcd for C18H14O5: C, 69.67; H, 4.55. Found: C, 69.45; H, 4.54. Methyl 5-diethoxymethyl-7-methoxy-2-phenylbenzofuran-3-carboxylate (6) Triethyl orthoformate (2.3 mL, 13.82 mmol) was added dropwise into a suspension of the ester 5 (1.4 g, 4.52 mmol) in absolute tOH (50 mL), followed by addition of p-TsOH (85 mg, 0.46 mmol) and the reaction mixture was stirred at room temperature for 1.5 hr, resulting into a clear solution. The bulk of the solvent was then vacuum-evaporated at low temperature, ethyl acetate was added to the residue and was extracted with a saturated aqueous KHCO3 solution. The organic layer was dried (Na2SO4) and concentrated to dryness to give pure 6 (1.71 g, 99%) as an oil. The majority of this ester was used to the next step with no further purifica-
Aminobenzofurans
tion, whereas an amount was purified by column chromatography (silica gel) using a mixture of cyclohexane/EtOAc (2/1, v/v) as the eluent. 1H-NMR (400MHz, CDCl3): 1.28 (6H, t, J = 7.05 Hz, (OCH2 CH3)2), 3.56 (2H, q, J = 7.06 Hz, OCH2CH3), 3.68 (2H, q, J = 7.05 Hz, OCH2CH3), 3.93 (3H, s, COOCH3), 4.04 (3H, s, OCH3), 5.56 (1H, s, CH(OCH2CH3)2), 7.06 (1H, d, J = 1.08 Hz, H-6), 7.48 (3H, m, H-3, H-4, H-5), 7.71 (1H, d, J = 1.08 Hz, H-4), 8.01 (2H, m, H-2, H-6). 13C-NMR (50MHz, CDCl3): 14.81 (CH(OCH2CH3)2), 53.25 (COOCH3), 57.00 (OCH3), 62.63 (CH(OCH2CH3)2), 102.95 (CH(OCH2CH3)2), 105.77 (C-6), 113.23 (C-4), 114.72 (C-3a), 129.21 (C-3, C-5), 129.72 (C1), 130.15 (C-2, C-6), 130.66 (C-5), 131.56 (C-4), 132.54 (C-3), 144.73 (C-7a), 146.60 (C-7), 163.48 (C-2), 165.36 (COOCH3). Anal. Calcd for C22H24O6: C, 68.74; H, 6.29. Found: C, 68.81; H, 6.32. 5-Diethoxymethyl-7-methoxy-2-phenylbenzofuran-3-methanol (7) A solution of the ester 6 (1.8 g, 4.6 mmol) in dry THF (20 mL) was added dropwise under Ar into a suspension of LiAlH4 (230 mgr, 5.6 mmol) in dry F (10 mL), and the mixture was refluxed for 1.5 hr. Upon cooling, the mixture was hydrolysed with water (0.3 mL) and a 15% NaOH solution (0.5 mL), the inorganic precipitate was filtered off and the filtrate was vacuum-evaporated. The residue was dissolved in EtOAc, extracted with 2 and the organic layer was dried (Na2SO4) and concentrated to dryness to give pure 7 (1.55 g, 94%) as an oil, which entered the next reaction without further purification, except from a small amount which was purified by column chromatography (silica gel) using CH2 Cl2 in order to characterize the compound. 1HNMR (400MHz, CDCl3): 1.24 (6H, t, J = 6.84 Hz, (OCH2CH3)2), 3.56 (2H, q, J = 6.60 Hz, OCH2CH3), 3.66 (2H, q, J = 6.84 Hz, OCH2CH3), 4.06 (3H, s, OCH3), 4.97 (2H, d, J = 5.5 Hz, CH2OH), 5.55 (1H, s, CH(OCH2CH3)2), 6.99 (1H, s, H-6), 7.39 (1H, m, H-4), 7.41 (1H, s, H-4), 7.47 (2H, m, H-3, H-5), 7.88 (2H, d, J = 7.63 Hz, H-2, H-6). 13 C-NMR (50MHz, CDCl3): 14.73 (CH(OCH2CH3)2), 55.99 (CH2OH), 56.93 (OCH3), 62.55 (CH(OCH2CH3)2), 101.94 (CH(OCH2CH3)2), 105.69 (C-6), 110.38 (C-4), 116.48 (C-3a), 128.67 (C-2, C-6), 129.13 (C-3, C-5), 129.61 (C-4), 130.55 (C-1), 131.00 (C-5), 131.49 (C-3), 144.62 (C-7a), 146.49 (C-7), 155.87 (C-2). Anal. Calcd for C21H24O5: C, 70.77; H, 6.79. Found: C, 70.99; H, 6.71. 3-Hydroxymethyl-7-methoxy-2-phenylbenzofuran-5-carboxaldehyde (8) p-TsOH monohydrate (80mg, 0.42mmol) and water (20 mL) were added to a solution of the carbinol 7 (1.55g, 4.35mmol) in acetone (50 mL) and the resulting solution was stirred at room temperature for 20 minutes. The solvent was vacuum-evaporated, the residue was diluted with ethyl acetate, extracted with a saturated Na2CO3 solution and the organic layer was dried (Na2SO4) and concentrated to dryness to give pure 8 (1.08 g, 97%) as a brown solid. mp 133-134 °C (EtOH). 1H-NMR (400MHz, CDCl3): 4.08 (3H, s, OCH3), 5.03 (2H, s, CH2OH), 7.39 (1H, s, H-6), 7.46 (1H, t, J = 7.50 Hz, H-4), 7.53 (2, t, J = 7.50 Hz, H-3, H-5), 7.87 (2, d, J = 7.50 Hz, -2, H-6), 7.90 (1H, s, H-4), 10.02 (1H, s, CH=O). 13C-NMR (50MHz, CDCl3): 56.05
Medicinal Chemistry, 2014, Vol. 10, No. ??
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(CH2OH), 57.00 (OCH3), 105.04 (C-6), 115.36 (C-3a), 118.17 (C-4), 127.54 (C-2, C-6), 128.10 (C-3, C-5), 128.48 (C-4), 129.42 (C-1), 131.30 (C-3), 146.30 (C-7), 147.24 (C-7a), 154.74 (C-2), 192.25 (C=O). Anal. Calcd for C17H14O4: C, 72.33; H, 5.00. Found: C, 72.56; H, 4.89. Ethyl (2E)-3-(3-hydroxymethyl-7-methoxy-2-phenylbenzofuran-5-yl)prop-2-enoate (9) Triethyl phosphonoacetate (1.7 mL, 8.57 mmol) was added dropwise under Ar into an ice-cold suspension of NaH (60% in hexanes, 225 mg, 9.36 mmol) in dry THF (10 mL), followed by addition of a solution of the aldehyde 8 (2.20g, 7.80mmol) in dry THF (15 mL). The ice-bath was removed and the reaction was stirred at room temperature for 45 minutes. Upon completion of reaction, the mixture was diluted with ethyl acetate and extracted with water. Combined organic layers were dried (Na2SO4) and concentrated to dryness and the residue was purified by column chromatography (silica gel) using a mixture of CH2Cl2 / EtOAc (9/1, v/v) as the eluent to give pure 9 (1.55 g, 56%) as a solid. mp 116117 °C (Et2O- n-hexane). 1H-NMR (400MHz, CDCl3): 1.35 (3H, t, J = 7.00 Hz, CH2CH3), 4.02 (3H, s, OCH3), 4.25 (2H, q, J = 7.00 Hz, CH2 CH3), 4.94 (2H, s, CH2OH), 6.38 (1H, d, J = 15.90 Hz, EtOCOCH=CH), 6.94 (1H, d, J = 1.37 Hz, H-6), 7.41 (1H, t, J = 7.17 Hz, H-4), 7.44 (1H, d, J = 1.37 Hz, H-4), 7.49 (2H, t, J = 7.17 Hz, H-3, H-5), 7.72 (1H, d, J = 15.90 Hz, EtOCOCH=CH), 7.85 (2H, d, J = 7.17 Hz, -2, H-6). 13C-NMR (50MHz, CDCl3): 13.69 (CH2CH3), 54.94 (CH2OH), 55.88 (OCH3), 60.57 (CH2CH3), 104.64 (C-6), 113.08 (C-4), 115.07 (C-3a), 116.83 (EtOCOCH=CH), 127.14 (C-2, C-6), 127.26 (C-3, C-5), 128.20 (C-4), 129.13 (C-1), 130.07 (C-5), 131.01 (C-3), 143.20 (C-7a), 144.96 (EtOCOCH=CH), 145.07 (C-7), 154.45 (C-2), 167.56 (C=O). Anal. Calcd for C21H20O5: C, 71.58; H, 5.72. Found: C, 71.73; H, 5.64. General Procedure for the Synthesis of Compounds 11a-c Thionyl chloride (1 mL) was added into a solution of the ester 9 (330 mg, 0.94 mmol) in dry toluene (10 mL) and the mixture was refluxed for 1 hr. The solvent was vacuumevaporated, the last traces of thionyl chloride were azeotropically removed by the addition of dry benzene and the resulting chloride 10, with no further purification, was dissolved in dry THF (10 mL) and an excess (50-fold) of the corresponding amine was added dropwise. The resulting mixture was refluxed for 1.5 hr and then the solvent was vacuum evaporated. The residue was purified by column chromatography (silica gel) to provide the amines 11a-c. Ethyl (2E)-3-(3-dimethylaminomethyl-7-methoxy-2-phenylbenzofuran-5-yl)prop-2-enoate (11a) This compound was prepared according to the general procedure described above and purified by column chromatography (silica gel) using a mixture of cyclohexane / EtOAc (2/1, v/v) as the eluent. Yield 93%. The oily product was converted to the corresponding hydrochloride. mp (HCl) 230-231 °C (EtOH). 1H-NMR (400MHz, CDCl3): 1.36 (3H, t, J = 7.17Hz, CH2 CH3), 2.32 (6H, s, (CH3)2), 3.64 (2H, s, CH2(CH3)2), 4.10 (3H, s, OCH3), 4.29 (2H, q, J = 7.17 Hz, CH2CH3), 6.42 (1H, d, J = 15.53 Hz, EtOCOCH=CH), 6.99 (1H, s, H-4), 7.39 (1H, t, J = 7.62 Hz, H-
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4), 7.46 (2H, t, J = 7.62 Hz, H-3, H-5), 7.50 (1H, s, H-6), 7.78 (1H, d, J = 15.53 Hz, EtOCOCH=CH), 7.95 (2H, d, J = 7.62 Hz, -2, H-6). 13C-NMR (50MHz, CDCl3): 14.38 (CH2CH3), 45.49 ((CH3)2), 53.24 (CH2(CH3)2), 56.17 (OCH3), 60.42 (CH2CH3), 105.47 (C-4), 113.90 (C-3a), 114.17 (C-6), 116.88 (EtOCOCH=CH), 127.72 (C-2, C-6), 128.59 (C-3, C-5), 128.71 (C-4), 129.35 (C-1), 130.33 (C5), 132.70 (C-3), 144.44 (C-7a), 145.41 (C-7), 145.52 (EtOCOCH=CH), 154.52 (C-2), 167.16 (C=O). Anal. Calcd for C23H26ClNO4: C, 66.42; H, 6.30; N, 3.37. Found: C, 66.33; H, 6.32; N, 3.26. Ethyl (2E)-3-[3-(pyrrolidin-1-ylmethyl)-7-methoxy-2phenylbenzofuran-5-yl]prop-2-enoate (11b) This compound was prepared according to the general procedure described above and purified by column chromatography (silica gel) using a mixture of CH2 Cl2 / MeOH (9/1, v/v) as the eluent. Yield 74%. The oily product was converted to the corresponding hydrochloride. mp (HCl) 212213 °C (EtOH). 1H-NMR (400MHz, CDCl3): 1.36 (3H, t, J = 6.95 Hz, CH2CH3), 1.79 (4H, s, pyrrolidine-H-3, pyrrolidine-H-4), 2.59 (4H, s, pyrrolidine-H-2, pyrrolidine-H-5), 3.83 (2H, s, CH2-pyrrolidine), 4.06 (3H, s, OCH3), 4.28 (2H, q, J = 6.95 Hz, CH2CH3), 6.43 (1H, d, J = 16.10 Hz, EtOCOCH=CH), 6.99 (1H, d, J = 1.46 Hz, H-4), 7.38 (1H, t, J = 7.32 Hz, H-4), 7.47 (2, t, J = 7.32 Hz, H-3, H-5), 7.53 (1, d, J = 1.46 Hz, -6), 7.80 (1H, d, J = 16.10 Hz, EtOCOCH=CH), 7.96 (2, d, J = 7.32 Hz, -2, H-6). 13CNMR (50MHz, CDCl3): 14.44 (CH2CH3), 23.71 (pyrrolidine-C-3, pyrrolidine-C-4), 49.28 (CH2-pyrrolidine), 54.27 (pyrrolidine-C-2, pyrrolidine-C-5), 56.20 (OCH3), 60.50 (CH2CH3), 105.36 (C-4), 114.23 (C-3a), 114.36 (C-6), 116.81 (EtOCOCH=CH), 127.79 (C-2, C-6), 128.60 (C-3, C-5), 128.68 (C-4), 130.24 (C-5), 130.42 (C-1), 132.61 (C3), 144.49 (C-7a), 145.42 (C-7), 145.64 (EtOCOCH=CH), 154.12 (C-2), 167.26 (C=O). Anal. Calcd for C25H28ClNO4: C, 67.94; H, 6.39; N, 3.17. Found: C, 68.11; H, 6.28; N, 3.02. Ethyl (2E)-3-[3-(piperidin-1-ylmethyl)-7-methoxy-2-phenylbenzofuran-5-yl]prop-2-enoate (11c) This compound was prepared according to the general procedure described above and purified by column chromatography (silica gel) using a mixture of cyclohexane / EtOAc (4/1, v/v) as the eluent. Yield 84%. The oily product was converted to the corresponding hydrochloride. mp (HCl) 203-204 °C (EtOH). 1H-NMR (400MHz, CDCl3): 1.32 (3H, t, J = 6.91 Hz, CH2 CH3), 1.34 (2H, s, piperidine-H-4), 1.53 (4H, s, piperidine-H-3, piperidine-H-5), 2.42 (4H, s, piperidine-H-2, piperidine-H-6), 3.55 (2H, s, CH2 piperidine), 3.98 (3H, s, OCH3), 4.25 (2H, q, J = 6.91 Hz, CH2CH3), 6.39 (1H, d, J = 15.36 Hz, EtOCOCH=CH), 6.92 (1H, s, H-4), 7.34 (1H, m, H-4), 7.41 (2H, t, J = 7.51 Hz, H3, H-5), 7.47 (1H, s, H-6), 7.76 (1H, d, J = 15.36 Hz, EtOCOCH=CH), 7.95 (2H, d, J = 7.51Hz, -2, H-6). 13C-NMR (50MHz, CDCl3): 14.43 (CH2CH3), 24.46 (piperidine-C4), 26.12 (piperidine-C3, piperidine-C-5), 52.80 (CH2 piperidine), 54.58 (piperidine-C-2, piperidine-C-6), 56.19 (OCH3), 60.49 (CH2CH3), 105.31 (C-4), 113.87 (C-3a), 114.59 (C-6), 116.75 (EtOCOCH=CH), 127.88 (C-2, C-6), 128.54 (C-3, C-5), 128.64 (C-4), 130.19 (C-5), 130.49 (C-
Daniilides et al.
1), 133.01 (C-3), 144.44 (C-7a), 145.39 (EtOCOCH=CH), 145.68 (C-7), 154.46 (C-2), 167.28 (C=O). Anal. Calcd for C26H30ClNO4: C, 68.49; H, 6.63; N, 3.07. Found: C, 68.71; H, 6.85; N, 3.26. General Procedure for the Synthesis of Compounds 12a-c A solution of each of the compounds 11a-c (1 mmol) in dry THF was added dropwise under Ar into an ice-cold suspension of LiAlH4 (3 mmol) in dry THF (5 ml) and the reaction mixture was stirred at 0 ºC for 45 minutes, followed by dropwise addition of water and a 15% aOH solution. The mixture was vacuum-evaporated and the residue was diluted with ethyl acetate and extracted with water. The organic layer was dried (Na2SO4) and concentrated to dryness and the crude product was purified by column chromatography to provide pure 12a-c. (2E)-3-(3-Dimethylaminomethyl-7-methoxy-2-phenylbenzofuran-5-yl)prop-2-en-1-ol (12a) This compound was prepared according to the general procedure described above and purified by column chromatography (silica gel) using a mixture of CH2Cl2 / MeOH (94/6, v/v) as the eluent. Yield 50%. The oily product was converted to the corresponding foumarate. mp (fumarate) 177-179 °C (EtOH). 1H-NMR (400MHz, CDCl3): 2.32 (6H, s, CH2N(CH3)2), 3.36 (1H, br s, CH2OH), 3.64 (2H, s, CH2N(CH3)2), 4.04 (3H, s, OCH3), 4.33 (2H, dd, J = 5.80 Hz, J = 1.11Hz, CH2OH), 6.36 (1H, m, HOCH2CH=CH), 6.70 (1H, d, J = 15.45 Hz, HOCH2CH=CH), 6.90 (1H, s, H6), 7.29 (1H, s, H-4), 7.39 (1H, m, H-4), 7.48 (2H, t, J = 7.51 Hz, H-3, H-5), 7.94 (2H, d, J = 7.51 Hz, H-2, H-6). 13 C-NMR (50MHz, CDCl3): 44.80 (CH2N(CH3)2), 52.30 (CH2N(CH3)2), 56.75 (OCH3), 64.25 (CH2OH), 105.51 (C6), 110.44 (C-4), 113.25 (C-3a), 127.32 (C-2, C-6), 128.01 (HOCH2CH=CH), 128.26 (C-3, C-5), 128.95 (C-4), 130.13 (C-1), 131.07 (HOCH2CH=CH), 132.01 (C-3), 132.94 (C-5), 142.32 (C-7a), 145.13 (C-7), 153.57 (C-2). Anal. Calcd for C25H27NO7: C, 66.21; H, 6.00; N, 3.09. Found: C, 66.30; H, 5.87; N, 2.91. (2E)-3-[3-(Pyrrolidin-1-ylmethyl)-7-methoxy-2-phenylbenzofuran-5-yl]prop-2-en-1-ol (12b) This compound was prepared according to the general procedure described above and purified by column chromatography (silica gel) using a mixture of CH2 Cl2 / MeOH (9/1, v/v) as the eluent. Yield 56%. The oily product was converted to the corresponding foumarate. mp (fumarate) 207208 °C (EtOH). 1H-NMR (400MHz, CDCl3): 1.74 (4H, s, pyrrolidine-H-3, pyrrolidine-H-4), 2.56 (4H, s, pyrrolidineH-2, pyrrolidine-H-5), 3.06 (1H, br s, OH), 3.79 (2H, s, CH2pyrrolidine), 4.00 (3H, s, OCH3), 4.27 (2H, d, J = 5.49 Hz, HOCH2CH=CH), 6.30 (1H, m, HOCH2CH=CH), 6.64 (1H , d , J = 16.28 Hz, HOCH2CH=CH), 6.84 (1, s, -6), 7.28 (1H, s, H-4), 7.34 (1H, m, H-4), 7.43 (2, t, J = 6.58 Hz, H3, H- 5), 7.93 (2, d, J = 6.58 Hz, -2, H-6). 13C-NMR (50MHz, CDCl3): 23.59 (pyrrolidine-C-3, pyrrolidine-C4), 48.70 (CH2-pyrrolidine), 53.91 (pyrrolidine-C-2, pyrrolidine-C-5), 56.15 (OCH3), 63.75 (HOCH2), 104.87 (C-6), 107.78 (HOCH2CH=CH), 111.18 (C-4), 111.69 (C-3a), 127.75 (C-2, C-6), 128.17 (HOCH2CH=CH), 128.59 (C-3, C-5), 128.62 (C-4), 131.13 (C-1), 132.66 (C-5), 132.84 (C-
Aminobenzofurans
3), 142.38 (C-7a), 145.11 (C-7), 153.63 (C-2). Anal. Calcd for C27H29NO7: C, 67.63; H, 6.10; N, 2.92. Found: C, 67.44; H, 6.29; N, 2.77. (2E)-3-[3-(Piperidin-1-ylmethyl)-7-methoxy-2-phenylbenzofuran-5-yl]prop-2-en-1-ol (12c) This compound was prepared according to the general procedure described above and purified by column chromatography (silica gel) using a mixture of CH2Cl2 / MeOH (95/5, v/v) as the eluent. Yield 62%. The oily product was converted to the corresponding foumarate. mp (fumarate) 188-189 °C (EtOH). 1-NMR (400MHz, CDCl3): 1.42 (2H, s, piperidine-H-4), 1.55 (4H, s, piperidine-H-3, piperidine-H5), 2.46 (4H, s, piperidine-H-2, piperidine-H-6), 3.62 (2H, s, CH2-piperidine), 4.01 (3H, s, OCH3), 4.31(2H, dd, J = 5.9 Hz, 0.8 Hz, HOCH2), 6.33 (1H, m, HOCH2 CH=CH), 6.67 (1H, d, J = 15.86 Hz, HOCH2 CH=CH), 6.86 (1H, s, H-6), 7.30 (1H, s, H-4), 7.35 (1H, t, J = 7.46 Hz, H-4), 7.44 (2H, t, J = 7.46 Hz, H-3, H-5), 7.98 (2, d, J = 7.46 Hz, H-2, H-6). 13C-NMR (50MHz, CDCl3): 22.67 (piperidine-C-4), 24.54 (piperidine-C-3, piperidine-C-5), 52.50 (CH2-piperidine), 54.18 (piperidine-C-2, piperidine-C-6), 56.25 (OCH3), 63.75 (CH2OH), 105.01 (C-6), 111.58 (C-4), 113.26 (C-3a), 127.32 (C-2, C-6), 127.51 (HOCH2CH=CH), 128.46 (C-3, C-5), 129.03 (C-4), 130.14 (C-1), 132.01 (C-5), 132.20 (HOCH2CH=CH), 132.95 (C-3), 142.33 (C-7a), 145.14 (C7), 153.58 (C-2). Anal. Calcd for C28H31NO7: C, 68.14; H, 6.33; N, 2.84. Found: C, 68.25; H, 6.21; N, 2.97. General Procedure for the Synthesis of Compounds 13a-c Triethylamine (10 equivalents) and acetic anhydride (10 equivalents) were added into an ice-cold solution of each of the alcohols 12a-c in dry CH2Cl2 and the reaction was stirred at room temperature for 2 hrs. The reaction mixture was then extracted with a concentrated Na2CO3 solution, the organic layer was dried (Na2SO4) and concentrated to dryness and the residue was purified by column chromatography to provide compounds 13a-c. (2E)-3-(3-Dimethylaminomethyl-7-methoxy-2-phenylbenzofuran-5-yl)prop-2-en-1-yl Acetate (13a) This compound was prepared according to the general procedure described above and purified by column chromatography (silica gel) using a mixture of CH2Cl2 / MeOH (98/2, v/v) as the eluent. Yield 65%. The oily product was converted to the corresponding hydrochloride. mp (HCl) 203-204 °C (EtOH). 1H-NMR (400MHz, CDCl3): 2.13 (3H, s, COCH3), 2.33 (6H, s, CH2N(CH3)2), 3.64 (2H, s, CH2N(CH3)2), 4.06 (3H, s, OCH3), 4.77 (2H, dd, J = 6.55 Hz, J = 1.08Hz, CH2OAc), 6.29 (1H, m, AcOCH2CH=CH), 6.77 (1H, d, J = 15.80 Hz, AcOCH2 CH=CH), 6.91 (1H, s, H6), 7.31 (1H, s, H-4), 7.38 (1H, t, J = 7.43 Hz, H-4), 7.48 (2H, t, J = 7.43 Hz, H-3, H-5), 7.97 (2H, d, J = 7.43 Hz, H2, H-6). 13C-NMR (50MHz, CDCl3): 21.34 (CH3CO), 45.72(CH2N(CH3)2), 50.95 (CH2N(CH3)2), 56.98(OCH3), 66.35 (CH2OAc), 104.80 (C-6), 112.30 (C-4), 113.30 (C-3a), 122.62 (AcOCH2CH=CH), 127.36 (C-2, C-6), 128.30 (C3, C-5), 128.78 (C-4), 130.18 (C-1), 132.05 (C-3), 135.75 (AcOCH2 CH=CH), 143.30 (C-7a), 145.18 (C-7), 153.62 (C2), 170.50 (C=O). Anal. Calcd for C23H26ClNO4: C, 66.42; H, 6.30; N, 3.37. Found: C, 66.50; H, 6.22; N, 3.15.
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(2E)-3-[7-Methoxy-2-phenyl-3-(pyrrolidin-1-ylmethyl)-5benzofuran-5-yl]prop-2-en-1-yl Acetate (13b) This compound was prepared according to the general procedure described above and purified by column chromatography (silica gel) using a mixture of CH2Cl2 / MeOH (98/2, v/v) as the eluent. Yield 75%. The oily product was converted to the corresponding foumarate. mp (fumarate) 186-187 °C (EtOH). 1H-NMR (400MHz, CDCl3): 1.73 (4H, s, pyrrolidine-H-3, pyrrolidine-H-4), 2.14 (3H, s, COCH3), 2.73 (4H, s, pyrrolidine-H-2, pyrrolidine-H-5), 4.05 (3H, s, OCH3), 4.08 (2H, s, CH2-pyrrolidine), 4.76 (2H, dd, J = 6.53 Hz, J = 1.04Hz, CH2OAc), 6.32 (1H, m, AcOCH2CH=CH), 6.77 (1H, d, J = 15.81 Hz, AcOCH2CH=CH), 6.91 (1H, d, J = 1.19 Hz, H-6), 7.36 (1H, d, J = 1.19 Hz, H-4), 7.40 (1H, m, H-4), 7.48 (2H, t, J = 7.16 Hz, H-3, H-5), 7.91 (2H, d, J = 7.16 Hz, H-2, H-6). 13C-NMR (50MHz) CDCl3: 20.03 (CH3CO), 21.90 (pyrrolidine-C-3, pyrrolidine-C-4), 47.22 (CH2-pyrrolidine), 53.29 (pyrrolidine-C-2, pyrrolidine-C-5), 55.66 (OCH3), 65.04 (CH2OAc), 104.47 (C-6), 110.05 (C-4), 111.43 (C-3a), 121.73 (AcOCH2CH=CH), 127.37 (C-2, C6), 128.05 (C-3, C-5), 128.30 (C-4), 130.18 (C-1), 132.06 (C-3), 134.86 (AcOCH2 CH=CH), 143.31 (C-7a), 145.18 (C7), 154.56 (C-2), 170.50 (C=O). Anal. Calcd for C29H31NO8: C, 66.78; H, 5.99; N, 2.69. Found: C, 66.91; H, 6.13; N, 2.57. (2E)-3-[7-Methoxy-2-phenyl-3-(piperidin-1-ylmethyl)-5benzofuran-5-yl]prop-2-en-1-yl Acetate (13c) This compound was prepared according to the general procedure described above and purified by column chromatography (silica gel) using a mixture of CH2Cl2 / MeOH (98/2, v/v) as the eluent, to result in an oil. Yield 70%. 1HNMR (400MHz, CDCl3): 1.44 (2H, s, piperidine-H-4), 1.57 (4H, s, piperidine-H-3, piperidine-H-5), 2.12 (3H, s, COCH3), 2.48 (4H, s, piperidine-H-2, piperidine-H-6), 4.06 (3H, s, OCH3), 4.07 (2H, s, CH2-piperidine ), 4.77 (2H, d, J = 6.36 Hz, CH2OAc), 6.31 (1H, m, AcOCH2 CH=CH), 6.76 (1H, d, J = 15.60 Hz, AcOCH2CH=CH), 6.91 (1H, s, H-6), 7.35 (1H, s, H-4), 7.40 (1H, t, J = 7.04 Hz, H-4), 7.48 (2H, t, J = 7.04 Hz, H-3, H-5), 7.89 (2H, d, J = 7.04 Hz, H-2, H-6). 13C-NMR (50MHz, CDCl3): 21.40 (CH3 CO), 23.80 (piperidine-C-4), 24.22 (piperidine-C-3, piperidine-C-5), 48.50 (CH2-piperidine), 52.35 (piperidine-C-2, piperidine-C6), 56.10 (OCH3), 64.54 (CH2OAc), 104.86 (C-6), 109.55 (C-3a), 111.42 (C-4), 121.74 (AcOCH2CH=CH), 127.37 (C2, C-6), 128.25 (C-3, C-5), 128.30 (C-4), 130.18 (C-1), 132.05 (C-3), 134.87 (AcOCH2CH=CH), 143.31 (C-7a), 145.18 (C-7), 155.50 (C-2), 170.50 (C=O). Anal. Calcd for C26H29NO4: C, 74.44; H, 6.97; N, 3.34. Found: C, 74.59; H, 7.03; N, 3.27. General Procedure for the Preparation of Fumarates To a stirred solution of the amine in anhydrous ethanol was added a slight excess (5%) of fumaric acid. The resulting solution was stirred at reflux temperature for 12-36 h and then allowed to cool at room temperature. The solid was collected by filtration, washed with absolute ethanol and diethyl ether, and dried under vacuum (yield 70-78%). Cytotoxicity Assay The human cancer cell lines MCF-7 and SKBR3 (breast), SKOV3 (ovarian), HCT-116 (colon) and HeLa (cervix) were
8
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maintained in RPMI-1640 (Lonza Ltd, Switzerland), supplemented with 10% heat inactivated fetal calf serum (Lonza), 10 mM Hepes (Lonza), 50 M -mercaptoethanol (Sigma-Aldrich Chemical Co., St Louis, MO, USA), 103 U/mL penicillin (Lonza), 1 mg/mL streptomycin (Lonza) and 5 g/mL gentamycin (Lonza) (referred to as complete medium). Cells were propagated at 37oC in a humidified CO2 incubator as monolayers and passaged by trypsinization (0.25% tryspin/0.25% EDTA) every 3-4 days. Cells were adjusted at 20-25 x 103 /mL, seeded in 96-well flat bottom microplates (Costar, Cambridge, MA, USA; 200 L/well) and pre-incubated for 24 h to adhere. Compounds 11-13 and phenoxodiol (kindly provided by Dr. A. Husband, NOVOGEN, North Ryde, NSW, Australia) were diluted in DMSO at 10 mg/mL and stored at -80oC. Doxorubicin (SigmaAldrich) and mitoxantrone (Novantrone, MEDA Pharmaceuticals Inc., Sweden) were diluted in d. H2O, aliquoted and stored at -20oC. Serial dilutions of all compounds were freshly prepared prior to use. Cultures set in complete medium or containing the equivalent amount of DMSO were used as negative controls. For each experiment, compounds were tested in triplicates. After 72 h of incubation, cytotoxicity was determined using the 3-(4,5-dimethyl-2-thiazyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) dye reduction assay, according to a previously reported procedure [22]. IC50 was calculated according to the formula 100(A0-A)/A0 = 50, where A and A0 are the optical densities of wells exposed to the compounds and control wells, respectively. CONFLICT OF INTEREST
Daniilides et al. [6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS
[14]
We thank Kyriaki Ioannou for her assistance in the cytotoxicity assays. This work was co-financed by European Union FP7 Capacities grant REGPOT-CT-2011-284460, INsPiRE. [15]
SUPPLEMENTARY MATERIAL Supplementary material is available on the publishers Web site along with the published article. [16]
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Received: April 30, 2013
Revised: September 14, 2013
Accepted: September 17, 2013
