Different 6-Aryl-Fulvenes Exert Anti-proliferative effects on Cancer Cells
Esther Sirignano1,§, Assunta Pisano2,§, Anna Caruso2,3, Carmela Saturnino1,*, Maria Stefania Sinicropi2,*, Rosamaria Lappano2, Antonio Botta1, Domenico Iacopetta2, Marcello Maggiolini2 and Pasquale Longo4
1Department of Pharmacy, University of Salerno, via Giovanni Paolo II, 132, 84084 Fisciano (SA), Italy; 2Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, (CS), Italy; 3Department of Computer Engineering, Modeling, Electronics and Systems, University of Calabria, 87036 Rende (CS), Italy; 4Department of Sciences, University of Salerno, via Giovanni Paolo II, 132, 84084 Fisciano (SA), Italy
Abstract: Fulvenes represent a class of molecules very interesting under a chemical point of view because are easily accessible starting materials and are still poorly characterized for their biological activities, with the exception of acylfulvene and irofulvenes which have been reported to exert cytotoxic properties. Here, we describe the synthesis and characterization of several aryl-fulvenes together with their effects on cancer cell growth by MTT method. The cytotoxic potential was investigated on a panel of tumor cell lines such as breast MCF7 and SkBr3, endometrial Ishikawa, prostate LnCaP and lung A549, in comparison with the cis-diamminedichloroplatinum(II) (cisplatin) which is largely used for the treatment of different types of cancer. The evaluation of the cytotoxic activity of these compounds indicated that they are able to inhibit the proliferation of the aforementioned cancer cell types. In particular, the compound 4 exhibited the most powerful antiproliferative activity on all tumor cells evaluated with higher inhibitory effects respect to cisplatin and without altering the proliferation of human mammary MCF-10A epithelial cells.
Fulvenes are molecules characterized by a "cross-conjugated" structure (Fig. 1a), with substituents (R groups) that can be alkyls, aryls, etc. [1]. The "cross-conjugation" represents a type of conjugation in which only two π bonds over three interact (while the third π bond is excluded from the conjugation [2]), moreover it influences the reactivity and molecular electronic transitions and is also typical of molecules such as benzophenones and p-quinones. Fulvenes are not aromatic, but the suitably substitution with electron-donor or electron-withdrawing groups on the five-membered ring allows to obtain the typical characteristics of an aromatic system [3] and the presence of a significant dipole moment (Fig. 1b).
Literature data regarding the biological properties of the fulvenes are, to date, quite scarce, only acylfulvene (Fig. 2a and 2b) [4-7] and irofulvenes [8-11] (Fig. 2d) have been shown to exhibit interesting cytotoxic properties.
The acylfulvene is a cytotoxic toxin correlated with Illudins, which are sesquiterpenes produced by mushroom Omphalotus illudens and related species of basidiomycetes, having antimicrobial and anticancer properties. In their isolated form, the Illudins have been shown to be particularly effective against hematopoietic and solid tumor cells [7].
Beside these positive effects, some Illudins, as for instance Illudin S (Fig. 2c), exerted a high toxicity which made useless their use in vivo [12], so that more efficient, and with lower side effects, analogues were synthesized, i.e. acylfulvenes. The latters have been used in a lung xenograft model and, moreover, 6-hydroxymethylacylfulvene has shown a marked efficacy in several in vivo models, as the MV522 lung carcinoma, MX1 breast
*Address correspondence to these authors at Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, (CS), Italy; Tel.: +39 0984 493200; Fax: +39 0984 493298; E-mail: [email protected] and Department of Sciences, University of Salerno, via Giovanni Paolo II, 132, 84084 Fisciano (SA), Italy; Tel: +39 089 969602; Fax: +39 089 969769; E-mail: [email protected] §These authors equally contributed to the manuscript
carcinoma, KB epidermic carcinoma, and HT29 colon carcinoma xenograft models [4-7], and is currently in phase II chemo- therapeutic clinical trials [13].
C C
a b Fig. (1). Cross conjugated (a) and aromatic (b) resonance structures of fulvene
The main mechanism by which Illudin S acts is not yet well known but is responsible of DNA transcription blockade [14], whereas acylfulvenes and derivatives most likely implicate DNA alkylation [15].
Another analogue of Illudin S is the Irofulven (hydroxymethylacylfulvene) (Fig. 2d), which is a novel pro-apoptotic antitumor agent active against a broad range of human tumor xenograft models [8, 9] and it has been shown also that the co-treatment of Irofulven with Topotecan or Irinotecan is able to synergistically reduce the MV522 lung carcinoma and HT29 colon carcinoma growth [16-18]. Additionally, even on MDR1 and MRP1 xenograft models of multidrug resistant tumors Irofulven still retained its antitumor activity [17, 19] and for several tumor types shrinkage and complete curative effects were observed [8, 15].
The potent induction of apoptosis is the most important feature of Irofulven activity in various tumor cell lines and is promoted through multiple mechanisms that include the alkylation of both DNA and proteins [10, 20-22]. The Irofulven (Fig. 2d) is currently undergoing a Phase III trial for gemcitabine-refractory pancreatic cancer and Phase II clinical trials for several tumor types, including advanced and metastatic breast cancer [8].
Furthermore, the fulvene reported in Fig. 3 (the so called "fulvene 5" synthesized and tested for its biological activity) potently inhibits NADPH oxidase 4 (NOX4), which is over-expressed in
2 Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 15, No. 0 Sirignano et al.
cancer cells respect to normal cells, and blocks the growth of endothelial tumors, including hemangiomas, in mice [23].
HN
NH
Fig. (3). Fulvene 5.
Given the interesting antitumor effectiveness of these fulvene derivatives, in this paper we report the synthesis and characterization of twelve fulvenes and some interesting preliminary data on their biological usefulness on selected cancer cell lines, i.e. MCF7, SkBr3, Ishikawa, LnCap and A549. The library of selected compounds possessing varied steric hindrance, different lipophilicity and different polar substituents may provide some valuable indications for a further development of this class of compounds which may represent a valid alternative in the arsenal of anticancer drugs.
MATERIAL AND METHODS
Chemistry
The elemental analyses for C, H, N, were recorded on a ThermoFinnigan Flash EA 1112 series and performed according to standard microanalytical procedures. 1H NMR, homodecoupled 1H NMR, 1H COSY and 13C NMR spectra were recorded at 298 K on a Bruker Avance 300 Spectrometer operating at 300 and 75 MHz for 1H and 13C, respectively. The 1H and 13C chemical shifts are referred to internal tetramethylsilane (δ = 0 ppm).
Molecular weights were determined by ESI mass spectrometry. ESI-MS analysis in positive and negative ion mode, were made using a Finnigan LCQ ion trap instrument, manufactured by Thermo Finnigan (San Jose, CA, USA), equipped with the Excalibur software for processing the data acquired. The sample
was dissolved in acetonitrile and injected directly into the electrospray source, using a syringe pump, which maintains constant flow at 5 µl/min. The temperature of the capillary was set at 220 °C. Melting point measurements have been carried out on a differential scanning calorimeter (DSC) 2920 apparatus manufactured by TA Instruments, calibrated against an indium standard (Tm = 156.6 8C), with heating scans from -10 to 200 °C, at a 10 °C/min heating rate, under a flowing nitrogen atmosphere. Specimens were sealed in aluminum pans.
All manipulations were carried out under oxygen- and moisture-free atmosphere in an MBraun MB 200 glove-box. All the solvents were thoroughly deoxygenated and dehydrated under argon by refluxing over suitable drying agents, while NMR deuterated solvents (Euriso-Top products) were kept in the dark over molecular sieves.
All chemicals were obtained from Aldrich chemical Co. and used without further purification. Cyclopentadiene was obtained by freshly cracked dicyclopentadiene. Compounds 1-8 were prepared following the procedures already published [6, 24-27].
General Procedure for the Preparation of Compounds 9 and 10
The synthesis was carried out under nitrogen. Pyrrolidine (2.5 ml, 30.0 mmol) was added to a solution of the suitable acetophenone (20 mmol) and cyclopentadiene (4.1 ml, 50.0 mmol) in 30 ml of methanol. After addition, the solution turned from colorless to red-orange color. After 20 hours, acetic acid (1.8 ml, 32.0 mmol) was added. The reaction mixture was diluted with 20 ml of a mixture of diethyl ether and water (1:1). The resultant organic layer was separated and the aqueous layer was washed with diethyl ether (3 x 20 ml). The combined organic extracts were washed with a saturated aqueous NaCl solution. The organic solution was dried over magnesium sulfate. Both products were purified by column chromatography over silica gel and a mixture of n-hexane/ethyl acetate (9:1) as the eluent. The yields are 20% for compound 9 and 31% for compound 10.
Fig. (2). Structures of (a) acylfulvene (R = H) and substituted (R =alkyl, aryl, etc.), (b) acylfulvene dimer (c) Illudin S and (d) Irofulven
Different 6-Aryl-Fulvenes Exert Anti-proliferative effects on Cancer Cells Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 15, No. 0 3
113.2 (C3’), 112.3 (C5’) 56.6 (OCH3), 22.5 (CH3). Mass (E.I., 70 eV., m/z): 183.2 [CpC(CH3)C6H4-O]•+. Calcd. for C14H14O (%): C 84.81, H 7.12. Found (%): C 84.56, H 6.98. m.p. 36.6 °C
6-Methyl-6-(2’,4’-dimethoxyphenyl)fulvene (10) 1H NMR (δ ppm, CDCl3): 7.04-6.56 (m, 3H, C6H3); 6.47-6.06 (m, 4H, C5H4); 3.85, 3.73 (s, 6H, 2 OCH3); 2.50 (s, 3H, CH3); 13 C NMR (δ ppm, CDCl3): 161.7 (C4’), 158.8 (C2’), 150.2 (C6), 144.7 (C1), 132.6 (C4), 132.1 (C3), 130.9 (C6’), 124.4 (C1’), 124.0 (C5), 121.2 (C2), 104.2 (C3’), 99.1 (C5’) 56.0 (OCH3), 22.5 (CH3). Mass (E.I., 70 eV., m/z): 198.3 [CpC(CH3)C6H3O2)]•+. Calcd. for C15H16O2 (%): C 78.92, H 7.06. Found (%): C 78.98, H 6.95. m.p. 72.4 °C
General procedure for the preparation of compounds 11 and 12
The synthesis was carried out under nitrogen at a -18 °C for compound 11 and at room temperature for compound 12. Pyrrolidine (2.5 ml, 30.0 mmol) was added to a solution of the opportune naphtaldehyde (20 mmol) and cyclopentadiene (4.1 ml, 50.0 mmol) in 30 ml of methanol. After addition, the solution turned from colorless to red-orange color. Large amounts of an orange solid precipitated out the solution and after 20 hours acetic acid (1.8 ml, 32.0 mmol) was added. The reaction mixture was diluted with 20 ml of a mixture of diethyl ether and water (1:1). The resultant organic layer was separated and the aqueous layer was washed with diethyl ether (3 x 20 ml). The combined organic extracts were washed with a saturated aqueous NaCl solution. The organic solution was dried over magnesium sulfate. When the solvent was removed under reduced pressure, an orange product was obtained. The yields are 99% for compound 11 and 30% for compound 12.
6-Naphtylfulvene (11) 1H NMR (δ ppm CDCl3): 8.04-7.54 (m, 7H, C10H7); 7.52 (s, 1H, Ph-CH-Cp); 6.83-6.40 (m, 4H, C5H4); 13 C NMR (δ ppm, CDCl3): 146.0 (C1), 138.8 (C4), 136.1 (C3), 134.9 (C1’), 134.0 (C9’ and C10’), 131.7 (C3’), 131.2 (C7’), 129.0 (C5’), 128.8 (C6’), 128.2 (C4’), 127.9 (C2’ and C8’), 127.7 (C6) 127.1 (C5), 120.8 (C2). Mass (E.I., 70 eV., m/z): 203.27 [CpCH(C10H7)]•+. Calcd. for C16H12 (%): C 94.08, H 5.92. Found (%): C 94.21, H 5.77. m.p. = 129.9 °C
6-(5’-Methoxynaphtyl)fulvene (12) 1H NMR (δ ppm CDCl3): 7.97-7.14 (m, 6H, C10H6); 7.34 (s, 1H, Ph-CH-Cp); 6.69-6.36 (m, 4H, C5H4); 3.95 (s, 3H, OCH3); 13 C NMR (δ ppm, CDCl3): 157.5 (C5’), 146.0 (C1), 138.8 (C4), 136.1 (C3), 134.9 (C10’), 134.7 (C1’), 133.0 (C7’), 132.7 (C9’), 130.8 (C3’), 128.4 (C2’), 127.7 (C6), 127.1 (C5), 125.1 (C8’), 120.8 (C2), 118.8 (C6’), 104.6 (C4’), 55.9 (OCH3). Mass (E.I., 70 eV., m/z): 219.30 [CpCH(C10H6O)]•+. Calcd. for C17H14O (%): C 87.15, H 6.02. Found (%): C 87.33, H 5.95. m.p. = 172.5 °C
Cell Culture
MCF7 breast and A549 lung cancer cells were maintained in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS), 100 mg/ml penicillin/streptomycin and 2 mM L-glutamine (Life Technologies, Milan, Italy). LNCaP prostate and SkBr3 breast cancer cells were cultured in RPMI-1640 medium, with and without phenol red respectively, supplemented with 10% FBS, 100 mg/ml penicillin/streptomycin and 2 mM L-glutamine (Life Technologies, Milan, Italy). Ishikawa endometrial cancer cells were maintained in MEM supplemented with 10% FBS, 100 mg/ml penicillin/ streptomycin, 2 mM L-glutamine and 1% Non-Essential Amino Acids Solution (Life Technologies, Milan, Italy). Human mammary MCF-10A epithelial cells were cultured in DMEM/F-12 medium supplemented with 5% Horse Serum (Eurobio, Les Ullis, Cedex, France) and 0.1 nmol/L nonessential amino acid, 2 mmol/L L-glutamine, and 50 units/ml penicillin/streptomycin.
Inhibition of Cell Proliferation
The effects of each compound on cell viability were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, which is based on the conversion of MTT to MTT-formazan by mitochondrial enzyme [28-36]. Cells were seeded in quadruplicate in 96-well plates in regular growth medium and grown until 70–80% confluence. Cells were washed once they had attached and then treated with increasing concentrations of each compound for 1 day in regular medium supplemented with 1% FBS. Relative cell viability was determined by MTT assay according to the manufacturer’s protocol (Sigma-Aldrich, Milan, Italy). Mean absorbance for each drug dose was expressed as a percentage of the control untreated well absorbance and plotted versus drug concentration. IC50 values represent the drugs concentration able to reduce the cells viability of 50% respect to the untreated control cells (vehicle) [37].
Immunoblotting
Cells were grown in 10-cm dishes, exposed to treatments and then lysed in 50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 0,1% SDS, and a mixture of protease inhibitors containing 1 mmol/L aprotinin, 20 mmol/L phenylmethylsulfonyl fluoride and 200 mmol/L sodium orthovanadate. Equal amounts of whole protein extract were resolved on a SDS/10% (w/v) polyacrylamide gel, transferred to a nitrocellulose membrane (GE Healthcare), probed with the antibodies against p-c-JunS73 and c-Jun, (Santa Cruz Biotechnology) and then revealed using the ECL System (GE Healthcare).
RESULTS
In Fig. 4 the structures of the synthesized compounds are reported: the fulvene 1 is the simple 6-phenyl-fulvene, while fulvenes 2-7 have, on the aryl group, substituents which make them more or less polar; fulvenes 8, 9 and 10 have in position 6 a methyl group that may improve the lipophilicity. Instead, the fulvenes 11 and 12 have as substituent in position 6 a ß-naphthyl and a 5-methoxy-ß-naphthyl, respectively.
The different fulvenes were synthesized by reaction between cyclopentadiene and the suitable carbonyl compounds (see Scheme 1). In particular, the cyclopentadiene was reacted with the appropriate aromatic aldehyde or methyl-aryl-ketone, in methanol, using pyrrolidine as base (see Matherial and Methods section) [25, 31, 32, 38].
All the synthesized compounds were characterized by 1H, homodecoupled 1H, and COSY NMR experiments, elemental analysis and mass spectrometry (see Material and Methods section).
With the aim to investigate the effects on cancer cell growth of the newly synthesized compounds, we evaluated by MTT test their cytotoxic potential against a panel of five human tumor cell lines. Cells were treated for 24 h with each compound and were also exposed to cis-diamminedichloroplatinum(II) (cisplatin), in order to compare the anticancer effects of the chemicals synthesized to this widely used anti-cancer drug. Cisplatin is used to treat several types of cancers as, testicular, ovarian, bladder, head and neck, esophageal, small and non-small cell lung, breast, cervical, stomach and prostate, therefore we employed breast MCF7 and SkBr3, endometrial Ishikawa, prostate LnCaP and lung A549 cancer cells as model systems. The IC50 values obtained as reported in Material and Method section are shown in Table 1. On the basis of the capability of fulvene 4 to exert the most powerful antiproliferative activity against all the human tumor cell lines evaluated, we extended our results investigating its effects on the proliferation of human mammary MCF-10A epithelial cells. Notably, we observed
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Scheme 1. Synthetic route for the preparation of 6-aryl-fulvenes.
Table 1. Cytotoxic activity of tested compounds on breast MCF7 and SkBr3, endometrial Ishikawa, prostate LnCaP and lung A549 cancer cells,
after 24 h treatment, as determined by using the MTT assay. IC50 values were calculated by probit analysis (P<0.05, χ2 test).
IC50 (µM)±S.D. Compound
MCF7 SkBr3 Ishikawa LnCaP A549
1 15(±3) 4(±1) 11(±3) 7(±1) 15(±4)
2 14(±2) 13(±4) 15(±2) 10(±4) 12(±4)
3 10(±1) 13(±2) 9(±3) 11(±2) 13(±5)
4 8(±2) 9(±2) 3(±1) 5(±2) 5(±1)
5 11(±3) 10(±2) 9(±3) 5(±2) 25(±4)
6 >50 >50 >50 >50 >50
7 41(±5) 8(±3) 5(±1) 5(±1) 15(±2)
8 15(±2) 35(±6) 10(±2) >50 >50
9 6(±1) 15(±2) 6(±2) >50 22(±4)
10 10(±1) >50 >50 >50 >50
11 5(±1) 15(±4) 8(±2) >50 >50
12 30(±5) >50 9(±1) 8(±1) 13(±2)
cisplatin 22 (±6) 12 (±1) 14(±3) 10(±1) 12(±2)
Different 6-Aryl-Fulvenes Exert Anti-proliferative effects on Cancer Cells Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 15, No. 0 5
that compound 4 did not elicit significant anti-proliferative effects on MCF-10A cells (data not shown). In order to provide mechanistic insights into the biological responses elicited by fulvene 4 in cancer cells, we evaluated c-Jun N-terminal phosphorylation at serine 73 (S73) (Fig. 5), which correlates with a pro-apoptotic activity [39-41]. The treatment for 2 h with 10 µM of fulvene 4 induced c-Jun N-terminal phosphorylation at serine 73 in MCF7, SkBr3, Ishikawa, LnCaP and A549 cancer cells (Fig. 5), suggesting that c-Jun phosphorylation is responsible of the antiproliferative effects of this compound, triggering the apoptosis response, in accord to previous data obtained using well known anticancer molecules [39].
Fig. (5). c-Jun (S73) phosphorylation and total c-Jun were evaluated in breast MCF7 and SkBr3, endometrial Ishikawa, prostate LnCaP and lung A549 cancer cells treated for 2 h with vehicle or compound 4.
DISCUSSION
The in vitro cytotoxic activity measurements indicated that most of the compounds studied are able to inhibit the proliferation of the five cell lines used in this study. In particular, as indicated in Table 1, it is possible to note that compound 1, having the simplest structure among all the synthesized 6-aryl-fulvenes, shows a good cytotoxic activity. This compound is planar and, evidently, this scaffold is already sufficiently able by itself to inhibit tumoral cell growth. The compounds 2-5 have methoxyl groups on the phenyl ring and show an interesting activity on all the tested cell lines. The fulvene 4 exhibits the most powerful antiproliferative activity against all the human tumor cell lines evaluated, while no effects were observed in mammary MCF-10A epithelial cells. Moreover, the treatment with fulvene 4 induced the phosphorylation of c-Jun N-terminal (S73), which has been previously correlated with a pro-apoptotic and antiproliferative activity [39-41]. The replacement of the methoxy group with a dimethylamino leads to a loss of the cytotoxic activity (compare the results of 2 with those of 6, Table 1), while the replacement of the same group with a SCH3 produces a fulvene having a good activity on SkBr3, endometrial Ishikawa,
prostate LnCaP and lung A549, but it is less effective on MCF7 cells (see results of 2 with those of 7, Table 1). The presence in position 6 of a methyl group produces a decrease of the antiproliferative activity on cells with the exception of endometrial Ishikawa (compare the data of 1 with those of 8). Instead the compound 9, compared to compound 2, retains a good activity on breast cancer cells and Ishikawa, while 10 is active only on MCF7 cells and loses the inhibitory activity on the other cell lines, in comparison with compound 4. In the compound 11, the substitution of a phenyl ring with a naphthyl ring makes it more active on cell lines MCF7 and Ishikawa, while it becomes less active on SKBr3 and inactive on LnCaP and A549 (compared to the compound 1). Finally, the substitution of a 4-methoxy-phenyl with a 5-methoxy-naphthyl leads to an increase of the activity of the compound 12 on cell lines Ishikawa, LnCaP and A549 but it diminishes or brings to a loss of activity on MCF7 and SKBr3 respectively (compared to the compound 2). Amongst the twelve compounds under investigation, the fulvene 4 exhibits the most powerful antiproliferative activity against all the human tumor cell lines evaluated, therefore it represents the most promising agent for the development of other analogues and for further investigations.
CONCLUSIONS
Summing up, 12 aryl-fulvenes possessing varied physico- chemical properties were synthesized, characterized and evaluated as potential anticancer agents on a panel of tumor cell lines, in comparison with one of the largely used anticancer drug such as cisplatin. The library of selected compounds possessing different steric hindrance, lipophilicity and polar substituents provided us novel informations regarding their cytotoxic activity in relationship with the different substituents.
Most of the aryl-fulvenes exhibited antiproliferative effects, however only the compound 4 showed the higher antigrowth activity on all the human tumor cell lines used, being even more active than cisplatin. Additionally, the results obtained highlight the importance of the 6-(2’,4’-dimethoxy)-phenyl-fulvene nucleus which is needed for the inhibitory effects elicited on cancer cells, as recently reported for the corresponding half-titanocene derivatives [24]. These studies may provide valuable indications for the development of new class of anticancer compounds useful as potentially valid therapeutic strategies.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no conflict of interest.
ACKNOWLEDGEMENTS
This work was supported by the Programma Operativo Nazionale (PON) Ricerca e Competività per le regioni della Convergenza-2007/2013-CCI: 2007IT161PO006 to Anna Caruso, and by Commissione Europea, Fondo Sociale Europeo (FSE 2007/ 2013-PROGRAMMA ARUE) and Regione Calabria to Domenico Iacopetta.
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Received: July 07, 2014 Revised: October 06, 2014 Accepted: October 09, 2014
