Synthesis and Evaluation of Cytotoxicity and ...

Advances in Research 6(6): 1-12, 2016, Article no.AIR.24265 ISSN: 2348-0394, NLM ID: 101666096

SCIENCEDOMAIN international www.sciencedomain.org

Synthesis and Evaluation of Cytotoxicity and Antioxidant Properties of Polyfluorinated Phosphorus-containing 1,4-Benzoquinones and 1,4-Naphthoquinones O. D. Zakharova1, L. P. Ovchinnikova2, S. I. Zhivetyeva3, L. I. Goryunov3†, V. D. Shteingarts3†, E. V. Tretyakov3* and G. A. Nevinsky1* 1

Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, 8 Lavrentiev Ave., 630090 Novosibirsk, Russia. 2 Institute of Cytology and Genetics, Siberian Division of Russian Academy of Sciences, 10 Lavrentiev Ave., 630090 Novosibirsk, Russia. 3 N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Division of Russian Academy of Sciences, 9 Lavrentiev Ave., 630090 Novosibirsk, Russia. Authors’ contributions This work was carried in collaboration between all authors. Authors ODZ and LPO carried out the biological studies. Authors SIZ and LIG preformed chemical synthesis of different compounds. Authors VDS, EVT and GAN supervised and directed the entire study and prepared manuscript. All authors read and approved the final manuscript. Article Information DOI: 10.9734/AIR/2016/24265 Editor(s): (1) Pradip K. Bhowmik, Department of Chemistry, University of Nevada Las Vegas Las Vegas, USA. Reviewers: (1) Dipak K. Raval, Sardar Patel University, India. (2) Anonymous, International Islamic University, Malaysia. (3) R. S. Rimal Isaac, Noorul Islam Centre for Higher Education, India. (4) Sule Erten Ela, Ege University, Turkey. Complete Peer review History: http://sciencedomain.org/review-history/13698

th

Original Research Article

Received 11 January 2016 Accepted 25th February 2016 th Published 15 March 2016

ABSTRACT Aim: Synthesis and analysis of antioxidant and antitumor properties of fluorinated phosphoruscontaining derivatives of tetrafluoro-1,4-benzoquinone and hexafluoro-1,4-naphthoquinone. Methodology: All compounds were synthesized by amino- and phosphanodefluorination with corresponding amines and phosphanes. The cytotoxicity of these fluorinated benzoquinones and _____________________________________________________________________________________________________ *Corresponding author: E-mail: [email protected], [email protected]; † Deceased

Zakharova et al.; AIR, 6(6): 1-12, 2016; Article no.AIR.24265

naphthoquinones was compared using human myeloma and human mammary adenocarcinoma as target tumors, as well as normal mouse fibroblasts cells. Their mutagenic and antioxidant properties in a Salmonella tester strain were also evaluated. Results: All the benzoquinones suppressed the growth of the two lines of tumor cells at approximately 3.2–90-fold higher concentrations than the naphthoquinones. In addition, the naphthoquinones were universal, while the benzoquinones were selective toward suppression of different tumor cells. At the same time, the benzoquinones and naphthoquinones demonstrated similar antioxidant properties, and protected bacterial cells against spontaneous and H2O2dependent mutagenesis at comparable concentrations. Conclusion: All compounds are effective antioxidants and suppress the growth of tumor cells.

Keywords: Polyfluorinated phosphorus-containing 1,4-benzoquinones and 1,4-naphthoquinones; nucleophilic substitution; antioxidant properties; cytotoxicity. 14 clinically used therapeutics [9]. Cpd5, or 2-(2mercaptoethanol)-3-methyl-1,4-naphthoquinone, was proven to be effective for inhibition of Cdc25 phosphatases. Although most quinones have been reported to inhibit Cdc25 by sulfhydryl arylation at the quinone nucleus, the redox properties of quinones can also generate toxic oxygen species [13], which may cause toxicity to normal tissues and thus reduce their therapeutic utility.

1. INTRODUCTION Mammalian cells commonly use reversible phosphorylation of proteins for intracellular signal transduction. Therefore, abnormalities in kinases and phosphatases, the key enzymes of the protein phosphorylation signaling pathways, are closely linked to many human diseases including cancer. Members of the Cdc25 family of phosphatases remove phosphates from phosphotyrosine and phosphothreonine residues in cyclin-dependent kinases (Cdk) and thus activate cyclin-Cdk complexes that control cell cycle progression [1-2]. Cdc25A and Cdc25B are overexpressed in a number of tumors of various origins, frequently showing correlations with higher-grade or more aggressive tumors and poor prognosis [3]. Together with the current evidence for their involvement in the DNA damage-activated G2/M checkpoint response, the putative involvement of Cdc25 phosphatases in tumorigenesis makes these cell cycle regulators potential targets for cancer therapy [4-5].

The rationale to study inhibitors of Cdc25 phosphatases is looking for better inhibitors of tumor cells growth having no toxic, mutagenic, or carcinogenic properties. One strategy for controlling the intrinsic toxicity of quinones might be to use derivatives that are more stable in their reduced state and thus are less likely to initiate formation of radicals and indiscriminately damage cells. Interestingly, a fluorinated quinone compound – 5,6,7,8-tetrafluoro-2-(2mercaptoethanol)-3-methyl-[1,4]-naphthoquinone (F-Cpd5), in contrast to its non-fluorinated analogue (Cpd5), was predicted not to generate reactive oxygen species [14]. Overall, the calculated reduction potential of F-Cpd5 was suggestive of its higher possible therapeutic index. Indeed, F-Cpd5 is three times more potent than Cpd5 in suppressing Hep3B cell growth [14–16], inhibiting, at that, the mitogen-induced DNA synthesis in normal rat hepatocytes 12-fold less than in Hep3B cells [15].

Among numerous analyzed substances several were found to inhibit Cdc25 family enzymes [6-7], and NSC 95397 (2,3-bis[2-hydroxyethyl)thio]-1,4naphthoquinone) from the National Cancer Institute Library was shown to be the most potent Cdc25 inhibitor [8]. p-Naphthoquinones and 7aminoquinoline-5,8-quinones are the core structures for the synthesis of potential inhibitors of Cdc25 phosphatases [8-9], including NSC 663284 [10]. Previous studies suggest that quinoline-5,8-quinones can inactivate Cdc25 family phosphatases by either Michael addition [11] or oxidation of the catalytic cysteine [12]. It was also shown that quinoline-5,8-quinone derivatives with substitutions at the C2 and C4 positions effectively inhibit Cdc25b and cancer cell growth [9]. The p-quinone core structure is fundamental to the biological activity of at least

Recently we have synthesized a set of polyfluorinated 1,4-naphthoquinones containing n-butylamino and sulfur-, alkylamino-, and phenylamino-groups, and amino acid substituents [17-23]. All these compounds were investigated for their mutagenic and antioxidant properties using a special type of Salmonella cells, as well as for their cytotoxicity in several tumor cells, and primary mouse fibroblast cells 2


[17-20]. Interestingly, all of these compounds demonstrated a high efficiency to suppress the growth of cancer cells and antioxidant properties in the bacterial system. Only several of many compounds possess a highly promising combination of these properties [17-20]. At the same time, the concentration ratio for suppressing cancer and normal cells, as well as antioxidant properties, significantly depend upon the structure of functional groups [17-20]. Therefore, in this work we concentrated on studying a broad series of fluorinated derivatives of phosphorus-containing benzoquinones (1-5) and naphthoquinones (6-9) concerning their cytotoxicity against cancer cells and ability to protect bacterial cells from mutagenesis (Table 1). The syntheses of compounds 1–3 and 6–9 were described in our previous articles [24,25], while the synthesis of compounds 4 and 5 is given in this article (vide infra).

dioxane (2 mL). The mixture was stirred at 20°C for 2 h. The precipitate was centrifuged off, washed with diethyl ether (2×1 mL) and water (2×2 mL). The diethyl ether, water and dioxane solutions were combined, the solvents were distilled off. The crude residue was purified by TLC (Cellulose, ethyl acetate, Rf = 0.9, four times; the solvent reached the middle of the plate, then the plate was cut and the fraction with Rf = 0.9 was eluted further with diethyl ether, Rf = 0.1, four times) and crystallized from benzene to yield 4 as bright purple crystals (0.077 g, 21%), m.p. 1 144.5°C. H NMR (300.13 MHz, CDCl3) δ: 5.80 (bs, 1H, NH), 4.30–4.18 (m, 4H, 2CH2), 1.29 [t, 3H, 3 13 1 JHH = 7.1 Hz, CH3] (Fig. 1). C{ H} NMR (100.61 2 3 MHz, CDCl3) δC: 175.57 [ddd, JCF = 21.7 Hz, JCF = 3 6 2 12.0 Hz, JCF = 8.0 Hz, C ], 169.64 [ddd, JCF = 2 3 3 23.0 Hz, JCF = 22.2 Hz, JCF = 5.3 Hz, C ], 168.64 5 1 [d, C=O, JCF = 2.9 Hz], 142.77 [ddd, JCF = 287.6 Hz, 2JCF = 8.9 Hz, 3JCF = 5.7 Hz, C4], 141.76 [dd, 1 2 5 JCF = 280.1 Hz, JCF = 8.7 Hz, C ], 137.17 [ddd, 1 3 4 JCF = 249.5 Hz, JCF = 7.6 Hz, JCF = 2.0 Hz, C2], 1 4 129.49 (m, C ), 62.21 (s, OCH2), 45.26 [d, JCF = 19 6.9 Hz, NHCH2], 14.04 (s, CH3). F NMR (282.36 MHz, CDCl3) δF: –166.33 (bs, 1F, F2), –148.88 [d, 3 5 3 JFF ~ 5.4 Hz, 1F, F ], –140.16 [d, JFF ~ 5.4 Hz, 1F, 4 + F ]. HRMS (EI) for С10H8F3NO4 (M ): calcd, 263.0399; found, 263.0398.

2. MATERIALS AND METHODS 2.1 General Methods Commercially available glycine ethyl ester hydrochloride was used without purification. Triphenylphosphane was recrystallized from Et2O; fluoranil 10 was recrystallized from CH2Cl2; solvents were distilled before use. Compounds 1-3 and 6-9 were synthesized as reported earlier 1 19 13 1 31 1 [24,25]. H, F, C{ H}, and P{ H} NMR spectra were recorded on a Bruker AV-300 (1H at 300.13, 19 31 1 F at 282.36, P{ H} at 121.49 MHz) and AV-400 13 1 ( C{ H} at 100.61 MHz) NMR spectrometers and calibrated relative to the residual proton chemical shifts of chloroform (δH 7.25 ppm, δC 77.00 ppm) in 1H NMR spectra, Me4Si in 13C{1H} NMR 19 spectra, external C6F6 (δF = –162.9 ppm) in F 31 1 NMR spectra, and H3PO4 in P{ H} spectra. High-resolution mass spectra (HRMS) were measured with a Thermo Scientific DFS instrument (EI, 70 eV). Melting points of quinones 4 and 5 were determined using a Mettler Toledo device with an FP 900 Thermosystem cell. TLC was performed using Whatman paper and cellulose (Merck). The isolated reaction products were found to be >95% purity by NMR analysis.

2.3 Synthesis of Compound 5 4-[(2-Ethoxy-2-oxoethyl)amino]-5-fluoro-2-oxido3,6-dioxocyclohexa-1,4-dien-1yl)triphenylphosphanium (5). A solution of PPh3 (0.055 g, 0.21 mmol) in benzene (1.5 mL) was added dropwise to a solution of quinone 4 (0.037 g, 0.14 mmol) in benzene or in toluene (1.7 mL). The mixture was stirred at 20°C for 1 h. Water (0.2 mL) was added and the obtained solution was stirred for another 72 h. The precipitate was centrifuged off and washed with benzene (3×0.5 mL). The benzene solutions were combined and the solvent was distilled off. The crude residue was purified by TLC (Whatman, diethyl ether, Rf = 0.9, four times; the solvent reached the middle of the plate, then the plate was cut and the fraction with Rf = 0.9 was eluted further with diethyl ether–hexane, 1:1, Rf = 0.1, four times). After double TLC in the same conditions compound 5 was obtained as red 1 crystals (0.015 g, 22%), m.p. 117.1°C. H NMR (300.13 MHz, CDCl3) δ: 7.67–7.55 (m, 9H, C6H5), 7.52–7.44 (m, 6H, C6H5), 5.12 (m, 1H, NH), 4.26– 3 4.17 [q, 2H, JHH 7.1 Hz, CH2], 4.16–4.11 [m, 2H, 3 JHH 6.3 Hz, 4JHH 3.3 Hz, CH2], 1.27 [t, 3H, 3JHH = 13 1 7.1 Hz, CH3] (Fig. 2). C{ H} NMR (100.61 MHz, 2 2 CDCl3) δC: 180.16 [dd, JCF = 15.1 Hz, JCP = 13.5

2.2 Synthesis of Compound 4 Ethyl 2-[(2,4,5-trifluoro-3,6-dioxocyclohexa-1,4dien-1-yl)amino]acetate (4). A mixture of glycine ethyl ester hydrochloride (0.194 g, 1.39 mmol), KOH (0.078 g, 1.39 mmol), H2O (1 mL) and dioxane (3 mL) was added dropwise to a suspension of quinone 10 (0.250 g, 1.39 mmol) in 3


7.0

2.4 Determination of Mutagenicity

5.7 p pm

4.30

4.2 8

6.5

4.26

4 .24

4.22

4.2

379.0

386.2

393.3

1258 .8

1265.0 126 2.8 2.3

1268.7

1272.6

1279.7

In the Ames test, a histidine-dependent strain of S. typhimurium TA102 was used, which carries a mutation at the histidine operon. The mutagenic activity of the samples was analyzed by the standard method without metabolic activation [26]. The Ames test was carried out using the

4.20

ppm

1.30

1.28

p pm

6.0

5.5

5.0

4.5

4.2

2.0 2. 3

5.8

1.0

5.9

С28H23FNO5P: C, 66.80; H, 4.60; N, 2.78; found: C, 66.58; H, 5.10; N, 3.23. HRMS (EI) for С28H23FNO5P (M+): calcd, 503.1292; found, 503.1281.

2.0

1.0

1286.9

5.8

Hz, C6], 177.79 [dd, 2JCF = 21.6 Hz, 2JCP = 6.3 Hz, 2 3 3 3 C ], 173.0 [dd, JCF = 4.8 Hz, JCP = 2.1 Hz, C ], 5 169.95 [d, C=O, JCF = 2.5 Hz], 142.44 1 3 5 [dd, JCF = 261.4 Hz, JCP = 16.4 Hz, C ], 134.0 [d, 2 4 3 JCF = 10.9 Hz, C ], 133.76 [d, JCP ~ 11 Hz, C3'], 4 4' 2 132.73 [d, JCP ~ 3 Hz, C ], 128.85 [d, JCP ~ 13 2' 1 1' Hz, C ], 123.18 [d, JCP = 92.7 Hz, C ], 79.08 1 3 1 [dd, JCP = 107.9 Hz, JCF = 3.6 Hz, C ], 61.46 (s, 4 OCH2), 45.66 [d, JCF = 7.0 Hz, NHCH2], 14.13 19 (s, CH3). F NMR (282.36 MHz, CDCl3) δF: – 148.29 [dq, 4JFP ~ 13.7 Hz, 4JFH ~ 5JFH 3.2 Hz]. 31P{1H} NMR (121.49 MHz, CDCl3) δP: 4 13.83 [d, JPF ~ 13.7 Hz]. Anal. calcd for

4.0

3.5

3.0

2.5

2.0

1.5

ppm

8.0

7.5

7.0

6.5

6.0

5.5

4.25

5.0

4.20

4.15

372.2

379.3 4.2

386.4

1234.7

1240.9 1238.0

1 244.2

1.8

1253.7

1 260.8 2.0

5.10 ppm

ppm

1.30

1.25

4.5

4.0

3.5

3.0

Fig. 2. 1H NMR spectrum for compound 5

4

pp m

4.2

5.15

126 8.0

127 5.1

1543.4 15 40.0 1537.1 1534.0 1530.8 15 27.9 0.8

ppm

2.0 1.8

7.50

0.8

7.55

6.0

7 .60

8.9

7.65

6.0

2253 .0 2249.6 2245.2 2 242.5 2238. 0 2236.7

2277.5 2275.8 22 70.2

2290.3 2288.8 22 84.2 8.9

2297.4

Fig. 1. 1H NMR spectrum for compound 4

2.5

2.0

1.5

ppm


Table 1. Structural formulas of phosphorus-containing derivatives of tetrafluoro-1,4benzoquinone 1-5 and hexafluoro-1,4-naphthoquinone 6-9 Comp. 1

Name (4,5-Difluoro-2-oxido-3,6-dioxocyclohexa-1,4dien-1-yl)triphenylphosphanium

2

(4-Fluoro-5-methoxy-2-oxido-3,6-dioxocyclohexa1,4-dien-1-yl)triphenylphosphanium

3

[2,4-Dioxido-3,6-dioxo-5(triphenylphosphaniumyl)cyclohexa-1,4-dien-1yl]triphenylphosphanium

4

Ethyl 2-[(2,4,5-trifluoro-3,6-dioxocyclohexa-1,4dien-1-yl)amino]acetate

5

{4-[(2-Ethoxy-2-oxoethyl)amino]-5-fluoro-2-oxido3,6-dioxocyclohexa-1,4-dien-1yl}triphenylphosphanium

6

Triphenyl(5,6,7,8-tetrafluoro-3-oxido-1,4-dioxo-1,4dihydronaphthalen-2-yl)phosphanium

7

(2,5-Difluorophenyl)diphenyl(5,6,7,8-tetrafluoro-3oxido-1,4-dioxo-1,4-dihydronaphthalen-2yl)phosphanium

8

Methyldiphenyl(5,6,7,8-tetrafluoro-3-oxido-1,4-dioxo1,4-dihydronaphthalen-2-yl)phosphanium

9

(6,11-Difluoro-9-oxido-7,10-dioxo-7,10-dihydro5,12-dioxatetracen-8-yl)triphenylphosphanium

described double-layer method [26,27]. An overnight culture of bacteria (100 µL) containing one of the tested compounds in different concentrations and, if required, 3 mM H2O2, were mixed at 42 °C with 2 mL of liquid 0.6% top agar. The mixture was poured onto plates with a minimal medium containing 0.2% glucose and

Structural formula

3% agar, taking care to distribute the mixture uniformly on the surface of the solid agar. The plates were incubated for 48 h at 37 °C, and the revertants were counted. The cells incubated with H2O2 in the absence of the analyzed compounds were used as positive controls, and the cells grown in the absence of H2O2 and 5


antioxidants served as negative controls for mutation induction. The results are expressed as mean±standard deviation of at least three independent experiments.

yl}triphenylphosphanium (5) in 22% isolated yield. The observed regioselectivity of fluoride-atom substitution with formation of 5 can be reasonably explained by a deactivation of position 5 in the primary product 4 due to π-donor effect of NHRgroup reducing the electron-acceptor effect of the adjacent C=O-group.

2.5 Cytotoxicity Assays Tumor cell lines from human myeloma RPMI 8226, human mammary adenocarcinoma MCF7, and LMTK cell line of mouse fibroblasts (LMTK) (~2000 cells per well) were incubated for 24 h at 37ºC in IMDM or RPMI 1640 medium (5% CO2). In several experiments hepatocellular carcinoma HepG2 epithelial tumor cells (HEP) and normal chinese hamster fibroblasts Ag17 (AG) cells were used. The cells were treated with benzoquinones 1-5 or naphthoquinones 6-9. After 72 h of cell incubation, the relative amount of live cells was determined using 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (a standard colorimetric MTT-test [28]) and the drug concentration that caused 50% cell growth inhibition (IC50) was determined. The results are expressed as mean±standard deviation of at least three independent experiments.

The structure of betaines 4 and 5 was confirmed 1 13 1 19 31 1 by H, C{ H}, F, and P{ H} spectra (in CDCl3) and high-resolution mass spectra. In the 19 F NMR spectrum of 4 there are three signals: a 2 broad singlet at –166.33 ppm belonging to atom F and doublets at –148.88 and –140.16 ppm with 3 5 spin coupling constants JFF ~ 5.4 Hz for atoms F 4 and F , respectively. A slight high-field shift of the signal for atom F4 as compared to atom F5 can be explained by an electron-releasing effect of the amino group. In the 19F NMR spectrum of 5 there is one signal: a double quartet at –148.29 ppm with 4 JFP ~ 13.7 Hz, 4JFH ~ 5JFH 3.2 Hz, belonging to 5 31 1 atom F . At the same time in the P{ H} NMR spectrum of 5 there is one doublet signal at 13.83 ppm with 4JPF ~ 13.7 Hz. The para-position of the triphenyl phosphine group with regard to the amino group is proved by the large value of spin 4 4 coupling constants JFP, which should be JFP ~ 12÷16 Hz [24,25].

3. RESULTS AND DISCUSSION 3.1 Synthesis of Compounds 4 and 5

3.2 Analysis of the Ability to Inhibit Cell Growth

Ethyl 2-[(2,4,5-trifluoro-3,6-dioxocyclohexa-1,4dien-1-yl)amino]acetate (4) was synthesized by interaction of tetrafluoro-1,4-benzoquinone (10) with ethyl 2-aminoacetate (Scheme 1). It is interesting to note that aminodefluorination of 10 by ethyl esters of glycine, α-alanine and βphenyl-α-alanine gave only the corresponding disubstituted compounds. The reaction of amine 4 with triphenylphosphane PPh3 (1:1.5) in benzene-H2O or in toluene-H2O solution gave a new polyfunctional betaine – {4-[(2-ethoxy-2oxoethyl)amino]-5-fluoro-2-oxido-3,6dioxocyclohexa-1,4-dien-1-

As the first step of evaluation of the biological properties of benzoquinones 1-5 and naphthoquinones 6-9, we have analyzed their ability to inhibit the growth of three mammalian cell lines: Tumor cell lines from human myeloma (RPMI 8226), human mammary adenocarcinoma (MCF-7), and normal mouse fibroblasts (LMTK). Several typical examples of cell growth inhibition are given in Fig. 3. The results obtained with all cells are summarized in Table 2.

Scheme 1. Synthesis of compounds 4 and 5

6


RA, %

RA, % 120

Compound 4, MLTK 100

Compound 5, MLTK 100

80 80 Compound 1, MLTK

60

Compound 4, MCF

60

40

Compound 5, MCF

40 Compound 2, MLTK

20

Compound 1, MCF

20

Compound 2, MCF 0

0 0

50

100

150

200

250

0

300

50

100

150

200

250

300

[Compound], µM

[Compound], µM

Fig. 3. Effects of benzoquinones 1, 2, 4 and 5 (relative activity, RA) on the growth of MCF-7 and MLTK cells. The average error in three experiments for any compound concentration did not exceed 7–10% Table 2. Cytotoxicity (IC50) of the derivatives of polyfluorinated phosphorus-containing 1,4-benzoquinones 1-5 and 1,4-naphthoquinones 6-9 Comp.

1 2 3 4 5 6 7 8 9

IC50 (µM) for different cell lines or % inhibition at maximal concentration, 300 µM* RPMI MCF-7 LMTK Benzoquinones 58.7±4.0 87.7±5.0 140±10 25.0±2.0 34.3±2.6 49.1±3.5 135.4±9.0 305.3±18.0 ≤ 8 %** ≤ 3 %** 109.0±8.0 ≤ 5 %** 20.5±1.5 261±20.0 51.0±4.0 Naphthoquinones 3.4±0.3 5.2±0.5 12.8±1.0 3.7±0.3 7.9±0.7 12.9±1.1 4.8±0.4 7.7±0.6 11.0±1.0 40±3.5 50.3±5.0 80.0±7.3

*Mean ± S.D. from three independent experiments;**Percent of cell, inhibition at high concentration, 300 µM

One can see that the benzoquinones vary greatly in their inhibition of tumor RPMI, MCF, and LMTK cells. Compound 4 does not inhibit remarkably the growth of tumor RPMI and normal LMTK cells, while benzoquinone 3 is inactive only in the case of LMTK cells. At the same time, compound 5 inhibits LMTK cells at about 12.7-fold lower concentration (IC50) than tumor MCF cells. It was previously shown [17-20] that all analyzed fluorinated 1,4-naphthoquinones usually inhibit the growth of all tumor cells at concentrations that are lower or at least comparable to concentrations observed for normal cells. Therefore, we analyzed the inhibition of cell growth at a high concentration of benzoquinones 1-5 (300 µM) not only in the case of RPMI, MCF,

LMTK, but also for human hepatocellular carcinoma HepG2 epithelial tumor cells (HEP) and normal chinese hamster fibroblasts Ag17 (AG) cells. Fig. 4 demonstrates survival of these cells at a high fixed concentration (300 µM) of compounds 1-5. One can see that each compound demonstrates a specific spectrum of its effect on the growth of the five types of cells. Compound 4 efficiently suppresses the growth of only HEP and MCF cells, while compounds 1 and 2 act on all cells except HEP. Compound 3 inhibits the growth of RPMI cells better than other cell types, and the best inhibition of all cell types is observed for compound 5.

7


demonstrated a widely variable IC50 from 0.4 to 275 µM, with alkylamino- and phenylaminocontaining derivatives possessing IC50 = 0.6–18.5 µM [21−23].

For comparison, we have analyzed the relative activity of four phosphorus-containing naphthoquinones 6-9. Fig. 5 demonstrates typical dependencies for these compounds 6-9 in the case of MCF-7 cells. The results obtained with all cells are also summarized in Table 2.

Certain previously studied polyfluorinated naphthoquinones were found to efficiently decrease the mutagenic effect of H2O2 [20−22]. In the present Ames assays, H2O2 was added to TA102 cells at the optimal concentration of 3 mM [20−22,26], and concentrations of the test compound were varied (Fig. 6).

3.3 Analysis of Antioxidants

Compound

Relative inhibition of cell growth , %

It can be seen that quinones 6-8 demonstrated a better inhibition of RPMI, MCF-7 cells (IC50 = 3.4– 7.9 µM) than compound 9 (40−50 µM). Overall, the estimated IC50 values for the benzoquinones varied from 20.5 to 305 µM (Table 2). For naphthoquinones 6-8 the IC50 values are 3.2−90fold lower than those for benzoquinones 1-5, and only naphthoquinone 9 demonstrates this value (40−50 µM) comparable to some of the benzoquinones.

as

It is known that certain compounds interacting with many cell targets at the same time may behave as polyfunctional and possess cytoprotective properties, or, on the contrary, may be cytotoxic, mutagenic, or carcinogenic. Obviously, drugs are more promising when they are not mutagenic, at least at therapeutic concentrations. The Salmonella typhimurium TA102 strain is often used both for evaluation of mutagenicity of different compounds and for detection of antioxidant properties, as judged from suppression of spontaneous mutagenesis in this strain and from a decrease in mutagenicity of oxidants, usually Н2O2. We evaluated the mutagenic activity of benzoquinones 1-5 and naphthoquinones 6-9 in the Ames test [26] using S. typhimurium TA102 as reported by Kemeleva et al. [27]. Mutation induction in the Ames assay was evaluated by calculating the frequency of reversion from histidine auxotrophy to prototrophy in response to the substance under test [26,27]. Fig. 6 shows representative data for benzoquinones 1-4 and naphthoquinones 6-9. The results obtained with all compounds are summarized in Table 3.

MCF RPMI HEP LMTK AG

100 80 60 40 20 0 4

1

2

3

5

Compound number

Fig. 4. Effects of benzoquinones 1-5 (relative activity, RA) on the growth of MCF, RPMI, HEP, LMTK, and AG cells at a high concentration of the compounds (300 µM)

RA,% 100 80 9 8

60 40 20

7 6

0

Interestingly, IC50 values for the benzoquinones (0.37−0.92 µM, average value 0.61±0.13 µM) and naphthoquinones (0.2−0.71 µM, average value 0.40±0.16 µM) in suppression of the spontaneous appearance of mutants are to some extent comparable. On the average, the naphthoquinones suppress spontaneous mutagenesis only 1.5–fold better than the benzoquinones. Previously analyzed polyfluorinated naphthoquinones

0

10

20

30

40

50

[Compound], µМ Fig. 5. Effects of naphthoquinones 6-9 (relative activity, RA) on the growth of MCF-7 cells. The average error in three experiments for any compound concentration did not exceed 5–10% 8


RA, %

RA, %

3 + H2O 2

120

120

4 + H2O2

100

4 - H2O2

80

1 + H2O2

60 3 - H2 O2

20

2 - H2O2

80

60 40

2 + H2O2

100

40

1 - H2O 2

20

0

0 0

1

2

3

4

0

1

RA, %

120 7 + H 2O 2

80

100 80

6 + H2O2

60

8 + H2O2 9 - H2O2

60

6 - H2O2

9 + H2O2

40

40

0 0.0

4

RA, %

120

20

3

[Compound], µM

[Compound], µM

100

2

7 - H2O 2

20

0.5

1.0

1.5

2.0

[Compound], µM

0 0.0

8 - H2O 2

0.5

1.0

1.5

2.0

2.5

[Compound], µM

Fig. 6. Analysis of the mutagenic and antioxidant activity of benzoquinones 1-4 (A, B) and naphthoquinones 6-9 (C, D) by a standard Ames test using the S. typhimurium strain TA102 in the absence and in the presence of 3 mM H2O2. The number of revertants in the absence of H2O2 was taken for 100%. The average error in three experiments for any compound concentration did not exceed 7–10% All the fluorinated benzoquinones efficiently suppressed the H2O2-dependent formation of mutants from 130 to 100% of revertants (range 0.12−0.40 µM, average value 0.27±0.11 µM; the number of revertants observed in the controls without H2O2 was taken for 100%) at approximately 2.5-fold higher concentration than the naphthoquinones (0.02–0.22 µM, average value 0.11±0.06 µM).

1.5-fold higher than for compounds 6-9 (0.29–0.58 µM, average value 0.47±0.09 µM). The data indicate that benzoquinones 1-5 and naphthoquinones 6-9 are not mutagenic themselves and decrease efficiently the level of spontaneous mutagenesis and the mutagenic effect of H2O2 at to a certain extent comparable concentrations.

The concentration causing an additional suppression of the H2O2-induced mutagenesis from 100 to 50% varied for benzoquinones 1-5 in the range 0.44–1.1 µM (average value 0.72±0.19 µM) and it was on the average only approximately

Interestingly, the potency of the majority of previously studied polyfluorinated naphthoquinones in suppressing spontaneous mutagenesis (IC50) in the absence of H2O2 was 1.8–7.0-fold lower than in decreasing H2O2-dependent mutagenesis, while

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Table 3. IC50 values characterizing suppression of spontaneous and H2O2-induced mutagenesis by polyfluorinated benzoquinones 1-5 and naphthoquinones 6-9 IC50, µM* Suppression of H2O2-induced and spontaneous mutagenesis, % From 130 to 100% From 100 to 50% Benzoquinones 0.4±0.03 0.8±0.06 0.18±0.02 0.63±0.05 0.12±0.01 0.44±0.03 0.4±0.03 1.1±0.08 0.24±0.02 0.62±0.05 0.27±0.11 0.72±0.19 Naphthoquinones 0.07±0.006 0.29±0.03 0.12±0.01 0.50±0.05 0.22±0.02 0.52±0.05 0.02±0.002 0.58±0.05 0.11±0.06 0.47±0.09

Comp.

Suppression of spontaneous mutagenesis (from 100 to 50%) 1 2 3 4 5 Average value

0.60±0.06 0.62±0.04 0.37±0.03 0.92±0.07 0.53±0.04 0.61±0.13

6 7 8 9 Average value

0.71±0.07 0.20±0.015 0.32±0.03 0.37±0.03 0.40±0.16

*Mean ± S.D. from three independent experiments is given for each compound

certain other compounds showed comparable IC50 values in the presence and in the absence of H2O2 [20−22]. For benzoquinones 2 and 4 the 50% suppression of spontaneous mutagenesis was observed at the same concentration as for H2O2-induced mutagenesis, while for benzoquinones 1, 3 and 5 – at approximately 1.2−1.3-fold lower concentrations. Naphthoquinones 7-9 suppressed spontaneous mutagenesis at 1.6−2.5 lower concentrations than H2O2-induced cell transformation. At the same time, there was a reverse situation for naphthoquinone 6, which suppressed mutagenesis in the presence of H2O2 at a concentration 2.4-fold lower than in the absence of the peroxide. Since benzoquinones 1-5 and naphthoquinones 6-9 did not enhance the spontaneous mutagenesis and effectively suppressed the mutagenic effect of H2O2, all of them can be considered as efficient antioxidants.

Table 3) than those providing a detectable effect on tumor and normal cells (benzoquinones, IC50 = 20.5−261 µM or higher; naphthoquinones, IC50 = 3.4–80 µM, Table 2). It means that the analyzed benzoquinones and naphthoquinones do not possess an appreciable general toxicity towards cells at low concentrations, when they protect the cells from mutagenesis. In addition, compounds 15 and 6-9 suppress the growth of normal LMTK cells at higher concentrations than tumor cells (Table 2). It is possible that all these compounds can play a double role, acting both as inhibitors of cell phosphatases and as antioxidants. Overall, fluorinated naphthoquinone derivatives are less active in generating reactive oxygen species and may be more promising inhibitors of Cdc phosphatases as compared to 1,4-naphthoquinone [15–17]. Since all of these compounds show to a certain extent comparable IC50 values in suppressing the spontaneous and H2O2-induced mutagenesis, they may be considered to be potentially about equally promising as antioxidants. Benzoquinones 1-5 are not universal in inhibition of tumor cells growth (Table 2). At the same time, benzoquinone 4 efficiently inhibits the growth of MCF and HEP cells tumor cells at lower concentration than normal MLTK and AG cells. All the fluorinated naphthoquinones are more promising as suppressors of all tumor cells, and they inhibit the growth of normal cells at higher concentrations.

It was not possible to exclude that the complete suppression of mutant cell appearance by these quinones at the observed concentration may result not only from their ability to suppress spontaneous and H2O2-induced mutagenesis, but also from their higher general toxicity towards bacterial cells. However, the 50% decrease in spontaneous and H2O2-dependent mutagenesis of bacterial cells including their possible general toxicity for the benzoquinones and naphthoquinones was observed at significantly lower concentrations (IC50 = 0.2–1.1 µM, 10


6.

4. CONCLUSION Our data indicate that compounds 5 and 6-8 are the most promising, demonstrating a better cytotoxic effect against all types of cancer cells as compared to normal mammalian cells. Quinones 68 are antioxidants protecting cells from spontaneous mutagenesis, and they can possess minimal general toxicity towards different cells. In addition, compounds 1-5 are the best protectors of bacterial cells from H2O2-dependent mutagenesis. Taken together, our data suggest that compounds 5 and 6-8 may be considered as potentially useful inhibitors of tumor cell growth, at the same time not excluding that derivatives 1-4 and 6 can also be used as promising suppressors of tumor cells.

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ACKNOWLEDGEMENTS 10.

This research was made possible by grants from the Presidium of the Russian Academy of Sciences (Molecular and Cellular Biology Program, No. 6.2), the interdisciplinary grant No. 59 from the Siberian Division of the Russian Academy of Sciences, the grants of Russian Foundation for Basic Research (nos 13-0400211, 14-03-00108, 16-33-00005) and Novosibirsk District Administration. Authors would like to acknowledge the Multi-Access Chemical Service Center SB RAS for spectral and analytical measurements.

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COMPETING INTERESTS Authors have interests exist.

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competing 13.

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