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. 2018 Nov 22;27(3):303–311. doi: 10.1016/j.jsps.2018.11.012

Triazene salts: Design, synthesis, ctDNA interaction, lipophilicity determination, DFT calculation, and antiproliferative activity against human cancer cell lines

Joanna Cytarska a,⁎, Artur Anisiewicz b, Angelika Baranowska-Łączkowska c, Adam Sikora d, Joanna Wietrzyk b, Konrad Misiura a, Krzysztof Z Łączkowski a
PMCID: PMC6438848  PMID: 30976172

Abstract

Synthesis, characterization and investigation of antiproliferative activity of nine triazene salts against human cancer cells lines (MV-4-11, MCF-7, JURKAT, HT-29, Hep-G2, HeLa, Du-145 and DAUDI), and normal human mammary epithelial cell line (MCF7-10A) is presented. The structures of novel compounds were determined using 1H and 13C NMR, and GC-APCI-MS analyses. Among the derivatives, compound 2c, 2d, 2e and 2f has very strong activity against biphenotypic B myelomonocytic leukemia MV4-11, with IC50 values from 5.42 to 7.69 µg/ml. The cytotoxic activity of compounds 2c-2f against normal human mammary gland epithelial cells MCF-10A is 6–11 times lower than against cancer cell lines. Our results also show that compounds 2c and 2f have very strong activity against DAUDI and HT-29 with IC50 4.91 µg/ml and 5.59 µg/ml, respectively. Their lipophilicity was determined using reversed-phase ultra-performance liquid chromatography and correlated with antiproliferative activity. Our UV–Vis spectroscopic results indicate also that triazene salts tends to interact with negatively charged DNA phosphate chain. To support the experiment, theoretical calculations of the 1H NMR shifts were carried out within the Density Functional Theory.

Keywords: Antiproliferative activity, Triazene, Nuclear Magnetic Resonance, Lipophilicity, DNA

1. Introduction

One of the most aggravating diseases in the present world is cancer. Each year more than ten million people are diagnosed with some type of cancer, and more than half of them can die of it. In many countries, cancer diseases occupy the second place immediately after cardiovascular diseases (Boyle and Levin, 2008). Although public awareness about the treatment and prevention of cancer is still growing, and although new anticancer drugs are still being developed, cancer remains the major health problem in the around the world (Ferlay et al., 2013). Many of the current anticancer drugs have very low selectivity, relatively high side effects, limited bioavailability and oral absorption or rapid metabolism (Zawilska et al., 2013). For this reason, many prodrug groups have been developed that are activated in the cancer cells. Such a group of prodrug alkylating agents are triazenes which are successfully used for the fight against many tumors, such as leukemia, lymphoma, melanoma, and sarcoma (Yahalom et al., 1983, Smith et al., 1990). Some triazenes have also been used as a prodrug candidate for melanocyte-directed enzyme prodrug therapy (MDEPT) (Monteiro et al., 2013).

Approved by the Food and Drug Administration (FDA) for medical use Dacarbazine (1) (5-(3,3-dimethyltriazene)imidazol-4-carboxamide, DTIC) and Temozolomide (2) (8-carbamoyl-3-methyl-imidazol[5,1-d]-1,2,3,5-tetrazin-4(3H)-one, TMZ) are the only triazenes used in the treatment of cancer (Meer et al., 1986, O’Reilly et al., 1993). DTIC requires activation by the cytochrome P450, resulting in the production of a very reactive methyldiazonium cations that can react with DNA O6-methylguanine, while Temozolomide does not require enzymatic activation and is hydrolysed to the active form already under physiological conditions (Meer et al., 1986, Friedman et al., 2000). However, many types of cancer cells have a mechanism to repair this type of damage by expressing a protein O6-alkylguanine DNA alkyltransferase thereby reducing the effectiveness of the drugs used (Happold et al., 2012, Kanugula and Pegg, 2003, Friedman et al., 2000).

These results encouraged us to continue our investigation on the synthesis and molecular properties of anticancer agents with divers mechanism of action (Łączkowski et al., 2014, Cytarska et al., 2015, Łączkowski et al., 2016, Łączkowski et al., 2018). Our research began with the design and synthesis of nine novel triazene salts and evaluation of their antiproliferative activity against human cancer cells lines (biphenotypic B myelomonocytic leukemia MV4-11, human breast carcinoma MCF-7, human leukemic T-cell lymphoblast JURKAT, human colon adenocarcinoma HT-29, human hepatocellular carcinoma Hep-G2, human cervical carcinoma HeLa, human prostate carcinoma Du-145, Burkitt lymphoma DAUDI, and normal human mammary epithelial cell line MCF7-10A using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliun bromide (MTT) or sulforhodamine B (SRB) assays. Moreover, for a better understanding of the mechanism of action we also performed interaction of triazenes with ctDNA using UV–Visible absorption spectroscopic (Sohrabi et al., 2018, Moosavi-Movahedi et al., 2004, Marouzi et al., 2017, Omidvar et al., 2013, Rashidipour et al., 2016, Bakaeean et al., 2012, Moosavi-Movahedi et al., 2003), as well as their lipophilicity parameter. To support the experiment, theoretical calculations of the 1H NMR shifts were carried out within the Density Functional Theory.

2. Experimental

2.1. Materials and methods

All experiments were carried out under air atmosphere unless stated otherwise. Reagents were generally the best quality commercial-grade products and were used without further purification. 1H NMR (700 and 400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker Avance III multinuclear instrument. MS spectra were recorded on triple quadrupole mass spectrometer detector LCMS-8040 (Shimadzu, Japan). Melting points were determined in open glass capillaries and are uncorrected. Analytical TLC was performed using Macherey-Nagel Polygram Sil G/UV254 0.2 mm plates. Bis(2-chloro-ethyl)amine hydrochloride, and appropriate anilines were commercial materials (Aldrich).

2.1.1. 3-(4-Acetylphenyl)-1-(2-chloroethyl)-4,5-dihydro-1H-1,2,3-triazol-3-ium chloride (2a). Typical procedure

1-(4-Aminophenyl)ethanone (1.50 g, 11.1 mmoles) was added to 6 M HCl (3.7 ml) and the reaction mixture was warmed until disappearance of the starting amine. The solution was cooled to 0 °C and sodium nitrite (0.80 g, 11.7 mmoles) in water (2 ml) was added dropwise during 10 min. Then bis(2-chloroethyl)amine hydrochloride (2.18 g, 12.2 mmoles) was slowly added, and next reaction mixture was alkalized with saturated NaHCO3 and left stirring for 15 min. Solid product was filtered, washed with water and dried. Yield: 2.70 g, 84%; mp 121–124 °C decomp., (dichloromethane/methanol, 80:20, Rf = 0.15). 1H NMR (DMSO‑d6, 400 MHz), δ (ppm): 2.64 (s, 3H, CH3); 4.19 (t, 2H, CH2, J = 5 Hz); 4.63 (t, 2H, CH2, J = 5 Hz); 4.73 (t, 2H, CH2, J = 14 Hz); 4.92 (t, 2H, CH2, J = 13 Hz); 7.75 (d, 2H, 2CH, J = 9 Hz); 8.17 (d, 2H, 2CH, J = 9 Hz). 13C NMR (DMSO‑d6, 100 MHz), δ (ppm): 27.31 (CH3); 40.64 (CH2); 52.22 (CH2); 55.43 (CH2); 55.36 (CH2); 118.47 (2CAr); 130.61 (2CAr); 136.58 (C); 139.89 (C); 197.43 (CO). GC-APCI-MS (m/z, %): 216 [(M+-2Cl), 100].

2.1.2. 1-(2-Chloroethyl)-3-phenyl-4,5-dihydro-1H-1,2,3-triazol-3-ium chloride (2b)

Yield: 1.67 g, 62%, (dichloromethane/methanol, 80:20, Rf = 0.20); mp 89–91 °C decomp. 1H NMR (DMSO‑d6, 700 MHz), δ (ppm): 4.21 (t, 2H, CH2, J = 5.5 Hz); 4.62 (t, 2H, CH2, J = 5 Hz); 4.74 (t, 2H, CH2, J = 14 Hz); 4.95 (t, 2H, CH2, J = 13 Hz); 7.52 (t, 1H, CH, J = 7 Hz); 7.63 (m, 2H, 2CH); 7.67 (m, 2H, 2CH). 13C NMR (DMSO‑d6, 100 MHz), δ (ppm): 40.78 (CH2); 52.48 (CH2); 54.94 (CH2); 54.66 (CH2); 118.55 (2CAr); 129.38 (C); 130.56 (2CAr); 136.71 (C). GC-APCI-MS (m/z, %): 174 [(M+-2Cl), 100].

2.1.3. 1-(2-Chloroethyl)-3-p-tolyl-4,5-dihydro-1H-1,2,3-triazol-3-ium chloride (2c)

Yield: 2.40 g, 83%, (dichloromethane/methanol, 95:5, Rf = 0.16); mp 107–108 °C decomp. 1H NMR (DMSO‑d6, 700 MHz), δ (ppm): 2.40 (s, 3H, CH3); 4.18 (t, 2H, CH2, J = 5.5 Hz); 4.57 (t, 2H, CH2, J = 6 Hz); 4.67 (t, 2H, CH2, J = 14 Hz); 4.91 (t, 2H, CH2, J = 12 Hz); 7.44 (d, 2H, 2CH, J = 8 Hz); 7.56 (d, 2H, 2CH, J = 8 Hz). 13C NMR (DMSO‑d6, 100 MHz), δ (ppm): 21.08 (CH3); 40.83 (CH2); 52.66 (CH2); 54.37 (CH2); 54.79 (CH2); 118.51 (2CAr); 130.96 (2CAr); 134.30 (C); 139.43 (C). GC-APCI-MS (m/z, %): 188 [(M+-2Cl), 100].

2.1.4. 1-(2-Chloroethyl)-3-(3-(trifluoromethyl)phenyl)-4,5-dihydro-1H-1,2,3-triazol-3-ium chloride (2d)

Yield: 1.30 g, 37%, (dichloromethane/methanol, 95:5, Rf = 0.18); mp 131–133 °C decomp. 1H NMR (DMSO‑d6, 400 MHz), δ (ppm): 4.20 (t, 2H, CH2, J = 5 Hz); 4.63 (t, 2H, CH2, J = 5.5 Hz); 4.75 (t, 2H, CH2, J = 14 Hz); 4.95 (t, 2H, CH2, J = 13 Hz); 7.88 (m, 2H, 2CH); 7.96 (m, 2H, 2CH). 13C NMR (DMSO‑d6, 100 MHz), δ (ppm): 40.67 (CH2); 52.46 (CH2); 55.51 (CH2); 55.27 (CH2); 115.37 (q, C, JC-F = 4 Hz); 120.70 (C); 125.60 (q, C, JC-F = 4 Hz); 131.06 (q, C, JC-F = 33 Hz); 131.98 (2C); 137.49 (C). GC-APCI-MS (m/z, %): 242 [(M+-2Cl), 100].

2.1.5. 1-(2-Chloroethyl)-3-(4-chlorophenyl)-4,5-dihydro-1H-1,2,3-triazol-3-ium chloride (2e)

Yield: 1.44 g, 46%, (dichloromethane/methanol, 80:20, Rf = 0.19); mp 151–156 °C decomp. 1H NMR (DMSO‑d6, 400 MHz), δ (ppm): 4.17 (t, 2H, CH2, J = 5 Hz); 4.58 (t, 2H, CH2, J = 5.5 Hz); 4.68 (t, 2H, CH2, J = 14 Hz); 4.88 (t, 2H, CH2, J = 13 Hz); 7.67 (m, 2H, 2CH); 7.71 (m, 2H, 2CH). 13C NMR (DMSO‑d6, 100 MHz), δ (ppm): 40.73 (CH2); 52.59 (CH2); 55.13 (2CH2); 120.40 (2CAr); 130.48 (2CAr); 133.50 (C); 135.64 (C). GC-APCI-MS (m/z, %): 208 [(M+-2Cl), 100].

2.1.6. 1-(2-Chloroethyl)-3-(4-(ethoxycarbonyl)phenyl)-4,5-dihydro-1H-1,2,3-triazol-3-ium chloride (2f)

Yield: 2.80 g, 80%, (dichloromethane/methanol, 80:20, Rf = 0.13); mp 153–154 °C decomp. 1H NMR (DMSO‑d6, 700 MHz), δ (ppm): 1.37 (t, 3H, CH3, J = 7 Hz); 4.21 (t, 2H, CH2, J = 5 Hz); 4.38 (t, 2H, CH2, J = 5 Hz); 4.65 (t, 2H, CH2, J = 13 Hz); 4.75 (t, 2H, CH2, J = 14 Hz); 4.93 (q, 2H, CH2, J = 8 Hz); 7.78 (d, 2H, 2CH, J = 9 Hz); 8.18 (d, 2H, 2CH, J = 9 Hz). 13C NMR (DMSO‑d6, 100 MHz), δ (ppm): 14.60 (CH3); 40.64 (CH2); 52.21 (CH2); 55.38 (CH2); 55.49 (CH2); 61.69 (CH2); 118.57 (2CAr); 129.89 (C); 130.47 (2CAr); 140.07 (C); 165.07 (CO). GC-APCI-MS (m/z, %): 246 [(M+-2Cl), 100].

2.1.7. 1-(2-Chloroethyl)-3-(4-nitrophenyl)-4,5-dihydro-1H-1,2,3-triazol-3-ium chloride (2g)

Yield: 2.90 g, 91%, (dichloromethane/methanol, 95:5, Rf = 0.12); mp 116–117 °C decomp. 1H NMR (DMSO‑d6, 700 MHz), δ (ppm): 4.24 (t, 2H, CH2, J = 5.5 Hz); 4.71 (t, 2H, CH2, J = 5 Hz); 4.83 (t, 2H, CH2, J = 13 Hz); 4.96 (t, 2H, CH2, J = 13 Hz); 7.89 (d, 2H, 2CH, J = 9 Hz); 8.48 (d, 2H, 2CH, J = 9 Hz). 13C NMR (DMSO‑d6, 100 MHz), δ (ppm): 40.57 (CH2); 52.15 (CH2); 55.68 (CH2); 56.13 (CH2); 119.30 (2CAr); 126.14 (2CAr); 141.37 (C); 146.81 (C). GC-APCI-MS (m/z, %): 219 [(M+-2Cl), 100].

2.1.8. 1-(2-Chloroethyl)-3-(4-(sodiumsulfonate)phenyl)-4,5-dihydro-1H-1,2,3-triazol-3-ium chloride (2h)

Yield: 2.00 g, 52%, (dichloromethane/methanol, 80:20, Rf = 0.10); mp 251–253 °C decomp. 1H NMR (DMSO‑d6, 700 MHz), δ (ppm): 4.17 (t, 2H, CH2, J = 6 Hz); 4.58 (t, 2H, CH2, J = 5.5 Hz); 4.65 (t, 2H, CH2, J = 14 Hz); 4.88 (t, 2H, CH2, J = 15 Hz); 7.58 (d, 2H, 2CH, J = 9 Hz); 7.89 (d, 2H, 2CH, J = 9 Hz). 13C NMR (DMSO‑d6), δ (ppm): 40.66 (CH2); 52.31 (CH2); 54.78 (CH2); 54.97 (CH2); 117.84 (2CAr); 127.59 (2CAr); 136.43 (C); 148.59 (C). GC-APCI-MS (m/z, %): 276 [(M+-2Cl), 100].

2.1.9. 1-(2-Chloroethyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-1,2,3-triazol-3-ium chloride (2i)

Yield: 1.30 g, 44%, (dichloromethane/methanol, 80:20, Rf = 0.11); mp 91–93 °C decomp. 1H NMR (DMSO‑d6, 400 MHz), δ (ppm): 4.16 (t, 2H, CH2, J = 6 Hz); 4.56 (t, 2H, CH2, J = 5.5 Hz); 4.67 (t, 2H, CH2, J = 13 Hz); 4.90 (t, 2H, CH2, J = 14 Hz); 7.50 (m, 2H, 2CH); 7.71 (m, 2H, 2CH). 13C NMR (DMSO‑d6, 100 MHz), δ (ppm): 40.78 (CH2); 52.88 (CH2); 54.82 (CH2); 54.92 (CH2); 117.52 (d, 2CAr, JC-F = 24 Hz); 121.27 (d, 2CAr, JC-F = 9 Hz); 133.30 (d, C, JC-F = 2 Hz); 160.80 (C). GC-APCI-MS (m/z, %): 192 [(M+-2Cl), 100].

2.2. Biological activity

2.2.1. Cells

Human cancer cell lines Du-145, HeLa, HepG2, HT-29, MCF-7, MV-4-11 and normal human mammary gland epithelial cells MCF-10A were obtained from American Type Culture Collection (Rockville, Maryland, USA). DAUDI and Jurkat cell lines were purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. All cell lines were maintained in liquid nitrogen at the Cell Culture Collection of Institute of Immunology and Experimental Therapy (Wroclaw, Poland). The Du-145 and MCF-7 cell lines were grown in Eagle’s medium (IIET, Wroclaw, Poland) with addition of 10% fetal bovine serum (Sigma-Aldrich, Steinheim, Germany). Medium of MCF-7 was supplemented with MEM Non-Essential Amino Acids Solution, 2.0 mM L-glutamine and 8 µg/ml of insulin (all Sigma-Aldrich, Steinheim, Germany) and medium of DU-145 was enriched by 1.0 mM sodium pyruvate and 4.0 mM L-glutamine (both Sigma-Aldrich, Steinheim, Germany). The HeLa and HT-29 cell lines were maintained in a mixture of RPMI + HEPES medium and Opti-MEM medium (1:1, IIET, Wroclaw, Poland) with the addition of 5% fetal bovine serum (HyClone, GE Healthcare, UK), 2.0 mM L-glutamine and 1.0 mM sodium pyruvate (both Sigma-Aldrich, Steinheim, Germany). HepG2 cells was cultured in Dulbecco medium DMEM (Gibco, Scotland, UK) with 10% fetal bovine serum (HyClone, GE Healthcare, UK) and 2.0 mM L-glutamine (Sigma-Aldrich, Steinheim, Germany). DAUDI, Jurkat and MV-4-11 cell lines were grown in RPMI 1640 medium with GlutaMAX (Gibco, Scotland, UK) with addition of 10% fetal bovine serum (Sigma-Aldrich, Steinheim, Germany). The medium of MV-4-11 cells was supplemented in 1 mM sodium pyruvate (Sigma-Aldrich, Germany). MCF-10A cells were maintained in Ham’s F-12 Nutrient Mixture with 5% horse serum (both Gibco, Scotland, UK). The MCF10A medium was enriched by 10 µg/ml of insulin, 0.5 µg/ml of hydrocortisone, 0.05 µg/ml of cholera toxin from Vibrio Cholerae and 20 ng/ml of human epidermal growth factor (all Sigma-Aldrich, Steinheim, Germany). All culture media were supplemented with 100 units/mL penicillin (Polfa Tarchomin S.A., Warsaw, Poland) and 100 µg/mL streptomycin (Sigma-Aldrich, Steinheim, Germany). The cell lines were cultured at 37 °C in a humid atmosphere saturated with 5% CO2.

2.2.2. Compounds

Prior to usage, the compounds were dissolved in DMSO and culture medium (1:9) to the concentration of 1 mg/ml, and subsequently diluted in culture medium to reach the required concentrations (0.1, 1, 10 and 100 µg/ml).

2.2.3. In vitro antiproliferative assay

The cells were plated in 96-well plates (Sarstedt, Germany) in an appropriate density: 1 × 104 per well for DAUDI, Du-145, Jurkat, Hep-G2, MV-4-11 and MCF-10A, 0.75 × 104 per well for HT-29 and MCF-7 and 0.25 × 104 per well for HeLa cell line. After twenty four hours the cultured cell lines were exposed to different concentrations of the tested agents for 72 h (total plate incubation time: 96 h). To determine cytotoxicity of tested agents, the antiproliferative tests were performed as previously described (Wietrzyk et al., 2007). The MTT assay was performed for DAUDI, Jurkat, MV-4-11 and the SRB assay was conducted for DU-145, HeLa, HepG-2, HT-29, MCF-7, MCF-10A cell lines. The results were presented as an IC50 values (inhibitory concentration 50) – the dose (μg/mL) of tested compounds that inhibits cell proliferation at 50%. Each concentration of examined agents was tested in triplicate in a single experiment, which was repeated at least 3 times (Rubinstein et al., 1990, Bramson et al., 1995). The activity of examined agents was compared to the activity of reference compound – cis-platin (Accord Healthcare Polska, Warsaw, Poland). The control of 99.8% ethanol that was the solvent of the tested agents was also performed.

2.2.4. SRB cytotoxic test

Cells were attached to the bottom of plastic wells by fixing them with cold 50% TCA (trichloroacetic acid, POCH, Gliwice, Poland) on top of the culture medium in each well. The plates were incubated at 4 °C for 1 h and then washed five times with tap water. The cellular material fixed with TCA was stained with 0.14% sulforhodamine B (SRB, Sigma-Aldrich, Germany) and dissolved in 1% acetic acid (POCH, Gliwice, Poland) for 30 min. Unbound dye was removed by rinsing (4X) in 1% acetic acid. The protein-bound dye was extracted with 10 mM unbuffered Tris base (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) for determination of the optical density (λ = 540 nm) in a computer-interfaced, 96-well Synergy H4 (BioTek Instruments USA) photometer microtiter plate reader (Sidoryk et al., 2012).

2.2.5. MTT cytotoxic test

20 µl of MTT solution (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, stock solution: 5 mg/ml) was added to each well and incubated for 4 h. After the incubation time was complete, 80 µl of the lysis mixture was added to each well (lysis mixture: 225 ml dimethylformamide, 67.5 g sodium dodecyl sulfate and 275 ml of distilled water). The optical densities of the samples were read after 24 h on a Synergy H4 (BioTek Instruments USA) photometer microtiter plate reader at 570 nm (Sidoryk et al., 2012). All of chemicals were obtained from Sigma-Aldrich, Germany.

2.3. Determiantion of lipophilicity by RP UPLC

The studies were performed on the UPLC–MS/MS system equipped with solvent delivery two pumps LC-30AD combined with gradient systems, degasser model DGU-20A5, an autosampler model SIL-30AC, a column oven model CTO-20AC, UV detector model SPD-M20A and triple quadrupole mass spectrometer detector LCMS-8040 (Shimadzu, Japan). Kinetex C18 (150 × 4.6 mm; 2.6 µm) column was purchased from Phenomenex Co. The methanol concentration, expressed in volumetric ratio v/v, ranged from 0.65 to 0.95 in constant steps of 0.05. Tested compounds were dissolved in methanol (10 µg/ml). The flow rate of the mobile phase was 0.5 ml/min. All analyses were carried out at 25 °C, and detection wavelength of 254 nm was chosen.

2.4. Spectroscopy

The UV absorption spectra were recorded on T60U spectrophotometer (PG Instruments) equipped with quartz cells of 1 cm path length; the pH value of the solutions were determined with CP-501 pH-meter (Elmetron). ctDNA, ethidium bromide dye (EB) and Tris were obtained from the Sigma-Aldrich Company. Tris-HCl buffer solution (concentration 10 mM) was prepared by dissolving solid substance in doubly distilled water and acidify by HCl to pH 7.4. The stock solution of ctDNA was prepared by dissolving solid substance in Tris-HCl buffer. EB solution was prepared by dissolving solid substance in ethanol and Tris-HCl solution. All solutions were stored at 4 °C. The concentrations of ctDNA and EB were determined by absorption spectroscopy using the molar extinction coefficient of 6600 M−1 cm−1 at 260 nm and 5800 M−1 cm−1 at 480 nm, respectively. The solutions of ctDNA had a ratio of UV absorbance at 260 and 280 nm larger than 1.8, which indicated that ctDNA was sufficiently free from protein. The stock solutions of substances of 2a-2i series at concentration 100 mM were prepared by dissolving solid substance in ethanol and Tris-HCl solution (1:10) (Charak et al., 2012).

2.5. Quantum mechanical calculations

Theoretical evaluation of NMR proton chemical shifts was carried out for all investigated compounds employing Density Functional Theory (DFT) approximation. As a first step, optimization of investigated systems geometrical parameters was carried out at the B3LYP/6-311G** level of theory and followed by frequency calculations to confirm that the resulting structures correspond to real minima on the potential energy surface. Single starting point per system was used. Next, chemical shifts were calculated with respect to tetramethylsilane (TMS) and compared to experimental data recorded in DMSO. Based on results of our earlier theoretical study of NMR shifts in similar compounds (Baranowska-Łączkowska et al., 2018) we used M06 and B3LYP functionals, combining them with the aug-pcS-1 basis set of Jensen (Jensen, 2008). London Atomic Orbitals (LAOs) (London, 1937) were employed to ensure gauge-origin independent results. Solvent effects were not included in the calculations, as the proton shifts of investigated compounds are not expected to be strongly solvent-dependent. All calculations were carried out using the Gaussian 09 package (Frisch et al., 2009). The aug-pcS-1 basis set was taken from the EMSL Basis Set Library (Feller, 1996, Schuchardt et al., 2007).

3. Results and discussion

3.1. Chemistry

The target triazene salts containing chloroethyl group were obtained in two steps, one-flask synthesis. In the first step, para- or meta-substituted benzenediazonium chlorides were prepared by diazotization reaction of appropriate anilines 1a-1i in the presence of sodium nitrite in 6 M hydrochloric acid (Scheme 1). In the next step, a series of triazene 2a-2i was synthesized by reaction between different substituted benzenediazonium chlorides and bis(2-chloroethyl)amine hydrochloride, followed by addition of sodium hydrogen carbonate, with good yield (37–91%) and chemical purity. All of the synthesized derivatives were characterized by spectroscopic methods 1H NMR (700 MHz) and 13C NMR (100 MHz), and GC-APCI-MS analyses. 1H NMR and 13C NMR spectra of triazenes 2a-2i showed four characteristic triplets at δ (4.16–4.96 ppm) and peaks at δ (40.57–56.13 ppm) due to the four methylene groups, which indicates the conversion of substrates to the expected products with the simultaneous creation of internal triazene salts 2a-2i. The mass spectra of all compounds showed (M+-2Cl) ion in the positive-ion mode which is fully consistent with the assigned structures.

Scheme 1.

Scheme 1

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Synthesis of triazene salts 2a-2i.

3.2. Calculations

To further confirm the structure of resulting triazene salts 2a-2i, theoretical evaluation of NMR proton chemical shifts was carried out for all investigated compounds employing Density Functional Theory (DFT) approximation. Resulting theoretical chemical shifts are presented in (Table 1) together with the corresponding experimental data. Values of the root mean square error (rmse) calculated with respect to experimental data are also printed. Complete set of geometrical parameters of investigated systems can be found in Supplementary Material. We note here that all calculations have been carried out employing frozen structures, and thus protons being chemically equivalent due to rotations around single bonds, and leading to single experimental signal, in our calculations have different chemical environment and appear at different chemical shifts.

Table 1.

DFT chemical shifts (ppm) of compounds 2a-2i, together with the experimental values recorded in DMSO.

Triazene 2a-2i RMSE
2a H18 H19 H20 H21 H22 H23 H24 H25 H26 H29 H27 H28 H30 H31 H32
δB3LYP 4.50 4.33 4.77 4.76 3.91 4.64 3.68 3.80 8.10 7.08 8.28 8.93 2.79 2.79 2.69 0.31
δM06 4.54 4.34 4.83 4.76 3.98 4.67 3.58 3.81 8.17 7.21 8.37 9.08 2.79 2.75 2.68 0.33
δexp 4.73 4.92 4.63 4.19 7.75 8.17 2.64
2b H15 H16 H17 H18 H19 H20 H21 H22 H23 H27 H24 H26 H25
δB3LYP 4.48 4.31 4.74 4.77 3.88 4.61 3.69 3.78 8.05 7.06 7.91 7.86 7.91 0.32
δM06 4.51 4.34 4.74 4.75 3.92 4.58 3.65 3.78 8.10 7.12 7.92 7.91 8.01 0.34
δexp 4.74 4.95 4.62 4.21 7.63 7.67 7.52
2c H20 H21 H22 H23 H24 H25 H26 H27 H16 H17 H18 H19 H28 H29 H30 . .
δB3LYP 4.44 4.27 4.71 4.73 3.83 4.57 3.66 3.76 6.87 7.91 7.71 7.62 2.77 2.77 2.36 0.28
δM06 4.45 4.28 4.71 4.73 3.89 4.57 3.61 3.76 6.96 8.02 7.73 7.64 2.73 2.71 2.32 0.28
δexp 4.67 4.91 4.57 4.18 7.44 7.56 2.40
2d H19 H20 H21 H22 H23 H24 H25 H26 H27 H28 H29 H30
δB3LYP 4.55 4.37 4.75 4.74 3.94 4.66 3.69 3.79 8.50 8.26 7.98 7.13
δM06 4.58 4.37 4.81 4.72 3.96 4.72 3.59 3.76 8.56 8.36 8.08 7.26
δexp 4.75 4.95 4.63 4.20 7.88, 7.96
2e H16 H17 H18 H19 H20 H21 H22 H23 H24 H27 H25 H26
δB3LYP 4.47 4.31 4.70 4.70 3.87 4.59 3.67 3.77 7.88 6.88 7.82 7.77 0.30
δM06 4.49 4.31 4.72 4.68 3.93 4.60 3.59 3.77 7.96 6.97 7.85 7.81 0.29
δexp 4.68 4.88 4.58 4.17 7.67 7.71
2f H20 H21 H22 H23 H24 H25 H26 H27 H28 H31 H29 H30 H32 H33 H34 H35 H36
δB3LYP 4.50 4.32 4.73 4.79 3.90 4.62 3.67 3.80 7.95 7.09 7.94 8.44 1.20 1.84 1.51 4.01 4.49 0.28
δM06 4.48 4.28 4.77 4.79 3.98 4.68 3.63 3.80 7.97 7.24 8.12 8.57 1.09 1.81 1.45 3.98 4.41 0.28
δexp 4.75 4.93 4.65 4.21 7.78 8.18 1.37 4.38
2g H18 H19 H20 H21 H22 H23 H24 H25 H26 H29 H27 H28
δB3LYP 4.58 4.40 4.77 4.74 3.97 4.68 3.70 3.82 8.07 7.07 8.97 8.96 0.38
δM06 4.61 4.41 4.81 4.73 4.04 4.71 3.60 3.82 8.13 7.16 9.05 9.11 0.40
δexp 4.83 4.96 4.71 4.24 7.89 8.48
2h H20 H21 H22 H23 H24 H25 H26 H27 H28 H31 H29 H30
δB3LYP 4.43 4.29 4.69 4.77 3.85 4.60 3.67 3.77 7.90 6.93 8.44 8.39 0.35
δM06 4.45 4.25 4.77 4.76 3.87 4.63 3.64 3.81 7.95 7.04 8.58 8.54 0.38
δexp 4.65 4.88 4.58 4.17 7.58 7.89
2i H16 H17 H18 H19 H20 H21 H22 H23 H24 H27 H25 H26
δB3LYP 4.48 4.31 4.70 4.73 3.87 4.59 3.67 3.77 7.99 7.00 7.56 7.49 0.27
δM06 4.51 4.33 4.70 4.72 3.94 4.59 3.61 3.77 8.09 7.10 7.57 7.54 0.27
δexp 4.67 4.90 4.56 4.16 7.50a 7.71a
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a

Opposite assignment is made based on the M06/aug-pc-1 results. See text for details.

For the purpose of comparison with experimental data average theoretical chemical shifts are thus calculated from chemical shifts of protons which would be chemically equivalent. Next experimental signals are assigned to the respective protons based on the comparison of theoretical and experimental values.

The assignments made based on the B3LYP results agree very well with those made on the M06 values, with the former functional leading in general to slightly smaller rmse values. An exception is observed in the case of molecule 2i, for which the two functionals yield opposite assignments of aromatic protons signals. Precisely, the B3LYP calculation allows to assign signal of 7.50 ppm to protons H24 and H27, and 7.71 ppm to protons H25 and H26, while opposite conclusion is made based on the M06 results. Both, theoretical and experimental spectra of all investigated systems confirm assumed structure of the products, and thus mechanism of reaction. Detailed analysis reveals that signals corresponding to protons of 2-chloroethyl group and 1,2,3-triazene ring appear in all investigated systems at approximately constant ppm values, maximum differences being in the order of 0.05–0.20 ppm. This is in line with our expectations, as all these protons are relatively far from the changing substituent whose influence is thus very small. In contrary, the signals of aromatic protons are much more sensitive to the change of the nearby substituent, and their position changes up to 0.9 ppm.

3.3. Lipophilicity determination

The ability to penetrate the drug through biological membranes is the decisive parameter responsible for its activity. Parameter describing this property is lipophilicity that is defined as the partition coefficient between the an aqueous phase and the non-aqueous phase usually 1-octanol and is expressed as log P (Arnott et al., 2012). One of the best methods to determine concentration of a compound in various solvents needed to determine lipophilicity is reversed-phase high performance liquid chromatography (RP-HPLC) (Marciniec et al., 2016). Therefore, for the determination of relative lipophilicity of triazene derivatives 2a-2i we used reversed-phase ultra-performance liquid chromatography (RP UPLC), based upon sub 3-µm porous particles. Chromatographic capacity factors (k) were calculated: k = (tR/tM) − 1, where tR [min] denotes retention time, and tM [min] is time for dead volume. The dead time was determined using uracil as a tM marker. Linear relationship between the log k and the concentration of the organic modifier in the mobile phase (methanol) was determined on the basis of the Soczewiński-Wachtmeister equation: log k = log kw + SΦ, where log kw denotes the capacity factor of the analyte in pure water, Φ is organic modifier concentration in the mobile phase, and S denotes slope of the regression curve. The lipophilicity parameter Φ0 was calculated using relationship Φ0 = −log kw/S, and R2 is correlation coefficient. Experimentally determined lipophilic parameters log kw are presented in (Table 2).

Table 2.

The lipophilicity parameters determined by RP UPLC-MS/MS analysis.

2a 2b 2c 2d 2e 2f 2g 2h 2i
log kw −1.478 −2.002 −2.675 −3.373 −3.503 −2.938 −1.504 −0.914 −1.873
-S 0.755 1.318 2.000 2.802 2.928 2.259 0.594 0.709 1.027
Φ0 1.958 1.519 1.338 1.204 1.196 1.301 2.532 1.289 1.824
R2 0.969 0.985 0.998 0.990 0.984 0.993 0.967 0.980 0.971
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In our research we have observed the linear dependence between log k values and concentration of organic modifier in the eluent with correlation coefficient (R2 = 0.967–0.998) value. The analysis shows that the value of the log kw is in the range from −3.503 to −0.914. From all tested compounds, the lowest log kw values, in the range from −3.503 to −2.675, were observed for compound 2c, 2d, 2e and 2f containing methyl, trifluoromethyl, chloride and carboxyethyl substituents respectively. The compounds 2a, 2b, 2g, and 2i containing acetyl, hydrogen, nitro, and fluoro substituents, showed average values of log kw, in the range from −2.002 to −1.478. The highest value of the log kw was found for compounds 2h containing sodium sulfonate group (log kw −0.914). Also, log kw of the derived compounds increases in the series of substituents: F (2e) < Cl (2i), and CH3 (2c) < COCH3 (2a).

3.4. Biological evaluation

All the synthesized compounds were investigated in vitro for their antiproliferative activity against eight human cancer cell lines (MV-4-11, MCF-7, JURKAT, HT-29, Hep-G2, HeLa, Du-145 and DAUDI) and normal human mammary gland epithelial cells MCF-10A using cis-platin as positive control and are summarized in (Table 3).

Table 3.

Antiproliferative activity of triazene nitrogen mustards 2a-2i against cancer cell lines and normal human mammary epithelial cells MCF-10A.

IC50 ± SD [µg/ml]
Triazene MV-4-11 MCF-7 JURKAT HT-29 Hep-G2 HeLa Du-145 DAUDI MCF-10A
2a 13.42 ± 5.937 45.07 ± 1.654 66.20 ± 13.428 49.32 ± 17.381 38.45 ± 12.341 39.42 ± 9.768 88.95 ± 9.309 16.07a ± 26.485 79.98 ± 8.431
2b 44.59 ± 4.369 50.44 ± 8.079 68.54 ± 6.356 48.88 ± 18.718 37.93 ± 17.106 39.41 ± 11.465 38.58a ± 8.824 40.19 ± 4.286 29.83a ± 13.201
2c 5.42 ± 2.523 19.75 ± 3.017 13.14 ± 0.503 16.25 ± 7.374 23.81 ± 5.363 18.11 ± 7.036 36.44 ± 5.287 4.91 ± 1.527 61.64 ± 13.066
2d 5.59 ± 3.033 16.47 ± 4.144 24.29 ± 1.221 31.74 ± 5.453 14.92 ± 1.079 22.70 ± 2.975 30.95 ± 3.146 17.63 ± 2.288 32.62 ± 2.787
2e 7.69 ± 3.169 15.09 ± 2.773 13.64 ± 3.002 31.94 ± 8.143 14.91 ± 4.658 12.53 ± 2.234 30.10 ± 10.156 13.64 ± 3.656 49.34 ± 15.485
2f 6.16 ± 1.763 20.87 ± 2.194 35.98 ± 7.111 5.59 ± 0.632 27.79 ± 10.104 23.51 ± 3.316 22.78 ± 10.062 61.89 ± 24.810 42.04 ± 17.314
2g 14.05 ± 7.746 23.88 ± 7.482 49.11 ± 14.143 47.64 ± 3.418 32.07 ± 1.399 28.97 ± 5.351 34.14 ± 1.350 −61.13a ± 60.483 36.61 ± 9.520
2h −50.8a ± 36.576 −5.5a ± 14.192 −7.92a ± 11.711 −13.65a ± 12.306 −27.2a ± 17.459 −6.65a ± 24.065 −11.95a ± 15.547 −89.97a ± 18.485 1.02a ± 0.478
2i 51.38 ± 11.917 61.40 ± 21.808 39.53a ± 10.080 45.17a ± 4.094 70.17 ± 13.874 51.26 ± 14.724 35.09a ± 7.588 75.60 ± 17.762 15.66a ± 4.010
cis-platin 0.76 ± 0.184 1.73 ± 0.443 0.24 ± 0.056 3.63 ± 0.715 0.68 ± 0.147 0.37 ± 0.137 0.59 ± 0.086 1.07 ± 0.208 4.65 ± 1.171
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a

Average proliferation inhibition at 100 μg/ml.

According to our results, compounds 2c, 2d, 2e and 2f have very strong activity against biphenotypic B myelomonocytic leukemia MV4-11, with IC50 values from 5.42 to 7.69 µg/ml, while their cytotoxic activity against normal human mammary gland epithelial cells MCF-10A is 6–11 times lower. Compound 2c showed also very strong activity against Burkitt lymphoma DAUDI while compound 2f showed very strong activity against human colon adenocarcinoma HT-29, with IC50 4.91 µg/ml and 5.59 µg/ml, respectively. Cytotoxic activity of compounds 2c and 2f against normal human MCF-10A cells is 8–13 times lower than against these cancer cell lines. Also, our data showed that compound 2a and 2g has good activity against MV4-11 cell line, with IC50 13.42 µg/ml and 14.05 µg/ml, respectively.

Compounds 2c, 2d, and 2e also show good activity in relation to other cell lines, MCF-7, JURKAT, HT-29, Hep-G2, HeLa, Du-145 and DAUDI with IC50 values from 12.53 to 36.44 µg/ml, and the cytotoxic activity of these compounds against normal human MCF-10A cells is 1–5 times lower than against cancer cell lines.

The structure-activity relationship (SAR) study revealed that triazene derivatives 2c, 2d, and 2e containing methyl, trifluoromethyl and chloro substituents showed the highest antiproliferative activity against all reference cancer cell lines. Also compound 2f, obtained from benzocaine – commonly used anesthetic, and containing carboxyethyl group, showed very high activity against some cancer cell lines. The most active compounds 2c, 2d, 2e and 2f simultaneously have the lowest lipophilicity, with the log kw values in the range from −3.503 to −2.675. Compound 2h containing the -SO3Na moiety did not show activity over any cancer line investigated, and is characterized by the highest lipophilicity (log kw −0.914) of all tested compounds. The highest lipophilicity of triazene salt 2h relative to other derivatives can be explained by the probable formation of zwitterion containing a positive quaternary ammonium cation and negative sulfonate anion. Additional forces in such salts probably play a significant role in the final lipophilicity profile of this compound, however, this problem requires further investigation (Mazák et al., 2011).

3.5. Spectroscopic properties

One of the most important targets of anti-cancer drugs is DNA, so understanding the mechanism of interaction with DNA provides further insight into the possible path of gene expression. Currently, the three main mechanisms of interaction of drugs with DNA are electrostatic interaction between the cationic species and the negatively charged DNA phosphate chain, which is on the outside of the helix, intercalation with the base pairs, and groove binding involving van der Waals bonds (Zhang et al., 2011, Rafique et al., 2013). In order to understand the mechanism of action of the triazenes 2a-2i, their interactions with calf-thymus DNA using UV–Vis spectroscopy were investigated. Calf-thymus DNA is currently the most commonly used DNA, which is derived from calf thymus tissue. It contains 41.9% G-C and 58.1% A-T base pairs. The UV–Vis absorbance spectra of pure triazenes exhibit two absorption bands in the 330–340 (nm) and 221–260 (nm) ranges (Table 4).

Table 4.

UV–Vis spectra of triazenes 2a-2i.

Triazene salts λ1 (nm) λ2 (nm)
2a 335 240
2b 331 236
2c 340 241
2d 326 234
2e 337 223
2f 333 235
2g 340 221
2h 330 –
2i 332 237
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For all compounds with increasing concentration of the triazene with constant DNA concentration, the hyperchromic effect was observed (Fig. 1). The absorption of DNA-triazene complexes at 258 nm showed a decrease in absorbance compared to the sum of the individual components, which clearly shows that the test compounds interact with DNA. With increasing concentration of the DNA at a constant triazene concentration, the hypsochromic shifts were observed relative to the sum of the absorbances of the individual components (Fig. 2). Both in experiments with constant DNA concentration and with a constant concentration of compounds, the incubation time does not play a key role in the formation of DNA linkage, suggesting a fast bonding to DNA.

Fig. 1.

Fig. 1

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The absorption spectrum of the solution containing 100 mM of DNA and increasing amounts of 2a.

Fig. 2.

Fig. 2

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The absorption spectrum of the solutions containing 30 μM of 2g and increasing amounts of DNA.

In the next step of our research we calculated the intrinsic binding constant Kb between triazene salts 2a-2i and DNA (Table 5) using the equation: [DNA]/(εa − εf) = [DNA]/(εb-εf) + 1/Kb(εb − εf), where [DNA] is the concentration of DNA in base pairs, while εa, εf and εb are the apparent, free and bound complex extinction coefficients, respectively (Pakravan et al., 2015). Plot of [DNA]/εa-εb × 108 vs. [DNA] for triazene 2g can be found in Supplementary Material.

Table 5.

The binding constant Kb between triazenes 2a-2i and DNA.

Triazene Binding constant Kb [M−1] Binding constant Kb
2a 55,330 5.53 × 104 M−1
2b 19,758 1.98 × 104 M−1
2c 23,248 2.32 × 104 M−1
2d 19,503 1.95 × 104 M−1
2e 24,968 2.50 × 104 M−1
2f 58,260 5.83 × 104 M−1
2g 26,037 2.60 × 104 M−1
2h 23,427 2.34 × 104 M−1
2i 19,254 1.92 × 104 M−1
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As we can see that the largest binding constant Kb equals 5.83 × 104 M−1 (2f) and 5.53 × 104 M−1 (2b) have compounds having the COOEt and COCH3 groups, which is probably caused by the formation of an additional hydrogen bond between the carbonyl group of these compounds and phosphate chain of DNA. The compounds containing NO2 and Cl groups are characterized by two times smaller binding constant Kb equals 2.60 × 104 M−1 and 2.50 × 104 M−1, respectively. An interesting observation is also that compounds containing strong electron-withdrawing CF3 and F groups are characterized by the smallest binding constant Kb = 1.95 × 104 M−1 and 1.92 × 104 M−1, respectively. This is probably due to the weakening of the formation of hydrogen bonds by these molecules.

The next experiment with the competitive replacement of ethidium bromide dye (EB) from its complex with DNA by the tested compounds showed no changes in absorbance spectra, suggesting non-intercalative mode of binding between triazenes and DNA (Fig. 3).

Fig. 3.

Fig. 3

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The absorption spectrum of the solutions containing 80 μM EB, 80 μM DNA and increasing concentration of 2f.

In conclusion, our research suggests that cationic triazene species interact fast with the negatively charged DNA phosphate chain outside of the helix.

4. Conclusion

In summary, we have developed an efficient method for the synthesis of triazene salts and confirmed their structure by spectroscopic methods and theoretical calculations. As a result of our research, we have identified new leading structures with very high activity against some types of cancer cells with IC50 values from 4.91 to 7.69 µg/ml, and with cytotoxic activity against normal human mammary gland epithelial cells MCF-10A from 6 to 11 times lower than against cancer cell lines. We have also demonstrated a good correlation between determined lipophilicity and the antiproliferative activity of obtained compounds. Our UV–Vis spectroscopic results indicate also that triazene salts tend to interact with negatively charged DNA phosphate chain. Additional calculations show that compounds 2f and 2b containing COOEt and COCH3 substituents bind more strongly to DNA than other compounds, their Kb values are 5.83 × 104 M−1 and 5.53 × 104 M−1, respectively. Moreover, the calculated binding constant Kb values indicates that the resulting derivatives could also interact with DNA in an in vivo situation, however, to confirm this further studies are required.

Conflict of interest

The authors confirm that this article content has no conflicts of interest.

Acknowledgements

This study was supported by the Nicolaus Copernicus University (project No. 786/2014).

Footnotes

Peer review under responsibility of King Saud University.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jsps.2018.11.012.

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (3.4MB, docx)

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