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

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.


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).

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. 1 H NMR (700 and 400 MHz) and 13 C 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/UV 254 0.2 mm plates. Bis(2-chloro-ethyl)amine hydrochloride, and appropriate anilines were commercial materials (Aldrich).

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 mg/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 mg/ml of insulin, 0.5 mg/ml of hydrocortisone, 0.05 mg/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 mg/mL streptomycin (Sigma-Aldrich, Steinheim, Germany). The cell lines were cultured at 37°C in a humid atmosphere saturated with 5% CO 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 mg/ml).

In vitro antiproliferative assay
The cells were plated in 96-well plates (Sarstedt, Germany) in an appropriate density: 1 Â 10 4 per well for DAUDI, Du-145, Jurkat, Hep-G2, MV-4-11 and MCF-10A, 0.75 Â 10 4 per well for HT-29 and MCF-7 and 0.25 Â 10 4 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 IC 50 values (inhibitory concentration 50) -the dose (lg/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.

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 (k = 540 nm) in a computerinterfaced, 96-well Synergy H4 (BioTek Instruments USA) photometer microtiter plate reader (Sidoryk et al., 2012).

MTT cytotoxic test
20 ml of MTT solution (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide, stock solution: 5 mg/ml) was added to each well and incubated for 4 h. After the incubation time was complete, 80 ml 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.

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 Â 4.6 mm; 2.6 mm) 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 mg/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.

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 À1 cm À1 at 260 nm and 5800 M À1 cm À1 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).

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-Łą czkowska 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 gaugeorigin 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).

Chemistry
The target triazene salts containing chloroethyl group were obtained in two steps, one-flask synthesis. In the first step, paraor 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 1 H NMR (700 MHz) and 13 C NMR (100 MHz), and GC-APCI-MS analyses. 1 H NMR and 13 C NMR spectra of triazenes 2a-2i showed four characteristic triplets at d (4.16-4.96 ppm) and peaks at d (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.

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.
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 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 H 24 and H 27 , and 7.71 ppm to protons H 25 and H 26 , 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.

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 nonaqueous 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-mm porous particles. Chromatographic capacity factors (k) were calculated: k = (t R /t M ) À 1, where t R [min] denotes retention time, and t M [min] is time for dead volume. The dead time was determined using uracil as a t M 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 Soczewiń ski-Wachtmeister equation: log k = log k w + SU, where log k w denotes the capacity factor of the analyte in pure water, U is organic modifier concentration in the mobile phase, and S denotes slope of the regression curve. The lipophilicity parameter U 0 was calculated using relationship U 0 = Àlog k w /S, and R 2 is correlation coefficient. Experimentally determined lipophilic parameters log k w are presented in (Table 2).
In our research we have observed the linear dependence between log k values and concentration of organic modifier in the eluent with correlation coefficient (R 2 = 0.967-0.998) value. The analysis shows that the value of the log k w is in the range from À3.503 to À0.914. From all tested compounds, the lowest log k w values, in the range from À3.503 to À2.675, 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 k w , in the range from À2.002 to À1.478. The highest value of the log k w was found for compounds 2h containing sodium sulfonate group (log k w À0.914). Also, log k w of the derived compounds increases in the series of substituents: F (2e) < Cl (2i), and CH 3 (2c) < COCH 3 (2a).

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).
According to our results, compounds 2c, 2d, 2e and 2f have very strong activity against biphenotypic B myelomonocytic leukemia MV4-11, with IC 50 values from 5.42 to 7.69 mg/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 IC 50 4.91 mg/ml and 5.59 mg/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 IC 50 13.42 mg/ml and 14.05 mg/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 IC 50 values from 12.53 to 36.44 mg/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 k w values in the range from À3.503 to À2.675. Compound 2h containing the -SO 3 Na moiety did not show activity over any cancer line investigated, and is characterized by the highest lipophilicity (log k w À0.914) 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).

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).  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.
In the next step of our research we calculated the intrinsic binding constant K b between triazene salts 2a-2i and DNA (Table 5)  As we can see that the largest binding constant K b equals 5.83 Â 10 4 M À1 (2f) and 5.53 Â 10 4 M À1 (2b) have compounds having the COOEt and COCH 3 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 NO 2 and Cl groups are characterized by two times smaller binding constant K b equals 2.60 Â 10 4 M À1 and 2.50 Â 10 4 M À1 , respectively. An interesting observation is also that compounds containing strong electron-withdrawing CF 3 and F groups are characterized by the smallest binding constant K b = 1.95 Â 10 4 M À1 and 1.92 Â 10 4 M À1 , 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).
In conclusion, our research suggests that cationic triazene species interact fast with the negatively charged DNA phosphate chain outside of the helix.

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 IC 50 values from 4.91 to 7.69 mg/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 COCH 3 substituents bind more strongly to DNA than other compounds, their K b values are 5.83 Â 10 4 M À1 and 5.53 Â 10 4 M À1 , respectively. Moreover, the calculated binding constant K b values indicates that the resulting derivatives could also interact with DNA in an in vivo situation, however, to confirm this further studies are required.