Synthesis, antiproliferative and antitrypanosomal activities, and DNA binding of novel 6-amidino-2-arylbenzothiazoles

Abstract A series of 6-amidinobenzothiazoles, linked via phenoxymethylene or directly to the 1,2,3-triazole ring with a p-substituted phenyl or benzyl moiety, were synthesised and evaluated in vitro against four human tumour cell lines and the protozoan parasite Trypanosoma brucei. The influence of the type of amidino substituent and phenoxymethylene linker on antiproliferative and antitrypanosomal activities was observed, showing that the imidazoline moiety had a major impact on both activities. Benzothiazole imidazoline 14a, which was directly connected to N-1-phenyl-1,2,3-triazole, had the most potent growth-inhibitory effect (IC50 = 0.25 µM) on colorectal adenocarcinoma (SW620), while benzothiazole imidazoline 11b, containing a phenoxymethylene linker, exhibited the best antitrypanosomal potency (IC90 = 0.12 µM). DNA binding assays showed a non-covalent interaction of 6-amidinobenzothiazole ligands, indicating both minor groove binding and intercalation modes of DNA interaction. Our findings encourage further development of novel structurally related 6-amidino-2-arylbenzothiazoles to obtain more selective anticancer and anti-HAT agents.


Introduction
The benzothiazoles are constituents of bioactive heterocyclic compounds that exhibit a wide spectrum of biological activities [1][2][3][4][5] . Electron deficient, bivalent sulphur atoms in sulphur-containing heterocycles were found to participate in attractive nonbonding sulphur aromatic and sulphur halogen interactions that proved to enhance drug À target binding affinity 6 . Functionalization of the benzothiazole scaffold at the C-2 and C-6 positions has been a key determinant for their enhanced biological activity, mainly antiproliferative and antiparasitic [7][8][9][10][11] . Thus, the antiproliferative activity of amidino-and amino-substituted 2-phenylbenzothiazole derivatives strongly depended on the position of the substituent in the benzothiazole skeleton, as well as on the type of amidino unit [12][13][14][15][16] . Anticancer effects of 2-arylbenzothiazoles involved metabolic activation by cytochrome P450 to electrophilic reactive species, which generated DNA adducts in sensitive tumour cells 17 . Polyhydroxylated 2-phenylbenzothiazoles were developed as surrogates for the naturally occurring bioactive flavonoid and isoflavone 18 .
Among C-2-arylbenzothiazoles, amino-substituted derivatives possessed excellent cytotoxicity in nanomolar concentrations against several breast cancer cell lines 19,20 . While the methylated analogue of C-2-arylbenzothiazole demonstrated superior in vivo efficacy, albeit, with metabolic instability, the fluorinated analogue exhibited enhanced stability with limited bio-availability 17 . The prodrug concept led to the development of the phortress compound, which had potent antitumor activity against human mammary tumour xenografts and is in clinical trials for the treatment of solid tumours 21 . 2-Piperazinyl benzothiazole derivatives were found to strongly inhibit the growth of hepatocellular, breast, and colorectal cancer cells 22 . Even though a number of structurally related benzothiazoles have been reported to exert antitumor effect, their mechanism is not fully evaluated, as a prelude to lead optimisation and clinical development. Some benzothiazole-based anticancer agents were found to target tyrosine kinase, topoisomerase, microtubule, cytochrome P450, heat shock protein 90 (Hsp90), epidermal growth factor receptor (EGFR) and apoptosis by activation of reactive oxygen species (ROS) 2, [23][24][25][26][27][28][29] . Amidino benzothiazoles that exhibited strong antiproliferative activity also exerted good DNA binding affinity having both helix groove binding and DNA intercalation properties 30 . Some benzothiazole sulphonamides were found to have a crucial role in the inhibition of the metalloenzyme carbonic anhydrase (CA IX and XII) that is overexpressed in hypoxic tumours 1,31,32 .
Human African trypanosomiasis (HAT,) or sleeping sickness, is a neglected tropical disease (NTD) caused by Trypanosoma brucei, a protozoan parasite transmitted to humans through the bite of a blood-sucking tsetse fly 33,34 . The drugs currently used to treat HAT are not effective against all stages and subspecies of the parasite, so further clinical investigation is needed to develop new antitrypanosomal drugs. Some 2-benzylsulfanyl-and 2-arylbenzothiazole derivatives have been found to exhibit good trypanocidal activity at low concentrations 35,36 . Optimisation of anti-parasite activity, physicochemical parameters and ADME properties afforded the fluoro-substituted benzothiazole, with a 2-cyclopropanecarboxamide at position 2, which displayed promising in vivo efficacy 37 .
In continuation of our recent work on the development of aromatic benzimidazole amidines as antitrypanosomal [38][39][40] and cytostatic agents 41 , we have now synthesised new chemical entities by the fusion of benzothiazole through a phenoxymethylene unit, or directly to 1,2,3-triazole ring with a p-substituted phenyl or benzyl subunit ( Figure 1).
In this context, the influence of linkers and the type of amidino substituents of the benzothiazole derivatives 10a-10d, 11a-11d, 12a-12d, 13a-13d, 14a-14d, 15a-15d on their antiproliferative and antiprotozoal activity has been explored. Compounds with potent antiproliferative and antitrypanosomal activities were selected for further investigation of their DNA binding affinities by UV-Vis and CD spectroscopy, as well as thermal denaturation experiments.

Materials and methods
General Melting points were determined by means of Original Kofler Mikroheitztisch apparatus (Reichert, Wien). 1 H NMR and 13 C NMR spectra were recorded with the Bruker Avance DPX-300 or Bruker AV-600 using TMS as an internal standard. Chemical shifts are reported in parts per million (ppm) relative to TMS. UPLC-MS spectra were recorded with Agilent 1290 Infiniti II/6120 Quadruple LC/ MS spectrometers using electrospray ionisation (ESI). Elemental analyses for carbon, hydrogen, and nitrogen were performed on a Perkin-Elmer 2400 elemental analyser. Analyses are indicated as symbols of elements, and the analytical results obtained are within 0.4% of the theoretical value.

Proliferation assay
Cells were seeded onto 96-well microtiter plates at a seeding density of 3000 cells/well for carcinoma cell lines, and 5000 cells/ well for normal human fibroblasts. The next day, cells were treated with test agents at five different concentrations (0.01-100 mM) and further incubated for 72 h. DMSO (solvent) was tested for potential cytotoxic effect but it did not exceed 0.1%. 5-Fluorouracil (5-FU, 0.384 M fluorouracilum, Pliva 500 mg/10 ml) dissolved in physiological solution was used as a positive control. Following 72 h incubation, the MTT assay was performed and measured absorbances were transformed into a percentage of cell growth as described previously 47 . Results were obtained from three independent experiments. IC 50 values were calculated using linear regression analysis (FORECAST option taking into account the concentration range of two experimental points above and below IC 50 ).

Antitrypanosomal screening and cytotoxicity assays
Antitrypanosomal screening Bloodstream form T. b. brucei (strain 221) were cultured in modified Iscove's medium 48 and assays were carried out in 96-well microtiter plates (200 ll volumes) to determine the IC 50 and IC 90 values of each compound. Parasites growth was initiated at 2.5 Â 10 4 ml À1 , compounds were added at a range of concentrations, and the plates were incubated at 37 C. Resazurin (20 ll at 0.125 mg ml À1 ) was added after 48 h, the plates were incubated for a further 16 h, and then read in a Spectramax plate reader, and data analysed using GraphPad Prism. Each drug was tested in triplicate.

L6 cell proliferation
For cytotoxicity assays, L6 cells (a rat myoblast line) were seeded into 96-well microtiter plates at 1 Â 10 4 ml À1 in 200 ll of the growth medium, and different compound concentrations were added. The plates were then incubated for 6 days at 37 C and 20 ll resazurin was added to each well. After a further 8 h incubation, the fluorescence was determined using a Spectramax plate reader, as outlined above.
The calf thymus ctDNA was additionally sonicated and filtered through a 0.45 mm filter 49 . Polynucleotide concentration was determined spectroscopically 50,51 as the concentration of phosphates.

UV/vis measurements
The UV/Vis spectra were recorded on a Varian Cary 100 Bio spectrophotometer using 1 cm path quartz cuvettes. Calibration experiments were performed at 25 C and pH ¼ 7 (I ¼ 0.05 mol dm À3 , sodium cacodylate buffer). Absorption maxima and corresponding molar extinction coefficients (e) of benzothiazole derivatives are given in Table S1 (Supplementary material). Thermal melting curves for DNA and their complexes with studied compounds were determined as previously described by following the absorption change at 260 nm as a function of temperature. The absorbance of the ligands was subtracted from every curve and the absorbance scale was normalised. T m values are the midpoints of the transition curves determined from the maximum of the first derivative and checked graphically by the tangent method. The DT m values were calculated by subtracting T m of the free nucleic acid from T m of the complex. Every DT m value reported here was the average of at least two measurements. The error in DT m is ±0.5 C.

Fluorimetric measurements
Fluorescence spectra were recorded on a Varian Cary Eclipse spectrophotometer at 25 C using appropriate 1 cm path quartz cuvettes. Fluorimetric experiments were performed at pH ¼ 7 (I ¼ 0.05 mol dm À3 , sodium cacodylate buffer) by adding portions of polynucleotide solution into the solution of the studied compound. In fluorimetric experiments, an excitation wavelength of k exc !300 nm was used to avoid the inner filter effect caused due to increasing absorbance of the polynucleotide. Emissions were determined in the range k em ¼ 350-650 nm. Values for K s were obtained by processing titration data using the Scatchard equation 52 . All had satisfactory correlation coefficients (>0.99).
CD measurements CD spectra were recorded on a JASCO J815 spectrophotometer in 1 cm path quartz cuvettes. CD parameters: range ¼ 500-220 nm, data pitch ¼ 2, standard sensitivity, scanning speed ¼ 200 nm/ min, accumulation ¼ 3-5. Titrations were performed at 25 C and pH ¼ 7 (I ¼ 0.05 mol dm À3 , sodium cacodylate buffer). CD experiments were done by adding portions of the compound stock solution into the polynucleotide solution.
It can be observed that the tested compounds exhibited strong to moderate inhibitory activity towards tumour cell lines. However, compounds with pronounced antiproliferative effects affected the proliferation of the non-tumour HFF-1 cell line. The type of amidino moiety at the benzothiazole influenced the antiproliferative activities (Figure 2). For example, among the 6-amidino benzothiazoles, imidazolino-substituted derivatives 11a-11d and 14a-14d showed the best inhibitory effects, particularly on colorectal adenocarcinoma (SW620) and cervical carcinoma (HeLa) cells. This correlates with our earlier findings for the benzimidazole series 41 . Unsubstituted amidino-benzothiazole 13a-13d exhibited up to 10-fold lower activities relative to their imidazolines congeners 14a-14d. The cytostatic effects decreased in the following order: imidazoline > pyrimidine > non-substituted amidine.
The effect of the aromatic substituent at N-1 of 1,2,3-triazole ring on antiproliferative activities varied depending on the type of 6-amidino moiety. In most cases, the p-chlorophenyl aromatic unit contributed to the enhanced antiproliferative effects of 10b, 12b, 13b, and 15b, except in the case of imidazolino-benzothiazoles. In benzothiazole imidazolines, the unsubstituted phenyl ring in 11a and 14a had the highest influence on inhibitory activity. The 2-imidazolino-substituted derivative 14a exhibited the highest activity on SW620 (IC 50 ¼ 0.25 mM) and CFPAC-1 (IC 50 ¼ 0.45 mM) cell lines, while 14c, which contains a p-methoxyphenyl ring, was the most potent against HeLa cells (IC 50 ¼ 0.38 mM). The benzothiazole imidazoline 11a was the most active representative of this class (IC 50 ¼ 0.49 mM) against MCF-7 cells. Comparison of the effect of the aryl substituents at the N-1 position of the 1,2,3-triazole ring on activity revealed that, among 2-imidazolino-substituted derivatives, phenyl and p-methoxyphenyl substituents caused better potencies than p-chlorophenyl and benzyl substituents.
Replacement of the phenoxymethylene linker in 10a-10d, 11a-11d, and 12a-12d with a direct fusion of benzothiazole to 1,2,3-triazole in 13a-13d, 14a-14d, and 15a-15d reduced antitrypanosomal activity against bloodstream-form T. brucei. Furthermore, the introduction of an electron-withdrawing p-chloro substituent in 10b-15b improved the inhibitory effect. The relationship between the type of aromatic substituents and their activities revealed that antitrypanosomal effects decreased in the following order: p-ClPh>p-OCH 3 Ph > Ph > Bn.

DNA binding study
As a carrier of genetic information and a great influence on vital processes in the cell, DNA is the main target of a large number of drugs with anticancer and antiprotozoal activity [55][56][57] . Among them, anthracyclines that target DNA topoisomerase II cause highly lethal DNA breaks in proliferating cancer cells, while some drugs, such as quinacrine, with a long history of clinical use in the treatment of malaria, modulate cellular pathways that lead to cellular toxicity and death 55 . With the aim of investigating possible mechanisms of antiproliferative and antitrypanosomal action, we examined the DNA interactions of compounds that had exhibited the most potent submicromolar activities, 10b, 11a, 11b, 12b, 14-14c, and 15b. The study was performed with a double-stranded (ds-) polynucleotide, calf-thymus (ct)DNA which represents a classical B-helix consisting of 58% AT base pairs (and 42% GC base pairs). Titration with ctDNA yielded fluorescence quenching of the studied compounds ( Figure 3, Table 3, Figures S19-S26, Supporting material). It can be observed that the addition of ctDNA caused a small redshift (D ¼ 1-5 nm) of emission maxima.
The binding constants, Ks obtained by processing of fluorimetric titration data with the Scatchard equation 52 are summarised in Table 3. Interestingly, the 6-amidinobenzothiazoles 10b, 11a, 11b, and 12b, which have a phenoxymethylene linker, showed higher binding affinities towards ds-DNA than compounds 14a-14c and 15b, which lack the linker. Non-covalent interaction of small molecules with DNA can affect the thermal stability of the double helix, which results in either an increase or decrease of the melting temperature (T m ) 58 . The intercalative mode of binding usually results in an increased T m value, while groove binding molecules can stabilise or destabilise the double-stranded structure, resulting in increased or decreased T m values, respectively. The majority of studied compounds showed a small stabilisation effect of ctDNA (58% AT). Moreover, all 6-amidinobenzothiazoles stabilised DNA consisted of only AT sequences. The best stabilisation effects of AT-DNA were observed for 11a, 14c, and 15b ( Table 4).
The formation of complexes between small molecules and DNA can be monitored using circular dichroism (CD) spectroscopy. A mutual orientation of achiral small molecule and polynucleotide chiral axis, which results in an induced CD (ICD) signal, could give us additional information about modes of interaction 59,60 . Hence, the most reliable insight into modes of interaction between small molecules and DNA can be retrieved at wavelength area, k > 300 nm, where ligands possess UV/Vis spectra, while DNA does not.
The addition of studied compounds to ctDNA resulted in a decreased intensity of the CD band of ds-polynucleotide, ctDNA (Figure 4, Figures S31 and S32).
Furthermore, compounds 12b and 15b exhibited positive induced CD spectra (ICD) with ctDNA around 300 nm and 345 nm, respectively, supporting a minor groove binding mode to ctDNA 39,40,61,62 . Similar changes in CD spectra were noticed for classical minor groove binders like DAPI or Hoechst 33258 44 .
The addition of compound 11a to a ctDNA solution resulted in the appearance of a bisignate signal with maxima at 317 and 360 nm. Such change suggests the binding of 11a in the form of a dimer within the minor groove ( Figure 4) 62 .
In the titration of ctDNA with 10b and 11b, a small negative ICD signal appeared, while 14a-14c caused the small positive ICD signal upon binding to ctDNA (Figure 4, Figures S31 and S32). Such changes, whether small negative, or positive, point to an intercalative way of binding. This is additionally supported by thermal stabilisation of ctDNA and/or AT-DNA (Table 4), as well as binding constants of $1 mM (Table 3), which were observed with the majority of studied benzothiazoles.
The positive sign of the ICD band observed with 14a-14c suggests that the long axis of the benzothiazole moiety is approximately perpendicular to the long axis of the basepair pocket, but still in the plane with the adjacent base pairs. The negative sign of the ICD band found for 10b and 11b indicates that the transition moment of the ligand is oriented "parallel" to the long axis of adjacent base pairs 42,63 .
At higher ratios, r ! 0.3, a big decrease of intensity of the CD bands and strong negative ICD spectra observed with 14a-14c can be attributed to non-specific aggregation of non-intercalated molecules along the DNA backbone, possibly within major grooves 43 .
We may conclude that the 6-amidinobenzothiazoles, which exhibit antiproliferative and antitrypanosomal potency in the submicromolar range and DNA interacting properties, warrant further structural optimisation to reduce their toxicity against normal cells, with the aim of developing novel antitumor or/and anti-HAT agents.

Disclosure statement
No potential conflict of interest was reported by the author(s). Table 3. Binding constants (logKs) a,b calculated from the fluorescence titrations of benzothiazole compounds with ds-DNA at pH ¼ 7.0 (buffer sodium cacodylate, I ¼ 0.05 mol dm À3 ).