The N’-Substituted Derivatives of 5-Chloro-3-Methylisothiazole-4-Carboxylic Acid Hydrazide with Antiproliferative Activity

Thanks to the progress in oncology, pharmacological treatment of cancer is gaining in importance and in the near future anti-cancer chemotherapeutics are expected to be the main method of treatment for cancer diseases. What is more, the search for new anti-cancer compounds with the desired application properties is constantly underway. As a result of designed syntheses, we obtained some new N’-substituted 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide derivatives with anticancer activity. The structure of new compounds was determined by mass spectrometry (MS), elemental analysis, proton nuclear magnetic resonance spectroscopy (1H-NMR), carbon nuclear magnetic resonance spectroscopy (13C-NMR), 1H-13C NMR correlations and infrared spectroscopy (IR). Moreover, the structures of the compounds were confirmed by crystallographic examination. The antiproliferative MTT tests for 11 prepared compounds was conducted towards human biphenotypic B cell myelomonocytic leukemia MV4-11. SRB test was used to examine their potential anticancer activity towards human colon adenocarcinoma cell lines sensitive LoVo, resistant to doxorubicin LoVo/DX, breast adenocarcinoma MCF-7 and normal non-tumorigenic epithelial cell line derived from mammary gland MCF-10A. The most active compound was 5-chloro-3-methyl-N′-[(1E,2E)-(3-phenyloprop-2-en-1-ylidene]isothiazole-4-carbohydrazide, which showed the highest antiproliferative activity against all tested cell lines.


Introduction
Chemotherapy is a method of systemic treatment of malignant tumors using cytostatic drugs, which stop the division and spread of cancer cells [1]. The basis of modern chemotherapy is the pairing of several cytostatics belonging to different classes. The simultaneous administration of several anticancer drugs reduces the risk of developing resistance to the treatment and leads to enhanced cytostatic activity. Cancer chemotherapy is difficult for many reasons. Due to the not fully understood causes of cancer, minimal biochemical differences between the cancer cell and the normal one, no specificity of antitumor drug action of cytostatic agents, their low therapeutic factor and toxicity to normal tissues [2][3][4][5][6], we decided to design new compounds with potential anticancer activity.

Results
The aim of our synthesis was to obtain new isothiazole derivatives with anticancer activity. The modification of the 4-position of isothiazole was characterized by the formation of N'-substituted 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazides with the chlorine substitution of the isothiazole ring in the 5-position. 5-Chloro-3-methyl-isothazole-4-carbohydrazide 2 has already been synthetized by Kuczyński et al. [19], whereas in this work we used another method of synthesizing hydrazides through an intermediate azide product (Scheme 1). The new method for preparing hydrazide 1 is more efficient, as the use of 2-PrOH as the solvent enables the product to crystallize out from the reaction mixture while cooling and can be obtained pure after a methanol wash. Moreover, we describe the spectral properties of 5-chloro-3-methylisothiazole-4-carbohydrazide 2, because these data have not been reported so far. Scheme 1 presents the synthesis of 5-chloro-3-methylisothazole-4-carbohydrazide 2 and the 4-substituted derivatives 3-11. Scheme 1. Synthesis of 5-chloro-3-methylizothiazole-4-carboxylic acid hydrazide derivatives. 5-Chloro-3-methylisothiazole-4-carboxylic acid 1 was prepared according to the method described by Machoń [20]. Substrate 1 and 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide 2 were also examined in terms of their antiproliferative activity.
The N'-substituted 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide derivatives 3-11 (Scheme 1) were obtained by the nucleophilic addition reaction in yields ranging from 50% to 86%. The new derivatives were obtained in the reaction 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide 2 with the corresponding carbonyl compounds heated at a temperature of 78 • C while being stirring vigorously for 4 h in ethanol. The synthesis took place according to the nucleophilic addition mechanism consisting of the attachment of 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide 2 to the carbon bond of the C=O carbonyl group to form a carbanion. The next step was the elimination of a water molecule and creation of an imine bond.
In the IR spectra of all the compounds, an absorption band was observed in the range of 1643-668 cm −1 corresponding to the C=O carbonyl bond, at 1551-1594 cm −1 corresponding to the N=CH azamethine bond and in the 3164-3271 cm −1 range for the amine NH group. The structures of new compounds were also determined by mass spectrometry (Figures S1-S10). In the 1 H-NMR (Figures S11-S30) and 13 C-NMR ( Figure S31-S40) spectra, double signals for each of the protons were present.
In the present study, the spectral data were measured in a DMSO-d 6 solution. For these compounds, we observed in 1 H-NMR and 13 C-NMR signals belonging to Z and E geometrical isomers about the C=N double bond, which is characteristic of arylidene-hydrazide structure [21][22][23][24][25]. In addition, the compounds with this structure may exist as cis-trans amide conformers [26][27][28]. What is more, we have done 1 H-13 C NMR correlations ( Figures S41-S50) for all compounds, which confirmed the presence of isomers. The correlation spectrum of the proton and carbon in the most active compound 3 (Figure 1) allows us to determine the presence of double signals from geometric isomers. Additionally, three isothiazole derivative compounds, denoted as 3, 4 and 8, were crystallized and X-ray crystallography confirmed their chemical structure with the expected trans (E) configuration in the solid form. The asymmetric unit of 3 consists of two independent molecules, denoted as A (and D in the case of disordered part attached to the atom C41A) and B (and C in the case of disordered part attached to the atom C41B), respectively (Figure 2a), whereas compounds 4 ( Figure 2b) and 8 ( Figure 2c) crystallize with one molecule in the asymmetric unit. In the first stage of biological research, 11 prepared compounds were tested for their antiproliferative activity towards human leukemia MV4-11 cells (Table 1). The highest activity was revealed by compound 3 with IC 50 4.4 µg/mL. Other tested compounds had lower activity, with 3-8 times smaller IC 50 values than compound 3. Three compounds (2, 9 and 10) had very low antiproliferative activity with IC 50 > 80 µg/mL, and two compounds 1 and 11 had no antiproliferative activity against leukemia cells. The MV4-11 leukemia line is often used in screening for antiproliferative activity as this cell line is very sensitive to the antiproliferative effect of various groups of compounds. By using it, inactive compounds can be eliminated from further research. On the other hand, we can be quite sure that no potentially active molecules are missed in this way.  In the next stage, five compounds with IC 50 values on MV4-11 cells lower than 30 µg/mL were tested for their antiproliferative activity against MCF-7 (breast cancer), LoVo (colon cancer) human cell lines and also against Doxorubicin-resistant colon cancer LoVo/DX (P-gp-dependent, MRP-, LRP-dependent multidrug resistance). The cytotoxicity experiments were also performed towards the MCF-10A cell line (normal breast epithelial).
The data for the in vitro anticancer activity (Tables 1 and 2) were expressed as the IC 50concentration of the compound (in µg/mL and µM) that inhibits proliferation of cells by 50% compared to the untreated control cells. Cisplatin and DMSO (in a concentration comparable to the one which was used at the highest concentration of compounds) were used as a positive control. Table 2. Antiproliferative activity of the most active compounds 9 against selected cell lines (human colon adenocarcinoma cell lines sensitive (LoVo) and multi-drug resistant (LoVo/DX), breast adenocarcinoma MCF-7 and normal non-tumorigenic epithelial cell line derived from mammary gland MCF-10A). Compound 3 also had the highest activity against breast MCF-7, colon LoVo and LoVo/DX cancer cells (IC 50 < 15 µg/mL). A similar activity was revealed by compound 4 with IC 50 < 20 µg/mL. Compound 7 had the lowest antiproliferative activity against colon cancer cell lines (non-resistant and resistant) with IC 50 > 30 µg/mL ( Table 2).

Name of Compound
The cytotoxicity study of compounds towards MCF-10A cell line showed that compounds 3 and 4 also had the highest activity against normal cells. Compounds 5, 8 and 9 were about three times less cytotoxic against normal MCF-10A cells than against cancer MCF-7 cells (Table 2).
We also calculated the resistance indexes (RI) by dividing the IC 50 values of the compounds tested against the cells of drug resistant cell LoVo/DX by respective values obtained against the drug sensitive LoVo cell line ( Table 2). All tested compounds were able to overcome the barrier of P-gp-dependent resistance. Compound 7 has the highest ability to overcome the barrier of resistance (RI = 0.72), and compound 3 had a lower ability (RI = 1.36).

Discussion
The main purpose of this work was to develop synthesis methods to obtain isothiazole derivatives with antitumor activity, in order to demonstrate the influence of the structure of the compounds obtained on antitumor activity as well as to determine the leading structure. Earlier, we prepared 5-hydrazino-3-methylisothiazole-4-carboxylic acid and its new 5-substituted derivatives, among which 13 compounds displayed strong antiproliferative activity. In this scientific work we obtained 3-methylisothiazole derivatives with modifications of position 4, which contained the same substituent as the most active 5-substituted derivatives of 3-methylisothiazole [29]. The same tests and cell lines as in the previously published work [29] were used. We chose different reference drugs to compare the results. In the previous studies, the reference medicine was 5-Fluorouracil, which is a small molecule therapeutic substance used in the treatment of colorectal cancer [29]. However, in this work, we chose Cisplatin as a reference medicine, which is also a small-molecule drug. It is the basis of many combination treatment regimens of various types of cancers, including breast cancer. The synthesized derivatives in position at 4 of the isothiazole moiety, including hydrazide groups, were selected on the basis of the most active derivatives from a series of derivatives substituted in the 5 position of the isothiazole containing the CH=N-Schiff's base group [29]. The activity of the substituent change in position at 4 of the isothiazole was assessed by preserving the 5-chloro-3-methylisothiazole fragment.
The highest activity towards all examined cancer and normal cell lines was demonstrated by compound 3, which contains a hydrazide group with a -3-phenylprop-2-en-1-ylidene substituent. Other compounds qualified for the second stage of studies are 5-7 times less active than compound 3 for all tested cell lines, except for the MCF-7 breast cancer line. Compounds 4-7 have similar IC 50 values for all tumor lines and a desirable weak antiproliferative activity relative to the normal line, except for compound 4, which has the 3-Cl substituent in the meta position. In this group of derivatives with lower IC 50 values, 4-7 dominated the compounds, which contained the substituent in the meta position, such as 4 (3-Cl), 5 (3-NO 2 ) and 7 (3-OMe). Compounds 2 (substrate for the synthesis of compounds 3-11), 9 (two Me groups, i.e., 2-Me and 4-Me) and 10 (2-Me) are characterized by very poor activity. IC 50 values were not determined, but only the inhibition of cell proliferation at a concentration of 80 µg/mL. Compound 1, the substrate for the synthesis of hydrazide 2 and 11, containing the ortho (2-Cl) substituted phenyl ring shows no antiproliferative activity. The compound that substitute aromatic rings with methoxy group 7 (3-OMe) exhibits 1.5-2 times higher antiproliferative activity than phenyl derivative 8. The ability of the obtained compounds to overcome drug resistance of the studied cancer cells was confirmed by low values of the resistance index, RI. RI values from 0 to 2 indicate the sensitivity of the cells tested to the compound used. RI values from 2 to 10 indicate moderate drug resistance of the cells in question to the test compound, and RI values > 10 indicate strong drug resistance. The activity against the LoVo/DX drug-resistant cell line and its equivalent LoVo sensitive line was calculated and compared. All compounds showed RI below 2. Compound 3, which is the most active, has over 2-fold higher RI index (1.37). Compound 7 showed the lowest RI index (0.72).
The most active compound of this series is 5-chloro-N'-[(1E, 2E)-3-phenylprop-2-en-1-ylidene]-3methylisothiazole-4-carbohydrazide 3, but it is less activity than compounds from the 5-substituted isothiazole Schiff base series, Cisplatin and 5-Fluorouracil towards sensitive (LoVo) and multi-drug resistant (LoVo/DX) human colon adenocarcinoma cell lines [29]. However, the activity of this series is relatively higher towards breast adenocarcinoma MCF-7 and comparable to the normal non-tumorigenic epithelial cell line derived from mammary gland MCF-10A. What is more, in both series 3-Cl (compound 4) with a monosubstituted benzene ring is in the group of most active compounds. In addition to this, we also examined the synthesis substrates 1 and 2, which are devoid of any anticancer activity. The results indicated that the activity of the synthesized derivatives 5-hydrazino-3-methylisothiazole-4-carboxylic acids [29] and new 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide derivatives 3-11 is mainly associated with the presence of the -N=CH group with a suitable size and shape substituent in 3-methyl-4-isothiazole derivatives.
The advantage of the obtained isothiazole derivatives is their low toxicity on healthy cells, and at the same time the selectivity in relation to colorectal cancer cells by the most active compound of this series. In addition, these compounds can be potentially one of the components of chemotherapeutic systems as a factor preventing the development of drug resistance during chemotherapy. Indeed, none of the compounds tested turned out to be more active with 5-Fluorouracil and Cisplatin. However, all of the tested compounds cross the cell-resistance barrier and their activity on LoVo/DX cells is in many cases higher than the activity of Doxorubicin.
Based on the results of biological studies of the 5-substituted 5-hydrazine-3-methylisothiazole-4carboxylic acid derivatives [29] and compounds from this work-N'-substituted derivatives of 5-chloro-3-methylisothiazole-4-carboxylic acid the lead structure of anti-cancer isothiazole derivatives was determined. The designated leading structure contains structural elements with anti-tumor effect, i.e., methylisothiazole, carboxyl and azomethine group, as well as the phenylprop-2-ene group located near the azomethine group of the 5-methylisothiazole derivatives, gives them probably potential selectivity towards tumor lines. In this scientific work the most active compound against all cancer cell lines is 5-chloro-N'-[(1E,2E)-3-phenylprop-2-en-1-ylidene]-3-methylisothiazole-4-carbohydrazide 3, which possesses almost two times lower activity towards the MCF-10A normal cell line than against cancer cells. This indicates the potential selectivity of the compound with regards to cancer cells such as leukemia, breast and colon cancer cell lines. In addition, the most active compounds in both series contain substituents at the meta position of the phenyl ring near the azomethine group. In our opinion, the research on low-molecular weight of isothiazole derivatives with antiproliferative activity is very desirable because of the demand for oncological drugs that break the increasing resistance of tumors to cytostatics currently used in therapy.

General Information
Commercially available reagents were used without further purification. Progress of the reaction was controlled by thin layer chromatography (TLC) on ALUGRAM SIL G/UV pre-coated TLC sheets (Macherey-Nagel, Dylan, Germany) and visualized by ultraviolet (UV) light at 254 nm (Bioblock Scientific lamp, Fisher, Hampton, NH, USA). Melting points of all new compounds were measured by a LLG uniMELT-2 apparatus (LLG). A Thermo Scientific Nicolet iS50 FT-IR spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to record infrared specta (IR). The samples were applied as solids and frequencies are given in cm −1 . Proton nuclear magnetic resonance ( 1 H-NMR), carbon nuclear magnetic resonance ( 13 C-NMR) and 2D 1 H-13 C NMR correlation spectra were recorded in deuterated dimethyl sulfoxide (DMSO-d 6   A 45% solution of thionyl chloride dissolved in benzene (90 mL) was added to 33.8 mmol of 5-chloro-3-methylisothiazole-4-carboxylic acid 1. The mixture was heating under reflux for 3 h to obtain a clear solution. After cooling, the solution was distilled in an evaporator to give an oily residue. Thereafter benzene (30 mL) was added twice and distilled off in each case to remove residual thionyl chloride.
In an ice-cold water bath cooled to 8 • C with a magnetic stirrer, 5-chloro-3-methylisothiazole-4carboxylic acid chloride (30 mmol, 5981.2 mg) was dissolved in acetone (240 mL). The reaction mixture was kept at a temperature of up to 8 • C at all times. At the same time, sodium azide (83 mmol, 539.5 mg) was dissolved in distilled water (18 mL) and then slowly added to the reaction mixture. After addition of the sodium azide solution, stirring was continued for 30 min. The separated salt was filtered off. The solution was distilled from the reaction mixture until the semi-liquid form. The suspension was cooled in an ice-cold water bath. The precipitate was filtered off and washed with 2-propanol.
To azide (

Single Crystal X-ray Structure Determination of 3, 4 and 8
Crystals of 3, 4 and 8 suitable for single-crystal X-ray diffraction analysis were obtained by dissolution in acetonitrile/2-propanol followed by slow evaporation of the solvent at room temperature. Crystallographic measurements for 3, 4 and 8 were collected with K-geometry diffractometers: Xcalibur R (Agilent Technologies, city, state abbrev if USA, country) with a Ruby CCD camera (3 and 4) and a Kuma KM-4 CCD with a Sapphire2 CCD camera (8), with graphite monochromatized Mo-Kα radiation (λ = 0.71073 Å) at 100(2) K, using an Oxford Cryosystems cooler. Data collection, cell refinement, data reduction and analysis were carried out with CrysAlisPro [33]. Analytical absorption correction was applied to data with the use of CrysAlisPro. The crystal structures were solved using SHELXS [34] and refined on F 2 by a full-matrix least squares technique with SHELXL-2014 [35] with anisotropic thermal parameters for all the ordered non-H atoms. In the final refinement cycles, H atoms were repositioned in their calculated positions and treated as riding atoms, with C-H = 0.95-0.98 Å, and N-H = 0.88 Å and with U iso (H) = 1.2U eq (C, N) for CH and NH or 1.5U eq (C) for CH 3 . All figures were made using DIAMOND program [36].  [21]. The asymmetric unit of 3 consists of two independent molecules and in both molecules (denoted as A and B), the isothiazole ring was found to be disordered over two sites and refined with the occupancy factors of 0.749 (11) and 0.251 (11) in molecule A, and 0.686 (10) and 0.314 (10) in molecule B. The atoms of lower occupancy were denoted as D (in case of disordered part attached to C41A) and C (in case of disordered part attached to C41B). Disordered atoms were refined with anisotropic displacement parameter using EADP instruction of SHELXL [35].
Twenty-four hours prior to the addition of the tested compounds, the cells were plated in 96-well plates (Sarstedt, Germany) at a density of 1 × 10 4 or 0.75 × 10 4 (MCF-7) cells per well. The assay was performed after 72 h of exposure to varying concentrations of the tested agents. The in vitro cytotoxic effect of all agents was examined using the MTT (MV4-11) or SRB assay [37]. The results were calculated as IC 50 (inhibitory concentration 50%) the concentration of tested agent, which is cytotoxic for 50% of the cancer cells. IC 50 values were calculated for each experiment separately and mean values ± SD are presented in Tables 1 and 2. Each compound in each concentration was tested in triplicate in a single experiment, which was repeated 3-4 times.
The resistance indexes (RI) were calculated by dividing the IC 50 values of the compounds tested against the cells of drug resistant cell LoVo/DX line by respective values obtained against the cells of drug sensitive LoVo line (Table 2). According to Harker et al. [38], three categories of the cells could be distinguished: (a) the cells are drug-sensitive if the ratio approaches 0-2; (b) the cells are moderately drug-resistant if the ratio ranges from 2 to 10; (c) the cells are markedly drug-resistant if the ratio is higher than 10.
MTT Assay (for MV4-11 Cell Line) This technique was applied for cells growing in suspension culture. An assay was performed after 72 h exposure to varying concentrations of the tested agents. For the last 4 h of incubation, 20 µL of MTT solution was added to each well (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; stock solution: 5 mg/mL, Sigma Aldrich). When the incubation time was completed, 80 µL of the lysing mixture was added to each well (lysing mixture: 225 mL dimethylformamide, POCh, Gliwice, Poland, 67.5 g sodium dodecyl sulfate, Sigma Aldrich, and 275 mL of distilled water). After 24 h, when formazan crystals had been dissolved, the optical densities of the samples were read on Synergy H4 photometer (BioTek Instruments, USA) at 570 nm wavelength. The background optical density was measured in the wells filled with culture medium, without the cells.

Sulforhodamine B Assay (for Other Cell Lines)
This technique was applied for the cytotoxicity screening against cells growing in adherent culture. The details of this technique were described by Skehan et. al. [39]. The cytotoxicity assay was performed after 72 h exposure of the cultured cells to varying concentrations of the tested agents. The cells attached to the plastic were fixed by gently layering cold 50% TCA (trichloroacetic acid, Sigma Aldrich) on the 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 0.1% sulforhodamine B (SRB, Sigma Aldrich) dissolved in 1% acetic acid (POCh) for 30 min. Unbound dye was removed by rinsing (4×) with 1% acetic acid. The protein-bound dye was extracted with 10 mM unbuffered Tris base (Sigma Aldrich) for determination of optical density (at 540 nm) on Synergy H4 photometer (BioTek Instruments). The background optical density was measured in the wells filled with culture medium, without the cells.

Conclusions
In recent decades, there has been great progress in designing and obtaining modern oncology drugs [40]. In this study we obtained nine compounds, which are the 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide derivatives 3-11. All the new compounds 3-11 and substrates 1 and 2 were tested for activity against the MV4-11 cell line. The substrates had no anticancer activity. The antiproliferative activity of the five most active compounds was examined against breast MCF-7, colon LoVo and LoVo/DX cancer cells and normal MCF-10A cells. 5-Chloro-N'-[(1E,2E)-3-phenylprop-2-en-1-ylidene]-3-methylisothiazole-4-carbohydrazide 3, which is the most active compound of this series, possessed almost two times lower activity (IC 50 value) towards the MCF-10A normal cell line than against cancer cells. This compound is characterized by potential selectivity toward cancer cells such as leukemia, breast and colon cancer cell lines and low toxicity against healthy cells. Other tested compounds displayed slightly weaker antiproliferative activity against tumor cells, but in the case of normal MCF-10A cells, their activity was about three times lower. On the basis of the conducted research, it can be concluded that the antiproliferative activity of this group of derivatives corresponds to the hydrazide moiety. The position of the azomethine group -N=CH at the 5-position of the isothiazole in Schiff bases [36] is more favorable than in the 4-position of the isothiazole ring on hydrazide derivatives 3-11.