Synthesis and characterization of a new series of thiadiazole derivatives as potential anticancer agents

Chemistry

All chemicals were obtained either from Merck (Merck KGaA, Darmstadt, Germany) or Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, MO, USA) and used without further chemical purification. Chemical purities of the compounds were checked by classical TLC applications performed on silica gel 60 F254 (Merck KGaA, Darmstadt, Germany). Melting points of the compounds were measured using an automatic melting point determination instrument (MP90, Mettler-Toledo, OH, USA) and were presented as uncorrected. ¹H and ¹³C NMR spectra were recorded in DMSO-d₆ using a Bruker digital FT-NMR spectrometer (Bruker Bioscience, MA, USA) at 300 MHz and 75 MHz, respectively. M+1 peaks were determined using a Shimadzu LC/MS IT-TOF system (Shimadzu, Tokyo, Japan).

Materials and methods

Cytotoxicity test

Metabolic activity of viable cells was measured by MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt to formazan product, which can be quantified spectrophotometrically to determine the percentage of viable cells. The anticancer activity of compounds 4a-4j were screened according to the MTT assay. The MTT assay was performed as previously described [16, 17, 18]. A459, MCF-7, HepG2 and C6 cancer cell lines were used in the MTT assay. Cisplatin was used as a reference drug.

Prediction of ADME (Absorption, Distribution, Metabolism, and Excretion) parameters and BBB (Blood–brain barrier) permeability

Online Molinspiration property calculation program was used to predict absorption, distribution, metabolism and excretion (ADME) parameters of compounds 3a-3j [19]. The blood brain barrier (BBB) permeability of the compounds was evaluated using an online BBB Predictor [20].

Chemistry

The compounds 4a-4j were synthesized as summarized in Scheme 1. For the synthesis of the compounds 1a-1j, a suitable isothiocyanate and an excess of hydrazine hydrate were reacted. Secondly, thiadiazole derivatives (2a-2j) were obtained by means of the reaction of thiourea derivatives (1a-1j) and carbon disulfide in the presence of NaOH. 2-Chloro-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl) acetamide (3a) was prepared via the acetylation reaction between 2-amino-5-trifluoromethyl-1,3,4-thiadiazole and chloroacetyl chloride. In the last step, the reaction of compounds 2a-2j and compound 3a afforded the target compounds (4a-4j).

Scheme 1

Synthesis route for the target compounds (4a-4j).

The final compounds were purified, and their structures were characterized by spectroscopic methods (¹H-NMR, ¹³C-NMR, and LC-MS/MS). In the ¹H NMR spectrum, mono-substituted benzene protons had triplet and doublet peaks between 6.98 ppm and 7.56 ppm. 1,4-disubstituted benzene protons had doublet peaks between 7.13 ppm and 7.43 ppm. Methylene protons had singlet peaks between 3.91 ppm and 4.21 ppm. The thiadiazole NH gave broad singlet peaks between 7.68 ppm and 7.99 ppm if it had an aliphatic substituent. The thiadiazole NH of compounds 4i and 4j, with aromatic substituents, gave broad singlet peaks between 10.31 ppm and 10.33 ppm. Compound 4f bearing an allyl moiety gave a double quartet peak at 5.15 ppm. Additionally, 4f had a triplet peak at 3.87 and multiplet peak between 5.80 ppm and 5.93 ppm. In the ¹³C NMR spectrum, aliphatic peaks belonging to substituents were observed between 11.81 ppm and 70.39 ppm. Aromatic carbons were observed between 116.69 ppm and 173.49 ppm. The trifluoromethyl carbon gave a peak between 120.38 ppm and 123.57 ppm as a quartet. The thiadiazole carbon to which the trifluoromethyl group was attached gave a peak between 146.25 ppm and 150.72 ppm. All masses were in accordance with the estimated M+H values.

In addition to structure elucidations using routine spectroscopic methods, 2D NMR studies including ¹H-¹³C HMBC and ¹H-¹³C HSQC were performed for compound 4a, as seen in Figure 1 and Figure 2. According to the information obtained in the HSQC, the peak at 31.49 ppm belongs to the methyl carbon and the peak at 38.86 ppm belongs to the methylene carbon. HMBC shows interactions between hydrogen and carbon at a distance of 2 to 4 bonds. Using the HMBC spectrum presented in Figure 2, it was determined that the peak at 149.30 ppm belongs to the thiadiazole carbon to which the sulphur is attached, the peak at 169.72 ppm belongs to the carbonyl carbon and the peak at 170.98 ppm belongs to the methylamino-bound carbon of the thiadiazole ring.

Figure 1

HSQC spectrum of compound 4a.

Figure 2

HMBC spectrum of compound 4a.

Biological Evaluations

All new synthesized thiadiazole compounds (4a-4j) were tested for cytotoxicity against A549 (lung) and MCF-7 (breast) human cancer lines. The IC₅₀ values of the compounds are presented in Table 1. Anticancer activities were tested using an MTT assay against human lung (A549), breast (MCF-7) liver (HepG2) and rat brain (C6) cancer cells; the assay is used to measure cell viability (the activity of living cells) via the mitochondrial dehydrogenase catalyzed conversion of tetrazolium MTT salt (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to formazan. These structures of thiadiazole derivatives did not prove to be significantly cytotoxic to the A549, MCF-7 and C6 cell lines at tested concentrations. Compound 4i (phenyl-substituted) was determined to have an IC₅₀ value of 0.097 mM against C6 cell line. In contrast, the p-tolyl-containing derivative (4j) showed no promising activity. Based on the activity results obtained on the C6 cell line, it can be assumed that incorporation of an aromatic ring into the structure increases the activity. However, when the non-substituted state of this aromatic ring was compared to the methyl-bearing state at position 4, it was found that the methyl group reduced activity.

Prediction of ADME parameters and BBB permeability

As an important drug discovery approach it is beneficial to evaluate the pharmacokinetic profiles of drug candidates during the early developmental phases. In recent years, with the help of combinatorial chemistry, the number of drug candidates for which early data on absorption, distribution, metabolism and excretion (ADME) are needed has significantly increased [21]. Thus, predictions of the ADME properties of the obtained compounds (4a-4j) were calculated using online Molinspiration chemical property software [19]. This program is based on Lipinski’s rule of five, which predicts the ADME properties of drug-like compounds, and is very important to optimize a biologically active compound. According to Lipinski’s rule, an orally active drug should not possess more than one violation of the criteria. The theoretical calculations of ADME parameters (topological polar surface area (TPSA), molecular volume (MV), number of hydrogen acceptors (nOHNH), number of hydrogen donors (nON), partition coefficient (log P), and molecular weight (MW)) are accessible in Table 2 along with the violations of Lipinski’s rule. In regard to these data, the obtained compounds (4a-4j) suited Lipinski’s rule by displaying no violation. As a result, it can be suggested that the synthesized compounds may have a good pharmacokinetic profile, increasing their pharmacological significance.

The activity of the compounds against brain cancer (C6) has prompted curiosity as to whether they can cross the blood brain barrier. Therefore, BBB permeability of the synthesized compounds (4a-4j) was calculated by a CBLigand-BBB prediction server [20]. This online predictor uses two different algorithms as AdaBoost and Support Vector Machine (SVM), combined with four different fingerprints, employed to predict if a compound can pass (+) or cannot pass (-) the BBB. Predictor scores higher than 0 indicate that a compound can pass the BBB. Table 2 demonstrates that calculations for all compounds resulted with BBB (+), which is essential for anticancer agents against brain cells to display their biological activity.