Synthesis and antitumor properties of some new N-(5-R-benzyl-1,3- thiazol-2-yl)-4,5-dihydro-1H-imidazole-2-carboxamides

1. Department of Pharmaceutical, Organic and Bioorganic Chemistry Danylo Halytsky Lviv National Medical University, Lviv, 79010, Ukraine 2. Department of General, Bioinorganic, Physical and Colloidal Chemistry, Danylo Halytsky Lviv National Medical University, 69 Pekarska, Lviv, 79010, Ukraine 3. Department of Organic Chemistry, Ivan Franko National University of Lviv, 6 Kyryla і Mefodia, Lviv, 79005, Ukraine

A suspension of 0.01mol of powdered sulfur in 10mL of morpholine was stirred for 5min.
The general procedure fot 4,5-dihydro-1Himidazole-2-carboxamides (3a-e) preparation Method A. 0.0015mol of the corresponding morpholin-4-yl-2-thioxoacetamide 2a-e and 4mL of ethylenediamine was stirred at 50ºС for 30min. The mixture was cooled and poured into the 30mL of water. The precipitate was filtered, washed with water, dried and recrystallized from an alcohol.
Method B. 1g of sulfur was dissolved in ethylenediamine (10 mL), and stirred for 30min. To the formed solution, 0.006mol of the corresponding chloroacetamide was added with constant stirring for 10min. The mixture was continued stirred for 30min, then cooled and poured into the 100mL of water and leave for 1 day. The precipitate was filtered, washed with water, dried and recrystallized from an alcohol.

Cytotoxic activity against malignant human tumor cells
The tested compounds were added to the culture at a single concentration (10 −5 M) and the cultures were incubated for 48 h. Endpoint determinations were made with a protein binding dye, sulforhodamine B (SRB). Results for each tested compound were reported as the percent growth of the treated cells when compared to the untreated control cells. The percent growth was evaluated spectrophotometrically versus not treated controls. The cytotoxic and/or growth inhibitory effects of the most active compounds were tested in vitro against the full panel of about 60 human tumor cell lines at 10-fold dilutions of five concentrations ranging from 10 −4 to 10 −8 M. The 48-h continuous drug exposure protocol was followed and an SRB protein assay was used to estimate cell viability or growth.
Using the seven absorbance measurements [time zero, (Tz), control growth in the absence of drug, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percent growth was calculated at each of the drug concentrations levels. Percent growth inhibition was calculated as: (Ti − Tz) × 100 for concentrations for which Ti ≥ Tz (C − Tz) (Ti − Tz) ×100 for concentrations for which Ti <Tz (Tz) Three dose-response parameters were calculated for each compound. Growth inhibition of 50% (GI50) was calculated from [(Ti − Tz)/(C − Tz)] ×100−50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment was calculated from [(Ti − Tz)/Tz] × 100 = -50. Values were calculated for each of these three parameters if the level of activity was reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as more or less than the maximum or minimum concentration was tested.
It well known that chloroacetanilides react with sulfur and morfoline to form corresponding monothiooxamides (Yarovenko et al., 1999). But chloracetamide derivatives of heterocyclic amines were not investigated in this reaction. So, we study the reaction of chloracetamides 1a-e, sulfur and morfoline. The optimal conditions for the synthesis of target monothiooxamides were the next: firstly, sulfur was stirred with morfoline for 30min. (this time is needed to obtain a sufficient amount of polysulfides in the reaction mixture); after, the corresponding chloroacetyl derivative was added and mixture and stirred for 1 hour. This protocol affords compounds 2a-e in a very high purity and in excellent yields (Scheme 2).
We exanimated the possibility of the synthesis of 4,5-dihydroimidazole-2-carboxamides 3a-e in one-pot reaction of 1, sulfur, and ethylenediamine (Scheme 2). The reaction was carried out by heating in DMF within 5-6 hours, but yields of the final products were lower and required additional crystallizations.  The structures of the obtained compounds were confirmed by 1 H NMR, mass spectroscopy and elemental analysis. Spectroscopic data of all compounds were in accordance to the proposed structures. In 1 H NMR spectra, signals of methylene group protons of N-[5-R-benzyl)-1,3-thiazol-2-yl]-4,5-dihydro-1H-imidazole-2-carboxamides appear as a singlets at 3.81-3.84 ppm. Such a character of the spectrum is due to a rapid tautomeric transformation (Scheme 3).
For the same reason, the NH protons of the amide group of the 4,5-dihydro-1H-imidazole ring do not appear at all. Signals of protons of the methylene group of the benzyl radical are at 4.00 -4.13 ppm. The 4-H proton signals of the thiazole moiety are at 7.12 -7.17 ppm. a Ratio between number of cell lines with percent growth from 0 to75 and total number of cell lines.

Pharmacology
Among newly synthesized compounds substances 2a-e and 3a-c were selected by the National Cancer Institute (NCI) Developmental Therapeutic Program for the in vitro cell line screening to investigate their anticancer activity. The human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers. Primary anticancer assays were performed according to the DTP protocol (NCI USA), which was described elsewhere (Boyd et al., 1995;Boyd et al., 1997;Shoemaker et al., 2006).The results of primary screening are reported as the percent of cancer cell line growth (GP%) ( Table I). The range of GP% shows the lowest and the highest values founded for different cancer cell lines.
Tested compounds 2a-e showed a low antitumor activity in the in vitro screening assay. For the compounds 3a and 3b the average levels of cell growth (GPmean) were 90.59% and 80.86 % respectively. It should be noted, that selective action of tested compounds was observed towards Leukemia cell lines (range of GP= 57.10-71.88% (compound 3a) and GP= 35.01-69.14% (3b). The most active compound 3c was found to be effective against 50 cell lines with the average cell growth Finally, compound 3c was selected for an advanced assay against a panel of approximately sixty tumor cell lines at 10-fold dilutions of five concentrations (100µM, 10µM, 1.0µM, 0.1µM and 0.01µM) ( Table II). The percentage of growth was evaluated spectrophotometrically versus controls not treated with test agents after 48h exposure and using SRB protein assay to estimate cell viability or growth.
Dose-response parameters were calculated for each cell line: GI50 -molar concentration of the compound that inhibits 50% net cell growth; TGI -molar concentration of the compound leading to the total inhibition; and LC50molar concentration of the compound leading to 50% net cell death. Furthermore, a mean graph midpoints (MG_MID) were calculated for GI50, giving an average activity parameter over all cell lines for the tested compound. For the MG_MID calculation, insensitive cell lines were included with the highest concentration tested.
The selectivity index (SI) obtained by dividing the full panel MG-MID (μM) of the compound 3c by its individual subpanel MG-MID (μM) was consideredas a measure of compound's selectivity. Ratios between 3 and 6 refer to moderate selectivity, ratios greater than 6 indicate high selectivity toward the corresponding cell line, while compounds not meeting ei ther of the criteria are rated non-selective (Rostom, 2006). In this context, the active compound 3с demonstrates moderate selectivity toward Leukemia cell lines at the GI50 levels (SI=3.08) and low selectivity toward Renal Cancer cell lines (SI=2.49) at the TGI levels (Table III).