The Novel [4,5-e][1,3]Diazepine-4,8-dione and Acyclic Carbamoyl Imino-Ureido Derivatives of Imidazole: Synthesis, Anti-Viral and Anti-Tumor Activity Evaluations

In the present paper, we report on the synthesis, and in vitro antiviral and cytostatic activities of a series of novel imidazole[4,5-e][1,3]diazepine-4,8-dione (compounds 9–11) and acyclic carbamoyl imino-ureido imidazole (compounds 12 and 13) derivatives. These new type of chemical entities showed no significant activity on the broad spectrum of DNA and RNA viruses. Results of antiproliferative assays performed on a panel of selected human tumor cell lines revealed that only compounds 1 and 5 showed moderate and selective cytostatic effect against HeLa cells (IC50 = 24 and 32 µM) with no concomitant cytotoxic effects on human normal fibroblasts (BJ). Importantly, an imidazole derivative containing a pyrrolidine moiety linked via an ethylenic spacer (3) showed a selective cytostatic effect toward cervical carcinoma (HeLa) cells (IC50 = 9.5 µM) with no apparent cytotoxicity on human normal fibroblasts (BJ). This compound can be therefore considered as a potential anti-tumor lead compound for further synthetic structure optimization.


Introduction
It has been found that infection with respiratory syncytial virus (RSV), which manifests primarily as bronchiolitis or viral pneumonia, is the leading cause of lower respiratory tract infections (LRTIs) in infants and young children [1]. Ribavirin still is the only antiviral agent approved for the treatment of RSV infection, but due to efficacy and toxicity issues, it has only limited utility [2]. There is a clear need for new anti-RSV therapeutics, with improved efficacy and safety for broader applications [3]. Powell and his colleagues have recently identified a new class of RSV inhibitors, namely 1,4-benzodiazepines [4], which eventually led to identification of RSV604 as a clinical candidate [5].
Furthermore, imidazole is an entity incorporated into many important biological molecules with a wide range of pharmacological activity. In the field of drug discovery the imidazole scaffold is widely used in the drug design strategy and imidazoles are generally well known as anticancer agents as well [6].

Structural Properties
The structures of 1-13 have been confirmed by 1 H and 13 C-NMR spectra (Experimental). The analysis of the spectra was achieved on the basis of the chemical shift, signal multiplicity and integral values. 19 F NMR resonances of 12 and 13 were well resolved (Experimental). 1 H decoupled 13 C-NMR showed C-F coupling constants that enabled straightforward identification of fluorinated carbon atoms and their neighbors. Two sets of signals were observed for 9 in the ratio 4:1 as was estimated from the integral values of 1 H-NMR signals (e.g., CH-5 proton at δ 8.05 and 7.69 ppm). Two species were most probably detected due to presence of dynamic equilibrium between amine and imine tautomers (C8-NH 2 and C8=NH). C8 chemical shifts of the major species showed value of 149.99 ppm, which is characteristic for the imine form. Therefore, imine form of 9 is the predominant species in solution. Likewise, broad 13 C-NMR signals suggested presence of tautomer forms for 10 and 11. Imine forms are favored according to C8 chemical shift (δ 149.70 and 150.12 ppm for 10 and 11, respectively).
The correlation signals observed in 1 H-13 C HSQC and HMBC spectra allowed assignment of C2, C3 and C5 atoms. Interestingly, major chemical shifts differences between 12 and 13 were observed for C2 and C3 atoms. The correlation signals between methylene protons CH 2 -1' and C2 and C5 in combination with chemical shift values of imidazole carbon atoms suggested that COOCH 3 group is attached to C2 (δ 121.43 ppm) and fluoro-phenyl-ureido-imino-methyl-carbamoyl moiety to C3 (δ 145.53) in 12. On the other hand, COOCH 3 in attached to C3 in 13, which is indicated by C2 and C3 chemical shift (δ 127.71 and 134.19 ppm).
These spectroscopic results indicate that compounds 12 and 13 exist as regioisomers with respect to substitution at positions C-2 and C-3 of the imidazole moiety. This implies that both regioisomers are formed as indicated in Figure 2 and their subsequent in situ reaction gave compounds 12 and 13. Structural differences between 12 and 13, which were suggested by distinct NMR chemical shifts of C2 and C3, were assessed by NOESY experiments. Unfortunately, only trivial NOESY cross-peaks were observed for 12 and 13. COOCH 3 group showed no NOESY signals with methylene protons and therefore no particular conformational preferences could be established for these compounds.

Antiviral Activity
Compounds 1-3, 5-7 and 9-13 were evaluated for their antiviral activity against a wide variety of DNA and RNA viruses, including herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus and vesicular stomatitis virus in HEL cells, parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus in Vero cells, vesicular stomatitis virus, Coxsackie virus B4, and respiratory syncytial virus in HeLa cells and influenza A (H1N1; H3N2) and influenza B viruses in MDCK cells. Unfortunately, none of the compounds showed pronounced antiviral activity at subtoxic concentrations. No cytotoxicity for all evaluated compounds on HEL, Vero, HeLa and MDCK cell cultures was observed (data not shown).

General Materials and Methods
All commercially available chemicals were purchased from Sigma Aldrich (Hamburg, Germany) and used without purification. All solvents were analytical grade purity and dried. Methanol (CH 3 OH) was stored over 3 Å molecular sieves. Dimethylformamide (DMF) was stored over 4 Å molecular sieves. Dichloromethane (CH 2 Cl 2 ) was refluxed over phosphorus pentoxide (P 2 O 5 ), distilled and stored over 4 Å molecular sieves. Merck silica gel 60 F 254 plates were used for thin-layer chromatography. Column chromatography was performed with Merck silica gel (0.063−0.200 mm), with dichloromethane/methanol as eluent. 1 H and 13 C-NMR spectra were recorded on a Varian Gemini 300 spectrometer (Institute Ruđer Bošković, Zagreb, Croatia) and Varian NMR System 600 and Varian Unity Inova 300 and Agilent Technologies DD2 300 MHz NMR spectrometers (National Institute of Chemistry, Ljubljana, Slovenia) Samples were measured in CDCl 3 and DMSO-d 6 solutions at 25 °C in 5 mm NMR tubes. Chemical shifts (δ) in ppm were referred to TMS. High performance LC was performed on Agilent 1100 series system with UV detection (photodiode array detector) using Zorbax C18 reverse-phase analytical column (2.1 × 30 mm; 3.5 µm). All compound used for biological evaluation showed >95% purity in this HPLC system. The electron impact mass spectra and the purity of compounds were assessed by using Agilent Technologies 6410 Triple Quad LC/MS instrument equipped with electrospray interface and triple quadrupole analyzer (LC-MS/MS).

General Procedure for Synthesis of 5-8
To a stirred solution of compounds 1-4 (500 mg; 1.68 mmol) in anhydrous CH 3 OH (15 mL) ammonia is introduced at 0 °C. After saturation a reaction mixture is stirred overnight at room temperature. The solvent and excess ammonia are removed under reduced pressure and the crude product purified by silica gel column chromatography (CH 2 Cl 2 /CH 3 OH = 2/1). (5) (6) (7)

General Procedure for Synthesis of 9-11
To a solution of guanidine hydrochloride (5.38 mmol) in anhydrous methanol (10 mL) cooled to 0 °C is added a solution of sodium methoxide (25 wt%; 13 mmol). The reaction mixture is stirred for 30 min at 0 °C. The sodium salt formed is removed by filtration, and the filtrate thus obtained is added to a solution of compounds 1-3 (1.35 mmol) in anhydrous methanol (5 mL). Reaction mixture is stirred for 72 h at room temperature. The solvent is evaporated and the crude product purified by silica gel column chromatography (CH 2 Cl 2 /CH 3 OH = 2/1).

General Procedure for Synthesis of 12-13
To a solution of guanidine hydrochloride (514 mg; 5.38 mmol) in anhydrous methanol (8 mL) cooled to 0 °C is added a solution of sodium methoxide (25 wt%; 0.75 mL; 13.12 mmol). The reaction mixture is stirred for 30 min at 0 °C. The sodium salt formed is removed by filtration, and the filtrate thus obtained is added to a solution of compound 1 or 2 (400 mg; 1.35 mmol) in anhydrous methanol (5 mL). Reaction mixture is stirred for 8 h at room temperature, solvent evaporated and in reaction mixture in situ dissolved in DMF (3 mL) is added 2-fluorophenyl isocyanate (0,02 mL; 0,18 mmol). Reaction mixture is stirred for 24 h at room temperature. The solvent is evaporated and the crude product purified by silica gel column chromatography (CH 2 Cl 2 /CH 3 OH = 60/1) to give:

Proliferation Assays
The panel cell lines were inoculated onto a series of standard 96-well microtiter plates on day 0, at 3,000 to 5,000 cells per well according to the doubling times of specific cell line. Test agents were then added in 10-fold dilutions (0,01 to 100 µM) and incubated for further 72 h. Working dilutions were freshly prepared on the day of testing in the growth medium. The solvent (DMSO) was also tested for eventual inhibitory activity by adjusting its concentration to be the same as in the working concentrations (DMSO concentration never exceeded 0.1%). After 72 h of incubation, the cell growth rate was evaluated by performing the MTT assay: experimentally determined absorbance values were transformed into a cell percentage growth (PG) using the formulas proposed by NIH and described previously [10]. This method directly relies on control of untreated cells at the day of substances addition because it compares the growth of treated cells with the growth of untreated cells in control wells on the same plate-the results are therefore a percentile difference from the calculated expected value. The IC 50 values for each compound were calculated from dose-response curves using linear regression analysis by fitting the mean test concentrations that give PG values above and below the reference value. If, however, all of the tested concentrations produce PGs exceeding the respective reference level of effect (e.g., PG value of 50) for a given cell line, the highest tested concentration is assigned as the default value (in the screening data report that default value is preceded by a ">" sign). Each test point was performed in quadruplicate in three individual experiments. The results were statistically analyzed (ANOVA, Tukey post-hoc test at p < 0.05). Finally, the effects of the tested substances were evaluated by plotting the mean percentage growth for each cell type in comparison to control on dose response graphs.

Antiviral Activity Assays
The antiviral assays, other than the anti-HIV assays, were based on inhibition of virus-induced cytopathicity in HEL [herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), Viral cytopathicity was recorded microscopically as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. The methodology of the anti-HIV assays was as follows: human CEM cells (~3 × 10 5 cells/mL) were infected with 100 CCID 50 of HIV(III B ) or HIV-2(ROD)/mL and seeded in 200-µL wells of a microtiter plate containing appropriate dilutions of the test compounds. After 4 days of incubation at 37 °C, HIV-induced giant cell formation was examined microscopically.

Conclusions
The main objective of this study was synthesis of a molecule that would be closely related to RSV604. None of the compounds showed pronounced antiviral activity at subtoxic concentrations. No cytotoxicity for all evaluated compounds on HEL, Vero, HeLa and MDCK cell cultures was observed.
From the structure-activity point of view, we believe that the main reason for lack of antiviral activity is probably the absence of either sugar moiety at N-1 of imidazole or a closer resemblance to the active compound RSV604, that was chemically caused and constrained by the formation of the inactive imino form of the cyclized product instead of the targeted amino form. The 1H-imidazole-4,5dicarboxylic acid dimethyl ester derivative having an ethyl morpholino ligand (compound 1) and the 1H-imidazole-4,5-diamide derivative with morpholino moiety bound to an imidazole ring (compound 5) have also exerted selective but modest cytostatic effect towards human cervix carcinoma (HeLa) cells (IC 50 = 24 and 32 µM), while moderate non-selective antiproliferative activity was observed for compound 10. The imidazole [4,5-e] [1,3]diazepine-4,8-dione derivative linked with a pyrrolidine ligand (11) showed only a modest cytostatic effect on colon cancer cells (SW620) (IC 50 = 20 µM). These compounds showed no cytotoxic effects on human normal fibroblasts.
Synthetic chemistry is often full of surprises and presented compounds containing a carbamoyl imino-ureido moiety are structurally very interesting, e.g., compounds 12 and 13. Moreover, 1H-imidazole derivative containing a pyrrolidine moiety linked via an ethylenic spacer to N-1 of the imidazole ring (compound 3) exerted a more pronounced selective cytostatic effect towards human cervix cancer (HeLa) cells (IC 50 = 9.5 µM) in comparison with other tested compounds with no apparent cytotoxicity on normal human skin fibroblasts (BJ) as well. This compound might therefore be suited for further exploration as a potential lead compound and chemical modifications.