Synthesis, Structure and Cytotoxicity Testing of Novel 7-(4,5-Dihydro-1H-imidazol-2-yl)-2-aryl-6,7-dihydro-2H-imidazo[2,1-c][1,2,4]triazol-3(5H)-Imine Derivatives

The appropriate 1-arylhydrazinecarbonitriles 1a–c are subjected to the reaction with 2-chloro-4,5-dihydro-1H-imidazole (2), yielding 7-(4,5-dihydro-1H-imidazol-2-yl)-2-aryl-6,7-dihydro-2H-imidazo[2,1-c][1,2,4]triazol-3(5H)-imines 3a–c, which are subsequently converted into the corresponding amides 4a–e, 8a–c, sulfonamides 5a–n, 9, ureas 6a–I, and thioureas 7a–d. The structures of the newly prepared derivatives 3a–c, 4a–e, 5a–n, 6a–i, 7a–d, 8a–c, and 9 are confirmed by IR, NMR spectroscopic data, as well as single-crystal X-ray analyses of 5e and 8c. The in vitro cytotoxic potency of these compounds is determined on a panel of human cancer cell lines, and the relationships between structure and antitumor activity are discussed. The most active 4-chloro-N-(2-(4-chlorophenyl)-7-(4,5-dihydro-1H-imidazol-2-yl)-6,7-dihydro-2H-imidazo[2,1-c][1,2,4]triazol-3(5H)-ylidene)benzamide (4e) and N-(7-(4,5-dihydro-1H-imidazol-2-yl)-2-(p-tolyl)-6,7-dihydro-2H-imidazo[2,1-c][1,2,4]triazol-3(5H)-ylidene)-[1,1′-biphenyl]-4-sulfonamide (5l) inhibits the growth of the cervical cancer SISO and bladder cancer RT-112 cell lines with IC50 values in the range of 2.38–3.77 μM. Moreover, N-(7-(4,5-dihydro-1H-imidazol-2-yl)-2-phenyl-6,7-dihydro-2H-imidazo[2,1-c][1,2,4]triazol-3(5H)-ylidene)-4-phenoxybenzenesulfonamide (5m) has the best selectivity towards the SISO cell line and induces apoptosis in this cell line.


Chemistry
Our research started with reactions of 1-arylhydrazinecarbonitriles 1a-c [42] with 2-chloro-4,5-dihydro-1H-imidazole (2) [43]. As outlined in Scheme 1, the treatment of 1a-c with an excess of 2-chloro-4,5-dihydro-1H-imidazole (2) in dichloromethane at ambient temperature yielded the desired The mechanism of the formation of 3a-c may be explained as follows. The nucleophilic attack of the NH2 group of 1-arylhydrazinecarbonitriles 1a-c at the carbon atom C-2 of 2-chloro-4,5-dihydro-1H-imidazole (2) leads to the formation of the intermediate A. The use of a molar excess of 2-chloro-4,5-dihydro-1H-imidazole (2) allows the compound A to attack the second molecule of 2 to yield intermediate B. It should be noted that 2-chloroimidazoline (2) acts as a base in this process. In turn, the resulting intermediate B undergoes intramolecular cyclization to form fused imidazo-triazole derivatives 3a-c (Scheme 1).
The imine moiety C=NH present in the structure of compounds 3a-c allowed for their further transformations. Thus, reactions of 3a-c carried out in chloroform with a variety of acyl chlorides or sulfonyl chlorides gave rise to the formation of the corresponding amides 4a-e and sulfonamides 5a-n in good yields (Scheme 2).
During the course of our experimental research, it was found that heating compounds 3a and 3b with a two-fold molar excess of acyl or sulfonyl chloride in the presence of triethylamine (TEA) leads to the formation of products substituted both at the nitrogen atom of the imine C=N-H moiety
In the 1 H-NMR spectra of compounds 4-7, a broad singlet corresponding to the proton of the N-H group of the imidazoline ring is present in the range of 5.45-6.33 ppm. The characteristic methylene protons CH2-CH2 of the fused imidazo-triazole moiety and 4,5-dihydro-1H-imidazole ring are found in the range of 3.38-4.79 ppm.
In the 1 H-NMR spectra of compounds 4-7, a broad singlet corresponding to the proton of the N-H group of the imidazoline ring is present in the range of 5.45-6.33 ppm. The characteristic methylene protons CH 2 -CH 2 of the fused imidazo-triazole moiety and 4,5-dihydro-1H-imidazole ring are found in the range of 3.38-4.79 ppm.
Moreover, the crystal structures of compounds 5e and 8c were determined by X-ray crystallography. The molecules of 5e and 8c contain a common 7-(4,5-dihydro-1H-imidazol-2-yl)-2,5,6,7-tetrahydro-3H-imidazo[2,1-c] [1,2,4]triazol-3-imine fragment that adopts the same configuration in both molecules (Figures 3 and 4). The amino N13 atom of the imidazolidine substituent shows a pyramidal arrangement of its bonds with the sum of valence angles equal to 343.4 • in 5e and 349.2 • in 8c. A weak intramolecular N13-H···N1 hydrogen-bond interaction with H···N1 distance of 2.4 Å is observed in 5e. In turn, introduction of the acyl group at N13 results in an intramolecular strain that leads to a short contact of 2.822 Å between the imino N1 atom of the bicyclic system and the carbonyl C23 atom of the acyl group.
Molecules 2020, 25, x FOR PEER REVIEW 7 of 23 the acyl group at N13 results in an intramolecular strain that leads to a short contact of 2.822 Å between the imino N1 atom of the bicyclic system and the carbonyl C23 atom of the acyl group.  The disordered water molecule is not shown.
First, primary screening of the new compounds was done to indicate whether a compound possesses enough activity at a concentration of 10 μM or 20 μM to inhibit cell growth by 50%. The human tumor cell lines used were: human non-small cell lung cancer LCLC-103H, human cervix cancer SISO, human bladder carcinoma 5637, and human bladder carcinoma epithelial RT-112. Compounds that inhibited cell growth by more than 50% at 10 or 20 μM in one or more cell line were further investigated.
It should be noted that all the imines 3a-c (Scheme 1), as well as the di-substituted amides 8a-c and benzenesulfonamide 9 (Scheme 4) were inactive. On the other hand, for amide 4e, sulfonamides 5e, 5i-m, ureas 6e-f, and thiourea 7c, which passed the preliminary test, a secondary screening to the acyl group at N13 results in an intramolecular strain that leads to a short contact of 2.822 Å between the imino N1 atom of the bicyclic system and the carbonyl C23 atom of the acyl group.  The disordered water molecule is not shown.
First, primary screening of the new compounds was done to indicate whether a compound possesses enough activity at a concentration of 10 μM or 20 μM to inhibit cell growth by 50%. The human tumor cell lines used were: human non-small cell lung cancer LCLC-103H, human cervix cancer SISO, human bladder carcinoma 5637, and human bladder carcinoma epithelial RT-112. Compounds that inhibited cell growth by more than 50% at 10 or 20 μM in one or more cell line were further investigated.
It should be noted that all the imines 3a-c (Scheme 1), as well as the di-substituted amides 8a-c and benzenesulfonamide 9 (Scheme 4) were inactive. On the other hand, for amide 4e, sulfonamides 5e, 5i-m, ureas 6e-f, and thiourea 7c, which passed the preliminary test, a secondary screening to determine their potency was performed on two human tumor cell lines: human cervix cancer SISO
First, primary screening of the new compounds was done to indicate whether a compound possesses enough activity at a concentration of 10 µM or 20 µM to inhibit cell growth by 50%. The human tumor cell lines used were: human non-small cell lung cancer LCLC-103H, human cervix cancer SISO, human bladder carcinoma 5637, and human bladder carcinoma epithelial RT-112. Compounds that inhibited cell growth by more than 50% at 10 or 20 µM in one or more cell line were further investigated.
It should be noted that all the imines 3a-c (Scheme 1), as well as the di-substituted amides 8a-c and benzenesulfonamide 9 (Scheme 4) were inactive. On the other hand, for amide 4e, sulfonamides 5e, 5i-m, ureas 6e-f, and thiourea 7c, which passed the preliminary test, a secondary screening to determine their potency was performed on two human tumor cell lines: human cervix cancer SISO and human bladder carcinoma epithelial RT-112. The results of the secondary screening are presented in Table 1 as the average IC 50 values calculated from dose-response data after 96 h of exposure to the tested compounds.  6.65 ± 0.55 >10 6f 3.75 ± 1.12 6.01 ± 0.85 7c 14.16 ± 0.80 nd cisplatin ref. [44] 0.24 ± 0.06 In the series of amides 4a-e (Scheme 2), only compound 4e bearing electron-withdrawing groups R = Cl at position 4 of the phenyl ring and R 1 = C 6 H 4 -Cl(4) of the amide functionality displayed growth inhibitory properties towards the two cell lines and showed slightly lower potency than the reference drug cisplatin (IC 50 values 2.87-3.06 µM vs. 0.24-1.22 µM, Table 1). Other compounds with R = H, CH 3 and R 1 = C 6 H 5 , C 6 H 4 -CH 3 (4), C 6 H 4 -F(4), and C 6 H 4 -Cl(4) did not pass the preliminary test (4a-d, Scheme 2). This may suggest that the presence of two electron-withdrawing substituents at both Rand R 1 -positions is important for the inhibitory activity of the tested compounds.
Similarly to amides 4a-e, in the series of ureas 6a-i, the best activity was found for compound 6f with two electron-withdrawing substituents: R = Cl at position 4 of the phenyl ring and R 1 = C 6 H 4 -Cl(4) of the urea moiety (IC 50 = 3.75-6.01 µM, Table 1). Replacement of the Cl-substituent at the R-position for the electron-donating methyl group yielded less active analogue 6e with selectivity to the cervical cancer cell line SISO (IC 50 = 6.65 µM) over the bladder cancer cell line RT-112 (IC 50 > 10 µM). Introduction of any of the substituents R = H, CH 3 and R 1 = C 6 H 5 , C 6 H 4 -CH 3 (4), and 1-naphthyl, SO 2 -C 6 H 4 -CH 3 (4), however, resulted in compounds that did not pass the preliminary test (6a-d, 6g-i, Scheme 3).
The sulfonamide 5m, which demonstrated pronounced cytotoxicity and selectivity for the cervical cancer cell line SISO (IC 50 = 5.37 µM) over the bladder cancer cell line RT-112 (IC 50 > 10 µM), was chosen to investigate whether it can induce apoptosis in the representative SISO cell line.

Induction of Apoptosis by Compound 5m
One of the most common methods used to detect apoptotic programmed cell death is to double stain treated cancer cells with the Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide, which together distinguish cells as normal and in early or late stage of apoptosis. The Annexin V assay allows the quantification of the relative number of cancer cells undergoing apoptosis; by use of fluorescent flow cytometry the distribution of cells in early and late stages of apoptosis can be measured. In Figure 5  The sulfonamide 5m, which demonstrated pronounced cytotoxicity and selectivity for the cervical cancer cell line SISO (IC50 = 5.37 μM) over the bladder cancer cell line RT-112 (IC50 > 10 μM), was chosen to investigate whether it can induce apoptosis in the representative SISO cell line.

Induction of Apoptosis by Compound 5m
One of the most common methods used to detect apoptotic programmed cell death is to double stain treated cancer cells with the Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide, which together distinguish cells as normal and in early or late stage of apoptosis. The Annexin V assay allows the quantification of the relative number of cancer cells undergoing apoptosis; by use of fluorescent flow cytometry the distribution of cells in early and late stages of apoptosis can be measured.. In Figure 5

Chemistry
The melting points were determined with a Boëtius apparatus and were uncorrected. The infrared spectra were recorded on a Nicolet 380 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Magnetic resonance spectra (NMR) (Agilent, Santa Clara, CA, USA) were recorded on a Varian Gemini 200 BB (200 MHz) spectrometer, a Varian Mercury-Vx300 spectrometer (300 MHz), and a Varian Unity Inova 500 (500 MHz) spectrometer in DMSO-d6 or CDCl3. The residual peaks of solvents were used as internal standards. Chemical shifts (δ) are given in ppm, and coupling constants (J) are given in Hz. Mass spectra were recorded on an LCMS 2010 spectrometer (Shimadzu, Tokyo, Japan). The compounds were identified based on their molecular ions obtained through electrospray ionization. Compounds were purified by the use of preparative chromatography. Thin-layer chromatography was performed on silica gel plates with fluorescence detection (Merck Silica Gel 254, Merck KGaA, Darmstadt, Germany). After, drying spots were detected under UV light (λ = 254 nm). The elemental analyses of carbon, hydrogen, and nitrogen

Chemistry
The melting points were determined with a Boëtius apparatus and were uncorrected. The infrared spectra were recorded on a Nicolet 380 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Magnetic resonance spectra (NMR) (Agilent, Santa Clara, CA, USA) were recorded on a Varian Gemini 200 BB (200 MHz) spectrometer, a Varian Mercury-Vx300 spectrometer (300 MHz), and a Varian Unity Inova 500 (500 MHz) spectrometer in DMSO-d 6 or CDCl 3 . The residual peaks of solvents were used as internal standards. Chemical shifts (δ) are given in ppm, and coupling constants (J) are given in Hz. Mass spectra were recorded on an LCMS 2010 spectrometer (Shimadzu, Tokyo, Japan).
The compounds were identified based on their molecular ions obtained through electrospray ionization. Compounds were purified by the use of preparative chromatography. Thin-layer chromatography was performed on silica gel plates with fluorescence detection (Merck Silica Gel 254, Merck KGaA, Darmstadt, Germany). After, drying spots were detected under UV light (λ = 254 nm). The elemental analyses of carbon, hydrogen, and nitrogen determined for the compounds were within ±0.4% of the theoretical values.

A General Procedure for the Preparation of Compounds 3a-c
To a stirred solution of 2.5 g of 2-chloro-4,5-dihydro-1H-imidazole (25 mmol) (2) in dichloromethane (25-30 mL), five millimoles of the appropriate 1-arylhydrazinecarbonitrile 1a-c were added. When the exothermic reaction subsided, the reaction mixture was stirred at room temperature for 12 h. The precipitate was filtered and washed with dichloromethane (1a, 1c) or the oily residue was separated by decantation and washed with dichloromethane (1b). After drying, the resulting precipitate or oily residue was mixed with cooled water (15 mL) and filtered. The cooled filtrate was basified with 15 mL of a 20% potassium carbonate solution. The precipitate (3b, 3c) was separated by suction, washed with a small amount of cooled water, and dried or the resulting oil (3a) was extracted with chloroform (4 × 20 mL). The combined organic extract was dried with anhydrous magnesium sulfate(VI), filtered, and concentrated under reduced pressure.

A General Procedure for the Preparation of Compounds 6a-i and 7a-d
To a stirring solution of compound 3a-c in anhydrous dichloromethane (5 mL), the appropriate aryl isocyanate or isothiocyanate was added (in the molar ratio of 1:1). The mixture was stirred at room temperature (20-22 • C) for 12 h. The progress of the reaction was controlled by TLC. After completion of the reaction, the precipitate was separated by suction, washed with a small amount of dichloromethane, and dried. The crude product was purified on silica gel by preparative thin-layer chromatography (chromatotron) or crystallization. In this manner, the following compounds were obtained.   [47]. Hydrogen atoms were placed in calculated positions and refined as riding on their carriers, except the N-H group H atom in 5e, which was freely refined. For 8c, the final difference Fourier map showed a residual electron density peak of ca. 1 e/Å3 close to the inversion center and at a distance of 2.97 Å from O16. This peak was interpreted as a water molecule with occupancy 0.25 disordered around the inversion center. The H atom positions of the disordered water molecule were not determined. Illustrations were prepared with the Mercury software [48].
Crystal data for 5e (C 20

In Vitro Anticancer Activity
All cell culture reagents were purchased from Sigma (Deisenhofen, Germany). Cancer cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The culture medium for cell lines was RPMI-1640 medium containing 2 g/L HCO 3 − and 10% FCS. Cells were incubated in a humid atmosphere of 5% CO 2 at 37 • C in 75 cm 2 plastic culture flasks (Sarstedt, Nümbrecht, Germany) and were passaged shortly before becoming confluent. For the cytotoxicity studies, one-hundred microliters of a cell suspension were seeded into 96 well microtiter plates (Sarstedt) at a density of 1000 cells per well except for the LCLC-103H cell line, which was plated out at 250 cells per well. One day after plating, the cells were treated with the test substance at five concentrations per compound. The 1000-fold concentrated stock solutions in DMF or DMSO were serially diluted by 50% in DMF or DMSO to give the feed solutions, which were diluted 500-fold into the culture medium. The controls received DMF or DMSO. Each concentration was tested in eight wells, with each well receiving 100 µL of the medium containing the substance. The concentration ranges were chosen to bracket the expected IC 50 values as best as possible. Cells were then incubated for 96 h, after which time, the medium was removed and replaced with 1% glutaraldehyde/PBS. Optical density (OD) was measured at λ = 570 nm by the use of a Sunrise plate reader (

Annexin V Assay
For this assay, the SISO cell line was used. Cells were detached by trypsinization and counted with a Coulter Counter Z2. Two-hundred fifty-thousand cells were seeded in 2 mL per well of a 6 well plate and allowed to attach overnight. The stock solutions of compound 5m dissolved in DMF were added to the culture medium to the desired end concentration of 10 µM. For the control, only the solvent was added. The old medium was removed, and 3 mL of fresh medium containing the test compound were added to each well. The plates were incubated for 24 h. After centrifugation, supernatants were removed, and the cells were washed once with PBS, then 500 µL of a 25% trypsin/EDTA/PBS solution were added to each well. Plates were incubated for 5 min, and 1.0 mL of medium was added per well. Cells were resuspended, transferred to 1.5 mL tubes and centrifuged for 5 min. The supernatant was discarded, and 500 µL of 1× binding buffer were added to each tube followed by a 5 min centrifugation. Afterward, the supernatant was removed and 50 µL of 1× binding buffer were added to resuspend the cells. Five microliters of Annexin V-FITC staining solution were pipetted into each tube. To obtain a homogenous suspension, tubes were vortexed and incubated in the dark for 15 min at room temperature. Afterward, 500 µL of 1× binding buffer were added per tube to wash the cells. Tubes were centrifuged for 5 min, and the supernatant was aspired completely. The cell pellet was resuspended in 250 µL 1× binding buffer. Immediately before measurement, two-point-five microliters of the PI solution were added. The prepared samples were analyzed by flow cytometry using the FITC signal detector (FL1) and the phycoerythrin emission signal detector (FL2).