Hybrid Molecules Composed of 2,4-Diamino-1,3,5-triazines and 2-Imino-Coumarins and Coumarins. Synthesis and Cytotoxic Properties

A series of 2-imino-2H-chromen-3-yl-1,3,5-triazine compounds 5–12, which are namely hybrids of 2,4-diamino-1,3,5-triazines and 2-imino-coumarins, was synthesized by reacting 2-(4,6-diamine-1,3,5-triazin-2-yl)acetonitriles 1–4 with 2-hydroxybenzaldehydes. After this, upon heating in aqueous DMF, 2-imino-2H-chromen-3-yl-1,3,5-triazines 10 and 12 were converted into the corresponding 2H-chromen-3-yl-1,3,5-triazines 13 and 14, which are essentially hybrids of 2,4-diamino-1,3,5-triazines and coumarins. The in vitro anticancer activity of the newly prepared compounds was evaluated against five human cancer cell lines: DAN-G, A-427, LCLC-103H, SISO and RT-4. The greatest cytotoxic activity displayed 4-[7-(diethylamino)-2-imino-2H-chromen-3-yl]-6-(4-phenylpiperazin-1-yl)-1,3,5-triazin-2-amine (11, IC50 in the range of 1.51–2.60 μM).


Introduction
It is a well-established fact that numerous medical disorders may be caused by defects in more than one specific biological target, such as a receptor and an enzyme. These disease states cannot be adequately addressed by the classical 'one target, one molecule' approach [1][2][3]. A promising strategy to tackle complex multifactorial diseases involves the design of hybrid molecules as a stable chemical combination of two drug molecules acting at different targets [4][5][6][7][8][9][10][11][12][13][14][15][16]. Such "dual-acting compounds" combine two distinct chemical entities.
In previous studies, we have synthesized a number of 1,3,5-triazine derivatives with pronounced in vitro antitumor properties [63][64][65][66]. Based on the ideas presented above, we reasoned that compounds incorporating both the 1,3,5-triazine and imino-coumarin pharmacophoric groups could be used in very effective antitumor agents as the hybridization of these two different bioactive molecules, which may lead to a synergistic effect. In this present study, we describe new hybrid compounds synthesized by linking 2,4-diamino-1,3,5-triazinyl moiety with coumarin or 2-iminocoumarin ring system ( Figure 1).
After this, the compounds bearing an active methylene group underwent a reaction with 2hydroxybenzaldehydes in the presence of piperidine to give the desired hybrid 2-imino-2H-chromen-3-yl-1,3,5-triazine compounds 5-12 (Scheme 2). The reactions were carried out in 98% ethanol at 20-40 °C in the presence of piperidine as a basic catalyst. The best yields (49-61%) were achieved by using 2-hydroxybenzaldehyde with electron-donating substituent, which was mainly 4-(diethylamino)-2-hydroxybenzaldehyde. After this, the compounds bearing an active methylene group underwent a reaction with 2-hydroxybenzaldehydes in the presence of piperidine to give the desired hybrid 2-imino-2H-chromen-3-yl-1,3,5-triazine compounds 5-12 (Scheme 2). The reactions were carried out in 98% ethanol at 20-40 • C in the presence of piperidine as a basic catalyst. The best yields (49-61%) were achieved by using 2-hydroxybenzaldehyde with electron-donating substituent, which was mainly 4-(diethylamino)-2-hydroxybenzaldehyde. After this, the compounds bearing an active methylene group underwent a reaction with 2hydroxybenzaldehydes in the presence of piperidine to give the desired hybrid 2-imino-2H-chromen-3-yl-1,3,5-triazine compounds 5-12 (Scheme 2). The reactions were carried out in 98% ethanol at 20-40 °C in the presence of piperidine as a basic catalyst. The best yields (49-61%) were achieved by using 2-hydroxybenzaldehyde with electron-donating substituent, which was mainly 4-(diethylamino)-2-hydroxybenzaldehyde. The structures of the 2-imino-coumarin derivatives 5-12 were confirmed by elemental analysis, IR and NMR spectra. In the IR spectra, N-H stretching vibrations of the C=N-H group and the primary amine group (NH 2 ) of the 1,3,5-triazine ring are observed in a range of 3500 to 3200 cm −1 . In turn, 1 H-NMR spectra contain a characteristic singlet representing the proton C4-H of the coumarin ring in a range of 7.43-8.57 ppm, while the proton signal of the imino group C=NH appears in the region of 10.45-11.00 ppm.
However, during spectroscopic characterization of the compounds 7 and 9 containing pyrazoline moiety, we noted the presence of two rotamers with doubled signals found for the protons of the C=NH group in the 1 H-NMR spectra, which were recorded in DMSO-d 6 solution at 20-22 • C (293. 15-295.15 K). The ratio of the rotamers, which was deduced from the integration of the C=NH proton signals, was 1:1 (see experimental section).
To obtain a better insight into the origin of signal doubling and the structures of the possible rotamers, we performed quantum chemical calculations for hybrid compound 7 [69]. The four possible rotamers A, B, C and D generated from the rotation around the bond axis C3-C4 (rotation of 2-imino-coumarin) and C6 -N1 (rotation pf pyrrazoline) are shown in Figure 2. The structures of the 2-imino-coumarin derivatives 5-12 were confirmed by elemental analysis, IR and NMR spectra. In the IR spectra, N-H stretching vibrations of the C=N-H group and the primary amine group (NH2) of the 1,3,5-triazine ring are observed in a range of 3500 to 3200 cm −1 . In turn, 1 H-NMR spectra contain a characteristic singlet representing the proton C4-H of the coumarin ring in a range of 7.43-8.57 ppm, while the proton signal of the imino group C=NH appears in the region of 10.45-11.00 ppm.
However, during spectroscopic characterization of the compounds 7 and 9 containing pyrazoline moiety, we noted the presence of two rotamers with doubled signals found for the protons of the C=NH group in the 1 H-NMR spectra, which were recorded in DMSO-d6 solution at 20-22 °C (293.15-295.15 K). The ratio of the rotamers, which was deduced from the integration of the C=NH proton signals, was 1:1 (see experimental section).
To obtain a better insight into the origin of signal doubling and the structures of the possible rotamers, we performed quantum chemical calculations for hybrid compound 7 [69]. The four possible rotamers A, B, C and D generated from the rotation around the bond axis C3-C4′ (rotation of 2-imino-coumarin) and C6′-N1′′ (rotation pf pyrrazoline) are shown in Figure 2.  The structure 7A was proven to be the lowest energy rotameric form both in DMSO and aqueous solution, while the energy differences between 7A and rotamers 7B, 7C and 7D were very low (0.52-1.53 kcal/mol). Therefore, we determined the barriers of C3-C4′ and C6′-N1′′ bond rotations ( Figure  3) and found that the barrier of C6′-N1′′ rotation was much higher (15.2 kcal/mol) than those of C3-C4′ rotation (8.5 kcal/mol). It is significantly easier to overcome the later barrier under normal conditions (14-20 kcal/mol), which suggests that the two separate pairs of rotamers (7A, 7B) and (7C, 7D) may exist in the solution at room temperature, leading to a doubling of the C2=NH proton signal. The structure 7A was proven to be the lowest energy rotameric form both in DMSO and aqueous solution, while the energy differences between 7A and rotamers 7B, 7C and 7D were very low (0.52-1.53 kcal/mol). Therefore, we determined the barriers of C3-C4 and C6 -N1 bond rotations ( Figure 3) and found that the barrier of C6 -N1 rotation was much higher (15.2 kcal/mol) than those of C3-C4 rotation (8.5 kcal/mol). It is significantly easier to overcome the later barrier under normal conditions (14-20 kcal/mol), which suggests that the two separate pairs of rotamers (7A, 7B) and (7C, 7D) may exist in the solution at room temperature, leading to a doubling of the C2=NH proton signal.

Synthesis of 2H-Chromen-3-yl-1,3,5-triazine Derivatives (Coumarin Derivatives)
In a series of experiments aimed at the purification of 2-imino-2H-chromen-3-yl-1,3,5-triazines 10 and 12 by means of crystallization, we observed that using dimethylformamide containing 10% of water results in the hydrolysis of the imino group, which results in the formation of coumarin derivatives 13 and 14 (Scheme 3). Thus, the imino-coumarins were proven to be rather unstable under aqueous conditions and the presence of a mineral acid is not required for the hydrolysis of iminocoumarin derivatives as described previously [72]. The structures of the newly prepared compounds 13 and 14 were confirmed by elemental analyses and spectroscopic data (IR and NMR). Thus, in the IR spectra, strong absorptions that are attributable to the carbonyl group (C=O) at 1735 cm −1 are observed. In turn, the most diagnostic feature of the 1 H-NMR spectra is the absence of signals corresponding to the protons of the C2=NH imino group. The characteristic C4-H proton signals of coumarin ring are found at 8.55 ppm (compound 13) and 8.54 ppm (compound 14).

Synthesis of 2H-Chromen-3-yl-1,3,5-triazine Derivatives (Coumarin Derivatives)
In a series of experiments aimed at the purification of 2-imino-2H-chromen-3-yl-1,3,5-triazines 10 and 12 by means of crystallization, we observed that using dimethylformamide containing 10% of water results in the hydrolysis of the imino group, which results in the formation of coumarin derivatives 13 and 14 (Scheme 3). Thus, the imino-coumarins were proven to be rather unstable under aqueous conditions and the presence of a mineral acid is not required for the hydrolysis of imino-coumarin derivatives as described previously [72].

Synthesis of 2H-Chromen-3-yl-1,3,5-triazine Derivatives (Coumarin Derivatives)
In a series of experiments aimed at the purification of 2-imino-2H-chromen-3-yl-1,3,5-triazines 10 and 12 by means of crystallization, we observed that using dimethylformamide containing 10% of water results in the hydrolysis of the imino group, which results in the formation of coumarin derivatives 13 and 14 (Scheme 3). Thus, the imino-coumarins were proven to be rather unstable under aqueous conditions and the presence of a mineral acid is not required for the hydrolysis of iminocoumarin derivatives as described previously [72]. The structures of the newly prepared compounds 13 and 14 were confirmed by elemental analyses and spectroscopic data (IR and NMR). Thus, in the IR spectra, strong absorptions that are attributable to the carbonyl group (C=O) at 1735 cm −1 are observed. In turn, the most diagnostic feature of the 1 H-NMR spectra is the absence of signals corresponding to the protons of the C2=NH imino group. The characteristic C4-H proton signals of coumarin ring are found at 8.55 ppm (compound 13) and 8.54 ppm (compound 14).
The compounds tested can be divided into two series: (1) derivatives 5-9 containing a small heterocyclic moiety at the position 6' of the triazine ring and (2) analogues 10-12 substituted with a bulky lipophilic 4-phenylpiperazine moiety. In the first series, the most potent substances were compounds 6 and 7 with the basic electron-donating diethylamino substituent at the position 7 of 2-iminocoumarin ring (IC 50 in the range of 5.67-9.21 µM and 8.16-15.02 µM, respectively). On the other hand, the lowest activity showed the analogue 8 bearing electron-withdrawing Br substituent at the position 6 (IC 50 in the range of 7.69-28.25 µM).
considerably reduced the cytotoxic activity (IC50 in the range of 8.35-21.12 μM vs. 26.32-37.19 μM), while the introduction of a basic electron-donating diethylamino group at the position 7 resulted in the most potent compound 11, which showed slightly lower cytotoxic activities than cisplatin (IC50 values 1.51-2.60 μM vs. 0.06-0.15 mM).
It should be noted that the transformation of 2-iminocoumarins 10 and 12 into the corresponding coumarins 13 and 14 did not improve their cytotoxic properties (see Table 1 and Figure 4). Table 1. Cytotoxic activity of 2-imino-2H-chromen-3-yl-1,3,5-triazines 5-12 and 2H-chromen-3-yl-1,3,5-triazines 13, 14 on five human tumor cell lines (IC50 ± SD, μM) compared to cisplatin (CDDP). The compounds tested can be divided into two series: (1) derivatives 5-9 containing a small heterocyclic moiety at the position 6' of the triazine ring and (2) analogues 10-12 substituted with a bulky lipophilic 4-phenylpiperazine moiety. In the first series, the most potent substances were compounds 6 and 7 with the basic electron-donating diethylamino substituent at the position 7 of 2iminocoumarin ring (IC50 in the range of 5.67-9.21 μM and 8.16-15.02 μM, respectively). On the other hand, the lowest activity showed the analogue 8 bearing electron-withdrawing Br substituent at the position 6 (IC50 in the range of 7.69-28.25 μM). The same pattern was seen in the second series of 4-phenylpiperazine-containing compounds. The substitution of 10 with a basic electron-withdrawing Cl substituent, which creates 12, considerably reduced the cytotoxic activity (IC50 in the range of 8.35-21.12 μM vs. 26.32-37.19 μM), while the introduction of a basic electron-donating diethylamino group at the position 7 resulted in the most potent compound 11, which showed slightly lower cytotoxic activities than cisplatin (IC50 values 1.51-2.60 μM vs. 0.06-0.15 mM).

Experimental Section
The melting points were determined with a Boëtius apparatus and are uncorrected. The infrared spectra were obtained on KBr pastilles using a Nicolet 380 FT-IR (Thermo Fisher Scientific, Waltham, MA, USA). Magnetic resonance spectra (NMR) were recorded with a Varian Gemini 200 BB (200 MHz) spectrometer (Varian Inc. Palo Alto, CA, USA) and Varian Unity Inova 500 (500 MHz) spectrometer in 200 MHZ in a DMSO-d6 solution. The residual peak of the solvent was used as an

Experimental Section
The melting points were determined with a Boëtius apparatus and are uncorrected. The infrared spectra were obtained on KBr pastilles using a Nicolet 380 FT-IR (Thermo Fisher Scientific, Waltham, MA, USA). Magnetic resonance spectra (NMR) were recorded with a Varian Gemini 200 BB (200 MHz) spectrometer (Varian Inc. Palo Alto, CA, USA) and Varian Unity Inova 500 (500 MHz) spectrometer in 200 MHZ in a DMSO-d 6 solution. The residual peak of the solvent was used as an internal standard. Chemical shifts (δ) are given in ppm. Coupling constants (J) are given in Hz. The elemental analyses of carbon, hydrogen and nitrogen determined for compounds were within ±0.4% of the theoretical values.
All cell culture reagents were purchased from Sigma (Deisenhofen, FRG). The cancer cell lines used included the following: human large cell lung carcinoma (LCLC-103H), human urinary bladder carcinoma (5637), human lung carcinoma (A-427), human uterine cervical adenocarcinoma (SISO), human bladder cell carcinoma (RT-4) and human pancreas cell adenocarcinoma (DAN-G). These cancer cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, FRG). The culture medium for cell lines was a 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, FRG) and were passaged shortly before becoming confluent. For the cytotoxicity studies, 100 µL of a cell suspension was seeded into 96-well microtiter plates (Sarstedt) at a density of 1000 cell 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 by 500-fold in 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. The cells were incubated for Cyanoguanidine (dicyandiamide) (2.86 g, 34 mmol) was added to an appropriate solution of amine hydrochloride (34 mmol) in anhydrous n-butanol (10 mL). The mixture was carefully heated until the exothermic reaction was initiated (ca. 90 • C) before being stirred at 122-123 • C for 8 h. After cooling, the mixture was stirred for a further 6 h at room temperature. The next day, the precipitate was filtered, washed with n-butanol and isopropanol and purified by crystallization using methanol.
Another method described in reference [74] involves the fusion of an equimolar mixture of an amine hydrochloride and cyanoguanidine at 130-150 • C for 0.5-2 h.   An appropriate amount of 2-hydroxybenzaldehyde derivative (13 mmol) was added gradually to a suspension of an appropriate amount of 1,3,5-triazineacetonitrile 1-4 (10 mmol) in 98% ethanol (30 mL). After 3 min of stirring, piperidine (0.2 mL) was added dropwise to the solution. The mixture was heated for 30 min at 40 • C and cooled. Stirring was continued at room temperature (20-22 • C) for 18 h. The precipitate was filtered, washed with anhydrous ethanol (3 × 2 mL) and dried. The imino-coumarin derivatives 5-12 were proven to be unstable upon heating in protic solvents. They also decomposed when we attempted chromatographic purification on silica gel. Therefore, the products washed with cold ethanol were used for structural and biological investigations.

Conclusions
The results of the biological studies indicate that hybrid compounds 5-14 have rather weak cytotoxic properties. However, significant antiproliferative potency is associated with a diethylamino substituent at the position 7 of the coumarin ring (compounds 6 and 11). Importantly, compound 11 with a bulky 4-phenylpiperazine moiety installed at the position 6' of the triazine ring showed similar potency to cisplatin against several of the cell lines. It is too early to speculate on the mechanism of action of the compound 11. However, it is well known that coumarins are minor groove binders and exhibit the intercalative mode of binding properties with DNA [75,76]. Therefore, the presence of diethylamino group may increase the efficiency of the intercalative binding due to the extra non-covalent force between the substituent and DNA grooves. The most potent 2,4-diamino-1,3,5-triazine-imino-coumarin 11 may serve as a lead structure for further development of new antitumor drugs. Thus, the hybrid molecule composed of 2,4-diamino-1,3,5-triazine and 2-iminocoumarin was proven to be a promising heterocyclic scaffold for the construction of novel cytotoxic compounds. The syntheses of analogues containing lipophilic substituents at the position 6 of the triazine ring and electron-donating substituents at the positions 5, 6, 7 or 8 of the imino-coumarin moiety, along with quantitative structure-activity relationship (QSAR) studies, are planned.