New Acetylenic Amine Derivatives of 5,8-quinolinediones: Synthesis, Crystal Structure and Antiproliferative Activity

Acetylenic amine derivatives of the 5,8-quinolinedione were synthesized and characterized by the 1 H and 13 C NMR, IR spectroscopy and MS spectra. Additionally, the 6-and 7-substituted allylamine-5,8-quinolinediones were synthesized for comparison purposes. The crystal structure was determined for the 6-chloro-7-propargylamine-5,8-quinolinedione and 7-chloro-6-propargylamine-5,8-quinolinedione. Additionally, the IR spectral analysis supplemented by the density functional theory (DFT) calculations were carried out. It was found that different positions of the propargylamine side chain had a distinct influence on crystal structure, formation of H-bonds and the carbonyl stretching IR bands. Correlation between the frequency separation ∆ν of the carbonyl IR bands and the position of the 6-and 7-substituents was found. The 7-substituted derivatives exhibited a higher frequency separation ∆ν. The observed correlation could provide an opportunity to use the IR spectroscopy to study substitution reactions. Cytotoxic activities against three human cancer cell lines for the 5,8-quinolinedione derivatives with different amine substituents, i.e., propargylamine, N-methylpropargylamine, 1,1-dimethylpropargylamine, allylamine and propylamine were also analysed with respect to their molecular structure.

There are many reports on the synthesis, structure and biological activity of the amine derivatives of 5,8-quinolinedione, whereas studies on the alkyne amino analogues are very scarce [14].Natural and synthetic acetylenic derivatives of the quinoline attract increasing attention since many of them display wide biological activity spectra [15][16][17][18][19][20][21][22].According to the literature data, introduction of the alkyne group may significantly improve biological activity of these compounds [21,22].
Our attention was also focused on the carbonyl stretching bands in the infrared spectra of the propargylamine-substituted 5,8-quinolinedione, which are known as bands very sensitive to morphology.For para-quinones, one or two carbonyl bands can be observed.This feature can be influenced by many factors: mainly intra-and intermolecular interactions and conformational changes [23,24].It can create the opportunity for additional structural investigations using IR carbonyl bands.For example, we recently found an interesting correlation between the frequency separation of carbonyl bands and the position of propylamine substituents on 5,8-quinolinediones [25].Therefore, in this report we also aimed to check whether a similar correlation exists for the propargylamine-substituted 5,8-quinolinediones.

Chemistry
The 6,7-dichloro-5,8-quinolinedione 1 was prepared by the oxidation of the 8-hydroxyquinoline [12] and used as a starting compound for the synthesis of the acetylenic derivatives 2-3 using procedures described in the literature [8,9,25].Treatment of compound 1 with the corresponding amine in tetrahydrofuran in the presence of potassium carbonate at room temperature gave a mixture of 7-and 6-aminosubstituted derivatives, 2a-e and 3a-e, respectively (Scheme 1).The obtained mixtures were separated by column chromatography to afford pure products 2a-e and 3a-e with the 68%-58% and 16%-21% yields, respectively.The structures of all derivatives 2-3 were determined by the 1 H, 13 C NMR, IR and MS spectra.
For both isomeric compounds 2 and 3 the 1 H NMR chemical shifts were similar, and therefore it was not possible to distinguish between 6-and 7-substituents based on the spectra (see Figure S1-S8 in supplement material).According to the literature data [8,12], such differentiation is possible when using the 13 C NMR spectra.It was found that the isomers 2 and 3 showed different signal intensities of the C-5, C-8, C-6 and C-7 atoms.For the 6-aminosubstituted derivatives, the signal intensities of the C-5 and C-7 atoms were higher than those for the C-8 and C-6 atoms.For the 7-aminosubstituted derivatives, the signal intensities followed the opposite relation, i.e., they were higher for the C-8 and C-6 atoms.Therefore, it was confirmed that compounds 2 and 3 possessed the amine group at the C-7 and C-6 positions, respectively.Additionally, for derivatives 2a and 3a, the X-ray diffraction analysis confirmed also substitution of the propargylamine chain at the C-7 and C-6 positions, respectively.Scheme 1. Synthesis of the 6-chloro-7-substituted 5,8-quinolinediones 2a-e and 7-chloro-6-substituted 5,8-quinolinediones 3a-e.
The obtained mixtures were separated by column chromatography to afford pure products 2a-e and 3a-e with the 68%-58% and 16%-21% yields, respectively.The structures of all derivatives 2-3 were determined by the 1 H, 13 C NMR, IR and MS spectra.
For both isomeric compounds 2 and 3 the 1 H NMR chemical shifts were similar, and therefore it was not possible to distinguish between 6-and 7-substituents based on the spectra (see Figure S1-S8 in supplement material).According to the literature data [8,12], such differentiation is possible when using the 13 C NMR spectra.It was found that the isomers 2 and 3 showed different signal intensities of the C-5, C-8, C-6 and C-7 atoms.For the 6-aminosubstituted derivatives, the signal intensities of the C-5 and C-7 atoms were higher than those for the C-8 and C-6 atoms.For the 7-aminosubstituted derivatives, the signal intensities followed the opposite relation, i.e., they were higher for the C-8 and C-6 atoms.Therefore, it was confirmed that compounds 2 and 3 possessed the amine group at the C-7 and C-6 positions, respectively.Additionally, for derivatives 2a and 3a, the X-ray diffraction analysis confirmed also substitution of the propargylamine chain at the C-7 and C-6 positions, respectively.

Crystal Structure and Formation of Hydrogen Bonds
The 6-chloro-7-propargylamine-5,8-quinolinedione 2a and7-chloro-6-propargylamine-5,8quinolinedione 3a crystallized in two different monocyclic space groups, i.e., Pc and P21/n, respectively.Figure 1 shows molecular structures and atom numbers of the compounds 2a and 3a.In Table 1, the crystal parameters, experimental data and refinement details are shown.The selected values of bond distances and angles are presented in Table S1 (supplement material).In terms of bond distances and angles, the geometry of molecules 2a and 3a shows typical  The selected values of bond distances and angles are presented in Table S1 (supplement material).In terms of bond distances and angles, the geometry of molecules 2a and 3a shows typical values [23,25].These are in good agreement with the calculated values.The observed discrepancies between experimental and calculated values are mainly due to the method of calculations.They were done for a single molecule in a vacuum, which means that intermolecular interactions were not taken into account.
The unit cell of 2a contains two molecules (Z = 2).The 5,8-quinolinedione rings accomplish a planar structure.In the unit cell these planes are arranged parallel to each other (see Figure S9 in supplement material).An angle between plane of rings and the propargylamine chain N2C9C10C11 is equal to 84.77 • .This conformation is very similar to that which occurred for the corresponding angle in the crystal structure of the 6-chloro-7-propylamine-5,8-quinolinedione (89.77 • ) described earlier by Jastrzebska et al. [25].Figure 2 depicts the hydrogen bonds found in the crystal structure of 2a.In Table 2 parameters of the hydrogen bonds for 2a are collected.
Crystals 2017, 7, 15 4 of 14 values [23,25].These are in good agreement with the calculated values.The observed discrepancies between experimental and calculated values are mainly due to the method of calculations.They were done for a single molecule in a vacuum, which means that intermolecular interactions were not taken into account.
The unit cell of 2a contains two molecules (Z = 2).The 5,8-quinolinedione rings accomplish a planar structure.In the unit cell these planes are arranged parallel to each other (see Figure S9 in supplement material).An angle between plane of rings and the propargylamine chain N2C9C10C11 is equal to 84.77°.This conformation is very similar to that which occurred for the corresponding angle in the crystal structure of the 6-chloro-7-propylamine-5,8-quinolinedione (89.77°) described earlier by Jastrzebska et al. [25].Figure 2 depicts the hydrogen bonds found in the crystal structure of 2a.In Table 2 parameters of the hydrogen bonds for 2a are collected.Both carbonyl groups of 2a participate in the formation of hydrogen bonds.The oxygen atom O1 forms the bifurcated hydrogen bond, which can be described as: 2).Two other short hydrogen bonds C11-H11•••N1 and C4-H4•••O2 have also been found in 2a with the H•••A distances equal to 2.373 and 2.300 Å, respectively (Table 3).According to the literature data [26,27], for the hydrogen bonds from the acidic C-H donors in the C≡C-H to the N acceptors, the mean H•••N distance is reported to be 2.40 Å.The reason for the shorter H•••N distance in 2a might be the higher basicity of the pyridyl N atom.

D-H•••A D-H H•••A D•••A <D-H•••A
6-chloro-7-propargylamine-5,8-quinolinedione 2a C4-H4 Both carbonyl groups of 2a participate in the formation of hydrogen bonds.The oxygen atom O1 forms the bifurcated hydrogen bond, which can be described as: N2-H2N•••O1•••H9A-C9 (Figure 2).Two other short hydrogen bonds C11-H11•••N1 and C4-H4•••O2 have also been found in 2a with the H•••A distances equal to 2.373 and 2.300 Å, respectively (Table 3).According to the literature data [26,27], for the hydrogen bonds from the acidic C-H donors in the C≡C-H to the N acceptors, the mean H•••N distance is reported to be 2.40 Å.The reason for the shorter H•••N distance in 2a might be the higher basicity of the pyridyl N atom.
For the 7-chloro-6-propargylamine-5,8-quinolinedione 3a, the crystal unit cell contains four molecules (Z = 4, Table 1).The molecules form two layers with the 5,8-quinolinedione rings located inside the unit (see Figure S10 in supplement material).An angle between the 5,8-quinolinedione rings' plane and the propargylamine chain is equal to 68.57• and is significantly smaller than that for 2a (84.77 • ).Simultaneously, this angle is very similar to the corresponding angle in the crystal structure of the 7-chloro-6-propylamine-5,8-quinolinedione 3e (68.57• ), which was described earlier [25].Figure 3 shows the unit cell and the hydrogen bonds identified in the crystal structure of 3a.All parameters of the H-bonds seen in Figure 3 are summarized in Table 2.For 3a crystal structure, the inter-and intra-molecular hydrogen bonds C11-H11•••O1 and N2-H2N•••N1 are observed, respectively.The N•••N distance between the donor and acceptor nitrogen nuclei for the 3a and 3e are equal to 2.957 Å and 3.151 Å, respectively [25].This pronounced difference could be explained by the higher basicity of the N-H donor group from the propargylamine chain in comparison to that from the propylamine.

IR Spectra
Analysis of the IR spectral bands, especially in the frequency ranges of the carbonyl and amine stretching vibrations, have been performed using the calculated harmonic vibrational spectra.Comparison of the experimental and the density functional theory (DFT)-calculated spectra allowed also to obtain information about an impact of the H-bond formation on the vibrational bands, e.g., νstr(N-H), νstr(C=O) or νstr(C≡C-H).
In Figures 4 and 5, the IR spectra for compounds 2a and 3a, both experimental and calculated, are presented.Assignments of the selected bands for all spectra are shown in Table 3.
As shown in Figures 4 and 5, the calculated spectra well reproduce these experimental.This also gives good agreement between calculated and experimental frequencies, which can be seen in Table 3.The observed differences are mainly due to the fact that we are comparing the theoretical spectra of a single molecule in a vacuum with the experimental spectra of crystalline substance.For 3a crystal structure, the inter-and intra-molecular hydrogen bonds C11-H11•••O1 and N2-H2N•••N1 are observed, respectively.The N•••N distance between the donor and acceptor nitrogen nuclei for the 3a and 3e are equal to 2.957 Å and 3.151 Å, respectively [25].This pronounced difference could be explained by the higher basicity of the N-H donor group from the propargylamine chain in comparison to that from the propylamine.

IR Spectra
Analysis of the IR spectral bands, especially in the frequency ranges of the carbonyl and amine stretching vibrations, have been performed using the calculated harmonic vibrational spectra.Comparison of the experimental and the density functional theory (DFT)-calculated spectra allowed also to obtain information about an impact of the H-bond formation on the vibrational bands, e.g., ν str (N-H), ν str (C=O) or ν str (C≡C-H).
In Figures 4 and 5, the IR spectra for compounds 2a and 3a, both experimental and calculated, are presented.Assignments of the selected bands for all spectra are shown in Table 3.
As shown in Figures 4 and 5, the calculated spectra well reproduce these experimental.This also gives good agreement between calculated and experimental frequencies, which can be seen in Table 3.The observed differences are mainly due to the fact that we are comparing the theoretical spectra of a single molecule in a vacuum with the experimental spectra of crystalline substance.
At lower wavenumbers, i.e., below 1300 cm −1 , the observed bands are mainly assigned to the aromatic C-C and C-H vibrations.One can also observe the C-C and C≡C-H aliphatic bend vibrations near 580-590 cm −1 and 650-660 cm −1 , respectively.For compound 2a the band at 1427 cm −1 is assigned to the C-H aliphatic stretching vibrations.As is seen in Figure 4a, its experimental and calculated band intensities show significant difference.The higher intensity of the experimental band is due to formation of the hydrogen bond C9-H9A•••O1.According to literature data [26][27][28], the enhancement of the band intensity for the stretching vibrations of the X-H group (H-bond donor group) is associated with the exceptionally great variation of the electric dipole moment of X-H•••Y.This enhancement of intensity is sometimes used to extract information on H-bond [28].
In Figures 4 and 5, the IR spectra for compounds 2a and 3a, both experimental and calculated, are presented.Assignments of the selected bands for all spectra are shown in Table 3.
As shown in Figures 4 and 5, the calculated spectra well reproduce these experimental.This also gives good agreement between calculated and experimental frequencies, which can be seen in Table 3.The observed differences are mainly due to the fact that we are comparing the theoretical spectra of a single molecule in a vacuum with the experimental spectra of crystalline substance.3 for band assignments.3 for band assignments.In Figure 6, the experimental and calculated IR spectra in the range of the carbonyl bands ~1600-1750 cm −1 are exposed.Each molecule of 2a and 3a possess two carbonyl groups in the para position.Stretching vibrations of two carbonyl groups are usually coupled into two vibrations located at different frequencies, i.e., asymmetric (out of phase) ν as at higher frequency and symmetric (in phase) ν s at lower frequency (see Table 3).At lower wavenumbers, i.e., below 1300 cm −1 , the observed bands are mainly assigned to the aromatic C-C and C-H vibrations.One can also observe the C-C and C≡C-H aliphatic bend vibrations near 580-590 cm −1 and 650-660 cm −1 , respectively.For compound 2a the band at 1427 cm −1 is assigned to the C-H aliphatic stretching vibrations.As is seen in Figure 4a, its experimental and calculated band intensities show significant difference.The higher intensity of the experimental band is due to formation of the hydrogen bond C9-H9A•••O1.According to literature data [26][27][28], the enhancement of the band intensity for the stretching vibrations of the X-H group (H-bond donor group) is associated with the exceptionally great variation of the electric dipole moment of X-H•••Y.This enhancement of intensity is sometimes used to extract information on H-bond [28].
In Figure 6, the experimental and calculated IR spectra in the range of the carbonyl bands ~1600-1750 cm −1 are exposed.Each molecule of 2a and 3a possess two carbonyl groups in the para position.Stretching vibrations of two carbonyl groups are usually coupled into two vibrations located at different frequencies, i.e., asymmetric (out of phase) νas at higher frequency and symmetric (in phase) νs at lower frequency (see Table 3).Analysis of the calculated spectra revealed the band νas is attributed mainly to the carbonyl vibration at the C-8 atom, whereas the νs band is attributed to the C=O vibrations at the C-5 atom.Furthermore, for the 7-substituted derivative, the N-H bending is involved in the νas carbonyl stretching, while for the 6-substituted derivative, the N-H bending is involved in the νs carbonyl Analysis of the calculated spectra revealed the band ν as is attributed mainly to the carbonyl vibration at the C-8 atom, whereas the ν s band is attributed to the C=O vibrations at the C-5 atom.Furthermore, for the 7-substituted derivative, the N-H bending is involved in the ν as carbonyl stretching, while for the 6-substituted derivative, the N-H bending is involved in the ν s carbonyl vibrations.A very similar situation occurred for the 6-and 7-propylamine-substituted 5,8-quinolinedione derivatives described previously by Jastrzebska et al. [25].As in this case, the C=O stretching and the N-H bending vibrations showed coupling effect if they were positioned in close proximity within the molecule.Moreover, there is a correlation between the frequency separation ∆ν = ν as − ν s of the carbonyl bands and the position of the substituent, i.e., the 7-substituted derivative shows higher value of ∆ν than the 6-substituted one.For the 7-propargylamine-substituted 5,8-quinolinedione the calculated and experimental separation values ∆ν are 57 cm −1 and 43 cm −1 versus 10 cm −1 and 15 cm −1 for the 6-substituted derivative, respectively (see Table 3).The similar situation occurred in the case of the 7-and 6-propylamine-substituted 5,8-quinolinediones described previously [25], for which the ∆ν were 59 cm −1 and 51 cm −1 versus 31 cm −1 and 7 cm −1 for the 7-and 6-substituted derivatives, respectively.
For the 7-propargylamino-5,8-quinolinedione 2a, the ν as stretching band shows two peaks at 1700 and 1680 cm −1 (see Figure 5), while for the 6-substituted derivative 3a only single peak at 1692 cm −1 is observed.This effect can be due to the formation of the bifurcated H-bond N2-H2N•••O1•••H9A-C9 described in the previous subsection.The N-H group of the propargylamine chain is involved in both the bifurcated H-bond and the ν as carbonyl stretching vibrations at the C-8 atom.It is also worth noting that the observed splitting into two peaks at 1700 and 1680 cm −1 for the ν as stretching band of the 7-substituted derivative is probably not associated with the type of interaction with the D-H system, but originates rather from the ν as distinctive characteristics.
The bifurcated H-bond also strongly influences the N-H stretching vibrations, giving two peaks at the 3315 and 3258 cm −1 .For the 6-substituted propargylamine derivative, the bifurcated H-bond is absent giving only single band at 3250 cm −1 due to the N-H stretching vibrations.

Antiproliferative Activity
Compounds 1, 2a-e and 3a-e were tested for the antiproliferative activity in vitro against the three human cancer cell lines: melanoma (C-32), glioblastoma (SNB-19) and breast cancer (T47D).Results of the analysis have been summarized in Table 4.It is seen that introduction of the alkynyl, allyl and propyl chains at the C-7 or C-6 position leads to an increase in the cytotoxic activity for the (C-32) and (SNB- 19)

cell lines in comparison to
Crystals 2017, 7, 15 9 of 14 the 6,7-dichloro-5,8-quinolinedione 1.Furthermore, the acetylenic amine derivatives 2a-c and 3a-c show higher activity than the reference compound cisplatin against the C-32 and T47D cell lines.All amino derivatives of the 5,8-quinolinedione show high cytotoxic activity against the melanoma (C-32) cell line, with the IC 50 varying in the range 0.58 to 0.75 µg/mL.Comparing the activity of compounds with alkane (2e and 3e), alkene (2d and 3d) and alkyne (2a and 3a) moiety, showed that the cytotoxic of derivatives depends on the type of bond in the substituent; the rank order of activity against the C-32 cell line, is as follows: propargyl > allyl > propyl.Moreover, for the other cell line (SNB-19 and T47D) the highest activity showed propargylamino compounds 2a and 3a.The activity of 3a and 2a against the glioblastoma (SNB-19) cell line for which the IC 50 parameters have the lowest values 0.09 ± 0.01 µg/mL and 0.26 ± 0.02 µg/mL, respectively.These results suggested that the triple bond seems to be essential for anticancer activity.
For compounds with the acetylenic amine substituents, the cytotoxic activity against the melanoma (C-32) and the breast cancer (T47D) cell lines follows the order: N-methylpropargylamine < 1,1-dimethylpropargylamine < propargylamine.As one can see, expansion of the acetylenic amine chain by binding methyl groups gives a reduction of the cytotoxic activity.

General Techniques
Melting points were measured in the open capillary tubes on a Boetius melting point apparatus.NMR spectra (600/150 MHz) were registered on a Bruker Avance 600 spectrometer (Bruker, Billerica, MA, USA).The spectra were recorded for 1 H and 13 C NMR at room temperature.Chemical shifts were reported in ppm (ν) and J values in Hz.Multiplicity was designated as the singlet (s), doublet (d), triplet (t) and multiplet (m).High-resolution mass spectral analysis was carried out on a Bruker Impact II instrument (Bruker, Billerica, MA, USA) The infrared spectra (IR) were registered using the IRAffinity 1 spectrometer (Shimadzu, Japan) and the KBr pellet method for the sample preparation.All spectra were recorded in the range of 400-4000 cm −1 at room temperature.TLC was carried out on silica gel plates (Merck, Darmstadt, Germany) using a mixture of chloroform and ethanol as an eluent.The visualization was accomplished with UV light and iodine vapour.Column chromatography was performed on silica gel (Merck) with the mixture of chloroform and ethanol (40:1, v/v) as an eluent.
The supplementary crystallographic data for 2a and 3a were deposited at the Cambridge Crystallographic Data Centre (CCDC) as CCDC-1047971 and CCDC-104797.These data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk/data_request/cif.

Density Functional Theory (DFT) Analysis
Harmonic vibrational spectra were calculated by the DFT method implemented in the Gaussian09 software package [30].Details have been described in the earlier work [25].Briefly, the ground state molecular structure was optimized in silico using the B3LYP exchange-correlation functional with the 6-31+G(d,p) basis set.The initial molecular structures of compounds 2a and 3a were taken from the X-ray crystallographic data.The obtained harmonic frequencies were scaled by a factor of 0.964 in accordance with [31].Calculated vibrational modes were also analyzed using the GaussView 5.0 visualization software (Gaussian, Inc., Wallingford, CT, USA).The effect of the position of 6-and 7-propargylamine chain on carbonyl vibrations was observed by taking into account the displacement vectors.

Analysis of Antiproliferative Activity
The cytotoxic activities of tested compounds were determined using the Cell Proliferation Reagent WST-1 assay (Roche Diagnostics, Mannheim, Germany).The entire procedure was previously described in detail in an earlier work [8].Cells were exposed to tested compounds for 24 h at indicated concentrations (in the rank of 0.1-100 µg/mL of dimethyl sulfoxide (DMSO)), and their viabilities were quantified using a cell proliferation assay.The WST-1-formazan was detected using a microplate reader at 450 nm with the reference wavelength of 600 nm.Results were expressed as a mean value of at least three independent experiments performed in triplicate.The cytotoxic activity of the tested compound was compared to the cisplatin.The experiments were repeated in triplicate for each concentration of the compound.The IC 50 parameter describes the concentration of compound (in µg/mL) that inhibits the proliferation rate of the tumor cells by 50% as compared to the control untreated cells.Calculation of the IC 50 was performed using the GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA).

Conclusions
New 6-and 7-propargylamine-substituted 5,8-quinolinediones were synthesized and examined using the X-ray diffraction and IR spectroscopy supplemented by the density functional theory (DFT) calculations.Different positions of the propargylamine chain influenced crystal structure and formation of H-bonds.It was found that the H-bond distinctly affected the ν as stretching band of the carbonyl groups only for the 7-propargylamine-substituted 5,8-quinolinedione.
Substantial changes in the frequency separation ∆ν of the carbonyl stretching bands for different positions of the propargylamine chain were found.Higher frequency separation ∆ν corresponds to the 7-substituted derivative.Correlation between the ∆ν and position of substituent may provide an opportunity to use the IR spectroscopy to study substitution reaction.
Cytotoxic activities against three human cancer cell lines for the 5,8-quinolinedione derivatives with different amine substituents, i.e., propargylamine, N-methylpropargylamine, 1,1-dimethylpropargylamine, allylamine and propylamine were analyzed with respect to their molecular structure.It was found that introduction of the acetylenic, allyl and propylamine chains at the C-7 or C-6 position led to an increase in the cytotoxic activity for the melanoma and glioblastoma cell lines in comparison to the starting compound 6,7-dichloro-5,8-quinolinedione 1.Furthermore, for the melanoma (C-32) and breast cancer (T47D) cell lines, the acetylenic amine derivatives showed higher activity than the reference compound cisplatin.The low IC 50 values for the 7-and 6-substituted propargylamine derivatives against the glioblastoma (SNB-19) cell line were observed.

Table 1 .
Crystal parameters, data collection and refinement details for compounds

Table 3
for band assignments.

Table 3 .
Experimental and calculated vibrational frequencies (cm −1 ) and band assignments for studied compounds 2a and 3a.

Table 3 .
Experimental and calculated vibrational frequencies (cm −1 ) and band assignments for studied compounds 2a and 3a.