Ultrasound-assisted multicomponent synthesis of 4H-pyrans in water and DNA binding studies

A simple approach to synthesize new highly substituted 4H-pyran derivatives is described. Efficient Et3N acts as a readily accessible catalyst of this process performed in pure water and with only a 20 mol% of catalyst loading. The extremely simple operational methodology, short reaction times, clean procedure and excellent product yields render this new approach extremely appealing for the synthesis of 4H-pyrans, as potentially biological scaffolds. Additionally, DNA interaction analysis reveals that 4H-pyran derivatives behave preferably as minor groove binders over major groove or intercalators. Therefore, this is one of the scarce examples where pyrans have resulted to be interesting DNA binders with high binding constants (Kb ranges from 1.53 × 104 M−1 to 2.05 × 106 M−1).


Results and discussion
Synthesis of 4H-pyrans in water. After an extensive screening of the reaction conditions such as solvents, base catalysts, the concentration of the reagents and time, among others (see supporting information for more details, Tables S1 and S2), the scope of this process was explored for the synthesis of highly substituted 4H-pyrans 3. Hence, in search of greener procedures, two new methods using pure water have been explored (Fig. 2, routes A and B). The first route involves the use of the preformed alkylidene malononitrile reagent 2. Additionally, more interesting was the development of a multicomponent approach using ultrasounds, also carried out in pure water (Fig. 2B).
With the best reaction conditions in hand [Et 3 N (20 mol%) and H 2 O (0.25 mL), at room temperature (Table S1, entry 38)], final products 3 were obtained with good yields (up to 92%) following route A after 24 h (Fig. 2, yields in blue). The addition of 50 μL of EtOH in those cases where poor yields were obtained, rendered better results, maybe due to an improved solubility of all reagents in the reaction medium (Fig. 2, yields in purple). It is worth noting that the multicomponent approach performed in pure water at room temperature ( Fig. 2B) gave rise to better results in shorter reaction times (2 h) (Fig. 2, yields in green). In this case, the use of ultrasounds as the activation way of the reaction was the key factor for the high yields obtained [not using ultrasounds under the same reaction conditions provides poorer yields (Table S2, entry 2)]. Moreover in this process, the bath temperature was not appreciably increased after 2 h. Therefore, it is expected that the reactions are activated by the ultrasound energy itself and no due to an increase in temperature of the reaction. In the multicomponent process, the reagents are used in equivalence as a clear example of atom economy 68 . The crudes of all these reactions are very clean and the purification and isolation of the products are carried out after a simple extraction from the same vessel and a fast column chromatography on silica gel.
Even though there is not a clear correlation between the substitution on the aromatic rings and the reactivity of the process following route A, it seems that for route B, electron-withdrawing groups in the aromatic ring render better yields in comparison with those bearing electron-donating groups or with heteroaromatic rings (Fig. 2 14 , III 15 , IV 16 , V 17 and VI 18 . Calculation of the binding constant: UV-Vis spectra. There are several ways to calculate binding constants (K b ) between drugs and DNA and, therefore, many techniques that allow to do so. Examples of these are fluorescence and absorption techniques [80][81][82][83][84] . We have selected UV-Vis because of its availability and straightforward handling. There are two procedures that can be used to perform the experiments. In the first one, the DNA is titrated with increasing amounts of the assayed compound. Then, the variations observed in the position and intensity of the DNA peak at 260 nm are measured and the data processed to obtain binding constants and hints about the plausible interaction modes 85 . However, this method presents a limitation. The small extinction coefficient of the DNA leads to a worse precision in the calculation of the resulting binding constant. In contrast, if the experiment is conducted taking as reference the peaks of the studied compounds, usually with stronger absorptions, bigger changes in the bands can be observed. Thus, better precisions on the K b are expected to be obtained 86,87 . Therefore, the second methodology was selected to elucidate the K b of compounds 3a-3o, 6-9 with ctDNA. An example of the titration experiments can be seen in Fig. S33 for compound 3n [88][89][90] .
In this case, 3n showed two intensive absorption bands at 242 and 295 nm, which are typically associated with π → π* and n → π* electronic transitions, respectively. The successive additions of DNA promoted a hypochromic effect in the peak at 295 nm and a hyperchromic effect in the peak at 242 nm, indicative of an interaction between compound 3n and ctDNA. Similarly, compounds 3 and 6-9 also showed diverse variations of the intensity of their absorption bands to different degrees, after subsequent addition of ctDNA, see Fig. S20-38. The binding constant was then calculated for all of them using the modified Benesi-Hildebrand equation (see Fig. S39 for an example) [91][92][93] .
K b values range from 1.53 × 10 4 M −1 to 2.05 × 10 6 M −1 , being the majority of them of the order of 10 5 M −1 . In Fig. 4, a summary of the binding constants obtained for every compound of this work is reported. The calculated K b evidence a high affinity of the new 4H-pyrans for ctDNA base pairs. The highest K b value presented by 9 (2.05 × 10 6 M −1 ) indicates a strong binding towards ctDNA. It is noteworthy that the binding constant for ethidium bromide (EtBr) 94 , a well-known intercalative agent, is 1.37 × 10 5 M −195 , suggests that these 4H-pyrans could have similar interaction with DNA. It is also known that typical K b values for intercalative compounds range from 10 4 to 10 6 M −1 , whereas for groove binders are between 10 5 M −1 to 10 9 M −196,97 . Therefore, further experiments were performed to elucidate the interaction mode with DNA. Additionally, a closer look at the K b values does not show a straightforward relationship between the strength of the interaction with ctDNA and the electronic properties or structure-property relationships of the products, which might indicate that the pyran core is the main responsible of such interaction. These high binding affinities could be due to the presence of the esters and the NH 2 groups in the pyran skeletons, which are able to establish additional interactions and hydrogen bonding forces with the base pairs of DNA molecule 78 .
Determination of the DNA binding type. The binding modes of a drug or a small organic molecule to DNA could be categorized into 98 : (1) a strong covalent union such as that exhibited by cisplatin 99 ; or (2) weaker unions through intermolecular forces 100 (such as van der Waals, hydrogen bonding, π stacking, etc.) in which intercalative molecules can be found (e.g. ethidium bromide) [101][102][103] and groove binding ones. Groove bindings are categorized into two subclasses, minor and major groove binding. Such variation refers to the differences found in the grooves of the macrostructure of DNA. Finally, (3) weakest union to DNA is driven by electrostatic interactions between the drug (or the studied molecule, in general) and the phosphorated scaffold of the double-strand. Many experiments could bring light upon the binding mechanism 104 , such as the study of the viscosity, circular dichroism or fluorescence quenching, among others. We have analyzed these properties in this work to shed light on the DNA binding type of our synthesized compounds.
Viscosity measurements. A very simple technique, such as viscometry, can provide a lot of information. It is considered as one of the best methods for studies in solution because of its high sensibility towards changes in the hydrodynamic properties of the DNA 105-107 . In these experiments, an increase of the viscosity is observed when an intercalative compound is measured. DNA length tends to increase due to the higher base pairs separa- www.nature.com/scientificreports/ tion promoted by the intercalative molecule inserted between them. In contrast, when a compound establishes covalent bonds with the DNA, its structure tends to bend and this leads to an average reduction of its length, causing a decrease of the viscosity. Any other interaction does not cause any significant influence [108][109][110] .
In the present work, the experiment is conducted with compound 3n as a model molecule. 3n has been selected because it shows one of the highest calculated binding constants (see Fig. 4) facilitating information gathering. Moreover, its structural similarity with the other compounds will allow an easy extrapolation of the results obtained in these studies. Specifically, ctDNA was placed in a thermostatic bath at 298 K with a www.nature.com/scientificreports/ Cannon-Fenske viscometer and successive additions of 3n were performed. In Fig. S40, a plot of ƞ/ƞ 0 vs the ratio of 3n to DNA concentration is presented. The values depicted in Fig. S40 support that the successive additions of 3n to the solution of ctDNA resulted in no significant change in the relative viscosity of the whole mixture. This finding might indicate that 3n interacts with ctDNA with either minor or major groove binding, discarding the intercalative hypothesis.
Circular dichroism (CD). This is a very sensitive technique that allows to see tiny changes in the secondary structure of DNA upon binding with a drug or a small organic molecule 111 . In the circular dichroism spectra of ctDNA, two bands can be seen at 243 (negative) and 277 (positive) nm caused by the helicity and base stacking, respectively. These bands are very sensitive towards binding molecules 112 . ctDNA spectra show no changes or small changes when a minor/major groove intercalation or electrostatic binding takes place. However, when an intercalative molecule is examined both bands should suffer considerable changes [113][114][115] . In Fig. 5, the experiments of CD conducted with compound 3n can be analyzed. In this case, the band at 243 nm does not give information due to the distortion caused upon addition of the increasing concentrations of compound 3n. Interestingly, the positive band at 277 nm shows no apparent changes, which is not compatible with an intercalation binding. This experiment supports the hypothesis raised from the viscosity experiment, suggesting a minor/major groove binding mode. Thereby, a more specific experiment is required to discern between both kinds of bindings.
Competitive assays of fluorescence quenching. In order finally to elucidate the binding mode of 4H-pyran 3n with ctDNA, three experiments of fluorescence quenching have been performed. Previous studies abovementioned have shown that, most likely, 3n and by structural analogy compounds 3a-3o and 6-9 bind to the minor/major groove of the DNA. Therefore, three commercially available luminescent model compounds such as ethidium bromide (EtBr, an intercalator) 116,117 , methyl green (MeGr, major groove binder) 118  www.nature.com/scientificreports/ behind this experiment is that when 4H-pyrans are added to a mixture of ctDNA and the model molecule (specially selected because of its strong emission), the fluorescence drops only when both compounds compete for the same binding position of the DNA. Higher concentrations of 4H-pyrans than those of the model molecules are used to grant their substitution from the DNA. Figures 6 and 7 show the results obtained in this study against the three model compounds.
In Fig. 6A, a slight decrease in the maximum intensity of EtBr takes place after the subsequent addition of compound 3n. This could indicate that there is a small interaction. However, previous experiments exclude such possibility and due to the slight change observed on the emission, it can be overseen. In the case of Fig. 6B, no quenching of the emission is detected when using MeGr. These results would discard a plausible major groove binding of the ctDNA and 3n. Similarly, the quenching experiment performed using Hoechst 33342 as a model molecule showed little diminution of the maximum emission intensity of the dye (Fig. 7).
Therefore, it seems that none of the three competition experiments showed the displacement of the model molecule (EtBr, MeGr or Hoechst) by the pyran derivative. However, it is known that emission from the synthesized pyran derivatives in Tris/HCl (0.1 M, pH 7.2) lies between 400 and 550 nm with a maximum intensity c.a. 455 nm [see plot (5) in Fig. 7]. Hence, emission from 3n could be masking the competition experiment of Hoechst, as both of them are excited and emit in the same area of the spectrum. Figure S41 showed the emission maxima of 3n and those of EtBr, MeGr and Hoechst, demonstrating that only the emission of Hoechst is affected by the presence of 3n.  www.nature.com/scientificreports/ plots (4), (5) and (6) in Fig. 7] a significant drop in the intensity for the emission of Hoechst was observed [see Fig. 7, plot (6)]. Such a decrease of the emission intensity suggests that 3n was displacing Hoechst from the minor groove of DNA 124 .
A new competition experiment was then designed considering a different pyran derivative, in order to extrapolate the behavior observed with compound 3n to their analogs. Thus, compound 3m was also examined as a possible minor groove binder using a competitive fluorescence experiment with Hoechst (Fig. 8). Similarly, quenching of the emission was observed after the addition of 3m to the mixture of ctDNA and Hoechst, demonstrating once again that these pyrans are minor groove binders.
As a summary, the high binding constants obtained from UV-Vis indicate that the synthesized pyran derivatives strongly interact with ctDNA, opening the scope to distinguish between intercalation or minor/major groove bindings. CD and viscometry show results not compatible with intercalation, whereas the fluorescence quenching profiles suggest a minor groove interaction as a plausible binding mode.

conclusion
A very powerful and sustainable multicomponent approach for the synthesis of new highly substituted 4H-pyran derivatives is described. Two different protocols using accessible and efficient Et 3 N as a simple catalyst in a 20 mol% in water have been developed. The use of the multicomponent approach for this process with ultrasounds affords excellent results, for the first time 125 . The extremely simple operational methodology, short reaction times, clean procedure and high product yields render this new protocol highly appealing for the synthesis of 4H-pyran derivatives, with high potential as therapeutic agents. DNA binding studies of the final products have been performed by viscosity measurements, circular dichroism, UV-visible absorption and fluorescence spectroscopy. These studies allow to conclude that the synthesized 4H-pyrans bind to DNA through the minor groove rather than to major groove or by intercalation, with a higher K b than those previously reported for a 4H-pyran 78,79 . This work represents one of the scarce studies carried out with pyrans and their DNA binding interactions, thus opening the door for the future development of these scaffolds as promising drugs.

Experimental section
General experimental methods and instrumentation 48 . Purification of reaction products was carried out by column chromatography using silica gel (0.063-0.200 mm). Analytical thin-layer chromatography was performed on 0.25 mm silica gel 60-F plates. ESI ionization method and mass analyzer type MicroTof-Q were used for the HRMS measurements. NMR spectra were recorded at room temperature on a Bruker ARX300 or AV400 instruments. 1 H-NMR spectra were recorded at 300 or 400 MHz, and 13 C-APT-NMR spectra were recorded at 75 or 100 MHz, using DMSO-d 6 as the deuterated solvent. Chemical shifts were reported in the δ scale relative to residual DMSO (2.50 ppm) for 1 H-NMR and to the central line of DMSO-d 6 (39.43 ppm) for 13 C-APT-NMR. A Branson 5510 ultrasonic bath is used in the synthesis of the final compounds. Melting points were determined on a Gallenkamp variable heating apparatus. IR spectra were recorded on a PerkinElmer FT-IR 2,400 microanalyzer.
All commercially available solvents and reagents were used as received.

Diethyl 6-amino-5-cyano-4-(thiophen-2-yl)-4H-pyran-2,3-dicarboxylate (3j).
Following the general procedures, compound 3j was obtained as a white solid in 23% yield ( (9). Following the general procedures, compound 9 was obtained as a white solid in 98% yield (28.9 mg), after 24 h of reaction at room temperature (route A); and in 98% yield (28.9 mg), after 2 h of reaction at room temperature (route B crystal structure determination. Crystals were mounted in inert oil on glass fibers and transferred to the cold gas stream of a Smart APEX CCD diffractometer equipped with a low-temperature attachment. Data were collected using monochromated MoKα radiation (λ = 0.71073 Å). Scan type ϖ. Absorption corrections based on multiple scans were applied using SADABS 126 . The structures were solved by direct methods and refined on F2 using the program SHELXT-2016 127 . All non-hydrogen atoms were refined anisotropically. CCDC deposition number 1982869 contains the supplementary crystallographic data. These data can be obtained free of charge by The Cambridge Crystallography Data Center.
Biological assays. Calf thymus DNA was purchased from SigmaAldrich. DNA solutions were prepared to dissolve the solid ctDNA in a buffer solution of Tris (tris(hydroxymethyl)aminomethane)/HCl (0.1 M, pH 7.2) at room temperature to a final concentration of 1 mg/mL, leaving the mixture stirring overnight. The purity of the DNA was determined by measuring the absorbance ratio at A260 nm/A280 nm, being in all cases between 1.8 and 1.9, no further purification was needed. The molar concentration of the solution was determined by using a mean extinction coefficient of 6600 M −1 cm −1 for a single nucleotide at 260 nm.
UV-Vis measurements. UV spectra were recorded using a Thermo Fisher Scientific Evolution 600 UV-Visible Spectrophotometer with 1 × 1 cm quartz cuvettes at 220-650 nm and 298 K. Stock solutions of the compounds 3a-o, 6-9 were prepared in DMSO to a final concentration of 0.1 M. The titration experiments performed to obtain the binding constants were conducted as follows. First, an intermedium solution of the compound must be prepared in DMSO to a final concentration of 2 mM. Then, the assay solution of the selected compound must be prepared in 2 mL with the Tris/HCl buffer to a final concentration of 20 µM (20 µL of the intermedium solution and 1,980 µL of the buffer solution) and its UV-Vis spectra must be recorded to obtain its extinction coefficient (a baseline correction must be done using the corresponding dimethyl sulfoxide (DMSO) solution in the Tris/HCl buffer). Then, small portions of the ctDNA solution must be added to both assay and reference cuvettes to correct the final spectra with a mixture time of 10 min after every addition (4 × 5 µL, 4 × 10 µL, 1 × 20 µL).
Viscosity measurements. Viscosity measurements were performed using a Cannon-Fenske viscometer (Afora, model 5354/2.50 series), submerged in a thermostatic bath at 298 K. The flow time was measured using a digital stopwatch. Each measure was repeated at least 4 times to obtain a mean time. The experiment was conducted as follows. 3.8 mL of a ctDNA solution in a buffer solution of Tris/HCl (0.1 M, pH 7.2) (1.14 mM measured by UV-Vis) was tested in the viscometer after 10 min to stabilize the temperature. Afterward, successive additions of 15 µL of a solution of 3n 0.1 M were performed and the resulting mixture measured, taking the same precautions from before, with concentrations corresponding to 0.393 mM, 0.783 mM and 1.17 mM. Data is represented as ƞ/ƞ0 vs the ratio of 3n to DNA concentration, being ƞ and ƞ0 the viscosity of the DNA solution with and without 3n. circular dichroism (cD). Circular dichroism (CD) measurements were recorded on a Jasco J-810 spectropolarimeter with a 1 cm path length quartz cuvette, using a scanning speed of 200 nm/min and a spectral bandwidth of 10 nm, each spectrum is the mean of 4 scans. The experiments were conducted at 298 K, covering the 240-300 nm range. The titration procedure starts by measuring a solution of ctDNA 28 µM in a buffer solution of Tris/HCl (0.1 M, pH 7.2), with a baseline correction of the buffer. Then, two samples more were measured containing also 30 µM and 60 µM of 3n, corrected with their respective baselines of 3n and buffer.