Su-Joung Ko

Tetsuo Yamaguchi

Fabrice Salles
email: f.sallesfabrice.salles@umontpellier.fr

Jae-Min Oh

Systematic utilization of layered double hydroxide nanosheets for effective removal of methyl orange from aqueous system by π-π stacking induced nanoconfinement

Keywords: Cationic dye removal, LDH, Monte-Carlo simulation, size effect, intercalation

Systematic utilization of carbonated Mg-Al layered double hydroxide (LDH) nanosheets on methyl orange removal was investigated with a respect to particle dimension. LDHs with smallest dimension were synthesized carefully to have a small lateral size as well as high dispersibility. The other particles with medium and large sizes were prepared by hydrothermal treatment and urea hydrolysis to have larger size and higher crystallinity. According to kinetics and isotherm analyses, the smallest sized LDH showed effective adsorption of methyl orange (1250 mg/g-LDH) which was remarkable higher than the other LDHs with the larger lateral sizes. Compared with the larger lateral sized LDHs, the small sized ones were verified to utilize all the accessible adsorption sites of the nanosheets generating nanoconfinement of the methyl orange molecules. Transmission electron microscopy (TEM) and powder X-ray diffraction (PXRD) patterns definitely indicated that the LDHs with ~40 nm of the lateral dimension could fully utilize the interlayer nanospace of the LDH. Monte-Carlo simulation suggested the intercalated methyl orange could be stabilized not only through electrostatic interaction with the LDH layer but also by π-π stacking between the methyl orange molecules, which is thought to be the driving force to replace carbonate anion.

Introduction

Water pollution is one of most serious global problems. In order to address the problem, removal of the pollutant is a secure way and thus environmental friendly adsorbent is important.

Various adsorbents have been developed to specifically remove cations [START_REF] Delkash | Using zeolitic adsorbents to cleanup special wastewater streams: A review[END_REF][START_REF] Leodopoulos | Adsorption of Cationic Dyes onto Bentonite[END_REF][START_REF] Liu | Adsorption of Dyes from Aqueous Solutions or Suspensions with Clay Nano-Adsorbents[END_REF][START_REF] Malamis | A review on zinc and nickel adsorption on natural and modified zeolite, bentonite and vermiculite: Examination of process parameters, kinetics and isotherms[END_REF]Mohanty et al., 2006bMohanty et al., , 2006a;;[START_REF] Srividya | Biosorption of hexavalent chromium from aqueous solutions by Catla catla scales: Equilibrium and kinetics studies[END_REF][START_REF] Teepakakorn | The Improved Stability of Molecular Guests by the Confinement into Nanospaces[END_REF][START_REF] Yamaguchi | Photochromic Reactions in Nanospace; Host-Guest Interactions and Opportunity[END_REF], anions of divalent (Mg, Mn, Fe, Co, Ni, Cu and Zn) and trivalent (Al, Mn, Fe, Co, Ni, Cr and Ga) metal cations are accepted for the nanosheets [START_REF] Rives | Layered double hydroxides: present and future[END_REF]. The interlayer anions, for example carbonate, halogens, sulfate, nitrate and so on can be directly accommodated [START_REF] Goh | Application of layered double hydroxides for removal of oxyanions : A review[END_REF]) by using corresponding metal salts during synthesis. The LDHs have strong points as anionic scavenger.

First, the LDHs can be synthesized in relatively mild conditions (e.g. at room temperature in water) with biocompatible metal cations [START_REF] Wijitwongwan | Preparation of layered double hydroxides toward precisely designed hierarchical organization[END_REF]. Second, various kinds of organic anions can be incorporated between the nanosheets through ion-exchange reaction [START_REF] Leroux | Fine tuning between organic and inorganic host structure: new trends in layered double hydroxide hybrid assemblies[END_REF][START_REF] Wang | Recent advances in the synthesis and application of layered double hydroxide (LDH) nanosheets[END_REF]; and high theoretical specific surface area of single LDH nanosheet (~2,000 m 2 g -1 ) calculated from unit cell of the LDH layer enables accommodation of a large amount of the anions. Third, the LDHs can be collected from aqueous systems by simple filtration or sedimentation. Fourth, dye selectivity based on molecular configuration and size is expected from the report of clays [START_REF] Atun | Adsorption equilibria between dye and surfactant in single and binary systems onto geological materials[END_REF][START_REF] Bée | Magnetic chitosan/clay beads: A magsorbent for the removal of cationic dye from water[END_REF][START_REF] Smith | Adsorptive selectivity of organic-cation-modified bentonite for nonionic organic contaminants[END_REF], although the anion selectivity for LDH is almost limited in inorganic cations so far [START_REF] He | High adsorption selectivity of ZnAl layered double hydroxides and the calcined materials toward phosphate[END_REF][START_REF] Li | Competitive adsorption behavior toward metal ions on nano-Fe/Mg/Ni ternary layered double hydroxide proved by XPS: Evidence of selective and sensitive detection of Pb(II)[END_REF].

In spite of above-mentioned advantages, the LDHs have some drawbacks as practical anionic adsorbents. Carbonate anion is extremely stable between the LDH layers. Thus, it is easy to prepare carbonated LDHs but their interlayer space cannot be fully exploited as adsorbent site due to the low ion-exchange susceptibility [START_REF] Choy | Clay minerals and layered double hydroxides for novel biological applications[END_REF][START_REF] Kim | Layered nanomaterials for environmental remediation applications[END_REF][START_REF] Oh | Layered nanomaterials for green materials[END_REF], which is a reason of the smaller empirical surface area than the theoretical one. On the other hand, exchanged LDHs, such as nitrated one, can effectively adsorb anions through ion-exchange, but the production of the nitrated LDHs requires much energy and the release nitrate after anion removal can induce secondary pollution by eutrophication. In this regard, it is strongly required to find a way to utilize as many adsorption sites of the carbonated LDHs as possible in order to obtain environmental-friendly and economical adsorbent.

There have been many reports on LDH based anion adsorbent. As it is thought that the LDHs form laminated particles in aqueous systems, maximum utilization of the laminated structure expects effective removal of the anionic pollutants [START_REF] Cheng | Simultaneous removal of phosphates and dyes by double hydroxide nanoflakes[END_REF]Zhu et al., 2014). In previous study, the adsorption of methyl orange (MO) onto carboxylate-CuZn-LDH (476 mg/g-LDH) (Zhu et al., 2014), Fe2O3 doped nitrate-MgAl-LDH (577 mg/g-LDH) [START_REF] Cheng | Simultaneous removal of phosphates and dyes by double hydroxide nanoflakes[END_REF] and Cl-Co4Al-LDH (801 mg/g-LDH) [START_REF] Chen | Acid-salt treated CoAl layered double hydroxide nanosheets with enhanced adsorption capacity of methyl orange dye[END_REF] was reported. In these cases, the adsorption followed the Langmuir isotherm, indicating that the intercalation of MO into the interlayer nanospace did not occur effectively despite of exchangeable carboxylate, nitrate and chloride. In order to solve the problem, ion-exchange behavior of the LDHs with respect to their physicochemical properties should be understood before adsorbent design. Sufficient comprehension would even enable the utility of the carbonated LDHs as an efficient adsorbent.

Although there have been extensive researches on the anion scavenging LDHs, to the best of our knowledge, there have been less interests in how the anions are removed by the LDHs and how the removal efficiency would be maximized. In addition, the elucidation of the adsorption or intercalation mechanisms by Monte Carlo simulations are scarce. Some examples for intercalation of antibiotics (cite here Zaher et al., 2020;10.100710. /s11356-020-07750-3 or Younes et al., 2019;;10.1016/j.jtice.2019.06.018) can be found in literature but only few articles deal with the use of simulations for dyes (cite Meng et al., 2019;10.101610. /j.clay.201910. .01.012 and Aguiar et al., 2013;;10.1080/01496395.2013.804837). Meng et al., 2018;10.1016/j.clay.2018.04.008 have already studied by Monte Carlo the methyl orange adsorption in Zn-Al samples but in order to extract the energy of interaction.

We chose MO due as a model adsorbate due to the following reasons: i) MO is the representative azobased dyes which are widely utilized in industry, ii) chemistry of MO is well comprehended so that the mechanistic study of MO adsorption could be effectively carried out, and iii) there exist many references to compare adsorption. In this regard, our final goal is to elucidate MO adsorption on the LDHs with respect to physicochemical properties and to find a way to exploit all the possible adsorption sites of the carbonated LDHs. In this study, we hypothesized that effective usage of the interlayer nanospace is possible when a LDH with a smaller lateral size (small aspect ratio) is dispersed in a pollutant solution thanks to the accessibility to the interlayer nanospace. In order to achieve the efficient adsorption of MO, the carbonated LDH with the lateral diameter of 40 nm was synthesized by two titration steps to regulate a number of crystal nuclei and a nucleation rate. Other LDHs with different lateral sizes were also carefully synthesized to have well-defined physicochemical properties. Time dependent MO adsorption and isotherms with respect to particle dimension were obtained and analyzed by mathematical modelling to show the effective adsorption of MO onto the smaller sized LDHs. In addition, the Monte-Carlo simulation was carried out, to investigate the plausible adsorption sites upon MO intercalation and therefore to elucidate the conformation of the guest molecules in the LDH structure. The microscopic simulations indicate that π-π stacking stabilized MO in the interlayer nanospace and thus the stabilization is thought to be driving force of the MO adsorption through replacing even the carbonate anions.

Experimental

Materials

Aluminum nitrate nonahydrate (Al(NO3)2•9H2O; 98%), urea (CH4N2O; 99.5%), methyl orange (C14H14N3NaO3S; 85%) were purchased from Sigma-Aldrich Co. LLC, St. Louis., USA.

Sodium hydroxide pellets (NaOH; 97.0%) and sodium bicarbonate (NaHCO3; 99.7%) were obtained from Daejung Chemicals & Metals Co., Ltd., Siheung, Korea. Magnesium nitrate hexahydrate (Mg(NO3)2•6H2O; 99.0%) was purchased from Junsei Chemical Co., Ltd., Tokyo, Japan. The deionized water was prepared from water purification system of Human power II+ (Human Corporation, Korea).

Syntheses of size-controlled LDHs

In order to obtain carbonated MgAl-LDHs with different lateral sizes, customized synthetic routes were applied. In order to obtain small size, a mixed metal solution (0.3 mol L -1 Mg 2+ and 0.15 mol L -1 Al 3+ ) was titrated with an alkaline solution (0.12 mol L -1 NaOH and 0.9 mol L -1 NaHCO3) until pH 9.5 for coprecipitation of both metal hydroxides. Especially, this process was carried out in two steps to produce small particles while preventing agglomeration among particles. The first step was fast titration until pH 7.0 to form as many nuclei as possible; through this step, the nucleation could be limited and the size of LDH would be small. The second step was slow titration until pH 9.5 and following aging at room temperature for 24 h. This process was expected to make particle surface smooth to reduce unintentional particle agglomeration. For the medium sized LDH, the metal solution was titrated by the alkaline solution until pH 9.5 and was hydrothermally treated at 150 °C for 24 h. For the largest LDH, urea hydrolysis method was applied; an aqueous solution (0.375 mol L -1 Mg 2+ , 0.075 mol L -1 Al 3+ and 1.5 mol L -1 urea) was mixed at 90 °C for 48 h with vigorous stirring.

All the white suspensions obtained from each reaction were centrifuged, thoroughly washed with deionized and lyophilized. Taking into account the lateral sizes of the LDHs, small, medium and large, the LDHs were designated as LDH-S, LDH-M and LDH-L, respectively.

Powder X-ray diffraction (XRD)

The crystallinity and crystal structure of the synthesized LDHs were evaluated by powder X-ray diffractometer (XRD) with a Bruker AXS D2 phaser (LYNXEYE™ detector; Bruker AXS GmbH, Karlsruhe, Germany) using Ni-filtered Cu-Kα radiation (λ= 1.5406 Å) with 1 mm of air-scattering slit and 0.1 mm of equatorial slit. XRD patterns were measured in a 2θ range from 5° to 80° with the time step increments of 0.02° and a scanning rate of 0.5 s/step. Crystallite sizes of samples were calculated by Scherrer's equation as follows; t= (0.9λ)/(Bcos) where λ: X-ray wavelength, B: fullwidth-at-half-maximum, : Bragg angle).

Inductively coupled plasma-optical emission (ICP-OES)

Compositions of metallic elements in the LDHs were analyzed by inductively coupled plasmaoptical emission spectrometer using plasma and spectrometer (ICP-OES; OPTIMA 7300DV, PerkinElmer). The powdered samples were thoroughly dissolved in hydrochloric acid and subsequently the solvent was evaporated on a hot plate. The remnants were added to 100 mL of deionized water and filtered with syringe filter (PVDF, 0.45 μm, 13 mm, Jaema Trade Inc.) for ICP measurement.

Scanning electron microscopy (SEM)

Morphologies and particle sizes of the LDHs were confirmed by scanning electron microscopy (SEM) using a Quanta 250 FEG (FEI Company, Hillsboro, OR, USA). For the SEM measurement, the samples were suspended in deionized water and then, the suspensions were dropped on a silicon wafer and dried in ambient conditions. The surface of the specimen was sputtered with Pt/Pd plasma for 60 s, and the images were obtained using a 30 kV accelerated electron beam.

N2 gas adsorption-desorption isotherm

N2 adsorption-desorption isotherms were obtained by Belsorp-mini (BEL, Japan, Inc, Osaka, Japan) after pre-treatment in vacuum for 12 h at 150 °C using BELPREP-vacⅡ (BEL, Japan, Inc, Osaka, Japan). Specific surface areas of the LDHs were calculated according to the Brunauer-Emmett-Teller (BET) theory and the porosities of the samples were estimated by Barret-Joyner-Halenda (BJH) analysis method.

Dynamic light scattering (DLS)

Zeta potential values and hydrodynamic radii were obtained by electrophoretic light scattering and dynamic light scattering (DLS) using an ELS-Z1000 (Otsuka Electronics, Osaka, Japan). For electrophoretic light scattering measurement, the LDHs were dispersed in deionized water, where the pH value was adjusted to ~8.4 with 0.1 mol L -1 HCl or NaOH. The DLS measurement was done with the suspension without pH adjustment.

Transmission Electron Microscopy (TEM)

Cross-sectional morphologies and interlayer distances of the LDHs before and after adsorption of MO were determined by Field Emission Transmission Electron Microscope (FE-TEM; Titan G2 ChemiSTEM Cs probe, FEI company) operated at an accelerating voltage of 200 kV. TEM specimens for cross-sectional observation were prepared using Cryo Ultramicrotome (CUMT; PT PC Ultramicrotome & Photographic).

Adsorption of methyl orange

Time-dependent MO adsorption was observed at 7.0×10 -3 mol L -1 MO solution. The LDHs were suspended in deionized water (143 mg 10 mL -1 ) and added to 100 mL of the MO solution.

Aliquots (1 mL) were collected at each time point until 48 h under stirring at room temperature, and then centrifuged and filtered by a syringe filter (PVDF, 0.45 μm, 13 mm, Jaema Trade Inc.). The adsorbed amount of MO was determined by quantifying un-adsorbed moiety in the supernatants and analyzed by using visible light spectroscopy (UV-1800, Shimadzu, Kyoto, Japan). The absorbance was measured at the wavelength of 464 nm which was an absorption peak of MO. The adsorbed amount, Qt (mg/g-LDH) was calculated as

𝑄 𝑡 = 𝑉(𝐶 0 -𝐶 𝑡 ) 𝑊 (eq. 1)
C0 and Ct were concentrations at time 0 and t of the MO solution (mol L -1 ), V was solution volume (mL) and W was mass of the absorbent (g). The time dependent adsorption was fitted by pseudo-first order, pseudo-second order and Elovich equations.

The concentration dependent of MO adsorption on each LDH was carried out using the batch method. The LDHs were dispersed in deionized water (10 mg mL -1 ) and 1.5 mL of each suspension was mixed with 10 mL MO solution with the concentration range of MO from 1.0×10 -5 to 1.5×10 -2 mol L -1 . The suspensions were shaken for 24 h with an orbital shaker and centrifuged at 3500 rpm for 5 min. The supernatant filtered with the syringe filter were analyzed by using UV-vis spectroscopy. The adsorption amount was calculated similarly. Obtained adsorption isotherms were analyzed by three models of the Langmuir, Freundlich, and Sips isotherms. The procedure was repeated three times.

Monte-Carlo simulations

In order to estimate the molecular configuration of MO in LDH interlayer space, Monte-Carlo simulations were performed by using a home-made code. The molecular structure of MO was built, and the partial charges were calculated by density functional theory (DFT) calculation with DMol 3 . In this case, the geometry optimization was performed using GGA/PW91 functional, DNP basis set and all electrons are considered for the core treatment. The convergence tolerances for Energy, maximum force and maximum force variation were the following 10 -5 Ha, 0.02 Ha/Å, 0.005 Å respectively (see Computational details in the Supporting Information).

Regarding LDH, the structure was taken from the methodology described the literature [START_REF] Newman | Interlayer arrangement of hydrated MgAl layered double hydroxides containing guest terephthalate anions: Comparison of simulation and measurement[END_REF] and by adapting the chemical composition and the unit cell parameters to the ones obtained experimentally. Then the partial charges of the LDH structure were calculated by a electronegativity equalization method. The Lennard-Jones parameters were issued from universal force field (UFF). A multi-cell formed by 3x3x1 unit cells for LDH was used for the Monte Carlo calculations in agreement with a cut-off for Lennard-Jones contributions fixed to 12.5 Å. The final parameters corresponded thus at a= b= 27.414 Å, c= 57 Å, α= β= 90° and χ= 120°. The parameters of the adsorbate/adsorbent Lonnard-Jones interatomic potential were then calculated using the Lorentz-Berthelot combining rule. Concerning the long-range electrostatic interactions, the summation Ewald technique was applied.

The simulations were performed in order to minimize the system energy by introducing a fixed number of anions in the structure. During these calculations, the motion of the anions included translation and rotation randomly considered during the equilibration steps. For that purpose, 10 7 Monte Carlo production steps following 2×10 7 equilibration steps were performed at 300 K to extract the distribution of interlayer anions.

Result and discussion

Characterization

For systematic optimization, we would like to fix all the other parameters same except the size.

Taking into account of a general chemical equation of LDHs et al., 2008). The basal spacings of the LDHs were estimated to be 0.76 nm, suggesting existence of interlayer carbonate anions. The lower intensity of the diffraction was observed in the smaller particle size. It indicates that the crystallinity of the LDHs decreased with the reduction in the particle size.

([M 2+ 1-xM 3+ x(OH)2] x (A n-x/n) X-•mH2O), chemical
We could also calculate the crystallite size along (003) plane to estimate the order of stacking along crystallographic c-axis. As summarized in Table 1, the crystallite sizes were 5.3, 42 and 36 nm for LDH-S, LDH-M, and LDH-L, respectively, showing that smaller particles tended to have less-ordered layer stacking. The XRD results confirmed that the prepared LDHs were same in crystal phase but different in crystallinity and crystallite size. Table 1. Physicochemical properties of LDHs a Crystallite sizes were calculated with Scherrer's equation utilizing (003) peak from XRD.

b Theoretical specific surface areas were determined by calculating surface area of one particle.

Particle dimensions obtained from SEM were utilized to compute particle volume and area. Particle density 2.06 g/cm 3 of hydrotalcite (Mg3Al(OH)6(CO3)0.50.1H2O) was utilized to calculate particle mass.

SEM

The SEM images showed that the three LDHs were successfully synthesized to have different particle dimensions as intended (Fig. 1). As summarized in Table 1, the lateral size and thickness were clearly different in each other, especially the degree of the difference was more significant for lateral size showing 40, 190, and 3000 nm for LDH-S, LDH-M and LDH-L, respectively.

Although the particles of LDH-S seemed to form agglomerates, we could distinguish the primary particle size of 40 nm. As described in the experimental section, we especially made efforts in preparing LDH-S by controlling the nucleation rate and the edge smoothness. As reported previously, controlling nucleus number and nucleation rate is critical in particle size control [START_REF] Nývlt | The Kinetics of Industrial Crystallization[END_REF].

In addition, the edge of the particle become ordered through aging while the particle dimension was generally preserved [START_REF] Nývlt | The Kinetics of Industrial Crystallization[END_REF]. Due to the customized synthesis control, LDH-S could avoid agglomeration among the particles, which were commonly found in small sized LDHs through edgeto-face interaction. [START_REF] Clark | Continuous synthesis of Zn2Al-CO3 layered double hydroxides: a comparison of bench, pilot and industrial scale syntheses[END_REF][START_REF] Jose | Hydrodynamic assembly of two-dimensional layered double hydroxide nanostructures[END_REF] In order to estimate the homogeneity in the particle size, we obtained size distribution from randomly obtained 200 particles from the SEM images (Fig. S1 in supporting information). The results revealed that the particles were fairly homogeneous in the size, which was further verified in suspension states through dynamic light scattering (DLS).

N2 adsorption-desorption isotherm

As the surface area and pore structure are the determining parameters for adsorbent, we then carried out N2 adsorption-desorption isotherms onto the LDHs (Fig. S2 in supporting information).

Both LDH-L and LDH-M showed Type III adsorption (weak adsorptive interaction), while LDH-S had some feature of Type IV adsorption (capillary condensation by mesopores) [START_REF] Sing | Reporting physisorption data for gas/solid systems with special reference to the determination of surface area and porosity[END_REF][START_REF] Thommes | Physisorption of gases, with special reference to the evaluation of surface area and pore size distribution (IUPAC Technical Report)[END_REF]. As it is obvious that the interlayer space of carbonated LDH does not serve as adsorption site for gases [START_REF] Olfs | Comparison of different synthesis routes for Mg-Al layered double hydroxides (LDH): Characterization of the structural phases and anion exchange properties[END_REF], we could expect that the Type IV in LDH-S might be attributed to interparticle pores. As it was confirmed from the SEM that the primary particle diameter was ~40 nm, we could geometrically expect the interparticle cavity of ~10 nm which lays within the mesoporous range to induce the capillary condensation. In terms of the desorption branch, LDH-S showed the H2 type hysteresis (IUPAC classification) while LDH-M and LDH-L did not show it; the difference, similar to the adsorption pattern, was attributed to the different particle size. Nevertheless, we did not seriously consider the difference in the N2 adsorption-desorption patterns, as the adsorption of MO in aqueous suspensions would be more governed by specific surface area. The calculated SBET from the early stage of adsorption was 53.2, 20.9 and 22.8 m 2 g -1 for LDH-S, LDH-M and LDH-L, respectively (Table 1). As the particle had only external surface as gas adsorption site, the difference in the particle size did not strikingly affect the specific surface area. As expected, the specific surface area was not significantly different in each other despite of the different N2 adsorption-desorption patterns. Furthermore, we calculated theoretical surface area (STHE) of the particles based on the size and density of the LDHs (Table 1). The theoretical value was fairly comparable with the experimental values, showing that most of the surface sites were exposed to adsorb molecules. From the N2 adsorption-desorption study, we could expect that LDH-S had slight advantage compared with the other two samples in terms of surface adsorption.

DLS

Although we confirmed the different primary particle sizes among the LDHs, we further characterized them in the aqueous state utilizing DLS (Fig. S3 in supporting information, Table 1) as our practical adsorption occurs in the water. The hydrodynamic size in the aqueous suspension showed average value of 128, 190 and 2863 nm. It was worthy to note here that the hydrodynamic radius from DLS had comparable tendency with primary particle sizes from SEM. Moreover, taking into account the peak shape and width, the hydrodynamic radius was considered fairly homogeneous.

We also checked the surface charge of the LDHs through zeta potential measurement, as the MO adsorption could be strongly affected by the charge-charge interactions between a sulfonate group of MO and positive LDH layer. We controlled the synthetic condition for the LDHs to have same composition and not to have any impurities; therefore the surface charge was successfully controlled same within 27.9 -30.6 mV in the same pH region.

According to the physicochemical characterizations, it was clearly confirmed that the three carbonated LDHs were prepared to have different properties in the particle size (both in powder and suspension) and crystallinity but comparable properties like crystalline phase, surface chemistry, pore structure and etc. Therefore, we proceeded the MO adsorption for the three LDHs in order to monitor the particle size effect on aqueous MO removal.

Methyl orange (MO) adsorption

For the first step, we investigated time-dependent MO adsorption at 7.0 × 10 -3 mol L -1 of an initial concentration. It was clearly shown that the adsorption of MO onto LDH-S continued to 50 h while the adsorption onto LDH-M and LDH-L reached equilibrium within 4 h shown in Fig. 2. The MO adsorption kinetic was fitted to well-established model like pseudo-first order (eq. 2), pseudosecond order (eq. 3) and the Elovich equation (eq. 4) (Table 2).

ln(𝑄 𝑒 -𝑄 𝑡 ) = 𝑙𝑛𝑄 𝑒 -𝑘 1 𝑡 (eq. 2)

𝑡 𝑄 𝑡 = ( 𝑡 𝑄 𝑒 ) + ( 1 𝑘 2 𝑄 𝑒 2 ) (eq. 3) 𝑄 𝑡 = ( 1 𝛽 ) ln(𝛼𝛽) + ( 1 𝛽 ) ln 𝑡 (eq. 4)
(Qe: equilibrium adsorption amount of MO (mg/g-LDH), Qt: adsorbed amount (mg/g-LDH) at time t (min), k1: pseudo-first order rate constant (min -1 ), k2: pseudo-second order rate constant (g (mg min) -1 ), α: initial adsorption rate (g (mg min) -1 ) and β: Elovich constant (g mg -1 ).

As shown in Fig. S4 in supporting information, the kinetics were not sufficiently explained by pseudo-first order but explained well by pseudo-second order kinetics. We could compare the adsorption kinetics of the three LDHs utilizing pseudo-second order (Table 2). It was clearly shown that Qe of LDH-S was much larger (more than twice) than that of LDH-L, suggesting its excellent adsorption ability. The adsorption rate k2 was following order: LDH-L > LDH-M > LDH-S. The calculated k2 value of LDH-S was about 1/4 for LDH-M and 1/20 for LDH-L. The difference in kinetic constants represented that smaller particles required more time to be equilibrated, suggesting that additional adsorption sites compared with LDH-M and LDH-L were activated in LDH-S. The Elovich model showed anomalous fitting results with respect to the dimension of the LDHs. The R 2 values obtained from the Elovich equation decreased with increase of the lateral dimension of the LDHs. Although the Elovich equation is empirical and it was hard to conclude the meaning of the fitting constants, the major hypothesis of this modelchemisorption and adsorption onto heterogeneous surface [START_REF] Ramachandran | Adsorption isotherms, kinetics, thermodynamics and desorption studies of reactive orange16 on activated carbon derived from Ananas comosus (L.) carbon[END_REF][START_REF] Turner | Kinetics of chemisorption: An examination of the Elovich equation[END_REF] implied distinguishable adsorption behavior of the small sized LDH compared with larger ones. In other words, the adsorption onto LDH-S could include the chemisorption or adsorption onto heterogeneous surface while that onto LDH-L preferred to the physisorption onto smooth surface. Possible explanation for the irreversible adsorption onto LDH-S is the electrostatic affinity of MO towards LDH layer and additional molecular attraction at the adsorbed state. Furthermore it can also be hypothesized that the global charge delocalization due to the substitution in the layer is weaker in the case of LDH-S than in LDH-L.

This should lead to enhance the electrostatic effect towards the larger MO anions. Qe

(mg/g) R 2 k2 (g (mg min) -1 ) Qe (mg/g) R 2 α (g (mg min) -1 ) β (g (mg) -1 ) R 2
LDH-S 1.4×10 -3 368.5 0.94 2.06×10 -5 1250 0.99 3.84×10 4 0.0105 0.99 LDH-M 1.0×10 -3 74.2 0.95 9.08×10 -5 555.6 0.99 7.38×10 12 0.062 0.96 LDH-L N.A. N.A. 0.007 4.37×10 -4 476.2 0.99 N.A. N.A. 0.5

As summarized in Table 2, the Qe of LDH-S (1250 mg/g-LDH) was larger than double of those of LDH-M and LDH-L (555 and 476 mg/g-LDH, respectively). Despite of comparable hydrodynamic radius of LDH-S with LDH-M, it showed significantly increase in adsorption. This implied that the agglomeration of LDH-S was fairly negligible in terms of the MO adsorption and that LDH-S would have different MO adsorption mechanism compared with larger LDHs. The adsorbed amount at 150 min was converted to %removal of MO showing 65, 31, and 30% for LDH-S, LDH-M and LDH-L, respectively. As shown in Fig. S3 in supporting information, the zeta potential of the LDHs was same. It indicated that the charge-charge interactions between MO and the LDHs were not the only reason of the larger adsorbed amount of MO onto LDH-S than the others. The adsorption amount of MO onto LDH-S was higher than that onto other materials (Table S1 in supporting information), expecting that LDH with small particle size is an excellent adsorbent of water pollution. Even the previously reported hybrid LDH adsorbent such as graphene-LDH [START_REF] Zheng | 3D hierarchical graphene oxide-NiFe[END_REF], carboxylate-LDH (Zhu et al., 2014), Fe2O3-doped LDH [START_REF] Cheng | Simultaneous removal of phosphates and dyes by double hydroxide nanoflakes[END_REF] and Cl-Co4Al-LDH [START_REF] Chen | Acid-salt treated CoAl layered double hydroxide nanosheets with enhanced adsorption capacity of methyl orange dye[END_REF] exhibited less adsorption amount (438, 476, 577 and 801 mg/g-LDH, respectively) than the LDH-S in current study.

Adsorption isotherm

Adsorption isotherms of MO onto the LDHs are shown in Fig. 3. The adsorbed amount (mg/g-LDH) of MO onto LDH-S was higher than those onto LDH-M and LDH-L in the equilibrium concentration from 0 to 0.010 mol L -1 . Similarly, the adsorbed amount based on the surface area of sample (mg m -2 ) was higher in LDH-S than the others. The adsorption isotherms were analyzed by the Langmuir (eq. 5), Freundlich (eq. 6) equations and the Sips isotherm (eq. 7). (Qmax: maximum amount of adsorbed MO (mg/g-LDH), Qe: adsorbed MO at equilibrium (mg/g-LDH),

Ce: concentration of MO at equilibrium (mol L -1 ), K: adsorption equilibrium constant (L mol -1 ), KF:

Freundlich adsorption capacity constant (mg g -1 L n mol -n ), KS: Sips equilibrium constant (L n mol -n )

and n: related value to the adsorption intensity with heterogeneity of the LDHs)

The R 2 values obtained from the fittings with the Freundlich equation were higher than those with the Langmuir and Sips equations in the three LDHs as shown in Fig. S5 in supporting information and as summarized in Table 3. The Freundlich model allows the multilayer adsorption, whereas the Langmuir and Sips equations are based on monolayer adsorption [START_REF] Tzabar | Adsorption isotherms and Sips models of nitrogen, methane, ethane, and propane on commercial activated carbons and polyvinylidene chloride[END_REF]. The molecular dimension of MO estimated from single crystal X-ray crystallography is a cylinder with 1.5 nm length and 0.5 nm diameter [START_REF] Burke | Incorporation of sulfonate dyes into hydrogen-bonded networks[END_REF]. The molecular density of the adsorbed MO on the LDH-S was estimated to be larger than 40 molecules nm -2 from SBET. Taking into account the two aspects, it is strongly suggested that the MO molecules existed in aggregates [START_REF] Grégoire | Multiscale mechanistic study of the adsorption of methyl orange on the external surface of layered double hydroxide[END_REF] or they were intercalated into the interlayer space [START_REF] Neumann | Influence of the layer charge and clay particle size on the interactions between the cationic dye methylene blue and clays in an aqueous suspension[END_REF].

Transmission electron microscopy (TEM)

The adsorption kinetics and isotherms suggested the possibility of the MO intercalation into LDH-S replacing pre-existing carbonate anion. In order to detect the intercalation directly, highresolution cross-section TEM was carried out (Fig. 4). The TEM image of LDH-S showed assembly of lamellar structure of which interlayer distance was estimated to be 0.76 nm (Fig. 4a), which was comparable to the PXRD pattern (Fig. 1). Although the morphology of LDH-S was not largely changed as shown in Fig. S9 in supporting information [START_REF] El Hassani | Effect of morphological properties of layered double hydroxides on adsorption of azo dye Methyl Orange: A comparative study[END_REF]Zhu et al., 2014), the adsorption of MO increased the interlayer distance to 1.9 nm (Fig. 4a), strongly indicating the intercalation of MO into the interlayer space despite of the stability of the interlayer carbonate anions [START_REF] Miyata | Anion-exchange properties of hydrotalcite-like compounds[END_REF]. New diffraction peak at 4.6° after the MO adsorption on LDH-S corresponded to the basal spacing of 1.92 nm in PXRD, corroborating the adsorption of MO even in the interlayer space of LDH-S (Fig. 5a). In case of LDH-M, three interlayer distances of 0.76, 0.93 and 1.9 nm were observed in the TEM image (Fig. 5b), suggesting that the MO was partially intercalated into the interlayer space. The PXRD pattern of LDH-M adsorbed MO showed both diffraction peaks at 4.6° and 11.7°, which supported the observation of TEM. The change of the interlayer distance of LDH-L was not observed by TEM after the adsorption of MO, while the relatively small but definite diffraction peak attributed to the LDH intercalated MO was observed at 4.6° in PXRD (Fig. 5c).

Monte-Carlo simulation

In order to speculate the geometry of MO in the LDHs and to address distinct MO intercalation behavior in a small sized carbonated LDH, the Monte-Carlo simulation was performed (see experimental section in the detail). The first step consisted to estimate the maximum MO amount that the interlayer space can accommodate by imposing the layer-layer distance obtained experimentally (d003). It followed from the calculations that 41 MO anions could enter in the interlayer space due to the steric repulsion and 20 CO3 2-were required to obtain a neutral charge in the multi-cell considered in our calculations. This theoretical maximum loading corresponded to a MO mass of around 1000 mg/g dry LDH. In comparison with the experimental results (see Fig. 3), in LDH-M and LDH-L, CO3 2-were not totally exchanged by MO while LDH-S was completely exchanged in the interlayer space and probably a non-negligible part of MO was also adsorbed on the external surface. As shown in Fig. 6a and 6b, CO3 2-were in strong interactions with the LDH layer through the three O but without interaction with other ions (MO or other CO3 2-), while MO anions could be closely packed in the interlayer space of the LDH taking advantage of nanoconfinement. If MO anions are located in the interlayer space of the LDH, its sulfonate group heads for the LDH layers and strongly interacts with the OH groups closed to substitutions. Various arrangements of the anions are obtained from the calculations illustrating that the anions are not strictly perpendicular to the layers but are tilted as observed in the case of amines in layered solids [START_REF] Alby | In fluence of the templating amine on the nanostructure and charge of layered vanadates for radioactive wastewater treatment[END_REF]. Different configurations are reported in Fig. 6b. Such configurations are mainly due to the size of the MO ion (14.5 Å) compared to the interlayer space (19.2 Å) and taking into account of the size of the LDH layer (~4 Å) and to the intermolecular interactions. Fig. 6c and 6d clearly describe that the strong ππ stacking between MO molecules adsorbed in the interlayer space was possible when the intermolecular distance is ~3.4-3.7 Å, following a sandwich configuration, where the benzene rings were parallel and could interact directly together.

It is generally accepted in clay intercalation chemistry that ionic moiety first interact with the surface of clay layers [START_REF] Neumann | Influence of the layer charge and clay particle size on the interactions between the cationic dye methylene blue and clays in an aqueous suspension[END_REF][START_REF] Valandro | Photophysics of auramine O adsorbed on solid clays[END_REF], and then the ions at the edge of the clay particle gradually migrate into the interlayer space through concentration gradient.

Similarly, the MO molecules first attached on the surface, and the molecules at the periphery of the particle would have a chance to move into gallery (Fig. 7 top sides). At this stage, usual carbonated LDH overcome the invasion of MO as the adsorption enthalpy calculated at the anhydrous state for carbonate (284 kcal/mol) is much larger than that determined for MO (122 kcal/mol). However, the impact of hydration shows clearly that the CO3 2-can interact with water while MO acts as an inhibitor of the hydration due to the strong hydrophobic character of this anion. Furthermore, LDH-S with the small lateral dimension is expected to provide short edge circumference for the MO moiety to easily get π-π stabilization (110 kcal/mol), giving rise to nanoconfinement (Fig. 7a). In large particles, due to the long circumference, there may not be appropriate formation of a band of MO at the edge to get efficient π-π interaction (Fig. 7b). In this way, small particles would provide a more chance for MO to get additional stabilization; as a consequence, electrostatic interaction and π-π attraction of MO overcome the stability of carbonate to be intercalated in LDH-S. On the other hand, due to the stability of the adsorbed MO, the regeneration of LDH-S might be lower than the other LDH-based adsorbent like previous report [START_REF] Gwak | Nanocomposites of magnetite and layered double hydroxide for recyclable chromate removal[END_REF].

Conflicts of interest

There are no conflicts to declare.

  formulas were determined almost same for the three LDHs by ICP measurement: LDH-S([Mg0.66Al0.34(OH)2] 0.34+ (CO3 2-0.17) 0.34-•mH2O), LDH-M ([Mg0.67Al0.33(OH)2] 0.33+ (CO3 2-0.16) 0.33-•mH2O)and LDH-L ([Mg0.66Al0.34(OH)2] 0.34+ (CO3 2-0.17) 0.34-•mH2O). Furthermore, we characterized the structural features such as phase, crystallinity and crystallite size of the LDHs. As shown in Fig.1(a), all diffractograms had well-developed (003) and (006) peaks at the same position 11.7° and 23.6° along with several characteristic lattices peaks corresponding to hydrotalcite (JCPDS 14-0191) (San Román

  Fig. 1 (A) Powder X-ray diffraction (PXRD) patterns and (B) scanning electron microscopic (SEM) images of (a) LDH-S, (b) LDH-M and (c) LDH-L. The XRD intensity for (a) was multiplied by 10 for peak comparison. The vertical lines in the bottom of XRD patterns are the reference peak from JCPDS 14-0191 (hydrotalcite) (San Román et al., 2008).

Fig. 2

 2 Fig. 2 Time-dependent MO adsorption onto the LDHs (--: LDH-S, --: LDH-M, --: LDH-L) under ambient conditions with the initial concentration of MO of 7.0 × 10 -3 mol L -1 .

Fig. 3

 3 Fig. 3 Methyl orange adsorption isotherms (a) adsorbed amount was determined based on mg-MO/g-LDH (b) adsorbed amount was determined based on mg-MO per surface area (m 2 ) of LDH. (--: LDH-S, --: LDH-M, --: LDH-L) For conversion of mg g -1 to mg m -2 , specific surface area values obtained from N2 adsorption-desorption isotherm were utilized.

Fig. 4

 4 Fig. 4 Transmission electron microscopic (TEM) images for (a) LDH-S, (b) LDH-M and (c) LDH-L before (left panels) and after (right panels) MO adsorption. The interlayer distance was visualized by the inset histograms obtained in the white rectangle region.

Fig. 5 X

 5 Fig. 5 X-ray diffraction patterns of (a) LDH-S, (b) LDH-M and (c) LDH-L before and after MO adsorption at various MO concentration. The concentration indicates the initial concentration of MO before adsorption.

  Fig. 6 (a) Global snapshot of the structure of MO containing LDH, (b) snapshot of the interactions for both MO CO3 2-and LDH layer (also showing the orientation of the intercalated MO), and (c and d) π-π stacking of MO in LDH simulated by Monte-Carlo method. The details of the simulation were mentioned in experimental section.

Table 3 .

 3 Isotherm parameters of MO adsorption on LDH adsorbent based on various adsorption

	models
		Langmuir			Freundlich			Sips
	Qmax (mg/	K (L mol -1	R 2	KF (mg	n	R 2	Qmax (mg/g)	n	KS (L n	R 2

Acknowledgement

This work was supported by Radiation Technology R&D program through the National Research Foundation of Korea funded by the Ministry of Science and ICT(2017M2A2A6A05016600, 2017M2A2A6A05093711)

composite with enhanced adsorption affinity to Congo red, methyl orange and Cr(VI) ions. J. Hazard. Mater. 369,[214][215][216][217][218][219][220][221][222][223][224][225]


Steve Saulnier

Rayane Ghoteimi

Christophe Mathé

Suzanne Peyrottes

Jean-Pierre Uttaro

2-(Substituted amino)-8-azachromones from 4,6-Diaryl-2-pyridones: a Synthetic Strategy toward Compounds of Broad Structural Diversity

3-Acetoacetyl-4,6-diaryl-2-pyridones are synthesized in three steps from chalcones and then condense with carbon disulfide to afford 8-azachromones containing a methylthio group at C2. This leaving group offers an entry point for the insertion of more complex moieties via nucleophilic substitution. For this purpose, N-nucleophiles are explored according to their positions in the Mayr's nucleophilicity scale (N parameter), and three main classes are distinguished depending on whether the substitution takes place from their neutral forms, from their deprotonated anionic forms, or under nucleophilic catalysis. A broad range of primary and secondary amines may be inserted by this method, including enantiomerically pure amino acids, enabling us to explore structural diversity.

INTRODUCTION

The 8-azachromone core was synthesized for the first time in 1967 from 1,3-diketone derivatives of 2-pyridones by an acid-catalyzed ring closure. 1 Since then, few methods have been developed in comparison with the isostere chromones, 2 although both scaffolds might have similar biological and pharmaceutical properties. 3 The electron-deficient nature of the pyridine ring is often pointed out as an issue to the challenging access to 8-azachromones. To date, most of the reported procedures still involve 1,3-diketones and fall into two approaches according to whether the ring closure takes place via intramolecular O-arylation or via condensation with carbon disulfide (Scheme 1). The intramolecular O-arylation has been the most explored and can be performed under acid catalysis for 2-pyridones 1,4 and 2-methoxypyridines, 5 under base catalysis for 2-halopyridines, 6 and via CH activation of N-oxides for 2-unsubstituted pyridine derivatives 7 (Scheme 1A). By contrast, the condensation with CS2 was reported solely in the case of 4,6-dimethyl-2pyridone derivatives (Scheme 1B) 8 and has not received further attention. Yet, the methylthio leaving group at the electrophilic site C2 on the cyclization product II would enable to access more complex derivatives III by simple nucleophilic substitution. Such derivatization would be more difficult from the O-arylation products I. The present work revisits the condensation with CS2 and further explores possibilities of nucleophilic substitution at C2 (Scheme 1B). We focus on 5,7-diaryl-8-azachromone derivatives because aryl substituents on the pyridine ring have not yet been considered by the synthetic methods mentioned above. We show that primary and secondary amines are well suited for the nucleophilic substitution and that they give rise to 2-(substituted amino)-8-azachromones under conditions that mainly depend on the nucleophile strength and position in the Mayr's nucleophilicity scale. 9 The synthesis of the 1,3-diketone cyclization precursors, namely 3-acetoacetyl-4,6-diaryl-2-pyridones, is also described, as they are not easily accessible.

RESULTS AND DISCUSSION

Synthesis of 3-Acetoacetyl-4,6-diaryl-2-pyridones. The 1,3-diketones cyclization precursors are commonly prepared by addition of a methyl Grignard reagent to the nitrile of 3-cyano-2-pyridones, followed by Claisen condensation of the resulting 3-acetyl-2-pyridones. This approach has been well described in the case of 4,5-dialkyl-2-pyridones but turned out to be more difficult with 4,5-diaryl derivatives.

Among the numerous methods for the synthesis of the starting material, 4,5-diaryl-3-cyano-2pyridones 1, we focused on the base-promoted condensation between chalcones and cyanoacetamide (Table 1). An effective reported procedure involves potassium t-butoxide in DMSO under oxygen atmosphere. 10 When conducted in open air, this procedure leads to a substantial amount of 3-unsubstituted 2-pyridones as by-products of decyanative aromatization, instead of the desired 3-cyano-2-pyridones. 11 This point is responsible for the modest yield of product 1a (40%) obtained in open air (Table 1, entry 1), whereas an 88% yield was reported when using an oxygen atmosphere (data from ref . 10). As other procedures using ethanol as solvent with different bases, and without the need for oxygen atmosphere were reported, 12 we evaluated the effect of replacing DMSO with ethanol, 2-propanol, and t-butyl alcohol, together with the corresponding potassium alkoxide bases in open air (entries [START_REF] Malets | Synthesis of azachromones and azachromanones[END_REF][3][START_REF]ESI/Q-TOF) m/z[END_REF][5]. Under these conditions, we did not observe the decyanative aromatization by-product. The reaction rate clearly depends on the base strength:

although the reaction in ethanol/KOEt was still incomplete after 72 hours at room temperature (entries 2 and

3), a 96% yield of 1a was obtained after 60 hours in 2-propanol/KOi-Pr (entry 4) and 92% after only 24 hours using the more basic t-butyl alcohol/KOt-Bu medium (entry 5). The desired product 1a is initially formed as potassium salt, and is soluble neither in 2-propanol nor in t-butyl alcohol under this form. We took advantage of this insolubility to isolate and characterize 1a as potassium salt 1a-K in a simplified reaction work-up. According to the same procedure, we prepared 3-cyano-2-pyridones 1b-e-K diversely substituted in the para position of the 4-phenyl ring (Table 1, entries [START_REF] Zhao | Transition-Metal-Free Intramolecular Ullmann-Type O-Arylation: Synthesis of Chromone Derivatives[END_REF][7][8][START_REF]ESI/Q-TOF) m/z[END_REF]. We assigned the position of the substituted phenyl ring on the basis of the probable reaction mechanism: the Michael addition of cyanoacetamide to the 4-substituted chalcone leads to a condensation product where the substituted phenyl ring would be adjacent to the 3-cyano group, i.e. at C4. This was later confirmed on the 3-acetyl derivative 2c by the NOESY correlation between the acetyl group and the ortho protons of the bromo-substituted phenyl ring (Figure 1). of cyanoacetamide in the procedure described above. However, the reaction requires heating under reflux and mainly gives the 3-unsubstituted products resulting from deacetylative aromatization. Therefore, the addition of a methyl Grignard reagent to 3-cyano-2-pyridones appears to be the most reliable strategy to access acetyl derivatives.

Alkyl-substituted cyanopyridones are known to give addition products with methylmagnesium bromide at room temperature. 13 For aryl-substituted derivatives 1a-e-K, the use of a large excess of MeMgBr and heating (65 °C) were required to reach a significant conversion. However, under these conditions, halogenated derivatives 1b (R 1 = Cl) and 1c (R 1 = Br) gave rise to unidentified by-products due to a possible halogen-magnesium exchange. We obtained better results with the more reactive dimethylmagnesium generated in situ by the dioxane precipitation method: addition of 1,4-dioxane to MeMgBr precipitates MgBr2 species as polymeric dioxane adducts and shifts the Schlenk equilibrium toward Me2Mg. 14 Thus, 3-cyano-2-pyridones 1a-e-K were converted to the corresponding Finally, the Claisen condensation between 2a-e and ethyl acetate in dioxane led to the 1,3-diketone derivatives 3a-e with yields of 72-84% (Scheme 2). These compounds were then considered as starting materials for their cyclization into 8-azachromones.

Cyclization into 2-(Methylthio)-8-azachromones.

The reported procedure uses potassium hydroxide (two equivalents) as base to promote the condensation with CS2 (three equivalents) in DMSO, followed by treatment with dimethyl sulfate (four equivalents). With the aim of maximizing the formation of 4a, the reaction conditions, including solvent nature, base strength, equivalents of the reagents, and reaction time were screened and revealed the following points. Firstly, the reaction requires polar solvents such as DMF, DMSO or acetonitrile. As solvent nucleophilicity may be problematic, anhydrous acetonitrile appears to be the solvent of choice, furthermore leading to the selective precipitation of product 4a in the reaction mixture. Secondly, the use of two equivalents of a base at least as strong as K3PO4 is required. In this respect, DBU increases the solubility of the deprotonated anionic species in acetonitrile, resulting in higher reaction rates. The third point concerns the amount of CS2. Although one equivalent would theoretically be enough for the reaction to proceed, we observed a decrease in the formation of the O-arylation by-product 5a when using higher amounts of CS2

(up to nine equivalents), thus resulting in a significant increase in the yield of 4a. On the basis of these observations, we developed an improved procedure affording the 2-(methylthio)-8-azachromone derivatives 4a-e with average yields of 87-91% (Scheme 4). Values of N and E parameters of many reagents have been determined, 16 so that these scales allow predicting the feasibility of a wide variety of combinations. A given electrophile such as 4a that reacts with a nucleophile referenced in the nucleophilicity scale (i.e. with a known N parameter), will be expected to react with other nucleophiles of similar or higher N parameters as well. Among the different π-, n-and σnucleophiles characterized in the Mayr's scales, we focused on neutral NH-type nucleophiles because they cover a wide range of nucleophilicity: basically, from N 7.69 for benzotriazole to N 18.52 for pyrrolidine (data in acetonitrile, which was chosen as reference solvent for this work). This means roughly eleven units.

In comparison, alcohols cover only six units and are significantly less nucleophilic.

Therefore, we explored the reactivity of 4a with some N-nucleophiles, starting from piperidine (N 17.35) and decreasing in nucleophilicity (Table 2). We observed the nucleophilic substitution in methylene chloride at room temperature until ethanolamine (N 14.11, entry 5) and glycine methyl ester (N 13.51 for potassium glycinate, data in water, entry 6). Under the same conditions, p-anisidine (N 13.42) only led to traces of the substitution product 6ag, and weaker nucleophiles such as aniline (N 12.64) and benzocaine did not react at all, even upon heating at 65 °C in THF. Thus, a first limit in reactivity is reached for N value of approximately 13.5. Nucleophiles located above this limit give substitution products with 4a whereas nucleophiles below do not, at least without further activation conditions.

To extend the scope of reactivity, we envisaged activation via nucleophilic catalysis. Because they

show N parameter values above the limit of 13.5, DBU (N 15.29), DMAP (N 15.51) and DABCO (N 18.80) were expected to react with 4a and to give rise to the corresponding positively charged intermediates of enhanced electrophile strengths. Indeed, all three catalysts enabled the nucleophilic substitution with panisidine and aniline, DMAP being the most effective with 90-91% yields of 6ag and 6ah after only two hours at 65 °C in THF (Table 2, entries 7 and8). However, the efficacy of nucleophilic catalysis is quite limited as benzocaine required 36 hours at 100 °C in dioxane to afford 61% of the substitution product 6ai (entry 9), and 2-methylimidazole (N 11.74) and imidazole (N 11.47) were completely unreactive under the same conditions (entries 10 and 11). Therefore, a second limit is reached for N value of about 12, above which the presence of DMAP enables the nucleophilic substitution. It is important to note that the products presented in Table 2 were obtained with one equivalent of nucleophile. With an excess, a second substitution takes place and leads to the 2-pyridone derivatives 7 by ring opening, due to the leaving group ability of the pyridone moiety. This second substitution works best with cyclic secondary amines and becomes more difficult when the nucleophile strength decreases. In the presence of three equivalents of piperidine, 4a was completely converted into the open-ring product 7a after 36 hours, whereas with morpholine only 72% of 7b were obtained after 60 hours (Scheme 6). The ring opening of the 8-azachromone scaffold in the presence of N-nucleophiles was reported in 2003 for 2polyfluoroalkyl derivatives. 17 Curiously, in that case the reaction worked well with primary amines whereas piperidine and morpholine did not give open-ring products but the addition products 2-morpholino-and 2piperidino-8-azachromanones instead.

Scheme 6. Formation of Open-Ring Products by Two Consecutive Nucleophilic Substitutions.

N Ph Ph O O O SMe 4a N H Ph Ph O O N N X X O HN X 3 equiv CH 2 Cl 2 rt, 36-60 h + 7 7a : X = CH 2 , 98% 7b : X = O, 72%
The N values of 13.5 and 12 mentioned above allow predicting the nucleophilic substitution with a given nucleophile and were exemplified for the 5,7-diphenyl derivative 4a, playing the role of constant electrophile strength. Nevertheless, these data may also be applied to the R 1 -substituted derivatives 4b-e as were observed that the presence of a para substituent on the 5-phenyl ring does not significantly affect the chemical properties of the azachromone core. This is revealed by comparing the 13 C chemical shifts of the azachromone scaffolds of compounds 4b-e with those of 4a (Table 3). Beside slight variations for carbon C5

(up to 1.3 ppm), the chemical shifts are almost identical, especially the chemical shifts of C2 and C4 (less than 0.4 ppm variations) which are directly connected to the electrophilicity. Therefore, compounds 4a-e can be considered of similar electrophile strengths and the limits in reactivity that were determined for 4a can be used with compounds 4b-e as well.

Table 3. Influence of the R 1 Substituents on the 13 C NMR Data of the 8-Azachromone Scaffold. Scheme 7 shows examples of substitution products that were obtained from 4a-e according to the reaction conditions developed. Briefly, aliphatic primary amines were inserted in methylene chloride at room temperature (conditions A) whereas aromatic primary amines were inserted in dioxane at 85-100 °C in the presence of DMAP (conditions B). Enantiomerically pure amino acid methyl esters (phenylalanine, tyrosine, tryptophan and histidine) afforded substitution products according to conditions A with yields ranging from 43% to 81%. Under the conditions of Scheme 7, no significant racemization was observed and the products 6aj-am and 6bb can almost be considered as enantiomerically pure (see specific rotations in the experimental section). This feature is interesting because most of amino acids and peptides have primary amino groups of similar nucleophilicities 18 and, therefore, may be inserted to the 8-azachromone scaffold in this way without loss of optical activity.

Scheme 7. Nucleophilic Substitution of 4a-e with Primary Aliphatic and Aromatic Amines. So far, only N-nucleophiles at least as nucleophilic as benzocaine enabled the formation of substitution products. When the N value falls below the limit of N 12, the nucleophilic substitution does not take place. Accordingly, imidazole with N 11.47 is unreactive. However, imidazole is relatively acidic with a pKa of 18.6 in DMSO 19 and, under anionic form, shows a significant increase in nucleophilicity to N 21.09

N Ph O O O SMe 4a-e N Ph O O O NHR 2 6 R 1 R 1 H 2 NR 2 Conditions A or B N Ph O O O NHMe Br N Ph Ph O O O NH MeO 2 C Ph N Ph Ph O O O NH MeO 2 C OH N Ph Ph O O O NH MeO 2 C NH N N Ph Ph O O O NH MeO 2 C NH N Ph O O O N H Cl Ph N Ph O O O NH MeO 2 C OH N Ph O O O N H Br Ph N Ph O O O NHMe Ot-Bu 6aj, 71% a,c 6ak, 75% a,c 6al, 81% a,c 6am, 43% a,d 6ba, 98% a 6bb, 74% a,c 6ca, 91% a 6cb, 89% a 6da, 95% a N Ph O O O NH Ot-Bu 6db, 38% b CO 2 Et N Ph O O O N H N Ph 6ea, 81% a O N Ph O O O NH Cl 6bc, 61% b
(data in DMSO), 20 which is above the limit of reactivity. Thus, after deprotonation in the presence of KOt-Bu in THF, the potassium salt of imidazole indeed reacts with 4a as predicted, but the product is quite unstable and unfortunately could not be isolated. However, primary amines of similar nucleophile strengths (i.e. unreactive under the conditions of Scheme 7), such as methyl and ethyl 5-aminopicolinate and 3aminoisoxazole, also react with 4a upon deprotonation by KOt-Bu in THF, but this time give stable products that were isolated and clearly identified as substitution products 6an-ap (Scheme 8). The reaction is actually complicated by two distinctive features that cap the yields at 50%: on the first hand, deprotonation of the substitution product by the amine anion that would require two equivalents for a complete conversion, but on the other hand the fast ring opening of the substitution product that prevents the use of a second equivalent of amine anion. Nevertheless, this strategy enables the formation of substitution products that would not be possible under neutral conditions, and was successfully applied to the R 1 -substituted derivatives 4b-e (Scheme 8). General procedure for the synthesis of 4-susbtituted chalcones. To a stirred solution of 4substituted benzaldehyde (6 mmol) and acetophenone (0.7 mL, 6 mmol) in absolute ethanol (18 mL), NaOH (15 M aq solution, 0.6 mL, 9 mmol) was added and the resulting reaction mixture was stirred 20 h at rt.

After completion, the reaction mixture was diluted with absolute ethanol (18 mL) and cooled to 0 °C. The precipitated product was recovered by filtration, washed with cold absolute ethanol and dried under vacuum.

4-Chlorochalcone was commercially available.

4-Bromochalcone was prepared from 4-bromobenzaldehyde according to the general procedure.

1.33 g (4.63 mmol, 77% yield), pale yellow solid. The spectroscopic data were consistent with those reported. 22 4-t-Butoxychalcone was prepared from 4-t-butoxybenzaldehyde according to the general procedure.

1.63 g (5.81 mmol, 97% yield), pale yellow solid. 1 8, 158.3, 144.8, 138.6, 132.8, 129.8, 129.6, 128.7, 128.6, 123.9, 120.8, 79.6, 29 8, 152.7, 145.1, 138.9, 132.6, 130.3, 128.7, 128.5, 126.2, 119.0, 115.0, 66.7, 48.3 General procedure for the synthesis of 4,6-diaryl-3-cyano-2-pyridones 1a-e-K (Table 1). To a stirred solution of chalcone (2 mmol) and cyanoacetamide (185 mg, 2.2 mmol) in the indicated alcohol (tbutyl alcohol or 2-propanol, 24 mL), KOt-Bu (898 mg, 8 mmol) was added in one portion and the resulting reaction mixture was stirred in open air under the indicated conditions. After completion, 2-propanol (24 mL) was added and the stirring was kept 15 min at rt. The precipitated product was recovered by filtration, washed with successively 2-propanol (3 x 24 mL), cyclohexane (3 x 12 mL) and Et2O (3 x 12 mL), and dried under vacuum. The products were characterized as potassium salts. The neutral forms 1a, 23,24 1b 23,25 and 1c 25,26 have been reported.

3-Cyano-4,6-diphenyl-2(1H)-pyridone, potassium salt (1a-K) was prepared from trans-chalcone

according to the general procedure (2-propanol, rt, 60 h). 595 mg (1.92 mmol, 96% yield), pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.97 (dm, J = 6.9 Hz, 2H), 7.57 (dm, J = 6.9 Hz, 2H), 1, 153.3, 140.2, 138.1, 133.1, 129.9, 128.6, 128.4, 128.2, 126.8, 121.3, 101.6, 91.0 159.5, 153.8, 140.1, 138.4, 131.6, 130.4, 129.0, 128.5, 127.1, 122.1, 121.2, 102.7, 91.4 2, 158.8, 155.4, 154.1, 140.4, 133.7, 128.8, 128.4, 128.1, 126.8, 122.9, 121.7, 101.7, 91.1, 78.3, 28.6. 4, 158.7, 154.4, 151.1, 140.5, 129.5, 128.8, 128.4, 128.1, 126.8, 121.9, 114.4, 101.6, 90.9, 66.1, 48. 0, 162.9, 152.6, 148.1, 136.8, 135.4, 132.7, 131.1, 129.42, 129.36, 129.1, 127.8 (br), 127. 4, 161.2, 150.2, 148.0 (br), 137.3, 133.0 (br), 131.6, 130.4 (2C), 130.2, 128.9, 127.4, 122.3, 106.7 (br) 163.2, 156.8, 152.9, 147.6, 133.0, 132.8, 130.8, 129.3, 129.0, 127.8, 127.2, 123.9, 107.8, 79.2, 31.8, 29.0. 2H),3H),7.31 (dm,J = 8.8 Hz,2H),6.92 (dm, J = 8.8 Hz, 2H), 6.60 (s, 1H), 3.90 -3.84 (m, 4H), 3.29 -3.19 (m, 4H), 2.38 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl3) δ 202. 9, 163.1, 152.8, 151.8, 147.5, 133.1, 130.7, 129.4, 129.3, 128.8, 127.3, 127.1, 115.1, 107.7, 66.9, 48.5, 31.9. HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C23H23N2O3 375.1703, found 375.1692.

HRMS (ESI/Q-TOF) m/z: [M -K] -calcd

HRMS (ESI

General procedure for the synthesis of 1,3-diketones 3a-e (Scheme 2). 3-Acetyl-2-pyridone 2 (2 mmol) and NaH (60% dispersion in mineral oil, 12 mmol, 480 mg) were suspended in anhydrous 1,4dioxane (8 mL) under argon atmosphere, and stirred 1 h at rt. Then, EtOAc (2 mL, 20.5 mmol, see note below) was added and the resulting reaction mixture was stirred 12 h at rt. After completion, the reaction was diluted with EtOAc and quenched at 0 °C by slow addition of an excess volume of saturated aq NH4Cl.

The organic layer was separated and the aqueous layer was extracted again with EtOAc. The combined organic layers were washed with saturated aq NH4Cl, dried over Na2SO4 and concentrated. The product was isolated by chromatography on silica gel and then precipitated by addition of the indicated solvent.

Enol/keto ratios were determined by 1 4, 184.9, 163.1, 154.9, 147.8, 139.0, 132.9, 130.9, 129.3, 129.0 (2C), 128.7, 127.9, 127.2, 108.1, 104.2, 25.3 6, 184.5, 162.9, 153.7, 148.0, 137.4, 135.2, 132.7, 131.0, 129.4, 129.3, 129.0, 127.4, 127.1, 107.7, 104.1, 25.4. HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C21H17ClNO3 366.0897, found 366.0894. 3, 185.4, 163.0, 156.6, 154.2, 147.7, 133.4, 133.0, 130.8, 129.3 (2C), 128.9, 127.1, 123.8, 107.9, 104.1, 79.2, 29.0, 25.3. HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C25H26NO4 404.1862, found 404.1859.

3-Acetoacetyl

3-Acetoacetyl-4-(4-morpholinophenyl)-6-phenyl-2(1H)-pyridone (3e) was prepared from 2e

according to the general procedure and was isolated by flash chromatography (DCM/EtOAc 70:30 then DCM/EtOAc/EtOH 70:25:5). 624 mg (1.50 mmol, 75% yield), orange solid (cyclohexane). Ratio enol/keto 3.9:1. 1 5, 185.7, 163.1, 154.2, 151.7, 147.4, 133.0, 130.7, 129.32, 129.28, 129.24, 127.1, 122.3, 114.9, 107.8, 104.2, 66.9, 48.5, 25.4. HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C25H25N2O4 417.1809, found 417.1808. NaHCO3 and brine, then dried over Na2SO4 and concentrated. The product was isolated by flash chromatography on silica gel (DCM/EtOAc 97:3). 28 mg (0.089 mmol, 89% yield), white solid (cyclohexane). 1 H NMR (400 MHz, CDCl3) δ 8.17 -8.08 (m, 2H), 7.66 (s, 1H), 7.55 -7.48 (m, 3H), 7.48 -7.42 (m, 3H), 7.42 -7.36 (m, 2H), 6.10 (d, J = 0.5 Hz, 1H), 2.45 (d, J = 0.5 Hz, 3H). 13 C{ 1 H} NMR ( 101MHz, CDCl3) δ 178. 3, 165.1, 162.2, 159.1, 154.6, 139.1, 137.0, 130.8, 129.1, 128.41, 128.38, 127.9, 127.8, 121.6, 114.1, 112.3, 20.4. HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C21H16NO2 314.1181, found 314.1180.

2-Methyl

General procedure for the synthesis of 2-(methylthio)-azachromones 4a-e (Scheme 4). To a suspension of 1,3-diketone 3 (1 mmol) in anhydrous acetonitrile (10 mL) under argon atmosphere, DBU (0.33 mL, 2.2 mmol) was added and the resulting mixture was stirred 30 min at rt while the starting diketone dissolved. Then, carbon disulfide (0.54 mL, 9 mmol) was first added, followed after 4 h by dimethyl sulfate (0.21 mL, 2.2 mmol). The reaction mixture was stirred 12 h at rt while product 4 slowly precipitated. After completion, 10 mL acetonitrile and 20 mL H2O were added, resulting in the complete precipitation of the product. After 15 min of additional stirring, the product was recovered by filtration, washed with successively H2O/acetonitrile mixture (1:1 v/v) and petroleum ether, and dried under vacuum. 9, 177.4, 174.6, 160.8, 159.0, 155.3, 139.0, 136.7, 131.1, 129.2, 128.7, 128.3, 128.1, 127.8, 122.6, 119.9, 114.2, 32.2, 14.6 8, 177.6, 174.6, 160.8, 159.3, 154.0, 137.4, 136.6, 134.9, 131.2, 129.7, 129.3, 128.5, 127.8, 122.4, 119.8, 114.1, 32.2, 14.6. HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C23H17ClNO3S 422.0612, found 422.0619. prepared from 3c according to the general procedure. 410 mg (0.88 mmol, 88% yield), pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 8.16 -8.09 (m, 2H), 7.65 (s, 1H), 7.60 (dm, J = 8.5 Hz, 2H), 7.55 -7.50 (m, 3H), 7.28 (dm, J = 8.5 Hz, 2H), 2.71 (s, 3H), 2.57 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl3) δ 197.7, 177.6, 174.5, 160.8, 159.3, 154.0, 137.9, 136.5, 131.4, 131.2, 130.0, 129.2, 127.8, 123.1, 122.3, 119.8, 114.0, 32.2, 14.6 197.9, 177.1, 174.7, 160.9, 158.9, 156.4, 155.1, 136.8, 133.2, 131.0, 129.4, 129.2, 127.8, 123.0, 122.7, 119.9, 114.2, 79.0, 32.1, 29.1, 14.6 was prepared from 3e according to the general procedure. 421 mg (0.89 mmol, 89% yield), yellowish solid. 1 H NMR (400 MHz, CDCl3) δ 8.16 -8.08 (m, 2H), 7.69 (s, 1H), 7.57 -7.47 (m, 3H), 7.38 (dm, J = 8.7 Hz, 2H), 6.98 (dm, J = 8.7 Hz, 2H), 3.95 -3.82 (m, 4H), 3.36 -3.21 (m, 4H), 2.71 (s, 3H), 2.61 (s, 3H).

3-Acetyl

13 C{ 1 H} NMR (101 MHz, CDCl3) δ 198.0, 177.1, 175.0, 161.1, 158.8, 155.1, 151.5, 136.9, 130.9, 130.0, 129.3, 129.2, 127.7, 122.5, 119.9, 114.4, 114.0, 67.0, 48.5, 32.3, 14.6. HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C27H25N2O4S 473.1530, found 473.1528.

General procedures for the synthesis of 2-(substituted amino)-8-azachromones 6aa-am, 6ba-bc, 6ca-cc, 6da-db and 6ea-eb (Table 2 andScheme 7 (11 μL, 0.11 mmol) according to procedure A (1 mL DCM, rt, 12 h), and was eluted with DCM/EtOAc 85:15. 41 mg (0.096 mmol, 96% yield), white solid (cyclohexane). 1 H NMR (400 MHz, CDCl3) δ 8.11 -8.06 (m, 2H), 7.57 (s, 1H), 7.54 -7.37 (m, 8H), 3.56 -3.49 (m, 4H), 2.54 (s, 3H), 1.83 -1.70 (m, 6H). 13 C{ 1 H} NMR (101 MHz, CDCl3) δ 197. 9, 176.7, 163.3, 158.5, 158.2, 154.6, 139.8, 137.3, 130.5, 129.1, 128.34, 128.29, 127.9, 127.6, 121.8, 114.0, 103.8, 50.3, 33.0, 26.0, 23.9 9, 176.8, 163.0, 158.4 (2C), 154.7, 139.5, 137.1, 130.6, 129.1, 128.4, 128.3, 128.0, 127.6, 122.0, 113.9, 104.1, 66.5, 49.3, 33 8, 175.3, 165.8, 158.03, 157.97, 155.2, 139.9, 137.0, 130.7, 129.1, 128.3, 128.2, 128.0, 127.6, 122.4, 114.0, 100.2, 32.6, 28.0 8, 175.4, 164.9, 158.0, 157.9, 155.1, 139.9, 137.0, 136.3, 130.7, 129.16, 129.13, 128.3, 128.23, 128.19, 127.98, 127.95, 127.6, 122.4, 114.0, 100.1, 45.5, 32.7 4, 174.7, 164.4, 157.5, 156.7, 154.2, 139.6, 136.2, 130.8, 129.1, 128.5, 127.8, 127.54, 127.45, 121.6, 113.5, 99.3, 59.2, 43.5, 32 0, 175.3, 168.7, 165.2, 158.2, 157.8, 155.2, 139.7, 136.9, 130.8, 129.2, 128.4, 128.2, 128.0, 127.6, 122.5, 114.1, 100.4, 53.0, 43.0, 32.6 162.6, 158.2, 158.1, 157.9, 155.0, 139.8, 136.8, 130.8, 129.1, 128.40, 128.36, 128.2, 128.0, 127.6, 124.8, 122.3, 114.8, 114.1, 100.1, 55.7, 32.8. HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C29H23N2O4 463.1652, found 463.1643. 39 mg (0.09 mmol, 90% yield), pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 13.61 (s, 1H), 8.15 -8.08 (m, 2H), 7.67 -7.61 (m, 2H), 7.64 (s, 1H), 7.54 -7.46 (m, 8H), 7.44 -7.38 (m, 2H), 7.32 (tm, J = 7.4 Hz, 1H), 2.68 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl3) δ 200. 3, 175.6, 162.8, 158.3, 157.9, 155.0, 139.7, 136.8, 135.8, 130.8, 129.7, 129.1, 128.4, 128.2, 128.0, 127.7, 126.4, 123.2, 122.4, 114.2, 100.3, 32.8 7, 175.5, 166.0, 162.7, 158.5, 157.8, 155.1, 139.8, 139.5, 136.6, 131.3, 131.0, 129.2, 128.5, 128.2, 128.1, 127.8, 127.7, 122.5, 122.3, 114.1, 100.7, 61.3, 32.8, 14.5 3.41 (dd, J = 13.9, 5.0 Hz, 1H), 3.24 (dd, J = 13.9, 8.1 Hz, 1H), 2.58 (s, 3H). 13 C{ 1 H} NMR (126 MHz, CDCl3) δ 199.8, 175.3, 170.6, 164.6, 157.9, 157.6, 155.0, 139.7, 136.9, 135.4, 130.7, 129.5, 129.1, 129.0, 128.3, 128.2, 128.0, 127.60, 127.55, 122.3, 113.9, 100.2, 56.0, 53.1, 39.0, 32.7. HRMS (ESI/Q-TOF) m/z: (s, 1H), 3H),3H),2H),7.11 (dm,J = 8.5 Hz,2H),6.72 (dm,J = 8.5 Hz,2H),5.93 (br s,1H),5.17 (td,J = 7.7,4.9 Hz,1H),3.82 (s,3H),3.31 (dd,J = 14.1,4.9 Hz,1H),3.18 (dd,J = 14.1,7.7 Hz,1H),2.58 (s,3H). 13 C{ 1 H} NMR (101 MHz, CDCl3) δ 200. 2, 175.4, 170.7, 164.6, 158.1, 157.7, 155.6, 155.1, 139.7, 136.9, 130.8, 130.7, 129.2, 128.4, 128.2, 128.0, 127.6, 126.8, 122.5, 115.9, 113.9, 100.3, 56.2, 53.1, 38.0, 32.5 1H),7.57 (s,1H),3H),3H),2H),1H),7.23 (br d,J = 2.4 Hz,1H),2H),5.26 (td,J = 7.3,5.2 Hz,1H) 199.8, 175.4, 171.1, 164.7, 157.9, 157.7, 155.0, 139.8, 136.9, 136.4, 130.7, 129.1, 128.3, 128.2, 128.0, 127.6, 127.1, 123.7, 122.5, 122.2, 119.9, 118.7, 113.9, 111.5, 109.4, 100.3, 55.3, 53.1, 32.6, 28.

3-Acetyl

  Scheme 1. Synthesis of 8-Azachromones from 1,3-Diketone Precursors.

Figure 1 .

 1 Figure 1. Selected NOESY Correlations (Arrows) Showing the Position of the Substituted Phenyl Ring.

  Scheme 4. Improved Conditions for the Cyclization of Diketones 3 into 2-(Methylthio)-8azachromones 4.

a

  Conditions A: 0.05-0.1 M 4, 1.1 equiv amine, DCM, rt. b Conditions B: 0.05 M 4, 1.1 equiv amine, 0.3 equiv DMAP, 1,4-dioxane, 85-100 °C. c Amino acid methyl ester generated from the corresponding hydrochloride (1.5 equiv) and Et3N (1.1 equiv). d Histidine methyl ester generated from the dihydrochloride (1.2 equiv) and Et3N (2 equiv).

Scheme 8 .a

 8 Scheme 8. Nucleophilic Substitution of 4a-e with Amine Anions. a

  0. HRMS (ESI/Q-TOF) m/z: [M -K] -calcd for C22H18N3O2 356.1405, found 356.1408.General procedure for the synthesis of 3-acetyl-4,6-diaryl-2-pyridones 2a-e (Scheme 2). To a stirred suspension of 3-cyano-2-pyridone 1-K (2 mmol) in anhydrous THF (10 mL) under argon atmosphere, anhydrous 1,4-dioxane (1 mL, 12 mmol) was added and the mixture was cooled to 0 °C. Then, MeMgBr (3 M solution in Et2O, 4 mL, 12 mmol) was slowly added and the resulting reaction mixture was stirred 48 h while allowed to reach rt. After completion, 4 mL H2O were added dropwise at 0 °C followed by 14 mL of 1 N aq HCl (14 mmol HCl). The resulting mixture was stirred 12 h at 40 °C. Finally, most of the THF was evaporated under reduced pressure, an excess volume of 0.1 N aq HCl was added and the product was extracted with DCM. The combined organic layers were washed with successively 0.1 N aq HCl, H2O and brine, dried over Na2SO4 and concentrated. The crude product containing 4-8% of the starting 3-cyano-2-pyridone (neutral form) was precipitated in petroleum ether, recovered by filtration, dried under vacuum and used without further purification (analytical samples were obtained by flash chromatography on silica gel).

  /Q-TOF) m/z: [M + H] + calcd for C23H24NO3 362.1751, found 362.1759. was prepared from 1e-K according to the general procedure. 454 mg (1.21 mmol, 61% yield), orange solid. An analytical sample was obtained by flash chromatography (DCM/EtOAc 70:30 then DCM/EtOAc/EtOH 70:25:5). 1 H NMR (400 MHz, CDCl3) δ 12.58 (br s, 1H),

  was prepared from 2c according to the general procedure and was isolated by flash chromatography (DCM/EtOAc 80:20 then DCM/EtOAc/EtOH 80:15:5). 591 mg (1.44 mmol, 72% yield), yellowish solid (cyclohexane). Ratio enol/keto 3.4:1. 1

- 5 , 7 -

 57 diphenyl-4H-pyrano[2,3-b]pyridin-4-one (5a) (Scheme 3). 1,3-Diketone 3a (33 mg, 0.1 mmol) was dissolved in H2SO4 (1 mL) and the resulting mixture was stirred 1 h at rt. The reaction mixture was then diluted with chloroform (3 mL), cooled to 0 °C, and quenched under vigorous stirring by dropwise addition of an excess volume of cold H2O. The organic layer was separated and the aqueous layer was extracted again with chloroform. The combined organic layers were washed with successively H2O, aq

  . HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C27H26NO4S 460.1577, found 460.1591.

  ). Note: safety precautions must be taken due to the release of toxic methanethiol. Procedure A: 2-(Methylthio)-8-azachromone 4 (0.1 mmol) and amine (amount indicated) were dissolved in anhydrous DCM (volume indicated) under argon atmosphere, and the resulting mixture was stirred under the indicated conditions. After completion, the crude reaction mixture was applied on top of a silica gel column and the product was eluted with the indicated solvent. Procedure B: 2-(Methylthio)-8-azachromone 4 (0.1 mmol), amine (0.11 mmol) and DMAP (4 mg, 0.03 mmol) were dissolved in the indicated anhydrous solvent (THF or 1,4-dioxane, 2 mL), and the resulting mixture was stirred in open air under the indicated conditions. After completion, the reaction mixture was cooled to rt and the product was precipitated by addition of the indicated solvents. After 15 min of additional stirring, the product was recovered by filtration, washed with the indicated solvents, and dried under vacuum.

  was prepared from 4a and aniline according to procedure B (2 mL THF, 65 °C, 2 h). The product was precipitated by addition of cyclohexane (4 mL), recovered by filtration, and washed with cyclohexane/THF 2:1 and petroleum ether.

  . HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C31H25N2O5 505.1758, found 505.1732. was prepared from 4a according to procedure A (1 mL DCM, rt, 84 h) using Lphenylalanine methyl ester hydrochloride (32 mg, 0.15 mmol) and triethylamine(15 μL, 0.11 mmol), and was eluted with DCM/EtOAc 49:1. 37 mg (0.071 mmol, 71% yield), white solid (MeOH). [α] D 20 +13.2° (c 1.0, CHCl3). 1 H NMR (500 MHz, CDCl3) δ 11.91 (d, J = 7.8 Hz, 1H), 8.11 -8.05 (m, 2H), 7.58 (s, 1H), 7.55 -7.42 (m, 6H), 7.39 -7.28 (m, 6H), 7.25 -7.20 (m, 1H), 5.21 (td, J = 8.0, 5.0 Hz, 1H), 3.83 (s, 3H),

  acetyl-4-oxo-5,7-diphenyl-4H-pyrano[2,3-b]pyridin-2-ylamino)-3-(4hydroxyphenyl)propanoate (6ak) was prepared from 4a according to procedure A (1 mL DCM, rt, 84 h) using L-tyrosine methyl ester hydrochloride (35 mg, 0.15 mmol) and triethylamine (15 μL, 0.11 mmol), and was eluted with DCM/EtOAc 85:15. 40 mg (0.075 mmol, 75% yield), white solid (cyclohexane). [α] D 20 +29.8° (c 1.0, CHCl3). 1 H NMR (400 MHz, CDCl3) δ 11.80 (d, J = 7.8 Hz, 1H), 8.12 -8.03 (m, 2H), 7.59

  . HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C32H27N2O6 535.1864, found 535.1864. was prepared from 4a according to procedure A (1 mL DCM, rt, 60 h) using Ltryptophan methyl ester hydrochloride (38 mg, 0.15 mmol) and triethylamine (15 μL, 0.11 mmol), and was eluted with DCM/EtOAc 9:1. 45 mg (0.081 mmol, 81% yield), pale yellow solid (MeOH). [α] D 20 -19.8° (c 1.0, CHCl3). 1 H NMR (400 MHz, CDCl3) δ 11.88 (d, J = 7.5 Hz, 1H), 8.20 (br s, 1H), 8.11 -8.04 (m, 2H),

  [START_REF]ESI/Q-TOF) m/z[END_REF]. HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C34H28N3O5 558.2023, found 558.2017. )propanoate (6am) was prepared from 4a according to procedure A (1 mL DCM, rt, 72 h) using Lhistidine methyl ester dihydrochloride (29 mg, 0.12 mmol) and triethylamine (28 μL, 0.2 mmol), and was eluted with DCM/EtOH 93:7. 22 mg (0.043 mmol, 43% yield), yellowish solid (Et2O). [α] D 20 -3.4° (c 0.5,

Table 1 . Synthesis of 3-Cyano-2-pyridones from 4-Substituted Chalcones and Cyanoacetamide.

 1

									R 1
			O
						O		base
			R 1	Ph	+	NC	NH 2	solvent, air	CN
								Ph	N H	O
									1a-e
	Entry a	R 1	Solvent			Base	Temperature Reaction time, h Product b Yield, % c
	1	H	DMSO		KOt-Bu		rt	2	1a	40
	2	H	ethanol		KOEt		rt	20	1a-K	37
	3	H	ethanol		KOEt		rt	72	1a-K	88
	4	H	2-propanol		KOi-Pr		rt	60	1a-K	96
	5	H	t-butyl alcohol		KOt-Bu	30 °C	24	1a-K	92
	6	Cl	2-propanol		KOi-Pr		rt	60	1b-K	93
	7	Br	2-propanol		KOi-Pr		rt	60	1c-K	96
	8	Ot-Bu	2-propanol		KOi-Pr		rt	60	1d-K	77
	9	Morpholino	t-butyl alcohol		KOt-Bu	30 °C	48	1e-K	93

a Reaction conditions: 0.083 M chalcone, 1.1 equiv cyanoacetamide, 4 equiv base, open air. b 1a-e-K refers to the potassium salts of products 1a-e.

c

Yields of isolated products.

Cyclization of Diketone 3a into 8-Azachromones According to Reported Procedures.

	Ph Scheme 3. N 3a H Ph	O O	O	1. KOH, CS 2 , DMSO, 4 h 2. Me 2 SO 4 , 2 h [Ref. 15] H 2 SO 4 , 1 h	Ph	N Ph 4a, 34% O O Ph O	O SMe	+	5a, 35%
				[Ref. 4]
					Ph	N	O
						5a, 89%

8,15

Under these conditions, diketone 3a gave rise to a mixture of the 2-(methylthio)-8-azachromone 4a (34%) and of the 2-methyl derivative 5a (35%) as intramolecular O-arylation by-product (Scheme 3). As discussed in the introduction, the O-arylation ring closure of 2-pyridones has been mainly reported through dehydration under acidic conditions, and 5a is indeed the main product observed (89%) after a one hour treatment of 3a in sulfuric acid (Scheme 3).

after methylation of the dithiocarbonate group, giving K and the corresponding anionic species L. The leaving group S-methyl dithiocarbonate at C2 enables the intramolecular O-arylation that leads to 5. Actually, this mechanism might involve more complex intermediate species as the above assumption

  about the absence of dianionic species may not be valid.

	(Conditions: 0.1 M 3, 2.2 equiv DBU, 9 equiv CS2, 2.2 equiv Me2SO4, rt)
		R 1			base +/-H +	R 1
					1. DBU, MeCN, 0.5 h
			O	O	2. CS 2 , 4 h 3. Me 2 SO 4 , 12 h		O	O	base = DBU or KOH
	Ph	N H	O		Ph	N	O	SMe	Z = Me or CS 2 Me
		3				4
				3a, 4a : R 1 = H	4a, 91%
				3b, 4b : R 1 = Cl 3c, 4c : R 1 = Br 3d, 4d : R 1 = Ot-Bu 3e, 4e : R 1 = Morpholino	4b, 87% 4c, 88% 4d, 89% 4e, 89%
				Path A
	To explain the formation of both cyclization products 4 and 5, we propose a mechanism in Scheme 5. This mechanism works on the assumption that dianionic species would not be formed under the conditions of Scheme 3 (KOH, DMSO) or Scheme 4 (DBU, acetonitrile). Deprotonation of diketone 3 gives rise to the multident nucleophilic species A and B in equilibrium. They may react with CS2 through the oxygen atom of the pyridone ring or through the enolate, leading to species C and D, respectively (among other possible reactive sites and intermediate species). The reaction via the enolate carbon atom may be considered as thermodynamically favored, so that the whole equilibrium is shifted toward D, which would then lead to product 4 through two possible paths depending on whether the ring closure takes place via a 6-exo-trig (path A) or a 6-endo-trig reaction (path B). In path B, the thioenolate of H is first alkylated by either Me2SO4 or CS2, the latter explaining the need for an excess of CS2. Product 5, however, would be formed E F G C Me 2 SO 4 base -MeSCOS -N H Ar Ph O O O -S SMe N H Ar Ph O O O S SMe Z base 4 CS 2 then Me 2 SO 4 (Z = CS 2 Me) or Me 2 SO 4 (Z = Me) -ZSH H J Path B from species C D Me 2 SO 4 Me 2 SO 4 -MeSH base
	K				L
	Nucleophilic Substitution of the Methylthio Group. Our approach to exploring the scope of the
	nucleophilic substitution is based on the Mayr's reactivity scales. These scales are constructed according to
	the equation log k = s (N + E), where the second-order rate constant k of a nucleophile-electrophile
	combination correlates with the nucleophilicity and electrophilicity parameters N and E of the reagents. 9

Table 2 . Nucleophilic Substitution of 4a with N-Nucleophiles Depending on the Nucleophilicity Parameter N.

 2

						Ph	O	O	HNR 2 R 3		Ph	O	O
				Ph	N	O 4a	SMe	solvent, conditions	Ph	N	6	O	R 3 N	R 2
	Entry a	HNR 2 R 3			N		Catalyst Solvent Temperature Time,	Product Yield,
						parameter b				h	% c
	1		HN				17.35	none	DCM		rt	12	6aa	96
	2		HN	O			15.65	none	DCM		rt	12	6ab	93
	3		MeNH 2			15.19	none	DCM		rt	12	6ac	97
	4	H 2 N	Ph			14.29	none	DCM		rt	12	6ad	94
	5	H 2 N	OH			14.11	none	DCM		rt	12	6ae	65
	6 d	H 2 N	CO 2 Me			13.51 e	none	DCM		rt	12	6af	63
	7 f	H 2 N		OMe		13.42	DMAP	THF		65 °C	2	6ag	91
	8 f	H	2 N				12.64	DMAP	THF		65 °C	2	6ah	90
	9 f	H 2 N		CO	2 Et		n/a g		DMAP dioxane		100 °C	36	6ai	61
			N
	10 f						11.74	DMAP dioxane		100 °C	12	0
			N
			H
			N
	11 f						11.47	DMAP dioxane		100 °C	12	0
			N
			H

a Reaction conditions: 0.1 M 4a and 1.1 equiv Nu. b N parameters are given in acetonitrile, data from ref. 16. c Yields of isolated products. d Glycine methyl ester was generated from the corresponding hydrochloride (1.5 equiv) and Et3N (1.1 equiv). e N parameter of the free carboxylate, data in water. f 0.05 M 4a.

g

Data not yet available.

  H NMR in CDCl3. Note: commercial grade EtOAc was dried over CaCl2 overnight, then filtered through a cotton plug and stored over 4 Ȧ molecular sieves until used.

	3-Acetoacetyl-4,6-diphenyl-2(1H)-pyridone (3a) was prepared from 2a according to the general
	procedure and was isolated by flash chromatography (DCM/EtOAc 85:15 then DCM/EtOAc/EtOH 85:10:5).
	557 mg (1.68 mmol, 84% yield), yellowish solid (cyclohexane). Ratio enol/keto 3.6:1. 1 H NMR (400 MHz,
	CDCl3, enol form) δ 15.41 (br s, 1H), 12.80 (br s, 1H), 7.89 -7.80 (m, 2H), 7.56 -7.36 (m, 8H), 6.63 (s,
	1H), 5.86 (s, 1H), 2.02 (s, 3H). 1 H NMR (400 MHz, CDCl3, keto form) δ 12.80 (br s, 1H), 7.89 -7.80 (m,

2H),

7.56 -7.36 (m, 8H), 6.60 (s, 1H)

, 3.96 (s, 2H), 2.00 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl3, enol form) δ 191.

  107.6, 104.1, 25.4. HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C21H17 79 BrNO3 410.0386, found 410.0388 ; calcd for C21H17 81 BrNO3 412.0372, found 412.0370.

	3-Acetoacetyl-4-(4-t-butoxyphenyl)-6-phenyl-2(1H)-pyridone (3d) was prepared from 2d
	according to the general procedure and was isolated by flash chromatography (DCM/EtOAc 80:20 then
	DCM/EtOAc/EtOH 80:15:5). 670 mg (1.66 mmol, 83% yield), yellowish solid (cyclohexane). Ratio
	enol/keto 3.8:1. 1 H NMR (400 MHz, CDCl3, enol form) δ 15.43 (br s, 1H), 12.58 (br s, 1H), 7.87 -7.80 (m,
	2H), 7.53 -7.44 (m, 3H), 7.34 (dm, J = 8.6 Hz, 2H), 7.01 (dm, J = 8.6 Hz, 2H), 6.63 (s, 1H), 5.72 (s, 1H),
	1.99 (s, 3H), 1.39 (s, 9H). 1 H NMR (400 MHz, CDCl3, keto form) δ 12.58 (br s, 1H), 7.87 -7.80 (m, 2H),
	7.53 -7.44 (m, 3H), 7.31 (dm, J = 8.6 Hz, 2H), 7.01 (dm, J = 8.6 Hz, 2H), 6.60 (s, 1H), 3.90 (s, 2H), 2.02
	(s, 3H), 1.39 (s, 9H). 13 C{ 1 H} NMR (101 MHz, CDCl3, enol form) δ 191.
	H NMR (400 MHz, CDCl3, enol form) δ 15.38 (br s, 1H), 11.94 (br s, 1H), 7.82 -7.73 (m,

2H),

7.55 (dm, J = 8.5 Hz, 2H), 3H), 7.30 (dm, J = 8.5 Hz, 2H), 6.55 (s, 1H)

, 5.89 (s, 1H), 2.05 (s, 3H).

1

H NMR (400 MHz, CDCl3, keto form) δ 11.94 (br s, 1H),

7.82 -7.73 (m, 2H), 7.58 -7.46 (m, 5H), 7.33 -7

.24 (m, 2H), 6.51 (s, 1H), 4.01 (s, 2H), 2.04 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl3, enol form) δ 191.

7, 184.5, 162.6, 153.7, 147.9, 137.9, 132.7, 131.9, 131.1, 129.51, 129.48, 127.2, 127.0, 123.5,

  . HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C23H19N2O3 371.1390, found 371.1409.

	3-Acetyl-2-(benzylamino)-5,7-diphenyl-4H-pyrano[2,3-b]pyridin-4-one (6ad) was prepared from
	4a and benzylamine (12 μL, 0.11 mmol) according to procedure A (1 mL DCM, rt, 12 h), and was eluted
	with DCM/EtOAc 19:1. 42 mg (0.094 mmol, 94% yield), white solid (cyclohexane). 1 H NMR (400 MHz,
	CDCl3) δ 11.91 (br t, J = 5.4 Hz, 1H), 8.17 -8.05 (m, 2H), 7.61 (s, 1H), 7.55 -7.37 (m, 12H), 7.37 -7.32
	(m, 1H), 4.91 (d, J = 5.8 Hz, 2H), 2.60 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl3) δ 199.

Methyl 2-(3-acetyl-4-oxo-5,7-diphenyl-4H-pyrano[2,3-b]pyridin-2-ylamino)acetate (6af) was

	prepared from 4a according to procedure A (1 mL DCM, rt, 12 h) using glycine methyl ester hydrochloride
	(19 mg, 0.15 mmol) and triethylamine (15 μL, 0.11 mmol), and was eluted with DCM/EtOAc 9:1. 27 mg
	(0.063 mmol, 63% yield), white solid (cyclohexane). 1 H NMR (400 MHz, CDCl3) δ 11.88 (br t, J = 5.3 Hz,
	1H), 8.12 -8.05 (m, 2H), 7.61 (s, 1H), 7.55 -7.43 (m, 6H), 7.42 -7.35 (m, 2H), 4.51 (d, J = 5.6 Hz, 2H),
	3.87 (s, 3H), 2.62 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl3) δ 200.

.1. HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C24H21N2O4 401.1501, found 401.1498.

(3-acetyl-4-oxo-5,7-diphenyl-4H-pyrano[2,3-b]pyridin-2-ylamino)benzoate (6ai) was

  . HRMS (ESI/Q-TOF) m/z: [M + H] + calcd for C28H21N2O3 433.1547, found 433.1551.prepared from 4a and ethyl 4-aminobenzoate according to procedure B (2 mL dioxane, 100 °C, 36 h). The product was precipitated by addition of dioxane (2 mL) and cyclohexane (8 mL), recovered by filtration, and washed with cyclohexane/dioxane 2:1 and petroleum ether. 31 mg (0.061 mmol, 61% yield), white solid.1 H NMR (400 MHz, CDCl3) δ 13.80 (s, 1H), 8.18 (dm, J = 8.7 Hz, 2H),8.16 -8.11 (m, 2H), 7.74 (dm, J = 8.7 Hz, 2H),7.67 (s, 1H), 7.56 -7.45 (m, 6H), 7.44 -7.38 (m, 2H), 4.42 (q, J = 7.1 Hz, 2H), 2.69 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl3) δ 200.

	Ethyl 4-

  ,3.78 (s, 3H), 3.55 (dd, J = 14.8, 5.2 Hz, 1H), 3.49 (dd, J = 14.8, 7.2 Hz, 1H), 2.57 (s, 3H). 13 C{ 1 H} NMR (101 MHz, CDCl3) δ

ACKNOWLEDGMENTS

This work was supported by institutional funds from the Institut National du Cancer (INCa 11560) and the University of Montpellier (R.G. PhD fellowship). The authors appreciate the contribution of C. Hennebert (Master student).

AUTHOR INFORMATION

*Corresponding author, email: jean-pierre.uttaro@umontpellier.fr