Ultrasound-Promoted Greener Synthesis of Novel Trifurcate 3-Substituted-chroman-2,4-dione Derivatives and Their Drug-Likeness Evaluation

An efficient and convenient approach for one-pot synthesis of 3-substituted chroman-2,4-diones via a three-component reaction of aromatic aldehydes, 4-hydroxy- coumarins and diverse pyrazolone derivatives was described. The combinatorial synthesis for this methodology was achieved by applying ultrasound irradiation in the absence of activator while making use of water as green solvent. Additionally, novel chroman-2,4-dione derivatives attached to an edaravone moiety represent an exploitable source of brand new anticancer agents. In comparison with conventional methods, experimental simplicity, good functional group tolerance, excellent yields, short routine, and atom efficiency are prominent features of this sonocatalyzed procedure.


Introduction
In recent years, ultrasound-assisted organic synthesis (UAOS) utilizing environmentally benign reagents and conditions has been one of the most fascinating developments in the arena of modern synthetic chemistry owing to its green credentials [1][2][3][4][5]. The notable features of the aqueous UAOS OPEN ACCESS come from its advantages such as easier manipulation, energy conservation, rate enhancement and insolubility of the final products which facilitates their isolation by simple filtration [6,7].
Coumarins, an old class of benzopyrene compounds, are widely used in the pharmaceutical industry as precursor molecules in the synthesis of various anticoagulant [8,9], anti-HIV [10,11], anti-tumor [12,13], anti-hypertension drugs [14]. Among these therapeutic properties, their cytotoxic effects on tumors were most extensively examined by a number of in vitro and in vivo experiments as well as clinical studies. Specifically, 4-hydroxycoumarin derivatives have attracted considerable attention since this class of compounds allows access to many demonstrated bioactive agents [15,16]. For example, 4-hydroxycoumarin offshoots ( Figure 1) have recently been identified as novel HIV protease inhibitors (compounds I,II,III) [17] and anti-tumor chemicals (compounds IV,V) [18]. Moreover, the pyrazolone moiety ( Figure 2) is an important pharmacophore which exhibits widespread pharmacological properties, such as antimicrobial (compound VI) [19], anticancer (compound VII) [20], analgesic (compound VIII) [21], and anti-inflammatory (compound IX) entities [22]. It is noteworthy that the free radical scavenger edaravone (X, 3-methyl-1-phenyl-2-pyrazolin-5-one, Radicut ® ) [23,24] enhances the anti-tumor effects of chemotherapeutics in murine colon cancer by increasing apoptosis via inhibition of nuclear factor kappa B (NF-κB) [25]. Meanwhile, renal toxicity (acute cisplatin-induced renal injury) was reversed by clinically available edaravone [26,27]. Therefore, in addition to its usefulness in the treatment of strokes, edaravone is expected to play an integral role in oncotherapy. From the viewpoint of rational drug design, the pharmacological properties and therapeutic applications of coumarins depend upon the pattern of substitution on the benzopyrene nucleus. Hence, novel chroman-2,4-dione (tautomer of 4-hydroxycoumarin) derivatives attached to an edaravone moiety represented an exploitable source of brand new anticancer agents, which might also help addressing side-toxicity. In the present research work, the objective was to develop new chemical entities (triplet 3-substituted chroman-2,4-diones interlinking 4-hydroxycoumarins and edaravone) as basic scaffolds, endowed with potential synergy in elevating antitumor effects as well as preventing the adverse effects and decreasing toxicity. Herein, we are delighted to afford a convenient, practical and efficient reaction for the synthesis of the target lead compounds via readily available building blocks under ultrasonic irradiation in water as shown in Scheme 1. The present research would significantly widen the versatility of the diversity-oriented aldehyde-based MCRs. Scheme 1. Synthesis of 3-substituted chroman-2,4-diones under ultrasonic irradiation.

Results and Discussion
To find out the suitable conditions for the reaction, our initial experiments were demonstrated by a series of experiments with the model reaction (Scheme 2) using different catalysts and solvents. In each case, the substrates were mixed together with 25 mL of solvent under ultrasonic irradiation (40 kHz) or high speed stirring with traditional heating. The screening results of the reaction are listed in Table 1.

Effects of the Catalysts under Ultrasound Irradiation
It was observed that when classical organic bases, such as morpholine and triethylamine, were used to mediate the reaction, low yields were obtained (entries 6, 7 and 10, 11). Further study with varying Lewis acids and BrØnsted acids revealed no yields of 4a compared with basic catalyst, while simultaneously, the byproduct 5a was predominantly afforded by a side-reaction (Scheme 2, entries 5, 8 and 9). Scheme 2. Side-reaction promoted by Lewis acids or BrØnsted acids.
As can be seen from Table 1, the reaction rendered moderate amounts of desired product 4a in the presence of L-proline (entry 12). When the reaction was performed in ethanol and acetonitrile in the absence of catalyst, 4a was obtained in 55% and 14% yield, respectively (entries 1 and 2). Specifically, the formation of product 4a was more facile and proceeded in pure water to give not only high yield, but also with high reaction rate (81% yield in 30 min, entry 4).

Effects of the Solvents under Ultrasound Irradiation
Polar protic solvents (ethanol and water) afforded moderate to high yields of the desired product. When the reaction was performed in acetonitrile, the desired product 4a was only obtained in 14% yield (entry 2) and took a comparatively longer reaction time. Disappointingly, when the reaction was conducted in the non-polar solvent toluene, only the byproduct 5a was obtained (entry 3, yield, 83%). In general, improvements in rates and yields of all trials are observed when reactions were carried out in polar protic solvents compared with polar aprotic and non-polar solvents.

Comparison of Ultrasonic Irradiation and Conventional Method
As depicted in Table 1, the ultrasound-assisted technique was proven to be excellent in all cases whereas traditional heating has low efficiency, even with prolonged reaction times. Based on the results from this study, it was attractive that the ultrasound improved both the purity and yield of products. In order to verify the effect of irradiation frequency, the reaction was also performed in 30, 40, and 50 kHz. When the frequency was 40 kHz, the yield of 4a (81%, Table 2, entry 2) was higher than that with 30 kHz irradiation within 30 min (67%, Table 2, entry 1). With an increase of irradiation frequency from 40 to 50 kHz (Table 2, entries 2, 3), the reaction yield did not change considerably (79%). The results showed that there was an optimum frequency for effective synthesis of 4a in the frequency of 40 kHz at 60 °C.

High Efficiency and Generality of Synthesis by Ultrasound Irradiation
With the optimized conditions in hand, the versatility of this protocol was investigated by a library of sixteen 3-substituted-chroman-2,4-diones combining substituted aromatic aldehydes 1, 4-hydroxy-coumarins 2 and pyrazolones 3. According to Table 3, 4-hydroxycoumarins and pyrazolones carrying either electron-releasing or electron-withdrawing substituents at the para-position of the aromatic ring proved to be suitable substrates for this reaction. On the other hand, the aromatic aldehydes bearing electron-donating groups are more reactive in this protocol, affording the desired products with excellent yields and after shortened reaction times (Table 3, entries 3, 5 and 7). It is noteworthy that the hydroxyl group of vanillin remains intact after the reaction, obviously, which leaves a useful handle for further synthetic diversity. The structures of isolated new products 4a-p were determined by physical and spectroscopic data such as: 1 H-NMR, 13 C-NMR and ESI-HRMS analysis. Taking compound 4a as a representative example, its 1 H-NMR spectrum revealed the presence of two singlet signals at δ = 2.41 and δ = 3.62 ppm corresponding to the -CH 3 group of edaravone and the -OCH 3 group adjacent to vanillin, respectively, and another singlet signal at δ = 5.66 ppm due to the -OH of the vanillin scaffold, which underwent a facile hydrogen exchange upon addition of deuterium oxide. In the HH-COSY spectroscopy of compound 4a, correlations between adjacent protons from three stereogenic centres were observed (4.05 ppm, 1.19 ppm and 1.17 ppm). This phenomenon suggested that the structure of compound 4a was depicted accurately, which could be proved by the other 2D-NMR spectra.

the Study of Acceleration Mechanism under Irradiation of Ultrasound
A plausible mechanism is proposed and shown in Scheme 3. The process represents a typical tandem of Knoevenagel condensation and Michael addition, which might initiate via two pathways, namely path A and path B.
Once the three components are mixed, aldehyde 1 undergoes a Knoevenagel condensation with edaravone (3) under ultrasonic irradiation to afford the relay moiety 5 after dehydratation, which is subsequently attacked by 4-hydroxycoumarin 2 to furnish the central target compound 4 (path A). Alternatively, intermediate 6' is likely formed from initial condensation of 1 with 2 to afford 6, followed by nucleophilic addition of edaravone 3 (path B).
Upon irradiation by high-intensity ultrasound, acoustic cavitation occurs and gives rise to the formation, growth, and implosive collapse of bubbles in water with consequent high local temperatures and pressures, which accelerates the mass transfer and shortens the reaction time.

Scheme 3.
Plausible mechanism for the formation of product 4.

Drug-Likeness Evaluation of Obtained 3-substituted-chroman-2,4-Diones
Drug-likeness may be defined as a complex balance of intriguing insights, including various molecular properties and structure features, which determine whether a particular molecule has potentiality to be an orally active drug. Lipinski et al. [28,29] examined the drugs on the market and established the so-called "Rules of Five", which have served as a guide in drug-like analysis and as an efficient filter in prioritizing compound libraries prior to high-throughput screening. With drug-like criteria in mind, the molecular mass of each obtained "drug-like" compound should be less than 500 Daltons. The partition coefficient logP values of the synthesized molecules were calculated (Table 3) and found to be between 2.45 and 4.34. This range is in agreement with estimated values for good lipophilicity and solubility (between 2.0 and 5.0).
Aqueous solubility of a solid chemical can be also applied as a rule to define drug-likeness. Melting point is usually an indicator of crystal lattice energy, which has a dramatic influence on solvation free energy (aqueous solubility). The ceiling melting point of all the trifurcate 3-substituted-chroman-2,4-diones discussed above is 201 °C, in which means favorable estimated solubility. We concluded that all the key parameters (predictors of molecular transport properties across cell membranes and indicators of protein binding characteristics) have no violation of Lipinski's Rules at all.

General
All reagents used in this work were obtained commercially available and were used without purification. 1 H-and 13 C-NMR spectra were recorded on a BRUKER AV-300 spectrometer at 300.13 and 75.47 MHz, respectively. 1 H and 13 C-NMR spectra were obtained from solutions of DMSO-d 6 and are reported as parts per million (ppm) downfield from a tetramethylsilane internal standard. The following abbreviations are used; singlet (s), doublet (d), triplet (t) and multiplet (m). The mass spectrometric analyses (HRMS) were performed using a JMS-700 MStation High Resolution JEOL Mass Spectrometer with a source temperature of 230 °C, an ionization energy of 70 eV and an ionization trap current of 300 A. Melting points were measured by a differential scanning calorimeter (Shimadzu DSC-50) and were uncorrected. The standard heating rate for all compounds was 10 °C/min. Sonication was performed in Shanghai Branson-CQX ultrasonic cleaner. The flask was located at the maximum energy area in the cleaner and addition or removal of water was used to control the temperature of the water bath.

Ultrasound-promoted Typical Procedure for Synthesis of 3-substituted-chroman-2,4-dione (4a)
A 50 mL flask was charged with vanillin (152 mg, 1 mmol), edaravone (174 mg, mmol) and 4-hydroxycoumarin (162 mg, 1mmol) in water (25 mL). The reaction flask was placed in the ultrasonic bath, where the surface of reactants is slightly lower than the level of the water, and irradiated separately under 30, 40, and 50 kHz at 50 °C (bath temperature, the temperature inside the reactor was also 50 °C) for the period of time as indicated in Tables 1-3. The reaction temperature was controlled by addition or removal of water from ultrasonic bath. After completion of the reaction (monitored by TLC), the crude mixture was purified by silica gel column chromatography to afford desired product 4a after solvent evaporation.