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Medicinal Attributes of Meldrum’s Acid

 

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About Authors: Sahil Sharma

Reference ID: PHARMATUTOR-ART-1083

Introduction
One hundred years ago Scottish chemist Andrew Norman Meldrum synthesized a substance [1] that later obtained his name. To date Meldrum’s acid is one of the most useful reagents in the synthesis of heterocycles. In contrast to the great popularity of Meldrum’s acid, its discoverer remains almost unknown for the majority of chemists.
Andrew Norman Meldrum [2] was born on 19th March, 1876, in a small burgh, Alloa, Scotland. In 1899 he received his B.Sc. with ?rst-class honors (chemistry) from the University of Aberdeen, where he worked as a research assistant with Francis Robert Japp [3].  
In his ?rst independent publication [1], he studied the reaction between acetone and malonic acid and, following the suggestion of Prof. Japp, employed a mixture of acetic anhydride and sulfuric acid as condensing agent. From elemental analysis data, in conjunction with previous results and the acidic properties of the ?nal compound, he formulated the structure of the product to be β-lactone of β-hydroxyisopropylmalonic acid 1 (Scheme 1).

Clearly, back then it was dif?cult to assign the correct chemical structure of product isolated from this reaction. For instance, the unusual C–H acidity of Meldrum’s acid (pKa    4.83 in water) continues to be the focus of attention for research [5,6]. However, it was not until 1948, when more experimental data on the chemistry of this product were collected, that its correct structure was determined to be 2,2-dimethyl-4,6-diketo-1,3-dioxane 2 [7].
The ?rst review on the chemistry of Meldrum’s acid by McNab [8] describes mostly the synthesis of derivatives via functionalization of the methylene group, and only a few reactions are mentioned where the 1,3-dioxane moeity is the reaction site. A more detailed literature survey [9] shows an enormous interest in Meldrum’s acid chemistry in the 1980s that continues to this century. Thus, recent reviews focused on more specialized subjects such as the role of Meldrum’s acid in multicomponent reactions [10], ?ash vacuum pyrolysis (FVP) techniques [11,12], and more recently in synthesis of natural products [13].
The aim of this review is to cover the role and impact of Meldrum’s acid and its derivatives when they have been used as building blocks for the construction of biologically active heterocyclic systems. To date Meldrum’s acid is one of the most useful reagents in the synthesis of heterocycles having various biological activities like antimalarial, antioxidant, antiparasitic, anti-HIV, anticancer etc. Synthesis and biological evaluation of arylidene analogues of Meldrum’s acid as a new class of antimalarial and antioxidant agents

Abstarct
A series of arylidene analogues of Meldrum’s acid were synthesized and evaluated for in vitro antimalarial and antioxidant activities for the ?rst time. The in?uence of various physico-chemical parameters such as dielectric constant (e), donor number (DN), acceptor number (AN), hydrogen bond donor (HBD), hydrogen bond acceptor (HBA), and solubilizing power of the solvents on Meldrum’s acid anion generation and thus on promoting the Knoevenagel condensation of Meldrum’s acid with aryl aldehydes has been discussed. Five compounds 9l, 9m, 9n, 9r, and 9s were found to be most active against Plasmodium falciparum with IC50values in the range of 9.68–16.11 lM. Compound 9l exhibited the most potent antimalarial activity (IC50 9.68 lM). The compounds were also found to possess antioxidant activity when tested against DPPH and ABTS free radicals.

1. Introduction of Malaria
Malaria is a tropical parasitic disease and one of the top three killers among communicable diseases.1It is a public health problem in more than 90 countries inhabited by about 40% of the world’s population. According to recent WHO reports, it affects approximately 250 million people and kills about one million people every year.2Among four species of human malarial parasite, Plasmodium falciparum is considered most fatal due to high mortality rate particularly in children. Several drugs are being used in malaria-endemic regions of the world to control, treat, and prevent malaria; however, the emergence and spread of antimalarial drug resistance has made the malaria treatments ineffective and is therefore, a global problem.3Drug resistance can be de?ned as the ability of a parasite strain to survive and/or multiply in the presence of a drug administered in doses equal to or higher than those usually recommended.4Resistance of    P. falciparum has emerged to all classes of antimalarial drugs (chloroquine (1), primaquine, sulfadoxine, pyrimethamine, etc.) except artemisinin (2)5–8(Fig. 1). The emergence of resistance of P. falciparum has provided a fresh impetus to the researchers for the development of a better and safe antimalarial drug. Recently, the use of antioxidants in the malaria as an adjunct treatment has generated a renewed interest. Several reports have evidenced that in patients with falci parum malaria infection oxidative stress is increased and relates to severity of disease and anemia.9,10    Therefore, the antimalarial drugs with antioxidant action are of considerable interest due to their additional bene?t to the treatment. The various classes such as pyrimidines,11indole,12chalcone,13 xanthone,14pyrazoline,15propenone,16etc., are reported to exhibit antimalarial activity. Licochalcone (3),17chalcones-like retinoid (4),18and various other chalcones (5 and 6)16,19(Fig. 1) are reported to possess promising antimalarial activity. It was noticed that chalcones with higher number of methoxyl groups exhibited potent antimalarial activity. In view of the dif?culty in making peroxides besides being more toxic it was thought worthwhile to design a compound which could react with Fe+2of heme or free Fe+2in a similar way, we conceptualized a molecule with structural features where important functionalities are equally placed as in artemisinin. The most suitable molecule we could design is in starting with an arylidene analogue of Meldrum’s acid where two oxygen and one carbon atoms of the six membered ring of Meldrum’s acid are exactly positioned with two oxygen and one carbon atoms of the six membered ring of artemisinin where one has to ignore the structural features while emphasizing on the shape of the molecule. We could visualize the ease of a ketal to bind with Fe+2and proved to be correct (Fig. 2). The novelty of this molecule appears to be that site of the molecule where it binds with Fe+2is symmetrical with the result that only one type radical is capable of interacting with Fe+2 while in artemisinin two types of radicals are possible.7 In the present communication the synthesis of various arylidene analogues of Meldrum’s acid, the corresponding epoxides (Fig. 3) and their biological evaluation as antimalarial and antioxidant agents are reported for the ?rst time.

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2. Results and discussion
2.1. Optimization of reaction conditions for the synthesis of arylidene analogues of Meldrum’s acid

Arylidene analogues (9) of Meldrum’s acid are reported as the key intermediates for cycloaddition reactions and for the synthesis of heterocyclic compounds of biological importance such as cardiotonic20aand HIV integrase inhibitory activities.20bThey are also reported to serve as the versatile substrates for various kinds of reactions.21
While planning for the synthesis of 9, it was realized that a convenient synthetic protocol is lacking. The synthesis of 9 via Knoevenagel condensation of 7 with aldehydes has been documented either catalyzed by bases such as pyridine under re?ux and water removal by Dean–Stark, 22a, b piperidine/glacial acetic acid in benzene, 22c, d piperidine22e, pyrrolidine/acetic acid in dry benzene, 22fNAP22g (3-aminopropylated silica gel), a potassium exchanged    zirconium    hydrogen phosphate    Zr(O3POK) 2a heterogeneous basic catalyst22hor by Lewis acid such as anhydrous ZnCl2, 23or in ionic liquids, 24or in solvents such as water25under heating for 2 h, DMF under heating at 80 °C26, 27 and DMSO, 27or using surfactants 28 or under microwave irradiation, 29 or melt condition. 30 However, these methodologies have one or more disadvantages such as the use of high boiling solvent (e.g., DMF, DMSO, and benzene) that are dif?cult to recover, use of acid or base catalyst, special efforts required to prepare the catalysts (e.g., NAP and Zr(O3POK)2) or to prepare the polar medium (e.g., ionic liquids), need to use special apparatus (e.g., microwave irradiation) and heating conditions. Thus, there is necessity to develop a more effective and convenient synthetic procedure. It is known that 7 exists in a boat form and its corresponding anion formed due to ease of deprotonation of one of the more acidic (?agpole) methylene hydrogens in a polar medium, is stabilized by extensive conjugation.31The reported condensation in polar medium24–27,31 encouraged us to screen various solvents of different dielectric constants32(e), (Scheme 1, Table 1). In a model reaction, 3,4,5-trimethoxybenzaldehyde (8i, 1 mmol) was treated with 7 (1 mmol) under solvent-free conditions at rt (    30–35 °C) for 12 h that did not result in the signi?cant formation (5%) of Knoevenagel product 2,2-dimethyl-5-(3,4,5-tri- methoxybenzylidene)-[1,3]-dioxane-4,6-dione (9i) (Scheme 1), indicating the requirement of a solvent for the reaction.
The reactions were performed in solvents of different polarity. The importance of solvent polarity was demonstrated by the obser- vations that excellent results were obtained in MeOH having the highest value of e (except water, MeCN, DMF and DMSO) of all the solvents used at rt after 15 min (96%) (Table 1). The product yield decreased with the decrease of e of the alcohols and a similar trend was also observed for the ethereal solvents. The Knoevenagel adduct 9i was formed in 18%, 24%, and 37% yields after 15 min in 1,4-dioxane, Et2O, and THF, respectively. The poor yields were obtained using the hydrocarbon and halogenated solvents due to their low e values and low solubility of starting materials in them. The use of MeOH under non-anhydrous and anhydrous conditions resulted in comparable yields and provided an added advantage to this new methodology in eliminating additional efforts required in drying the solvent. Lesser yields obtained with the use of MeCN, DMF, DMSO, and water signi?ed the role of various factors other than e in promoting the condensation (Table 1). The involvement of various factors such as donor number33(DN), acceptor number33(AN), hydrogen bond donor34(HBD, a), and hydrogen bond acceptor35(HBA, b) of the solvents and their in?uence in Meldrum’s acid anion generation and hence on Knoevenagel condensation was also considered. Solvent MeCN, in spite of having higher e (36.6) than MeOH (32.7), afforded only 32% product which could be probably due to its low DN, poor AN, poor HBD, and nil HBA. Similarly, DMF and DMSO have higher e, higher DN but lesser AN, comparable HBA, and nil HBD than MeOH; whereas the water has highest e, higher AN, comparable HBD but lesser DN (data not reported), lesser HBA and poor solubility of starting materials at rt and hence retarded the rate of reaction. This was further supported by the fact that the formation of the condensed product was enhanced from 35% to 66% and 68% while carrying out the reaction under re?ux for 2 h or in surfactant (sodium dodecyl sulfate; 100 mol %) at rt for 2 h, respectively, indicating the in?uence of solubility. The results were found in good agreement with the previously reported studies.25,28 Thus we can anticipate that the high e of MeOH makes it a polarmedium with the ability to donate hydrogen bond (HBD), accept hydrogen bond (HBA), optimum Lewis acidity (AN 41.5), Lewis basicity (DN 20.0) and solubility of the starting materials at rt during Knoevenagel condensation make it an ideal coordinating solvent with the 7 and that results into Meldrum’s acid anion generation 11 via a polar six membered transitions state TS (Scheme 2) followed by condensation with aldehyde 8 to yield the dehydrated product 9 as the target compound.

2.2. Synthesis of arylidenes and the corresponding epoxides
By using optimized procedure, we synthesised compounds 9a and 9d–w (85–96%) via Knoevenagel condensation of 7 (1 equiv) with aldehydes (8a and 8d w; 1 equiv) in MeOH at rt (Scheme 3, Fig. 4) in 15–45 min. Various aromatic and heteroaromatic aldehydes condensed smoothly. The crude products were puri?ed by column chromatography and were characterized by using spectroscopic techniques such as IR, NMR, and mass spectrometry. The reaction was found to be compatible with diverse functional groups such as ether, nitro, hydroxyl and halogens. It was noticed that the reaction of    7 with aldehydes bearing electron withdrawing group(s) irrespective of their position on aromatic ring (8b and 8c) in MeOH yielded a mixture of Knoevenagel product (9b and 9c), aldol product (12b and 12c) and Michael adduct (13b and 13c) (Scheme 2). Similar results were obtained in carrying out the reaction with water under heating for 2 h which were in accordance with earlier observations reported by Bigi et al.25The reason why electron donating substituents on aromatic ring accelerate the last step of Knoevenagel condensation and electron withdrawing groups accelerate the ?rst step of Knoevenagel condensation is still unclear and is a question of study.22e,25 Though the various approaches such as methoxide,36amines,37 and thiols38are utilized to minimize the formation of Michael adduct 13 but this is an unsatisfactory solution in terms of atom and step economy. However we succeeded to synthesize the desired compounds 9b and    9c with excellent selectivity (>96) when the reaction of 7 (1 equiv) with 8b and 8c (1 equiv each) was carried out separately under the catalytic in?uence of type 3 Å MS (50 mol %) in MeOH under re?ux for 3 h. Two epoxide derivatives 10a and 10b (Scheme 4, Fig. 4) and one 2-benzylidenecyclohexane-1,3-dione (14), respectively, were synthesized as per the reported procedures.39,40

2.3. Biological evaluation of synthesized compounds for antimalarial activity
In vitro screening of all the compounds (9a–x, 10a, 10b, and 14) was carried out for antimalarial activity using histidine-rich protein 2 (HRP-2) double site sandwich enzyme-linked immunosorbent assay (ELISA) with the previously reported method.41Each compound was tested in triplicate. None of the compounds showed any cytotoxic activity (IC50value higher than 100 lM after 48 h). Among the series of 27 compounds, ?ve compounds 9l, 9m, 9n, 9r, and 9s were found to be most active against the resistant strain of P. falciparum with IC50ranging from 9.68 lM to 16.11 lM(Table 2). Compound 9l exhibited the most potent antimalarial activity (IC509.68 lM).

2.4. Structure–activity relationship (SAR)
The compounds bearing electron withdrawing substituents on phenyl moiety of ring A irrespective of their position, were found to be inactive or less active than electron donating substituents (9b, 9c, and 9d–p). Replacement of phenyl group with more bulky groups such as 1-naphthyl (9s) and 2-naphthyl (9r), resulted in enhancement of antimalarial activity (except 2-methoxy-1-naphthyl; 9u and anthranyl; 9t). The compounds with hydroxyl group on phenyl ring at p-position (9l) showed better activity than those with m-position (9n). The replacement of the phenyl ring with 2-theinyl (9v) or 3-indolyl (9w) did not result in increase of the activity. The conversion of the arylidene analogues into their corresponding epoxides (9a and 9b) resulted in loss of the antimalarial activity indicating the importance of ole?nic linkage. Further no activity was observed with 14 highlighting the signi?cance of two oxygen atoms. These observations helped us to formulate a basic pharmacophore with the structural features required for antimalarial activity (Fig. 5).

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2.5. Biological evaluation for antioxidant activity
1,1 - Diphenyl - 2 - picrylhydrazyl (DPPH) and 2, 20 - azino bis (3 - ethylbenzothiazoline - 6 - sulfonic acid) (ABTS) radicals 42, 43    are commonly used for assessing the antioxidant property of substances of natural or synthetic origin because of their good reproducibility. Both assays measure the decrease in absorbance of radicals at a characteristic wavelength during the reaction with antioxidant; which reverses the formation of ABTS radical cation and DPPH radical: DPPH þ AH ! DPPH þ A

ABTSþþ AH ! ABTSþþ A
It is well known that ABTS activity is closely related with DPPH44because both are responsible for the same chemical property of hydrogen or electron donation.

2.5.1. Free radical scavenging effects in DPPH assay
The free radical scavenging effect of the synthesized compounds were tested in DPPH assay.45,46 Initially, all the compounds were tested at 100 lM concentration and their results are shown in Table 3. Three compounds 9l, 9m, and 9n were found to be strong free radical scavengers with 76.0%, 66.1%, and 40.2% scavenging of the DPPH activity. All other compounds were found to have very weak radical scavenging activity. The study was further extended to determine the scavenging effects of 9l, 9m, and 9n at different concentrations and to calculate the IC50values and various other parameters (Table 3 and Table 4). Compound 9l was found to exhibit comparable IC50and ED50 values (Table 4) with that of ascorbic acid while it was less active than that of trolox. Compounds 9m and 9n were less active than 9l. Compound 9l was found to exhibit the reduction of more than one molecule of DPPH per molecule of 9l; whereas 9m and 9n demonstrated the reduction of less than one molecule of DPPH/molecule. The standard trolox demonstrated the reduction of about two molecules of DPPH/molecule. Thus, it can be concluded that compounds 9l showed the comparable activity as that of ascorbic acid.

2.5.2. Free radical scavenging effects in ABTS assay
ABTSÅ+ cation radical is commonly used to determine the hydrogen-donating ability of antioxidants.47,48    Initially, all the compounds were tested at 100 lM concentration and their results are shown inTable 3. The similar results were also obtained in this assay. Compound 9l,    9m, and 9n showed the 92.5%, 95.5%, and 52.7% scavenging of the ABTS free radical, respectively; while the other compounds were weak radical scavengers. The IC50values of    9l, 9m, and    9n    were found as 30.4, 30.6, and 72.1 lM, respectively (Table 4).

3. Conclusions
The present study was initiated with the aim of providing an ef?cient and convenient method for the synthesis of arylidene analogues of Meldrum’s acid. The optimization of the synthetic procedure was carried out and the effect of the various factors such as donor number (DN), acceptor number (AN), hydrogen bond donor (HBD, a), and hydrogen bond acceptor (HBA, b) and the solubility on the reaction conditions was also studied. The MeOH emerged as the best suitable solvent for carrying out the Knoevenagel condensation of Meldrum’s acid with aldehydes. Following the optimized procedure twenty four analogues of Meldrum’s acid were synthesized out of which six were found as new. The synthesized compounds were evaluated for antimalarial (against P. falciparum) and free radical (DPPH and ABTS) scavenging activities. SAR study revealed that: (i) nature of substituents present on phenyl ring, (ii) an ole?nic double bond, and (iii) two oxygen atoms are critical requirements for high antimalarial activity. Further the determination of mode of action of arylidene analogues of Meldrum’s acid and a detailed target analysis is currently in progress.

4. Experimental
The reagents were purchased from Sigma–Aldrich, Loba and CDH, India and used without further puri?cation. All yields refer to isolated products after puri?cation. Products were characterized by comparison with authentic samples and by spectroscopic data (IR,1H NMR,13C NMR spectra). The NMR spectra were recorded on a Bruker Avance DEX 400 MHz instrument. The spectra were measured in CDCl3relative to TMS (0.00 ppm). IR (KBr pellets) spectra were recorded on a Fourier transform infrared (FT-IR) Thermo spectrophotometer. Melting points were determined in open capillaries and were uncorrected.

4.1. Typical experimental procedure for the synthesis of 9a and 9d–w: 2,2-Dimethyl-5-(3,4,5-trimethoxybenzylidene)-[1,3]dioxane-4,6-dione (9i)21d
A mixture of 7 (1 mmol, 1 equiv, 0.14 g) and 8i (1 equiv, 0.19 g) in methanol (1 mL) was stirred at room temperature. The progress of the reaction was monitored by TLC (15 min). After the comple- tion of reaction, the precipitate was ?ltered-off and dried to afford 9i (0.30 g, 96%). The remaining reactions were carried out following these general procedures. In each occasion, the spectral data (IR,1H NMR and13C NMR) of known compounds such as 5-benzylidene-2,2-dimethyl-[1,3]dioxane-4,6-dione (9a), 285-benzylidene-2,2-dimethyl - [1,3] dioxane-4, 6-dione (9a), 285-(4-hydroxybenzylidene)-2, 2-dimethyl - [1,3] dioxane-4, 6-dione (9d), 255-(4-methoxybenzylidene)-2, 2-dimethyl - [1,3] dioxane-4, 6-dione(9e), 25 5 - (4-dimethylaminobenzylidene) - 2, 2-dimethyl - [1,3] dioxane-4, 6-dione (9f), 24a5 - (3,4-dimethoxybenzylidene) -2, 2-dimethyl - [1,3] - dioxane-4, 6-dione (9g), 24a5 - (4-hydroxy-3-methoxybenzylidene) - 2, 2-dimethyl - [1,3] dioxane-4, 6-dione (9l), 22f5 - (3-hydroxy-4-methoxybenzylidene) - 2, 2-dimethyl - [1,3] dioxane-4, 6-dione (9n), 20b5-benzo [1,3] dioxol-5-ylmethylene-2, 2-dimethyl - [1,3] dioxane-4, 6-dione (9q), 22c5 - (2,3,4-trimethoxybenzylidene) - 2, 2-dimethyl - [1,3] dioxane-4, 6-dione (9k), 21e5 - (2,4,5-trimethoxybenzylidene) - 2, 2-dimethyl - [1,3] dioxane-4, 6-dione (9j), 21e2, 2-dimethyl-5-naphthalen-2-ylmethylene - [1,3] dioxane-4, 6-dione (9r), 22f2, 2-dimethyl-5-naphthalen-1-ylmethylene - [1,3] dioxane-4, 6-dione (9s), 22f2, 2-dimethyl-5-thiophen-2-ylmethylene - [1,3] dioxane-4, 6-dione (9v), 21d 5 - (1H-Indol-3-ylmethylene) - 2, 2-dimethyl - [1,3] dioxane-4, 6-dione (9w), 20b2, 2-dimethyl-5 - (3-phenyl-allylidene) - [1,3]dioxane-4, 6-dione (9x), 256, 6-dimethyl-2-phenyl-1,5,7-trioxaspiro [2.5] octane-4, 8-dione (10a), 396, 6-dimethyl-2 - (2-nitro-phenyl) - 1,5,7-trioxaspiro [2.5] octane-4, 8-dione (10b), 392-benzylidenecyclohexane-1, 3-dione (14), 40 were found to be identical with those reported in the literature. The physical data of new compounds are provided below.

4.2. Typical experimental procedure for the synthesis of 9b and 9c
To a mixture of 7 (1 mmol, 1 equiv, 0.14 g) and 8b (1 equiv, 0.15 g) in methanol (1 mL) was added MS 3 Å (50 mol %). The reaction mixture was re?uxed for 3 h (TLC) The reaction mixture was ?ltered, dried under reduced pressure and washed with ether and hexane mixture to afford pure 9b (0.22 g, 81%). Mp 116–118 °C (Lit.24a117–118 °C).

4.3. Biology
4.3.1. Antimalarial assay
4.3.1.1. P. falciparum in vitro culture
Parasites were maintained in in vitro culture using the method of Trager and Jensen.41a Brie?y, an equal volume of sterile 3.5% sodium chloride was added to the infected blood samples, and the mixture was centrifuged at 1000g    for 5 min. The pellet was washed three times and re-suspended with RPMI-1640 medium supplemented with 10% human serum and placed in a 25 mL tissue culture ?ask in a total volume of 10 mL containing a 5% RBC suspension. The ?ask was ?ushed with a gas mixture of 5% CO2, 5% O2, and 90% N2for 30 s and incubated at 37 °C. The culture medium was changed once a day; group O red blood cells were added to maintain the 5% cell suspension. Parasite growth was monitored by thin smear examination with Field stain (A and B).

4.3.1.2. Hrp2 ELISA
A commercial HRP2 ELISA kit41bwas used for the quanti?cation of HRP2 in the culture samples. One hundred microliters of each of the hemolyzed culture samples was transferred to the ELISA plates, which are precoated with monoclonal antibodies against P. falciparum    HRP2 (capture antibody of the immunoglobulin M class; code CPF4), and the plates were incubated at room temperature for 1 h. Subsequently, the plates were washed four times with the washing solution provided with the test kit, and 100 lL of the diluted antibody conjugate (an indicator antibody of the immunoglobulin G1 isotype; code CPF6) was added to each well. After incubation for an additional 1 h, the plates were washed four times and 100 lL of diluted (1:20) chromogen (tetramethylbenzidine) was added to each well. The plates were then incubated for another 15 min in the dark, and 50 lL of the stock solution was added. Spectrophotometric analysis was performed with a Multiscan Ex ELISA reader (Thermo Scienti?c) at an absorbance maximum of 450 nm. The optical density values correspond to the amount of HRP2 found in the culture samples and provide consistent indicators of parasite growth.41c

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4.3.2. Antioxidant assay
4.3.2.1. DPPH radical scavenging assay
The free radical scavenging activities of compounds were determined by the widely used 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay employing trolox as reference standard.45,46 Brie?y, to a methanolic solution of DPPH (60 lM, 3.9 mL) was added    the different concentrations of test compound (0.1 mL), the resulting mixture was incubated at room temperature for 1 h and then the absorbance was read at 517 nm. The experiment was done in triplicate for each concentration and the results are expressed as the% of the DPPH radical scavenged by the test compounds. The IC50is de ?ned as the concentration of the test compound that causes 50% scavenging of DPPH radical and was calculated from the regression analysis obtained by plotting the scavenging effect of compounds at ?ve different concentrations. ED50, ef?cient dose is de?ned as micromoles of compound able to consume half the amount of free radical divided by micromoles of initial DPPH and the value is obtained by dividing IC50value by 60. The inverse of ED50is the measure of the antiradical power (ARP). By multiplying the ED50by two, the stoichiometric value (theoretical concentration of antioxidant to reduce 100% of the DPPH) is obtained. The inverse of this value represents the moles of DPPH reduced by one mole of antioxidant and gives an estimate of the number of hydrogen atoms involved in the process.

4.3.2.2. ABTS radical scavenging assay
2,20-Azino-bis (3- ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity of the compounds were determined using an ABTS radical cation decolorization assay.47,48    ABTS was dissolved in water to a concentration of 7 mM. The ABTSÅ+ cation radical was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate and by allowing the mixture to stand in the dark for 12 h. To an aliquot (5 lL) of ethanol solution of test compounds was added ABTS solution (0.1 mL) and the absorbance was read at 734 nm after mixing up to 6 min. All the determinations were carried out in triplicate and the results are expressed as the% of the ABTS radical scavenged by the test compounds. The IC50values were calculated by regression analysis in the similar manner as described above.

Synthesis and biological evaluation of new heterocyclic quinolinones as anti-parasite and anti-HIV drug candidates

Abstract
We have synthesized quinolinones with potential antiparasitic and anti-HIV activities by an original two-step method involving microwave irradiation and have evaluated their activities against Plasmodium falciparum, Leishmania donovani, Trichomonas vaginalis, and HIV. None of the tested compounds had been previously described using this method of synthesis. One of the compounds had interesting antiparasitic and anti-HIV activity, which could be improved by substitution with different radicals.

Treatment of prominent parasitic diseases remains challenging due to parasite’s resistance to available drugs. Malaria has been known since antiquity, but it is still responsible for the death of more than one million people every year, mostly in Africa.1Parasite’s resistance to drugs continues to undermine efforts for malaria control. During the past decade, antileishmanial therapy has become a bewildering subject due to the complexity of the disease2 and the continuous appearance of glucantime-resistant Leishmania strains responsible for therapeutic failures in immuno-competent patients. Another parasitic disease such as urogenital trichomonosis, previously considered as a benign sexually transmitted disease (STD), has received more attention following the spread of the AIDS epidemic given that STDs are favouring factors for the transmission of human immunode?ciency virus (HIV).3Infection by HIV is still a major worldwide concern. Despite availability of effective drugs, HIV becomes resistant to medications over time, thus allowing the virus to replicate. Moreover, co-infections with parasites and HIV are frequently occurring, making their treatment even morechallenging. Increasing drug resistance or sparse therapeutic options urge the need for developing new and ef?cient molecules to complete the therapeutic armamentarium. Acridine derivatives and heterocyclic compounds such as benzothiazoles have anti-parasite activity but most of them are toxic.4 The quinolone scaffold and its different substituted arylquinolinone derivatives have anticancer (Fig. 1A and B),5–9antimicrobial (Fig. 1C),10or neuroprotective (Fig. 1D)11properties. We have synthesized quinolinone with antiparasitic and anti-HIV activities by an original two-step method involving microwave irradiation(MWI). Then, we have evaluated biological activities of both intermediates and corresponding quinolinones against Plasmodium falciparum, Leishmania donovani, Trichomonas vaginalis, and HIV. Descriptions of quinolinone synthesis using microwave irradiation (MWI) in various media12are numerous. We prepared our tricyclic compounds by condensation of the appropriate aminoheterocycle with Meldrum’s acid derivatives (Scheme 1), followed by thermal cyclization of the resulting synthetic intermediate. The conventional heating methodology involves a 5 h re?ux step for the Meldrum acid intermediate’s 1a (Scheme 1), using p-nitroaniline as starting material.13We optimized the protocol on a milligram scale, and performed a successful MWI-scale-up to gram quantities with comparable yields. We have applied this method to various heterocycles (Table 1) successfully (compounds 1a–o) with good yields (39–92%). None of our compounds has been previously described using this method of synthesis. The second step was a thermal cyclization usually done in diphenyl ether at 240 °C for 20 min. Using MWI requires a solvent with a high dielectric constant to take advantage of MW heating. Diphenyl ether can rise without degradation to 200 °C in 30 min at full power MWI (300 W). Ionic liquids enhance MW heating,14thus we added bmimPF6 (50 mg) into diphenyl ether (2g). It allowed reaching 220 °C in 3 min with full power MWI (300 W) (Scheme 2). The cyclization of our compounds occurred by precipitating the product in a mixture of diethyl ether/acetonitrile and removing ionic liquid and diphenyl ether. Products obtained were bent or linear tricyclic quinolinones, depending on the starting heterocycle. Structures and isomeric ratio of bent and linear derivatives (Table 2) were determined without ambiguity by1H and13C NMR spectroscopy. There was an AB system for protons H4 and H5 (JAB= 9 Hz) for the bent structure. For the linear one, two singlets appeared, that corresponded to pro tons H4 and H9 of the structure. In our case, MWI was a clean, simple and faster protocol compared to the conventional heating method. We have obtained nine new quinolinone (2a–j) within 5 min. of reaction time. Human    therapies    already use    benzothiazole    derivatives although they have a potential toxicity due to the presence of a benzyl moiety in their structure. Our goal was to synthesize similar products with less toxicity, by suppressing an aromatic nucleus while maintaining the antiparasitic activity. This synthesis was already effective with quinolones.15 Most of our products (Table 3) were not cytotoxic (IC50THP1 >250 lM), and only ?ve had low to moderate cytotoxicity. Compounds 1a and 1d were even less toxic than their respective cyclized derivatives, compounds 2a and 2d. Although some had a structural analogy with chloroquine, 16 compounds had no antimalarial activity while the others had a moderate activity. Cyclization did not increase activity seeing that intermediate compounds had antimalarial activities similar to those of their respective cyclized derivatives. Cyclized compounds are usually more active than their corresponding intermediate structures. It was not the case with these series, moreover, intermediate compounds were less toxic than their cyclized derivatives.
Therefore, it is relevant to measure activity of both intermediate and cyclized compounds, rather than that of only the cyclized compounds. None of the products had a signi?cant antileishmanial activity on either promastigote or amastigote forms, from low to moderate concentrations. None had any antitrichomonal activity either. Compounds    1m and 1k were active on HIV-1, while compounds 1c, 2j, and 3a were active on HIV-2. Globally, non-cyclized compounds seemed more active than their respective cyclized derivatives on HIV. Within our products,    1k    was the only one combining activity against both malaria and HIV, having also a low cytotoxicity and the highest speci?city index (see ‘Supplementary data’ for detailed results on HIV strains).16,17 Although higher than that of chloroquine, IC50of compound 1k was close to that of doxycycline, another antimalarial drug of reference, both experimentally (6.5 lM) (Table 3) and as reported (8.5 lM).19Moreover, compound 1k was less toxic than doxycycline and had a better speci?city index on THP1 (Table 3).18,19 We are studying structures differing by a single moiety to explain the differences of activity between a compound and its cyclized derivative. A concomitant goal is to improve the structure of our most interesting drug candidate. All of our compounds are new chemical structures deriving from acridine and heterocyclic structures. Suppressing an aromatic nucleus from the acridine nucleus only reduced toxicity. Considering that this third aromatic nucleus is required for biological activity, we are trying to synthesize new products with a third benzyl nucleus that would not be adjacent to the quinolinone structure. We have validated an innovative method for chemical synthesis based on microwave irradiation and allowing the preparation of new chemical structures with potential antiparasitic activity.

Synthesis and HIV-1 integrase inhibitory activity of spiroundecane(ene) derivatives

Abstract
Fifteen 2, 4-dioxaspiro [5.5] undecane ketone and 2, 4-dioxa-spiro [5.5] undec-8-ene (spiroundecane(ene)) derivatives were synthesized using the Diels–Alder reaction. Inhibition of human immunode?ciency virus integrase (IN) was examined. Eight spiroundecane(ene) derivatives inhibited both 30-processing and strand transfer reactions catalyzed by IN. SAR studies showed that the undecane core with at least one furan moiety is preferred for IN inhibition. Moreover, crosslinking experiments showed that spiroundecane derivatives did not a?ect IN–DNA binding at concentrations that block IN catalytic activity, indicating spiroundecane derivatives inhibit preformed IN–DNA complex. The moderate toxicity of spiroundecane(ene) derivatives encourages the further design of therapeutically relevant analogues based on this novel chemotype of IN inhibitors.

Human immunode?ciency virus (HIV) enzymes are targets for antiretroviral therapy due to their requirement for the HIV life cycle. At the present time, only inhibitors of HIV reverse transcriptase and HIV protease are approved for AIDS therapy.1However, the third viral enzyme—integrase (IN) is also a promising target because of its non-homology to mammalian enzymes.2 In contrast, toxicity of compounds that inhibit HIV reverse transcriptase and protease is believed to be due to their homology to the host cell’s enzymes. Promising results of clinical trials for two new IN inhibitors—a derivative from quinolone antibiotics (JTK-303/GS-9137, Gilead Sciences, Inc.) and the compound MK-0518 from ‘Merck & Co’ were announced recently, providing the proof of concept for IN inhibitors as anti-retroviral therapy.3

The joining (integration) of the viral cDNA to host cellular DNAs is performed by IN whose catalytic site is characterized by the D,D-35-E motif.4The ?rst IN-catalyzed reaction, 30-processing (30-P), consists of the cleavage of the viral cDNA immediately 30from the conserved CA-sequences at the 30-ends of the HIV long terminal repeats (LTRs). 30-P occurs in the cytoplasm after viral reverse transcription. It is still unclear whether 30-P takes place before or after preintegration complex (PIC) formation and whether PIC formation requires the catalytic activity of IN. As almost all viral cDNA within the PICs consists of 30-processed ends and viral DNA is not protected from nucleases after isolation of PICs with mutant IN,530-P probably precedes and may be required for the formation of PICs. The second IN-catalyzed reaction, strand transfer (ST), consists of joining of viral cDNA to cellular DNA. ST is therefore contingent of the 30P and migration of the PIC into the nucleus.

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Recently the development of HIV integration inhibitors has focused on inhibitors of the ST reaction.3However, equal importance of 30-P for HIV integration as well as its possible involvement in PIC formation make 30-P a rational approach to inhibit HIV integration. It might also be logical to combine 30-P inhibitors with the currently developed ST inhibitors.

In our systematic search for novel IN inhibitors, we have identi?ed spirocyclic ketone derivatives (Scheme 1) as compounds that e?ectively block recombinant HIV IN. Spirocyclic ketones are employed as intermediates in the total stereoselective synthesis of natural products such as gymnodimine (marine toxin from oysters) and laxaphycins A (cytotoxic compounds from marine cyanobacterium).6

The synthesis of the above compounds 1–11 is outlined in Scheme 1. Diels–Alder reaction of 1 - (2-furyl) - 3-trimethylsiloxy-butadiene 127and 5-aryl (hetaryl) methylene-2, 2-dimethyl-1, 3-dioxane-4, 6-diones (13a–k) with a catalytic amount of L-proline in acetonitrile at ambient temperature proceeds in the regioselective fashion and furnished the corresponding spirodioxane triones (1–11) (yield 63–92%).8Compound 4 was also obtained by the three-component reaction of diene 12, 4-methoxy-benzaldehyde, and Meldrum’s acid 14 in the presence of L-proline in methanol solution (yield 56%).

The reaction of 1 - (2-furyl) - 2-ethoxycarbonyl-3-trimethylsiloxy-butadiene 159with methylene Meldrum’s acid (13l R1= H) (obtained in situ from Meldrum’s acid 14 and formaldehyde with L-proline in equivalent acetonitrile)    gave 7 - (furan-2-yl) - 9-hydroxy-3, 3-dimethyl-1, 5-dioxa-spiro [5,5] undec-8-ene-8-carboxylic acid ethyl esters 16.10Compounds 17–19, 10containing aryl substituents in the C-7 position, were obtained as follows. Cycloaddition reaction of 1 - (2-methoxyphenyl) - 3-trimethylsiloxy-butadiene 2011 with 5 - [1 - (3-hydroxy-4-methoxy-phenyl) - ethylidene] - 2, 2-dimethyl - [1,3] dioxane-4, 6-dione 13h leads to compound 17. By three-component reaction of 1 - (4-methoxyphenyl) - 2-ethoxycarbonyl-3-trimethylsiloxy-butadiene 219 with Meldrum’s acid and formaldehyde compound 18 was obtained. The reaction of 1 - (2-methoxyphenyl) - 2-ethoxycarbonyl-3-trimethylsiloxy-butadiene 2211 with Meldrum’s acid and formaldehyde yielded the dioxaspiro-undec-8-ene derivative 19.

All compounds were formed as single diastereomers. The stereochemistry of products was established by NMR analysis. Relative stereochemistry of cyclohexanone derivatives 1–11 and 17 was determined by analysis of the vicinal coupling constants for protons at C-7 and C-11. The syn-arrangement of aryl(hetaryl) and furyl substituents follows from the axial–axial coupling constants between 7-H and 8-H (J = 13.4–14.8 Hz) and 10-H and 11H (J = 13.3–15.0 Hz). The axial–axial coupling constants between 7-H and 8-H were also observed in the case of compound 18.

The structure of compound 1 was determined by single crystal X-ray di?raction analysis (Fig. 112). The bond lengths in the molecule are close to the statistical mean values. In the Cambridge structural database (University of Cambridge, UK. Version 5.26) we found only two compounds 6a, 13 in which the cyclohexane ring was spirofused to the 1, 3-dioxane ring. The most closest structural analogue was 3, 3-dimethyl-7 - (4-nitrophenyl) - 11-phenyl-2, 4-dioxaspiro [5,5] undecane-1, 5, 9-trione.6a

All compounds were tested against recombinant IN using a 21 bp substrate that allows determination of 30-P, as the release of the terminal dinucleotide, and ST, as the generation of DNA molecules larger than the starting substrate (Fig. 2 and Table 1).14Substitutions on the R1position of the spiroundecane core (Scheme 1, Table 1) highlight the importance of this position for IN inhibition. The most active spiroundecane ketone derivative (2) contains 3-indolyl moiety at R1, and is approximately twice more active than the symmetric 7,11-bis-furan-2yl substituted compound (1). Substitution to the dimethoxy-phenyl moiety (11) at R1failed to increase potency compared to the symmetrical compound (1). Comparison of four compounds with di?erent substitutions on the phenyl ring (11,9,4,3) demonstrates the importance of the ortho-methoxy substitution in compound 11 for 30-P inhibition. Additionally, halogen (5–7) or para-hydroxy (17 and 8) substitutions to R1tend to decrease inhibitory potency with exception for compound 4. We tested two spiroundecene derivatives (16 and 19) and found that the furan moiety is preferred for IN inhibition compared with methoxyphenyl for the spiroundecene core (compare 16 and 19). We also found that the spiroundecane core is more potent as a sca?old for IN inhibitors than the spiroundecene core (compare 18 and 19) (Table 1).

To characterize IN inhibition by spiroundecane(ene) derivatives, we compared the e?ect of the two most inhibitory spiroundecane (2 and 1) and of one spiroundecene (16) derivative on the three reactions catalyzed by IN (30-P, ST, and disintegration). As shown in Figure 2 these compounds show similar inhibition for 30-P (21bp duplex as a substrate, Fig. 2A, Table 1) and ST (precleaved substrate),    Figure 2B, IC50are 10.3 ± 5.0 lM (compound 2), 13.7 ± 4.4 lM (compound 1), and 35.7 ± 16.3 lM (compound 16). Therefore, spiroundecane(ene) derivatives are dual inhibitors of IN-mediated 30P and ST. Spiroundecane(ene) derivatives also inhibit disintegration, which corresponds to the reverse reaction of strand transfer,3with comparable potency as for 30-P or ST for undecane derivatives (Fig. 2C), IC50are 19.2 ± 1.2 lM (compound 2), 81.4 ± 10.8 lM (compound 1), and 213.0 ± 11.2 lM (compound 16).

To explore whether spiroundecane(ene) derivatives’ inhibitory properties were based on their ability to prevent DNA binding to IN, we investigated the e?ects of compound 1 on IN–DNA binding using two crosslinking strategies. First, we evaluated the ability of compound 1 to inhibit a crosslinking reaction between the cytosine in the 50-AC overhang of the viral DNA and glutamine 148 of IN (Fig. 3A). A Q148C mutant form of HIV-1 IN allows speci?c covalent interaction with a thiol-modi?ed cytosine in the 50-AC overhang.15Figure 3 shows minimal interference with this speci?c IN–DNA contact. Marginal inhibition was only observed at the highest concentration (333 lM).

To further determine whether compound 1 could interfere with the IN–DNA binding at other sites, we used the Schi?-base assay.16This assay measures crosslinking between IN and a DNA substrate mimicking the viral U5 LTR end containing a single abasic site. We examined the e?ects of compound 1 on IN crosslinking at three di?erent positions in DNA substrates (Fig. 3C). Compound    1 only marginally blocked the Schi?-base IN–DNA interactions (Fig. 3D), which is consistent with the results of the disul?de crosslinking assay. Together, the crosslinking results indicate that the spiroundecane derivative (compound 1) has no signi?cant interference with IN–DNA binding at concentrations that block IN catalytic activity.

None of the compounds that inhibit HIV integrase displayed cytoprotective activity for MT-2 cells infected by HIVIIIB, but all of them had moderate toxicity in this type of cells (CC50> 111 lM). Probably, low cell penetration ability leads to failure of antiviral activity. Therefore, the design of analogues of this novel chemotype will be necessary for antiviral activity. Such work is currently in progress and will be reported elsewhere.

In conclusion, we have synthesized and evaluated a series of original spiroundecane (ene) derivatives as HIV-1integrase inhibitors. Spiroundecane (ene) derivatives are dual inhibitors of both reactions (30-processing and strand transfer) catalyzed by IN. An undecane core with at least one furan moiety is preferred for IN inhibition. Structure–activity comparison provides evidence that the presence of an oxygen-containing substitution in the benzene is important for inhibition of IN. Crosslinking data suggest that spiroundecane derivatives interfere with the IN catalytic activity without signi?cantly a?ecting IN–DNA binding. The moderate toxicity of spiroundecane(ene) derivatives encourages the further design of therapeutically relevant analogues based on of this novel chemotype of IN inhibitors. General procedure for the synthesis of the 2, 4-dioxa-spiro [5,5] undecane-1,5,9-triones 1–11. A solution of 3-trimethylsiloxy-1,3-butadiene 12 (1.14 g, 5.5 mmol) in 5 ml acetonitrile was added under stirring to a suspension of dienophile (5 mmol) and 0.03 g L-proline in 50 ml acetonitrile. The mixture was stirred at rt for 25–40 h. Upon evaporation of solvent, a residue was treated with 10 ml of 3% NH4OH. The mixture was stirred for 10 min and then treated with 50 ml methylene chloride. The organic layer was separated, while the water one was extracted with methylene chloride. The collected extracts are washed (with water and brine) and dried (MgSO4). The solvent was evaporated and the residue was puri?ed by column chromatographies. The eluates, containing the products, are evaporated, and residues are recrystallized from the corresponding solvent.

Synthesis of trans-caffeate analogues and their bioactivities against HIV-1integrase and cancer cell lines

Abstract
Forty caffeate analogues were synthesized via a convenient method starting from vanillin with moderate to good yields. The testing of biological activity of these compounds against HIV-1 integrase indicates that four compounds: bornyl caffeate, bornyl 2-nitrocaffeate, 5-nitrocaffeic acid and 5-nitrocaffeic acid phenethyl ester (5-nitroCAPE) possess a good HIV integrase inhibitory activity, IC5019.9, 26.8, 25.0 and 13.5 lM, respectively. Twelve caffeate analogues were tested by MTT assay on growth of human hepatocellular carcinoma BEL-7404, human breast MCF-7 adenocarcinoma, human lung A549 adenocarcinoma and human gastric cancer BCG823 cell lines, respectively. And the best result is IC505.5 lM for CAPE against BEL-7404.

Some naturally occurring caffeates are widely distributed in plant kingdom. 1Most of them have bioactivities such as antibacterial, 2antiviral, 3anti-in?ammatory, 4antiatherosclerotic, 5anti-HIV, 6antitumor 7and so on. Epidemiological studies indicate that a diet, rich in fruits and vegetables reduces cancer risk in humans, suggesting that certain dietary constituents may be effective in preventing cancer.8Especially, caffeic acid phenethyl ester (CAPE) has been identi?ed as the major biologically active compounds.9 Encouraged by the aforementioned information and as a part of our new drug discovery efforts, it was valuable to synthesize caffeate analogues and study the structure–activity relationship (SAR) on caffeate analogues. Caffeate analogues have been synthesized previously by methods such as: acid-catalyzed esteri?cation, alkylation of caffeic acid with halohydrocarbons, esteri?cation via acyl chlorides, coupling reaction with DCC as coupling agent, transesteri?cation and Wittig reaction.10 Herein, we have developed a more convenient, one-pot method to prepare the caffeate analogues with moderate to good yields shown as Scheme 1. The synthesized forty compounds were summarized in Table 1. In addition, all caffeate analogues are trans (E) con?guration con?rmed by the1H NMR spectra in that the coupling constants of a-H and b-H on double bonds were 15.9–16.4 Hz.11Furthermore, it was con?rmed caffeate analogues are trans (E) con?guration by X-ray analysis of compound III-26 (Fig. 1).12 The anti-HIV integrase activities of caffeates were evaluated by the Biotin-Avidin ELISA method. The results were summarized in Table 1. From Table 1, it has been found that compound III-21, III-25, III-26 and III-40 possess a good HIV-1 integrase inhibitory activity. In part of esteri?able site, aryl ring or multi-ring compound seemed to be required because of all alkyl esters being inactive; replacement of 3-hydroxyl groups with methyl ether, such as III-25 to III-22, III-26 to III-23, and III-40 to III-37, resulted completely loss of potency whether adding a third group to the mother phenyl ring or not. So the presence of a catechol entity seems to be of importance.6a Adding a third strong election-withdrawing group NO2on 3, 4-dihydroxyl pattern resulted in potential activity, such as compound III-25, III-26 and III-40. These evidences would reveal that analogues with resonance electron-drawing group decrease election density to stabilize the corresponding conformers responsible for the higher activity. Unfortunately, although the lead compound CAPE (III-19) was tested twice by the same ELISA method, it showed no activity against HIV integrase, which was not consistent with the results of the literature.6aThese different results may be somewhat different from the Burke’s method, and it need more work to identify. The antitumor activities in vitro for these compounds were evaluated by the MTT method for BEL-7404, MCF-7, A549 and BCG823 cell lines. The results are summarized in Table 2. All compounds except for III-23 and III-33 possessed potent activity. III-19 and III-37 possessed stronger BEL-7404 activities than positive control cisplatin. On the MCF-7 examination, III-17 showed good anti-breast tumor potency. III-39 showed good anti-lung cancer potency. But on the BCG832 examination, activities of all twelve compounds were not better than that of positive control. Comparing with the anti-HIV-1 integrase activities, replacement of the OH group with CH3O group, such as    III-40 to III-37, the antitumor activities did not lost. Adding NO2group as a third group, such as III-19 to III-26 and III-39, III-21 to III-40, the antitumor activities did not increase much. 5-Br (III-23) or 5-NO2(III-33) phenethyl ferulate was completely inactive. Comparing with all anti-cancer data, the best result is IC505.5 lM for CAPE against BEL-7404. These results indicate that in our case there is no evidence for direct correlation between HIV-1 integrase and antitumor inhibition. In summary, 40 caffeate analogues synthesized by the simple one-pot method with moderate to good yields, were tested by Biotin-Avidin ELISA method for the activities against HIV-1 integrase. It has been found there are four compounds possessing good activities. In part of esteri?able site, aryl or multi-ring group seems important for the activity; in the mother phenyl part, the catechol entity was important, and the replacement of 3-OH by CH3O group will lost activity; the NO2 group was assign to the mother phenyl ring could increase the activity. Some of compounds were tested by MTT method for antitutor activities. Most of them possessed antitumor activities. The best result is IC505.5 lM for CAPE against BEL-740. Considering the structure–activity relationship, the ring substitutions and ester groups in the structure are probably important determinants for some potent biological activities which are worth to research further. General    procedure:Preparation of caffeic acid3,4 Dihydroxybenzaldehyde (9.0 g, 65 mmol), malonic acid (7.3 g, 70 mmol) were added to a mixture of toluene (15 ml), pyridine (100 mmol) and aniline (0.7 ml). The solution was stirred at re?uxing temperature 2 h. When the mixture was cooled to room temperature, yellow precipitation was ?ltrated, then washed with 50 ml 3 M HCl solution and 50ml water twice, respectively. The crude product was recrystallized from EtOH to give the light beige powderPreparation of Caffeic acid phenylethyl ester (CAPE)The Meldrum’s acid (3.6 g, 25 mmol) was added into toluene (50 ml), and then added b-phenethanol (3.05 g, 25 mmol). The mixture was heated and re?uxed for 4 h. When the mixture was cooled to room temperature, added 3,4-dihydroxybenzaldehyde (1.4 g, 10 mmol), pyridine (2.5 ml) and piperidine (0.25 ml). The stirring continued at room temperature 22 h, using TLC to trace the reaction until the reaction completely ?nished. The solvents were distilled out in vacuum; the residue was dissolved in diethyl ether (30 ml), washed with saturated solution of sodium bicarbonate 20 ml twice, then diluted hydrochloride 20 ml and distilled water 20 ml, respectively. The ether phase was dried by anhydrous MgSO4 overnight. After removal of the drier, the solvent was distilled out to get yellow crude solid. The crude solid was recrystallized from a mixture of benzene and diethyl ether (8:2) to afford pale white needle crystal.

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