2,3-Dihydroquinazolin-4(1H)-one as a privileged scaffold in drug design

2,3-Dihydroquinazolin-4-one (DHQ) belongs to the class of nitrogen-containing heterocyclic compounds representing a core structural component in various biologically active compounds. In the past decades, several methodologies have been developed for the synthesis of the DHQ framework, especially the 2-substituted derivatives. Unfortunately, multistep syntheses, harsh reaction conditions, and the use of toxic reagents and solvents have limited their full potential as a versatile fragment. Recently, use of green chemistry and alternative strategies are being explored to prepare diverse DHQ derivatives. This fragment is used as a synthon for the preparation of biologically active quinazolinones and as a functional substrate for the synthesis of modified DHQ derivatives exhibiting different biological properties. In this review, we provide a comprehensive assessment of the synthesis and biological evaluations of DHQ derivatives.


Introduction
Nitrogen-containing heterocyclic scaffolds are quite common fragments in drugs and biologically active compounds. 1,2 The 2,3-dihydroquinazolin-4(1H)-one (DHQ) is an important nitrogen-containing motif consisting of a phenyl ring condensed with a six-membered ring with two nitrogen atoms on positions 1 and 3, and a keto group on carbon 4 ( Fig. 1). Most of the DHQ derivatives are substituted on the carbon 2 chiral center. Due to their attractive properties, 2-substituted DHQs are becoming a prominent synthetic intermediate for organic chemists and various methodologies are reported in the literature for their preparation as racemic mixtures. Although some asymmetric strategies have been attempted, the aminal chiral center is sensitive to racemization, making it difficult to Mariateresa Badolato is currently pursuing her PhD degree in Translational Medicine under the supervision of Dr Francesca Aiello and Professor Antonio Garofalo at the University of Calabria, Italy. She received her Master's degree in Pharmaceutical Technology and Chemistry at the University of Calabria in 2013. She worked for one year as a visiting scholar in Professor Neamati's laboratory at the Department of Medicinal Chemistry, College of Pharmacy, University of Michigan. With the project "RYGoldZip" guided by Dr Francesca Aiello, as a member of the team she won the Clinic Center Life Sciences award. Among various projects, she designed and synthesized several ligands for TRPV1 receptors. Her PhD dissertation focuses on the preclinical and mechanistic studies of small-molecule drugs targeting STAT3 and other signaling pathways for the treatment of various cancers.
synthesize pure enantiomers. 3 The aim of the present review is to discuss reported methods for the synthesis of DHQ derivatives, highlighting their evolution towards alternative approaches and enantioselective strategies, and to summarize their use as synthons in organic chemistry and their important biological activities.

2,3-Dihydroquinazolin-4(1H)-one: a privileged scaffold
In 1988, Evans et al. introduced for the rst time the concept of "privileged structures". They are useful tools in the eld of drug discovery since they represent suitable lead compounds for diverse receptors and the rational optimization of such structures could provide new receptor modulators and potential drugs. 4 Medicinal chemists exploit the "privileged structures" to synthesize new libraries of compounds based on a central scaffold and screen them against various receptors implicated in different pathways, in some cases yielding biologically active compounds. In this regard, the DHQ core is emerging as a "privileged scaffold" and a variety of its derivatives, having diverse mechanism of action, are currently used for the treatment of various diseases. [5][6][7][8][9][10][11] A panel of marketed drugs with the DHQ core is shown in Fig. 2. Besides these marketed drugs, a number of new DHQ derivatives have been designed that exhibit a wide range of pharmacological properties. Because of their importance, the synthesis of substituted DHQ derivatives has attracted much attention and different synthetic strategies have been developed. Since the classical protocols involved the use of toxic reagents and solvents in harsh reaction conditions, the evolution towards simple, clean, environmentally benign and high-yielding methods is gaining momentum (Fig. 3).
Conventional procedure: cyclocondensation of anthranilamide and an aldehyde Although numerous strategies have been developed for the construction of the DHQ core, the most common and simple synthetic route for the preparation of DHQs is the direct cyclocondensation of anthranilamide and an aldehyde (Scheme 1). In the past decades, different catalysts and organic solvents have been used to speed up and improve the general yield of the reaction ( Table 1). Regardless of the used catalyst and/or solvent, the most presumed mechanism of cyclocondensation is shown in Scheme 2. 12 The rst step involves the nucleophilic attack of the nitrogen of the amino group of the anthranilamide on the carbonyl carbon of the aldehyde, promoted by the catalyst, resulting in the formation of hydroxyl intermediate I. Next, the catalyst promotes the formation of the Schiff base (II) from I through the removal of a water molecule. Finally, the imine undergoes intramolecular cyclization by nucleophilic attack of the nitrogen of the amide group on the imine carbon, to furnish the corresponding DHQ derivative.
A variation of the traditional cyclocondensation of anthranilamide and an aldehyde is represented by the intramolecular cyclization of a Schiff base. As shown in Scheme 3, the nucleophilic attack of the nitrogen of the amide group on the imine carbon leads to the cyclic DHQ derivative.

Basic catalysis
The base-catalyzed cyclocondensation of anthranilamide and an aldehyde was the rst proposed strategy for the synthesis of DHQ derivatives. In 1967, Yale et al. prepared a new class of DHQs in the presence of 20% aqueous NaOH in reuxing EtOH. These compounds were tested for their inhibition of cell proliferation. 13 Later, Bonola et al. used NaOH or NaOEt in absolute EtOH to prepare DHQs with antibacterial and antifungal activities. 14 Ericsson et al. patented DHQ derivatives as anti-fertility agents; these compounds were prepared from anthranilamide and 1naphthaldehyde in the presence of NaOH in reuxing absolute EtOH. 15 The NaOH-catalyzed reactions gave moderate to high yields (60-85%) in 1 h compared to NaOEt, which took 3-4 h to complete the formation of DHQs (Table 1). Depending on the available aldehyde, the use of a strong base as catalyst was not always successful. Because of this limitation, different catalysts were commonly used for the synthesis of DHQs starting from anthranilamide and an aldehyde.

Acid catalysis
As shown in Table 1, more frequently, the cyclocondensation of anthranilamide and an aldehyde is performed in acid catalysis. HCl in EtOH 14 or in association with concentrated HNO 3 (ref. 16) was used as catalyst under reux conditions. As reported, DHQ derivatives were oen synthesized in the presence of a catalytic amount of H 2 SO 4 (ref. 17) or by using catalysts bearing sulfonic acid functionality. Among them, p-toluenesulfonic acid was used in different solvents, including boiling chlorobenzene, 13 benzene, 18 N,N-dimethylacetamide 19,20 and EtOH. 21 2-(N-Morpholino)ethanesulfonic acid, commonly used as a buffering agent in biology, also stood out as a mild acid catalyst for the cyclocondensation of anthranilamide and an aldehyde. 22 The use of sulfanilic acid, 23 NaHSO 4 , 24,25 and SOCl 2 , 26 as a catalyst in EtOH has also been described. Moreover, formic acid 12 as catalyst and solvent, as well as malonic acid 27 in aqueous EtOH (50%), were used for the synthesis of DHQs. Furthermore, boric acid (H 3 BO 3 ), 28 and propylphosphonic anhydride, 29 usually employed in the Fisher indole synthesis and the Pictet-Spengler reaction, efficiently catalyzed the cyclocondensation of anthranilamide and aldehydes.

Iodine and metal salts as catalysts
Molecular iodine (I 2 ) emerged as a versatile, inexpensive and non-toxic catalyst, which serves as a Lewis acid. It is suitable in the cyclocondensation of anthranilamide and aldehydes, in ionic liquids, 30,31 or in EtOAc. 32 A synthesis in aqueous medium was also attempted. Due to poor solubility of I 2 in water, 1 mol% of I 2 as Lugol's solution (I 2 /KI) was used to prepare DHQs. 33 In the presence of I 2 as catalyst, DHQ derivatives were obtained with 66-95% yield, but the reactions required up to 15 h to complete.  Transition metal salts are excellent catalysts due to their kinetic stability, low toxicity and intrinsic metallic Lewis acidity. They were used in various organic transformations, including the cyclocondensation of anthranilamide and aldehydes. 34,40 In particular, the use of transition metal salts reduced reaction time from hours to minutes without decreasing the yield. Compared to conventional Lewis acids, metal triates are better catalysts in organic synthesis because of their chemical and physical properties, such as moisture and air-stability, recoverability, operational simplicity, and a strong tolerance to oxygen, nitrogen, phosphorus, and sulfur-containing reaction substrates and functional groups. 41 Among them, scandium, 42-44 ytterbium, 45,46 and yttrium 47 triates were extensively used in various conditions to catalyze the cyclocondensation of anthranilamide and aldehydes, leading to DHQs with high yields (78-99%) in 0.3-8 h.
As shown in Table 1, even ammonium salts stood out as good catalysts for the preparation of DHQ derivatives. [48][49][50][51] The reactions required short time and yields were up to 60%.

Intramolecular cyclization of a Schiff base
The synthesis of DHQs through intramolecular cyclization of a Schiff base was performed under different conditions, providing DHQs with 42-99% yield in 1-16 h ( Table 2). While strong basic conditions gave the nal product in long time, 52 the use of molecular nitrogen shortened the completion of the reaction. 53 A rapid synthesis was obtained using metal oxide nanoparticles, 54 and under catalyst-free conditions. 55,56 An atom-efficient method: one-pot three-component synthesis Although the cyclocondensation of anthranilamide and aldehydes is a facile and simple approach to obtain the DHQ core, more advantageous strategies to synthesize DHQ derivatives remain a desired goal in organic chemistry. The rst evolution towards a more convenient approach was moving from a twocomponent reaction (anthranilamide and an aldehyde) to a one-pot three-component reaction (isatoic anhydride, ammonium acetate and an aldehyde) (Scheme 4). Initially, Staiger et al. suggested this strategy about half a century ago, 57 and recently the advantages of multi-component reactions have been highlighted. Compared to conventional synthesis, the onepot three-component reaction represents an attractive and atom-efficient method to efficiently prepare the DHQ framework.
Multi-component reactions are characterized by (i) atom economy, incorporating all materials used in the process into the nal product; (ii) high levels of diversity achieved simply by varying the reaction components; (iii) time-efficiency, since products are formed in a single step, allowing a fast probe of a chemical hypothesis; and (iv) simple experimental procedures, ideally there is no need to isolate the intermediates. Obviously, the adoption of such a strategy minimizes both waste production and cost. 58 The plausible mechanism of the one-pot three-component synthesis of DHQs is shown in Scheme 5. Initially, the catalyst facilitates the nucleophilic attack of NH 4 OAc on the carbonyl carbon of the isatoic anhydride. Nucleophilic addition of ammonium leads to Scheme 2 Presumed mechanism of the cyclocondensation of anthranilamide and an aldehyde. intermediate I while the following decarboxylation produces anthranilamide (II). Then, the reaction proceeds similarly to the cyclocondensation. The catalyst promotes the nucleophilic attack of the amino group of II on the carbonyl carbon of the aldehyde, resulting in the formation of the Schiff base (III) by removing a water molecule. Finally, the imine intramolecularly cyclizes by nucleophilic attack of the nitrogen of the amide group on the imine carbon, to furnish the corresponding DHQ derivative. Examples of various catalysts and organic solvents used in the reaction of isatoic anhydride, NH 4 OAc and an aldehyde are listed in Table 3.

Conventional reaction conditions
In general, the one-pot three-component syntheses of DHQs were performed under acid-catalyzed conditions (Table 3). BrØnsted acid catalysts, such as ethylenediamine diacetate 59 and pentauorophenylammonium triate 60 were used under reux conditions, giving DHQ derivatives with 86-95% yield in about 10 h or less. Catalysts bearing sulfonic acid functionalities were also widely used in the multi-component reactions. [61][62][63][64][65][66] They signicantly reduced the reaction time, generating DHQs with 50-98% yield. Recently, the use of N-halo sulfonamides as catalysts, broadly reported in organic synthesis, has been attempted in the one-pot three-component reaction. 67 Metal-catalyzed multi-component synthesis of DHQ derivatives was also reported. [68][69][70][71][72][73] Generally, the use of metals or Lewis acids as catalysts did not lead to great yields (<90%), except when I 2 is used under solvent-free conditions at 115 C 69 (Table 3).

Recyclable catalysts
To improve the one-pot three-component reaction using green chemistry, the use of recyclable catalysts was introduced to further minimize waste production (Table 3). Some Lewis acid catalysts had the advantage of being easily recovered aer the reaction and recycled several times without considerable loss of reactivity. 63,64,66,71 b-Cyclodextrin (b-CD) is a cyclic oligosaccharide, composed of seven glucose units connected "head-to-tail" by 1,4-links. This cyclic heptamer has a truncated cone shape, with a hydrophobic cavity in the center. Thus, b-CD is used in numerous applications in drug formulation. It is well known that hydrophobic and van der Waals interactions are involved in the inclusion of complex formation between guest molecules and b-CD. 74 Due to its hydrophobic cavity, b-CD is widely used as a catalyst for a variety of organic reactions, providing a microenvironment whereby it catalyzes reactions through the formation of non-covalent interactions. 75 b-CD is a suitable catalyst in the one-pot three-component synthesis of various DHQs producing 78-92% yield in aqueous media. 76-78 DHQs were also obtained in less than half an hour in the presence of an inexpensive, safe and recyclable sulfonic acid-functionalized b-CD (b-CD-SO 3 H) as an efficient catalyst in green media. 79 Starch is a renewable, biodegradable, and relatively inexpensive material. A starch aqueous solution in EtOH was employed as safe, non-toxic and reusable catalyst for the preparation of DHQ derivatives with 73-94% yields. 80 Starch sulfate also reduced the reaction time to less than 1 hour. 81 Furthermore, alum (KAl(SO 4 ) 2 $12H 2 O), 82 citric acid, 83 and thiamine hydrochloride (VB 1 ), 84 catalyzed the one-pot threecomponent reaction in aqueous media. The protocols were longer (up to 7 h) but reported to be environmentally safe.
Amberlyst-15 is a cationic exchange resin. These types of resins, especially the macroporous ones, are recyclable catalysts for various organic syntheses, including the preparation of DHQs. Amberlyst-15 presents several advantages over conventional catalyst with respect to corrosion, product recovery, and selectivity. 85

Heterogeneous and reusable catalysts
Recently, the development of heterogeneous organic reactions has been gaining popularity. They are characterized by ease of handling, separation, recycling, and environmentally safe disposal. 86 In the last decade, the eld of nanoscience and nanotechnology has had tremendous growth. Nanoparticles (NPs) are dened as materials having 1-50 nm diameter, a size range where metals can show size-dependent properties. Because of their interesting structures and high catalytic activities, due to the wide surface/volume ratio that provides many active sites per unit area, NPs and particularly magnetic particles have emerged as useful heterogeneous catalysts in terms of selectivity, reactivity, and improved yields of products. In addition, the magnetic properties of NPs make complete recovery of the catalyst possible by means of an external magnetic eld. 87 As reported in Table 3, some metal and metal oxide NPs, exhibiting high surface/volume ratio, quantum size and quantum tunnel effects, efficiently catalyzed the one-pot three-component synthesis of DHQs, in 0.5-6 h. 88-91 Nanosized aluminum nitride, a non-toxic, low-cost and highly pure powder, was used as a solid source of ammonia in the multicomponent synthesis of DHQs. 92 Hydroxyapatite (HAP) NPs show ion-exchange ability, adsorption capacity, and acid-base properties. Because of their higher surface areas and lower particle size, HAP NPs provide greater catalytic activity in the synthesis of DHQs. 93 Although NPs have advantages such as simplied isolation of the product, easy recyclability and recovery of the catalyst, the naked NPs could aggregate into large clusters, limiting their use. This problem could be solved by immobilizing the NPs on mesoporous substrates characterized by large surface area, high chemical and thermal stability and good compatibility, such as silica, polymers and carbon. 94 The integration of mesoporous silica with magnetic NPs is certainly of great interest for practical applications. As shown in Table 3, different silica-supported NPs were used as catalysts for the one-pot three-component reaction, leading to 45-97% yield in 1-8 h. [95][96][97][98][99] The use of solid acids, as non-toxic, low-cost and reusable catalysts, has also emerged in the synthesis of DHQs (Table 3). Among them, several acids bearing sulfonic moiety supported on silica catalyzed the multi-component synthesis under different conditions, providing DHQs with >70% yield in 0.5-7 h. 100-104 MCM-41 is another type of ordered mesoporous silica material. Various acid-functionalized MCM-41, 105-107 cellulose, 108 and alumina, 109,110 were used as solid recyclable acidic catalysts for the one-pot three-component synthesis of DHQs, under solvent-free conditions, affording 70-98% yield in less than 1 hour. On the other hand, solid acids used in reuxing EtOH lengthened the reaction time to 5-7 hours. 111,112 Montmorillonite K-10 is one of the most important smectites, a phyllosilicate mineral species, used as a catalyst in organic synthesis. It is a clay with both BrØnsted and Lewis acid sites, with a high cation-exchange capacity. Montmorillonite K-10 is considered a solid acid that acts as heterogeneous catalyst for diverse syntheses, including the one-pot three-component cyclocondensation, and can be easily removed from the reaction mixture. 113,114 Other interesting solid supports used in heterogeneous catalysis are carbon nanotubes (CNTs), due to the porosity, inertness, and low interactions between catalyst and support and good mechanical strength. 115 The deposition of metal NPs on the external surface of CNTs and multi-walled CNTs (MWCNTs) are attractive for catalysis, since they increase the reactive surface area. As reported in Table 3, CNTs supporting transition metals efficiently catalyzed the one-pot threecomponent synthesis of DHQs, obtained with 76-99% yield in less than half an hour. [116][117][118][119] About the same yield was obtained using acid-surfactant-combined catalysts. 120,121 A strong acidic cation-exchange resin (732-resin), 122 and a 4Å molecular sieve modied with lanthanum(III) (La 3+ /4Å), 123 were employed as heterogeneous and recyclable catalysts for the multi-component synthesis of DHQs.

Greener and convenient approaches to obtain DHQ derivatives
In the new century, there has been an increasing demand for the development of sustainable chemistry. In 1998, Anastas and Warner published the "Twelve Principles of Green Chemistry", 124 whose main purpose was the pollution prevention. In this regard, green chemistry was committed to (i) decrease pollution-generating chemicals; (ii) limit the use of dangerous chemicals and exhaustible feedstock materials and scarce resources; and (iii) reduce the harmful effects of nal products. [125][126][127] The development of cleaner and safer chemical processes started with the use of heterogeneous and recyclable catalysts or alternative solvents, which are not volatile, ammable or toxic. Then, it moved to performing reactions under catalyst-and/or solvent-free conditions. Finally, it incorporated the replacement of conventional thermal equipment by nonconventional sources, such as microwave or ultrasound irradiations.
Enzymes have received great attention as sustainable and biodegradable catalysts for the synthesis of biologically active compounds. Among them, a-chymotrypsin rapidly catalyzes the cyclocondensation of anthranilamide and aldehydes with 90-98% yield. 153 Transition metal-based heterogeneous systems were efficiently used as recyclable catalysts for the cyclocondensation of anthranilamide and aldehydes, yielding > 70% DHQs. [154][155][156][157][158][159][160][161] Alternative solvents The choice of the solvent for a desired chemical process can have profound economic and environmental consequences. For this reason, there has been signicant interest in using alternative "clean" solvents, mostly aqueous media and ionic liquids. Water is readily available, cheap, non-toxic, non-ammable and is very attractive from both an economical and environmental point of view. 162 The use of ionic liquids as reaction media and catalysts also gives a solution to solvent emission and catalyst-recycling problems. Ionic liquids present many important features, such as negligible vapor pressure, non-inammability, immiscibility with non-polar solvents, reasonable thermal and chemical stability and recyclability. 163 Various catalysts were used in aqueous media to synthesize DHQ derivatives, both from the cyclocondensation of anthranilamide and aldehydes, and the one-pot three-component reaction. A hydrotropic solution 164 and a deep eutectic solvent 165 were used under catalyst-free conditions (Table 5). Ionic liquids were also widely employed for the synthesis of DHQ derivatives, in different reaction conditions (Table 6). Imidazolium-based ionic liquids 30,31,46,[166][167][168][169][170][171] afforded DHQ derivatives with 70-99% yield, in 0.5-10 h unless used under microwave irradiation (MWI). 172 The triazolium-based reactions take less than half an hour to complete the one-pot threecomponent reaction. 173 Ionic liquids bearing sulfonic acid functionality were used under solvent-free conditions and rapidly catalyzed the synthesis of DHQs. 174,175 Although basic ionic liquids 171-178 and a glycerol based ionic liquid with a boron core 179 were also used under solvent-free conditions, they catalyzed the reaction in a longer time (10-90 min).

Catalyst-and solvent-free reactions
The challenge for a sustainable environment requires the development of greener and cleaner chemical processes that can avoid the use of harmful solvents and catalysts. In this sense, new strategies have been developed to synthesize DHQ derivatives in solvent-and/or catalyst-free conditions (  New energy sources: microwave and ultrasound irradiations MWI in organic synthesis is commonly used because it facilitates heat transfer better in chemical reactions. The efficiency of MWI heating results in a dramatic reduction in reaction times to minutes as compared to conventional heating methods taking several hours. From an economic and environmental viewpoint, the use of MWI provides unique chemical processes, characterized by enhanced reaction rates, sometimes higher yields, greater selectivity, and ease of manipulation. 201,202 Previously, efforts were made to synthesize DHQs under MWI (Table 8). Among them, the cyclocondensation of anthranilamide, under acidic catalysis, 203 as well as the one-pot threecomponent reaction, in the presence of L-proline in water. 204 The ultrasound irradiation (USI)-assisted reactions have also become increasingly popular in organic synthesis. Due to faster reactions, MWI and USI allow the elimination or minimization of side products formation. They are frequently used in the pharmaceutical industry and may pave the way towards a greener and more sustainable approach to chemical synthesis. 205 A variety of organic reactions were carried out  (Table 8).

Alternative synthetic strategies
Although the cyclocondensation of anthranilamide and the one-pot three-component reaction of isatoic anhydrides, ammonium acetates and aldehydes are the main ways to synthesize DHQ derivatives, other synthetic strategies have also been developed ( Table 9).

Cyclocondensation of anthranilamide and different substrates
Anthranilamide was the most common starting material for the preparation of DHQs. Other than aldehydes, other substrates were used for the cyclocondensation with anthranilamide in different conditions to give DHQ derivatives (Scheme 6). First, the cyclocondensation of anthranilamide and oxocompounds, such as benzil, 207 2-oxo(alkyl)acetates, 208 and 4 0bromoacetophenone, 209 were attempted to give DHQs in different conditions. The obtained 2,2-disubstituted DHQ was then cleaved in ethanolic/methanolic hydroxide to give the respective derivative. Alcohols were used in a rutheniumcatalyzed cyclocondensation with anthranilamide. 210 Cyclocondensations of anthranilamide and gem-dibromomethylarenes, as aldehyde equivalents, were performed in the presence of potassium tert-butoxide (t-BuOK), in anhydrous pyridine and N,N-dimethylformamide (DMF). 211 Anthranilamide also reacts with dicyanoepoxide to give DHQs in reuxing CH 3 CN. 212   The direct hydroamination/hydroarylation and double hydroamination of alkynes, followed by the cyclocondensation with anthranilamide was also exploited for the synthesis of DHQ derivatives. 213,214 These alternative strategies afforded DHQs with 45-97% yield in 1-24 h (Table 9).

Other strategies
Other strategies have been developed to synthesize DHQ derivatives starting from various substrates (Scheme 10) giving 39-94% yield in 0.5-48 h (Table 9). One such method employed 2-aminobenzoic acid as a starting material to prepare DHQ derivatives. The condensation of the 2-aminobenzoic acid with amines in the presence of SiCl 4 led to the corresponding substituted anthranilamides, subsequently reduced with sodium bis(2-methoxyethoxy)-aluminum hydride to o-aminobenzylamines. Then, the latter compounds were cyclized into DHQs by means of ethylchloroformate/pyridine. 230 Another strategy converted the 2-aminobenzoic acid in isatoic anhydride by means of triphosgene in dry THF, then converted to anthranilamide using 28% NH 4 OH solution. The cyclocondensation with the aldehyde in the presence of p-toluenesulfonic acid, in reuxing MeOH, gave the respective DHQ. 231 The copper-catalyzed cyclocondensation of 2-halobenzamide and aldehyde in the presence of aqueous ammonia, 232 and 2halobenzamide with anilines, 233 also gave DHQs derivatives. DHQs could also be prepared by reduction from 4(3H)-quinazolinones (QZs) with NaBH 4 , 234,235 or NaBH 4 CN. 236 DHQs were also obtained by reductive desulfurization of 2-thioxo-4(3H)quinazolinones with nickel boride, using nickel(II) chloride (NiCl 2 ) and NaBH 4 . 237 The intramolecular cyclization of 2phenyl-ethyl anthranilate in the presence of NH 4 OAc led to the respective DHQs. 238 The oxidation of benzylamines to the corresponding N-benzylbenzaldimines was also investigated and used for the synthesis of DHQ derivatives. 239

Enantioselective synthesis of DHQ derivatives
Most of the reported methods allow the synthesis of DHQ derivatives as racemic mixtures. In certain examples, the (S)enantiomer of DHQs had better antiproliferative activity, compared to the (R)-enantiomer, even though the racemic mixture showed a similar potency to the pure S-enantiomer. 3 Chinigo et al. rst proved that a (S)-enantiomer of DHQ binds to tubulin, showing antiproliferative activity in different cancer cell lines. They obtained the pure (S)-enantiomer using tri-uoracetic acid in CH 3 CN with 34-86% yield and 79-91% enantiomeric excess percentage (ee%) in 1.5 h. 3 Although the enantioselective synthesis of DHQs is difficult due to an unstable aminal stereo-center that is sensitive to racemization, some asymmetric strategies have been developed to obtain pure (S)-enantiomers of DHQs (Table 10). Mainly, they consist of the cyclocondensation of anthranilamide and aldehydes in the presence of chiral catalysts that promote the formation of pure enantiomers, or the intramolecular amidation of N-Boc imines and anthranilamide (Scheme 11). The use of chiral phosphoric acidic catalysts is common in the enantioselective synthesis of DHQs, although they need a longer time (15-48 h) to complete the reaction. Various chiral phosphoric acids were employed to catalyze the cyclocondensation of anthranilamide and aldehydes, 240-243 affording 67-99% yield and 26-99 ee%. An amidation of N-Boc imines and anthranilamide, 244,245 gave pure (S)enantiomer in 10-96 ee%. Scandium(III)-catalytic systems were also effectively employed for the enantioselective cyclocondensation of anthranilamide and an aldehyde (Table  10). 246,247

DHQ as an intermediate in organic chemistry
In addition to their many signicant pharmacological activities, DHQs also play a central role as intermediates in organic synthesis. In particular, they can be easily oxidized to the biologically active QZs. The quinazolinone ring (Fig. 4) is frequently encountered in organic chemistry as well as in medicinal chemistry. [248][249][250][251][252][253][254] The QZ core is present in the structure of numerous natural products, especially alkaloids, 255,256 and in some drugs, 257 exhibiting various pharmacological properties. QZs also represent a privileged scaffold and many protocols are reported in literature for the synthesis of this important synthon. Among them, the dehydrogenation of DHQ derivatives (Scheme 12) has emerged as an easy and fast strategy to prepare QZs under different oxidant conditions (Table 11). Initially, QZs were obtained by dehydrogenation of DHQs by means of ZnCl 2 in the presence of air, with 42% yield and in 10 h. 202 A catalystfree reaction in an open ask in reuxing EtOH was completed in few hours. 258 The oxidation of DHQs by the addition of 2,3dichloro-5,6-dicyano-1,4-benzoquinone also gave the corresponding oxidized derivatives with 83% yield. 231 Various oxidizing agents were used for the dehydrogenation of DHQs to efficiently generate QZs, with 26-92% yield. [259][260][261][262] Metalcatalyzed dehydrogenation was also attempted, affording the oxidized derivatives in moderate to good yields (18-85%) but in 16-24 h. 263-265 Biocatalysis using laccase/N-hydroxybenzotriazole gave QZs in 62-87% yield. 266

DHQ as a versatile fragment in drug design
The purpose of drug discovery has always been the design and development of "magic bullets" targeting a single key biomolecule in a central pathway of a specic disease. This led to the dominant paradigm "one target, one drug", which might be inadequate to achieve a therapeutic effect for complex diseases. 267,268 For this reason, the polypharmacology research and the design of multitarget compounds is considerably emerging, 269,270 contributing to overcome some of the limitations of classical approach, in term of risks and costs. 271 DHQ is a versatile fragment that can be easily functionalized at different positions. The introduction of specic moieties in the DHQ nucleus leads to the ability to interact with multiple targets, ensuring diverse pharmacological properties that make it a privileged scaffold (Fig. 5).

2-Aryl DHQ derivatives
The anticancer activity of DHQ derivatives was one of the rst to be discovered. In 1967, Yale et al. identied DHQs as a new class of inhibitors of cell proliferation of the Earle's L cells. In particular, 2-aryl derivatives showed signicant in vitro activity with low median effective dose (ED 50 ) values of 0.1-6 mg ml À1 . 13 The antitumor activity of various 2-aryl DHQs against different cancer cell lines was then conrmed by several laboratories. 26,36,38,109,272 Although many efforts to explain their cytotoxicity have been made, the target of the DHQ framework remains unknown. Almost three decades later, Hamel et al. used COMPARE algorithm to suggest that the antitumor effect of 2aryl DHQs resulted from interactions with tubulin. Some derivatives inhibited the polymerization of tubulin at low micromolar concentrations and the binding of radiolabeled colchicine to tubulin at higher doses. 273 Furthermore, based on the crystal structure of a,b-tubulin in complex with colchicine, through computational docking experiments and molecular dynamics, it was rationalized that (S)-DHQs may bind to tubulin better than the (R)-enantiomers, showing a better antitumor activity. 3 In the same study, the accumulation of DHQs in the cytoplasm of MDA-MB-435 cells was observed through the inherent uorescent properties of DHQs. 3 In order to improve the antitumor effect of DHQs with antimitotic properties, a tumor-targeting liposomal delivery system that incorporates an anti-transferrin receptor single-chain antibody fragment was used, showing preferential targeting of tumor cells. 274 2-Quinolin-5-yl DHQ induced cytochrome c mediated apoptosis and autophagy in human leukemia MOLT-4 cells as demonstrated by ow cytometry, microscopy, LC3 immunouorescence, and western blot analysis. 275 Other pathways have been suggested to clarify the antitumor activity of DHQ derivatives. DHQs bearing a phenyl substituent and piperidine/piperazine moiety on C5 and C7 respectively were identied as selective inhibitors of p38 MAP kinase. They efficiently repressed the production of TNF-a in monocyte, THP-1 cells and LPSstimulated whole blood (IC 50 values in the nanomolar range). These analogs had good clearance but low oral bioavailability in rats. However, the introduction of a bulky t-butyl substituent on the piperidine nitrogen signicantly increased the oral exposure in rats. 276 Other DHQ derivatives were discovered by a highthroughput screening as inhibitors of the Hedgehog pathway, involved in embryonic development and oncogenesis. The biochemical mechanism of action of these DHQs was the inhibition of the AAA+ ATPase motor cytoplasmic dynein that converts chemical energy into mechanical force and regulates many cellular processes, including ciliary trafficking, formation of mitotic spindle and organelle transport. These AAA+ ATPase inhibitors could be useful to study cellular processes that require microtubule motor. 277 280 N-indolylmethyl substituted spiroindoline-3,2 0 -DHQ were hypothesized as potential sirtuin inhibitors. Sirtuins, whose family consists of seven members (SIRT1-7), are important targets for cancer therapy, being upregulated in several types of cancer. In particular, SIRT1 has several substrates, including p53 and NF-kB, and its inhibition leads to the re-expression of silenced tumor suppressor genes and the subsequent decrease of cancer cell growth. These new DHQ derivatives were obtained through Pd/C-Cu-mediated coupling cyclization and tested in vitro using a yeast homologue of mammalian SIRT1, Sir 2 protein, showing dosedependent inhibition. Molecular docking analysis showed that the benzene ring of the DHQ occupied the deep hydrophobic pocket of the protein, while the NH and the sulfonyl groups form H-bonding interaction with select amino acid residues (Val412 and Gly415). 281 Over half a century ago, DHQ derivatives bearing a sulfonamide moiety on C7 showed diuretic activity, causing natriuresis and chloruresis and a slight increase of potassium excretion aer oral administration. 282 Among them, fenquizone is a FDA-approved drug for the treatment of edema and hypertension. [283][284][285][286] The substitution of C8 with N in the benzene ring of DHQ and the introduction of    289 Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, is a worldwide leading cause of death. Multiple antibiotics are needed to treat tuberculosis over a long period of time, but the development of multiple drug-resistant tuberculosis limits complete recovery. Some spiro-DHQ derivatives were tested for their in vitro inhibitory activity against Mycobacterium tuberculosis H37Rv chorismite mutase, a promising target for the identication of new antitubercular drugs. These derivatives showed a moderate inhibition at relatively high doses (30 mM). 290 2-Aryl DHQs also show moderate to good antifungal activities, in particular against Candida albicans and Aspergillus niger. 43 A class of 2-thioxo DHQ derivatives showed an excellent antimicrobial activity by inhibiting myeloperoxidase. This enzyme plays an important role in host defense and contributes to inammation. They reversibly inhibited myeloperoxidase, exhibiting IC 50 values in the nanomolar range. 291 4-Alkynyl-3,4dihydro-2(1H)-quinazolinones, bearing a second substituent at position 4, such as cycloalkyl, aryl and triuoromethyl were identied as potent HIV-1 non-nucleoside reverse transcriptase inhibitors, inhibiting wild-type and various mutant forms of HIV-1. These compounds also showed a good oral bioavailability. 292-294 2-Aryl DHQ derivatives, with or without substituents on N1 and N3, were found to interact with human serum albumin (HSA), using uorescence spectroscopy. The therapeutic effects of drugs depend on their absorption, distribution, metabolism and excretion, and can be inuenced by the binding affinities of drugs with HSA. In particular, strong binding can reduce free drug concentrations in plasma whereas weak binding can decrease lifetime and/or distribution of drugs. Results of site marker competitive experiments revealed that DHQs spontaneously bind to HSA on site II, subdomain IIIA, though hydrophobic forces. Various substituents in the benzene ring of DHQ could increase the interactions with HSA, forming additional van der Waals forces and H-bonds. [295][296][297] Transition metals, such as iron, zinc and copper, play important roles in the human body. Specically, copper is a catalytic cofactor for a variety of metalloenzymes and physiological processes. Increased levels of copper in the body could be involved in the production of reactive oxygen species (ROS), causing imbalance in cellular functions and several diseases. For this reason, the development of uorescent chemosensors for biologically active transition metal ions are becoming attractive. In this regard, 2-aryl DHQs were efficiently used as uorescent probes to selectively detect Cu 2+ ions. 298 A series of 2-disubstituted DHQ derivatives showed inhibitory activity against cholinesterases (ChE), involved in the lysis of choline-based esters that act as neurotransmitters. Acetylcholinesterase is present in chemical synapses and in red blood cell membranes, and butyrylcholinesterase is found in blood plasma. ChE inhibitors are the only effective therapeutic approach for the symptomatic treatment of Alzheimer's disease. These derivatives inhibited both enzymes (IC 50 values in micromolar range), better than or comparable to the standard drug galantamine. Molecular docking studies revealed that the benzene ring of DHQs can ts into the choline-binding site while the phenyl ring at position 2 is oriented towards the peripheral anionic site. The NH group of DHQs and Asp72 form an ion-dipole interaction, while substituents in the benzene ring are involved in H-bonding interactions. 16 Mono-and di-substituted DHQ derivatives As a privileged scaffold, the DHQ nucleus is amenable to modication. The most common functionalization was the single introduction of different chemical groups on heterocyclic N1 and mainly on N3, and substitutions on both positions. The resulting mono-and di-substituted derivatives showed specic activities depending on the substituents (Table 12).

Conclusions
The importance of the DHQ nucleus has emerged due to its versatility as suitable substrate for functionalization and its remarkable bioactivities. Many procedures have been reported for the synthesis of DHQ derivatives as racemic mixture. Although the cyclocondensation of anthranilamide and an aldehyde and the one-pot three-component reaction of isatoic anhydride, ammonium acetate and an aldehyde represent the most common ways to obtain DHQ derivatives, several other methods have been suggested. Many approaches have been investigated, from classical to greener and more sustainable reaction conditions. Recently, enantioselective strategies have been attempted in order to obtain pure (S)-enantiomers. Furthermore, the DHQ scaffold is an important intermediate in organic chemistry and can easily be oxidized into QZ scaffold. On the other hand, various important bioactivities have been associated to the DHQ scaffold and reported in the literature. On this basis, the DHQ nucleus imposes itself as a privileged scaffold in drug design and an interesting fragment for drug discovery.

Conflicts of interest
There are no conicts to declare.