Allosteric GABAA Receptor Modulators—A Review on the Most Recent Heterocyclic Chemotypes and † Their Synthetic Accessibility

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Review Allosteric GABAA Receptor Modulators—A Review on the Most Recent Heterocyclic Chemotypes and † Their Synthetic Accessibility

1, 1, 2 Blanca Angelica Vega Alanis ‡ , Maria Teresa Iorio ‡, Luca L. Silva , Konstantina Bampali 3 , Margot Ernst 3,* , Michael Schnürch 1,* and Marko D. Mihovilovic 1

1 Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9/193, 1060 Vienna, Austria; [email protected] (B.A.V.A.); [email protected] (M.T.I.); [email protected] (M.D.M.) 2 Department of Anesthesiology and Intensive Care Medicine, Charité–Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany; [email protected] 3 Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Spitalgasse 4, 1090 Vienna, Austria; [email protected] * Correspondence: [email protected] (M.E.); [email protected] (M.S.) Dedicated to Prof. Fritz Sauter on the occasion of his 90th birthday. † These authors have contributed equally to this manuscript. ‡ Academic Editors: Antimo Gioiello and Bruno Cerra Received: 31 January 2020; Accepted: 19 February 2020; Published: 24 February 2020

Abstract: GABAA receptor modulators are structurally almost as diverse as their target protein. A plethora of heterocyclic scaﬀolds has been described as modulating this extremely important receptor family. Some made it into clinical trials and, even on the market, some were dismissed. This review focuses on the synthetic accessibility and potential for library synthesis of GABAA receptor modulators containing at least one heterocyclic scaﬀold, which were disclosed within the last 10 years.

Keywords: GABAA modulators; allosteric modulators; heterocyclic modulators; heterocyclic synthesis; nitrogen heterocycles

1. Introduction Heterocycles are an extremely diverse compound class since any cyclic system in which at least one carbon is exchanged for any other atom belongs to it. Consequently, they occur abundantly in nature, where they exhibit important functions. For example, the purine and pyrimidine bases in DNA, are heterocyclic, or the amino acids proline, histidine, and tryptophan contain heterocyclic motifs. Naturally, many heterocyclic scaffolds can be found in biologically active natural products such as coniine, atropine, tetrodotoxin, etc. This list could be extended ad infinitum. Hence, it is not surprising that drug development also makes excessive use of heterocyclic scaffolds. Their use enabled us to explore and expand the drug-like chemical space. Some privileged structures contain heterocyclic moieties, which are most frequently nitrogen, oxygen, and sulfur. Heterocycles are useful structures to optimize ADME/toxicity features of drug candidates, such as solubility, lipophilicity, or their hydrogen bond donor/acceptor properties. The presence of heterocyclic moieties in recently developed drugs has increased due to advances in synthetic chemistry such as the development of cross-coupling or hetero-coupling reactions, which allow synthetic accessibility to molecules containing functionalized heterocycles. Additionally, some heterocyclic drug compounds have been developed to be inspired by natural product isolates [1].

Molecules 2020, 25, 999; doi:10.3390/molecules25040999 www.mdpi.com/journal/molecules Molecules 2020, 25, 999 2 of 47

Even though many small synthetic organic molecules with high medicinal potential contain heterocyclic rings, the range of easily accessible and suitably functionalized heterocyclic building blocks is surprisingly limited, and the construction of even a small array of relevant heterocyclic compounds is far from trivial. Heterocyclic chemistry, therefore, continues to attract the attention of medicinal and synthetic chemists. The development of novel methodologies allowing for efficient access to heterocycles is still highly beneficial [2]. In particular, synthesis of heterocyclic scaffolds has to implement novel reaction technologies for expanding the chemical space of biologically-active molecules such as biocatalytic methods [3], electrochemical methods, flow chemistry [4], and photochemistry [5] to expedite medicinal chemistry programs [6,7]. Within this review, heterocyclic compounds acting as GABAA receptor modulators will be revised. First, an introduction to this specific receptor family and the biological responses related to modulation will be provided. Subsequently, synthetic methods, which have been disclosed in the last 10 years to access various heterocyclic GABAA receptor modulators, will be highlighted. The selection criteria for the examples of compounds included was mainly based on synthetic originality and versatility, and not on any specific pharmacological property. Scaffolds are arranged according to their heterocyclic motif rather than according to their activity profile or binding site.

1.1. The Receptor Family

Among the mammalian ligand-gated ion channels, the GABAA receptor family comprises the largest family with subunits encoded by 19 different genes. Some of these undergo alternative splicing, and, thereby, increase the variety [8,9]. Their endogenous ligand known as the gamma-aminobutyric acid (GABA) has been established as the main inhibitory neurotransmitter in the central nervous system [10]. These chloride channels are homopentamers or heteropentamers, whereby the subunits share a 30–80% sequence identity [11]. It is generally accepted (though not proven) and suggested by the bulk of experimental evidence that the majority of GABAA receptors in the adult brain are pentamers with the subunit composition two α1, two β2, or β3 and one γ2 subunits, i.e., (α1)2(β2 or β3)2(γ2)1 [9]. The subunit arrangement of this type of receptor is depicted in Figure1. Each subunit is defined by its position in the pentamer and by its interfaces perceived from the extracellular side, going into counter-clockwise directions. A principal (or plus, +) face is in contact with the next complementary (or minus, ) face, i.e., the canonical orthosteric β+/α interface. − − For heteromeric receptors of different compositions (such as αβ or αβδ combinations), the arrangements are debated controversially and have not yet been defined conclusively [12,13]. The number of pentameric assemblies that exist has also not been determined [9]. Figure1 depicts a generic subunit interface, which features sites known or proposed at and near a subunit interface. Regions 1 and 1a form the extracellular domain (ECD) interface site which harbors, e.g., GABA or benzodiazepine sites (see panel b). Region 2 is a cation site while region 3 corresponds with the etomidate or barbiturate sites in the upper transmembrane domain (TMD) (see panel c), while region 4 at the lower TMD is the site where positive modulatory steroids have been observed [14]. Sites more remote to the interface (intrasubunit-sites and lipid-associated sites) for which ligands are confirmed include site 9 [14] for negative modulatory steroids, and tentative sites in positions 5, 6, 7, and 8 [15]. One α subunit shows the remaining sites with the numbers to illustrate their approximate localization from this perspective. For the major receptor subtypes that have been observed in the mammalian central nervous system, subtype-selective targeting to address specific circuitry and physiology selectively has been attempted and is still an ongoing major direction of ligand development [16]. Knowledge of binding sites is crucial for subtype-selective targeting so that pockets that feature sequence differences can be taken into consideration as discussed for some relevant examples below. Molecules 2020, 25, 999 3 of 47 Molecules 2019, 24, x FOR PEER REVIEW 3 of 48

Figure 1. Overview of pentamer structure and localization of orthosteric and allosteric binding sites. (a):Figure Generic 1. Overview subunit interface,of pentamer featuring structure ECD, and localization TMD, and of lower orthosteric TMD. and (b) Viewallosteric of the binding ECD. sites. GABA sites,(a): theGeneric benzodiazepine subunit interface, (BZD) feat site,uring and ECD, the modulatoryTMD, and lower pyrazoloquinolinone TMD. (b) View of (PQ) the ECD. site are GABA shown explicitly.sites, the ( cbenzodiazepine) View of the TMD (BZD) of site, a canonical and the receptormodulatory with pyrazoloquinolinone etomidate (Eto) and (PQ) barbiturate site are (Bbt)shown sites shownexplicitly. explicitly. (c) View of the TMD of a canonical receptor with etomidate (Eto) and barbiturate (Bbt) sites shown explicitly. 1.2. Allosteric Binding Sites For the major receptor subtypes that have been observed in the mammalian central nervous system,GABA subtype-selectiveA receptors are targets targeting of manyto address heavily spec usedific pharmaceuticalscircuitry and physiology such as benzodiazepine-basedselectively has been sedatives,attempted hypnotics, and is still and an ongoing anxiolytics, major barbiturate-based direction of ligand anticonvulsants, development [16]. and Knowledge general anesthetics of binding of a broadsites rangeis crucial of chemotypesfor subtype-selective [16,17]. Nearlytargeting all so pharmaceuticals that pockets that and feature a wide sequence range differences of toxins that can target be memberstaken into of consideration the GABAA receptoras discussed family for some do not relevant interact examples with the below. binding site for the physiological agonist (GABA) as orthosteric ligands, but rather with one or several allosteric-binding sites [12,15,17]. Figure1.2. Allosteric1 provides Binding an overview Sites of the described allosteric sites. Of the sites depicted in Figure1, some display a high degree of variability, while others are fairly GABAA receptors are targets of many heavily used pharmaceuticals such as benzodiazepine- conservedbased sedatives, throughout hypnotics, the family and [15 ].anxiolytics, The extracellular barbiturate-based interfaces have anticonvulsants, large variable pocket-formingand general regions and are, thus, particularly suited for the development of selective agents. The ECD α+/γ anesthetics of a broad range of chemotypes[16,17]. Nearly all pharmaceuticals and a wide range of − interfacetoxins that features target potentially members of 18 the subtypes GABAA (sixreceptorα and family three doγ subunitnot interact isoforms) with the and binding is the site major for sitethe of actionphysiological of high-a agonistffinity benzodiazepine(GABA) as orthosteric effects ligands, [18]. but rather with one or several allosteric-binding sitesSince [12,15,17]. their serendipitousFigure 1 provides discovery an overview and of the the introduction described allosteric of chlordiazepoxide sites. (Librium®) in 1960, benzodiazepinesOf the sites depicted have in Figure spurred 1, enormoussome display eff aorts high to degree develop of variability, benzodiazepine while others site ligands are fairly with furtherconserved improved throughout properties the family compared [15]. The to classicalextracellu benzodiazepines.lar interfaces have large Dozens variable of chemotypes, pocket-forming among themregionsβ-carbolines, and are, thus, pyrazoloquinolinones, particularly suited andfor imidazopyridines,the development of have selective been agents. identified The as ECD high-a α+/ffiγ−nity ligandsinterface of benzodiazepinefeatures potentially binding 18 subtypes sites. The(six α exact and numberthree γ subunit of high isoforms) and low-a andffi nityis the benzodiazepine major site of bindingaction sitesof high-affinity is still not benzodiazepine completely defined, effects and [18]. even high affinity sites that are not localized to the canonicalSinceα+/ theirγ interfacesserendipitous have discovery been described and the introduction [18]. of chlordiazepoxide (Librium®) in 1960, − benzodiazepinesDue to the high have homology spurred enormous of the α+/ effortsβ interfaces to develop with benzodiazepine the α+/γ2 siteinterfaces ligands with (see Figurefurther1 ), − − benzodiazepineimproved properties binding compared site ligands to classical such as benz theodiazepines. class of pyrazoloquinolinones Dozens of chemotypes, can bindamong at them both theseβ- carbolines, pyrazoloquinolinones, and imidazopyridines, have been identified as high-affinity interfaces, eliciting different functional effects [19]. While still being in an early stage, the α+/β ligands of benzodiazepine binding sites. The exact number of high and low-affinity benzodiazepine − interfaces are considered to be highly promising targets for novel therapeutic principles as well as a wide range of tool compounds to be utilized in (human brain) imaging or research applications [20].

Molecules 2019, 24, x FOR PEER REVIEW 4 of 48 binding sites is still not completely defined, and even high affinity sites that are not localized to the canonical α+/γ− interfaces have been described [18] Due to the high homology of the α+/β− interfaces with the α+/γ2− interfaces (see Figure 1), benzodiazepine binding site ligands such as the class of pyrazoloquinolinones can bind at both these interfaces, eliciting different functional effects [19]. While still being in an early stage, the α+/β− interfaces are considered to be highly promising targets for novel therapeutic principles as well asMolecules a wide2020 range, 25, 999 of tool compounds to be utilized in (human brain) imaging or research applications4 of 47 [20]. Some benzodiazepines also bind at non-homologous sitessites locatedlocated inin the TMD [[21,22].21,22]. These sites are thought to be the main site of action of intravenousintravenous anesthetics such as as etomidate or or propofol. propofol. Promiscuous bindingbinding ofof chemotypes chemotypes that that bind bind at theat the high-a high-affinityﬃnity benzodiazepine benzodiazepine binding binding site atsite both at bothextracellular extracellular and TMD and TMD sites hassites been has observedbeen observed unexpectedly unexpectedly often [often22]. In[22]. this In review, this review, ligands ligands of the +/ − ofhigh-a the ﬃhigh-affinitynity benzodiazepine benzodiazepine sites, the sites, extracellular the extracellularα+/β αinterfaces,β interfaces, and the and TMD the sitesTMD with sites which with − whichbarbiturates barbiturates and etomidate and etomidate interact interact are covered. are covered.

1.3. Allosteric Allosteric Ligands and Their Action on GABA A ReceptorsReceptors Ligands thatthat bindbind toto allostericallosteric sites sites can can have have a multitudea multitude of of eﬀ effectsects on on the the receptor receptor complex complex in the in presencethe presence or absence or absence of orthosteric of orthosteric ligands. Theligands. structural The basisstructural of the basis GABA ofA receptorthe GABA pharmacologyA receptor pharmacologyhas been reviewed has been recently reviewed based recently on a surge based of structural on a surge data of structural of heteropentameric data of heteropentameric receptors with variousreceptors ligands with vari presentous ligands in ortho- present and allosteric in ortho- sites and [allosteric23]. sites [23]. Many compoundscompounds cancan interact interact with with several several binding binding sites sites that that are are contained contained within within a receptor. a receptor. The Theones ones that that bind bind at the at the location location of theof the endogenous endogenous ligand ligand are are known known as as orthosteric orthosteric ligands,ligands, whereas compounds that interact in other binding sites that are different diﬀerent from the orthosteric binding site are known as allosteric ligands.

1.3.1. Orthosteric Ligands Orthosteric ligands can be classified classified into agonists, antagonists, or inverse agonists, based on the response that their binding causes to to the the receptor receptor (definitions (definitions were were taken taken from from NIH, NIH, NIC NIC dictionary). dictionary). Agonists causecause the the same same effect effect as the as endogenous the endogeno ligandus thatligand normally that normally binds to the binds receptor. to the Antagonists receptor. Antagonistsattenuate or attenuate block the or eff ectblock of anthe agonist.effect of Inversean agonist. agonists Inverse bind agonists to the receptorbind to the and receptor produce and the produceopposite the pharmacological opposite pharma effectcological that would effect be that produced would be by produc the endogenoused by the endogenous ligand [24]. ligand [24]. An example example of ofa GABA a GABAA agonistA agonist is muscimol, is muscimol, whereas whereas a well-known a well-known antagonist antagonistis bicuculline is [25–27].bicuculline Figure [25– 227 depicts]. Figure the2 depictsstructure the of structure examples of of examples an agonist of anand agonist an antagonist/inverse and an antagonist agonist/inverse of theagonist GABA ofA the receptor. GABAA receptor.

Figure 2. Examples of a GABAA receptorreceptor agonist agonist and and antagonist. antagonist. 1.3.2. Allosteric Ligands 1.3.2. Allosteric Ligands When allosteric ligands interact with a receptor, they can either exert an effect in the receptor’s When allosteric ligands interact with a receptor, they can either exert an effect in the receptor’s physiology or just bind but produce no modification on the receptor’s physiology. In the former case, physiology or just bind but produce no modification on the receptor’s physiology. In the former case, some allosteric ligands can directly exert their effects without the orthosteric ligand present. On the some allosteric ligands can directly exert their effects without the orthosteric ligand present. On the other hand, allosteric ligands that require the presence of the endogenous ligand to exert an effect are other hand, allosteric ligands that require the presence of the endogenous ligand to exert an effect are commonly referred to as allosteric modulators. commonly referred to as allosteric modulators. Allosteric ligands that produce an enhanced response when interacting with the receptor in the absence of the endogenous ligand are known as allosteric functional agonists or termed GABAmimetics. Examples of these kinds of allosteric functional agonists are etomidate, barbiturates, or neuroactive steroids [28,29]. Similarly, allosteric ligands can reduce or block channel activity in non-competitive ways. An example of a GABAA receptor blocker is picrotoxin, which has a binding site located within the channel pore [30]. Functional allosteric antagonism that is not based on a channel block can occur at a variety of allosteric sites, such as the effects elicited by inhibitory steroids [14]. Figure3 depicts the Molecules 2019, 24, x FOR PEER REVIEW 5 of 48

Allosteric ligands that produce an enhanced response when interacting with the receptor in the absence of the endogenous ligand are known as allosteric functional agonists or termed GABAmimetics. Examples of these kinds of allosteric functional agonists are etomidate, barbiturates, or neuroactive steroids [28,29]. Similarly, allosteric ligands can reduce or block channel activity in non-competitiveMolecules 2020, 25, 999 ways. An example of a GABAA receptor blocker is picrotoxin, which has a binding5 of 47 site located within the channel pore [30]. Functional allosteric antagonism that is not based on a channel block can occur at a variety of allosteric sites, such as the effects elicited by inhibitory steroids [14].structure Figure of 3 some depicts examples the structure of allosteric of some ligands, examples which of impactallosteric the ligands, receptor which function impact in the the absence receptor or functionpresence in of the GABA. absence or presence of GABA.

FigureFigure 3. 3. ExamplesExamples of of allosteric allosteric GABA GABAAA receptorreceptor ligands. ligands.

AllostericAllosteric modulators modulators (in (in the the strict strict sense) sense) requir requiree the the presence presence of of the the endogenous endogenous ligand ligand to to exert exert theirtheir effects. effects. In In other other words, words, they they modulate modulate the the GABA-elicited GABA-elicited channel channel response. response. In In general, general, allosteric allosteric modulatorsmodulators are are classified classified in in the the following following way. way. • Positive allosteric modulators (PAM): they enhance the effect of the endogenous ligand Positive• allosteric modulators (PAM): they enhance the effect of the endogenous ligand • Negative allosteric modulators (NAM): they diminish the effect of the endogenous Negative allosteric modulators (NAM): they diminish the effect of the endogenous ligand • ligand Silent• allostericSilent allosteric modulators modulators (SAM): they(SAM): do notthey influence do not influence the effects the of theeffects endogenous of the endogenous ligand, but • they canligand, compete but with they acan PAM compete or a NAM with for a PAM the occupation or a NAM offor a the binding occupation site [31 of]. a binding site [31]. Figure4 shows a schematic representation of the e ffect that a GABAA receptor PAM, NAM, and SAM Figure 4 shows a schematic representation of the effect that a GABAA receptor PAM, NAM, and Moleculescan produce 2019, 24 in, x comparisonFOR PEER REVIEW to the response that GABA produces. 6 of 48 SAM can produce in comparison to the response that GABA produces.

Figure 4. Figure 4. EffectEﬀect on on the the response response current current caused caused by by a aGABA GABAA Apositivepositive allosteric allosteric modulators modulators (PAM), (PAM), negative allostericnegative modulatorsallosteric modulators (NAM), and (NAM), silent and allosteric silent modulatorsallosteric modulators (SAM). (SAM).

Since some ligands can fall into more than one category of these classifications, each receptor subtype must be considered separately for each case. Some ligands can act as a SAM in a certain receptor subtype, whereas, in other receptor subtypes, it can show a PAM behavior. Flumazenil is an example of a GABAA receptor SAM [18]. Etomidate is another example of a GABAA receptor ligand with a mixed behavior. Depending on the concentration that is being studied, etomidate can behave as an allosteric modulator or as an allosteric functional agonist. The next chapter presents the most commonly used assays, which are used to characterize GABAA receptor ligands. In terms of the specific activity of any ligand, it is important to note that terminology is very heterogeneous and has developed historically, and terms such as “modulator” and “agonist” are often used synonymously and without clear experimental evidence for one or the other mechanism of action.

2. Assays to Probe Interactions of Small Molecules with the Receptors, or with Specific Binding Sites Historically, radioligands were used to test substances’ ability to displace or modulate a known ligand, where usually tritiated ligands are used and where brain membranes are used as a preparation of all receptors found in the brain. To test specific receptor subtypes, membrane preparations from cells used for recombinant expression are employed (typically HEK, COS, Ltk cells, etc.). The most commonly used radioligands interact with the orthosteric site, with the high- affinity benzodiazepine sites, and with the channel pore. Ligands of the orthosteric site (GABA sites) such as muscimol, GABA, gabazine (and likely many others) were and still are used. They display different degrees of specificity for different isoforms (subtypes). To indicate the challenges and limitations, in a recent study, it was shown that the

Molecules 2020, 25, 999 6 of 47

Since some ligands can fall into more than one category of these classiﬁcations, each receptor subtype must be considered separately for each case. Some ligands can act as a SAM in a certain receptor subtype, whereas, in other receptor subtypes, it can show a PAM behavior. Flumazenil is an example of a GABAA receptor SAM [18]. Etomidate is another example of a GABAA receptor ligand with a mixed behavior. Depending on the concentration that is being studied, etomidate can behave as an allosteric modulator or as an allosteric functional agonist. The next chapter presents the most commonly used assays, which are used to characterize GABAA receptor ligands. In terms of the speciﬁc activity of any ligand, it is important to note that terminology is very heterogeneous and has developed historically, and terms such as “modulator” and “agonist” are often used synonymously and without clear experimental evidence for one or the other mechanism of action.

2. Assays to Probe Interactions of Small Molecules with the Receptors, or with Specific Binding Sites Historically, radioligands were used to test substances’ ability to displace or modulate a known ligand, where usually tritiated ligands are used and where brain membranes are used as a preparation of all receptors found in the brain. To test specific receptor subtypes, membrane preparations from cells used for recombinant expression are employed (typically HEK, COS, Ltk cells, etc.). The most commonly used radioligands interact with the orthosteric site, with the high-affinity benzodiazepine sites, and with the channel pore. Ligands of the orthosteric site (GABA sites) such as muscimol, GABA, gabazine (and likely many others) were and still are used. They display different degrees of specificity for different isoforms (subtypes). To indicate the challenges and limitations, in a recent study, it was shown that the majority of high-affinity muscimol sites in brain membranes are not the most expressed αβγ receptors, but are a smaller population of δ- containing receptors [32]. In this scenario, we are interested chiefly in allosteric ligands. Historically, radioligands for the high-affinity benzodiazepine sites predate the cloning of receptor subunits and were originally considered as ligands of an unidentified “benzodiazepine receptor.” Thus, ligands that displaced radiolabeled benzodiazepines were termed “agonists,” “antagonists,” and “inverse agonists” of the “benzodiazepine receptor.” This leads to the unfortunate situation that benzodiazepine site ligands are often referred to as “GABAA receptor agonists or inverse agonists,” which is rather misleading. Several radioligands for high-affinity benzodiazepine sites exist and are widely used: flunitrazepam, flumazenil, Ro-15 4513 are the most commonly used ones. They do not show complete overlap in the subtypes to which they are binding with high affinity. Therefore, some radioligands bind to more receptor subtypes than others due to differential interactions with isoforms of α or γ subunits. Radioligands that bind in a state-dependent way to the ion channel also exist, where a prominent example is EBOB. EBOB binding is state-dependent. Therefore, its binding is dependent on the absence or presence of GABA and often even responds to allosteric modulators. Thus, modulation (rather than displacement) of EBOB binding can also be used to detect ligands that bind at sites other than the channel lumen, but these sites then remain unspecified [33]. Limitations of radioligand based assays include the following. Competitive displacement is the gold standard to confirm binding site usage. Radioligand modulation helps to detect ligands that use unknown sites [33], but may fail to respond to sites that cause only small or no conformational changes (silent binders that may not be silent in the presence of other agents). As discussed in the previous chapter, ligands not only bind to the receptors, but they exert complex effects such as negative or positive modulation, or direct channel block, or allosteric “activation” on them. These effects can only be detected with functional assays. This more comprehensive approach directly assesses the effects of ligands on channel activity, or on agonist-elicited channel activity. The biggest disadvantage of functional studies is that the binding sites cannot be identified by the assay. Molecules 2020, 25, 999 7 of 47

Competitive functional assays are exceedingly challenging and often allosteric and competitive effects cannot be reliably distinguished. The most commonly used methods to determine ligand function comprise low and high throughput electrophysiological assays and diverse methods that are based on fluorescent probes that are charge-sensitive or, otherwise, coupled to changes in membrane potential. Functional assays are performed in recombinantly expressing cell systems to determine the effects of ligands on defined receptor species or on known mixtures of subunits that are expressed, or on ex vivo preparations such as acute brain slices, synaptosome or membrane preparations, or on primary cultured cells (typically hippocampal neurons). It has to be noted that the large diversity of receptor subtypes renders every assay incomplete in the sense that effects only on those receptors that are present in a given preparation can be captured. Due to the big complexity of GABAA receptor-mediated pharmacology and the wide diversity of assays, which are used to investigate ligand properties such as affinity, potency, electrophysiological efficacy, and in vivo effects and efficacy, it is challenging to compare ligand characteristics across different studies. Even though, by now, it is well acknowledged that radioligand-based competition assays can fail to detect modulatory interactions, there is still a lack of consensus concerning functional testing. The large number of receptor subunits, the lacking knowledge about the precise number of existing subtypes, and the observation that neurons, glia cells, and non- neuronal cells express a broad and heterogeneous mixture of subunits documents that no single in vitro or ex vivo protocol can be designed and research relies on combining multiple assay types to define ligand effects on molecular species. A recent review illustrates the complexity and discusses many of the problems that are faced on the path toward receptor subtype-specific ligand characterization [16]. Generally, after binding or functional effects are confirmed in either heterologously expressed protein or suitable ex vivo preparations (such as brain membranes, acute brain slices, or neuronal cell cultures), a broad range of toxicological and behavioral testing will follow for the most promising compounds. The behavioral animal experiments are out of the scope of this review. As a very broad rule, compounds that are chiefly GABAmimetic or positive allosteric modulators display sedative-like and anticonvulsant-like effects while ortho- and allosteric antagonists and blockers will be anxiogenic and pro-convulsant. If individual subtypes respond differently, very complex combinations of effects can occur and raise hopes for the development of totally novel therapeutics that combine silent, PAM, and NAM characteristics toward different receptor subtypes [34].

3. Clinical Pharmacology of Allosteric Ligands Used in Human Medicine

Three large groups of heavily used GABAergics are allosteric modulators of GABAA receptors, and additional indication groups exist and are summarized in this case as a fourth heterogeneous group.

3.1. Sedative General Anesthetics Drugs with reliable sedative-hypnotic, amnestic, anxiolytic, antinociceptive, and immobilizing properties represent an indispensable component of state-of-the-art general anesthesia. After the introduction of diethyl ether as the first viable anesthetic for surgery in the middle of the 19th century, drug discovery became a prime focus of research in the field of anesthesia [35]. The ongoing quest for novel compounds with rapid-onset, potent sedative-hypnotic effects, predictable clearance to inactive metabolites and minimal side effects has yielded a range of clinically established drugs. The vast majority of anesthetic compounds in use today interact with GABAA receptors [36]. In this scenario, we provide a glance at the clinical roles of four important anesthetics, with the primary effects to rely predominantly on positive allosteric modulation of GABAA receptors: Propofol, Etomidate, Midazolam, and Thiopental (Figure5). Molecules 2019, 24, x FOR PEER REVIEW 8 of 48 compounds. The behavioral animal experiments are out of the scope of this review. As a very broad rule, compounds that are chiefly GABAmimetic or positive allosteric modulators display sedative- like and anticonvulsant-like effects while ortho- and allosteric antagonists and blockers will be anxiogenic and pro-convulsant. If individual subtypes respond differently, very complex combinations of effects can occur and raise hopes for the development of totally novel therapeutics that combine silent, PAM, and NAM characteristics toward different receptor subtypes [34].

3. Clinical Pharmacology of Allosteric Ligands Used in Human Medicine

Three large groups of heavily used GABAergics are allosteric modulators of GABAA receptors, and additional indication groups exist and are summarized in this case as a fourth heterogeneous group.

Moleculeseffects to2020 rely, 25, 999predominantly on positive allosteric modulation of GABAA receptors: Propofol,8 of 47 Etomidate, Midazolam, and Thiopental (Figure 5).

Figure 5. AnestheticAnesthetic drugs drugs with different diﬀerent allostericallosteric mechanisms of action.

Propofol is a hydrophobic compound for intravenousintravenous application in a lipid-solution. It was first first approved for use in the 1980s and has quickly advanc advanceded to become the most frequently administered intravenous drugdrug forfor thethe inductioninduction of of general general anesthesia anesthesia and and a a common common choice choice for for the the maintenance maintenance of generalof general anesthesia anesthesia as wellas well as proceduralas procedural sedation sedation [37]. [37]. Rapid Rapid inductions, inductions, rapid rapid terminal terminal half-life half-life time, lowtime, incidence low incidence of postoperative of postoperative nausea nausea and vomiting, and vomiting, as well asas rapidwell as psychomotor rapid psychomotor recovery makerecovery it a verymake versatile it a very hypnoticversatile drughypnotic [38]. drug [38]. Propofol substantially substantially inhibits inhibits sympathetic sympathetic nerve nerve activity, activity, contri contributingbuting to a significant, to a significant, dose- dose-dependentdependent decrease decrease of mean of mean blood blood pressure pressure [39] [39 as] aswell well as asmoderate moderate resp respiratoryiratory depression depression [40]. [40]. While these cardiopulmonary side-eside-effectsffects are predictablepredictable and well manageable in most settings, they often present a challenge in hemodynamically constrainedconstrained patient populations. Other adverse events include pain on the the injection injection site site and and less less commonl commonlyy hyperlipidemia hyperlipidemia resulting resulting from from lipid lipid formulation. formulation. Etomidate is an imidazole derivative, where its an anestheticesthetic activity was coincidentally coincidentally discovered during animal testing as an antifungal agent in the 1960s. Its rapid onset of effects effects can be explained by the compound’s lipid solubility and large non-ionized fraction at physiological pH and make it a common choicechoice forfor induction induction of of general general anesthesia anesthesia and and short short procedures procedures requiring requiring deep deep sedation. sedation. The prolongedThe prolonged application application of etomidate, of etomidate, however, however, is not feasible is not due feasible to the incidencedue to the of adrenocorticalincidence of suppression,adrenocortical which suppression, negatively which impacts negatively the outcome, impacts especially the inoutcome, critically-ill especially populations. in critically-ill Compared topopulations. propofol, etomidateCompared onlyto propofol, causes a etomidate modest decrease only causes of arterial a modest blood decrease pressure of and arterial leaves blood the respiratorypressure and system leaves largely the respiratory unaffected system [41]. largely unaffected [41]. Midazolam is a short-acting, rapid-onset benzodiazepine primarily used during procedural sedation. Its low hemodynamic side-effects also make it a common choice for general anesthesia during cardiac surgery. Its amnestic effects are advantageous in treating intraoperative awareness [42] and its application may be correlated with a decreased likelihood of delayed recall of procedural sedation [43]. As with other benzodiazepines, paradoxical excitement, cognitive impairment, and prolonged recovery time present known complications. Thiopental is an ultra-short acting barbiturate derivative. Barbiturates were used extensively over several decades to induce and maintain general anesthesia. They have been largely replaced by newer compounds with safer pharmacokinetic and pharmacodynamic profiles. Thiopental remains the only anesthetically used barbiturate with a firm place in today’s surgery rooms and emergency departments. Many additional anesthetic compounds such as inhalational fluranes and ketamine also allosterically modulate GABAA receptors but rely on other primary mechanisms of action.

3.2. Prescription Hypnotics and Sleeping Aids

GABAA receptors present the prime molecular targets in the pharmacological treatment of insomnia. Barbiturates undoubtedly played a historically signiﬁcant role as prescription hypnotics but have been largely replaced by safer options over the years. Their low therapeutic index paired with high tolerance formation and addiction potential contribute to the signiﬁcant risks associated with their use [44]. Benzodiazepines are known for their ability to promote sleep, dampen anxiety, and provide muscle relaxation with a much broader therapeutic index than barbiturates. Their aggressive marketing and extensive prescription during recent decades after their market introduction are now being increasingly criticized after tremendous evidence for risks associated with long-term usage emerged. Molecules 2020, 25, 999 9 of 47

Prescription of benzodiazepines such as temazepam and nitrazepam still constitute a significant fraction of prescribed hypnotics [45]. The largest fraction of prescribed treatments for insomnia today is newer non-benzodiazepines such as zolpidem and zopiclone (Z-Drugs). While they are promoted and attributed to possessing greater benefits and fewer side effects than benzodiazepines, there is no evidence of significant differences in clinical effectiveness and safety [46]. Many nature-derived compounds of which some are used as OTC sleep-aids, also seem to mediate their sleep-promoting effects via interactions with GABAA receptors [47]. The development of new drugs with high efficacy, lower addiction liability, and lower incidence of paradoxical reactions remains imperative in order to create safer long-term treatment options for insomnia.

3.3. Anticonvulsants Several diﬀerent antiepileptic drugs with various known and unknown mechanisms of action are in use for long-term prophylactic treatment. The treatment of acute seizures and status epilepticus, however, requires potent and fast-acting drugs capable of eﬀectively disrupting the convulsive state. Many of these compounds are modulators of GABAA receptors. Parenteral benzodiazepines such as midazolam, lorazepam, and diazepam are recommended during the initial phase of therapy, which begins after 5 min of a persistent seizure. If the seizure continues after a duration of 20 min, a second treatment phase begins, which consists of, not predominantly, GABAergic drugs such as fosphenytoin, valproic acid, or levetiracetam. Due to a higher incidence of adverse events, phenobarbital is only recommended in case the other options are unavailable. At the 40-min mark, a third treatment phase begins, where anesthetic doses of thiopental, midazolam, or propofol should be considered [48].

3.4. Additional Indication Groups for Therapeutically Used Allosteric Modulators of GABAA Receptors Exist Other indication groups for benzodiazepines include anxiolysis and central muscle relaxation. In such cases, sedative effects are unwanted and tolerance formation limits the duration of effective treatment. Since there are differences in the effect profiles of benzodiazepines, specific derivatives are recommended for specific indications, which are not only based on their pharmacokinetic profiles [45]. Barbiturates are still regularly used in the management of severe traumatic brain injury, where lowering intracranial pressure and optimization of cerebral oxygenation are critical [49].

3.5. PET and SPECT Imaging Ligands

Allosteric ligands of specific groups of GABAA receptor subtypes are not only in use as therapeutics but also as isotope-labeled imaging ligands for positron emission tomography (PET) or single-photon computed tomography (SPECT) to investigate altered receptor expression patterns in a wide range of neuropsychiatric disorders [50]. At this time, all commonly used ligands for human brain imaging are ligands of some of the high-affinity benzodiazepine binding sites. Prominent examples are flumazenil, iomazenil, and Ro 15-4513. Imaging ligands that interact with other receptor subtypes and do not rely on the presence of a γ-subunit would be highly desired.

4. Allosteric Ligands and Their Synthetic Accessibility

Many heterocyclic scaffolds have been investigated as GABAA receptor ligands. In addition, it turns out that many of them contain nitrogen heterocycles. In fact, a large part contains even fused heterocycles, which represent scaffolds that are often complex to synthesize. Within this chapter, the more recent (last 10 years) synthetic developments in the large field of GABAA receptor ligands are summarized. This means that the selection of examples is based on new or improved synthetic methods that have been applied, and not on improved biological activity. Hence, examples synthesized via long-standing synthetic procedures are not included even though they might show interesting properties. For completion, the pharmacological data reported along with the synthesis are briefly Molecules 2020, 25, 999 10 of 47 summarized. Additionally, the subchapters are ordered according to the chemical relation, and not according to activity, a binding site, or factors alike. The chemotypes herein covered are a non-limiting list of the GABAA receptor modulators that have been, so far, discovered and synthesized via new or improved routes within the last 10 years. For this reason, synthetic accessibility of heterocycles is reviewed.

4.1. Monocyclic GABAA Receptor Modulators

4.1.1. Monocyclic GABAA Receptor Modulators with 5-Membered Rings

Imidazole Derivatives

Etomidate is a general anesthetic that not only positively allosterically modulates the GABAA receptor, but also, at subclinical doses, it can activate the GABAA receptor in the absence of GABA. β2 and or β3-subunit containing receptors are much more sensitive to etomidate than β1 containing receptors. Another relevant etomidate derivative is methoxycarbonyl metomidate (MOC-etomidate), which modulates GABAA receptors with similar potency, but it is quickly metabolized and inactivated [51,52]. Azietomidate also enhances the GABAA receptor function whereas TFD-etomidate has a low GABAA receptor activity. Achiral etomidate analogs have been designed and tested, such as dihydrogen etomidate and cyclopropyl etomidate, but both of them have low potencies, comparable to S-etomidate [53]. The structures of these etomidate derivatives are depicted in Figure6. Molecules 2019, 24, x FOR PEER REVIEW 11 of 48

Figure 6. Structures of etomidate enantiomers and analogs.

The synthesis synthesis of ofetomidate etomidate was reported was reported for the first for time the in ﬁrst the US time patent in the3,354,173. US patentIn this 3,354,173.process, methyl In benzylamine this process, (compound methyl 9) benzylamineis treated with (compound ethyl chloroacetate9) is treated to yield with N- ethylethoxycarbonylmethyl- chloroacetateN to-1-phenylethylamine yield N-ethoxycarbonylmethyl- (compound 10N).-1-phenylethylamine This product is subsequently (compound 10formylated). This with product formic is subsequentlyacid and the formylated resulting withN-ethoxycarbonylmethyl- formic acid and theN-formyl- resultingN-l- Nphenylethylamine-ethoxycarbonylmethyl- is againN -formyl-C-formylatedN-l-phenylethylamine with ethyl formate is and again sodium C-formylated ethoxide, which with yields ethyl formatecompound and 11 sodium as a result. ethoxide, The product which yields(compound compound 11) is 11treatedas a result.with thiocyanate The product in hydrochloric (compound 11acid.) is This treated causes with the thiocyanate N-formamide in protecting hydrochloric group acid. to hydrolize This causes and the resultingN-formamide product protecting with an groupaldehyde to hydrolizemoiety will and undergo the resulting a Marckwald product heterocyclization, with an aldehyde which moiety forms will compound undergo 12 a Marckwald. In the final heterocyclization,step, the thiol group which is removed forms compoundunder oxidative12. In cond theitions ﬁnal with step, nitric the thiol and groupnitrous is acids removed and further under oxidativetreated with conditions IPA HCl with to nitric achieve andnitrous the correspon acids andding further hydrochloride treated with salt IPA HClof the to achieveetomidate. the correspondingNeutralization hydrochlorideyielded etomidate salt of(compound the etomidate. 1 or 2 Neutralization) as a free base yielded (Scheme etomidate 1). [54,55]. (compound 1 or 2) asSubsequently, a free base (Scheme a slightly1). [54 modified,55]. approach was described in which the last step is performed by using hydrogen peroxide as an oxidizing agent to perform the final desulfurization in order to avoid highly exothermic conditions and the generation of nitrous oxide gas. This modified approach is only reported with the R-enantiomer of etomidate (compound 1), whereas the original procedure was performed with the corresponding racemate (Scheme 1) [54].

Scheme 1. a) Ethyl chloroacetate, TEA, DMF, rt., overnight, yield not reported. b) HCOOH, xylene, reflux, few hours, quant. yield. c) 1. NaOEt/HCOOEt, EtOH, r.t., 2. KSCN/HCl. d) 1. HNO3/NaNO2, 25–35 °C, 2 h, r.t. 2. Na2CO3, yield not reported. e) 30% H2O2, Toluene, H2O, 40–45 °C, yield not reported.

An alternative approach was reported by using a Mitsunobu alkylation of ethyl imidazole carboxylate (compound 13), which was followed by a palladium-catalyzed cyclization in very good yields. Racemization was avoided by performing the reaction at low temperatures (Scheme 2) [56].

Scheme 2. a) 1. PPh3, THF, −40 °C, 70%.

Molecules 2019, 24, x FOR PEER REVIEW 11 of 48

Figure 6. Structures of etomidate enantiomers and analogs.

The synthesis of etomidate was reported for the first time in the US patent 3,354,173. In this process, methyl benzylamine (compound 9) is treated with ethyl chloroacetate to yield N- ethoxycarbonylmethyl-N-1-phenylethylamine (compound 10). This product is subsequently formylated with formic acid and the resulting N-ethoxycarbonylmethyl-N-formyl-N-l- Figure 6. Structures of etomidate enantiomers and analogs. phenylethylamine is again C-formylated with ethyl formate and sodium ethoxide, which yields compoundThe synthesis 11 as a ofresult. etomidate The product was reported (compound for the 11 )first is treated time in with the thiocyanateUS patent 3,354,173. in hydrochloric In this process,acid. This methyl causes thebenzylamine N-formamide (compound protecting 9 group) is treated to hydrolize with andethyl the chloroacetate resulting product to yield with Nan- ethoxycarbonylmethyl-aldehyde moiety will undergoN-1-phenylethylamine a Marckwald heterocyclization, (compound 10 which). This forms product compound is 12subsequently. In the final formylatedstep, the thiol withgroup isformic removed acid under and oxidative the resultingconditions withN-ethoxycarbonylmethyl- nitric and nitrous acidsN-formyl- and furtherN-l- treatedphenylethylamine with IPA isHCl again to C-formachieveylated the withcorrespon ethyl dingformate hydrochloride and sodium saltethoxide, of the which etomidate. yields compoundNeutralization 11 asyielded a result. etomidate The product (compound (compound 1 or 2) 11as) ais free treated base with(Scheme thiocyanate 1). [54,55]. in hydrochloric acid. Subsequently,This causes the a Nslightly-formamide modified protecting approach group was to described hydrolize in and which the theresulting last step product is performed with an aldehydeby using hydrogenmoiety will peroxide undergo as a Marckwaldan oxidizing heterocyclization, agent to perform which the final forms desulfurization compound 12 . inIn orderthe final to step,avoid the highly thiol exothermic group is removed conditions under and oxidative the generati condonitions of nitrous with nitricoxide and gas. nitrous This modified acids and approach further Moleculestreatedis only reported2020with, 25 ,IPA 999 with HCl the Rto-enantiomer achieve the of etomidatecorrespon (compoundding hydrochloride 1), whereas salt the oforiginal the etomidate.procedure11 of 47 Neutralizationwas performed yielded with the etomidate corresponding (compound racemate 1 or (Scheme 2) as a free 1) [54]. base (Scheme 1). [54,55]. Subsequently, a slightly modified approach was described in which the last step is performed by using hydrogen peroxide as an oxidizing agent to perform the final desulfurization in order to avoid highly exothermic conditions and the generation of nitrous oxide gas. This modified approach is only reported with the R-enantiomer of etomidate (compound 1), whereas the original procedure was performed with the corresponding racemate (Scheme 1) [54].

Scheme 1. a) Ethyl chloroacetate, TEA, DMF, rt., overnight,overnight, yield not reported.reported. b) HCOOH, xylene, reflux, few hours, quant. yield. c) 1. NaOEt/HCOOEt, EtOH, r.t., 2. KSCN/HCl. d) 1. HNO3/NaNO2, reﬂux, few hours, quant. yield. c) 1. NaOEt/HCOOEt, EtOH, r.t., 2. KSCN/HCl. d) 1. HNO3/NaNO2, 25–35 °C,◦C, 2 h, r.t. 2. 2. Na Na2CO2CO3, 3yield, yield not not reported. reported. e) 30% e) 30% H2O H22, OToluene,2, Toluene, H2O, H 240–45O, 40–45 °C, ◦yieldC, yield not notreported. reported.

Subsequently,An alternative aapproach slightly modified was reported approach by using was described a Mitsunobu in which alkylation the last of step ethyl is performedimidazole Scheme 1. a) Ethyl chloroacetate, TEA, DMF, rt., overnight, yield not reported. b) HCOOH, xylene, bycarboxylate using hydrogen (compound peroxide 13), which as an was oxidizing followed agent by toa palladium-catalyzed perform the final desulfurization cyclization in invery order good to reflux, few hours, quant. yield. c) 1. NaOEt/HCOOEt, EtOH, r.t., 2. KSCN/HCl. d) 1. HNO3/NaNO2, avoidyields. highly Racemization exothermic was conditions avoided by and performing the generation the reaction of nitrous at oxidelow temperatures gas. This modified (Scheme approach 2) [56]. is 25–35 °C, 2 h, r.t. 2. Na2CO3, yield not reported. e) 30% H2O2, Toluene, H2O, 40–45 °C, yield not only reported with the R-enantiomer of etomidate (compound 1), whereas the original procedure was reported. performed with the corresponding racemate (Scheme1)[54]. An alternative approach was reported by using a Mitsunobu alkylation of ethyl imidazole An alternative approach was reported by using a Mitsunobu alkylation of ethyl imidazole carboxylate (compound 13), which was followed by a palladium-catalyzed cyclization in very good carboxylate (compound 13), which was followed by a palladium-catalyzed cyclization in very good yields. Racemization was avoided by performing the reaction at low temperatures (Scheme 2)[56]. yields. Racemization was avoided by performing the reaction at low temperatures (Scheme 2) [56]. Scheme 2. a) 1. PPh3, THF, −40 °C, 70%.

Molecules 2019, 24, x FOR PEER REVIEW 12 of 48 SchemeScheme 2.2. a)a) 1.1. PPh3,, THF, THF, −4040 °C,C, 70%. 70%. 3 − ◦ The synthesis of etomidate derivatives with a space linker (MOC-etomidate, (compound 3), The synthesis of etomidate derivatives with a space linker (MOC-etomidate, (compound 3), cyclopropyl etomidate (compound 4), and azietomidate (compound 5)) can be achieved from the cyclopropyl etomidate (compound 4), and azietomidate (compound 5)) can be achieved from the desired etomidate enantiomer (compound 1 or 2) in a two-step procedure (Scheme 3). Azietomidate desired etomidate enantiomer (compound 1 or 2) in a two-step procedure (Scheme3). Azietomidate (compound 15) can be easily obtained by treating etomidate in its chirally pure desired form (compound 15) can be easily obtained by treating etomidate in its chirally pure desired form (compound (compound 1 or 2) with an excess of a base in order to hydrolyze the ester moiety. The resulting 1 or 2) with an excess of a base in order to hydrolyze the ester moiety. The resulting carboxylic acid was carboxylic acid was re-esterified with 3-azibutanol in the presence of dicyclohexylcarbodiimide re-esteriﬁed with 3-azibutanol in the presence of dicyclohexylcarbodiimide (DCC) and a nucleophilic (DCC) and a nucleophilic catalyst, 4-(dimethylamino)pyridine (DMAP), to yield azietomidate catalyst, 4-(dimethylamino)pyridine (DMAP), to yield azietomidate (compound 5) as a product [57]. (compound 5) as a product [57]. This same approach is also reported to obtain the MOC-Etomidate This same approach is also reported to obtain the MOC-Etomidate (compound 3) and cyclopropyl (compound 3) and cyclopropyl methoxycarbonyl metomidate (compound 4), but using the methoxycarbonyl metomidate (compound 4), but using the corresponding alcohol instead [58]. corresponding alcohol instead [58].

1 Scheme 3. a) NaOH 10 M, 100 °C, 1 h. b) R -OH, 4-(dimethylamino)pyridine,1 dicyclohexyl- Scheme 3. a) NaOH 10 M, 100 ◦C, 1 h. b) R -OH, 4-(dimethylamino)pyridine, carbodiimide, or 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride. dicyclohexyl-carbodiimide, or 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride.

TheThe dihydrogen dihydrogen derivative derivative of etomidate of etomidate (compound (compound 7) was 7synthesized) was synthesized with a phase with transfer– a phase catalyzedtransfer–catalyzed N-alkylationN -alkylationof imidazole of ethyl imidazole ester (compound ethyl ester 13) (compound with benzyl13 bromide) with (compound benzyl bromide 17) and(compound tetra-n-butylammonium17) and tetra-n-butylammonium bromide. This procedure bromide. yielded This procedure the desired yielded dihydrogen the desired etomidate dihydrogen as welletomidate as its respective as well as isomer its respective (compound isomer 18) (compound (Scheme 4)18 [59].) (Scheme 4)[59].

Scheme 4. a) tetra-n-butyl-ammonium bromide, NaOH aq 20%, toluene, yield not reported.

Arylalkylazoles

Compounds containing an azolic ring have been reported as ligands for the GABAA receptors. Among them, compounds such as loreclezole (compound 21), nafimidone (compound 20), and denzimol (compound 19) belong to the class of arylalkylazoles, which is a class of experimental anticonvulsants (Figure 7).

Figure 7. Arylalkylazoles.

Loreclezole (compound 21) was shown to be anticonvulsant in animals, and features β-isoform selectivity [60]. In particular, selectivity towards β2 and β3 over β1 containing receptors has been demonstrated with mutational studies and points to an overlapping site of action with etomidate [61]. Loreclezole (compound 21) is synthesized in a two-step synthesis, as outlined in Scheme 5. The first step, an N1-alkylation of the 1H-1,2,4-triazole with compound 22 is performed under microwave irradiation to yield compound 23 [62,63]. In the second step, 1-(3,5-dichlorophenyl)-2-(1H-1,2,4- triazol-1-yl)ethan-1-one (compound 23) is transformed to loreclezole (compound 21) upon treatment with phosphorus pentachloride.

MoleculesMolecules 2019 2019, ,24 24, ,x x FOR FOR PEER PEER REVIEW REVIEW 1212 ofof 4848

TheThe synthesissynthesis ofof etomidateetomidate derivativesderivatives withwith aa spacespace linkerlinker (MOC-etomidate,(MOC-etomidate, (compound(compound 33),), cyclopropylcyclopropyl etomidateetomidate (compound(compound 44),), andand azietomidateazietomidate (compound(compound 55)))) cancan bebe achievedachieved fromfrom thethe desireddesired etomidateetomidate enantiomerenantiomer (compound(compound 1 1 oror 22)) in in a a two-step two-step procedureprocedure (Scheme(Scheme 3).3). AzietomidateAzietomidate (compound(compound 1515)) cancan bebe easilyeasily obtainedobtained byby treatingtreating etomidateetomidate inin itsits chirallychirally purepure desireddesired formform (compound(compound 11 oror 22)) withwith anan excessexcess ofof aa basebase inin orderorder toto hydrolyzehydrolyze thethe esterester moiety.moiety. TheThe resultingresulting carboxyliccarboxylic acidacid waswas re-esterifiedre-esterified withwith 3-azibutan3-azibutanolol inin thethe presencepresence ofof dicyclohexylcarbodiimidedicyclohexylcarbodiimide (DCC)(DCC) andand aa nucleophilicnucleophilic catalyst,catalyst, 4-(dimethyla4-(dimethylamino)pyridinemino)pyridine (DMAP),(DMAP), toto yieldyield azietomidateazietomidate (compound(compound 55)) asas aa productproduct [57].[57]. ThisThis samesame approachapproach isis alsoalso reportedreported toto obtainobtain thethe MOC-EtomidateMOC-Etomidate (compound(compound 33)) andand cyclopropylcyclopropyl methoxycarbonylmethoxycarbonyl metomidatemetomidate (compound(compound 44),), butbut usingusing thethe correspondingcorresponding alcohol alcohol instead instead [58]. [58].

SchemeScheme 3.3. a)a) NaOHNaOH 1010 M,M, 100100 °C,°C, 11 h.h. b)b) RR11-OH,-OH, 4-(dimethylamino)pyridine,4-(dimethylamino)pyridine, dicyclohexyl-dicyclohexylcarbodiimide,carbodiimide, or or 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride. hydrochloride.

TheThe dihydrogendihydrogen derivativederivative ofof etomidateetomidate (compound(compound 77)) waswas synthesizedsynthesized withwith aa phasephase transfer–transfer– catalyzedcatalyzed N N-alkylation-alkylation ofof imidazoleimidazole ethylethyl esterester (compound (compound 13 13)) withwith benzylbenzyl bromidebromide (compound (compound 17 17))

Moleculesandand tetra-tetra-2020nn-butylammonium,-butylammonium25, 999 bromide.bromide. ThisThis procedureprocedure yieldedyielded thethe desireddesired dihydrogendihydrogen etomidateetomidate12 of as47as wellwell as as its its respective respective isomer isomer (compound (compound 18 18)) (Scheme (Scheme 4) 4) [59]. [59].

SchemeScheme 4.4. a) a) tetra- tetra-nn-butyl-ammonium-butyl-ammonium bromide, bromide, NaOHNaOH aq aq 20%, 20%, toluene,toluene, yieldyield notnot reported. reported.

ArylalkylazolesArylalkylazoles

CompoundsCompounds containingcontaining anan azolicazolic ringring havehave beenbeen reportedreported asas ligandsligands forfor thethe GABA GABAGABAAA receptors.receptors. AmongAmong them,them, compoundscompounds suchsuch asas loreclezoleloreclezoleloreclezole (compound(compound(compound 2121),),), naﬁmidonenafimidonenafimidone (compound(compound(compound 2020),), andandand denzimoldenzimol (compound(compound 1919)) belongbelong toto thethe classclassclass ofofof arylalkylazoles,arylalkylazoles,arylalkylazoles, whichwhich isis aa classclassclass ofofof experimentalexperimentalexperimental anticonvulsantsanticonvulsants (Figure(Figure7 7). ).7).

FigureFigure 7. 7. Arylalkylazoles. Arylalkylazoles.

LoreclezoleLoreclezole (compound(compound 21 21)) was was shownshown toto bebebe anticonvulsant anticonvulsantanticonvulsant inin animals, animals,animals, and andand features featuresfeatures β ββ-isoform-isoform selectivityselectivity [60]. [[60].60]. InIn In particular,particular, particular, selectivityselectivity selectivity towardstowards towards ββ22 β andand2 and ββ33 overβover3 over ββ11 containingcontainingβ1 containing receptorsreceptors receptors hashas beenbeen has beendemonstrateddemonstrated demonstrated withwith mutationalmutational with mutational studiesstudies studies andand pointspoints and toto points anan overlappingoverlapping to an overlapping sitesite ofof actionaction site with ofwith action etomidateetomidate with etomidate[61].[61]. Loreclezole Loreclezole [61]. (compound Loreclezole(compound 21 21 (compound)) is is synthesized synthesized21) is in in synthesizeda a two-step two-step synthesis, synthesis, in a two-step as as outlined outlined synthesis, in in Scheme Scheme as outlined 5. 5. The The 1 infirstfirst Scheme step, step, an an5. N N11-alkylation The-alkylation ﬁrst step, of of the the an 1 1HH-1,2,4-triazole-1,2,4-triazoleN -alkylation with with of thecompound compound 1H-1,2,4-triazole 22 22 is is performed performed with under compoundunder microwave microwave22 is performedirradiationirradiation to underto yieldyield microwave compoundcompound irradiation 2323 [62,63].[62,63]. toInIn yieldthethe secondsecond compound step,step, 1-(3,5-dichlorophenyl)-2-(1231-(3,5-dichlorophenyl)-2-(1[62,63]. In the secondHH-1,2,4--1,2,4- step, 1-(3,5-dichlorophenyl)-2-(1triazol-1-yl)ethan-1-onetriazol-1-yl)ethan-1-one (compound (compoundH-1,2,4-triazol-1-yl)ethan-1-one 23 23)) is is transformed transformed to to (compoundloreclezole loreclezole (compound 23(compound) is transformed 21 21)) upon upon to loreclezole treatment treatment MoleculesMolecules(compoundwithwith phosphorus phosphorus 20192019,, 242124,, ) xx upon FORFOR pentachloride. pentachloride. PEERPEER treatment REVIEWREVIEW with phosphorus pentachloride. 1313 ofof 4848

SchemeScheme 5.a5.a 5. )a)) KK K22COCO2CO33,, 3 11,HH 1H-1,2,4-triazole,-1,2,4-triazole,-1,2,4-triazole, CHCH CH33CN,CN,3CN, MWMW MW 8585 85 °C°C◦C (P(P (P 5050 50 W),W), W), 5050 50 min,min, min, 98%.98%. 98%. b)b) b) 1,2-dichloroethane,1,2-dichloroethane, 1,2-dichloroethane, PClPCl55,, , reflux,reflux, reﬂux, 1.31.3 1.3 h.h. h.

ModificationsModificationsModifications ofof thethe loreclezoleloreclezole scaffoldscaffold scaffold have have been been investigated investigated toto discover discover newnew potential potential anticonvulsants.anticonvulsants. TheThe The synthesynthe synthesississis ofof ofthesethese these derivativesderivatives derivatives isis shownshown is shown inin SchemeScheme in Scheme 6.6. AfterAfter6. After alkylationalkylation alkylation ofof 1,2,4-1,2,4- of triazole1,2,4-triazoletriazole withwith with2,42,4′′-dichloroacetophenone-dichloroacetophenone 2,40-dichloroacetophenone (compound(compound (compound 2424).).24 TheThe). The hydroxylaminehydroxylamine hydroxylamine 2626 26 waswas was obtainedobtained obtained byby by refluxingrefluxingrefluxing withwith hydroxylaminehydroxylamine hydrochloridehydrochloride under under basicbasic conditions.conditions. Lastly,Lastly, Lastly, esterificationesterification esterification usingusing DCCDCC asas aa couplingcoupling agentagent yieldedyielded the the final finalfinal compound compound 27. 27.

SchemeScheme 6.6. a)a)a) 1,2,4-triazole,1,2,4-triazole, Na, Na, CH CH33OH,OH, reflux.reflux. reﬂux. b)b) NHNH 22OH·HCl,OH·HCl,OH HCl, CC 22HHH55OH,OH,OH, pHpH pH 14,14, 14, reflux.reflux. reﬂux. c)c) c) R-COOH,R-COOH, R-COOH, 3 2 · 2 5 DCC,DCC, DMAP,DMAP, DCM, DCM, ethereal ethereal gg HCl.HCl.

Similarly,Similarly, nafimidonenafimidone (compound(compound 2020)) isis synthesized,synthesized, asas shownshown inin SchemeScheme 77 [64–66],[64–66], andand alsoalso denzimoldenzimol (compound(compound 1919)) isis synthesizedsynthesized inin aa similarsimilar fashion,fashion, startingstarting fromfrom thethe correspondingcorresponding αα-- haloalkylhaloalkyl ketonesketones [66].[66].

SchemeScheme 7.7. a)a) BrBr2,2, glacialglacial aceticacetic acid,acid, ice,ice, overnightovernight 80%.80%. b)b) 1,2,4-triazole,1,2,4-triazole, DMF,DMF, 00 °C,°C, 22 h,h, 45%.45%.

4.1.2.4.1.2. MonocyclicMonocyclic GABAGABAAA ReceptorReceptor ModulatorsModulators withwith Six-MemberedSix-Membered RingsRings

BarbituratesBarbiturates BarbituratesBarbiturates (structures(structures 3030 andand 3131)) areare derivativesderivatives ofof barbituricbarbituric acid,acid, whichwhich cancan positivelypositively modulatemodulate thethe GABAGABAAA receptorreceptor and,and, atat higherhigher concentrations,concentrations, cancan activateactivate thethe receptorreceptor withoutwithout thethe endogenousendogenous ligandligand [67,68].[67,68].

TheThe synthesissynthesis ofof thisthis familyfamily ofof compoundscompounds hahass beenbeen reviewedreviewed byby VardanyanVardanyan andand HrubyHruby [67].[67]. Thus,Thus, onlyonly contributionscontributions sincesince thenthen areare discusseddiscussed inin thisthis chapter.chapter. Traditionally,Traditionally, barbituratesbarbiturates cancan bebe preparedprepared byby aa condensationcondensation reactionreaction ofof ureaurea andand thethe correspondingcorresponding malonicmalonic esterester withwith sodiumsodium alkoxidealkoxide inin ethanolethanol oror DMSODMSO [2,67].[2,67]. AA disadvantagedisadvantage ofof thisthis traditionaltraditional procedureprocedure isis thatthat itit yieldsyields barbituratebarbiturate derivativesderivatives inin aa modestmodest yieldyield duedue toto seveseveralral well-knownwell-known sideside reactions.reactions. Additionally,Additionally, thisthis procedureprocedure requiresrequires drydry solvents,solvents, highhigh temperaturestemperatures,, inertinert atmospheres,atmospheres, andand metallicmetallic sodium,sodium, whichwhich limitslimits thethe scopescope ofof thethe derivativesderivatives thatthat cancan bebe obtainedobtained [2].[2]. VolonterioVolonterio andand ZandaZanda developeddeveloped aa one-potone-pot procedureprocedure toto synthesizesynthesize 1,3,5-substituted1,3,5-substituted andand 1,3,5,5-1,3,5,5- substitutedsubstituted barbituratesbarbiturates (Scheme(Scheme 8).8). InIn ththisis procedure,procedure, carbodiimidescarbodiimides (structure(structure 3232)) reactedreacted withwith malonicmalonic acidacid monoethylmonoethyl estersesters (structure(structure 33)33) inin orderorder toto yieldyield NN-acylurea-acylurea intermediatesintermediates (structure(structure 3434)) thatthat wouldwould be,be, afterward,afterward, cyclizedcyclized byby addingadding aa basebase andand thethe desireddesired electrophileelectrophile (structure(structure 3030).). BesidesBesides beingbeing aa single-stepsingle-step strategy,strategy, thethe advantageadvantage ofof thisthis procedureprocedure isis thatthat allall reactionsreactions werewere

Molecules 2019, 24, x FOR PEER REVIEW 13 of 48

Scheme 5.a) K2CO3, 1H-1,2,4-triazole, CH3CN, MW 85 °C (P 50 W), 50 min, 98%. b) 1,2-dichloroethane,

PCl5, reflux, 1.3 h.

Modifications of the loreclezole scaffold have been investigated to discover new potential anticonvulsants. The synthesis of these derivatives is shown in Scheme 6. After alkylation of 1,2,4- triazole with 2,4′-dichloroacetophenone (compound 24). The hydroxylamine 26 was obtained by refluxing with hydroxylamine hydrochloride under basic conditions. Lastly, esterification using DCC as a coupling agent yielded the final compound 27.

Scheme 6. a) 1,2,4-triazole, Na, CH3OH, reflux. b) NH2OH·HCl, C2H5OH, pH 14, reflux. c) R-COOH, MoleculesDCC,2020 DMAP,, 25, 999 DCM, ethereal g HCl. 13 of 47

Similarly, nafimidone (compound 20) is synthesized, as shown in Scheme 7 [64–66], and also Similarly, naﬁmidone (compound 20) is synthesized, as shown in Scheme7[ 64–66], and also denzimol (compound 19) is synthesized in a similar fashion, starting from the corresponding α- denzimol (compound 19) is synthesized in a similar fashion, starting from the corresponding α-haloalkyl haloalkyl ketones [66]. ketones [66].

2, SchemeScheme 7. 7.a) a) BrBr2, glacial acetic acid, ice, overnight 80%. b) 1,2,4-triazole, DMF, 00 ◦°C,C, 2 h, 45%.

4.1.2.4.1.2. MonocyclicMonocyclic GABAGABAAA Receptor Modulators with Six-Membered Rings

Molecules 2019, 24, x FOR PEER REVIEW 14 of 48 BarbituratesBarbiturates performedBarbituratesBarbiturates under (structuresmild (structures conditions.30 30and and The31) are31solvents) derivativesare derivatives employed of barbituric wereof barbituric THF, acid, dioxane, which acid, can acetonitrile,which positively can positively modulateor DCM, the GABA receptor and, at higher concentrations, can activate the receptor without the endogenous evenmodulate though Athe dioxane GABA provided,A receptor in and, general, at higher the best concentrations, results. With can this activateprotocol, the the receptor yields mostly without were the superiorligandendogenous [67 to,68 60% ].ligand [2]. [67,68]. AThe limiting synthesis step ofof thisthis familyprocedure of compounds was identified has for been dialkyl reviewed carbodiimides by Vardanyan that were and Hruby employed [67]. asThus, reagentsThe only synthesis (i.e., contributions diisopropyl of this since family carbodiimide). then of arecompounds discussed In thes hae incases,s thisbeen decarboxylation chapter. reviewed Traditionally, by Vardanyan of the malonate barbiturates and Hruby (structure can [67]. be 33)preparedThus, occurred. only by contributions a Thus, condensation reactions since reaction with then this of are ureatype discussed andof carbodiimides the correspondingin this chapter. were malonic performedTraditionally, ester in with a barbiturates less sodium polar alkoxidesolvent can be (DCM)inprepared ethanol and by or in DMSOa thecondensation presence [2,67]. A of disadvantagereaction TMP in of order urea of this andto traditionalenhance the corresponding the procedure O-N acyl malonic is migration that it ester yields process. with barbiturate sodium This modificationderivativesalkoxide in in ethanolsatisfactorily a modest or yieldDMSO resolved due [2,67]. to the several A issue disadvantagewell-known and yielded ofside respectivethis reactions. traditional desired Additionally, procedure products is thisin that yields procedure it yieldsover 67%requiresbarbiturate [2]. dry derivatives solvents, high in a temperatures,modest yield due inert to atmospheres,several well-known and metallic side reactions. sodium, Additionally, which limits thethis scopeprocedureC-monosubstituted of the requires derivatives dry thatsolvents,barbiturates can be high obtained (structuretemperatures [2]. 30), inertwere atmospheres,obtained by andquenching metallic thesodium, barbituric which carbanionlimitsVolonterio the scopewith andofprotic the Zanda derivativesacids developed and that using can a one-pot2Nbe obtained NaOH procedure [2].as a base. to synthesizeHowever, 1,3,5-substitutedin the case of andthe tetrasubstituted1,3,5,5-substitutedVolonterio barbiturates,and barbiturates Zanda developed it (Scheme was found a8 ).one-pot In that this some procedure procedure, carbanions to carbodiimides synthesize of barbiturat 1,3,5-substituted (structurees failed32 )to reacted andreact 1,3,5,5- with with theirmalonicsubstituted respective acid barbiturates monoethyl electrophiles. esters(Scheme Thus, (structure 8).a slightly In 33)this in modifiedprocedure, order to procedure yield carbodiimidesN-acylurea was developed, intermediates(structure where 32) (structurereacted anhydrous with34) that would be, afterward, cyclized by adding a base and the desired electrophile (structure 30). Besides Kmalonic2CO3 was acid employed monoethyl as aesters base. (structure ACN became 33) in the order solvent to yield of choice N-acylurea and the intermediates reaction was (structure carried out 34 ) atbeingthat 120 would °C a single-step in abe, sealed afterward, strategy,tube. This cyclized the protocol advantage by yieldedadding of this thea base proceduredesired and tetrasubstitutedthe is desired that all electrophile reactions barbiturates were (structure performed(structure 30). 31underBesides) in yields mild being conditions.superior a single-step to The65% solvents(Scheme strategy, employed 8). the It is advantage noteworthy were THF, of to dioxane,this mention procedure acetonitrile, that this is alkylationthat or DCM,all reactions protocol even though werewas dioxane provided, in general, the best results. With this protocol, the yields mostly were superior to also directly tested with barbituric acid as a starting point with satisfactory yields (Scheme not shown)60% [2]. [2].

Scheme 8. a) Solvent, r.t., 12 h, b) 2 N NaOH, H3O++, solvent, c) ACN, r.t., overnight, sealed tube, and Scheme 8. a) Solvent, r.t., 12 h, b) 2 N NaOH, H3O , solvent, c) ACN, r.t., overnight, sealed tube, and d) K2CO3, R4-X,4 ACN, 120 °C, sealed tube. d) K2CO3,R -X, ACN, 120 ◦C, sealed tube.

Additionally, another synthesis of heterocyclic-substituted barbiturates (structure 36) was reported by performing a Knoevenagel condensation of barbituric acid and/or thiobarbituric acid (structure 37) and differently substituted aldehydes (structure 38). This procedure was performed either in the presence of acetic acid or in the presence of sodium acetate. Modified conditions were also performed under microwave irradiation, which substantially improved yields. This procedure enabled the access to barbituric acid derivatives (structure 36) with different aromatic or heteroaromatic substituents and, thus, represents a methodology that enhances the diversity of this scaffold (Scheme 9) [69].

Molecules 2020, 25, 999 14 of 47

A limiting step of this procedure was identified for dialkyl carbodiimides that were employed as reagents (i.e., diisopropyl carbodiimide). In these cases, decarboxylation of the malonate (structure 33) occurred. Thus, reactions with this type of carbodiimides were performed in a less polar solvent (DCM) and in the presence of TMP in order to enhance the O-N acyl migration process. This modification satisfactorily resolved the issue and yielded respective desired products in yields over 67% [2]. C-monosubstituted barbiturates (structure 30) were obtained by quenching the barbituric carbanion with protic acids and using 2N NaOH as a base. However, in the case of the tetrasubstituted barbiturates, it was found that some carbanions of barbiturates failed to react with their respective electrophiles. Thus, a slightly modified procedure was developed, where anhydrous K2CO3 was employed as a base. ACN became the solvent of choice and the reaction was carried out at 120 ◦C in a sealed tube. This protocol yielded the desired tetrasubstituted barbiturates (structure 31) in yields superior to 65% (Scheme8). It is noteworthy to mention that this alkylation protocol was also directly tested with barbituric acid as a starting point with satisfactory yields (Scheme not shown) [2]. Additionally, another synthesis of heterocyclic-substituted barbiturates (structure 36) was reported by performing a Knoevenagel condensation of barbituric acid and/or thiobarbituric acid (structure 37) and differently substituted aldehydes (structure 38). This procedure was performed either in the presence of acetic acid or in the presence of sodium acetate. Modified conditions were also performed under microwave irradiation, which substantially improved yields. This procedure enabled the access to barbituric acid derivatives (structure 36) with different aromatic or heteroaromatic substituents and, thus,Molecules represents 2019, 24, x a FOR methodology PEER REVIEW that enhances the diversity of this scaffold (Scheme9)[69]. 15 of 48

SchemeScheme 9. 9.a) a)CH CH33COOHCOOH oror CHCH33COONa, reﬂux,reflux, 12–3612–36 h.h.

4.2. Fused Bicyclic GABAA Receptor Modulators 4.2. Fused Bicyclic GABAA Receptor Modulators

4.2.1. Fused Bicyclic GABAA Receptor Modulators Containing One Nitrogen 4.2.1. Fused Bicyclic GABAA Receptor Modulators Containing One Nitrogen Quinolones Quinolones 4-Quinolinone derivatives have been reported as high-affinity ligands at the BZD binding site 4-Quinolinone derivatives have been reported as high-affinity ligands at the BZD binding site of GABAA receptors [70]. More recently, this scaffold was used for the synthesis of fluoro-labeled of GABAA receptors [70]. More recently, this scaffold was used for the synthesis of fluoro-labeled compounds (compounds 39 and 40) as PET radiotracers for the GABAA receptors [71]. Starting from 41compounds, the addition (compounds of diethyl 39 ethoxymethylenemalonate and 40) as PET radiotracers yielded for the42 GABAthat wasA receptors heated in[71]. the Starting presence from of diphenyl41, the addition ether to of deliver diethyl43 .et Hydrolysishoxymethylenemalonate of the ester 43 andyielded amide 42 formationthat was heated in thepresence in the presence of HATU of diphenyl ether to deliver 43. Hydrolysis of the ester 43 and amide formation in the presence of HATU led to (compounds 39 and 40) (Scheme 10). Among these, compound 45 (structure 39,R=CH2Ph) was foundled to to(compounds be a potent 39 ligand and 40 of) the(Scheme GABA 10).receptors Among these, (Ki = 0.70compound0.66 nM).45 (structure 39, R=CH2Ph) was A ± foundOther to be quinolinone a potent ligand derivatives of the GABA haveA receptors been reported (Ki = 0.70 as ± anticonvulsants. 0.66 nM). Starting from the pharmacophore of nitrocoumarine-derivatives (structure 46, Figure8), 4 amino-3-nitro-quinolin-2-one derivatives were designed (structure 47, Figure8)[ 72]. These structures are synthesized as displayed in Scheme 11 the reaction of malonic acid ester (compound 49) and aniline (compound 48) yielded compound 50. Compound 51 was obtained via cyclo-condensation of compound 50 in the presence of polyphosphoric acid. In the following steps, nitration and chlorination were performed to deliver

Scheme 10. a) diethyl ethoxymethylenemalonate, 3 h, 120 °C, 80%. (b) diphenyl ether, 1 h, reflux, 50%. (c) NaOH (2 N), ethanol, 2 h, reflux, 90%. (d) 2-fluoroethylamine/3-fluoropropylamine, trifluoroacetate, HATU, DIPEA, DMF, 12 h, rt.

Other quinolinone derivatives have been reported as anticonvulsants. Starting from the pharmacophore of nitrocoumarine-derivatives (structure 46, Figure 8), 4 amino-3-nitro-quinolin-2- one derivatives were designed (structure 47, Figure 8) [72]. These structures are synthesized as displayed in Scheme 11 the reaction of malonic acid ester (compound 49) and aniline (compound 48) yielded compound 50. Compound 51 was obtained via cyclo-condensation of compound 50 in the presence of polyphosphoric acid. In the following steps, nitration and chlorination were performed to deliver compound 53. Lastly, the reaction with GABA methyl ester and the following hydrolysis delivered compound 55 (Scheme 11). Additionally, structure 47 showed anticonvulsant activity. In the same work, 4-amino-3-nitro-1-thiocoumarins were investigated. However, the thio-coumarin derivatives were found to have a lower anticonvulsant activity compared to the coumarin derivatives (O-containing).

Molecules 2019, 24, x FOR PEER REVIEW 15 of 48

Scheme 9. a) CH3COOH or CH3COONa, reflux, 12–36 h.

4.2. Fused Bicyclic GABAA Receptor Modulators

4.2.1. Fused Bicyclic GABAA Receptor Modulators Containing One Nitrogen

Quinolones

Molecules4-Quinolinone2020, 25, 999 derivatives have been reported as high-affinity ligands at the BZD binding15 ofsite 47 of GABAA receptors [70]. More recently, this scaffold was used for the synthesis of fluoro-labeled compounds (compounds 39 and 40) as PET radiotracers for the GABAA receptors [71]. Starting from compound41, the addition53. Lastly, of diethyl the reaction ethoxymethylenemalonate with GABA methyl yielded ester and 42 thethat following was heated hydrolysis in the presence delivered of compounddiphenyl ether55 (Scheme to deliver 11). 43 Additionally,. Hydrolysis structureof the ester47 43showed and amide anticonvulsant formation activity. in the presence In the same of HATU work, 4-amino-3-nitro-1-thiocoumarinsled to (compounds 39 and 40) (Scheme were 10). investigated. Among these, However, compound the thio-coumarin 45 (structure 39 derivatives, R=CH2Ph) were was found to havebe a potent a lower ligand anticonvulsant of the GABA activityA receptors compared (Ki = to0.70 the ± coumarin0.66 nM). derivatives (O-containing).

Molecules 2019, 24, x FOR PEER REVIEW 16 of 48

Scheme 10. a)a) diethyl diethyl ethoxymethylenemalonate, ethoxymethylenemalonate, 3 h, 120 °C◦C,, 80%. 80%. (b) (b) diphenyl diphenyl ether, ether, 1 1 h, h, reflux, reflux, 50%. 50%. (c)(c) NaOHNaOH (2 N),(2 ethanol,N), ethanol, 2 h, reflux, 2 h, 90%. reflux, (d) 2-fluoroethylamine 90%. (d) 2-fluoroethylamine/3-fluoropropylamine,/3-fluoropropylamine, trifluoroacetate, MoleculesHATU,trifluoroacetate, 2019, DIPEA, 24, x FORDMF, HATU, PEER 12 REVIEW DIPEA, h, rt. DMF, 12 h, rt. 16 of 48

Other quinolinone derivatives have been reported as anticonvulsants. Starting from the pharmacophore of nitrocoumarine-derivatives (structure 46, Figure 8), 4 amino-3-nitro-quinolin-2- one derivatives were designed (structure 47, Figure 8) [72]. These structures are synthesized as displayed in Scheme 11 the reaction of malonic acid ester (compound 49) and aniline (compound 48) yielded compound 50. Compound 51 was obtained via cyclo-condensation of compound 50 in the presence of polyphosphoric acid. InFigure the following 8. Coumarin steps, derivatives. nitration and chlorination were performed to deliver compound 53. Lastly, the reaction with GABA methyl ester and the following hydrolysis delivered compound 55 (Scheme 11). Additionally, structure 47 showed anticonvulsant activity. In the same work, 4-amino-3-nitro-1-thiocoumarins were investigated. However, the thio-coumarin Figure 8. Coumarin derivatives. derivatives were found to have a lower anticonvulsant activity compared to the coumarin derivatives (O-containing).

Scheme 11. a) 210 °C, 5 h, 76%. b) Polyphosphoric acid, 150 °C, 5 h, 73. c) HNO3, 75 °C, 12 min, Scheme 11. a) 210 ◦C, 5 h, 76%. b) Polyphosphoric acid, 150 ◦C, 5 h, 73. c) HNO3, 75 ◦C, 12 min, 93%. 3 d)93%. POCl d)3 POCl, TBBAB,, TBBAB, MeCN, MeCN, 40 ◦C, 40 30 °C, min, 30 reflux,min, reflux, 22 min, 22 60%.min, 60%. e) Py, e) reflux, Py, reflux, 2 h, 63%. 2 h, 63%. (f) NaOH, (f) NaOH, H2O, MeOH, reflux, 40 min, 64%. H2O, MeOH, reflux, 40 min, 64%.

Scheme 11. a) 210 °C, 5 h, 76%. b) Polyphosphoric acid, 150 °C, 5 h, 73. c) HNO3, 75 °C, 12 min, Indol-3-yl glyoxylglyoxyl DerivativesDerivatives 93%. d) POCl3, TBBAB, MeCN, 40 °C, 30 min, reflux, 22 min, 60%. e) Py, reflux, 2 h, 63%. (f) NaOH, The indol-3-yl glyoxyl scascaffoldﬀoldH (structure2O, MeOH, 56 reflux,) has 40 also min, been 64%. explored as a template for designing GABAA receptorreceptor ligands. Efficacy Eﬃcacy on on co-application co-application with with GABA GABA has has been been screened for several derivativesIndol-3-yl glyoxyl and some Derivatives derivatives behaved as positive modulators with efficacies comparable to diazepam.The indol-3-yl The general glyoxyl structure scaffold of this(structure scaffold 56 is) hasdepicted also been in Figure explored 9. as a template for designing GABAA receptor ligands. Efficacy on co-application with GABA has been screened for several derivatives and some derivatives behaved as positive modulators with efficacies comparable to diazepam. The general structure of this scaffold is depicted in Figure 9.

Figure 9. Structure of Indol-3-yl-glyoxyl scaffold.

Synthesis of this scaffold (structure 56) is reported by first performing an acylation of the accordingly substituted indole (structure 57) with oxalyl chloride in anhydrous ether [73] and, Figure 9. Structure of Indol-3-yl-glyoxyl scaffold. afterward, treating the resulting compound with the desired glyoxalyl chloride (structure 58) with eitherSynthesis the corresponding of this scaffold hydrazine (structure hydrochloride 56) is reported (structure by 61 first) in theperf presenceorming anof aacylation base [74] ofor the correspondingaccordingly substituted amine (structure indole 62(structure) [75]. Alternatively, 57) with oxalyl derivatives chloride with in a heterocyclicanhydrous ethertriazole [73] moiety and, (structureafterward, 59 treating) can be the prepared resulting by compound reacting the with corresponding the desired glyoxalylindole-3-glyoxalyl chloride (structurechloride (structure 58) with 58either) with the propargyl corresponding amine hydrazine to afford hydrochloridethe corresponding (structure derivative 61) in with the presencea terminal of alkynea base [74](structure or the 60corresponding). The alkyne amine moiety (structure was 62cyclized) [75]. Alternatively,with click chemistry derivatives conditions with a heterocyclic using the triazole accordingly moiety substituted(structure 59 azide) can (Schemebe prepared 12) [76]. by reacting the corresponding indole-3-glyoxalyl chloride (structure 58) with propargyl amine to afford the corresponding derivative with a terminal alkyne (structure 60). The alkyne moiety was cyclized with click chemistry conditions using the accordingly substituted azide (Scheme 12) [76].

Molecules 2019, 24, x FOR PEER REVIEW 16 of 48

Figure 8. Coumarin derivatives.

Scheme 11. a) 210 °C, 5 h, 76%. b) Polyphosphoric acid, 150 °C, 5 h, 73. c) HNO3, 75 °C, 12 min, 93%. d) POCl3, TBBAB, MeCN, 40 °C, 30 min, reflux, 22 min, 60%. e) Py, reflux, 2 h, 63%. (f) NaOH,

H2O, MeOH, reflux, 40 min, 64%.

Indol-3-yl glyoxyl Derivatives Molecules 2020, 25, 999 16 of 47 The indol-3-yl glyoxyl scaffold (structure 56) has also been explored as a template for designing GABAA receptor ligands. Efficacy on co-application with GABA has been screened for several derivatives and some derivatives behaved as positivepositive modulators with eefficaciesﬃcacies comparable to diazepam. The general structure of this scascaffoldﬀold is depicted in Figure9 9..

Figure 9. StructureStructure of Indol-3-yl-glyoxyl scaffold. scaﬀold.

Synthesis ofof this this sca scaffoldffold (structure (structure56) is56 reported) is reported by first by performing first perf anorming acylation an ofacylation the accordingly of the accordinglysubstituted indolesubstituted (structure indole57) with(structure oxalyl 57 chloride) with inoxalyl anhydrous chloride ether in [anhydrous73] and, afterward, ether [73] treating and, afterward,the resulting treating compound the resulting with the desired compound glyoxalyl with chloridethe desired (structure glyoxalyl58) withchloride either (structure the corresponding 58) with eitherhydrazine the corresponding hydrochloride hydrazine (structure hydrochloride61) in the presence (structure of a 61 base) in [the74] presence or the corresponding of a base [74] amineor the (structurecorresponding62)[ 75amine]. Alternatively, (structure 62 derivatives) [75]. Alternatively, with a heterocyclic derivatives with triazole a heterocyclic moiety (structure triazole 59moiety) can (structurebe prepared 59 by) can reacting be prepared the corresponding by reacting indole-3-glyoxalylthe corresponding chlorideindole-3-glyoxalyl (structure 58chloride) with propargyl(structure 58amine) with to propargyl afford the correspondingamine to afford derivative the corresponding with a terminal derivative alkyne with (structure a terminal60). The alkyne alkyne (structure moiety was60Molecules). cyclizedThe 2019alkyne, with24, x FOR clickmoiety PEER chemistry REVIEWwas cyclizedconditions with using click the chemistry accordingly conditions substituted using azide the (Scheme accordingly 1217)[ of76 ].48 substituted azide (Scheme 12) [76].

SchemeScheme 12. 12.a) a) Oxalyl Oxalyl chloride, chloride, dry dry diethyl diethyl ether, ether, r.t., r.t., 30 30 min, min, quant. quant. b) b) NH-Aryl, NH-Aryl, TEA, TEA, Toluene, Toluene, r.t. r.t. c) c) ArylhydrazineArylhydrazine hydrochloride, hydrochloride, TEA, TEA, toluene, toluene,r.t., r.t., 24–36 24–36 h. h.d) d)Propargyl Propargylamine, amine,dry dryPyridine, Pyridine, r.t.,r.t.,8 8 h, h, quant.quant. e) e) Aryl-azide, Aryl-azide, CuSO CuSO4H·H2O,O, sodiumsodium ascorbate,ascorbate,t t-BuOH-BuOH/H/H 2OO (1:1),(1:1), r.t.,r.t., 88 h.h. 4· 2 2

4.2.2.4.2.2. Fused Fused Bicyclic Bicyclic GABA GABAAA ReceptorReceptor ModulatorsModulators ContainingContainingTwo Two Nitrogens Nitrogens and and 5–6 5–6 Membered Membered Rings Rings Benzimidazoles Benzimidazoles Benzimidazoles are a scaffold that was first reported in 1999 as modulators of the GABAA receptor Benzimidazoles are a scaffold that was first reported in 1999 as modulators of the GABAA with the aim to develop functionally selective ligands [77]. Figure 10 depicts examples of this family of receptor with the aim to develop functionally selective ligands [77]. Figure 10 depicts examples of compounds are NS 2710 (compound 63), NS 11,394 (compound 64), and NS 16,085 (compound 65). this family of compounds are NS 2710 (compound 63), NS 11,394 (compound 64), and NS 16,085 NS 11,394 (compound 64) is a positive allosteric modulator for which functional selectivity has been (compound 65). NS 11,394 (compound 64) is a positive allosteric modulator for which functional explored in a small panel of α5-, α3-, α2-, and α1-containing GABAA receptors. [78,79]. The profiles of selectivity has been explored in a small panel of α5-, α3-, α2-, and α1-containing GABAA receptors. NS 2710 (compound 63) and NS16085 (compound 65) showed subtle differences in comparison [79,80]. [78,79]. The profiles of NS 2710 (compound 63) and NS16085 (compound 65) showed subtle differences in comparison [79,80].

Figure 10. Structure of benzimidazole derivatives.

Synthetic accessibility of this scaffold is reported in general via a Suzuki-Miyaura coupling, using 3-bromophenyl-1H-benzo[d]imidazole with the desired substituent in position 5, the accordingly substituted aryl boronic acid, sodium carbonate as a base, and 5 mol% of the catalyst (Scheme 13) [81]. This procedure is compatible with several functional groups in position 5 (alkyl alcohols, trifluoromethyl, ketones, aldehydes, nitriles, amides, and heterocyclic rings such as oxazoles, pyrazoles, and imidazoles). Additionally, the corresponding arylboronic acids can be substituted with halogens, methoxy groups, short-chain alkyl groups such as methyl or ethyl, sulfonamides, or even a heterocyclic ring such as a pyridine.

Molecules 2019, 24, x FOR PEER REVIEW 17 of 48

Scheme 12. a) Oxalyl chloride, dry diethyl ether, r.t., 30 min, quant. b) NH-Aryl, TEA, Toluene, r.t. c) Arylhydrazine hydrochloride, TEA, toluene, r.t., 24–36 h. d) Propargyl amine, dry Pyridine, r.t., 8 h,

quant. e) Aryl-azide, CuSO4·H2O, sodium ascorbate, t-BuOH/H2O (1:1), r.t., 8 h.

4.2.2. Fused Bicyclic GABAA Receptor Modulators Containing Two Nitrogens and 5–6 Membered Rings

Benzimidazoles

Benzimidazoles are a scaffold that was first reported in 1999 as modulators of the GABAA receptor with the aim to develop functionally selective ligands [77]. Figure 10 depicts examples of this family of compounds are NS 2710 (compound 63), NS 11,394 (compound 64), and NS 16,085 (compound 65). NS 11,394 (compound 64) is a positive allosteric modulator for which functional selectivity has been explored in a small panel of α5-, α3-, α2-, and α1-containing GABAA receptors. [78,79]. The profiles of NS 2710 (compound 63) and NS16085 (compound 65) showed subtle differences in comparison [79,80]. Molecules 2020, 25, 999 17 of 47

Figure 10. Structure of benzimidazole derivatives.

Synthetic accessibilityaccessibility of of this this sca scaffoldﬀold is reportedis reported in generalin general via avia Suzuki-Miyaura a Suzuki-Miyaura coupling, coupling, using 3-bromophenyl-1using 3-bromophenyl-1H-benzo[H-benzo[d]imidazoled]imidazole with the with desired the substituentdesired substituent in position in 5, position the accordingly 5, the substitutedaccordingly aryl substituted boronic acid,aryl sodiumboronic carbonateacid, sodium as a carbonate base, and 5as mol% a base, of theand catalyst 5 mol% (Scheme of the catalyst13)[81]. This(Scheme procedure 13) [81]. is compatible This procedure with several is compatible functional with groups several in position functional 5 (alkyl groups alcohols, in position triﬂuoromethyl, 5 (alkyl ketones,alcohols, aldehydes, trifluoromethyl, nitriles, amides,ketones, and aldehydes, heterocyclic nitriles, rings suchamides, as oxazoles, and heterocyclic pyrazoles, andrings imidazoles). such as Additionally,oxazoles, pyrazoles, the corresponding and imidazoles). arylboronic Additionally, acids can bethe substituted corresponding with halogens,arylboronic methoxy acids groups,can be short-chainsubstituted alkylwith groupshalogens, such methoxy as methyl groups, or ethyl, short-chain sulfonamides, alkyl orgroups even asuch heterocyclic as methyl ring or such ethyl, as Moleculesasulfonamides, pyridine. 2019, 24, orx FOR even PEER a heterocyclic REVIEW ring such as a pyridine. 18 of 48

SchemeScheme 13. 13.a) a)(Ph (Ph33P)P)22PdCl2,, Na Na2COCO33, ,DME/H DME/H22O.O.

Compounds such as NS11394 (compound 64) and NS16085 (compound 65) can be obtained using intermediate 7272 asas a a starting starting point point (Scheme (Scheme 14). 14). The The synthesis synthesis of of7272 beginsbegins with with anan SNAr SNAr of of4- fluoro-3-nitro-acetophenone4-ﬂuoro-3-nitro-acetophenone (compound (compound 6969) )with with 3-bromoaniline 3-bromoaniline (compound (compound 7070)) giving giving compound 71. Compound Compound 7171 isis converted converted to to the the key key intermediate intermediate 7272 viavia reduction reduction of of the nitro group to the corresponding ortho-dianiline and cyclization to thethe benzimidazole using formic acid as a methylene source in a a one-step one-step fashion. fashion. In In order order to to obtain obtain compound compound NS NS 2710 2710 (compound (compound 63),63 intermediate), intermediate 72 undergoes72 undergoes a Suzuki-Miyaura a Suzuki-Miyaura coupling coupling using using pyridine-4-ylboronic pyridine-4-ylboronic acid acid and, and, subsequently, subsequently, the resulting product (compound 73) is treated with o-ethylhydroxylamine-ethylhydroxylamine hydrochloridehydrochloride to obtain the corresponding oxime (compound 63). Further treatment of intermediate 72 with methylmagnesium chloride yielded the tertiary alcohol 74. The The synthesis of both NS11394 (compound 64) and NS1685 (compound 65) requires the Suzuki-Miyaura coupling of intermediate 74 with the corresponding aryl boronic acid.

Scheme 14. a) NMP, 80 °C, N2 atm, 72 h, 95% yield. b) Fe, formic acid, acetic acid, EtOAc, 70 °C, 30 h, 88% yield. c) Suzuki coupling, conditions not specified, yield not reported. d) o-ethylene hydroxylamine HCl, EtOH abs, reflux, 1.5 h, 60% e) MeMgCl, THF, rt, overnight, 57% f) 2- cyanophenyl boronic acid, sodium carbonate, dichlorobis(triphenylphosphine)palladium, 1,2- Dimethoyxethane, 90 °C, 6–8 h, yield not reported. g) (2-Chloro-5-cyanophenyl)boronic acid, sodium carbonate, dichlorobis(triphenylphosphine)palladium, 1,2-Dimethoyxethane, 90 °C, 6–8 h, yield not reported [81–83].

Molecules 2019, 24, x FOR PEER REVIEW 18 of 48

Scheme 13. a) (Ph3P)2PdCl2, Na2CO3, DME/H2O.

Compounds such as NS11394 (compound 64) and NS16085 (compound 65) can be obtained using intermediate 72 as a starting point (Scheme 14). The synthesis of 72 begins with an SNAr of 4- fluoro-3-nitro-acetophenone (compound 69) with 3-bromoaniline (compound 70) giving compound 71. Compound 71 is converted to the key intermediate 72 via reduction of the nitro group to the corresponding ortho-dianiline and cyclization to the benzimidazole using formic acid as a methylene source in a one-step fashion. In order to obtain compound NS 2710 (compound 63), intermediate 72 undergoes a Suzuki-Miyaura coupling using pyridine-4-ylboronic acid and, subsequently, the resulting product (compound 73) is treated with o-ethylhydroxylamine hydrochloride to obtain the corresponding oxime (compound 63). Further treatment of intermediate 72 with methylmagnesium chloride yielded the tertiary alcohol 74. The synthesis of both NS11394 (compound 64) and NS1685 Molecules(compound2020, 2565,) 999 requires the Suzuki-Miyaura coupling of intermediate 74 with the corresponding18 ofaryl 47 boronic acid.

SchemeScheme 14. a)a) NMP, NMP, 80 80°C,◦ NC,2 atm, N2 atm, 72 h, 7295% h, yield. 95% b) yield. Fe, formic b) Fe, acid, formic acetic acid, acid, acetic EtOAc, acid, 70 °C, EtOAc, 30 h, 7088%◦C, yield. 30 h, c) 88% Suzuki yield. coupling, c) Suzuki conditions coupling, not conditions specified, not yield speciﬁed, not reported. yield not reported.d) o-ethylene d) ohydroxylamine-ethylene hydroxylamine HCl, EtOH HCl, abs, EtOHreflux, abs, 1.5 reﬂux,h, 60% 1.5e) h,MeMgCl, 60% e) THF, MeMgCl, rt, overnight, THF, rt, overnight,57% f) 2- 57%cyanophenyl f) 2-cyanophenyl boronic boronicacid, sodium acid, sodium carbonate, carbonate, dichlorobis(triphenylphosphine)palladium, dichlorobis(triphenylphosphine)palladium, 1,2- 1,2-Dimethoyxethane,Dimethoyxethane, 90 °C, 90 6–8◦C, h, 6–8 yield h, yieldnot reported. not reported. g) (2-Chlor g) (2-Chloro-5-cyanophenyl)boronico-5-cyanophenyl)boronic acid, sodium acid, sodiumcarbonate, carbonate, dichlorobis(triphenylphosphine)palladi dichlorobis(triphenylphosphine)palladium,um, 1,2-Dimethoyxethane, 1,2-Dimethoyxethane, 90 °C, 6–8 90 h,◦ C,yield 6–8 not h, yieldreported not reported[81–83]. [81–83]. Imidazopyridines

Zolpidem (compound 75) (Ambien ®) is the most prominent representative of the imidazopyridine compound class. It belongs to the “Z-Drugs,” which take their name from the initial letter of the drugs name such as zolpidem (Ambien ®), zaleplon (Sonata®), and zopiclone (Imovane, Zimovane, Dopareel®). [84] Z-drugs are non-benzodiazepines that act as positive modulators at some of the same binding sites as the benzodiazepines, but with a different selectivity profile toward the α-isoforms. They are mostly prescribed in treating insomnia and sleeping disorders [85]. Zolpidem has a short elimination half-life and a rapid onset of effect, which makes it a short-acting sleeping aid [86,87]. Different strategies have been investigated for the synthesis of Zolpidem [88]. The most common routes involve the synthesis of the acetonitrile intermediate 76 [89–91]: treatment of 2-bromo-1-(p-tolyl)ethan-1-one (compound 77) with 2-amino-5-methylpyridine yielded compound 78. The addition of formalin, dimethylamine, and acetic acid lead to the dimethylamino-imidazopyridine (compound 70), which, upon addition of methyl iodide, followed by the addition of sodium cyanate delivers intermediate 76. In the last two steps, hydrolysis and amidation are performed to obtain zolpidem (compound 75), as depicted in Scheme 15. Other strategies involve a formylation approach for the synthesis of the acetonitrile intermediate 76 (Scheme 16)[91]. Formylation is obtained by the treatment of 78 with oxalyl chloride and dimethylformamide, which is followed by a reduction to yield alcohol 83. This is, subsequently, transformed to the pyridinium tosylate salt 84. The addition of sodium cyanide to 84 leads to the formation of 76. However, both procedures are not suitable for large-scale production due to the toxicity of the reagents (oxalyl chloride, methyl iodide), their moisture sensitivity (carbonyldiimidazole), their volatility (methyl iodide), and unsatisfactory yields. Therefore, different strategies have been investigated. As reported by Eswaraiah et al. [92], intermediate 76 can be obtained using ethyl chloroformate for the synthesis of the quaternary salt that, upon treatment with sodium cyanide, delivered the key intermediate 76 (scheme not shown). Furthermore, the treatment of acid 81 with Molecules 2019, 24, x FOR PEER REVIEW 19 of 48

Imidazopyridines

tolyl)ethan-1-oneMolecules 2020, 25, 999 (compound 77) with 2-amino-5-methylpyridine yielded compound 7819. ofThe 47 addition of formalin, dimethylamine, and acetic acid lead to the dimethylamino-imidazopyridine (compound 70), which, upon addition of methyl iodide, followed by the addition of sodium cyanate pivaloyldelivers chlorideintermediate in triethylamine 76. In the last followed two steps, by the hydrolysis additionof and aqueous amidation dimethylamine are performed solution to obtain led to zolpidemthe formation (compound of zolpidem 75), as in depicted good yields. in Scheme 15.

SchemeScheme 15. 15.a) a) 2-Amino-5-methylpyridine, 2-Amino-5-methylpyridine, NaHCONaHCO33, EtOH, ∆.. (b) (b) Acetic Acetic acid, acid, aq. aq. dimethylamine, dimethylamine, formalin,formalin, rt (c)rt (c) Acetone, Acetone, CH CH3I, 3∆I, (d)∆ (d) NaCN, NaCN, EtOH, EtOH,∆ (e)∆ (e) KOH, KOH, EtOH EtOH//∆ (f)∆ (f) Carbonyldiimidazole, Carbonyldiimidazole, THF, MoleculesMoleculesdimethylamine, 20192019,, 2424,, xx FORFOR rt. PEERPEER REVIEWREVIEW THF, dimethylamine, rt. 2020 ofof 4848

Other strategies involve a formylation approach for the synthesis of the acetonitrile intermediate 76 (Scheme 16) [91]. Formylation is obtained by the treatment of 78 with oxalyl chloride and dimethylformamide, which is followed by a reduction to yield alcohol 83. This is, subsequently, transformed to the pyridinium tosylate salt 84. The addition of sodium cyanide to 84 leads to the formation of 76. However, both procedures are not suitable for large-scale production due to the toxicity of the reagents (oxalyl chloride, methyl iodide), their moisture sensitivity (carbonyldiimidazole), their volatility (methyl iodide), and unsatisfactory yields. Therefore, different strategies have been investigated. As reported by Eswaraiah et al. [92], intermediate 76 can be obtained using ethyl chloroformate for the synthesis of the quaternary salt that, upon treatment with sodium cyanide, delivered the key intermediate 76 (scheme not shown). Furthermore, the treatment

of acid 81 with pivaloyl chloride in triethylamine followed by the addition of aqueous dimethylamine SchemeSchemeScheme 16. 16.16.a) a) Oxalyla) OxalylOxalyl chloride, chloride,chloride, DMF DMFDMF30 −−3030C °C°C to to 0to 0C,0 °C,°C, 30 30 min,30 min,min, yield yieldyield not notnot reported. reported.reported. b) NaBH b)b) NaBHNaBHrt,4,4, rt, 8rt, h, 88 yieldh,h, solution led to the formation of zolpidem− ◦in good◦ yields. 4, yieldnotyield reported. notnot reported.reported. c) p-toluenesulfonyl c)c) pp-toluenesulfonyl-toluenesulfonyl chloride, chloride,chloride, pyridine, pyridine,pyridine, rt, 8 h, rt,rt, yield 88 h,h, not yieldyield reported. notnot reported.reported. d) NaCN, d)d) NaCN,NaCN, H2O, reﬂux, HH22O,O, 2-5 h, yield not reported. reflux,reflux, 2-52-5 h,h, yieldyield notnot reported.reported.

AnotherAnother reported reported strategy strategy involves involves the the condensation condensation of of the the αα-tosyloxy-tosyloxy ketone ketone 8686,, obtained obtained from from N,NN,N-dimethyl-4-oxo-4-tolylbutanamide-dimethyl-4-oxo-4-tolylbutanamide-dimethyl-4-oxo-4-tolylbutanamide85 85upon85 uponupon treatment treatmenttreatment with HTIB, withwith with HTIB,HTIB, 2-amino-5-methylpyridine withwith 2-amino-5-2-amino-5- methylpyridine(Schememethylpyridine 17)[88 ]. (Scheme(Scheme 17)17) [88].[88].

Scheme 17. p 3 SchemeScheme 17.17. a)a) Iodosobenzene Iodosobenzene (PhIO), (PhIO), pp-TsOH/CH-TsOH-TsOH/CH/CH33CN,CN, reflux reﬂuxreflux 2 2 h. h. b)b) 2-Amino-5-methyl 2-Amino-5-methyl pyridine, pyridine, refluxreﬂuxreflux 2 22 h, h, 47%. 47%.

Other structures containing the imidazopyridine scaffold, such as TP-003 (compound 87) are OtherOther structuresstructures containingcontaining thethe imidazopyridineimidazopyridine scaffold,scaffold, suchsuch asas TP-003TP-003 (compound(compound 8787)) areare described as GABAA modulators. This chemotype reflects an effort to achieve anxio-selective describeddescribed asas GABAGABAAA modulators.modulators. ThisThis chemotypechemotype reflectsreflects anan eeffortffort toto achieveachieve anxio-selectiveanxio-selective activityactivity activity (with reduced sedation) by functional selectivity toward receptor subtypes containing specific (with(with reducedreduced sedation)sedation) byby functionalfunctional selectivityselectivity towardtoward receptorreceptor subtypessubtypes containingcontaining specificspecific αα-- α-isoforms. TP-003 (compound 87) was initially proposed as an α3-selective modulator with anxiolytic isoforms.isoforms. TP-003TP-003 (compound(compound 8787)) waswas initiallyinitially proposedproposed asas anan αα3-selective3-selective modulatormodulator withwith anxiolyticanxiolytic effects, but the in vitro selectivity and the in vivo effects were discussed controversially in subsequent effects,effects, butbut thethe inin vitrovitro selectivityselectivity andand thethe inin vivovivo effectseffects werewere discusseddiscussed controversiallycontroversially inin subsequentsubsequent follow-upfollow-up studiesstudies [93–95].[93–95]. TheThe synthesissynthesis ofof TP-003TP-003 (compound(compound 8787)) isis describeddescribed inin WOWO 03/09981603/099816 andand depicteddepicted inin SchemeScheme 1818 [96,97].[96,97]. 2-Bromo-5-Fluorobenzonitrile2-Bromo-5-Fluorobenzonitrile (compound(compound 8888)) isis coupledcoupled withwith compoundcompound 8989 toto yieldyield compoundcompound 9090.. TheThe additionaddition ofof SnClSnCl22 andand sodiumsodium hydroxidehydroxide inin THFTHF affordedafforded compoundcompound 9191.. Bromo-deaminationBromo-deamination ledled toto thethe formationformation ofof compoundcompound 9292,, whichwhich isis transformedtransformed inin compoundcompound 9393 byby treatmenttreatment withwith bis(pinacolato)diboron.bis(pinacolato)diboron. TheThe followingfollowing couplingcoupling ledled toto thethe protectedprotected precursorprecursor (compound(compound 9595),), whichwhich waswas de-protectedde-protected toto obtainobtain TP-003TP-003 (compound(compound 8787).).

Molecules 2020, 25, 999 20 of 47 follow-up studies [93–95]. The synthesis of TP-003 (compound 87) is described in WO 03/099816 and depicted in Scheme 18[ 96,97]. 2-Bromo-5-Fluorobenzonitrile (compound 88) is coupled with compound 89 to yield compound 90. The addition of SnCl2 and sodium hydroxide in THF aﬀorded compound 91. Bromo-deamination led to the formation of compound 92, which is transformed in

Moleculescompound 2019,93 24,by x FOR treatment PEER REVIEW with bis(pinacolato)diboron. The following coupling led to the protected21 of 48 precursorMolecules 2019 (compound, 24, x FOR PEER95), REVIEW which was de-protected to obtain TP-003 (compound 87). 21 of 48

Scheme 18. a) KOAc, Pd(DPPF)Cl2, 2% DMSO in dioxane, 90 °C, 14 h, 100%. b) 1. SnCl2·2H2O, EtOH:THFSchemeScheme 18.18. (1:1), a)a) KOAc,KOAc, rt, 12 h; Pd(DPPF)ClPd(DPPF)Cl 2. 2 N NaOH,2,, 2%2% rt, DMSODMSO 1 h, 75%. inin dioxane,c)dioxane, 1. HBr 90 90(aq. C,°C, 48%),14 14 h, h,NaNO 100%. 100%.2, b)0b) °C, 1. 1. 2 SnClSnCl h, 2.2 2H·2HCuBr,2O, 2 ◦ 2· 2 NHEtOH:THFEtOH:THF4OH 0 °C (1:1),(1:1), to 50 rt,rt, °C,1212 h; h;10 2.2. min, 22 NN NaOH,64%.NaOH, d) rt,KOAc,rt, 11 h,h, 75%.75%.bis(pinacolato)diboron, c)c) 1.1. HBrHBr (aq.(aq. 48%),48%), PdCl NaNONaNO2[P(cy)22, 0 3◦°C,],C, 1% 2 h,DMSO 2.2. CuBr, in dioxane,NHNH44OH 90 0 ◦ °C°C,C toto 16 5050 h, ◦ 100%.°C,C, 1010 min, e)min, Pd(PPh 64%.64%.3) d)4d) (5 KOAc, KOAc,mol%), bis(pinacolato)diboron, bis(pinacolato)diboron,2 M Na2CO3, THF, 75 °C, PdClPdCl 16 h,22[P(cy)[P(cy) 60%. 3f)3], HCl 1%1% DMSO(aq.37%),DMSO inin EtOH,dioxane,dioxane, 95%. 9090◦ °C,C, 1616 h,h, 100%.100%. e)e) Pd(PPhPd(PPh33))44 (5 mol%), 22 MM NaNa22CO3,, THF, THF, 75 ◦°C,C, 16 h, 60%. f) HCl (aq.37%), EtOH,EtOH, 95%.95%. Among the imidazopyridines, not only ligands of the high-affinity binding site have been described.AmongAmong DS1 thethe imidazopyridines,(compound imidazopyridines, 96) and not not onlyDS2 only ligands(compound ligands of the of97 high-a )the have ﬃhigh-affinitynity been binding introduced binding site have as site been somewhat have described. been δ- DS1 (compound 96) and DS2 (compound 97) have been introduced as somewhat δ-selective modulators selectivedescribed. modulators DS1 (compound of GABA 96)A andreceptors DS2 (compound(Figure 11) 97[98,99].) have δbeen-Subunits introduced are present as somewhat in certain δ- of GABA receptors (Figure 11)[98,99]. δ-Subunits are present in certain extrasynaptic GABA extrasynapticselective Amodulators GABAA receptorsof GABA andA receptors contribute (Figure to pentamers 11) [98,99]. devoid δ-Subunits of high-affinity are present benzodiazepine in certainA bindingreceptorsextrasynaptic sites and [100,101]. contributeGABAA receptors to pentamers and contribute devoid of to high-a pentamersﬃnity devoid benzodiazepine of high-affinity binding benzodiazepine sites [100,101]. binding sites [100,101].

Figure 11. Structures of DS1, DS2, 98, and 99. Figure 11. Structures of DS1, DS2, 98, and 99. Even though DS2 (compound 97) cannot be considered fully δ selective [102], [102], it shows a Even though DS2 (compound 97) cannot be considered fully δ selective [102], it shows a considerable functional preference for δ-containing-containing GABAGABAA receptors and, therefore, different diﬀerent derivativesconsiderable have functional been investigated. preference Among Amongfor δ-containing them, them, compound compound GABA 9898A receptorsand compound and, 99therefore, (Figure 11)11different) have derivatives have been investigated. Among them, compound 98 and compound6 2/3 99 (Figure 11) have shown promising pharmacological profiles proﬁles with potential selectivity for α6βß2/3δ GABAA receptors,receptors, 6 2/3 besidesshown promisingthe δ selectivity, pharmacological whichwhich isis anan profilesinterestinginteresting with selectivityselectivity potential forfor selectivity thetheα α66 subunitsubunit for α ß observed.observed.δ GABA A receptors, besidesGenerally, the δ selectivity, the synthesis which of issuch an interestingderivatives selectivity can be obtained for the in α 6two subunit steps observed.[102] by reacting with an aromaticGenerally, aldehyde the synthesis with 2-amino of such pyridines derivatives in cana multi-component be obtained in two fashion, steps [102]which by is reacting followed with by thean aromaticformation aldehyde of an amide with bond. 2-amino pyridines in a multi-component fashion, which is followed by the formation of an amide bond.

Molecules 2020, 25, 999 21 of 47

Generally, the synthesis of such derivatives can be obtained in two steps [102] by reacting with an aromatic aldehyde with 2-amino pyridines in a multi-component fashion, which is followed by the formation of an amide bond. As shown in Scheme 19, compound 102 is obtained by the reaction of thiophene-2-carbaldehyde (compound 100) with 2-amino-5-bromopyridine (compound 101). The reaction of compound 102 with 4-butoxybenzoyl chloride yielded compound 98. For the synthesis of compound 99, thiophene-2-carbaldehyde (compound 100) is reacted with 2-amino-3,5-dibromopyridine (compound 104) in the presence of 2-isocyano-2-methylpropane to yield compound 105. Upon treatment of compound 105 with HBr (5 M), intermediate 106 was formed. In the following step, the amide formation was performed via reaction with 4-((tert-butyldiphenylsilyl)oxy)benzoyl chloride (compound 107). Deprotection with TBAF followed by a Williamson ether formation delivered compound 99 in a moderate yield (35%). These two syntheses already show that the substitution pattern on the core scaMoleculesﬀold can2019,be 24, easilyx FOR PEER varied REVIEW by using diﬀerently substituted starting materials and reagents. 22 of 48

Scheme 19. a): KCN, aq. Na2S2O5, 100 °C to 25 °C, 4 h, 56%, 3.5 h. b) Toluene/pyridine, 25 °C, 1 h, 57%. Scheme 19. a): KCN, aq. Na2S2O5, 100 ◦C to 25 ◦C, 4 h, 56%, 3.5 h. b) Toluene/pyridine, 25 ◦C, 1 h, c) 2-isocyano-2-methylpropane, 1 M HClO4, MeOH, 25 °C, 24 h, 52%. d): 5 M HBr, 80–110 °C, 3.5 h, 57%. c) 2-isocyano-2-methylpropane, 1 M HClO4, MeOH, 25 ◦C, 24 h, 52%. d): 5 M HBr, 80–110 ◦C, 3.5 98%. e) Toluene/pyridine, 25 °C, 1 h, 75%. f) TBAF, THF, 25 °C, 24 h, 88%, g) K2CO3, acetone, 60 °C, h, 98%. e) Toluene/pyridine, 25 ◦C, 1 h, 75%. f) TBAF, THF, 25 ◦C, 24 h, 88%, g) K2CO3, acetone, 60 ◦C, 4848 h,h, 35%.35%.

4.2.3.As Fused shown Bicyclic in Scheme GABA 19,A Receptor compound Modulators 102 is obtained Containing by the Tworeaction Nitrogens of thiophene-2-carbaldehyde and 6–7 Membered Rings(compound 100) with 2-amino-5-bromopyridine (compound 101). The reaction of compound 102 with 4-butoxybenzoyl chloride yielded compound 98. For the synthesis of compound 99, thiophene- Benzodiazepines 2-carbaldehyde (compound 100) is reacted with 2-amino-3,5-dibromopyridine (compound 104) in the presenceBenzodiazepines of 2-isocyano-2-methy have dominatedlpropane the to yield scene compound of “tranquilizers” 105. Upon since treatment the first of representativecompound 105 chlordiazepoxidewith HBr (5 M), (Libriumintermediate®, 1960) 106 waswas introducedformed. In intothe following the market step, [103 the]. Benzodiazepines amide formation are was a largeperformed group ofvia compounds reaction with widely 4-(( usedtert-butyldiphenylsilyl)oxy)benzoyl in clinical treatment. They are usedchloride for a range(compound of diff erent107). indicationsDeprotection such with as insomnia,TBAF followed anxiety, by anda Will depression,iamson ether but alsoformation as anticonvulsants delivered compound and anesthetics. 99 in a Suchmoderate a versatile yield (35%). use requires These ditwofferent syntheses characteristics. already show For example,that the substitution when used inpattern the treatment on the core of anxietyscaffold or can depression, be easily varied the sedation by using and differently the hypnotic substituted effect should starting be materials reduced, and while, reagents. in procedural anesthesia, rapid sedation is highly desired. Pharmacologically, benzodiazepines can be classified according4.2.3. Fused to Bicyclic their elimination GABAA Receptor half-life Modulators time in short-, Containing intermediate-, Two Nitrogens and long-acting. and 6–7 Membered Short-acting Rings

Benzodiazepines Benzodiazepines have dominated the scene of “tranquilizers” since the first representative chlordiazepoxide (Librium®, 1960) was introduced into the market [103]. Benzodiazepines are a large group of compounds widely used in clinical treatment. They are used for a range of different indications such as insomnia, anxiety, and depression, but also as anticonvulsants and anesthetics. Such a versatile use requires different characteristics. For example, when used in the treatment of anxiety or depression, the sedation and the hypnotic effect should be reduced, while, in procedural anesthesia, rapid sedation is highly desired. Pharmacologically, benzodiazepines can be classified according to their elimination half-life time in short-, intermediate-, and long-acting. Short-acting benzodiazepines, such as midazolam, are mostly used in anesthesia or insomnia due to their rapid onset. Long-acting benzodiazepines such as diazepam are preferred in the treatment of alcohol or drug withdrawal [104].

Classical Benzodiazepines

Molecules 2020, 25, 999 22 of 47 benzodiazepines, such as midazolam, are mostly used in anesthesia or insomnia due to their rapid onset. Long-acting benzodiazepines such as diazepam are preferred in the treatment of alcohol or drug withdrawal [104].

ClassicalMolecules 2019 Benzodiazepines, 24, x FOR PEER REVIEW 23 of 48

The ﬁrstfirst benzodiazepine,benzodiazepine, chlordiazepoxidechlordiazepoxide (compound (compound111 111),), was was synthesized synthesized serendipitously, serendipitously, as depictedas depicted in Schemein Scheme 20. 20.

Scheme 20. A)a) HydroxylammoniumHydroxylammonium chloride,chloride, EtOH,EtOH, reﬂux,reflux, 88 h,h, 75%.75%. b)B) chloroacetylchloroacetyl chloride,chloride, acetic

acid, 50 ◦°C,C, 1010 min,min, 91%.91%. C) c) NaOH,NaOH, d)d) Methylamine,Methylamine, MeOH,MeOH, rt,rt, 33 d,d, 36%.36%. e)E) HH22O,O, f)f) 1.1. PCl3,, 2. 2. CH3I/base.I/base.

The additionaddition of of hydroxylamine hydroxylamine to 2-amino-5-chlorobenzophenoneto 2-amino-5-chlorobenzophenone (compound (compound112) yielded112) yielded oxime 113oxime, which, 113, which, upon chloro-acetylation, upon chloro-acetylation, yielded yielded compound compound114. The 114 cyclized. The cyclized benzodiazepine benzodiazepineN-oxide N- (compoundoxide (compound115) is formed115) is formed by the treatment by the treatment of 114 with of 114 NaOH. with Interestingly, NaOH. Interestingly, methylamine methylamine did not attack did thenot methyleneattack the methylene group carrying group the carrying Cl, but thethe equallyCl, but the electron-deﬁcient equally electron-deficient C2-position ofC2-position the heterocyclic of the coreheterocyclic unexpectedly core leadingunexpectedly to compound leading111 ,to via compound the proposed 111 rearrangement, via the proposed mechanism rearrangement displayed in Schememechanism 20. Todisplayed further in investigate Scheme 20. the To interesting further inve sedativestigate properties, the interesting many sedative more compounds properties, ofmany the samemore classcompounds were then of the synthesized same class purposely. were then Thesynthesized synthetic purposely. routes via The new synthetic synthetic routes approaches via new of selectedsynthetic examples approaches are of described selected inexamples the following are described paragraphs. in the following paragraphs. The structure of diazepam (compound 117) can be obtained from the chlordiazepoxidechlordiazepoxide 111 (Scheme(Scheme 2121)) uponupon oxidativeoxidative deamination,deamination, whichwhich isis followedfollowed byby aa reductionreduction ofof thethe NN-oxide-oxide withwith PClPCl33 and N-methylation with methylmethyl iodideiodide [[103].103].

Molecules 2020, 25, 999 23 of 47 Molecules 2019, 24, x FOR PEER REVIEW 24 of 48 Molecules 2019, 24, x FOR PEER REVIEW 24 of 48

Scheme 21; a) 1. Chloroacetyl chloride, toluene, rt, 2 h. 2. NH4OH/K2CO3, r.t, 3 h. b) dimethyl sulfate, Scheme 21; a) 1. Chloroacetyl chloride, toluene, rt, 2 h. 2. NH4OH/K2CO3, r.t, 3 h. b) dimethyl sulfate, SchemeK2CO3, r.t, 21. 2a) h. 1. c) Chloroacetyl bromoacetyl chloride, bromide, toluene, toluene, rt, refl 2 h.ux, 2. 30 NH min,4OH 88%./K2CO D)3 NaN, r.t, 33, h.EtOH, b) dimethyl reflux, 30 sulfate, min, K2CO3, r.t, 2 h. c) bromoacetyl bromide, toluene, reflux, 30 min, 88%. D) NaN3, EtOH, reflux, 30 min, K90%.2CO E)3, r.t,PhP 23 h., Toluene, c) bromoacetyl rt. F) reflux, bromide, 1 h, 63%. toluene, reflux, 30 min, 88%. d) NaN3, EtOH, reflux, 30 min, 90%. E) PhP3, Toluene, rt. F) reflux, 1 h, 63%. 90%. e) PhP3, Toluene, rt. f) reflux, 1 h, 63%. Alternatively, diazepam can also be obtained starting from 2-amino-5-chlorobenzophenone (compoundAlternatively,Alternatively, 112 or structure diazepamdiazepam 118 cancan R1 : alsoalso Cl) with bebe obtainedobtained chloroacetyl startingstarting chloride fromfrom and 2-amino-5-chlorobenzophenone2-amin hydroxylamine,o-5-chlorobenzophenone followed by a 1 (compound(compoundmethylation 112 (Scheme or structure 21) [105]. 118 RSimilar:: Cl) Cl) with withsynt chloroacetylheses chloroacetyl are used chloride chloride for nitrazepam and and hydroxylamine, hydroxylamine, (Mogadon followed ® followed), used byas by a ®® amethylationhypnotic methylation agent, (Scheme (Scheme delorazepam 21) 21 )[[105].105 (En ].Similar® Similar), used synt synthesesas hesesanxiolytic, are are used and used fornordazepam for nitrazepam nitrazepam (Nordaz (Mogadon (Mogadon®)(Scheme ), ),used used21). as An as a ® ® ® ® ahypnoticalternative hypnotic agent, agent,synthesis delorazepam delorazepam for nitrazep (En (En),am used), usedinvolves as asanxiolytic, anxiolytic, azide andintermediate and nordazepam nordazepam 121 (Nordaz, which (Nordaz )(Schemeis )(Schemereacted 21). with 21An). Analternativetriphenylphosphine alternative synthesis synthesis towards for fornitrazep nitrazepam122 includingam involves involves the substrate azide azide intermediate intermediatefor cyclization 121121 to, , whichnitrazepam which is is reacted (compound withwith triphenylphosphinetriphenylphosphine123, Scheme 21) [106]. towards In principle, 122 includingincluding the same thethe rout substratesubstratee was applied forfor cyclizationcyc forlization the synthesis toto nitrazepamnitrazepam of the bromazepam (compound(compound 123123(Lexotan,, SchemeScheme®), which 2121))[ [106].106 contains]. InIn principle,principle, a 2-pyridyl thethe sameringsame instead routeroute waswas of a applied appliedphenyl forringfor thethe in synthesispositionsynthesis 5 ofof[107]. thethe bromazepamAdditionally,bromazepam ®® (Lexotan(Lexotansubstitution),), which which with different contains contains halogensa a 2-pyridyl 2-pyridyl on ringboth instead rings of of the a phenylbenzophenones ring in position is reported. 5 [107]. [107 However,]. Additionally, Additionally, other substitutionsubstitutionsubstituents withwithshould, didifferentff erentin principle, halogenshalogens be onontolerated. bothboth ringsring s ofof thethe benzophenonesbenzophenones isis reported.reported. However, otherother substituentssubstituents should,should, inin principle,principle, bebe tolerated.tolerated. Acetylated Benzodiazepines AcetylatedAcetylated BenzodiazepinesBenzodiazepines Position 3 of the general scaffold was modified to increase the polarity and, therefore, to increase the speedPositionPosition of 3 3metabolic of the general elimination. scascaffoldffold waswas A hydroxyl modifiedmodified totogroup increase is introduced the polarity in and, the therefore, case of totolorazepam increase the speed of metabolic elimination. A hydroxyl group is introduced in the case of lorazepam (compound the(compound speed of 128 metabolic) (Tavor ®elimination.) and oxazepam A hydroxyl (compound group 127 is ) introduced(Alepam®, inMurelax the case®, Serepax of lorazepam®). The ® ® ® ® ® ® ® ® 128(compoundgeneral) (Tavor synthesis )128 and) oxazepam(Tavorof these) derivativesand (compound oxazepam is127 depicted )(compound (Alepam in Scheme, Murelax127) (Alepam 22 ,via Serepax an, Murelaxoxidation). The general, followedSerepax synthesis ).by The an ofgeneralacetylation these derivativessynthesis step [108–110]. of is depictedthese Alternatively,derivatives in Scheme is 22 acetylatiodepicted via an oxidation nin using Scheme lead followed 22(IV) via acetate by an an oxidation acetylation and potassium followed step [ 108iodide by–110 an in]. Alternatively,acetylationacetic acid stepis acetylationreported [108–110]. (Scheme usingAlternatively, lead 22, (IV)conditions acetylatio acetate e andn andusing potassium f) lead without (IV) iodide acetatethe isolation in and acetic potassium of acid the is iodinated reportediodide in (Schemeaceticintermediate acid 22, conditionsis [111,112]. reported e and(Scheme f) without 22, conditions the isolation e and of the f) iodinatedwithout intermediatethe isolation [111of ,the112 ].iodinated intermediate [111,112].

SchemeScheme 22.22. a)A) R R= =H: H: 30%30% H H22OO22,, AcOH,AcOH, NaNa22WO44 cat., 70–100 ◦°C,C, 6 h, 84%. b)B) R == Cl, m-CPBA, DCM, Scheme 22. A) R = H: 30% H2O2, AcOH, Na2WO4 cat., 70–100 °C, 6 h, 84%. B) R = Cl, m-CPBA, DCM, rt,rt, 1010 h,h, 77%.77%. c)C) R R= =H: H: acetic acetic anhydride, anhydride, 100 100◦C, °C 78%,, 78%, 20 20 min min or or R R= Cl:= Cl: acetic acetic anhydride, anhydride, 70 70◦C; °C; 79%, 79%, 3 rt, 10 h, 77%. C) R = H: acetic anhydride, 100 °C, 78%, 20 min or R = Cl: acetic anhydride, 70 °C; 79%, h.3 h. d) d) NaOH, NaOH, reﬂux. reflux. 80%. 80%. e) E) 1.1 1.1 eq. eq. KI, KI, 2 eq. 2 eq. Cu(Oac) Cu(Oac)2, 22, eq.2 eq. KOAc, KOAc, HOAc, HOAc, 80 80◦C, °C, 5 h 5(73%). h (73%). f) 1F) eq. 1 eq. I2, 3 h. d) NaOH, reflux. 80%. E) 1.1 eq. KI, 2 eq. Cu(Oac)2, 2 eq. KOAc, HOAc, 80 °C, 5 h (73%). F) 1 eq. 2I2 eq., 2 eq. NH NH4S24OS28,O28, 2 eq. eq. KOAc, KOAc, HOAc, HOAc, 80 80◦ C,°C, 6 6 h h (84%). (84%). I2, 2 eq. NH4S2O8, 2 eq. KOAc, HOAc, 80 °C, 6 h (84%). Diazolobenzodiazepines Diazolobenzodiazepines

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DiazolobenzodiazepinesMolecules 2019, 24, x FOR PEER REVIEW 25 of 48

Among thethe di ffdifferenterent synthetic synthetic strategies strategies developed developed after the discoveryafter the of thediscovery first benzodiazepines, of the first thebenzodiazepines, introduction of the new introduction rings annellated of new to therings benzodiazepine annellated to scatheff oldbenzodiazepine was investigated. scaffold In 1982,was ® midazolaminvestigated. (compound In 1982, midazolam129) wasmarketed (compound as Versed129) was andmarketed was initially as Versed used ® asand a generalwas initially anesthetic used inas procedurala general anesthetic sedation andin procedural in the treatment sedation of epileptic and in the seizures treatment due to of its epileptic rapid onset seizures [113]. due Its waterto its solubilityrapid onset makes [113]. it suitableIts water for solubility oral or buccal makes administration it suitable for and, oral therefore, or buccal it administration makes it suitable and, to administertherefore, it to makes children it suitable [114]. to administer to children [114]. The synthesissynthesis of of midazolam midazolam (compound (compound129) is129 achieved) is achieved as depicted as depicted in Scheme in 23 Scheme. The nitrosation 23. The ofnitrosation amidine 130of amidineafforded 130 compound afforded131 compoundin good yields.131 in good Compound yields.132 Compoundwas obtained 132 was by the obtained treatment by ofthe nitroso-amidine treatment of nitroso-amidine131 with nitromethane 131 with innitromethane the presence in of the potassium presencet-butoxide. of potassium The t-butoxide. hydrogenation The overhydrogenation Raney nickel over of Raney compound nickel132 of compoundyielded compound 132 yielded133 compoundin high yields. 133 in Acetylation high yields. of Acetylation compound 133of compoundfollowed by 133 a thermal followed cyclization by a thermal delivered cyclization compound delivered134. Alternatively, compound compound 134. Alternatively,134 could be obtainedcompound directly 134 could from be133 obtainedupon treatment directly from with 133 triethyl upon orthoacetate. treatment with In thetriethyl laststep, orthoacetate. oxidation In with the 129 [113] MnOlast step,2 aff ordedoxidation compound with MnO2 afforded. compound 129 [113].

Scheme 23. A)a) NaNONaNO22,, acetic acid, rt, 3 h, 60%. B) b) CH 3NONO22, ,Ko KotBu,tBu, DMF, DMF, rt, rt, 1 1 h, h, 53%. 53%. C) c) Ni/H Ni/H2,, THF, THF, 155 psi,psi, 2424 h,h, 83%. 83%. d) D) CH CH3C(Oet)3C(Oet)3,3 xylene,, xylene, reﬂux, reflux, 2 h,2 68%.h, 68%. f) ACF) 2ACO,2 pyridine,O, pyridine, 100 100◦C, 4°C, h, 43%.4 h, 43%. g) PPA, G)

150–170PPA, 150–170◦C, 10 °C, min., 10 23%.min., e)23%. MnO E)2 MnO, toluene,2, toluene, reﬂux, reflux, 40 min., 40 58%.min., 58%.

In a didifferentﬀerent strategy, thethe methylmethyl groupgroup cancan bebe insertedinserted in position 2 of the imidazole via a lithiation, as outlined in Scheme 2424.. This This method method allows allows the the insertion insertion of of different diﬀerent residues on the imidazole ring independently ofof thethe cyclizationcyclization stepstep [[115].115].

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Molecules 2020, 25, 999 25 of 47 Molecules 2019, 24, x FOR PEER REVIEW 26 of 48

Scheme 24. A) 1. KotBu, THF, 0 °C. 2. ClPO(Oet)2, −20–0 °C. b) 1. TosMIC, 2. KotBu, −20 °C–rt, 4 h, 80%.Scheme C) 1. BuLi,24. A) − 1.78 Ko °Ct Bu,2. MeI, THF, − 780 °C. °C–rt, 2. ClPO(Oet) 90%. D) Na-Hg2, −20–0 amalgam,°C. b) 1. TosMIC, K2HPO 42., EtOH/HKotBu, −220O, °C–rt,ultrasound, 4 h, Scheme 24. a) 1. KotBu, THF, 0 ◦C. 2. ClPO(Oet)2, 20–0 ◦C. b) 1. TosMIC, 2. KotBu, 20 ◦C–rt, 4 h, 72%.80%. E) C)1. CNCH1. BuLi,2COOEt, −78 °C 2. 2. MeI, Kot Bu,−78 4°C–rt, h, 60%. 90%. F) D)NaOH, Na-Hg− EtOH, amalgam, rt, 4 h, K 298%.HPO 4G), EtOH/H HCl, EtOH,2O,− ultrasound, rt, 4 h, 83%. 80%. c) 1. BuLi, 78 ◦C 2. MeI, 78 ◦C–rt, 90%. d) Na-Hg amalgam, K2HPO4, EtOH/H2O, ultrasound, 72%. E) 1. CNCH− 2COOEt, 2. Ko−tBu, 4 h, 60%. F) NaOH, EtOH, rt, 4 h, 98%. G) HCl, EtOH, rt, 4 h, 83%. 72%.H) NMP, e) 1. 200 CNCH °C, 2COOEt, h, 70%. 2.I) 1. Ko BuLi,tBu, 4− h,78 60%.°C 2. f)MeI, NaOH, −78 °C EtOH, to rt, rt, overnight, 4 h, 98%. 90%. g) HCl, EtOH, rt, 4 h, 83%. H) NMP, 200 °C,2 2 h, 70%. I) 1. BuLi, −78 °C 2. MeI, −78 °C to rt, overnight, 90%. h) NMP, 200 ◦C, 2 h, 70%. i) 1. BuLi, 78 ◦C 2. MeI, 78 ◦C to rt, overnight, 90%. Triazolobenzodiazepines − − TriazolobenzodiazepinesTriazolobenzodiazepines Drugs containing a 1,3,4-triazole moiety, such as triazolam (Halcion ®), estazolam (Prosom®), Drugs containing a 1,3,4-triazole moiety, such as triazolam (Halcion ®), estazolam (Prosom®), and alprazolamDrugs containing (Xanax a®)[116] 1,3,4-triazole were synthesized moiety, such and as introduced triazolam to (Halcion the market®), estazolam to treat insomnia (Prosom and®), and alprazolam (Xanax®)[116] were synthesized and introduced to the market to treat insomnia and andpanic alprazolam attacks. The (Xanax general®)[ synthesi116] weres of synthesized alprazolam and (compound introduced 144 to) is the depicted market in to Scheme treat insomnia 25 starting and panic attacks. The general synthesis of alprazolam (compound 144) is depicted in Scheme 25 starting panicfrom diazepam attacks. The (compound general synthesis 117) [105]. of alprazolam After the acetylation, (compound the144 imide) is depicted (compound in Scheme 145) is25 cyclizedstarting from diazepam (compound 117) [105]. After the acetylation, the imide (compound 145) is cyclized from diazepam (compound 117)[105]. After the acetylation, the imide (compound 145) is cyclized withwith hydrazine hydrazine hydrate hydrate to to the the 1,3,4-triazole 1,3,4-triazole motif. motif. Variation inin thethe phenyl phenyl ri ringsngs can can again again be be obtained obtained withby byusing hydrazineusing differently differently hydrate substituted tosubstituted the 1,3,4-triazole benzophenonesbenzophenones motif. Variation as starting in the materialsmaterials phenyl rings forfor thecanthe againbenzodiazepine benzodiazepine be obtained bysynthesis.synthesis. using di ﬀ erently substituted benzophenones as starting materials for the benzodiazepine synthesis.

SchemeSchemeScheme 25.25. 25. a)A) A) AC AC AC2O,O,2O, KK K2COCO2CO3/KOH,/3KOH,/KOH, r.t, r.t, 33 3 h, h,h, 80%.80%.80%. b)C) NHNH22NHNH22·H·HH22O,O,O, NaOAc, NaOAc, NaOAc, reflux, reflux, reﬂux, 12 12 12h, h, h,75%. 75%. 75%. 2 2 3 2 2· 2

TheThe synthesis synthesis of of a a1,2,3-triazolobe 1,2,3-triazolobenzodiazepine 1,2,3-triazolobenzodiazepinenzodiazepine scaffold scaffold (structure(structure 146) 146) is is outlined outlined in in Scheme Scheme Scheme 26. 26.26 . CompoundCompound 148 148 was was obtained obtained via via a a Buchwald-Hartwig Buchwald-Hartwig coupling from from 1,2-diiodobenzene 1,2-diiodobenzene (compound (compound 147147)) andand) and aniline.aniline. aniline. Subsequent Subsequent Sonogashira Sonogashira coupling, coupling,coupling, which which waswas followed followed by by acetylation, acetylation,acetylation, yielded yieldedyielded compound 115 in low yields. Even though this step represents a drawback of this synthesis, for compound 115 inin lowlow yields.yields. Even Even though though this this step step repr representsesents a drawback of this synthesis, for similar derivatives (e.g., with a bromine atom in position 9), better yields could be achieved with the similar derivatives (e.g., with a bromine atom in positionposition 9), better yields couldcould be achieved with the same conditions. Lastly, a cyclo-addition with sodium azide and a carbonyl reduction with BH3 were same conditions. Lastly, a cyclo-addition withwith sodiumsodium azideazide andand aa carbonyl reductionreduction withwith BHBH33 were performed to obtain the triazolobenzodiadepine scaffold 146 [117]. The cyclo-addition in this specific performed to obtain the triazolobenzodiadepine scascaffoldffold 146146 [[117].117]. The cyclo-addition in this specific specific example gave only a poor yield of 13% due to unoptimized reaction conditions. example gave only a poor yield of 13% due toto unoptimizedunoptimized reactionreaction conditions.conditions.

Molecules 2019, 24, x FOR PEER REVIEW 27 of 48 Molecules 2020, 25, 999 26 of 47 Molecules 2019, 24, x FOR PEER REVIEW 27 of 48

Scheme 26. A) Aniline, Pd(dba)2, Xantphos, Cs2CO3, toluene, 110 °C, overnight, 27%. B) Scheme 26. a) Aniline, Pd(dba)2, Xantphos, Cs2CO3, toluene, 110 ◦C, overnight, 27%. b) trimethyl(prop-1-yn-1-yl)sScheme 26. A) Aniline,ilane, Pd(dba) TBAF,2, Xantphos,CuI, Pd(PPh Cs3)24CO , THF,3, toluene, 70 °C, overnight, 110 °C, 84%;overnight, c) 2-chloroacetyl 27%. B) trimethyl(prop-1-yn-1-yl)silane, TBAF, CuI, Pd(PPh3)4, THF, 70 ◦C, overnight, 84%; c) 2-chloroacetyl chloride,trimethyl(prop-1-yn-1-yl)s K2CO3, DCM, 45 °C,ilane, overnight, TBAF, CuI, 64%. Pd(PPh D) NaN3)4 3,, THF,DMF, 70 150 °C, °C, overnight, 2 h, 13%. 84%;E) BH c)3 ,2-chloroacetyl THF, 70 °C, 2 chloride, K2CO3, DCM, 45 ◦C, overnight, 64%. d) NaN3, DMF, 150 ◦C, 2 h, 13%. e) BH3, THF, 70 ◦C, 2 h,h, 88%.chloride, 88%. K2CO3, DCM, 45 °C, overnight, 64%. D) NaN3, DMF, 150 °C, 2 h, 13%. E) BH3, THF, 70 °C, 2 h, 88%. DiDifferentlyfferently substituted substituted anilinesanilines areare tolerated, such such as as pp-chloroaniline-chloroaniline or orm-bromoaniline.m-bromoaniline. Additionally,Additionally,Differently a slightlya slightly substituted modified modified anilines procedure procedure are startingtolerated,starting from such substituted substituted as p-chloroaniline 2-iodoanilines 2-iodoanilines or (Rm (R-bromoaniline. = Br,= Br, COOMe) COOMe) insteadinsteadAdditionally, of of 1,2-diiodobenzene, 1,2-diiodobenzene, a slightly modified allows allows procedurethe the introductionintroduction starting from of of substituents substituents substituted on 2-iodoanilines on ring ring A A of of the the (R benzodiazepine = benzodiazepine Br, COOMe) scascaffoldinsteadffold (scheme of(scheme 1,2-diiodobenzene, not not shown). shown).These Theseallows bromobromo the introduction or ester derivatives derivatives of substituents can can be beon used usedring for A for offurther furtherthe benzodiazepine modifications modifications (e.g.,(e.g.,scaffold with with (scheme coupling coupling not reactions). reactions). shown). These Furthermore,Furthermore, bromo or in ester th thee derivativeslast last few few years, years, can bemulti-component multi-component used for further reactions modifications reactions have have been reported for the synthesis of various triazolobenzodiazepine scaffolds (A, B, C), which deliver been(e.g., reported with coupling for the reactions). synthesis ofFurthermore, various triazolobenzodiazepine in the last few years, multi-component scaffolds (A, B, C), reactions which deliverhave compoundbeen reported libraries for the quickly synthesis (Scheme of various 27). Multi-cotriazolobenzodiazepinemponent reactions scaffolds involving (A, B,aldehydes, C), which amines, deliver compound libraries quickly (Scheme 27). Multi-component reactions involving aldehydes, amines, and andcompound acylating libraries agents quickly are a suitable (Scheme and 27). versatile Multi-co stmponentrategy to reactionsaccess polycyclic involving heterocyclic aldehydes, scaffolds amines, acylating agents are a suitable and versatile strategy to access polycyclic heterocyclic scaffolds [118]. In [118].and acylating In particular, agents structures are a suitable containing and versatile both th est rategy1,4 benzodiazepine to access polycyclic ring system heterocyclic and the scaffolds triazole particular, structures containing both the 1,4 benzodiazepine ring system and the triazole system are of system[118]. In are particular, of high interest structures because containing of their both medica the l1,4 potential benzodiazepine [119]. All ring thre esystem strategies and requirethe triazole a 2- high interest because of their medical potential [119]. All three strategies require a 2-azidobenzaldehyde azidobenzaldehydesystem are of high interest (structure because 154 )of and their a medica propargylaminel potential [119].derivative All thre (structuree strategies 154 require) as keya 2- (structurecompoundsazidobenzaldehyde154) with and athe propargylamine addition(structure of 154other) derivative andreagents. a propargylamine (structure 154) asderivative key compounds (structure with 154 the) additionas key of othercompounds reagents. with the addition of other reagents.

Scheme 27 a) 1. AcOH, NaB(Oac)3H, DCE, rt, 3 h. 2. Toluene, 100 °C, 4.5 h. b) 1. Amine and aldehyde,

DMF,Scheme rt, 272.5 a) h, 1. 2. AcOH, TosMIC, NaB(Oac) K2CO3, 3rt,H, 17DCE, h. c) rt, InCl 3 h.3, 2.(10 Toluene, mol%), 100MeOH, °C, 4.5 60–100 h. b) °C,1. Amine 10–72 andh. aldehyde, Scheme 27. a) 1. AcOH, NaB(Oac)3H, DCE, rt, 3 h. 2. Toluene, 100 ◦C, 4.5 h. b) 1. Amine and aldehyde, DMF, rt, 2.5 h, 2. TosMIC, K2CO3, rt, 17 h. c) InCl3, (10 mol%), MeOH, 60–100 °C, 10–72 h. DMF, rt, 2.5 h, 2. TosMIC, K CO , rt, 17 h. c) InCl (10 mol%), MeOH, 60–100 C, 10–72 h. For example, Donald et2 al. 3[120,121] demonstrated3, that the triazolobenzodiazepine◦ scaffold (structureForFor example, example, 156) is Donaldeasily Donald accessible etet al.al. [120[120,121] via,121 a reductive] demonstrateddemonstrated amination that that and the the an triazolobenzodiazepine triazolobenzodiazepine azide-alkyne cycloaddition scaffold sca ffasold (structureshown(structure in156 Scheme156) is) is easily easily27 (route accessible accessible A). The viavia scaffold aa reductivereductive obtained aminationamination can be and andfurther an an azide-alkyne azide-alkynemodified to cycloadditionrapidly cycloaddition access asa as shownvarietyshown in Schemeofin compoundsScheme 27 (route27 (routewith A). different A). The The sca ffNscaffoldold-substituents. obtained obtained can can be further be further modified modified to rapidly to rapidly access access a variety a ofvariety compounds of compounds with diff erentwith differentN-substituents. N-substituents. Another azide-alkyne cycloaddition was investigated by Djuric et al. as a modification of the van Leusen imidazole synthesis for the synthesis of imidazole and triazole-fused benzodiazepines [122]. Molecules 2019, 24, x FOR PEER REVIEW 28 of 48 Molecules 2020, 25, 999 27 of 47 Another azide-alkyne cycloaddition was investigated by Djuric et al. as a modification of the van Leusen imidazole synthesis for the synthesis of imidazole and triazole-fused benzodiazepines [122].As shown As shown in Scheme in Scheme 27, after27, after the th formatione formation of of the the imidazole imidazole (structure (structure152 152),), anan intramolecular cycloaddition is performed. The The different different fused triazole benzodiazepines accessible with this route have the generalgeneral formulaformula157 157,, with with R R11being being -H -H or or F-, F-, R R2 2being being H- H- or or dioxolane dioxolane and and R 3Rbeing3 being either either -H, - -MeH, -Me or -Ph.or -Ph. A third third variation variation was was reported reported by by Nguyen Nguyen et et al al.. [123] [123 (Scheme] (Scheme 27C) 27C) when when taking taking advantage advantage of anof anintramolecular intramolecular 1,3-dipolar 1,3-dipolar Huisgen Huisgen cyclo-addition. cyclo-addition. The The triazolobenzodiazepine triazolobenzodiazepine scaffold scaffold is obtained in a one-pot reactionreaction usingusing aa 1,2-diketone1,2-diketone (structure (structure160 160),), an an azido-aldehyde azido-aldehyde (structure (structure154 154),), a apropargylamine propargylamine (structure (structure155 155), and), and an ammonia an ammonia reactant reactant in the in presence the presence of a Lewis of a acid. Lewis This acid. method This 1 1 methodtolerates tolerates a variety a of variety 1,2-diketones of 1,2-diketones (R : Ph, CH(R1:3 ,Ph, H, CHp-BrPh,3, H, pp-MeOPh,-BrPh, p-MeOPh,p-MePh) p as-MePh) well as as di wellfferent as 2 2 3 3 differentazo-benzaldehydes azo-benzaldehydes (R : CO2 Me,(R2: Br,CO dioxolane2Me, Br, dioxolane and -H) and and propargylic -H) and propargylic amines (R amines: H, Et, Ph)(R3: withH, Et, a Ph)yield with range a yield of 18–72%. range of 18–72%. Recently, a one-pot synthesis of triazolobenzodiazepinestriazolobenzodiazepines (structure 164) has been accomplished using a [3 + 2] cycloadditio cycloadditionn of azomethine yield and N-ethylmaleimide (structure (structure 163),), which is followed by an N-propargylation and a click reaction [[124]124] (Scheme(Scheme 2828).). The method accommodates 22 33 2 33 the use ofof readilyreadily availableavailable aminoamino acidsacids (R(R2 andand RR3 == CH3 or R 2 == H andand RR3 = iPr.iPr. Or Or Ph), Ph), different different 11 44 azido-aldehydes (R(R1 = H,H, CF CF33, ,Br, Br, Cl), Cl), and and different different maleimides (R(R4 == Et,Et, Me, Me, Bn, Bn, Ph), Ph), which which generates generates as a byproduct of only H2OO and and CO CO22..

Scheme 28. 1. AcOH, CH3CN, 110 °C 6 h. 2. K2CO3, µw, 1 h, 35–65%. SchemeScheme 28.28. 1.1. AcOH, CHCH33CN, 110 °C◦C 6 h. 2. 2. K K22COCO3,3 µw,, µw, 11 h, h, 35–65%. 35–65%.

4.2.4. Fused Fused Bicyclic Bicyclic GABA GABAAA ReceptorReceptor Modulators Modulators Containing Containing Three Three Nitrogens Nitrogens Pyrazolopyrimidine Pyrazolopyrimidine Zaleplon (compound 165), also belonging to the Z-drugs, is a pyrazolopyrimidine used as a Zaleplon (compound 165), also belonging to the Z-drugs, is a pyrazolopyrimidine used as a short-acting hypnotic [125,126]. The synthesis of zaleplon (compound 165) is performed as outlined in short-acting hypnotic [125,126]. The synthesis of zaleplon (compound 165) is performed as outlined Scheme 29 with the reaction of acetamide 167 with 5-amino-1H-pyrazole-4-carbonitrile (compound in Scheme 29 with the reaction of acetamide 167 with 5-amino-1H-pyrazole-4-carbonitrile (compound 168. One major drawback of the synthesis of zaleplon (compound 165) is the formation of the isomer Z 168. One major drawback of the synthesis of zaleplon (compound 165) is the formation of the isomer (compound 169) as a side-product. Diﬀerent acidic conditions are reported. Aqueous acetic or formic Z (compound 169) as a side-product. Different acidic conditions are reported. Aqueous acetic or acids are preferred over anhydrous acetic acid, which leads to the formation of a high amount of the formic acids are preferred over anhydrous acetic acid, which leads to the formation of a high amount desired isomer (compound 165)[89,127]. of the desired isomer (compound 165) [89,127].

Scheme 29. A)a) 1.1. DMF-DMAc, reﬂux,reflux, 8 h, 2. NaH, EtBr, rt, 0.5 h, yield not reported, b) 30% acetic acid in H2O, 50 °C, 2 h, 86%. in H 2O,O, 50 50 °C,◦C, 2 2 h, h, 86%. 86%.

Molecules 2019, 24, x FOR PEER REVIEW 29 of 48

Among the pyrazolopyrimidines investigated as sedative-hypnotic agents, Indiplon [128] received an initial approval letter from the United States Food and Drug Administration (FDA). However, after further investigations, it remained unapproved unless more clinical studies are conducted.Molecules 2020 ,Other25, 999 structures containing a pyrazolopyrimidine core (structure 172) were investigated28 of 47 Molecules 2019, 24, x FOR PEER REVIEW 29 of 48 as potential ligands for the GABAA receptors [129,130]. One compound of this class (Ar: 3′-thienyl, R1: 2′-pyridyl) showed a promising selectivity for α1 containing receptors [129]. These structures can be Among thethe pyrazolopyrimidines pyrazolopyrimidines investigated investigated as sedative-hypnotic as sedative-hypnotic agents, agents, Indiplon Indiplon [128] received [128] obtained in a one-step reaction as displayed in general Scheme 30, which allows a rapid synthesis of receivedan initial an approval initial letterapproval from letter the United from Statesthe Unit Fooded andStates Drug Food Administration and Drug Administration (FDA). However, (FDA). after a variety of derivatives [131]. However,further investigations, after further itinvestigations, remained unapproved it remained unless unapproved more clinical unless studies more are clinical conducted. studies Other are conducted.structures containing Other structures a pyrazolopyrimidine containing a pyrazolopyrimidine core (structure 172) core were (structure investigated 172) as were potential investigated ligands 1 asfor potential the GABA ligandsA receptors for the GABA [129,130A receptors]. One compound [129,130]. One of this compound class (Ar: of this 30-thienyl, class (Ar: R 3:′-thienyl, 20-pyridyl) R1: 2showed′-pyridyl) a promising showed a selectivitypromising for selectivityα1 containing for α1 receptors containing [129 receptors]. These structures[129]. These can structures be obtained can in be a obtainedone-step in reaction a one-step as displayed reaction as in displayed general Scheme in general 30, which Scheme allows 30, which a rapid allows synthesis a rapid of synthesis a variety of of aderivatives variety of derivatives [131]. [131]. Scheme 30. Reflux, diglyme/ethylene glycol, 8 h, yield not reported.

Cyclopyrrolones The last Z-drug presented here is Zopiclone (compound 173) [132], which is commercially available in Europe as a racemic mixture (Imovane ®), while, in the USA, the active S-enantiomer ® (compound 173) [133]Scheme is marketed 30. Reflux, as diglyme/ethyleneLunesta in an glycol,optically 8 h, pure yield formnot reported. (Scheme 31). Like the two other drugs in this class,Scheme it is 30. prescribedReﬂux, diglyme for the/ethylene treatment glycol, of insomnia. 8 h, yield notChemically, reported. zopiclone belongs to the class of cyclopyrrolones. Cyclopyrrolones The synthesis of zopiclone is performed via the reaction of compound 178 and compound 179 The last Z-drug presented here is Zopiclone (compound 173) [132], which is commercially or itsThe hydrochloride last Z-drug presentedin the presence here is of Zopiclone a base [134–136] (compound (Scheme173)[ 13231).], The which precursor is commercially (alcohol available178) can available in Europe as a racemic mixture® (Imovane ®), while, in the USA, the active S-enantiomer bein Europeprepared as by a racemicstarting mixturefrom the (Imovane reaction between), while, compound in the USA, 174 the and active compoundS-enantiomer 175. The (compound resulting (compound 173) [133] is marketed® as Lunesta ® in an optically pure form (Scheme 31). Like the two acid173)[ is133 refluxed] is marketed in thionyl as Lunesta chloride into anyield optically compound pure form 177. (SchemeThe latter 31 ).upon Like addition the two otherof potassium drugs in other drugs in this class, it is prescribed for the treatment of insomnia. Chemically, zopiclone belongs borohydridethis class, it isin prescribed an aqueous for solution the treatment is converted of insomnia. to compound Chemically, 178. Final zopiclone esterification belongs delivers to the class the to the class of cyclopyrrolones. targetof cyclopyrrolones. R/S-Zopiclone (compound 173). The synthesis of zopiclone is performed via the reaction of compound 178 and compound 179 or its hydrochloride in the presence of a base [134–136] (Scheme 31). The precursor (alcohol 178) can be prepared by starting from the reaction between compound 174 and compound 175. The resulting acid is refluxed in thionyl chloride to yield compound 177. The latter upon addition of potassium borohydride in an aqueous solution is converted to compound 178. Final esterification delivers the target R/S-Zopiclone (compound 173).

SchemeScheme 31. A)a) I,I, reﬂux,reflux, 1.51.5 h.h. b) SOClSOCl22, reflux. reﬂux. c) c) KBH KBH44, ,dioxane/water, dioxane/water, 13 13 °C,◦C, 6 6 min. min. d) d) base base (NaH. (NaH.

KK22COCO33, ,Et Et3N,3N, DBU), DBU), solvent solvent (DMF, (DMF, DCM, DCM, benzene, benzene, 50 50 °C).◦C).

178 179 Alternatively,The synthesis compound of zopiclone 177 is performedcan be synthesized via the reaction starting of from compound pyrazine-2,3-dicarboxylicand compound acid 178 andor its 5-chloropyridine-2-amine hydrochloride in the presence in a one-step of a base conversion [134–136] in (Scheme acetic anhydride 31). The precursor (scheme not (alcohol shown) [137].) can be prepared by starting from the reaction between compound 174 and compound 175. The resulting 177 4.2.5.acidScheme isFused reﬂuxed Bicyclic31. A) in I, thionyl GABAreflux, 1.5A chloride Receptor h. b) SOCl to Modulators yield2, reflux. compound c) KBHContaining4, dioxane/water,. The Four latter Nitrogens 13 upon °C, 6 additionmin. d) base of (NaH. potassium borohydrideK2CO3, Et in3N, an DBU), aqueous solvent solution (DMF, DCM, is converted benzene, to50 compound°C). 178. Final esteriﬁcation delivers the Pyrazolotriazinestarget R/S-Zopiclone (compound 173). Alternatively, compoundcompound177 177can can be be synthesized synthesized starting starting from from pyrazine-2,3-dicarboxylic pyrazine-2,3-dicarboxylic acid acid and Different structures are reported as being active on GABAA receptors with a pyrazolotriazine and5-chloropyridine-2-amine 5-chloropyridine-2-amine in a in one-step a one-step conversion conversion in acetic in acetic anhydride anhydride (scheme (scheme not not shown) shown) [137 [137].]. (PT) scaffold (Figure 12).

4.2.5. Fused Bicyclic GABAA Receptor Modulators Containing Four Nitrogens

Pyrazolotriazines

Different structures are reported as being active on GABAA receptors with a pyrazolotriazine (PT) scaffold (Figure 12).

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4.2.5. Fused Bicyclic GABAA Receptor Modulators Containing Four Nitrogens

Pyrazolotriazines Molecules 2019, 24, x FOR PEER REVIEW 30 of 48 MoleculesDiﬀ 2019erent, 24 structures, x FOR PEER are REVIEW reported as being active on GABAA receptors with a pyrazolotriazine30 of (PT) 48 scaﬀold (Figure 12).

FigureFigure 12.12. GABAAA receptorreceptor ligands ligands containing containing th thee pyrazolotriazine pyrazolotriazine scaffold. scaﬀold. Figure 12. GABAA receptor ligands containing the pyrazolotriazine scaffold.

PT structuresstructures were synthesizedsynthesized and investigated as aza-isomers of pyrazolopyrimidines [138]. [138]. PT structures were synthesized and investigated as aza-isomers of pyrazolopyrimidines [138]. Pyrazolotriazines are prepared starting from diff diﬀerently substituted 3-aminopyrazoles (structure 183) Pyrazolotriazines are prepared starting from differently substituted 3-aminopyrazoles (structure 183) (Scheme(Scheme 32 32).). A A diazotization diazotization to structureto structure184 followed 184 followed by azo-coupling by azo-coupling to structure to structure186 and a cyclization186 and a (Scheme 32). A diazotization to structure 184 followed by azo-coupling to structure 186 and a withcyclization ethyl acetoacetate with ethyl yieldedacetoacetate 8-substituted yielded structure 8-substituted187. Compounds structure 187 of general. Compounds structure of187 generalwere cyclization with ethyl acetoacetate yielded 8-substituted structure 187. Compounds of general thenstructure further 187 transformed were then further into structure transformed188 with into astructure mixture of188 dimethylformamide-dimethylacetamide with a mixture of dimethylformamide- structure 187 were then further transformed into structure 188 with a mixture of dimethylformamide- (DMF-DMAc).dimethylacetamide Lastly, (DMF-DMAc). the addition of La hydrazinestly, the hydrate addition in theof hydrazine presence of hydrate sodium acetatein the inpresence acetic acid of dimethylacetamide (DMF-DMAc). Lastly, the addition of hydrazine hydrate in the presence of ledsodium to structure acetate189 in acetic. This synthesisacid led to could structure be used 189 for. This a broad synthesis range could of substituted be used aminopyrazolesfor a broad range with of sodium acetate in acetic acid led to structure 189. This synthesis could be used for a broad range of Rsubstituted being 2- or aminopyrazoles 3-pyridyl, 2-thienyl, with 1- R or being 2-naphthyl 2- or 3-pyri and substituteddyl, 2-thienyl, aryl 1- rings or 2-naphthyl carrying electron-donating and substituted substituted aminopyrazoles with R being 2- or 3-pyridyl, 2-thienyl, 1- or 2-naphthyl and substituted oraryl withdrawing rings carrying substituents. electron-donating or withdrawing substituents. aryl rings carrying electron-donating or withdrawing substituents.

Scheme 32. a) HCl, NaNO2/H2O, rt. b) NaOAc, ethyl acetoacetate, EtOH, rt, 1 h. c) DMF-DMAc, Scheme 32.32. a) HCl,HCl, NaNONaNO22/H/H2O,O, rt. b) b) NaOAc, NaOAc, ethyl ethyl acetoacetate, EtOH, rt, 1 h. c) DMF-DMAc, toluene, piperidine cat., 90 °C. d) hydrazine hydrate 60%, acetic acid, sodium acetate, reflux. toluene, piperidinepiperidine cat.,cat., 9090 ◦°CC.. d) hydrazine hydrate 60%, acetic acid, sodium acetate,acetate, reﬂux.reflux.

Compounds with structure 189 were found to have a Ki in the nM range for the benzodiazepine Compounds with structure 189189 werewere foundfound toto havehave aa KKii in the nM range for the benzodiazepine binding site. The product with R = 3-trifluoromethylphenyl showed selectivity towards α1. Of the binding site. The product with RR == 3-triﬂuoromethylphenyl3-trifluoromethylphenyl showed selectivity towards α1. Of the same class of compounds, MRK-016 (compound 181) [139] is reported to be an “α5-selective inverse same class of compounds, MRK-016 (compound 181)[) [139]139] is reported to bebe anan ““α5-selective inverse agonist,” which is the historical term for a negative allosteric modulator at the benzodiazepine agonist,” whichwhich is is the the historical historical term term for afor negative a nega allosterictive allosteric modulator modulator at the benzodiazepineat the benzodiazepine binding binding site of receptors that contain α5 subunits. sitebinding of receptors site of receptors that contain thatα contain5 subunits. α5 subunits.

The synthesis of this compound is described by Chambers et al. [140]. Compound 190 was heated The synthesis of this compound is described by Chambers et al. [140]. Compound 190 was withThe hydrazine synthesis hydrate of this to compound yield pyrazole is descri191.bed After by protecting Chambers the et hydroxylal. [140]. Compound moiety, the primary190 was heated with hydrazine hydrate to yield pyrazole 191. After protecting the hydroxyl moiety, the alcoholheated with of structure hydrazine192 hydratewas oxidized to yield to pyrazole aldehyde 191193. .After Condensation protecting between the hydroxyl the aldehyde moiety, 193the primary alcohol of structure 192 was oxidized to aldehyde 193. Condensation between the aldehyde andprimary the isoxazolealcohol of194 structureaﬀorded 192 compound was oxidized195 .to After aldehyde deprotection, 193. Condensation compound between196 was the reacted aldehyde with 193 and the isoxazole 194 afforded compound 195. After deprotection, compound 196 was reacted 5-chloromethyl-1-methyl-1193 and the isoxazole 194 Hafforded-1,2,4-triazole compound to yield 195 MRK-016. After deprotection, (compound compound181) (Scheme 196 33 was). reacted with 5-chloromethyl-1-methyl-1H-1,2,4-triazole to yield MRK-016 (compound 181) (Scheme 33). with 5-chloromethyl-1-methyl-1H-1,2,4-triazole to yield MRK-016 (compound 181) (Scheme 33).

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Scheme 33. (a) NH2NH2‚H2O, EtOH, reflux, quant. (b) p-toluenesulfonyl chloride, Et3N, DCM, 67%. (c) MnO2, CHCl3, reflux, 95%. (d) Xylene, reflux, 67%. (e) 4 N NaOH, MeOH, 65 °C, 85%. (f) K2CO3, SchemeScheme 33.33. a)(a) NH NH2NH2NH2‚H2‚H22O,O, EtOH,EtOH, reflux,reflux, quant. quant. b) (b)p-toluenesulfonyl p-toluenesulfonyl chloride, chloride, Et 3EtN,3N, DCM, DCM, 67%. 67%. c) DMF, 5-chloromethyl-1-methyl-1H-1,2,4-triazole, 76%. MnO(c) MnO2, CHCl2, CHCl3, reflux,3, reflux, 95%. 95%. d) Xylene,(d) Xylene, reflux, reflux, 67%. 67%. e) 4 N(e) NaOH, 4 N NaOH, MeOH, MeOH, 65 ◦C, 65 85%. °C, f)85%. K2CO (f)3 K, DMF,2CO3, 5-chloromethyl-1-methyl-1DMF, 5-chloromethyl-1-methyl-1H-1,2,4-triazole,H-1,2,4-triazole, 76%. 76%. Triazolopyridazines Triazolopyridazines TriazolopyridazinesTriazolopyridazines are a scaffold from which compounds MRK-409 (compound 197), TPA023 (compoundTriazolopyridazinesTriazolopyridazines 198), and TPA023B areare aa sca(compoundscaffoldffold fromfrom 200 whichwhich) have compoundscompounds progressed MRK-409 MRK-409into clinical (compound(compound trials. This 197 scaffold),), TPA023TPA023 was (compounddeveloped(compound under 198), and andthe TPA023B TPA023Bcontroversially (compound (compound debated 200200 )hypothesis have) have progressed progressed that compounds into into clinical clinical thattrials. trials. show This This ascaffold functional scaff wasold waspreferencedeveloped developed towardunder under thethe the controversiallyα2- controversially or α3-subunits debated debated of the hypothesis GABA hypothesisA receptors that that compounds compounds should have that that selectiveshow show a functionalfunctionalanxiolytic preference toward the α2- or α3-subunits of the GABA receptors should have selective anxiolytic effectspreference [141,142]. toward The the structures α2- or α3-subunits of some highlighted of the GABA derivativesAA receptors of should this family have selectiveof compounds anxiolytic are eshowneffectsffects [ 141in[141,142]. Figure,142]. The13. The structures structures of of some some highlighted highlighted derivatives derivatives of this of familythis family of compounds of compounds are shown are inshown Figure in 13 Figure. 13.

Figure 13. Structure of most studied triazolopyridazine derivatives. Figure 13. Structure of most studied triazolopyridazine derivatives. Synthesis of this scascaffoldffold (structure 204) hashas beenbeen reported from three didifferentfferent precursorsprecursors (structures(structuresSynthesis 201 ,, of202 this,, andand scaffold 203203)) forfor (structure thethe finalfinal step.204step.) has The been synthesissynthesis reported of intermediates from three different201, 202, precursors and 203 isis depicted(structures inin Scheme 201Scheme, 202 34, 34and and and 203 it beginsit) forbegins the with finalwith 3,6-dichloropyridazine step. 3,6-dichloropyridazine The synthesis (compoundof intermediates (compound205), which201205,), 202 which can, and be treatedcan 203 be is withtreateddepicted the with desiredin Scheme the hydrazidedesired 34 and hydrazide andit begins further and with cyclized further 3,6-dichloropyridazine incy oneclized step in to one obtain (compoundstep the to accordinglyobtain 205 the), which accordingly substituted can be triazolopyridazinesubstitutedtreated with triazolopyridazine the desired scaffold (structurehydrazide scaffold206 and (structure). Thefurther desired 206 cy).clized ether The indesiredin position one etherstep 6 can toin bepositionobtain easily the prepared6 can accordingly be easily using Williamsonpreparedsubstituted using etherificationtriazolopyridazine Williamson conditions. etherification scaffold Additionally, (structure conditio 206ns. a). substituent Additionally, The desired of ether choicea substituent in can position be introducedof 6choice can be can toeasily thebe initialintroducedprepared 3,6-dichloropyridazine using to the Williamson initial 3,6-dichloropyridazine (compoundetherification207 conditio) using (compound eitherns. Additionally, a homolytic 207) using alkylationa eithersubstituent a homolytic in the of heteroaromatic choice alkylation can be ringinintroduced the or heteroaromatic by synthesizing to the initial ring the 3,6-dichloropyridazinepyridazine or by synthesizing using the the substituted (compound pyridazine maleic 207using) anhydrideusing the substituted either (structure a homolytic maleic204) asanhydridealkylation a source of(structurein thethe requiredheteroaromatic 204) as 1,4-diketone, a source ring of or the hydrazine, by required synthesizing and 1,4-diketone, subsequent the pyridazine hydrazine, halogenation using and the subsequent with substituted phosphorus halogenation maleic oxychloride. anhydride with Afterward,phosphorus(structure 204 the oxychloride.) as substituted a source ofAfterward, dichloropyridazine the required the 1,4-diketone, substituted (structure hydrazine, dichloropyridazine207) can and be subsequent treated (structure again halogenation either 207) with can with thebe sametreatedphosphorus conditions again oxychloride. either mentioned with theAfterward, above same to conditions obtain the substituted the triazolopyridazinementioned dichloropyridazine above moietyto obtain or (structure anthe alternative triazolopyridazine 207) way can in be a stepwisemoietytreated oragain manner, an alternativeeither using with hydrazine way the insame a and stepwise conditions the corresponding manner, mentioned using acyl above chloride.hydrazine to Lastly,obtain and the intermediatethe corresponding triazolopyridazine202 can acyl be obtainedchloride.moiety or fromLastly, an alternative201 intermediatejust by refluxingway in202 a it stepwisecan with be sodium obtainedmanner, hydroxide fromusing 201 inhydrazine the just presence by and refluxing ofthe 1,4-dioxane corresponding it with [143 sodium– 145acyl]. hydroxidechloride. Lastly, in the presenceintermediate of 1,4-dioxane 202 can [143–145].be obtained from 201 just by refluxing it with sodium hydroxide in the presence of 1,4-dioxane [143–145].

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Scheme 34. a) Ar-CO-NH-NH2, TEA, xylene or 1,4-dioxane, reflux. b) Het-CH2-OH, Base (NaH or Scheme 34. a) Ar-CO-NH-NH2, TEA, xylene or 1,4-dioxane, reflux. b) Het-CH2-OH, Base (NaH or LiHMDS),LiHMDS),Scheme DMf.34. DMf. a) c) Ar-CO-NH-NH c)(NH (NH4)2S)2OS8,O AgNO,2, AgNO TEA,3, R xylene1,R-COOH,-COOH, or H1,4-dioxane,2SO H 4SO, H2,HO, reflux.70O, °C. 70 d)C.b) d)Het-CH1. NH 1. NH2-NH2-OH,-NH2·H Base2O,H AcOH,O, (NaH AcOH, or 4 2 2 8 3 1 2 4 2 ◦ 2 2· 2 NaOAc, reflux. 2. POCl3. e) 4Ar-CO-NH-NH2 2 8 3 2, 1TEA, xylene2 or 41,4-dioxane,2 reflux. f) NH2 2-NH2 2,2 solvent NaOAc,LiHMDS), reflux. DMf. 2. c) POCl (NH3.) e)S Ar-CO-NH-NHO , AgNO , R -COOH,2, TEA, xylene H SO or, H 1,4-dioxane,O, 70 °C. d) reflux. 1. NH f) NH-NH2-NH·H O,2, solventAcOH, notnotNaOAc, reported. reported. reflux. g) Ar-CO-Cl, g) 2. Ar-CO-Cl, POCl3 .solvent e) solventAr-CO-NH-NH not not reported. reported.2, TEA,h) NaOH, h) xylene NaOH, 1,4-dioxane, or 1,4-dioxane, 1,4-dioxane, reflux. reflux. reflux. f) NH2-NH2, solvent not reported. g) Ar-CO-Cl, solvent not reported. h) NaOH, 1,4-dioxane, reflux. IntermediatesIntermediates 201-203201-203 areare then then converted converted to tothe the target target scaffold scaffold 204204 viavia the the three three methods methods shown shown in inScheme SchemeIntermediates 35. 35 Conditions. Conditions 201-203 a) and a) are and b) then correspond b) correspond converted to a to classical athe classical target Williamson scaffold Williamson 204 etheri etherificationviafication the three protocol, methods protocol, where shown where eithereitherin Scheme the the necessary necessary 35. Conditions alcohol alcohol ora) orandthe the halideb) halidecorrespond is attached is attached to a to classical tothe the main main Williamson heterocyclic heterocyclic etheri core. core.fication The The third protocol, third reported reported where strategystrategyeither wasthe was necessaryperformed performed alcohol by bya homolytic a or homolytic the halide alkylation alkylation is attached of the of to the heteroaromatic the heteroaromatic main heterocyclic ring, ring, generating core. generating The the third the required requiredreported alkylalkylstrategy radical radical was from performed from a silver-catalyzed a silver-catalyzed by a homolytic decarboxylat decarboxylation alkylationion of theof oftheheteroaromatic the corresponding corresponding ring, carboxylic generating carboxylic acid the acid requiredusing using ammoniumammoniumalkyl radical persulphate. persulphate. from a silver-catalyzed The The synthesi synthesiss of decarboxylat the of the required requiredion intermediates intermediatesof the corresponding 201-203201-203 is shownis carboxylic shown in Scheme in Schemeacid using35. 35 . ammonium persulphate. The synthesis of the required intermediates 201-203 is shown in Scheme 35.

Scheme 35. a) Het-CH2-OH, base (NaH or LiHMDS), DMf. b) Het-CH2-Cl, base (NaH or LiHMDS),

DMf.SchemeScheme c) (NH 35.35.4)2 Sa)a)2O Het-CHHet-CH8, AgNO22-OH,3, R1-COOH, base (NaH(NaH acidic oror LiHMDS),conditionsLiHMDS), DMf.DMf.(H2SO b)4 aq, Het-CHHet-CH CH3CN2-Cl,-Cl, aq), base reflux. (NaH or LiHMDS),

DMf.DMf. c) (NH4))22SS22OO8,8 ,AgNO AgNO3,3 R,R1-COOH,1-COOH, acidic acidic conditions conditions (H (H2SO2SO4 aq,4 aq, CH CH3CN3CN aq), aq), reflux. reﬂux. 4.3. Fused Tricyclic GABAA Receptor Modulators 4.3. Fused Tricyclic GABAA Receptor Modulators 4.3.1. Fused Tricyclic GABAA Receptor Modulators Containing Two Nitrogens 4.3.1. Fused Tricyclic GABAA Receptor Modulators Containing Two Nitrogens β-Carbolines β-Carbolines β-Carbolines were identified in the 1980s as potential endogenous ligands for the benzodiazepineβ-Carbolines binding were site identified(Figure 14). in In thisthe versatile1980s as scaffold, potential compounds endogenous with diverseligands profiles for the benzodiazepine binding site (Figure 14). In this versatile scaffold, compounds with diverse profiles

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4.3. Fused Tricyclic GABAA Receptor Modulators

4.3.1. Fused Tricyclic GABAA Receptor Modulators Containing Two Nitrogens Molecules 2019, 24, x FOR PEER REVIEW 33 of 48 Molecules 2019, 24, x FOR PEER REVIEW 33 of 48 β-Carbolines ranging from strong to moderate unselective negative modulation DMCM (compound 210) and FG- 7142ranging β(compound-Carbolines from strong 211 were )to to moderate identified mixed NAM/weak inunselective the 1980s PAM negative as potential subtype modulation endogenousprofiles ( DMCMβ-carboline-3-carboxylate- ligands (compound for the benzodiazepine 210) andt-butyl FG- ester,binding7142 (compound βCCt) site were (Figure 211 identified, )14 to). mixed In thiswhich NAM/weak versatile makes scathem PAMffold, heavily subtype compounds used profiles research with (β-carboline-3-carboxylate- diverse tools in profiles rodent rangingstudiest -butyl[141– from 144].strongester, β toCCt) moderate were identified, unselective which negative makes modulation them heavily DMCM used (compound research 210tools) and in rodent FG-7142 studies (compound [141– 211144].) to mixed NAM/weak PAM subtype profiles (β-carboline-3-carboxylate-t-butyl ester, βCCt) were identified, which makes them heavily used research tools in rodent studies [141–144].

Figure 14. Chemical structure of carbolines as GABAA modulators. Figure 14.14. Chemical structure of carbolines as GABAAA modulators. The synthetic accessibility of derivatives such as βCCT (compound 209) is outlined in Scheme The syntheticsynthetic accessibilityaccessibility ofof derivatives derivatives such such as asβ CCTβCCT (compound (compound209 209) is) outlinedis outlined in Schemein Scheme 36. 36. As the first step, tetrahydro-β-carboline (compound 213) was obtained from D,L-tryptophan As the first step, tetrahydro-β-carboline (compound 213) was obtained from d,l-tryptophan (compound (compound36. As the first212 ) step,via atetrahydro- Pictet–Spenglerβ-carboline reaction (compound on a kilogram 213) was scale. obtained The tetrahydro- from D,L-tryptophanβ-carboline 212(compound) via a Pictet–Spengler 212) via a Pictet–Spengler reaction on a kilogramreaction scale.on a Thekilogram tetrahydro- scale. βThe-carboline tetrahydro- (compoundβ-carboline213) (compound 213) underwent a Fischer esterification, and, afterward, oxidation with activated MnO2, underwent a Fischer esterification, and, afterward, oxidation with activated MnO , which yielded which(compound yielded 213 )the underwent product aβ FischerCCE (compound esterification, 215 and,). The afterward, ester was oxidation further withhydrolyzed 2activated and MnO the2, the product βCCE (compound 215). The ester was further hydrolyzed and the corresponding acid correspondingwhich yielded acidthe productre-esterified βCCE with (compound t-butanol 215and). CDIThe esterto yield was β CCTfurther (compound hydrolyzed 209 and) [146]. the re-esterified with t-butanol and CDI to yield βCCT (compound 209)[146]. Compound FG 7142 Compoundcorresponding FG 7142acid (compoundre-esterified 211 with) can t -butanolbe easily achievedand CDI byto treating yield β intermediateCCT (compound (compound 209) [146]. 216) (compound 211) can be easily achieved by treating intermediate (compound 216) with methylamine withCompound methylamine FG 7142 overnight, (compound which 211) yields can be the easily product achieved in a byperiod treating of 12 intermediate h. (compound 216) overnight, which yields the product in a period of 12 h. with methylamine overnight, which yields the product in a period of 12 h.

Scheme 36. a)a) HCHO, HCHO, NaOH, 37 °C,◦C, 95%. b) b) EtOH, EtOH, 2 eq H 2SOSO44, ,reflux, reflux, 83%. 83%. c) c) MnO MnO22, ,benzene, benzene, reflux, reflux, Scheme 36. a) HCHO, NaOH, 37 °C, 95%. b) EtOH, 2 eq H2SO4, reflux, 83%. c) MnO2, benzene, reflux, 80%. d) d) NaOH, NaOH, H H22O,O, reflux, reflux, 90%. 90%. e) e) CDI/DMF, CDI/DMF, DBU, DBU, 50 50 eq. eq. tt-BuOH,-BuOH, 85 85 °C,◦C, 75%. 75%. 80%. d) NaOH, H2O, reflux, 90%. e) CDI/DMF, DBU, 50 eq. t-BuOH, 85 °C, 75%. Alternatively, an an improved improved large- large-scalescale two-step two-step route route for for carboline carboline βCCTβCCT was was developed developed by performingby performingAlternatively, a Buchwald-Hartwig a Buchwald-Hartwig an improved large- coupling couplingscale oftwo-step t of-butylt-butyl route 5-bromopicolinate 5-bromopicolinate for carboline β(compoundCCT (compound was developed 217217) and) and by2- chloroaniline.2-chloroaniline.performing a Buchwald-HartwigThe The resulting resulting diarylamine diarylamine coupling (compound of (compound t-butyl 5-bromopicolinate218218) )underwent underwent an(compound an intramolecular intramolecular 217) and Heck 2- cyclization,chloroaniline. whichwhich The waswas resulting performedperformed diarylamine on on a a 10-g 10-g scale (compoundscale and and aff affordedorded 218)a underwent mixture a mixture of β ofCCTan β CCTintramolecular (compound (compound209 Heck) 209 and) anditscyclization, regioisomer, its regioisomer, whichδCCT was δ (compoundCCT performed (compound219 on), a in 21910-g a ratio), scalein 2:1,a ratioand respectively. afforded2:1, respectively. a Despite mixture this,Despite of overallβCCT this, (compound yields overall improved yields 209) improvedsignificantly,and its regioisomer, significantly, from the δCCT previous from (compound the 35% previous to 52%219 35%), on in a toa 10-g ratio52% scale on2:1, a andrespectively.10-g authors scale and declared Despite authors thatthis, declared theoverall separation that yields the separationofimproved regioisomers significantly,of regioisomers was performed from was the with performedprevious simple 35% with flash to 52%simple chromatography on flasha 10-g chromatography scale (Scheme and authors 37). (Scheme This declared protocol 37). that This was the protocolalsoseparation applied was of to alsoregioisomers derivatives applied to withwas derivatives performed different with alkoxy with different simple substituents alkoxyflash orthochromatography substituents to the pyridyl ortho (Scheme nitrogento the 37). pyridylatom, This nitrogenwhichprotocol yielded atom,was also which the desiredapplied yielded couplingto the derivatives desired product coupling with in highdifferent product yields. alkoxyin Whenhigh yields.substituents the subsequent When orthothe subsequent Heck to the cyclization pyridyl Heck cyclizationwasnitrogen performed, atom, was which performed, the ratios yielded betweenthe the ratios desired ß-carbolinesbetween coupling ß-carbolines andproductδ-carbolines inand high δ-carbolines yields. seemed When seemed to bethe influenced subsequent to be influenced by Heck the bysubstituentscyclization the substituents was located performed, ortholocated tothe ortho the ratios pyridyl to between the nitrogen pyridyl ß-carbolines atom.nitrogen Smaller and atom. δ-carbolines substituents Smaller seemed substituents provided to be influenced equimolarprovided equimolarby the substituents amounts of located both isomer orthos, tobut the bulkier pyridyl substituents nitrogen (iatom.-Pr, t-Bu) Smaller afforded substituents β-carbolines provided as the mainequimolar product amounts (scheme of bothnot shown) isomer s,[147]. but bulkier substituents (i-Pr, t-Bu) afforded β-carbolines as the main product (scheme not shown) [147].

Molecules 2020, 25, 999 33 of 47

amounts of both isomers, but bulkier substituents (i-Pr, t-Bu) aﬀorded β-carbolines as the main product (schemeMolecules 2019 not, 24 shown), x FOR [PEER147]. REVIEW 34 of 48

Scheme 37. a) 2-chloroaniline, 5 mol% Pd(OAC)2, 7.5 mol% Xantphos, 1.5 eq. Cs2CO3, toluene, 100 °C, Scheme 37. a) 2-chloroaniline, 5 mol% Pd(OAC)2, 7.5 mol% Xantphos, 1.5 eq. Cs2CO3, toluene, 100 ◦C, 15 h, 94%. B) Pd(OAc)2, (t-Bu)3P HBF4, K2CO3, DMAc, 120 °C, 16 h, 83%. 15 h, 94%. b) Pd(OAC)2,(t-Bu)3P HBF4,K2CO3, DMAc, 120 ◦C, 16 h, 83%.

4.3.2. Fused Tricyclic GABAA Receptor Modulators Co Containingntaining Three Nitrogens

Pyrazoloquinolinones Pyrazoloquinolinones (PQs) (structure 220)) were reported for the firstfirst time as ligands of thethe “benzodiazepine“benzodiazepine receptor” receptor” (which (which is ais historical a historical term term for the for high-a the high-affinityffinity benzodiazepine benzodiazepine sites present sites atpresent GABA atA GABAreceptors)A receptors) in 1982 [ 148in 1982] when [148] the when synthesis the ofsynthesis three PQs of wasthree reported, PQs was and reported, these derivatives and these werederivatives presented were as presented useful compounds as useful compounds to study the to complex study the pharmacology complex pharmacology of this receptor. of this The receptor. three compounds,The three compounds, in fact, showed in fact, three showed different threein different vivo pharmacological in vivo pharmacological behaviors, behaviors, which were which ascribed were toascribed the benzodiazepine to the benzodiazepine binding site. binding While the site. unsubstituted While the derivative,unsubstituted CGS8216 derivative, (compound CGS8216225), showed(compound benzodiazepine-antagonist 225), showed benzodiazepine-antagonist properties, the p-chloro properties, derivative the p-chloro (CGS9896) derivative (compound (CGS9896)226) showed(compound benzodiazepine 226) showed agonistic benzodiazepine properties andagonistic the p-methoxy properties derivative and the (CGS9895) p-methoxy (compound derivative227) behaved(CGS9895) consistently (compound with 227 benzodiazepine) behaved consistently partial with agonists. benzodiazepine Among these partial three, agonists. the agonist Among CGS9896 these (compoundthree, the agonist226)(p CGS9896-chloro) was (compound claimed to226 be) a(p safe-chloro) anti-anxiety was claimed agent to due be to a thesafe lack anti-anxiety of neurological agent deficitsdue to the and lack its non-sedativeof neurological behavior. deficits Furthermore,and its non-sedative functional behavior. studies Furthe revealedrmore, that functional PQs interact studies with additionalrevealed that sites PQs of GABAinteractA withreceptors additional [149], sites which of makesGABA itA receptors impossible [149], to assign which their makesin vivo it impossibleeffects of anyto assign single their binding in vivo site. effects of any single binding site. The synthetic strategy for the synthesis of PQ derivativesderivatives remained unchanged with few modificationsmodifications and still follows the route displayed in SchemeScheme 3838.. The quinoline derivative (structure 222) is obtainedobtained starting from thethe correspondingcorresponding p-anisidine-anisidine (structure 221)) uponupon condensationcondensation withwith diethyl ethoxymethylenemalonate (DEEMM), which was followedfollowed byby aa thermalthermal cyclization.cyclization. The chlorination of of the the quinoline quinoline derivative derivative with with POCl POCl3 (ii)3 (ii) yields yields the theprecursor precursor of the of PQ the (structure PQ (structure 223). 223The). required The required phenylhydrazine phenylhydrazine (structure (structure 224)224 is )used is used for for the the formation formation of of PQs PQs with with didifferentfferent substituents in the D ring. Different Different A-ring substitution patterns are obtained using didifferentfferent aniline derivatives as starting materials.materials. Replacing the A-ring with electron-rich heterocyclic scaffolds (structure 223) requires modification of the synthetic sequence (Scheme 39). Thiophene-3-amine would be too electron-rich to be a stable starting material. Hence, compound 229 is used as starting material and in situ hydrolyzed, decarboxylated, and reacted with DEEMM as reported in the supplementary information of Varagic et al. [149]. From then on, the sequence follows the same steps as with carbocyclic A-rings.

Scheme 38. a) DEEMM, Ph2O, reflux. b) POCl3, reflux. c) arylhydrazine, DMAc, 140 °C. d) xylene, 120 °C.

Molecules 2019, 24, x FOR PEER REVIEW 34 of 48

Scheme 37. a) 2-chloroaniline, 5 mol% Pd(OAC)2, 7.5 mol% Xantphos, 1.5 eq. Cs2CO3, toluene, 100 °C,

15 h, 94%. B) Pd(OAc)2, (t-Bu)3P HBF4, K2CO3, DMAc, 120 °C, 16 h, 83%.

4.3.2. Fused Tricyclic GABAA Receptor Modulators Containing Three Nitrogens

Pyrazoloquinolinones Pyrazoloquinolinones (PQs) (structure 220) were reported for the first time as ligands of the “benzodiazepine receptor” (which is a historical term for the high-affinity benzodiazepine sites present at GABAA receptors) in 1982 [148] when the synthesis of three PQs was reported, and these derivatives were presented as useful compounds to study the complex pharmacology of this receptor. The three compounds, in fact, showed three different in vivo pharmacological behaviors, which were ascribed to the benzodiazepine binding site. While the unsubstituted derivative, CGS8216 (compound 225), showed benzodiazepine-antagonist properties, the p-chloro derivative (CGS9896) (compound 226) showed benzodiazepine agonistic properties and the p-methoxy derivative (CGS9895) (compound 227) behaved consistently with benzodiazepine partial agonists. Among these three, the agonist CGS9896 (compound 226) (p-chloro) was claimed to be a safe anti-anxiety agent due to the lack of neurological deficits and its non-sedative behavior. Furthermore, functional studies revealed that PQs interact with additional sites of GABAA receptors [149], which makes it impossible to assign their in vivo effects of any single binding site. The synthetic strategy for the synthesis of PQ derivatives remained unchanged with few modifications and still follows the route displayed in Scheme 38. The quinoline derivative (structure 222) is obtained starting from the corresponding p-anisidine (structure 221) upon condensation with diethyl ethoxymethylenemalonate (DEEMM), which was followed by a thermal cyclization. The chlorination of the quinoline derivative with POCl3 (ii) yields the precursor of the PQ (structure 223). The required phenylhydrazine (structure 224) is used for the formation of PQs with different substituents in the D ring. Different A-ring substitution patterns are obtained using different aniline Moleculesderivatives2020, 25as, 999starting materials. 34 of 47

Molecules 2019, 24, x FOR PEER REVIEW 35 of 48

Replacing the A-ring with electron-rich heterocyclic scaffolds (structure 223) requires modification of the synthetic sequence (Scheme 39). Thiophene-3-amine would be too electron-rich to be a stable starting material. Hence, compound 229 is used as starting material and in situ hydrolyzed, decarboxylated, and reacted with DEEMM as reported in the supplementary information of Varagic et al. [149]. From then on, the sequence follows the same steps as with SchemeScheme 38. 38.a) a) DEEMM,DEEMM, PhPh22O, reflux. reﬂux. b) b) POCl POCl3,3 reflux., reﬂux. c) c)arylhydrazine, arylhydrazine, DMAc, DMAc, 140 140 °C.◦ d)C. xylene, d) xylene, 120 carbocyclic A-rings. 120°C.◦ C.

SchemeScheme 39. 39. a)a) 1 1 M, M, NaOH, NaOH, reflux. reflux. b) b) DEEMM, DEEMM, AcOH, AcOH, toluene, toluene, reflux. reflux. c) c) Ph Ph22O,O, reflux. reflux. d) d) POCl POCl33, ,reflux. reflux. e)e) arylhydrazine, arylhydrazine, DMAc, DMAc, 140 140 °C.◦C.

DueDue to thethe inconsistentinconsistent results results obtained obtained during during the the investigation investigation of their of their potential potential anxiolytic anxiolytic effect, effect,PQs were PQs notwere further not further investigated investigated as potential as potentia therapeuticl therapeutic agents agents [150,151 [150,151],], but they but keptthey beingkept being used usedas pharmacological as pharmacological tools tools in the in studies the studies of GABA of GABAA receptors.A receptors. OverOver the the years, years, the the general general scaffold scaffold of of PQ PQ has been extensively modified, modified, especially especially on on ring ring A A andand ring ring D D [152–154], [152–154], which which results results in in compounds compounds wi withth different different pharmacological pharmacological profiles.[155,156]. profiles [155,156]. Additionally,Additionally, studiesstudies onon thethe binding binding site site of of PQs PQs were were performed. performed. While While displaying displaying a highera higher affi affinitynity for forthe the BZD-site, BZD-site, PQs PQs such such as as CGS9895 CGS9895 (compound (compound227 227) were) were reported reported to to have have a a modulatory modulatory e ffeffectect at at a abinding binding site site situated situated between between the theα+/ αβ+/β−interfaces, interfaces, where where it displaysit displays a lower a lower potency potency compared compared to the to − theBZD-site BZD-site [19 ,[19,157].157]. Replacement Replacement of ringof ring A withA with an aliphatican aliphatic ring ring resulted resulted in ain null a null modulator modulator at the at thebenzodiazepine benzodiazepine (BZD) (BZD) binding binding site, and site, the and replacement the replacement with a thiophene with a ringthiophene led to thering synthesis led to ofthe a synthesisnon-binder of [a149 non-binder]. Additionally, [149]. theAdditionally, relationship the between relationship the substitution between the pattern substitution and binding pattern activity and bindingwas investigated. activity was It was investigated. demonstrated It was that demonstrat substituentsed inthat positions substituents 7 and in 9 causedpositions a lack 7 and of a9ffi causednity at athe lack BDZ of affinity site, while at the substitution BDZ site, inwhile position substitution 8 associated in position with a 8 substituent associated onwith the a phenylsubstituent ring on on theN2 phenylincreased ring the on activity N2 increased [156]. the activity[156].

4.3.3.4.3.3. Fused Fused Tricyclic Tricyclic GABA GABAAA ReceptorReceptor Modulators Modulators Containing Containing Four Four Nitrogens Nitrogens

TriazoloquinazolinesTriazoloquinazolines 220 AfterAfter the the discovery discovery of of PQ PQ derivatives derivatives (structure (structure 220),), and and their their modulatory modulatory activity, activity, more more compoundscompounds with with heterotricyclic heterotricyclic structures structures became became appealing appealing chemotypes. chemotypes. The The replacement replacement of of ring ring 234 CC with with a a triazole triazole led led to to structure structure 234 (Figure(Figure 15).15). However, However, it it did did not not lead lead to to an an improvement improvement in in the the activityactivity in in comparison comparison with with the the PQ PQ scaffold. scaﬀold. Th Thee only only exception exception was was the the ortho fluorophenyl, ﬂuorophenyl, which which hadhad a a comparable comparable activity activity [158,159]. [158,159].

Figure 15. Triazoloquinazolines.

Triazole containing structures, such as compound 235 (Figure 15), were initially reported as antagonists or partial inverse agonists for the BZR (the historical term for the high-affinity benzodiazepine binding sites) [160]. The synthesis of such compounds is achieved by starting from the arylhydrazines (structure 237) upon treatment with aryl chlorides, which was followed by a chlorination. Structure 239 was then treated with ammonia to yield structure 241. The reaction of the

Molecules 2019, 24, x FOR PEER REVIEW 35 of 48

Scheme 39. a) 1 M, NaOH, reflux. b) DEEMM, AcOH, toluene, reflux. c) Ph2O, reflux. d) POCl3, reflux. e) arylhydrazine, DMAc, 140 °C.

Due to the inconsistent results obtained during the investigation of their potential anxiolytic effect, PQs were not further investigated as potential therapeutic agents [150,151], but they kept being used as pharmacological tools in the studies of GABAA receptors. Over the years, the general scaffold of PQ has been extensively modified, especially on ring A and ring D [152–154], which results in compounds with different pharmacological profiles.[155,156]. Additionally, studies on the binding site of PQs were performed. While displaying a higher affinity for the BZD-site, PQs such as CGS9895 (compound 227) were reported to have a modulatory effect at a binding site situated between the α+/β− interfaces, where it displays a lower potency compared to the BZD-site [19,157]. Replacement of ring A with an aliphatic ring resulted in a null modulator at the benzodiazepine (BZD) binding site, and the replacement with a thiophene ring led to the synthesis of a non-binder [149]. Additionally, the relationship between the substitution pattern and binding activity was investigated. It was demonstrated that substituents in positions 7 and 9 caused a lack of affinity at the BDZ site, while substitution in position 8 associated with a substituent on the phenyl ring on N2 increased the activity[156].

4.3.3. Fused Tricyclic GABAA Receptor Modulators Containing Four Nitrogens

Triazoloquinazolines After the discovery of PQ derivatives (structure 220), and their modulatory activity, more compounds with heterotricyclic structures became appealing chemotypes. The replacement of ring C with a triazole led to structure 234 (Figure 15). However, it did not lead to an improvement in the Moleculesactivity 2020in comparison, 25, 999 with the PQ scaffold. The only exception was the ortho fluorophenyl, 35which of 47 had a comparable activity [158,159].

Figure 15. Triazoloquinazolines.

Triazole containing structures, structures, such such as as compound compound 235235 (Figure(Figure 15), 15 ),were were initially initially reported reported as asantagonists antagonists or orpartial partial inverse inverse agonists agonists for for the the BZR BZR (the (the historical historical term term for for the high-ahigh-affinityﬃnity benzodiazepine binding binding sites) sites) [160]. [160]. The The synthesis synthesis of such of such compounds compounds is achieved is achieved by starting by starting from from the arylhydrazines (structure 237) upon treatment with aryl chlorides, which was followed by a Moleculesthe arylhydrazines 2019,, 24,, xx FORFOR (structurePEERPEER REVIEWREVIEW 237 ) upon treatment with aryl chlorides, which was followed36 byof 48 a chlorination. Structure 239 was then treated with ammonia toto yieldyield structurestructure 241241.. TheThe reactionreaction ofof thethe latter with ethyl oxalyl chloride led directly or upon further heating to structure 240. In the last step, a latterlatter withwith ethylethyl oxalyloxalyl chloridechloride ledled directlydirectly oror uponupon furtherfurther heatingheating toto structurestructure 240.. InIn thethe lastlast step,step, reduction performed with Fe and acetic acid led to the ﬁnal structure 235 (Scheme 40)[161]. a reduction performed with Fe and acetic acid led to the final structure 235 (Scheme(Scheme 40)40) [161].[161].

R H R 1 a) 1 l H R1 NHNH2 a) R1 NHNHCOR b) Rl N R NHNH2 R NHNHCOR b) R N d) N Cl

NO2 NO2 NO2 NO2 NO2 NO2 R = H, Cl, Br (237) (238) (239) (237) (238) (239) c) R 1 R R1 R N R R 1 H R N R 1 N 1 H d) N R N N R1 N d) N e) R N N N NH 2 N 2 N O NO2 COOEt N O NO 2 NO2 COOEt H 2 (240) (234) (241) (240) (234) (241)

Scheme 40. (a) RCOCl, EtOH/pyridine, reflux, 1 h. (b) POCl3, reflux, 1 h (c) NH3 (g), (d) Scheme 40.40. a)(a) RCOCl, RCOCl, EtOH EtOH/pyridine,/pyridine, reﬂux, reflux, 1 h. b) 1 POCl h. 3(b), reﬂux, POCl 13 h., reflux, c) NH 3 1(g), h d)(c) CICOCOOEI. NH3 (g), (d) (e) CICOCOOEI(e) Fe/CH3COOH. CICOCOOEI(e)Fe/CH3COOH. Fe/CH3COOH.

Looking at the structures of the GABA A receptorsreceptors ligands,ligands, PQs,PQs, andand phenyltriazoloquinoxalonephenyltriazoloquinoxalone (structure 234), the designdesign of triazoloquinazolinedionestriazoloquinazolinediones (structure (structure 236236)) was was the the logical next step (Figure 1616))[ [162].162].

Figure 16. DesignDesign of of 2-Aryltri 2-Aryltriazoloquinazolinediones.azoloquinazolinediones.

According to pharmacophore studies, this type of structure fulfills fulﬁlls the requirements for a strong affinityaﬃnity to the benzodiazepine binding site [163]. [163]. The The synthesis synthesis of of triazoloquinazolinedione triazoloquinazolinedione (structure (structure 254) was achievedachieved starting with the anthranilicanthranilic acid derivative (structure 242), which was treated with ethoxycarbonyl isothiocyanate. This This is is followed by a cyclization in acetic anhydride (structure 244). Deprotection Deprotection and and methylation methylation yielded yielded the the precursor precursor (structure (structure 246246), which), which was was treated treated with with 3- nitro-13-nitro-1H-1,2,4-triazoleH-1,2,4-triazole to yield to yield structure structure 247247.. TheThe. The reactionreaction reaction betweenbetween between structurestructure structure 247 andand247 compoundsandcompounds compounds 249- 252249 - 252delivereddelivereddelivered (structure(structure (structure 243243). Triazoloquinazolinediones). Triazoloquinazolinediones (structure (structure 254254) are) are then then obtained obtained upon treatmenttreatment of of (structure (structure 243243)) withwith mm--CPBACPBA (Scheme(Scheme 4141).).

Molecules 20192020,, 2425,, x 999 FOR PEER REVIEW 3736 of of 48 47 Molecules 2019, 24, x FOR PEER REVIEW 37 of 48

Scheme 41. a) Ethoxycarbonyl isothiocyanate, MeCN, 80 °C, 4 h, 91–95%. b) Ac2O, 60 °C, 1 h, 93–100%.

c)Scheme 0.5 M NaOMe41. a)a) Ethoxycarbonyl Ethoxycarbonyl in MeOH, THF, isothiocyanate isothiocyanate, reflux, 90 min,, MeCN, 88–100%. 80 ◦°C, d)C, DMF,4 h, 91–95%. 0.5 M NaOMeb) Ac Ac22O,O, in 60 60 MeOH, °C,◦C, 1 1 h, h, then93–100%. 93–100%. MeI, rt,c) 0.5 10 M h 77–90%. NaOMe e) in 3-nitro-1 MeOH,MeOH, THF,THF,H-1,2,4-triazole, reﬂux, reflux, 90 90 min, min, PPh 88–100%. 88–100%.3, I2, toluene, d) d) DMF, DMF, DIPEA, 0.5 0.5 M M95 NaOMe NaOMe°C, 1 h, in 89%.in MeOH, MeOH, f) DIPEA, then then MeI, MeI,249- rt,

252,rt,10 10 h 77–90%. 110h 77–90%. °C, e)60 3-nitro-1 e)h, 3-nitro-1 51–70%.H-1,2,4-triazole,H -1,2,4-triazole,g) m-CPBA PPh (77%), PPh3,I23, toluene,ICH2, toluene,2Cl2, DIPEA,rt, DIPEA,6-h 95quantitative ◦95C, °C, 1 h, 189%. h, yield.89%. f) DIPEA, f)h) DIPEA, CuI, 249-252, 1,10-249- phenantrolein,252,110 ◦C,110 60 °C, h, 51–70%.60 ethyl h, 51–70%.carbazate, g) m-CPBA g) Cs m (77%),2CO-CPBA3, DMF, CH (77%),2Cl 802, °C, rt,CH 6-h 162Cl h, quantitative2 ,yields rt, 6-h compound quantitative yield. h) 249: CuI, yield.83% 1,10-phenantrolein, compound h) CuI, 1,10- 250: 76%,phenantrolein,ethyl compound carbazate, ethyl Cs251:2CO carbazate,40%,3, DMF, compound 80Cs◦2C,CO 16 252:3, DMF, h, yields22%. 80 compound°C, 16 h, yields 249: compound 83% compound 249: 250:83% 76%,compound compound 250: 76%,251: 40%,compound compound 251: 40%, 252: 22%.compound 252: 22%. Alternatively, structure 246 can be activated with phosphorous oxychloride before the addition of compoundAlternatively, 250 structurestructureto yield 246 structure246can can be be 253 activated activated with withyields with phosphorous phosphorous comparable oxychloride oxychloridewith the nitrotriazole before before the the addition additionstrategy of (Schemeofcompound compound 42).250 250to yield to yield structure structure253 with 253 yields with comparable yields comparable with the nitrotriazolewith the nitrotriazole strategy (Scheme strategy 42). (Scheme 42).

Scheme 42. a) POCl 33,, pyridine, pyridine, 110 110 °C,◦C, 18 18 h, 75–77%. b) b) DIPEA, DIPEA, 110 110 °C,◦C, 60 h, 71–75%. c) c) m-CPBA-CPBA (77%), CH Cl , rt, 6 h, quantitative yields. (77%),Scheme CH 42.2Cl a)22, rt,POCl 6 h,3, quantitativepyridine, 110 yields. °C, 18 h, 75–77%. b) DIPEA, 110 °C, 60 h, 71–75%. c) m-CPBA

(77%), CH2Cl2, rt, 6 h, quantitative yields. The compounds compounds of of this this class class display display good good affinities affinities for for the the BZD BZD site, site, determined determined in vitroin vitro by 3 displacementby displacementThe compounds of 3 ofH-Flumazenil H-Flumazenilof this class in display inrat ratcortical corticalgood affinitiestissue, tissue, according accordingfor the BZD to to thesite, the pharmacophore pharmacophoredetermined in vitromodel. model. by Compound 256 can be further modified with cross-coupling reactions. For example, copper-free Compounddisplacement 256 of can3H-Flumazenil be further modified in rat cortical with cross-coup tissue, accordingling reactions. to the For pharmacophore example, copper-free model. SonogashiraCompound 256 coupling can be conditionsconditions further modified followedfollowed bywithby hydrogenation hydr cross-coupogenationling (Scheme (Scheme reactions. 43 43)) are Forare used usedexample, for for the the copper-free synthesis synthesis of 258, which displayed high-affinity and a certain subtype selectivity for α1ß3γ2 GABA receptors. ofSonogashira 258, which coupling displayed conditions high-affinity followed and a bycertain hydr subtypeogenation selectivity (Scheme for43) α are1ß3 usedγ2 GABA forA theA receptors. synthesis of 258, which displayed high-affinity and a certain subtype selectivity for α1ß3γ2 GABAA receptors.

Molecules 2020, 25, 999 37 of 47

MoleculesMolecules 2019 2019, ,24 24, ,x x FOR FOR PEER PEER REVIEW REVIEW 3838 ofof 4848

Scheme 43. a) Pd(OAc)2, PPh3, RcCH, NEt3, DMF, 100 °C, 18 h, 50%. b) H2 (1 atm) 10% Pd/C, CH2Cl2, Scheme 43.43. a) Pd(OAC)Pd(OAc)22,, PPh PPh33, ,RcCH, RcCH, NEt NEt33, ,DMF, DMF, 100 100 °C,◦C, 18 h, 50%. b) H2 (1(1 atm) 10% PdPd/C,/C, CHCH22Cl22, MeOH,MeOH, rt, rt, 18 1818 h, h,h, 91%. 91%.91%.

PyrazolobenzotriazinesPyrazolobenzotriazines

TheThe pyrazolobenzotriazine pyrazolobenzotriazine scaffold scaffold was was first first reported reported as as a a GABA GABAA receptorreceptor ligand ligand in in 1998 1998 [164]. [164]. The pyrazolobenzotriazine scaffold was first reported as a GABAAA receptor ligand in 1998 [164]. DuringDuring the the past past 10 10 10 years, years, years, N NN-oxide-oxide-oxide derivatives derivatives derivatives of of of this this this scaffold scaffold scaffold were were were synthesized synthesized synthesized and and and studied studied studied both both both in in vitroinvitro vitro and andand in in vivo invivo vivo with withwith some some some derivatives derivatives derivatives having having having binding binding binding affi affi affinitiesnitiesnities to to to the the the benzodiazepine benzodiazepine benzodiazepine binding binding site site lowerlowerlower than thanthan 10 1010 nM nMnM [165,166]. [[165,166].165,166]. Some Some of of them them possess possess anxiolytic anxiolytic activity activity [166], [166], [166], which, which, together, together, suggests suggestssuggests positivepositivepositive modulatory modulatorymodulatory effects eeffectsffects elicited elicited via via the the benzodiazepine benzodiazepine binding binding site.site. ThereThere are areare several severalseveral strategies strategiesstrategies reported reportedreported to toto access accessaccess th thisthisis scaffold. scascaffold.ffold. An An example example of of how how to to synthetically synthetically accessaccess the the main main scaffold scascaffoldffold is isis shown shown in inin Scheme Scheme 44. 4444.. The The accordingly accordingly substituted substituted 2-nitrophenylhydrazine 2-nitrophenylhydrazine (structure(structure(structure 260 260)) )was was was reacted reacted reacted with with with 2-oxosuccinonitrile 2-oxosuccinonitrile 2-oxosuccinonitrile (structure (structure (structure 261 261)261) to to )obtain obtain to obtain the the corresponding corresponding the corresponding 2-(2- 2-(2- nitrophenyl)-5-aminopyrazole-4-acetonitrile2-(2-nitrophenyl)-5-aminopyrazole-4-acetonitrilenitrophenyl)-5-aminopyrazole-4-acetonitrile (structure(structure (structure 262262262).). AcidicAcidic). Acidic hydrolysishydrolysis hydrolysis ofof of thisthis this compound compound gave,gave, asas as aa aresult,result, result, thethe the correspondingcorresponding corresponding acetamideacetamide acetamide derivative,derivative, derivative, andand furtherfurther and further treatmenttreatment treatment inin alkalinealkaline in alkaline media-media- initiatedmedia-initiatedinitiated cyclization cyclization cyclization to to the the benzotriazine benzotriazine to the benzotriazine system system system(structure (structure (structure 259 259).). 259).

Scheme 44. a) EtOH. b) H2SO4. c) 10% aq NaOH. SchemeScheme 44.44. a)a) EtOH.EtOH. b)b) HH22SO44. c) c) 10% 10% aq aq NaOH. NaOH.

PyrazolobenzotriazinePyrazolobenzotriazine (structure (structure(structure259 259259) derivatives)) derivativesderivatives containing containingcontaining halogenated halohalogenatedgenated substituents substituentssubstituents were usefulwerewere usefulbuildinguseful building building blocks blocks blocks to perform to to perform perform subsequent subsequent subsequent Suzuki-Miyaura Suzuki-Miyaura Suzuki-Miyaura cross-coupling cross-coupling cross-coupling reactions reactions reactions with with with several several several aryl aryloraryl heteroaryl or or heteroaryl heteroaryl boronic boronic boronic acids acids acids by by incorporatingby incorporating incorporating these th theseese cyclic cyclic cyclic substituents substituents substituents into into into the the the mainmain main core.core. core. Further FurtherFurther 4 modificationsmodiﬁcationsmodifications can can be be performed performedperformed as as well wellwell to toto the thethe R R44 substituentssubstituentssubstituents in inin order orderorder for forfor convenient convenientconvenient functional functionalfunctional groupsgroups such suchsuch as as carboxylic carboxylic acids, acids, amides, amides, or or other other functional functional groups groups to to introduce introduce several several heterocyclic heterocyclic five-memberedﬁve-memberedfive-membered rings ringsrings (Scheme (Scheme(Scheme not notnot shown). shown).shown).

5.5. Conclusions Conclusions ItItIt was waswas shown shownshown in inin the thethe previous previousprevious sections sectionssections that that a a larg largelargee number number of of heterocyclic heterocyclic scaffolds scascaffoldsﬀolds are, are, or or have havehave been, under investigation as GABAA receptor modulators. It can be observed that the number of been, underunder investigationinvestigation asas GABAGABAAA receptor modulators. It It can can be observed that the numbernumber ofof examplesexamples for for a a given given scaffold scascaffoldﬀold that thatthat underwent underwentunderwent biological biologicalbiological testing testing is is directly directly proportional proportional to toto the the ease ease of of accessaccess of of the the corresponding corresponding structures. structures. However, However, focu focusingfocusingsing on onon easily easily accessible accessible chemotypes chemotypes is is not not how how medicinalmedicinal chemistry chemistry will will experience experience progress progress in in the the future. future. Hence, Hence, it it is is the the task task of of synthetic synthetic chemists chemists toto develop develop methods methods to to make make any any scaffold scaffold accessible accessible within within only only a a few few synthetic synthetic steps steps and and to to develop develop thethe potential potential to to deliver deliver compounds compounds with with good good diversity diversity regarding regarding substitution substitution patterns. patterns. Thus, Thus, there there

Molecules 2020, 25, 999 38 of 47 medicinal chemistry will experience progress in the future. Hence, it is the task of synthetic chemists to develop methods to make any scaffold accessible within only a few synthetic steps and to develop the potential to deliver compounds with good diversity regarding substitution patterns. Thus, there is still an infinite playground to develop new and exciting heterocyclic chemistry in order to enable the discovery of improved or completely new biological activity, especially within the pending challenges in GABAA pharmacology toward improved subtype-selective compounds. New (or re-discovered) developments in synthesis will be important contributors to reach this goal. In particular, it will be interesting to see how a broader implementation of biocatalysis, electrochemistry, continuous-flow chemistry, and photo(redox)chemistry will impact drug development in the future.

Author Contributions: B.A.V.A. writing—original draft preparation, writing—review and editing. M.T.I. writing—original draft preparation, writing—review and editing. L.S. writing—original draft preparation. K.B. writing—original draft preparation, writing—review and editing. M.E. conceptualization, writing—original draft preparation, writing—review and editing. M.S. conceptualization, writing—original draft preparation, writing—review and editing. M.D.M. conceptualization, writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding: The Austrian Science Fund FWF grant W1232 (graduate school program MolTag) financially supported this work. Acknowledgments: Open Access Funding by the Austrian Science Fund (FWF). Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ADME Absorption, Distribution, Metabolism, and Excretion ACN Acetonitrile Bbt Barbiturate BZD Benzodiazepine(s) BZR Benzodiazepine receptor CDI 1,10-Carbonyldiimidazole DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCC N,N0-Dicyclohexylcarbodiimide DCE 1,2-dichloroethane DCM Dichloromethane DMAc Dimethylacetamide DMAP 4-Dimethylaminopyridine DMF Dimethylformamide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DEEMM Diethyl ethoxymethylenemalonate DIPEA N,N-Diisopropylethylamine EBOB Ethynylbicycloorthobenzoate ECD Extracellular domain Eto Etomidate GABA Gamma-aminobutyric acid GABAA GABAA receptors HATU Hexaﬂuorophosphate Azabenzotriazole Tetramethyl Uronium HTIB [Hydroxy(tosyloxy)iodo]benzene IPA HCl Isopropanol hydrochloride KOtBu Potassium tert-butoxide NAM Negative Allosteric Modulator NMP N-Methyl-2-Pyrrolidone OTC Over-the-counter PAM Positive Allosteric Modulator PET Positron Emmission Tomography PQ Pyrazoloquinolinone Molecules 2020, 25, 999 39 of 47

SAM Silent Allosteric Modulator TBBAB Tetra-n-butylammonium bromide TBAF Tetrabutylammonium ﬂuoride TEA Triethylamine THF Tetrahydrofuran TMD Transmembrane domain TMP 2,2,6,6-Tetramethylpiperidine TosMIC p-Toluenesulfonylmethyl isocyanide

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