pharmaceuticals

Review BACE-1 and γ-Secretase as Therapeutic Targets for Alzheimer’s Disease

Miguel A. Maia 1 and Emília Sousa 1,2,* 1 Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; [email protected] 2 Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal * Correspondence: [email protected]; Tel.: +351-220428689



Received: 15 February 2019; Accepted: 15 March 2019; Published: 19 March 2019


Abstract: Alzheimer’s disease (AD) is a growing global health concern with a massive impact on affected individuals and society. Despite the considerable advances achieved in the understanding of AD pathogenesis, researchers have not been successful in fully identifying the mechanisms involved in disease progression. The amyloid hypothesis, currently the prevalent theory for AD, defends the deposition of β-amyloid protein (Aβ) aggregates as the trigger of a series of events leading to neuronal dysfunction and dementia. Hence, several research and development (R&D) programs have been led by the pharmaceutical industry in an effort to discover effective and safety anti-amyloid agents as disease modifying agents for AD. Among 19 drug candidates identified in the AD pipeline, nine have their mechanism of action centered in the activity of β or γ-secretase proteases, covering almost 50% of the identified agents. These drug candidates must fulfill the general rigid prerequisites for a drug aimed for central nervous system (CNS) penetration and selectivity toward different aspartyl proteases. This review presents the classes of γ-secretase and beta-site APP cleaving enzyme 1 (BACE-1) inhibitors under development, highlighting their structure-activity relationship, among other physical-chemistry aspects important for the successful development of new anti-AD pharmacological agents.

Keywords: Alzheimer’s disease; amyloid hypothesis; γ-secretase; BACE-1

1. Introduction

1.1. Alzheimer’s Disease–Epidemiology AD is recognized by the World Health Organization (WHO) as a global public health priority [1]. Notwithstanding the advances in the understanding of AD pathogenesis since Alois Alzheimer reported the first case in 1907 [2], there are still a lot of uncertainties regarding the mechanisms involved in disease progression. AD is the most prevalent cause of dementia-acquired progressive cognitive impairment sufficient to impact on activities of daily living, being a major cause of dependence, disability and mortality. The WHO estimated that in 2010, 35.6 million worldwide were living with dementia [3]. This figure is projected to almost double every 20 years, reaching 65.7 million by 2030 and 115.4 million by 2050. Europe, with an estimated 10 million cases of dementia in 2010 and acquiring progressively an old population structure [4], has a projected increase to 14 million cases in 2030 [1]. In the United States of America (USA), the total annual payments for health care, long-term care and hospice care for people with AD or other dementias are projected to increase from $259 billion in 2017 to more than $1.1 trillion in 2050 [5]. These numbers show the dramatic impact that AD and the other dementias will have in the health care systems in the future.

Pharmaceuticals 2019, 12, 41; doi:10.3390/ph12010041 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2018,, 11,, 2 of 31 Pharmaceuticals 2018, 11, 2 of 31 in 2017 to more than $1.1 trillion in 2050 [5]. These numbers show the dramatic impact that AD and inin 20172017 toto moremore thanthan $1.1$1.1 trilliontrillion inin 20502050 [5].[5]. ThesThese numbers show the dramatic impact that AD and the other dementias will have in the health care systems in the future. the otherPharmaceuticals dementias2019 will, 12 have, 41 in the health care systems in the future. 2 of 31

1.2. Pathology 1.2. Pathology1.2. Pathology The main characteristics of Alzheimer’s pathology are the presence of amyloid plaques and The main characteristics of Alzheimer’s pathology are the presence of amyloid plaques and neurofibrillaryThe tangles main characteristics (aggregates of of hyperphosphorylated Alzheimer’s pathology tau are protein) the presence [5]. In addition, of amyloid neuropil plaques and neurofibrillary tangles (aggregates of hyperphosphorylated tau protein) [5].. In addition, neuropil treads,neurofibrillary dystrophic neurites, tangles associated (aggregates astrogliosis, of hyperphosphorylated and microglial activation tau protein) frequently [5]. In addition,coexist. The neuropil treads, dystrophic neurites, associated astrogliosis, and microglial activation frequently coexist. The downstreamtreads, consequences dystrophic neurites, of these pathological associated astrogliosis, processes include and microglial neurodegeneration activation with frequently synaptic coexist. downstream consequences of these pathological processes include neurodegeneration with synaptic and neuronalThe downstream loss leading consequences to macroscopic of these atrophy pathological [6]. Research processes suggests include that neurodegenerationthe brain changes with and neuronal loss leading to macroscopic atrophy [6]. Research suggests that the brain changes associatedsynaptic with andAD neuronalmay begin loss 20 leadingor more to years macroscopic before symptoms atrophy [ 6appear]. Research [7,8]. suggestsWhen the that initial the brain associated with AD may begin 20 or more years before symptoms appear [7,8]. When the initial changeschanges occur, associatedthe brain withcompensates AD may beginfor them, 20 or enabling more years individuals before symptoms to continue appear to [7function,8]. When the changes occur, the brain compensates for them, enabling individuals to continue to function normally.initial As changesneuronal occur, damage the brainincreases, compensates the brain forcan them, no longer enabling compensate individuals for tothe continue changes toand function normally. As neuronal damage increases, the brain can no longer compensate for the changes and individualsnormally. show As subtle neuronal cognitive damage decline. increases, Later, theneuronal brain candamage no longer is so significant compensate that for individuals the changes and individualsindividuals showshow subtlesubtle cognitivecognitive decline.decline. Later,Later, neneuronal damage is so significant that individuals show obviousindividuals cognitive show decline, subtle cognitive including decline. symptoms Later, such neuronal as memory damage loss isor so confusion significant as thatto time individuals or show obvious cognitive decline, including symptoms such as memory loss or confusion as to time or place [5].show obvious cognitive decline, including symptoms such as memory loss or confusion as to time or place [5].place [5]. 1.3. Current Pharmacology and Drug Development 1.3. Current1.3. Current Pharmacology Pharmacology and Drug and Development Drug Development Currently, five drugs are approved for the treatment of AD (Table 1). These include four Currently,Currently, five drugs five drugsare approved are approved for the for treatment the treatment of AD of (Table AD (Table 1). These1). These include include four four acetylcholinesterase inhibitors (AChEi) - tacrine (approved in 1993), donepezil (approved in 1996), acetylcholinesteraseacetylcholinesterase inhibitors inhibitors (AChEi) (AChEi) - tacrine - tacrine(approved (approved in 1993), in donepezil 1993), donepezil (approved (approved in 1996), in 1996), rivastigmine (1998), and galantamine (approved in 2001) [9]. This class of drugs act by blocking the rivastigminerivastigmine (1998), (1998),and galantamin and galantaminee (approved (approved in 2001) in[9]. 2001) This [cl9].ass This of drugs class act of drugsby blocking act by the blocking process that downregulate the neurotransmitter acetylcholine (ACh), a key signaling agent for nerve processthe that process downregulate that downregulate the neurotransmitter the neurotransmitter acetylcholine acetylcholine (ACh), a key (ACh), signaling a key agent signaling for nerve agent for cells communication. Although tacrine has been the first AChEi approved by Food and Drug cells communication.nerve cells communication. Although tacrine Although has tacrinebeen the has first been AChEi the first approved AChEi approved by Food by and Food Drug and Drug Administration (FDA), it is currently not used due to its hepatotoxicity [10]. Donepezil, rivastigmine, AdministrationAdministration (FDA), (FDA), it is currently it is currently not used not due used to dueits hepatotoxicity to its hepatotoxicity [10]. Donepezil, [10]. Donepezil, rivastigmine, rivastigmine, and galantamine belong to the same therapeutic class, but present different pharmacodynamic (PD) and galantamineand galantamine belong belongto the same to the therapeutic same therapeutic class, but class, present but present different different pharmacodynamic pharmacodynamic (PD) (PD) and pharmacokinetic (PK) profiles: donepezil is a noncompetitive reversible AChEi; galantamine is and pharmacokineticand pharmacokinetic (PK) profiles: (PK) profiles: donepezil donepezil is a noncompetitive is a noncompetitive reversible reversible AChEi; AChEi; galantamine galantamine is is a selective reversible AChEi and a positive allosteric modulator of nicotinic ACh receptors; and a selectivea selective reversible reversible AChEi AChEi and a andpositive a positive allosteric allosteric modulator modulator of nicotinic of nicotinic ACh receptors; ACh receptors; and and rivastigmine acts as an inhibitor of both acetylcholinesterase (AChE) and butyrylcholinesterase rivastigminerivastigmine acts as acts an asinhibitor an inhibitor of both of acetylcholinesterase both acetylcholinesterase (AChE) (AChE) and butyrylcholinesterase and butyrylcholinesterase (BuChE) [11]. (BuChE)(BuChE) [11]. [11].

Table 1.. ApprovedApproved drugsdrugs forfor thethe treatmenttreatment ofof Alzheimer’sAlzheimer’s disease.disease. Table 1Table. Approved 1. Approved drugs for drugs the fortreatment the treatment of Alzheimer’s of Alzheimer’s disease. disease. Name Structure Name NameStructure Structure

Tacrine Tacrine Tacrine

Donepezil Donepezil Donepezil

Rivastigmine RivastigmineRivastigmine

Galantamine GalantamineGalantamine

Memantine MemantineMemantine

  Pharmaceuticals 20192018,, 1211,, 41 3 of 31

An alternative therapy to AChEi is the administration of memantine, a non-competitive antagonistAn alternative of N-methyl-D-aspartate therapy to AChEi is(NMDA) the administration receptor, approved of memantine, in 2004. a non-competitive This drug regulates antagonist the ofactivityN-methyl-D-aspartate of glutamate in the (NMDA) brain. Attachment receptor, approvedof the neurotransmitter in 2004. This glutamate drug regulates to the the cell activity surface of glutamateNMDA receptors in the brain.allows Attachment calcium to enter of the the neurotransmitter cell. This is an important glutamate event to the in cell cell surface signaling, of NMDA as well receptorsas in learning allows and calcium memory. to However, enter the cell.in AD This patients, is an important excess glutamate event in released cell signaling, from damaged as well ascells in learningleads to andoverexposure memory. However, to calcium in and AD patients,accelerates excess cell glutamatedamage. Memantine released from disrupts damaged this cells chain leads of toevents overexposure by blocking to calciumthe NMDA and receptors accelerates [11], cell [12]. damage. In Table Memantine 1 are comprised disrupts the this current chain of drugs events used by blockingin the therapy the NMDA of AD. receptors [11,12]. In Table1 are comprised the current drugs used in the therapy of AD. 1.4. Disease-Modifying Therapies and Drug Development 1.4. Disease-Modifying Therapies and Drug Development The drugs currently available for the treatment of AD only relieve symptoms, not being able to The drugs currently available for the treatment of AD only relieve symptoms, not being able impact in the progression of the disease. There is an urgent need for the development of disease to impact in the progression of the disease. There is an urgent need for the development of disease modifying therapies or treatments (DMT’s) able to prevent, delay, or slow the progression and target modifying therapies or treatments (DMT’s) able to prevent, delay, or slow the progression and target the primary pathophysiology mechanisms of AD. It has been estimated that the overall frequency of the primary pathophysiology mechanisms of AD. It has been estimated that the overall frequency of the disease would be decreased by 40% if the onset of the disease could be delayed by 5 years [13]. the disease would be decreased by 40% if the onset of the disease could be delayed by 5 years [13]. Unfortunately, drug development for AD has proven to be very difficult. The high cost of drug Unfortunately, drug development for AD has proven to be very difficult. The high cost of drug development, the relatively long time needed to observe whether an investigational treatment affects development, the relatively long time needed to observe whether an investigational treatment affects disease progression and the needed capacity of the drug to cross the blood-brain barrier (BBB) are disease progression and the needed capacity of the drug to cross the blood-brain barrier (BBB) are some some factors that contribute to the absence of new approved drugs for AD. Even considering the factors that contribute to the absence of new approved drugs for AD. Even considering the compounds compounds that reach clinical trials, many of them fail to prove efficacy or present unacceptable that reach clinical trials, many of them fail to prove efficacy or present unacceptable toxicity, both small toxicity, both small molecules and immunotherapies [9]. Considering the decade between 2002 and molecules and immunotherapies [9]. Considering the decade between 2002 and 2012, 244 compounds 2012, 244 compounds were assessed in 413 trials for AD. Of the agents which advanced to phase III, were assessed in 413 trials for AD. Of the agents which advanced to phase III, only one was advanced only one was advanced to FDA and approved for marketing (memantine). This represents an overall to FDA and approved for marketing (memantine). This represents an overall success rate for approval success rate for approval of 0.4%, which is among the lowest for any therapeutic area [9]. of 0.4%, which is among the lowest for any therapeutic area [9]. However, besides the mentioned difficulties in the development of new therapies for AD, there However, besides the mentioned difficulties in the development of new therapies for AD, there are currently large research programs for the drug development of new anti-AD agents. In 2018, there are currently large research programs for the drug development of new anti-AD agents. In 2018, there were 112 agents in the AD pipeline, of which 23 in phase I, 63 in phase II and 26 in phase III. Across were 112 agents in the AD pipeline, of which 23 in phase I, 63 in phase II and 26 in phase III. Across all all stages, 63% are DMTs, 34% symptomatic agents for neuropsychiatric and behavioral changes, and stages, 63% are DMTs, 34% symptomatic agents for neuropsychiatric and behavioral changes, and 3% 3% have undisclosed mechanism of action (MoA) [14] have undisclosed mechanism of action (MoA) [14] Figure 1 summarizes the MoA of the agents in clinical trials in 2018. Twenty five percent of the Figure1 summarizes the MoA of the agents in clinical trials in 2018. Twenty five percent of the agents have an amyloid-related MoA (12% immunotherapy and 14% small molecules), 14% anti-tau, agents have an amyloid-related MoA (12% immunotherapy and 14% small molecules), 14% anti-tau, 22% neurotransmitter-based (symptomatic treatment agents), 18% neuroprotection/ anti- 22% neurotransmitter-based (symptomatic treatment agents), 18% neuroprotection/ anti-inflammatory, inflammatory, 18% metabolic and 3% with other mechanisms [14] 18% metabolic and 3% with other mechanisms [14].

Others 3%

Anti-amyloid (small Neurotransmitter molecules & based immunotherapy) 22% 25%

Neuroprotection / anti-inflammatory 18% Anti-tau Metabolic 14% 18%

Figure 1. Mechanism of action of AD agents in clinical trials in 2018. Adapted from [14]. Figure 1. Mechanism of action of AD agents in clinical trials in 2018. Adapted from [14]. As depicted in Figure1,A β appears as the main therapeutic target for AD, with several pharma/biopharmaAs depicted in industries Figure 1, trying AΆ appears to develop as agentsthe main (as immunotherapiestherapeutic target or for small AD, molecules) with several able topharma/biopharma decrease Aβ accumulation/aggregation industries trying to develop and to agents show (as a positive immunotherapies effect on AD or pathology.small molecules) able to decrease AΆ accumulation/aggregation and to show a positive effect on AD pathology.

 Pharmaceuticals 2018, 11, 4 of 31

Pharmaceuticals2. The Amyloid2019, 12 Hypothesis, 41 of AD 4 of 31 Pharmaceuticals 2018, 11, 4 of 31 The amyloid hypothesis, the current prevalent theory of AD pathogenesis, suggests that the 2.accumulation The Amyloid of Hypothesis pathological of ADforms of AΆ, due to an increase of production and/or decreased clearance, is the primary pathological process in AD [3,15]. The accumulation of AΆ peptides leads The amyloid hypothesis, the current prevalent theory of AD pathogenesis, suggests that the to theirThe oligomerizationamyloid hypothesis, and formation the current of AprevalentΆ plaques. theory These of plaques AD pathogenesis, generate an anti-inflammatorysuggests that the accumulation of pathological forms of Aβ, due to an increase of production and/or decreased clearance, accumulationresponse causing of pathological oxidative stress forms in neurons of AΆ, anddue disrupting to an increase normal of kinase production and phosphatase and/or decreased activity, is the primary pathological process in AD [3,15]. The accumulation of Aβ peptides leads to their clearance,resulting inis thehyperphosphorylation primary pathological of processtau protein in AD and [3,15]. subsequent The accumulation neurofibrillary of A tangleΆ peptides formation. leads oligomerization and formation of Aβ plaques. These plaques generate an anti-inflammatory response toThis their cascade oligomerization of events leads and formation to abnormal of AsignalingΆ plaques. and These synaptic plaques impairment, generate resultingan anti-inflammatory ultimately in causing oxidative stress in neurons and disrupting normal kinase and phosphatase activity, resulting responseneuronal causing dead and oxidative dementia stress in the in neuronsAD patient and [16]. disrupting This is normalschematized kinase in and Figure phosphatase 2. activity, inresulting hyperphosphorylation in hyperphosphorylation of tau protein of tau and protein subsequent and neurofibrillarysubsequent neurofibrillary tangle formation. tangle This formation. cascade ofThis events cascade leads of toevents abnormal leads signaling to abnormal and signaling synaptic impairment, and synaptic resulting impairment, ultimately resulting in neuronal ultimately dead in andneuronal dementia dead in and the dementia AD patient in the [16]. AD This patient is schematized [16]. This inis schematized Figure2. in Figure 2.

Figure 2. Cascade of events according the amyloid hypothesis. Adapted from [16].

AΆ is produced by a two-step sequential cleavage of amyloid precursor protein (APP): first beta- site APP-cleavingFigure enzyme 2. Cascade (BACE-1) of events cleaves according APP the to amyloid generate hypothesis. soluble AdaptedAPPΆ (sAPP from Ά [[16].16) and]. a 99 amino acid fragment (C99), which then suffers several cleavage events by ·-secretase to produce peptides Aβ is produced by a two-step sequential cleavage of amyloid precursor protein (APP): first of differentAΆ is produced lengths byfrom a two-step 38 to 43 sequential amino acids, cleavage being of A amyloidΆ40 and precursorAΆ42 the proteinmain products (APP): first and beta- both beta-site APP-cleaving enzyme (BACE-1) cleaves APP to generate soluble APPβ (sAPPβ) and a siteplaying APP-cleaving a key role enzyme in the aggregation (BACE-1) cleaves of neuritic APP plaques to generate [15], [17]soluble (Figure APP 3).Ά (sAPPThe longerΆ) and peptide a 99 amino AΆ42 99 amino acid fragment (C99), which then suffers several cleavage events by γ-secretase to produce acidhas beenfragment shown (C99), to be which the most then prone suffers to severalaggregation, cleavage with events an increased by ·-secretase AΆ42:A toΆ40 produce ratio observed peptides in peptides of different lengths from 38 to 43 amino acids, being Aβ40 and Aβ42 the main products ofthe different familial lengthsform of fromthe disease 38 to 43 [17,18]. amino acids, being AΆ40 and AΆ42 the main products and both and both playing a key role in the aggregation of neuritic plaques [15,17] (Figure3). The longer playingThe aactivity key role of in Ά -cleavagethe aggregation is considered of neuritic to determine plaques [15], the [17] total (Figure amount 3). of The AΆ longer production, peptide and AΆ the42 peptide Aβ42 has been shown to be the most prone to aggregation, with an increased Aβ42:Aβ40 ratio hasefficiency been shown of the to successive be the most ·-cleavage prone to impacts aggregation, the production with an increased ratio of toxicAΆ42:A AΆΆ4240 to ratio total observed AΆ [15]. in observedthe familial in form the familial of the disease form of [17,18]. the disease [17,18]. The activity of Ά-cleavage is considered to determine the total amount of AΆ production, and the efficiency of the successive ·-cleavage impacts the production ratio of toxic AΆ42 to total AΆ [15].

Figure 3. Scheme of the production of Aβ by the two step sequential cleavage of APP by β-secretase

and γ-secretase. Adapted from [15]. Figure 3. Scheme of the production of AΆ by the two step sequential cleavage of APP by Ά-secretase Theand activity·-secretase. of β Adapted-cleavage from is considered [15]. to determine the total amount of Aβ production, and the efficiency of the successive γ-cleavage impacts the production ratio of toxic Aβ42 to total Aβ [15]. TheFigureThe amyloidamyloid 3. Scheme hypothesishypothesis of the production isis stronglystrongly of AΆ supported bysupported the two step by by geneticsequential genetic findings. findings.cleavage of AllA APPll thethe by known knownΆ-secretase familialfamilial Alzheimer’sAlzheimer’sand ·-secretase. disease disease Adapted (fAD) (fAD) mutations mutations from [15]. are are involved involved in in either either A AβΆincrease increase generation generation or or processing processing and and  The amyloid hypothesis is strongly supported by genetic findings. All the known familial Alzheimer’s disease (fAD) mutations are involved in either AΆ increase generation or processing and  Pharmaceuticals 2019, 12, 41 5 of 31 result in relative overproduction of toxic forms of Aβ, namely Aβ42 [3]. On the other hand, it was reported that a mutation in the APP gene (A673T), identified from a set of whole-genome sequence data of 1795 Iceland people, results in a lifelong decrease in APP cleavage by β-secretase conferring a reduced clinical risk of AD and age-related decline [19,20]. Besides the fact that occurrence of AD and genetic mutations associated with Aβ formation supports the amyloid hypothesis, some observations remains without explanation, with histological studies being controversial in the correlation between the formation of Aβ plaques and cognitive decline [21]. Karran et al. [22] defend the prevention of Aβ deposition in patients that are in risk of developing AD as a robust test of the amyloid hypothesis. If individuals are at risk of developing AD but Aβ deposition has not yet occurred, then a significant reduction of Aβ42 production or a decrease in the longer amyloid-β/shorter amyloid-β ratio would be expected to prevent the onset of disease within a normal lifetime [22]. Although there are further questions concerning the exact neuronal toxicity of Aβ, the amyloid hypothesis is still broadly accepted as the general pathological cascade of events in AD [22,23].

3. Amyloid Targeting Strategies Following the amyloid hypothesis, where a high brain Aβ level is observed as an important factor in AD pathogenesis, pharmacological intervention to reduce its production or improve the clearance has become a logical approach for AD therapy development. Considering the 112 agents in the AD pipeline in 2018 [14], there are 29 with a mechanism of action amyloid related, 13 corresponding to immunotherapeutic agents (Table2) and 16 to small molecules (Table3) involved in phase I, II and III clinical trials.

Table 2. Immunotherapeutic agents (amyloid related) in the AD pipeline (2018).

Phase Agent Mechanism of Action Sponsor (Clinical Trial(s)) I, III Aducanumab Monoclonal antibody Biogen Albumin + III Polyclonal antibody Grifols immunoglobulin I, II, III Crenezumab Monoclonal antibody Roche/Genentech II, III Gantenerumab Monoclonal antibody Roche Washington University, Eli Lilly, Roche, NIA, Alzheimer’s III Solanezumab Monoclonal antibody Association, ATRI (Alzheimer’s Therapeutic Research Institute) II, III CAD106 Amyloid vaccine Novartis, Amgen, NIA, University of Colorado, Denver, II Sargramostim (GM-CSF) Immunostimulator The Dana Foundation II BAN2401 Monoclonal antibody Eisai II UB-311 Monoclonal antibody United Neuroscience I, II LY3002813 Monoclonal antibody Eli Lilly and Company I LY3303560 Monoclonal antibody Eli Lilly and Company I Lu AF20513 Polyclonal antibody H. Lundbeck A/S I KHK6640 Monoclonal antibody Kyowa Hakko Kirin Pharma Pharmaceuticals 2019, 12, 41 6 of 31

Table 3. Small molecules (amyloid related) in AD pipeline (2018).

Phase Agent Mechanism of Action Sponsor Clinical Trial(s) NeuroGenetic I NGP 555 GSM Pharmaceuticals Phosphatidylinositol II ID1201 3-kinase/Akt pathway II Dong Pharmaceutical Co activation II Nilotinib Tyrosine kinase inhibitor Georgetown University III CNP520 (γ-secretase modulator) Alzheimer’s Association ALZT-OP1a (cromolyn)+ III BACE1 inhibitor AZTherapies ALZT-OP1b (ibuprofen) Sodium Oligo-mannurarate III Increases amyloid clearance Shanghai Green Valley (GV-971) RAGE1) (Receptor for advanced III TTP488 (Azeliragon) glycation end products) vTv Therapeutics antagonist II, III JNJ-54861911 BACE1 inhibitor Janssen II, III E2609 (Elenbecestat) BACE1 inhibitor Eisai, Biogen II LY3202626 BACE1 inhibitor Eli Lilly Adrenergic uptake inhibitor, II Atomoxetine Emory University, NIA SNRI II AZD0530 (Saracatinib) Kinase inhibitor Yale University, ATRI, Sigma-2 receptor II CT1812 Cognition Therapeutics competitive inhibitor2) Selective inhibitor of APP II Posiphen QR Pharma, ADCS production II Valacyclovir Antiviral agent 4) Umea University III AZD3293 (LY3314814) BACE1 inhibitor AstraZeneca, Eli Lilly

With regard to small molecules, different targets have been identified for activity modulation hoping to get a marked impact in the amyloid cascade and in disease progression. Among 16 anti-amyloid agents identified in the AD pipeline, six have their mechanism of action centered in the activity of β or γ-secretase proteases, covering almost 40% of the identified agents. In fact, in the last years several pharmaceutical industries and other research centers have led research programs to discovered potent compounds for the modulation or inhibition of these two targets [16,17,24–26]. The discovery and optimization activities which lead to the development of these compounds will be herein detailed.

3.1. Gamma Secretase Inhibitors and Modulators Gamma secretase (GS) (Figure4) is an aspartyl protease composed by a complex of four different membrane proteins: presenilin (PS), presenilin enhancer 2 (Pen- 2), nicastrin (Nct), and anterior pharynx-defective 1 (Aph-1) [27]. PS is the catalytic component of γ-secretase. In humans, PS is encoded by the PSEN1 (PS-1) gene on chromosome 14 or the PSEN2 (PS-2) gene on chromosome 1, and mutations in both genes have been found to cause fAD [28]. The products of these genes (PS-1 and PS-2) are nine transmembrane domain (TMD) proteins that form the catalytic subunit of GS [17]. GS cleaves several type-I transmembrane proteins (over 90 reported substrates), being APP and Notch the best characterized substrates [29]. The activity of GS on the substrate APP occurs after the cleavage performed by β-secretase (BACE-1). Then, GS perform a series of cleavages within the transmembranar domain of the remaining fragment (C99), termed epsilon (ε), zeta (ζ), and gamma (γ) cleavages, allowing the generation of Aβ peptides of different lengths (Figure5). Pharmaceuticals 2018, 11, 7 of 31 and mutations in both genes have been found to cause fAD [28]. The products of these genes (PS-1 and PS-2) are nine transmembrane domain (TMD) proteins that form the catalytic subunit of GS [17]. GS cleaves several type-I transmembrane proteins (over 90 reported substrates), being APP and Notch the best characterized substrates [29]. Pharmaceuticals 2018, 11, 7 of 31

and mutations in both genes have been found to cause fAD [28]. The products of these genes (PS-1 and PS-2) are nine transmembrane domain (TMD) proteins that form the catalytic subunit of GS [17].

GSPharmaceuticals cleaves several2019, 12 ,type-I 41 transmembrane proteins (over 90 reported substrates), being APP 7and of 31 Notch the best characterized substrates [29].

Figure 4. Crystal structure of human ·-secretase. Adapted by permission from Xiao-chen Bai et al. Nature [30], Copyright 2015.

The activity of GS on the substrate APP occurs after the cleavage performed by Ά-secretase (BACE-1). Then, GS perform a series of cleavages within the transmembranar domain of the remaining fragment (C99), termed epsilon (Ή), zeta (Ί), and gamma (·) cleavages, allowing the Figure 4. Crystal structure of human ·γ-secretase. Adapted by permission from Xiao-chen Bai et al. generationFigure of 4. ACrystalΆ peptides structure of different of human lengths-secretase. (Figure Adapted 5). by permission from Xiao-chen Bai et al. Nature [30], Copyright 2015. Nature [30], Copyright 2015.

The activity of GS on the substrate APP occurs after the cleavage performed by Ά-secretase (BACE-1). Then, GS perform a series of cleavages within the transmembranar domain of the remaining fragment (C99), termed epsilon (Ή), zeta (Ί), and gamma (·) cleavages, allowing the generation of AΆ peptides of different lengths (Figure 5).

FigureFigure 5. Cleavage 5. Cleavage steps steps of C99of C99 by GS.by GS. Adapted Adapted with with permission permission from from [17]. [17]. Copyright Copyright 2016 2016 American Chemical Society. American Chemical Society The ε cleavage (1) releases the APP intracellular domain (AICD) and produces Aβ49 or The Ή cleavage (1) releases the APP intracellular domain (AICD) and produces AΆ49 or AΆ48 Aβ48 [28,31]. Then, the carboxypeptidase cleavages ζ (2) and γ (3 and 4) progressively trims these [28,31]. Then, the carboxypeptidase cleavages Ί (2) and · (3 and 4) progressively trims these longer longer Aβ forms in both Aβ40 and Aβ42 [31,32]. The successive cleavage events performed by GS AΆ forms in both AΆ40 and AΆ42 [31], [32]. The successive cleavage events performed by GS consists consistsFigure in four 5. cycles Cleavage to generatesteps of C99 Aβ by40 GS. (49-46-43-40) Adapted with and permission Aβ38 (48-45-42-38). from [17]. Copyright Further 2016 cleavage will in four cycles to generate AΆ40 (49-46-43-40) and AΆ38 (48-45-42-38). Further cleavage will subsequently generate the shorter isoformsAmerican Aβ Chemical39and A Societyβ37 [33 ]. subsequently generate the shorter isoforms AΆ39and AΆ37 [33]. Many fAD-causing mutations in PS have been found to decrease the catalytic activity of GS [34,35] Many fAD-causing mutations in PS have been found to decrease the catalytic activity of GS [34], withThe the Ή most cleavage pronounced (1) releases effect the on APP the fourth intracellular cleavage domain cycle [ 36(AICD),37]. This and loss produces of function AΆ49 contributes or AΆ48 [35] with the most pronounced effect on the fourth cleavage cycle [36,37]. This loss of function [28,31].to the increasedThen, the Acarboxypeptidaseβ42:Aβ40 ratio observed cleavages in Ί the (2) familialand · (3 form and of4) theprogressively disease [38 trims]. Aβ 42these is consider longer contributes to the increased AΆ42:AΆ40 ratio observed in the familial form of the disease [38]. AΆ42 AtheΆ forms most in toxic both A βAΆisoform40 and A dueΆ42 to [31], its high [32]. propensityThe successive to form cleavage fibrillary events and performed non-fibrillary by GS aggregates. consists inOn four the othercycles hand, to generate shorter A AβΆpeptides40 (49-46-43-40) are speculated and A toΆ be38 less(48-45-42-38). toxic or even Further neuroprotective cleavage [33will]. subsequently generate the shorter isoforms AΆ39and AΆ37 [33]. 3.1.1.Manyγ-Secretase fAD-causing Inhibitors mutations in PS have been found to decrease the catalytic activity of GS [34], [35] with the most pronounced effect on the fourth cleavage cycle [36,37]. This loss of function Based on the genetic evidence of the role of GS and Aβ formation on AD, the pharmaceutical contributes to the increased AΆ42:AΆ40 ratio observed in the familial form of the disease [38]. AΆ42 industry made efforts in developing compounds able to inhibit this protease and, consequently, reduce  the amount of Aβ peptide formation. Two key γ-secretase inhibitors (GSIs), semagacestat (1) from Eli Lilly & Co and later avagacestat (2) from Bristol-Myers Squibb (Figure6) advanced in late-stage clinical trials for AD (phase III and phase II, respectively) [39,40]. Pharmaceuticals 2018, 11, 8 of 31 is consider the most toxic AΆ isoform due to its high propensity to form fibrillary and non-fibrillary aggregates. On the other hand, shorter AΆ peptides are speculated to be less toxic or even neuroprotective [33].

3.1.1. ·-Secretase Inhibitors Based on the genetic evidence of the role of GS and AΆ formation on AD, the pharmaceutical industry made efforts in developing compounds able to inhibit this protease and, consequently, reduce the amount of AΆ peptide formation. Two key ·-secretase inhibitors (GSIs), semagacestat (1) fromPharmaceuticals Eli Lilly2019 & ,Co12, 41and later avagacestat (2) from Bristol-Myers Squibb (Figure 6) advanced in8 late- of 31 stage clinical trials for AD (phase III and phase II, respectively) [39,40].

&O

2 6 2 2 1 1+ )  ) ) ) 1 1 2 

 

Figure 6. ·γ-Secretase-Secretase inhibitors inhibitors ( 1–2) advanced in late-stage clinical trials.

Unfortunately, due to GS high promiscuity in terms of substrates, several side effects were observed throughout the clinical trials with these compounds [25]. [25]. The main responsible pointed for these adverse effects was Notch, a single-pass single-pass type I transmembrane transmembrane receptor [[41].41]. Notch Notch is is processed processed in a similar similar way way as as C99, C99, where where the the Notch Notch intrace intracellularllular domain domain (NICD) (NICD) is is released released after the Ήε-cleavage-cleavage and translocate to the nucleus to regulate gene expressionexpression and to mediate important intercellular communication functions as cellular differentiation. Additionally, Additionally, Notch Notch pathway pathway is is involved in neurogenesis, neuritic neuritic growth growth and and long-term long-term memory memory [41]. [41]. The The inhibitory inhibitory effect effect of ofGSIs GSIs blocks blocks the the Ή- cleavageε-cleavage of of ·-secretaseγ-secretase affecting affecting the the Notch Notch signaling, signaling, leading leading to to several several adverse adverse events events as as skin skin cancer, cancer, decreased lymphocyte count and/or memory memory loss. loss. These These adverse adverse effects effects were were observed observed in in phase III clinical trial of semagacestat (1)[) [40].40]. Consequently, subsequent drug-development programs programs have have aimed aimed to achieve a greater separation between between APP APP and and Notch Notch inhibition. inhibition. Bristol-Myers Bristol-Myers Squibb Squibb developed developed avagacestat avagacestat (2) and (2) reportedand reported it as having it as having a 137-fold a 137-fold selectivity selectivity for APP for over APP Notch over Notchin cell culture, in cell culture, and to robustly and to robustly reduce cerebrospinalreduce cerebrospinal fluid (CSF) fluid (CSF)AΆ levels Aβ levels without without causing causing Notch-related Notch-related toxicity toxicity in inrats rats and and dogs dogs [42]. [42]. However, aa phasephase II II clinical clinical trial trial performed performed with with avagacestat avagacestat demonstrated demonstrated a side-effect a side-effect profile similarprofile similarto the one to the found one withfound semagacestat with semagacestat (1)[39]. (1 These) [39]. resultsThese results were attributed were attributed to an overestimation to an overestimation of the ofselectivity the selectivity of avagacestat of avagacestat between between Notch Notch and APP, and withAPP, other with researchersother researchers indicating indicating an only an 3-fold only 3-foldselectivity selectivity for cleavage for cleavage of an APP of an substrate APP substrate compared compared with a with Notch a Notch substrate substrate [43]. [43]. The disappointingdisappointing results results obtained obtained with with these twothese GSI two led toGSI discontinuation led to discontinuation of the development of the developmentof GSIs as therapeutic of GSIs strategy as therapeutic against AD. strategy Additionally, against other AD. options Additionally, as GS modulators other options arose asas safer GS modulatorsdisease modifying arose as therapies safer disease for AD. modifying therapies for AD.

3.1.2. Gamma secretase Modulators A γ·-secretase modulatormodulator (GSM)(GSM) isis defined defined as as a a compound compound that that changes changes the the relative relative proportion proportion of ofthe the Aβ AisoformsΆ isoforms generated generated while while maintaining maintaining the the rate rate at whichat which APP APP is processedis processed [33 [33].].

Nonsteroidal Anti-inflammatory Drug Derived GSM’s The first generation of GSMs was discovered from an epidemiological study documenting a reduced prevalence of AD among users of nonsteroidal anti-inflammatory drugs (NSAIDs) [44]. Then, in 2001 Weggen et al. [44] reported that NSAIDs (Figure7) were able to decrease the A β42 peptide accompanied by an increase in the Aβ38 isoform, indicating that NSAIDs modulate γ-secretase activity without significantly perturbing other APP processing pathways or Notch cleavage [44]. PharmaceuticalsPharmaceuticals 20182018,, 1111, , 99 ofof 3131

NonsteroidalNonsteroidal Anti-inflammatoryAnti-inflammatory DrugDrug DerivedDerived GSM’sGSM’s TheThe firstfirst generationgeneration ofof GSMsGSMs waswas discovereddiscovered fromfrom anan epidemiologicalepidemiological studystudy documentingdocumenting aa reducedreduced prevalenceprevalence ofof ADAD amongamong usersusers ofof nonstenonsteroidalroidal anti-inflammatoanti-inflammatoryry drugsdrugs (NSAIDs)(NSAIDs) [44].[44]. Then,Then, inin 20012001 WeggenWeggen etet alal.. [44][44] reportedreported thatthat NSAIDsNSAIDs (Figure(Figure 7)7) werewere ableable toto decreasedecrease thethe AAΆΆ4242 peptidepeptide accompaniedaccompanied byby anan increaseincrease inin thethe AAΆΆ3838 isoform,isoform, indicatingindicating thatthat NSAIDsNSAIDs modulatemodulate ··-- Pharmaceuticals 2019, 12, 41 9 of 31 secretasesecretase activityactivity withoutwithout significantlysignificantly perturbingperturbing otherother APPAPP processingprocessing pathwayspathways oror NotchNotch cleavagecleavage [44].[44].

33 44 55 IbuprofenIbuprofen IndomethacinIndomethacin SulindacSulindac sulfidesulfide AAΆΆ4242 IC IC5050 = = 200-300200-300 μμMM AAΆΆ4242 IC IC5050 = = 25-5025-50 μμMM AAΆΆ4242 IC IC5050 = = 3434 μμMM

FigureFigure 7.7. NSAIDsNSAIDsNSAIDs GSMsGSMs GSMs 33––553 and–and5 and respectiverespective respective AAΆΆ4242 A half-maximalβhalf-maximal42 half-maximal inhibitoryinhibitory inhibitory concentrationconcentration concentration (IC(IC5050)) valuesvalues (IC50) [44].values[44]. [44].

ThisThis change change in in the the cleavage cleavage pattern pattern could could be be explainedexexplainedplained byby (1)1)1) aa decreasedecrease inin the the probability probability of of releasingreleasing longer longer A AAΆβΆ fromfrom the the enzyme-substrate enzyme-substrate complex complex (defined (defined(defined as as dissociation dissociation constant, constant, ΎκΎdd))) ofof of GS,GS, GS, oror 2) (2)2) anan an increaseincrease increase inin in thethe the cleavagecleavage cleavage acac activitytivitytivity (defined(defined (defined byby by thethe the catalyticcatalytic catalytic constant,constant, constant, ΎΎcatκcatcat)) ofof) of GS.GS. GS. UsingUsing Using C99C99 C99 asas as aa substrate,asubstrate, substrate, Chavez-GutierrezChavez-Gutierrez Chavez-Gutierrez etet et alal al... [36][36] [36 ] showedshowed showed that that thethe AAAβΆΆ40/A40/AΆΆβ434343 and and AAβΆΆ38/A38/AΆΆβ424242 ratiosratios were were decreaseddecreased byby allall thethethe PS PSPS mutations mutationsmutations studied, studied,studied, suggesting suggestingsuggesting an anan deterioration deteriorationdeterioration of of theof thethe four fourfour cleavage cleavagecleavage cycle cyclecycle of GS ofof GS(GSγ’ cleavage).((··’’ cleavage).cleavage). Additionally, Additionally,Additionally, using usingusing Aβ42 AA asΆΆ42 a42 substrate, asas aa substrate,substrate, Okochi OkochiOkochi et al. [35 etet] measuredal.al. [35][35] measuredmeasured the kinetic thethe constants kinetickinetic constantsforconstants the γ cleavage forfor thethe and·· cleavagecleavage reported andand that reportedreported GSMs decreased thatthat GSMsGSMsκd and decreaseddecreased increased ΎΎdκd catandand, while increasedincreased PS mutations ΎΎcatcat,, whilewhile caused PSPS mutationsthemutations opposite causedcaused effect. thethe In oppositeopposite sum, these effect.effect. results InIn sum,sum, suggest thesthesee resultsresults that the suggestsuggest modulation that that thethe effect modulationmodulation of GSMs effect effect could of of GSMsGSMs be an couldeffectivecould bebe an approachan effectiveeffective to approachapproach reverse the toto effect reversereverse of PSthethe mutations effecteffect ofof PSPS by mutationsmutations restoring by theby restoringrestoring normal ratios thethe normalnormal of Aβ peptidesratiosratios ofof Aformed,AΆΆ peptidespeptides and formed, informed, this way andand modifying inin thisthis wayway the modifyinmodifyin diseasegg pathology thethe diseasedisease and pathologypathology progression andand [ progression17progression]. [17].[17]. Though,Though, andand asas expected, expected, thethe useuse ofof NSAIDNSAID asas GSMs GSMs present present some some issues issues in in terms terms of of gastrointestinalgastrointestinal and and renal renal toxicity toxicity due due to to its its acti acti activityvityvity against against cyclooxygenase cyclooxygenase 1 1 (COX-1), (COX-1), compromising compromising itsits use use asas aa a long-termlong-term long-term therapeutictherapeutic therapeutic solutionsolution solution [45].[45]. [45 ]. FoFo Fortunately,rtunately,rtunately, COX-1COX-1 COX-1 inhibitioninhibition inhibition activityactivity activity waswas was shownshown shown toto betobe beindependentindependent independent ofof of··-secretase-secretaseγ-secretase modulationmodulation modulation activity.activity. activity. ForFor For inin instance,stance,stance, flurbiprofenflurbiprofen flurbiprofen isis is aa a COX-1COX-1 inhibitor inhibitor administeredadministered ininin the thethe clinic clinicclinic as asas a racemate. aa racemate.racemate. However, However,However, the R thethe-enantiomer, RR-enantiomer,-enantiomer, tarenflurbil tarenflurbiltarenflurbil (6) (Figure ((66)) 8(Figure(Figure), showed 8),8), showedreducedshowed reducedreduced activity inactivityactivity terms inin of termsterms COX-1 ofof inhibition COX-1COX-1 inhibitiinhibiti whileonon maintaining whilewhile maintainingmaintaining its action itsits as actionaction a GSM asas [ 45 aa GSM].GSM [45].[45].

66

FigureFigure 8.8. TarenflurbilTarenflurbilTarenflurbil (R-flurbiprofen) (R-flurbiprofen)(R-flurbiprofen) ((66).).).

TarenflurbilTarenflurbilTarenflurbil ( (66,,, Flurizan™, FlurizanFlurizan™,™, Myriad MyriadMyriad Genetics GeneticsGenetics & & Laboratories) Laboratories) reachedreached phasephase IIIIII III clinicalclinical trials.trials. trials. Unfortunately,Unfortunately, results results showed showed no no difference difference between between 66 andand the the placebo, placebo, the the failure failure being being attributed attributed toto thethe the insufficientinsufficient insufficient PDPD PD propertiesproperties properties ofof ofFlurizan™,Flurizan™, Flurizan ™ namelynamely, namely itsits inadequateinadequate its inadequate capacitycapacity capacity toto penetratepenetrate to penetrate thethe brainbrain the andbrainand engageengage and engage itsits targettarget its target atat dosesdoses at doses sufficientsufficient sufficient toto yiyi toeldeld yield anan effect.effect. an effect. Actually,Actually, Actually, poorpoor poor CNSCNS CNS penetrationpenetration penetration ofof of tarenflurbil (6) was previously reported in preclinical studies in rodents, with a CSF/plasma ratio of 1.3%. The unsatisfactory results obtained led to the discontinuation of tarenflurbil (6) clinical development [46]. Later, two NSAID carboxylic acid derivatives developed by Chiesi (CHF5074, 7) and FORUM Pharmaceuticals (EVP-0015962, 8) (Figure9) were also tested in clinical trials. Pharmaceuticals 2018, 11, 10 of 31

tarenflurbil (6) was previously reported in preclinical studies in rodents, with a CSF/plasma ratio of 1.3%. The unsatisfactory results obtained led to the discontinuation of tarenflurbil (6) clinical development [46]. PharmaceuticalsLater, two2019 ,NSAID12, 41 carboxylic acid derivatives developed by Chiesi (CHF5074, 7) and FORUM10 of 31 Pharmaceuticals (EVP-0015962, 8) (Figure 9) were also tested in clinical trials.

7 7 8 8 CHF5074CHF5074 (Chiesi) (Chiesi) EVP-00150962EVP-00150962 (FORUM (FORUM Pharmaceuticals) Pharmaceuticals) AΆ42 IC50=40 μM AΆ42 IC50= 67 μM Aβ42 IC50=40 µM Aβ42 IC50= 67 µM

Figure 9.9. CHF5074 (7)) andand EVP-0015962EVP-0015962 ((88).).

Chiesi Farmaceutici Farmaceutici developed developed CHF5074 CHF5074 (7) ( 7based) based on onthe thescaffold scaffold of tarenflurbil of tarenflurbil (6). The (6). Thereplacement replacement of the of R the-methylR-methyl substituent substituent by a cyclopropyl by a cyclopropyl group groupled to a led complete to a complete removal removal of COX ofinhibition COX inhibition (at 100 μ (atM for 100 COX-1µM for and COX-1 300 μ andM for 300 COX-2).µM for A COX-2).Ά42 production Aβ42 production inhibition inhibitionwere improved were improvedwith the addition with the of addition chlorine ofsubstituents chlorine substituents on the terminal on the phenyl terminal ring, phenyl allowing ring, a allowing7-fold comparing a 7-fold comparingwith tarenflurbil with tarenflurbil (6) [47] (Figure (6)[47 10).] (Figure The carboxylic 10). The carboxylic acid function acid functionwas proposed was proposed to interact to interact with a withlysine a residue lysine residue of APP of located APP located close to close the membrane to the membrane interface, interface, with the with lipophilic the lipophilic substituents substituents serving servingas membrane as membrane anchors anchors[48]. [48].

Figure 10. Improvement activity and selectivity ofof CHF5074CHF5074 ((77)) againstagainst tarenflurbiltarenflurbil ((66).).

Despite the increaseincrease inin potencypotency andand selectivityselectivity accomplishedaccomplished with CHF5074 (7), nono sufficientsufficient improvementsimprovements were were achieved achieved in in terms terms of PK of PKparameters. parameters. CHF5074 CHF5074 (7) failed (7) to failed demonstrate to demonstrate efficacy efficacydue to its due poor to druglike its poor properties druglike properties and extremely and extremelypoor CNS penetration poor CNS penetration (brain to plasma (brain ratio to plasma = 0.03– ratio0.05) =[49]. 0.03–0.05) [49]. EVP-0015962 (8()8 was) was developed developed by FORUM by FORUM Pharmaceutical Pharmaceutical (ex Envivo) (ex through Envivo) the through introduction the ofintroduction additional substituentsof additional on substituent the biphenylacetics on the acid biphenylacetic core of R-flurbiprofen. acid core Thisof R compound-flurbiprofen. showed This improved in vitro potency (Aβ42 IC = 67 µM) with reproducible animal efficacy, with a reduction of compound showed improved in vitro50 potency (AΆ42 IC50 = 67 μM) with reproducible animal efficacy, 22%with anda reduction 38% with of oral 22% doses and of38% 10 with and 30oral mg/kg, doses respectivelyof 10 and 30 [33mg/kg,]. However, respectively as general [33]. characteristicHowever, as ofgeneral NSAID characteristic derived GSMs, of it presentsNSAID suboptimalderived GSMs, properties it presents as high lipophilicity,suboptimal whichproperties could resultas high in lowlipophilicity, free fraction which and could poor solubilityresult in low [17, 33free]. Additionally, fraction and thepoor presence solubility of the[17,3 carboxylic3]. Additionally, moiety canthe lead to the formation of reactive metabolites such as acyl glucuronides, compounds chemically reactive leading to covalent binding with macromolecules and toxicity [49,50]. EVP-0015962 (8) move on to a phase II clinical trial in 2012 but results were not publicly reported. The latest update information in the platform clinicaltrials.org was in January 2014. Additionally, EVP- 0015962 (8) is not part of

1

Pharmaceuticals 2018, 11, 11 of 31 Pharmaceuticals 2018, 11, 11 of 31 presencepresence ofof thethe carboxyliccarboxylic moietymoiety cancan leadlead toto thethe formationformation ofof reactivereactive metabolitesmetabolites suchsuch asas acylacyl glucuronides, compounds chemically reactive leading to covalent binding with macromolecules and Pharmaceuticalsglucuronides,2019 compounds, 12, 41 chemically reactive leading to covalent binding with macromolecules11 ofand 31 toxicitytoxicity [49],[49], [5[50].0]. EVP-0015962EVP-0015962 ((88)) movemove onon toto aa phasephase IIII clinicalclinical trialtrial inin 20122012 butbut resultsresults werewere notnot publiclypublicly reported. reported. The The latest latest update update information information in in the the platform platform clinicaltrials.org clinicaltrials.org was was in in January January 2014. 2014. Additionally,theAdditionally, AD pipeline EVP- EVP- in 0015962 2018,0015962 leading ( (88)) is is not tonot the part part assumption of of the the AD AD pipeline thatpipeline the clinicalin in 2018, 2018, development leading leading to to the the of assumption assumption this compound that that thewasthe clinical clinical discontinued. development development of of this this compound compound was was discontinued. discontinued.

Non-NSAIDNon-NSAID Derived Derived GSM’s GSM’s OneOne of of the the first first first GSM GSM series series not not presenting presenting a a ca ca carboxylicrboxylicrboxylic acid acid moiety moiety (non-NSAID) (non-NSAID) was was developed developed byby Neurogenetics Neurogenetics in in 2004, 2004, leading leading to toto the thethe discovery discoverydiscovery of ofof NGP555 NGP555NGP555 (Figure (Figure(Figure 11) 1111))[ [33]. [33].33].

1 + 1 + 11 11 11

66 ))

1111

FigureFigure 11. 11. Structure Structure of of NGP555 NGP555 ( (1111).).

ThisThis class class of of compounds compounds share share a a scaffold scaffold consisting consisting of of four four consecutives consecutives linked linked (hetero)aromatic (hetero)aromatic ringsrings identified identifiedidentified as as A, A, B, B, C C and and and D D D which which which focus focus focus on on on ar ar aryl-yl-yl- or or or heteroarylimidazoles heteroarylimidazoles heteroarylimidazoles with with with an an an anilinothiazole. anilinothiazole. anilinothiazole. FigureFigure 121212 represents representsrepresents the thethe basic basicbasic scaffold scaffoldscaffold and and theand structure-activity thethe structure-activitystructure-activity relationship relationshiprelationship established. established.established. The addition TheThe additionofaddition a methyl ofof a ora methylmethyl a halogen oror aa at halogenhalogen the 4-position atat thethe 4-posit4-posit of theionion imidazole ofof thethe imidazoleimidazole ring does ring notring have doesdoes a notnot significant havehave aa significantsignificant impact on impact on potency, while the addition of a CF3 substituent in ring B, ortho to the thiazole, leads to a potency,impact on while potency, the addition while the of addition a CF3 substituent of a CF3 substituent in ring B, ortho in ringto the B, ortho thiazole, to the leads thiazole, to a decrease leads to in a decreaseactivity.decrease Ain in pyridineactivity. activity. A orA pyridine pyridine pyrimidine or or pyrimidine pyrimidine ring at B ring ring ring increases at at B B ring ring potency, increases increases as po po welltency,tency, as theas as well well addition as as the the of addition addition an ortho ofmethylof an an ortho ortho substituent methyl methyl substituent substituent in the aniline in in the [the51 ].aniline aniline [51]. [51].

Figure 12. Structure-activity relationship (SAR) of non-NSAID GSMs four rings scaffold. FigureFigure 12. 12. Structure-activity Structure-activity relationship relationship (SAR) (SAR) of of non-NSAID non-NSAID GSMs GSMs four four rings rings scaffold. scaffold. Compound NGP555 (11) from Neurogenetics (Figure 11) showed a decrease in CSF Aβ between Compound NGP555 (11) from Neurogenetics (Figure 11) showed a decrease in 42CSF AΆ42 Compound NGP555 (11) from Neurogenetics (Figure 11) showed a decrease in CSF AΆ42 20–40% and an increase of the shorter forms in rodent studies. Additionally, it demonstrated protection betweenbetween 20–40% 20–40% and and an an increase increase of of the the shorter shorter forms forms in in rodent rodent studies. studies. Additionally, Additionally, it it demonstrated demonstrated from cognitive decline in two independent mouse studies using different memory and learning protectionprotection fromfrom cognitivecognitive declinedecline inin twotwo independentindependent mousemouse studiesstudies usingusing differentdifferent memorymemory andand tasks [51]. NGP 555 (11) entered in phase I clinical trials in 2015. According to a press release learninglearning tasks tasks [51]. [51]. NGP NGP 555 555 ( (1111)) entered entered in in phase phase I I clinical clinical trials trials in in 2015. 2015. According According to to a a press press release release from Neurogenetics on January 2017, NGP555 showed to be safe and well-tolerated in healthy fromfrom NeurogeneticsNeurogenetics onon JanuaryJanuary 2017,2017, NGP555NGP555 showedshowed toto bebe safesafe andand well-toleratedwell-tolerated inin healthyhealthy volunteers [52]. Detailed results and future clinical studies with this compound have not been volunteersvolunteers [52].[52]. DetailedDetailed resultsresults andand futurefuture clinicclinicalal studiesstudies withwith thisthis compoundcompound havehave notnot beenbeen disclosed yet. discloseddisclosed yet. yet. Based on this A-D scaffold, Eisai Pharmaceuticals developed a series of patented diarylcinnamide BasedBased onon thisthis A-DA-D scaffold,scaffold, EisaiEisai PharmaceuticalsPharmaceuticals developeddeveloped aa seriesseries ofof patentedpatented derivatives (12–15, Figure 13)[53], where the aminothiazole group present in Neurogenetics series was diarylcinnamidediarylcinnamide derivativesderivatives ((1212––1515, , FigureFigure 13)13) [53],[53], wherewhere thethe aminothiazoleaminothiazole groupgroup presentpresent inin replaced by an α, β-unsaturated amide or a piperidone. NeurogeneticsNeurogenetics series series was was replaced replaced by by an an ΅ ΅, ,Ά Ά-unsaturated-unsaturated amide amide or or a a piperidone. piperidone.  Pharmaceuticals 2019, 12, 41 12 of 31 Pharmaceuticals 2018, 11, 12 of 31 Pharmaceuticals 2018, 11, 12 of 31

12 13

14 14 15 Figure 13. Examples of Eisai cinnamides. Figure 13. Examples of Eisai cinnamides. Figure 13. Examples of Eisai cinnamides. Beyond the common A-D scaffold, the molecules 12–15 developed shared an hydrogen bond Beyond the common A-D scaffold, the molecules 12–15 developed shared an hydrogen bond donorBeyond (as atheα, βcommon-unsaturated A-D amidescaffold, or the a piperidone) molecules suggesting12–15 developed the importance shared an of hydrogen a hydrogen bond bond donor (as a ΅, Ά-unsaturated amide or a piperidone) suggesting the importance of a hydrogen bond donordonor (as in a this΅, Ά-unsaturated region [33]. The amide work or arounda piperidone) this cinnamide suggesting series the fromimportance Eisai lead of a to hydrogen the discovery bond of donor in this region [33]. The work around this cinnamide series from Eisai lead to the discovery of donorthe clinicalin this region compounds [33]. The E2012 work (16 )around and E2212 this (cinn17) (Figureamide series14)[33 from]. Eisai lead to the discovery of the clinical compounds E2012 (16) and E2212 (17) (Figure 14) [33].

16 17 E2012 E2212 (predicted structure) Figure 14. Structures of E2012 (16) and E2212 (17, predicted structure). FigureFigure 14.14. StructuresStructures of E2012 of E2012 (16 ()16 and) and E2212 E2212 (17 (,17 predicted, predicted structure). structure).

E2012E2012 (16 ()16 decreased) decreased levels levels of ofAΆ A40β 40and and AΆ A42β 42in rat in ratCSF, CSF, brain brain and and plasma plasma in invivo vivo in ina dose a dose dependent manner. The reported IC50 values of E2012 (16) for AΆ40 and AΆ42 were 330 and 92 nM, dependentdependent manner. manner. The The reported reported IC50 ICvalues50 values of E2012 of E2012 (16) for (16 A) forΆ40 Aandβ40 A andΆ42 were Aβ42 330 were and 330 92 andnM, 92 respectivelynM, respectively [54]. In [ 54rat]. CSF, In rat E2012 CSF, E2012(16) significantly (16) significantly decreased decreased AΆ42 Alevelsβ42 levelsby 16.6% by 16.6% and 47.2% and 47.2% at dosesat doses of 10 ofand 10 30 and mg/kg, 30 mg/kg, respectively. respectively. It was also It was revealed also revealed that E2012 that (16 E2012) reduced (16) reduced AΆ40 and Aβ A40Ά42 and andA βincreased42 and increased shorter A shorterΆ peptides, Aβ peptides, such as suchAΆ37 as and Aβ 37AΆ and38, without Aβ38, without changing changing total amount total amount of AΆ of peptidesAβ peptides [55]. E2012 [55]. E2012(16) was (16 )the was first the non-carboxylic first non-carboxylic acid to acid enter to enterin clinical in clinical trials trials in 2006 in 2006and andit showedit showed to be to efficacious be efficacious in reduce in reduce plasma plasma levels levels of A ofΆ42 Aβ of42 ~ of 50% ~ 50% in a in phase a phase I clinical I clinical trial trial [56]. [56 ]. However,However, lenticular lenticular opacity opacity was was observed observed in in a ahigh-dose high-dose group group of of a a 13-week 13-week preclinical preclinical safety study in in rats, runningrunning inin parallelparallel to thethe phasephase II study leading to the suspension ofof the clinical study. Follow-upFollow- upstudies studies up up to to the the highest highest dose dose tolerated tolerated in in monkeys monkeys for for E2012 E2012 (16 (16) did) did not not show show ocular ocular toxicity toxicity [57 ]. [57].However, However, Eisai Eisai decided decided to developto develop their their improved improved E2212 E2212 compound compound (17 ),(17 instead), instead [17 ].[17]. E2212E2212 (17 ()17 entered) entered in ina phase a phase I clinical I clinical trial trial in in2010 2010 (clinicaltrials.gov (clinicaltrials.gov Ident Identifier:ifier: NCT01221259). NCT01221259). It demonstratedIt demonstrated to tohave have a similar a similar pharmacological pharmacological profile profile as asE2012 E2012 (16 ()16 and) and a better a better safety safety profile, profile, withwith no no clinically clinically significant significant ophthalmologic ophthalmologic findings findings [58]. [58 The]. The PD PD response response measured measured in inplasma plasma increasedincreased with with the the dose dose and and was was shown shown to toperform perform 54% 54% AΆ A42β42 reduction reduction at atthe the 250 250 mg mg dose. dose. No No furtherfurther development development has beenbeen reported reported to dateto date for E2212 for (E221217) by Eisai(17) andby theEisai platform and theclinicaltrials.gov platform clinicaltrials.govdoes not present does new not studies.present Thenew predictedstudies. The structure predicted of E2212 structure (17), of possessing E2212 (17), four possessing aromatic four rings, aromatic rings, a high molecular weight (480 g/mol) and a high cLogP (5.5) [17] could have conditioned its further development due to its poor drug-like properties.

  Pharmaceuticals 2019, 12, 41 13 of 31

Pharmaceuticalsa high molecular 2018, 11 weight, (480 g/mol) and a high cLogP (5.5) [17] could have conditioned its further13 of 31 development due to its poor drug-like properties. OtherOther pharmaceutical pharmaceutical companies, companies, namely namely Schering, Schering, Roche, Roche, AstraZeneca, AstraZeneca, Bristol-Myers Bristol-Myers Squibb, Squibb, Amgen,Amgen, between others, hadhad theirtheir own own programs programs for for the the development development of non-NSAIDof non-NSAID derived derived GSM GSM [59]. [59].The scaffoldThe scaffold of the of four the consecutivefour consecutive linked linked rings firstlyrings firstly reported reported by Neurogenetics by Neurogenetics can be foundcan be infound most inof themost non-NSAID of the non-NSAID compounds developedcompounds [33 developed]. Consequently, [33]. theyConsequently, share the same they suboptimal share the drug-like same suboptimalproperties asdrug-like the first properties non-NSAID as the derived first non-NSAID GSM, leading derived to a GSM, lack of leading compounds to a lack reaching of compounds clinical reachingevaluation. clinical The lackevaluation. of information The lack about of inform the structuralation about characteristics the structural of characteristicsγ-secretase has of been ·-secretase pointed hasas anbeen important pointed as factor an important for the difficulty factor for in the developing difficulty in potent developing GSM compounds potent GSM [ 33compounds]. The recently [33]. Thesolved recently structure solved of gamma-secretase structure of gamma-secretase bound to the C99 bound fragment to couldthe C99 help fragment the development could help of potent the developmentand selective of GSM potent compounds and selective [60]. GSM compounds [60].

3.2.3.2. BACE-1 BACE-1 Inhibitors Inhibitors BACE-1BACE-1 is is a a type-1 type-1 membrane-anchored membrane-anchored aspartyl aspartyl protease protease responsible responsible for for the the first first step step of of the the proteolysisproteolysis of of APP, APP, identified identified in 1999 1999 [61]. [61]. BACE-1 BACE-1 cleaves cleaves APP APP in in the the luminal luminal surface surface of of the the plasma plasma membranemembrane and and releases releases the the soluble soluble ectodomain ectodomain of of APP, APP, leaving leaving C99 C99 (A (AΆβ plusplus AICD) AICD) in in the the membrane membrane toto be be subsequently subsequently cleave cleavedd by by GS GS to to generate generate A AΆβ peptidespeptides of of different different lengths lengths as as previously previously described described (Figure(Figure 15)15)[ [61,62].61,62].

Figure 15. Processing steps steps of of APP APP by by ( (AA)) BACE-1 BACE-1 and and (B (B) )GS. GS. Adapted Adapted with with permission permission from from [17]. [17 ]. Copyright 2016 American ChemicalCopyright Society. 2016 American Chemical Society

APPAPP mutations mutations close close to to the the Άβ-cleavage-cleavage site site that that increase increase the the efficiency efficiency of of Άβ-cleavage-cleavage and and result result inin overproduction overproduction of of AΆ A peptidesβ peptides strongly strongly influence influence the risk the of risk AD of [52]. AD On [52 the]. other On the and, other a mutation and, a adjacentmutation to adjacent the Ά-cleavage to the β -cleavagesite that reduces site that the reduces formation the formation of amyloidogenic of amyloidogenic peptides peptides has a strong has a protectivestrong protective effect against effect against AD [19]. AD [Considering19]. Considering this thisgenetic genetic information, information, the the inhibition inhibition of of APP APP proteolysisproteolysis byby BACE-1BACE-1 to to lower lower the the concentration concentration of neurotoxic of neurotoxic Aβ peptides AΆ peptides became became a rational a rational strategy strategyfor clinical for intervention.clinical intervention. BACE-1BACE-1 protease protease is is characterized characterized by by a a large large catalytic catalytic domain domain which which is is marked marked by by the the centrally centrally locatedlocated catalytic catalytic aspartates aspartates Asp32 Asp32 and and Asp228. Asp228. Fr Freeee BACE-1 BACE-1 features features a a flap-open flap-open conformation conformation that that isis energetically energetically stable stable due due to to the the multiple multiple hydrogen hydrogen bonds bonds in in the the flap flap region region of of the the enzyme. enzyme. When When a substrate is bound, BACE-1 assumes a flap-closed or a flap-open conformation, depending on the characteristics of the substrate [16], [63]. The catalytic domain of BACE-1 contain eight pockets consisted of different amino acid residues (Table 4).  Pharmaceuticals 2019, 12, 41 14 of 31 a substrate is bound, BACE-1 assumes a flap-closed or a flap-open conformation, depending on the characteristics of the substrate [16,63]. The catalytic domain of BACE-1 contain eight pockets consisted of different amino acid residues (Table4).

Table 4. Pockets and their amino acid residues on the catalytic domain of BACE-1 [64].

Pocket Amino Acid Residues Pocket Amino Acid Residues S1 Leu30, Asp32, Tyr71, Leu119, Gln73, Phe108 S10 Val31, Tyr71, Thr72, Asp228 S2 Asn233, Arg235, Ser325 S20 Ser35, Val69, Pro70, Tyr198 S3 Leu133, Ile110, Ser229 S30 Arg128, Tyr198 S4 Gln73, Thr232, Arg307 S40 Glu125, Ile126, Tyr197, Tyr198

3.2.1. BACE-1 Inhibitor Development Initially, the development of BACE-1 inhibitors appeared to be a relatively simple approach. First, the development of successful clinical aspartic proteases inhibitors for other therapeutic areas, namely human immunodeficiency virus (HIV) and hypertension, had established an important knowledge for the development of others aspartic protease inhibitors [20]. Second, the first crystal structure of this secretase, elucidated in 2000, provided powerful information for the structure-based drug design of BACE-1 inhibitors [65]. However, progress has been difficulted by combination of properties needed for being efficacious: compounds must fulfill the general rigid prerequisites for a drug aimed for CNS penetration and at the same time be compatible with the large and hydrophobic catalytic pocket of BACE-1. Moreover, selectivity toward different aspartyl proteases have been an additional attrition factor in BACE-1 drug discovery. Nevertheless, despite the many challenges in the design of selective and effective BACE-1 inhibitors, several pharmaceutical industries have made impressive efforts for the improvement of BACE-1 inhibitors, with several compounds currently under clinical development. Peptidomimetics, compounds mimicking the sequences of BACE-1 substrates were the first BACE-1 inhibitors developed and showed potent activity in vitro [15]. However, these large compounds suffer from poor PK properties, such as low bioavailability and low penetrance across the BBB, leading to the unsuccess of their development. Consequently, the design of BACE-1 inhibitors has focused on small molecules with nonpeptidic nature with improved PK properties and BBB penetration. Herein, a general overview of the structural evolution of BACE-1 inhibitors with a focus on the medicinal chemistry aspects of drug development programs will be provided.

Acyl-Guanidine-Based Inhibitors A series of acyl guanidine-based BACE-1 inhibitors were discovered by high-throughput screening (HTS) by Wyeth (acquired by Pfizer in 2009) [63] represented by compounds 18–21 in Figure 16. An X-ray crystal structure of compound 18 complexed with the catalytic domain of BACE-1 revealed that the acyl guanidine moiety forms four key hydrogen interactions with the catalytic aspartic acids Asp32 and Asp228 [63]. A structural change in BACE-1 upon binding with the compound was observed, in which the flap region adopts an “open conformation”, due to stabilized interactions between Tyr71 and the π-system of the diarylpyrrole ligand 18 [63]. Additionally, it was observed that the two aryl groups extend into the S1 and S20 pockets and the para position of the P1 phenyl group projects directly to an unoccupied S3 subsite, allowing the addition of substituents in the P1 phenyl and potentially increasing binding affinity. In contrast to the S1-S3 pockets, S20 provides access to more polar/charged groups, allowing to explore analogues of compound 18 able to form hydrogen-bonds in this region [63]. Pharmaceuticals 2018, 11, 14 of 31

Table 4. Pockets and their amino acid residues on the catalytic domain of BACE-1 [64].

Pocket Amino Acid Residues Pocket Amino Acid Residues S1 Leu30, Asp32, Tyr71, Leu119, Gln73, Phe108 S1’ Val31, Tyr71, Thr72, Asp228 S2 Asn233, Arg235, Ser325 S2’ Ser35, Val69, Pro70, Tyr198 S3 Leu133, Ile110, Ser229 S3’ Arg128, Tyr198 S4 Gln73, Thr232, Arg307 S4’ Glu125, Ile126, Tyr197, Tyr198

3.2.1. BACE-1 Inhibitor Development Initially, the development of BACE-1 inhibitors appeared to be a relatively simple approach. First, the development of successful clinical aspartic proteases inhibitors for other therapeutic areas, namely human immunodeficiency virus (HIV) and hypertension, had established an important knowledge for the development of others aspartic protease inhibitors [20]. Second, the first crystal structure of this secretase, elucidated in 2000, provided powerful information for the structure-based drug design of BACE-1 inhibitors [65]. However, progress has been difficulted by combination of properties needed for being efficacious: compounds must fulfill the general rigid prerequisites for a drug aimed for CNS penetration and at the same time be compatible with the large and hydrophobic catalytic pocket of BACE-1. Moreover, selectivity toward different aspartyl proteases have been an additional attrition factor in BACE-1 drug discovery. Nevertheless, despite the many challenges in the design of selective and effective BACE-1 inhibitors, several pharmaceutical industries have made impressive efforts for the improvement of BACE-1 inhibitors, with several compounds currently under clinical development. Peptidomimetics, compounds mimicking the sequences of BACE-1 substrates were the first BACE-1 inhibitors developed and showed potent activity in vitro [15]. However, these large compounds suffer from poor PK properties, such as low bioavailability and low penetrance across the BBB, leading to the unsuccess of their development. Consequently, the design of BACE-1 inhibitors has focused on small molecules with nonpeptidic nature with improved PK properties and BBB penetration. Herein, a general overview of the structural evolution of BACE-1 inhibitors with a focusPharmaceuticals on the medicinal2019, 12, 41 chemistry aspects of drug development programs will be provided. 15 of 31

Pharmaceuticals 2018, 11, 15 of 31 18 19

Acyl-guanidine-BasedIC50 (BACE-1) Inhibitors = 3700 nM IC50 (BACE-1) = 110 nM A series of acyl guanidine-based BACE-1 inhibitors were discovered by high-throughput screening (HTS) by Wyeth (acquired by Pfizer in 2009) [63] represented by compounds 18–21 in Figure 16. An X-ray crystal structure of compound 18 complexed with the catalytic domain of BACE- 1 revealed that the acyl guanidine moiety forms four key hydrogen interactions with the catalytic aspartic acids Asp32 and Asp228 [63]. A structural change in BACE-1 upon binding with the compound was observed, in which the flap region adopts an “open conformation”, due to stabilized interactions between Tyr71 and the Δ-system of the diarylpyrrole ligand 18 [63]. Additionally, it was observed that the two aryl groups extend into the S1 and S2’ pockets and the para position of the 20P1 phenyl group projects directly to an unoccupied21 S3 subsite, allowing the IC50 (BACE-1) = 5.0 nM IC50 (BACE-1) = 9.5 nM addition of substituents in the P1 phenyl and potentially increasing binding affinity. In contrast to the S1-S3 pockets,FigureFigure S2’ 16. 16.provides StructuresStructures access and and activity to activity more of ofacyl polar/charged acyl guanidine-based guanidine-based groups, BACE-1 BACE-1 allowing inhibitors inhibitors to explore18–2118–21. . analogues of compound 18 able to form hydrogen-bonds in this region [63].  AccordingAccording withwith thethe informationinformation substitutionssubstitutions werewere mademade toto compoundcompound 1818 inin orderorder toto improveimprove potency.potency. Compound Compound19, 19,containing containing a apara parapropyloxyphenyl propyloxyphenyl moietymoiety inin thethe unsubstitutedunsubstituted arylaryl ringring andand aa propylpropyl alcoholalcohol inin thethe thirdthird guanidineguanidine nitrogen,nitrogen, allowedallowed anan improvementimprovement ofof approximatelyapproximately 30-fold30-fold inin potencypotency comparingcomparing withwith compoundcompound 1818.. TheThe 2-chloro2-chloro groupgroup inin PP22 doesdoes notnot appearappear toto contributecontribute significantlysignificantly toto potencypotency (Figure(Figure 17 17))[ 63[63].].

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Figure 17. Optimization of 18 to 19. Figure 17. Optimization of 18 to 19. Bristol-Myers Squibb (BMS) also worked in an acyl guanidine series (Figure 18). Starting with hit Bristol-Myers Squibb (BMS) also worked in an acyl guanidine series (Figure 18). Starting with compound 22 it was developed compound 20 with a good potency against BACE-1 (IC50 = 5.0 nM) hit compound 22 it was developed compound 20 with a good potency against BACE-1 (IC50 = 5.0 nM) and inactive against other aspartyl proteases tested, namely cathepsin D (CatD), cathepsin E (CatE) and inactive against other aspartyl proteases tested, namely cathepsin D (CatD), cathepsin E (CatE) and pepsin (IC50 > 100.000 nM) [66]. and pepsin (IC50 > 100.000 nM) [66]. The optimized compound 20 was evaluated in rats in order to access its effect on Aβ40 levels in plasma, brain and CSF. Although a marked and dose-dependent reduction was observed in plasma 1+ Aβ40 (about 80%) no significant reduction in brain and in CSF was achieved (<20%). This lack of efficacy on brain and CSF was ascribed to P-glycoprotein (P-gp)1 efflux.1 Further improvement2 was + necessary to maximize its PK properties [66]. 2 1 6 + 1 &O 1

0H 2 22 20 BACE-1 hit compound BACE-1 optimized compound

IC50 (BACE-1) = 3900 nM IC50 (BACE-1) = 5 nM

IC50 (Cathepesin D) = 14000 nM IC50 (Cathepesin D) = > 100 000 nM

IC50 (Cathepesin E) = 17000 nM IC50 (Cathepesin E) = > 100 000 nM IC50 (Pepsin) = 11000 nM IC50 (Pepsin) = > 100 000 nM

Figure 18. Acyl guanidine-based BACE-1 inhibitors developed by BMS [66].

 Pharmaceuticals 2018, 11, 15 of 31

Acyl-guanidine-Based Inhibitors A series of acyl guanidine-based BACE-1 inhibitors were discovered by high-throughput screening (HTS) by Wyeth (acquired by Pfizer in 2009) [63] represented by compounds 18–21 in Figure 16. An X-ray crystal structure of compound 18 complexed with the catalytic domain of BACE- 1 revealed that the acyl guanidine moiety forms four key hydrogen interactions with the catalytic aspartic acids Asp32 and Asp228 [63]. A structural change in BACE-1 upon binding with the compound was observed, in which the flap region adopts an “open conformation”, due to stabilized interactions between Tyr71 and the Δ-system of the diarylpyrrole ligand 18 [63]. Additionally, it was observed that the two aryl groups extend into the S1 and S2’ pockets and the para position of the P1 phenyl group projects directly to an unoccupied S3 subsite, allowing the addition of substituents in the P1 phenyl and potentially increasing binding affinity. In contrast to the S1-S3 pockets, S2’ provides access to more polar/charged groups, allowing to explore analogues of compound 18 able to form hydrogen-bonds in this region [63]. According with the information substitutions were made to compound 18 in order to improve potency. Compound 19, containing a para propyloxyphenyl moiety in the unsubstituted aryl ring and a propyl alcohol in the third guanidine nitrogen, allowed an improvement of approximately 30-fold in potency comparing with compound 18. The 2-chloro group in P2 does not appear to contribute significantly to potency (Figure 17) [63].

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Figure 17. Optimization of 18 to 19.

Bristol-Myers Squibb (BMS) also worked in an acyl guanidine series (Figure 18). Starting with hit compound 22 it was developed compound 20 with a good potency against BACE-1 (IC50 = 5.0 nM) andPharmaceuticals inactive against2019, 12, 41other aspartyl proteases tested, namely cathepsin D (CatD), cathepsin E (CatE)16 of 31 and pepsin (IC50 > 100.000 nM) [66].

1+ 1 1 2 + 2 1 6 + 1 &O 1

0H 2 22 20 BACE-1 hit compound BACE-1 optimized compound

Pharmaceuticals 2018, 11, 16 of 31 IC50 (BACE-1) = 3900 nM IC50 (BACE-1) = 5 nM The optimizedIC50 (Cathepesin compound D) 20 = was14000 evaluated nM in rats in orderIC to50 (Cathepesinaccess its effect D) = >on 100 A Ά00040 nMlevels in plasma, brain andIC50 CSF.(Cathepesin Although E) = 17000a marked nM and dose-dependentIC 50reduction (Cathepesin was E) observed = > 100 000 in nM plasma AΆ40 (about 80%)IC no50 (Pepsin)significant = 11000 reduction nM in brain and in CSF wasIC50 achieved (Pepsin) = (<> 10020%). 000 ThisnM lack of efficacy on brain FigureandFigure CSF 18. 18. AcylwasAcyl guanidine-basedascribed guanidine-based to P-glycoprotein BACE-1 BACE-1 inhibitors inhibitors (P-gp) developed developed efflux. Further by by BMS BMS improvement[66]. [66]. was necessary to maximize its PK properties [66]. ArrayArray BioPharma BioPharma together together with with Genentech Genentech develope developedd a series a series of ofchroma chromane-basedne-based spirocyclic spirocyclic acylacyl guanidine-derived guanidine-derived BACE-1 BACE-1 inhibitors inhibitors leading leading to to compound compound 2121 (Figure(Figure 19). 19). Although Although it it showed showed a  a goodgood selectivity to BACE-1BACE-1 andand waswas ableable to to reduce reduce CSF CSF A βA40Ά40 levels levels from from 53% 53% to to 63% 63% in ratin rat and and cyno, cyno,respectively, respectively, this this compound compound also also showed showed a high a high efflux efflux ratio ratio by P-gp by P-gp [67]. [67].

21 IC50 = 9.5 nM CSF AΆ40 reduction (Cyno): 63% CatD /BACE-1 affinity: 1/88

FigureFigure 19.19. Chromane-basedChromane-based spirocyclic spirocyclic acyl acyl guanidine-derived guanidine-derived BACE-1 BACE-1 inhibitor inhibitor 21 21developdevelop by byArray Array BioPharmaBioPharma together together with with Genentech Genentech [67]. [67 ].

2-Aminopyridine-Based2-Aminopyridine-Based Inhibitors Inhibitors A Akey key advance advance in the in the development development of small of small molecule molecule BACE-1 BACE-1 inhibitors inhibitors was wasthe discovery the discovery of 2- of amino2-amino heterocycles heterocycles that, that, comparing comparing with with the theinit initialial acyl acyl guanidine guanidine compo compounds,unds, generally generally enable enable a a betterbetter physico-chemical physico-chemical profile, profile, improved improved brain brain penetration penetration and and in invivo vivo efficacy efficacy [68]. [68 ]. WyethWyeth developed developed a series a series of ofpyrrolyl pyrrolyl 2-aminopyridines, 2-aminopyridines, as asan anextension extension of oftheir their work work on on acyl acyl guanidineguanidine inhibitors inhibitors represented represented in inFigure Figure 16. 16 Acyl. Acyl guanidine guanidine inhibitors inhibitors are are polar polar compounds compounds as as suggestedsuggested by by the the high high total total polar polar surface surface area area (TPSA) (TPSA) ƿ80≈ 80particularly particularly due due to tothe the acyl acyl guanidine guanidine moiety,moiety, which which was was associatedassociated to to the the poor poor BBB BBB permeation permeation (<5%) (<5%) observed observed with this with class this of compounds. class of compounds.The bioisosteric The bioisosteric replacement replacement of the acyl guanidineof the acyl moiety guanidine in compound moiety in18 compoundby an aminopyridine 18 by an aminopyridine(compound 23 (compound) was performed 23) was to improve performed compound to improve permeability, compound maintaining permeability, the hydrogen-bondingmaintaining the hydrogen-bondinginteractions with interactions the aspartic with acids th ine theaspartic catalytic acids site in ofthe BACE-1 catalytic [69 site] (Figure of BACE-1 20). [69] (Figure 20).

&\FOL]DWLRQ

18 23 Acyl guanidine-based compound Amynopiridine-based compound

IC50 (BACE-1) = 3.7 μM IC50 (BACE-1) = 5.2 μM TPSA = 86.4 TPSA = 43.8

Figure 20. Bioisosteric replacement of the acyl guanidine moiety of compound 18.

The similarity of the hydrogen interaction between these two compounds was confirmed by X- ray studies which demonstrate the practically total sobreposition of the two moieties interacting with the aspartic acids Asp228 and Asp32 of the catalytic site of BACE-1 [69]. The modulation of the TPSA parameter enhanced the permeability, with compound 23 showing a good central drug exposure with a brain to plasma ratio of 1.7, compared with 0.04 achieved with  Pharmaceuticals 2018, 11, 16 of 31

The optimized compound 20 was evaluated in rats in order to access its effect on AΆ40 levels in plasma, brain and CSF. Although a marked and dose-dependent reduction was observed in plasma AΆ40 (about 80%) no significant reduction in brain and in CSF was achieved (< 20%). This lack of efficacy on brain and CSF was ascribed to P-glycoprotein (P-gp) efflux. Further improvement was necessary to maximize its PK properties [66]. Array BioPharma together with Genentech developed a series of chromane-based spirocyclic acyl guanidine-derived BACE-1 inhibitors leading to compound 21 (Figure 19). Although it showed a good selectivity to BACE-1 and was able to reduce CSF AΆ40 levels from 53% to 63% in rat and cyno, respectively, this compound also showed a high efflux ratio by P-gp [67].

21 IC50 = 9.5 nM CSF AΆ40 reduction (Cyno): 63% CatD /BACE-1 affinity: 1/88

Figure 19. Chromane-based spirocyclic acyl guanidine-derived BACE-1 inhibitor 21 develop by Array BioPharma together with Genentech [67].

2-Aminopyridine-Based Inhibitors A key advance in the development of small molecule BACE-1 inhibitors was the discovery of 2- amino heterocycles that, comparing with the initial acyl guanidine compounds, generally enable a better physico-chemical profile, improved brain penetration and in vivo efficacy [68]. Wyeth developed a series of pyrrolyl 2-aminopyridines, as an extension of their work on acyl guanidine inhibitors represented in Figure 16. Acyl guanidine inhibitors are polar compounds as suggested by the high total polar surface area (TPSA) ƿ80 particularly due to the acyl guanidine moiety, which was associated to the poor BBB permeation (<5%) observed with this class of compounds. The bioisosteric replacement of the acyl guanidine moiety in compound 18 by an aminopyridinePharmaceuticals 2019 (compound, 12, 41 23) was performed to improve compound permeability, maintaining17 the of 31 hydrogen-bonding interactions with the aspartic acids in the catalytic site of BACE-1 [69] (Figure 20).

&\FOL]DWLRQ

18 23 Acyl guanidine-based compound Amynopiridine-based compound

IC50 (BACE-1) = 3.7 μM IC50 (BACE-1) = 5.2 μM TPSA = 86.4 TPSA = 43.8

Figure 20. Bioisosteric replacement of the acyl guanidine moiety of compound 18. Figure 20. Bioisosteric replacement of the acyl guanidine moiety of compound 18. The similarity of the hydrogen interaction between these two compounds was confirmed by X-ray The similarity of the hydrogen interaction between these two compounds was confirmed by X- studies which demonstrate the practically total sobreposition of the two moieties interacting with the ray studies which demonstrate the practically total sobreposition of the two moieties interacting with aspartic acids Asp228 and Asp32 of the catalytic site of BACE-1 [69]. the aspartic acids Asp228 and Asp32 of the catalytic site of BACE-1 [69]. The modulation of the TPSA parameter enhanced the permeability, with compound 23 showing a PharmaceuticalsPharmaceuticalsThe modulation 2018 2018, 11, 11 ,of , the TPSA parameter enhanced the permeability, with compound 23 showing1717 of of 31 31 good central drug exposure with a brain to plasma ratio of 1.7, compared with 0.04 achieved with the a good central drug exposure with a brain to plasma ratio of 1.7, compared with 0.04 achieved with acylthethe acyl guanidine-basedacyl guanidine-based guanidine-based compound compound compound18 [69 18 18]. [69]. Further[69]. Further Further development development development of 23 of byof 23 23 Wyeth by by Wyeth Wyeth led to led led compound to to compound compound24 in  Figure2424 in in Figure Figure 21. 21. 21.

FigureFigure 21. 21. Structure Structure of of the the improved improved aminopyridine-base aminopyridine-base compound compound 24 24 developed developed by by Wyeth. Wyeth.

TheThe pyrimidine pyrimidine linked linked by by an an oxygen oxygen at at para para position position of of the the P P11 1phenyl phenyl allow allow the the establishment establishment of of hydrogen-bondinghydrogen-bonding interactions interactions with with Ser229 Ser229 at at S3 S3 region. region. The The oxo-ethyl-hydroxyl oxo-ethyl-hydroxyl at at position-3 position-3 of of the the 0 aminopyridineaminopyridine moiety moiety allow allow an an improved improved ligand ligand affi affinityaffinitynity in in S1S1’ S1’ region region and and selectivity selectivity against against other other proteasesproteases (Figure(Figure (Figure 2222) 22))[ [69]. 69[69].].

66 6¶6¶

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FigureFigure 22.22 22. .Interactions Interactions of of the the improved improved aminopyridine-base aminopyridine-base compound compound 24 24 developed developed by by Wyeth Wyeth with with thethe catalytic catalytic sitesite site ofof of BACE-1.BACE-1. BACE-1. Adapted Adapted from from [[16]. 16[16].].

TheThe aromatic aromatic rings rings linked linked to to the the pyrrole pyrrole ring ring established established van van der der Waals Waals interactions interactions at at the the S1 S1 andand S2’ S2’ pockets, pockets, while while the the pyrrole pyrrole ring ring of of the the liga ligandnd points points toward toward the the FLAP FLAP region, region, presumably presumably makingmaking a a Δ Δ-edge-edge stacking stacking interaction interaction with with Tyr71 Tyr71 [69]. [69]. Compound Compound 24 24 showed showed an an IC IC5050 (BACE-1) (BACE-1) = = 40 40 nMnM and and >100-fold >100-fold and and >500 >500 fold fold selectivity selectivity against against BACE-2 BACE-2 and and CatD, CatD, respectively. respectively.

2525 ICIC5050 (BACE-1) (BACE-1) = = 41.2 41.2 μ μMM FigureFigure 23. 23. HTS HTS aminothiazine aminothiazine fragment fragment hit hit 25 25. .

  Pharmaceuticals 2018, 11, 17 of 31 the acyl guanidine-based compound 18 [69]. Further development of 23 by Wyeth led to compound 24 in Figure 21.

Figure 21. Structure of the improved aminopyridine-base compound 24 developed by Wyeth.

The pyrimidine linked by an oxygen at para position of the P1 phenyl allow the establishment of hydrogen-bonding interactions with Ser229 at S3 region. The oxo-ethyl-hydroxyl at position-3 of the aminopyridine moiety allow an improved ligand affinity in S1’ region and selectivity against other proteases (Figure 22) [69].

6 6¶ Pharmaceuticals 2019, 12, 41 18 of 31 3RWHQF\ The aromatic rings linked to the pyrrole ring established van der Waals interactions6HOHFWLYLW\ at the S1 and S20 pockets, while the pyrrole ring of the ligand points toward the FLAP region, presumably making a π-edge stacking interaction with Tyr71 [69]. Compound 24 showed an IC50 (BACE-1) = 40 nM and >100-fold and >5003RWHQF\ fold selectivity against BACE-2 and CatD, respectively.

Aminothiazine- and Aminooxazoline-Based Inhibitors Figure 22. Interactions of the improved aminopyridine-base compound 24 developed by Wyeth with F.the Hoffmann-La catalytic site of RocheBACE-1. (Roche) Adapted developed from [16]. a series of aminothiazine-based inhibitors starting with the aminothiazine fragment 25 as a hit (Figure 23). X-ray analysis of hit 25 co-crystallized withThe BACE-1 aromatic showed rings that linked both to nitrogens the pyrrole of thering protonated established amidine van der moietyWaals interactions form tight hydrogen at the S1 bondsand S2’ to pockets, the catalytic while aspartates. the pyrrole Interactive ring of the three-dimensional ligand points toward (3D) modelingthe FLAP revealedregion, presumably that the S3 pocketmaking of a theΔ-edge catalytic stacking site ofinteraction BACE-1 couldwith Tyr71 be best [69]. reached Compound by meta-substitution 24 showed an onIC50 the (BACE-1) phenyl ring.= 40 ThisnM and meta-substitution >100-fold and >500 was fold made selectivity with an amideagainst linker BACE-2 to enable and CatD, the formation respectively. of a hydrogen bond between the NH of the amide bond with the backbone carbonyl oxygen of Gly291 (Figure 24).

Pharmaceuticals 2018, 11, 18 of 31

Aminothiazine- and Aminooxazoline-Based Inhibitors F. Hoffmann-La Roche (Roche) developed a series of aminothiazine-based inhibitors starting with the aminothiazine fragment 25 as a hit (Figure 23). X-ray analysis of hit 25 co-crystallized with BACE-1 showed that both nitrogens of the proton25ated amidine moiety form tight hydrogen bonds to the catalytic aspartates. Interactive three-dimensionalIC50 (BACE-1) = 41.2 (3D) μ Mmodeling revealed that the S3 pocket of the catalytic site of BACE-1 could be best reached by meta-substitution on the phenyl ring. This meta- Figure 23. HTS aminothiazine fragment hit 25. substitution was made with anFigure amide 23. linkerHTS aminothiazine to enable the fragmentformation hit of25 a. hydrogen bond between the NH of the amide bond with the backbone carbonyl oxygen of Gly291 (Figure 24). Additionally, it leads to a conformationally favorable fixation of the two aromatic rings in an Additionally, it leads to a conformationally favorable fixation of the two aromatic rings in an almost planar arrangement. Extensive SAR studies led to the discovery of inhibitor 26, a highly active almost planar arrangement. Extensive SAR studies led to the discovery of inhibitor 26, a highly active in vitro compound (BACE-1 enzyme IC50 = 27 nM, cellular assay IC50 = 2 nM). However, it turned out in vitro compound (BACE-1 enzyme IC50 = 27 nM, cellular assay IC50 = 2 nM). However, it turned out to be a good P- gp substrate (P-gp efflux ratio (P-gp ER) = 5.5), leading to unsatisfactory results in the to be a good P- gp substrate (P-gp efflux ratio (P-gp ER) = 5.5), leading to unsatisfactory results in the in vivo model (<10% reduction of Aβ40 levels with an oral dose of 30 mg/kg) [26]. in vivo model (<10% reduction of AΆ40 levels with an oral dose of 30 mg/kg) [26].

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26 Figure 24. SAR of optimized inhibitor 26 developed by Roche. Figure 24. SAR of optimized inhibitor 26 developed by Roche. In order to improve brain penetration, Roche continued developing compound 26. A set of In order to improve brain penetration, Roche continued developing compound 26. A set of modifications and SAR studies led to the discovery of an aminooxazine-based inhibitor containing a modifications and SAR studies led to the discovery of an aminooxazine-based inhibitor containing a CF3 group. Compound 26 has a high basicity (pKa = 9.8), giving rise to potential phospholipidosis and CF3 group. Compound 26 has a high basicity (pKa = 9.8), giving rise to potential phospholipidosis and leading to the high efflux rate by P-gp. The introduction of fluorines into the headgroup showed to be efficacious in reducing pKa about 3.5 log units and improving brain penetration.

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27

IC50 (BACE-1) = 12 nM

Cell IC50 (AΆ40) = 2 nM P-gp ER = 1.9

Figure 25. Oxazine-based compound 27 with a trifluoromethyl group developed by Roche.

Compound 27 (Figure 25) was the most potent compound, combining favorable in vitro properties (cellular IC50 (BACE-1) = 2 nM, P-gp ER = 1.9) with a reduction of AΆ40 of 78% at a dose of

 Pharmaceuticals 2018, 11, 18 of 31

Aminothiazine- and Aminooxazoline-Based Inhibitors F. Hoffmann-La Roche (Roche) developed a series of aminothiazine-based inhibitors starting with the aminothiazine fragment 25 as a hit (Figure 23). X-ray analysis of hit 25 co-crystallized with BACE-1 showed that both nitrogens of the protonated amidine moiety form tight hydrogen bonds to the catalytic aspartates. Interactive three-dimensional (3D) modeling revealed that the S3 pocket of the catalytic site of BACE-1 could be best reached by meta-substitution on the phenyl ring. This meta- substitution was made with an amide linker to enable the formation of a hydrogen bond between the NH of the amide bond with the backbone carbonyl oxygen of Gly291 (Figure 24). Additionally, it leads to a conformationally favorable fixation of the two aromatic rings in an almost planar arrangement. Extensive SAR studies led to the discovery of inhibitor 26, a highly active in vitro compound (BACE-1 enzyme IC50 = 27 nM, cellular assay IC50 = 2 nM). However, it turned out to be a good P- gp substrate (P-gp efflux ratio (P-gp ER) = 5.5), leading to unsatisfactory results in the in vivo model (<10% reduction of AΆ40 levels with an oral dose of 30 mg/kg) [26].

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Pharmaceuticals 2019, 12, 41 19 of 31

3RWHQF\ leading to the high efflux rate by P-gp. The introduction+\GURJHQERQGZLWK*O\ of fluorines into the headgroup showed to be efficacious in reducing pKa about 3.5 log units and improving$OORZVDIDYRUDEOHFRQIRUPDWLRQ brain penetration.  Compound 27 (Figure 25) was the most potent compound, combining favorable in vitro properties 26 (cellular IC50 (BACE-1) = 2 nM, P-gp ER = 1.9) with a reduction of Aβ40 of 78% at a dose of 1 mg/kg in mice. With regards to its toxicological profile, compound 27 did not inhibit cytochromes P450 (CYP450) Figure 24. SAR of optimized inhibitor 26 developed by Roche. 3A4, 2D6, and 2C9 (IC50 > 25 µM) and showed to be selective against other aspartyl proteases such as humanIn order CatD to /E improve and the peptidasesbrain penetration, renin and Roche pepsin continued (IC50 > 200 developingµM) [70]. compound Amgen reported 26. A aset series of modificationsof small-molecule and SAR BACE-1 studies inhibitors led to the including discovery a xantheneof an aminooxazine-based core with a spirocyclic inhibitor aminooxazoline containing a CFhead3 group. group. Compound The company 26 hasstarted a high basicity with lead (pK compounda = 9.8), giving28 (Figurerise to potential 26) which phospholipidosis showed a good and Aβ leadinglowering to activitythe high in efflux a rat PD rate model, by P-gp. however The introd it alsouction has a lowof fluorines therapeutic into window the headgroup to QTc prolongation, showed to beconsistent efficacious with in inreducing vitro activity pKa about on the 3.5 human log units ether-a-go-go and improving gene brain (hERG) penetration. ion channel [68,71].

%UDLQSHQHWUDWLRQ Pharmaceuticals 2018, 11, 5HGXFHVS.DDQG3JS(5 19 of 31

1 mg/kg in mice. With regards to its toxicological profile,27 compound 27 did not inhibit cytochromes P450 (CYP450) 3A4, 2D6, and 2C9 (IC50 > 25 μM) and showed to be selective against other aspartyl proteases such as human CatD /E and the peptidases renin and pepsin (IC50 > 200 μM) [70]. Amgen reported a series of small-molecule BACE-1 ICinhibit50 (BACE-1)ors including = 12 nM a xanthene core with a spirocyclic aminooxazoline head group. The company startedCell IC 50with (AΆ 40)lead = 2compound nM 28 (Figure 26) which showed a good AΆ lowering activity in a rat PD model, howeverP-gp ER it= 1.9also has a low therapeutic window to QTc prolongation,FigureFigure consistent 25. 25. Oxazine-basedOxazine-based with in vitro compound compound activity 27 27on withwith the a a humantrifluoromethyl trifluoromethyl ether-a-go-go group group developed developedgene (hERG) by by Roche. Roche. ion channel [68], [71]. Compound 27 (Figure 25) was the most potent compound, combining favorable in vitro properties (cellular IC50 (BACE-1) = 2 nM, P-gp ER = 1.9) with a reduction of AΆ40 of 78% at a dose of



28

IC50 (BACE-1) = 2nM

Cell IC50 (BACE-1) = 25 nM hERG Ki= 660 nM

Figure 26. Spirocyclic aminooxazoline lead compound 28. Figure 26. Spirocyclic aminooxazoline lead compound 28. Introduction of polar groups is used as a common strategy to reduce hERG binding affinity. Introduction of polar groups is used as a common strategy to reduce hERG binding affinity. However, it increases TPSA, which is strongly correlated with increased P-gp recognition. A series However, it increases TPSA, which is strongly correlated with increased P-gp recognition. A series of SAR studies were conducted in order to determinate how to balance hERG, P-gp ER and BACE-1 of SAR studies were conducted in order to determinate how to balance hERG, P-gp ER and BACE-1 potency by identifying regions of the molecule where polarity could be incorporated to minimize hERG potency by identifying regions of the molecule where polarity could be incorporated to minimize activity without leading to a significant efflux or a potency decrease. This balance was accomplished by hERG activity without leading to a significant efflux or a potency decrease. This balance was introducing polarity at the P20 site and at the same time reducing the TPSA of the P3 group (Figure 27). accomplished by introducing polarity at the P2’ site and at the same time reducing the TPSA of the The introduction of a fluorine in position 4 of the xanthene ring also improved BACE-1 enzymatic and P3 group (Figure 27). The introduction of a fluorine in position 4 of the xanthene ring also improved cellular potencies, attributed to a favorable hydrogen-bond interaction between the 4-fluor atom and BACE-1 enzymatic and cellular potencies, attributed to a favorable hydrogen-bond interaction NH of Trp76 of BACE-1 [71]. between the 4-fluor atom and NH of Trp76 of BACE-1 [71].

+1 2 1 3 3

< 2 ;

(28)   (30)

IC50 (BACE-1) = 2nM IC50 (BACE-1) = 0.3nM

Cell IC50 (BACE-1) = 25 nM Cell IC50 (BACE-1) = 4.0 nM

hERG Ki= 660 nM hERG Ki= >15 000 nM

Figure 27. Spirocyclic aminooxazoline developed by Amgen [68,71].

Inhibitor 30 was orally administered to a number of species and showed a robust reduction of CNS AΆ40 levels (74 % and 75 % for CSF and brain, respectively) when orally administered in Dawley rats. It showed a bioavailability of 50% and 43% in rat and Cynomolgus monkey, respectively, and no significant effect on the QTc interval at a maximum dose of 12 mg/kg [72]. Among the several aminothiazine/aminooxazoline based inhibitors developed by different pharmaceutical research teams, just a limited number of compounds were able to reach clinical evaluation. Figure 28 depicted the structure of two aminothiazines (31–32) and one aminooxazine (33) based compounds with BACE-1 inhibitory activity that entered in clinical trials.

 Pharmaceuticals 2018, 11, 19 of 31

1 mg/kg in mice. With regards to its toxicological profile, compound 27 did not inhibit cytochromes P450 (CYP450) 3A4, 2D6, and 2C9 (IC50 > 25 μM) and showed to be selective against other aspartyl proteases such as human CatD /E and the peptidases renin and pepsin (IC50 > 200 μM) [70]. Amgen reported a series of small-molecule BACE-1 inhibitors including a xanthene core with a spirocyclic aminooxazoline head group. The company started with lead compound 28 (Figure 26) which showed a good AΆ lowering activity in a rat PD model, however it also has a low therapeutic window to QTc prolongation, consistent with in vitro activity on the human ether-a-go-go gene (hERG) ion channel [68], [71].

28

IC50 (BACE-1) = 2nM

Cell IC50 (BACE-1) = 25 nM hERG Ki= 660 nM

Figure 26. Spirocyclic aminooxazoline lead compound 28.

Introduction of polar groups is used as a common strategy to reduce hERG binding affinity. However, it increases TPSA, which is strongly correlated with increased P-gp recognition. A series of SAR studies were conducted in order to determinate how to balance hERG, P-gp ER and BACE-1 potency by identifying regions of the molecule where polarity could be incorporated to minimize hERG activity without leading to a significant efflux or a potency decrease. This balance was accomplished by introducing polarity at the P2’ site and at the same time reducing the TPSA of the P3 group (Figure 27). The introduction of a fluorine in position 4 of the xanthene ring also improved BACE-1Pharmaceuticals enzymatic2019 ,and12, 41 cellular potencies, attributed to a favorable hydrogen-bond interaction 20 of 31 between the 4-fluor atom and NH of Trp76 of BACE-1 [71].

+1 2 1 3 3

< 2 ;

(28)   (30)

IC50 (BACE-1) = 2nM IC50 (BACE-1) = 0.3nM

Cell IC50 (BACE-1) = 25 nM Cell IC50 (BACE-1) = 4.0 nM

hERG Ki= 660 nM hERG Ki= >15 000 nM

Figure 27. Spirocyclic aminooxazoline developed by Amgen [68,71]. Figure 27. Spirocyclic aminooxazoline developed by Amgen [68,71]. Inhibitor 30 was orally administered to a number of species and showed a robust reduction of Inhibitor 30 was orally administered to a number of species and showed a robust reduction of CNS Aβ40 levels (74 % and 75 % for CSF and brain, respectively) when orally administered in Dawley CNS AΆ40 levels (74 % and 75 % for CSF and brain, respectively) when orally administered in Dawley rats. It showed a bioavailability of 50% and 43% in rat and Cynomolgus monkey, respectively, and no rats. It showed a bioavailability of 50% and 43% in rat and Cynomolgus monkey, respectively, and significant effect on the QTc interval at a maximum dose of 12 mg/kg [72]. no significant effect on the QTc interval at a maximum dose of 12 mg/kg [72]. AmongAmong the theseveral several aminothiazine/aminooxazoline aminothiazine/aminooxazoline based basedinhibitors inhibitors developed developed by different by different pharmaceuticalpharmaceutical research research teams, teams, just a just limited a limited number number of compounds of compounds were able were to ablereach to clinical reach clinical evaluation.Pharmaceuticalsevaluation. Figure 2018 Figure, 11 28, depicted 28 depicted the thestructure structure of two of two aminothiazines aminothiazines (31– (3231)– and32) andone oneaminooxazine aminooxazine20 of 31 ( 33) (33)based basedcompounds compounds with BACE-1 inhibitory inhibitory ac activitytivity that that entered entered in inclinical clinical trials. trials.



31 32 33

LY2811376 (Eli Lilly) LY2886721 (Eli Lilly) RG7129 (Roche) IC50 (BACE-1) = 240 nM IC50 (BACE-1) = 20.3 nM IC50 (BACE-1) = 30 nM

Figure 28. Aminothiazines 31–32 and aminooxazine 33 based compounds evaluated in clinical trials. Figure 28. Aminothiazines 31-32 and aminooxazine 33 based compounds evaluated in clinical trials. 31 LY2811376LY2811376 (31 () is) an is anaminothiazine aminothiazine based based compound compound and and was was the thefirst first clinical clinical BACE-1 BACE-1 inhibitor inhibitor developeddeveloped by byEli EliLilly, Lilly, which which began began phase phase I clinical I clinical trials trials in 2009. in 2009. The inhibitor The inhibitor was given was givento 61 to healthy61 healthy individuals individuals to investigate to investigate the safety the and safety tolerability and tolerability at single atdoses single from doses 5 to from500 mg 5 toas 500oral mg capsulesas oral [73]. capsules LY2811376 [73]. LY2811376(31) showed (31 to) be showed well tolerated to be well with tolerated no serious with adverse no serious effects adverse reported. effects Thereported. maximum The concentration maximum concentration was reached was2 h post reached dose 2 in h postplasma dose and in 5 plasma hours andpost 5dose h post in CSF, dose and in CSF, β β a dose-dependentand a dose-dependent reduction reduction of AΆ40 of Aand40 A andΆ42 Awas42 observed. was observed. With Witha single a single dose dose of 90 of mg 90 mgit was it was β observedobserved a A aΆ A40 40reduction reduction of of 80% 80% after after 7 hhours and 54and % after54 % 12–14 after h,12–14 in plasma h, in andplasma CSF, and respectively. CSF, respectively.However, inHowever, a rat toxicologic in a rat studytoxicologic performed study inperformed parallel with in parallel the phase with I clinical the phase studies, I clinical a retinal ≥ studies,pathology a retinal at doses pathology30 mg/kg at doses was observed,ǃ 30 mg/kg characterized was observed, by cytoplasmic characterized accumulations by cytoplasmic of finely accumulationsgranular autofluorescent of finely granular material autofluorescent dispersed withinmaterial the dispers retinaled epithelium.within the retinal As a epithelium. consequence, Asthe a consequence,undergoing clinicalthe undergoing trials of LY2811376clinical trials (31 of) wereLY2811376 terminated (31) were and theterminated compound and did the notcompound proceed to didphase not proceed II. This toxicto phase effect II. was This attributed toxic effect to off-targetwas attributed effects to of off-target LY2811376 effects (31) againstof LY2811376 other aspartic (31) againstacid proteases,other aspartic namely acid CatD, proteases, as demonstrated namely CatD, by aas subsequent demonstrated study by using a subsequent LY2811376 study (31) in using BACE1 _/_ LY2811376mice ( [3174)]. in As BACE1 a safety _/_ mice procedure, [74]. Asall a safety the study procedure, participants all the werestudy contacted participants for were follow-up contacted exams for 6–100follow-up months exams after 6-10 conclusion months after of theconclusion trial. Fortunately, of the trial. Fortunately, all the volunteers all the volunteers revealed no revealed clinically no significantclinically significant observations observations [74]. [74]. LY2886721LY2886721 (32 (),32 also), also an anaminothiazine aminothiazine based based inhibitor, inhibitor, was was the the second second clinical clinical candidate candidate tested tested in humanin human subjects subjects and and the the first first BACE-1 BACE-1 inhibitor inhibitor to reach to reach phase phase II clinic II clinicalal trials. trials. LY2886721 LY2886721 (32 ()32 was) was testedtested in six in sixphase phase I clinical I clinical studies studies for forthe theevaluat evaluationion of its of safety, its safety, tolerability, tolerability, and and pharmacology, pharmacology, evolving a total of 150 healthy volunteers. Fourteen days of daily dosing of a 70 mg strength reduced CSF AΆ40 by 74 % and CSF AΆ42 by 71 %. No safety concerns were apparent in dosing up to six weeks [75]. Based on the satisfactory results found in the phase I clinical trials, in March 2012 Lilly started a phase II study to examine the safety, tolerability, and PD effects of LY2886721 (32) in patients with mild AD [76]. During the study, routine safety monitoring detected abnormal liver enzyme elevations in 4 patients of 70. Consequently, Lilly voluntarily terminate the phase II study and the clinical development of LY2886721 (32) was halted. Again, the toxicity of this BACE-1 inhibitor was considered to be an off-target effect of the compound unrelated to BACE-1 inhibition [75], [77]. RG7129 (RO5598887, 33) is an aminooxazine-based compound developed by Roche which entered in clinical trials phase I in September 2011. Preclinical evaluation showed high potency against BACE-1 (IC50 = 30 nM). It also showed selectivity against CatD and CatE, pepsin and renin (>1000 fold) but not against BACE-2 (IC50 = 40 nM). Three phase I clinical trials have been completed between 2012 and 2013, however no results were reported. In October 2013, Roche terminated the development of RG7129 (33) but did not provide an explanation for its cessation [78]. In 2014, in a peer reviewed paper Roche reported a mouse study supporting the use of a combination treatment of BACE-1 inhibitor RG7129 (33) and the anti-AΆ antibody gantenerumab, suggesting a future clinical evaluation of these two compounds combined [79]. 

 Pharmaceuticals 2019, 12, 41 21 of 31 evolving a total of 150 healthy volunteers. Fourteen days of daily dosing of a 70 mg strength reduced CSF Aβ40 by 74 % and CSF Aβ42 by 71 %. No safety concerns were apparent in dosing up to six weeks [75]. Based on the satisfactory results found in the phase I clinical trials, in March 2012 Lilly started a phase II study to examine the safety, tolerability, and PD effects of LY2886721 (32) in patients with mild AD [76]. During the study, routine safety monitoring detected abnormal liver enzyme elevationsPharmaceuticals in 2018 4 patients, 11, of 70. Consequently, Lilly voluntarily terminate the phase II study and21 of the 31 clinical development of LY2886721 (32) was halted. Again, the toxicity of this BACE-1 inhibitor was consideredAminoimidazole-Based to be an off-target Inhibitors effect of the compound unrelated to BACE-1 inhibition [75,77]. RG7129 (RO5598887, 33) is an aminooxazine-based compound developed by Roche which entered Starting with the aminoimidazole HTS hit 34 (Figure 29) Merck developed a series of in clinical trials phase I in September 2011. Preclinical evaluation showed high potency against BACE-1 aminoimidazole-based inhibitors represented in Figure 30 [16]. (IC50 = 30 nM). It also showed selectivity against CatD and CatE, pepsin and renin (>1000 fold) but not against BACE-2 (IC50 = 40 nM). Three phase I clinical trials have been completed between 2012 and 2013, however no results were reported. In October 2013, Roche terminated the development of RG7129 (33) but did not provide an explanation for its cessation [78]. In 2014, in a peer reviewed paper Roche reported a mouse study supporting the use of a combination treatment of BACE-1 inhibitor RG7129 (33) and the anti-Aβ antibody gantenerumab,34 suggesting a future clinical evaluation of these two compounds combined [79]. Pharmaceuticals 2018, 11, Figure 29. Merck’s aminoimidazole HTS hit. 21 of 31 Aminoimidazole-Based Inhibitors Aminoimidazole-BasedMolecular modeling Inhibitors studies showed that the amino group of the imidazole heterocycle was responsibleStarting for with the binding the aminoimidazole with the catalytic HTS aspart hit 34ic acid(Figure residues 29) of Merck BACE-1. developed It was also a seriesobserved of Starting with the aminoimidazole HTS hit 34 (Figure 29) Merck developed a series of aminoimidazole-basedthat 2-methoxy-5-nitro inhibitorssubstituents represented on the benzyl in Figure subunit 30[ led16]. to potent inhibitors, such as compound aminoimidazole-based inhibitors represented in Figure 30 [16]. 35 (Figure 30), however, their presence was also responsible for a high P-gp ER. The following synthesis of compound 36 allowed a significant reduction in P-gp ER and Merck moved forward to improve the potency of this inhibitor. The introduction of a conformational constraint in compound 37 allowed an increase in potency of 5 fold due to additional hydrophobic interactions with the flap region of BACE-1 [80]. Additionally, the introduction of a fluor group in compound 38 allowed

additional hydrophobic interactions and increased 34the potency in about 6-fold to an IC50 (BACE-1) = 63 nM. This inhibitor has a reduced P-gp ER of 3.6, suggesting viable brain penetration [80]. Figure 29. Merck’s aminoimidazole HTS hit. Figure 29. Merck’s aminoimidazole HTS hit.

Molecular modeling studies showed that the3RWHQ amino group of the imidazole3RWHQ heterocycle was %UDLQ Conformational Additional responsible for the binding with the catalytic aspartic acid residues of BACE-1. It was also observed constraint hydrophobic that 2-methoxy-5-nitro substituents on the benzyl subunit led to potent inhibitors, such as compound 35 (Figure 30), however, their presence was also responsible for a high P-gp ER. The following synthesis of compound 36 allowed a significant reduction in P-gp ER and Merck moved forward to improve the potency of this inhibitor. The introduction of a conformational constraint in compound 37 allowed an increase in potency of 5 fold due to additional hydrophobic interactions with the flap region of BACE-1 [80]. Additionally, the introduction of a fluor group in compound 38 allowed 35 36 37 38 additional hydrophobic interactions and increased the potency in about 6-fold to an IC50 (BACE-1) = IC50 (BACE-1) = 470 nM IC50 (BACE-1) = 1800 nM IC50 (BACE-1) = 350 IC50 (BACE-1) = 63 nM 63 nM. This inhibitor has a reduced P-gp ER of 3.6, suggesting viable brain penetration [80]. P-gp ER = 20 P-gp ER = 4.0 nM P-gp ER = 3.6 P-gp ER = 4.0 3RWHQ 3RWHQ Figure 30. Merck’s aminoimidazole-based inhibitors. %UDLQ Figure 30. Merck’s aminoimidazole-basedConformational inhibitors. Additional constraint hydrophobic MolecularWyeth reported modeling the studies design showed and synthesis that the aminoof potent group BACE-1 of the imidazoleinhibitors heterocyclewith a bicyclic was responsibleaminoimidazole for the scaffold, binding based with theon catalyticthe HTS aspartichit compound acid residues 39 (Figure of BACE-1. 31). It Itrepresents was also observeda bicyclic thataminoimidazole 2-methoxy-5-nitro core substituentsand a biphenyl on the mo benzyliety, subunitand a ledsuboptimal to potent inhibitors,potency (IC such50 as(BACE-1)) compound in 35micromolar(Figure 30 ),range. however, X-ray their studies presence show was that also the responsible hit occupies for the a highcenter P-gp of BACE-1 ER. The followingbinding pocket synthesis (S1, ofS2’ compound regions) in36 anallowed orientation a significant where the reduction aminoimidaz in P-gpole ER portion and Merck of the moved ligand forward directly to interacts improve with the potencythe catalytic-site of this inhibitor. aspartic Theacids introduction (Asp32 and Asp228) of a conformational via hydrogen constraint interactions. in compound 37 allowed 35 36 37 38 an increase in potency of 5 fold due to additional hydrophobic interactions with the flap region of IC50 (BACE-1) = 470 nM IC50 (BACE-1) = 1800 nM IC50 (BACE-1) = 350 IC50 (BACE-1) = 63 nM  P-gp ER = 20 P-gp ER = 4.0 nM P-gp ER = 3.6 P-gp ER = 4.0  Figure 30. Merck’s aminoimidazole-based inhibitors.

Wyeth reported the design and synthesis of potent BACE-1 inhibitors with a bicyclic aminoimidazole scaffold, based on the HTS hit compound 39 (Figure 31). It represents a bicyclic aminoimidazole core and a biphenyl moiety, and a suboptimal potency (IC50 (BACE-1)) in micromolar range. X-ray studies show that the hit occupies the center of BACE-1 binding pocket (S1, S2’ regions) in an orientation where the aminoimidazole portion of the ligand directly interacts with the catalytic-site aspartic acids (Asp32 and Asp228) via hydrogen interactions.



 Pharmaceuticals 2019, 12, 41 22 of 31

BACE-1 [80]. Additionally, the introduction of a fluor group in compound 38 allowed additional hydrophobic interactions and increased the potency in about 6-fold to an IC50 (BACE-1) = 63 nM. This inhibitor has a reduced P-gp ER of 3.6, suggesting viable brain penetration [80]. Wyeth reported the design and synthesis of potent BACE-1 inhibitors with a bicyclic aminoimidazole scaffold, based on the HTS hit compound 39 (Figure 31). It represents a bicyclic aminoimidazole core and a biphenyl moiety, and a suboptimal potency (IC50 (BACE-1)) in micromolar range. X-ray studies show that the hit occupies the center of BACE-1 binding pocket (S1, S20 regions) in an orientation where the aminoimidazole portion of the ligand directly interacts with the catalytic-site Pharmaceuticals 2018, 11, 22 of 31 Pharmaceuticalsaspartic acids 2018 (Asp32, 11, and Asp228) via hydrogen interactions. 22 of 31

39 39 IC50 (BACE-1) = 38 μm IC50 (BACE-1) = 38 μm )LJXUH%LF\FOLFDPLQRLPLGD]ROHKLWFRPSRXQG Figure)LJXUH 31. Bicyclic%LF\FOLFDPLQRLPLGD]ROHKLWFRPSRXQG aminoimidazole hit compound39. The structure also shows that the S3 region could be explored through the substitution of the TheThe structure structure also also shows shows that that the the S3 S3 region region coul couldd be be explored explored through through th thee substitution substitution of of the the moiety occupying the S1 pocket, indicating an opportunity to build off the phenyl moiety into this S3 moietymoiety occupying occupying the the S1 S1pocket, pocket, indicating indicating an oppo an opportunityrtunity to build to off build the offphenyl the phenylmoiety into moiety this intoS3 region and improve binding affinity [81]. In order to achieve the unoccupied S3 region the meta- regionthis S3 and region improve and improvebinding bindingaffinity [81]. affinity In [order81]. In to order achieve to achievethe unoccupied the unoccupied S3 region S3 the region meta the- substitution of the phenyl ring was explored with benzyl analogues allowing an improvement of substitutionmeta-substitution of the ofphenyl the phenyl ring ringwas wasexplored explored with with benzyl benzyl analogues analogues allowing allowing an an improvement improvement of of ligand potency about 5-fold. Greater results were obtained with a pyridine moiety where the pyridine ligandligand potency potency about about 5-fold. 5-fold. Greater Greater results results were were obtained obtained with with a apyridine pyridine moiety moiety where where the the pyridine pyridine nitrogen interacts with Ser229 through the conserved water at S3 pocket (Figure 32). The introduction nitrogennitrogen interacts interacts with with Ser229 Ser229 through through the the conserved conserved water water at at S3 S3 pocket pocket (Figure (Figure 32). 32). The The introduction introduction of a 2-fluorine was centered on subtle metabolic properties (additional information not disclosed). In ofof a a2-fluorine 2-fluorine was was centered centered on on subtle subtle metabolic metabolic properties properties (additional (additional information information not disclosed). not disclosed). In additional SAR studies exploring the phenyl ring that projects into the S2´pocket,0 it was discovered additionalIn additional SAR SAR studies studies exploring exploring the thephenyl phenyl ring ring that that projects projects into into the the S2´pocket, S2 pocket, it itwas was discovered discovered that the trifluoromethoxy substituted compound 40 shows an approximate 15-fold improvement of thatthat the the trifluoromethoxy trifluoromethoxy substituted substituted compound compound 4040 showsshows an an approximate approximate 15-fold 15-fold improvement improvement of of BACE-1 potency (comparing with compound 39). The improved compound 40 (R-enantiomer) BACE-1BACE-1 potency potency (comparing(comparing withwith compound compound39 ).39 The). The improved improved compound compound40 (R -enantiomer)40 (R-enantiomer) showed showed a high potency for BACE-1 inhibition and >100-fold selectivity over the other structurally showeda high potencya high potency for BACE-1 for BACE-1 inhibition inhibition and >100-fold and >100-fold selectivity selectivity over the ov otherer the structurallyother structurally related related aspartyl proteases BACE-2, CatD, renin, and pepsin [81]. relatedaspartyl aspartyl proteases proteases BACE-2, BACE-2 CatD,, renin,CatD, andrenin, pepsin and pepsin [81]. [81].

- Improve metabolic - Improve metabolic properties properties

3RWHQF\ 3RWHQF\ 3RWHQF\ 3RWHQF\ - Additional interactions - Improve interactions in S2’ - Additional interactions - Improve interactions in S2’ in S3 pocket pocket in S3 pocket pocket 40 40 IC50 (BACE-1) = 20 nM IC50 (BACE-1) = 20 nM IC50 (BACE-2) = 3600 nM IC50 (BACE-2) = 3600 nM IC50 (cathepsin D) = 6900 nM IC50 (cathepsin D) = 6900 nM IC50 (renin)= 2080 nM IC50 (renin)= 2080 nM

Figure 32. Optimized aminoimidazole-based inhibitor 40 from Wyeth. Figure 32. Optimized aminoimidazole-based inhibitor 40 from Wyeth. IndependentlyFigure from 32. Wyeth, Optimized AstraZeneca aminoimidazole-based also developed inhibitor a series 40 of from inhibitors Wyeth. based on compound 39, herein represented by compounds 41 and 42 in Figure 33. Independently from Wyeth, AstraZeneca also developed a series of inhibitors based on Independently from Wyeth, AstraZeneca also developed a series of inhibitors based on compound 39, herein represented by compounds 41 and 42 in Figure 33. compound 39, herein represented by compounds 41 and 42 in Figure 33.

  Pharmaceuticals 2018, 11, 23 of 31 Pharmaceuticals 2019, 12, 41 23 of 31 Pharmaceuticals 2018, 11, 23 of 31

42 41 (R -enantiomer)42 pIC50 (BACE-1)=41 7.11 pIC(50R (BACE-1)=-enantiomer) 7.50

CellpIC pIC50 50(BACE-1)= (sAPPΆ) =7.11 7.46 CellpIC pIC50 50(BACE-1)= (sAPPΆ) =7.50 8.10 CellEfflux pIC50 ratio(sAPP = Ά3.5) = 7.46 CellEfflux pIC50 ratio(sAPP = Ά0.8) = 8.10 Figure 33.Efflux Aminoimidazole-based ratio = 3.5 inhibitors 41–42 developedEfflux by ratioAstraZeneca. = 0.8 FigureFigure 33. 33. Aminoimidazole-basedAminoimidazole-based inhibitors inhibitors 4141–42–42 developeddeveloped by by AstraZeneca. AstraZeneca. Compound 41, with a p-difluoromethyl ether substitution on one of the phenyl rings and an m- alkynylCompoundCompound substituent 41 41,on with, the with a other, p a-difluoromethylp-difluoromethyl showed good ether pote ether substitutionncy substitution in both on enzymatic one on oneof the ofand phenyl the cellular phenyl rings assays rings and anwith and m- an pICalkynylm50-alkynyl values substituent of substituent 7.11 andon the7.46, on other, therespectively. other, showed showed goodCell membrane goodpotency potency in permeability, both in enzymatic both enzymatic as determinedand cellular and cellularby assays a Caco-2 assayswith -6 assay,pICwith50 valueswas pIC 8.450 of xvalues 7.1110 cm/s,and of 7.46,7.11 and respectively. andthe efflux 7.46, ratio respectively. Cell was membrane 3.5, indicating Cell permeability, membrane potential permeability,as for determined BBB penetration. as by determined a Caco-2 The -6 crystalassay,by a wasstructure Caco-2 8.4 x assay, 10of-6 cm/s,compound was and 8.4 the x41 10efflux in cm/s,complex ratio andwas with 3.5, the indicating effluxBACE-1 ratio displayedpotential was 3.5, forthe indicating BBB interaction penetration. potential of theThe for aminoimidazolecrystalBBB penetration.structure moiety of Thecompound with crystal the structure 41catalytic in complex ofAsp32 compound withand BACE-1Asp22841 in complex residues,displayed with binding the BACE-1 interaction in a displayed flap-open of the the conformation,aminoimidazoleinteraction of allowing the moiety aminoimidazole Trp76 with theto be catalytic moiety in position with Asp32 the for catalyticand hydrogen Asp228 Asp32 bondingresidues, and Asp228 to binding the residues,oxygen in a bindingofflap-open the p in- a difluoromethylconformation,flap-open conformation, ether.allowing The Trp76 alkynyl allowing to substituent be Trp76 in position to beof inthe positionfor second hydrogen forring hydrogen extends bonding into bonding to the the S3 tooxygen pocket the oxygen [16,82].of the of p the- difluoromethylp-difluoromethylReplacement ether. of ether. the The alkyne The alkynyl alkynyl chain substituent substituentof inhibitor of the of 41 thesecond with second ringa fluorinated ring extends extends into propyl into the theS3 ether pocket S3 pocketresulted [16,82]. [16 in,82 ]. inhibitorReplacementReplacement 42. This inhibitor of ofthe the alkyne showed alkyne chain similar chain ofof potencyinhibitor inhibitor as 41 compound41 withwith a afluorinated fluorinated41 but an propylenhanced propyl ether ether efflux resulted resulted ratio ofin in 0.8,inhibitorinhibitor which 42 42is. This .4 This times inhibitor inhibitor lower. showed showed However, similar similar an potency potency in vivo as as compound assessment 4141 in butbut a ananmouseenhanced enhanced model efflux efflux using ratio ratio oral of of 0.8, administration0.8,which which is 4 is times of4 timescompound lower. lower. However, 42 However,showed an in vivoa lowanassessment inbrain/plasma vivo assessment in a mouseratio of modelin 0.18, a mouse usingshowing oralmodel that administration theusing results oral of obtainedadministrationcompound in the42 ofshowedCaco-2 compound cells a low efflux brain/plasma42 showed test were a low ratio underes brain/plasma of 0.18,timated. showing ratio This that of lack the0.18, resultsof showing brain obtained penetration that inthe the results Caco-2was attributedobtainedcells efflux into the testP-gp Caco-2 were efflux, underestimated. cells on ceefflux the testcoadministration This were lack underes of brain timated.of penetration 42 with This a was lackP-gp attributed of inhibitor brain topenetration P-gpresulted efflux, in was oncea brain/plasmaattributedthe coadministration to ratio P-gp of efflux, 2.34 of [82].42 onwith ce the a P-gp coadministration inhibitor resulted of in42 a with brain/plasma a P-gp inhibitor ratio of 2.34 resulted [82]. in a brain/plasmaCompoundCompound ratio AZD3293 of AZD3293 2.34 [82]. (LY3314814, (LY3314814, Lanabecestat, Lanabecestat, 43,43 Figure, Figure 34) 34 is) isan anaminoimidazole aminoimidazole based based compoundcompoundCompound developed developed AZD3293 by byAstraZeneca AstraZeneca(LY3314814, which whichLanabecestat, reache reachedd phase 43 phase, FigureI clinical I clinical 34) tr ialsis trials an in in2012aminoimidazole 2012 [83]. [83 ]. based compound developed by AstraZeneca which reached phase I clinical trials in 2012 [83].

FigureFigure 34. 34.AZD3293AZD3293 (LY3314814, (LY3314814, lanabecestat, lanabecestat, 43)43 from) from AstraZeneca. AstraZeneca. Figure 34. AZD3293 (LY3314814, lanabecestat, 43) from AstraZeneca. TheThe results results from from the the firstfirst twotwo phase phase I clinicalI clinical studies studies are currentlyare currently available available [84]. It [84]. was composedIt was composedof (1)The a results singleof 1) a ascending fromsingle the ascending first dose two study dose phase evaluating study I clinical evaluating doses studies of doses 1–750 are of mgcurrently 1–750 with mg aavailable food-effect with a [84].food-effect component It was componentcomposed(n = 72), and (ofn =1) (2) 72), a a single 2-weekand 2) ascendinga multiple 2-week ascendingmultipledose study ascending dose ev studyaluating dose evaluating dosesstudy evaluatingof doses 1–750 of 15mg doses or with 50 of mg a15 oncefood-effect or 50 daily mg or oncecomponent70 daily mg once or ( n70 weekly = mg72), once and in elderly 2)weekly a 2-week subjects in elderly multiple (Part subjects 1, ascendingn = 31), (Part and dose1, 15,n = study 50,31), or and 150evaluating 15, mg 50, once or doses daily150 mgof in 15 once patients or 50daily mg with inonce mildpatients daily to moderate orwith 70 mgmild AD once to (Part moderateweekly 2, n =in 16). ADelderly Results (Part subjects 2, showed n = (Part 16). that 1,Results AZD3293n = 31), showed and (43 15,) was that50, generally orAZD3293 150 mg well once(43 tolerated) dailywas generallyinup patients to the well highestwith tolerated mild doses to up given.moderate to the No highest notableAD (Part doses food 2, given. effectsn = 16). No were Resultsnotable observed. showedfood For effects singlethat were AZD3293 doses observed.≥5 ( mg,43) Forwas≥70% singlegenerallyreduction doses well wasǃ5 toleratedmg, observed ǃ70% up reduction in to mean the plasmahighest was observed Adosesβ40 and given. in Ameanβ 42No concentrations, plasmanotable AfoodΆ40 effectsand with A prolonged Άwere42 concentrations, observed. suppression For withsinglefor prolonged up doses to 3 ǃ weeks5 suppressionmg, atǃ70% the highestreduction for up doseto was 3 weeks level observed studied. at the in highest mean Following plasmadose multiplelevel AΆ studied.40 and doses, A FollowingΆ robust42 concentrations, reductions multiple in doses,withplasma prolonged robust (≥64% reductions suppression at 15 mg in andplasma for≥ up78% (ǃ to64% at3 ≥weeks at50 15 mg) mg at andtheand highest CSFǃ78% (≥ at dose51% ǃ50 atmg)level 15 and mgstudied. CSF and (≥ ǃFollowing51%76% at 15≥ 50 multiplemg mg) and A β ǃdoses,76%peptides at robust ǃ50 weremg) reductions A seen,Ά peptides including in plasma were prolonged seen,(ǃ64% including at suppression 15 mg andprolonged ǃ even78% atwith suppression ǃ50 a mg) once and weekly even CSF with ( dosingǃ51% a onceat regimen 15 weeklymg and [84 ]. dosingǃ76% at regimen ǃ50 mg) [84]. AΆ peptides were seen, including prolonged suppression even with a once weekly dosing regimen [84].  Pharmaceuticals 2018, 11, 24 of 31

In 2015 and 2016, four additional phase I trials with a total of 175 healthy volunteers were conducted. They evaluated a new tablet formulation and possible interactions of this inhibitor with several drugs commonly prescribed in the elderly, namely warfarin and dabigatran, midazolam, as well as simvastatin and donepezil [85]. Currently, AZD3293 (43) is being evaluated in two phase III clinical trials (NCT02245737 and NCT02783573) sponsored by an alliance between AstraZeneca and PharmaceuticalsEli Lilly [83].2019 These, 12, 41 multi-center, randomized, double-blind, placebo-controlled studies are testing24 of 31 Pharmaceuticalsthe disease-modifying 2018, 11, potential of AZD3293 (43) at daily doses 20 or 50 mg for 18 to 24 months,24 of 31in overIn 4,000 2015 patients and 2016, with four mild additional cognitive im phasepairment I trials due with to AD a total and ofmild 175 AD healthy [84]. volunteers were In 2015 and 2016, four additional phase I trials with a total of 175 healthy volunteers were conducted. They evaluated a new tablet formulation and possible interactions of this inhibitor with conducted.Iminothiadiazinane They evaluated Dioxide-Based a new tablet Inhibitors formulation and possible interactions of this inhibitor with several drugs commonly prescribed in the elderly, namely warfarin and dabigatran, midazolam, as several drugs commonly prescribed in the elderly, namely warfarin and dabigatran, midazolam, as well asMerck simvastatin developed and donepezila series of [85 iminothiadiazinane]. Currently, AZD3293 dioxide (43) is based being evaluatedinhibitors inrepresented two phase IIIby well as simvastatin and donepezil [85]. Currently, AZD3293 (43) is being evaluated in two phase III clinicalcompound trials 45 (NCT02245737, verubecestat.and The NCT02783573) starting point was sponsored an iminopyrimidinone by an alliance between scaffold AstraZeneca (44) which andwas clinical trials (NCT02245737 and NCT02783573) sponsored by an alliance between AstraZeneca and Elimodified Lilly [83 in]. Theseorder multi-center,to improve binding randomized, affinity double-blind, and explore placebo-controlled a new intellectual studies property are testingspace [86] the Eli Lilly [83]. These multi-center, randomized, double-blind, placebo-controlled studies are testing disease-modifying(Figure 35). potential of AZD3293 (43) at daily doses 20 or 50 mg for 18 to 24 months, in over the disease-modifying potential of AZD3293 (43) at daily doses 20 or 50 mg for 18 to 24 months, in 4,000Although patients with the mildPD acti cognitivevities impairmentof compound due 44 to were ADand considered mild AD favorable [84]. for further clinical over 4,000 patients with mild cognitive impairment due to AD and mild AD [84]. development, this compound did not advance. Metabolite profiling following oral administration to Iminothiadiazinanerats revealed biliary Dioxide-Based excretion of Inhibitorsmetabolites corresponding to direct glutathione addition and Iminothiadiazinane Dioxide-Based Inhibitors glutathioneMerck developedadducts derived a series from of oxidative iminothiadiazinane metabolism. dioxideAdditionally, based the inhibitors modest representedCatD selectivity by compound(onlyMerck 21 fold) 45developed, was verubecestat. considered a series The to beof starting inadequateiminothiadiazinane point wasand ana major iminopyrimidinone dioxide limitation based to inhibitorsits scaffoldprogression represented (44) which[87]. In was thisby compoundmodifiedway, an effort in 45 order, wasverubecestat. tomade improve in orderThe binding starting to remove affinity point the was and metabolically an explore iminopyrimidinone a newlabile intellectual propynylpyridine scaffold property (44 and) which space improve was [86] (FiguremodifiedCatD selectivity. 35 ).in order to improve binding affinity and explore a new intellectual property space [86] (Figure 35). Although the PD activities of compound 44 were considered favorable for further clinical development, this compound did not advance. Metabolite profiling following oral administration to rats revealed biliary excretion of metabolites corresponding to direct glutathione addition and glutathione adducts derived from oxidative metabolism. Additionally, the modest CatD selectivity (only 21 fold) was considered to be inadequate and a major limitation to its progression [87]. In this way, an effort was made in order to remove the metabolically labile propynylpyridine and improve CatD selectivity.

44 45 Iminopyrimidinone based compound Verubecestat

IC50 (AΆ40) = 11 nM IC50 (AΆ40) = 2.1 nM BACE-1/CatD = 21 BACE-1/CatD = >10.000

FigureFigure 35.35. StructuresStructures ofof iminopyrimidinoneiminopyrimidinone basedbased compoundcompound44 44and and verubecestatverubecestat( (4545).).

AlthoughMerck had the previously PD activities disclosed of compound the iminohydantoin44 were 46 considered (Figure 36) favorable which although for further represents clinical a development,suboptimal activity, this compound it displayed did an not enhanced advance. selectivity Metabolite for profilingBACE-1 over following CatD. oral administration to rats revealed biliary excretion44 of metabolites corresponding to45 direct glutathione addition and glutathione adductsIminopyrimidinone derived from based oxidative compound metabolism. Additionally,Verubecestat the modest CatD selectivity (only 21 fold) was consideredIC50 (AΆ to40) be = inadequate11 nM and a majorIC limitation50 (AΆ40) = to2.1 its nM progression [87]. In this way, an effort was madeBACE-1/CatD in order to remove = 21 the metabolicallyBACE-1/CatD labile propynylpyridine = >10.000 and improve CatD selectivity.Figure 35. Structures of iminopyrimidinone based compound 44 and verubecestat (45). Merck had previously disclosed the iminohydantoin 46 (Figure 36) which although represents a suboptimalMerck had activity, previously it displayed disclosed an enhanced the iminohydantoin selectivity46 for 46 BACE-1(Figure 36) over which CatD. although represents a suboptimal activity, it displayed an enhancedBACE-1/vCatD selectivity for = 83BACE-1 over CatD.

Figure 36. Iminohydantoin 46 previously developed by Merck.

In an X-ray cocrystal structure of compound 46 with BACE-1, the interaction of the phenyl and furanyl groups with the S1 and S3 pockets, respectively, and the amide N-H engaged in a hydrogen- bonding interaction with the carbonyl of Gly230 were observed. Additionally, comparing the

 46 BACE-1/vCatD = 83

Figure 36. Iminohydantoin 46 previously developed by Merck. Figure 36. Iminohydantoin 46 previously developed by Merck.

In an X-ray cocrystal structure of compound 46 with BACE-1, the interaction of the phenyl and furanyl groups with the S1 and S3 pockets, respectively, and the amide N-H engaged in a hydrogen- bonding interaction with the carbonyl of Gly230 were observed. Additionally, comparing the

 Pharmaceuticals 2018, 11, 25 of 31 Pharmaceuticals 2019, 12, 41 25 of 31 interactions of the iminopyrimidinone based compound 44 and the iminohydantoin 46 with BACE- 1, it was observed that the furanyl ring of the iminohydantoin is projected deeper into the S3 subpocketIn an X-ray (S3sp cocrystal) of BACE-1 structure in comparison of compound with 46thewith pyridyl BACE-1, ring of the the interaction iminopyrimidinone of the phenyl based andcompound furanyl groups 44 [87]. with the S1 and S3 pockets, respectively, and the amide N-H engaged in a hydrogen-bondingComparing interactionthe crystal structures with the carbonyl of BACE-1 of Gly230 and CatD were it observed.was observed Additionally, that their comparingS3SP differ in thetopology, interactions where of the CatD iminopyrimidinone S3SP in slightly basedsmaller compound than the one44 ofand BACE-1. the iminohydantoin Hence, it was postulated46 with BACE-1,the differences it was observed in CatD that selectivity the furanyl of these ring two of the compounds iminohydantoin is related is projectedwith their deeper interaction into thewith S3 the subpocketS3 domains (S3sp of) the of BACE-1 two proteases in comparison [87]. with the pyridyl ring of the iminopyrimidinone based compoundIn this44 [way,87]. Merck conducted a series of SAR studies in order to improve selectivity over CatD whileComparing maintaining the crystalhigh BACE-1 structures affinity, of BACE-1 leading and to the CatD discovery it was observed of verubecestat that their (45, S3 FigureSP differ 37). in The topology,pyridyl wheremotif thein verubecestat CatD S3SP in (45 slightly) allowed smaller an improved than the oneselectivity of BACE-1. over Hence,CatD >10 it was 000. postulated The 6-fluoro thesubstituent differences in in the CatD phenyl selectivity group of allowed these two an compounds improvement is related of 5-fold with in their potency interaction comparing with thewith S3 the domainsdefluoro of theanalogues two proteases and the [87 iminothiadiazinane]. dioxide core enhanced permeability and reduced effluxIn this ratio, way, improving Merck conducted in vivo potency a series [87]. of SAR studies in order to improve selectivity over CatD while maintainingVerubecestat high (45) BACE-1showed affinity,good PK/PD leading properties to the discovery in preclinical of verubecestat species, allowing (45, Figure a profound 37). Theand pyridyl sustained motif reduction in verubecestat of CSF (45 A) allowedΆ40 levels an in improved cynomolgus selectivity monkeys over (72% CatD and >10 000.81% Thereduction 6-fluoro at 3 substituentand 10 mg/kg, in the respectively). phenyl group In allowed a single-dose an improvement cardiovascular of 5-foldstudy in telemetered potency comparing monkeys with it showed the defluorono effect analogues on the QT and interval the iminothiadiazinane and no induction of dioxide CYP 3A4 core or enhanced 1A2 expression permeability in human and hepatocytes reduced efflux[87]. ratio, improving in vivo potency [87].

1+ Selectivity over CatD

+1 1 6 1 2 Permeability and efflux ratio

&O Potency

44  45 Iminopyrimidinone based Verubecestat compound

Figure 37. Drug development and SAR of verubecestat (45). Figure 37. Drug development and SAR of verubecestat (45). Verubecestat (45) showed good PK/PD properties in preclinical species, allowing a profound and Despite the fact verubecestat (45) has selectivity over the proteases CatD, CatE, pepsin and renin sustained reduction of CSF Aβ40 levels in cynomolgus monkeys (72% and 81% reduction at 3 and 10 (>45,000, >45,000, >45,000, and 15,000, respectively) it showed to be a potent inhibitor of BACE-2 (Ki mg/kg, respectively). In a single-dose cardiovascular study in telemetered monkeys it showed no (BACE-2) = 0.38 nM against Ki (BACE-1) = 2.2 nM). Although the specific functions of BACE-2 are effect on the QT interval and no induction of CYP 3A4 or 1A2 expression in human hepatocytes [87]. currently not well known, recent reports associate BACE-2 with the process of pigmentation, Despite the fact verubecestat (45) has selectivity over the proteases CatD, CatE, pepsin and renin consistent with the lighter coat color observed in BACE-2 knockout mice [88]. This phenomenon was (>45,000, >45,000, >45,000, and 15,000, respectively) it showed to be a potent inhibitor of BACE-2 observed in mice and rats treated with verubecestat (45), but not in the chronic toxicology studies (Ki (BACE-2) = 0.38 nM against Ki (BACE-1) = 2.2 nM). Although the specific functions of BACE-2 performed in monkeys. Overall, the relatively benign phenotypes of BACE-2 knockout mice, current are currently not well known, recent reports associate BACE-2 with the process of pigmentation, understanding of the role of BACE-2 processing of its endogenous substrates, and the outcome of consistent with the lighter coat color observed in BACE-2 knockout mice [88]. This phenomenon was preclinical toxicology studies has mitigated concerns related to the lack of selectivity of verubecestat observed in mice and rats treated with verubecestat (45), but not in the chronic toxicology studies (45) over BACE-2 [87]. performed in monkeys. Overall, the relatively benign phenotypes of BACE-2 knockout mice, current Verubecestat (45) advanced to phase I clinical trials in 2011 to assess safety, PK and PD in healthy understanding of the role of BACE-2 processing of its endogenous substrates, and the outcome of volunteers and in mild to moderate AD patients. Single and multiple doses were generally well preclinical toxicology studies has mitigated concerns related to the lack of selectivity of verubecestat tolerated and produced reductions in AΆ levels in the CSF >80% of both healthy human subjects and (45) over BACE-2 [87]. AD patients. Notably, there were no reports of changes in skin or hair pigmentation as a potential Verubecestat (45) advanced to phase I clinical trials in 2011 to assess safety, PK and PD in healthy consequence of BACE-2 inhibition in any of the phase I studies; however, longer treatment time volunteers and in mild to moderate AD patients. Single and multiple doses were generally well would likely be required for pigmentation changes to manifest [87]. In 2012, verubecestat (45) entered tolerated and produced reductions in Aβ levels in the CSF > 80% of both healthy human subjects and into phase II/III clinical trials in people with mild to moderate AD (EPOCH trial), and in 2013 a phase AD patients. Notably, there were no reports of changes in skin or hair pigmentation as a potential III trial for prodromal AD (APECS trial) was also started [68]. consequence of BACE-2 inhibition in any of the phase I studies; however, longer treatment time would  Pharmaceuticals 2019, 12, 41 26 of 31 likely be required for pigmentation changes to manifest [87]. In 2012, verubecestat (45) entered into phase II/III clinical trials in people with mild to moderate AD (EPOCH trial), and in 2013 a phase III trial for prodromal AD (APECS trial) was also started [68]. EPOCH trial was discontinued on February 2017 due to lack of efficacy, with researchers defending that it is very unlikely to observe a clinical benefit in using a BACE-1 inhibitor in cases that a substantial synaptic and neuron loss is already installed [89]. In February 2018, APECS clinical trial was also discontinued and Merck no longer listed verubecestat in its research pipeline. Participants taking verubecestat scored worse than the placebo group on the cognitive test Alzheimer’s Disease Cooperative Study-Activities of Daily Living (ADCS-ADL), in a small but significant way. Further research in needed to understand the origin of this negative effect, if it is reversible and if can be associated with certain patient populations or stages of disease [90,91].

4. Conclusions The discovery of disease modifying agents for AD has shown to be very challenging for medicinal chemists. Compounds should exhibit improved CNS penetration by enhanced BBB permeability and reduced P-gp ER. The physical chemistry characteristics needed for this PK properties need to be balanced with target requirements, in which the improvements of these properties sometimes lead to a reduction on binding affinity and potency. Additionally, off-target effects also need to be addressed, being these features one of the principal causes for the discontinuation of clinical programs. Medicinal chemistry teams have utilized high throughput and virtual screening followed by chemical optimization trying to accommodate several physico-chemical nuances and discover new compounds able to be safety and efficacy in modifying AD progression. Taking into consideration the actual AD pipeline, a special focus has been given to BACE-1 as a target for a disease modifying therapy for AD. The information currently available pointed the inhibition of BACE-1 as a safety and efficacy target for Aβ reduction. All the adverse effects identified up to date in the use of BACE-1 inhibitors were pointed as off-target effects, confirming BACE-1 as a suitable target to explore. Nevertheless, medicinal chemists should perform extended SAR studies, not only for potency and PK properties exploration, but also in terms of selectivity, providing the discovery of compounds highly selective for BACE-1 against other related proteases such as CatD. Another question stands on the adequacy of the pharmacological therapy with the stage of Alzheimer disease (AD). The use of disease modifying therapies as BACE-1 inhibitors should be a suitable option for early stages of AD where minimum neuronal loss and synaptic dysfunction are observed. On the other hand, in a mild-to-moderate AD scenario it would possibly be too late in the disease process for BACE-1 inhibition to be effective and a symptomatically therapeutic should be preferred. In sum, the future of AD will rely on the development of potent, selective and safety compounds able to delay AD progression and neuronal impairment. At the same time, it is essential the identification of specific biomarkers to allow an early and efficacious pharmacological intervention.

Funding: This work was partially supported through national funds provided by FCT/MCTES - Foundation for Science and Technology from the Minister of Science, Technology and Higher Education (PIDDAC) and European Regional Development Fund (ERDF) through the COMPETE – Programa Operacional Factores de Competitividade (POFC) programme, under the Strategic Funding UID/Multi/04423/2013. Acknowledgments: The authors thanks to Tino Rossi, László E. Kiss and Patrício Soares-da-Silva (Laboratory of Chemistry, Research Department, BIAL-Portela & Cª, S.A., À Avenida da Siderurgia Nacional, 4745-457 Coronado (S. Romão and S. Mamede, Portugal)) for their support on the development of this work. Conflicts of Interest: The authors declare no conflict of interest. Pharmaceuticals 2019, 12, 41 27 of 31

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