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Gamma Secretase Modulators: New Alzheimer's Drugs on the Horizon? Matthew G Bursavich, Bryce A Harrison, and Jean-François Blain J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01960 • Publication Date (Web): 23 Mar 2016 Downloaded from http://pubs.acs.org on March 29, 2016

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Journal of Medicinal Chemistry

Gamma Secretase Modulators: New Alzheimer’s Drugs on the Horizon? Matthew G. Bursavich,* Bryce A. Harrison and Jean-François Blain FORUM Pharmaceuticals, 225 2nd Avenue, Waltham, MA 02451

Abstract The rapidly aging population desperately requires new therapies for Alzheimer disease. Despite years of pharmaceutical research, limited clinical success has been realized with several failed disease modification therapies in recent years. Due to compelling genetic evidence, the pharmaceutical industry has put a large emphasis on brain beta amyloid (Aβ) either through its removal via antibodies or by targeting the proteases responsible for its production. In this perspective we focus on the development of small molecules that improve the activity of one such protease, gamma secretase, through an allosteric binding site to preferentially increase the concentration of the shorter non-amyloidogenic Aβ species. After a few early failures due to poor drug-like properties, the industry is now on the cusp of delivering gamma secretase modulators (GSMs) for clinical proof of mechanism studies that combine potency and efficacy with improved drug-like properties such as lower cLogP, high CNS multiparameter optimization (MPO) scores and high sp3 character.

ACS Paragon Plus Environment

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Introduction Alzheimer’s disease (AD) is thought to affect about 5.3 million people in the United States and is currently ranked as the 6th leading cause of death. Costs of care for these patients are estimated at $226 billion and expected to rise to as much as $1.1 trillion by 2050.1 These figures clearly highlight the urgent need for therapies that will affect the course of the disease to prevent, or at least delay, the onset of cognitive decline. The two classes of drugs approved for AD treatment aim to alleviate the symptoms of the disease by modulating the action of neurotransmitters, either acetylcholine or glutamate. Standard of care therapy consists of an acetylcholinesterase inhibitor (donepezil, rivastigmine, or galantamine) and/or a partial N-methyl-D-aspartic acid (NMDA) receptor antagonist (memantine). Unfortunately, these symptomatic treatments do not show long term efficacy, nor do they change the course of disease progression. Despite decades of research and many failed clinical trials, the field has very little progress to show in terms of therapeutic approaches.2 The two characteristic neuropathological markers of AD are amyloid plaques, composed mainly of Aβ peptides, and neurofibrillary tangles, composed of hyperphosphorylated tau. The Aβ peptides which form the core of the amyloid plaques are produced by the sequential proteolytic cleavage of the amyloid precursor protein (APP) by beta amyloid cleavage enzyme 1 (BACE1) and gamma secretase (GS). The discovery that mutations in APP cause familial Alzheimer’s disease (FAD)3 led to the formulation of the amyloid hypothesis of AD.4-6 Originally, this hypothesis stated that amyloid deposits in the brain are the primary cause of the disease and that through a gain of toxic function, they instigate a cascade of events that drive the pathology. The hypothesis has been further refined over many years, and it is now believed that a form of soluble Aβ rather than the Aβ aggregates found in plaques, is responsible for neurotoxicity.7 The precise


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identity of the toxic form of Aβ and its kinetic dynamics with other Aβ forms are however still not clear. Importantly, additional support for the amyloid hypothesis comes from a recently reported APP mutation in an Icelandic population that provides a protective effect against AD by potentially reducing Aβ production by ~20%.8 It is however still unclear if Aβ drives the disease pathophysiology, if an Aβ threshold mediates toxicity, or if Aβ is merely a trigger for downstream events.9 The processing of APP follows this sequence: first, BACE1 cleaves APP to generate a 99 amino acid fragment (C99; Figure 1a) which then undergoes a series of cleavage events by GS to produce Aβ peptides of different lengths (Figure 1b), of which Aβ38, 40 and 42 are the most abundant in the cerebrospinal fluid (CSF).10, 11 The longer peptides, particularly Aβ42, have been shown to be most prone to aggregation with an increased Aβ42:Aβ40 ratio observed in the familial form of the disease.12 In 1999, the protein responsible for the proteolytic activity of gamma secretase, presenilin (PS), was formally identified.13, 14 Interestingly, mutations in both genes for PS-1 and PS-2 had been discovered in FAD populations and suggested to be causative of the disease,15, 16 further supporting the amyloid hypothesis.


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Figure 1. Processing of APP. a) In step 1, APP is processed by BACE1 which releases the soluble APPβ, leaving C99 (Aβ plus AICD) in the membrane. b) In step 2, GS releases AICD by cleaving C99 at the ε cleavage site (endopeptidase activity) followed by ζ and γ cleavages (carboxypeptidase activities) which further process the Aβ substrate into peptides of different lengths (see figure 2 for details).

Not surprisingly, the pharmaceutical industry became interested in developing drugs to prevent the production of toxic Aβ peptides in the hopes of reducing amyloid plaque buildup and thus, delaying the progression of AD symptoms. Therefore, considerable work has been performed to develop BACE inhibitors and gamma secretase inhibitors (GSIs). Several BACE inhibitors are presently under evaluation in the clinic and have been reviewed elsewhere.17-20 GSIs have been abandoned as a treatment for AD because of mechanism-based toxicities (weight loss, skin cancers, infections, etc.) exemplified in a Phase 3 clinical trial with semagacestat.21-23 These toxicities are believed to arise from the inhibition of the processing of other GS substrates such as Notch.


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The finding in 2001 by Weggen et al. that certain compounds could modify the activity of GS to produce less Aβ42 without affecting the total amount of Aβ produced or the processing of other GS substrates offered an additional therapeutic approach.24 These compounds were termed gamma secretase modulators (GSMs) and hypothesized to provide a safer alternative to GSIs for the reduction of toxic Aβ42 in the treatment of AD. In this review we will focus on the evolution of GSMs and the potential path for their improvement and development.

Gamma secretase – An unusual protease Gamma secretase, an aspartyl protease, is a member of the intramembrane cleaving protease family (I-CLiPs) that also includes signal peptide peptidases, rhomboid proteases and site2 peptidases. These enzymes have their active site within the lipid bilayer of cellular membranes and cleave their substrates through regulated intramembrane proteolysis (RIP). Gamma secretase itself is a complex composed of four different proteins: presenilin (PS), nicastrin (Nct), anterior pharynx-defective 1 (Aph-1) and presenilin-enhancer 2 (Pen-2) present in a 1:1:1:1 stoichiometry.25 In humans, PS is encoded by either the PSEN1 gene on chromosome 14 or the PSEN2 gene on chromosome 1, and mutations in both genes have been found to cause FAD.15, 16 Today, more than 180 mutations have been reported in PSEN1, making it the most common cause of early onset FAD.26 The products of these genes, namely PS-1 and PS-2 are 9 transmembrane domain (TMD) proteins that form the catalytic subunit of GS. PS undergoes endoproteolysis in an intracellular loop between TMD6 and TMD7 to produce the fragments that comprise its active form.27 The two aspartic acids forming the catalytic site are located in TMD6 and TMD7, and mutation of either one of these aspartic acids renders GS inactive.13 As for the other components of the GS complex,


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it is suggested that Nct is involved in maturation of the complex28 and binding of the substrate,29, 30 Aph-1 is involved in the assembly and activity of the complex31,

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and Pen-2 appears to be

important for activity,33 complex stability and trafficking.34 Most importantly, the four components need to be expressed together for maximal GS activity.35 The atomic structure of human GS has recently been solved at 3.4 Å resolution via single-particle cryo-electron microscopy.36 While this new 3D structure does not include a GSI or GSM, it provides a more thorough understanding of this enzyme on a molecular basis. GS cleaves type I transmembrane proteins, with over 90 reported substrates,37 earning it the nickname ‘proteasome of the membrane’.38 APP and Notch are the best characterized of these substrates. GS cleavage of APP is unusual in that the cleavage site is loosely defined. Additionally, GS harbors both endopeptidase and carboxypeptidase activities39, 40 which allow for generation of Aβ peptides of different lengths. After APP is cleaved by BACE (Figure 1a), GS catalyzes a series of cleavages within the transmembrane domain of the remaining fragment (C99), termed epsilon (ε), zeta (ζ) and gamma (γ) cleavages (Figure 1b). The ε cleavage releases the intracellular domain of APP (AICD) and produces Aβ49 or Aβ48.41 Then, in a stepwise manner, the carboxypeptidase cleavages ζ and γ take place to generate both Aβ40 and Aβ42.40, 42 In a series of elegant studies, Ihara and colleagues have shown two major lines of Aβ production: ε cleavage between Leu49 and Val50 of C99 preferentially produces Aβ40 and cleavage between Thr48 and Leu49 preferentially produces Aβ42.43, 44 They also showed that the successive cleavage events occur every 3-4 amino acid, consistent with the α-helical structure of the transmembrane domain of APP (3.6 amino acids/turn).45, 46 More recently, the tri- and tetra-peptide model has been refined to include other cleavage events that happen to a lesser extent, including those forming shorter Aβ peptides.47 These results suggest that GS performs 4 cycles to generate Aβ40 (49464340) and 3 cycles


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to generate Aβ42 (484542) (Figure 2a). Many FAD-causing mutations in PS have been found to decrease the catalytic activity of GS39, cleavage cycle (Figure 2b).39,

50

48, 49

with the most pronounced effect on the fourth

This loss-of-function contributes to the increased ratio of

Aβ42:Aβ40 observed in the familial form of the disease.51

Figure 2. Step-wise cleavage model for Aβ generation and potential effect of PS FAD mutations and GSMs. a) In a normal situation, C99 is sequentially cleaved by GS ε−,ζ- and γ-cleavages. The first cleavage cycle (ε) between amino


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acids 50/49 or 49/48 will determine the Aβ peptide production line (49464340 or 484542). b) Many PS mutations affect the fourth cleavage cycle of the complex leading to the increased Aβ42:Aβ40 ratio observed in FAD. c) GSMs increase the processivity of the complex leading to an increased production of the shorter, nonamyloidogenic peptides Aβ37 and Aβ38. Thickness of the arrows depicts the cleavage efficiency.

Pharmacological targeting of gamma secretase As mentioned earlier, the pharmaceutical industry became interested in targeting GS because of the genetic evidence suggesting a role for Aβ in the pathophysiology of AD. Two key GSIs, semagacestat and avagacesat, have been tested in late stage clinical trials for AD.21, 52 Given the large number of substrates cleaved by GS, it is not surprising that many side effects were observed following treatment with these GSIs. One of the main culprits thought to be responsible for the majority of side effects is Notch. Indeed, Notch is processed in a similar way as APP where the ε cleavage event releases the Notch intracellular domain (NICD) that then translocates to the nucleus to regulate gene expression and mediates important intercellular communication functions such as cellular differentiation.53 By blocking the ε cleavage step, GSIs impair Notch signaling which likely led to several adverse events such as skin cancer and/or decreased lymphocyte counts, which were both observed in the semagacestat Phase 3 trial.21 In that trial, semagacestat was also reported to worsen memory and, in addition to inhibition of Notch processing, it is possible that the accumulation of C99 in the membrane54 or the inhibition of EphA4 processing55 may have contributed to this effect. Alternatively, given its pharmacokinetic profile, the concentrationdependent change in Aβ produced by semagacestat56 might have been responsible for the apparent increase in the Aβ42:Aβ40 ratio21 observed in the study, which could have also precipitated the memory decline.23 While avagacestat claims a reported window of 137 to 193-fold selectivity for APP processing over Notch,57, 58 it demonstrated a similar side effect profile to semagacestat in the


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clinic.52 This result could be explained by an overestimation of the apparent selectivity between Notch and APP due to the use of different assays (reporter assay for Notch and Aβ production for APP) to measure compound potency. In fact, measurement of ε and γ cleavages in a single assay revealed the selectivity window to be no more than 3-fold.39, 59, 60

Figure 3. GSIs: Semagacestat and Avagacestat

Another way of targeting GS was discovered in 2001 when it was reported that a subset of non-steroidal anti-inflammatory drugs (NSAIDs) could specifically lower Aβ42 and increase Aβ38 by a mechanism independent of cyclooxygenase inhibition.24 Sulindac sulfide, indomethacin, ibuprofen, and Flurbiprofen were all shown to mediate this effect at extremely high concentrations (25-300 µM). Modulation of GS in this manner was especially promising given that Aβ42 is considered to be the most aggregation prone Aβ peptide and thus the most toxic. Changing the ratio of longer to shorter Aβ peptides has since been shown to prevent the aggregation of Aβ42 in vitro61 and plaque formation in vivo.62


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Figure 4. Early NSAID GSMs

Using this idea, NSAID derivatives were quickly developed with increased potency towards GS compared to the original NSAIDs.63,

64

These molecules, however, possessed poor

drug-like properties, requiring very high doses to achieve the appropriate unbound drug concentration at the target in the central nervous system (CNS). Proof-of-mechanism (POM) studies in man with these NSAID derivatives have been further complicated by dose-limiting toxicity of the molecules. Non-NSAID GSMs were later discovered and found to have a mechanism of action slightly different than the NSAID-like molecules. In addition to lowering Aβ42 and increasing Aβ38, these second generation compounds also increase Aβ37 and decrease Aβ40, albeit with lower potency.65 Many groups have investigated the mechanism by which GSMs exert their effect. Beher et al. were the first to report that NSAID-based GSMs are non-competitive inhibitors of Aβ42 production and non-competitive antagonists of transition state GSIs.66 This suggested the presence of an allosteric binding site that, when occupied, could change the conformation of the enzyme with specific effects on Aβ42 production. Recently, a more definitive binding site for GSMs has been uncovered via molecular photoprobes. Multiple groups have demonstrated that GSMs interact with the N-terminal fragment of presenilin (PS-NTF).67-73 However, different scaffolds appear to bind in different locations since imidazole-based GSMs have limited ability to displace NSAID


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GSMs and vice versa.69, 72 Indeed, it has been proposed that a photoprobe based on NSAID GSM-1 binds within the TMD1 of PS68 while an aryl imidazole GSM photoprobe based on ST1120 GSMs binds in the extracellular pocket formed by hydrophilic loop 1 (HL1)/TMD2/TMD5.73 Interestingly, occupation of the binding site appears to change the active site conformation in such a manner to modify the Aβ generation pattern by increasing shorter peptides and decreasing the longer ones. This change in the cleavage pattern could be explained by two possible mechanisms: a decreased probability of releasing longer Aβ from the enzyme–substrate complex defined by the dissociation constant (kb) or an increase in the processive cleavage activity defined by the catalytic constant (kcat) of GS. As discussed earlier, GS harbors both endo- and carboxypeptidase activities and performs multiple cleavage cycles to generate its product. Chavez-Gutierrez et al. showed that the kcat of the ε cleavage event was not affected by GSMs but was reduced by certain FAD-causing PS mutations.39 By using C99 as a substrate they also demonstrated that the ratios of Aβ40 to Aβ43 and Aβ38 to Aβ42 were decreased by all mutations studied, suggesting an impairment of the fourth cleavage cycle of GS (Figure 2b). Moreover, GSMs mostly increased the ratio of Aβ38 to Aβ42, again suggesting an effect on the fourth cleavage step. By using Aβ42 as a substrate, Okochi et al. measured the kinetic constants of the γ cleavage and reported that GSMs increased the kcat and decreased the kb of the γ cleavage, whereas FAD-causing PS mutations had the opposite effect (Figure 2c).49 Taken together, this evidence suggests that GSMs could reverse the effect of PS mutations to restore the normal balance of GS activity to treat the underlying disease pathology.50 While it has been challenging, there are now many examples of very potent GSMs in the literature targeting this unique intramembrane cleaving protease. Unfortunately these highly potent


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compounds also typically possess poor drug-like properties borne out in their high aromatic ring count or low fraction sp3 character (Fsp3 values),74 high molecular weight (MW), high lipophilicity (cLogP) and low multi-parameter optimization (MPO) scores.75 The CNS MPO score76 has been proposed by Pfizer researchers as a method to identify compounds with increased probability of success, that relies upon several commonly used physicochemical properties. A CNS MPO value is given for the alignment of six key physicochemical parameters (calculated octanol-water partition coefficient (cLogP), calculated distribution coefficient at pH = 7.4 (cLogD), molecular weight, topological polar surface area, number of hydrogen bond donors and the pKa of the most basic center). Each parameter provides a value between 0 and 1 to afford a possible summary score of 6, with 74% of marketed CNS drugs possessing a MPO score ≥ 4. Increasing the CNS MPO scores also appears to correlate with an improvement in a number of other in vitro properties (permeability, P-glycoprotein (P-gp) efflux, metabolic stability, and safety). Historical GSMs have been extensively reviewed elsewhere, thus we will particularly focus on compounds disclosed since 2011.63, 77 There are many hurdles remaining in this field to deliver safe, efficacious GSMs and it appears several research groups have subsequently moved on from pursuing GSMs in favor of more traditional targets. However, we believe endeavoring to persevere in the development of potent and efficacious GSMs that possess good drug-like properties will soon enable POM studies in human clinical trials.

Carboxylic-acid GSMs The first generation GSMs based on NSAID derivatives have been extensively reviewed.63, 78, 79

A few novel carboxylic acids (CHF507480 and EVP-001596281) have been investigated in

clinical trials. CHF5074 failed to demonstrate efficacy due to limited potency (EC50 = 40 µM),


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poor drug-like properties and extremely poor CNS penetration (B:P = 0.03-0.05).80,

82, 83

This

compound required very high doses to achieve the necessary efficacious unbound drug concentration in the CNS which was complicated by dose-limiting toxicity. Detailed results from the EVP-0015962 trial have yet to be reported. While it showed improved in vitro potency with reproducible animal efficacy, it suffers from the same poor drug-like properties as other NSAIDlike GSMs..

Figure 5. NSAID-based GSMs investigated in clinical trials

Alkenyl GSMs Recent non-carboxylic acid GSMs have been inspired by the work of the Eisai group and their discovery of very important GSMs in 2005.84 These compounds possess the key arylimidazole group now found in nearly all GSMs. Here, the arylimidazole is linked via an olefin to a lactam (Compound 1: E2012)85 or heterocycle (Compound 2: E2212 structure not disclosed) possessing a key hydrogen bond acceptor. Compound 1 was shown to be a potent early GSM (EC50 = 83 nM) and was the first non-carboxylic acid GSM to enter clinical trials in 2006. Compound 1 demonstrated lenticular opacity in a 13 week rat safety study which caused concerns


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for this class of GSMs. Recently, Eisai has established that 1 inhibits 24-dehydrocholesterol reductase, leading to increased desmosterol and decreased cholesterol in the lens.86 These sustained and prolonged sterol changes produce cataracts and can be monitored by preceding changes in the plasma. Follow-up studies up to the highest dose tolerated in monkeys for 13 weeks did not show ocular toxicity as the elevation of desmosterol in the plasma was very mild. While the Phase 1 studies with 1 demonstrated dose dependent plasma reductions of Aβ40/42,87 Eisai decided to develop their improved compound 2 instead. In a phase 1 clinical trial, 2 was demonstrated to be safe and well tolerated at single oral doses from 10 to 250 mg, the maximum dose studied.88 The pharmacodynamic response measured in plasma increased with the dose and was shown to be 54% Aβ42 reduction (AUC0-24h of 44%) at the 250 mg dose. While the structure of E2212 has never been disclosed, it has been speculated89 to be compound 2 based on several Eisai process chemistry patents.90-93 This very potent compound (EC50 = 17 nM) still possesses a centralized double bond, 4 aromatic rings, high MW (480), high cLogP (5.5) and a low MPO score (3.1). No further development of 2 has been reported to date, and there remained much need to optimize the drug-like properties after this initial effort to develop non-carboxylic acid GSMs.

Figure 6. Eisai GSMs investigated in clinical trials


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The Schering-Plough group described a series of oxadiazolines based on the structure of compound 1.94 A conformational constraint was engineered between the amide carbonyl and the αmethyl carbon via a cyclized hydroxy amidine (oxadiazoline) which maintains the potential hydrogen bond acceptor of the amide. Rewardingly, compound 3 had good potency in cells (EC50 = 58 nM). Oral dosing in rats at 3 and 10 mg/kg resulted in total brain exposures of 0.8 and 5 µM at 3 hours post dose and demonstrated CSF Aβ42 reductions of 30% and 51% respectively. Compound 3 demonstrated good ADME properties including no cytochrome P450 (CYPs) inhibition (3A4, 2D6 and 2C9), but a high cLogP (5.2) and moderate hERG inhibition (hERG IC50 = 740 nM in a voltage clamp cellular assay) led to considerable analog design. One of the more interesting and well characterized analogs was compound 4. Introduction of a hydroxyl group improves the potency (EC50 = 29 nM) with a lower cLogP (4.4 vs. 5.2) and also improves the potential hERG issue (hERG IC50 = 3.3 µM in a voltage clamp cellular assay). Dosing rats with compound 4 at 10 mg/kg p.o. resulted in total brain exposures of 0.54 µM at 3 hours post dose and demonstrated Aβ42 reductions in the CSF and cortex of 66% and 37% respectively. While this compound demonstrated both good potency and efficacy, it suffers from poor CNS drug-like properties as measured by a cLogP of 4.4 and a MPO score of 3.3.

Figure 7. Schering-Plough oxadiazoline GSMs


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A ring expansion of the oxadiazolines by the Schering group led to the discovery of the oxadiazine scaffold.94, 95 A trifluorophenyl was substituted onto either the C3 or C4 position of the fused oxadiazine to afford potent compounds with unfortunately high cLogPs (4.6) and low MPO scores (3.4). Substitution at the C3 position afforded compound 5 which demonstrated good in vitro potency (EC50 = 48 nM). Dosing rats at 10 mg/kg p.o. resulted in CSF Aβ42 reductions of 45% at 3 hours post dose. While the unbound brain concentration at 3 hours is not reported, pharmacokinetic (PK) assessment in rats demonstrated sustained exposures with 4 µM observed in the brain at 6 hours. Compound 5 did not demonstrate a CYP liability, but a potential hERG issue was identified (83% hERG inhibition in a functional assay at 10 µM). Substitution at the C4 position afforded compound 6 which also demonstrated good in vitro potency (EC50 = 33 nM). Dosing rats with compound 6 at 10 mg/kg p.o. resulted in robust CSF Aβ42 reductions of 62% at 3 hours post dose. Again, the unbound brain concentration at 3 hours was not reported, but PK assessment in rats demonstrated sustained exposures with 6.2 µM observed in the brain at 6 hours. Compound 6 also did not inhibit CYPs but still showed a potential hERG issue (76% hERG inhibition in a functional assay at 10 µM). Further investigation demonstrated there was no effect with either compound 5 or 6 on QTc intervals in dogs at sufficient exposure multiples over the required exposures for pharmacological effects. Compound 6 was further profiled in cynomologous monkeys to demonstrate a robust 70% and 44% Aβ42 reduction in CSF and cortex with approximately 8.4 µM in the brain at 4 hours following a 30 mg/kg dose. While this compound demonstrated good potency, efficacy, and initial safety relative to both CYPs and hERG, follow up studies have not yet been reported. However, this compound still suffers from poor drug-like properties as measured by a cLogP of 4.6 and a MPO score of 3.4.


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Figure 8. Schering-Plough oxadiazine GSMs

Further ring expansion by the Schering group led to the discovery of the azepane oxadiazine 7 which demonstrated 2-3 fold weaker in vitro potency than the related oxadiazine 6 (EC50 = 33 vs 88 nM) while also increasing the cLogP (5.0) and decreasing the MPO score (3.1).96 Another ring expansion led to the discovery of the oxadiazepine 8 which demonstrated better in vitro potency (EC50 = 39 nM) and slightly better cLogP (4.6) and MPO score (3.3). Dosing rats with compound 8 at 3, 10 and 30 mg/kg p.o. resulted in dose dependent reductions of Aβ42 in the CSF by 43, 61 and 78% at 3 hours post dose. Unfortunately, the unbound brain concentration at 3 hours is not reported, but PK assessment in rats demonstrated sustained exposures with 3.7 µM observed in the brain at 6 hours. Compound 8 also did not inhibit CYPs and demonstrated a reduced potential hERG issue (33% inhibition of hERG in a functional assay at 10 µM). Once again, this compound demonstrated good potency, efficacy, and safety relative to CYPs and hERG, but follow up studies have not yet been reported. Like other analogs in this series, this compound still suffers from poor drug-like properties as measured by the high cLogP of 4.6 and low MPO score of 3.3.


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Figure 9. Schering-Plough oxadiazine and oxadiazepine GSMs

Anilines In order to remove the potential liability of the olefin linker in compound 1, many groups have replaced the olefin with a nitrogen linker.97 The Janssen group has described a series of Nlinked bicyclic heterocycles designed to mimic the structure of 1.98 Compound 9 was designed to contain an intramolecular hydrogen bond between the aniline and amide carbonyl along with a steric clash between the carbonyl and the α-methyl carbon to pre-organize the molecule into the proposed bioactive conformation. Unfortunately, compound 9 was determined to have an EC50 of 550 nM (~7 times weaker than 1) and did not demonstrate any in vivo efficacy at a dose of 30 mg/kg in mice. To further improve these designs, compound 10 was engineered with a conformational constraint between the same amide carbonyl and the α-methyl carbon. Compound 10 led to a further reduction in potency (EC50 = 813 nM) and only a modest Aβ42 reduction of 12% in mouse brain when dosed p.o. at 30 mg/kg. The regioisomeric compound 11 was ~5 times more potent (EC50 = 182 nM) and demonstrated a more pronounced Aβ42 reduction (35%) in mouse brain following a 30 mg/kg oral dose. Several analogs related to compound 11 showed


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improved potency and demonstrated 30-40% Aβ42 reduction in mouse brain. While compound 11 possesses a respectable cLogP (3.6) and MPO score (4.1), the in vitro potency and associated brain exposure (total and/or unbound) for this class of compounds do not correlate with in vivo efficacy. For most compounds in this class, fu,brain was less than 0.1%, and many compounds demonstrated in vivo efficacy at estimated unbound brain concentration much lower than their respective in vitro Aβ42 EC50. These confounding results, seemingly inconsistent with the free drug principle, have led many research groups to discontinue their GSM programs in favor of more traditional targets. Furthermore, many of the analogs in this class also demonstrated low solubility, CYP inhibition and potential hERG liabilities. This class of compounds suffers from poor drug-like properties as measured by the number of aromatic rings, high cLogP, and low MPO score.

Figure 10. Janssen aniline GSMs

The Janssen group investigated other bicyclic heterocycles replacing the central core of these molecules. Benzimidazole 12 led to a further improvement in potency (EC50 = 17 nM) and


19


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demonstrated a robust 43% Aβ42 reduction in mouse brain when dosed p.o. at 30 mg/kg. Similarly, benzimidazole 13 demonstrated good potency and efficacy (EC50 = 43 nM; 45% Aβ42 reduction in mouse brain at 30 mg/kg p.o.). Of note, an analog of 12 with the benzimidazole free N-H (compound not shown) was 12x less potent (EC50 = 203 nM). Indazole 14 with R = H was moderately potent (EC50 = 131 nM), but compound 15 with R = Me demonstrated high potency (EC50 = 18 nM) and a robust 41% Aβ42 reduction in mouse brain following oral treatment at 30 mg/kg. Unfortunately, the benzoxazoles, which cannot incorporate the added methyl group pushing the aromatic group out of the plane via steric clash, possess only limited potency as evidenced by compound 16 (EC50 = 5.3 µM). Again, many of these analogs also demonstrated low solubility, CYP inhibition and potential hERG liabilities. This class of compounds also possesses poor drug-like properties as measured by the number of aromatic rings (Fsp3 values as low as 0.08), higher cLogPs, and lower MPO scores.

Figure 11. Janssen aniline GSMs


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Further optimization of the series by the Janssen group, with an effort to improve the druglike properties by changing the central aromatic ring to a pyridine, afforded compound 17 which maintained high potency (EC50 = 16 nM) and demonstrated a robust 62% Aβ42 reduction in mouse brain when dosed p.o. at 30 mg/kg. This compound also showed improved in vitro ADME properties with only moderate CYP inhibition observed (low micromolar for 3A4, 2D6 and 2C19) and minimal hERG channel binding (IC50 = 8.6 µM). Compound 17 was also shown to be highly selective in a CEREP panel and negative in both Ames and GreenScreen genotoxicity assays. Unfortunately in subsequent studies, signs of liver toxicity, not uncommon for highly planar, lipophilic compounds,74 were observed in dogs dosed with 20 mg/kg of compound 17, halting all further development. Again, this compound suffers from poor drug-like properties as measured by the number of aromatic rings (Fsp3 = 0.13), cLogP (4.3), and MPO score (3.5).

Figure 12. Optimized Janssen aniline GSM


21


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Three Boehringer Ingelheim patents describe a series of aniline linked pyrimidine compounds with fused 5 or 6-membered rings.99-101 Compound 18 is the most potent from a series of thienopyrimidines and only demonstrated a modest EC50 of 90 nM in H4 cells. A series of dihydropteridinones, such as compounds 19 and 20, are interesting due to their good predicted physicochemical properties and improved potency (22 nM and 46 nM respectively). While there appears to be an overall improvement in reducing the cLogP and increasing the MPO score for compound 19 and 20, no ADME or in vivo efficacy data have been provided for these compounds.

Figure 13. Boehringer Ingelheim pyrimidine GSMs

Three recent FORUM Pharmaceuticals patent applications describe a series of aniline linked pyrimidomorpholines. In the first application, compound 21 represents the basic structure and possesses good potency (EC50 = 39 nM) with a low cLogP (2.7) and good MPO score (4.8).102 Introduction of fluorine atoms on the phenyl ring affords compound 22 with improved potency (EC50 = 14 nM), a good cLogP value (3.0) and still good MPO score (4.5). While other substituents on the aromatic ring maintain or improve the potency, these new analogs also possess higher cLogPs and lower MPO scores. Compound 23 moves away from the N-aryl group with the


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introduction of a trifluoroethyl to nearly double the sp3 character (21: Fsp3 = 0.17 vs. 23: Fsp3 = 0.32). Compound 23 demonstrates modest potency (EC50 = 101 nM) with a low cLogP (2.5) and even better MPO score (4.9). While no in vivo efficacy is reported in this patent application, several potent compounds possessing higher sp3 character, lower cLogPs and higher MPO scores have been identified within this scaffold.

Figure 14. FORUM N-substituted pyrimidomorpholine GSMs

To further expand upon these compounds and increase the sp3 character, the FORUM group has focused on the aniline portion of the scaffold.103 Here compound 24 represents the basic structure and possesses good potency (EC50 = 25 nM) with a good cLogP (3.3), good MPO score (4.4) and high sp3 character (Fsp3 = 0.35). Again, other substituents on the phenyl ring maintain or improve the potency, but these new analogs also possess higher cLogPs and lower MPO scores. Interestingly, compound 25 utilizes a tetrahydropyran as a non-aromatic replacement in this region of the molecule that maintains high sp3 character (Fsp3 = 0.35). This design motif has not previously been described in this region of a GSM and possesses good potency (EC50 = 41 nM), but also leads to increasing cLogP (3.9) and decreasing MPO score (3.7). Compound 26 also


23


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moves away from the N-aryl group with the introduction of a trifluoroethyl which increases the sp3 character to over half the atoms in the compound (Fsp3 = 0.53). Compound 26 demonstrates modest potency (EC50 = 105 nM) with low cLogP (2.5) and an excellent MPO score (4.8). While there appears to be a significant improvement in the sp3 character of these GSMs, the challenge remains to identify compounds with excellent potency, low cLogP values and high MPO scores.

Figure 15. FORUM N-substituted pyrimidomorpholines with increased sp3 character

In a third patent application, the FORUM group has investigated the vector of the aryl group via substitution on the C7 of the pyrimidomorpholine core structure.104 Compound 27 represents the basic structure and possesses increased potency (EC50 = 16 nM) with a respectable cLogP value (3.4) and good MPO score (4.3). Again, other substituents on the phenyl group maintain or improve the potency, but these new analogs also possess higher cLogPs and lower MPO scores. In an effort to improve the drug-like properties by removing one of the aromatic rings (increases the sp3 character, lowers the molecule weight, lowers the cLogP and increases the MPO score), several non-aromatic right hand side groups have been explored. The spirocyclopropyl


24

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analog 28 possesses good potency (EC50 = 49 nM) with a low cLogP (1.6), excellent MPO score (5.4) and increased Fsp3 (0.35). The fused pyrrolidine analog 29 also possesses good potency (EC50 = 71 nM) with a low cLogP (1.9), excellent MPO score (5.4) and high Fsp3 (0.35). While there appears to be a significant improvement in this GSM scaffold with respect to high sp3 character, lower molecular weights, lower lipophilicity and higher MPO scores, the challenge remains to combine these good drug-like properties with excellent potency.

Figure 16. FORUM C-substituted pyrimidomorpholines with increased sp3 character

The Bristol-Meyers Squibb group has described a series of compounds based on the optimization of compound 30 (EC50 = 29 nM). One of their lead compounds, 31 (BMS-869780),105 has been described as their first optimized candidate. This GSM replaces the typical methyl imidazole with a chloroimidazole. Compound 31 possesses excellent in vitro potency (EC50 = 6 nM) but a high cLogP (4.4) and a low MPO score (3.3). Compound 31 demonstrated robust in vivo efficacy in both mouse and rat brain. In mice, 30 and 100 mg/kg oral doses produced Aβ42 AUC024h

reductions of 31 and 55% respectively and a 10 mg/kg oral dose in rats produced an Aβ42

AUC0-24h reduction of 47%. Safety studies eventually revealed activity in the human PXR (pregnane X receptor) transcriptional reporter assay at all concentrations greater than 0.3 ߤM.


25


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Further experiments confirmed the induction of CYP3A4 and the potential risk of drug-drug interactions. Finally, liver lipidosis was observed in rats after four daily doses each of 10, 30 and 100 mg/kg, indicating hepatotoxicity at the exposures required for Aβ42 lowering. Further studies with 31 have been discontinued due to potential safety concerns at the projected human daily dose (700 mg) required to afford a desired Aβ42 AUC0-24h reduction of 25%. Efforts to optimize solubility, hERG, CYP3A4 and PXR afforded compound 32 (BMS-932481).106,

107

This GSM

deviates from the typical methyl imidazole with the inclusion of the methyl triazole. Compound 32 demonstrates excellent potency (EC50 = 7 nM) and very robust in vivo activity. A phase 1 clinical trial with 32 reportedly demonstrated reductions in CSF Aβ42 with a single dose of 900 mg.108 While this compound apparently demonstrated efficacy, it still suffers from poor drug-like properties as measured by both high cLogP (4.5) and low MPO score (3.0).

Figure 17. Bristol-Meyers Squibb aniline GSMs

The Bristol-Meyers Squibb group has also recently described a series of tricyclics designed to further optimize compound 30 via a conformational restriction linking the key aniline to the central heterocycle.109 The first set of compounds explored a thiazole ring system to form the key


26

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tricyclic. Compound 33 possessed similar potency (EC50 = 29 nM) but at the expense of a higher cLogP (4.4 vs. 3.7) relative to compound 30. Unfortunately, compound 34 demonstrated lower potency (EC50 = 140 nM) with even worse calculated properties. Compound 33 was further profiled to show good metabolic stability and no appreciable hERG activity, but significant CYP3A4 inhibition (IC50 = 0.7 µM) was identified as a potential issue. Dosing compound 33 in 3xTg-AD mice at 30 and 100 mg/kg p.o. resulted in brain Aβ42 reductions of 40% and 50% respectively at 3 hours post dose. The 30 mg/kg dose afforded 8 µM and 8.9 µM exposures in the plasma and brain respectively at 3 hours and the 100 mg/kg dose afforded 25 µM in the plasma at 3 hours. The measured human and mouse plasma protein binding was extremely high (>99.8%), thus the associated unbound brain concentrations in the 30 mg/kg dose that affords 40% Aβ42 reduction is less than 18 nM (assuming equal brain and plasma binding, whereas the brain binding is often higher). Here, the calculated unbound brain exposure is roughly 2x lower than the in vitro EC50 for Aβ42, producing an apparent disconnect with the in vitro – in vivo correlation as is often observed for this target.

Figure 18. Bristol-Meyers Squibb tricyclic thiazole GSMs


27


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The Bristol-Meyers Squibb team then explored both pyrazole and imidazole tricyclic designs. While pyrazole compound 35 was equipotent to the thiazole (EC50 = 40 nM), both the CYP3A4 inhibition (IC50 = 0.3 µM) and the CNS drug-like properties as measured by the cLogP (4.6) and MPO score (3.6) were worse. Replacing the pyrazole with an imidazole and the central aniline with a ketone afforded compound 36 with significantly lower cLogP (2.9) and better MPO score (5.0). Unfortunately, compound 36 also possessed much lower potency (EC50 = 1.1 µM) and still demonstrated CYP3A4 inhibition (IC50 = 0.3 - 0.5 µM).

Figure 19. Bristol-Meyers Squibb tricyclic pyrazole and imidazole GSMs

To improve the potency and potentially address the CYP3A4 inhibition issue, a series of tricyclic diazepinones were also designed. Compound 37 was demonstrated to be equipotent to thiazole 33 (EC50 = 50 nM), but still exhibited CYP3A4 inhibition (IC50 = 0.15 µM). Rewardingly, introduction of the chloroimidazole in compound 38 resulted in a moderately potent analog (EC50 = 190 nM) and dramatically improved the problematic CYP3A4 inhibition (IC50 > 13 µM). The data suggest the CYP3A4 inhibition closely tracks with the basicity of the imidazole –the basic methyl imidazole is a substantially more potent inhibitor than the weakly basic chloroimidazole. Unfortunately compound 37 did not demonstrate any Aβ42 reduction when tested in 3xTg-AD


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mice at an oral dose of 30 mg/kg. These various tricyclic compounds provide new avenues for structure activity relationship (SAR) exploration but have not yet delivered potent, efficacious compounds without ADMET issues and good predicted CNS drug-like properties.

Figure 20. Bristol-Meyers Squibb tricyclic pyridine GSMs

Researchers at Amgen carried out a high throughput screen (HTS) for modulators of Aβ42 production in a HEK293 cell line stably expressing guinea pig Swedish mutant SFV-APP695sw. This screen identified oxazole 39 as a novel GSM chemotype.110 While the imidazole-based GSMs nearly universally require a methoxy group on the aniline for potency, this same methoxy substitution resulted in a loss in potency for this series. Optimization of 39 ultimately led to compound 40. Replacement of the oxazole with a methyl pyridine improved the potency and reduced the CYP inhibition. Conversion of the sulfonamide to a carboxamide improved brain exposure, although this modification also required the introduction of a methylene spacer between the carbonyl and terminal aryl group to maintain potency. Compound 40 (EC50 = 0.26 µM) is 6 times more potent than compound 39, but also significantly more lipophilic (cLogP = 5.9). While compound 40 demonstrated 33% Aβ42 reduction in rat brain at a dose of 100 mg/kg, other


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compounds in this series with increased in vitro potency failed to demonstrate improved in vivo efficacy. The authors claim various issues with this scaffold, including high cLogPs, low passive membrane permeability and “flat,” “shallow”, “non-additive” SAR.

Figure 21. Amgen aniline GSMs

Removal of the Aniline The Torrey Pines Therapeutics group also focused early on the arylimidazole group.111 Compound 41 was identified via a HTS screening campaign of 80,000 compounds and determined to possess low micromolar activity (EC50 = 1.5 µM). Further optimization afforded compound 42 with greatly improved potency (EC50 = 29 nM) that demonstrated 30-40% Aβ42 brain reductions after 3 days of repeated oral doses of 50 and 100 mg/kg in Tg2576 female mice.112 While compound 42 represents a significant potency improvement upon the initial HTS hit, it still suffers from poor drug-like properties as measured by the 3 contiguous aromatic rings, high cLogP (6.0) and low MPO score (3.2).


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Figure 22. Torrey Pines early GSMs

Wagner et al. have worked to improve the properties of these early thiazole GSMs by addition of heteroatoms to lower the cLogP and improve the solubility.113 The introduction of an N-ethyl pyrazole ring afforded compound 43 which demonstrated moderate potency (EC50 = 347 nM) and reduced lipophilicity (cLogP = 3.7). The kinetic solubility of compound 43 was measured as 2.3 µM at pH 6.6. A pyridine was then introduced to afford compound 44 (cLogP = 3.1) which demonstrates only weak in vitro potency (EC50 = 1.3 µM).. Combination of the pyridine and a methoxy provided compound 45, the most potent compound reported in this effort. This analog demonstrated good in vitro potency (EC50 = 30 nM) with a cLogP of 3.3 and a calculated MPO score of 4.3. Unfortunately, no kinetic solubility measurement is provided to directly compare this improved compound with the others in the series. Compound 45 was shown to decrease Aβ42 levels and increase Aβ33, Aβ34, Aβ37 and Aβ38 peptides in vitro. The potential for this class of GSMs remains to be seen since no efficacy or ADMET data has been published. While compound 45 still has 4 aromatic rings, it does appear to possess better drug-like properties as measured by its cLogP and MPO score. A related set of compounds in a recent patent application further engineered 5 and 6-membered non-aromatic rings to provide a series of compounds represented by


31


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compound 46.114 Compound 46 demonstrated excellent potency (EC50 = 6 nM) with good predicted drug-like properties as evidenced by a cLogP of 3.3 and an MPO score of 4.5. The ADMET properties and in vivo efficacy for this class of compounds also remain to be seen.

S N

S NH N N

N N

N

N N

N

N A

43 EC 42 50 = 347 nM MW = 405 cLogP = 3.7 MPO = 4.0

A

44 EC 42 50 = 1.3 M MW = 406 cLogP = 3.1 MPO = 4.6

S

S MeO

N

N

NH

MeO N N

N N

NH

N

N N

45 A 42 EC50 = 30 nM MW = 436 cLogP = 3.3 MPO = 4.3

N

A

N N N

46 EC 42 50 = 6 nM MW = 447 cLogP = 3.3 MPO = 4.5

Figure 23. Improvements to the early thiazole GSMs

The Merck group has described a series of triazole-based GSMs. Their recent efforts have focused on converting triazole 47 into triazoloamides 48 to increase the potency while reducing the potential hERG liabilities.115, 116 The optimized amide 49 possesses moderate drug-like properties as measured by the cLogP (3.7) and MPO score (3.8), modest cellular potency (EC50 = 90 nM), but demonstrated a potential hERG liability (IC50 = 0.8 µM). Various conformational constraints led to the triazololactams 50 and 51. Rewardingly, compound 50 possesses even better drug-like


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properties, better cellular potency (EC50 = 20 nM) and very low hERG activity (IC50 = 32 µM). Compound 51 also possesses good predicted CNS drug-like properties and excellent cellular potency (EC50 = 9 nM) with low hERG activity (IC50 = 5.4 µM). Several compounds in this series were dosed at 50 mg/kg p.o. but demonstrated no efficacy due to minimal brain exposures and B:P ratios less than 0.1. Clearly this GSM scaffold combines good potency with acceptable predicted drug-like properties, but a real need to improve the PK properties remains.

Figure 24. Merck triazole GSMs

Towards the goal of improving the PK properties, a variety of substitutions were explored by the Merck team with the optimized 6-fluoro benzazepinone.117,


118

Optimized compound 52 33


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demonstrated good cellular potency (EC50 = 58 nM), no potential hERG issue as measured in a binding assay (IC50 = 8.3 µM) and an improved B:P of 1. Dosing compound 52 in YAC-APP mice at 100 mg/kg p.o. resulted in robust brain Aβ42 reduction of 74% at 6 hours post dose with 14 µM central exposure. The methoxy analog 53 demonstrated even better cellular potency (EC50 = 13 nM), no potential hERG issue as measured in a binding assay (IC50 = 6.5 µM) and a B:P of 0.65. Dosing compound 53 in YAC-APP mice at 100 mg/kg p.o. resulted in brain Aβ42 reduction of 68% at 6 hours post dose with 8.5 µM central exposure. The methyl triazole analog 54 also demonstrated good cellular potency (EC50 = 42 nM), no potential hERG issue as measured in a binding assay (IC50 = 19 µM), and an improved B:P of 1. Dosing compound 54 in YAC-APP mice now at 50 mg/kg p.o. resulted in brain Aβ42 reduction of 38% at 6 hours post dose with 4.5 µM central exposure. Reverting back to the methyl imidazole provided compound 55, which also possessed good cellular potency (EC50 = 31 nM), but a now potential hERG issue as measured in a binding assay (IC50 = 1.9 µM). Follow-up functional hERG assay in a CHO cell line demonstrated a significantly reduced hERG liability (EC50 = 13 µM). Dosing compound 55 in YAC-APP mice now at 30 mg/kg p.o. resulted in brain Aβ42 reductions of 30% at 6 hours post dose with 1.6 µM central exposure and a measured B:P of 0.31. Given the observed Aβ42 reduction at limited exposure in mice, dosing 55 in rats at 100 mg/kg was explored and resulted in robust brain Aβ42 reduction of 92% at 6 hours post dose with 35 µM central exposure. Based on these promising results, the efficacy of compound 55 was further explored in non-human primates. Unfortunately, this compound displayed no oral bioavailability in rhesus monkeys so an alternative dosing strategy had to be undertaken. Dosing via i.v. infusion (6 mg/kg bolus followed by 44 mg/kg infusion over 4 hr) demonstrated significant Aβ42 lowering in rhesus CSF – up to 90% with sustained Aβ42 reductions over a 20 hour period. Upon further profiling, compound 55 was


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determined to possess significant activity at the serotonin re-uptake transporter (IC50 = 32 nM), so this off-target activity will need to be monitored for future compounds from this class.

Figure 25. Merck triazole GSMs with improved DMPK properties

The Takeda group has described a series of piperazine ureas and amides which demonstrate both in vitro potency and in vivo efficacy.119 Here, the in vitro potency is reported as an inflection point (IP) and maximum inhibition at 3 µM because the compounds only partially modulate Aβ42 production in the primary rat cortical neuron assay. These compounds, similar to NSAID-like GSMs, do not modulate Aβ40 production. The initial compound 56 demonstrates an IP of 56 nM with a maximum inhibition of 57% at 3 µM. Dosing mice at 100 mg/kg resulted in only minimal reductions of Aβ42 levels in the plasma (16%) and brain (4%) at 3 hours post dose. Compound 56 also possesses high in vitro metabolic clearance (150 µL/min/mg) as measured in mouse hepatic


35


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microsomes. SAR exploration of the methyl imidazole led to the identification of oxazole 57 which demonstrates an improved IP of 34 nM, a maximum inhibition of 67% at 3 µM but a similar in vitro metabolic clearance (130 µL/min/mg). Incorporation of a methyl group onto the piperazine linker and replacement of the α-methyl naphthalene with a trifluoromethyl benzimidazole led to the improved compound 58. Compound 58 demonstrated an improved IP of 21 nM, a maximum inhibition of 60% at 3 µM and a significantly improved in vitro metabolic clearance (67 µL/min/mg). Rewardingly, dosing mice with compound 58 at 100 mg/kg p.o. resulted in Aβ42 reductions in the plasma and brain by 64% and 59% respectively at 3 hours post dose. The potential for this class of GSMs remains to be seen, and while the MW is still high (514), compound 58 appears to possess improved drug-like properties as measured by the cLogP and MPO score.

O N N

O N H

N N O

N N

N H

OMe 56 A 42 IP = 56 nM (57%) MW = 470 cLogP = 3.7 MPO = 3.7

OMe 57 A 42 IP = 34 nM (67%) MW = 471 cLogP = 3.7 MPO = 3.7

N

O N

N

N CF3

N O N

OMe

A

42

58 IP = 21 nM (60%) MW = 514 cLogP = 3.1 MPO = 4.4

Figure 26. Takeda piperazine GSMs


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Researchers at Dainippon Sumitomo designed and synthesized amides based on 1 with the goal of removing the cinnamide olefin.120 A series of cyclic amines were coupled with 3-methoxy4-imidazole phenyl carboxylic acid to afford a library of 200 compounds. From this initial library, the aminopiperidine motif 59 was identified. Further optimization of the aminopiperidine led to compound 60 which produced a 97% reduction of Aβ42 at 2.5 µM in rat primary cortical neurons, while the enantiomer only showed 55% reduction in the same assay. Compound 60 demonstrated a 55% Aβ42 reduction in wild-type mouse hippocampus with an oral dose of 100 mg/kg. Further work from the Dainippon Sumitomo group in this class of molecules remains to be seen.

Figure 27. Dainippon Sumitomo piperidine amide GSMs

The Janssen group has also described a series of bicyclic heterocycles designed from the simple amide 61 – a weakly active (EC50 = 2.6 µM), low MW, low lipophilicity, high MPO starting point.121 To increase the potency and brain penetration potential, the amide was cyclized into triazole 62. While this led to a two-fold increase in potency (EC50 = 1.3 µM), it was proposed that the remaining N-H hydrogen bond donor might limit brain penetration. Furthermore, this compound possesses 4 contiguous aromatic rings with very low sp3 character (Fsp3 = 0.11). The methylated triazole compound 63 demonstrated a two-fold improvement in potency (EC50 = 0.62


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µM).122 Based on the hypothesis that the fluorophenyl should not be co-planar with the triazole, a methylene spacer was introduced to provide compound 64. While this did not afford an improvement in potency (EC50 = 0.85 µM), it provided the design concept for compound 65 which demonstrated a significant increase in potency (EC50 = 0.22 µM) and improvement in the sp3 character (Fsp3 = 0.26). Unfortunately, the change also led to an increase in cLogP and considerable lowering of the MPO score.

Figure 28. Janssen triazole GSMs


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The Janssen group also investigated substituents on the phenyl ring and heteroatoms in the saturated carbocycle.121 For these compounds, the absolute configuration was determined using vibrational circular dichroism. Ortho substituents on the aromatic ring provided a significant boost in potency as highlighted by compound 66 (EC50 = 24 nM) which demonstrated a 46% Aβ42 reduction in rat brain at 4 hours following a 30 mg/kg oral dose. Unfortunately, this compound was shown to be a highly potent CYP3A4 inhibitor (IC50 = 0.1 µM). Replacement of the CF3 with an OCF3 and incorporation of a pyridine provided the very potent and efficacious compound 67. This compound was shown to have an EC50 of 28 nM and a robust 66% Aβ42 reduction in rat brain 4 hours following a 30 mg/kg oral dose. Furthermore, this compound had no activity against CYP3A4 (IC50 > 10 µM). Compound 67 was further profiled in dogs to demonstrate a 50% Aβ42 reduction in CSF following an oral dose of 20 mg/kg. It was also shown to have no effects on liver function in the canine model, a clear improvement over some of Janssen’s earlier compounds. While the in vivo efficacy and the initial safety data look promising, these compounds still possess relatively high cLogP values and low MPO scores. Further work from the Janssen group in this class of compounds remains to be seen.


39


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Figure 29. Janssen triazole GSMs with improved properties

The Janssen team continued to explore this series of compounds with incorporation of a cyanoindole. Key compounds in the patent application appear to be ortho-phenyl substituted compounds 68–70.123 While the stereochemistry is undefined, the enantiomer demonstrating a negative optical rotation in DMF is preferred. Compound 68 is both very potent and efficacious demonstrating an EC50 of 20 nM and a robust 66% Aβ42 reduction and 40% increase of Aβ38 in mouse brain 4 hours following a 30 mg/kg oral dose. This compound was also recently highlighted to have no effects on liver function in follow up safety studies.124 Compound 69 demonstrated an EC50 of 21 nM and robust 73% Aβ42 reduction, but only a modest 10% increase of Aβ38 in mouse brain 4 hours following a 30 mg/kg oral dose. Compound 70 is also both potent and efficacious demonstrating an EC50 of 45 nM and robust 57% Aβ42 reduction and 41% increase of Aβ38 in mouse brain 4 hours following a 30 mg/kg oral dose. While the potency, in vivo efficacy and initial safety data look promising, these compounds still possess relatively high cLogPs and low MPO scores again demonstrating the difficulty obtaining compounds with good predicted CNS drug-like properties.


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Figure 30. Janssen cyanoindole GSMs

The Takeda group has also published a series of related compounds incorporating an oxazole in place of the typical methyl imidazole.125 In comparison with the data from Janssen, the oxazole 71 appears to be less active than imidazole 66 (EC50 = 0.38 µM) with a slight improvement in the drug-like properties as measured by the cLogP and MPO score. Insertion of an ether to form a triazolooxazine core (compound not shown but analogous to 67) leads to a ~2.5fold reduction in potency. Optimization of the aryl group afforded more potent compound 72 which was shown to have an EC50 of 60 nM at the expense of a higher cLogP and lower MPO score. Dosing mice at 10 mg/kg p.o. resulted in Aβ42 reductions in the plasma and brain by 69% and 50% respectively at 3 hours post dose with exposures of 13.2 and 4.8 µM measured in plasma and brain respectively. Unfortunately, like many GSMs, compound 72 does not appear to possess good CNS drug-like properties as measured by higher a cLogP value (4.3) and a lower MPO score (3.4).


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Figure 31. Takeda oxazole GSMs

Pfizer has reported their efforts to generate GSMs with improved properties relative to 1 by removing the cinnamide olefin and reducing the lipophilicity. They were able to achieve these aims by attaching an amide directly to the methoxyphenyl ring to afford compound 73, which showed an improved clogP (2.8) and modest in vitro potency (EC50 = 0.32 µM).126 Various amide groups were tolerated and further optimization led to dihydrobenzofuran compound 74 (EC50 = 0.19 µM). Compound 74 possesses good drug-like properties as measured by MW (403), cLogP (1.9) and MPO score (5.2). It demonstrated 36% Aβ42 lowering in guinea pig brain with a 100 mg/kg oral dose affording 89 nM unbound brain exposure at 4 hours post dose. However, efforts to further improve the potency of this series proved unsuccessful with CYP inhibition issues encountered for the more potent analogs.


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Figure 32. Pfizer amide GSMs

To improve on these compounds, the Pfizer group designed a series of pyridopyrazine-1,6diones in which the amide nitrogen was tied back onto the pyridine ring to generate a bicyclic core.127 These compounds demonstrate good physicochemical properties and improved in vitro potency. Various groups could be incorporated into the right hand side of the molecule to generate potent analogs. For example, ether linked analog 75 demonstrated an EC50 of 100 nM with low rat and human liver microsome clearance as well as minimal CYP inhibition. Compound 75 also demonstrated good bioavailability (88%) and achieved 2.1 µM brain exposure at 1 hour following an oral dose of 5 mg/kg in rats. Compound 75 exhibited a dose-dependent reduction of Aβ42 in guinea pig brain – with an observed reduction of 45% at 4 hours post 30 mg/kg p.o. dose. Even more potent compounds were identified by incorporating an indole in compound 76 (EC50 = 6 nM).128 While this compound had acceptable apparent intrinsic clearance (28.3 mL/min/kg) in human liver microsomes, oral dosing in rats at 30 mg/kg only resulted in 21% reduction of brain Aβ42 at 4 hours post dose. This limited reduction can be attributed to extremely low unbound compound exposure in rat brain (5 nM). One reason for the low brain exposure was the measured Cb,u/Cp,u of 0.2. The relative brain to plasma exposure was restored in P-gp knockout mice, indicating the compound was a P-gp efflux substrate. Further optimization of compound 75


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produced compound 77 with the stereochemistry of the methyl group adjacent to the trifluoromethyl established by X-ray crystallography.129 This compound was the first Pfizer compound with good physicochemical properties reported to have excellent potency (EC50 = 6 nM) along with low clearance in human liver microsomes (12.7 mL/min/kg) and low potential for multidrug resistance protein efflux (ratio = 1.4). Dosing compound 77 at 60 mg/kg p.o. in guinea pigs produced a 41% brain Aβ42 reduction with 225 nM unbound brain exposure at 3 hours post dose. The success of this compound has been attributed to maintaining good sp3 character (Fsp3 = 0.38) and the ability of fluorine to prevent metabolism of the pendant phenyl ring while also maintaining good physicochemical properties.129 Clickable photoaffinity probes related to compound 75 and 76 have demonstrated these pyridopyrazine-1,6-diones bind to the PS1-NTF.128


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Figure 33. Pfizer pyridopyrazine-1,6-dione GSMs

In a related patent application, researchers at Pfizer report engineering conformational restrictions in the alkyl chain via 4, 5, and 6-membered rings.130 Several compounds in this patent demonstrate high potency (EC50 < 20 nM) with good predicted drug-like properties. Compound 78 demonstrates high in vitro potency (EC50 = 14 nM) with a MW of 486, cLogP of 2.8 and a calculated MPO score of 4.7. The slightly more lipophilic compound 79 possesses even better in vitro potency (EC50 = 9 nM) albeit at the expense of a higher MW (521), higher cLogP (3.4) and slightly lower calculated MPO score (4.1). Adding a single fluorine atom onto compound 79 provides compound 80 which also demonstrates excellent potency (EC50 = 8 nM) with a MW of 538, cLogP of 3.5 and a calculated MPO score of 4.0. Even though the molecular weights for these compounds are higher, the compounds are predicted to possess good drug-like properties as measured by their cLogPs, MPO scores and high sp3 character (Fsp3 = 0.4). While no in vivo or other ADME data has been published on this tetrahydrofuran scaffold, it appears the Pfizer group has succeeded in combining excellent potency with good predicted CNS drug-like properties, a notable milestone for GSM drug design.


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Figure 34. Pfizer pyridopyrazine-1,6-dione GSMs with conformational restrictions

Several patent applications from the Janssen group also describe a series of pyridopyrazine1,6-diones that succeed in combining excellent potency and efficacy with good predicted CNS drug-like properties as well as high sp3 character (Fsp3 up to 0.4).131, 132 For example, compound 81 combines excellent potency (EC50 = 6 nM) with a cLogP of 3.0 and MPO score of 4.5 and demonstrates 42% Aβ42 lowering in mouse brain at 4 hours post 10 mg/kg p.o. dose. A number of different substituents could also be incorporated onto the right side of the molecule. In addition to benzofuran 81, compounds such as indole 82 (EC50 = 10 nM, 61% Aβ42 lowering in mouse brain at 30 mg/kg p.o.) appear very interesting. Tricyclic structures such as compound 83 (EC50 = 50 nM) were also investigated, although these compounds tended to be slightly larger and less potent.133 Further work from the Janssen group in this class of compounds has yet to be reported.


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Figure 35. Janssen pyridopyrazine-1,6-dione GSMs

Natural Product GSMs A few groups have investigated natural product based GSMs. Natural products and their derivatives have historically been an invaluable source of therapeutic agents. While many natural products and their derivatives possess higher molecular weights, more rotatable bonds and more stereogenic centers than a typical small molecule drug, their chemical structures have been selected by evolution for the structural prerequisites for binding proteins and crossing biological membranes. Consequently, natural products may represent privileged chemical scaffolds for drug discovery, although it remains to be seen if they will succeed in delivering a promising GSM. Satori Pharmaceuticals reported the discovery of a novel class of triterpene glycoside GSMs obtained from screening a library of natural product extracts. Bioassay guided fractionation


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identified compound 84 as the active component from a black cohash extract.134 In a different profile to other GSMs, compound 84 lowered Aβ42 (EC50 = 100 nM) and Aβ38, while increasing both Aβ39 and Aβ37 and having little effect on Aβ40 and total Aβ. Like all GSMs, the natural product also did not alter Notch processing. Unfortunately, compound 84 was highly cleared in vivo and demonstrated very low brain exposures. The high clearance results from the instability of the enol ether, the glycoside and the acetate ester. Several rounds of optimization led to compound 85 obtained via reduction of the enol ether double bond, glycoside replacement with a substituted morpholine, and conversion of the acetate to an ethyl group.135-137 This compound maintained modest potency (EC50 = 110 nM) and demonstrated improved clearance (0.50 mL/min/kg), good oral bioavailability (50%) and low CYP3A4 inhibition (IC50 = 37 µM). When dosed at 100 mg/kg p.o., compound 85 reduced Aβ42 by 50% in rat brain. This compound was tolerated at 90 mg/kg/day in a 14 day rat toxicology study and selected as the development candidate. This work has since been discontinued, and Satori Pharmaceuticals subsequently shut down after this class of compounds was found to disrupt adrenal function in monkeys.138

Figure 36. Satori natural product GSMs


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Researchers at the University of Florida have evaluated the ability of steroids to reduce Aβ42 by screening 170 natural and synthetic steroids at 10 µM in CHO cells.139 Many non-acidic steroids were found to increase Aβ42 while acidic steroids were found to lower Aβ42. Of the steroids that lowered Aβ42, the most potent were 5β-cholanic acid 86 (EC50 = 5.7 µM) and its endogenous analog lithocholic acid 87 (62% Aβ42 reduction at 10 µM). Compound 86 was shown to have a similar profile to the early carboxylic acid GSMs by lowering Aβ42 and increasing Aβ38 without changing Aβ40 or total Aβ. The compounds also had no effect on Notch processing. The authors hypothesized that various endogenous steroids could play a role in modulating Aβ production in vivo, but more evidence is needed to establish this hypothesis. Similar to the Satori natural products, when measured against typical small molecules, these steroids possess high cLogPs and low MPO scores. It is unclear if these compounds will be pursued any further as therapeutics.

Figure 37. University of Florida steroid GSMs


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Conclusions There is a desperate need for drugs that alter the course of AD and prevent, or at least delay, the onset of cognitive decline. The pharmaceutical industry became interested in GS many years ago due to the genetic evidence ascribing a central role for Aβ in the development of AD. Identifying potent, efficacious and safe agents targeting this unique intramembrane protease for POM studies has been very elusive. After the failure of GSIs in the clinic, it became clear that GSMs would be a safer approach. The inhibition of GS may not be tolerable in a chronic dosing paradigm due to its many substrates, but increasing its processivity with GSMs may address the underlying molecular deficit in FAD. GSMs provide the potential to reduce the accumulation of the toxic Aβ species by changing the ratio of Aβ42:Aβ40 and increasing the shorter nonamyloidogenic Aβ species, such as Aβ38 and Aβ37. This approach is not unlike statins where the changing the LDL:HDL ratio impacts cardiovascular disease. Initially the challenge was to improve on the potency of compounds, something that was achieved rather quickly. Unfortunately, most of these GSMs have not yet been able to combine excellent in vitro potency and in vivo efficacy with good drug-like properties. There are several reports of moderately active GSMs with respectable drug-like properties, but the challenge remains to further increase the potency without a significant increase in the lipophilicity (cLogP) and aromatic ring count (i.e. lower Fsp3). This challenge may be especially daunting due to the hydrophobic nature of this intramembrane protease. Only compounds with highly desirable drug-like properties will address the solubility, permeability, metabolic stability, CYP inhibition, hERG liability, P-gp efflux, and toxicity issues observed for so many GSMs.140 Delivering a safe and efficacious GSM for POM studies in human clinical trials will absolutely require the combination of potency/efficacy with good drug-like properties. It appears, for the first time, that GSMs may be on the cusp of this goal. We believe


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continued focus to reduce the aromaticity (increase the Fsp3), molecular weight, lipophilicity, and increase the CNS MPO score will be key in the generation of a drug in this class. As some GSMs have demonstrated promise in reversing the effects of PS1 mutations, the initial proof of concept studies may come from a prevention trial in asymptomatic FAD patients similar to the planned DIAN trials. Furthermore it will be exciting to consider GSMs used in combination with other emerging Alzheimer’s therapies.

Abbreviations AD, Alzheimer’s disease; ADMET, Absorption, Distribution, Metabolism, Elimination, and Toxicity; Aβ, amyloid β peptide; AICDs, amyloid precursor protein intracellular domains; APP, amyloid precursor protein; Aph-1, anterior pharynx-defective 1; AUC, area under the curve; BACE1, β-amyloid cleavage enzyme 1; brain to plasma ratio, B:P; cLogP, calculated logarithm of octanol/water partition coefficient; CNS, central nervous system; CSF, cerebrospinal fluid; CTF, C-terminal fragment; CYP, cytochrome P450, FAD, familial Alzheimer’s disease; GS, γ-secretase; GSI, γ-secretase inhibitor; GSM, γ-secretase modulator; hERG, human ether-a-go-go-related gene; HTS, high-throughput screening; I-CLiPs, intramembrane cleaving protease family; MPO, multiparameter optimization; MW, molecular weight; Nct, nicastrin; NICD, Notch intracellular domain; NMDA, N-methyl-D-aspartic acid; NSAID, nonsteroidal anti-inflammatory drug; P-gp, Pglycoprotein; PK, pharmacokinetic; POM, proof-of-mechanism; PS, presenilin; Pen-2, presenilin enhancer protein 2; PSEN, presenilin gene; NTF, N-terminal fragment; PXR, pregnane X receptor; RIP, regulated-intramembrane proteolysis; sAPP, soluble APP fragment; SAR, structure activity relationship; TMD, transmembrane domain.


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Acknowledgements The authors thank our many, many colleagues at FORUM Pharmaceuticals for their sustained contribution to our efforts to deliver a safe, efficacious GSM. We especially appreciate Drs Duane A. Burnett and Gerhard Koenig for their insightful discussions in preparation of this document.

Biographies Matthew G. Bursavich obtained his B.S. degree in chemistry from Louisiana State University. He completed his Ph.D. studies in organic chemistry at the University of Wisconsin-Madison under the direction of Prof. Daniel H. Rich, where he designed, synthesized and developed strategies targeting aspartic protease inhibitors, including BACE. He began his industrial career at Wyeth developing hit generation strategies and leading projects to develop new leads for Oncology, Inflammation and Neuroscience targets. He then joined Myriad Pharmaceuticals in Utah leading teams to identify new hits for Oncology targets and progress them into late stage studies. In 2011, he joined the Department of Medicinal Chemistry at FORUM Pharmaceuticals, where he is an associate director leading neuroscience teams in pursuit of medicines for serious brain diseases.

Bryce A. Harrison obtained his B.S. degree in chemistry from Brigham Young University. He continued his education at Harvard University where he completed his Ph.D. in organic chemistry in the lab of Prof. Gregory L. Verdine, investigating diversity-based approaches to peptide mimetics. This work was followed by post-doctoral studies towards the synthesis of marine natural products with Prof. Erik J. Sorensen at Princeton University. Upon completion of his post-doctoral studies, he joined Lexicon Pharmaceuticals in Princeton, NJ, working as a


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medicinal chemist in the areas of diabetes and ophthalmology. In 2014, he moved to FORUM Pharmaceuticals and joined the medicinal chemistry team in pursuit of treatments for serious brain diseases.

Jean-François Blain obtained his B.Sc. in Biochemistry from Université de Sherbrooke, Canada, where he also obtained his M.Sc. in Pharmacology under the supervision of Dr. Pierre Sirois. He subsequently moved to Montreal, Canada, to pursue his Ph.D in Neurological Sciences at McGill University under the supervision of Dr. Judes Poirier, where he worked on components of the brain cholesterol metabolism, apolipoprotein E and Aβ. Before joining FORUM in 2010, he held positions at Merck Research Laboratories in Boston, MA and a joint appointment at the Harvard School of Public Health and the Broad Institute in Cambridge, MA. He has been working on Alzheimer’s disease for over 15 years.

Corresponding Author *M.G.B, Email: [email protected].

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