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. 2012 Aug 29;3(11):908–913. doi: 10.1021/ml300256p

Initial Optimization of a New Series of γ-Secretase Modulators Derived from a Triterpene Glycoside

Nathan O Fuller 1,*, Jed L Hubbs 1, Wesley F Austin 1, Steffen P Creaser 1, Timothy D McKee 1, Robyn M B Loureiro 1, Barbara Tate 1, Weiming Xia 1, Jeffrey L Ives 1, Mark A Findeis 1, Brian S Bronk 1
PMCID: PMC4025870  PMID: 24900406

Abstract

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The discovery of a new series of γ-secretase modulators is disclosed. Starting from a triterpene glycoside γ-secretase modulator that gave a very low brain-to-plasma ratio, initial SAR and optimization involved replacement of a pendant sugar with a series of morpholines. This modification led to two compounds with significantly improved central nervous system (CNS) exposure.

Keywords: Alzheimer’s disease, amyloid β, γ-secretase, modulator, black cohosh

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is the leading form of dementia and affects an estimated 5.4 million Americans1 and 35.6 million people worldwide.2 AD is the sixth leading cause of death in the United States, and with the aging baby boomer generation and increasing life expectancy, the prevalence and mortality rates associated with this disease are projected to rise dramatically. Healthcare costs associated with AD are already costing the United States government close to $200 billion annually, and recent estimates project that by 2050 the numbers of individuals afflicted with AD will increase to between 11 and 16 million in the United States1 and 115.4 million people worldwide.2 These cumulative social and economic factors make this one of the major healthcare crises facing the world today. Despite significant research efforts, there are currently no treatments which effectively slow or stop the progression of AD. Several symptomatic treatments have been approved, but these only temporarily improve symptoms of AD patients.3,4 Thus, there is an urgent unmet medical need for a disease-modifying therapy for AD that can slow, halt, or reverse disease progression.

The pathology of the Alzheimer’s brain is characterized by amyloid plaques and neurofibrillary tangles.5−7 A prevailing viewpoint for the underlying cause of the disease is the amyloid hypothesis, which contends that amyloid β peptide (Aβ) dysregulation initiates a cascade of neuropathological changes—formation of amyloid plaques, neurofibrillary tangles, synaptic loss, and neurodegeneration—that ultimately result in the precipitous decline in cognition and ability to function in daily life that define AD dementia.8 Amyloid plaques consist of Aβ peptides which are formed by the processing of amyloid precursor protein (APP). Aβ peptides are produced through a series of sequential cuts by two membrane-bound enzymes: first, β-secretase cleaves APP into the β-C-terminal fragment; then, γ-secretase makes further cuts to generate Aβ peptides ranging from 37 to 49 amino acids in length.9,10 The main component of the amyloid plaques is the aggregation-prone 42 amino acid form of amyloid β (Aβ42), a relatively minor component in the total Aβ pool but particularly neurotoxic.11−13 As a result, there has been much focus on both β-secretase and γ-secretase as therapeutic targets to interrupt the amyloid cascade by decreasing the amount of Aβ42 and thereby preventing the buildup of the amyloid plaques that initiate the disease.

Significant drug discovery research efforts have focused on inhibiting γ-secretase with small molecules to reduce the overall amount of amyloid production. Several different classes of these γ-secretase inhibitors (GSIs) have been reported in the literature and been shown to be potent inhibitors of γ-secretase activity, lowering Aβ levels in vitro and in vivo.14−16 However, in addition to its role in the cleavage of APP, γ-secretase also cleaves multiple essential proteins, including Notch.17 Thus, by inhibiting γ-secretase, GSIs also interfere with Notch processing, which leads to toxicities that include severe gastrointestinal abnormalities and skin cancer.18,19 Several GSIs have been advanced to the clinic, but most clinical GSI studies have failed or been halted early due to observed toxicity, likely associated with the inhibition of Notch.20,21

In order to develop drugs with a safer profile, recent efforts have shifted from γ-secretase inhibition to γ-secretase modulation.22−24 With the role of Aβ42 in the initiation of the disease process,10 there is growing evidence that suggests it may be more important to lower the ratio of Aβ42/Aβ40 (Aβ40 being the most prevalent Aβ peptide residue) or Aβ42/total Aβ rather than reduce the total amount of Aβ.25 γ-Secretase modulators (GSMs) achieve this profile by shifting the sites of APP cleavage away from the more neurotoxic Aβ42 to shorter, nontoxic peptides such as Aβ37, Aβ38, and Aβ39. Importantly, GSMs have an added advantage in that they do not affect the release of the Notch intracellular domain (NICD) following the processing of Notch by γ-secretase, a vital factor in developing safe and tolerable therapeutics for AD.26,27 The first reported GSMs were NSAIDs that were found to decrease the amounts of Aβ42 while increasing Aβ38.28,29 A representative example is (R)-flurbiprofen, which advanced to clinical trials but failed due to lack of potency and poor brain exposure, a common problem among the NSAID GSMs.30,31 There have been more recent disclosures of carboxylic acid GSMs and aryl imidazole GSMs which more potently reduce levels of Aβ42 while raising levels of Aβ38.22,23 A recent report of three distinct pyrimidine-based GSMs showed reduction of both Aβ40 and Aβ42 in vitro but different effects on Aβ(37–39), with some examples raising Aβ38 and some having no effect on Aβ38 production.32 These “second generation” GSMs have achieved much higher activities toward Aβ42 reduction than NSAIDs, and several examples have shown efficacy and selectivity both in vitro and in vivo, supporting modulation of γ-secretase as an effective approach to decreasing toxic species of Aβ without preventing the processing of other proteins, including Notch. Thus, modulation of γ-secretase has great potential as a therapeutic approach for the treatment of Alzheimer’s disease.

As part of a program aimed at developing a series of novel γ-secretase modulators (GSMs) as therapeutics for AD, Satori identified an initial lead compound via screening of natural product extracts for selective reduction of Aβ42.33 This initial lead (1, Scheme 1) was isolated from extracts of the black cohosh plant (Actaea racemosa) and represents the first entry in a unique class of GSMs that features a complex molecular architecture characterized by a plant sterol core structure. Most importantly, 1 exhibits a unique and compelling profile toward γ-secretase modulation, selectively reducing levels of Aβ42 and Aβ38 in vitro, while raising levels of Aβ37 and Aβ39. The levels of Aβ40 and total Aβ levels were maintained, and 1 also displayed pronounced selectivity over Notch processing in vitro.

Scheme 1.

Scheme 1

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While 1 was an intriguing initial hit, examination of its structure quickly revealed a number of potential metabolic and chemical liabilities that limited its potential usefulness as a therapeutic drug. In particular, the C3 glycoside, C16 enol ether, and C24 acetate stood out as areas of concern. Indeed, the concerns over the metabolic stability of 1 were confirmed upon dosing in CD1 mice. Rapid clearance of 1 precluded its therapeutic usefulness, as both the C3 sugar and the C24 acetate were readily metabolized in vivo.33 In addition, the high MW and tPSA of 1 lay well outside the range typical for CNS drugs, causing concerns that we would not be able to prepare compounds that gave adequate CNS exposure to be therapeutically useful. Therefore, an important milestone in this project would be to prepare compounds that gave sufficient brain-to-plasma ratios in vivo.

Recognizing the need to resolve the issues of intrinsic liability presented by the functionality native to this triterpene glycoside, the primary focus of initial medicinal chemistry efforts was placed on exploring the SAR around the core structure of the lead compound. Thus, we investigated the effects of removal or modification of selected structural features of 1 on the overall profile of modulation of Aβ processing by γ-secretase. In our initial exploratory forays into the chemistry of 1, we exposed the compound to aqueous HCl, which resulted in acyl migration and subsequent formation of bicyclic ketal 2 and a 30-fold loss in potency toward Aβ42 reduction (Scheme 1). The bicyclic ketal was found to be quite stable and was resistant to a variety of reaction conditions, including many attempts to reopen the ketal. Thus, we turned our attention to exploring transformations that could be carried out on the enol ether and would not result in this irreversible cyclization. While many attempts at direct reduction of the C16–C17 olefin were unfruitful, we discovered that ZrCl4 catalyzed the isomerization of the enol ether of 1 in CH2Cl2 to provide the C15 ketone 3 with cis-configuration between the C16 and C23 of the resulting trans-fused tetrahydropyran ring. While ketone 3 saw a 6-fold diminution of potency relative to 1, subsequent treatment with NaBH4 to reduce the C15 ketone and provide the corresponding C15 hydroxyl 4 moderately improved upon the potency of the initial lead (Aβ42 IC50 = 60 nM). The ketone reduction proceeded with excellent stereoselectivity (>95:5, only one product observed by 1H NMR), with the angular methyl group at C14 presumably creating the steric environment that accounts for the high degree of selectivity in the reaction. The stereochemistry resulting from the two-step reduction process was confirmed by obtaining an X-ray crystal structure (see Supporting Information). Thus, in the process of removing one of the more troubling structural features from a metabolic and chemical perspective, the primary pharmacology of the lead compound was improved upon.

More extensive testing was carried out on 4, and it was found to have excellent selectivity in transporter, off-target, and safety assays. However, following dosing in CD1 mice, the compound was found to have an insufficient pharmacokinetic profile for advancement, displaying moderate-to-high clearance and poor brain exposure (data not shown). This poor in vivo performance was not entirely unexpected, for the native C3 sugar appendage and the C24 acetate had been shown to be vulnerable to metabolism when 1 had been dosed in vivo.33 In addition, little had been done to address other parameters, such as number of hydrogen bond donors (HBD = 5) and topological polar surface area (tPSA = 155 Å2) that could preclude a molecule from achieving acceptable levels of brain exposure.34−36 Thus, we sought to improve the overall CNS disposition by further modification of the structure of 4 that would address these design parameters.

Interrogation of the additional metabolic and chemical soft spots of 4 revealed some interesting trends in the SAR of this lead series. Hydrolysis of the C24 acetate to unmask the C24,C25 diol 5 resulted in a 50-fold reduction in potency in lowering Aβ42 (Scheme 2). Cleavage of the glycoside to provide triol 6 resulted in only a 10-fold loss of activity, while further treatment with K2CO3/MeOH to remove the C24 acetate and provide the tetrol 7 caused the potency to decrease by more than 17-fold compared to triol 6. These results identified the C24 acetate as a more critical pharmacophore to the Aβ42 pharmacology than the C3 glycoside. As a result, the focus of these investigations shifted to carrying out further SAR studies at the C3 position of the scaffold through which we hoped to identify a glycoside surrogate that maintained the overall pharmacological profile while improving on the physicochemical properties of the series. These improvements would then hopefully translate into an improved pharmacokinetic profile.

Scheme 2.

Scheme 2

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While part of our early work was focused on discovering a suitable replacement for the C3 glycoside via initial cleavage of the sugar followed by functionalization or chemical displacement of the exposed C3 alcohol,37 we were also interested in exploiting rather than replacing the native sugar in our synthetic endeavors. To this aim, by treating 4 with NaIO4 or Pb(OAc)4, we were able to carry out a double oxidative cleavage event on the sugar to provide dialdehyde 8 (Scheme 3), which served as a versatile intermediate.38,39 Reduction of dialdehyde 8 with NaBH4 provided tetrol substrate 9, which possessed good pharmacology but did not offer any advantages over 4 from a physicochemical properties standpoint. Dual reductive amination with dimethylamine provided the diamine 10, which, although lowering the tPSA, also realized a diminution in potency. When we carried out the reductive amination of dialdehyde 8 with methylamine, after undergoing an initial reductive amination, the intermediate amino-aldehyde species participated in a second intramolecular reductive amination event to provide C3 morpholine 11. This two-step chemical transformation of the native sugar into a morpholine provided a substrate which not only maintained the primary pharmacology (Aβ42 IC50 = 130 nM) but offered a significant improvement from a physicochemical properties perspective, lowering the tPSA to 98 Å2 and the HBD count to two. Thus, by unlocking the reactive potential of the 1,2,3-triol of the sugar to synthesize the C3 morpholine as a replacement for the C3 glycoside, we had discovered a new lead series of compounds possessing a modified headpiece which maintained high potency in lowering Aβ42 in vitro and provided a versatile handle for further derivatization to allow for engineering of the overall physicochemical properties.

Scheme 3.

Scheme 3

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To fully exploit the medicinal and synthetic chemistry potential of this C3 morpholine series of compounds, a focused but diverse range of morpholine compounds was designed and synthesized, and representative examples from this series of compounds are shown in Table 1. A scan of the SAR of this C3 morpholine series reveals that a wide variety of substitution is tolerated on the morpholine nitrogen. The simple N–H morpholine 12 was particularly potent with an Aβ42 IC50 of 70 nM. Small aliphatic substituents were also tolerated, and the general trends suggest that polar substituents on a basic morpholine are preferred over more lipophilic substituents, a trend that was in concert with one of our key design elements. Hence, while N-methyl (11) and N-ethyl morpholines (13) maintained potency, imparting more lipophilic character by functionalization of the morpholine nitrogen with propyl, benzyl, or cycloalkyl groups resulted in a loss of activity (14–17). A decrease in potency was also realized for N-trifluoroethyl morpholine 18. Incorporating a heteroatom into the morpholine substituents and thereby increasing the polarity in this region of the molecule resulted in improved Aβ42 activity relative to the purely aliphatic analogs. For example, replacing the terminal methyl of the N-propyl derivative 14 with a hydroxyl (19) or a methyl ether (21) resulted in much improved potency, and the N-oxetane derivative 22 was 2-fold more potent than the corresponding N-cyclobutyl analog 17. Both hydroxyalkyl and ether appendages on the morpholine nitrogen showed a strong propensity for reduction of Aβ42 (19–23). A similar effect was seen upon incorporation of aminoalkyls into the morpholine side chain, providing compounds with strong Aβ42 lowering capabilities (24–26). Amide appendages on the morpholine nitrogen also resulted in extremely potent compounds (27–28), but carboxylic acids were not as active (29–30). Both diastereomers of the γ-lactam moiety showed good activity (31 and 32), as did the imidazole derivative 33. Whether or not the improved potency results from the ability of the heteroatoms to pick up additional interactions or is simply due to increased polarity is not clear.

Table 1. SAR of Representative Examples from the Satori C3 Morpholine Series of GSMs.

graphic file with name ml-2012-00256p_0006.jpg

graphic file with name ml-2012-00256p_0007.jpg

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a

IC50 reported as an average of multiple determinations (n ⩾ 2).

The synthesis of acyl morpholines and related species also furnished interesting and highly potent analogs. Conversion of the morpholine nitrogen to sulfonamide 34, urea 35, and amide derivatives (36–40) provided a series of compounds with excellent activity toward Aβ42 lowering. Although these acylated derivatives were intriguing, the physicochemical properties of these compounds were less compelling than morpholines that maintained a basic center (Supporting Information). The morpholine headpiece proved to be a very versatile handle for incorporating a diverse range of functional groups into the scaffold, and it proved advantageous for tuning the overall molecular properties and the potential to improve the CNS disposition. In addition, the selectivity for reduction of Aβ42 versus Aβ40 was maintained across the series.

The overall profile of two C3 morpholine derivatives, N–H morpholine 12 and the N-oxetane analog 22, allowed these to emerge as candidates for further evaluation. While all molecules had a higher molecular weight than is usually targeted for a CNS drug, the balance of potency, HBD, tPSA, and lipophilicity of analogs 12 and 22 along with the potential benefits provided by having a basic center in the molecule to assist in permeating the blood–brain barrier elevated these over our initial leads (1 and 4) and other potential candidates in the series (Supporting Information).

The C3 morpholines 12 and 22 were dosed in CD1 mice to evaluate in vivo pharmacokinetics (Figure 1). The results showed that attenuating amine basicity in N-oxetane morpholine species 22 (pKa = 4.6) decreases the volume of distribution (0.6 L/kg versus 12.1 L/kg for 12) while modestly increasing the clearance (3.3 L/(h kg) for 22 versus 3.1 L/(h kg)) compared to the more basic N–H morpholine 12 (pKa = 7.7). Most importantly, we found that these compounds gave encouraging brain-to-plasma ratios (1.7 for 12, 0.23 for 22). While the compounds exhibited acceptable brain-to-plasma ratios, the bioavailability values were low (%F = 11% for 22 and 37% for 12, respectively) and low levels of brain exposure were observed. Still, this murine PK data represented a significant improvement over the previously disclosed lead compounds from the C3 glycosidic series of GSMs (1 and 4), and we felt this disposition and overall profile were sufficient to assess a PK/PD response in vivo.

Figure 1.

Figure 1

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Full Aβ production and pharmacokinetic profiles (CD1 mouse) for compounds 12 and 22.

After comparing the in vivo PK and predicted microsomal stability of 12 versus 22 in mouse (MLM, % remaining after 60 min ± NADPH: 12 = 20%/25%; 22 = 1%/1%), we decided to focus on 12, which was taken forward for in vivo efficacy evaluation in CD1 mice.40 Unfortunately, due to the high clearance of the molecules, we were unable to obtain sufficient brain exposure to elicit a robust pharmacodynamic response in vivo (see Supporting Information). Results of further studies aimed at improving clearance of this series in order to obtain adequate exposure for a PD response will be presented in due course.

Evaluation of the Aβ profile in our cell-based assay showed that both N–H morpholine 12 and N-oxetane analog 22 exhibited the ability to lower Aβ42 levels in cells, with similar activity versus Aβ38, good selectivity for lowering Aβ42 versus Aβ40 (ratio of Aβ40 IC50/Aβ42 IC50 = 25 for 12 and 52 for 22), and preserved levels of total Aβ in cells42 (Figure 1). The molecules showed excellent selectivity for lowering Aβ42 versus Notch processing (>70-fold selective for both 12 and 22). This level of Notch selectivity is consistent with a GSM profile and, due to the toxicities associated with inhibition of Notch processing, offers a distinct advantage over the lack of Notch selectivity exhibited by GSIs which have advanced to clinical trials.43 The overall modulation profile of the series, reducing Aβ42 and Aβ38 while maintaining Aβ40 and total Aβ, represents a different GSM profile when compared to most other GSMs reported in the literature.

In summary, the Satori GSM program progressed from an intriguing initial hit, triterpene glycoside 1, identified in a targeted bioassay screening, to a new lead compound (4) with a modified core. Application of medicinal chemistry design principles in conjunction with innovative synthetic chemistry approaches led to the discovery of a new series of C3 morpholine compounds. This series represents a new structural class of GSMs that maintained the compelling pharmacological profile of the initial leads, lowering Aβ42 and Aβ38 levels in cells, preserving Aβ40 and total Aβ, and showing no inhibition of Notch activity. This GSM profile differs from most other GSMs reported in the literature, which lower Aβ42 but raise levels of Aβ38. In the process of design, we significantly improved druglike properties of the lead compounds by decreasing both the tPSA and the overall count of hydrogen bond donors (HBD) while keeping the lipophilicity in an acceptable range (<5). This led to compounds 12 and 22, which have significantly improved CNS penetration relative to glycoside 4, which more closely resembles our natural product lead 1. In addition, these compounds retained an excellent profile in standard transporter, off-target, and safety assays in vitro, and showed improved chemical stability. The morpholine nitrogen provides a convenient and versatile handle by which the overall molecular properties of the compounds can be readily manipulated. The discovery of this new series of C3 morpholine GSMs with a plant sterol-derived core structure represents a novel and exciting entry into the arena of γ-secretase modulation as a potential therapeutic approach for Alzheimer’s disease.

Acknowledgments

The authors would like to thank Ruichao Shen for his comments on the paper.

Glossary

Abbreviations

Aβ

amyloid beta

AD

Alzheimer’s disease

APP

amyloid precursor protein

cLogP

calculated lipophilicity

CH2Cl2

dichloromethane

CNS

central nervous system

EtOAc

ethyl acetate

GSM

γ-secretase modulator

GSI

γ-secretase inhibitor

HCl

hydrochloric acid

HBD

hydrogen bond donors

K2CO3

potassium carbonate

Me2NH

dimethylamine

MeNH2

methylamine

MLM

mouse liver microsomes

MeOH

methanol

NaBH4

sodium borohydride

NaCNBH3

sodium cyanoborohydride

NaIO4

sodium periodate

NSAID

nonsteroidal anti-inflammatory drug

Pb(OAc)4

lead tetraacetate

PK

pharmacokinetic

PO

by mouth

tPSA

topological polar surface area

SAR

structure–activity relationship

ZrCl4

zirconium tetrachloride

Supporting Information Available

Calculated physicochemical properties of high-interest compounds from the Satori C3 morpholine series of GSMs, exemplary procedures for the synthesis of compounds of interest, characterization data of key compounds 12 and 22, X-ray crystal structure of compound 4, and assay protocols. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Present Address

† Resilientx Therapeutics, 431 School Street, Belmont, MA 02478, USA.

Author Present Address

‡ Genzyme Corporation, 500 Kendall Street, Cambridge, MA 02142.

Author Present Address

§ Department of Veterans Affairs, Boston, MA.

Author Contributions

All authors have given approval to the final version of the manuscript.

The authors note the following relevant financial interests: the authors are named as inventors on one or more patents and patent applications related to compounds discussed in this paper and either hold equity and/or options on equity in Satori Pharmaceuticals.

Supplementary Material

ml300256p_si_001.pdf (828.7KB, pdf)

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  43. Known GSIs which have advanced to clinical trials show no selectivity for Notch inhibition (ratio of Aβ IC50/Notch IC50 for semagecestat = 0.6, for begacestat = 0.8, and for avagecestat = 0.6. Known GSMs in the literature show much better selectivity toward Notch inhibition (Aβ42 IC50/Notch IC50 for Merck GSM-1 = >350, for JNJ-40418677 = >20, for Eisai E-2012 = >400). See “An improved cell-based method for determining the gamma-secretase enzyme activity against both NOTCH and APP substrates”, T. D. McKee, et al., AAIC 2012. poster #P2-095, http://www.satoripharma.com/news-events/publications-and-presentations.php.

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ml300256p_si_001.pdf (828.7KB, pdf)

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