Discovery of cyclopropyl chromane-derived pyridopyrazine-1,6-dione γ-secretase modulators with robust central efficacy

γ-Secretase modulators for the treatment of Alzheimer's disease.

Abstract

Herein we describe the discovery of a novel series of cyclopropyl chromane-derived pyridopyrazine-1,6-dione γ-secretase modulators for the treatment of Alzheimer's disease (AD). Using ligand-based design tactics such as conformational analysis and molecular modeling, a cyclopropyl chromane unit was identified as a suitable heterocyclic replacement for a naphthyl moiety that was present in the preliminary lead 4. The optimized lead molecule 44 achieved good central exposure resulting in robust and sustained reduction of brain amyloid-β42 (Aβ42) when dosed orally at 10 mg kg^–1 in a rat time-course study. Application of the unpaced isolated heart Langendorff model enabled efficient differentiation of compounds with respect to cardiovascular safety, highlighting how minor structural changes can greatly impact the safety profile within a series of compounds.

Introduction

Alzheimer's disease (AD) is the most common form of dementia among the elderly population, affecting approximately 1 in 5 people over the age of 65.1 It is a debilitating neurodegenerative disease that presents with gradual loss of memory, impaired speech, inability to carry out tasks of daily living, and is ultimately fatal.1 AD pathology is characterized by the accumulation of amyloid plaques consisting primarily of Aβ42, which is a neurotoxic peptide produced via sequential processing of the amyloid precursor protein (APP) by the β-secretase (BACE) and γ-secretase enzymes. In an effort to develop disease-modifying therapies that can slow or halt the progression of AD, inhibition or modulation of these enzymes has been aggressively pursued to reduce formation of Aβ42.2

γ-Secretase modulators (GSMs) have emerged as a promising therapeutic approach for AD.3,4 This class of compounds has a mechanism of action that is distinct from that of γ-secretase inhibitors (GSIs). While GSIs inhibit processing of all γ-secretase substrates including notch, which is critical for normal cell signaling, GSMs alter the cleavage site of APP to reduce formation of longer, aggregation-prone peptides in favor of shorter, more benign species. Data suggests that Aβ40, which is present at ∼10 times the concentration of Aβ42 in a healthy human brain, can sequester Aβ42 in stable mixed tetramers, potentially preventing further oligomerization of Aβ42.5 GSMs therefore hold the promise of selectively reducing levels of the toxic species without inhibiting notch signaling, and avoiding the side effects observed in GSI clinical trials.6,7 γ-Secretase is comprised of four subunits: presenilin, Aph-1, nicastrin, and Pen-2.8 Chemical biology studies using photoaffinity probes have established that GSMs bind to allosteric sites on presenilin that are different from those targeted by GSIs.9,10

Analysis of the GSM literature clearly reveals the challenge of identifying potent GSMs within favorable CNS physicochemical property space.11 γ-Secretase is an intra-membrane cleaving aspartyl protease, which may underlie the apparent requirement for compounds to have increased lipophilicity in order to achieve robust potency.12 Our initial efforts led to the discovery of dihydrobenzofuran amide GSMs as exemplified by 1 (Scheme 1).13,14 Because of difficulties in further improving potency while maintaining acceptable physicochemical properties and ADME (absorption, distribution, metabolism, and excretion) characteristics, we shifted our efforts toward the design of novel, conformationally restricted heterocyclic cores with increased polarity. As previously described, this led to the discovery of the pyridopyrazine-1,6-dione series (e.g., 2 and 3), which had several advantages such as improved potency, good metabolic stability, low MDR efflux ratio, and reduced inhibition of cyctochrome P450 (CYP450) enzymes.15,16 During this time, the team had also identified naphthyl derivative 4, which had one of the highest ligand efficiency values (LE = 0.34) thus far, and it was therefore selected for further optimization (Scheme 1). A rapid survey of diverse chemical space using parallel medicinal chemistry (PMC) led to identification of an indole as a suitable heterocyclic replacement for the naphthyl moiety.17,18 Further optimization eventually delivered 5, which was a highly potent modulator of γ-secretase in vitro in Chinese hamster ovary cells overexpressing wild-type human APP (CHO APP) (Aβ42 IC₅₀ = 6 nM, clog P = 3.1).19 However, poor central Aβ42-lowering activity was observed with this compound in an acute in vivo efficacy study in rat (in part due to reduced central exposure), prompting further investigation into alternative heterocyclic replacements for the naphthyl group.17 Herein we describe the design and synthesis leading to the discovery of cyclopropyl chromane-derived GSMs, which overcame significant safety hurdles and achieved excellent Aβ42-lowering activity in vivo.

Scheme 1. Design of pyridopyrazine-1,6-dione GSMs.

Results and discussion

Given the poor exposure and efficacy of the indole series, we envisioned that a chromene might serve as a more suitable replacement for the naphthyl unit. Chromenes feature prominently in bioactive natural products, and incorporation of this ring system would result in reduced aromatic ring-count and increased polarity while avoiding the addition of basic centers that could serve as recognition elements for P-glycoprotein (P-gp)-mediated efflux. Several synthetic routes were developed to facilitate efficient evaluation of structure–activity relationships (SAR). Our general strategy involved the use of lactone 7 as a common starting material17 in combination with a variety of chromene-derived amines 8 (Scheme 2). We envisioned that the chromene motif could be accessed from an intramolecular hydroarylation/amination sequence. The substrate required for the hydroarylation chemistry could be derived from readily available starting materials such as appropriately substituted phenols and alkynes. Given that a large number of substituted phenol monomers are readily available, this strategy would enable facile evaluation of the chromene SAR.

Scheme 2. Overview of synthetic strategy.

4-(Trifluoromethyl)phenol (12) was alkylated with propargyl bromide (11, Scheme 3). The resultant alkynyl arene 13 was deprotonated and then reacted with paraformaldehyde to install the hydroxymethyl moiety. Cyclization of 14 to generate the chromene ring system 15 was initially performed using indium triiodide.20 The primary alcohol of 15 was then converted to the amine 16via a two-step process involving Mitsunobu reaction with phthalimide followed by hydrazine-mediated deprotection. The corresponding saturated chromane amine 17 was readily accessed via hydrogenation of 16. The chromene/chromane amines 16 and 17 were then coupled to lactone 7via a DABCO bis-trimethylaluminum (DABAL-Me₃)-mediated amidation to afford 18 and 19 (Scheme 4),17 whereupon cyclization to the pyridopyrazine-1,6-dione core could be accomplished via in situ activation of the primary alcohol as the mesylate, followed by intramolecular alkylation to afford 20 and 21. Note that low yields were observed in the synthesis of compound 20 due to its susceptibility to air oxidation.

Scheme 3. Synthesis of chromene amine 16 and chromane amine 17. Reagents and conditions: (a) propargyl bromide, K₂CO₃, DMF, quantitative; (b) n-BuLi, –78 °C then paraformaldehyde, 56%; (c) InI₃, THF, 73%; (d) phthalimide, DIAD, PPh₃, 58%; (e) hydrazine hydrate, 57%; (f) Pd/C, 40 psi H₂, 88%.

Scheme 4. Synthesis of chromene GSM 20 and chromane GSM 21. Reagents and conditions: (a) DABAL-Me₃, THF, 40 °C; (b) Ms₂O, –20 °C then TBD, 28% over two steps for 20, and PPh₃, DIAD, 57% over two steps for 21.

The hydroarylation strategy also proved valuable for the preparation of more sterically demanding chromene analogs. By simply varying the alkyne unit of the cyclization precursor, we were able to readily access gem-dimethyl propargyl alcohols such as 23 (Scheme 5). In this case, the subsequent cyclization was carried out using a gold-based catalyst.21 It was found that protection of the free alcohol with a silyl group prior to cyclization provided extremely clean reaction profiles and high yields; however, protection was not necessary in all cases. Following cyclization and deprotection, the resultant allylic alcohol intermediates 24 could be converted to their corresponding amines 25 and then coupled to lactone 7 in a sequence similar to that shown in Scheme 4, affording gem-dimethyl-substituted chromene GSMs such as 28 and 29 (Table 1). Alternatively, oxidation of the allylic alcohol 24 to the corresponding aldehyde, followed by addition of methylmagnesium bromide, afforded secondary alcohol intermediate 26. Mitsunobu reaction with phthalimide followed by hydrazine-mediated deprotection afforded amine 27, which smoothly underwent lactam formation to afford alpha-methyl substituted GSMs 30–32.

Scheme 5. Synthesis of gem-dimethyl-substituted amine intermediates 25 and 27. Reagents and conditions: (a) 2-methyl-3-butyn-2-ol, TFAA, CuBr, –5 °C; (b) n-BuLi, –78 °C; then paraformaldehyde; (c) TBSCl, imidazole; (d) (JohnPhos)Au(MeCN)SbF₆ (1–5 mol%); (e) TBAF; (f) phthalimide, DIAD, PPh₃; (g) hydrazine hydrate (h) Dess–Martin periodinane; (i) MeMgBr, –78 °C to r.t. Note: R = Cl, CF₃, OCF₃; see Table 1 for details.

Table 1. In vitro pharmacology and disposition data for pyridopyrazine-1,6-dione GSMs 20–32.

	R	IC50 (Aβ42, nM) a	clog P	MDR ER b	HLM CLint,app (μL/min/mg) c
20		30	2.4	1.7	108
21		322	2.5	1.9	37.6
28		13	3.4	2.0	47.8
29		53	3.1	1.9	n.d. d
30		4.9	3.7	1.9	14.3
31		801	3.7	1.8	242
32		6.0	3.8	1.9	25.7

^aAβ42 IC₅₀ values were obtained in a whole-cell assay using CHO APP_wt cells. Aβ42 IC₅₀ values are the geometric mean of at least three experiments except for compound 20 (n = 2).

^bMDR efflux ratio using an MDR1/MDCK assay utilizing MDCK cells transfected with the gene that encodes human P-glycoprotein.27

^cHuman liver microsome-derived intrinsic clearance.22,23

^dNot determined.

The initial chromene analog 20 exhibited an encouraging level of potency (Aβ42 IC₅₀ = 30 nM, clog P = 2.4), but poor metabolic stability was observed in human liver microsomes (HLM CL_int,app = 108 μL/min/kg).22,23 This was not entirely surprising, as we suspected that the methylene between the olefin and the chromene oxygen may be susceptible to CYP-mediated oxidative metabolism. In fact, metabolite ID studies indicated that this site is indeed rapidly oxidized to afford the corresponding chromen-2-one. The related saturated chromane 21 (racemic) showed improved metabolic stability, but this structural modification resulted in a significant loss of potency. We therefore sought to block the site of metabolism on the chromene. Efforts to introduce gem-difluoro substitution were met with failure owing to instability of several of the synthetic intermediates. In fact, examples in the literature describing the synthesis of gem-difluorochromenes are very limited.24 We therefore opted to block the metabolically labile site with gem-dimethyl substitution. Interestingly, the gem-dimethyl chromene moiety is commonly observed in natural products.25 Although this modification would lead to an increase in log P, we hoped for an improvement in lipophilic metabolism efficiency (LipMetE).26 Toward this end, 28 was prepared; the gem-dimethyl substitution was found to be well tolerated with respect to potency (Aβ42 IC₅₀ = 13 nM). Although lipophilicity was increased from a clog P value of 2.4 to 3.4, metabolic stability was significantly improved (CL_int,app values of 108 and 47.8 μL/min/mg for 20 and 28, respectively), indeed generating an increase in LipMetE, from 0.6 to 1.3.

At this point, we observed that SAR trends from the indole series in many cases were applicable to the chromene series.17 For example, replacing the critical trifluoromethyl substituent with a less lipophilic chlorine atom, as in 29, led to a reduction in activity. Introduction of a conformational constraint via insertion of an (S)-methyl substituent on the methylene linker to afford 30 delivered an almost three-fold improvement in potency while maintaining good metabolic stability (Aβ42 IC₅₀ = 4.9 nM, CL_int,app = 14.3 μL/min/mg). In contrast, the enantiomer 31 was approximately 160-fold less active than 30. Finally, a survey of alternative lipophilic, non-metabolizable replacements for the CF₃ substituent proved fruitful, with the trifluoromethoxy analogue 32 exhibiting a favorable profile (Aβ42 IC₅₀ = 6.0 nM, CL_int,app = 25.7 μL/min/mg). Notably, the two chromenes 30 and 32 maintained MDR ER values less than 2.0, suggesting a low risk for P-gp-mediated efflux.

An analysis of the aryl-imidazole GSM literature provides numerous examples of rigid and/or constrained analogues with excellent potency, suggesting that the orientation of the lipophilic group is rather well-defined in the active conformation, and that this substituent prefers to be oriented in a “turned” conformation below the plane of the core, as drawn in Fig. 1.11 To understand the effect of the (S)-methyl substituent on conformation of the chromene series, we applied molecular modeling and conformational analysis to both 28 and 30 (see ESI† for details on the computational work-flow). The analysis revealed that the lowest energy conformation of 28 does indeed prefer a “turned” conformation consistent with the putative bioactive conformation and conformationally restricted GSMs reported in the patent literature (Fig. 1a).11 However, given the pseudo-symmetry above and below the planar pyridopyrazine core and the absence of a conformational control element, there are two possible conformers of equal probability (Fig. 1b). Consistent with our observations in the indole series,17 introduction of the (S)-methyl substituent (30) reinforces the chromene group in the putative bioactive conformation, below the plane of the core, with a probability >99% (Fig. 1c). Conversely, modeling predicted that the (R)-methyl analogue 31 would be locked in the undesired conformation above the plane of the core. We hypothesize that this results in the remarkable difference in potency between 30 and 31 (Table 1).

Fig. 1. a) The lowest energy conformer of 28 adopts a turned conformation that places the chromene moiety below the plane of the pyridopyrazine core; b) lacking a conformational control element, chromene 28 is expected to exist in a 1 : 1 ratio of two conformers; c) chromene 30, bearing the (S)-methyl group, is predicted to exist primarily in a single conformer – the putative bioactive orientation.

While exploring geminal dimethyl substitution on the chromene as a means of improving metabolic stability, an alternative approach was pursued in parallel. The key design objective was to identify additional heterocyclic systems that would force the terminal aryl ring to adopt the putative bioactive conformation. Analysis of compounds 20 and 21, among other SAR trends, led us to conclude that sp² hybridization was required at the center connecting the methylene linker to the terminal heterocycle to achieve good Aβ42-lowering activity. With this in mind, we envisioned that the chromene olefin could be replaced with a cyclopropyl ring; we anticipated that this modification would both maintain sufficient sp² character and serve as an efficient conformational control element (Scheme 6). Application of the conformational analysis workflow described above to cyclopropyl chromane 33 supported this hypothesis, suggesting that this (S,S) diastereomer, with a 92% probability, would position the cyclopropyl chromane moiety in the requisite bioactive conformation, below the plane of the core (Fig. 2). Furthermore, we hypothesized that this design may lead to improved metabolic stability, as the resultant chromane methylene is no longer allylic.

Scheme 6. Design of cyclopropyl chromane series.

Fig. 2. Conformational analysis of cyclopropyl chromane 33 indicates that the desired bioactive orientation – below the plane of the core – is the predominant conformation. a) Extended view of 33; b) end-to-end view of 33.

The initial synthetic approach to cyclopropyl chromane-derived GSMs such as 33 relied on allylic alcohol intermediate 15 (Scheme 7). This material was subjected to a Simmons–Smith cyclopropanation to afford cyclopropyl alcohol 34. A subsequent two-step alcohol-to-amine inter-conversion delivered 35, which was then reacted with lactone 7 as previously described. The resulting racemic cyclopropyl chromane GSM (not shown) was separated via chiral HPLC to afford the enantiomerically pure analogs 33 and 40 (Table 2).

Scheme 7. First-generation synthesis of cyclopropyl chromane amines. Reagents and conditions: (a) ZnEt₂, CH₂I₂, 88%; (b) CBr₄, PPh₃, 70%; (c) NH₃/MeOH, 93%.

Table 2. In vitro pharmacology and disposition data for cyclopropyl pyridopyrazine-1,6-dione GSMs 33–45.

	R	IC50 (Aβ42, nM) a	clog P	MDR ER b	HLM CLint,app (μL/min/mg) c
33		5.6	2.2	2.4	37.9
40		129	2.2	1.9	70.5
41		52	2.2	1.7	14.6
42		63	1.9	1.8	43.9
43		181	1.2	2.2	33.7
44		4.9	3.2	2.5	34.5
45		5.1	3.3	2.1	42.4

^aAβ42 IC₅₀ values were obtained in a whole-cell assay using CHO APP_wt cells. Aβ42 IC₅₀ values are the geometric mean of at least three experiments.

^bMDR efflux ratio using an MDR1/MDCK assay utilizing MDCK cells transfected with the gene that encodes human P-glycoprotein.27

^cHuman liver microsome-derived intrinsic clearance.22,23

We quickly realized that routes relying on Simmons–Smith cyclopropanation of chromene alcohols such as 15 were not ideal as the cyclopropanation was not suitable for large-scale chemistry due to the safety concerns associated with diethylzinc. This problem was addressed by turning to the Corey–Chaykovsky cyclopropanation, a more benign and scalable method, to access the cyclopropyl chromane system. In order to generate the requisite α,β-unsaturated ester 37, the synthetic sequence was modified slightly (Scheme 8). This route also accommodated installation of the gem-dimethyl substituents in analogy with chromene GSMs 28–32. The ester moiety was installed by lithiation of the previously described alkynyl derivative 22, followed by reaction with ethyl chloroformate to afford 36, which in turn underwent gold-catalyzed cyclization to provide 37. We were initially concerned that 1,4-addition of the Corey–Chaykovsky reagent might be impeded due to steric hindrance imparted by the gem-dimethyl substituents. However, the reaction proceeded smoothly to provide the corresponding cyclopropyl chromane intermediate in good yield. Subsequent reduction of the ester with DIBAL-H provided the alcohol 38, which was then converted to amine 39. The previously employed two-step process involving Mitsunobu reaction with phthalimide and deprotection with hydrazine was replaced with a more step- and atom-efficient one-pot process involving tosylation followed by displacement with ammonia to afford amine 39. Finally, condensation/cyclization with lactone 7 and chiral separation delivered the desired analogs such as 44 and 45 (Table 2).

Scheme 8. Second-generation synthesis of cyclopropyl chromane amines. Reagents and conditions: (a) n-BuLi, –78 °C, then ethyl chloroformate; (b) (JohnPhos)Au(MeCN)SbF₆ (1–5 mol%); (c) trimethylsulfoxonium iodide, t-BuOK; (d) DIBAL-H; (e) Ts₂O, NEt₃, then 7 N NH₃/MeOH. Note: R = Cl, CF₃, OCF₃, OMe; see Table 2 for details.

The cyclopropyl chromane strategy actually led to an improvement in Aβ42-lowering activity, with 33 having an IC₅₀ value of 5.6 nM, versus 30 nM for 20. Notably, the opposite enantiomer 40 was 23-fold less potent. In accordance with the design objectives, metabolic stability of 33 was significantly improved compared to 20 (HLM CL_int,app values of 37.9 and 108 μL/min/mg, respectively). Furthermore, cyclopropyl chromane 33 exhibited an MDR ER of less than 2.5 despite being one of our most polar GSMs under 10 nM made to date (clog P = 2.2, LipE = 5.0).28 SAR of the cyclopropyl chromanes tracked very closely with both the chromene and the indole series. For example, CF₃-substitution in the 6-position was critical for potency: moving this substituent to the 7-position (i.e., 41) or replacing it with a methoxy group (43) led to significant loss in Aβ-lowering activity.

At this stage, cyclopropyl chromane 33 appeared to be the optimal compound based on its exquisite in vitro potency, improved metabolic stability, and reduced lipophilicity, and it was therefore selected for evaluation in a rat efficacy model. However, as seen with several earlier compounds such as 3 and 5, poor central Aβ42-lowering activity was observed, which was attributed in part to inadequate central exposure (data not shown). The challenge of achieving robust central activity with γ-secretase modulators of reduced lipophilicity has been well documented in the literature.11,12 We therefore hypothesized that the in vitro/in vivo disconnect observed with 33 potentially could be rescued by a slight increase in lipophilicity. Taking a cue from the chromene series, we therefore installed gem-dimethyl substitution on the cyclopropyl chromane methylene. Gratifyingly, this structural modification had little impact on potency, and 44 maintained acceptable metabolic stability and MDR ER, with Aβ42 IC₅₀ = 4.9 nM, CL_int,app = 34.5 μL/min/mg, and MDR ER = 2.5. Likewise, the corresponding trifluoromethoxy substituted compound 45 was equipotent with a similar ADME profile.

At this juncture, chromenes 30 and 32 and cyclopropyl chromanes 44 and 45 were selected for in-depth profiling in vivo based on their superior in vitro potency and favorable ADME profile. However, lethality was observed with chromene 30 at 40 mg kg^–1 in a rat time-course efficacy study, whereas cyclopropyl chromanes 44 and 45 were well tolerated at the same dose. Given that this unexpected adverse event, which was confirmed in a rat dose-escalation study (vide infra), appeared rapidly and seemed to be C_max-driven, we hypothesized that it could be attributed to an acute cardiovascular (CV) safety liability. To test this hypothesis, we turned to the use of the rat unpaced isolated heart Langendorff assay, which allows measurement of left ventricle pressure/contractility, heart rate, and coronary vascular perfusion pressure.29,30 This model can help determine whether there is a direct adverse effect on the heart, versus a centrally mediated event; it has the additional advantage of higher throughput, as compared to conducting CV studies with telemetered animals.

As shown in Table 3, a remarkable difference in the activity of chromenes 30/32 and cyclopropyl chromanes 44/45 was observed in the Langendorff assay. Chromenes 30 and 32 exhibited potent and rapid effects on left ventricular pressure/contractility and perfusion pressure, whereas the corresponding cyclopropyl chromanes 44 and 45 were devoid of activity against these endpoints at concentrations >30 μM. The chromenes completely inhibited left ventricular contractility in this preparation while dramatically increasing coronary perfusion pressure. These results supported the hypothesis that the observed lethality may be attributed to a CV-mediated adverse event. Rat dose-escalation studies were subsequently conducted with all four lead compounds to establish translation from the Langendorff assay to in vivo tolerability. As indicated in Table 3, lethality was observed with both chromenes 30 and 32, at 30 mg kg^–1 and 100 mg kg^–1, respectively, whereas the corresponding cyclopropyl chromanes 44 and 45 exhibited excellent tolerability up to and including a 300 mg kg^–1 dose. The in vivo findings observed with the chromenes 30 and 32 occurred at unbound C_max exposures of 205 nM and 620 nM, respectively. A similar rank order of potency was observed in the Langendorff assay, where effects on contractility were observed, albeit with IC₅₀ values that were approximately 4.0 to 6.6-fold higher (IC₅₀ values of 1355 nM and 2477 nM for 30 and 32, respectively). It is interesting to note that this relatively small structural modification (replacement of an olefin with a cyclopropyl ring and removal of the chiral methyl group) had such a dramatic impact on cardiovascular safety. Notably, the specific target responsible for this effect was not identified, in spite of broad off-target pharmacology screening at Cerep. The Langendorff assay served as an efficient phenotypic counter-screen; it was subsequently used prospectively in the screening funnel to prioritize compounds for advancement.

Table 3. Correlation between activity in the Langendorff assay and in vivo toleration studies in rats.

R Compound	30	32	44	45
Aβ42 (IC50, nM)	4.9	6.0	4.9	5.1
Contractility (IC50, nM)	1355	2477	>30 000	>30 000
Left ventricle pressure (IC50, nM)	1031	1625	>30 000	>30 000
Perfusion pressure (EC50, nM)	576	1369	>30 000	>30 000
Observation/dose in rat dose-escalation study	Lethality (1/6) a at 30 mg kg–1	Lethality (1/6) b at 100 mg kg–1	Well tolerated at 300 mg kg–1	Well tolerated at 300 mg kg–1
C max at indicated dose	5.84 μM total 205 nM free	17.7 μM total 620 nM free	90.4 μM total 1620 nM free	60.9 μM total 670 nM free

^aObserved with 1/6 rats when dosed at 30 mg kg^–1 p.o. and 6/6 rats when dosed at 100 mg kg^–1 p.o.

^bObserved with 1/6 rats when dosed at 100 mg kg^–1 p.o. (highest dose for this compound).

Having established that the cyclopropyl chromane series was devoid of activity in the Langendorff assay while exhibiting excellent tolerability in rat dose-escalation studies at 300 mg kg^–1, this series was selected for further profiling in vitro and in vivo. Compound 44 exhibited the best overall profile; key in vitro and in vivo data is given in Table 4. Excellent selectivity was achieved over notch (notch intracellular domain, NICD)31 and hERG32 (>3200-fold and 3180-fold, respectively), while acceptable physicochemical properties were maintained. As noted earlier, the lead series was specifically designed to target moderate lipophilicity space, given the suboptimal in vitro/in vivo correlation that had been observed with earlier, more polar compounds in similar in vitro potency space. Nevertheless, compound 44 demonstrated favorable in vitro ADME characteristics, including good microsomal stability and passive permeability and adequate MDR efflux ratio (HLM CL_int,app = 34.5 μL/min/mg, RRCK P_{app, A→B} = 5.3 × 10^–6 cm s^–1,33 MDR ER = 2.5). This, in turn, resulted in an encouraging rat pharmacokinetic profile (CL = 26.4 mL/min/kg, F = 67%) and acceptable rodent brain penetration as indicated by an unbound brain-to-plasma ratio (C_b,u/C_p,u) of 0.23.

Table 4. In vitro and in vivo profile of compound 44.

Parameter	Measurement
In vitro potency/selectivity
Aβ42 (IC50, nM)	4.9
NICD (IC50, μM)	>15.8
hERG (IC50, μM)	15.6
Physicochemical properties
clog P/log D a	3.2/3.8
LipE/LipMetE	4.5/1.5
CNS MPO b	3.9
Solubility (pH 6.5) c	63 μM
In vitro ADME
HLM CLint,app	34.5 μL/min/mg
RRCK Papp, A→B	5.3 × 10–6 cm s–1
MDR ER	2.5
CYP 1A2 IC50	>30 μM
CYP 2C19 IC50	16.9 μM
CYP 2C8 IC50	7.2 μM
CYP 2C9 IC50	22.7 μM
CYP 2D6 IC50	14.2 μM
CYP 3A4 IC50	16.3 μM
Rat PK profile
CL d	26.4 mL/min/kg
T 1/2 d	1.6 h
%F e	67%
B/P f	0.76
C b,u/Cp,u g	0.23

^aShake flask log D.34

^bCalculated CNS MPO desirability score was obtained using the published algorithm.35

^cKinetic solubility was measured at Analiza Inc.36

^dDetermined from 1 mg kg^–1 intravenous dose.

^eDetermined from 1 mg kg^–1 intravenous dose and 5 mg kg^–1 oral dose.

^fDetermined from the brain and plasma exposure at the 1, 2, 4, and 7 h time-points following a 5 mg kg^–1 subcutaneous dose.

^gPlasma and brain free fractions of 44 in rat were 1.8% and 0.54%, respectively.

Compound 44 was examined in rat time-course studies at oral doses of 10 and 40 mg kg^–1 to assess its effect on de novo synthesis of brain Aβ42. As shown in Fig. 3, robust, dose- and time-dependent reductions were achieved as compared to vehicle treatment, and at the 4 h time-point, the levels of brain Aβ42 were maximally reduced by 40% and 62% at the 10 mg kg^–1 and 40 mg kg^–1 dose, respectively. The corresponding unbound brain concentrations at this time-point were 11 and 67 nM for the two doses, respectively (see ESI† for details on Aβ42 reductions at each time point and the corresponding brain and plasma exposure). Notably, the robust central efficacy of 44 was achieved at a significantly lower dose and with reduced unbound brain drug concentrations as compared to earlier GSMs such as 3. Compound 3 required an unbound brain concentration of 225 nM to afford a 40% maximal reduction of brain Aβ42 at the 3 h time-point.17,37 Compound 44, on the other hand, achieved the same level of Aβ42 reduction at an unbound brain drug level of only 11 nM, despite similar in vitro IC₅₀ values for these two compounds. The significantly improved in vivo efficacy of 44 relative to earlier compounds, in concert with its maintaining a favorable physicochemical property profile and overcoming the safety hurdles observed in the rat dose-escalation studies, marked an important milestone for the program.

Fig. 3. Rat in vivo efficacy of cyclopropyl chromane 44.

Conclusion

Herein we have described the design and synthesis of a novel series of GSMs that incorporate chromene- and cyclopropyl chromane-derived heterocyclic systems as bioisosteres for a naphthyl moiety that was present in preliminary lead 4. Molecular modeling played a key role in rationalizing the 3D conformation of the series and subsequently evaluating and prioritizing new design ideas. The observation of apparent C_max-driven lethality in subsequent in vivo rat efficacy studies led to the hypothesis that chromenes 30 and 32 may carry a CV liability. This prompted the use of the Langendorff isolated heart model to examine a potentially direct CV effect on the heart. Chromenes 30 and 32 displayed dose-dependent effects on left ventricular pressure/contractility and perfusion pressure, whereas compounds in the closely related cyclopropyl chromane series (44 and 45) were devoid of activity in the Langendorff assay. This translated into excellent tolerability up to 300 mg kg^–1 in rat dose-escalation studies with 44 and 45. Taken together, these studies highlight how subtle structural modifications can have a profound effect on the safety profile of a given series. Lead cyclopropyl chromane 44 demonstrated excellent in vitro potency (Aβ42 IC₅₀ = 4.9 nM) while maintaining acceptable lipophilicity (clog P = 3.2) and ADME parameters. This led to robust and sustained reductions of brain Aβ42 in rat time-course studies when dosed orally at 10 and 40 mg kg^–1. In vivo efficacy of 44 was achieved at significantly lower doses and free drug levels relative to earlier compounds such as 3 thus meeting a key design objective. In conclusion, the overall profile of 44 supports further advancement and in-depth characterization of its in vivo efficacy and safety.

Experimental section

General information

All solvents and reagents were obtained from commercial sources and were used as received. Reactions were monitored by TLC (TLC plates F254, Merck) or UPLC-MS analysis (Waters Acquity, ESCI +/–, APCI +/–). Gas chromatography-mass spectrometry (GC-MS) was performed with an Agilent 5890 GC Oven and an Agilent 5973 Mass Selective Detector. Melting points were obtained with a Thomas-Hoover melting point apparatus and are uncorrected. ¹H NMR and ¹³C NMR spectra were obtained using deuterated solvent on a Varian 400 MHz instrument. All ¹H NMR shifts are reported in δ units (ppm) relative to the signals for chloroform (7.27 ppm) and methanol (3.31 ppm). All ¹³C shifts are reported in δ units (ppm) relative to the signals for chloroform (77.0 ppm) and methanol (49.1 ppm) with ¹H-decoupled observation. All coupling constants (J values) are reported in hertz (Hz). NMR abbreviations are as follows: br, broadened; s, singlet; d, doublet; t, triplet; q, quartet; p, pentuplet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublets. High-resolution mass spectra (HRMS) were acquired on an Agilent model 6220 MS (TOF). Optical rotations were determined with a Jasco P-2000 polarimeter. Column chromatography was carried out on silica gel 60 (32–60 mesh, 60 Å) or on pre-packed Biotage™ or ISCO columns. HPLC purity analysis of the final test compounds was carried out using one of five methods. Method A: UPLC/UV/MS using a Waters Acquity CSH C18 column, 2.1 × 50 mm, with 1.7 μm particles; UV purity detected at 215 nm; mass spectrometer ESI positive/negative switching, acquiring from m/z 150 to 1000; mobile phase A = 0.1% formic acid in water (v/v); mobile phase B = 0.1% formic acid in acetonitrile (v/v); gradient beginning at 95% A, 5% B, increasing to 100% B over 1.2 min, and remaining at 100% B until 1.5 min; flow rate: 1.0 mL min^–1. Method B: column: Waters Atlantis dC18 4.6 × 50 mm, 5 μm; mobile phase A: 0.05% TFA in water (v/v); mobile phase B: 0.05% TFA in acetonitrile (v/v); gradient: 95.0% H₂O/5.0% acetonitrile, linear to 5% H₂O/95% acetonitrile in 4.0 min, HOLD at 5% H₂O/95% acetonitrile to 5.0 min. Flow rate: 2 mL min^–1. Purity detected at 215 nm. Mass spectrometer ESI positive acquiring from m/z 160 to 2000 Da. Method C: UPLC/UV. Chembiotek Research International, Kolkata, India. Column: Agilent Zorbax SB C18, 50 × 4.6 mm, 1.8 μm; UV purity detected at 220 nm; mobile phase A = 0.05% TFA in water; mobile phase B = acetonitrile. Method D: UPLC/UV. Chembiotek Research International, Kolkata, India. Column: Waters Atlantis dC18, 50 × 4.6 mm, 1.8 μm; UV purity detected at 220 nm; mobile phase A = 0.05% TFA in water; mobile phase B = acetonitrile. Method E: UPLC/UV/MS using Chiral Technologies CHIRALPAK® AS-H column, 4.6 × 100 mm, 5 μm; mass spectrometer ESI positive, acquiring from m/z 160 to 650; mobile phase A = CO₂; mobile phase B = methanol; 80 : 20 A/B hold for 10 min; column temperature: 40 °C; back pressure: 120 Bar; flow rate: 1.5 mL min^–1. All final compounds were determined to have a purity of >95% by one of the aforementioned methods unless stated otherwise.

Synthesis of 2-{[(1aS,7bS)-2,2-dimethyl-6-(trifluoromethyl)-1a,2-dihydrocyclopropa[c]chromen-7b(1H)-yl]methyl}-7-(4-methyl-1H-imidazol-1-yl)-3,4-dihydro-2H-pyrido[1,2-a]pyrazine-1,6-dione (44)

N-{[2,2-Dimethyl-6-(trifluoromethyl)-1a,2-dihydrocyclopropa[c]chromen-7b(1H)-yl]methyl}-1-(2-hydroxyethyl)-5-(4-methyl-1H-imidazol-1-yl)-6-oxo-1,6-dihydropyridine-2-carboxamide

1-[2,2-Dimethyl-6-(trifluoromethyl)-1a,2-dihydrocyclopropa[c]chromen-7b(1H)-yl]methanamine (see ESI†) (3.60 g, 13.3 mmol) was dissolved in THF (50 mL), and DABAL-Me₃ (97%, 4.5 g, 17 mmol) was added portion-wise. The reaction mixture was warmed to 45 °C for 45 min, whereupon lactone 7 (4.63 g, 18.9 mmol) was added. The resulting mixture was heated at reflux for 2 h, cooled in an ice bath and quenched via slow addition of water (10 mL). Aqueous 1 M NaOH solution (50 mL) was introduced and the mixture was stirred at room temperature for 15 min before being extracted with EtOAc. The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure; the residue was suspended in diethyl ether and collected via filtration to afford the product as an off-white solid. Yield: 6.10 g, 11.8 mmol, 89%. LCMS m/z 517 [M + H]⁺. ¹H NMR (400 MHz, CD₃OD) δ 8.16 (d, J = 1.3 Hz, 1H), 7.85 (br d, J = 2.0 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.37 (ddq, J = 8.4, 2.2, 0.7 Hz, 1H), 7.21–7.23 (m, 1H), 6.86–6.90 (m, 1H), 6.37 (d, J = 7.5 Hz, 1H), 4.32–4.39 (m, 3H), 3.72–3.82 (m, 2H), 2.22 (d, J = 1.0 Hz, 3H), 1.90 (dd, J = 8.6, 5.9 Hz, 1H), 1.50 (s, 3H), 1.23 (s, 3H), 1.18 (dd, J = 8.7, 4.9 Hz, 1H), 1.07 (dd, J = 5.7, 5.1 Hz, 1H).

rel-2-{[(1aS,7bS)-2,2-Dimethyl-6-(trifluoromethyl)-1a,2-dihydrocyclopropa[c]chromen-7b(1H)-yl]methyl}-7-(4-methyl-1H-imidazol-1-yl)-3,4-dihydro-2H-pyrido[1,2-a]pyrazine-1,6-dione

Triethylamine (2.5 mL, 18 mmol) was added to a 0 °C suspension of N-{[2,2-dimethyl-6-(trifluoromethyl)-1a,2-dihydrocyclopropa[c]chromen-7b(1H)-yl]methyl}-1-(2-hydroxyethyl)-5-(4-methyl-1H-imidazol-1-yl)-6-oxo-1,6-dihydropyridine-2-carboxamide (6.10 g, 11.8 mmol) in THF (100 mL). Methanesulfonic anhydride (2.5 g, 14 mmol) was then added portion-wise, and the reaction mixture was stirred under ice cooling for 45 min. Additional triethylamine (1 mL, 7 mmol) and methanesulfonic anhydride (1 g, 6 mmol) were introduced, and stirring was continued for 2 h. After addition of triethylamine (1 mL, 7 mmol) and methanesulfonic anhydride (0.5 g, 3 mmol) and a further 30 min of stirring, TBD (6.0 g, 42 mmol) was added and the reaction was allowed to continue for 30 min at 0 °C. Additional TBD (2 g, 14 mmol) was introduced, and after 30 min at 0 °C, another charge of TBD (3 g, 21 mmol) was added. After 30 min, the reaction mixture was partitioned between water (100 mL) and EtOAc (750 mL). The organic layer was washed with water (100 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (gradient: 0% to 10% MeOH in CH₂Cl₂) which provided partially purified material (4.8 g); this was treated with diethyl ether (50 mL), warmed to reflux for 10 min, cooled to room temperature, and filtered to provide the racemic title compound as a pale yellow solid (3.5 g, 7.0 mmol, 59%). ¹H NMR (400 MHz, CDCl₃) δ 8.21 (d, J = 0.9 Hz, 1H), 7.74 (d, J = 1.8 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H), 7.33–7.37 (m, 1H), 7.30 (d, J = 7.8 Hz, 1H), 7.12 (br s, 1H), 6.86 (d, J = 8.6 Hz, 1H), 4.96 (d, J = 14.8 Hz, 1H), 4.35 (ddd, J = 14.3, 6.7, 4.0 Hz, 1H), 4.19 (ddd, J = 14.3, 8.1, 4.2 Hz, 1H), 3.64–3.79 (m, 2H), 3.20 (d, J = 14.7 Hz, 1H), 2.28 (s, 3H), 1.80 (dd, J = 8.7, 5.8 Hz, 1H), 1.53 (s, 3H), 1.27 (s, 3H), 1.17 (dd, J = 5.7, 5.4 Hz, 1H), 1.08 (dd, J = 8.8, 5.3 Hz, 1H). The racemate was separated into its enantiomers via supercritical fluid chromatography (column: Phenomenex Lux Cellulose-1, 250 × 21.2 mm, 5 μm; isocratic conditions: mobile phase A: 60% carbon dioxide; mobile phase B: 40% methanol + 0.2% ammonium hydroxide; detection 210 nm; flow: 80.0 mL min^–1; back pressure: 120 Bar).

2-{[(1aS,7bS)-2,2-Dimethyl-6-(trifluoromethyl)-1a,2-dihydrocyclopropa[c]chromen-7b(1H)-yl]methyl}-7-(4-methyl-1H-imidazol-1-yl)-3,4-dihydro-2H-pyrido[1,2-a]pyrazine-1,6-dione (44)

The first-eluting enantiomer (1.8 g) was suspended in ethyl acetate (25 mL), heated to reflux and treated with additional ethyl acetate (10 mL). After cooling to room temperature, a solid was removed via filtration, and the filtrate was concentrated under reduced pressure to provide an off-white solid. This was dissolved in ethyl acetate (10 mL), heated to reflux and treated with heptane (20 mL); the mixture was cooled to room temperature and the resulting crystalline solid was isolated via filtration and washed with heptane (1.36 g, 2.73 mmol, 23%). m.p. 198–202 °C softens, 203–204 °C melts and decomposes; LCMS m/z 499.3 [M + 1]⁺; ¹H NMR (400 MHz, CD₃OD) δ 8.27–8.28 (m, 1H), 7.86–7.89 (m, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.31–7.35 (m, 1H), 7.26–7.30 (m, 2H), 6.86 (d, J = 8.4 Hz, 1H), 5.17 (d, J = 14.8 Hz, 1H), 4.28–4.36 (m, 1H), 4.14–4.22 (m, 1H), 3.73–3.85 (m, 2H), 3.05 (d, J = 14.6 Hz, 1H), 2.23 (s, 3H), 2.05 (dd, J = 8.6, 5.9 Hz, 1H), 1.52 (s, 3H), 1.30 (s, 3H), 1.10 (dd, J = 8.6, 5.0 Hz, 1H), 1.06 (dd, J = 5.5, 5.4 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 160.0, 157.4, 155.6, 138.9, 138.1, 136.4, 131.1, 130.4, 128.4, 126.1 (q, ¹J_CF = 270.7 Hz), 125.1 (q, ³J_CF = 3.7 Hz), 124.5 (q, ²J_CF = 32.3 Hz), 124.4 (q, ³J_CF = 3.7 Hz), 119.8, 116.6, 110.1, 73.8, 51.0, 45.2, 41.2, 35.8, 28.5, 26.6, 21.3, 16.0, 13.3; [α]22D –15.0 (c 0.99, MeOH); HRMS m/z, calcd [M + H]⁺ for C₂₆H₂₅F₃N₄O₃ 499.1952, observed 499.1956; retention time: 8.35 minutes (column: Phenomenex Lux Cellulose-1, 4.6 × 250 mm, 5 μm; mobile phase A: carbon dioxide; mobile phase B: 0.2% [7 M solution of ammonia in ethanol] in methanol; gradient: 5% B from 0 to 1.0 minute, then linear from 5% to 60% B for 8.5 minutes; flow rate: 3.0 mL per minute). The indicated stereochemistry was assigned on the basis of single crystal X-ray determination of compound 44 (see ESI† for details).

2-{[(1aR,7bR)-2,2-dimethyl-6-(trifluoromethyl)-1a,2-dihydrocyclopropa[c]chromen-7b(1H)-yl]methyl}-7-(4-methyl-1H-imidazol-1-yl)-3,4-dihydro-2H-pyrido[1,2-a]pyrazine-1,6-dione (ent-44)

The second-eluting enantiomer yielded the enantiomer of compound 44 (ent-44) (1.8 g, 3.6 mmol, 30%). LCMS m/z 499.3 [M + H]⁺. ¹H NMR (400 MHz, CD₃OD) δ 8.27 (br s, 1H), 7.86–7.89 (m, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.31–7.35 (m, 1H), 7.26–7.30 (m, 2H), 6.86 (d, J = 8.6 Hz, 1H), 5.17 (d, J = 14.6 Hz, 1H), 4.28–4.36 (m, 1H), 4.14–4.22 (m, 1H), 3.73–3.85 (m, 2H), 3.05 (d, J = 14.6 Hz, 1H), 2.23 (s, 3H), 2.05 (dd, J = 8.5, 6.0 Hz, 1H), 1.52 (s, 3H), 1.30 (s, 3H), 1.04–1.13 (m, 2H). Retention time: 9.56 min, using conditions identical to those described above for compound 44.

All animal experiments were carried out in strict accordance with federal, state, local and institutional guidelines governing the use of laboratory animals in research and were reviewed and approved by Pfizer Institutional Animal Care and Use Committee.

Abbreviations used

Aβ

Amyloid-β peptide

AD

Alzheimer's disease

ADME

Absorption, distribution, metabolism, and excretion

APP

Amyloid precursor protein

AB

Apical to basolateral

BA

Basolateral to apical

CL_int,app

Apparent intrinsic clearance

CNS MPO

Central nervous system multiparameter optimization

CSF

Cerebrospinal fluid

CV

Cardiovascular

CYP

Cytochrome P450

GSI

γ-Secretase inhibitor

GSM

γ-Secretase modulator

HHEP

Human hepatocytes

HLM

Human liver microsomes

LipE

Lipophilic efficiency

LipMetE

Lipophilic metabolism efficiency

MDCK

Madin–Darby canine kidney

MDR1

Multidrug resistance protein (P-glycoprotein, P-gp)

PK

Pharmacokinetic

PD

Pharmacodynamic

PS

Presenilin

RLM

Rat liver microsomes

SAR

Structure–activity relationship

TPSA

Topological polar surface area