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

Article abstract of DOI:10.1039/c6md00406g

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 molecul

Full text of DOI:10.1039/c6md00406g

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RESEARCH ARTICLE
Discovery of cyclopropyl chromane-derived
pyridopyrazine-1,6-dione γ-secretase modulators
with robust central efficacy†
Cite this: DOI: 10.1039/
c6md00406g
a
a
d
*
*
Martin Pettersson, Douglas S. Johnson, Danica A. Rankic,
Gregory W. Kauffman,a Christopher W. am Ende,d Todd W. Butler,d Brian Boscoe,d
Edelweiss Evrard,a Christopher J. Helal,d John M. Humphrey,d Antonia F. Stepan,a
Cory M. Stiff,d Eddie Yang,d Longfei Xie,d Kelly R. Bales,b Eva Hajos-Korcsok,b
Stephen Jenkinson,g Betty Pettersen,f Leslie R. Pustilnik,e David S. Ramirez,g
Stefanus J. Steyn,c Kathleen M. Woodb and Patrick R. Verhoesta
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 tac-
tics 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 reduc-
tion 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.
Received 18th July 2016,
Accepted 5th October 2016
DOI: 10.1039/c6md00406g
www.rsc.org/medchemcomm
β-secretase (BACE) and γ-secretase enzymes. In an effort to de-
velop disease-modifying therapies that can slow or halt the
Introduction
Alzheimer's disease (AD) is the most common form of demen-
tia among the elderly population, affecting approximately 1 in
5 people over the age of 65.1 It is a debilitating neurodegener-
ative disease that presents with gradual loss of memory, im-
paired speech, inability to carry out tasks of daily living, and
is ultimately fatal.1 AD pathology is characterized by the accu-
mulation of amyloid plaques consisting primarily of Aβ42,
which is a neurotoxic peptide produced via sequential pro-
cessing of the amyloid precursor protein (APP) by 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 promis-
ing 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 fa-
vor 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 tet-
ramers, potentially preventing further oligomerization of
Aβ42.5 GSMs therefore hold the promise of selectively reduc-
ing levels of the toxic species without inhibiting notch signal-
ing, and avoiding the side effects observed in GSI clinical tri-
als.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 allo-
steric sites on presenilin that are different from those
targeted by GSIs.9,10
a Neuroscience and Pain Medicinal Chemistry, Cambridge, Massachusetts 02139,
USA. E-mail: martin.pettersson@pfizer.com; Tel: +(617) 395 0705
b Neuroscience and Pain Research Unit, Cambridge, Massachusetts 02139, USA
c Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and
Development, Cambridge, Massachusetts 02139, USA
d Neuroscience and Pain Medicinal Chemistry, Groton, Connecticut 06340, USA.
E-mail: danica.rankic@pfizer.com; Tel: +(860) 441 4354
e Primary Pharmacology Group, Groton, Connecticut 06340, USA
f Drug Safety R&D, Pfizer Worldwide Research and Development, Groton,
Connecticut 06340, USA
g Global Safety Pharmacology, Pfizer Worldwide Research and Development, La
Jolla, California 92121, USA
Analysis of the GSM literature clearly reveals the challenge
of identifying potent GSMs within favorable CNS physico-
chemical property space.11 γ-Secretase is an intra-membrane
† Electronic supplementary information (ESI) available. CCDC 1452774. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c6md00406g
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cleaving aspartyl protease, which may underlie the apparent Results and discussion
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 exempli-
fied by 1 (Scheme 1).13,14 Because of difficulties in further im-
proving potency while maintaining acceptable physico-
chemical properties and ADME (absorption, distribution,
metabolism, and excretion) characteristics, we shifted our ef-
forts toward the design of novel, conformationally restricted
heterocyclic cores with increased polarity. As previously de-
scribed, 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 there-
fore selected for further optimization (Scheme 1). A rapid sur-
vey of diverse chemical space using parallel medicinal chem-
istry (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 po-
tent 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 alter-
native heterocyclic replacements for the naphthyl group.17
Herein we describe the design and synthesis leading to the
discovery of cyclopropyl chromane-derived GSMs, which over-
came significant safety hurdles and achieved excellent Aβ42-
lowering activity in vivo.
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 promi-
nently 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 combina-
tion 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.
4-(Trifluoromethyl)phenol (12) was alkylated with
propargyl bromide (11, Scheme 3). The resultant alkynyl ar-
ene 13 was deprotonated and then reacted with paraformal-
dehyde 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 16 via a two-step process
involving Mitsunobu reaction with phthalimide followed by
hydrazine-mediated deprotection. The corresponding satu-
rated chromane amine 17 was readily accessed via hydroge-
nation of 16. The chromene/chromane amines 16 and 17
Scheme 1 Design of pyridopyrazine-1,6-dione GSMs.
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Scheme 2 Overview of synthetic strategy.
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%.
were then coupled to lactone
7
via
a
DABCO bis-
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 alco-
hols such as 23 (Scheme 5). In this case, the subsequent cy-
clization 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
trimethylaluminum (DABAL-Me3)-mediated amidation to af-
ford 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 com-
pound 20 due to its susceptibility to air oxidation.
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.
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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)AuIJMeCN)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.
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 methyl-
magnesium bromide, afforded secondary alcohol intermedi-
ate 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.
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 CLint,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 satu-
rated chromane 21 (racemic) showed improved metabolic sta-
bility, but this structural modification resulted in a signifi-
cant 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 in-
stability of several of the synthetic intermediates. In fact, ex-
amples 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 substitu-
tion. 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 sta-
bility was significantly improved (CLint,app values of 108 and
47.8 μL/min/mg for 20 and 28, respectively), indeed generat-
ing 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 se-
ries.17 For example, replacing the critical trifluoromethyl sub-
stituent with a less lipophilic chlorine atom, as in 29, led to a
reduction in activity. Introduction of a conformational con-
straint via insertion of an (S)-methyl substituent on the meth-
ylene linker to afford 30 delivered an almost three-fold im-
provement in potency while maintaining good metabolic
stability (Aβ42 IC50 = 4.9 nM, CLint,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, CLint,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 confor-
mation, 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 ap-
plied 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
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Table 1 In vitro pharmacology and disposition data for pyridopyrazine-1,6-dione GSMs 20–32
R
IC50 (Aβ42, nM)a
clog P
MDR ERb
1.7
HLM CLint,app (μL/min/mg)c
20
21
28
30
2.4
108
322
13
2.5
3.4
1.9
2.0
37.6
47.8
29
30
31
32
a
53
3.1
3.7
3.7
3.8
1.9
1.9
1.8
1.9
n.d.d
14.3
242
4.9
801
6.0
25.7
Aβ42 IC50 values were obtained in a whole-cell assay using CHO APPwt cells. Aβ42 IC50 values are the geometric mean of at least three experi-
b
ments except for compound 20 (n = 2). MDR efflux ratio using an MDR1/MDCK assay utilizing MDCK cells transfected with the gene that en-
codes human P-glycoprotein.27 c Human liver microsome-derived intrinsic clearance.22,23 d Not determined.
conformation of 28 does indeed prefer a “turned” conforma-
tion consistent with the putative bioactive conformation and
conformationally restricted GSMs reported in the patent lit-
erature (Fig. 1a).11 However, given the pseudo-symmetry
above and below the planar pyridopyrazine core and the ab-
sence of a conformational control element, there are two
possible conformers of equal probability (Fig. 1b). Consis-
tent with our observations in the indole series,17 introduc-
tion of the (S)-methyl substituent (30) reinforces the
chromene group in the putative bioactive conformation, be-
low 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
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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.
the remarkable difference in potency between 30 and 31
(Table 1).
tone 7 as previously described. The resulting racemic cyclo-
propyl chromane GSM (not shown) was separated via chiral
HPLC to afford the enantiomerically pure analogs 33 and 40
(Table 2).
While exploring geminal dimethyl substitution on the
chromene as a means of improving metabolic stability, an al-
ternative 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 bioac-
tive 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 ac-
tivity. With this in mind, we envisioned that the chromene
olefin could be replaced with a cyclopropyl ring; we antici-
pated that this modification would both maintain sufficient
sp2 character and serve as an efficient conformational control
element (Scheme 6). Application of the conformational analy-
sis workflow described above to cyclopropyl chromane 33
supported this hypothesis, suggesting that this (S,S) diaste-
reomer, with a 92% probability, would position the cyclo-
propyl chromane moiety in the requisite bioactive conforma-
tion, below the plane of the core (Fig. 2). Furthermore, we
hypothesized that this design may lead to improved meta-
bolic stability, as the resultant chromane methylene is no
longer allylic.
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 diethyl-
zinc. 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 syn-
thetic sequence was modified slightly (Scheme 8). This route
also accommodated installation of the gem-dimethyl substitu-
ents 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 cycliza-
tion to provide 37. We were initially concerned that 1,4-addi-
tion of the Corey–Chaykovsky reagent might be impeded due
to steric hindrance imparted by the gem-dimethyl substitu-
ents. 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 displace-
ment with ammonia to afford amine 39. Finally,
The initial synthetic approach to cyclopropyl chromane-
derived GSMs such as 33 relied on allylic alcohol intermedi-
ate 15 (Scheme 7). This material was subjected to
Simmons–Smith cyclopropanation to afford cyclopropyl alco-
hol 34. subsequent two-step alcohol-to-amine inter-
a
A
conversion delivered 35, which was then reacted with lac-
Scheme 6 Design of cyclopropyl chromane series.
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served, which was attributed in part to inadequate central ex-
posure (data not shown). The challenge of achieving robust
central activity with γ-secretase modulators of reduced lipo-
philicity has been well documented in the literature.11,12 We
therefore hypothesized that the in vitro/in vivo disconnect ob-
served with 33 potentially could be rescued by a slight in-
crease in lipophilicity. Taking a cue from the chromene se-
ries, we therefore installed gem-dimethyl substitution on the
cyclopropyl chromane methylene. Gratifyingly, this structural
modification had little impact on potency, and 44 maintained
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.
acceptable metabolic stability and MDR ER, with Aβ42 IC50
=
4.9 nM, CLint,app = 34.5 μL/min/mg, and MDR ER = 2.5. Like-
wise, the corresponding trifluoromethoxy substituted com-
pound 45 was equipotent with a similar ADME profile.
condensation/cyclization with lactone 7 and chiral separation
delivered the desired analogs such as 44 and 45 (Table 2).
The cyclopropyl chromane strategy actually led to an im-
provement 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 signifi-
cantly improved compared to 20 (HLM CLint,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 sig-
nificant loss in Aβ-lowering activity.
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 ad-
verse event, which was confirmed in a rat dose-escalation
study (vide infra), appeared rapidly and seemed to be Cmax
-
driven, we hypothesized that it could be attributed to an
acute cardiovascular (CV) safety liability. To test this hypothe-
sis, we turned to the use of the rat unpaced isolated heart
Langendorff assay, which allows measurement of left ventri-
cle 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.
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 ob-
As shown in Table 3, a remarkable difference in the activ-
ity of chromenes 30/32 and cyclopropyl chromanes 44/45 was
Scheme 7 First-generation synthesis of cyclopropyl chromane amines. Reagents and conditions: (a) ZnEt2, CH2I2, 88%; (b) CBr4, PPh3, 70%; (c)
NH3/MeOH, 93%.
Scheme 8 Second-generation synthesis of cyclopropyl chromane amines. Reagents and conditions: (a) n-BuLi, −78 °C, then ethyl chloroformate;
(b) (JohnPhos)AuIJMeCN)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.
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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 ERb
2.4
HLM CLint,app (μL/min/mg)c
33
40
5.6
2.2
37.9
129
2.2
1.9
70.5
41
42
52
63
2.2
1.9
1.7
1.8
14.6
43.9
43
44
181
4.9
1.2
3.2
2.2
2.5
33.7
34.5
45
5.1
3.3
2.1
42.4
a
Aβ42 IC50 values were obtained in a whole-cell assay using CHO APPwt cells. Aβ42 IC50 values are the geometric mean of at least three experi-
ments. MDR efflux ratio using an MDR1/MDCK assay utilizing MDCK cells transfected with the gene that encodes human P-glycoprotein.27
b
c
Human liver microsome-derived intrinsic clearance.22,23
observed in the Langendorff assay. Chromenes 30 and 32
exhibited potent and rapid effects on left ventricular pres-
sure/contractility and perfusion pressure, whereas the corre-
sponding cyclopropyl chromanes 44 and 45 were devoid of
activity against these endpoints at concentrations >30 μM.
The chromenes completely inhibited left ventricular contrac-
tility in this preparation while dramatically increasing coro-
nary perfusion pressure. These results supported the hypoth-
esis that the observed lethality may be attributed to a CV-
mediated adverse event. Rat dose-escalation studies were sub-
sequently conducted with all four lead compounds to estab-
lish translation from the Langendorff assay to in vivo tolera-
bility. 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
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and including a 300 mg kg−1 dose. The in vivo findings
observed with the chromenes 30 and 32 occurred at unbound
Cmax 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, respec-
tively). 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. Nota-
bly, the specific target responsible for this effect was not
identified, in spite of broad off-target pharmacology screen-
ing at Cerep. The Langendorff assay served as an efficient
phenotypic counter-screen; it was subsequently used pro-
spectively in the screening funnel to prioritize compounds
for advancement.
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. Excel-
lent selectivity was achieved over notch (notch intracellular
domain, NICD)31 and hERG32 (>3200-fold and 3180-fold, re-
spectively), 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 po-
tency space. Nevertheless, compound 44 demonstrated favor-
able in vitro ADME characteristics, including good micro-
somal stability and passive permeability and adequate
MDR efflux ratio (HLM CLint,app
RRCK Papp, A→B = 5.3 × 10−6 cm s−1 33
=
34.5 μL/min/mg,
,
MDR ER = 2.5). This,
in turn, resulted in an encouraging rat pharmacokinetic
profile (CL = 26.4 mL/min/kg, F = 67%) and acceptable ro-
dent brain penetration as indicated by an unbound brain-
to-plasma ratio (Cb,u/Cp,u) of 0.23.
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 com-
pared 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). Nota-
bly, 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.
Table 3 Correlation between activity in the Langendorff assay and in vivo toleration studies in rats
45
30
32
44
R Compound
Aβ42 (IC50, nM)
4.9
6.0
4.9
5.1
Contractility (IC50, nM)
Left ventricle pressure (IC50, nM)
Perfusion pressure (EC50, nM)
Observation/dose in rat
dose-escalation study
1355
1031
576
2477
1625
1369
>30 000
>30 000
>30 000
Well tolerated
at 300 mg kg−1
90.4 μM total
1620 nM free
>30 000
>30 000
>30 000
Well tolerated
at 300 mg kg−1
60.9 μM total
670 nM free
Lethality (1/6)a
at 30 mg kg−1
5.84 μM total
205 nM free
Lethality (1/6)b
at 100 mg kg−1
17.7 μM total
620 nM free
Cmax at indicated dose
a
b
Observed 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. Observed with 1/6 rats when dosed at
100 mg kg−1 p.o. (highest dose for this compound).
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Table 4 In vitro and in vivo profile of compound 44
conformation of the series and subsequently evaluating and
prioritizing new design ideas. The observation of apparent
Cmax-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 iso-
lated 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 cyclo-
propyl chromane series (44 and 45) were devoid of activity in
the Langendorff assay. This translated into excellent tolerabil-
ity 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 reduc-
tions 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 rela-
tive to earlier compounds such as 3 thus meeting a key de-
sign objective. In conclusion, the overall profile of 44 sup-
ports further advancement and in-depth characterization of
its in vivo efficacy and safety.
Parameter
Measurement
In vitro potency/selectivity
Aβ42 (IC50, nM)
NICD (IC50, μM)
4.9
>15.8
15.6
hERG (IC50, μM)
Physicochemical properties
clog P/log Da
3.2/3.8
4.5/1.5
3.9
LipE/LipMetE
CNS MPOb
Solubility (pH 6.5)c
63 μM
In vitro ADME
HLM CLint,app
RRCK Papp, A→B
MDR ER
CYP 1A2 IC50
CYP 2C19 IC50
CYP 2C8 IC50
CYP 2C9 IC50
CYP 2D6 IC50
CYP 3A4 IC50
34.5 μL/min/mg
5.3 × 10−6 cm s−1
2.5
>30 μM
16.9 μM
7.2 μM
22.7 μM
14.2 μM
16.3 μM
Rat PK profile
CLd
26.4 mL/min/kg
d
T1/2
1.6 h
67%
0.76
0.23
%Fe
B/Pf
g
Cb,u/Cp,u
a
b
Shake flask log D.34 Calculated CNS MPO desirability score was
obtained using the published algorithm.35 Kinetic solubility was
c
Experimental section
General information
measured at Analiza Inc.36 d Determined from 1 mg kg−1 intravenous
dose. Determined from 1 mg kg−1 intravenous dose and 5 mg kg−1
e
f
oral dose. Determined from the brain and plasma exposure at the
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 (Wa-
ters Acquity, ESCI +/−, APCI +/−). Gas chromatography-mass
spectrometry (GC-MS) was performed with an Agilent 5890
GC Oven and an Agilent 5973 Mass Selective Detector. Melt-
ing points were obtained with a Thomas-Hoover melting
1, 2, 4, and 7 h time-points following a 5 mg kg−1 subcutaneous dose.
g
Plasma and brain free fractions of 44 in rat were 1.8% and 0.54%,
respectively.
1
point apparatus and are uncorrected. H 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)
1
and methanol (49.1 ppm) with H-decoupled observation. All
coupling constants (J values) are reported in hertz (Hz). NMR
abbreviations are as follows: br, broadened; s, singlet; d, dou-
blet; t, triplet; q, quartet; p, pentuplet; m, multiplet; dd, dou-
blet 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 pu-
rity detected at 215 nm; mass spectrometer ESI positive/
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. Mo-
lecular modeling played a key role in rationalizing the 3D
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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. Col-
umn: 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;
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-
dihydrocyclopropaijc]chromen-7bIJ1H)-yl]methyl}-7-(4-methyl-
1H-imidazol-1-yl)-3,4-dihydro-2H-pyridoij1,2-a]pyrazine-1,6-di-
one. Triethylamine (2.5 mL, 18 mmol) was added to a 0 °C
suspension
of
N-{[2,2-dimethyl-6-(trifluoromethyl)-1a,2-
dihydrocyclopropaijc]chromen-7bIJ1H)-yl]methyl}-1-(2-hydroxy-
ethyl)-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 intro-
duced, and stirring was continued for 2 h. After addition of
triethylamine (1 mL, 7 mmol) and methanesulfonic anhy-
dride (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
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 fi-
nal 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-dihydrocyclopropaijc]chromen-7bIJ1H)-yl]methyl}-7-(4-
methyl-1H-imidazol-1-yl)-3,4-dihydro-2H-pyridoij1,2-a]-
pyrazine-1,6-dione (44)
1
mmol, 59%). H 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% am-
monium hydroxide; detection 210 nm; flow: 80.0 mL min−1;
back pressure: 120 Bar).
2-{[(1aS,7bS)-2,2-Dimethyl-6-(trifluoromethyl)-1a,2-
dihydrocyclopropaijc]chromen-7bIJ1H)-yl]methyl}-7-(4-methyl-
1H-imidazol-1-yl)-3,4-dihydro-2H-pyridoij1,2-a]pyrazine-1,6-di-
one (44). The first-eluting enantiomer (1.8 g) was suspended
in ethyl acetate (25 mL), heated to reflux and treated with ad-
ditional ethyl acetate (10 mL). After cooling to room tempera-
ture, 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
N - { [ 2 , 2 - D i m e t h y l - 6 - ( t r i f l u o r o m e t h y l ) - 1 a , 2 -
dihydrocyclopropaijc]chromen-7bIJ1H)-yl]methyl}-1-(2-hydroxy-
ethyl)-5-(4-methyl-1H-imidazol-1-yl)-6-oxo-1,6-dihydropyridine-
2-carboxamide. 1-[2,2-Dimethyl-6-(trifluoromethyl)-1a,2-
dihydrocyclopropaijc]chromen-7bIJ1H)-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, where-
upon 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 col-
lected 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,
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(1.36 g, 2.73 mmol, 23%). m.p. 198–202 °C softens, 203–204
GSI
γ-Secretase inhibitor
γ-Secretase modulator
Human hepatocytes
Human liver microsomes
Lipophilic efficiency
1
°C melts and decomposes; LCMS m/z 499.3 [M + 1]+; H NMR
GSM
HHEP
HLM
LipE
(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,
LipMetE Lipophilic metabolism efficiency
MDCK
MDR1
Madin–Darby canine kidney
Multidrug resistance protein (P-glycoprotein, P-
gp)
Pharmacokinetic
Pharmacodynamic
PK
PD
PS
RLM
SAR
TPSA
3
2
1JCF = 270.7 Hz), 125.1 (q, JCF = 3.7 Hz), 124.5 (q, JCF = 32.3
3
Hz), 124.4 (q, JCF = 3.7 Hz), 119.8, 116.6, 110.1, 73.8, 51.0,
Presenilin
45.2, 41.2, 35.8, 28.5, 26.6, 21.3, 16.0, 13.3; [α]2D2 −15.0 (c 0.99,
Rat liver microsomes
Structure–activity relationship
Topological polar surface area
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 Acknowledgements
solution of ammonia in ethanol] in methanol; gradient: 5% B
We thank Stacey Becker, Emily Miller, Michael Marconi,
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 stereo-
chemistry was assigned on the basis of single crystal X-ray de-
termination of compound 44 (see ESI† for details).
Emily Sylvain, Don Tyszkicwicz, and Karin Wallace for their
contributions to the in vivo studies. We also thank the Pfizer
ADME technology group for generating the in vitro pharmaco-
kinetic data to support the SAR efforts and Scott Obach and
Loretta Cox for metabolite ID studies. We would like to thank
Qingli Zhang and her team at WuXi Apptec and Sajal Mal at
Chembiotek for synthetic contributions. Finally we thank
Brian Samas for X-ray crystallographic analysis and Katherine
Brighty for critical review of the manuscript.
2-{[(1aR,7bR)-2,2-dimethyl-6-(trifluoromethyl)-1a,2-
dihydrocyclopropaijc]chromen-7bIJ1H)-yl]methyl}-7-(4-methyl-
1H-imidazol-1-yl)-3,4-dihydro-2H-pyridoij1,2-a]pyrazine-1,6-di-
one (ent-44). The second-eluting enantiomer yielded the
enantiomer of compound 44 (ent-44) (1.8 g, 3.6 mmol, 30%).
1
LCMS m/z 499.3 [M + H]+. H 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 accor-
dance 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.
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