Application of the bicyclo[1.1.1]pentane motif as a nonclassical phenyl ring bioisostere in the design of a potent and orally active γ-secretase inhibitor

Article abstract of DOI:10.1021/jm300094u

Replacement of the central, para-substituted fluorophenyl ring in the γ-secretase inhibitor 1 (BMS-708,163) withthe bicyclo[1.1.1]pentane motif led to the discovery of compound 3, an equipotent enzyme inhibitor with significant improvements in passive permeability and aqueous solubility. The modified biopharmaceutical properties of 3 translated into excellent oral absorption characteristics (～4-fold C_max and AUC values relative to 1) in a mouse model of γ-secretase inhibition. In addition, SAR studies into other fluorophenyl replacements indicate the intrinsic advantages of the bicyclo[1.1.1]pentane moiety over conventional phenyl ring replacements with respect to achieving an optimal balance of properties (e.g., γ-secretase inhibition, aqueous solubility/permeability, in vitro metabolic stability). Overall, this work enhances the scope of the [1.1.1]-bicycle beyond that of a mere spacer unit and presents a compelling case for its broader application as a phenyl group replacement in scenarios where the aromatic ring count impacts physicochemical parameters and overall drug-likeness.

Full text of DOI:10.1021/jm300094u

Article
pubs.acs.org/jmc
Application of the Bicyclo[1.1.1]pentane Motif as a Nonclassical
Phenyl Ring Bioisostere in the Design of a Potent and Orally Active
γ-Secretase Inhibitor
Antonia F. Stepan,* Chakrapani Subramanyam, Ivan V. Efremov, Jason K. Dutra, Theresa J. O’Sullivan,
Kenneth J. DiRico, W. Scott McDonald, Annie Won, Peter H. Dorff, Charles E. Nolan, Stacey L. Becker,
Leslie R. Pustilnik, David R. Riddell, Gregory W. Kauffman, Bethany L. Kormos, Liming Zhang,
Yasong Lu, Steven H. Capetta, Michael E. Green, Kapil Karki, Evelyn Sibley, Kevin P. Atchison,
Andrew J. Hallgren, Christine E. Oborski, Ashley E. Robshaw, Blossom Sneed,
and Christopher J. O’Donnell
Pfizer Worldwide Research & Development, Eastern Point Road, Groton, Connecticut 06340, United States
S
* Supporting Information
ABSTRACT: Replacement of the central, para-substituted
fluorophenyl ring in the γ-secretase inhibitor 1 (BMS-708,163) with
the bicyclo[1.1.1]pentane motif led to the discovery of compound 3,
an equipotent enzyme inhibitor with significant improvements in
passive permeability and aqueous solubility. The modified bio-
pharmaceutical properties of 3 translated into excellent oral
absorption characteristics (∼4-fold ↑ Cmax and AUC values relative
to 1) in a mouse model of γ-secretase inhibition. In addition, SAR
studies into other fluorophenyl replacements indicate the intrinsic
advantages of the bicyclo[1.1.1]pentane moiety over conventional
phenyl ring replacements with respect to achieving an optimal
balance of properties (e.g., γ-secretase inhibition, aqueous solubility/
permeability, in vitro metabolic stability). Overall, this work en-
hances the scope of the [1.1.1]-bicycle beyond that of a mere “spacer” unit and presents a compelling case for its broader
application as a phenyl group replacement in scenarios where the aromatic ring count impacts physicochemical parameters and
overall drug-likeness.
in differentiation and proliferation of embryonic cells, T-cells,
and splenic B-cells.5 Consequently, achieving APP selectivity
over Notch signaling protein has become an overarching goal in
the design of γ-secretase inhibitors (GSIs), and new inhibitor
classes have been shown to selectively lower Aβ production
without modulating Notch signaling. Some of these agents,
such as 1 (BMS-708,163, Figure 1), have progressed into advanced
clinical trials.6,7
INTRODUCTION
■
Alzheimer’s disease (AD) is the most common cause of dementia
in the elderly, with a prevalence of 5% after 65 years of age,
increasing to about 30% in people aged 85 years or older. It is
characterized clinically by progressive cognitive impairment,
often accompanied, in later stages, by psychobehavioral dis-
turbances as well as language impairment.1 Although the cause
of AD is not known, genetic and experimental evidence sug-
gests that β-amyloid peptide (Aβ) chains of 40−42 amino acids
are plausible contributors to the pathogenesis of the disease.2
The APP (β-amyloid precursor protein) is found in many types
of cell membranes, and the action of secretases (β and γ) on
this precursor protein releases the β-amyloids into the plasma
and the cerebrospinal fluid.3 Subsequent aggregation of these
peptides to oligomers and plaques are believed to be the key
event in disease progression. γ-Secretase represents a poten-
tially attractive drug target because the enzyme dictates the
solubility of the generated Aβ fragment by creating peptides of
various lengths, ranging from 37 (Aβ37) to 42 (Aβ42) amino
acid residues.4 γ-Secretase catabolizes several endogenous sub-
strates, including the Notch signaling protein, which is involved
As part of our efforts to identify novel chemotypes,8 we have
recently reported our structure−activity relationship (SAR) find-
ings on a series of N-substituted arylsulfonamides, which cul-
minated in the novel oxetane derivative 2 (Figure 1), a GSI with
Notch-sparing activity.9 To further improve upon the potency of
2 (IC50 (Aβ42) = 16.9 nM), we explored nonclassical bioisosteric
replacements of the central fluorophenyl ring in 1, which resulted
in the identification of compound 3 (Figure 1), a bicyclo[1.1.1]-
pentane derivative with subnanomolar γ-secretase inhibitory
potency in vitro and a robust pharmacological response in vivo.
Received: January 20, 2012
Published: March 15, 2012
© 2012 American Chemical Society
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Figure 1. Structures of 1, its corresponding bicyclo[1.1.1]pentane analogue 3, and the oxetane-containing GSI 2 (a) and comparison of spacial
features between a para-substituted phenyl ring and a disubstituted bicyclo[1.1.1]pentane system (b).
An additional finding resulting from this work is the significant
changes in physicochemical attributes upon introduction of the
bicyclo[1.1.1]pentane unit, most notably manifested by signif-
icant improvements in passive permeability and aqueous solu-
bility relative to 1. As such, our work provides a compelling case
for applications of this bicyclo[1.1.1]pentane system in drug
discovery as a strategy to “escape the flatland” imposed by aryl
systems10 and alter the overall physicochemical characteristics
during lead optimization.
in 4 with lithium borohydride (THF, 0 → 23 °C) furnished
cyanoalcohol 5, which was coupled to known sulfonamide 67,13
under Mitsunobu conditions to provide 7 (vide infra). Nitrile 7
was converted to the title oxadiazole 3 via a two-step sequence
involving treatment with hydroxylamine to generate an inter-
mediate amide-oxime, which was then transformed to 3 in the
presence of triethyl orthoformate and a catalytic amount of
boron trifluoride etherate.
The synthesis of analogues 8−25 (vide infra) is outlined in
Scheme 2 and relied on standard alkylation reactions using either
alkyl halides or alcohols. In some cases, the alkylation products
were obtained as a diastereomeric mixture, which was separated
chromatographically to afford the individual stereoisomers. The
desired oxadiazoles were accessed via a two-step protocol in-
volving addition of hydroxylamine to the corresponding nitriles,
followed by treatment with triethyl orthoformate and boron
trifluoride etherate. Alcohol 26 was accessed from nitrile 27
through α-methylation with methyl iodide in the presence of
LDA, followed by hydroboration with 9-BBN (Scheme 3).
Comparison of the in Vitro Pharmacology and
Biopharmaceutical Properties of 1 and 3. The rationale
for the use of the disubstituted bicyclo[1.1.1]pentane motif as a
bioisostere for the central fluorophenyl ring in compound 1
derived from the commonalities in generic spatial features of a
para-substituted phenyl ring and a di-substituted bicyclo[1.1.1]-
pentane system, which include comparable dihedral angles and
similar distances between substituents (“distances between key
carbon atoms”, Figure 1). This draws parallel with the previous
report by Pellicciari et al., who utilized this unusual motif as a
phenyl group replacement in the design of a glutamate receptor
antagonist.11 Consistent with this hypothesis, the bicyclo[1.1.1]-
pentane analogue 3 exhibited similar degree of γ-secretase
inhibition (IC50 (Aβ42) = 0.178 nM, Table 1) in our whole cell
assay when compared with 1 (IC50 (Aβ42) = 0.225 nM). Under
our experimental conditions, the γ-secretase inhibitory potency
and Notch selectivity for 1 were comparable to the previously
published values.7 As the stereoelectronic characteristics of
the bicyclo[1.1.1]pentane moiety are different from those of a
phenyl ring,11 the similarity in γ-secretase inhibitory potencies
of 1 and 3 suggests that the main role of the fluorophenyl
group in 1 is that of a spacer and that the bicyclo[1.1.1]pentane
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of analogue 3 commenced with
the preparation of known nitrile-ester 4 (Scheme 1)11,12 using a
a
Scheme 1. Synthesis of Bicyclo[1.1.1]pentane Analogue 3
a
Reagents and conditions: (i) LiBH4, THF, 0 → 23 °C, 18 h (98%
yield); (ii) 6, DIAD, PPh3, THF, 0 → 23 °C, 18 h (50% yield); (iii)
NH2OH/H2O (1/1), EtOH, reflux, 3 h; (iv) BF3·Et2O, CH(OMe)3,
(CHCl2)2, 70 °C, 9 h (42% yield over two steps).
slightly modified literature procedure (see Supporting Informa-
tion for details). Chemoselective reduction of the ester moiety
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a
Scheme 2. Synthesis of Compounds 8−25
a
Reagents and conditions: (i) alkyl bromide, TBAI, Cs2CO3, DMF, 23 °C; (ii) alkyl bromide, TBAB, K2CO3, EtOAc/H2O (5/1), 50 °C; (iii) alkyl
bromide, Cs2CO3, DMF, 80 °C, 23−48% yield; (iv) alkyl alcohol, DIAD, PPh3, THF, 23 °C; (v) alkyl alcohol, DEAD, PPh3, 23 °C; (vi)
(a) NH2OH·HCl, NEt3, EtOH, reflux; (b) (MeO)3CH, BF3·OEt, py, 80 °C; (vii) (a) NH2OH·HCl, K2CO3, EtOH, reflux; (b) (MeO)3CH,
b
BF3·OEt, THF, 80 °C; (viii) (a) aq NH2OH, EtOH, 80 °C; (b) (MeO)3CH, BF3·OEt, (CH2Cl2)2, 70 °C. The absolute configuration around the
c
cyclopropane ring was randomly assigned. The absolute configuration around the cyclobutane ring was determined using a series of 2-D NMR
d
e
spectroscopy experiments (COSY, HSQC, NOESY). The absolute configuration around the cyclobutane ring was randomly assigned. The
absolute configuration around the cyclohexane ring was randomly assigned.
a
Scheme 3. Synthesis of Alcohol 26
Table 1. In Vitro Pharmacology, Biopharmaceutical, and
Disposition Attributes of Compounds 1 and 3
1
3
a,b
IC50 (Aβ42, nM)
0.225 (52)
350 (14)
15.0
0.178 (4)
178 (4)
<3.80
b
a
Notch selectivity
Reagents and conditions: (i) MeI, LDA, THF, −78 → 23 °C, 16 h,
human hepatocytes CLint,app (μL/min/million
54% yield; (ii) 9-BBN, sodium borate, THF, −78 → 23 °C, 16 h, 30%
yield.
c
cells)
b,d
HLM CLint,app (mL/min/kg)
RRCK Papp (A to B) (10−6 cm/s)
<16.2 (4)
5.52
<8.17 (2)
19.3
e
moiety in 3 orients the oxadiazole and the sulfonamide scaffold
in the same well-defined, coplanar orientation found in 1, as evident
from the X-ray structures of the two compounds (Figure 2).
Introduction of the bicyclo[1.1.1]pentane moiety also resulted
in significant improvements in the aqueous solubility of 3 (Table 1).
In contrast to 1 (kinetic and thermodynamic solubility at pH
6.5 aqueous buffer was measured to be 0.60 and 1.70 μM,
respectively), 3 was relatively more soluble with measured
kinetic and thermodynamic solubilities of 216 and 19.7 μM,
respectively, in pH 6.5 aqueous buffer. We attribute this
improvement in solubility to the increased 3-dimensionality
of the bicyclo[1.1.1]pentane system relative to a phenyl ring,
a characteristic that can possibly prevent intermolecular π-
stacking. Besides the improvement in aqueous solubility, 3 also
demonstrated a considerable increase in intrinsic permeability in
the RRCK assay (1, Papp = 5.52 × 10−6 cm/s; 3, Papp = 19.3 ×
10−6 cm/s)14 and was not a substrate for multidrug resistant
gene (MDR1)-mediated efflux (Papp basolateral to apical (BA)/
f
ElogD
4.70
3.80
kinetic solubility (pH = 6.5, μM)
0.60
216
thermodynamic solubility (pH = 6.5, μM)
thermodynamic solubility (pH = 7.4, μM)
1.70
19.7
0.90
29.4
g
MDR1/MDCK BA/AB ratio
1.72
1.66
a
IC50 values were obtained in a whole cell assay using hu APPwt cells
by measuring Aβ1−42 as previously described.26 Number of repetitions
indicated in parentheses. CLint,app refers to total apparent intrinsic
b
c
clearance obtained from scaling in vitro half-lives in human
d
hepatocytes.15 CLint,app refers to total intrinsic clearance obtained
from scaling in vitro half-lives in human liver microsomes.15 RRCK
e
cells with low transporter activity were isolated from Madine−Darby
canine kidney cells and were used to estimate intrinsic absorptive
f
g
permeability.14 ELogD was measured at pH 7.4.16 MDR1/MDCK
assay utilized MDCK cells transfected with the gene that encodes
human p-glycoprotein.14
apical to basolateral (AB) ratio ∼1.66) in the MDR1/MDCK
assay.14
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Figure 2. ORTEP representation of the X-ray crystal structures of compounds 1 (a) and 3 (b).
In addition to the altered biopharmaceutical properties, 3 ex-
hibited a greater resistance toward metabolic turnover in cryo-
preserved human hepatocytes (3, apparent intrinsic clearance
(CLint,app) < 3.8 μL/min/million cells; 1, CLint,app = 15 μL/
min/million cells) and a comparable metabolic stability in human
liver microsomes (3, HLM CLint,app < 8.17 mL/min/kg; 1,
HLM CLint,app < 16.2 mL/min/kg).15 The lower CLint,app of 3
(versus 1) in cryopreserved human hepatocytes is most likely a
reflection of its lower lipophilicity (3, ELogD = 3.80; 1, ELogD =
4.70).16 The ElogD values of 1 and 3 are consistent with the
observations correlating lipophilicity reductions with higher
degrees of bond saturation (described by parameters such as
the aromatic ring count (N) and the ratio of sp3-hybridized car-
bon atoms to the total carbon count (Fsp3)4)10,17 and translate
into clear improvements in lipophilic efficiency (LiPE: 1, LipE =
4.76; 3, LipE = 6.55)18 and the central nervous system multi-
parameter optimization (CNS MPO: 1, CNS MPO = 2.49; 3,
CNS MPO = 2.97) desirability score.19 Furthermore, like 1,
compound 3 was devoid of inhibitory effects on the major
human cytochrome P450 (CYP) enzymes.20 Finally, no other
adverse safety risks of compound 3 (relative to 1) were ob-
served in in vitro safety screens (e.g., dofetilide binding,21
reactive metabolite assay,22 and CEREP promiscuity panel).
To further validate the observation that the bicyclo[1.1.1]-
pentane system can function as a nonclassical bioisostere for
para-substituted phenyl rings within this series of GSIs, the in
vitro pharmacologic potency of bicyclo[1.1.1]pentane-contain-
ing nitrile analogue 7 was compared to the known phenyl and
fluorophenyl derivatives 813a and 9.13b As shown in Table 2,
compound 7 (IC50 (Aβ42) = 0.99 nM) achieved the same
degree of γ-secretase inhibitory potency discerned with the
corresponding aryl analogues 8 (IC50 (Aβ42) = 1.04 nM) and 9
(IC50 (Aβ42) = 1.77 nM). As noted with compounds 1 and 3,
compound 7 also exhibited an increase in aqueous solubility (7,
kinetic solubility (pH = 6.5) = 272 μM; 8, kinetic solubility
(pH = 6.5) = 44.5 μM; 9, kinetic solubility (pH = 6.5) = 6.8 μM)
and a decrease in lipophility (7, ELogD = 3.80; 8, ELogD = 4.10;
9, ELogD = 4.30] relative to 8 and 9. Passive RRCK permeability
and metabolic stability were comparable for all three analogues.
In a parallel series of investigations, the para-substituted aryl
rings in compounds 1 and 8 were also replaced with simple alkyl/
cycloalkyl spacers to ensure that the bicyclo[1.1.1]pentane system
is the optimum replacement for this disubstituted aromatic ring.
The resulting compounds 10−25 were evaluated for γ-secretase
inhibition and in vitro biopharmaceutical properties. As seen in
Table 3, none of the compounds 10−25 exhibit the appropriate
balance between inhibitory potency, metabolic stability, and/or
Table 2. In Vitro Pharmacology and Disposition Data for
Compounds 7−9
8
9
7
a,b
IC50 (Aβ42, nM)
1.04 (4)
570 (3)
13.3
1.77 (4)
nd
0.99 (4)
467 (4)
<8.00
30.2
b
Notch selectivity
c
HLM CLint,app (mL/min/kg)
RRCK Papp (A to B) (10−6 cm/s)
<8.00
18.7
d
19.7
e
ElogD
4.10
4.30
3.80
f
MDR1/MDCK BA/AB
2.05
1.71
2.04
kinetic solubility (pH = 6.5, μM)
44.5
6.80
272
a
IC50 values were obtained in a whole cell assay using hu APPwt cells
by measuring Aβ1−42 as previously described.21 Number of repetitions
b
c
indicated in parentheses. CLint,app refers to total intrinsic clearance
obtained from scaling in vitro half-lives in human liver microsomes.15
d
RRCK cells with low transporter activity were isolated from Madine−
Darby canine kidney cells and were used to estimate intrinsic absorp-
tive permeability.14 ELogD was measured at pH 7.4.16 MDR1/MDCK
assay utilized MDCK cells transfected with the gene that encodes
human p-glycoprotein.14
e
f
passive permeability observed with 3 and 7. Although limited in
scope, these selected examples highlight the advantages of the
bicyclo[1.1.1]pentane system over conventional phenyl group
replacements23 with respect to achieving an ideal balance be-
tween in vitro inhibitory potency for γ-secretase and disposition
properties.
Having established the bicyclo[1.1.1]pentane system as the
ideal phenyl replacement, the pharmacokinetic (PK) parame-
ters of 3 were assessed in female rats following intravenous (iv)
administration at a dose of 1 mg/kg and oral (po) admin-
istration at a dose of 5 mg/kg (Table 4). Female rats were used
in this PK assessment due to gender differences in the
pharmacokinetic profile of 1 in male versus female rats.7 Plasma
exposure post iv dosing was characterized by a multiexponential
elimination. In line with the reported PK for compound 1
in the same species,7 compound 3 exhibited a low plasma
clearance (CLp) of 13.3
0.35 mL/min/kg, a moderate
volume of distibution (Vss = 2.12 0.30 L/kg) and complete
oral absorption resulting in an oral bioavailibity of ∼100%.
Compound 3 also demonstrated sufficient brain partitioning
as indicated by its total brain to total plasma (B/P) ratio of 0.61
and its brain availability (BA, calculated as (fu,brain[brain])/
(fu,plasma[plasma]) of 0.34.
in Vivo Pharmacology Studies on Compounds 1 and 3.
Because of its favorable disposition parameters upon po
administration, we next examined whether the good in vitro
potency of 3 translated into a robust pharmacodynamic (PD)
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Table 3. In Vitro Pharmacology and Disposition Data for Compounds 10−25
a
b
IC50 values were obtained in a whole cell assay using hu APPwt cells by measuring Aβ1−42 as previously described.26 Number of repetitions
indicated in parentheses. CLint,app refers to total intrinsic clearance obtained from scaling in vitro half-lives in human liver microsomes.15 ELogD
c
d
was measured at pH 7.4.16 RRCK cells with low transporter activity were isolated from Madine−Darby canine kidney cells and were used to
e
estimate intrinsic absorptive permeability.14
a
Table 4. Single-Dose Pharmacokinetic Parameters of Compound 3 in Female Rats
route
dose (mg/kg)
Cmax (μM)
Tmax (h)
AUC (0−24 h) (μM·h)
T1/2 (h)
CL (mL/min/kg)
13.3 0.35
Vss (L/kg)
F (%)
b
iv
1
5
2.54 0.06
2.18 0.32
2.12 0.30
100
b
po
1.86 0.09
2.00 0.00
19.4 0.46
a
b
Data represent means standard errors of the mean (n = 3). Vehicle for both iv (2 mL/kg, 0.5 mg/mL) and po (10 mL/kg, 0.5 mg/mL) studies
was 40% (w/v) hydroxypropyl-β-cyclodextrin.
response in vivo. Compound 1 was also included as a positive
control.7 Following po administration of compound 3 at 10
and 30 mg/kg and of compound 1 at 30 mg/kg to wild-type
male 129/sve mice,9,24 Aβ lowering was measured in brain,
cerebrospinal fluid (CSF), and plasma over a time course of
0−30 and 0−18 h, respectively. The corresponding exposures
were also measured in these matrixes to gain an understanding
of the PK/PD relationship for γ-secretase inhibition. From a PK
perspective, it is noteworthy to comment on the increases in
plasma, brain, and CSF concentration of 3 (versus 1) at the
identical 30 mg/kg doses (Table 5, the individual exposure and
Aβ values can be found in the Supporting Information), which
is most likely a reflection of the improvements in passive
permeability and solubility of the [1.1.1] analogue.
From a PD perspective (Figure 3), a significant reduction of
Aβ levels relative to the vehicle treated animals was discerned
with compound 3 in all three compartments and at both doses.
For example, maximum Aβ40 and Aβ42 reductions in brain were
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a
Table 5. Single-Dose Pharmacokinetic Parameters of Compounds 1 and 3 in 129/sve Mice Following 30 mg/kg po Dosing
b
b
b
Cmax (μM),
plasma
Tmax (h), AUC (0−last)
Cmax (μM),
Tmax (h), AUC (0−last)
Cmax (μM),
Tmax (h), AUC (0−last)
c
c
d
e
c
compd
plasma (μM·h), plasma
brain
brain
(μM·h), brain B/P
BA
CSF
CSF
(μM·h), CSF
1
3
1.41 0.24
6.48 1.42
1
1
3.63
14.3
2.08 0.45
5.60 1.69
1
1
5.18
12.0
1.43
0.84
0.72
0.62
0.012 0.002
0.053 0.017
1
1
0.024
0.118
a
b
Formulation of compound 1 and 3: 98% (v/v) Phosal 50 PG and 2% Tween (Polysorbate 80). Data represent means standard deviations of the
c
mean (compound 1, n = 4; compound 3, n = 5). AUC values based on mean matrix concentrations at each time point because this is a nonserial
study (compound 1, AUC (0−18 h); compound 2, AUC (0−14 h)]. B/P values calculated as AUCbrain/AUCplasma. The brain availability is
calculated as (fu,brain[brain])/(fu,plasma[plasma]). Compound 1: fu,brain = 0.005, fu,plasma = 0.01. Compound 3: fu,brain = 0.026, fu,plasma = 0.0354.
d
e
Figure 3. Time course changes of brain Aβ40 (a), brain Aβ42 (b), CSF Aβ40 (c), and plasma Aβ40 (d) levels and corresponding exposures (total) of
compound 3 following a single dose in wild-type 129/sve mice. Data shown are the mean and standard error of the mean (SEM) as % of vehicle Aβ
levels (n = 5 per time point).
observed at 5 h postdose at 30 mg/kg (Figure 3a,b: Aβ40
reduction, 64%; Aβ42 reduction, 31%). In CSF and plasma
(Figure 3c,d), the maximum reductions of Aβ40 were 64% and
59%, respectively, and were observed at the 1 h time point post
dosing 30 mg/kg. The increase of Aβ40 levels at the later time
points in both CSF and plasma has been widely observed for
multiple GSIs25 and is probably due to the inhibitor causing an
increase in Aβ at low concentrations but inhibition at higher
concentrations.25 The precise mechanisms leading to this rise in
Aβ are unknown.25 At 30 mg/kg, compound 1 demonstrated a
comparable PD profile in this preclinical model and induced a
maximum reduction of Aβ40 and Aβ42 in brain at the 4 h time
point (Figure 4: Aβ40 reduction, 53%; Aβ42 reduction, 42%).
Maximum reduction in Aβ40 levels in CSF (65%) and plasma
(55%) were discerned at the 2 and 0.5 h time points,
respectively.
in the required coplanar orientation. A favorable outcome
of this exercise was the increase in both passive permeability
and aqueous solubility characteristics of 3 relative to 1, a find-
ing that we attribute to the increased three-dimensionality
and concomitant disruption of planarity and intermolecular
π-stacking of the two aromatic rings in 1. These altered
physicochemical and biopharmaceutical properties manifested
in a significantly improved oral absorption of 3 (relative to 1)
in the mouse model of pharmacology. A such, the attractive
in vitro potency, in vivo oral efficacy, and favorable human
disposition attributes makes 3 a potential candidate for fur-
ther preclinical evaluation. Although limited in scope, our
SAR studies into alternative replacements of the fluorophenyl
group in 1 indicate the superiority of the bicyclo[1.1.1]-
pentane functionality over conventional phenyl ring replace-
ments (e.g., alkyl/cycloalkyl spacers) with respect to achieving
the desired balance between γ-secretase inhibition, aqueous
solubility/permeability, and in vitro metabolic liability. This
work therefore highlights the physicochemical changes in-
duced by this unusual phenyl bioisostere and showcases its
potential use as a tactic to “escape the flatland” of multiple
aryl systems in drug discovery.
CONCLUSION
■
The identification of compound 3, an equipotent γ-secretase
inhibitor relative to clinical candidate 1, indicates the fluorophenyl
moiety in 1 is not necessarily involved in any enzyme−inhibitor
specific interactions and the bicyclo[1.1.1]pentane motif is ideally
suited to dispose the oxadiazole and N-arylsulfonamide moieties
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Figure 4. Time course changes of brain Aβ40 (a), brain Aβ42 (b), CSF Aβ40(c), and plasma Aβ40 (d) levels and corresponding exposures (total) of
compound 1 levels following a single dose in wild-type 129/sve mice. Data shown are the mean and standard error of the mean (SEM) as % of
vehicle Aβ levels (n = 8 per time point).
Diisopropyl azodicarboxylate (470 μL, 2.40 mmol) was then added
EXPERIMENTAL SECTION
■
dropwise, and the reaction mixture was stirred at 23 °C for 12 h. The
Chemistry. Unless specified otherwise, starting materials were
reaction mixture was loaded directly onto a silica gel cartridge and was
available from commercial sources. Dry solvents and reagents were of
purified by flash column chromatography using a 0→75% gradient of
commercial quality and were used as purchased. 1H NMR spectra were
ethyl acetate/heptane to afford 460 mg (64% yield) of 7 as a white
recorded on Varian INOVA spectrometers (300, 400, or 500 MHz)
(Varian Inc., Palo Alto, CA) at room temperature. Chemical shifts are
expressed in parts per million δ relative to residual solvent as an
internal reference. Peak multiplicity are expressed as follows: singlet
(s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad
singlet (br s). The purity of title compounds used in pharmacology
testing was verified by HPLC-MS using the following method: 12 min
gradient on a HP1100C pump of increasing concentrations of ace-
tonitrile in water (5→95%) containing 0.1% formic acid with a flow
rate of 1 mL/min and UV detection at λ 220 and 254 nm on a Gemini
C18 150 mm × 4.6 mm, 5 μm column (Phenomenex, Torrance, CA).
Title compounds used in pharmacology testing were >95% pure.
Electrospray ionization mass spectra were obtained on a Waters ZQ
and ZMD instrument (Milford, MA) mass spectrometer operated in
positive or negative ionization mode using nitrogen as a carrier gas.
1
solid. H NMR (500 MHz, CDCl3) δ 7.78−7.70 (m, 2H), 7.60−7.51
(m, 2H), 6.48 (br s, 1H), 5.44 (br s, 1H), 4.15 (dd, J = 9.76, 5.13 Hz,
1H), 3.41 (d, J = 16.1 Hz, 1H), 3.24 (d, J = 16.1 Hz, 1H), 2.27−2.18
(m, 6H), 2.17−2.06 (m, 1H), 2.01−1.89 (m, 1H), 1.82−1.69 (m, 1H),
1.24−1.11 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 170.5, 140.5,
137.2, 129.9 (2 C), 128.4 (2 C), 126.3 (q, J = 276.1 Hz), 117.4, 57.72,
54.50 (3 C), 44.91, 42.99, 30.34 (q, J = 29.5 Hz), 23.29, 20.86 (q, J =
2.80 Hz). LCMS m/z 450 (M + H+); [α]D24 = +9.80° (c 1.1, CHCl3).
(R)-2-{N-{[3-(1,2,4-Oxadiazol-3-yl)bicyclo[1.1.1]pentan-1-yl]-
methyl}-4-chlorophenylsulfonamido}-5,5,5-trifluoropentanamide
(3). To a solution of 7 (1.35 g, 3.00 mmol) in ethanol (30 mL) was
added hydroxylamine (50 wt % in water, 793 mg, 12.0 mmol). The re-
sulting reaction mixture was heated at 80 °C for 90 min and then
cooled to 23 °C and concentrated in vacuo. Dichloromethane (250 mL)
and water (60 mL) were added, the phases were separated, and the
Gas chromatography−mass spectroscopy (GC/MS) was performed
on a HP 5988A instrument (Palo Alto, CA). All synthetic reactions
organic layer was washed with brine, dried (Na2SO4), filtered, and
concentrated in vacuo to afford the intermediate amide-oxime as an
were carried out under nitrogen atmosphere or in sealed vials. The
off-white solid, which was used in the next step without further puri-
chemical yields reported below are unoptimized specific examples of
fication. To a mixture of the crude amide-oxime (1.39 g, 2.88 mmol)
one preparation.
and trimethyl orthoformate (920 mg, 8.67 mmol) in 1,2-dichloro-
3-(Hydroxymethyl)bicyclo[1.1.1]pentane-1-carbonitrile (5). To a
solution of 4 (190 mg, 1.26 mmol) in tetrahydrofuran (6.0 mL) at
0 °C was added lithium borohydride (2.0 M in tetrahydrofuran,
ethane (30 mL) was added boron trifluoride diethyl etherate (62.1 mg,
0.15 mmol) at 23 °C. The resulting mixture was stirred at 80 °C under
a water-cooled reflux condenser for 10 h, cooled to 23 °C, dry-loaded,
630 μL, 1.26 mmol). The reaction mixture was stirred at 23 °C for 12 h.
and then purified by flash column chromatography using a 0→70%
The cloudy mixture was then cooled to 0 °C (external), and methanol
gradient of ethyl acetate/heptane to afford 618 mg (42% yield) of 3 as
(12 mL) was added dropwise. This mixture was stirred at 23 °C for 2 h,
a white solid; mp = 163−165 °C (recrystallized from a mixture of ethyl
and the solvent was removed in vacuo. The crude product was purified
by flash column chromatography using a 0→100% gradient of ethyl
acetate/heptane to afford 166 mg (100% yield) of 5 as a colorless oil. 1H
NMR (500 MHz, CDCl3) δ 3.62 (d, J = 5.61 Hz, 2H), 2.21 (s, 6H),
1.32 (t, J = 5.74 Hz, 1H). GCMS m/z 122 (M − H+).
(R)-2-{4-Chloro-N-[(3-cyanobicyclo[1.1.1]pentan-1-yl)methyl]-
phenylsulfonamide-5,5,5-trifluoropentanamide (7). To a solution of
6 (552 mg, 1.60 mmol) and 5 (197 mg, 1.60 mmol) in tetrahydrofuran
(8.0 mL) at 0 °C was added triphenylphosphine (639 mg, 2.40 mmol).
1
acetate and heptane). H NMR (500 MHz, CDCl3) 8.64 (s, 1H),
7.83−7.78 (m, 2H), 7.60−7.51 (m, 2H), 6.62 (br s, 1H), 5.53 (br s,
1H), 4.18 (dd, J = 9.52, 5.61 Hz, 1H), 3.45 (q, J = 15.9 Hz, 2H), 2.27−
2.13 (m, 7H), 2.02−1.89 (m, 1H), 1.89−1.74 (m, 1H), 1.35−1.26 (m,
1H). 13C NMR (125 MHz, CDCl3) δ 170.5, 167.2, 164.7, 140.4, 137.5,
129.9, 128.6, 126.4 (q, J = 275.0 Hz), 58.1, 52.8, 45.8, 40.8, 32.9,
30.5 (q, J = 28.8 Hz), 21.1 (q, J = 2.5 Hz). LCMS m/z 493 (M + H+);
[α]24 +13.2° (c 0.78, CHCl3).
D
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(R)-2-[4-Chloro-N-(4-cyanobenzyl)phenylsulfonamido]-5,5,5-tri-
fluoropentanamide (8). To a solution of 6 (34.0 mg, 0.10 mmol) in
dimethylformamide (172 μL) at 23 °C was added 4-(bromomethyl)-
benzonitrile (19.6 mg, 0.10 mmol), followed by cesium carbonate
(103 mg, 0.31 mmol). The reaction mixture was stirred at that tem-
perature for 48 h. Ethyl acetate (2 mL) and water (1 mL) were then
added. The biphasic mixture was loaded onto a Varian ChemElut
(Hydromatrix) cartridge (6 cm3, 1 g sorbent). After 5 min, the car-
tridge was eluted with ethyl acetate (2 × 3 mL) and the solvent was
removed. The crude material was dissolved in dimethyl sulfoxide
(1 mL) and purified by reversed phase HPLC (Waters XBridge C18
column; mobile phase: 55/45→0/100 gradient of 0.1% NH4OH in
water/0.1% NH4OH in acetonitrile). Removal of the solvent in vacuo
(R)-2-[4-Chloro-N-(4-cyanobutyl)phenylsulfonamido]-5,5,5-tri-
fluoropentanamide (12). Compound 6 (400 mg, 1.16 mmol), bro-
mopentanenitrile (282 mg, 1.74 mmol), cesium carbonate (680 mg,
2.10 mmol), and tetrabutylammonium iodide (100 mg, 0.27 mmol)
were used to synthesize 12 using the procedure described for the
preparation of 11. The crude product was purified by flash column
chromatography using a 25→50% gradient of ethyl acetate/heptane to
afford 156 mg (31% yield) of 12 as a clear gum. 1H NMR (400 MHz,
CDCl3) δ 7.73 (d, J = 8.00 Hz, 2H), 7.52 (d, J = 8.59 Hz, 2H), 6.50
(br s, 1H), 5.47 (br s, 1H), 4.22 (dd, J = 9.18, 6.05 Hz, 1H), 3.42−3.31
(m, 1H), 3.19−3.09 (m, 1H), 2.36 (t, J = 6.93 Hz, 2H), 2.21−2.08 (m,
1H), 2.03−1.86 (m, 1H), 1.84−1.59 (m, 5H), 1.38−1.25 (m, 1H).
LCMS m/z 426 (M + H+).
1
(R)-2-{4-Chloro-N-{[(1S,2S)-2-cyanocyclopropyl]methyl}-
phenylsulfonamido}-5,5,5-trifluoropentanamide (13) and (R)-2-{4-
C h l o r o - N - { [ ( 1 R , 2 R ) - 2 - c y a n o c y c l o p r o p y l ] m e t h y l } -
phenylsulfonamido}-5,5,5-trifluoropentanamide (14). Compound 6
(593 mg, 1.72 mmol), trans-2-(hydroxymethyl)cyclopropanecarbonitrile
(racemic) (167 mg, 1.72 mmol), triphenylphosphine (729 mg, 2.76
mmol), and diethyl azodicarboxylate (443 μL, 2.41 mmol) were used to
synthesize 13 and 14 using the procedure described for the preparation
of 7. The crude product was purified by supercritical fluid chiral chro-
matography (MiniGram-2 Chiralcel OJ-H column; mobile phase: 90/10
CO2/MeOH) to afford 10.0 mg (1.4% yield) of 13 as a white solid and
10.0 mg (1.4% yield) of 14 as a white solid. The absolute configuration
afforded 22.2 mg (48% yield) of 8 as a white foam. H NMR (500
MHz, CDCl3) δ 7.74−7.68 (m, 2H), 7.65−7.60 (m, 2H), 7.57−7.52
(m, 2H), 7.49 (d, J = 8.54 Hz, 2H), 6.20 (br s, 1H), 5.25 (br s, 1H),
4.64 (d, J = 16.1 Hz, 1H), 4.42 (d, J = 15.9 Hz, 1H), 4.36 (dd, J = 8.91,
6.22 Hz, 1H), 2.20−2.08 (m, 1H), 2.08−1.93 (m, 1H), 1.88−1.71 (m,
1H), 1.47−1.36 (m, 1H). LCMS m/z 460 (M + H+).
(R)-2-[4-Chloro-N-(4-cyano-2-fluorobenzyl)phenylsulfonamido]-
5,5,5-trifluoropentanamide (9). To a solution of 6 (1.18 g, 3.42
mmol) in ethyl acetate (12 mL) and water (2.4 mL) at 23 °C was
added 4-(bromomethyl)-3-fluorobenzonitrile (0.73 mg, 3.42 mmol),
followed by potassium carbonate (946 mg, 6.85 mmol) and
tetrabutylammonium bromide (0.17 mg, 0.51 mmol). The reaction
mixture was stirred at 50 °C for 48 h. To the cooled reaction mixture
(23 °C), ethyl acetate (100 mL) and water (100 mL) were added. The
phases were separated, the organics were washed with water (100 mL)
and brine, dried (Na2SO4), filtered, and concentrated in vacuo. The
crude product was purified by flash column chromatography using a
0→25% gradient of ethyl acetate/dichloromethane to afford 1.26 g
1
of both isomers was randomly assigned. 13: H NMR (400 MHz),
CDCl3) δ 7.76−7.72 (m, 2H), 7.58−7.54 (m, 2H), 6.63 (s, 1H), 5.73
(s, 1H), 4.31−4.27 (m, 1H), 3.30 (dd, J = 15.4, 5.85 Hz, 1H), 3.12
(dd, J = 15.4, 8.78 Hz, 1H), 2.28−2.18 (m, 1H), 2.06−1.92 (m, 1H),
1.90−1.70 (m, 2H), 1.56−1.51 (m, 1H), 1.39−1.30 (m, 2H), 1.09−
1
1.04 (m, 1H); LCMS m/z 424 (M + H+). 14: H NMR (400 MHz,
CDCl3) δ 7.80−7.77 (m, 2H), 7.59−7.55 (m, 2H), 6.58 (s, 1H), 5.79
(s, 1H), 4.28 (dd, J = 9.17, 6.05 Hz, 1H), 3.43 (dd, J = 15.6, 5.66 Hz,
1H), 2.98 (dd, J = 15.8, 8.00 Hz, 1H), 2.21−2.12 (m, 1H), 2.06−1.91
(m, 1H), 1.86−1.76 (m, 2H), 1.47−1.42 (m, 1H), 1.34−1.23 (m, 2H),
1.03−0.97 (m, 1H); LCMS m/z 424 (M + H+).
1
(77% yield) of 9 as a white foam. H NMR (500 MHz, CDCl3) δ
7.76−7.71 (m, 2H), 7.68 (t, J = 7.69 Hz, 1H), 7.57−7.52 (m, J = 8.79
Hz, 2H), 7.46 (d, J = 8.05 Hz, 1H), 7.32 (d, J = 9.52 Hz, 1H), 6.29 (br
s, 1H), 5.35 (br s, 1H), 4.67 (d, J = 16.1 Hz, 1H), 4.49 (d, J = 16.1 Hz,
1H), 4.39 (dd, J = 9.15, 5.98 Hz, 1H), 2.18 (dtd, J = 14.2, 8.79, 5.86
Hz, 1H), 2.07−1.93 (m, 1H), 1.90−1.75 (m, 1H), 1.48−1.38 (m, 1H).
(R)-2-{4-Chloro-N-{[(1S,3S)-3-cyanocyclobutyl]methyl}-
phenylsulfonamido}-5,5,5-trifluoropentanamide (15) and (R)-2-{4-
Chloro-N-{[(1R,3R)-3-cyanocyclobutyl]methyl}phenylsulfonamido}-
5,5,5-trifluoropentanamide (16). Compound 6 (300 mg, 0.87 mmol),
hydroxymethyl-cyclobutanecarbonitrile (106 mg, 0.96 mmol), triphe-
nylphosphine (280 mg, 1.10 mmol), and diisopropyl azodicarboxylate
(280 mg, 1.30 mmol) were used to synthesize 15 and 16 using the
procedure described for the preparation of 7. The crude product was
purified by supercritical fluid chiral chromatography (Chiralpak AD-H
column; mobile phase: 85/15 CO2/methanol) to afford 69.0 mg (52%
yield) of 15 (cis) as a clear glass and 25.0 mg (6.6% yield) of 16 (trans)
as a clear glass. The relative configuration across the cyclopropane ring
was determined using a series of 2D NMR experiments (COSY,
HSQC, NOESY). 15: 1H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.59
Hz, 2H), 7.53 (d, J = 8.59 Hz, 2H), 6.51 (br s, 1H), 5.48 (br s, 1H),
4.20 (dd, J = 9.37, 5.85 Hz, 1H), 3.44 (dd, J = 14.64, 8.98 Hz, 1H),
3.07 (dd, J = 14.7, 5.56 Hz, 1H), 2.91 (quintet, J = 8.98 Hz, 1H),
2.68−2.53 (m, 1H), 2.52−2.37 (m, 2H), 2.29−2.18 (m, 1H), 2.18−
1.83 (m, 3H), 1.73 (ddd, J = 15.2, 9.90, 5.56 Hz, 1H), 1.33−1.14 (m,
[α]20 +33.0° (c 1.00, DMSO).
D
(R)-2-[4-Chloro-N-(2-cyanoethyl)phenylsulfonamido]-5,5,5-tri-
fluoropentanamide (10). To a solution of 6 (500 mg, 1.45 mmol) in
dimethylformamide (2.0 mL) at 23 °C was added 3-bromopropaneni-
trile (388 mg, 2.90 mmol), followed by cesium carbonate (945 mg,
2.90 mmol). The reaction mixture was stirred at 80 °C for 12 h. The
reaction mixture was then filtered through Celite, and the resulting
filtrate was concentrated in vacuo. The crude product was purified
by flash column chromatography using a 0→7.5% gradient of 7 N
methanolic NH3/dichloromethane to afford 132 mg (22% yield) of 10
1
as a colorless oil. H NMR (400 MHz, CDCl3) δ 7.83−7.77 (m, 2H)
7.61−7.56 (m, 2H), 6.41 (br s, 1H), 5.51 (br s, 1 H), 4.28 (dd, J =
9.39, 6.06 Hz, 1H), 3.68−3.58 (m, 1H), 3.50 (ddd, J = 15.1, 8.22, 5.28
Hz, 1H), 2.92−2.73 (m, 2H), 2.24−2.11 (m, 1H), 2.09−1.84 (m, 2H),
1.44 (td, J = 14.3, 6.85 Hz, 1H). LCMS m/z 398 (M + H+).
(R)-2-[4-Chloro-N-(3-cyanopropyl)phenylsulfonamido]-5,5,5-tri-
fluoropentanamide (11). To a solution of 6 (300 mg, 0.87 mmol) in
dimethylformamide (8 mL) at 23 °C was added 4-bromo-butyronitrile
(193 mg, 1.30 mmol) followed by cesium carbonate (511 mg, 1.57
mmol) and tetrabutylammonium iodide (73.8 mg, 0.20 mmol). The
reaction mixture was stirred at that temperature for 12 h. Ethyl acetate
(20 mL) was then added, and the organic layer was washed with water
(10 mL), a saturated solution of sodium bicarbonate (10 mL), and
brine (10 mL). The organics were dried (MgSO4), filtered, and con-
centrated in vacuo. The crude product was purified by flash column
chromatography using a 25→50% gradient of ethyl acetate/heptane to
afford 192 mg (53% yield) of 11 as a clear gum. 1H NMR (400 MHz,
CDCl3) δ 7.76−7.70 (m, 2H), 7.55−7.50 (m, 2H), 6.44 (br s, 1H),
5.53 (br s, 1H), 4.27 (dd, J = 9.18, 6.05 Hz, 1H), 3.50−3.39 (m, 1H),
3.22−3.12 (m, 1H), 2.38 (t, J = 7.42 Hz, 2H), 2.22−2.09 (m, 1H),
2.01−1.91 (m, 3H), 1.85−1.71 (m, 1H), 1.32 (dd, J = 14.3, 8.20 Hz,
1H). LCMS m/z 412 (M + H+).
1
1H); LCMS m/z 438 (M + H+). 16: H NMR (400 MHz, CDCl3) δ
7.70 (d, J = 8.59 Hz, 2H), 7.51 (d, J = 8.59 Hz, 2H), 6.51 (br s, 1H),
5.72 (br s, 1H), 4.17 (dd, J = 9.37, 5.66 Hz, 1H), 3.47 (dd, J = 14.6,
9.96 Hz, 1H), 3.15−3.04 (m, 1H), 2.89−2.75 (m, 1H), 2.50−2.34 (m,
2H), 2.34−2.25 (m, 1H), 2.17−1.99 (m, 2H), 1.99−1.85 (m, 1H),
1.78−1.62 (m, 1H), 1.28−1.12 (m, 2H); LCMS m/z 438 (M + H+).
(R)-2-{4-Chloro-N-{[(1R,3R)-3-cyano-3-methylcyclobutyl]methyl}-
phenylsulfonamido}-5,5,5-trifluoropentanamide (17) and (R)-2-{4-
Chloro-N-{[(1S,3S)-3-cyano-3-methylcyclobutyl]methyl}-
phenylsulfonamido}-5,5,5-trifluoropentanamide (18). Compound 6
(90.0 mg, 0.26 mmol), 3-(hydroxymethyl)-1-methylcyclobutanecarbo-
nitrile (26, 42.4 mg, 0.34 mmol), triphenylphosphine (88.9 mg, 0.34
mmol), and diisopropyl azodicarboxylate (78.5 mg, 0.37 mmol) were
used to synthesize 17 and 18 using the procedure described for the
preparation of 7. The crude product was purified by flash column
chromatography using a 0→50% gradient of ethyl acetate/heptane.
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The obtained material was further purified by supercritical fluid chiral
chromatography (Chiralpak OD-H column; mobile phase: 90/10
CO2/methanol) to afford 1.50 mg (1.3% yield) of 17 (trans) as a clear
glass and 7.10 mg (6.0% yield) of 18 (cis, as a 82:12 mixture of 18:17)
as a clear glass. The absolute configuration of both isomers was
randomly assigned. 17: 1H NMR (400 MHz, CDCl3) δ 7.72−7.70 (m,
2H), 7.53−7.51 (m, 2H), 6.50 (br s, 1H), 5.41 (br s, 1H), 4.20−4.12
(m, 1H), 3.42−3.31 (m, 1H), 3.17−3.06 (m, 1H), 2.87−2.74 (m, 1H),
2.65−2.52 (m, 2H), 2.20−2.06 (m, 1H), 2.00−1.86 (m, 2H), 1.78−
1.65 (m, 2H), 1.46 (s, 3H), 1.27−1.15 (m, 1H). 18, title compound
peaks only: 7.74−7.72 (m, 2H), 7.55−7.52 (m, 2H), 6.51 (br s, 1H),
5.47 (br s, 1H), 4.27−4.19 (m, 1H), 3.57−3.47 (m, 1H), 3.15−3.06
(m, 1H), 2.75−2.63 (m, 1H), 2.50−2.40 (m, 1H), 2.26−2.17 (m, 1H),
2.17−2.06 (m, 3H), 2.05−1.87 (m, 1H), 1.80−1.64 (m, 1H), 1.50 (s,
3H), 1.33−1.18 (m, 1H). Mixture of 17 and 18: LCMS m/z 452
(M + H+).
(R)-2-{4-Chloro-N-[(1-cyanocyclopropyl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (19). Compound
6 (1.00 g, 2.90 mmol), 1-hydroxymethyl-cyclopropanecarbonitrile
(366 mg, 3.77 mmol), triphenylphosphine (989 mg, 3.77 mmol), and
diisopropyl azodicarboxylate (811 mg, 3.77 mmol) were used to syn-
thesize 19 using the procedure described for the preparation of 7. The
crude product was purified by preparative reversed phase HPLC
(Phenomenex Gemini C18 column; mobile phase: 75:25→0:100
gradient of 0.1% NH4OH in water/0.1% NH4OH in methanol) to
afford 650 mg (52% yield) of 19 as a white solid. 1H NMR (400 MHz,
CDCl3) δ 7.86 (dd, J = 8.59, 1.76 Hz, 2H), 7.54 (dd, J = 8.49, 1.66 Hz,
2H), 6.62 (br s, 1H), 5.50 (br s, 1H), 4.28 (dd, J = 8.20, 6.83 Hz, 1H),
3.50 (d, J = 15.4 Hz, 1H), 3.28 (d, J = 15.4 Hz, 1H), 2.28−2.14 (m,
1H), 2.05−1.83 (m, 2H), 1.60−1.48 (m, 1H), 1.31 (s, 2H), 1.15−1.08
(m, 1H), 1.01−0.93 (m, 1H). LCMS m/z 424 (M + H+).
mixture was stirred at 70 °C and stirred for 12 h. The solvent was
removed in vacuo, and the resulting crude product was purified by
flash column chromatography using a 20→60% gradient of ethyl
acetate/heptane to afford 68.0 mg (95% yield) of 22 as a colorless
1
solid. H NMR (400 MHz, MeOD) δ 9.15 (s, 1H), 7.91−7.85 (m,
2H), 7.62−7.57 (m, 2H), 4.42 (dd, J = 8.30, 6.35 Hz, 1H), 3.88 (ddd,
J = 15.4, 9.37, 6.05 Hz, 1H), 3.72−3.61 (m, 1H), 3.29−3.22 (m, 1H),
3.18−3.07 (m, 1H), 2.22−2.00 (m, 3H), 1.77−1.63 (m, 1H). LCMS
m/z 441 (M + H+).
(R)-2-{N-[3-(1,2,4-Oxadiazol-3-yl)propyl]-4-chlorophenylsulfona-
mido}-5,5,5-trifluoropentanamide (23). To a solution of 11 (100 mg,
0.24 mmol) in ethanol (1.5 mL) at 23 °C was added hydroxylami-
ne·HCl (34.5 mg, 0.49 mmol), followed by triethylamine (0.05 mL,
0.36 mmol). The reaction mixture was stirred at 80 °C for 16 h, after
which time the solvent was removed in vacuo. To this crude product
was added pyridine (1.50 mL, 19.0 mmol), followed by trimethyl
orthoformate (2.00 mL, 18.3 mmol) and boron trifluoride diethyl
etherate (3.40 mg, 0.024 mmol). The reaction mixture was stirred at
80 °C for 16 h. The solvent was removed in vacuo, and the crude
product was purified by preparative reversed phase HPLC
(Phenomenex Gemini C18 column; mobile phase: 95/5→5/95
gradient of 0.1% NH4OH in water/0.1% NH4OH in methanol) to
afford 15.0 mg (15% yield) of 23 as a clear gum. 1H NMR (400 MHz,
CDCl3) δ 8.62 (s, 1H), 7.74−7.72 (m, 2H), 7.59−7.52 (m, 2H), 6.52
(br s, 1H), 5.49 (br s, 1H), 4.24 (dd, J = 8.40, 6.64 Hz, 1H), 3.49−3.36
(m, 1H), 3.23 (ddd, J = 14.8, 9.81, 4.88 Hz, 1H), 2.80 (t, J = 7.22 Hz,
2H), 2.23−1.69 (m, 4H), 1.43−1.28 (m, 2H). LCMS m/z 456 (M + H+).
(R)-2-{N-{[1-(1,2,4-Oxadiazol-3-yl)cyclopropyl]methyl}-4-chloro-
phenylsulfonamido}-5,5,5-trifluoropentanamide (24). Compound
19 (50.0 mg, 0.12 mmol), hydroxylamine (50 wt % in water, 32.3 mg,
0.49 mmol), trimethyl orthoformate (23.9 mg, 0.23 mmol), trifluoride
diethyl etherate (2.10 mg, 0.015 mmol), and boron trifluoride diethyl
etherate (2.10 mg, 0.015 mmol) were used to synthesize 24 using the
procedure described for the preparation of 3. The crude product was
purified by flash column chromatography using a 0→80% gradient of
ethyl acetate/heptane to afford 22.0 mg of 24 and 19 as a 9:1 mixture.
This material was repurified by preparative HPLC (Princeton 2-Ethyl
Pyridine column; mobile phase: 95/5 → 5/95 gradient of heptane/
(R)-2-{4-Chloro-N-{[(1R,4R)-4-cyanocyclohexyl]methyl}-
phenylsulfonamido}-5,5,5-trifluoropentanamide (20) and (R)-2-{4-
Chloro-N-{[(1R,4R)-4-cyanocyclohexyl]methyl}phenylsulfonamido}-
5,5,5-trifluoropentanamide (21). Compound 6 (414 mg, 1.20 mmol),
4-(hydroxymethyl)cyclohexanecarbonitrile (mixture of cis- and trans-
isomers) (250 mg, 1.50 mmol), triphenylphosphine (470 mg, 1.88
mmol), and diethyl azodicarboxylate (283 μL, 1.80 mmol) were used
to synthesize 20 and 21 using the procedure described for the prepara-
tion of 6. The crude product was purified by supercritical fluid chiral
chromatography (MiniGram-1 Chiralpak AS-H column; mobile phase:
85/15 CO2/ethanol) to afford 10.0 mg (1.8% yield) of 20 as a white
solid and 22.0 mg (3.9% yield) of 21 as a white solid. The absolute
1
ethanol) to afford 3.00 mg (8.6% yield) of 24 as a white solid. H
NMR (500 MHz, CDCl3) δ 8.54 (s, 1H), 7.87−7.69 (m, 2H), 7.57−
7.45 (m, 2H), 6.96 (br s, 1H), 5.36 (br s, 1H), 4.30 (dd, J = 9.03, 5.86
Hz, 1H), 3.82−3.69 (m, 2H), 2.44−2.35 (m, 1H), 2.17−2.03 (m, 1H),
1.91−1.79 (m, 1H), 1.55−1.47 (m, 1H), 1.44 (ddd, J = 9.33, 7.02, 4.64
Hz, 1H), 1.34−1.23 (m, 3H). LCMS m/z 467 (M + H+).
1
configuration of both isomers was randomly assigned. 20: H NMR
(400 MHz, CDCl3) δ 7.77−7.74 (m, 2H), 7.58−7.55 (m, 2H), 6.69 (s,
1H), 5.44 (s, 1H), 4.26 (dd, J = 9.76, 5.46 Hz, 1H), 3.34 (dd, J = 14.4,
10.2 Hz, 1H), 2.96−2.91 (m, 2H), 2.22−2.12 (m, 1H), 2.08, 1.87 (m,
4H), 1.82−1.58 (m, 4H), 1.52−1.23 (m, 4H); LCMS m/z 466 (M +
(R)-2-{N-{[(1S,3S)-3-(1,2,4-Oxadiazol-3-yl)cyclobutyl]methyl}-4-
chlorophenylsulfonamido}-5,5,5-trifluoropentanamide (25). To a
solution of 15 (120 mg, 0.27 mmol) in ethanol (2.5 mL) at 23 °C was
added hydroxylamine·HCl (19.4 mg, 0.27 mmol), followed by potas-
sium carbonate (18.9 mg, 0.14 mmol). The reaction mixture was
stirred at 80 °C for 48 h. Ethyl acetate (10 mL) was then added at
23 °C, the mixture was filtered, and the filtrate was concentrated in
vacuo. To this crude product in tetrahydrofuran (1.0 mL) was added
trimethyl orthoformate (1.50 mL, 14.0 mmol) and boron trifluoride
diethyl etherate (1.40 μL, 0.05 mmol). The reaction mixture was
stirred at 80 °C for 48 h. The solvent was removed in vacuo, and the
crude product was purified by preparative HPLC (Phenomenex
Phenyl Hexyl column; mobile phase: 95:5 → 0:100 gradient of 0.1%
formic acid in water/0.1% formic acid in methanol) to afford 1.60 mg
(2.5% yield) of 25 as a pale-white solid. 1H NMR (400 MHz, MeOD)
δ 9.10−9.07 (m, 1H), 7.87−7.86 (m, 2H), 7.61−7.55 (m, 2H), 4.36
(t, J = 8.00 Hz, 1H), 3.56−3.45 (m, 2H), 2.77−2.65 (m, 1H), 2.48−
2.38 (m, 2H), 2.12−1.94 (m, 6H), 1.61−1.49 (m, 1H). LCMS m/z
503 (M + Na).
1
H+). 21: H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 8.59 Hz, 2H),
7.55 (d, J = 8.59 Hz, 2H), 6.64 (s, 1H), 5.53 (s, 1H), 4.25−4.21 (m,
1H), 3.26−3.20 (m, 1H), 2.94−2.89 (m, 1H), 2.41−1.96 (m, 6H),
1.76−1.47 (m, 6H), 1.02−0.76 (m, 2H); LCMS m/z 466 (M + H+).
(R)-2-{N-[2-(1,2,4-Oxadiazol-3-yl)ethyl]-4-chlorophenylsulfona-
mido}-5,5,5- trifluoropentanamide (22). To a solution of 10 (70.0 mg,
0.18 mmol) in ethanol (2.0 mL) at 23 °C was added hydroxylamine
(50% v/v in water, 46.5 mg, 0.70 mmol). The reaction mixture was
stirred at 80 °C for 1 h. The solvent was then removed in vacuo, and
the resulting material was diluted with dichloromethane (5.0 mL) and
washed with water (5.0 mL). The organic layer was dried (MgSO4),
filtered, and concentrated in vacuo. The crude product was purified by
flash column chromatography using a 25 → 50% gradient of ethyl
acetate/heptane to afford 72.0 mg (96% yield) of the intermediate
1
amide-oxime as a colorless solid. H NMR (400 MHz, MeOD) δ
7.93−7.88 (m, 2H), 7.62−7.57 (m, 2H), 4.39−4.34 (m, 1H), 3.68−
3.59 (m, 1 H), 3.48 (ddd, J = 15.2, 9.88, 5.28 Hz, 1H), 2.57−2.39
(m, 2H), 2.18−2.04 (m, 3H), 1.83−1.74 (m, 1 H). LCMS m/z 431
(M + H+). To a solution of the above amide-oxime intermediate
(70.0 mg, 0.16 mmol) in 1,2-dichloroethane (2.0 mL) at 23 °C was
added trimethyl orthoformate (51.6 mg, 0.48 mmol), followed by
boron trifluoride diethyl etherate (3.40 mg, 0.02 mmol). The reaction
3-(Hydroxymethyl)-1-methylcyclobutanecarbonitrile (26). To a
solution of 3-methylenecyclobutanecarbonitrile (27, 1.00 g, 9.33
mmol) in tetrahydrofuran (40 mL) at 0 °C was added a solution of
9-borabicyclo[3.3.1]nonane (0.50 M in THF, 26.1 mL, 13.1 mmol)
dropwise over a period of 10 min. The reaction mixture was stirred
warmed to room temperature and stirred at that temperature for 2 h.
Water (50 mL) and sodium borate (4.30 g, 11.3 mmol) were then
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Journal of Medicinal Chemistry
Article
added, and the resulting mixture was stirred at room temperature for
12 h. The mixture was then filtered, and the filtrant was washed with
diethyl ether (20 mL). The layers of the filtrate were separated, and
the organic layer was extracted with diethyl ether (2 × 20 mL). The
combined organic extracts were washed with brine (50 mL), dried
(MgSO4), and filtered. The solvent was removed in vacuo, and the
crude product was purified by flash column chromatography using a
25 → 50% gradient of ethyl acetate/heptane to afford 192 mg (53%
(4) Pettersson, M.; Kauffman, G. W.; am Ende, C. W.; Patel, N. C.;
Stiff, C.; Tran, T. P.; Johnson, D. S. Novel γ-secretase modulators: a
review of patents from 2008 to 2010. Expert Opin. Ther. Pat. 2011, 21,
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Sanders, C. R. Substrate specificity of gamma-secretase and other
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advances in the identification of γ-secretase inhibitors to clinically test
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(7) Gillman, K. W.; Starrett, J. E.; Parker, M. F.; Xie, K.; Bronson,
J. J.; Marcin, L. R.; McElhone, K. E.; Bergstrom, C. P.; Mate, R. A.;
Williams, R.; Meredith, J. E.; Burton, C. R.; Barten, D. M.; Toyn, J. H.;
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Decicco, C. P.; Macor, J. E.; Olson, R. E. Discovery and Evaluation of
BMS-708163, a Potent, Selective and Orally Bioavailable γ-Secretase
Inhibitor. ACS Med. Chem. Lett. 2010, 1, 120−124.
1
yield) of 26 (as a 2:1 ratio of cis- and trans-isomers). H NMR (400
MHz, CDCl3) δ 3.70−3.54 (m, 2H), 2.81−2.33 (m, 3H), 2.16−1.87
(m, 2H), 1.60−1.40 (m, 3H). APCI m/z 126 (M + H).
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Synthesis of compound 4, H and 13C NMR spectra of com-
pound 3, X-ray crystallography data for compounds 1 and 3,
and biology protocols. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
(8) (a) Brodney, M. A.; Auperin, D. D.; Becker, S. L.; Bronk, B. S.;
Brown, T. M.; Coffman, K. J.; Finley, J. E.; Hicks, C. D.; Karmilowicz, M.
J.; Lanz, T. A.; Liston, D.; Liu, X.; Martin, B.-A.; Nelson, R. B.; Nolan,
C. E.; Oborski, C. E.; Parker, C. P.; Richter, K. E. G.; Pozdnyakov, N.;
Sahagan, B. G.; Schachter, J. B.; Sokolowski, S. A.; Tate, B.; Van Deusen,
J. W.; Wood, D. E.; Wood, K. M. Diamide amino-imidazoles: a novel
series of γ-secretase inhibitors for the treatment of Alzheimer’s disease.
Bioorg. Med. Chem. Lett. 2011, 21, 2631−2636. (b) Brodney, M. A.;
Auperin, D. D.; Becker, S. L.; Bronk, B. S.; Brown, T. M.; Coffman, K. J.;
Finley, J. E.; Hicks, C. D.; Karmilowicz, M. J.; Lanz, T. A.; Liston, D.;
Liu, X.; Martin, B.-A.; Nelson, R. B.; Nolan, C. E.; Oborski, C. E.; Parker,
C. P.; Richter, K. E. G.; Pozdnyakov, N.; Sahagan, B. G.; Schachter, J. B.;
Sokolowski, S. A.; Tate, B.; Wood, D. E.; Wood, K. M.; Van Deusen,
J. W.; Zhang, L. Design, synthesis, and in vivo characterization of a novel
series of tetralin amino imidazoles as γ-secretase inhibitors: discovery of
PF-3084014. Bioorg. Med. Chem. Lett. 2011, 21, 2637−2640.
(9) Stepan, A. F.; Karki, K.; McDonald, W. S.; Dorff, P. H.; Dutra,
J. K.; DiRico, K. J.; Won, A.; Subramanyam, C.; Efremov, I. V.;
O’Donnell, C. J.; Nolan, C. E.; Becker, S. L.; Pustilnik, L. R.; Sneed, B.;
Sun, H.; Lu, Y.; Robshaw, A. E.; Riddell, D.; O’Sullivan, T. J.; Sibley,
E.; Capetta, S.; Atchison, K.; Hallgren, A. J.; Miller, E.; Wood, A.;
Obach, R. S. J. Med. Chem. 2011, 54, 7772−7783.
Corresponding Author
*Phone: (860) 686-3247. E-mail: Antonia.Stepan@pfizer.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Drs. Amit S. Kalgutkar, Anabella Villalobos, and
Joshua R. Dunetz for helpful suggestions in preparing this
article, Drs. Anthony Kreft, David Rotella, and Alexander Porte
for fruitful discussions during the project transition from Wyeth,
and Brian Samas and Ivan Samardjiev for the single crystal X-ray
structure elucidation of compounds 1 and 3. We also thank James
Bradow, Robert P. Depianta, and Drs. Qi Yan and Laurence
Philippe-Venec from the purification group and Brendon Kapinos,
Walter E. Mitchell, Jillian B. Van Hausen, James J. Frederico, III,
John P. Umland, Mark W. Snyder, and Dr. Marina Y. Shalaeva
from the ADME high-throughput screening group at Pfizer,
Groton.
ABBREVIATIONS USED
■
(10) Lovering, F.; Bikker, J.; Humblet, C. Escape from Flatland:
Increasing Saturation as an Approach to Improving Clinical Success.
J. Med. Chem. 2009, 52, 6752−6756.
AD, Alzheimer’s disease; Aβ, amyloid-β; APP, β-amyloid precursor
protein; BA, brain availability; B/P, brain to plasma ratio; CLint,app
,
(11) (a) Pellicciari, R.; Raimondo, M.; Marinozzi, M.; Natalini, B.;
Costantino, G; Thomsen, C. (S)-(+)-2-(3′-Carboxybicyclo[1.1.1]-pentyl)-
glycine, a structurally new group 1 metabotropic glutamate receptor
antagonist. J. Med. Chem. 1996, 39, 2874−2876. (b) Costantino, G.;
Maltoni, K.; Marinozzi, M.; Camaioni, E.; Prezeau, L.; Pin, J.-P.;
Pellicciari, R. Synthesis and biological evaluation of 2-(3′-(1H-tetrazol-
5-yl)bicyclo[1.1.1]pent-1-yl)glycine (S-TBPG), a novel mGlu1
receptor antagonist. Bioorg. Med. Chem. 2001, 9, 221−227.
(12) Della, E. W.; Taylor, D. K. Synthesis of Some Bridgehead−
Bridgehead-Disubstituted Bicyclo[1.1.1]pentanes. J. Org. Chem. 1994,
59, 2986−2996.
(13) (a) Parker, M. F.; McElhone, K. E.; Mate, R. A.; Bronson, J. J.;
Gai, Y.; Bergstrom, C. P.; Marcin, L. R.; Macor, J. E. Preparation of α-
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WO 2003053912, A1 20030703, CAN 139:85645, AN 2003:511281,
2003; (b) Starrett, J. E.; Gillman, K. W.; Olson, R. E. A novel alpha-(n-
sulfonamido)acetamide compound as an inhibitor of beta amyloid
peptide production. PCT Int. Appl. WO 2010107435, A1 20100923,
2010.
apparent intrinsic clearance; CNS MPO, central nervous system
multiparameter optimization; CSF, cerebrospinal fluid; CYP,
cytochrome P450; fu, fraction unbound; GSI, γ-secretase inhibitor;
HHEP, human hepatocytes; HLM, human liver microsomes; iv,
intravenous; MDCK, Madine Darby canine kidney; MDR1,
multidrug resistance protein (p-glycoprotein); PD, pharmaco-
dynamics; PK, pharmacokinetic; po (per os), oral admin-
istration; SAR, structure−activity relationship
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