Metabolism-directed design of oxetane-containing arylsulfonamide derivatives as γ-secretase inhibitors

Article abstract of DOI:10.1021/jm200893p

A metabolism-based approach toward the optimization of a series of N-arylsulfonamide-based -secretase inhibitors is reported. The lead cyclohexyl analogue 6 suffered from extensive oxidation on the cycloalkyl motif by cytochrome P450 3A4, translating into poor human liver microsomal stability. Knowledge of the metabolic pathways of 6 triggered a structure activity relationship study aimed at lowering lipophilicity through the introduction of polarity. This effort led to several tetrahydropyran and tetrahydrofuran analogues, wherein the 3- and 4-substituted variants exhibited greater microsomal stability relative to their 2-substituted counterparts. Further reduction in lipophilicity led to the potent -secretase inhibitor and 3-substituted oxetane 1 with a reduced propensity toward oxidative metabolism, relative to its 2-substituted isomer. The slower rates of metabolism with 3-substituted cyclic ethers most likely originate from reductions in lipophilicity and/or unfavorable CYP active site interactions with the heteroatom. Preliminary animal pharmacology studies with a representative oxetane indicate that the series is generally capable of lowering A in vivo. As such, the study also illustrates the improvement in druglikeness of molecules through the use of the oxetane motif.

Full text of DOI:10.1021/jm200893p

Article
pubs.acs.org/jmc
Metabolism-Directed Design of Oxetane-Containing Arylsulfonamide
Derivatives as γ-Secretase Inhibitors
Antonia F. Stepan,* Kapil Karki, W. Scott McDonald, Peter H. Dorff, Jason K. Dutra, Kenneth J. DiRico,
Annie Won, Chakrapani Subramanyam, Ivan V. Efremov, Christopher J. O’Donnell, Charles E. Nolan,
Stacey L. Becker, Leslie R. Pustilnik, Blossom Sneed, Hao Sun, Yasong Lu, Ashley E. Robshaw,
David Riddell, Theresa J. O'Sullivan, Evelyn Sibley, Steven Capetta, Kevin Atchison, Andrew J. Hallgren,
Emily Miller, Anthony Wood, and R. Scott Obach
Pfizer Worldwide Research & Development, Eastern Point Road, Groton, Connecticut 06340, United States
S
* Supporting Information
ABSTRACT: A metabolism-based approach toward the optimization of a series of N-arylsulfonamide-based γ-secretase
inhibitors is reported. The lead cyclohexyl analogue 6 suffered from extensive oxidation on the cycloalkyl motif by cytochrome
P450 3A4, translating into poor human liver microsomal stability. Knowledge of the metabolic pathways of 6 triggered a
structure−activity relationship study aimed at lowering lipophilicity through the introduction of polarity. This effort led to several
tetrahydropyran and tetrahydrofuran analogues, wherein the 3- and 4-substituted variants exhibited greater microsomal stability
relative to their 2-substituted counterparts. Further reduction in lipophilicity led to the potent γ-secretase inhibitor and 3-
substituted oxetane 1 with a reduced propensity toward oxidative metabolism, relative to its 2-substituted isomer. The slower
rates of metabolism with 3-substituted cyclic ethers most likely originate from reductions in lipophilicity and/or unfavorable CYP
active site interactions with the heteroatom. Preliminary animal pharmacology studies with a representative oxetane indicate that
the series is generally capable of lowering Aβ in vivo. As such, the study also illustrates the improvement in druglikeness of
molecules through the use of the oxetane motif.
γ-secretase in Notch processing,4,5 identification of inhibitors
with minimal impact on Notch signaling is an important
INTRODUCTION
■
Alzheimer's disease (AD) is the most prevalent form of senile
consideration from a drug discovery perspective. Indeed, a
dementia in the elderly population.1 Current treatment options
number of structurally diverse γ-secretase inhibitors with
are only symptomatic, and thus, disease-modifying anti-
Notch-sparing activity have been reported in the primary
Alzheimer's drugs that slow or reverse neuronal loss and the
literature,6 and several (e.g., BMS-708,163, LY-450,139, GSI-
underlying cognitive decline are urgently needed. Histopatho-
953, PF-3,084,014, and BMS-299,897) have also advanced into
logical and genetic evidence suggests that the formation of the
clinical trials for the treatment of AD (Figure 1).7
amyloid-β (Aβ) peptide and its subsequent aggregation to
As part of ongoing efforts to identify novel chemotypes,6k,l
we report our structure−activity relationship (SAR) findings on
a series of N-substituted arylsulfonamides as potent γ-secretase
inhibitors. The incorporation of in vitro metabolite identi-
fication studies in human hepatic tissue at the lead optimization
stage greatly facilitated our efforts in the modulation of
physicochemical property space and led to the rational design
oligomers and plaques are key events in disease progression.2
Aβ peptides are produced by sequential proteolytic cleavage of
the Aβ precursor protein by two aspartyl proteases, β- and γ-
secretases.3 Heterologous cleavage by γ-secretase creates Aβ
isoforms ranging from 37 (Aβ 37) to 42 (Aβ42) amino acid
residues. Aβ40 is the most abundant isoform, while it is thought
that Aβ42 is closely associated with the pathogenesis of AD. As
a result, reduction in the formation of Aβ peptides via
inhibition of γ-secretase by small molecule agents appears to be
a viable option for the treatment of AD. Because of the role of
Received: July 7, 2011
Published: October 13, 2011
© 2011 American Chemical Society
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Figure 1. γ-Secretase inhibitors in clinical trials.
Figure 2. Design progression toward compounds 1 and 2.
a
Scheme 1. Synthesis of Compounds 1−23
a
Reagents and conditions: (i) (Oxetan-3-yl)methanol (for 1)/(tetrahydofuran-3-yl)methanol (for 16 and 17)/(tetrahydrofuran-2-yl)methanol (for
18 and 19)/(oxetan-2-yl)methanol (for 20 and 21), DEAD, PPh3, THF, 23 °C. (ii) Cyclohexylmethanol (for 6)/cyclopentylmethanol (for 7)/
cyclobutylmethanol (for 8), DCAD, PPh3, THF, 23 °C. (iii) (a) (Bromomethyl)tetrahydro-2H-pyran (for 9 and 10), Cs2CO3, DMF, 80 °C. (iv) (a)
(Tetrahydro-2H-pyran-3-yl)methanol (for 11 and 12)/(S)-(1,4-dioxan-2-yl)methanol (for 15)/(R)-(1,4-dioxan-2-yl)methanol (for 14), CH3SO2Cl,
NEt3, CH2Cl2, 0 °C then 24, Cs2CO3, DMF, 80 °C. (v) 4-(Iodomethyl)tetrahydro-2H-pyran, Cs2CO3, DMF, 80 °C. (vi) Compound 25, Cs2CO3,
DMAc, 60 °C. (vii) Compound 26, Cs2CO3, DMAc, 90 °C. (viii) (3,3-Dimethyloxetan-2-yl)methanol, DBAD, PPh3, THF, 23 °C, 16 h.
of oxetane derivatives 1 and 2 (Figure 2) with potent γ-
secretase inhibitory activity, Notch selectivity, and superior
oxidative metabolic stability. A prominent highlight of the SAR
studies was the discovery of the oxetane ring system with the
appropriate balance of potency, metabolic stability, and
permeability, relative to simple cycloalkyl rings and other cyclic
ethers. These observations are compatible with the recent
reports by Carreira and co-workers, which highlight the
attractive physicochemical and disposition attributes of the
oxetane functionality in drug design.8
RESULTS AND DISCUSSION
■
Chemistry. Title compounds 1−23 were prepared via
alkylation of sulfonamide 247c with the appropriate alkyl
alcohol or alkyl halide (Scheme 1). In the case of several cyclic
ethers, the alkylation products were obtained as diastereomeric
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Table 1. In Vitro Pharmacology and Disposition Data for a Representative Set of N-Arylsulfonamides
mixtures, which were separated chromatographically to afford
the individual stereoisomers (Table 1). The alkyl alcohols and
alkyl iodides required for the synthesis of compounds 2−5 were
not commercially available and were prepared as outlined in
Scheme 2. Iodo-containing oxetanes 25 and 26 were prepared
via an iodocylization of homoallylic alcohols 27 and 28 using
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Table 1. continued
a
b
IC50 values were obtained in a whole cell assay using hu APPwt cells by measuring Aβ1−42 as previously described.12 Number of repetitions
c
d
indicated in parentheses. CLint,app refers to total intrinsic clearance obtained from scaling in vitro HLM half-lives. Elog D was measured at pH 7.4 as
previously described.13 RRCK cells with low transporter activity were isolated from Madine−Darby canine kidney cells and were used to estimate
e
intrinsic absorptive permeability.14 MDR1/MDCK assay utilized MDCK cells transfected with the gene that encodes human p-glycoprotein.14 The
f
g
absolute configuration was arbitrarily assigned.
a
Scheme 2. Synthesis of Substituted Oxetanes 25, 26, and 30
a
Reagents and Conditions: (i) Ag+(sym-collidine)ClO4 (29), I2, 23 °C, CH2Cl2, 16 h. (ii) LiAlH4, THF, −30 °C, 1.5 h.
−
Figure 3. Sites of oxidative metabolism in HLM for representative N-arylsulfonamides.
the I+ transfer reagent bis(sym-collidine)iodine perchlorate,
generated in situ from silver bis(sym-collidine)iodine per-
chlorate (29) and iodine.9 The crude products obtained in
these reactions were used in the alkylation step without further
purification. 3,3-(Dimethyloxetan-2-yl)methanol (30) was
accessed from known ester 3110 via reduction to the
corresponding alcohol.
In Vitro Pharmacology and Metabolism SAR. The
starting point of our study was the identification of compound
6 (Figure 2), obtained by replacement of the biaryl core in
BMS-708,163 with a smaller cyclohexyl group.11 Although 6
demonstrated good γ-secretase inhibition [IC50 (Aβ42) = 12.3
nM, Table 1] in our whole cell assay,12 the compound suffered
from high oxidative metabolic turnover in human liver
microsomes (HLM; apparent intrinsic clearance, CLint,app
=
176 mL/min/kg), a phenomenon that is likely related to the
lipophilic nature of the molecule (Elog D = 5.60).13 In addition
to its metabolic instability, the moderate permeability of 6 (Papp
= 4.42 × 10−6 cm/s) in the RRCK assay14 indicates that the
compound might suffer from poor oral bioavailability.
Replacement of the cyclohexane ring in 6 with smaller
cycloalkyl rings (compounds 7 and 8) had minimal effect on γ-
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Figure 4. HPLC-UV chromatograms of metabolites of compounds 1 (A) and 20 (B) in NADPH-supplemented HLM.
secretase potency [IC50 (Aβ42) = 16.6 and 11.7 nM] but,
unfortunately, did not improve the metabolic liability (CLint,app
= 229 and 207 mL/min/kg). To elucidate the source(s) of the
metabolic instability in the cycloalkyl derivatives, the
biotransformation pathways of representative members 6 and
8 were examined in NADPH-supplemented HLM. The
formation of multiple cytochrome P450 (CYP)-mediated
hydroxylated metabolites derived exclusively from oxidations
of the cycloalkyl moiety was noted for both compounds (Figure
3). No metabolism was discerned on the aryl chloride or
trifluoropropane substituents in compounds 6 and 8,
respectively.
With the source of metabolic instability identified, we
decided to introduce polarity into the cycloalkyl ring system
as a tactic to potentially reduce oxidative metabolism, while
retaining pharmacologic activity. As illustrated with compounds
9−19, incorporation of oxygen into the cycloalkyl ring system
reduced Elog D but did not substantially impact γ-secretase
inhibitory potency noted with 6, suggesting that the overall
approach was a viable one to reduce HLM clearance. Of much
interest in this SAR study was the finding that the 3- and 4-
substituted tetrahydropyran analogues 11−13 displayed greater
resistance toward oxidative metabolism in HLM as judged by a
greater than 2-fold decrease in CLint,app relative to the 2-
substituted tetrahydropyran counterparts (compounds 9 and
10). A similar trend with respect to HLM stability was also
noted with the 3-substituted tetrahydrofurans (compounds 16
and 17) relative to the 2-substituted derivatives (compounds 18
and 19). Compound 14 displayed a greater than 2-fold
improvement in γ-secretase inhibition [IC50 (Aβ42) = 5.25
nM], relative to lead compound 6 [IC50 (Aβ42) = 12.3 nM].
However, metabolic turnover in HLM remained relatively high
(CLint,app = 117 mL/min/kg), despite a net reduction in ELog
D value as compared with 6.
studies were performed in HLM with representative members
of the 2- and 3-substituted tetrahydropyrans (compounds 10
and 12) and tetrahydrofurans (compounds 17 and 19). As seen
in Figure 3, oxidative metabolism on all compounds still
occurred primarily on the cycloether ring. For the tetrahy-
dropyran and tetrahydrofuran rings, the characterization of
stable hydroxy-carboxylic acid metabolites is consistent with an
initial CYP-catalyzed oxidation(s) on the carbon α to the
oxygen atom, which would lead to unstable hemiacetal
intermediates followed by hydrolysis to the corresponding
aldehyde derivatives and further oxidation to the corresponding
hydroxy-acid metabolites. On the basis of these observations,
we speculate that the greater stability of the 3-substituted cyclic
ethers (relative to the 2-substituted variants) results from a
slower overall rate of metabolism caused by a diminished
affinity for CYP binding due to a reduction in Elog D and/or
unfavorable active site interactions with the 3-oxo substituent.
It is possible that net reductions in Elog D values for the 3-
substituted cyclic ethers arises from a greater degree of
solvation of the more exposed oxygen atom in the 3-substituted
cyclic ethers (relative to the 2-substituted analogues).
Encouraged by these findings, we decided to further
investigate whether such metabolic differences could be
preserved with the even less lipophilic oxetane ring system.
We were pleased to find out that, relative to the other cyclic
ethers, the oxetane-containing sulfonamides (compounds 20,
21, and 1) possessed the highest degree of HLM stability, while
retaining γ-secretase inhibitory potency in the low nanomolar
range. While the increased metabolic stability is most likely a
reflection of reduced lipophilicity [Elog D = 2.50−2.60
(compounds 1, 20, and 21)], there is a distinct possibility
that the greater chemical stability of the oxetane ring (relative
to the higher cyclic ether homologues) in 20, 21, and 1 also
induces metabolic resistance. Certainly, using tert-butoxyl
radicals, it has been demonstrated that the rate of hydrogen
abstraction α to oxygen atoms is lower in oxetanes relative to
tetrahydrofurans and tetrahydropyrans.15
To gain an insight into the biochemical basis for the greater
metabolic resistance of 3-substituted cyclic ethers as opposed to
the 2-substituted analogues, additional metabolite identification
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Table 2. In Vitro Pharmacology and Disposition Data for Substituted Oxetanes
a
b
IC50 values were obtained in a whole cell assay using hu APPwt cells by measuring Aβ1−42 as previously described.12 Number of repetitions
c
d
indicated in parentheses. CLint,app refers to total intrinsic clearance obtained from scaling in vitro HLM half-lives. Elog D was measured at pH 7.4 as
previously described.13 RRCK cells with low transporter activity were isolated from MDCK cells and were used to estimate intrinsic absorptive
e
permeability.14 MDR1/MDCK assay utilized MDCK cells transfected with the gene that encodes human p-glycoprotein.14 The relative
f
g
configuration was assigned using a set of two-dimensional NMR spectroscopy experiments (COSY and NOE); the absolute configuration was
h
arbitrarily assigned. The absolute configuration was arbitrarily assigned.
The relationship between oxetane ring substitution and
oxidative metabolic stability followed similar trends as seen with
the tetrahydropyran and tetrahydrofuran derivatives. Thus, 3-
substituted oxetane 1 (CLint,app = 28.6 mL/min/kg) was
relatively more stable in HLM than the two 2-substituted
oxetanes (compounds 20 and 21, CLint,app = 62.1 and 63.8 mL/
min/kg, respectively). Examination of the metabolic profile of
oxetanes 1 and 20 in NADPH-supplemented HLM (Figure 4)
revealed N-dealkylation as an additional metabolic pathway (via
oxidation of the bridging methylene carbon) that was not
observed with 2- and 3-substituted tetrahydropyrans and
tetrahydrofurans. For compound 1, which was the most
metabolically stable among all of the tested analogues, N-
dealkylation was a dominant biotransformation pathway, while
for compound 20, this reaction was observed but was not the
major metabolic route (see Figure 4). Compound 20 was
mainly metabolized in HLM via oxetane ring scission leading to
the formation of the hydroxy acid and diol metabolites (see
Figure 4). Through the use of selective CYP inhibitors, it was
shown that oxidative metabolism of both compounds in HLM
was predominantly catalyzed by CYP3A4 because coincubation
of HLM with ketoconazole, a selective CYP3A4 inhibitor,
decreased metabolite formation in compounds 1 and 20 by
greater than 80%. Finally, it is also worth noting that oxetanes
1, 20, and 21 demonstrated excellent absorptive permeability
(Papp > 25 × 10−6 cm/s) in the RRCK assay, and no multidrug
resistant gene (MDR1)-mediated efflux [Papp basolateral to
apical (BA)/apical to basolateral (AB) ratio <2.0] as measured
in the MDR1/Madine Darby canine kidney (MDCK) assay.14
Besides regiochemical manipulations on oxetane ring attach-
ment, the effect of methyl substitution on the oxetane ring in 2-
and 3-substituted oxetanes was also examined with respect to
pharmacology and metabolism characteristics. As shown in
Table 2, monomethyl substitution in 2-substituted oxetanes
resulted in compounds that retained good γ-secretase potency
of the nonmethylated analogues [IC50 (Aβ42) = 10.8 and 6.46
nM for compounds 22 and 23, respectively] but demonstrated
relatively high turnover in HLM (CLint,app = 167 and 76.9 mL/
min/kg). Introduction of the gem-dimethyl substituent adjacent
to the oxetane oxygen, however, led to a 2-fold improvement in
metabolic stability (compounds 2 and 3, CLint,app = 25.9 and
29.0 mL/min/kg, respectively). In addition, oxetane 2 [IC50
(Aβ42) = 16.5 nM] had an inhibitory potency comparable to
the initial oxetane leads [compounds 20 and 1: IC50 (Aβ42) =
7.99 and 16.9 nM, respectively; see Table 1]. As such, overall
improvement in the disposition attributes of oxetane derivatives
1 and 2, relative to the initial lead compound 6, is also reflected
by clear improvements in topological polar surface area
(TPSA),16 lipophilic efficiency (LiPE),17 and central nervous
system multiparameter optimization (CNS MPO) desirability
score18 (compound 6: TPSA = 88.9 Å2, LiPE = 2.24, and CNS
MPO = 3.22; compound 1: TPSA = 98.1 Å2, LiPE = 4.97, and
CNS MPO = 4.71; and compound 2: TPSA = 98.1 Å2, LiPE =
3.71, and CNS MPO = 3.91). LipE and CNS MPO are key
parameters to evaluate the quality and guide the selection of
compounds with favorable physicochemical properties and
safety profiles. As such, the clear improvement of both values
suggests that the introduction of oxetanes into lead matter can
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Figure 5. Aβ lowering efficacy of compound 20 in brain (a and b) and CSF (c) in wild-type male 129/sve mice at 1 and 4 h after subcutaneous
dosing (100 mg/kg). Aβ levels were quantified by ELISA and reported as a percent of vehicle-treated mice. Data represent means ± standard errors
of the mean (n = 5). *P < 0.05 and **P < 0.01 between vehicle and treated mice.
be used as a tactic to increase the overall druglikeness of
molecules.
h postdose. Corresponding exposures were also measured in
these matrices to gain preliminary insight into the PK/PD
relationships for γ-secretase inhibition. As shown in Figure 5,
statistically significant lowering of brain Aβ 40 (at 1 h, 32%; at 4
h, 18%) and Aβ42 (at 1 h, 19%; at 4 h, 14%) was discerned at
these two time points. Although unbound brain concentrations
declined sharply from 1 (1256 nM) to 4 h (141 nM) postdose,
they significantly exceeded the in vitro whole cell IC50 value of
7.99 nM for 20. Significant lowering of CSF Aβ 40 (48%) at 1 h
postdose was also noted at a CSF concentration of 447 nM.
CSF efficacy was lost at the 4 h time point (CSF concentration
= 59.6 nM) with a trend toward increased Aβ 40 levels, which
was not statistically significant (a scatter plot showing individual
mouse CSF levels is depicted in the Supporting Information).
The position of the gem-dimethyl group on the oxetane ring
appeared to be crucial for metabolic stability. For example, the
two diastereomers 4 and 5 (in which the gem-dimethyl group
occupied the C4 position on the oxetane ring) exhibited high
CLint,app in HLM (compound 4, CLint,app > 293 mL/min/kg;
compound 5, CLint,app = 262 mL/min/kg). As such, metabolite
profiling of representative members of the methylated oxetanes
(compounds 2, 4, and 5) in HLM revealed that oxidative
metabolism continued to occur on the oxetane portion of the
molecules (Figure 3) implying that differences in CLint,app arose
via individual differences in binding site affinity to the CYP
enzyme(s) responsible for their metabolism.
In Vivo Pharmacology. In addition to their favorable
HLM metabolic stability and in vitro γ-secretase potency
described above, the oxetane derivatives also met our general
criteria for Notch selectivity.4,5 For example, the Notch
selectivity of compounds 1, 2, and 20 was 187-, 439-, and
523-fold (Tables 1 and 2), respectively, and was comparable to
the Notch selectivity of 347-fold measured for the clinical
candidate BMS-708,163. We therefore assessed whether the in
vitro γ-secretase inhibitory potency of the oxetanes translated
into a pharmacodynamic (PD) response in vivo. Acute γ-
secretase inhibition was examined in wild-type male 129/sve
mice, a validated preclinical model for evaluating pharmacoki-
netic (PK)/PD relationships of γ-secretase inhibitors.19 As the
in vitro CLint,app of oxetanes 1, 2, and 20 in NADPH-
supplemented rat liver microsomes was found to be
considerably higher (CLint,app = 187, 439, and 523 mL/min/
kg, respectively) than the corresponding CLint,app values in
HLM (CLint,app = 16.9, 16.5, and 62.1 mL/min/kg, Tables 1
and 2), poor oral bioavailability (<5%) was discerned in both
mice and rats.20 Consequently, we chose the subcutaneous
route of administration to bypass first pass metabolism by CYP
enzymes in the mouse model. Compound 20 was used as a
representative oxetane for in vivo profiling based upon its
somewhat greater in vitro potency relative to 1 and 2
[compound 20, IC50 (Aβ42) = 7.99 nM, vs compounds 1 and
2, IC50 (Aβ42) = 16.8 and 16.5 nM, respectively].
CONCLUSION
■
Via the use of in vitro metabolite identification studies, we were
successful in improving the metabolic stability of the lead N-
cyclohexylmethyl arylsulfonamide 6 in human hepatic tissue,
which led to the identification of oxetane-based γ-secretase
inhibitors 1 and 2. A prominent highlight in the SAR exercise
leading to oxetane-based derivatives 1 and 2 was the
serendipitous discovery of the structure−metabolism differ-
ences between the 2-, 3-, and 4-substituted cycloalkyl ethers,
which translated into an overall reduction in HLM clearance for
the 3- and 4-substituted analogues. Metabolism differences for
the 2- and 3-substituted oxetanes (compounds 1 and 20,
respectively) in HLM were rationalized in terms of differences
in active site binding to CYP3A4, the principal human CYP
responsible for their metabolism in liver microsomes. Whether
the differences in metabolic clearance between the 2- and the 3-
substituted cycloalkyl ethers observed in our studies represent a
general phenomenon remains to be examined with additional
examples. As such, our findings on the metabolic stability of the
oxetane ring system are in good agreement with the recent
reports by Carreira et al., which demonstrated the favorable
disposition characteristics of oxetanes relative to certain other
functional groups.8 Furthermore, while Carreira et al. high-
lighted the bioisosteric properties of oxetanes over carbonyl and
gem-dimethyl groups, our SAR data suggest that oxetanes can
offer a unique advantage over the corresponding 5- and 6-
membered cycloalkanes and/or cycloalkyl ethers in terms of
metabolic resistance, a finding that could be used as a general
Following subcutaneous administration of 20 at a dose of
100 mg/kg (chosen to ensure adequate exposure), Aβ lowering
was measured in brain and cerebrospinal fluid (CSF) at 1 and 4
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afford 6.00 mg (3.1% yield) of 2 as a white solid and 5.00 mg (2.6%
yield) of 3 as a white solid. The absolute configuration of 2 and 3 was
arbitrarily assigned. Compound 2: H NMR (400 MHz, CDCl3): δ
tactic to reduce metabolic instability and improve the
physicochemical properties of lead compounds. To the best
of our knowledge, our metabolism studies on the oxetane ring
system constitute the first report on the biotransformation of
these four-membered cycloalkyl ethers. Finally, our preliminary
preclinical studies with 20, a representative member of this
series, indicate that this series of γ-secretase inhibitors induces a
pharmacological response in vivo by lowering Aβ in both brain
and CSF. Further SAR and preclinical profiling on the oxetane
derivatives was suspended due to the parallel discovery of a
superior chemical series. These findings will be reported in due
course.
1
7.82−7.72 (m, 2H), 7.54−7.44 (m, 2H), 6.69 (br s, 1H), 5.33 (br s,
1H), 4.74 (m, 1H), 4.15 (t, J = 7.4 Hz, 1H), 3.56−3.46 (m, 1H), 3.35
(dd, J = 15.0, 9.4 Hz, 1H), 2.42 (dd, J = 11.3, 7.8 Hz, 1H), 2.22−2.08
(m, 1H), 2.04−1.91 (m, 2H), 1.90−1.68 (m, 2H), 1.41 (s, 3H), 1.34
(s, 3H). LC-MS (ES−) m/z 441 (M − H−). Compound 3: 1H NMR
(400 MHz, CDCl3): δ 7.81−7.74 (m, 2H), 7.52−7.46 (m, 2H), 7.37
(br s, 1H), 5.31 (br s, 1H), 4.91−4.82 (m, 1H), 4.26 (t, J = 7.5 Hz,
1H), 3.54 (dd, J = 14.4, 4.1 Hz, 1H), 3.27 (dd, J = 14.4, 9.0 Hz, 1H),
2.43 (dd, J = 11.2, 7.7 Hz, 1H), 2.34−2.22 (m, 1H), 2.21−2.06 (m,
2H), 2.03−1.85 (m, 1H), 1.65−1.55 (m, 1H), 1.39 (s, 3H) 1.34 (s,
3H). LC-MS (ES−) m/z 441 (M − H−).
(R)-2-{4-Chloro-N-[((S)-3,3-dimethyloxetan-2-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (4) and (R)-2-
{4-Chloro-N-[((R)-3,3-dimethyloxetan-2-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (5). Com-
pound 24 (289 mg, 0.84 mmol), (3,3-dimethyloxetan-2-yl)methanol
(100 mg, 0.86 mmol), triphenylphosphine (336 mg, 1.26 mmol), and
t-butyl azodicarboxylate (242 mg, 1.05 mmol) were used to synthesize
4 and 5 using the procedure described for the preparation of 1. The
crude product was purified by flash chromatography over silica using 0
→ 50% gradient of ethyl acetate in heptanes to afford 33.0 mg (8.9%
yield) of 4 as a white solid and 26.0 mg (7.0% yield) of 5 as a white
solid. The absolute configuration of 4 and 5 was arbitrarily assigned.
EXPERIMENTAL SECTION
■
Chemistry. Unless specified otherwise, starting materials were
available from commercial sources. Dry solvents and reagents were of
commercial quality and were used as purchased. 1H NMR spectra were
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 multiplicities 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
acetonitrile 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. All synthetic reactions were carried out under nitrogen
atmosphere or in sealed vials. The chemical yields reported below are
unoptimized specific examples of one preparation.
1
Compound 4: H NMR (400 MHz, CDCl3): δ 7.80−7.73 (m, 2H),
7.54−7.46 (m, 2H), 6.71−6.62 (m, 1H), 4.54 (dd, J = 9.7, 2.4 Hz,
1H), 4.25 (d, J = 5.7 Hz, 1H), 4.16−4.09 (m, 2H), 3.54 (dd, J = 15.2,
2.3 Hz, 1H), 3.45 (dd, J = 15.4, 9.8 Hz, 1H), 32.20−2.08 (m, 1H),
2.01−1.88 (m, 1H), 1.86−1.69 (m, 2H), 1.27 (s, 3H), 1.14 (s, 3H).
LC-MS (ES+) m/z 443 (M + H+); mp 153.5−154.5 °C. Compound 5:
1H NMR (400 MHz, CD3OD): δ 7.91−7.83 (m, 2H), 7.61−7.53 (m,
2H), 4.60 (dd, J = 8.4, 3.3 Hz, 1H), 4.30 (dd, J = 8.0, 6.6 Hz, 1H), 4.22
(d, J = 5.7 Hz, 1H), 4.09 (d, J = 5.7 Hz, 1H), 3.60 (dq, J = 15.7, 5.9
Hz, 2H), 2.18−1.94 (m, 3H), 1.76−1.56 (m, 1H), 1.24 (s, 3H), 1.18
(s, 3H). LC-MS (ES+) m/z 443 (M + H+).
(R)-2-[4-Chloro-N-(cyclohexylmethyl)phenylsulfonamido]-
5,5,5-trifluoropentanamide (6). Compound 24 (282 mg, 0.82
mmol), cyclohexylmethanol (112 mg, 0.98 mmol), triphenylphosphine
(260 g, 0.98 mmol), and (E)-bis(4-chlorobenzyl)-diazene-1,2-
dicarboxylate (361 mg, 0.98 mmol) were used to synthesize 6 using
the procedure described for the preparation of 1. The crude product
was purified by preparative reverse phase HPLC (Phenomenex
Gemini C18 column 5 μ, 150 mm × 21.2 mm; 5−100% methanol/
water with 0.1% NH4OH; flow rate = 28 mL/min) to afford 52.0 mg
(14% yield) of 6 as an off-white solid. 1H NMR (500 MHz, CDCl3): δ
7.80−7.72 (m, 2H), 7.59−7.50 (m, 2H), 6.70 (br s, 1H), 5.58 (br s,
1H), 4.24 (dd, J = 9.4, 5.7 Hz, 1H), 3.21 (dd, J = 14.2, 9.8 Hz, 1H),
2.92 (dd, J = 14.2, 4.6 Hz, 1H), 2.25−2.13 (m, 1H), 2.06−1.90 (m,
1H), 1.88−1.56 (m, 7H), 1.36−1.09 (m, 4H), 1.01−0.80 (m, 2H).
LC-MS (ES+) m/z 441 (M + H+); mp 170.8−171.8 °C.
(R)-2-[4-Chloro-N-(oxetan-3-ylmethyl)phenylsulfonamido]-
5,5,5-trifluoropentanamide (1). To a solution of 24 (100 mg, 0.29
mmol) and oxetan-3-ylmethanol (38.3 mg, 0.43 mmol) in
tetrahydrofuran (1.5 mL) at 23 °C was added triphenylphosphine
(115 mg, 0.43 mmol). Diethylazodicarboxylate (0.29 μL, 0.29 mmol)
was then added dropwise, and the reaction mixture was stirred at that
temperature for 16 h. The solvent was removed in vacuo, and the
crude product was purified by preparative reverse phase HPLC
[Phenomenex hydrophilic interaction liquid chromatography (HILIC,
diol), 250 mm × 21.2 mm 5 μ; 5−100% ethanol/heptanes; flow rate =
28 mL/min] to afford 21.0 mg (17% yield) of 1 as an off-white solid.
1H NMR (500 MHz, CDCl3): δ 7.79−7.68 (m, 2H), 7.54 (d, J = 7.8
Hz, 2H), 6.45 (br s, 1H), 5.57 (br s, 1H), 4.71 (q, J = 6.3 Hz, 2H),
4.50 (t, J = 6.1 Hz, 1H), 4.33−4.17 (m, 2H), 3.91−3.79 (m, 1H),
3.32−3.18 (m, 2H), 2.22−2.08 (m, 1H), 2.07−1.88 (m, 1H), 1.84−
1.67 (m, 1H), 1.32−1.18 (m, 1H). LC-MS (ES+) m/z 415 (M + H+);
mp 142.7−144.2 °C.
(R)-2-{4-Chloro-N-[((R)-4,4-dimethyloxetan-2-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (2) and (R)-2-
{4-Chloro-N-[((S)-4,4-dimethyloxetan-2-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (3). To a sus-
pension of 24 (150 mg, 0.43 mmol) and cesium carbonate (255 g, 0.78
mmol) in dimethylacetamide (1.0 mL) at 23 °C was added 26 (98.3
mg, 0.43 mmol), and the resulting reaction mixture was heated at 90
°C for 16 h. The reaction mixture was then cooled to 23 °C, water (10
mL) and ethyl acetate (10 mL) were added, and the phases were
separated. The aqueous phase was extracted with ethyl acetate (3 × 10
mL), dried (MgSO4), filtered, and concentrated in vacuo. The crude
product was purified by flash column chromatography using a 0 →
40% gradient of ethyl acetate in heptanes to give an isomeric mixture
of 2 and 3. The isomers were separated by supercritical
chromatography (Chiralpak AD-H 250 mm × 10 mm, 5 μ; mobile
phase, carbon dioxide/methanol (80/20); flow rate = 10 mL/min) to
(R)-2-[4-Chloro-N-(cyclopentylmethyl)phenylsulfonamido]-
5,5,5-trifluoropentanamide (7). Compound 24 (282 mg, 0.82
mmol), cyclopentylmethanol (90.1 mg, 0.90 mmol), triphenylphos-
phine (238 mg, 0.90 mmol), and (E)-bis(4-chlorobenzyl)-diazene-1,2-
dicarboxylate (300 mg, 0.90 mmol) were used to synthesize 7 using
the procedure described for the preparation of 1. The crude product
was purified by flash chromatography over silica using a gradient of 10
→ 40% ethyl acetate in heptanes to afford 39.0 mg (11% yield) of 7 as
1
a white solid. H NMR (500 MHz, CDCl3): δ 7.75−7.68 (m, 2H),
7.54−7.47 (m, 2H), 6.67 (br s, 1H), 5.65 (br s, 1H), 4.20 (dd, J = 9.4,
5.7 Hz, 1H), 3.26 (dd, J = 14.1, 10.5 Hz, 1H), 2.97 (dd, J = 14.1, 5.1
Hz, 1H), 2.23−2.09 (m, 2H), 2.05−1.86 (m, 1H), 1.80−1.44 (m, 7H),
1.43−1.32 (m, 1H), 1.32−1.20 (m, 1H), 1.10−1.01 (m, 1H). LC-MS
(ES+) m/z 427 (M + H+); mp 151.2−152.0 °C.
(R)-2-[4-Chloro-N-(cyclobutylmethyl)phenylsulfonamido]-
5,5,5-trifluoropentanamide (8). Compound 24 (282 mg, 0.82
mmol), cyclobutylmethanol (84.6 mg, 0.98 mmol), triphenylphos-
phine (238 mg, 0.90 mmol), and (E)-bis(4-chlorobenzyl) diazene-1,2-
7779
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dicarboxylate (361 mg, 0.98 mmol) were used to synthesize 8 using
the procedure described for the preparation of 1. The crude product
was purified by preparative reverse phase HPLC (Princeton 2-ethyl
pyridine 250 mm × 21.2 mm 5 μ; 5−100% ethanol/heptanes; 28 mL/
min) to afford 223 mg (66% yield) of 8 as an off-white solid. 1H NMR
(500 MHz, CDCl3): δ 7.84−7.71 (m, 2H), 7.61−7.49 (m, 2H), 6.59
(br s, 1H), 5.72 (br s, 1H), 4.22 (dd, J = 8.8, 6.1 Hz, 1H), 3.45 (dd, J =
14.3, 9.6 Hz, 1H), 3.09 (dd, J = 14.4, 5.6 Hz, 1H), 2.63−2.51 (m, 1H),
2.24−2.13 (m, 1H), 2.09−1.92 (m, 3H), 1.92−1.68 (m, 4H), 1.66−
1.56 (m, 1H), 1.40−1.23 (m, 1H). LC-MS (ES+) m/z 413 (M + H+);
mp 154.8−155.8 °C.
(R)-2-{4-Chloro-N-[((R)-tetrahydro-2H-pyran-2-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (9) and (R)-2-
{4-Chloro-N-[((S)-tetrahydro-2H-pyran-2-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (10). To a
solution of 24 (630 mg, 1.80 mmol) in dimethylformamide (3.0
mL) at 23 °C was added 2-(bromomethyl)tetrahydro-2H-pyran (654
mg, 3.60 mmol) followed by cesium carbonate (1.19 g, 3.60 mmol).
The reaction mixture was heated at 80 °C for 16 h, then cooled to 23
°C, and filtered through Celite. The resulting filtrate was concentrated
in vacuo, and the crude product was purified by flash chromatography
over silica using a 30 → 50% gradient of ethyl acetate in heptanes to
afford 89.0 mg (11% yield) of 9 as a white solid and 36.0 mg (4.5%
yield) of 10 as a white solid. The absolute configuration of 9 and 10
was arbitrarily assigned. Compound 9: 1H NMR (400 MHz, CD3OD):
δ 7.91−7.87 (m, 2H), 7.63−7.59 (m, 2H), 4.40 (dd, J = 8.5, 6.4 Hz,
1H), 3.79 (dd, J = 10.7, 2.1 Hz, 1H), 3.64−3.56 (m, 1H), 3.42 (dd, J =
15.0, 3.0 Hz, 1H), 3.25−3.14 (m, 2H), 2.27−2.08 (m, 3H), 2.07−1.95
(m, 1H), 1.86−1.73 (m, 2H), 1.61 (d, J = 12.7 Hz, 1H), 1.54−1.46
(m, 2H), 1.24−1.16 (m, 1H). LC-MS (ES+) m/z 443 (M + H+).
Compound 10: 1H NMR (400 MHz, CD3OD): δ 7.86−7.82 (m, 2H),
7.60−7.56 (m, 2H), 4.36−4.32 (m, 1H), 3.87 (dd, J = 11.6, 2.8 Hz,
1H), 3.61 (ddt, J = 11.1, 8.6, 2.4 Hz, 1H), 3.41−3.33 (m, 3H), 2.39−
2.31 (m, 1H), 2.05−1.91 (m, 3H), 1.89−1.83 (m, 1H), 1.65−1.58 (m,
1H), 1.57−1.48 (m, 3H), 1.15 (dd, J = 11.4, 4.3 Hz, 1H). LC-MS
(ES+) m/z 443 (M + H+); mp 187.8−188.7 °C.
3.68 (m, 1H), 3.28−3.20 (m, 1H), 3.05−2.97 (m, 2H), 2.19 −2.09 (m,
2H), 2.02−1.93 (m, 2H), 1.89−1.80 (m, 1H), 1.74 (d, J = 9.6 Hz,
1H), 1.59−1.35 (m, 3H), 1.18−1.07 (m, 2H). LC-MS (ES+) m/z 443
(M + H+); mp 126.0−127.0 °C.
(R)-2-{4-Chloro-N-[(tetrahydro-2H-pyran-4-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (13). To a
solution of 24 (300 mg, 0.87 mmol) and 4-(iodomethyl)tetrahydro-
2H-pyran (295 mg, 1.30 mmol) in dimethylformamide (4.4 mL) at 23
°C was added cesium carbonate (510 mg, 1.57 mmol). The reaction
mixture was then heated at 80 °C for 16 h. Ethyl acetate (20 mL) and
water (10 mL) were then added, the phases were separated, and the
organic phase was washed with a saturated aqueous solution of sodium
bicarbonate (10 mL), water (10 mL), and brine (10 mL). The organic
phase was dried (Na2SO4), filtered, and concentrated in vacuo. The
crude product was purified by flash chromatography over silica using a
10 → 100% gradient of ethyl acetate in heptanes to afford 223 mg
1
(58% yield) of 13 as a white solid. H NMR (400 MHz, CDCl3): δ
7.74−7.69 (m, 2H), 7.53−7.49 (m, 2H), 6.63 (br s, 1H), 5.44 (br s,
1H), 4.21 (dd, J = 9.5, 5.6 Hz, 1H), 3.98−3.88 (m, 2H), 3.38−3.19
(m, 3H), 2.91 (dd, J = 14.4, 4.8 Hz, 1H), 2.21−2.09 (m, 1H), 2.02−
1.80 (m, 2H), 1.78−1.64 (m, 2H), 1.47 (d, J = 13.1 Hz, 1H), 1.37−
1.13 (m, 3H). LC-MS (ES−) m/z 441 (M − H−).
(R)-2-{N-[((R)-1,4-Dioxan-2-yl)methyl]-4-chlorophenylsulfo-
namido}-5,5,5-trifluoropentanamide (14). (R)-(1,4-Dioxan-2-
yl)methanol (257 mg, 2.18 mmol), triethylamine (330 mg, 3.26
mmol), methane sulfonyl chloride (299 mg, 2.61 mmol), compound
24 (250 mg, 0.72 mmol), and cesium carbonate (472 mg, 1.45 mmol)
were used to synthesize 14 using the procedure described for the
preparation of 11 and 12. The crude product was purified by flash
chromatography over silica using a 0 → 5% gradient of methanol in
dichloromethane to afford 39.0 mg (12% yield) of 14 as a white solid.
1H NMR (400 MHz, CDCl3): δ 7.80−7.75 (m, 2H), 7.54−7.49 (m,
2H), 7.00 (br s, 1H), 5.38 (br s, 1H) 4.35 (t, J = 7.5 Hz, 1H), 3.89−
3.81 (m, 1H), 3.78 (dd, J = 11.4, 2.5 Hz, 1H), 3.69−3.61 (m, 2H),
3.54 (dt, J = 11.1, 2.1 Hz, 1H), 3.48−3.41 (m, 1H), 3.34−3.20 (m,
2H), 3.09 (dd, J = 14.8, 8.7 Hz, 1H), 2.35−2.24 (m, 1H), 2.15−2.03
(m, 1H), 1.89 (dtd, J = 15.4, 10.3, 5.2 Hz, 1H), 1.54−1.48 (m, 1H).
LC-MS (ES+) m/z 445 (M + H+).
(R)-2-{N-[((S)-1,4-Dioxan-2-yl)methyl]-4-chlorophenylsulfo-
namido}-5,5,5-trifluoropentanamide (15). (S)-(1,4-Dioxan-2-yl)-
methanol (257 mg, 2.18 mmol), triethylamine (330 mg, 3.26 mmol),
methane sulfonyl chloride (299 mg, 2.61 mmol), compound 24 (250
mg, 0.72 mmol), and cesium carbonate (472 mg, 1.45 mmol) were
used to synthesize 15 using the procedure described for the
preparation of 11 and 12. The crude product was purified by flash
chromatography over silica using a 0 → 5% gradient of methanol in
dichloromethane to afford 86.0 mg (27% yield) of 15 as a white solid.
1H NMR (400 MHz, CDCl3): δ 7.77−7.73 (m, 2H), 7.53−7.48 (m,
2H), 6.55 (br s, 1H), 5.41 (br s, 1H), 4.18 (dd, J = 8.4, 6.1 Hz, 1H),
3.82 (tt, J = 9.4, 2.7 Hz, 1H), 3.74−3.68 (m, 2H), 3.68−3.64 (m, 1H),
3.56−3.51 (m, 2H), 3.24−3.21 (m, 1H), 3.20−3.11 (m, 2H), 2.13−
2.03 (m, 1H), 1.97−1.66 (m, 3H). LC-MS (ES+) m/z 445 (M + H+).
(R)-2-{4-Chloro-N-[((R)-tetrahydrofuran-3-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (16) and (R)-
2-{4-Chloro-N-[((S)-tetrahydrofuran-3-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (17). Com-
pound 24 (345 mg, 1.00 mmol), (tetrahydrofuran-3-yl)methanol
(128 mg, 1.25 mmol), triphenylphosphine (333 mg, 1.25 mmol), and
diethyl azodicarboxylate (0.20 mL, 1.25 mmol) were used to
synthesize 16 and 17 using the procedure described for the
preparation of 1. The crude product was purified by flash
chromatography over silica using a 20 → 100% gradient of ethyl
acetate in heptanes to give an isomeric mixture of 16 and 17. The
isomers were separated by supercritical fluid chiral chromatography
(10 mm × 250 mm Chiralpak AD-H column; 80/20 CO2/methanol
isocratic gradient over 7.5 min; flow rate = 10 mL/min) to afford 34.0
mg (7.9% yield) of 16 as a white foam and 30.0 mg (7.0% yield) of 17
as a white foam. The absolute configuration of 17 was determined
using X-ray crystallography. Compound 16: 1H NMR (500 MHz,
CDCl3): δ 7.81−7.70 (m, 2H), 7.62−7.48 (m, 2H), 6.69 (br s, 1H),
(R)-2-{4-Chloro-N-[((R)-tetrahydro-2H-pyran-3-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (11) and (R)-
2-{4-Chloro-N-[((S)-tetrahydro-2H-pyran-3-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (12). To a
solution of (tetrahydro-2H-pyran-3-yl)methanol (505 mg, 4.35
mmol) and triethylamine (660 mg, 6.52 mmol) in dichloromethane
(10 mL) at 0 °C was added methane sulfonyl chloride (598 mg, 5.22
mmol), and the reaction mixture was stirred at that temperature for 0.5
h. A 1 M aqueous solution of hydrochloric acid (10 mL) was then
added, the phases were separated, and the organic phase was extracted
with dichloromethane (2 × 10 mL). The combined organic phases
were washed with a saturated aqueous solution of sodium bicarbonate
(10 mL) and brine (10 mL). The organic phase was dried (MgSO4),
filtered, and concentrated in vacuo to give a clear oil. To a solution of
this crude product in dimethylformamide (3.0 mL) at 23 °C was
added 24 (500 mg, 1.45 mmol) followed by cesium carbonate (945
mg, 2.90 mmol). The resulting reaction mixture was heated at 80 °C
for 16 h. The reaction mixture was then cooled to 23 °C and filtered
over Celite, and the resulting filtrate was concentrated in vacuo. The
crude product was purified by flash chromatography over silica using a
0 → 2.5% gradient of methanol in dichloromethane to give an isomeric
mixture of 11 and 12 as a white solid. The isomers were separated by
supercritical chromatography (Chiralpak AD-H 250 mm × 10 mm;
20% methanol/carbon dioxide; 10 mL/min) to afford 36.0 mg (5.6%
yield) of 11 white solid and 35.0 mg (5.5% yield) of 12 as a white
solid. The absolute configuration of 11 and 12 was arbitrarily assigned.
1
Compound 11: H NMR (400 MHz, DMSO-d6): δ 7.90−7.86 (m,
2H), 7.70−7.66 (m, 2H), 7.32 (br s, 1H), 7.24 (br s, 1H), 4.30 (dd, J =
7.5, 7.5 Hz, 1H), 3.77 (dd, J = 10.9, 2.5 Hz, 1H), 3.72−3.66 (m, 1H),
3.31−3.23 (m, 2H), 3.07−2.93 (m, 2H), 2.16−2.05 (m, 2H), 2.02−
1.93 (m, 1H), 1.58−1.51 (m, 1H), 1.49−1.32 (m, 3H), 1.21−1.11 (m,
1
2H). LC-MS (ES+) m/z 443 (M + H+). Compound 12: H NMR
(400 MHz, DMSO-d6): δ 7.89−7.85 (m, 2H), 7.69−7.65 (m, 2H),
7.30 (br s, 1H), 7.24 (br s, 1H), 4.29 (dd, J = 7.4, 7.4 Hz, 1H), 3.79−
7780
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5.50 (br s, 1H), 4.29 (dd, J = 9.6, 5.3 Hz, 1H), 4.04−3.86 (m, 1H),
3.83−3.68 (m, 2H), 3.56−3.35 (m, 2H), 3.00 (dd, J = 14.4, 4.9 Hz,
1H), 2.66−2.49 (m, 1H), 2.30−2.13 (m, 1H), 2.08−1.85 (m, 3H),
1.85−1.64 (m, 1H), 1.34−1.18 (m, 1H). LC-MS (ES+) m/z 429 (M +
acetate (2 × 20 mL), and the combined organic phases were dried
(MgSO4), filtered, and concentrated in vacuo. The crude product was
purified by chiral HPLC [Chiralpak AD-H 250 mm × 10 mm, 5 μ;
mobile phase, carbon dioxide/methanol (80/20), 10 mL/min] to
afford 6.00 mg (0.8% yield) of 22 as a white solid and 14.0 mg (1.9%
yield) of 23 as a white solid. The relative configuration of 22 and 23
was assigned using a set of two-dimensional NMR spectroscopy
experiments (COSY and NOE), and the absolute configuration was
1
H+). Compound 17: H NMR (500 MHz, CDCl3): δ 7.79−7.72 (m,
2H), 7.59−7.52 (m, 2H), 6.62 (br s, 1H), 5.47 (br s, 1H), 4.27 (dd, J =
9.8, 5.4 Hz, 1H), 3.90 (dt, J = 8.4, 5.0 Hz, 1H), 3.77−3.68 (m, 3H),
3.44 (dd, J = 14.3, 10.6 Hz, 1H), 3.05 (dd, J = 14.5, 5.0 Hz, 1H), 2.63−
2.54 (m, 1H), 2.26−2.17 (m, 1H), 2.07−1.97 (m, 2H), 1.84−1.71 (m,
1H), 1.52−1.44 (m, 1H), 1.29 (dt, J = 14.5, 5.9 Hz, 1H). LC-MS (ES
+) m/z 429 (M + H+); mp 151.3−153.2 °C.
1
arbitrarily assigned. Compound 22: H NMR (400 MHz, CDCl3): δ
7.81−7.73 (m, 2H), 7.55−7.45 (m, 2H), 6.66 (br s, 1H), 5.43 (br s,
1H), 4.90−4.76 (m, 2H), 4.19−4.10 (m, 1H), 3.52 (dd, J = 15.2, 2.9
Hz, 1H), 3.33 (dd, J = 15.1, 9.5 Hz, 1H), 2.73 (dt, J = 11.3, 7.2 Hz,
1H), 2.21−2.07 (m, 1H), 2.05−1.90 (m, 1H), 1.88−1.69 (m, 3H),
1.32 (d, J = 6.1 Hz, 3H). LC-MS (ES−) m/z 427 (M − H−); mp
(R)-2-{4-Chloro-N-[((R)-tetrahydrofuran-2-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (18) and (R)-
2-{4-Chloro-N-[((S)-tetrahydrofuran-2-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (19). Com-
pound 24 (500 mg, 1.45 mmol), (tetrahydrofuran-2-yl)methanol
(141 μL, 1.45 mmol), triphenylphosphine (579 mg, 2.18 mmol), and
diethyl azodicarboxylate (286 μL, 1.45 mmol) were used to synthesize
18 and 19 using the procedure described for the preparation of 1. The
crude product was purified by preparative reverse phase HPLC
(Phenomenex Kinetex C18 column, 50 mm × 3.0 mm 2.6 μ; 0−100%
methanol/water with 0.1% formic acid; flow rate = 1.0 mL/min) to
afford 110 mg (18% yield) of 18 as a white solid and 15 mg (2.4%
yield) of 19 as a white solid. The absolute configuration of 18 and 19
was arbitrarily assigned. Compound 18: 1H NMR (500 MHz, CDCl3):
δ 7.82 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 6.98 (s, 1H), 5.66
(s, 1H), 4.22−4.13 (m, 2H), 3.84−3.71 (m, 2H), 3.42 (dd, J = 15.0,
2.3 Hz, 1H), 3.13 (dd, J = 15.0, 10.0 Hz, 1H), 2.22−2.14 (m, 1H),
2.07−1.72 (m, 6H), 1.49−1.41 (m, 1H). LC-MS (ES+) m/z 429 (M +
H+); mp 150.0−151.0 °C. Compound 19: 1H NMR (500 MHz,
CDCl3): δ 7.85−7.82 (m, 2H), 7.53−7.50 (m, 2H), 7.46 (s, 1H), 5.48
(s, 1H), 4.35−4.25 (m, 2H), 3.81−3.67 (m, 2H), 3.41 (dd, J = 14.6,
3.5 Hz, 1H), 3.09 (dd, J = 14.6, 9.6 Hz, 1H), 2.37−2.27 (m, 1H),
2.24−1.88 (m, 6H), 1.57−1.48 (m, 1H). LC-MS (ES+) m/z 429 (M +
H+).
( R ) - 2 - { 4 - C h l o r o - N - [ ( S ) - o x e t a n - 2 - y l m e t h y l ] -
phenylsulfonamido}-5,5,5-trifluoropentanamide (20) and (R)-
2-{4-Chloro-N-[(R)-oxetan-2-ylmethyl]phenylsulfonamido}-
5,5,5-trifluoropentanamide (21). Compound 24 (3.14 g, 9.10
mmol), oxetan-2-ylmethanol (960 mg, 10.9 mmol), triphenylphos-
phine (3.61 g, 13.6 mmol), and diethyl azodicarboxylate (2.31 mL,
13.6 mmol) were used to synthesize 20 and 21 using the procedure
described for the preparation of 1. The crude product was purified by
preparative reverse phase HPLC (Phenomenex Amylose-2 250 mm ×
21.2 mm, 5 μ; 5−100% ethanol/heptanes; flow rate = 28 mL/min) to
afford 698 mg (19% yield) of 20 as an off-white solid and 425 mg
(11% yield) of 21 as an off-white solid. The absolute configuration of
20 and 21 was determined using X-ray crystallography. Compound 20:
1H NMR (500 MHz, CDCl3): δ 7.82−7.70 (m, 2H), 7.58−7.41 (m,
2H), 6.63 (br s, 1H), 5.45 (br s, 1H), 5.03−4.94 (m, 1H), 4.61 (ddd, J
= 8.5, 7.3, 6.1 Hz, 1H), 4.47 (dt, J = 9.1, 6.1 Hz, 1H), 4.14 (t, J = 7.4
Hz, 1H), 3.52−3.49 (m, 2H), 2.75−2.64 (m, 1H), 2.33−2.21 (m, 1H),
2.19−2.08 (m, 1H), 2.01−1.65 (m, 3H). LC-MS (ES+) m/z 415 (M +
H+); mp 134.8−137.6 °C. Compound 21: 1H NMR (500 MHz,
CDCl3): δ 7.84−7.71 (m, 2H), 7.54−7.45 (m, 2H), 7.22−7.11 (m,
1H), 5.34 (br s, 1H), 5.13−4.99 (m, 1H), 4.66−4.59 (m, 1H), 4.51
(dt, J = 9.2, 6.2 Hz, 1H), 4.27 (t, J = 7.5 Hz, 1H), 3.56−3.43 (m, 2H),
2.78−2.68 (m, 1H), 2.38−2.22 (m, 2H), 2.21−2.03 (m, 1H), 2.01−
1.82 (m, 1H), 1.63−1.50 (m, 1H). LC-MS (ES+) m/z 415 (M + H+);
mp 146.6−148.9 °C.
(R)-2-{4-Chloro-N-[((2S,4S)-4-methyloxetan-2-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (22) and (R)-
2-{4-Chloro-N-[((2S,4R)-4-methyloxetan-2-yl)methyl]-
phenylsulfonamido}-5,5,5-trifluoropentanamide (23). To a
solution of 24 (580 mg, 1.68 mmol) in dimethylacetamide (1.0 mL)
at 23 °C was added cesium carbonate (1.10 g, 3.36 mmol), and the
resulting mixture was heated at 60 °C for 15 min. Compound 25 (357
mg, 1.68 mmol) was then added, and the resulting mixture was heated
at this temperature for 16 h. The reaction mixture was then cooled to
23 °C, water (20 mL) and ethyl acetate (20 mL) were added, and the
phases were separated. The aqueous phase was extracted with ethyl
1
107.0−109.0 °C. Compound 23: H NMR (400 MHz, CDCl3): δ
7.83−7.68 (m, 2H), 7.56−7.44 (m, 2H), 6.68 (br s, 1H), 5.42 (br s,
1H), 4.88−4.70 (m, 2H), 4.14 (t, J = 7.4 Hz, 1H), 3.54−3.48 (m, 2H),
2.40−2.23 (m, 2H), 2.22−2.08 (m, 1H), 2.04−1.87 (m, 1H), 1.88−
1.65 (m, 2H), 1.39 (d, J = 6.2 Hz, 3H). LC-MS (ES−)m/z 427 (M −
H−).
2-(Iodomethyl)-4-methyloxetane (25). To a slurry of bis(sym-
collidine)silver(I) perchlorate (7.55 g, 16.3 mmol) in dichloromethane
(75 mL) at 23 °C was added iodine (4.21 g, 16.3 mmol) in one
portion, and the resulting mixture was stirred at that temperature for
20 min (during which time AgI precipitated from the reaction
mixture). 2-Methylpent-4-en-2-ol (1.00 g, 11.6 mmol) was then added
in one portion at that temperature, and the reaction mixture was
stirred for 16 h. The reaction mixture was then filtered through Celite,
and the crude product was washed with dichloromethane (20 mL).
The resulting filtrate was washed with a 10% aqueous solution of
sodium sulfite (2 × 30 mL), a 10% aqueous solution of hydrochloric
acid (2 × 50 mL), dried (K2CO3), filtered, and concentrated in vacuo.
The crude product was used in the next step without purification.
4-(Iodomethyl)-2,2-dimethyloxetane (26). bis(sym-Collidine)-
silver(I) perchlorate (6.50 g, 14.0 mmol), iodine (3.63 g, 14.0 mmol),
and 2-methylpent-4-en-2-ol (1.00 g, 10.0 mmol) were used to
synthesize 26 using the procedure described for the preparation of
25. The crude product was used in the next step without purification.
(3,3-Dimethyloxetan-2-yl)methanol (30). To a solution of
methyl 3,3-dimethyloxetane-2-carboxylate (2.90 g, 20.1 mmol) in
tetrahydrofuran (58 mL) was added lithium aluminum hydride (783
mg, 20.6 mmol) at −30 °C, and the resulting mixture was stirred at
this temperature for 1.5 h. Water (50 mL) and dichloromethane (100
mL) were then added, the phases were separated, and the aqueous
phase was extracted with dichloromethane (2 × 100 mL). The
combined organic phases were dried (Na2SO4), filtered, and
concentrated in vacuo to afford 1.20 g (51% yield) of 30 as a yellow
oil. 1H NMR (400 MHz, CDCl3): δ 4.38 (s, 1H), 4.26 (d, J = 5.6 Hz,
1H), 4.21 (d, J = 5.6 Hz, 1H), 3.83−3.78 (m, 1H), 3.69−3.65 (m,
1H), 1.26 (s, 3H), 1.14 (s, 3H). LC-MS (ES−) m/z 99.1 (M − 17,
loss of OH).
In Vitro Metabolism Studies. Test compounds (10 μM) were
incubated with pooled HLM (1.0 mg/mL; BD Gentest, Woburn, MA)
in 1 mL of potassium phosphate (100 mM, pH 7.4) containing MgCl2
(3.3 mM) and NADPH (1.3 mM). Incubations were carried out in a
shaking water bath set at 37 °C open to air. At 5 and 30 min, 0.5 mL of
the incubation mixture was added to 5 mL of acetonitrile to terminate
the reactions. The mixtures were centrifuged (1700g) for 5 min, and
the supernatants were evaporated in a vacuum centrifuge. The residues
were reconstituted in 0.2 mL of 0.1% formic acid in water and injected
(0.06 mL) onto an HPLC system. The HPLC system consisted of a
ThermoFinnigan Surveyor system interfaced with a ThermoFinnigan
LTQ mass spectrometer. Separation was effected on a Varian Polaris
C18 column (4.6 mm × 250 mm; 5 μ particle size) equilibrated in
0.1% formic acid in 10% acetonitrile at a flow rate of 0.8 mL/min. This
mobile phase composition was maintained for 5 min followed by a
linear gradient of increasing acetonitrile to 90% at 50 min. This was
followed by a 10 min of reequilibration at initial conditions. The eluent
was passed through a photodioade array UV detector followed by
introduction of a split of ∼50 μL/min into the mass spectrometer.
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Article
Analysis was done in both positive and negative ion mode in separate
injections. Tune file settings in each mode were optimized for the
signal for compound 6. MS2 and MS3 data were gathered with data-
dependent scanning of the three most intense ions. To verify the
identity of metabolites, samples were also analyzed on a
ThermoFinnigan Orbitrap mass spectrometer operated at a resolution
setting of 30000 to gather high-resolution mass spectral data. HPLC
conditions used were the same as above. The metabolism of
compounds 1 and 20 was also examined in the presence of CYP-
specific inhibitors. Incubations were carried out as above, in the
presence and absence of furafylline (20 μM), sulfaphenazole (5 μM),
N-benzylnirvanol (20 μM), quinidine (1 μM), or ketoconazole (1
μM), except that incubations were carried out for 40 min. Samples
were processed as above and analyzed by HPLC-MS for metabolite
formation.
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T. M.; Coffman, K. J.; Finley, J. E.; Hicks, C. D.; Karmilowicz, M. J.;
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ASSOCIATED CONTENT
* Supporting Information
■
S
X-ray crystallography data for compounds 17 and 20, mass
spectral fragmentation data for compounds 1 and 20, 1H NMR
spectra of compounds 1, 2, and 6, experimental details for in
vivo pharmacology, and scatter plots for Figure 5. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 860-686 3247. E-mail: Antonia.Stepan@pfizer.com.
■
ACKNOWLEDGMENTS
■
We thank Drs. Amit S. Kalgutkar, Robert Dow, and Patrick R.
Verhoest for helpful suggestions in preparing this article and
Brian Samas and Ivan Samardjiev for the single crystal X-ray
structure elucidation of compounds 17 and 20. 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
■
Aβ, amyloid-β peptide; AD, Alzheimer's disease; BA, basolateral
to apical; CLint,app, apparent intrinsic clearance; CNS MPO,
central nervous system multiparameter optimization; CSF,
cerebrospinal fluid; CYP, cytochrome P450; HLM, human liver
microsomes; LiPE, lipophilic efficiency; MDCK, Madine Darby
canine kidney; MDR1, multidrug resistance protein (p-
glycoprotein); PK, pharmacokinetic; PD, pharmacodynamic;
SAR, structure−activity relationship; TPSA, topological polar
surface area
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