Spirocyclic sulfamides as β-secretase 1 (BACE-1) inhibitors for the treatment of alzheimers disease: Utilization of structure based drug design, watermap, and cns penetration studies to identify centrally efficacious inhibitors

Article abstract of DOI:10.1021/jm3009426

β-Secretase 1 (BACE-1) is an attractive therapeutic target for the treatment and prevention of Alzheimers disease (AD). Herein, we describe the discovery of a novel class of BACE-1 inhibitors represented by sulfamide 14g, using a medicinal chemistry strategy to optimize central nervous system (CNS) penetration by minimizing hydrogen bond donors (HBDs) and reducing P-glycoprotein (P-gp) mediated efflux. We have also taken advantage of the combination of structure based drug design (SBDD) to guide the optimization of the sulfamide analogues and the in silico tool WaterMap to explain the observed SAR. Compound 14g is a potent inhibitor of BACE-1 with excellent permeability and a moderate P-gp liability. Administration of 14g to mice produced a significant, dose-dependent reduction in central Aβ_X-40 levels at a free drug exposure equivalent to the whole cell IC₅₀ (100 nM). Furthermore, studies of the P-gp knockout mouse provided evidence that efflux transporters affected the amount of Aβ lowering versus that observed in wild-type (WT) mouse at an equivalent dose.

Full text of DOI:10.1021/jm3009426

Article
pubs.acs.org/jmc
Spirocyclic Sulfamides as β‑Secretase 1 (BACE-1) Inhibitors for the
Treatment of Alzheimer’s Disease: Utilization of Structure Based
Drug Design, WaterMap, and CNS Penetration Studies To Identify
Centrally Efficacious Inhibitors
Michael A. Brodney,*,† Gabriela Barreiro,† Kevin Ogilvie,† Eva Hajos-Korcsok,† John Murray,†
Felix Vajdos,‡ Claude Ambroise,† Curt Christoffersen,† Katherine Fisher,† Lorraine Lanyon,†
JianHua Liu,† Charles E. Nolan,† Jane M. Withka,‡ Kris A. Borzilleri,‡ Ivan Efremov,†
Christine E. Oborski,† Alison Varghese,‡ and Brian T. O’Neill†
‡
†Department of Neuroscience and Center of Chemistry Innovation and Excellence, Pfizer Worldwide Research and Development,
Eastern Point Road, Groton, Connecticut 06340, United States
S
* Supporting Information
ABSTRACT: β-Secretase 1 (BACE-1) is an attractive therapeutic target for the treatment and prevention of Alzheimer’s disease
(AD). Herein, we describe the discovery of a novel class of BACE-1 inhibitors represented by sulfamide 14g, using a medicinal
chemistry strategy to optimize central nervous system (CNS) penetration by minimizing hydrogen bond donors (HBDs) and
reducing P-glycoprotein (P-gp) mediated efflux. We have also taken advantage of the combination of structure based drug design
(SBDD) to guide the optimization of the sulfamide analogues and the in silico tool WaterMap to explain the observed SAR.
Compound 14g is a potent inhibitor of BACE-1 with excellent permeability and a moderate P-gp liability. Administration of 14g
to mice produced a significant, dose-dependent reduction in central AβX‑40 levels at a free drug exposure equivalent to the whole
cell IC50 (100 nM). Furthermore, studies of the P-gp knockout mouse provided evidence that efflux transporters affected the
amount of Aβ lowering versus that observed in wild-type (WT) mouse at an equivalent dose.
Proteolytic cleavage of APP by the β-site APP-cleaving enzyme
(BACE-1) takes place within the endosome at low pH and
generates a soluble N-terminal ectodomain of APP (sAPPβ)
and the C-terminal fragment C99.4 Subsequent cleavage of the
membrane-bound C99 fragment by γ-secretase liberates the
various Aβ isoforms, of which Aβ40 and Aβ42 are the
predominant forms.5 In vitro characterization suggests that
BACE-1 is the rate-limiting enzyme for the generation of Aβ
peptides and therefore an attractive target for the development
of AD therapeutics.
INTRODUCTION
■
Alzheimer’s disease (AD), the most common neurodegener-
ative disorder, is defined clinically by a slowly progressing loss
of cognitive function, ultimately leading to dementia and death.
AD pathology is characterized by the presence of extracellular
plaques in the hippocampal and cortical regions of the brain,
accompanied by intraneuronal neurofibrillary tangles and
extensive neuronal loss. The primary components of plaques
are insoluble aggregates of amyloid-β (Aβ) peptides of 38−43
amino acids in length. According to the amyloid hypothesis,
increased production and/or decreased clearance and degrada-
tion of Aβ initiate a molecular cascade leading to neuro-
degeneration and AD.1,2 Aβ is derived from sequential cleavage
of the type I integral membrane protein, amyloid precursor
protein (APP), by two proteases, β- and γ-secretase.3
Special Issue: Alzheimer's Disease
Received: July 3, 2012
Published: September 17, 2012
© 2012 American Chemical Society
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Early work on β-secretase inhibitors focused on peptidic
transition state mimics that took advantage of lipophilic
interactions with binding pockets on the N-terminal (“non-
prime”) or C-terminal (“prime”) sides of the scissile bond.
Unfortunately, the majority of these inhibitors failed to reduce
brain Aβ levels because of poor exposure in the CNS.6 In most
cases, significant P-gp mediated efflux likely prevented adequate
levels of drug from crossing the blood−brain barrier (BBB)
because of the high molecular weight, large total polar surface
area, and undesirable number of hydrogen bond donors
(HBDs) characteristic of these compounds.7−10 To identify
inhibitors with robust CNS penetration, novel scaffolds with
improved properties for a CNS drug candidate were sought.11
In recent years, alternative chemotypes such as acylguanidine,
aminothiazine, and aminoimidazole based inhibitors were
discovered that bind directly to the catalytic aspartic acids
(Asp32 and Asp228) in BACE-1 by displacing the tightly
bound catalytic water.12−16 Efficient ligand binding requires
compensation for the high desolvation costs by binding
interactions between the catalytic aspartates and a strongly
basic ligand via multiple hydrogen bond interactions. If the
tightly bound catalytic water is considered part of the binding
site, an indirect ligand−protein contact could occur via a water
mediated hydrogen bond to the inhibitor. In this manner, the
protein desolvation penalty would be greatly reduced and a less
basic inhibitor with fewer H-bond donors could be envisaged.
Recent reports in the literature described several classes of
BACE-1 inhibitors (1−3) that do not displace the catalytic
water but rather make a hydrogen bond via the water to the
catalytic aspartates (Figure 1). Of particular interest were a class
and explain new SAR that ultimately led to the discovery of a
potent, selective, and centrally efficacious BACE inhibitor.
CHEMISTRY
■
The synthesis of spirocyclic sulfamides (4) as represented by
analogues 11a−h is illustrated in Scheme 1. The key step in the
synthetic route is the cyclization of diamine 6 to generate
sulfamide 9. The synthesis of diamine 6 began with a Strecker
reaction of chiral piperidinone 5, which proceeded with modest
stereoselectivity (3:2) favoring the desired product.22−24
Following separation of the diastereomers, the nitrile was
reduced to diamine 6 using Raney nickel. Initial attempts to
cyclize diamine 6 by treatment with sulfamide/pyridine at 120
°C provided the desired sulfamide 9 in only 6% isolated yield.
In order to improve the yield, we sought a more reactive source
of the sulfonyl (SO2) group. Reaction of 6 with sulfuryl
chloride (SO2Cl2) was next attempted, but only trace product
was observed; the major product was dimerized starting
material. Attempts to carry out the ring closure in a two-step
process by treatment with sulfamide (NH2SO2NH2) or
dimethylsulfamoyl chloride (ClSO2NMe2) gave cyclization
precursor 8 (where R = NH2 or NMe2); however, further
attempts to cyclize these intermediates to sulfamide 9 failed to
provide the desired material. Following the conditions of
Beaudoin, using the triflate salt of mono-N-methyl-N,N′-
sulfuryldiimidazole (7), the ring closure was successfully
achieved in 30% yield.25 Further optimization by the dropwise
addition of 6 to a solution of triflate salt 7 in acetonitrile
decreased side product formation and provided 9 in 44% yield.
Conversion of 9 into the desired piperidine analogues 11a−h
was accomplished by deprotection of the Cbz group using H2/
Pd(OH)2 followed by reductive amination with an appropriate
benzaldehyde. Further functionalization of the sulfamide 11 at
sulfamide nitrogen was accomplished via N-alkylation with an
appropriate alkyl halide or N-arylation using copper(I)-
mediated coupling to provide sulfonamides 12a−h.26 Alter-
natively, the sulfamide 9 was first functionalized using N-
alkylation or N-arylation conditions followed by deprotection
and reductive amination to furnish 12a−h. Sulfamide 9 was also
alkylated with methyl iodide; removal of the Cbz protecting
group provided N-methylsulfamide 13. Reductive amination
with an appropriate benzaldehyde or alkylation with a benzylic
bromide provided piperidine analogues 14a−g.
Figure 1. Examples of BACE inhibitors that do not displace catalytic
water.
RESULTS AND DISCUSSION
■
We began our SAR exploration by varying the substituents on
the piperidine nitrogen (R1, see Table 1). The benzyl analogue
11a was prepared first and showed weak binding activity (IC50
= 20.9 μM) in our BACE-1 cell-free assay (CFA), with a
moderate MDR efflux ratio (MDR Er) of 2.5 in an MDR1-
transfected MDCK cell line; this suggested the potential for this
compound to be a substrate for the P-gp efflux transporter.27
To rapidly expand the SAR, the effect of a wide range of aryl,
heteroaryl, and cycloalkyl R1 substituents was investigated using
plate-based synthesis. Alkyl ethers at the ortho or para positions
of aryl rings were inactive (IC50 > 300 μM) in the CFA (data
not shown), but meta substituents such as ethers (11c−g)
improved BACE-1 potency relative to 11a. Within this series of
analogues, the isopropyl congener (11d) appeared to be
optimal for potency relative to methyl ether (11b), ethyl ether
(11c), branched alkyls (11f), or cycloalkyls (11g). Potency was
not directly correlated to lipophilicity (clogP) or molecular
of tyramine derivatives (2) and iminohydantoin spiropiper-
idines (3) that were confirmed through X-ray analysis to bind
to the active site via a water mediated hydrogen bond with a
protonated basic amine.17−20 The tyramine derivatives (2) bind
to the catalytic aspartates with the protonated primary amine
which places the cyclohexyl group in the S3 pocket.20 Key
features of the iminohydantoin (3) complex with BACE-1 were
a hydrogen bond between the N−H of Gln73 of the protein
flap and the carbonyl group of the hydantion and the
arylacetamide binding in the S1 pocket.17 Inspired by these
structures, we designed a series of spirocyclic sulfamides (4)
aiming to make further interactions with the protein flap via the
sulfone group. Described in this paper is the optimization of a
class of spirocyclic sulfamides 4 using a combination of
structure based drug design (SBDD) and WaterMap21 to guide
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a
Scheme 1
a
Reagents and conditions: (a) Zn(CN)2, 3-fluoroaniline, CH2Cl2, 80−90%, then chiral HPLC; (b) Raney Ni, 2 N NH3, MeOH, 50−60%; (c) 7,
CH3CN, rt, 44%; (d) NaH, DMF, 0 °C, then MeI, rt, 95%; (e) Pd(OH)2, 2 N NH3, MeOH, H2, 50 psi; (f) RCHO, Cl(CH2)2Cl, AcOH, then
NaBH(OAc)3, rt, 40−80% over two steps; (g) Cs2CO3, DMF, R1CH2Br, rt, 35−60%; (h) R2X, CuI, K2CO3, N,N’-dimethylethane-1,2-diamine,
dioxane, 105 °C, 12−24 h, 30−68%; (i) NaH, DMF, 0 °C then R2X, rt, 20−93%.
weight (MW). In addition, analogue 11d showed minimal
difference between the cell-free assay (IC50 = 1.3 μM) and
whole-cell assay (WCA IC50 = 2.9 μM), indicating the absence
of barriers to cellular penetration. Further profiling for potential
P-gp mediated efflux demonstrated that 11d was a substrate,
with MDR Er = 3.5. Replacing the ether group of 11b−g with a
biaryl substituent (11h) led to a significant improvement in
enzyme inhibition but at the expense of significant P-gp
mediated efflux (MDR Er = 16). We postulated that the high
predicted P-gp efflux may be due in part to the combination of
high molecular weight and the presence of a hydrogen bond
donor (HBD) in the sulfamide. Thus, the follow-up strategy to
analogues 11a−h was to reduce efflux from the CNS by
capping the N−H sulfamide to reduce the HBD count.
To investigate the sulfamide substituent (R2), a series of
alkyl- and heteroaryl-substituted compounds were prepared and
evaluated in the CFA and WCA and for efflux potential (Table
2). The methyl analogue 12a showed comparable cellular
potency with reduced P-gp mediated efflux, compared to the
N−H analogue 11d (MDR Er = 1.7 vs 3.8). Attempts to
improve potency by replacing the methyl of sulfamide 12a with
ethyl (12b) or isopropyl (12c) resulted in loss of ligand
efficiency (LE) and were not further pursued. We next
investigated heterocyclic R2 substituents, which we anticipated
would make unique interactions with the protein flap and
potentially increase potency. To this end, a set of heteroaryls
including pyridines 12d−e, pyrimidine 12f, thiazole 12g, and
oxazole 12h were prepared (see Chemistry section). In general,
the potency was not significantly improved by varying the
heterocycle off the sulfonamide (R2). Relative to the alkyl
analogues 12a−c, the heterocyclic variants also suffered from
reduced passive permeability in the RRCK cell line.28 Despite
the increase in MW for analogues 12b−h relative to 12a, the
projected P-gp efflux values remained low underscoring the
importance of reducing the number of HBDs when designing
in a higher range of MW (Table 2).11
Relative to most peptidomimetic BACE-1 inhibitors,
compound 12a possesses unique physicochemical properties.
The compound lacks a hydrogen bond donor, has moderate
TPSA of 69, and has a clogP of 3.9. This physicochemical
property space represents a significant improvement over
classical BACE-1 inhibitors.6−9 To further understand the brain
penetration of this series, a mouse CNS pharmacokinetic study
was conducted with analogues 11d and 12a (Table 4).29 Both
analogues showed good brain to plasma ratio (11d, 0.67; 12a,
0.97); however, compound 12a also displayed favorable
unbound brain to unbound plasma ratios (Cu,b/Cu,p of 0.67 vs
0.2 for 11d), suggesting that no efflux transporters were
limiting access to the central compartment.
On the basis of the attractive binding, selectivity, and CNS
penetration, the structure of 12a bound to BACE-1 was
determined by X-ray crystallography (Figure 2). The
compound adopts a folded conformation that places the
fluorophenyl and isopropoxyphenyl rings in proximity to one
another, presumably stabilized by π−π stacking interactions
and/or hydrophobic collapse (see Supporting Information
Table 1 for refinement statistics). The protonated nitrogen of
the piperidine directly hydrogen-bonds to the catalytic water
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Table 1. In Vitro Pharmacology, MDR Efflux Ratio, and Physicochemical Properties for 11a−h
a
b
IC50 values obtained from BACE-1 cell free assay (CFA). IC50 values obtained from BACE-1 whole cell assay (WCA); ND = not determined.
c
MDR efflux ratio (MDR Er) from MDR1-transfected MDCK line.
situated between the catalytic aspartates Asp32 and Asp228 in a
manner similar to that reported in the literature for compounds
2 and 3.17,20 One sulfamide oxygen accepts two hydrogen
bonds, one from the backbone N−H of Thr72 and the other
from the backbone N−H of Gln73. The methyl group of the
spiropiperidine inserts into a small cleft at the back of the
binding pocket. The fluorophenyl ring occupies what we
termed the S1a site, as this pocket is primarily an extension of
the S1 pocket. The isopropoxyphenyl group is situated squarely
in the S1 pocket, with the isopropoxy group extending into the
S3 pocket. Several waters, both crystallographic and modeled,
are displaced by the isopropoxy group.
Although crystal structures provide information regarding
steric and electrostatic factors, some aspects of ligand SAR
cannot be accounted for, so additional computational analysis is
required for a comprehensive explanation of the available SAR.
One such method, applied in this work to help explain the SAR
shown in Table 1, is the use of WaterMap.21 This method
computes the free energy of hydration (ΔGhyd) for binding site
waters, that is, the energy difference associated with the
displacement of water molecules from the protein binding site
into bulk solution.30 WaterMap is well-suited to the analysis of
congeneric molecules where small structural modifications can
result in changes in activity.
The ΔGhyd was computed for the crystal structure of
compound 12a as described in the Computational Details
section. The hydration sites can be seen in Figure 3. Stable
hydration sites (ΔGhyd < 0.0 kcal/mol) with respect to bulk
water are shown in green, unstable hydration sites (ΔGhyd
>
+1.0 kcal/mol) in red, and those that are moderately unstable
(0.0 < ΔGhyd < +1.0 kcal/mol) in yellow. These ranges were
assigned based on the ΔGhyd values obtained for the water
molecules in the BACE binding site. The core of the
spiropiperidine analogues overlaps with eight hydration sites,
all of which are unfavorable with free energies ranging from
+0.5 to +5 kcal/mol. Hydration site 2, one of the most unstable
of all 22 hydration sites computed in the BACE-1 binding
pocket, showed a ΔGhyd of +4.94 kcal/mol and overlaps with
the fluorine atom of the phenyl ring. All the remaining
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Table 2. In Vitro Pharmacology, MDR Efflux Ratio, and Passive Permeability for 12a−h
a
b
c
IC50 values obtained from BACE-1 CFA. IC50 values obtained from BACE-1 WCA; ND = not determined. MDR efflux ratio (MDR Er) from
d
MDR1-transfected MDCK line. RRCK cells with low transporter activity were isolated from Madin−Darby canine kidney cells and were used to
estimate intrinsic absorptive permeability.28
Figure 3. X-ray crystal structure and overlapping hydration sites for
compound 12a bound to BACE-1.
Figure 2. X-ray structure of compound 12a bound to BACE-1.
As shown in Table 1, the unsubstituted phenyl compound
11a has an IC50 of 20.9 μM. The addition of the isopropoxy
(11d) and biaryl (11h) groups resulted in almost 20-fold
hydration sites (1,3−8) display ΔGhyd values between +0.5 and
+2.5 kcal/mol.
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increase in potency (IC50 of 1.3 and 1.7 μM, respectively). This
can be understood by considering the displacement of water
from hydration sites 9, 10, and 11 with ΔGhyd of +5.7, +3.2, and
+0.8 kcal/mol, respectively. Hydration site 9 showed the
second most unfavorable free energy of all hydration sites
computed. Compound 11d displays a binding mode very
similar to the one observed in the X-ray structure for
compound 12a, with the isopropoxy group overlapping very
well with the three unfavorable hydration sites. A similar
binding mode is predicted by modeling of the biaryl derivative
11h. The significant increase in potency seen for compounds
11d and 11h may be attributable to the free energy gain from
release of these three waters into bulk solution. The reduced
affinity for the methoxy analogue (11b) is probably related to
its inability to displace water from these unfavorable hydration
sites (9, 10, and 11). A more flexible alkoxy side chain present
in compounds 11e and 11f and a more rigidified one present in
compound 11g might be preventing an ideal overlap with the
same unstable water molecules and could be responsible for the
weak potency observed for these compounds. Compound 11b
may also suffer an increased desolvation penalty when
compared to compound 11a caused by the presence of the
ether oxygen. Although the same argument can be extended to
the ether analogues in Table 1, the free energy gain from the
liberation of waters 9, 10, and 11 largely offsets the increased
desolvation penalty for the analogues able to displace the
unstable hydration sites.
examined are not likely differentiated on the basis of formation
of specific H-bonds with the protein.
The discovery of 12a indicated that the spirocyclic sulfamide
template was a viable starting point for further optimization of
potency and ADME properties. Analysis of the X-ray structure
of 12a suggested that interactions with the backbone carbonyl
of residue Phe108 could potentially provide improved potency.
An extensive SAR effort was undertaken to replace the benzyl
group of 12a with an aromatic group lacking additional HBDs;
however, we were unable to identify alternative analogues with
improved binding potency and ADME properties relative to
12a. Despite the inherent risk of adding back a HBD into the
R1 group due to the potential for increased P-gp efflux, we
synthesized and profiled a series of analogues containing an
acetamide, phenol, or N−H heterocycle (Table 3). Installation
of an acetamide in the para positon of the benzyl group in
(14a) resulted in reduced potency relative to 12a and
significant P-gp mediated efflux (MDR Er = 7.4). Indole 14b
indicated that heteroaromatics in the S1 pocket gave reduced P-
gp efflux but additional substituents off the heteroaryl ring
would be required for improved binding activity. To this end,
ethylindazole 14c was designed to make an interaction with
Phe108 and displace unstable water molecules in hydration
sites 9−11 (Figure 3); this yielded improved cellular potency
relative to 14b. We next postulated that a phenol might
efficiently interact with Phe108, which proved to be the case
(Table 3). Isothermal titration calorimetry (ITC) data for
compound 12a showed a free energy of binding of −8.15 kcal/
mol with an enthalpic contribution of −10.3 kcal/mol. In the
case of the unsubstituted phenol analogue (14d), a free energy
of binding of −8.6 kcal/mol with an enthalpic contribution of
−10.7 kcal/mol was obtained suggesting that the phenol OH is
engaged in favorable interactions with the protein. The entropic
contributions for 12a and 14d were essentially equivalent.
Inspired by this observation and the earlier ether SAR (Table
1), a series of disubstituted analogues 14e−g were designed and
synthesized (see Chemistry section). As was previously
observed (Table 1), the isopropyl analogue 14g provided
superior potency relative to a range of other ethers including
the ethyl ether 14f as well as the chloro analogue 14e.
Compound 14g exhibited a ∼10-fold improvement in free
energy of binding (−9.88 kcal/mol) relative to 12a. In addition,
14g showed improved binding kinetics, with a measurable off
rate of 0.034 (1/s) compared with the equilibrium binding for
14d as measured by surface plasmon resonance (SPR)
techniques. Interestingly, analogues 14e−g showed reduced
P-gp mediated efflux relative to phenol 14d. We speculated that
an ortho alkoxy substituted phenol such as analogues 14e−g
may engage in intermolecular hydrogen bonding, effectively
blocking recognition from the P-gp efflux transporter.31,32
Analogue 14g also exhibited a superior balance of microsomal
stability and MDR Er (3.4) relative to other analogues. The
structure of isopropoxyphenol (14g) bound to BACE-1 is
depicted in Figure 5 (see Supporting Information Table 1 for
refinement statistics). The structure is essentially identical to
that of the parent compound 12a, with the exception that the
phenolic OH is clearly donating a hydrogen bond to the
backbone carbonyl of Phe108.
The important role that binding-site waters play in the
binding affinity differences for the compounds in Table 1 can
be further supported by the good correlation (R2 = 0.82)
between the experimental data and the WaterMap free energy
liberation (ΔGWM) (Figure 4). ΔGWM estimates the free energy
Figure 4. Correlation between the experimental activities and the
WaterMap free energy liberation of binding site waters for
spiropiperidines analogues in Table 1 (see Supporting Information
Table 2 for the ΔGWM, ΔHWM, and −TΔSWM values).
gained when a ligand that is suitably complementary to the
binding site displaces the waters therein into bulk solution.28
Therefore, the R1 groups that displace the most unfavorable
binding-site waters result in the most potent compounds. The
WaterMap results can also help rationalize, from protein-
hydration observations, the flat SAR observed for compounds
in Table 2. First, the waters in that region of the binding site are
not particularly unfavorable (0.0 < ΔG < +1.0 kcal/mol), as
that site is somewhat more solvent-exposed. Second, the
analogues are similar in size, except for 12a, and displace
essentially the same water molecules. Also, the R2 substituents
To further understand the in vitro selectivity profile of
compounds 12a and 14g, screening against the related aspartyl
protease cathepsin D (CatD) was initiated. At doses up to 100
μM, compounds 12a and 14g showed no inhibition of CatD. In
addition, compound 14g was screened in a broad pharmaco-
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Table 3. In Vitro Pharmacology, MDR Efflux Ratio, and Microsomal Stability for 14a−g
a
b
c
IC50 values obtained from BACE-1 CFA. IC50 values obtained from BACE-1 WCA. Predicted MDR efflux ratio (MDR Er) from MDR1-
d
transfected MDCK line. Predicted human intrinsic clearance, scaled (h-CLint, scaled) from human liver microsomal stability assay.
logical panel (Cerep) and exhibited greater than 100-fold
selectivity versus other receptors and enzymes with the
exception of L-type Ca2+ channel activity (see Supporting
Information Table 3).
(plasma, brain, CSF). At 3 h following subcutaneous
administration of a single dose of 14g to wild-type FVB mice
(WT), significant dose-dependent reductions were observed in
both brain and CSF Aβ40 levels at the two highest doses (100
and 300 mg/kg; Figure 6a and Figure 6c). The changes in Aβ42
paralleled those of Aβ40 in the brain, with statistically
significant lowering reached at the 30−300 mg/kg doses
(Figure 6b). The compound also elicited a robust decrease in
plasma Aβ40 (average 55%) compared to vehicle control across
the doses examined (Figure 6d). The non-dose-dependent
changes in the plasma suggest that maximum reductions likely
have been achieved in this compartment at all doses tested,
consistent with the 55% Aβ40 reduction reported in the plasma
of BACE-1(−/−) vs BACE-1(+/+) mice.33
Analogous to compounds 11d and 12a, isopropoxyphenol
14g underwent brain penetration studies in the mouse with
Cu,b/Cu,p = 0.27 (Table 4). The Cu,b/Cu,p ratio for 14g showed
asymmetry between unbound drug concentrations in the
plasma and brain compartment, suggesting involvement of an
efflux transporter. The MDR Er data support the conclusion
that P-gp is involved in the observed asymmetric distribution of
14g in plasma and brain. Despite the reduced brain penetration
of 14g relative to 12a, the higher free fraction (fu plasma),
excellent potency, and selectivity of 14g suggested this was an
excellent tool to advance our understanding of the PK−PD
relationship between unbound drug concentrations and Aβ
reductions in relevant peripheral and central compartments
In vivo efficacy of 14g was also tested in P-gp knockout mice,
an animal model that has functional deficiency at the blood−
brain barrier due to disruption of the MDR1a efflux
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CONCLUSION
■
Described herein is the identification and optimization of a
novel series of spirocyclic sulfamide BACE-1 inhibitors that
bind to the catalytic aspartic acids via water mediated hydrogen
bond. A combination of SBDD and computational modeling
using WaterMap guided the rational design of new analogues
and explained the obatined SAR. Excellent in vitro potency was
achieved in the series by combining a substituent in the S3
pocket with a phenol of the inhibitor, resulting in favorable
interactions with the protein. Improved brain penetration
relative to classical BACE-1 inhibitors was achieved by reducing
the number of hydrogen bonds, lipophilicity, and molecular
weight. Despite the presence of a HBD and modest predicted
P-gp efflux, analogue 14g achieved unbound drug concen-
trations in brain well above the cell free or whole cell binding
affinities. Compound 14g displays key characteristics of a
BACE-1 inhibitor in vivo, significantly reducing β-cleavage of
APP and Aβ production in brain, plasma, and CSF in WT mice
after an acute dose. The Cu,b/Cu,p ratio for 14g in WT mice
coupled with the difference in doses required for Aβ lowering
in the P-gp KO relative to WT mice suggests that this
compound is a substrate for the Pgp transporter. Future effort
will focus on identification of BACE-1 inhibitors with reduced
asymmetry between the central and peripheral compartments
to optimize the therapeutic index.
Figure 5. X-ray structure of compound 14g bound to BACE-1.
Table 4. In Vitro and in Vivo PK Properties of
Representative Compounds
EXPERIMENTAL SECTION
PK properties
11d
12a
7.5
14g
■
a
MDCK AB (10−6 cm/s)
10.2
3.8
17.6
3.3
Biology. BACE-1 Enzyme Cell-Free Assay (CFA). BACE-1 activity
was measured using a polyclonal antibody, p1007 (Pfizer), that
specifically recognizes the C-terminus of APP695swe created after
cleavage by BACE-1. To generate p1007, rabbits were immunized with
the peptide NH2-EISEVNL-COOH conjugated to keyhole limpet
hemacyanin. Concentrated conditioned medium from cells secreting
human recombinant soluble BACE-1 was titrated to provide a source
of BACE-1 enzyme. The reaction is initiated by the addition of soluble
BACE-1 enzyme to a mixture containing 0.05 M sodium acetate (pH
4.5), compound inhibitor, 2% DMSO, and 3 μM APP substrate
(K(biotin)RGLTTRPGSGLTNIKTEEISEVNLDAEFRHDSGA,
American Peptide). The reaction is incubated at 37 °C for 1 h and
quenched by the addition of an equivalent volume of 0.1 M Tris, pH
8.0. Cleaved product is measured via an ELISA with streptavidin-
coated plates (Greiner Bio-One), using p1007 reporter antibody
(Pfizer) and anti-rabbit horseradish peroxidase (GE Healthcare, U.K.).
sAPPβ Whole Cell Assay (WCA). sAPPβ, the primary cleavage
product of BACE-1, was determined in H4 human neuroglioma cells
overexpressing the wild-type human APP695. Cells were treated for 18
h with compound in a final concentration of 1% DMSO. sAPPβ levels
were measured by ELISA with a capture APP N-terminal antibody
(Affinity BioReagents, OMA1-03132), wild-type sAPPβ-specific
reporter antibody p192 (Elan), and tertiary anti-rabbit-HRP (GE
Healthcare). The colorimetric reaction was read by an EnVision
(Perkin-Elmer) plate reader.
Animals and in Vivo Protocols. All animal procedures were
approved by the Institutional Animal Care and Use Committee and
were compliant with animal welfare act regulations. Female FVB/N
and male P-glycoprotein knockout (P-gp KO) mice (Taconic,
Germantown, NY) aged 2−4 months received a single subcutaneous
injection of test compound or vehicle (20% DMSO, 20% ethanol in
polyethylene glycol), N = 5−6 mice/treatment group. At 3 h following
dosing, animals were euthanized with CO2, and brain, CSF, and blood
samples were collected for measurement of Aβ and drug
concentrations.
b
MDR BA/AB
1.7
c
in vitro h-CLh (mL min−1 kg−1
)
>19
>19
15.4
59.7
0.35
0.17
0.12
0.27
d
in vitro r-CLh (mL min−1 kg−1
)
>65.5
0.67
0.041
0.018
0.22
>65.5
0.97
0.036
0.024
0.65
e
B/P (mouse)
f
Fu (plasma)
f
Fu (brain)
Cub/Cup
a
MS-based quantification of the basal/apical transfer rate of a test
compound at 2 μM across contiguous monolayers from MDCK
(Madin−Darby canine kidney) cells. Ratio from the MS-based
quantification of apical/basal and basal/apical transfer rates of a test
compound at 2 μM across contiguous monolayers from MDR1-
transfected MDCK cells. Hepatic clearance predicted from in vitro
human microsomal stability study. Hepatic clearance predicted from
in vitro rat microsomal stability study. Determined from plasma and
brain exposures in mice 60 min after 10 mg/kg subcutaneous dosing.
b
c
d
e
f
Determined from equilibrium dialysis.
transporter.34 At 3 h after sc administration of 14g, robust brain
Aβ40 lowering was achieved even at low doses, with statistically
significant effects starting at the 3 mg/kg sc dose (Figure 6e).
At the 10−300 mg/kg dose range, brain drug exposures were
about 7- to 12-fold higher in P-gp KO mice compared to the
WT strain (see Supporting Information Table 4). Importantly,
brain Aβ-lowering efficacy of 14g shows a consistent relation-
ship with brain exposures across the two mouse strains (Figure
6f), with significant Aβ reductions associated with a range of
drug concentrations. These data also suggest that maximum Aβ
reductions are about 65% of vehicle control under the present
experimental conditions, and no further Aβ lowering can be
achieved despite robust increases in brain exposures. The
reason for this residual Aβ is not clear, but could be due, at least
in part, to the limitations of single time-point data collection in
the present study.
β-Amyloid ELISA. Sample preparation and assay protocols for
determination of Aβ40 and Aβ42 were as described previously.35
Briefly, brains were homogenized in 9 volumes of 5 M guanidine HCl/
50 mM Tris-HCl (pH 8.0), extracted for 3 h, and centrifuged at
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Figure 6. Effect of 14g on Aβ peptide levels in mice at 3 h following acute sc administration. Brain Aβ40 (a), brain Aβ42 (b), CSF Aβ40 (c), and
plasma Aβ40 (d) were determined in wild-type (FVB/N) mice, N = 5−6 per treatment group by ELISA. Brain Aβ40 lowering was measured in P-gp
KO mice using the same experimental conditions for (e) brain exposure−response relationship in WT and P-gp KO mice (f). Statistical analysis was
conducted using one-way ANOVA, post hoc Dunnett’s test: (∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001 vs vehicle.
135000g. Aliquots (200 μL) were analyzed for Aβ40 by a standard
Delfia immunoassay using 4 μg/mL monoclonal antibody 1219 (Rinat,
Pfizer) for capture and 0.2 μg/mL biotinylated 4G8 antibody
(Covance) for detection. Fluorescence of the Delfia europium label
was measured by an EnVision reader.
Measurement of Fractions Unbound in Brain. The unbound
fraction of each compound was determined in brain tissue homogenate
using a 96-well equilibrium dialysis method as described by Kalvass et
al.29 with the following exceptions. Brain homogenates were prepared
from freshly harvested rat brains following dilution with a 4-fold
volume of phosphate buffer and spiked with 1 μM compound. The
homogenates were dialyzed against an equal volume (150 μL) of
phosphate buffer at 37 °C for 6 h. Following the incubation, equal
volumes (50 μL) of brain homogenate and buffer samples were
collected and mixed with 50 μL of buffer or control homogenate,
respectively, for preparation of mixed matrix samples. All samples were
then precipitated with internal standard in acetonitrile (200 μL),
vortexed, and centrifuged. Supernatants were analyzed using an LC−
MS/MS assay. A dilution factor of 5 was applied to the calculation of
brain fraction unbound.
X-ray and Biophysics. Crystallization of BACE. Crystals of BACE
were prepared as previously described.36 Briefly, recombinant BACE
lacking the prosegment was concentrated to 10−13 mg/mL in 0.1 M
sodium borate, pH 8.5. Crystallization was done by the vapor diffusion
method, using 2 μL of protein solution and 2 μL of reservoir solution
(30% PEG 200, 0.1 M sodium acetate, pH 5.2−5.4). Drops were
microseeded 24 h after setup with 0.3−0.5 μL of seed stock made from
a previously grown BACE-1 crystal. Crystals generally grew to ∼0.1 ×
0.1 × 0.05 mm3 in 2 weeks. The crystal form is C2221, with a single
molecule of BACE-1 in the asymmetric unit, and diffracts to high
resolution (dmin < 2.0 Å) with typical X-ray exposure times.
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Soaking of Crystals. Soaking solutions were made by diluting the
concentrated stock compound mixtures 1:10 with 30% PEG 200 and
0.1 M sodium acetate, pH 5.2. Crystals were transferred individually
from their mother drops to a 5 μL drop of soaking solution and
allowed to equilibrate at 21 °C for ∼24 h. Following this equilibration,
crystals were flash cooled for data collection by harvesting in rayon
loops and plunging directly into liquid nitrogen.
Data Collection. X-ray diffraction data were collected in house on a
Rigaku MicroMax007HF X-ray generator outfitted with a RaxisIV++
image plate detector. For each crystal a total of 120° of data were
collected in 0.5° oscillations, generally yielding ∼100% complete data
coverage at 4−5× redundancy for accurate scaling and outlier
rejection. Data processing was performed using D*TREK37 for
compound 12a or XDS38 for compound 14g. All data manipulations
were performed using the CCP4 suite of programs,39 care being taken
to preserve the Rfree test set for each data set.
Structure Solution. Structures were determined by rigid body
refinement using the program REFMAC.40 The initial model was a 1.5
Å crystal structure of apo BACE-1 that was solved as part of an
ongoing structure-based drug design effort. As we expected conforma-
tional variability in the “flap” region of BACE, residues 68−75 were
excised from the model prior to refinement to minimize difficulty in
interpretation of the mFo − dFc σA-weighted difference maps. Binding
of compounds to the BACE-1 active site was assessed by visual
inspection of the resulting difference maps using the program
COOT.41 Final refinement was done using AUTOBUSTER.42
Refinement statistics are reported in Supporting Information Table 3.
Isothermal Titration Calorimetry. Isothermal titration calorimetry
(ITC) experiments were performed using a VP-ITC calorimetric
system from MicroCal Inc. Data collection, analysis, and plotting were
performed using a Windows-based software package (Origin, version
7.0) supplied by Microcal. All titrations were performed by adding
titrant in steps of 8 μL and were conducted at a constant temperature
of 25 °C. All solutions contained within the calorimetric cell and
injector syringe were prepared in the same buffer: 50 mM sodium
acetate, pH 5.0, 50 mM NaCl, 0.02% sodium azide, and 0.5% DMSO.
To assess binding of the compounds to BACE, 10 μM protein was
titrated with 100 μM compound contained in the injector syringe. All
solutions were properly degassed to prevent bubble formation in the
calorimetric cell during stirring. The heat evolved upon each injection
of compound was obtained from the integral of the calorimetric signal.
The heat of dilution was subtracted from the heat of reaction to obtain
the heat associated with binding of a ligand to the protein in the cell.
Nonlinear regression of the data provided the enthalpy change (ΔH)
and association constant (Ka) 1/Kd.
Compounds were presalted as TFA salts and diluted with 1 mL of
dimethylsulfoxide. Samples were purified by mass triggered collection
using a mobile phase of 0.1% trifluoroacetic acid in water and
acetonitrile with a starting gradient of 100% aqueous to 100%
acetonitrile over 10 min at a flow rate of 20 mL/min. Elemental
analyses were performed by QTI, Whitehouse, NJ. All target
compounds were analyzed using ultrahigh performance liquid
chromatography/ultraviolet/evaporative light scattering detection
coupled to time-of-flight mass spectrometry (UHPLC/UV/ELSD/
TOFMS). Unless otherwise noted, all tested compounds were found
to be >95% pure by this method.
UHPLC/MS Analysis. The UHPLC was performed on a Waters
ACQUITY UHPLC system (Waters, Milford, MA), which was
equipped with a binary solvent delivery manager, column manager,
and sample manager coupled to ELSD and UV detectors (Waters,
Milford, MA). Detection was performed on a Waters LCT premier XE
mass spectrometer (Waters, Milford, MA). The instrument was fitted
with an Acquity BEH (bridged ethane hybrid) C18 column (30 mm ×
2.1 mm, 1.7 μm particle size, Waters, Milford, MA) operated at 60 °C.
Benzyl (2S,4R)-4-Cyano-4-[(3-fluorophenyl)amino]-2-meth-
ylpiperidine-1-carboxylate. To a solution of benzyl (2S)-2-
methyl-4-oxopiperidine-1-carboxylate22,23 (20.0 g, 80.9 mmol) in
acetic acid (162 mL) were added 3-fluoroaniline (18.0 g, 162
mmol) and zinc cyanide (23.7 g, 202 mmol). The mixture was allowed
to stir overnight at room temperature. The mixture was cooled to 0 °C
and quenched with NH4OH until the mixture was basic. The mixture
was filtered to provide a yellow solid, which was combined with further
extractions with CH2Cl2, dried with Na2SO4, and concentrated. The
material was purified using silica gel chromatography to afford the title
compound (29.7 g, 72%). MS (LCMS) m/z 368.1 (M + 1). The
diastereomeric mixture of benzyl (2S)-4-cyano-4-[(3-fluorophenyl)-
amino]-2-methylpiperidine-1-carboxylates was subjected to super-
critical fluid chromatography to separate the diastereomeric products
(column, Chiralcel OJ-H; eluent, 80:20 CO2/MeOH). The (2S,4S)
isomer eluted first, followed by the title product (17 g, 47%). MS
(LCMS) m/z 368.1 (M + 1). 1H NMR (400 MHz, CDCl3) δ 1.49 (d,
J = 7.2 Hz, 3H), 1.70 (ddd, J = 13.1, 13.2, 4.4 Hz, 1H), 1.89 (dd, J =
13.9, 6.5 Hz, 1H), 2.42−2.49 (m, 2H), 3.35 (ddd, J = 14.6, 13.0, 2.4
Hz, 1H), 3.74 (s, 1H), 4.27 (m, 1H), 4.63 (m, 1H), 5.14 (d, half of AB
quartet, J = 12.3 Hz, 1H), 5.18 (d, half of AB quartet, J = 12.3 Hz, 1H),
6.60−6.68 (m, 3H), 7.21 (m, 1H), 7.32−7.41 (m, 5H); 13C NMR
(100 MHz, CDCl3) observed peaks, δ 17.4, 35.9, 36.8, 38.8, 40.3, 46.0,
46.8, 48.5, 50.5, 67.7, 104.8, 107.9, 113.4, 121.4, 128.2, 128.8, 130.7,
136.6, 144.8, 162.5, 164.9. HRMS calcd for C21H23N3O2F (M + 1)
368.1768, found 368.1766.
Surface Plasmon Resonance (SPR) Experiments. Experiments
were performed on a Biacore 3000 instrument (GE Healthcare).
BACE-1 was immobilized on a CM5 biosensor surface to levels
ranging from 4000 to 5000 RU using standard amine coupling
procedures. Compound binding experiments were performed in 50
mM sodium acetate, pH 5.0, 150 mM NaCl, 0.05% P20, and 3%
DMSO at 25 °C. A series of various compound concentrations were
injected over the BACE surface (flow rate 50 μL/min, contact time
100 s, dissociation time 600 s). Binding responses were processed
using Scrubber 2 (BioLogic Software, Inc.) to zero, x-align, double-
reference, and correct for excluded volume effects in the data. Scrubber
2 was also used to obtain affinity from steady-state equilibrium data.
Kinetic parameters were obtained from global fits of the data to a
simple 1:1 interaction model using Biaeval (GE Healthcare).
Chemistry. General Methods. Solvents and reagents were of
reagent grade and were used as supplied by the manufacturer. All
reactions were run under a N2 atmosphere. Organic extracts were
routinely dried over anhydrous Na2SO4. Concentration refers to rotory
evaporaration under reduced pressure. Chromatography refers to flash
chromatography using disposable RediSepRf 4−120 g silica columns or
Biotage disposable columns on a CombiFlash Companion or Biotage
Horizon automatic purification system. Microwave reactions were
carried out in a SmithCreator microwave reactor from Personal
Chemistry. Purification by mass-triggered HPLC was carried out using
Waters XTerra PrepMS C18 columns, 5 μm, 30 mm × 100 mm.
Benzyl (2S,4R)-4-(Aminomethyl)-4-[(3-fluorophenyl)amino]-
2-methylpiperidine-1-carboxylate (6). To a solution of benzyl
(2S,4R)-4-cyano-4-[(3-fluorophenyl)amino]-2-methylpiperidine-1-car-
boxylate (8.0 g, 21.8 mmol) in a 1 M solution of NH3 in MeOH (109
mL, 218 mmol) was added Raney nickel in water (1.87 g, 21.8 mol).
The mixture was hydrogenated (50 psi) for 12 h, filtered through
Celite, and concentrated. Purification by silica gel chromatography
(gradient 1−4% MeOH in CH2Cl2) provided title compound 6 (7.8 g,
77%). MS (LCMS) m/z 372.1 (M + 1). 1H NMR (400 MHz, CDCl3)
δ 1.17 (d, J = 6.5 Hz, 3H), 1.56 (dd, J = 14.4, 8.3 Hz, 1H), 1.70 (m,
1H), 1.85 (ddd, J = 13.9, 11.3, 6.2 Hz, 1H), 2.04 (dd, J = 14.4, 6.3 Hz,
1H), 2.87 (d, half of AB quartet, J = 14 Hz, 1H), 2.91 (d, half of AB
quartet, J = 14 Hz, 1H), 3.05 (ddd, J = 13.9, 11.5, 4.7 Hz, 1H), 3.93
(ddd, J = 14.0, 6.0, 2.8 Hz, 1H), 4.12 (m, 1H), 5.08 (s, 2H), 6.31−6.40
(m, 3H), 6.99 (m, 1H), 7.23−7.31 (m, 5H); 13C NMR (100 MHz,
CDCl3) observed peaks, δ 19.9, 36.6, 37.6, 46.0, 46.1, 55.6, 56.6, 67.2,
103.1, 105.3, 112.1, 127.9, 128.0, 128.2, 128.7, 130.6, 137.0, 147.5,
155.5, 155.7, 162.7. HRMS calcd for C21H27N3O2F (M + 1) 372.2081,
found 372.2094.
Benzyl (5R,7S)-1-(3-Fluorophenyl)-7-methyl-2-thia-1,3,8-
triazaspiro[4.5]decane-8-carboxylate 2,2-Dioxide (9). The tri-
flate salt of mono-N-methyl-N,N′-sulfuryldiimidazole25 (compound 7,
2.45 g, 6.7 mmol) was dissolved in acetonitrile (20 mL). To this stirred
solution was added a solution of 6 (2.0 g, 5.4 mmol) in acetonitrile (10
mL) dropwise via an addition funnel. The solution was stirred at room
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temperature for 18 h. The mixture was concentrated under reduced
pressure and purified via silica gel chromatography (gradient, 0−35%
ethyl acetate in heptane) to provide the product as a white solid (1.0 g,
43%). MS (LCMS) m/z 434.4 (M + 1). 1H NMR (400 MHz, CDCl3)
δ 1.16 (d, J = 7.2 Hz, 3H), 1.58 (m, 1H), 1.66−1.75 (m, 2H), 2.02 (br
d, J = 12 Hz, 1H), 2.94 (m, 1H), 3.60 (dd, J = 11.6, 9.2 Hz, 1H), 3.72
(dd, J = 12.0, 6.9 Hz, 1H), 4.08 (v br s, 1H), 4.50 (v br s, 1H), 5.05 (d,
half of AB quartet, J = 12.5 Hz, 1H), 5.09 (d, half of AB quartet, J =
12.8 Hz, 1H), 5.56 (dd, J = 8.7, 7.2 Hz, 1H), 7.07−7.18 (m, 3H),
7.29−7.37 (m, 5H), 7.39 (ddd, J = 8.2, 8.2, 6.5 Hz, 1H); 13C NMR
(100 MHz, CDCl3) observed peaks, δ 32.7, 36.1, 38.8, 46.3, 51.2, 66.1,
67.6, 117.0, 117.2, 120.1, 120.3, 128.0, 128.1, 128.4, 128.8, 130.8,
133.7, 136.5, 161.7, 164.2. HRMS calcd for C21H24N3O4FS (M + 1)
434.1544, found 434.1542.
sodium triacetoxyborohydride (10.2 mg, 0.048 mmol) and stirred
overnight. The reaction was quenched with aqueous NaHCO3
solution, extracted with CH2Cl2, dried over Na2SO4, filtered, and
concentrated. Purification by silica gel chromatography using a 0−3%
gradient of methanol/dichloromethane provided the first eluent (6.6
mg, 38%) of the title compound 11c. MS (LCMS) m/z 434 (M + 1).
1H NMR (400 MHz, CDCl3) δ 7.34 (td, J = 8.1, 6.4 Hz, 1H), 7.02−
7.16 (m, 4H), 6.67−6.80 (m, 3H), 4.63 (br s, 1H), 3.96 (q, J = 6.9 Hz,
2H), 3.59 (d, J = 13.5 Hz, 1H), 3.48 (q, J = 12.2 Hz, 2H), 3.25 (d, J =
13.5 Hz, 1H), 2.61−2.74 (m, 1H), 2.49 (td, J = 8.4, 4.2 Hz, 1H),
2.17−2.30 (m, 1H), 1.90−2.01 (m, 2H), 1.72−1.86 (m, 1H), 1.58 (dd,
J = 13.6, 6.4 Hz, 1H), 1.36 (t, J = 6.9 Hz, 3H), 1.02 (d, J = 6.6 Hz,
3H). HRMS calcd for C22H29N3O3FS (M + 1) 434.1908, found
434.1911.
(5R,7S)-8-Benzyl-1-(3-fluorophenyl)-7-methyl-2-thia-1,3,8-
triazaspiro[4.5]decane 2,2-Dioxide (11a). To a solution of 9 (90
mg, 0.2 mmol) in MeOH (2 mL) in a Parr bottle was added palladium
hydroxide (15 mg, 0.1 mmol). The mixture was hydrogenated on a
Parr shaker at 50 psi overnight. The mixture was filtered and then
concentrated to afford (5R,7S)-1-(3-fluorophenyl)-7-methyl-2-thia-
1,3,8-triazaspiro[4.5]decane 2,2-dioxide (60 mg, 96%) as a colorless
oil. MS (LCMS) m/z 299.37 (M + 1). 1H NMR (400 MHz, CDCl3) δ
0.98 (d, J = 6.3 Hz, 3H), 1.37 (dd, J = 14.3, 10.9 Hz, 1H), 1.69 (ddd, J
= 14.4, 12.2, 4.7 Hz, 1H), 2.17−2.22 (m, 2H), 2.46 (ddd, J = 12.5,
12.5, 2.8 Hz, 1H), 2.56 (m, 1H), 2.80 (ddd, J = 12.8, 4.6, 3.5 Hz, 1H),
3.29 (d, half of AB quartet, J = 11.9 Hz, 1H), 3.34 (d, half of AB
quartet, J = 12.0 Hz, 1H), 7.08−7.14 (m, 2H), 7.19 (ddd, J = 8.0, 2.0,
1.0 Hz, 1H), 7.36 (dddd, J = 8.0, 8.0, 6.4, 0.8 Hz, 1H).
(5R,7S)-1-(3-Fluorophenyl)-8-(3-isopropoxybenzyl)-7-meth-
yl-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Dioxide (11d). To a
solution of (5R,7S)-1-(3-fluorophenyl)-7-methyl-2-thia-1,3,8-
triazaspiro[4.5]decane 2,2-dioxide (12 mg, 0.040 mmol) and 3-
isopropoxybenzaldehyde (12 mg, 0.08 mmol) in dichloroethane (0.4
mL) was added triethylamine (8.1 mg, 0.08 mmol). The mixture was
allowed to stir at room temperature for 30 min, then was charged with
sodium triacetoxyborohydride (10.2 mg, 0.048 mmol) and stirred
overnight. The reaction was quenched with aqueous NaHCO3
solution, extracted with CH2Cl2, dried over Na2SO4, filtered, and
concentrated. Purification by silica gel chromatography using a 0−3%
gradient of methanol/dichloromethane provided the first eluent (11.6
mg, 65.2%) of the title compound 11d. MS (LCMS) m/z 448.6 (M +
1
1). H NMR (400 MHz, CDCl3) δ 7.30 (d, J = 7.4 Hz, 1H), 7.03−
To a solution of (5R,7S)-1-(3-fluorophenyl)-7-methyl-2-thia-1,3,8-
triazaspiro[4.5]decane 2,2-dioxide (28 mg, 0.095 mmol) and
benzaldehyde (19.8 mg, 0.187 mmol) in dichloroethane (0.94 mL)
was added acetic acid (5.6 mg, 0.095 mmol). The mixture was allowed
to stir at room temperature for 5 h, then was charged with sodium
triacetoxyborohydride (40 mg, 0.18 mmol) and stirred overnight. The
reaction was quenched with aqueous NaHCO3 solution, extracted with
CH2Cl2, dried over Na2SO4, filtered, and concentrated. Purification by
silica gel chromatography using a 20−100% gradient of ethyl acetate/
heptane provided the title compound 11a (34 mg, 86%). MS (LCMS)
7.18 (m, 4H), 6.67−6.78 (m, 3H), 5.50−5.87 (m, 1H), 4.40−4.53 (m,
1H), 3.58 (d, J = 13.5 Hz, 1H), 3.45−3.52 (m, 1H), 3.37−3.44 (m,
1H), 3.30 (d, J = 13.5 Hz, 1H), 2.64−2.76 (m, 1H), 2.44−2.55 (m,
1H), 2.18−2.27 (m, 1H), 1.87−2.00 (m, J = 4.5 Hz, 2H), 1.77−1.87
(m, 1H), 1.55−1.66 (m, 1H), 1.26 (d, J = 6.0 Hz, 6H), 1.01 (d, J = 6.8
Hz, 3H). HRMS calcd for C23H30N3O3FS (M + 1) 448.2081, found
448.2064.
(5R,7S)-1-(3-Fluorophenyl)-7-methyl-8-(3-propoxybenzyl)-2-
thia-1,3,8-triazaspiro[4.5]decane 2,2-Dioxide (11e). To a sol-
ution of (5R,7S)-1-(3-fluorophenyl)-7-methyl-2-thia-1,3,8-triazaspiro-
[4.5]decane 2,2-dioxide (12 mg, 0.040 mmol) and 3-propoxybenzal-
dehyde (12 mg, 0.08 mmol) in dichloroethane (0.4 mL) was added
triethylamine (8.1 mg, 0.08 mmol). The mixture was allowed to stir at
room temperature for 30 min, then was charged with sodium
triacetoxyborohydride (10.2 mg, 0.048 mmol) and stirred overnight.
The reaction was quenched with aqueous NaHCO3 solution, extracted
with CH2Cl2, dried over Na2SO4, filtered, and concentrated.
Purification by silica gel chromatography using a 0−3% gradient of
methanol/dichloromethane provided the first eluent (8.5 mg, 48%) of
1
m/z 390.1 (M + 1). H NMR (400 MHz, CDCl3) δ 7.36 (m, 1H),
7.07−7.31 (m, 8H), 4.96 (br s, 1H), 3.63 (d, J = 13.7 Hz, 1H), 3.42−
3.58 (m, 2H), 3.31 (d, J = 13.3 Hz, 1H), 2.64−2.76 (m, 1H), 2.43−
2.58 (m, 1H), 2.19−2.32 (m, 1H), 1.92−2.07 (m, 2H), 1.75−1.88 (m,
1H), 1.61 (dd, J = 5.8, 13.7 Hz, 1H), 1.04 (d, J = 6.2 Hz, 3H). HRMS
calcd for C20H25N3O2FS (M + 1) 390.1632, found 390.1646.
(5R,7S)-1-(3-Fluorophenyl)-8-(3-methoxybenzyl)-7-methyl-
2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Dioxide (11b). To a
solution of (5R,7S)-1-(3-fluorophenyl)-7-methyl-2-thia-1,3,8-
triazaspiro[4.5]decane 2,2-dioxide (16 mg, 0.053 mmol) and 3-
methoxybenzaldehyde (14.4 mg, 0.106 mmol) in dichloroethane (0.53
mL) was added acetic acid (3.2 mg, 0.053 mmol). The mixture was
allowed to stir at room temperature for 5 h, then was charged with
sodium triacetoxyborohydride (22.5 mg, 0.106 mmol) and stirred
overnight. The reaction was quenched with aqueous NaHCO3
solution, extracted with CH2Cl2, dried over Na2SO4, filtered, and
concentrated. Purification by silica gel chromatography using a 20−
100% gradient of ethyl acetate/heptane provided the first eluent (6
mg, 25%) of the title compound 11b. MS (LCMS) m/z 420.0 (M +
1). 1H NMR (400 MHz, CDCl3) δ 7.32−7.45 (m, 1H), 7.06−7.24 (m,
4H), 6.71−6.81 (m, 3H), 4.61−4.74 (m, 1H), 3.77 (s, 3H), 3.44−3.65
(m, 3H), 3.28 (d, J = 13.3 Hz, 1H), 2.64−2.76 (m, 1H), 2.47−2.57
(m, 1H), 2.19−2.32 (m, 1H), 1.93−2.05 (m, 2H), 1.77−1.87 (m, 1H),
1.55−1.65 (m, 1H), 1.05 (d, J = 6.6 Hz, 3H). HRMS calcd for
C2H26N3O3FS (M + 1) 420.1743, found 420.1751.
1
the title compound 11e. MS (LCMS) m/z 448.5 (M + 1). H NMR
(400 MHz, CDCl3) δ 7.31−7.44 (m, 1H), 7.05−7.20 (m, 4H), 6.70−
6.81 (m, 3H), 4.80 (br s, 1H), 3.87 (t, J = 6.6 Hz, 2H), 3.60 (d, J =
13.7 Hz, 1H), 3.43−3.59 (m, 2H), 3.27 (d, J = 13.3 Hz, 1H), 2.63−
2.77 (m, 1H), 2.51 (dt, J = 3.9, 8.4 Hz, 1H), 2.19−2.32 (m, 1H),
1.92−2.04 (m, 2H), 1.72−1.87 (m, 3H), 1.60 (dd, J = 5.8, 13.7 Hz,
1H), 0.96−1.08 (m, 6H). HRMS calcd for C23H30N3O3FS (M + 1)
448.2060, found 448.2064.
(5R,7S)-1-(3-Fluorophenyl)-8-(3-isobutoxybenzyl)-7-methyl-
2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Dioxide (11f). To a
solution of (5R,7S)-1-(3-fluorophenyl)-7-methyl-2-thia-1,3,8-
triazaspiro[4.5]decane 2,2-dioxide (12 mg, 0.040 mmol) and 3-
isobutoxybenzaldehyde (12 mg, 0.08 mmol) in dichloroethane (0.4
mL) was added triethylamine (8.1 mg, 0.08 mmol). The mixture was
allowed to stir at room temperature for 30 min, then was charged with
sodium triacetoxyborohydride (10.2 mg, 0.048 mmol) and stirred
overnight. The reaction was quenched with aqueous NaHCO3
solution, extracted with CH2Cl2, dried over Na2SO4, filtered, and
concentrated. Purification by silica gel chromatography using a 0−3%
gradient of methanol/dichloromethane provided the first eluent (8.5
mg, 46%) of the title compound 11f. MS (LCMS) m/z 462.3 (M + 1).
1H NMR (400 MHz, CDCl3) δ 7.27−7.40 (m, 1H), 7.04−7.17 (m,
(5R,7S)-8-(3-Ethoxybenzyl)-1-(3-fluorophenyl)-7-methyl-2-
thia-1,3,8-triazaspiro[4.5]decane 2,2-Dioxide (11c). To a
solution of (5R,7S)-1-(3-fluorophenyl)-7-methyl-2-thia-1,3,8-
triazaspiro[4.5]decane 2,2-dioxide (12 mg, 0.040 mmol) and 3-
ethoxybenzaldehyde (12 mg, 0.08 mmol) in dichloroethane (0.4 mL)
was added triethylamine (8.1 mg, 0.08 mmol). The mixture was
allowed to stir at room temperature for 30 min, then was charged with
4H), 6.68−6.78 (m, 3H), 4.73 (br s, 1H), 3.61−3.67 (m, 2H), 3.59 (d,
9234
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J = 13.5 Hz, 1H), 3.48 (q, J = 12.1 Hz, 2H), 3.25 (d, J = 13.5 Hz, 1H),
2.61−2.73 (m, 1H), 2.43−2.55 (m, 1H), 2.17−2.30 (m, 1H), 1.90−
2.08 (m, 3H), 1.73−1.86 (m, 1H), 1.59 (dd, J = 13.7, 6.0 Hz, 1H),
1.02 (d, J = 6.6 Hz, 3H), 0.98 (dd, J = 6.6, 2.0 Hz, 6H). HRMS calcd
for C24H33N3O3FS (M + 1) 462.2221, found 448.2212.
mg, 0.061 mmol) in DMF (0.2 mL) was added. The mixture was
allowed to gradually warm to room temperature and stirred overnight.
The mixture was diluted with water and extracted with ethyl acetate (3
× 10 mL). The combined organics were dried over Na2SO4,
concentrated, and purified by silica gel chromatography to yield the
1
(5R,7S)-8-[3-(Cyclopentyloxy)benzyl]-1-(3-fluorophenyl)-7-
methyl-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Dioxide (11g).
To a solution of (5R,7S)-1-(3-fluorophenyl)-7-methyl-2-thia-1,3,8-
triazaspiro[4.5]decane 2,2-dioxide (12 mg, 0.040 mmol) and 3-
cyclopentyloxybenzaldehyde (15.2 mg, 0.08 mmol) in dichloroethane
(0.4 mL) was added triethylamine (8.1 mg, 0.08 mmol). The mixture
was allowed to stir at room temperature for 30 min, then was charged
with sodium triacetoxyborohydride (10.2 mg, 0.048 mmol) and stirred
overnight. The reaction was quenched with aqueous NaHCO3
solution, extracted with CH2Cl2, dried over Na2SO4, filtered, and
concentrated. Purification by silica gel chromatography using a 0−3%
gradient of methanol/dichloromethane provided the first eluent (6.2
mg, 30%) of the title compound 11g. MS (LCMS) m/z 474.0 (M +
1). 1H NMR (400 MHz, CDCl3) δ 7.30−7.43 (m, 1H), 7.03−7.18 (m,
4H), 6.65−6.78 (m, 3H), 4.59−4.74 (m, 2H), 3.58 (d, J = 13.5 Hz,
1H), 3.43−3.55 (m, 2H), 3.25 (d, J = 13.5 Hz, 1H), 2.64−2.77 (m,
1H), 2.50 (ddd, J = 12.4, 8.6, 3.5 Hz, 1H), 2.16−2.32 (m, 1H), 1.90−
2.01 (m, 2H), 1.69−1.89 (m, 7H), 1.50−1.64 (m, 3H), 1.02 (d, J = 6.8
Hz, 3H). HRMS calcd for C25H32N3O3FS (M + 1) 474.2221, found
474.2211.
(5R,7S)-8-[(2′-Chlorobiphenyl-3-yl)methyl]-1-(3-fluorophen-
yl)-7-methyl-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Dioxide
(11h). To a solution of (5R,7S)-1-(3-fluorophenyl)-7-methyl-2-thia-
1,3,8-triazaspiro[4.5]decane 2,2-dioxide (40 mg, 0.13 mmol) and 3-
iodobenzaldehyde (62.2 mg, 0.268 mmol) in dichloroethane (1.34
mL) was added triethylamine (27.1 mg, 0.268 mmol). The mixture
was allowed to stir at room temperature for 30 min, then was charged
with sodium triacetoxyborohydride (34.1 mg, 0.161 mmol) and stirred
overnight. The reaction was quenched with aqueous NaHCO3
solution, extracted with CH2Cl2, dried over Na2SO4, filtered, and
concentrated. Purification by silica gel chromatography using a 0−3%
gradient of methanol/dichloromethane provided 43.6 mg (63%) of
(5R,7S)-8-[3-iodobenzyl]-1-(3-fluorophenyl)-7-methyl-2-thia-1,3,8-
triazaspiro[4.5]decane 2,2-dioxide. MS (LCMS) m/z 516.1 (M + 1).
1H NMR (400 MHz, CDCl3) δ 7.48−7.58 (m, 2H), 7.29−7.42 (m,
title compound 12a (15 mg, 67%). H NMR (400 MHz, CD3OD) δ
7.45 (dt, J = 6.4, 8.1 Hz, 1H), 7.12−7.29 (m, 4H), 6.72−6.87 (m, 3H),
4.56 (spt, J = 6.0 Hz, 1H), 3.69 (d, J = 13.3 Hz, 1H), 3.57 (d, J = 9.4
Hz, 1H), 3.49 (d, J = 13.7 Hz, 1H), 3.29−3.34 (m, 1H), 2.81 (s, 3H),
2.65−2.76 (m, 2H), 2.32−2.43 (m, 1H), 2.06−2.21 (m, 2H), 1.92−
2.02 (m, 1H), 1.73 (dd, J = 7.0, 14.0 Hz, 1H), 1.31 (dd, J = 1.2, 5.8 Hz,
6H), 1.16 (d, J = 6.6 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 163.8,
161.3, 157.9, 135.3, 130.2, 129.0, 121.3, 120.1, 116.6, 114.6, 69.4, 61.3,
60.3, 56.9, 51.3, 44.5, 41.5, 33.6, 31.8, 21.0, 15.0; LCMS m/z 462.3 (M
+ 1); HRMS calcd for C24H32N3O3FS (M + 1) 462.2221, found
462.2224.
(5R,7S)-3-Ethyl-1-(3-fluorophenyl)-8-(3-isopropoxybenzyl)-
7-methyl-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Dioxide
(12b). (5R,7S)-1-(3-Fluorophenyl)-8-(3-isopropoxybenzyl)-7-methyl-
2-thia-1,3,8 triazaspiro[4.5]decane 2,2-dioxide (25 mg, 0.061 mmol)
was added to a slurry of NaH (4.9 mg, 0.09 mmol) in DMF (0.61 mL)
at 0 °C. The mixture was stirred at 0 °C for 2 h. Then iodoethane (8.7
mg, 0.061 mmol) in DMF (0.2 mL) was added. The mixture was
allowed to gradually warm to room temperature and stirred overnight.
The mixture was diluted with water and extracted with ethyl acetate (3
× 10 mL). The combined organics were dried over Na2SO4 and
concentrated to yield the title compound 12b (20 mg, 93%). LCMS
1
m/z 476.3 (M + 1). H NMR (400 MHz, CDCl3) δ 1.03 (d, J = 6.6
Hz, 3H), 1.28 (d, J = 6.1 Hz, 6H), 1.30 (t, J = 7.2 Hz, 3H), 1.62 (ddd,
J = 13.6, 5.5, 1.4 Hz, 1H), 1.85−1.97 (m, 2H), 2.03 (dd, J = 13.4, 5.0
Hz, 1H), 2.23−2.34 (m, 1H), 2.48−2.57 (m, 1H), 2.73−2.80 (m, 1H),
3.09 (dd, J = 12.9, 7.2 Hz, 1H), 3.18−3.31 (m, 3H), 3.42 (d, J = 9.2
Hz, 1H), 3.56 (d, J = 13.5 Hz, 1H), 4.43−4.53 (m, 1H), 6.68−6.76
(m, 3H), 7.06−7.21 (m, 4H), 7.30−7.41 (m, 1H), 476.3 (M + 1).
HRMS calcd for C25H34N3O3FS (M + 1) 476.2388, found 476.2377.
(5R,7S)-1-(3-Fluorophenyl)-8-(3-isopropoxybenzyl)-3-iso-
propyl-7-methyl-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Diox-
ide (12c). (5R,7S)-1-(3-Fluorophenyl)-8-(3-isopropoxybenzyl)-7-
methyl-2-thia-1,3,8 triazaspiro[4.5]decane 2,2-dioxide (25 mg, 0.045
mmol) was added to a slurry of NaH (4.9 mg, 0.1 mmol) in DMF (0.1
M, 0.45 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h. Then 2-
iodopropane (8.7 mg, 0.045 mmol) in DMF (0.2 mL) was added. The
mixture was allowed to gradually warm to room temperature and
stirred overnight. The mixture was diluted with water and extracted
with ethyl acetate (3 × 10 mL). The combined organics were dried
over Na2SO4, concentrated, and purified by silica gel chromatography
to yield the title compound 12c (5 mg, 20%). MS (LCMS) m/z 490.3
(M + 1). 1H NMR (400 MHz, CDCl3) δ 1.03 (d, J = 6.6 Hz, 3H), 1.28
(d, J = 6.0 Hz, 6H), 1.31 (d, J = 6.6 Hz, 6H), 1.55 (s, 1H), 1.62 (dd, J
= 12.5, 6.0 Hz, 1H), 1.82−1.89 (m, 1H), 1.94 (dd, J = 8.2, 4.3 Hz,
1H), 2.05 (dd, J = 14.1, 4.3 Hz, 1H), 2.22−2.32 (m, 1H), 2.45−2.56
(m, 1H), 2.70−2.76 (m, 1H), 3.23−3.30 (m, 2H), 3.34−3.41 (m, 1H),
3.58 (d, J = 13.5 Hz, 1H), 3.75−3.84 (m, 1H), 4.44−4.52 (m, 1H),
6.72 (d, J = 2.0 Hz, 1H), 6.73 (s, 2H), 7.09−7.19 (m, 4H), 7.30−7.39
(m, 1H). HRMS calcd for C26H36N3O3FS (M + 1) 490.2541, found
490.2534.
(5R,7S)-1-(3-Fluorophenyl)-3,7-dimethyl-2-thia-1,3,8-
triazaspiro[4.5]decane 2,2-Dioxide (13). A solution of 9 (611 mg,
1.41 mmol) in DMF (7 mL) was added to a slurry of NaH (110 mg,
2.8 mmol) in DMF (7 mL) at 0 °C. The mixture was stirred at 0 °C
for 30 min. Methyl iodide (202 mg, 1.41 mmol) was then added. The
mixture was allowed to gradually warm to room temperature and
stirred overnight. The mixture was diluted with water and extracted
with ethyl acetate (3 × 10 mL). The combined organics were dried
over Na2SO4, concentrated, and purified by silica gel chromatography
to yield benzyl (5R,7S)-1-(3-fluorophenyl)-3,7-dimethyl-2-thia-1,3,8-
triazaspiro[4.5]decane-8-carboxylate 2,2-dioxide (560 mg, 89%). MS
(LCMS) m/z 448.0 (M + 1). 1H NMR (400 MHz, CDCl3) δ 1.21 (d,
J = 7.2 Hz, 3H), 1.66 (m, 1H), 1.73−1.82 (m, 2H), 2.10 (br d, J = 13
Hz, 1H), 2.85 (s, 3H), 3.03 (v br dd, J = 14, 14 Hz, 1H), 3.38 (br d, J
1H), 7.05−7.18 (m, 4H), 6.91−7.01 (m, 1H), 4.97−5.14 (m, 1H),
3.38−3.61 (m, 3H), 3.20 (d, J = 13.7 Hz, 1H), 2.56−2.69 (m, 1H),
2.45 (ddd, J = 3.4, 8.2, 12.2 Hz, 1H), 2.12−2.24 (m, 1H), 1.87−2.05
(m, 2H), 1.73−1.85 (m, 1H), 1.58 (dd, J = 6.2, 13.7 Hz, 1H), 1.00 (d,
J = 6.6 Hz, 3H).
To a solution of (5R,7S)-8-[3-iodobenzyl]-1-(3-fluorophenyl)-7-
methyl-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-dioxide (40 mg, 0.078
mmol) were added Na2CO3 (41.3 mg, 0.39 mmol), 2-chlorobenzyl-
boronic acid (24.4 mg, 0.156 mmol), ethanol (0.78 mL), and water
(0.2 mL). Dry nitrogen was bubbled through the reaction mixture for
5 min in order to degas the solvent. Tetrakis(triphenylphosphine)-
palladium(0) (9.4 mg, 10 mol %) was then added. The vial was sealed,
and the mixture was heated to 50 °C for 18 h. The reaction was
quenched with aqueous NaHCO3 solution, extracted with CH2Cl2,
dried over Na2SO4, filtered, and concentrated. Purification by silica gel
chromatography using a 0−4% gradient of methanol/dichloromethane
provided the first eluent (8.4 mg, 22%) of the title compound 11h. MS
1
(LCMS) m/z 500.2 (M + 1). H NMR (400 MHz, CDCl3) δ 7.40−
7.46 (m, 1H), 7.24−7.37 (m, 7H), 7.06−7.20 (m, 4H), 4.67 (t, J = 8.0
Hz, 1H), 3.68 (d, J = 13.7 Hz, 1H), 3.42−3.59 (m, 2H), 3.32 (d, J =
13.5 Hz, 1H), 2.66−2.75 (m, 1H), 2.54 (ddd, J = 12.4, 8.3, 3.5 Hz,
1H), 2.24−2.32 (m, 1H), 1.93−2.01 (m, 2H), 1.76−1.86 (m, 1H),
1.57−1.63 (m, 1H), 1.04 (d, J = 6.6 Hz, 3H). HRMS calcd for
C26H27N3O2FSCl (M + 1) 500.1569, found 500.1558.
(5R,7S)-1-(3-Fluorophenyl)-8-(3-isopropoxybenzyl)-3,7-di-
methyl-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Dioxide (12a).
(5R,7S)-1-(3-Fluorophenyl)-8-(3-isopropoxybenzyl)-7-methyl-2-thia-
1,3,8-triazaspiro[4.5]decane 2,2-dioxide 11d (20 mg, 0.045 mmol) was
added to a slurry of NaH (3.6 mg, 0.09 mmol) in DMF (0.6 mL) at 0
°C. The mixture was stirred at 0 °C for 2 h. Then methyl iodide (8.7
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= 9.3 Hz, 1H), 3.62 (d, J = 9.3 Hz, 1H), 4.16 (v br s, 1H), 4.53 (v br s,
1H), 5.06 (br s, 2H), 7.10 (ddd, J = 9.3, 2.2, 2.2 Hz, 1H), 7.15−7.21
(m, 2H), 7.28−7.37 (m, 5H), 7.41 (ddd, J = 8.2, 8.2, 6.4 Hz, 1H).
HRMS calcd for C22H26N3O4FS (M + 1) 448.1700, found 448.1680.
To a solution of benzyl (5R,7S)-1-(3-fluorophenyl)-3,7-methyl-2-
thia-1,3,8-triazaspiro[4.5]decane-8-carboxylate 2,2-dioxide (549 mg,
1.23 mmol) in MeOH (10 mL) was added palladium hydroxide (150
mg, 0.21 mmol). The mixture was hydrogenated at 50 psi for 3 h. The
mixture was filtered and then concentrated to afford the title
compound 13 as a white foam (405 mg, quantitative). MS (LCMS)
(br s, 1H), 8.55 (br s, 1H). HRMS [M + H]+ calcd for C28H34N4O3FS
525.2330, found 525.2324.
(5R,7S)-1-(3-Fluorophenyl)-8-(3-isopropoxybenzyl)-7-meth-
yl-3-(pyridin-4-yl)-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Diox-
ide (12d). To a sealed, dry flask were added 9 (53.3 mg, 0.123 mmol),
4-bromopyridine hydrochloride (71.8 mg 0.369 mmol), copper(I)
iodide (71.0 mg, 0.369 mmol), potassium carbonate (102.0 mg, 0.738
mmol), N,N′-dimethylethane-1,2-diamine (52 μL, 0.492 mmol), and
dioxane (3 mL). The suspension was heated overnight at 105 °C, then
cooled to room temperature and filtered through Celite. The filter pad
was washed with ethyl acetate (20 mL), and the combined filtrates
were washed with saturated aqueous sodium bicarbonate solution (15
mL), dried over sodium sulfate, filtered, and concentrated in vacuo.
The residue was purified by chromatography on silica gel (gradient, 0−
100% ethyl acetate in heptane) to provide benzyl (5R,7S)-1-(3-
fluorophenyl)-7-methyl-3-(pyridin-4-yl)-2-thia-1,3,8-triazaspiro[4.5]-
decane-8-carboxylate 2,2-dioxide as a white solid (42.8 mg, 68%). MS
(ESI) m/z: 511.2 (M + 1). 1H NMR (400 MHz, CDCl3) δ 8.03−9.25
(m, 2H), 7.43 (td, J = 8.2, 6.4 Hz, 1H), 7.18−7.34 (m, 9H), 7.14 (dt, J
= 9.0, 2.2 Hz, 1H), 5.04 (s, 2H), 4.51−4.63 (m, 1H), 4.19 (d, J = 12.5
Hz, 1H), 4.06 (d, J = 8.8 Hz, 1H), 3.94 (d, J = 8.4 Hz, 1H), 3.04 (t, J =
13.3 Hz, 1H), 2.21 (d, J = 13.9 Hz, 1H), 1.70−1.85 (m, 3H), 1.25 (d, J
= 7.2 Hz, 3H).
1
m/z 314.0 (M + 1). H NMR (400 MHz, CDCl3) δ 1.00 (d, J = 6.4
Hz, 3H), 1.41 (dd, J = 14.0, 10.0 Hz, 1H), 1.76 (ddd, J = 14.1, 11.2, 4.6
Hz, 1H), 2.22−2.31 (m, 2H), 2.55 (ddd, J = 12.8, 11.3, 3.0 Hz, 1H),
2.66 (m, 1H), 2.80−2.86 (m, 1H), 2.83 (s, 3H), 3.21 (d, half of AB
quartet, J = 9.3 Hz, 1H), 3.28 (d, half of AB quartet, J = 9.3 Hz, 1H),
7.13−7.22 (m, 2H), 7.27 (ddd, J = 8.0, 1.9, 1.1 Hz, 1H), 7.40 (ddd, J =
8.1, 8.1, 6.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) observed peaks, δ
21.5, 33.4, 35.1, 41.0, 43.4, 47.5, 61.0, 62.7, 117.3, 120.7, 129.2, 130.7,
161.6, 164.1. HRMS calcd for C14H20N3O2FS (M + 1) 314.1333,
found 314.1346.
(5R,7S)-1-(3-Fluorophenyl)-8-(3-isopropoxybenzyl)-7-meth-
yl-3-(1,3-oxazol-2-yl)-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-
Dioxide (12h). To a dry sealed flask were added 11d (35.8 mg,
0.080 mmol), 2-iodooxazole (46.5 mg, 0.239 mmol), copper(I) iodide
(46.0 mg, 0.239 mmol), potassium carbonate (33.0 mg, 0.239 mmol),
N,N′-dimethylethane-1,2-diamine (34 μL, 0.319 mmol), and dioxane
(2 mL). The suspension was heated overnight at 105 °C, then cooled
to room temperature and filtered through Celite. The filter pad was
washed with ethyl acetate (20 mL), and the combined filtrates were
washed with saturated aqueous sodium bicarbonate solution (15 mL),
dried over sodium sulfate, filtered, and concentrated in vacuo. The
residue was purified by chromatography on silica gel (gradient, 0−
100% ethyl acetate in heptane) to provide 12h as a white solid (12.5
To a solution of benzyl (5R,7S)-1-(3-fluorophenyl)-7-methyl-3-
(pyridin-4-yl)-2-thia-1,3,8-triazaspiro[4.5]decane-8-carboxylate 2,2-di-
oxide (42 mg, 0.082 mmol) in MeOH (5 mL) was added 20%
palladium hydroxide on carbon (25 mg, 0.036 mmol). The mixture
was hydrogenated at 40 psi overnight. The mixture was filtered and
concentrated to afford (5R,7S)-1-(3-fluorophenyl)-7-methyl-3-(pyr-
idin-4-yl)-2-thia-1,3,8-triaza-spiro[4.5]decane 2,2-dioxide as a colorless
oil, which was used in the next reaction without further purification
(31 mg, 0.082 mmol). To a suspension of (5R,7S)-1-(3-fluorophenyl)-
7-methyl-3-(pyridin-4-yl)-2-thia-1,3,8-triaza-spiro[4.5]decane 2,2-diox-
ide (0.082 mmol) and cesium carbonate (80.2 mg, 0.246 mmol) in
DMF (1.5 mL) was added 1-(bromomethyl)-3-isopropoxybenzene
(37.6 mg, 0.164 mmol) in DMF (0.5 mL). The suspension was stirred
overnight at room temperature. The reaction mixture was diluted with
water (20 mL) and extracted with ethyl acetate (3 × 20 mL). The
combined organic extracts were dried over sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified by silica gel
chromatography (gradient, 0−100% ethyl acetate in heptanes) to
1
mg, 30%). H NMR (500 MHz, CDCl3) δ 1.11 (d, J = 6.7 Hz, 3H),
1.32 (d, J = 6.1 Hz, 6H), 1.71 (br dd, J = 13.6, 6.0 Hz, 1H), 2.01 (m,
1H), 2.07−2.14 (m, 2H), 2.37 (ddd, J = 12.8, 6.7, 3.7 Hz, 1H), 2.62
(ddd, J = 12.6, 8.4, 3.5 Hz, 1H), 2.82 (m, 1H), 3.34 (d, half of AB
quartet, J = 13.5 Hz, 1H), 3.64 (d, half of AB quartet, J = 13.5 Hz, 1H),
4.08 (d, half of AB quartet, J = 9.9 Hz, 1H), 4.24 (d, half of AB quartet,
J = 9.8 Hz, 1H), 4.51 (septet, J = 6.1 Hz, 1H), 6.75−6.78 (m, 3H),
7.01 (d, J = 1.0 Hz, 1H), 7.15−7.24 (m, 4H), 7.43 (ddd, J = 8.1, 8.1,
6.2 Hz, 1H), 7.51 (d, J = 1.1 Hz, 1H). 13C NMR (CDCl3) δ 15.87,
22.22, 22.25, 33.64, 41.59, 44.57, 51.33, 57.33, 57.98, 62.85, 69.87,
114.31, 116.52, 117.54, 120.62, 121.01, 127.11, 129.13, 129.38, 130.68,
134.04, 136.81, 140.22, 153.29, 158.11, 161.61. HRMS [M + H]+ calcd
for C26H32N4O4FS 515.2122, found 515.2121.
(5R,7S)-1-(3-Fluorophenyl)-8-(3-isopropoxybenzyl)-7-meth-
yl-3-(1,3-thiazol-2-yl)-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-
Dioxide (12g). Application of the method used to make 12h from
11d, using 2-iodothiazole in place of 2-iodooxazole, afforded 12g as a
white solid (17.6 mg, 41%). 1H NMR (500 MHz CDCl3) δ 7.46 (d, J
= 3.4 Hz, 1H), 7.43 (td, J = 8.1, 6.5 Hz, 1H), 7.14−7.25 (m, 4H), 7.01
(d, J = 3.4 Hz, 1H), 6.73−6.79 (m, 3H), 4.51 (spt, J = 6.0 Hz, 1H),
4.33 (d, J = 10.0 Hz, 1H), 4.13 (d, J = 10.0 Hz, 1H), 3.63 (d, J = 13.7
Hz, 1H), 3.34 (d, J = 13.4 Hz, 1H), 2.79−2.87 (m, 1H), 2.62 (ddd, J =
12.5, 8.6, 3.3 Hz, 1H), 2.37 (ddd, J = 12.5, 6.6, 4.0 Hz, 1H), 2.04−2.16
(m, 2H), 1.95−2.03 (m, 1H), 1.73 (ddd, J = 13.7, 5.8, 1.2 Hz, 1H),
1.31 (d, J = 6.1 Hz, 6H), 1.12 (d, J = 6.8 Hz, 3H). HRMS [M + H]+
calcd for C26H32N4O3FS2 531.1894, found 531.1897.
(5R,7S)-1-(3-Fluorophenyl)-8-(3-isopropoxybenzyl)-7-meth-
yl-3-(pyridin-3-yl)-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Diox-
ide (12e). Application of the method used to make 12h from 11d,
using 3-bromopyridine in place of 2-iodooxazole, afforded 12e as a
white solid (10.2 mg, 49%). 1H NMR (500 MHz CDCl3) δ 1.14 (br s,
3H), 1.32 (d, J = 5.9 Hz, 3H), 1.71−1.84 (m, 1H), 2.03−2.26 (m,
1H), 2.34−2.41 (m, 1H), 2.57−2.68 (m, 1H), 2.79−2.87 (m, 2H),
3.29−3.39 (m, 1H), 3.62−3.73 (m, 1H), 3.89 (d, half of AB quartet, J
= 8.8 Hz, 1H), 3.93 (d, half of AB quartet, J = 8.8 Hz, 1H), 4.48−4.55
(m, 1H), 6.75−6.81 (m, 3H), 7.14−7.25 (m, 4H), 7.35 (dd, J = 4.6
Hz, 8.3 Hz, 1H), 7.39−7.45 (m, 1H), 7.81 (d, J = 9.5 Hz, 1H), 8.47
1
provide 12d as a white solid (18 mg, 42%). H NMR (500 MHz
CDCl3) δ 1.10−1.19 (br s, 3H), 1.32 (d, J = 6.1 Hz, 6H), 1.71−1.81
(m, 1H), 1.98−2.10 (m, 1H), 2.12−2−25 (m, 1H), 2.33−2.40 (m,
1H), 2.59−2.67 (m, 1H), 2.76−2.86 (m, 1H), 3.29−3.40 (m, 1H),
3.66−3.74 (m, 1H), 3.84 (d, J = 8.5 Hz, 1H), 3.93 (d, J = 8.5 Hz, 1H),
4.48−4.55 (m, 1H), 6.72−6.83 (m, 3H), 7.10−7.25 (m, 6H), 7.4−7.47
(m, 1H), 8.54 (d, J = 5.9 Hz, 2H). 13C NMR (CDCl3) δ 16.35, 22.23,
22.25, 33.81, 41.97, 44.90, 51.36, 56.26, 57.94, 61.46, 69.90, 110.93(2),
114.29, 116.63, 117.55, 120.58, 121.04, 129.09, 129.41, 130.68, 134.22,
14005, 144.97, 150.99(2), 158.12, 161.61. HRMS [M + H]+ calcd for
C28H34N4O3FS 525.2330, found 525.2318.
(5R,7S)-1-(3-Fluorophenyl)-8-(3-isopropoxybenzyl)-7-meth-
yl-3-(pyrimidin-2-yl)-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-
Dioxide (12f). Application of the procedure used to make 12d,
using 2-bromopyrimidine in place of 4-bromopyridine hydrochloride,
afforded benzyl (5R,7S)-1-(3-fluorophenyl)-7-methyl-3-(pyrimidin-2-
yl)-2-thia-1,3,8-triazaspiro[4.5]decane-8-carboxylate 2,2-dioxide as a
white solid (36.7 mg, 52%). 1H NMR (500 MHz, CDCl3) δ 8.62 (d, J
= 4.9 Hz, 2H), 7.43−7.49 (m, 1H), 7.26−7.38 (m, 6H), 7.18−7.26 (m,
2H), 7.03 (t, J = 4.9 Hz, 1H), 5.08 (br s, 2H), 4.59 (br s, 1H), 4.54 (d,
J = 10.0 Hz, 1H), 4.21 (br s, 1H), 4.16 (d, J = 10.0 Hz, 1H), 3.12 (t, J
= 13.4 Hz, 1H), 2.26 (d, J = 12.9 Hz, 1H), 1.72−1.86 (m, 3H), 1.31
(d, J = 7.3 Hz, 3H). MS (ESI) m/z: 512.1 (M + 1).
Application of the procedure used to make 12d, employing (5R,7S)-
1-(3-fluorophenyl)-7-methyl-3-(pyrimidin-2-yl)-2-thia-1,3,8-triaza-
spiro[4.5]decane 2,2-dioxide, afforded 12f as a white solid (10 mg,
40%). 1H NMR (500 MHz, CDCl3) δ 1.03−1.14 (br s, 3H), 1.28 (d, J
= 6.1 Hz, 6H), 1.61−1.71 (m, 1H), 1.92−2.02 (m, 1H), 2.04−2.16 (m,
2H), 2.29−2.38 (br s, 1H), 2.53−2.63 (m, 1H), 2.73−2.83 (br s, 1H),
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3.30 (d, J = 13.3 Hz, 1H), 3.62 (d, J = 14.3 Hz, 1H), 4.02 (d, J = 9.6
Hz, 1H), 4.27 (d, J = 9.9 Hz, 1H), 4.42−4.53 (m, 1H), 6.69−6.78 (m,
3H), 6.96 (t, J = 4.9 Hz, 1H), 7.10−7.19 (m, 2H), 7.20−7.28 (m, 1H,
assumed, partially obscured by solvent peak), 7.33−7.42 (m, 2H), 8.57
(d, J = 4.9 Hz, 2H); 13C NMR (CDCl3) δ 16.15, 22.22, 22.25, 33.45,
41.52, 44.84, 51.47, 56.18, 57.96, 60.92, 69.90, 114.34, 115.68, 116.65,
117.18, 120.67, 121.06, 129.17, 129.39, 130.44, 134.84, 140.35, 157.28,
158.12, 158.56 (2), 161.56. HRMS [M + H]+ calcd for C27H33N5O3FS
526.2282, found 526.2275.
4-{[(5R,7S)-1-(3-Fluorophenyl)-3,7-dimethyl-2,2-dioxido-2-
thia-1,3,8-triazaspiro[4.5]dec-8-yl]methyl}phenol (14d). To a
solution of (5R,7S)-1-(3-fluorophenyl)-3,7-dimethyl-2-thia-1,3,8-
triazaspiro[4.5]decane 2,2-dioxide (20 mg, 0.064 mmol) and 4-
hydroxybenzaldehyde (11.7 mg, 0.096 mmol) in dichloroethane (0.5
mL) was added acetic acid (3.8 mg, 0.064 mmol). The mixture was
allowed to stir at room temperature for 5 h, then was charged with
sodium triacetoxyborohydride (27.1 mg, 0.128 mmol) and stirred
overnight. The reaction was quenched with aqueous NaHCO3
solution, extracted with CH2Cl2, dried over Na2SO4, filtered, and
concentrated. Purification by silica gel chromatography using 20−70%
gradient of ethyl acetate/heptane provided the first eluent (20.9 mg,
54%) of the title compound 14d. MS (LCMS) m/z 420.1 (M + 1). 1H
NMR (400 MHz, CDCl3) δ 1.07 (d, J = 6.6 Hz, 3H), 1.66 (dd, J =
14.3, 5.7 Hz, 1H), 1.92−1.99 (m, 2H), 2.02−2.10 (m, 1H), 2.29−2.36
(m, 1H), 2.48−2.58 (m, 1H), 2.74−2.81 (m, 1H), 2.83 (s, 3H), 3.27
(d, J = 9.3 Hz, 1H), 3.33 (d, J = 13.2 Hz, 1H), 3.44 (d, J = 9.3 Hz,
1H), 3.55 (d, J = 13.4 Hz, 1H), 5.10 (br s, 1H), 6.72 (d, J = 8.5 Hz,
2H), 7.05 (d, J = 8.3 Hz, 2H), 7.10−7.15 (m, 1H), 7.15−7.21 (m,
2H), 7.34−7.43 (m, 1H). HRMS calcd for C21H26N3O3FS (M + 1)
420.1756, found 420.1751.
2-Chloro-4-{[(5R,7S)-1-(3-fluorophenyl)-3,7-dimethyl-2,2-di-
oxido-2-thia-1,3,8-triazaspiro[4.5]dec-8-yl]methyl}phenol
(14e). To a solution of (5R,7S)-1-(3-fluorophenyl)-3,7-dimethyl-2-
thia-1,3,8-triazaspiro[4.5]decane 2,2-dioxide (27 mg, 0.086 mmol) and
3-chloro-4-hydroxybenzaldehyde (15.5 mg, 0.086 mmol) in dichloro-
ethane (0.5 mL) was added acetic acid (5.2 mg, 0.086 mmol). The
mixture was allowed to stir at room temperature for 5 h, then was
charged with sodium triacetoxyborohydride (36.5 mg, 0.172 mmol)
and stirred overnight. The reaction was quenched with aqueous
NaHCO3 solution, extracted with CH2Cl2, dried over Na2SO4, filtered,
and concentrated. Purification by silica gel chromatography using 0−
5% gradient of MeOH/CH2Cl2 provided the first eluent (29.9 mg,
68%) of the title compound 14e. MS (LCMS) m/z 454.2 (M + 1). 1H
NMR (400 MHz, CDCl3) δ 1.04 (d, J = 6.6 Hz, 3H), 1.24−1.32 (m,
2H), 1.64 (dd, J = 13.7, 5.4 Hz, 1H), 1.89−1.97 (m, 1H), 2.05 (dd, J =
13.5, 4.8 Hz, 1H), 2.22−2.33 (m, 1H), 2.44−2.56 (m, 1H), 2.71−2.79
(m, 1H), 2.82 (s, 3H), 3.22−3.31 (m, 2H), 3.43 (d, J = 9.5 Hz, 1H),
3.47−3.54 (m, 1H), 5.51 (br s, 1H), 6.87−6.93 (m, 1H), 6.96−7.02
(m, 1H), 7.10−7.21 (m, 4H), 7.34−7.43 (m, 1H). HRMS calcd for
C21H25N3O3FSCl (M + 1) 454.1361, found 454.1357.
2-Ethoxy-4-{[(5R,7S)-1-(3-fluorophenyl)-3,7-dimethyl-2,2-di-
oxido-2-thia-1,3,8-triazaspiro[4.5]dec-8-yl]methyl}phenol
(14f). To a solution of (5R,7S)-1-(3-fluorophenyl)-3,7-dimethyl-2-
thia-1,3,8-triazaspiro[4.5]decane 2,2-dioxide (25 mg, 0.08 mmol) and
3-ethoxy-4-hydroxybenzaldehyde (13.3 mg, 0.08 mmol) in dichloro-
ethane (0.5 mL) was added acetic acid (4.8 mg, 0.08 mmol). The
mixture was allowed to stir at room temperature for 5 h, then was
charged with sodium triacetoxyborohydride (33.9 mg, 0.160 mmol)
and stirred overnight. The reaction was quenched with aqueous
NaHCO3 solution, extracted with CH2Cl2, dried over Na2SO4, filtered,
and concentrated. Purification by silica gel chromatography using a 0−
4% gradient of MeOH/CH2Cl2 provided the first eluent (9.7 mg,
24%) of the title compound 14f. MS (LCMS) m/z 464.3 (M + 1). 1H
NMR (400 MHz, CDCl3) δ 1.01 (d, J = 6.8 Hz, 3H), 1.38 (t, J = 7.0
Hz, 3H), 1.61 (dd, J = 13.6, 5.4 Hz, 1H), 1.87−1.93 (m, 2H), 2.00
(dd, J = 13.7, 4.9 Hz, 1H), 2.23−2.32 (m, 1H), 2.44−2.53 (m, 1H),
2.72−2.78 (m, 1H), 2.79 (s, 3H), 3.22 (d, J = 9.4 Hz, 1H), 3.26 (d, J =
13.3 Hz, 1H), 3.41 (d, J = 9.2 Hz, 1H), 3.44−3.50 (m, 1H), 4.01 (q, J
= 7.0 Hz, 1H), 5.58 (br s, 1H), 6.62 (dd, J = 8.0, 2.0 Hz, 1H), 6.66 (d,
J = 1.8 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 7.07−7.18 (m, 3H), 7.31−
7.39 (m, 1H). HRMS calcd for C23H30N3O4FS (M + 1) 464.2008,
found 464.2013.
N-(4-{[(5R,7S)-1-(3-Fluorophenyl)-3,7-dimethyl-2,2-dioxido-
2-thia-1,3,8-triazaspiro[4.5]dec-8-yl]methyl}phenyl)acetamide
(14a). To a solution of (5R,7S)-1-(3-fluorophenyl)-3,7-dimethyl-2-
thia-1,3,8-triazaspiro[4.5]decane 2,2-dioxide (20 mg, 0.064 mmol) and
N-(4-formylphenyl)acetamide (10.4 mg, 0.064 mmol) in dichloro-
ethane (0.5 mL) was added acetic acid (3.8 mg, 0.064 mmol). The
mixture was allowed to stir at room temperature for 5 h, then was
charged with sodium triacetoxyborohydride (27.1 mg, 0.128 mmol)
and stirred overnight. The reaction was quenched with aqueous
NaHCO3 solution, extracted with CH2Cl2, dried over Na2SO4, filtered,
and concentrated. Purification by silica gel chromatography using 20−
70% gradient of ethyl acetate/heptane provided the first eluent (15
mg, 47%) of the title compound 14a. MS (LCMS) m/z 461.3 (M +
1). 1H NMR (400 MHz, CDCl3) δ 1.01 (d, J = 6.8 Hz, 3H), 1.61 (dd,
J = 13.4, 5.4 Hz, 1H), 1.87−1.92 (m, 1H), 2.00 (dd, J = 13.5, 4.9 Hz,
1H), 2.11 (s, 3H), 2.22−2.30 (m, 1H), 2.44−2.53 (m, 1H), 2.70−2.77
(m, 1H), 2.79 (s, 3H), 3.23 (d, J = 9.2 Hz, 1H), 3.29 (d, J = 13.5 Hz,
1H), 3.41 (d, J = 9.2 Hz, 1H), 3.45 (q, J = 7.0 Hz, 1H), 3.52 (d, J =
13.5 Hz, 1H), 7.05−7.17 (m, 5H), 7.26 (s, 1H), 7.31−7.39 (m, 3H).
HRMS calcd for C23H29N4O3FS (M + 1) 461.2026, found 461.2017.
(5R,7S)-1-(3-Fluorophenyl)-8-(1H-indol-5-ylmethyl)-3,7-di-
methyl-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Dioxide (14b).
To a solution of (5R,7S)-1-(3-fluorophenyl)-3,7-dimethyl-2-thia-1,3,8-
triazaspiro[4.5]decane 2,2-dioxide (18 mg, 0.057 mmol) and 1H-
indole-5-carbaldehyde (12.5 mg, 0.086 mmol) in dichloroethane (0.5
mL) was added acetic acid (3.4 mg, 0.057 mmol). The mixture was
allowed to stir at room temperature for 5 h, then was charged with
sodium triacetoxyborohydride (24.2 mg, 0.114 mmol) and stirred
overnight. The reaction was quenched with aqueous NaHCO3
solution, extracted with CH2Cl2, dried over Na2SO4, filtered, and
concentrated. Purification by silica gel chromatography using 20−
100% gradient of ethyl acetate/heptane provided the first eluent (16
mg, 59%) of the title compound 14b. MS (LCMS) m/z 443.5 (M +
1
1). H NMR (400 MHz, CDCl3) δ 1.07 (d, J = 6.6 Hz, 3H), 1.57−
1.66 (m, 1H), 1.86−1.95 (m, 2H), 1.99−2.09 (m, 1H), 2.26−2.39 (m,
1H), 2.48−2.59 (m, 1H), 2.74−2.78 (m, 1H), 2.79 (s, 3H), 3.22 (d, J
= 9.4 Hz, 1H), 3.39 (d, J = 9.2 Hz, 1H), 3.42−3.48 (m, 1H), 3.69 (d, J
= 13.1 Hz, 1H), 6.44−6.48 (m, 1H), 6.51−6.55 (m, 1H), 6.99−7.30
(m, 4H), 7.33−7.43 (m, 2H), 7.62 (d, J = 0.8 Hz, 1H), 8.22 (br s, 1H).
HRMS calcd for C23H27N4O2FS (M + 1) 443.1916, found 443.1911.
(5R,7S)-8-[(3-Ethyl-1H-indazol-5-yl)methyl]-1-(3-fluorophen-
yl)-3,7-dimethyl-2-thia-1,3,8-triazaspiro[4.5]decane 2,2-Diox-
ide (14c). To a solution of (5R,7S)-1-(3-fluorophenyl)-3,7-dimethyl-
2-thia-1,3,8-triazaspiro[4.5]decane 2,2-dioxide (20 mg, 0.064 mmol)
and 3-ethyl-1H-indazole-5-carbaldehyde43 (22.3 mg, 0.128 mmol) in
dichloroethane (0.5 mL) was added acetic acid (3.8 mg, 0.063 mmol).
The mixture was allowed to stir at room temperature for 5 h, then was
charged with sodium triacetoxyborohydride (27.1 mg, 0.128 mmol)
and stirred overnight. The reaction was quenched with aqueous
NaHCO3 solution, extracted with CH2Cl2, dried over Na2SO4, filtered,
and concentrated. Purification by silica gel chromatography using 0−
50% gradient of acetone/ethyl acetate provided the first eluent (6 mg,
20%) of the title compound 14c. MS (LCMS) m/z 472.5 (M + 1). 1H
NMR (400 MHz, CDCl3) δ 1.11 (d, J = 6.6 Hz, 3H), 1.27 (t, J = 7.1
Hz, 3H), 1.64−1.71 (m, 1H), 2.06 (s, 3H), 2.32−2.37 (m, 1H), 2.55−
2.62 (m, 1H), 2.79−2.82 (m, 1H), 2.83 (s, 3H), 3.01−3.06 (q, 2H),
3.28 (d, J = 9.3 Hz, 1H), 3.44−3.50 (m, 2H), 3.73 (d, J = 13.7 Hz,
1H), 4.82 (s, 3H), 7.09−7.15 (m, 2H), 7.19 (d, J = 8.5 Hz, 1H), 7.31−
7.36 (m, 2H), 7.40−7.46 (m, 2H), 7.48 (s, 1H), 7.72 (s, 1H). HRMS
calcd for C24H30N5O2FS (M + 1) 472.2177, found 472.2172.
4-{[(5R,7S)-1-(3-Fluorophenyl)-3,7-dimethyl-2,2-dioxido-2-
thia-1,3,8-triazaspiro[4.5]dec-8-yl]methyl}-2-isopropoxyphe-
nol (14g). To a solution of (5R,7S)-1-(3-fluorophenyl)-3,7-dimethyl-
2-thia-1,3,8-triazaspiro[4.5]decane 2,2-dioxide (27 mg, 0.086 mmol)
and 4-hydroxy-3-isopropoxybenzaldehyde44 (15.5 mg, 0.086 mmol) in
dichloroethane (0.5 mL) was added acetic acid (5.2 mg, 0.086 mmol).
The mixture was allowed to stir at room temperature for 5 h, then was
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charged with sodium triacetoxyborohydride (36.5 mg, 0.172 mmol)
and stirred overnight. The reaction was quenched with aqueous
NaHCO3 solution, extracted with CH2Cl2, dried over Na2SO4, filtered,
and concentrated. Purification by silica gel chromatography using 0−
5% gradient of methanol/dichloromethane provided the first eluent
(29.9 mg, 68%) of the title compound 14g. MS (LCMS) m/z 464.3
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1
(M + 1). H NMR (400 MHz, CD3OD) δ 7.41 (dt, J = 6.63, 8.2 Hz,
1H), 7.11−7.28 (m, 3H), 6.69−6.80 (m, 2H), 6.62 (dd, J = 1.0, 8.2
Hz, 1H), 4.43−4.58 (m, 1H), 3.49−3.62 (m, 2H), 3.38−3.49 (m, 1H),
3.28−3.33 (m, 1H), 2.80 (s, 3H), 2.54−2.71 (m, 2H), 2.23−2.38 (m,
1H), 2.05−2.18 (m, 2H), 1.86−2.00 (m, 1H), 1.68 (dd, J = 7.4, 13.7
Hz, 1H), 1.32 (dd, J = 1.4, 6.0 Hz, 6H), 1.14 (d, J = 6.6 Hz, 3H). 13C
NMR (100 MHz, CD3OD) δ 163.8, 161.3, 146.8, 145.2, 135.2, 130.1,
129.2, 122.3, 120.1, 119.9, 116.1, 116.3, 114.8, 71.2, 61.4, 60.4, 56.6,
50.5, 44.4, 41.7, 33.9, 31.8, 20.9, 15.3. HRMS calcd for C24H33N3O4FS
(M + 1) 478.2162, found 478.2170.
Computational Details. The crystal structure for BACE-1
complexed with inhibitor 12a was employed in the WaterMap
calculation. The complex was prepared with the Protein Preparation
Wizard in Maestro (version 9.2, Schrodinger, LLC, New York, NY,
̈
2011), where it was submitted to a series of restrained, partial
minimizations using the OPLS-AA force field.45 Ligands were
generated with LigPrep (version 2.5, Schrodinger, LLC, New York,
̈
NY, 2011) and their cores superimposed to the one from compound
12a, followed by conformational search for the R groups and energy
minimization using eMBrAcE (within Macromodel, version 9.9,
Schrodinger, LLC, New York, NY, 2011). WaterMap was run in the
̈
default mode using the crystal structure ligand to define the binding
site but removed in the MD simulation. The poses obtained for the
ligands with the eMBrAcE procedure were scored using the ab initio
form of the displaced-solvent functional as described by Abel and co-
workers that estimates the free energy of liberation (ΔGWM).21
ASSOCIATED CONTENT
* Supporting Information
■
S
Exposure data from in vivo mouse experiments, the Cerep
profile of lead compound 14g, spectral data for 12a and 14g,
and the WaterMap entropy, enthalpy, and free energy liberation
of binding site waters for analogues in Table 1. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
Atomic coordinates and structure factors for the following
BACE cocrystal structures have been deposited with the RCSB:
compound 12a (PDB code 4FM8) and compound 14g (PDB
code 4FM7).
AUTHOR INFORMATION
Corresponding Author
*Phone: 617 395 0706. E-mail: michael.a.brodney@pfizer.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(16) Stachel, S. J.; Coburn, C. A.; Rush, D.; Jones, K. L.; Zhu, H.;
Rajapakse, H.; Graham, S. L.; Simon, A.; Katharine Holloway, M.;
Allison, T. J.; Munshi, S. K.; Espeseth, A. S.; Zuck, P.; Colussi, D.;
Wolfe, A.; Pietrak, B. L.; Lai, M. T.; Vacca, J. P. Discovery of
aminoheterocycles as a novel beta-secretase inhibitor class: pH
dependence on binding activity part 1. Bioorg. Med. Chem. Lett.
2009, 19 (11), 2977−2980.
■
The authors thank Luis Martinez-Alsina for synthetic expertise,
and Dan Virtue and Bob Depianta for chiral HPLC separations.
We also gratefully acknowledge Christopher Butler, Jennifer
Davoren, Shawn Doran, and Yasong Lu for helpful discussion
of the manuscript.
(17) Barrow, J. C.; Stauffer, S. R.; Rittle, K. E.; Ngo, P. L.; Yang, Z.;
Selnick, H. G.; Graham, S. L.; Munshi, S.; McGaughey, G. B.;
Holloway, M. K.; Simon, A. J.; Price, E. A.; Sankaranarayanan, S.;
Colussi, D.; Tugusheva, K.; Lai, M. T.; Espeseth, A. S.; Xu, M.; Huang,
Q.; Wolfe, A.; Pietrak, B.; Zuck, P.; Levorse, D. A.; Hazuda, D.; Vacca,
J. P. Discovery and X-ray crystallographic analysis of a spiropiperidine
iminohydantoin inhibitor of β-secretase. J. Med. Chem. 2008, 51 (20),
6259−6262.
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