Design and validation of bicyclic iminopyrimidinones as beta amyloid cleaving enzyme-1 (BACE1) inhibitors: Conformational constraint to favor a bioactive conformation

Article abstract of DOI:10.1021/jm301039c

On the basis of our observation that the biaryl substituent of iminopyrimidinone 7 must be in a pseudoaxial conformation to occupy the contiguous S1-S3 subsites of BACE1, we have designed a novel fused bicyclic iminopyrimidinone scaffold intended to favor this bioactive conformation. Strategic incorporation of a nitrogen atom in the new constrained ring allowed us to develop SAR around the S2′ binding pocket and ultimately resulted in analogues with low nanomolar potency for BACE1. In particular, optimization of the prime side substituent led to major improvements in potency by displacement of two conserved water molecules from a region near S2′. Further optimization of the pharmacokinetic properties of this fused pyrrolidine series, in conjunction with facile access to a rat pharmacodynamic model, led to identification of compound 43, which is an orally active, brain penetrant inhibitor that reduces Aβ₄₀ in the plasma, CSF, and cortex of rats in a dose-dependent manner.

Full text of DOI:10.1021/jm301039c

Article
pubs.acs.org/jmc
Design and Validation of Bicyclic Iminopyrimidinones As Beta
Amyloid Cleaving Enzyme‑1 (BACE1) Inhibitors: Conformational
Constraint to Favor a Bioactive Conformation
Mihirbaran Mandal,*,† Zhaoning Zhu,† Jared N. Cumming,† Xiaoxiang Liu,† Corey Strickland,§
Robert D. Mazzola,† John P. Caldwell,† Prescott Leach,‡ Michael Grzelak,‡ Lynn Hyde,‡ Qi Zhang,‡
Giuseppe Terracina,‡ Lili Zhang,‡ Xia Chen,‡ Reshma Kuvelkar,‡ Matthew E. Kennedy,‡
Leonard Favreau,# Kathleen Cox,# Peter Orth,§ Alexei Buevich,∥ Johannes Voigt,§ Hongwu Wang,§
Irina Kazakevich,⊥ Brian A. McKittrick,† William Greenlee,† Eric M. Parker,‡ and Andrew W. Stamford†
‡
§
∥
†Department of Medicinal Chemistry, Department of Neuroscience, Global Structural Chemistry, Department of Analytical
⊥
#
Chemistry, Department of Basic Pharmaceutical Sciences, and Department of Exploratory Drug Metabolism, Merck Research
Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United States
ABSTRACT: On the basis of our observation that the biaryl
substituent of iminopyrimidinone 7 must be in a pseudoaxial
conformation to occupy the contiguous S1−S3 subsites of
BACE1, we have designed a novel fused bicyclic iminopyr-
imidinone scaffold intended to favor this bioactive con-
formation. Strategic incorporation of a nitrogen atom in the
new constrained ring allowed us to develop SAR around the
S2′ binding pocket and ultimately resulted in analogues with
low nanomolar potency for BACE1. In particular, optimization
of the prime side substituent led to major improvements in
potency by displacement of two conserved water molecules from a region near S2′. Further optimization of the pharmacokinetic
properties of this fused pyrrolidine series, in conjunction with facile access to a rat pharmacodynamic model, led to identification
of compound 43, which is an orally active, brain penetrant inhibitor that reduces Aβ40 in the plasma, CSF, and cortex of rats in a
dose-dependent manner.
scoring the critical role of Aβ in AD.4 While the approach of γ-
secretase inhibition had appeared promising, progression
INTRODUCTION
■
Notwithstanding enormous progress made in science and
through the clinic has been hampered by mechanism-based
side effects largely mediated by concomitant inhibition of
Notch processing.5 On the other hand, Aβ lowering through
inhibition of BACE16 represents an excellent alternative
therapeutic strategy because BACE1 knockout mice show
complete absence of Aβ production coupled with relatively
moderate phenotypes.7,8 Furthermore, crossing BACE1 knock-
out mice with PDAPP mice, which overproduce a mutant form
of human APP, reversed the synaptic deficits and neuro-
degenerative pathologies characteristic of AD in the resulting
biogenic mice.9,10 From these studies, only a moderate
reduction of CNS Aβ observed in BACE1 heterozygous/
PDAPP mice resulted in significantly less plaque accumulation
over time. These data suggest that inhibition of BACE1 could
be an excellent strategy for the treatment and prevention of AD
by providing potent inhibition of Aβ peptide formation in
medicine around Alzheimer’s disease (AD), a treatment to slow
or halt the progression of this devastating illness is still out of
reach. To date, the only therapies available for AD patients are
cholinesterase inhibitors such as donepezil and the NMDA
receptor antagonist memantine.1 With the emergence of the
amyloid hypothesis based on discoveries using biochemistry,
human genetics, and pathology,2 vigorous research in both
academia and industry was undertaken to address this unmet
medical need. The presence of β-amyloid plaques and
neurofibrillary tangles of tau protein in the brain are key
hallmarks of AD. The plaques are mainly composed of fibrillar
aggregates of insoluble β-amyloid peptides (Aβ40,42), and it is
now recognized that the soluble, lower molecular weight
oligomers of Aβ40,42 are also key culprits contributing to the
cognitive decline in Alzheimer’s patients.3 The Aβ40,42 peptides
are generated from intraneuronal amyloid precursor protein
(APP) through sequential cleavage by β-secretase (BACE1)
and by γ-secretase, and thus agents that inhibit these enzymes
would be beneficial as disease modifying treatments for AD.
Indeed, human genetic mutations such as the Swedish mutation
lead to enhanced β-secretase processing of APP, excess CNS
Aβ levels, and ultimately aggressive early onset AD, under-
Special Issue: Alzheimer's Disease
Received: July 16, 2012
© XXXX American Chemical Society
A
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Figure 1. Potent peptidomimetic BACE1 inhibitors.
combination with the anticipation of an acceptable tolerability
profile.
BACE1 is a membrane bound aspartyl protease expressed in
the CNS. Early BACE1 inhibitor designs were inspired by the
success of transition state mimetics in other drug targets from
the aspartyl protease family such as HIV protease and
renin.11,12 Following that approach, Ghosh and Tang were
the first to report on potent peptidic inhibitors including several
crystal structures of these potent ligands complexed with
BACE1.13 Subsequent efforts produced many potent inhibitors
containing statine (cf. 1),14 hydroxyethylene (cf. 2),15 and
hydroxyethyl amine (cf. 3)16 cores (Figure 1). However, unlike
HIV protease and renin, BACE1 is not a peripheral target. The
high molecular weight, low permeability, and high susceptibility
to P-glycoprotein (P-gp) efflux of these peptidomimetics made
them unsuitable for further development as brain penetrant
drugs.17
In parallel with the peptidomimetic approach, we and
others18,19 pursued fragment-based lead generation (FBLG)
strategies to identify lower molecular weight (<300 Da) leads.
From our FBLG efforts, HSQC screening using 15N-BACE1,18
we identified isothiourea 4 as a weak inhibitor of BACE1 that,
based on NMR chemical shift perturbations, had interactions
with both aspartic acids of the catalytic dyad as well as with
other residues in the S1 and S3 subsites of BACE1. The X-ray
cocrystal structure of this fragment with BACE1 showed that
the amidine portion of isothiourea 4 formed a network of
interactions with the catalytic aspartic acids by displacing a
conserved water molecule. Recently, we have described the
progression of 4 to a potent iminohydantoin 5 as a part of an
affinity-driven optimization campaign (Figure 2).18 Subse-
quently, with a focus on ligand efficiency, improved oral
bioavailability, and improved selectivity, we developed a highly
optimized iminohydantoin inhibitor 620 and discovered a
structurally distinct chemical series, the iminopyrimidinones,
represented by compound 7.21 These structure-based design
efforts led to the discovery of the first orally available brain
penetrant small molecule BACE1 inhibitors.
Figure 2. Evolution of iminopyrimidinone 7 through iminohydantoin
5 and 6 from the FBLG lead 4.
this novel bicyclic series resulted in discovery of the potent,
centrally efficacious lead compound 43.
CHEMISTRY
■
The synthesis of key intermediate 22 is depicted in Scheme 1.
Commercially available 4-bromothiophene-2-carbaldehyde 8
was reduced by sodium borohydride, and the corresponding
alcohol was converted to 4-bromo-2-(chloromethyl)thiophene
9 using thionyl chloride. Conversion of chloride 9 to the
corresponding cyanide 10 was complicated due to the likely
formation of condensed products from the reaction of product
with the starting material. After evaluation of several reaction
conditions, we found that oven-dried potassium cyanide was
critical to minimize the formation of these side products. Once
obtained, the 2-(4-bromothiophen-2-yl)acetonitrile 10 was
condensed with glyoxalic acid, and product 11 precipitated
from the reaction mixture.22 Refluxing compound 11 in a
mixture of sulfuric and formic acid hydrolyzed the nitrile and
formed an intermediate anhydride that itself was hydrolyzed to
diacid 12. Formation of the key pyrrolidine ring was mediated
through a [2 + 3] cycloaddition reaction between compound
12 and the dipolarophile N-benzyl-1-methoxy-N-
((trimethylsilyl)methyl)-methanamine 13. The resulting prod-
uct 14 was then treated directly without purification with acetic
To further improve the potency of iminopyrimidinone series,
we have now designed a bicyclic iminopyrimidinone core to
introduce conformational constraint and to facilitate access to
the prime side of the BACE1 active site. Details of the
structure−activity relationships (SAR) of prime side substitu-
tion, including discovery of a new binding pocket, will be
reported in this article. Ultimately, structure-based evolution of
B
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Scheme 1. Synthesis of (4aR,7aR)-2-(Trimethylsilyl)ethyl 7a-(4-Bromothiophen-2-yl)-2-((tert-butoxycarbonyl)imino)-3-methyl-
a
4-oxohexahydro-1H-pyrrolo[3,4-d]pyrimidine-6(2H)-carboxylate 22, Key Intermediate for SAR Studies
a
Reagents and conditions: (a) NaBH4, MeOH, 0 °C; (b) SOCl2, CH2Cl2, room temperature (rt); (c) KCN, acetonitrile, reflux; (d) 50% glyoxalic
acid in water, K2CO3, MeOH, rt; (e) 10% concentrated H2SO4 in formic acid, 100 °C; (f) water, acetonitrile, 40 °C; (g) THF, 0 °C; (h) acetic
anhydride, 90 °C; (i) MeOH, 0 °C; (j) diphenyl phosphorazidate, Et3N, toluene, then TMS−ethanol, 120 °C; (k) 4N HCl in dioxane, rt; (l) t-butyl
N-[(methylamino)thioxomethyl]carbamate, EDCI, DIEA, DMF, rt; (m) chloroethyl chloroformate, K2CO3, dichloromethane, rt; (n) 2,5-
dioxopyrrolidin-1-yl 2-(trimethylsilyl)ethyl carbonate, Et3N, dichloromethane, rt; (o) chiral HPLC (details in Experimental Section).
Table 1. Selected SAR of Amides
a
b
BACE1 Ki and cell IC50 values are the average of a minimum of two independent determinations. Selectivity ratios were derived from cathepsin-D
c
Ki values (average of a minimum of two independent determinations). Oral dose 10 mg/kg.
anhydride to reform an anhydride 15. This anhydride
underwent solvolysis with methanol at the less hindered center
to give compound 16 with excellent regioselectivity. This
monoacid was converted to protected amine 17 by a Curtius
rearrangement followed by treatment with 2-(trimethylsilyl)-
ethanol, and the amine functionality was subsequently
unmasked by treatment with HCl to provide compound 18.
Coupling of this material with t-butyl N-[(methylamino)-
thioxomethyl]-carbamate using standard peptide coupling
conditions followed by in situ intramolecular ring closure
provided protected racemic iminopyrimidinone 19. To facilitate
downstream resolution of the enantiomers, the benzyl
protecting group of compound 19 was swapped for a Teoc
protecting group. Thus, treatment of compound 19 with
chloromethyl chloroformate produced free amine 20 that was
reprotected using N-[2-(trimethylsilyl)ethoxycarbonyloxy]-
succinimide (Teoc-OSu) to provide penultimate intermediate
21. The enantiomers of compound 21 were separated on
C
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a
multihundred gram scale using chiral HPLC to provide desired
isomer 22 and its enantiomer ent-22.
Scheme 4. Synthesis of Compounds 36−45
The amides shown in Table 1 were synthesized through the
reaction sequence described in Scheme 2. Suzuki reactions
a
Scheme 2. Synthesis of Amides 26−28
a
Reagents and conditions: (a) R1-boronic acid, dichloro[1,1′-
bis(diphenylphosphino)ferrocene]-palladium(II) dichloromethane,
tert-butanol, K2CO3, 60 °C; (b) TBAF, THF, 0 °C; (c) R2-CO2H,
EDCI, HOBt, DIEA, dichloromethane, rt; (d) trifluoroacetic acid,
dichloromethane, rt.
between bromide 22 and various boronic acids installed the
biaryl moiety, and subsequent treatment of the products with
TBAF resulted in Teoc removal from the pyrrolidine nitrogen.
Coupling of these free amines (compounds 23−25) with
various acids followed by treatment of the resulting compounds
with TFA provided analogues 26−28.
As shown in Scheme 3, the 2,5-thiophene isomer could be
accessed easily from the same advanced intermediate as the 2,4-
thiophene isomers above. To that end, debromination of
compound 21 followed by treatment of the resulting
compound with NBS installed the bromide in the 5 position
of the thiophene. Following similar protocols as those described
above for biaryl formation and pyrrolidine deprotection,
compound 29 was converted to free amine 30. In this case,
the free pyrrolidine core was subjected to palladium-mediated
arylation that, following removal of the Boc protecting group,
provided analogues 31 and 32.
The remainder of the analogues discussed in this manuscript
were synthesized from intermediates 23−25 using a variety of
N-arylation conditions (Scheme 4) followed by TFA-mediated
deprotection of the core. Compounds 33−35 were obtained
through copper acetate mediated coupling with an appropriate
boronic acid.23 On the other hand, compounds 36−45 were
made with palladium mediated N-arylation chemistry24 using
the appropriate pyridine or pyrimidine halide.
a
Reagents and conditions: (a) aryl boronic acid, Cu(OAc)2, Et3N,
dichloromethane, rt; (b) TFA in dichloromethane, rt; (c) 2-bromo-
substituted pyridine, Tris(dibenzylideneacetone)dipalladium(0), race-
mic-2,2′-bis-(diphenylphosphino)-1,1′-binaphthyl, NaOt-Bu, 80 °C;
(d) 2-halo-substituted pyrimidine, palladium acetate, 2′-(di-tert-
butylphosphino)-N,N-dimethylbiphenyl-2-amine, NaOt-Bu, 80 °C.
RESULTS AND DISCUSSION
■
The X-ray cocrystal structure of iminopyrimidinone 721 bound
to BACE1 showed that the biaryl substituent is oriented in a
pseudoaxial conformation to occupy the continuous S1−S3
hydrophobic pocket. In an attempt to bias the energetics of the
molecule toward this active conformer, a C5−C6 cycle (cf. 7a)
was predicted to stabilize the pseudoaxial conformation by 0.92
kcal/mol (Figure 3). In addition, modeling indicated that
substitution of the new fused ring structures would provide
access to space in or near the S2′ pocket. On the basis of these
a
Scheme 3. Synthesis of N-Phenyl Analogue in the 2,5-Thiophene Series (31, 32)
a
Reagents and conditions: (a) 10% Pd/C, H2, methanol, rt; (b) NBS, DMF, rt; (c), (3-cyano-4-fluorophenyl)boronic acid, dichloro[1,1′-
bis(diphenylphosphino)-ferrocene]palladium(II) dichloromethane, tert-butanol, K2CO3; 60 °C; (d) TBAF, THF, 0 °C; (e) Tris-
(dibenzylideneacetone)dipalladium, tri-t-butylphosphonium tetrafluoroborate, toluene, ArBr, NaOt-Bu, 80 °C; (f) trifluoroacetic acid,
dichloromethane, rt.
D
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Figure 3. Conformational analysis.
computational results, development of a bicyclic version of 7
became a high priority.
Figure 5. Co-crystal structure of 26 (green, PDB ID code: 4H1E) with
BACE1 (catalytic dyad in red). One of the three water molecules
moved 1.8 Å away from its original position (gray sphere) to reside
2.8 Å away from the amide carbonyl and 2.7 Å away from the NH of
Trp76.
Modeling of possible rings to fuse to the C5−C6 position of
the iminopyrimidinone core suggested that a 3,4-fused
pyrrolidine would provide the optimal trajectory for sub-
stituents on the nitrogen to reach the S2′ subsite. Overlay of a
model of benzamide 26 with the X-ray structure of compound
7 revealed the likelihood that the carbonyl oxygen of the
benzamide would perturb or even displace one of the three
conserved water molecules due to their close proximity (Figure
4a). Because these water molecules form a hydrogen bonding
network among Trp76, Asn37, Ile126, Ser35, and Asp32 in the
protein surface (Figure 4b), the potential impact of disturbing
one such conserved water molecule was unclear. In fact, upon
synthesis, compound 26 showed a 19-fold improvement of
potency (BACE1 Ki = 2.0 nM) over compound 7.
The X-ray crystal structure of compound 26 bound to
BACE1 (Figure 5) showed that the core and biaryl substituent
bind in a similar fashion as was observed for the nonfused
iminopyrimidinones (e.g., 7). Additionally, none of the three
conserved water molecules adjacent to the S2′ binding pocket
were actually displaced but instead were reorganized to
accommodate the new benzamide substituent. The water
molecule that was nearest (1.1 Å) the amide oxygen of
compound 26 in docking studies was perturbed by 1.8 Å from
its original position to maintain the hydrogen bonding network
observed with compound 7. From its new position, this water
molecule was also within hydrogen bonding distance (2.8 Å) of
the amide oxygen of 26 (Figure 5).
The significant improvement in affinity for this first bicyclic
iminopyrimidinone 26 established this fused pyrrolidine series
as an alternative BACE1 inhibitor scaffold. The compound was
very potent in the cell-based assay, with an Aβ IC50 of 19 nM
representing only a 6-fold cell shift (Table 1). Replacement of
the P3 cyanophenyl group with an optimized 3-propynlpyr-
idine20,21 improved the affinity and cell potency, producing an
inhibitor 28 with a single-digit nanomolar IC50 (8.0 nM) in the
cell-based assay. Unfortunately, many of these analogues (e.g.,
compounds 26−28, Table 1) displayed only modest plasma
exposures in rat PK studies,25 possibly due to their
susceptibility to P-glycoprotein (P-gp) efflux26 or high hepatic
clearance (e.g., 27, Caco-2 efflux ratio = 17, rat hepatocyte
clearance = 46 μL/min/106 cells).
To improve the PK profile of this new series, we focused on
SAR exploration near the S2′ subsite while retaining the
previously optimized substituents in the S1 and S3 subsites. In
particular, we envisioned that replacements for the amide
linkage would be beneficial to the overall properties of the
Figure 4. (a) Superimposition of docking conformation of proposed compound 26 (green) with X-ray conformation of 7 (yellow, PDB ID code:
4HA5) showing the pseudoaxial orientation of the biaryl substituent spanning from S1−S3 to S3sp. Three conserved water molecules (yellow
spheres) are in or near the S2′ pocket site when 7 is bound. Docking studies show that one of the water molecules would be ∼1.1 Å from the
modeling position of the amide oxygen of target 26. (b) Details of the hydrogen bonding network of the three conserved water molecules with the
protein backbone of BACE1.
E
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molecule.27 Docking studies showed that a phenyl group
directly linked to the pyrrolidine nitrogen would occupy a
pocket that we have termed S2″ that is distinct from the S2′
binding pocket occupied by the amides and would also provide
opportunities to reach the S2′ site with appropriate
substitution. In this direction, the first two examples
(compounds 31 and 32, Figure 6) were prepared as racemates
structure of compound 31 complexed with BACE1 (Figure 7),
the N-phenyl group occupies the S2″ site. Interestingly, one of
Figure 7. Superimposition of X-ray crystal structures of 31 (yellow,
PDB ID code: 4H3J) and 26 (green, PDB ID code: 4H1E) complexed
with BACE1. The gray ball represents the displaced water molecule,
which is 2.4 Å away from the ortho position of the phenyl ring, and its
coordinates are taken from the crystal structure of 26 complexed with
BACE1. The active site flap residues of BACE1 are omitted for ease of
viewing, but despite significant movement or displacement of water
molecules in the S2″ region, no significant movement of the flap was
observed.
Figure 6. BACE1 affinity of racemic 31 and 32.
using a 2,5-disubstituted thiophene as the S1 substituent and
were found to have BACE1 affinities of 90 nM and 124 nM,
respectively. Subsequent investigations using both P1 2,5- and
2,4-disubstituted thiophenes showed that the 2,4-substitution
pattern provided slightly superior BACE1 inhibition. For
example, as shown in Table 2, N-fluorophenyl analogue 33 in
the 2,4-disubstituted thiophene series was 2.6-fold more potent
than 32. Although compound 33 was 9-fold less potent than
benzamide 26, we were quickly able to restore single-digit
nanomolar BACE1 affinity by introduction of an m-isopropoxy
group on the phenyl in combination with the P3 3-
propynlpyridine moiety (cf. 35). Gratifyingly, compound 35
possessed significantly improved oral rat PK properties (rat
AUC0−6 h = 3.7 μM·h) compared to its amide counterpart 26.
On the basis of the observation that inhibitors with lower
ClogP generally have lower cell shifts, we next sought to
incorporate heteroatoms into the molecule. Strategically, the
new N-aryl substituent in the S2′ region provided an excellent
opportunity for this effort. As shown in the X-ray crystal
the conserved water molecules positioned between the carbonyl
of amide 26 and Trp76 was displaced in the crystal structure of
compound 31. The proximity of this water molecule to the
ortho position of the N-phenyl group (2.4 Å) suggested that
replacement of the N-phenyl with a N-pyridin-2-yl would allow
for a hydrogen bonding interaction and thus re-establish the
original network of water molecules.
To test this proposal, N-pyridyl compound 36 was prepared,
and it showed improvements in both BACE1 Ki and cell Aβ
IC50 values over N-phenyl analogue 33 (Table 3). Both the 3-
methoxypyridyl and 3-cyanopyridyl analogues 37 and 38 were
well tolerated, and the X-ray crystal structure of compound 37
Table 2. SAR of N-Phenyl Derivatives 33−35
a
b
BACE1 Ki and cell IC50 values are the average of a minimum of two independent determinations. Selectivity ratios were derived from cathepsin-D
c
d
Ki values (average of a minimum of two independent determinations). Oral dose 10 mg/kg. Not determined.
F
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Table 3. Selected SAR of Pyridine Derivatives 36−41
a
a
b
compd
R1
R2
R3
MW (ClogP)
BACE1 Ki (nM)
HEK293 IC50 (nM)
LE
Cath-D/BACE1
36
37
38
39
40
41
H
H
H
H
F
H
428 (2.7)
458 (3.1)
454 (2.3)
446 (2.9)
458 (3.5)
496 (3.7)
8
3
50
23
48
22
18
0.36
0.35
0.34
0.35
0.37
0.28
23
42
39
15
89
H
OMe
CN
H
H
6
H
6
OMe
CF3
H
H
H
1
c
c
H
64
nd
nd
a
b
BACE1 Ki and cell IC50 values are the average of a minimum of two independent determinations. Selectivity ratios were derived from cathepsin-D
c
Ki values (average of a minimum of two independent determinations). Not determined.
Figure 8. (a) Co-crystal structure of 37 (yellow, PDB ID code: 4H3I) in BACE1; the emerging water molecule is 2.8 Å away from the pyridine
nitrogen and 2.9 Å away from the NH of the Trp76. For clarity, the rest of the H-bonding network depicted in Figure 4b is omitted. (b) X-ray crystal
structure of 38 (PDB ID code: 4H3G) complexed with BACE1; the cyano group displaces one of the conserved water molecules and makes direct
contact the Trp76. (c) X-ray crystal structure of 40 (green, PDB ID code: 4H3F) complexed with BACE1; the methoxy group occupies the S2″
subsite, displacing two of the conserved water molecules. Superimposition with X-ray conformation of 37 (yellow) shows that 40 moves almost 1.0 Å
toward the S2″ binding pocket. As in Figure 7, the flap residues are omitted from these figures, but again no significant movement of the flap of was
observed despite changes in the binding mode and water molecule organization.
(Figure 8a) demonstrated reincorporation of the water
molecule connecting the pyridine nitrogen and the NH of
Trp76, recapitulating the network previously observed with
compound 26. In contrast, the 3-cyanopyridyl analogue 38
bound in the opposite orientation with the nitrile projected
deep into the S2″ binding pocket. This conformation again
displaced a conserved water molecule from the hydrogen-bond
network to establish a direct hydrogen bond between the nitrile
and the NH of Trp76 (Figure 8b).
Turning to the 6-position of pyridyl, while introduction of a
lipophilic group like trifluoromethyl (e.g., 41) was not
tolerated, incorporation of a methoxy group resulted in an 8-
fold boost in affinity, with compound 40 having a BACE1 Ki of
1 nM. The X-ray crystal structure of 40 complexed with BACE1
showed that the inhibitor shifted almost 1.0 Å toward the
nonprime side of the enzyme to bury the methoxy pyridine into
the S2″ subpocket and displace a second conserved water
molecule from this region (Figure 8c). The in-plane anti-
oriented methoxy group, a result of lone pair/lone pair
repulsion between the pyridine nitrogen and the ether
oxygen,28 positions the methoxy group in the deepest part of
S2″. Overall, replacement of the N-(4-fluoro)phenyl group with
an appropriately substituted pyridyl resulted in a 26-fold
improvement in BACE1 affinity (c.f., 33 vs 40).
While beneficial for affinity and cellular potency, introduction
of the S2″ pyridyl group consistently resulted in inhibition of
CYP450 enzymes. For example, compound 39 inhibited 2D6
and 3A4 with IC50 values of 6 μM each. Thus, we next focused
on less basic heterocyclic replacements of the S2″ pyridine ring
in an attempt to improve the CYP inhibition profile. The X-ray
structures of analogues 38 and 40 complexed with BACE1
(Figure 8b,c) demonstrated that compounds with the N-pyridyl
nitrogen oriented either toward S2′ (e.g., 38) or toward S2″
(e.g., 40) were essentially equipotent, suggesting that an N-
pyrimidyl group should itself be well tolerated. In fact,
pyrimidine 42 was equipotent with the analogous pyridine 36
against BACE1 with no CYP450 inhibition. In a rat oral PK
screen with 10 mg/kg of compound 42, a Cmax of 1 μM was
observed at 1 h with plasma levels decreasing to 0.06 μM at 6 h
and a 6 h brain-to-plasma ratio of 1. On the basis of its
promising profile, albeit with low plasma and brain
concentrations at the 6 h time point, compound 42 was
G
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Table 4. Selected SAR of Pyrimidine Derivatives 42−45
d
MW
(ClogP)
BACE1 Ki
HEK293 IC50
rat AUC0−6h
brain 6 h
(μM)
rat PPB (%
a
a
b
c
compd
R1
(nM)
(nM)
LE
Cath-D/BACE1
(μM·h)
brain/plasma
unbound)
42
43
44
45
H
429 (1.9)
447 (2.1)
464 (2.6)
443 (2.4)
11
4
31
16
51
29
0.35
0.36
0.35
0.34
28
94
81
67
2.0
31
0.058
0.463
0.965
0.232
1.0
0.1
2.2
2.4
0.4
F
Cl
Me
7
90
0.1
e
9
111
<0.1
nd
a
b
BACE1 Ki and cell IC50 values are the average of a minimum of two independent determinations. Selectivity ratios were derived from cathepsin-D
c
d
e
Ki values (average of a minimum of two independent determinations). Oral dose 10 mg/kg. Plasma protein binding. Not determined.
Figure 9. (a) Dose-dependent inhibition of Aβ40 production in the plasma, CSF, and cortex compartments of rats by compound 43 measured at 3 h
period after oral administration. (b) Post dose (30 mg/kg) measurements in plasma, CSF, and cortex revealed that the level of Aβ40 remained
inhibited in cortex and CSF until 9 h after dosing and in plasma until 24 h after dosing.
screened in vivo for central efficacy in rats. However,
compound 42 did not produce a significant reduction of CSF
Aβ40 in the CSF following a single oral 10 mg/kg dose. This
lack of efficacy is likely due to a very low level of compound in
the brain considering that, from the rat PK data in Table 4,
there is a free concentration of only approximately 1 nM in the
brain at 6 h.29 This result suggested that further improvement
in potency and PK to increase t1/2 and brain exposure was
necessary.
Through introduction of a substituent at the 5-position of the
pyrimidine, we identified three compounds (43−45), bearing
5-fluoro, -chloro, and -methyl groups, respectively, with good
affinity as well as improved oral exposure and 6 h absolute brain
levels in rat. Compounds 43 and 44 were selected over 45 for
in vivo screening due to their higher 6 hour brain levels (Table
4). Of these two compounds, only fluoropyrimidine 43 showed
robust lowering of CSF Aβ40 following a 10 mg/kg oral dose
(52% Aβ40 lowering for 43 versus 15% for 44), even though
both compounds had similar total brain levels. To explain this
discrepancy, we speculate that again free fraction plays an
important role: chloropyrimidine 44 has a significantly lower
unbound fraction (0.4%) than fluoropyrimidine 43 (2.4%).
When these free fractions are applied to the total brain levels
from the rat PK data in Table 4,29 the free drug level for
compound 44 is only 4 nM versus 11 nM for compound 43.
The latter free concentration is close to the cellular IC50 of the
compound. This delicate balance underscores the critical
importance of physicochemical properties, particularly the
interplay of lipophilicity and free fraction, on PK and thus
CNS efficacy.
Upon further in vivo evaluation, compound 43 was found to
dose-dependently reduce Aβ40 in plasma, CSF, and cortex of
rats. Complete inhibition of Aβ40 in plasma was observed with
oral doses as low as 3 mg/kg (Figure 9a). In the central
compartments, administration of compound 43 (10 mg/kg
PO) produced 50% reduction of CSF Aβ40 at 3 h postdose,
while 30 mg/kg produced 73% and 42% reduction of CSF and
cortical Aβ40, respectively (Figure 9a). In a time course study
(Figure 9b) using a single oral 30 mg/kg dose of compound 43,
the levels of Aβ40 in the CSF and cortex remained inhibited for
9 h before returning to baseline at 15 and 12 h, respectively.
Maximal inhibition of Aβ40 in both CNS compartments
occurred at tmax for plasma concentration (3 h post dose)
and continued for approximately one-half-life of exposure (i.e.,
out to 9 h post dose).
In terms of its broader profile, compound 43 is nearly 100-
fold selective for BACE1 over cathepsin-D, making it the most
selective of the analogues discussed herein from this new series
H
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Table 5. Rat Pharmacokinetic Properties of 43
P450 enzyme inhibition μM-co, μM-po
a
AUC0−24 h
31
PO Cmax μM
B/P (6 h)
Papp (nm/s)
efflux ratio
3A4
2D6
2C9
h-ERG patch clamp
70% @ 1 μM
6.1
0.1
151
1.9
>30, 1.9
30, 19.7
22.4, 30
hepatocyte Clint μL·min−1·10−6 cells
b
rat
human
2.0
cyno monkey
5.7
F%
rat fupl
0.024
h-PXR (over rifampicin)
0.2X @ 10 μM
13.5
97
a
b
Oral dose 10 mg/kg. Fraction unbound in plasma.
Relative Energy Calculations. Conformational searches for
compounds 7 and 7a were carried out using the MacroModel method
(Tables 1−4). While this compound does not cause induction
of human h-PXR, it is a time-dependent inhibitor of CYP450
3A4 and showed significant inhibition of the h-ERG channel in
a patch clamp assay (Table 5). Compound 43 showed a
significantly reduced efflux ratio in Caco-2 cells (Table 5)
compared to amide-containing analogue 27 (1.9 vs 17),
although even with this lower ratio the compound may be a
substrate for P-gp given its low brain-to-plasma ratio in rats.
This compound also showed low to moderate in vitro
hepatocyte clearance across species. Overall, while still
requiring further optimization to address these ancillary issues,
compound 43 provided a key proof-of-concept for this new
series, showing that this significantly modified core structure
could confer robust central efficacy.
in the Schrodinger modeling package.30 Each compound was subjected
̈
to 3000 steps of mixed torsional/low-mode sampling using MMFFs
force field to find the low energy conformations within 10 kcal/mol
above the global energy minimum. Quantum mechanics (QM) energy
optimizations were then carried out on these low energy
conformations. QM method Jaguar in Schrodinger package was used
for the QM calculations. The B3LYP hybrid density functional and the
6-31G** basis set were used in geometry optimizations. The lowest
QM energy of the pseudoaxial conformation and that of the
pseudoequatorial conformation for each compound were used to
calculated the energy differences.
Synthesis. Unless otherwise mentioned, all the reagents and
solvents were obtained from commercial sources and used without
further purification. Air sensitive chemistries were performed under an
atmosphere of nitrogen or argon. Purification of the final compounds
to >95% purity were carried our either using prepacked silica gel
cartridge (Analogix or Biotage or ISCO) or reverse phase C18 column
(mobile phase, A = 0.05% TFA in water and B = 0.05% TFA in
acetonitrile, gradient = 10−95% B over 10 min). All NMR data were
collected using 400 MHz NMR spectrometry, and the chemical shifts
are listed as ppm relative to tetramethylsilane. Microwave assisted
reactions were performed in a Smith synthesizer from Personal
Chemistry. The purity of final compounds was analyzed on two
independent reverse phase HPLC systems using different gradients.
LC-electrospray-mass spectroscopy with a C-18 column using a
gradient of 5−95% MeCN in water as the mobile phase was used to
determine the molecular mass and retention time. The purity of the
samples was assessed using a mass detector and a UV detector at 254
nm. An additional analytical reverse phase HPLC system was used to
assess the purity of final compounds using a UV detector monitored at
both 220 and 254 nm and an ELSD detector.
SUMMARY AND CONCLUSION
■
As part of efforts to further optimize a lead iminopyrimidinone
BACE1 inhibitor 7, a structure-based design approach enabled
discovery of a structurally distinct fused pyrrolidine series.
Although the conformational constraint of this novel bicyclic
core was designed both to favor the bioactive conformer and to
explore SAR in the S2′ region, we found that gains in potency
did not have accompanying gains in ligand efficiency. This
disconnect may be due to the only modest bias for the
pseudoaxial conformation conferred by the fused five-
membered ring, which was predicted to favor the bioactive
conformation by roughly 1 kcal/mol. On the other hand, the
fused ring provided an ideal platform to reach into and study
the SAR of both the S2′ and the S2″ binding pockets,
ultimately providing a number of potent analogues. Initial N-
carboxamide compounds in this series (e.g., compound 26)
showed good binding affinity but suffered from poor
pharmacokinetic properties. Moving to N-aryl analogues
ultimately provided compounds that had excellent potency
and demonstrated significantly improved PK, validating an
emphasis on optimization of drug-like properties during SAR
development. A close collaboration with structural chemistry
and chemical modeling and informatics facilitated our under-
standing of the importance of a network of hydrogen bound
waters in the S2″ subpocket and drove additional targeted
modifications. Ultimately, these investigations, in conjunction
with the efforts from in vitro and in vivo pharmacology, led to
identification of compound 43, an orally active brain penetrant
inhibitor that potently reduced central Aβ40 in rats. Further
modifications to this series aimed at improving the pharmaco-
logical and off-target profile will be the subject of a future
communication.
(4aR,7aR)-2-(Trimethylsilyl)ethyl 7a-(4-Bromothiophen-2-
yl)-2-((tert-butoxycarbonyl)imino)-3-methyl-4-oxohexahydro-
1H-pyrrolo[3,4-d]pyrimidine-6(2H)-carboxylate (22). To a sol-
ution of aldehyde 8 (20 g, 105 mmol) in methanol (100 mL) was
added NaBH4 (4.0 g, 108 mmol) portionwise at 0 °C. The resulting
solution was stirred at 0 °C for 2 h. Water (100 mL) was added slowly,
and the resulting mixture was stirred at 0 °C until gas evolution had
ceased. The ice bath was removed, and the solution was stirred at
room temperature for 30 min before evaporation to dryness. The
resulting slurry was extracted with ethyl acetate (3 × 100 mL). The
organic layers were combined and washed with brine, dried with
MgSO4, and evaporated to provide the corresponding alcohol. The
1
crude alcohol was >95% pure by H NMR and used directly in next
step. 1H NMR (400 MHz, CDCl3) δ 7.17 (s, 1H), 6.93 s, 1H), 4.80 (d,
J = 6.0 Hz, 2H), 1.84 (t, J = 6.0 Hz, 1H). To a solution of this alcohol
(20 g 103 mmol) in CH2Cl2 (10 mL) was added SOCl2 (14.5 g, 124
mmol) slowly at 0 °C. The resulting reaction mixture was stirred at 0
°C until the gas evolution had ceased. The ice bath was removed, and
the reaction mixture was slowly warmed to room temperature and
stirred at room temperature for 12 h. The reaction mixture was
evaporated to dryness, and the resulting slurry was diluted with ethyl
acetate and washed with brine. The organic layer was dried with
MgSO4 and evaporated to yield the chloride 9 (20 g, 98%). The crude
chloride was >95% pure by 1H NMR and directly used in the next step
EXPERIMENTAL SECTION
Details of the protocols for X-ray crystallography, aspartyl protease Ki,
and cell Aβ40 IC50 assays and in vivo rat CSF studies can be found in
ref 21.
■
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without further purification. 1H NMR of 9 (400 MHz, CDCl3) δ
7.15−7.30 (m, 2H), 4.73 (s, 2H).
To a solution of 17 (35.2 g, 65 mmol) in dioxane (200 mL) was
added 4N HCl (20 mL) in dioxane at 0 °C, and the resulting solution
was allowed to warm to room temperature over 14 h. Ether (400 mL)
was added to form a white precipitate. This precipitate was collected,
washed with ether (200 mL), and dried in vacuo for 12 h to give 18
(28 g, 100%) as a HCl salt, which was used without further
purification. 1H NMR of 18 (400 MHz, CDCl3) of the product amine
HCl salt: δ 7.20−7.40 (m, 5H), 7.17(d, J = 1.5 Hz, 1H), 6.86 (d, J =
6.86 Hz, 1H), (3.65 (m, 1H), 3.45−3.31 (m, 6H), 3.29 (s, 3H).
To a solution of 18 (32 g, 76 mmol) in DMF (300 mL) was added
DIEA (66 mL, 380 mmol), t-butyl N-[(methylamino)thioxomethyl]-
carbamate (14.4 g, 76 mmol) and followed by EDCI·HCl (14.5 g, 76
mmol), and the resulting mixture was stirred at room temperature for
48 h. It was then diluted with ethyl acetate (150 mL), washed with
brine, and water. The organic layer was dried with Na2SO4 and
concentrated. The crude product was purified by silica gel
chromatography using 0−100% ethyl acetate in hexanes to provide
19 (23 g, 60%). 1H NMR of 19 (400 MHz, CDCl3) δ 7.25−7.34 (m, 5
H), 7.15 (d, J = 1.5 Hz, 1H)), 6.91 (d, J = 1.5 Hz, 1H), 3.75 (m, 2H),
3.42 (m, 1H), 3.34 (m, 1H), 3.31 (s, 3H), 3.22 (d, J = 10 Hz, 1H),
3.09, (d, J = 10 Hz, 1H), 3.02 (m, 1H), 1.54 (s, 9H).
To a solution of chloride 9 (4.4 g, 21 mmol) in 50 mL of
acetonitrile was added oven-dried KCN (5 g, 76 mmol) portionwise.
The resulting mixture was stirred at room temperature for 1 h and
then heated under reflux for 20 h until the reaction was completed.
The reaction mixture was diluted with ethyl acetate (100 mL) and
filtered, the solid was washed with ethyl acetate (3 × 50 mL), and the
combined filtrate was evaporated. The residue was purified by silica gel
chromatography using 10% ethyl acetate/hexanes to give nitrile 10
1
(3.1 g, 75%). H NMR of 10 (400 MHz, CDCl3) δ 7.18 (s, 1H), 7.0
(s, 1H), 3.89 (s, 2H).
Compound 11 was prepared following a literature procedure.22 To
a solution of 10 (4 g, 20 mmol) and glyoxaldehyde (4.2 g, 30 mmol,
50% aq solution) in MeOH (50 mL) was added K2CO3 (6.9 g. 50
mmol), and the reaction mixture was stirred at room temperature for 4
h. The reaction mixture was filtered, and the solid was washed with
ether. The solid was suspended in cold water and stirred vigorously for
1 h. The solid was filtered and dried to give crude product 11 (5.12 g,
100%). A small quantity of 11 was dissolved in water, neutralized with
1N HCl, and extracted with EtOAc. The organic solution was
1
evaporated to give the corresponding free acid of 11. H NMR (400
To a solution of 19 (1.0 g, 1.9 mmol) in DCM (10 mL) was added
potassium carbonate (0.3 g, 2.0 mmol) followed by 1-chloroethyl-
chloroformate (0.269 g, 1.9 mmol) at −15 °C, and the resulting
solution was slowly warmed to room temperature and stirred for 90
min. The reaction mixture was filtered, and the filtrate was evaporated.
The residue was dissolved in methanol (10 mL) and stirred overnight.
After removal of methanol in vacuo, the residue was purified by silica
gel chromatography using 10% methanol in dichloromethane to give
MHz, CDCl3) of 11 as a free acid: δ 7.69 (s, 1H), 7.56 (s, 1H), 6.96 (s,
1H).
To a solution of 11 (50 g, 193 mmol) in formic acid (mL) was
added concd sulfuric acid (40 mL). The resulting solution was heated
under reflux for 2 h. The reaction was cooled to room temperature and
poured into ice water, and the solid was filtered to give the anhydride
of product 12. This anhydride was dissolved in the mixture of
CH3CN−H2O (9:1) was heated at 40 °C for 12 h. The reaction
solution was concentrated and the residue dried to give compound 12
(56 g, 100%). 1H NMR of 12 (400 MHz, CDCl3) δ 7.66 (s, 1H), 7.40
(s, 1H), 6.42 (s, 1H).
1
20 (0.570 g, 70% yield). H NMR of 20 (400 MHz, CDCl3) δ 10.30
(br s, 1H), 7.17 (d, J = 1.5 Hz, 1H), 6.86 (d, J = 1.5 Hz, 1H), 3.68 (m,
1H), 3.42 (d, J = 12 Hz, 1H), 3.31−3.40 (m, 3H), 3.29 (s, 3H), 1.52
(s, 9H).
To a solution of 12 (5 g, 18.17 mmol) in anhydrous THF (72 mL)
was added N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)-
methanamine (8.61 g, 37 mmol) at 0 °C, and the resulting mixture
was stirred at 0 °C for 90 min. It was then quenched with 1N HCl (10
mL) and extracted with ethyl acetate (3 × 50 mL). The organic layers
were combined, dried with anhydrous Na2SO4, and concentrated to
provide 14 (6 g, 82%), which was used in the next step without further
To a solution of 20 (13 g, 30 mmol) in DCM (100 mL) was added
Teoc-OSu (7.7 g, 30 mmol) followed by DIEA (5.2 mL, 30 mmol) at
0 °C. The reaction was stirred for 6 h. The reaction mixture was
diluted with ethyl acetate and washed with water and brine. The
organic layer was dried and evaporated, and the residue was purified by
silica gel chromatography using 20% ethyl acetate in hexanes to give
1
21 (13.7 g, 80%). H NMR (400 MHz, CDCl3) δ 10.40 (br m, 1H),
1
purification.. H NMR of 14 (400 MHz, CDCl3) δ 7.45−7.60 (m,
7.22 (br s, 1H), 6.90 (br s, 1H), 4.20 (m, 2H), 3.68−4.06 (m, 4H),
3.47 (m, 1H), 3.30 (s, 3H), 1.29 (s, 9H), 1.01 (m, 2H), 0.03 (s, 9H).
m/z: 595.22. 21 was resolved using a semipreparative ChiralPak AS
column eluted with 50% isopropyl alcohol in hexane (50 mL/min).
7H), 4.49 (m, 2H), 4.30 (d, J = 12.0 Hz, 1H), 3.8−4.0 (m, 4H).
To an oven-dried flask containing 14 (6 g, 14.6 mmol) was added
acetic anhydride (100 mL) at room temperature. The resulting
solution was heated for 30 min at 90 °C and then allowed to cool to
room temperature. The reaction mixture was evaporated to dryness to
provide crude anhydride 15, which was poured into a flask containing
methanol (500 mL) at 0 °C. The resulting solution was slowly warmed
to room temperature and evaporated to dryness. The crude product
was dissolved in CH2Cl2 (200 mL) and washed with 1H HCl (50 mL),
followed by with brine, and then dried with Na2SO4 and concentrated
to provide 16 (6 g, 96%), which was used in the next step without
purification. 1H NMR of 16 (400 MHz, CDCl3) δ 7.10−7.60 (m, 7H),
4.3 (m, 2H), 4.12 (d, J = 12.0 Hz, 1H), 3.7−3.9 (m, 4H), 3.6 (s, 3H).
To an oven-dried flask containing 16 (4.67 g, 11 mmol) in toluene
(10 mL) was added DPPA (3 mL, 14.18 mmol), followed by triethyl
amine (2.16 mL, 24.2 mmol) at 0 °C. The resulting mixture was slowly
warmed to room temperature and stirred for 12 h. To the reaction
mixture was added trimethylsilylethanol (4 mL, 44 mmol), and the
reaction mixture was heated under reflux for 1 h, then it was cooled to
room temperature, diluted with ethyl acetate (150 mL), and washed
with brine and water. The organic layer was dried with Na2SO4,
filtered, and concentrated in vacuo. The crude product was purified by
silica gel chromatography using 30% ethyl acetate in hexanes to
provide 17 (1.5 g, 40%). 1H NMR of 17 (400 MHz, CDCl3) δ 7.20−
7.40 (m, 5H), 7.07 (d, J = 1.5 Hz, 1H), 6.94 (d, J = 1.5 Hz, 1H), 4.11
(m, 2H), 3.79 (d, J = 11 Hz, 1H), 3.70 (s, 3H), 3.69 (d, J = 11 Hz,
1H), 3.54, (d, J = 10 Hz, 1H), 3.32 (t, J = 8 Hz, 1H), 3.21 (d, J = 10
Hz, 1H), 3.16 (t, J = 8 Hz, 1H), 2.88 (t, J = 8 Hz, 1H), 0.97 (m, 2H),
0.03 (s, 9H).
Enantiomer A, 22: tR = 19.3 min, [α]25 = −94° (MeOH, C = 1, 23
D
°C). Enantiomer B, ent-22: tR = 39.5 min, [α]25D = +105° (MeOH, C
= 1, 23 °C).
General Procedure A for Synthesis of Pyrrolidine 23−25 and
30. To a solution of bromide 22 (1.74 mmol) and boronic acid (2.61
mmol) in t-BuOH (7 mL) was added dichloro[1,1′-bis-
(diphenylphosphino)-ferrocene]palladium(II) dichloromethane
(0.190 g, 0.261 mmol) followed by aqueous 1N K2CO3 (2.61 mL,
2.61 mmol). The resulting mixture was heated at 60 °C for 1 h before
it was cooled, diluted with ethyl acetate, and washed with water. The
organic layer was dried and concentrated, and the residue was purified
by silica gel chromatography using 0−100% ethyl acetate in hexanes to
provide the product. To this product (0.963 g, 1.56 mmol) was added
1 M TBAF (5 mL, 5 mmol) in THF at 0 °C, and the solution was
stirred at room temperature for 4 h. The reaction mixture was poured
into a saturated solution of NaHCO3 and extracted with ethyl acetate.
The organic layer was concentrated, and residue was purified by silica
gel chromatography using 100% ethyl acetate to give pyrrolidines 23−
25.
General Procedure B for Synthesis of Compounds 26−28.
To a solution of pyrrolidine (1 mmol) in dichloromethane (5 mL)
were added HOBt (1 mmol), benzoic acid (1 mmol), and DIEA (1
mmol) followed by EDCI·HCl (1 mmol) and the solution was stirred
for 3 h. The reaction mixture was diluted with water and extracted with
EtOAc (3 × 50 mL). The organic layer was dried and concentrated,
and the residue was purified by silica gel chromatography using 0−
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100% ethyl acetate in hexanes to give the product, which was
subsequently deprotected with 20% TFA/DCM (2 mL). The
deprotected compounds were purified by reverse phase C18 column
using 0−90% water (0.05% TFA) in acetonitrile (0.05% TFA).
General Procedure C for Synthesis of Compounds 33−35.
To a solution of pyrrolidine (1 mmol) in DCM (5 mL) was added
boronic acid (1 mmol), Cu(OAc)2 (1 mmol), triethylamine (2 mmol),
and preactivated 4 Å molecular sieves (5 μm, 200 mg). The resulting
mixture was stirred for 48 h, whereupon the solid was filtered and the
organic solution was concentrated. The residue was purified by silica
gel chromatography using 0−100% ethyl acetate in hexanes to give the
product which was deprotected with 20% TFA/DCM (2 mL). The
deprotected compounds were purified by reverse phase C18 column
using 0−90% water (0.05% TFA) in acetonitrile (0.05% TFA).
General Procedure D for the Synthesis of Compounds 31−
32 and 36−45. To a solution of pyrrolidine (1 mmol) and
corresponding hetero halide (1 mmol) in toluene (2 mL) was added
tris(dibenzylidene-acetone)dipalladium(0) (0.07 mmol), BINAP (0.14
mmol), and NaOt-Bu (1.2 mmol). The resulting solution was degassed
and heated at 70 °C for 3 h and then cooled to room temperature and
filtered through a pad of Celite, and the crude material was purified by
silica gel chromatography using 0−100% EtOAc/hexanes to give the
product which was treated with 20% TFA/DCM (2 mL). The
deprotected compounds were purified by reverse phase C18 column
using 0−90% acetonitrile (0.05% TFA) in water (0.05% TFA).
tert-Butyl ((4aR,7aR)-7a-(4-(3-Cyanophenyl)thiophen-2-yl)-
3-methyl-4-oxohexahydro-1H-pyrrolo[3,4-d]pyrimidin-2(3H)-
ylidene)carbamate (23). Compound 23 was prepared from 22 in
ution of 22 (1.8 g, 3.14 mmol) in methanol (15 mL) was added 10%
Pd/C (200 mg), and the resulting solution was stirred for 90 min
under an atmosphere of hydrogen. Upon completion of the reaction
the reaction mixture was basified using Et3N, and then it was filtered
and concentrated. The residue was purified by silica gel chromatog-
raphy using 0−100% ethyl acetate in hexanes to provide the
1
debrominated product (1.3 g, 69%). H NMR (400 MHz, CDCl3) δ
10.40 (m, 1H), 7.31 (m, 1H), 6.97−6.99 (m, 2H), 4.20 (m, 2H),
3.68−4.06 (m, 4H), 3.50 (m, 1H), 3.30 (s, 3H), 1.53 (s, 9H), 1.01 (m,
2H), 0.03 (s, 9H). To a solution of the above debrominated product
(0.3 g, 2 mmol) in DMF (6 mL) was added NBS (0.42 g, 2.4 mmol),
and the reaction was stirred for 12 h. The reaction mixture was diluted
with water and extracted with ethyl acetate. The organic layer was
dried and concentrated. The residue was purified by silica gel
chromatography using 0−100% ethyl acetate in hexanes to provide 29
1
(0.916 g, 80%). H NMR (400 MHz, CDCl3) δ 10.39 (m, 1H), 6.94
(d, J = 4 Hz, 1H), 6.76 (m, 1H), 4.20 (m, 2H), 3.68−4.04 (m, 4H),
3.43 (m, 1H), 3.29 (s, 3H), 1.53 (s, 9H), 1.01 (m, 2H), 0.03 (s, 9H).
2-Fluoro-5-(5-((4aR,7aR)-2-imino-3-methyl-4-oxo-6-phenyl-
octahydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-2-yl)-
benzonitrile (31). Compound 31 was prepared from 29 in four steps
following the general procedure A and followed by general procedure
1
D. H NMR (400 MHz, CD3OD) δ 8.03 (dd, J = 2.6, 5.9 Hz, 1 H),
7.95 (ddd, J = 2.4, 5.0, 8.7 Hz, 1 H), 7.44−7.38 (m, 2 H), 7.26−7.19
(m, 3 H), 6.74 (t, J = 7.3 Hz, 1 H), 6.65 (d, J = 8.1 Hz, 2 H), 4.11−
4.02 (m, 2 H), 3.95 (d, J = 10.6 Hz, 1 H), 3.89 (m, 2 H), 3.35 (s, 3 H).
m/z: 446.2
2-Fluoro-5-(5-((4aR,7aR)-6-(4-fluorophenyl)-2-imino-3-meth-
yl-4-oxooctahydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)-
thiophen-2-yl)benzonitrile (32). Compound 32 was prepared from
29 in four steps following the general procedure A and followed by
1
two steps following the general procedure A. H NMR (400 MHz,
CDCl3) δ 10.54 (br s, 1H), 7.78 (m, 1H), 7.73−7.71 (m, 1H), 7.59−
7.57 (m, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.43 (m, 1H), 7.18 (m, 1H),
3.73 (m, 1H), 3.49 (m, 2H), 3.36 (m, 2H), 3.32 (s, 3H), 1.54 (s, 9H).
m/z: 452.3.
tert-Butyl ((4aR,7aR)-7a-(4-(3-Cyano-4-fluorophenyl)-
thiophen-2-yl)-3-methyl-4-oxohexahydro-1H-pyrrolo[3,4-d]-
pyrimidin-2(3H)-ylidene)carbamate (24). Compound 24 was
prepared following the general procedure A from 22 in two steps.
1H NMR (400 MHz, CDCl3) δ 10.4 (br s, 1H), 7.72 (m, 2H), 7.34 (br
1
general procedure D. H NMR (400 MHz, CD3OD) δ 8.03 (dd, J =
2.2, 5.9 Hz, 1 H), 7.95 (ddd, J = 2.2, 4.8, 8.8 Hz, 1 H), 7.45−7.40 (m,
2 H), 7.21 (d, J = 4.0 Hz, 1 H), 6.99 (t, J = 8.8 Hz, 2 H), 6.65−6.60
(m, 2 H), 4.08−4.02 (m, 2 H), 3.94 (d, J = 10.6 Hz, 1 H), 3.90−3.85
(m, 2 H), 3.35 (s, 3 H). m/z: 464.3.
3-(5-((7aR)-6-(4-Fluorophenyl)-2-imino-3-methyl-4-oxoocta-
hydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)-
benzonitrile (33). Compound 33 was prepared from 23 in two steps
s, 1H), 7.25 (m, 2H), 7.13 (m, 1H), 3.72 (m, 1H), 3.46 (m, 7H), 1.53
(s, 9H). m/z: 470.2.
1
following the general procedure C. H NMR (400 MHz, CD3OD) δ
8.03 (m, 1H), 7.95 (m, 1H), 7.86 (m, 1H), 7.65 (m, 2H), 7.58 (t, J =
8.0 Hz, 1H), 6.98 (m, 2H), 6.63 (m, 2H), 4.07 (m, 2H), 3.91 (m, 3H),
3.34 (s, 3H). m/z: 446.2.
tert-Butyl ((4aR,7aR)-3-Methyl-4-oxo-7a-(4-(5-(prop-1-yn-1-
yl)pyridin-3-yl)thiophen-2-yl)hexahydro-1H-pyrrolo[3,4-d]-
pyrimidin-2(3H)-ylidene)carbamate (25). Compound 25 was
prepared following the general procedure A in two steps from
3-(5-((7aR)-2-Imino-6-(3-isopropoxyphenyl)-3-methyl-4-ox-
ooctahydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)-
benzonitrile (34). Compound 34 was prepared from 23 in two steps
1
bromide 22. H NMR (400 MHz, CDCl3) δ 10.36 (s, 1H), 8.65(s,
1H), 8.53 (s, 1H), 7.77 (s, 1H), 7.43 (br s, 1H), 7.18 (m, 1H), 3.73
(m, 1H), 3.49 (m, 2H), 3.40 (m, 2H), 3.32 (s, 3H), 2.09 (s, 3H), 1.54
(s, 9H).
1
following the general procedure C. H NMR (400 MHz, CD3OD) δ
8.02 (m, 1H), 7.94 (m, 1H), 7.86 (m, 1H), 7.64 (m, 2H), 7.58 (t, J =
8.0 Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 6.33 (m, 1H), 6.22 (m, 1H),
6.11 (m, 1H), 4.55 (m, 1H), 4.07 (m, 2H), 3.91 (m, 3H), 3.34 (s, 3H),
1.28 (d, J = 6.0 Hz, 6H). m/z: 486.3.
(7aR)-2-Imino-6-(3-isopropoxyphenyl)-3-methyl-7a-(4-(5-
(prop-1-yn-1-yl)pyridin-3-yl)thiophen-2-yl)hexahydro-1H-
pyrrolo[3,4-d]pyrimidin-4(4aH)-one (35). Compound 35 was
prepared from 25 in two steps following the general procedure C.
1H NMR (400 MHz, CD3OD) δ 8.87 (br s, 1H), 8.49 (s, 1H), 8.05 (s,
3-(5-((4aR,7aR)-6-Benzoyl-2-imino-3-methyl-4-oxooctahy-
dro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)-
benzonitrile (26). Compound 26 was prepared in two steps from 23
1
following the general procedure B. H NMR (400 MHz, CD3OD) δ
7.94 (m, 2H), 7.57 (m, 9H), 4.47 (m, 0.4H from one rotamer), 4.11
(m, 4.6H), 3.3 (m, 3H). m/z: 456.3.
2-Fluoro-5-(5-((4aR,7aR)-6-(3-fluorobenzoyl)-2-imino-3-
methyl-4-oxooctahydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)-
thiophen-3-yl)benzonitrile (27). Compound 27 was prepared from
1H), 7.72 (s, 1H), 7.09 (t, J = 8 Hz, 1H), 6.32 (m, 1H), 6.23 (m, 1H),
6.16 (m, 1H), 4.56 (m, 1H), 4.04 (m, 2H), 3.99 (m, 3H), 3.33 (s, 3H),
2.12 (s, 3H), 1.28 (d, J = 6.0 Hz, 6H). m/z: 500.3
1
24 in two steps following the general procedure B. H NMR (400
MHz, CD3OD)) δ 7.97 (m, 2H), 7.63 (m, 1H), 7.45 (m, 6H), 4.43−
3.84 (m, 5H), 3.24 (m, 3H). m/z: 492.3.
3-(5-((7aR)-2-Imino-3-methyl-4-oxo-6-(pyridin-2-yl)-
octahydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)-
benzonitrile (36. Compound 36 was prepared from 23 in two steps
(7aR)-6-(3-Fluorobenzoyl)-2-imino-3-methyl-7a-(4-(5-(prop-
1-yn-1-yl)pyridin-3-yl)thiophen-2-yl)hexahydro-1H-pyrrolo-
[3,4-d]pyrimidin-4(4aH)-one (28). Compound 28 was prepared
1
following the general procedure D. H NMR (400 MHz, CD3OD)
1
from 25 in two steps following the general procedure B. H NMR
8.06 (m, 3H), 7.97 (m, 1H), 7.94 (m, 1H), 7.71 (m, 1H), 7.67 (m,
1H), 7.59 (t, J = 7.8 Hz, 1H), 7.15 (m, 1H), 7.06 (t, J = 7.0 Hz, 1H),
4.48 (d, J = 11.0 Hz, 1H), 4.24 (m, 4H), 3.35 (s, 3H). m/z: 429.2.
3-(5-((7aR)-2-Imino-6-(3-methoxypyridin-2-yl)-3-methyl-4-
oxooctahydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-
yl)benzonitrile (37). Compound 37 was prepared from 23 in two
steps following the general procedure D. 1H NMR (400 MHz,
CD3OD) δ 8.04 (m, 1H), 7.96 (m, 1H), 7.92 (m, 1H), 7.62 (m, 5H),
(400 MHz, CD3OD)) 8.83 (br s, 1H), 8.79 (br s, 1H), 8.25 (br s,
0.5H), 8.20 (br s, 0.5H), 7.88 (br s, 0.5H), 7.84 (br s, 0.5H), 7.55−
7.25 (m, 5H), 4.50−4.05 (m, 5H), 3.30 (m, 3H), 2.16 (m 3H). m/z:
488.3.
(4aR,7aR)-2-(Trimethylsilyl)ethyl 7a-(5-Bromothiophen-2-
yl)-2-((tert-butoxycarbonyl)imino)-3-methyl-4-oxohexahydro-
1H-pyrrolo[3,4-d]pyrimidine-6(2H)-carboxylate (29). To a sol-
K
dx.doi.org/10.1021/jm301039c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
6.99 (m, 1H), 4.65 (d, J = 12 Hz, 1H), 4.42 (m, 3H), 4.1 (t, J = 8.7 Hz,
1H), 3.9 (s, 3H), 3.34 (s, 3H). m/z: 459.3.
AUTHOR INFORMATION
Corresponding Author
*Phone: 732-594-3369. Fax: 732-594-1185. E-mail: mihirbaran.
mandal@merck.com.
■
2-((7aR)-7a-(4-(3-Cyanophenyl)thiophen-2-yl)-2-imino-3-
methyl-4-oxohexahydro-1H-pyrrolo[3,4-d]pyrimidin-6(2H)-yl)-
nicotinonitrile (38). Compound 38 was prepared from 23 in two
steps following the general procedure D. 1H NMR (400 MHz,
CD3OD) δ 8.35 (m, 1H), 8.03 (m, 1H), 7.97 (m, 1H), 7.95 (m, 1H),
7.92 (m, 1H), 7.89 (m, 2H), 7.66 (m, 2H), 7.58 (m, 1H), 6.85 (m,
1H), 4.64 (d, J = 12 Hz, 1H), 4.38 (m, 3H), 4.14 (t, J = 8.42 Hz, 1H),
3.34 (s, 3H). m/z: 454.2
3-(5-((4aR,7aR)-6-(5-Fluoropyridin-2-yl)-2-imino-3-methyl-4-
oxooctahydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-
yl)benzonitrile (39). Compound 39 was prepared from 23 in two
steps following the general procedure D. 1H NMR (400 MHz,
CD3OD) δ 8.08 (m, 1H), 8.03 (m, 1H), 7.95 (m, 1H), 7.88 (m, 1H),
7.65 (m, 2H), 7.60 (m, 2H), 6.63 (d, J = 9.0 Hz, 1H), 4.32 (d, J = 11.0
Hz, 1H), 4.12 (m, 3.0 Hz), 3.98 (m, 1H), 3.34 (s, 3H). m/z: 447.2.
3-(5-((7aR)-2-Imino-6-(6-methoxypyridin-2-yl)-3-methyl-4-
oxooctahydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-
yl)benzonitrile (40). Compound 40 was prepared from 23 in two
steps following the general procedure D. 1H NMR (400 MHz,
CD3OD) δ 8.04 (m, 1H), 7.97 (m, 1H), 7.91 (m, 1H), 7.76−7.65 (m,
3H), 7.59 (t, J = 7.6 Hz, 1H), 6.33 (m, 2H), 4.4 (d, J = 11.7 Hz, 1H),
4.13 (m, 4H), 3.97 (s, 3H), 3.35 (s, 3H). m/z: 459.3.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank T.-M.Chan for his help in compound character-
ization, Drs. Jim Tata and Ann Weber for their strong support,
and Dr. Eric Gilbert for helpful feedback on the manuscript.
Use of the IMCA-CAT beamline 17-ID (or 17-BM) at the
Advanced Photon Source was supported by the companies of
the Industrial Macromolecular Crystallography Association
through a contract with Hauptman-Woodward Medical
Research Institute. Use of the Advanced Photon Source was
supported by the U.S. Department of Energy, Office of Science,
Office of Basic Energy Sciences, under Contract No. DE-AC02-
06CH11357.
ABBREVIATIONS USED
■
AUC, area under the curve; BACE, β-site APP-cleaving
enzyme; AD, Alzheimer's disease; Aβ, β-amyloid; SAR,
structure−activity relationship; CNS, central nervous system;
CSF, cerebrospinal fluid; Pgp, P-glycoprotein; HTS, high
throughput screening; ER, efflux ratio; LE, ligand efficiency; Kd,
dissociation constant; PK, pharmacokinetic; PD, pharmacody-
namic; hERG, the human ether-a-go-go-related gene; CYP,
̀
cytochrome P450; hPXR, human pregnane X receptor; EDC, 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
3-(5-((7aR)-2-Imino-3-methyl-4-oxo-6-(6-(trifluoromethyl)-
pyridin-2-yl)octahydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)-
thiophen-3-yl)benzonitrile (41). Compound 41 was prepared from
1
23 in two steps following the general procedure D. H NMR (400
MHz, CD3OD) δ 8.04 (m, 1H), 7.96 (m 1H), 7.88 (m, 1H), 7.74 (t, J
= 8.0 Hz, 1H), 7.66 (m, 2H), 7.58 ( t, J = 8.0 Hz, 1H), 7.05 (d, J = 7.3
Hz, 1H), 6.80 (d, J = 7.3 Hz, 1H), 4.42 (d, J = 12 Hz, 1H), 4.15 (m,
3H), 4.02 (m, 1H), 3.34 (s, 3H). m/z: 497.3.
3-(5-((7aR)-2-Imino-3-methyl-4-oxo-6-(pyrimidin-2-yl)-
octahydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)-
benzonitrile (42). Compound 42 was prepared from 23 in two steps
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1
following the general procedure D. H NMR (400 MHz, CD3OD) δ
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7.66 (m, 2H), 7.58 (t, J = 8.0 Hz, 1H), 4.45 (d, J = 12.0 Hz, 1H),
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yl)benzonitrile (45). Compound 45 was prepared from 23 in two
steps following the general procedure D. 1H NMR (400 MHz,
CD3OD) δ 8.31 (m, 2H), 8.01 (m, 1H), 7.95 (m, 1H), 7.87 (m, 1H),
7.65 (m, 2H), 7.57 (t, J = 7.7 Hz, 1H), 4.46 (d, J = 12.0 Hz, 1H),
4.25−4.03 (m, 4H), 3.34 (s, 3H), 2.18 (s, 3H). m/z: 444.2.
3-(5-((7aR)-6-(5-Fluoro-4-methoxypyrimidin-2-yl)-2-imino-3-
methyl-4-oxooctahydro-1H-pyrrolo[3,4-d]pyrimidin-7a-yl)-
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1
27 in two steps following the general procedure D. H NMR (400
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(s, 3H). m/z: 478.3.
L
dx.doi.org/10.1021/jm301039c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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