Synthesis of potent BACE-1 inhibitors incorporating a hydroxyethylene isostere as central core

Article abstract of DOI:10.1016/j.ejmech.2009.11.013

We herein describe the design and synthesis of a series of BACE-1 inhibitors incorporating a P1-substituted hydroxylethylene transition state isostere. The synthetic route starting from commercially available carbohydrates yielded a pivotal lactone intermediate with excellent stereochemical control which subsequently could be diversified at the P1-position. The final inhibitors were optimized using three different amines to provide the residues in the P2′-P3′ position and three different acids affording the residues in the P2-P3 position. In addition we report on the stereochemical preference of the P1′-methyl substituent in the synthesized inhibitors. All inhibitors were evaluated in an in vitro BACE-1 assay where the most potent inhibitor, 34-(R), exhibited a BACE-1 IC₅₀ value of 3.1 nM.

Full text of DOI:10.1016/j.ejmech.2009.11.013

European Journal of Medicinal Chemistry 45 (2010) 870–882
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of potent BACE-1 inhibitors incorporating a hydroxyethylene isostere
as central core
,
Fredrik Wångsell a c, Karin Gustafsson a, Ingemar Kvarnstro¨m a, Neera Borkakoti b, Michael Edlund b,
,
b
,
b,d,
*
Katarina Jansson b, Jimmy Lindberg b, Anders Hallberg c, Åsa Rosenquist a , Bertil Samuelsson
*
a Department of Chemistry, Linko¨ping University, SE-581 83 Linko¨ping, Sweden
b Medivir AB, Lunastigen 7, SE-141 44 Huddinge, Sweden
c Department of Organic Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
d Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
We herein describe the design and synthesis of a series of BACE-1 inhibitors incorporating a P1-
substituted hydroxylethylene transition state isostere. The synthetic route starting from commercially
available carbohydrates yielded a pivotal lactone intermediate with excellent stereochemical control
which subsequently could be diversified at the P1-position. The final inhibitors were optimized using
three different amines to provide the residues in the P20–P30 position and three different acids affording
the residues in the P2-P3 position. In addition we report on the stereochemical preference of the P10-
methyl substituent in the synthesized inhibitors. All inhibitors were evaluated in an in vitro BACE-1 assay
where the most potent inhibitor, 34-(R), exhibited a BACE-1 IC50 value of 3.1 nM.
Received 21 April 2009
Received in revised form
28 October 2009
Accepted 5 November 2009
Available online 12 November 2009
Keywords:
Alzheimer’s disease
BACE-1 inhibitors
Hydroxylethylene
Transition state isostere
Ó 2009 Published by Elsevier Masson SAS.
1. Introduction
found in the brain of AD patients. From the amyloid cascade
hypothesis, supported by genetic and pathological evidence, the
Alzheimer’s disease (AD) is a progressive neurodegenerative
disease of the brain and is the most common form of dementia
among the aging population in the world [1,2]. Symptoms of the
disease are behavioral disturbances and loss of cognitive function.
AD is the biggest unmet medicinal need in neurology with over 30
million people suffering from the disease worldwide with death
occurring, on average, nine years after diagnosis [2,3]. Current
available therapies only treat the symptoms and do not affect the
underlying progression of the disease [4]. AD is characterized by
the progressive formation of insoluble amyloid plaques and fibril-
lary tangles in the brain. Plaques, relatively specific for AD, are
primarily a result from extracellular accumulation of aggregated
formation of A
[6,9]. BACE-1 (
b
42 plays an early and vital role in all cases of AD
b
-site APP cleaving enzyme) was identified in 1999
independently by several groups [10–14] as a novel membrane-
bound aspartic protease belonging to the pepsin family. BACE-1 is
responsible for the N-terminal cleavage of APP leading to A
C-terminal intramembrane proteolysis of the formed fragment,
C99, by -secretase [15–17]. BACE-1 knockout homozygote mice
show complete absence of A production and the animals appear to
b after
g
b
develop normally and have no discernable abnormalities [18].
Tissue cultures and animal studies indicated that BACE-1 is
expressed in all tissues but at highest levels in the neurons in the
brain. Therefore, in vivo inhibition of BACE-1 is likely to reduce the
amyloid
proteolytic cleavage of the large transmembrane amyloid precursor
protein (APP) [5–8]. The A peptides ranges from 40 to 42 amino
acids in length where amyloid- 42 (A 42) dominates in the plaque
b
-peptide (A
b
). A
b
is a peptide fragment formed by
production of Ab and is considered to be an attractive therapeutic
target for the treatment and prevention of AD [19].
b
Analogues of peptides in which the scissile bond is replaced
with a transition state isostere have proven to be effective inhibi-
tors of this protease [20]. A key structural element in most transi-
tion state isosteres is a secondary hydroxyl moiety that interacts
with the catalytic aspartic acids side chains of BACE-1 via hydrogen
bonds.
b
b
* Corresponding authors. Medivir AB, Lunastigen 7, SE-141 44 Huddinge, Sweden.
Tel.: þ46 8 54683100; fax: þ46 8 54683199.
A number of hydroxyl containing BACE-1 inhibitors have been
reported[15,21–25] that contains e.g. statine [26], tert-hydroxy
E-mail addresses: asa.rosenquist@medivir.se (Å. Rosenquist), bertil.samuelsson@
medivir.se (B. Samuelsson).
0223-5234/$ – see front matter Ó 2009 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2009.11.013

F. Wångsell et al. / European Journal of Medicinal Chemistry 45 (2010) 870–882
871
Fig. 1. Examples of transition state isosteres with a hyrdoxylgroup incorporated as stable peptide bond replacement.
statine [27], hydroxyethylene [28] (HE) and hydroxyethylamine
[29] (HEA) as non-cleavable transition state isosteres (Fig. 1) and
crystal structures have been reported revealing that the hydroxyl
group of the inhibitors forms hydrogen bonds with the side chains
of Asp32 and Asp228 in the active site [30].
Previously reports have shown that a 10-fold increase in BACE-1
inhibitory activity can be achieved using an HE template over sta-
tine motifs [31]. Tang and co-workers have reported on an eight
residue peptidic transition state inhibitor, OM99-2, having an HE
central core displaying a BACE-1 IC50 value of 2 nM [28]. Using X-
ray crystal structure information a lower molecular weight inhib-
itor could be designed (I [30], Fig. 2) displaying an IC50 value of
2.5 nM. Recently a low nanomolar inhibitor II (Fig. 2) containing
a C-terminal truncated HEA moiety was disclosed by Stachel et al.
[29].
Previous work from our group has demonstrated that insertion
of an oxygen atom in the appropriate position of a peptidometic or
dipeptidometic structure not only simplified synthesis of diversi-
fied templates but also can provide increased potency against the
desired proteases [32]. Encouraged by these results and based on
molecular modeling we set out to explore the P1-position within
the HE motif to identify easily diversified central cores for future
use in the development of BACE inhibitors. In this paper we report
on the development of a synthetic route from commercially avail-
able carbohydrates to yield potent HE containing BACE-1 inhibitors
comprising O-benzyl- or O-phenyl-moieties in the tether.
a- and b-anomers of the methyl glycoside 2 in a ratio of 11:2 [37].
The hydroxyl group in 2 was then deoxygenated as described by
Barton[38,39] using thiocarbonyldiimidazole in refluxing THF
yielding the thiocarbonyl derivative 3 (99% yield) which was
subsequently reduced using tributyl tin hydride in refluxing
toluene rendering the methyl glycosides 4 [40] in 72% yield.
Oxidation of the glycoside 4 (anomeric mixture) applying the
procedure reported by Grieco [41] using m-chloroperbenzoic acid
in methylene chloride at 0 ꢀC in the presence of boron trifluoride
etherate gave the corresponding g-lactone 5 in quantitative yield.
Stereoselective methylation at the C2 position was achieved by
treating
g
-lactone 5 with LDA for 30 min at ꢁ78 ꢀC followed by
addition of methyl iodide [42] furnishing the desired alkylated
lactone 6 in 66% yield. Only one epimer of compound 6 was
observed after column chromatographic purification, probably due
to steric hindrance from the bulky benzyloxy groups at C5 and C6.
Catalytic hydrogenolysis of compound 6 gave the diol 7 in almost
quantitative yield. At this stage the stereochemical assignment was
confirmed by NOESYexperiments on 7 [43]. The NOESY spectrum of
a
the diol 7 affirmed that the hydrogen H3 was in proximity to the
hydrogen atoms in both the methyl group and H4 (Fig. 3).
Furthermore H3b was spatially correlated with H2, which indicated
that the methyl group at C2 in 7 has the desired (R) configuration
[43].
Synthesis of final products was performed according to two
similar protocols starting from the diol 7 furnishing compounds
with either O-benzyl- or O-phenyl-moieties in the C6 position
(Scheme 2). For the synthesis of the O-benzyl substituted deriva-
tives regioselective O-alkylation of the primary hydroxyl group in
compound 7 was achieved by reacting the diol with dibutyltin
oxide in toluene to form the corresponding tin acetal, which was
allowed to react with 3,5-difluorobenzylbromide or benzylbromide
in the presence of tetrabutylammonium bromide to afford the
desired 6-O-benzylated compounds 8 and 9 in 81% and 79% yield
[44]. Compounds 8 and 9 were then converted to their corre-
sponding azides 10 and 11 with inversion of the configuration at C5
2. Result and discussion
2.1. Chemistry
The synthesis of the pivotal intermediate 7 is outlined in
Scheme 1. Starting from commercially available 1,2:5,6-di-O-iso-
propylidene-a-D-glucofuranose compound 1 was generated in an
overall yield of 73% over four steps [33–36]. The isopropylidene
group in 1 was removed under acidic conditions to give a mixture of
Fig. 2. Inhibitors from the literature containing the HE-(I), and the HEA-isostere (II).

872
F. Wångsell et al. / European Journal of Medicinal Chemistry 45 (2010) 870–882
Scheme 1. Reagents and conditions. (a) H2SO4, MeOH, 0 ꢀC; (b) thiocarbonyldiimidazole, THF, reflux; (c) tributyl tin hydride, toluene, reflux; (d) m-CPBA, BF3OEt, DCM; (e) LDA, THF,
ꢁ78 ꢀC; (f) MeI; (g) Pd/C, H2, EtOH (95%).
using Mitsunobu conditions with DIAD, PPh3, and DPPA in THF in
93% and 90% yield [45].
or
5-(methanesulfonyl-methyl-amino)-N-methyl-isophthalamic
acid (F), in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N’,N0-
tetramethyluronium hexafluorophosphate (HATU) and DIPEA in
DMF to yield the final products 28–35 in 17–77% yield. The acid E
was synthesized from commercially available amino acids (Boc–
Val–OH and Met–OMe) and the acids D and F were synthesized
according to previously reported literature procedure [29,43,49].
For the synthesis of the O-phenyl substituted derivatives the
diol 7 was reacted with DIAD and PPh3 in refluxing chloroform to
provide the epoxide 12 in 63% yield [37,38]. Regioselective opening
of the epoxide 12 at the least hindered position by 3,5-difluor-
ophenol in warm DMF in the presence of potassium carbonate
furnished mixture 13 in 91% yield [45]. This clean reaction was
however accompanied by epimerization at the C2 position of 13.
Due to difficulties in separation at this stage, using either flash
column chromatography or HPLC, the diastereomeric mixture was
taken to the next reaction step where separation could be achieved.
The mixture 13 was thus converted to the corresponding azides
with inversion of configuration at C5 under Mitsunobu-like
conditions, vide supra, to yield the separable diastereomers 14-(R)
and 15-(S) in 32% and 42% yield [45]. To corroborate the assigned
stereochemistry 1H–1H-NOESY experiments were performed on
both 14-(R) and 15-(S). The NOESY spectrum of 14-(R) revealed that
H4 is spatially correlated with the methyl hydrogen at C2, which is
consistent with the (R) configuration of the methyl group in the
lactone 14-(R) (Fig. 4). In the NOESY spectrum of 15-(S), H4 is
correlated with H2, which indicates that the lactone 15-(S) has the
presumed (S) configuration [43].
The lactones 10, 11, 14-(R), and 15-(S) were then further reacted
with a selected set of amines (A–C) and carboxylic acid derivatives
(D–F). Two different protocols were used for the conversion of the
lactones to the amides 16–21. For the synthesis of 16 and 18–21 the
lactones 10, 11, 14-(R) and 15-(S) were ring opened with (S)-2-
amino-N-benzyl-3-methyl-butyramide (A) or 4-fluorobenzylamine
(C) in the presence of diisopropylethylamine (DIPEA) and 2-
hydroxypyridine in refluxing THF to give amides 16 and 18–21 in
36–88% yield [46]. For the synthesis of 17 a protocol described by
Weinreb et al. was used [47]. Reacting trimethylaluminium with
cyclopropylamine (B) in methylene chloride afforded the corre-
sponding dimethylaluminium amide in situ. The lactone 10 was
then added to the solution to furnish amide 17 in 53% yield. The
amine A was synthesized from Boc–Val–OH and benzylamine
whereas the amines B and C were commercially available [30,43].
Reduction of the azide group in 16–21 with PPh3 in methanol con-
taining a few drops of water provided the corresponding amines 22–
27 in 62–100% yield [48]. These amines, 22–27, were then subjected
to standard peptide coupling chemistry with three different
carboxylic acids, (S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-
butyrylamino)-4-methylsulfanyl-butyric acid (E), 5-(methanesul-
2.2. Biological data and structure activity relationships
All synthesized inhibitors were screened against BACE-1 to
determine IC50 values (Table 1) [32]. For the most potent inhibitors
the BACE cellular activities were determined measuring production
of secreted soluble Ab-40 in cultured HEK-293 cells (Table 2). In
parallel the inhibitors were screened for inhibition of the anti-
target human cathepsin D [32].
All BACE-1 inhibitors, vide infra, contain the novel HE moiety
containing a methyl substituent in the P10-position previously
reported to be the preferred side chain [31]. To optimize the central
core, Val–benzylamide (A) were incorporated as P20–P30 groups
and combined with a selected set of P2–P3 substituents (Table 1).
The first inhibitor, 33, carrying benzyloxymethyl as P1-substituent
furnished a BACE-1 IC50 value of 3.6 nM and a cathepsin D IC50
value of 41 nM. When the benzyloxymethyl group in the P1-posi-
tion was replaced with a difluorobenzyloxymethyl group as in 28
the BACE-1 IC50 value increases marginally from 3.6 nM to 6.6 nM.
A small improvement in selectivity over cathepsin D was observed,
i.e. 33 and 28 furnishing cathepsin D IC50 values of 41 and 210 nM.
Contraction of the P1-substituent with one carbon to a difluor-
ophenyloxymethyl group as in 34-(R) gave a small improvement in
BACE-1 activity, displaying an IC50 value of 3.1 nM.
In addition we studied the effect of the stereochemistry of the
substituent at the P10-position. Whilst the R stereochemistry of the
methyl group in the P10-position of 34-(R) is more potent than
diastereomer 35-(S) the potency loss of 35-(S) is quite modest, IC50
fonyl-methyl-amino)-N-((R)-1-phenyl-ethyl)-isophthalamide
(D)
Fig. 3. Relevant NOE correlations in the diol 7.

Scheme 2. Reagents and conditions. (a) Bu2SnO, toluene, reflux; (b) tetrabutylammonium bromide, benzylbromide or 3,5-difluorobenzylbromide, toluene, 90 ꢀC; (c) PPh3, DIAD,
DPPA, THF; (d) DIAD, PPh3, CHCl3, reflux; (e) 3,5-difluorophenol, K2CO3, DMF, 110 ꢀC; (f) R2NH2 (A or C), DIPEA, 2-hydroxypyridine, THF (dry), reflux; (g) R2NH2 (B), Me3Al, DCM; (h)
PPh3, MeOH, H2O; (i) R3COOH (D, E or F), DIPEA, HATU, DMF.
values of 3.1 nM and 10 nM for 34-(R) and 35-(S). This indicates that
the S10-pocket could be further explored for improvements in both
potency and selectivity.
In the exploration of this HE moiety we have evaluated
a selected set of P2–P3 groups and modifications in the P20-posi-
tion. Introducing the Met–Val [50] (E) moiety in the P2–P3 posi-
tion, 29, surprisingly resulted in
a BACE-1 IC50 of 10 mM.
Interestingly, 29 is a highly potent cathepsin D inhibitor with an
IC50 value of 0.63 nM. Removal of the P3 methylbenzylamine
group of inhibitor 28 furnishing the truncated compound 30
resulted in loss of activity and a BACE-1 IC50 value of 1260 nM
compared to 6.6 nM for 28, validating that the P3 methylbenzyl-
amine substituent is essential for activity. When replacing the Val–
benzylamide (A) in the P20–P30 position with cyclopropylamine (B)
Fig. 4. Relevant NOE correlations in compound 14-(R) and 15-(S).

Table 1
BACE-1 and cathepsin D enzyme data.
Compound
Structure
IC50 (nM) BACE-1
Ki (nM) Cat D
33
3.6
41
28
6.6
210
34-(R)
35-(S)
29
3.1
59
120
0.63
10
10 000
30
1260
>5000
31
>10 000
>5000

F. Wångsell et al. / European Journal of Medicinal Chemistry 45 (2010) 870–882
875
Table 1 (continued)
Compound
Structure
IC50 (nM) BACE-1
Ki (nM) Cat D
32
3600
>5000
or 4-fluorobenzylamine (C), important interactions with BACE-1
were lost, delivering 31 and 32 displaying BACE-1 IC50 values of
>10 mM and 3.6 mM.
Some BACE-1 inhibitors were evaluated for their activity in a cell
culture assay. Whereas the inhibitors showed good inhibition of
BACE-1 in cell-free systems, they all lost considerable activity in the
cell-based assay (Table 2). Most promising was inhibitor 34-(R)
furnishing a BACE-1 IC50 value of 3.1 nM and an IC50 value of
160 nM in the cell-based assay.
providing an X-ray crystal structure determined to 2.8 Å resolution
(PDB-code: 3IXK, Fig. 5) [32]. Structural analysis reveals a compact
binding including numerous hydrogen bonds that coordinates
inhibitor 28 with the BACE-1 enzyme active site. The hydroxyl
group of the hydroxyethylene transition state isostere in the
inhibitor is positioned between the two catalytic residues Asp32
and Asp228, forming a hydrogen bond network. Furthermore the
inhibitor makes several hydrogen bonds to the backbone of BACE-1.
These bonds are; P3 NH to Gly230, P3 carbonyl to Thr232, P2
carbonyl to the backbone of the flap residue Gln73, P1 NH to
Gly230, P10 carbonyl to the flap residue Thr72, P20 NH to Gly34, P20
carbonyl to the side chain hydroxyl of Tyr198 and the P30 NH to
Pro70. The sulphonamide of the P2 substituent makes hydrogen
bond interactions with the backbone NH of Thr232 and Asn233 and
with the side chain of Arg235.
2.3. X-ray crystal structure results
To characterize the binding mode of these novel BACE-1 inhib-
itors compound 28 was co-crystallized with human BACE-1
Table 2
Cellular inhibition data.
The P20 valine side chain interacts mainly with Ser35, Val69,
Ile126, Arg128 in the S20 pocket and the P30 capping benzyl is
binding on top of the flap and interacts with Pro70, Tyr71 and
Thr72.
Compound Structure
Cell IC50
(HEK-293) (nM)
Hydrophobic interactions are additionally an important
component of the potency of 28. The P3 capping phenyl group
makes close interactions with several residues in the S3 sub pocket.
It is stacked between Thr232 and Gly13 from top to bottom and has
edge on close contact interactions with Gly11, Tyr14, Ser229,
Gly230, and Arg307. The P3 methyl group has close contact inter-
actions with Gln12, Gly13, Leu30, Ile110 and Trp115. The aromatic
ring of P2 is stacked between Thr231 and with the side chain of the
flap residue Gln73.
33
1000
The aromatic moiety of the P1 side chain in 28 is surrounded by
both aromatic and hydrophobic residues in the S1 pocket, notably
Phe108, Ile110, Trp115 and the flap residue Tyr71. Additionally
there is a close contact interaction between one of the P1 meta-
28
4500
34-(R)
160
35-(S)
340
Fig. 5. X-ray crystal structure of compound 28 in the active site of BACE-1.

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F. Wångsell et al. / European Journal of Medicinal Chemistry 45 (2010) 870–882
fluoro substituents with the backbone amide of Gln73 and Gly74 of
the flap inducing an amide flip whereby these more extended P1
side chain of e.g. inhibitor 28 also can be accomodated into the S1
pocket of BACE-1.
CDCl3 or methanol-d4 (CD3OD) with TMS as an internal standard.
NOESY spectra that were used to determine the structure of
compound 7, 14-(R) and 15-(S) were recorded on Varian 500
instrument using CDCl3 or CD3OD with TMS as an internal standard.
In the recorded NMR spectra of the diastereomeric mixture 13 the
presumed diastereomeric peaks were put into brackets with the
general formula [nn.n & nn.n] for 13C NMR and [n.nn & n.nn, (x, yH)]
for 1H NMR. TLC was carried out on Merck precoated 60 F254 plates
using UV-light and charring with ethanol/sulfuric acid/acetic acid/
p-aninsaldehyde (90:3:1:2) or a solution of 0.5% ninhydrin in
ethanol (95%) for visualization. Flash column chromatography was
performed using silica gel 60 (0.040–0.063 mm, Merck). Prepara-
tive isocratic HPLC was performed on a Gynkotek (pump: P580,
detector: UVD 170S, software: Chromeleon) using a Kromasil 100-
10-C18 (250 ꢂ 20 mm) column. Mixtures of deionized water and
methanol with 0.1% TFA or 0.1% TEA were used as mobile phase.
Gradient LC/MS was performed on a Gilson system (Column: Phe-
nomenex C18 250 ꢂ 15 mm for preparative and Phenomenex C18
150 ꢂ 4.6 mm for analytical runs; Pump: Gilson gradient pump
322; UV/VIS-detector: Gilson 155; MS detector: Thermo Finnigan
Surveyor MSQ; Gilson Fraction Collector FC204, software: Gilson
UniPoint ver. 4.0 and Xcalibur ver. 1.3) using methanol with 0.1%
formic acid and deionized water with 0.1% formic acid as mobile
phase. Optical rotations were measured using a Perkin–Elmer 141
polariometer. Concentrations were performed in vacuo below
40 ꢀC. All low degree temperature reactions were accomplished by
submerging the reaction vessels into an ethanol bath that had been
cooled by additions of liquid nitrogen. All degassing of solvents
were achieved using ultra sonification (Ultrasonik 104ꢂ) for at least
1 h. Drying of solvents: THF was refluxed over sodium/benzophe-
none and distilled onto 4 Å MS, toluene and dichloromethane was
refluxed over calcium hydride and distilled onto 4 Å MS. Organic
extracts were dried over magnesium sulfate monohydrate and
filtered. Filtrating was achieved using filter paper Munktell, OOH
quality. Combustion analysis was performed by Analytische Labo-
ratorien, Lindlar, Germany.
3. Conclusion
We have developed a promising class of BACE-1 inhibitors
comprising the HE as transition state isostere with different
extensions in the P1-position readily available from an inexpensive
carbohydrate starting material. The synthetic route yielded the
pivotal intermediate 7 with excellent stereochemical control which
was corroborated by NOESY experiments. Variations in the P1-
position with three different substituents were evaluated and
revealed that the 3,5-difluorophenyl moiety was the most suitable
in combination with this HE isostere. The two stereochemical
configurations at the methyl substituent in the P10-position were
investigated and from the BACE-1 inhibitory data the (R) stereo-
chemistry of the methyl group is preferred. Furthermore, the HE
isostere was evaluated together with different P2–P3 and P20–P30
substituents selected from the literature based on good inhibitory
properties for other transition state isosteres. The most potent
inhibitor 34-(R) exhibits a promising BACE-1 IC50 value of 3.1 nM
(Fig. 6). The described inhibitors provides a novel and easily
diversified HE central core that can be further used use for the
development BACE-1 inhibitors as well as inhibitors of other
aspartic proteases.
4. Experimental section
4.1. A-beta cell-based assay
Human embryonic kidney 293 cells (HEK-293) stably expressing
Swedish mutant APP (swAPP751)[51] were cultured in DMEM
supplemented with 10% FCS, PEST (50 U penicillin/50
m
g/mL
streptomycin) and 200
m
g Hygromycin/mL at 37 ꢀC, 5% CO2. Cells
were seeded at a density of 2 ꢂ 104 cells/well into 96-well plates
and left overnight to settle. Thereafter cells were washed with fresh
media once to reduce background. Inhibitor was added (max 1%
DMSO final conc.). After 24 h cell culture media was harvested and
4.2.1. 5,6-Di-O-benzyl-3-deoxy-1,2-O-isopropylidene-a-D-
glucofuranoside (1)
Compound 1 was synthesized from commercially available
1,2:5,6-di-O-isopropylidene- -glucofuranose in four-step
a
-D
a
analysed by
Ab 1–40 ELISA according to the manufacturer
protocol according to literature procedure in an overall yield of 73%
[33–36].
instruction (The Genetics Company, Schwitzerland). The results
were treated with the curve fitting package in GraphPad Prism 5.
4.2.2. Methyl 5,6-di-O-benzyl-3-deoxy-
a
-
D
-glucofuranoside (2
a)
4.2. General methods
and methyl 5,6-di-O-benzyl-3-deoxy-
b-
D-glucofuranoside (2
b)
Compound 1 (6.21 g, 16.2 mmol) was dissolved in methanol and
cooled in an ice bath (0 ꢀC) and conc. H2SO4 (2.69 mL, 48.5 mmol)
was slowly added to the solution. The reaction mixture was stirred
overnight at room temperature and then neutralized with NaHCO3
and concentrated. The residue was extracted with ethyl acetate
(3 ꢂ 50 mL) and H2O (50 mL). The combined organic layers were
dried, filtered and concentrated. Purification by flash column
All chemicals were purchased from commercial suppliers and
used directly without further purification. Standard 1H NMR and
13C NMR spectra were recorded on a Varian 300 instrument using
chromatography (toluene/ethyl acetate 3:1) yielded the
anomers in an 11:2 ratio (2 :2 ) as transparent oils (4.69 g, 80% and
0.84 g, 14% for the - and -anomers). 2
1H NMR (300 MHz,
CDCl3):
a- and b-
a
b
a
b
a:
d
2.0 (dd, J ¼ 6.6, 13.7 Hz, 1H), 2.14 (dq, J ¼ 4.7, 13.5 Hz, 1H),
3.30 (s, 3H), 3.57–3.69 (m, 2H), 3.80 (dd, J ¼ 2.7, 10.2 Hz, 1H), 4.23 (t,
J ¼ 4.9 Hz, 1H), 4.39–4.47 (m, 1H), 4.53–4.66 (m, 3H), 4.75–4.81 (m,
2H), 7.22–7.38 (m, 10H); 13C NMR (75.5 MHz, CDCl3):
d 35.5, 54.9,
71.3, 73.2, 73.7, 76.0, 79.0, 81.8,109.8,127.8,127.9,128.1, 128.6, 128.7,
138.7, 139.0. 2 1.76–1.88 (m, 1H),
b:
1H NMR (300 MHz, CDCl3):
d
2.20–2.30 (m, 1H), 2.40 (d, J ¼ 11.8 Hz, 1H), 3.47 (s, 3H), 3.56 (d,
J ¼ 5.5 Hz, 1H), 3.68–3.75 (m, 1H), 4.20–4.30 (m, 1H), 4.31–4.40 (m,
1H), 4.54 (s, 2H), 4.57–4.64 (m, 1H), 4.70 (d, J ¼ 17.9 Hz, 1H), 4.75–
Fig. 6. BACE inhibition value for the most potent inhibitor, compound 34-(R) from this
series.

F. Wångsell et al. / European Journal of Medicinal Chemistry 45 (2010) 870–882
877
4.85 (m, 1H), 7.23–7.39 (m, 10H); 13C NMR (75.5 MHz, CDCl3):
32.8, 55.2, 70.4, 72.0, 73.3, 73.4, 76.0, 79.6, 102.5, 127.3, 127.5,
127.6, 127.7, 128.2, 128.3, 128.4, 129.0, 138.1, 138.7.
(4 mL). The mixture was diluted with H2O (50 mL) and extracted with
ethyl acetate (3 ꢂ 50 mL). The combined organic layers were dried,
filtered and concentrated. Purification by flash column chromatog-
raphy (toluene/ethyl acetate 18:1) gave compound 6 (899 mg, 66%) as
d
4.2.3. Methyl 5,6-di-O-benzyl-3-deoxy-2-O-
an pale yellow oil. 1H NMR (300 MHz, CDCl3):
d
1.23 (d, J ¼ 7.42, 3H),
(imidazolethiocarbonyl)-
D-glucofuranoside (3)
1.78–1.91 (m,1H), 2.43–2.53 (m,1H), 2.66–2.81(m,1H), 3.53–3.65 (m,
2H), 3.81–3.90 (m, 1H), 4.47–4.73 (m, 5H), 7.23–7.42 (m, 10H); 13C
NMR (75.5 MHz, CDCl3): d 16.2, 30.4, 34.0, 68.9, 73.4, 73.5, 77.5, 78.5,
127.6, 127.7, 127.8, 127.9, 128.3, 128.4, 137.7, 137.8, 180.3. MS m/z 341.4
[(M þ H)þ calcd for C21H25Oþ4 341.17].
The alcohol 2 ( - and
a
b
-anomer mixture) (2.24 g, 6.25 mmol)
and 1,10-thiocarbonyldiimidazole (1.67 g, 9.37 mmol) were dis-
solved in THF (31 mL) and the mixture was refluxed overnight. The
crude residue was concentrated and purified by flash column
chromatography (toluene/ethyl acetate 3:1) to yield compound 3
(2.92 g, 99%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3):
d
2.29–
4.2.7. 2,3-Dideoxy-2-methyl-D-glucono-1,4-lactone (7)
2.50 (m, 2H), 3.37 (s, 3H), 3.61–3.73 (m, 2H), 3.76–3.84 (m, 1H),
4.43–4.53 (m, 1H), 4.60 (dd J ¼ 7.4, 12.1 Hz, 2H), 4.64 (d, J ¼ 11.5 Hz,
1H), 4.80 (d, J ¼ 11.5 Hz, 1H), 5.10 (s, 1H), 5.74 (d, J ¼ 4.1 Hz, 1H),
7.05–7.07 (m, 1H), 7.27–7.39 (m, 10H), 7.60–7.62 (m, 1H), 8.33–8.34
Compound 6 (174 mg, 0.51 mmol) was dissolved in ethanol
(2.5 mL, 95%) and Pd-C (w5 mg) was added. The reaction was per-
formed under H2-atmosphere, which was refilled until the reaction
was complete. The mixture was filtered though a pad of celite and
concentrated. This gave after co-concentration with methanol and
toluene the diol 7 (83 mg, quant.) as white crystals. 1H NMR
(m, 1H); 13C NMR (75.5 MHz, CDCl3):
d 32.1, 54.9, 70.1, 72.7, 73.4,
79.2, 80.8, 86.1, 106.0, 117.7, 127.7, 127.5, 127.6, 128.2, 128.3, 130.9,
136.7, 138.0, 138.3, 183.0. MS m/z 469.3 [(M þ H)þ calcd for
C25H29N2O5Sþ 469.18].
(300 MHz, CD3OD):
d
1.23 (d, J ¼ 7.4 Hz, 3H),1.85–1.98 (m,1H), 2.45–
2.57 (m, 1H), 2.72–2.86 (m, 1H), 3.52–3.59 (m, 2H), 3.74–3.84 (m,
1H), 4.53–4.61 (m, 1H); 13C NMR (75.5 MHz, CD3OD):
d 15.3, 30.0,
4.2.4. Methyl 5,6-di-O-benzyl-2,3-dideoxy-D-glucofuranoside (4)
34.3, 62.7, 72.4, 78.7,181.8. HRMS m/z 183.0631 [(M þ Na)þ calcd for
Tributyl tin hydride (5.18 g, 17.79 mmol) was dissolved in dry
toluene (35 mL) under N2-atmosphere and refluxed for 5 min.
Compound 3 (5.56 g, 11.86 mmol) dissolved in dry toluene (35 mL)
was added drop wise to the solution during 30 min. The combined
solution was stirred at 110 ꢀC and after 2 h the mixture was
concentrated. Purification by flash column chromatography
(toluene/ethyl acetate 18:1) yielded compound 4 (2.94 g, 72%) as
C7H12NaOþ4 183.0628].
4.2.8. 2,3-Dideoxy-6-O-(3,5-difluorobenzyl)-2-methyl-D-glucono-
1,4-lactone (8)
The diol 7 (201 mg, 1.25 mmol) and dibutyltin oxide (405 mg,
1.63 mmol) was dissolved in toluene (7 mL). The mixture was
refluxed for 5 h before the temperature was lowered to 90 ꢀC and
tetrabutylammonium bromide (464 mg, 1.44 mmol) and 3,5-
difluorobenzylbromide (0.186 mL, 1.44 mmol) were added. The
mixture was allowed to stir at 90 ꢀC overnight and thereafter
concentrated. Purification by flash column chromatography
(toluene/ethyl acetate 6:1) gave compound 8 (291 mg, 81%) as
colourless semi-crystals. 1H NMR (300 MHz, CD3OD):
d 1.87–1.98
(m, 4H), 3.25 (s, 3H), 3.55–3.66 (m, 2H), 3.81 (dd, J ¼ 2.2, 9.9 Hz,
1H), 4.10 (dt, J ¼ 6.5, 8.2 Hz, 1H), 4.45 (s, 1H), 4.61 (d, J ¼ 11.6 Hz,
1H), 4.74 (d, J ¼ 11.6 Hz, 1H), 4.80 (s, 1H), 4.92 (t, J ¼ 2.4 Hz, 1H),
7.24–7.38 (m, 10H); 13C NMR (75.5 MHz, CD3OD):
d 27.4, 33.5, 55.0,
71.8, 73.8, 74.4, 80.7, 82.7, 106.7, 128.6, 128.7, 128.8, 129.0, 129.2,
129.3, 139.8, 140.0. MS m/z 365.4 [(M þ Na)þ calcd for C21H26NaOþ4
365.17].
a colourless oil. 1H NMR (300 MHz, CD3OD):
d
1.22 (d, J ¼ 7.3 Hz,
3H), 1.92 (dt, J ¼ 8.6,13.0 Hz,1H), 2.52 (ddd, J ¼ 3.6, 9.5, 13.0 Hz, 1H),
2.79 (ddq, J ¼ 7.3, 8.6, 9.5 Hz, 1H), 3.54 (dd, J ¼ 5.7, 10.8 Hz, 1H), 3.58
(dd, J ¼ 5.0, 10.8 Hz, 1H), 3.95 (dt, J ¼ 4.7, 5.7 Hz, 1H), 4.55 (s, 2H),
4.59 (ddd, J ¼ 3.6, 4.7, 8.6 Hz, 1H), 6.78–6.86 (m, 1H), 6.92–7.00 (m,
4.2.5. 5,6-Di-O-benzyl-2,3-dideoxy-D-glucono-1,4-lactone (5)
The methyl-acetal 4 (2.79 g, 8.16 mmol) was dissolved in dry
CH2Cl2 (50 mL) and cooled to 0 ꢀC in an ice bath. BF3OEt (0.52 mL,
2.04 mmol) and m-chloroperbenzoic acid (2.20 g, 9.79 mmol) were
added to the solution and the mixture was kept at 0 ꢀC for 2 h
before it was allowed to reach room temperature. After 4 h the
mixture was concentrated and extracted with ethyl acetate
(3 ꢂ 50 mL) and saturated NaHCO3 (50 mL). The combined organic
layers were dried, filtered and concentrated. The crude residue was
purified by flash column chromatography (toluene/ethyl acetate
18:1) to yield the lactone 5 (2.66 g, quant.) as white crystals. 1H
2H); 13C NMR (75.5 MHz, CD3OD):
d 16.4, 31.3, 35.3, 71.9, 72.5, 72.9,
79.7, 103.4 (t, JCF ¼ 25.8 Hz), 110.9 (d, JCF ¼ 25.5 Hz, 2C), 144.4 (t,
JCF ¼ 8.9 Hz),164.3 (d, JCF ¼ 247.5 Hz),164.5 (d, JCF ¼ 247.5 Hz),182.8.
MS m/z 287.2 [(M þ H)þ calcd for C14H17F2Oþ4 287.11].
4.2.9. 2,3-Dideoxy-6-O-benzyl-2-methyl-D-glucono-1,4-lactone (9)
Compound 9 (413 mg, 79%) was synthesized from 7 (336 mg,
2.09 mmol) according tothemethod of thepreparation of8, with the
exception that benzylbromide was used instead of 3,5-difluor-
obenzylbromide. After purification compound 9 was collected as
NMR (300 MHz, CD3OD):
d
2.14–2.25 (m, 2H), 2.44–2.50 (m, 2H),
a colourless oil.1H NMR (300 MHz, CDCl3):
d
1.23 (d, J ¼ 7.28 Hz, 3H),
3.60 (d, J ¼ 5.3 Hz, 2H), 3.86 (dt, J ¼ 3.6, 5.3 Hz, 1H), 4.49 (d,
J ¼ 12.0 Hz, 1H), 4.53 (d, J ¼ 12.0 Hz, 1H), 4.56 (d, J ¼ 11.5 Hz, 1H),
4.67 (d, J ¼ 11.5 Hz, 1H), 4.68–4.75 (m, 1H), 7.25–7.34 (m, 10H); 13C
1.86 (dt, J ¼ 8.3,13.0 Hz,1H), 2.49 (ddd, J ¼ 3.9, 9.4,13.0 Hz,1H), 2.72
(ddq, J ¼ 7.3, 8.5, 9.4, 1H), 2.94 (bs, 1H), 3.51 (dd, J ¼ 5.2, 9.8 Hz, 1H),
3.58 (dd, J ¼ 4.5, 9.8 Hz, 1H), 3.90 (dt, J ¼ 4.5, 5.6 Hz, 1H), 4.47 (ddd,
J ¼ 3.9, 5.6, 8.3 Hz, 1H), 4.51 (d, J ¼ 11.8 Hz, 1H), 4.56 (d, J ¼ 11.8 Hz,
NMR (75.5 MHz, CD3OD):
d 23.2, 29.3, 69.9, 74.3, 74.5, 80.0, 81.9,
128.7, 128.8, 128.9, 129.0, 129.3, 129.4, 139.4, 139.6, 180.2. MS m/z
1H), 7.28–7.38 (m, 5H); 13C NMR (75.5 MHz, CDCl3):
d 16.0, 30.6, 33.7,
327.3 [(M þ H)þ calcd for C20H23Oþ4 327.39].
70.5, 70.7, 73.4, 77.5, 127.7, 127.8, 128.4, 137.5, 180.3.
4.2.6. 5,6-Di-O-benzyl-2,3-dideoxy-2-methyl-D-glucono-1,4-
4.2.10. 5-Azido-2,3,5-trideoxy-6-O-(3,5-difluorobenzyl)-2-methyl-
lactone (6)
L
-iodono-1,4-lactone (10)
Thelactone 5 (1.31 g, 4.01 mmol) wasdissolvedindry THF(40 mL)
and cooled to ꢁ78 ꢀC. After 15 min a solution of 2.0 M LDA (2.47 mL,
4.01 mmol) was added drop wise. After 30 min at ꢁ78 ꢀC methyl
iodide (2.5 mL, 40.1 mmol) dissolved in dry THF (5 mL) was slowly
added. After further 2 h at ꢁ78 ꢀC the reaction was allowed to attain
roomtemperature and quenched withsaturatedammonium chloride
Compound 8 (150 mg, 0.53 mmol) was dissolved in dry THF
(5 mL) and cooled to ꢁ15 ꢀC (ice/acetone 1:1). Triphenyl phosphine
(207 mg, 0.79 mmol) and diisopropyl azodicarboxylate (0.156 mL,
0.79 mmol) were added. After 10 min the mixture was allowed to
reach 0 ꢀC and diphenylphosphoryl azide (217 mg, 0.79 mmol) was
added. After 30 min of stirring at 0 ꢀC the mixture was allowed to

878
F. Wångsell et al. / European Journal of Medicinal Chemistry 45 (2010) 870–882
attain room temperature and was stirred overnight. The solvent
was evaporated, and the crude mixture was purified by flash
column chromatography (toluene/ethyl acetate 18:1) yielding the
azide 10 (152 mg, 93%) as a colourless oil. 1H NMR (300 MHz,
temperature was raised to 0 ꢀC and diphenylphosphoryl azide
(142 L, 0.64 mmol) was added. Thereaction mixturewas allowed to
attain room temperature and was stirred overnight. The solvent was
evaporated and the crude diastereomeric mixture was purified by
flash column chromatography (toluene/ethyl acetate 18:1). The two
diastereomers 14-(R) (40 mg, 32%) and 15-(S) (53 mg, 42%) were
separated and collected as transparent oils. 14-(R): 1H NMR
m
CD3OD):
d
1.22 (d, J ¼ 7.4 Hz, 3H), 2.05 (dt, J ¼ 8.4, 13.2 Hz, 1H), 2.42
(ddd, J ¼ 4.1, 9.6, 13.2 Hz, 1H), 2.83 (ddq, J ¼ 7.4, 8.4, 9.6 Hz, 1H), 3.73
(dd, J ¼ 8.0, 9.9 Hz, 1H), 3.79 (dd, J ¼ 4.0, 9.9 Hz, 1H), 3.80–3.88 (m,
1H), 4.58 (s, 2H), 4.65 (dt, J ¼ 4.1, 8.4 Hz, 1H), 6.77–6.86 (m, 1H),
6.91–6.99 (m, 2H); 13C NMR (75.5 MHz, CD3OD): 16.4, 33.5, 34.9,
65.4, 71.6, 72.8, 72.9, 103.6 (t, JCF ¼ 25.8 Hz), 110.9 (d, JCF ¼ 25.2 Hz,
2C), 143.9 (t, JCF ¼ 8.9 Hz), 164.4 (d, JCF ¼ 247.2 Hz), 164.6 (d,
JCF ¼ 247.2 Hz), 182.0.
(300 MHz, CDCl3):
d
1.29 (d, J ¼ 7.1 Hz, 3H), 2.02–2.16 (m, 1H), 2.40–
2.53 (m,1H), 2.82–2.98 (m,1H), 3.86–3.95 (m,1H), 4.21 (d, J ¼ 6.3 Hz,
2H), 4.63–4.71 (m, 1H), 6.38–6.52 (m, 3H); 13C NMR (75.5 MHz,
CDCl3):
d
16.2, 32.6, 33.4, 63.3, 68.4, 75.2, 97.4 (t, JCF ¼ 25.8 Hz), 98.5
(d, JCF ¼ 28.9 Hz, 2C),159.6 (t, JCF ¼ 13.7 Hz),163.5 (d, JCF ¼ 247.2 Hz),
22
163.7 (d, JCF ¼ 247.2 Hz) 179.1. [
a
]
¼ þ63.5 (chloroform). MS m/z
D
4.2.11. 5-Azido-2,3,5-trideoxy-6-O-benzyl-2-methyl-
L
-idono-1,4-
319.6 [(M þ Na)þ calcd for C13H13F2N3NaOþ3 320.08].15-(S): 1H NMR
lactone (11)
(300 MHz, CDCl3):
d
1.33 (d, J ¼ 4.1 Hz, 3H), 1.87–2.01 (m, 1H), 2.43–
Compound 11 (403 mg, 90%) was synthesized from 9 (408 mg,
1.63 mmol) according to the method of the preparation of 10. After
purification compound 11 was collected as a colourless oil. 1H NMR
2.56 (m, 1H), 2.66–2.80 (m, 1H), 3.80–3.87 (m, 1H), 4.17 (m, 2H),
4.52–4.62 (m, 1H), 6.41–6.52 (m, 3H); 13C NMR (75.5 MHz, CDCl3):
d
15.1, 32.9, 35.0, 62.0, 68.2, 76.0, 97.4 (t, JCF ¼ 26.1 Hz), 98.5 (d,
(300 MHz, CD3OD):
d
1.22 (d, J ¼ 7.4 Hz, 3H), 2.02 (dt, J ¼ 8.4,
JCF ¼ 29.2 Hz, 2C), 163.5 (d, JCF ¼ 247.4 Hz), 163.8 (d, JCF ¼ 247.4 Hz),
22
13.1 Hz, 1H), 2.39 (ddd, J ¼ 4.1, 9.6, 13.1 Hz, 1H), 3.83 (ddq, J ¼ 7.4,
177.9. [
a]
¼ þ46.3 (chloroform). MS m/z 319.6 [(M þ Na)þ calcd for
D
8.4, 9.6 Hz, 1H), 3.67–3.82 (m, 3H), 4.51–4.66 (m, 3H), 7.26–7.38 (m,
C13H13F2N3NaOþ3 320.08].
5H); 13C NMR (75.5 MHz, CD3OD):
78.2, 128.8, 129.4, 139.2, 182.1.
d 16.4, 33.5, 35.0, 65.4, 71.1, 74.4,
4.2.15. (2R,4S,5S)-5-Azido-6-(3,5-difluoro-benzyloxy)-4-hydroxy-
2-methyl-hexanoic acid ((S)-1-benzylcarbamoyl-2-methyl-propyl)-
amide (16)
4.2.12. 5,6-Anhydro-2,3-dideoxy-2-methyl-D-glucono-1,4-lactone
(12)
Compound 16 (133 mg, 54%) was synthesized from 10 (141 mg,
0.51 mmol) according to the method of the preparation of 19.
Compound 16 was collected as crystals after purification. 1H NMR
The diol 7 (119 mg, 0.75 mmol) was dissolved in chloroform
(37 mL), triphenyl phosphine (293 mg, 1.12 mmol) and diisopropyl
azodicarboxylate (220
m
L, 1.12 mmol) were added. The mixture
(300 MHz, CDCl3):
d
0.90–0.99 (m, 6H), 1.16 (d, J ¼ 7.01 Hz, 3H),
was refluxed overnight before the solvent was evaporated. The
crude mixture was purified by flash column chromatography
(toluene/ethyl acetate 9:1) to give the epoxide 12 (66 mg, 63%) as
1.67–1.77 (m, 2H), 2.06–2.19 (m, 1H), 2.58–2.72 (m, 1H), 3.05 (bs,
1H), 3.39–3.47 (m, 1H), 3.64–3.78 (m, 3H), 4.17–4.24 (m, 1H), 4.32–
4.50 (m, 2H), 4.52 (s, 2H), 6.30–6.38 (m, 1H), 6.41–6.49 (m, 1H),
6.67–6.77 (m, 1H), 6.81–6.89 (m, 2H), 7.20–7.36 (m, 5H); 13C NMR
a colourless oil. 1H NMR (300 MHz, CDCl3):
d
1.13 (d, J ¼ 6.32 Hz,
3H), 1.76–1.87 (m, 1H), 2.06–2.17 (m, 1H), 2.50–2.56 (m, 1H), 2.57–
(75.5 MHz, CDCl3): d 17.0, 17.2, 17.9, 29.7, 36.5, 37.2, 42.1, 58.3, 65.1,
2.68 (m, 1H), 2.74 (t, J ¼ 4.67 Hz, 1H), 3.06–3.12 (m, 1H), 4.40–4.49
68.1, 69.8, 70.9, 101.5 (t, JCF ¼ 25.2 Hz), 108.9 (d, JCF ¼ 25.2 Hz, 2C),
126.2, 126.5, 127.5, 137.5, 141.9 (t, JCF ¼ 8.8 Hz), 162.4 (d,
JCF ¼ 251.2 Hz), 162.6 (d, JCF ¼ 251.2 Hz), 171.5, 176.7. MS m/z 518.4
[(M þ H)þ calcd for C26H34F2N5Oþ4 518.26].
(m, 1H); 13C NMR (75.5 MHz, CDCl3):
76.5, 179.1.
d 15.3, 30.2, 33.3, 44.2, 51.5,
4.2.13. 2,3-Dideoxy-6-O-(3,5-difluorophenyl)-2-methyl-D-glucono-
1,4-lactone and 2,3-dideoxy-6-O-(3,5-difluorophenyl)-2-methyl-
D
-
4.2.16. (2R,4S,5S)-5-Azido-6-(3,5-difluoro-benzyloxy)-4-hydroxy-
manono-1,4-lactone (13)
2-methyl-hexanoic acid cyclopropylamide (17)
To the epoxide 12 (66 mg, 0.47 mmol) in DMF (2.4 mL) were 3,5-
difluorophenol (92 mg, 0.71 mmol) and K2CO3 (37 mg, 0.24 mmol)
added. The reaction mixture was heated to 110 ꢀC for 4 h. The DMF
was removed byco-evaporationwith toluene (3 ꢂ15 mL). The crude
residue was purified by flash column chromatography (toluene/
ethyl acetate 9:1) to yield a diastereomeric mixture of compound 13
Cyclopropylamine (B) (49
CH2Cl2 (2 mL) and 2 M Me3Al (710
m
L, 0.71 mmol) was dissolved in
mL, 1.41 mmol) was added drop
wise to the solution. The reaction was stirred for 15 min before the
lactone 10 (110 mg, 0.35 mmol) dissolved in CH2Cl2 (1 mL) slowly
was added. The reaction mixture was stirred at 40 ꢀC for 1 h and
then quenched with 1 M HCl to pH 5. The crude mixture was
extracted with H2O (10 mL) and ethyl acetate (3 ꢂ 10 mL). The
combined organic layers were dried, filtered and concentrated.
Purification by flash column chromatography (toluene/ethyl
acetate 2:1) gave the azide 17 (69 mg, 53%) as a colourless oil. 1H
(116 mg, 91%) as a colourless oil. 1H NMR (300 MHz, CD3OD):
d 1.21–
1.29 (m, 3H),1.87–2.02 (m,1H), 2.41–2.63 (m,1H), 2.64–2.85 (m,1H),
[3.31 (d, J ¼ 4.94 Hz, 0.5H) & 3.47 (d, J ¼ 5.22 Hz, 0.5 Hz, 0.5H)], 3.98–
4.04 (m, 2H), 4.10–4.20 (m, 1H), [4.46–4.53 (m, 0.5H), 4.55–4.63 (m,
0.5H)], 6.38–6.48 (m, 3H); 13C NMR (75.5 MHz, CD3OD):
d
[15.4 &
NMR (300 MHz, CDCl3):
d
0.98 (d, J ¼ 6.9 Hz, 3H), 1.17 (s, 2H), 1.25 (s,
16.4], [31.5 & 32.8], [35.3 & 36.5], 70.8, [71.2 & 71.4], 79.3, 97.2 (t,
JCF ¼ 25.7 Hz), 99.5 (d, JCF ¼ 28.9 Hz, 2C),162.3 (t, JCF ¼ 13.7 Hz),165.0
(d, JCF ¼ 247.1 Hz), 165.5 (d, JCF ¼ 247.1 Hz), [181.7 & 182.6]. MS m/z
272.8 [(M þ H)þ calcd for C13H15F2Oþ4 273.09].
2H), 1.51–1.62 (m, 1H), 1.64–1.78 (m, 2H), 2.38–2.48 (m, 1H), 2.43
(bs, 1H), 3.54 (ddd, J ¼ 3.6, 4.7, 6.6 Hz, 1H), 3.75 (dd, J ¼ 4.7, 9.9 Hz,
1H), 3.79 (dd, 6.6, 9.9 Hz, 1H), 3.86–3.92 (m, 1H), 4.56 (s, 2H), 6.68–
6.77 (m, 1H), 6.84–6.92 (m, 2H); 13C NMR (75.5 MHz, CDCl3):
d 16.0,
25.6, 29.1, 36.8, 40.4, 65.1, 69.7, 71.4, 72.3, 73.3, 103.0 (t,
JCF ¼ 25.2 Hz), 109.9 (d, JCF ¼ 25.2 Hz, 2C), 141.8 (t, JCF ¼ 8.9 Hz),
163.0 (d, JCF ¼ 249.0 Hz), 163.2 (d, JCF ¼ 249.0 Hz), 184.7.
4.2.14. 5-Azido-2,3,5-trideoxy-6-O-(3,5-difluorobenzyl)-2-methyl-
L
-idono-1,4-lactone (14-(R)) and 5-azido-2,3,5-trideoxy-6-O-(3,5-
difluorobenzyl)-2-methyl- -gulono-1,4-lactone (15-(S))
L
A diastereomeric mixture of 13 (116 mg, 0.42 mmol) and tri-
phenyl phosphine (168 mg, 0.64 mmol) was dissolved in dry THF
(4.3 mL). The mixture was cooled to ꢁ15 ꢀC (acetone/ice 1:1) and
4.2.17. (2R,4S,5S)-5-Azido-6-(3,5-difluoro-benzyloxy)-4-hydroxy-
2-methyl-hexanoic acid 4-fluoro-benzylamide (18)
Compound 18 (188 mg, 88%) was synthesized from 10 (152 mg,
0.49 mmol) according to the method of the preparation of 19 using
the amine 4-fluorobenzylamine (C) instead of the amine (S)-2-
diisopropyl azodicarboxylate (126 mL, 0.64 mmol) was added. The
mixturewas stirred for 30 min at ꢁ15 ꢀC (acetone/ice 1:1) before the

F. Wångsell et al. / European Journal of Medicinal Chemistry 45 (2010) 870–882
879
Amino-N-benzyl-3-methyl-butyramide (A). Compound 18 was
160.6 (t, JCF ¼ 13.5 Hz), 163.8 (d, JCF ¼ 246.3 Hz), 164.0 (d,
JCF ¼ 246.3 Hz), 172.4, 178.2. MS m/z 504.5 [(M þ H)þ calcd for
C25H32F2N5Oþ4 504.24].
collected as white crystals after purification. 1H NMR (300 MHz,
CDCl3):
d
1.21 (d, J ¼ 6.7 Hz, 3H), 1.56–1.59 (m, 1H), 1.70–1.79 (m,
2H), 2.52–2.67 (m, 1H), 2.89–2.95 (m, 1H), 3.41–3.48 (m, 1H), 3.61–
3.79 (m, 2H), 4.40 (d, J ¼ 5.8, 2H), 4.54 (s, 2H), 6.69–6.78 (m, 1H),
6.82–6.91 (m, 2H), 6.95–7.04 (m, 2H), 7.20–7.26 (m, 3H). 13C NMR
4.2.21. (2R,4S,5S)-5-Amino-6-(3,5-difluoro-benzyloxy)-4-hydroxy-
2-methyl-hexanoic acid ((S)-1-benzylcarbamoyl-2-methyl-propyl)-
amide (22)
(75.5 MHz, CDCl3): d 18.3, 37.7, 38.4, 42.9, 65.8, 69.1, 71.5, 72.4,103.3
(t, JCF ¼ 25.3 Hz, 1C), 110.1 (d, JCF ¼ 25.3 Hz, 2C), 115.7 (d,
JCF ¼ 21.5 Hz, 2C), 129.5 (d, JCF ¼ 8.0 Hz, 2C), 134.3 (d, JCF ¼ 3.3 Hz,
1C), 141.9 (t, JCF ¼ 8.9 Hz, 1C), 162.3 (d, JCF ¼ 245.9 Hz, 1C), 163.2 (d,
JCF ¼ 248.8 Hz, 1C), 163.4 (d, JCF ¼ 248.8 Hz, 1C), 176.7. MS m/z 437.1
[(M þ H)þ calcd for C21H24F3N4Oþ3 437.2].
The azide 16 (42 mg, 0.08 mmol) and triphenyl phosphine
(32 mg, 0.12 mmol) was dissolved in MeOH (4 mL). Three drops of
water were added and the reaction mixture was stirred overnight
at room temperature. Removal of the solvent and purification by
LC/MS (50% MeOH, 6 min ramp time to 100% MeOH, 10 min) gave
the amine 22 (34.8 mg, 87%) as white crystals. 1H NMR (300 MHz,
4.2.18. (2R,4S,5S)-5-Azido-6-benzyloxy-4-hydroxy-2-methyl-
hexanoic acid ((S)-1-benzylcarbamoyl-2-methyl-propyl)-amide
(19)
Compound 11 (59 mg, 0.21 mmol) and (S)-2-amino-N-benzyl-3-
methyl-butyramide (A) (71 mg, 0.34 mmol) were dissolved in dry
CDCl3):
d
0.88–1.01 (m, 6H), 1.15 (d, J ¼ 7.01 Hz, 3H), 1.42–1.54 (m,
1H), 1.85–1.97 (m, 1H), 1.99–2.11 (m, 1H), 2.69–2.84 (m, 1H), 3.09–
3.19 (m, 1H), 3.53–3.73 (m, 3H), 4.16 (d, J ¼ 7.76 Hz, 1H), 4.37 (d,
J ¼ 2.68 Hz, 2H), 4.56 (s, 2H), 6.80–6.90 (m, 1H), 6.95–7.05 (m, 2H),
7.20–7.34 (m, 5H); 13C NMR (75.5 MHz, CDCl3):
d 17.9, 18.1, 18.6,
THF (2 mL). DIPEA (74
m
L, 0.43 mmol) and 2-hydroxy-pyridine
30.6, 37.1, 37.8, 42.8, 56.2, 59.4, 67.0, 68.4, 71.9, 102.5 (t,
JCF ¼ 26.1 Hz), 110.1 (d, JCF ¼ 25.5 Hz, 2C), 127.0, 127.4, 128.3, 138.6,
142.6 (t, JCF ¼ 8.9 Hz), 163.3 (d, JCF ¼ 247.4 Hz), 163.5 (d,
JCF ¼ 247.4 Hz), 172.4, 177.5. MS m/z 477.9 [(M þ H)þ calcd for
C25H34F2N3Oþ4 478.3].
(81 mg, 0.86 mmol) were added and the mixture was refluxed for 4
days. Purification by HPLC chromatography (80% MeOH, 20%
H2O þ 0.2% TFA) gave 19 (37 mg, 36%) as a colourless oil. 1H NMR
(300 MHz, CD3OD):
d
0.92 (d, J ¼ 6.8 Hz, 3H), 0.94 (d, J ¼ 6.8 Hz, 3H),
1.13 (d, J ¼ 7.0 Hz, 3H), 1.55 (ddd, J ¼ 3.9,10.2, 13.9 Hz,1H),1.82 (ddd,
J ¼ 2.9, 10.4, 13.9 Hz, 1H), 1.98–2.11 (m, 1H), 2.70 (ddd, J ¼ 3.9, 7.0,
10.4 Hz, 1H), 3.42–3.49 (m, 1H), 3.59 (ddd, J ¼ 2.9, 3.9, 10.2 Hz, 1H),
3.64 (dd, J ¼ 7.2,10.1 Hz,1H), 3.70 (dd, J ¼ 4.6,10.1 Hz,1H), 4.12–4.19
(m, 1H), 4.35 (dd, J ¼ 5.8, 15.0 Hz, 1H), 4.55 (dd, J ¼ 6.0, 15.0 Hz, 1H),
4.55 (s, 2H), 7.20–7.37 (m, 10H); 13C NMR (75.5 MHz, CD3OD):
4.2.22. (2R,4S,5S)-5-Amino-6-(3,5-difluoro-benzyloxy)-4-hydroxy-
2-methyl-hexanoic acid cyclopropylamide (23)
Compound 23 (58 mg, 92%) was synthesized from 17 (69 mg,
0.19 mmol) according to the method of the preparation of 22. After
purification compound 23 was collected as a colourless oil. 1H NMR
d
19.0, 19.1, 19.8, 31.8, 38.5, 39.2, 44.1, 60.7, 67.2, 70.3, 71.4, 74.3,
(300 MHz, CD3OD):
d
0.99 (d, J ¼ 6.9 Hz, 3H), 1.05–1.12 (m, 1H), 1.11
128.2, 128.6, 128.7, 128.8, 129.4, 129.5, 139.4, 139.8, 173.8, 179.0. MS
(s, 2H), 1.19 (s, 2H), 1.23–1.35 (m, 2H), 1.58–1.66 (m, 1H), 1.83–1.92
(m, 1H), 3.67–3.73 (m, 2H), 3.81–3.88 (m, 1H), 4.59 (s, 2H), 6.82–
m/z 482.4 [(M þ H)þ calcd for C26H36N5Oþ4 482.6].
6.90 (m, 1H), 6.97–7.04 (m, 2H); 13C NMR (75.5 MHz, CD3OD):
d 17.1,
4.2.19. (2R,4S,5S)-5-Azido-6-(3,5-difluoro-phenoxy)-4-hydroxy-2-
methyl-hexanoic acid ((S)-1-benzylcarbamoyl-2-methyl-propyl)-
amide (20-(R))
Compound 20-(R) (94 mg, 70%) was synthesized from 14-(R)
(77 mg, 0.26 mmol) according to the method of the preparation of
19. Compound 20-(R) was collected as crystals after purification by
flash column chromatography (toluene/ethyl acetate 1:1). 1H NMR
24.8, 28.8, 37.5, 42.2, 56.9, 68.8, 69.3, 73.1, 73.9, 103.7 (t,
JCF ¼ 25.7 Hz), 110.3 (d, JCF ¼ 25.2 Hz, 2C), 144.0 (t, JCF ¼ 8.8 Hz),
163.5 (d, JCF ¼ 247.2 Hz), 163.7 (d, JCF ¼ 247.2 Hz), 178.4.
4.2.23. (2R,4S,5S)-5-Amino-6-(3,5-difluoro-benzyloxy)-4-hydroxy-
2-methyl-hexanoic acid 4-fluoro-benzylamide (24)
Compound 24 (96.5 mg, 62%) was synthesized from 18 (149 mg,
0.57 mmol) according to the method of the preparation of 22. After
purification compound 24 was collected as a colourless oil. 1H NMR
(300 MHz, CD3OD/CDCl3 (1:1, v/v)):
d
0.92 (d, J ¼ 6.8 Hz, 3H), 0.93
(d, J ¼ 6.6 Hz, 3H), 1.14 (d, J ¼ 6.9 Hz, 3H), 1.56–1.68 (m, 1H), 1.78–
1.91 (m, 1H), 1.97–2.10 (m, 1H), 2.62–2.75 (m, 1H), 3.60–3.70 (m,
2H), 4.02–4.20 (m, 3H), 4.33 (d, J ¼ 14.9 Hz, 1H), 4.40 (d, J ¼ 14.9 Hz,
1H), 6.39–6.52 (m, 3H), 7.16–7.31 (m, 5H); 13C NMR (75.5 MHz,
(300 MHz, CDCl3):
d
1.16 (d, J ¼ 6.9, 3H),1.43–1.54 (m, 1H),1.66–1.87
(m, 1H), 2.32–2.53 (m, 3H), 2.53–2.67 (m, 1H), 2.73–2.82 (m,1H),
3.30–3.42 (m, 2H), 3.48 (dd, J ¼ 4.1, 9.4 Hz, 1H), 4.36 (t, J ¼ 5.4 Hz,
1H), 4.44 (s, 2H), 6.41–6.50 (m, 1H), 6.66–6.75 (m, 1H), 6.77–6.86
(m, 2H), 6.88–7.02 (m, 2H), 7.14–7.31 (m, 2H). 13C NMR (75.5 MHz,
CD3OD/CDCl3 (1:1, v/v)): d 18.2, 18.3, 19.1, 30.8, 37.6, 38.1, 43.2, 59.2,
65.4, 68.8, 68.9, 96.6 (t, JCF ¼ 26.1 Hz), 98.5 (d, JCF ¼ 28.6 Hz, 2C),
127.3, 127.5, 128.5, 138.3, 160.6 (t, JCF ¼ 13.7 Hz), 163.8 (d,
JCF ¼ 264.1 Hz), 164.0 (d, JCF ¼ 264. 1 Hz), 172.3, 177.5. MS m/z 504.5
[(M þ H)þ calcd for C25H32F2N5Oþ4 504.24].
CDCl3):
d 18.6, 37.8, 39.1, 42.9, 55.4, 69.2, 72.3, 73.6, 103.2 (t,
JCF ¼ 25.4 Hz, 1C), 110.1 (d, JCF ¼ 25.1, 2C), 115.6 (d, JCF ¼ 21.5 Hz, 2C),
129.6 (d, JCF ¼ 8.2 Hz, 2C), 134.6 (d, JCF ¼ 3.3 Hz, 1C), 142.3 (t,
JCF ¼ 8.8 Hz, 1C), 162.3 (d, JCF ¼ 246.2 Hz, 1C), 163.2 (d,
JCF ¼ 248.9 Hz, 1C), 163.4 (d, JCF ¼ 248.9 Hz, 1C), 176.6. MS m/z 411.1
[(M þ H)þ calcd for C21H26F3N2Oþ3 411.2].
4.2.20. (2S,4S,5S)-5-Azido-6-(3,5-difluoro-phenoxy)-4-hydroxy-2-
methyl-hexanoic acid ((S)-1-benzylcarbamoyl-2-methyl-propyl)-
amide (21-(S))
Compound 21-(S) (93 mg, 62%) was synthesized from 15-(S)
(86 mg, 0.29 mmol) according to the method of the preparation of
19. Compound 21-(S) was collected as crystals after purification by
flash column chromatography (toluene/ethyl acetate 1:1). 1H NMR
4.2.24. (2R,4S,5S)-5-Amino-6-benzyloxy-4-hydroxy-2-methyl-
hexanoic acid ((S)-1-benzylcarbamoyl-2-methyl-propyl)-amide
(25)
Compound 25 (35 mg, quant.) was synthesized from 19 (37 mg,
0.077 mmol) according to the method of the preparation of 22.
After purification compound 25 was collected as white powder. 1H
(300 MHz, CD3OD/CDCl3 (1:1, v/v)):
d
0.91 (d, J ¼ 6.9 Hz, 3H), 0.92
(d, J ¼ 6.9 Hz, 3H), 1.15 (d, J ¼ 6.9 Hz, 3H), 1.49–1.60 (m, 1H), 1.82–
1.96 (m, 1H), 2.05–2.19 (m, 1H), 2.50–2.64 (m, 1H), 3.61–3.70 (m,
1H), 3.70–3.79 (m, 1H), 3.99 (m, 3H), 4.35 (s, 2H), 6.38–6.51 (m, 3H),
7.15–7.32 (m, 5H); 13C NMR (75.5 MHz, CD3OD/CDCl3 (1:1, v/v)):
NMR (300 MHz, CD3OD):
d
0.91 (d, J ¼ 6.8 Hz, 3H), 0.93 (d,
J ¼ 6.8 Hz, 3H), 1.11 (d, J ¼ 7.0 Hz, 3H), 1.48 (ddd, J ¼ 4.1, 10.2, 13.9 Hz,
1H), 1.84 (ddd, J ¼ 3.3, 10.3, 13.9 Hz, 1H), 1.98–2.10 (m, 1H), 2.63–
2.81 (m, 2H), 3.43 (dd, J ¼ 6.3, 9.4 Hz, 1H), 3.50–3.58 (m, 2H), 4.16
(d, J ¼ 7.8 Hz, 1H), 4.34 (d, J ¼ 14.9 Hz, 1H), 4.40 (d, J ¼ 14.9 Hz, 1H),
d
17.7, 17.8, 19.1, 30.5, 37.7, 43.3, 58.9, 65.1, 69.1, 69.2, 96.7 (t,
JCF ¼ 26.1 Hz), 98.6 (d, JCF ¼ 28.9 Hz, 2C), 127.3, 127.6, 128.6, 138.3,

880
F. Wångsell et al. / European Journal of Medicinal Chemistry 45 (2010) 870–882
4.50 (s, 2H), 7.23–7.34 (m, 10H); 13C NMR (75.5 MHz, CD3OD):
19.0, 19.8, 31.8, 38.6, 39.3, 44.0, 56.4, 60.5, 70.6, 72.9, 74.3, 128.2,
850.3675 [(M þ H)þ calcd for C44H54F2N5O8Sþ 850.3656]. Anal.
d
(C44H53F2N5O8S) C, H, N.
128.6, 128.8, 128.9, 129.4, 129.5, 139.6, 139.8, 173.7, 179.1. MS m/z
456.4 [(M þ H)þ calcd for C26H37N3Oþ4 456.3].
4.2.28. ((S)-1-[(S)-1-[(1S,2S,4R)-4-((S)-1-Benzylcarbamoyl-2-
methyl-propylcarbamoyl)-1-(3,5-difluoro-benzyloxymethyl)-2-
hydroxy-pentylcarbamoyl]-3-methylsulfanyl-propylcarbamoyl]-2-
methyl-propyl)-carbamic acid tert-butyl ester (29)
4.2.25. (2R,4S,5S)-5-Amino-6-(3,5-difluoro-phenoxy)-4-hydroxy-
2-methyl-hexanoic acid ((S)-1-benzylcarbamoyl-2-methyl-propyl)-
amide (26-(R))
Compound 26-(R) (77 mg, 82%) was synthesized from 20-(R)
(97 mg, 0.19 mmol) according to the method of the preparation of
22, white powder after purification. 1H NMR (300 MHz, CD3OD):
Compound 29 (27 mg, 71%) was synthesized from 22 (25 mg,
0.051 mmol) according to the method of the preparation of 28 using
(S)-2-((S)-2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-4-
methylsulfanyl-butyric acid (E) instead of 5-(methanesulfonyl-
methyl-amino)-N-((R)-1-phenyl-ethyl)-isophthalamide (D). Com-
pound 29 was collected as white powder after lyophilization. 1H
NMR (300 MHz, CD3OD/CDCl3 (2:1, v/v)): 0.88 (d, J ¼ 6.8 Hz, 3H),
0.89 (d, J ¼ 6.8 Hz, 6H), 0.90 (d, J ¼ 6.8 Hz, 3H),1.10 (d, J ¼ 6.9 Hz, 3H),
1.42 (s, 9H), 1.49 (dq, J ¼ 4.8, 13.9 Hz, 1H), 1.71 (ddd, J ¼ 3.8, 5.8,
13.9 Hz,1H),1.91–2.10 (m, 4H), 2.05 (s, 3H), 2.47–2.54 (m, 2H), 2.55–
2.67 (m, 1H), 3.50 (dd, J ¼ 6.0, 9.6 Hz, 1H), 3.56 (dd, J ¼ 6.6, 9.6 Hz,
1H), 3.76–3.83 (m, 1H), 3.83 (d, J ¼ 6.6 Hz, 1H), 3.95–4.04 (m, 1H),
4.12 (d, J ¼ 7.8 Hz, 1H), 4.32 (d, J ¼ 14.9 Hz, 1H), 4.40 (d, J ¼ 14.9 Hz,
1H), 4.43 (d, J ¼ 12.4 Hz, 1H), 4.48 (d, J ¼ 12.4 Hz, 1H), 4.48–4.53 (m,
1H), 6.68–6.74 (m, 1H), 6.82–6.86 (m, 2H), 7.17–7.31 (m, 5H); 13C
d
0.93 (d, J ¼ 6.7 Hz, 3H), 0.95 (d, J ¼ 6.7 Hz, 3H), 1.14 (d, J ¼ 7.14 Hz,
3H), 1.51–1.63 (m, 1H), 1.82–1.93 (m, 1H), 1.98–2.12 (m, 1H), 2.66–
2.79 (m, 1H), 2.90–2.98 (m, 1H), 3.58–3.65 (m, 1H), 3.85 (dd, J ¼ 6.3,
9.3 Hz, 1H), 3.99 (dd, J ¼ 5.8, 9.3 Hz, 1H), 4.19 (d, J ¼ 7.7 Hz, 1H), 4.37
(d, J ¼ 2.48 Hz, 2H), 6.44–6.60 (m, 3H), 7.16–7.32 (m, 5H); 13C NMR
(75.5 MHz, CD3OD):
d 17.8, 18.7, 30.7, 37.5, 38.0, 42.9, 54.5, 59.3,
69.0, 70.4, 95.8 (t, JCF ¼ 26.3 Hz), 98.3 (d, JCF ¼ 28.9 Hz, 2C), 127.0,
127.4, 128.3, 138.6, 161.3 (t, JCF ¼ 13.7 Hz), 163.9 (d, JCF ¼ 244.8 Hz),
164.1 (d, JCF ¼ 244.8 Hz), 172.5, 177.9. MS m/z 477.9 [(M þ H)þ calcd
for C25H34F2N3Oþ4 478.25].
4.2.26. (2S,4S,5S)-5-Amino-6-(3,5-difluoro-phenoxy)-4-hydroxy-
2-methyl-hexanoic acid ((S)-1-benzylcarbamoyl-2-methyl-propyl)-
amide (27-(S))
Compound 27-(S) (66 mg, 99%) was synthesized from 21-(S)
(70 mg, 0.14 mmol) according to the method of the preparation of
22, white powder after purification. 1H NMR (300 MHz, CD3OD):
NMR (75.5 MHz, CD3OD/CDCl3 (2:1, v/v)): d 15.3,18.1, 18.2,18.7, 19.5,
28.5, 30.5, 31.2, 31.6, 37.8, 38.7, 43.7, 53.2, 53.5, 59.5, 60.8, 68.1, 70.4,
72.3, 80.6, 103.1 (t, JCF ¼ 25.5 Hz), 110.3 (d, JCF ¼ 25.2 Hz, 2C), 127.7,
128.0, 129.0, 138.5, 142.9 (t, JCF ¼ 8.9 Hz), 157.1, 161.9 (d,
JCF ¼ 248.2 Hz),165.2 (d, JCF ¼ 247.9 Hz),172.5,173.4,177.8. HRMS m/
z 822.4293 [(M þ H)þ calcd for C41H62F2N5O8Sþ 822.4282].
d
0.94 (d, J ¼ 6.9 Hz, 6H), 1.16 (d, J ¼ 6.9 Hz, 3H), 1.52–1.63 (m, 1H),
1.80–1.92 (m, 1H), 2.07–2.21 (m, 1H), 2.59–2.71 (m, 1H), 2.93–3.01
(m, 1H), 3.67–3.75 (m, 1H), 3.84 (dd, J ¼ 9.1, 6.6 Hz, 1H), 3.96 (dd,
J ¼ 9.1, 5.2 Hz, 1H), 4.20 (d, J ¼ 6.9 Hz, 1H), 4.36 (s, 2H), 6.44–6.60
4.2.29. N-[(1S,2S,4R)-4-((S)-1-Benzylcarbamoyl-2-methyl-
propylcarbamoyl)-1-(3,5-difluoro-benzyloxymethyl)-2-
hydroxy-pentyl]-5-(methanesulfonyl-methyl-amino)-
N0-methyl-isophthalamide (30)
(m, 3H), 7.16–7.32 (m, 5H); 13C NMR (75.5 MHz, CD3OD):
d
17.3,18.6,
30.4, 37.6, 37.8, 42.9, 54.5, 59.0, 69.6, 70.5, 95.8 (t, JCF ¼ 26.6 Hz),
98.3 (d, JCF ¼ 29.2 Hz, 2C), 127.0, 127.4, 128.3, 138.3, 161.4 (t,
JCF ¼ 13.7 Hz), 163.9 (d, JCF ¼ 244.7 Hz), 164.1 (d, JCF ¼ 244.7 Hz),
172.6, 178.7. MS m/z 477.9 [(M þ H)þ calcd for C25H34F2N3Oþ4
478.25].
Compound 30 (18 mg, 52%) was synthesized from 22 (25 mg,
0.051 mmol) according to the method of the preparation of 28 using
5-(methanesulfonyl-methyl-amino)-N-methyl-isophthalamic acid
(F) instead of 5-(methanesulfonyl-methyl-amino)-N-((R)-1-phenyl-
ethyl)-isophthalamide (D). Compound 30 was collected as white
powder after lyophilization. 1H NMR (300 MHz, CD3OD):
d 0.87 (d,
4.2.27. N-[(1S,2S,4R)-4-((S)-1-Benzylcarbamoyl-2-methyl-
propylcarbamoyl)-1-(3,5-difluoro-benzyloxymethyl)-2-hydroxy-
pentyl]-5-(methanesulfonyl-methyl-amino)-N0-((R)-1-phenyl-
ethyl)-isophthalamide (28)
The amine 22 (17 mg, 0.034 mmol) was dissolved in DMF (2 mL)
and cooled to 0 ꢀC. 5-(Methanesulfonyl-methyl-amino)-N-
((R)-1-phenyl-ethyl)-isophthalamide (D) (25 mg, 0.064 mmol),
J ¼ 6.8 Hz, 6H), 1.14 (d, J ¼ 6.9 Hz, 3H), 1.48–1.58 (m, 1H), 1.83–1.92
(m, 1H), 1.93–2.05 (m, 1H), 2.72–2.80 (m, 1H), 2.92 (s, 3H), 2.95 (s,
3H), 3.37 (s, 3H), 3.69 (dd, J ¼ 7.0, 9.8 Hz,1H), 3.78 (dd, J ¼ 6.3, 9.8 Hz,
1H), 3.85–3.92 (m, 1H), 4.14 (d, J ¼ 7.7 Hz, 1H), 4.33 (d, J ¼ 15.0 Hz,
1H), 4.41 (d, J ¼ 15.0 Hz, 1H), 4.34–4.45 (m, 1H), 4.56 (s, 2H), 6.76–
6.84 (m, 1H), 6.92–6.97 (m, 2H), 7.21–7.32 (m, 5H), 8.02–8.05 (m,
2H), 8.23–8.25 (m, 1H); 13C NMR (75.5 MHz, CD3OD):
d 18.5, 19.0,
diisopropylethylamine (12
m
L, 0.067 mmol) and O-(7-Azabenzo-
19.8, 27.0, 31.9, 35.9, 38.4, 39.5, 40.4, 44.0, 55.0, 60.4, 69.5, 70.7, 72.6,
103.4 (t, JCF ¼ 25.8 Hz),111.0 (d, JCF ¼ 25.5 Hz, 2C),125.9,128.2,128.6,
129.1, 129.5, 137.1, 137.2, 139.8, 143.8, 144.5 (t, JCF ¼ 9.0 Hz), 162.9 (d,
JCF ¼ 247.3 Hz), 166.1 (d, JCF ¼ 247.1 Hz), 168.9, 173.7, 180.0. HRMS m/
z 760.3196 [(M þ H)þ calcd for C37H48F2N5O8Sþ 760.3186].
triazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate
(25 mg, 0.067 mmol) were added and the reaction was stirred at
room temperature overnight. The crude product was purified with
LC/MS (50% MeOH, 15 min ramp time to 100% MeOH, 10 min) to
give 28 (10 mg, 35%) as white crystals after lyophilization. 1H NMR
(300 MHz, CD3OD):
d
0.86 (d, J ¼ 6.8 Hz, 3H), 0.87 (d, J ¼ 6.8 Hz, 3H),
4.2.30. N-((1S,2S,4R)-1-Benzyloxymethyl-4-cyclopropylcarbamoyl-
2-hydroxy-pentyl)-5-(methanesulfonyl-methyl-amino)-N0-((R)-1-
phenyl-ethyl)-isophthalamide (31)
Compound 31 (13 mg, 17%) was synthesized from 23 (36 mg,
0.105 mmol) according to the method of the preparation of 28.
Compound 31 was collected as white powder after lyophilization.
1.14 (d, J ¼ 6.9 Hz, 3H), 1.48–1.57 (m, 1H), 1.58 (d, J ¼ 7.0 Hz, 3H),
1.84–2.05 (m, 2H), 2.70–2.81 (m, 1H), 2.95 (s, 3H), 3.36 (s, 3H), 3.68
(dd, J ¼ 7.0, 9.8 Hz, 1H), 3.77 (dd, J ¼ 6.3, 9.8 Hz, 1H), 3.85–3.91 (m,
1H), 4.14 (d, J ¼ 7.7 Hz, 1H), 4.32–4.40 (m, 1H), 4.33 (d, J ¼ 15.0 Hz,
1H), 4.39 (d, J ¼ 15.0 Hz, 1H), 4.56 (s, 2H), 5.25 (q, J ¼ 7.1 Hz, 1H),
6.76–6.84 (m, 1H), 6.91–6.96 (m, 2H), 7.19–7.42 (m, 10H), 8.02–8.07
1H NMR (300 MHz, CD3OD):
d
0.99 (d, J ¼ 6.9 Hz, 3H), 1.11 (d,
(m, 2H), 8.27 (t, J ¼ 1.58 Hz, 1H); 13C NMR (75.5 MHz, CDCl3):
d
17.6,
J ¼ 11.1 Hz, 4H), 1.23–1.33 (m, 2H), 1.58 (d, J ¼ 7.1 Hz, 3H), 1.59–1.68
(m, 1H), 1.83–1.92 (m, 1H), 2.96 (s, 3H), 3.37 (s, 3H), 3.68 (dd, J ¼ 6.8,
9.6 Hz, 1H), 3.77 (dd, J ¼ 6.7, 9.6 Hz, 1H), 4.01–4.09 (m, 1H), 4.41 (dt,
J ¼ 2.4, 6.6 Hz, 1H), 4.55 (d, J ¼ 12.9 Hz, 1H), 4.61 (d, J ¼ 12.9 Hz, 1H),
5.25 (q, J ¼ 7.1 Hz, 1H), 6.77–6.82 (m, 1H), 6.90–6.99 (m, 2H), 7.22–
7.44 (m, 5H), 8.02 (d, J ¼ 1.6 Hz, 2H), 8.21 (t, J ¼ 1.6 Hz, 1H); 13C NMR
18.3, 19.3, 21.7, 30.8, 35.5, 37.7, 37.9, 38.5, 43.5, 49.7, 52.9, 59.0, 69.7,
71.2, 72.3, 103.2 (t, JCF ¼ 25.2 Hz), 110.0 (d, JCF ¼ 24.9 Hz, 2C), 124.2,
126.2, 127.5, 127.5, 127.6, 127.8, 128.0, 128.6, 128.7, 135.3, 135.9,
137.8, 141.6 (t, JCF ¼ 8.6 Hz), 142.3, 142.9, 163.0 (d, JCF ¼ 249.4 Hz),
163.2 (d, JCF ¼ 249.4 Hz), 164.6, 166.0, 171.2, 176.9. HRMS m/z

F. Wångsell et al. / European Journal of Medicinal Chemistry 45 (2010) 870–882
881
(75.5 MHz, CD3OD):
d
15.7, 22.1, 26.3, 27.4, 35.9, 37.4, 38.3, 42.1,
(d, JCF ¼ 245.9 Hz), 166.0, 167.3, 172.2, 177.5. HRMS m/z 836.3476
50.9, 53.6, 70.3, 71.3, 72.6, 73.8, 103.4 (t, JCF ¼ 25.8 Hz), 110.9 (d,
JCF ¼ 25.2 Hz, 2C), 125.8, 127.3, 128.2, 129.2, 129.3, 129.6, 137.2, 137.5,
143.8, 144.6, 145.0, 164.4 (d, JCF ¼ 247.5 Hz), 164.6 (d, JCF ¼ 247.5 Hz),
167.8, 167.9, 168.9. HRMS m/z 676.2863 [(M þ H)þ calcd for
C37H48F2N5O8Sþ 676.2842].
[(M þ H)þ
calcd
for
C43H52F2N5O8Sþ
836.3499].
Anal.
(C43H51F2N5O8S$½H2O) C, H, N: calcd, 8.29; found, 7.36.
4.2.34. N-[(1S,2S,4S)-4-((S)-1-Benzylcarbamoyl-2-methyl-
propylcarbamoyl)-1-(3,5-difluoro-phenoxymethyl)-2-hydroxy-
pentyl]-5-(methanesulfonyl-methyl-amino)-N0-((R)-1-phenyl-
ethyl)-isophthalamide (35-(S))
Compound 35-(S) (28 mg, 64%) was synthesized from 27-(S)
(25 mg, 0.052 mmol) according to the method of the preparation of
28. White powder after lyophilization. 1H NMR (300 MHz, CD3OD/
4.2.31. N-[(1S,2S,4R)-1-(3,5-Difluoro-benzyloxymethyl)-4-(4-
fluoro-benzylcarbamoyl)-2-hydroxy-pentyl]-5-(methanesulfonyl-
methyl-amino)-N0-((R)-1-phenyl-ethyl)-isophthalamide (32)
Compound 32 (9.4 mg, 18%) was synthesized from 24 (27.9 mg,
0.068 mmol) according to the method of the preparation of 28.
Compound 32 was collected as white powder after lyophilization.
CDCl3 (1:1, v/v)):
d
0.92 (d, J ¼ 6.8 Hz, 6H), 1.14 (d, J ¼ 6.9 Hz, 3H),
1.48–1.56 (m, 1H), 1.58 (d, J ¼ 7.0 Hz, 3H), 1.85–1.97 (m, 1H), 2.05–
2.19 (m, 1H), 2.55–2.67 (m, 1H), 2.93 (s, 3H), 3.33 (s, 3H), 3.96–4.03
(m, 1H), 4.05–4.12 (m, 2H), 4.13–4.22 (m, 1H), 4.28–4.32 (m, 2H),
4.40–4.47 (m, 1H), 5.25 (q, J ¼ 7.0 Hz, 1H), 6.36–6.55 (m, 3H), 7.11–
7.41 (m, 10H), 7.98–8.01 (m, 2H), 8.23 (t, J ¼ 1.58 Hz, 1H); 13C NMR
1H NMR (300 MHz, CD3OD):
d
1.17 (d, J ¼ 7.1 Hz, 3H), 1.21–1.54 (m,
3H), 1.58 (d, J ¼ 6.9 Hz, 3H), 1.86–1.99 (m, 1H), 2.65 (s, 3H), 2.95 (s,
3H), 3.61–3.86 (m, 4H), 4.19–4.40 (m, 2H), 4.49–4.62 (m, 2H), 5.19–
5.32 (m, 1H), 6.74–6.86 (m, 1H), 6.87–7.04 (m, 4H), 7.06–7.50 (m,
10H), 7.95–8.09 (m, 2H), 8.20–8.23 (m, 1H). 13C NMR (75.5 MHz,
(75.5 MHz, CD3OD/CDCl3 (1:1, v/v)): d 17.7,18.2,19.0, 21.3, 30.5, 35.4,
CD3OD):
d
17.8, 20.9, 34.7, 37.1, 37.6, 38.0, 42.1, 49.8, 54.2, 68.5, 69.5,
37.7, 38.0, 38.1, 43.2, 49.8, 53.6, 58.9, 67.6, 68.3, 96.4 (t, JCF ¼ 26.1 Hz),
98.5 (d, JCF ¼ 28.9 Hz, 2C), 124.8, 126.2, 127.2, 127.3, 127.5, 128.4,
128.5, 128.6, 128.7, 135.7, 136.3, 138.3, 142.4, 143.6, 160.9 (t,
JCF ¼ 13.6 Hz), 163.8 (d, JCF ¼ 245.9 Hz), 164.0 (d, JCF ¼ 245.9 Hz),
165.6, 166.2, 167.6, 178.4. HRMS m/z 836.3502 [(M þ H)þ calcd for
C43H52F2N5O8Sþ 836.3499]. Anal. (C43H51F2N5O8S$H2O) C, H, N:
calcd, 8.20; found, 7.58.
71.5, 102.3 (t, JCF ¼ 28.8 Hz, 1C), 109.8 (d, JCF ¼ 25.5 Hz, 2C), 114.9 (d,
JCF ¼ 21.8 Hz, 2C), 124.7, 126.1, 127.0, 128.0, 128.1, 128.4, 129.1 (d,
JCF ¼ 8.0 Hz, 2C), 135.0, 136.1, 142.6, 143.3 (t, JCF ¼ 9.2 Hz, 1C), 143.8,
162.1 (d, JCF ¼ 243.9, 1C), 163.2 (d, JCF ¼ 247.1, 1C), 163.4 (d,
JCF ¼ 247.1, 1C), 166.5, 167.8, 177.7. HRMS m/z 769.2868 [(M þ H)þ
calcd for C39H44F3N4O7Sþ 769.2877].
4.2.32. N-[(1S,2S,4R)-4-((S)-1-Benzylcarbamoyl-2-methyl-
propylcarbamoyl)-1-benzyloxymethyl-2-hydroxy-pentyl]-5-
(methanesulfonyl-methyl-amino)-N0-((R)-1-phenyl-ethyl)-
isophthalamide (33)
Acknowledgement
We gratefully thank Dr Tatiana Maltseva at Medivir AB for per-
forming 2D-NMR experiments, and Dr Kurt Benkestock and for
conducting HRMS data. We also thank Elizabeth Hamerlink and Dr
Ian Henderson for BACE-I and CathD enzyme data, Alexandra
Johansson, and Elisabet Lilja for excellent technical assistance in the
production of BACE-1, Dr Anders Blomqvist for the original cloning
of the human gene for BACE-1. We also thank Prof Torsten Unge and
Dr Dean Derbyshire for helpful comments and insights. Finally, we
would like to acknowledge Medivir AB for financial support.
Compound 33 (27 mg, 77%) was synthesized from 25 (20 mg,
0.044 mmol) according to the method of the preparation of 28.
Compound 33 was collected as white powder after lyophilization.
1H NMR (300 MHz, CD3OD):
d 0.92 (d, 6.8 Hz, 3H), 0.93 (d, 6.8 Hz,
3H), 1.18 (d, J ¼ 6.9 Hz, 3H), 1.51–1.62 (m, 1H), 1.61 (d, J ¼ 7.1 Hz, 3H),
1.89–1.99 (m, 1H), 2.00–2.10 (m,1H), 2.74–2.84 (m, 1H), 3.00 (s, 3H),
3.40 (s, 3H), 3.69–3.82 (m, 2H), 3.90–3.97 (m, 1H), 4.19 (d, J ¼ 7.7 Hz,
1H), 4.38 (d, J ¼ 15.0 Hz, 1H), 4.38–4.44 (m, 1H), 4.44 (d, J ¼ 15.0 Hz,
1H), 4.57 (d, J ¼ 11.9 Hz, 1H), 4.62 (d, J ¼ 11.9 Hz, 1H), 5.30 (q,
J ¼ 7.0 Hz, 1H), 7.25–7.48 (m, 15H), 8.07–8.11 (m, 2H), 8.31 (t,
Appendix. Supplementary data
J ¼ 1.58 Hz, 1H); 13C NMR (75.5 MHz, CD3OD):
d 18.4, 19.0, 19.8, 22.1,
Experimental details and spectroscopic data for the compound
A and D. 1H and 2D-NMR (COSY and NOESY) on compound 7, 14-(R)
and 15-(S). MS, HPLC purity on all the final compounds 29–32,
together with combustion analysis of the final compounds 28, 34-
(R), 35-(S) and 33. This material is available at Science Direct.
Supplementary data associated with this article can be found in
online version at doi:10.1016/j.ejmech.2009.11.013.
31.9, 36.0, 38.4, 38.5, 39.5, 44.0, 50.9, 55.0, 60.4, 69.7, 70.4, 74.1,
126.0, 127.3, 128.2, 128.6, 128.7, 128.9, 129.4, 129.5, 129.6, 137.2,
137.4, 139.5, 139.8, 143.8, 145.0, 167.7, 168.9, 173.7, 179.0. HRMS m/z
814.3847 [(M þ H)þ calcd for C44H56N5O8Sþ 814.3844]. Anal.
(C43H55N5O8S) C, H, N.
4.2.33. N-[(1S,2S,4R)-4-((S)-1-Benzylcarbamoyl-2-methyl-
propylcarbamoyl)-1-(3,5-difluoro-phenoxymethyl)-2-hydroxy-
pentyl]-5-(methanesulfonyl-methyl-amino)-N0-((R)-1-phenyl-
ethyl)-isophthalamide (34-(R))
References
Compound 34-(R) (34 mg, 74%) was synthesized from 26-(R)
(26 mg, 0.055 mmol) according to the method of the preparation of
28. White powder after lyophilization. 1H NMR (300 MHz, CD3OD/
[1] D.K. Lahiri, M.R. Farlow, N.H. Greig, K. Sambamurti, Current drug targets for
Alzheimer’s disease treatment. Drug Dev. Res. 56 (3) (2002) 267–281.
[2] D.J. Selkoe, Alzheimer disease: mechanistic understanding predicts novel
therapies. Ann. Intern. Med. 140 (8) (2004) 627–638.
[3] L.E. Hebert, P.A. Scherr, J.L. Bienias, D.A. Bennett, D.A. Evans, Alzheimer disease
in the US population – prevalence estimates using the 2000 census. Arch.
Neurol. 60 (8) (2003) 1119–1122.
[4] M. Citron, Strategies for disease modification in Alzheimer’s disease. Nat. Rev.
Neurosci. 5 (9) (2004) 677–685.
[5] J. Hardy, K. Duff, K.G. Hardy, J. Perez-Tur, M. Hutton, Genetic dissection of
Alzheimer’s disease and related dementias: amyloid and its relationship to
tau. Nat. Neurosci. 1 (5) (1998) 355–358.
[6] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer’s disease: progress
and problems on the road to therapeutics. Science 297 (5580) (2002) 353–356.
[7] D.J. Selkoe, Translating cell biology into therapeutic advances in Alzheimer’s
disease. Nature 399 (6738) (1999) A23–A31.
[8] D.H. Small, C.A. McLean, Alzheimer’s disease and the amyloid beta protein:
what is the role of amyloid? J. Neurochem. 73 (2) (1999) 443–449.
CDCl3 (1:1, v/v)):
d
0.84 (d, J ¼ 6.9 Hz, 3H), 0.87 (d, J ¼ 6.9 Hz, 3H),
1.13 (d, J ¼ 6.9 Hz, 3H), 1.56 (d, J ¼ 7.2 Hz, 3H), 1.50–1.64 (m, 1H),
1.77–1.88 (m, 1H),1.90–2.03 (m, 1H), 2.62–2.75 (m, 1H), 2.92 (s, 3H),
3.33 (s, 3H), 3.89–3.97 (m, 1H), 4.05–4.20 (m, 3H), 4.29–4.37 (m,
2H), 4.40–4.45 (m, 1H), 5.25 (q, J ¼ 6.9 Hz, 1H), 6.34–6.51 (m, 3H),
7.16–7.39 (m, 10H), 7.99 (t, J ¼ 1.6 Hz, 1H) 8.01 (t, J ¼ 1.6 Hz, 1H), 8.26
(t, J ¼ 1.6 Hz, 1H); 13C NMR (75.5 MHz, CD3OD/CDCl3 (1:1, v/v)):
d
17.7, 18.2, 19.1, 21.4, 31.0, 35.6, 37.6, 37.9, 38.5, 43.3, 49.8, 53.4, 59.1,
67.4, 67.7, 96.5 (t, JCF ¼ 26.1 Hz), 98.5 (d, JCF ¼ 28.9 Hz, 2C), 125.0,
126.3, 127.3, 127.4, 127.6, 128.5, 128.6, 128.7, 135.6, 136.3, 138.2,
142.4, 143.5, 160.8 (t, JCF ¼ 14.0 Hz), 163.8 (d, JCF ¼ 245.9 Hz), 164.0

882
F. Wångsell et al. / European Journal of Medicinal Chemistry 45 (2010) 870–882
[9] M. Citron, Beta-secretase inhibition for the treatment of Alzheimer’s disease –
[30] A.K. Ghosh, G. Bilcer, C. Harwood, R. Kawahama, D. Shin, K.A. Hussain, L. Hong,
J.A. Loy, C. Nguyen, G. Koelsch, J. Ermolieff, J. Tang, Structure-based design:
potent inhibitors of human brain memapsin 2 (beta-secretase). J. Med. Chem.
44 (18) (2001) 2865–2868.
[31] R.K. Hom, A.F. Gailunas, S. Mamo, L.Y. Fang, J.S. Tung, D.E. Walker, D. Davis,
E.D. Thorsett, N.E. Jewett, J.B. Moon, V. John, Design and synthesis of
hydroxyethylene-based peptidomimetic inhibitors of human beta-secretase. J.
Med. Chem. 47 (1) (2004) 158–164.
promise and challenge. Trends Pharmacol. Sci. 25 (2) (2004) 92–97.
[10] I. Hussain, D. Powell, D.R. Howlett, D.G. Tew, T.D. Week, C. Chapman,
I.S. Gloger, K.E. Murphy, C.D. Southan, D.M. Ryan, T.S. Smith, D.L. Simmons,
F.S. Walsh, C. Dingwall, G. Christie, Identification of a novel aspartic protease
(Asp 2) as beta-secretase. Mol. Cell. Neurosci. 14 (6) (1999) 419–427.
[11] X.L. Lin, C. Koelsch, S.L. Wu, D. Downs, A. Dashti, J. Tang, Human aspartic
protease memapsin
2 cleaves the beta-secretase site of beta-amyloid
precursor protein. Proc. Natl. Acad. Sci. U.S.A. 97 (4) (2000) 1456–1460.
[12] S. Sinha, J.P. Anderson, R. Barbour, G.S. Basi, R. Caccavello, D. Davis, M. Doan,
H.F. Dovey, N. Frigon, J. Hong, K. Jacobson-Croak, N. Jewett, P. Keim, J. Knops,
I. Lieberburg, M. Power, H. Tan, G. Tatsuno, J. Tung, D. Schenk, P. Seubert,
S.M. Suomensaari, S.W. Wang, D. Walker, J. Zhao, L. McConlogue, V. John,
Purification and cloning of amyloid precursor protein beta-secretase from
human brain. Nature 402 (6761) (1999) 537–540.
[32] M. Ba¨ck, J. Nyhlen, I. Kvarnstro¨m, S. Appelgren, N. Borkakoti, K. Jansson,
J. Lindberg, S. Nystro¨m, A. Hallberg, S. Rosenquist, B. Samuelsson, Design,
synthesis and SAR of potent statine-based BACE-1 inhibitors: exploration of P1
phenoxy and benzyloxy residues. Bioorg. Med. Chem. 16 (21) (2008)
9471–9486.
[33] T. Arslan, A.T. Abraham, S.M. Hecht, Structurally altered substrates for DNA
topoisomerase I. Effects of inclusion of a single 3’-deoxynucleotide within the
[13] R. Vassar, B.D. Bennett, S. Babu-Khan, S. Kahn, E.A. Mendiaz, P. Denis,
D.B. Teplow, S. Ross, P. Amarante, R. Loeloff, Y. Luo, S. Fisher, L. Fuller,
S. Edenson, J. Lile, M.A. Jarosinski, A.L. Biere, E. Curran, T. Burgess, J.C. Louis,
F. Collins, J. Treanor, G. Rogers, M. Citron, Beta-secretase cleavage of Alz-
heimer’s amyloid precursor protein by the transmembrane aspartic protease
BACE. Science 286 (5440) (1999) 735–741.
[14] R.Q. Yan, M.J. Bienkowski, M.E. Shuck, H.Y. Miao, M.C. Tory, A.M. Pauley,
J.R. Brashler, N.C. Stratman, W.R. Mathews, A.E. Buhl, D.B. Carter,
A.G. Tomasselli, L.A. Parodi, R.L. Heinrikson, M.E. Gurney, Membrane-anchored
aspartyl protease with Alzheimer’s disease beta-secretase activity. Nature 402
(6761) (1999) 533–537.
scissile strand. Nucleosides Nucleotides
515–530.
[34] J.R. Rasmussen, C.J. Slinger, R.J. Kordish, D.D. Newman-Evans, Synthesis of deoxy
sugars. Deoxygenation by treatment with N,N’-thiocarbonyldiimidazole/tri-n-
butylstannane. J. Org. Chem. 46 (24) (1981) 4843–4846.
[35] B. Rondot, T. Durand, J.P. Girard, J.C. Rossi, L. Schio, S.P. Khanapure, J. Rokach, A
free-radical route to syn lactones and other prostanoid intermediates in Iso-
prostaglandin synthesis. Tetrahedron Lett. 34 (51) (1993) 8245–8248.
[36] A. Sakurai, H. Horibe, N. Kuboyama, Y. Hashimoto, Y. Okumura, Absolute-
configuration of urothion, a pigment of urine – asymmetric-synthesis of 2-
amino-6-(3,4-dihydroxybutyl)pteridin-4(3H)-one the desulfurization product.
Tetrahedron Lett. 36 (15) (1995) 2631–2632.
[37] A.K. Sanki, T. Pathak, Synthesis of anomerically pure vinyl sulfone-modified
pent-2-enofuranosides and hex-2-enopyranosides: a group of highly reactive
Michael acceptors for accessing carbohydrate based synthons. Tetrahedron 59
(36) (2003) 7203–7214.
[38] D.H.R. Barton, W.B. Motherwell, New and selective reactions and reagents in
carbohydrate-chemistry. Pure Appl. Chem. 53 (1) (1981) 15–31.
[39] D.H.R. Barton, R. Subramanian, Reactions of relevance to the chemistry of
aminoglycoside antibiotics. Part 7. Conversion of thiocarbonates into deoxy-
sugars. J. Chem. Soc., Perkin Trans. 1 (1977) 1718–1723.
[40] S. Takahashi, N. Ogawa, H. Koshino, T. Nakata, Total synthesis of the proposed
structure for pyragonicin. Org. Lett. 7 (13) (2005) 2783–2786.
[41] P.A. Grieco, T. Oguri, Y. Yokoyama, One-step conversion of protected lactols
into lactones. Tetrahedron Lett. 19 (5) (1978) 419–420.
& Nucleic Acids 17 (1–3) (1998)
[15] V. John, J.P. Beck, M.J. Bienkowski, S. Sinha, R.L. Heinrikson, Human beta-se-
cretase (BACE) and BACE inhibitors. J. Med. Chem. 46 (22) (2003) 4625–4630.
[16] R. Vassar, beta-Secretase (BACE) as a drug target for alzheimer’s disease. Adv.
Drug Delivery Rev. 54 (12) (2002) 1589–1602.
[17] M.S. Wolfe, Secretase targets for Alzheimer’s disease: identification and
therapeutic potential. J. Med. Chem. 44 (13) (2001) 2039–2060.
[18] S.L. Roberds, J. Anderson, G. Basi, M.J. Bienkowski, D.G. Branstetter, K.S. Chen,
S.B. Freedman, N.L. Frigon, D. Games, K. Hu, K. Johnson-Wood,
K.E. Kappenman, T.T. Kawabe, I. Kola, R. Kuehn, M. Lee, W.Q. Liu, R. Motter,
N.F. Nichols, M. Power, D.W. Robertson, D. Schenk, M. Schoor, G.M. Shopp,
M.E. Shuck, S. Sinha, K.A. Svensson, G. Tatsuno, H. Tintrup, J. Wijsman,
S. Wright, L. McConlogue, BACE knockout mice are healthy despite lacking the
primary beta-secretase activity in brain: implications for Alzheimer’s disease
therapeutics. Hum. Mol. Genet. 10 (12) (2001) 1317–1324.
[19] R. Vassar, The beta-secretase, BACE – a prime drug target for Alzheimer’s
disease. J. Mol. Neurosci. 17 (2) (2001) 157–170.
[20] J. Eder, U. Hommel, F. Cumin, B. Martoglio, B. Gerhartz, Aspartic proteases in
drug discovery. Curr. Pharm. Des. 13 (3) (2007) 271–285.
[42] J.A. Marshall, B.A. Johns, Stereoselective synthesis of C5-C20 and C21-C34
subunits of the core structure of the aplyronines. Applications of enantiose-
lective additions of chiral allenylindium reagents to chiral aldehydes. J. Org.
Chem. 65 (5) (2000) 1501–1510.
[21] E.W. Baxter, A.B. Reitz, BACE inhibitors for the treatment of Alzheimer’s
disease. Annu. Rep. Med. Chem. 40 (2005) 35–48.
[22] L.A. Thompson, J.J. Bronson, F.C. Zusi, Progress in the discovery of BACE
inhibitors. Curr. Pharm. Des. 11 (26) (2005) 3383–3404.
[43] Supporting information.
[44] M. Muller, U. Huchel, A. Geyer, R.R. Schmidt, Efficient intramolecular glyco-
sylation supported by a rigid spacer. J. Org. Chem. 64 (17) (1999) 6190–6201.
[45] G. Zuccarello, A. Bouzide, I. Kvarnstro
¨m, G. Niklasson, S.C.T. Svensson,
[23] T. Guo, D.W. Hobbs, Development of BACE1 inhibitors for Alzheimer’s disease.
Curr. Med. Chem. 13 (15) (2006) 1811–1829.
[24] Z. Ziora, T. Kimura, Y. Kiso, Small-sized BACE1 inhibitors. Drugs Future 31 (1)
(2006) 53–63.
[25] R. Silvestri, Boom in the development of non-peptidic beta-secretase (BACE1)
inhibitors for the treatment of Alzheimer disease. Med. Res. Rev. 29 (2) (2009)
295–338.
[26] J.N. Cumming, U. Iserloh, M.E. Kennedy, Design and development of BACE-1
inhibitors. Curr. Opin. Drug Discov. Devel. 7 (4) (2004) 536–556.
[27] F. Wangsell, F. Russo, J. Savmarker, A. Rosenquist, B. Samuelsson, M. Larhed,
Design and synthesis of BACE-1 inhibitors utilizing a tertiary hydroxyl motif as
the transition state mimic. Bioorg. Med. Chem. Lett. 19 (16) (2009) 4711–4714.
[28] L. Hong, G. Koelsch, X.L. Lin, S.L. Wu, S. Terzyan, A.K. Ghosh, X.C. Zhang, J. Tang,
Structure of the protease domain of memapsin 2 (beta-secretase) complexed
with inhibitor. Science 290 (5489) (2000) 150–153.
M. Brisander, H. Danielsson, U. Nillroth, A. Karlen, A. Hallberg, B. Classon,
B. Samuelsson, HIV-1 protease inhibitors based on acyclic carbohydrates. J.
Org. Chem. 63 (15) (1998) 4898–4906.
[46] H.T. Openshaw, N. Whittaker, Synthesis of emetine and related compounds.
VII. Utility of bifunctional catalysts in amine–ester interactions. J. Chem. Soc. 1
(1969) 89–91.
[47] A. Basha, M. Lipton, S.M. Weinreb, A mild, general method for conversion of
esters to amides. Tetrahedron Lett. 18 (48) (1977) 4171–4174.
[48] S. Nagarajan, B. Ganem, Chemistry of naturally-occurring polyamines .11.
unsaturated spermidine and spermine derivatives. J. Org. Chem. 52 (22)
(1987) 5044–5046.
[49] S.J. Stachel, C.A. Coburn, T.G. Steele, M.C. Crouthamel, B.L. Pietrak, M.T. Lai,
M.K. Holloway, S.K. Munshi, S.L. Graham, J.P. Vacca, Conformationally biased
P3 amide replacements of beta-secretase inhibitors. Bioorg. Med. Chem. Lett.
16 (3) (2006) 641–644.
[29] S.J. Stachel, C.A. Coburn, T.G. Steele, K.G. Jones, E.F. Loutzenhiser, A.R. Gregro,
H.A. Rajapakse, M.T. Lai, M.C. Crouthamel, M. Xu, K. Tugusheva, J.E. Lineberger,
B.L. Pietrak, A.S. Espeseth, X.P. Shi, E. Chen-Dodson, M.K. Holloway, S. Munshi,
A.J. Simon, L. Kuo, J.P. Vacca, Structure-based design of potent and selective
cell-permeable inhibitors of human beta-secretase (BACE-1). J. Med. Chem. 47
(26) (2004) 6447–6450.
[50] A.K. Ghosh, L. Hong, J. Tang, Beta-secretase as a therapeutic target for inhibitor
drugs. Curr. Med. Chem. 9 (11) (2002) 1135–1144.
[51] M. Citron, T. Oltersdorf, C. Haass, L. McConlogue, A.Y. Hung, P. Seubert, C. Vigo-
Pelfrey, I. Lieberburg, D.J. Selkoe, Mutation of the beta-amyloid precursor
protein in familial Alzheimer’s disease increases beta-protein production.
Nature 360 (6405) (1992) 672–674.