Design, synthesis and SAR of potent statine-based BACE-1 inhibitors: Exploration of P1 phenoxy and benzyloxy residues

Article abstract of DOI:10.1016/j.bmc.2008.09.041

Several BACE-1 inhibitors with low nanomolar level activities, encompassing a statine-based core structure with phenyloxymethyl- and benzyloxymethyl residues in the P1 position, are presented. The novel P1 modification introduced to allow the facile exploration of the S1 binding pocket of BACE-1, delivered highly promising inhibitors.

Full text of DOI:10.1016/j.bmc.2008.09.041

Bioorganic & Medicinal Chemistry 16 (2008) 9471–9486
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and SAR of potent statine-based BACE-1 inhibitors:
Exploration of P1 phenoxy and benzyloxy residues
Marcus Bäck a, Jonas Nyhlén a, Ingemar Kvarnström a, Sara Appelgren b, Neera Borkakoti b,
Katarina Jansson b, Jimmy Lindberg b, Susanne Nyström b, Anders Hallberg c, Åsa Rosenquist a,b,
,
*
Bertil Samuelsson b,d,
*
a Department of Chemistry, Linköping University, S-581 83 Linköping, Sweden
b Medivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden
c Department of Organic Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, S-751 23 Uppsala, Sweden
d Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-10691 Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2008
Revised 10 September 2008
Accepted 16 September 2008
Available online 19 September 2008
Several BACE-1 inhibitors with low nanomolar level activities, encompassing a statine-based core struc-
ture with phenyloxymethyl- and benzyloxymethyl residues in the P1 position, are presented. The novel
P1 modification introduced to allow the facile exploration of the S1 binding pocket of BACE-1, delivered
highly promising inhibitors.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Alzheimer’s disease
BACE-1 inhibitors
P1 modifications
Peptidomimetics
1. Introduction
rise to any major adverse effects.10–12 Moreover, BACE-1 appears
to have a limited number of substrates13 whereas
c-secretase is in-
Alzheimer’s disease (AD) is a neurodegenerative disorder char-
acterized by loss of memory and cognition. It is the most common
form of dementia among elderly afflicting up to 30 million people
worldwide,1 with the suffering amount expected to grow due to an
aging population.2 The current therapy, mainly consisting of cho-
linesterase inhibitors for mild to moderate AD, has only modest
symptomatic effects. Other pharmaceutical treatments address
mood disorders, agitation and psychosis in the later stages of the
disease.3 In all, these drugs are safe but cannot offer disease mod-
ifying treatment regimens to the AD patient population.4
volved in numerous transmembrane cleavages.14,15 Taken to-
gether, these studies portray BACE-1 as a promising target for
the treatment of AD.16
Previously BACE-1 inhibitors encompassing the central cores I
(Fig. 1), characterized by a methylphenyl moiety in the P1 position,
have been presented.17,18 Herein we disclose inhibitors comprising
OH
O
OH
O
O
R
OH
O
O
H
H
N
H
N
N
The disease is associated with accumulation and aggregation of
amyloid plaques, consisting of Ab40 and Ab42 peptides along with
neurofibrillary tangles, in the brain. This overproduction of
Ab40,42 within the neurons is believed to be central for the devel-
opment of the disease.5–8 The peptides result from cleavage of the
amyloid precursor protein (APP) by b-secretase, or beta-site APP
R
R
R
R
R
cleaving enzyme (BACE-1), and c
-secretase, respectively.9
I
II
III
BACE-1 knockout mice have been shown to display greatly
diminished Ab40,42 production and the phenotype does not give
R = H or F
* Corresponding authors. Tel.: +46 8 54683100; fax: +46 8 54683199.
E-mail addresses: asa.rosenquist@medivir.se (Åsa Rosenquist), bertil.samuels-
son@medivir.se (B. Samuelsson).
Figure 1. A comparison of three statine-based central cores incorporating different
P1 residues. Central core I has been used recently in inhibitors targeting BACE-1 and
the modified central cores II and III are described in this report.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.041

9472
M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
R
R
O
R
R
OH
O
O
R
O
N3
O
OH
HO
N3
e, f, g
O
O
d
O
b or c
O
O
O
O
O
O
a
HO
HO
R
2 (67 %)
3 R = F (76%)
6 R = H (90%)
4 R = F (92%)
7 R = H (84%)
5 R = F (52 %)
8 R = H (19%)
O
O
O
1
h, i or j
R
R
OH
N3
O
O
O
OH
R
R
HO
N3
O
O
e, f, g
d
O
R
O
O
O
O
9 R = F (94%)
12 R = H (88%)
10 R = F (77%)
13 R = H (99%)
11 R = F (51%)
14 R = H (29%)
R
Scheme 1. Reagents and conditions: (a) PPh3, DIAD, CHCl3; (b) 3,5-difluorophenol, K2CO3, DMF, 100 °C; (c) phenol, K2CO3, DMF, 100 °C; (d) PPh3, DIAD, DPPA, THF (dry); (e)
HOAc/H2O 1:1, 110 °C; (f) NaIO4, KMnO4, t-BuOH/H2O 2:3; (g) 1 M NaOH; (h) Bu2SnO, toluene, reflux; (i) tetrabutylammonium bromide (TBAB), 3,5-difluoroBnBr, toluene,
90 °C; (j) TBAB, BnBr, toluene, 90 °C.
the central cores II with a phenyloxymethyl P1 group for which the
norstatine thio analog was recently reported.19 In addition we
present inhibitors encompassing the extended central core III20,21
with a benzyloxymethyl residue in the P1 position.
Flexible synthesis provided compounds exhibiting IC50 values in
the low nanomolar range.
and diisopropyl azodicarboxylate (DIAD) in refluxing chloroform,28
furnishing 2 in 67% yield. Subsequent reaction of epoxide 2 at
100 °C in the presence of 3,5-difluorophenol or phenol and potas-
sium carbonate in DMF29 delivered compounds 3 and 6 in 76%
and 90% yields, respectively. Phenoxy ethers 3 and 6 were then
subjected to PPh3, DIAD and diphenyl phosphoryl azide (DPPA) in
dry THF29 affording azides 4 and 7, with inversion of configuration,
in 92% and 84% yields, respectively. The statine-based templates 5
and 8 were then delivered in a three-step reaction sequence start-
ing with hydrolysis of the isopropylidene groups of 4 and 7 using
50% acetic acid at 110 °C, producing the corresponding diols, fol-
lowed by oxidative cleavage of the 1,2-diols using sodium perio-
2. Results and discussion
Previous reports from our laboratories have shown that inser-
tion of oxygen in the appropriate position of peptidomimetic struc-
tures, not only provides new centers for diversification, but also
simplified synthesis and may when appropriately positioned pro-
vide increased potency against the desired proteases.20–23
The syntheses of the central core building blocks, comprising
the different phenyloxy-, and bensyloxy P1 residues, were per-
formed as presented in scheme 1. The couplings of various amines
and carboxylic acids to these building blocks furnished the target
compounds. In Figure 2 the general structure of the inhibitors are
presented.
date and potassium permanganate, rendering
a mixture of
carboxylic acids 5 and 8 along with their corresponding formic es-
ters. Treating the mixtures with 1 M aqueous sodium hydroxide
provided compounds 5 and 8 in 52% and 19% yields, respectively,
over three steps.20,30
For the synthesis of compounds 11 and 14, diol 1 was first
transformed into its corresponding tin acetal by reaction with
dibutyltin oxide in refluxing toluene followed by treatment with
tetrabutylammonium bromide and 3,5-difluorobenzyl bromide or
benzyl bromide to furnish the selectively alkylated31 benzyl ethers
9 and 12 in 94% and 88% yields, respectively. Azides 10 and 13
were obtained in 77% and 99% yields, respectively, using the same
procedure as employed for the synthesis of compounds 4 and 7,
and templates 11 and 14 were delivered in 51% and 29% yields,
respectively, according to the same three-step procedure used
when synthesizing templates 5 and 8.
2.1. Chemistry
Amines C and D are commercially available whereas substitu-
ents A–B, E–G, L, N, and P were synthesized from suitably pro-
tected commercially available precursors. Sulfonamides M, O, and
Q were obtained as described by Stachel et al.24
The synthesis of the statine-based templates is outlined in
The synthesis of target compounds 16 and 17 (Table 1) was per-
formed as outlined in Scheme 2. Template 5 was coupled to amine
A (Fig. 2) employing O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetram-
ethyluronium hexafluorophosphate (HATU) and N,N-diisopropyl-
ethylamine (DIPEA) in DMF to give compound 15 in 76% yield.
Reduction of the azide group was achieved using PPh3 and metha-
nol containing a few drops of water. Subsequent coupling of the
Scheme 1. 3-Deoxy-1,2-O-isopropylidene-a-D-glucose (1) was syn-
thesized in three steps according to literature procedures.25–27 In
order two obtain the phenyloxymethyl templates 5 and 8 and
the benzyloxymethyl templates 11 and 1420 two slightly different
routes were employed.
For the synthesis of templates 5 and 8 diol 1 was first converted
into the corresponding epoxide28 using triphenylphosphine (PPh3)

M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
9473
OH
O
H
N
R3
R1
N
H
O
O
R2
H2N-R1
R2
R3-CO2H
O
S
O
H
N
O
H
H2N
O
N
OH
N
H
O
F
F
A
O
O
H
L
O
H
N
O
S
H2N
O
O
O
N
O
B
H
O
O
O
N
OH
OH
I
M
O
H2N
C
O
N
F
F
J
N
O
O
O
H2N
O
S
D
O
N
O
H
N
H
N
H2N
OH
OH
O
K
E
O
O
O
O
H
N
H2N
H
N
O
F
O
O
P
O
S
H
N
H2N
N
O
O
G
O
OH
O
Q
Figure 2. General picture of the different R-moieties connected or coupled to the central core.
corresponding amine to carboxylic acid M (Fig. 2) using HATU and
DIPEA in DMF furnished target compound 16 in 35% yield over the
two steps. Finally, methyl ester hydrolysis using lithium hydroxide
in THF/MeOH/H2O 2:1:1 provided target compound 17 in 74%
yield.
Scheme 3 depicts the synthesis of building block 19 having the
alcohol of the central core with inverted configuration. The carbox-
ylic acid 14 was treated with thionyl chloride in MeOH to afford
methyl ester 18 in 85% yield. Oxidation of the hydroxyl group using
Dess–Martin periodinane furnished the corresponding ketone,
which after workup was subsequently reduced using NaBH4 in
MeOH. Workup followed by ester hydrolysis using LiOH provided
the corresponding acid, which was subsequently coupled to amine
A to give a diastereomeric mixture (19) (R/S ꢀ 1:1) in 74% yield
over four steps. The diastereomers were separated on HPLC and
the desired R-isomer was confirmed by HPLC retention times-
and NMR analysis. Target compound 25 (Table 1) with R-configu-
ration of the hydroxyl was synthesized in three steps according
to Scheme 2, from the R-isomer of compound 19.
All target compounds were synthesized according to Scheme 2,
starting from templates 5, 8, 11 or 14, employing the appropriate
amines and carboxylic acids from Figure 2. In Tables 1–3 are given
the structures of all target compounds synthesized and enzyme
inhibition data.

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M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
O
OH
O
O
F
OH
O
O
F
O
H
N3
N3
N
a
b, c
OH
N
H
O
5
15 (76%)
F
F
O
O
OH
O
F
OH
OH
O
O
F
O
H
N
H
H
N
H
R3
N
R3
N
d
N
H
N
H
O
O
O
O
O
17 (74%)
16 (35%)
F
F
R3 = M
R3 = M
Scheme 2. Reagents and conditions: (a) A, DIPEA, HATU, DMF; (b) PPh3, MeOH, H2O; (c) M, DIPEA, HATU, DMF; (d) 1 M LiOH, THF/MeOH/H2O 2:1:1.
O
OH
O
O
OH
O
O
OH
O
O
O
H
N3
N3
N3
N
OH
O
N
H
a
b, c, d, e
O
14
18 (85%)
19 (74%) R/S ~1:1
Scheme 3. Reagents and conditions: (a) SOCl2, MeOH; (b) Dess–Martin periodinane, DCM; (c) NaBH4, MeOH, ꢁ15 °C; (d) LiOH, dioxane/H2O 1:1; (e) A, DIPEA, HATU, DMF.
2.2. Biological data and structure–activity relationships
Studies on HIV protease inhibitors have shown that the (R)-hy-
droxyl stereochemistry are preferred for rather small HEA motif
inhibitors,33 whereas the opposite S stereochemistry is preferred
for hydroxyethylene and statine-based isosteres. In order to con-
firm the preference for S-configuration of the hydroxyl group when
employing these statine cores, we prepared compound 25 having
the R-configuration, and as expected this resulted in a large drop
All target compounds were screened against BACE-1 and the
IC50 values are shown in Tables 1–3 along with Ki values for the
inhibition of human cathepsin D.
Potent BACE-1 inhibitors incorporating methionine and valine
in P2 and P3 positions, respectively, have previously been re-
ported.32 Moreover, statine-based inhibitors carrying carboxylic
acid moieties on the P0 side have resulted in highly potent inhibi-
tors.18 These results prompted the synthesis of compounds 20
and 21 (Table 1) with the 3,5-difluorophenyloxymethyl residue
in the P1 position where both methyl ester 20 and carboxylic acid
in activity delivering an IC50 value of 1.2 lM (Table 1).
Further explorations into interactions with the S30 pocket re-
sulted in Kiso34 analog inhibitors 26–32, carrying an isophthalic-
diacid group (Table 2). The diester 26 was found to have an activity
of 0.22
0.037
logue 17 (IC50 value of 0.013
l
M while the target diacid 27 displays an IC50 value of
M; thus making it slightly less active than monoacid ana-
M). When extending the P1 moiety
21 turned out to be inactive with IC50 values of >10
lM.
l
Replacing the P2–P3 groups with isophthalamide M, reported
l
by Vacca and co-workers,24 (Fig. 2) furnished methyl ester 16
from 3,5-difluorophenyloxymethyl to 3,5-difluorobenzyloxymeth-
yl as in compound 28, no change in activity was observed com-
pared to 27, whereas enhanced activity results from the non-
fluorinated extended P1 group as seen for compound 29, delivering
and carboxylic acid 17 with promising IC50 values of 0.2
0.013 M, respectively.
lM and
l
Exploration of the SAR of the P1 residue resulted in compounds
22–24, where compound 22, incorporating a phenyloxymethyl P1
residue, is equipotent with the corresponding 3,5-difluorophenyl-
an IC50 value of 0.012 lM.
Replacing the M P2–P3 group in this series with simplified iso-
phathalamides furnished inhibitors 30–32 displaying considerable
lower potency (Table 2).
oxymethyl analog 17 displaying an IC50 value of 0.014
lM. Com-
pound 23 with P1 3,5-difluorobenzyloxymethyl residue is
a
about nine times less active, whereas compound 24 incorporating
a benzyloxymethyl residue is only slightly less active than com-
pound 17, with IC50 values of 0.12 lM and 0.024 lM for 23 and
24, respectively (Table 1). This indicates that there is limited size
of the S1 pocket, as it is accessed and utilized by the statine-based
inhibitor cores.
In Table 3 are depicted inhibitors featuring the central core of
the potent inhibitor 17. Truncation of the P20–P30 portion and
replacing it with the small cyclopropyl residue or p-carboxybenzyl
residue or removing the P2’ side chain, in compound 17, (com-
pounds 33–35) renders essentially inactive inhibitors of BACE-1.
Notably, compound 36, which lacks the carboxylate functionality

M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
9475
Table 1
Target compounds and inhibition data
O
R1
R4
O
O
H
N
H
R3
N
N
H
O
O
O
R2
Compound
20
R1
R2
R3
R4
IC50
(
l
M) BACE-1
Ki (
l
M) Cat D
S
O
O
Me
H
(S)-OH
(S)-OH
(S)-OH
>10
0.015
0.024
0.73
O
N
N
H
F
F
F
F
O
O
S
21
16
H
H
N
>10
O
N
H
F
F
O
S
O
N
N
N
N
Me
0.20
H
N
O
O
O
S
17
22
23
H
H
H
(S)-OH
(S)-OH
(S)-OH
0.013
0.014
0.12
0.51
0.36
0.43
H
N
F
F
O
O
O
S
H
N
O
O
O
S
F
H
N
F
O
O
O
S
N
N
24
25
H
H
(S)-OH
(R)-OH
0.024
0.18
H
N
O
O
O
S
1.2
>5
H
N
O
of 17, is almost equipotent to the methyl ester analog 16, with an
293 cells. Whereas many compounds showed good inhibition of
BACE-1 in cell-free systems, they lost all activity in the cell-based
assay resulting in IC50 values above 10 lM.
IC50 value of 0.21 lM indicating the importance of the acidic func-
tion in this position of the S30 pocket. A slightly less extended P30
group as in compound 37 leads to a 20-fold drop in activity com-
pared to 17, that is, 0.26
l
M versus 0.013
l
M. An exploration in
2.2.1. X-ray crystal structure results of inhibitor 27 (Fig. 3)
Compound 27 was co-crystallized with BACE-1 (PDB code,
3dm6) and the key binding interactions, obtained from the X-ray
crystal structure, are described below. The hydrogen bonds from
the nonprime side inhibitor backbone are made up by the P3 NH
to the carbonyl of Gly230, the two P2 carbonyls that bind to the
truncation of the P2–P3 M group resulted in inactive inhibitors,
that is, compounds 38 and 39, confirming the importance of both
the S2 and S3 groups for these BACE-1 inhibitors (Table 3).
For all inhibitors the BACE cellular activities were determined
measuring production of secreted soluble Ab40 in cultured HEK-

9476
M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
Table 2
Target compounds and inhibition data
OH
O
O
H
H
R3
N
N
R1
N
O
H
O
O
O
R2
O
R1
O
Compound
26
R1
R2
R3
IC50
(
lM) BACE-1
Ki (lM) Cat D
O
S
O
N
Me
0.22
1.95
0.78
0.44
0.63
H
N
F
F
F
O
O
O
S
N
N
N
27
28
29
H
H
H
0.037
0.040
0.012
H
N
F
O
O
O
S
F
H
N
F
O
O
O
S
H
N
O
F
30
31
H
H
2.23
1.89
0.93
0.35
N
O
F
N
O
N
O
S
F
O
O
32
H
1.6
>5
H
N
F
side chain of Thr232 and to the back bone of the flap residue Gln73,
respectively, and the P1 NH that (similar to the P3 NH) also inter-
acts with the carbonyl of Gly230. The hydrogen bond interactions
of the backbone prime side are composed of the P10 carbonyl to the
back bone NH of the flap residue Thr72, the P20 NH to the carbonyl
of Gly34, the P20 carbonyl to the side chain hydroxyl of Tyr198, and
the P30 NH to the carbonyl of Pro70. The hydroxyl group of the sta-
tine-based transition-state isostere is positioned between the two
catalytic residues Asp32 and Asp228, forming a hydrogen bond
network.
The P3 capping phenyl makes close interactions with several
residues in the S3 subpocket. It is stacked between Thr232 and
Gly13 from top and bottom and has edge on close contact interac-
tions with Gly11, Tyr14, Ser229, Gly230, and Arg307. The P3
methyl group has close contact interactions with Gln12, Gly13,
Leu30, and Ile110 in the S3 pocket. The P2 aromatic ring is stacked
between Thr231 and the side chain of the flap residue Gln73. The
sulfonamide makes hydrogen bond interactions with the back
bone NH of Thr232 and Asn233 and with the side chains of
Ser325 and Arg235. The P1 side chain is contributing considerably
to the activity of inhibitor 27 with aromatic stacking interactions
with the side chains of Tyr71, Phe108 and Trp115, and close con-
tact interactions with Gln73, Gly74, Lys107, and Ile110 in the
S1–S3 pocket. The P20 valine side chain interacts mainly with
Ser35, Val69, Ile126, and Arg128 in the S20 pocket. The P30 iso-
phthalic acid group interacts weakly with the solvent exposed
S30 pocket and appears less hindered to rotate then any of the other
side groups. However, in one out of three observations in the
asymmetric unit, the isophthalic acid group is locked in position
by two hydrogen bonds formed between the carboxylates and

M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
9477
Table 3
Target compounds and inhibition data
OH
O
O
F
H
N
R3
R1
N
H
O
F
Compound
33
R1
R3
IC50
(
lM) BACE-1
Ki (
lM) Cat D
O
S
O
N
>10
>5
H
N
O
O
O
S
N
N
N
N
OH
34
35
36
37
4.0
2.3
>5
>5
H
N
O
O
O
O
S
O
OH
H
N
H
N
O
O
O
O
O
O
S
H
N
0.21
0.26
0.57
H
N
O
O
O
S
H
N
1.5
OH
H
N
O
O
O
O
H
N
OH
OH
38
39
H
N
>10
>5
O
O
O
O
S
O
N
H
N
>10
>5
the side chains of Thr72 and Arg128. This gain in interactions with
the S30 pocket may relate to the observed increase in efficacy of
this class of inhibitors upon addition of carboxylate functionalities
in P30.
the carboxyl. Moreover, according to modeling results, none of
the carboxyl groups of compounds 27 and 37 interact with Lys75.
Modeling of compounds 23 and 24 could not account for the
differences in potency seen from the change in substitution of
the P1 substituent when going from difluorobenzyloxymethyl ana-
log 23 to the corresponding benzyloxymethyl analog 24.
All the described inhibitors were also tested for inhibition of hu-
man cathepsin D. Generally, the explored variations of the P1 and
P20–P30 side chains in this series of inhibitors are having minor ef-
fects on the cathepsin D selectivity. The variations of P2–P3 side
chains are on the other hand of great importance for the cathepsin
D activity as can be seen for compounds 20, 21, 30, and 31 display-
2.2.2. Modeling of selected compounds in BACE-1 and
cathepsin D
Modeling of compounds 17, 36, and 37, using the structural
information obtained from the crystal structure of 27, revealed
information that can rationalize some of the inconsistency found
in potency. Especially modeling of compound 17 showed a possi-
bility of an additional strong interaction between the P30 p-car-
boxyl group and the side chain of Lys75. This explains the
increased activity of compound 17 over compound 36, lacking
ing cathepsin D IC50 values of 0.015, 0.024, 0.93, and 0.35
lM,
respectively. Compounds 20 and 21 are completely inactive on

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M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
4.1.1. Growth of cells and collection of inclusion bodies
Escherichia coli cells (BL21(DE3)) expressing human BACE-1
were grown in Terrific Broth with 50 g/mL carbenicillin added.
l
When cell density reached OD600 ꢂ 1.3, 1 mM IPTG was added
and the growth was continued for 3 h. The cells were harvested
by centrifugation at 6000g for 10 min at 4 °C. Cell pellets were
stored at ꢁ20 °C. The cells were solubilized in a glass homogenizer
in lysis buffer (50 mM Tris–HCl, pH 7.6, 10 mM EDTA, 50 mM
MgCl2, and 0.1% (w/v) TX-100) with DNAse and Complete protease
inhibitor cocktail (Merck) added. The cells were disrupted in a cell
disrupter (Constant cell system) at 1.7 kbar. The inclusion bodies
were collected from the lysate by centrifugation at 20,000g for
15 min at 4 °C and washed with wash buffer (50 mM Tris–HCl,
pH 7.6, and 100 mM NaCl) with 0.5% Triton X-100 added twice
and twice with wash buffer without the addition of Triton X-100.
Washed inclusion bodies were stored at ꢁ70 °C.
Figure 3. X-ray crystal structure of inhibitor 27 and BACE-1. Depicted are the key
interactions between the inhibitor and the active site.
4.1.2. Denaturing and refolding of BACE-1
Inclusion bodies were dissolved in denaturing buffer (50 mM
Tris–HCl, pH 9, 8 M urea, and 10 mM b-mercaptoethanol) 35 mL
buffer/g cell pellet). The sample was further diluted 1:20 in refold-
ing buffer (20 mM Tris–HCl, pH 9, 0.5 mM oxidized glutathione,
and 1.25 mM reduced glutathione) at room temperature. It was
important that the solutions were thoroughly degassed before
the reducing agents were added. The sample was stirred at 4 °C
for 4 h, the pH was adjusted to 8.0 and the solutions were stirred
for 48–72. The pH was further adjusted to 6.8 and the solutions
were stirred for an additional 48–72 h.
BACE-1 while compounds 30 and 31 are weak BACE-1 inhibitors
with IC50 values of 2.2 and 1.9 lM, respectively.
The excellent potency of compound 20 for cathepsin D can be
explained from docking of compound 20 with the crystal struc-
ture of cathepsin D (1lyb).35 Accordingly, compound 20 shows an
extensive hydrogen bonding network to the active site in
cathepsin D including four strong hydrogen bonds to the flap.
The methionine side chain in the P2 position of the inhibitor is
interacting closely with the side chains of methionine 307 and
309 in the S2 pocket. The difluorophenyloxy P1 group also fits
well having favorable aromatic stacking interactions in the S1
pocket.
4.1.3. Chromatography of BACE-1
Precipitate was removed by centrifugation at 18,500g for 1 h
followed by filtration through a 0.8/02 lm filter. The sample was
loaded onto an equilibrated 5 mL HiTrap Q-sepharose (GE Health
Care) overnight. The column was washed with A-buffer (20 mM
Tris–HCl, pH 7.6, 0.4 M urea) until the absorbance was stabilized.
BACE-1 was eluted by A-buffer with 0.25 M NaCl added and the
peak was collected and concentrated in a 20 mL Viva Science spin
concentrator (MWCO 10 000). The concentrated sample was ap-
plied onto a HiLoad Superdex 200 26/60 (GE Health Care) previ-
ously equilibrated with 20 mM Tris–HCl, pH 7.6. The eluted
monomeric form of BACE-1 could easily be distinguished from
multi-meric forms by running native PAGE. Fractions containing
monomer BACE-1 were pooled and applied to a 1 mL HiTrap Q-se-
pharose equilibrated with 20 mM Tris–HCl buffer, pH 7.6. Pure
BACE-1 was eluted by a gradient of 0–0.25 M NaCl for 20 CV. The
total yield of pure BACE-1 from 1 L of bacterial cell culture was
generally 1–2 mg.
A model of compound 20 in BACE-1 on the other hand reveals
that the large hydrophobic P2 methionine does not accommodate
well to the polar environment of the BACE-1 S2 pocket. The lack
of BACE-1 activity of compound 20 may also be a result of the
bulky and unflexible tert-butoxycarbonyl group that does not fit
well in the narrow path between the flap of Gln73 and the S3
and S4 pockets of BACE-1.
3. Conclusion
Several potent BACE-1 inhibitors have been synthesized com-
prising a statine-based central core containing novel phenyloxy-
methyl- and benzyloxymethyl residues in the P1 position. These
novel templates were obtained by employing an efficient synthesis
route starting from 3-deoxy-1,2-O-isopropylidene-a-D-glucose (1).
Different substituents were evaluated in order to study the SAR
for this inhibitor class. Carboxylate functionalities in P30 position
were found to be of importance in order to obtain inhibitors of high
potency, for example, 17 (13 nM), 22 (14 nM), 27 (37 nM), and 29
(12 nM). X-ray crystal data revealed the binding mode of inhibitor
27.
4.2. BACE-1 assay
The inhibition of BACE-1 was determined in a homogeneous
time resolved fluorescence (TRF) assay (True Point kit, Perkin-El-
mer). The assay buffer contained sodium acetate, CHAPS, Triton-
X 100, and EDTA, pH 4.5. The substrate used was the Swedish mu-
tant sequence Eu-EVNLDAEFK-Quencher.
4. Experimental
Inhibitor dilutions were made in DMSO at 30ꢃ final assay con-
centration. 1
ll per well of inhibitor dilutions were added to 15 ll
4.1. Production of soluble BACE-1
per well of 20 nM BACE-1 in assay buffer on a black half area 96-
well plate. The plate was covered and incubated at room tempera-
Construction of soluble BACE-1. The cDNA for human BACE-1 was
isolated from human brain. Two different expression constructs
were made; one containing residues 42–446 and one containing
residues 42–454. Both were cloned into the expression vector
pET11a. In order to improve protein expression the first 205 bp
of the expression constructs were changed into more common
Escherichia coli codons.
ture for 30 min. The assay was initiated by the addition of 15 ll per
well of 400 nM substrate in assay buffer (final concentration in as-
say 200 nM) and the plate was read for 60 min at room tempera-
ture in
a Victor-2 (Wallac). The excitation wavelength was
340 nm and the emission was monitored at 615 nm. The percent
inhibition was calculated from initial velocities of the inhibited
reactions relative to the uninhibited control. IC50 values were

M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
9479
determined using the following relationship: % inhibition = 100[I]/
([I] + IC50).
nomenex C-18 250 ꢃ 15 mm and Phenomenex C-18 150 ꢃ 4.6 mm
for preparative and analytical runs, respectively; Pump: Gilson gra-
dient pump 322; UV/vis-detector: Gilson 155; MS detector: Ther-
mo Finnigan Surveyor MSQ; Gilson Fraction Collector FC204)
using methanol with 0.1% formic acid and deionized water with
0.1% formic acid as mobile phase.
4.3. Cathepsin D assay
Cathepsin D, purified from human liver, was purchased from
Sigma. The assay buffer was 125 mM sodium acetate, 12.5% glyc-
erol, and 0.0125% Tween 20, pH 3.5. The substrate used was DAB-
CYL-Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS, purchased from
Anaspec. Inhibitor dilutions were made in DMSO at 100ꢃ final as-
say concentration. One microliter per well of inhibitor dilutions
were added to 95 ll per well of 175 ng/mL cathepsin D in assay
buffer on a 96-well plate. The plate was covered and incubated
at 37 °C for 20 min. The assay was initiated by the addition of
4.7. LC–MS purity measurements
4.7.1. Chromatography system A
Column: Phenomenex C-18 150 ꢃ 4.6 mm; Pump: Gilson gra-
dient pump 322; UV/vis-detector: Gilson 155; MS detector:
Thermo Finnigan Surveyor MSQ; Software: Gilson UniPoint 4.0
and Xcalibur 1.3. Gradient: methanol 40–100% over 10 min at
1 mL/min followed by 100% for 5 min at 1 mL/min. To all sol-
vents formic acid (0.1% v/v) was added. Peaks were detected at
254 nm.
5
l
l per well of 40
lM substrate in assay buffer (final concentra-
tion in assay 2 M) and the plate was read for 20 min at 37 °C in
l
a Fluoroskan Ascent (Thermo Labsystems). The excitation wave-
length was 355 nm and the emission was monitored at 500 nm.
Data were fit to the competitive inhibition equation by non-linear
4.7.2. Chromatography system B
regression with the substrate concentration and KM fixed at 2
and 2.7 M, respectively.
lM
As system A except: Gradient: acetonitrile 60–100% over 10 min
at 1 mL/min followed by 100% for 5 min at 1 mL/min.
l
4.4. Crystallography
4.8. General procedures (used in the synthesis and
deprotections of building blocks A–B, E–G, and L)
The crystals were grown using the hanging drop vapor diffusion
method. The protein was concentrated to 5 mg/mL in 20 mM Tris,
pH 7.6, 200 mM sodium chloride. Inhibitory compounds (dissolved
at 10 mM in 16% DMSO and 33% isopropanol) were added to the
concentrated protein in twofold molar excess and allowed for com-
plex formation over 30 min at 4 °C. Hanging drops were formed by
4.8.1. Peptide coupling
To a cooled (0 °C) solution of the acid (4.95 mmol), the amine
(5.44 mmol), and DIPEA (14.84 mmol) in DMF (20 mL) was added
HATU (5.94 mmol). The solution was stirred for 0.5 h and for an
additional 2 h at room temperature. EtOAc and brine were added
and the organic phase was separated and washed once more with
brine, dried and concentrated. The crude peptide was purified by
flash column (FC) chromatography or HPLC.
the addition of 1
5.0, 3–15% PEG8000, and 300 mM lithium sulphate) to 1
tein–compound complex. Typically, single crystals appeared after
one day and grew to dimensions of 200 lm
lL reservoir solution (100 mM sodium citrate, pH
lL of pro-
lm ꢃ 150
lm ꢃ 80
over a period of two weeks. Prior to diffraction experiments, single
crystals were transferred to a cryo-protecting solution that con-
tained the reservoir solution plus 25% glycerol, soaked briefly
and frozen in liquid nitrogen. Crystals diffracted to 2.2–2.8 Å and
belonged to the monoclinic space group P21 with cell dimensions
4.8.2. Boc deprotection
To a solution of the protected compound (0.167 mmol) and tri-
ethylsilane (0.419 mmol) in DCM (3 mL) was added TFA (1 mL).
The solution was stirred for 2 h at room temperature and then con-
centrated and co-concentrated with toluene.
of
a = 83.435°, b = 103.017°, c = 103.110°, and d = 103.50°.
4.8.3. Methyl ester deprotection
4.5. Experimental modeling
To a cooled (0 °C) solution of the ester (0.021 mmol) in dioxane/
water 1:1 (1 mL) was added 1 M LiOH (0.031 mmol) and the mix-
ture was stirred for 0.5 h and for an additional 0.5 h in room tem-
perature. The solution was neutralized with 1 M HCl and
concentrated and co-concentrated with toluene.
Inhouse cocrystal structure of compound 27 in BACE-1 (PDB
code, 3dm6) was used for the analysis of the binding interactions.
Inhibitor 20 was docked and minimized in the crystal structure of
cathepsin D (1lyb).35 All modeling experiments were performed
using SYBYL 7.3 (Tripos Inc. 1699 South Hanley Road, St. Louis,
Missouri, 63144, USA).
4.9. Synthetic procedures
4.9.1. 5-(Methanesulfonyl-methyl-amino)-N-((R)-1-phenyl-
ethyl)-isophthalamic acid (M)
4.6. General methods
Synthesized according to Ref. 24.
NMR spectra were recorded on a Varian 300 MHz instrument
using CDCl3, CD3OD and (CD3)2SO as solvent. TLC was carried out
on Merck precoated 60 F254 plates using UV-light and charring
with ethanol/sulfuric acid/p-anisaldehyde/acetic acid 90:3:2:1,
and a solution of 0.5% ninhydrin in ethanol for visualization. Flash
column (FC) chromatography was performed using silica gel 60
(0.040–0.063 mm, Merck). Organic phases were dried over anhy-
drous magnesium sulfate. Concentrations were performed under
diminished pressure (1–2 KPa) at a bath temperature of 40 °C.
Optical rotations were measured using a Perkin-Elmer 141 polar-
imeter. Isocratic HPLC was performed on a preparative C-18
column.
4.9.2. N,N-Dipropyl-isophthalamic acid (N)
Synthesized in 50% yield over three steps from commercially
available isophthalic acid performing in sequential order: (1)
monoester hydrolysis according to the monoester hydrolysis step
in Ref. 24; (2) peptide coupling using dipropyl amine according
to General procedure 1 (Section 4.8.1); (3) ester hydrolysis accord-
ing to General procedure 3 (Section 4.8.3).
4.9.3. 5-(Methanesulfonyl-methyl-amino)-N-methyl-
isophthalamic acid (O)
Synthesized according to Ref. 24 except for the amide formation
Gradient HPLC-MS (used mainly for mass detection and purity
measurements) was performed on a Gilson system (Column: Phe-
step where methyl amine was coupled instead of R-(+)-
a-
methylbenzylamine.

9480
M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
4.9.4. N-((R)-1-Phenyl-ethyl)-isophthalamic acid (P)
4.9.10. (3S,4S)-4-Azido-5-(3,5-difluorophenoxy)-3-hydroxy-
pentanoic acid (5)
Synthesized in 50% total yield over three steps from commer-
cially available isophthalic acid performing in sequential order:
(1) monoester hydrolysis according to the monoester hydrolysis
step in Ref. 24; (2) coupling of (R)-(ꢁ)-1-phenylethylamine using
General Synthetic Procedure 1 (Peptide coupling); (3) ester hydro-
lysis using General Synthetic Procedure 3 (Section 4.8.3).
Compound 4 (185 mg, 0.542 mmol) was refluxed (115 °C) in
acetic acid/water (1:1, 4 mL) for 1 h. The mixture was concentrated
and the residual was dissolved in tert-butyl alcohol/water (2:3,
4 mL) and sodium periodate (NaIO4) (581 mg, 2.72 mmol) was
added. After stirring for 20 min potassium permanganate (KMnO4)
(12.8 mg, 0.081 mmol) was added and the reaction mixture was al-
lowed to stir for an additional 1.5 h. The mixture was extracted
three times with CHCl3 and the combined organic extracts were
dried and concentrated.
The crude residual was dissolved in water (5 mL) and the mix-
ture was cooled to 0 °C. One molar NaOH (2.2 mL) was added drop
wise and the solution was stirred at 0 °C for 30 min and for an
additional 1 h at room temperature. The reaction mixture was
washed twice with diethyl ether and was then acidified with 6 M
HCl to pH 1–2. The acidified water phase was extracted three times
with CHCl3 and the combined organic extracts were dried and con-
4.9.5. 3-(Methanesulfonyl-methyl-amino)-benzoic acid (Q)
Commercially available 3-amino-benzoic acid methyl ester was
used as starting material. Mesylation and methylation were per-
formed according to Ref. 24 and the final hydrolysis step was per-
formed according to General procedure 3 (Section 4.8.3).
4.9.6. 3-Deoxy-1,2-O-isopropylidene-a-D-glucose (1)
The diol 1 was synthesized from 1,2,5,6-diisopropylidene-D-glu-
cose in 60% yield over three steps according to Refs. 25–27.
4.9.7. 5,6-Anhydro-3-deoxy-1,2-O-isopropylidene-
(2)
a
-
D
-glucose
centrated. FC gradient (toluene/EtOAc 6:1 + 1% HOAc and toluene/
22
EtOAc 2:1 + 1% HOAc) gave 5 (52%) as white crystals. [
a]
D
ꢁ14.0
To a mixture of diol 1 (1.49 g, 7.27 mmol) and triphenylphos-
phine (2.29 g, 8.73 mmol) in chloroform (120 mL) were added
diisopropyl azodicarboxylate (DIAD) (1.72 mL, 8.28 mmol) and
the mixture was stirred at reflux overnight. Concentration and
flash column (FC) chromatography gradient (toluene/EtOAc 15:1
and 9:1) gave 2 (0.91 g, 67%) as a colorless oil. Analytical data in
accordance with Ref. 28.
(c 0.1, MeOH); 1H NMR (300 MHz, CD3OD) d 2.58 (dd, J = 7.7,
15.9 Hz, 1H), 2.65 (dd, J = 5.2, 15.9 Hz, 1H), 3.81–3.88 (m, 1H),
4.14–4.31 (m, 3H), 4.94 (br s, 1H), 6.47–6.64 (m, 3H); 13C NMR
(75.5 MHz, CD3OD) d 39.7, 65.4, 68.7, 70.1, 97.5 (t, JCF = 26.5 Hz),
99.5 (d, JCF = 29.2 Hz, 2C), 161.9, 165.0 (d, JCF = 245.4 Hz), 165.2
(d, JCF = 245.4 Hz), 174.7.
4.9.11. 6-Phenoxy-3-deoxy-1,2-O-isopropylidene-a-D-glucose
(6)
4.9.8. 6-(3,5-Difluorophenoxy)-3-deoxy-1,2-O-isopropylidene-
a
-
D
-glucose (3)
Compound 6 was synthesized in 90% yield (colorless oil)
To epoxide 2 (0.893 g, 4.80 mmol), and 3,5-difluorophenol
according to the synthesis method of compound 3 using phenol in-
22
(0.690 g, 5.30 mmol) in DMF (20 mL) was added K2CO3 (0.332 g,
2.40 mmol). The mixture was heated to 100 °C and stirred over-
night. After cooling the solution aqueous NH4Cl (20 mL) was added
and the reaction mixture was extracted with diethyl ether. The or-
ganic phase was washed twice with water, dried and concentrated.
stead of 3,5-difluorophenol. [
a]
D
ꢁ26.0 (c 0.1, MeOH); 1H NMR
(300 MHz, CDCl3) d 1.27 (s, 3H), 1.49 (s, 3H), 1.81–1.93 (m, 1H),
2.12 (dd, J = 4.5, 13.5 Hz, 1H), 3.45 (d, J = 4.2 Hz, 1H), 3.92 (dd,
J = 6.6, 9.9 Hz, 1H), 4.01 (dd, J = 3.9, 9.9 Hz, 1H), 4.07–4.16 (m,
1H), 4.28–4.37 (m, 1H), 4.64 (app. t, J = 4.1 Hz, 1H), 5.76 (d,
J = 3.6 Hz, 1H), 6.84–6.94 (m, 3H), 7.06–7.14 (m, 2H); 13C NMR
(75.5 MHz, CDCl3) d 25.7, 26.3, 33.4, 68.9, 70.1, 78.1, 80.2, 105.0,
110.8, 114.2, 120.6, 129.0, 158.2.
FC gradient (toluene/EtOAc 15:1, 9:1 and 6:1) gave 3 (1.16 g, 76%)
as a colorless oil. [
22
a
]
D
ꢁ17.0 (c 0.1, MeOH); 1H NMR (300 MHz,
CDCl3) d 1.33 (s, 3H), 1.52 (m, 3H), 1.81–1.96 (m, 1H), 2.16 (dd,
J = 4.2, 13.5 Hz, 1H), 2.62 (d, J = 4.2 Hz, 1H), 3.94 (dd, J = 6.6,
9.6 Hz, 1H), 4.05 (dd, J = 3.9, 9.6 Hz, 1H), 4.09–4.17 (m, 1H), 4.26–
4.35 (m, 1H), 4.74–4.79 (m, 1H), 5.82 (d, J = 3.9 Hz, 1H), 6.38–
6.49 (m, 3H); 13C NMR (75.5 MHz, CDCl3) d 26.1, 26.8, 34.1, 69.9,
70.1, 78.1, 80.6, 96.9 (t, JCF = 25.9 Hz), 98.5 (d, JCF = 27.8 Hz, 2C),
105.5, 111.5, 160.4, 163.6 (d, JCF = 246.6 Hz), 163.8 (d,
JCF = 246.6 Hz).
4.9.12. 5-Azido-6-phenoxy-3,5 dideoxy-1,2-O-isopropylidene-
a-
L
-idose (7)
Compound 7 was synthesized in 84% yield (colorless oil) from 6
22
according to the synthesis method of compound 4. [
a
]
D
ꢁ44.0 (c
0.1, MeOH); 1H NMR (300 MHz, CDCl3) d 1.29 (s, 3H), 1.49 (m, 3H),
1.82–1.96 (m, 1H), 2.07 (dd, J = 4.7, 13.4 Hz, 1H), 3.63–3.73 (m, 1H),
4.10–4.22 (m, 2H), 4.32–4.41 (m, 1H), 4.65–4.72 (m, 1H), 5.79 (d,
J = 3.6 Hz, 1H), 6.87–6.98 (m, 3H), 7.08–7.16 (m, 2H); 13C NMR
(75.5 MHz, CDCl3) d 26.4, 27.1, 35.6, 62.3, 68.7, 77.6, 80.5, 105.8,
111.8, 114.9, 120.5, 129.8, 158.4.
4.9.9. 5-Azido-6-(3,5-difluorophenoxy)-3,5 dideoxy-1,2-O-
isopropylidene-
a
3
-
L
-idose (4)
Compound
(1.16 g, 3.65 mmol) and triphenylphosphine
(1.44 g, 5.64 mmol) were dissolved in dry THF (19 mL). The mix-
ture was cooled to ꢁ15 °C and diisopropyl azodicarboxylate
(DIAD) (1.80 mL, 9.13 mmol) was added. After stirring the solu-
tion for 10 min at ꢁ15 °C the temperature was raised to 0 °C
and diphenylphosphoryl azide (DPPA) (1.23 mL, 5.48 mmol) was
added. The solution was stirred for 30 min at 0 °C and then over-
night at room temperature. Concentration and FC gradient (tolu-
4.9.13. (3S,4S)-4-Azido-3-hydroxy-5-phenoxy-pentanoic acid
(8)
Compound 8 was synthesized in 19% yield (white crystals) from
22
7 according to the synthesis method of compound 5. [
a]
ꢁ15.0
D
(c 0.1, MeOH); 1H NMR (300 MHz, CDCl3) d 2.65 (dd, J = 4.2,
16.6 Hz, 1H), 2.77 (dd, J = 8.6, 16.6 Hz, 1H), 3.78 (ddd, J = 3.5, 5.0,
6.9 Hz, 1H), 4.22 (dd, J = 6.9, 9.9 Hz, 1H), 4.27 (dd, J = 5.0, 9.9 Hz,
1H), 4.27–4.33 (m, 1H), 6.90–7.02 (m, 3H), 7.26–7.33 (m, 2H);
13C NMR (75.5 MHz, CDCl3) d 38.3, 63.5, 67.7, 67.9, 114.6, 121.6,
129.6, 158.0, 177.0.
ene, toluene/EtOAc 24:1, 15:1, 9:1) gave 4 (1.15 g, 92%) as a
22
colorless oil. [
a
]
D
ꢁ21.0 (c 0.1, MeOH); 1H NMR (300 MHz,
CDCl3) d 1.33 (s, 3H), 1.51 (s, 3H), 1.92–2.03 (m, 1H), 2.13 (dd,
J = 4.7, 13.4 Hz, 1H), 3.70–3.78 (m, 1H), 4.17–4.22 (m, 2H),
4.36–4.44 (m, 1H), 4.74–4.80 (m, 1H), 5.84 (d, J = 3.6 Hz, 1H),
6.41–6.51 (m, 3H); 13C NMR (75.5 MHz, CDCl3) d 26.2, 26.8,
35.3, 61.6, 69.2, 77.1, 80.2, 97.2 (t, JCF = 25.8 Hz), 98.6 (d,
JCF = 28.9 Hz, 2C), 105.6, 111.7, 160.0, 163.6, (d, JCF = 247.1 Hz)
163.8 (d, JCF = 246.8 Hz).
4.9.14. 6-(3,5-Difluorobenzyloxy)-3-deoxy-1,2-O-
isopropylidene-
Compound
8.48 mmol) were refluxed in toluene (46 mL) for 5 h. (The water
a-
D-glucose (9)
1
(1.50 g, 7.35 mmol) and Bu2SnO (2.11 g,

M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
9481
produced from the acetal formation was removed using a Dean-
Stark trap.) The mixture was allowed to cool down to 90 °C and tet-
rabutylammonium bromide and 3,5-difluorobenzyl bromide were
added. The solution was stirred at 90 °C overnight and concen-
1.263 mmol) in DMF (4 mL) was added HATU (193 mg,
0.508 mmol) and the mixture was stirred for 0.5 h and for an addi-
tional 2 h at room temperature. EtOAc and brine were added and
the organic phase was separated and washed once more with
brine, dried and concentrated. Purification by HPLC (MeOH/H2O
85:15 + 0.2% TFA) gave 15 (170 mg, 76%) as a colorless solid. 1H
NMR (300 MHz, CDCl3/CD3OD 1:1) d 0.93 (d, J = 6.9 Hz, 6H), 2.00–
2.15 (m, 1H), 2.50 (dd, J = 7.7, 14.6 Hz, 1H), 2.57 (dd, J = 5.1,
14.6 Hz, 1H), 3.71–3.79 (m, 1H), 3.87 (s, 3H), 4.09–4.25 (m, 4H),
4.43 (d, J = 6.0 Hz, 2H), 6.38–6.54 (m, 3H), 7.34 (d, J = 8.1 Hz, 2H),
7.72 (b, 1H), 7.87 (d, J = 8.1 Hz, 2H), 8.32 (b, 1H); 13C NMR
(75.5 MHz, CDCl3/CD3OD 1:1) d 18.4, 19.5, 40.6, 42.1, 43.5, 52.4,
63.6, 64.8, 68.4, 69.3, 97.2 (t, JCF = 26.2 Hz), 99.0 (d, JCF = 27.8 Hz,
2C), 128.0, 129.5, 130.3, 144.4, 160.9, 164.2 (d, JCF = 246.5 Hz),
164.4 (d, JCF = 246.5 Hz), 167.9, 172.6, 172.9.
trated. FC (toluene/EtOAc 2:1) gave 9 (2.28 g, 94%) as a colorless
22
oil. [a]
ꢁ24.0 (c 0.1, MeOH); 1H NMR (300 MHz, CDCl3) d 1.30
D
(s, 3H), 1.49 (s, 3H), 1.78–1.90 (m, 1H), 2.07 (dd, J = 4.6, 13.8 Hz,
1H), 2.52 (br s, 1H), 3.49 (dd, J = 6.8, 9.9 Hz, 1H), 3.59 (dd, J = 3.9,
9.9 Hz, 1H), 3.94–4.02 (m, 1H), 4.17–4.26 (m, 1H), 4.51 (s, 2H),
4.73 (app. t, J = 4.2 Hz, 1H), 5.78 (d, J = 3.9 Hz, 1H), 6.64–6.74 (m,
1H), 6.79–6.89 (m, 2H); 13C NMR (75.5 MHz, CDCl3) d 26.1, 26.7,
33.9, 70.9, 71.7, 72.1, 78.2, 80.5, 102.9 (t, JCF = 25.3 Hz), 105.3,
109.9 (d, JCF = 25.2 Hz, 2C), 111.3, 142.0, 163.0 (d, JCF = 248.5 Hz),
163.1 (d, JCF = 248.5 Hz).
4.9.15. 5-Azido-6-(3,5-difluorobenzyloxy)-3,5 dideoxy-1,2-O-
isopropylidene-
a
-L
-idose (10)
4.10.1.2. Synthetic protocol B. Azide reduction and amide bond
Compound 10 was synthesized in 77% yield (colorless oil) from
formation.
4-[((S)-2-{(3S,4S)-5-(3,5-Difluoro-phenoxy)-3-
22
9 according to the synthesis method of compound 4. [
a
]
D
ꢁ35.0
hydroxy-4-[3-(methanesulfonyl-methyl-amino)-5-((R)-1-phenyl-
ethylcarbamoyl)-benzoylamino]-pentanoylamino}-3-methyl-
butyrylamino)-methyl]-benzoic acid methyl ester (16). Azide 15
(160 mg, 0.300 mmol) and PPh3 (118 mg, 0.450 mmol) were dis-
solved in MeOH (15 mL). A few drops of water were added and
the mixture was stirred overnight. The solvent was evaporated
and the residual was purified by HPLC (MeOH/H2O 80:20 + 0.2%
TEA).
(c 0.1, MeOH); 1H NMR (300 MHz, CDCl3) d 1.31 (s, 3H), 1.49 (s,
3H), 1.84–1.96 (m, 1H), 2.07 dd, J = 4.5, 13.2 Hz, 1H), 3.52–3.60
(m, 1H), 3.71–3.78 (m, 2H), 4.32 (app. dt, J = 4.2 Hz, 1H), 4.55 (s,
2H), 4.71–4.77 (m, 1H), 5.81 (d, J = 3.6 Hz, 1H), 6.67–6.77 (m,
1H), 6.83–6.91 (m, 2H); 13C NMR (75.5 MHz, CDCl3) d 26.2, 26.8,
35.9, 62.5, 71.0, 72.3, 77.3, 80.3, 103.1 (t, JCF = 25.3 Hz), 105.5,
109.9 (d, JCF = 25.2 Hz, 2C), 111.6, 141.9, 163.1 (d, JCF = 248.8 Hz),
163.3 (d, JCF = 248.8 Hz).
The amine was subsequently coupled to acid M (102 mg,
0.271 mmol) according to synthetic protocol A. Purification by
4.9.16. (3S,4S)-4-Azido-5-(3,5-difluorobenzyloxy)-3-hydroxy-
pentanoic acid (11)
HPLC (MeOH/H2O 85:15 + 0.2% TEA) afforded 16 (colorless solid)
22
in 35% yield over two steps. [
a
]
D
ꢁ6.4 (c 0.14, MeOH); 1H NMR
Compound 11 was synthesized in 51% yield (white crystals)
(300 MHz, CDCl3) d 0.86 (app. t, J = 6.9 Hz, 6H), 1.52 (d, J = 6.9 Hz,
3H), 1.95–2.10 (m, 1H), 2.24–2.34 (m, 2H), 2.35–2.49 (m, 1H),
2.79 (s, 3H), 3.23 (s, 3H), 3.84 (s, 3H), 4.06–4.17 (m, 2H), 4.21–
4.49 (m, 5H), 4.82 (br s, 1H), 5.15–5.28 (m, 1H), 6.30–6.45 (m,
3H), 7.14–7.29 (m, 5H), 7.30–7.36 (m, 2H), 7.43–7.51 (m, 1H),
7.53–7.64 (m, 2H), 7.85 (d, J = 8.1 Hz, 2H), 7.92 (d, J = 7.8 Hz, 2H),
8.24 (s, 1H); 13C NMR (75.5 MHz, CDCl3) d 18.3, 19.3, 21.7, 30.8,
35.7, 37.9, 40.2, 43.1, 49.9, 52.1, 53.3, 59.2, 67.3, 67.7, 96.5 (t,
JCF = 26.2 Hz), 98.5 (d, JCF = 28.3 Hz, 2C), 124.7, 126.2, 127.4,
127.5, 127.9, 128.2, 128.7, 129.2, 129.8, 135.2, 136.1, 142.1,
143.1, 143.2, 160.2, 163.5 (d, JCF = 246.5 Hz), 163.7 (d,
JCF = 246.8 Hz), 165.0, 166.5, 166.8, 171.8, 172.2. HRMS calcd
(M+H)+: 866.3246; found 866.3271. LC–MS Purity System A:
tR = 9.20 min, 98%; System B: tR = 8.96 min, 97%.
from 10 according to the synthesis method of compound 5.
22
[
a]
D
ꢁ16.0 (c 0.1, MeOH); 1H NMR (300 MHz, CD3OD) d 2.53
(dd, J = 8.1, 15.8 Hz, 1H), 2.60 (dd, J = 5.1, 15.8 Hz, 1H), 3.62–3.69
(m, 1H), 3.70–3.83 (m, 2H), 4.16 (ddd, J = 3.6, 5.1, 8.1 Hz, 1H),
4.57 (s, 2H), 6.77–6.87 (m, 1H), 6.91–7.01 (m, 2H); 13C NMR
(75.5 MHz, CD3OD)
d 39.8, 66.0, 68.8 71.8, 72.7, 103.5 (t,
JCF = 25.8 Hz), 110.8 (d, JCF = 25.2 Hz, 2C), 144.2, 164.4 (d,
JCF = 247.1 Hz), 164.5 (d, JCF = 247.1 Hz), 174.8.
4.9.17. 6-Benzyloxy-3-deoxy-1,2-O-isopropylidene-a-D-glucose
(12)
Compound 12 was synthesized in 88% yield (colorless oil)
according to the synthesis method of compound 9 using benzyl
bromide. Analytical data in accordance with Ref. 20.
4.10.1.3. Synthetic protocol C. Hydrolysis of ester. 4-[((S)-2-
{(3S,4S)-5-(3,5-Difluoro-phenoxy)-3-hydroxy-4-[3-(methane-
sulfonyl-methyl-amino)-5-((R)-1-phenyl-ethylcarbamoyl)-ben-
zoylamino]-pentanoylamino}-3-methyl-butyrylamino)-methyl]-
4.9.18. 5-Azido-6-benzyloxy-3,5-dideoxy-1,2-O-isopropylidene-
a-
L-idose (13)
Compound 13 was synthesized in 99% yield (colorless oil) from
12 according to the synthesis method of compound 4. Analytical
data in accordance with Ref. 20.
benzoic acid (17).
(22 mg, 0.025 mmol) in THF/MeOH/H2O 2:1:1 (2 mL) was added
1 M LiOH (100 L, 0.100 mmol). The solution was stirred overnight
To a cooled (0 °C) solution of ester 16
l
4.9.19. (3S,4S)-4-Azido-5-benzyloxy-3-hydroxy-pentanoic acid
(14)
Compound 14 was synthesized in 29% yield (white crystals)
from 13 according to the synthesis method of compound 5. Analyt-
ical data in accordance with Ref. 20.
and acidified to approximately, pH 3–4, with 1 M HCl. Concentra-
tion and purification by HPLC twice using MeOH/H2O
80:20 + 0.2% TEA as the first eluent and MeOH/H2O 80:20 + 0.2%
TFA as the second eluent gave 17 (16 mg, 74%) as a colorless solid.
22
[a
]
D
ꢁ11 (c 0.1, MeOH); 1H NMR (300 MHz, CD3OD) d 0.94 (d,
J = 6.8 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H), 1.57 (d, J = 6.9 Hz, 3H),
2.09–2.24 (m, 1H), 2.48–2.65 (m, 2H), 2.96 (s, 3H), 3.35 (s, 3H),
4.11–4.28 (m, 3H), 4.39–4.55 (m, 4H), 5.19–5.31 (m, 1H), 6.44–
6.59 (m, 3H), 7.18–7.35 (m, 4H), 7.36–7.44 (m, 3H), 7.90 (d,
J = 8.4 Hz, 2H), 8.02–8.09 (m, 2H), 8.28 (s, 1H); 13C NMR
(75.5 MHz, CD3OD) d 17.2, 18.6, 20.9, 30.2, 34.8, 37.2, 40.1, 42.7,
49.8, 52.9, 59.6, 67.4, 67.7, 96.0 (t, JCF = 26.2 Hz), 98.4 (d,
JCF = 28.1 Hz, 2C), 125.0, 126.1, 127.0, 127.3, 128.1, 128.4, 129.5,
129.7, 130.5, 135.8, 136.2, 142.5, 143.8, 144.2, 161.0, 163.9 (d,
4.10. Target compounds
4.10.1. Synthetic protocols for target compounds
4.10.1.1. Synthetic protocol A. Peptide coupling. 4-({(S)-2-
[(3S,4S)-4-Azido-5-(3,5-difluoro-phenoxy)-3-hydroxy-pentanoyl-
amino]-3-methyl-butyrylamino}-methyl)-benzoic acid methyl
ester (15). To a cooled (0 °C) solution of acid 5 (121 mg, 0.421
mmol), amine
A (175 mg, 0.480 mmol) and DIPEA (220 lL,

9482
M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
JCF = 245.6 Hz), 164.1 (d, JCF = 245.6 Hz), 167.8, 168.4, 172.6,172.7,
172.9. HRMS calcd (M+H)+: 852.3090; found 852.3109. LC–MS Pur-
ity System A: tR = 8.33 min, 100%; System B: tR = 8.17 min, 100%.
816.3309. LC–MS Purity System A: tR = 7.67 min, 100%; System B:
tR = 7.87 min, 99%.
4.10.5. 4-[((S)-2-{(3S,4S)-5-(3,5-Difluoro-benzyloxy)-3-hydroxy-
4-[3-(methanesulfonyl-methyl-amino)-5-((R)-1-phenyl-ethyl-
carbamoyl)-benzoylamino]-pentanoylamino}-3-methyl-
butyrylamino)-methyl]-benzoic acid (23)
4.10.1.4. Synthetic protocol D. Hydrolysis of diester. Diesters
were hydrolyzed according to General protocol C using eight equiv-
alents of LiOH instead of four.
Compound 23 (colorless solid) was synthesized in four steps
4.10.2. 4-({(S)-2-[(3S,4S)-4-[(S)-2-((S)-2-tert-Butoxycarbonyl-
amino-3-methyl-butyrylamino)-4-methylsulfanyl-butyryl-
amino]-5-(3,5-difluoro-phenoxy)-3-hydroxy-pentanoylamino]-
3-methyl-butyrylamino}-methyl)-benzoic acid methyl ester (20)
Compound 20 (colorless solid) was synthesized in three steps
from central core 11 according to synthetic protocols A, B, and C
22
using amine A and acid M. [
a]
ꢁ17 (c 0.15, MeOH); 1H NMR
D
(300 MHz, CD3OD) d 0.94 (d, J = 6.8 Hz, 3H), 0.96 (d, J = 6.8 Hz,
3H), 1.58 (d, J = 7.2 Hz, 3H), 2.10–2.24 (m, 1H), 2.51 (dd, J = 6.9,
14.6 Hz, 1H), 2.60 (dd, J = 7.1, 14.6 Hz, 1H), 2.95 (s, 3H), 3.48 (s,
3H), 3.64–3.78 (m, 2H), 4.24 (d, J = 6.0 Hz, 1H), 4.33–4.43 (m,
2H), 4.95 (s, 4H), 5.19–5.31 (m, 1H), 6.72–6.82 (m, 1H), 6.84–
6.94 (m, 2H), 7.19–7.26 (m, 1H), 7.28–7.36 (m, 2H), 7.37–7.44
(m, 4H), 7.92 (d, J = 8.4 Hz, 2H), 8.01–8.05 (m, 2H), 8.26 (s, 1H);
13C NMR (75.5 MHz, CD3OD) d 18.4, 19.7, 22.1, 31.4, 36.0, 38.4,
41.3, 43.7, 51.0, 54.2, 60.7, 68.8, 71.0, 72.5, 103.4 (t, JCF = 25.8 Hz),
111.0 (d, JCF = 25.5 Hz, 2C), 126.1, 127.3, 128.2, 128.5, 129.2, 129.5,
129.6, 130.7, 130.9, 137.2, 137.4, 143.7, 144.4, 145.0, 145.5, 164.4 4
(d, JCF = 246.8 Hz), 164.6 4 (d, JCF = 247.4 Hz), 167.8, 169.0, 169.6,
173.9, 174.0. HRMS calcd (M+H)+: 866.3246; found 866.3276.
LC–MS Purity System A: tR = 8.19 min, 99%; System B:
tR = 8.12 min, 100%.
from central core 5 according to synthetic protocols A and B using
22
amine A and acid L. [
a
]
D
ꢁ30 (c 0.05, MeOH); 1H NMR (300 MHz,
CDCl3) d 0.86–1.02 (m, 12H), 1.40 (s, 9H), 1.88–2.10 (m, 3H), 2.02
(s, overlapped, 3H), 2.11–2.27 (m, 1H), 2.39–2.56 (m, 4H), 3.79–
3.88 (m, 1H), 3.82 (s, overlapped, 3H), 3.89–3.96 (m, 2H), 3.97–
4.11 (m, 2H), 4.16 (d, J = 6.0 Hz, 1H), 4.22–4.38 (m, 3H), 6.30–
6.48 (m, 3H), 7.35 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 8.6 Hz, 2H); 13C
NMR (75.5 MHz, CDCl3) d 15.3, 18.1, 18.3, 19.4, 19.6, 28.5, 30.4,
30.6, 30.9, 38.8, 40.6, 43.2, 52.3, 54.5, 60.3, 61.4, 67.7, 68.0, 68.7,
81.1, 96.8 (t, JCF = 25.9 Hz), 98.7 (d, JCF = 28.1 Hz, 2C), 127.8,
129.2, 130.1, 144.7, 151.9, 160.9, 164.1 (d, JCF = 244.8 Hz), 164.3
(d, JCF = 244.8 Hz), 167.8, 172.3, 173.0, 173.5, 174.0. HRMS calcd
(M+H)+: 838.3872; found 838.3906. LC–MS Purity System A:
tR = 10.25 min, 98%; System B: tR = 3.41 min, 99%.
4.10.6. 4-[((S)-2-{(3S,4S)-5-Benzyloxy-3-hydroxy-4-[3-
(methanesulfonyl-methyl-amino)-5-((R)-1-phenyl-ethyl-
carbamoyl)-benzoylamino]-pentanoylamino}-3-methyl-
butyrylamino)-methyl]-benzoic acid (24)
4.10.3. 4-({(S)-2-[(3S,4S)-4-[(S)-2-((S)-2-tert-Butoxycarbonyl-
amino-3-methyl-butyrylamino)-4-methylsulfanyl-butyryl-
amino]-5-(3,5-difluoro-phenoxy)-3-hydroxy-pentanoylamino]-
3-methyl-butyrylamino}-methyl)-benzoic acid (21)
Compound 24 (colorless solid) was synthesized in four steps
from central core 14 according to synthetic protocols A, B, and C
22
Compound 21 (colorless solid) was synthesized in four steps
using amine A and acid M. [
a]
ꢁ20 (c 0.14, MeOH); 1H NMR
D
from central core 5 according to synthetic protocols A, B, and C
(300 MHz, CD3OD) d 0.92 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.9 Hz,
3H) 1.57 (d, J = 7.1 Hz, 3H), 2.09–2.23 (m, 1H), 2.48 (dd, J = 7.1,
14.6 Hz, 1H), 2.57 (dd, J = 6.7, 14.6 Hz, 1H), 2.94 (s, 3H), 3.34 (s,
3H), 3.65–3.72 (m, 2H), 4.23 (d, J = 6.2 Hz, 1H), 4.32–4.40 (m,
2H), 4.46 (s, 2H), 4.49 (d, J = 11.8 Hz, 2H), 5.24 (q, J = 7.1 Hz, 1H),
7.17–7.36 (m, 7H), 7.36–7.42 (m, 5H), 7.92 (d, J = 8.5 Hz, 2H),
8.00–8.02 (m, 2H), 8.25 (s, 1H); 13C NMR (75.5 MHz, CD3OD) d
18.4, 19.7, 22.1, 31.4, 36.0, 38.4, 41.4, 43.8, 51.0, 54.3, 60.7, 69.0,
70.7, 74.0, 126.1, 127.3, 128.2, 128.4, 128.5, 128.7, 128.9, 129.1,
129.1, 129.2, 129.3, 129.4, 129.6, 130.6, 130.9, 137.2, 137.3,
139.4, 143.7, 145.0, 145.4, 167.8, 168.9, 169.6, 173.9, 174.0. HRMS
calcd (M+H)+: 830.3435; found 830.3445. LC–MS Purity System A:
tR = 7.66 min, 100%; System B: tR = 7.88 min, 100%.
22
using amine A and acid L. [
a]
D
ꢁ20 (c 0.05, MeOH); 1H NMR
(300 MHz, CD3OD) d 0.90–1.06 (m, 12H), 1.43 (s, 9H), 1.91–2.12
(m, 3H), 2.06 (s, overlapped, 3H), 2.14–2.24 (m, 1H), 2.45–2.57
(m, 3H), 2.58–2.66 (m, 1H), 3.85 (d, J = 6.6 Hz, 1H), 3.92–4.10 (m,
2H), 4.12–4.26 (m, 2H), 4.28–4.38 (m, 1H), 4.44–4.55 (m, 3H),
6.45–6.57 (m, 3H), 7.39 (d, J = 8.3 Hz, 2H), 7.92 (d, J = 8.3 Hz, 2H);
13C NMR (75.5 MHz, CD3OD) d 15.3, 18.4, 18.7, 18.8, 19.7, 19.8,
28.7, 31.1, 31.5, 31.5, 32.1, 41.2, 43.7, 53.3, 54.6, 60.9, 62.1, 68.2,
68.8, 80.9, 97.2 (t, JCF = 27.3 Hz), 99.5 (d, JCF = 28.1 Hz, 2C), 128.4,
130.9, 145.0, 154.5, 158.4, 165.0 (d, JCF = 245.5 Hz), 165.2 (d,
JCF = 245.5 Hz), 170.4, 170.9, 173.6, 174.0, 175.1. HRMS calcd
(M+H)+: 824.3716; found 824.3741. LC–MS Purity System A:
tR = 9.22 min, 100%; System B: tR = 7.01 min, 97%.
4.10.7. 4-[((S)-2-{(3R,4S)-5-Benzyloxy-3-hydroxy-4-
[3-(methanesulfonyl-methyl-amino)-5-((R)-1-phenyl-ethyl-
carbamoyl)-benzoylamino]-pentanoylamino}-3-methyl-
butyrylamino)-methyl]-benzoic acid (25)
4.10.4. 4-[((S)-2-{(3S,4S)-3-Hydroxy-4-[3-(methanesulfonyl-methyl-
amino)-5-((R)-1-phenyl-ethylcarbamoyl)-benzoyl- amino]-5-
phenoxy-pentanoylamino}-3-methyl-butyrylamino)-methyl]-
benzoic acid (22)
Compound 25 (colorless solid) was synthesized in three steps
Compound 22 (colorless solid) was synthesized in four steps
from compound (R)-19 (below) according to synthetic protocols
22
from central core 8 according to synthetic protocols A, B, and C
B and C using acid M. [
a
]
D
ꢁ11 (c 0.14, MeOH); 1H NMR
22
using amine A and acid M. [
a]
ꢁ7.1 (c 0.1, MeOH); 1H NMR
(300 MHz, CD3OD) d 0.90 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.9 Hz,
3H), 1.56 (d, J = 7.2 Hz, 3H), 2.06–2.19 (m, 1H), 2.55–2.58 (m,
2H), 2.93 (s, 3H) 3.34 (s, 3H), 3.74–3.79 (m, 2H), 4.16–4.25 (m,
1H), 4.21 (d, overlapped, J = 6.6 Hz, 1H), 4.25–4.33 (m, 1H), 4.41–
4.47 (m, 2H), 4.52 (s, 2H), 5.23 (q, J = 7.2 Hz, 1H), 7.19–7.34 (m,
8H), 7.36–7.41 (m, 4H), 7.91 (s, 1H), 7.94 (s, 1H) 7.98–8.02 (m,
2H) 8.21 (s, 1H); 13C NMR (75.5 MHz, CD3OD) d 18.4, 19.7, 22.1,
31.4, 36.0, 38.4, 41.0, 43.7, 51.0, 55.6, 60.6, 69.8, 70.0, 74.2, 126.1,
127.3, 128.2, 128.5, 128.7, 129.0, 129.2, 129.3, 129.4, 129.6,
130.7, 131.0, 137.2, 137.4, 139.5, 143.7, 145.0, 145.3, 167.8,
168.5, 169.6, 173.9, 174.6. HRMS calcd (M+H)+: 830.3435; found
D
(300 MHz, CD3OD) d 0.94 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.9 Hz,
3H), 1.57 (d, J = 7.1 Hz, 3H), 2.10–2.23 (m, 1H), 2.54 (dd, J = 7.3,
14.6 Hz, 1H), 2.62 (dd, J = 6.7, 14.6 Hz, 1H), 2.96 (s, 3H), 3.35 (s,
3H), 4.15–4.27 (m, 3H), 4.42–4.53 (m, 4H), 5.25 (q, J = 7.1 Hz,
1H), 6.86–6.98 (m, 3H), 7.17–7.43 (m, 8H), 7.92 (d, J = 8.4 Hz,
2H), 8.01–8.06 (m, 3H), 8.26 (s, 1H); 13C NMR (75.5 MHz, CD3OD)
d 18.4, 19.7, 22.1, 31.4, 36.0, 38.4, 41.3, 43.8, 51.0, 54.4, 60.7,
68.2, 68.8, 115.8, 122.1, 126.1, 127.3, 128.2, 128.5, 129.2, 129.6,
130.5, 130.9, 137.1, 137.4, 143.7, 145.0, 145.4, 160.1, 167.7,
169.0, 173.9, 174.0. HRMS calcd (M+H)+: 816.3278; found

M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
9483
830.3450. LC–MS Purity System A: tR = 7.87 min, 100%; System B:
tR = 7.94 min, 100%.
6H), 4.15–4.27 (m, 2H), 4.31 (d, J = 6.6 Hz 1H), 4.40–4.48 (m, 1H),
4.50–4.58 (m, 1H), 5.25 (q, J = 7.0 Hz, 1H), 6.36–6.45 (m, 1H),
6.46–6.56 (m, 2H), 7.18–7.26 (m, 1H), 7.27–7.34 (m, 2H), 7.37–
7.43 (m, 2H), 8.04 (s, 1H), 8.10 (s, 1H), 8.28–8.33 (m, 2H), 8.53 (s,
2H); 13C NMR (75.5 MHz, CDCl3) d 18.7, 19.7, 22.1, 31.8, 36.1,
38.4, 41.3, 51.0, 52.9, 54.1, 61.6, 68.7, 68.9, 97.2 (t, JCF = 26.3 Hz),
99.5 (d, JCF = 28.9 Hz, 2C), 126.2, 126.2, 126.7, 127.3, 128.2, 129.5,
129.5, 129.5, 132.3, 132.4, 137.2, 137.4, 140.4, 143.8, 145.0,
162.2, 164.9 (d, JCF = 245.1 Hz), 165.2 (d, JCF = 244.7 Hz), 167.3,
167.8, 169.2, 172.8, 173.9. HRMS calcd (M+H)+: 910.3145; found
910.3165. LC–MS Purity System A: tR = 9.84 min, 100%; System B:
tR = 9.39 min, 100%.
4.10.7.1. (3S,4S)-4-Azido-5-benzyloxy-3-hydroxy-pentanoic
acid methyl ester (18).
(550 mg, 2.07 mmol) in MeOH (10 mL) was added thionyl chloride
(226 L, 3.11 mmol) and the mixture was stirred for 16 h and con-
centrated. Purification by FC (toluene/ethyl acetate 4:1) gave
methyl ester 18 (438 mg, 76%) as
colorless oil. 1H NMR
To a cooled solution (0 °C) of acid 14
l
a
(300 MHz, CD3OD) d 2.53 (dd, J = 7.6, 15.6 Hz, 1H), 2.58 (dd,
J = 5.6, 15.6 Hz, 1H), 3.57 (ddd, J = 3.8, 4.9, 6.9 Hz, 1H), 3.66 (s,
3H), 3.69 (dd, J = 6.9, 10.1 Hz, 1H), 3.74 (dd, J = 4.9, 10.1 Hz, 1H),
4.14 (ddd, J = 3.8, 5.6, 7.6 Hz, 1H), 4.52 (d, J = 11.9 Hz, 1H), 4.55
(d, J = 11.9 Hz, 1H), 7.23–7.37 (m, 5H); 13C NMR (75.5 MHz, CD3OD)
d 39.8, 52.2, 66.0, 68.9, 71.2, 74.2, 128.7, 128.8, 129.4, 139.2, 173.3.
4.10.9. 5-((S)-2-{(3S,4S)-5-(3,5-Difluoro-phenoxy)-3-hydroxy-4-
[3-(methanesulfonyl-methyl-amino)-5-((R)-1-phenyl-ethyl-
carbamoyl)-benzoylamino]-pentanoylamino}-3-methyl-
butyrylamino)-isophthalic acid (27)
4.10.7.2. 4-{[(S)-2-((3R,4S)-4-Azido-5-benzyloxy-3-hydroxy-
pentanoylamino)-3-methyl-butyrylamino]-methyl}-benzoic
Compound 27 (colorless solid) was synthesized in four steps
acid methyl ester ((R)-19).
To a stirred solution of Dess–Martin
from central core 5 according to synthetic protocols A, B, and D
22
periodinane (167 mg, 0.394 mmol) in dry DCM (2.5 mL) was added
a solution of compound 18 (100 mg, 0.358 mmol) in dry DCM
(2 mL). After stirring for 40 min the solution was diluted with ether
(5 mL) and poured into a solution of saturated NaHCO3 (15 mL)
containing a sevenfold excess of Na2S2O3 (435 mg, 2.75 mmol).
Stirring was continued until the solid was dissolved and the layers
were separated. The water phase was extracted once more with
ether and the combined ether layers were washed twice with
water, dried and concentrated. The crude ketone was dissolved in
MeOH (6 mL). The solution was cooled to ꢁ15 °C before adding
NaBH4 (25 mg, 0.658 mmol). After 10 min the reaction was
quenched with brine (10 mL) and the MeOH was evaporated. The
remaining aqueous phase was extracted three times with EtOAc
and the combined organic phases were dried and concentrated.
The diastereomeric mixture of alcohols was dissolved in dioxane/
using amine B and acid M. [
a
]
D
ꢁ25 (c 0.12, MeOH); 1H NMR
(300 MHz, CD3OD) d 1.01 (d, J = 6.6 Hz, 3H), 1.02 (d, J = 6.6 Hz,
3H), 1.57 (d, J = 7.2 Hz, 3H), 2.12–2.27 (m, 1H), 2.56 (dd, J = 7.1,
14.6 Hz, 1H), 2.64 (dd, J = 6.5, 14.6 Hz, 1H), 2.97 (s, 3H), 3.36 (s,
3H), 4.14–4.27 (m, 2H), 4.28–4.36 (m, 1H), 4.40–4.50 (m, 1H),
4.51–4.60 (m, 1H), 5.25 (q, J = 6.9 Hz, 1H), 6.38–6.48 (m, 1H),
6.49–6.59 (m, 2H), 7.17–7.26 (m, 1H), 7.27–7.36 (m, 2H), 7.37–
7.44 (m, 2H), 8.03 (s, 1H), 8.09 (s, 1H), 8.29 (s, 1H), 8.35 (s, 1H),
8.26 (s, 2H); 13C NMR (75.5 MHz, CD3OD) d 18.7, 19.7, 22.1, 31.9,
36.0, 38.4, 41.2, 51.0, 54.3, 61.5, 68.6, 68.8, 97.2 (t, JCF = 26.2 Hz),
99.6 (d, JCF = 28.9 Hz, 2C), 126.1, 126.5, 127.3, 127.4, 128.2, 129.4,
129.6, 133.0, 137.2, 137.4, 140.2, 143.8, 145.0, 162.2, 165.0 (d,
JCF = 245.1 Hz), 165.2 (d, JCF = 245.4 Hz), 167.8, 168.5, 169.1,
172.8, 173.9. HRMS calcd (M+H)+: 882.2832; found 882.2856.
LC–MS Purity System A: tR = 8.14 min, 100%; System B:
tR = 8.05 min, 100%.
water 1:1 (4 mL) and cooled to 0 °C before adding LiOH (580 lL,
0.580 mmol). After stirring for 1 h the solution was neutralized
with 1 M HCl and concentrated and co-concentrated with toluene.
The crude carboxylic acid and amine A (141 mg, 0.387 mmol) was
dissolved in DMF (3 mL) and the solution was cooled to 0 °C. DIPEA
4.10.10. 5-((S)-2-{(3S,4S)-5-(3,5-Difluoro-benzyloxy)-3-
hydroxy-4-[3-(methanesulfonyl-methyl-amino)-5-((R)-1-
phenyl-ethylcarbamoyl)-benzoylamino]-pentanoylamino}-3-
methyl-butyrylamino)-isophthalic acid (28)
(168 lL, 0.967 mmol) and HATU (140 mg, 0.368 mmol) were added
and the mixture was stirred for 0.5 h and for an additional 2 h at
room temperature. Concentration and purification by HPLC
(MeOH/H2O 80:20 + 0.2% TFA) gave 77 mg (white solid) of the first
eluted compound (the S-isomer) and 64 mg (white solid) of the
second eluted compound (R)-19, altogether in 74% total yield over
four steps. 1H NMR (R-isomer) (300 MHz, CDCl3) d 0.92 (d,
J = 6.7 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H), 2.03–2.17 (m, 1H), 2.39
(dd, J = 9.1, 14.8 Hz, 1H), 2.51 (dd, J = 2.8, 14.8 Hz, 1H), 3.50–3.57
(m, 1H), 3.58–3.65 (m, 1H), 3.75 (dd, J = 3.7, 9.8 Hz, 1H), 3.88 (s,
3H), 3.93–4.02 (m, 1H), 4.26–4.36 (m, 2H), 4.44 (dd, J = 6.0,
15.4 Hz, 1H), 4.53 (s, 2H), 5.20 (br s, 1H), 7.01 (b, 1H), 7.20–7.38
(m, 8H), 7.93 (d, J = 8.2 Hz, 2H); 13C NMR (R-isomer) (75.5 MHz,
CDCl3) d 18.2, 19.2, 30.1, 39.1, 43.2, 52.1, 59.0, 64.6, 68.8, 69.8,
73.5, 127.4, 127.6, 127.9, 128.5, 129.3, 129.9, 137.4, 142.8, 166.7,
171.7, 172.4.
Compound 28 (colorless solid) was synthesized in four steps
from central core 11 according to synthetic protocols A, B, and D
22
using amine B and acid M. [
a
]
D
ꢁ39 (c 0.1, MeOH); 1H NMR
(300 MHz, CD3OD) d 1.02 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 6.8 Hz,
3H), 1.58 (d, J = 6.9 Hz, 3H), 2.13–2.26 (m, 1H), 2.52 (dd, J = 7.1,
14.6 Hz, 1H), 2.62 (dd, J = 6.8, 14.6 Hz, 1H), 2.96 (s, 3H), 3.36 (s,
3H), 3.69 (dd, J = 7.2, 10.0 Hz, 1H), 3.78 (dd, J = 6.5, 10.0 Hz, 1H),
4.28–4.40 (m, 2H), 4.43–4.47 (m, 1H), 4.47 (s, overlapped, 2H),
5.25 (q, J = 6.9 Hz, 1H), 6.69–6.78 (m, 1H), 6.81–6.86 (m, 2H),
7.20–7.25 (m, 1H), 7.28–7.35 (m, 2H), 7.38–7.43 (m, 2H), 8.02 (s,
1H), 8.09 (s, 1H), 8.28 (s, 1H), 8.34 (s, 1H), 8.52 (s, 2H); 13C NMR
(75.5 MHz, CD3OD) d 18.7, 19.7, 22.1, 31.9, 36.0, 38.4, 41.5, 51.0,
54.3, 61.5, 68.6, 70.8, 72.4, 101.3, 103.4 (t, J = 25.6 Hz), 110.9 (d,
J = 25.2 Hz, 2C), 126.1, 126.5, 127.3, 128.2, 129.3, 129.6, 137.3,
137.4, 140.2, 143.8, 144.4, 145.0, 164.3 (d, JCF = 247.1 Hz), 164.5
(d, JCF = 247.1 Hz), 168.3, 169.1, 169.4, 172.8, 174.0. HRMS calcd
(M+H)+: 896.2988; found 896.3002. LC–MS Purity System A:
tR = 7.98 min, 99%; System B: tR = 8.00 min, 98%.
4.10.8. 5-((S)-2-{(3S,4S)-5-(3,5-Difluoro-phenoxy)-3-hydroxy-4-
[3-(methanesulfonyl-methyl-amino)-5-((R)-1-phenyl-ethyl-
carbamoyl)-benzoylamino]-pentanoylamino}-3-methyl-
butyrylamino)-isophthalic acid dimethyl ester (26)
4.10.11. 5-((S)-2-{(3S,4S)-5-Benzyloxy-3-hydroxy-4-[3-(methane-
sulfonyl-methyl-amino)-5-((R)-1-phenyl-ethylcarbamoyl)-
benzoylamino]-pentanoylamino}-3-methyl-butyrylamino)-
isophthalic acid (29)
Compound 26 (colorless solid) was synthesized in three steps
from central core 5 according to synthetic protocols A and B using
22
amine B and acid M. [
a]
ꢁ23 (c 0.11, MeOH); 1H NMR (300 MHz,
D
CDCl3) d 1.02 (d, J = 6.6 Hz, 3H), 1.03 (d, J = 6.6 Hz, 3H), 1.58 (d,
J = 6.9 Hz, 3H), 2.14–2.28 (m, 1H), 2.57 (dd, J = 7.1, 14.6 Hz, 1H),
2.66 (dd, J = 6.9, 14.6 Hz, 1H), 2.98 (s, 3H), 3.37 (s, 3H), 3.88 (s,
Compound 29 (colorless solid) was synthesized in four steps
from central core 14 according to synthetic protocols A, B, and D
22
using amine B and acid M. [
a
]
D
ꢁ38 (c 0.1, MeOH); 1H NMR

9484
M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
(300 MHz, CD3OD) d 1.02 (d, J = 6.8 Hz, 3H), 1.04 (d, J = 6.8 Hz, 3H),
1.58 (d, J = 7.1 Hz, 3H), 2.14–2.24 (m, 1H), 2.52 (dd, J = 7.3, 14.4 Hz,
1H), 2.62 (dd, J = 6.5, 14.4 Hz, 1H), 2.97 (s, 3H), 3.36 (s, 3H), 3.68
(dd, J = 7.0, 10.1 Hz, 1H), 3.75 (dd, J = 6.2, 10.1 Hz, 1H), 4.32 (d,
J = 6.7 Hz, 1H), 4.34–4.46 (m, 2H), 4.48 (s, 2H), 5.26 (q, J = 7.1 Hz,
1H), 7.18–7.25 (m, 6H), 7.29–7.35 (m, 3H), 7.38–7.43 (m, 2H),
8.02–8.04 (m, 1H), 8.07–8.10 (m, 1H), 8.28 (s, 1H), 8.36 (s, 1H),
8.54 (s, 1H); 13C NMR (75.5 MHz, CD3OD) d 18.7, 19.7, 22.1, 31.9,
36.0, 38.4, 41.5, 51.0, 54.4, 61.5, 69.0, 70.4, 73.8, 126.1, 126.3,
126.6, 127.3, 127.4, 128.2, 128.6, 128.9, 129.2, 129.3, 129.6,
133.1, 137.3, 139.4, 140.2, 143.8, 145.0, 167.8, 168.5, 169.1,
172.8, 174.0. HRMS calcd (M+H)+: 860.3177; found 860.3201. LC–
MS Purity System A: tR = 7.62 min, 100%; System B: tR = 7.80 min,
100%.
3H), 2.14–2.27 (m, 1H), 2.54 (dd, J = 7.2, 14.4 Hz, 1H), 2.64 (dd,
J = 6.9, 14.4 Hz, 1H), 2.95 (s, 3H), 2.97 (s, 3H), 3.37 (s, 3H), 3.71
(dd, J = 7.1, 10.2 Hz, 1H), 3.78 (dd, J = 6.5, 10.2 Hz, 1H), 4.32 (d,
J = 6.9 Hz, 1H), 4.35–4.41 (m, 1H), 4.42–4.46 (m, 1H), 4.47 (s, 2H),
6.71–6.79 (m, 1H), 6.81–6.87 (m, 2H), 8.02 (d, J = 3.6 Hz, 1H),
8.08 (d, J = 3.6 Hz, 1H), 8.27 (s, 1H), 8.35 (s, 1H), 8.54 (s, 2H); 13C
NMR (75.5 MHz, CD3OD) d 18.7, 19.7, 27.0, 31.9, 36.0, 38.4, 41.5,
54.2, 61.5, 68.6, 70.8, 72.4, 103.4 (t, J = 26.0 Hz), 110.9 (d,
J = 24.9 Hz, 2C), 126.0, 126.6, 127.4, 129.1, 129.3, 133.1, 137.1,
137.4, 140.2, 143.8, 164.3 (d, J = 247.1 Hz), 164.5 (d, J = 247.1 Hz),
168.5, 168.9, 169.2, 172.8, 174.0. HRMS calcd (M+H)+: 806.2519;
found 806.2529. LC–MS Purity System A: tR = 4.83 min, 100%; Sys-
tem B: tR = 6.74 min, 99%.
4.10.15. N-[(1S,2S)-3-Cyclopropylcarbamoyl-1-(3,5-difluoro-
phenoxymethyl)-2-hydroxy-propyl]-5-(methanesulfonyl-
methyl-amino)-N0-((R)-1-phenyl-ethyl)-isophthalamide (33)
Compound 33 (colorless solid) was synthesized in three steps
4.10.12. 5-{(S)-2-[(3S,4S)-5-(3,5-Difluoro-benzyloxy)-4-(3-
dipropylcarbamoyl-benzoylamino)-3-hydroxy-pentanoyl-
amino]-3-methyl-butyrylamino}-isophthalic acid (30)
Compound 30 (colorless solid) was synthesized in four steps
from central core 5 according to synthetic protocols A and B using
22
from central core 11 according to synthetic protocols A, B, and D
amine C and acid M. [
a]
ꢁ43 (c 0.1, MeOH); 1H NMR (300 MHz,
D
22
using amine B and acid N. [
a
]
D
ꢁ13 (c 0.1, MeOH); 1H NMR
CD3OD) d 0.44–0.52 (m, 2H), 0.64–0.74 (m, 2H), 1.59 (d, J = 7.1 Hz,
3H), 2.37–2.45 (m, 2H), 2.59–2.68 (m, 1H), 2.98 (s, 3H), 3.38 (s, 3H),
4.16 (dd, J = 6.9, 9.6 Hz, 1H), 4.25 (dd, J = 6.6, 9.6 Hz, 1H), 4.39–4.46
(m, 1H), 4.47–4.55 (m, 1H), 5.25 (q, J = 7.1 Hz, 1H), 6.47–6.57 (m,
1H), 6.58–6.67 (m, 2H), 7.20–7.28 (m, 1H), 7.29–7.38 (m, 2H),
7.39–7.45 (m, 2H), 8.04 (s, 1H), 8.06 (s, 1H), 8.25 (s, 1H); 13C
NMR (75.5 MHz, CDCl3) d 5.2, 5.3, 20.9, 22.1, 34.7, 37.2, 40.2,
49.8, 53.2, 67.0, 67.4, 96.0 (t, JCF = 26.5 Hz), 98.4 (d, JCF = 29.2 Hz,
2C), 124.9, 126.1, 127.0, 128.2, 128.2, 128.4, 135.8, 136.2, 142.6,
143.9, 161.1, 163.9 (d, JCF = 245.1 Hz), 164.1 (d, JCF = 245.1 Hz),
166.6, 167.8, 173.4. HRMS calcd (M+H)+: 659.2351; found
659.2367. LC–MS Purity System A: tR = 7.88 min, 100%; System B:
tR = 8.20 min, 100%.
(300 MHz, CD3OD) d 0.71 (t, J = 7.2 Hz, 3H), 0.98 (t, J = 7.2 Hz,
3H), 1.02 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 6.8 Hz, 3H), 1.47–1.62 (m,
2H), 1.64–1.77 (m, 2H), 2.16–2.28 (m, 1H), 2.52 (dd, J = 7.1,
14.4 Hz, 1H), 2.63 (dd, J = 6.8, 14.4 Hz, 1H), 3.14–3.24 (m, 2H),
3.43–3.52 (m, 2H), 3.71 (dd, J = 7.1, 10.0 Hz, 1H), 3.79 (dd, J = 6.3,
10.0 Hz, 1H), 4.32 (d, J = 6.6 Hz, 1H), 4.34–4.41 (m, 1H), 4.43–4.48
(m, 1H), 4.49 (s, 2H), 6.70–6.79 (m, 1H), 6.83–6.88 (m, 2H), 7.51–
7.60 (m, 2H), 7.89–7.91 (m, 1H), 7.97–8.02 (m, 1H), 8.36 (s, 1H),
8.54 (s, 2H); 13C NMR (75.5 MHz, CD3OD) d 11.3, 11.7, 18.6, 19.7,
21.7, 22.9, 31.8, 41.5, 52.3, 54.2, 61.4, 68.9, 70.8, 72.4, 103.3 (t,
J = 25.8 Hz), 110.9 (d, J = 25.2 Hz, 2C), 126.6, 126.8, 127.4, 129.6,
130.5, 133.1, 136.1, 138.4, 140.2, 144.4, 164.3 (d, J = 247.4 Hz),
164.5 (d, J = 247.1 Hz), 168.5, 169.7, 172.8, 173.2, 174.1. HRMS
calcd (M+H)+: 769.3260; found 769.3286. LC–MS Purity System
A: tR = 8.41 min, 97%; System B: tR = 7.93 min, 97%.
4.10.16. 4-({(3S,4S)-5-(3,5-Difluoro-phenoxy)-3-hydroxy-4-[3-
(methanesulfonyl-methyl-amino)-5-((R)-1-phenyl-ethyl-
carbamoyl)-benzoylamino]-pentanoylamino}-methyl)-benzoic
acid (34)
4.10.13. 5-{(S)-2-[(3S,4S)-5-Benzyloxy-4-(3-dipropylcarbamoyl-
benzoylamino)-3-hydroxy-pentanoylamino]-3-methyl-butyryl-
amino}-isophthalic acid (31)
Compound 34 (colorless solid) was synthesized in four steps
from central core 5 according to synthetic protocols A, B, and C
22
Compound 31 (colorless solid) was synthesized in four steps
using amine D and acid M. [
a]
ꢁ51 (c 0.1, MeOH); 1H NMR
D
from central core 14 according to synthetic protocols A, B, and D
(300 MHz, CD3OD) d 1.57 (d, J = 7.1 Hz, 3H), 2.52–2.58 (m, 2H),
2.96 (s, 3H), 3.35 (s, 3H), 4.19 (dd, J = 6.7, 9.8 Hz, 1H), 4.27 (dd,
J = 6.5, 9.8 Hz, 1H), 4.43 (d, J = 6.6 Hz, 2H), 4.45–4.52 (m, 1H),
4.56 (dt, J = 2.6, 6.6 Hz, 1H), 5.34 (q, J = 7.1 Hz, 1H), 6.45–6.53 (m,
1H), 6.57–6.64 (m, 2H), 7.14–7.26 (m, 1H), 7.27–7.35 (m, 2H),
7.36–7.42 (m, 4H), 7.94 (d, J = 8.4 Hz, 2H), 8.03 (s, 1H), 8.06 (s,
1H), 8.26 (s, 1H); 13C NMR (75.5 MHz, CD3OD) d 22.1, 36.0, 38.3,
41.6, 43.8, 51.0, 54.6, 64.3, 68.3, 68.7, 97.2 (t, J = 26.3 Hz), 99.6 (d,
J = 29.2 Hz, 2C), 126.0, 127.2, 127.3, 128.2, 128.4, 129.4, 129.6,
130.7, 131.0, 137.0, 137.4, 143.8, 145.0, 145.3, 162.3,165.1 (d,
J = 245.1 Hz), 165.3 (d, J = 245.1 Hz), 167.7, 169.0, 169.6, 173.6.
HRMS calcd (M+H)+: 753.2406; found 753.2399. LC–MS Purity Sys-
tem A: tR = 7.96 min, 100%; System B: tR = 8.16 min, 100%.
22
using amine B and acid N. [
a
]
D
ꢁ15 (c 0.1, MeOH); 1H NMR
(300 MHz, CD3OD) d 0.71 (t, J = 7.4 Hz, 3H), 0.99 (t, J = 7.4 Hz,
3H), 1.02 (d, J = 6.8 Hz, 3H), 1.04 (d, J = 6.8 Hz, 3H), 1.48–1.60 (m,
2H), 1.65–1.77 (m, 2H), 2.14–2.28 (m, 1H), 2.52 (dd, J = 7.1,
14.8 Hz, 1H), 2.62 (dd, J = 6.9, 14.8 Hz, 1H), 3.15–3.23 (m, 2H),
3.43–3.52 (m, 2H), 3.69 (dd, J = 6.6, 10.2 Hz, 1H), 3.76 (dd, J = 6.5,
10.2 Hz, 1H), 4.33 (d, J = 6.5 Hz, 1H), 4.35–4.47 (m, 2H), 4.48 (s,
2H), 7.19–7.27 (m, 5H), 7.48–7.60 (m, 2H), 7.87–7.91 (m, 1H),
7.96–8.01 (m, 1H), 8.36 (s, 1H), 8.54 (s, 2H); 13C NMR (75.5 MHz,
CD3OD) d 11.3, 11.7, 18.6, 19.7, 21.7, 22.9, 31.8, 41.4, 52.3, 54.2,
61.4, 69.0, 70.4, 73.4, 126.6, 126.7, 127.4, 128.6, 128.8, 129.1,
129.3, 129.6, 129.9, 130.5, 133.1, 136.2, 138.4, 139.4, 140.2,
168.6, 169.7, 172.9, 173.2, 174.1.HRMS calcd (M+H)+: 733.3449;
found 733.3441. LC–MS Purity System A: tR = 7.76 min, 99%; Sys-
tem B: tR = 7.63 min, 98%.
4.10.17. 4-[(2-{(3S,4S)-5-(3,5-Difluoro-phenoxy)-3-hydroxy-4-
[3-(methanesulfonyl-methyl-amino)-5-((R)-1-phenyl-ethyl-
carbamoyl)-benzoylamino]-pentanoylamino}-acetylamino)-
methyl]-benzoic acid (35)
4.10.14. 5-((S)-2-{(3S,4S)-5-(3,5-Difluoro-benzyloxy)-3-hydroxy-4-
[3-(methanesulfonyl-methyl-amino)-5-methylcarbamoyl-
benzoylamino]-pentanoylamino}-3-methyl-butyrylamino)-
isophthalic acid (32)
Compound 35 (colorless solid) was synthesized in four steps
from central core 5 according to synthetic protocols A, B, and C
22
using amine E and acid M. [
a]
ꢁ49 (c 0.1, MeOH); 1H NMR
D
Compound 32 (colorless solid) was synthesized in four steps
(300 MHz, CD3OD) d 1.56 (d, J = 7.1 Hz, 3H), 2.54 (d, J = 7.2 Hz,
2H), 2.95 (s, 3H), 3.35 (s, 3H), 3.79 (d, J = 16.8 Hz, 1H), 4.01 (d,
J = 16.8 Hz, 2H), 4.11–4.25 (m, 2H), 4.39–4.54 (m, 4H), 5.23 (q,
J = 7.1 Hz, 1H), 6.44–6.52 (m, 1H), 6.53–6.58 (m, 2H), 7.17–7.24
from central core 11 according to synthetic protocols A, B, and D
22
using amine B and acid O. [
a]
+7.0 (c 0.1, MeOH); 1H NMR
D
(300 MHz, CD3OD) d 1.03 (d, J = 6.9 Hz, 3H), 1.04 (d, J = 6.9 Hz,

M. Bäck et al. / Bioorg. Med. Chem. 16 (2008) 9471–9486
9485
(m, 1H), 7.26–7.41 (m, 6H), 7.90 (d, J = 8.1 Hz, 2H), 8.02 (s, 1H), 8.03
(s, 1H) 8.25 (s, 1H); 13C NMR (75.5 MHz, CD3OD) d 22.1, 36.0, 38.4,
41.4, 43.7, 43.8, 51.0, 54.0, 68.5, 68.9, 97.2 (t, J = 26.3 Hz), 99.6 (d,
J = 28.9 Hz, 2C), 126.1, 127.2, 128.2, 128.3, 129.3, 129.6, 130.7,
131.0, 137.0, 137.4, 143.7, 145.0, 145.3, 162.2, 165.0 (d,
J = 244.8 Hz), 165.2 (d, J = 245.1 Hz), 167.7, 169.0, 169.6, 172.0,
174.0. HRMS calcd (M+H)+: 810.2620; found 810.2622. LC–MS Pur-
ity System A: tR = 7.51 min, 100%; System B: tR = 7.73 min, 100%.
(75.5 MHz, CD3OD) d 18.4, 19.7, 22.2, 31.4, 41.2, 43.7, 50.8, 53.9,
60.8, 68.6, 68.9, 97.2 (t, JCF = 26.5 Hz), 99.6 (d, JCF = 28.9 Hz, 2C),
127.2, 127.5, 128.1, 128.5, 129.5, 129.8, 130.7, 130.9, 131.5,
131.6, 135.9, 136.3, 145.2, 145.4, 162.3, 165.0 (d, JCF = 245.1 Hz),
165.3 (d, JCF = 244.2 Hz), 168.8, 169.6, 169.9, 173.8, 174.0. HRMS
calcd (M+H)+: 745.3049; found 745.3045. LC–MS Purity System
A: tR = 8.47 min, 100%; System B: tR = 8.15 min, 100%.
4.10.21. 4-[((S)-2-{(3S,4S)-5-(3,5-Difluoro-phenoxy)-3-hydroxy-
4-[3-(methanesulfonyl-methyl-amino)-benzoylamino]-
pentanoylamino}-3-methyl-butyrylamino)-methyl]-benzoic
acid (39)
4.10.18. N-[(1S,2S)-3-((S)-1-Benzylcarbamoyl-2-methyl-propyl-
carbamoyl)-1-(3,5-difluoro-phenoxymethyl)-2-hydroxy-propyl]-
5-(methanesulfonyl-methyl-amino)-N0-((R)-1-phenyl-ethyl)-
isophthalamide (36)
Compound 39 (colorless solid) was synthesized in four steps
Compound 36 (colorless solid) was synthesized in three steps
from central core 5 according to synthetic protocols A, B, and C
ꢁ31 (c 0.1, MeOH); 1H NMR
22
from central core 5 according to synthetic protocols A and B using
using amine A and acid Q. [
a
]
D
22
amine F and acid M. [
a]
ꢁ41 (c 0.1, MeOH); 1H NMR (300 MHz,
(300 MHz, CD3OD) d 0.94 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.9 Hz,
3H), 2.12–2.25 (m, 1H), 2.53 (dd, J = 7.3, 14.8 Hz, 1H), 2.61 (dd,
J = 6.9, 14.8 Hz, 1H), 2.92 (s, 3H), 3.32 (s, 3H), 4.05–4.26 (m, 4H),
4.39–4.51 (m, 3H), 6.44–6.52 (m, 1H), 6.53–6.60 (m, 2H), 7.39 (d,
J = 8.3 Hz, 2H), 7.45–7.52 (m, 1H), 7.58–7.64 (m, 1H), 7.68–7.73
(m, 1H), 7.75–7.81 (m, 1H), 7.91 (d, J = 8.3 Hz, 2H); 13C NMR
(75.5 MHz, CD3OD) d 18.3, 19.7, 31.4, 35.7, 38.5, 41.2, 43.7, 53.9,
60.7, 68.6, 68.9, 97.2 (t, JCF = 26.2 Hz), 99.6 (d, JCF = 29.2 Hz, 2C),
126.7, 127.5, 128.4, 129.9, 130.4, 130.9, 132.4, 136.7, 143.4,
145.2, 161.1, 163.9 (d, JCF = 244.9 Hz), 164.1 (d, JCF = 244.9 Hz),
168.2, 169.7, 173.8, 174.0. HRMS calcd (M+H)+: 705.2406; found
705.2406. LC–MS Purity System A: tR = 7.80 min, 99%; System B:
tR = 7.44 min, 99%.
D
CD3OD) d 0.92 (d, J = 6.9 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H), 1.57 (d,
J = 6.9 Hz, 3H), 2.07–2.20 (m, 1H), 2.47–2.60 (m, 2H), 2.95 (s, 3H),
3.34 (s, 3H), 4.12–4.28 (m, 3H), 4.35–4.46 (m, 3H), 4.47–4.55 (m,
1H), 5.24 (q, J = 6.9 Hz, 1H), 6.44–6.55 (m, 1H), 6.55–6.64 (m,
2H), 7.12–7.35 (m, 8H), 7.40 (d, J = 7.1 Hz, 2H), 8.01–8.07 (m,
2H), 8.29 (s, 1H); 13C NMR (75.5 MHz, CD3OD) d 18.4, 19.7, 22.1,
31.5, 36.0, 38.4, 41.3, 44.1, 51.0, 54.2, 60.6, 68.5, 68.8, 97.2 (t,
JCF = 26.5 Hz), 99.6 (d, JCF = 29.2 Hz, 2C), 126.2, 127.3, 128.1,
128.2, 128.6, 129.3, 129.4, 129.6, 129.6, 137.0, 137.4, 139.9,
143.7, 145.0, 162.3, 165.0 (d, JCF = 245.1 Hz), 165.2 (d,
JCF = 245.3 Hz), 167.7, 168.9, 173.7, 173.7. HRMS calcd (M+H)+:
808.3192; found 808.3212. LC–MS Purity System A: tR = 9.55 min,
100%; System B: tR = 9.07 min, 100%.
Acknowledgments
4.10.19. 4-((S)-2-{(3S,4S)-5-(3,5-Difluoro-phenoxy)-3-hydroxy-
4-[3-(methanesulfonyl-methyl-amino)-5-((R)-1-phenyl-ethyl-
carbamoyl)-benzoylamino]-pentanoylamino}-3-methyl-
butyrylamino)-benzoic acid (37)
We gratefully thank the following personnel at Medivir AB: Mr.
Kurt Benkestock for conducting HRMS data, Elizabeth Hamelink
and 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 at Uppsala University for helpful discussions during develop-
ment of the expression of soluble BACE-1. Finally, we acknowledge
Medivir AB for financial support.
Compound 37 (colorless solid) was synthesized in four steps
from central core 5 according to synthetic protocols A, B, and C
22
using amine G and acid M. [
a
]
D
ꢁ20 (c 0.1, MeOH); 1H NMR
(300 MHz, (CD3)2SO) d 0.89 (d, J = 6.6 Hz, 6H), 1.49 (d, J = 6.9 Hz,
3H), 1.95–2.11 (m, 1H), 2.30–2.40 (m, 1H), 2.43–2.54 (m, over-
lapped, 1H), 3.01 (s, 3H), 3.29 (s, 3H), 4.10–4.19 (m, 1H), 4.20–
4.35 (m, 3H), 4.36–4.46 (m, 1H), 5.11–5.23 (m, 1H), 6.66–6.80
(m, 3H), 7.20 (t, J = 7.3 Hz, 1H), 7.25–7.34 (m, 2H), 7.38 (d,
J = 7.7 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.5 Hz, 2H),
7.98–8.08 (m, 3H), 8.34 (s, 1H), 8.52 (b, 1H), 9.04 (b, 1H), 10.3
(br s, 1H); 13C NMR (75.5 MHz, CD3)2SO) d 18.2, 19.2, 21.1, 22.0,
30.5, 35.7, 37.7, 48.7, 53.1, 58.8, 66.8, 67.6, 96.2 (t, JCF = 26.3 Hz),
98.8 (d, JCF = 28.3 Hz, 2C), 118.6, 125.2, 126.1, 126.4, 126.7, 127.9,
128.2, 130.2, 135.3, 135.6, 141.7, 142.4, 144.6, 160.6, 162.9 (d,
JCF = 243.9 Hz), 163.2 (d, JCF = 244.2 Hz), 164.5, 165.7, 167.2,
170.8, 172.0. HRMS calcd (M+H)+: 838.2933; found 838.2948.
LC–MS Purity System A: tR = 8.52 min, 100%; System B:
tR = 8.39 min, 100%.
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