Investigation of α-phenylnorstatine and α-benzylnorstatine as transition state isostere motifs in the search for new BACE-1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.11.042

Inhibition of the BACE-1 protease enzyme has over the recent decade developed into a promising drug strategy for Alzheimer therapy. In this report, more than 20 new BACE-1 protease inhibitors based on α-phenylnorstatine, α-benzylnorstatine, iso-serine, an

Full text of DOI:10.1016/j.bmc.2010.11.042

Bioorganic & Medicinal Chemistry 19 (2011) 145–155
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Investigation of a-phenylnorstatine and a-benzylnorstatine as transition
state isostere motifs in the search for new BACE-1 inhibitors
Fredrik Wångsell a, Patrik Nordeman a, Jonas Sävmarker a, Rikard Emanuelsson a, Katarina Jansson b,
Jimmy Lindberg b, Åsa Rosenquist b,c, Bertil Samuelsson b,d, Mats Larhed a,
⇑
a Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
b Medivir AB, Lunastigen 7, SE-141 44 Huddinge, Sweden
c Department of Chemistry, Linköping University, SE-581 83 Linköping, 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:
Received 8 March 2010
Revised 12 November 2010
Accepted 18 November 2010
Available online 26 November 2010
Inhibition of the BACE-1 protease enzyme has over the recent decade developed into a promising drug
strategy for Alzheimer therapy. In this report, more than 20 new BACE-1 protease inhibitors based on
a
-phenylnorstatine,
inhibitors were synthesized by applying Fmoc solid phase methodology and evaluated for their inhibitory
properties. The most potent inhibitor, tert-alcohol containing (R)-12 (IC50 = 0.19 M) was co-crystallized
a-benzylnorstatine, iso-serine, and b-alanine moieties have been prepared. The
l
in the active site of the BACE-1 protease, furnishing a novel binding mode in which the N-terminal amine
makes a hydrogen bond to one of the catalytic aspartic acids.
Keywords:
Alzheimer’s disease
BACE-1 inhibitors
tert-Hydroxyl
Ó 2010 Elsevier Ltd. All rights reserved.
Transition state mimic
a
a
-Phenylnorstatine
-Benzylnorstatine
1. Introduction
sec-alcohol functionality, for example, norstatine, statine, hydroxy-
ethylene, and hydroxyethylamine structures as non-cleavable
Inhibition of the human aspartic protease BACE-1 (b-secretase
or memapsin-2) has emerged as a promising therapeutic target
for the treatment of Alzheimer’s disease (AD).1,2 AD is the most
common form of dementia among the elderly and it is believed
that approximately 37 million people worldwide have the dis-
ease.3,4 Up to today the available treatment can only partly reduce
the symptoms and do not affect the underlying progression of the
disease.5 Two hallmarks for AD are accumulation and aggregation
of insoluble extracellular Ab-plaques, along with intracellular neu-
rofibrillary tangles in the brain.6 The human aspartic protease
BACE-1 is the initial protease that process amyloid precursor pro-
tein (APP) in the pathway leading to Ab proteins.7 Thus, inhibition
of BACE-1 has emerged as a promising pharmaceutical target for
combating AD. Peptide based inhibitors in which the scissile bond
has been replaced with a transition state (TS) isostere have proven
to be effective inhibitors of this protease.8,9 A key structural ele-
ment in most of the successful TS-isosteres is a sec-alcohol moiety
that interacts with the catalytic aspartic acids (Asp32 and Asp228)
of the BACE-1 protease via hydrogen bonds. Hence, a number of
potent BACE-1 inhibitors have been reported that contain a
TS-moieties.2,10,11 Kiso and co-workers have recently reported a
series of potent BACE-1 inhibitors containing a phenyl norstatine
moiety as the TS-isostere, for example, inhibitor A (Fig. 1).12–14
Ph
O
O
O
O
H
N
H2N
OH
N
H
N
H
N
H
O
OH
O
NH
A
N
O
O
BACE-1
NH
IC50 = 0.0082 µM
N
N
O
O
H
N
HO
H2N
OH
O
N
H
N
H
N
H
O
NH
Ph
N
(R)-12
BACE-1
NH
N
IC50 = 0.19 µM
N
Figure 1. Previously reported phenylnorstatine (sec-alcohol) inhibitor A12, together
⇑
Corresponding author. Tel.: +46 18 471 4667; fax: +46 18 471 4474.
E-mail address: mats.larhed@orgfarm.uu.se (M. Larhed).
with one of the novel
in this report.
a-benzylnorstatine (tert-alcohol) inhibitors, (R)-12, discussed
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.042

146
F. Wångsell et al. / Bioorg. Med. Chem. 19 (2011) 145–155
In the literature there are few examples reported where tert-
nia in DMF and diversified with different Fmoc protected amino
acids by solid phase chemistry. Finally the tetrazole 924 was at-
tached and the final BACE-1 inhibitors 10–23 were cleaved of the
resin and purified by HPLC in a yield of 3–17% (calculated from
5, 6, (R)-6 or (S)-6).
alcohols have been used as parts of TS-isosteres in protease inhib-
itors.15,16 Previous research on HIV-1 protease inhibitors compris-
ing a tert-alcohol TS-mimetic indicates the possibility to retain
high potency, while improving the membrane permeability.17–19
Inspired by these results we sought to develop and evaluate a ser-
ies of new tert-alcohol containing BACE-1 inhibitors20,21 utilizing
Inhibitors with secondary alcohols were also synthesized in or-
der to evaluate the importance of the phenyl- and benzyl-group
(Scheme 2). The commercially available acryloyl chloride was re-
acted with tert-butyl-3-aminobenzoate to give the alkene 24
(82%). The alkene was treated with mCPBA which gave epoxide
25 (racemate) and after subsequent opening with 25% aqueous
ammonia the primary amine (26) was protected with Fmoc-Cl.25
After removal of the tert-butyl protecting group the corresponding
acid was coupled to the resin (2-chlorotrityl chloride resin) to give
27. The immobilized 27 was treated with 20% piperidine in DMF to
give the corresponding amine. Using Fmoc protected amino acids
the inhibitors 28–33 were produced and purified with HPLC
(9–16% by yield calculated from structure 26).
either a
a-phenylnorstatine or a a-benzylnorstatine moiety as
the central core.
2. Results and discussion
2.1. Chemistry
The synthesis of the final compounds containing the
a-phenyl-
norstatine (n = 1) or the -benzylnorstatine (n = 2) TS-mimic is de-
a
picted in Scheme 1. The pivotal intermediates 5, 6, (R)-6, and (S)-6
were synthesized using two different protocols, starting from the
alkene 1, epoxide 3, 4, (R)-4 or (S)-4. The acrylic acid 1 was synthe-
sized from diethyl-2-phenethylmalonate in a two step protocol
according to a literature procedure.22
Next, 1 was attached to tert-butyl-3-aminobenzoate by peptide
coupling to render 2 in 40% yield. Epoxidation of the alkene 2 with
mCPBA furnished the racemic epoxide 6 in a yield of 80%. The
epoxides 3 and 4 were synthesized in a similar fashion23 and access
to the enantiomers (R)-4 and (S)-4 were achieved by chiral pre-
parative HPLC. Hydrolysis of the esters 3, 4, (R)-4, and (S)-4 fol-
lowed by peptide coupling with tert-butyl-3-aminobenzoate gave
the racemic compounds 5 and 6, or the pure enantiomers (R)-6
and (S)-6 (37–46%). The esters were hydrolyzed to the correspond-
ing acids and attached to the resin (2-chlorotrityl chloride resin) to
furnish 7, 8, (R)-8, and (S)-8. The epoxide was opened with ammo-
To further evaluate the
a-phenylnorstatine and the a-benzyl-
norstatine scaffold, inhibitors without the tert-alcohol and the
aromatic tether were synthesized (Scheme 3). Readily available
Fmoc-3-Abz-OH was attached to the resin to furnish 34. The inhibi-
tors 35–38 were synthesized by solid phase chemistry as described
above in a yield of 12–35% (calculated from Fmoc-3-Abz-OH).
Two compounds (39, 40) with reduced C-terminal were pre-
pared using solid phase synthesis, beginning from Fmoc protected
iso-serine and leucine (Scheme 4). The protected acid was first cou-
pled to the resin (2-chlorotrityl chloride resin) with subsequent re-
moval of the Fmoc group by 20% piperidine in DMF. After the
subsequent coupling cycles the peptide was liberated 95% aqueous
TFA with 5% Et3SiH, and purified with HPLC (39, 12% and 40, 23%
yield from respective acid before coupling).
2.2. X-ray crystal structure results
O
O
i
O
N
H
OH
One inhibitor, (R)-12, was co-crystallized together with BACE-1
providing an X-ray crystal structure (PDB-code: 3KYR, Fig. 2). Anal-
O
1
2
Ph
Ph
O
ii
O
O
O
i
ii
O
Cl
N
H
O
O
O
iv, v
iii, i
O
24
O
N
H
O
n
n
Ph
Ph
3
5
, n=1
O
O
, n=1
O
4
6
, (R)-6 or (S)-6, n=2
, (R)-4 or (S)-4, n=2
O
iv-vi
iii
O
N
H
H2N
N
H
O
O
OH
O
25
26
N
OH
10
HN
xi
Fmoc-R3-OH
Fmoc-R2-OH
N
(R)-11
(S)-12
13
N
O
x
9
N
OH
28
(R)-14
(S)-15
16
HN
ix
xii
Fmoc-R1-OH
Fmoc-R3-OH
Fmoc-R2-OH
N
29
30
31
32
33
N
viii
xi
9
17
O
n
O
18
vi, vii
x
O
Fmoc-R1-OH
19
N
H
20
ix
O
O
21
Ph
vii, viii
22
O
7, n=1
8, (R)-8 or (S)-8, n=2
FmocHN
N
23
H
OH
O
27
Scheme 1. Reagents and conditions: (i) tert-butyl-3-aminobenzoate, HATU, DIPEA,
CH2Cl2 (dry), 40 °C, 37–46%; (ii) mCPBA, CH2Cl2, reflux, 80%; (iii) KOH, EtOH (99%) rt,
quant.; (iv) TFA, CH2Cl2, rt, quant.; (v) 2-chlorotrityl chloride resin, DIPEA, CH2Cl2
Scheme 2. Reagents and conditions: (i) tert-butyl-3-aminobenzoate, CH2Cl2 (dry),
0 °C, 82%; (ii) mCPBA, CH2Cl2, reflux, 42%; (iii) NH3 (25% aq), 60 °C, quant.; (iv)
Fmoc-Cl, NaHCO3, dioxane, 0 °C, 95%; (v) TFA, CH2Cl2, rt, quant.; (vi) 2-chlorotrityl
(dry); (vi) NH3 (g) DMF; (vii) Fmoc-
DMF; (viii) (1) 20% piperidine in DMF; (2) Fmoc-
-Nle-OH or Fmoc- -Phe-OH, PyBOP, DIPEA, DMF; (ix) (1) 20% piperidine in DMF; (2)
Boc-
L
-Leu-OH or Fmoc-L-Cha-OH, PyBOP, DIPEA,
L
-Val-OH, Fmoc-
L-b-Leu-OH, Fmoc-
chloride resin, DIPEA, CH2Cl2 (dry); (vii) 20% piperidine in DMF; (viii) Fmoc-
OH or Fmoc- -Val-OH, PyBOP, DIPEA, DMF; (ix) (1) 20% piperidine in DMF; (1)
Fmoc- -Val-OH, PyBOP, DIPEA, DMF; (x) (1) 20% piperidine in DMF; (2) Boc-
DAP(Fmoc)-OH, Boc- -DAP(Fmoc)-OH or Fmoc-
-Ser(tBu)-OH, PyBOP, DIPEA, DMF;
(xi) 9, PyBOP, DIPEA, DMF, DMSO, H2O; (xii) TFA (95% aq), Et3SiH.
L-Leu-
L
L
L
L
-DAP(Fmoc)-OH, Boc-
D
-DAP(Fmoc)-OH, Fmoc-
L
-Ser(tBu)-OH or Fmoc-
D
-
L
L-
Ser(tBu)-OH, PyBOP, DIPEA, DMF; (x) 9, PyBOP, DIPEA, DMF, DMSO, H2O; (xi) TFA
D
L
(95% aq), Et3SiH.

F. Wångsell et al. / Bioorg. Med. Chem. 19 (2011) 145–155
147
O
N
OH
35
36
37
38
HN
vii
N
Fmoc-R4-OH
N
9
vi
Fmoc-R3-OH
Fmoc-R2-OH
v
iv
Fmoc-R1-OH
iii
i
ii
OH
O
FmocHN
FmocHN
34
O
O
Scheme 3. Reagent and conditions: (i) 2-chlorotrityl chloride resin, DIPEA, CH2Cl2
(dry); (ii) (1) 20% piperidine in DMF; (2) Fmoc-b-Ala-OH, PyBOP, DIPEA, DMF;
(iii) (1) 20% piperidine in DMF; (2) Fmoc-
piperidine in DMF; (2) Fmoc- -Val-OH, PyBOP, DIPEA, DMF; (v) (1) 20% piperidine in
DMF; (2) Boc- -DAP(Fmoc)-OH, Boc- -DAP(Fmoc)-OH, Fmoc-
-Ser(tBu)-OH or
Fmoc-
-Ser(tBu)-OH, PyBOP, DIPEA, DMF; (vi) 9, PyBOP, DIPEA, DMF, DMSO, H2O;
L-Leu-OH, PyBOP, DIPEA, DMF; (iv) (1) 20%
L
L
D
L
D
(vii) TFA (95% aq), Et3SiH.
OH
OH
H
N
iii-vi
i,ii
FmocHN
OH
OH
O
39
FmocHN
FmocHN
O
O
O
O
iii-vi
i
O
40
FmocHN
O
Scheme 4. Reagent and conditions: (i) 2-chlorotrityl chloride resin, DIPEA, CH2Cl2
(dry); (ii) (1) 20% piperidine in DMF; (2) Fmoc- -Leu-OH, PyBOP, DIPEA, DMF; (iii)
(1) 20% piperidine in DMF; (2) Fmoc- -Val-OH, PyBOP, DIPEA, DMF; (iv) (1) 20%
piperidine in DMF; (2) Boc- -DAP(Fmoc)-OH, PyBOP, DIPEA, DMF; (v) 9, PyBOP,
L
L
L
DIPEA, DMF, DMSO, H2O; (vi) TFA (95% aq), Et3SiH.
Figure 2. X-ray crystal structure of inhibitor (R)-12 (green) and BACE-1 (PDB-code:
3KYR) showing the polar key interactions between the inhibitor and enzyme. Flap
residues (66–77) are marked in magenta.
ysis of the crystal structure revealed a number of interactions. The
N-terminal amine, and not the tertiary alcohol, makes a hydrogen
bond to Asp228. Further the tetrazole carbonyl (a) is in proximity
to Arg235 and one of the nitrogens in the tetrazole makes a hydro-
gen bond to Tyr198. The N-terminal carbonyl in the inhibitor inter-
acts with the flap residue Thr72. Additionally the Val-NH
(b) coordinates to Gly34 and the Val-carbonyl with Thr198. As de-
duced from the X-ray crystallography, the Leu-NH (c) hydrogen
bonds to Pro70 (flap) and the Leu-carbonyl is closely situated to
Thr198. The C-terminal carboxylic functionality (e) in (R)-12 makes
polar interactions with Thr68 and Lys75 and the carbonyl next to
the tert-alcohol makes a hydrogen bond to Arg128. Several internal
interactions in (R)-12 lock the inhibitor in its arrangement. Inter-
a slight change in activity (14, IC50 = 0.68 lM) was observed. The
alteration from the valine (b) to Phe, Nle or Leu resulted in complete
loss in inhibitory properties: the inhibitors showed no inhibition
against the BACE-1 protease, exemplified by comparing 11 with
(R)-15 and (S)-16 (Phe, IC50 >10
(Leu, IC50 >10 M). Introduction of b-Leu (19, IC50 >10
as reversing the stereochemistry ( - to - in the a-position, cf. 11
and 20, IC50 >10 M) in the N-terminal resulted in inactive inhibi-
tors. Important interactions with the enzyme were apparently lost
by removal of the tetrazole moiety (a) (21, IC50 >10 M). The X-ray
lM), 17 (Nle, IC50 >10
lM) and 18
l
lM) as well
L
D
l
l
crystallographic data revealed that the N-terminal (R-NH2) was
positioned close to one of the catalytic aspartic residues (Asp228),
and that the tert-hydroxy group not served as a functional TS-isoste-
re. From these findings inhibitors with a serine analogue in the N-
terminal were evaluated with the aim of providing closer contact
with the aspartic residues. This attempt resulted in the inactive
estingly, the phenylethyl side chain (d) of the
a-benzylnorstatine
isostere extends beyond the protein surface; therefore this moiety
might not be necessary for protein interaction.
2.3. Biological evaluation
inhibitors 22 and 23 (L- and D-serine, IC50 >10 lM). Notably, a one
The structures of all the inhibitors together with the inhibitory
data (BACE-1 IC50) are summarized in Table 1. Compared to A12
(IC50 = 8.2 nM, Fig. 1) the interchange from the phenylnorstatine
carbon contraction in the tetrazole moiety (a) could also explain
the loss in activity. Further conclusions from the X-ray crystal struc-
ture of (R)-12 disclosed that the aromatic tether (d) was exposed to
the solvent and the tert-alcohol moiety did not contributed with
important interactions with the BACE-1 enzyme (Fig. 2). On the basis
of this molecular insight, the inhibitors lacking the tert-alcohol were
designed and produced. Removing the phenyl- and benzylnorstatine
gave access to iso-serine comprising sec-alcohols 28–33. Compound
(sec-alcohol) TS-mimic to the
taining core structure gave a compound with declined inhibitory
properties (10, epimeric mixture (1:1), IC50 = 0.43 M). Elongation
of the tether by one carbon ( -benzylnorstatine) gave an equipotent
inhibitor (11, IC50 = 0.40 M) and the corresponding diastereomers
(R)-12 and (S)-13 provided IC50 values of 0.19 and 1.4 M, respec-
a-phenylnorstatine (tert-alcohol) con-
l
a
l
l
28 (IC50 = 0.44 lM) showed almost equal inhibitory properties as
tively. As mentioned vide supra, (R)-12 was co-crystallized together
with the BACE-1 enzyme. The disclosed information from this X-ray
structure was used in the search for inhibitors with more desirable
inhibitory properties. By replacing the leucine (c) to the larger Chg,
the inhibitors 10 and 11, proving that the substituents in d-position
do not participate in the enzyme binding. By shortening (c) from leu-
cine to valine a loss of activity was observed (29, IC50 = 3
lM).
Removing the tetrazole moiety or incorporating the serine analogue

148
F. Wångsell et al. / Bioorg. Med. Chem. 19 (2011) 145–155
Table 1
Table 1 (continued)
BACE-1 inhibition data of the inhibitors
Compd Structure
IC50
IC50
M)
Compd Structure
(
l
(lM)
N
N
b
NH
O
N
e
O
O
O
H
N
HO
HO
HO
HO
HO
NH
H2N
H2N
H2N
H2N
H2N
OH
OH
OH
OH
OH
O
O
N
H
N
H
N
H
H
H
N
HO
10
0.43a
19
>10a
N
OH
O
O
H2N
N
H
N
H
NH
O
O
O
O
N
O
c
NH
d
N
a
N
O
O
O
O
O
O
O
O
O
H
N
O
O
O
H
N
HO
N
H
N
H
N
H
H2N
OH
N
H
NH
N
H
N
H
O
O
O
O
O
11
0.40a
0.19
1.4
NH
O
O
O
20
21
22
23
28
>10a
>10a
>10a
>10a
0.44a
N
O
O
O
O
NH
N
O
N
N
NH
N
N
O
H
N
O
O
H
N
HO
N
H
N
H
N
H
H2N
OH
N
H
NH2
N
H
N
H
O
(R)-12
(S)-13
14
NH
O
N
NH
N
N
O
H
O
O
O
N
H
HO
N
H
N
H
N
H
N
OH
HO
O
N
N
N
O
H
H
H
NH
NH
O
O
O
N
NH
N
N
HN
N
N
N
O
H
N
O
O
O
H
N
HO
N
N
H
N
H
OH
H
HO
O
N
H
N
H
N
H
O
0.68a
NH
NH
N
O
N
NH
N
HN
N
N
N
O
O
O
O
O
H
O
O
O
H2N
H2N
H2N
N
OH
H
N
HO
N
H
NH
N
N
H2N
OH
H
H
N
H
NH
N
H
N
H
O
OH
O
O
O
(R)-15
>10
O
O
N
O
O
NH
N
O
N
NH
N
N
N
O
O
H
N
OH
OH
N
H
NH
N
H
N
H
O
O
O
O
OH
3a
H
N
HO
29
H2N
OH
N
H
NH
N
H
N
H
N
(S)-16
>10
O
O
NH
N
N
N
O
O
O
NH
H
N
N
N
30
31
>10a
>10a
N
H
NH2
N
H
N
H
O
OH
O
O
O
O
HO
HO
H
N
H2N
OH
O
O
O
H
N
N
H
NH
N
H
N
H
>10a
OH
17
O
O
HO
N
N
H
N
H
H
NH2
O
O
OH
O
N
O
NH
N
N
O
O
H
N
H2N
OH
N
H
NH
N
H
N
H
O
O
O
OH
O
32
>10a
H
N
H2N
OH
N
H
NH
N
H
N
H
N
O
18
>10a
O
O
NH
N
N
N
O
NH
N
N

F. Wångsell et al. / Bioorg. Med. Chem. 19 (2011) 145–155
149
promising activity against the BACE-1 enzyme. More than twenty
Table 1 (continued)
inhibitors were synthesized and the most potent inhibitor, (R)-12
(IC50 = 0.19 lM), was co-crystallized (PDB-code: 3KYR) together
Compd Structure
IC50
M)
(
l
with the BACE-1 enzyme. From the solved structure a novel bind-
ing mode for this class of inhibitors were disclosed, in which
the N-terminal amine and not the tert-hydroxy group served as
the TS-isostere. Subtraction of the hydroxyl group also furnished
active compound 35 which strengthens the theory of the new
binding mode. Removal of the benzoic acid moiety decreased the
activity 10-fold (39, 40). All these findings might be of importance
for further drug discovery research. Nevertheless, the search for
potent BACE-1 inhibitors continues to be a difficult challenge.
O
O
O
H
N
OH
HO
O
N
H
N
H
N
H
NH
O
OH
O
33
35
>10a
N
N
N
NH
O
O
O
O
O
H
H2N
N
OH
N
H
NH
N
N
H
H
O
O
O
0.87
4. Experimental
4.1. General
N
O
O
NH
N
N
O
H
N
H2N
OH
Standard 1H NMR (399.9 MHz or 300.0 MHz if stated) and 13C
NMR (100.6 MHz or 75.4 MHz if stated) spectra were recorded
using (CD3)2SO, CDCl3 or methanol-d4 (CD3OD) as d values (ppm)
referenced to TMS (0.00 ppm) or the solvent residual signal. In
the recorded NMR spectra of diastereomeric mixtures the pre-
sumed diastereomeric peaks were put into brackets with the gen-
eral formula [nn.n & nn.n] for 13C NMR and [n.nn & n.nn, (x, yH)]
for 1H NMR, respectively. TLC was carried out on Merck precoated
60 F254 aluminum plates (0.2 mm) using UV-light (254 nm) and
charring with ethanol/sulfuric acid/acetic acid/p-anisaldehyde
(90:3:1:2) or a solution of 0.5% ninhydrin in ethanol (95%) for visu-
alization. Flash column chromatography was performed using
Silica Gel 60 (0.040–0.063 mm, Merck). Analytical LC/MS was per-
formed on a Gilson HPLC system with a Finnigan AQA quadropole
N
N
H
N
H
H
O
36
37
38
39
>10
>10
>10
6.1a
NH
N
NH
N
N
O
O
O
H
N
OH
HO
O
N
N
H
N
H
H
NH
O
O
O
N
N
N
NH
O
O
O
H
N
OH
HO
O
N
H
N
H
N
H
NH
O
mass spectrometer using a C18 (5
lm 4.6 ꢀ 100 mm) column, with
N
N
N
NH
a gradient of CH3CN in 0.05% aqueous HCOOH as mobile phase at a
flow rate of 4 mL/min. Dry dichloromethane (CH2Cl2) was achieved
by refluxing the solvent over calcium hydride and distilled onto 4 Å
molecular sieves. Optical rotations were obtained on a Perkin–El-
mer 241 polarimeter. Specific optical rotations (½aꢁTD) are reported
in deg/dm at ambient temperature (T) in the specified solvent
and the concentration (c) is given as g/100 mL. Evaporations were
performed under diminished pressure (7–10 mbar) at a water bath
temperature of 35 °C. Organic extracts were dried over magnesium
sulfate monohydrate and filtered. Starting materials and reagents
were purchased from commercial suppliers and used without fur-
ther purification. Compound 2426 is known and the spectroscopic
data are in agreement with the literature values. Tetrazole 9 was
synthesized according to the literature procedure.24
O
O
O
O
H
H2N
N
N
H
NH
N
OH
H
O
OH
N
O
O
NH
N
N
O
H
N
H2N
N
H
NH
OH
O
40
4.4
N
NH
N
N
a
Diastereomeric mixture (1:1).
4.1.1. General procedure for the loading of substrate onto resin
A tube was charged with 2-chlorotrityl chloride resin (substitu-
tion: 1.41 mmol/g resin, the polymer matrix was copoly(styrene-
1% DVB), 100–200 mesh) (1.5 equiv relative to the loading acid
substrate) and dry CH2Cl2 (substrate concentration 0.1 M) and
N,N-diisopropylethylamine (DIPEA) (4 equiv) was added and the
tube was capped. After the mixture had been agitated for 3 h, unre-
acted linkers was capped with MeOH (0.5 mL) during 15 min be-
fore the resin was filtered off and washed with several portions
of DMF, CH2Cl2, and finally MeOH. The resin was dried in vacuo
overnight yielding the corresponding substrate attached to the 2-
chlorotrityl chloride resin.
also resulted in reduced activity (30–33, IC50 >10 lM). To further ex-
pand the investigation, compounds lacking both hydroxyl group and
phenyl- and benzylstatine moiety were synthesized. Interestingly,
one of these compounds, 35, showed inhibitory properties
(IC50 = 0.87
structure. The inhibitors comprising the Ser moiety as well as the
-configuration in the N-terminal residue furnished inactive inhibi-
tors (36, 37 and 38, IC50 >10 M). Deletion of the benzoic acid group
(e) and the -phenylethyl moiety (d) and/or the tert/sec-alcohol
functionality resulted in less potent compounds 39 and 40 (6.1 and
4.4 M).
lM), supporting the X-ray enzyme–ligand complex
D
l
a
l
4.1.2. General procedure for the epoxide opening with ammonia
on solid support
3. Conclusion
To a falcon tube charged with the epoxide attached to the resin,
DMF was added and the solution was cooled to 0 °C. NH3 (g) was
added via a needle into the solution. After bubbling NH3 (g) for
15 min the tube was capped and agitated for 4 h at ambient tem-
The tert-alcohol containing
a-phenylnorstatine or a-benzyl-
norstatine moieties were evaluated as the central core structures
in the search for drug-like BACE-1 inhibitors. Also inhibitors
lacking the tert-alcohol moiety were produced, which showed

150
F. Wångsell et al. / Bioorg. Med. Chem. 19 (2011) 145–155
perature. The solution was then re-cooled and the procedure re-
peated until the reaction went to completion. Monitoring and anal-
ysis were performed by analytical LC/MS after removal of a small
portion of the resin and subsequent release of the product by addi-
the fractions were performed using analytical HPLC, 30–60% over
5 min at a wavelength of 220 nm.
4.1.7. General procedure for the HPLC purity analysis of the final
inhibitors
tion of TFA (100 lL, 95% aq) and triethylsilane (Et3SiH) (10 lL). The
Purity analysis was performed on two different HPLC systems
with UV- and ELSD-detector (Sedex 55), respectively or by using
different columns (C4 and C18). The purity for the corresponding
final compound together with the retention time (rt, min) is re-
ported. HPLC analysis with UV (254 nm) detection was performed
on a Gilson-Finnegan ThermoQuest AQA system equipped with a
resin was filtered off and washed with several portions of DMF,
CH2Cl2 and finally MeOH. Thereafter, the resin was dried in vacuo
overnight yielding the corresponding amine compound attached to
the resin.
4.1.3. General procedure for the synthesis of BACE-1 inhibitors
on solid support
C18 (50 ꢀ 4.6 mm) and C4 (ACT C4 4.6 ꢀ 50 mm, 5
lm) column,
using CH3CN (or MeOH if stated) in 0.05% aqueous HCOOH as mobile
phase at a flow rate of 4 mL/min, 10 min gradient from 10% to 100%
MeCN (or MeOH if stated). HPLC analysis with ELSD detection was
performed on a Gilson-Finnigan MSQ system equipped with a C18
(50 ꢀ 4.6 mm) column, using CH3CN (or MeOH if stated) in 0.05%
aqueous HCOOH as mobile phase at a flow rate of 4 mL/min,
10 min gradient from 10% to 100% MeCN (or MeOH if stated).
The substrate attached to the resin (mmol calculated from the
substrate loading procedure) was weighted into a 2 mL syringe fit-
ted with porous polyethylene filter. The Fmoc group was removed
(if necessary) by treatment with 20% piperidine in DMF
(2 ꢀ 1.5 mL, 10 + 15 min) and the resin was washed with DMF
(4 ꢀ 1.5 mL, 4 ꢀ 1 min). Coupling of the appropriate (Fmoc pro-
tected) amino acid was performed by agitation overnight in DMF
(2 mL), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexa-
fluorophosphate (PyBOP) (2 equiv) and DIPEA (6 equiv). The resin
was washed with DMF (4 ꢀ 1.5 mL, 4 ꢀ 1 min) and subsequently
Fmoc-deprotected and washed as described above. Finally, after
three or four coupling procedures, the tetrazole derivative (9)
was attached as described in the General procedure for attaching
the tetrazole carboxylate. After completion of the coupling cycles,
the resin was washed with several portions of DMF, CH2Cl2, and
finally MeOH before it was dried in vacuo. The peptide was cleaved
of the resin by treatment with TFA (95% aq, 2.5 mL) and Et3SiH
4.1.7.1. 3-(2-Methylene-4-phenyl-butyrylamino)-benzoic acid
tert-butyl ester (2). The acid 1 (0.50 g, 2.84 mmol), prepared
according to literature procedure21 starting from diethyl 2-phen-
ethylmalonate instead of diethyl 2-benzylmalonate, was dissolved
in CH2Cl2. tert-Butyl-3-aminobenzoate (0.55 g, 2.84 mmol), DIPEA
(1.52 mL, 8.52 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-
tetra-methyluronium hexafluorophosphate (HATU) (1.18 g,
3.12 mmol) was added. The reaction mixture was stirred at 40 °C
in 14 h. The reaction mixture was concentrated and subsequent
purification by flash column chromatography (iso-hexane/ethyl
acetate, 9:1) afforded the desired compound 2 (0.36 g, 36%) as a
colorless oil. 1H NMR (CDCl3) d 1.60 (s, 9H), 2.71 (d, J = 8.4 Hz,
1H), 2.72 (d, J = 9.4 Hz, 1H), 2.82 (d, J = 8.4 Hz, 1H), 2.83 (d,
J = 9.4 Hz, 1H), 5.37 (s, 1H), 5.74 (s, 1H), 7.17–7.21 (m, 3H), 7.25–
7.30 (m, 2H), 7.32 (m, 1H), 7.72 (m, 1H), 7.94 (m, 1H), 8.06 (m,
1H), 8.08 (br s, 1H); 13C NMR (CDCl3) d 28.1, 34.2, 34.5, 81.2,
118.9, 121.1, 124.4, 125.3, 126.1, 128.4, 128.5, 128.9, 132.7,
138.0, 141.1, 145.2, 165.3, 167.2; MS m/z 352.1 [(M+H)+ calcd
(100 lL) for 2 h in a capped tube. The resin was filtered off and
washed with TFA (3 ꢀ 0.5 mL). The filtrate was collected in a cen-
trifuge tube and concentrated in a stream of N2 (g). Cold diethyl
ether (12 mL) was used to precipitate the product as a TFA-salt,
which was collected by centrifugation, washed with cold diethyl
ether (2 ꢀ 3 mL) and dried.
4.1.4. General procedure for the attachment of the tetrazole
carboxylate
The dilithium or dipotassium salt (generally the dilithium salt
was more soluble) of the tetrazole carboxylate24 (2 equiv relative
to substrate loading) was dissolved in DMF (0.75 mL), DMSO
(0.75 mL), DIPEA (6 equiv) and minimum amount of water
þ
for: C22H26NO3 352.19].
4.1.7.2. 3-[(2-Benzyl-oxiranecarbonyl)-amino]-benzoic acid tert-
butyl ester (5). To the ester 323 (136 mg, 0.66 mmol) was 0.5 M
(99% EtOH) KOH (1.32 mmol, 2.63 mL) added at 0 °C. The mixture
was allowed to attainroom temperature, stirred for 2 h, and the mix-
ture was concentrated. The residue was dissolved in CH2Cl2 (2 mL),
water (2 mL), and 1 M (aq) HCl (0.5 mL) was added. The reaction
mixture was extractedwith CH2Cl2 (3 ꢀ 2 mL). Thecombined organ-
ic layers were dried with MgSO4, filtered and concentrated to afford
the corresponding acid which was used in the next step without fur-
ther purification. The acid was dissolved in dry CH2Cl2 (3.3 mL) and
3-amino-benzoic acid tert-butyl ester (127 mg, 0.658 mmol), DIPEA
(ꢂ150–200
ultrasonic treatment to dissolve the tetrazole salt. PyBOP (2 equiv)
L)
lL). The solution was gently heated and subject to
was separately dissolved in minimum amount of DMF (ꢂ200
l
and both solutions added to the syringe via the needle containing
the resin and agitated overnight at ambient temperature. The resin
was filtered off and washed with several portions of DMF, CH2Cl2,
and finally MeOH. Thereafter, the resin was dried in vacuo over-
night yielding the corresponding product.
(344 lL, 1.97 mmol) and HATU (275 mg, 0.724 mmol) was added.
4.1.5. General procedure for the purification of the compounds
(R)-4 and (S)-4
Chiral LC–MS was performed on the Gilson HPLC system in
straight-phase using 2-Propanol and iso-Hexane using isocratic
elution (iso-hexane/2-propanol, 99.25:0.75) and Reprosil Chiral
After 3 h at 30 °C the crude mixture was concentrated. Purification
by flash column chromatography (iso-hexane/ethyl acetate, 6:1)
yielded the desired 5 (103 mg, 44%) as a pale yellow oil. 1H NMR
(CDCl3) d 1.56 (s, 9H), 2.85 (d, J = 4.7 Hz, 1H), 2.93 (d, J = 4.7 Hz,
1H), 3.02 (d, J = 14.7 Hz, 1H), 3.67 (d, J = 14.7 Hz, 1H), 7.18–7.29
(m, 5H), 7.34 (t, J = 8.0 Hz, 1H), 7.70 (dt, J = 7.8, 1.4 Hz, 1H), 7.82–
7.88 (m, 2H), 8.09 (br s, 1H); 13C NMR (CDCl3) d 28.1, 35.9, 52.7,
59.9, 81.3, 120.4, 123.6, 125.5, 127.0, 128.4, 129.0, 129.8, 132.8,
NR columns 8
l
m 250 ꢀ 4.6 mm and 8 m 250 ꢀ 20 mm (Dr. Mai-
l
sch GmbH) with flow rate of 10 mL/min. Several repetitions were
needed in order to achieve sufficient separation.
135.1, 136.9, 165.1, 167.7; MS m/z 354.1 [(M+H)+ calcd for
þ
4.1.6. General procedure for the purification of the final
inhibitors
C
21H24NO4 354.42].
4.1.7.3. 3-[(2-Phenethyl-oxiranecarbonyl)-amino]-benzoic acid
tert-butyl ester (6, (R )-6, and (S)-6). From 4, (R)-4 or (S)-4:
compound 6 (103 mg, yield 44%), (R)-6 (98 mg, yield 37%) and
The TFA-salt of the crude BACE-1 inhibitor was dissolved in
MeCN/H2O (2 mL, 3:1). Preparative HPLC was performed using
5–65% gradient (MeCN containing 0.09% TFA and H2O containing
0.09% TFA) over 60 min with UV-detection at 254 nm. Analysis of
(S)-6 (121 mg, yield 46%) were prepared from
4 (136 mg,

F. Wångsell et al. / Bioorg. Med. Chem. 19 (2011) 145–155
151
0.658 mmol), (R)-4 (160 mg, 0.727 mmol) or (S)-4 (158 mg,
0.716 mmol) according to the same procedure as for compound
5. Compound 6: colorless oil. From 2: The alkene 2 (0.105 g,
0.299 mmol) was dissolved in CH2Cl2 (5 mL), and mCPBA (70%,
221 mg, 0.896 mmol) was added. The stirred solution was heated
at reflux temperature. After 4 h, additional mCPBA (111 mg,
0.448 mmol) was added, and the reaction mixture was stirred for
20 h. The reaction mixture was concentrated and dissolved in
CH2Cl2 (20 mL), washed with 10% (aq) Na2SO3 (25 mL) and satd
(aq) NaHCO3 (3 ꢀ 25 mL). The organic layer was dried with MgSO4,
filtered and concentrated. Purification by flash column chromatog-
raphy (iso-hexane/ethyl acetate, 6:1) yielded 6 (0.087 g, 80%) as a
clear oil. 1H NMR (CDCl3) d 1.60 (s, 9H), 1.87 (m, 1H), 2.72–2.86
(m, 3H), 2.96 (s, 2H), 7.16–7.31 (m, 5H), 7.40 (t, J = 7.8 Hz, 1H),
7.76 (dt, J = 7.8, 1.2 Hz, 1H), 7.91–7.95 (m, 2H), 8.20 (br s, 1H);
13C NMR (CDCl3) d 28.1, 30.9, 32.7, 53.3, 59.4, 81.3, 120.4, 123.6,
125.5, 126.1, 128.4 (2C), 129.0, 132.8, 136.9, 140.7, 165.1, 167.9;
4.1.7.7.
3-{3-[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-5-car-
bonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-4-me
thyl-pentanoylamino]-2-benzyl-2-hydroxy-propionylamino}-be
nzoic acid (10). Compound 10 (16 mg, 15% yield) was prepared
according to the General procedures from 5 (51 mg, 0.15 mmol) in
the following order: Fmoc-L-Leu-OH, Fmoc-L-Val-OH, Boc-L-DAP(F-
moc)-OH, and finally tetrazole 9. Compound 10: white powder after
lyophilization. 1H NMR (CD3OD) d 0.73–0.81 (m, 6H), 0.93–0.98 (m,
6H), 1.41–1.65 (m, 3H), 2.09 (m, 1H), 2.93 (m, 1H), 3.15 (m, 1H), 3.47
(m, 1H), 3.72 (m, 1H), 3.84 (m, 1H), 3.96 (m, 1H), 4.15–4.22 (m, 2H),
4.41 (m, 1H), 7.12–7.26 (m, 4H), 7.35 (m, 1H), 7.63 (m, 1H), 7.71 (m,
1H), 8.11–8.33 (m, 2H); 13C NMR (CD3OD) d [18.6 & 18.7], [19.7 &
19.8], 22.1, [23.1 & 23.2], [25.7 & 25.8], [31.5 & 31.6], 41.4, 42.0,
[43.9 & 44.0], [53.3 & 53.6], 54.4, [61.0 & 61.2], [80.5 & 80.9],
[122.9 & 123.1], [126.0 & 126.2], [126.7 & 126.8], 127.8, [128.9 &
129.0], [129.7 & 129.8], 131.5, 132.5, [137.0 & 137.1], 139.0,
154.73, [159.8 & 159.9], [168.5 & 168.7], 169.4, [173.4 & 173.5],
[173.9 & 174.3], [176.1 & 176.7]; HRMS m/z 709.3422 [(M+H)+ calcd
MS m/z 368.2 [(M+H)+ calcd for: C22H26NO4 368.19].
þ
þ
for C33H45N10O8 709.3416]; HPLC purity: >99% (UV, rt 2.34), 98.9%
4.1.7.4. 3-Acryloylamino-benzoic acid tert-butyl ester (24). tert-
Butyl-3-aminobenzoate (1.06 g, 5.5 mmol) was dissolved in dry
CH2Cl2 (30 mL) under nitrogen atmosphere and stirred at 0 °C. Acry-
loyl chloride (0.9 mL, 11 mmol, 2 equiv) was slowly added and the
solution was stirred for 2 h. The cloudy mixture was then washed
two times with brine (2 ꢀ 20 mL), dried over MgSO4, filtered and
concentrated to give product 24 as white powder (1.11 g, 82%) and
used without further purification. 1H NMR (CDCl3) d 1.59 (s, 9H),
5.80 (d, J = 10.2 Hz, 1H), 6.26 (dd, J = 16.8, 10.2 Hz, 1H), 6.46 (d,
J = 16.8 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H),
7.92–7.24 (m, 1H), 7.99–8.03 (m, 1H); 13C NMR (CDCl3) d 28.3,
81.5, 120.7, 124.2, 124.7, 125.7, 128.4, 129.3, 131.1, 133.0, 137.9,
163.7, 165.4; MS m/z 495.70 [(2M+H)+ calcd for C14H18NO3þ 248.13].
(ELSD, rt 2.42); BACE-1 IC50 = 0.43 lM.
4.1.7.8. 3-(2-{[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-5-car-
bonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-4-met
hyl-pentanoylamino]-methyl}-2-hydroxy-4-phenyl-butyrylami-
no)-benzoic acid (11). Compound 11 (16 mg, 14% yield) was pre-
pared according to the General procedures from
0.19 mmol) in the following order: Fmoc- -Leu-OH, Fmoc-
OH, Boc- -DAP(Fmoc)-OH, and finally tetrazole 9. Compound 11:
6
(70 mg,
L
L-Val-
L
white powder after lyophilization. 1H NMR (CD3OD) d 0.72–0.80
(m, 6H), 0.92–0.97 (m, 6H), 1.41–1.64 (m, 3H), 1.90 (m, 1H), 2.04–
2.24 (m, 2H), 2.51 (m, 1H), 2.84 (m, 1H), [3.34 & 3.88 (d,
J = 13.8 Hz, 1H), 3.55 & 3.61 (d, J = 13.8 Hz, 1H)], 3.82- 4.00 (m, 2H),
4.17 (m, 1H)], 4.22 (m, 1H), 4.41 (m, 1H), 7.10 (m, 1H), 7.14–7.22
4.1.7.5. 3-(Oxiranecarbonyl-amino)-benzoic acid tert-butyl ester
(25). Compound 24 (1.11 g, 4.5 mmol) was dissolved in CH2Cl2
(20 mL) and mCPBA (12.6 g, 45 mmol) was added to the solution.
The mixture was refluxed for 24 h, then additional mCPBA (2.8 g,
10 mmol) was added and the reaction was then further refluxed over
night. The yellow solution was washed with 10% (aq) Na2S2O3
(50 mL), washed three times with satd NaHCO3 (30 mL) solution
and then one time with brine (20 mL). The aqueous NaHCO3 and
brine was extracted once with CH2Cl2 and all the organic layers were
dried over MgSO4, filtered and concentrated. The mixture was puri-
fied by flash column chromatography (iso-hexane/ethyl acetate
2.5:1.5) yielding 25 (506 mg, 42%) as white powder. 1H NMR (CDCl3)
d 1.57 (s, 9H), 2.92 (dd, J = 5.4, 2.6 Hz, 1H), 3.09 (t, J = 5.6 Hz, 1H), 3.57
(dd, J = 4.6, 2.6, 1H), 7.38 (t, J = 8.3 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H),
7.96–7.98 (m, 2H); 13C NMR (CDCl3) d 28.3, 47.9, 50.1, 81.5 120.6,
123.9, 125.9, 129.3, 133.1, 136.9, 165.2, 166.7; MS m/z 527.45
(m, 4H), 7.41 (m, 1H), 7.76 (m, 1H), 7.85 (m, 1H), 8.32 (m, 1H); 13
C
NMR (CD3OD) d [18.6 & 18.7], [19.7 & 19.8], [22.0 & 22.1], [23.0 &
23.1], [25.7 & 25.8], 30.6, [31.5 & 31.6], 40.3, 41.4, [41.8 & 42.0],
[53.3 & 53.5], 54.3, [61.1 & 61.3], [79.7 & 80.1], [122.8 & 123.0],
[125.9 & 126.1], [126.7 & 126.8], 126.9, 129.3, 129.4, [129.8 &
129.9], [132.5 & 132.6], [139.2 & 139.3], [142.9 & 143.0], 153.9,
158.3, [168.5 & 168.6], 169.4, [173.3 & 173.4], [174.5 & 174.9],
[175.9
&
176.5]; HRMS m/z 723.3578 [(M+H)+ calcd for
þ
C34H47N10O8 723.3573]; HPLC purity: 97.2% (UV, rt 2.54), >99%
(ELSD, rt 2.69); BACE-1 IC50 = 0.40 lM.
4.1.7.9. 3-(R)-(2-{[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-5-
carbonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-4-
methyl-pentanoylamino]-methyl}-2-hydroxy-4-phenyl-butyryla
mino)-benzoic acid ((R)-12). Compound (R)-12 (17.1 mg, 17%
yield) was prepared according to the General procedures from (R)-
[(2M+H)+ calcd for C14H18NO4 264.12].
þ
6 (50 mg, 0.14 mmol) in the following order: Fmoc-
L
-Leu-OH,
4.1.7.6. 3-(3-Amino-2-hydroxy-propionylamino)-benzoic acid
tert-butyl ester (26). Compound 25 (100 mg, 3.8 mmol) was dis-
solved in tert-BuOH (10 mL) and slowly added to a 25% aqueous
ammonia solution (100 mL) stirred at 60 °C over a period of
30 min. The reaction was left for 6 h and then the yellow solution
was evaporated and dried in vacuo. The amine could be retrieved
in quantitative yield and was used in the next step without purifi-
cation. 1H NMR (DMSO) d 1.54 (s, 9H), 2.73 (dd, J = 12.7, 6.0 Hz,
1H), 2.85 (d, J = 12.7 Hz, 1H), 3.96–4.00 (m, 1H), 7.41 (t, J = 8.0,
1H), 7.58 (t, J = 7.4 Hz, 1H), 7.93 (d, J = 8.6 Hz, 1H), 8.31–8.34 (m,
1H); 13C NMR (DMSO) d 27.8, 45.7, 73.7, 80.7, 120.2, 123.9,
124.0, 128.8, 131.8, 138.9, 164.9, 172.4; MS m/z 281.25 [(M+H)+
Fmoc- -Val-OH, Boc-
L
L-DAP(Fmoc)-OH, and finally tetrazole 9. Com-
pound (R)-12: white powder after lyophilization. 1H NMR (CD3OD) d
0.73 (d, J = 6.2 Hz, 3H), 0.74 (d, J = 6.2 Hz, 3H), 0.95 (d, J = 8.0 Hz, 6H),
1.39–1.61 (m, 3H), 1.89 (m, 1H), 2.07 (m, 1H), 2.20 (m, 1H), 2.51 (dt,
J = 12.5, 5.3 Hz, 1H), 2.84 (dt, J = 13.5, 4.5 Hz, 1H), 3.35 (m, 1H), 3.80–
4.00(m, 3H), 4.14–4.23(m, 2H), 4.41 (m, 1H), 7.10(m, 1H), 7.15–7.24
(m, 4H), 7.41 (m, 1H), 7.77 (m, 1H), 7.87 (m, 1H), 8.33 (m, 1H); 13
C
NMR (CD3OD) d 18.7, 19.7, 22.1, 23.1, 25.7, 30.7, 31.5, 40.2, 41.4,
41.8, 53.5, 54.3, 61.2, 79.7, 122.8, 125.9, 126.7, 126.9, 129.4 (2C),
129.9, 132.6, 139.2, 143.0, 154.5, 159.6, 168.7, 169.5, 173.5, 174.5,
175.9; ½a 2D3
ꢁ
= ꢃ12.6 (c 0.76, MeOH); HRMS m/z 723.3578 [(M+H)+
þ
calcd for C34H47N10O8 723.3573]; HPLC purity: >99% (UV, rt 2.58),
þ
calcd for C14H21N2O3 281.15].
>99% (ELSD, rt 2.68); BACE-1 IC50 = 0.19 lM.

152
F. Wångsell et al. / Bioorg. Med. Chem. 19 (2011) 145–155
4.1.7.10. 3-(S)-(2-{[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-5-
carbonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-
4-methyl-pentanoylamino]-methyl}-2-hydroxy-4-phenyl-but-
yrylamino)-benzoic acid ((S)-13). Compound (S)-13 (21 mg, 17%
yield) was prepared according to the General procedures from (S)-
4.1.7.13. 3-(S)-(2-{[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-5-
carbonyl)-amino]-propionylamino}-3-phenyl-propionylamino )-
4-methyl-pentanoylamino]-methyl}-2-hydroxy-4-phenyl-but-
yrylamino)-benzoic acid ((S)-16). Compound (S)-16 (16 mg, 12%
yield) was prepared according to the General procedures from (S)-
6 (63 mg, 0.17 mmol) in the following order: Fmoc-
L-Leu-OH,
6 (59 mg, 0.16 mmol) in the following order: Fmoc-
L-Leu-OH,
Fmoc- -Val-OH, Boc- -DAP(Fmoc)-OH, and finally tetrazole 9.
L
L
Fmoc- -Phe-OH, Boc- -DAP(Fmoc)-OH, and finally tetrazole 9.
L
L
Compound (S)-13: white powder after lyophilization. 1H NMR
(CD3OD) d 0.77 (d, J = 6.3 Hz, 3H), 0.78 (d, J = 6.3 Hz, 3H), 0.93
(d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 1.49 (m, 1H), 1.55–
1.66 (m, 2H), 1.92 (m, 1H), 2.05 (m, 1H), 2.16 (m, 1H), 2.51 (dt,
J = 13.6, 4.9 Hz, 1H), 2.83 (dt, J = 12.7, 4.4 Hz, 1H), 3.55 (d,
J = 13.7 Hz, 1H), 3.62 (d, J = 13.7 Hz, 1H), 3.87 (dd, J = 14.6,
5.6 Hz, 1H), 3.95 (dd, J = 14.6, 5.6 Hz, 1H), 4.16 (d, J = 6.8 Hz,
1H), 4.22 (dd, J = 5.4, 4.4 Hz, 1H), 4.41 (m, 1H), 7.10 (m, 1H),
7.14–7.23 (m, 4H), 7.43 (m, 1H), 7.79 (m, 1H), 7.84 (m, 1H),
8.32 (m, 1H); 13C NMR (CD3OD) d 18.6, 19.7, 22.0, 23.2, 25.8,
30.7, 31.6, 40.3, 41.4, 42.0, 53.3, 54.3, 61.1, 80.1, 123.0, 126.1,
126.8, 126.9, 129.3, 129.4, 129.9, 132.6, 139.2, 143.0, 154.6,
Compound (S)-16: white powder after lyophilization. 1H NMR
(CD3OD) d 0.76 (d, J = 6.2 Hz, 3H), 0.79 (d, J = 6.2 Hz, 3H), 1.44–
1.62 (m, 3H), 1.91 (m, 1H), 2.16 (m, 1H), 2.52 (dt J = 12.3, 5.1 Hz,
1H), 2.79–2.89 (m, 2H), 3.13 (dd, J = 14.0, 5.3 Hz, 1H), 3.54 (d,
J = 13.9 Hz, 1H), 3.62 (d, J = 13.9 Hz, 1H), 3.85 (dd, J = 14.8, 5.6 Hz,
1H), 3.94 (dd, J = 14.8, 4.4 Hz, 1H), 4.08 (m, 1H), 4.39 (m, 1H),
4.64 (q, J = 5.1 Hz, 1H), 7.10 (m, 1H), 7.14–7.29 (m, 9H), 7.36–
7.42 (m,1H), 7.77 (m, 1H), 7.84 (m, 1H), 8.30 (m, 1H); 13C NMR
(CD3OD) d 22.0, 23.2, 25.8, 30.7, 38.4, 40.3, 41.3, 42.0, 53.4, 54.3,
56.7, 80.0, 123.0, 126.2, 126.8, 126.9, 127.9, 129.4 (2C), 129.6,
129.9, 130.2, 132.6, 138.0, 139.1, 143.0, 154.5, 159.6, 168.4,
169.5, 173.4, 174.9, 176.3; HRMS m/z 771.3578 [(M+H)+ calcd for
159.7, 168.5, 169.5, 173.4, 174.9, 176.5; ½a D23
ꢁ
= ꢃ46.8 (c 1.00,
C
38H47N10O8 771.3573]; ½a D23
ꢁ
= -36.7° (c 0.58, MeOH); HPLC pur-
M.
þ
MeOH); HRMS m/z 723.3578 [(M+H)+ calcd for C34H47N10O8
ity: 98.8% (UV, rt 2.85), >99% (ELSD, rt 3.02); BACE-1 IC50 >10
l
þ
723.3573]; HPLC purity: 97.3% (UV, rt 2.72), >99% (ELSD, rt
2.61); BACE-1 IC50 = 1.4 lM.
4.1.7.14.
3-(2-{[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-5-
carbonyl)-amino]-propionylamino}-hexanoylamino)-4-methyl-
pentanoylamino]-methyl}-2-hydroxy-4-phenyl-butyrylamino)-
benzoic acid (17). Compound 17 (5 mg, 3% yield) was prepared
according to the General procedures from 6 (92 mg, 0.25 mmol)
4.1.7.11. 3-{3-[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-5-car
bonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-3-
cyclohexyl-propionylamino]-2-benzyl-2-hydroxy-propionyla
mino}-benzoic acid (14). Compound 14 (16.6 mg, 14% yield) was
prepared according to the General procedures from 5 (54 mg,
in the following order: Fmoc-L-Leu-OH, Fmoc-L-Nle-OH, Boc-L-
DAP(Fmoc)-OH, and finally tetrazole 9. Compound 17: white pow-
der after lyophilization. 1H NMR (CD3OD) d 0.72–0.89 (m, 9H),
1.22–1.34 (m, 4H), 1.44–1.78 (m, 6H), 1.90 (m, 1H), 2.17 (m, 1H),
2.52 (m, 1H), 2.84 (m, 1H), 3.58 (m, 1H), 3.77–3.96 (m, 2H), 4.16
(m, 1H), 4.24 (m, 1H), 4.37 (m, 1H), 7.10 (m, 1H), 7.14 (m, 3H),
7.40 (m, 1H), 7.74–7.81 (m, 2H), 7.86 (m, 1H), 8.27 (m, 1H); 13C
NMR (CD3OD) d 14.2, [21.9 & 22.0], [23.2 & 23.3], [23.4 & 23.5],
[25.8 & 25.9], 29.1, 29.5, 30.7, [32.5 & 32.6], 40.3, 41.8, [53.5 &
53.6], [54.7 & 54.8], [55.7 & 55.8], [79.8 & 79.9], [123.1 & 123.2],
125.7, [126.6 & 126.7], 126.8, 129.3, 129.4, 129.7, [134.1 &
134.4], [139.1 & 139.2], [143.0 & 143.1], 153.7, 158.5, 163.7,
[169.4 & 169.5], [174.0 & 174.1], [174.6 & 174.9], [176.1 &
0.15 mmol) in the following order: Fmoc-
L-Cha-OH, Fmoc-L-Val-
OH, Boc- -DAP(Fmoc)-OH, and finally tetrazole 9. Compound 14:
L
white powder after lyophilization. 1H NMR (CD3OD) d 0.69–0.89
(m, 2H), 0.93–0.99 (m, 6H), 1.04–1.17 (m, 3H), 1.30 (m, 1H), 1.43–
1.70 (m, 7H), 2.08 (m, 1H), 2.93 (m, 1H), 3.14 (m, 1H), 3.39 (m, 1H),
3.64 (m, 1H), 3.79–3.99 (m, 2H), 4.14–4.24 (m, 2H), 4.44 (m, 1H),
7.11–7.25 (m, 5H), 7.35 (m, 1H), 7.51–7.79 (m, 2H), 8.12 (m, 1H);
13C NMR (CD3OD) d [18.6 & 18.7], [19.6 & 19.7], 27.0, [27.2 & 27.4],
[31.6 & 31.7], 33.3, 34.6, [35.1 & 35.2], [40.4 & 40.6], 41.4, 44.0,
[52.6 & 52.8], 54.4, [61.1 & 61.3], [80.5 & 80.9], [122.9 & 123.1],
[126.0 & 126.2], [126.7 & 126.8], 127.8, [128.9 & 129.0], [129.7 &
129.8], 131.5, [132.4 & 132.5], [137.0 & 137.1], [138.9 & 139.0],
154.7, [159.6 & 159.8], [168.5 & 168.7], 169.4, [173.3 & 173.5],
[173.9 & 174.3], [176.3 & 176.8]; HRMS m/z 749.3735 [(M+H)+ calcd
for C36H49N10O8þ 749.3729]; HPLC purity: 96.5% (UV, rt 2.69), 98.7%
176.5]; HRMS m/z 737.3735 [(M+H)+ calcd for C35H49N10O8
þ
737.3729]; HPLC purity: 98.4% (UV, rt 5.37, MeOH), 98.7% (ELSD,
rt 5.89, MeOH); BACE-1 IC50 >10
lM.
(ELSD, rt 2.85); BACE-1 IC50 = 0.68 lM.
4.1.7.15. 3-(2-{[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-5-
carbonyl)-amino]-propionylamino}-4-methyl-pentanoylamino)-
4-methyl-pentanoylamino]-methyl}-2-hydroxy-4-phenyl-but-
yrylamino)-benzoic acid (18). Compound 18 (8 mg, 4% yield) was
prepared according to the General procedures from 6 (92 mg,
4.1.7.12. 3-(R)-(2-{[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-
5-carbonyl)-amino]-propionylamino}-3-phenyl-propionylamino)-
4-methyl-pentanoylamino]-methyl}-2-hydroxy-4-phenyl-but-
yrylamino)-benzoic acid ((R)-15). Compound (R)-15 (16 mg, 15%
yield) was prepared according to the General procedures from (R)-
0.25 mmol) in the following order: Fmoc-L-Leu-OH, Fmoc-L-Leu-
OH, Boc- -DAP(Fmoc)-OH, and finally tetrazole 9. Compound 18:
L
6 (50 mg, 0.14 mmol) in the following order: Fmoc-
L-Leu-OH,
white powder after lyophilization. 1H NMR (CD3OD) d 0.69–0.79
(m, 6H), 0.86–0.93 (m, 6H), 1.42–1.70 (m, 7H), 1.91 (m, 1H), 2.19
(m, 1H), 2.52 (m, 1H), 2.85 (m, 1H), 3.58 (m, 1H), 3.81–3.97 (m,
2H), 4.20 (m, 1H), 4.33–4.41 (m, 2H), 7.09 (m, 1H), 7.14–7.23 (m,
4H), 7.40 (m, 1H), 7.76 (m, 1H), 7.85 (m, 1H), 8.30 (m, 1H); 13C
NMR (CD3OD) d 21.7, [21.9 & 22.0], [23.2 & 23.3], [23.4 & 23.5],
25.8, 25.9, 30.7, [40.2 & 40.3], [41.5 & 41.6], 41.7, [41.8 & 41.9],
[53.4 & 53.6], 54.0, [54.7 & 54.8], [79.6 & 80.0], [123.0 & 123.2],
[125.9 & 126.0], [127.0 & 126.8], 126.9, 129.4, 129.5, [129.7 &
129.8], [133.6 & 133.4], [139.1 & 139.2], [143.0 & 143.1], 152.7,
158.5, 163.7, [169.0 & 169.1], [174.4 & 174.5], [174.6 & 174.9],
Fmoc- -Phe-OH, Boc- -DAP(Fmoc)-OH, and finally tetrazole 9.
L
L
Compound (R)-15: white powder after lyophilization. 1H NMR
(CD3OD) d 0.73 (d, J = 6.3 Hz, 3H), 0.75 (d, J = 6.3 Hz, 3H), 1.41–
1.58 (m, 3H), 1.91 (m, 1H), 2.21 (dt, J = 13.7, 5.1 Hz, 1H), 2.52 (dt,
J = 12.3, 5.1 Hz, 1H), 2.80–2.93 (m, 2H), 3.19 (dd, J = 14.4, 4.9 Hz,
1H), 3.81–4.00 (m, 4H), 4.09 (m, 1H), 4.40 (m, 1H), 4.66 (q,
J = 4.9 Hz, 1H), 7.10 (m, 1H), 7.16–7.29 (m, 9H), 7.38 (m, 1H),
7.76 (m, 1H), 7.86 (m, 1H), 8.28 (m, 1H); 13C NMR (CD3OD) d
22.1, 23.1, 25.7, 30.7, 38.4, 40.2, 41.3, 41.9, 53.6, 54.3, 56.8, 79.6,
122.9, 126.0, 126.7, 126.9, 127.9, 129.4, 129.5, 129.6, 129.9,
130.2, 132.5, 138.1, 139.2, 143.0, 154.1, 159.2, 168.5, 169.5,
[175.9
&
176.5]; HRMS m/z 737.3735 [(M+H)+ calcd for
173.5, 174.6, 175.8;
½
a 2D3
ꢁ
= -0.6° (c 0.59, MeOH); HRMS m/z
þ
771.3578 [(M+H)+ calcd for C38H47N10O8 771.3573]; HPLC purity:
þ
C35H49N10O8 737.3729]; HPLC purity: >99% (UV, rt 5.53, MeOH),
98.8% (ELSD, rt 5.50, MeOH); BACE-1 IC50 >10 lM.
>99% (UV, rt 2.86), >99% (ELSD, rt 3.03); BACE-1 IC50 >10 lM.

F. Wångsell et al. / Bioorg. Med. Chem. 19 (2011) 145–155
153
4.1.7.16.
3-(2-{[(S)-2-((R)-3-{(S)-2-Amino-3-[(2H-tetrazole-5-
4.1.7.19. 3-(2-Hydroxy-2-{[(S)-2-((S)-2-{(R)-3-hydroxy-2-[(2H-
tetrazole-5-carbonyl)-amino]-propionylamino}-3-methyl-buty-
rylamino)-4-methyl-pentanoylamino]-methyl}-4-phenyl-buty-
rylamino)-benzoic acid (22). Compound 22 (9 mg, 5% yield) was
prepared according to the General procedures from 6 (81 mg,
carbonyl)-amino]-propionylamino}-4-methyl-pentanoylamino)-
4-methyl-pentanoylamino]-methyl}-2-hydroxy-4-phenyl-but-
yrylamino)-benzoic acid (19). Compound 19 (15 mg, 8% yield)
was prepared according to the General procedures from
(92 mg, 0.25 mmol) in the following order: Fmoc- -Leu-OH,
Fmoc- -b-Leu-OH, Boc- -DAP(Fmoc)-OH, and finally tetrazole 9.
6
L
0.22 mmol) in the following order: Fmoc-
L-Leu-OH, Fmoc-L-Val-
L
L
OH, Fmoc-
D
-Ser(tBu)-OH, and finally tetrazole 9. Compound 22:
Compound 19: white powder after lyophilization. 1H NMR
(CD3OD) d 0.62–0.8 (m, 6H), 0.87–1.02 (m, 6H), 1.35–1.90 (m,
5H), 2.08–2.21 (m, 2H), 2.40–2.55 (m, 2H), 2.77 (m, 1H), [3.25 &
3.90 (d, J = 13.7 Hz, 1H), 3.50 & 3.64 (d, J = 13.7 Hz, 1H)], 3.77 (m,
1H), 3.98–4.13 (m, 3H), 4.47 (m, 1H), 7.08–7.23 (m, 5H), 7.42 (m,
1H), 7.74–7.89 (m, 2H), [8.26 & 8.36 (m, 1H)]; 13C NMR (CD3OD)
d 18.5, [19.5 & 19.6], [22.0 & 22.1], [23.1 & 23.2], [25.7 & 25.8],
29.4, [30.6 & 30.7], [33.7 & 33.8], [39.1 & 39.4], [40.1 & 40.2],
[41.4 & 41.6], 53.4, 53.9. [54.5 & 54.7], [79.7 & 79.8], [122.6 &
123.0], 125.7, 126.2, [126.6 & 126.8], [126.8 & 126.9], 129.3,
[129.8 & 129.9], 132.6, [139.1 & 139.3], [143.0 & 143.1], 154.0,
[158.9 & 159.4], [167.3 & 167.4], 169.5, [174.0 & 174.3], [174.4 &
white powder after lyophilization. 1H NMR (CD3OD) d 0.77–0.80
(m, 6H), 0.92–0.95 (m, 6H), 1.44–1.60 (3H), 1.91 (m, 1H), 2.08–
2.25 (m, 2H), 2.52 (m, 1H), 2.85 (m, 1H), [3.38 & 3.77 (d,
J = 13.8 Hz, 1H), 3.51 & 3.67 (d, J = 13.8 Hz, 1H)], 3.86–4.01 (m,
2H), 4.15 (m, 1H), 4.33 (m, 1H), 4.80 (m, 1H), 7.10 (m, 1H), 7.17–
7.22 (m, 4H), 7.42 (m, 1H), 7.77 (m, 1H), 7.87 (m, 1H), 8.33 (m,
1H); 13C NMR (CD3OD) d [18.2 & 18.3], 19.6, [21.6 & 21.7], 23.3,
[25.8 & 25.9], [30.6 & 30.7], [31.2 & 31.3], 40.1, [41.8 & 41.9],
[53.3 & 53.4], [56.4 & 56.5], [60.9 & 61.0], [63.0 & 63.2], [79.8 &
80.0], [123.0 & 123.1], [126.1 & 126.2], [126.6 & 126.7], 126.9,
129.3, 129.5, [129.8 & 129.9], [132.5 & 132.6], [139.1 & 139.2],
[143.0 & 143.1], 153.8, 158.8, [169.2 & 169.3], [171.3 & 171.4],
[173.4 & 173.5], [174.5 & 174.6], [175.9 & 176.2]; HRMS m/z
174.6], 176.5; HRMS m/z 737.3735 [(M+H)+ calcd for
C
35H49N10O8 737.3729]; HPLC purity: 99.0% (UV, rt 2.50), >99%
724.3418 [(M+H)+ calcd for C34H46N9O9 724.3413]; HPLC purity:
þ
þ
(ELSD, rt 2.63); BACE-1 IC50 >10
lM.
99.0% (UV, rt 3.03), >99% (ELSD, rt 2.85); BACE-1 IC50 >10 lM.
4.1.7.17. 3-(2-{[(S)-2-((S)-2-{(R)-2-Amino-3-[(2H-tetrazole-5-
4.1.7.20. 3-(2-Hydroxy-2-{[(S)-2-((S)-2-{(S )-3-hydroxy-2-[(2H-
tetrazole-5-carbonyl)-amino]-propionylamino}-3-methyl-buty-
rylamino)-4-methyl-pentanoylamino]-methyl}-4-phenyl-buty-
rylamino)-benzoic acid (23). Compound 23 (7 mg, 4% yield) was
prepared according to the General procedures from 6 (81 mg,
carbonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-4-
methyl-pentanoylamino]-methyl}-2-hydroxy-4-phenyl-butyry-
lamino)-benzoic acid (20). Compound 20 (13 mg, 7% yield) was
prepared according to the General procedures from 6 (92 mg,
0.25 mmol) in the following order: Fmoc-
L-Leu-OH, Fmoc-L-Val-
0.22 mmol) in the following order: Fmoc-
L-Leu-OH, Fmoc-L-Val-
OH, Fmoc-
L
-Ser(tBu)-OH, and finally tetrazole 9. Compound 23:
OH, Boc- -DAP(Fmoc)-OH, and finally tetrazole 9. Compound 20:
D
white powder after lyophilization. 1H NMR (CD3OD) d 0.72–0.82
(m, 6H), 0.87–0.94 (m, 6H), 1.42–1.64 (m, 3H), 1.89 (m, 1H), 2.04
(m, 1H), 2.17 (m, 1H), 2.52 (m, 1H), 2.85 (m, 1H), [3.33 & 3.83 (d,
J = 14.0 Hz, 1H), 3.51 & 3.65 (d, J = 14.0 Hz, 1H)], 3.83–3.89 (m,
2H), 4.15 (m, 1H), 4.23 (m, 1H), 4.36 (m, 1H), 7.11 (m, 1H), 7.16–
7.25 (m, 4H), 7.44 (m, 1H), 7.77–7.90 (m, 2H), 8.34 (m, 1H); 13C
NMR (CD3OD) d [18.6 & 18.7], [19.7 & 19.8], [21.9 & 22.0], [23.1
& 23.2], [25.6 & 25.7], 30.6, 31.9, 40.3, 41.4, [42.0 & 42.2], [53.1 &
53.2], [54.2 & 54.3], [60.7 & 60.8], [79.7 & 79.9], [123.0 & 123.1],
[126.0 & 126.1], [126.7 & 126.8], 126.9, 129.2, 129.3, 129.9,
[132.6 & 132.7], [139.1 & 139.2], [142.9 & 143.0], 154.2, 158.4,
168.2, [169.4 & 169.5], [173.3 & 173.4], [174.5 & 174.8], [175.7 &
white powder after lyophilization. 1H NMR (CD3OD) d 0.76–0.79
(m, 6H), 0.91–0.95 (m, 6H), 1.43–1.62 (m, 3H), 1.85 (m, 1H),
2.07–2.18 (m, 2H), 2.48 (m, 1H), 2.78 (m, 1H), [3.28 & 3.75 (d,
J = 13.7 Hz, 1H), 3.45 & 3.56 (d, J = 13.7 Hz, 1H)], 3.92–3.95 (m,
2H), 4.15 (m, 1H), 4.34 (m, 1H), 4.70 (m, 1H), 7.11 (m, 1H), 7.15–
7.23 (m, 4H), 7.43 (m, 1H), 7.78 (m, 1H), 7.86–7.92 (m,1H), 8.35
(m, 1H); 13C NMR (CD3OD) d 18.5, [19.7 & 19.8], [21.6 & 21.8],
[23.2 & 23.3], [25.7 & 25.8], 30.6, [31.3 & 31.4], [40.0 & 40.2],
[41.6 & 41.7], [53.4 & 53.5], [57.1 & 57.2], [60.7 & 61.0], 62.8,
[79.6 & 79.8], 123.0, 126.1, [126.6 & 126.7], 126.9, 129.4, 129.5,
[129.8 & 129.9], [132.5 & 132.6], [139.1 & 139.2], [143.0 &
143.1], 154.1, 158.6, [169.5 & 169.6], 170.9, [172.4 & 172.6]
[173.4 & 173.8], [174.6 & 174.9]; HRMS m/z 724.3418 [(M+H)+
176.2]; HRMS m/z 723.3578 [(M+H)+ calcd for C34H47N10O8
þ
723.3573]; HPLC purity: >99% (UV, rt 2.56), 98.1% (ELSD, rt 2.73);
BACE-1 IC50 >10 lM.
þ
calcd for C34H46N9O9 724.3413]; HPLC purity: 99.0% (UV, rt
2.84), 98.3% (ELSD, rt 2.97); BACE-1 IC50 >10 lM.
4.1.7.18. 3-[2-({(S)-2-[(S)-2-((S)-2,3-Diamino-propionylamino)-
3-methyl-butyrylamino]-4-methyl-pentanoylamino}-methyl)-2-
hydroxy-4-phenyl-butyrylamino]-benzoic acid (21). Com-
pound 21 (10 mg, 10% yield) was prepared according to the Gen-
eral procedures from 6 (70 mg, 0.19 mmol) in the following
4.1.7.21. 3-{3-[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-5-car
bonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-4-me
thyl-pentanoylamino]-2-hydroxy-propionylamino}-benzoic acid
(28). Compound 28 (10.5 mg, 15% yield) was prepared according to
the General procedures from 26 (31 mg, 0.11 mmol) in the following
order: Fmoc-L-Leu-OH, Fmoc-L-Val-OH, and finally Boc-L-DAP(F-
order: Fmoc-L-Leu-OH, Fmoc-L-Val-OH, Boc-L-DAP(Fmoc)-OH, and
moc)-OH. Compound 21: white powder after lyophilization. 1H
NMR (CD3OD) d 0.72–0.81 (m, 6H), 0.91–1.01 (m, 6H), 1.39–1.65
(m, 4H), 2.11–2.24 (m, 2H), 2.53 (m, 1H), 2.85 (m, 1H), [3.22 &
3.91 (d, J = 13.8 Hz, 1H), 3.50 & 3.64 (d, J = 13.8 Hz, 1H)], 3.36 (m,
1H), 3.50 (m, 1H), 4.23 (m, 1H), 4.37–4.45 (m, 2H), 7.11 (m, 1H),
7.16–7.24 (m, 4H), 7.44 (m, 1H), 7.78–7.87 (m, 2H), 8.33 (m, 1H);
13C NMR (CD3OD) d [16.7 & 16.9], [18.5 & 18.6], [20.7 & 20.8],
[21.9 & 22.0], [24.5 & 24.6], 29.5, 30.2, [39.0 & 39.1], 40.0, [41.0
& 41.2], [52.0 & 52.1], [53.0 & 53.1], 60.1, [78.3 & 78.7], [121.6 &
121.7], [124.7 & 124.9], [125.5 & 125.6], 125.7, 128.2, 128.3,
128.8, 131.5, [138.0 & 138.1], [141.7 & 141.8], 168.3, [172.6 &
finally tetrazole 9. Compound 28: white powder after lyophilization.
1H NMR (CD3OD) d0.77–1.00 (m, 12 H), 1.45–1.69 (m, 3H), 2.04–2.14
(m, 1H), 3.48–3.57 (m, 1H), 3.62–3.79 (m, 1H), 3.86–3.99 (m, 2H),
4.18–4.24 (m, 2H), 4.26–4.32 (m, 1H), 4.39–4.44 (m, 1H), 7.39–
7.44 (m, 1H), 7.75–7.78 (m, 1H), 7.84–7.89 (m, 1H), 8.29–8.31 (m,
1H); 13C NMR (CD3OD) d [18.6 & 18.7], 19.7, 22.0, [23.2 & 23.3],
[25.8 & 25.9], 31.6, 41.4, [44.2 & 44.4], [53.4 & 53.5], 54.3, [61.2 &
61.3], [72.5 & 72.6], [122.7 & 122.8], [125.8 & 125.9], [126.6 &
126.7], 129.9, 132.6, [139.3 & 139.4], [154.9 & 155.0], 160.0, 168.6,
169.5, 173.2, [173.3 & 173.4], [175.4 & 175.5]; HRMS m/z 619.2961
[(M+H)+ calcd for C26H39N10O8 619.2952]; HPLC (10–100% MeOH)
þ
172.7], [173.3
&
173.7], 174.2, 174.9; HRMS m/z 623.3506
purity: 99.0% (C4, rt 1.27), >99% (C18, rt 2.84); BACE-1
IC50 = 0.44 lM.
[(M+H)+ calcd for C32H47N6O7 627.3501]; HPLC purity: 97.8%
þ
(UV, rt 2.17), >99% (ELSD, rt 2.08); BACE-1 IC50 >10 lM.

154
F. Wångsell et al. / Bioorg. Med. Chem. 19 (2011) 145–155
4.1.7.22. 3-{3-[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-5-car-
bonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-3-met
J = 8.2 Hz, 1H), 7.76–7.80 (m, 1H), 7.82–7.89 (m, 1H), 8.29–8.32 (m,
1H); 13C NMR (CD3OD) d 18.7, 19.7, 21.9, [23.2 & 23.4], [25.7 &
25.8], 31.8, 41.4, [44.2 & 44.3], 53.2, [54.2 & 54.3], [61.0 & 61.2],
[72.4 & 72.5], [122.7 & 122.8], [125.8 & 125.9], 126.7, 129.9, 132.6,
139.3, 154.4, 159.3, [168.4 & 168.6], 169.5, 173.2, 173.4, 175.2;
hyl-butyrylamino]-2-hydroxy-propionylamino}-benzoic
acid
(29). Compound 29 (6.0 mg, 9% yield) was prepared according to
the General procedures from 26 (31 mg, 0.11 mmol) in the following
HRMS m/z 619.2943 [(M+H)+ calcd for C26H39N10O8 619.2952];
þ
order: Fmoc-
L
-Val-OH, Fmoc-
L
-Val-OH, Boc-
L-DAP(Fmoc)-OH, and
finally tetrazole 9. Compound 29: white powder after lyophilization.
1H NMR (CD3OD) d0.82–1.01 (m, 12H), 1.96–2.15 (m, 2H), 3.54–3.60
(m, 1H), 3.64–3.75 (m, 1H), 3.94–4.00 (m, 1H), 4.17–4.25 (m, 3H),
4.24–4.32 (m, 1H), 7.42 (t, J = 8.2 Hz, 1H), 7.76–7.79 (m, 1H), 7.84–
7-89 (m, 1H), 8.29–8.31 (m, 1H); 13C NMR (CD3OD) d 18.7, [18.8 &
18.9], 19.7, 31.6, 31.9, 41.7, [44.2 & 44.3], 53.4, 60.5, [61.1 & 61.2],
72.3, [122.7 & 122.8], [125.8 & 125.9], 126.7, 139.5, 154.7, 159.9,
[168.3 & 168.4], 169.5, [173.1 & 173.2], [173.4 & 173.5], 174.2; HRMS
m/z 605.2805 [(M+H)+ calcd for C25H37N10O8þ 605.2796]; HPLC (10–
100% MeOH) purity: >99% (C4, rt 1.49), 99.0% (C18, rt 3.03); BACE-1
HPLC (10–100% MeOH) purity: 99.0% (C4, rt 1.60), >99% (C18, rt
3.28); BACE-1 IC50 >10
lM.
4.1.7.26. 3-{2-Hydroxy-3-[(S)-2-((S)-2-{(R)-3-hydroxy-2-[(2H-
tetrazole-5-carbonyl)-amino]-propionylamino}-3-methyl-buty-
rylamino)-3-methyl-butyrylamino]-propionylamino}-benzoic
acid (33). Compound 33 (6.0 mg, 9% yield) was prepared accord-
ing to the General procedures from 26 (31 mg, 0.11 mmol) in the
following order: Fmoc-L-Val-OH, Fmoc-L-Val-OH, Fmoc-D
-Ser(t-
Bu)-OH, and finally tetrazole 9. Compound 33: white powder after
lyophilization. 1H NMR (CD3OD) d 0.86–0.97 (m, 12H), 1.99–2.17
(m, 2H), 3.47–3.65 (m, 2H), 3.90–3.97 (m, 2H), 4.10–4.15 (m,
1H), 4.22–4.27 (m, 2H), 4.67–4.72 (m, 1H), 7.42 (t, J = 7.9 Hz, 1H),
7.76–7.79 (m, 1H), 7.84–7.90 (m, 1H), 8.29–8.32 (m, 1H); 13C
NMR (CD3OD) d 18.6, [18.8 & 18.9], 19.8, [31.6 & 31.7], 44.2,
57.0, [60.5 & 60.6], 60.8, 62.9, 72.6, 122.8, 125.9, 126.7, 129.9,
132.6, 139.3, 154.5, 158.2, 169.5, 172.1, 173.1, [173.5 & 173.6],
IC50 = 3 lM.
4.1.7.23. 3-(3-{(S)-2-[(S)-2-((S)-2,3-Diamino-propionylamino)-3-
methyl-butyrylamino]-4-methyl-pentanoylamino}-2-hydroxy-
propionylamino)-benzoic acid (30). Compound 30 (9.2 mg, 13%
yield) was prepared according to the General procedures from 26
(31 mg, 0.11 mmol) in the following order: Fmoc-
L-Leu-OH, Fmoc-
174.2; HRMS m/z 606.2639 [(M+H)+ calcd for C25H36N9O9
þ
L
-Val-OH, and finally Boc- -DAP(Fmoc)-OH. The Fmoc group was
L
then removed according to the General procedure. Compound 30:
white powder after lyophilization. 1H NMR (CD3OD) d 0.80–1.03
(m, 12 H), 1.46–1.70 (m, 3H), 2.13–2.22 (m, 1H), 3.34–3.36 (m,
4H), 4.22–4.30 (m, 2H), 4.36–4.45 (m, 2H), 7.44 (t, J = 8.1 Hz, 1H),
7.77–7.81 (m, 1H), 7.83–7.88 (m, 1H), 8.23–8.32 (m, 1H); 13C NMR
(CD3OD) d [18.0 & 18.1], 19.7, [21.8 & 21.9], 23.3, 25.8, 31.3, 41.2,
42.1, 44.3, 51.4, 53.4, 61.5, [72.4 & 72.6], [122.6 & 122.7], [125.8 &
125.9], 126.7, 130.0, 132.7, 139.3, 168.1, 169.5, 173.2, [175.0 &
606.2636]; HPLC (10–100% MeOH) purity: 98.5% (C4, rt 1.05),
97.4% (C18, rt 3.07); BACE-1 IC50 >10
lM.
4.1.7.27. 3-{3-[(S)-2-((S)-2-{(S)-2-Amino-3-[(2H-tetrazole-5-car-
bonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-4-meth
yl-pentanoylamino]-propionylamino}-benzoic acid (35). Com-
pound 35 (57 mg, 32% yield) was prepared according to the General
procedures from Fmoc-3-Abz-OH (108 mg, 0.30 mmol) in the fol-
175.1]; HRMS m/z 523.2893 [(M+H)+ calcd for C24H39N6O7
lowing order: Fmoc-b-Ala-OH, Fmoc-L-Leu-OH, Fmoc-L-Val-OH,
þ
523.2880]; HPLC (10–100% MeOH) purity: 98.3% (C4, rt 0.65),
Boc- -DAP(Fmoc)-OH, and finally tetrazole 9. Compound 35: white
L
96.1% (C18, rt 2.52); BACE-1 IC50 >10
lM.
powder after lyophilization. 1H NMR (CD3OD) d 0.81–0.89 (m, 6H),
0.93–0.97 (m, 6H), 1.50–1.68 (m, 3H), 2.11 (m, 1H), 2.57–2.63 (m,
2H), 3.46–3.62 (m, 2H), 3.86–3.97 (m, 2H), 4.20–4.27 (m, 2H), 4.38
(m, 1H), 7.38 (m, 1H), 7.72 (m, 1H), 7.9 (m, 1H), 8.22 (m, 1H); 13C
NMR (CD3OD) d 17.4, 18.5, 20.9, 22.1, 24.7, 30.4, 35.6, 36.2, 40.2,
40.6, 52.3, 53.1, 60.0, 121.1, 124.2, 125.0, 128.7, 131.3, 139.0,
153.3, 158.3, 167.5, 168.3, 171.1, 172.1, 173.4; HRMS m/z 603.3003
4.1.7.24. 3-(3-{(S)-2-[(S)-2-((R)-2-Amino-3-hydroxy-propionyla-
mino)-3-methyl-butyrylamino]-4-methyl-pentanoylamino}-2-
hydroxy-propionylamino)-benzoic acid (31). Compound 31
(9.2 mg, 16% yield) was prepared according to the General proce-
dures from 26 (31 mg, 0.11 mmol) in the following order: Fmoc-
-Ser(tBu)-OH. The
[(M+H)+ calcd for C26H39N10O7 603.2998]; HPLC purity: 95.9%
þ
L
-Leu-OH, Fmoc-
L
-Val-OH, and finally Fmoc-
D
Fmoc group then removed according to General procedure. Com-
pound 31: white powder after lyophilization. 1H NMR
(300.0 MHz, CD3OD) d 0.81–0.97 (m, 12H), 1.50–1.62 (m, 3H),
2.03–2.15 (m, 1H), 3.45–3.75 (m, 2H), 3.80–3.90 (m, 1H), 3.91–
3.99 (m, 1H), 4.03–4.12 (m, 1H), 4.16–4.25 (m, 1H), 4.30–4.45
(m, 2H), 7.48 (t, J = 8.1 Hz, 1H), 7.77–7.90 (m, 2H), 8.29–8.32 (m,
1H); 13C NMR (75.4 MHz, CD3OD) d 18.6, 19.7, 21.9, 23.3, 25.8,
31.9, 42.0, [44.3 & 44.4], 53.2, 56.2, [60.6 & 60.7], 61.8, 72.5,
[122.7 & 122.9], [125.8 & 126.1], [126.6 & 126.7], 129.0, 132.6,
139.3, [168.6 & 168.8], 169.5, [173.1 & 173.2], 173.4, [175.1 &
(UV, rt 1.32), 96.7% (ELSD, rt 1.31); BACE-1 IC50 = 0.87
lM.
4.1.7.28. 3-{3-[(S)-2-((S)-2-{(R)-2-Amino-3-[(2H-tetrazole-5-car
bonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-4-meth
yl-pentanoylamino]-propionylamino}-benzoic acid (36). Com-
pound 36 (45 mg, 25% yield) was prepared according to the General
procedures from Fmoc-3-Abz-OH (108 mg, 0.30 mmol) in the fol-
lowing order: Fmoc-b-Ala-OH, Fmoc-L-Leu-OH, Fmoc-L-Val-OH,
Boc- -DAP(Fmoc)-OH, and finally tetrazole 9. Compound 36: white
D
powder after lyophilization. 1H NMR (CD3OD) d 0.86–0.97 (m,
12H), 1.53–1.66 (m, 3H), 2.08 (m, 1H), 2.60–2.64 (m, 2H), 3.47–
3.58 (m, 2H), 3.86–3.89 (m, 2H), 4.17 (m, 1H), 4.28–4.38 (m, 2H),
7.40 (m, 1H), 7.71–7.81 (m, 2H), 8.26 (m, 1H); 13C NMR (CD3OD) d
18.7, 19.7, 22.0, 23.3, 25.8, 31.8, 36.7, 37.2, 41.4, 41.8, 53.4, 54.4,
61.0, 122.4, 125.5, 126.3, 129.9, 132.5, 140.1, 153.0, 160.8, 168.6,
175.2]; HRMS m/z 524.2717 [(M+H)+ calcd for C24H38N5O8
þ
524.2720]; HPLC (10–100% MeOH) purity: 98.5% (C4, rt 1.01),
97.0% (C18, rt 2.49); BACE-1 IC50 >10 lM.
4.1.7.25. 3-{3-[(S)-2-((S)-2-{(R)-2-Amino-3-[(2H-tetrazole-5-car
bonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-4-met
hyl-pentanoylamino]-2-hydroxy-propionylamino}-benzoic acid
(32). Compound 32 (10.0 mg, 15% yield) was prepared according
to the General procedures from 26 (31 mg, 0.11 mmol) in the follow-
169.6, 172.3, 173.3, 174.6; HRMS m/z 603.3003 [(M+H)+ calcd for
þ
C
26H39N10O7 603.2998]; HPLC purity: 96.2% (UV, rt 3.25, MeOH),
96.4% (ELSD, rt 3.14, MeOH); BACE-1 IC50 >10 lM.
ing order: Fmoc-
L
-Leu-OH, Fmoc-
L
-Val-OH, Boc-
D
-DAP(Fmoc)-OH,
4.1.7.29. 3-{3-[(S)-2-((S)-2-{(R)-3-Hydroxy-2-[(2H-tetrazole-5-
andfinallytetrazole9. Compound32: whitepowderafter lyophiliza-
tion. 1H NMR (CD3OD) d 0.80–0.99 (m, 12 H), 1.48–1.69 (m, 3H),
2.00–2.20 (m, 1H), 3.47–3.74 (m, 2H), 3.82–3.95 (m, 2H), 4.12–
4.17 (m, 1H), 4.23–4.32 (m, 2H), 4.35–4.41 (m, 1H), 7.43 (t,
carbonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-
4-methyl-pentanoylamino]-propionylamino}-benzoic
acid
(37). Compound 37 (26 mg, 14% yield) was prepared according
to the General procedures from Fmoc-3-Abz-OH (108 mg,

F. Wångsell et al. / Bioorg. Med. Chem. 19 (2011) 145–155
155
0.30 mmol) in the following order: Fmoc-b-Ala-OH, Fmoc-
L
-Leu-
0.95 (m, 3H), 0.98–1.04 (m, 6H), 1.62–1.75 (m, 3H), 2.08–2.18
(m, 1H), 3.72–3.83 (m, 1H), 3.89–3.95 (m, 1H), 3.21–3.34 (m,
2H), 4.41–4.48 (m, 1H); 13C NMR (CD3OD) d 17.7, 19.7, 21.9, 23.4,
26.0, 31.8, 41.6, 41.8, 52.4, 54.8, 60.9, 158.4, 163.7, 168.5, 173.2,
OH, Fmoc- -Val-OH, Fmoc-
L
D
-Ser(tBu)-OH, and finally tetrazole 9.
Compound 37: white powder after lyophilization. 1H NMR
(CD3OD) d 0.84–0.90 (m, 6H), 0.94–0.98 (m, 6H), 1.53–1.64 (m,
3H), 2.16 (m, 1H), 2.60–2.64 (m, 2H), 3.51–3.56 (m, 2H), 3.87–
3.98 (m, 2H), 4.20 (m, 1H), 4.32 (m, 1H), 4.79 (m, 1H), 7.41 (m,
1H), 7.72 (m, 1H), 7.80 (m, 1H), 8.23 (m, 1H); 13C NMR (CD3OD)
d 18.3, 19.6, 21.7, 23.4, 25.9, 31.3, 36.9, 37.3, 41.6, 53.4, 56.3,
61.0, 63.1, 122.3, 125.5, 126.2, 129.9, 132.5, 140.1, 154.1, 157.6,
169.5, 172.2, 172.6, 173.5, 174.8; HRMS m/z 604.2843 [(M+H)+
176.2; HRMS m/z 413.2256 [(M+H)+ calcd for C16H29N8O5
þ
413.2261]; HPLC purity: >99.0% (C4, rt 0.78), 97.6% (C18, rt 1.33);
BACE-1 IC50 = 4.4 lM.
Acknowledgments
þ
calcd for C26H38N9O8 604.2838]; HPLC purity: 95.1% (UV, rt
Dr. Elizabeth Hamerlink and Dr. Ian Henderson for the BACE-1
enzyme data. Alexandra Johansson, and Elisabet Lilja for excellent
technical assistance in the production of BACE-1. Dr. Aleh Yahorau
for conducting the HRMS experiments. Finally, we would like to
acknowledge the Swedish Research Council, Alice and Knut Wal-
lenberg’s Foundation and Medivir AB for financial support.
1.67), 98.1% (ELSD, rt 1.66); BACE-1 IC50 >10 lM.
4.1.7.30.
3-{3-[(S)-2-((S)-2-{(S)-3-Hydroxy-2-[(2H-tetrazole-5-
carbonyl)-amino]-propionylamino}-3-methyl-butyrylamino)-
4-methyl-pentanoylamino]-propionylamino}-benzoic
acid
(38). Compound 38 (22 mg, 12% yield) was prepared according
to the General procedures from Fmoc-3-Abz-OH (108 mg,
References and notes
0.30 mmol) in the following order: Fmoc-b-Ala-OH, Fmoc-
L-Leu-
-Ser(tBu)-OH, and finally tetrazole 9.
1. Hamada, K.; Kiso, Y. Expert Opin. Drug Discov. 2009, 4, 391.
2. Silvestri, R. Med. Res. Rev. 2009, 29, 295.
3. Evans, D. A.; Funkenstein, H. H.; Albert, M. S.; Scherr, P. A.; Cook, N. R.; Chown,
M. J.; Hebert, L. E.; Hennekens, C. H.; Taylor, J. O. JAMA 1989, 262, 2551.
4. Melnikova, I. Nat. Rev. Drug Discov. 2007, 6, 341.
5. Citron, M. Nat. Rev. Neurosci. 2004, 5, 677.
6. Hardy, J.; Selkoe, D. J. Science 2002, 297, 353.
7. Masters, C. L.; Cappai, R.; Barnham, K. J.; Villemagne, V. L. J. Neurochem. 2006,
97, 1700.
8. Ghosh, A. K.; Kumaragurubaran, N.; Hong, L.; Koelsh, G.; Tang, J. Curr. Alzheimer
Res. 2008, 5, 121.
OH, Fmoc- -Val-OH, Fmoc-
L
L
Compound 38: white powder after lyophilization. 1H NMR
(CD3OD) d 0.83–0.89 (m, 6H), 0.93–0.97 (m, 6H), 1.53–1.66 (m,
3H), 2.14 (m, 1H), 2.54–2.59 (m, 2H), 3.42–3.59 (m, 2H), 3.92–
3.96 (m, 2H), 4.17 (m, 1H), 4.34 (m, 1H),4.69–4.72 (m,1H), 7.41
(m, 1H), 7.72 (m, 1H), 7.80 (m, 1H), 8.23 (m, 1H); 13C NMR (CD3OD)
d 18.5, 19.7, 21.7, 23.3, 25.8, 31.3, 36.9, 37.4, 41.6, 53.4, 57.0, 60.9,
62.9, 122.3, 125.4, 126.2, 129.8, 132.5, 140.1, 154.7, 158.0, 169.5,
172.1, 172.5, 173.5, 174.7; HRMS m/z 604.2843 [(M+H)+ calcd for
9. Hills, I. D.; Vacca, J. P. Curr. Opin. Drug Discov. Devel. 2007, 10, 383.
10. John, V. Curr. Top. Med. Chem. 2006, 6, 569.
þ
C
26H38N9O8 604.2838]; HPLC purity: 95.5% (UV, rt 1.64), 95.6%
11. Thompson, L. A.; Bronson, J. J.; Zusi, F. C. Curr. Pharm. Des. 2005, 11, 3383.
12. Kimura, T.; Shuto, D.; Hamada, Y.; Igawa, N.; Kasai, S.; Liu, P.; Hidaka, K.;
Hamada, T.; Hayashi, Y.; Kiso, Y. Bioorg. Med. Chem. Lett. 2005, 15, 211.
13. Hamada, Y.; Abdel-Rahman, H.; Yamani, A.; Nguyen, J.; Stochaj, M.; Hidaka, K.;
Kimura, T.; Hayashi, Y.; Saito, K.; Ishiura, S.; Kiso, Y. Bioorg. Med. Chem. Lett.
2008, 18, 1649.
14. Hamada, Y.; Ohta, H.; Miyamoto, N.; Yamaguchi, R.; Yamani, A.; Hidaka, K.;
Kimura, T.; Saito, K.; Hayashi, Y.; Ishiura, S.; Kiso, Y. Bioorg. Med. Chem. Lett.
2008, 18, 1654.
(ELSD, rt 1.63); BACE-1 IC50 >10 lM.
4.1.7.31. (4S,7S,10S)-4-Amino-14-hydroxy-10-isobutyl-7-isopro-
pyl-1,5,8,11-tetraoxo-1-(2H-tetrazol-5-yl)-2,6,9,12-tetraazapen-
tadecan-15-oic acid (39). Compound 39 (6 mg, 12% yield) was
prepared according to the General procedures from Fmoc-
ine-OH (36.0 mg, 0.11 mmol) in the following order: Fmoc-
OH, Fmoc- -Val-OH, Boc- -DAP(Fmoc)-OH, and finally tetrazole 9.
D,L
-Isoser-
L
-Leu-
15. Rich, D. H. J. Med. Chem. 1985, 28, 263.
L
L
16. Agarwal, N. S.; Rich, D. H. J. Med. Chem. 1986, 29, 2519.
17. Ekegren, J. K.; Ginman, N.; Johansson, S. A.; Wallberg, H.; Larhed, M.;
Samuelsson, B.; Unge, T.; Hallberg, A. J. Med. Chem. 2006, 49, 1828.
18. Ekegren, J. K.; Unge, T.; Safa, M. Z.; Wallberg, H.; Samuelsson, B.; Hallberg, A. J.
Med. Chem. 2005, 48, 8098.
19. Mahalingam, A. K.; Axelsson, L.; Ekegren, J. K.; Wannberg, J.; Kihlström, J.;
Unge, T.; Wallberg, H.; Samuelsson, B.; Larhed, M.; Hallberg, A. J. Med. Chem.
2010, 53, 607.
Compound 39: white powder after lyophilization. 1H NMR (CD3OD)
d0.88–0.91 (m, 3H), 0.93–0.96 (m, 3H), 0.98–1.03 (m, 6H), 1.54–1.73
(m, 3H), 2.09–2.17 (m, 1H), 3.01–3.08 (m, 2H), 3.49–3.74 (m, 1H),
3.89–4.00 (m, 2H), 4.20–4.25 (m, 2H), 4.42–4.48 (m, 1H); 13C NMR
(CD3OD) d 18.7, 19.8, [22.0 & 22.1], 23.4, 25.6, 31.7, 41.5, 41.8, 44.1,
[53.4 & 53.5], 54.5, 61.2, [70.6 & 70.7], 165.5, 161.7, 168.7, 173.3,
20. Wångsell, F.; Russo, F.; Sävmarker, J.; Rosenquist, Å.; Samuelsson, B.; Larhed, M.
Bioorg. Med. Chem. Lett. 2009, 19, 4711.
175.1, 175.7; HRMS m/z 500.2577 [(M+H)+ calcd for C19H34N9O7
þ
21. Russo, F.; Wångsell, F.; Sävmarker, J.; Jacobsson, M.; Larhed, M. Tetrahedron
2009, 65, 10047.
22. Liu, X.; Hu, X. E.; Tian, X.; Mazur, A.; Ebetino, F. H. J. Organomet. Chem. 2002,
646, 212.
500.2581]; HPLC purity: >99.0% (C4, rt 0.69), 97.9% (C18, rt 0.91);
BACE-1 IC50 = 6.1 lM.
23. Orrling, K. M.; Marzahn, M. R.; Gutiérrez-de-Terán, H.; Åqvist, J.; Dunn, B. M.;
Larhed, M. Bioorg. Med. Chem. 2009, 17, 5933.
4.1.7.32. (S)-2-((S)-2-((S)-2-Amino-3-(2H-tetrazole-5-carboxa-
mido)propanamido)-3-methylbutanamido)-4-methylpentanoic
acid (40). Compound 40 (11 mg, 24% yield) was prepared accord-
24. Ford, R. E.; Knowles, P.; Lunt, E.; Marshall, S. M.; Penrose, A. J.; Ramsden, C. A.;
Summers, A. J.; Walker, J. L.; Wright, D. E. J. Med. Chem. 1986, 29, 538.
25. Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404.
26. Al-Muaikel, N. S.; Al-Diab, S. S.; Al-Salamah, A. A.; Zaid, A. M. A. J. Appl. Polym.
Sci. 2000, 77, 740.
ing to the General procedures from Fmoc-
L-Leu-OH (38.9 mg,
0.11 mmol) in the following order: Fmoc- -Val-OH, Boc-
L
L
-DAP(F-
moc)-OH, and finally tetrazole 9. Compound 40: white powder
after lyophilization. 1H NMR (CD3OD) d 0.87–0.90 (m, 3H), 0.92–