P3 cap modified Phe*-Ala series BACE inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.09.085

With the aim of reducing molecular weight and adjusting log D value of BACE inhibitors to more favorable range for BBB penetration and better bioavailability, we synthesized and evaluated several series of P3 cap modified BACE inhibitors obtained via replacement of the P3 NHBoc moiety as seen in 3 with other polar functional groups such as amino, hydroxyl and fluorine. Several promising inhibitors emerging from this P3 cap SAR study (e.g., 15 and 19) demonstrated good enzyme inhibitory potencies (BACE-1 IC₅₀ <50 nM) and whole cell activities (IC₅₀ ～1 μM).

Full text of DOI:10.1016/j.bmcl.2003.09.085

Bioorganic & Medicinal Chemistry Letters 14 (2004) 245–250
P3 cap modified Phe*-Ala series BACE inhibitors
Shu-Hui Chen,* Jason Lamar, Deqi Guo, Todd Kohn, Hsiu-Chiung Yang, James McGee,
David Timm, Jon Erickson, Yvonne Yip, Patrick May and James McCarthy
Eli Lilly and Company, Lilly Research Laboratories, Discovery Chemistry Division and Technology, Lilly Corporate Center,
Indianapolis, IN 46285, USA
Received 22 July 2003; accepted 9 September 2003
Abstract—With the aim of reducing molecular weight and adjusting log D value of BACE inhibitors to more favorable range for
BBB penetration and better bioavailability, we synthesized and evaluated several series of P3 cap modified BACE inhibitors
obtained via replacement of the P3 NHBoc moiety as seen in 3 with other polar functional groups such as amino, hydroxyl and
fluorine. Several promising inhibitors emerging from this P3 cap SAR study (e.g., 15 and 19) demonstrated good enzyme inhibitory
potencies (BACE-1 IC50 <50 nM) and whole cell activities (IC50 ꢀ1 mM).
# 2003 Elsevier Ltd. All rights reserved.
Almost a century after its initial description, Alzhei-
mer’s disease (AD) has been recognized as an emerging
healthcare problem.1 Although the actual cause of the
disease has not been identified with certainty, numerous
theories have been formulated to explain the pathology
of AD with amyloid cascade hypothesis being the most
popular one.2 According to this b-amyloid hypothesis,
Ab42 produced in excessive quantities, or cleared too
slowly, or in the presence of aggregation factors, would
form aggregates that lead to senile plaques, which may
then initiate an immunologic and neurotoxic cascade
resulting in the pathology of AD.3 Since Ab (40/42)
peptides are produced by two consecutive protease
cleavage events (by b- and g-secretases)4,5 of the b-
amyloid precursor protein (APP), thus inhibition of the
enzyme activity of either secretase involved could in
theory slow down Ab formation and thereby progres-
sion of Alzheimer’s disease. Furthermore, a recent
report from Vassar showed that BACE KO mice are
healthy, fertile, and appear normal in gross anatomy,
tissue histology, hematology, and clinical chemistry.6
When taken together as a whole, the information pre-
sented herewith suggests that b-secretase is a prime drug
target for the treatment of AD disease. Prompted by the
mounting evidences supporting b-amyloid hypothesis,7
we8 and others9 embarked on the design and evaluation
of peptidyl mimetic BACE inhibitors. To build upon the
Leu*-Ala series inhibitors reported recently by Tang’s
group,10 we focused on both C- and N-termini SAR
modifications surrounding Phe*-Ala based inhibitors.
As a result of our effort, many inhibitors bearing C-ter-
mini Val-Pyridine moieties (as P20–P20cap) demon-
strated excellent whole cell activities with IC50 values
less than 1 uM.8a
During the course of our crystal structure based inhi-
bitor design effort, we discovered that the N-termini
Boc groupsuch as that seen in 1 or 2 was not involved
in the intimate contact with the BACE enzyme, sug-
gesting that this functional groupmay be modified
without significant loss of enzyme inhibitory activity.8a
To take advantage of this observation, we decided to
synthesize several novel P3 capbearing inhibitors
4
through 7 endowed with reduced MW and possibly
altered physical chemical properties (e.g., Log D, aqu-
eous solubility, and tissue distribution) in comparison to
compound 3 (Fig. 1).
The synthetic routes utilized for the preparation of
inhibitors 5 and 7 are outlined in Scheme 1. PyBOP
mediated coupling11 reaction between AA and alanine
methylester CC afforded the expected adduct EE (55%),
which was then subjected to NaOH mediated ester
hydrolysis to give the desired hydroxyacid GG (60%).
Final coupling of GG with the known building block
II8a was promoted by HOAt/EDCI,12 providing the
desired diol 5 in 30% yield after reverse-phase HPLC
purification. Following essentially identical sequence as
* Corresponding author. Tel.: +1-317-276-2076; fax: +1-317-276-
1177; e-mail: chen_shu-hui@lilly.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.085

246
S.-H. Chen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 245–250
described for inhibitor 5, the P3 NHBoc truncated inhi-
bitor 7 was synthesized in three steps with 32% overall
yield (see Scheme 1).
of (s)-4-benzyl-2-oxazolidinone JJ with isovaleryl
chloride provided the adduct LL (93%). Treatment of
LL with LiHMDS, followed by N-fluorodibenzene-
sulfonamide thus afforded the fluorinated intermediate
MM (91%), which was then converted to the desired
acid NN (71%) according to a published procedure.13
The synthetic route employed for the preparation of the P3
fluorinated inhibitor 6 is shown in Scheme 2. N-Acylation
Figure 1. P3 capmodified BACE inhibitors.
Scheme 1. Syntheses of BACE inhibitors 5 and 7. Reagents and conditions: (i) PyBOP/CH2Cl2/THF/i-Pr2EtN; (ii) 1 N NaOH/MeOH, then 1 N
HCl; (iii) 4 N HCl in dioxane/CH2Cl2; (iv) HOAt/EDCI/i-Pr2EtN/THF+DMF.
Scheme 2. Synthesis of BACE inhibitor 6. Reagents and conditions: (i) LiHMDS/THF/KK; (ii) LiHMDS/THF/F-N(SO2Ph)2; (iii) LiOH/30%
H2O2/THF/H2O; (iv) HOAt/EDCI/CH2Cl2+DMF, then l-Ala-OBu(t) and i-Pr2EtN; (v) 4 N HCl in dioxane+CH2Cl2; (vi) HOAt/EDCI/
THF+DMF, then II/i-Pr2EtN.

S.-H. Chen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 245–250
247
HOAt/EDCI mediated coupling12 of NN with alanine
t-butylester provided the expected adduct OO (60%),
which was further converted to its corresponding acid
PP (100%) via saponification. Finally, coupling of the
fluorinated acid PP with the requisite amine II provided
the final product 6 in 36% yield.
nificant biological activity with whole cell IC50 value
ranging from 49 to >100 uM. When evaluated in the
enzyme assay, inhibitors 6 and 7 were found to be at
least 13-fold less potent than the P3 NHBoc bearing
inhibitor 3. Thus, in view of the data presented in Table
1, it is clear that replacing the P3 NHBoc moiety in 3
with a hydroxyl groupresulted in only minimal loss of
inhibitory activity (for 5) in both the enzyme and whole
cell assays. In addition, inhibitor 5 was found to be
most selective against BACE-2 within the BACE inhi-
bitors included in Table 1. Furthermore, when com-
pared with the P3-NHBoc inhibitor 3, the newly
synthesized P3 hydroxylated inhibitor 5 had lower Mr
(ꢀ100 mass units) as well as more favorable Log D
value of 2.88 for brain penetration.18 To build upon
these results, we decided to pursue additional inhibitor 5
based SAR investigation to further optimize this novel
series inhibitors. The syntheses and evaluation of these
new inhibitors (8–22) are discussed in Tables 2–5.
It should be mentioned that the synthesis of the P3-
amino moiety bearing inhibitor 4 was accomplished by
treatment of the known inhibitor 38a with 4N HCl in
dioxane at 0 ꢁC. We noticed that prolonged acid treat-
ment of 3 resulted in expected N-deprotection along
with undesirable dehydration.
Four P3 capmodified BACE inhibitors 4–7 along with
the P3 NHBoc containing derivative 3 were evaluated in
the following assays: (1) in vitro homogenous BACE
FRET (fluorescent resonance energy transfer) enzyme
assay;14 and (2) whole cell Ab lowering assay in
HEK293/APP751swe cells.15 In addition, inhibitors 3–7
were also evaluated for BACE-2 selectivity16 and Log D
determination at neutral pH.17 The results of these test-
ing are summarized in Table 1.
1. Second round inhibitor evaluation
Following the same synthetic route described for 5, five
additional P2 modified inhibitors 8–12 were prepared
starting with their corresponding P2–P3 hydroxyacids
(EE0). As can be seen in Table 2, replacement of the P2
Ala. in 5 with either Met., Nva., or Ser. led to three new
inhibitors 8, 9, and 10 exhibiting 3- to 5-fold reduced
enzyme inhibitory activity and whole cell activity rela-
tive to inhibitor 5. Furthermore, inhibitors containing
either bulky (e.g., 11) or branched alkyl (e.g., 12) at the
P2 pocket were much less potent than 5 in the enzyme
assay.
As shown in Table 1, when compared with the NHBoc
bearing inhibitor 3, the P3 amino analogue 4 displayed
slightly reduced enzyme activity (2-fold) along with
rather significant reduction in whole cell activity (ꢀ20-
fold). The poor cellular activity observed with 4 was
likely attributed to its high hydrophilicity. On the other
hand, the P3 hydroxyl bearing inhibitor 5 exhibited
comparable enzyme inhibitory potency and slightly
reduced whole cell activity (2–3-fold) relative to the
P3 NHBoc containing counterpart 3. Replacement of
the P3 NHBoc in 3 with a fluorine or a hydrogen led to
6 or 7, respectively. Judging from the data shown in
Table 1, it is evident that neither 6 nor 7 showed sig-
Upon completion of our limited P2 SAR effort, we then
embarked on P3 and C-termini optimizations. The P3
Table 1. P3 capmodified BACE inhibitors
Compd
Mr
BACE-1 IC50 (nM)
BACE-2 selectivity
Abeta (sw) IC50 (mM)
Log D @ pH=7
3
697
597
598
600
582
82
190
130
1130
1740
0.4
0.52
2.8
1.35
0.72
2.1
38.4
4.8–7.5
49
>100
4.36
Not tested
2.88
4
5 (2070103)
6
7
3.45
3.2
Table 2. LY2070103 (5) based P2 modifications
Compd
P2
BACE-1 IC50 (mM)
Abeta (sw) IC50 (mM)
Log D (pH=7)
5
8
9
10
11
12
Me (Ala)
CH2CH2SMe (Met)
CH2CH2Me (Nva)
CH2OH (Ser)
0.13
0.35
0.55
0.35
4.49
4.64
4.8
25.2
30.4
20
2.88
3.64
3.46
2.73
CH2OBu-t
CHMe2 (Val)
Not tested
Not tested
Not tested
3.69

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S.-H. Chen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 245–250
modified inhibitors 13–15 were synthesized according to
the procedure outlined in Scheme 1 using either (s)-3-
trifluromethyl lactic acid19 or commercial available (s)-
2-hydroxyl-3,3-dimethylbutyric acid, or (s)-2-hydroxyl-
(s)-3-methyl pentanoic acid as their respective P3 resi-
due. When evaluated in the BACE FRET and whole
cell assays, whilst the P3 trifluoromethyl bearing inhi-
bitor 13 showed similar activity to that of 5, the P3 i-Bu
bearing inhibitor 14 was found to be ꢀ3-fold less
potent than compound 5 in the cellular assay. To our
satisfaction, the P3 Ile mimetic 15 demonstrated 4-fold
enhanced enzyme and cellular potency in comparison to
inhibitor 5. It is worthwhile to mention that the SAR
trend observed with 15 is in good agreement with that
found with its corresponding P3-NHBoc bearing inhi-
bitor.8a
Parallel with the P3 optimization, we also modify the C-
termini via either benzyl moiety incorporation or ste-
reospecific installation of a methyl group at the benzylic
position (see Table 4). All of the C-termini modified
inhibitors 16–18 were prepared via coupling of the P2–
P3 hydroxyacid EE with the appropriate II-like P1–P20
units. On the basis of the biological activities deter-
mined for these new analogues, it is clear that (1) no
stereochemistry preference was observed at the benzylic
position (P20 cap); (2) the C-termini Bn bearing inhi-
bitor 16 displayed 4-fold better enzyme potency than 5.
Table 3. LY2070103 (5) based P3 modifications
Compd
P3
BACE-1 IC50 (mM)
Abeta (sw) IC50 (mM)
Log D (pH=7)
5
–CHMe2 (Val)
–CH2CF3
–CH2CHMe2 (Leu)
–(s)-CHMeEt (i-Leu)
0.13
0.22
0.23
0.035
4.8
3.9
16.9
1.23
2.88
Not tested
3.36
13
14
15
3.21
Table 4. LY2070103 (5) based P10 and P20 capmodifications
Compound
P10
R
X
BACE-1 IC50 (mM)
Abeta (sw) IC50 (mM)
Log D (pH=7)
5
Me
Me
Et
H
H
N
0.13
0.037
0.06
0.10
4.8
6.3
19.8
13.6
2.88
3.91
Not tested
4.46
16
17
18
CH
CH
CH
(R)-Me
(S)-Me
Et
Table 5. LY2090089 (13) based modifications
Compd
Structural modification
BACE-1 IC50 (mM)
Abeta (sw) IC50 (mM)
Log D (pH=7)
15
19
20
21
22
None
CH2CHF2 as P2
Leu as P1
CH2CH2CH2F as P10
CH2CHF2 as P20
0.035
0.045
0.176
0.067
3.18
1.23
1.08
3.7
0.97–1.35
>37.5
3.21
3.71
Not tested
3.56
3.69

S.-H. Chen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 245–250
249
However, this enhanced enzyme potency did not trans-
late into any improvement in cellular assay. The lack of
correlation between enzyme and whole cell assays
observed for 16–18 (>100 fold) is likely due to high
lipophilicity of these inhibitors.8a This is due at least in
part to the absence of the polar C-termini pyridyl moi-
ety as seen in inhibitor 5.
with inhibitor 2.8a It is also evident that the N-Boc
moiety in 2 pointed away from the enzyme surface.
Consistent with this finding, the enzyme inhibitory
potency detected with 5 is very similar to that observed
with 3 (see Table 1).
Guided by the insights gained through detailed analysis
of the co-crystal structures of 1 and 2 with human
BACE enzyme, we designed and synthesized several
novel series P3 capmodified inhibitors including the P3
hydroxylated inhibitor 5. Compared with the P3 NHBoc
bearing compound 3, inhibitor 5 had lower molecular
weight (ꢀ100 mass units) and more favorable Log D
value for BBB penetration,18 albeit with slightly reduced
biological activity. Subsequently, we carried out com-
pound 5 based systematic SAR modifications in hopes
of identifying inhibitors with enhanced activity. As a
result of this effort, we discovered several P3 hydro-
xylated inhibitors (e.g., 15, 19, and 21) showing
impressive enzyme (IC50 range: 35–67 nM) and cellular
activities (IC50ꢀ1 uM).
With the aim of further improving upon the potent
inhibitor 15 (BACE IC50=35 nM) identified via P3
SAR optimization, we decided to incorporate fluori-
nated amino acids into various position such as P2, P10
and P20. The syntheses of 19 and 22 were accomplished
in accordance with the reaction sequence outlined in
Scheme 1 using the appropriate P2 or P20 residue. On
the other hand, inhibitor 21 was prepared via incor-
poration of the fluorinated n-Pr moiety into the P10
position.8c In order to synthesize compound 20, we uti-
lized the Leu. P1 bearing version of II as the building
block, which was in turn prepared following a published
procedure.10a With the new inhibitors in hand, we car-
ried out comparative evaluation against inhibitor 15.
Careful inspection of the testing result shown in Table 5
reveals the following SAR trends: (1) fluorine incor-
poration at either P2 or P10 position did not impact
BACE inhibitory activity; (2) replacing the P1 Phe with
Leu resulted in 3–4-fold dropin both the enzyme and
cellular potency; and (3) introduction of the difluor-
oaminobutyric acid at the P20 site resulted in significant
loss of enzyme inhibitory activity and cellular potency
(ꢀ100ꢂ).
Acknowledgements
We are indebted to R. Brier, D. K. Laigle, J. Huang, T.
Yin for performing bioassays. We shall also thank J.
Copp, L. Patterson, and T. Kohn for providing various
fluorinated building blocks as well as S. Semones, L.
Zornes for Log D measurements. Acknowledgments
should be made to Drs. T. Burkholder, Y.-C. Xu, J.
Munroe, and J. Audia for helpful discussions and
encouragement.
2. Enzyme–inhibitor interaction
The co-crystal of the P3 hydroxylated inhibitor 5 with
human BACE enzyme was solved using standard pro-
tocol.20,21 Inspection of the structure shown in Figure 2
reveals that the P3 isopropyl group was indeed posi-
tioned at the P3 binding pocket. We also notice that the
types of interactions made by the backbone and other
side chains of 5 overlapped very well with that found
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Figure 2. Crystal structures of inhibitors 2 and 5 binding to BACE-1.

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the majority of the FDA approved drugs for the treat-
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14. BACE FRET assay uses 20 nM purified recombinant
human BACE/Fc. The standard assay format contains
100 M ELGY-9 (an aminobenzoate based FRET peptide
containing Swedish mutation) in 50 mM ammonium ace-
tate, pH 4.6, 1 mg/mL BSA and 1 mM Triton X-100 at
room temperature for 4 h. The enzyme activity is deter-
mined by an increase in the relative fluorescent of reaction
mixture, with umbilliferone excitation/emission filter set.
15. Whole cell Ab lowering assay measures intracellular inhi-
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Testing subjects are incubated with cells for 4 h at 37 ꢁC
20. Co-crystal of 5 with BACE: Crystals of the BACE cata-
lytic domain were grown in the presence of a 5-fold molar
excess of LY2070103 (5) using minor modifications of
previously described methods cited in ref 21. Diffraction
data were collected at 100 K using a synchrotron source
on beamline 17ID at the Advanced Photon Source. The
LY2070103 (5) model was built into active site electron den-
sity using QUANTA (Accelrys, Inc) and O (Acta Crystal-
logr. 1991, A47, 110). The structure of BACE bound to
2070103 has been determined at 2.05 Ang. resolution and
refined to a crystallographic R-factor of 0.201 (Rfree=0.227).
The figure shows a superposition of the 2070103 structure
with the OM99-2 inhibitor structure (see ref 21).
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