BACE-1 inhibitors part 2: Identification of hydroxy ethylamines (HEAs) with reduced peptidic character

Article abstract of DOI:10.1016/j.bmcl.2007.12.019

This paper describes the discovery of non-peptidic, potent, and selective hydroxy ethylamine (HEA) inhibitors of BACE-1 by replacement of the prime side of a lead di-amide 2. Inhibitors with nanosmolar potency and high selectivity were identified. Dependi

Full text of DOI:10.1016/j.bmcl.2007.12.019

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1017–1021
BACE-1 inhibitors part 2: Identification of hydroxy
ethylamines (HEAs) with reduced peptidic character
Brian Clarke, Emmanuel Demont,* Colin Dingwall, Rachel Dunsdon, Andrew Faller,
Julie Hawkins, Ishrut Hussain, David MacPherson, Graham Maile, Rosalie Matico,
Peter Milner, Julie Mosley, Alan Naylor, Alistair O’Brien, Sally Redshaw, David Riddell,
Paul Rowland, Virginie Soleil, Kathrine J. Smith, Steven Stanway, Geoffrey Stemp,
Sharon Sweitzer, Pam Theobald, David Vesey, Daryl S. Walter,
John Ward and Gareth Wayne
Neurology and Gastrointestinal Centre of Excellence for Drug Discovery, GlaxoSmithKline R&D, New Frontiers Science Park,
Third Avenue, Harlow, Essex CM19 5AW, United Kingdom
Received 30 October 2007; revised 7 December 2007; accepted 11 December 2007
Available online 15 December 2007
Abstract—This paper describes the discovery of non-peptidic, potent, and selective hydroxy ethylamine (HEA) inhibitors of BACE-
1 by replacement of the prime side of a lead di-amide 2. Inhibitors with nanosmolar potency and high selectivity were identified.
Depending on the nature of the P01 and P20 substituents, two different binding modes were observed in X-ray co-crystal structures.
Ó 2007 Elsevier Ltd. All rights reserved.
The preceding paper in this series1 described how a fo-
cused library of potential BACE-1 inhibitors delivered
a hit (compound 1, Table 1), which was then optimised
to give compounds such as 2, a sub-micromolar inhibi-
tor of amyloid-b (Ab40 and Ab42) production in cells
expressing amyloid precursor protein (APP) Wild-Type.
A precise combination of meta-substituents on the phe-
nyl ring of the non-prime side benzamide was found to
be important for good potency and selectivity.
commercially available epoxide 3 or reductive amina-
tions using amine 4.3
In total, 150 potential inhibitors were prepared and four
were found to have IC50s of less than 200 nM (Fig. 2).
The first of these, compound 6, which incorporates a
cyclohexylamine at the prime side, was especially inter-
esting since it is only 10-fold less potent than lead com-
pound 2, despite significant truncation (Table 2).
Analogues containing cyclopentylamine or cycloheptyl-
amine were ꢁ 10-fold less potent (compounds 7 and
8). Introduction of a heteroatom into the ring (com-
pound 9) or replacing the cyclohexyl with cyclohexylm-
ethyl (compound 10) was also detrimental to activity.
Compounds in which the cyclohexyl ring was replaced
with smaller alkyl fragments were also more than 10-
fold less potent than 6 (data not shown).
The next round of optimisation focused on modifica-
tions to the prime side. The strategy was to replace the
amide bond-containing P01 ꢀ P20 fragment by groups with
lower molecular weight, fewer heteroatoms and a lower
polar surface area, in order to obtain inhibitors with im-
proved potential for oral absorption and brain
penetration.
The routes used to generate a library of such compounds
are depicted in Figure 1 and involved either opening of
The co-crystal structure of inhibitor 6 with the enzyme4
showed that the cyclohexyl moiety was binding into the
S01 pocket which contrasts with the observation that the
peptidic prime side of inhibitor 2 binds into the S02 pocket
(Fig. 3). It was difficult to pinpoint additional potential
interactions within the pocket and consequently further
derivatization of the cyclohexyl ring was not explored.
Keywords: Alzheimer; BACE-1; Aspartic protease; Hydroxy
ethylamine.
*
Corresponding author. Tel.: +44 1279643468; fax: +44
1279622727; e-mail: emmanuel.h.demont@gsk.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.019

1018
B. Clarke et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1017–1021
Table 1. Activity and selectivity of inhibitors 1 and 2
O
H
R1
N
N
H
N
H
OH
O
R2
Compound R1
R2
H
BACE-1a,b IC50 (nM) BACE-2 IC50 (nM) Cat-D IC50 (nM) Ab40b,c IC50 (lM) Ab42b,c IC50 (lM)
1
CH3SO2-
5400 (3)
41770
1810
7700
2695
—
—
O
2
-NHEt 13 (2)
0.31
0.36
N
a In all tables, IC50s reported are means of the values of n different experiments, n being reported in bracket and identical for BACE-1, BACE-2 and
Cat-D. Each IC50 is within 3-fold of the mean value.
b See Ref. 2 for protocol.
c IC50 values are means of at least two separate experiments. Each IC50 is within 3-fold of the mean value.
O
O
N
R2
O
N
H
N
a-c
OH R1
N
H
O
O
NH
50-70%
3
g
d, e, b
65%
50-90%
O
O
N
OH
O
5
O
NH
N
N
H
NH2
Z
OH
H2N
N
H
OH
c, f
NH
4
70%
Figure 1. Conditions and reagents: (a) NHR1R2, EtOH, reflux; (b) TFA, CH2Cl2, 0 °C or HCl, CH3CN or Dioxan, 25 °C; (c) 5, EDAC.HCl, HOBT,
CH2Cl2 or DMF, 25 °C; (d) NH3, MeOH/H2O, 25 °C; (e) Z-Cl, NEt3, DMF, 25 °C; (f) NH4COOH, Pd/C, EtOH/H2O, 60 °C; (g) aldehyde or ketone,
AcOH, (CHCl2)2, NaHB(OAc)3, 0 °C.
The second hit (compound 11, Table 3) was tested as a
mixture of diastereoisomers.5 However, the analogous
O
O
dimethyl derivative 12 proved to be equipotent and the
co-crystal structure of this inhibitor with the enzyme
showed that the prime side adopted an extended confor-
mation when interacting with the enzyme and could be
superimposed on the peptidic prime side of compound
2 (Fig. 4).
6
N
R1
BACE-1 IC50 =180 nM
N
N
H
H
OH
NH
11
BACE-1 IC50 =33 nM
The SAR around inhibitor 12 was investigated. Re-
moval of both methyl groups from the carbon adjacent
to the nitrogen led to a 10-fold decrease in potency
(compound 13). Removal of one of the terminal methyl
substituents (compound 14) also resulted in a significant
reduction in potency. Incorporating a heteroatom in the
R2
16 R2 = CF3
BACE-1 IC50 =40 nM
BACE-1 IC50 = 43 nM
17 R2 = OCH3
Figure 2. Activity of the four hits from the prime side replacement
library.

B. Clarke et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1017–1021
Table 3. Prime side SAR from inhibitor 11
1019
Table 2. Prime side SAR from inhibitor 6
O
O
O
O
N
N
H
R
N
R
N
H
N
N
H
H
OH
OH
NH
NH
Compound R
BACE-1 BACE-2 Cat-D
IC50 (nM) IC50 (nM) IC50 (nM)
Compound
R
BACE-1 BACE-2 Cat-D
IC50 (nM) IC50 (nM) IC50 (nM)
H
N
H
N
2
13 (2)
1810
2695
780
2
13 (2)
33 (2)
33 (1)
305 (2)
550 (1)
790 (2)
1810
2695
1095
2510
970
O
O
6
180 (1)
1120 (1)
2450 (1)
650 (1)
5890 (1)
5010
11
12
13
14
15
3090
7
12,020
41,690
5890
2400
10,960
1550
12,020
5890
8
3610
S
9
30,200
69,010
10,720
22,325
10
57,540
O
Figure 4. Superimposition of compounds 2 (red) and 12 (blue) prime
sides bound to BACE-1.
Figure 3. Superimposition of inhibitors 2 (red) and 6 (yellow) prime
sides bound to BACE-1.
alkyl chain also led to at least a 10-fold reduction in
potency (see compound 15 for a representative example)
and replacement of the alkyl prime side of compound 12
by shorter branched alkyl chains always resulted in at
least an order of magnitude loss of potency (data not
shown).
The similar potencies of compounds 11 and 12 were dif-
ficult to rationalize until the co-crystal structures of
these compounds were superimposed onto those of the
remaining hits from the initial array (compounds 16
and 17, Table 4). These inhibitors were as potent and
selective as compound 12 but, interestingly, removal of

1020
B. Clarke et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1017–1021
Table 4. Prime side SAR from inhibitors 16 and 17
O
O
N
R
N
N
H
H
OH
NH
Compound
R
BACE-1 BACE-2 Cat-D
IC50 (nM) IC50 (nM) IC50 (nM)
H
N
2
13 (2)
33 (1)
40 (1)
43 (1)
760 (1)
17 (1)
1810
5890
4070
7590
63,100
600
2695
2510
5750
5010
19,050
930
O
12
16
17
18
19
Figure 5. Superimposition of compounds 12 (blue) and 16 (red) prime
sides bound to BACE-1.
CF
3
potency of compounds lacking the meta- substituent or
those in which the substituent is moved to the ortho-
or para-position (compare activities of 20 and 21 versus
17).
O
Having identified benzylamines and, specifically, meta-
substituted benzylamines as suitable prime side replace-
ments, variations in the linker between the amine and
the aromatic ring were explored. As shown in Table 5,
compound 22, incorporating a phenyl prime side, was
somewhat less potent than compound 18 whilst com-
pound 23, with a phenethyl residue, had similar potency
to 18. However, the potency of compound 23 was not
significantly increased by substitution in the ortho- meta-
or para-positions (data not shown) and further efforts
focused on the benzylamine series. Modelling based on
co-crystal structures suggested that conformational
restriction of the benzylic residue should be possible
CF3
O
20
21
1350 (1)
2090
44,670
>106
9770
18,620
O
Table 5. Chain length modification and constrained derivatives
Compound Ra
BACE-1
IC50 (nM) IC50 (nM) IC50 (nM)
BACE-2
Cat-D
the meta-substituent from the phenyl ring gave a 50-fold
reduction in potency (compound 18). Di-methylation of
the benzylic position of compound 16 gave compound
19 with similar potency but slightly lower selectivity.
22
23
1700 (1)
600 (1)
21380
3390
4680
1780
Figure 5 shows the enzyme-bound conformations of the
prime side of inhibitors 12 and 16 superimposed. The
meta-CF3 group of compound 16 fills the same sub-
pocket as the terminal methyl substituent of compound
12 which may explain the similar loss of potency when
these substituents are removed. The 10-fold difference
in potency between dimethyl compound 12 and the
unsubstituted analogue 13 is not replicated in com-
pounds with a benzylic prime side. Here, compound 16
and the dimethyl analogue 19 show similar potency. It
may be hypothesized that in the former case, the substit-
uents are necessary to orientate the alkyl chain towards
the crucial S02 pocket: filling this pocket is essential for
good activity as shown by the significantly reduced
24
160 (1)
3720
5250
25
26
34,670 (1) 93,330
10,230
380
14 (2)
370
O
a See structure above in Table 4 for definition of R.

B. Clarke et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1017–1021
Table 6. Optimisation of the prime side substituent
1021
ring resulted in no significant benefit in terms of potency
or selectivity (compare for example 29 versus 17).
Replacement of the meta-substituted phenyl ring by
meta-substituted heteroaromatic rings led to compounds
such as 30 and 31 with decreased potency and selectivity.
Compound Ra
BACE-1 BACE-2 Cat-D
IC50 (nM) IC50 (nM) IC50 (nM)
O
O
17
42 (1)
7590
5010
In summary, it was shown that reduction in the peptidic
nature of the prime side of compound 2 led to a range of
nanomolar inhibitors of BACE-1 with two different
binding modes and with improved cell potency when
compared to 2 (Table 7).
CF3
27
28
20 (1)
83 (1)
2000
3550
5620
Cl
O
15,140
The absorption and brain penetrance of these com-
pounds were predicted to be sub-optimal (high PSA ,
CMR and MW, large number of HBAs and HBDs).8
We postulated that, with modest levels of CNS penetra-
tion, amyloid lowering in an animal model would most
likely be achieved by a compound with low nanomolar
potency in a cell-based assay.9 The further optimisation
of this series of inhibitors towards highly potent, orally
active inhibitors will form the subject of a subsequent
publication.
29
100 (1)
6920
5890
O
N
O
30
31
330 (1)
230 (1)
33,100
33,110
26,300
46,770
N
N
a See structure above in Table 4 for definition of R.
Table 7. Cellular activity of representative inhibitors
Compound
BACE-1
IC50 (nM)
Ab40a,b
IC50 (lM)
Ab42a,b
IC50 (lM)
References and notes
2
13 (2)
180 (1)
33 (1)
40 (1)
0.31
—
0.36
—
6
1. See the preceding paper.
12
16
0.23
0.18
0.21
0.20
2. Hussain, I.; Hawkins, J.; Harisson, D.; Hille, C.; Wayne,
G.; Cutler, L.; Buck, T.; Walter, D.; Demont, E.; Howes,
C.; Naylor, A.; Jeffrey, P.; Gonzalez, M. I.; Dingwall, C.;
Michel, A.; Redshaw, S.; Davis, J. B. J. Neurochem. 2007,
100, 802.
a See Ref. 2 for protocol.
b IC50 values are means of at least two separate experiments. Each IC50
is within 3-fold of the mean value.
3. The synthesis of acid 5 is described in the preceding paper.
4. See preceding paper for references on BACE-1 construct.
The X-ray data were collected at ESRF beamline ID14-4.
The PDB deposition codes and refinement details for the
BACE-1 complex crystal structures are: compound 2 (2vj6,
and that the S-indane should be more potent than the
corresponding R-isomer. This was confirmed experi-
mentally (compounds 24 and 25) with the S-indane 24
showing a 5-fold improvement in potency compared
with 18.6,7
˚
1.8A resolution, R = 0.187, Rfree = 0.212), compound 6
˚
(2vj9, 1.6A resolution, R = 0.203, Rfree = 0.232), compound
˚
12 (2vie, 1.9A resolution, R = 0.191, Rfree = 0.216) and
˚
compound 16 (2vj7, 1.6A resolution, R = 0.209,
Rfree = 0.231).
Combining some of the observations described above, it
was hoped that filling both the S01 and S20 pockets simul-
taneously might lead to increased potency but com-
pound 26, incorporating both the S01 cyclohexyl group
and the S02 meta-methoxy benzyl group, was not signifi-
cantly more potent than inhibitor 17 (3-fold) which is
able to interact with only one of the prime-side pockets.
Compound 26 was also less selective versus BACE-2 and
Cat-D than compound 17.
5. The stereoisomers of compound 11 were not separated.
6. Similar conformational constraint of the phenethyl prime
side did not give an improvement in potency (data not
shown).
7. In a further optimised series of compounds, introduction of
a meta-substituent into the S-indane did not lead to a
significant improvement in potency. See following paper.
8. For discussion on absorption and brain-penetration, see for
examples: (a) Lipinski, C. A.; Lombardo, F.; Dominy, B.
W.; Feeney, P. J. Adv. Drug Deliv. Rev. 1997, 23, 3; (b)
Clark, D. E. J. Pharm. Sci. 1999, 88, 807; (c) Seelig, A. Eur.
J. Biochem. 1998, 251, 252.
9. It was felt that IC50 ꢂ 10 nM could be achieved in a cell-
based assay, as it has been in the HIV protease field. See for
example: Ami, E.; Nakahara, K.; Sato, A.; Nguyen, J.-T.;
Hidaka, K.; Hamada, Y.; Nakatani, S.; Kimura, T.; Hay-
ashi, Y.; Kiso, Y. Bioorg. Med. Chem. Lett 2007, 17, 4213.
Further exploration of different meta-substituents on the
phenyl ring did not lead to more potent inhibitors. A
large range of lipophilic substituents was tolerated but
no significant improvement in potency or selectivity
was obtained compared with the initial hit 17 (Table 6,
see representative examples 27 and 28). Moreover,
incorporation of additional substituents in the aromatic