Evaluation of a Series of β-Secretase 1 Inhibitors Containing Novel Heteroaryl-Fused-Piperazine Amidine Warheads

Article abstract of DOI:10.1021/acsmedchemlett.9b00181

Despite several years of research, only a handful of β-secretase (BACE) 1 inhibitors have entered clinical trials as potential therapeutics against Alzheimer's disease. The intrinsic basic nature of low molecular weight, amidine-containing BACE 1 inhibitors makes them far from optimal as central nervous system drugs. Herein we present a set of novel heteroaryl-fused piperazine amidine inhibitors designed to lower the basicity of the key, enzyme binding, amidine functionality. This study resulted in the identification of highly potent (IC₅₀ ≤ 10 nM), permeable lead compounds with a reduced propensity to suffer from P-glycoprotein-mediated efflux.

Full text of DOI:10.1021/acsmedchemlett.9b00181

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Letter
Evaluation of a series of BACE 1 inhibitors containing
novel het-eroaryl-fused-piperazine amidine warheads
Daniel Oehlrich, Aldo Peschiulli, Gary Tresadern, Michiel Van Gool, Juan Antonio Vega,
Ana Isabel de Lucas, Sergio A. Alonso de Diego, Hana Propkopcova, Nigel Austin,
Sven Van Brandt, Michel Surkyn, Michel De Cleyn, Ann Vos, Frederik J.R. Rombouts,
Gregor J. Macdonald, Diederik Moechars, Harrie J.M. Gijsen, and Andrés A. Trabanco
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00181 • Publication Date (Web): 02 Jul 2019
Downloaded from http://pubs.acs.org on July 5, 2019
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Evaluation of a series of BACE 1 inhibitors containing novel
heteroaryl-fused-piperazine amidine warheads
Daniel Oehlrich,*† Aldo Peschiulli,† Gary Tresadern,# Michiel Van Gool,‡ Juan Antonio Vega,‡ Ana Isabel De Lucas,‡
Sergio A. Alonso de Diego,‡ Hana Prokopcova,† Nigel Austin,# Sven Van Brandt,† Michel Surkyn,† Michel De Cleyn,†
Ann Vos,# Frederik J. R. Rombouts,† Gregor Macdonald,† Dieder Moechars,§ Harrie J.M. Gijsen,† and Andrés A. Trabanco
*‡.
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†Medicinal chemistry; #Discovery Sciences, §Neuroscience biology, Janssen Research & Development, Janssen
Pharmaceutica N.V., Turnhoutseweg 30, B-2340, Beerse, Belgium.
‡Discovery Sciences Medicinal Chemistry, Janssen Research & Development, Janssen−Cilag S.A., C/Jarama 75A, 45007,
Toledo, Spain.
KEYWORDS: BACE inhibitor, β-secretase 1, Alzheimer’s disease, cyclic sulfamidates, pKa.
ABSTRACT: Despite several years of research, only a handful of BACE 1 inhibitors have entered clinical trials as potential
*
therapeutics against Alzheimer’s disease. The intrinsic basic nature of low MW, amidine-containing
BACE 1 inhibitors makes them far from optimal as CNS drugs. Herein we present a set of novel
heteroaryl-fused piperazine amidine inhibitors designed to lower the basicity of the key, enzyme
binding, amidine functionality. This study resulted in the identification of highly potent (IC50 ≤ 10
nM), permeable lead compounds with a reduced propensity to suffer from Pgp-mediated efflux.
*
Bio-isosteric
replacements
O
N
*
N
H2N
N
H2N
N
Ar
Ar
Genetics has suggested β-secretase 1 (BACE 1) as a key target
to attenuate the formation of amyloid beta (Aβ) in Alzheimer’s
disease (AD).1 Since the discovery of BACE 1 in 1999,2 many
potent BACE 1 inhibitors have been described,3 but relatively
few successfully combine in vitro potency and the necessary
pharmacokinetic (PK) properties/parameters to achieve in vivo
efficacy. The amidine class of BACE 1 inhibitors have become
vivo efficacy. Thus, the challenge of BACE 1 inhibition is
revealed: liabilities are inherent to the amidine, yet it is crucial
for key binding motifs. To be capable of identifying BACE 1
clinical candidates, there is a need to efficiently synthesize and
evaluate many novel but synthetically challenging amidine
containing heterocycles. Here we describe a convergent
approach to meet this goal.
the main focus.3, and contain a protonated amidine warhead
4
We have previously described our approach to assess new
candidate warheads with computational approaches leading to
series of cyclic amidines,8 , acylguanidines,9, and
necessary for an optimal interaction with the aspartyl catalytic
dyad of BACE 1.3 The high intrinsic basicity of amidine
heterocycles presents multiple liabilities for their development,
including high Pgp efflux and hERG inhibition, and low
permeability. Several compounds have unfortunately failed
clinical trials, including MK-8931 (1)5 and LY-2886721 (2)
(Figure 1).6
aminopiperazinones such as
3
(Figure 1).10 The
aminopiperazinones suffered from poor brain penetration which
halted their progress. However, they served as inspiration for
further research, such as a series of aminomorpholine
containing BACE 1 inhibitors. The morpholine series,
exemplified by JNJ-50138803, (4) (Figure 1) was also
supported by in-silico work, and their optimization has been
Figure 1. Representative BACE 1 inhibitors.
F
F
,
described.11 12 The initial prototypes, displayed high pKa values
O
O
O
N
H
F
S
O
N
N
(9 < pKa < 9.6) and a strong Pgp-mediated efflux liability.11 To
attenuate the issues attributed to the elevated pKa, the amidine
warhead was carefully modified with fluorine containing
electron withdrawing substituents at position C-2 of the
morpholine ring. This optimization focused on achieving a pKa
within the range of 6.5-7.5. The evolution of the series
ultimately culminated with the identification of 4 (Figure 1), a
recently disclosed clinical candidate.12
N
S
F
O
H2N
F
F
F
H2N
N
H2N
N
H2N
HN
HN
HN
HN
O
1
2
3
4
O
O
O
N
N
N
N
F
F
Cl
Aminopiperazinone
N
MK-8931
LY-2886721
JNJ-50138803
Our interest in amidine BACE 1 inhibitors can be traced back
to early reports of aminodihydroquinazolines.7 Since then we
have been dedicated to identifying novel warheads with
adequate substitution to control the pKa of the
guanidine/amidine and as such balance the liabilities with in
Besides the exploration of the morpholine series, here we
described a parallel effort using bioisosteric replacements of the
amide in the aminopiperazinone warhead (3). We focused on
replacing this amide functional group by nitrogen-containing
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fused five membered heteroaromatic rings, where a bridge head
nitrogen was maintained.
in 93% yield over 2 steps. This was then oxidized to the target
sulfamidate 8 using NaIO4 and a catalytic amount of RuCl3 in
acetonitrile/water with excellent yield (Scheme 1).14
1
2
To validate our approach, we initially synthesized the
imidazolopiperazine warhead (5) with identical decoration to
the reported aminopiperazinone (Table 1). Similar potency in
both the BACE 1 cellular and enzymatic assays was observed
giving an immediate indication this could offer a promising
avenue for deeper exploration. Therefore, an efficient and
versatile synthetic route based on the ring opening of a cyclic
sulfamidate intermediate (8) was developed, enabling the
synthesis of the bicyclic warheads X1 – X8, shown in Figure 2.
3
4
5
6
7
8
9
Scheme 1. Synthesis of cyclic sulfamidate 8.
HO
F
F
O
F
O
O
S
(a) - (b)
(c)
S
H2N
N
O
O
N
Boc
Boc
Br
Br
Br
6
7
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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31
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40
41
42
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45
46
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60
Reagents and conditions: (a) (Boc)2O, NaHCO3 aq, THF, rt, 2.5 d; (b)
SOCl2, MeCN, 2 h, rt (93% yield over 2 steps); (c) RuCl3, NaIO4, CAN,
MeCN/H2O, 2 h, rt; (95% yield).
Table 1. BACE 1 cellular data for aminopiperazinone 3 and
imidazolopiperazine 5.
With sulfamidate 8 in hand, we studied its nucleophile induced
ring opening reaction with several nitrogen-containing
heteroaromatics. Sulfamidate 8 was successfully ring opened
using methyl 1H-pyrrole-2-carboxylate in the presence of
cesium carbonate in acetonitrile, to afford 9 in 84% yield
(scheme 2). The resulting intermediate 9 was then deprotected
and cyclized by sequential treatment with HCl in dioxane,
followed by sodium methoxide to afford lactam 10, in 22%
yield for the two steps. Lactam 10 was then elaborated to the
targeted warhead by initial alkylation to form an imino-ether
intermediate, and subsequent reaction with ammonia in EtOH
to form the desired amidine 11, in 33% yield over the two
transformations. While there are many techniques to convert
arylbromides to the corresponding anilines, and alternatives are
described vide infra, on this occasion we used a Büchwald
coupling using the benzophenone imine; which was
subsequently hydrolyzed to the corresponding aniline 12.
Subsequent chemo-selective coupling of the carboxylic acid
was mediated by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride (DMTMM).15 This reagent is
designed to activate the carboxylic acid and concomitantly
protonate the most basic center, subsequently promoting the
chemo-selective amide formation on the least basic center,
giving 13, in 50% yield.15
N
N
O
N
N
N
N
N
F
F
H2
H2
HN
HN
O
O
N
N
Cl
Cl
Compd #
3
5
BACE1
enz pIC50
7.63
7.55
7.46
7.98
hAβ42
pIC50
Figure 2. Proposed aminopiperazinone bisosteric replacements X1-X8
from common sulfamidate intermediate.
N
N
N
N
N
H2N
N
N
N
Ar
H2N
N
X3
Ar
X4
H2N
Ar
N
X2
N
N
Scheme 2. Synthesis of pyrrolopiperazine BACE 1 inhibitor 13.16
H2N
N
O
N
Ar
O
O
S
N
N
X5
Ar
MeO2C
Boc
N
N
F
O
N
H2N
H
F
(a)
Ar
F
O
N
N
S
(b)-(c)
X1
MeO2C
N
O
O
N
H
HN
N
N
Boc
Boc
Br
8
H2N
10
Br
9
Ar
Br
N
N
N
N
N
X6
N
N
H2N
N
N
N
H2N
N
Ar
F
Ar
X8
N
N
N
N
H2N
F
F
X7
(d)-(e)
(h)
(f)-(g)
H2N
H2N
O
NH
N
11
12
Chemistry: Cyclic sulfamidates have emerged as important
and versatile intermediates in organic synthesis because of their
established ability to undergo highly selective reactions with O-
, S-, N-, C-, P- and F-based nucleophiles.13 We reasoned that
nitrogen-containing heteroaromatics could be suitable
nucleophiles to ring open adequately substituted sulfamidates
leading to intermediates which could then be further elaborated
to yield the targeted warheads (Figure 2).
13
Br
NH2
CN
Reagents and conditions: (a) Cs2CO3, MeCN, 130 °C MW, 30 min,
(84% yield); (b) HCl (4 N in 1,4-dioxane), rt, 18 h; (c) MeONa, MeOH,
60 °C, 16 h, (22% yield over 2 steps); (d) OMe3BF4, DCM, rt, 4 d; (e)
NH4Cl, NH3 (2 M in MeOH), EtOH, 80 °C, 48 h (33% yield from 10);
(f) Pd2(dba)3, BINAP, NatBuO, benzophenone-imine, 100 °C, 2 h; (g)
HCl (6 N in iPrOH) 1 h, rt; (h) 5-cyanopyridine-2-carboxylic acid,
DMTMM, MeOH, 0 °C to rt, 3 h, (50% yield from 11).
Key cyclic sulfamidate 8 was by Boc protection of the β-amino
alcohol 6,11 followed by cyclisation with thionyl chloride in
acetonitrile at -40 ºC afforded the corresponding sulfamidite 7
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MeOH, 55 °C, 5 h, (55% yield over 3 steps); (d) 1 N NaOH, MeOH, rt,
The regioisomeric imidazolepiperazine warheads 5, 17 and 21
were also accessed using cyclic sulfamidate 8. Reaction of 2-
cyanoimidazole with 8 in the presence of DBU afforded 14 in
97% yield Scheme 3). HCl promoted cleavage of the Boc group
in 14 occurred with concomitant cyclisation to amidine 15, in
71% yield. The aryl bromide was then converted to aniline 16
using sodium azide and copper iodide in a solution of
dimethylene ethylene diamine and DMSO in 75% yield. The
regioselective amide formation was again achieved using
DMTMM to afford the final products 5 and 17 in 45% and 57%
yield, respectively.
1 h; (e) DMSO, 120 °C, 2 h (62% yield over 3 steps); (f) P2S5, pyridine,
110 °C, 16 h; (g) MeI, K2CO3, acetone, 16 h, rt; (h) NH4Cl, NH3 (7 M
in MeOH), 80 °C, 48 h, (35% yield over 3 steps); (i) NaN3, DMEDA,
CuI, Na2CO3, DMSO, 110 °C, 4 h; (j) 5-cyanopyridine-2-carboxylic
acid, DMTMM, MeOH, 0 °C to rt, 24 h, (33% yield over 2 steps).
1
2
3
4
5
6
7
8
Pyrazolopiperazine analog 26 could be prepared by reaction of
cyclic sulfamidate 2218 with 4-bromopyrazole; this was
achieved in good yield using cesium carbonate in dimethyl
sulfoxide to yield 23 (Scheme 5). Cyclic sulfamidate 22 was
synthesized in an analogous way to cyclic sulfamidate 8,
previously described in Scheme 1, albeit racemically, thus
requiring that the final compound undergo chiral separation,
which in the case of 26 was achieved using chiral SFC.
Intermediate 23 was deprotonated with LDA and reacted with
ethylchloroformate to form the lactam precursor. This was then
cyclized, after TFA-mediated Boc deprotection, by treatment
with KOAc in refluxing ethanol. Lactam 24 was elaborated to
the amidine via generation of the thioamide and subsequent
reaction with ammonia. The aromatic nitro group in 24 was
reduced to the corresponding aniline using heterogeneous
catalysis which went with concomitant bromide removal to
afford 25 in excellent yields over three reaction steps (94%).
The chemoselective amide formation was facilitated by
DMTMM to afford the desired amide 26 in 10% yield after
chiral separation.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
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36
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Synthesis of imidazolopiperazine BACE 1 inhibitors 5 and
17.
N
N
N
F
O
N
N
N
(a)
N
NH
F
F
O
S
(b)
NC
N
O
H2N
HN
Boc
8
Boc
15
14
Br
Br
Br
N
N
N
F
N
N
F
(d)
H2N
(c)
H2N
N
HN
O
N
16
NH2
5
17
= X = Cl
= X = CN
X
Reagents and conditions: (a) DBU, ACN, 90 °C, 16 h, (97% yield); (b)
HCl (4 N in 1,4-dioxane), 70 °C, 4 h, (71% yield); (c) NaN3, DMEDA,
CuI, Na2CO3, DMSO, 110 °C, 24 h, (75% yield); (d) (5) 5-Chloro-
pyridine-2-carboxylic acid or 5-cyanopyridine-2-carboxylic acid,
DMTMM, MeOH, 0 °C to rt, 24 h, (45-57% yield).
Scheme 5. Synthesis of pyrazolopiperazine BACE 1 inhibitor 26.
N
N
N
Br
N
Br
O
NH
Br
(a)
O
S
(b) - (d)
N
N
O
O
N
HN
H
Boc
Boc
24
23
NO2
22
NO2
NO2
For the preparation of 21, due to the lack of symmetry and
difference in reactivity, ethylimidazole-4-carboxylate could not
be used to open the cyclic sulfamidate. Instead, reaction of 8
with symmetrical diethyl-1H-imidazole-4,5-dicarboxylate
yielded the expected product in 88% yield (Scheme 4).
Subsequent Boc deprotection, followed by sodium methoxide
mediated intramolecular cyclisation afforded lactam 18 (55%
yield over 3 steps). Decarboxylation by treatment with NaOH
and heating in DMSO led to 19. Treatment of 19 with
phosphorus pentasulfide to the thio-lactam and subsequent S-
methylation followed by treatment with ammonia yielded
amidine 20. Target compound 21 was obtained from 20
following analogous conditions to those described in Schemes
2 and 3.17
N
N
H2N
N
N
N
(e) - (g)
(h) - (i)
O
NH
N
H2N
N
26
25
NH2
CN
Reagents and conditions: (a) Na2CO3, DMSO, 130 °C, 2 h, (79%
yield); (b) ethylchloroformate, LDA, THF, -78 °C to -50 °C, 2 h (66%
yield); (c) TFA, DCM, rt, 3 h; (d) KOAc, EtOH, reflux, 4 h, ( 87% yield
over 2 steps); (e) P2S5, pyridine, 110 °C, 24 h; (f) NH4Cl, NH3 (7 M in
MeOH), 100 °C, 16 h; (g) Zn, AcOH, EtOH, reflux, 24 h, (37% yield
over 3 steps); (h) 5-cyanopyridine-2-carboxylic acid, DMTMM,
MeOH, 0 °C to rt, 4 h; (i) Chiral SFC purification, (10% yield over 2
steps).
Scheme 4. Synthesis of imidazolopiperazine BACE 1 inhibitor 21.
To access 1,2,4-triazolopiperazine 30, reaction of cyclic
sulfamidate 8 with 1,2,4-triazole-3-carbonitrile afforded 27 in
92% yield (Scheme 6). This was then elaborated to the final
compound following a very similar sequence to that previously
described in Scheme 4 for the imidazolopiperazine analog 21.
This started with formic acid-mediated Boc cleavage and
subsequent intramolecular lactamization facilitated by
trimethyl aluminum. Then, amination of the bromophenyl ring
in 28 by treatment with sodium azide and CuI afforded 29 in
92% yield. Regioselective amide formation led to the desired
BACE 1 inhibitor 30 in 52% yield.
N
HN
EtO2
(a)
N
N
N
HO2
C
F
O
N
C
CO2Et
F
F
O
S
(e)
N
O
O
N
H
O
N
H
(b) - (d)
Boc
Br
8
18
Br
19
Br
N
N
F
N
N
H2N
N
F
(f) - (h)
(i) - (j)
O
NH
N
H2N
N
20
21
Br
CN
Scheme 6. Synthesis of 1,2,4-triazolopiperizine BACE 1 inhibitor 30.
Reagents and conditions: (a) DBU, ACN, 90 °C, 5 h, (88% yield); (b)
HCl (4 M in 1,4-dioxane), 1,4-dioxane, 70°C, 15 h; (c) NaOMe,
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N
N
N
The 1,2,3-triazolopiperazine analogue 39 was prepared from
HN
N
N
N
F
N
O
(a)
F
F
O
1
intermediate 31 as outlined in Scheme 8. Boc deprotection of
the amine group in 31 followed by coupling with propiolic acid
led to amide 36; the resulting intermediate was double cyclized
under thermal conditions to afford 37. Lactam 37 was
elaborated to amidine 38 following a similar procedure as
described in Scheme 5. Subsequent chemoselective amide
coupling with 5-cyano-pyridine-2-carboxylic acid led to BACE
1 inhibitor 39.19
S
N
N
(b) - (c)
N
O
H2N
N
HN
N
2
3
Boc
Boc
28
Br
8
27
Br
Br
N
N
4
5
N
F
N
N
H2N
N
N
F
6
7
(e)
(d)
H2N
N
HN
O
N
29
30
8
NH2
9
Scheme 8. Synthesis of 1,2,3-triazolo-piperizine BACE 1 inhibitor 39.
CN
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
-N
N
N
-N
N+
N+
Reagents and conditions: (a) KOtBu, 60 °C, 1,4-dioxane, 16 h, (99%
yield); (b) HCO2H, rt, 2 h; (c) AlMe3, toluene, 60 °C, 1 h, (53% yield
over 3 steps) (d) NaN3, DMEDA, Na2CO3, CuI, DMSO, 100 °C, 2 h,
(50% yield); (e) 5-cyano-pyridine-2-carboxylic acid, EDCI, HCl (6 N
in iPrOH), MeOH, rt, 2 h, (52% yield).
N
N
N
F
F
F
(c)
(a) - (b)
O
N
HN
HN
Boc
H
O
Br
Br
37
Br
31
36
N
N
N
N
N
N
N
F
F
1,3,4-Triazolopiperazine 35 was accessed via ring opening of
cyclic sulfamidate 8 with sodium azide to afford alkylazide 31,
which, after subsequent hydrogenation, afforded diamine 32,
with concomitant and undesired bromide removal (Scheme 7).
(d) - (e)
(f) - (g)
H2N
N
H2N
Br
HN
O
N
39
38
Intermediate
32
was
then
treated
with
N,N’-
bis(dimethylaminomethylene)hydrazine in the presence of a
catalytic amount of p-TSA to afford the desired 1,3,4-triazole
33. The introduction of a nitrile functional group in the
heteroaromatic ring was achieved in a 4-step sequential
sequence. First, a hydroxymethyl substituent was introduced
with formaldehyde in the presence of triethylamine; this
primary alcohol was then selectively oxidized to the
corresponding aldehyde using manganese dioxide. Finally,
aldehyde condensation with hydroxylamine followed by
dehydration of the intermediate oxime using oxalyl chloride
afforded the desired nitrile containing intermediate 34. Boc
deprotection of 34 under acidic conditions and concomitant
cyclisation led to the expected cyclic amidine intermediate.
Nitration of the aromatic ring under standard conditions,
followed by hydrogenation of the nitro group resulted in the
corresponding aniline intermediate. Final amide coupling with
5-cyanoyridine-2-carboxylic acid afforded BACE 1 inhibitor 35
in 58% yield from nitrile containing intermediate 34.
CN
Reagents and conditions: (a) HCl (4 N in 1,4-dioxane), rt, 24 h; (b)
propiolic acid, DCC, DCM, 0 °C, 2 h; (c) toluene, 70 °C, 18 h; (78%
yield over 3 steps); (d) P2S5, 1,4-dioxane, 95 °C, 2 h; (e) NH3 (7 M in
MeOH) 75 °C, 2 h; (f) NaN3, DMEDA, Na2CO3, CuI, DMSO, 110 °C,
5 h; (g) 5-cyanopyridine-2-carboxylic acid, EDCI, HCl (6 N in iPrOH),
MeOH, rt, 3 h, (39% yield over 4 steps).
Compound 43 containing a fused tetrazole ring was again
generated from intermediate 31, as shown in Scheme 9.
Intermediate 31 initially underwent a 1,3-dipolar cyclisation
with ethyl cyanoformate to afford tetrazole 40. Acid-mediated
Boc deprotection, followed by cyclisation gave lactam 41.
Lactam 41 was converted to the corresponding amidine 42, as
described in Schemes 5 and 7. Bromide 42 was converted to the
aniline, which underwent chemoselective amide formation to
yield BACE 1 inhibitor 43.
Scheme 9. Synthesis of tetrazolo-piperizine BACE 1 inhibitor 43.
Scheme 7. Synthesis of 1,3,4-triazolo-piperizine BACE 1 inhibitor 35.
N
-N
N
-N
N
N
N
N+
N
N+
N
N
N
F
F
O
N
N
H2N
HN
N
N
N
F
F
O
F
F
EtO2C
(c)
S
(a)
(a)
(b)
(b) - (c)
HN
Boc
N
O
O
N
H
HN
Boc
HN
Boc
Boc
Boc
Br
Br
8
31
32
Br
40
Br
F
Br
41
31
N
N
N
N
F
N
N
N
N
N
N
N
N
H2N
N
N
N
N
F
(d) - (e)
(d) - (g)
(f) - (g)
N
N
(h) - (k)
H2N
N
F
F
HN
N
O
H2N
N
HN
HN
N
HN
O
N
Boc
Boc
Br
35
34
43
33
42
CN
CN
Reagents and conditions: (a) NaN3, DMF, 80 °C, 2 h; (quantitative);
(b) H2, Pd/C, TEA, MeOH, rt, 16 h; (87% yield) (c) p-TSA, toluene,
130 °C, 16 h, (47% yield) (d) CH2O, TEA, 100 °C, 2 h; (e) MnO2,
CHCl3, rt, 1 h; (f) NH2OH.HCl, NaOAc, EtOH, 80 °C, 1.5 h; (g)
CO2Cl2, TEA, EtOAc, 0 °C to rt, 2 h, (31% yield over 4 steps); (h)
HCO2H, 50 °C, 1 h; (i) HNO3, H2SO4 -10 °C, 30 min; (j) H2, Pd/C,
MeOH, rt, 4 h; (k) 5-cyanopyridine-2-carboxylic acid, EDCI, HCl (6 N
in iPrOH), MeOH, rt, 16 h, (58% yield over 4 steps).
Reagents and conditions: (a) Ethylcyanoformate, 120 °C, 20 h; (b)
TFA, DCM, rt, 2 h ; (c) K2CO3, EtOH, reflux, 20 h, (75% yield over 3
steps); (d) P2S5, 1,4-dioxane, 95 °C, 2 h; (e) NH3, (7 M in MeOH), 70
°C, 18 h; (f) NaN3, DMEDA, Na2CO3, CuI, DMSO, 110 °C, 2 h; (g) 5-
cyanopyridine-2-carboxylic acid, EDCI, HCl (6 N, iProH), MeOH, rt,
2h, (5% yield over 4 steps).
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The resulting eight bicyclic heteroaryl-fused aminopiperazine
unfavorable explaining the lower activity. The rest of the fused
cycle is solvent exposed. Although not attempted here,
additional substituents to the fused bicycle may allow further
modulation of the potency by forming additional interactions
with the receptor.
1
2
3
4
5
6
7
8
BACE 1 inhibitors 13, 17, 21, 26, 30, 35, 39, 43 provide an
excellent opportunity for the side-by-side comparison of the
effect of the fused heterocycle. They were profiled for their
BACE 1 and 2 inhibitory activity in both our enzymatic assay
and also cellular BACE1 assay, Table 2. Cathepsin D was not
measured for this series of compound as it known that the
expansion to the S3 pocket via the amide linked heteroaromatic
removes the Cathepsin D liability.20
Figure 3. Crystal structure of 13 in BACE 1 showing key interactions.
9
Most compounds showed high cellular potency in the range of
pIC50 7.7-8.9. The low activity observed for the tetrazole
derivative 43 (hAβ42 cell pIC50 = 7.01) was attributed to the
low basicity of the amidine function (pKa = 5.1). In general, we
have observed a dependency for BACE 1 cellular potency with
pKa. This is rationalized by the BACE 1 cellular location in the
endosomes, an acidic compartment that favors basic compound
distribution.21
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Our working hypothesis is that the optimal pKa for a BACE 1
inhibitor should be in the range of 6.5-8.5. Higher pKa´s are
tolerated and, in some cases, result in increased cellular
potency. As demonstrated with compounds 13 and 43,
compound 13 has a pKa of 10.3 and this is translated into a
cellular potency of pIC50 of 8.88, which compound 43 has the
lowest pKa of 5.1 and has a cellular potency of pIC50 of 7.1.
However, the higher basicity can also be responsible for off-
target related complications, such as hERG and Pgp-efflux
liabilities, this is not always the case and Pgp liability is not
resultant on one factor and multiple properties of the molecule
can affect this value. The measured pKa for compounds 13 (pKa
= 10.3) and 21 (pKa = 9.0) was found to be higher than the
optimal range. This high basicity could contribute to the high
Pgp efflux observed for both compounds (13, AB+/AB- = 4.6
and 21, AB+/AB- = 5.3), but a clear outlier is compound 39 that
has an ideal pKa and suffers from significant Pgp efflux. We
did see in this series of compounds a clear correlation between
hERG and pKa, with compound 13 with the highest pKa having
a significant hERG liability and compound 43 not showing any
hERG inhibition. Therefore, further work around these warhead
modifications should aim at lowering the high pKa of the
amidine function and modulating the vitro and vivo profile.
Compounds 17, 26, 30, 35 and 39 possess pKas in an almost
optimal range between 6.4-8.5 and show good to excellent
intrinsic permeability.
Unfortunately, despite their good pKa´s, the Pgp-efflux liability
measured for 35 (AB+/AB- = 4.8) and 39 (AB+/AB- = 9.8)
precluded further investigation around these two chemotypes.
On the other hand, compounds 17, 26 and 30 exhibit the desired
in vitro profile combining potency, with good intrinsic
permeability a reduced Pgp-efflux and hERG liability and the
heteroaryl-fused aminopiperazine warheads they represent have
been selected for further profiling and optimization.
The crystal structure of 13 with BACE 1 was solved at 2 Å
resolution (PDB code 6OD6, Figure 3). The result confirmed
the binding mode to be similar to those seen for analogous
amidine containing BACE 1 inhibitors.11, This includes the
22
key interactions between the protonated amidine and the
catalytic dyad (Asp32 and Asp228). The quaternary center is
optimal for the orientation of the aryl ring and the methyl group,
allowing the methyl to protrude into the S2’ pocket without
clashing with the protein. The quaternary center favorably
positions the F-aryl ring in an axial orientation to occupy the S1
pocket. The amide linker then optimally orientates the pyridine
ring in the S3 pocket of BACE 1, while generating a favorable
H-bond interaction with Gly230. Most of the compounds have
a similar enzymatic activity with pIC50 approx. 8-8.5, with
examples 17 and 39 showing the biggest difference: pIC50 7.54
and 8.70 respectively. Comparing pairs 13 and 17, 21 and 35,
21 and 35, 26 and 30, 39 and 43, all demonstrate that
incorporation of a nitrogen at position 2’ led to a loss of activity.
The crystal structure suggests that this nitrogen points towards
the oxygen of Thr231, and is therefore electrostatically
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cyclic sulfamidates we have developed a convergent synthesis
Page 6 of 7
1
2
3
4
5
6
7
8
to explore the replacement of the aminopiperazinone warhead
by bioisosteric replacements in the form of fused
heteroaromatics. Several new series of BACE 1 inhibitors have
been identified, and efforts are currently focused on the
optimization of properties and progression of key compounds.
These BACE 1 inhibitors represent a unique opportunity, as the
fused 5-membered heterocyclic ring offers additional handles
for modification to enhance their overall profile.
Table 2. Overview of BACE 1 inhibitor in vitro profile.
*
*
*
N
F
H2N
N
HN
O
N
CN
9
ASSOCIATED CONTENT.
Supporting Information.
N
N
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
N
N
*
N
*
N
N
*
N
N
N
N
N
N
N
N
N
N
N
N
*
The Supporting Information is available free of charge on the ACS
Publications website.
Compound
#
13
17
21
26
30
35
39
43
Experimental procedures for the synthesis and the analytical
characterization of all intermediates and final compounds are included.
X
F
F
F
H
F
F
F
F
AUTHOR INFORMATION.
BACE1
enz
8.21
7.54
8.40
8.34
8.22
7.97
8.70
8.04
Corresponding Author.
* (D. O.) Phone: +3214603100. Email: doehlric@its.jnj.com
* (A. A.T.) Phone: +34925245771. Email: atrabanc@its.jnj.com
pIC50
hAβ42 cell
8.88
7.60
8.20
6.85
7.74
7.72
8.66
7.58
8.01
7.43
8.18
7.25
8.92
8.26
7.01
7.57
ORCID
pIC50 nM
Daniel Oehlrich: 0000-0002-3392-1952
Gary Tresadern: 0000-0002-4801-1644
Andrés A. Trabanco: 0000-0002-4225-758X
Harrie J.M. Gijsen: 0000-0001-8020-4828
Frederik J.R. Rombouts: 0000-0003-1986-0476
Author Contributions.
BACE2
enz
pIC50
hERG IC50
µM
0.49
4.17
3.72
1.78 11.75
>10
7.24
>50
Pgp
AB+
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
11.1
4.6
17.2
2.0
1.5
5.3
15.6
3.6
24.8
2.0
5.9
4.8
11.6
9.8
22.1
1.9
AB+/AB-
ACKNOWLEDGMENT.
We thank the analytical departments in Beerse and Toledo for helping
with compound characterization and purification and the DMPK and
toxicology departments for their contributions in profiling the
compounds.The authors would also like to thank Claudio Meyer for
reviewing the manuscript.
pKa
10.3
8.2
9.0
8.5
6.5
6.4
7.1
5.1
See supplementary information for assay details
In summary, an efficient evaluation of multiple novel BACE 1
inhibitors has been achieved. Exploiting the reactivity of the
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