Carbazole-containing arylcarboxamides as BACE1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.09.064

β-Secretase (BACE1) is widely recognized as a prime drug target for the treatment of Alzheimer's disease (AD). In this Letter, we report the synthesis and the BACE1 inhibitory activity of novel, variously substituted N-[3-(9H-carbazol-9-yl)-2-hydroxypropyl]-arylcarboxamides. The best results have been obtained with the introduction of a 4-OMe substituent (IC₅₀ = 3.8 μM) or a 3,4-dichloro substituent (IC₅₀ = 2.5 μM) in the amidic aromatic ring. The blood-brain barrier penetration predictions resulted to be promising for this type of compounds. To better understand the structure-activity relationships (SAR) of the new derivatives, a docking study procedure has been applied exploiting different conformational and ionic states of BACE1.

Full text of DOI:10.1016/j.bmcl.2011.09.064

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6657–6661
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Carbazole-containing arylcarboxamides as BACE1 inhibitors
Simone Bertini a, Valentina Asso a, Elisa Ghilardi a, Carlotta Granchi a, Clementina Manera a,
Filippo Minutolo a, Giuseppe Saccomanni a, Andrea Bortolato b, Jonathan Mason b,d, Stefano Moro c,
Marco Macchia a,
⇑
a Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
b Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 RAX, UK
c Molecular Modeling Section (MMS), Dipartimento di Scienze Farmaceutiche, Università di Padova, Via Marzolo 5, I-35131 Padova, Italy
d Lundbeck A/S, Ottiliavej 9, DK-2500 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2011
Revised 15 September 2011
Accepted 16 September 2011
Available online 21 September 2011
b-Secretase (BACE1) is widely recognized as a prime drug target for the treatment of Alzheimer’s disease
(AD). In this Letter, we report the synthesis and the BACE1 inhibitory activity of novel, variously substi-
tuted N-[3-(9H-carbazol-9-yl)-2-hydroxypropyl]-arylcarboxamides. The best results have been obtained
with the introduction of a 4-OMe substituent (IC50 = 3.8 lM) or a 3,4-dichloro substituent (IC50 = 2.5 lM)
in the amidic aromatic ring. The blood–brain barrier penetration predictions resulted to be promising for
this type of compounds. To better understand the structure–activity relationships (SAR) of the new deriv-
atives, a docking study procedure has been applied exploiting different conformational and ionic states of
BACE1.
Keywords:
Alzheimer
BACE1
Carbazole
Molecular docking
Flap
Ó 2011 Elsevier Ltd. All rights reserved.
BACE1 (b-secretease) is a transmembrane aspartic protease in-
volved in Alzheimer’s disease (AD). It has been reported to be
responsible of the cleavage of the Amyloid precursor protein
(APP) to generate the amyloid b peptide (Ab). Ab is the principal
constituent of extracellular senile plaques found in the brain of
AD patients and seems to be linked to intracellular neurofibrillary
tangles caused by the aggregation of tau proteins.1,2a In fact, Ab has
been shown to induce stress-activated protein kinases (SAPK)
which predominantly can contribute to the phosphorylation of
tau proteins; this in turn stimulates the self-assembly of tau.2b Also
reactive oxygen species (ROS), in particular H2O2, referred as
‘oxidative stress’ mediators, are able to stimulate protein cascade
pathways that induce the expression of inflammatory genes such
as SAPK.2b BACE1 is widely recognized as a prime drug target for
the treatment of AD.3,4
of a flexible antiparallel b-harpin region called flap covering the ac-
tive site.9 This region has been reported to be able to close in a
tightly packed state to optimize the interactions with inhibitors,
while tending to adopt an open conformation in the active apo
structure10 promoting substrate binding. In particular functionally
essential residues and water molecules have been identified to
play a key role in the activation of BACE1 and large scale conforma-
tional changes seem to be involved between the active and inactive
forms.10
Many BACE1-inhibitors so far discovered incorporate the
hydroxyethylamine (HEA) transition state isoster moiety and pos-
sess peptidomimetic structures.3,4,11 In a previous work12 we re-
ported the synthesis and the BACE1 inhibitory activity of some
a-naphthylaminopropan-2-ol derivatives, that are non-peptidomi-
metic compounds, and in which the HEA scaffold is structurally
Different studies suggest a localization of BACE1 within the
endosomal system and trans-golgi network. Subcellular acid com-
partments like lysosomes are likely to be major sites for APP cleav-
age by BACE1 leading to Ab production.5–7 The low pH
environment of these organelles results in an optimal activity of
the protease.8 Different crystallographic structures are available
of BACE1 complexes showing the ability of this enzyme to adopt
different conformations in particular as result of the movement
recognisable in the central ‘linker’ of the molecules.
In particular, structure A (Fig. 1), bearing an unsubstituted car-
bazole group, showed an appreciable BACE1 inhibitory activity
(IC50 = 2.99 l
M).12 On these basis, we developed some new deriv-
atives (1–18, Fig. 1), where the unsubstituted carbazole-methyl
carbinol portion was kept constant and the N-atom was acylated
with different groups. The effects of these structural modifications
on the BACE1 inhibitory activity of the resulting compounds 1–18
were evaluated.
These novel derivatives were synthesized as shown in Scheme 1.
Commercially available carbazole 19 was treated with KOH
⇑
Corresponding author. Tel./fax: +39 050 2219553.
E-mail address: mmacchia@farm.unipi.it (M. Macchia).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.064

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S. Bertini et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6657–6661
Table 1
Structures, BACE1 inhibitory activities and blood–brain barrier penetration
predictions of novel variously substituted arylcarboxamides 1–18
2
R
O
N
OH
OH
N
N
N
1
2
R
H
1-18
R
A
O
OH
N
Figure 1. Structure of
structure of novel variously substituted N-[3-(9H-carbazol-9-yl)-2-hydroxypropyl]-
arylcarboxamides (1–18, see also Table 1).
a
-naphthylaminopropan-2-ol-carbazole (A) and general
N
1
R
R1
R2
IC50
(lM)
logBBpred
a
b
Compd
1
2
3
4
5
6
7
8
Ph
Ph
Ph
Ph
4-CF3-Ph
4-NO2-Ph
4-Cl-Ph
4-F-Ph
4-CH3-Ph
Bn
H
4-Cl
4-OCH3
3,4-Cl2
H
H
H
H
>10
4.7
3.8
À0.2
À0.1
À0.3
À0.1
0.1
(1.2 equiv) in DMF and then alkylated with epichlorohydrin
(2.5 equiv) in DMF, (added dropwise at 0 °C).
The resulting epoxide (20) was treated with the appropriate
amine (2.0 equiv) in EtOH to obtain aminoalcohols 21–32. Reaction
of these derivatives with substituted acyl-chlorides (1.1 equiv) in
the presence of PS-DIEA (1.2 equiv), DMAP (catalytic amount) in
CH2Cl2 afforded final compounds 1–18.
BACE1 inhibitory activities of the newly synthesized compounds
were determined by a previously reported fluorescence-based
assay13 and the results are reported in Table 1.
The presence of a phenyl substituent on the amidic nitrogen
atom (R1), together with the presence of a substituent (R2) on the
amidic aromatic ring (compounds 2–4), led to a preservation of
the BACE1 inhibitory activity, compared to that of reference com-
2.5
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.8
À0.4
À0.1
À0.1
À0.2
À0.4
À0.3
À0.5
À0.3
À0.3
À0.4
À0.5
À0.4
À0.3
9
H
H
10
11
12
13
14
15
16
17
18
Bn
Bn
Bn
4-Cl
4-OCH3
3,4-Cl2
H
H
H
H
H
4-Cl-Bn
4-CH3-Bn
4-OCH3-Bn
Phenethyl
Cyclohexyl
>10
pound A (IC50 = 2.99 lM). If the amidic aromatic ring is unsubsti-
tuted (R2 = H), the activity is completely lost (compound 1).
Compound 4, bearing two chloro-substituents in positions 3 and
4 of the amidic aromatic ring, proved to be the most active inhib-
a
IC50 measurements were performed as reported in Ref. 13. Data represent mean
values for at least three separate experiments. Standard errors are not shown for the
sake of clarity and were never higher than 15% of the means.
Predicted blood–brain barrier permeation (logBBpred = log[Brain]/[Blood]).14
b
itor of this series, with an IC50 value of 2.5 lM. The introduction of
a 4-substituent in the phenyl portion (R2) of the amidic moiety,
leaving unsubstituted the amidic aromatic ring (R2 = H), always
led to inactive compounds (5–9). The presence of a benzyl portion
(R2), either substituted, or unsubstituted on the amidic nitrogen
caused a loss in BACE1 inhibitory activity in all cases (compounds
10–16). When R1 is a phenethyl and the amidic aromatic ring is
unsubstituted (R2 = H), an appreciable level of activity
the efficiency and hence vulnerability to metabolism, which is addi-
tional information over the regioselectivity of the molecular
portions affected by metabolism.15
Pharmacokinetics predictions suggested that all the compounds
included in this study may have a good human intestinal absorp-
tion, at least higher than 30%, but there is a risk of high plasma pro-
tein binding, probably higher than 80%. The BBB penetration
predictions did not detect potential problems and in particular
for the most active compound of the series, that is, compound 4,
the brain concentration has been predicted to be approximately
equal to the blood concentration (Table 1). The prediction of cyto-
chrome metabolism for the three predominant drug metabolizing
P450 enzymes, that is, CYP3A4, CYP2D6 and CYP2C9, has been
performed for the best two derivatives using the Stardrop soft-
ware.14 For compound 3 the methoxy moiety resulted in a moder-
ate labile position suggesting a potentially low metabolic stability
of the molecule. The prediction of the most probable site of P450
metabolism of compound 4 resulted in position 1 of the carbazole
(IC50 = 4.8 lM) is observed (compound 17). The activity is com-
pletely lost again when R1 is a completely aliphatic substituent
(such as a cyclohexyl in the case of compound 18) and the amidic
aromatic ring is unsubstituted.
Cytochrome P450 metabolism (3A4, 2D6 and 2C9), blood–brain
barrier (BBB) penetration, plasma protein binding and human intes-
tinal absorption have been predicted using the StarDrop software.14
StarDrop uses a quantum mechanical approach to calculate the elec-
tronic reactivity of each site and ligand-based descriptors to capture
steric and orientation effects of the enzyme active site on the pattern
of metabolism. The advantage of this approach is its ability to calcu-
late the site liability and composite site liability, that are measures of
a
b
OH
O
N
N
N
H
NH
R
21-32
1
20
19
2
R
O
N
OH
c
N
1
R
1-18
Scheme 1. Reagents and conditions: (a) epichlorohydrin, KOH, DMF, 0 °C, 5 h, 50%; (b) R1-NH2, EtOH, 65 °C, overnight, 40–79%; (c) ArC(O)Cl, PS-DIEA, DMAP, CH2Cl2, rt,
overnight, 11–90%.

S. Bertini et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6657–6661
6659
moiety (Fig. 2). Nevertheless this position has been predicted to be
quite stable (only 30%), similarly to all the other potential meta-
bolic sites, thus indicating a promising metabolic stability of this
inhibitor.
To better understand the structure-activity relationships (SAR)
of the novel carboxamido-derivatives a molecular docking study
was carried out to predict their binding modes to BACE1. BACE1
crystal structures in a flap-closed (PDB code 1W5116) conformation
and in a flap-open state (PDB code 2ZHU10) were downloaded from
the Protein Data Bank.17 The proteins were prepared with the Pro-
tein Preparation Wizard. Since BACE1 is believed to be active prin-
cipally in subcellular environments at low pH values, the ionic
state of the residues especially in the active pocket were conse-
quently further analyzed using the Discovery Studio 2.5.5 Suite.18
In particular different ionic states of Asp228 were considered, as
well as the presence of
2 water molecules (WAT412 and
WAT437) in the active pocket of the flap-open conformation.
The small organic molecules were prepared for the docking
study exploiting a Knime Workflow. In the first step 2D coordi-
nates were generated from the original molecular SMILES (Simpli-
fied Molecular Input Line Entry Specification). Tautomeric states
with a 10% minimum abundance were then predicted using the
TAUTHOR component of the MoKa Suite from Molecular Discov-
ery.19,20 The program BLABBER was applied to the resulting tau-
tomers to create multiple molecular ionic states populated at pH
7.4 or at the lower pH of subcellular acid compartments where
BACE1 is localized. The workflow continues creating enantiomeric
states and a starting low energy 3D conformation exploiting the
software CORINA.21 Since docking software are known to poorly
explore the conformational space of aliphatic ring, all the possible
low energy conformations of the cyclohexyl ring derivative in
study were generated in CORINA and included in the molecular
docking calculation. The final step of the workflow consists in the
molecular docking of the prepared ligands using Glide SP22,23 to
different grids corresponding to different ionic or conformational
states of BACE1, either including or not including up to two water
molecules in the active pocket. An additional docking run was per-
formed using the Induced Fit procedure24 to allow the comparison
to a series of congeric ligands.12 A maximum of five docking poses
were generated per ligand state. The visual inspection and evalua-
tion of the predicted binding modes were supported by GRID maps
generated using different molecular probes.25
The computational protocol herein applied takes into account
the flexibility of the flap region exploiting two crystallographic
structures of BACE1: a flap-closed state (PDB code 1W5116) and a
flap-open conformation (PDB code 2ZHU10). This strategy aimed
to test the possibility that the presence of bulky moieties, resulting
from sterically hindered carboxamides, would favour the binding
to a fully flap-open BACE1 state. In this conformation the effect
of two crystallographic water molecules (WAT412 and WAT437
in the PDB 2ZHU atom numbering) in the active pocket on the pre-
dicted ligands binding mode was analyzed. The prediction of the
titration curve of Asp228 suggested a high probability that this res-
idue is in a neutral state in the lysosome acidic environment18 and,
therefore, both the neutral and anionic states of this residue were
considered in this study. The results of the different molecular
docking studies suggested that the carboxamido-derivatives can
adopt a favourable binding mode in agreement with the SAR of this
series of inhibitors only in complexes with flap-closed BACE1 (PDB
code 1W51).
In the predicted binding orientation, the carbazole moiety of
molecule 4 creates hydrophobic interactions with Tyr198, Ile226,
Thr329 and Val332 (Fig. 3 and 4A). This compound is anchored
to the opposite side of the active pocket through additional apolar
interactions between its N-phenylbenzamide region and Phe108,
Ile118, Trp115, Tyr72, Gly11 and Thr232. The ligand creates two
crucial hydrogen bonds: one between its hydroxyl group and one
of the two catalytic aspartic residues of BACE1 (Asp228) and the
other between the carbonyl moiety and Thr71 of the flap loop.
The enantiomeric configuration of the hydroxyl group only caused
minor changes in the binding mode (Fig. 4A). The obtained docking
result was compared to the crystallographic binding mode of
hydroxyethylamine inhibitor 3-[({(1S,2R)-1-benzyl-2-hydroxy-3-
[(3-methoxybenzyl)amino]propyl}amino)(hydroxy)-methyl]-N,N-
dipropylbenzamide 33 (PDB code 1W51, Fig 4B and C). This ligand
Figure 2. Cytochrome metabolism predictions for the two most active molecules of
the series, compounds 3 (on the right) and 4 (on the left), for the three P450
enzymes: CYP3A4, CYP2D6 and CYP2C9. All the predicted potential sites of
metabolism are shown. They were ranked as stable for compound 4, while the
methoxy moiety in compound 3 resulted in a moderate labile site. In the case that
the molecules are substrates for one of the isoforms modeled, the potential relative
proportion of products at each position is shown when resulting equal or higher
than 5%.

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S. Bertini et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6657–6661
bridge between the phenyl ring and the nitrogen of the amide moi-
ety can orient this group in a position far enough from Trp115 to
produce an active inhibitor (compound 17).
In a previous study12 a series of related
a-naphthylaminopro-
pan-2-ol derivatives have been predicted to affect the side-chain
orientations of Gln73 and Arg128. These aminoacids are part of
the most flexible residues in the flap-closed conformation, as the
superimposition of the available BACE1 X-ray structures would
confirm.26,27 A similar conformational state was also predicted by
means of a comparable approach based on an induce-fit docking
for a series of substituted N-(3-(4-benzhydrylpiperazin-1-yl)-2-
hydroxypropyl)-arylsulfonamides.28 On the contrary Gln73 is not
impairing the predicted binding mode to the ligands considered
in this study. This is due principally to the different predicted ori-
entation of the molecules’ benzamide group. Therefore, the inclu-
sion of an induced fit analysis allowing small changes in
aminoacid side-chains orientation and protein backbone relaxation
did not improve the obtained results.
Figure 3. Schematic representation of the principal interactions resulting from the
docking study of compound 4 in the flap-closed conformation of BACE1. The length
of the polar interactions are shown in Angstrom (Å).
In conclusion, we have synthesized a new series of BACE1 inhib-
itors possessing a N-[3-(9H-carbazol-9-yl)-2-hydroxypropyl]-aryl-
carboxamido structure. The most active compounds 3 and 4
showed promising IC50 values (3.8 and 2.5 lM, respectively). Phar-
macokinetics predictions suggested that all the new compounds
may have a good human intestinal absorption. The blood–brain
barrier penetration resulted to be promising in particular for the
most active compound (4). Docking of 4 showed that the carbazole
and the substituted benzamide moieties create hydrophobic inter-
actions with the BACE1 active site, while the OH of the central
hydroxyethylamine portion creates a crucial hydrogen bond with
one of the catalytic aspartic residues (Asp228). Finally, the amidic
carbonyl participates to another crucial hydrogen bond with Thr71
of the flap loop. In general, the docking results were in good agree-
ment with the biological data obtained in the enzyme inhibition
assays.
Acknowledgments
The authors are thankful to Siena Biotech SpA – Italy, for scien-
tific and financial support. The molecular modeling work coordi-
nated by S.M. has been carried out with financial support from
the University of Padova, Italy, and the Italian Ministry for Univer-
sity and Research, Rome, Italy (MIUR, PRIN2008: protocol number
200834TC4L_002).
Figure 4. (A) Docking result of compound 4 in BACE1. The (R)-stereoisomer is
shown in cyan and the S in pink. (B) The obtained docking result is compared to the
crystallographic binding mode of a reference hydroxyethylamine inhibitor (PDB
code 1W51). (C) Molecular structure of the reference hydroxyethylamine ligand 33.
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