BACE1 inhibitory activities of enantiomerically pure, variously substituted N-(3-(4-benzhydrylpiperazin-1-yl)-2-hydroxypropyl) arylsulfonamides

Article abstract of DOI:10.1016/j.bmc.2010.09.032

β-Secretase (BACE1) has been widely recognized as one of the possible therapeutic targets for the treatment of Alzheimer's disease. In this paper, we report the synthesis and the BACE1 inhibitory activity of new, variously substituted N-(3-(4-benzhydrylpi

Full text of DOI:10.1016/j.bmc.2010.09.032

Bioorganic & Medicinal Chemistry 18 (2010) 7991–7996
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
BACE1 inhibitory activities of enantiomerically pure, variously substituted
N-(3-(4-benzhydrylpiperazin-1-yl)-2-hydroxypropyl) arylsulfonamides
Simone Bertini a, Elisa Ghilardi a, Valentina Asso a, Carlotta Granchi a, Filippo Minutolo a, Mauro Pineschi a,
Valeria Di Bussolo a, Andrea Bortolato b, Stefano Moro b, Alessandro Saba c, Marco Macchia a,
⇑
a Department of Pharmaceutical Sciences, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
b Molecular Modeling Section, Department of Pharmaceutical Sciences, University of Padova Via Marzolo 5, I 35131 Padova, Italy
c Department of Chemistry and Industrial Chemistry, University of Pisa, Via Risorgimento 35, 56126 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 April 2010
Revised 4 September 2010
Accepted 14 September 2010
Available online 18 September 2010
b-Secretase (BACE1) has been widely recognized as one of the possible therapeutic targets for the treatment
of Alzheimer’s disease. In this paper, we report the synthesis and the BACE1 inhibitory activity of new,
variously substituted N-(3-(4-benzhydrylpiperazin-1-yl)-2-hydroxypropyl) arylsulfonamides. Each enan-
tiomeric form was separately evaluated in BACE1 inhibition assays and IC50 values were obtained in the low
micromolar range. According to our biological results and docking studies, it can be asserted that the stereo-
chemistry around the OH group in the central hydroxyethylamino linker does not significantly influence the
BACE1 inhibitory activity of this type of molecules.
Keywords:
Alzheimer
BACE1
Ó 2010 Elsevier Ltd. All rights reserved.
Enzyme inhibition
Docking
Benzhydryl-piperazines
1. Introduction
R2
O
Alzheimer’s disease (AD) is the most common form of senile
dementia and is characterized by a neurodegenerative disorder that
causes a progressive damage to the cholinergic and dopaminergic
systems responsible for memory, learning and behaviour. In the
brain neurons of AD patients there are relevant amounts of extracel-
lular senile plaques, mainly constituted by amyloid b (Ab) peptide
aggregates1 and intracellular neurofibrillary tangles made of tau
proteins,2 both of which play a possible role in the progression of
the pathology.3
S
N
OH
N
O
N
R1
Figure 1. General structure of chiral N-(3-(4-benzhydrylpiperazin-1-yl)-2-
hydroxypropyl) arylsulfonamides (1–4a,b; see Table 1).
of which incorporate the hydroxyethylamine transition state iso-
stere moiety (HEA)6 and have a peptidomimetic structure.
In this paper, we report the synthesis and the BACE1 inhibitory
activity of new non-peptidomimetic derivatives characterized
by a N-substituted arylsulfonamido moiety linked, through a
2-hydroxypropyl portion, to a benzhydryl-piperazine (Fig. 1).
Although an hydroxyethylamino scaffold is structurally recognis-
able, these type of compounds cannot be considered as ‘classical’
hydroxyethylamine (HEA) isosteres, because of the alkylation of both
nitrogen-atoms of the central linker. To evaluate if and how the ste-
reochemistry around the OH group could influence the BACE1 inhib-
itory activity, each new compound was synthesized and assayed in
both enantiomeric forms, starting from optically pure precursors.
The Ab peptide is generated from proteolytic processing of the
amyloid precursor protein (APP) first by b-secretase (BACE1)
followed by
c-secretase. BACE1 catalyses the first rate-limiting
step of Ab production. Furthermore, it has been discovered that
BACE1 knockout (BACE1 ꢀ/ꢀ) mice are devoid of the ability to
generate Ab, but they are healthy.4 It is therefore considered that
BACE1 inhibition will be well tolerated.
Therefore, BACE1 is now considered as the preferred therapeu-
tic target for lowering brain Ab levels in the treatment or preven-
tion of AD.5
The identification of this promising pharmacological target has
so far led to the development of selective BACE1 inhibitors, many
2. Results and discussion
Compounds 1a,b, 2a,b, 3a,b and 4a,b were synthesized from
commercially available 1-benzhydryl-piperazine 5, as outlined in
⇑
Corresponding author. Tel./fax: +39 050 2219553.
E-mail address: mmacchia@farm.unipi.it (M. Macchia).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.032

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S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 7991–7996
Scheme 1. Reaction of enantiomerically pure (S)-glycidyl tosylate 6a
or (R)-glycidyl tosylate 6b with 1-benzhydryl-piperazine 5, in the
presence of K2CO3 in DMSO, yielded epoxide intermediates 7a and
7b, respectively. These were treated directly with phenethyl-,
benzyl-, or cyclopropylmethyl-amine in ethanol, affording interme-
diates 8a,b, 9a,b and 10a,b. Reaction of these amines with the appro-
priately substituted phenylsulfonyl chloride in DCM, using catalytic
amounts of DMAP, yielded final products 1a,b, 2a,b, 3a,b and 4a,b.
BACE1 inhibitory activities of the newly synthesized com-
pounds were determined by a previously reported fluorescence-
based assay7 and the results are reported in Table 1.
Enantiomers 1a and 1b, possessing a p-chlorophenyl-(N-phen-
ethyl)-sulphonamido moiety, are the most potent inhibitors of the
series, with IC50 values of, respectively, 2.3 and 1.9 lM. Substitution
of the phenethyl group (R1) with a benzyl group (maintaining
R2 = 4-Cl), as in compounds 2a and 2b, or the replacement of the
chlorine atom (R2) in the p-chlorophenyl-sulphonamido moiety
with an hydrogen (maintaining R1 = phenetyl), as in 3a and 3b, re-
sults in about a twofold decrease of BACE1 inhibition. Likewise, the
simultaneous introduction of a 3,4-chloro substituent (R2) and of a
N-cyclopropylmethyl group (R1), as in compounds 4a and 4b, results
again in a significant drop of activity. Finally, for each couple of enan-
tiomers, the IC50 values of R- and S-isomers are approximately the
same, suggesting that the stereochemistry around the OH group
does not significantly influence the BACE1 inhibitory activity of this
type of molecules (indeed, this was previously hypothesized by
Fujimoto et al. for related HEA-inhibitors12). However, it is impor-
tant to underline that in previous works on hydroxyethylamine-
based aspartyl protease inhibitors, the hydroxyl stereochemistry
was found to be tremendously important, leading oftentimes to as
much as 1000-fold difference.6b
The predicted in silico Log BB values (Table 1),8–11 defined as
the ratio of the steady-state concentrations of a compound in the
brain to those in the blood (Log BB = Log [brain]/[blood]), are quite
favourable for the whole series, with values ranging from ꢀ0.5 to
+0.1, with negligible differences between the enantiomeric forms.
Therefore, these derivatives are predicted to be able to efficiently
cross the blood-brain barrier and then reach satisfactory concen-
trations in the central nervous system, comparable to those pres-
ent in the blood stream.
N
HN
5
O
O
TsO
TsO
6a
6b
(i)
(i)
N
N
O
O
N
N
7a
7b
(ii)
N
R1NH2
R1NH2
(ii)
OH
OH
N
N
H
H
N
N
N
R1
R1
8a: R1 = phenethyl
9a: R1 = benzyl
10a: R1 = cyclopropylmethyl
8b: R1 = phenethyl
9b: R1 = benzyl
10b: R1 = cyclopropylmethyl
R2
R2
(iii)
(iii)
SO2Cl
SO2Cl
R2
R2
O
O
S
N
OH
N
S
N
OH
N
O
O
N
N
R1
R1
1b: R1 = phenethyl, R2 = 4-Cl
2b: R1 = benzyl, R2 = 4-Cl
3b: R1 = phenethyl, R2 = H
1a: R1 = phenethyl, R2 = 4-Cl
2a: R1 = benzyl, R2 = 4-Cl
3a: R1 = phenethyl, R2 = H
4b: R1 = cyclopropylmethyl, R2 = 3,4-Cl
4a: R1 = cyclopropylmethyl, R2 = 3,4-Cl
Scheme 1. Reagents and conditions: (i) K2CO3, DMSO; (ii) EtOH; (iii) CH2Cl2, DMAP, pyridine.

S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 7991–7996
7993
Table 1
Structures, BACE1 inhibitory activity and predicted Log BB values of benzhydryl-piperazines 1a,b, 2a,b, 3a,b and 4a,b
a
b
Compd
R1
R2
Stereo chemistry
IC50
(lM)
Log BBpred
1a
1b
2a
2b
3a
3b
4a
4b
Phenethyl
Phenethyl
Benzyl
Benzyl
Phenethyl
Phenethyl
Cyclopropylmethyl
Cyclopropylmethyl
4-Cl
4-Cl
4-Cl
4-Cl
H
H
3,4-Cl
3,4-Cl
R
S
R
S
R
S
R
S
2.3
1.9
6.3
5.4
5.1
6.9
5.4
4.7
ꢀ0.2
ꢀ0.3
0
+0.1
ꢀ0.3
ꢀ0.5
0
ꢀ0.1
a
IC50 measurements were performed as reported in Ref. 7. 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.
b
Predicted blood-brain barrier permeation (Log BB = Log [brain]/[blood]).8
The most active compounds (1a and 1b), have been submitted to
molecular docking studies in an attempt to understand the
interactions of these molecules with the catalytic site of BACE1 (Figs.
2 and 3). The computational protocol used in this analysis has been
recently exploited with promising results for the evaluation of
atom of the sulfonyl portion by means of a hydrogen bond through
its phenol OH group. Finally, the secondary alcohol present on the
chiral carbon participates to a hydrogen bond network with
Asp228 and Arg235. In this binding orientation the protonation
of the piperazine seems to have a marginal effect on the ligand–
protein interaction. Indeed the nearer residue is the oxygen of
the backbone of Gly230 at a distance of about 4 Å from the proton-
ated nitrogen. Furthermore they are not correctly oriented to cre-
ate a strong interaction.
The comparison of the predicted binding mode with a standard
orientation of a HEA inhibitor (Fig. 3) points out the peculiarities of
the scaffold in study. Even if there are common steric and electro-
static similarities to the known HEA ligands the resulting most
promising pose for this new class of inhibitors can be considered
‘upside-down’ to the reference. Principally for steric reasons this
class of inhibitors seems to not adopt the standard HEA binding
mode. The principal reason is the bulky N-substituted arylsulpho-
nylamino moiety that in the proposed binding mode creates good
van der Waals interactions, while in the standard HEA orientation
it would result in not favourable interactions or steric clashes with
the protein. However further studies are needed to validate this
preliminary hypothesis. In case of a confirmation of this ligand–
protein complex conformation, this study will represent an impor-
tant starting point to optimize the lead to fully exploit its peculiar
binding mode.
a
-naphthylaminopropan-2-ol derivatives.15
As we can see from the superimpositions of inhibitors 1a and 1b
(in cyan and magenta, respectively, Fig. 2), the binding modes of
the two enantiomeric forms are comparable. In particular, both R
and S enantiomers are able to create a strong hydrogen bond with
only one of the two catalytic aspartic acids, Asp228. These results
are in agreement with the very similar inhibitory activity values
found with both the enantiomers. Consequently, it is deducible
that the chirality of the C–OH in the hydroxyethylamine isoster
portion of this kind of structures does not have any significant
influence on the BACE1 inhibitory activity. In details (Fig. 3), the
two rings of the benzhydryl-moiety are respectively placed into
the S2-pocket (surrounded by Gly230, Thr231 and Thr232), and
S3-pocket (Ile230). The piperazine portion lies on the S1-pocket,
interacting with Tyr51 and Phe108. The S20-pocket host a big part
of the sulfonamido-terminal portion of the inhibitor. In fact, the
aromatic ring of the phenylethyl-substituent interacts with Val69
and Ile126, whereas other hydrophobic interactions occur between
the p-chlorophenyl ring and Ile226, Thr329, and Val332. The S20-
pocket delimitation is completed by Ser35, Gly34, Tyr71 and, in
particular, by Tyr198 which closely interacts with one oxygen
3. Conclusion
In conclusion, we have synthesized a new series of chiral
BACE1 inhibitors possessing a N-(3-(4-benzhydrylpiperazin-1-yl)-
2-hydroxypropyl)arylsulfonamido structure. The most active enan-
tiomeric compounds 1a and 1b showed promising IC50 values (2.3
and 1.9 lM, respectively). Some derivatives are predicted to be
able to cross the blood-brain barrier, suggesting that they may
reach and inhibit BACE1, inside the central nervous system.
According to our biological results and docking studies, it can be
asserted that the BACE1 inhibitory activity of this type of com-
pounds does not show a significant dependency upon the absolute
configuration of the chiral carbon in the central hydroxyethyl-
amino linker.
4. Experimental section
Purity of the final products was determined by HLPC technique.
Preparative HLPC was run using a Waters 2767 system with a binary
GradientModule Waters 2525 pump and coupled to a Waters Micro-
mass ZQ (ES) or Waters 2487 DAD, using a Supelco Discovery HS C18
Figure 2. Superimposition of Induce Fit13 docking poses for inhibitors 1a (cyan) and
1b (magenta). The molecule co-crystalized with the BACE1 X-ray crystal structure
(PDB code 1W5114) used in this study is shown as reference in green. BACE1 active
site is shown and the FLAP region is highlighted together with the two catalytic
aspartic acids (Asp32 and Asp228).
5.0
lm 10 ꢁ 21.2 mm column. Gradients were run using 0.1% formic
acid/water and 0.1% formic acid/acetonitrile with gradient 5:95–
95:5. In all cases, purity of analyzed compounds was P98%. The
characterization by mass spectrometry was carried out by a Perkin

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S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 7991–7996
S2'
(Val69,Ile126)
Tyr198
S2
S1
(Ser35,Gly34,Tyr71)
(Gly230, Thr231, Thr232)
(Tyr71, Phe108)
HO
1.8
O
(Ile226, Thr329, Val332)
N
N
N
S
Cl
O
O
1.8
1.7
H
NH2
S3
O
O
(Ile110)
HN
NH
Asp228
Arg235
Figure 3. Principal interactions scheme of 1b with BACE1 active pocket as resulting from the docking study. Hydrogen bonding are shown as dotted lines, important distances
in Å are quoted, and the principal amino acids creating van der Waals interactions with the inhibitors for every active sitesubpocket are shown in parentheses.
Elmer/Sciex (Concord, ON, Canada) API 365 triple quadrupole mass
spectrometer, equipped with a Turbo ionspray ion source and cou-
pled to a Perkin Elmer (Waltham, MA, USA) 200 Series HPLC system,
including a binary micro pump system, an high pressure mixer, and
an autosampler. Experiments were performed either in full scan
mode, to get the molecular weight, or in Product ion scan mode
(MS–MS), to obtain structural information. Analytical data are
shown for only one enantiomer of each couple. Chiral HPLC analysis:
Analytical HPLC were performed on a Waters 600E equipped with a
Varian Prostar 325 detector with detection at 254 nm and 220 nm.
The stationary phase used was an analytical chiral column (Chiralcel
OD-H column, Daicel Chemical, Tokyo, Japan). HPLC grade 2-propa-
nol and n-hexane were purchased from Sigma–Aldrich and used as
received. The mobile phase was in all cases n-hexane/2-propanol
(75:25), with a flow rate of 0.5 mL/min.
mmol) in EtOH (50 mL) was heated to reflux overnight. Then solvent
was removed under reduced pressure and the crude obtained
was purified by flash chromatography (eluent: EtOAc/MeOH 95:5)
to afford 0.83 g of (R)-1-(4-Benzhydryl-piperazin-1-yl)-3-pheneth-
ylamino-propan-2-ol (8b) (yield: 57%). To
a mixture of 8b
(0.10 g, 0.23 mmol), 4-dimethylaminopyridine (DMAP) (0.003 g,
0.02 mmol) and pyridine (0.04 mL, 0.48 mmol), 4-chlorobenzenesulfo-
nylchloride(0.05 g, 0.24 mmol)wasadded. After 16 h stirring at room
temperature, 1 N HCl (2 mL) was added, then left stirring for 5 min.,
and the organic layer was recovered, dried over Na2SO4, filtered and
evaporated under reduced pressure. The resulting crude was puri-
fied by flash chromatography (eluent: EtOAc/MeOH 95:5) to obtain
0.04 g of the title compound (yield: 30%).1H NMR (400 MHz, CDCl3) d
(ppm): 2.56–2.95 (m, 13H), 3.09 (dd, 1H, J = 14.8, 6.0 Hz), 3.33 (dd,
1H, J = 14.8, 5.6 Hz), 3.30–3.50 (m, 2H), 4.00 (br, 1H), 4.26 (s, 1H),
7.16–7.21 (m, 5H), 7.24–7.30 (m, 6H), 7.39–7.41 (m, 4H), 7.46
(AA0XX0, 2H, JAX = 8.8 Hz, JAA/ XX = 2.0 Hz), 7.71 (AA0XX0, 2H, JAX
=
4.1. 1-Benzhydryl-4-(S)-1-oxiranylmethyl-piperazine (7b)
0
0
8.4 Hz, JAA/ XX = 2.0 Hz). MS: m/z 604 (M+, 30), 438 (M+-benzhydryl,
0
0
To
a
solution of 1-benzhydryl-piperazine (5) (4.20 g,
100). Chiral HPLC: retention time 28.7 min, 98.6% ee.
16.64 mmol) in DMSO (30 mL), K2CO3 (2.99 g, 21.64 mmol) and
toluene-4-sulfonic acid (R)-1-oxiranylmethyl ester (6b) (3.80 g,
16.64 mmol) were added. The mixture was stirred at room temper-
ature for 24 h, poured into water (300 mL) and extracted with
EtOAc (3 ꢁ 200 mL). The organic phase was dried over Na2SO4, fil-
tered and concentrated under reduced pressure to obtain a residue
that was purified by flash chromatography (eluent: EtOAc/CH2Cl2
8:2) to afford 1.81 g of the title compound (yield: 37%). 1H NMR
(400 MHz, CDCl3) d ppm): 2.32 (dd, 1H, J = 13.2, 6.8 Hz), 2.35–
2.65 (m, 8H), 2.47 (dd, 1H, J = 5.2, 2.8 Hz), 2.71 (dd, 1H, J = 13.2,
3.6 Hz), 2.76 (t, 1H, J = 4.6 Hz), 3.05–3.12 (m, 1H), 4.23 (s, 1H),
7.15–7.19 (m, 2H), 7.24–7.28 (m, 4H), 7.40–7.42 (m, 4H). MS: the
experiment in full scan mode showed a pseudo-molecular ion
([M+H]+) at m/z 309 Th; MS–MS of the ion at m/z 309 Th (20 eV)
provided a spectrum constituted by three main ions: m/z 167
(benzhydryl+, 100), 309 (0.9), 141 (M+-benzhydryl, 1.2).
4.4. N-[(R)-3-(4-Benzhydryl-piperazin-1-yl)-2-hydroxy-propyl]-
4-chloro-N-phenethyl-benzenesulfonamide (1a)
Same procedure as described above starting from 1-benzhydryl-
4-(R)-1-oxiranylmethyl-piperazine 7a, through the intermediate
(S)-1-(4-Benzhydryl-piperazin-1-yl)-3-phenethylamino-propan-2-
ol (8a), to obtain 0.68 g of the title compound (yield: 51%). Chiral
HPLC: retention time 21.3 min, >99% ee. Analytical data (1H-NMR
and MS) are identical to those of enantiomer 1b.
4.5. N-[(S)-3-(4-Benzhydryl-piperazin-1-yl)-2-hydroxy-propyl]-
N-benzyl-4-chloro-benzenesulfonamide (2b)
A mixture of 1-benzhydryl-4-(S)-1-oxiranylmethyl-piperazine
(7b) (1.05 g, 3.41 mmol) and benzylamine (1.86 mL, 17.04 mmol)
in EtOH (50 mL) was heated to reflux overnight. Then solvent was re-
moved under reduced pressure and the crude obtained was purified
by flash chromatography (eluent: EtOAc/MeOH 95:5) to afford
0.64 g of (R)-1-(4-Benzhydryl-piperazin-1-yl)-3-benzylamino-
propan-2-ol (9b) (yield:45%). To a mixtureof 9b(0.10 g, 0.24 mmol),
4-dimethylaminopyridine (DMAP) (0.003 g, 0.02 mmol) and pyri-
dine (0.04 mL, 0.48 mmol), 4-chlorobenzenesulfonyl chloride
(0.05 g, 0.24 mmol) was added. After 16 h stirring at room tempera-
ture 1 N HCl (2 mL) was added, then left stirring for 5 min, and the
organic layer was recovered, dried over Na2SO4, filtered and evapo-
rated under reduced pressure. The resulting crude was purified by
flash chromatography (eluent: EtOAc/MeOH 95:5) to obtain 0.01 g
(yield: 6%) of the title compound. 1H NMR (400 MHz, CDCl3) d
4.2. 1-Benzhydryl-4-(R)-1-oxiranylmethyl-piperazine (7a)
Same procedure as described above starting from toluene-
4-sulfonic acid (S)-1-oxiranylmethyl ester (6a), to obtain 2.03 g
of the title compound (yield: 40%). Analytical data (1H NMR and
MS) are identical to those of enantiomer 7b.
4.3. N-[(S)-3-(4-Benzhydryl-piperazin-1-yl)-2-hydroxy-propyl]-
4-chloro-N-phenethyl benzenesulfonamide (1b)
A mixture of 1-benzhydryl-4-(S)-1-oxiranylmethyl-piperazine
(7b) (1.05 g, 3.41 mmol) and phenethylamine (2.14 mL, 17.04

S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 7991–7996
7995
(ppm): 2.03–2.44 (m, 10H), 3.06 (dd, 1H, J = 14.8, 7.2 Hz), 3.27 (dd,
1H, J = 14.8, 4.8 Hz), 3.50–3.60 (m, 1H), 4.18 (s, 1H), 4.33 (d, 1H,
J = 14.8 Hz), 4.50 (d, 1H, J = 14.8 Hz), 7.15–7.19 (m, 2H), 7.24–7.28
shaken overnight at room temperature. Then 1 N HCl (2 mL) was
added, the organic layer was recovered, dried over Na2SO4, filtered
and evaporated under reduced pressure to get a crude that was
purified by flash chromatography (eluent: CH2Cl2/MeOH 95:5) to
obtain 0.12 g (yield: 52%) of the title compound. 1H NMR
(400 MHz, CDCl3) d (ppm): 0.02–0.03 (m, 2H), 0.29–0.33 (m, 2H),
0.65 (m, 1H), 2.63 (m, 4H), 2.76–2.78 (m, 2H), 2.95–3.04 (m, 5H),
3.15–3.24 (m, 4H), 4.14 (s, 1H), 4.35 (br s, 1H), 6.97–7.00 (m,
2H), 7.05–7.09 (m, 5H), 7.17–7.19 (m, 3H), 7.39 (m, 2H), 7.66 (m,
1H). MS: m/z 588 (M+, 35), 422 (M+-benzhydryl, 100). Chiral HPLC:
retention time 13.2 min, 97% ee.
(m, 9H), 7.37–7.39 (m, 4H), 7.47 (AA0XX0, 2H, JAX = 8.8 Hz, JAA/ XX
=
0
0
2.0 Hz), 7.76 (AA0XX0, 2H, JAX = 8.4 Hz, JAA/ XX = 2.0 Hz). MS: m/z 590
(M+, 35), 424 (M+-benzhydryl, 100). Chiral HPLC: retention time
34.4 min, 95% ee.
0
0
4.6. N-[(R)-3-(4-Benzhydryl-piperazin-1-yl)-2-hydroxy-propyl]-
N-benzyl-4-chloro-benzenesulfonamide (2a)
Same procedure as described above starting from 1-benzhydryl-4-
(R)-1-oxiranylmethyl-piperazine (7a), through the intermediate (S)-
1-(4-Benzhydryl-piperazin-1-yl)-3-benzylamino-propan-2-ol (9a), to
obtain 0.02 g of the title compound (yield: 14%). Chiral HPLC: reten-
tion time 21.4 min, >99% ee. Analytical data (1H NMR and MS) are
identical to those of enantiomer 2b.
4.10. N-[(R)-3-(4-Benzhydryl-piperazin-1-yl)-2-hydroxy-
propyl]-3,4-dichloro-N-cyclopropylmethyl-
benzenesulfonamide (4a)
Same procedure as described above starting from 1-benzhy-
dryl-4-(R)-1-oxiranylmethyl-piperazine (7a), through the interme
diate (S)-1-(4-Benzhydryl-piperazin-1-yl)-3-(cyclopropylmethyl-
amino)-propan-2-ol (10a) to obtain 0.09 g of the title compound
(yield: 39%). Chiral HPLC: retention time 14.4 min, 98% ee. Analyt-
ical data (1H NMR and MS) are identical to those of enantiomer 4b.
4.7. N-[(S)-3-(4-Benzhydryl-piperazin-1-yl)-2-hydroxy-propyl]-
N-phenethyl-benzenesulfonamide (3b)
A mixture of 1-benzhydryl-4-(S)-1-oxiranylmethyl-piperazine
7b (1.05 g, 3.41 mmol) and phenethylamine (2.14 mL, 17.04 mmol)
in EtOH(50 mL) was heated to reflux overnight. Then solvent was re-
moved under reduced pressure and the crude obtained was purified
by flash chromatography (eluent: EtOAc/MeOH 95:5) to afford
0.83 g of (R)-1-(4-Benzhydryl-piperazin-1-yl)-3-phenethylamino-
propan-2-ol(8b) (yield:57%). Toa mixtureof 8b (0.10 g, 0.23 mmol),
4-dimethylaminopyridine (DMAP) (0.003 g, 0.02 mmol) and pyri-
dine (0.04 mL, 0.48 mmol), benzenesulfonyl chloride (0.03 mL,
0.24 mmol) was added. After 16 h stirring at room temperature
1 NHCl (2 mL)was added, thenleftstirringfor5 min, andtheorganic
layer was recovered, dried over Na2SO4, filtered and evaporated
under reduced pressure. The resulting crude was purified by flash
chromatography (eluent: EtOAc/MeOH 95:5) to obtain 0.02 g (yield:
16%) of the title compound. 1H NMR (400 MHz, CDCl3) d (ppm):
2.35–2.41 (m, 8H), 2.61–2.62 (m, 2H), 2.91–2.93 (m, 2H), 3.07 (dd,
1H, J = 14.6, 6.6 Hz), 3.35–3.43 (m, 4H), 3.79 (br s, 1H), 4.21 (s, 1H),
7.15–7.20 (m, 5H), 7.24–7.29 (m, 6H), 7.40–7.41 (m, 4H), 7.47–
7.51 (m, 2H), 7.54–7.57 (m, 1H), 7.79–7.81 (m, 2H). MS: m/z 570
(M+, 30), 404 (M+-benzhydryl, 100). Chiral HPLC: retention time
28.1 min, 98.7% ee.
5. Computational methods
The protocol used in this study was the same applied in Ref. 16.
Briefly, the target structure has been based on coordinates of the
X-ray diffraction BACE1 crystal structure freely available in the
RSCB Protein Data Bank16 (PDB code 1W5114). Different docking
software have been used: GOLD,17 FlexX,18 Glide19 and Induced
Fit Docking.13 All the inhibitors were built using MOE,20 energy
minimized using MMFF94x force field21 until a 0.01 energy gradi-
ent was attained, and atomic charges assigned using AM1-BCC
semi-empirical partials charge.22 The top scored final docking
poses for all of the inhibitors were compared with the pharmaco-
phore proposed by Limongelli et al.23 created using the MOE com-
putational suite.
Acknowledgements
The authors are thankful to Siena BiotechSpA—Italy, for scientific
and financial support. The molecular modeling work coordinated
by S.M. has been carried out with financial supports of the Italian
Ministry for University and Research (MIUR), Rome, Italy and of
the University of Padova, Italy. S.M. is very grateful to Chemical
Computing Group for the long and fruitful collaboration. Ph.D.
fellowships from SienaBiotech SpA—Italy (V.A. and E.G.) and MIUR—
‘grandi programmi strategici’ (C.G.) are gratefully acknowledged.
4.8. N-[(R)-3-(4-Benzhydryl-piperazin-1-yl)-2-hydroxy-propyl]-
N-phenethyl-benzenesulfonamide (3a)
Same procedure as described above starting from 1-benzhydryl-
4-(R)-1-oxiranylmethyl-piperazine (7a), through the intermediate
(S)-1-(4-benzhydryl-piperazin-1-yl)-3-phenethylamino-propan-
2-ol (8a), to obtain 0.02 g of the title compound (yield: 13%). Chiral
HPLC: retention time 22.4 min, >99% ee. Analytical data (1H NMR
and MS) are identical to those of enantiomer 3b.
References and notes
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