Synthesis and biological evaluation of 3-aryltyramines as fragments binding to BACE-1 and BACE-2

Article abstract of DOI:10.1016/j.tetlet.2012.02.070

3-Aryltyramines were prepared in one single step from tyramine and various arenediazonium salts by radical arylation. Binding as well as enzyme inhibition data of the 14 compounds do not prove true interaction with BACE-1. In contrast, with BACE-2 inhibition and binding could be confirmed indicating that 3-aryltyramines are potential starting points for a drug discovery effort.

Full text of DOI:10.1016/j.tetlet.2012.02.070

Tetrahedron Letters 53 (2012) 2189–2194
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and biological evaluation of 3-aryltyramines as fragments binding
to BACE-1 and BACE-2
Stefanie K. Fehler a, Gerald Pratsch a, Walter Huber b, Alain Gast b, Remo Hochstrasser b,
a,
Michael Hennig b, , Markus R. Heinrich
⇑
⇑
a Department für Chemie und Pharmazie, Pharmazeutische Chemie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schuhstraße 19, 91052 Erlangen, Germany
b F. Hoffmann-La Roche Ltd., Discovery Technologies, 4070 Basel 65/319, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
3-Aryltyramines were prepared in one single step from tyramine and various arenediazonium salts by
radical arylation. Binding as well as enzyme inhibition data of the 14 compounds do not prove true inter-
action with BACE-1. In contrast, with BACE-2 inhibition and binding could be confirmed indicating that
3-aryltyramines are potential starting points for a drug discovery effort.
Received 18 January 2012
Revised 9 February 2012
Accepted 14 February 2012
Available online 21 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Radical reactions
Tyramine
BACE
Biaryl
Arylation
Tyramine1 and tyrosine2 derivatives structurally modified by
ring-arylation have recently become of interest as new building
blocks for pharmaceutical research. In fragment screening experi-
ments, Kuglstatter et al.1 observed that compounds possessing a
(2-hydroxybiphen-5-yl)ethylamine substructure bind to the b-site
amyloid precursor protein cleavage enzyme 1 (BACE-1) and act as
inhibitors. Due to its essential role in the amyloid cascade hypoth-
esis, BACE-1 today represents one of the most attractive drug tar-
gets for the treatment of Alzheimer’s disease. According to the
hypothesis, BACE-1 is the enzyme responsible for the formation
of the pathological 40 or 42 amino acid containing b-amyloid pep-
tides. The cerebral plaques resulting from the deposition of b-amy-
loid peptides are considered to be responsible for Alzheimer’s
disease and the related symptoms of dementia.3,4 To show activity
in vivo, potential BACE-1 inhibitors have to be able to pass the
blood–brain barrier. For this purpose, molecular properties like
low molecular weight and positive charge are beneficial. Both
requirements are fulfilled by tyramine (1) and its arylated deriva-
tives 2a and 2b, which have so far been identified as inhibitors for
BACE-1 (Fig. 1).1 BACE-2, which is structurally a close homolog of
BACE-1,5 is expressed in beta cells and involved in the cleavage
OH
OH
OH
NH2
tyramine (1)
BACE-1 KD (µM) 2000
NH2
NH2
2a
2b
350
60
Figure 1. 3-Aryltyramines 2a and 2b acting as BACE-1 inhibitors.
of TMEM27. Consequently, inhibition of BACE-2 could be of thera-
peutic use for the treatment of diabetes.
Based on the crystal structure of the enzyme,6 in silico docking
experiments have been performed with a number of substituted
tyramines.7 Among the compounds investigated, especially those
tyramines bearing a meta-substituted aromatic phenyl group as
substituent show a good interaction with the active site of BACE-
1 with the potential to reach out to the S3 pocket (Fig. 2). This
structure based molecular design study encouraged us to synthe-
size a number of such compounds and test their binding as well
as biological activity to BACE-1 and BACE-2.
⇑
Corresponding authors. Tel.: +49 9131 85 24115; fax: +49 9131 85 22585
(M.R.H.); tel.: +41 616886046 (M.H.).
Our interest in 3-aryltyramines such as 2a and 2b was further
driven by recent developments in the field of radical arylation of
E-mail addresses: michael.hennig@roche.com (M. Hennig), Markus.Heinrich@
medchem.uni-erlangen.de (M.R. Heinrich).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.02.070

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S. K. Fehler et al. / Tetrahedron Letters 53 (2012) 2189–2194
Table 1
3-Aryltyramines 2c–2t prepared by radical arylation of tyramine (1)
R2
R3
R4
R1
R2
R3
R4
R1
N2 Cl
OH
OH
3c-3t
TiCl3
(H2O/HCl)
NH2
NH2
1
2c-2t
Product (diazonium salt)
R1
R2
R3
R4
Yielda,b (%)
2c (3c)
2d (3d)
2e (3e)
2f (3f)
2g (3g)
2h (3h)
2i (3i)
2j (3j)
2k (3k)
2l (3l)
2m (3m)
2n (3n)
2o (3o)
2p (3p)
2q (3q)
2r (3r)
2s (3s)
2t (3t)
H
H
H
H
H
H
H
H
H
Cl
Br
H
H
H
H
H
H
H
H
H
Cl
F
H
Br
CN
F
CF3
H
H
H
I
Cl
H
H
H
OMe
Cl
F
H
H
Br
H
H
F
H
H
H
OMe
H
Cl
I
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
42
43
41
40
46
61
35
39
40
Figure 2. Tyramine derivative with meta-substituted phenyl substituent docked to
the active site of BACE-1.
40c
27c
10d
41d
41d
26d
14d
7d
non-protected phenols and anilines.8–10 Especially through the use
of arenediazonium salts,11 such as 3c, as aryl radical source, tyra-
mine (1) can be converted into its 3-aryl derivative 2c in a single
step without the need for protecting groups (Scheme 1).10b,12,13
The existing access, which has been exploited for the synthesis of
the BACE-1 inhibitors 2a and 2b, comprises four steps with a Suzu-
ki coupling as the key reaction.1
COMe
COOMe
H
22d
With the perspective of applying radical arylation reactions in
automated parallel syntheses, we decided to evaluate the scope
of this new methodology by preparing a small library of 3-aryl-
tyramines in combination with biological testing as BACE-1 and
BACE-2 inhibitors. The radical reactions for the library synthesis
were carried out using titanium(III)-chloride as the reductant in
diluted hydrochloric acid and acetonitrile. Diazonium salts were
prepared as chlorides and were added to a solution containing
tyramine (1) and TiCl3. Since the 3-aryltyramines 2c–2t are less po-
lar than tyramine (1), product separation from excess 1 was largely
possible by simple extraction of the basified aqueous reaction mix-
ture with diethyl ether.14 The compounds synthesized through the
radical arylation reactions are summarized in Table 1.
a
For reaction conditions, see Experimental section.
Yield after extraction with Et2O (corrected for minor amounts of 1, regioisomer
b
and by-products, extraction not quantitative).
c
Separation of regioisomer not possible by HPLC.
3-Aryltyramines accompanied by further by-products (see text).
d
different side-reactions. Due to its electron-donating substituent,
the diazonium salt 3n was reduced too slowly to effectively give
aryl radicals at ambient temperature.15 In the cases of 3h, 3k, 3o,
3p, 3r, and 3s, additional acetonitrile had to be added to achieve
solubility in the diazotization step. Larger amounts of this solvent
usually lead to increased hydrogen abstraction.16,17 The radical
arylations with the iodinated diazonium salts 3o and 3q gave
1,3- and 1,4-diiodobenzene as by-products, which suggest the
occurrence of intramolecular iodine transfer reactions.18 Signifi-
cant homocoupling, which means the addition of aryl radicals to
their original diazonium salts, was observed in the arylation with
salt 3t. Due to the fact that especially well stabilized cyclohexan-
dienyl radicals (captodative effect) arise from the addition step,19
Aryltyramines 2c–2k were accessible without any complica-
tions. Products 2l and 2m were obtained in lower yields, which
are most probably due to the steric hindrance caused by their
ortho-substituents. In addition, we were unable to fully separate
2l and 2m from their regioisomers by HPLC.
The syntheses starting from the diazonium salts 3n–3t gave the
desired products in lower yields, since they were complicated by
1) Boc2O
2) chloramine-T,NaI
3) R
Cl
R
Cl
OH
OH
OH
B(OH)2
Pd(OAc)2
4) HCl
N2
TiCl3
3c
for R = H, CH3
NH2
NH2
tyramine (1)
NH2
tyramine (1)
2a :R = H
2b : R = CH3
2c : R = Cl
Scheme 1. Radical and organometallic access to 3-aryltyramines 2a–2c.

S. K. Fehler et al. / Tetrahedron Letters 53 (2012) 2189–2194
2191
Table 2
BACE-2 can tolerate such substitution much better indicating more
flexibility of the pocket. Third, overall BACE-2 shows a better abil-
ity for interaction with 3-aryltyramines compared to BACE-1. Con-
sequently, the results reported here are encouraging for the further
evaluation of tyramine derivatives for the drug discovery of BACE-
2 inhibitors.
In-vitro binding and inhibition data
Compound
BACE-1 IC50
BACE-1 KD
BACE-2 IC50
BACE-2 KD
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
2000
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>2000
1300
1300
2200
2500
800
1300
>2000
2000
>2000
500
400
600
500
400
400
300
300
300
300
500
150
400
n.d.
500
Experimental section
General remarks
Solvents and reagents were degassed with nitrogen prior to use
in radical reactions. 1H NMR spectra were recorded on 360 and
600 MHz spectrometers using CDCl3 and CD3OD as solvents refer-
enced to CHCl3 (7.26 ppm) and CHD2OD (3.31 ppm). Chemical
shifts are reported in parts per million (ppm). Coupling constants
are in Hertz (J Hz). The following abbreviations are used for the
description of signals: s (singlet), d (doublet), dd (double doublet),
t (triplet), q (quadruplet), m (multiplet). 13C NMR spectra were re-
corded at 90.6 and 150.9 MHz in CDCl3 and CD3OD using CDCl3
(77.0 ppm) and CD3OD (49.5 ppm) as standard. Chemical shifts
are given in parts per million (ppm). Purification by preparative
HPLC was carried out using Varian 940-LC with a Pursuit XRs C8
2o
2p
2q
2t
>2000
>2000
>2000
>2000
1900
1400
>2000
IC50 and KD values are in
>2000 = no detection of inhibition at 2000
independent experiments. As the Km values of the substrates are 28
for BACE-1 and BACE-2, respectively, and the concentration used is 300 nm, IC50 is
about equivalent to Ki20KD’s are estimated values as no full saturation of the binding
could be observed at concentrations used for the dose response experiments.
l
m. n.d. = not determined due to solubility problems,
m. IC50 values are the mean of two
m and 35
l
l
lm
homocoupling is now able to compete with the desired addition of
the 3-methoxyphenyl radical to tyramine (1).
column (RP-8, 150 ꢀ 21.2 mm, 5
lm particle size) and PDA detec-
tion (Varian, 254 and 280 nm).
In conclusion, structurally diverse 3-aryltyramines could be ob-
tained through the simple standard procedure without adaption to
a particular diazonium salt. If possible in parallel synthesis, slightly
modified conditions are recommended for diazonium salts 3n, 3p,
3r, and 3s.15,17 From the set of compounds available, 14 aryltyram-
ines (2c–2l, 2o–2q, and 2t) were chosen for biological testing. Ta-
ble 2 summarizes the results observed for in vitro binding data and
biochemical enzyme inhibition assays for BACE-1 and BACE-2.
Overall, all data observed suggest no or very weak interaction of
the ligands with the active site of the enzyme. For BACE-1 with its
less sensitive assay we could not measure binding in the surface
plasmon resonance assay due to the solubility problems for any li-
gand and no inhibition could be detected. The only exception is
compound 2l with some activity at 2 mM. For BACE-2 we could de-
tect some binding as well as inhibitory activity for a number of li-
gands. Especially 2h and 2o are interesting as they show a good
correlation of binding and inhibition with the lowest values mea-
sured in the SPR and enzymatic assay.
General procedure for the radical arylation
Preparation of the arenediazonium chloride
To an ice-cooled degassed solution of aniline (5.00 mmol) in 3 N
hydrochloric acid (5 mL) and water (5 mL) was dropwise added a
degassed solution of sodium nitrite (5.00 mmol, 0.35 g) in water
(2.5 mL) over a period of 10 min. After stirring for 20 more minutes
at 0 °C, the clear solution was used for the aryl-aryl coupling reac-
tions (5 mmol/12.5 mL = 0.4 M).
Biaryl synthesis
A 5 mL aliquot of the 0.4 M arenediazonium chloride solution
(2.00 mmol) was added dropwise by a syringe pump to a vigor-
ously stirred solution of tyramine hydrochloride (6.00 mmol,
1.04 g) in water (6 mL), acetonitrile (4 mL), and titanium(III)-chlo-
ride (4.00 mmol, 4.00 mL, ca. 1 M solution in 3 N hydrochloric acid)
under nitrogen atmosphere over 10–15 min. After the addition was
complete, the mixture was left to stir for 10 more minutes and a
solution of sodium hydroxide (2 g) and sodium sulfite (2 g) in
water (20 mL) was added. After extraction with diethylether
(3 ꢀ 75 mL), the combined organic phases were washed with satd.
aqueous sodium chloride and dried over sodium sulfate. The sol-
vent was removed under reduced pressure. Prior to purification
by HPLC, the crude product was dissolved in dichloromethane
(10 mL) and TFA (1.5 equiv) was added. After concentration in va-
cuo, the trifluoroacetate salt was submitted to preparative HPLC
(gradient: CH3CN/(H2O + 0.1% TFA) = 10:90?95:5 over 8 min,
flow: 20 mL/min). Additional analytic data obtained from the char-
acterization of the compounds can be found in the Supplementary
data.
From a preparative point of view, radical arylations of tyramine
can provide a fast and simple access to 3-aryltyramines from
readily available starting materials. Since the best results were ob-
tained from the reactions with halogen-substituted arenediazoni-
um salts the method represents
a valuable complement to
known organometallic protocols, which would mostly be difficult
to conduct with comparable substitution patterns.12c C–H activa-
tion of aromatics, as which this type of radical arylation can also
be classified, has recently been identified as a priority area for fu-
ture green chemistry research.21 Evaluation of the binding and
inhibitory activity of the 3-aryltyramines synthesized in the con-
text of this study gives no encouraging results for BACE-1. With
the given solubility of the compounds and the sensitivity of the as-
says an interaction of the ligands with the BACE-1 target protein
can not be measured with confidence. On the other hand, for most
of the molecules binding activity could be detected against BACE-2.
Here, compounds 2h and 2o with iodo and bromo substituents in
the R2 position show the lowest IC50 and KD values. Comparison
of the data with the unsubstituted 3-phenyltyramine1 offers a
number of interpretations. First, it seems that BACE-1 does not tol-
erate substitutions at the phenyl ring in any positions indicating a
steep structure–activity relationship of this binding pocket. Sec-
ond, besides the high degree of similarity between the enzymes,
2-(40-Chloro-6-hydroxybiphen-3-yl)ethylamine (2c): From 4-
chloroaniline and tyramine hydrochloride according to the general
procedure described above. Purification of the crude product
(0.84 mmol, 208 mg, 42%) by preparative HPLC gave 2-(40-chloro-
6-hydroxybiphen-3-yl)ethylamine (2c) as a yellow oil: 1H NMR
(360 MHz, CD3OD): d (ppm) = 2.90 (t, J = 7.6 Hz, 2 H), 3.16 (t,
J = 7.6 Hz, 2 H), 6.89 (d, J = 8.2 Hz, 1 H), 7.09 (dd, J = 2.3 Hz,

2192
S. K. Fehler et al. / Tetrahedron Letters 53 (2012) 2189–2194
J = 8.2 Hz, 1H), 7,17 (d, J = 2.3 Hz, 1 H), 7.37 (d, J = 8.6 Hz, 2 H), 7.55
(d, J = 8.6 Hz, 2 H); MS (EI): m/z (%): 248 (2) [M+], 247 (12), 220
(42), 219 (30), 218 (100), 217 (36), 183 (15), 182 (17), 181 (39),
165 (11), 154 (12), 153 (25), 152 (36), 127 (13), 77 (11), 69 (48),
51 (27), 46 (53).
(36), 262 (100), 261 (21), 183 (30), 182 (27), 181 (76), 165 (10),
154 (14), 153 (26), 152 (37), 69 (33), 51 (22), 46 (39).
2-(30-Cyano-6-hydroxybiphen-3-yl)ethylamine (2i): From 3-cya-
noaniline and tyramine hydrochloride according to the general
procedure described above. Purification of the crude product
(0.70 mmol, 167 mg, 35%) by preparative HPLC gave 2-(30-cyano-
6-hydroxybiphen-3-yl)ethylamine (2i) as an orange solid: 1H
NMR (360 MHz, CD3OD): d (ppm) = 2.91 (t, J = 7.6 Hz, 2 H), 3.17
(t, J = 7.6 Hz, 2 H), 6.92 (d, J = 8.3 Hz, 1 H), 7.14 (dd, J = 2.3 Hz,
J = 8.3 Hz, 1 H), 7.21 (d, J = 2.2 Hz, 1 H), 7.57 (t, J = 7.8 Hz, 1 H),
7.65 (td, J = 1.3 Hz, J = 7.7 Hz, 1 H), 7.89 (ddd, J = 1.2 Hz, J = 1.7 Hz,
J = 7.9 Hz, 1 H), 7.93 (t, J = 1.4 Hz, 1 H); MS (EI): m/z (%): 238 (16)
[M+], 210 (16), 209 (100), 208 (34), 153 (12), 152 (15), 151 (9),
77 (9), 69 (21), 51 (15), 46 (24).
2-(40-Fluoro-6-hydroxybiphen-3-yl)ethylamine (2d): From 4-fluo-
roaniline and tyramine hydrochloride according to the general pro-
cedure described above. Purification of the crude product
(0.86 mmol, 199 mg, 43%) by preparative HPLC gave 2-(40-fluoro-
6-hydroxybiphen-3-yl)ethylamine (2d) as a brown oil: 1H NMR
(360 MHz, CD3OD): d (ppm) = 2.89 (t, J = 7.7 Hz, 2 H), 3.15 (t,
J = 7.7 Hz, 2 H), 6.88 (d, J = 8.2 Hz, 1 H), 7.07 (dd, J = 2.3 Hz,
J = 8.2 Hz, 1 H), 7.11 (t, JHF = 8.9 Hz, J = 8.9 Hz, 2 H), 7.15 (d,
J = 2.3 Hz, 1 H), 7.56 (dd, JHF = 5.5 Hz, J = 8.9 Hz, 2 H); MS (EI): m/
z (%): 231 (18) [M+], 203 (27), 202 (100), 201 (67), 199 (12), 183
(12), 181 (17), 171 (12), 170 (12), 152 (16), 133 (11), 69 (29), 51
(17), 46 (33).
2-(30,40,50-Trifluoro-6-hydroxybiphen-3-yl)ethylamine (2j): From
3,4,5-trifluoroaniline and tyramine hydrochloride according to
the general procedure described above. Purification of the crude
product (0.78 mmol, 208 mg, 39%) by preparative HPLC gave 2-
(30,40,50-trifluoro-6-hydroxybiphen-3-yl)ethylamine (2j) as an or-
2-(30-Chloro-6-hydroxybiphen-3-yl)ethylamine (2e): From 3-
chloroaniline and tyramine hydrochloride according to the general
procedure described above. Purification of the crude product
(0.82 mmol, 203 mg, 41%) by preparative HPLC gave 2-(30-chloro-
6-hydroxybiphen-3-yl)ethylamine (2e) as an orange solid: 1H
NMR (360 MHz, CD3OD): d (ppm) = 2.90 (t, J = 7.6 Hz, 2 H), 3.16
(t, J = 7.6 Hz, 2 H), 6.90 (d, J = 8.3 Hz, 1 H), 7.10 (dd, J = 2.3 Hz,
J = 8.25 Hz, 1 H), 7.18 (d, J = 2.2 Hz, 1 H), 7.30 (ddd, J = 1.2 Hz,
J = 2.1 Hz, J = 8.0 Hz, 1 H), 7.36 (t, J = 7.8 Hz, 1 H), 7.48 (dd,
J = 1.8 Hz, J = 3.3 Hz, 1 H), 7.58 (dd, J = 1.1 Hz, J = 2.7 Hz, 1 H); MS
(EI): m/z (%): 248 (5) [M+], 247 (27), 221 (14), 220 (100), 219
(68), 218 (100), 217 (78), 183 (31), 182 (18), 181 (66), 165 (15),
154 (22), 153 (42), 152 (59), 151 (16), 139 (12), 127 (13), 77
(13), 69 (49), 51 (31), 50 (11), 46 (57).
ange solid: 1H NMR (600 MHz, CD3OD):
d (ppm) = 2.90 (t,
J = 7.6 Hz, 2 H), 3.16 (t, J = 7.6 Hz, 2 H), 6.90 (d, J = 8.3 Hz, 1 H),
7.12 (dd, J = 2.3 Hz, J = 8.3 Hz, 1 H), 7.20 (d, J = 2.2 Hz, 1 H), 7.26
(dd, JHF = 6.8 Hz, JHF = 9.4 Hz, 2 H); MS (EI): m/z (%): 267 (12)
[M+], 239 (14), 238 (100), 237 (44), 219 (9), 217 (9), 189 (11),
188 (17), 169 (14), 77 (9), 69 (41), 51 (25), 50 (9), 46 (44).
2-(30-Trifluoromethyl-6-hydroxybiphen-3-yl)ethylamine
(2k):
From 4-(3-trifluoromethyl)aniline and tyramine hydrochloride
according to the general procedure described above. Purification
of the crude product (0.80 mmol, 225 mg, 40%) by preparative
HPLC gave 2-(30-trifluoromethyl-6-hydroxybiphen-3-yl)ethyla-
mine (2k) as
a d
beige solid: 1H NMR (600 MHz, CD3OD):
2-(30-Fluoro-6-hydroxybiphen-3-yl)ethylamine (2f): From 3-fluo-
roaniline and tyramine hydrochloride according to the general pro-
cedure described above. Purification of the crude product
(0.80 mmol, 185 mg, 40%) by preparative HPLC gave 2-(30-fluoro-
6-hydroxybiphen-3-yl)ethylamine (2f) as an orange solid: 1H
NMR (360 MHz, CD3OD): d (ppm) = 2.91 (t, J = 7.6 Hz, 2 H), 3.16
(t, J = 7.6 Hz, 2 H), 6.90 (d, J = 8.3 Hz, 1 H), 6.99–7.05 (m, 1 H),
7.10 (dd, J = 2.3 Hz, J = 8.3 Hz, 1 H), 7.19 (d, J = 2.3 Hz, 1 H), 7.30–
7.34 (m, 1 H), 7.35–7.42 (m, 2 H); MS (EI): m/z (%): 231 (20)
[M+], 203 (37), 202 (100), 201 (87), 199 (13), 183 (15), 181 (30),
171 (14), 170 (17), 152 (23), 146 (13), 133 (15), 69 (43), 51 (26),
50 (10), 46 (50).
(ppm) = 2.92 (t, J = 7.6 Hz, 2 H), 3.17 (t, J = 7.6 Hz, 2 H), 6.92 (d,
J = 8.3 Hz, 1 H), 7.13 (dd, J = 2.3 Hz, J = 8.3 Hz, 1 H), 7.21 (d,
J = 2.2 Hz, 1 H), 7.56–7.60 (m, 2 H), 7.80–7.83 (m, 1 H), 7.87 (s, 1
H); MS (EI): m/z (%): 281 (9) [M+], 262 (9), 252 (100), 251 (16),
231 (28), 183 (18), 181 (10), 152 (11), 69 (23), 51 (13), 46 (28).
2-(20-Chloro-6-hydroxybiphen-3-yl)ethylamine (2l): From 2-chlo-
roaniline and tyramine hydrochloride according to the general pro-
cedure described above. Purification of the crude product
(0.80 mmol, 198 mg, 40%) by preparative HPLC gave a mixture of
the regioisomers 2-(20-chloro-6-hydroxybiphen-3-yl)ethylamine
(2l) and 2-(20-chloro-5-hydroxybiphen-2-yl)ethylamine (ratio
5:1) as a brown oil: 1H NMR (360 MHz, CD3OD, major isomer): d
(ppm) = 2.89 (t, J = 7.6 Hz, 2 H), 3.14 (t, J = 7.6 Hz, 2 H), 6.89 (d,
J = 8.3 Hz, 1 H), 7.01 (d, J = 2.2 Hz, 1 H), 7.13 (dd, J = 2.4 Hz,
J = 8.3 Hz, 1 H), 7.28–7.31 (m, 3 H), 7.43–7.46 (m, 1 H); 1H NMR
(360 MHz, CD3OD, minor isomer): d (ppm) = 2.63–2.75 (m, 2 H),
2.84–2.93 (m, 2 H), 6.58 (d, J = 2.6 Hz, 1 H), 6.83 (dd, J = 2.6 Hz,
J = 8.4 Hz, 1 H), 7.19 (d, J = 8.6 Hz, 1 H), 7.27–7.30 (m, 1 H), 7.38
(dd, J = 3.6 Hz, J = 5.7 Hz, 2 H), 7.49–7.52 (m, 1 H).
2-(40-Bromo-6-hydroxybiphen-3-yl)ethylamine (2g): From 4-bro-
moaniline and tyramine hydrochloride according to the general
procedure described above. Purification of the crude product
(0.92 mmol, 269 mg, 46%) by preparative HPLC gave 2-(40-bromo-
6-hydroxybiphen-3-yl)ethylamine (2g) as a yellow solid: 1H NMR
(360 MHz, CD3OD): d (ppm) = 2.90 (t, J = 7.6 Hz, 2 H), 3.16 (t,
J = 7.5 Hz, 2 H), 6.89 (d, J = 8.2 Hz, 1 H), 7.09 (dd, J = 2.3 Hz,
J = 8.2 Hz, 1 H), 7.17 (d, J = 2.0 Hz, 1 H), 7.48 (d, J = 8.8 Hz, 2 H),
7.53 (d, J = 8.74 Hz, 2 H); MS (EI): m/z (%): 292 (3) [M+], 291 (13),
265 (18), 264 (100), 263 (41), 262 (100), 261 (25), 183 (30), 182
(29), 181 (84), 168 (11), 165 (10), 154 (14), 153 (29), 152 (41),
69 (31), 51 (22), 46 (39).
2-(20-Bromo-6-hydroxybiphen-3-yl)ethylamine (2m): From 2-
bromoaniline and tyramine hydrochloride according to the general
procedure described above. Purification of the crude product
(0.54 mmol, 158 mg, 27%) by preparative HPLC gave a mixture of
the regioisomers 2-(20-bromo-6-hydroxybiphen-3-yl)ethylamine
(2m) and 2-(20-bromo-5-hydroxybiphen-2-yl)ethylamine (ratio
3:1) as a brown oil: 1H NMR (360 MHz, CD3OD, major isomer): d
(ppm) = 2.90 (t, J = 7.6 Hz, 2 H), 3.14 (t, J = 7.6 Hz, 2 H), 6.88 (d,
J = 8.3 Hz, 1 H), 6.99 (d, J = 2.3 Hz, 1 H), 7.13 (dd, J = 2.3 Hz,
J = 8.3 Hz, 1 H), 7.21 (ddd, J = 1.8 Hz, J = 7.4 Hz, J = 8.0 Hz, 1 H),
7.28–7.30 (m, 1 H), 7.35 (dt, J = 1.2 Hz, J = 7.4 Hz, 1 H), 7.64 (dd,
J = 1.0 Hz, J = 8.0 Hz, 1 H); 1H NMR (360 MHz, CD3OD, minor iso-
mer): d (ppm) = 2.62–2.78 (m, 2 H), 2.83–2.98 (m, 2 H), 6.57 (d,
J = 2.6 Hz, 1 H), 6.84 (dd, J = 2.6 Hz, J = 8.4 Hz, 1 H), 7.18 (d,
J = 8.3 Hz, 1 H), 7.27–7.29 (m, 2 H), 7.42 (dd, J = 1.2 Hz, J = 7.6 Hz,
1 H), 7.69 (ddd, J = 0.7 Hz, J = 1.1 Hz, J = 7.9 Hz, 1 H).
2-(30-Bromo-6-hydroxybiphen-3-yl)ethylamine (2h): From 3-bro-
moaniline and tyramine hydrochloride according to the general
procedure described above. Purification of the crude product
(1.22 mmol, 356 mg, 61%) by preparative HPLC gave 2-(30-bromo-
6-hydroxybiphen-3-yl)ethylamine (2h) as a brown solid: 1H NMR
(360 MHz, CD3OD): d (ppm) = 2.90 (t, J = 7.6 Hz, 2 H), 3.16 (t,
J = 7.6 Hz, 2 H), 6.90 (d, J = 8.3 Hz, 1 H), 7.10 (dd, J = 2.3 Hz,
J = 8.3 Hz, 1 H), 7.17 (d, J = 2.3 Hz, 1 H), 7.30 (t, J = 7.9 Hz, 1 H),
7.44 (ddd, J = 1.0 Hz, J = 2.0 Hz, J = 8.0 Hz,
1 H), 7.53 (ddd,
J = 1.1 Hz, J = 1.6 Hz, J = 7.7 Hz, 1 H), 7.7 (t, J = 7.6 Hz, 1 H); MS
(EI): m/z (%): 293 (8) [M++H], 291 (10), 265 (15), 264 (100), 263

S. K. Fehler et al. / Tetrahedron Letters 53 (2012) 2189–2194
2193
2-(40-Methoxy-6-hydroxybiphen-3-yl)ethylamine (2n): From 4-
methoxyaniline and tyramine hydrochloride according to the gen-
eral procedure described above. Purification of the crude product
(0.20 mmol, 49 mg, 10%) by preparative HPLC gave 2-(40-meth-
oxy-6-hydroxybiphen-3-yl)ethylamine (2n) as a brown oil: 1H
NMR (360 MHz, CD3OD): d (ppm) = 2.89 (t, J = 7.6 Hz, 2 H), 3.15
(t, J = 7.6 Hz, 2 H), 3.82 (s, 3 H), 6.87 (d, J = 8.2 Hz, 1 H), 6.94 (d,
J = 8.9 Hz, 2 H), 7.03 (dd, J = 2.3 Hz, J = 8.2 Hz, 1 H), 7.14 (d,
J = 2.3 Hz, 1 H), 7.48 (d, J = 8.9 Hz, 2 H); MS (EI): m/z (%): 243
(14) [M+], 215 (16), 214 (100), 213 (31), 199 (13), 141 (11), 137
(14), 120 (14), 115 (11), 108 (81), 107 (55), 91 (10), 77 (31), 69
(61), 51 (36), 50 (12), 46 (67), 45 (10).
12000 RU’s and 6000 RU’s of protein were immobilized in case of
BACE-1 and BACE-2, respectively. Acetate buffer (10 mM acetate,
pH 4.6, 150 mM NaCl, 3 mM EDTA, 0.01% P20, 4% DMSO) was used
as the running buffer during binding experiments. Compounds
were dissolved and diluted in pure DMSO and these DMSO solu-
tions added to acetate buffer to obtain buffer solutions of the com-
pounds of desired concentration. The immobilized BACE-1 and
BACE-2 were contacted with these solutions and the response at
the end of the injection phase after solvent correction taken as
the measure for compound binding. These responses were used
to estimate the KD values.
2-(30-Iodo-6-hydroxybiphen-3-yl)ethylamine (2o): From 3-iodo-
aniline and tyramine hydrochloride according to the general proce-
dure described above. Purification of the crude product
(0.82 mmol, 278 mg, 41%) by preparative HPLC gave 2-(30-iodo-6-
hydroxybiphen-3-yl)ethylamine (2o) as a red-brown oil: 1H NMR
(360 MHz, CD3OD): d (ppm) = 2.91 (t, J = 7.6 Hz, 2 H), 3.16 (t,
J = 7.6 Hz, 2 H), 6.90 (d, J = 8.2 Hz, 1 H), 7.09–7.17 (m, 3 H), 7.56
(ddd, J = 1.1 Hz, J = 1.7 Hz, J = 7.8 Hz, 1 H), 7.63 (ddd, J = 1.0 Hz,
J = 1.8 Hz, J = 7.9 Hz, 1 H), 7.94 (t, J = 1.6 Hz, 1 H); MS (EI): m/z
(%): 339 (10) [M+], 311 (24), 310 (100), 309 (14), 183 (37), 182
(40), 181 (74), 168 (14), 165 (10), 153 (22), 152 (36), 115 (10),
77 (11), 76 (11), 69 (40), 51 (27), 46 (45).
Enzyme inhibition assay
The fluorescent substrate human BACE-1 and BACE-2 kinetic as-
says were performed at room temperature in 384-well microtiter
plates (black with clear flat bottom, non binding surface plates
from Corning, Cat. No.: 3655) in a final volume of 51 lL. The
recombinant enzymes (BACE-1 expressed in SF9 cells and BACE-2
in E. coli and purified at F. Hoffmann-La Roche, Basel) and the sub-
strate were diluted in assay buffer (100 mM sodium acetate,
20 mM EDTA, 0.05% BSA, pH 4.5). Tyramines 100 mM DMSO stock
solutions were serially diluted in DMSO (15 concentrations, 1/3
dilution steps; final concentration range: 2000–0.0004
L of diluted compounds were mixed for 10 min with 40
BACE-1 (final concentration: 30 nM) or with 40 L of BACE-2 (final
concentration: 125 nM). After addition of 10 L of the substrate
l
M) and
2-(30,40-Dichloro-6-hydroxybiphen-3-yl)ethylamine (2p): From
3,4-dichloroaniline and tyramine hydrochloride according to the
general procedure described above. Purification of the crude prod-
uct (0.82 mmol, 231 mg, 41%) by preparative HPLC gave 2-(30,40-di-
chloro-6-hydroxybiphen-3-yl)ethylamine (2p) as a brown solid: 1H
NMR (600 MHz, CD3OD): d (ppm) = 2.90 (t, J = 7.6 Hz, 2 H), 3.16 (t,
J = 7.6 Hz, 2 H), 6.90 (d, J = 8.3 Hz, 1 H), 7.11 (dd, J = 2.3 Hz,
J = 8.3 Hz, 1 H), 7.19 (d, J = 2.1 Hz, 1 H), 7.49–7.53 (m, 2 H), 7.75
(dd, J = 0.4 Hz, J = 1.2 Hz, 1 H); MS (EI): m/z (%): 283 (7) [M++H],
181 (11), 256 (11), 255 (12), 254 (62), 253 (29), 252 (100), 251
(26), 217 (12), 215 (19), 182 (11), 181 (24), 153 (20), 152 (42),
151 (13), 69 (25), 51 (17), 46 (29).
1
l
lL of
l
l
WSEVNLDAEFRC-MR121 (final concentration: 300 nM), the plates
were strongly shaked for 2 min. The enzymatic reaction was fol-
lowed in a plate vision reader (Perkin–Elmer) (excitation wave-
length: 630 nm; emission: 695 nm) for at least 30 min in a
kinetic measurement detecting an increase of MR121 fluorescence
during the reaction time. The slope in the linear range of the kinetic
was calculated and the IC50 of the test compounds were deter-
mined using a four parameter equation for curve fitting.
2-(40-Iodo-6-hydroxybiphen-3-yl)ethylamine (2q): From 4-iodo-
aniline and tyramine hydrochloride according to the general proce-
dure described above. Purification of the crude product
(0.52 mmol, 176 mg, 26%) by preparative HPLC gave 2-(40-iodo-6-
hydroxybiphen-3-yl)ethylamine (2q) as a yellow oil: 1H NMR
(600 MHz, CD3OD): d (ppm) = 2.90 (t, J = 7.6 Hz, 2 H), 3.12 (t,
J = 7.6 Hz, 2 H), 6.90 (d, J = 8.2 Hz, 1 H), 7.04 (dd, J = 2.3 Hz,
J = 8.2 Hz, 1 H), 7.09 (d, J = 2.3 Hz, 1 H), 7.33 (d, J = 8.5 Hz, 2 H),
7.72 (d, J = 8.5 Hz, 2 H); MS (EI): m/z (%): 339 (9) [M+], 311 (14),
310 (100), 309 (10), 183 (25), 182 (29), 181 (56), 168 (13), 153
(17), 152 (25), 69 (35), 51 (24), 46 (43).
Acknowledgments
The generous financial support of our research by the Deutsche
Forschungsgemeinschaft (DFG) and the FAU Erlangen-Nürnberg is
gratefully acknowledged. We thank Francis Hermann and Roger
Werder for their contribution to the in vitro characterization of
the compounds, Jens-Uwe Peters and Martin Stahl for discussion.
Supplementary data
2-(30-Methoxy-6-hydroxybiphen-3-yl)ethylamine (2t): From 3-
methoxyaniline and tyramine hydrochloride according to the gen-
eral procedure described above. Purification of the crude product
(0.44 mmol, 107 mg, 22%) by preparative HPLC gave 2-(30-meth-
oxy-6-hydroxybiphen-3-yl)ethylamine (2e) as a brown oil: 1H
NMR (360 MHz, CD3OD): d (ppm) = 2.90 (t, J = 7.6 Hz, 2 H), 3.15
(t, J = 7.6 Hz, 2 H), 3.81 (s, 3 H), 6.85 (ddd, J = 1.0 Hz, J = 2.6 Hz,
J = 8.3 Hz, 1 H), 6.89 (d, J = 8.3 Hz, 1 H), 7.07 (dd, J = 2.3 Hz,
J = 8.2 Hz, 1 H), 7.09–7.12 (m, 1 H), 7.12 (d, J = 1.5 Hz, 1 H), 7.17
(d, J = 2.2 Hz, 1 H), 7.29 (dt, J = 1.3 Hz, J = 8.2 Hz, 1 H).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2012.02.070.
References and notes
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in acetate buffer (10 mM acetate, pH 4.6) during immobilization.

2194
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