Potent transglutaminase inhibitors, aryl β-aminoethyl ketones

Article abstract of DOI:10.1016/j.bmcl.2009.12.011

Aryl β-aminoethyl ketones were discovered as potent inhibitors of tissue transglutaminase. Heteroaryl-like thiophene groups and N-benzyl N-t-butyl aminoethyl group are critical to the strong inhibitory activity of aryl β-aminoethyl ketones.

Full text of DOI:10.1016/j.bmcl.2009.12.011

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1141–1144
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent transglutaminase inhibitors, aryl b-aminoethyl ketones
Shoichiro Ozaki a, Etsuko Ebisui a, Kozo Hamada a,b, Jun-Ichi Goto a, Akinobu Z. Suzuki a,
Akiko Terauchi a,b, Katsuhiko Mikoshiba a,b,
*
a Laboratory for Developmental Neurobiology, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
b Calcium Oscillation Project, ICORP-SORST, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Aryl b-aminoethyl ketones were discovered as potent inhibitors of tissue transglutaminase. Heteroaryl-
like thiophene groups and N-benzyl N-t-butyl aminoethyl group are critical to the strong inhibitory activ-
ity of aryl b-aminoethyl ketones.
Received 20 October 2009
Revised 30 November 2009
Accepted 3 December 2009
Available online 22 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Transglutaminase inhibitor
Protein cross-link inhibitor
Alzheimer’s disease
Huntington’s disease
Parkinson’s disease
Celiac disease
Transglutaminases (TGases)1 are calcium-dependent enzymes
that catalyze various post-translational modifications including
protein cross-linking, amine incorporation, and deamidation.
Protein cross-links are formed by the creation isopeptide bonds
between the carboxamide group of glutamine residues and the
regulation should have pivotal pathophysiological roles. Therefore,
the development of TGase inhibitors could result in novel preven-
tions or curing for these implicated diseases.
Many chemical inhibitors of TGase such as thienopyrimidonine-
4-one acylhydrazides,25 peptidylmethyl ketone,26 dihydrooxaz-
oles,27 and cinnamoyl derivatives,28 have been reported. Lai et al.29
recently carried out a study on TGase inhibitors by screening exist-
ing drug libraries in which 2-naphtyl 2-(N-isopropyl N-benzyl)-ami-
noethyl ketone showed the highest activity. This report helped us to
postulate that the important functional groups that are responsible
for the inhibitory activity of TGase are carbonyl and amine moieties
proximal to the aromatic ring.
e
-amino group of lysine residues to form N-(
with the loss of ammonia.2 The intra- and inter-molecular cross-
links with the -bond are physically robust to serve physiological
c-glutamyl)-L-lysine
r
roles. These include the blood clotting by fibrin cross-linking and
the cornified envelope formation in which TGase1 (a genetic factor
for lamellar ichthyosis disease) cross-links insoluble amalgam of
proteins.3 Tissue TGase is also implicated in various neurodegener-
ative diseases including Alzheimer’s disease,4,5 Huntington’s
disease,6–9 Parkinson’s disease, and progressive supranuclear palsy.
We synthesized about 200 amino ketones and discovered that
5-bromo-2-thienyl-(N-t-butyl N-benzyl)-aminoethyl ketone (1)
was a potent TGase inhibitor.
Unregulated high activity of TGase and many N-(c-glutamyl)-L-ly-
sine isopeptide bonds in protein aggregates are observed in these
neurodegenerative diseases10–12 as well as in vitro amyloid b
O
deposits,13 tau protein,4,12 polyglutaminylated proteins, and
a-syn-
N
Br
S
uclein are all potential substrates of tissue TGase. TGase is also
implicated in the pathogenesis of ear comedogenesis,14,15 catarac-
tous lenses,16 psoriasis,17 cancer metastasis,18 liver injury,19 fibrin
injury,20,21 and damage to the immune system.22 The deamidation
of ingested gluten peptide by TGase is a pathogenic modification in
celiac disease.23,24 Altogether, these reports suggest that TGase
1
In the course of synthesis, we found that only b-aminoethyl
ketones strongly inhibited TGase; - and -aminoalkyl ketones
showed very weak TGase inhibition (IC50 >30 M). Therefore, we
a
c
l
present the TGase inhibitory activities of only b-aminoethyl
ketones in this Letter.
* Corresponding author. Tel.: +81 48 467 9745; fax: +81 48 467 9744.
E-mail address: mikosiba@brain.riken.jp (K. Mikoshiba).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.011

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S. Ozaki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1141–1144
Table 1
IC50 values of aryl b-aminoethyl ketones (4)
R1
R2
0.83
1.4
1.4
2.0
2.5
4.8
2.5
3.5
11
7.9
5
6
7
8
9
10
11
12
13
14
0.83
0.47
1.7
4.0
2.3
7.4
2.7
3.3
7.8
5.2
15
16
17
18
19
20
21
22
23
24
1.1
1.0
2.7
17
4.9
5.4
10
5.1
12
>30
25
26
27
28
29
30
31
32
33
34
0.53
0.57
0.67
1.2
1.5
6.9
1.7
1.2
2.7
9.9
35
36
37
38
39
40
41
42
43
44
0.92
1.7
18
7.7
15
45
46
47
48
49
0.32
0.65
0.55
1.8
1.3
0.12
0.84
0.36
4.5
1.2
50
51
52
53
54
55
56
57
58
59
0.32
2.3
2.8
4.3
6.6
0.42
0.80
6.5
0.68
1.4
60
61
62
63
64
65
66
67
68
69
1.4
0.44
2.1
10
10
6.5
19
8.3
6.8
4.0
70
71
72
73
74
75
76
77
78
79
0.19
80
0.081
1
0.61
81
1.4
82
0.23
0.10
0.42
1.0
1.5
5.4
1.9
83
84
85
86
87
88
89
8.6
11
12
10
12
11
12
13
14
90
91
92
93
94
95
96
97
98
0.98
2.7
2.5
99
100
101
0.77
0.21
9.4
102
103
104
3.2
1.4
2.6
4.3
8.5
16
6.9
10
4.7
105
106
107
108
109
110
111
112
113
0.85
1.8
2.3
114
115
116
5.6
1.7
11
10
117
118
119
120
0.59
0.17
6.1
10
121
122
123
124
1.4
0.7
2.2
125
126
127
10
1.1
13
10
128
129
130
131
4.0
21
16
22
132
133
134
135
1.7
1.5
1.9
136
137
138
2.6
139
12
3.6
140
26
141
18
7.4
142
143
144
In each box, the upper and lower values are the IC50 value and compound number, respectively.
Aryl (N,N-disubstituted) b-aminoethyl ketone 4, 5–144 (Table 1)
were prepared by the conventional Mannich reaction30–32 of the
corresponding aryl methyl ketones (2), secondary amines (3) and
formaldehyde (paraformaldehyde) by heating from room tempera-
ture to 133 °C in 1 h, and then keeping at 133 °C for 15 min.

S. Ozaki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1141–1144
1143
The TGase inhibitors described in this Letter are readily synthe-
sized and yet are very strong inhibitors of TGase. Therefore, they
may be strong candidates as therapeutic drugs for many diseases
caused by abnormal protein cross-linking (isopeptide bonds). They
may slow or block the progression of diseases (e.g., Alzheimer’s,
Huntington’s, celiac, and cataract), and various disorders in the
skin, liver, and immune system.
The products were purified by alumina column and were ob-
tained as viscous oils. The inhibitory activity against tissue TGase
was evaluated by our improved assay.33 The resultant IC50 values
of aminoketones are summarized in Table 1.
Among b-aminoketones, when R1 of compound 4 was aliphatic,
TGase inhibitory activity was very weak (IC50 >30 lM). We there-
fore showed only TGase inhibitory activities of aryl b-aminoketones.
Among aryl b-aminoketones, when a single hydrogen atom was
attached at N,N-positions of the amino group (NHR), TGase inhibi-
References and notes
tory activities were very weak (IC50 >30
only aryl N,N-disubstituted b-aminoethyl ketone 4 as shown in this
Letter.
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When we compared the aryl group R1 of 4 by looking vertically
at the top left line of Table 1, heteroaryls were more potent than
phenyls and naphthyls. Among heteroaryl groups, thienyl ap-
peared the most potent. Among thienyls, 5-bromo-2-thienyl was
the most potent, followed by 5-chloro-2-thienyl and 2-benzothie-
nyl, 2-thienyl in that order. The second most potent in the hete-
roaryls was the furyl group. Among the furyl group, the 2-furyl
group was better than the 3-furyl group and the 5-methyl-2-furyl
group. The pyridyl group was also effective. The 3-pyridyl group
was potent, followed by 4-pyridyl and 2-pyridyl. The 2-pyrazinyl
group and 6-ethyl 2-pyrazinyl group were also effective. 2-Thiazol-
yl groups were not very effective. Phenyl groups were quite
effective. 4-Cyanophenyl had potent activity, followed by phenyl,
4-chlorophenyl, and 4-fluorophenyl in that order. 2-Naphtyl,
chloronaphtyl, diphenyl methane, phenoxyphenyl groups also gave
active compounds.
When we compared R2 (NR3R4 in the reaction scheme) by
looking horizontally at the top line of Table 1, N-t-butyl N-benzyl
amino showed the most potent activity followed by N-isopropyl
N-benzylamino, N-isopropyl, and N,N-bis-2-hydroxyethylamino
groups. The N,N-dibenzylamino, and N,N-diphenylamino group
showed very weak activity (IC50 >30
pounds are not shown in Table 1.
lM). Therefore these com-
We synthesized 10 types of N-monosubstituted b-aminoethyl
ketones. These compounds showed very weak activities (IC50
>30
Letter.
Duval et al.25 reported that thienopyrimidines LDDN-80042 has
an IC50 value of 0.13 M. This was the highest value that was found
in our literature search. Griffin et al.26 reported on peptidyl methyl
ketones which had an IC50 value of 3
M. Watts et al.27 had re-
lM). Therefore these compounds are also not shown in this
l
l
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ported on dihydroisozazoles earlier, but the IC50 value was not re-
ported. Pardin et al.28 studied cinnamoyl benzotriazoyl amide
which had an IC50 value of 18 l
M. Lai et al.29 reported that 2-naph-
tyl (N-isopropyl N-benzyl)-aminoethyl ketone was the most potent
in existing drug libraries. The IC50 value of this compound (132)
was 4 lM by our assay. The most potent compound presented in
this Letter, 5-bromo-2-thienyl-(N-t-butyl N-benzyl)-aminoethyl
ketone (1),35 had an IC50 value of 0.081
lM. This value is currently
32. Mannkind cooporation. Intracellular Kinase Inhibitors. PCT WO 2007/136790
A2.
the best.
33. The inhibitory activities of compounds against TGase were evaluated by our
improved high-throughput screening. We applied the method established for
the measurement of TGase activity34 to a high-throughput screening using the
functional drug screening system, FDSS 3000 (Hamamatsu Photonics,
Shizuoka, Japan). The synthesized compounds were added to 0.1 ml enzyme
reaction solutions containing 100 mM HEPES-NaOH (pH 7.5), 1 mM CaCl2,
Interestingly, our best compound had a thiophene ring. This
supports the theory that the thiophene ring is critical to inhibitory
activity, as reported by Duval et al.25 Our best compound had a
bromine atom. Watts et al.27 also reported that compounds with
bromine atoms were potent. In this way, these two structures
make it feasible for us to obtain the strongest TGase inhibitors.
Hartley et al.13 reported that TGase induced Ab oligomerization.
The TGase inhibitor LDDN-80042 (a gift from Cuny laboratory) was
highly effective in attenuating Ab aggregation. Aggregation was
20 lM monodansyl cadaverine, 0.05 mg/ml N,N-dimethylcasein, 5 lg/ml
guinea-pig liver TGase in a 96-well plate (Nunc, 96-Well Black Plate with
clear bottom). We measured fluorescence emissions at 510 nm after excitation
at 340 nm. We calculated the velocity of the fluorescent increase to evaluate
enzyme activity. Compounds were dissolved and diluted with DMSO to
prepare final concentrations of 3.0, 10, and 30 lM. When the compounds were
effective at these concentrations, we diluted the compounds to determine the
IC50 values at submicromolar concentrations. DMSO was added to the reaction
mixture as a control in the same 96-well plate and enzyme activity expressed
as a percentage of control. The IC50 values were calculated by nonlinear
blocked at concentrations ranging from 0.1 to 10 lM, suggesting
that TGase may constitute a specific therapeutic target for slowing
(or blocking) the progression of Alzheimer’s disease.

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S. Ozaki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1141–1144
regression analysis to fit experimental data to the logistic function with 4 or 3
parameters using SigmaPlot.
34. Lorand, L.; Lockridge, O. M.; Campbell, L. K.; Myhrman, R.; Bruner-Lorand, J.
Anal. Biochem. 1971, 44, 221.
J = 7.5 Hz, 2H), 2.60 (t, J = 7.5 Hz, 2H), 1.18 (s, 9H). 13C NMR (CDCl3, 270 MHz)
189.5, 145.9, 142.7, 132.4, 131.9, 131.2, 128.4, 128.3, 128.1, 127.8, 122.7, 54.6,
51.0, 46.7, 40.8, 28.9; HREMS m/e; found 380.0694; calcd for C18H22BrNOS,
380.0684.
35. Spectroscopic data for 5-bromo-2-thienyl-(N-t-butyl N-benzyl)-aminoethyl
ketone (1). 1H NMR (CDCl3, 270 MHz) 7.0–7.8 (m, 7H), 3.73 (m, 2H), 2.97 (t,