Structure-activity relationship, kinetic mechanism, and selectivity for a new class of ubiquitin C-terminal hydrolase-L1 (UCH-L1) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.04.027

3-Amino-2-keto-7H-thieno[2,3-b]pyridin-6-one derivatives were discovered as moderately potent inhibitors of ubiquitin C-terminal hydrolase-L1 (UCH-L1) utilizing an assay that measures hydrolysis of the fluorogenic substrate Ub-AMC. SAR studies revealed that both the carboxylate at the 5-position and the 6-pyridone ring were critical for inhibitory activity. Furthermore, activity was dependent on the nature of the ketone substituent at the 2-position, with 4-Me-Ph and 2-naphthyl being best. Kinetic mechanism studies revealed that these compounds were uncompetitive inhibitors of UCH-L1, binding only to the Michaelis-complex and not to free enzyme. The active compounds were selective for UCH-L1, exhibiting neither inhibition of other cysteine hydrolases (e.g., UCH-L3, papain, isopeptidase T, caspase-3, and tissue transglutaminase) nor cytotoxicity in N2A cells.

Full text of DOI:10.1016/j.bmcl.2007.04.027

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3729–3732
Structure–activity relationship, kinetic mechanism,
and selectivity for a new class of ubiquitin C-terminal
hydrolase-L1 (UCH-L1) inhibitors
Ara H. Mermerian, April Case, Ross L. Stein and Gregory D. Cuny*
Laboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neurodegeneration and Repair,
Brigham & Women’s Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA
Received 16 March 2007; accepted 6 April 2007
Available online 10 April 2007
Abstract—3-Amino-2-keto-7H-thieno[2,3-b]pyridin-6-one derivatives were discovered as moderately potent inhibitors of ubiquitin
C-terminal hydrolase-L1 (UCH-L1) utilizing an assay that measures hydrolysis of the fluorogenic substrate Ub-AMC. SAR studies
revealed that both the carboxylate at the 5-position and the 6-pyridone ring were critical for inhibitory activity. Furthermore, activ-
ity was dependent on the nature of the ketone substituent at the 2-position, with 4-Me-Ph and 2-naphthyl being best. Kinetic mech-
anism studies revealed that these compounds were uncompetitive inhibitors of UCH-L1, binding only to the Michaelis-complex and
not to free enzyme. The active compounds were selective for UCH-L1, exhibiting neither inhibition of other cysteine hydrolases
(e.g., UCH-L3, papain, isopeptidase T, caspase-3, and tissue transglutaminase) nor cytotoxicity in N2A cells.
Ó 2007 Elsevier Ltd. All rights reserved.
Deubiquitinating enzymes (DUBs) comprise a large
family of enzymes that specifically cleave ubiquitin-
derived substrates of general structure Ub1–72-Leu73-
Arg74-Gly75-Gly76-X, where X can be any number of
leaving groups ranging from small thiols and amines
to ubiquitin (Ub) and other proteins.1–4 While at least
five families of DUBs1 have been identified,4 the two
largest subsets and best characterized are the ubiquitin
C-terminal hydrolases (UCHs) and the ubiquitin prote-
ases (UBPs). UBPs are generally high molecular weight
enzymes (ꢀ100 kDa) that cleave substrates where X is a
protein or another molecule of Ub. These enzymes are
thought to have domains adjacent to the primary
Ub-substrate binding site that can recognize and bind
these proteinaceous leaving groups. Perhaps the most
thoroughly studied UBP is isopeptidase T,5–7 which
cleaves substrates where X is Ub. In contrast, UCHs
are lower molecular weight enzymes (ꢀ30 kDa) that
hydrolyze Ub derivatives where X is a thiol or an amine
and are thought not to have a binding domain for
protein leaving groups.
Both UBPs and UCHs are cysteine hydrolases and
catalyze the hydrolysis of amide bonds of their
substrates according to a mechanism in which substrate
and enzyme combine to form a non-covalent Michaelis-
complex, from within which the sulfhydryl of the active-
site cysteine attacks the carbonyl carbon of the amide
bond of the substrate to generate an acyl-enzyme inter-
mediate and liberate the first product. Hydrolysis of the
acyl-enzyme produces the reaction’s second product Ub
and regenerates free enzyme.
Our interest in these enzymes originates in the potential
involvement of UCH-L1 in Parkinson’s disease8–10 and
cancer.11–13 In the course of screening for UCH-L1
inhibitors, we discovered that the 3-amino-2-keto-7H-
thieno[2,3-b]pyridin-6-one derivative 1 was a moderately
potent inhibitor (Ki app = 2.8 lM). Herein, we report an
initial structure–activity relationship (SAR), kinetic
mechanism, and selectivity studies for this class of
UCH-L1 inhibitors.
O
NH2
O
HO
Keywords: Ubiquitin C-terminal hydrolase-L1; UCH-L1; Inhibitor;
Kinetic mechanism; Selectivity.
S
O
N
H
*
Corresponding author. Tel.: +1 617 768 8640; fax: +1 617 768
8606; e-mail: gcuny@rics.bwh.harvard.edu
1
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.027

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A. H. Mermerian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3729–3732
Most of the 3-amino-2-keto-7H-thieno[2,3-b]pyridin-6-
one derivatives were prepared according to the general
procedure outlined in Scheme 1.14 Meldrum’s acid, 2,
was treated with triethyl orthoformate and aniline to
give derivative 3. Cyclization of 3 in the presence of cya-
nothioacetamide under basic conditions gave the 1-pyri-
done 4. Alkylation with an a-bromoketone under basic
conditions followed by acidification with HCl gave 5.
to give 8. Finally, treatment with KOH in DMF gave
the methyl ester 9.
A derivative containing a thieno[2,3-b]pyridine in place of
the 7H-thieno[2,3-b]pyridin-6-one was prepared accord-
ing to the procedure outlined in Scheme 3. Methyl aceto-
acetate, 10, was converted to 11 with dimethylformamide
dimethyl acetal. Cyclization in the presence of cya-
nothioacetamide gave pyridine 12.16 Again, alkylation
with a-bromo-4-methylacetophenone followed by base
cyclization utilizing sodium methoxide gave methyl ester
14. Ester hydrolysis followed by acidification gave pyri-
dine-5-carboxylate 15.
Initial attempts to prepare the methyl ester of 3-amino-2-
keto-7H-thieno[2,3-b]pyridin-6-one derivatives by esteri-
fication (e.g., with methanol or diazomethane) or by
alkylation (e.g., with MeI) of the corresponding carbox-
ylic acids failed to yield the desired products. Instead an
alternative synthetic procedure was utilized as depicted
in Scheme 2. The dimethyl malonate derivative 6 was
allowed to react with cyanothioacetamide in the presence
of N-methylmorpholine in ethanol to give 7.15 This mate-
rial was alkylated with a-bromo-4-methylacetophenone
The prepared compounds were evaluated for UCH-L1
inhibitory activity utilizing an assay in which hydrolysis
of the fluorogenic substrate Ub-AMC, the 7-amido-4-
methylcoumarin C-terminus derivative of ubiquitin, is
measured in the presence of different compound concen-
trations.7 Kiapp values were calculated using a four-
parameter fit from the dependence of the initial velocity
values on inhibitor concentration.
Me
Me
Me
Me
O
O
O
O
O
a
b
Esterification of the carboxylic acid in the 5-position of
the 3-amino-2-keto-7H-thieno[2,3-b]pyridin-6-one ring
(9) or replacing the 6-pyridinone portion with a pyridine
(15) resulted in complete loss in UCH-L1 inhibitory
activity (Table 1). Furthermore, the inhibitory activity
was dependent on the nature of the ketone substituent
at the 2-position. Substituents at the ortho-position
(17, 20, and 24) or electron-donating substituents at
the para-position of the aromatic ketone (19) were detri-
mental to activity. Introduction of alkyl (16) or electron-
withdrawing groups at the para-position of the aromatic
ketone (23) improved activity. Also, introduction of a 2-
naphthyl ketone (26) resulted in improved inhibitory
activity. Heterocyclic ketones (27 and 28) or aliphatic
ketones (29 and 30) with the exception of cyclohexyl ke-
tone (31) were detrimental to activity. Replacement of
O
O
NHPh
O
2
3
O
O
NH2
CN
SH
c
R
O
HO
HO
S
O
N
H
O
N
H
4
5
Scheme 1. Reagents and conditions: (a) HC(OEt)3, PhNH2, D until
solution formed then allowed to cool (85%); (b) NCCH2C(S)NH2,
KOH, EtOH, rt, 16 h (69%); (c) RC(O)CH2Br, KOH, EtOH, rt, 16 h,
then HCl (34–84%).
O
MeO2C
O
MeO2C
O
CN
S_
7
O
MeO2C
Me
CN
SH
a
b
a
OMe
CO2Me
+
CO2Me
NMe2
Me
MeO2C
Me
N
N
N
H
H Me
6
10
11
12
MeO2C
Me
CN
S
Me
MeO2C
O
CN
S
Me
d
c
c
b
N
N
H
O
O
13
8
NH2
RO2C
Me
O
14 R = Me
15 R = H
NH2
O
e
MeO2C
S
N
S
O
N
H
Me
Scheme 3. Reagents and conditions: (a) Me2NC(OMe)2, DMF, rt,
16 h; (b) NCCH2C(S)NH2, NaH, DMF, rt, 16 h (30% yield over two
steps); (c) p-Me–C6H4C(O)CH2Br, NaOMe (2 equiv), MeOH, rt, 16 h
(62%); (d) NaOMe, DMF, MeOH, rt, 16 h (82%); (e) LiOH, THF/H2O
(1:1), D, 1 h then HCl (58%).
9
Me
Scheme 2. Reagents and conditions: (a) NCCH2CSNH2, N-meth-
ylmorpholine, EtOH, rt, 30 min (45%); (b) p-Me–C6H4C(O)CH2Br,
MeOH, D, 1 h (79%); (c) KOH, DMF, rt, 2 h (31%).

A. H. Mermerian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3729–3732
3731
Table 1. 7H-Thieno[2,3-b]pyridin-6-one derivatives prepared for struc-
ture–activity relationship study and Ki app values for UCH-L1
inhibition
1.5
1.0
0.5
1.0
(Vmax/Km)i
Vmax/Km
O
NH2
R2
R1O
S
O
O
N
H
0.8
0.6
0.4
Compounda
R1
R2
Ph
Ki app (lM)
b
Vmax,i
Vmax
1
9
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2.8
>30
0.91
10
4-Me-Ph
4-Me-Ph
2-Me-Ph
4-t-Bu-Ph
4-OMe-Ph
2-OMe-Ph
4-CF3-Ph
2-CF3-Ph
4-Cl-Ph
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
0.2
0.0
1.4
30
1
10
5.3
3.6
30
[LDN-91946] (μM)
Figure 1. Uncompetitive inhibition of UCH-L1 by 1 (LDN-91946). At
seven fixed concentrations of 1 that ranged from 0.3 to 20 lM, we
determined the dependence of initial reaction velocity on substrate
concentration for the UCH-L1-catalyzed hydrolysis of Ub-AMC.
Each of these data sets, comprising no less than six (vo, [S]) pairs, was
then fit by non-linear least squares to the Michaelis–Menten equation
to provide apparent values of Vmax and Vmax/Km, which are plotted
here as a function of 1. The solid line through the data set for the
dependence of Vmax,i/Vmax on [1] was drawn using a simple binding
isotherm and the best fit Ki value of 2.8 0.1 lM.
1.2
11
2-Cl-Ph
1-Naphthyl
2-Naphthyl
4-Pyridyl
2-Thienyl
Me
1.5
0.74
30
20
>30
20
t-Bu
c-Hexyl
NH2
N-Piperidinyl
2.0
>30
>30
a HCl salt.
b Standard deviation <10%.
Table 2. Activity of 7H-thieno[2,3-b]pyridin-6-one derivatives against
other cysteine hydrolases
Compound
Enzyme
Result (lM)
1
UCH-L3
TGase 2
Papain
No activity at 20
No activity at 40
No activity at 40
No activity at 40
30% inhibition at 20
No activity at 40
No activity at 40
No activity at 40
the ketone with a primary (32) or tertiary amide (33) was
also not tolerated.
Caspase-3
UCH-L3
Papain
16
23
The kinetic mechanism of inhibition for these com-
pounds was elucidated in experiments in which we
determined Vmax and Vmax/Km values for the UCH-L1-
catalyzed hydrolysis of Ub-AMC at various fixed
concentrations of inhibitor. An example is shown in
Figure 1 for inhibition by 1, where we observed that
while Vmax titrates with a Ki value of 2.8 lM, values of
Vmax/Km were independent of inhibitor concentration.
Such behavior is diagnostic of uncompetitive inhibition
where inhibitor binds not to free enzyme but to some
form of the enzyme that is complexed with substrate.
For cysteine and serine hydrolases, the form of the en-
zyme can be either the Michaelis-complex or the acyl-en-
zyme. To probe this, we conducted transient-state kinetic
experiments and found that the acyl-enzyme does not
accumulate in the steady-state; that is, acylation of the
active-site cysteine is rate-limiting (data not shown). This
indicates that 1 must bind to the Michaelis-complex.
However, we cannot exclude the possibility that the
inhibitor also stays bound to the acyl-enzyme and per-
haps even to forms of the enzyme with product bound.
Isopeptidase T
Papain
(Table 2). Compounds 16 and 23 were also inactive
against papain at 40 lM. Compound 16 demonstrated
very weak activity (30% inhibition at 20 lM) against
UCH-L3 and no activity against isopeptidase T at
40 lM. In addition, we observed no cytotoxicity when
serum-starved N2A cells were treated with 1 at concen-
trations as high as 0.1 mM.
In conclusion, a class of 3-amino-2-keto-7H-thieno[2,3-
b]pyridin-6-one derivatives were discovered as moder-
ately potent UCH-L1 inhibitors. A preliminary SAR
study revealed that both the carboxylate at the 5-posi-
tion and the 6-pyridinone ring were necessary for inhib-
itory activity. Furthermore, inhibitory activity was
dependent on the nature of the ketone substituent at
the 2-position, with 4-Me-Ph and 2-naphthyl being best.
A kinetic mechanism study revealed that these com-
pounds were uncompetitive inhibitors of UCH-L1,
binding only to the Michaelis-complex and not to free
enzyme. Finally, the active compounds were selective
for UCH-L1, exhibiting no inhibition of other cysteine
hydrolases (e.g., UCH-L3, papain, isopeptidase T,
The selectivity of 1 toward UCH-L1 was demonstrated
in experiments in which we observed no inhibition of
other cysteine hydrolases, including UCH-L3, tissue
transglutaminase (TGase 2), papain, and caspase-3

3732
A. H. Mermerian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3729–3732
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herein provide useful tools for investigating the role of
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pathological conditions, such as Parkinson’s disease
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Acknowledgments
We thank the Harvard Center for Neurodegeneration
and Repair (HCNR) for financial support and Kai Ding
for conducting the cytotoxicity experiments.
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