SAR development of lysine-based irreversible inhibitors of transglutaminase 2 for huntington's disease

Article abstract of DOI:10.1021/ml300241m

We report a series of irreversible transglutaminase 2 inhibitors starting from a known lysine dipeptide bearing an acrylamide warhead. We established new SARs resulting in compounds demonstrating improved potency and better physical and calculated properties. Transglutaminase selectivity profiling and in vitro ADME properties of selected compounds are also reported.

Full text of DOI:10.1021/ml300241m

Letter
pubs.acs.org/acsmedchemlett
SAR Development of Lysine-Based Irreversible Inhibitors of
Transglutaminase 2 for Huntington's Disease
John Wityak,*,† Michael E. Prime,‡ Frederick A. Brookfield,‡ Stephen M. Courtney,‡ Sayeh Erfan,‡
Siw Johnsen,‡ Peter D. Johnson,‡ Marie Li,‡ Richard W. Marston,‡ Laura Reed,‡ Darshan Vaidya,‡
Sabine Schaertl,§ Anna Pedret-Dunn,‡ Maria Beconi,† Douglas Macdonald,† Ignacio Munoz-Sanjuan,†
̃
and Celia Dominguez†
†CHDI Management/CHDI Foundation, 6080 Center Drive, Suite 100, Los Angeles, California 90045, United States
‡Evotec (U.K.) Ltd., 114 Milton Park, Abingdon, OX14 4SA, United Kingdom
§Evotec AG, Manfred Eigen Campus, Essener Bogen 7, 22419 Hamburg, Germany
S
* Supporting Information
ABSTRACT: We report a series of irreversible transglutaminase 2 inhibitors starting from a
known lysine dipeptide bearing an acrylamide warhead. We established new SARs resulting in
compounds demonstrating improved potency and better physical and calculated properties.
Transglutaminase selectivity profiling and in vitro ADME properties of selected compounds are
also reported.
KEYWORDS: plasma stability, polar surface area, acrylamides, celiac disease, in vitro ADME
issue transglutaminase 2 (TG2) is a multifunctional
enzyme primarily known for its calcium-dependent
T
protein cross-linking activity.1 Less well-studied functions
include simple amidase, GTPase, ATPase, and protein disulfide
isomerase activities.2−4 TG2 has been characterized in at least
three forms, including open,5 closed,6 and an open-inactive
form.7 Genetic deletion of TG2 in mice suggests a role for TG2
activity in mitochondrial energy function,8 and its overactivity
has been most closely associated with celiac disease and
Huntington's disease (HD). In addition, there is growing
support for roles in inflammation and cancer.9−12
HD is an autosomal dominant, progressive, neurodegener-
ative disease that is characterized clinically by motor, cognitive,
and behavioral deficits.13 TG2 expression and transglutaminase
activity have been shown to be increased in the brains of HD
patients,14 and in vitro and in vivo models have implicated TG2
in HD pathophysiology,15−18 although more recent contra-
dictory animal data have appeared.19
The subject of irreversible inhibitors of TG2 has been
recently reviewed.20 Our studies have focused on irreversible
inhibitors bearing an acrylamide warhead,21,22 during which we
became interested in dipeptides A and B23 (Figure 1) as leads
due to their attractive potency and specificity for TG2. In a
prior report,21 we established that an excellent correlation exists
for several transglutaminase isoforms between the IC50 values
using a 30 min compound incubation and the irreversible
inhibition constants, kinact/Ki. With this correlation in hand, we
relied on the IC50 values to guide our medicinal chemistry
effort. We began by benchmarking dipeptide A, resulting in the
selectivity profile illustrated in Figure 1. We also resynthesized
and tested several compounds from the Marrano paper,23 the
Figure 1. Lysine-based irreversible inhibitors of TG2 from Marrano et
al.23
results of which are found in the Supporting Information. To
summarize, the results confirmed that dipeptide A was one of
the most potent TG2 inhibitors from this report. As shown in
Figure 1, ADME profiling studies on this compound indicated
good solubility, low permeability potentially accompanied by
efflux, and rapid metabolism in mouse liver microsomes
(mLM). Our goal was to identify a tool molecule from this
series for in vivo proof of concept studies in HD, where brain is
the target organ. Therefore, we focused on increasing potency,
improving the absorption profile, and increasing microsomal
stability. Lowering the polar surface area (PSA), the number of
hydrogen bond donors, and the number of rotatable bonds
Received: August 15, 2012
Accepted: October 4, 2012
Published: October 4, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
were the main tactics employed. The synthesis of new
compounds for this report was accomplished using well-
known literature methods, the details of which may be found in
the Supporting Information.
potency, while the 4-fluoro (3g) was 2−3-fold less potent. A
series of 4-alkyl substituents were next examined (3h−j);
however, these were also of lower potency relative to B. Similar
results were found with the 2,6-difluoro (3k) and with the 1-
and 2-naphthyl derivatives 3l and 3m.
Because of its lower molecular weight, lower PSA, and
equipotency, we selected compound B from the Marrano paper
as our starting point. Our prior reports described a structural
biology/computational chemistry-driven approach to struc-
ture−activity relationship (SAR) construction;21,22 however, in
the present series, we were unable to successfully use these
models to explain the known (or new) SAR. We instead
focused on an empirical approach based on improvement of
calculated properties. As the carboxylic acid was viewed as an
impediment to permeability, we began by replacement with a
hydroxmethyl (1) or deletion of this group entirely as in 2
(Table 1). While these modifications resulted in approximately
After preliminary investigation of the SAR with respect to the
carbamate moiety, we chose to keep the benzyl carbamate
constant and turned our attention to replacing the carboxylate
with a series of tertiary amides (Table 2). Relatively low
molecular weight aliphatic amides as exemplified by 4a−h
nearly all showed significantly improved potency relative to B,
with several compounds showing submicromolar potency. It
was noteworthy that this improvement did not require an
increase in PSA, bulking up the molecular weight, or the
addition of hydrogen bond donors relative to 1. Observing that
a more lipophilic pyrrolidine (4i) afforded 2-fold improved
potency relative to 4e, we next prepared 4-phenylpiperidine 4j,
which had a TG2 IC50 of 0.054 μM, and proved to be one of
the more potent compounds from this series. Phenylpiperidine
4j also had a favorable selectivity profile, with IC50 values of 9.3,
> 80, and 21 μM against TG1, TG3, and FXIIIa, respectively.
We followed this with a series of piperazines, which had
superior synthetic accessibility facilitating SAR development,
but at the cost of increased PSA. Judicious choice of
functionality could, in principle, keep the PSA of the
piperazines in a more acceptable range. The potency of
methylpiperazine 4k (IC50 = 0.61 μM) was in line with the
earlier simple tertiary aliphatic amides, indicating that addition
of the basic piperazine nitrogen atom was well tolerated. The
additional substitution on the terminal phenyl moiety of
piperazines 4l and 4m did not affect potency relative to 4j,
while a slight loss of potency was observed with 2-naphthyl 4n.
Next investigated was a series of 2-piperazinylpyridines (4o−s).
As a baseline, pyridine 4o recorded an IC50 of 0.26 μM. The
addition of a methyl group at either the 6- (4p) or the 3-
position (4q) resulted in improved potency, with 4p having an
IC50 of 0.055 μM. Substitution of 4o with a trifluoromethyl
group at either the 3- (4r) or the 5-position (4s) resulted in
approximately 2-fold improvement in potency relative to 4o.
Interestingly, the 3-methyl (4q) and electron-withdrawing 3-
trifluoromethyl (4r) and 5-trifluoromethyl (4s) analogues all
had similar potency. Favorable transglutaminase selectivity was
also retained; 4l had IC50 values of 0.062, 12.9, >80, 69, and 67
μM against TG2, TG1, TG3, TG6, and FXIIIa, respectively.
Further profiling also demonstrated that compounds from this
series were 2−7-fold less potent against mouse TG2 than they
were against the human form (shown in the Supporting
Information). This species difference may be an indication of a
somewhat different binding mode as compared with our earlier
chemotypes,21,22 which did not show this effect.
a
Table 1. TG2 Activity of Carbamate Derivatives
compd
no.
PSA
b
R
R′
(Å2)
TG2 IC50 SD (μM)
B
C6H5
C6H5
C6H5
H
CO2H
CH2OH
H
105
88
2.7 0.32
5.4 0.54
7.6 0.78
>80
1
2
67
3a
3b
3c
3d
3e
3f
3g
3h
3i
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
105
105
105
105
105
148
105
105
105
105
105
105
105
2−Cl-C6H4
2-CF3-C6H4
3-CF3-C6H4
4-CF3-C6H4
4-NO2-C6H4
4−F-C6H4
4-CH3-C6H4
4-n-Bu-C6H4
4-t-Bu-C6H4
2,6-F,F-C6H3
1-naphthyl
1.9 0.19
3.5 0.62
10 0.028
33
62
7.9 0.075
14 0.064
5.9 0.68
21
3j
3k
3l
10 3.7
14 0.39
9.1 1.3
3m
2-naphthyl
a
Values accompanied by standard deviations were averaged from at
least two independent experiments; they were otherwise obtained in a
b
single determination. PSA calculations were obtained using the
Dotmatics mol2image property calculation engine (http://www.
dotmatics.com/).
2- and 4-fold losses in potency relative to B, the PSAs of these
compounds (88 and 67, respectively) were significantly
improved and in a range more compatible with blood−brain
barrier (BBB) permeability. In addition, the relatively modest
loss in potency suggested that the carboxylate was not making a
productive interaction with TG2.
We next established the SAR with respect to the carbamate
moiety (Table 1). With B again as a comparator, the methyl
carbamate (3a) resulted in a large loss in potency. Turning
again to the benzyl carbamate motif, the 2-chloro (3b) was
slightly more potent than B, the 2-trifluoromethyl (3c) was
equipotent, and the 3- or 4-trifluoromethyl (3d and 3e,
respectively) resulted in 3- and 10-fold losses in potency,
respectively. Continuing with 4-substituted benzyl carbamates,
a 4-nitro substituent, as in 3f, resulted in a 20-fold loss in
At this point, we had significantly improved potency while
keeping transglutaminase selectivity and the hydrogen bond
donor count in favorable ranges. However, the large number of
rotatable bonds inherent in the lysine-based scaffold and PSA
exceeding 100 for some potent compounds (particularly the 2-
piperazinylpyridines) were concerns for oral bioavailability.24
A
key tactic employed to reduce the number of rotatable bonds
was lysine scaffold replacement. Early attempts resulted in 4-
aminophenylalanine-based 5 and piperidinylalanine-based 6
(Figure 2); however, both were inactive against TG2 when
tested at 80 μM, and comparisons with the potencies of B, 4p,
and 8a argued against synthesis of additional analogues.
Improved potency was observed for proline-based 7 (IC50
=
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ACS Medicinal Chemistry Letters
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a
Table 2. TG2 Activity of Tertiary Amide Derivatives
a
Values accompanied by standard deviations were averaged from at least two independent experiments; they were otherwise obtained in a single
b
determination. PSA calculations were obtained using the Dotmatics mol2image property calculation engine (http://www.dotmatics.com/).
liabilities in terms of permeability/efflux and microsomal
stability, we elected not to continue the SAR development
required to optimize this scaffold.
Another round of SAR development led to the series of 2-
piperazinyl-6-methylpyridines shown in Table 3. The bulky Boc
derivative 8a was ca. 15-fold less potent than benzyl carbamate
4p. A further loss in activity resulted from incorporation of a 2-
naphthylamide (8b). Interestingly, improved potency was seen
with acetamide 8c, and an additional improvement was
observed from phenylacetamide 8d, which was the most potent
compound of the series. Favorable transglutaminase selectivity
was also retained; 8d had IC50 values of 0.014, 15, >80, >80,
and 35 μM against TG2, TG1, TG3, TG6, and FXIIIa,
respectively. Benzamide 8e was equipotent with 8c, demon-
Figure 2. Lysine scaffold replacements 5−7.
strating the importance of the methylene linker in positioning
and orienting the phenyl substituent.
Illustrated in Table 4 are the results of in vitro metabolism
profiling of 4l and 8d. In a kinetic solubility assay, both showed
1.8 μM), which was prepared as a diastereomeric mixture.
However, given this potency, that it shared many structural
features with 4p and would be expected to carry similar
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ACS Medicinal Chemistry Letters
Letter
further optimization of the ADME profile will be required to
achieve brain exposure in vivo.
Table 3. TG2 Potency of 2-Piperazinyl-6-methylpyridine
Derivatives
a
ASSOCIATED CONTENT
■
S
* Supporting Information
Table of in-house-generated TG2 activity of compounds from
Marrano et al.;22 synthetic disclosures comprising schemes and
full experimental procedures; and characterization of key
compounds, supporting figures, details of the ADME profiling
assays, and transglutaminase selectivity profiling data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
b
compd no.
R
PSA (Å2)
TG2 IC50 SD (μM)
4p
8a
8b
8c
8d
8e
PhCH2O
tert-BuO
2-naphthyl
CH3
104
104
95
0.055 0.013
0.85 0.34
1.6 0.021
95
0.19 0.064
0.014 0.0082
0.19 0.073
AUTHOR INFORMATION
PhCH2
Ph
95
■
95
Corresponding Author
a
Values accompanied by standard deviations were averaged from at
least two independent experiments; they were otherwise obtained in a
single determination. PSA calculations were obtained using the
Dotmatics mol2image property calculation engine (http://www.
dotmatics.com/).
*Tel: 310-342-5518. Fax: 310-342-5519. E-mail: john.wityak@
chdifoundation.org.
b
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
good solubility and were stable in both mouse and human
plasma with a half-life >24 h. In addition, 4l showed no
evidence of conjugation to the prototypical biological
nucleophile glutathione (GSH) when tested in vitro over a
period of 68 h (data not shown).25 In liver microsomal stability
testing, metabolic stability was poor, with a short half-life and
rapid intrinsic clearance in both mouse and human. The high
rate of metabolism was likely due to the benzyl and piperazinyl
moieties, which are susceptible to oxidation at multiple sites.
To be effective agents for HD, it is preferable that
compounds possess a high rate of permeability and a low
efflux rate. P-glycoprotein (P-gp) is one of the main efflux
transporters in brain; the potential for 4l and 8d to be effluxed
by P-gp was assessed in an MDCK-MDR1 transfected cell line.
The results of this study indicated that both had good
permeability but suffered high active efflux via P-gp. Combining
this result with the results of microsomal stability testing
indicated that these compounds were not suitable candidates
for in vivo evaluation in the context of HD. The result of this
profiling suggests that they may be better suited as treatments
for celiac disease, where BBB permeability would be a
disadvantage and 24 h systemic coverage may not be required
for efficacy.
We are indebted to Ina Sternberger for outstanding technical
assistance in conducting transglutaminase biochemical assays.
We thank Andreas Ebneth for valuable discussions.
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a
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a
a
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