Irreversible 4-aminopiperidine transglutaminase 2 inhibitors for Huntington's disease

Article abstract of DOI:10.1021/ml3001352

A new series of potent TG2 inhibitors are reported that employ a 4-aminopiperidine core bearing an acrylamide warhead. We establish the structure-activity relationship of this new series and report on the transglutaminase selectivity and in vitro ADME properties of selected compounds. We demonstrate that the compounds do not conjugate glutathione in an in vitro setting and have superior plasma stability over our previous series.

Full text of DOI:10.1021/ml3001352

Letter
pubs.acs.org/acsmedchemlett
Irreversible 4‑Aminopiperidine Transglutaminase 2 Inhibitors for
Huntington's Disease
Michael E. Prime,† Frederick A. Brookfield,† Stephen M. Courtney,† Simon Gaines,† Richard W. Marston,†
Osamu Ichihara,† Marie Li,† Darshan Vaidya,† Helen Williams,† Anna Pedret-Dunn,† Laura Reed,†
Sabine Schaertl,‡ Leticia Toledo-Sherman,§ Maria Beconi,§ Douglas Macdonald,§
Ignacio Munoz-Sanjuan,§ Celia Dominguez,§ and John Wityak*,§
̃
†Evotec (U.K.) Ltd., 114 Milton Park, Abingdon OX14 4SA, United Kingdom
‡Evotec AG, Manfred Eigen Campus, Essener Bogen 7, 22419 Hamburg, Germany
§CHDI Management/CHDI Foundation, 6080 Center Drive, Suite 100, Los Angeles, California 90045, United States
S
* Supporting Information
ABSTRACT: A new series of potent TG2 inhibitors are reported that employ a
4-aminopiperidine core bearing an acrylamide warhead. We establish the
structure−activity relationship of this new series and report on the trans-
glutaminase selectivity and in vitro ADME properties of selected compounds. We
demonstrate that the compounds do not conjugate glutathione in an in vitro
setting and have superior plasma stability over our previous series.
KEYWORDS: plasma stability, computer-aided drug design, SAR, acrylamides, Celiac disease, ADME
issue transglutaminase 2 (TG2) is a multifunctional
enzyme primarily known for its calcium-dependent
T
cross-linking activity via isopeptide bond formation.1 Less
well-studied activities of TG2 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 TG2 overactivity has been most closely associated
with Celiac disease and Huntington's disease (HD), and 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. TG2 expression and transglutaminase
activity have been shown to be increased in the brains of HD
patients,13 and in vitro and in vivo studies have implicated TG2
in HD pathophysiology.14−17
Figure 1. Plasma stability of 1 and putative metabolite 2.
progressed to clinical trials,20 suggesting that this moiety is not
especially susceptible to metabolism and enabling compounds
to reach acceptable plasma exposures in vivo. Thus, we present
here a structure−activity relationship (SAR) study of a new
class of acrylamide-bearing TG2 inhibitors that show improved
plasma stability.
Our approach to stabilize the compounds to plasma exposure
initially focused on replacing the problematic anilide with a
more robust alkylamide. The compounds in Table 1 were
prepared mainly to expand rapidly the SAR with respect to
TG2 as this series of compounds still possessed an anilide-
linked Cbz group expected to be susceptible to plasma cleavage.
Upon identification of a suitable replacement, a second round
of SAR would be required to replace the remaining Cbz-anilide.
Our working hypothesis regarding the mode of binding of these
Several classes of TG2 inhibitors have been reported, most of
which are irreversible in nature.18 In addition, we have reported
potent and selective covalent inhibitors bearing an acrylamide
warhead as in 1 (Figure 1).19 Compound 1 has TG2 IC50 of
0.11 μM; however, in vitro metabolism profiling studies
identified plasma stability as an issue cautioning against in
vivo evaluation. Compound 1 was one of the examples showing
better stability in plasma, having a half-life of 209 min in mouse
plasma. Evidence supporting the acrylamide amide linkage as
the site of metabolic instability was obtained by testing the
plasma stability of aniline analogue 2 (Figure 1) and detecting
its formation in plasma incubation of cyclopropylamide 1.
Aniline 2 showed no indication of metabolism over a 24 h
period in mouse plasma. The acrylamide warhead has been
featured in several EGFR irreversible inhibitors that have
Received: May 30, 2012
Accepted: August 9, 2012
Published: August 9, 2012
© 2012 American Chemical Society
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a
Table 1. Acrylamide Anilide Linkage Replacements
a
Values accompanied by standard deviations were averaged from at
least two independent experiments.
compounds remained consistent with our prior modeling and
SAR studies (Figure 2).19 Key interactions are capture of the
acrylamide by Cys277 accompanied by additional acrylamide
hydrogen-bonding interactions with the side chains of Trp241
and Gln276. In addition, there is a potential hydrogen-bonding
interaction of the sulfonyl with the side chain of Asn333, which
may facilitate further interactions with residues Leu312,
Phe316, Ile331, and Leu420 that line an adjacent lipophilic
pocket.
Figure 2. (a) Docking pose of analogue 8a (tan) covalently bound to
Cys-277 within the active site of TG2. (b) Overlay of 8a with the
diazoketone-containing pentapeptide inhibitor A (magenta) described
by Pinkas et al.5
In our prior report,19 we established that for several human
transglutaminase isoforms, a correlation exists (r2 = 0.95)
between the IC50 values using a 30 min compound incubation
and the irreversible inhibition constants, kinact/Ki. With this in
hand, we relied on the IC50 values to guide the medicinal
chemistry effort. In addition, all activity data in this report are
for the human form of these enzymes unless expressly stated.
As shown in Table 1, we began by preparing the 4-
aminopiperidine (8a), which appeared to be of the appropriate
shape and size to allow the acrylamide moiety to reach Cys277,
while the sulfonamide provided a kink necessary to direct the
Cbz group toward the lipophilic pocket. We were additionally
intrigued that the apparent proximity of Asn333 to the
sulfonamide might also contribute toward orienting the Cbz
group. The TG2 activity of 8a was 2−3-fold lower with respect
to anilide 1. Not surprisingly, 8a had an unacceptably short (4
h) half-life in mouse plasma. Adding a methylene as in 8b,
reducing the overall distance between the sulfonamide and the
acrylyl moiety as in 8c, or opening the piperazine as in 8d
resulted in a 10−20-fold loss in activity. In the next set of
analogues, the attachment point of the acrylamide moiety on
the piperidine was changed from the 4-position to the 3-
position in 8e and 8f, resulting in a complete loss of activity
when tested at 80 μM. Reducing the ring size resulted in the 3-
aminopyrrolidines 8g and 8h. These showed slightly improved
activity, but these analogues were 100−200-fold less active
when compared to 8a. Interestingly, the activity improved
somewhat upon further reduction in ring size to the 3-
aminoazetidine (8i). We've previously noted an inverse
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Letter
a
correlation of TG2 activity with the magnitude of acrylamide
electronegativity. While we suspect that electronegativity
(reactivity) is a factor contributing to TG2 potency, it is likely
not an overriding feature given the 2 orders of magnitude range
in potency observed within the aryl acrylamide SAR.19
Transglutaminase selectivity profiling of 8a afforded the
following IC50 values: TG2, 0.28 μM; TG1, 0.91 μM; TG3,
>80 μM; TG6, 8.9 μM; and FXIIIa, 3.6 μM.
Table 2. Reversed Amide TG2 Inhibitors
The importance of the geometry and the presumed
interaction of the sulfonyl moiety with Asn333 was probed
using compounds 14 and 15 (Figure 3). Replacing the sulfonyl
Figure 3. Structures of Asn333 probes 14 and 15.
with an sp2-hybridized carbonyl resulted in 14, which had a
TG2 IC50 of 8.9 μM, a 30-fold loss in activity when compared
to 8a. Further reduction of the carbonyl to an sp3-hybridized
methylene resulted in 15, which had a TG2 IC50 of 1.6 μM, a 6-
fold loss relative to 8a. These results indicate that the effect of
sp3 hybridization at this center dominates any benefit of the
putative hydrogen bond with Asn333 in orienting the
benzylcarbamate moiety toward the lipophilic pocket.
Keeping the 4-aminopiperidinyl moiety of 8a, further
modifications were focused on replacement of the remaining
anilide linkage. As shown in Table 2, this was accomplished by
reversing the sense of the amide linkage in compounds 9a−m
and ensuring in the design that only aliphatic amines were
incorporated for improved stability and optimal interactions
with residues Leu312, Phe316, Ile331, and Leu420 that line this
portion of the binding site. Our initial compound in this study,
phenethyl amide 9a, had a TG2 IC50 of 0.18 μM. Reducing the
length of the ethyl linker as in 9b resulted in little change in
activity; however, lengthening this linker by one methylene as
in 9c resulted in a 4-fold activity loss. Reducing the number of
rotatable bonds by incorporating the ethyl linker in a
cyclopropane (9d) or a tetrahydoisoquinoline (9e) caused a
2−3-fold loss in TG2 activity, as did N-methylation of the
amide (9f and 9g). Highlighting the lipophilic nature of this
portion of the site, a small but significant improvement in
activity relative to 9a was achieved by 9h, which bears a
cyclohexyl moiety in place of the terminal phenyl; however,
significant activity was lost upon incorporation of a larger
adamantyl group (9i). This improvement was also lost in the 4-
tetrahydropyranyl and morpholinyl analogues (9j and 9k), but
it was nonetheless interesting to observe that the basic
morpholinyl nitrogen and polar oxygen atoms were well-
tolerated. The final three compounds in this set (9l−n) also
attempted to reduce the number of rotatable bonds relative to
9a. These changes were mostly well-tolerated, although none
possessed improved TG2 activity.
a
Values accompanied by standard deviations were averaged from at
least two independent experiments.
replacement of the terminal phenyl with either a 2-naphthyl
(9t) or a 7-quinolinyl moiety (9u) resulted in roughly
equipotent activity. Following 9q, several alkoxy-substituted
analogues (9v−y) were investigated; however, none was as
potent as 9q. Of the remaining compounds, 3-chloro 9z and
3,4-dimethyl 9aa were equipotent with 9a.
Transglutaminase selectivity profiles against mouse TG2
(mTG2) and human isoforms of TG1, TG6, and FXIIIa were
obtained on the more potent compounds. The IC50 values for
compound 9q were typical: TG2, 0.11 μM; mTG2, 0.055 μM;
TG1, 1.2 0.047 μM; TG6, 34 0.062 μM; and FXIIIa, 6.4
A further expansion of the SAR around the phenethyl amide
is shown in Table 3. When compared to 9a, the addition of a 4-
trifluoromethyl substituent as in 9o resulted in a 3-fold loss of
activity. This activity was mostly recovered in 4-chloro 9p, and
a small improvement was observed in 4-methoxy 9q, which had
a TG2 IC50 of 0.11 μM. The incorporation of large lipophilic
groups at the 4-position, as in 9r and 9s, was disfavored, while
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Letter
a
of HD. The result of this profiling suggests that the compounds
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.
Table 3. Phenethylamide Derivatives
In summary, we have developed a series of covalent TG2
inhibitors having selectivity against closely related trans-
glutaminase isoforms exhibiting low GSH reactivity and
excellent plasma stability. In vitro ADME profiling indicated
rapid rates of oxidative metabolism and high P-gp efflux,
suggesting that further optimization of ADME profiles will be
required to achieve brain exposure in vivo.
compd
R
TG2 IC50 SD (μM)
9a
9o
9p
9q
9r
C6H5
0.18 0.029
0.55 0.049
0.28 0.016
0.11 0.0028
22 15
4-CF3-C6H4
4-Cl-C6H4
4-CH3O-C6H4
4-t-Bu-C6H4
ASSOCIATED CONTENT
■
9s
4-Ph-C6H4
57 8.8
S
* Supporting Information
9t
2-naphthyl
0.28 0.0071
0.31 0.016
0.23 0.016
0.46 0.093
0.28 0.052
0.26 0.081
0.17 0.021
0.18 0.044
Synthetic disclosures including schemes and full experimental
procedures and characterization of key compounds, supporting
figures, details of the ADME profiling assays, and trans-
glutaminase selectivity profiling data. This material is available
free of charge via the Internet at http://pubs.acs.org.
9u
9v
9w
9x
9y
9z
9aa
7-quinolinyl
2-CH3O-C6H4
3,4-(CH3O)2-C6H3
benzo[d][1,3]dioxol-4-yl
2,3-dihydrobenzofuran-7-yl
3-Cl-C6H4
AUTHOR INFORMATION
3,4-(CH3)2-C6H3
■
a
Values accompanied by standard deviations were averaged from at
least two independent experiments.
Corresponding Author
*Tel: 310-342-5518. Fax: 310-342-5519. E-mail: john.wityak@
chdifoundation.org.
Notes
0.11 μM. It is important to note that all of the compounds
tested against mTG2 had activity comparable to their human
TG2 values, confirming the species-independent nature of this
activity. Typical selectivity against TG1 was 3−10-fold, and
>100-fold selectivity was routinely observed against TG6, while
10−20-fold selectivity was typical against FXIIIa. The TG3
activity was not tested since the compounds were expected to
be inactive, this based on a large body of testing.19 The
selectivity profiling revealed that further effort might be needed
to improve selectivity against TG1 and FXIIIa; selectivity
profiles would need to be factored into any decision to consider
these compounds for in vivo studies.
Several compounds were profiled in in vitro metabolism
assays of kinetic solubility, plasma stability, liver microsomal
stability, and permeability/efflux. The results for compound 9a
were typical; it showed aqueous solubility of <0.01 mg/mL and
mouse and human liver microsomal intrinsic clearance values of
99 and 595 μL/min/mg, respectively. The high rate of
metabolism was likely due to the phenethyl and amino-
piperidinyl moieties, which are susceptible to oxidative
metabolism at multiple sites. All compounds tested were stable
in both mouse and human plasma, with a half-life >6 h and
several showing a half-life >24 h. In addition, 8a and 9z showed
no evidence of conjugation to the prototypical biological
nucleophile glutathione (GSH) when tested in vitro over a
period of 120 h.21
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are indebted to Ina Sternberger for outstanding technical
assistance in conducting the transglutaminase biochemical
assays. We also thank Andreas Ebneth for valuable discussions.
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