Fused 3-hydroxy-3-trifluoromethylpyrazoles inhibit mutant huntingtin toxicity

Article abstract of DOI:10.1021/ml400251g

Here, we describe the selection and optimization of a chemical series active in both a full-length and a fragment-based Huntington's disease (HD) assay. Twenty-four thousand small molecules were screened in a phenotypic HD assay, identifying a series of compounds bearing a 3-hydroxy-3- trifluoromethylpyrazole moiety as able to revert the toxicity induced by full-length mutant Htt by up to 50%. A chemical exploration around the series led to the identification of compound 4f, which demonstrated to be active in a Htt171-82Q rat primary striatal neuron assay and a PC12-Exon-1 based assay. This compound was selected for testing in R6/2 mice, in which it was well-tolerated and showed a positive effect on body weight and a positive trend in preventing ventricular volume enlargment. These studies provide strong rationale for further testing the potential benefits of 3-hydroxy-3-trifluoromethylpyrazoles in treating HD.

Full text of DOI:10.1021/ml400251g

Letter
pubs.acs.org/acsmedchemlett
Fused 3‑Hydroxy-3-trifluoromethylpyrazoles Inhibit Mutant
Huntingtin Toxicity
,
†,§
†
†
†
†
Salvatore La Rosa,* Tiziana Benicchi, Laura Bettinetti, Ilaria Ceccarelli, Enrica Diodato,
†
†
†
†
‡
†,∥
Cesare Federico, Pasquale Fiengo, Davide Franceschini, Ozgun Gokce, Freddy Heitz,
Giulia Lazzeroni, Ruth Luthi-Carter, Letizia Magnoni, Vincenzo Miragliotta, Carla Scali,†
†
‡,⊥
†,○
and Michela Valacchi†
†
Siena Biotech SpA, Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy
‡
́
Brain Mind Institute, Ecole Polytechnique Fed
́ ́
erale de Lausanne (EPFL), Lausanne, Switzerland
*
S Supporting Information
ABSTRACT: Here, we describe the selection and optimization
of a chemical series active in both a full-length and a fragment-
based Huntington’s disease (HD) assay. Twenty-four thousand
small molecules were screened in a phenotypic HD assay,
identifying a series of compounds bearing a 3-hydroxy-3-
trifluoromethylpyrazole moiety as able to revert the toxicity
induced by full-length mutant Htt by up to 50%. A chemical
exploration around the series led to the identification of
compound 4f, which demonstrated to be active in a Htt171−
82Q rat primary striatal neuron assay and a PC12-Exon-1 based assay. This compound was selected for testing in R6/2 mice, in
which it was well-tolerated and showed a positive effect on body weight and a positive trend in preventing ventricular volume
enlargment. These studies provide strong rationale for further testing the potential benefits of 3-hydroxy-3-
trifluoromethylpyrazoles in treating HD.
KEYWORDS: Huntington’s disease, mutant Htt toxicity, phenotypic screening, 3-hydroxy-3-trifluoromethylpyrazoles, ADME,
R6/2 mouse model
untington’s disease (HD) is an autosomal-dominant
neurodegenerative brain disorder caused by a CAG
Current target-based drug discovery strategies for HD are of
limited use, and alternative approaches for which an a priori
knowledge of molecular targets is not necessary are desirable.
Toward this goal, several groups have developed phenotypic
strategies to identify small molecules to be further developed
H
trinucleotide repeat expansion (>35 repeats), which encodes an
abnormally long polyglutamine (polyQ) tract in the N-terminal
1
part of a large protein called huntingtin (Htt). Mutant Htt is
1
subject to cleavage by proteolytic enzymes and to aberrant pre-
8
2
into potential drug candidates (Figure 1). The antiaggregation
RNA splicing, which results in the generation of N-terminal
1
fragments containing the expanded polyQ. These fragments
are able to aggregate with themselves and other proteins and
3
form large nuclear and cytoplasmatic inclusions. Although the
scientific community is still debating the role of these
aggregates, the affected neurons suffer from many different
dysfunctions altering the normal cell equilibrium and leading to
4
apoptotic or necrotic cell death. Among these dysfunctions,
2
+
reduced ATP levels, decreased Ca uptake, oxidative stress,
excitotoxicity, altered transcription, and impaired autophagy are
5
reported as major pathogenic mechanisms. Taken together,
the pathophysiological picture of HD still remains very complex
and poorly understood, lacking validated target proteins for
therapeutic intervention. Furthermore, despite significant
research efforts in the last 20 years since the mutated
Figure 1. Chemical structures of hit compounds identified in HTS HD
screenings.
6
huntingtin gene was discovered, none of the drug discovery
programs aimed at tackling a single target or a single pathway
involved in the disease pathology has yet yielded a molecule
capable of interfering with disease progression.
Received: July 3, 2013
Accepted: August 8, 2013
Published: August 8, 2013
7
©
2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
activity of the Rho-associated protein kinase 1 (Rock1)
inhibitor Y-27632 1, identified in a HEK-293 cell system, was
subsequently demonstrated to be active in HD-relevant cell and
and stability to dehydration (data not shown). We speculate
that in this particular assembly the carbonyl oxygen atom could
stabilize the 3-hydroxyl group on the pyrazole ring from
dehydration by an intramolecular hydrogen bond interaction
(see compound 4a in Figure 1).
In the optimization program, we opted for maintaining the
main structural features of the molecules in order to keep the
general pharmacophore shape and focused on the exploration
of three main points: (a) the carbocyclic ring, (b) the linker,
and (c) the R1 ring (see Figure 1).
Initial hit 4a showed acceptable solubility and permeability,
but a far too high metabolism rate in human and mouse. In an
effort to improve the overall profile of 4a, mitigating its
metabolic stability and moving to a IP-free chemical space, we
decided to explore the insertion of different heterocycle rings in
R1 position, and few analogues were synthesized (see Scheme
9
animal models. Compounds 2 and 3 were identified in cell
screenings respectively as selective enhancer of mutant Htt
1
0
clearance and active in a cell viability assay against HD-
11
induced neurodegeneration. Both compounds resulted also
active in follow-up animal models, including mouse and
1
2,13
C.elegans models of HD.
Our approach aimed at identifying a class of compounds
displaying activity in both full-length and Exon-1 mutant
huntingtin-based HD assays, thus enabling us to recapitulate
the animal models we planned to use for preclinical compound
profiling (R6/2, Exon-1 based) and the human version of the
disease. Although not exhaustive, we sought to build a paradigm
to maximize the chance for effective translation of preclinical
results toward clinical trials (Figure 2).
1
). The presence of an heterocycle in R1 not only produced a
Scheme 1. General Synthetic Route for the Fused 3-
a
Hydroxy-3-trifluoromethylpyrazole Derivatives
Figure 2. General workflow.
An HTS method was developed in-house creating a stable
recombinant 293/T-Rex cell line generated with both a CRE-
luciferase (CRE-LUC) reporter gene and with the full-length
mutant Htt gene under control of an inducible CMV promoter;
it has been shown that mutated Htt sequesters the cAMP
response element-binding protein (CREB) coactivator, CREB-
binding protein (CBP) through direct protein interactions,
a
Reagents and conditions: (a) ethyl trifluoroacetate, NaOMe, Et O,
2
14
which leads to decreased CREB-mediated transcription. In
addition to this, we planned to use another in vitro model of
HD based on Htt expression via LV infection on primary
striatal rat neurons as a secondary screening assay. This assay
relies on the incorporation of a Htt-derived sequence
expressing an N-terminal 171 aa fragment of mutant or wild-
type Htt (Htt171−82Q or Htt171−18Q, respectively; see
−10 °C to RT; (b) glacial AcOH; (c) acylhydrazide, pyrrolidine, mol
sieves, 0 °C to RT, THF; (d) sulphonylhydrazide, pyrrolidine, mol
sieves, 0 °C to RT, THF.
general improvement in the metabolic stability of the
compounds but also favored solubility and permeability across
the series (see Table 1).
15
Supporting Information).
As shown in Scheme 1, the fused 3-hydroxy-3-trifluorome-
thylpyrazoles 4 and 8 were prepared starting from the
corresponding cyclic ketones 6. Various cyclic rings were
explored starting from commercially available cyclohexanone,
cycloheptanone, and 4-pyranone. Upon treatment with sodium
methoxide and ethyl trifluoroacetate in diethyl ether, the
diketone 7 was isolated as the corresponding sodium salt, or as
a mixture in the keto−enol form. Cyclization of the diketone
with a substituted acylhydrazide or sulphonylhydrazide in the
presence of pyrrolidine yielded exclusively the 2-substituted
acyl- or sulphonyl-hexahydroindazoles with yields ranging 10−
70% and no trace of the N1-substituted isomer.
Noncommercially available acylhydrazides 11 and sulpho-
nylhydrazides 13 (Scheme 2) were generally obtained following
a published procedure from corresponding carboxylic acids 9
by transformation into the methyl ester 10 and treatment with
hydrazine monohydrate;
For the HTS screening campaign we selected 24,000 small
organic molecules from the diverse Siena Biotech compound
collection. Among the most promising hit compounds, a small
set of molecules containing a fused 3-hydroxy-3-trifluorome-
thylpyrazole moiety, initially consisting of 4 compounds and
exemplified by compound 4a, displayed an activity range
between 5.9 and 18 μM with fold increase (FI) values between
30% and 50% as a measure of efficiency of the compound to
restore the CREB-mediated transcriptional activity in cells
expressing mutant Htt. A set of nonfused analogues represented
by compound 5 proved inactive in the screening when tested
up to 50 μM, showing the selectivity of this specific chemotype
only when fused to a cyclic ring.
17
A major concern of this series was the presence of the
geminal 3-hydroxy-3-trifluoromethyl functionality and its
stability to dehydration. Indeed, it is reported in the literature
that 2-aryl or 2-alkyl substituted 3-hydroxy-3-trifluoromethyl
hexahydroindazoles undergo dehydration in acidic conditions
to afford the corresponding 3-trifluoromethyl tetrahydroinda-
18,19
sulphonylhydrazides 13 were
obtained from corresponding sulphonyl chlorides 12 by
treatment with hydrazine monohydrate.
Information for details.
The stereospecificity of the reaction was confirmed by X-ray
diffraction data obtained for compound 4f (see Supporting
Information for full data and 3D representations). The data
2
0,21
See Supporting
16
zoles derivatives. After retest from a new batch and a
preliminary stability test conducted at pH = 7.4 and pH = 3, the
2-acyl and 2-sulphonyl hexahydroindazoles confirmed activity
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Table 1. CRE-LUC Activity and ADME Properties of Fused 3-Hydroxy-3-trifluoromethylpyrazole Derivatives
a
Cellular assay. EC50 values represent arithmetic means of n reported determinations. These assay generally produced results within 2-fold of the
b
reported mean. Compound 1 (Y-27632) was used as a standard and positive control for the assay. Fold increase is considered as the measure of the
difference of maximum efficacy calculated from the concentration response curve fit of the normalized CRE-LUC activity of the mutant Htt
c
−6
d
expressing cells (Emax‑ind) and of the noninduced cells (E
permeability assay (B:A/A:B). Kinetic solubility.
). Permeability assay A:B (10 cm/s). Efflux ratio measured in MDCK-MDR1
max‑nonind
e
confirmed the presence of a racemate having the −H and −OH
potency, with compounds 4j−4l showing double digit EC s
5
0
substituents, respectively, on the C1 and C2 chiral centers trans
and fold-increase values below 30%.
22
to each other as a mixture of (R,S) and (S,R). No cis
diastereomers analogues were consistently detected. Carbocy-
clic ring expansion to a 7-membered ring did not improve
Furthermore, cycloheptane derivatives showed increased
metabolism rates, such as for compound 4j, mainly due to
carbocyclic chain hydroxylation (data not shown). No
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Scheme 2. General Synthetic Route for Acylhydrazide and
a
Sulphonylhydrazide Derivatives
Figure 3. (A) Effects of 4f treatment on LV-Htt (wild-type htt171−
1
8Q and mutant htt171−82Q) infected striatal neurons. *p < 0.05
Student’s t test vs 82Q Ctrl. (B) Protection against cell death in Doxy-
induced Exon 1-mutHTT expressing cells (Doxy). *p < 0.05 vs Doxy-
induced Ctrl group (Two-Way ANOVA, Tukey’s posthoc test). Data
are expressed as mean and SEM values.
a
Reagents and conditions: (a) thionyl chloride, MeOH, 0 °C; (b)
hydrazine monohydrate, 50 °C; (c) hydrazine monohydrate, THF, 0
°
C.
Following the achievement of a proof of activity in a full-
length, an Exon-1-based and an N171-based HD cell model, it
was decided to progress investigation of the series in an in vivo
model of HD. Selection criteria were based on compounds with
the best achievable systemic exposure in the animal. Selected
compounds were progressed to PK experiments (see Table S1
in Supporting Information) to determine whether a suitable
candidate for animal model testing (R6/2 mice) could be
identified. All analyzed compounds showed very short half-lives
with no remaining compound detected at 24 h after
administration. The Sulphone derivative 8b showed a high
clearance in mouse and very low levels after PO administration,
while the carbonyl derivatives 4f and 4c showed medium to
optimum bioavailability values (see Table S1 in Supporting
Information). Compound 4f was selected as a lead compound
after a dose escalating PK and an MTD experiment in which it
showed suitable systemic exposure and tolerability properties in
mouse (data not shown) making it compatible with a chronic
administration protocol. Additional studies on 4f determined a
B/P ratio of 0.34, no hERG activity detected up to 100 μM
concentration, negative mini-Ames test, and no CYP inhibition
(3A4 TST > 30 μM; 3A4 NIF > 30 μM; 1A2 > 30 μM; 2D6 >
30 μM; 2C9 > 30 μM; 2C19 = 24.4 μM).
R6/2 mice were treated with 4f at 3, 10, and 30 mg/kg, PO,
QD at Psychogenics (Tarrytown, NY), starting from 4 weeks of
age. Effects of compound treatment were assessed with a motor
test battery comprising open field, grip strength, and rotarod.
Drug effects were also evaluated on BW and followed-up with
survival assessment. Compound 4f showed a significant effect
on the clinically relevant parameter of BW, counteracting the
characteristic decrease in BW at doses of 10 and 30 mg/kg (see
Figure S3 in Supporting Information). No significant effects of
treatment were detected on motor tests or survival.
improvements were detected with the insertion of a pyrane
ring, with compound 4n showing comparable cellular potency
to initial hit 4a. The best results in terms of potency and
efficiency were obtained with acyl derivatives 4f and sulphonyl
derivative 8b exibiting a 6-membered carbocyclic ring and a 5-
membered heterocycle in R1 position as the most favorable
combination with both linkers. Our phenotypic assay approach
has thus lead to the selection of a subseries of molecules that
showed FI and EC values feasible for further evaluation in the
5
0
primary rat striatal neuron secondary assay.
Compounds 4b, 4c, 4f, and 8b were tested at EPFL in
Lausanne at 1, 10, and 30 μM. In this primary rat striatal
neuron-based model, Htt171−18Q or Htt171−82Q expression
is driven by a tetracycline responsive element (TRE) promoter
(
TRE-htt171−18Q/82Q) in a lentiviral expression vector,
producing a rapid polyQ-dependent pathology that leads to
formation of intracellular Htt inclusions starting at 1−2 weeks
and decreased expression of the neuronal marker NeuN (a
readout of viability) as early as 2 weeks after infection. All four
compounds were shown to significantly improve NeuN counts
relative to vehicle (DMSO-treated) counterparts at 10 and 30
μM concentrations (see Figure S1 in Supporting Information).
Figure 3A shows the effect on survival of compound 4f as
representative of the series. The effect was not completely
specific, with an increased NeuN count also detectable in the
htt171−18Q expressing cells. Our deduction was that the
potential target(s) of this series of compounds is involved in a
general cell viability pathway. To further build upon this result,
the cytoprotection of compound 4f was tested in a PC12 cell
model in which Exon-1 mut-Htt expression is under control of
a tetracycline (Tet-on) system. Again, the compound was able
to protect from doxycycline (Doxy)-induced cell death by
reducing LDH release, a readout of mortality (Figure 3B). In
this model no effects of the compound were evident in
uninduced cells.
More generally, all compounds examined did not show any
toxicity on normal cells (or neurons) even for a long period of
treatment (results not shown). Potential off-target effects of the
series were evaluated in an early representative, compound 4b,
which was tested in a CEREP panel. The CEREP express
screening was performed on 71 receptors and 62 enzymes in
which 4b was profiled at 20 μM concentration. Compound 4b
showed no significant activity on any target (full list and result
values available in Supporting Information) providing us with
enough information and confidence to consider the series
potentially free from most common off-target effect and ready
to enter into animal studies.
23
A small satellite group of animals was sacrificed at 12 weeks
of age (after 8 weeks of treatment) to perform brain
morphological studies. Effects of 4f was assessed on the
neuropathological hallmarks of brain atrophy and ventricular
enlargement, using both ex vivo MRI and histological measures.
The quantification of EM48-positive nuclear Htt aggregates
were also analyzed by immuno-histochemistry. Results of
morphological studies (Figure 4) showed a severe enlargement
of cerebral ventricular in R6/2 animals as compared to the wild-
type strain (C57Bl6). Furthermore, compound 4f showed a
trend toward decreasing the size of the cerebral ventricles.
These results were also confirmed by ex vivo MRI performed in
fixed brain (see Figure S3 in Supporting Information).
In conclusion, we have identified a class of compounds active
in three different HD cell assays comprising different
phenotypes, readouts, and promoters of toxicity induced by
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ACKNOWLEDGMENTS
■
We thank Dr. Francesco Berrettini (Universita
̀
degli Studi di
Siena) for the X-ray diffraction data collection and analysis; Dr.
Carol Murphy and Dr. Sylvie Ramboz at Psychogenics,
Tarrytown, NY, for in life phase of R6/2 experiments.
ABBREVIATIONS
■
Htt, Huntingtin; CRE, cAMP response element; BW, body
weight; HTS, high throughput screening; LDH, lactate
dehydrogenase; B/P, brain/plasma
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