Discovery, synthesis, and biological evaluation of novel SMN protein modulators

Article abstract of DOI:10.1021/jm200497t

Spinal muscular atrophy (SMA) is an autosomal recessive disorder affecting the expression or function of survival motor neuron protein (SMN) due to the homozygous deletion or rare point mutations in the survival motor neuron gene 1 (SMN1). The human genome includes a second nearly identical gene called SMN2 that is retained in SMA. SMN2 transcripts undergo alternative splicing with reduced levels of SMN. Up-regulation of SMN2 expression, modification of its splicing, or inhibition of proteolysis of the truncated protein derived from SMN2 have been discussed as potential therapeutic strategies for SMA. In this manuscript, we detail the discovery of a series of arylpiperidines as novel modulators of SMN protein. Systematic hit-to-lead efforts significantly improved potency and efficacy of the series in the primary and orthogonal assays. Structure-property relationships including microsomal stability, cell permeability, and in vivo pharmacokinetics (PK) studies were also investigated. We anticipate that a lead candidate chosen from this series may serve as a useful probe for exploring the therapeutic benefits of SMN protein up-regulation in SMA animal models and a starting point for clinical development.

Full text of DOI:10.1021/jm200497t

ARTICLE
pubs.acs.org/jmc
Discovery, Synthesis, and Biological Evaluation of Novel SMN Protein
Modulators
Jingbo Xiao,*,† Juan J. Marugan,*,† Wei Zheng,† Steve Titus,† Noel Southall,† Jonathan J. Cherry,‡,§
Matthew Evans,‡ Elliot J. Androphy,‡,§ and Christopher P. Austin†
†NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center
Drive, Rockville, Maryland 20850, United States
‡Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, LRB 328, Worcester,
Massachusetts 01605, United States
S Supporting Information
b
ABSTRACT: Spinal muscular atrophy (SMA) is an autosomal
recessive disorder affecting the expression or function of
survival motor neuron protein (SMN) due to the homozygous
deletion or rare point mutations in the survival motor neuron
gene 1 (SMN1). The human genome includes a second nearly
identical gene called SMN2 that is retained in SMA. SMN2
transcripts undergo alternative splicing with reduced levels of
SMN. Up-regulation of SMN2 expression, modification of its
splicing, or inhibition of proteolysis of the truncated protein
derived from SMN2 have been discussed as potential therapeu-
tic strategies for SMA. In this manuscript, we detail the
discovery of a series of arylpiperidines as novel modulators of
SMN protein. Systematic hit-to-lead efforts significantly im-
proved potency and efficacy of the series in the primary and orthogonal assays. Structureꢀproperty relationships including
microsomal stability, cell permeability, and in vivo pharmacokinetics (PK) studies were also investigated. We anticipate that a lead
candidate chosen from this series may serve as a useful probe for exploring the therapeutic benefits of SMN protein up-regulation in
SMA animal models and a starting point for clinical development.
’ INTRODUCTION
correlation between SMA severity and the amount of SMN
protein have shown an inverse relationship.5 Humans possess an
additional paralogous gene, survival motor neuron 2 (SMN2), a
centromeric copy gene that differs from the SMN1 gene at a
critical nucleotide at position six in exon 7 (C-T).6 While full
length 38 kDa SMN protein can be produced from the transcrip-
tion of both SMN1 and SMN2 genes, the small variation in the
CꢀT nucleotide has important consequences for alternative
splicing of the SMN2 transcript. The pre-mRNA transcripts from
SMN1 gene produce full-length mRNA with nine exons encod-
ing full-length of SMN protein, while the C- to T- transition at
position six in exon 7 of SMN2 induces mostly alternative pre-
mRNA splicing lacking exon 7, which results in the truncated
SMN protein (termed as SMNΔ7) as its major product. SMNΔ7
protein has a reduced ability to self-oligomerize, leading to
protein instability and rapid degradation.7 SMN2 has been
genetically validated as a target for SMA therapy, and there is a
striking correlation between SMA type and SMN2 gene copy
numbers.8 From the functional point of view, SMN associates
Spinal muscular atrophy (SMA), an inherited autosomal
neurodegenerative disease, is the leading genetic disorder affect-
ing infant mortality.1 SMA is a relatively “common” rare disease,
affecting approximately 1 in 6000 newborns. Approximately 1 in
40 individuals are genetic carriers.2 Clinically, there are four types
of SMA (types I, II, III, and IV). The determination of the type
of SMA is based upon the physical milestones achieved. Usu-
ally, children with the most severe form of the disease (type 1;
WerdnigꢀHoffmann disease) die before the age of two years in
the absence of supportive respiratory care.3 In fact, SMA is the
number one genetic cause of death in children under the age of
two, and many of those children who make it through infancy are
confined to wheelchairs for their entire lives. There is currently
no cure or effective therapy for SMA.
SMA is caused by deficiency in survival motor neuron (SMN)
protein due to homozygous mutations or deletion of the survival
motor neuron 1 telomeric gene (SMN1) that is located on
chromosome 5q13.4 The SMN protein is expressed in all tissues,
although there are broad variations regarding levels. Deficiencies
in SMN lead to degeneration and dysfunction of alpha motor
neurons in the anterior horn of the spinal cord. Studies of the
Received: April 22, 2011
Published: August 05, 2011
r
2011 American Chemical Society
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with proteins Gemin 2, Gemin 3, and Gemin 4, forming a large
complex that plays a role in snRNP assembly, pre-mRNA splic-
ing, and transcription.9
its strong safety profile, high pediatric bioavailability, and its
known gene up-regulation.16 Other bioactive agents,17 such as
valproic acid,18 aclarubicin (aclacinomycin A),19 tobramycin and
amikacin,20 Trichostatin A (TSA),21 suberoylanilide hydroxamic
acid (SAHA),22 EIPA [5-(N-ethyl-N-isopropyl)-amiloride],23
LBH589,24 and (E)-Resveratrol,25 have been shown to increase
levels of exon 7-containing SMN transcript and/or overall SMN
protein. All of these repurposed agents have significant liabilities
that include gastrointestinal bleeding, short half-life in human serum,
high toxicity, and lack of target specificity; additionally, valproic acid,
PBA, and hydroxyurea were not therapeutic in human trials. Lunn
et al. identified indoprofen as having an effect on full length
SMN2 expression in a cell-based reporter assay that up-regulated
the SMN protein through a cyclooxygenase-independent me-
chanism, but it was not potent (AC50 >1 μM) and increased
protein expression by only 13% (p-value <0.0139).26 Recently,
a tetracycline compound, PTK-SMA1, was found to have the
activity to promote SMN2 exon 7 splicing. However, this com-
pound could not penetrate the bloodꢀbrain barrier or increase
SMN protein concentration in the central nervous system.27 An-
other previous high-throughput screen identified two novel
chemical series as HDAC inhibitors that can modulate reporter
activity in a cell-based SMN2 promoter assay.28 Another quinazo-
line series probe, identified as D156844, targets DcpS, which
appears to modulate SMN protein levels by stabilizing the SMN2
mRNA transcript.29 Oral administration of D156844 significantly
increased the mean lifespan of SMNΔ7 SMA mice by approxi-
mately 21ꢀ30% when given prior to motor neuron loss.30 After
removal of the compound’s off-target activity, reducing its toxicity
and improving its potency and brain permeability, it produced the
current clinical candidate, D157495.31 However, it has not been
shown whether this series of molecules acts specifically on SMN2
mRNA transcript stability or what the safety profile of this
molecule might be.32
There are several SMA therapeutic strategies under investi-
gation, including gene therapy,10 antisense oligonucleotides
(ATO),11 and stem cells.12 However, increasing SMN protein
levels by altering expression of the SMN2 mRNA by small
molecules has been proposed as a promising potential therapeu-
tic strategy for SMA. Over the past several years, several classes of
small molecules have been identified that increase SMN tran-
script and/or protein levels in SMA patient-derived cell lines
through a variety of mechanisms.13 Chang et al. reported that
sodium butyrate effectively increased the amount of exon 7-
containing SMN protein in SMA lymphoid cell lines by changing
the alternative splicing pattern of exon 7 in the SMN2 gene. In
vivo, sodium butyrate treatment of SMA-like mice resulted in
increased expression of SMN protein in motor neurons of the
spinal cord and resulted in significant improvement of SMA
clinical symptoms.14 Sodium 4-phenylbutyrate (PBA), an ana-
logue of sodium butyrate, was also found to increase SMN
expression in vitro. PBA has a much longer half-life time (1.5
h) in human serum compared with sodium butyrate (6 min),
which suggested that PBA, also due to its favorable pharmaco-
logical properties, could be a candidate for the treatment of
SMA.15 The FDA approved antineoplastic drug hydroxyurea
was recently reported as a candidate SMA therapeutic due to
We are particularly interested in identifying small molecules
that can increase the level of SMN transcript harboring the full
length of SMN protein for the therapeutic goal. This may be
achieved by modulating splicing, increasing the level of transcript
Figure 1. SMN2-luciferase reporter assay.
Figure 2. Overview of screening and hits selection process.
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Figure 4. SAR strategy.
protein. The NIH Molecular Libraries Small Molecule Reposi-
tory (MLSMR),35 containing 210386 compounds, was screened
in the primary assay during the course of this screen campaign
(Figure 2). We tested 1229 1536-well plates in the primary
screen. The signal to background averaged 9-fold, and the
average Z0 for the screen was 0.47 ( 0.12, excluding 8 plates
which failed visual quality control. 6128 compounds were found
to give significant concentrationꢀresponses in the primary
screen. Compounds were deprioritized for confirmation if they
were suspected to interfere with assay readout or cellular viability
using internal SAR data from other related assays of the MLPCN
using the same chemical library.36 Recently, it has been brought
to our attention that certain classes of luciferase inhibitors can
stabilize the luciferase protein and therefore appear as com-
pounds that are able to increase the signal.37 Compounds that act
as luciferase inhibitors could score in the SMN2 assay as false
positives due to this phenomenon. Indeed, more than 98% of hits
in the primary screen were identified as luciferase inhibitors,
which presumably stabilized the SMN2-luciferase fusion protein
and prevented cellular degradation, although this has not been
explicitly proven. Select samples active in the primary screen
were obtained in DMSO stock solutions from the MLSMR and/
or as powders from compound vendors to confirm activity in the
original assay. A homologous cell line harboring the SMN1 gene
fused to luciferase was used as a counterscreen. The SMN1
reporter recapitulates the splicing pattern typically seen with the
endogenous SMN1 gene and predominately includes exon 7.
Follow-up compounds that exhibit increased signal in the SMN1
cell line were flagged as nonspecific activators (i.e., transcription
or protein stability). Purified luciferase enzyme was used in
another counterscreen of the active compounds to filter out
the potential false positives. A complete cheminformatics analysis
of the follow-up screening data included chemotype clustering,
singleton identification, and structural considerations, including
physical properties and optimization potential. In addition, potency
range, maximum response, and the curve class38 were also con-
sidered. Ultimately, this analysis led to selection of 21 candidates for
further confirmation. The results of this qHTS screening campaign
are detailed in Figure 2.
Figure 3. (A) Chemical structures of 1 and 2 from qHTS. (B) Activity
of compounds 1 and 2 in SMN1 (cyan) and SMN2 (blue) reporter
assays, indicating the rate of SMN induction (ROI) at different
concentrations, and luciferase inhibition assay (black). (C) Quantifica-
tion by Western blot of SMN levels after treatment with compounds
1 and 2 at different concentrations, as indicated.
produced, or stabilizing the SMN protein. To achieve this goal of
looking for molecules able to elevate SMN levels by any of those
mechanisms, we developed a new high-throughput screening
methodology that allows us to test a large number of compounds
in a quantitative manner. In this manuscript, we present some of
the results of our screening and optimization campaign.
’ RESULTS AND DISCUSSION
qHTS Results. Using a luciferase reporter gene assay that
combines the promoter and splicing based cassettes in tandem
with the major portion of the native SMN2 cDNA with luciferase,
we were able to screen for modulators of all of these mechanisms
in a high-throughput manner. Earlier screening assays for SMA
only targeted compounds that either stimulate the SMN2 pro-
moter or decrease exon 7 skipping. In this reporter assay, the
SMN2 promoter drives expression of luciferase reporter that is
fused to an SMN splicing reporter (Figure 1). By including our
SMN splicing cassette,33 luciferase is only expressed when exon 7
is included in the mRNA transcript and the full-length SMN
protein is generated. This new assay can identify compounds that
increase SMN protein levels by three mechanisms: modulating
alternative splicing of SMN2 exon 7, increasing transcription
from the SMN2 promoter or, due to the presence of the native
SMN2 cDNA, and stabilizing the SMN protein. The assay
measures the luminescent signal produced by luciferase, and
compounds that increase luminescence are flagged as potential
actives.
Ideally, compounds modulating SMN protein production in
the SMN2-luciferase reporter line would also modulate SMN
protein production in primary fibroblasts directly derived from
SMA individuals. Therefore, 21 compounds representing the
diverse set of chemotypes39 were tested in a SMA-derived patient
fibroblast assay for up-regulation of SMN protein production by
Western blot in a luciferase-free setting. Ultimately, this
campaign led us to focus on two lead series represented by
the 5-methoxy-3,6-diphenyl-1,2,4-triazine (1) and 1-(4-(4-bromo-
phenyl)thiazol-2-yl)piperidine-4-carboxamide (2), which were able
to increase the luciferase signal in the SMN2 reported assay and
increase SMN protein production in a dose dependent manner in the
We performed quantitative high-throughput screen (qHTS)34
using this cell-based SMN2-luciferase reporter assay to identify
compounds that increase expression of the full-length SMN
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Scheme 1. Synthesis of the Hit Compound 2 and 4-Arylthiazolyl Piperidine Analoguesa
a Conditions and reagents: (i) triethylamine, μW, 100 °C; (ii) arylboronic acids or pinacol esters, 2.0 N Na2CO3, 5% Pd(PPh3)4, μW, 100 °C; (iii) (a)
4-bromophenylboronic acid, 2.0 N Na2CO3, 5% Pd(PPh3)4, μW, 100 °C, (b) MCPBA; (iv) triethylamine, μW, 100 °C.
Scheme 2. Modifications at the Thiazole Core Templatea
a Conditions and reagents: (i) piperidine-4-carboxamide, triethylamine, μW, 100 or 120 °C; (ii) arylboronic acids or pinacol esters, 2.0 N Na2CO3, 5%
Pd(PPh3)4, μW, 100 °C; (iii) piperidine-4-carboxamide, N,N-diisopropylethylamine, 0 °C.
Western blot (Figure 3). In addition, although we do not know their
specific mechanism of action, we noticed that two active compounds
1 and 2 appear to have distinct mechanisms of action. Compound 1
increased the expression of SMN2 and did not affect the expression of
SMN1. On other hand, compound 2 increased the expression of
SMN2 but reduced the expression of SMN1. This reduction of SMN1
expression would not be relevant in vivo due to the fact that the
SMN1 gene is not functional at all in SMA patients. The data in
Figure 3 also shows the differences in luciferase inhibitory capacity of
1 and 2. It can be seen that compound 1 was able to both increase the
production of SMN protein (western) and inhibit luciferase. So
without the creation of another cell line with a different reporter
format, such as β-lactamase, GFP, or Renilla luciferase, it would be
impossible to discriminate between SNM up-regulation and luciferase
stabilization effects using our firefly luciferase reporter assay. In
addition, the first triazine chemotype was found to have a very narrow
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Scheme 3. Synthesis of the Piperidine Modification Analoguesa
a Conditions and reagents: (i) triethylamine, μW; (ii) arylboronic acids or pinacol esters, 2.0 N Na2CO3, 5% Pd(PPh3)4, μW, 100 °C; (iii) LiOH, THF,
H2O; (iv) EDC, DMAP, μW, 100 °C; (v) KOCN, H2O, or R5COCl, triethylamine, or R6SO2Cl, triethylamine; (vi) trifluoroacetic acid; (vii) acetyl
chloride, triethylamine; (viii) NaN3, ZnBr2, NaOH, dioxane, H2O, 120 °C.
SAR. Therefore, we chose compound 2 as the prioritized series for
further SAR optimization and biological evaluation because it pos-
sessed a more tractable SAR and drug-like properties. Furthermore, as
reported in later section, this series displayed high cell permeability in
Caco-2 cell permeability assay40 and it had with no indication of being
a glycoprotein (P-gp) substrate.41
Chemistry. Figure 4 shows the initial SAR strategy designed
for the study of compound 2, including evaluation of the nature
and position of the aromatic substituents, the geometry and
substituents at the thiazole core, as well as the modification of the
piperidine functional group.
In a similar reaction fashion, if the starting material, 2,4-
dibromothiazole is replaced by 2,5-dibromothiazole,44 2,5-
dibromothiadiazole,45 5-bromo-2-chloropyrimidine,46 2,4-
dichloropyrimidine,47 4,6-dichloropyrimidine,48 or 2,4-dichloro-
1,3,5-triazine,49 the corresponding modifications at the thiazole
core template can be accomplished as detailed in Scheme 2. It
was also interesting to note that the replacement of thiazole with
imidazole or N-methylimidazole was unsuccessful under the
same reaction conditions. All aminated building blocks under-
went the same type of Suzuki coupling procedure to give the
desired final analogues.
Similar procedures were utilized to further explore modifica-
tions of the piperidine-4-carboxamide moiety (Scheme 3). In the
process of typical aminationꢀSuzuki steps, route (a), piperidine-
4-carboxamide can be successfully switched to ethyl piperidine-
4-carboxylate, piperazine, tert-butyl piperidin-4-ylcarbamate,
piperidine-4-carbonitrile, 1-(piperidin-4-yl)ethanone as well as
4-(1H-imidazol-2-yl)piperidine, but not piperazine-1-carboxa-
mide. Within our SAR studies, we were interested in examining
several related substituted amide derivatives. Saponification of
the ethyl ester afforded the appropriate acid, which can be readily
coupled with amines to provide the final substituted amide
modification analogues. We were also interested in replacing
the piperidine with N-substituted piperazines. To access these
derivatives, the free secondary amine of the piperazine ring can be
easily converted into corresponding urea, amides, and sulfony-
lamides. Finally, a reverse amide bond analogue and a tetrazole
analogue were successfully synthesized, as detailed in Scheme 3.
Most of the final compounds were purified by preparative scale
HPLC with reasonable yields.
We developed convergent synthetic routes toward the original
hit compound 2 and its 4-arylthiazolyl piperidine analogues
(Scheme 1). Route (a) was designed to prepare analogues with
the modification of the phenyl ring. Starting with 2,4-dibro-
mothiazole, selective amination occurred at the 2-position of the
thiazole ring to give the key building block 1-(4-bromothiazol-
2-yl)piperidine-4-carboxamide.42 Then, a mixture of an arylboro-
nic acid or its corresponding pinacol ester and 1-(4-bromothia-
zol-2-yl)piperidine-4-carboxamide was irradiated under micro-
waves to induce a Suzuki type of cross-coupling reaction43 us-
ing tetrakis(triphenylphosphine)palladium(0) as a catalyst to
furnish the final 4-arylthiazolyl piperidine analogues. Alterna-
tively, the route (b), a Suzuki coupling at the 4-position of the
2-methylthio-4-bromothiazole, followed by MCPBA oxidation
of the methylthioyl group to the methylsulfonyl moiety, provided
another building block 4-aryl-2-(methylsulfonyl)thiazole. How-
ever, the displacement of the methylsulfonyl group of the 4-aryl-
2-(methylsulfonyl)thiazole with a secondary amine proved to be
very difficult. Thus, all of the analogues described in this article
were prepared through the aminationꢀSuzuki coupling ap-
proach, route (a). This synthetic route was straightforward and
provided for rapid SAR studies.
SAR Discussion. Tables 1ꢀ7 disclose all of the analogues
that were generated for this series to derive a SAR. The activity
was reported showing two parameters: AC50 (concentration
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Table 1. SAR of 2,4-Substituted Thiazole Analogues Containing an Aromatic Substituent
necessary to reach 50% of maximum luciferase signal) and rate of
induction (ROI) (efficacy indicated as maximum percentage of
luciferase signal with respect to basal levels), which were used to
conduct SAR analysis. Both parameters, ROI and AC50, were
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Table 2. SAR of 2,4-Substituted Thiazole Analogues Containing a Phenoxy Substituent
evaluated and optimized, as the ideal compound should provide
maximum SMN protein production with minimal concentration
of a given compound.
ROI = 357%), meta-N,N-dimethylamino substituted analogue 3f
(AC50 = 0.97 μM, ROI = 504%), and para-methyl substituted
analogue 3m (AC50 = 1.22 μM, ROI = 439%). Regarding
heteroaromatic ring substituents, none of analogues 4sꢀ4u
had a positive impact on either parameter compared to the
parent compound 2. We also studied several polycyclic aromatic
ring analogues (4e, 4f, and 4vꢀ4x). However, most had either no
activity or decreased activity and efficacy.
Given the improved potency and efficacy observed with the
electron-donating substituents, we chose to screen a large
number of phenoxy substitutents (Table 2). Several additional
electron-donating substituents were tolerated including meta-
isopropoxyphenyl (5c, AC50 = 0.77 μM, ROI = 605%). We also
examined the 3,5- or 3,4-disubstituted analogues (5h and 5i,
AC50 = 7.72 and 12.24 μM, ROI = 45% and 59%, respectively)
and 3,4,5-trisubstituted derivative (5m, AC50 = 19.4 μM, ROI =
73%). Surprisingly, these modifications resulted in a huge loss of
both activity and efficacy. Interestingly, this loss could be
regained by bridging two substituents together. Furthermore,
the resulting cyclic poly phenolic-ethers demonstrated a clear
trend of improvement in activity in line with ring size (5 < 6 < 7,
5j, 5k, 5l, AC50 = 2.44, 0.77, and 0.49 μM, respectively).
The hit compound 2 identified from the primary screen was
resynthesized and found to possess an AC50 value of 1.22 μM
with a ROI of 268%. Our early efforts were focused on improving
both parameters in order to further explore the SAR for this
chemotype. To conduct a comprehensive SAR (Table 1) for the
aromatic group at the 4-position of the thiazole ring while
exploring the steric and electronic effects of the substituents,
the thiazole and piperidine rings were held constant, as shown in
Table 1. We first examined the effect of the substituent position
at the phenyl ring on potency (AC50) and efficacy (ROI). With
the exception of the ortho-fluoro substituted analogue 3r (AC50
=
2.44 μM, ROI = 274%), all other ortho-substituents were either
inactive (3b, 3e, 3o, 4a, 4c, 4e, 4g, 4m, and 4p) or were signi-
ficant less active (3u and 4x) versus compound 2. These data
indicated the potential necessity of having a coplanar conforma-
tion between the two aromatic rings to maintain good activity. In
contrast to ortho-substitutions, substituents at meta or para
positions were generally well tolerated. Regarding the electronic
nature of the substituents, an electron-donating functional group
at the meta or para position tended to provide better activity and
efficacy than the corresponding electron-withdrawing group (3c,
3f vs 4h, 4n, 4q and 3d vs 4i, 4o, 4r). In addition, halo, methyl, or
trifluoromethyl substitutions at para and meta position were also
tolerated. Several substituents slightly increased or maintained
the activity compared to the efficacy of the hit compound 2, such
as the meta-methoxy substituted analogue 3c (AC50 = 1.94 μM,
With several improved analogues in hand (analogues 5c, 5k,
and 5l), we further studied the SAR of the piperidine ring moiety
as shown in Table 3. In general, mono- or disubstituted amide
derivatives 6aꢀ6i had worse potency and efficacy, with the
exception of the phenylamide derivative (6e, AC50 = 2.44 μM,
ROI = 262%), which was about the same as the hit compound 2.
It is also important to remark that both the carboxylic acid 6k and
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Table 3. SAR of 2,4-Substituted Thiazole Analogues with Modifications of the Primary Amide Moiety and Piperidine Ring
ARTICLE
the corresponding ethyl ester 6j derivatives were about 4- and
6-fold less active, respectively, compared to compound 2, while
both showed improved efficacy (6k, AC50 = 4.87 μM, ROI =
554%, and 6j, AC50 = 7.72 μM, ROI = 550%, respectively). This
result indicated that only the carbonyl functional group might be
involved in a potential hydrogen bond interaction. This was
further supported by significant loss of potency of the noncar-
bonyl bearing analogues 6qꢀ6s. When the amide moiety was
replaced by cyano or tetrazole groups, the resulting derivatives
(analogues 6l and 6m) also showed a loss of potency. Re-
verse amide 6o and carbamate 6p analogues were not tolerated
either. Surprisingly, a free amine moiety seemed to be tolerated
(6n, AC50 = 2.44 μM, ROI = 182%). In contrast to the modifica-
tion of the amide moiety, the piperidine ring was found to be
necessary tomaintain potency. Interestingly, a piperazineanalogue
6t, which represents another retro-amide variation, displayed
reasonable good activity. In contrast, all of the other piperazine
derivatives, 6uꢀ6y, were essentially inactive.
substituent between the two heteroaromatic nitrogen atoms and
aromatic substituent at the 4-position increased the potency of the
molecule by 2ꢀ3-fold (7d, AC50 = 0.387 μM, ROI = 387%, and 7i,
AC50 = 0.772 μM, ROI = 446%). The aromatic substituent at the 5
position of the pyrimidine was also tolerated as represented by
analogue 7b (AC50 = 0.613 μM, ROI = 236%), although its efficacy
was only half in comparison with the above-described pyrimidine
analogues. The meta-isopropoxyphenyl substituted derivative 7g
was found to be the best analogue in this series, with about 9-fold
increase in potency (7g, AC50 = 0.154 μM, ROI = 298%) versus the
hit compound 2. Derivatives bearing a monofluoro substituent at 5
position of the pyrimidine gave an additional 2ꢀ4-fold improvement
in potency (7j vs 7i and 7l vs 7n). Replacement of the pyrimidine for
1,3,5-triazine resulted in analogues with decreased potency (7i vs 7k
and 7h vs 7p). These results indicated that the potency of these
compounds would not be improved by the introduction of additional
nitrogens in the pyrimidine core ring. An ortho-methyl substituted
pyrimidine derivative 7o was found to be totally inactive, which
further supported an earlier observation for requirement of a coplanar
conformation for this portion of the molecule for activity. On the
other hand, when the pyrimidine substitution patterns were reversed,
In general, replacement of the 2,4-disubstituted thiazole ring
with a 2,4-disubstituted pyrimidine maintained or improved potency
and efficacy (Table 4). Placement of the piperidine-4-carboxamide
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Table 4. SAR of Analogues with Modifications of Thiazole Core
with the aromatic substituent between the two heteroaromatic
nitrogen atoms and the piperidine-4-carboxamide substituent at the
position 4, the resulting derivatives decreased activity by 8ꢀ16-fold
(7i vs 7n and 7j vs 7l). In addition, moving two nitrogen atoms
around the pyrimidine also decreased the potency by 5-fold (7i vs 7m
and 7h vs 7q). These findings lead to the hypothesis that the sterically
accessible nitrogen atom adjacent to the piperidine group is involved
in a hydrogen bond interaction, serving as a hydrogen bond acceptor.
Table 5 shows modifications for the thiazole portion of the
molecule. Switching the aromatic substituent from the 4 position
to 5 position of the thiazole core ring increased the potency by
5-fold and efficacy by 2-fold (8g, AC50 = 0.224 μM, ROI =
599%). We speculated that this increment was due to accessi-
bility of the thiazole nitrogen for involvement in hydrogen bond
interactions with the putative molecular target. Replacement of a
CH with a N atom in the thiazole core resulting in 1,3,4-
thiadiazole led to a 3ꢀ10-fold loss of potency (8a vs 8b; 8c vs
8d; 8e vs 8f; and 8g vs 8h). These results are analogous to
replacement of the pyrimidine with the triazine and are due to the
electronic effect of the core ring.
With the rationale that the SAR of the aromatic substituents
from the 2,4-substituted thiazole could be translated to a 2,5-
substituted thiazole series, the SAR of the different electron-
donating groups on the phenyl moiety was explored (Table 5 and
Table 6). Gratifyingly, several electron-donating groups on the
phenyl ring indeed produced a synergistic effects on both
potency and efficacy (8a, AC50 = 0.038 μM, ROI = 370%; 8e,
AC50 = 0.039 μM, ROI = 550%; 8l, AC50 = 0.077 μM, ROI =
709%; 8m, AC50 = 0.031 μM, ROI = 576%). Again, the 3,4-
disubstituted derivative 8n again was less active than the corre-
sponding ring closed analogues 8a and 8oꢀ8q. Several cyclic
meta-N,N-disubstituted aminophenyl derivatives, 8rꢀ8t, were
examined and were found to be less active than the parent meta-
N,N-dimethylaminophenyl derivative 8e.
The modification of the amide moiety in the 2,5-substituted
thiazole system was further explored, as shown in Table 7. For
these studies, 3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl or
meta-N,N-dimethylaminophenyl were chosen as the two aro-
matic substituents at the C5 position of thiazole core ring. The
results demonstrated that the replacement of amide with an acid
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Table 5. SAR of Analogues Having 2,5-Substituted Thiazole and Thiadiazole Cores
had a clear advantage for potency with a 3-fold improvement, but
with a slight loss of efficacy (9a with best AC50 = 0.012 μM,
ROI = 325%). Ethyl ester or phenylamide derivatives were also
Other selected agents (9c, 8c, and 8l) all showed an up-regulation of
SMN protein at concentrations ranging between 37 and 333 nM.
Furthermore, the increase in SMN protein production led us
to explore whether the arylpiperidine analogues had an effect on
the overall number of SMN positive foci or gems in the nucleus.
This would corroborate that the increased protein amounts
represent functional SMN protein. SMN protein is predomi-
nately a cytoplasmic protein, but in the nucleus, SMN localizes to
distinct punctuate bodies, often referred to as gems (gemini of
coiled [Cajal] bodies). There is a direct correlation between the
number of gems and total SMN protein production in the cell.50
Gem counts are commonly used as another metric to score the
amount of total SMN protein expressed on a cell-to-cell basis.
The number of nuclei with gems and the number of gems per cell
were both significantly reduced in type I SMA cells. Only 3.3% of
nuclei have gems in fibroblast cells from SMA type I patients (cell
line 3813), while 24.8% of nuclei have gems in fibroblast from a
carrier parent (3814 cell line). We treated human type I SMA
fibroblasts with increasing concentrations (37ꢀ3000 nM) of
arylpiperidine analogues 8a, 8m, and 9a for 3 days, and the
number of gems per 100 nuclei were examined (Figure 6).
Analogue 8m treatment yielded more than a 2-fold of increase
tolerated (9b, AC50 = 0.031 μM, ROI = 320%, and 9c, AC50
=
0.049 μM, ROI = 353%). However, the imidazole and methylk-
etone replacements were not tolerated (9d, 9e, 9f, and 9g).
Secondary Validation Assays. With the SAR assessment
established for this series, a number of selected analogues
featuring desirable potency below 150 nM in the reporter assay
were then examined to evaluate the effect on the human SMN
protein expression using fibroblasts from SMA patients. We
incubated a fibroblast cell line from a type I SMA patient
(3813 cell line) with different doses of analogues and assessed
the SMN protein level by quantitative Western blotting
(Figure 5). We observed that analogue 8m at 37 nM increased
the SMN protein level by 2ꢀ3-fold. The SMN protein level was
decreased with increasing drug concentration. This result
matched with the bell-shaped doseꢀresponse curve observed
in the SMN2-luciferase reporter assay. This could be due to
decreased compound solubility at higher concentrations or it may
reflect the intrinsic mechanism of action. Analogue 9a showed a
dose-dependent trend between 37 nM toand 1 μM concentration.
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Table 6. SAR of 2,5-Substituted Thiazole Analogues Containing an Aromatic Substituent
of the number of the gems at the low 37 nM dose. With
increasing concentration, the number of gems was reduced,
which agreed with the previous findings on our reporter and
westerns experiments. Analogue 9a also showed an 80% increase
of the gem numbers at 37ꢀ1000 nM concentrations and aslightly
decrease at 3000 nM. However, analogue 8a did not show any
activity in this assay.
Mechanism of Action. Compounds were initially character-
ized using RT-PCR to measure RNA expression levels and exon 7
inclusion within our reporter cell line (not endogenous SMN).
Some analogues (2, 3f, and 8e) showed a slight increase in total
SMN-luciferase transcripts, but there was almost no increase in
exon 7 mRNA (data not shown). Therefore, the mechanism of
action of our series is likely post-transcriptional, in contrast to the
HDAC and DcpS inhibitors previously identified. Perhaps this
mechanism includes increasing the stability of the SMN protein
and/or affecting its degradation, but it is distinct from D156844,
a deCode compound,29a which had no activity in the present screen-
ing assay. So far, no other SMN modulator has been described as
having this type of activity, and therefore it will be interesting for the
scientific community to have a better understanding of its proper-
ties, mechanism, and potential pharmaceutical application. The
SMNΔ7 protein, the primary product from SMN2, is functional
in the context of low levels of exon-7 included SMN protein but it is
proteolytically unstable. It will be interesting to explore whether the
present series has an impact on the stability of the SMNΔ7 derived
truncated SMN protein, thereby establishing a new paradigm for the
treatment of SMA.
ADME Properties and Pharmacokinetics. In addition, sev-
eral of the most potent analogues were profiled for mouse liver
microsomal stability and cell permeability in Caco-2 cells
(Table 8). For the microsomal stability tests, we decided to
evaluate the amount of parent compound remaining following
incubation at three different time periods: 10, 15, and 60 min.
This allowed us to both calculate both intrinsic clearance and
model half-lives. The original hit compound 2 was found to have
a relatively short half-life (12 min). Analogues 8c, 8m, and 9c all
had a very short half-lives <10 min. Analogues 8b, 8l, and 9a had
half-lives of more than 30 min. In the bidirectional Caco-2 cell
permeability assay, analogues 7g, 8b, and 9a were found to
exhibit high permeability, while analogues 8b and 9a had efflux
ratios of 2.4 and 1.3 respectively and analogue 8l had a promising
efflux ratio of 0.05. The cell permeability of analogue 8m was not
great, although the efflux ratio was acceptable (0.125).
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Table 7. SAR of 2,5-Substituted Thiazole Analogues with Modifications of the Primary Amide
In consideration of a balance of potency, efficacy, and
ADME properties of all current analogues, we selected analo-
gues 8l and 9a for full mouse in vivo pharmacokinetic (PK)
studies to evaluate the central nervous system (CNS) penetra-
tion of this series (Table 9). The plasma and brain concentra-
tion of analogues 8l and 9a in male Swiss Albino mice after a
single oral gavage administration at a dose of 30 mg/kg were
measured. As shown in Table 9, the brain to plasma ratio of
analogues 8l and 9a in male Swiss Albino mice was found to be
0.29 and 0.028, respectively. Both analogues 8l and 9a possessed
a reasonable long half-life in brain (t1/2 = 5.44 and 13.69 h) as
well as in plasma (t1/2 = 5.78 and 11.18 h). The concentration
of analogue 8l in brain reached at 1.28 μmol/kg (Cmax) in only
5 min, and a concentration above its AC50 of 0.077 μM
was maintained for more than 4 h. Despite its high plasma
protein binging (94.9%) for analogue 9a, its concentration in
brain reached 1.23 μmol/kg (Cmax) in 15 min and a concentra-
tion above AC50 concentration of 0.012 μM for over 24 h. In
addition, no behavioral disturbance was observed in animals
administered with both tested compounds throughout the whole
study period. Oral administration with good brain exposure will
be preferred for demonstrating in vivo activity, as many of the
current animal models require chronic administration of
the drug.
’ CONCLUSION
In summary, after several rounds of medicinal chemistry
SAR studies, we were able to improve the potency of the hit
compound by ∼100-fold from μM to low double-digit nM in
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Figure 5. Quantification by Western blot of SMN levels after treatment with drug compounds at different concentrations, as indicated.
Figure 6. Number of gems per 100 nuclei after treatment with drug compounds at different concentrations, as indicated.
our cell-based SMN2-luciferase reporter assay, we were also
able to increase the rate of SMN protein production 3ꢀ7-fold.
From this study, analogues 8a (AC50 = 0.038 μM, ROI =
tested in our primary reporter assay, as well as in relevant
orthogonal assays such as Western blot and gems assays
with patient fibroblasts. In consideration of all the aspects
including ADME properties, analogues 8l and 9a possessed
the best combination of potency, efficacy, mouse liver micro-
somal stability, and cell permeability of all the analogues
presented in this study. Moreover, analogues 8l and 9a showed
good oral absorption and CNS penetration upon oral gavage
administration at a reasonable dose of 30 mg/kg, provid-
ing levels of exposure above their AC50 for more than 4 h
in brain with no sign of toxicity or behavior disturbance in
animals.
370%), 8e (AC50 = 0.039 μM, ROI = 550%), 8l (AC50
=
0.077 μM, ROI = 709%), 8m (AC50 = 0.031 μM, ROI = 576%),
and 9a (AC50 = 0.012 μM, ROI = 325%) were identified as
novel SMN protein modulators. In the field, it is generally
believed that an up-regulation of SNM production of at least
20% above patient basal levels will be necessary to have a
therapeutic impact. Our best molecules clearly surpass that
mark, in vitro, increasing levels of SMN protein between 100%
and 300% at low nanomolar concentrations when they were
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Table 8. Selected ADME Properties Assessment for Chosen Lead Compoundsa
luciferase inh.
mouse liver
Caco-2 permeability
mean Papp A f B (10ꢀ6 cm sꢀ1
Caco-2 permeability mean
Papp B f A (10ꢀ6 cm sꢀ1
)
no.
SMN2 AC50 (fiM)
ROI (%)
IC50 (μM)
microsome T1/2 (min)
)
2
1.22
268
554
298
370
257
609
709
576
325
353
inactive
1.60
10
12
30.7
32.6
40.1
13.1
21.2
ND
6.1
12.1
12.0
17.3
9.9
6k
7g
8a
8b
8c
8l
4.87
ND
18
0.154
0.038
0.122
0.077
0.077
0.031
0.012
0.049
3.2
30
3.2
>60
7
51.2
ND
0.3
6.4
0.80
40
30
8m
9a
9c
2
1.6
0.2
0.005
4.0
>60
<15
33.8
ND
44.1
ND
a Microsomal stability analysis was performed by Cerep and was based upon duplicate incubations of test reagent with mouse liver microsomes at 37 °C
as described (www.cerep.com). LC/MS/MS was utilized to quantitate remaining test compound. Test compounds were applied at standard
concentrations (1 μM). Caco-2 permeability analysis was performed by Cerep as described (www.cerep.com). Data are expressed in Papp (apparent
permeability) in 10ꢀ6 cm sꢀ1. Colchicine and ranitidine were used as a low permeability controls (colchicine: mean AfB < 0.1 and mean BfA = 7.9;
ranitidine mean AfB = 0.2 and mean BfA = 2.8), propranolol was used as a high permeability control (mean AfB = 41.0 and mean BfA = 39.4), and
labetalol was used as a P-gp substrate control (mean AfB = 7.3 and mean BfA = 36.6). Test compounds were applied at standard concentrations
(10 μM).
’ EXPERIMENTAL SECTION
incubation, cells were harvested, washed with cold PBS, and lysed as
above. We have determined that 10 μg total protein per lane is within the
linear range for immunoblot detection of SMN and R-tubulin. Western
blots were probed for SMN with the 4f11 mouse monoclonal antibody
and R-tubulin. Quantification of protein was performed with Fujifilm
LAS-4000 multifunctional imaging System. The signal intensity was
measured for each band on an immunoblot, normalized to the loading
control, and the fold increase was determined in relation to the
appropriate DMSO treated control.
Biology Reagents. All cell culture reagents were purchased from
Invitrogen. The white solid-bottom, 1536-well, tissue culture treated
plates came from Greiner. The OneGlo luciferase detection reagent was
from Promega. The luciferase enzyme and buffers came from Sigma
Aldrich. The 4f11 and 4B7 SMN antibodies were provided to EJA as a
generous gift from Christian Lorson (University of Missouri). Goat
antimouse HRP-conjugated secondary and R-tubulin antibodies
(DM1R) were supplied by Sigma.
Luciferase Reporter Assay. HEK-293 cells stably expressing the
SMN1 or SMN2 reporter construct were maintained and passaged in
complete DMEM media containing phenol red, glutamax, sodium
pyruvate, 10% FCS, 1ꢁ pen/strep, and 200 μg/mL hygromycin. Cells
were assayed in the same media lacking only hygromycin. For the HTS,
cells were plated at a density of 2000 cells/well in 5 μL of media and were
allowed to adhere overnight at 37 °C in 90% humidity with 5% CO2.
After incubation, cells received 23 nL of compound library (in 100%
DMSO) by pintool addition (Kalypsys). The final concentration of
DMSO was 0.46%. The screen was conducted in a 7-point qHTS
titration with a final concentration of 50 μM to 25 nM. The positive
control compound used was sodium butyrate (4.5 mM final con-
centration) and the negative control compound was DMSO. Plates
were incubated in the presence of compounds for 30ꢀ36 h in the
humidified incubator. After incubation, cells were treated with 3 μL of
OneGlo luciferase detection reagent and incubated at room temperature
for 5ꢀ15 min. Luminescence was detected on a Viewlux CCD based
instrument (Perkin-Elmer) with settings of 60 s integration and high
speed 2ꢁ binning. Data were normalized against sodium butyrate
(100%) and DMSO (0%) treatments.
Chemistry General Methods. All solvents were of anhydrous
quality, purchased from Aldrich Chemical Co., and used as received.
Commercially available starting materials and reagents were purchased
from Aldrich, TCI, and Acros and were used as received. Analytical thin
layer chromatography (TLC) was performed with Sigma Aldrich TLC
plates (5 cm ꢁ 20 cm, 60 Å, 250 μm). Visualization was accomplished by
irradiation under a 254 nm UV lamp. Chromatography on silica gel was
performed using forced flow (liquid) of the indicated solvent system on
Biotage KPSil prepacked cartridges and using the Biotage SP-1 auto-
1
mated chromatography system. H NMR spectra were recorded on a
Varian Inova 400 MHz spectrometer. Chemical shifts are reported
in ppm with the solvent resonance as the internal standard (CDCl3
1
7.27 ppm, DMSO-d6 2.49 ppm, for H NMR). Data are reported as
follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet, sep = septet, quin = quintet, br = broad, m = multiplet),
coupling constants, and number of protons. Low resolution mass spectra
(electrospray ionization) were acquired on an Agilent Technologies
6130 quadrupole spectrometer coupled to an Agilent Technologies
1200 series HPLC. The HPLC retention time were recorded through
standard gradient 4% to 100% acetonitrile (0.05% TFA) over 7 min
using Luna C18 (3 μm, 3 mm ꢁ 75 mm) column with a flow rate of
0.800 mL/min. High resolution mass spectral data were collected in-
house using an Agilent 6210 time-of-flight mass spectrometer coupled to
an Agilent Technologies 1200 series HPLC system. If needed, products
were purified via a Waters preparative HPLC equipped with a Phenom-
enex Luna or Gemini C18 reverse phase (5 μm, 30 mm ꢁ 75 mm)
column having a flow rate of 45 mL/min. The mobile phase was a mix-
ture of acetonitrile (0.1% TFA) and H2O (0.1% TFA) for acidic
conditions and a mixture of acetonitrile and H2O (0.1% NH4OH)
for basic conditions. Samples were analyzed for purity on an Agilent
1200 series LC/MS equipped with a Luna C18 reverse phase (3 μm,
Firefly Luciferase Enzyme Assay. The firefly luciferase enzyme
was assayed at 5 nM in buffer consisting of 50 mM Tris-acetate pH 7.6,
10 mM mg acetate 0.05% BSA, 0.01% Tween. Enzyme (2.5 μL/well)
was dispensed in a white solid-bottom 1536-well plate, and compounds
(23 nL) were added using a pintool. Plates were incubated at room
temperature for 5 min, followed by the addition of 2.5 μL of OneGlo
luciferase detection reagent. Luminescence was detected in the Viewlux
with a 1 s integration time, no binning, and medium camera sensitivity.
SMN Protein Detection. For detection of SMN protein in patient
fibroblasts, 8000 cells per cm2 were plated 24 h prior to drug addition.
Fresh media and compound were added every 24 h. After 72 h of
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Table 9. Pharmacokinetic Data for Analogues 8l and 9a Following Oral Gavage Administration Dosing to Mice
oral administration of 30 mg/kg of 8l in male Swiss Albino mice
mean plasma concentration
mean (ng/mL)
mean brain concentration
sampling time (h)
mean (μM)
mean (ng/g)
mean (μmol/kg)
0
0
1286
416
0
0
444
109
138
207
85
0
0.083
0.25
0.5
1
3.72
1.2
1.28
0.31
0.4
416
1.2
439
1.27
0.6
0.6
2
206
0.23
0.083
0
4
0.98
8.49
6.71
20.64
0.0028
0.025
0.019
0.06
29
8
0
12
24
0
0
7.78
0.023
PK parameters
unit
estimate
PK parameters
Tmax
unit
estimate
Tmax
h
0.083
3.72
5.78
1857
1429
29
h
0.083
1.28
5.44
546
Cmax
μM
h
Cmax
μmol/kg
T1/2
T1/2
h
AUCplasma(AUClast
AUCINF
)
h ng/mL
AUCplasma(AUClast
AUCINF
)
h ng/mL
3
3
h ng/mL
h ng/mL
607
3
3
AUCbrain/AUCplasma
%
oral administration of 30 mg/kg of 9a in male Swiss Albino mice
mean plasma concentration
mean (ng/mL) mean (μM)
mean brain concentration
mean (ng/g) mean (μmol/kg)
sampling time (h)
free concentration (μM)a
0
0.083
0.25
0.5
1
0
15990
15038
14822
7575
5990
3890
4537
1645
1570
0
0
0
173
442
409
214
157
129
114
47
0
44.4
41.7
41.1
21
2.26
2.13
2.1
0.48
1.23
1.14
0.59
0.43
0.36
0.32
0.13
0.13
1.07
0.85
0.55
0.64
0.23
0.22
2
16.6
10.8
12.6
4
8
12
24
4.57
4.36
48
PK parameters
unit
estimate
PK parameters
unit
estimate
Tmax
Cmax
T1/2
h
0.083
44.4
Tmax
h
0.25
μM
h
Cmax
μmol/kg
1.23
11.18
77768
103094
2.8
T1/2
h
13.69
2167
3115
AUCplasma(AUClast
AUCINF
)
h ng/mL
AUCplasma(AUClast
AUCINF
)
h ng/mL
3
3
h ng/mL
h ng/mL
3
3
AUCbrain/AUCplasma
a Based on 94.9% of plasma protein binding for 9a
%
3 mm ꢁ 75 mm) column having a flow rate of 0.800 mL/min over a
7.0 min gradient and a run time of 8.5 min. Purity of final compounds
was determined to be >95%, using a 3 μL injection with quantitation by
AUC at 220 and 254 nm (Agilent diode array detector).
General Protocol A. A mixture of bromo- or chlorosubstituted
building block (0.100 mmol), boronic acid or pinacol ester (0.200
mmol), and tetrakis(triphenylphosphine)palladium (5.00 μmol) in
DMF (1.50 mL) or CH3CN (1.50 mL), and 2.0 M Na2CO3 aqueous
solution (0.50 mL) was heated in a microwave at 100 °C for 30 min. The
reaction mixture was cooled to room temperature and treated with a
small portion of Si-THIOL to get rid of palladium. The mixture was
filtered through a frit to give a light-yellow solution. The crude material
was purified by preparative HPLC under acidic or basic condition to give
the final product.
General Protocol B. A mixture of carboxylic acid (0.082 mmol),
EDC (0.082 mmol), HOBt (0.082 mmol), DMAP (0.082 mmol), and
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amine (0.163 mmol) in DMF (1.50 mL) was heated in a microwave at
100 °C for 1.5 h. The crude material was purified by preparative HPLC
to give the final product.
General Protocol C. A solution of 4-(4-bromophenyl)-2-(piperazin-1-
yl)thiazole (0.093 mmol) and Et3N (0.139 mmol) in CH2Cl2 (2.00mL) was
treated at 0 °C with carboxylic chloride or sulfonyl chloride (0.111 mmol).
The reaction mixture was allowed to warm to room temperature, stirred for
1 h, and concentrated in vacuo. The crude material was purified by
preparative HPLC to give the final product.
1-(4-(4-Bromophenyl)thiazol-2-yl)piperidine-4-carboxa-
mide (2). A general protocol for amination: A mixture of 2,4-
dibromothiazole (2.06 g, 8.48 mmol), piperidine-4-carboxamide
(1.30 g, 10.1 mmol), and Et3N (2.50 mL) in ethanol (5.00 mL) was
heated in a microwave at 100 °C for 1 h. The reaction mixture was
cooled to room temperature, diluted with water, and extracted with a
mixture of MeOH and CH2Cl2. The organic layer was separated, dried
with Na2SO4, and concentrated as a light-brown solid. The crude
mixture was purified by Biotage with 0ꢀ10% of MeOH in CH2Cl2 with
1% Et3N to give 2.08 g (85%) of 1-(4-bromothiazol-2-yl)piperidine-4-
carboxamide as a white solid. 1H NMR (400 MHz, DMSO-d6) δ
ppm 7.31 (br s, 1 H), 6.85 (s, 1 H), 6.82 (br s, 1 H), 3.77ꢀ3.90 (m, 2
H), 3.03 (td, J = 12.5, 2.8 Hz, 2 H), 2.29ꢀ2.39 (m, 1 H), 1.78 (dd, J =
13.5, 3.1 Hz, 2 H), 1.50ꢀ1.63 (m, 2 H). LCMS RT = 4.13 min, m/z
289.9 [M + H+]. The title compound was prepared according to the
general protocol A: 1H NMR (400 MHz, DMSO-d6) δ 7.83ꢀ7.79 (m,
2 H), 7.59ꢀ7.55 (m, 2 H), 7.33 (s, 1 H), 7.32 (br s, 1 H), 6.82 (br s, 1
H), 3.96 (br. d., J = 12.8 Hz, 2 H), 3.06 (td, J = 12.6, 2.9 Hz, 2 H), 2.36
(tt, J = 11.6, 3.8 Hz, 1 H), 1.82 (dd, J = 13.0, 2.6 Hz, 2 H), 1.61 (qd, J =
12.4, 4.0 Hz, 2 H); LCMS RT = 5.16 min, m/z 366.0 [M + H+]; HRMS
(ESI) m/z calcd for C15H1779BrN3OS [M + H+] 366.0276, found
366.0272.
60 h. Another aliquot of reagents was added to the reaction solution, and
the mixture was reheated at 120 °C for additional 60 h. The reaction
mixture was cooled to room temperature, diluted with EtOAc, and
washed with H2O. The organic layer was separated, dried with Na2SO4,
and concentrated in vacuo. The crude product was purified by pre-
parative HPLC under basic condition to give 15.0 mg (22%) of product.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.76ꢀ7.87 (m, 2 H), 7.53ꢀ7.62
(m, 2 H), 7.33 (s, 1 H), 3.97 (dt, J = 13.2, 3.7 Hz, 2 H), 3.20ꢀ3.30 (m, 2
H), 3.14 (tt, J = 11.1, 3.6 Hz, 1 H), 2.04 (dd, J = 13.3, 3.1 Hz, 2 H),
1.71ꢀ1.87 (m, 2 H). LCMS RT = 5.62 min, m/z 391.0 [M + H+]. HRMS
(ESI) m/z calcd for C15H1679BrN6S [M + H+] 391.0341, found391.0338.
1-(4-(4-Bromophenyl)thiazol-2-yl)piperidin-4-amine (6n).
A solution of tert-butyl 1-(4-(4-bromophenyl)thiazol-2-yl)piperidin-4-
ylcarbamate (78.7 mg, 0.180 mmol) in CH2Cl2 (2.00 mL) was treated at
0 °C with TFA (1.00 mL). The reaction mixture was stirred at 0 °C for
30 min and at room temperature for additional 10 min. The solvents
were removed in vacuo. The crude product was filtered through a short
cartridge column to remove TFA to give 43.4 mg (72%) of product as a
white solid. A small portion of sample was repurified by preparative
HPLC under acidic condition to give the final product as a TFA salt. 1H
NMR (400 MHz, DMSO-d6) δ ppm 7.93 (br s, 2 H), 7.72ꢀ7.85 (m, 2
H), 7.50ꢀ7.65 (m, 2 H), 7.37 (s, 1 H), 4.00 (d, 2 H), 3.25ꢀ3.35 (m, 1
H), 3.14 (td, J = 12.8, 2.6 Hz, 2 H), 1.99 (dd, J = 12.6, 3.0 Hz, 2 H), 1.57
(qd, J = 12.4, 4.4 Hz, 2 H). 19F NMR (376 MHz, DMSO-d6) δ
ppm ꢀ73.54 (s). LCMS RT = 4.66 min, m/z 338.0 [M + H+]. HRMS
(ESI) m/z calcd for C14H1779BrN3S [M + H+] 338.0327, found
338.0323.
1-(4-(4-(4-Bromophenyl)thiazol-2-yl)piperazin-1-yl)ethan-
one (6t). Thecompoundwas preparedaccording tothe generalprotocol
C as a TFA salt. 1H NMR (400 MHz, CDCl3) δ ppm 7.64ꢀ7.74 (m, 2
H), 7.47ꢀ7.55 (m, 2 H), 6.82 (s, 1 H), 3.77ꢀ3.92 (m, 2 H), 3.66 (br s, 4
H), 3.48ꢀ3.58 (m, 2 H), 2.20 (s, 3 H). 19F NMR (376 MHz, DMSO-d6)
δ ppm ꢀ76.04 (s). LCMS RT = 5.89 min, m/z 366.0 [M + H+]. HRMS
(ESI) m/z calcd for C15H1779BrN3OS [M + H+] 366.0276, found
366.0274.
4-(4-(4-Bromophenyl)thiazol-2-yl)piperazine-1-carboxa-
mide (6u). A solution of 4-(4-bromophenyl)-2-(piperazin-1-yl)thia-
zole (40.0 mg, 0.123 mmol) in H2O (2.00 mL) was treated at room
temperature with KOCN (20.0 mg, 0.247 mmol). The reaction mixture
was stirred at room temperature overnight and extracted with EtOAc.
The organic layer was separated, dried with Na2SO4, and concentrated
in vacuo. The crude mixture was purified by preparative HPLC under
basic condition to give 14.2 mg (31%) of product. 1H NMR (400 MHz,
DMSO-d6) δ ppm 7.82 (d, J = 8.4 Hz, 2 H), 7.57 (d, J = 8.4 Hz, 2 H),
7.37 (s, 1 H), 6.11 (br s, 2 H), 3.37ꢀ3.57 (m, 8 H). LCMS RT = 5.33
min, m/z 367.0 [M + H+]. HRMS (ESI) m/z calcd for C14H1679BrN4OS
[M + H+] 367.0228, found 367.0215.
1-(4-(4-Bromophenyl)thiazol-2-yl)-N-methylpiperidine-4-
carboxamide (6a). The compound was prepared according to the
1
general protocol B as a TFA salt. H NMR (400 MHz, CDCl3) δ
ppm 7.60ꢀ7.71 (m, 2 H), 7.47ꢀ7.57 (m, 2 H), 6.72 (s, 1 H), 5.78 (br s,
1 H), 4.15 (dt, J = 13.3, 3.5 Hz, 2 H), 3.10ꢀ3.30 (m, 2 H), 2.84 (d, J = 5.1
Hz, 3 H), 2.41 (tt, J = 11.1, 3.8 Hz, 1 H), 1.95ꢀ2.05 (m, 2 H), 1.81ꢀ1.95
(m, 2 H). 19F NMR (376 MHz, CDCl3) δ ppm ꢀ75.97 (s). LCMS RT =
5.34 min, m/z 380.0 [M + H+]. HRMS (ESI) m/z calcd for
C16H1979BrN3OS [M + H+] 380.0432, found 380.0425.
1-(4-(4-Bromophenyl)thiazol-2-yl)piperidine-4-carboxylic
acid (6k). A solution of ethyl 1-(4-(4-bromophenyl)thiazol-2-yl)-
piperidine-4-carboxylate (3.34 g, 8.45 mmol) in THF (24.0 mL) and
H2O (8.00 mL) was treated at room temperature withLiOH (1.01 g, 42.2
mmol). The reaction mixture was stirred at room temperature for 24 h,
diluted with 100 mL of CH2Cl2, and washed with 2.0 N HCl (25.0 mL).
The organic layer was separated, dried with Na2SO4, and concentrated to
give a yellow oil. The crude product was purified by Biotage with 0ꢀ15%
of MeOH in CH2Cl2 to give 2.79 g (90%) of product as a white solid. A
small amount of sample was repurified by preparative HPLC under acidic
condition to give the product as a TFA salt. 1H NMR (400 MHz, DMSO-
d6) δ ppm 7.74ꢀ7.88 (m, 2 H), 7.50ꢀ7.64 (m, 2 H), 7.33 (s, 1 H), 3.90
(dt, J = 12.7, 3.5 Hz, 2 H), 3.04ꢀ3.23 (m, 2 H), 2.51ꢀ2.59 (m, 1 H), 1.94
(dd, J = 13.1, 3.7 Hz, 2 H), 1.51ꢀ1.75 (m, 2 H). 19F NMR (376 MHz,
DMSO-d6) δ ppm ꢀ74.87 (s). LCMS RT = 5.84 min, m/z 367.0 [M +
H+]. HRMS (ESI) m/z calcd for C15H1679BrN2O2S [M + H+] 367.0116,
found 367.0103.
2-(4-(1H-Tetrazol-5-yl)piperidin-1-yl)-4-(4-bromophenyl)-
thiazole (6m). A solution of 1-(4-(4-bromophenyl)thiazol-2-yl)-
piperidine-4-carbonitrile (59.5 mg, 0.171 mmol) in water (1.00 mL)
and 1,4-dioxane (0.58 mL) was treated at roomtemperature with sodium
azide (33.3 mg, 0.513 mmol) and zinc bromide (57.7 mg, 0.256 mmol).
The pH value of the solution was adjusted to about 7 with 1 N NaOH
(∼6 drops). The reaction mixture was heated in oil bath at 120 °C for
1-(4-(3-Isopropoxyphenyl)pyrimidin-2-yl)piperidine-4-
carboxamide (7g). The compound was prepared according to the
general protocol A as a TFA salt. 1H NMR (400 MHz, DMSO-d6) δ
ppm 8.41 (d, J = 5.1 Hz, 1 H), 7.66 (ddd, J = 8.1, 1.3, 1.0 Hz, 1 H),
7.57ꢀ7.63 (m, 1 H), 7.41 (t, J = 7.9 Hz, 1 H), 7.29 (br s, 1 H), 7.19 (d,
J = 5.3 Hz, 1 H), 7.03ꢀ7.13 (m, 1 H), 6.78 (br s, 1 H), 4.55ꢀ4.84 (m,
3 H), 2.88ꢀ3.10 (m, 2 H), 2.41 (tt, J = 11.5, 3.8 Hz, 1 H), 1.80 (dd,
J = 12.7, 2.9 Hz, 2 H), 1.50 (qd, J = 12.4, 3.9 Hz, 2 H), 1.30 (d, J = 6.1
Hz, 6 H). 19F NMR (376 MHz, DMSO-d6) δ ppm ꢀ74.96 (s). LCMS
RT = 4.45 min, m/z 341.2 [M + H+]. HRMS (ESI) m/z calcd for
C19H25N4O2 [M + H+] 341.1978, found 341.1983.
1-(4-(3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)pyrimidin-
2-yl)piperidine-4-carboxamide (7h). The compound was prepared
according to the general protocol A. 1H NMR (400 MHz, DMSO-d6) δ
ppm 8.36 (d, J = 5.1 Hz, 1 H), 7.62ꢀ7.80 (m, 2 H), 7.29 (br s, 1 H),
7.01ꢀ7.14(m, 2H), 6.78(brs, 1H), 4.63ꢀ4.84(m, 2H), 4.10ꢀ4.26 (m, 4
H), 2.93 (td, J = 12.7, 2.5 Hz, 2 H), 2.40 (tt, J = 11.5, 3.7 Hz, 1 H), 2.14
6230
dx.doi.org/10.1021/jm200497t |J. Med. Chem. 2011, 54, 6215–6233

Journal of Medicinal Chemistry
ARTICLE
(quin, J= 5.6 Hz, 2 H), 1.79 (dd, J= 12.9, 2.7 Hz, 2 H), 1.49 (qd, J= 12.4, 4.3
Hz, 2 H). LCMS RT = 3.84 min, m/z 355.2 [M + H+]. HRMS (ESI) m/z
calcd for C19H23N4O3 [M + H+] 355.1770, found 355.1770.
Hz, 1 H), 6.97ꢀ7.03 (m, 1 H), 6.89ꢀ6.96 (m, 1 H), 4.12 (dt, J = 9.2, 5.5
Hz, 4 H), 3.83 (ddd, J = 13.2, 3.7, 3.5 Hz, 2 H), 2.98ꢀ3.20 (m, 2 H),
2.51ꢀ2.57 (m, 1 H), 2.04ꢀ2.17 (m, 2 H), 1.87ꢀ1.98(m, 2 H), 1.49ꢀ1.69
(m, 2 H). LCMS RT = 4.18 min, m/z 361.1 [M + H+]. HRMS (ESI) m/z
calcd for C18H21N2O4S [M + H+] 361.1222, found 361.1221.
1-(5-(3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)thiazol-
2-yl)-N-phenylpiperidine-4-carboxamide (9c). The compound
was prepared according to the general protocol A. 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.95 (s, 1 H), 7.60 (dd, J = 8.7, 1.1 Hz, 2 H), 7.49 (s, 1
H), 7.24ꢀ7.33 (m, 2 H), 6.97ꢀ7.11 (m, 3 H), 6.92ꢀ6.97 (m, 1 H), 4.13
(dt, J = 9.4, 5.5 Hz, 4 H), 3.90ꢀ4.03 (m, 2 H), 3.10 (td, J = 12.6, 2.7 Hz, 2
H), 2.62 (tt, J = 11.5, 3.7 Hz, 1 H), 2.10 (quin, J = 5.4 Hz, 2 H), 1.90 (dd,
J = 13.0, 2.8 Hz, 2 H), 1.71 (qd, J = 12.4, 4.3 Hz, 2 H). LCMS RT = 4.94
min, m/z 436.2 [M + H+]. HRMS (ESI) m/z calcd for C24H26N3O3S
[M + H+] 436.1695, found 436.1699.
1-(5-(3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)thiazol-
2-yl)piperidine-4-carboxamide (8a). The compound was pre-
pared according to the general protocol A. 1H NMR (400 MHz,
DMSO-d6) δ ppm 7.47 (s, 1 H), 7.31 (br s, 1 H), 7.07 (d, J = 2.3 Hz,
1 H), 6.97ꢀ7.04 (m, 1 H), 6.89ꢀ6.96 (m, 1 H), 6.82 (br s, 1 H), 4.12
(dt, J = 9.3, 5.5 Hz, 4 H), 3.89 (dt, J = 12.8, 3.3 Hz, 2 H), 3.04 (td, J =
12.5, 2.9 Hz, 2 H), 2.36 (tt, J = 11.5, 3.7 Hz, 1 H), 2.02ꢀ2.15 (m, 2 H),
1.79 (dd, J = 13.2, 2.8 Hz, 2 H), 1.50ꢀ1.68 (m, 2 H). LCMS RT = 3.74
min, m/z 360.1 [M + H+]. HRMS (ESI) m/z calcd for C18H22N3O3S
[M + H+] 360.1382, found 360.1379.
1-(5-(3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)-1,3,4-
thiadiazol-2-yl)piperidine-4-carboxamide (8b). The com-
pound was prepared according to the general protocol A as a TFA
1
salt. H NMR (400 MHz, DMSO-d6) δ ppm 7.23ꢀ7.52 (m, 3 H),
’ ASSOCIATED CONTENT
6.95ꢀ7.11 (m, 1 H), 6.84 (br s, 1 H), 4.19 (t, J = 5.6 Hz, 4 H), 3.88 (dt,
J = 12.8, 3.5 Hz, 2 H), 3.19 (td, J = 12.4, 2.9 Hz, 2 H), 2.35 (tt, J = 11.4,
4.0 Hz, 1 H), 2.14 (dt, J = 11.2, 5.6 Hz, 2 H), 1.82 (dd, J = 13.5, 2.9 Hz,
2 H), 1.65 (qd, J = 12.4, 3.9 Hz, 2 H). 19F NMR (376 MHz, DMSO-d6)
δ ppm ꢀ74.79 (s). LCMS RT = 4.21 min, m/z 361.1 [M + H+].
HRMS (ESI) m/z calcd for C17H21N4O3S [M + H+] 361.1334, found
361.1332.
S
Supporting Information. Assay protocols, experimental
b
procedures, and spectroscopic data (1H NMR, LCMS, and
HRMS) are listed for all compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
1-(5-p-Tolylthiazol-2-yl)piperidine-4-carboxamide (8c). The
compound was prepared according to the general protocol A as a TFA salt.
1H NMR (400 MHz, DMSO-d6) δppm7.58(s, 1H), 7.34ꢀ7.45 (m, 2 H),
7.32 (br s, 1 H), 7.18 (d, J = 8.0 Hz, 2 H), 6.84 (br s, 1 H), 3.91 (dt, J = 12.9,
3.2 Hz, 2 H), 3.12 (td, J = 12.6, 2.8 Hz, 2 H), 2.38 (tt, J = 11.4, 3.8 Hz, 1 H),
2.29 (s, 3 H), 1.82 (dd, J = 13.2, 2.8 Hz, 2 H), 1.48ꢀ1.72 (m, 3 H). 19F
NMR (376 MHz, DMSO-d6) δ ppm ꢀ74.86 (s). LCMS RT = 3.91 min,
m/z 302.1 [M + H+]. HRMS (ESI) m/z calcd for C16H20NOS [M + H+]
302.1327, found 302.1328.
Corresponding Author
*For J.J.M.: phone, 301-217-9198; fax, 301-217-5736; E-mail,
maruganj@mail.nih.gov. For J.X.: E-mail, xiaoj@mail.nih.gov.
Present Addresses
§Department of Dermatology, Indiana University School of
Medicine, 980 West Walnut Street, Indianapolis, Indiana 46202.
1-(5-(3-Isopropoxyphenyl)thiazol-2-yl)piperidine-4-carboxa-
mide (8l). The compound was prepared according to the general protocol
A as a TFA salt. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.65 (s, 1 H), 7.32
(br s, 1 H), 7.24 (t, J = 8.0 Hz, 1 H), 6.93ꢀ7.03 (m, 2 H), 6.83 (br s, 1 H),
6.76ꢀ6.81 (m, 1 H), 4.55ꢀ4.75 (m, 1 H), 3.91 (dt, J = 12.8, 3.4 Hz, 2 H),
3.11 (td, J = 12.5, 2.9 Hz, 2 H), 2.37 (tt, J = 11.4, 3.7 Hz, 1 H), 1.81 (dd,
J = 13.3, 2.9 Hz, 2 H), 1.52ꢀ1.68 (m, 2 H), 1.26 (d, J = 6.1 Hz, 6 H). 19F
NMR (376 MHz, DMSO-d6) δ ppm ꢀ74.91 (s). LCMS RT = 4.30 min,
m/z 346.1 [M + H+]. HRMS (ESI) m/z calcd for C18H24N3O2S [M + H+]
346.1589, found 346.1593.
’ ACKNOWLEDGMENT
We thank William Leister, Jim Bougie, Chris LeClair, Paul
Shinn, Danielle van Leer, Thomas Daniel, and Jeremy Smith for
assistance with compound purification and management. We
thank Dr. Marc Ferrer for helpful suggestions and Allison
Mandich for critical review of the manuscript. This research
was supported by the Molecular Libraries Program of the
National Institutes of Health Roadmap for Medical Research
(U54MH084681), the Intramural Research Program of the
National Human Genome Research Institute, National Institutes
of Health, and R03MH084179-01 to E.J.A.
1-(5-(4-(Methylthio)phenyl)thiazol-2-yl)piperidine-4-car-
boxamide (8m). The compound was prepared according to the
1
general protocol A as a TFA salt: H NMR (400 MHz, DMSO-d6) δ
ppm 7.58 (s, 1 H), 7.37ꢀ7.45 (m, 2 H), 7.32 (br s, 1 H), 7.21ꢀ7.29 (m,
2 H), 6.83 (br s, 1 H), 3.91 (dt, J = 12.9, 3.0 Hz, 2 H), 3.09 (td, J = 12.5,
2.8 Hz, 2 H), 2.48 (s, 3 H), 2.37 (tt, J = 11.6, 4.0 Hz, 1 H), 1.81 (dd, J =
13.5, 3.5 Hz, 2 H), 1.54ꢀ1.67 (m, 2 H). 19F NMR (376 MHz, DMSO-
d6) δ ppm ꢀ74.56 (s); LCMS RT = 4.07 min, m/z 361.1 [M + H+].
HRMS (ESI) m/z calcd for C16H20N3OS2 [M + H+] 334.1048, found
334.1048.
1-(5-(3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)thiazol-2-
yl)piperidine-4-carboxylic acid (9a). A solution of ethyl 1-(5-(3,4-
dihydro-2H-benzo[b][1,4]dioxepin-7-yl)thiazol-2-yl)piperidine-4-carbox-
ylate (284 mg, 0.72 mmol) in THF (6.00 mL) and H2O (2.00 mL) was
treated at room temperature with LiOH (86.0 mg, 3.60 mmol). The
reaction mixture was stirred at room temperature for 24 h, diluted with
100 mL of CH2Cl2, and washed with 2 N HCl (25.0 mL). The organic
layer was separated, dried with Na2SO4, and concentrated as a yellow oil.
The crude product was purified by Biotage with 0ꢀ15% of MeOH in
CH2Cl2 to give 233 mg (90%) of product as a white solid. 1H NMR (400
MHz, DMSO-d6) δ ppm 12.32 (br s, 1 H), 7.48 (s, 1 H), 7.07 (d, J = 2.2
’ ABBREVIATIONS USED
SMA, spinal muscular atrophy; SMN, survival motor neuron; SMN1,
survival motor neuron gene 1; SMN2, survival motor neuron gene
2; qHTS, quantitative high-throughput screen; ROI, rate of induc-
tion; NIH, National Institutes of Health; RNA, ribonu-
cleic acid; snRNP, small nuclear ribonucleoproteins; mRNA,
messenger RNA; SAR, structureꢀactivity relationships; SPR,
structureꢀproperty relationships; PK, pharmacokinetics; PBA,
sodium 4-phenylbutyrate; FDA, U.S. Food and Drug Adminis-
tration; cDNA, complementary DNA; DMSO, dimethyl sulfox-
ide; MLSMR, Molecular Libraries-Small Molecule Reposito-
ry; GFP, green fluorescent protein; P-gp, P-glycoprotein; MCPBA,
meta-chloroperoxybenzoic acid; HPLC, high-performance liquid
chromatography; RT-PCR, real-time polymerase chain reaction;
HDAC, histone deacetylases; DcpS, decapping enzyme scavenger;
SMNΔ7, delete exon 7 SMN protein; ADME, absorption, distribu-
tion, metabolism, and excretion; CNS, central nervous system;
6231
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Journal of Medicinal Chemistry
ARTICLE
HRP, horseradish peroxidase; HEK-293, human embryonic kid-
ney 293; DMEM, Dulbecco’s Modified Eagle Medium; FCS, fetal
calf serum; CCD, charge-coupled device; TLC, thin layer chroma-
tography; DMF, dimethylformamide; EDC, 1-ethyl-3-(3-dimethyla-
minopropyl)carbodiimide; HOBt, hydroxybenzotriazole; DMAP, 4-
dimethylaminopyridine; TEA, triethylamine; TFA, trifluoroacetic acid.
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C. Phenylbutyrate increases SMN expression in vitro: relevance for
treatment of spinal muscular atrophy. Eur. J. Hum. Genet. 2004, 12,
59–65.
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