Specific Correction of Alternative Survival Motor Neuron 2 Splicing by Small Molecules: Discovery of a Potential Novel Medicine to Treat Spinal Muscular Atrophy

Article abstract of DOI:10.1021/acs.jmedchem.6b00459

Spinal muscular atrophy (SMA) is the leading genetic cause of infant and toddler mortality, and there is currently no approved therapy available. SMA is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. These mutations or deletion

Full text of DOI:10.1021/acs.jmedchem.6b00459

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Article
Specific correction of alternative survival motor neuron
2 (SMN2) splicing by small molecules: Discovery of a
potential novel medicine to treat spinal muscular atrophy
Hasane Ratni, Gary M. Karp, Marla Weetall, Nikolai N. Naryshkin, Sergey V. Paushkin, Karen S. Chen,
Kathleen D. McCarthy, Hongyan Qi, Anthony Turpoff, Matthew G Woll, Xiaoyan Zhang, Nanjing Zhang, Tianle
Yang, Amal Dakka, Priya Vazirani, Xin Zhao, Emmanuel Pinard, Luke Green, Pascale David-Pierson, Dietrich
Tuerck, Agnès Poirier, Wolfgang Muster, Stephan Kirchner, Lutz Mueller, Irene Gerlach, and Friedrich Metzger
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00459 • Publication Date (Web): 14 Jun 2016
Downloaded from http://pubs.acs.org on June 15, 2016
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Specific correction of alternative survival motor
neuron 2 (SMN2) splicing by small molecules:
Discovery of a potential novel medicine to treat
spinal muscular atrophy
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Hasane Ratni1*, Gary M. Karp2*, Marla Weetall2, Nikolai A. Naryshkin2, Sergey V. Paushkin3,
Karen S. Chen3, Kathleen D. McCarthy3, Hongyan Qi2, Anthony Turpoff2, Matthew G. Woll2,
Xiaoyan Zhang2, Nanjing Zhang2, Tianle Yang2, Amal Dakka2, Priya Vazirani2, Xin Zhao2,
Emmanuel Pinard1, Luke Green1, Pascale David-Pierson1, Dietrich Tuerck1, Agnes
Poirier1,Wolfgang Muster1, Stefan Kirchner1, Lutz Mueller1, Irene Gerlach1, Friedrich Metzger1
1F. HoffmannꢀLa Roche Ltd., pRED, Pharma Research & Early Development, Roche Innovation
Center Basel, Grenzacherstrasse 124, 4070 Basel, Switzerland
2PTC Therapeutics, Inc., 100 Corporate Court, South Plainfield, NJ 07080, USA
3SMA Foundation, 888 Seventh Avenue, Suite 400, New York, NY 10019, USA
ABSTRACT
Spinal muscular atrophy (SMA) is the leading genetic cause of infant and toddler mortality, and
there is currently no approved therapy available. SMA is caused by mutation or deletion of the
survival motor neuron 1 (SMN1) gene. These mutations or deletions result in low levels of
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functional SMN protein. SMN2, a paralogous gene to SMN1, undergoes alternative splicing and
exclusion of exon 7, producing an unstable, truncated SMN∆7 protein. Herein, we report the
identification of pyridoꢀpyrimidinone series of small molecules that modify the alternative
splicing of SMN2, increasing the production of full length SMN2 mRNA. Upon oral
administration of our small molecules, the levels of full length SMN protein were restored in two
mouse models of SMA. Inꢀdepth lead optimization in the pyridoꢀpyrimidinone series culminated
in the selection of compound 3 (RG7800), the first small molecule SMN2 splicing modifier to
enter human clinical trials.
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INTRODUCTION
Spinal muscular atrophy (SMA) is the most common genetic cause of death of infants
and toddlers with an incidence of 1 in 11,000 live births.1 The disease varies in severity and
presentation. There are three most common types of SMA. Patients with severe type I SMA can
never sit and have a median survival around 2 years of age. Intermediately affected type II SMA
patients can never stand unaided and commonly die in early adulthood. Those with the mildest
form, type III SMA, have reduced motor function and may lose ambulation later in life.2
Survival motor neuron protein (SMN) is expressed in all body tissues, and is essential for
normal development and functional homeostasis in all species.3 Reduced levels of SMN protein
result in loss of alpha motor neurons and progressive muscular atrophy. There is an inverse
correlation between the quantity of fullꢀlength SMN protein expressed and disease severity.4 In
humans, SMN protein is produced by two paralogous genes: SMN1 and SMN2. SMN protein
expression is primarily driven by the SMN1 gene. SMN2 produces only low levels of full length
mRNA due to alternative splicing of exon 7.
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SMA results from a loss of function mutation or deletion of the SMN1 gene, and the
disease manifests by the insufficient generation of full length SMN protein by the SMN2 gene.
All SMA patients have at least one SMN2 copy, and some may have up to six copies in a somatic
cell.4b SMN protein levels generally correlate with SMN2 copy number.5 Relative to the levels of
SMN in healthy individuals, SMN protein levels in SMA patients are reduced by approximately
70% in SMA type I, 50% in type II and 30% in type III.4 This suggests that moderate increases
in SMN protein levels can modify the disease severity. Therefore, our strategy to treat SMA has
been to identify small molecules that shift the outcome of the alternative splicing of SMN2 exon
7 towards the production of full length SMN mRNA and consequently functional SMN protein
expression.
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Over the last few years, a number of therapeutic options for SMA have been evaluated5
and among them splicing correction of SMN2 appears highly promising. It can be achieved either
by intrathecal administration of antisense oligonucleotides (ASOs),6 or advantageously by small
molecules upon oral administration leading to both central and peripheral SMN protein increase
as initially reported by us7 and recently by others.8
We have reported the discovery of highly specific, orally available small molecules
(coumarin 1,9 isoꢀcoumarin 210 and pyridoꢀpyrimidinone derivatives) that specifically modify
SMN2 splicing in SMA patientꢀderived cells and in two SMA mouse models, resulting in a clear
therapeutic benefit in mice that model a severe SMA form.7 However, the clinical development
of the coumarin and isoꢀcoumarin compounds 1 and 2 was mainly hampered by an in vitro flag
in the Ames assay indicative of genotoxicity, phototoxicity, and chemical instability in plasma or
aqueous buffers. These findings shifted our focus to the pyridoꢀpyimidinone series (Figure 1).
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pyridoꢀpyrimidinone derivatives
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Figure 1. Coumarin 1, isoꢀcoumarin 2, and pyridoꢀpyrimidinone derivatives.
We describe here the initial optimization attempts on the coumarin and isoꢀcoumarin
series, then we report the lead optimization strategy for the pyridoꢀpyrimidinone series
addressing multiple issues that led to the discovery of orally active compounds 3-5 (Figure 2).
Compound 3 was selected as our clinical candidate. It became the first orally active small
molecule SMN2 splicing modulator to enter human trials for the potential treatment of spinal
muscular atrophy.
,
3 RG7800
4
5
Figure 2. Structures of compounds 3, 4 and 5.
RESULTS AND DISCUSSION
Chemistry. The pyridoꢀpyrimidinone derivatives were prepared using two synthetic
routes (Schemes 1 and 2).11 The first route (Scheme 1) was used specifically for the pyrazoloꢀ
pyrazine subclass. Nꢀalkylation of the pyrazole 6 with chloroacetone followed by an
intramolecular cyclisation in the presence of ammonium acetate gave the pyrazoloꢀpyrazine 7.
Chlorination with POCl3 afforded 8. A Suzuki or a Negishi crossꢀcoupling with either dimethyl
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or diethyl zinc led to 9a/b. A Claisen condensation with tꢀbutyl acetate followed by a transꢀ
esterification with ethanol provided the βꢀketo esters 10a/b. The key step is a condensation of the
7
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9
βꢀketo ester 10a/b with 5ꢀfluoropyridinꢀ2ꢀamine to construct the 2ꢀsubstitutedꢀ7ꢀfluoroꢀ
pyrido[1,2ꢀa]pyrimidinꢀ4ꢀone intermediates 11a/b. A nucleophilic aromatic substitution reaction
between 11a and methyl piperazine gave 12 whereas 13 was obtained by reaction between 11b
and (S)ꢀoctahydropyrrolo[1,2ꢀa]pyrazine. Alternatively, the condensation of 10a/b with the
substituted 2ꢀaminoꢀpyridines 14 and 19 provided 15, 16 and 20, respectively. A reductive
amination of 16 with formaldehyde concluded the synthesis of 3, while a reductive amination of
15 with either formaldehyde or acetaldehyde gave 17 and 18, respectively.
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Scheme 1. Synthesis of the pyrazoloꢀpyrazine derivatives 3, 12, 13, 15, 16, 17, 18, and 20a
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a
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: R = Me
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: R = Et
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f
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: R = Me
: R = Me
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: R = Et
: R = Et
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R = Me
R1 = Et; R2 = Me
R = Me, R = Me
15:
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R = Et
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R = Me, R = Et
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a Conditions: a) i. 1ꢀChloropropanꢀ2ꢀone (1 equiv), K2CO3 (1.1 equiv), acetone, 30 °C, 6 h; ii.
NH4OAc (20 equiv), acetic acid, reflux, 48 h, 64% over 2 steps; b) POCl3, reflux, 15 h, 85%; c)
Me2Zn (1.0 equiv), NiCl(dppp) (0.1 equiv), THF, 0 °C, 1 h, 82%; or Et2Zn (1.0 equiv),
NiCl(dppp) (0.05 equiv), THF, 0 °C, 1 h, 80%; d) EtOAc (5 equiv), LiHMDS (2.5 equiv), 77ꢀ
90%; e) 2ꢀaminoꢀ5ꢀfluoroꢀpyridine (1.2 equiv), PPTS (0.05 equiv), 130 °C, 8 h, 71ꢀ86%; f) 1ꢀ
methylpiperazine (10 equiv), DMA, 120 °C, 15 h, 80%; g) (S)ꢀoctahydropyrrolo[1,2ꢀa]pyrazine
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(2.0 equiv), K2CO3 (1.0 equiv), DMSO, 110 °C, 48 h, 63%; h) PPTS (1.1 equiv), 3ꢀmethylꢀ1ꢀ
butanol, 150 °C, 45 h, 25ꢀ39%; i) PPTS (1.0 equiv), 3ꢀmethylꢀ1ꢀbutanol, 150 °C, 24 h, 30%; j)
HCHO (3.0 equiv), NaBH(OAc)3 (1.5 equiv), CH2Cl2, 10 °C, 0.5 h, 47ꢀ70%; or acetaldehyde
(3.0 equiv), NaBH(OAc)3 (1.5 equiv), CH2Cl2, 10 °C, 0.5 h, 90%.
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An alternative synthetic route was used to prepare 2ꢀsubstituted pyridoꢀpyrimidinones
containing a fused 6,5ꢀbiaryl system (e.g. imidazoꢀpyridine, benzoxazole, benzotriazole or
benzimidazole) (Scheme 2). A condensation between 5ꢀfluoropyridinꢀ2ꢀamine 21 and dimethyl
malonate, followed by chlorination with POCl3 afforded 22. The right hand side moiety was then
introduced by means of a Suzuki crossꢀcoupling between 22 and a wide range of aromatic
boronic acids or esters to give 23a-h. The boronic acid/ester derivatives were commercially
available or readily prepared from the corresponding aromatic halide. Finally, an aromatic
nucleophilic substitution with a range of secondary amines led to the final derivatives 4, 5, and
24-31.
Scheme 2. Synthesis of derivatives 4, 5, and 24-31a
a
b
c
Ar = g; R = Me
Ar = h; R = Me
: Ar = a; R = Me
: Ar = b; R = Me
: Ar = c; R = Me
Ar = d; R = Me
Ar = d; R = H
: Ar = e; R = Me
: Ar = f; R = Me
: Ar = h; R = H
4:
5:
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23a-h
Ar
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h
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f
g
a Conditions: a) i. Dimethyl malonate (5 equiv), 230 °C, 1.5 h; ii. POCl3, iꢀPr2NEt (1.0 equiv),
110 °C, 15 h, 50% over 2 steps; b) Pd(dppf)Cl2 or Pd(PPh3)4 (0.05 equiv), aqueous K2CO3 (2M,
3.0 equiv), CH3CN, 50 °Cꢀreflux, 31ꢀ97%; c) piperazine or methylꢀpiperazine (5 equiv), DMA
or DMSO, 50ꢀ140 °C, 31ꢀ66%.
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Lead optimization. As spinal muscular atrophy is driven by reduced levels of SMN
protein, we aimed to identify a small molecule capable of increasing the level of SMN protein in
multiple tissues, including peripheral and central nervous system, upon oral administration.
Therefore, in addition to a suitable PK profile, the compounds should not be Pꢀgp substrates.
From a safety perspective, although SMA is a terminal disease for its severe form (type 1)
without approved therapy, it was important to select a development candidate with no potential
for genotoxicity as indicated by the standard Ames assay. The Ames test was considered
positive, indicating mutagenicity, when a dose dependent increase in the number of colonies
reached at least a 2ꢀfold (strains TA1535, TA98) or 1.5ꢀfold (strains TA97, TA100, TA102) over
the background level.
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To prevent the genotoxocity observed with the coumarin 1 and its corresponding Nꢀ
dealkylated metabolite 32, we either modulated the electronic density of the central core by
preparing the azaꢀcoumarin analog 33 or we replaced the right hand side fragment as exemplified
by the selected examples 34 and 35 (Figure 3). The azaꢀcoumarin 33 had similar in vitro potency
as 1, however it was also found positive in the Ames assay. The replacement of the imidazoꢀ
pyrazine moiety (right hand side fragment) led to derivatives that were negative in the Ames
assay. However, both 34 and 35 had poor in vitro potency. Further efforts to identify an Amesꢀ
negative compound with high in vitro potency remained unsuccessful in that series.
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Figure 3. Ames SAR in the coumarin series.
The isoꢀcoumarin 2 and its Nꢀdealkylated metabolite were also found to be positive in the
Ames assay. Although not confirmed, we hypothesized that this was most likely due to the
presence of the identical imidazoꢀpyrazine moiety found in coumarin compounds 1, 32, and 33.
Compound 2 additionally exhibited pronounced human plasma instability (Figure 4). After a five
hour incubation of 2 in human plasma, approximately 55% percent of the parent molecule
remained, in contrast to the analogous coumarin compound 1 (Figure 4). This observation
pointed towards a ringꢀopening of the isoꢀcoumarin as the most likely degradation pathway. This
hypothesis was strengthened upon replacement of the piperazine moiety by a more electron
withdrawing fluorine atom that increased the instability (derivative 36, 30% remaining). The
exchange of the imidazoꢀpyrazine moiety for an acetyl further increased the instability
(derivative 37, 16%). Our knowledge of the structure activity relationship of the isoꢀcoumarin
series indicated that it would not be possible to add an electron rich group on the isoꢀcoumarin
core, and therefore we concluded that it would be difficult to increase the plasma stability while
maintaining high in vitro potency.
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% parent molecule
remaining after 5 h
incubation in
55%
30%
16%
93%
human plasma
Figure 4. Human plasma stability of representative compounds from coumarin and isocoumarin
series.
At this stage, it appeared very challenging to further optimize the coumarin and the isoꢀ
coumarin series, due to the Ames flag and plasma instability. Additionally, since both coumarin
and isoꢀcoumarin moieties are known chromophores that confer phototoxicity,12 we investigated
the selection of an alternative central core (Figure 5). Several bioisosteres were evaluated, such
as the isoquinolinꢀ1ꢀone 38 and quinazolinꢀ4ꢀone 39, and most of them were found to be inactive
in vitro. However, the pyridoꢀpyrimidinone 12 had high in vitro potency (SMN HTRF EC1.5x
=
170 nM). Compound 12 was chemically stable in human plasma and aqueous buffer between pH
1 and 12. And while the coumarin 1 and isoꢀcoumarin 2 exhibited strong in vitro phototoxicity
with IC50 of 80 nM and 150 nM respectively (3T3 assay in the presence of UVA irradiation), the
pyridoꢀpyrimidinone 12 had markedly reduced phototoxicity potential with an IC50 of 2,900 nM.
The pyridoꢀpyrimidinone moiety was therefore selected as the central core of choice.
2
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Figure 5. Evaluation of alternative central cores.
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Several right hand side fragments in place of the imidazoꢀpyrazine were evaluated for their in
vitro potency and potential mutagenicity (Table 1). As previously observed with the coumarin
series, the outcome of the Ames assay was dependent on the right hand side fragment. With the
pyridoꢀpyrimidinone central core, we were able to identify compounds negative in the Ames
assay that also demonstrated good in vitro potency by incorporating a pyrazoloꢀpyrazine moiety
as in 12 or a substituted imidazoꢀpyridine (e.g. 4, 5 and 31) as right hand side fragments.
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Table 1. Ames assay results and in vitro potency of compounds with a pyridoꢀpyrimidinone
central core and various right hand side fragments R2.
Activity in
Ames assay
SMN2 splicing SMN protein
Compd
R1
R2
EC1.5x [nM]
EC1.5x [nM]
Me
Negative
Positive
Positive
Positive
92
170
12
Me
Me
Me
Inactive
314
Inactive
820
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148
395
Me
Positive
3
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H
Positive
Positive
Positive
Negative
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Me
Me
Me
582
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3400
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Me
H
Negative
Negative
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31
Additional analogs around the pyrazoloꢀpyrazine subseries (analogs of 12) were prepared and
assessed for their in vitro activity and evaluated in the Ames assay (Table 2). Each compound
from this subseries was negative in the Ames assay.
Table 2. Ames assay results and in vitro potency within the pyrazoloꢀpyrazine subseries.
Activity in
SMN2 splicing SMN protein
EC1.5x [nM]
Compd
R1
R2
H
R3
Ames
assay
EC1.5x [nM]
Me
Negative
92
170
12
H
N
H
Et
Negative
40
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13
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Me
Me
Me
Et
Negative
Negative
30
6
84
15
18
20
An additional objective was to design efficacious compounds leading to increased SMN
protein levels in multiple tissues, including peripheral and central nervous system in vivo.
Therefore, an important consideration was to prepare derivatives that were not Pꢀglycoprotein
(Pꢀgp) substrates. We observed the lipophilicity to be a strong predictor of the efflux ratio (ER, a
measure of Pꢀgp efflux; assessed using both human and mouse cells). Compounds were not or
only weak Pꢀgp substrates when the measured LogD was greater than 1.6 (Table 2). Compound
18 was described in an earlier manuscript as compound SMNꢀC3.7
Table 2. Pꢀgp efflux ratio and lipophilicity.
Hum
Pꢀgp
ER
Mouse
Pꢀgp
ER
SMN protein
EC1.5x [nM]
Compd
R1
R2
H
R3
LogD
1.45
8.5
14.5
76
40
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H
1.53
1.82
1.87
6.6
10.6
2.6
46
77
84
41
17
18
Me
Me
1.2
1.4
10
11
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18
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2.6
Me
2.26
2.0
2.5
87
3
H
H
H
2.30
2.46
2.49
1.9
1.1
2.4
2.6
1.3
3.2
40
13
55
5
27
4
In vivo pharmacokinetics and efficacy. At this stage, several compounds that were not
Pꢀgp substrates, had good in vitro potency (SMN HTRF EC1.5x < 100 nM), and were negative in
the Ames assay were identified. Additional characterization for chemical and human plasma
stability, aqueous solubility (Thesa, FaSSIF and FeSSIF), permeability (Pampa), DDI (drugꢀdrug
interaction CYP3A4, 1A2, 2C9, 2C19 and 2D6), TDI (time dependent inhibition, CYP3A4), in
vitro clearance (liver microsomes and hepatocytes) and absence of covalent binding (CVB)
prompted us to evaluate compounds 3, 4 and 5 further. The three compounds demonstrated a
favorable drug metabolism and pharmacokinetics (DMPK) profile in the rat and in cynomolgus
monkey with good oral bioavailability. A much longer halfꢀlife for compound 3 relative to 4 and
5 was observed, consistent with a higher volume of distribution (Table 3). Compounds 4 and 5
have a similar fraction unbound in plasma across species (fup for 4: human, cyno, rat, mouse:
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11%, 8%, 10% and 16%, respectively, and fup for 5: human, cyno, rat, mouse: 22%, 18%, 16%
and 17%, respectively) whereas for 3 a markedly lower free fraction in mouse was measured (fup
human, cyno, rat, mouse: 13%, 16%, 17% and 2%, respectively).
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18
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Table 3. In vivo SDPK profile for compounds 3 ꢀ 5
rat
Cyno
b
d
Cla
Vssa
T1/2
(h)
Fb
(%)
Clc
Vssc
(l/kg)
T1/2
(h)
Fd
(%)
Compd
(mL/min/kg) (l/kg)
(mL/min/kg)
18
38
61
20
14
5
12
83
5
20
4.3
2.4
40ꢀ60
~ 8
52
~
100
84
3
4
5
~ 6
4
55ꢀ100
~ 100
7.5
9.7
2.9
a) iv: 2 mg/kg; b) PO: 10 mg/kg; c) iv: 0.3 mg/kg; d) PO: 1 mg/kg.
We next evaluated the small molecule splicing modifiers 3, 4 and 5 in disease models of SMA.
For this purpose we used the adult C/Cꢀallele SMA mouse model, in which the animals have a
mild SMA phenotype, a normal life span, but show muscle weakness, reduced body weight gain
and peripheral necrosis. Compounds 3ꢀ5 were administered orally once daily (qd) for 10 days at
three different doses (1, 3 and 10 mg/kg). One hour after the final dose tissues were collected
from the mice and the level of the SMN protein was determined in brain and quadriceps muscle.
All three compounds showed a clear dose dependent increase in SMN protein levels (Table 4).
Table 4. Pharmacokinetics and SMN induction in adult C/Cꢀallele mouse model
Total Plasmaa
AUC
(ꢀg·h/mL)
Free Plasma AUC
Dose
(mg/kg PO)
% SMN indu. % SMN indu.
Compd
in brain
in quadriceps
(ꢀg·h/mL)
Vehicle
0
1
0
0
0
7
0
30
3
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4
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5
5
33
2.5
2.7
0.66
0.40
0.45
28
3
0
70
10
1
39
42
60
141
133
40
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3
81
10
126
a Plasma AUC determined in a satellite group
These splicing modifiers were then tested in ꢁ7 SMA mice, a model of severe SMA, where
animals die within three weeks after birth.13 Dosing was initiated with compounds 3-5 by
intraperitoneal (i.p.) injection once daily starting on postnatal day 3 (P3) and continued through
day 9 (P9). One hour after the last dose, the level of the SMN protein was determined in brain
and quadriceps muscle (Table 5). All three compounds doseꢀdependently increased SMN protein
level in the brain and in peripheral tissue (quadriceps muscle).
Table 5. Pharmacokinetics and SMN induction in neonatal ꢁ7 mouse model
Total Plasmaa
AUC
(ꢀg·h/mL)
Free Plasma AUC
Dose
(mg/kg ip)
% SMN indu. % SMN indu.
Compd
in brain
in quadriceps
(ꢀg·h/mL)
Vehicle
0
0.3
1
0
0
0
0
20
40
3
3
3
4
4
4
5
55
57
3
5.9
1.3
0.118
0.208
135
39
160
31
0.3
1
130
162
72
115
130
107
3
0.1
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0.3
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189
260
5
5
4.3
0.721
a Plasma AUC determined in a satellite group of neonatal (P10) wildꢀtype mice. (The freeꢀ
7
8
9
fraction measured using serum from adult mice was used to calculate the free plasma AUC.
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Finally, we assessed the impact of treatment with SMN2 splicing modifiers on the
lifespan and body weight of ꢁ7 SMA mice. Mice were treated with compounds 3 and 4 at 0.3, 1
and 3 mg/kg/day by i.p. injections from P3 through P23, and thereafter at 1, 3 and 10 mg/kg/day,
respectively, by oral gavage. Compound 5 was administered at 0.03, 0.1, 0.3 and 1 mg/kg/day by
i.p injections from P3 through P23, and thereafter at 0.1, 0.3, 1 and 3 mg/kg/day, respectively, by
oral gavage (Figure 7). Survival and body weight were assessed daily. While all vehicleꢀtreated
mice died less than three weeks after birth, mice treated with 3 and 4 demonstrated a doseꢀ
dependent increase in survival beginning at the low dose (0.3/1 mg/kg). In the mid and high dose
groups (1/3 and 3/10 mg/kg, respectively), approximately 80 to 90% of animals survived beyond
P50/P60 with profound body weight gain when the study was terminated. Compound 5 showed a
similar effect on survival and body weight gain, initiating at an even lower dose.
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Figure 7. Survival and body weight gain of ꢁ7 mice treated with compounds 3, 4 and-5.
Animals were treated with vehicle or compound at doses of 0.3, 1, or 3 mg/kg/day from PND3ꢀ
PND23 by IP injection, and thereafter by oral gavage at doses of 1, 3, or 10 mg/kg/day from
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PND24 through PND65. Data represent means ± SEM of an initial group size of 10 (HET), 15
(SMNꢁ7 SMA mice, vehicle or compound ꢀtreated).
7
8
9
Further characterization of compound 3 was then performed to demonstrate that those
small molecules are increasing the SMN protein level via inducing alternative splicing of the
SMN2 mRNA both in vitro and in vivo. Compound 3 was shown to promote the inclusion of
exon 7 in SMN2 mRNA generating full length (FL) mRNA in vitro using fibroblasts from an
SMA type 1 patient (Figure 8, panel A). To investigate SMN protein production as a
consequence of splicing correction, an in vitro assay assessed the levels of SMN protein in
fibroblasts and in spinal motor neurons derived from SMA Type I and II patient induced
pluripotent stem cells (iPSC). The maximal increase in SMN protein produced, compared to
untreated controls, was similar in both cell types (60ꢀ80%; Figure 8, panels B and C), suggesting
that in different cell types derived from SMA patients, compound 3 increases SMN protein level
as a result of correcting the dysfunctional SMN2 splicing in vitro.
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[ꢀM]
[ꢀM]
[ꢀM]
Figure 8: Compound 3 tested in: A. SMN2 splicing in SMA type 1 fibroblasts. B. SMN protein
in SMA type 1 fibroblasts. C. SMN protein in SMA type I and II motor neurons. FL, full length,
ꢁ7, mRNA lacking exon 7.
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For assessment of the in vivo effects of compound 3 on SMN2 splicing, adult SMA model mice
(C/Cꢀallele) were treated for 10 days with vehicle or compound 3 (3 or 10 mg/kg po once daily).
Compound 3 dose dependently corrected SMN2 splicing by including exon 7 to create FL
mRNA (Figure 9, panel A), suggesting that compound 3 corrects alternative splicing of the
human SMN2 gene in the brain of transgenic SMA mice model, leading to an increase of the
SMN protein in brain (Figure 9, panel B).
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A
B
Brain hSMN2 mRNA
1.6
Brain SMN protein
150
FL
ꢁ7
1.4
1.2
1.0
0.8
0.6
0.4
100
50
0
Veh
1
3
10
Veh
3
10
RO6885247 [mg/kg]
RO6885247 [mg/kg]
Compound 3 [mg/kg]
Compound 3 [mg/kg]
Figure 9: Compound 3 induces SMN2 splicing (panel A) and SMN protein increase (panel B) in
C/Cꢀallele mice.
CONCLUSION
In summary, chemical optimization of the pyridoꢀpyimidinone class for safety parameters
(Ames), DMPK profile (Pꢀgp, plasma and chemical stability) and pharmacology (in vitro / in
vivo potency) has led to the discovery of orally available small molecule compounds that
specifically modify the alternative splicing of SMN2 exon 7 in SMA patientꢀderived cells and in
two SMA mouse models. This splicing modification results in a clear functional benefit in mice
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that model a severe SMA form. Compounds 3ꢀ5 exhibited excellent pharmacokinetic and in vivo
5
6
efficacy and had a favorable safety profile.
7
8
9
These results suggested that our splicing modifier compounds could provide a therapeutic
benefit for SMA patients. On this basis, compound 3 from the pyrazoloꢀpyrazine subclass
entered clinical development. This represents the first clinical trial with a small molecule splicing
modifier in SMA, enlarging the path for the treatment of other diseases via modulation of preꢀ
mRNA splicing. An eye finding in a nonꢀhuman primates safety study led us to put on hold our
clinical trial with that molecule. The complete safety characterization of 3 and the outcome of the
clinical trials will be reported in future publications.
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EXPERIMENTAL SECTION
Compound Synthesis and Characterization. Chemistry.
Reactions were carried out under argon atmosphere. Unless otherwise mentioned, all
reagents and chemicals were obtained from commercial suppliers and used without further
purification. All reactions were followed by TLC (TLC plates F254, Merck) or LCMS (liquid
chromatographyꢀmass spectrometry) analysis. The purity of final compounds as measured by
HPLC was at least above 95%. Flash column chromatography was carried out either using
cartridges packed with silica gel (Isolute Columns, Telos Flash Columns) or on glass columns on
silica gel 60 (32ꢀ60 mesh, 60Å). LC high resolution spectra were recorded with a Agilent LCꢀ
system consisting of Agilent 1290 high pressure system, a CTC PAL auto sampler and a Agilent
6520 QTOF. The separation was achieved on a Zorbax Eclipse Plus C18 1,7 µm 2.1*50mm
column at 55°C; A=0.01% formic acid in Water; B= 0.01% formic acid in acetonitrile at flow 1
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mL/min. gradient: 0 min 5%B, 0.3 min 5%B, 4.5 min 99 %B 5 min 99%B. The NMR spectra
were measured on a Bruker 600 MHz machine in a 5 mm TCI cryoprobe at 298 K. TMS was
used for referencing for experiment done in CDCl3. The deuterated DMSOꢀd6 solvent signal was
used as reference with 2.50 ppm.
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2-(4-Ethyl-6-methyl-pyrazolo[1,5-a]pyrazin-2-yl)-9-methyl-7-(1-methyl-4-piperidyl)
pyrido[1,2-a]pyrimidin-4-one (3). According to scheme 1: Step A. Preparation of 7. (i) To a
solution of diethyl 1Hꢀpyrazoleꢀ3,5ꢀdicarboxylate 6 (10.0 g, 47 mmol) and 1ꢀchloropropanꢀ2ꢀone
(3.76 mL, 47 mmol) in acetone (200 mL) was added K2CO3 (7.2 g, 52 mmol). The reaction
mixture was heated at 30 °C for 6 hours, and concentrated in vacuo to remove the volatiles. The
residue was dissolved in EtOAc and washed with water. The organic phase was dried over
MgSO4 and concentrated to give diethyl 1ꢀ(2ꢀoxopropyl)ꢀ1Hꢀpyrazoleꢀ3,5ꢀdicarboxylate as a
light brown solid, which was used directly in the next step. (ii) The crude product was dissolved
in acetic acid (300 mL) and NH4OAc (72 g, 0.940 mol) was added. After refluxing for 48 h, the
mixture was concentrated and diluted with water. The precipitate was collected by filtration,
washed with cold water and CH3CN to give ethyl 4ꢀhydroxyꢀ6ꢀmethylꢀpyrazolo[l,5ꢀ a]pyrazineꢀ
2ꢀcarboxylate 7 (6.7 g, 64%) as an offꢀwhite solid. 1H NMR (600 MHz, DMSOꢀd6) δ ppm: 11.51
(br s, 1H), 7.62 (d, J=0.9 Hz, 1H), 7.32 (d, J=0.7 Hz, 1H), 4.32 (q, J=7.2 Hz, 2H), 2.14 (d, J=1.1
Hz, 3H), 1.31 (t, J=7.1 Hz, 3H); LCꢀHRMS: m/z=221.08093 [(M+H)+ calcd for C10H11N3O3:
221.08004; Diff 0.9 mDa].
Step B. Preparation of 8. Ethyl 4ꢀhydroxyꢀ6ꢀmethylpyrazolo [1,5 ꢀa]pyrazineꢀ2ꢀ
carboxylate 7 (7.18 g, 32.5 mmol) was dissolved in POC13 (80 mL) and heated at reflux for 15
hours. The dark mixture was cooled to RT, concentrated in vacuo to form a precipitate which
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was collected by filtration and washed with CH3CN to give ethyl 4ꢀchloroꢀ6ꢀmethylpyrazolo[1,5ꢀ
a]pyrazineꢀ2ꢀ carboxylate 8 (5.197 g) as an offꢀwhite solid. The filtrate was concentrated and
purified by column chromatography to give an additional fraction (1.42 g) of 8 and a combined
yield of 85%. 1H NMR (600 MHz, DMSOꢀd6) δ ppm: 8.80 (t, J=1.0 Hz, 1H), 7.41 (d, J=1.0 Hz,
1H), 4.38 (q, J=7.2 Hz, 2H), 2.45 (d, J=0.9 Hz, 3H), 1.34 (t, J=7.1 Hz, 3H); LCꢀHRMS:
m/z=239.0461 [(M+H)+ calcd for C10H10ClN3O2: 239.04615; Diff ꢀ0.1 mDa].
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Step C. Preparation of 9-b. To a solution of 4ꢀchloroꢀ6ꢀmethylpyrazolo[1,5ꢀa]pyrazineꢀ2ꢀ
carboxylate
8
(10 g, 41 mmol) in THF (150 mL) was added [1,3ꢀ
bis(diphenylphosphino)propane]nickel(II) chloride (1.13 g, 2.08 mmol). The yellowish
suspension was cooled to 0 °C and diethyl zinc (43.2 mL, 1.1 M in toluene, 47.5 mmol) was
added over 40 minutes and stirring continued for an additional 30 minutes. Water (5 mL) diluted
in THF (20 mL) was added dropwise at 0 °C (very exothermic reaction) and the reaction mixture
concentrated in vacuo. The residue was dissolved in EtOAc (150 mL) and washed with brine
(150 mL). The organic phase was dried over MgSO4 and the product was purified by column
chromatography (SiO2, EtOAc/Heptane 1:1) to give ethyl 4ꢀethylꢀ6ꢀmethylꢀpyrazolo[1,5ꢀ
a]pyrazineꢀ2ꢀcarboxylate 9-b (7.65 g, 80%) as a light yellow solid. 1H NMR (600 MHz, DMSOꢀ
d6) δ ppm: 8.54 (s, 1H), 7.50 (d, J=1.0 Hz, 1H), 4.36 (q, J=7.2 Hz, 2H), 3.06 (q, J=7.5 Hz, 2H),
2.44 (d, J=0.9 Hz, 3H), 1.34 (t, J=7.1 Hz, 3H), 1.30 (t, J=7.5 Hz, 3H); LCꢀHRMS:
m/z=233.1178 [(M+H)+ calcd for C12H15N3O2: 233.11643; Diff 1.4 mDa].
Step D. Preparation of 10-b. To a solution of ethyl 4ꢀethylꢀ6ꢀmethylꢀpyrazolo[1,5ꢀ
a]pyrazineꢀ2ꢀcarboxylate 9-b (6.6 g, 28.3 mmol) in THF (370 mL) was added EtOAc (13.5 mL,
141 mmol). The reaction mixture was cooled down to ꢀ30 °C before LiHMDS (70.7 mL, 1M in
THF, 70.7 mmol) was added slowly over 20 minutes. Stirring was continued for an additional 30
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minutes at ꢀ30 °C, and the reaction mixture was quenched by addition of an aqueous saturated
NH4Cl solution (50 mL). The reaction mixture was poured in EtOAc (300 mL) and washed with
water. The organic phase was dried over Na2SO4 and concentrated in vacuo. The crude material
was purified by chromatography (SiO2, CH2Cl2/EtOAc: 9:1 to 4:1) to give ethyl 3ꢀ(4ꢀethylꢀ6ꢀ
methylꢀpyrazolo[1,5ꢀa]pyrazinꢀ2ꢀyl)ꢀ3ꢀoxoꢀpropanoate 10-b (7.01 g, 90%) as a light yellow
solid.
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Step H. Preparation of 16. A stirred solution of ethyl 3ꢀ(4ꢀethylꢀ6ꢀmethylpyrazolo[1,5ꢀ
a]pyrazinꢀ2ꢀyl)ꢀ3ꢀoxoꢀpropanoate 10-b (8.14 g, 29.6 mmol), tertꢀbutyl 4ꢀ(6ꢀaminoꢀ5ꢀ
methylpyridinꢀ3ꢀyl)piperidineꢀ1ꢀcarboxylate (8.62 g, 29.6 mmol) and pyridinium pꢀtoluene
sulfonate (8.17 g, 32.5 mmol) in 3ꢀmethylꢀ1ꢀbutanol (82 mL) was heated at 150 °C for 45 hours.
The volatiles were removed in vacuo and the residue poured into 350 mL CH2Cl2 and washed
with an aqueous solution of NaOH (0.5 M, 200 mL). The organic phase was dried over Na2SO4
and concentrated in vacuo. The crude product was purified by column chromatography (SiO2,
CH2Cl2/MeOH 95:5 to 9:1) to give 2ꢀ(4ꢀethylꢀ6ꢀmethylꢀpyrazolo[1,5ꢀa]pyrazinꢀ2ꢀyl)ꢀ9ꢀmethylꢀ
1
7ꢀ(4ꢀpiperidyl)pyrido[1,2ꢀa] pyrimidinꢀ4ꢀone 16 (3.05 g, 25%) as an offꢀwhite solid. H NMR
(600 MHz, DMSOꢀd6) δ ppm: 8.68 (s, 1H), 8.56 (s, 1H), 7.92 (s, 1H), 7.60 (d, J=0.9 Hz, 1H),
6.99 (s, 1H), 3.07 ꢀ 3.14 (m, 3H), 3.07 ꢀ 3.14 (m, 1H), 2.74 ꢀ 2.84 (m, 1H), 2.63 ꢀ 2.67 (m, 3H),
2.62 ꢀ 2.70 (m, 2H), 2.45 (s, 3H), 1.83 (br d, J=13.0 Hz, 2H), 1.58 (qd, J=12.3, 3.8 Hz, 2H), 1.34
(t, J=7.6 Hz, 3H); LCꢀHRMS: m/z=402.21862 [(M+H)+ calcd for C23H26N6O: 402.21681; Diff
1.8 mDa].
Step J. To a suspension of 2ꢀ(4ꢀethylꢀ6ꢀmethylꢀpyrazolo[1,5ꢀa]pyrazinꢀ2ꢀyl)ꢀ9ꢀmethylꢀ7ꢀ
(4ꢀpiperidyl)pyrido[1,2ꢀa]pyrimidinꢀ4ꢀone 16 (5.79 g, 14.4 mmol) in CH2Cl2 (91 mL) cooled
down to 5ꢀ10 °C, was added sodium triacetoxyborohydride (4.7 g, 21.6 mmol) and a
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formaldehyde solution (3.22 mL, 37 wt% in H2O, 43.2 mmol) dropwise over 5ꢀ10 minutes. After
an additional 30 minutes, the reaction was quenched by addition of an aqueous saturated solution
of K2CO3 (30 mL). The reaction mixture was poured into 500 mL of CH2Cl2 and washed with
brine. The organic phase was dried over Na2SO4 and concentrated in vacuo. The crude material
was purified by column chromatography (SiO2, CH2Cl2/MeOH 9:1 to 4:1) to give 2ꢀ(4ꢀethylꢀ6ꢀ
methylꢀpyrazolo[1,5ꢀa]pyrazinꢀ2ꢀyl)ꢀ9ꢀmethylꢀ7ꢀ(1ꢀmethylꢀ4ꢀpiperidyl)pyrido[1,2ꢀa]pyrimidinꢀ
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4ꢀone 3 (4.05 g ,69%) as an offꢀwhite solid. H NMR (600 MHz, CHLOROFORMꢀd) δ ppm:
8.86 (d, J=1.7 Hz, 1H), 8.12 (s, 1H), 7.59 (s, 1H), 7.44 (d, J=1.0 Hz, 1H), 7.27 (s, 1H), 3.00ꢀ3.15
(m, 4H), 2.72 (s, 3H), 2.57 ꢀ 2.67 (m, 1H), 2.54 (d, J=0.9 Hz, 3H), 2.39 (br s, 3H), 2.16 (br s, 2H)
1.93 (br s, 4H), 1.45 (t, J=7.6 Hz, 3H); LCꢀHRMS: m/z=416.23403 [(M+H)+ calcd for
C24H28N6O: 416.23357; Diff 1.1 mDa].
2-(2,8-Dimethylimidazo[1,2-a]pyridin-6-yl)-7-(4-methylpiperazin-1-yl)pyrido[1,2-
a]pyrimidin-4-one (4). According to scheme 2: Step A. Preparation of 22. (i) A mixture of 2ꢀ
aminoꢀ5ꢀfluoropyridine 21 (11.20 g, 0.10 mol) and dimethyl malonate (57.0 mL, 0.50 mol) was
heated at 230 °C for 1.5 hours. After cooling to RT, the precipitate was collected by filtration and
washed with CH3CN to give 7ꢀfluoroꢀ2ꢀ hydroxyꢀ4Hꢀpyrido[1,2ꢀa]pyrimidinꢀ4ꢀone as a light
brown solid, which was used directly in the next step. (ii) A mixture of crude 7ꢀfluoroꢀ2ꢀ
hydroxyꢀ4Hꢀpyrido[1,2ꢀ a]pyrimidinꢀ4ꢀone in POC13 (50 mL) and iꢀPr2NEt (13.3 mL, 77 mmol)
was heated at 110 °C for 15 hours. The volatiles were removed and the dark residue was treated
with iceꢀwater, washed with water (3x) and dried to give a brown solid. The crude brown solid
was purified by column chromatography (SiO2, CH2C12/MeOH 95:5) to give 2ꢀchloroꢀ7ꢀfluoroꢀ
1
pyrido[1,2ꢀa]pyrimidinꢀ4ꢀone 22 (9.84 g, 50%) as a yellow solid. H NMR (600 MHz, DMSOꢀ
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d6) δ ppm: 8.99 (dd, J=4.7, 2.9 Hz, 1H), 8.25 (ddd, J=9.8, 7.1, 2.9 Hz, 1H), 7.85 (dd, J=9.7, 5.3
Hz, 1H), 6.56 (s, 1H); LCꢀHRMS: m/z=198.00032 [(M+H)+ calcd for C8H4ClFN2O: 197.99962;
Diff 0.7 mDa].
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Step B. Preparation of 23-g. To a solution of 2,8ꢀdimethylꢀ6ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
dioxaborolanꢀ2ꢀyl)imidazo[1,2ꢀa]pyridine (6.91 g, 25.4 mmol) in CH3CN (240 mL) was added
Pd(Ph3P)4 (1.22 g, 1.06 mmol), 2ꢀchloroꢀ7ꢀfluoroꢀpyrido[1,2ꢀa]pyrimidinꢀ4ꢀone 22 (4.2 g, 21.1
mmol) and an aqueous solution of K2CO3 (21.1 mL, 2M, 21.1 mmol). The reaction mixture was
heated at 80 °C for 7 hours and concentrated in vacuo. Purification of the residue by column
chromatography (SiO2, CH2Cl2/MeOH 98:2 to 95:5) gave 2ꢀ(2,8ꢀdimethylimidazo[1,2ꢀa]pyridinꢀ
6ꢀyl)ꢀ7ꢀfluoroꢀpyrido[1,2ꢀa]pyrimidinꢀ4ꢀone 23-g (4.57 g, 70%) as a light yellow solid.
Preparation
of
2,8ꢀdimethylꢀ6ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)imidazo[1,2ꢀa]
pyridine: (i) A solution of 5ꢀbromoꢀ3ꢀmethylpyridinꢀ2ꢀamine (10 g, 51.9 mmol) and
chloroacetone (5.22mL, 62.2 mmol) in CH3CN (100 mL) was heated at 110°C for 6 hours and
then at 100 °C overnight. The reaction was cooled down to RT before an aqueous saturated
solution of NaHCO3 (200 mL) was added. The product was extracted several times with EtOAc,
the combined organic phases were dried over MgSO4 and purification by column
chromatography (SiO2, EtOAc/Heptane 1:2) gave 6ꢀbromoꢀ2,8ꢀdimethylꢀimidazo[1,2ꢀa]pyridine
(9.46 g, 81%) as a light brown solid. (ii) A solution of 6ꢀbromoꢀ2,8ꢀdimethylimidazo[1,2ꢀ
a]pyridine (1.00 g, 4.44 mmol), 4,4,4',4',5,5,5',5'ꢀoctamethylꢀ2,2'ꢀbi(1,3,2ꢀdioxaborolane) (1.24
g, 4.89 mmol), potassium acetate (872 mg, 8.89 mmol) and PdCl2(DPPF)ꢀCH2Cl2 (163 mg,
0.222 mmol) in dioxane (35 mL) was heated at 80 °C overnight. The reaction mixture was
cooled down to RT, filtered through a pad of celite, concentrated under vacuo and the residue
was used directly in the next step without further purification.
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Step C. To a solution of 2ꢀ(2,8ꢀdimethylimidazo[1,2ꢀa]pyridinꢀ6ꢀyl)ꢀ7ꢀfluoroꢀpyrido[1,2ꢀ
a]pyrimidinꢀ4ꢀone 23-g (6.4 g, 20.8 mmol) in DMSO (120 mL) was added K2CO3 (2.87 g, 20.8
mmol) and 1ꢀmethylpiperazine (6.91 mL, 62.3 mmol). The reaction mixture was heated at 110
°C for 48 hours. The reaction was concentrated under vacuo and the residue purified by column
chromatography (SiO2, CH2Cl2/MeOH 98:2 to to 9:1) to give 2ꢀ(2,8ꢀdimethylimidazo[1,2ꢀ
a]pyridinꢀ6ꢀyl)ꢀ7ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)pyrido[1,2ꢀa]pyrimidinꢀ4ꢀone 4 (4.76 g, 59%) as a
yellow solid. 1H NMR (600 MHz, DMSOꢀd6) δ ppm 9.20 (s, 1 H), 8.25 (d, J=2.6 Hz, 1 H), 8.10
(dd, J=9.7, 2.8 Hz, 1H), 7.78 (d, J=4.1 Hz, 1H), 7.77 ꢀ 7.79 (m, 1H), 7.70 (d, J=9.7 Hz, 1H), 6.94
(s, 1H), 3.22 ꢀ 3.29 (m, 4H), 2.53 ꢀ 2.66 (m, 4H), 2.52 (s, 3 H), 2.36 (s, 3 H), 2.24 ꢀ 2.34 (m, 3
H); LCꢀHRMS: m/z=388.19897 [(M+H)+ calcd for C22H24N6O: 388.201159448; Diff ꢀ2.2 mDa].
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2-(8-Fluoro-2-methyl-imidazo[1,2-a]pyridin-6-yl)-7-(4-methylpiperazin-1-yl)pyrido
[1,2-a]pyrimidin-4-one (5). According to scheme 2: Step B. Preparation of 23ꢀh. To a solution
of
8ꢀfluoroꢀ2ꢀmethylꢀ6ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)imidazo[1,2ꢀa]pyridine
(5.18 g, 18.8 mmol) in acetonitrile (160 mL) was added Pd(Ph3P)4 (1.08 g, 0.94 mmol), 2ꢀchloroꢀ
7ꢀfluoroꢀpyrido[1,2ꢀa]pyrimidinꢀ4ꢀone 22 (4.47 g, 22.5 mmol) and an aqueous solution of K2CO3
(18.8 mL, 2M, 37.6 mmol). The reaction mixture was heated at 80 °C for 5 hours and
concentrated in vacuo. Purification of the residue by column chromatography (SiO2,
CH2Cl2/MeOH 98:2 to 95:5) gave 7ꢀfluoroꢀ2ꢀ(8ꢀfluoroꢀ2ꢀmethylꢀimidazo[1,2ꢀa]pyridinꢀ6ꢀ
yl)pyrido[1,2ꢀa]pyrimidinꢀ4ꢀone 23ꢀh (3.59 g, 51%) as a light yellow solid.
Preparation of 8ꢀfluoroꢀ2ꢀmethylꢀ6ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)imidazo[1,2ꢀ
a]pyridine: (i) An orange solution of 5ꢀbromoꢀ3ꢀfluoropyridinꢀ2ꢀamine (5.20 g, 27.2 mmol),
PPTS (0.684 g, 2.72 mmol) and 1ꢀbromoꢀ2,2ꢀdimethoxypropane (4.05 mL, 29.9 mmol) in 2ꢀ
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propanol (65 mL) was heated at 80 °C overnight. The reaction was cooled down to RT before an
aqueous saturated solution of NaHCO3 (100 mL) was added. The product was extracted several
times with EtOAc, the combined organic phases were dried over MgSO4 and purification by
column chromatography (SiO2, EtOAc/Heptane 1:4 to 1:1) gave 6ꢀbromoꢀ8ꢀfluoroꢀ2ꢀmethylꢀ
imidazo[1,2ꢀa]pyridine (4.80 g ,77%) as a yellow solid. (ii) A solution of 6ꢀbromoꢀ8ꢀfluoroꢀ2ꢀ
methylꢀimidazo[1,2ꢀa]pyridine (1.58 g, 6.90 mmol), 4,4,4',4',5,5,5',5'ꢀoctamethylꢀ2,2'ꢀbi(1,3,2ꢀ
dioxaborolane) (2.21 g, 8.69 mmol), potassium acetate (2.03 g, 20.7 mmol) and PdCl2(DPPF)ꢀ
CH2Cl2 (282 mg, 0.345 mmol) in dioxane (32 mL) was heated at 90 °C for 5 hours. The reaction
mixture was cooled down to RT, filtered through a pad of celite, concentrated in vacuo and the
residue was used directly in the next step without further purification.
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Step C. To a solution of 7ꢀfluoroꢀ2ꢀ(8ꢀfluoroꢀ2ꢀmethylꢀimidazo[1,2ꢀa]pyridinꢀ6ꢀ
yl)pyrido[1,2ꢀa]pyrimidinꢀ4ꢀone 23-h (145 mg, 0.46 mmol) in DMSO (2.0 mL) was added
K2CO3 (96 mg, 0.697 mmol) and 1ꢀmethylpiperazine (0.15 mL, 1.39 mmol). The reaction
mixture was heated at 120 °C for 7 hours. The reaction was concentrated in vacuo and the
residue purified by column chromatography (SiO2, CH2Cl2/MeOH 98:2 to to 9:1) to give 2ꢀ(8ꢀ
fluoroꢀ2ꢀmethylꢀimidazo[1,2ꢀa]pyridinꢀ6ꢀyl)ꢀ7ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)pyrido[1,2ꢀa]pyrimidinꢀ
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4ꢀone 5 (75 mg, 42%) as a yellow solid. H NMR (600 MHz, CHLOROFORMꢀd) δ ppm: 8.77
(d, J=1.4 Hz, 1H), 8.43 (d, J=2.6 Hz, 1H), 7.70 (d, J=2.7 Hz, 1H), 7.61 – 7.67 (m, 1H), 7.46 –
7.52 (m, 2H), 6.75 (s, 1H), 3.24 ꢀ 3.38 (m, 4H), 2.65 (br s, 4H), 2.51 (d, J=0.7 Hz, 3H), 2.40 (d,
3H); LCꢀHRMS: m/z=392.17532 [(M+H)+ calcd for C21H21FN6O: 392.17609; Diff 0.77 mDa].
2-(4,6-Dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(4-methylpiperazin-1-yl)pyrido[1,2-
a]pyrimidin-4-one (12). According to scheme 1: Step C. Preparation of 9-a. To a suspension of
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ethyl 4ꢀchloroꢀ6ꢀmethylpyrazolo[1,5ꢀa]pyrazineꢀ2ꢀcarboxylate 8 (6.7 g, 28.0 mmol) in THF (265
5
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mL) at
0
°C was added dimethylzinc (28.0 mL, 28.0 mmol) and 1,3ꢀ
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bis(diphenylphosphino)propaneꢀnickel(II) chloride (1.52 g, 2.8 mmol). The temperature was
raised to RTand the reaction stirred one additional hour. The reaction mixture was poured into
EtOAc (500 mL) and washed with H2O400 mL. The organic phase was dried over Na2SO4 and
concentrated in vacuo. The crude material was purified by column chromatography (SiO2,
EtOAc/Heptane 1:1) to give ethyl 4,6ꢀ dimethylpyrazolo[1,5ꢀa]pyrazineꢀ2ꢀcarboxylate 9-a (5.01
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g, 82%) as a yellow solid. H NMR (600 MHz, DMSOꢀd6) δ ppm: 8.54 (d, J=0.7 Hz, 1H), 7.48
(d, J=1.0 Hz, 1H), 4.36 (q, J=7.1 Hz, 2H), 2.65 ꢀ 2.73 (m, 3H), 2.42 (d, J=0.9 Hz, 3H), 1.34 (t,
J=7.1 Hz, 3H); LCꢀHRMS: m/z=219.10176 [(M+H)+ calcd for C11H13N3O2: 219.10078; Diff 1
mDa].
Step D. Preparation of 10-a. A solution of ethyl 4,6ꢀdimethylpyrazolo[1,5ꢀa]pyrazineꢀ2ꢀ
carboxylate 9-a (25.12 g, 115 mmol)and EtOAc (50.5 g, 56.1 mL, 575 mmol) in THF (1.50 L)
was cooled down to ꢀ40 °C. LiHMDS (286 mL, 1M in THF/Ethylbenzene, 286 mmol) was
slowly added over 1 hour and stirring was continued for one additional hour at ꢀ40 °C (dark
solution). The reaction was quenched by addition of an aqueous saturated NH4Cl solution (200
mL). The reaction mixture was concentrated in vacuo, poured into EtOAc and then washed
several times with water. The organic phase was dried over Na2SO4, and concentrated in vacuo.
The residue was purified by column chromatography (SiO2, CH2Cl2/EtOAc 9:1 to 8:2) to give
ethyl 3ꢀ(4,6ꢀdimethylpyrazolo[1,5ꢀa]pyrazinꢀ2ꢀyl)ꢀ3ꢀoxoꢀpropanoate 10-a (23.02 g, 77%) as a
yellow solid.
Step E. Preparation of 11-a. A mixture of ethyl 3ꢀ(4,6ꢀdimethylpyrazolo [1,5ꢀa]pyrazinꢀ2ꢀ
yl)ꢀ3ꢀoxoꢀpropanoate 10-a (261 mg, 1.0 mmol), 2ꢀaminoꢀ5ꢀfluoropyridine (134 mg, 1.2 mmol)
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