Discovery of a Novel Class of Survival Motor Neuron 2 Splicing

Article pubs.acs.org/jmc

Discovery of a Novel Class of Survival Motor Neuron 2 Splicing Modifiers for the Treatment of Spinal Muscular Atrophy Emmanuel Pinard,*,† Luke Green,† Michael Reutlinger,† Marla Weetall,‡ Nikolai A. Naryshkin,‡ John Baird,‡ Karen S. Chen,§ Sergey V. Paushkin,§ Friedrich Metzger,† and Hasane Ratni*,† †

F. Hoffmann-La Roche Ltd., pRED, Pharma Research & Early Development, Roche Innovation Center Basel, Grenzacherstrasse 124, 4070 Basel, Switzerland ‡ PTC Therapeutics, Inc., 100 Corporate Court, South Plainfield, New Jersey 07080, United States § SMA Foundation, 888 Seventh Avenue, Suite 400, New York, New York 10019, United States S Supporting Information *

ABSTRACT: Spinal muscular atrophy (SMA) is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene, resulting in low levels of functional SMN protein. We have reported recently the identification of small molecules (coumarins, iso-coumarins and pyrido-pyrimidinones) that modify the alternative splicing of SMN2, a paralogous gene to SMN1, restoring the survival motor neuron (SMN) protein level in mouse models of SMA. Herein, we report our efforts to identify a novel chemotype as one strategy to potentially circumvent safety concerns from earlier derivatives such as in vitro phototoxicity and in vitro mutagenicity associated with compounds 1 and 2 or the in vivo retinal findings observed in a long-term chronic tox study with 3 at high exposures only. Optimized representative compounds modify the alternative splicing of SMN2, increase the production of full length SMN2 mRNA, and therefore levels of full length SMN protein upon oral administration in two mouse models of SMA.

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INTRODUCTION Spinal muscular atrophy (SMA)1 is an orphan disease and the leading genetic cause of mortality in children and toddlers.2 Survival motor neuron protein (SMN) is ubiquitously present in periphery and central nervous system (CNS), with a minimal level mandatory for normal development in all species.3 In humans, two paralogous genes, the SMN1 and SMN2 produces this protein, however, its level is mainly driven by the SMN1 gene. The only functionally relevant difference between the two genes is a silent C to T mutation in exon 7 of SMN2, which results in alternative splicing, i.e., exclusion of exon 7 from a large fraction of mRNA transcript and production of an unstable, truncated SMNΔ7 protein. SMA is caused by genetic defect in the SMN1 gene, rendering it incapable of producing the SMN protein. A lower level of SMN protein (produced only by the SMN2 gene) results in loss of alpha motor neurons and progressive muscular atrophy.4 Humans usually have two or more copies of the SMN2 gene in a somatic cell.4b Our strategy to find a treatment for SMA has focused on the discovery of small molecules that potently move the alternative splicing of SMN2 exon 7 toward the production of full length SMN mRNA and protein. In the past decade, a number of therapeutic approaches to treat SMA have been considered.5 Among them, the promising splicing correction of SMN2 was achieved either by intrathecal administration of antisense oligonucleotides (ASOs)6 (nusinersen recently approved by the FDA, December 2016) or by small molecules. Small molecules possess the inherent © 2017 American Chemical Society

advantage of oral administration and broad distribution increasing the SMN protein both centrally and peripherally as initially reported by us7 and by others (Novartis, 5-(1Hpyrazol-4-yl)-2-[6-[(2,2,6,6-tetramethyl-4-piperidyl)oxy]pyridazin-3-yl]phenol (LMI070), currently in phase 2 clinical trials).8 We recently described three classes of small molecules (coumarin 1, iso-coumarin 2, and pyrido-pyrimidinone derivatives)9 with high in vitro and in vivo potency, leading to functional improvement in mice that model a severe SMA form.7 Further development of the compounds 1 and 2 was prevented due to in vitro safety flags.9a For instance, 1 and 2 were found positive in the Ames assay, as well as in tests for phototoxicity, and suboptimal physicochemical properties. Putting our attention on the pyrido-pyrimidinone series has led to the discovery of compound 3 (RG7800),9a which was the first orally active small molecule SMN2 splicing modifier to enter human trials for the potential treatment of SMA (Figure 1). This compound was recently put on clinical hold due to a retinal finding observed in the long term chronic tox study at high exposures only. We hypothesized that the liabilities associated with 1 and 2 (in vitro phototoxicity and genotoxicity) were at least partly caused by the high aromaticity and electronic conjugation of the central fused bicyclic cores. Polycyclic aromatic systems are Received: March 14, 2017 Published: April 25, 2017 4444

DOI: 10.1021/acs.jmedchem.7b00406 J. Med. Chem. 2017, 60, 4444−4457

Journal of Medicinal Chemistry

Article

Figure 1. Coumarin 1, iso-coumarin 2, and pyrido-pyrimidinone 3.

Figure 2. Structure of compounds 2 and 4.

Scheme 1. Synthesis of the Benzamide Derivatives 9−12a

a Conditions: (a) 6,8-dimethylimidazo[1,2-a]pyrazin-2-amine hydrochloride (1 equiv), i-Pr2NEt (5 equiv), dioxane, RT, 1 h, 83−89%; (b) amine (1−10 equiv), DMA, 100−180 °C, 18−40%.

Scheme 2. Synthesis of Benzamide Derivative 15a

a Conditions: (a) 6-bromo-2-methyl-imidazo[1,2-a]pyrazine (1.0 equiv), CuI (0.1 equiv), 1,10-phenantroline (0.2 equiv), K3PO4 (2.1 equiv), dioxane, sealed tube, 120 °C, 15%; (b) (i) tert-butyl piperazine-1-carboxylate (2.0 equiv), Pd2(dba)3 (0.08 equiv), Xantphos (0.12 equiv), NaOtBu (1.5 equiv), toluene,120 °C, (ii) HCl, dioxane, RT, 16% over two steps.

considered as an Ames toxicophore.10 Moreover, multiple analyses showed that the fewer aromatic rings contained in an oral drug candidate, the more developable that candidate is predicted to be.11 To potentially circumvent the issue associated with 3, two options were offered to us: one would be to remain in the same chemical class and further increase the potency on target to increase the safety window while a second option would be to aim for a novel chemotype with a maximum of diversity. We describe here our strategy following the second option, aiming for a novel chemical class and with a reduced electronic conjugation and lower aromatic rings while keeping an overall flat structure to maintain the potency. This effort led us to the identification through rational drug design of a novel

series having a benzamide as a core. Optimal derivatives, such as compound 4, demonstrate an excellent in vitro and in vivo profile (Figure 2).

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RESULTS AND DISCUSSION

Chemistry. The benzamide derivatives were prepared in a straightforward manner using three synthetic routes (Schemes 1−3).12 The first route (Scheme 1) was used specifically for the compounds bearing an imidazopyrazine moiety as the righthand side fragment. A substituted benzoyl chloride 5 or 6 was coupled with 6,8-dimethylimidazo[1,2-a]pyrazin-2-amine to produce the amide compounds 7 and 8. Those intermediates 4445



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Scheme 3. Synthesis of Benzamide Derivatives 4, 23, and 24a

Conditions: (a) amine (1.1 equiv), Cs2CO3 (1.5 equiv), Pd2(dba)3, or Pd(OAc)2 (0.02 equiv), rac-BINAP (0.08 equiv), toluene, 100 °C, 55−89%; (b) (i) NaOH (2.5 equiv), MeOH, H2O, RT to 50 °C, (ii) CDI (1.5 equiv), NH4OH (3.0 equiv), DMF, RT, 60−85%; (c) (i) 6-bromo-2-methylimidazo[1,2-a]pyrazine (1.2 equiv), Xantphos (0.01 equiv), Pd2(dba)3 (0.25 equiv), Cs2CO3 (1.2 equiv), dioxane, 100 °C 60−77%, (ii) HCl, dioxane, RT, quantitative. a

Figure 3. Two approaches for the design of alternative central cores.

Fortunately, the second approach provided us with our first compound displaying moderate activity in the micromolar range (compound 9, Table 2). Its potency was further corroborated with the analogous benzamide derivatives 10 and 37 (entries 10 and 11, Table 2). This first prototype compound 9 was subjected to multiple safety and physicochemical properties assessment in vitro. In agreement with our initial hypothesis, 9 was devoid of any phototoxicity and had no flag in the Ames assay unlike the coumarin 1 or isocoumarin 2. In addition, 9 was also found negative in the GSH assay (indicative of covalent binding), had no affinity for the hERG channel (IC20 > 10 μM), and did not inhibit the cytochrome P450 enzymes (IC50 > 50 μM for 3A4, 2D6, 2C9). With respect to physicochemical properties, 9 displayed a high aqueous solubility (Lysa: 111 μg/mL), is highly permeable (Pe: 2.38 × 10−6 cm/s), has a good lipophilicity for a CNS drug (log D: 2.0), and is in vitro metabolically stable. Finally, it is both chemically stable at various pH levels as well as in plasma (human and rodent), excluding a potential amide bond hydrolysis. Interestingly, the analogous tertiary amide 36 was found completely inactive (Table 2, entry 9). A gas phase analysis of the torsional angle of the bond between the phenyl and the carbonyl amide showed that while the minimal energy conformation for the secondary amide 9 leads nearly to a planar configuration with two minima (Figure 4B), for the tertiary amide 36, the carbonyl group clearly adopts an out of plane conformation (Figure 4C). This further emphasized the importance of the planarity of the central core. We therefore decided to lock the putative planar active conformation. We prepared compound 11 containing a fluorine atom on the phenyl ring to form an intramolecular H-bond and the pyridyl analogue 38 to generate a lone pair repulsion between the pyridyl nitrogen and the

can subsequently undergo a nucleophilic aromatic substitution with various piperazines, leading to derivatives 9−12. The preparation of compound 15 is conveniently carried out in only two steps (Scheme 2). It started with a copper catalyzed Ullman type coupling to generate the versatile intermediate 14, albeit with a low yield due to side products formations (homocouplings). This derivative 14 could undergo palladium catalyzed Buchwald type coupling with a wide range of amines or, more specifically, with the mono boc-protected piperazine to form 15 after deprotection. Alternatively, the sequence of Ullman/Buchwald couplings could be interchanged to prepare the derivatives 4, 23, and 24 (Scheme 3) in a higher overall yield. A Buchwald type coupling between the methyl 4-bromo-2-fluoro-benzoate 16 and a bocprotected substituted piperazine gave the intermediates 17−19. The ester function was converted into the corresponding primary amides 20−22, which subsquetly were submitted to a copper catalyzed coupling with 6-bromo-2-methyl-imidazo[1,2a]pyrazine, affording 4, 23, and 24. Lead Optimization. To break the electronic conjugation of the compounds 1−3 (and reduce the aromaticity), the rational replacement of the coumarin, isocoumarin, or pyridopyrimidinone core was done following two strategies. The first approach (Figure 3) was to use an aromatic ring as the right-hand side ring of the isocoumarin core while its left-hand side phenyl ring was replaced by a bioisostere. In the second approach, the replacement was introduced in place of the righthand side part of the original core (Figure 3). Following the first approach, we evaluated both saturated cyclic and acyclic replacements while preserving the planarity of the central core and the geometry of the exit vectors. Unfortunately, all modifications led to a complete loss of activity and this approach was no longer considered (Table 1). 4446



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Table 1. In Vitro Potency of Compounds with the First Type of Alternative Central Core

compound with high permeability and limited P-gp efflux, good brain penetration (brain/plasma ratio of 3.4) was observed in a mouse in vivo PK study. Because the plasma fraction unbound was found to be high (human, mouse: 18.6%, 19.7%), the predicted free plasma concentration were high. We next evaluated the pharmacodynamic profile of the benzamide derivative 12 in adult C/C-allele SMA mouse model. The compound 12 was given orally (qd) for 10 days at three doses. We assessed the level of the SMN protein in the brain and quadriceps muscle for which a dose dependent increase in SMN protein levels was observed (Table 4). Compound 12 was then tested in Δ7 SMA mice, a model of severe SMA.14 The compound was administered by intraperitoneal (ip) injection once a day, from postnatal day 3 (P3) until day 9 (P9). We then assessed the SMN protein level in brain and quadriceps muscle (Table 5 and also observed a dose depenant increase. No further survival efficacy assessment was performed, as we have already demonstrated that significant survival benefit can be clearly expected from any compounds raising brain SMN protein beyond 50%.9a,15 Although all benzamide derivatives tested in vitro for chemical and plasma stability did not show any hydrolysis of the amide bond, this compound class still bears an intrinsic risk of formation of an aromatic amine in humans (Figure 7).

carbonyl of the amide and a favorable electrostatic interaction with the amide NH (Figure 5).13 Encouragingly, this improved the potency significantly, particularly for the compound 11, being now in a submicromolar range. This is in line with a perfect planarity and a locked conformation into a single conformer in the putative active mode for compounds 11 and 38 as shown by the in silico analysis (Figure 6). We subsequently turned our attention to the basic amine side chain and prepared a small set of analogues (Table 3). While a wide range of amines were tolerated, a substantial increase of potency was achieved with compounds 12 and 41 (SMN protein EC1.5x of 98 and 86 nM) both as active as our starting points: iso-coumarin 2 or 3 (SMN protein EC1.5x of 120 and 87 nM). A full in vitro characterization of compound 12 in terms of safety and physicochemical properties was performed. Satisfyingly, the promising profile of the first in vitro active benzamide prototype compound 9 was maintained (no flag on Ames, phototoxicity, GSH, hERG, CYP inhibition, and with high solubility, permeability, and stability). Compound 12 was further assessed for interaction with P-glycoprotein (P-gp). While not a human P-gp substrate, it was only a moderate mouse P-gp substrate (efflux ratio of 4.1). As expected for a 4447



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Table 2. In Vitro Potency of Compounds with the Second Type of Alternative Central Core

Primary aromatic amines have the potential to cause toxicity through mutagenicity. The toxicity can arise through a series of metabolic steps leading to a reactive electrophilic nitrenium cation intermediate that reacts with DNA nucleotides causing mutation. Therefore, we decided to focus on benzamide compounds for which the potentially formed corresponding

primary aromatic amine is negative in the Ames assay (Table 6). Strinkingly, with respect to the SAR for SMN in vitro potency, with 5,6 fused bicyclics, small modifications such as exchange of carbon with nitrogen on the five-membered ring cycle (e.g., 12 to 44) was detrimental. In contrast, with 6,5 4448



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Figure 4. (A) Overlay of 9 in orange and 36 in gray; gas phase QM analysis and CSD statistics of the torsional angle of the phenyl-carbonyl bond: (B,C) compound 9; (D,E) compound 36.

monofluorinated central phenyl was the best choice. We have observed in that series as well as with our earlier series,9a a good correlation between the lipophilicity and the p-gp efflux ratio. The more polar the compound, the higher the efflux ratio was. The basic amine side chain was then functionalized to increase the lipophilicity in order to prevent the interaction with P-gp (entries 8−11). The most potent alkyl groups were either a methyl or ethyl (R and S). The compounds 4 and 24 offered the best compromise in terms of potency, lipophilicity (not a P-gp substrate), as well as an excellent overall profile. These results supported their pharmacodynamic evaluation in our two in vivo SMA mouse models (Table 8). Both compounds demonstrated a very strong and a clear dose dependent increase in SMN protein in the adult C/Callele and neonatal Δ7 mouse model.

fused systems, much higher structural diversity was tolerated as seen with 45, 46, 47, and 15. Disappointingly, the corresponding primary amine of 12, the 6,8-dimethylimidazo[1,2-a]pyrazin-2-amine (Table 6, entry 1) was found positive. However, we could identify alternative aromatic amines that were negative in the Ames test leading to potent final benzamide derivatives. Of particular interest was 15, being even more potent than compound 12 (entries 6 vs 1, Table 6). Next, we maintained this right-hand side moiety, the imidazopyrazine, and fine-tuned the substitution pattern of the central benzamide moiety and the amine side chain (Table 7). As expected, disrupting the planarity by removing the fluorine atom (entry 2) or adding an extra chlorine atom (entry 3) led to a loss of potency. Replacing the fluorine atom by a methoxy group, capable of retaining planarity by forming an intramolecular H-bond, maintained potency (entry 4). Difluorination led to a loss of potency (entries 6 and 7). The 4449



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Figure 5. Locking of the putative active conformation.

Figure 6. Gas phase QM analysis of the torsional angle of the bond between the phenyl and the carbonyl: (A) compound 9; (B) compound 11; (C) compound 38.

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CONCLUSION

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 chromatography−mass 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 (32−60 mesh, 60 Å). LC-MS high resolution spectra were recorded with an Agilent LC-system consisting of an Agilent 1290 high pressure system, an Agilent 1290 multisampler, and an Agilent 6545 QTOF. The separation was achieved on a Zorbax Eclipse Plus C18 1.7 μm 2.1 mm × 50 mm column at 55 °C; A = 0.02% formic acid in water; B = acetonitrile with 0.01% formic acid at flow 0.8 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 DMSO-d6 solvent signal was used as reference with 2.50 ppm. 4-[(3R)-3-Ethylpiperazin-1-yl]-2-fluoro-N-(2-methylimidazo[1,2a]pyrazin-6-yl)benzamide (4). According to Scheme 3. Step A: Preparation of 18. To a solution of methyl 4-bromo-2-fluoro-benzoate (450 mg, 1.93 mmol) in dioxane (8.0 mL) was added tert-butyl (2R)2-ethylpiperazine-1-carboxylate (497 mg, 2.32 mmol), Pd(OAc)2 (89 mg, 5.79 mmol), rac-BINAP (240 mg, 0.386 mmol), and Cs2CO3

In summary, a novel chemical class of orally bioavailable SMN2 splicing modifiers has been discovered by rational drug design. Reducing the aromaticity and the electronic conjugation of the fused bicyclic central core of the derivatives 1−3 was critical to address successfully safety concerns related to the coumarin 1 and isocoumarin 2. Chemical optimization of this new benzamide series for in vitro potency was mostly achieved by designing key intramolecular H-bonding interaction. A careful selection of the right-hand side aromatic amine fragment was performed to prevent any potential late stage issue (in case it is formed upon amide hydrolysis). Finally, optimizing the amine side chain by incorporating small alkyl residues allowed us to maintain the lipophilicity in a range reducing the likelihood of P-gp efflux. This effort culminated in the discovery of the orally bioavailable SMN2 splicing modifiers 4 and 24. Both exhibited excellent pharmacokinetic profiles, in vivo pharmacodynamic profile in two SMA mouse models (SMN protein increase in brain and in periphery) and have favorable safety profiles. These results suggested that our splicing modifier compounds could provide a therapeutic benefit for SMA patients. 4450



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Table 3. In Vitro Potency of Compounds with Improved Basic Amine Side Chains

Table 4. Pharmacokinetics and SMN Induction Adult C/C-Allele SMA Mice

a

compd

dose (mg/kg PO)

vehicle 12 12 12

0 1 3 10

total plasmaa AUC (μg·h/mL)

free plasma AUC (μg·h/mL)

0

0

7.9

1.58

% SMN ↑ in brain

% SMN ↑ in quadriceps

0 0 24 102

0 13 54 152

% SMN ↑ in brain

% SMN ↑ in quadriceps

0 24 75 150

0 14 24 140

Plasma AUC determined in a satellite group. Brain/plasma ratio of 3.4.

Table 5. Pharmacokinetics and SMN Induction in Neonatal Δ7 Mouse Model compd

dose (mg/kg ip)

vehicle 12 12 12

0 0.3 1 3

total plasmaa AUC (μg·h/mL)

free plasmab AUC (μg·h/mL)

0

0

3.9

0.79

a

Plasma AUC determined in a satellite group of neonatal (P10) wild-type mice. bThe free-fraction measured using serum from adult mice was used to calculate the free plasma AUC.

Figure 7. Potential hydrolysis products arising from the amide bond cleavage. (1.89 g, 5.79 mmol). The reaction mixture was heated at 100 °C for 2 h and cooled to RT. The solvent was removed in vacuum and the residue taken up in EtOAc and washed with H2O. The organic phase was dried over Na2SO4 and concentrated under vacuo, and a purification by column chromatography (SiO2, EtOAc/heptane) gave tert-butyl (2R)-2-ethyl-4-(3-fluoro-4-methoxycarbonyl-phenyl)-

piperazine-1-carboxylate 18 (630 mg, 89%) as a light-yellow oil. 1H NMR (300 MHz, chloroform-d) δ ppm: 7.82 (t, J = 8.80 Hz, 1H), 6.57 (dd, J = 2.52, 8.98 Hz, 1H), 6.45 (dd, J = 2.52, 14.63 Hz, 1H), 4.05−4.19 (m, 1H), 3.92−4.05 (m, 1H), 3.87 (s, 3H), 3.56−3.69 (m, 2H), 3.19 (ddd, J = 3.83, 11.50, 14.00 Hz, 1H), 3.11 (dd, J = 3.83, 12.92 Hz, 1H), 2.96 (dt, J = 4.00, 11.90 Hz, 1H), 1.67−1.80 (m, 1H), 4451



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Table 6. in Vitro Potency of the Parent Compounds 12, 15, and 44−47 and Ames Assessment of the Primary Aromatic Amine Residues

1.53−1.63 (m, 1H), 1.48 (s, 9H), 0.91 (t, J = 7.27 Hz, 3H). LC-HRMS (m/z): [M + H]+ calcd for C19H27N2O4 367.2028, found 367.2031; Diff 0.3 mDa. Step B: Preparation of 21. To a solution of tert-butyl (2R)-2-ethyl-4(3-fluoro-4-methoxycarbonyl-phenyl)piperazine-1-carboxylate 18 (870 mg, 2.37 mmol) in MeOH (15 mL) was added an aqueous NaOH solution (0.475 mL, 2.85 mmol). The resulting mixture was heated at reflux for 12 h, cooled to RT, and acidified with Amberlite IR120 resin. The resin was filtered off and the solvent concentrated under vacuo. The residue was dissolved in DMF (15 mL), and CDI (443 mg, 2.73 mmol) was added. After 1 h at RT, NH4OH (4.09 mL, 105 mmol) was added and stirring was further continued for an additional 1 h. The reaction mixture was then diluted with water and the product extracted with EtOAc. The organic phase was dired over Na2SO4 and concentrated under vacuo. A purification by column chromatography (SiO2, EtOAc/heptane) gave tert-butyl (2R)-4-(4-carbamoyl-3-fluorophenyl)-2-ethyl-piperazine-1-carboxylate 21 (708 mg, 85%) as a colorless oil. 1H NMR (300 MHz, chloroform-d) δ ppm: 7.99 (t, J = 9.10 Hz, 1H), 6.66 (dd, J = 2.42, 9.08 Hz, 1H), 6.56 (br s, 1H), 6.45 (dd, J = 2.42, 16.15 Hz, 1H), 5.56 (br s, 1H), 4.06−4.19 (m, 1H), 4.00 (br d, J = 12.51 Hz, 1H), 3.62 (br d, J = 12.31 Hz, 2H), 3.14−3.25 (m, 1H), 3.10 (dd, J = 3.94, 12.82 Hz, 1H), 2.95 (dt, J = 4.20, 11.70 Hz, 1H), 1.66−1.82 (m, 1H), 1.52−1.65 (m, 1H), 1.56 (s, 9H), 0.91 (t, J = 7.47 Hz, 3H). LC-HRMS (m/z): [M + H]+ calcd for C18H26N3O3 352.2031, found 352.2032; Diff 0.1 mDa.

Step C: Preparation of 4. In a sealed tube, tert-butyl (2R)-4-(4carbamoyl-3-fluoro-phenyl)-2-ethyl-piperazine-1-carboxylate 21 (820 mg, 2.33 mmol), 6-bromo-2-methyl-imidazo[1,2-a]pyrazine (594 mg, 2.8 mmol), Pd2(dba)3 (107 mg, 0.117 mmol), Xantphos (203 mg, 0.350 mmol), and Cs2CO3 (1.14 g, 3.5 mmol) were suspended in dioxane (15 mL). The reaction mixture was heated at 100 °C for 24 h and cooled to RT. The crude mixture was purified by column chromatography (SiO2, CH2Cl2/MeOH) to give the boc-protected derivative 4. Removal of the boc-protecting group was performed by dissolving the solid residue in dioxane (5 mL) and adding a 4 M HCl solution in dioxane (2.5 mL). After 1 h at RT, the solvent was removed under vacuo and the solid was triturated in Et2O and dried under vacuo to afford 4-[(3R)-3-ethylpiperazin-1-yl]-2-fluoro-N-(2methylimidazo[1,2-a]pyrazin-6-yl)benzamide hydrochloride 4 (585 mg, 60%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.53 (d, J = 3.63 Hz, 1H), 9.51 (d, J = 1.21 Hz, 1H), 9.31(br s, 2H), 9.06 (s, 1H), 8.23 (s, 1H), 7.69 (t, J = 9.00 Hz, 1H), 6.99 (dd, J = 2.42, 13.53 Hz, 1H), 6.95 (dd, J = 2.22, 7.47 Hz, 1H), 3.94−4.07 (m, 2H), 3.29−3.43 (m, 1H), 3.04−3.27 (m, 3H), 2.97 (dd, J = 11.10, 13.52 Hz, 1H), 1.58−1.80 (m, 2H), 1.03 (t, J = 7.57 Hz, 3H). LC-HRMS (m/z): [M + H]+ calcd for C20H23FN6O 383.1990, found 383.1994; Diff 0.4 mDa. N-(6,8-Dimethylimidazo[1,2-a]pyrazin-2-yl)-4-(4-methylpiperazin-1-yl)benzamide (9). According to Scheme 1. Step A: Preparation of 7. To a mixture of 6,8-dimethylimidazo[1,2-a]pyrazin-2-amine trihydrochloride (134 mg, 0.494 mmol) and iPr2NEt (0.420 mL, 2.47 4452



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Table 7. in Vitro Potency of the Fully Optimized Benzamide Derivatives

The reaction mixture was heated in a microwave at 160 °C for 90 min. The mixture was cooled to RT, and the resulting precipitate was collected by filtration, washed with Et2O, and dried to provide N-(6,8dimethylimidazo[1,2-a]pyrazin-2-yl)-4-(4-methylpiperazin-1-yl)benzamide 9 (4.5 mg, 18%) as an off-white solid. 1H NMR (300 MHz, chloroform-d) δ ppm: 8.60 (s, 1H), 8.20 (s, 1H), 7.84 (d, J = 8.88 Hz, 2H), 7.77 (s, 1H), 6.71−7.02 (m, 2H), 3.33−3.41 (m, 4H), 2.81 (s, 3H), 2.54−2.61 (m, 4H), 2.48 (s, 3H), 2.37 (s, 3H). LC-HRMS (m/ z): [M + H]+ calcd for C20H24N6O 365.2085, found 365.2091; Diff 0.6 mDa. N-(6,8-Dimethylimidazo[1,2-a]pyrazin-2-yl)-4-piperazin-1-ylbenzamide (10). In analogy to the preparation of 9 step B, from N(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-4-fluoro-benzamide 7 (20 mg, 0.07 mmol) and piperazine (60.6 mg, 0.70 mmol) was prepared

mmol) in dioxane (2.0 mL) was added dropwise a solution of 4fluorobenzoyl chloride (80 mg, 0.494 mmol) in dioxane (0.5 mL) at RT. The mixture was stirred for 1 h, and the solvent was removed in vacuum. The solid was taken in water, and the resulting suspension was stirred for 15 min. The solid was collected by filtration and dried to afford N-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-4-fluoro-benzamide 7 (125 mg, 89%) as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ ppm: 11.49 (s, 1H), 8.27−8.53 (m, 2H), 7.97−8.22 (m, 2H), 7.35 (t, J = 8.88 Hz, 2H), 2.63−2.81 (m, 3H), 2.37 (s, 3H). LCHRMS (m/z): [M + H]+ calcd for C15H13FN4O 285.1146, found 285.1152; Diff 0.6 mDa. Step B: Preparation of 9. To a solution of N-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-4-fluoro-benzamide 7 (20 mg, 0.070 mmol) in DMA (0.2 mL) was added 1-methylpiperazine (70.1 mg, 0.70 mmol). 4453



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Table 8. Pharmacokinetics and SMN Induction in C/C-Allele and Neonatal Δ7 Mouse Models neonatal Δ7 mouse model

adult C/C-allele mouse model

a

compd

dose (mg/kg PO)

total plasmaa

% SMN ↑ in brain

dose (mg/kg ip)

vehicle 4 4 4

0 1 3 10

0

19.0

0 19 45 217

0 0.3 1 3

24 24 24

1 3 10

27.1

22 81 260

0.3 1 3

total plasmaa 0

% SMN ↑ in brain

3.73

0 0 66 117

4.45

26 53 100

AUC in μg·h/mL; plasma AUC determined in a satellite group. (300 MHz, chloroform-d) δ ppm: 9.28 (d, J = 1.41 Hz, 1H), 8.89 (br d, J = 13.52 Hz, 1H), 8.75 (s, 1H), 8.05 (t, J = 8.50 Hz, 1H), 7.53 (s, 1H), 7.50 (dd, J = 2.00, 10.30 Hz, 1H), 7.44 (dd, J = 1.82, 11.50 Hz, 1H), 2.52 (s, 3H). LC-HRMS (m/z): [M + H]+ calcd for C14H10BrFN4O 349.0095, found 349.0099; Diff 0.4 mDa. Step B: Preparation of 15. To a solution of 4-bromo-2-fluoro-N-(2methylimidazo[1,2-a]pyrazin-6-yl)benzamide 14 (30 mg, 0.086 mmol) in toluene (2 mL) was added tert-butyl piperazine-1-carboxylate (32 mg, 0.172 mmol), Pd2(dba)3 (6.7 mg, 0.0068 mmol), Xantphos (5.9 mg, 0.0103 mmol), and NaOtBu (12.4 mg, 0.129 mmol). The reaction mixture was heated to reflux overnight, cooled to RT, filtered through Celite, and concentrated under vacuo. The residue was dissolved in dioxane and MeOH (2 mL), and HCl in dioxane (1 mL) was added. After 1 h at RT, the reaction mixture was concentrated under vacuo and a purification by preparative HPLC gave the title compound 2fluoro-N-(2-methylimidazo[1,2-a]pyrazin-6-yl)-4-piperazin-1-yl-benzamide 15 (4.5 mg, 16%) as a white solid. 1H NMR (300 MHz, DMSOd6) δ ppm: 10.51 (br d, J = 3.10 Hz, 1H), 9.50 (s, 1H), 9.23 (br s, 1H), 9.05 (s, 1H), 8.22 (s, 1H), 7.69 (t, J = 9.10 Hz, 1H), 6.95 (br dd, J = 1.82, 8.48 Hz, 1H), 6.91 (br s, 1H), 4.02−5.48 (m, 4H), 3.58−3.61 (m, 2H), 3.19−3.25 (m, 2H), 2.42−2.44 (m, 1H). LC-HRMS (m/z): [M + H]+ calcd for C18H19FN6O 355.1677, found 355.1680; Diff 0.3 mDa. 2-Fluoro-N-(2-methylimidazo[1,2-a]pyrazin-6-yl)-4-[(3R)-3-methylpiperazin-1-yl]benzamide (23). In analogy to the preparation of 4, using (2R)-2-methylpiperazine was prepared the title compound 23 as a white solid. 1H NMR (600 MHz, DMSO-d6) δ ppm: 10.52 (br s, 1H), 9.50 (s, 1H), 9.34−9.44 (m, 1H), 9.12−9.25 (m, 1H), 9.05 (s, 1H), 8.23 (s, 1H), 7.69 (t, J = 8.87 Hz, 1H), 6.90−7.00 (m, 2H), 3.95−4.09 (m, 2H), 3.27−3.40 (m, 2H), 3.13−3.23 (m, 2H), 3.03− 3.12 (m, 1H), 2.95 (dd, J = 10.73, 13.65 Hz, 1H), 2.49 (s, 3H). LCHRMS (m/z): [M + H]+ calcd for C19H21FN6O 369.1834, found 369.1836; Diff 0.2 mDa. 4-[(3S)-3-Ethylpiperazin-1-yl]-2-fluoro-N-(2-methylimidazo[1,2a]pyrazin-6-yl)benzamide (24). In analogy to the preparation of 4, using tert-butyl (2S)-2-ethylpiperazine-1-carboxylate prepared the title compound 24 as a white solid. 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.57 (d, J = 3.43 Hz, 1H), 9.53 (s, 1H), 9.38 (br s, 2H), 9.08 (s, 1H), 8.25 (s, 1H), 7.69 (t, J = 8.98 Hz, 1H), 6.99 (dd, J = 2.02, 13.32 Hz, 1H), 6.95 (dd, J = 2.02, 7.27 Hz, 1H), 3.91−4.09 (m, 2H), 3.30− 3.40 (m, 1H), 3.04−3.28 (m, 3H), 2.98 (br dd, J = 10.70, 13.32 Hz, 1H), 1.60−1.80 (m, 2H), 1.03 (t, J = 7.57 Hz, 3H). LC-HRMS (m/z): [M + H]+ calcd for C20H23FN6O 383.1990, found 383.1992; Diff 0.2 mDa. Ames Mutagenicity Test. The profiling of test compounds for their mutagenic potential was performed using an AMES bacterial reverse mutation test essentially as described previously. In brief, Salmonella typhimurium strains TA1535, TA97, TA98, TA100, and TA102 were obtained from B. N. Ames (University of California, Berkeley, USA). S9 rat liver mixtures were freshly prepared for each experiment by mixing 0.1 mL of S9 preparation (Molecular Toxicology Inc., Boone, NC, USA), 0.2 mL of a 165 mM KCl solution, 0.2 mL of a 40 mM MgCl2 solution, 0.2 mL of 200 mM sodium phosphate buffered saline, pH 7.4, 3.2 mg of NADP (Roche Diagnostics,

the title compound 10 (6 mg, 24%) as an off-white solid. 1H NMR (300 MHz, chloroform-d) δ ppm: 8.65 (s, 1H), 8.24 (s, 1H), 7.88 (d, J = 8.88 Hz, 2H), 7.81 (s, 1H), 6.98 (d, J = 9.08 Hz, 2H), 3.31−3.40 (m, 4H), 3.04−3.13 (m, 4H), 2.85 (s, 3H), 2.52 (s, 3H). LC-HRMS (m/ z): [M + H]+ calcd for C19H22N6O 351.1928, found 351.1932; Diff 0.4 mDa. N-(6,8-Dimethylimidazo[1,2-a]pyrazin-2-yl)-2-fluoro-4-(4-methylpiperazin-1-yl)benzamide (11). According to Scheme 1. Step A: Preparation of 8. To a mixture of 6,8-dimethylimidazo[1,2-a]pyrazin2-amine trihydrochloride (200 mg, 0.736 mmol) and iPr2NEt (0.626 mL, 3.68 mmol) in dioxane (5.0 mL) was added dropwise a solution of 2,4-difluorobenzoyl chloride (133 mg, 0.736 mmol) in dioxane (1.25 mL) at RT. The mixture was stirred for 2 h at 60 °C, and the solvent was removed in vacuum. The solid was taken in water, and the resulting suspension was stirred for 15 min. The solid was collected by filtration and dried to afford N-(6,8-dimethylimidazo[1,2-a]pyrazin-2yl)-2,4-difluoro-benzamide 8 (184 mg, 83%) as a white solid and used in the next step directly. Step B: Preparation of 11. To a solution of N-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2,4-difluoro-benzamide 8 (30 mg, 0.099 mmol) in DMA (0.3 mL) was added 1-methylpiperazine (10 mg, 0.099 mmol). The reaction mixture was heated in a microwave at 130 °C for 15 min and then at 180 °C for 30 min. The mixture was cooled to RT, and purification by preparative HPLC provided N-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-fluoro-4-(4-methylpiperazin-1-yl)benzamide 11 (7 mg, 18%) as a white foam. 1H NMR (300 MHz, chloroform-d) δ ppm: 9.10 (br d, J = 14.73 Hz, 1H), 8.19 (s, 1H), 8.05 (t, J = 9.18 Hz, 1H), 7.75 (s, 1H), 6.77 (d, J = 6.66 Hz, 1H), 6.58 (br d, J = 14.13 Hz, 1H), 3.32−3.43 (m, 4H), 2.82 (s, 3H), 2.51−2.59 (m, 4H), 2.48 (s, 3H), 2.36 (s, 3H). LC-HRMS (m/z): [M + H]+ calcd for C20H23FN6O 383.1990, found 383.1989; Diff −0.1 mDa. N-(6,8-Dimethylimidazo[1,2-a]pyrazin-2-yl)-2-fluoro-4-(3-methylpiperazin-1-yl)benzamide (12). In analogy to the preparation of 11 step B, from N-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2,4-difluorobenzamide 8 (182 mg, 0.602 mmol) and 2-methylpiperazine (302 mg, 3.01 mmol) was prepared the title compound 12 (70 mg, 30%) as a white solid. 1H NMR (300 MHz, chloroform-d) δ ppm: 9.11 (br d, J = 15.14 Hz, 1H), 8.19 (s, 1H), 8.04 (t, J = 9.28 Hz, 1H), 7.76 (s, 1H), 6.76 (dd, J = 2.22, 9.08 Hz, 1H), 6.57 (dd, J = 2.22, 16.35 Hz, 1H), 3.61−3.75 (m, 2H), 3.10−3.18 (m, 1H), 2.83−3.05 (m, 3H), 2.82 (s, 3H), 2.55 (br d, J = 12.11 Hz, 1H), 2.48 (s, 3H), 1.16 (d, J = 6.26 Hz, 3H). LC-HRMS (m/z): [M + H]+ calcd for C20H23FN6O 383.1990, found 383.1992; Diff 0.2 mDa. 2-Fluoro-N-(2-methylimidazo[1,2-a]pyrazin-6-yl)-4-piperazin-1yl-benzamide (15). According to Scheme 2. Step A: Preparation of 14. In a sealed tube, to a solution of 4-bromo-2-fluoro-benzamide 13 (150 mg, 0.688 mmol) in dioxane (5.0 mL) was added 6-bromo-2-methylimidazo[1,2-a]pyrazine (146 mg, 0.688 mmol), CuI (13.1 mg, 0.0688 mmol), K3PO4 (307 mg, 1.44 mmol), and 1,10-phenantroline (14.9 mg, 0.138 mmol). The mixture was heated at 120 °C overnight, cooled down to RT, and filtered through Celite. The solvent was removed in vacuum, and a purification by column chromatography (SiO2, CH2Cl2/MeOH) afforded 4-bromo-2-fluoro-N-(2-methylimidazo[1,2a]pyrazin-6-yl)benzamide 14 (35 mg, 15%) as a white solid. 1H NMR 4454



Article

Rotkreuz, Switzerland), and 1.53 mg of glucose 6-phosphate (Roche Diagnostics). Bacterial growth media and agar, supplements, and tetracycline were obtained from Sigma (Buchs, Switzerland). Cultures of the strains were grown overnight at 37 °C in a shaking water bath in a nutrient broth (NB) liquid medium to which 0.3 μg/mL tetracycline was added for strain TA102 in order to maintain a stable plasmid copy number. The bacterial density was checked photometrically, and cultures were diluted in 0.85% NaCl as needed. The sensitivity of the Salmonella typhimurium strains was verified using the following positive controls: NaN3 with strains TA1535 and TA100, ICR 191 with strain TA97, 2-nitrofluorene with strain TA98, and MMC with strain TA102. Moreover, 2-aminoanthracene was used with all strains, with and without metabolic activation, to confirm the activity of the S9 mix. For testing of compounds, test tubes containing 2 mL of 0.7% agar medium were autoclaved and kept in a prewarmed water bath at 42−45 °C, and the following solutions were added: (a) 0.2 mL of a histidine/biotin mixture corresponding to 21 μg of L-histidine and 24.4 μg of biotin, (b) 0.1 mL solutions of test compound (20−2000 μg/ plate) and positive controls, (c) 0.1 mL of bacterial overnight liquid cultures, (d) 0.5 mL of the S9 mixture where metabolic activation was needed, or 0.5 mL of 200 mM sodium phosphate buffered saline, pH 7.4, where no metabolic activation was needed. The contents of the tubes were mixed and poured immediately onto Vogel−Bonner minimal agar plates, allowed to solidify, and incubated at 37 °C upside down for 2 days. Bacterial colonies were counted electronically using a DOMINO automatic image analysis system (Perceptive Instruments, Haverhill, UK) after inspection of the background lawn for signs of toxicity. The outcome of the test was considered a positive result indicating mutagenicity when a dose dependent increase in the number of colonies was observed reaching at least a 2-fold (strains TA1535, TA98) or 1.5-fold (strains TA97, TA100, TA102) increase over the background level. Lipophilicity (log D) Determination by High-Throughput Shake Flask. The applied methods called CAMDIS (Carrier Mediated Distribution System) for the determination of distribution coefficients are derived from the conventional “shake flask” method. CAMDIS was carried out in 96-well microtiterplates in combination with the novel DIFI-tubes constructed by Roche, which provided a hydrophobic layer for the octanol phase. The experiment started with the accurate coating of the hydrophobic layer (0.45 mm PVDF membranes), which was fixed on the bottom of each DIFI-tube: Each membrane was impregnated with exactly 1.0 mL of 1-octanol by a robotic system (Microfluidic Dispenser BioRAPTR, Bechman Coulter). To expand the measurement range down to log D = −0.5, the procedure was carried at two different octanol/water ratios: one with an overplus of octanol for hydrophilic compounds (log D < 1) and one with a low volume of octanol for the lipophilic compounds (log D > 1). Therefore, some DIFI-tubes were filled with 15 μL of 1octanol. The coated membranes were then connected to a 96-well plate which had been prefilled with exactly 150 mL of the selected aqueous buffer solution (25 mM Phosphate, pH 7.4). The buffer solution already contained the compound of interest with a starting concentration of 100 mM. The resulting sandwich construct guaranteed that the membrane was completely dipped in the buffered sample solution. The plate was then sealed and shaken for 24 h at room temperature (23 °C). During this time, the substance was distributed between the layer, the octanol, and the buffer solution. After distribution, equilibrium was reached and the DIFI-tubes were easily disassembled from the top of the 96-well plate, so that the remaining sample concentration in the aqueous phase could be analyzed by LC/MS. To know the exact sample concentration before incubation with 1-octanol, a part of the sample solution was connected to DIFI-tubes without impregnation. The distribution coefficient was then calculated from the difference in concentration in the aqueous phase with and without impregnation and the ratio of the two phases. The preparation of the sample solutions was carried out by a TECAN robotic system (RSP 100, eight channels). Solubility Determination (Lysa Assay). Samples were prepared in duplicate from 10 mM DMSO stock solutions. After evaporation (1 h) of DMSO with a centrifugal vacuum evaporator (Genevac

Technologies), the compounds were dissolved in 0.05 M phosphate buffer (pH 6.5), stirred for 1 h, and shaken for 2 h. After one night, the solutions were filtered using a microtiter filter plate (Millipore MSDV N65), and the filtrate and its 1/10 dilution were then analyzed by direct UV measurement or by HPLC-UV. In addition, a four-point calibration curve was prepared from the 10 mM stock solutions and used for the solubility determination of the compounds. Starting from 10 mM stock solution, the measurement range for MW 500 was 0− 666 μg/mL. Permeability (PAMPA) Assay. Parallel Artificial Membrane Permeability Assay (PAMPA) is an automated assay based on 96well microplates. The permeation of drugs is measured using a “sandwich” construction. A filter plate was coated with phospholipids (membrane) and placed into a donor plate containing a compound/ buffer solution. Finally, the filter plate was filled with buffer solution (acceptor). The donor concentration was measured at t-start (reference) and compared with the donor and acceptor concentration after a certain time (t-end) to calculate the extent of passage of the compound through the membrane. The main readout of the PAMPA assay was the permeability value Pe expressed in 10−6·cm/s. Secondary readouts determined are the amount of compound in the donor and acceptor compartments as well as the retention in the membrane. Depending on the permeation rate and the membrane retention, the compounds were classified as low (Pe < 0.2 and membrane 72,400 specimens. Eur. J. Hum. Genet. 2012, 20, 27−32. (3) (a) Fallini, C.; Bassell, G. J.; Rossoll, W. Spinal muscular atrophy: The role of SMN in axonal mRNA regulation. Brain Res. 2012, 1462, 81−92. (b) Schrank, B.; Gotz, R.; Gunnersen, J. M.; Ure, J. M.; Toyka, K. V.; Smith, A. G.; Sendtner, M. Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 9920−9925. (c) Paushkin, S.; Gubitz, A. K.; Massenet, S.; Dreyfuss, G. The SMN complex, an assemblyosome of ribonucleoproteins. Curr. Opin. Cell Biol. 2002, 14, 305−312. (4) (a) Kolb, S. J.; Gubitz, A. K.; Olszewski, R. F., Jr.; Ottinger, E.; Sumner, C. J.; Fischbeck, K. H.; Dreyfuss, G. A novel cell immunoassay to measure survival of motor neurons protein in blood cells. BMC Neurol. 2006, 6, 6. (b) Crawford, T. O.; Paushkin, S. V.; Kobayashi, D. T.; Forrest, S. J.; Joyce, C. L.; Finkel, R. S.; Kaufmann, P.; Swoboda, K. J.; Tiziano, D.; Lomastro, R.; Li, R. H.; Trachtenberg, F. L.; Plasterer, T.; Chen, K. S. Evaluation of SMN protein, transcript, and copy number in the biomarkers for Spinal Muscular Atrophy (BforSMA) clinical study. PLoS One 2012, 7, e33572. (5) (a) Kaczmarek, A.; Schneider, S.; Wirth, B.; Riessland, M. Investigational therapies for the treatment of spinal muscular atrophy. Expert Opin. Invest. Drugs 2015, 24, 867−881. (b) Calder, A. N.; Androphy, E. J.; Hodgetts, K. J. Small molecules in development for the treatment of spinal muscular atrophy. J. Med. Chem. 2016, 59, 10067−10083. (6) (a) Hua, Y.; Sahashi, K.; Rigo, F.; Hung, G.; Horev, G.; Bennett, C. F.; Krainer, A. R. Peripheral SMN restoration is essential for longterm rescue of a severe spinal muscular atrophy mouse model. Nature (London, U. K.) 2011, 478, 123−126. (b) Passini, M. A.; Bu, J.; Richards, A. M.; Kinnecom, C.; Sardi, S. P.; Stanek, L. M.; Hua, Y.; Rigo, F.; Matson, J.; Hung, G.; Kaye, E. M.; Shihabuddin, L. S.;

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S Supporting Information *

This material is free of charge via the Internet at The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00406. Dose−response curves of compounds 4, 12, and 24 for the SMN protein EC1.5x determination; 13C NMR spectra of compounds 4 and 24 (PDF) Molecular formula strings (CSV)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*For H.R.: phone, (+41) 61-688-2748; E-mail, hasane.ratni@ roche.com. *For E.P.: phone, (+41) 61-688-4388; Email, emmanuel. [email protected]. ORCID

Hasane Ratni: 0000-0002-8151-8295 4456



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Discovery of a Novel Class of Survival Motor Neuron 2 Splicing

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