Discovery of small molecule splicing modulators of survival motor

Subscriber access provided by Kaohsiung Medical University

Drug Annotation

Discovery of small molecule splicing modulators of survival motor neuron-2 (SMN2) for the treatment of Spinal Muscular Atrophy (SMA) Atwood K. Cheung, Brian Hurley, Ryan Kerrigan, Lei Shu, Donovan N Chin, Yiping Shen, Gary O'Brien, Moo Je Sung, Ying Hou, Jake Axford, Emma Cody, Robert Sun, Aleem Fazal, Ronald C Tomlinson, Monish Jain, Lin Deng, Keith Hoffmaster, Cheng Song, Mailin Van Hoosear, Youngah Shin, Rebecca Servais, Christopher S. Towler, Marc Hild, Daniel Curtis, William F Dietrich, Lawrence G. Hamann, Karin Briner, Karen S. Chen, Dione Kobayashi, Rajeev Sivasankaran, and Natalie A. Dales J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01291 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 9, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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

Journal of Medicinal Chemistry

Discovery

of

Small

Molecule

Splicing

Modulators of Survival Motor Neuron-2 (SMN2) for the Treatment of Spinal Muscular Atrophy (SMA) Atwood K. Cheung†,*, Brian Hurley†, Ryan Kerrigan†, Lei Shu†, Donovan N. Chin†,±, Yiping Shen†, Gary O’Brien†, Moo Je Sung†, Ying Hou†, Jake Axford†, Emma Cody†, Robert Sun†,«, Aleem Fazal†, Ronald C. Tomlinson†,€ Monish Jain†, Lin Deng†, Keith Hoffmaster†, Cheng Song†,¢, Mailin Van Hoosear†,£, Youngah Shin†, Rebecca Servais†, Christopher Towler‡, Marc Hild†, Daniel Curtis†,$, William F. Dietrich†, Lawrence G. Hamann†,, Karin Briner†, Karen S. Chen§, Dione Kobayashi§,¥, Rajeev Sivasankaran†, and Natalie A. Dales†,*

†Novartis

Institutes for BioMedical Research and ‡Novartis Pharmaceuticals, 250 Massachusetts

Ave, Cambridge, MA 02139. §SMA Foundation, 888 Seventh Avenue, Suite 400, New York, NY 10019

ABSTRACT Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene

ACS Paragon Plus Environment

1

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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

Page 2 of 54

SMN2 exists, however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070 / branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased fulllength SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA. INTRODUCTION SMA is a rare autosomal recessive neuromuscular disorder, occurring in ca. 1 in every 11,000 live births worldwide, however, this deadly disease is the number one monogenic cause of death in infants and toddlers.1 SMA is caused by the deletion or mutation of the survival motor neuron 1 (SMN1) gene.2 A closely related gene, survival of motor neuron 2 (SMN2), exists in humans which can partially compensate for the loss of SMN1; however, a single nucleotide mutation in exon7 of SMN2 leads to the exclusion of exon7 from the mature RNA transcript, resulting in a truncated and unstable SMN protein.3 The loss of SMN protein leads to premature degeneration of motor neurons and progressive loss of peripheral and central motor control.2a Infants with the most severe form of SMA (Type 1) will never sit, and require interventions such as ventilator assistance and feeding tubes. Milder forms include SMA Type 2 (patients sit up, but never walk), and Type 3 (patients walk, but may eventually lose that ability), which manifest at later ages (6-36 mo.).2 The recently approved (December 2016) splice correcting antisense oligonucleotide (ASO) nusinersen is the only marketed disease-modifying treatment for SMA.4 Nusinersen is administered via an intrathecal injection and although it provides a valuable


2

Page 3 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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


treatment option for SMA patients, an orally bioavailable small molecule treatment would have several advantages, including delivery route and wider tissue distribution. Recently, progress has been made towards gene-replacement therapy,5 and small molecule disease modifying treatments for SMA.6 Publications from PTC Therapeutics and F. Hoffmann-La Roche and our laboratories have described promising orally available small molecule therapies.7,8,9 We have demonstrated that LMI070 / branaplam (1, NVS-SM1, Fig. 1) can correct the splicing defect in the SMN2 gene through stabilizing the interaction between the U1-snRNP complex and SMN2 pre-messenger RNA, leading to generation of functionally competent full length SMN protein.9 Additionally, when administered to transgenic mice bearing the human SMN2 gene, 1 increased levels of full length SMN protein in the central nervous system and extended the lifespan of the diseased mice to match that of their healthy littermates. Clinical trials in Type-1 SMA patients are ongoing to evaluate the safety, tolerability, PK/PD, and efficacy of orally dosed 1. Herein, we describe the identification, via high-throughput phenotypic screening, of a pyridazine SMN2 pre-mRNA splicing modulator and medicinal chemistry optimization to the clinical compound branaplam, the first oral small molecule splicing modulator tested in SMA Type I patients.10 O N

N

NH

OH

HN N

Figure 1. Structure of SMN2 splicing modulator NVS-SM1 / LMI070 / branaplam (1) RESULTS AND DISCUSSION Identification of the SMA lead scaffold For our high-throughput screening campaign, we utilized an NSC34 motor neuron cell line expressing an SMN2 minigene reporter. A pair of SMN2 matched reporter constructs were


3


Page 4 of 54

designed such that one would express luciferase in-frame reporting on exon7 inclusion (fulllength) and the other on exon7 exclusion (Delta7).9 Hits were selected based upon complementary changes between the inclusion and exclusion reporter-gene assay systems. Initially, hit molecules were evaluated via qPCR for dose responsiveness and to confirm the desired splicing activity. Once the desired splicing pattern was confirmed, SMN protein levels were assessed by ELISA in SMNΔ7 mouse myoblasts to confirm effect on the endogenous gene. The 3,6-disubstituted pyridazine compound 2 (Fig. 2a) was an attractive starting point confirmed through the hit finding efforts. Compound 2 demonstrated activation of the SMN2 reporter of 1700% over DMSO control with an EC50 of 3.5 M. Moreover, in a mouse SMN ELISA assay, 2 displayed significant increase in SMN protein levels (EC50 = 0.6 M, 2.5 fold increase), and 1.5 fold increase in human patient-derived fibroblasts.9 Based on the promising activity data and favorable physicochemical and in vitro profiling data (Table 1), we assessed the in vivo pharmacokinetics and distribution of 2 in mice. The compound demonstrated high in vivo plasma clearance (113 mL/min/kg), modest bioavailability (%F = 18), and low brain exposure. Notably, 2 also displays potent human-ether-a-go-go-related gene (hERG) inhibitory activity (IC50 = 0.6 M, [3H] dofetilide binding) which is associated with cardiotoxicity caused by QT interval prolongation.11 With a clear understanding of parameters for improvement, we initiated an optimization program based on pyridazine 2. We focused our efforts on maximizing cellular potency, improving in vivo exposure through reducing clearance and increasing permeability, and addressing the hERG channel binding liability. With respect to assessing cellular potency, we utilized the SMN protein ELISA assay, as we anticipated that SMN protein would be a relevant and translatable biomarker from the cellular setting through to in vivo settings. Table 1. ADME / PK Profile of Compound 2.


4

Page 5 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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


Physicochemical properties

In vitro Safety

MW

366.5

hERG binding IC50

PSA, cLogD (7.4)

49.8, 2.67

Mouse PKa

pKa

10.0

CL (mL/min/kg)

113

oral BAV

18%

ADME profile Sol pH 6.8 (mM)

>1

AUC 0-7 hrs (M.hr)

Clint (L/min/mg)

22.6 (R) 23.5 (M)

Plasma / Brain

0.6 M

0.40 / BQLb

a

C57BL/6 mouse PK. Animals were dosed i.v. at 1 mg/kg; p.o. at 5 mg/kg, n = 2; 10% 0.1 N HCl, 10% PG, 25 (20%) Solutol, 100 mM pH 5 citrate buffer formulation. bBQL 10 M*

Pyridazine core and linker

a.

N

N

N

NH

S

4

N

S N

3.5 M* 2

N N 0.02 - 0.1 M*

Figure 2. (a) HTS hit compound 2, and (b) SAR of core modifications. *SMN Elisa EC50. Early work focused on the basic amine and linker atom, and highlights are described as follows. We found replacement of the eastern tetramethylpiperidine with the less lipophilic piperidine resulted in a decrease in SMN protein levels and reduced potency relative to 2. Replacement of the basic amine with an ether, such as pyran, resulted in a complete loss of SMN activity. Of note, both of these modifications significantly reduce hERG binding affinities, highlighting that the basicity and lipophilicity of the tetramethylpiperidine were key contributors to SMN potency as well as the undesired hERG activity. In the linker region, methylation of the amine (3) or modification to an ether linker (not shown) were both well-tolerated, with potency and fold activation equivalent to 2. The western aromatic region was tolerant of a wide variety of aromatic and heteroaromatic modifications, and proved to be a useful handle for the modulation of potency as well as physicochemical and ADME properties (Table 2). Although truncation of the benzothiophene ring to the thiophene rendered the compound inactive, replacement of the benzothiophene with the


6

Page 7 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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


isosteric 2-naphthyl group (4) retained activity, whereas the 1-naphthyl group (5), which cannot maintain planarity with the pyridazine ring, was inactive. Based on the naphthyl result, further exploration evolved to substituted phenyl rings in the western aromatic region. While we found that substitution at the ortho position of a western aryl ring generally resulted in inactive analogs, introduction of an ortho-hydroxy group significantly improved potency. As an illustration, both o-hydroxyphenyl (6, EC50 = 0.15 M, 2.5 fold) and ohydroxynaphthyl aromatic (7, EC50 = 0.016 M, 2.4 fold) analogs were greater than 10-fold more potent than their des-hydroxy counterparts (data not shown). Combination of the ortho-hydroxy functionality with a 4’-cyano group provided 8 (EC50 = 0.031 M, 3.1 fold) a highly potent compound with, however, significant hERG binding (IC50 = 0.3 M). Masking of the phenol with a methoxy group (9) resulted in an over 50-fold reduction in potency. Taken in sum, the data suggest that the potency improvement of the phenolic hydroxyl group is due to an internal hydrogen bond with the proximal nitrogen of the pyridazine ring system. This would result in a low-energy near planar conformation of the phenyl-pyridazine biaryl system, presumably representing the preferred bioactive conformation. The phenolic hydroxyl functionality as a conformational constraint was unique in its ability to improve potency as alternative hydrogen bond donor groups such as amino (-NH2), methylsulfonamide (-NHSO2Me), and the homologated hydroxymethyl (-CH2OH) analogs were inactive (data not shown).

Table 2. Pyridazine SMN ELISA and hERG SAR data. X R

N

N

NH


7


Page 8 of 54

SMN ELISA X

EC50 (M)

Fold

hERG IC50 (M)

NH

0.6

2.5

0.6

NMe

1.1

2.4

0.11

4

NH

0.35

2.3

1.5

5

NH

-

~1

>30

NMe

0.15

2.5

2.2

NMe

0.016

2.4

0.08

NMe

0.031

3.1

0.3

NMe

1.69

2.3

-

Compound

R

2

S

3

S

6 F

7

OH

OH

8 N

9 N

OH

O

Small Molecule X-ray Crystal Structures of Pyridazine Analogs Evidence in favor of the bioactive conformation hypothesis was provided by an X-ray crystal structure (Fig. 3a) in which the pyridazine ring and phenyl ring of 8 are almost coplanar with a dihedral angle of 3°. The phenolic hydroxyl group oxygen was observed to be within hydrogen bonding distance of the pyridazine nitrogen (2.54 Å). In contrast, the phenyl and pyridazine rings are twisted 29° out of plane with the less active o-methoxy analog 9 (Fig. 3b), due to repulsion between the oxygen and nitrogen lone pair electrons.13 Further analysis of the crystal structures revealed the expected chair conformation for the piperidine ring, however due to


8

Page 9 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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


flexibility at the amine linker, two distinct conformations were observed for the piperidine group relative to the pyridazine nitrogens (an anti and a syn orientation). As there is a strong conformational preference for heterocyclic ethers to minimize lone pair repulsion,14 we postulate that the syn orientation of the pyridazinyl nitrogen and tetramethylpiperidinyl groups may be the preferred conformation (Fig. 3b).

3°

29°

a.

b.

Figure 3. Small molecule X-ray crystal structures (ORTEP plots). (a) Compound 8 (anti orientation of the tetramethylpiperidinyl group relative to the pyridazine nitrogen), (b) Compound 9 (syn orientation). Development Toward In Vivo Tool Compounds Since in vivo validation of the SMN pre-mRNA splicing approach was critical to confidently advance the project, we began assessing the ADME and pharmacokinetic profiles of the benzothiophene and ortho-hydroxy-aryl analogs. A subset of compounds was chosen for mouse PK evaluation (10 mg/kg p.o., 1 mg/kg i.v.) with brain exposures evaluated from the oral arm (Table 3). Benzothiophene 3, differs from 2 by addition of a single methyl group on the amine linker. This small modification, while having little effect on in vivo clearance, provided significant improvements in both plasma (AUC, 4.9 M.h) and brain exposure (AUC, 17.4 M.h), as well as


9


Page 10 of 54

oral bioavailability (%F = 100), as compared to 2, potentially due to improved permeability. Reducing the number of hydrogen bond donors is known to improve diffusion through the blood brain barrier15,16 and likely contributes to the improved brain distribution of 3. Based on this result, methylation of the linker amine became an early strategy for improving exposure and brain distribution. The

ortho-hydroxyphenyl

and

ortho-hydroxynaphthalene

derivatives

offered

improvements in potency and intrinsic clearance. For example, the hydroxynaphthalene 7 displayed an IC50 of 16 nM and in vitro rat Clint of 17 L/min/mg, and the cyanophenol 8 displayed an IC50 of 31 nM and Clint of 7 L/min/mg. As with many of our early tool compounds, we found that both 7 and 8 had acceptable distribution into the brain compartment, and moderate in vivo CL (Table 3). Compound 8 was selected for in vivo efficacy studies due to its favorable PK properties, potency (SMN ELISA EC50 = 31 nM), and fold activation (3.1 fold). Table 3. Comparing potential pyridazine in vivo tool compounds.

Compound

SMN EC50 (M), fold

AUC po mouse^ cLogD RLM Clint Plasma (M.h) (pH 7.4) (L/min/mg) Brain (M.h)

CL (mL/mi n/kg)

T1/2 (hr)

%F

3

0.6 2.4

3.4

40

P: 4.9 B: 17.4

100

3.2

100%

7

0.016 2.5

4.0

17

P: 16.9 B: 36.7

56

3.7

100%

8

0.031 3.1

3.2

7

P: 5.2 B: 2.0 M*

23

4.5

37%

^Mouse PK: 10 mg/kg PO. *Brain concentration determined at a single time point of 7h.


10

Page 11 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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


To validate splicing correction as a viable approach for treating SMA patients, 8 was tested in SMN delta-7 mice,17 which display a severe phenotype with mortality seen at approximately postnatal day 15. Initially, the delta-7 mice were used to establish the PK/PD relationship, and determine the magnitude of SMN protein increase. The mice were treated orally with doses of 1, 3, 10, and 30 mg/kg of 8 daily, starting at postnatal day 3. After ten days of dosing, a concentration dependent increase in SMN protein levels was seen in the brain, with significant increases observed at each dose level (Figure 4). At 4 hours post last dose, mean brain concentration of 1.55 M and 61.7 M were observed for the 1 and 30 mg/kg doses, respectively. The apparent plateau in SMN protein levels between 10 and 30 mg/kg dose levels suggested the maximal threshold was reached. Next, we sought to determine if the increases in SMN protein levels induced by 8 were therapeutically relevant, i.e. improved overall survival. Gratifyingly, the increased SMN protein levels translated to increased overall survivals (Figure 5), and improvement in body weight as compared to the vehicle treated mice (S.Fig. 1). Efficacy was similar at 3 and 10 mg/kg dose levels, while reduced survival at the highest dose suggested tolerability issues. Encouraged by this exciting dataset, we concluded that splicing modulation via oral administration of small molecule SMN2 splicing modulators was a viable therapeutic strategy for the treatment of SMA.


11


3

SMN protein elevation

2.5 2 1.5 1 0.5 0 Vehicle

1 mpk

3 mpk

10 mpk

30 mpk

Figure 4. PD results for 8 in SMN delta7 mice.

Survival 100

Percent survival

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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

Page 12 of 54

Vehicle 3 mpk, 8 10 mpk, 8

50

30 mpk, 8

0 0

2

4

6

8 10 12 14 16 18 20 22 24 26 28 30

Postnatal Days

Figure 5. In vivo efficacy study for 8 in SMN delta7 mice. Reducing hERG binding


12

Page 13 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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


From our early work, we understood that the basicity of the tetramethylpiperidine group, in combination with the hydrophobicity imparted by the neighboring gem-dimethyl groups, contributed significantly to SMN potency. Unfortunately, this motif is shared with potent hERG binders. QSAR models show that the general features of the hERG binding pharmacophore consist of an amine which is protonated at physiological pH and adjacent hydrophobic centers.18 Thus, a series of compounds was prepared (Table 4) to investigate the effect of reducing both overall lipophilicity and the pKa of the amine, both of which are established tactics for decreasing hERG binding.17 We found that the nitrile of 8 could be replaced by a wide range of heterocycles, leading to the discovery of compounds such as pyrazole 10. Although 10 did not provide a hERG improvement, heteroaryl moieties in this position reduced other off-target liabilities (such as PDE3/4, data not shown). Using 10 as a template, we then explored incorporation of different eastern amine groups and linker functionalities distal to the pyrazole. We found that replacing the N-methyl linker with an oxygen linker (11) did not improve the hERG activity; however a small boost in fold induction was observed which we felt may have an impact in vivo. We then focused on reducing hERG activity by modifying lipophilicity and pKa. Removal of methyl groups from the tetramethylpiperidine reduced lipophilicity and led to lower potency and hERG binding. The des-methyl piperidine analog 12 showed a two unit decrease in cLogD (0.69) as compared to 11, without a significant change in amine pKa (9.8, calculated). 12 improved hERG binding to IC50 = 6 M, but saw a 10-fold reduction is SMN potency. Its difluoro matched pair, analog 13, reduced the basicity of the piperidine (pKa 6.8, calculated), eliminated hERG binding, but also eliminated SMN activity (EC50 = 3.3 M, 1.2 fold). Previous SAR had shown that N-methylation of the piperidine was tolerated with modest reductions in potency. In another


13


Page 14 of 54

example, 14 was prepared with the N-trifluoroethyl substitution, which profoundly reduced the piperidine basicity (calculated pKa 3.9), and resulted in a complete loss of both SMN and hERG activity. These examples illustrated that the effect of reducing lipophilicity was much less profound than that of reducing pKa. Unfortunately, both strategies led to reductions in SMN potency which could not be decoupled from hERG binding, thus an alternate approach to reduction of hERG binding was needed. Table 4. Modifying eastern amine to reduce hERG. A N N N

Compound

*

10

11

Me N NH

*

*

O NH

13

*

cLogD (pH 7.4)

pKa

Calc pKa

hERG IC50 (M)

0.020

3.1

3.11

10.3

10.4

0.3

0.030

3.3

2.79

10.1

10.7

0.23

0.34

2.6

0.69

-

9.8

6

3.3

1.2

2.59

5.89

6.8

>30

>10

1.1

4.65

-

3.9

>30

O NH

12

OH

SMN ELISA EC50 Fold (M)

A

N

O

F F NH

14

*

O N

CF3


14

Page 15 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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


In a parallel effort, we examined modulation of physicochemical properties through modification of the western aromatic region as a hERG mitigation strategy (Table 6). Early SAR suggested that introducing polarity through aza substitution in the distal aromatic region reduced hERG binding while retaining good SMN potency. For example, the hydroxyquinoline and hydroxyisoquinoline analogs (15, 16) retained high SMN potency (EC50 = 0.02 M and 0.006 M, respectively) with reduced hERG binding (IC50 = 4.5 M and 14 M, respectively). Interestingly, 15 and 16 have similar physicochemical properties (cLogD, tPSA), but differ in hERG binding affinity, suggesting that the position of the nitrogen can be tuned to decrease hERG interaction. Both of these analogs had desirable physicochemical profiles which led us to evaluate their in vivo pharmacokinetics (vide infra). Table 5. Reducing hERG in pyridazines. Me N

A N N

NH

Compound

A *

15 16

SMN ELISA EC50 Fold (M) OH

N

*

N

OH

cLogD (pH 7.4)

tPSA

logPAMPA (cm/s)

hERG IC50 (M)

0.020

2.9

3.23

74

-4.5

4.5

0.006

2.6

3.07

74

-4.3

14

0.29

2.8

2.52

105

-5.2

16

0.023

2.8

2.16

99

-5.5

16

0.004

3.1

2.92

90

-5.4

6.0

*

17

H 2N

N

N

OH

* N

18

N

OH

HO

*

19

N HN

OH


15


Page 16 of 54

A strategy for hERG attenuation centered on incorporating polar heteroaromatic substituents to the 4-position of the phenyl ring to increase PSA. The most successful analogs from this effort were derived from the N-linked pyrazole (10). Incorporation of the aminopyrazole to give 17 reduced hERG binding, however at the expense of SMN potency and permeability likely due to increased PSA. However, addition of the 4-hydroxymethyl pyrazole in 18 which had a more moderate effect on PSA provided a reduction in hERG binding without the loss of SMN potency. A significant improvement on hERG binding came when regioisomeric carbon-linked pyrazoles were explored. Pyrazole 19 displayed excellent potency and reduced hERG binding (IC50 = 6.0 M), while maintaining moderate PSA and bioavailability of 53% (Table 5). These changes in hERG binding affinity were largely unconnected to LogD of the analogs, and mainly driven by addition of polar groups in the western aromatic region. Within this sub-series, increases in polarity reduced hERG binding; however, decreased permeability and bioavailability were also observed with increasing PSA, thus careful modulation was required to maintain optimal pharmacokinetics, reduce hERG binding, and maintain high SMN potency.

Amine Linker Optimization As the carbon-linked pyrazole provided significant improvement in potency and hERG binding, we therefore looked to re-optimize the amine linker and tetramethylpiperidine regions with the western pyrazole fixed (Table 6). We found that bicyclic, and spirocyclic motifs for the eastern amine were well tolerated, and maintained the desired level of potency. However, as evidenced by 20 and 21, these diamines were more potent hERG binders. Modification of the amine linker to a methylene (22) led to a slight decrease in potency, but a significantly improved hERG binding affinity of 17 M. As we had observed previously, nitrogen to oxygen linker


16

Page 17 of 54 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 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


modification as in 1 showed a significant increase in fold induction to 3.6. This value was the highest seen at that juncture, and combined with a >30 fold improvement in hERG binding affinity relative to 11, compound 1 became an instant frontrunner.

Table 6. Optimizing basic region of pyridazines. A N HN

SMN ELISA Fold

cLogD (pH 7.4)

tPSA

logPAMPA (cm/s)

hERG IC50 (M)

0.156

2.32

1.20

89

-

0.27

0.058

2.54

0.58

89

-

2.5

NH

0.060

3.17

2.89

86

-5.3

17

NH

0.020

3.6

2.51

90

-4.9

6.3

Compound

Structure A

20

N N

NH

N H

21

N N

22

N

OH

EC50 (M)

NH H

N

N

O

1 branaplam

N

N

In vivo PK and PD Optimization of Lead Compounds An accepted assertion within the SMA field is that efficacy is driven by exposure in the brain and spinal cord.19 Early compounds such as 8 and 7 showed good brain exposures due to balancing lipophilicity, polar surface area, and number of hydrogen bond donors, which are parameters which govern blood brain barrier penetration.20 In general, the pyridazine series


17


Page 18 of 54

compounds showed moderate brain tissue and plasma protein binding (typically

Discovery of small molecule splicing modulators of survival motor

Recommend Documents