Synthesis and SAR of novel isoxazoles as potent c-jun N-terminal kinase (JNK) Inhibitors

Abstract

The design and synthesis of isoxazole 3 is described, a potent JNK inhibitor with two fold selectivity over p38. Optimization of this scaffold led to compounds 27 and 28 which showed greatly improved selectivity over p38 by maintaining the JNK3 potency of compound 3. Extensive SAR studies will be described as well as preliminary in vivo data of the two lead compounds.

As a member of the mitogen-activated protein kinase (MAPK) family, the c-Jun N-terminal kinases (JNKs) regulate the serine/threonine phosphorylation of several transcription factors when they are activated in response of a variety of stress-based stimuli such as cytokines, ultraviolet irradiation, heat shock, fatty acids and osmotic shock.^{1, 2} Three distinct genes (jnk1, jnk2 and jnk3) encoding the ten splice variants of JNK have been identified.³ These isoforms differ in their tissue distribution profile and functions, with JNK1 and JNK2 being ubiquitously expressed, whereas JNK3 is expressed predominantly in the brain and at lower levels in the heart and testis.⁴

In recent studies, JNK-1, often in concert with JNK-2, has been suggested to play a central role in the development of obesity-induced insulin resistance which implies therapeutic inhibition of JNK1 may provide a potential solution in type-2 diabetes mellitus.^{5, 6} JNK2 has been described in the pathology of autoimmune disorders such as rheumatoid arthritis and asthma, and it also has been implicated to play a role in cancer, as well as in a broad range of diseases with an inflammatory component.^7–11 JNK3 has been shown to play important roles in the brain to mediate neurodegeneration, such as beta amyloid processing, Tau phosphorylation and neuronal apoptosis in Alzheimer’s disease, as well as the mediation of neurotoxicity in a rodent model of Parkinson’s disease.^12–14 JNK3 is almost exclusively found in the brain. Identifying potent and selective inhibitors of JNK3 may contribute towards neuroprotection therapies with reduced side effect profiles. JNK Inhibitors may have implications in many therapeutic areas. ^{15, 16} Therefore, developing JNK inhibitors as therapeutic agents has gained considerable interest over the past few years. ^{15, 17–36}

Given the significant body of evidence supporting the role of JNK3 in neurodegenerative disorders, our interest is in discovering potent, selective JNK3 inhibitors with good in vivo profiles as potential therapeutics for Parkinson’s disease. We previously reported on the synthesis and SAR of 4-phenyl substituted pyrimidines.³¹ Compounds in this class had moderate to good in vitro potency, but only modest in vivo profiles (rodent pk and brain penetration). Our ongoing research is focused on the discovery of novel chemical entities with improved potency and in vivo profiles.

The aminopyrimidine 1 (figure 1, JNK1 IC₅₀ = 26 nM) was previously reported in the literature.³⁷ While 2-amino-4-(hetero)aryl pyrimidine compounds are widely described in the literature, their pyridine equivalents are far less investigated, both synthetically and biologically. We anticipated to discover molecules with promising biological properties and yet patentable by replacing the pyrimidine- by a pyridine core. The very potent dual JNK3/p38 Inhibitor 2 (figure 1) was discovered in our lab,³⁸ with an IC₅₀ = 7 nM for JNK3 and IC₅₀ = 4 nM for p38 in the biochemical assays. While we were intrigued by the potency of compound 2 in the JNK biochemical assay, selectivity over p38 is highly desirable because of potential adverse effects, namely liver toxicity of p38 inhibitors.^39–41 In order to dial out p38 inhibitory properties, we looked at the crucial binding interactions of these types of molecules with the protein. The aryl-heteroaryl moiety is found in numerous p38 inhibitors. Crystal structures⁴² and molecular modeling^{43, 44} of similar molecules suggest two critical interactions of the pyrazole mojety with the p38 kinase, one being the aryl moiety in the 3-position extending into the deep hydrophobic pocket and the other one the lone pair of the pyrazole nitrogen forming a hydrogen bond to Lys53. We pursued several strategies to achieve selectivity over p38. Our efforts to achieve selectivity in aryl substituted pyrazole analogs are described elsewhere.^34,45 In this letter we describe modifications on the heterocyclic core. It was shown that replacing the nitrogen with oxygen, a weaker H bond acceptor, will result in reduced p38 potency,⁴⁶ thus we substituted the pyrazole moiety with an isoxazole group. Compound 3 (figure 1, table 1) indeed showed much reduced p38 potency; the JNK3 potency was also reduced, to a lesser extent however. Surprisingly, 4-(isoxazol-3-yl)pyridin-2 -amino derivatives are not described in the literature. It is also noteworthy that the isomeric isoxazole, in which the nitrogen and oxygen position were permutated, resulted in a compound with equal JNK3 potency as compared to compound 3 but loss in selectivity over p38. Encouraged by these results, we explored substitutions in the 5 position of the isoxazole group. Indeed we were able to further improve the selectivity over p38 (table 1).⁴⁷

Figure 1.

Potent novel JNK inhibitors

Table 1.

SAR of 4-Fluorophenyl isoxazoles^a

compound	R	JNK3 IC50 (μM)	JNK1 IC50 (μM)	p38 IC50 (μM)
3	H	0.026	0.160	0.057
4	Ph	>20	nt	nt
5		0.070	nt	0.563
6	CH3	0.032	nt	0.265
7	CN	0.126	nt	nt
8		0.523	nt	>20
9	N(Me)2	0.366	nt	5.16
10	NHMe	0.027	0.152	0.180
11	NHBn	0.134	nt	0.519
12	NH(CH2)2N(CH3)2	0.216	nt	0.174
13	OH	0.001	0.033	0.020

^aJNK3 biochemical IC₅₀ values are the averages of four or more experiments, and the JNK1 and p38 values are the averages of two or more experiments. All standard deviations are ≤30%

By introducing a methyl group in the 5-position (6) we maintained the JNK3 potency but further increased the selectivity to >8 fold. While cyclopropyl (5) and mono-methyl amine (10) groups are well tolerated, larger groups decreased potency. Interestingly, compound 13 containing a hydroxyl group was found to be exquisitely potent in the JNK3 biochemical assay with approximately 20 fold selectivity over p38 and 30 fold selectivity over JNK1. The stability of compound 13 in human microsoms was very poor however (data not shown). The synthesis of highly substituted isoxazoles via electrophilic cyclisation is reported in the literature^48–50 and is outlined in scheme 1.

Scheme 1.

Reagents and conditions: (a) NH₂OMe*HCl, Na₂CO₃, EtOH, 120° C, 1h; (b) ICl, DCM, rt; (c) Boronic acid, Pd(PPh₃)₄, Na₂CO₃ (aq.), DME, 120° C, 20′; (d) 4-morpholinoaniline, Pd₂(dba)₃, Xantphos, Cs₂CO₃, Dioxane, 90°C, 1–24 h.

The chloro-pyridines 14 were synthesized in two steps from the commercially available 2-Chloro-4-pyridinecarboxaldehyde and the respective acetylenes using a known procedure⁵¹. This approach allowed for the easy modifications of the 4 and 5 positions of the isoxasole ring and thus efficient exploration of a variety of groups and their effect on the biochemical potency. Alternatively by using the 2-bromopyridine analogs in scheme 1, the reaction sequence of the last two steps may be reversed. Compound 3 as well as the compounds from table 2 were synthesized by using TMS-acetylene, the trimethylsilyl group was cleaved during the Suzuki coupling giving the desired bis-substituted isoxazoles. For a more efficient synthesis of tri-substituted isoxazoles we developed the reaction sequence outlined in scheme 2. Good regioselectivity was achieved. The 5 position may be diversified in the last step by replacing the chloro- group with nucleophiles such as basic amines, alkoxides or nitriles.

Table 2.

SAR of different modifications in the 4 position^a

compound	R	R′	JNK3 IC50 (μM)	JNK1 IC50 (μM)	p38 IC50 (μM)
24	CH3		0.435	>20	>20
25			0.019	nt	19.3
26			0.074	nt	4.15
27			0.052	nt	>20
28			0.016	0.013	5.37
29			0.187	0.098	8.95
30			0.024	0.027	7.57
31			0.587	0.469	>20

^aJNK3 biochemical IC₅₀ values are the averages of four or more experiments, and the JNK1 and p38 values are the averages of two or more experiments. All standard deviations are ≤30%

Scheme 2.

Reagents and conditions: (a) NH₂OMe*HCl, Na₂CO₃, EtOH, 120° C, 1 h; (b) ICl, DCM, rt; (c) 4-morpholinoaniline, Pd₂(dba)₃, Xantphos, Cs₂CO₃, Dioxane, 90 °C,1–24 h; (d) Boronic acid, Pd(PPh₃)₄, Na₂CO₃ (aq.), DME, 120 °C, 20′; (e) NaOMe/MeOH or R′R″NH, TEA, DMF, 80 °C, 15 h.

Based on the literature, it was concluded that the phenyl group is optimal for p38 potency. As the hydrophobic pocket in JNK is smaller we then went on to explore different substitutions in the 4 position (table 2).

Replacing the aryl with a methyl group (24) reduced the JNK potency 10 fold while no inhibition of p38 was observed. By replacing the fluoro phenyl group in compound 3 with an N-methyl pyrazole group (25) we maintained the JNK3 potency but greatly increased the selectivity over p38. Sterically very hindered pyrazole groups (29) reduced JNK potency 10 fold and reduced selectivity over p38. Isomeric pyrazole groups (31) reduced JNK3 potency even further while this compound was inactive in the p38 assay. In summary, by introducing substituted or un-substituted pyrazole groups in the 4 position we were able to maintain the good JNK potency but dramatically reduce the p38 potency. Compound 27 was analyzed in a cell based JNK assay (IC₅₀ = 1.5 μM) and in vivo (table 3).⁵² This compound had good oral exposure but suffered from a short half live and poor brain penetration. Compound 29, which has a non-substituted pyrazole moiety, was found to be more potent than the N-methyl analog 27 in the biochemical assay (IC₅₀ = 24 nm vs. 42 nM), but slightly less potent in the cell based JNK assay (IC₅₀ = 1.5 μM vs. 1.9 μM). The N-unsubstituted analog 28 (cell based JNK assay IC₅₀ = 1.8 μM) was found to have poor in vivo properties (table 3).

Table 3.

PK properties of the two lead compounds

a rat pk							b mouse [μM]
compound	CIp (mL/min/kg)	t1/2 (h)	Vd (L/kg)	oral AUC (μM*h)	oral Cmax (μM)	%F	[plasma]	[brain]
27	6	1.5	0.4	5.0	1.5	37	13.4	0.6
28	13	1.8	0.4	0.7	0.3	12	nt	nt

^a1 mg/kg IV, 2 mg/kg po;

^b10 mg/kg ip 2h.

Thus far we focused on modifications on the isoxazole group for potency and selectivity. We have not yet extensively explored the aniline portion of the molecule. Published data of JNK inhibitors containing the 2-aminopyrimidine or 2-aminopyridine motif^{35, 37, 53} suggest that a variety of different groups will be tolerated in the 2-position. Modifications in this part of the molecule may allow to modulate the physicochemical properties and thus potentially to improve the in vivo properties and cellular potency.

In summary, we discovered a novel class of isoxazole derivatives as highly potent JNK1/3 inhibitors and greatly improved on the selectivity over the p38 kinase of the lead compound. The cellular potency and in vivo properties need to be improved. Through our extensive structure activity (SAR) studies we identified the crucial groups for potency and selectivity. Systematic exploration of diversified 2-anilino groups was not done and may yield compounds maintaining the favorable properties and potentially improving the cellular potency and in vivo profile.