Design and Synthesis of Soluble and Cell-Permeable PI3Kδ Inhibitors

Article pubs.acs.org/jmc

Design and Synthesis of Soluble and Cell-Permeable PI3Kδ Inhibitors for Long-Acting Inhaled Administration Matthew W. D. Perry,*,†,‡ Karin Björhall,†,∥ Britta Bonn,†,§ Johan Carlsson,⊥ Yunhua Chen,# Anders Eriksson,†,‡ Linda Fredlund,⊥ Hai’e Hao,# Neil S. Holden,†,∥,∇ Kostas Karabelas,† Helena Lindmark,⊥ Feifei Liu,# Nils Pemberton,†,‡ Jens Petersen,⊥ Sandra Rodrigo Blomqvist,†,∥ Reed W. Smith,†,‡ Tor Svensson,†,‡ Ina Terstiege,†,‡ Christian Tyrchan,†,‡ Wenzhen Yang,# Shuchun Zhao,# and Linda Ö ster⊥ †

Respiratory, Inflammation and Autoimmunity, Innovative Medicines and Early Development, AstraZeneca Gothenburg, Pepparedsleden 1, SE-431 83 Mölndal, Sweden ‡ Medicinal Chemistry, §DMPK, ∥BioScience, and ⊥Discovery Sciences, Innovative Medicines and Early Development, AstraZeneca Gothenburg, Pepparedsleden 1, SE-431 83 Mölndal, Sweden # Pharmaron Beijing Co., Ltd., No. 6 Taihe Road, BDA, Beijing 100176, P.R. China S Supporting Information *

ABSTRACT: PI3Kδ is a lipid kinase that is believed to be important in the migration and activation of cells of the immune system. Inhibition is hypothesized to provide a powerful yet selective immunomodulatory effect that may be beneficial for the treatment of conditions such as asthma or rheumatoid arthritis. In this work, we describe the identification of inhibitors based on a thiazolopyridone core structure and their subsequent optimization for inhalation. The initially identified compound (13) had good potency and isoform selectivity but was not suitable for inhalation. Addition of basic substituents to a region of the molecule pointing to solvent was tolerated (enzyme inhibition pIC50 > 9), and by careful manipulation of the pKa and lipophilicity, we were able to discover compounds (20b, 20f) with good lung retention and cell potency that could be taken forward to in vivo studies where significant target engagement could be demonstrated.

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INTRODUCTION Phosphoinositide 3-kinases (PI3Ks) are a group of enzymes that are involved in the phosphorylation of the membranebound inositol phospholipids in the 3-position of the inositol moiety. The eight known members are divided into three classes, I, II, and III. Class I PI3Ks have been the most extensively studied of these subclasses; the class is further subdivided into IA (PI3Kα, β, and δ) and IB (PI3Kγ) based upon the types of regulatory subunits with which the catalytic domains combine in the active heterodimeric forms. Class 1A PI3Ks mediate the signal transduction from receptor tyrosine kinases, while PI3Kγ is principally activated by GPCRs. PI3Kδ and γ are restricted to cells of the hematopoietic system, while PI3Kα and β are ubiquitous. Dysregulation of the PI3K system (the kinases and the associated phosphatases) is often observed in cancers, and there has been a great deal of interest in developing pan or selective inhibitors of PI3Ks for applications in oncology.1,2 Evidence is growing that inhibition of PI3Kδ and/or γ can have powerful yet selective effects on the immune © 2017 American Chemical Society

response and inhibitors have attracted interest as potential treatments for diseases with a significant immunological component such as rheumatoid arthritis and asthma.3−6 Understanding of the biology of PI3Ks is developing fast, however, there are still details and subtleties to be worked out. Several reviews describe the current state of knowledge.7−10 Recently the PI3Kδ selective inhibitor idelalisib (Figure 1A) has been launched as the first PI3K inhibitor to market for the treatment of B-cell cancers.11 The drug is effective but shows a number of significant side effects.12 We were interested in PI3Kδ as a potential treatment for lung conditions such as asthma. We have previously described an earlier series of PI3Kδ inhibitors that we investigated but which showed disappointing cell potencies.13 We selected an inhaled approach with a view to minimizing the systemic exposure and thus the potential for side effects from either Received: March 22, 2017 Published: May 18, 2017 5057

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nones and quinazolinones. We identified the thiazolopyridone ring system as a potentially suitable core for our explorations. Since the completion of the work described herein but prior to the submission of this article, the same core has been described in the patent literature for some PI3Kδ inhibitors.18

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CHEMISTRY

Synthesis of the compounds described in this paper followed a general route with late-stage diversification in order to introduce changes to the aryl substituents. The route (Scheme 1) started by following the procedures described by the Almqvist group for the synthesis of 6-bromothiazolidinopyridones.19,20 Initially we used a racemic synthesis with late-stage chiral separation, but the majority of work was carried out starting with chiral (R)-2-chloropropionic acid. Coupling of this acid with Meldrum’s acid gave dioxinone 3. This dioxinone reacted thermally with thiazoline 2 to give the core ring system 4 as a mixture of diastereomers. Ring bromination of 4 was followed by sulfoxidation, Pummerer rearrangement, and elimination to deliver the aromatized compound 7. Displacement of the chlorine with sodium azide followed by reduction gave amine 9. CBZ protection of the free amine produced carbamate 10. Suzuki coupling of racemic 10 from the initial synthesis with phenyl boronic acid gave 11 that was deprotected to amine 12, which on heating with 4-amino-6-chloropyrimidine-5-carbonitrile, followed by separation of the enantiomers by chiral HPLC, produced initial lead 13 (Scheme 2). Chiral analysis of 10 produced using (R)-2-chloropropionic acid showed that some loss of stereochemical purity had occurred during the synthesis. 10, however, proved to be suitable for chiral SFC and could be purified to >99% enantiopurity. Cleavage of the carbamate returned chirally pure amine 9 that was used as the starting point for subsequent exploration (Scheme 3). Heating amine 9 and 4-amino-6chloropyrimidine-5-carbonitrile in n-butanol gave the bromide 14 that was coupled with substituted phenyl boronic acids to give, after deprotections and, where required, reductive amination, compounds 15a and 17−19. Alternatively, use of

Figure 1. Reference PI3Kδ inhibitors.

target-related or off-target interactions. This early decision to follow an inhaled approach mandated several aspects of our medicinal chemistry strategy: (i) The limitations on dose deliverable through standard inhalers meant that achieving subnanomolar potency was crucial. (ii) Soluble compounds are typically cleared from the lungs in a matter of minutes,14 while inflammatory cells are recruited to the lung continuously, so sufficient exposure and retention in the lung is required. (iii) To minimize systemic exposure, oral bioavailability should be low and plasma clearance high. Multiple chemotypes have been described as PI3Kδ selective inhibitors.3 Broadly speaking, they fall into two categories, “propeller” shaped compounds, such as idelalisib, that access an induced pocket,15 and other chemotypes that all appear to derive selectivity by a combination of optimizing the interactions in the affinity pocket for PI3Kδ and accessing the “tryptophan shelf”. The “tryptophan shelf” describes the face of Tyr760 (PI3Kδ numbering), which is accessible in PI3Kδ owing to the small size of Thr750, whereas larger residues in the corresponding positions of PI3Kα, β and γ occlude this surface.16 Since the disclosure of the structure of IC8711417 (Figure 1B) in 2003, there has been extensive medicinal chemistry exploration of chemotypes that can give “propeller” type structures.3 We started our search for PI3Kδ inhibitors by looking for a novel propeller-shaped chemotype in view of the heavy patenting around 6,6-fused systems such as isoquinoliScheme 1a

(i) Lawesson’s reagent, THF, 65 °C; (ii) Meldrum’s acid, DCC, DMAP, CH2Cl2; (iii) TFA, TFAA, ClCH2CH2Cl; (iv) isoamyl nitrite, HBr (40% aq), CH2Cl2, < −5 °C; (v) mCPBA, CH2Cl2, 0 °C; (vi) TFAA, PhMe, 60 °C then cH2SO4, CH2Cl2, 0 °C to RT; (vii) NaN3, DMF, 45 °C; (viii) Ph3P, CH2Cl2, reflux; (ix) CbzCl, K2CO3, THF:water 10:1, 0 °C.

a

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Scheme 2a

could obtain duration. Two main strategies have been described to produce a prolonged effect in lung tissue: low solubility21,22 or basicity.23 Solubility-driven approaches require that material dissolves slowly over the time after dosing to produce the effect while a pH-driven approach relies on dissolved compound being trapped in acidic organelles, such as lysosomes, which are prevalent in lung tissue, to provide a depot that is then released slowly over an extended period of time. We prioritized the addition of basic centers as a potentially predictable method to modulate lung retention. Crystallization of 13 in a murine PI3Kδ construct (Figure 2) showed that the compound bound in a similar conformation and into the same induced pocket in the enzyme that has been described previously.15 Examination of the crystal structure revealed that there might be scope to extend from the meta position of the phenyl ring toward solvent if the phenyl ring made a small rotation, enabling modulation of the physical properties of the compounds without affecting the enzyme affinity. Addition of a benzylic amine to the meta position of the pendant phenyl in 13 gave compounds (15a−c) that retained the PI3Kδ potency of 13 but exhibited differing selectivities toward the other isoforms (Table 1). Addition of the basic center increased selectivity toward PI3Kγ by reducing potency at that isoform, particularly for 15b,c. Selectivity toward PI3Kβ, on the other hand, was reduced for 15a,b, though not for 15c. Selectivity against PI3Kα was slightly increased, with 15c again the most selective. All three compounds had low lipophilicity, particularly 15a,b, and these two showed a significant potency drop-off in the cell assay, probably reflecting poor cell permeability.

a (i) PhB(OH)2, PdCl2(dppf)·CH2Cl2, DME−water, 100 °C; (ii) BBr3, CH2Cl2, 0 °C to RT; (iii) 4-amino-6-chloropyrimidine-5-carbonitrile, DIEA, n-butanol, 120 °C then chiral HPLC.

an aldehyde functionalized boronic acid gave intermediate 16 that could be reductively aminated with a range of amines to give, in some cases after additional manipulation, compounds 15b−g and 20a−l.

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RESULTS AND DISCUSSION Initial thiazolopyridone 13 met our criteria for a lead with excellent PI3Kδ potency in both biochemical and cellular assays, very good selectivity against PI3Kα, and acceptable selectivity against PI3Kβ and γ (Table 1). As a model for inhalation, we dosed compounds by intra tracheal (i.t.) administration of a solution of compound to rats followed by termination at time points up to 24 h and subsequent analysis of the residual compound in the lungs. Compound 13, a moderately soluble (77 μM) neutral molecule, was cleared extremely rapidly from lung; compound could only be detected at the first time point (3 min after dosing, Table 2). Given the rapid clearance from the lung of the parent compound, we needed to find a mechanism by which we Scheme 3a

(i) BBr3, CH2Cl2, 0 °C to RT; (ii) 4-amino-6-chloropyrimidine-5-carbonitrile, DIEA, n-butanol, 120 °C; (iii) ArB(OH)2, PdCl2(dppf)·CH2Cl2, Cs2CO3, DME−water, 100 °C then TFA, CH2Cl2; (iv) ArB(OH)2 PdCl2(dppf)·CH2Cl2, Cs2CO3, DME−water, 100 °C; (v) HNRR, NaBH3CN, MeOH−CH2Cl2; (vi) (a) ArB(pinacol), third-generation Pd precatalyst, P(Cy)3·HBF4, K3PO4, dioxane−water, 130 °C, (b) TFA, CH2Cl2, (c) CH2O, NaBH3CN, AcOH, MeOH; (vii) ArB(pinacol), third-generation Pd precatalyst, P(Cy)3·HBF4, K3PO4, dioxane−water, 130 °C then DEAD, Me2NCH2CH2OH, Ph3P, THF, 0 °C; (viii) (a) ArB(pinacol), Pd dppf, Cs2CO3, dioxane−water, 130 °C, (b) TFA, CH2Cl2, (c) acetone, NaBH3CN, AcOH, CH2Cl2−MeOH; (ix) NaBH3CN, acetaldehyde or CH2O, AcOH, CH2Cl2−MeOH; (x) (a) CH2O, NaBH3CN, AcOH, MeOH, (b) TFA/ CH2Cl2−water, (c) amine, NaBH3CN, AcOH, CH2Cl2−MeOH. a

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Table 1. SAR of m-Benzylaminesa

a

All biological values are mean of ≥3 replicates.

Table 2. Lung PK of Compounds 13, 15a−ga compd

lung t1/2 (h)

residual dose at 4 h (%)

residual dose at 24 h (%)

13 15a 15b 15c 15d 15e 15f 15g

NAb 1.6 4.9 2.3 1.3 NAb 1.1 3.5

0.0 2.0 2.5 0.2 0.8 0.0 0.5 4.8

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

a

All time points were a mean from three animals. bCompounds 13 and 15e were only detected in lung tissue at the first time-point of the experiment (3 min after dosing) and then only 2% and 6.5% of the total dose, respectively.

Figure 3. View of 15c (gray) overlaid with 13 (blue) showing the very similar conformations adopted by the ligands and the almost identical protein conformations. Note the movement of the side chain amide of Asn836 permitting the dimethylamino group to be accommodated.

rotation of the phenyl ring; the dimethylamino group, as expected, pointed into the solvent channel. The dimethylamino group lies in the proximity of Asn836 (PI3Kδ), the amide moiety of which can be seen to rotate slightly to accommodate the ligand; this is one of the few highly variable residues in the vicinity of the ATP binding site of the highly homologous PI3K isoforms. The corresponding residues in the other isoforms PI3Kα Gln859, PI3Kβ Asp856, and PI3Kγ Lys890 are all different. These differences can in part explain the observed selectivity changes, thus the positively charged protonated amines can make a favorable interaction with the aspartate anion of PI3Kβ that is reflected in the increased potency at this isoform but make an unfavorable one with the cation of Lys890 in PI3Kγ, accounting for the big drop in potency observed. The neutral glutamine in PI3Kα has a more modest effect but is a

Figure 2. Compound 13 bound in murine PI3Kδ showing the ATP binding pocket with the induced pocket between Trp760 and Met752 and with hydrogen bonds to the NH of Val828 and the carbonyl of Glu826 making the hinge interaction.

Crystallization of 15c with PI3Kδ (Figure 3) showed an almost complete superposition with the structure of 13 without 5060

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Table 3. SAR of Alternate Positions for the Basea

larger group than the asparagine in δ and thus the slight loss of potency as the size of the substituent increases from 15a to 15b to 15c can be understood. These effects were seen consistently for further examples (15d−f), and consequently we shifted our focus to tertiary amines in this position to ensure we obtained sufficient selectivity vs PI3Kβ. Larger N-substituents were tolerated, as expected based on this group pointing to solvent (Figure 4), and enzyme affinities

All biological values are means of ≥3 replicates except †, n = 1, and ‡, n = 2. a

In contrast to compound 13, i.t. dosing of 15a−g to rat lung showed measurable half-lives for all except 15e, but the vast majority of compound was still cleared within a short period and negligible compound was present 24 h after dosing for any of the compounds (Table 2). We focused on the amount of compound retained at 4 and 24 h to guide our design rather than the half-life, as the latter reflected the terminal β-phase only and did not account for differences in the amount of compound lost in the rapid initial α-phase, which was often >99% (Figure 6). Compounds 15e and g both met the criteria

Figure 4. Docked structure of 20f (gray) overlaid with 13 (blue) in murine PI3K showing the large substituted aminopiperidine accommodated in the solvent channel.

at all four isoforms were remarkably consistent across the series 15a−g, barring the effect already mentioned with PI3Kβ. More lipophilic compounds, however, showed improved cell potencies, consistent with a potential favorable effect on membrane permeability (Table 1, Figure 5). We investigated

Figure 6. Rat ITPK time-course profiles of selected examples.

of Austin et al.24 for obtaining duration with inhaled β-agonists (pKa > 8, Log D > 2; pKa could not be determined for 15e but related tertiary benzylamines had pKa > 9; pKa 15g = 9.4). The long phenylethoxyalkyl chain of 15g was modeled on that used for β-agonists like Salmeterol and Sibenadet.25 Despite these precedents, 15e was barely retained at all in lung, while 15g, though having the greatest retention of this group of compounds, still exhibited insufficient lung residence for our purposes. In parallel with our investigations of basic side chains, we sought to investigate dibasic analogues (Table 4). The first two compounds synthesized (20a,b) were highly potent PI3Kδ inhibitors. As with the monobases, the secondary amine 20a was less selective against PI3Kβ than the tertiary amine 20b.

Figure 5. Enzyme and cell potencies for compounds 13, 15a−g, 17, 18, and 19 showing cell drop-off at low lipophilicity.

the preferred location of the basic center by making a series of changes to the linker length between the amine and the aromatic ring (17, 18, 19, Table 3). As the distance between the amine and the aromatic ring increased beyond two atoms the potency at PI3Kβ reduced, consistent with loss of a favorable interaction with Asp856; activity against the other isoforms was little changed. Once again cell potency dropped off for compounds with low Log D. 5061

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Table 4. Data for Dibasic Compounds 20a−la

a

compound

PI3Kδ pIC50

PI3Kβ pIC50

PI3Kδ cell pIC50

Log D

ClogP

pKa B1

pKa B2

lung t1/2 (h)

residual dose at 24 h (%)

20a 20b 20c 20d 20e 20f 20g 20h 20i 20j 20k 20l

9.2 9.3 9.2 9.1 8.8 9.2 9.1 9.1 9.2 8.9 8.9 9.3

7.7 7.2 7.3 7.0 6.6 7.1 7.2 6.6 7.9 6.9 7.1 7.8

7.9 8.0 8.6 9.0 7.9 8.9 8.7 8.9 8.5 8.3 8.2 8.7

0.6 0.5 2.1 2.5 1.1 1.7 2.0 1.4 3.2 1.5 1.5 2.2

2.7 2.7 4.8 3.5 2.6 4.2 3.8 3.8 6.1 4.4 4.3 5.1

9.6 9.5 9.8 8.4 7.3 9.7 9.0 8.0 8.8 8.5 8.9 9.1

4.7 6.5 8.0 5.4 6.0 6.8 7.0 4.0 7.5 5.7 6.3 7.0

4.2 23.2 12.0 4.5 8.7 9.9 10.9 0.0 13.0 5.9 7.6 17.6

0.1 4.8 3.3 0.2 3.5 2.8 5.2 0.0 3.3 0.8 3.9 7.4

pIC50 values for PI3Kα and γ not shown; these were consistently low (α ≤ 6.2, γ ≤ 6.7) All biological values are means of ≥3 replicates.

this effect further, we synthesized a set of compounds with a range of pKas for both basic centers (Table 4). Inevitably, these compounds (20c−l) also had varying lipophilicities; thus we also investigated the role of lipophilicity on both cell potency and PK (Figures 7 and 8). Examination of Figure 7 shows that, for the range of variables tested, the second (weaker) basic pKa is the most significant factor (6b,e). Thus, for compounds with a second pKa less than 5 (20a, 20h), half-life is short (∼6, only a small fraction is retained in the lung after 24 h. With a low second pKa, changes in lipophilicity have little effect (20a vs 20h; 20d vs 20j). For compounds where the second pKa is greater than 6, further increases provide, in

Both compounds showed reduced cell potency, consistent with their low lipophilicities. When dosed IT to rats 20b, but not 20a, showed excellent lung retention with both a long half-life and a substantial proportion of the dose remaining in the lung 24 h after dosing (Table 4, Figure 6). For 20b, there was still an initial α-phase with a rapid decrease of compound over the first 30 min, followed by a prolonged β-phase where the levels declined slowly however dibase 20b exhibited the base-driven PK profile that we were seeking for our compounds. This left the question of why 20b but not 20a was retained in the lung. Previous work23 has described the benefits of dibases for lung retention, but the precise basicities required have not been defined. Measured pKas for the two compounds were surprisingly different, with the second pKa for 20b being markedly higher than that of 20a (6.5 vs 4.7). To investigate 5062

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Figure 7. Relationships between lung retention (% remaining at 24 h; half-life) and the first and second pKas or lipophilicity (Log D) for dibases.

escape from the lysosome and hence half-life is driven by the fraction of monocation, affected by the second pKa, and lipophilicity. The effect of lipophilicity on half-life probably principally derives from the greater membrane affinity of the more lipophilic compounds, thus the longest half-life belongs to the least lipophilic dibase 20b. Additional structural features, like the long aralkyl chain of 20l, also affect the rate of membrane permeability. The most important driver of cell potency appears to be the lipophilicity (Table 4, Figure 8) (compounds where the second pKa is less than 6 are excluded from this analysis as they behave more like monobases). If Log D is greater than approximately 1.6, then a compound with good enzyme inhibition will also have good cell potency. When a larger and more diverse set of dibases were examined (data not shown), the same trend was still apparent, with a lower cutoff of Log D ca. 1.4 and an upper cutoff of ca. 2.8, although structural variations did have an additional effect. Compounds 20b and 20f were selected for further study based on their good lung retention, high potency coupled with isoform selectivity, and, for 20f, excellent cell potency. Both of these compounds were screened against a panel of 345 kinases; neither showed >50% activity at any other kinase at 1 μM. Neither compound showed significant inhibition of the hERG ion channel despite containing basic centers (