Development of potent and selective pyrazolopyrimidine IRAK4

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Development of potent and selective pyrazolopyrimidine IRAK4 inhibitors Marian C. Bryan, Joy Drobnick, Alberto Gobbi, Aleksandr Kolesnikov, Yongshen Chen, Naomi Rajapaksa, Chudi O Ndubaku, Jianwen A. Feng, Willy Chang, Ross Francis, Christine Yu, Edna F. Choo, Kevin DeMent, Yingqing Ran, Le An, Claire Emson, Zhiyu Huang, Swathi Sujathabhaskar, Hans Brightbill, Antonio DiPasquale, Jonathan Maher, John Wai, Brent McKenzie, Patrick J. Lupardus, Ali Zarrin, and James R. Kiefer J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00439 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 13, 2019

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Journal of Medicinal Chemistry

Development of potent and selective pyrazolopyrimidine IRAK4 inhibitors

Marian C. Bryan,*,A Joy Drobnick,A Alberto Gobbi,A Aleksandr Kolesnikov,A Yongsheng Chen,B Naomi Rajapaksa,A Chudi Ndubaku,A° Jianwen Feng,A∆ Willy Chang,A# Ross Francis,A Christine Yu,A Edna F. Choo,A Kevin DeMent,A≠ Yingqing Ran,A Le An,A Claire Emson,A□ Zhiyu Huang,A Swathi SujathaBhaskar,A Hans Brightbill,A Antonio DiPasquale,A Jonathan Maher,A John Wai,B Brent S. McKenzie,A Patrick J. Lupardus,A● Ali A. Zarrin,A James R. KieferA

A Genentech B

Inc., One DNA Way, South San Francisco, CA 94080

WuXi Apptec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, P. R. China

KEYWORDS Kinase, inhibitor, drug discovery, lupus, inflammation, interleukin-1 receptor activated kinase, IRAK, structure based drug design

ACS Paragon Plus Environment

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ABSTRACT: A series of pyrazolopyrimidine inhibitors of IRAK4 were developed from a high throughput screen (HTS). Modification of an HTS hit led to a series of bicyclic heterocycles with improved potency and kinase selectivity but lacking sufficient solubility to progress in vivo. Structure-based drug design, informed by co-crystal structures with the protein and small molecule crystal structures yielded a series of dihydrobenzofurans. This semi-saturated bicycle provided superior drug-like properties while maintaining excellent potency and selectivity. Improved physicochemical properties allowed for progression into in vivo experiments, where lead molecules exhibited low clearance and showed targetbased inhibition of IRAK4 signaling in an inflammation-mediated PK/PD mouse model.

INTRODUCTION The innate immune system forms a first line of response to infection in mammals. This system is required to sensitively distinguish between self and non-self antigens and immediately respond to infections and injuries. Receptors, both on the cell surface and in endosomal compartments, are critical sensors of innate immunity, recognizing pathogens and initiating signaling cascades to mount immune responses.1 Two such examples are the IL-1 receptor (IL-1R) family and toll-like receptors (TLRs), transmembrane pattern recognition receptors (PRRs) critical in the detection of pathogen-associated molecular patterns (PAMPs) of foreign microbes. Together with the IL-1R family, TLRs enable cells to recognize molecular structures present in pathogens and quickly respond to them by initiating and amplifying the pro-inflammatory cytokine response. While this system is critically important to the innate immune system, dysregulation of TLR signaling in particular has been implicated in autoinflammatory diseases such as rheumatoid arthritis (RA) and several others.2-3 In RA for example, multiple TLR family members are upregulated when compared to healthy individuals. Stimulation of these receptors leads to production of the inflammatory cytokines such


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as IL-6 and tissue-destructive matrix metaloproteins.4 TLR family members are also implicated in the pathophysiology of systemic lupus erythematosus (SLE), another debilitating autoimmune disease. SLE is characterized by chronic systemic inflammation of the joints and kidneys among other organs. Ligand binding to either IL-1R or TLR family members leads to dimerization and recruitment of adaptor molecules to a conserved motif on the cytoplasmic domain of the receptor called the Toll/IL-1R (TIR) domain.5 Binding then leads to recruitment of MyD88 and formation of the myddosome complex which includes the interleukin-1 receptor activated kinase (IRAK) family.4-9 This family of kinases then plays the crucial role of signal transduction propagating both the innate immune response. Autophosphorylation of IRAK4, the first step in the pathway, leads to IRAK1 phosphorylation which in turn leads to downstream signaling through multiple cascades, ultimately altering gene transcription and initiation of the inflammatory response. The IRAK kinase family is comprised of four members, IRAK1 and IRAK4 which perform scaffolding functions in addition to being active kinases, and IRAK2 and IRAK3 (also called IRAKm), which are catalytically inactive.9 Of the two family members with kinase activity, IRAK4 has been called the “master IRAK” and is the most upstream kinase required for IL-1R and TLR signaling.8, 10 IRAK4’s role in the innate immune response has led to both interest in it as a target for therapeutic intervention in autoinflammatory diseases as well as concerns of safety due to its role as a critical node. While IRAK4-deficient patients show increased susceptibility to certain pyogenic infections, adults show no increased risk de-risking IRAK4 as a therapeutic target to an extent and interest in targeting IRAK4 has grown. Several reports of small molecule kinase inhibitors targeting the IRAK4 kinase domain, including those shown in Figure 1, have been recently published, including the clinical candidate PF06650833 (1), pyridine indazole 2, BMS-986126 (3) and aminopyridine 4.2, 9, 11 Lactam 1 was advanced



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into Phase II in RA though results have not yet been disclosed (NCT02996500). This molecule and others continue to undergo clinical evaluation.

O O

NH2 N O

O Me

NH F

O

PF-06650833, 1

N NH

N N

O

O

N

Me

F

F F

F HO

N H N

H N

N

O

N O

HN

S O

N NH

N

N

NH2

N N

2

3

4

Figure 1. Structures of IRAK4 kinase inhibitors PF-06650833 (1), indazole 2, 1,6-naphthyridine 3 and aminopyridine 4.

RESULTS AND DISCUSSION From a high throughput screening campaign, we identified hits 5 and 6, differing only in piperidine substitution, as starting points from which to design potent and selective IRAK4 inhibitors. Both of these quinolines showed excellent biochemical potency against IRAK4 (Table 1) and reasonable LLE based on cLogP.12-13 Both molecules possessed selectivity over IRAK1. While IRAK1 plays important scaffolding roles in the cell, its kinase activity has been reported as unnecessary for homeostasis, and several studies have highlighted the integral role of IRAK4 alone in Toll-like receptor (TLR)-mediated immune responses.3, 14 As there is a high degree of sequence homology between the ATP binding sites of IRAK1 and IRAK4,2 we hypothesized that measured selectivity of greater than 200-fold against IRAK1 would provide a useful initial kinase selectivity triage parameter. Evaluation of compounds 5 and 6 against a broad kinase panel showed compound 5 inhibited ≥70% of the activity of 34 out of 236 kinases at 1 µM inhibitor concentration, while compound 6 inhibited 22 out of 220 kinases tested.


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Table 1. Profile of high throughput screening hits, quinolines 5 and 6. N N

O NH N

N N

O

N

NH

N

N

N

N

NH2

5

OH

6

5

6

LLEcLogP13 / Calc’d pKaa

6.78 / 9.3

6.54 / 7

LogD / K. Sol.b (μM)

0.79 / 22

3.1 / 1

IRAK4 Ki (nM)c

2 ± 0.71

1.7 ± 0.73

(n=183)

(n=6)

510 ± 110

400 ± 110

(n=172)

(n=5)

IRAK1 Ki (nM)c

41 ± 2.5 NFkB Cell IC50 (nM)c H/R/MLMd a

35 ± 4.1 (n=2) (n=133) 4.5 / 14 / 51

12 / 43 / 75

Moka v.2.6.515 bKinetic Solubility: experimental procedures for kinetic solubility experiments can be

found in the Supporting Information; cData are the geometric mean of at least two independent experiments ± standard deviation of the average for the number (n) of experiments conducted.; dH/R/MLM defined as predicted hepatic clearance in liver microsomes for human (H), rat (R) and mouse (M). Reported in units of mL/min/kg. Both compounds showed strong inhibition of NFkB reporter gene activity (41 and 35 nM, respectively, Table 1) in a THP1-XBlue stable cell line. As a secondary readout, cell viability was assayed in this cell line, and no inhibition of proliferation was seen at up to 20 µM concentrations for either compound. The



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proliferation assay was also used to give a preliminary indication of kinase selectivity, as inhibition of IRAK4 alone should not lead to target-related cytotoxicity due to the previously mentioned safety rationale. While both compounds were potent in the biochemical and cellular assays, the scaffold presented challenges that needed to be addressed, including kinase selectivity compatible with chronic dosing and in vitro ADME properties. In order to understand their binding mode to enable SAR development, we obtained a 2.5Å resolution x-ray co-crystal structure of 5 with fully phosphorylated IRAK4 (Figure 2A, Figure S2 in the Supporting Information). Compound 5 binds to the hinge through the backbone of Met265 and Val263 via the amide carbonyl and the C2 hydrogen of the pyrazolopyrimidine, referred to as the “hinge binding element” (Figure 2B). 16This was of interest to the team as the pyrazolopyrimidine moiety is known to bind to hinge residues in an alternate configuration. In an example from the JAK kinase family, the N1-nitrogen of the pyrazolopyrimidine core makes a hydrogen bond with a backbone NH and the amide carbonyl forms a hydrogen bond with a water molecule.17 This binding configuration has also been reported for other IRAK4 inhibitors.16 The leading edge of this fused 5,6 ring system forms van der Waals contacts and potentially -stacking interactions with the kinase “gatekeeper” residue Tyr262, an amino acid relatively unique to the IRAK family among protein tyrosine kinases.18 The quinoline system, referred to as the “linker” in Figure 2B, stacks under Met192 and forms multiple additional van der Waals interactions to residues of the P-loop and floor of the kinase binding site (Figure 2C). Finally, the methylamine substitution on the piperidine, occupying the ATP ribose pocket, forms a hydrogen bond with the backbone carbonyl of Ala315 and proximal solvent molecules.


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Figure 2. Co-crystal structures of IRAK4 initial lead inhibitors. The 2.5Å resolution co-crystal structure of 5 (A, PDB ID 6O9D) bound in the IRAK4 active site reveals three polarized functional groups forming hydrogen bonds to the protein. Further annotation (B) shows the sub-regions together with key molecule features and their interactions. The solvent accessible surface of the binding site (C) shows substantial van der Waals contacts made between nonpolar (green mesh) protein residues and the inhibitor as well as focal polar (pink mesh) contact points. Regions above and below the plane of the quinoline ring (dotted circles) are potentially accessible through elaboration of the inhibitor linker. A 2.0Å resolution co-crystal structure of 17 (D, PDB ID 6O94) shows the inhibitor benzimidazole forms an additional polar interaction with the protein.

Analysis of the protein-inhibitor contacts in the co-crystal structure of 5 and comparison of them to known IRAK4 small molecule inhibitors (SMI) revealed additional potential interactions and vectors towards the solvent front that could be exploited for optimization of the linker region of the compound.2



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As such, we synthesized a set of 6,6- and 5,6- ring systems (Table 2). Truncation of the ribose pocket group to methyl ether 7 led to a 30-fold loss in activity and a 5-fold loss in selectivity when compared to 6 though allowed for rapid development of linker SAR. Replacement or movement of the quinoline N to yield naphthalene 8 and isomeric quinoline 9, respectively, lost significant potency when compared to parent 7. The dramatic erosion in potency of 9 was presumably due to a repulsion between the N lone pair and a backbone carbonyl. Substitution at R1 was then undertaken with morpholine 11. The morpholine moiety was selected due to its presence in a number of known IRAK SMI.2 This compound lost potency compared to parent 10. In order to understand if this was due to steric interference, we generated the smaller dimethylamino analog (12) and modified the angle by introduction of a second nitrogen to the linker to give quinazoline 13. While removal of some of the morpholine bulk was able to regain some of the potency (26 nM for 12 versus 8 nM for 10), quinazoline 13 was equipotent to quinoline 11 and none of these changes to the 6,6biaryl systems were able to improve LLEcLogP compared to the initial HTS hit 6.

Table 2. 6,6 versus 5,6 linkers.


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IRAK4 Ki

Cmpd R1

IRAK1

(nM)a

R2

Core

NFkB (nM)a

# (LLEcLogP13)

6

NH R

N

7

(n=6) (6.5)

(n=2) 2,800 ±

N N N

52 ± 15 (n=2)

370 ± 71 600

O

(5.6)

R2

(n=4) (n=2)

N N

O

-

35 ± 4.1 110 (n=5)

NH N

8

OH

2

O

-

1.7 ± 0.73 N

N

(nM)a 400 ±

N N

O

-

Cell IC50

NH

N

O

610b (3.6)

ND

ND

O

>30,000b (ND)

ND

ND

8b (5.0)

ND

ND

R2

9

-

N N

O N

NH

N

R2

10

-

NH N

11

N N

O

290 ± 130

R2

4,700 ± 620

N

(n=3) (3.1)

0 (n=3) (n=4) 500 ±

N N NH

R1 N

N

R2

O N

1,300 ±

N N NH

O

R1 N

12

N

R2

O

N

N

N

26 ± 9.8 (n=6)

98 ± 24 230

N

(3.8)

(n=4) (n=6)



13

O

N O

NH

N R1 N

14

-

N N

5,400 ±

3,000 ±

860

2,000

(n=4)

(n=4)

ND

ND

170 ± 300 N

N

(n=4) (3.8)

R2

N N

O H N

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NH

N

790b (4.4)

O

R2

15

16

17

18

19

20

-

O H N

NH

N

R2

O

R1

O

R1

R1

R1

R2

R1

OH

N

NH

N

R2

N

NH

N

R2

OH

N

NH

N

R2

OH

N

NH

N

R2

(n=2)

N

1.7 ± 0.6 (n=5)

>5,500

250 ± 33

(7.2)

(n=5)

(n=3)

1.8 ± 0.9 (n=9)

>6,100

97 ± 97

(7.1)

(n=9)

(n=7)

95 ± 22 (n=4)

>10,000

910 ± 50

(4.9)

(n=4)

(n=2)

N OH

340 ± 1.2 ± 0.5 (n=6) OH

60 ± 19 94

N

(6.8)

(n=6) (n=4)

N N

S

(6.4)

N

N N

O

150

N

N N

N

>5,700 (n=4)

N N

O

N O

N

O

N O

NH

O

N O

H N

O

N

6.7 ± 0.8 (n=2) N

N

N N

O

N

330 ±

N N

N

0.5 ± 0.2 (n=6)

24 ± 5.9

8 ± 2.1

(6.9)

(n=5)

(n=3)

N OH


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21

N

R1

22

aData

N N

O NH

N

2

R

R1

N

OH

S

NH

N

2

R

N

95 ± 36

53 ± 20

(6.3)

(n=5)

(n=3)

190 ±

N N

O

N O

S

0.5 ± 0.2 (n=5) N

0.8 ± 1.0 (n=5) O

76 ± 18 12

N

(7.1)

(n=5) (n=4)

are the geometric mean of at least two independent experiments ± standard deviation of the average

for the number (n) of experiments conducted. ND = not determined. bAssay conditions and experimental datafor these molecules were reported in WO2012007375A1.19

At this point we shifted focus to optimize the linker to 5,6-ring systems. Indole 14 was analogously to naphthalene 8 highlighting the same trend towards a requirement for a critical H-bond acceptor in the quinoline linker. Unsubstituted benzimidazole 15 was 4x less potent than the quinolone 6 in the biochemical assay with similar LLEcLogP but continued to experience a larger cell shift (45x vs 20x). Appending a morpholine R1 substituent resulted in compound 16 that was equipotent to 6 in a biochemical context with improved LLEcLogP but continues to show a sizeable shift in cellular potency. Capping the NH of the benzimidazole with a methyl group (17) led to a compound that was both potent and selective over IRAK1. This kinase selectivity was recapitulated when the molecule was assessed against a broader kinase panel as shown in Table 3, where the molecule inhibited to ≥70% the activity of only 13 out of 220 kinases when measured at 1 µM (Supporting Information Table S1). Introduction of an additional methylene (18) eroded both of these potency and selectivity gains, presumably due to steric clashes with the protein backbone. Alternate 5,6-linker systems such as benzoxazole 19 and benzothiazole 20 either maintained or improved potency relative to initial molecule 6 with morpholine 20 being 3-4x more potent in the assays. Trimming the morpholine to a dimethylamine (21) retained enzymatic potency but



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negatively impacted the cellular potency (52 nM versus 8 nM). In addition, while selectivity over IRAK1 was improved with compound 21 relative to 20, evaluation in broader panels revealed dimethylamine 21 inhibited 23 kinases (in addition to IRAK4) in a panel of 41 to ≥70% activity at 1 µM, while morpholine 20 inhibited only 15 additional kinases to the same degree against a much broader panel of 220 at the same concentration (Supporting Information Table S1). Analysis of the 2.0Å resolution co-crystal structure of benzimidazole 17 (Figure 2D, Figure S2 in the Supporting Information) shows that the compound maintains the same key hinge interactions of 5 and projects its morpholine towards solvent, forming additional van der Waals interactions with the protein. The bis-morpholine inhibitor 22 lost cellular potency compared to 20 but importantly improved kinase selectivity, reducing the number of off-target kinases with activity inhibited ≥70% to 9 out of 221 at 1 µM including IRAK4 (Supporting Information Table S1). With comparably promising biochemical and cellular potency, as well as reasonable kinase selectivity, molecules 17, 20, and 22 were examined in in vitro metabolic stability assays. These analogs were predicted to be stable-to-moderate in mouse and human based on liver microsomes (Table 3: 7.4 to 8.5 mL/min/kg in HLM and 33 to 46.4 mL/min/kg in MLM) but differentiated when advanced to hepatocytes. Benzimidazole 17 and benzothiazole 20 both showed heightened lability in human hepatocytes with predicted clearances of >70% of liver blood flow (LBF)(15.3 and 17.8 mL/min/kg, respectively) though 17 appeared stable in mouse hepatocytes at 200°C), resulting in poor solubility across a range of pH,20 and several crystal packing interactions for the fused biaryl linker leading to extensive -stacking interactions in the crystal lattice (see small molecule crystal structures in Figure S4 in the Supporting Information). Despite many changes probing exploration of the solvent front and ribose pocket of this and related biaryl systems, we were not able to see measurable improvements in solubility. Therefore, we next designed molecules incorporating a reduction in linker aromaticity and planarity to drive solubility and bioavailability improvements.

Table 3. In vitro and in vivo PK of 17, 20 and 22. Mouse Mouse PO PK IV PK H/MLMa

MDCK

Cmpd H/M # CLhepb

Papp (A-B)c

PPB

Kinase

(H / M)

countd

CLp / Vss

Dose

Cmax

AUC∞

F

(mg/kg) (μM)

(μM*h)

(%)

(L/kg)e



7.4 / 33 17

13 / 2.4

88 / 95

15 / 21

220

8.5 / 42

16 /

20

8.4

98 / 99

18 / 47

23 / 0.4

5f

18 / 0.4

5f

12 7.5 / 21

aH/MLM

0.2

0.5

3%

BLODh

220

7.6 / 46 22

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95 / 99

9 / 221

5f

0.2

0.6

5%

10g

1.3

12

25%

30g

2.0

11

12%

100g

3.3

40

6%

17 / 0.9

defined as predicted hepatic clearance in liver microsomes for human (H) and mouse (M).

Reported in units of mL/min/kg. bH/M CLhep defined as predicted hepatic clearance in hepatocytes for human (H) and mouse (M). Reported in units of mL/min/kg. cA-B, apical-to-basolateral permeability as measured in Madin–Darby canine kidney (MDCK) cells. Reported in units of ×10−6 cm/s; PPB = plasma protein binding, % bound. dRatio of number of kinases inhibited at ≥70% against the total number of kinases assessed at 1 μM. eClp = blood clearance. Reported in units of mL/min/kg. Compound dosed IV (1 mg/kg) as a solution in DMSO:Cremophor:Saline (30:10:60). fCompound dosed po as a suspension in MCT; gCompound dosed po as a nanosuspension in MCT. hBLOD = Below the Limit of Detection

Similar to other kinases, most of the active site of IRAK4 is a relatively flat, narrow pocket; however, the solvent front adjacent to the hinge region (Figure 2C) broadens and can accommodate threedimensional ligand topology; thus, we sought to introduce more sp3 character into this region of the molecules in order to improve solubility.21-22 Docking studies with semi-saturated systems suggested that they would complement the protein shape, and their enhanced 3-dimensionality could disrupt the crystal packing observed in the small molecule x-ray structures, while maintaining the key vectors to the ribose


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pocket and solvent front regions. Additionally, for dihydrobenzofuran linkers, the docking pose suggested that the oxygen atom would occupy a comparable position to the conserved hydrogen bond acceptor functionality observed in the previous scaffolds, such as 17 (Figure 2D). This feature could maintain a water mediated hydrogen bond with the protein, previously observed to enhance potency (e.g. the pyridine N of 7 compared with the CH of 8, Ki = 52 nM and 613 nM, respectively, Table 2). The design also provided flexibility to introduce modifications to the hinge and ribose pocket groups. The first molecule synthesized in this series validated the hypothesis by demonstrating that a dihydrobenzofuran linker provided biochemical and cellular potency within an order of magnitude of previous leads 20 and 22 though with lower efficiency (LLEcLogP = 5.7 and 5.6 vs 6.9 and 7.1, respectively). Both 23 and 24 also maintained or improved in vitro permeability. Although 23 remained poorly soluble (kinetic sol. = 1 µM), modification of the ribose pocket group in 24 improved kinetic solubility (kinetic sol. = 95 µM) while maintaining potency and improving permeability. Furthermore, these molecules maintained excellent selectivity over IRAK1 (220x and 210x, respectively). In order to swiftly assess kinase selectivity without requiring a full screen of >200 kinases, we profiled off-target kinases that either routinely showed significant inhibition with our core scaffold, were also involved in the cytokine production pathway which would complicate on-target efficacy evaluation, or whose inhibition would lead to known safety liabilities. This group formed a core panel of 27 kinases that could be rapidly screened. A kinase selectivity ratio (KS27) was developed for rank-ordering molecules on selectivity, with a lower value correlating to inhibition of fewer off-target kinases inhibited at ≥70% at 1μM. Evaluation of 23 and 24 along with the previous lead molecules provided a range of KS27 ratios with initial quinoline 6 showing a KS27 = 0.52 reflecting its inhibition of 14 kinases out of 27 and 22 out of 220 when screened at 1 μM. Improvements in kinase selectivity for benzothiazoles 20



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and 22 also translated to an improved KS27 (KS27 = 0.44 and 0.26, respectively). Gratifyingly, 23 and 24 demonstrated improved kinase selectivity with a KS27’s of 0.15 and 0.22, respectively. Table 4. Initial efforts toward reducing planarity with semi-saturated 5,6-ring systems and modification of the pyrazolopyrimidine hinge binding element. N

N N

O NH O

N

NH O

N

N

O

N

NH O

N

N

OH

N

O

N

NH O

N

OH

O

23

N N

O

N

O

24

O

25

R= 26

OH

27

OCF2H

28

CF2H

29

IRAK4 Ki

K. Cell

(nM)a # (LLEcLogP) 5.3 ± 2.1

MDCK KS27

Sol.b IC50

Ratioc

Papp(A-

H/MLMe

B)d

(μM)

(nM)a 170 ± 38

23 (n=4) (5.7)

(n=4)

1.5 ± 0.5

38 ± 6.7

(n=5) (5.6)

(n=4)

>8,400

>20,000

(n=2)

(n=2)

3.2 ± 0.4

330 ± 34

(n=4) (6.3)

(n=4)

34 ± 11

>20,000

(n=4) (4.4)

(n=4)

24

25

26

27

R= 30

1

0.15

14

5.3 / 81

95

0.22

16

9.5 / 74

77

--

--

13 / 26

3.1

0.15

14

3.9 / 44

1

--

0.6

15 / 58


CN O

31 NH2

32

NFkB Cmpd

R

N

OH

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31 ± 11

1,200 ± 1,000

28 (n=4) (5.4)

--

--

10 / 56

1

--

--

7.4 / 54

1

0.19

2.7

14 / 47

1

0.19

0.7

12 / 49

15

0.074

18

3.9 / 57

(n=4)

35 ± 9.0

>20,000

(n=7) (4.0)

(n=2)

6.1 ± 0.4

84 ± 10

(n=4) (5.9)

(n=4)

1.1 ± 0.4

56 ± 9.7

(n=4) (7.5)

(n=4)

6.5 ± 0.9

66 ± 5.2

(n=8) (6.4)

(n=4)

29

30

31

32 aData

2.5

are the geometric mean of at least two independent experiments ± standard deviation of the average

for the number (n) of experiments conducted. ND = not determined; bKinetic Solubility: experimental procedures for kinetic solubility experiments in the Supporting Information; cKS27 Ratio defined in Supporting Information. dA-B, apical-to-basolateral permeability as measured in Madin–Darby canine kidney (MDCK) cells. Reported in units of ×10−6 cm/s. eH/MLM defined as predicted hepatic clearance in liver microsomes for human (H) and mouse (M). Reported in units of mL/min/kg. To this point, no changes had been undertaken on the pyrazolopyrimidine. However, we were interested in the ability of pyrazolopyrimidine substitution to impact selectivity given the relative rarity of the tyrosine gatekeeper. Modification of the pyrazolopyrimidine of 23, selected for synthetic feasibility over hydroxymethyl piperidine 24, was then undertaken (Table 4). The only example of substitution at C7, hydroxypyridine 25, lost measurable potency while C6 substitution was well tolerated in the biochemical assay with hydroxyl-substituted pyrimidine 26 being equipotent to the parent pyrimidine 23



Page 18 of 67

with the lowered lipophilicity reflected in the improved LLEcLogP. Moieties with increased lipophilicity at R4 led to 6-fold loss in biochemical potency and lower lipophilic efficiency as seen with 27-29 despite differences in steric bulk or hydrogen bond donating / accepting capabilities. This is particularly interesting for 26 when compared to 28. Both have the potential for hydrogen bond donation but 28 loses an order of magnitude in biochemical potency. Modifications with comparable or improved lipophilicity, such as nitrile 30 and primary amide 31, maintained biochemical potency with superior cellular potency. However, these groups led to poor permeability with low solubility and no improvement on KS27 (Table 4). The one modification that appeared universally beneficial was the hydroxymethyl substituent in 32. This molecule maintained biochemical potency and permeability while improving solubility, in vitro stability and kinase selectivity (K. Sol. = 15 μM, H/MLM =

Development of potent and selective pyrazolopyrimidine IRAK4

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