Fragment-Based Discovery of a Small Molecule Inhibitor of Bruton's

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Fragment-Based Discovery of a Small Molecule Inhibitor of Bruton’s Tyrosine Kinase Christopher Ronald Smith, Douglas R Dougan, Mallareddy Komandla, Toufike Kanouni, Beverly Michelle Knight, John David Lawson, Mark Sabat, Ewan R Taylor, Phong Vu, and Corey Wyrick J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 18 Jun 2015 Downloaded from http://pubs.acs.org on June 18, 2015

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Journal of Medicinal Chemistry

Fragment-Based Discovery of a Small Molecule Inhibitor of Bruton’s Tyrosine Kinase. Christopher R Smith*, Douglas R Dougan, Mallareddy Komandla, Toufike Kanouni, Beverly Knight, J. David Lawson, Mark Sabat, Ewan R Taylor, Phong Vu, Corey Wyrick. Takeda California, 10410 Science Center Drive, San Diego, CA 92121 KEYWORDS: Fragment-Based Drug Discovery (FBDD), Bruton’s Tyrosine Kinase (Btk), X-ray crystallography

ABSTRACT: The discovery and optimization of a series of 4-aminocinnoline-3-carboxamide inhibitors of Bruton’s tyrosine kinase are reported. A fragment-based screening approach incorporating X-ray co-crystallography was used to identify a cinnoline fragment and characterize its binding mode in the ATP binding site of Btk. Optimization of the fragment hit resulted in the identification of a lead compound which reduced paw swelling in a dose- and exposure-dependent fashion in a rat model of collagen-induced arthritis.

Introduction Rheumatoid arthritis (RA) is a progressive autoimmune disease characterized by swelling and erosion of the joints.1 In 2013, tofacitinib,2 a JAK1/JAK3 inhibitor, was the first kinase inhibitor to be approved for the treatment of RA. We sought to identify a Bruton’s tyrosine kinase (Btk) inhibitor, an alternative kinase implicated in autoimmune disorders. Btk is a member of the Tec family of nonreceptor tyrosine kinases and is expressed in B cells,3 mast cells,4 monocytes/macrophages,4b,5 neutrophils,6 dendritic cells,7 erythroid cells,4b,8 platelets,9 hematopoietic stem cells and multipotent progenitors.10 Initial studies using both human and mouse B cells identified Btk as a key signaling protein in B cell antigen receptor (BCR) signaling and activation, transferring signals from intracellular immunoreceptor tyrosine-based activation motifs (ITAMs) to phospholipase C, gamma 2 (PLCγ2) which in turn mobilizes Ca2+ and activates NF-κB.11 Examination of patients with the inherited immunodeficiency disease Xlinked agammaglobulinaemia (XLA) revealed that loss of function mutations in the Btk gene prevent the development of B cells, causing an almost complete depletion of B cells and circulating immunologloblins.3 These findings indicate Btk inhibition could be an attractive approach for the treatment of autoimmune diseases such as RA. Recently, several groups have published reports on small molecule Btk inhibitors. These studies demonstrated efficacy in rodent models of collagen-induced arthritis (CIA) and systemic lupus erythematosus (SLE)12 providing additional support for the role of Btk in autoimmune disease. The closely related protein kinase Lck (Lymphocytespecific protein tyrosine kinase) plays a key role in T-cell activation,13 hence competitors have focused on achieving selectivity for Btk over Lck. Ibrutinib14 1 is the most ad-

vanced Btk inhibitor in the clinic, approved for the treatment of mantle cell lymphoma in 2013 and chronic lymphocytic leukemia in 2014. Ibrutinib is an irreversible covalent inhibitor of Btk. Herein we report our approach to identify a new series of non-covalent potent Btk inhibitors with a targeted enzyme selectivity over Lck of ≥ 100-fold.

Figure 1. Btk inhibitor Ibrutinib 1

The concept of drug-like chemical space has been the focus of a large number of analyses, discussions and publications.15 This knowledge led us to focus our molecular designs within the following physical chemical criteria: MW < 380 Da (heavy atoms (HA) ≤ 29) and 0.5 ≤ clogP ≤ 3, as a strategy to provide the best opportunity to discover an orally available, low clearance Btk inhibitor. In addition to potency, we envisioned tracking ligand efficiency (LE)16 and ligand lipophilicity efficiency (LLE)17 metrics during the optimization process. With our focus to identify a lead molecule with MW < 380 Da, a fragment-based drug discovery (FBDD) approach was selected as the method of choice as hits are often highly ligand efficient and form high quality interactions with the protein.

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Results and Discussion Identification and Characterization of Fragment Hit 2. A library of 11,098 compounds (11 ≤ HA ≤ 19; –1.0 ≤ clogP ≤ 3) was screened biochemically at 200µM using a Caliper mobility shift assay (Caliper Life Sciences, Hopkinton, Massachusetts). A 4.6% hit rate was observed by defining a hit as > 40% inhibition at 200 µM. Hits were selected for crystallography based on a subjective assessment of fragment structure novelty, LE and after confirmation of binding by saturated transfer difference 1HNMR. A total of 20 X-ray co-crystallography structures (soaking−see supporting information for procedure) were identified. Fragment hit 2 was selected for optimization on account of its LE (0.53), accessible vectors for growth towards the P-loop and floor pocket, synthetic tractability (vide infra) and a water-mediated H-bonding interaction with the side chain of gatekeeper residue Thr474 (Table 1 and Figure 2). Significance was placed on the fragment interacting with gatekeeper residue Thr474 as only 20% of the kinome have either a Ser or Thr gatekeeper.

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by forming a diazonium salt of the readily available 2bromo aniline 4 and converting to (E)-2-amino-N’-(2-

Table 1. Biochemical and Biophysical Characterization of Fragment Hit 2 NH2 O NH2 N

N 2

HA 14 a

a

clogP 0.2

b

Btk IC50 c (µM)

LE

d

LLE

3.5

0.53

5.3

e

Lck IC50 c (µM) 38

b

HA = heavy atoms. Calculated logarithm of the octanol/water partition coefficient using JChem version 5.11.2. c Enzyme IC50 was determined in duplicate as described in the d e experimental section. LE = (1.36*pIC50)/HA. LLE = pIC50 – clogP.

Fragment Growing. Considering our starting fragment 2 (HA = 14 and clogP = 0.2) and our design goals for a lead molecule (HA ≤ 29, clogP ≤ 3.0, Btk IC50 ≤ 5 nM), a maximal addition of 15 HAs and 2.8 units of clogP were available to add during the optimization process. A 5 nM Btk inhibitor at the maximal boundaries of HA and clogP described above, translates to an LE = 0.39 and LLE = 5.3, thus providing 0.14 LE units to trade during the optimization process. Any trade in LE during the optimization process would need to be offset by gains in potency, selectivity and ADME properties. Of the possible vectors for fragment growth, the vector off the 8-position of the cinnoline ring was selected (Figure 2A). We reasoned the 8position vector pointed towards the center of the active site and provided opportunity to grow the fragment towards the P-loop (glycine rich loop) and floor pocket shown in Figure 2B. Chemistry. Compounds 7–11 described herein were prepared as illustrated in Scheme 1. 8-bromocinnoline intermediate 3 was prepared in 63% yield over two steps

Figure 2. (A) X-ray crystal structure of fragment 2 binding in the ATP binding site of Btk making three hydrogen bonds with the hinge and a water mediated hydrogen bond with the gatekeeper side chain residue Thr474. H-bond distances are measured from heavy atom to heavy atom. The red arrow highlights the vector off the 8-position of the cinnoline scaffold to grow towards the P-loop and floor pocket. (B) Alternative view of fragment 2 highlighting the hinge, P-loop (glycine-rich loop) and floor pockets regions. The water molecule binding to the gate keeper and ligand was omitted for clarity.

bromophenyl)-2-oxoacetohydrazonyl cyanide18 5 via the addition of a solution of sodium acetate and 2cyanoacetamide in water followed by Friedel-Craft alkylation using AlCl3 and chlorobenezene as the solvent to form intermediate 3. The synthesis was completed using catalytic 1,1'-bis(diphenylphosphino)ferrocenepalladium(II)dichloride dichloromethane complex (PdCl2•(dppf)∙CH2Cl2), sodium carbonate as the base in 1,4-dioxane at 140°C in a microwave to complete Suzuki-


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Miyaura19 cross coupling between aryl bromide 3 and boronic acids/esters 6a-e.

Table 2. In-vitro ADME and Rat In-Vivo PK Characterization of 7 Human liver microsomes (Eh)

0.8 a

a

Rat liver microsomes (Eh)

0.5

a

Membrane Permeability

b

IV clearance

c

Vd -1 e

-1

-1

Papp(A-B) -1 (nm sec )

Efflux c ratio

(mL min kg )

In-vivo d Eh

288

0.7

22 ± 5.7

0.3

b

c

t1/2

F e

(L kg )

(IV, h)

0.7 ± 0.15

1.3 ± 0.45

(%)

e

35 ± 30

d

Eh extraction ratio. Membrane permeability in LLC-PK1-MDR1 cell line. Efflux ratio is Papp(B-A)/Papp(A-B) In-vivo Eh ex-1 -1 e traction ratio = IV clearance/liver blood flow, where liver blood flow in rat = 70 mL min kg . Male Sprague-Dawley rat (n=2) -1 -1 dosed 1 mg kg IV (30% β-cyclodextrin in 0.05 M methanesufonic acid, pH3) and n=3 rats dosed 4 mg kg PO (0.5% methylcellulose suspension).

Scheme 1. Synthesis of compounds 7–11a

cy, increased selectivity over Lck and the PK attributes of 7 while retaining the option to add additional heavy atoms and lipophilicity.

a

Reagents and conditions: (a) (i) HCl(c), NaNO2, H2O, 0°C, 15 min (ii) NaOAc, 2-cyanoacetamide, H2O, 0°C-rt; 16 h (b) (i) AlCl3, chlorobenzene, 115°C, 16 h (ii) 2N HCl, 100°C, 1 h (c) 10% PdCl2 (dppf)∙CH2Cl2, 2M Na2CO3, 1,4-dioxane, µW, 140°C, 45 min. Structures of R1 are reported in table 3.

Analogs designed to explore the SAR off the 8-position of the cinnoline were synthesized using Suzuki-Miyaura19 methodology, coupling bromo intermediate 3 to a series of monocyclic boronic acids/esters (Scheme 1, step c). An exemplar 7 Table 3, demonstrated that growing off the 8position vector was a viable option for fragment growth in an LE and LLE-efficient manner. Progressing from 2 to 7 increased potency by a factor of 15, LE decreased from 0.53 to 0.45, LLE improved by 1.0 and Lck selectivity increased from 11-fold to 54-fold. We concluded that the increase in potency, improved Lck selectivity and increased LLE was an acceptable gain/trade for a 0.08 reduction in LE. The pharmacokinetic (PK) attributes of 7 were investigated in preparation for a lead optimization campaign and a summary of the key in-vitro and in-vivo PK data for 7 is presented in Table 2. The rat liver microsome (RLM) extraction ratio (Eh – see supplemental information) of 0.5 correlated with the rat in-vivo Eh of 0.3 consistent with a hepatic route of clearance and the invitro human liver microsome (HLM) Eh was 0.8. We concluded that this series warranted further investment in lead optimization on account of the increased Btk poten-

Lead Optimization. The goals of lead optimization were to improve Btk potency to IC50 ≤ 5nM, reduce HLM Eh to ≤ 0.5, increase Lck/Btk selectivity ratio to ≥ 100-fold and maintain the membrane permeability and in-vivo PK profile of 7. An X-ray co-crystal structure of 4methylpyridin-3-yl analog 8 with Btk revealed the pyridine lone pair makes a bifurcated water-mediated hydrogen bond with P-loop residue Phe412 and Gly414, Figure 3A. A series of bicyclic heterocycles were designed to displace this water, 2 of which are shown in Table 3. Indol-6yl 9 inhibited Btk with an IC50 850 nM causing a reduction in LE and LLE whereas indazol-6-yl analog 10 inhibited Btk with an IC50 of 12 nM and was the first analog in this series to exhibit ≥ 100-fold selectivity for Btk over Lck. Addition of a methyl group, resulting in 5-methylindazol-6-yl analog 11, further improved the Btk IC50 to 4 nM while maintaining selectivity ≥ 100-fold over Lck. Compounds 10 and 11 had cellular potencies of 92 nM and 28 nM respectively, by measuring inhibition of BTK autophosphorylation in RAJI cells stimulated with anti-IgM. An X-ray co-crystal structure of 11 bound in the ATP binding site of Btk, Figure 3B, revealed the indazole moiety oriented between the two strands of the P-loop making two H-bonds, locking down and stabilizing the P-loop conformation. The hydrogen bonds (measured heavy atom to heavy atom) with the P-loop are between indazoleN1-H and the backbone carbonyl of Gly414 (2.9Å) and indazole-N2 lone pair and backbone N-H of Phe413 (3.0Å). The 4-amino and 3-carboxamide motifs make three H-bond interactions with the hinge and a water mediated interaction with the gatekeeper side chain as seen with the initial fragment 2, thus conserving the binding mode of the initial fragment in the lead molecule. The methyl substituent aids in creating the observed 104° dihedral angle between the indazole and the cinnoline ring and points into a small pocket on the floor of the active site making efficient van de Waals interactions with lipophilic residue Leu528. Encouraged by the potency of 11 and the selectivity over Lck the kinome selectivity profile



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and Src family kinases is shown in table 4 and the full was investigated by determining % inhibition at 1 µM of dataset is available in the supporting information. 303 kinases. Follow-up IC50 data was generated for the kinases that inhibited > 75% at 1 µM. The data for the Tec Table 3. Heavy atom count, clogP, Btk enzyme inhibition, LE, LLE, Lck selectivity, cellular potency and HLM Eh data for compounds 7–11 NH2 O

5

NH2 N 1

R1

N

7 - 11

Compound

a

R1

HA

a

clogP

b

Btk IC50 c (nM)

LE

d

LLE

e

Lck IC50 c (µM)

pBtk EC50 c (µM)

HLM f (Eh)

7

20

0.3

240 ± 8

0.45

6.3

13 ± 1.7

>50.0

0.77

8

21

1.2

100 ± 46

0.45

5.9

5.9 ± 0.57

28.6 ± 2

0.24

9

23

2.0

850 ± 11

0.36

4.2

6.4 ± 0.04

ND

0.65

10

23

1.2

12 ± 0.2

0.47

6.7

1.3 ± 0.05

0.092 ± 0.01

98%. tR=3.77 min. 4-amino-8-(1H-indazol-6-yl)cinnoline-3carboxamide (10). To a mixture of 4-amino-8bromocinnoline-3-carboxamide hydrochloride 3 (0.1 g, 0.374 mmol), (1H-indazol-6-yl) boronic acid 7d (0.079 g, 0.487 mmol), and 2M aqueous Na2CO3 (0.749 mL, 1.498 mmol) in 1,4-dioxane (10 mL) was added PdCl2•(dppf)∙CH2Cl2 (0.014 g, 0.019 mmol) and heated in a microwave at 140°C for 1 h. The catalyst was removed by filtration and the filtrate evaporated to dryness. The residue was taken up in DMSO and purified by Waters- ZQLCMS system (Phenomenex Gemini Prep 5 μm C18, 75 x 30 mm column) eluting with a gradient of 25-25% in 20/80(v/v) water/acetonitrile (containing 10 mMol NH4HCO3) and water (containing 10 mM NH4HCO3). The pure fractions were collected, acetonitrile evaporated under reduced pressure and extracted in to EtOAc. The organic layer was washed with water and dried over Na2SO4 then filtered and concentrated by rotary evaporator to return 4-amino-8-(1H-indazol-6-yl) cinnoline-3carboxamide (68 mg, 0.22 mmol, 60 % yield) as light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.34 7.52 (m, 1 H) 7.64 (br, 1 H) 7.73 - 7.89 (m, 3 H) 7.94 (dd, J=7.20, 1.14 Hz, 1 H) 8.14 (s, 1 H) 8.22 (br, 1 H) 8.45 (dd, J=8.59, 1.01 Hz, 1 H) 8.55 (br, 1 H) 9.20 (br, 1 H) 13.16 (s, 1 H). 13C NMR (101 MHz, DMSO-d6) δ ppm 112.2, 116.3, 119.3, 121.9, 122.0, 124.0, 127.7, 128.4, 132.5, 133.2, 136.1, 139.9, 140.3, 144.1, 146.2, 170.1. LC-MS m/z 305 [M+H]+. HPLC purity (Method A): 98%. tR=3.49 min. 4-amino-8-(5-methyl-1H-indazol-6-yl)cinnoline-3carboxamide (11). A mixture of 4-amino-8bromocinnoline-3-carboxamide hydrochloride 3 (1.0 equiv, 0.100 g, 0.374 mmol), (5-methyl-1H-indazol-6yl)boronic acid 6e (1.0 equiv, 0.066 g, 0.374 mmol) was suspended in THF (2.8 mL, 0.135 molar) then added 2M aqueous solution of sodium carbonate (0.375 mL). PdCl2•(dppf)∙CH2Cl2 (0.07 equiv, 0.021 g, 0.026 mmol) was added last and the mixture heated in a microwave at 130°C for 1 h. The catalyst was removed by filtration and the filtrate evaporated to dryness. The residue was taken up in a DMSO/methanol mixture (9:1, 5 mL), passed through a syringe filter and purified by Waters- ZQ-LCMS system (Phenomenex Gemini 5 μm C18, 75 x 30 mm column) eluting with a gradient of 15-25% ACN (containing 0.035% TFA) in water (containing 0.05% TFA). The pure fractions were collected, evaporated to a minimal volume, and lyophilized to give the title compound, 11 as a yellow solid (81 mg, 0.25 mmol, 68 % yield). Conversion of the TFA salt to the free base; suspended 11 in water (M = 6.2), heated at 500C and stirred for 4 h (slurry to slurry change from yellow to white was observed). After 4 h, aqueous 1M NaOH (1.0 equiv) was added and the suspension was stirred for an additional 1 h with heating and then cooled to RT. The white solid was collected by vacuum filtration, washed with additional water and dried in a vacuum oven for 24 h at 40°C. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.99 (s, 3 H) 7.36 (s, 1 H) 7.58 - 7.64 (m, 2 H) 7.71 - 7.84 (m, 2 H) 8.04 (d, J=1.01 Hz, 1 H) 8.23 (br, 1 H) 8.40 (d, J=2.02



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Hz, 1 H) 8.47 (dd, J=8.46, 1.39 Hz, 1 H) 9.16 (br, 1 H) 12.95 (br, 1 H). 13C NMR (101 MHz, DMSO-d6) δ ppm 20.5, 111.1, 116.1, 119.5, 122.1, 122.6, 127.6, 128.3, 128.9, 132.3, 132.6, 138.2, 138.7, 141.1, 144.1, 146.9, 170.1. LC-MS m/z 319 [M+H]+. HPLC purity (Method B): 100%. tR=2.54 min. HRMS (ESI) m/z calcd for C17H14N6O [M+H]+, 319.1302; found, 319.1310. Anal. Calcd for C17H14N6O: C, 64.14; H, 4.43; N, 26.40. Found: C, 64.23; H, 4.41; N, 26.47.

ASSOCIATED CONTENT Supporting Information 1 H-NMR spectra, broad kinase inhibition data for 11, biological and PK experimental procedures are available free of charge via the Internet at http://pubs.acs.org. Accession codes Compound 2 4ZLY; compound 8 4ZLZ; compound 11 4Z3V.

AUTHOR INFORMATION Corresponding Author * C.R.S (+1) 858-205-0419; [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT The staff of the Berkeley Center for Structural Biology is gratefully acknowledged for support of beam line 5.0.3 at the Advanced Light Source. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC0205CH11231. We aknowledge the contributions of the following Takeda California scientists: Jacques Ermolieff, Petro Halkowycz, Ched Grimshaw, Gavin Hirst, Weston Lane, Michael Klein, Matthew Marx, Loui Madakamutil, Lisa Rahbaek, Bi-Ching Sang, Lihong Shi, Gyorgy Snell, Isabelle Tcholakov and Haihong Wu.

ABBREVIATIONS Btk; Bruton’s tyrosine kinase, Btk-KD; kinase domain of Btk, Cl; clearance; Da; Daltons, Eh; extraction ratio, JAK; Janus Kinase, Lck; Lymphocyte-specific protein tyrosine kinase, HLM; human liver microsomes, RLM; rat liver microsomes, Vd; volume of distribution.

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