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Article Cite This: J. Med. Chem. 2017, 60, 10071−10091

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Discovery and Optimization of Pyrrolopyrimidine Inhibitors of Interleukin‑1 Receptor Associated Kinase 4 (IRAK4) for the Treatment of Mutant MYD88L265P Diffuse Large B‑Cell Lymphoma James S. Scott,*,† Sébastien L. Degorce,† Rana Anjum,‡ Janet Culshaw,§ Robert D. M. Davies,§ Nichola L. Davies,† Keith S. Dillman,‡ James E. Dowling,‡ Lisa Drew,‡ Andrew D. Ferguson,‡ Sam D. Groombridge,§ Christopher T. Halsall,§ Julian A. Hudson,§ Scott Lamont,† Nicola A. Lindsay,† Stacey K. Marden,∥ Michele F. Mayo,‡ J. Elizabeth Pease,† David R. Perkins,§ Jennifer H. Pink,§ Graeme R. Robb,† Alan Rosen,‡ Minhui Shen,‡ Claire McWhirter,† and Dedong Wu∥ J. Med. Chem. 2017.60:10071-10091. Downloaded from pubs.acs.org by DURHAM UNIV on 11/24/18. For personal use only.

†

Oncology, IMED Biotech Unit, AstraZeneca, Cambridge CB4 0FZ, United Kingdom Oncology, IMED Biotech Unit, AstraZeneca, Boston, Massachusetts 02451, United States § Oncology, IMED Biotech Unit, AstraZeneca, Macclesfield SK10 4TG, United Kingdom ∥ Pharmaceutical Sciences, IMED Biotech Unit, AstraZeneca, Boston, Massachusetts 02451, United States ‡

S Supporting Information *

ABSTRACT: Herein we report the optimization of a series of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated kinase 4 (IRAK4) using X-ray crystal structures and structure based design to identify and optimize our scaffold. Compound 28 demonstrated a favorable physicochemical and kinase selectivity profile and was identified as a promising in vivo tool with which to explore the role of IRAK4 inhibition in the treatment of mutant MYD88L265P diffuse large B-cell lymphoma (DLBCL). Compound 28 was shown to be capable of demonstrating inhibition of NF-κB activation and growth of the ABC subtype of DLBCL cell lines in vitro at high concentrations but showed greater effects in combination with a BTK inhibitor at lower concentrations. In vivo, the combination of compound 28 and ibrutinib led to tumor regression in an ABC-DLBCL mouse model.

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homeostasis of human B-cells.6 After activation of TLRs, MYD88 is phosphorylated and subsequently recruits IRAKs and other downstream proteins such as TRAF6, resulting in NF-κB, JAK kinase/STAT3 activation, and secretion of IL6, IL10, and interferon-β.7,8 In ABC-DLBCL cells, an L265P mutation of MYD88 leads to a spontaneous assembly of a stable signaling complex involving IRAK4, IRAK1, and TRAF6 that leads to an increase in kinase activity of IRAK4, resulting in constitutive NF-κB activation, promoting cell proliferation and survival.4 MYD88 has also been found to be mutated at different frequencies in several lymphoid neoplasms including Waldenström’s macroglobulinemia (90%), primary cutaneous leg type DLBCL (69%), central nervous system lymphomas (38−50%), and primary testicular lymphomas (68%).9−13 IRAK4-mediated signaling is also important for inflammation and bridges innate and adaptive immunity. IRAK4 or MYD88 deficiency has been linked to susceptibility to bacterial

INTRODUCTION Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell non-Hodgkin lymphoma (NHL) comprising 30−35% of all NHLs.1 More than half of DLBCL patients can be cured with immunochemotherapy, however, approximately 30−40% of patients will develop relapsed/refractory disease that remains a major cause of morbidity and mortality due to the limited therapeutic options.2 The molecularly distinct subtype “activated B-cell like” (ABC) DLBCL has particularly poor clinical outcomes with this standard treatment regimen when compared to the “germinal center B cell-like” (GCB) DLBCL, which has a favorable prognosis.3 In ABC-DLBCL patients, recurrent gain of function mutations in the essential adaptor protein MYD88 is seen in 39% of patients, with one dominant mutation of MYD88, L265P, occurring in 29% of cases.4 Occurrence of L265P mutation in DLBCL patients has been linked to poor prognosis on standard immunochemotherapy regimen.5 MYD88 is an adaptor protein of the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) that participates in the innate immune response and plays a crucial role in the © 2017 American Chemical Society

Received: August 31, 2017 Published: November 27, 2017 10071

DOI: 10.1021/acs.jmedchem.7b01290 J. Med. Chem. 2017, 60, 10071−10091

Journal of Medicinal Chemistry

Article

Scheme 1. Core Variation with SNAr Displacementa

infections in children, particularly recurrent pyogenic bacterial infections.14,15 Given the critical role of IRAK4 in inflammatory processes and MYD88 driven cell proliferation, the inhibition of IRAK4 activity presents an attractive therapeutic approach for the treatment of inflammatory diseases and cancer. A number of groups have reported chemical equity capable of inhibiting this target. This area has recently been reviewed,16 with notable contributions published in the literature from groups including Amgen,17 UCB,18 Astellas,19 Merck,20 Pfizer,21 and Nimbus.22 In the context of oncology, the contribution made by Nimbus was particularly significant in that it showed IRAK4 inhibition, together with inhibition of B-cell receptor (BCR) signaling, could have synergistic effects on ABC DLBCL killing.22 This was also demonstrated in vivo, where a modest tumor growth inhibition effect in OCI-LY10 xenografts with an IRAK4 inhibitor was significantly enhanced by cotreatment with ibrutinib, an inhibitor of Bruton’s tyrosine kinase (BTK).22 As a starting point for our program, we were intrigued by the tricyclic scaffold reported by Nimbus and profiled a representative example (1) in terms of biological activity, selectivity, and physicochemical properties.23 This compound showed potency against IRAK4 (Table 1) and a good level of selectivity against a panel of kinases.23 A crystal structure (Figure 1) showed that this compound binds at the ATP site,

a

Reagents and conditions: (a) trans-H2NCyNMe2, DIPEA, iPrOH, MW, 85−150 °C, 4−14 h, 17−61%.

through an SNAr reaction with trans-N,N-dimethylcyclohexane1,4-diamine and the corresponding chloroheterocycle under microwave conditions (Scheme 1). In cases where the requisite chloroheterocycle was not available, these were constructed as shown in Scheme 2. Tricyclic compound 6 was constructed through formation of cyclopentyl hydrazone 6a followed by a Fischer indole synthesis to 6b, which proceeded in good yield (79% over 2 steps). Chlorination to 6c and an SNAr reaction with trans-N,Ndimethylcyclohexane-1,4-diamine gave 6. In the case of dimethyl substituted 7, the required chloroheterocycle was prepared through assembly of benzyl protected pyrrole 7a, followed by construction of the pyrimidine ring 7b using formic acid. Chlorination gave 7c, which was displaced with transdimethylcyclohexane-1,4-diamine to give 7d. Removal of the benzyl protecting group using aluminum trichloride gave 7. Variation of the 5-position was achieved via the 5-bromo substituted key intermediate 11a, prepared by SNAr reaction on the 4-chloro group of SEM protected 4-chloro-5-bromopyrrolopyrimidine with trans-dimethylcyclohexane-1,4-diamine (Scheme 3). The alkyl group of compounds 11 and 15−17 could then be installed by Negishi reaction with organozincates followed by deprotection of the SEM group (steps b,c) or alternatively, in the case of compounds 12 and 18−21, through Suzuki reaction with alkenyl organoboronates followed by hydrogenation and removal of the SEM protection25 (steps d,e,c). The 5-trifluoromethyl group was introduced using a trifluoromethylsulfonium salt26 from the protected iodo intermediate to give 14a, followed by SNAr reaction and removal of the SEM group to give 14. Attempts to introduce the analogous 5-tert-butyl substituent directly from the 5-bromo precursor 11a were unsuccessful, therefore this analogue was synthesized by condensation of 6-aminopyrimidine-2,4-diol with 2-bromo-3,3-dimethylbutanal to construct the pyrrole ring of 13a. Bis-chlorination with phosphorus oxychloride gave 13b, which underwent a regioselective SNAr reaction in which the 4chloro group was preferentially displaced to give 13c. Hydrogenation to remove the 2-chloro substituent gave 13. Variation of the linker atom was carried out using a similar approach to that described above by SNAr displacement of the 4-chloro group of SEM protected 4-chloro-5-bromo-pyrrolopyrimidine (Scheme 4). In the case of the NMe linked compound 22, the conditions were similar to those used for the NH linker. For the oxygen linked analogue 23, the trans-4(dimethylamino)cyclohexanol was treated with sodium hydride before reaction with the chloroheterocycle, using DMF as solvent. The 5-THP substituent was introduced in each case by

Figure 1. X-ray crystal structure of tricylic inhibitor 1 in complex with IRAK4 (PDB 5K75). Green dotted lines indicate intermolecular interactions involved in binding.

forming interactions with the kinase “hinge” (Val263 and Met265) and a salt bridge with Asp272. The hinge-binding is unusual in that it involves an interaction between the sulfur atom and the carbonyl of Val263, which can be understood as the donation of electron density from the carbonyl to a chargedepleted region in the valence shell of the aromatic sulfur.24 We therefore embarked on a medicinal chemistry program to investigate and understand the structure−activity relationship (SAR) surrounding this scaffold and to develop and optimize novel chemical equity in order to validate IRAK4 as an oncology target.

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RESULTS AND DISCUSSION The synthesis of compounds is described below (Schemes 1−6). Bicyclic compounds 2−5 and 8−10 were prepared 10072



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Scheme 2. Core Variation through Heterocycle Constructiona

Reagents and conditions: (a) cyclopentanone, EtOH, 80 °C, 4 h, 89%; (b) Ph2O, 250 °C, 6 h, 89%; (c) POCl3, 100 °C, 45 min, 85%; (d) transH2NCyNMe2, DIPEA, iPrOH, MW, 160 °C, 9 h, 39%; (e) HCl, PhMe, Δ, 24 h, 36%; (f) HCOOH, Δ, 7 h, 90%; (g) POCl3, PhNMe2, Δ, 3.5 h, 100%; (h) trans-H2NCyNMe2, DIPEA, iPrOH, MW, 150 °C, 10 h, 52%; (i) AlCl3, PhMe, Δ, 135 min, 32%. a

Scheme 3. Variation of the 5-Position of the Pyrrolopyrimidinea

Reagents and conditions: (a) trans-H2NCyNMe2, DIPEA, iPrOH, MW, 130 °C, 5 h, 70%; (b) R2Zn or RZnBr, Pd(dppf)Cl2·CH2Cl2, THF, 80 °C, 3−16 h, 55−100%; (c) TBAF, THF, 80 °C, 16−72 h, 9−35% or TFA, CH2Cl2, 20 °C, 3−24 h then NH3/MeOH, 1−24 h, 44−81%; (d) RBpin, Pd(PPh3)4 or Pd(dtbpf)Cl2, Na2CO3 or K2CO3, DME/H2O, 85−90 °C, 5−16 h, 41−67%; (e) Pd/C, H2, EtOH/THF or MeOH, rt, 3−16 h, 28− 100% (alkenes) or Pd/C, NH4HCO2, MeOH, Δ, 4−8 h, 57−82% (furyls); (f) Cu, Ph2SCF3·CF3SO2, DMF, 80 °C, 16 h, 44%; (g) transH2NCyNMe2, K2CO3, MeCN, 80 °C, 4 h, 51%; (h) NaOAc, H2O, 20 °C, 3 d, 72%; (i) POCl3, DIPEA, Δ, 16 h, 100%; (j) trans-H2NCyNMe2, K2CO3, MeCN, MW, 140 °C, 16 h, 84%; (k) Pd/C, H2, K2CO3, MeOH, 20 °C, 16 h, 16%. a

poorly controlled and so separation of the isomers was required. Hydrogenation and SEM deprotection gave the desired products (route II; 24, 30, 31). For the oxygen linked ether 33, an analogous approach to that described above was used (Scheme 6). For the thioether 34, the 4-chloro group was first converted to the thiol 34a, which wa then used in an SN2 reaction with the mesylate of cis4-morpholinocyclohexanol, proceeding with inversion of stereochemistry to set up the desired trans-stereochemistry of 34d. The 5-THP group was introduced under standard conditions in the presence of the sulfur, however the reduction of the alkene required Wilkinson’s catalyst at elevated

Suzuki coupling with cyclohexenyl boronate and hydrogenation, followed by removal of the SEM protection. For variation of the amine capping group, two complementary strategies were employed (Scheme 5). The first was to install the entire aminocyclohexanamine fragment with defined stereochemistry through SNAr reaction, followed by Suzuki coupling with cyclohexenyl boronate, hydrogenation, and removal of the SEM group (route I; 25−29, 32). The alternative approach was to use the SNAr to introduce an aminocyclohexanone fragment at C4, followed by a Suzuki reaction to install a cyclohexenyl group at C5. Reductive amination could then be employed to install the desired amine capping group, although the cis/trans stereochemistry was 10073



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Scheme 4. Variation of the 4-Linkage of the Pyrrolopyrimidine (NMe2)a

Scheme 6. Variation of the 4-Linkage of the Pyrrolopyrimidine (Morpholine)a

a

Reagents and conditions: (a) trans-HXCyNMe2, (DIPEA, iPrOH, 90 °C, 12 h, 75% for 22 or NaH, DMF, 20 °C, 24 h, 63% for 23); (b) dihydropyranylBpin, Pd(PPh3)4 or Pd(dtbpf)Cl2, K2CO3, 1,4-dioxane or DME, H2O, 85−100 °C, 1−6 h, 60−85%; (c) Pd/C, H2, MeOH or EtOH/THF, rt, 3−16 h, 86−93%; (d) TFA, CH2Cl2, rt, 1−3 h then NH3/MeOH, 1−24 h, 32−67%.

Scheme 5. Variation of the 4-Substituent of the Pyrrolopyrimidinea

a

Reagents and conditions: (a) trans-4-morpholinocyclohexanol, NaH, DMF, 20 °C, 24 h, 61%; (b) dihydropyranylBpin, Pd(dtbpf)Cl2, K2CO3, DME or 1,4-dioxane, H2O, 80−85 °C, 2−6 h, 73−100%; (c) Pd/C, H2, EtOH/THF, rt, 3 h, 97%; (d) TFA, CH2Cl2, rt, 1−3 h then NH3/MeOH, 1−24 h, 25−56%; (e) Na2S, DMSO, rt, 2 h, 78%; (f) cis4-morpholinocyclohexanol mesylate, K2CO3, MeCN, 80 °C, 16 h, 92%; (g) Rh(PPh3)3Cl, H2, THF, 50 °C, 7 d, 30%.

Scheme 7. Variation of the Six-Membered Ringa

a

Reagents and conditions: (a) H2NCyY or H2NCyX, where X = N[(CH2)2]2NBz for 25/26 and N[(CH2)2]2CHCO2Me for 29, DIPEA, iPrOH or nBuOH, MW, 80−150 °C, 3−18 h, 53−100%; (b) dihydropyranylBpin, Pd(PPh3)4 or Pd(dtbpf)Cl2, K3PO4 or K2CO3, THF or 1,4-dioxane or DME, H2O, 80−100 °C, 3−16 h, 52−100%; (c) Pd/C, H2, MeOH or EtOAc or EtOH/THF, rt, 11−18 h, 29−100%; (d) for 29 (i) LiOH, H2O, THF, rt, 2 h, 86%, (ii) Me2NH, HATU, DIPEA, DMF, rt, 2 h, 24%; (e) TFA, CH2Cl2, rt, 1− 3 h then NH3/MeOH, 1−24 h, or Me2NCOCl for 25/MeSO2Cl for 26, DIPEA, CH2Cl2, rt, 1 h then TFA, CH2Cl2, rt, 1 h, NH3/MeOH, 1−24 h, 39−74%; (f) 4-H2N-cyclohexanone, DIPEA, iPrOH, MW, 120 °C, 36 h, 34%; (g) HY, NaBH(OAc)3, AcOH, CH2Cl2 or MeCN, 15−25 °C, 16−18 h, 52−100%.

a Reagents and conditions: (a) dihydropyranylBpin, Pd(dtbpf)Cl2, K2CO3, 1,4-dioxane/H2O, 80 °C, 1−2 h, 76% for 35a, 100% for 36a; (b) SEMCl, NaH, THF, rt, 4−16 h, 48% for 35b, 63% for 36b; (c) LHMDS, trans-H2NCyMorph, BrettPhos Pd G3, THF, 80 °C, 16 h, 56%; (d) Pd/C, H2 (2 atm), MeOH, rt, 2−3 h, 84% for 35, 100% for 36; (e) TFA, CH2Cl2, rt, 3−4 h then NH3/MeOH, 3−4 h, 61% for 35, 70% for 36; (f) NBS, CH2Cl2, rt, 16 h, 100% (g) trans-H2NCyMorph, DIPEA, iBuOH, MW, 180 °C, 5 h, 81%.

pyridine, SEM protection, and an SNAr reaction to install the C4 amine of 36c. A subsequent Suzuki reaction, hydrogenation, and deprotection gave 36. Examination of the SAR, through deconstruction of the cyclopentyl ring of 1 revealed that opening the ring to 5,6dimethyl substituted 2 reduced potency ∼10× (Table 1). Further truncation of the 5-substituent 3, 6-substituent 4 or both groups 5 reduced potencies in line with lipophilicity with ligand lipophilic efficiency (LLE) scores remaining around 5. We hypothesized that replacement of the thiophene sulfur with a pyrrole NH could make a more traditional, and potentially stronger, hydrogen bond with the carbonyl and introduce polarity to the hinge binding motif. The fully analogous tricycle

temperature. Removal of the SEM group under acidic conditions gave 34. For variations of the six-membered ring of the core (Scheme 7), the corresponding pyridine was made by a Suzuki reaction on the unprotected azaindole core followed by SEM protection of the NH to give 35b. A Buchwald−Hartwig amination of the 4-chloro group with trans-4-morpholinocyclohexanamine using a third-generation Brettphos precatalyst27 proceeded in moderate yield (56%), then hydrogenation and removal of the SEM protection gave 35. The 3-cyano derivative 36 was synthesized by bromination of the available 3-cyano pyrrolo10074



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Table 1. Effect of Substitution on Thienopyrimidine and Pyrrolopyrimidine Cores

compd

A

R1

R2

IRAK4 Enz IC50 (μM)a

IRAK4 Cell IC50 (μM)b

log D7.4c

IRAK4 Cell LLEd

1 2 3 4 5 6 7 8 9 10

S S S S S NH NH NH NH NH

−(CH2)3− Me Me H H −(CH2)3− Me Me H H

−(CH2)3− Me H Me H −(CH2)3− Me H Me H

0.14 1.5 1.6 2.4 4.3 0.41 9.0 >10 0.005e 0.64

0.49 2.5 3.6 4.3 8.4f 1.7f 23f >30f 0.14 1.4f

1.2 0.7 0.6 0.3 0.1 0.5 0.6 −0.2 0.1 −0.6

5.2 4.9 4.8 5.1 5.0 5.3 4.0 6.6 6.5

Enzyme inhibition based on n ≥ 3 with SEM within 0.3 units unless otherwise stated. bCell inhibition based on n ≥ 2 with SEM within 0.3 units unless otherwise stated. clog D7.4 determined by shake flask method. dCell LLE calculated from pIC50-log D. eSEM = 0.28. fn = 1. a

Figure 2. Plot of IRAK4 enzyme pIC50 (A), cell pIC50 (B), cell LLE (C) against scaffolds with A = S or N showing divergent SAR between the cores. X = out of range value.

(19a/b), a clear preference for one isomer was observed.29 Unfortunately, despite having low log D7.4 values, the compounds were rapidly turned over by rat hepatocytes. Expansion to the tetrahydropyrans showed an increase in LLE for both 3- and 4-substituted isomers (20 and 21), with the 4isomer (21) having an advantage of increased stability in rat hepatocytes. The 4-THP substituent was considered to offer an attractive balance of cellular potency/low lipophilicity/low turnover in rat hepatocytes, and a crystal structure of 21 in IRAK4 was obtained (Figure 3). Comparison of the crystal structures of 21 and 1 showed a clear difference in positioning of the two scaffolds with the pyrrolopyrimidine core being held closer into the hinge by virtue of the tighter hydrogen bond between the pyrrolo NH and the carbonyl of Val262 (O--N distance is 2.9 Å, cf. O--S distance for the tricyclic compound at 3.7 Å). Closer inspection of the structures suggested that 6substituents on the pyrrolopyrimidine ring may clash with the protein surface, potentially explaining the reduced potency observed with 7 and 8 and that the enhancement in potency seen with 5-subsitution may be rationalized by the placement of lipophilicity in a similar region occupied by the cyclopentyl ring of the tricyclic core. The 4-THP of 21 shows a lipophilic stacking interaction with Tyr262 as well as a hydrogen bond to Lys213.

6 had reduced potency, albeit with lower lipophilicity and similar LLE. Opening of the ring to 5,6-dimethyl 7 reduced potency and LLE further, and the 6-methyl analogue 8 had even lower activity. By contrast, however, the 5-methyl bicycle 9 had improved enzyme potency relative to the original tricycle start point 1 and at significantly lower lipophilicity (log D7.4 0.1) with consequently higher cellular LLE (6.6), suggesting the 5-substituted pyrrolopyrimidine core as a potentially interesting template for further optimization. Removal of all substitution 10 reduced potency, but the unsubstituted core remained at higher LLE than the original tricyclic core. The divergent SAR between the S and N cores is shown graphically in Figure 2. Exploration of the 5-substitution28 with larger alkyl groups (11, 12) showed cell potency increasing in line with lipophilicity; unfortunately, metabolism in rat hepatocytes was also seen to increase (Table 2). Tertiary butyl (13) and trifluoromethyl (14) substituents had reduced potency and LLE values. Cyclic secondary alkyl substituents (15−17) showed a similar trend of potency increasing in line with lipophilicity as the ring size increased from 3 to 5, but LLE remained constant and rat hepatocyte metabolism increased. In an attempt to increase polarity, cyclic ethers were investigated. In the tetrahydrofurans, both 2-substituted stereoisomers (18a/ b) showed similar potency, but with the 3-substituted isomers 10075



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Table 2. Exploration of 5-Substituent

compd

R

IRAK4 Enz IC50 (μM)

9 11 12 13 14 15 16 17 18a 18b 19a 19b 20 21

Me Et iPr tBu CF3 cPr cBu cPn 2-THFa 2-THFa 3-THFa 3-THFa 3-THPb 4-THP

0.005d 0.013 0.005 0.14 0.099 0.011 0.003 0.006e 0.27 0.28 0.12 0.008 0.016 0.006

IRAK4 cell IC50 (μM) 0.14 0.063 0.029 0.81 0.25 0.075 0.042 0.023 1.2 2.1 0.54 0.034 0.066 0.059

Table 3. Variation of 4-Linker

log D7.4

IRAK4 cell LLE

rat heps Clintc

0.1 0.6 1.0 1.7 1.5 0.9 1.5 1.7 0.4 0.3 0.1 0.1 0.3 0.2

6.6 6.6 6.5 4.4 5.1 6.2 5.9 5.9 5.5 5.4 6.2 7.5 6.9 7.1

9 29 40 25 66 15 52 71 59 36 120 73 30 14

compd

X

IRAK4 Enz IC50 (μM)

21 22 23

NH NMe O

0.006 0.19 0.044

IRAK4 cell IC50 (μM) 0.059 0.40 0.28

log D7.4

IRAK4 cell LLE

rat heps Clint

0.2 0.6 1.2

7.1 5.8 5.4

14 8.2 100

concerns. We attributed this to the strength of the basic nitrogen (cpKA(BH+) = 10.6) and overall polarity (log D7.4 = 0.2). To address this, we designed analogues with increased lipophilicity and attenuated basicity (Table 4). Capping the basic nitrogen with various groups such as amide (24), urea (25), and sulphonamide (26) increased the log D7.4 and gave modest gains in permeability, although efflux remained an issue. In contrast, capping with a methyl carbamate (27) gave significantly enhanced permeability and removed the efflux liability observed with previous compounds, although the potency was slightly reduced compared to 21. Introduction of a morpholine was also observed to enhance permeability and reduce efflux, with 28 exhibiting high potency in both enzyme and cellular assays and good stability in rat hepatocytes. Piperidines with amides in the 4-position (29) were also found to have activity, and attempts were made to conformationally rigidify and increase lipophilicity with lactams such as 30 and 31. Intriguingly, both enantiomers of the lactam were very potent in both enzyme and cell although low permeability and high efflux ratios precluded further progression of these compounds. Our interpretation was that these groups project toward bulk solvent, and hence, in the absence of a preference from the protein, both enantiomers are accommodated equally well. The crystal structure of 28 revealed a near identical binding mode to compound 21 and a highly complementary shape to the pocket (Figure 4). The basic morpholine moiety is larger

a

Separated enantiomers of unknown stereochemistry. bRacemic mixture. cRate of metabolism (μL/min/106 cells) determined from DMSO stock solution in isolated rat hepatocytes diluted to 1 × 106 cells/mL. dSEM = 0.28. eSEM = 0.33.

The effect of changes to the atom linking the 4-position of the pyrrolopyrimidine to the cyclohexyl motif was examined (Table 3). Methylation of the nitrogen (22) resulted in a reduction in enzyme and cell potency relative to the unsubstituted amine (21) and an increase in log D7.4 (+0.4). Switching to an ether linkage (23) also reduced potency, despite a sizable increase in log D7.4 (+1.0), and showed increased turnover in rat hepatocytes. On balance, the 4-NH linker was viewed as optimal and variations in the amine group were subsequently investigated. More detailed profiling of compound 21 highlighted poor permeability (Papp 50%inh) but showed inhibition of the CLK family (×3) and haspin kinase. These hits were followed up with concentration response data (Table 6) and confirmed inhibition of CLK1, 2, and 4 and haspin together with other members of the IRAK family. We also generated Kd data (DiscoverX KdELECT)30 for the IRAK isoforms, which showed >30× selectivity for IRAK4 over IRAK1 and >100× selectivity over IRAK2 and 3. Plasma pharmacokinetic parameters for compound 28 were determined in Han Wistar rats (male; intravenous and oral) and beagle dogs (male; intravenous only). The compound was administered orally (PO) in rats as a suspension in 0.5% hydroxypropylmethylcellulose, 0.1% Tween in water at a dose of 5 mg/kg and intravenously (IV) as a solution in 5% DMSO:95% hydroxylpropyl β-cyclodextrin (30% w/v) in water at doses of 1 and 2 mg/kg in dog and rat, respectively, as shown in Table 7. The compound was characterized by high clearance (Cl) in rat (75 mL/min/kg) despite moderate predictions based on hepatocyte data (Clint 15 μL/min/106 cells, predicted clearance 42 mL/min/kg) with low bioavailability consistent with a high first pass effect. In dog, the clearance was lower (29 mL/min/kg) and in better accordance with the in vitro predictions based on hepatocyte data (Clint 5 μL/min/106 cells, predicted clearance 24 mL/min/kg). No discernible turnover was observed in human hepatocytes (Clint < 1.1 μL/min/106 cells; n = 8), translating through to a low to moderate predicted clearance in vivo (30 μM) of five cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) in a high throughput human liver microsome assay. The compound showed no discernible activity (IC50 > 100 μM) against the hERG ion channel and was inactive (IC50 > 30 μM) against a wider selection (NaV1.5, Ito Kv4.3, Iks) of ion channels. Selectivity 10078



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Figure 7. Millipore kinome selectivity for 28 (%inhibition @ 0.1 μM).

Table 6. Inhibitory Concentrations and Selected Kd Values for 28 in Kinases Identified in the Kinome Selectivity Profile As Having %Inhibition >75% kinase

IC50 (μM)

selectivity

Kd (μM)

selectivity

IRAK4 IRAK3 IRAK2 IRAK1 CLK1 CLK2 CLK3 CLK4 haspin

0.005

1.0×

0.023 0.050 0.005 >1 0.008 0.004

4.6× 10.0× 1.0× >200× 1.6× 0.8×

0.0007 0.093 0.170 0.026

1.0× >130× >200× 37×

inhibition (Figure 8B). Similar data was obtained in other ABC and GCB-DLBCL cell lines (data not shown). While compound 28 inhibits phosphorylation of IκBα and shows a concentration dose response, we did not observe complete inhibition of IκBα phosphorylation at concentrations up to 3.3 μM. In addition to MYD88, the OCI-LY10 cell line has a mutation in CD79A. ABC-DLBCL cell lines, like OCILY10, are characterized by genetic aberrations in both the BCR signaling (CD79A or B, CARD11) and the MYD88 pathways, suggesting that dual inhibition of these two pathways could be required for complete inhibition of NF-κB signaling.31 Bruton’s tyrosine kinase (BTK) is a crucial kinase in the BCR signaling pathway that maintains NF-κB signaling, and it has been shown to be a promising therapeutic target in ABC-DLBCL.32 Addition of 3 nM of the BTK inhibitor ibrutinib33 to compound 28 inhibits NF-κB signaling (Figure 9A) at much lower concentrations than either compound 28 or ibrutinib alone and leads to complete inhibition of IκBα phosphorylation at concentrations above 0.37 μM (compare Figure 8A with Figure 9A). Inhibition of NF-κB activity translates to synergistic effects on the loss of cell viability in cells treated with a combination of compound 28 and ibrutinib (Figure 9B). There is an increase in cell death in OCI-LY10 cells upon increasing concentrations of compound 28 and BTK inhibitor ibrutinib (Figure 9B,C). In contrast, no increased cell killing was observed for SUDHL2, a GCB-DLBCL cell line that is not dependent on MYD88 or BCR signaling (Figure 9B). Induction of cell death upon combination of compound 28 and ibrutinib in OCI-LY10 cells was also confirmed by detection of cleaved caspase 3 in cellular lysates which is a known marker of apoptosis (Figure 9D). We subsequently investigated the antitumor efficacy of compound 28 in combination with ibrutinib in OCI-LY10 xenograft studies in mouse. As shown in Figure 10 and Table 8, both compound 28 (12.5 mg/kg) and ibrutinib (12 mg/kg) dosed orally (qd) had modest antitumor activity as single agents, but a combination of both agents led to tumor regression. All three regimens were well tolerated throughout

study (2 mg/kg IV, using the same formulation as the rat IV study) was carried out to investigate the under prediction observed when scaling clearance from rat hepatocytes. A significant proportion of compound was excreted as a parent in the urine (∼27%) with low levels of parent eliminated in the bile (∼2%). The amount eliminated in the urine was in excess of what would be expected from passive diffusion alone, suggesting active secretion was occurring. By considering the active renal secretion component, clearance due to metabolism was calculated as 50 mL/min/kg and in much closer agreement with the prediction from hepatocytes (42 mL/min/kg). This data suggested that the amount of active renal secretion occurring in the dog was low, given the good prediction of clearance when scaling from dog hepatocytes. Oncogenic activation of NF-κB pathway via MYD88 involves IκB kinase β (IKKβ)-mediated phosphorylation and degradation of IκB proteins.4 Treatment of OCI-LY10 cells, an ABCDLBCL cell line harboring a MYD88 mutation, with compound 28 suppressed IKK activation, as indicated by decreased phosphorylation of IκBα (Figure 8A). The inhibition of NF-kB signaling by compound 28 also inhibited growth of OCI-LY10 cells in a dose-dependent manner, whereas SUDHL2, a GCB-cell line, was not sensitive to IRAK4 Table 7. Pharmacokinetic Parameters for Compound 28a species

dose (mg/kg)

Cl (mL/min/kg)

Vss (L/kg)

PO half-life (h)

IV half-life (h)

Fabs (%)

F (%)

rat dog

2, 5 1

75 29

2.1 3.0

2.0

0.8 3.3

100

28

a Compounds were dosed intravenously at either 1 (dog) or 2 mg/kg (rat) in 5% DMSO:95% hydroxylpropyl β-cyclodextrin (30% w/v) in water and orally at 5 mg/kg (rat) using a 0.5% hydroxypropylmethylcellulose, 0.1% Tween suspension in water, at a volume of 2 and 4 mL/kg (rat, intravenous and orally respectively) and 1 mL/kg (dog).

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Article

Figure 8. Compound 28 inhibits NF-κB signaling and growth of ABC-DLBCL cell line. (A) Dose-dependent inhibition of IκBα phosphorylation in OCI-LY10 cells upon treatment of cells with compound 28 for 14 h. (B) OCI-LY10 and SUDHL2 were monitored for growth in the presence of various concentrations of compound 28 in a 3-day growth assay.

Figure 9. Compound 28 combines synergistically with ibrutinib to inhibit growth and NF-κB signaling in vitro in OCI-LY10 cells. (A) Effects of compound 28 upon combination with ibrutinib (3 nM) on IκBα phosphorylation in OCI-LY10 cells. (B) Percent growth inhibition of compound 28 on proliferation of OCI-LY10 or SUDHL2 cells upon combination with ibrutinib in a 5 × 5 dose matrix in a 3-day growth assay. (C) Schematic to show the typical scale in a combination growth assay where % growth inhibition is measured from 0 to 200, and 100 is regarded as zero growth or stasis. Increasing numbers from 100 to 200 exemplifies increase in cell death. (D) Western blots showing induction of apoptosis, as measured by cleavage of caspase 3 upon treatment of OCI-LY10 cells with a combination of 10 nM ibrutinib and either 0.3 or 3 μM of compound 28.

Figure 10. Antitumor activity of compound 28 in combination with ibrutinib in OCI-LY10, an ABC-subtype DLBCL xenograft. (A) Daily oral dosing of Vehicle (×), ibrutinib 12 mg/kg (■), compound 28 12.5 mg/kg (●), and combination (▲) in female SCID mice (n = 10 per data point) with blue coloring representing duration of dosing. (B) Body weight data for the four dose groups.

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Journal of Medicinal Chemistry Table 8. Antitumor Activity of Compound 28, Ibrutinib, and Combination in the OCI-LY10 Xenograft Model treatment

dose (mg/kg)

Vehicle ibrutinib compound 28 ibrutinib compound 28

12 12.5 12 12.5

schedule qd qd qd qd qd

(po) (po) (po) (po) (po)

tumor growth inhibition (day 51) (%)

p-value (day 51)

56 60 >100 >100

0.0079 0.0009

IRAK4 - ACS Publications - American Chemical Society

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