Discovery and Optimization of Pyrrolopyrimidine Inhibitors of

Subscriber access provided by READING UNIV

Article

Discovery and Optimisation of Pyrrolopyrimidine Inhibitors of Interleukin-1 Receptor Associated Kinase 4 (IRAK4) for the Treatment of Mutant MYD88 Diffuse Large B-Cell Lymphoma. L265P

James S. Scott, Sébastien L. Degorce, Rana Anjum, Janet Culshaw, Robert M. D. 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., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01290 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Discovery and Optimisation 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,♣ Dedong Wu.♥ ♣

Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom.

♠

Oncology, IMED Biotech Unit, AstraZeneca, Boston, United States.

♦

Oncology, IMED Biotech Unit, AstraZeneca, Macclesfield, United Kingdom.

♥

Pharmaceutical Sciences, IMED Biotech Unit, AstraZeneca, Boston, United States.

ABSTRACT

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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, combination of compound 28 and ibrutinib led to tumor regression in an ABC-DLBCL mouse model.

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 to 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 centre B-like’ (GCB) DLBCL which has 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


Page 2 of 72

Page 3 of 72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 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 NFκB, JAK kinase / STAT3 activation and secretion of IL6, IL10 and interferon-β.7,8 In ABCDLBCL 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 signalling is also important for inflammation and bridges innate and adaptive immunity. IRAK4 or MYD88 deficiency has been linked to susceptibility to bacterial 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 reviewed16 with notable contributions published in the literature from groups including Amgen,17 UCB,18 Astellas,19 Merck,20 Pfizer21 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) signalling could have synergistic effects on ABC DLBCL killing.22 This was also demonstrated in vivo, where a modest tumour growth inhibition effect in OCI-Ly10 xenografts with an IRAK4 inhibitor was significantly enhanced by co-treatment



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, 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 charge-depleted region in the valence shell of the aromatic sulfur.24

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


Page 4 of 72

Page 5 of 72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The synthesis of compounds is described below (Schemes 1-6). Bicyclic compounds 2–5 and 8–10 were prepared through an SNAr reaction with trans-N,N-dimethylcyclohexane-1,4diamine and the corresponding chloroheterocycle under microwave conditions (Scheme 1). Scheme 1. Core variation with SNAr displacementa

(a)

X=S X=NH 2 R1=Me; R2=Me 8 R1=Me; R2=H 3 R1=Me; R2=H 9 R1=H; R2=Me 4 R1=H; R2=Me 10 R1=H; R2=H 1 2 5 R =H; R =H

a

Reagents and Conditions: (a) trans-H2NCyNMe2, DIPEA, iPrOH, MW, 85-150 oC, 4-14 h,

17-61%. 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 trans-dimethylcyclohexane-1,4-diamine to give 7d. Removal of the benzyl protecting group using aluminium trichloride gave 7. Scheme 2. Core variation through heterocycle constructiona



(a)

(b)

(d)

6a

(e)

Page 6 of 72

6

X=OH 6b X=Cl 6c

(c)

(h)

(f)

H2NBn 7a

a

(g)

X=OH 7b X=Cl 7c

(i)

Y=Bn 7d Y=H 7

Reagents and Conditions: (a) cyclopentanone, EtOH, 80 oC, 4 h, 89%; (b) Ph2O, 250 oC, 6 h,

89%; (c) POCl3, 100 oC, 45 min, 85%; (d) trans-H2NCyNMe2, DIPEA, iPrOH, MW, 160 oC, 9 h, 39%; (e) HCl, PhMe, ∆, 24 h, 36%; (f) HCOOH, ∆, 7 h, 90%; (g) POCl3, PhNMe2, ∆, 3.5 h, 100%; (h) trans-H2NCyNMe2, DIPEA, iPrOH, MW, 150 oC, 10 h, 52%; (i) AlCl3, PhMe, ∆, 135 min, 32%. 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, 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, 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 synthesised by condensation of


Page 7 of 72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6-aminopyrimidine-2,4-diol with 2-bromo-3,3-dimethylbutanal to construct the pyrrole ring of 13a. Bis-chlorination with phosphorous oxychloride gave 13b, which underwent a regioselective SNAr reaction in which the 4-chloro group was preferentially displaced to give 13c. Hydrogenation to remove the 2-chloro substituent gave 13. Scheme 3. Variation of the 5-position of the pyrrolopyrimidinea

(b,c) 11 R=Et 15 R=c Pr 16 R=c Bu 17 R=c Pn

(b,c) or

(a)

(d,e,c)

(d,e,c) 12 R=iPr 18 R=2-THF 19 R=3-THF 20 R=3-THP 21 R=4-THP

11a

(g,c)

(f)

14a

14

(j)

(h)

(i)

a

X=OH 13a X=Cl 13b

(k)

Y=Cl 13c Y=H 13

Reagents and Conditions: (a) trans-H2NCyNMe2, DIPEA, iPrOH, MW, 130 oC, 5 h, 70%;

(b) R2Zn or RZnBr, Pd(dppf)Cl2.CH2Cl2, THF, 80 oC, 3-16 h, 55-100%; (c) TBAF, THF, 80 o

C, 16-72 h, 9-35% or TFA, CH2Cl2, 20 oC, 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 oC, 5-16 h, 41-67%; (e) Pd/C, H2, EtOH/THF or MeOH, r.t., 3-16 h, 28-100% (alkenes) or Pd/C, NH4HCO2, MeOH, ∆, 4-8h, 57-82% (furyls); (f) Cu, Ph2SCF3.CF3SO2, DMF, 80 oC, 16 h, 44%; (g)

trans-H2NCyNMe2, K2CO3, MeCN, 80 oC, 4 h, 51%; (h) NaOAc, H2O, 20 oC, 3 d, 72%; (i) POCl3, DIPEA, ∆, 16 h, 100%; (j) trans-H2NCyNMe2, K2CO3, MeCN, MW, 140 oC, 16 h, 84%; (k) Pd/C, H2, K2CO3, MeOH, 20 oC, 16 h, 16%.



Page 8 of 72

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-bromopyrrolopyrimidine (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 Suzuki coupling with cyclohexenyl boronate and hydrogenation, followed by removal of the SEM protection. Scheme 4. Variation of the 4-linkage of the pyrrolopyrimidine (NMe2)a

(a)

(b,c,d) 22 X=NMe 23 X=O

a

Reagents and Conditions: (a) trans-HXCyNMe2, (DIPEA, iPrOH, 90 oC, 12 h, 75% for 22 or

NaH, DMF, 20 oC, 24 h, 63% for 23); (b) DihydropyranylBpin, Pd(PPh3)4 or Pd(dtbpf)Cl2, K2CO3, 1,4-dioxane or DME, H2O, 85-100 oC, 1-6 h, 60-85%; (c) Pd/C, H2, MeOH or EtOH/THF, r.t., 3-16 h, 86-93%; (d) TFA, CH2Cl2, r.t., 1-3 h then NH3/MeOH, 1-24 h, 3267%. 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


Page 9 of 72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


employed to install the desired amine capping group, although the cis/trans stereochemistry was poorly controlled and so separation of the isomers was required. Hydrogenation and SEM deprotection gave the desired products (Route II; 24, 30, 31). Scheme 5. Variation of the 4-substituent of the pyrrolopyrimidinea

Route I (b,c,e) (+d for 29) (a)

(g,c,e)

(f,b) Route II

Route I

Route II

Y=

Y= 27

25

a

29

26

24

28

32

30

31

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 oC, 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 oC, 3-16 h, 52-100%; (c) Pd/C, H2, MeOH or EtOAc or EtOH/THF, r.t., 11-18 h, 29-100%; (d) for 29 (i) LiOH, H2O, THF, r.t., 2 h, 86%; (ii) Me2NH, HATU, DIPEA, DMF, r.t., 2 h, 24%; (e) TFA, CH2Cl2, r.t., 1-3 h then NH3/MeOH, 1-24 h, or Me2NCOCl for 25 / MeSO2Cl for 26, DIPEA, CH2Cl2, r.t., 1 h then TFA, CH2Cl2, r.t., 1 h,



Page 10 of 72

NH3/MeOH, 1-24 h, 39-74%; (f) 4-H2Ncyclohexanone, DIPEA, iPrOH, MW, 120 oC, 36 h, 34%; (g) HY, NaBH(OAc)3, AcOH, CH2Cl2 or MeCN, 15-25 oC, 16-18 h, 52-100%. 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 then used in an SN2 reaction with the mesylate of cis-4-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 temperature. Removal of the SEM group under acidic conditions gave 34. Scheme 6. Variation of the 4-linkage of the pyrrolopyrimidine (morpholine)a

(a)

(b,c,d) 33 (X=O) 34 (X=S)

33a (b,g,d)

(e)

(f)

34d

34a

a

Reagents and Conditions: (a) trans-4-morpholinocyclohexanol, NaH, DMF, 20 oC, 24 h,

61%; (b) DihydropyranylBpin, Pd(dtbpf)Cl2, K2CO3, DME or 1,4-dioxane, H2O, 80-85 oC, 26 h, 73-100%; (c) Pd/C, H2, EtOH/THF, r.t., 3 h, 97%; (d) TFA, CH2Cl2, r.t., 1-3 h then NH3/MeOH,

1-24 h,

25-56%;

(e)

Na2S,

DMSO,

r.t.,

2

h,

78%;

(f)

cis-4-

morpholinocyclohexanol mesylate, K2CO3, MeCN, 80 oC, 16 h, 92%; (g) Rh(PPh3)3Cl, H2, THF, 50 oC, 7 d, 30%.


Page 11 of 72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For variations of the 6-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 3rd generation Brettphos precatalyst27 proceeded in moderate yield (56%), then hydrogenation and removal of the SEM protection gave 35. The 3-cyano derivative 36 was synthesised by bromination of the available 3-cyano pyrrolopyridine, SEM protection and an SNAr reaction to install the C4 amine of 36c. A subsequent Suzuki reaction, hydrogenation and deprotection gave 36. Scheme 7. Variation of the 6-membered ringa

(a)

(c,d,e)

35 (b) Y=

X=H 35a X=SEM 35b

(f,b,g)

(a,d,e) 36c

a

36

Reagents and Conditions: (a) DihydropyranylBpin, Pd(dtbpf)Cl2, K2CO3, 1,4-dioxane/H2O,

80 oC, 1-2 h, 76% for 35a, 100% for 36a; (b) SEMCl, NaH, THF, r.t., 4-16 h, 48% for 35b, 63% for 36b; (c) LHMDS, trans-H2NCyMorph, BrettPhos Pd G3, THF, 80 oC, 16 h, 56%; (d) Pd/C, H2 (2 atm), MeOH, r.t., 2-3 h, 84% for 35, 100% for 36; (e) TFA, CH2Cl2, r.t., 3-4 h then NH3/MeOH, 3-4 h, 61% for 35, 70% for 36; (f) NBS, CH2Cl2, r.t., 16 h, 100% (g) transH2NCyMorph, DIPEA, iBuOH, MW, 180 oC, 5 h, 81%.



Page 12 of 72

Examination of the SAR, through deconstruction of the cyclopentyl ring of 1, revealed that opening the ring to 5,6-dimethyl substituted 2 reduced potency ∼10x (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 hypothesised 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 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 (logD7.4 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. Table 1. Effect of substitution on thienopyrimidine and pyrrolopyrimidine cores.

Cpd

A

1

S

2

S

R1

R2

-(CH2)3Me

Me

logD7.4c

IRAK4

IRAK4

Enz IC50

Cell IC50

Cell

(µM)a

(µM)b

LLEd

0.14

0.49

1.2

5.2

1.5

2.5

0.7

4.9


IRAK4

Page 13 of 72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a

3

S

Me

H

1.6

3.6

0.6

4.8

4

S

H

Me

2.4

4.3

0.3

5.1

5

S

H

H

4.3

8.4f

0.1

5.0

6

NH

0.41

1.7f

0.5

5.3

7

NH

Me

Me

9.0

23f

0.6

4.0

8

NH

Me

H

>10

>30f

-0.2

-

9

NH

H

Me

0.005e

0.14

0.1

6.6

10

NH

H

H

0.64

1.4f

-0.6

6.5

-(CH2)3-

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; clogD7.4 determined by shake flask method; dcell LLE calculated from pIC50-logD; eSEM=0.28; fn=1.

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 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 (19a/b) a clear preference for one isomer was observed.29 Unfortunately, despite having low logD7.4 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 4-isomer (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, c.f. O--S distance for the tricyclic compound at 3.7 Å). Closer inspection of the structures suggested that 6-substituents 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 rationalised 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.


Page 14 of 72

Page 15 of 72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. X-ray crystal structure of pyrrolopyrimidine inhibitor 21 (5K72) in complex with IRAK4 and overlaid with tricyclic inhibitor 1 (5K75). Table 2. Exploration of 5-substituent.

Cpd

a

R

IRAK4

IRAK4

Enz IC50

logD7.4

IRAK4

Rat heps

Cell IC50

Cell

Clintc

(µM)

(µM)

LLE

9

Me

0.005d

0.14

0.1

6.6

9

11

Et

0.013

0.063

0.6

6.6

29

12

iPr

0.005

0.029

1.0

6.5

40

13

tBu

0.14

0.81

1.7

4.4

25

14

CF3

0.099

0.25

1.5

5.1

66

15

cPr

0.011

0.075

0.9

6.2

15

16

cBu

0.003

0.042

1.5

5.9

52

17

cPn

0.006e

0.023

1.7

5.9

71

18a

2-THFa

0.27

1.2

0.4

5.5

59

18b

2-THFa

0.28

2.1

0.3

5.4

36

19a

3-THFa

0.12

0.54

0.1

6.2

120

19b

3-THFa

0.008

0.034

0.1

7.5

73

20

3-THPb

0.016

0.066

0.3

6.9

30

21

4-THP

0.006

0.059

0.2

7.1

14

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 1x106 cells/mL; dSEM=0.28; d

SEM=0.33.



Page 16 of 72

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 logD7.4 (+0.4). Switching to an ether linkage (23) also reduced potency, despite a sizeable increase in logD7.4 (+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. Table 3. Variation of 4-linker.

Cpd

X

IRAK4

IRAK4

Enz IC50

logD7.4

IRAK4

Rat heps

Cell IC50

Cell

Clint

(µM)

(µM)

LLE

21

NH

0.006

0.059

0.2

7.1

14

22

NMe

0.19

0.40

0.6

5.8

8.2

23

O

0.044

0.28

1.2

5.4

100

More detailed profiling of compound 21 highlighted poor permeability (Papp 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 against other kinase targets was investigated by profiling 28 in a panel (Millipore kinase selectivity panel) of 275 kinases at a concentration of 0.1 µM (Figure 7). The profile was generally clean (IRAK4 + 4/275 >75%inh; 8/275 >50%inh) but showed inhibition of the CLK family (x3) and haspin kinase. These hits were followed up with concentration response



data (Table 6) and confirmed inhibition of CLK1, 2 & 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 >30x selectivity for IRAK4 over IRAK1 and >100x selectivity over IRAK2 and 3.

Figure 7. Millipore kinome selectivity for 28 (%inhibition @ 0.1 µM) Table 6. Inhibitory concentrations & 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

0.005

1.0x

0.0007

1.0x

IRAK3

-

-

0.093

>130x

IRAK2

-

-

0.170

>200x

IRAK1

0.023

4.6x

0.026

37x

CLK1

0.050

10.0x

-

-

CLK2

0.005

1.0x

-

-

CLK3

>1

>200x

-

-

CLK4

0.008

1.6x

-

-

haspin

0.004

0.8x

-

-


Page 22 of 72

Page 23 of 72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 75%inh). 24) Destro, R.; Soave, R.; Barzaghi, M.; Lo Presti, L.; Progress in the understanding of drug–receptor interactions, part 1: Experimental charge-density study of an angiotensin II receptor antagonist (C30H30N6O3S) at T=17 K. Chem. Eur. J. 2005, 11, 4621–4634. 25) Under acidic conditions, treatment with TFA/DCM afforded partial deprotection of the SEM group, with LC-MS showing the desired product mass in addition to variable amounts of an M+30 impurity, corresponding to product with the CH2OH group remaining. Full conversion to the desired product was achieved by allowing the crude product following SCX purification to stand in NH3/MeOH solution for 1-24 h.


Page 68 of 72

Page 69 of 72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


26) Zhang, C.; Wang, Z.; Chen, Q.; Zhang, C.; Gu, Y.; Xiao, J. Copper-mediated trifluoromethylation of heteroaromatic compounds by trifluoromethyl sulfonium salts. Ang. Chem., Int. Ed. Engl. 2011, 50, 1896–1900. 27) Bruno, N. C.; Tudge, M. T.; Buchwald, S. L. Design and preparation of new palladium precatalysts for C-C and C-N cross-coupling reactions. Chem. Sci. 2013, 4, 916–920. 28) During the evaluation of this series, a patent application (Harriman, G. C.; Romero, D. L.; Masse, C. E.; Robinson, S.; Wessel, M. D.; Greenwood, J. R. Irak inhibitors and uses thereof. Patent application WO2014011911, January 16, 2014) on IRAK inhibitors was published containing nine examples of pyrrolopyrimidines. Two of the most potent examples described had substitution at the 5-position with cyano (I-1) and fluoro (I-3). These were made and profiled in our assays and IRAK4 potency was found to be consistent with the binned data reported in the patent (0.1 < IC50 < 1 µM) and less potent than many examples reported in Table 2.

R=CN (I-1) IRAK4 IC50 164 nM R=F (I-3) IRAK4 IC50 264 nM

Compound 8 (I-9) was also included in the application and was reported to have IC50 >10 µM in an IRAK4 enzyme assay, also consistent with the results obtained in our assay (Table 2). This patent application was subsequently abandoned. 29) For compounds 18-20 with chiral groups in the 4-position, we observed a doubling up of some signals in the

13

C spectra of the cyclohexyl ring which we attributed to the

chiral centre imparting diastereotopicity to the cyclohexyl carbons. This phenomenon was not observed with achiral 4-substituents.



30) Kd values were determined using the DiscoverX KdELECT® platform. Data was generated in duplicate using an 11 point dose response curve with experimental details described on the company website (https://www.discoverx.com/home). 31) Davis, R. E.; Ngo, V. N.; Lenz, G.; Tolar, P.; Young, R. M.; Romesser, P. B.; Kohlhammer, H.; Lamy, L.; Zhao, H.; Yang, Y.; Xu, W.; Shaffer, A. L.; Wright, G.; Xiao, W.; Powell, J.; Jiang, J. K.; Thomas, C. J.; Rosenwald, A.; Ott, G.; MullerHermelink, H. K.; Gascoyne, R. D.; Connors, J. M.; Johnson, N. A.; Rimsza, L. M.; Campo, E.; Jaffe, E. S.; Wilson, W. H.; Delabie, J.; Smeland, E. B.; Fisher, R. I.; Braziel, R. M.; Tubbs, R. R.; Cook, J. R.; Weisenburger, D. D.; Chan, W. C.; Pierce, S. K.; Staudt, L. M. Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature 2010, 463, 88–92. 32) Wilson, W. H.; Young, R. M.; Schmitz, R.; Yang, Y.; Pittaluga, S.; Wright, G.; Lih, C. J.; Williams, P. M.; Shaffer, A. L.; Gerecitano, J.; de Vos, S.; Goy, A.; Kenkre, V. P.; Barr, P. M.; Blum, K. A.; Shustov, A.; Advani, R.; Fowler, N. H.; Vose, J. M.; Elstrom, R. L.; Habermann, T. M.; Barrientos, J. C.; McGreivy, J.; Fardis, M.; Chang, B. Y.; Clow, F.; Munneke, B.; Moussa, D.; Beaupre, D. M.; Staudt, L. M. Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma. Nat. Med. 2015, 21, 922–926. 33) Davids, M. S.; Brown, J. R. Ibrutinib: a first in class covalent inhibitor of Bruton's tyrosine kinase. Future Oncol. 2014, 10, 957-967. 34) The roles of haspin and CLK in oncology are the subject on ongoing research. For recent work on haspin inhibition see; Cuny, G. D.; Ulyanova, N. P.; Patnaik, D.; Liu, J.-F.; Lin, X.; Auerbach, K.; Ray, S. S.; Xian, J.; Glicksman, M. A.; Stein, R. L.; Higgins, J. M. G., Structure–activity relationship study of beta-carboline derivatives as haspin kinase inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 2015-2019; For recent


Page 70 of 72

Page 71 of 72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


work on CLK inhibitors see; Araki, S.; Dairiki, R.; Nakayama, Y.; Murai, A.; Miyashita, R.; Iwatani, M.; Nomura, T.; Nakanishi, O. Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by modulating pre-mRNA splicing. PLoS ONE 2015, 10, e0116929. 35) Goedken E. R.; Devanarayan V.; Harris C. M.; Dowding L. A.; Jakway J. P.; Voss J. W.; Wishart N.; Jordan D. C.; Talanian R. V. Minimum significant ratio of selectivity ratios (MSRSR) and confidence in ratio of selectivity ratios (CRSR): quantitative measures for selectivity ratios obtained by screening assays. J. Biomol. Screen. 2012, 17, 857-867.



Graphical Abstract


Page 72 of 72

Discovery and Optimization of Pyrrolopyrimidine Inhibitors of

Recommend Documents