Fragment-Based, Structure-Enabled Discovery of Novel Pyridones

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Fragment-based, structure-enabled discovery of novel pyridones and pyridone macrocycles as potent bromodomain and extra-terminal domain (BET) family bromodomain inhibitors. Le Wang, John K. Pratt, Todd Soltwedel, George S. Sheppard, Steven D. Fidanze, Dachun Liu, Lisa A. Hasvold, Robert A. Mantei, James H. Holms, William J. McClellan, Michael D. Wendt, Carol K. Wada, Robin R Frey, T. Matthew Hansen, Robert Hubbard, Chang H. Park, Leiming Li, Terrance J. Magoc, Daniel H Albert, Xiaoyu Lin, Scott E. Warder, Peter Kovar, Xiaoli Huang, Denise Wilcox, Rongqi Wang, Ganesh Rajaraman, Andrew M. Petros, Charles W. Hutchins, Sanjay C. Panchal, Chaohong C. Sun, Steven W. Elmore, Yu Shen, Warren M Kati, and Keith F. McDaniel J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00017 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 3, 2017

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Journal of Medicinal Chemistry

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Fragment-based, structure-enabled discovery of novel pyridones and pyridone macrocycles as potent bromodomain and extra-terminal domain (BET) family bromodomain inhibitors.

Le Wang,a,* John K. Pratt,a,* Todd Soltwedel,b George S. Sheppard,a Steven D. Fidanze,a Dachun Liu,a Lisa A. Hasvold,a Robert A. Mantei,a James H. Holms,a William J. McClellan,b Michael D. Wendt,a Carol Wada,a Robin Frey,a T. Matthew Hansen,a Robert Hubbard,b Chang H. Park,a Leiming Li,a Terrance J. Magoc,b Daniel H. Alberta, Xiaoyu Lina, Scott E. Wardera, Peter Kovara, Xiaoli Huanga, Denise Wilcox,a Rongqi Wang,a Ganesh Rajaraman,b Andrew M. Petros,a Charles W. Hutchins,a Sanjay C. Panchal,a Chaohong Sun,a Steven W. Elmore,a Yu Shen,a Warren M. Katia and Keith F. McDaniela a

AbbVie Inc., 1 North Waukegan Rd., North Chicago, IL 60064, USA

b

Former AbbVie employee

Abstract Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A 2dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenyl pyridazinone fragment with a weak binding affinity (1, Ki = 160 µM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series

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exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.

Introduction Reversible lysine acetylation plays a central role in the regulation of chromatin structure and transcription of genes via modification of histone proteins and transcription factors.1-2 These changes in gene expression are modulated by three kinds of epigenetic regulatory proteins, “writers”, “erasers”, and “readers”. Histone acetyltransferases (HATs) function as “writers” to acetylate lysine. Histone deacetylases (HDACs) remove the acetyl group from acetylated lysine, acting as “erasers”.3-5 The third kind of epigenetic regulatory proteins are the bromodomain (BRD) family of proteins that selectively bind to acetylated lysines,6 thus functioning as “readers” of the lysine acetylation state.7-8 Bromodomains consist of approximately 110 amino acid residues and can be found in 42 proteins in the human proteome. The Bromodomain and Extraterminal (BET) family is a subset of related bromodomain-containing proteins that includes BRD2, BRD3, BRD4, and BRDT. All BET family members share the common configuration of two N-terminal bromodomains (BDI and BDII) and a C-terminal extra terminal (ET) domain. The ET domain interacts with other cellular proteins that include cyclin-dependent kinase 9 (CDK9) and cyclin T to activate transcription of proximal genes into RNA. The BDI and BDII domains, respectively, are highly conserved across BET family members (>70% identity), whereas BDI and BDII from the same BET protein exhibit a larger degree of divergence (~40% identity).



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The inhibition of bromodomain proteins was largely unknown until 2009 when Mitsubishi disclosed the first small molecule BET inhibitor 2a (MS417)9 shown in Figure 1. Two additional BET inhibitors, 2b (JQ1)10 and 2c (I-BET762)11 were published in late 2010. 2b is closely related to the molecules described in the initial Mitsubishi patent whereas 2c was discovered at GlaxoSmithKline (GSK) via a phenotypic screening campaign. All three compounds are potent and selective BET family protein inhibitors that were found to occupy the acetyl lysine (KAc) binding site,12 a well-defined pocket in the BET bromodomains. RVX-208 from Resverlogix Corp, a molecule in Phase II clinical trials for cardiovascular disease, was also found to be a BET inhibitor, albeit with rather weak activity.13 Based on these early BET inhibitors, rapid and significant progress has been made in understanding the role of BET proteins in multiple disease areas including oncology,14 inflammation,15 immunology,16 and cardiovascular disease.17 From an oncology standpoint, the mechanisms-of-action for BET inhibitors fall into three broad categories: 1) disruption of cell cycle control leading to G1 arrest, 2) inhibition of genetic

drivers

of

cytokine/chemokine

oncogenesis signaling

leading pathways

to

apoptosis

that

maintain

and a

3)

inhibition

of

tumor-supportive

microenvironment.18 From an immunology disease standpoint, the inhibition of NF-kB driven pathways contributes, at least in part, to anti-inflammatory properties exhibited by BET inhibitors.19 For example, the BET inhibitor 2d (I-BET151) (Figure 1) has been shown to down-regulate expression of inflammatory genes and matrix degrading enzymes in rheumatoid arthritis synovial fibroblasts.20


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N O

S

N N

N N

N

O O

N

N N

N

O

R

N H

N

MeO

S

N N

N

O

S N H

N

N

N

O NH2

N

N H

Cl 1

Cl

Cl

2c (I-BET762)

2a (MS417) R= Me, 2b (JQ1) R= But

N

Cl OTX-015 /MK-8628

N O

N

O

O

N

O

O

HN N O

N

HN

HO2C

Cl CPI-610

2d (I-BET151)

THQ

TEN010

O NH

N NH2

N

Cl

OH

O MeO

HN O S O MeO

OH

N NH OMe O

PFI-1

RVX-208

Figure 1. Representative BET inhibitors.

The impressive biological activities of 2b-2d in a variety of therapeutic areas spurred the pharmaceutical industry to develop clinical quality BET inhibitors.21-22 The triazole diazepine binding motif found in 2b is contained in clinical compounds developed by GSK (2c), OncoEthix/Merck (OTX-015/MK8628),23 Tensha/Roche (TEN010),23 and Constellation (CPI-610).23 The 3,5-dimethylisoxazole motif, a KAc binding mimic, was first published by GSK as shown in 2d.24 Later, this moiety appeared in a number of BET inhibitor patent applications filed by several companies such as Incyte,25 Gilead,26 and Boehringer-Ingelheim.27 In addition, GSK published a tetrahydroquinoline based BET inhibitor, THQ,28 while Pfizer disclosed its tool BET inhibitor PFI-1.29 In search of novel BET inhibitor chemotypes, we initiated a proteinbased NMR fragment screen30 carried out against the second bromodomain of BRD4 (BRD4-BDII). This fragment screen yielded a number of structurally distinct hits,



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including compound 1, a novel small molecule which had not been previously reported to bind to bromodomains. Compound 1 showed a strong spectral shift in this NMR screen (see Supporting Information). Subsequent testing revealed that compound 1 was a weak BRD4 inhibitor with a Ki of 160 µM and its target binding was confirmed by a proteininhibitor X-ray co-crystal structure. Medicinal chemistry efforts directed toward the generation of BET bromodomain inhibitors based on unique fragment 1 are reported in this publication. The design, syntheses, and SAR investigation of novel pyridones/ pyridazinones and pyridone macrocycles as potent BET inhibitors will be discussed. The pharmacokinetics and in vivo anti-tumor activity of selected compounds will also be presented.

Chemistry The general methodology for the syntheses of the pyridazinone and pyridone BET inhibitors described for this publication utilized a convergent synthetic approach. A key transformation in many of the synthetic sequences to prepare the acyclic inhibitors utilized a Suzuki coupling reaction, allowing flexibility in the choice of boronate/halide coupling partners.

Scheme 1a


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a

Reagents and conditions: (i) MeI, Cs2CO3, DMF, 91%; (ii) Na,

MeOH, 50 ºC, 89%; (iii) phenol, Cs2CO3, DMSO, 100 ºC, 95%; (iv) Fe0, NH4Cl, EtOH/THF/water, 95 ºC, 97%; (v) 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane), KOAc, Pd2(dba)3, (1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6phosphaadamantane), 1,4-dioxane, 80 ºC, 70%; (vi) 5, Pd(PPh3)4, CsF, DME/MeOH, 120 ºC, 57-62%; (vii) (13a) AcCl, Et3N, DCM, 99%; (13b) MsCl, Et3N, DCM, then aq. NaOH/1,4dioxane, 90 ºC, 68%; (13c) ClCOOEt, Et3N, DCM, 81%; (13d) EtNCO, Et3N, DCM, 51%.

As shown in Scheme 1, methylation of commercially available 3 followed by nucleophilic displacement of the more reactive of the two aryl chlorides with sodium methoxide provided intermediate 5. The aryl boronate 10 was prepared from commercially available 2-bromo-1-fluoro-4-nitrobenzene 6 in three steps. Nucleophilic



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aromatic substitution of the aryl fluoride of 6 with phenol using cesium carbonate in DMSO at elevated temperature provided 7. Iron reduction of 7 in the presence of ammonium chloride provided aniline 8. Conversion of the aryl bromide to the boron pinacolate ester gave Suzuki coupling partner 10. Suzuki coupling of 5 with either commercially available boronate 9 or 10 provided 11 and 12, respectively. Either Pd(PPh3)4

in

the

presence

of

cesium

fluoride,

or

the

combination

of

tris(dibenzylideneacetone)dipalladium(0) as the palladium catalyst and 1,3,5,7tetramethyl-6-phenyl-2,4,8-trioxa-6-phosphaadamantane as the phosphine ligand were employed for the Suzuki coupling reactions. Aniline 12 was then treated with various electrophilic reagents to obtain final derivatives 13a-d. Sulfonylation of anilines such as 12 often provided bis-sulfonylated intermediates. Removal of one sulfonyl group with aqueous hydroxide at elevated temperature gave the desired mono-sulfonylated products in good yield. Scheme 2 a

a

Reagents and conditions: (i) NaNO2, H2SO4, NH4OH,

water, 0 ºC, quant.; (ii) MeI, Cs2CO3, DMF, 91%; (iii)


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NaOMe in MeOH, or NaOEt in EtOH, 60 ºC, 82-89%; (iv) 9 or 10 Pd(PPh3)4, CsF, 48-65%; (v) MsCl or ethanesulfonyl chloride, Et3N, DCM, then aq NaOH/1,4-dioxane, 90 ºC, 3556%.

Synthesis of the acyclic pyridone core (Scheme 2) was very similar in nature to that described for the pyridazinone core. Diazotization of commercially available 5bromo-4-chloropyridin-2-amine 14 followed by N-methylation gave the dihalogenated pyridone 16. Nucleophilic displacement of the pyridone chloride with either methoxide or ethoxide provided compounds 17a and 17b. Suzuki couplings using boronate 10 followed by sulfonylation of the resulting aniline intermediates provided analogs 20a, 20b and 21. Scheme S1 (Supporting Information) is an expansion of Scheme 2 highlighting chemistry used to explore the SAR of the aryl ether region of the inhibitors (Table 2).

Scheme 3a



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Br

Br F

O

i

O2N

O

F

F

H2N

F 27

O N

N Cl O

iv

F

Cl O

v O

H2N

Et

F

O

R

S

N H

F

30d: R =

O S

N H

vi F

29

30a: R = Me 30b: R = CH2CF3 30c: R = isoBu

N O O

F

O

28

O

F O

26 O

Et

O

ii, iii

O2N 6

B

O OH

F 30a-f

30e: R = 30f: R =

N O

a

Reagents and conditions: (i) 2,4-difluorophenol, Cs2CO3, DMSO,

110 ºC, quant.; (ii) Fe0, NH4Cl, EtOH/THF/water, 100 ºC, 82%; (iii) 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane), KOAc, Pd2(dba)3, (1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6phosphaadamantane), 1,4-dioxane, 80 ºC, quant,; (iv) 16, K3PO4, Pd2(dba)3, (1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6phosphaadamantane), 1,4-dioxane /water, 60 ºC, 86%; (v) ethanesulfonyl chloride, Et3N, DCM, then aq NaOH/1,4-dioxane, 90 ºC, 81%; (vi) NaOMe in MeOH, 60 ºC for 30a; NaH, ROH, 1,4-dioxane, 70 ºC for 30b-f, 11-87%.

Exploration of various ethers incorporated on the pyridone core is shown in Scheme 3. Nucleophilic displacement of the fluorine of compound 6 followed by boronate ester formation provided 27. Suzuki coupling gave 28 which was subsequently


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sulfonylated providing the common intermediate 4-chloropyridone 29. Nucleophilic displacement of the chloride with commercially available or prepared alkoxides gave the final aliphatic ether derivatives 30a-f. Schemes S2-S5 in Supporting Information illustrate synthetic methods that were used to prepare individual analogs found in Table 3, generally following the routes described above in Schemes 1-3. One of the challenges associated with the exploration of macrocyclic frameworks for drug discovery is the difficulty in synthesizing such structures.31 Several cyclization strategies employing the SNAr displacement reaction developed for the synthesis of acyclic pyridone BET inhibitors were utilized successfully to generate macrocycles. In addition, two new macrocyclization routes using intramolecular Heck32 and Mitsunobu33 reactions were also employed with modest to good yields. Syntheses of macrocycles 60ac and 61a-b are described in Scheme 4. The key transformation was an intramolecular SNAr displacement to form the macrocycle. Suzuki coupling between compound 16 and compound 6a yielded compound 56. Compounds 53 and 56 were reacted with various 2iodo phenols via nucleophilic displacement to give compounds 57a-d in good yields. Sonogashira coupling between compounds 57a-d and selected alkynyl alcohols gave compounds 58a-e in excellent yields. Alkynes 58a-e were then reduced in the presence of Pt and hydrogen to give compounds 59a-e, which were subjected to intramolecular cyclization using sodium hydride to afford macrocycles 60a-e. It is worth pointing out that this cyclization reaction usually gave modest yields with rather limited functional compatibility. For the larger ring sizes, such as a 13-membered ring, the cyclization failed to give the desired product. Compounds 60d and 60e were allowed to react with ethyl



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sulfonyl chloride followed by treatment with aqueous sodium hydroxide to provide compounds 61a-b. Scheme 4a O

O O N

N N

i

ii

Cl

Cl

I

Cl O

F

Br R1 R2 X X 56: X = NO2 57a: X = SO2Et, R1 = R2 = H 53: X = SO2Et 57b: X = SO2Et, R1 = Cl, R2 = H 57c: X = NO2, R1 = R2 = F 57d: X = NO2, R1 = Cl, R2 = H

16

O

HO

O HO

N

n

N

n Cl

iii

Cl

iv

O R1

O

R2

X

R2

R1

58a: X = SO2Et, R1 = R2 = H, n=1 58b: X = SO2Et, R1 = R2 = H, n=2 58c: X = SO2Et, R1 = Cl, R2 = H, n=2 58d: X = NO2, R1 = R2 = F, n=2 58e: X = NO2, R1 = Cl, R2 = H, n=2

O

O N

N O O

v

n

O O

vi O

X

R2

R1 60a: X = SO2Et, R1 = R2 = H, n=1 60b: X = SO2Et, R1 = R2 = H, n=2 60c: X = SO2Et, R1 = Cl, R2 = H, n=2 60d: X = NH2, R1 = R2 = F n=2 60e: X = NH2, R1 = Cl, R2 = H, n=2

a

X

59a: X = SO2Et, R1 = R2 = H, n=1 59b: X = SO2Et, R1 = R2 = H, n=2 59c: X = SO2Et, R1 = Cl, R2 = H, n=2 59d: X = NH2, R1 = R2 = F, n=2 59e: X = NH2, R1 = Cl, R2 = H, n=2

Et

O S

N H

R2

61a: R1 = F, R2 = F 61b: R1 = Cl, R2 = H

R1

Reagents and conditions: (i) 6a or 52, Pd2(dba)3, CsF, THF,

tri-tert-butylphosphonium tetrafluoroborate, 45 ºC, 79%; (ii) (a) 53: phenols, Cs2CO3, DMSO, 100 ºC, 6-62%; (b) 56: phenols, Cs2CO3, 70-85 ºC, DMSO, 69-75%; (iii) Et3N, CuI, but-3-yn-1-ol or prop-2-yn-1-ol, Pd(PPh3)2Cl2, DMF, 47-100%; (iv) Pt, H2, THF, 80-95%; (v) NaH, 1,4-dioxane, 90 ºC, 12-48%; (vi) EtSO2Cl, Et3N, DCM, then aq NaOH/1,4-dioxane,


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90 ºC, 58-81%.

Although the intramolecular displacement successfully yielded the 11- and 12membered macrocycles described for Scheme 4, this strategy failed to form the 13membered ring found in compound 66. After several cyclization attempts, the intramolecular Heck reaction was found to provide a successful approach to this 13membered ring, as described in Scheme 5. The nitro group of compound 57d was reduced to the aniline 62, which was then converted to the ethyl sulfonamide 63. In order to generate the iodoalkene 64 required for macrocycle formation, the chloride of compound 63 was displaced by the hydroxyl of pent-4-en-1-ol. The macrocycle formation was accomplished by an intramolecular Heck reaction between the terminal double bond and the iodide, catalyzed by Pd(PPh3)4 and triethylamine in DMF, to give compounds 65a and 65b in roughly equal amounts. Hydrogenation of compound 65a resulted in formation of compound 66 in excellent yield. This intramolecular Heck reaction was also applied to the formation of certain 12-membered ring analogs, such as 61b, in reasonable yield.

Scheme 5a



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O

O

N

N

I

i

Cl

I O

Cl

NO2 Cl

NH2

57d

62

O

O

N

N

I

Cl

I

iii

O

O

O O

Cl

N H

63

O S

O

Et Cl 64

O

iv

Et

O O

O

O N H

Et

N O

S

N H

O S

O

N

O

ii

Cl

O

O Et

65a

O S

N H

Cl 65b

Cl O N

v

O O O Et

a

O S

N H

Cl 66

Reagents and conditions: (i) Fe0, NH4Cl, THF/EtOH/

water, 95 °C, 90%; (ii) EtSO2Cl, Et3N, DCM then aq NaOH/ 1,4-dioxane, 90 ºC, 80%; (iii) NaH, pent-4-en-1-ol, 1,4-dioxane, 90 ºC, 75%; (iv) Pd(PPh3)4, Et3N, DMF, 100 ºC, 65a: 29%; 65b: 30%; (v) Pt, H2, THF, 85%.

Scheme 6a


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OH OH X R3

O

i

R1

R2

ii

O

R3

R1

R1

R2

67a: R1 = R2 = F, R3 = Me, X= Br, 67b: R1 = H, R2 = Cl R3 = Me, X = I 67c: R1 = R2 = F, R3 = Bn, X = Br

iii

O

R3

R2

69a: R1 = R2 = F, R3 = Me, 69b: R1 = H, R2 = Cl, R3 = Me, 69c: R1 = R2 = F, R3 = H

68a-c

O

O N

N

O O

(CH2)4

O O

iv

Br

O

70

B

O

(CH2)4

71

Cl O

O

N

N

O

v

O Cl

O Cl Cl

Cl

73a: R = NO2 73b: R = SO2Et 73c: R = CO2Me

O

O

N

N O O

vii

viii

N

R Cl

74a: R = NO2 74b: R = SO2Et 74c: R= CO2Et a

(CH2)4

N

R

72a: R = NO2 72b: R = SO2Et 72c: R= CO2Me

R

OH

vi

(CH2)4

N

R

Cl

O O N H

ix

N Cl 75: R = H 76: R = SO2Et

Reagents and conditions: (i) Et3N, CuI, but-3-yn-1-ol,

Pd(PPh3)2Cl2, DMF, 83-96%; (ii) Pd/C, H2, THF, 68-100%; (iii) 16, KOBut, 1,4-dioxane, 75%; (iv) 4,4,4',4',5,5,5',5'-octamethyl -2,2'-bi(1,3,2-dioxaborolane), KOAc, dicyclohexyl(2',4',6'triisopropyl-[1,1'-biphenyl]-2-yl)phosphine, Pd2(dba)3, 1,4-dioxane, 80 ºC, 61%; (v) for 72a: 77a, Pd2(dba)3, K3PO4, 1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6phosphaadamantane, 1,4-dioxane, water, 50 ºC, 32%; for 72b and 72c: 77b or 77c respectively, CsF, Pd2(dba)3, tri-tert-



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butylphosphonium tetrafluoroborate, THF, 50 ºC, 49-70%; (vi) BBr3, DCM, -78 ºC to rt, 83-88%; (vii) Cs2CO3, MeCN, 75-95 ºC, overnight, 77-90%; (viii) Fe0, NH4Cl, THF/EtOH/water, 90 °C, 94%; (ix) EtSO2Cl, Et3N, DCM then aq NaOH/1,4-dioxane, 85 ºC, 80%.

Although 12-membered ring macrocycles could be generated by intramolecular displacement of the chloropyridones 59 (Scheme 4), the yields were modest at best. Because a strong base such as sodium hydride was required, functional group tolerability was limited. Therefore, an alternative route which relied on an intramolecular SNAr reaction to form the biaryl ether moiety was developed for the preparation of the 12membered macrocycles (Scheme 6). Sonogashira coupling of compounds 67a-c followed by hydrogenation resulted in compounds 69a-c. Pyridone 70 was prepared in good yield by reaction of compound 16 and compound 69b. Palladium-catalyzed borylation of compound 70 resulted in the formation of boronate ester 71, which was allowed to react with three 3-bromo-2-chloro-5-subtituted-pyridines (77a-c) to give pyridines 72a-c. The required phenols 73a-c were generated by reaction of compounds 72a-c with boron tribromide. Intramolecular nucleophilic substitution mediated by Cs2CO3 in acetonitrile successfully converted compounds 73a-c to the macrocycles 74a-c in excellent yields. Compound 74a was then reduced to aniline 75 and converted to ethyl sulfonamide 76. Macrocycle 86 was prepared by an analogous route, as described in the Supporting Information (Scheme S7).

Scheme 7a


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OH Br Cl O2N

Br

i

+ 69c

N

O N

O2N

77a

F

F

78

iv

O

O ii, iii

N

N

O O

Cl Br

B

O

16

O

O

79

O O O

N

v

O O N

O2N

OH

F

F

80 O

O

OH N

N

N

O2N

F

F

81

O O

vi

OH O

N F

O2N

82

O

F

N vii

O O O

Et

a

O S

N H

N F 83

F

Reagents and conditions: (i) Cs2CO3, DMSO, 65 ºC, 93%;

(ii) 2,4-dimethoxybenzyl alcohol, KOBut, THF, 55%; (iii) KOAc, dicyclohexyl(2',4',6'-triisopropyl-[1,1'-biphenyl]-2yl)phosphine, Pd2(dba)3, 4,4,4',4',5,5,5',5'-octamethyl-2,2'bi(1,3,2-dioxaborolane), 1,4-dioxane, 80 ºC, 60%; (iv) CsF, THF, Pd2(dba)3, tri-tertbutylphosphonium tetrafluoroborate, 50 ºC, 37%; (v) TFA, DCM, 100%; (vi) 2(tributylphosphoranylidene)acetonitrile, toluene, 80 ºC, 33%;



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(vii) (a) Fe0, NH4Cl, THF/EtOH/water, 90 °C, 81%; (b) EtSO2Cl, Et3N, DCM then aq NaOH/1,4-dioxane, 80 ºC, 67%.

Synthesis of macrocycle 83 is shown in Scheme 7. Because nitropyridine 81 was likely to decompose under the strongly basic conditions required for displacement of a chloropyridone, as described above in Scheme 4, this particular cyclization was accomplished using an intramolecular Mitsunobu reaction, albeit in modest yield. Biaryl ether 78 was prepared by the reaction between chloropyridine 77a and phenol 69c. Boronate 79 was prepared by displacement of the chloride of pyridone 16 by the hydroxyl group of 2,4-dimethoxybenzyl alcohol followed by palladium-catalyzed boronate formation. Suzuki coupling between bromide 78 and boronate 79 provided compound 80. The latent hydroxyl group of pyridone 81 was unmasked by removal of the 2,4-dimethoxybenzyl group of compound 80 upon treatment with trifluoroacetic acid. Intramolecular

Mitsunobu

reaction

of

alcohol

81

in

the

presence

of

cyanomethylenetributylphosphorane in toluene gave to macrocycle 82 in modest yield. Reduction of the nitro group followed by ethyl sulfonamide formation resulted in compound 83. Schemes and experimental procedures for the preparation of macrocycles 55, 86, and 94 can be found in Supporting Information. Macrocycles 55 (Scheme S6) and 94 (Scheme S8) were generated by an intramolecular Mitsunobu reaction sequence related to the approach utilized in Scheme 7.

Results and Discussion


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A crucial component of the development of this novel series of pyridazinone- and pyridone-based BET inhibitors was the utilization of X-ray structure information to guide compound design. Although pyridazinone fragment 1 exhibited only weak binding to BRD4 (Ki = 160 µM), an X-ray co-crystal structure was solved (Figure 2a) confirming that fragment 1 (PDB code: 5UFO) occupied the acetylated lysine binding pocket of BRD4 BDII. Two key interactions were observed between 1 and the protein (Figure 2b). The first crucial interaction is a 2.8 Å hydrogen bond between the carbonyl oxygen of the pyridazinone and the amino group of conserved Asn433 in the BET protein. The second interaction involves the N-methyl group of the pyridone situated in an amphoteric pocket created by a network of ordered water molecules. Together these two interactions function as an anchor for the N-methylpyridazinone core, and served as a common binding feature upon which all subsequent SAR was based.

Figure 2a (left panel). X-ray co-crystal structure of 1 in the binding pocket of the BRD4 BDII protein. Figure 2b (right panel). Illustrates hydrogen bonding of pyridone carbonyl oxygen to Asn433 and pyridone N-methyl substituent in amphoteric pocket.

An overlay of the crystal structures of fragment 1 and 2a (PDB code: 5UEU) (Figure 3) confirmed that both molecules occupied the same binding site in the BRD4 BDII protein. However, fragment 1 lacked many of the important structural features of the more potent inhibitor 2a (Ki = 16.7 nM). Analysis of the X-ray structure overlay



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revealed several potential vectors that might be useful for the incorporation of additional binding moieties leading to improved potency of fragment 1 analogs. The first vector was directed toward the hydrophobic WPF shelf of the protein, which is occupied by the pchlorophenyl ring of 2a. A second vector radiating away from the phenyl ring of 1 was directed towards an area of the protein with mixed hydrophilic/hydrophobic amino acid side chains. A third vector was directed towards a groove in the protein coming off of the pyridazinone ring in the same general direction as the methyl amine of fragment 1. This third vector essentially points toward the pocket occupied by the methyl ester of 2a.

Figure 3. Overlay of X-ray co-crystal structures 1 in blue and 2a in yellow bound to the BRD4 BDII protein. Vectors show opportunities for additional SAR exploration.

Novel compounds were evaluated using a TR-FRET binding assay and two cellular assays, which are described in detail in Supporting Information. A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the


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affinities (Ki) of compounds for the tandem bromodomain of BRD4. The impact of compounds on cancer cell proliferation was determined using the triple negative breast cancer cell line MX-1 (ATCC) in a 3-day proliferation assay. Target engagement in cells was measured with a luciferase reporter assay. A luciferase signal occurs only when a BET inhibitor blocks BRD4, thus providing a measure of target engagement. Good correlation was observed between the two cellular assays indicating anti-proliferation is due to target engagement. Efforts to improve the potency of 1 were initiated by adding a phenyl ether at the ortho position of the phenyl ring attached to the pyridazinone heterocycle. To avoid confusion in discussing these molecules a lettering system was implemented for the three rings present in the inhibitors (Table 1 header structure). The pyridazinone ring system was assigned the letter A. The phenyl ring attached to the pyridazinone was assigned letter B, and the newly introduced phenyl ether ring was assigned letter C. Through molecular modeling it was anticipated that the aromatic C-ring would be directed toward the WPF shelf of the protein. Simultaneous replacement of the methyl amine substituent on the pyridazinone core with a methyl ether gave compound 11 (Table 1) These modifications improved the binding potency of 11 compared to fragment 1, representing nearly a 170-fold improvement. Compound 11 also exhibited significant cellular activity, having an EC50 of 4.3 µM in the MX-1 proliferation assay and 3 µM in the BRD4 engagement assay. The X-ray co-crystal structure of compound 11 (PDB code: 5UEZ) (Figure 4) clearly illustrates the phenyl ether C-ring occupying the hydrophobic WPF pocket.



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Figure 4. X-ray co-crystal structure of 11 in the BRD4 BDII protein illustrating the C-ring phenyl ether sitting in the WPF shelf pocket.

Using the biochemical and cellular potencies of 11 as a benchmark, polar substituents were explored para to the phenyl ether to reach the aforementioned mixed hydrophilic/hydrophobic region of the BRD4 protein (Table 1). Aniline 12 was slightly weaker than 11 in the biochemical and MX-1 proliferation assays and equipotent in the BRD4 engagement cellular assay. Acylation of the aniline to give compound 13a resulted in a small improvement in MX-1 cellular potency and a larger potency increase in the cellular proliferation assay. Methyl sulfonylation 13b resulted in a 7-fold potency improvement over compound 11 in the biochemical assay (140 nM) and a 10-fold improvement in the cellular assays, with an EC50 of 730 nM in the MX-1 proliferation assay and 230 nM in the BRD4 engagement assay. The ethyl carbamate 13c and the ethyl urea 13d did not show potency improvements when compared to 11.


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Table 1. Biochemical and cellular potency of pyridazinone and pyridone phenyl ether derivatives.

a

R

MX-1

BRD4

TR-FRET

Proliferation

Engagement

Ki (nM)a

EC50 (nM)b

EC50 (nM)b

ID

X

11

N

-H

970 ± 200

4300

3000

12

N

-NH2

1700 ± 120

7200

2900

13a

N

-NHAc

900 ± 80

1300

370

13b

N

-NHSO2Me

140 ± 10

730

230

13c

N

-NHCO2Et

1100 ± 50

4500

4900

13d

N

-NHCONHEt

1900 ± 450

13000

2800

18a

CH

-H

450 ± 30 (2)

1500

2800

20a

CH

-NHSO2Me

58 ± 3

130

77

20b

CH

-NHSO2Et

8.2 ± 0.5

16

67

TR-FRET BRD4 Ki values are reported as the geometric mean derived from 3 independent measurements

except where indicated; bEC50 values are reported as the mean derived from two measurements.



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A modification of the pyridazinone core of 11 to the pyridone core homolog 18a (Scheme 2) provided a slight improvement in the biochemical and MX-1 proliferation assays, but no improvement in the BRD4 engagement assay. Similar to what was observed in the pyridazinone series, potency of the pyridone series was improved through methyl sulfonylation (compound 20a, Ki = 58 nM). Adding an additional carbon to form the ethyl sulfonamide 20b further improved the binding potency (Ki = 8.2 nM) and had a larger impact on MX-1 proliferation cellular potency (EC50 = 16 nM). An X-ray cocrystal structure of 20a (PDB code: 5UEW) bound to BRD2 BDII (Figure 5) illustrates key interactions of the inhibitor with the protein resulting in the large potency increase observed compared to compound 18a. One of the sulfonamide oxygen atoms of 20a replaces a water molecule in the water network surrounding the binding pocket and simultaneously forms a hydrogen bond with the backbone nitrogen of Asp377 in BRD2 BDII. The key pyridone carbonyl hydrogen bond to Asn429 and the position of the Nmethyl group in the amphoteric pocket are maintained.

Figure 5. X-ray co-crystal structure of 20a bound to BRD2 BDII highlighting the sulfonamide oxygen hydrogen bond to Asp377 and the retained hydrogen bond to Asn429.


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SAR of the phenyl ether portion of the molecule was investigated next, with an emphasis on reducing the potential for oxidative metabolism of the phenyl ring while maintaining biochemical and cellular potency. Using pyridone 21 as a starting point, derivatives 25a-e with electron withdrawing groups attached at likely sites of oxidation were prepared (Table 2). Addition of these substituents had little effect on the binding affinity with the exception of the 4-cyano analog 25c which was less potent in the biochemical assay. However, the cellular potencies of these analogs were weaker compared to 21. The unbound clearances in human and mouse liver microsomes for 25ae were comparable to 21 with the exception of the trifluoromethoxy analog 25d, which had much higher clearance in mouse liver microsomes. A positive attribute for this set of analogs was the dramatic improvement in rat microsomal stability for 25a-e with clearance reduced to single digit values. Once again 25d was the exception showing no improvement. The 3-pyridyl analog 25f was weaker than 21 in both the binding and cellular assays, but exhibited excellent microsomal stability in all three species. An aliphatic derivative 25g was substantially less potent in all three assays. Compounds 25b and 25e demonstrated the best overall combination of potency and microsomal stability of the compounds shown. Further SAR studies based upon the 2,4-difluorophenyl ether derivative 25e were conducted.

Table 2. SAR and microsomal stability of substituted phenyl ether pyridone inhibitors. O N

O Et

O S

OEt O R

N H



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MX-1

BRD4

TR-FRET

Proliferation

Engagement

Page 26 of 90

Liver Microsomes Human

Mouse

Rat

ID

R

Ki (nM)a

EC50 (nM)b

EC50 (nM)b

21

-Ph

4.4 ± 0.1

4.7

39

4.0

5.5

180

25a

4-F-Ph

5.6 ± 0.4

16

69

Fragment-Based, Structure-Enabled Discovery of Novel Pyridones

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