Article pubs.acs.org/jmc
Optimization of a Series of Bivalent Triazolopyridazine Based Bromodomain and Extraterminal Inhibitors: The Discovery of (3R)‑4[2-[4-[1-(3-Methoxy-[1,2,4]triazolo[4,3‑b]pyridazin-6-yl)-4piperidyl]phenoxy]ethyl]-1,3-dimethyl-piperazin-2-one (AZD5153) Robert H. Bradbury,† Rowena Callis,† Gregory R. Carr,† Huawei Chen,‡ Edwin Clark,*,‡ Lyman Feron,† Steve Glossop,† Mark A. Graham,† Maureen Hattersley,‡ Chris Jones,† Scott G. Lamont,† Gilles Ouvry,§ Anil Patel,† Joe Patel,‡ Alfred A. Rabow,† Craig A. Roberts,† Stephen Stokes,† Natalie Stratton,† Graeme E. Walker,† Lara Ward,† David Whalley,† David Whittaker,† Gail Wrigley,† and Michael J. Waring*,†,∥ †
AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K. AstraZeneca, Gatehouse Park, Waltham, Massachusetts 02451, United States § AstraZeneca, Chemin du Moulin de Vrilly, 51100 Reims, France ∥ Northern Institute for Cancer Research, School of Chemistry, Newcastle University, Bedson Building, Newcastle upon Tyne NE1 7RU, U.K. ‡
S Supporting Information *
ABSTRACT: Here we report the discovery and optimization of a series of bivalent bromodomain and extraterminal inhibitors. Starting with the observation of BRD4 activity of compounds from a previous program, the compounds were optimized for BRD4 potency and physical properties. The optimized compound from this campaign exhibited excellent pharmacokinetic profile and exhibited high potency in vitro and in vivo effecting c-Myc downregulation and tumor growth inhibition in xenograft studies. This compound was selected as the development candidate, AZD5153. The series showed enhanced potency as a result of bivalent binding and a clear correlation between BRD4 activity and cellular potency.
■
INTRODUCTION
The BET family proteins regulate gene expression through binding of acetylated lysine residues on histone proteins followed by activation of transcription elongation driven by RNA-PolII. BET inhibitors inhibit this gene transcription and, as a consequence, exhibit growth inhibition in a number of hematological and solid tumor models. This action is believed, at least in part, to be due to downregulation of critical oncogenes such as c-Myc, leading to great interest in BET inhibitors in the oncology field, and a number are currently progressing in early clinical trials. From the initial ApoA1 screen, pioneering work at GlaxoSmithkline led to the discovery of the triazolobenzodiazepine 1 (iBET-762, Figure 1),3,4 which has become one of the leading clinical BET inhibitors. Work carried out by Bradner and the Structural Genomics Consortium identified the structurally related compound 2 (JQ1).5 Compounds 1 and 2 have become key standard compounds in the field with many
The bromodomains (BRD) are a family of proteins responsible for binding acetylated lysine residues of proteins. The BRDs are rapidly emerging as a target class amenable to pharmacological intervention with small molecules.1 The bromodomain and extraterminal (BET) subfamily are perhaps the most widely explored of the class.2 Initially identified as a result of phenotypic screening for modulation of ApoA1, subsequent target identification in tandem with optimization has revealed multiple small molecule inhibitors of BET bromodomains, which have progressed to preclinical and clinical evaluation (Figure 1).3−5 The BET family consists of four proteins, termed BRD2, BRD3, BRD4, and BRDT. Each of these proteins contains two separate bromodomains. BRD4 in particular has been lauded as a potential drug target,6 although it is worth remembering that there is a high degree of homology within this class and the majority of compounds, particularly those studied most extensively in biological elucidation, do not exhibit selectivity within the class. © 2016 American Chemical Society
Received: January 18, 2016 Published: August 15, 2016 7801
DOI: 10.1021/acs.jmedchem.6b00070 J. Med. Chem. 2016, 59, 7801−7817
Journal of Medicinal Chemistry
Article
Figure 1.
Figure 2.
Scheme 1. Synthesis of 5a
Reagents and conditions: (i) DIPEA, MeCN, rt, 66%; (ii) ethylene carbonate, K2CO3, DMF, 80 °C, 72%; (iii) mesyl chloride, Et3N, DCM, 0 °C, 100%; (iv) N-acetylpiperazine, DIPEA, DMA, 110 °C, 73%. Compounds 6 and 7 were prepared in an analogous manner.
a
activity of the compounds and resulting effects on ER and cMyc DR were discovered during the course of this work.11 Later chemistry focused on the BRD4 potency and ER DR data to develop SAR and was generated retrospectively on the earlier compounds. The AR, ER, and c-Myc DR data (measured in LNCaP, MCF7, and MM1.S cell lines, respectively) are tightly correlated and can be used reasonably interchangeably to interpret the SAR. The data from these assays are also predictive of growth inhibitory effects measured in MM1.S cells (see later and Supporting Information for more detail). In order to assess the effect of the bivalent binding, an assay employing a protein construct containing both bromodomains was required. Hence, a fluorescence polarization (FP) assay for tandem domain BRD4 protein (residues 44−460) was developed using a fluorescently labeled small molecule probe derived from 2. It should be noted throughout that while data for BRD4 are quoted, no selectivity of these molecules within the BET family is anticipated due to their close homology. The synthetic route that could be used to prepare analogues with modifications to the acylpiperazine is exemplified with the synthesis of 5 (Scheme 1). S N Ar addition of 4-(4hydroxyphenyl)piperidine to chlorotriazolopyridazine afforded the corresponding phenol. The phenol was alkylated with ethylene carbonate to give the ethyl alcohol, which was derivatized as the mesylate, and this was used to alkylate the required amine, in this case acetylpiperazine, to give 5 in 35%
studies using these compounds as probes to understand BET biology. More recently, OTX015, 3,7 the corresponding phydroxyanilide derivative of 2 has been reported to show encouraging clinical results in AML patients.8 We have recently described the discovery that compounds from the androgen receptor (AR) downregulator (DR) program9,10 were found to be potent BET inhibitors that are demonstrated to act through an in-cis bivalent binding mode in which a single inhibitor molecule simultaneously engages both bromodomains of a single protein via two acetyl lysine mimicking moieties.11 This activity is postulated to explain the effects of the compounds observed in AR and estrogen receptor (ER) DR assays, as well as their effects on c-Myc levels. Here we describe the medicinal chemistry program that led to the identification of these compounds and their optimization, leading to the discovery of a clinical candidate bivalent BET inhibitor.
■
RESULTS AND DISCUSSION The campaign of optimization was initiated with the objective of improving the potency of the previous clinical AR downregulator AZD3514, 4, and the corresponding unsaturated analogue 5 while maintaining their excellent ADMET properties.9 These compounds exhibited modest cellular AR DR potencies (pIC50 5.8 and 6.5 for 4 and 5, respectively, Figure 2), and subsequent chemistry was focused on cellular AR DR potency to guide the chemistry campaign. The BET inhibitory 7802
DOI: 10.1021/acs.jmedchem.6b00070 J. Med. Chem. 2016, 59, 7801−7817
Journal of Medicinal Chemistry
Article
Scheme 2. Synthesis of 8a
Reagents and conditions: (i) Et3N, BOC2O, DCM, 0 °C, 88%; (ii) 1-bromo-2-chloroethane, K2CO3, acetone, 90 °C, 97%; (iii) DIPEA, KI, DMA, 110 °C, 90%; (iv) HCl, Et2O, quant; (v) DIPEA, DMA, 80 °C, 68%. Compounds 9 to 20 were prepared in a similar fashion.
a
Scheme 3. Synthesis of 22a
a Reagents and conditions: (i) Et3N, NaBH(OAc)3, DCM, 56%; (ii) TFA, DCM, quant; (iii) DIPEA, DMF, 80 °C, 34%; (iv) Ruphos palladium(II) phenethylamine chloride, dicyclohexyl(2′,6′-diisopropoxy-[1,1′-biphenyl]-2-yl)phosphine, Cs2CO3, toluene, 90 °C, 28%. Compounds 9−20 were prepared in a similar fashion.
Table 1. Effects of Structural Modifications of the Acylpiperazine
compd no.
BRD4 FP pKia
AR DR pIC50a
ER DR pIC50a
5 6 7 8
6.2 6.7 5.6 8.1
6.5 6.5
6.3 6.3 6 7.6
7.4
MycDR pIC50a
8
MM1.S pGI50a
log D7.4
solubility (μM)
Hu heps Clint (mL min−1 × 10−6 cells)
8.3
3.2 3 3.1 3.3
5.4 65 52 63
12 18 26 70
a
Measurements reported are the mean of at least three determinations. The standard deviation is not shown, because all three assays were found to be highly reproducible on repeat testing (see Experimental Section for more detail).
piperazinone by reductive amination with an appropriate ketone, exemplified with N-BOC-azetidine-3-one (Scheme 3). Removal of the BOC group with trifluoroacetic acid gave the intermediate amine. Arylation of 4-(4-bromophenyl)piperidine with 6-methoxy-3-(trifluoromethyl)-[1,2,4]triazolo[4,3-b]pyridazine gave the aryl bromide, which was coupled with the amine under Buchwald−Hartwig conditions to give the final compound 22 (10% overall yield for the lowest yielding sequence). Compounds 23 and 24 were prepared in an analogous manner. In exploring the structure−activity relationships in relation to the potential bromodomain inhibitory activity, we considered that both the triazolopyridazine and acylpiperazine moieties of the molecule could act as acetyl lysine mimetics (subsequently, we discovered that in fact both do, resulting in the in-cis binding mode and accounting for the SAR we observed).11 Modifications to both of these regions were designed with consideration to the overlay of the compounds with acetyl
overall yield for the four steps. Compounds 6 and 7 were prepared in an analogous manner. Alternatively, the amine substituent could be introduced before the triazolopyridazine allowing more effcient synthesis of compounds with different triazolopyridazine substituents. This is exemplified by the synthesis of 8 (Scheme 2). BOC protection of 4-(4-hydroxyphenyl)piperidine was followed by alkylation of the phenol with 1-bromo-2-chloroethane to give the alkyl halide. This underwent displacement with R-1,3dimethylpiperazin-2-one to afford the BOC protected intermediate. Removal of the BOC group with ethereal HCl afforded the piperazine, which could be arylated with a variety of substituted chlorotriazolopyridazines, in this case 6-chloro-3(trifluoromethyl)-[1,2,4]triazolo[4,3-b]pyridazine to give the final compound 8 in five steps and 52% overall yield. Compounds 9−20 were prepared in a similar fashion. Analogues with variations to the ethoxy linker of the piperazinone were prepared by initial alkylation of the 7803
DOI: 10.1021/acs.jmedchem.6b00070 J. Med. Chem. 2016, 59, 7801−7817
Journal of Medicinal Chemistry
Article
Table 2. Effect of Changing the Triazolopyridazine Trifluoromethyl Substituent
compd no.
R
BRD4 pKia
AR DR pIC50a
ER DR pIC50a
Myc DR pIC50a
MM1.S pGI50a
log D7.4
solubility (μM)
Hu heps Clint (mL min−1 × 10−6 cells)
8 9 10 11 12 13 14
-CF3 -H -Me -Cl -SMe -OMe -NMe2
8.1 1600 >1600
70 180 7.2 24 21 8.7
8.3 >8.7
7.9
8.2
2.3 2.2 2.5 3.4 2.3
>1600 900 >1000 280 750
1600 >1000 470 570 870 760 >1000
