Identification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the

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Identification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the Bromodomain and Extra-Terminal (BET) Family as a Candidate for Human Clinical Trials Brian Kevin Albrecht, Victor S Gehling, Michael Charles Hewitt, Rishi G Vaswani, Alexandre Cote, Yves Leblanc, Christopher G Nasveschuk, Steve Bellon, Louise Bergeron, Robert Campbell, Nico Cantone, Michael R Cooper, Richard T Cummings, Hariharan Jayaram, Shivangi Joshi, Jennifer A Mertz, Adrianne Neiss, Emmanuel Normant, Michael O'Meara, Eneida Pardo, Florence Poy, Peter Sandy, Jeffrey Supko, Robert J Sims, Jean-Christophe P Harmange, Alexander Merton Taylor, and James E. Audia J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01882 • Publication Date (Web): 27 Jan 2016 Downloaded from http://pubs.acs.org on January 29, 2016

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Journal of Medicinal Chemistry

Identification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the Bromodomain and ExtraTerminal (BET) family as a Candidate for Human Clinical Trials Brian K. Albrecht†, Victor S. Gehling, Michael C. Hewitt, Rishi G. Vaswani, Alexandre Côté, Yves Leblanc†, Christopher G. Nasveschuk†, Steve Bellon, Louise Bergeron†, Robert Campbell†, Nico Cantone, Michael R. Cooper, Richard T. Cummings, Hariharan Jayaram†, Shivangi Joshi†, Jennifer A. Mertz, Adrianne Neiss†, Emmanuel Normant†, Michael O’Meara, Eneida Pardo†, Florence Poy, Peter Sandy†, Jeffrey Supko†, Robert J. Sims, III, Jean-Christophe Harmange, Alexander M. Taylor*, James E. Audia* Constellation Pharmaceuticals, 215 First St., Suite 200, Cambridge, MA 02142, United States. Abstract In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates anti-tumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clinical trials for hematological malignancies (CPI-0610). Introduction

ACS Paragon Plus Environment


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Bromodomains are relatively small protein domains (~110 residues) that recognize acetylated lysine residues, namely on histone tails, via a set of key hydrogen-bonding interactions between highly-conserved asparagine and tyrosine residues and the acetylated lysine. When incorporated into a larger protein scaffold, these rigid four-helical bundles1 can aid in the localization of transcriptional machinery to specific gene loci, in some cases regulating gene expression. As such, the disruption of an important bromodomain/acetyl lysine interaction by a small molecule presents a potential opportunity to modulate disease-relevant gene expression. One such example is the bromodomain and extra terminal domain (BET) family, whose members (BRD2, BRD3, BRD4, and BRDT) each contain two highly homologous bromodomains (BD-1 and BD-2) that serve to localize transcriptional co-regulators to acetylated histone tails. We and others have reported the discovery of small molecule BET bromodomain inhibitors2that has enabled rapid deconvolution of the role of the protein family in diverse disease states, most notably

in

oncology

and

immuno-inflammation.3,4,5,6,7,8,9,10,11,12

Mechanistically,

the

bromodomains of the BET proteins link chromatin to the CDK9-containing complex P-TEFb, which phosphorylates the large subunit of RNAPII and facilitates pause-release and transcript elongation.13,14,15 Small molecule inhibitors block this critical function of BET proteins, leading to attenuated expression of key oncogenes such as MYC, BCL-2, and NF-ĸB target genes, ultimately resulting in cancer cell death.16,5,9,10,4,12,17 Because drugging MYC directly was previously intractable,18 the BET bromodomain represents a potential new modality for targeting MYC-dependent cancers. Indeed, in recent reports, BET inhibitors have shown activity in numerous cancer cell lines, xenograft mouse models, and patient samples of diverse origin, with hematologic malignancies among the most sensitive.19,20,9,21 Our belief in the therapeutic


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potential of the BET bromodomains prompted us to initiate a drug discovery effort aimed at enabling studies of their inhibition in human cancer. Here, we report the discovery of a series of benzoisoxazoloazepine BET bromodomain inhibitors and the identification of a candidate for human clinical studies in oncology. Proof of concept: combination of hydrogen bonding and hydrophobic interactions Our drug discovery effort began with a thermal shift assay of ~1200 low molecular weight compounds. Molecules that induced a >1 °C shift in the melting temperature of isolated BRD4 BD-122 were subsequently tested in a biochemical assay to evaluate their potency (initially with an AlphaLisa assay involving the isolated bromodomain, later with a similar time-resolved fluorescence resonance energy transfer [TR-FRET] assay). Co-crystallization of a number of compounds chosen for their potency and favorable molecular properties resulted in several biophysically-validated bromodomain inhibitors, all of which bound in the acetyl-lysine recognition site and made direct hydrogen-bonding interactions with Asn140 and, in some cases, hydrophobic interactions with Ile146 (a key region of the bromodomain that has been shown to improve binding of diacetylated histone tail peptides through contacts with the second acetyl group).1 As shown in Figure 1a, an overlay of eight of these fragments mapped out the available bonding and space filling interactions in the recognition site and suggested that an ideal bromodomain inhibitor would engage Asn140 and complement Ile146. (Potency and crystallographic data for individual fragments not reported.)



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A)

Tyr97

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B) Tyr97

Asn140 Asn140 Ile146

C)

Trp81

Ile146

D)

Figure 1: Co-crystal structures in BRD4 BD-1. Dashed lines indicate hydrogen bonds; spheres represent water molecules. A) An overlay of multiple fragments maps out key hydrogen bonding and lipophilic interactions in the acetylated lysine recognition site. B) Amino isoxazole fragment 1 (1.3 Å, PDB 4LR6) makes multiple hydrogen bonds with Asn140 and Tyr97 (through water) but does not complement Ile146. C) Benzotriazoloazepine iBET-762 (1.6 Å PDB 3P5O). D) Proof of concept benzoisoxazoloazepine 3 recapitulates the hydrogen-


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bonding interactions of 1 and introduces hydrophobic interactions with Ile146 (1.4 Å, PDB 5HMO). Among the fragments examined was aminoisoxazole 1, a lipophilic ligand efficient inhibitor (BRD4 BD-1 TR-FRET IC50 = 20 µM, LLE = 3.123) that was appealing because of its ability to form two hydrogen bonds with Asn140, along with a third, through-water hydrogen bond to Tyr97. (Table 1, Figure 1b) Whereas some fragments made hydrophobic interactions with Ile146, 1 did not extend into that pocket, leading us to hypothesize that incorporation of such an interaction would substantially increase binding affinity for the bromodomain. Around the same time that we were exploring 1, GSK10 and the SGC/Bradner6 separately reported the diazepine-derived BET inhibitors iBET-762 and JQ-1, respectively. Based on an overlay of the co-crystal structures of our collection of fragments, 1, and iBET-762, we hypothesized that incorporation of a sevenmembered ring into our aminoisoxazole fragment might suitably constrain the fragment and provide a vector from which to complement the hydrophobic region of the BRD4 BD-1 binding pocket. To that end, a number of isoxazole-derived compounds with fused ring systems were prepared. As shown in Table 1, the sub-micromolar TR-FRET IC50 of benzoisoxazolodiazepine 2 illustrated the potential improvement in potency that was to be gained from this approach. The related benzoisoxazoloazepine 3 demonstrated comparable potency, as did its regioisomer 4. A co-crystal structure of 3 in BRD4 BD-1 established the binding mode of this series (Figure 1d). As anticipated, the isoxazole engaged in hydrogen-bonding interactions with Asn140 and, through water, with Tyr97, although the scaffold was shifted away from Asn140 slightly, relative to 1, such that it did not form a second interaction with Asn140. Importantly, the compound adopted a cup shape that complemented the aggregate space filling observed with our



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fragment binders, with the lipophilic chlorophenyl group of 3 positioned over the hydrophobic Ile146 residue, likely accounting for the improved binding affinity. Interestingly, the greater potency of all of the isoxazole-derived benzoazepine-type compounds when compared to the corresponding triazole analog, 5, suggested that the isoxazole was especially well-suited to engage in the key interaction with Asn140 and Tyr97. We were particularly interested in the combination of biochemical activity and lipophilic ligand efficiency of 3 and so chose this scaffold to optimize for cellular potency and in vivo metabolic stability. Table 1: Structure-activity relationship of fragment-derived azepine isoxazoles and triazoles

1

2

3

4

5

1.3 ± 0.09

1.8 ± 0.22

2.3

3.2

BRD4-BD1 0.44 ± TR-FRET IC50

20 ± 1.1

0.88 ± 0.03 0.02

(µ µM)a LLEb

3.1

1.8

2.8

a

Time-resolved fluorescence energy transfer binding assay with the isolated BRD4 BD-1

bromodomain. N = 3. bLLE = lipophilic ligand efficiency = pIC50 – cLogP (ChemDraw 14). Development of the structure-activity relationship of the benzoisoxazoloazepine lead series


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We first turned our attention to understanding the effect of substituents at the methylene of the azepine ring of 3, hypothesizing that incorporation of a second hydrogen-bonding interaction with Asn140 would further improve potency. As shown in Table 2, the tert-butyl acetate derivative 6 did not deliver a meaningful improvement relative to 3, which stood in contrast to the reported structure-activity relationships of JQ-1 analogs.6 Testing of the opposite enantiomer, 7, demonstrated that essentially all of the binding affinity resided in the S-enantiomer, a trend that carried through all of our studies (data not shown). Interestingly, Nsubstituted acetamides such as 8 and 9 were markedly more active than 6, providing a roughly ten-fold improvement in potency. Their biochemical activity translated well into a cellular readout of MYC inhibition. Substitution on the amide nitrogen was not required for potency, as illustrated by 10 (CPI-0610), which also demonstrated greater in vitro metabolic stability than the secondary amides. Certain more-dramatic changes, such as incorporation of an amide isostere (11) or removal of the acetamide methylene linker (12) led to a substantial loss in potency. Modification of the side chain to form urea 13 was tolerated, demonstrating comparable potency to the corresponding acetamide 8, in both biochemical and in cellular assays. We recently reported that an (S)-methyl substituent off of the azepine ring was also sufficient to provide substantial potency and metabolic stability.24 A broad range of substituents was tolerated at different positions of the lipophilic phenyl ring, some of which are highlighted in Table 2. But whereas electron-neutral, -withdrawing, and – donating substituents at the para-position (compounds 14, 15, 16, and 17, respectively) all demonstrated favorable biochemical activity, cellular potency, or in vitro metabolic stability, no single compound possessed overall composite properties as favorable as the corresponding



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chlorophenyl analog, 10. In general, aliphatic rings and N-linked heterocycles were not tolerated as replacements for the chlorophenyl group (data not shown). A number of substituents on the benzo ring were explored, as well, several of which led to an improvement in the BRD4 BD-1 binding affinity of this series (Table 2). One example was 2methyl-substituted 18, which was three-fold more potent in the biochemical assay than its parent, 10, perhaps because of the introduction of a periplanar clash between the 2-methyl and 4-chlorophenyl groups that would serve to reinforce the cup shape of the compound. The increase in biochemical potency, however, failed to translate in a cellular context. In the neighboring 3-position, a number of substituents led to increased potency, including methoxy (19), aminopyridine (20), and a C-linked imidazole (21). As evidenced by co-crystal structures of related compounds (not shown here), such compounds extend over Trp81, in some cases making favorable edge-face interactions that increase binding affinity. Table 2: Structure-activity relationships of substitutions to the benzoisoxazoloazepine core. BRD4-BD1 TR-FRET Compound

R IC50

Clint

PPB

(m/r/d/h)

(m/r/d/h)

(mL/min/kg)c

(%)d

MYC EC50 b

(µ µM)

(µ µM)a 99/ 99/ >99/ 3

H

0.44 ± 0.02

ND

52/ 40/ 29/15 99

6

0.58e ND


ND

ND

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7

>80

0.052 ±

0.21 ±

61/ 42/ 29/

99/ 96/ 98/

0.002

0.001

18

98

82/ 45/ 30/

99/ 93/ 97/

19

>99

8

0.056 ± 9

0.27 0.002

10 (CPI-

0.039 ±

0.18 ±

30/ 21/ 20/

97/ 93/ 96/

0610)

0.001

0.036

10

95

41/ 38/ 27/

99/ 99/ 99/

13

99

11

11 ± 0.45

>4

32/ 27/ 29/ 12

15 ± 0.70

4

99/ -/ 98/ 98 20

0.033 ±

0.10 ±

38/ 29/ 21/

97/ 98/ 98/

0.001

0.035

13

97

10/ 28/ 17/

84/ 80/ 88/

20 µM, >80 µM, >20 µM, >20 µM, >20 µM, >80 µM, >20 µM, and >15 µM, respectively), among others. In a CEREP express panel of about fifty GPCRs, ion channels, and transporters, the compound displayed no meaningful inhibition. 10 also displayed negligible inhibition of cytochrome P450 activity when tested at 10 µM against a number of common isoforms, and it also showed no time-dependent inhibition. Overall, these and other data painted a convincing picture of 10 as an inhibitor of the BET family of bromodomains with no significant off-target activity.


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In vivo pharmacodynamics and anti-tumor activity of 10 Next, we evaluated 10 in several models to assess how its MYC cellular transcriptional suppression translated in vivo. In one study, the compound was dosed subcutaneously at 15 mg/kg twice daily in a mouse xenograft model using MV-4-11 acute myeloid leukemia cells. At multiple time points following the second dose, we assessed the levels of MYC mRNA, as well as the concentration of 10 in plasma and tumor. As shown in Figure 2, MYC mRNA levels were substantially reduced at 4 h compared to the vehicle control and recovered toward the control level at the later time points, which corresponded with decreasing free concentrations of compound in plasma. (Concentrations of 10 in tumor were within 25% of those measured in the plasma.) Significant suppression of MYC was observed only when free plasma concentration was above the cellular MYC EC50, consistent with the free drug hypothesis. 1.5

10

1 1

0.75

0.1

0.5

Compound 10 conentration (µ µM)

MYC Cmpd 10, Plasma Cmpd 10, Tumor Cmpd 10, Plasma (free)

1.25

MYC RNA expression

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


0.01 0.25

0

0.001 Vehicle

4h

8h

Time post last dose (BID 15 mg/kg)


12 h


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Figure 2. Pharmacodynamic effect of 10 on MYC expression. (Dotted line represents cellular MYC EC50.) The same MV-4-11 mouse xenograft model was used to explore the effect of prolonged treatment with 10 on tumor growth. For twenty eight days, the compound was dosed orally as a single agent at 30 mg/kg once daily, 30 mg/kg twice daily, or 60 mg/kg once daily. As shown in Figures 3a and b, BET bromodomain inhibition resulted in substantial suppression of tumor growth over the time period examined (41%, 80%, and 74% tumor growth inhibition, respectively), without any significant body weight loss in the animals. In a separate experiment with the same model, the effect of combination dosing with doxorubicin (a commonly used chemotherapeutic for hematological malignancies) was examined. There, both 10 and doxorubicin were dosed at sub-optimal levels, 10 mg/kg subcutaneously twice daily and 2 mg/kg twice weekly, respectively, for twenty eight days. Subcutaneous dosing was chosen to better mimic the target coverage expected from lower clearance rates in higher species. As shown in Figure 3c, treatment with individual compounds at those levels resulted in only modest reduction of tumor growth, whereas in combination complete inhibition of tumor growth was observed. Upon subsequent removal of therapy, however, tumor growth appeared to resume. Again, the efficacious treatment regimen did not lead to body weight loss in the animals (Figure 3d), suggesting that it was well tolerated. Taken together, the reduction of MYC expression at free plasma concentrations of 10 above its cellular MYC EC50 and the ability of the compound to substantially inhibit tumor growth at well-tolerated doses in a mouse xenograft model, both alone and in combination, supported BET bromodomain inhibition as a therapeutically relevant target.


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20

Vehicle PO BID 10 (30 mpk PO, QD) 10 (60 mpk PO, QD) 10 (30 mpk PO, BID)

1000

Body weight change (%)

Tumor volume (mm3)

1500

500

0

10 0 -10 -20

0

10 20 Treatment (day)

2,500

30

1,500

10 20 Treament (day)

30

20

Vehicle 10 (10 mpk SC, BID) Dox. 2 mg/kg IV BIW 10 + Dox.

2,000

0

Body weight Change (%)

Tumor volume (mm3)

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


1,000 500 0

10 0 -10 -20

0

10 20 Treatment (day)

30

0

7 14 21 28 Days after the start of treatment

Figure 3. A) Effect of 10 on MV-4-11 tumor growth in mouse xenograft. B) Corresponding body weight change. C) Effect of 10 in combination with doxorubicin on MV-4-11 tumor growth in mouse xenograft. D) Corresponding body weight change. Toxicology of 10 To determine whether 10 was safe for repeated dosing in human clinical trials, toxicity studies with the compound were conducted in rat and dog. The compound was administered as an oral suspension (rat: 2 mg/kg, 6 mg/kg, and 20 mg/kg; dog: 2 mg/kg, 4 mg/kg, 6 mg/kg) once daily for fourteen consecutive days as part of a dose range-finding study and in GLP-compliant repeat-dose studies that also included an additional 2-week recovery period. A common set of toxicities was observed in both species, which we believe to be the result of exacerbated target



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engagement: lymphoid depletion; hypocellularity of the bone marrow with associated anemia and thrombocytopenia; GI mucosal atrophy, erosion, and ulceration; degeneration of the testicular seminiferous epithelium; and mild to moderate hyperglycemia. The GLP-compliant studies demonstrated these toxicities to be reversible, with the exception of the testicular findings, for which 14 days is likely insufficient to demonstrate recovery.28 Pharmacokinetic results in human Having established that 10 was a potent, selective, and cell-active BET bromodomain inhibitor with significant exposure in multiple species and an acceptable toxicity profile, clinical trials were initiated to determine its pharmacokinetics and maximum tolerated dose in patients with hematologic malignancies. In advance of those studies, a process chemistry synthesis of 10 was developed that allowed for a more stereoselective, convergent preparation of crystalline material with no chromatography, as was an immediate-release drug product that provided oral bioavailability in dogs comparable to dosing with a solution formulation. Those efforts, as well as a detailed accounting of the human pharmacokinetics and pharmacodynamics of the drug in humans, will be the subject of future reports, but interim data regarding the exposure of 10 bear mentioning here. In an ongoing study, 10 has been administered to patients with progressive lymphoma once daily orally for fourteen consecutive days, followed by a seven-day period without treatment. Cycles are repeated, at escalating doses, as tolerated by the patients. To date, nine dose levels have been examined. Highlighted in Figure 4 are the plasma concentrations observed at various time points following dosing at three representative levels (24 mg, 120 mg, and 300 mg).


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Calculated pharmacokinetic parameters for the 300 mg dose at day 14 are shown in Table 5. (See supporting information for data on all doses.) As shown, both maximum concentration and total exposure increase with escalating dose across the cohorts evaluated. Importantly, neither parameter changes significantly from day 1 to day 14 [cf. 300 mg vs. 300 mg (Day 14), Figure 4 and accumulation factor (X) = 1.1 (Table 5)]. An estimation of the free plasma concentration of 10 suggests that doses above 230 mg lead to transient levels of the compound at or above the cellular MYC EC50 (indicated by the dotted line in Figure 4). Initial results indicate that those doses cause substantial modulation of biomarkers linked to BET and even to clinically meaningful effects.29 Anti-tumor responses have been observed following 10 treatment in patients with heavily pre-treated diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma, specifically in patients with T-cell/histiocyte rich, activated B-cell, and germinal center B cell subtypes of DLCBL, which will be the subject of future reports. Taken together, these preliminary human pharmacokinetic data are consistent with expectations based on rat and dog studies and demonstrate that oral dosing of 10 achieves plasma concentrations that are meaningful in the context of BET bromodomain inhibition. Figure 4. Day one (except where noted) plasma concentration of 10 following oral dosing in human. Dashed line indicates cellular MYC EC50.



10

300 mg (Day 14) 300 mg 120 mg

Compound 10 plasma concentration (µ µM)

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24 mg 300 mg (free fraction)

1

0.1

0.01 0

5

10

Time (h)

15

20

25

Table 5: Day 14 steady state human pharmacokinetic parameters of 10 (QD, po, 300 mg). N = 3. Cmax (µ µM) a

tmax (h)

1.8 ± 0.19

C0 (µ µM)

AUC0-24

CL/F

e

f

b

Xg

t1/2 (h)

(Cmax, unbound)c

(C0, unbound)d

5.9 ± 0.8

0.22 ± 0.08

23 ± 11 (0.30)

(µ µM·h)

(L/h)

49 ± 31

21 ± 9.7

1.1 ± 0.04

(0.011)

a

Time at which the maximum concentration was observed. bTerminal half-life

of 10. cMaximum concentration observed. Cmax,

unbound

= (free fraction)∙Cmax.

d

Concentration at 24 h (trough concentration). eExposure over 24 h. fClearance

on day 14 based on oral bioavailability (Dose/AUC0-24). gAccumulation factor. X = AUC0-24(day 14)/AUC0-24(day 1).


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Conclusion In summary, we report here the results of combining insights from a fragment discovery effort with the binding mode of reported literature probes, which gave rise to a series of benzoisoxazoloazepines that were potent inhibitors of the BET family of bromodomains. Subsequent optimization of cell activity and metabolic stability led to 10, which was suitable for in vivo studies in mouse and which demonstrated a correlation between BET-driven reduction in MYC gene expression and tumor growth inhibition in a xenograft study. Based on those data and an acceptable toxicity profile in rat and dog, 10 was chosen as a candidate for human clinical studies to evaluate the impact of BET inhibition in oncology. 10 is currently undergoing evaluation in multiple phase I clinical trials, the results of which will be described in due course. Associated Content Supporting Information. Assay details; descriptions of the MYC cellular assay and mouse xenograft study; synthetic and characterization data for most of the compounds reported; cocrystal structures of 1, 3, and 10; crystallographic methods and data; human PK profiles for multiple doses; other bromodomain potency data for 10. This material is available free of charge via the Internet at http://pubs.acs.org. PDB ID Codes: Coordinates have been deposited in the PDB with accession code 4LR6 for 1, 5HMO for 3, and 5HLS for 10. Author Information



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Corresponding authors: [email protected]; [email protected]. Present Addresses Brian K. Albrecht: Third Rock Ventures, 29 Newbury Street, #301, Boston, Massachusetts 02116, United States; Yves Leblanc: Paraza Pharma Inc., 7171 Frederick-Banting, Montreal, Quebec, Canada H4S 1Z9; Christopher G. Nasveschuk: Broad Institute, 415 Main Street, Cambridge, Massachusetts

02142,

United

States;

Louise

Bergeron:

Bergeron

Drug

Discovery,

www.bergerondrugdiscovery.com; Robert Campbell: Blueprint Medicines, 38 Sidney Street, #200, Cambridge, Massachusetts 02139, United States; Hariharan Jayaram, Shivangi Joshi: Editas Medicine, 300 Third Street, Cambridge, Massachusetts 02142, United States; Adrianne Neiss: Huntsman Cancer Institute, 2000 Circle of Hope Drive, Salt Lake City, Utah 84112, United States; Emmanuel Normant: Surface Oncology, 215 First Street, Suite 400-S, Cambridge, Massachusetts 02142, United States; Eneida Pardo: Forma Therapeutics, 500 Arsenal Street, Watertown, Massachusetts 02472, United States; Peter Sandy: Evelo Therapeutics, 620 Memorial Drive, Suite 200 West, Cambridge, Massachusetts 02139, United States; Jeffrey Supko: Massachusetts General Hospital, Dana-Farber/Harvard Cancer Center, 55 Fruit Street, GRJ1025, Boston, Massachusetts 02114, United States; Jean-Christophe Harmange: Third Rock Ventures, 29 Newbury Street, #301, Boston, Massachusetts 02116, United States. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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Notes The authors declare no competing financial interest. Acknowledgments We are grateful to Ted Peters and Christina Lee in the Lead Discovery group for plating compounds used in these studies; PPD BioDuro, WuXi AppTec, and Custom NMR Services for the synthesis and analysis of some of the compounds presented here; Jeremy W. Setser for assistance with the co-crystallographic images. Abbreviations Used BET, bromodomain and extraterminal domain; BD, bromodomain; TR-FRET, time-resolved fluorescence resonance energy transfer; LEAN, ligand efficiency adjusted for atom number; LLE, lipophilic ligand efficiency; Clint, intrinsic clearance; PPB, plasma protein binding; CL, clearance; Q, liver blood flow; IV, intravenous; sc, subcutaneous; po, per os; VSS, volume of distribution at steady state; AUC, area under the curve; F, oral bioavailability; DMA, dimethylacetamide; PEG, polyethylene glycol; SBECD, sulfobutylether-β-cyclodextrin; NMP, 1-methyl-2-pyrrolidinone; Boc2O, di-tert-butyl dicarbonate; DMAP, 4-(dimethylamino)pyridine; THF, tetrahydrofuran; TFA, trifluoroacetic

acid;

DMF,

N,N-dimethylformamide;

HPLC,

high-performance

liquid

chromatography; SFC, super-critical fluid chromatography; GPCR, G-protein coupled receptor. Table of contents graphic



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OTX015 targets BRD2 and BRD4 and decreases c-MYC in acute leukemia cells. Oncotarget 2015, 6, 17698-17712. 21 Herrmann, H.; Blatt, K.; Shi, J.; Gleixner, K. V.; Cerny-Reiterer, S.; Mullauer, L.; Vakoc, C. R.; Sperr, W. R.; Horny, H. P.; Bradner, J. E.; Zuber, J.; Valent, P. Small-molecule inhibition of BRD4 as a new potent approach to eliminate leukemic stem- and progenitor cells in acute myeloid leukemia AML. Oncotarget 2012, 3, 1588-1599. 22 Throughout this work, BRD4 BD-1 was used as a surrogate for binding to both bromodomains of the entire BET family. For certain compounds, the binding of individual compounds to bromodomains 1 and 2 of BRD2, 3, and T was determined and was, in general, very similar to that of BRD4 BD-1 (data not shown), which supported that assumption. 23 Ryckmans, T.; Edwards, M. P.; Horne, V. A.; Correia, A. M.; Owen, D. R.; Thompson, L. R.; Tran, I.; Tutt, M. F.; Young, T. Rapid Assessment of a novel series of selective CB2 agonists using parallel synthesis protocols: a lipophilic efficiency (LipE) analysis. Bioorg. Med. Chem. Lett. 2009, 19, 4406-4409. 24 Hewitt, M. C.; Leblanc, Y.; Gehling, V. S.; Vaswani, R. G.; Côte,́ A.; Nasveschuk, C. G.; Taylor, A. M.; Harmange, J.-C.; Audia, J. E.; Pardo, E.; Cummings, R.; Joshi, S.; Sandy, P.; Mertz, J. A.; Sims, R. J., III; Bergeron, L.; Bryant, B. M.; Bellon, S.; Poy, F.; Jayaram, H.; Tang, Y.; Albrecht, B. K. Development of methyl isoxazoleazepines as inhibitors of BET. Bioorg. Med. Chem. Lett. 2015, 25, 1842−1848. 25 Robak, M. T.; Herbage, M. A.; Ellman, J. A. Synthesis and applications of tertbutanesulfinamide. Chem. Rev. 2010, 110, 3600–3740. 26 Williams, M. J.; Jobson, R. B.; Yasuda, N.; Marchesini, G.; Dolling, U. H.; Grabowski, E. J. J. A new general method for preparation of N-methoxy-N-methylamides. Application in direct conversion of an ester to a ketone. Tetrahedron Lett. 1995, 36, 5461–5464. 27 Muñoz, J. d. M.; Alcázar, J.; de la Hoz, A.; Díaz-Ortiz, Á.; Alonso de Diego, S.-A. Preparation of amides mediated by isopropylmagnesium chloride under continuous flow conditions. Green Chem. 2012, 14, 1335– 1341. 28 Berkovits, B. D.; Wolgemuth, D. J. The role of the double bromodomain-containing BET genes during mammalian spermatogenesis. Curr. Top. Dev. Biol. 2013, 102, 293-326. 29 Abramson, J. S.; Blum, K. A.; Flinn, I. W.; Gutierrez, M.; Goy, A.; Maris, M.; Cooper, M.; O'Meara, M.; Borger, D.; Mertz, J. A.; Sims, R. J. III; Supko, J. G.; Younes, A. BET inhibitor CPI0610 is well tolerated and induces responses in diffuse large B-cell lymphoma and follicular lymphoma: preliminary analysis of an ongoing phase 1 study. 57th ASH Annual Meeting & Exposition, 5-8 December, 2015, Orlando, FL.


Identification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the

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