Diving into the Water: Inducible Binding Conformations for BRD4

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Diving into the water: Inducible binding conformations for BRD4, TAF1(2), BRD9, and CECR2 bromodomains Terry D. Crawford, Vickie Tsui, E. Megan Flynn, Shumei Wang, Alexander M. Taylor, Alexandre Côté, James E. Audia, Maureen H. Beresini, Daniel J. Burdick, Richard T. Cummings, Les A. Dakin, Martin Duplessis, Andrew C Good, Michael C. Hewitt, Hon-Ren Huang, Hariharan Jayaram, James R. Kiefer, Ying Jiang, Jeremy M. Murray, Christopher G. Nasveschuk, Eneida Pardo, Florence Poy, F. Anthony Romero, Yong Tang, Jian Wang, Zhaowu Xu, Laura E. Zawadzke, Xiaoyu Zhu, Brian K. Albrecht, Steven R. Magnuson, Steven F. Bellon, and Andrea G. Cochran J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00264 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 25, 2016

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Journal of Medicinal Chemistry

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Diving into the water: Inducible binding conformations for BRD4, TAF1(2), BRD9, and CECR2 bromodomains Terry D. Crawford,*† Vickie Tsui,† E. Megan Flynn,† Shumei Wang,† Alexander M. Taylor,‡ Alexandre Côté,‡James E. Audia,‡Maureen H. Beresini,† Daniel J. Burdick,†Richard Cummings,‡ Les A. Dakin,‡ Martin Duplessis,‡ Andrew C. Good,‡Michael C. Hewitt,‡ Hon-Ren Huang,‡ Hariharan Jayaram,‡ James R. Kiefer,†Ying Jiang,§ Jeremy Murray,† Christopher G. Nasveschuk,‡ Eneida Pardo,‡ Florence Poy,‡F. Anthony Romero,† Yong Tang,‡ Jian Wang,§ Zhaowu Xu,§ Laura E. Zawadzke,‡ Xiaoyu Zhu,§ Brian K. Albrecht,‡ Steven R. Magnuson,† Steve Bellon,‡ Andrea G. Cochran† †

Genentech, Inc. 1 DNA Way, South San Francisco, CA 94080

‡

Constellation Pharmaceuticals, Inc. 215 First Street, Suite 200, Cambridge, MA 02142

§

Wuxi AppTec Co., Ltd., 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai

200131, People’s Republic of China

RECEIVED DATE

Abstract

The biological role played by non-BET bromodomains remains poorly understood, and it is therefore imperative to identify potent and highly selective inhibitors to effectively explore the biology of individual bromodomain proteins. A ligand-efficient non-selective bromodomain inhibitor was identified from a 6-methyl pyrrolopyridone fragment.

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substituents replacing the N-methyl group were designed directing towards the conserved bromodomain water pocket, and two distinct binding conformations were then observed. The substituents either directly displaced and rearranged the conserved solvent network, as in BRD4(1) and TAF1(2), or induced a narrow hydrophobic channel adjacent to the lipophilic shelf, as in BRD9 and CECR2.

The preference of distinct substituents for individual

bromodomains provided selectivity handles useful for future lead optimization efforts for selective BRD9, CECR2, and TAF1(2) inhibitors.

Introduction Bromodomains are a family of 61 protein modules present in 46 distinct human proteins.1-2 These modules consist of roughly 110 amino acid residues and serve as epigenetic readers via the recognition of acetylated lysine residues on histone tails as well as other proteins.3-7 The most prominently studied of these bromodomains to date are members of the bromodomain and extra terminal domain (BET) family, consisting of dual bromodomains in bromodomain-containing protein 2 (BRD2), bromodomain-containing protein 3 (BRD3), bromodomain-containing protein 4 (BRD4), and bromodomain testis-specific protein (BRDT). Inhibition of the BET family bromodomains has been shown to have potential therapeutic benefit in cancer, inflammation, immunology, and male contraception.8a-f While the function of BET inhibitors has been broadly studied, the role of non-BET bromodomains remains uncertain. To elucidate the biological function of these bromodomains, it is critical to identify potent inhibitors suitable for phenotypic profiling that are highly selective over other bromodomain family members. The recent disclosure of a number of non-BET bromodomain inhibitors highlights the strong level of interest in these types of molecules.9-19



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Figure 1. Tertiary structure of the BRD9 bromodomain. Notable residues include the conserved asparagine and tyrosine required for binding acetylated lysine (Asn100 and Tyr57 in BRD9), the gatekeeper (Tyr106 in BRD9), the lipophilic shelf adjacent to the ZA channel (Gly43, Phe44, Phe45 in BRD9), as well as the conserved water network. The bromodomain tertiary structure consists of four left-handed alpha-helices (αA, αB, αC, αZ) packed in an antiparallel bundle, with two loop regions between helices αA and αZ (ZA loop) and αB and αC (BC loop) (Figure 1).20 Acetylated lysine residues bind in the hydrophobic pocket created by these two loop regions, with the amide usually forming a direct hydrogen bond to a conserved asparagine located at the beginning of the BC loop (Asn100 in BRD9), as well as a water mediated hydrogen bond interaction with a conserved tyrosine located on the ZA loop (Tyr57). This solvent molecule is also part of a network of five highly conserved water


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molecules in the binding pocket, which often extends into a channel formed by the ZA loop, called the ZA channel. A lipophilic shelf21 of three residues adjacent to the ZA channel as well as a “gatekeeper” residue located at beginning of the C helix help to further define the structure of the binding pocket. In BRD9, this lipophilic shelf is comprised of Gly43, Phe44, and Phe45, and Tyr106 is the gatekeeper. While the asparagine and tyrosine residues are highly conserved throughout most bromodomains, there is significant variation within the ZA channel, lipophilic shelf, and gatekeeper residues.22 We hypothesized that we could exploit the moderate degree of homology found among bromodomain binding pockets to quickly identify new selective lead molecules from a shared scaffold. Indeed, we observed that modest changes to the substituents of a common bromodomain inhibitor core induces novel binding conformations, either through reorganization and stabilization of the conserved water network, as in the BRD4(1) and the second bromodomain of human transcription initiation factor TFIID subunit 2 [TAF1(2)] binding sites, or through the formation of a hydrophobic channel between the lipophilic shelf and gatekeeper residues, as in bromodomain containing protein 9 (BRD9) and cat eye syndrome critical region protein 2 (CECR2). These distinct structural changes for different bromodomains provide a new means of achieving selectivity starting from a non-selective bromodomain inhibitor. Results and Discussion The N-methyl pyrrolopyridone 1 was a fragment-derived lead molecule with strong binding across bromodomain family members (Table 1), with ligand efficiencies23 (LE) > 0.54 for all eight bromodomains screened.24 We observed from a structure of compound 1 bound to BRD9 that the pyrrolopyridone serves as the acetyl-lysine mimetic, with the pyridone carbonyl and pyrrole nitrogen forming a 2-point interaction with Asn100 (Figure 2). The pyrrolopyridone core



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forms a π−π stacking interaction with the BRD9 Tyr106 gatekeeper. The conserved solvent network is labeled as in Flynn et al.25, which uses the labels 1–4 from Hewings et al.8a for the four canonical binding site waters, and 0 for the additional conserved solvent molecule utilized in a computational study by Vidler et al.26 It is noteworthy that water 0 forms a stable tetracoordinated network with residues corresponding to Asn95, Tyr57, Met65 in BRD9 as well as an additional solvent molecule. In order to evaluate the structural importance of the observed solvent molecules, we used the algorithm described in Amadasi et al.27 as implemented in Proasis v.3.342 (Desert Scientific Software, Pty, Ltd.) to calculate water scores. Water 0 receives a score of 5.4 while waters 1, 2, 3 and 4 receive scores of 2.2, 3.9, 2.3 and 2.3, respectively. According to the classification scheme in Proasis, a water score of >4.0 is “highly stable.”

Figure 2. A) Crystal structure of pyrrolopyridone fragment 1 bound to BRD9 (PDB Code: 5I40, 1.04 Å resolution, with conserved waters (0–4) and corresponding hydrogen bonding network. B) Binding pocket features with ZA channel, gatekeeper Y106, and lipophilic shelf formed by G43, F44, and F45.


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With the pyrrolopyridone proving to be a highly ligand efficient fragment scaffold, we next targeted substitution at the 4-position to occupy the ZA channel with the aim of improving our potency profile. A library utilizing the Suzuki-Miyaura reaction and 4-bromo-6-methyl-1-tosylpyrrolopyridone intermediate 17a (Scheme 1) yielded the non-selective bromodomain inhibitor 2 which displayed low to sub-micromolar affinity across a bromodomain panel (Table 1). The LEs of 2 suffer slightly for each bromodomain when compared to fragment 1, however all remain greater than 0.35.

Scheme 1. Synthesis of pyrrolopyridone scaffold and representative example. Reagents and conditions: (i) NaOMe, MeOH, reflux; quantitative (ii) NaOAc, AcOH, Br2, 80 °C, 82%; (iii)



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DMF-DMA, DMF, 95 °C, quantitative; (iv) Fe, NH4Cl, MeOH/H2O, reflux, 99%; (v) NaH, THF, tosyl chloride, 0 °C–r.t., 89%; (vi) HBr, EtOH, 90 °C, 89%; (vii) NaH, DMF, R-Br or R-I, 0

°C–r.t.;

(viii)

[3-(dimethylcarbamoyl)phenyl]boronic

acid,

bis(di-tert-butyl(4-

dimethylaminophenyl)phosphine) dichloropalladium(II), K3PO4, dioxane/H2O, 90 °C; (ix) KOH, MeOH, H2O, 45 °C, 7–70% over 3 steps. Table 1. Binding potenciesa and ligand efficienciesb for fragment lead 1 and compounds 2–5.c

a

All bromodomain assays were run in TR-FRET format as described in Experimental

Procedures. IC50 data are an average of at least 2 independent experiments. bLE = –RTln(IC50)/N heavy atoms. cA full table of data with standard deviations for these and other N-alkyl substituents can be found in the Supporting Information. Co-crystal structures of 2 bound to BRD9, the first bromodomain of BRD4 [(BRD4(1)] and TAF1(2) indicate that in each case the pyrrolopyridone core maintains the 2-point interaction with the conserved asparagine residue that was observed with the fragment 1 (Figure 3 A, C, and


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E).

In contrast, the meta-substituted dimethylbenzamide substituent engages in different

interactions with the individual bromodomains (Figure 3 B, D, and F). When bound to BRD9, the dimethylbenzamide makes van der Waals contacts with Ile53 and lipophilic shelf Phe44 of the ZA loop. In BRD4(1), the benzamide forms an edge-to-face π−π interaction with Trp81 of the WPF shelf, while the phenyl ring is involved in hydrophobic contact with ZA loop Leu92. The benzamide is flipped in the TAF1(2) complex, with the carbonyl oxygen making a direct hydrogen bond to the backbone NH of Asn1533 of the ZA loop, while the phenyl ring makes an edge-to-face π−π interaction with Trp1526 of the WPF shelf.



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Figure 3. (A,B) Compound 2 in complex with BRD9 (PDB Code: 5I7X, 1.15 Å resolution). In these panels and subsequent pairs, a stick representation is shown at left and in the right panel there is a 90° rotation of the view with the protein surface depicted. The metadimethylbenzamide maintains van der Waals contacts with Phe44 from the lipophilic shelf and Ile53 on the ZA loop. (C,D) Compound 2 bound to BRD4(1) (PDB Code: 5I80, 1.45 Å resolution). The benzamide forms an edge-to-face π−π interaction with Trp81 from the lipophilic shelf, while the phenyl ring is involved in hydrophobic contact with Leu92 on the ZA loop. (E,F) Compound 2 bound to TAF1(2) (PDB Code: 5I29, 1.21 Å resolution). The benzamide was found to be flipped to make a direct hydrogen bond to the backbone NH of Asn1533 of the ZA loop, while the phenyl ring is involved in an edge-to-face π−π interaction with Trp1526 from the WPF shelf. While the potent non-selective bromodomain inhibitor 2 is potentially a useful tool, it remained to be seen whether selectivity for individual family members could be identified from this scaffold. We next explored the vector extending from the pyridone methyl towards the four conserved water molecules in the binding pocket. The x-ray co-crystal structures of 2 appear to indicate that substitution beyond N-methyl would be unproductive, as perturbation of the these waters in the bromodomain binding pocket has been viewed as exceedingly difficult.16,

26

However, we have observed that hydrophobic substituents larger than methyl are tolerated in other chemical series (data not shown), as well as in peptides with larger acylated lysine residues such as propionyl, crotyl, and butyryl.25, 28 Huang et al. have also performed solvent molecular dynamic simulations which suggests that the conserved water network may be disrupted.29 We therefore synthesized a series of analogues varying the length and saturation of the N-alkyl tail to further probe the accessibility of the pocket along this vector.



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Interestingly, we found the binding specificity among bromodomains to be very sensitive to the nature of these extended substituents. BRD4(1), BRD9, CECR2, and TAF1(2) each bind preferentially to a distinct unsaturated chain as an optimal hydrophobic substituent. Incorporation of the allyl in compound 3 only yielded improved potency against CECR2, while potency decreases were observed across the other panel bromodomains ranging from a minor decrease for BRD9 to >10-fold losses for the other members (Table 1). Extending one methyl from allyl, the unsaturated trans-crotyl 4 gives slightly improved potency for BRD9 relative to compound 2, while BRD4(1) and TAF1(2) tolerate this substituent with only 5–10-fold potency decreases. LE for 4 remained high (≥ 0.36) for all three bromodomains. Transitioning from crotyl 4 to 1-butene 5 resulted in a large potency drop across the majority of bromodomains, with the exception of TAF1(2), against which 5 maintains excellent potency compared to 2 and now demonstrates selectivity across the entire panel (> 30 fold over BRD9, >50 fold over BRD4). In each example of extended substituents, there was selectivity against CREB binding protein bromodomain (CBP), bromodomain and PHD finger containing, 1 (BRPF1), and the first bromodomain of human transcription initiation factor TFIID subunit 1 [TAF1(1)] with negligible binding at 20 µM. Additionally, we further investigated substituents which included branched (cyclic or acyclic) or heteroatom containing chains. However, potency dropped in all cases at least 10-fold for each bromodomain and more often >30 fold (data not shown). Co-crystal structures of compounds 4 and 5 confirmed that these small molecules induce binding conformations in TAF1(2) and BRD9 consistent with previously reported observations for crotonylated or butyrylated lysines on histone peptides.25 In the crystal structure of 5 bound to TAF1(2), the 1-butenyl substituent extends into the water channel between Tyr1540 and the lipophilic shelf residues Pro1527 and Phe1528 (Figures 4 A,B). The butenyl group displaces


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water 4 in the conserved water network and induces a rearranged solvent network around the butenyl group which includes two tetra-coordinated water molecules, labeled as waters 0’ and 3’. While water 0’ is only 1.2 Å from the location where water 0 is located in the 2-bound structure of TAF1(2), the position of water 3’ is 3.0 Å from water 3 in 2/TAF1(2). Water 3’ is now resides closer to Asn1578 to create new interactions with the amide sidechain, as well as water 0’. Water scores from Proasis were 5.2 for 0’ and 5.7 for 3’, indicating they are highly stabilized. On the other hand, the solvent molecules corresponding to this water channel in other ligandbromodomain co-crystal structures described herein, aside from water 0, have scores of 4.0 or less (Table 2), underscoring the significance of the two stabilized waters shown in Figure 4B.

Table 2. Proasis water scores of waters 0-4 in ligand-bound crystal structures.



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Protein\ Compound

1

BRD4(1)

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2

4

wat 0: 5.2

wat 0': 4.0

wat 1: 2.1

wat 1: 2.1

wat 2: 2.3

wat 2: 4.0

wat 3: 2.3

wat 3': 2.3

5

wat 4: 2.3

BRD9

wat 0: 5.4

wat 0: 5.3

wat 0: 5.4

wat 0: 5.4

wat 1: 2.2

wat 1: 2.2

wat 1: 2.2

wat 1: 2.2

wat 2: 3.9

wat 2: 3.9

wat 2: 3.9

wat 2: 3.9

wat 3: 2.3

wat 3: 2.3

wat 3: 2.3

wat 3: 2.3

wat 4: 2.3

wat 4: 3.7

wat 4: 3.7

wat 4: 2.3

wat 0: 5.4

wat 0': 5.5

wat 0': 5.2

wat 1: 2.1

wat 1: 1.0

wat 1: 1.0

wat 2: 2.3

wat 2: 2.3

wat 2: 2.3

TAF1(2)

wat 3: 2.3

wat 3': 5.7

wat 4: 2.3 While other factors such as differential degrees of protein flexibility or long-range effects of residue differences away from the binding site may be important, we believe the formation of these two stable water molecules when 5 is bound to TAF1(2) offers insight into the interplay between the affinity of functional groups residing in the water channel and the ability of remaining water molecules to re-organize.


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Figure 4. A) Compound 2 bound to TAF1(2), with water network depicted (small spheres). B) Compound 5 in complex with TAF1(2), with rearranged water network including tetracoordinated waters 0’ and 3’. Water 0’ forms a fourth interaction with an additional solvent molecule in the binding pocket. Water 3’ forms new interactions with the sidechain of N1578 and water 0’ (PDB Code: 5I1Q, 1.50 Å resolution). C) Compound 2 bound to BRD4(1), with water network indicated as in A. D) Compound 4 bound to BRD4(1), with rearranged water network. Water 4 is displaced, with water 0’ failing to form the fully coordinated hydrogen bond network (PDB Code: 5I88, 1.4 Å resolution).



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We also found that the crotyl substituent from 4 is able to disrupt the conserved solvent network of BRD4(1) (Figures 4 C, D). As in the structure of 5 bound to TAF1(2), water 4 is displaced while the remaining solvent molecules consist of unmoved waters 1 and 2, and rearranged waters labeled as 0’ and 3’. The Proasis scores for waters 0’, 1, 2 and 3’ when 4 is bound to BRD4(1) are 4.0, 2.1, 4.0 and 2.3, respectively.

While multiple parameters may contribute to the

decreased potency of 4, the lack of any water with scores >5, indicating close to ideal tetracoordination based on geometry, may play a role. We next investigated the binding conformation of compound 4 bound to BRD9. From the cocrystal structure, we observed that the crotyl group of 4 leaves the canonical water network intact, and instead is directed orthogonal to the pyrrolopyridone plane into a narrow induced hydrophobic channel (Figure 5A). Critical residues for the crotyl 4 binding to BRD9 are Phe45 of the lipophilic shelf, gatekeeper Tyr106, as well as Met92 (residing behind Phe45), which we have labelled as the “backstop” residue (Table 3). The flexible Met92 allows Phe45 from the lipophilic shelf to shift to accommodate the crotyl group, while the gatekeeper Tyr106 also moves slightly to expand the hydrophobic channel (Figure 5B). This movement increases the distance between the Cζ of Phe45 and the Cβ of Tyr106 from 5.3 Å to 7.0 Å. We believe that more sterically hindered beta-branched residues at the gatekeeper (such as valine or isoleucine found in BRD4(1), BRD4(2), CBP, and TAF1(1)), or the backstop (isoleucine in BRPF1 and both TAF1 bromodomains), prevent this channel from opening, resulting in selectivity over these bromodomains.


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Table 3:

Lipophilic shelf, gatekeeper, and backstop residue differences between selected

bromodomains.a

Bromodomain BRD4-BD1 BRD4-BD2 CBP BRPF1 TAF1-BD1 TAF1-BD2 BRD9 CECR2 a

"Lipophilic shelf" residues W W L N Y W G W

P P P I P P F P

F F F F F F F F

"Gatekeeper" residue

"Backstop" Residue

I V V F L Y Y Y

M M M I I I M M

Lipophilic shelf corresponding to the “WPF shelf” in BRD4(1), gatekeeper, and backstop

residue differences between BRD4 and non-BET bromodomains. Residues that favorably contribute to the ability to form the hydrophobic channel are highlighted in green.



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Figure 5. A) Compound 2 (yellow, conserved waters in red) and 4 (cyan, conserved waters in cyan, PDB Code: 5I7Y, 1.41 Å resolution) bound to BRD9. The crotyl group is positioned orthogonal to the pyrrolopyridone core, leaving the conserved waters unaffected. B) Compound 2 (yellow, solvent-accessible surface in green, residues in green) and 4 (cyan, solvent accessible surface in gray, residues in brown) bound to BRD9. Lipophilic shelf Phe45, gatekeeper Tyr106, and backstop Met92 provide the ability to form the induced hydrophobic channel, as indicated by the increase in solvent-accessible volume visualized by the gray surface. The binding site of CECR2 can be viewed as a hybrid between those of BRD9 and TAF1(2). While all three bromodomains have a tyrosine gatekeeper, CECR2 shares the methionine backstop residue with BRD9 while sharing the same lipophilic shelf motif as TAF1(2). While we were unable to obtain a co-crystal structure of allyl 3 bound to CECR2, modeling of 3 in the CECR2 apo structure provided a plausible rationale for the preference of the allyl group (Figure 6). As with BRD9, the allyl is hypothesized to reside in the channel between the lipophilic shelf and gatekeeper Tyr520. While Phe459 of the CECR2 lipophilic shelf appears capable of shifting slightly towards the Met506 backstop to accommodate the allyl group, the Pro458 effectively seals deeper penetration into this channel, preventing access of longer substituents such as the 1butene or crotyl. The allyl fits well into this more shallow space, and one would expect it to make hydrophobic interactions with Pro458, Phe459, and Tyr520. While the model in Figure 6 may not be suitable for atomic-scale structure-based design, it helps conceptualize the predicted binding mode of 3.


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Figure 6. Model of allyl 3 in CECR2 (brown) utilizing structure of unbound CECR2 (PDB code: 3NXB) with accessibility surface shown in grey. CECR2’s lipophilic shelf residues Pro458 and Phe459, backstop Met506, and gatekeeper Tyr520 are able to form a smaller induced hydrophobic pocket relative to BRD9, with Pro458 blocking deeper penetration. To verify the importance of the tyrosine gatekeeper and methionine backstop residues for the ability to create the hydrophobic channel in BRD9 and CECR2, we compared the binding of compounds to mutant proteins using differential scanning fluorimetry (DSF) (Table 4).30 For both wild-type BRD9 and wild-type CECR2, thermal stability shifts (∆Tm) were similar for binding of the parent N-methyl compound 2 and the most potent N-substituted analogue (4 or 3, respectively), consistent with the similar potencies measured for the relevant pairs in TR-FRET competition assays (Table 1). Replacing either the gatekeeper (Y-to-I) or the backstop (M-to-I) in CECR2 resulted in a modest loss of binding affinity for 2, as evidenced by a decrease in ∆Tm, while only the gatekeeper mutation likewise affected binding of 2 by BRD9. This decrease may



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reflect loss of the π−π interaction between the tyrosine and pyrrolopyridone core. However, the loss of affinity for the appropriate analogue 3 or 4 was much larger for the CECR2 and BRD9 mutant proteins. While the backstop mutants retained some residual binding (~2 °C ∆Tm), the gatekeeper mutants appeared to be incapable of binding 3 or 4. These data confirm that, as was the case for the butyryl-lysine peptides, gatekeeper and backstop residues are important for access to the hydrophobic channel leading in the direction of the lipophilic shelf phenylalanine. Table 4. Stability of wild type and mutated BRD9 and CECR2 bromodomains in the presence of compound 2 or the appropriate selective analogue 3 or 4.a

a

Protein only

Protein with 2

Protein with 4

Tm (°C +/- SD)

∆Tm bound-free (°C +/- SD)

∆Tm bound-free (°C +/- SD)

WT BRD9

49.5 ± 0.5

4.6 ± 0.8

4.4 ± 0.8

BRD9 Y106I

51.7 ± 0.7

2.3 ± 0.8

0 ± 0.9

BRD9 M92I

47.3 ± 0.7

5±1

2±1

Protein only

Protein with 2

Protein with 3

WT CECR2

42.1 ± 1

6±1

6±1

CECR2 Y520I

45.1 ± 0.6

3±1

−1 ± 1

CECR2 M506I

45.5 ± 0.9

3±1

2±1

Mean, apparent Tm, and standard deviation were calculated from measurements from DSF

measurements on at least five (CECR2) or ten (BRD9) individual wells for each sample. Positive ∆Tm reflects enhanced protein stability in the presence of bound compound, with larger values indicating greater affinities.


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In order to further discern the impact of these hydrophobic substituents on selectivity against bromodomain family members, compounds 3, 4, and 5 were profiled in a BROMOscan panel of 40 bromodomains (Figure 7).33 As was observed by Humphreys et al,16 the potencies from the BROMOscan were found to be generally higher compared to those from the TR-FRET assays (~2-10 fold), with the exception of compounds binding to CECR2 which were either equipotent or slightly more potent in our TR-FRET assay (see Supporting Information for full BROMOscan assay data and IC50/Kd graphical analysis). The non-selective compound 2 exhibits bromodomain binding consistent with the IC50 values observed in our TR-FRET assays, as well as activity across the majority of the broader bromodomain panel. This includes sub-micromolar affinity for bromodomain adjacent to zinc finger domain, 2A (BAZ2A, 0.88 µM), tripartite motif 24 protein (TRIM24, 0.91 µM), and the CBP homolog E1A Binding Protein P300 (EP300, 0.27 µM). Low micromolar affinity was observed for 2 binding to bromodomain adjacent to zinc finger domain, 2B (BAZ2B, 3.6 µM), protein polybromo-1 bromodomain 2 [PBRM1(2), 7.6 µM], SMARCA2 (6.7 µM), and SMARCA4 (9.0 µM). The more selective compounds 3, 4, and 5 also have potencies that correlate well with the TR-FRET data, re-enforcing the importance of the hydrophobic substituent to bromodomain binding specificity.



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Figure 7. BROMOscan data for compounds 2 (7A), 3 (7B), 4 (7C), and 5 (7D). Compound Kd values are the average of Kds from 2 independent experiments.

Chemistry The pyrrolopyridone scaffold was accessed via the Leimgruber-Batcho indole synthesis (Scheme 1).31 Addition of sodium methoxide to 2-chloro-4-methyl-3-nitropyridine 10 afforded methoxypyridine 11, which was brominated to give 12. Formation of enamine 13 followed by


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cyclization via iron-mediated nitro reduction afforded pyrrolopyridone 14. Pyrrole tosyl protection (compound 15) followed by deprotection of the methoxy group with aqueous HBr provided pyridone 16. The pyridone was then alkylated with alkyl bromides or iodides using sodium hydride to provide the desired N-alkyl substituents 17 a–h. Final targets were then obtained by Suzuki coupling followed by tosyl deprotection. Conclusion From the N-methyl pyrrolopyridone fragment hit 1 we first identified the non-selective bromodomain inhibitor 2, which proved useful for assay development for multiple bromodomains. We then probed SAR for the space near the conserved water residues and identified two distinct, inducible binding conformations. This led to the discovery of highly ligand-efficient lead chemical matter suitable for further progression toward selective inhibitors of BRD9, CECR2, and TAF1(2). We observed that the butenyl substituents of compounds 4 and 5 were able to displace conserved water molecules, inducing a rearranged water network in TAF1(2) and BRD4(1). The nature of the rearranged water molecules appears to correlate with potency, in that the unique potency and selectivity of 5 for TAF1(2) corresponds to the only structure in which disturbance of the conserved water molecules resulted in two highly stabilized waters with Proasis scores >5. Binding experiments using BRD9 and CECR2 mutants revealed the importance of their tyrosine gatekeeper and methionine backstop residues to the ability of allyl 3 in CECR2 and crotyl 4 in BRD9 to enter a narrow hydrophobic channel adjacent to the conserved waters. The identification of these unique selectivity substituents enabled us to rapidly pursue selective chemical matter for all three bromodomains from a common core scaffold. Experimental Procedures:



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General Methods. All solvents and reagents were used as obtained. NMR analysis performed in a deuterated solvent with a Varian Avance 300-MHz or Bruker Avance 400- or 500-MHz NMR spectrometers, referenced to trimethylsilane (TMS). Chemical shifts are expressed as δ units using TMS as the external standard (in NMR description, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad peak). All coupling constants (J) are reported in Hertz. Mass spectra were measured with a Finnigan SSQ710C spectrometer using an ESI source coupled to a Waters 600MS high performance liquid chromatography (HPLC) system operating in reverse-phase mode with an X-bridge Phenyl column of dimensions 150 mm by 2.6 mm, with 5 µm sized particles. Preparatory-scale silica gel chromatography was performed using mediumpressure liquid chromatography (MPLC) on a CombiFlash Companion (Teledyne ISCO) with RediSep normal phase silica gel (35−60 µm) columns and UV detection at 254 nm. Reversephase (HPLC) was used to purify compounds as needed by elution from a Phenomenex GeminiNX C18 column (20.2 x 50 mm, 5 micron) as stationary phase using mobile phase indicated, and operating at a 35 mL/min flow rate on a Waters 3100 mass-directed prep instrument. Chemical purities were >95% for all final compounds as assessed by LC/MS analysis. The following analytical method was used to determine chemical purity of final compounds: HPLC-Agilent 1200, water with 0.05% TFA, acetonitrile with 0.05% TFA (buffer B), Agilent SB-C18, 1.8 µM, 2.1 x 30 mm, 25 ºC, 3–95% buffer B in 8.5 min, 95% in 2.5 min, 400 µL/min, 220 nm and 254 nm, equipped with Agilent quadrupole 6140, ESI positive, 90-1300 amu. Differential Scanning Fluorimetry The stability of all WT and mutant bromodomains was assessed in the presence and absence of small molecules using differential scanning fluorimetry (DSF) measurements with SYPRO Red dye (Molecular Probes).30 Protein aliquots stored at –80 °C were gently thawed at room


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temperature, any protein aggregate was removed by centrifugation at 16,100 X g for 10 min at 4 °C, and the bromodomain concentration was determined using a NanoDrop spectrophotometer (NanoDrop Instruments). Compound stocks (10 mM) in 100% DMSO were diluted 500-fold with DSF buffer (20 mM HEPES, pH 7.7, 100 mM NaCl) and 20 µL aliquots transferred to individual wells of a mixing plate. For control samples without compound, 20 µL of DSF buffer + 0.2% DMSO was added to the mixing plate. Proteins were diluted to a final concentration of 16 µM in DSF buffer. SYPRO Red dye (5000X stock in DMSO as supplied by manufacturer) was diluted 250-fold with DSF buffer. Diluted SYPRO Red dye was added to diluted protein in a 1:1 ratio, and the solution was mixed well followed by centrifugation at 16,100 X g for 1 min at room temperature. The protein/dye mixture (20 µL) was then transferred to individual wells of the mixing plate to yield final concentrations of 4 µM protein, 10X dye, 10 µM compound, and 0.2% DMSO in DSF buffer. The mixing plate was centrifuged at 1000 rpm for 1 min. The compound/dye/protein mixture was mixed well, and 30 µL was transferred to a 96-well optical qPCR plate (Applied Biosystems), followed by centrifugation at 1000 rpm for 1 min to remove any bubbles. The samples were heated from 25 °C to 95 °C in an AB7500 qPCR machine, and fluorescence measurements were taken at 1 °C increments. Thermal melting curves were plotted, and the ThermoFluor analyzer software was used to generate apparent Tm values according to published methods.30 Mean, apparent Tm, and standard deviations were calculated from measurements of at least five (CECR2) or ten (BRD9) individual wells for each protein. Time-Resolved Fluorescence Resonance Energy Transfer Assays Compound potencies were evaluated in a panel of biochemical bromodomain binding assays. Binding of biotinylated small-molecule ligands to recombinant His-tagged bromodomains was assessed by time-resolved fluorescence resonance energy transfer (TR-FRET) as previously



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described.32 Test compounds that compete with the biotinylated ligand for bromodomain binding reduce the TR-FRET signal. Assays were conducted in a total volume of 15 µL in white 384-well plates with the DMSO concentration held constant at 0.2%. Unless otherwise specified,32 reagents were prepared in assay buffer (50 mM HEPES pH 7.5, 1 mM TCEP, 0.069 mM Brij-35, 50 mM NaCl, and 0.1 mg/mL bovine serum albumin). Compounds in DMSO were added to empty assay plates using an Echo 550 acoustic dispenser (Labcyte, Santa Clara, CA). Bromodomain was added followed by biotinylated ligand, and the plates were incubated at room temperature for 10 min after each addition. Subsequently, the TR-FRET detection reagents, antiHis-europium (0.2 nM) and streptavidin-allophycocyanin (SA-APC) (Perkin Elmer, Waltham, MA) were added and incubated for an additional 40 min. Compounds were evaluated as 10point titrations with N = 2. Each compound was assayed in at least 2 independent assays. Results were analyzed with XLFit (IDBS, London, UK) beginning with a 4-parameter Hill fit and, in some cases, constraining the upper or lower baseline to generate a suitable fit. In cases where > 50% inhibition was not achieved, the data were not fit, and the IC50 is instead reported as a lower limit. All IC50 values are reported in units of micromolar. Biotinylated probe preparation is described in Supporting Information. For those assays not described previously,32 reagent concentrations are reported in Table S3. Modeling Methods. The figures of crystal structures were created using MOE version 2014.09 (Chemical Computing Group, Inc.) with the exception of Figures 1 and 9, which were created using PyMol version 1.5.0.5 (Schrodinger, LLC). The Proasis water scores were computed using Proasis version 3 (Desert Scientific Software). The model of 3 bound to CECR2 was created in MOE, starting from a crystal structure of CECR2 with ethylene glycol bound in the Kac-binding pocket (PDB: 3NXB). Chain A of this structure was superimposed to the structure of 4 in


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BRD9, and 3 was manually built starting from the aligned small molecule 4. Ethylene glycol and water were removed from the CECR2 pocket, resulting in a complex of 3 and the bromodomain of CECR2. This complex was minimized using the MMFFs force field with default minimization parameters but with protein residues other than those contacting 3 fixed. Residues in the pocket were tethered with default restraints, and no restraints were used on 3 during minimization. Computation of water scores The waters scores were calculated using the software Proasis3 v.3.342 (Desert Scientific Software, Pty, Ltd.). Each co-crystal structure reported in this manuscript was used as input, and the water scores were computed using default parameters, and the output scores were included in a PyMol file that could be readily visualized by the program PyMol v.1.5.0.5 (Schrodinger, Inc.). General procedure for 4-Bromo-1-(p-tolylsulfonyl)-6H-pyrrolo[2,3-c]pyridin-7-one (16): 2-Methoxy-4-methyl-3-nitropyridine (11). A solution of 2-chloro-4-methyl-3-nitropyridine 10 (commercially available, 250 g, 1.45 mol) in methanol (1.0 L) was added dropwise (2 h) to a stirred and cooled (0 °C) solution of sodium methoxide (250 g, 4.63 mol) in methanol (850 mL). After addition, the mixture was heated to reflux for 23 h, at which time thin layer chromatography (TLC) indicated the reaction had gone to completion. The mixture was concentrated under reduced pressure to a volume of approximately 900 mL, and quenched by addition of water (1.5 L). The resulting solid was collected by filtration, washed with water and dried under reduced pressure to give the title compound (250 g, 100% yield) as a brown solid. 1H NMR (400 MHz, DMSO-d6): δ 8.22 (d, J = 5.2 Hz, 1 H), 7.10 (d, J = 5.6 Hz, 1 H), 3.92 (s, 3 H), 2.26 (s, 3 H).



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Page 28 of 40

5-Bromo-2-methoxy-4-methyl-3-nitropyridine (12). Sodium acetate (365 g, 5.37 mol) was added to a stirred solution of 2-methoxy-4-methyl-3-nitropyridine (250 g, 1.49 mol) in acetic acid (1.5 L) at ambient temperature, and then Br2 (639 g, 4.00 mol) was added drop-wise (30 min). After addition, the mixture was heated at 80 °C for 12 h, at which time TLC indicated the reaction had gone to completion. The mixture was cooled (0 °C) and quenched by sequential addition of 10% aqueous (1.5 L) and saturated aqueous sodium sulfate (1.5 L). The resulting solid was collected by filtration, washed with water, and dried under reduced pressure to give the title compound (302 g, 82% yield) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 8.25 (s, 1 H), 3.94 (s, 3 H), 2.29 (s, 3 H). (E)-2-(5-Bromo-2-methoxy-3-nitro-4-pyridyl)-N,N-dimethyl-ethenamine

(13).

N,N-

Dimethylformamide dimethyl acetal (600 mL) was slowly added to a stirred and heated (80 °C) solution of 5-bromo-2-methoxy-4-methyl-3-nitropyridine (134 g, 0.54 mol) in DMF (1.1 L). After addition, the mixture was heated at 95 °C for 5 h. The mixture was cooled to room temperature and poured into ice-cold water (3 L). The resulting red solid was collected by filtration, washed with water, and dried under reduced pressure to give the title compound (167 g, 100% yield) as red solid. 1H NMR (400 MHz, DMSO-d6): δ 8.24 (s, 1 H), 7.05 (d, J = 13.6 Hz, 1 H), 7.05 (d, J = 13.6 Hz, 1 H), 4.80 (d, J = 13.2 Hz, 1 H), 3.88 (s, 3 H), 2.90 (s, 6 H). 4-Bromo-7-methoxy-1H-pyrrolo[2,3-c]pyridine (14). A mixture of 2-(5-bromo-2-methoxy3-nitropyridin-4-yl)-N,N-dimethylethenamine (50.0 g, 165 mmol), Fe (50.0 g, 893 mmol) and NH4Cl (50.0 g, 943 mmol) in methanol/H2O (1900/250 mL) was heated at reflux for 7 h, at which time LC/MS analysis indicated that the reaction had gone to completion. The mixture was filtered while hot and the solid was washed with methanol (3 x 200 mL). The combined filtrates were concentrated under reduced pressure, and the resulting residue was purified by silica gel


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chromatography (petroleum ether : ethyl acetate=5:1) to yield the crude product. This crude material was triturated with acetonitrile to give the title compound (37.4 g, 99% yield) as a light brown solid. LC/MS M/Z (M+H) 226.7, 228.7. 4-Bromo-7-methoxy-1-(p-tolylsulfonyl)pyrrolo[2,3-c]pyridine (15). A solution of 4-bromo7-methoxy-1H-pyrrolo[2,3-c]pyridine (34.3 g, 0.15 mol) in THF (700 mL) was added dropwise to a stirred and cooled (0 °C) solution of sodium hydride (60%, 19.2 g, 0.48 mol) in THF (700 mL). After addition, the mixture was stirred at room temperature for 1 h, and then cooled again to 0 °C. Tosyl chloride (38.0 g, 0.20 mol) in THF (700 mL) was added drop-wise and the resulting mixture was stirred at ambient temperature for 2 h. The reaction was quenched by addition of saturated aqueous ammonium chloride (1.0 L), and then extracted with ethyl acetate (3 x 600 mL). The combined organic extracts were dried over sodium sulfate and concentrated under reduced pressure. The residue was triturated with acetonitrile to give the title compound (51.2 g, 89% yield) as a brown solid. This crude material was used in the next step without further purification. 4-Bromo-1-(p-tolylsulfonyl)-6H-pyrrolo[2,3-c]pyridin-7-one (16). HBr (40% aqueous, 1.1 L) was added to a solution of 4-bromo-7-methoxy-1-(p-tolylsulfonyl)pyrrolo[2,3-c]pyridine (102.5 g, 0.27 mol) in ethanol (200 mL). After addition, the mixture was heated at 90 °C for 2h, at which time TLC indicated that the reaction had gone to completion. The mixture was cooled to 0 °C and the resulting white solid was collected by filtration. This solid was washed with water and dried under vacuum to give the title compound (87.5 g, 89% yield) as a light brown solid. 1H NMR (400 MHz, DMSO-d6): δ 11.48 (s, 1 H), 8.01 (d, J = 3.6 Hz, 1 H), 8.90 (d, J = 8.0 Hz, 2 H), 7.38 (d, J = 8.0 Hz, 2 H), 7.32 (s, 1 H), 6.57 (d, J = 3.2 Hz, 1 H), 2.34 (s, 3 H).



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Page 30 of 40

6-methyl-4-bromo-1-(p-tolylsulfonyl)pyrrolo[2,3-c]pyridin-7-one (17a). To a cooled (0 °C) solution of 4-bromo-1-(p-tolylsulfonyl)-6H-pyrrolo[2,3-c]pyridin-7-one (7.0 g, 19.1 mmol) in DMF (70 mL) was added sodium hydride (60% in mineral oil, 953 mg, 23.8 mmol). The mixture was stirred for 15 min and then methyl iodide (5.4 g, 38.1 mmol) was added. The reaction mixture was allowed to warm to room temperature and stirring was continued for 2 h. The reaction was quenched with water (20 mL) and then extracted with ethyl acetate (3 x 20 mL). The combined organic extracts were washed with brine (2 x 20 mL), dried over sodium sulfate and concentrated under reduced pressure to yield the title compound (6.9 g, 95% yield) as a yellow solid. This crude material was used in the next step without further purification. LCMS M/Z (M+H) 381, 383. 3-(6-Methyl-7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl)-N,N-dimethyl-benzamide

(2).

To

a

mixture of 6-methyl-4-bromo-1-(p-tolylsulfonyl)pyrrolo[2,3-c]pyridin-7-one (17a, 50 mg, 0.13 mmol) and [3-(dimethylcarbamoyl)phenyl]boronic acid (50 mg, 0.26 mmol) in 1,4-dioxane (0.5mL) and 2 M potassium phosphate tribasic in water (0.2 mL, 0.40 mmol) was added bis(ditert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) (5 mg, 0.007 mmol). The reaction mixture was shaken at 80 °C overnight. The mixture was filtered through a pad of Celite, using ethyl acetate (3 mL) to rinse. The filtrate was concentrated under reduced pressure to yield the title compound, and the crude was carried on without purification. The crude product was taken up in methanol (1 mL) and potassium hydroxide (43 mg, 0.66 mmol) was added. The reaction was stirred at 50 °C for 1h. After cooling, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in water and extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (25-


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35%ACN/0.1%NH4OH in H2O) to give 3-(6-Methyl-7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl)-N,Ndimethyl-benzamide.

13

C NMR (101 MHz, DMSO) δ 170.44, 154.55, 137.65, 137.61, 129.26,

128.80, 128.64, 128.53, 127.65, 125.94, 125.66, 123.84, 114.38, 102.41, 35.99, 35.19.1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 7.67 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.57 (t, J = 1.5 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.44 (s, 1H), 7.39 – 7.31 (m, 2H), 6.43 (d, J = 2.8 Hz, 1H), 3.59 (s, 3H), 3.02 – 2.95 (m, 6H). LCMS M/Z (M+H) 296. 3-(6-Allyl-7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl)-N,N-dimethyl-benzamide

(3).

To

a

mixture of 6-allyl-4-bromo-1-(p-tolylsulfonyl)pyrrolo[2,3-c]pyridin-7-one (17d, prepared in the same manner as 17a, using allyl bromide as alkylating agent, 350 mg, 0.86 mmol) and [3(dimethylcarbamoyl)phenyl]boronic acid (220 mg, 1.1 mmol) in acetonitrile (2 mL) and 1 M potassium

carbonate

in

water

(2

mL)

was

added

[1,1'-

bis(diphenylphosphino)ferrocene]palladium(ii) dichloride (63 mg, 0.086 mmol). The reaction mixture was subjected to microwave irradiation at 100 °C for 10 min. The mixture was filtered through a pad of Celite, using ethyl acetate (30 mL) to rinse. The filtrate was washed with brine (10 mL), dried over sodium sulfate and concentrated under reduced pressure to yield the title compound (300 mg, 70% yield) as a yellow solid. This crude material was used in the next step without further purification. LC/MS M/Z (M+H) 476.4. A mixture of 3-[6-allyl-7-oxo-1-(p-tolylsulfonyl)pyrrolo[2,3-c]pyridin-4-yl]-N,N-dimethylbenzamide (Step 2, 35 mg, 0.074 mmol) in methanol (1 mL) and 10 M potassium hydroxide in water (0.8 mL) was stirred at 50 °C for 1h. After cooling, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in water (5 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (25-



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35%ACN/0.1%NH4OH in H2O) to give the title compound (13 mg, 65% yield) as a white solid. 13

C NMR (101 MHz, DMSO) δ 170.41, 153.90, 137.64, 137.59, 134.80, 129.26, 128.62, 128.56,

127.86, 127.58, 125.96, 125.72, 123.75, 117.38, 114.83, 102.52, 49.51, 35.20. 1H NMR (400 MHz, DMSO-d6) δ 7.66 (dt, J = 7.7, 1.4 Hz, 1H), 7.58 – 7.49 (m, 2H), 7.41 – 7.31 (m, 3H), 6.44 (d, J = 2.7 Hz, 1H), 6.02 (ddt, J = 17.2, 10.7, 5.5 Hz, 1H), 5.21 – 5.08 (m, 2H), 4.68 (dt, J = 5.5, 1.6 Hz, 2H), 2.99 (d, J = 11.6 Hz, 6H). LCMS M/Z (M+H) 322. 3-(6-But-2-enyl-7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl)-N,N-dimethyl-benzamide

(4).

Prepared in the same manner as 3 starting from 4-bromo-1-(p-tolylsulfonyl)-6H-pyrrolo[2,3c]pyridin-7-one (16) using crotyl bromide as alkylating agent.

13

C NMR (101 MHz, DMSO) δ

170.42, 153.88, 137.63, 129.26, 128.84, 128.56, 127.79, 127.51, 127.44, 125.96, 125.69, 123.81, 114.77, 102.46, 48.92, 35.20, 17.90. 1H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 7.70 – 7.63 (m, 1H), 7.58 – 7.46 (m, 2H), 7.40 – 7.31 (m, 3H), 6.45 – 6.39 (m, 1H), 5.67 (d, J = 4.8 Hz, 1H), 4.64 – 4.56 (m, 2H), 2.99 (d, J = 10.1 Hz, 7H), 1.68 – 1.61 (m, 3H). LCMS M/Z (M+H) 336. 3-(6-But-3-enyl-7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl)-N,N-dimethyl-benzamide

(5).

Prepared in the same manner as 3 starting from 4-bromo-1-(p-tolylsulfonyl)-6H-pyrrolo[2,3c]pyridin-7-one (16) using 4-bromobut-1-ene as alkylating agent. 13C NMR (101 MHz, DMSO) δ 170.44, 154.08, 137.71, 137.61, 135.70, 129.22, 128.58, 128.53, 128.01, 127.72, 125.98, 125.63, 123.85, 117.50, 114.40, 102.40, 47.12, 35.20, 34.08.1H NMR (400 MHz, DMSO-d6 ) δ 12.14 (s, 1H), 7.67 (dt, J = 7.8, 1.4 Hz, 1H), 7.57 (t, J = 1.7 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.42 (s, 1H), 7.38 – 7.32 (m, 2H), 6.43 (d, J = 2.8 Hz, 1H), 5.99 – 5.75 (m, 1H), 5.17 – 4.94 (m, 2H), 4.16 – 4.08 (m, 2H), 2.99 (d, J = 13.3 Hz, 6H). LCMS M/Z (M+H) 336. 3-(6-Ethyl-7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl)-N,N-dimethyl-benzamide (6).

Prepared

in the same manner as 3 starting from 4-bromo-1-(p-tolylsulfonyl)-6H-pyrrolo[2,3-c]pyridin-7-


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one (16) using ethyl bromide as alkylating agent. 1H NMR (400 MHz, DMSO-d6 ) δ 12.11 (s, 1H), 7.68 (dt, J = 7.8, 1.5 Hz, 1H), 7.58 (t, J = 1.7 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.42 (s, 1H), 7.35 (dd, J = 8.3, 1.9 Hz, 2H), 6.42 (d, J = 2.8 Hz, 1H), 4.08 (q, J = 7.1 Hz, 2H), 2.99 (d, J = 7.7 Hz, 6H), 1.29 (t, J = 7.1 Hz, 3H). LCMS M/Z (M+H) 310. 3-(6-Propyl-7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl)-N,N-dimethyl-benzamide (7). Prepared in the same manner as 3 starting from 4-bromo-1-(p-tolylsulfonyl)-6H-pyrrolo[2,3-c]pyridin-7one (16) using 1-bromopropane as alkylating agent. 1H NMR (400 MHz, DMSO-d6 ) δ 7.68 (dt, J = 7.9, 1.4 Hz, 1H), 7.60 – 7.48 (m, 2H), 7.42 (s, 1H), 7.40– 7.29 (m, 2H), 6.43 (t, J = 2.3 Hz, 1H), 4.00 (dd, J = 8.0, 6.6 Hz, 2H), 2.99 (d, J = 13.3 Hz, 6H), 1.84 – 1.62 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H). LCMS M/Z (M+H) 324. 3-(6-Butyl-7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl)-N,N-dimethyl-benzamide (8). Prepared in the same manner as 3 starting from 4-bromo-1-(p-tolylsulfonyl)-6H-pyrrolo[2,3-c]pyridin-7-one (16) using 1-bromobutane as alkylating agent. 1H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 7.67 (dt, J = 7.8, 1.4 Hz, 1H), 7.57 (t, J = 1.7 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.41 (s, 1H), 7.35 (dt, J = 7.7, 1.6 Hz, 2H), 6.43 (d, J = 2.8 Hz, 1H), 4.04 (t, J = 7.3 Hz, 2H), 3.06 – 2.89 (m, J = 11.1 Hz, 6H), 1.83 – 1.62 (m, 2H), 1.33 (h, J = 7.4 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H). LCMS M/Z (M+H) 338. 3-[6-(2-Methoxyethyl)-7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl]-N,N-dimethyl-benzamide (9). Prepared in the same manner as 3 starting from 4-bromo-1-(p-tolylsulfonyl)-6H-pyrrolo[2,3c]pyridin-7-one (16) using 1-bromo-2-methoxyethane as alkylating agent. 1H NMR (400 MHz, DMSO-d6 ) ) δ 12.17 (s, 1H), 7.70 – 7.62 (m, 1H), 7.60 – 7.49 (m, 2H), 7.43 – 7.31 (m, 3H), 6.44 (d, J = 2.8 Hz, 1H), 4.22 (t, J = 5.6 Hz, 2H), 3.64 (t, J = 5.5 Hz, 2H), 3.26 (s, 3H), 2.99 (d, J = 13.1 Hz, 6H). LCMS M/Z (M+H) 340.



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Supporting Information Protein expression and purification; crystallography methods; Expanded biochemical assay data with standard deviations; Biotinylated probe preparation; Molecular modeling coordinates for model of compound 3 bound to CECR2.

Acknowledgements The authors thank Mengling Wong, Michael Hayes, and Amber Guillen for compound purification. Baiwei Lin, Yanzhou Liu, Deven Wang, and Yutao Jiang are acknowledged for analytical support. Phil Bergeron and Tom Pillow for assistance during manuscript preparation. Grady Howes, Jan Seerveld, Hao Zheng, Ted Peters, Gigi Yuen, and Jeff Blaney for help with compound management and logistics are also recognized. Ancillary Information: PDB ID Codes: 5I80 (BRD4-2), 5I88 (BRD4-4), 5I7X (BRD9-2), 5I7Y (BRD9-4), 5I40 (BRD9- 1), 5I29 (TAF1(2)-2), and 5I1Q (TAF1(2)- 5). 3NXB (CECR2). Authors will release the atomic coordinates an experimental data upon article publication. Author Information: Corresponding Author: *Tel.: 650-467-2210. Email: [email protected]

Abbreviations BAZ2A: bromodomain adjacent to zinc finger domain; BAZ2B: bromodomain adjacent to zinc finger domain; BET: bromodomain and extra terminal domain; BRD2: Bromodomain-containing


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protein 2; BRD3: Bromodomain-containing protein 3; BRD4(1): The first bromodomain of bromodomain-containing protein 4; BRD4(2): The second bromodomain of bromodomaincontaining protein 4; BRD9: Bromodomain containing protein 9; BRDT: Bromodomain testisspecific protein; BRPF1: Bromodomain and PHD finger containing, 1; CBP: CREB binding protein bromodomain; CECR2: Cat eye syndrome critical region protein 2; EP300: E1A Binding Protein P300; PBRM1(2): protein polybromo-1 bromodomain 2; TAF1(1): The first bromodomain of human transcription initiation factor TFIID subunit 1; TAF1(2): The second bromodomain of human transcription initiation factor TFIID subunit 2; TRIM24: tripartite motif 24 protein; References

1) Haynes, S. R.; Dollar, C.; Winston, F.; Beck, S.; Trowsdale, J.; Dawid, I.B. The bromodomain: a conserved sequence found in human, Drosophila and yeast proteins. Nucleic Acids Res. 1992, 20, 2603. 2) Filippakopoulos, P.;Picaud, S.; Mangos, M.; Keates, T.; Lambert, J.-P., Barsyte-Lovejoy, D.; Felletar, I.; Volkmer, R.; Müller, S.; Pawson, T.; Gingras, A.-C.; Arrowsmith, C. H.; Knapp, S. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 2012, 149, 214−231. 3) Gershey, E. L.; Vidali, G.; Allfrey, V. G. Chemical studies of histone acetylation. J. Biol. Chem. 1968, 243, 5018−5022. 4) Dhalluin, C.; Carlson, J. E.; Zeng, L.; He, C;, Aggarwal, A. K.; Zhou, M.-M. Structure and ligand of a histone acetyltransferase bromodomain. Nature 1999, 399, 491−496. 5) Winston, F.; Allis, C. D. The bromodomain: a chromatin-targeting module? Nat. Struct. Biol. 1999, 6, 601−604.



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6) Choudhary, C.; Kumar, C.; Gnad, F.; Nielsen, M. L.; Rehman, M.; Walther, T. C.; Olsen, J. V.; Mann, M. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 2009, 325, 834−840. 7) Sanchez, R.; Zhou, M. M. The role of human bromodomains in chromatin biology and gene transcription. Curr. Opin. Drug Discovery Dev. 2009, 12, 659−665. 8) For recent reviews on biological relevance of BET bromodomains as well as BET inhibitors see: a) Hewings, D. S.; Rooney, T. P. C.; Jennings, L. E.; Hay, D. A.; Schofield, C. J.; Brennan, P. E.; Knapp, S.; Conway, S. J. Progress in the development and application of small molecule inhibitors of bromodomain-acetyl-lysine interactions. J. Med. Chem. 2012, 55, 9393−9413. B) Müller, S.; Lingard, H.; Knapp, S. Selective targeting of protein interactions mediated by BET bromodomains. Concepts Case Stud. Chem. Biol. 2014, 255-307. C) Filippakopoulos, P.; Knapp, S. Targeting bromodomains: epigenetic readers of lysine acetylation. Nat. Rev. Drug Discovery. 2014, 13, 337-356 D) Garnier, J. M.; Sharp, P. P.; Burns, C. J. BET bromodomain inhibitors: a patent review. Expert Opin. Ther. Pat. 2014, 24, 185−199. E) Brand, M.; Measures, A. M.; Wilson, B.G.; Cortopassi, W.A.; Alexander, R.; Höss, M.; Hewings, D.; Rooney, T.; Paton, R.; Conway, S. Small molecule inhibitors of bromodomain-acetyl-lysine interactions. ACS Chem. Biol. 2015, 10, 22−39. F) Romero, F. A.; Taylor, A. M.; Crawford, T. D.; Tsui, V.; Côté, A.; Magnuson S. Disrupting acetyl-lysine recognition: Progress in the development

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A.; Yapp, C.; Filippakopoulos, P.; Bunnage, M. E.; Müller, S.; Knapp, S.; Schofield, C. J.; Brennan, P. E. Discovery and optimization of small-molecule ligands for the CBP/ P300 bromodomains. J. Am. Chem. Soc. 2014, 136, 9308−9319. 10) Xu, M.; Unzue, A.; Dong, J.; Spiliotopoulos, D.; Nevado, C.; Caflisch, A. Discovery of CREBBP bromodomain inhibitors by high throughput docking and hit optimization guided

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10.1021/acs.jmedchem.5b00171. 11) Piatnitski Chekler, E.L.; Pellgrino, J.A.; Lanz, T.A.; Denny, R.A.; Flick, A.C.; Coe, J.; Langille, J.; Basak, A.; Liu, S.; Stock, I.A.; Sahasrabudhe, P.; Bonin, P.D.; Lee, K.; Pletcher, M.T.; Jones, L.H. Transcriptional profiling of a selective CREB binding protein bromodomain inhibitor highlights therapeutic opportunities. Chem. Biol. 2015, 22, 15881596. 12) Drouin, L.; McGrath, S.; Vidler, L. R.; Chaikuad, A.; Monteiro, O.; Tallant, C.; Philpott, M.; Rogers, C.; Fedorov, O.; Liu, M.; Akhtar, W.; Hayes, A.; Raynaud, F.; Müller, S.; Knapp, S.; Hoelder, S. Structure enabled design of BAZ2-ICR, a chemical probe targeting the bromodomains of BAZ2A and BAZ2B. J. Med. Chem. 2015, 58, 2553−2559. 13) Chen, P.; Chaikuad, A.; Bamborough, P.; Bantscheff, M.; Bountra, C.; Chung, C.-W.; Fedorov, O.; Grandi, P.; Jung, D.; Lesniak, R.; Lindon, M.; Müller, S.; Philpott, M.; Prinjha, R.; Rogers, C.; Selenski, C.; Tallant, C.; Werner, T.; Willson, T. M.; Knapp, S.; Drewry, D. H. Discovery and characterization of GSK2801, a selective chemical probe for the bromodomains BAZ2A and

BAZ2B. J. Med. Chem.

10.1021/acs.jmedchem.5b00209.


2015, DOI:


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14) Clark, P. G. K.; Vieira, L. C. C.; Tallant, C.; Fedorov, O.; Singleton, D. C.; Rogers, C. M.; Monteiro, O. P.; Bennett, J. M.; Baronio, R.; Müller, S.; Daniels, D. L.; Mendez, J.; Knapp, S.; Brennan, P.; Dixon, D. J. LP99: Discovery and synthesis of the first selective BRD7/9 bromodomain inhibitor. Angew. Chem., Int. Ed. 2015, 54, 6217−6221. 15) Hay, D. A.; Rogers, C. M.; Fedorov, O.; Tallant, C.; Martin, S.; Monteiro, O. P.; Müller, S.; Knapp, S.; Schofield, C. J.; Brennan, P. E. Design and synthesis of potent and selective inhibitors of BRD7 and BRD9 bromodomains. MedChemComm 2015, 6, 1381−1386. 16) Theodoulou, N. H.; Bamborough, P.; Bannister, A. J.; Becher, I.; Bit, R. A.; Che, K. H.; Chung, C.-W.; Dittmann, A.; Drewes, G.; Drewry, D. H.; Gordon, L.; Grandi, P.; Leveridge, M.; Lindon, M.; Michon, A.- M.; Molnar, J.; Robson, S. C.; Tomkinson, N. C. O.; Kouzarides, T.; Prinjha, R. K.; Humphreys, P. G. Discovery of I-BRD9, a selective cell active chemical probe for bromodomain containing protein 9 inhibition. J. Med. Chem. 2015, DOI: 10.1021/acs.jmedchem.5b00256. 17) Bamborough, P.; Chung, C.-W.; Furze, R. C.; Grandi, P.; Michon, A.-M.; Sheppard, R. J.; Barnett, H.; Diallo, H.; Dixon, D. P.; Douault, C.; Jones, E. J.; Karamshi, B.; Mitchell, D. J.; Prinjha, R. K.; Rau, C.; Watson, R. J.; Werner, T.; Demont, E. H. Structure-based optimization of naphthyridones into potent ATAD2 bromodomain inhibitors. J. Med. Chem. 2015, 58, 6151−6178. 18) Demont, E. H.; Bamborough, P.; Chung, C.-W.; Craggs, P. D.; Fallon, D.; Gordon, L. J.; Grandi, P.; Hobbs, C. I.; Hussain, J.; Jones, E. J.; Le Gall, A.; Michon, A.-M.; Mitchell, D. J.; Prinjha, R. K.; Roberts, A. D.; Sheppard, R. J.; Watson, R. J. 1,3-Dimethyl


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benzimidazolones are potent, selective inhibitors of the BRPF1 bromodomain. ACS Med. Chem. Lett. 2014, 5, 1190−1195. 19) Bennett, J.; Fedorov, O.; Tallant, C.; Monteiro, O.; Meier, J.; Gamble, V.; Savitsky, P.; Nunez-Alonso, G.A.; Haendler, B.; Rogers, C.; Brennan, P.E.; Müller, S.; Knapp, S. Discovery of a chemical tool inhibitor targeting the bromodomains of TRIM24 and BRPF. J. Med. Chem. 2015, DOI: 10.1021/acs.jmedchem.5b00458 20) Dhalluin, C.; Carlson, J. E.; Zeng, L.; He, C.; Aggarwal, A. K.; Zhou, M.-M. Structure and ligand of a histone acetyltransferase bromodomain. Nature 1999, 399, 491-496. 21) Jeanmougin, F.; Wurtz, J.-M.; Le Douarin, B.; Chambon, P.; Losson, R. The bromodomain revisited. Trends Biochem. Sci. 1997, 22, 151-153. 22) For consistency, we will use the term “lipophilic shelf” when referring to the BC loop region in all bromodomains that correlates to the WPF residues in BRD4(1&2) (W81, P82, F83 in BRD4(1)), as the actual residues will vary between family members CBP (L1109, P1110, F1111), BRPF1 (N651, I652, F653); TAF1(BD2) (Y1526, P1527, F1528) and BRD9 (G43 ,F44, F45). 23) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a useful metric for lead selection. Drug Discovery Today 2004, 9, 430− 431. 24) AbbVie also identified the pyrrolopyridone scaffold, with their BET inhibitors patented concurrent to our work. a) Wang, L.; Pratt, J. K.; McDaniel, K.F. Bromodomain inhibitors. WO2013097052(A1), 2013. b) Wang, L.; Pratt, J. K.; McDaniel, K. F.; Dai, Y.; Fidanze, S. D.; Hasvold, L.; Holms, J. H.; Kati, W. M.; Liu, D.; Mantei, R. A.; McClellan, W. J.; Sheppard, G. S.; Wada, C. K.; Bromodomain inhibitors WO2013097601 (A1), 2013. c) Incyte also recently patented BET inhibitors using the



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pyrrolopyridone scaffold. Combs, A. P.; Maduskuie, T. P. Jr.; Falahatpisheh, N. 1H[2,3c]pyridine-7-(6H)-ones and pyrazolo[3,4-c]pyridine-7(6H)-ones as inhibitors of BET proteins. WO2015164480 (A1), 2015. 25) Flynn, M.; Huang, O.; Poy, F.; Oppikofer, M.; Bellon, S.; Tang, Y.; Cochran, A.G. A subset

of

human

bromodomains

recognizes

butyryllysine

and

crotonyllysine

histonepeptide modifications. Structure 2015, 23, 1801-1814 26) Vidler, L.; Brown, N.; Knapp, S.; Hoelder, S. Druggability analysis and structural classification of bromodomain acetyl-lysine binding sites. J. Med. Chem. 2012, 55, 73467359. 27) Amadasi, A.; Surface, A.; Spyrakis, F.; Cozzini, P.; Mozzarelli, A.; Kellogg, G.E. Robust classification of “relevant” water molecules in putative protein binding sites. J. Med. Chem. 2008, 51, 1063-1067. 28) Vollmuth, F.; Geyer, M. Interaction of propionylated and butyrylated Histone H3 lysine marks with Brd4 bromodomains. Angew. Chem. Int. Ed. 2010, 49, 6768 –6772 29) Huang, D.; Rossini, E.; Steiner, S.; Caflisch, A. Structured water molecules in the binding site of bromodomains can be displaced by cosolvent. ChemMedChem 2014, 9, 573 – 579 30) Pantoliano, M. W.; Petrella, E. C.; Kwasnoski, J. D.; Lobanov, V. S.; Myslik, J.; Graf, E.; Carver, T.; Asel, E.; Springer, B. A.; Lane, P.; Salemme, F. R. High-density miniaturized thermal shift assays as a general strategy for drug discovery. J. Biomol. Screen. 2001, 6, 429–440. 31) Batcho, A. D.; Leimgruber, W. Indoles from 2-Methylnitrobenzenes by condensation with formamide acetals followed by reduction. Org. Synth. 1985, 63, 214–220


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32) Albrecht, B. K.; Gehling, V. S.; Hewitt, M. C.; Vaswani, R. G.; Côté, A.; Leblanc, Y.; Nasveschuk, C. G.; Bellon, S.; Bergeron, L.; Campbell, R.; Cantone, N.; Cooper, M. R.; Cummings, R. T.; Jayaram, H.; Joshi, S.; Mertz, J. A.; Neis, A.; Normant, E.; O'Meara, M.; Pardo, E.; Poy, F.; Sandy, P.; Supko, J.; Sims, R. J. 3rd; Harmange, J. C.;Taylor, A. M.; Audia, J. E. Identification of a benzoisoxazoloazepine inhibitor (CPI-0610) of the bromodomain and extra-terminal (BET) family as a candidate for human clinical trials. J Med Chem. 2016, 59, 1330–1339. 33) BROMOscan bromoKdMAX recombinant protein binding assays performed at DiscoveRx. https://www.discoverx.com

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Diving into the Water: Inducible Binding Conformations for BRD4

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