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Discovery of Orally Active Inhibitors of Brahma Homolog (BRM)/ SWI/SNF Related Matrix Associated Actin Dependent Regulator Of Chromatin Subfamily A Member 2 (SMARCA2) ATPase Activity for the Treatment of Brahma Related Gene 1 (BRG1)/ SMARCA4-Mutant Cancers Julien P.N. Papillon, Katsumasa Nakajima, Christopher D Adair, Jonathan Hempel, Andriana Olga Jouk, Rajeshri Karki, Simon Mathieu, Henrik Moebitz, Rukundo Ntaganda, Troy Smith, Michael Visser, Susan E. Hill, Felipe Kellermann Hurtado, Gregg Chenail, Hyo-Eun C Bhang, Anka Bric, Kay Xiang, Geoffrey Bushold, Tamara Gilbert, Anthony Vattay, Julia Dooley, Emily A Costa, Isabel Park, Ailing Li, David Farley, Eugen Lounkine, Q. Kimberley Yue, Xiaoling Xie, Xiaoping Zhu, Raviraj Kulathila, Daniel King, Tiancen Hu, Katarina Vulic, John Cantwell, Catherine Luu, and Zainab Jagani J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01318 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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Journal of Medicinal Chemistry

King, Daniel; Novartis Institutes for Biomedical Research Hu, Tiancen; Novartis Institutes for Biomed. Research Vulic, Katarina; Novartis Cantwell, John; Novartis Institutes for BioMedical Research Emeryville, Protein Sciences Luu, Catherine; Novartis Institutes for BioMedical Research Emeryville Jagani, Zainab; Novartis Institutes for BioMedical Research Inc

ACS Paragon Plus Environment

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

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Discovery of Orally Active Inhibitors of Brahma Homolog (BRM)/ SWI/SNF Related Matrix Associated Actin Dependent Regulator Of Chromatin Subfamily A Member 2 (SMARCA2) ATPase Activity for the Treatment of Brahma Related Gene 1 (BRG1)/ SMARCA4-Mutant Cancers Julien P. N. Papillon,◊,* Katsumasa Nakajima,◊ Christopher D. Adair,◊,† Jonathan Hempel,◊ Andriana O. Jouk,◊,† Rajeshri G. Karki,◊ Simon Mathieu,◊,† Henrik Möbitz,^ Rukundo Ntaganda,◊ Troy Smith,◊ Michael Visser,◊ Susan E. Hill,∥ Felipe Kellermann Hurtado,∥ Gregg Chenail,‡ HyoEun C. Bhang,‡ Anka Bric,‡,† Kay Xiang,‡ Geoffrey Bushold,‡ Tamara Gilbert,‡ Anthony Vattay,‡ Julie Dooley,‡ Emily A. Costa,‡,† Isabel Park,‡ Ailing Li,‡ David Farley,§ Eugen Lounkine, § Q. Kimberley Yue,§ Xiaoling Xie,§ Xiaoping Zhu, § Raviraj Kulathila,§ Daniel King,§ Tiancen Hu,§ Katarina Vulic,⊥ John Cantwell,# Catherine Luu# and Zainab Jagani‡


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◊Global

Discovery Chemistry, ‡Oncology Disease Area, §Chemical Biology and Therapeutics,

∥Pharmacokinetics

Sciences, Novartis Institutes for Biomedical Research, and ⊥Chemical and

Pharmaceutical Profiling, Novartis Pharmaceuticals, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States. ^Global Discovery Chemistry, Novartis Institutes for Biomedical Research, 4002 Basel, Switzerland. #Novartis

Institutes for Biomedical Research, 5300 Chiron Way, Emeryville, California 94608,

United States

ABSTRACT

SMARCA2 (SWI/SNF Related Matrix Associated Actin Dependent Regulator Of Chromatin Subfamily A Member 2), also known as BRM (Brahma homolog) is a Snf2-family DNAdependent ATPase. BRM, and its close homolog Brahma related gene 1 (BRG1), also known as SMARCA4, are mutually exclusive ATPase members of the large ATP-dependent SWI/SNF chromatin remodeling complexes involved in transcriptional regulation of gene expression. No small molecules modulating SWI/SNF chromatin remodeling activity via inhibition of its ATPase activity have been reported, an important goal given the well-established dependence of BRG1deficient cancers on BRM. Here, we describe allosteric dual BRM and BRG1 inhibitors that downregulate BRM-dependent gene expression and show anti-proliferative activity in a BRG1mutant lung tumor xenograft model upon oral administration. These compounds represent useful tools for understanding the functions of BRM in BRG1 loss-of-function settings, and should enable probing the role of SWI/SNF function more broadly in different cancer contexts and other diseases.


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INTRODUCTION Aberrant regulation of transcriptional programs through defects in chromatin remodeling activity has emerged as an important feature of many different cancers. Notably, tumor exome sequencing studies have revealed the importance of the multi-subunit SWI/SNF chromatin remodeling complexes in cancer, with mutations in its various subunits detected at a collective frequency of approximately 20% across various cancers. 1-3 Mutations in BRG1/SMARCA4, one of the catalytic ATPase subunits of SWI/SNF complexes, occur most prominently in Non-Small Cell Lung Cancer (NSCLC) with a 7-10% frequency, but also in melanoma, liver and pancreatic cancers among others.4 RNAi-based genetic screening has revealed a synthetic lethal relationship between loss of BRG1/SMARCA4 and BRM/SMARCA2, the other mutually exclusive ATPase of the complex.5, 6 In particular, cancer cells deficient in BRG1, such as through homozygous loss of function mutations, are exquisitely dependent on BRM for their growth. Such findings have driven great interest in targeting BRM, or the SWI/SNF complexes through small molecule inhibition. Cancer dependence on SWI/SNF activity appears to arise in other settings as well, such as in acute myeloid leukemias (AML), where dependence on BRG1 has been reported.7, 8 BRM and BRG1 are multi-domain proteins that share high protein sequence homology (76% identity). Both proteins contain a DNA-stimulated conserved ATPase domain (92% identity) which imparts a core catalytic function in driving chromatin remodeling activity, 9-13 as well as bromodomains which engage with acetylated histones on nucleosomes.14, 15 While inhibitors targeting bromodomains of both BRM and BRG1 have been described,16 genetic studies have indicated that the ATPase but not the bromodomain is essential for cancer dependence.17 We therefore set out to develop small molecule inhibitors of BRM ATPase activity. BRM and BRG1 belong to the SF2 family of helicases, although, like other proteins within the Snf2 sub-family,


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they do not possess duplex unwinding activity, but instead remodel chromatin via an ATPdependent translocation mechanism.13 Much research has been conducted targeting SF2 ATPases, starting with viral NS3 targets in the 1990’s, but few validated inhibitors have been identified, with most screening hits found to bind to the nucleic acid substrate or interfere with assay read-outs.18-20 Robust counter-screening and biophysical hit validation are therefore essential, and several groups have recently demonstrated that SF2 ATPases could be successfully targeted.18 Here we describe for the first time the discovery, optimization and characterization of potent allosteric small molecule inhibitors of BRM and BRG1 ATPase activity (dual inhibitors) that recapitulate the genetic synthetic lethality in BRG1-deficient lung cancer models. RESULTS The dependence of BRG1-mutant cancer cells on BRM ATPase activity17 led us to focus on developing an ATP turnover assay to identify inhibitors of SWI/SNF function. Full length BRM is a 180 kDa protein, including a sizeable unstructured region at the N terminus. In order to facilitate establishing binding assays for hit validation, several truncated constructs were evaluated. Of those several constructs, a protein containing the ATPase and the SnAC domain (636-1331), from here on referred to as ATPase-SnAC, proved to be a suitable workhorse for screening and assay development. A focused screen of about 72,000 compounds was conducted against ATPase-SnAC using an ADP-Glo assay. The assay was run at an ATP concentration equal to the Km, and at a concentration of plasmid DNA equal to half the concentration determined to elicit maximal activation of ATPase activity. Compounds that showed inhibition were assayed against the closely related BRG1 ATPase-SnAC (658-1361), evaluated in a differential scanning fluorimetry (DSF) assay followed by assessment of binding in an SPR assay using Avi-tagged BRM ATPase-SnAC. This screening strategy led to the identification of


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1, with an IC50 = 3.7 µM (Table 1). Initial evidence of the compound binding to BRM was provided by DSF, with measured positive thermal shifts (Tm) of 1.6 °C against the apo protein and 1.5 °C in the presence of ADP. The inhibitory activity of this series was confirmed with an isomeric analog, compound 2, which gave an IC50 = 1.1 µM for BRM ATPase-SnAC, and IC50 = 8.1 µM for full length BRM. These compounds were not selective for BRM over BRG1, with BRG1 ATPase-SnAC IC50 = 0.77 µM for compound 2. Multiple lines of evidence of specific binding of 2 to the protein were obtained. In a DSF experiment using BRG1, 2 showed a positive Tm of 3.8 °C against the apo protein and 2.7 °C in the presence of ADP (Figure 1A). Additional hit validation was provided by SPR using BRM, which displayed dose-dependent saturable binding, with KD = 0.087 and 0.45 µM in the absence and presence of ADP, respectively (Figure 1B). Although SPR data suggest 2 is partially competitive with ADP, no IC50 shifts were observed when the BRM ATPase-SnAC assay was run at 10x ATP Km. A similar KD (0.28 µM) was obtained against BRG1 ATPase-SnAC by ITC, N=0.83, consistent with stoichiometric binding (Figure 1C). In view of this strong hit validation data package, combined with high ligand efficiency (LE = 0.35) and good aqueous solubility (0.33 and 3.1 mM at pH 6.8 and 1.0, respectively for 2), this scaffold was taken into an optimization program. Figure 1. Thermodynamic characterization of 2. (A) Determination of BRG1 melting temperatures (Tm) by DSF. (top) Melt curve overlay; (middle) Melt peak overlay; (bottom) Plot of Tm; Spectra obtained with BRG1 ATPase-SnAC (1 μM) only (red); BRG1 ATPase-SnAC (1 μM) + 2 (100 μM) (blue); BRG1 ATPase-SnAC (1 μM) + ADP (500 μM) (green); BRG1 ATPase-SnAC (1 μM) + 2 (100 μM) + ADP (500 μM) (orange). (B) SPR dose response obtained by injection of 2 over N-terminal Avi-tagged BRM ATPase-SnAC immobilized on SA chip with (top panel) and without (bottom panel) 500 µM ADP. Double referenced data (colored traces) were fit with 1:1


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binding model (black traces). (C) Isothermal calorimetry of 2 and BRG1 ATPase-SnAC. (top) Baseline corrected data obtained at 25 °C for injections of inhibitor 2 (200 μM) into the sample cell containing BRG1 ATPase-SnAC (18.7 μM); (middle) Calculated enthalpies after subtraction of the reference heat of dilution as determined by the later injections. Data were fit to a simple one binding site model to give a KD value of 0.28 μM; (bottom) Plot of G, H, -TS.

B

C

Response (RU)

A

Response (RU)

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


KD = 0.28 μM N = 0.8

Cl N

N

O N H

N H 2

We initially focused on generating matched pairs of 2 with single modifications in the di-pyridyl urea series. We found that the 2-halo-4-substituted pyridine motif was essential for inhibitory activity, although the chlorine could be replaced with bromine or fluorine. Likewise, no replacement for the urea could be identified, except for thiourea, which showed comparable potency. On the other hand, we found the SAR on the opposite pyridine to be more forgiving. 2Methylpyridine, as in 5 gave a 5-fold improvement in potency, with further gains in potency


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achieved with difluoromethyl (7) and trifluoromethyl (6) substitutions, with IC50 = 0.20, 0.11 and 0.06 µM, respectively (Table 1). Isosteric replacement of the pyridine with isothiazole afforded further potency gain, as shown with the 5/8, 6/9 and 7/10 matched pairs. All compounds similarly inhibited BRM and BRG1 ATPase-SnAC ATPase activity. Analysis of the Cambridge Structural Database (CSD) data for related sulfur-carbonyl motifs as shown in Figure 2 (218 compounds at the time this analysis was conducted) revealed an overwhelming preference for urea trans conformation, as assessed by querying the sulfur-oxygen distance. Indeed, any heteroaromatic replacements of the pyridine/isothiazole that might favor the cis urea conformation via intramolecular hydrogen bonding, or that might twist the ring out-of-plane due to unfavorable electrostatic interaction, were detrimental to potency. Compounds 3 and 4 are illustrative, the former with an IC50 = 0.97 µM, i.e. a 30-fold drop compared to 8, and the latter with an IC50 >50 µM. This combined data set was indicative of a preferred flat trans urea binding conformation, which, along with an absolute requirement for the urea was suggestive that both N-Hs were engaged in hydrogen bonding. This hypothesis led us to explore addition of a third hydrogen bond donor. Although the biochemical potency of the resulting compounds, such as 11 and 12 appear to be only marginally improved, it became evident that the biochemical assay limit had been reached, and we could no longer reliably rank-order compound potency based on inhibitory activity in biochemical assays alone. These potent compounds could be clearly differentiated based on their cellular activity in BRM-dependent gene expression assays and BRG1-mutant cell proliferation assays. Compound 11, which includes an amine group on the chloropyridine, and 12, which includes a hydroxymethyl group have AAC50 = 0.026 and 0.042 µM, respectively, compared with AAC50 = 0.36 µM for 10 in an endogenous reporter gene assay measuring KRT80 expression in BRG1-mutant H1299 cells (Table 2). Inhibition of KRT80


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expression with compounds was consistent with previously established data showing modulation of this gene upon genetic depletion of BRM.21 In proliferation assays, 11 and 12 also showed increased potency over 10 in a BRM-dependent BRG1-mutant cutaneous melanoma cell line (SKMEL5).22 Importantly, no significant anti-proliferative activity of these compounds was detected in a BRM/BRG1 target deficient cell line SBC5 (Table 2).5 Additional gains in potency in the KRT80 reporter gene assay could be achieved by replacing the chlorine with fluorine, i.e. AAC50 = 0.026 and 0.010 µM were measured for the 11/13 matched pair, and AAC50 = 0.042 and 0.010 µM were measured for the 12/14 matched pair (Table 2). The chlorine-fluorine switch also afforded small increase in thermodynamic solubility, as measured by shake-flask assay at pH 6.8, i.e. 0.092 mM for 13 versus 0.040 mM for 11, and 0.034 mM for 14 versus 0.014 mM for 12. Compounds 11-14 were all crystalline as established by optical microscopy and differential scanning calorimetry (these compounds were found to crystallize even upon lyophilization in acetonitrile/water solution). Table 1. Inhibition of ATPase activity of BRM and BRG1 ATPase-SnAC Cl

N

N

O N H

N H

Ar

*

N 1

*

2

S

HN N

*

*

3

S N

N R

* R=CH3

5

8

R=CF3

6

9

R=CHF2

7

10

*

R

N 4

X N

S N

O Y

N H

N H

F F

11 12 13 14

X=Cl, Y=NH2 X=Cl, Y=CH2OH X=F, Y=NH2 X=F, Y=CH2OH


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Compound

BRM IC50 (µM)

BRG1 IC50 (µM)

Compound

BRM IC50 (µM)

BRG1 IC50 (µM))

1

3.8 ± 0.7

1.7

8

0.033 ± 0.007

0.03 ± 0.01

2

1.1 ± 0.2

0.77

9

0.010 ± 0.003

0.010

3

0.97 ± 0.03

0.57

10

0.005 ± 0.002

0.006

4

> 50

n.d.

11

10 μM.

Figure 4. Determination of the binding mode of the urea series. (A) 2.8 Å crystal structure of MBP-hBRM[705-960] in complex with 15 (PDB code 6EG3). (B) 3.0 Å crystal structure of


11

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MBP-hBRM[705-960] in complex with 16 (PDB code 6EG2). Hydrogen bonding interactions are shown as black dotted lines. The ARL loop (Glu852-His860) is colored in yellow.

HO2C

Cl N

N

O N H

(A)

N H 15

Cl N NH2

(B)

S N

O N H

N H 16

OH

Br

In view of its robust cellular activity and favorable pharmaceutical properties, 14 was progressed into in vivo studies. A xenograft model was developed using BRG1-mutant RERF-LC-AI human lung cancer cells subcutaneously implanted into athymic nude mice. Consistent with the H1299 data, 14 inhibited KRT80 expression in RERF-LC-AI cells in vitro with an AAC50 = 0.01 µM.


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Compound 14 was well tolerated up to 20 mg/kg upon daily oral administration for 3 weeks, resulting in unbound plasma concentration above the cellular EC50 (KRT80) for approximately 12 h (Figure 5A). Compound 14 drove tumor growth inhibition of 21% and 55% with 7.5 and 20 mg/kg daily dose, respectively (Figure 5B). Dose-dependent modulation of KRT80 expression was observed, up to 90% inhibition 7 h post-dose at 20 mg/kg, with mRNA levels rebounding by the 16 h time point and beyond (Figure 5C). Our ability to drive more sustained PD inhibition was limited by tolerability, and significantly higher doses were not investigated in multi-dose efficacy studies. We were able to conduct a 30 mg/kg study using a 4 days on-3 days off dosing regimen, which resulted in increased plasma exposure, more sustained PD inhibition and slightly improved tumor growth inhibition (T/C = 30%). However, despite the 4 days on-3 days off dosing schedule, 30 mg/kg showed tolerability issues, as 3 out of 12 animals in the treatment group exhibited significant body weight loss after four daily doses and were excluded from the study. Figure 5. Data obtained from oral dosing of 14 (suspension vehicle: 0.5% methyl cellulose, 0.5% tween 80 in water) in RERF-LC-AI tumor bearing nude mice. (A) Unbound plasma concentrations of 14; plasma concentrations denoted with an asterisk indicates that 2 out of 3 animals were below the lower limit of quantification. (B) Tumor volume measurement. (C) KRT80 mRNA modulation upon compound treatment, which was measured following the last dose (% vehicle control).


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A

B

*

* *

C

Ureas 2-6 and 8 were prepared in low yields from commercially available amines using triphosgene in the presence of triethylamine in either DCM or THF under various conditions as shown in Schemes 1, 2 and 3. Compound 7 was obtained by Curtius rearrangement of acid 25, which was derived from saponification of ester 24. Ester 24 could be conveniently prepared using Baran’s difluoromethylation reagent23 (Scheme 4). Scheme 1. Synthesis of pyridine analogs 2, 5 and 6. Cl

Cl N

+ NH2 17

a (2)

N H 2N

R

N

b (5 and 6)

18: R = H 19: R = CH3 20: R = CF3

N

O N H

N H

R 2: R = H 5: R = CH3 6: R = CF3


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(a) Triphosgene (1.0 equiv), Et3N (4.0 equiv), DCM, 0 °C, 30 min, then chloropyridin-4-amine (2.0 equiv), 0 °C to r.t., 2%. (b) Triphosgene (0.35-0.45 equiv), Et3N (1.1-1.2 equiv), THF, r.t., 30 min, then chloropyridin-4-amine (0.9-1 equiv), r.t., 18 h, 17% (5), 16% (6) Scheme 2. Synthesis of compounds 3 and 4. Cl

Cl N

a

N

+

17

O

X

H 2N

NH2

N N H

N N H

X

21: X=S 22: X=NH

3: X=S 4: X=NH

(a) Triphosgene (0.4 equiv), Et3N (2.0 equiv), THF, DCM, –78 °C to r.t., 15 min, then 21 (1.1 equiv.) or 22 (1.0 equiv), r.t., 2-18 h, 5%. Scheme 3. Synthesis of isothiazole 8. Cl

Cl N NH2

a

S N

+

N

H 2N .HCl

17

S N

O N H

23

8

N H

(a) Triphosgene (0.45 equiv), Et3N (1.0 equiv), THF, r.t., 20 min, 23 (1.0 equiv), Et3N (1.0 equiv), r.t., 18 h, 3% Scheme 4. Synthesis of pyridine 7. Cl N EtO2C

N

a RO2C

24

b

F

c, d

F 25: R=Et 26: R=H

N

N

O N H

7

N H

F F


15

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(a) Zinc(II) difluoromethanesulfinate (2.0 equiv), tert-butylhydroperoxide (3.0 equiv), TFA (1.0 equiv), 2.5:1 DCM-water, r.t., 2 h, 44%. (b) KOH (4.0 equiv), MeOH, H2O, r.t., 3 h, 98%. (c) Diphenyl phosphoryl azide (1.1 equiv), Et3N (1.2 equiv), acetone, r.t., 24 h. (d) Toluene, 100°C, 1 h then 17 (1.2 equiv), Et3N (2 equiv), 80°C, 18 h, 8%. The corresponding trifluoromethyl and difluoromethyl isothiazoles required novel syntheses, as depicted in Schemes 5 and 6. Isothiazole 31 was prepared by an oxidative ring closure of thioamide 30, which could itself be obtained by magnesium chloride-mediated addition of sodium sulfide into nitrile 29. Nitrile 29 was prepared in two steps starting with the addition of potassium cyanomethanide to ethyl trifluoracetate (27) followed by acid-catalyzed amination of the resulting ketone (28). The yield for this 4 steps sequence was 15%. Amine 31 was converted to the lithium amide and reacted with carbamate 32 to afford 9 in 23% yield. As shown in Scheme 6, the key amine intermediate 39 was prepared by an initial amine to nitro oxidation of 33, followed by manipulation of the oxidation state of the methyl in 34, leading to aldehyde 37, which could be converted to the difluoromethyl group with DAST. Reduction of the nitro group under standard conditions completed the synthesis of 39 in good overall yields. Good yields for the urea formation leading to 10 were obtained by the reaction of 39 with nitrophenyl carbamate 40. Scheme 5. Synthesis of isothiazole 9. O O

O

a

NC

CF3 27

NH2

b

NC

CF3 28

S

c

NH2

H 2N 30

S N

d

CF3

CF3 29

H 2N

CF3

31


16


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Cl

Cl

e

Cl

N

N

f

O N H 32

NH2 17

N

S N

O N H

OPh

CF3

N H 9

(a) t-BuOK (1.4 equiv), CH3CN (0.7 equiv), 0 °C to r.t., 24 h. (b) NH4HCO2 (3 equiv), acetic acid (0.1 equiv), toluene, reflux, Dean-Stark, 18 h. (c) MgCl2 (1 equiv), NaSH (2 equiv), DMF, r.t., 24 h. (d) 30% aqueous H2O2 (4 equiv), pyridine, 0 °C to r.t., 2 h, 15% (4 steps). (e) Phenyl chloroformate (1.0 equiv), pyridine (1.0 equiv), DCM, r.t., 2 h, 99%. (f) 31 (1.0 equiv), LHMDS (1.0 equiv), DMF, r.t., 16 h, 23% Scheme 6. Synthesis of isothiazole 10. S N H 2N

O 2N

33

O 2N S N 38

F

S N

d O 2N S N

f H 2N

39

CO2H

35

H

e

O 37

F F

Cl

Cl g

N NH2 17

O 2N

36

F

S N

b

34

OH

S N

c

O 2N

S N

a

Cl

N

NO2

O N H

O 40

h

N

S N

O N H

N H

F F

10

(a) Cu (3.0 equiv), NaNO2 (3.0 equiv), HCl (1.5 equiv), water, r.t., 3 h, 36%. (b) CrO3 (3.0 equiv), sulfuric acid, r.t., 3 days, 28%. (c) BH3.THF (1.3 equiv), THF, r.t., 16 h, 89%. (d) DessMartin periodinane (1.1 equiv), r.t., 20 min. (e) DAST (3.0 equiv), DCM, r.t., 2 h. (f) Fe (3.0


17

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equiv), acetic acid, 50 °C, 2 h, 72% (3 steps). (g) 4-nitrophenyl chloroformate (1.05 equiv), pyridine (1.4 equiv), DCM, r.t., 20 min. (h) 39 (0.87 equiv), DIPEA (3.0 equiv), dioxane, 60 °C, 3 h, 40% (2 steps). Synthesis of urea 11 was conveniently carried out starting from commercially available pyridine 41, which was converted to carbamate 42 (Scheme 7). Reaction with 39 followed by nitro group reduction afforded 11 proceeded in 43% yield. Urea 12 was also prepared via reaction of a carbamate intermediate (48) with amine 39 (Scheme 8). Carbamate 48 was made starting from pyridine 44, with a regioselective SNAr to give 45 in 90% yield as the key step. Intermediate 43 from the synthesis of 11 was used to access 13 (Scheme 9). Fluoride displacement24 of the chlorine in 43 proceeded efficiently providing dry TMAF was used, which could be easily achieved with azeotropic removal of water in commercial batches (See Experimentals). Scheme 7. Synthesis of isothiazole 11. Cl

Cl a

N

Cl

N

NH2 NO2 41

NO2

N H

N

S N

O NH2

N H

N H

N

S N

O

O NO2 42

Cl c

b

O

N H

N H

F F

43 F F

11

(a) Phenyl chloroformate (1.05 equiv), pyridine (1.05 equiv), dioxane, 80 ºC, 18 h, 70%. (b) 39 (0.87 equiv), DIPEA (2.2 equiv), dioxane, 85 ºC, 16 h, 43%. (c) Iron (4.0 equiv), ammonium chloride (8.0 equiv), ethanol, water, 50 ºC, 1 h, 78%.


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Scheme 8. Synthesis of isothiazole 12. Cl

Cl

Cl a

N Cl CO2Me

N

c, d

N

NH2

N H

44

R 45: R = CO2Me 46: R = CH2OH

b

TBSO

O

Cl

Cl

N

e

47

f

O N H

TBSO

N N H

O TBSO

48

S N

O N H

F F

49

Cl N

g

N H HO

F

S N

O

F

N H 12

(a) 4-methoxybenzylamine (1.2 equiv), trimethylamine (1.35 equiv), acetonitrile, r.t., 96 h, 90%. (b) LAH (1.1 equiv), THF, r.t., 30 min, 81%. (c) TFA, 60°C, 18 h, 79%. (d) TBSCl (1.1 equiv), imidazole (2.5 equiv), DMF, r.t., 1 h, 79%. (e) phenyl chloroformate (1.05 equiv), pyridine (1.1 equiv), DCM, r.t., 2 h, 82%. (f) 39 (0.87 equiv), LHMDS (1.04 equiv), DMF, r.t., 30 min, 35%. (g) TBAF (1.1 equiv), THF, r.t., 15 min, 70%. Scheme 9. Synthesis of isothiazole 13. F

Cl N

S N

O NO2

N H

N H 43

F

a, b

N

S N

O

F NH2

N H

N H

F F

13


19

Page 21 of 66 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a) TMAF (4.5 equiv), DMF, 75 ºC, 1 h. (b) Iron (6.0 equiv), ammonium chloride (7.0 equiv), 5:1 ethanol/water, 45 ºC, 30 min, 56% (2 steps). For the synthesis of 14, we also relied on nucleophilic aromatic fluorination of a chloropyridine, as shown on Scheme 10. Anhydrous TMAF reacted with 44 at the 2-position, followed by ammonia displacement of the second chlorine to give 50 in 70% yield. Ester 50 was then transformed into carbamate 53 in three steps under standard conditions. Coupling with amine 39 was carried out using LHMDS in DMF in good yields (43%). TBAF-mediated removal of the TBS group afforded 14. Scheme 10. Synthesis of isothiazole 14. Cl

F

F a

N Cl CO2Me 44

N

d

N

N H

NH2 R 50: R = CO2Me 51: R = CH2OH 52: R = CH2OTBS

b c

TBSO

F e

O 53

F

N

S N

O N H

TBSO

O

N H 54

F

f

N

F

N H HO

S N

O N H

F F

14

(a) TMAF (2.6 equiv), DMF, r.t., 1.5 h, then 2M NH3 in iPrOH (2.1 equiv), r.t., 20 h, 70%. (b) LAH (2.0 equiv), THF, r.t., 2 h, 89%. (c) TBSCl (1.1 equiv), imidazole (2.5 equiv), DMF, r.t., 1 h, 73%. (d) pyridine (1.0 equiv), phenylchloroformate (1.0 equiv), dioxane, r.t., 1 h, 93%. (e) 39 (1.0 equiv), LHMDS (1.5 equiv), DMF, r.t., 30 min, 43%. (f) TBAF (1.0 equiv), THF, r.t., 2 h, 51%.


20


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Syntheses of alkynes 15 and 16 were both conducted using Sonogashira chemistry to couple methyl 3-ethynylbenzoate with amine 55 and 58, respectively (Scheme 11 and 12). Urea 15 was then prepared from coupling amine 56 with carbamate 32 under microwave irradiation conditions in the presence of DIPEA, followed by ester hydrolysis. Urea 16 was made from coupling amine 59 with carbamate 42 in the presence of LHMDS. Nitro reduction, followed by ester reduction under standard conditions afforded 16. Scheme 11. Synthesis of isothiazole 15. N N

H 2N

a

CO2Me

Br

H 2N 55

56

Cl N

O N H

c

b

N N H

CO2R

57: R=CO2Me 15: R=CO2H

(a) CuI (0.05 equiv), Pd(PPh3)2Cl2 (0.05 equiv), methyl 3-ethynylbenzoate (1.3 equiv), THF, Et3N, 60 °C, 18 h, 42%. (b) 32 (1.1 equiv), DIPEA (2.0 equiv), THF, microwave, 110 °C, 45 min, 16%. (c) KOH (4.0 equiv), 2:1 MeOH/water, r.t., 3 h, 15%. Scheme 12. Synthesis of isothiazole 16.


21

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S N

H 2N

H 2N

a Br

Br

b

58

N

59

N H

Cl

CO2Me

S N

O N H

CO2Me

Br

Cl

R

S N

d

c

S N

O NH2

Br

OH

N

60: R=NO2 61: R=NH2

N H

N H

Br

16

(a) CuI (0.15 equiv), Pd(PPh3)2Cl2 (0.15 equiv), Et3N (10 equiv), methyl 3-ethynylbenzoate (1.2 equiv), dioxane, 90 °C, 16 h, 46%. (b) LHMDS (1.5 equiv), 42 (1.1 equiv), DMF, r.t., 16 h, 42%. (c) Fe (4 equiv), NH4Cl (8 equiv), 2:1 EtOH/water, 75 °C, 1 h, 44%. (d) LAH (5 equiv), THF, 0 °C to r.t., 10 min, 8%. DISCUSSION AND CONCLUSIONS Members of the SWI/SNF multi-subunit complexes are among the most frequently mutated chromatin modifying genes in cancer. In particular, the discovery of a synthetic lethal relationship between SWI/SNF catalytic subunits BRM and BRG1 in which cancer cells harboring mutations in BRG1/SMARCA4 are exquisitely dependent on the ATPase activity of BRM for their growth has made BRM an attractive cancer target. The use of a truncated BRM protein which includes the ATPase and SnAC domains allowed assembly of a robust biophysical validation package for screening hits. The potency of screening hit 1 could be optimized, and the resulting ATPase inhibitors demonstrated activity in biochemical, cellular pharmacodynamic and growth assay systems in vitro, therefore validating our hit finding approach. Furthermore, X-ray crystallographic studies of 15 and 16 bound to MBP-hBRM[705-960] elucidated the binding mode of these compounds and revealed an allosteric pocket in the vicinity of the ATP binding site. Compound


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14 demonstrated activity in a BRG1-mutant lung tumor xenograft model upon oral administration, with a consistent dose-response relationship between PD modulation measured by changes in KRT80 gene expression and anti-proliferative effect. Importantly however, these studies to our knowledge provide the first demonstration of small molecule modulation in vitro and in vivo of BRM and BRG1, both in the context of isolated proteins and incorporated into the large SWI/SNF complex. Although this series exhibited excellent cellular potency and oral exposure, our ability to fully explore the potential of BRM inhibition on tumor growth in vivo was hampered by doselimiting tolerability issues. It is notable that this series of compounds are dual inhibitors of BRM and BRG1, with no observed meaningful separation of inhibitory activity against the two proteins. Animal models exploring the role of BRM and BRG1 in normal tissues have uncovered partly redundant roles and reveal lethality upon dual BRM and BRG1 knockout.25-27 In addition, tissue specific deletion of BRG1 demonstrates crucial roles for BRG1 in intestinal homeostasis.28 Nevertheless, given the overall importance of SWI/SNF function in development and disease, the availability of such small molecule inhibitors not only provides an important avenue for further developing novel SWI/SNF based targeted therapies, but will also be invaluable for probing the breadth of functions of this chromatin remodeling machinery in cancer and yet unanticipated contexts.

EXPERIMENTAL SECTION Compound synthesis and characterization. All solvents employed were commercially available anhydrous grade, and reagents were used as received unless otherwise noted. A Biotage Initiator Sixty system was used for microwave heating. NMR spectra were recorded on a Bruker AV400 (Avance 400 MHz) or AV600 (Avance 600 MHz) instruments. Compound purity was


23

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assessed by high performance liquid chromatography (HPLC) to confirm >95% purity. Purity of all tested compounds was determined by LC/ESI-MS data recorded using an Agilent 6220 mass spectrometer with electrospray ionization source and Agilent 1200 liquid chromatography. Flash column chromatography was performed on an ISCO system (32−63 μm particle size, KP-Sil, 60 Å pore size). Preparative HPLC was performed using a Waters 2525 pump with 2487 dual wavelength detector and 2767 sample manager. Two methods were employed both using XBridge 5 μm column at 75 mL/min with 1.5 mL injections: basic method (5 mM NH4OH in acetonitrile and water) and formic acid method (0.1% formic acid in acetonitrile and water). These two HPLC methods run a focused gradient from the starting % acetonitrile to the final % acetonitrile. The initial and final conditions for each gradient are as follows: 10-30% acetonitrile (method 2); 15-40% acetonitrile (method 3); 25-50% acetonitrile (method 4); 35-60% acetonitrile (method 5). 1-(2-chloropyridin-4-yl)-3-(pyridin-2-yl)urea (1). Compound 1 was taken from the Novartis compound archive. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.00 (s, 1H), 9.71 (s, 1H), 8.32 (dd, J = 5.1, 1.9 Hz, 1H), 8.23 (d, J = 5.5 Hz, 1H), 7.79 (ddd, J = 8.9, 7.4, 2.0 Hz, 1H), 7.75 (d, J = 1.5 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.44 (dd, J = 5.8, 1.9 Hz, 1H), 7.08 (dd, J = 7.3, 5.0 Hz, 1H). 1-(2-chloropyridin-4-yl)-3-(pyridin-4-yl)urea (2). Triethylamine (2.15 g, 21.2 mmol) and DCM (50 mL) were added to 18 (0.50 g, 5.3 mmol). The mixture was cooled to 0 °C. Triphosgene (1.58 g, 5.3 mmol) was added, and after 30 min, 2-chloropyridin-4-amine 17 (1.58 g, 10.6 mmol) was added and the cooling bath was removed. The mixture was concentrated to dryness and taken up in water. The white solid was filtered off and washed with water. Purification by reverse phase HPLC (basic method 2) afforded 2 as a white solid (28 mg, 2%). 1H

NMR (400 MHz, DMSO-d6) δ ppm 9.53 (s, 1 H), 9.41 (s, 1 H), 8.33 - 8.47 (m, 2 H), 8.22 (d,


24


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J=5.6 Hz, 1 H), 7.65 (d, J=1.6 Hz, 1 H), 7.41 - 7.51 (m, 2 H), 7.36 (dd, J=5.6, 2.0 Hz, 1 H). HRMS calculated for C11H10N4OCl (M+H): 249.0543. Found: 249.0545. 1-(2-chloropyridin-4-yl)-3-(4-methylthiazol-2-yl)urea (3). To a solution of triphosgene (92 mg, 0.31 mmol) in DCM (3 mL) at –78°C was added 17 (100 mg, 0.78 mmol) in 1:5 THF-DCM (0.8 mL) dropwise. Triethylamine (0.22 mL, 1.6 mmol) was then added dropwise and the cooling bath was removed. After 15 min, 21 (98 mg, 0.86 mmol) was added and the mixture was stirred for 2 h. The mixture was then filtered through Celite, the filtrate was concentrated and the residue was purified by HPLC (basic method 3) to give 3 (10 mg, 5% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 8.19 (d, J = 5.7 Hz, 1H), 7.71 (s, 1H), 7.44 (d, J = 5.6 Hz, 1H), 6.63 (s, 1H), 2.22 (s, 3H); HRMS calculated for C10H10N4OClS (M+H): 269.0264. Found: 269.0264. 1-(2-chloropyridin-4-yl)-3-(4-methyl-1H-imidazol-2-yl)urea (4). To a solution of triphosgene (92 mg, 0.31 mmol) in DCM (3 mL) at –78°C was added 17 (100 mg, 0.78 mmol) in 1:5 THF-DCM (0.8 mL) dropwise. Triethylamine (0.22 mL, 1.56 mmol) was then added dropwise and the cooling bath was removed. After 15 min, 22 (76 mg, 0.78 mmol) was added and the mixture was stirred for 18 h. The mixture was then filtered through celite, the filtrate was concentrated and the residue was purified by HPLC (basic method 3) to give 4 as a yellow powder (10 mg, 5% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 9.41 (s, 1H), 8.05 (d, J = 5.7 Hz, 1H), 7.79 (d, J = 1.8 Hz, 1H), 7.42 (dd, J = 5.8, 1.9 Hz, 1H), 6.43 (s, 1H), 2.07 (s, 3H); HRMS calculated for C10H11N5OCl (M+H): 252.0652. Found: 252.0679. 1-(2-chloropyridin-4-yl)-3-(2-methylpyridin-4-yl)urea (5). To a solution of 19 (93 mg, 0.86 mmol) in THF (2 mL) was added dropwise a solution of triphosgene (115 mg, 0.39 mmol) in


25

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THF (2 mL). Triethylamine (0.13 mL, 0.93 mmol) was added and the reaction was stirred at room temperature for 30 min. 17 (100 mg, 0.78 mmol) was then added and the reaction was stirred at room temperature for 18 h. The reaction mixture was diluted with saturated aqueous NaHCO3 and the product was extracted with EtOAc. The organics were combined, dried with Na2SO4, filtered, and volatiles were removed in vacuo. The crude residue was purified by reversed-phase HPLC (basic method 3) to give 5 as a white solid after lyophilization (35 mg, 17% yield). 1H NMR (400 MHz, methanol-d4) δ 8.23 (d, J = 5.8 Hz, 1H), 8.16 (d, J = 5.8 Hz, 1H), 7.71 (d, J = 1.9 Hz, 1H), 7.46 - 7.33 (m, 3H), 2.48 (s, 3H). MS (ESI) m/z 263.2 [M + H]+. 1-(2-chloropyridin-4-yl)-3-(2-(trifluoromethyl)pyridin-4-yl)urea (6). To a solution of 20 (300 mg, 1.85 mmol) in THF (2 mL) was added dropwise a solution of triphosgene (192 mg, 0.65 mmol) in THF (2 mL). Triethylamine (0.31 mL, 2.22 mmol) was added and the reaction was stirred at room temperature for 30 min. 17 (238 mg, 1.85 mmol) was then added and the reaction was stirred at room temperature for 18 h. The reaction mixture was diluted with saturated aqueous NaHCO3 and the product was extracted with EtOAc. The organics were combined, dried with Na2SO4, filtered, and volatiles were removed in vacuo. The crude residue was purified by HPLC (basic method 4) to give 6 as a white solid after lyophilization (92 mg, 16% yield). 1H NMR (400 MHz, methanol-d4) δ 8.50 (d, J = 5.6 Hz, 1H), 8.17 (d, J = 5.8 Hz, 1H), 8.02 (d, J = 2.0 Hz, 1H), 7.78 - 7.63 (m, 2H), 7.41 (dd, J = 5.8, 2.0 Hz, 1H). MS (ESI) m/z 317.1 [M + H]+. 1-(2-Chloropyridin-4-yl)-3-(2-(difluoromethyl)pyridin-4-yl)urea (7). Step 1. To a solution of 24 (300 mg, 2.0 mmol) and zinc difluoromethanesulfinate (1.17 g, 4.0 mmol) in dichloromethane (5 mL) and water (2 mL) at r.t. was added TFA (0.15 mL, 2.0 mmol) followed by slow addition of tert-butylhydroperoxide (0.82 mL, 5.9 mmol). The reaction was stirred for 2


26


Page 28 of 66

h, whereupon it was partitioned between DCM and saturated aqueous NaHCO3. The aqueous layer was extracted with DCM and the combined organic fraction was dried with Na2SO4, filtered, and volatiles were removed in vacuo. The crude residue was purified by silica gel chromatography (EtOAc/heptane) to give ethyl 2-(difluoromethyl)isonicotinate 25 (178 mg, 44 % yield) as a colorless oil. MS (ESI) m/z 202.1 [M + H]+. Step 2. To a solution of 25 (178 mg, 0.88 mmol) in water (1 mL) and methanol (2 mL) was added KOH (199 mg, 3.5 mmol). The reaction was stirred at room temperature for 3 h. The reaction was neutralized with aqueous 2M HCl until the pH of the solution was approximately 7. Volatiles were removed in vacuo to give 2-(difluoromethyl)isonicotinic acid 26 as a white solid (200 mg, 98% yield). MS (ESI) m/z 174.1 [M + H]+. Step 3. To a solution of 26 (250 mg, 1.4 mmol) in acetone (5 mL) were added diphenyl phosphoryl azide (437 mg, 1.6 mmol) and triethylamine (0.24 mL, 1.7 mmol). The mixture was stirred at room temperature for 24 h. Volatiles were removed in vacuo and the residue was taken up in toluene (5 mL). After heating at 100°C for 1 h, 17 (223 mg, 1.7 mmol) and triethylamine (292 mg, 2.9 mmol) were added. The reaction was stirred at 80°C for 18 h. The crude residue was purified by silica gel chromatography (MeOH/DCM) followed by HPLC (formic acid method 3) to give 7 as a white solid after lyophilization (36 mg, 8% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.94 - 9.58 (m, 2H), 8.49 (d, J = 5.6 Hz, 1H), 8.22 (d, J = 5.6 Hz, 1H), 7.87 (d, J = 1.9 Hz, 1H), 7.67 (d, J = 1.9 Hz, 1H), 7.52 (dd, J = 5.6, 2.0 Hz, 1H), 7.38 (dd, J = 5.6, 2.0 Hz, 1H), 6.90 (t, J = 55.0 Hz, 1H). MS (ESI) m/z 299.2 [M + H]+. 1-(2-chloropyridin-4-yl)-3-(3-methylisothiazol-5-yl)urea (8). A solution of 17 (101 mg, 0.79 mmol) in THF (2 mL) was added slowly to a solution of triphosgene (101 mg, 0.34 mmol)


27

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in THF (2 mL) at r.t. Triethylamine (0.11 mL, 0.79 mmol) was then added. After the mixture was stirred at room temperature for 20 min, a mixture of 23 (120 mg, 0.79 mmol) and triethylamine (0.12 mL, 0.86 mmol) in THF (2 mL) was added. The mixture was stirred at room temperature for 18 h and partitioned between EtOAc and aqueous KOH. The combined organic extract was dried over MgSO4 and concentrated. The residue was purified by HPLC (basic method 3) to give 8 as an off-white solid (7 mg, 3% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 9.87 (s, 1H), 8.24 (d, J = 4 Hz, 1H), 7.66 (s, 1H), 7.43 (d, J = 4 Hz, 1H), 6.73 (s, 1H), 2.30 (s, 3H). HRMS calculated for C10H10ClN4OS (M+H): 269.0264. Found: 269.0276. 1-(2-chloropyridin-4-yl)-3-(3-(trifluoromethyl)isothiazol-5-yl)urea (9). Step 1. To a dry 100 mL flask charged with potassium tert-butoxide solution (1M in THF, 85 mL, 85 mmol) and cooled on an ice bath was slowly added a mixture of 27 (7.27 mL, 60.9 mmol) in acetonitrile (3.18 mL, 60.9 mmol) resulting in a suspension. The mixture was allowed to warm to room temperature, then stirred for 24 h. The mixture was quenched with 1M HCl, then extracted with ether and washed with water. The organic layer was separated, dried over magnesium sulfate, filtered and concentrated in vacuo to furnish 4,4,4-trifluoro-3-oxobutanenitrile 28 which was used in the next step with no further purification. 1H NMR (400 MHz, DMSO-d6) δ 2.99 (s, 2H). Step 2. A mixture of 28 (1.1 g, 8.0 mmol), ammonium formate (1.52 g, 24.0 mmol) and acetic acid (0.046 mL, 0.80 mmol) in toluene (100 mL) was heated to reflux under a Dean-Stark trap for 18 h. The mixture was then concentrated in vacuo to give 29, which was taken up in DMF (20 mL). MgCl2 (0.70 g, 7.3 mmol) and NaSH (0.82 g, 14.7 mmol) were added and the mixture was stirred at room temperature for 24 h. Ethyl acetate and water were added. The organic portion was separated and washed with water followed by brine. The organic portion was dried


28


Page 30 of 66

over magnesium sulfate, filtered and concentrated in vacuo to furnish 3-amino-4,4,4-trifluorobut2-enethioamide 30 which was used in the following step without purification. Step 3. To an ice-cold mixture of 30 (1.2 g, 7.0 mmol) in pyridine (24 mL) was added a 30% solution of hydrogen peroxide (3 mL, 29 mmol). The mixture was allowed to warm to room temperature and stirred for 2 h. It was then concentrated in vacuo, and the residue was purified by silica gel chromatography (EtOAc /heptane) to give 3-(trifluoromethyl)isothiazol-5-amine 31 (182 mg, 15% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.21 (s, 2H), 6.44 (s, 1H). 19F NMR (376 MHz, DMSO) δ -63.92. MS (ESI) m/z 169.0 [M + H]+. Step 4. To a solution of 17 (12.9 g, 101 mmol) and pyridine (8.1 mL, 101 mmol) in DCM (300 mL) at 0 °C was added phenyl chloroformate (13.3 mL, 106 mmol). The mixture was allowed to warm to r.t. over 2 h, then concentrated in vacuo. Water was added and the mixture was stirred at r.t. resulting in precipitation. The solids were filtered over a fritted funnel, rinsed with water and dried under high vacuum at 40 °C for 24 h to give phenyl (2-chloropyridin-4-yl)carbamate 32 (25.0 g, 99% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 8.27 (d, J = 5.6 Hz, 1H), 7.58 (d, J = 1.8 Hz, 1H), 7.51 - 7.41 (m, 3H), 7.34 - 7.23 (m, 3H). MS (ESI) m/z 249.2 [M + H]+. Step 5. To an ice-cold mixture of 31 (70 mg, 0.41 mmol), and 32 (104 mg, 0.41 mmol) in DMF (1.2 mL) was added a solution of LHMDS (1M in THF, 0.41 mL, 0.41 mmol). The mixture was allowed to warm to r.t. and stirred for 16 h. The mixture was concentrated in vacuo, then purified by HPLC (basic method 3) to obtain 9 (32 mg, 23% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.21 (br s, 1H), 10.18 (br s, 1H), 8.23 (d, J = 5.6 Hz, 1H), 7.69 (d, J = 1.8 Hz, 1H), 7.46 (dd, J = 5.7, 1.9 Hz, 1H), 7.22 (s, 1H). 19F NMR (376 MHz, DMSO) δ -63.48. HRMS calculated for C10H7ClF3N4OS (M+H) 322.9981, found 322.9986.


29

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1-(2-chloropyridin-4-yl)-3-(3-(difluoromethyl)isothiazol-5-yl)urea (10). Step 1. Cu powder (96 g, 1.5 mol) was placed in a 5 L reactor. Water (1 L) was added followed by NaNO2 (104 g, 1.5 mol). Aqueous HCl (12 M, 1.5 mL, 18 mmol) was added, and the reaction mixture was stirred for 20 min. A solution of 33 (58 g, 507 mmol) in 500 mL of water and aqueous HCl (12 M, 65 mL, 0.78 mol) was added dropwise via addition funnel maintaining the temperature below 30 °C. An additional 100 mL of water was added. The reaction mixture was allowed to stir for 3 h after addition. The reaction mixture was filtered through Celite with water and MTBE. The filtrate was transferred to the reactor and the layers were separated. The aqueous layer was washed twice with MTBE. The combined organic layers were dried over MgSO4, filtered and concentrated to give 3-methyl-5-nitroisothiazole 34 as a solid (26.5 g, 36% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H), 2.50 (s, 3H). Step 2. To a 1L 3-neck round bottom flask in a water bath equipped with a mechanical stirrer and a temperature monitor was added 34 (26.5 g, 184 mmol), then H2SO4 (350 mL) at a rate to keep the temperature below 30 °C. CrO3 (55.1 g, 552 mmol) was added in 6 portions every 20 min, ensuring the temperature remained below 24 °C. The reaction was left stirring in the presence of the water bath for 3 days. The reaction mixture was poured into ice water (total of 1.4 L) and was extracted 3 times with Et2O (1 L). The combined organic layers were washed with brine, then dried over MgSO4, filtered and concentrated to provide a yellow solid. The solid was taken up in heptane (80 mL) and Et2O (20 mL) and triturated. After 2 min of vigorous stirring, the mixture was filtered, and then rinsed with a 5:1 heptane/ether mixture (minimal amount) to provide 5-nitroisothiazole-3-carboxylic acid 35 as a beige solid (9.13 g, 28%). 1H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 8.57 (s, 1H).


30


Page 32 of 66

Step 3. A flask was charged with 35 (3.0 g, 17.2 mmol) in THF (50 mL), cooled on an ice bath, then borane tetrahydrofuran complex (1M in THF) (22.4 mL, 22.4 mmol) was added dropwise over 30 min and the reaction was allowed to warm overnight. The reaction mixture was recooled to 0 °C, then methanol (20 mL) was added dropwise. The reaction was vigorously stirred at 0 °C for 5 min, then allowed to warm to r.t. and stirred for another 15 min. The reaction mixture was concentrated in vacuo to half volume, then was diluted with EtOAc (100 mL), saturated aqueous NH4Cl (50 mL) and water (50 mL). The layers were separated and the aqueous portion was extracted with EtOAc (2x100 mL). Organics were combined and washed with brine, then dried over sodium sulfate, filtered and concentrated in vacuo. Purification via silica gel chromatography (EtOAc/DCM) gave 5-nitroisothiazol-3-yl)methanol 36 as a yellow oil (2.46 g, 89% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.09 (s, 1H), 5.77 (t, J = 6.1 Hz, 1H), 4.58 (d, J = 6.1 Hz, 2H). MS (ESI) m/z 161.0 [M + H]+. Step 4. Dess-Martin periodinane (2.23 g, 5.2 mmol) was added in small portions over 5 min to 36 (767 mg, 4.8 mmol) in DCM (25 mL) at 0 °C. The mixture was stirred at 0 °C for 10 min, warmed to r.t. and stirred at r.t. for 20 min. The mixture was diluted with DCM. Saturated aqueous NaHCO3 and saturated aqueous sodium thiosulfate were added. The mixture was vigorously stirred for 10 min, and the two layers were separated. The aqueous layer was extracted with DCM. The combined organic extract was washed with brine, dried over sodium sulfate and concentrated in vacuo to give 5-nitroisothiazole-3-carbaldehyde 37. The product was used directly in the next step without purification. 1H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 8.58 (s, 1H). Step 5. DAST (0.20 mL, 1.52 mmol) was added at 0 °C in a dropwise fashion to a solution of 37 (80 mg, 0.50 mmol) in DCM (3 mL). The mixture was stirred at 0 °C for 25 min, warmed to


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r.t. and stirred at r.t. for 2 h. The mixture was quenched at 0 °C with saturated aqueous NaHCO3, and diluted with DCM. The mixture was vigorously stirred for 1 min, and the two layers were separated. The aqueous layer was extracted with DCM. The combined organic extract was washed with brine, dried over sodium sulfate and concentrated in vacuo to give 3(difluoromethyl)-5-nitroisothiazole 38. The product was used immediately in the next step without purification. 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 7.16 (t, J = 52 Hz, 1H). Step 6. A mixture of 38 (49 mg, 0.27 mmol), iron powder (46 mg, 0.81 mmol) and acetic acid (1.5 mL) was heated to 50 °C for 2 h. The mixture was diluted with ethyl acetate and basified with 30 % ammonium hydroxide. The organic layer was separated and concentrated in vacuo, then purified by silica gel chromatography (EtOAc/heptane) to obtain 3(difluoromethyl)isothiazol-5-amine 39 (29 mg, 72 % yield, 3 steps from 36). 1H NMR (400 MHz, methanol-d4) δ 6.46 (t, J = 55.0 Hz, 1H), 6.39 (s, 1H). 19F NMR (376 MHz, MeOD) δ 116.06. MS (ESI) m/z 151.2 [M + H]+. Step 7. To a mixture of 17 (1.57 g, 12.2 mmol) and pyridine (1.4 mL, 17.0 mmol) in DCM (100 mL) at 0 °C was added 4-nitrophenyl chloroformate (2.6 g, 12.9 mmol). The mixture was maintained at 0 °C for 2 min, then warmed up at rt. After another 20 min, the mixture was concentrated in vacuo to obtain 40 as a white solid, which was taken up in dioxane (80 mL). A solution of 39 (1.60 g, 10.6 mmol) in dioxane (10 mL) was rapidly added, followed by DIPEA (6.5 mL, 37.3 mmol). The mixture was heated to 60 °C. After 3 h, the mixture was cooled to r.t., then water and EtOAc were added. The organic layer was washed repeatedly with water, saturated aqueous sodium bicarbonate and brine. The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo to obtain a residue which was purified by flash chromatography (EtOH/EtOAc/heptane). The partially purified residue was triturated with ether,


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and the obtained solid was taken up in water and lyophilized to remove residual ether. 10 was obtained as a white solid (1.3 g, 40%). 1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 10.15 (s, 1H), 8.26 (d, J = 4 Hz, 1H), 7.68 (d, J = 2 Hz, 1H), 7.46 (dd, J = 4.2 Hz, 1H), 7.15 (s, 1H), 6.97 (t, J = 52 Hz, 1H). MS (ESI) m/z 305.1 [M + H]+. 1-(5-Amino-2-chloropyridin-4-yl)-3-(3-(difluoromethyl)isothiazol-5-yl)urea (11). Step 1. To a solution of 41 (1.0 g, 5.7 mmol) and pyridine (0.93 mL, 11.5 mmol) in dioxane (12 mL) at 0 °C was added phenyl chloroformate (0.95 mL, 6.0 mmol). The mixture was heated at 80 °C for 18 h, then cooled to r.t. and concentrated in vacuo. The crude residue was purified by silica gel chromatography (EtOAc / heptane) to give phenyl (2-chloro-5-nitropyridin-4-yl)carbamate 42 (1.20 g, 70% yield). MS (ESI) m/z 294.1 [M + H]+. Step 2. A solution of 42 (4.5 g, 15.3 mmol), 39 (2.0 g, 13.3 mmol) and DIPEA (5.8 mL, 33.3 mmol) in dioxane (60 mL) was heated at 85 ºC for 16 h. The mixture was concentrated in vacuo and the residue was purified by silica gel chromatography (EtOAc/heptane) to give 1-(5-nitro-2chloropyridin-4-yl)-3-(3-(difluoromethyl)isothiazol-5-yl)urea 43 (2.43 g, 43%). MS (ESI) m/z 350.1 [M + H]+. Step 3. A mixture of 43 (7.63 g, 21.8 mmol), iron (4.87 g, 87 mmol) and ammonium chloride (9.34 g, 175 mmol) in ethanol (84 mL) and water (25 mL) was heated for 1 h at 50 ºC. The mixture was filtered over Celite, the filter cake was rinsed with MeOH, and the filtrate was concentrated in vacuo. The residue was taken up in EtOAc and washed with brine. The organic fraction was dried over magnesium sulfate and concentrated in vacuo to give a residue which was purified by silica gel chromatography (EtOAc/heptane) followed by purification using ISCO reverse phase purification on a C18 column eluting with water + 0.1% formic acid and


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acetonitrile + 0.1% formic acid. 11 was obtained as a white powder (5.5 g, 78%). 1H NMR (400 MHz, methanol-d4) δ 7.88 (s, 1H), 7.84 (s, 1H), 6.99 (s, 1H), 6.66 (t, J = 54.9 Hz, 1H). HRMS calculated for C10H9ClF2N5OS (M+H) 320.0184, found 320.0185. 1-(2-Chloro-5-(hydroxymethyl)pyridin-4-yl)-3-(3-(difluoromethyl)isothiazol-5-yl)urea (12). Step 1. A mixture of 4-methoxybenzylamine (19.0 mL, 146 mmol), 44 (25 g, 121 mmol), and triethylamine (20.3 mL, 146 mmol) in acetonitrile (60 mL) was stirred at room temperature for 24 h. An additional portion of 4-methoxybenzylamine (2.5 mL, 18 mmol) was added and the mixture was stirred at room temperature for 72 h. The mixture was concentrated and the residue was partitioned between EtOAc and saturated aqueous NH4Cl. The aqueous layer was extracted with EtOAc (2x500mL). The organics were combined, dried with Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by silica gel chromatography (EtOAc/heptane) to give methyl 6-chloro-4-((4-methoxybenzyl)amino)nicotinate 45 as an offwhite solid (28.5 g, 90% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H), 8.48 (t, J = 5.9 Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 6.76 (s, 1H), 4.47 (d, J = 5.9 Hz, 2H), 3.84 (s, 3H), 3.74 (s, 3H). MS (ESI) m/z 307.2 [M + H]+. Step 2. To a solution of LAH (2M in THF, 21.5 mL, 43.0 mmol) in THF (150 mL) stirring at 0°C was added dropwise a solution of 45 (12.0 g, 39.1 mmol) in THF (100 mL). The reaction was allowed to warm to room temperature and was stirred for 30 min. The reaction was quenched with slow addition of EtOAc in an ice bath, followed by Steinhardt conditions for quenching LAH (2 mL H2O, followed by 2 mL of 15% NaOH and 6 mL of H2O). The resulting solution was stirred for 15 min and was allowed to warm to room temperature. The mixture was filtered over Celite and the filtrate was transferred to a separatory funnel. The product was further diluted with water and was extracted with EtOAc (3x500mL). The organics were


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combined, dried with Na2SO4, filtered, and volatiles were removed in vacuo to give (6-chloro-4((4-methoxybenzyl)amino)pyridin-3-yl)methanol 46 as an off-white solid (9.82 g, 81% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.79 (s, 1H), 7.33 - 7.20 (m, 2H), 6.95 - 6.86 (m, 2H), 6.82 (s, 1H), 6.38 (s, 1H), 5.23 (t, J = 5.4 Hz, 1H), 4.44 (d, J = 5.4 Hz, 2H), 4.34 (d, J = 6.0 Hz, 2H), 3.72 (s, 3H). MS (ESI) m/z 279.3 [M + H]+. Step 3. A solution of 46 (9.82 g, 35.2 mmol) in TFA (2.7 ml, 35.2 mmol) was heated at 60°C for 18 h. The reaction was neutralized with 10% aqueous K2CO3 to pH 7. The mixture was then transferred to a separatory funnel and was extracted with DCM. The organics were combined, dried with Na2SO4, filtered, and volatiles were removed in vacuo to give a portion of intermediate alcohol. The aqueous layer was concentrated in vacuo, and the resulting solid was diluted in isopropanol. Insoluble salts were filtered off, and the solution was cooled on ice. Additional salts that precipitated out were filtered off. Volatiles were removed in vacuo to give the intermediate alcohol (5.5 g, 79% yield). MS (ESI) m/z 159.1 [M + H]+. A portion (5 g, 32 mmol) was taken up in DMF (10 mL) and tert-butyldimethylsilyl chloride (5.2 g, 34.7 mmol) and imidazole (5.3 g, 79 mmol) were added. After stirring for 1 h, the reaction mixture was poured into water and was extracted with EtOAc. The organics were combined, dried with Na2SO4, filtered, and volatiles were removed in vacuo. The crude residue was purified by silica gel chromatography (EtOAc/heptane) to give 5-(((tert-butyldimethylsilyl)oxy)methyl)-2chloropyridin-4-amine 47 as a white solid (6.88 g, 79% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.82 (s, 1H), 6.53 (s, 1H), 6.16 (s, 2H), 4.62 - 4.50 (m, 2H), 0.88 (s, 9H), 0.07 (s, 6H). MS (ESI) m/z 273.3 [M + H]+. Step 4. To a solution of 47 (5.55 g, 20.3 mmol) and pyridine (1.8 mL, 22.4 mmol) in DCM (75 mL) was added phenyl chloroformate (2.68 mL, 21.4 mmol) dropwise. The reaction was stirred


35

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at room temperature for 2 h. Volatiles were removed in vacuo and the residue was diluted with 1:1 EtOAc/saturated aqueous NaHCO3. The layers were separated and the aqueous layer was extracted with EtOAc. The organics were combined, dried with Na2SO4, filtered, and volatiles were removed in vacuo. The crude residue was purified by silica gel chromatography (EtOAc/heptane) to give phenyl (5-(((tert-butyldimethylsilyl)oxy)methyl)-2-chloropyridin-4yl)carbamate 48 as a white solid (7.31 g, 82% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.32 (s, 1H), 7.88 (s, 1H), 7.52 - 7.41 (m, 2H), 7.34 - 7.22 (m, 3H), 4.89 (s, 2H), 0.92 (s, 9H), 0.11 (s, 6H). MS (ESI) m/z 393.2 [M + H]+. Step 5. LHMDS (1M in THF, 9.2 mL, 9.2 mmol) was added dropwise to a solution of 48 (3.46 g, 8.81 mmol) and 39 (1.15 g, 7.66 mmol) in DMF (30 mL), and the reaction was stirred at room temperature for 30 min. The reaction was quenched with MeOH (10 mL) and volatiles were removed in vacuo. The residue was taken up in 1:1 EtOAc/saturated aqueous NH4Cl and the layers were separated. The aqueous layer was extracted with EtOAc. The organics were combined, dried with Na2SO4, filtered, and volatiles were removed in vacuo. The crude residue was purified by silica gel chromatography (EtOAc/heptane) to give 1-(5-(((tertbutyldimethylsilyl)oxy)methyl)-2-chloropyridin-4-yl)-3-(3-(difluoromethyl)isothiazol-5-yl)urea 49 as an off-white solid (1.31 g, 35% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 8.93 (s, 1H), 8.29 (s, 1H), 8.14 (s, 1H), 7.19 (s, 1H), 6.96 (t, J = 54.5 Hz, 1H), 4.79 (s, 2H), 0.86 (s, 9H), 0.07 (s, 6H). MS (ESI) m/z 449.2 [M + H]+. Step 6. TBAF (1M in THF, 2.4 mL, 2.4 mmol) was added to a solution of 49 (1.3 g, 2.19 mmol) in THF (15 mL) and the reaction was stirred at room temperature for 15 min. The reaction mixture was poured into saturated aqueous NH4Cl and was extracted with EtOAc. The organics were combined, dried with Na2SO4, filtered, and volatiles were removed in vacuo. The crude


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residue was purified by silica gel chromatography (EtOAc/heptane), followed with (MeOH/DCM), followed by triturating the compound with EtOAc to give 12 (520 mg, 1.54 mmol) as a white solid after lyophilization. 1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 9.22 (s, 1H), 8.23 (s, 1H), 8.15 (s, 1H), 7.20 (s, 1H), 6.97 (t, J = 56 Hz, 1H), 5.79 (t, J = 5 Hz, 1H), 4.59 (d, J = 5 Hz, 2H). MS (ESI) m/z 335.1 [M + H]+. 1-(5-amino-2-fluoropyridin-4-yl)-3-(3-(difluoromethyl)isothiazol-5-yl)urea (13). In our hands, it was critical that TMAF be a free-flowing powder (presumed to be anhydrous) for this reaction to be successful. Commercial sources vary in quality and batches from the same vendor also vary in quality. If the TMAF sample is not a free-flowing white powder, azeotropic removal of water with isopropanol must be conducted as follows (see EP0457966A1): a 5 g bottle of Aldrich 107212-5G (colorless syrup) was transferred to a 200 mL round-bottom flask and iPrOH (100 mL) was added. The iPrOH was removed in vacuo, then iPrOH (100 mL) was added. This sequence was repeated twice and the rotoevaporator bath temperature was increased to 75°C for the last evaporation. TMAF was then subjected to high vacuum for 1.5 h to give a white freeflowing powder (2.98 g). A mixture of 43 (3.77 g, 8.62 mmol), TMAF (3.61 g, 38.8 mmol) and DMF (83 mL) was heated at 75 °C for 1 h. The reaction was quenched with water and extracted with EtOAc. The combined organic fractions were washed with water, brine, then dried with sodium sulfate, filtered and concentrated in vacuo. The residue was then purified by flash chromatography (EtOAc/heptane) to give partially purified product (2.73 g) which was taken up in ethanol (100 mL) and water (20 mL). Ammonium chloride (2.13 g, 39.8 mmol) and iron (1.91 g, 34.2 mmol) were added and the mixture was heated to 45 °C for 30 min. The reaction mixture was then filtered on a short pad of celite, which was washed with MeOH. The filtrate was concentrated


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down, and then diluted with EtOAc and water. The aqueous layer was extracted with EtOAc. The combined organic fractions were washed with brine, then dried with sodium sulfate, filtered and concentrated in vacuo. The residue was sequentially purified by silica gel chromatography (EtOAc/heptane), followed by ISCO reverse phase purification on a C18 column eluting with water + 0.1% formic acid and acetonitrile + 0.1% formic acid. A small number of impure fractions were finally purified by HPLC (formic acid method 3) and the combined fractions afforded 13 (984 mg, 56%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 1H), 8.92 (s, 1H), 7.63 (d, J = 0.9 Hz, 1H), 7.46 (d, J = 0.8 Hz, 1H), 7.14 (s, 1H), 6.95 (t, J = 54.5 Hz, 1H), 4.82 (s, 2H). HRMS calculated for C10H8F3N5OS (M+H) 304.0480, found 304.0475. 1-(3-(difluoromethyl)isothiazol-5-yl)-3-(2-fluoro-5-(hydroxymethyl)pyridin-4-yl)urea (14). Step 1. To a 500 mL flask containing 44 (6.75 g, 32.8 mmol) and free flowing tetramethylammonium fluoride (8.0 g, 86 mmol) was added DMF (100 mL) and the mixture was stirred at room temperature for 1.5 h. To this reaction mixture was then added a solution of 2M ammonia in isopropanol (35 mL, 70 mmol) and stirred at room temperature for 20 h. The reaction mixture was quenched with water and extracted with EtOAc. The organic layer was washed with brine, then dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / heptane) to obtain methyl 4-amino6-fluoronicotinate 50 (3.88 g, 70% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.43 (s, 1H), 7.52 (s, 2H), 6.30 (s, 1H), 3.82 (s, 3H). MS (ESI) m/z 174 [M + H]+. Step 2. To an ice-cold solution of LAH (2M in THF, 25 mL, 50 mmol) diluted with THF (200 mL) was added a solution of 50 (4.22 g, 24.8 mmol) in THF (100 mL) via addition funnel over 45 min, resulting in a suspension. The mixture was allowed to warm to r.t. for 2 h after which it was diluted with THF (200 mL) and cooled again on ice. Solid sodium sulfate decahydrate was


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added in portions to the mixture until the bubbling ceased. The mixture was stirred for 18 h, then filtered and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (EtOAc / heptane) to afford (4-amino-6-fluoropyridin-3-yl)methanol 51 (3.13 g, 89% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.63 (s, 1H), 6.20 (s, 2H), 6.09 (s, 1H), 5.04 (t, J = 5.5 Hz, 1H), 4.35 (d, J = 5.5 Hz, 2H). MS (ESI) m/z 143 [M + H]+. Step 3. A mixture of 51 (1.52 g, 10.7 mmol), TBSCl (1.77 g, 11.8 mmol), and imidazole (1.82 g, 26.7 mmol) in DMF (50 mL) was stirred at r.t. for 1 h. The reaction mixture was concentrated in vacuo, then purified by silica gel chromatography (EtOAc / heptane) to afford 5-(((tertbutyldimethylsilyl)oxy)methyl)-2-fluoropyridin-4-amine 52 (1.99 g, 73% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.60 (s, 1H), 6.11 (s, 2H), 6.04 (s, 1H), 4.50 (s, 2H), 0.81 (s, 9H), 0.00 (s, 6H). MS (ESI) m/z 257 [M + H]+. Step 4. To a mixture of 52 (1.45 g, 5.66 mmol) and pyridine (0.46 mL, 5.66 mmol) in dioxane (30 mL) was added phenyl chloroformate (0.71 mL, 5.66 mmol) and the resulting mixture was stirred for 1 h. The mixture was then diluted with EtOAc, then washed with sodium bicarbonate followed by water. The organic portion was dried over sodium sulfate and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / heptane) to afford phenyl (5-(((tert-butyldimethylsilyl)oxy)methyl)-2-fluoropyridin-4-yl) carbamate 53 (1.97 g, 93%). 1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.03 (s, 1H), 7.41 (s, 1H), 7.39 - 7.31 (m, 2H), 7.16 - 7.09 (m, 2H), 6.74 - 6.52 (m, 1H), 4.78 (s, 2H), 0.80 (s,9 H), 0.00 (s, 6H). MS (ESI) m/z 377 [M + H]+ Step 5. To a solution of 39 (48 mg, 0.32 mmol) and 53 (135 mg, 0.32 mmol) in DMF (2 mL) was added LHMDS (1M in THF, 0.48 mL, 0.48 mmol) and the resulting mixture was stirred at


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room temperature for 30 min. The mixture was concentrated in vacuo and the product purified by silica gel chromatography (EtOAc / heptane) to give 1-(5-(((tertbutyldimethylsilyl)oxy)methyl)-2-fluoropyridin-4-yl)-3-(3-(difluoromethyl)isothiazol-5-yl)urea 54 as a white solid (62 mg, 43% yield). MS (ESI) m/z 433.2 [M + H]+ Step 6. To a solution of 54 (119 mg, 0.27 mmol) in THF (5 mL) was added TBAF (1M in THF, 0.27 mL, 0.27 mmol) and the resulting mixture was allowed to stir at r.t. for 2 h. The mixture was concentrated in vacuo and purified by silica gel chromatography (MeOH / DCM with ammonium hydroxide as the modifier) to give 14 as a white solid (46 mg, 51% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H), 9.26 (s, 1H), 8.06 (s, 1H), 7.77 (s, 1H), 7.22 - 6.70 (m, 2H), 5.73 (t, J = 5.4 Hz, 1H), 4.59 (d, J = 5.1 Hz, 2H). 19F NMR (376 MHz, DMSO-d6) δ 68.97, -113.82. HRMS calculated for C11H10F3N4O2S (M+H) 319.0475, found 319.0477. 3-((4-(3-(2-chloropyridin-4-yl)ureido)pyridin-2-yl)ethynyl)benzoic acid (15). Step 1. To a solution of 55 (500 mg, 2.89 mmol) in THF (3 mL) purged with nitrogen gas were added copper(I) iodide (27 mg, 0.14 mmol) and bis(triphenylphosphine)palladium dichloride (101 mg, 0.14 mmol). The reaction was purged again with nitrogen gas and was stirred at r.t. for 10 min. A solution of methyl 3-ethynylbenzoate (602 mg, 3.76 mmol) and triethylamine (2.0 mL, 14.4 mmol) in THF (3 mL) was then added dropwise. The reaction was stirred at 60°C for 18 h. The reaction mixture was poured into water was extracted with EtOAc. The organics were combined, dried with Na2SO4, filtered, and volatiles were removed in vacuo. The crude residue was purified by silica gel chromatography (MeOH/DCM) to give methyl 3-((4-aminopyridin-2yl)ethynyl)benzoate 56 as a brown oil (304 mg, 42 % yield). MS (ESI) m/z 253.3 [M + H]+.


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Step 2. To a solution of 56 (334 mg, 1.32 mmol) in THF (2 mL) were added DIPEA (342 mg, 2.65 mmol) and 32 (362 mg, 1.46 mmol). The mixture was irradiated in the microwave to 110 °C for 45 min, whereupon the mixture was concentrated in vacuo. The residue was purified by silica gel chromatography (ethyl acetate/heptane) followed by HPLC (basic method 5) to obtain methyl 3-((4-(3-(2-chloropyridin-4-yl)ureido)pyridin-2-yl)ethynyl)benzoate 57 (90 mg, 16% yield). 1H NMR (400 MHz, Methanol-d4) δ 8.36 (d, J = 5.8 Hz, 1H), 8.24 – 8.19 (m, 1H), 8.16 (d, J = 5.8 Hz, 1H), 8.09 – 8.02 (m, 1H), 7.86 – 7.79 (m, 2H), 7.71 (d, J = 2.0 Hz, 1H), 7.58 – 7.49 (m, 2H), 7.40 (dd, J = 5.8, 2.0 Hz, 1H), 3.93 (s, 3H). MS (ESI) m/z 407.1 [M + H]+. Step 3. To a solution of 57 (70 mg, 0.172 mmol) in water (1 mL) and methanol (2 mL) was added KOH (38 mg, 0.69 mmol). The reaction was stirred at r.t. for 3 h. The reaction was neutralized with 2M aqueous HCl until the pH of the solution was approximately 7 and volatiles were removed in vacuo. The crude residue was purified by reversed-phase HPLC (basic method 2) to give 15 as a white solid after lyophilization (10 mg, 15% yield). 1H NMR (400 MHz, methanol-d4) δ 8.40 – 8.30 (m, 2H), 8.19 – 8.11 (m, 2H), 8.08 (dd, J = 5.8, 2.2 Hz, 1H), 7.88 (d, J = 1.9 Hz, 1H), 7.78 – 7.71 (m, 1H), 7.63 (d, J = 2.2 Hz, 1H), 7.59 (dd, J = 5.8, 1.9 Hz, 1H), 7.53 – 7.43 (m, 1H). MS (ESI) m/z 393.3 [M + H]+. Step 1. A solution of 58 (0.40 g, 1.55 mmol) (prepared according to the procedure described in WO2008/103185) in dioxane (13 mL) was purged with nitrogen gas. To this was added copper(I) iodide (0.044 g, 0.233 mmol) and bis(triphenylphosphine) palladium dichloride (0.163 g, 0.233 mmol). The mixture was purged again with nitrogen gas and was stirred at r.t. for 10 min. To the mixture was added a solution of methyl 3-ethynylbenzoate (0.298 g, 1.86 mmol) in triethylamine (2.2 mL, 15.5 mmol) in a dropwise manner. The mixture was stirred at 90 °C for 16 h and then concentrated in vacuo. The residue was purified by silica gel chromatography


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(EtOAc / heptane) to give methyl 3-((5-amino-4-bromoisothiazol-3-yl)ethynyl)benzoate 59 (243 mg, 46% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.08 (t, J = 1.4 Hz, 1H), 8.05 - 8.03 (m, 1H), 7.88 (dt, J = 7.7, 1.3 Hz, 1H), 7.64 (t, J = 7.9 Hz, 1H), 7.07 (s, 2H), 3.89 (s, 3H). MS (ESI) m/z 339.1 [M + H]+. 1-(5-amino-2-chloropyridin-4-yl)-3-(4-bromo-3-((3-(hydroxymethyl)phenyl)ethynyl) isothiazol-5-yl)urea (16). Step 1. To an ice-cold mixture of 59 (190 mg, 0.56 mmol) and 42 (182 mg, 0.62 mmol) in DMF (5 mL) was added LHMDS (1M in THF, 0.84 mL, 0.84 mmol). The mixture was allowed to warm to r.t. and stirred for 16 h, whereupon it was quenched with water and extracted with EtOAc. The combined organic portion was washed with brine and dried over MgSO4. The crude residue obtained after filtration and concentration in vacuo was purified by silica gel chromatography (EtOAc / heptane) to give methyl 3-((4-bromo-5-(3-(2-chloro-5nitropyridin-4-yl)ureido)isothiazol-3-yl)ethynyl)benzoate 60 (259 mg, 42% yield). MS (ESI) m/z 536.0 [M + H]+. Step 2. A mixture of 60 (152 mg, 0.283 mmol), iron powder (63 mg, 0.113 mmol) and ammonium chloride (121 mg, 2.26 mmol) in ethanol (6 mL) and water (3 mL) was heated to 75 °C for 1 h. The mixture was filtered through Celite, washed with methanol and concentrated in vacuo. The crude residue was dissolved in EtOAc and washed with water, then the organic layer was dried over brine, followed by sodium sulfate and filtered. The filtrate was concentrated in vacuo, then purified by silica gel chromatography (EtOAc/heptane) to obtain methyl 3-((5-(3-(5amino-2-chloropyridin-4-yl)ureido)-4-bromoisothiazol-3-yl)ethynyl)benzoate 61 (64 mg, 44 % yield). MS (ESI) m/z 506.0 [M + H]+.


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Step 3. To a solution of LAH (1M in THF) (0.63 mL, 0.63 mmol) in THF (3 mL) stirring at 0 °C was added dropwise a solution of 61 (64 mg, 0.126 mmol) in THF (1 mL). The reaction was allowed to warm to r.t. over 10 min, after which methanol was added dropwise until bubbling stopped. The mixture was concentrated in vacuo and purified by HPLC (basic method 3) to obtain 16 (5 mg, 8% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.17 (s, 1H), 7.62 (s, 1H), 7.52 (s, 1H), 7.47 - 7.36 (m, 3H), 5.30 (t, J = 5.8 Hz, 1H), 5.18 (s, 2H), 4.53 (d, J = 5.6 Hz, 2H). HRMS calculated for C18H14BrClN5O2S (M+H) 477.9740, found 477.9738. Thermodynamic solubility assay. Thermodynamic solubility was determined using a shake flask method. Samples were prepared by weighing dry powder in a glass vial and adding buffer (0.067 M potassium phosphate buffer, pH 6.8 or 0.1 N HCl, pH 1.0) to create a saturated solution with a 1 mg/mL final target concentration. The solution was continuously agitated at room temperature for 16-24 h. Solution was filtered through 0.45 micron PVDF filters. Quantification of filtered solute was performed by HPLC-DAD using a four-point calibration curve. No to little sample loss to filters is assumed based on internal comparisons of filtration versus centrifugation and external validations. Isolation of recombinant BRM ATPase-SnAC domain. Cloning of His10-ZZ-HCV3C-BRM(636-1331) into pFastBac1. Sequences encoding a His10 tag (SEQ ID NO: 1), the immunoglobulin G (IgG) binding ZZ domain of protein A (Staphylococcus aureus) and a human rhinovirus 3C protease site were fused upstream of BRM residues 636-1331 using standard DNA synthesis methods. The synthesized construct was cloned into the MCS of pFastBac1 (Life Technologies) by PCR amplification using the following 5’ and 3’ primers: 5’-GACCGAACTAGTATGGCTTCTCACCACCAT-3’ (SEQ ID NO: 2) and 5’AGCGTTAAGCTTTTAATCCTCGATGGCGCG-3’ (SEQ ID NO: 3) to include a stop codon and


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ligated into SpeI and HindIII sites using standard molecular biology techniques. The final recombinant vector, pFB1-His10-ZZ-HCV3C-BRM (636-1331), results in the expression of a HCV3C protease-cleavable His10-ZZ tag upstream of native BRM sequences encoding the ATPase and SnAC domains. MASHHHHHHHHHHAQHDEAVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDD PSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDP SQSANLLAEAKKLNDAQAPKVDANGGGGSGGGGSLEVLFQGPEESDSDYEEEDEEEES SRQETEEKILLDPNSEEVSEKDAKQIIETAKQDVDDEYSMQYSARGSQSYYTVAHAISER VEKQSALLINGTLKHYQLQGLEWMVSLYNNNLNGILADEMGLGKTIQTIALITYLMEHK RLNGPYLIIVPLSTLSNWTYEFDKWAPSVVKISYKGTPAMRRSLVPQLRSGKFNVLLTTY EYIIKDKHILAKIRWKYMIVDEGHRMKNHHCKLTQVLNTHYVAPRRILLTGTPLQNKLP ELWALLNFLLPTIFKSCSTFEQWFNAPFAMTGERVDLNEEETILIIRRLHKVLRPFLLRRL KKEVESQLPEKVEYVIKCDMSALQKILYRHMQAKGILLTDGSEKDKKGKGGAKTLMNT IMQLRKICNHPYMFQHIEESFAEHLGYSNGVINGAELYRASGKFELLDRILPKLRATNHR VLLFCQMTSLMTIMEDYFAFRNFLYLRLDGTTKSEDRAALLKKFNEPGSQYFIFLLSTRA GGLGLNLQAADTVVIFDSDWNPHQDLQAQDRAHRIGQQNEVRVLRLCTVNSVEEKILA AAKYKLNVDQKVIQAGMFDQKSSSHERRAFLQAILEHEEENEEEDEVPDDETLNQMIAR REEEFDLFMRMDMDRRREDARNPKRKPRLMEEDELPWIIKDDAEVERLTCEEEEEKIFG RGSRQRRDVDYSDALTEKQWLRAIED (SEQ ID NO: 4) Expression of BRM (636-1331). The recombinant vector generated above was used to make recombinant bacmid by transforming to DH10Bac cells using standard protocols as detailed by the manufacturer (Life Technologies). High titer P3 virus was generated by transfecting the bacmid to Spodoptera frugiperda 9 (Sf9) cells and amplifying the virus using standard methods as detailed


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by Life Technologies. His10-ZZ-HCV3C-BRM (636-1331) was expressed from 25 L of Sf9 cells in log phase growth (1.5x106 cells/mL) in a WAVE bioreactor (GE Healthcare Life Sciences) at a 1:100 v/v of virus. The infection was allowed to proceed on the rocking incubator at 27 °C and harvested three days post infection after cell viability had dropped to 80% with an increase in the overall cell diameter consistent with infection. Cells were harvested at 4,000xg for 20 min, flash frozen and stored at –80 °C until use. Purification of BRM (636-1331). Sf9 cells expressing recombinant His10-ZZ-HCV3CBRM(636-1331) were lysed in 50 mM Tris (8.0), 300 mM NaCl, 10% glycerol and 2 mM TCEP supplemented with a protease inhibitor cocktail (Roche cOmplete) , using 7.5 mL lysis buffer per gram of cell paste. Cells were lysed upon thawing, homogenized and subsequently clarified in a JA25.50 rotor at 50,000xg for 30 min to remove insoluble material. The clarified lysate was applied to a 5 ml His-Trap HP column (GE Healthcare Life Sciences), washed rigorously in lysis buffer without protease inhibitors supplemented with 25 mM imidazole. Bound protein was eluted over a fifteen column volume gradient against lysis buffer supplemented with 500 mM imidazole. Fractions containing His10-ZZ-HCV3C-BRM (636-1331) were pooled and dialyzed overnight against 50 mM Tris (8.0), 300 mM NaCl, 10% glycerol and 2 mM TCEP supplemented with HCV3C protease to effect removal of the His10-ZZ tag. Cleavage was monitored by coomassiestained SDS/PAGE and LC/MS. The intact mass was consistent with BRM residues 636-1331 proceeded by two non-native amino acids, Gly-Pro, a residual of the HCV3C cleavage site. The expected mass was 160 Da greater than predicted, consistent with two phosphorylation sites. The cleaved product was diluted in dialysis buffer not supplemented with salt to a final NaCl concentration of 100 mM, passed thru a 0.2 μm syringe filter and immediately loaded to a 1 mL HiTrap Q HP column (GE Health Biosciences) previously equilibrated in 50 mM Tris (8.0), 100


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mM NaCl, 10% glycerol & 1 mM TCEP. Following capture, the bound protein was competed against the same buffer supplemented with 1 M NaCl. Fractions containing BRM (636-1331) were pooled and loaded to a S200 16/60 size exclusion column equilibrated in 50 mM Tris (8.0), 200 mM NaCl, 10% glycerol & 2 mM TCEP. The purified construct was concentrated to 2.5 mg/mL, flash frozen and stored at –80 °C until used in downstream assays. BRM ATPase-SnAC ATPase inhibition assay. Compound inhibition of ATPase activity of Brm ATPase-SnAC (636-1331) was measured by using the ADP-Glo assay kit from Promega (V6930). 120 nL of compound in 100% DMSO were transferred to a white 384 well microtiter assay plate using an ATS Acoustic Transfer System from EDC Biosystems. All subsequent reagent additions were performed using a MultiFlo FX Multi-Mode Dispenser. Assay buffer was 20 mM HEPES pH 7.5, 1 mM MgCl2, 20 mM KCl, 1 mM DTT, 0.01% BSA, 0.005% Tween 20. 4 µL of 7.5 nM Brm ATPase-SnAC in assay buffer was added to the assay plate and incubated at room temperature for 5 min with compound. 2 µL of 255 µM ATP and 6 nM pCMV-dR8.91 plasmid in assay buffer was added to assay plate to initiate the reaction. The final concentrations of reagents were 5 nM Brm ATPase-SnAC, 85 µM ATP, and 2nM pCMV-dR8.91 plasmid. The ATPase reaction was incubated at room temperature for 60 min. 3 µL of ADP-Glo reagent was added to stop the reaction and was incubated for 30 min at room temperature. 3 µL of kinase detection reagent was added to the assay plate which was incubated for 90 min at room temperature. Plates were read with a 2103 Multilabel Envision reader using ultrasensitive luminescence detection. IC50 values were determined from the average of duplicate data points by non-linear regression analysis of percent inhibition values plotted versus compound concentration. Isolation of recombinant BRG1 ATPase-SnAC Domain. Cloning of BRG1(658-1361)-His6 into pDEST8. The construct BRG1(658-1361)-His6 for expression in insect cells was sub-cloned


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from a full length BRG1 plasmid, pDONR221-BRG1-His6 (OPS7173) by PCR as follows. An ATTB flanked PCR fragment encoding BRG1(658-1361)-His6 was generated using the following primers:

Forward,

ATTB1

BRG1(658-x)

5’-

GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATGGA AGAAAGTGGCTCAGAAGAAGAGGAAG

(SEQ

ID

NO:

5);

Reverse,

1361)HISstpATTB2rev,

BRG1(x5’-

GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGTGATGATGATGATGATGCTCCTC GATGGCCTTGAGCCACTGC (SEQ ID NO: 6). This PCR fragment was recombined into the vector pDEST8 using the Gateway® method following the manufacturer’s protocol (Life Technologies). The insertion was confirmed by sequencing and entered into the OPS database (OPS8023) before proceeding to bacmid generation. MEESGSEEEE IENAKQDVDD

EEEEEEQPQA EYGVSQALAR

AQPPTLPVEE

KKKIPDPDSD

DVSEVDARHI

GLQSYYAVAH

AVTERVDKQS

ALMVNGVLKQ

YQIKGLEWLV SLYNNNLNGI LADEMGLGKT IQTIALITYL MEHKRINGPF LIIVPLSTLS NWAYEFDKWA KHILAKIRWK

PSVVKVSYKG YMIVDEGHRM

SPAARRAFVP KNHHCKLTQV

QLRSGKFNVL

LTTYEYIIKD

LNTHYVAPRR

LLLTGTPLQN

KLPELWALLN FLLPTIFKSC STFEQWFNAP FAMTGEKVDL NEEETILIIR RLHKVLRPFL LRRLKKEVEA KGKGGTKTLM RASGKFELLD

QLPEKVEYVI NTIMQLRKIC RILPKLRATN

KCDMSALQRV NHPYMFQHIE HKVLLFCQMT

LYRHMQAKGV

LLTDGSEKDK

ESFSEHLGFT

GGIVQGLDLY

SLMTIMEDYF

AYRGFKYLRL

DGTTKAEDRG

MLLKTFNEPG

SEYFIFLLST

RAGGLGLNLQ

SADTVIIFDS

DWNPHQDLQA

QDRAHRIGQQ

NEVRVLRLCT

VNSVEEKILA

AAKYKLNVDQ

KVIQAGMFDQ

KSSSHERRAF

LQAILEHEEQ

DEEEDEVPDD

ETVNQMIARH


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Page 49 of 66 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


EEEFDLFMRM

DLDRRREEAR

NPKRKPRLME

EDELPSWIIK

DDAEVERLTC

EEEEEKMFGR GSRHRKEVDY SDSLTEKQWL KAIEEHHHHH H (SEQ ID NO: 7) Expression of BRG1(658-1361)-His6. The recombinant vector generated above was used to make recombinant bacmid by transforming to DH10Bac cells using standard protocols as detailed by the manufacturer (Life Technologies). High titer P3 virus was generated by transfecting the bacmid to Spodoptera frugiperda 9 (Sf9) cells and amplifying the virus using standard methods as detailed by Life Technologies. BRG1 (658-1361)-His6 was expressed from Sf9 cells in log phase growth (1.5-3.9x106 cells/mL) at 15 virus/cell. The infection was allowed to proceed on the rocking incubator at 27 °C and harvested three days post infection after cell viability had dropped to 80% with an increase in the overall cell diameter consistent with infection. Cells were harvested at 4,000xg for 20 min, flash frozen and stored at –80 °C until use. Purification of BRG1(658-1361)-His6. Sf9 cells expressing recombinant BRG1(658-1361)-His6 were lysed in 50 mM Tris (8.0), 300 mM NaCl, 5% glycerol and 1 mM TCEP supplemented with a protease inhibitor cocktail (Roche cOmplete) , using 7.5 mL lysis buffer per gram of cell paste. Cells were lysed upon thawing, homogenized and subsequently clarified in a JA25.50 rotor at 50,000xg for 30 min to remove insoluble material. The clarified lysate was applied to a 5 mL HisTrap HP column (GE Healthcare Life Sciences), washed rigorously in lysis buffer without protease inhibitors supplemented with 20 mM imidazole. Bound protein was eluted over a ten column volume gradient against lysis buffer supplemented with 250 mM imidazole. Fractions containing BRG1(658-1361)-His6 were pooled and diluted till conductivity reached about 6 mS/cm (~60 mM NaCl) using 50 mM Tris pH 8.0, 5% glycerol, and 1 mM TCEP, passed thru a 0.2 μm filter and immediately loaded to a 5 ml HiTrap Q HP column (GE Health Biosciences) previously equilibrated in 50 mM Tris (pH 8.0), 100 mM NaCl, 5% glycerol, and 1 mM TCEP. Following


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capture, the bound protein was competed against the same buffer supplemented with 1 M NaCl. Fractions containing BRG1(658-1361)-His6 were pooled and loaded to a S200 16/60 size exclusion column equilibrated in 50 mM Tris (8.0), 200 mM NaCl, 5% glycerol, and 1 mM TCEP. The purified construct was concentrated to 1 to 2.5 mg/mL, flash frozen and stored at –80 °C until used in downstream assays. BRG1 ATPase-SnAC ATPase inhibition assay. Compound inhibition of ATPase activity of Brg1 ATPase-SnAC (658-1361) was measured by using the ADP-Glo assay kit from Promega (V6930). 120 nL of compound in 100% DMSO were transferred to a white 384 well microtiter assay plate using an ATS Acoustic Transfer System from EDC Biosystems. All subsequent reagent additions were performed using a MultiFlo FX Multi-Mode Dispenser. Assay buffer was 20 mM HEPES pH 7.5, 1 mM MgCl2, 20 mM KCl, 1mM DTT, 0.01% BSA, 0.005% Tween 20. 4 µL of 7.5 nM Brg1 ATPase-SnAC in assay buffer was added to the assay plate and incubated at room temperature for 5 min with compound. 2 µL of 195 µM ATP and 6 nM pCMV-dR8.91 plasmid in assay buffer was added to assay plate to initiate the reaction. The final concentrations of reagents were 5 nM BRG1 ATPase-SnAC, 65 µM ATP, and 2 nM pCMV-dR8.91 plasmid. The ATPase reaction was incubated at room temperature for 60 min. 3 µL of ADP-Glo reagent was added to stop the reaction and was incubated for 30 min at room temperature. 3 µL of kinase detection reagent was added to the assay plate which was incubated for 90 min at room temperature. Plates were read with a 2103 Multilabel Envision reader using ultrasensitive luminescence detection. IC50 values were determined from the average of duplicate data points by non-linear regression analysis of percent inhibition values plotted versus compound concentration. Expression and purification of BRM full-length. Full-length BRM construct (C-terminal 6xHis and C-terminal FLAG-6xHis tag) was produced in Spodoptera frugiperda (Sf9) cells by


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infecting 1-5 L of cells at a density of 1.5x10^6 cells/mL with 3% volume of baculovirus encoding BRM. Infected cells were incubated at 27°C and harvested in 48 h. Cell pellets were stored at – 80°C. Cell pellets containing full-length BRM were lysed by Dounce disruption in buffer consisting of 50 mM Tris pH 8.0, 300 mM NaCl, 10% glycerol, 2 mM TCEP, 1 mM PMSF, 150 µM TPCK, 2 x Proteases Inhibitor Tablets-EDTA free (Thermo). Purification was initially performed by either Ni-Sepharose affinity column (GE Healthcare) via a 25-500 mM imidazole gradient over 15 CV or by Anti-FLAG affinity column (Sigma) by batch mode with a 2 h incubation at 4°C on a Nutator mixer, followed by 200 µM 3xFLAG peptide (Sigma) elution. Each mode was followed by Q-Sepharose anion exchange (0.1-1M NaCl gradient over 20CV), and Superdex-200 or Superose-6 Size Exclusion Chromatography (GE Healthcare). With the exception of the Ni-Sepharose step, all buffers contained 1-5 mM EDTA BRM Full length ATPase inhibition assay. Compound inhibition of ATPase activity of full length BRM (1-1572) was measured using the ADP-Glo assay kit from Promega (V6930). 120 nL of compound in 100% DMSO were transferred to a white 384 well microtiter assay plate using an ATS Acoustic Transfer System from EDC Biosystems. All subsequent reagent additions were performed using a MultiFlo FX Multi-Mode Dispenser. Assay buffer was 20 mM HEPES pH 7.5, 1 mM MgCl2, 20 mM KCl, 1 mM DTT, 0.01% BSA, 0.005% Tween 20. 4 µL of 7.5 nM full length BRM in assay buffer was added to the assay plate and incubated at room temperature for 5 min with compound. 2 µL of 270 µM ATP and 0.03 nM pCMV-dR8.91 plasmid in assay buffer was added to assay plate to initiate the reaction. The final concentrations were 5 nM enzyme, 90 µM ATP, and 0.01 nM pCMV-dR8.91 plasmid. The ATPase reaction was incubated at room temperature for 60 min. 3 µL of ADP-Glo reagent was added to stop the reaction and was incubated for 30 min at room temperature. 3 µL of kinase detection reagent was added to the assay


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plate which was incubated for 90 min at room temperature. Plates were read with a 2103 Multilabel Envision reader using ultrasensitive luminescence detection.

IC50 values were

determined from the average of duplicate data points by non-linear regression analysis of percent inhibition values plotted versus compound concentration. Differential scanning fluorimetry. Screening of compounds was carried out under the following assay conditions: 1 μM BRG1 ATPaseSnAC, 5× SYPRO Orange dye (5000× concentrate in DMSO; Life Technologies), 100 mM HEPES (pH 7.0), 100 mM NaCl, 2 mM MgCl2, 5% Glycerol, 0.5 mM TCEP, and 5% DMSO. The final compound concentration evaluated was 100 μM. Screening of compounds was also done with additional 500 μM ADP using the same assay conditions. To carry out the experiment, the DSF assay solution (9.5 μL) was dispensed into an assay plate (LightCycler; 480 Multiwell Plate 384 White) containing 500 nL of compound dissolved in DMSO then mixed. The final assay volume was 10 μL per well in a 384-well format. Plates were then sealed after reagent addition, centrifuged at 1000 rpm for 1 minute, and read on a Bio-Rad C1000 Thermal Cycler with a CFX384 Real Time System using an excitation of 465 nm and an emission at 580 nm. The temperature was ramped from 25 °C to 80 °C and measurements were taken at 0.5 °C increments. The melting temperature (Tm) of the raw fluorescence data was identified as the midpoint of the transitions via a semi-parametric fit. The ΔTm was determined by comparing the individual Tm values for each compound with the mean Tm of the apo protein controls (32 per plate) containing DMSO only. Surface plasmon resonance assay. SPR experiments were performed using a Biacore T100 instrument at 10 °C. Approximately 2000 SPR response units (RU) of N-terminal Avi-tagged BRM ATPase-SnAC was captured onto a Biacore streptavidin (SA) chip sensor surface in assay buffer containing 20 mM HEPES pH 7.5, 1 mM MgCl2, 200 mM KCl, 1 mM DTT, 0.005% Tween


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20, 2% DMSO, and +/- 500 µM ADP depending on the experimental format. Flow cell 1 was left blank as a reference. Compound 1 was injected for 60 sec at a flow rate of 100 µL/min, with a 120 sec dissociation. All data were solvent corrected and double referenced using a blank injected immediately prior to the compound dose response. The data were analyzed using Biacore T100 evaluation software and fit to a simple 1:1 binding model. Isothermal Titration Calorimetry. ITC experiments were performed using an Auto ITC titration calorimetric system (MicroCal ITC from Malvern Panalytical). For the determination of the direct binding of compounds to various BRM and BRG1 proteins, the calorimetric cell, which contained purified protein in 100 mM HEPES (pH 7.0), 100 mM NaCl, 2 mM MgCl2, 5% Glycerol, 0.5 mM TCEP, and 5% DMSO, was titrated with compounds dissolved in the same buffer. The titration experiments were performed by adding the titrant in 13 steps of 3 μL or 20 steps of 2 μL at 25 °C. Expression and purification of MBP fusion N-terminal RecA domain of BRM (abbreviation MBP- hBRM[705-960]). The N-terminal RecA domain of human BRM (containing residues 705 to 960) bearing a C-terminal 6xHis tag was sub-cloned into pMAL-c5X vector (New England BioLabs Inc.) with residues NAAA as linker between maltose-binding protein (MBP) and BRM.29 E. coli strain BL21 Star™ (DE3) (ThermoFisher) transformed with the MBP-hBRMNRecA-6His expression construct was grown at 37 °C in shaker flasks to an OD600 of 1.6 in Terrific Broth (Teknova) with 100 μg/ml of Carbenicillin, then cooled down to below 16 °C. Protein expression was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to 1 mM for 18 hours at 16 °C. The harvested cells were resuspended in lysis buffer (50mM Mes pH 6.5, 500 mM NaCl, 2 mM MgCl2, 20 mM imidazole, 5% Glycerol, 1 mM TCEP) containing DNAse I and protease inhibitors (cOmplete EDTA-free protease inhibitor tablets, 1


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tablet per 50 mL of buffer), and lysed on ice using a microfluidizer (M-110L, Microfluidics). The cleared lysate containing MBP-hBRMNRecA[705-960] fusion protein was then loaded onto a 5 mL HisTrap column (GE Healthcare), and eluted with Ni Elution Buffer (50 mM Mes pH6.5, 500 mM NaCl, 400 mM imidazole, 5% Glycerol, 1 mM TCEP). Peak eluted fractions were pooled and dialyzed against S-A buffer (50 mM Mes pH 6.5, 200 mM NaCl, 5% Glycerol, 1 mM) for 18 hours at 4 °C. The dialyzed material was then diluted with S-0 buffer (50 mM Mes pH 6.5, 5% Glycerol, 1 mM TCEP) to a final NaCl concentration of 100 mM and loaded onto a 5 mL HiTrap S FF column (GE Healthcare) equilibrated with S-A buffer. Following capture, the bound protein was eluted using the same buffer supplemented with 1 M NaCl. Fractions containing MBPhBRM[705-960] were pooled and loaded to a HiLoad 16/60 Superdex 200 column (GE Healthcare) equilibrated in S-A buffer. The purified MBP-hBRM[705-960] was concentrated to 12 mg/ml, flash frozen and stored at –80 °C until use in downstream assays. Crystallization, data collection and structure determination. To obtained crystals of BRM:compound complex, MBP-hBRM[705-960] at 12 mg/mL was incubated with compound at 2-5x molar excess of protein concentration. The complexes were crystallized using sitting drop vapor diffusion method at 4 °C by mixing equal volumes (200 nL + 200 nL) of protein:compound mixture and crystallization solution. Crystallization solution containing 0.1 M Tris pH 7.0 and 14% (v/v) ethanol yielded MBP-hBRM[705-960]:15 crystals and crystallization solution containing 0.1 M Hepes pH 7.5, 0.2 M NaCl and 8% (v/v) isopropanol produced MBP-hBRM[705960]:16 crystals. For the diffraction experiment, crystals were transferred briefly to a solution containing 30% glycerol in their corresponding crystallization solution then flash frozen in liquid nitrogen for X-ray diffraction data collection. Crystal of MBP-hBRM[705-960]:compound complex was determined to have the tetragonal space group P42212 with one complex in the


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asymmetric unit. Each complex comprises a polypeptide chain of MBP fusion protein and one compound molecule. A 2.84Å data set was collected and used for structure solution of MBPhBRM[705-960]:15. A 2.98Å data set was collected and used for structure solution of MBPhBRM[706-960]:16. All diffraction data were collected at the X-ray Operations and Research beamline 17-ID at the Advanced Photon Source, Argonne National Laboratory, with the crystal kept at 100 °K and wavelength of X-ray beam at 1.0 Å. The diffraction data from all crystals were integrated and scaled using autoPROC.30 The structures were solved by molecular replacement with PHENIX31,32 using MBP (PDB entry 1FQA)33 and Myceliophthora thermophile SNF2 (PDB entry 5HZR)34 starting models. Model building and refinement was performed using COOT35 and PHENIX.32,36 Statistics for the collected data and refined model are summarized in the Table (Supporting Information). PDB coordinates and accompanying structure factors have been deposed under PDB code 6EG3 for MBP-hBRM[705-960]:15 and 6EG2 for MBP-hBRM[705960]:16. KRT80-Luc reporter gene Assay. NCI- H1299 (CRL-5803) engineered to express luciferase under the control of the endogenous KRT80 promoter (CRISPR mediated Knock-In of NanoLuc) were maintained in RPMI-1640 + L-glutamine (Lonza) with 10 % Fetal Bovine Serum (Thermo Scientific) at 37°C with 5% CO2. The cells were split twice a week with dissociation reagent (0.05% Trypsin-EDTA Gibco Life technologies) and re-plated into 75 cm flasks (Corning). For the assay cells were plated at 500 cells per well in a 384 well plate (Corning), and incubated overnight at 37 °C 5% CO2 in 50 µL. The following day cells were treated with 50 nL compound, 10 point 3 fold dilution starting down from 10 µM using an acoustic dispenser. After 24 h incubation, plates were read with Nano-Glo Luciferase Assay System (Promega) according to manufactures instructions. Essentially 25 µL of reagent are added to each well and incubated for


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15 min room temperature and protected from light. Luminescence was then recorded (Wallac Envision plate reader) and the IC50 determined using an in house statistics package (HELIOS). Compound Treatments for Gene Expression Changes. Cell lines were plated 24 h prior to compound treatment at 150,000 cells/well in a 12-well plate. Compound was diluted in DMSO and then added to RPMI-1640+10% FBS. Using NCI-H1299 or RERF-LC-AI cell lines, compound was added to each plate in a 10-point dose response using a 1:3 dilution starting from 1 µM. Each compound was pre-diluted in DMSO, and subsequently added to RPMI-1640+10% FBS to a 20X working concentration. A total of 50 µL of each was added to 1 mL of growth media on the cells to achieve a final 1X concentration. DMSO (1%) was used as a negative control. Cells were treated with compound for 24 h, media was aspirated, and cells were washed with PBS. Final plates were frozen for future RNA Isolation. Preparation of RNA and cDNA. Each well was lysed using 350 µL Buffer RLT+BME and column purified using the RNeasy Plus protocol (Qiagen, #74134), Final RNA was eluted with 50 µL RNase/DNase-free water and quantitated using the Nanodrop to check for RNA purity. Using the ABI High Capacity cDNA synthesis kit (Applied Biosystems, #4368813), approximately 1 µg of Total RNA was used as input for a 20 µL cDNA synthesis reaction. Final cDNA product was then diluted 1:5 with Milli-Q water for a final yield of 100 µL of cDNA. Tumor samples were first homogenized using the Precellys-24 (Bertin) in combination with 600 µL buffer RLT+ BME and lysing matrix B (MP Biomedicals, #116913050). Final homogenate was spun at 10,000Xg for 10 min. RNA and cDNA was then isolated following the protocol listed above. Real Time PCR of KRT80. Each cDNA sample was used in subsequent Taqman Real Time PCR assays to verify KRT80 (IDT, Hs.PT.58.27334718.g) gene expression. A total of 3 µL of


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cDNA was used for each 12.5 µL Taqman Real Time PCR reaction on a 384-well plate with Fast Start Universal Master Mix (Roche, #4914058001). TBP (Applied Biosystems, #4326322E) was run independently as the housekeeping control for input normalization. Samples were run on the Applied Biosystems 7900HT or Biorad CFX Real Time PCR instrument. Cell Proliferation Assays. SKMEL5 (ATCC HTB-7) melanoma cells and SBC-5 small cell carcinoma (JCRB0819) were grown in RPMI-1640 + L-glutamine (Lonza) with 10 % Fetal Bovine Serum (Thermo Scientific) at 37 °C with 5% CO2. The cells were split twice a week with dissociation reagent (0.05% Trypsin-EDTA Gibco Life technologies) and re-plated into 75 cm flasks (Corning). For proliferation inhibition analysis cells were plated in a 384-well plate (Corning) at 2000 cells per well in 50 µL. The following day treated with compound, 10-point 3fold dilution starting down from 10 µM then assayed after 6 days. Growth inhibition was determined by quantifying the ATP concentration by the addition of Cell Titer-Glo® Luminescent Cell Viability Assay (Promega), 25 µL per well, followed by luminescence acquisition (Wallac Envision plate reader). IC50 for compounds were determined using an in house statistics package (HELIOS). In vivo efficacy and pharmacodynamics. Mice were maintained and handled in accordance with the Novartis Institutes for BioMedical Research (NIBR) Institutional Animal Care and Use Committee (IACUC) and all studies were approved by the NIBR IACUC. RERF-LC-AI tumor xenografts were generated by implanting 1x107 cells in 50% Matrigel subcutaneously into the right flank of female athymic nude mice (6–8 weeks old, Charles River). Mice were randomized into treatment groups. Tumor volumes and body weights were monitored twice per week and the general health condition of mice was monitored daily. Tumor volume was determined by measurement with calipers and calculated using a modified ellipsoid formula, where tumor


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volume (TV) (mm3) = [((l × w2) × 3.14159))/6], where l is the longest axis of the tumor and w is perpendicular to l. At the end of the efficacy study, tumor samples were collected at 7, 16, and 24 h post last treatment and analyzed for KRT80 mRNA expression as described above. For the 7.5 mg/kg and 20 mg/kg treatment groups, tumor samples were collected for PD analysis post 3 weeks of daily dosing. For the 30 mg/kg group, tumor samples were collected post four daily doses in the third week of compound treatment. All dosing groups were formulated in a suspension vehicle (0.5% methyl cellulose, 0.5% tween 80 in water). ASSOCIATED CONTENT Supporting Information Summary of X-ray diffraction data collection and structure refinement, difference density maps and HPLC traces for compounds 2 and 5-14 (PDF); molecular formula strings (CSV) Accession Codes PDB codes are the following: 6EG2 (MBP-hBRM[705-960] in complex with compound 16); 6EG3 (MBP-hBRM[705-960] in complex with compound 15). Authors will release the atomic coordinates and experimental data upon article publication. AUTHOR INFORMATION Corresponding Author * Phone: +1 617 871 7653, e-mail: [email protected] ORCID Julien P.N. Papillon: 0000-0002-8952-5289


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Katsumasa Nakajima: 0000-0001-8295-5086 Present Affiliations † (C.D.A.) Canopy Growth Corporation, Smiths Falls, Ontario K7A 3L1, Canada; (A.O.J.) Marcus Evans Group, Toronto, Ontario M5C 2W7, Canada; (S.M.) Kallyope, New York, New York 10016, United States; (A.B.) Twyrl Pasta Bistro, Arlington, Massachusetts 02474, United States; (E.A.C.) Memorial Sloan Kettering Cancer Center, New York, New York 10065, 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. Notes All animal experiments performed in the manuscript were conducted in compliance with institutional guidelines. The authors declare no competing financial interest. ACKNOWLEDGMENT Lukas Leder provided the ATPase-SnAC construct. Suzanne Skolnik and Gina Geraci conducted shake-flask assay solubility measurement. Pete Delgado, Cary Fridrich and Carina Sanchez contributed to material scale-up. ABBREVIATIONS AAC50, absolute AC50; CSD, Cambridge Structure Database; DAST, diethylaminosulfur trifluoride; DIPEA, Diisopropylethylamine; DSF, Differential Scanning Fluorimetry; ITC,


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