âOptimization of potent and selective Ataxia Telangiectasia Mutated

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“Optimization of potent and selective Ataxia Telangiectasia Mutated (ATM) inhibitors suitable for a proof-ofconcept study in Huntington’s disease models” Leticia Marisel Toledo-Sherman, Perla Breccia, Roger Cachope, Jennifer Ruth Bate, Ivan AnguloHerrera, Grant Wishart, Kim Lisa Matthews, Sarah L Martin, Helen C Cox, George McAllister, Stephen D Penrose, Huw Vater, William Esmieu, Amanda Van de Poël, Rhea Van de Bospoort, Annelieke Strijbosch, Marieke B. A. C. Lamers, Philip M. Leonard, Rebecca Jarvis, Wesley Blackaby, Karen Barnes, Maria Eznarriaga, Simon Dowler, Graham D Smith, David Fischer, Ovadia Lazari, Dawn Yates, Mark Rose, Sung-Wook Jang, Ignacio Muñoz-Sanjuan, and Celia Dominguez J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01819 • Publication Date (Web): 06 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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Journal of Medicinal Chemistry

“Optimization of potent and selective Ataxia Telangiectasia Mutated (ATM) inhibitors suitable for a proof-of-concept study in Huntington’s disease models” Leticia Toledo-Sherman,*‡ Perla Breccia,*† Roger Cachope,‡ Jennifer R. Bate,† Ivan AnguloHerrera,† Grant Wishart,† Kim L. Matthews,† Sarah L. Martin,† Helen C. Cox,† George McAllister,‡ Stephen D. Penrose,† Huw Vater,† William Esmieu,† Amanda Van de Poël,† Rhea Van de Bospoort,† Annelieke Strijbosch,† Marieke Lamers,† Philip Leonard,† Rebecca E. Jarvis,†,1 Wesley Blackaby,†,2 Karen Barnes,† Maria Eznarriaga,† Simon Dowler,† Graham D. Smith,† David F. Fischer, † Ovadia Lazari,† Dawn Yates,† Mark Rose,‡ Sung-Wook Jang,‡ Ignacio Muñoz-Sanjuan,‡ Celia Dominguez.‡ † Charles River, Chesterford Research Park, CB10 1XL, United Kingdom & Leiden, 2333 CR, Netherlands. ‡CHDI Management/CHDI Foundation, 6080 Center Drive, Los Angeles, CA 90045, United States 1

Current address: Neuroscience, IMED Biotech Unit, AstraZeneca, Cambridge CB21

6GH, United Kingdom. 2

Current address: Storm Therapeutics Ltd., Babraham Research Campus, Cambridge,

United Kingdom. 1 ACS Paragon Plus Environment

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*These authors contributed equally to the manuscript KEYWORDS: ATM, DNA Damage, HD, CNS kinase inhibitor, HTT

Abstract

Genetic and pharmacological evidence indicates that reduction of ataxia telangiectasiamutated (ATM) kinase activity can ameliorate mutant huntingtin (mHTT) toxicity in cellular and animal models of Huntington’s disease (HD), suggesting that selective inhibition of ATM could provide a novel clinical intervention to treat HD. Here we describe the development and characterization of ATM inhibitor molecules to enable in vivo proof-of-concept studies in HD animal models. Starting from previously reported ATM inhibitors, we aimed with few modifications to increase brain exposure by decreasing P-glycoprotein (P-gp) liability, while maintaining potency and selectivity. Here we report brain-penetrant ATM inhibitors that have robust pharmacodynamic (PD) effects consistent with ATM kinase inhibition in mouse brain and an understood pharmacokinetic/pharmacodynamic (PK/PD) relationship. Compound 17, engages ATM kinase and shows robust dose-dependent inhibition of X-ray irradiation-induced KAP1 phosphorylation in mouse brain. Furthermore, compound 17 protects against mHTT (Q73)induced cytotoxicity in a cortical-striatal cell model of HD.

INTRODUCTION Ataxia telangiectasia-mutated kinase (ATM) is a large (350 kDa) serine/threonine kinase that belongs to the phosphatidylinositol-3-kinase-like kinase (PIKK) family that includes ATR, mTOR, DNA-PK and SMG-1. ATM is involved in the DNA damage response and plays a central role in maintaining genome integrity by regulating the detection and repair of DNA double-strand breaks. ATM is predominantly localized in the cell nucleus as a 2 ACS Paragon Plus Environment

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homodimer and is subject to tight regulation. In response to DNA damage, ionizing radiation or oxidative stress, ATM auto-phosphorylates and is converted to a monomeric active form that initiates the phosphorylation of a number of substrates involved in cell DNA repair, cell-cycle checkpoints or apoptosis, including H2AX, KAP1, p53, Mdm2, CHK2, NSB1, BRAC1 and FancD2.1,2,3 More recently, other roles for ATM have been proposed, suggesting an involvement of ATM in several cellular homeostasis pathways (protein synthesis, glucose uptake in response to insulin, mitochondrial homeostasis, mitophagy, modulation of the pentose phosphate cycle and inhibition of mTOR).3 Functions of ATM in the brain have also been investigated,4 suggesting that ATM is required for apoptosis of post-mitotic neurons under genotoxic or oxidative stress.5 The ATM signalling pathway has been reported to be dysregulated in several neurodegenerative disorders6 including Huntington’s disease (HD).7 HD is an autosomal dominant, progressive neurodegenerative disease caused by an expanded polyglutamine (polyQ) tract in the N-terminal region of mutant huntingtin (mHTT).8 Despite extensive efforts aimed at understanding how the polyQ expansion leads to HD pathology, no disease-modifying therapies and only limited symptomatic treatments are available for HD.9 Evidence that ATM inhibition could be therapeutically relevant in HD has recently emerged. HTT has also been proposed as a scaffolding protein in the ATM oxidative DNA damage response complex.10 Cells transfected with mHTT fragments showed elevated DNA damage and ATM activation.11 ATM signalling is higher in cells derived from HD patients12 and from HD mouse models compared to controls.13 Genetic reduction of ATM gene dosage by one copy (ATM+/- mice) ameliorated multiple behavioural deficits and partially improved neuropathology when such mice were crossed with the BacHD mouse model7 and, consistent with a benefit from a reduction of ATM kinase activity, a small-molecule ATM kinase inhibitor 3 ACS Paragon Plus Environment


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ameliorated mHTT-induced cell death in both rat primary striatal neurons and in induced pluripotent stem cells derived from HD patients.7 These two studies support the idea that inhibition of ATM activity could offer a novel clinical intervention strategy to treat HD.7 Selective and brain-penetrant inhibitors of ATM kinase could therefore have utility in the treatment of HD. Additionally, identifying a pharmacodynamics (PD) biomarker in the central nervous system would enable monitoring ATM inhibition in vivo and aid the selection of molecules for further efficacy testing. Several selective ATM kinase inhibitors14, including KU-6001915 and CP-46672216, have been reported (Figure 1). AstraZeneca (AZ) has described a series of 3-quinoline carboxamides (3) and more recently 3-cinnoline carboxamides as new classes of potent, selective and orally bioavailable ATM kinase inhibitors.17 Further optimisation of this series led to the identification of a recently disclosed clinical candidate AZD0156,18 currently in Phase I clinical trials as a mono and combination therapy for solid tumour indications. More recently ATM brain penetrant inhibitors for glioblastoma were reported by the same group.19

4 ACS Paragon Plus Environment

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O

O

O N

O

N

S

O

S

O S

O

O

KU-55933 1a

KU-60019 1b

O

N

N

N

N

N H

CP-466722 2 O

N N

N N N

O

O

O

H 2N

NH

O

O

N

N N

O NH2

N

AZ 3-quinoline carboxamide 3

N

AZD0156 clinical candidate 4

Figure 1. Known ATM inhibitors At the start of our program, only the thianthrene 1a (biochemical IC50 value of 13 nM for ATM),20 thioxanthene 1b (biochemical IC50 value of 6 nM)21 and triazoloquinazoline 2 (cell IC50 0.48 µM for ATM in ICW cell MCF7 pKAP1)22 had been disclosed. We performed kinome scan assays to evaluate the selectivity (data not shown) of these compounds over other kinases, a key requirement of a proof-of-concept (PoC) tool for pharmacological validation of ATM kinase as HD target. We confirmed the high selectivity of KU-60019 for ATM, but found the compound to be equipotent against Vps34, a member of the class III PI3K kinases involved in cell autophagy which represented a potentially detrimental off target liability. However, evaluation of brain penetration in mouse revealed poor brain exposure. Consistent with the above, KU-60019 was shown to be poorly permeable (Papp 70 nm/s) and a P-glycoprotein (P-gp) substrate with an effective efflux ratio (EER) of 8.8 in an MDCK/MDR1 in vitro ADME assay. Nonetheless, given its superior selectivity profile and potency we chose KU-60019 as a starting point for optimization of central nervous system (CNS) properties to attain a brain penetrant ATM inhibitor tool compound for proof-of-concept (PoC) studies in mouse models of HD. In 5 ACS Paragon Plus Environment


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agreement with a recent perspective paper23 on small molecule kinase inhibitors for brain cancer treatment, which highlighted the challenge of identifying brain penetrant kinase inhibitors, we focused on optimizing permeability and decreasing P-gp efflux to maximize brain penetration. An atomic resolution structure of ATM did not exist at the onset of our program, therefore we created a homology model of the ATM kinase domain based on the human mTOR X-ray structure 4JSX.24 Using this model we proposed a binding mode for KU-60019 in order to identify the portions of the molecule not important for ATM activity and selectivity that are likely to impart poor passive permeability and P-gp efflux. Passive permeability and transportermediated efflux may be modulated by changes in a number of calculated physicochemical properties. Topological polar surface area (TPSA) and/or the number of hydrogen bond donors (HBD) have been correlated with the probability of P-gp mediated efflux.25 High molecular weight (MW) is also associated with P-gp recognition.26 Thus our optimization approach comprised a combination of computer-aided drug design and physicochemical property optimization to create compounds predicted to occupy CNS property space. The probability that a compound may be a substrate for P-gp was assessed by measuring effective efflux ratios in wild type Madin-Darby Canine Kidney (MDCK) cells and MDCK cells transfected with the human MDR1 gene encoding P-gp. The effective efflux ratio (EER), determined by comparing compound efflux ratios (ER) in the two cell types, was used to confirm the involvement of P-gp in compound efflux. Using this data we iteratively refined our guidelines for physicochemical parameters for compound prioritization and optimization. Prospective generation of physicochemical properties and target prioritisation based upon these parameters was fundamental to the identification of the brain penetrant analogues of KU-60019 described herein. RESULTS AND DISCUSSION 6 ACS Paragon Plus Environment

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Compounds reported herein were synthesized as shown in Schemes 1–2. Close analogs of KU-60019, amine- and amide-substituted thioxanthenes were prepared by the route in Scheme 1 adapted from precedent literature reports.15 Bromide intermediate 7 was coupled to 2chloro-6-morpholino-4H-pyran-4-one15 which, following Boc deprotection, afforded key amino intermediate 9. Amides 10-12 were obtained after reaction of intermediate 9 with the corresponding acid chloride according to the described procedure.15 Analog 13 was synthesized via reductive amination of 3-methyloxetane-3-carbaldehyde and 5-bromo-9H-thioxanthen-2amine, followed by boronate formation and Suzuki coupling with 2-chloro-6-morpholino-4Hpyran-4-one. Scheme 1a

NO2

NO2 a

Br

b,c

S

CO2H

Br

6

O f, g

h,i or h,j

O

7

O

O N

N

R1 = H; R2=

O

O

S O

S

Br

5

N

d,e

S

CO2H

Br

NHBoc

NO2

11

S O

NHBoc 8

9, R1= R2= H

N R1

R2

O

12

O

7

h, k, f, g

O

13

R1 = Me; R2=

10

13 14

aReagents

and conditions: (a) 2-bromothiophenol, KOH, H2O, reflux, 3 h, 97%; (b) MsOH, 150 °C, 1.5 h, 92%; (c) BH3.DMS, 0 °C to rt, 16 h, 83%; (d) Zn, AcOH, 0 °C to rt, 2 h, 87%; (e) Boc2O, THF, 50 °C, 48 h, 92%; (f) B2pin2, [Pd(dppf)(Cl)2][CH2Cl2], KOAc, dioxane, 100 °C, 24 h, 94%; (g) 2-chloro-6-morpholino-4H-pyran-4-one, Pd(PPh3)4], Cs2CO3, dioxane, 100 °C, 74%; (h) TFA, CH2Cl2, rt, 80%; (i) RCOCl, Et3N, DMA, rt , 17 h; (j) 1. bromoacetyl chloride, Et3N, DMA, rt, 16 h; 2. pyrrolidine, Et3N, rt, 17 h, 14%; (k) 3-methyloxetane-3carbaldehyde, MgSO4, 1,2-DCE, rt, 4 d, then NaBH3CN, rt, 24 h, 18%.



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Ether-substituted thioxanthenes were prepared by the route in Scheme 2. Ullmann-type coupling of 2-bromothiophenol and 2-bromo-5-methoxybenzoic acid gave the sulfide product 15 in moderate yield. Treatment with trifluoroacetic anhydride produced the tricyclic ketone intermediate in excellent yield. Reduction of the carbonyl with borane followed by borylation and Suzuki reaction gave pyranone 17. Demethylation with boron tribromide at 0 °C liberated the phenolic oxygen 18, which was alkylated either through Mitsunobu coupling with aliphatic alcohols or by reaction with alkyl halides to afford 19 to 47 (Table 2, 3 and 4). Alkylation of the thioxanthene benzylic position of 16a followed by Suzuki coupling afforded compounds 48 to 51 (Table 5). Scheme 2a

a

b,c

S O

15

O

16a

17

O

O N f

O

O

S

R1

R

O N

Br

O

19-47 O

S

O

O

OH

18

k

N

g or h-i or j

S

16a

O

Br

CO2H

Br

CO2H

N

d,e

S

S

Br

O

O

O

O

d,e

R2 16b

R1

O S O

R2

O

48-51

aReagents

and conditions: (a) 2-bromothiophenol, K2CO3, CuO, Cu powder, methoxyethanol, reflux, 3 h, 33%; (b) TFAA, BF3.OEt2, CH2Cl2; 0 °C- rt, 20 h, 87%; (c) BH3.DMS, 0 °C- rt, 16 h, 78%; (d) B2pin2, [Pd(dppf)Cl2][CH2Cl2], KOAc, dioxane, 100 °C, 24 h, 100%; (e) 2-chloro-6-morpholino-4H-pyran-4-one, [Pd(PPh3)4], Cs2CO3, dioxane, 95 °C; 60%; (f) BBr3, CH2Cl2, 0 °C- rt, 83%; (g) ROH, PPh3, DIAD, THF, DMF, rt to 70 °C; (h) NaH, dibromoethane, DMA, 0 °C- rt, 16 h, 84%; (i) amine, DMA, DIPEA, rt, 48 h; (j) alkyl halide, Cs2CO3, DMA, 60 °C, 16 h; (k) NaH, DMSO, 0 °C- 65 °C then alkyl halide, THF, rt, 2 h. 8 ACS Paragon Plus Environment

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We evaluated each compound’s ability to inhibit ATM kinase using biochemical and cellular assays that monitored phosphorylation of ATM substrates. The biochemical assay measured phosphorylation of full length p53 at Ser15, while the cell-based assay measured inhibition of etoposide-induced phosphorylation of KAP1 at Ser824.27 Hypotheses for compound binding to ATM and selectivity against other kinases were deemed critical for guiding the optimization process in order to maintain appropriate compound potency and kinase selectivity. Homology models for human ATR and DNA-PK were also generated from the human mTOR X-ray structure 4JSX and used alongside the published mTOR X-ray structures and ATM homology model to generate docking-based compound mode of binding hypotheses and kinase selectivity. A binding mode of the benchmark KU-55933 1a, established by Induced Fit Docking using Glide28 was consistent with existing SAR (see Supporting Information). In our working hypothesis the morpholine moiety of 1a accepts a hydrogen bond from the backbone NH of hinge residue Cys2770. The pyran-4-one carbonyl is orientated towards the back of the pocket in the vicinity of Lys2717. The proximal phenyl ring A of the thianthrene is out of plane of the pyran-4-one and forms hydrophobic contacts with residues in the phosphate binding loop. The curvature of the thianthrene system places the distal phenyl C ring between Trp2769 and the hydrophobic residues at the base of the binding site Pro2775 and Leu2877 (Figure 2).



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Figure 2. Preferred docked pose of 1a within human ATM homology model Comparing the ATP binding site of ATM kinase to DNA-PK, ATR and mTOR reveals two key regions that vary across the family that may contribute to the selectivity of the thianthrene and thioxanthene series, respectively. Leu2715 within the phosphate binding loop of ATM is conserved in DNA-PK and mTOR, but not in ATR, which has a methionine residue at this position and is likely to pose a steric clash with the first phenyl ring of the tricycle, and possibly with the pyran-4-one moiety (Figure 3).

Figure 3. (A) Protein superposition of the human ATM homology model and preferred docked pose of 1a (cyan ribbon, grey residues) with human ATR homology model (orange ribbon and residues) highlighting potential selectivity residues in thick sticks and steric clashes in red. (B) Protein superposition of the human ATM homology model and preferred docked pose of 1a 10 ACS Paragon Plus Environment

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(cyan ribbon, grey residues) with the human DNA-PK homology model (magenta ribbon and residues) highlighting potential selectivity residues in thick sticks and steric clashes in red

We hypothesize that for this compound series the presence of this methionine is key for compound selectivity against ATR. Additionally, a three residue motif towards the tip of the phosphate binding loop varies considerably across the PIKK family, where Ala2693-Gly2694Gly2695 is unique for ATM. Although these residues do not make direct contact with compound 1a in the preferred docked pose, it is reasonable to hypothesise that Gly2694-Gly2695 play a role in conformational flexibility of the loop. It is reasonable that potential direct contacts can be made from substituents on the proximal phenyl ring A of compound 1a, as well with alternative ATM inhibitors series such as compound 4. The second region that diverges within the family forms the base of the pocket in ATM with pairing of Leu2877 and Pro2775. Leu2877 is replaced by a methionine in DNA-PK and mTOR, and potentially involved in a steric clash with the distal phenyl ring C of the tricycle. Furthermore, Pro2775 in ATM, is able to form favourable hydrophobic contacts with this distal phenyl of the tricycle, but in ATR it is replaced by a glycine and a threonine in DNA-PK and mTOR. Therefore, the combination of a potential steric clash with methionine in place of Leu2877, and the loss of a favourable hydrophobic contact with Pro2775, may account for the reported selectivity of tricycles 1a and 1b for ATM over DNA-PK and mTOR. KU-60019 has high MW, relatively high LogP and one hydrogen bond donor (LogD7.4 4.5, LogP 4.5, TPSA 80.3, MW 548, 1 HBD and 8 HBA)29 and thus was predicted to have low CNS exposure. From the docked pose of compound 1a and SAR on early analogs, we hypothesized that changes to the amide portion of the KU-60019 would not affect binding



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affinity and selectivity, thus we targeted this region of the molecule to reduce MW and LogP (Figure 4). Hinge binder kept for activity & selectivity

O N

O

X S

O

Thioxanthene moiety kept for activity & selectivity

R X= C, N

Changes to modulate properties - explore linker - explore small capping groups - explore solubilising groups

Figure 4. SAR strategy to identify a brain penetrant analogue of KU-60019

Pyrrolidine 10, cyclopropyl 11 and methyl 12 amides were prepared (Table 1), and, in agreement with our binding hypothesis, all maintained potency at ATM inhibition at the biochemical and cellular levels. However, all three compounds were found to be P-gp substrates (EER > 4) when tested in the MDR1/MDCK assay. Suspecting that presence of a hydrogen bond donor on the amide had a detrimental effect on efflux, we prepared N-methyl analog of 12, compound (14). Unfortunately, the addition of the methyl group was detrimental to biological activity and did not improve efflux. In agreement with a detrimental effect of a HB donor on Pgp, both oxetane 13 carrying an NH moiety, and hydroxyl 18 retained ATM potency, but were substrates of P-gp. Although both compounds had reduced MW compared to KU-60019, carrying an HB donor was sufficient to maintain P-gp mediated efflux. In agreement with the above, methoxy analog 17, with equivalent MW, but devoid of an HB donor had an effective efflux ratio of 1.5. While 17 had decreased ATM potency compared to hydroxyl 18, it displayed favorable in vitro ADME properties.


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Table 1. Structures, biological activity and properties of 1, 10-14, 17-18a O N

O S O

ATM IC50 (µM)

Structure R=

O

1b

O N

N H O

11

N H

O

12

N H N H

13

Biochem

Cell

Kinetic solubility (mg/mL)

0.002

0.26

0.046

9

70

4.5, 80, 548

0.001

0.47

0.081

22

140

4.4, 71, 504

0.0006

0.57

0.005

91

295

4.3, 68, 461

0.0006

0.37

0.034

31

141

4.0, 68, 435

0.0005

0.11

0.014

> 50

183

4.5, 60, 477

0.016

4.0

0.077

35

191

4.1, 59, 449

0.005

0.91

0.011

1.5

218

4.9, 48, 408

0.0005

2.2

ND

31.8

265

4.5, 59, 393

EERb

MDCK Papp (nm/s)

LogP, TPSA, MWc

O N

N H

10

R

O O

14

N

17

O

18

OH

a

SD for figures ±5−10%, IC50 values are the geometric mean of two tests. b An effective efflux ratio (EER)

value > 4 predicts that a compound is a substrate for P-gp, whereas values < 2 suggest the compound is not a P-gp substrate.c Physicochemical properties pKa, LogP, TPSA (Å2) and MW were calculated using ACD Percepta Client (Version 1.7.2. Communication Module Version 1.3. Calculation Kernel Version 1.38.0, www.acdlabs.com; [email protected]).

Having identified HB donor containing groups as potential culprits for increased efflux, ether linked analogs were evaluated (Table 2). Initially, amino groups were included to retain solubility; we explored the effect of modulating basicity on P-gp driven efflux with a small range of oxy-ethyl linked cyclic amines. Lowering basicity (predicted pKa values ≤7) as in analogs 20 13 ACS Paragon Plus Environment


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and 22 helped to reduce the effective efflux ratio. Unfortunately, compound 20 showed low passive permeability and, not surprisingly, most compounds in this series showed high intrinsic clearance in mouse liver microsomes. Table 2. Structures, biological activity and properties of 19-26a O N

O S O

ATM IC50 µM

Structure R= O

19

N

O

R

Biochem

Cell

Kinetic solubility (mg/mL)

EER

MDCK Papp (nm/s)

MLM Clint (mL/min/kg bodyweight)

pKa, LogP, tPSA, MW

0.0005

0.21

0.082

64

507

170

7.4, 4.0, 61, 519

0.0007

0.35

0.003

2.7

54

261

6.8, 5.2, 51, 541

0.0014

0.38

0.047

25

202

464

7.4, 5.2, 61, 547

0.0005

0.26

0.003

1.7

170

566

5.4, 4.8, 51, 527

0.0006

0.67

0.046

19

298

1297

7.3, 4.5, 51, 495

0.003

0.86

0.101

4.8

145

92

8.4, 5.5, 72, 563

0.0007

0.25

0.092

24.1

316

201

8.0, 4.2, 55, 520

0.0005

0.52

0.0425

4.6

151

352

7.1, 5.0, 61, 535

F F

20

N

N

21

O F

22

F

N F

23

N

OH

24

N

N

25

N

O

26

N

a

SD for figures ±5−10%, IC50 values are the geometric mean of at least two tests.

A metabolite ID study comparing KU-60019 and ether analog 26 confirmed Ndealkylation occurs on both of the morpholine moieties and O-dealkylation takes place on the 14 ACS Paragon Plus Environment

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ether linker of 26. Therefore, our efforts focused on blocking these metabolically labile sites (Table 3). Alpha substitution on the oxy-ethyl linker did not affect the high intrinsic clearance measured in mouse liver microsomes, but appeared to have a positive effect in removing P-gp associated efflux. All compounds displayed very good activity in both ATM biochemical and cellular assays, with 29 and 33 showing good potency in the cellular assay (cell IC50 < 100 nM), and over 3-fold increase in cellular potency when compared to KU-60019. The stereochemistry of the alpha-methyl group, introduced on the oxy-ethyl linker, appears to have little effect on potency (28, 29 & 33, 34). Shifting the methyl substitution to the beta position of the oxy-ethyl linker, such as in 35 and 36, did not show a beneficial effect on ATM activity or in vitro ADME data. However, incorporation of a more rigid linker to the amino group as in pyrrolidine 38, showed good EER, high passive permeability, and good stability in mouse hepatocytes.

Table 3. Structures, biological activity and properties of 27-41a O N

O S O

ATM IC50 µM

Structure R=

27

28

N O

Kinetic solubility mg/mL

O

EER

Biochem

Cell

0.001

0.2

0.089

31

0.002

0.34

0.027

1.7

MDCK Papp (nm/s)

R

Clint (mL/min/kg bodyweight)

pKa,, LogP, tPSA, MW

MLM

Mhep

227

218

ND

7.4, 4.3, 61, 533

192

697

ND

7.1, 5.2, 61, 549

N O



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N

29

30

O

0.002

0.07

0.03

1.3

150

297

187

O

0.002

0.34

0.026

2.7

227

678

584

7.1, 4.9, 61, 547

0.001

0.23

0.029

11

360

329

475

5.2, 4.4, 70, 563

0.003

0.14

0.021

1.5

321

1297

1643

7.4, 5.2, 61, 547

0.0006

0.07

0.049

6.4

370

200

ND

N

O

31

N O O

32

N

33

N

34

N

35

N

O

7.1, 4.4, 61, 521 O

O

0.001

0.31

0.056

ND

ND

ND

ND

0.001

0.23

0.013

14

385

ND

158 7.1, 4.4, 61, 521

36

N O

37

N

F

38

39 N

F N

0.16

0.009

4.8

529

ND

137

0.003

0.10

0.084

17

321

312

ND

8.9, 4.4, 51, 476

0.002

0.18

0.007

2.3

327

408

38

5.4, 4.9, 51, 527

0.007

0.32

0.089

75

170

138

ND

9.3, 4.9, 51, 477

0.004

0.25

0.012

6.4

331

164

36

5.5, 5.1, 51, 513

0.001

0.27

0.024

2.4

186

ND

431

7.1, 5.3, 61, 549

F

N

40

0.0007

F

N

41

O

a

ND = not determined, SD for figures ±5−10%, IC50 values are the geometric mean of two tests.


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Replacement of the amino group by neutral substituents was also investigated (Table 4). These compounds were predicted to have lower pKa, TPSA and MW, offset by being more lipophilic and possibly less soluble than the amino compounds.

Table 4. Structures, biological activity and properties of 17, 42-47a O N

O S O

Structure R= 17

Me O

42

OH

43 44

O

O

45

O

46 47

O a

O

MDCK Papp (nm/s)

Clint (mL/min/kg body weight) MLM Mheps

Biochem

Cell

Kinetic solubility mg/mL

0.005

0.91

0.011

1.5

218

122

33

4.9, 48, 408

0.004

0.66

0.014

1.8

312

118

< 34

5.1, 57, 466

0.0006

0.45

0.03

33

623

< 65

ND

4.3, 68, 452

0.001

0.21

0.022

1.6

396

150

72

4.9, 57, 466

0.0032

0.23

0.01

1.5

349

121

< 25

4.8, 57, 478

0.001

0.38

0.025

4.7

574

105

68

4.1, 57, 450

0.0005

0.89

30

0.029

0.285

29

0.002

0.07

> 30

0.024

0.198

33

0.0006

0.07

ND

0.014

0.082

38

0.002

0.18

> 30

0.067

ND

44

0.001

0.21

> 30

0.041

0.153

0.001

0.38

> 30

0.020

0.278

46

ND = not determined SD for figures ±5−10%, IC50 values are the geometric mean of two tests. a



Page 20 of 70

As a number of compounds from the series showed high affinity and potency at Vps34, literature ligand bound human Vps34 X-ray structures (e.g. 4UWH) were compared with our human ATM homology model.31 Several key similarities within the proposed ligand binding site emerged, most notably including the conservation of the paired ATM residues Leu2877 and Pro2775. Despite significant effort, a human ATM construct suitable for crystallization and Xray diffraction could not be generated to confirm the ligand binding mode for the series. Based upon the binding site similarity between ATM and Vps34, combined with the activity of the series at both proteins and the literature precedence for human, Vps34 X-ray crystallography, an alternative strategy was adopted. Five mutations and a residue insertion were introduced into human Vps34 to create a Vps34 construct with key residues of the ATM ligand binding site in the vicinity of the thioxanthene series. The gatekeeper mutation Met682Leu, hinge mutation Phe684Trp, phosphate binding loop mutations Ile634Leu and Leu616Asn in addition to Phe612Ala which is the first residue of the ATM unique Ala2693-Gly2694-Gly2695 triad were all incorporated into human Vps34. Furthermore it was noted that ATM has a single glycine residue insertion at the C-terminus of the hinge region, so the Vps34 Gln686-Ser687 motif was replaced by Gln686-Gly-Ser687 in the mutant construct (see Supporting Information). To test whether this Vps34-ATM construct binds to ATM compounds, we solved the crystal structure of Vps34-ATM in complex with 33 at 2.09 Å resolution. The co-crystal structure confirmed the binding mode predicted from the ATM homology model (Figure 5A). The pyranone morpholine substituent accepts a hydrogen bond from the backbone NH of Ile685 (residue numbering refers to wild type human Vps34). The pyranone 20 ACS Paragon Plus Environment

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carbonyl participates in a water-mediated interaction with Tyr670 and Asp644. The sidechain morpholine is well resolved, packing against Trp684 and can form a water-mediated hydrogen bond in either the neutral or protonated state with the backbone carbonyl of Ser687 (Thr2773 for ATM). The methyl substituent is within van der Waals contact distance of Pro689 and the sidechain methylenes of Glu692. Furthermore the sidechain morpholine oxygen is highly solvated and in the region of Gln686 and Gln620 for mutant Vps34. The equivalent residues for ATM are Thr2771 and Ile2701, however the nearby ATM residues Arg2713 and Asp2703 are likely to provide a similar polar environment for ATM. Protein structure superposition of 33 bound to mutant Vps34 X-ray structure showed a good concordance between the overall orientation of the ligands and the hinge region around the glycine insertion (Figure 5B). Although there is a general translation within the ligand binding region, the overall orientation of the backbone and sidechains are highly similar. It is anticipated that interrogation of the mutant Vps34 X-ray structure in combination with literature wildtype Vps34 structures will be used to optimize the ATM over Vps34 selectivity for future compound design.

Figure 5. (A) 2.09 Å X-ray structure of 33 bound to the mutant Vps34 construct, mutated residues shown as thick sticks. (B) Protein superposition of 33 bound mutant Vps34 X-ray structure (yellow ribbon, orange ligand) with ATM homology containing preferred docked pose of 1a (cyan ribbon, green ligand) 21 ACS Paragon Plus Environment


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A set of compounds with favorable in vitro profiles was selected for pharmacokinetic profiling in mice in order to establish in vitro/in vivo relationships. The PK of neutral compounds 17, 44, 46, 49, 51 and amine 24, 29 and 38 was assessed following a single intravenous injection of a 3 mg/kg compound dose (Figure 6). a)

b)

Figure 6. PK profiles evaluated following intravenous administration (normalized to 3 mg/kg) to male mice a) plasma concentrations and b) brain concentration profiles with KU60019 shown for reference Table 7. PK parameters evaluated following intravenous administration to male mice AUCnorm (nM.h.kg/mg) Brain 3500

CLp (L/h/kg)

Vdss (L/kg)

Half-life (h)

Br:Pl (AUC ratio)

Kpu,u

17

AUCnorm (nM.h.kg/mg) Plasma 2700

0.9

1.6

1.1

1.3

0.44

24

950

120

1.9

4.2

1.9

0.1

0.05

29

1100

110

1.7

2.3

1

0.1

0.03

38

1600

380

1.2

2.6

1.8

0.2

0.05

44

1600

680

1.3

1.9

1.1

0.4

0.14

46

1900

450

1.2

1.6

1.2

0.2

0.07


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49

1500

700

1.4

3

1.6

0.5

0.12

51

960

430

2.3

3

1

0.4

0.18

Comparable plasma exposures were observed across the complete set of selected compounds, however brain exposure varied. Overall a good relationship between Kpu,u and EER was observed. In addition compounds with higher permeability (Papp) and lower MW showed greater brain exposure than compounds with similar EER (Figure 7a). Comparison of measured in vivo clearance with predicted clearance from microsomal and/or hepatocyte in vitro assays, showed that both in vitro methods overestimate in vivo plasma clearance. Of the two methods, in vitro hepatocytes data were the better predictor of in vivo clearance (Figure 7b). a)

b)

Figure 7. In vivo/in vitro correlations a) Kpu,u plotted vs EER; b) In vivo measured plasma clearance (CLp) plotted vs predicted plasma clearance from in vitro mouse microsomal and mouse hepatocyte data



Page 24 of 70

Compound 17 had the highest plasma and brain exposures with a total brain to plasma ratio 1.3 to 1, and Kpu,u of 0.44. Although it was not one of the most potent ATM inhibitors, we decided to advance this compound as a tool to develop in vivo PK/PD relationships for ATM kinase inhibition. The compound was advanced for an oral dosing study in mice at 10, 30 and 100 mg/kg. Compound 17 showed good bioavailability (F) and linear pharmacokinetics at 10 and 30 mg/kg (F: 89% and 111%, AUCnorm: 2400 and 3000 nM∙h∙kg/mg respectively), but showed some saturation of absorption at 100 mg/kg (F: 67%, AUCnorm: 1800 nM∙h∙kg/mg) (Figure 8). Since the total concentration of 17 in brain was maintained above cell IC50 for 8 h at the 10 mg/kg dose, we selected 17 as a tool compound to explore the effect of ATM inhibition in vivo.

Figure 8. Plasma and brain concentration profiles of compound 17 following oral administration (10, 30 and 100 mg/kg) to male mice

Table 8. PK parameters of compound 17 following oral administration (10, 30 and 100 mg/kg) to male mice Plasma PK PARAMETER

Brain

10 mg/kg

30 mg/kg

100 mg/kg

10 mg/kg

30 mg/kg

100 mg/kg

AUCnorm (nM hkg/mg)

2400

3000

1800

3200

3300

2600

Oral Bioavailability (%)

89

111

67

NA

NA

NA

Cmax norm (nM kg/mg)

850

890

450

950

880

850


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Tmax (h)

1

0.25

0.25

1

0.5

1

Half-life (h)

1.6

2.4

1.8

1.6

2.7

2.1

Br:pl (AUC ratio)

NA

NA

NA

1.3

1.1

1.4

Kpu,u

NA

NA

NA

0.45

0.37

0.49

NA: Not applicable PK parameters due to route and/or matrix

With the aim of finding an appropriate PD biomarker for assessing ATM activity, we monitored phosphorylation of several ATM substrates including H2AX (Ser139), KAP1 (Ser824) ATM (Ser1981), p53 (Ser15), SMC1 (Ser957), NBS1 (Ser343) and 53BP1 (Ser25). Antibodies against each protein, as well as antibodies that specifically recognize their respective ATM phosphorylation sites were validated through western blot analysis in a Neuro2A mouse cell line using DNA damaging agents in combination with ATM inhibitors. C57Bl/6J or Q175HET mice were orally dosed with vehicle or 17 (10 mg/kg), and brain samples were collected at 0.25, 1, 2, 4, 6, 12 and 24 h after dosing. However, western blot analysis was not able to detect significant levels of phosphorylation of the ATM substrates (H2AX, KAP1, ATM, p53, SMC1, NBS1 and 53BP1), and no reduction in phosphorylation signal following compound treatment (data not shown). These results suggested that enhancing in vivo ATM activation by means of external factors (i.e., gamma- or X-ray irradiation, or hydrogen peroxide) was needed, as it has been previously reported,2 in order to enable quantification of compound effect. To test that hypothesis, we examined the effects of two different X-ray doses in C57Bl/6J mice over time using previously described conditions.32 Analysis of γH2AX and pKAP1 brain levels by western blot shows that, consistent with the literature, increases in H2AX and KAP1 phosphorylation are triggered by X-ray irradiation in a dose and time dependent manner (Figure 9). Interestingly, levels were higher at 1h than 3h post-dose, suggesting that rapid induction of DNA repair was induced in these mice by irradiation. 25 ACS Paragon Plus Environment


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Figure 9. Effects of X-ray dose over time on γH2AX and pKAP1 levels in C57Bl/6J mouse brains: western blots and quantification of western blot immunoreactivity. Phosphorylated target protein is shown in green, total target protein in red. Mice were treated with 3 Gy or 5 Gy X-ray (or control); animals were euthanized at 1h or 3h post-irradiation and tissues collected. Immunoreactivity was quantified and the ratio of phosphorylated:total target protein calculated To test the responsiveness of γH2Ax and pKAP1 to ATM inhibition, an acute PD biomarker study was performed evaluating the effect of multiple concentrations of 17 on H2AX and KAP1 phosphorylation in the presence of 5 Gy irradiation at 30 and 60 min post-irradiation. Significant effects of irradiation on KAP1 and H2AX phosphorylation were observed at both time points, consistent with the results described above. More importantly, significant dosedependent inhibition of irradiation-induced KAP1 phosphorylation by 17 was observed at 30 and 60 min post-irradiation (Figure 10). In contrast, no significant decrease in H2AX phosphorylation (Ser139) was observed at any concentration or time point at any dose of 17. The lack of a significant decrease in H2AX phosphorylation in response to compound may be due to the fact that other PI3K-like kinases, ATR and DNA-PK, as well as ATM are activated by irradiation and can also contribute to the phosphorylation of H2AX (at residue Ser139) whereas 26 ACS Paragon Plus Environment

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KAP1 phosphorylation (at residue Ser824) seems to be predominantly regulated by ATM alone.33 a) 30 min post-irradiation

b) 60 min post-irradiation

Figure 10. Effect of 17 on phosphorylation of KAP1: quantification of western blot immunoreactivity in C57Bl/6J mouse brains. a) Effect of 17 at 3, 10, 30, 100 mg/kg dose levels at 30 min post irradiation; b) Effect of 17 at 3, 10, 30, 100 mg/kg dose levels at 60 min post irradiation; phosphorylated KAP1 is shown in green, total KAP1 in red. Mice were dosed with vehicle and 17 (3, 10, 30 or 100 mg/kg) for 60 min prior to exposure to sham or 5 Gy irradiation. Brain hemispheres were collected and analyzed 30 and 60 min post-irradiation as described previously. Immunoreactivity was quantified and the ratio of phosphorylated:total target protein calculated and analysed using GraphPad Prism. One-way ANOVA was performed with a Dunnett’s multiple comparison test. * 95% confidence, ** 99% confidence, ***99.9% confidence PK/PD relationships were interrogated using a direct-effect PD model (inhibitory effect sigmoid Imax model) which considered the effect of concentrations of 17 on irradiation-induced KAP1 phosphorylation. The free plasma concentration necessary to achieve 50% blockade of ATM activity in the brain were estimated to be between 105 and 140 nM.



Altered ATM function has been described in human HD cells and tissue,7, 34 as well as in animal models of HD,7 which points to a potential differential response to irradiation and to ATM inhibition in HD model as compared to WT mice. To investigate such possibility, an acute PD study was designed to evaluate the effect of 17 (100 mg/kg) on 5 Gy irradiation in 6 and 12 month old Q175 neo minus mice and WT littermate controls. As shown in Figure 11, compound 17 (100 mg/kg) showed significant inhibition of irradiation-induced KAP1 phosphorylation in both WT and Q175 neo minus mice. However, there was no consistent difference between the response of Q175 neo minus mice and WT littermate controls at either age, post-irradiation or +/compound 17.

Figure 11. Effect of 17 on irradiation induced KAP1 phosphorylation in Q175 neo minus and WT (C57Bl/6J) animals after 60 min. Effect of 17 at 100 mg/kg dose level at 60 min post irradiation; phosphorylated KAP1 is shown in green, total KAP1 in red. Mice were dosed with vehicle and 17 (100 mg/kg) for 60 min prior to exposure to sham or 5 Gy irradiation. Brain hemispheres were collected and analyzed 60 min post-irradiation as described previously 28 ACS Paragon Plus Environment

Page 28 of 70

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Immunoreactivity was quantified and the ratio of phosphorylated:total target protein calculated and analysed using GraphPad Prism, unpaired t-test were performed for comparison of the different groups ns= not significant, ** 99% confidence, ***99.9% confidence

With the purpose of monitoring in vivo target kinase engagement and broad panel selectivity, the chemical proteomics technology developed by ActivX Bioscience35 was utilized to demonstrate the ability of 17 to engage ATM in the rodent central nervous system. Brain tissue from non-irradiated and irradiated mice treated with 17 was processed using a biotintagged irreversible ADP probe that covalently binds to a wide range of kinases.36 Brain samples (from 3 animals) were selected for KiNativ™ profiling to ensure that a range of brain levels and predicted target engagement would be covered. As expected, results demonstrated robust concentration dependent target engagement of ATM by 17 in both WT non-irradiated and WT irradiated animals (Figure 12). a)

b)

Figure 12 a) Target engagement/inhibition of 17 at ATM in WT non-irradiated brain tissues. b) Target engagement/inhibition of 17 at ATM in WT irradiated brain tissues. Mean (SD) values used n=3



Page 30 of 70

The free plasma concentration necessary to achieve 50% occupancy of ATM activity in the brain were estimated, using a direct effect PD model (Inhibitory effect Sigmoid Imax model), to be 34 nM IC50 (irradiated samples), in agreement with the PD biomarker data (pKAP1 IC50 137 nM) and suggesting a direct correlation between level of target occupancy and ATM inhibition with this compound. Taken together, these data can be used to guide dose selection to maintain defined ATM inhibition in the brain in subsequent efficacy studies. KiNativ™ profiling also generated a selectivity profile of 17. In both WT non-irradiated and irradiated mouse brain tissue (100 mg/kg dose, 2 h post dose), occupancy at Vps34 (PIK3C3 non irradiated 69%, irradiated 96%) and PIK3CB (non irradiated 61%, irradiated 57%) was observed as well as ATM (non irradiated 59%, irradiated 82%), with no inhibition detected at ATR (non irradiated 14%, irradiated 22%), as expected from 17, consistent with its in vitro selectivity profile (data in Supporting Information). ATM function is quite complex,37 and while its activation has been identified as proapoptotic,38 its inhibition has, in apparent contradiction, been linked to neuroprotective effects.7 In order to assess the neuroprotective role of ATM inhibition in an HD context, the effect of compound application on cell survival was tested in a primary neuronal cortico-striatal co-culture system,39 which allows for differential analysis of mHTT fragment-induced cytotoxicity in synaptically connected cortical and striatal neurons. Co-cultured rat striatal and cortical neurons were labeled independently with fluorescent reporters and transfected with a mHTT-exon1-Q73 construct in order to induce the mHTT phenotype. Both ATM inhibitors 17 and 29 rescued the mHTT-induced loss of striatal and cortical neurons with a more pronounced neuroprotective effect in striatal neurons (Figure 13, A & B). Compound 29 was more potent compared to 17 (Compound 29: striatal EC50 0.005, cortical EC50 0.0006 µM, max effect 223 %; Compound 17: 30 ACS Paragon Plus Environment

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striatal EC50 1.1, cortical EC50 0.6 µM, max effect 254 %). Lower potency and efficacy of either the Vps34 inhibitor, SAR405,40 (striatal EC50: 2.4 µM, max effect 116 %) or the DNA-PK inhibitor NU744141 (striatal EC50: 1.2 µM, max effect 98 %) (Figure 13, C & D) was observed in this assay format suggesting that the effect of the tested ATM inhibitors is very likely due to its on-target effects.

Figure 13. Neuroprotective effect of compound 29 (A) and 17 (B) in a primary rat corticostriatal co-culture system expressing mHTT. Vps34 inhibitor SAR405 (C) and DNA-PK inhibitor NU7441 (D) showed lower potency and efficacy. Representative concentrationresponse curves of the observed effect are shown for at least n=3 experiments for 29 (A) and 17 (B). Due to an incomplete curve, a top constraint was set for 17. Representative concentrationresponse curves are shown for at least n=2 experiments for NU7441 (D). SAR405 (C) did not show any response in a repeat experiment. Neuroprotection assay window is defined by the number of neurons present when neurons are transfected with a control plasmid (100% rescue) compared to neurons transfected with a mHTT plasmid (exon1-Q73 N-terminal HTT fragment) and treated with DMSO (0% rescue). Data is plotted as means ± SEM of n=6 replicates and analyzed with Dunnett’s t-test following ANOVA (P < 0.05)



Page 32 of 70

CONCLUSIONS We report here the generation and characterization of brain-penetrant ATM kinase inhibitors by optimizing permeability and reducing P-gp mediated efflux. Several compounds had favorable PK profiles and displayed appropriate brain exposure. Compound 17 was chosen as a tool to investigate in vivo effects of ATM kinase inhibition in mouse brain, and was shown to engage ATM kinase in the KiNativ™ profiling target engagement platform using an ADPprobe. In agreement with the observed ATM kinase target engagement, compound 17 showed a robust dose-dependent inhibition of X-ray irradiation-induced KAP1 phosphorylation in WT mouse brain and the effect at highest dose was confirmed in an HD mouse model. These data can be used to select doses for chronic efficacy studies that will maintain brain ATM inhibition at defined levels throughout such studies. Furthermore, compound 17 showed protection of rat cortical and striatal cells against mHTT (Q73) induced cytotoxicity with EC50 in agreement with the ATM cell IC50. Further in vivo chronic efficacy studies and subchronic PK/PD studies in HD mouse models are planned to investigate ATM kinase inhibition in mitigating HD pathology. EXPERIMENTAL SECTION General procedure All chemicals were purchased from commercial suppliers and used as received. All reactions involving air or moisture sensitive reagents were performed under a nitrogen atmosphere using dried solvents and glassware. Flash chromatography was carried out with prepacked SiO2 SNAP cartridges (KP-SIL) from Biotage using a Biotage Isolera Four system using gradient elution. Analytical thin-layer chromatography (TLC) was performed on silica 32 ACS Paragon Plus Environment

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using Polygram®SIL G/UV254 with fluorescent indicator (200 μm thickness), and visualized under UV light. NMR spectra were recorded on a Bruker AV 400 (1H = 400.13 MHz, 13C = 100.60 MHz) instrument spectrometer and referenced to tetramethylsilane. The following abbreviations are used: br = broad signal, s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Preparative HPLC was performed on a Waters Sunfire OBD C18 10 μm column (150 mm × 19 mm), Phenomenex Luna phenylhexyl 10 μm column (150 mm × 21.2 mm) or a Waters Xbridge phenyl 5 µm column (100 mm × 19 mm), eluting with mixtures of water−acetonitrile or water−methanol, optionally containing a modifier (0.1% v/v formic acid or 10 mM ammonium bicarbonate). Low-resolution mass spectra were recorded on a Waters ZQ single quadrapole LCMS in ESCi+, ESCi− mode, or a Quattro Micro LC− MS−MS in ESCi+, ESCi− mode). High-resolution mass spectra were recorded on a Waters Acquity UPLC system and Waters Xevo G2 TOF mass spectrometer. HPLC purity was assessed using one of four systems (see analytical methods in Supporting Information). All final compounds were purified to >95% chemical and optical purity (analytical methods detailed in compound experimental). Supercritical Fluid Chromatography (SFC) used a Waters Thar Prep100 preparative SFC system (P200 CO2 pump, 2545 modifier pump, 2998 UV/VIS detector, 2767 liquid handler with Stacked Injection Module). The Waters 2767 liquid handler acted as both auto-sampler and fraction collector. Chiral HPLC purification used either Daicel Chiralpak IA or IC columns. Each sample was run under both un-modified and basic conditions (5.0 μL injection, 5/95 gradient for 5 minutes) across ethanol, methanol and isopropanol. Compounds were named with the aid of the Cambridgesoft Chemistry Cartridge (version 15.1.0.144) software. Racemic mixtures are denoted using asterisks e.g. (1R*,2R*). Enantiomerically pure compounds of known



Page 34 of 70

stereochemistry are denoted without asterisks e.g. (1R,2R). Enantiomerically pure compounds of unknown absolute stereochemistry are denoted by the prefix abs e.g. (abs1). All IC50 data are quoted as geometric mean values, and statistical analysis is available in the Supporting Information. All experimental activities involving animals were carried out in accordance with Charles River animal welfare protocols which are consistent with The American Chemical Society Publications rules and ethical guidelines. Methods and data for specific compounds 2-(7-methoxy-9H-thioxanthen-4-yl)-6-morpholino-4H-pyran-4-one (17) Step 1: 2-((2-bromophenyl)thio)-5-methoxybenzoic acid A suspension of 2-bromo-5-methoxybenzoic acid (10.0 g, 43.3 mmol), 2bromothiophenol (10.6 g, 56.2 mmol), K2CO3 (5.98 g, 43.3 mmol), copper powder (255 mg) and copper (II) oxide (190 mg) in degassed ethoxyethanol (15 mL) was stirred at 135 °C under nitrogen for 3 h. After cooling to rt, the green solution was diluted with 50 mL water and filtered through Celite, washing with 1 M NaOH (2 x 75 mL) and EtOAc (2 x 75 mL). The filtrate was separated into its constituent phases and the aqueous acidified to pH 3 using 1 M HCl. The acidic mixture was extracted with EtOAc (100 mL). The combined organics were washed with brine (100 mL), dried (Na2SO4) and concentrated to give an orange residue. The residue was triturated with a mixture of MeOH and iso-hexane to give the title compound as a pale yellow solid (4.80 g, 14.2 mmol, 33%). ¹H NMR (400 MHz, DMSO-d6) 13.30 (s, 1H), 7.74 (dd, J=1.4, 7.9 Hz, 1H), 7.42 - 7.36 (m, 2H), 7.30 - 7.21 (m, 2H), 7.11 (dd, J=3.0, 8.8 Hz, 1H), 6.94 (d, J=8.8 Hz, 1H), 3.81 (s, 3H). 34 ACS Paragon Plus Environment

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Step 2: 5-bromo-2-methoxy-9H-thioxanthen-9-one A suspension of 2-((2-bromophenyl)thio)-5-methoxybenzoic acid (14.0 g, 41.3 mmol) in dry CH2Cl2 (100 mL) at 0 °C was treated with trifluoroacetic anhydride (6.40 mL, 43.4 mmol) over 30 min. BF3·Et2O (510 µL, 4.13 mmol) was added to the colorless solution and the mixture stirred at rt for 16 h. The mixture was poured into saturated aqueous NaHCO3 and stirred for 15 min. The layers were separated and the aqueous extracted with CH2Cl2 (2 x 100 mL). The combined organics were washed with brine (75 mL), dried (Na2SO4) and concentrated. Recrystallisation from EtOAc gave two crops of the title compound as yellow plates (11.5 g, 35.8 mmol, 87%). ¹H NMR (400 MHz, DMSO-d6) 8.53 (d, J=7.9 Hz, 1H), 8.14 (d, J=7.7 Hz, 1H), 7.94 - 7.89 (m, 2H), 7.56 (t, J=7.9 Hz, 1H), 7.49 (dd, J=2.2, 8.7 Hz, 1H), 3.92 (s, 3H). Step 3: 5-bromo-2-methoxy-9H-thioxanthene A solution of 5-bromo-2-methoxy-9H-thioxanthen-9-one (2.20 g, 6.88 mmol) in dry THF (40 mL) was cooled to 0 °C under nitrogen. Borane-DMS complex (2 M in THF, 10.3 mL, 20.6 mmol) was added dropwise. The solution was stirred at 0 °C for 7 h – the yellow colour dissipated. Additional borane-DMS complex (2 M in THF, 10 mL) was added and the mixture stirred at rt for 48 h. The reaction was quenched by dropwise addition into MeOH at 50 °C, then concentrated to a pale yellow solid. Purification by silica gel chromatography (gradient elution, 0-35% EtOAc/iso-hexane) gave the title compound as a colourless solid (1.65 g, 5.37 mmol, 78%). ¹H NMR (400 MHz, CDCl3) 7.43 - 7.40 (m, 1H), 7.13 - 7.08 (m, 2H), 6.87 (t, J=7.7 Hz, 1H), 6.77 (dd, J=3.0, 8.8 Hz, 1H), 6.69 (d, J=3.0 Hz, 1H), 4.04 (s, 2H), 3.79 (s, 3H). Step 4: 2-(7-methoxy-9H-thioxanthen-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 35 ACS Paragon Plus Environment


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A flask containing a suspension of 5-bromo-2-methoxy-9H-thioxanthene (2.00 g, 6.51 mmol), bis(pinacolato)diboron (1.83 g, 7.21 mmol) and KOAc (1.62 g, 16.5 mmol) in dry dioxane (30 mL) was evacuated and backfilled with N2 three times. PdCl2(dppf) (266 mg, 0.33 mmol) was added and the flask evacuated and backfilled with N2 twice more. The mixture was stirred at 100 °C for 4 h. After cooling to rt, the mixture was filtered through Celite, washing with CH2Cl2. The filtrate was concentrated and purified by silica gel chromatography (gradient elution, 0-15% EtOAc/iso-hexane) to give the title compound as white solid (1.58 g, 4.46 mmol, 69%). ¹H NMR (400 MHz, CDCl3) 7.59 (dd, J=1.4, 7.5 Hz, 1H), 7.38 - 7.33 (m, 2H), 7.16 (t, J=7.5 Hz, 1H), 6.88 (d, J=2.8 Hz, 1H), 6.74 (dd, J=2.7, 8.5 Hz, 1H), 3.81 - 3.78 (m, 5H), 1.41 (s, 12H). Step 5: 2-(7-methoxy-9H-thioxanthen-4-yl)-6-morpholino-4H-pyran-4-one (17) A flask containing a suspension of 2-chloro-6-morpholino-4H-pyran-4-one (809 mg, 3.75 mmol, 1 equiv), 2-(7-methoxy-9H-thioxanthen-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.58 g, 4.46 mmol, 1.2 equiv) and K2CO3 (1.28 g, 9.26 mmol, 2.5 equiv) in dry dioxane (20 mL) was evacuated and backfilled with N2 three times. Pd(PPh3)4 (223 mg, 0.19 mmol, 0.05 equiv) was added and the flask evacuated and backfilled with N2 twice more. The mixture was then stirred at 100 °C for 16 h. After cooling to rt the mixture was filtered through Celite, washing with DCM. The filtrate was concentrated to give a brown residue. The residue was purified by silica gel chromatography (gradient elution, 0-20% MeOH/DCM), giving the title compound containing some triphenylphosphine oxide, which was removed by SFC to give the title compound as an off-white powder (852 mg, 2.09 mmol, 56%). ¹H NMR δ (ppm) (400 MHz, DMSO-d₆): 7.59 (d, J = 7.4 Hz, 1H), 7.52 (d, J = 6.4 Hz, 1H), 7.42 - 7.37 (m, 2H), 7.10 (d, J = 36 ACS Paragon Plus Environment

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2.8 Hz, 1H), 6.83 (dd, J = 2.8, 8.5 Hz, 1H), 6.20 (d, J = 1.9 Hz, 1H), 5.51 (d, J = 1.9 Hz, 1H), 3.93 (s, 2H), 3.77 (s, 3H), 3.71 (q, J = 4.8 Hz, 4H), 3.41 (q, J = 4.8 Hz, 4H); 13C NMR δ (ppm) (100 MHz, DMSO-d₆): 178.3, 163.4, 159.5, 159.0, 138.9, 137.8, 133.9, 130.9, 130.8, 128.2, 127.8, 127.3, 123.5, 114.1, 113.9, 113.2, 89.8, 65.7 (2C), 55.8, 45.0 (2C), 39.3. HRMS (ESI) calculated for C23H21NO4S ([M+H]+), 408.1269; found, 408.1278. RT 3.22 min (Analytical method 10cm_Formic_AQ). 2-(7-hydroxy-9H-thioxanthen-4-yl)-6-morpholino-4H-pyran-4-one (18) A solution of 17 (1.10 g, 2.70 mmol, 1 equiv) in dry DCM (40 mL) was cooled to 0 °C and treated with BBr3 (520 µL, 5.40 mmol, 2 equiv). The mixture was allowed to warm to rt over 1.5 h, then cooled to 0 °C and quenched by cautious addition of MeOH (10 mL). The solvents were evaporated to give an orange residue. This was azeotroped with MeOH (4 x 15 mL), then redissolved in the minimum volume of MeOH (~5 mL) and precipitated by addition of EtOAc (20 mL). The solids were collected by filtration, washed with cold Et2O and air dried to give the title compound as a yellow solid (912 mg, 86%). ¹H NMR (400 MHz, CDCl3) 7.45 (dd, J = 1.3, 8.1 Hz, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.24 (dd, J = 1.1, 7.5 Hz, 1H), 7.06 (t, J = 7.7 Hz, 1H), 6.84 (d, J = 2.5 Hz, 1H), 6.70 (dd, J = 2.8, 8.3 Hz, 1H), 4.71 (s, 1H), 3.82 (s, 2H); RT 2.95 min (Analytical method 10cm_ESCI_Bicarb); m/z (ESI) [M+1]+ 394. 2-(7-(((R)-1-((2S,6R)-2,6-dimethylmorpholino)propan-2-yl)oxy)-9H-thioxanthen-4yl)-6-morpholino-4H-pyran-4-one (29) Step 1: (S)-1-((2S,6R)-2,6-dimethylmorpholino)propan-2-ol



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A solution of (S)-propylene oxide (1.22 mL, 17.4 mmol) in water (14 mL) was treated with cis-2,6-dimethylmorpholine (2.00 g, 17.4 mmol) at 0 oC in a sealed reaction vessel. After stirring at rt for 24 h, the reaction mixture was diluted with CH2Cl2 (25 mL). The organic layer was dried by passage through a hydrophobic frit and concentrated. The desired amino alcohol was obtained as a colourless liquid (2.42 g, 14.0 mmol, 80%) in sufficient purity to proceed to the next step without further purification. ¹H NMR (400 MHz, CDCl3) , 3.89 - 3.80 (m, 1H), 3.74 - 3.60 (m, 2H), 3.40 (s, 1H), 2.83 (td, J =1.8, 11.1 Hz, 1H), 2.62 (td, J =1.8, 10.8 Hz, 1H), 2.28 (dd, J =3.8, 12.6 Hz, 1H), 2.21 (dd, J =10.0, 12.3 Hz, 1H), 2.03 (t, J =10.6 Hz, 1H), 1.70 (t, J =10.6 Hz, 1H), 1.16 (d, J =6.2 Hz, 6H), 1.13 (d, J =6.1 Hz, 3H). Step 2: (2S,6R)-4-((R)-2-((5-bromo-9H-thioxanthen-2-yl)oxy)propyl)-2,6dimethylmorpholine A solution of 18 (3.84 g, 9.76 mmol) and (S)-1-((2S,6R)-2,6-dimethylmorpholino)propan2-ol (2.35 g, 26.1 mmol) in dry THF (50 mL) was treated at 0 oC with PPh3 (6.85 g, 26.1 mmol) and a solution of DIAD (3.70 mL, 18.9 mmol) in dry THF (15 mL), added dropwise over 10 min. The mixture was stirred at 0 oC for 2 h and then at rt for 4 days. The mixture was concentrated and the residue was passed through a 70 g SCX cartridge, eluting with CH2Cl2 (200 mL), then MeOH (200 mL), then 4:1 CH2Cl2:[7 M NH3/MeOH]. The ammonia fractions were combined and concentrated. The residue was purified by silica gel column chromatography (gradient elution, 0-100% EtOAc/iso-hexane) to give the title compound as a yellow oil (3.31 g, 7.38 mmol, 76%). ¹H NMR (400 MHz, CDCl3) 7.45 (dd, J =1.1, 8.0 Hz, 1H), 7.38 (d, J =8.6 Hz, 1H), 7.25 (d, J =9.0 Hz, 1H), 7.06 (dd, J =7.7, 7.7 Hz, 1H), 6.91 (d, J =2.8 Hz, 1H), 6.79 (dd, J =2.7, 8.5 Hz, 1H), 4.53 (tq, J =6.0, 5.8 Hz, 1H), 3.84 (s, 2H), 3.69 - 3.60 (m, 2H), 2.79 - 2.71 38 ACS Paragon Plus Environment

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(m, 2H), 2.66 (dd, J =7.0, 13.2 Hz, 1H), 2.45 (dd, J =4.8, 13.1 Hz, 1H), 1.90 - 1.80 (m, 2H), 1.29 (d, J =6.2 Hz, 3H), 1.14 (d, J =4.8 Hz, 3H), 1.12 (d, J =4.7 Hz, 3H). Step 3: (2S,6R)-2,6-dimethyl-4-((R)-2-((5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)-9H-thioxanthen-2-yl)oxy)propyl)morpholine A solution of (2S,6R)-4-((R)-2-((5-bromo-9H-thioxanthen-2-yl)oxy)propyl)-2,6dimethylmorpholine (3.31 g, 7.38 mmol) in dry dioxane (35 mL) was treated at rt with bis(pinacolato)diboron (1.90 g, 7.48 mmol) and KOAc (2.21 g, 22.5 mmol). The mixture was sparged with N2 for 50 min, then PdCl2(dppf) (599 mg, 0.73 mmol) was added and the reaction heated to reflux under N2 for 4 h. After cooling to rt the mixture was filtered through Celite, washing with CH2Cl2. The filtrate was concentrated and the residue purified by silica gel column chromatography (gradient elution, 0-100% EtOAc/iso-hexane) to give impure title compound as a yellow oil (3.18 g), which was used without further purification. Step 4: 2-(7-(((R)-1-((2S,6R)-2,6-dimethylmorpholino)propan-2-yl)oxy)-9Hthioxanthen-4-yl)-6-morpholino-4H-pyran-4-one (29) A mixture of (2S,6R)-2,6-dimethyl-4-((R)-2-((5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)-9H-thioxanthen-2-yl)oxy)propyl)morpholine (3.18 g from previous step), 2-chloro-6morpholino-4H-pyran-4-one (1.39 g, 6.45 mmol) and K2CO3 (2.26 g, 16.4 mmol) was prepared in dry dioxane (30 mL). The flask was evacuated and backfilled with N2 three times. Pd(PPh3)4 (367 mg, 0.32 mmol) was added and the flask evacuated and backfilled with N2 once more. The reaction was stirred at reflux for 21 h. After cooling to rt the mixture was filtered through Celite, washing with CH2Cl2 and MeOH. The filtrate was concentrated and the residue purified by 39 ACS Paragon Plus Environment


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silica gel column chromatography (gradient elution, 0-100% EtOAc/iso-hexane, then 0-20% MeOH/CH2Cl2). The peak containing the desired compound was concentrated and purified by chiral SFC to give the title compound as a pale yellow powder (463 mg, 11% over two steps). ¹H NMR δ (ppm) (400 MHz, DMSO-d6) 7.58 (d, J =7.4 Hz, 1H), 7.52 (d, J =6.4 Hz, 1H), 7.42 7.36 (m, 2H), 7.11 (d, J =2.6 Hz, 1H), 6.83 (dd, J =8.6, 2.7 Hz, 1H), 6.20 (d, J =1.9 Hz, 1H), 5.51 (d, J =1.9 Hz, 1H), 4.68 - 4.62 (m, 1H), 3.91 (s, 2H), 3.73 - 3.68 (m, 4H), 3.54 - 3.46 (m, 2H), 3.43 - 3.39 (m, 4H), 2.81 - 2.74 (m, 2H), 2.57 - 2.52 (m, 1H), 2.41 (dd, J =13.2, 5.2 Hz, 1H), 1.70 (q, J =10.5 Hz, 2H), 1.21 (d, J =6.0 Hz, 3H), 1.02 (d, J =6.3 Hz, 6H). 13C NMR δ (ppm) (100 MHz, DMSO-d6) 178.3, 163.4, 159.0, 157.8, 138.9, 137.8, 133.9, 131.0, 130.8, 128.2, 127.8, 127.3, 123.4, 116.0, 114.8, 114.0, 89.8, 71.8, 71.5, 71.4, 65.7 (2C), 63.3, 60.3, 59.9, 44.9 (2C), 39.3, 19.4 (2C), 18.7. HRMS (ESI) calculated for C31H36N2O5S ([M+H]+), 549.2423; found, 549.2408. RT 2.62 min (Analytical Method 10cm_Formic_AQ), 4.64 min (Analytical Method SFC1, Lux Cellulose-3, 15/85 MeOH (0.1% DEA)/CO2). (R)-2-(7-((1-(2,2-difluoroethyl)pyrrolidin-3-yl)oxy)-9H-thioxanthen-4-yl)-6morpholino-4H-pyran-4-one (38) A solution of 18 (44.4 mg, 0.11 mmol), (S)-1-(2,2-difluoroethyl)pyrrolidin-3-ol (106 mg, 0.70 mmol), PPh3 (134 mg, 0.51 mmol) and DIAD (71 µL, 0.36 mmol) in dry THF (1 mL) was stirred for 71 h at rt. Dry DMF (1 mL) was added, followed by additional PPh3 (130 mg, 0.50 mmol) and DIAD (71 µL, 0.36 mmol). The mixture was heated to 60 °C for 18 h. LCMS analysis indicated no reaction had occurred, so further PPh3 (130 mg, 0.50 mmol) and DIAD (71 µL, 0.36 mmol) were added and heating continued at 70 °C for 22 h. After this time LCMS analysis indicated reaction was complete. The mixture was concentrated and the residue was 40 ACS Paragon Plus Environment

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filtered through an SCX cartridge, eluting with 3 CV CH2Cl2, 5 CV MeOH, then 5 CV 6:1 CH2Cl2:[7 M NH3/MeOH]. The ammonia fraction was concentrated and purified by reverse phase HPLC to give the title compound as an off-white powder (32 mg, 61 µmol, 54%). ¹H NMR δ (ppm) (400 MHz, DMSO-d6) 7.58 (d, J =7.5 Hz, 1H), 7.52 (dd, J =7.4, 1.3 Hz, 1H), 7.42 - 7.37 (m, 2H), 7.05 (d, J =2.6 Hz, 1H), 6.78 (dd, J =8.5, 2.6 Hz, 1H), 6.20 (d, J =1.8 Hz, 1H), 6.11 (tt, J =55.9, 4.4 Hz, 1H), 5.51 (d, J =1.9 Hz, 1H), 4.92 - 4.87 (m, 1H), 3.92 (s, 2H), 3.73 3.68 (m, 4H), 3.43 - 3.39 (m, 4H), 2.98 - 2.74 (m, 5H), 2.59 - 2.53 (m, 1H), 2.34 - 2.22 (m, 1H), 1.81 - 1.73 (m, 1H). HRMS (ESI) calculated for C28H28F2N2O4S ([M+H]+), 527.1816; found, 527.1815. RT 2.63 min (Analytical Method 10cm_Formic_AQ). (R)-2-(7-((1-methoxypropan-2-yl)oxy)-9H-thioxanthen-4-yl)-6-morpholino-4Hpyran-4-one (44) A solution of 18 (40 mg, 0.10 mmol, 1 equiv), (S)-1-methoxypropan-2-ol (63.1 mg, 0.70 mmol), PPh3 (184 mg, 0.70 mmol) and DIAD (137 µL, 0.69 mmol) in dry THF (1 mL) was stirred at rt for 24 h. The mixture was concentrated and the residue was filtered through an SCX cartridge, eluting with 3 CV CH2Cl2, 5 CV MeOH, then 5 CV 6:1 CH2Cl2:[7 M NH3/MeOH]. Purification by preparative HPLC gave the title compound as a colourless solid (10.0 mg, 21%). ¹H NMR δ (ppm) (DMSO-d₆): 7.58 (d, J = 7.4 Hz, 1H), 7.53 - 7.51 (m, 1H), 7.42 - 7.36 (m, 2H), 7.11 (d, J = 2.5 Hz, 1H), 6.83 (dd, J = 2.7, 8.6 Hz, 1H), 6.20 (d, J = 1.9 Hz, 1H), 5.51 (d, J = 1.9 Hz, 1H), 4.65 - 4.54 (m, 1H), 3.92 (s, 2H), 3.71 (q, J = 4.8 Hz, 4H), 3.51 - 3.38 (m, 6H), 3.28 (s, 3H), 1.20 (d, J = 6.3 Hz, 3H). HRMS (ESI) calculated for C26H27NO5S ([M+H]+), 466.1688; found, 466.1685. RT 3.53 min (Analytical method 10cm_esci_bicarb). 2-Morpholino-6-(7-(oxetan-3-yloxy)-9H-thioxanthen-4-yl)-4H-pyran-4-one (46) 41 ACS Paragon Plus Environment


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A solution of 18 (396 mg, 1.01 mmol) and oxetan-3-ol (547 mg, 7.38 mmol) in dry DMF (10 mL) was treated at rt with PPh3 (1.31 g, 4.99 mmol) and DIAD (0.72 mL, 5.06 mmol). The mixture was stirred at 70 °C for 2 h, then at 60 °C for 17 h. LCMS analysis indicated the reaction was incomplete, so further PPh3 (1.3 g, 5.0 mmol) and DIAD (0.72 mL, 5.06 mmol) were added. Stirring at 70 °C was continued for 67 h, where upon LCMS analysis indicated reaction was complete. The mixture was concentrated and the residue purified by silica gel column chromatography (gradient elution, 0-10% MeOH/CH2Cl2). A second batch of material, from a reaction conducted on the same scale, was combined with this material and purified further by reverse phase HPLC and normal phase HPLC. The foamy material was dried from CH2Cl2 to give the title compound as an off-white powder (71 mg, 8%). ¹H NMR δ (ppm) (400 MHz, DMSO-d6) 7.58 (d, J =7.5 Hz, 1H), 7.52 (dd, J =7.8, 1.6 Hz, 1H), 7.43 - 7.38 (m, 2H), 6.95 (d, J =2.6 Hz, 1H), 6.69 (dd, J =8.5, 2.8 Hz, 1H), 6.20 (d, J =1.9 Hz, 1H), 5.51 (d, J =1.9 Hz, 1H), 5.31 - 5.25 (m, 1H), 4.93 (t, J =6.8 Hz, 2H), 4.53 (dd, J =7.6, 5.0 Hz, 2H), 3.93 (s, 2H), 3.73 - 3.68 (m, 4H), 3.43 - 3.38 (m, 4H). HRMS (ESI) calculated for C25H23NO5S ([M+H]+), 450.1375; found, 450.1366. RT 3.02 min (Analytical Method 10cm_Formic_AQ). 2-(2'-methoxy-2,3,5,6-tetrahydrospiro[pyran-4,9'-thioxanthen]-5'-yl)-6-morpholino4H-pyran-4-one (49) Step 1: 5'-bromo-2'-methoxy-2,3,5,6-tetrahydrospiro[pyran-4,9'-thioxanthene] A suspension of NaH (60 wt% in oil, 130 mg, 3.25 mmol, 5 equiv) in dry DMSO (2.6 mL) was heated at 65 °C for 15 min. After cooling to rt, it was added to a solution of 16a (200 mg, 0.65 mmol, 1 equiv) and 1-chloro-2-(2-chloroethoxy)ethane (0.10 mL, 0.85 mmol, 1.3 equiv) in dry THF (2.5 mL). The reaction mixture was stirred at rt for 2 h, then poured into water 42 ACS Paragon Plus Environment

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and extracted with ethyl acetate. The organic phase was washed with water, dried with MgSO4, filtered and concentrated to afford a crude material that was purified by silica gel chromatography (gradient elution, 2-20% EtOAc/iso-hexane) to give the title compound (175 mg, 71%). ¹H NMR δ (ppm) (400 MHz, CDCl3) 7.45 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.1 Hz, 1H), 7.12 (t, J = 8.2 Hz, 1H), 7.04 (d, J = 2.7 Hz, 1H), 6.77 (dd, J = 2.6, 8.9 Hz, 1H), 3.89-3.73 (m, 4H), 3.80 (s, 3H), 2.44 (t, J = 5.1 Hz, 4H). Step 2: 2-(2'-methoxy-2,3,5,6-tetrahydrospiro[pyran-4,9'-thioxanthen]-5'-yl)-4,4,5,5tetramethyl-1,3,2-dioxaborolane A stirred suspension of 5'-bromo-2'-methoxy-2,3,5,6-tetrahydrospiro[pyran-4,9'thioxanthene] from the previous step (175 mg, 0.46 mmol), bis(pinacolato)diboron (137 mg, 0.54 mmol, 1.3 equiv) and KOAc (137 mg, 1.4 mmol, 2.9 equiv) in dry dioxane (2.0 mL) was degassed five times, backfilling with N2. Pd(dppf)Cl2·CH2Cl2 (20 mg, 0.024 mmol, 0.06 equiv) was added and the mixture stirred at 110 °C for 16 h. After cooling to rt, the mixture was filtered through Celite, washing with DCM. The filtrate was concentrated and the residue purified by silica gel chromatography (gradient elution, 0-20% EtOAc/iso-hexane) to give 150 mg (77%) of title compound, which was used without further purification. Step 3: 2-(2'-methoxy-2,3,5,6-tetrahydrospiro[pyran-4,9'-thioxanthen]-5'-yl)-6morpholino-4H-pyran-4-one (49) A solution of 2-(2'-methoxy-2,3,5,6-tetrahydrospiro[pyran-4,9'-thioxanthen]-5'-yl)4,4,5,5-tetramethyl-1,3,2-dioxaborolane (150 mg, 0.35 mmol, 1 equiv) in dry dioxane (2.5 mL) was treated with 2-chloro-6-morpholino-4H-pyran-4-one (77 mg, 0.35 mmol, 1 equiv) and 43 ACS Paragon Plus Environment


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K2CO3 (145 mg, 1.05 mmol, 2.5 equiv). The reaction tube was evacuated and backfilled with N2 three times. Pd(PPh3)4 (40 mg, 35 µmol, 0.08 equiv) was added and the tube was evacuated and backfilled with N2 three times more, before being sealed. The reaction mixture was stirred at 100 oC for 16 h. After cooling to rt, the mixture was filtered through Celite, washing with DCM. The filtrate was concentrated and the residue purified by reverse phase HPLC to give the title compound as an off-white powder (37.9 mg, 47%). ¹H NMR δ (ppm) (400 MHz, DMSO-d6) 7.72 (d, J = 7.1 Hz, 1H), 7.52 (d, J = 7.2 Hz, 1H), 7.49-7.41 (m, 2H), 7.10 (d, J = 3.1 Hz, 1H), 6.84 (dd, J = 2.8, 8.5 Hz, 1H), 6.21 (d, J = 1.8 Hz, 1H), 5.50 (d, J = 2.0 Hz, 1H), 3.77 (s, 3H), 3.75 - 3.62 (m, 8H), 3.43-3.37 (m, 4H), 2.49-2.36 (m, 4H). HRMS (ESI) calculated for C27H27NO5S ([M+H]+), 478.1688; found, 478.1685. RT 3.06 min (Analytical Method 10cm_Bicarb_AQ). (abs1)-2-(7-methoxy-9-(methoxymethyl)-9H-thioxanthen-4-yl)-6-morpholino-4H-pyran4-one (50) and (abs2)-2-(7-methoxy-9-(methoxymethyl)-9H-thioxanthen-4-yl)-6-morpholino4H-pyran-4-one (51) Step 1: (±)-5-bromo-2-methoxy-9-(methoxymethyl)-9H-thioxanthene A solution of 16a (214 mg, 0.70 mol) in dry THF (20 mL) was treated with MOM chloride (247 µL, 3.25 mmol) at 0 oC. The flask was evacuated and backfilled with N2 four times. KHMDS (1 M in THF, 1.3 mmol) was added dropwise over 90 s, allowing the brown coloration to dissipate between drops. The mixture was then stirred at 0 oC for 1 h and for a further hour at rt before being quenched with 2 mL sat. aq. NH4Cl. The mixture was diluted with water (10 mL) and extracted with EtOAc (3 x 30 mL); the combined organics were dried (Na2SO4) and concentrated. The orange residue was purified by silica gel column 44 ACS Paragon Plus Environment

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chromatography (gradient elution, 0-30% EtOAc/iso-hexane), to give the title compound as a pale yellow oil (177 mg, 0.50 mmol, 72%). ¹H NMR δ (ppm) (400 MHz, CDCl3) 7.47 (dd, J =1.2, 8.0 Hz, 1H), 7.34 (d, J =8.5 Hz, 1H), 7.26 - 7.23 (m, 1H), 7.07 (t, J =7.7 Hz, 1H), 6.89 (d, J =2.8 Hz, 1H), 6.82 (dd, J =2.7, 8.6 Hz, 1H), 4.25 (t, J =7.6 Hz, 1H), 3.81 (s, 3H), 3.59 - 3.51 (m, 2H), 3.22 (s, 3H). Step 2: (±)-2-(7-methoxy-9-(methoxymethyl)-9H-thioxanthen-4-yl)-4,4,5,5tetramethyl-1,3,2-dioxaborolane A suspension of (±)-5-bromo-2-methoxy-9-(methoxymethyl)-9H-thioxanthene (228 mg, 0.65 mmol), bis(pinacolato)diboron (177 mg, 0.70 mmol) and KOAc (168 mg, 1.71 mmol) in dry dioxane (6 mL) was deoxygenated by five cycles of evacuation and N2 backfill. PdCl2(dppf) (43.2 mg, 53 µmol) was added and the mixture deoxygenated twice more by evacuation and N2 backfill. The tube was sealed and stirred at 100 oC for 4 h. After cooling to rt, the mixture was filtered through Celite, washing with CH2Cl2. The filtrate was concentrated and purified by silica gel column chromatography (gradient elution, 0-20% EtOAc/iso-hexane) to give the title compound as a colourless oil (203 mg, 0.51 mmol, 79%). ¹H NMR δ (ppm) (400 MHz, CDCl3) 7.62 (dd, J =1.5, 7.3 Hz, 1H), 7.36 - 7.30 (m, 2H), 7.18 (t, J =7.4 Hz, 1H), 6.87 (d, J =2.6 Hz, 1H), 6.78 (dd, J =2.7, 8.6 Hz, 1H), 4.22 (t, J =7.7 Hz, 1H), 3.80 (s, 3H), 3.62 - 3.52 (m, 2H), 3.20 (s, 3H), 1.41 (s, 6H), 1.39 (s, 6H). Step 3: (abs1)-2-(7-methoxy-9-(methoxymethyl)-9H-thioxanthen-4-yl)-6morpholino-4H-pyran-4-one (50) and (abs2)-2-(7-methoxy-9-(methoxymethyl)-9Hthioxanthen-4-yl)-6-morpholino-4H-pyran-4-one (51)



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A solution of (±)-2-(7-methoxy-9-(methoxymethyl)-9H-thioxanthen-4-yl)-4,4,5,5tetramethyl-1,3,2-dioxaborolane (203 mg, 0.51 mmol) in dry dioxane (3 mL) was treated with 2chloro-6-morpholino-4H-pyran-4-one (110 mg, 0.51 mmol) and K2CO3 (179 mg, 1.30 mmol). The reaction tube was evacuated and backfilled with N2 three times. Pd(PPh3)4 (45.6 mg, 39 µmol) was added and the tube was evacuated and backfilled with N2 three times more, before being sealed. The reaction mixture was stirred at 100 oC for 4 h. After cooling to rt, the mixture was filtered through Celite, washing with CH2Cl2. The filtrate was concentrated and the residue purified by silica gel column chromatography (gradient elution, 0-100% EtOAc/iso-hexane, then 0-20% MeOH/CH2Cl2). Additional purification by reverse phase HPLC and chiral SFC gave the title compounds as an off-white solid (50: 40 mg, 17%; 51: 38 mg, 17%). Absolute stereochemistry was not established for these two compounds. Data for 50: ¹H NMR δ (ppm) (400 MHz, DMSO-d6) 7.56 - 7.53 (m, 2H), 7.40 (t, J =7.6 Hz, 1H), 7.38 (d, J =8.7 Hz, 1H), 7.06 (d, J =2.8 Hz, 1H), 6.88 (dd, J =2.8, 8.7 Hz, 1H), 6.20 (d, J =1.8 Hz, 1H), 5.50 (d, J =1.9 Hz, 1H), 4.53 (t, J =7.7 Hz, 1H), 3.77 (s, 3H), 3.73 - 3.68 (m, 4H), 3.60 (dd, J =8.4, 9.8 Hz, 1H), 3.53 (dd, J =7.3, 9.5 Hz, 1H), 3.43 - 3.38 (m, 4H), 3.23 (s, 3H). HRMS (ESI) calculated for C25H25NO5S ([M+H]+), 452.1531; found, 452.1543. RT 3.11 min (Analytical Method 10cm_Formic_AQ), 3.12 min (Analytical Method SFC1, YMC Cellulose-CISO, 30/70 MeOH/CO2); Data for 51: ¹H NMR δ (ppm) (400 MHz, DMSO-d6) 7.56 - 7.53 (m, 2H), 7.40 (t, J =7.6 Hz, 1H), 7.38 (d, J =8.7 Hz, 1H), 7.06 (d, J =2.8 Hz, 1H), 6.88 (dd, J =2.8, 8.7 Hz, 1H), 6.20 (d, J =1.8 Hz, 1H), 5.50 (d, J =1.9 Hz, 1H), 4.53 (t, J =7.7 Hz, 1H), 3.77 (s, 3H), 3.73 - 3.68 (m, 4H), 3.60 (dd, J =8.4, 9.8 Hz, 1H), 3.53 (dd, J =7.3, 9.5 Hz, 1H), 3.43 - 3.38 (m, 4H), 3.23 (s, 3H). HRMS (ESI) calculated for C25H25NO5S ([M+H]+) 452.1531; found, 452.1524. RT 3.11 min (Analytical Method 10cm_Formic_AQ). 46 ACS Paragon Plus Environment

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Enzymatic assay ATM: Recombinant, full length human FLAG-tagged ATM activity (Eurofins 14-933) was measured in an ELISA for p53 S15 phosphorylation, in a 384 well V-bottom polypropylene plate (Greiner Bio One 781280). Reactions contained 0.75 nM ATM, 25 nM full length myc tagged p53 (Eurofins 23-034), 10 µM UltraPure ATP (Promega V915B) and serial dilutions of inhibitors (0.1% DMSO final) in 25 mM HEPES, pH 7.5. 5 mM MgCl2, 2.5 mM MnCl2, 0.006% Brij-35, 0.5% glycerol, 0.1mg/ml BSA, 0.5 mM DTT and were allowed to proceed for 30 min at 20°C. 70 mM EDTA final terminated the kinase reactions. An aliquot of the terminated reaction (25 µl) was transferred to a 384-well, high binding microplate (Greiner Bio One 781061), precoated with anti-myc capture antibody overnight (Millipore 05-724) diluted in 1:1000 in PBS, then blocked with Odyssey blocking buffer (LI-COR Biosciences 92740000) for 60 min at 20°C. The terminated reaction was allowed to capture for 2h at 20°C. Phosphorylation was measured using an antibody specific to p53 S15 phosphorylation (1:5000 Abcam ab38497), anti-rabbit HRP (1:1000 Cell Signalling Technology, 7074) and TMB (ab171522). Antibodies were diluted in Odyssey blocking buffer and incubated for 60 min at 20°C. Three washes with PBS containing 0.05% (v/v) Tween 20 (PBS-T) were performed between each incubation. The TMB reaction was stopped with an equal volume of 0.2 M sulfuric acid and absorbance read at 450nm on the Perkin Elmer Envision within 30 min of terminating the reaction. The percentage of inhibition was calculated for each concentration of compound using 0.1% DMSO control wells as 0% inhibition and 1 µM compound 1a as 100% inhibition. IC50 values represent the mean from at least two independent experiments.



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Inhibition of cellular ATM activity was determined by changes in etoposide-stimulated KAP1 phosphorylation, based on the work of Guo et al.27 Phosphorylation of KAP1, ATM, H2AX and p53 were evaluated in-house with comparable results, but the greater assay window was observed with KAP1. Cryopreserved U20S cells were plated overnight at 6k cells/well in black walled, clear bottomed 384 well plates (Greiner Bio One 781090). After overnight incubation (37ºC, 5% CO2), cells were incubated with titrations of test compounds (0.3% DMSO final) in the presence of 100 µM etoposide (Sigma E1383) to stimulate ATM via induction of double stranded DNA breaks. After 60 min, cells were fixed using 100% cold methanol and incubated at -20 °C for 10 min. Fixed cells were washed with PBS containing 0.05% (v/v) Tween 20 (PBS-T) prior to a 60 minute blocking step in Odyssey blocking buffer (LI-COR Biosciences 92740000). Primary antibodies (1:1000 phosphorylated KAP1: Cell Signaling Technology 4127, 1:5000 total KAP1: Cell Signaling Technology 5868) were diluted in Odyssey blocking buffer and incubated overnight at 4°C, before addition of IRDye(R) conjugated secondary antibodies (LI-COR Biociences 926-32211, LI-COR Biosciences 926-68070) diluted 1:2000 in Odyssey blocking buffer for 60 min at room temperature and detection by LI-COR Odyssey (ImageStudio version 2.1.10, focus offset 4 mm, resolution 169 µm, lowest quality, intensities of 1 and 3 for 700 and 800 channels respectively). Immunoreactivity was quantified and the ratio of phosphorylated KAP1 calculated by dividing by the total KAP1 immunoreactivity. The percentage of inhibition was calculated for each concentration of compound using 100% (100 µM etoposide + 30 µM compound 1a) and 0% effect controls (100 µM etoposide + DMSO). IC50 values represent the mean from at least two independent experiments.


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Vps34, DNA-PK and ATR: Measurement of cellular ATR activity was performed using HT29 cells. Cells were seeded at 25 k/well in black walled, clear bottomed 384 well plates (Greiner Bio One 781090). After overnight incubation, cells were incubated with titrations of test compounds (0.3% DMSO final) in the presence of 10 µM gemcitabine (Sigma G6423) to stimulate ATR. After 4h, cells were fixed using 3.7% paraformaldehyde for 10 min and permeabilised using 0.2% Triton-X for 10 min. Fixed and permeabilised cells were washed with PBS-T prior to a 60 min blocking step in Odyssey blocking buffer (LI-COR Biosciences 92740000). Primary antibodies (1:500 phosphorylated Chk1: Cell Signalling Technology 2348, 1:2000 total Chk1: Cell Signaling Technology 2360) were diluted in Odyssey blocking buffer and incubated overnight at 4°C, before addition of IRDye(R) conjugated secondary antibodies (LI-COR Biosciences 926-32211, LI-COR Biosciences 926-68070) diluted 1:1000 in Odyssey blocking buffer for 60 min at room temperature and detection by LI-COR Odyssey (ImageStudio version 2.1.10, focus offset 4 mm, resolution 169 µm, lowest quality, intensity of 5 for both channels). Immunoreactivity was quantified and the ratio of phosphorylated Chk1 calculated by dividing by the total Chk1 immunoreactivity. The percentage of inhibition was calculated for each concentration of compound using 100% (10 µM gemcitabine + 30 µM AZ-20) and 0% effect controls (100 µM gemcitabine + DMSO). IC50 values represent the mean from at least two independent experiments. Recombinant, full length baculovirus produced Vps34 activity (Life Technologies PV5126) was measured using the ADP-Glo™ kinase assay as per the manufacturer’s instructions. Reaction contained 4 nM Vps34, 50 µM PI:PS (PV5122), 30 µM ATP (1 x Km, Promega V915B) and serial dilutions of inhibitors in 50 mM HEPES, pH 7.5, 5 mM MnCl2, 0.1% CHAPS and 2 mM DTT and were allowed to proceed for 60 min at 20°C. Luminescence was measured using the Perkin Elmer Envision. The percentage of inhibition was 49 ACS Paragon Plus Environment


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calculated for each concentration of compound using 0.1% DMSO control wells as 0% inhibition and 1 µM SAR405 (533063, Merck)31 as 100% inhibition. IC50 values represent the mean from at least two independent experiments. Activity of DNA-PK (Promega V5811) purified from HeLa cells was measured using a HTRF assay detecting phosphorylation of serine 15 on full length p53, the same substrate used for the biochemical ATM assay. Reactions contained 0.5 units per well DNA-PK, 25 nM full length his-tagged p53 (Eurofins 23-034), 2.5 µM ATP (1 x Km , Promega V915B) and serial dilutions of inhibitors in 50 mM HEPES, pH 7.5, 5 mM MgCl2, 0.01% Brij35, 1 mM DTT and 1 µg/ml sheared calf thymus DNA. After 75 min at 20°C the reaction was terminated by addition of 0.001 mg/ml final d2-labelled anti-6HIS and 0.125 mg/ml final cryptate labelled anti phospho p53 antibodies in 50 mM HEPES pH 7.5, 5 mM MgCl2, 0.01% Brij-35, 200 mM KF, 200 mM EDTA. The signals of fluorescence at 665 and 620 nm were recorded after 7 h incubation at 20°C on the Perkin Elmer Envision using a 337 nm excitation laser. The percentage of inhibition was calculated for each concentration of compound using 0.1% DMSO control wells as 0% inhibition and 1 µM NU744141 (Cat. No. 3712, Tocris) as 100% inhibition. IC50 values represent the mean from at least two independent experiments. Pharmacokinetics Animal Studies Animals and Housing: Male C57BL/6 mice obtained from Hilltop Laboratories (Scottdale, PA) or Charles River UK Ltd. were individually identified by tail markings and acclimated for 6 or 8 days prior to dose administration Mice were either individually housed in 50 ACS Paragon Plus Environment

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suspended wire caging or housed in groups of 6 per individually vented cage. Mice were kept kept on a 12/12 hour light/dark cycle at 18 to 26 °C. Temperature and humidity in the animal room were regulated and continuously monitored. Animals were fed commercially available rodent food ad libitum and had access to water ad libitum. Preparation of Dose Formulations: Compounds were dosed intravenously (iv) as a solution in 10:50:40 DMSO:PEG400:H2O, by volume. Compound 17 was dosed orally (po) as a fine suspension in 10% or 20% hydroxypropyl-beta-cyclodextrin in water. The dose volume administered was 10 mL/kg for po doses and 5 mL/kg for iv doses. Dose formulation aliquots (100 µL; n=3 per formulation) were collected and submitted to bioanalysis for quantification of analyte concentrations in the administered doses. Sample Collection: Blood for plasma and brain tissues (n=3 per time-point and dose group) were collected at selected time-points post-dose. Terminal blood samples were collected via cardiac puncture at each time point into tubes containing Na heparin held on wet ice and centrifuged at 8200rcf for 5 minutes at 5°C ± 3°C to separate the plasma. Plasma samples were stored at 20°C ± 5°C until bioanalysis. Immediately after collection, each brain was rinsed with saline, snap frozen in liquid nitrogen, and stored individually at -70°C to -80oC. Sample Processing for Bioanalysis: At the time of bioanalysis each brain was homogenized in acetonitrile:water (3:1, v/v) using an Precellys tissue homogenizer resulting in a brain:solvent ratio of 1:3 (w/v) or 1: 2.536 (w/w). Plasma (50 µL) and brain homogenate (50 µL) from study samples, controls and blanks were dispensed into 96 well plates. Extracting solution (200 µL) consisting of 0.1% formic acid 51 ACS Paragon Plus Environment


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in acetonitrile containing 200 ng/mL diclofenac as the internal standard (IS) was added to all samples except to matrix blanks and solvent blanks, followed by vortexing and centrifugation (30 min). Supernatants (100 µL) were transferred to a new plate, an aliquot (200 µL) of Milli-Q water was added to the samples, covered and vortexed for 5 min prior to liquid chromatography mass spectrometry (LC-MS/MS) analysis. Bioanalytical Methods: Concentrations in mouse plasma, brain or muscle were determined using LC-MS/MS assays developed at Charles River Discovery Service UK. Reverse phase separation was performed in a Waters Acquity UPLC with a UPLC Kinetix XBC18 100A column (50 x 2.1 mm, 2.6 µm) using a mobile phase gradient consisting of 0.01% formic acid (v/v) in milliQ water and 0.01% formic acid (v/v) in acetonitrile. The entire LC eluent was directly introduced to an electrospray ionization (ESI) source operating in the positive ion mode for LC MS/MS analysis on Waters TQD or Xevo triple quadrupole mass spectrometers with a source temperature of 150°C and a desolvation temperature of 500°C. The mass spectrometer ion optics were set in the multiple reaction monitoring (MRM) mode. The data was processed using QuanLynx software from Waters. Calibration standards were prepared in duplicate at each concentration. At a minimum, 75% of all the calibration standards and at least two calibration standards per concentration met the accuracy and precision of ±30%. The coefficient of variation (CV%) of the IS signal/area response for the entire run was within ±30%. There was no bias in the accuracy or precision of the calibration standards and no bias or trend in the IS signal/area response for the run to be acceptable. The mean concentrations of the calibration standards at the front end versus those at


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the back end of calibration curve were within ±15% of each other. All samples were analyzed within 10-days of sample collection. The assay lower limits of quantitation for compound 17 were as follows: 6 nM in plasma and 21 nM in brain. Pharmacokinetic Analysis: Composite non-compartmental pharmacokinetic parameters were calculated from the mean concentrations (n=3) obtained for each time point using Phoenix WinNonlin, version 5.2.1 (Pharsight Corporation, Cary, NC). For the iv dose the plasma concentration at Time = 0 was back extrapolated from the first two post dose plasma concentrations. For the po dose the concentration at Time = 0 was assumed to be zero. Plasma and tissue concentrations below the lower limit of quantification were treated as absent samples. The area under the concentration versus time curve (AUC) was calculated using the linear trapezoidal method when appropriate, the elimination rate constant (kel), was estimated using at least the last three observed concentrations. The portion of the AUC from Ct to infinity (AUClast-inf) was extrapolated from the ratio of Ct/kel where Ct represents the last measurable concentration. The AUCinf was calculated as AUClast + AUClast-inf. The AUC determined were normalized to the actual dose administered. The oral bioavailability (%F) was calculated by dividing the dose normalized po AUCinf over the dose normalized iv AUCinf. In vitro ADME assays Kinetic solubility assay



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Kinetic solubility assays were performed as described in the Charles River Standard Operating Procedure. Using a 10 mM stock solution of each test and control compound (hydrocortisone, reserpine) in 100% DMSO, dilutions were prepared to a theoretical concentration of 200 µM in both 0.1 M potassium phosphate buffer containing 0.8% NaCl (PBS), pH 7.4 (2% DMSO final), and in 100% DMSO. An aliquot of the 200 µM DMSO solution was then further diluted to 10 µM and all dilutions (n=2, in 96-well plates) allowed to equilibrate at room temperature on an orbital shaker for two hours. The PBS dilutions were filtered using a Multiscreen HTS solubility filter plate (Millipore) and filtrate was analysed by LC-UV with confirmation of the peak of interest by mass spectrometry. The concentration of compound in PBS filtrate was determined by comparing the UV absorbance peak with that of the two DMSO dilutions as calibration standards. Liver microsomal stability Incubations of test compound (1 µM, n=2, 37 °C) in pooled liver microsomes (0.25 mg protein/mL in 0.1 M phosphate buffer pH7.4) were initiated with the addition of NADPH (1 mM). Samples (100 µL) were obtained at 0, 5, 10, 20 and 40 min and added to 100 µL of acetonitrile containing carbamazepine as analytical internal standard (IS), centrifuged and the supernatants analyzed by LC MS/MS. Permeability and Effective Efflux Ratio in MDCK-MDR1 MDCKII (MDR1 and WT) cell lines were cultured in 24-well Transwell plates (Corning, catalogue number 3397) following the guidelines provided by SOLVO Biotechnology (Budapest, Hungary). The seeding density was 2×105 cells/well and the culture period 3 days. 54 ACS Paragon Plus Environment

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Test compounds (10 μM) were dissolved in Hanks’ Balanced Salt Solution containing 25 mM HEPES (pH 7.4) and added to either the apical or basolateral chambers of the Transwell plate assembly in duplicate. Lucifer Yellow was added to the apical buffer in all wells to assess integrity of the cell layers; wells with Lucifer Yellow permeability above 100 nm/s were rejected. After a 1h incubation at 37 °C, aliquots were taken from both chambers of each Transwell and added to acetonitrile containing IS (carbamazepine). Analyte concentrations were measured by LC-MS/MS. The apparent permeability (Papp) values of test compound were determined for both the apical to basal (A>B) and basal to apical (B>A) permeation and the efflux ratio (B>A: A>B) determined in both the wild type MDCK and MDR1-MDCK cells. Apparent permeability (Papp) values were calculated from the relationship:

Where V = chamber volume and Tinc = incubation time in seconds. Donor = Chamber of Transwell to which compound is dosed: apical for A>B experiments and basal for B>A experiments. Acceptor = Chamber of Transwell in to which permeation of compound is measured: basal for A>B experiments and apical for B>A experiments. The efflux ratios, as an indication of active efflux from the apical cell surface, were calculated using the ratio of Papp B>A/ Papp A>B.



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The effective efflux ratio was also determined from the ratio observed in MDCK-MDR1 cells relative to the ratio observed in wild-type cells. Brain tissue binding Brain tissue binding was determined in vitro by equilibrium dialysis. Briefly, compounds were added (5 µM, 0.5% DMSO final) to brain homogenate (n = 2) prepared from control mouse (C57/Bl6) brains and dialysed against 0.01M phosphate buffered saline (pH 7.4) for 6 hours at 37ºC. After incubation, the contents of each brain homogenate and buffer compartment were removed and mixed with equal volumes of control buffer or brain homogenate as appropriate to maintain matrix equivalence for analysis. Proteins were then precipitated by the addition of acetonitrile containing carbamazepine as IS, centrifuged and the supernatant removed for analysis by LC-MS/MS. The percentage of drug bound was determined using the following relationship, where total brain homogenate concentration and free concentration are the MS responses obtained from analysis of the brain homogenate and buffer compartments, respectively. Fraction bound = Total brain homogenate concentration – free concentration Total brain homogenate concentration

Fraction unbound in the homogenate and then the fraction unbound in the brain were derived using the following equations:Free fraction in homogenate (ƒu homogenate) = 1 – fraction bound 1

Free fraction in brain (ƒu brain) =

D

× 100

((1 fuhomogenate) - 1) +

1 D


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D = Dilution factor during homogenisation In vivo PK/PD studies Male C57BL/6 J Q175 Neo-WT and Neo-Het mice (B6 J .129S1-Htttm1Mfc/190Chdi J; Jackson Laboratories) were approximately six or twelve months old on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o’cobs™ bedding in static microisolators on a 12hour light cycle at 20 – 22 °C (68 – 72 °F) and 40 – 60% humidity. Charles River Discovery Services North Carolina (CR Discovery Services) specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, which assures compliance with accepted standards for the care and use of laboratory animals. Radiation experiments were performed at Charles River Laboratories (Morrisville, NC) Mice were dosed with 0, 3, 10, 30 or 100 mg/kg 17 in 20% HP-beta-CD. One hour post oral dose a Faxitron model CP-160 X-ray system was employed to administer radiation therapy. Each animal was immobilized in a live restrainer. Therapy was administered for 6.5 min at 160 kV and 6.0 mA, to provide a dose of 5 gray (Gy). The beam was filtered with a 0.2 mm Copper sheet. Animals not receiving radiation were placed in the live restrainer for 6.5 minutes with no radiation treatment given (sham). Thirty or sixty minutes post irradiation, animals were euthanized by terminal cardiac puncture under isoflurane anaesthesia and plasma and hemisected brains collected and snap-frozen for analysis. Brain 57 ACS Paragon Plus Environment


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hemispheres (~200mg) were homogenised in 1 ml RIPA buffer containing 2x HALT protease (Thermo Fisher Scientific 1861278) and phosphatase (Thermo Fisher Scientific 1861277) inhibitors (and 1 mM PMSF (Sigma 93482) using 2.8 mm ceramic beads at 5000 rpm for 15 seconds on the Precellys. For histone extraction, 50 µl of 0.2M HCl was added to 200 µl homogenate and the samples left overnight at 4°C with continual inversion. Samples were clarified using 13000 rpm for 20 min at 4 °C. Normalised samples were denatured in sample buffer containing 5% β-mercaptoethanol for 10 min at 70°C before separation by electrophoresis as per the manufacturer’s instructions. Following electrophoresis, samples were transferred onto 0.45 µm Immobilon-FL PVDF membrane (Fisher Scientific 10452792) and blocked for 1 hour at RT in Odyssey blocking buffer before addition of primary antibodies (4 °C overnight). The blots were imaged using IRDye® secondary antibodies (RT 1 h) diluted in Odyssey blocking buffer on the LI-COR Odyssey (ImageStudio version 2.1.10, focus offset 0 mm, resolution 169µm, lowest quality, optimized intensities for membrane). Phosphorylated and total KAP1 and H2AX antibodies were purchased from Abcam (ab70369, ab25553, ab26350, ab140498), secondary antibodies were purchased from LI-COR Biosciences (926-32211, 926-68070, 926-32212, 92668074).

Abbreviations used: 53BP1, p53-binding protein 1; ANOVA, analysis of variance ATM, Ataxia telangiectasia mutated serine/threonine kinase; ATR, ataxia telangiectasia and Rad3-related protein; B2pin2, bis(pinacolato)diboron; Br:Pl, brain:plasma ratio; CLp, plasma clearance; DIAD, diisopropyl azodicarboxylate; DIPEA, diisopropylethylamine; DMS, dimethyl sulfide; DNA-PK, DNA-dependent protein kinase; dppf, 1,1’-bis(diphenylphosphino)ferrocene; 58 ACS Paragon Plus Environment

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EER, effective efflux ratio; ER, efflux ratio; FancD2, Fanconi anemia group D2 protein; Gy, Gray; H2AX, H2A histone family member X; HD, Huntington’s disease; ICW, in-cell Western; KAP1, KRAB-associated protein 1; Kpu,u, unbound partition coefficient; logD, logarithm of distribution coefficient; MDCK, Madin-Darby Canine Kidney; Mdm2, Mouse double minute 2 homolog; mHTT, mutant huntingtin protein; mTOR, mammalian target of rapamycin; Papp, apparent permeability; PIKK, phosphatidylinositol-3-kinase-like kinase; PoC, proof of concept; SMG-1, serine/threonine-protein kinase SMG1; Tmax, time to reach maximum concentration; TPSA, topological polar surface area; Vdss, volume of distribution at steady state; VPS34, phosphatidylinositol 3-kinase VPS34. Acknowledgments The authors would like to thank Paula Miliani de Marval from Charles River Morrisville site for undertaking the in vivo irradiation studies, Mark Sandle and Nicholas Richards for analytical support. ASSOCIATED CONTENT Supporting Information: The supporting information is available free of charge via the Internet at http://pubs.acs.org. Molecular formula strings (CSV). General chemistry methods and methods for specific compounds and intermediates, methods and solvent gradients; preparative HPLC instrument and solvent gradients; ATM, DNA-PK, ATR homology model building (coordinates of the ATM, ATR and DNA-PK homology models are uploaded in separate files as part of the Supporting Information). VPS34-ATM surrogate crystal structures, DiscoverX selectivity screening profile, in vitro in vivo correlation plots. 59 ACS Paragon Plus Environment


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ACCESSION CODES Atomic coordinates and structure factors for X-ray crystal structure of 33 have been deposited in the Protein Data Bank with PDB ID 6I3U. Authors will release the atomic coordinates and experimental data upon article publication. Author information Corresponding authors Perla Breccia e-mail, [email protected] Leticia Toledo-Sherman e-mail, [email protected] ORCID Perla Breccia: 0000-0002-8552-9012


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a

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Atm and Bax cooperate in ionizing radiation-induced

apoptosis in the central nervous system. Proc. Natl. Acad. Sci. U S A. 2000, 97, 889-94. (39)

L. S. Kaltenbach, M. M. Bolton, B. Shah, P. M. Kanju, G. M. Lewis, G. J. Turmel, J. C.

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(DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. Bioorg. Med. Chem. Lett. 2004, 14, 6083-6087.



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TOC GRAPHIC

Superposition of 33 in mutant Vps34 X-ray structure (yellow ribbon, orange ligand) with ATM homology 1a (cyan ribbon, green ligand)

Effect of 17 on pKAP1 at 3, 10, 30, 100 mg/kg at 60 min post irradiation in mouse brain tissues; pKAP1 is shown in green, total KAP1 in red.


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