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Rational design of 5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4((methylamino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-amine (VX-970, M6620): Optimization of intra- and inter-molecular polar interactions of a new ataxia telangiectasia mutated and Rad3-related (ATR) kinase inhibitor Ronald M. A. Knegtel, Jean-Damien Charrier, Steven John Durrant, Chris Davis, Michael O'Donnell, Pierre Storck, Somhairle MacCormick, David Kay, Joanne Pinder, Anisa Virani, Heather Twin, Matthew Griffiths, Philip Reaper, Peter Littlewood, Steve Young, Julian M Golec, and John Pollard J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00426 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 10, 2019

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Journal of Medicinal Chemistry

Rational

design

of

5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-

((methylamino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-amine (VX-970, M6620): Optimization of intra- and inter-molecular polar interactions of a new ataxia telangiectasia mutated and Rad3-related (ATR) kinase inhibitor

Ronald Knegtel*, Jean-Damien Charrier, Steven Durrant, Chris Davis, Michael O’Donnell, Pierre Storck, Somhairle MacCormick†, David Kay, Joanne Pinder, Anisa Virani, Heather Twin, Matthew Griffiths, Philip Reaper, Peter Littlewood, Steve Young‡, Julian Golec, John Pollard

Vertex Pharmaceuticals (Europe) Ltd, 86-88 Jubilee Avenue, Milton Park, Abingdon, Oxfordshire OX14 4RW, United Kingdom †Present

Address: PharmaCytics Operations BV, Novio Tech Campus, Office 3.12,

Transistorweg 5v, 6534 AT Nijmegen, The Netherlands ‡Present

Address: Sygnature Discovery Limited, BioCity, Pennyfoot Street,

Nottingham, NG1 1GF, United Kingdom

ABSTRACT

The DNA damage response (DDR) is a DNA damage surveillance and repair mechanism that can limit the effectiveness of radiotherapy and DNA-damaging chemotherapy, commonly used treatment modalities in cancer. Two related

ACS Paragon Plus Environment

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kinases, ataxia telangiectasia mutated (ATM) and ATM and Rad3-related kinase (ATR), work together as apical proteins in the DDR to maintain genome stability and cell survival in the face of potentially lethal forms of DNA damage. However, compromised ATM signaling is a common characteristic of tumor cells, which places greater reliance on ATR to mediate the DDR. In such circumstances, ATR inhibition has been shown to enhance the toxicity of DNA damaging chemotherapy to many cancer cells in multiple pre-clinical studies, while healthy tissue with functional ATM can tolerate ATR inhibition. ATR therefore, represents a very attractive anti-cancer target. Herein we describe the discovery of VX-970/M6620, the first ATR inhibitor to enter clinical studies, which is based on a 2-aminopyrazine core first reported by Charrier et al. 1


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INTRODUCTION

DNA-damaging agents, such as cisplatin, etoposide, gemcitabine and ionizing radiation (IR) are important and commonly used agents in cancer therapy. Their mechanism of action is to induce potentially lethal DNA damage including double strand breaks and replication stress (RS). RS commonly arises when the replication machinery encounters a lesion in the DNA, resulting in a stalled and unstable replication fork that in turn can collapse to form a double strand break. The effectiveness of DNA damaging chemotherapy can be limited by the cell’s ability to detect and repair the DNA damage.2 The related kinases ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) kinase are apical regulators of the DNA damage response (DDR), a surveillance and repair process that enables cells to survive double strand breaks and replication stress. Although each kinase recognizes different DNA damage structures (double strand breaks in the case of ATM and single stranded DNA within double stranded DNA in the case of ATR) they share many downstream substrates and have overlapping roles in maintaining cell survival. ATR and ATM share a high degree of sequence homology and are members of the phosphoinositol 3-kinaselike kinase (PIKK) family of serine/threonine protein kinases3, which includes other important DNA repair kinases such as DNA-dependent protein kinase (DNA-PK). Despite the importance of the DDR for cell survival following DNA damage, ATM function is often found to be impaired in tumor cells (from defects in activation, expression or downstream signaling proteins such as p53). Loss of ATM or its effectors is thought to enable a genomically unstable environment, which is advantageous to the developing tumor.4 However, impaired ATM


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function places additional burden on remaining repair proteins, such as ATR, to maintain survival. This Achilles’ heel reliance on ATR may be exploited with drugs. Consistent with this hypothesis, a series of preclinical studies have demonstrated that cancer cells treated with DNA damaging drugs are especially sensitive to ATR inhibition when they harbor defects in ATM signalling.5-7 In contrast, normal tissue can tolerate ATR inhibition by activating an ATMmediated compensatory DDR.5 Furthermore, several studies have shown that some tumors are sensitive to ATR inhibition as a single agent, and to combinations of ATR inhibitors with inhibitors of other repair proteins such as poly (ADP-ribose) polymerase (PARP) and Chk1. As such, ATR inhibition represents an attractive anti-cancer target.5,8-20 The first reported series of potent and selective ATR inhibitors, typified by compound 1 (VE-821, Figure 1) is based on a 2-aminopyrazine amide hinge binding motif.1 Compound 1 has proven to be a useful tool to assess the biology of ATR in cells and the potentially beneficial profile for ATR inhibition. However, certain properties of 1 were not fully optimized in the initial chemistry campaign. These include enzyme and cell potency, physical properties such as solubility and the metabolic profile, specifically the potential to form aniline metabolites.1 Here we present the optimization of the 2-aminopyrazine series to a more potent and stable hetero-aromatic scaffold from which compound 2 (VX970, M6620, Figure 1) the first ATR inhibitor to enter clinical studies21 is derived.


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NH 2 O N

NH 2 O N N

N H

N

Compound 1 VE-821 SO2Me

NHMe

N

Compound 2 VX-970/M6620 SO 2iPr

Figure 1. Chemical structures of compound 1 (VE-821) and compound 2 (VX970/M6620).

In our initial optimization efforts of 1, replacement of the anilide group with a range of fused bicyclics such as benzimidazole, benzoxazole, benzothiazole and indole1 was well tolerated by ATR but resulted in reduced selectivity for ATR against ATM. The loss of selectivity over ATM was attributed to a rotation of the bound inhibitor (Figure 2A) caused by a steric clash between the bulkier fused bicyclic aromatic systems with the Tyrosine gatekeeper residue of the PIKK family. This movement in turn resolves a second steric clash between Pro2775 of ATM (which is a smaller Glycine residue in ATR) and the isopropylsulfone of the inhibitor, leading to tighter binding to ATM.


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Figure 2a-b. (A) Comparison of anilide 1 (in green) versus a benzimidazole isostere (in cyan) in a model of the active site of ATM based on PDB entry 1E7V. The benzimidazole is thought to experience steric hindrance from Tyr2755 in ATM. In response it rotates away from Tyr2755, which allows it to escape a steric clash with Pro2775 (Gly in ATR). (B) Replacing the benzimidazole with a phenyl substituted monocyclic heteroaryl (in cyan) yields a better mimic of the original anilide and avoids movement of the inhibitor due to steric hindrance with Tyr2755.

Based on homology models (derived from a crystal structure of the homologous kinase PI3K, PDB entry 1E7V)1 we hypothesized that phenyl substituted 5membered heteroaromatic groups can bind without causing steric clashes with the gatekeeper residue as it mimics the shape of the original anilide more closely. Thus we expected selectivity for ATR over ATM to be retained (Figure 2B). Furthermore, we speculated that functionalization of the phenyl ring would enable exploitation of a highly negatively charged area of the ATP binding site. This would have potential to increase potency and selectivity and also provide an opportunity to modulate physical properties to improve solubility. In keeping with this, substitution with a 4-benzylamine on the 5-membered heteroaryl core yielded compounds with picomolar ATR inhibition, good retention of selectivity and potent cellular activity, culminating in the discovery of the clinical candidate 2 (Figure 1).


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RESULTS

The commercially available methyl 3-amino-6-bromopyrazine-2-carboxylate 21 was readily converted into the 4-isopropylsulfonylphenyl using Suzuki crosscoupling conditions to provide 22.1,22,23 The carboxylic acid in 22 was then transformed into the anilide 324 or used in a sequence leading to 5phenylisoxazole 7, via formation of a Weinreb amide, methyl ketone or benzylidene ketone intermediate.25 Acid 22 was also engaged in a cyclodehydration with N-hydroxybenzamidine to furnish 3-phenyl-1,2,4oxadiazole 10.26

Compound 25 was used as a versatile intermediate in the preparation of 1phenyl-1,2,3-triazole 6 and 3-phenylisoxazole 4 (as well as analogues 2, 14-20) (Scheme 2). Commercially available 5-bromopyrazin-2-amine 23 was subjected to Suzuki palladium cross-coupling with 4-isopropylsulfonylphenyl boronic acid and then brominated at position 3 using NBS to provide intermediate 24 in high yield.27 Copper assisted Sonogashira coupling with TMS-acetylene28, followed by di-BOC protection of the amino group and TMS deprotection afforded advanced intermediate 25. The acetylene functional group in 25 was then used as a coupling partner for click chemistry with azidobenzene to lead to the 1,2,3triazole 6 after BOC removal.29 [3+2] Cycloaddition between 25 and phenylnitrile oxides, generated in situ from the corresponding N-hydroxyimidoyl chlorides, delivered the 3-aryl isoxazoles 4 (as well as 2 and 14-20 after additional steps, see experimental).30


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Compound 24 was also used as a coupling partner to 2- and 5-phenyloxazoles under CH insertion conditions to produce inhibitors 11 and 12 respectively (Scheme 3).31-33 The 2-vinyl intermediate, 26, prepared from the bromo precursor, 24, and vinyl potassium trifluoroborate under palladium catalysis34, underwent [3+2] cycloaddition with phenylnitrile oxide, generated in situ from the corresponding N-hydroxyimidoyl chloride, to provide the 3-phenyl-4,5dihydro isoxazole 5.35

The carboxylic acid group in the commercial starting point, 27, was used to build the 1,3,4-oxadiazole and the 1,3,4-thiadiazole rings in intermediates 28 and 29, via cyclodehydration with benzohydrazide and benzothiohydrazide, respectively (Scheme 4).36 These intermediates were then functionalized using Suzuki crosscouplings to afford 2-phenyl-1,3,4-oxadiazole 8 and 2-phenyl-1,3,4-thiadiazole 13. Using a similar strategy, building block 30 was converted into the 5-phenyl1,2,4-oxadiazole, 9. The nitrile at position 2 in 30 was transformed into a hydroxyimidamide with hydroxylamine and subsequently O-benzoylated prior to a cyclodehydration under acidic conditions.37


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Scheme 1a

Reagents and conditions: (a) (4-isopropylsulfonylphenyl)boronic acid, K3PO4, Pd[P(tBu)3]2, MeCN:H2O (4:1), 60 C, 2 h, 97%; (b) LiOH, THF, rt, 2 h, 96%; (c) diethoxyphosphorylformonitrile, aniline, Et3N, DME, 120 C, 18 h, 96%; (d) N-methoxymethanamine hydrochloride, HOBt, DIPEA, 3(ethyliminomethylene amino)-N,N-dimethyl-propan-1-amine, THF, RT, 18 h, 77%; (e) MeMgBr 3 M in THF, THF, -20 C, 1 h, 80%; (f) benzaldehyde, NaOH, MeOH:DCM (1:1), rt, 18 h, 70%; (g) hydroxylamine hydrochloride, NaOAc, EtOH/AcOH (10/3), 130 C, 1 h, 17%; (h) CDI, Nhydroxybenzamidine, RT to 120 C, 5 h, 67%. a

Scheme 2a

Reagents and conditions: (a) (4-isopropylsulfonylphenyl)boronic acid, K3PO4, Pd[P(tBu)3]2, MeCN/H2O (4/1), 60 C, 1 h, 87%; (b) NBS, DMF, rt, 3 h, 84 %; (c) (trimethylsilyl)acetylene, CuI, Et3N, Pd(PPh3)4, DMF, 0 C to rt, 1 h, 81%; (d) Boc2O, DMAP, Et3N, DCM, RT, 18 h, 100%; (e) a


9


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Na2CO3, MeOH, RT, 1 h, 97%; (f) azidobenzene (0.5M in MTBE), (+)-sodium L-ascorbate, CuSO4.5H2O, t-BuOH: H2O (1:1), rt to 40 C, 12 h, 41%; (g) TFA/DCM (1/3), rt, 1h, 80%; (h) Nhydroxybenzimidoylchloride, Et3N, DMF, 60 C, 45 min then TFA/DCM, rt, 1 h, 52%.

Scheme 3a

Reagents and conditions: (a) 2-phenyloxazole, Ag2CO3, PPh3, Pd(dppf)Cl2.DCM, water, 70°C, 24 h, 29%; (b) 5-phenyloxazole, t-BuOLi, Pd(PPh3)4, dioxane, 140 °C in wave, 3 h, 46%; (c) vinyl potassium trifluoroborate, Pd(dppf)Cl2, Et3N, n-PrOH, 100 °C in wave, 40 min, 57%; (d) Nhydroxybenzimidoyl chloride, Et3N, DMF, 65 °C, 1h, 58%. a


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Scheme 4a

Reagents and conditions: (a) benzohydrazide, PPh3Br2, MeCN, rt, 1 h, then DIPEA, 0 °C, 1 h, 80%; (b) benzothiohydrazide, PPh3Br2, MeCN, RT, 3 h, 39%; (c) hydroxylamine hydrochloride, Et3N, MeOH, 0 C to RT, 3 h, 76%; (d) benzoyl chloride, Et3N, DCM, RT, 16 h, 93%; (e) PPA, 70 C, 7 h, 88%; (f) (4-isopropylsulfonylphenyl) boronic acid, Na3CO3, Pd(dppf)Cl2.DCM, dioxane, 100 C in wave, 25 min, 34-69%.

a

The preparation of intermediates 22, 24-26, 28-29, 31 is described in the Supporting Information.

DISCUSSION

The main aim of the optimization of the 2-aminopyrazine series was to identify a more potent and water soluble analog of 1 without the potential to form an aniline metabolite. The primary approach was to mimic the anilide of 1 with an optimally functionalized saturated or unsaturated ring.


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In our earlier work, a 3-fold gain in ATR inhibitory potency was achieved by replacing the methylsulfone of 1 with an isopropyl sulfone.1 Therefore compound 3 (Table 1) was used as a more potent starting point for exploring biaryl replacements of the anilide. Docking analogues of 3 with 5- and 6membered heteroaromatic replacements for the anilide into an ATR homology model1 suggested that phenyl substituted 5-membered heteroaromatics would fit well in the ATP binding site and mimic the shape and planar nature of the original anilide (Figure 2B). Previously reported initial studies had established that the amide of 1 linking the phenyl and amino pyrazine was not making any specific H-bonding interactions with the ATP binding site.1 However, it had not been determined whether replacement linkers needed to retain a flat profile or whether a degree of 3-dimensionality could be accommodated. To test this we prepared a matched pair with either an unsaturated and planar isoxazole (4) or the partially saturated 4,5-dihydroisoxazole analog (5, Table 1). Compound 4 was approximately 90-fold more potent than 5, indicating that a flat linker is required for an optimal steric fit in the ATP binding pocket. The energy required to flatten the 4,5-dihydroisoxazole ring system of 5 (R-isomer) from its minimized, more 3-dimensional conformation in vacuum to its docked conformation is estimated to be approximately 2 kcal/mol in the gas phase. This would equate to about a 30-fold reduction of its inhibition constant, which is consistent with the observed data.


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Table 1. Enzyme inhibition constants (Ki) and cell assay IC50 values for aminopyrazine ATR inhibitors. Compound 5 is a racemic mixture. NH 2 R

N N

SO 2iPr

Compounds

R

ATM

DNA-PK

Cellular potency IC50 (M) pH2AX

0.004

4

2.8

1.03

0.001

1.4

>4

1.1

0.090

>4

>4

>2.5

Enzyme inhibition Ki (M) ATR

O

3

4

5

N H O N

O N

A selection of diverse 5-membered aromatic heterocycles were considered as possible amide replacements and were prioritized by assessing their conformational energies (as outlined below), followed by docking into our ATR kinase homology model.1 As a consequence of conjugation, the 5-membered heteroayl ring can assume only two orientations with respect to the 2aminopyrazine hinge binder, as illustrated in Figure 3. We will refer to the orientation that best mimics the anilide of 1 as the bioactive conformation.


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H

N

H

X Z

H

Y

N

N N

N

H

Page 14 of 73

Y X

Z

N Bioactive conformation

SO2iPr

Alternative conformation

SO2iPr

Figure 3. Intra-molecular polar interactions within heteroaryl ATR inhibitors. Polar interactions between the central 5-membered heteroaryl ring atoms X and Y and the 2-aminopyrazine hinge binder determine whether the bioactive conformation (left) is energetically preferred.

Complementary intra-molecular interactions between the heteroaryl rings of the inhibitor would assure that the proposed bioactive conformation is energetically favored. Atoms X and Y in the central ring are either opposite the 2-amino group or the 4-sp2 nitrogen atom of the pyrazine ring and the nature of their mutual interactions determines the preferred orientation of the 5-membered heteroaryl ring. While hydrogen bond acceptors (atom X) are preferred opposite the pyrazine NH2 and an aromatic CH (atom Y) opposite the pyrazine ring sp2 nitrogen for reasons of electrostatic complementarity, it is less clear which conformation is preferred when both X and Y are hydrogen bond acceptors. Aromatic nitrogen, oxygen and sulfur atoms vary in their effectiveness as hydrogen bond acceptors38 and will be tolerated to different degrees opposite the sp2 nitrogen at position 4 of the pyrazine ring. Therefore the nature and


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shape of the 5-membered aryl ring determine how readily a compound can assume the proposed bioactive conformation. In order to predict which 5membered heteroaryl rings favor the proposed bioactive conformation, the gas phase energies of the two accessible conformations shown in Figure 3 from a library of putative heteroaryl rings were calculated using Density Functional Theory (DFT) using the B3LYP functional and a 6-31G** basis set.38-41 Although calculated rotational barriers can give insight into how readily a certain conformation can be assumed, they provide no information on which conformation is more stable and tracked poorly with the binding data when calculated for compounds 8, 9 and 10 (results not shown). Calculating the enthalpy difference between two rigid conformers is both computationally less demanding and a better estimator of the free energy of the binding state function. Nine phenyl-substituted 5-membered heteroaryl replacements of anilide 3 were prioritized and synthesized. Table 2 shows the calculated energy differences between the two accessible conformations for each of these compounds in kcal/mol (a negative energy signifying a preference for the proposed bioactive conformation), along with inhibition constants (Ki) against recombinant full-length ATR, ATM and DNA-PK and where available IC50 values from an ATR-mediated cell based biomarker assay (measuring phosphorylation of the ATR substrate variant histone H2A (H2AX) at Serine 139 in human colorectal carcinoma HCT116 cells).1


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Table 2. Enzyme inhibition constants (Ki), pH2AX cell assay IC50 values and conformational energy differences for aminopyrazine ATR inhibitors.

NH 2 R

N N

SO 2iPr

Compounds

R

DNA-PK


Enzyme inhibition Ki (M) ATR

ATM

Energy difference (kcal/mol)

O

3

4

6

7

8

N H O N

N N N

N O

N N O

0.004

4

2.8

1.03

0.001

1.4

>4

1.1

-4.59

0.006

1.3

>4

>2.5

-8.94

0.002

1.3

>4

0.8

-8.74

0.001

>10

8.9

0.41

-7.76

0.005

0.4

4

2.3

-0.87

0.026

1.35

>1.6

>2.5

3.80

0.021

>10

>4

0.004

>10

>4

>2.5

-6.73

0.001

>4

>4

1.7

-12.47

N O

9

10

N O N N N

11

O

1.65

N

12

O N N

13

S


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Table 2 shows that aromatic 5-membered rings are generally well tolerated as an amide replacement with many compounds showing similar ATR inhibition potency to the original amide 3. The isoxazole 4, oxadiazole 8 and thiadiazole 13 show improvements in potency (Ki ~1 nM vs ~4 nM for the starting compound, 3). Encouragingly, these analogues retained the >1000-fold selectivity for ATR over the homologous kinases ATM and DNA-PK, and the cell activity of 3. The improvement in affinity for ATR observed for some compounds cannot be attributed to new hydrogen bonding interactions formed between the 5membered heteroaryl rings and the ATR active site. For example, the O-1 and the N-2 atoms of compound 10 that are accessible to the enzyme are the same as those of compound 4 yet these compounds have substantially different inhibition constants (26 nM vs 1 nM respectively). Instead, the observed differences in affinity can be predicted by the conformational energy differences between the bioactive and alternative conformations listed in Table 2. Compound 4 favors the bioactive conformation as this aligns the isoxazole oxygen and CH with the 2aminopyrazine NH2 and ring nitrogen, respectively. For compounds 10 and 11, the alternative conformation is favored by 3.8 and 1.7 kcal/mol respectively over the bioactive conformation and these energetic penalties are reflected in their elevated Ki values. For compound 11 to assume the bioactive conformation the aromatic proton of the oxazole ring would need to sit opposite a proton of the hinge binder amine. For compound 10, the electrostatic misalignment is less obvious since in both conformations a hydrogen bond acceptor, either the O-1 or N-4 of the 1,2,4-oxadiazole, sits opposite the 2-aminopyrazine 4-nitrogen or amine. The calculated preference for the alternative conformation is in agreement with the observation that aromatic oxygen atoms are weaker


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hydrogen

bond

acceptors

than

aromatic

nitrogens.38

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The

alternative

conformation, which places the oxadiazole O-1 opposite the pyrazine ring nitrogen, is thus preferred as a consequence of the relatively lower electrostatic repulsion between these two acceptors compared to the repulsion between two nitrogen atoms in the bioactive conformation. Compounds 4, 8 and 12 maintain low nanomolar potency against ATR as predicted by the negative relative conformational energies of -4.6, -7.8 and -6.7 kcal/mol, respectively, favoring the bioactive conformation. In compound 9, an aromatic nitrogen atom of the 1,2,4-oxadiazole is always positioned opposite the pyrazine ring acceptor. The energy difference between the two conformations is small but the bioactive conformation is still preferred as confirmed by its low nanomolar Ki. Aromatic sulfur atoms carry a small positive charge and are known to interact favorably with hydrogen bond acceptors.42 This favorable interaction is reflected in the largest negative energy difference calculated for the thiadiazole 13, indicating a strong preference for the bioactive conformation. Although ligand pre-organization is only one of several factors contributing to the free energy of binding, the calculated conformational energy differences for fully aromatic 5-membered rings still show a reasonable trend when plotted against the measured inhibition constants (R2 = 0.72). Overall, the calculated energy differences provided useful guidance for the design of heteroaryl amide replacements. The isoxazole 4 was selected as a starting point for further optimization because of its superior selectivity profile against unrelated kinases, for example GSK3 (4 Ki = 650 nM, 7 Ki = 530 nM, 8 Ki = 22 nM). The phenyl ring of the isoxazole 4 is predicted to extend deep under the P-loop of ATR near a negatively charged area


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in our ATR kinase homology model (Figure 4). We speculated that this negatively charged environment could be exploited by amines projected from the para position to improve both binding affinity and aqueous solubility. Table 3 shows enzyme inhibition constants and cell based IC50 values for a selection of compounds containing para amine substituted benzyl rings.

Figure 4a-b. (A) Compound 4 docked in a homology model of the ATR active site based on PDB entry 1E7V with ATR represented by its electrostatic potential map. An area of high negative charge (in red) is located to the right of the phenyl ring of compound 4. (B) Compound 2 docked into the active site of ATR. Hydrogen bonds to the hinge, the unique residue Gly2385 as well as Asn2480 and Asp2494 that are part of the conserved magnesium binding site and salt bridge are indicated. Polar residues around the phenyl ring are shown as sticks.


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Table 3. Enzyme inhibition constants, pH2AX cell assay IC50s and aqueous solubility for 3-isoxazole-2-aminopyrazine ATR inhibitors. NH 2 O N N

R

N

SO 2iPr

ATM

DNA-PK


1.3

1400

>4000

1.1

4000

>25

12.5

0.17

44

>4000

0.019

52

0.21

47

3350

0.076

72

0.14

189

>4000

0.013

0.8

Enzyme inhibition Ki (nM) Compounds

R ATR

4 NH 2

14

N H

2

N H

15

Aqueous solubility (M)

O

16

N H

17

NH 2

1.24

82

785

0.3

27.9

18

NH 2

0.53

105

1700

0.13

45.1

19

N H

2.40

190

>4000

0.13

1.36

0.75

93

2610

0.21

4.4

Cl

N H

20 Cl

Introduction of a benzylamine motif, 14, resulted in improved ATR affinity (Ki 270 pM cf 1.3 nM for 3), however cell activity was compromised. This may be attributed to poor cell penetration associated with a highly solvated primary


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amine with a low calculated logD of 0.76. Small amine substituents, to form a secondary amine such as in 2, improved cell activity substantially (e.g. 0.019 µM for 2 vs 1.1 µM for 4). The introduction of secondary amines also led to a significant reduction in calculated logD (from 3.6 for compound 4 to 1.1 for 2) with a concomitant increase in aqueous solubility (cf. Table 3). Larger amine substituents such as the ethyl in 15 and pyranyl in 16 provided only limited improvement in terms of affinity and cell potency over the methylamine 2. As an alternative approach to modulate the properties of the primary amine we considered alkylation of the benzylic position and prepared the two stereoisomers of a methyl substituent on the benzylamine methylene. This was most effective for the R-stereoisomer, which retained good ATR affinity and a substantial improvement in cell potency compared with 4. Although introduction of the -methyl group markedly improved the potency in the pH2AX cell assay compared to 14, N-alkylation, as in 2, yielded lower cell assay IC50s (19 nM for 2 vs 130 nM for 18). Further hydrophobic substitutions on the ortho and meta positions of the phenyl ring in 19 and 20 lost potency in both the ATR enzyme and pH2AX cell assays. We speculate that this may be due to the polar local environment and limited available space around the phenyl ring in the ATP binding site. In general little correlation was observed between measured and calculated logD and pKa values and activity in cellular assays. For example, although compound 16 had a markedly lower measured pKa value of 8.2 versus 9.7 for compound 2 both compounds are essentially equipotent in the pH2AX cell assay. Despite its relatively high pKa, compound 2 showed good permeability in a Caco2 assay (AB 7.2 10-6 cm/s with an efflux ratio of 2.9). From the set of hetero-alkyl


21


Page 22 of 73

secondary amines explored on the isoxazole scaffold, only compounds 15 and 16 showed activity comparable to that of compound 2 in the pH2AX cell assay and out of these only 2 and 15 combined this with good aqueous solubility. Introduction of primary or secondary amines had no impact on affinity for DNAPK but did increase affinity for ATM kinase (Ki = 44 nM for 2, cf 1400 nM for 4) as the amino acids near the benzyl amine are conserved between ATR and ATM. For DNAPK, any favorable effects of introducing the benzyl amine on binding affinity may be cancelled out by the proximity of the positive charge of the unique Arg3733 residue in the P-loop of DNAPK. Despite this increase in ATM enzyme affinity, selectivity for ATR over ATM is maintained at >200-fold. The overall selectivity profile of 2 was established by testing the compound against a large panel of unrelated kinases at a concentration of 400 nM. 278 out of 291 kinases tested showed 200 nM, and > 1000 fold selectivity for ATR). Inhibition constants for the 13 remaining kinases were determined and are listed in Table 4. Of these only Flt4 kinase showed less than 100-fold selectivity, with an estimated Ki of 8 nM, which corresponds to a selectivity of ~50-fold).


22

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Table 4. Human kinases with enzyme inhibition constants Ki < 200 nM for compound 2.

a

Kinase

Ki (nM)

ATR

0.165

Abl(T315I)

59a

cKit(D816H)

39a

DYRK2

26a

Flt3(D835Y)

18a

Flt3

130

Flt4

8a

GSK3a

33a

GSK3b

140

JAK2

150

Mer

30a

MLK1

15a

PI3Kinase (p110/p85)

140

SYK

190

Ki estimated based on the ATP concentration in the assay being equal to the

Michaelis constant (Km) for ATP and inhibition is through an ATP-competitive mechanism of action. All data was obtained from a minimum of 2 replicates.

Compound 2 was selected as the clinical candidate based on its overall profile in terms of potency, selectivity and solubility. A ligand interaction diagram detailing nearby residues and key hydrogen bonds involving compound 2 as docked into our ATR homology model is shown in Figure 5. This compound showed >200-fold selectivity over Cytochrome P450’s (IC50s against Cyp3A4,


23


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2C9 and 2D6 all > 30 M) and the hErg potassium ion channel (IC50 = 3 M). Compound 2 had a favorable whole blood IV pharmacokinetic profile in Male Sprague-Dawley rats (Cl = 26 mL/min/kg; Vss = 21 L/kg; T1/2 = 11.6 h) and oral bioavailability to support in vivo efficacy studies.8, 12

Figure 5.

Ligand interaction diagram showing nearby residues, aromatic

stacking (green lines) and key hydrogen bonds (purple lines) involving compound 2 as docked into our ATR homology model derived from PDB entry 1E7V.

In cell experiments, compound 2 markedly sensitized the HCT116 colorectal cell line to the DNA cross-linking agent cisplatin, at concentrations of 2 100 fold selectivity over related kinases and unrelated proteins such as hErg and P450 enzymes and had PK properties suitable for IV administration. Three factors helped to optimize the potency and physical properties of compound 1. The first was to maximize steric fit to the active site of ATR. While flat, 5-membered heteroaryl linkers maintained good ATR affinity, a significant loss of potency was observed for the partially saturated 4,5-dihydroisoxazole 5


26

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compared to its fully aromatic isoxazole counterpart 4. The more 3-dimensional shape of 5, due to its sp3 carbon linking the 4,5-dihydroisoxazole to the 2aminopyrazine, results in the compound incurring an energy penalty when binding to the ATR binding site in a flattened conformation. A second factor was the degree of electrostatic complementarity between the 5membered heteroaryl ring and the 2-aminopyrazine hinge binder. In all cases, except for the 1,2,4-oxadiazole 10 and the oxazole 11, the interactions between the two rings favor the bound conformation as predicted by quantum chemical calculations. Furthermore, the magnitude of the gas phase energy differences between the two possible conformations for each of the inhibitors correlated with ATR enzyme affinity and as such this calculation provided a convenient tool for predicting optimal heterocyclic scaffold modifications. Thirdly, we optimized polar interactions between the inhibitor and the ATP binding site of ATR by substituting the phenyl isoxazole of 4 at the para position with amines. Compound 2, with a simple N-methyl benzylamine motif, achieved an 8-fold improvement in affinity compared to its unsubstituted counterpart 4 along with greatly improved cell potency, aqueous solubility and pharmacokinetics. The 5-heteroaryl containing inhibitors reported in this study present a new chemical motif for potent ATR kinase inhibition. The resulting compound, 2 (VX970/M6620), is currently being evaluated in Phase I/II clinical studies in combination with several DNA damaging chemotherapies and PARP inhibitors, and as monotherapy for the treatment of solid tumours.


27


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EXPERIMENTAL SECTION

General synthetic methods All commercially available solvents and reagents were used as received. Microwave reactions were carried out using a CEM Discovery microwave. Analytical thin layer chromatography was carried out using glass-backed plates coated with Merck Kieselgel 60 GF240. Plates were visualized using UV light (254 nm or 366 nm) and/or by staining with potassium permanganate followed by heating. Flash chromatography was carried out on an ISCO© CombiflashR CompanionTM system eluting with a 0 to 100% EtOAc/petroleum ether gradient. Samples were applied pre-absorbed on silica. Semi-preparative reverse phase HPLC was carried out on a Waters autopurification HPLC-MS system equipped with a waters C-18 sunfire reverse phase column (19 mm x 150 mm, 5 mm). The mobile phases were acetonitrile (0.1 % trifluoroacetic acid) and water (0.1 % trifluoroacetic acid). 1H-NMR and 13C-NMR spectra were recorded at 400 MHz or 500 MHz and 125 MHz or 100 MHz, using a Bruker DPX 400 instrument or a Bruker DPX 500 instrument. MS samples were analyzed on a MicroMass Quattro Micro mass spectrometer operated in single MS mode with electrospray ionization.

Samples were introduced into the mass spectrometer using

chromatography. All final products had a purity ≥95%, unless specified otherwise in the experimental details. The purity of the final compounds was determined using HPLC method: analytical reverse phase HPLC-MS was carried out on an agilent 100 HPLC with a waters Quattro MS system equipped with an ACE 5mm C-18 reverse phase column (4.6 mm x 150 mm, 5 mm). The mobile phases were acetonitrile/methanol (1:1) and water (10 mm ammonium acetate).


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5-(4-(Isopropylsulfonyl)phenyl)-3-(3-(4((methylamino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-amine (2). To a solution

of

N,N-di(tert-butoxycarbonyl)-5-(4-(isopropylsulfonyl)phenyl)-3-

ethynylpyrazin-2-amine 25 (6.8 g, 13.56 mmol) and N-hydroxy-4-methylbenzimidoyl chloride (2.71 g, 13.56 mmol) in THF (140 mL) at room temperature was added triethylamine (1.65 g, 2.27 mL, 16.27 mmol) dropwise over l0 min. The mixture was stirred at room temperature overnight then at 60 °C for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was partitioned between CH2Cl2 (30 mL) and brine (50 mL). The organic layer was washed with an aqueous saturated solution of NaHCO3 (50 mL), dried over MgSO4, filtered and evaporated under reduced pressure. The residue was purified by chromatography on silica gel, eluting with 20% EtOAc/petroleum

ether

to

give

N,N-di(tert-butoxycarbonyl)-5-(4-

(isopropylsulfonyl)phenyl)-3-(3-(4-methyl)phenylisoxazol-5-yl)pyrazin-2-amine as an off-white solid (7.1 g, 82% Yield); 1H NMR (400 MHz, DMSO-d6)  9.51 (s, IH), 8.66 (m, 2H), 8.07 (m, 2H), 8.01 (s, 1H), 7.92 (m, 2H), 7.39 (m, 2H), 3.55 (m, 1H), 3.34 (s, 3H), 1.33 (s, 18H), 1.21 (m, 6H); MS (ES+) m/z 635.3 (M + H)+. To a solution of N,N-di(tert-butoxycarbonyl)-5-(4-(isopropylsulfonyl)phenyl)3-(3-(4-methyl)phenylisoxazol-5-yl)pyrazin-2-amine (3.5 g, 5.51 mmol) in ethyl acetate (35 mL) was added N-bromosuccinimide (1.28 g, 7.17 mmol), followed by AIBN (180 mg, 1.1 mmol). The resulting mixture was placed under a bright lamp and heated to 85 °C for l h. The reaction mixture was allowed to cool to ambient temperature, washed sequentially with brine (50 mL) and an aqueous saturated solution of NaHCO3 (50 mL), then filtered, dried over MgSO4 and concentrated

in

vacuo

to

afford



29


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(isopropylsulfonyl)phenyl)-3-(3-(4-bromomethyl)phenylisoxazol-5-yl)pyrazin2-amine as a pale brown foam (4.56 g), which was used directly in the next stage without further purification. 1H NMR (400 MHz, DMSO-d6)  9.52 (s, 1H), 8.65 (m, 2H), 8.03-8.09 (m, 4H), 7.66 (m, 2H), 4.80 (s, 2H), 3.57 (m, 1H), 1.31 (s, 18H), 1.21 (m, 6H),; MS (ES+) m/z 715.3 (M + H)+. A

solution

of

crude


(isopropylsulfonyl)phenyl)-3-(3-(4-bromomethyl)phenylisoxazol-5-yl)pyrazin2-amine (60 mg, 0.084 mmol) in CH2Cl2 (5 mL) was added to methylamine (791 mg, 8.41 mmol) in ethanol (3 mL). The reaction mixture was stirred at room temperature for 1 h. The solvent was then removed in vacuo to provide an oil, which was dissolved in a mixture of CH2C12 (5 mL) and TFA (479 mg, 4.2 mmol). The reaction mixture was stirred at room temperature for 1 h, then concentrated in vacuo. The residue was purified by reverse phase preparative HPLC [Waters Sunfire C18, 10 M, 100 A column, gradient 10% - 95% B (solvent A: 0.05% TFA in water; solvent B: CH3CN)]. The product fractions were eluted through a bicarbonate cartridge and lyophilized to give 5-(4-(isopropylsulfonyl)phenyl)-3(3-(4-((methylamino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-amine

2

as

a

yellow solid (13.6 mg, 28 % yield over two steps); 1H NMR (400 MHz, DMSOd6)  8.92 (s, 1 H), 8.85 (br s, 2H), 8.43 (m, 2H), 8.12 (m, 2H), 7.85 (m, 2H), 7.82 (s, 1H), 7.65 (m, 2H), 7.26 (br s, 2H), 4.21-4.25 (m, 2H), 3.55 (m, J 7.5 Hz, 1H), 2.61-2.65 (m, 3H), 1.22 (d, J 7.5 Hz, 6H);

13C

NMR (125 MHz, DMSO-d6) 

168.35, 162.38, 152.08, 143.00, 141.66, 138.04, 136.14, 134.81, 131.27, 129.64, 129.10, 127.43, 126.17, 124.86, 102.76, 54.68, 51.10, 32.25, 15.56; MS (ES+) 464.4; HRMS (ES+) calcd for C24H25N5O3S (M + H)+ 464.1751; found: 464.1754.


30

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3-Amino-6-(4-(isopropylsulfonyl)phenyl)-N-phenylpyrazine-2carboxamide (3). 3-Amino-6-(4-isopropylsulfonylphenyl)pyrazine-2-carboxylic acid 22 (0.8 g, 2.5 mmol), diethoxyphosphorylformonitrile (677 mg, 620 µL, 3.73 mmol), aniline (348 mg, 340 µL, 3.73 mmol) and triethylamine (504 mg, 694 µL, 5 mmol) were stirred in 1,2-dimethoxyethane (10 mL) at 120 °C for 18 hours. Water (20 mL) was added and the solid was collected by filtration. The residue was washed with diethyl ether (20 mL) and dried in vacuo to afford 3 as a yellow solid. (950 mg, 96% Yield). 1H NMR (400 MHz, DMSO-d6)  10.47 (s, 1H), 9.05 (s, 1H), 8.53 (d, J 8.5 Hz, 2H), 7.93 (d, J 8.5 Hz, 2H), 7.82 (d, J 7.8 Hz, 2H), 7.41 (t, J 7.8 Hz, 2H), 7.18 (m, 1H), 3.48 (m, J 6.8 Hz, 1H), 1.19 (d, J 6.8 Hz, 6H); 13C NMR (100 MHz, DMSO-d6)  164.72, 155.14, 145.80, 141.17, 138.20, 136.74, 136.16, 129.36, 128.99, 126.56, 124.87, 124.68, 121.62, 54.56, 15.59; MS (ES+) m/z 397.3 (M + H)+; HRMS (ES+) calcd for C20H20N4O3S (M + H)+ 397.1334; found: 397.1343.

5-(4-(Isopropylsulfonyl)phenyl)-3-(3-phenylisoxazol-5-yl)pyrazin-2-amine (4). To

a

solution

of

N,N-bis-tert-butoxycarbonyl-3-ethynyl-5-(4-

(isopropylsulfonyl)phenyl)pyrazin-2-amine 25 (300 mg, 0.6 mmol) and Nhydroxybenzimidoyl chloride (93.0 mg, 0.6 mmol) in N,N-dimethylformamide (3 mL) was added triethyl amine (60.5 mg, 83.4 µL, 0.6 mmol). The mixture was warmed to 60 °C for 45 minutes. The mixture was diluted with ethyl acetate (20 mL), washed sequentially with water (20 mL) and brine (20 mL). The organic layer was separated


31


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and dried over MgSO4, filtered and concentrated in vacuo. The residue was dissolved in CH2Cl2 (5 mL) and TFA (2.05 g, 1.4 mL, 17.94 mmol) was added. The mixture was stirred at ambient temperature for 1 hour and then concentrated in vacuo. The residue was purified by reverse phase HPLC [Waters Sunfire C18, 10 μM, 100 Å column, gradient 10% - 95% B (solvent A: 0.05% TFA in water; solvent B: CH3CN)] to give 5-(4(isopropylsulfonyl)phenyl)-3-(3-phenylisoxazol-5-yl)pyrazin-2-amine 4 as a yellow solid (136 mg, 52% Yield). 1H NMR (400 MHz, DMSO-d6)  8.95 (s, 1H), 8.44 (m, 2H), 8.01 - 8.06 (m, 2H), 7.95 (m, 2H), 7.67 (s, 1H), 7.55 - 7.63 (m, 2H), 7.22 - 7.35 (m, 2H), 3.47 (m, J 6.9 Hz, 1H), 1.14 (d, J 6.9 Hz, 6H); 13C NMR (100 MHz, DMSO-d6)  168.93, 163.52, 153.04, 143.78, 142.32, 138.82, 137.00, 131.76, 131.74, 130.44, 129.54, 128.08, 126.96, 125.72, 103.46, 55.46, 16.47; MS (ES+) m/z 421.3 (M + H)+; HRMS (ES+) calcd for C22H20N4O3S (M + H)+ 421.1334; found: 421.1339.

5-(4-(Isopropylsulfonyl)phenyl)-3-(3-phenyl-4,5-dihydroisoxazol-5yl)pyrazin-2-amine (5). To a solution of 5-(4-isopropylsulfonylphenyl)-3vinyl-pyrazin-2-amine 26 (20 mg, 0.066 mmol) in N,N-dimethylformamide (2 mL) was added N-hydroxybenzimidoyl chloride (13.3 mg, 0.086 mmol), followed by dropwise addition of triethylamine (20 mg, 28 µL, 0.198 mmol). The mixture was stirred at ambient temperature for 20 mins, then heated to 65 °C for 1 hour. The mixture was cooled to room temperature and diluted with ethyl acetate (5 mL) and water (5 mL) and the layers separated. The aqueous layer was extracted further with ethyl acetate (5 mL x2) and the combined


32

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organic extracts washed with water (10 mL x3), dried over MgSO4 and concentrated in vacuo. The product was purified by reverse phase HPLC [Waters Sunfire C18, 10 µM, 100 Å column, gradient 10% - 95% B (solvent A: 0.05%

TFA

in

water;

solvent

B:

CH3CN)]

to

give

5-(4-

(isopropylsulfonyl)phenyl)-3-(3-phenyl-4,5-dihydroisoxazol-5-yl)pyrazin-2amine 5 as a white solid (16 mg, 58% Yield). 1H NMR (400 MHz, DMSO-d6)  8.75 (s, 1H), 8.22 (m, 2H), 7.78 - 7.85 (m, 4H), 7.48-7.53 (m, 3H), 6.95 (br s, 2H), 6.02 (m, 1H), 4.28 (m, 1H), 3.75 (m, 1H), 3.44 (m, J 6.8 Hz, 1H), 1.15 (d, J 6.8 Hz, 6H);

13C

NMR (125 MHz, DMSO-d6)  157.78, 154.40, 142.19, 141.24, 136.48,

136.02, 135.65, 130.70, 129.67, 129.56, 129.37, 127.26, 125.53, 78.88, 54.65, 37.03, 15.66; MS (ES+) m/z 423.4 (M + H)+; HRMS (ES+) calcd for C22H22N4O3S (M + H)+ 423.1491; found: 423.1487.

5-(4-(Isopropylsulfonyl)phenyl)-3-(1-phenyl-1H-1,2,3-triazol-4-yl)pyrazin2-amine

(6).

N,N-Bis-tert-butoxycarbonyl-3-ethynyl-5-(4-

(isopropylsulfonyl)phenyl)pyrazin-2-amine 25 (380 mg, 0.76 mmol) and azidobenzene (0.5 M in MTBE) (3.03 mL of 0.5 M, 1.52 mmol) were suspended in t-BuOH/water (1/1; 15 mL). (+)-Sodium L-ascorbate (30 mg, 0.15 mmol), followed by CuSO4.5H2O (3.8 mg, 0.02 mmol) were added and the reaction allowed to stir at 40 °C for 48 hours. The reaction mixture was cooled to ambient temperature, diluted with ethyl acetate (50 mL), then washed sequentially with water (50 mL) and brine (50 mL x 2). The organic layer was


33


Page 34 of 73

dried over MgSO4, filtered and concentrated in vacuo. The residue was purified

by

column

chromatography

on

silica,

using

an

ethyl

acetate/petroleum ether gradient as eluent, to provide crude tert-butyl N-tertbutoxycarbonyl-N-[5-(4-isopropylsulfonylphenyl)-3-(1-phenyltriazol-4yl)pyrazin-2-yl]carbamate as an off-white solid. (193 mg, 41% Yield). 1H NMR (400 MHz, DMSO-d6)  9.73 (s, 1H), 9.36 (s, 1H), 8.68 (d, J 8.4 Hz, 2H), 8.09 (d, J 8.4 Hz, 2H), 8.04-8.08 (m, 2H), 7.66-7.70 (m, 2H), 7.57 (m, 1H), 3.55 (m, J 6.8 Hz, 1H), 1.27 (s, 18H), 1.191 (d, J 6.8 Hz, 6H); MS (ES+) m/z 465.1. To

a

solution

of

tert-butyl

N-tert-butoxycarbonyl-N-[5-(4-

isopropylsulfonylphenyl)-3-(1-phenyltriazol-4-yl)pyrazin-2-yl]carbamate (193 mg, 0.31 mmol) in CH2Cl2 (3 mL) was added TFA (1 mL). The reaction mixture was stirred at ambient temperature for 1 hour. The reaction mixture was concentrated in vacuo and the residue was sonicated in acetonitrile (5 mL). The solid was collected by filtration and dried by suction providing 5-(4(isopropylsulfonyl)phenyl)-3-(1-phenyl-1H-1,2,3-triazol-4-yl)pyrazin-2-amine 6 as a pale yellow solid. (105 mg, 80% Yield); 1H NMR (400 MHz, DMSO-d6)  9.66 (s, 1H), 8.85 (s, 1H), 8.46 (m, 2H), 8.12 (m, 2H), 7.91 (m, 2H), 7.74 (br s, 2H), 7.67 (m, 2H), 7.58 (m, 1H), 3.47 (m, J 6.8 Hz, 1H), 1.19 (d, J 6.8 Hz, 6H); 13C

NMR (125 MHz, DMSO-d6)  152.28, 148.04, 142.08, 140.53, 137.22, 136.85,

135.86, 130.40, 129.74, 129.48, 128.22, 125.99, 123.44, 121.08, 54.72, 15.71; MS (ES+) m/z 421.1 (M + H)+; HRMS (ES+) calcd for C21H20N6O2S (M + H)+ 421.1447; found: 421.1445.


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5-(4-(Isopropylsulfonyl)phenyl)-3-(5-phenylisoxazol-3-yl)pyrazin-2-amine (7). 3-Amino-6-(4-isopropylsulfonylphenyl)pyrazine-2-carboxylic acid 22 (10 g, 31.1 mmol) was dissolved in tetrahydrofuran (80 mL) and the mixture was cooled in an ice-bath. N-methoxymethanamine hydrochloride (3.6 g, 37.3 mmol), 1-hydroxybenzotriazole hydrate (5.2 g, 34.2 mmol), DIPEA (8 g, 10.8 mL, 62.2 mmol) and 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1amine (5.3 g, 34.2 mmol) were added to the cooled solution. The mixture was allowed to warm to ambient temperature and stirred for 18 hours. The mixture was concentrated in vacuo and the residue separated between ethyl acetate (200 mL) and water (200 mL). The organic phase was washed sequentially with saturated bicarbonate (200 mL) and brine (200 mL), dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica, eluting with 30% ethylacetate/petroleum ether to afford 3-amino-6-(4-isopropylsulfonylphenyl)-N-methoxy-N-methylpyrazine-2-carboxamide (8.8 g, 77% Yield). 1H NMR (400 MHz, DMSO-d6)  8.80 (s, 1H), 8.22 (d, 2H), 7.92 (m, 2H), 6.95 (br s, 2H), 3.65-3.71 (m, 3H), 3.413.50 (m, 1H), 2.73 (s, 3H), 1.21 (d, 6H); MS (ES+) m/z 365.4 (M + H)+. 3-Amino-6-(4-isopropylsulfonylphenyl)-N-methoxy-N-methyl-pyrazine-2carboxamide (5 g, 13.72 mmol) was dissolved in tetrahydrofuran (60 mL) and cooled to -20 °C. Methylmagnesium bromide (25.2 mL of 3 M, 75.46 mmol) was added dropwise and the mixture was stirred at -20 °C for 1 h. The


35


Page 36 of 73

reaction was quenched by dropwise addition of saturated ammonium chloride solution (3 mL), then was allowed to warm to ambient temperature. The mixture was concentrated in vacuo to an oil, diluted with ethyl acetate (50 mL) and washed sequentially with water (50 mL) and brine (50 mL). The organic layer was separated, dried over MgSO4, filtered and concentrated in vacuo. The residue was purifed by column chromatography on silica, eluting with 50-80% ethyl acetate/petroleum ether to afford 1-[3-amino-6-(4isopropylsulfonylphenyl)pyrazin-2-yl]ethanone as a yellow solid (3.5 g, 80% Yield). 1H NMR (400 MHz, DMSO-d6)  9.12 (s, 1H), 8.35 (d, 2H), 8.02-7.85 (m, 2H), 3.50-3.42 (m, 1H), 2.71 (s, 3H), 1.21 (d, 6H); MS (ES+) m/z 320.1 (M + H)+. To a solution of benzaldehyde (332 mg, 318 µL, 3.13 mmol) and 1-[3-amino6-(4-isopropylsulfonylphenyl)pyrazin-2-yl]ethanone (1 g, 3.13 mmol) in a mixture of MeOH/CH2Cl2 (1/1; 20 mL) was added sodium hydroxide (12.5 mL of 10 %w/v, 3.13 mmol) in methanol. The mixture was stirred at ambient temperature for 18 hours and then concentrated in vacuo. The residue was diluted with ethyl acetate (30 mL), washed sequentially with water (30 mL) and brine (30 mL). The organic layer was separated, dried over MgSO4, filtered and concentrated in vacuo. The residue was purifed by column chromatography on silica, eluting with ethyl acetate to afford 1-[3-amino-6-(4isopropylsulfonylphenyl)pyrazin-2-yl]-3-phenyl-prop-2-en-1-one as a yellow solid (900 mg, 70% Yield). 1H NMR (400 MHz, CDCl3)  8.82 (s, 1H), 8.50 (d, J 16 Hz, 1H), 8.22 (d, J 8.8 Hz, 2H), 8.06 (d, J 8.8 Hz, 2H), 7.94 (d, J 16 Hz, 1H),


36

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7.74-7.77 (m, 2H), 7.48-7.50 (m, 3H), 3.28 (m, J 7.2 Hz, 1H), 1.38 (d, J 7.2 Hz, 6H); MS (ES+) m/z 408.1 (M + H)+. To a solution of 1-[3-amino-6-(4-isopropylsulfonylphenyl)pyrazin-2-yl]-3phenyl-prop-2-en-1-one (50 mg, 0.12 mmol) in ethanol (1 mL) was added hydroxylamine hydrochloride (8.5 mg, 0.12 mmol) and a solution of sodium acetate (10.1 mg, 0.12 mmol) in warm acetic acid (0.3 mL). The reaction was heated under microwave conditions for 1 hour at 130 °C. The mixture was concentrated in vacuo, the residue diluted with ethyl acetate (10 mL) and washed sequentially with water (10 mL) and brine (10 mL). The organic layer was separated, dried over MgSO4, filtered and concentrated in vacuo. The residue was re-dissolved in methanol and then purified by reverse phase HPLC [Waters Sunfire C18, 10 µM, 100 Å column, gradient 10% - 95% B (solvent A: 0.05% TFA in water; solvent B: CH3CN)] to give 5-(4(isopropylsulfonyl)phenyl)-3-(5-phenylisoxazol-3-yl)pyrazin-2-amine 7 as a yellow solid (8 mg, 17% Yield). 1H NMR (400 MHz, DMSO-d6)  8.95 (s, 1H), 8.42 (m, 2H), 8.02 - 8.05 (m, 2H), 7.93 (m, 2H), 7.78 (s, 1H), 7.55 - 7.61 (m, 3H), 7.27 (br s, 2H), 3.55 (m, J 6.9 Hz, 1H), 1.22 (d, J 6.9 Hz, 6H);

13C

NMR (100

MHz, DMSO-d6)  166.65, 161.23, 150.76, 141.50, 140.03, 136.52, 134.71, 129.45, 128.17, 128.15, 127.25, 125.79, 124.67, 123.43, 101.18, 53.18, 14.18; MS (ES+) m/z 421.1 (M + H)+; HRMS (ES+) calcd for C22H20N4O3S (M + H)+ 421.1334; found: 421.1354.


37


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5-(4-(Isopropylsulfonyl)phenyl)-3-(5-phenyl-1,3,4-oxadiazol-2-yl)pyrazin2-amine (8). To a mixture of (4-isopropylsulfonylphenyl)boronic acid (47.8 mg, 0.21 mmol) and 5-bromo-3-(5-phenyl-1,3,4-oxadiazol-2-yl)pyrazin-2amine 28 (66.5 mg, 0.21 mmol) in dioxane (3 mL) was added Pd(dppf)Cl2.CH2Cl2 (171 mg, 0.21 mmol), followed by sodium carbonate (2.1 mL of 2 M, 4.2 mmol). The reaction mixture was subjected to microwave irradiation at 100 °C for 25 minutes. The mixture was then diluted with water (10 mL) and extracted with EtOAc (10 mL x2). The combined organic extracts were washed with brine (10 mL), dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica, using a

diethyl

ether/Petroleum

ether

gradient

as

eluent,

to

give

5-(4-

(isopropylsulfonyl)phenyl)-3-(5-phenyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine 8 as an orange solid (60 mg, 69% Yield). 1H NMR (400 MHz, DMSO-d6)  9.06 (s, 1H), 8.40 (m, 2H), 8.19 - 8.17 (m, 2H), 7.98 (m, 4H), 7.72 - 7.66 (m, 3H), 3.47 (m, J 6.8 Hz, 1H), 1.20 (d, J 6.8 Hz, 6H); MS (ES+) m/z 422.0 (M + H)+; HRMS (ES+) calcd for C21H19N5O3S (M + H)+ 422.1287; found: 422.1283.

5-(4-(Isopropylsulfonyl)phenyl)-3-(5-phenyl-1,2,4-oxadiazol-3-yl)pyrazin2-amine (9). Prepared using the experimental procedure described for 8, using

5-bromo-3-(5-phenyl-1,2,4-oxadiazol-3-yl)pyrazin-2-amine

31.

5-(4-

(Isopropylsulfonyl)phenyl)-3-(5-phenyl-1,2,4-oxadiazol-3-yl)pyrazin-2-amine 9 was obtained as a pale orange solid (44.9 mg, 34% Yield). 1H NMR (400


38

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MHz, DMSO-d6)  8.86 (s, 1H), 8.18 (m, 2H), 8.12 (m, 2H), 7.79 (m, 2H), 7.61 (m, 1H), 7.53 (m, 2H), 7.42 (br s, 2H), 3.29 (m, J 6.8 Hz, 1H), 1.02 (d, J 6.8 Hz, 6H);

13C

NMR (100 MHz, DMSO-d6)  175.45, 167.51, 153.68, 144.06, 141.50,

138.15, 136.14, 134.00, 129.99, 129.64, 128.60, 126.09, 123.50, 123.21, 54.60, 15.59; MS (ES+) m/z 422.2 (M + H)+; HRMS (ES+) calcd for C21H19N5O3S (M + H)+ 422.1287; found: 422.1298.

5-(4-(Isopropylsulfonyl)phenyl)-3-(3-phenyl-1,2,4-oxadiazol-5-yl)pyrazin2-amine

(10).

To

a

solution

of

3-amino-6-(4-

isopropylsulfonylphenyl)pyrazine-2-carboxylic acid 22 (250 mg, 0.77 mmol) in N,N-dimethyl formamide (7.5 mL) was added carbonyl diimidazole (249.2 mg, 1.54 mmol) and the resulting solution was stirred at ambient temperature for 1 hour. N'-hydroxybenzamidine (86.6 mg, 0.64 mmol) was added and the reaction stirred at ambient temperature for a further 2.5 hours then heated to 120 °C for 1.5 hours. The reaction mixture was cooled to ambient temperature and added slowly to water (20 mL). The resulting precipitate was filtered, triturated from CH2Cl2/methanol (1/1, 10 mL), isolated by filtration and dried in vacuo at 40 °C to give 5-(4-(isopropylsulfonyl)phenyl)-3-(3-phenyl-1,2,4oxadiazol-5-yl)pyrazin-2-amine 10 as a yellow solid (177 mg, 67% Yield). 1H NMR (400 MHz, DMSO-d6)  9.16 (s, 1H), 8.37 (m, 2H), 8.26 (m, 2H), 8.07 (br s, 2H), 7.98 (m, 2H), 7.68 - 7.61 (m, 3H), 3.48 (m, J 6.8 Hz, 1H), 1.20 (d, J 6.8 Hz, 6H);

13C

NMR (100 MHz, DMSO-d6)  172.90, 168.02, 153.71, 146.42, 141.05,


39


Page 40 of 73


5-(4-(Isopropylsulfonyl)phenyl)-3-(2-phenyloxazol-5-yl)pyrazin-2-amine (11). To a mixture of 3-bromo-5-(4-isopropylsulfonylphenyl)pyrazin-2-amine 24 (88.3 mg, 0.248 mmol) and 2-phenyloxazole (30 mg, 0.21 mmol) in water (3 mL) was added Ag2CO3 (114 mg, 0.41 mmol), followed by PPh3 (5.4 mg, 0.021 mmol) and Pd(dppf)Cl2.CH2Cl2 (8.4 mg, 0.010 mmol). The reaction mixture was heated at 70 °C for 24 h before being cooled down and partitioned between CH2Cl2 (20 mL) and water (20 mL). The aqueous layer was extracted further with CH2Cl2 (20 mL x 2) and the combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by reverse phase preparative HPLC [Waters Sunfire C18, 10 µM, 100 Å column, gradient 10% - 95% B (solvent A: 0.05% TFA in water; solvent B: CH3CN)]. The solvents were evaporated to give 5-(4-(isopropylsulfonyl)phenyl)-3-(2phenyloxazol-5-yl)pyrazin-2-amine 11 as an off-white solid (25 mg, 29% Yield). 1H NMR (500 MHz, DMSO-d6)  8.45 (s, 1H), 8.12 - 8.07 (m, 2H), 8.06 8.01 (m, 2H), 7.97 - 7.87 (m, 3H), 7.56 - 7.25 (m, 3H), 3.17 (m, J 6.9 Hz, 1H), 1.27 (d, J 6.9 Hz, 6H);

13C

NMR (125 MHz, DMSO-d6)  158.88, 153.04, 151.78,

143.07, 141.75, 137.82, 136.18, 129.76, 129.73, 129.67, 127.47, 126.16, 124.93,


40

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124.16, 122.96, 54.75, 15.74; MS (ES+) m/z 421.0 (M + H)+; HRMS (ES+) calcd for C22H20N4O3S (M + H)+ 421.1334; found: 421.1335.

5-(4-(Isopropylsulfonyl)phenyl)-3-(5-phenyloxazol-2-yl)pyrazin-2-amine (12). To a mixture of 3-bromo-5-(4-isopropylsulfonylphenyl)pyrazin-2-amine 24 (147 mg, 0.413 mmol) and 5-phenyloxazole (49.9 mg, 0.344 mmol) in dioxane (2 mL), was added Pd(PPh3)4 (39.7 mg, 0.034 mmol), followed by lithium tert-butoxide (55 mg, 0.688 mmol). The reaction mixture was heated at 140 °C under microwave irradiation for 3 h. The reaction was cooled to ambient temperature and the mixture was purified by reverse phase chromatography [Waters Sunfire C18, 10 µM, 100 Å column, gradient 10% 95% B (solvent A: 0.05% TFA in water; solvent B: CH3CN)] to afford 5-(4(isopropylsulfonyl)phenyl)-3-(5-phenyloxazol-2-yl)pyrazin-2-amine 12 as an off-white solid (66 mg, 46% Yield). 1H NMR (500 MHz, DMSO-d6)  8.96 (s, 1H), 8.40 (d, J 8.5 Hz, 2H), 8.07 (s, 1H), 7.97 (d, J 8.5 Hz, 2H), 7.94 - 7.88 (m, 2H), 7.56 (m, 2H), 7.47 (m, 1H), 3.47 (m, J 6.8 Hz, 1H), 1.21 (d, J 6.8 Hz, 6H); 13C

NMR (125 MHz, DMSO-d6)  161.47, 151.60, 148.70, 141.84, 140.80, 137.91,



41


Page 42 of 73

5-(4-(Isopropylsulfonyl)phenyl)-3-(5-phenyl-1,3,4-thiadiazol-2-yl)pyrazin2-amine (13). Prepared using the experimental procedure described for 8, using 5-bromo-3-(5-phenyl-1,3,4-thiadiazol-2-yl)pyrazin-2-amine 29. 5-(4(Isopropylsulfonyl)phenyl)-3-(5-phenyl-1,3,4-thiadiazol-2-yl)pyrazin-2-amine 13 was obtained as a yellow solid (44.9 mg, 35% Yield). 1H NMR (400 MHz, DMSO-d6)  9.03 (s, 1H), 8.32 (m, 2H), 8.12 - 8.18 (m, 3H), 7.98 (m, 2H), 7.61 7.65 (m, 3H), 2.72 (m, J 6.8 Hz, 1H), 1.75 (d, J 6.8 Hz, 6H); 13C NMR (100 MHz, DMSO-d6)  171.60, 169.50, 152.86, 144.74, 141.73, 138.76, 137.06, 132.88, 130.68, 130.43, 130.37, 128.95, 126.74, 126.2, 54.62, 15.63; MS (ES+) m/z 438.0 (M + H)+; HRMS (ES+) calcd for C21H19N5O2S2 (M + H)+ 438.1058; found: 438.1056.

3-(3-(4-(Aminomethyl)phenyl)isoxazol-5-yl)-5-(4(isopropylsulfonyl)phenyl) pyrazin-2-amine (14). Prepared using the experimental procedure described for 2, using a methanolic solution of ammonia instead of methylamine. 3-(3-(4-(Aminomethyl)phenyl)isoxazol-5yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-amine 14 was obtained as a yellow solid (42% Yield over two steps). 1H NMR (400 MHz, DMSO-d6)  8.97 (s, 1H), 8.21 (br s, 2H), 8.43 (m, 2H), 8.14 (m, 2H), 7.95 (m, 2H), 7.82 (s, 1H), 7.65 (m, 2H), 7.25 (br s, 2H), 4.21-4.25 (m, 2H), 3.55 (m, J 6.8 Hz, 1H), 1.22 (d, J 6.8 Hz, 6H);

13C

NMR (125 MHz, DMSO-d6)  168.42, 162.25, 152.27, 143.09,

141.52, 138.12, 136.71, 136.31, 130.06, 129.63, 128.86, 127.49, 126.17, 124.85,


42

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102.70, 54.71, 42.51, 15.70; MS (ES+) m/z 450.4 (M + H)+; HRMS (ES+) calcd for C23H23N5O3S (M + H)+ 450.1600; found: 450.1603.

3-(3-(4-((Ethylamino)methyl)phenyl)isoxazol-5-yl)-5-(4(isopropylsulfonyl) phenyl)pyrazin-2-amine (15). Prepared using the experimental procedure described for 2, using ethylamine instead of methylamine.

3-(3-(4-((Ethylamino)methyl)phenyl)isoxazol-5-yl)-5-(4-

(isopropylsulfonyl)phenyl)pyrazin-2-amine 15 was obtained as a yellow solid (46% Yield over two steps). 1H NMR (400 MHz, DMSO-d6)  8.97 (s, 1H), 8.82 (br m, 1H), 8.41 (m, 2H), 8.10 (m, 2H), 7.95 (m, 2H), 7.82 (s, 1H), 7.65 (m, 2H), 7.25 (br s, 2H), 4.23 (m, 2H), 3.55 (m, J 6.9 Hz, 1H), 3.05 (m, 2H), 1.25 (t, J 7.5 Hz, 3H), 1.22 (d, J 6.9 Hz, 6H); 13C NMR (125 MHz, DMSO-d6)  168.44, 162.21, 152.27, 143.10, 141.52, 138.14, 136.32, 134.81, 131.04, 129.63, 129.29, 127.58, 126.18, 124.83, 102.71, 54.71, 49.74, 42.50, 15.70, 11.45; MS (ES+) m/z 478.4 (M + H)+; HRMS (ES+) calcd for C25H27N5O3S (M + H)+ 478.1913; found: 478.1915.

5-(4-(Isopropylsulfonyl)phenyl)-3-(3-(4-(((tetrahydro-2H-pyran-4yl)amino)methyl)phenyl)isoxazol-5-yl)pyrazin-2-amine (16). Prepared using the experimental procedure described for 2, using 4-aminotetrahydro-2Hpyran

instead

of

methylamine.

5-(4-(Isopropylsulfonyl)phenyl)-3-(3-(4-

(((tetrahydro-2H-pyran-4-yl)amino)methyl)phenyl)isoxazol-5-yl)pyrazin-2amine 16 was obtained as a yellow solid (87% Yield over two steps). 1H NMR


43


Page 44 of 73

(400 MHz, DMSO-d6)  8.93 (s, 1H), 8.37 (m, 2H), 7.93 - 7.98 (m, 4H), 7.74 (s, 1H), 7.52 (m, 2H), 7.18 (br s, 2H), 3.79-3.82 (m, 4H), 3.44 (m, J 6.8 Hz, 1H), 3.25 (m, 2H), 2.59 (m, 1H), 2.12 (br s, 1H), 1.78 (m, 2H), 1.29 (m, 2H), 1.19 (d, J 6.8 Hz, 6H);

13C

NMR (125 MHz, DMSO-d6)  167.56, 162.13, 151.71, 144.04,

142.38, 141.04, 137.57, 135.74, 129.10, 128.45, 126.52, 126.37, 125.65, 124.50, 102.06, 65.79, 54.22, 52.58, 49.18, 33.16, 15.17; MS (ES+) m/z 534.2 (M + H)+; HRMS (ES+) calcd for C28H31N5O4S (M + H)+ 534.2175; found: 534.2173.

(S)-3-(3-(4-(1-Aminoethyl)phenyl)isoxazol-5-yl)-5-(4(isopropylsulfonyl)phenyl)pyrazin-2-amine (17). To a solution of tert-butyl N-[(1S)-1-[4-(hydroxymethyl)phenyl]ethyl]carbamate (840 mg, 3.34 mmol) in THF (17 mL) was added manganese dioxide (2.9 g, 33.4 mmol). The reaction mixture was stirred at room temperature overnight, then filtered through a pad of celite, washing through with EtOAc. The filtrate was concentrated in vacuo to afford the crude tert-butyl (S)-(1-(4-formylphenyl)ethyl)carbamate as a colourless oil. This oil was dissolved in ethanol (8 mL) and hydroxylamine (approximately 441 µL of 50 %w/v, 6.68 mmol) was added. The resulting solution was stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuo and partitioned between ethyl acetate (10 mL) and water (10 mL). The aqueous layer was extracted further with ethyl acetate (2 x 10 mL) and the combined organic extracts dried over MgSO4 and concentrated

in

vacuo

to

provide


tert-butyl

(S)-(1-(444

Page 45 of 73 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


((hydroxyimino)methyl)phenyl)ethyl)carbamate as a colourless oil, which was used in the next step without further purification (844 mg, 96%); MS (ES-) m/z 263.0 (M - H)+. To

a

solution

of

tert-butyl

(S)-(1-(4-

((hydroxyimino)methyl)phenyl)ethyl)carbamate (300 mg, 1.14 mmol) in DMF (1.5 mL) was added N-chloro succinimide (152 mg, 1.14 mmol) and the reaction mixture was heated at 55 °C for 15 min. The mixture was allowed to cool to room temperature, diluted with ethyl acetate (5 mL) and water (5 mL) and the layers separated. The aqueous layer extracted further with ethyl acetate (2 x 5 mL) and the combined organic extracts washed with water (3 x 5 mL), dried over MgSO4 and concentrated in vacuo. The residue was dissolved in THF (7.5 mL) and tert-butyl N-tert-butoxycarbonyl-N-[3-ethynyl-5-(4isopropylsulfonylphenyl)pyrazin-2-yl]carbamate (569.3 mg, 1.14 mmol) was added, followed by Et3N (190 µL, 1.36 mmol). The mixture was stirred at room temperature for 45 mins followed by heating to 65 °C for 4 h. The mixture was cooled to room temperature and diluted with ethyl acetate (5 mL) and water (5 mL) and the layers separated. The aqueous layer was extracted further with ethyl acetate (2 x 5 mL) and the combined organic extracts washed with water (3 x 10 mL), dried over MgSO4 and concentrated in vacuo. The residue was pre-absorbed onto silica and purified by column chromatography, eluting with ethyl acetate/ petroleum ether (0-80% EtOAc in petroleum ether, 24 g SiO2) to give tert-butyl N-tert-butoxycarbonyl-N-[3-[3-


45


Page 46 of 73

[4-[(1S)-1-(tert-butoxycarbonylamino)ethyl]phenyl]isoxazol-5-yl]-5-(4isopropylsulfonylphenyl)pyrazin-2-yl]carbamate as a pale yellow solid (645 mg, 74%); 1H NMR (400 MHz, CDCl3)  9.08 (s, 1H), 8.37 (d, 2H), 8.11 (d, 2H), 7.89 (d, 2H), 7.46 (d, 2H), 7.37 (s, 1H), 4.87 (br s, 1H), 4.14 (q, 1H), 3.55 (m, 1H), 1.43 (s, 9H), 1.42 (s, 18H), 1.38 (d, 6H), 1.29 (d, 3H). To a solution of tert-butyl N-tert-butoxycarbonyl-N-[3-[3-[4-[(1S)-1-(tertbutoxycarbonylamino)ethyl]phenyl]isoxazol-5-yl]-5-(4isopropylsulfonylphenyl)pyrazin-2-yl]carbamate (644 mg, 0.843 mmol) in DCM (13 mL) was added TFA (1.3 mL, 16.9 mmol) and the resulting solution was stirred overnight at room temperature. The mixture was partitioned between dichoromethane and saturated aqueous sodium hydrogen carbonate solution and the layers separated once effervescence has stopped. The organic extracts were dried over MgSO4 and concentrated in vacuo to leave a solid. The residue was purified by column chromatography on silica eluting with 020% MeOH/ DCM to afford 3-[3-[4-[(1S)-1-aminoethyl]phenyl]isoxazol-5-yl]5-(4-isopropylsulfonylphenyl)pyrazin-2-amine 17 as a yellow solid (387 mg, 99%); 1H NMR (400 MHz, DMSO-d6)  8.95 (s, 1H), 8.39 (d, J 8.5 Hz, 2H), 7.96 7.93 (m, 4H), 7.77 (s, 1H), 7.56 (d, J 8.3 Hz, 2H), 7.20 (s, 2H), 4.07 (q, J 6.6 Hz, 1H), 3.47 (m, J 6.8 Hz, 1H), 1.95 (br s, 2H), 1.29 (d, J 6.6 Hz, 3H), 1.20 (d, J 6.8 Hz, 6H);

13C

NMR (125 MHz, DMSO-d6)  168.02, 162.66, 152.23, 151.87,

142.91, 141.55, 138.08, 136.25, 129.63, 127.08, 127.05, 126.68, 126.18, 125.01,


46

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102.60, 54.72, 50.97, 26.55, 15.70; MS (ES+) 464.3; HRMS (ES+) calcd for C24H23N4O3S (M –NH2 )+ 447.1485; found: 447.1491.

(R)-3-(3-(4-(1-Aminoethyl)phenyl)isoxazol-5-yl)-5-(4(isopropylsulfonyl)phenyl)pyrazin-2-amine

(18).

Prepared

using

the

experimental procedure described for 17, using tert-butyl N-[(1R)-1-[4(hydroxymethyl)phenyl]ethyl]carbamate instead of tert-butyl N-[(1S)-1-[4(hydroxymethyl)phenyl]ethyl]carbamate.

(R)-3-(3-(4-(1-

Aminoethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2amine 18 (TFA salt) was obtained as a yellow solid; 1H NMR (400 MHz, DMSO-d6)  8.96 (s, 1H), 8.39 (m, 3H), 8.38 (d, J 8.5 Hz, 2H), 8.11 (d, J 8.2 Hz, 2H), 7.94 (d, J 8.5 Hz, 2H), 7.83 (s, 1H), 7.69 (d, J 8.3 Hz, 2H), 7.22 (br s, 2H), 4.54 (m, 1H), 3.47 (q, J 6.8 Hz, 1H), 1.56 (d, J 6.8 Hz, 3H) and 1.20 (d, J 6.8 Hz, 6H);

13C

NMR (125 MHz, DMSO-d6) δ 168.38, 162.22, 152.26, 143.07, 141.83,

141.52, 138.12, 136.30, 129.63, 128.86, 128.03, 127.64, 126.18, 124.85, 102.71, 54.71, 50.18, 21.02, 15.70; MS (ES+) 464.1; HRMS (ES+) calcd for C24H23N4O3S (M –NH2 )+ 447.1485; found: 447.1497.

3-(3-(3-Chloro-4-((methylamino)methyl)phenyl)isoxazol-5-yl)-5-(4(isopropylsulfonyl)phenyl)pyrazin-2-amine

(19).


Prepared

using

the 47


Page 48 of 73

experimental procedure described for 17, using tert-butyl (2-chloro-4formylbenzyl)(methyl)carbamate

instead

of

formylphenyl)ethyl)carbamate.

tert-butyl

(S)-(1-(4-

3-(3-(3-Chloro-4-

((methylamino)methyl)phenyl)isoxazol-5-yl)-5-(4(isopropylsulfonyl)phenyl)pyrazin-2-amine 19 was obtained as a yellow solid. 1H

NMR (400 MHz, DMSO-d6)  8.98 (br s, 2H), 8.38 (m, 2H), 8.25 (m, 1H),

8.12 (m, 1H), 7.94-7.96 (m, 3H), 7.79 (m, 1H), 7.24 (br s, 2H), 4.37 (s, 2H), 3.48 (m, 1H), 2.71 (s, 3H), 1.19 (d, 6H);

13C

NMR (125 MHz, DMSO-d6) δ 168.64,

161.19, 158.24, 152.27, 143.20, 141.49, 138.14, 136.32, 134.83, 132.67, 132.47, 131.18, 129.64, 128.16, 126.17, 124.66, 102.86, 54.71, 48.85, 33.30, 15.69; MS (ES+) 498.2; HRMS (ES+) calcd for C24H24ClN5O3S (M + H)+ 498.1361; found: 498.1360.

3-(3-(2-Chloro-4-((methylamino)methyl)phenyl)isoxazol-5-yl)-5-(4(isopropylsulfonyl)phenyl)pyrazin-2-amine

(20).

Prepared

using

the

experimental procedure described for 17, using tert-butyl (3-chloro-4formylbenzyl)(methyl)carbamate

instead

of

formylphenyl)ethyl)carbamate.

tert-butyl

(S)-(1-(4-

3-(3-(2-Chloro-4-

((methylamino)methyl)phenyl)isoxazol-5-yl)-5-(4(isopropylsulfonyl)phenyl)pyrazin-2-amine 20 (TFA salt) was obtained as a yellow solid; 1H NMR (400 MHz, DMSO-d6)  8.98 (s, 1H), 8.87 (br s, 2H), 8.36


48

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(m, 2H), 7.90-7.96 (m, 3H), 7.85 (m, 1H), 7.63-7.65 (m, 2H), 7.25 (br s, 2H), 4.26 (m, 2H), 3.49 (m, 1H), 2.63 (t, 3H), 1.18 (d, 6H); 13C NMR (125 MHz, DMSO-d6) δ 167.99, 160.97, 158.32, 152.30, 143.24, 141.44, 138.19, 136.32, 132.46, 132.18, 132.06, 129.66, 129.56, 128.46, 126.19, 124.62, 105.16, 54.70, 50.71, 32.81, 15.69; MS (ES+) 498.2; HRMS (ES+) calcd for C24H24ClN5O3S (M + H)+ 498.1361; found: 498.1379.

Molecular modeling. Energy minimization was performed with Jaguar version 8.7.013 as implemented in Maestro v10.1.013, release 2015-1 (Schrödinger Inc., New York, USA) using Density Functional Theory (DFT) with the B3LYP functional40,41 and a 6-31G** basis set.39 Default settings were used with the exception of the accuracy level, which was set to “accurate” and the maximum number of iterations, which was increased to 200. All energy minimizations converged within 100 iterations. In order to minimize noise in the calculated gas phase energies the conformationally flexible isopropyl sulfone moieties were replaced by a proton prior to minimization. Starting conformations were obtained from the top ranking docking solutions generated with Glide v6.6.013 against our ATR homology model (see below). Alternative conformations were generated from the docked conformation by changing the dihedral angle of the bond connecting the amino pyrazine and the 5-membered hetero aryl ring by 180°. The energy required for the 4,5-dihydroisoxazole 5 to assume its flatter, bound conformation was estimated by subtracting the gas phase energy of the freely minimized compound from the energy after minimization with the 4


49


Page 50 of 73

dihedral angles of the single bonds in the 4,5-dihydroisoxazole ring constrained to their values in the docked conformation. Homology models for ATR, ATM and DNA-PK were generated using a phosphatidylinositol 3-kinase  (PI3K-) crystal structure as a template as described previously1 using the Prime v 3.9.013 homology building tool in Maestro and further optimized by manual manipulation and energy minimization with Macromodel version 10.7.013 using the OPLS2.1 force field. Kinase Inhibition Assays. HEPES, Tris-HCl, NaCl, KCl, MnCl2, MgCl2, EGTA, EDTA, BSA, ATP, phosphoric acid, calf thymus DNA, and DMSO were supplied by SigmaAldrich. DTT was from Melford Laboratories. Target peptides were synthesized at Biomol International LP. Stock 3 mCi/mmol [-33P]ATP and Optiphase Supermix scintillation cocktail were supplied by Perkin-Elmer. Phosphocellulose capture plates (MSPHNXB) were from Millipore. Full-length ATR and ATM kinases were produced in-house using methods based on published protocols43 and DNA-PK was purchased from Promega. The ability of compounds to inhibit ATR, ATM or DNA-PK kinase activity was tested using a radiometric phosphate incorporation assay44 as described previously.1 A stock solution was prepared consisting of the appropriate buffer, kinase, and target peptide. To this was added the compound of interest, at varying concentrations in DMSO to a final DMSO concentration of 7%. Assays were initiated by addition of an appropriate [-33P]ATP solution and incubated at 25 °C. Assays were stopped, after the desired time course, by addition of phosphoric acid and ATP to a final concentration of 100 mM and 0.66 M, respectively. Peptides were captured on a phosphocellulose membrane, prepared as per manufacturer's instructions, and washed six times with 200 L


50

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of 100 mM phosphoric acid, prior to the addition of 100 L of scintillation cocktail and scintillation counting on a 1450 Microbeta Liquid Scintillation Counter (Perkin- Elmer). Dose-response data were analyzed using GraphPad Prism software (Version 3.0cx for Macintosh, GraphPad Software). Additional kinase inhibition data for kinases unrelated in sequence to ATR was obtained from MDS Pharma services. Cellular Assays. Cell lines were purchased from ATCC and maintained according to the distributor's instructions. Cell assays were performed using exponentially growing cultures. For H2AX phosphorylation analysis using immunofluorescence (IF) microscopy, cells were fixed in 4% formaldehyde, permeabilized with 0.5% Triton X-100, and stained with mouse H2AX pS139 antibody (Upstate), AlexaFluor 488 goat antimouse antibody (Invitrogen), and Hoechst (Invitrogen). The cells were then analysed using the BD Pathway 855 bioimager and BD Attovision software.

The cell density was analyzed using the CellTiter96

AQueous Cell Proliferation (MTS) assay (Promega). Cells were plated in 96-well plates and allowed to adhere overnight. The following day, compounds were added at the indicated concentrations in a final volume of 200 μL, and the cells were then incubated for 96 hours. MTS reagent (40 μL) was then added, and 1 hour later absorbance at 490 nm was measured using a SpectraMax Plus 384 plate reader (Molecular Devices). Synergy and antagonism were assessed using Macsynergy software.44 Solubility assays. The solubility of compounds was assessed by dissolving them in 100 mL of DMSO or 100 mL of PBS buffer (Dulbecco’s Phosphate-Buffered Saline Solution, DPBS 1X, cat. No. 21-031 from www.cellgro.com, containing 10 mM phosphate and 140 mM NaCl) to a maximum concentration of 200 M in


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Costar 250 µl 384-well plates. At this concentration most compounds absorb sufficient amount of UV light and signal response is still linearly proportional to concentration. The DMSO standards were run on UPLC-MS using a reverse phase column and chromatograms were analysed by determining the area under the UV curve (AUC). Mass spectrometry was used for mass confirmation only. The PBS 384-well plates were sealed and shaken overnight at room temperature and subsequently centrifuged for 45 min at 4000 rpm and 22 C before being analysed by determining the AUCs of the UPLC-MS traces as before. The solubility of compounds in PBS was then determined by using the formula: Solubility = (AUC(PBS)/AUC(standard))*[standard] Intravenous rat pharmacokinetics. For IV dosing Male Sprague-Dawley rats (Charles River, UK) selected at a weight of approximately 200-350 g were used. Animals were group housed in cages of 3 in the animal facility at the Northwick Park Institute for Medical Research and maintained under a 12 hr light/dark cycle with free access to food and water. Temperature and humidity were controlled according to Home Office regulations. On the first day of the study, indwelling catheters were implanted into each animal’s jugular and femoral veins under aseptic conditions using gaseous anaesthetic (isoflurane). For IV dosing, 3 rats were administered a single infusion of 3.33 mg/kg/h of 2 for 3 h. This was equivalent to a nominal dose of 10 mg/kg. All doses of 2 were expressed as free base equivalent and were formulated as a solution in 5% Captisol:2.5 % Mannitol in deionized water. Blood samples were collected via the jugular vein catheter at the following time-points: 0 (pre-dose), 1.5, 3, 4, 6, 9, 12, 15, 27, 39 and 51h post dose. Blood was immediately centrifuged (3,000 G for 10 minutes at 4°C) and the resultant plasma transferred into clean, labeled Eppendorf tubes. Samples


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were stored frozen at -20 °C prior to dispatch to the bio-analytical laboratory. At all times, these studies were carried out in accordance with the requirements of the UK national legislation and conducted using the appropriate guidelines.


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ANCILLARY INFORMATION

Supporting information: Analytical data for compounds 2-20 (page S3), HPLC characterization of purity for compounds 2-20 (page S13), preparation of key intermediates (page S23), enzyme assays (page S29) and cellular assays (page S30). A PDB file with atomic coordinates of an ATR kinase domain homology model based on PDB entry 1E7V with bound compound 2. Molecular formula strings are available separately in comma-separated values file format. Authors will release the atomic coordinates and experimental data upon article publication.

Corresponding author Dr. Ronald Knegtel, e-mail: [email protected]

Author Contributions All authors were employees of Vertex Pharmaceuticals at the time of the described work and have contributed to and approved the final version of the manuscript.

Acknowledgements We thank our colleagues at Vertex and Merck KGaA for their helpful suggestions and critical reading of this manuscript.


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Abbreviations The following abbreviations are used: Abl, Abelson murine leukemia viral oncogene homolog 1; AcOH, acetic acid; ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3-related; B3LYP, Becke, three-parameter, Lee-Yang-Parr; BSA, bovine serum albumin; CDI, 1,1'-carbonyldiimidazole; Cl, drug clearance; dppf, 1,1'-bis(diphenylphosphino)ferrocene, DDR, DNA damage response; DIPEA, N,Ndiisopropyl ethylamine; DNA-PK, DNA-dependent protein kinase; DFT, density functional theory; DPBS, Dulbecco’s phosphate-buffered saline solution; DYRK2, Dual specificity tyrosine-phosphorylation-regulated kinase 2; EGTA, egtazic acid; Et3N, triethylamine; EtOAc, ethyl acetate; EtOH, ethanol; Flt4, Fms Related Tyrosine Kinase 4; GSK3, Glycogen synthase kinase 3; H2AX; variant histone H2A; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; HOBt, hydroxybenzotriazole; IR, ionizing radiation; JAK2, Janus kinase 2; MeCN, acetonitrile; MeOH, methanol; MLK1, Mitogen-activated protein kinase kinase kinase 1; n-PrOH; 1-Propanol; PARP, poly (ADP-ribose) polymerase; PI3K, Phosphoinositide 3-kinase; PIKK, phosphoinositol 3-kinase-like kinase; PPA, Phenylpropanolamine; PPh3, triphenylphosphine; RS, replication stress; Syk, Spleen tyrosine kinase; t-BuOH, tert-butyl alcohol; t-BuOLi, lithium tertbutoxide; Vss, volume of distribution at steady state.

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TABLE OF CONTENTS GRAPHIC

NH 2 O N

NH 2 O N N

N H

N N

SO2Me Compound 1 VE-821

NHMe

SO2iPr Intra-molecular electrostatics

Inter-molecular electrostatics


Compound 2 VX-970/M6220

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Figure 1. Chemical structures of Compound 1 (VE-821) and compound 2 (VX-970/M6620). 120x49mm (300 x 300 DPI)



Figure 2a-b. (A) Comparison of anilide 1 (in green) versus a benzimidazole isostere (in cyan) in a model of the active site of ATM based on PDB entry 1E7V. The benzimidazole is thought to experience steric hindrance from Tyr2755 in ATM. In response it rotates away from Tyr2755, which allows it to escape a steric clash with Pro2775 (Gly in ATR). (B) Replacing the benzimidazole with a phenyl substituted monocyclic heteroaryl (in cyan) yields a better mimic of the original anilide and avoids movement of the inhibitor due to steric hindrance with Tyr2755. 60x45mm (300 x 300 DPI)


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Figure 2a-b. (A) Comparison of anilide 1 (in green) versus a benzimidazole isostere (in cyan) in a model of the active site of ATM based on PDB entry 1E7V. The benzimidazole is thought to experience steric hindrance from Tyr2755 in ATM. In response it rotates away from Tyr2755, which allows it to escape a steric clash with Pro2775 (Gly in ATR). (B) Replacing the benzimidazole with a phenyl substituted monocyclic heteroaryl (in cyan) yields a better mimic of the original anilide and avoids movement of the inhibitor due to steric hindrance with Tyr2755. 60x45mm (300 x 300 DPI)



Figure 3. Intra-molecular polar interactions within heteroaryl ATR inhibitors. Polar interactions between the central 5-membered heteroaryl ring atoms X and Y and the 2-aminopyrazine hinge binder determine whether the bioactive conformation (left) is energetically preferred. 96x63mm (300 x 300 DPI)


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Figure 4a-b. (A) Compound 4 docked in the ATR active site with ATR represented by its electrostatic potential map. An area of high negative charge (in red) is located to the right of the phenyl ring of compound 4. (B) Compound 2 docked into the active site of ATR. Hydrogen bonds to the hinge, the unique residue Gly2385 as well as Asn2480 and Asp2494 that are part of the conserved magnesium binding site and salt bridge are indicated. Polar residues around the phenyl ring are shown as sticks. 60x45mm (300 x 300 DPI)



Figure 4a-b. (A) Compound 4 docked in a homology model of the ATR active site based on PDB entry 1E7V with ATR represented by its electrostatic potential map. An area of high negative charge (in red) is located to the right of the phenyl ring of compound 4. (B) Compound 2 docked into the active site of ATR. Hydrogen bonds to the hinge, the unique residue Gly2385 as well as Asn2480 and Asp2494 that are part of the conserved magnesium binding site and salt bridge are indicated. Polar residues around the phenyl ring are shown as sticks. 60x45mm (300 x 300 DPI)


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Figure 5. Ligand interaction diagram showing nearby residues, aromatic stacking (green lines) and key hydrogen bonds (purple lines) involving compound 2 as docked into our ATR homology model derived from PDB entry 1E7V. 83x60mm (300 x 300 DPI)



Figure 6. Macsynergy plots for compounds 1 and 2 dosed in combination with cisplatin in an HCT116 MTS cell assay. Synergistic or antagonistic interactions between ATR inhibitors and cisplatin are represented as deviations above or below the plane (at z=0) that corresponds to additivity. Compound 2 achieves similar synergy with cisplatin as 1 at a ~10-fold lower concentration. 60x45mm (300 x 300 DPI)


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Table of Contents Graphic 236x60mm (300 x 300 DPI)


Rational design of 5-(4-(isopropylsulfonyl)phenyl ... - ACS Publications

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