Discovery and Characterization of AZD6738, a Potent Inhibitor of Ataxia Telangiectasia Mutated and Rad3 Related (ATR) Kinase with Application as an Anticancer Agent

Article abstract of DOI:10.1021/acs.jmedchem.8b01187

The kinase ataxia telangiectasia mutated and rad3 related (ATR) is a key regulator of the DNA-damage response and the apical kinase which orchestrates the cellular processes that repair stalled replication forks (replication stress) and associated DNA double-strand breaks. Inhibition of repair pathways mediated by ATR in a context where alternative pathways are less active is expected to aid clinical response by increasing replication stress. Here we describe the development of the clinical candidate 2 (AZD6738), a potent and selective sulfoximine morpholinopyrimidine ATR inhibitor with excellent preclinical physicochemical and pharmacokinetic (PK) characteristics. Compound 2 was developed improving aqueous solubility and eliminating CYP3A4 time-dependent inhibition starting from the earlier described inhibitor 1 (AZ20). The clinical candidate 2 has favorable human PK suitable for once or twice daily dosing and achieves biologically effective exposure at moderate doses. Compound 2 is currently being tested in multiple phase I/II trials as an anticancer agent.

Full text of DOI:10.1021/acs.jmedchem.8b01187

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Drug Annotation
Discovery and characterization of AZD6738, a potent
inhibitor of ataxia telangiectasia mutated and rad3 related
(ATR) kinase with application as an anti-cancer agent
Kevin Michael Foote, J. Willem M. Nissink, Thomas M. McGuire, Paul Turner,
Sylvie Guichard, James T. Yates, Alan Lau, Kevin Blades, Dan Heathcote, Rajesh
Odedra, Gary Wilkinson, Zena Wilson, Christine Wood, and Philip J Jewsbury
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01187 • Publication Date (Web): 22 Oct 2018
Downloaded from http://pubs.acs.org on October 23, 2018
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Discovery and characterization of AZD6738, a potent inhibitor
of ataxia telangiectasia mutated and rad3 related (ATR) kinase
with application as an anti-cancer agent
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Kevin M. Footea#, J. Willem M. Nissink a*, Thomas McGuirea, Paul Turnera, Sylvie
Guichardb£, James W. T. Yatesc, Alan Laub, Kevin Bladesa$, Dan Heathcoted, Rajesh Odedrad^,
Gary Wilkinsona&, Zena Wilsonb, Christine M. Wood a, and Philip J Jewsburya
a Chemistry, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge Science Park, 310
Milton Rd, Milton, Cambridge, CB4 0WG, UK
b Bioscience, Oncology, IMED Biotech Unit, AstraZeneca, Chesterford Research Park,
Little Chesterford, Cambridge, CB10 1XL, UK
c DMPK, Oncology, IMED Biotech Unit, AstraZeneca, Chesterford Research Park,
Little Chesterford, Cambridge, CB10 1XL, UK
d Discovery Sciences, IMED Biotech Unit, AstraZeneca, Cambridge Science Park, 310 Milton
Rd, Milton, Cambridge CB4 0WG , UK
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# Current affiliation: Pharmaron, Drug Discovery Services Europe, Hertford Road,
Hoddesdon, Hertfordshire, EN11 9BU , UK
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£ Current affiliation: Forma Therapeutics, 500 Arsenal Street, Watertown, MA
02472, Boston, USA
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$ Current affiliation: AMR Centre Ltd, 19B70, Mereside Alderley Park, Alderley Edge, SK10
4T, UK
& Current affiliation: Bayer, Müllerstraße 178, 13353, Berlin, Germany
^ Current affiliation: Evotec, 114 Innovation Drive, Milton Park, Abingdon Oxfordshire
OX14 4RZ, UK
ABSTRACT
The kinase ataxia telangiectasia mutated and rad3 related (ATR) is a key regulator of the
DNA-damage response and the apical kinase which orchestrates the cellular processes that
repair stalled replication forks (replication stress) and associated DNA double-strand breaks.
Inhibition of repair pathways mediated by ATR in a context where alternative pathways are
less active is expected to aid clinical response by increasing replication stress. Here we describe
the development of the clinical candidate 2 (AZD6738), a potent and selective sulfoximine
morpholino-pyrimidine ATR inhibitor with excellent preclinical physicochemical and
pharmacokinetic (PK) characteristics. Compound 2 was developed improving aqueous
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solubility and eliminating CYP3A4 time-dependent inhibition starting from the earlier
described inhibitor 1 (AZ20). The clinical candidate 2 has favorable human PK suitable for
once or twice daily dosing and achieves biologically effective exposure at moderate doses.
Compound 2 is currently being tested in multiple Phase I/II trials as an anti-cancer agent.
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INTRODUCTION
Human cells are constantly exposed to DNA-damage events as a result of environmental and
endogenous factors. In order to suppress genomic instability, an integrated group of biological
pathways collectively called the DNA-damage response (DDR), has evolved to recognize,
signal, and promote the repair of damaged DNA.1, 2 DNA-damage leads to cell death if
sufficiently high and left unrepaired and is the concept behind DDR inhibition for cancer
therapy. Tumor cells are sensitized to DDR based therapies through a combination of relatively
rapid proliferation and DDR pathways that may already be functionally compromised. Ataxia
telangiectasia and Rad3-related (ATR) is a serine/threonine-protein kinase belonging to the
phosphatidylinositol 3-kinase-related kinase (PIKK) family of proteins and is a key regulator
4
of DNA replication stress response (RSR) and DNA-damage activated checkpoints.3,
Replication stress, a hallmark of cancer,5 may occur in tumors through oncogene drivers or
induced exogenously through treatment with DNA-damaging drugs or ionizing radiation (IR).
Persistent replication stress leads to DNA breaks which if left unresolved are highly toxic to
cells. In recent years potent and selective inhibitors of ATR (Scheme 1) have been developed
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from orthogonal chemical series demonstrating preclinical in vivo proof of concept. These
pivotal compounds and studies have been extensively reviewed,6-9 and reveal synthetic
lethality of ATR inhibitors on tumors with p53-mutations or Ataxia telangiectasia mutated
(ATM) loss-of-function,10, 11 as well as synergy in combination with a broad range of
replication stress inducing chemotherapy agents such as platinums,12 ionizing radiation,13, 14
and with novel agents such as the PARP inhibitor olaparib.15
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Scheme 1. ATR inhibitors; 2, 3 and 4 are undergoing clinical testing
O
N
O
N
Optimization
O
O
N
N
HN
O
NH
NH
S
S
N
N
N
1
2
; AZD6738
; AZ20
N
O
NH2
N
O
N
HN
N
N
N
N
H
N
N
N
S
O
O
3
4
; BAY 1895344
; Berzosertib (M-6620 / VX-970)
We have previously described a series of potent and selective ATR inhibitors, exemplified
by 1 (AZ20), from the sulfonylmethyl morpholino-pyrimidine series.16 Compound 1, and close
analogues,17 were shown to inhibit the growth of ATM-deficient xenograft models at well
tolerated doses. However, we did not consider compound 1 of sufficient quality for further
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development due to low aqueous solubility and high-risk for drug-drug interactions (DDI)
resulting from Cytochrome P450 3A4 (CYP3A4) time-dependent inhibition (TDI). In this
report we describe our further studies to identify ATR inhibitors with the requisite properties
suitable for clinical development that led to the discovery of 2 (AZD6738). The sulfoximine
morpholino-pyrimidine 2, along with the aminopyrazine 3 (berzosertib, M-6620 / VX-970),
originating from Vertex and licensed to Merck KGaA, and most recently the naphthyridine 4
(BAY 1895344) from Bayer,18 have entered human studies. These compounds are being
explored in early-phase clinical trials as single-agents and in combination with standard of care
(SOC) and novel agents.8, 9
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RESULTS
Compounds 1, 5, 6, 8, 9 and 10 (Table 1) and intermediates 39, 46 – 49, 70 were prepared as
described previously.16 Compounds 7, 17 and 18 were prepared as shown in Scheme 2 and
compounds 11 – 16 were prepared as shown in Scheme 3. Intermediates 43 – 45 were prepared
starting from the dichloropyrimidine 39. Suzuki coupling with 2-cyclohexenyl-4,4,5,5-
tetramethyl-1,3,2-dioxaborolane led to 40 while SNAr reaction with 8-oxa-3-
azabicyclo[3.2.1]octane and 3-oxa-8-azabicyclo[3.2.1]octane afforded 41 and 42 respectively
with no evidence of substitution at the 2-position. Cyclopropanation with 1,2-dibromoethane
and strong base afforded 43 – 45. The 2-arylpyrimidine test compounds were synthesized by
Suzuki coupling between the 2-chloropyrimidine substrates 43 – 48 and the corresponding
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boronic acid or esters which were either purchased or prepared from the corresponding aryl
bromides using literature methods.19
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1-H-benzimidazole test compounds 19 – 30 (Table 4) were made as shown in Scheme 4 and
5. Cyclopropanation of 49 led to 50 where reaction with 3(R)-methyl morpholine followed by
sodium tungstate catalysed oxidation of the sulfide proceeded well to afford 51 without over-
oxidation of the pyrimidine ring. Reaction with 1H-benzo[d]imidazol-2-amine or N-methyl-
1H-benzo[d]imidazol-2-amine led directly to compounds 19 and 21 respectively; compound
19 was derivatized to the N-acetyl 20. Benzimidazoles 22 – 30 were made starting from the 2-
chloropyrimidine intermediate 47 (Scheme 5). Buchwald-Hartwig coupling with the
appropriate substituted 2-nitroaniline utilising the xantphos ligand system afforded
intermediates 52 – 60. Reduction of nitro to amino was achieved under indium catalyzed
transfer hydrogenation conditions or with zinc in acetic acid to give the corresponding anilines
61 – 69 which were then cyclized using cyanogen bromide to afford compounds 22 – 30.
The iodobenzyl compound 70 was used as the starting point for the synthesis of sulfoxides
31, 32 and sulfoximines 2, 33 – 36 (Scheme 6). Displacement of the iodide in 70 with
sodiumthiomethoxide gave sulfide 71 in high yield which was then oxidized to the
corresponding sulfoxide R/S-72 with sodium metaperiodate. Compounds were either made as
a mixture of diastereoisomers and separated by chiral chromatography or prepared starting
with the appropriate chirally pure sulfoxide. The mixture of sulfoxide diastereoisomers R/S-72
were readily separable by chiral chromatography or vapor diffusion crystallization to afford
R-72 as a white crystalline solid and S-72 as an oil. The stereochemistry of R-72 was confirmed
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by X-ray structure (Supplementary Figure S2). Later, a biocatalytic process to the chiral
sulfoxide R-72 was developed from 71 on large scale.20 Cyclopropanation led to R/S-73
followed by Suzuki coupling to afford the test compounds 31 and 32. The sulfoximine moiety
was introduced starting from the sulfoxides via rhodium catalyzed nitrene insertion.21 This
proceeded with complete retention of stereochemistry and worked equally well either starting
from the mixture of sulfoxide diastereoisomers to afford R/S-74 or from single diastereoisomer
R-72 to afford R-74. Cyclopropanation resulted in rapid removal of the trifluoroamide group,
to give R/S-75 followed by Suzuki reaction led to test compounds 2, 33 and 34 or reaction with
N-methyl-benzo[d]imidazole-2-amine led to 35 and 36.
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Scheme 2a
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Cl
R1
N
a
N
N
O O
S
O O
S
N
Cl
Cl
O
7
8
9
39
[
[
40
R1 =
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O
N
N
41
42
R1 =
R1 =
O
[
b
R1
c
N
O O
S
7, 17, 18
N
Cl
[
[
43
O
O
R1 =
N
44
45
R1 =
R1 =
O
[
N
a
Reagents: (a) compound 40: (Ph3P)4Pd, 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-
tetramethyl-1,3,2-dioxaborolane, Cs2CO3, 1,4-dioxane, water, rt; compounds 41 and 42: 8-oxa-
3-azabicyclo[3.2.1]octane or 3-oxa-8-azabicyclo[3.2.1]octane respectively, Et3N, DCM, rt; (b)
compound 43: 1,2-dibromoethane, NaH, DMF, 0 °C → rt; compounds 44 and 45: 1,2-
dibromoethane, 50% NaOH (aq.), tetraoctylammonium bromide, DCM, rt; (c) compound 7:
(Ph3P)2PdCl2, 1H-indol-4-yl boronic acid, Na2CO3 (aq.), 4:1 DME:water, microwave, 110 °C;
compounds 17 and 18: (Ph3P)4Pd, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrrolo[2,3-c]pyridine, Na2CO3 (aq.), 1,4-dioxane, 95 °C.
Scheme 3a
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[
46
47
48
N
N
O
O
R1 =
R1 =
R1 =
R1
N
a
[
[
N
O O
S
11 - 16
Cl
N
O
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a
Reagents: (a) compound 11: (Ph3P)4Pd, 1H-pyrrolo[2,3-b]pyridine-4-ylboronic acid,
Na2CO3 (aq.), 1,4-dioxane, 90 °C; compound 12: 1H-benzo[d]imidazol-2-amine, Na2CO3,
DMA, 160 °C, microwave; compound 13: (Ph3P)2PdCl2, 1H-pyrrolo[2,3-b]pyridine-4-
ylboronic acid, Na2CO3 (aq.), 4:1 DME:water, 110 °C, microwave; compound 14: 2-
dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl, bis(dibenzylideneacetone)palladium(0),
4-bromo-1H-pyrrolo[2,3-c]pyridine, KOAc, bis(pinacolato)diboron, dioxane, 100°C followed
by compound 46, (Ph3P)4Pd, Na2CO3 (aq.), 100 °C; compound 15: 1,1'-
bis(diphenylphosphino)ferrocenedichloropalladium,
4-bromo-1H-pyrrolo[2,3-c]pyridine,
KOAc, bis(pinacolato)diboron, dioxane, 95 °C followed by compound 47, (Ph3P)4Pd, Na2CO3
(aq.), 95 °C; compound 16: (Ph3P)4Pd, Na2CO3 (aq.), 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrrolo[2,3-c]pyridine, dioxane, 95 °C.
Scheme 4a
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Cl
N
Cl
N
a
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b, c
O
O
N
N
N
N
O O
S
d
N
O O
S
N
N
S
N
HN
R1
O O
51
19
20
21
R1 = H
e
R1 = Ac
R1 = Me
a Reagents: (a) 1,2-dibromoethane, 50% NaOH (aq), tetraoctylammonium bromide, toluene,
60 °C; (b) (R)-3-methylmorpholine, DIPEA, 1,4-dioxane, 80 °C; (c) NaO4W·2H2O, Bu4NHSO4,
EtOAc, H2O2, 0 °C → rt; (d) compound 19: 1H-benzo[d]imidazol-2-amine, Cs2CO3, DMA, 110
°C, microwave; compound 21: N-methyl-1H-benzo[d]imidazol-2-amine, Cs2CO3, DMA, 90 °C;
(e) DMAP, Ac2O, 90 °C.
Scheme 5a
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52
R1 = 3-F
R1 = 4-F
R1 = 5-F
R1 = 6-F
R1 = 3-Cl
R1 = 3-OMe
R1 = 4-Cl
R1 = 4-CN
R1 = 4-OMe
O
N
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a
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6
47
N
O O
S
R1
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N
N
1
2
H
NO2
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b
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R1 = 3-F
R1 = 4-F
R1 = 5-F
R1 = 6-F
R1 = 3-Cl
R1 = 3-OMe
R1 = 4-Cl
R1 = 4-CN
R1 = 4-OMe
O
N
5
c
4
6
22 30
-
N
O O
S
R1
3
N
N
1
2
H
NH2
a
Reagents: (a) Substituted 2-nitroaniline, Pd(OAc)2, xantphos, Cs2CO3, 1,4-dioxane, 80 °C,
microwave; (b) Zn, AcOH, rt or In, NH4Cl (aq.), EtOH, reflux; (c) Cyanogen bromide, MeOH,
rt.
Scheme 6a
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O
N
O
N
O
O
N
N
N
a
b
c
O-
N
N
N
O-
N
+
*
S
+
*
S
I
S
N
Cl
N
Cl
Cl
N
Cl
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S-72
R-72
S-73
R-73
e
d
O
O
N
N
N
N
g
f
O
2 33 34 35 36
31 32
,
,
,
,
,
N
HN O
* S
N
N O
F3C * S
Cl
Cl
S-75
R-75
S-74
R-74
a Reagents: (a) NaSMe, DMF, rt; (b) NaIO4, EtOAc, MeOH, H2O, rt; (c) 1,2-dibromoethane,
50% NaOH (aq.), tetraoctylammonium bromide, 2-Me-THF, 60 °C; (d) X-Phos 2nd gen.
precatalyst, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine,
Cs2CO3, 1,4-dioxane:H2O (4:1), 90 °C; (e) trifluoroacetamide, iodobenzene diacetate, Rh(OAc)2
dimer, MgO, iso-propylacetate, 80 °C; then 7M NH3 in MeOH, rt; (f) NaOH (50% aq.), 1,2-
dibromoethane, tetra-octylammonium bromide, mTHF, rt; (g) compound 33: (Ph3P)2PdCl2,
2M
Na2CO3,
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-pyrrolo[2,3-
b]pyridine, DME:H2O (4:1), 90 °C then 2M NaOH (aq.), 50 °C; compound 34: 1H-pyrrolo[2,3-
b]pyridin-4-ylboronic acid, (Ph3P)2PdCl2, 2M Na2CO3, DME:H2O (4:1), 90 °C; compound 35
and 36: Cs2CO3, N-methyl-1H-benzo[d]imidazol-2-amine, DMA, 80 °C.
DISCUSSION
Compound 1 is a potent ATR inhibitor with excellent kinase selectivity and free exposure
and is a useful tool compound to explore ATR pharmacology in vivo.16 However, compound 1
was also found to be a time-dependent inhibitor of CYP3A4 and to suffer from low aqueous
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solubility. Inhibition of CYP3A4, particularly mechanism based inhibition, is a concern for
clinical DDI,22-24 whereas low solubility limits the maximum absorbable dose (Dabs).25 These
properties increase risk of failure in clinical development and compound 1 was therefore not
considered a suitable candidate for clinical-enabling studies. Eliminating CYP3A4 TDI and
achieving high aqueous solubility at the same time maintaining high ATR potency, excellent
specificity and the attractive pharmacokinetic properties exhibited by 1, were the key
medicinal-chemistry design goals in the optimization phase.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
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34
35
36
37
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50
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52
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57
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59
60
CYP TDI has been observed as a common feature in kinase inhibitors with CYP3A4 being
the most commonly inhibited isoform.26 CYP TDI is associated with the formation of covalent
(or reversible-covalent) adducts to heme or protein following metabolic activation.23, 24 In
addition to the risk of DDIs, formation of reactive metabolites is a causative factor for
idiosyncratic drug toxicity.23, 27 The risk and impact of clinical DDI will be determined by
overall drug disposition, dose, regimen and target patient population. While compound 1
clearly demonstrated TDI of CYP3A4 when incubated at 10 µM in human microsomes,16 we
did not fully characterize this activity or model in detail the human PK and predicted clinical
dose to understand the magnitude of the expected clinical DDI. We anticipated ATR inhibitors
would be combined with cytotoxic and targeted drugs in the clinic. As DDI arising from
inhibition of CYP3A4 would complicate co-dosing of such agents and many likely co-
medicants,28 we set out to remove this undesirable activity.
The indole and morpholine groups were thought particularly vulnerable to metabolic
activation. The susceptibility of cyclic tertiary amines such as morpholine to -carbon
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oxidation, generating reactive iminium ion intermediates is well described.29-31 Indoles are
known to undergo ring hydroxylation particularly at C-5 and/or C-6 positions;32 these species
could arise via reactive epoxides and could also lead to quinone-like reactive intermediates
following additional bioactivation. Oxidation of indole has also been observed at C-2 or C-3,
presumably via the corresponding epoxide, and further oxidation and/or oxidative ring
cleavage can lead to anthranilic acid products. The putative metabolic vulnerability of
morpholine and indole presented us with a potentially insoluble problem as our previous work
clearly demonstrated the importance of both groups to ATR potency.16
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
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44
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48
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50
51
52
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56
57
58
59
60
Reactive metabolites formed from bioactivation are generally electrophilic in character and
highly unstable. Trapping experiments can be used to detect and characterize metabolites
whereby a compound is incubated in human liver microsomal preparations and any reactive
metabolites generated are trapped by specific added nucleophiles. Orthogonal nucleophiles are
used to trap the different electrophilic species arising from bioactivation of chemical
substrates. The nucleophiles commonly used are glutathione (GSH), a soft nucleophile efficient
at trapping soft electrophiles such as epoxides, cyanide to trap iminium species arising from
oxidation of tertiary amines and methoxyamine to effectively trap aldehyde products as Schiff
bases.33 These screens provide valuable mechanistic information to support rational medicinal
chemistry design. When 1 was incubated with human liver microsomes, adducts with GSH
but not cyanide were detected. Whilst the mechanisms of bioactivation and TDI may not
necessarily overlay, the formation of GSH adducts implicates the indole group, likely through
ring oxidation.
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We had systematically explored the structure activity relationships (SAR) in the
morpholino-pyrimidine pharmacophore, varying each of the substituents on the pyrimidine
core. These compounds now allowed facile investigation into the molecular features in 1
responsible for CYP3A4 TDI, independent of ATR potency.
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
Table 1. Morpholine and C-2 heterocycle CYP3A4 TDI SAR
R1
N
O O
S
N
R2
ATR
ATR cell
CYP3A4 %
c
Compound
R1
R2
LogD7.4
2.5#
IC50 (μM)a IC50 (μM)b
TDI, 10 μMd
NH
NH
NH
[
[
[
[
[
N
O
1
0.005
0.008
0.007
0.018
0.061*
0.29
50
38
53
49
[
N
N
O
5
6
7
2.3
2.6
2.9
O
[
0.11
NH
NH
[
[
O
O
0.27
[
N
8
0.015
0.13
ND
61
F
NH
[
[
[
N
N
O
O
9
0.14
0.18
16.5
6.2
1.6
2.2
<9
O
NH
N
NH
[
10
<11
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NH
[
[
[
N
N
O
O
11
12
0.037
0.003
0.35
2.2
2.2
<11
17†
N
[
N
0.051
N
H2N
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
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54
55
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57
58
59
60
aUncertainty (95% confidence) for pIC50 measurements is 0.38 (2.4-fold) based on an average
b
of two repeat occasions per compound.
Uncertainty (95% confidence) for pIC50
measurements is 0.7 (4.6-fold) based on at least two repeat measurements per compound
(median number of repeats across tables is 3). The data presented is from a variation of the
16
ATR cell assay described in Ref. (see experimental section). A good correlation is observed
between ATR cell assay versions; N=41 compounds from morpholino-pyrimidine series,
correlation: 0.94, mean difference: -0.084 pIC50. *Compound 1 IC50 = 0.050 µM in the earlier
version reported.16 LogD7.4 assay: lipophilicity was determined using the ‘shake-flask’
c
method. Plated aliquots of sample are dried down and octanol and water are added. Sample
content of the phases is determined by LC/MS/MS after stirring, equilibration, and separation
of phases by centrifuge. Full description of the assay can be found in the Supplementary
Materials. An excellent correlation is observed between versions of the LogD7.4 methods; N=23
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compounds from the morpholino-pyrimidine series, correlation: 0.96, mean difference: -0.075.
16
d
#Compound 1 LogD7.4 = 2.7 using the earlier methodology (Ref. ). ND = not determined.
Mean value (N≥2) unless otherwise stated. Compounds were pre-incubated at 10 μM with
human liver microsomes (1 mg/mL) with and without NADPH (5 mM) for 30 min at 37 °C
followed by 15 min incubation with 10 μM midazolam; analysis of 1-hydroxymidazolam was
performed using liquid chromatography-tandem mass spectrometry.34 No activity detected vs.
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
†
control for 1A2, 2C19, 2C9, and 2D6. Result for compound 12 was just above background
level in test 1 and below background level (<11%) in test 2; result for test 1 shown.
The unsubstituted and 3(R)-methyl substituted morpholines (compounds 5 and 1
respectively) both show clear CYP3A4 TDI activity when incubated at 10 µM in human liver
microsomes using a standard liquid chromatography-tandem mass spectrometric endpoint
(Table 1).34 Introducing structural architecture to eliminate reactive moieties resulting from
oxidation on the morpholine ring, for example by modification of the methylene adjacent to
the oxygen atom using the bridged morpholine 6 or removal of the nitrogen heteroatom by
substitution of morpholine for the 3,6-dihydro-2H-pyran 7, did not eliminate CYP3A4 TDI
activity compared with morpholine. The dihydropyran and bridged morpholines, both of
which have been described as morpholine isosteres in mTOR inhibitor series,35, 36 are
equivalent in ATR potency to unsubstituted morpholine but at the price of higher
lipophilicity. The impact of blocking and/or deactivating substituents on the indole ring was
investigated. Simple ring substituents that retain ATR potency, for example the 6-fluoro indole
8, did not reduce CYP3A4 TDI. In contrast, addition of a polar and deactivating group such as
acetamido 9 and replacement of the indole, for example with benzimidazole 10, which has
equivalent lipophilicity to the indole 5, led to undetectable CYP3A4 TDI activity. In both cases
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ATR potency was also significantly reduced but these results supported the notion that the
indole ring was the likely key contributor to CYP3A4 TDI. We discovered the 7-azaindole
(1H-pyrrolo[2,3-b]pyridine) 11 retains the ATR potency of the indole while effectively
eliminating CYP3A4 TDI. In addition, through a wider campaign to identify ATR-active
indole isosteres, the 2-amino-N1-substituted benzimidazole 12 was found to have no or weak
detectable 3A4 TDI. This variant was also found to possess superior cellular potency without
increasing lipophilicity compared with the indole 5. Unknown to us at the time, Safina et al.37
had developed a series of PI3K-specific morpholinopyrimidines substituted with 4-indole
that were also found to be potent CYP3A4 TDIs and further determined that the 4-indole
group was associated with this activity. In contrast to our own findings, replacement of the
indole with 7-azaindole in the PI3K series did not attenuate CYP3A4 TDI. The apparent
conflicting results are a reminder that metabolic activation is complex, and SAR may not be
simplified to contributions of individual functional groups. It is whole-molecule structure and
properties that determine metabolic fate. However, in our efforts towards identifying an ATR
inhibitor candidate for human studies, both the 7-azaindole 11 and 2-aminobenzimidazole 12
became productive leads for optimization.
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
In the 7-azaindole series, the SAR was found to mirror that determined for indole with
cellular potency increasing for the corresponding 3-(R) methyl morpholine 13 (Table 2).
Pleasingly we could not detect CYP3A4 TDI activity for this compound. Measured
lipophilicity for 13 is unchanged in comparison to 1 and unsurprisingly solubility remains low.
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Table 2. Azaindole SAR
5
6
7
R1
8
9
N
O O
S
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
N
R2
ATR
ATR cell
Solubility
CYP3A4 %
Compound
R1
R2
LogD7.4 LLEc
IC50 (μM)a IC50 (μM)b
pH7.4 (µM)d TDI, 10 μM
NH
NH
NH
[
[
[
[
N
O
1
0.005
0.005
0.005
0.061
0.10
2.5
2.6
1.8
4.7
4.4
5.3
10
8
50
N
O
13
14
<10
<15
N
[
[
N
N
O
O
0.085
130
N
N
N
N
N
NH
NH
NH
NH
[
[
[
[
[
[
15
16
17
18
0.002
ND
0.012
0.36
2.1
2.2
2.2
2.1
5.8
4.2
4.9
4.5
108
165
<20
ND
ND
ND
N
O
O
[
N
ND
0.086
0.26
>1510
98
ND
O
[
N
a,b For assay uncertainty, see footnotes in Table 1 c Lipophilicity ligand efficiency (LLE): ATR
cell pIC50 – LogD7.4. d Solid material was agitated in 0.1 M pH 7.4 phosphate buffer for 24 h,
double centrifuged, and the supernatant analyzed for compound concentration by
LC−UV−MS. Crystallinity assessed by polarized light microscopy of remaining solid. Full
description is provided in Supplementary Materials. Lowest solubility given in the table where
multiple measurements were taken: N≥3 for compounds 1, 13, 15; N=1 for compounds 14, 16,
17, 18. ND: not determined.
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Substitution with the 6-azaindole (1H-pyrrolo[2,3-c]pyridine) isomer surprisingly resulted in
compounds with much improved cellular potency compared with the corresponding indole
and 7-azaindole. Moreover the 6-azaindole series combines improved potency with reduced
lipophilicity and improved aqueous solubility (compounds 14 – 18, Table 2; note: the very high
solubility result for compound 17 is most likely an outlier that we speculate is due to low
crystallinity) while also having no detectable CYP3A4 TDI. It is interesting to observe a
seemingly small structural change delivering such a significant effect on both potency and
physicochemical properties.38 Directionality of the hydrogen bond acceptor nitrogen in the
azaindole isomers is changed relative to the morpholine hinge binder offering the potential to
impact key interactions to the protein. Recent structures of ATR-mimicking PI3K mutants,39
and an ATR cryo-EM,40 structure suggest that the difference in potency between the
azaindoles isomers can be explained by their interaction with Asp2335, potentially mediated
by a water molecule. 6-Azaindole is also considerably more basic than 7-azaindole (pKa 7.9
and 4.6 respectively41) resulting in an expected change in ionization state at physiological pH
and concomitant effects on LogD7.4 and solubility. The unsubstituted morpholine 7-azaindole
14 demonstrated excellent ATR cellular potency (IC50 <100 nM) and again the established
morpholine SAR translated, with a significant increase in potency observed when 3-(R)
methyl morpholine was employed over the 3-(S) methyl isomer (compounds 15 and 16
respectively). The bridged morpholines, compounds 17 and 18, retained potency compared to
morpholine 14 and the reduced lipophilicity provided by the 6-azaindole group led to
compounds with a good overall balance of properties. It is noticeable from the compounds
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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shown in Table 2 that the hitherto excellent ATR enzyme to cell correlation breaks down. We
had high confidence in the cellular assay measuring inhibition of CHK1 phosphorylation, a
direct substrate of ATR, in response to a DNA-damage stimulus. Presumably, with the more
potent compounds, the IC50 is below the detection threshold (tight binding limit) of the
enzyme assay. Therefore, at this stage of the optimization phase the cellular assay was
primarily used to drive chemistry in conjunction with lipophilicity and physicochemical
driven properties of aqueous solubility, permeability and metabolic stability. The 3-(R)-methyl
morpholine 6-azaindole 15 stood out in combining excellent cellular potency and moderate
lipophilicity (LLE = 5.8) with improved aqueous solubility and no measureable CYP3A4 TDI
and represented a significant advancement over the indole lead 1.
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
The benzimidazole head group utilized in compound 12, substituted at the 2-position with
amino or alkyl substituents on a morpholino-pyrimidine core was described in a series of anti-
tumor agents with PI3K activity from Zenyaku Kogyo.42 We discovered the novel
methylsulfonylmethyl morpholino-pyrimidine 12 to possess excellent ATR potency (Table 1)
albeit with some class 1 PI3K inhibitory activity (e.g. PI3K IC50 = 0.24 µM). Substitution on
the morpholine hinge binder has been shown to affect PI3K activity,16, 36 thus the 3(R)-methyl
morpholine 2-aminobenzimidazole (compound 19, Table 3) became a key compound to make
that was subsequently shown to possess greatly improved ATR potency and selectivity over
PI3K (ATR cell IC50 = 0.015 µM, PI3K IC50 = 9 µM).
Table 3. Benzimidazole C2 SAR
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2
3
4
5
6
7
8
O
N
9
N
O O
S
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
N
N
N
HN
R1
ATR
ATR cell
Solubility
CYP3A4 %
Compound R1
LogD7.4 LLE
IC50 (μM)a IC50 (μM)b
pH7.4 (μM)c TDI, 10 μM
19
20
21
H
Ac
Me
0.001
0.014
0.002
0.015
0.017
0.008
2.5
1.8
2.8
5.3
6.0
5.3
20
7
49
20†
<17
<18
a,b
c
For assay uncertainty, see footnotes in Table 1 Lowest solubility result is given in the
table where multiple measurements were taken: N=2 for compound 21; N=1 for compounds
19, 20. Result for compound 19 was just above background level in test 1 and below
background level (<17%) in test 2; result shown for test 1.
†
The potency improvement relative to indole is achieved without increasing lipophilicity;
compound 19 shows borderline CYP3A4 TDI and aqueous solubility is not significantly
improved compared to indole 1. Substitution on the amino group with acetyl (compound 20)
or methyl (compound 21) were well tolerated with no loss of cellular potency and in the case
of the N-acetyl 20, led to higher LLE. Moreover, we did not detect CYP3A4 TDI for either of
the substituted 2-amino-benzimidazole compounds. While a relatively modest improvement
in aqueous solubility was perhaps evident, particularly for N-methyl 21, aqueous solubility was
not robustly improved compared with indole.
Substitution on the benzimidazole aryl ring was explored for effects on potency and to
address a theoretical concern that the naked aryl ring might be susceptible to metabolic
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instability. The result of systematic substitution on the aryl ring of the benzimidazole group
with fluorine is shown in Table 4.
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
Table 4. Benzimidazole ring SAR
O
N
6
7
N
O O
S
5
R1
N
N
4
N
H2N
ATR
ATR cell
Compound
R1
IC50 (μM)a
IC50 (μM)b
19
22
23
24
25
26
27
28
29
30
H
4-F
5-F
6-F
7-F
0.001
0.010
0.002
0.008
0.090
0.017
0.27
0.021
4.0
0.55
0.015
0.13
0.035
0.12
5.1
5.3
5.3
0.87
>30
15
4-Cl
4-OMe
5-Cl
5-CN
5-OMe
a,b
See footnotes a,b in Table 1 for assay uncertainty; repeat measurements: N=2 for
compound 19; N=1 for compounds 22 – 30.
A 5-F substituent (compound 23) retains the same level of ATR potency compared with
unsubstituted benzimidazole 19. While 4-F and 6-F (compounds 22 and 24 respectively) retain
a degree of ATR potency the 7-F analogue (compound 25) is significantly less potent. A broader
range of substituents in positions 4- and 5- (exemplified by compounds 26 – 30, Table 4) were
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then made to probe into the protein further but for all these examples reduced ATR potency
was seen.
7
8
9
In the solid state, methylsulfone compounds such as 1 display a centrosymmetric
methylsulfone to methylsulfone contact (in addition to ring-ring stacking and a hydrogen
bonding network between indole N-H and sulfone oxygen) associated with high melting
points and low solubility.16 Our SAR study of the indole series demonstrated that specific
changes around the methyl sulfonyl moiety (e.g. addition of charged substituents) could
improve physicochemical properties, but these changes ultimately led to reduced potency
and/or attenuation of other fundamental properties such as permeability leading to low oral
exposure. However, we had yet to attempt specific changes to the sulfone group, for example
replacement with sulfoxide or sulfoximine whereby one of the oxygen atoms is replaced with
nitrogen. We hypothesized that such changes could disrupt the observed solid-state contacts
albeit at the complication of introducing additional chirality and with uncertain impact on
target potency.
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
Table 5. Sulfoxide and sulfoximine SAR
O
N
N
R1
N
R2
Compound
R1
R2
ATR
ATR cell
LogD7.4 LLE
Solubility
CYP3A4 %
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5
6
7
IC50 (μM)a IC50 (μM)b
pH7.4 (μM)c TDI, 10 μM
O
O
NH
[
S
]
13
0.005
0.10
2.6
4.4
8
<10
N
8
9
O
S
NH
NH
NH
NH
[
[
[
[
]
]
31
32
2
0.016
0.011
0.004
ND
0.18
0.090
0.074
0.24
2.5
2.5
1.9
1.8
4.2
4.6
5.2
4.8
240
198
661
179
ND
ND
<20
<11
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
N
N
N
N
O
S
HN O
S
]
]
HN O
S
33
HN O
S
NH
[
]
34
35
ND
0.014
0.009
1.4
2.1
6.5
6.0
>2120
>780
ND
<20
N
HN O
S
[
N
N
]
]
0.006
N
N
HN
HN O
S
[
36
ND
0.023
2.1
5.5
933
ND
HN
a,b See footnotes in Table 1 for assay uncertainty. ND = not determined. c Lowest solubility
result is given in the table where multiple measurements were taken: N≥2 for compound 13,
2, 33, 35; N=1 for compounds 31, 32, 34, 36.
The diastereomeric sulfoxides in the 7-azaindole series, compounds 31 and 32 (Table 5), were
found to retain ATR potency compared with the corresponding sulfone 13 with no change in
measured lipophilicity. These sulfoxides were also found to have high aqueous solubility
though it proved challenging to generate stable crystalline forms and therefore the apparent
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improvement compared with the corresponding sulfones was treated with caution. The
sulfoxides are highly permeable and have high exposure from oral doses in rodents. However,
the sulfoxide group is prone to oxidation in vivo and a significant level of the corresponding
sulfone was observed in rodent PK studies; for this reason, despite other properties being
generally attractive, we discounted the sulfoxides from further progression. The sulfoximines
(compounds 2, 33 – 36 Table 5) achieved both of the key aims we set at the start of the lead
optimization campaign. The R-stereochemistry of the sulfoximine 2 was initially inferred from
the stereochemistry of the sulfoxide precursor, with imination to the sulfoximine proceeding
with retention of configuration, and later confirmed from an X-ray structure (Figure 1).
Interestingly, hydrogen bonds are observed only between the azaindole substituents of
adjacent molecules for 2, whereas the indole NH donor in the structure of 1 does not form a
hydrogen bond. The centrosymmetric packing characteristic for methylsulfones as seen for 1
is prevented in the structure of 2 by introduction of the sulfoximine (Figure 1).
10
11
12
13
14
15
16
17
18
19
20
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31
32
33
34
35
36
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Figure 1. Small-molecule crystal structures of 1 and 2. Short contacts of sulfonomethyl (left-
5
6
7
hand panel) and azaindole (right-hand panel) moieties are indicated by dotted lines.
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9
10
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12
13
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21
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60
The R-isomer of the sulfoximine group showed slight but consistent greater potency than
the corresponding S-isomer (compare 2 with 33, Table 5) and similar magnitude compared
with the corresponding sulfone. The sulfoximines also benefit from a significant reduction in
lipophilicity and greatly improved aqueous solubility compared to the sulfones. Data from
molecular matched pairs clearly show consistent retention of ATR cellular potency for the R-
isomer, concomitant reduction in lipophilicity and improved solubility of sulfoximine
analogues compared with the corresponding sulfones (Figure 2). While the sulfoximine shows
a different solid-state structure, the melting point of sulfoximine 2 was measured at 222 °C and
is similar to sulfone 1 (204 °C). Therefore, the observed improvement in solubility appears to
be driven mainly through reduced lipophilicity, though we speculate that solvation and the
removal of the methylsulfones’ centrosymmetric organization may contribute additional
effects which are difficult to quantify. Replacement of methylsulfones has been shown to
improve solubility in other contexts,43 and sulfoximines may have wider utility in such cases.
A further lipophilicity-related benefit of the sulfoximine group was realized in reduced hERG
activity and was particularly advantageous for the 2-N-methylamino benzimidazole analogues
35 and 36 (Figure 2). Several reviews on the properties of the sulfoximine moiety in drug
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discovery have since appeared elsewhere which observe the same broad trends as described
here.44-46
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
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49
50
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53
54
55
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57
58
59
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Figure 2. Sulfone and sulfoximine matched-pairs. Data for a) ATR cell pIC50; b) LogD7.4; c)
hERG pIC50 and d) aqueous solubility. Numbers refer to the compounds shown in Tables 2 –
5.
Compounds that addressed the dual risks of low solubility and CYP3A4 TDI activity were
further characterized as a shortlist to identify a candidate molecule for clinical-enabling
studies. Physicochemical and ADME properties of compounds 2, 15, 21 and 35 are presented
in Table 6.
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Table 6. Physicochemical and ADME characterization for preclinical candidate shortlist
8
9
compared with lead 1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
35
36
37
38
39
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1
2
15
21
35
LogD7.4
2.5
10
14, 9.2
>10
50
50
23, 37
25, <3
1.0
1.9
661
54, 26
>10
<20
166
6.8, 12
<3.5, <3
0.8
2.1
108
48, 26
>10
2.8
49
2.1
>780
Solubility pH7.4 (μM)a
PPB mouse, hu (%free)b
CYP3A4 IC50 (μM)c
CYP3A4 %TDI, 10 M
hERG IC50 (μM)d
4.4, 2.2 15, 4.3
>10
<18
>10
<20
15
-, 8
11, <3
0.35
<20
†
15
4.6
Caco-2 Papp A-B (pH6.5, 7.4)e
8, -
10, <3
0.08
-, 53
25, <3
0.4
f
Rat, hu CLint
Rat AUC (µM.hr)g
a Lowest solubility result is given in the table where multiple measurements were taken: N=5
for compound 1, N=3 for compound 15, N=2 for compound 21, N=7 for compound 2; N=2 for
compound 35. b Measured in 10% plasma; % free calculated for 100% plasma assuming a single-
site binding model. c >10 µM against 1A2, 2C19, 2C9, and 2D6. d Average activity against the
human ether-a-go-go-related gene (hERG) encoded potassium channel was determined using
automated whole-cell electrophysiology.47 e Median A to B Papp (1 × 10−6 cm/s), 10 μM
compound concentration. f Median CLint: intrinsic clearance from hepatocytes (μL/min per 1
× 106 cells, 1 μM compound concentration). g Plasma exposure; data normalised to 1 μmol/kg;
compounds were dosed orally to male Han−Wistar rats at either 4 (compounds 1 and 15) or 10
(compounds 21, 2 and 35) μmol/kg formulated in propylene glycol; compounds 1, 2, 15, 35
were dosed as solutions and compound 21 as a suspension.
†
The most potent of 3
measurements is shown.
All compounds have equivalent or improved cellular potency combined in most cases with
reduced lipophilicity relative to the lead 1. The sulfoximines in particular have very high
aqueous solubility, high unbound fraction and none of this set of compounds exhibited
CYP3A4 reversible inhibitory or TDI activity against the five major human P450 isoforms. The
benzimidazole sulfone 21 has the highest lipophilicity and hERG potency. Employment of the
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