Structure-Based Exploration of Selectivity for ATM Inhibitors in Huntington's Disease

Article abstract of DOI:10.1021/acs.jmedchem.1c00114

Our group has recently shown that brain-penetrant ataxia telangiectasia-mutated (ATM) kinase inhibitors may have potential as novel therapeutics for the treatment of Huntington's disease (HD). However, the previously described pyranone-thioxanthenes (e.g., 4) failed to afford selectivity over a vacuolar protein sorting 34 (Vps34) kinase, an important kinase involved with autophagy. Given that impaired autophagy has been proposed as a pathogenic mechanism of neurodegenerative diseases such as HD, achieving selectivity over Vps34 became an important objective for our program. Here, we report the successful selectivity optimization of ATM over Vps34 by using X-ray crystal structures of a Vps34-ATM protein chimera where the Vps34 ATP-binding site was mutated to approximate that of an ATM kinase. The morpholino-pyridone and morpholino-pyrimidinone series that resulted as a consequence of this selectivity optimization process have high ATM potency and good oral bioavailability and have lower molecular weight, reduced lipophilicity, higher aqueous solubility, and greater synthetic tractability compared to the pyranone-thioxanthenes.

Full text of DOI:10.1021/acs.jmedchem.1c00114

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Article
Structure-Based Exploration of Selectivity for ATM Inhibitors in
Huntington’s Disease
Amanda Van de Poël,§ Leticia Toledo-Sherman,§ Perla Breccia,§ Roger Cachope, Jennifer R. Bate,
Ivan Angulo-Herrera, Grant Wishart, Kim L. Matthews, Sarah L. Martin, Marcus Peacock, Amy Barnard,
Helen C. Cox, Graham Jones, George McAllister, Huw Vater, William Esmieu, Cole Clissold,
Marieke Lamers, Philip Leonard, Rebecca E. Jarvis, Wesley Blackaby, Maria Eznarriaga, Ovadia Lazari,
̃
Dawn Yates, Mark Rose, Sung-Wook Jang, Ignacio Munoz-Sanjuan, and Celia Dominguez*
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ABSTRACT: Our group has recently shown that brain-penetrant ataxia telangiectasia-mutated (ATM) kinase inhibitors may have
potential as novel therapeutics for the treatment of Huntington’s disease (HD). However, the previously described pyranone-
thioxanthenes (e.g., 4) failed to afford selectivity over a vacuolar protein sorting 34 (Vps34) kinase, an important kinase involved
with autophagy. Given that impaired autophagy has been proposed as a pathogenic mechanism of neurodegenerative diseases such
as HD, achieving selectivity over Vps34 became an important objective for our program. Here, we report the successful selectivity
optimization of ATM over Vps34 by using X-ray crystal structures of a Vps34-ATM protein chimera where the Vps34 ATP-binding
site was mutated to approximate that of an ATM kinase. The morpholino-pyridone and morpholino-pyrimidinone series that
resulted as a consequence of this selectivity optimization process have high ATM potency and good oral bioavailability and have
lower molecular weight, reduced lipophilicity, higher aqueous solubility, and greater synthetic tractability compared to the pyranone-
thioxanthenes.
mutant HTT (mHTT) can disrupt the functional integrity of
the TCR complex, which in turn leads to an increase in DNA
strand breaks and subsequently increased ATM activity.5
Several ATM kinase inhibitors have been described
previously (Figure 1), including those by AstraZeneca, who
identified 3-quinoline carboxamides and 3-cinnoline carbox-
amides as novel classes of potent, selective, and orally
bioavailable ATM kinase inhibitors.6 The AstraZeneca
program ultimately provided the clinical candidates
AZD0156 as a potential treatment for solid tumor indications
INTRODUCTION
■
Ataxia telangiectasia-mutated (ATM) kinase is a large serine/
threonine kinase that is involved in the DNA damage response.
It belongs to the phosphatidyl inositol-3-kinase-like (PIKK)
family that includes other members such as ataxia- and rad3-
related (ATR), mammalian target of rapamycin (mTOR), and
DNA-dependent protein (DNA-PK) kinase. ATM plays a key
role in maintaining genetic integrity by regulating the detection
and repair of DNA double-stranded breaks. In response to
DNA damage, ATM autophosphorylates and consequently
phosphorylates approximately 700 substrates, including H2AX,
p53, and KAP1.1,2 Other roles for ATM have also been
identified, including involvement in cellular homeostasis and
neurodegenerative diseases.3,4 A recent publication demon-
strated increased DNA damage in a human post-mortem
Huntington’s disease (HD) brain (Q58); the protein central to
HD pathogenesis, huntingtin (HTT), forms a transcription-
coupled DNA repair (TCR) complex, and polyQ expansions in
Received: January 21, 2021
Published: March 30, 2021
https://doi.org/10.1021/acs.jmedchem.1c00114
© 2021 American Chemical Society
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Figure 1. Known ATM inhibitors.
a
Scheme 1. Syntheses of 7, 8, and 11
a
Reagents and conditions: (a) 12, Pd(PPh3)4, K2CO3, dioxane, 100 °C, 47%; (b) morpholine or (R)-2-methylmorpholine, Pd(OAc)2, tBu3PHBF4,
NaOtBu, toluene, 110 °C, 14−56%; (c) K2CO3, MeI, acetone, r.t., 100%.
a
Scheme 2. Synthesis of 16
a
Reagents and conditions: (a) morpholine, 1,4-dioxane, 110 °C, 70%; (b) 12, Pd(PPh3)4, Na2CO3 (aq.), DME, 85 °C, 57%; (c) H2, Pd/C, r.t.,
45%.
and the brain-penetrant AZD1390 for glioblastoma (Figure
1).7,8
(PD) effects consistent with ATM inhibition in a mouse brain
after oral dosing.9
Our group recently developed brain-penetrant ATM
inhibitors that may have potential as novel therapeutics for
the treatment of HD, which were derived from a series of ATM
inhibitors identified by Kudos.9 Starting from the ATM
inhibitor KU-60019, we developed a brain-penetrant ATM
inhibitor (4) that demonstrated robust pharmacodynamic
However, compound 4 was not an optimal compound,
having some drawbacks for proof-of-concept (PoC) studies of
ATM inhibition in HD, given it also inhibited vacuolar protein
sorting 34 kinase (Vps34) with less than 10-fold selectivity.
This lack of selectivity against Vps34, a critical kinase involved
in autophagy, was a serious concern for the program.10
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Impaired autophagy has been proposed as a pathogenic
mechanism of neurodegenerative diseases such as HD;
therefore, achieving selectivity over Vps34 became a key goal
for the program.
Scheme 5. Syntheses of N-Linked Pyrimidinones 30−32 and
a
34
Here, we describe the optimization of 4 toward a novel
series of potent ATM inhibitors with high selectivity over
Vps34.
CHEMISTRY
■
The thioxanthenes 7, 8, and 11 were prepared according to
Scheme 1, starting from 4,6-dichloropyridin-2(1H)-one (5).
The 4-pyridone 16 was synthesized by SNAr reaction of 4-
(benzyloxy)-2,6-dichloropyridine 13 with morpholine, fol-
lowed by Suzuki coupling with 12 and subsequent benzyl
deprotection (Scheme 2).
As shown in Scheme 3, hydrolysis of 17 followed by Suzuki
coupling with 12 afforded compound 19.
a
Scheme 3. Synthesis of Pyridone 19
a
Reagents and conditions: (a) 2-( )-benzylpyrrolidine or 2-(R)-
benzylazepane, DIPEA, DMF, 90 °C, 54−69%; (b) (i) TFA, DCM,
r.t., 11−17%; (ii) For 30 and 31 SFC separation; (c) 12, K2CO3,
Pd(PPh3)4, dioxane, 70 °C.
a
t
For synthesis of the 2-amino-linked 4-pyrimidinone
analogues, a regioselective route was developed, as shown in
Scheme 7. Chloro displacement of 57 with morpholine, and
subsequent reaction with 4-methoxybenzyl alcohol, afforded
the O-protected pyrimidine 58. In the case of the methylated
morpholine 59, the reaction order was reversed, with (R)-2-
methylmorpholine being introduced after the initial displace-
ment with 4-methoxybenzyl alcohol. Transformation to the
sulfones 60 and 61 and subsequent reaction with tetrabuty-
lammonium fluoride afforded the corresponding fluorides in
situ. Finally, deprotection provided the target compounds 62−
68.
Reagents and conditions: (a) KOH, BuOH, 82 °C, 18%; (b) 12,
Pd(PPh3)4, Na2CO3 (aq.), DME, 85 °C, 61%.
As outlined in Scheme 4, the thioxanthene-pyrimidinones 25
and 26 were prepared by initially reacting 2,4-dichloro-6-
methoxypyrimidine 20 with morpholine. This reaction
produced two pyrimidine regioisomers (21 and 22), which
were separated and converted through to the pyrimidinones 25
and 26.
As shown in Scheme 5, pyrimidinones 30−32 and 34 were
synthesized according to published procedures, starting from
the regioisomers of 27.11 In the case of the (R)-2-
benzylpyrrolidine analogues 30 and 31, the individual
enantiomers were isolated from the racemate using super-
critical fluid chromatography (SFC).
Scheme 6 describes the syntheses of thioxanthene 40 and
pyridones 43−56. Intermediate 37 was prepared by reaction of
35 with 1-chloro-2-(2-chloroethoxy)ethane in DMF. For
syntheses of the 2-methylmorpholines 51, 53, and 56,
intermediate 38 was prepared via a palladium-catalyzed
coupling of (R)-2-methylmorpholine with 36.
In the case of compounds 45 and 55, the absolute
stereochemistry was confirmed by chiral synthesis, using (S)-
2-benzylpyrrolidine and (R)-2-benylazepane, respectively.12
RESULTS AND DISCUSSION
■
The approach that we adopted to optimize selectivity for ATM
over Vps34 was to take advantage of the published structure of
Vps34 and use X-ray crystal structures of Vps34-ATM chimera
where the Vps34 ATP-binding site was mutated to
approximate that of ATM.
A comparison of the ATP-binding site of the human Vps34
X-ray crystal structures (pdb code 4UWH) with a homology
model of ATM identified key differences between these two
kinases, helping us focus our optimization strategy to drive
selectivity for ATM over Vps34. The ATM hydrophobic
gatekeeper residue Leu2767 corresponds to Met682 in Vps34;
a
Scheme 4. Syntheses of Pyrimidinones 25 and 26
a
Reagents and conditions: (a) morpholine, K2CO3, DMF, r.t., major 67%, minor 8%; (b) 12, Pd(PPh3)4, Na2CO3 (aq.), DME, 85 °C, 20−49%; (c)
TMSCl, KI, CH3CN, 80 °C, 58−100%.
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a
Scheme 6. Syntheses of Compounds 40 and 43−56
a
Reagents and conditions: (a) NaH, 1-chloro-2-(2-chloroethoxy)ethane, DMF, 0 °C-r.t., 65%; (b) (R)-2-methylmorpholine or morpholine,
PdCl2(dtbpf)2 NaOtBu, toluene, 50 °C; 78%; (c) NaOMe, methanol, 70 °C, 81%; (d) 12, Pd(PPh3)4, Na2CO3 (aq.), DME, 85 °C, 39%; (e) 4-
methoxybenzylalcohol, NaH, THF, 0 °C, then reflux; (f) Cs2CO3, Pd2(dba)3, BINAP or RuPhos, RuPhos Pd G1, NaOtBu, amine, 1,4-dioxane, 100
°C; (g) (i) 1-methylcyclohexa-1,4-diene, 5% Pd/C, EtOH; (ii) SFC chiral separation.
in the p-loop, Ala2693 in ATM is replaced by Phe612 in
Vps34; and in the hinge region, there is a key Gly hinge
insertion in ATM (Figure 2) that creates a small shallow
groove in the binding site that differs from that of Vps34.
These changes create subtle, but important topological
differences in the two sites that indicated attaining selectivity
were possible. We had postulated that the distal phenyl ring of
the thioxanthene group was important to maintain selectivity
across the closely related family kinases and sought to preserve
this feature while exploring modifications of hinge binding
groups and replacements of the pyranone core.
thioxanthene tricycle and replace the pyranone core to reduce
lipophilicity (compound 4 cLogP = 4.9) and improve aqueous
solubility (compound 4 aqueous solubility pH 7.4 = 0.001 mg/
mL).13,14 Importantly, given our goal to generate brain-
penetrant compounds, keeping the topological polar surface
area (TPSA) <80, molecular weight < 500, cLogP <5, and a
minimal number of hydrogen bond donors were important
parameters for our medicinal chemistry strategy.15,16
A
schematic summary of the structural approach adopted toward
increasing ATM selectivity over Vps34 and improving
compound properties is shown in Figure 3.
To improve the physicochemical properties associated with
Replacement of the pyranone core group of 4 with a
the pyranone-thioxanthene series, we sought to open up the
pyridone decreases biochemical potency by 8-fold but retains
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a
Scheme 7. Regioselective Synthesis of the Pyrimidinone Scaffold
a
Reagents and conditions: (a) morpholine or (R)-2-methylmorpholine, DIPEA, THF, 60 °C, 57−82%; (b) 4-methoxybenzyl alcohol, NaH or
K2CO3, DMF, 53−57%; (c) m-CPBA, DCM, r.t., 45−100%; (d) amine, TBAF·H2O, DIPEA, DMSO, 80 °C; (e) TFA, DCM, r.t.
activity exhibiting a 28-fold decrease in ATM biochemical
potency when compared to its 2-pyridone isomer (7). In
contrast to 7, the alternative 2-pyridone isomer 19 has no
discernible potency. As with the 2-pyridones, the pyrimidinone
isomers 25, 26, and 34 showed marked differences in
biochemical activity. While isomer 26 (bearing the hetero-
aromatic nitrogen atoms adjacent to the morpholino) showed
increased biochemical inhibitory potency (Ki 25 nM), 34 was
less potent (Ki 160 nM), and 25 was not active (Ki > 1.6 μM).
These differences are likely the result of a complex
combination of tautomeric preferences, water network hydro-
gen bonding, and the conformational impact upon the tricyclic
group in relation to the core. In the structures of a Vps34
kinase, a conserved water residue makes a key bridging
hydrogen bond between the conserved catalytic lysine residue
Lys636, a conserved Asp644 and the ligands. We hypothesize
that a similar water molecule could be situated in the active site
of ATM and could help the ligand bridge the equivalent ATM
Asp and Lys residues. This was corroborated by the
comparable potencies that ATM inhibitors 7 and 26 had for
ATM and Vps34. This also indicated that a simple change to
the pyranone core would not be sufficient to attain selectivity
over Vps34. Based on the biochemical and cellular potency
values highlighted in Table 1, we decided to optimize
Figure 2. Superposition of the homology model of human ATM with
proposed docked pose of 4 (green ribbon, grey residues, green ligand)
over the crystal structure of human Vps34 (pdb code: 4UWH, yellow
ribbon, yellow residues) highlighting proposed key residue differences
in thick sticks.
cellular potency (Table 1). N-Methylation (11) and O-
methylation (40) of the pyridone ring obliterates activity,
suggesting that the carbonyl and/or hydrogen bond donor
components of 7 are important to maintain potency toward
ATM inhibition. The 4-pyridone (16) core was detrimental to
Figure 3. Medicinal chemistry strategy toward identifying novel, selective, and brain-penetrant ATM inhibitors.
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compounds containing the 2-pyridone core with an emphasis
on finding a suitable replacement for the thioxanthene group.
Having identified a potential replacement for the pyranone,
our attention turned to the exploration of the tricyclic region,
seeking replacements for the thioxanthene moiety that would
maintain the conformation of the distal aromatic ring of the
thioxanthene. We particularly focused on substituted saturated
cyclic amines as potential replacements for thioxanthene with
the view that increasing the fraction of sp3 centers and
decreasing cLogP would improve the overall properties of the
compounds (Table 2). Pleasingly, the 2-benzylpyrrolidine 43
showed promising biochemical and cellular activity with
significantly improved solubility compared to 7. Separation
of the enantiomers of 43 afforded 44 and 45, which showed
that the (R)-enantiomer 44 was preferred for potency.
Table 1. Structures, Biological Activity, and Properties of
Pyranone Core Replacements
a
Moving the benzyl substituent to the 3-position (46) was
detrimental to both biochemical and cellular activity,
presumably due to the benzylic moiety being unable to occupy
similar space as the distal phenyl ring of the thioxanthene
group. However, ring expansion of 44 to afford the (R)-2-
benzylpiperidine (54) or (R)-2-benzylazepane (55) retained
ATM biochemical activity and provided an improved
selectivity profile compared to 44 and comparable to the
pyranone-thioxanthene 7. We postulate that the improved
selectivity of 54 and 55 for ATM over Vps34 compared to
compound 44 could arise from the increase in the size of the
heterocyclic ring, positioning the phenyl group in a steric clash
with p-loop residue Phe612 in Vps34 but not with the small
Ala2693 in ATM. In both cases, the (R)-enantiomer was much
more active over the (S)-enantiomer, with the (S)-enantiomer
of 54 and 55 displaying Ki values for ATM of >1.4 and 1.3 μM,
respectively (data not shown).17 The (R)-stereochemistry of
the benzylic group in 45 and 55 was confirmed by chiral
synthesis, beginning with the appropriate chiral starting
materials. Overall, the 2-benzyl-substituted amino-linked series
a
b
Ki and IC50 values are the geometric mean of at least two tests. n.d.
= not determined.
a
Table 2. Structures, Biological Activity, and Properties of N-Linked Heterocycles as Thioxanthene Replacements
a
Ki and IC50 values are the geometric mean of at least two tests.
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Figure 4. (A) X-ray structure (3.12 Å) of 44 bound to the mutant Vps34 construct, mutated residues shown as thick sticks. (B) Protein
superposition of 44 bound mutant Vps34 X-ray structure (cyan ribbon, green ligand) with published tricycle bound mutant Vps34 X-ray structure
6I3U (yellow ribbon, orange ligand).
a
Table 3. Structures, Biological Activity, and Properties of Pyrrolidino-Substituted Pyridones
a
b
Ki and IC50 values are the geometric mean of at least two tests. EER is the effective efflux ratio as measured in MDCK cells over-expressing
c
d
MDR1. Papp is the permeability value measured in the apical-to-basal direction using MDCK cells. Intrinsic clearance measured in mouse liver
microsomes.
had reduced lipophilicity and greater solubility compared to
the thioxanthene 7 and was therefore regarded as being a
promising series for further modifications.
substituent mimics the third ring of the tricyclic thioxanthene
and maintains a hydrophobic interaction with Pro690 of the
mutant Vps34 construct. The equivalent interaction with
Pro2775 in ATM was previously hypothesized to contribute to
selectivity over DNA-PK and mTOR.9
Having established the likely binding mode of 44 within
ATM and Vps34, we turned our attention toward introducing
substitution directed toward the p-loop difference between
ATM (Ala2693) and Vps34 (Phe612).
Substitution at the 3-position of the (R)-2-benzylpyrrolidine
ring 44 with difluoro (47) resulted in a small increase in
selectivity over Vps34 (Table 3), but a more pronounced
improvement was forthcoming with the 3-fluoropyrrolidine
(48), exhibiting a selectivity value over Vps34 of 43-fold. The
3-fluoropyrrolidine 48 also exhibited high potency (Ki 3 nM),
demonstrating 8-fold higher activity than its 3-methoxy
To better understand the binding mode and ligand protein
interactions of the amino-linked series, the co-crystal X-ray
structure of 44 in the previously described mutant Vps34
construct was solved (Figure 4).9 In addition to confirming the
stereochemistry of the benzylic group, the crystal structure
demonstrated a binding mode consistent with that observed
for the tricyclic series. In this structure, the morpholino moiety
acts as a hydrogen bond acceptor to the backbone NH of the
hinge residue Ile685, while the pyridone carbonyl forms the
predicted water-mediated hydrogen bond network with
Tyr670 and Asp644. Lys636 extends toward the pyranone
carbonyl at a distance of 3.76 Å, suggesting a weaker
interaction with the conserved lysine. As predicted, the benzyl
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a
Table 4. Structures, Biological Activity, and Properties of Core Replacements in the N-Linked Series
a
b
Ki and IC50 values are the geometric mean of at least two tests. EER is the effective efflux ratio as measured in MDCK cells over-expressing
c
d
MDR1. Papp is the permeability value measured in the apical-to-basal direction using MDCK cells. Intrinsic clearance measured in mouse liver
microsomes.
a
Table 5. Substituted and Unsubstituted Morpholino Derivatives in the Pyridone and 2-Pyrimidinone Series
a
Ki and IC50 values are the geometric mean of at least two tests
counterpart 49. Introduction of the fused cyclopropyl
pyrrolidine in 50 and 52 provided increased potency for
ATM compared to 44 and improved selectivity against Vps34.
Most notably, compound 52 showed a 33-fold selectivity gain
for ATM over Vps34, relative to compound 44. As the data in
Table 3 demonstrate, the lower molecular weight and less
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lipophilic character of the pyridone derivatives with the N-
linked pyrrolidine substituent showed high cell permeability,
much improved aqueous solubility compared to 4 and, in most
instances, greater stability in mouse liver microsomes (4, Clint
MLM = 122 mL/min/kg body weight).9
DNA-PK and ATR (Table 6). Generally, the analogues
evaluated in the PIKK family screening panel had low activity
Table 6. PIKK Selectivity Profile of Selected Pyridones and
a
Pyrimidinones
Having identified 2-substituted N-linked saturated hetero-
cycles as a replacement for the thioxanthene, we revisited some
of the more promising core replacements previously high-
lighted in Table 1, incorporating (R)-2-benzylpyrrolidine and
(R)-2-benzylazepanyl as core substituents. As shown in Table
4, the 2-((R)-2-benzylpyrrolidinyl)pyrimidinone (31) and 2-
((R)-2-benzylazepanyl)pyrimidinone (62) analogues demon-
strated high ATM biochemical potency, with Ki values of 1 and
0.1 nM, respectively. This was in sharp contrast to the lack of
measurable activity determined for the same pyrimidinone
scaffold in the thioxanthene series (25). We postulate that this
difference arises from the thioxanthene’s orientation that has
the more rigid tricyclic moiety that requires optimal placement
over Pro690, whereas the more flexible pyrrolidine is better at
adapting to the binding pocket and can tolerate the nitrogen
atoms either side of the N-linked heterocycle. As the data in
Table 4 demonstrate, 31 provided significantly higher ATM
biochemical potency (87-fold) and increased cellular potency
(17-fold) compared to its regioisomeric scaffold 30, and within
this favored scaffold type, it was the azepanyl substituent (62)
that offered a 10-fold potency enhancement compared to the
corresponding pyrrolidine (31). The pyrimidinones 30, 31,
and 62 and pyridone 55 exhibit improved aqueous solubility
compared to 4 and have similar high cell permeability and
efflux ratio to those pyridones shown in Table 3. In contrast,
the mouse microsomal turnover for the pyrimidinones was
greater than the pyridones.
ATM
ATR
cell
biochem (IC50;
no. (Ki; μM) μM)
Vps34 biochem
DNA-PK
Cell
(IC50;
μM)
(Ki μM) (fold biochem (Ki μM)
selectivity)
(fold selectivity)
51
52
55
68
0.004
0.002
0.001
0.0004
0.52
0.29
0.23
0.09
0.75 (188)
0.098 (49)
0.050 (50)
0.006 (15)
0.007 (2)
0.16 (80)
0.18 (180)
0.11 (275)
13
>30
>30
5.5
a
Ki and IC50 values are the geometric mean of at least two tests.
in the ATR cellular assay, with values ranging from 5.5 μM to
greater than 30 μM. With respect to ATM selectivity versus
DNA-PK, the picture is more complex, with 55, 52, and 68
demonstrating high ATM selectivity against DNA-PK, and
greater than was seen against Vps34. In contrast, 51 exhibited
essentially no ATM selectivity against DNA-PK.
As a representative example of the pyridone class, compound
55 was profiled against a panel of 263 kinases at DiscoverX at 1
μM compound concentration (Figure 5), and its TREEspot
interaction map is shown in Figure 5. Vps34 was identified as
the main “off-target” kinase, which in a follow-on assay
delivered a Kd value of 11 nM. mTOR was also identified as a
hit in the DiscoverX panel, but this was subsequently shown to
have significantly weaker affinity with a Kd value of 4.6 μM.
However, compound 52 shows selectivity over Vps34 of 49-
fold and over DNA-PK of 80-fold.
The pyridone and pyrimidinone templates were found to be
suitable cores in the N-linked series, and having attained
improved selectivity over Vps34, we decided to explore
substitution on the hinge-binding morpholino group to
improve selectivity against Vps34. Specifically, we considered
that the glycine insertion in ATM provided an opportunity to
introduce increased steric bulk on the morpholine substituent
to provide additive increases in ATM selectivity against Vps34.
In accordance with our hypothesis, the introduction of a
methyl adjacent to the oxygen of the morpholino substituent
did indeed provide notably improved selectivity against Vps34.
The α-methylated morpholines in Table 5 exhibited exquisite
ATM selectivity values against Vps34, ranging from 190-fold to
1700-fold, significantly higher than the 30-fold selectivity value
demonstrated by the previously described pyranone 4. While
the α-methylated morpholines have slightly weaker ATM
activity compared to their unsubstituted counterparts, they are,
nevertheless, high potency ATM inhibitors with Ki values in
the single digit nanomolar range. It was rewarding to see that
our strategy to address selectivity by simultaneously targeting
differences in the p-loop and the linker region provided
compounds with synergistic and enhanced selectivity. As
shown above with related analogues, these high potent
selective N-linked substituted pyridones and pyrimidinones
have lower molecular weight, significantly lower lipophilicity,
higher aqueous solubility, and are much more synthetically
tractable compared to the pyranone 4.
Three of the compounds screened in the selectivity panel,
52, 55, and 68, were progressed to PK studies in mice.
Compounds 52 and 55 were dosed at 2 mg/kg IV, and
compound 68 dosed at 0.8 mg/kg IV, and in each case, the
brain and plasma concentrations determined over an 8-hour
period (Table 7 and Figure 6).
Pyrimidinone 68 exhibits very good brain penetration, with a
brain/plasma ratio of 1.00 (Table 7). In contrast, the pyridones
55 and 52 had more modest brain penetration, with brain/
plasma ratios of 0.41 and 0.23, respectively. Kpuu values for
these three analogues ranged from 0.18 to 0.47, somewhat
lower than their corresponding Kp values, as a consequence of
having higher binding to brain protein compared to that of
plasma protein. Plasma clearance for these novel core
derivatives was moderate to high (2.0−6.9 L/h/kg), and all
four analogues demonstrated high volumes of distribution
ranging from 3.4 to 11 L/kg, culminating in the modest half-
life values ranging from 0.75 to 2.4 h.
Compounds 55 and 68 were also progressed to a mouse PK
study and demonstrated high oral bioavailability values of 68
and 78%, respectively.18 The plasma clearance and volume of
distribution are perhaps higher than anticipated for compound
68; however, data for compounds containing this core scaffold
were limited, and it was not clear if the PK result is a feature
relating to this core structure. Figure 7 shows the plasma and
brain concentration profiles after oral administration of 55 to
mice at 10 and 30 mg/kg.19
For a more complete picture of the selectivity profile against
other PIKK family members, we evaluated a selection of
analogues with varying Vps34 selectivity and either a
morpholine or α-methylated morpholine substituent against
After dosing at 10 mg/kg PO, the total plasma and brain
concentrations of 55 are sustained at a level that is above the
cellular IC50 (230 nM) for approximately 9 and 5 h,
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Figure 5. TREEspot interaction map for compound 55.
Table 7. PK Parameters for the N-Linked Analogues 52 (2 mg/kg), 55 (2 mg/kg), and 68 (0.8 mg/kg) Following IV
Administration to Male Mice
a
no.
AUCnorm (nM·h·kg/mg) plasma
AUCnorm (nM·h·kg/mg) brain
CLp (L/h/kg)
Vdss (L/kg)
Half-life (h)
Kp/Kpuu (AUC ratio)
52
55
68
1400
1300
410
320
530
400
2.0
2.1
6.9
4.7
3.4
11
2.4
1.6
0.75
0.23/0.12
0.41/0.18
1.0/0.47
a
The brain/plasma ratio shown is based on total concentrations (Kp) and free concentrations (Kpuu) after dosing IV to mice at 2 mg/kg
(compounds 55 and 52) or 0.8 mg/kg (compound 68).
Figure 6. Mouse IV PK profile for compounds 55 and 52 (dosed at 2 mg/kg) and 68 (dosed at 0.8 mg/kg).
respectively (Figure 7). With respect to free plasma and free
brain levels (Kpuu 0.20 after dosing at either 10 or 30 mg/kg
PO), at 10 mg/kg PO, the concentrations are maintained
above the biochemical Ki (1.5 nM) for 12 and 9 h, respectively.
In contrast, after dosing at 30 mg/kg PO, the total and free
plasma and brain concentrations remain above the biochemical
Ki for the entire 12 h duration of the study. Furthermore, at 30
mg/kg PO, free plasma concentrations are maintained at levels
above the cellular IC50 for up to 4 h, whereas the brain free
concentrations rise above the cellular IC50 at Cmax approx-
imately 0.5 h after dosing.
biochemical and cellular activity. Using structure-based design
strategies, we have established that the novel α-methylated
morpholino-substituted pyridones and pyrimidinones demon-
strate good levels of selectivity against Vps34, at significantly
greater selectivity than that of the previously described
thioxanthenes. Furthermore, these novel pyridones and
pyrimidinones have superior drug properties, with lower
molecular weight, significantly reduced lipophilicity, higher
aqueous solubility, and greater synthetic tractability compared
to the thioxanthene series exemplified by 4. Moreover,
representative compounds from this series have high oral
bioavailability and, as compound 55 demonstrates, can attain
free plasma and brain levels above the biochemical Ki and
cellular IC50 values. Consequently, we consider these selective
N-linked heterocyclyl pyridones and pyrimidinones a starting
point for further optimization to provide a brain-penetrant,
CONCLUSIONS
■
In summary, using the previously described pyranone 4 as a
starting point, we have developed a series of brain-penetrant
pyridone and pyrimidinone ATM inhibitors that exhibit high
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Figure 7. Mouse PK profile for compound 55 after dosing at 10 and 30 mg/kg PO.
The different analytical methods A−F, SFC1, and SFC4 used in
this paper are described in detail in the Supporting Information, as are
the general synthetic methods A-L. Intermediate synthesis is also
described in the Supporting Information. The Supporting Information
also describes synthesis of key Intermediates used in the construction
of the final compounds.
All IC50 and Ki data are quoted as geometric mean values, and
statistical analysis is available in the Supporting Information. All
experimental activities involving animals were carried out in
accordance with the Charles River Animal Welfare and Humane
Treatment of Animals policies, which are consistent with The
American Chemical Society Publications rules and ethical guidelines.
ATM-selective PoC compound to evaluate the effects of ATM
inhibition in HD.
EXPERIMENTAL SECTION
■
General Procedure. All chemicals were purchased from
commercial suppliers and used as received. All reactions involving
air or moisture-sensitive reagents were performed under a nitrogen
atmosphere using dried solvents and glassware. Flash chromatography
was carried out with prepacked SiO2 SNAP cartridges (KP-SIL) from
Biotage using a Biotage Isolera Four system using gradient elution.
Analytical thin-layer chromatography (TLC) was performed on silica
using PolygramSIL G/UV254 with a fluorescent indicator (200 μm
thickness) and visualized under UV light. NMR spectra were recorded
on a Bruker AV 400 (1H = 400.13 MHz, 13C = 100.60 MHz)
instrument spectrometer and referenced to tetramethylsilane. The
following abbreviations are used: br = broad signal, s = singlet, br s =
broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet.
Preparative HPLC was performed on a Waters Sunfire OBD C18 10
μm column (150 mm × 19 mm), Phenomenex Luna phenylhexyl 10
μm column (150 mm × 21.2 mm), or a Waters Xbridge phenyl 5 μm
column (100 mm × 19 mm), eluting with mixtures of water−
acetonitrile or water−methanol, optionally containing a modifier
(0.1% v/v formic acid or 10 mM ammonium bicarbonate). Low-
resolution mass spectra were recorded on a Waters ZQ single
quadrapole LCMS in ESCi+, ESCi− mode, or a Quattro Micro LC−
MS−MS in ESCi+, ESCi− mode. High-resolution mass spectra were
recorded on a Waters Acquity UPLC system and Waters Xevo G2
TOF mass spectrometer. HPLC purity was assessed using one of four
systems (see analytical methods table in the Supporting Information).
All final compounds were purified to >95% chemical and optical
purity (analytical methods for individual compounds are specified in
the experimental writeup for each molecule). Supercritical fluid
chromatography (SFC) used a Waters Thar Prep100 preparative SFC
system (P200 CO2 pump, 2545 modifier pump, 2998 UV/VIS
detector, 2767 liquid handler with stacked injection module). The
Waters 2767 liquid handler acted as both an auto-sampler and fraction
collector. Chiral HPLC purification used either Daicel Chiralpak IA or
IC columns. Each sample was run under both unmodified and basic
conditions (5.0 μL injection, 5/95 gradient for 5 min) across ethanol,
methanol, and isopropanol. Compounds were named with the aid of
Cambridgesoft Chemistry Cartridge (version 15.1.0.144) software.
Racemic mixtures are denoted using asterisks, e.g., (1R*,2R*).
Enantiomerically pure compounds of known stereochemistry are
denoted without asterisks, e.g., (1R,2R).
METHODS AND DATA FOR SPECIFIC
COMPOUNDS
■
General Methods. Method A: To a mixture of alcohol (1.1
equiv) in THF (0.4 M) at 0 °C was added portionwise NaH (2 eq,
60% mineral oil). The reaction was stirred for 15 min. Aryl chloride (1
equiv) was added dropwise as a solution in THF (0.7 M). After
complete addition, the reaction was warmed to r.t. and heated at
reflux for 19 h. The reaction mixture was cooled to 0 °C and
cautiously quenched with water. The THF was removed under
reduced pressure. The residue was dissolved in EtOAc and washed
sequentially with water and brine. The organic extract was dried
(MgSO4), filtered, and concentrated under reduced pressure. The
crude product was either triturated or purified by silica gel column
chromatography.
Method B: A mixture of amine (1.0 equiv), 2,6-dichloro-4-
iodopyridine (1.0 equiv), NaOt Bu (2 equiv), and
PdCl2(tBu2Pferrocene)2 (0.025 equiv) in toluene (0.3 M) was placed
in a sealed reaction tube under nitrogen and heated at 50 °C for 20 h.
The reaction mixture was cooled to r.t. and filtered through Celite.
The mixture was concentrated under reduced pressure and purified by
silica gel chromatography.
Method E: To a solution of PMB-protected compound (1.0 equiv)
in ethanol (0.15 M) was added 5% palladium on carbon (100% by
weight of PMB compound), and the reaction mixture purged with N2
for 20 min. 1-Methylcyclohexa-1,4-diene (10 equiv) was added, and
the reaction heated at 75 °C for 1−18 h. The reaction was cooled to
r.t. and filtered through Celite, washing with methanol. The solution
was concentrated under reduced pressure.
Method H: TFA (0.24 M) was added to a solution of the PMB-
protected compound in DCM (0.24 M). The reaction was stirred at
r.t. for 1−4 h before concentrating under reduced pressure. The
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1
residue was dissolved in DCM, washed with saturated sodium
bicarbonate solution, dried (phase separator), and concentrated under
reduced pressure. The crude material was purified by reverse-phase
preparative HPLC.
compound. H NMR (400 MHz, CDCl3): δ 6.57 (2H, s), 4.02 (1H,
ddt, J = 1.3, 5.1, 5.8 Hz), 3.74−3.63 (2H, m), 3.58−3.49 (2H, m),
3.03 (1H, dt, J = 4.4, 12.4 Hz), 2.67 (1H, dd, J = 10.4, 12.4 Hz), 1.26
(3H, d, 6.4 Hz).
6-((1S,2S,5R)-2-Benzyl-3-azabicyclo[3.1.0]hexan-3-yl)-4-((R)-2-
methylmorpholino)pyridin-2(1H)-one (51). Step 1: tert-Butyl 3-
Azabicyclo[3.1.0]hexane-3-carboxylate. Boc anhydride (2.6 g, 11.9
mmol) and a solution of NaHCO3 (3.32 g, 39.5 mmol) in water (78
mL) were added to a suspension of 3-azabicyclo[3.1.0]hexane
hydrochloride (950 mg, 7.9 mmol) in THF (26 mL). The resulting
biphasic mixture was stirred vigorously overnight at r.t. The reaction
mixture was diluted with water and DCM. The layers were separated,
and the organic phase dried (phase separator) and concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography (gradient elution, 0−10% EtOAc/iso-hexane) to
Step 5: (R)-4-(2-Chloro-6-((4-methoxybenzyl)oxy)pyridin-4-yl)-2-
methylmorpholine (Intermediate 42). Following Method A using
(R)-4-(2,6-dichloropyridin-4-yl)-2-methylmorpholine (1.6 g, 6.4
mmol) and para-methoxybenzyl alcohol (0.87 mL, 7.0 mmol).
Purified by silica gel column chromatography (gradient elution, 5−
40% EtOAc/iso-hexane) to afford the title compound. 1H NMR (400
MHz, CDCl3): δ 7.39−7.36 (2H, m), 6.91−6.88 (2H, m), 6.40 (1H,
d, J = 2.0 Hz), 5.98 (1H, d, J = 2.0 Hz), 5.24 (2H, s), 4.00−3.95 (1H,
m), 3.81 (3H, s), 3.71−3.61 (2H, m), 3.52−3.42 (2H, m), 2.96−2.88
(1H, m), 2.58 (1H, dd, J = 10.4, 12.4 Hz), 1.24−1.21 (3H, m).
Step 6: (R)-4-(2-((1R*,2R*,5S*)-2-Benzyl-3-azabicyclo[3.1.0]-
hexan-3-yl)-6-((4-methoxybenzyl)oxy)pyridin-4-yl)-2-methylmor-
pholine. A mixture of (R)-6-chloro-4-(2-methylmorpholino)pyridin-
2(1H)-one (178 mg, 0.50 mmol), (1R*,2R*,5S*)-2-benzyl-3-
azabicyclo[3.1.0]hexane (88 mg, 0.50 mmol), NaOtBu (55 mg, 0.55
mmol), RuPhos (12 mg, 0.025 mmol), and RuPhos palladacycle G1
methyl tert-butyl ether adduct (20 mg, 0.025 mmol) was placed in a
stem block tube and then sealed. The tube was evacuated and refilled
with nitrogen (×3). 1,4-Dioxane (3.3 mL) was added, and the
reaction heated at 85 °C for 16 h. The reaction mixture was cooled to
r.t. and filtered through Celite. The mixture was concentrated under
reduced pressure and purified by silica gel column chromatography
(gradient elution, 5−40% EtOAc/iso-hexane) to afford the title
compound as a yellow oil (230 mg, 95%). LCMS (ES+) 486 (M +
H)+, RT 1.77 min (Analytical Method F).
1
afford the title compound as a mixture of rotamers. H NMR (400
MHz, CDCl3): δ 3.55 (1H, d, J = 11.0 Hz), 3.47 (1H, J = 11.0 Hz),
3.35−3.27 (2H, m), 1.42 (9H, s), 1.21−1.14 (1H, m), 0.93−0.84
(1H, m), 0.67−0.61 (1H, m), 0.16−0.13 (1H, m).
Step 2: tert-Butyl (1R*,2R*,5S*)-2-Benzyl-3-azabicyclo[3.1.0]-
hexane-3-carboxylate and tert-Butyl (1R*,2S*,5S*)-2-Benzyl-3-
azabicyclo[3.1.0]hexane-3-carboxylate. To a solution of tert-butyl
3-azabicyclo[3.1.0]hexane-3-carboxylate (1.50 g, 7.9 mmol) in diethyl
ether (27 mL), under an N2 atmosphere, was added tetramethyle-
thylenediamine (1.23 mL, 8.2 mmol), and the reaction mixture cooled
to −78 °C. sec-BuLi (11.8 mL, 10.6 mmol, 0.9 M in cyclohexane) was
added, and the mixture was stirred at −78 °C for 30 min. Benzyl
bromide (1.95 mL, 16.4 mmol) was added dropwise at −78 °C. After
complete addition, the reaction was allowed to slowly warm to r.t.
overnight. The reaction was quenched with saturated aqueous NH4Cl
solution, the layers separated, and the aqueous further extracted with
Et2O (×2). The combined organic extracts were dried (MgSO4) and
concentrated under reduced pressure. The crude material was purified
by silica gel column chromatography (gradient elution, 0−10%
EtOAc/iso-hexane) to afford the title compounds as separate
diastereomers tert-butyl (1R*,2S*,5S*)-2-benzyl-3-azabicyclo[3.1.0]-
hexane-3-carboxylate and tert-butyl (1R*,2R*,5S*)-2-benzyl-3-
azabicyclo[3.1.0]hexane-3-carboxylate.
Step 7: 6-((1S,2S,5R)-2-Benzyl-3-azabicyclo[3.1.0]hexan-3-yl)-4-
((R)-2-methylmorpholino)pyridin-2(1H)-one (51). (R)-4-(2-
((1R*,2R*,5S*)-2-Benzyl-3-azabicyclo[3.1.0]hexan-3-yl)-6-((4-
methoxybenzyl)oxy)pyridin-4-yl)-2-methylmorpholine (220 mg, 0.45
mmol) was suspended in EtOH (4.5 mL), and the reaction was
warmed to obtained a homogeneous solution. Five percent Pd/C
(220 mg, 100% by wt.) was added followed by 1-methylcyclohexyl-
1,4-diene (425 mg, 4.50 mmol). The reaction was heated at 75 °C for
3 h. The reaction was cooled to r.t. and filtered through a plug of
cotton to give a residue, which was purified by chiral SFC to yield 29
mg (17%) of the title compound. Absolute configuration was assigned
following determination of biological activity, the (1S) isomer being
the more active. LCMS (ES+) 366 (M + H)+, RT 3.05 min
(Analytical Method A); RT 2.43 min (Analytical Method SFC4, YMC
amylose-C, 20/80 MeOH + 0.1% diethylisopropylamine (DEAISO)/
CO2); 1H NMR (400 MHz, CDCl3): δ 7.35−7.23 (5H, m), 5.28 (1H,
d, J = 2.3 Hz), 5.02 (1H, d, J = 2.0 Hz), 4.00−3.92 (2H, m), 3.88
(1H, d, J = 9.1 Hz), 3.73−3.63 (2H, m), 3.54−3.42 (3H, m), 3.25
(1H, dd, J = 2.8, 13.6 Hz), 2.97 (1H, dt, J = 3.6, 12.3 Hz), 2.65−2.51
(2H, m), 1.73−1.67 (2H, m), 1.23 (3H, d, J = 6.1 Hz), 0.78 (1H, dt, J
= 5.1, 8.0 Hz), 0.62 (1H, q, J = 4.4 Hz), NH not observed. HRMS
(ESI+) (M + H)+: calcd for C22H28N3O2, 366.2181; found, 366.218.
6-((1R,2S,5S)-2-Benzyl-3-azabicyclo[3.1.0]hexan-3-yl)-4-mor-
pholinopyridin-2(1H)-one (52). Step 1: (1R*,2S*,5S*)-2-Benzyl-3-
azabicyclo[3.1.0]hexane. To tert-butyl (1R*,2S*,5S*)-2-benzyl-3-
azabicyclo[3.1.0]hexane-3-carboxylate (Isomer B from Step 2 of
compound 51, 400 mg, 1.46 mmol) were added DCM (3.5 mL) and
TFA (3.5 mL), and the resulting mixture was stirred at r.t. for 1 h.
The solvent was evaporated and purified by a SCX cartridge eluting
sequentially with DCM/MeOH, 1:1 then 3:1 DCM/7 N ammonia in
methanol. The ammonia in methanol fractions were combined and
concentrated under reduced pressure to afford the title compound.
nOe experiments allowed determination of the stereochemistry of the
Isomer A (592 mg, later determined to be the (1R*, 2R*, 5S*)
isomer, see next step): LCMS (ES+) 218 (M-tBu)+, RT 1.77 min
1
(Analytical Method F); H NMR (400 MHz, CDCl3): δ 7.31−7.18
(5H, m), 4.12−3.96 (1H, m), 3.61−3.39 (1H, m), 3.22−2.91 (2H,
m), 2.77−2.64 (1H, m), 1.48−1.42 (9H, m), 1.38−1.28 (1H, m),
0.95−0.87 (1H, m), 0.59−0.53 (1H, m), 0.12−0.09 (1H, m),
rotamers observed.
Isomer B (420 mg, later determined to be the (1R*, 2S*, 5S*)
isomer, see next step): LCMS (ES+) 218 (M-tBu)+, RT 1.83 min
1
(Analytical Method F); H NMR (400 MHz, CDCl3): δ 7.33−7.27
(4H, m), 7.23−7.17 (1H, m), 4.04−3.95 (1H, m), 3.86−3.44 (3H,
m), 2.40 (1H, dd, J = 10.8, 12.4 Hz), 1.57−1.34 (11H, m), 0.69−0.63
(1H, m), 0.47−0.43 (1H, m), rotamers observed.
Step 3: (1R*,2R*,5S*)-2-Benzyl-3-azabicyclo[3.1.0]hexane. To
tert-butyl (1R*,2R*,5S*)-2-benzyl-3-azabicyclo[3.1.0]hexane-3-car-
boxylate (Isomer A from the previous step, 570 mg, 2.09 mmol)
were added DCM (5 mL) and TFA (5 mL), and the resulting mixture
was stirred at r.t. for 1 h. The solvent was evaporated and purified by a
SCX cartridge eluting sequentially with DCM/MeOH, 1:1 then 3:1
DCM/7 N ammonia in methanol. The ammonia in methanol
fractions were combined and concentrated under reduced pressure to
afford the title compound. nOe experiments allowed the determi-
nation of the stereochemistry of the benzyl group relative to the
1
cyclopropyl group. H NMR (400 MHz, CDCl3): δ 7.32−7.17 (5H,
1
benzyl group relative to the cyclopropyl group. H NMR (400 MHz,
m), 3.41 (1H, ddd, J = 3.0, 5.5, 8.1 Hz), 2.94 (2H, d, J = 1.8 Hz), 2.82
(1H, dd, J = 5.9, 13.0 Hz), 2.61 (1H, dd, J = 7.8, 13.1 Hz), 1.38−1.32
(1H, m), 1.30−1.23 (1H, m), 0.40−0.34 (1H, m), 0.34−0.30 (1H,
m). NH not observed.
CDCl3): δ 7.32−7.17 (5H, m), 3.29 (1H, t, J = 7.2 Hz), 3.00 (1H, dd,
J = 3.2, 11.2 Hz), 2.87 (1H, d, J = 11.0 Hz), 2.69 (1H, dd, J = 7.2,
13.5 Hz), 2.60 (1H, dd, J = 7.5, 13.4 Hz), 1.43−1.36 (1H, m), 1.29
(1H, ddd, J = 4.0, 6.2, 7.9 Hz), 0.45 (1H, dt, J = 5.0, 7.7 Hz), 0.15
(1H, dd, J = 4.0, 8.7 Hz). NH not observed.
Step 2: 4-(2,6-Dichloropyridin-4-yl)morpholine. 2,6-Dichloropyr-
idin-4-amine (10 g, 61.3 mmol) was dissolved in DMF (200 mL) and
Step 4: (R)-4-(2,6-Dichloropyridin-4-yl)-2-methylmorpholine (In-
termediate 38). Following Method B using (2R)-methyl morpholine
(0.5 g, 5 mmol). Purified by silica gel column chromatography
(gradient elution, 10−50% EtOAc/iso-hexane) to afford the title
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cooled to 0 °C. Sodium hydride (6.13 g, 153 mmol) was added
portionwise over 20 min. After 15 min, 1-chloro-2-(2-chloroethoxy)-
ethane (8.63 mL, 73.6 mmol) was added dropwise. The reaction
mixture was warmed to r.t. and stirred for 21.5 h. After this time, the
reaction was cooled to 0 °C and cautiously quenched with water. The
DMF was removed under reduced pressure. The resultant residue was
dissolved in DCM and washed with water. The organic layers were
dried (MgSO4), filtered, and concentrated under reduced pressure.
The crude product was triturated with EtOAc and iso-hexane to afford
°C. Acetic acid (3.16 mL, 55.1 mmol) was added followed by
portionwise addition of NaBH4 (759 mg, 20.05 mmol). The reaction
was stirred at 0 °C for 3.5 h. The reaction mixture was cautiously
quenched by dropwise addition of water (5 mL) followed by brine
(∼100 mL). The layers were separated, and the aqueous layer was
extracted with DCM. The combined layers were washed with brine,
dried (MgSO4), filtered, and concentrated under reduced pressure to
afford the title compound. Then, it was used without further
purification in the next step.
Step 3: tert-Butyl (R)-6-((tert-Butoxycarbonyl)amino)-7-phenyl-
heptanoate. tert-Butyl (R)-(5-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-
yl)-1-phenylpentan-2-yl)carbamate (1.86 g, 4.59 mmol) was dissolved
in toluene (5 mL) and t-BuOH (5 mL). The reaction mixture was
refluxed for 6 h. After this time, the solvents were removed under
reduced pressure. The resultant oil was dissolved in toluene and
heated at reflux for 25 h. The reaction mixture was concentrated
under reduced pressure. The oil was dissolved in DCM and NaHCO3
(saturated aqueous solution) added. The layers were inseparable. A
small quantity of brine was added to separate the layers. The aqueous
layer was extracted with EtOAc. The combined organic layers were
dried (phase separator) and concentrated under reduced pressure.
The crude oil was purified by silica gel column chromatography
(gradient elution 0−30% EtOAc/iso-hexane) to afford the title
compound. 1H NMR (400 MHz, CDCl3): δ 7.32−7.14 (5H, m), 4.32
(1H, d, J = 0.8 Hz), 3.78 (1H, br s), 3.32 (2H, s), 2.80−2.71 (2H, m),
2.60 (2H, t, J = 0.8 Hz), 1.83−1.81 (1H, m), 1.45 (9H, s), 1.39 (9H,
s).
1
the title compound as a white solid (9.27 g, 65%). H NMR (400
MHz, CDCl3): δ 6.59 (2H, s), 3.84−3.80 (4H, m), 3.33−3.29 (4H,
m).
Step 3: 4-(2-Chloro-6-((4-methoxybenzyl)oxy)pyridin-4-yl)-
morpholine. Following Method A using 4-(2,6-dichloropyridin-4-
yl)morpholine (9.27 g, 39.8 mmol) and PMBOH (6.05 g, 43.8
mmol). The crude product was triturated with EtOAc and iso-hexane
1
to afford the title compound as an off-white solid (11.4 g, 86%). H
NMR (400 MHz, CDCl3): δ 7.39−7.35 (2H, m), 6.91−6.88 (2H, m),
6.40 (1H, d, J = 2.0 Hz), 5.99 (1H, d, J = 2.0 Hz), 5.25 (2H, s), 3.81−
3.77 (7H, m), 3.25−3.21 (4H, m).
Step 4: 4-(2-((1R*,2S*,5S*)-2-Benzyl-3-azabicyclo[3.1.0]hexan-3-
yl)-6-((4-methoxybenzyl)oxy)pyridin-4-yl)morpholine. A mixture of
4-(2-chloro-6-((4-methoxybenzyl)oxy)pyridin-4-yl)morpholine (150
mg, 0.45 mmol), NaOtBu (48 mg, 0.50 mmol), RuPhos (11 mg,
0.023 mmol), and a RuPhos Pd G1 catalyst (methyl t-butyl ether
adduct, 18 mg, 0.023 mmol) was placed in a tube and sparged with
N2. A solution of (1R*,2S*,5S*)-2-benzyl-3-azabicyclo[3.1.0]hexane
(78 mg, 0.45 mmol) in anhydrous dioxane (3 mL) was added, the
tube was sealed, and stirring commenced at 85 °C for 16 h. The
reaction was cooled to r.t. and filtered through Celite, washing with
DCM, and the reaction concentrated under reduced pressure. The
crude mixture was purified by silica gel column chromatography
(gradient elution, 0−15% EtOAc/iso-hexane) to afford the title
compound as a green solid (197 mg, 93%). LCMS (ES+) 472 (M +
H)+, RT 1.48 min (Analytical Method F).
Step 4: (R)-6-Amino-7-phenylheptanoic Acid. TFA (2 mL) was
added to tert-butyl (R)-6-((tert-butoxycarbonyl)amino)-7-phenyl-
heptanoate (457 mg, 1.21 mmol) in DCM (2 mL) at r.t. with
stirring. After 1.5 h, the reaction mixture was concentrated under
reduced pressure to afford a yellow oil. The oil was dissolved in 4 M
HCl in dioxane (10 mL) and stirred at r.t. for 4 h. The reaction
mixture was concentrated under reduced pressure and dissolved in 4
M HCl in dioxane (10 mL). After 6 h, the reaction mixture was
concentrated under reduced pressure and used without further
purification in the next step.
Step 5: 6-((1R,2S,5S)-2-Benzyl-3-azabicyclo[3.1.0]hexan-3-yl)-4-
morpholinopyridin-2(1H)-one (52). Following Method E from 4-(2-
((1R*,2S*,5S*)-2-benzyl-3-azabicyclo[3.1.0]hexan-3-yl)-6-((4-
methoxybenzyl)oxy)pyridin-4-yl)morpholine (190 mg, 0.40 mmol).
Purification by silica gel column chromatography (gradient elution,
0−20% MeOH/EtOAc) and chiral SFC yielded the title compound
and its enantiomer. Absolute configuration was assigned following
determination of biological activity, the (1R) isomer being the more
active. LCMS (ES+) 352 (M + H)+, RT 2.83 min (Analytical Method
A); RT 1.65 min (Analytical Method SFC4,YMC amylose-C, 40/60
Step 5: (R)-7-Benzylazepan-2-one. EDC (348 mg, 1.82 mmol)
and HOPO (201 mg, 1.82 mmol) were added to a solution of (R)-6-
amino-7-phenylheptanoic acid (1.21 mmol) and triethylamine (1.01
mL, 7.26 mmol) in DMF (13 mL) at r.t. with stirring. After 54.5 h,
the reaction mixture was transferred to a separating funnel and diluted
with DCM. The reaction mixture was washed sequentially with 1 M
HCl (aq), NaHCO3 (saturated aqueous solution), and brine. The
organics were dried (MgSO4), filtered, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (gradient elution 50−70% EtOAc/iso-hexane) to
afford the title compound. 1H NMR (400 MHz, CDCl3): δ 7.34−7.17
(5H, m), 5.54 (1H, brs), 3.68−3.55 (1H, m), 2.87−2.72 (2H, m),
2.51−2.38 (2H, m), 1.98−1.81 (3H, m), 1.61−1.32 (3H, m).
Step 6: (R)-2-Benzylazepane. Lithium aluminum hydride (0.59
mL, 0.59 mmol, 1 M solution in THF) was added dropwise to an ice-
cooled solution of (R)-7-benzylazepan-2-one (60 mg, 0.30 mmol) in
THF (2 mL). The reaction mixture was warmed to r.t. and heated at
reflux for 3 h. The reaction mixture was cooled to r.t., followed by 0
°C, and cautiously quenched with water. The THF was removed
under reduced pressure. The mixture was diluted with water and
EtOAc. The layers were separated, and the organic extract dried
(MgSO4), filtered, and concentrated under reduced pressure. The
resultant oil was purified by a SCX cartridge. The sample was loaded
in DCM and eluted with three column volumes of DCM, three
column volumes of methanol, and three column volumes 7 N NH3 in
MeOH/DCM (1:9). The basic fractions were combined and
concentrated under reduced pressure to afford the title compound.
It was used without further purification in the next step.
1
IPA + 0.1% DEAISO/CO2); H NMR (400 MHz, CDCl3): δ 7.27−
7.26 (3H, m), 7.19−7.15 (2H, m), 5.22 (1H, d, J = 2.0 Hz), 4.82 (1H,
d, J = 2.0 Hz), 4.22 (1H, dd, J = 3.4, 7.7 Hz), 3.81−3.77 (4H, m),
3.33 (1H, d, J = 9.3 Hz), 3.27−3.23 (4H, m), 3.12 (1H, dd, J = 4.0,
9.1 Hz), 2.99 (1H, dd, J = 3.3, 13.6 Hz), 2.81 (1H, dd, J = 8.0, 13.5
Hz), 1.56−1.39 (2H, m), 0.66 (1H, dt, J = 5.2, 7.8 Hz), 0.19 (1H, q, J
= 4.3 Hz), NH not observed. HRMS (ESI+) (M + H)+: calcd for
C21H26N3O2, 352.2025; found, 352.203.
(R)-6-(2-Benzylazepan-1-yl)-4-morpholinopyridin-2(1H)-one
(55). Step 1: tert-Butyl (R)-(5-(2,2-Dimethyl-4,6-dioxo-1,3-dioxan-5-
yl)-5-oxo-1-phenylpentan-2-yl)carbamate. (R)-4-((tert-
Butoxycarbonyl)amino)-5-phenylpentanoic acid (3.0 g, 10.2 mmol)
was dissolved in DCM (50 mL) and cooled to 0 °C. Meldrum’s acid
(1.47 g, 10.2 mmol), EDC hydrochloride (2.94 g, 15.3 mmol), and
DMAP (1.87 g, 15.3 mmol) were added sequentially to the reaction.
The reaction mixture was stirred for 19 h, removing the ice-bath after
1 h. The reaction was transferred to a separating funnel and washed
with 1 M potassium bisulfate aqueous solution. The organic extracts
were dried (phase separator) and concentrated under reduced
pressure to afford the title compound. It was used without further
purification in the next step.
Step 7: (R)-4-(2-(2-Benzylazepan-1-yl)-6-((4-methoxybenzyl)-
oxy)pyridin-4-yl)morpholine. A mixture of 4-(2-chloro-6-((4-
methoxybenzyl)oxy)pyridin-4-yl)morpholine (64 mg, 0.19 mmol),
(R)-2-benzylazepane (40 mg, 0.21 mmol), NaOtBu (20 mg, 0.0.21
mmol), RuPhos (4 mg, 0.01 mmol), and a RuPhos Pd G1 catalyst
Step 2: tert-Butyl (R)-(5-(2,2-Dimethyl-4,6-dioxo-1,3-dioxan-5-
yl)-1-phenylpentan-2-yl)carbamate. tert-Butyl (R)-(5-(2,2-dimeth-
yl-4,6-dioxo-1,3-dioxan-5-yl)-5-oxo-1-phenylpentan-2-yl)carbamate
(2.1 g, 5.01 mmol) was dissolved in DCM (50 mL) and cooled to 0
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(methyl t-butyl ether adduct, 8 mg, 0.01 mmol) was placed in a tube
and sparged with N2. Anhydrous dioxane (2.5 mL) was added, the
tube was sealed, and stirring commenced at 85 °C for 16 h. The
reaction was cooled to r.t. and filtered through Celite, washing with
DCM, and the reaction concentrated under reduced pressure. The
crude mixture was purified by silica gel column chromatography
(gradient elution, 10−100% Et2O/iso-hexane) to afford the title
compound as a colorless oil (31 mg, 33%).
dried (MgSO4), filtered, and concentrated under reduced pressure.
Purified by silica gel column chromatography (gradient elution, 10−
50% EtOAc/iso-hexane) to afford the title compound. LCMS (ES+)
1
348 (M + H)+, RT 1.69 min (Analytical Method F); H NMR (400
MHz, CDCl3): δ 7.37−7.33 (2H, m), 6.91−6.87 (2H, m), 5.53 (1H,
s), 5.30 (2H, s), 3.80 (3H, s), 3.76−3.72 (4H, m), 3.55−3.51 (4H,
m), 2.51 (3H, s).
Step 4: 4-(6-((4-Methoxybenzyl)oxy)-2-(methylsulfonyl)-
pyrimidin-4-yl)morpholine. m-CPBA (759 mg, 4.4 mmol, 50−55
wt % in H2O) was added portionwise to a solution of 4-(6-((4-
methoxybenzyl)oxy)-2-(methylthio)pyrimidin-4-yl)morpholine (695
mg, 2.0 mmol) in DCM (20 mL) at r.t. After 2 h, the reaction was
filtered, washing the precipitate with DCM. The filtrate was washed
successively with saturated NaHCO3 (aqueous solution), brine, dried
(phase separator), and concentrated under reduced pressure to afford
the product, which was used without further purification in the next
step. 1H NMR (400 MHz, CDCl3): δ 7.38−7.35 (2H, m), 6.91−6.88
(2H, m), 5.88 (1H, s), 5.36 (2H, s), 3.81 (3H, s), 3.78−3.74 (4H, m),
3.63−3.57 (4H, m), 3.26 (3H, s).
1H NMR (400 MHz, CDCl3): δ 7.39 (2H, m), 7.54−7.15 (5H, m),
6.91−6.87 (2H, m), 5.57 (1H, d, J = 1.8 Hz), 5.48 (1H, d, J = 1.8
Hz), 5.31 (1H, dd, J = 12.2, 18.0 Hz), 3.82−3.76 (7H, m), 3.22−3.17
(4H, m), 3.00−2.89 (2H, m), 2.68 (1H, dd, J = 8.6, 12.9 Hz), 1.99−
1.90 (1H, m), 1.81−1.55 (5H, m), 1.46−1.36 (1H, m), 1.30−1.09
(3H, m).
Step 8: (R)-6-(2-Benzylazepan-1-yl)-4-morpholinopyridin-2(1H)-
one (55). Following Method E from (R)-4-(2-(2-benzylazepan-1-yl)-
6-((4-methoxybenzyl)oxy)pyridin-4-yl)morpholine (31 mg, 0.06
mmol). Purification by silica gel column chromatography (gradient
elution, 0−20% MeOH/EtOAc) yielded the title compound as an off-
white solid (11 mg, 0.03 mmol, 50%).
Step 5: 4-(2-((2R*,3S*)-2-Benzyl-3-methylpyrrolidin-1-yl)-6-((4-
methoxybenzyl)oxy)pyrimidin-4-yl)morpholine. A suspension of 4-
(6-((4-methoxybenzyl)oxy)-2-(methylsulfonyl)pyrimidin-4-yl)-
morpholine (180 mg, 0.48 mmol), (2R*,3S*)-2-benzyl-3-methyl-
pyrrolidine (90 mg, 0.51 mmol), DIPEA (166 μL, 0.95 mmol), and
TBAF hydrate (249 mg, 0.95 mmol) in DMSO (0.95 mL) was stirred
at 80 °C for 17 h. After cooling to RT, the mixture was dissolved in 20
mL of 1:1 DCM/H2O. The layers were separated; the organic layer
dried by passing through a hydrophobic frit and concentrated. The
residue was used without further purification in the next step.
Step 6: 2-((2S,3R)-2-Benzyl-3-methylpyrrolidin-1-yl)-6-morpholi-
nopyrimidin-4(3H)-one (68). Following Method H from 4-(2-
((2R*,3S*)-2-benzyl-3-methylpyrrolidin-1-yl)-6-((4-methoxybenzyl)-
oxy)pyrimidin-4-yl)morpholine (the crude material from the previous
step), TFA (1.9 mL), and DCM (1.9 mL). Purification by silica gel
chromatography (gradient elution, 2−20% MeOH/EtOAc), followed
by chiral SFC, gave the title compound (23 mg, 13% over two steps)
as a white solid, plus its enantiomer. Absolute configuration was
assigned following determination of biological activity, the (2S)
isomer being the more active. LCMS (ES+) 355 (M + H)+, RT 3.18
min (Analytical Method B), RT 2.71 min (Analytical Method SFC4,
LCMS (ES+) 368 (M + H)+, RT 2.91 min (Analytical Method A);
RT 2.02 min (Analytical Method SFC1, YMC amylose-C, 35/65 IPA
1
+ 0.1% DEAISO/CO2); H NMR (400 MHz, CDCl3): δ 7.31−7.21
(3H, m), 7.16−7.12 (2H, m), 5.17 (1H, d, J = 2.0 Hz), 4.89 (1H, d, J
= 2.0 Hz), 3.80−3.75 (6H, m), 3.40−3.35 (1H, m), 3.22−3.18 (4H,
m), 2.99 (1H, dd, J = 11.7, 16.0 Hz), 2.87−2.76 (2H, m), 2.15−2.05
(1H, m), 1.84−1.70 (2H, m), 1.57−1.45 (2H, m), 1.31−1.13 (2H,
m). HRMS (ESI+) (M + H)+: calcd for C22H30N3O2, 368.2338;
found, 368.2346.
2-((2S,3R)-2-Benzyl-3-methylpyrrolidin-1-yl)-6-morpholinopyri-
midin-4(3H)-one (68). Step 1: tert-Butyl (2R*,3S*)-2-Benzyl-3-
methylpyrrolidine-1-carboxylate. Palladium (II) acetate (18 mg,
0.08 mmol, Dpe-phos (86 mg, 0.16 mmol) and sodium tert-butoxide
(884 mg, 9.2 mmol) were placed in a reaction tube sealed, evacuated,
and re-filled with nitrogen (×3). 3-Methylpent-4-en-1-yl)carbamate
(798 mg, 4.0 mmol) was dissolved in dioxane (16 mL) and degassed.
The solution was added to the reaction tube followed by
bromobenzene (0.25 mL, 4.8 mmol), and the reaction heated at
100 °C for 17.5 h. The reaction mixture was cooled to r.t. and diluted
with NH4Cl. The reaction mixture was extracted with EtOAc (×3).
The organic extracts were dried (phase separator) and concentrated
under reduced pressure. The crude product was purified by silica gel
column chromatography (gradient elution, 2−20% EtOAc/iso-
hexane) to afford the title compound. 1H NMR (400 MHz,
CDCl3): δ 7.31−7.13 (5H, m), 3.68−3.36 (2H, m), 3.27−2.98
(2H, m), 2.79−2.57 (1H, m), 2.11−1.76 (2H, m), 1.61−1.46 (9H,
m), 0.95−0.81 (4H, m), rotamers observed.
1
YMC amylose-C + 0.1% DEAISO 25% IPA); H NMR (400 MHz,
DMSO-d6): δ 10.22 (1H, br s), 7.31 (2H, dd, J = 7.4, 7.4 Hz), 7.20
(3H, dd, J = 7.0, 13.2 Hz), 4.81 (1H, s), 3.93 (1H, br d, J = 7.9 Hz),
3.67−3.62 (4H, m), 3.48−3.42 (6H, m), 3.08 (1H, dd, J = 2.6, 13.1
Hz), 2.68 (1H, dd, J = 9.2, 12.9 Hz), 2.08−1.94 (2H, m), 1.51−1.44
(1H, m), 0.80 (3H, d, J = 6.8 Hz). HRMS (ESI+) (M + H)+: calcd for
C20H27N4O2, 355.2134; found, 355.2133.
Enzymatic Assay. Ataxia Telangiectasia-Mutated. Recombi-
nant, full length human FLAG-tagged ATM activity (Eurofins 14−
933) was measured in an ELISA for p53 S15 phosphorylation, in a
384 well V-bottom polypropylene plate (Greiner Bio One 781280).
Reactions contained 0.75 nM ATM, 25 nM full length myc-tagged
p53 (Eurofins 23-034), 10 μM UltraPure ATP (Promega V915B),
and serial dilutions of inhibitors (0.1% DMSO final) in 25 mM
HEPES, pH 7.5. 5 mM MgCl2, 2.5 mM MnCl2, 0.006% Brij-35, 0.5%
glycerol, 0.1 mg/mL BSA, 0.5 mM DTT and were allowed to proceed
for 30 min at 20 °C. A 70 mM EDTA final terminated the kinase
reactions. An aliquot of the terminated reaction (25 μL) was
transferred to a 384-well, high binding microplate (Greiner Bio One
781061), precoated with anti-myc capture antibody overnight
(Millipore 05-724), diluted in 1:1000 in PBS, and then blocked
with Odyssey blocking buffer (LI-COR Biosciences 92740000) for 60
min at 20 °C. The terminated reaction was allowed to capture for 2 h
at 20 °C. Phosphorylation was measured using an antibody specific to
p53 S15 phosphorylation (1:5000 Abcam ab38497), anti-rabbit HRP
(1:1000 Cell Signaling Technology, 7074), and TMB (ab171522).
Antibodies were diluted in Odyssey blocking buffer and incubated for
60 min at 20 °C. Three washes with PBS containing 0.05% (v/v)
Tween 20 (PBS-T) were performed between each incubation. The
TMB reaction was stopped with an equal volume of 0.2 M sulfuric
Step 2: (2R*,3S*)-2-Benzyl-3-methylpyrrolidine. tert-Butyl
(2R*,3S*)-2-benzyl-3-methylpyrrolidine-1-carboxylate (785 mg, 2.85
mmol) was dissolved in 4 M HCl in dioxane (7.1 mL) at r.t. with
stirring. After 2 h, the reaction mixture was concentrated under
reduced pressure. The residue was dissolved in DCM and washed
with saturated NaHCO3 solution. The organic extract was dried
(phase separator) and concentrated under reduced pressure to afford
the title compound. 1H NMR (400 MHz, CDCl3): δ 7.32−7.16 (5H,
m), 3.00−2.79 (3H, m), 2.77−2.68 (1H, m), 2.59 (1H, dd, J = 8.5,
13.3 Hz), 2.08−1.96 (1H, m), 1.76−1.66 (1H, m), 1.42−1.31 (1H,
m), 1.00 (3H, d, J = 6.6 Hz), NH not observed.
Step 3: 4-(6-((4-Methoxybenzyl)oxy)-2-(methylthio)pyrimidin-4-
yl)morpholine. NaH (3.43 g, 85.8 mmol, 60% dispersion in mineral
oil) was suspended in DMF (70 mL) and cooled to 0 °C. 4-Methoxy
benzyl alcohol (6.52 g, 47.2 mmol) in DMF (30 mL) was added
dropwise to the suspension over 15 min. After stirring at 0 °C for 15
min, a solution of 4-(6-chloro-2-(methylthio)pyrimidin-4-yl)-
morpholine (10.54 g, 42.9 mmol, prepared according to WO 2008/
125839) in DMF (90 mL) was added. The reaction was allowed to
warm to r.t. and stirred for 4.5 h. The reaction was cooled to 0 °C and
cautiously quenched with water. The reaction mixture was
concentrated under reduced pressure. The residue was dissolved in
EtOAc and washed with brine and water. The organic extracts were
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acid and absorbance read at 450 nm on the PerkinElmer Envision
within 30 min of terminating the reaction. The percentage of
inhibition was calculated for each concentration of compound using
0.1% DMSO control wells as 0% inhibition and 1 μM KU60019 as
100% inhibition. Ki values represent the mean of at least two
independent experiments.
Inhibition of cellular ATM activity was determined by changes in
etoposide-stimulated KAP1 phosphorylation, based on the work of
Guo et al.20 Phosphorylation of KAP1, ATM, H2AX, and p53 was
evaluated in-house with comparable results, but the greater assay
window was observed with KAP1.
Cryo-preserved U20S cells were plated overnight at 6 k cells/well
in black walled, clear bottomed 384 well plates (Greiner Bio One
781090). After overnight incubation (37 °C, 5% CO2), cells were
incubated with titrations of test compounds (0.3% DMSO final) in
the presence of 100 μM etoposide (Sigma E1383) to stimulate ATM
via induction of double stranded DNA breaks. After 60 min, cells were
fixed using 100% cold methanol and incubated at −20 °C for 10 min.
Fixed cells were washed with PBS containing 0.05% (v/v) Tween 20
(PBS-T) prior to a 60 min blocking step in Odyssey blocking buffer
(LI-COR Biosciences 92740000). Primary antibodies (1:1000
phosphorylated KAP1: Cell Signaling Technology 4127, 1:5000
total KAP1: Cell Signaling Technology 5868) were diluted in Odyssey
blocking buffer and incubated overnight at 4 °C, before addition of
IRDye(R) conjugated secondary antibodies (LI-COR Biosciences
926-32211, LI-COR Biosciences 926-68070) diluted 1:2000 in
Odyssey blocking buffer for 60 min at r.t. and detection by LI-
COR Odyssey (ImageStudio version 2.1.10, focus offset 4 mm,
resolution 169 μm, lowest quality, intensities of 1 and 3 for 700 and
800 channels, respectively). Immunoreactivity was quantified, and the
ratio of phosphorylated KAP1 was calculated by dividing by the total
KAP1 immunoreactivity. The percentage of inhibition was calculated
for each concentration of compound using 100% (100 μM etoposide
+30 μM KU-60019) and 0% effect controls (100 μM etoposide +
DMSO). IC50 values represent the mean of at least two independent
experiments.
inhibition. Ki values represent the mean from at least two independent
experiments.
Activity of DNA-PK (Promega V5811) purified from HeLa cells
was measured using a HTRF assay detecting phosphorylation of
serine 15 on full length p53, the same substrate used for the
biochemical ATM assay. Reactions contained 0.5 units per well DNA-
PK, 25 nM full-length His-tagged p53 (Eurofins 23-034), 2.5 μM ATP
(1× Km, Promega V915B), and serial dilutions of inhibitors in 50
mM HEPES, pH 7.5, 5 mM MgCl2, 0.01% Brij35, 1 mM DTT, and 1
μg/mL sheared calf thymus DNA. After 75 min at 20 °C, the reaction
was terminated by the addition of 0.001 mg/mL final d2-labeled anti-
6HIS and 0.125 mg/mL final cryptate-labeled anti-phospho p53
antibodies in 50 mM HEPES pH 7.5, 5 mM MgCl2, 0.01% Brij-35,
200 mM KF, and 200 mM EDTA. The signals of fluorescence at
665and 620 nm were recorded after 7 h of incubation at 20 °C on the
PerkinElmer EnVision using a 337 nm excitation laser. The
percentage of inhibition was calculated for each concentration of a
compound using 0.1% DMSO control wells as 0% inhibition and 1
μM NU744122 (cat. no. 3712, Tocris) as 100% inhibition. Ki values
represent the mean from at least two independent experiments.
Pharmacokinetics. Animal Studies. All experimental activities
involving animals were carried out in accordance with the Charles
River Animal Welfare and Humane Treatment of Animals policies,
which are consistent with The American Chemical Society
Publications rules and ethical guidelines.
Animal Housing. Male C57BL/6 mice, obtained at between 8 and
12 weeks of age from Charles River UK Ltd., were individually
identified by tail markings and acclimated for at least 8 days prior to
dose administration. Mice were individually housed in suspended
polycarbonate/polypropylene cages with stainless steel wire mesh
tops and solid bottoms. Mice were kept on a 12/12 h light/dark cycle,
except when interrupted for study procedures. Temperature and
humidity were constantly monitored to maintain approximately 20 °C
(ranging from 19 to 23 °C) and humidity of around 55% (ranging
from 40 to 60%) in the animal room. Animals were fed a pellet diet
(RM-1) (Special Diet Services, Witham, Essex, UK) food ad libitum
and had access to water ad libitum.
Vps34, DNA-PK, and ATR. The measurement of cellular ATR
activity was performed using HT29 cells. Cells were seeded at 25 k/
well in black-walled, clear-bottomed 384 well plates (Greiner Bio-One
781,090). After overnight incubation, cells were incubated with
titrations of test compounds (0.3% DMSO final) in the presence of 10
μM gemcitabine (Sigma G6423) to stimulate ATR. After 4 h, cells
were fixed using 3.7% paraformaldehyde for 10 min and permeabilized
using 0.2% Triton-X for 10 min. Fixed and permeabilized cells were
washed with PBS-T prior to a 60 min blocking step in Odyssey
blocking buffer (LI-COR Biosciences 92740000). Primary antibodies
(1:500 phosphorylated Chk1: Cell Signaling Technology 2348,
1:2000 total Chk1: Cell Signaling Technology 2360) were diluted
in Odyssey blocking buffer and incubated overnight at 4 °C, before
the addition of IRDye(R)-conjugated secondary antibodies (LI-COR
Biosciences 926-32211, LI-COR Biosciences 926-68070) diluted
1:1000 in Odyssey blocking buffer for 60 min at r.t. and detection by
LI-COR Odyssey (Image Studio version 2.1.10, focus offset 4 mm,
resolution 169 μm, lowest quality, intensity of 5 for both channels).
Immunoreactivity was quantified, and the ratio of phosphorylated
Chk1 was calculated by dividing the total Chk1 immunoreactivity.
The percentage of inhibition was calculated for each concentration of
the compound using 100% (10 μM gemcitabine +30 μM AZ-20) and
0% effect controls (100 μM gemcitabine + DMSO). IC50 values
represent the mean from at least two independent experiments.
Recombinant, full-length baculovirus-produced Vps34 activity (Life
Technologies PV5126) was measured using the ADP-Glo kinase assay
as per the manufacturer’s instructions. Reaction contained 4 nM
Vps34, 50 μM PI/PS (PV5122), and 30 μM ATP (1× Km, Promega
V915B) and serial dilutions of inhibitors in 50 mM HEPES, pH 7.5, 5
mM MnCl2, 0.1% CHAPS, and 2 mM DTT and were allowed to
proceed for 60 min at 20 °C. Luminescence was measured using the
PerkinElmer EnVision. The percentage of inhibition was calculated
for each concentration of the compound using 0.1% DMSO control
well as 0% inhibition and 1 μM SAR405 (533,063, Merck)21 as 100%
Preparation of Dose Formulations. Compounds 52, 55, and 68
were dosed intravenously (IV) as a 0.4 mg/mL solution in 10:40:50
DMSO/PEG414/H2O, by volume. The intravenous formulation was
administered as a slow bolus (ca. 15 s) via the tail vein at a target dose
volume of 5 mL/kg to achieve a target dose level of 2 mg/kg, except
compound 55, which was dosed at 2 mL/kg giving 0.8 mg/kg.
Compound 55 was gavage-dosed orally (PO) as a solution as 10 mL/
kg of either 1 or 3 mg/mL in 10% hydroxypropyl-beta-cyclodextrin
(10% HP-β-CD). Compound 68 was dosed orally as a 10 mL/kg
gavage of a 1 mg/mL solution in 10% HP-β-CD targeting 10 mg/kg.
Dose formulation aliquots (100 μL; n = 3 per formulation) were
collected and submitted to bioanalysis for quantification of analyte
concentrations in the administered doses.
Sample Collection. Blood for plasma and brain tissues (n = 3 per
time-point and dose group) were collected at selected time-points
post-dose. Terminal blood samples were collected via orbital sinus at
each time point into tubes containing Na heparin held on wet ice (no
longer than 30 min) and centrifuged at 2200×g for 10 min at 5 °C
3 °C to separate the plasma. Plasma samples were transferred to
micronic tubes and stored at approximately −80 °C until bioanalysis.
Immediately after collection, each brain was rinsed with saline, snap
frozen in liquid nitrogen, and stored individually at approximately
−80 °C.
Sample Processing for Bioanalysis. At the time of bioanalysis,
each brain was homogenized in water (3:1, v/v) using a Precellys
tissue homogenizer resulting in a brain/solvent ratio of 1:3 (w/v) or
1: 2.536 (w/w).
Plasma (50 μL) and brain homogenate (50 μL) from study
samples, controls, and blanks were dispensed into 96-well plates.
Extracting solution (200 μL) consisting of 0.1% formic acid in
acetonitrile containing 200 ng/mL diclofenac and 200 ng/mL
reserpine as the internal standards (IS) was added to all samples
except to matrix blanks and solvent blanks, followed by vortexing and
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centrifugation (30 min). Supernatants (100 μL) were transferred to a
new plate; an aliquot (200 μL) of Milli-Q water was added to the
samples, covered, and vortexed for 5 min prior to liquid
chromatography mass spectrometry (LC−MS/MS) analysis.
Bioanalytical Methods. Concentrations in mouse plasma and
brain were determined using LC−MS/MS assays developed at
Charles River UK. Reverse-phase separation was performed in a
Waters Acquity UPLC with a UPLC Kinetix XB-C18 100A column
(50 × 2.1 mm, 2.6 μm) using a mobile phase gradient consisting of
0.01% formic acid (v/v) in milliQ water and 0.01% formic acid (v/v)
in acetonitrile, except for compound 55, which utilized a mobile phase
gradient consisting of 2.5 mM ammonium formate in milliQ water
and methanol.
The entire LC eluent was directly introduced to an electrospray
ionization (ESI) source operating in the positive ion mode for LC
MS/MS analysis on a Xevo triple quadrupole mass spectrometer with
a source temperature of 150 °C and a desolvation temperature of 500
°C. The mass spectrometer ion optics were set in the multiple
reaction monitoring (MRM) mode. The data were processed using
QuanLynx software from Waters.
Calibration standards were prepared in duplicate at each
concentration. At a minimum, 75% of all the calibration standards
and at least two calibration standards per concentration met the
accuracy and precision of 30%. The coefficient of variation (CV%)
of the IS signal/area response for the entire run was within 30%.
There was no bias in the accuracy or precision of the calibration
standards and no bias or trend in the IS signal/area response for the
run to be acceptable. The mean concentrations of the calibration
standards at the front end versus those at the back end of calibration
curve were within 15% of each other.
The respective assay lower limits of quantitation in plasma and
brain homogenate were 14 and 11 nM for compound 55, 7 and 11
nM for compound 52, and 1.4 and 7 nM for compound 68.
Pharmacokinetic Analysis. Composite noncompartmental phar-
macokinetic parameters were calculated from the mean concen-
trations (n = 3) obtained for each time point using Phoenix
WinNonlin, version 5.2.1. For the IV dose, the plasma concentration
at Time = 0 was back extrapolated from the first two post-dose plasma
concentrations. For the PO dose, the concentration at Time = 0 was
assumed to be zero. Plasma and tissue concentrations below the lower
limit of quantification were treated as absent samples.
The area under the concentration versus time curve (AUC) was
calculated using the linear trapezoidal method. When appropriate, the
elimination rate constant (kel) was estimated using at least the last
three observed concentrations. The portion of the AUC from Ct to
infinity (AUClast-inf) was extrapolated from the ratio of Ct/kel where
Ct represents the last measurable concentration. The AUCinf was
calculated as AUClast + AUClast-inf. The AUC determined were
normalized to the actual dose administered. The oral bioavailability
(%F) was calculated by dividing the dose-normalized PO AUCinf
over the dose-normalized IV AUCinf.
In Vitro ADME Assays. Kinetic Solubility Assay. Kinetic solubility
assays were performed, as described in the Charles River Standard
Operating Procedure. Using a 10 mM stock solution of each test and
control compound (hydrocortisone, reserpine) in 100% DMSO,
dilutions were prepared to a theoretical concentration of 200 μM in
both 0.1 M potassium phosphate buffer containing 0.8% NaCl (PBS),
pH 7.4 (2% DMSO final), and in 100% DMSO. An aliquot of the 200
μM DMSO solution was then further diluted to 10 μM, and all
dilutions (n = 2, in 96-well plates) allowed to equilibrate at room
temperature on an orbital shaker for 2 h. The PBS dilutions were
filtered using a MultiScreen HTS solubility filter plate (Millipore),
and the filtrate was analyzed by LC-UV with confirmation of the peak
of interest by mass spectrometry. The concentration of the compound
in PBS filtrate was determined by comparing the UV absorbance peak
with that of the two DMSO dilutions as calibration standards.
Liver Microsomal Stability. Incubations of test compound (1 μM,
n = 2, 37 °C) in pooled liver microsomes (0.25 mg protein/mL in 0.1
M phosphate buffer pH 7.4) were initiated with the addition of
NADPH (1 mM). Samples (100 μL) were obtained at 0, 5, 10, 20,
and 40 min and added to 100 μL of acetonitrile containing
carbamazepine as analytical IS and centrifuged, and the supernatants
were analyzed by LC MS/MS.
Permeability and EER in MDCK/MDR1. MDCKII (MDR1 and
WT) cell lines were cultured in 24-well Transwell plates (Corning,
catalogue number 3397), following the guidelines provided by
SOLVO Biotechnology (Budapest, Hungary). The seeding density
was 2 × 105 cells/well with the culture period of 3 days. Test
compounds (10 μM) were dissolved in Hanks’ balanced salt solution
containing 25 mM HEPES (pH 7.4) and added to either the apical or
basolateral chambers of the Transwell plate assembly in duplicate.
Lucifer yellow was added to the apical buffer in all wells to assess the
integrity of cell layers; wells with Lucifer yellow permeability above
100 nm/s were rejected. After a 1 h incubation at 37 °C, aliquots were
taken from both chambers of each Transwell and added to acetonitrile
containing IS (carbamazepine). Analyte concentrations were meas-
ured by LC−MS/MS.
The apparent permeability (Papp) values of a test compound were
determined for both apical to basal (A > B) and basal to apical (B >
A) permeation, and the ER (B > A/A > B) was determined in both
wild-type MDCK and MDR1/MDCK cells.
Apparent permeability (Papp) values were calculated from the
relationship
P (cm/s × 10−6)
app
Ä
Å
Å
Å
Å
Å
Å
É
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
compoundacceptor final × V
× V
acceptor
donor
−6
=
× 10
Å
Ñ
Å
Ñ
Åcompound
× V
× T × surface area Ñ
Å
donor
inc
Ñ
donor initial
Å
Ç
Ñ
Ö
where V is the chamber volume and Tinc is the incubation time in
seconds. The donor is the chamber of Transwell to which the
compound is dosed: apical for A > B experiments and basal for B > A
experiments. The acceptor is the chamber of Transwell to which
permeation of compound is measured: basal for A > B experiments
and apical for B > A experiments. The ERs, as an indication of active
efflux from the apical cell surface, were calculated using the ratio of
Papp B > A/Papp A > B.
The EER was also determined from the ratio observed in MDCK/
MDR1 cells relative to the ratio observed in wild-type cells.
Brain Tissue Binding. Brain tissue binding was determined in vitro
by equilibrium dialysis. Briefly, compounds were added (5 μM, 0.5%
DMSO final) to brain homogenate (n = 2) prepared from control
mouse (C57/Bl6) brains and dialyzed against 0.01 M phosphate
buffered saline (pH 7.4) for 6 h at 37 °C. After incubation, the
contents of each brain homogenate and buffer compartment were
removed and mixed with equal volumes of control buffer or brain
homogenate as appropriate to maintain matrix equivalence for
analysis. Proteins were then precipitated by the addition of
acetonitrile containing carbamazepine as IS and centrifuged, and the
supernatant was removed for analysis by LC−MS/MS. The
percentage of drug bound was determined using the following
relationship, where total brain homogenate concentration and free
concentration are the MS responses obtained from the analysis of the
brain homogenate and buffer compartments, respectively.
fraction bound
total brain homogenate concentration − free concentration
=
total brain homogenate concentration
Fraction unbound in the homogenate and then the fraction
unbound in the brain were derived using the following equations
free freaction in homogenate (fu hom ogenate)
= 1 − fraction bound
free fraction in brain (fu brain)
1
D
× 100
=
1
D
((1/fu hom ogenate) − 1) +
5033
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
where D is the dilution factor during homogenization.
Graham Jones − Charles River, Saffron Walden CB10 1XL,
Plasma Protein Binding. Test and control compounds (warfarin,
metoprolol, propranolol, and nicardipine), prepared in DMSO, were
U.K.
George McAllister − Charles River, Saffron Walden CB10
1XL, U.K.
̈
added (10 μM, 1% DMSO final) to naive plasma (C57BL/6 plasma
on lithium heparin as anticoagulant) supplied by Marshall
Bioresources, Grimston, HU11 4QE, and dialyzed (n = 2) against
1× phosphate buffered saline (PBS), pH 7.4 for 4 h at 37 °C using a
Rapid Equilibrium Device (RED, ThermoFisher Scientific). After
incubation, the contents of each plasma and buffer compartment were
removed and mixed with a balancing volume of control dialyzed
buffer or plasma as appropriate to maintain matrix similarity for
analysis. Plasma proteins were then precipitated by the addition of
acetonitrile containing an analytical internal standard and centrifuged,
and the supernatant was removed for analysis by mass spectrometry
(LC−MS/MS). The fraction of drug unbound (Fu) was determined
using the following relationship, where total plasma concentration and
free concentration are the MS responses (normalized for internal
standard) obtained from analysis of the plasma and buffer
compartments, respectively.
Huw Vater − Charles River, Saffron Walden CB10 1XL,
U.K.; orcid.org/0000-0002-2904-1848
William Esmieu − Charles River, Saffron Walden CB10 1XL,
U.K.
Cole Clissold − Charles River, Saffron Walden CB10 1XL,
U.K.
Marieke Lamers − Charles River, Saffron Walden CB10 1XL,
U.K.
Philip Leonard − Charles River, Saffron Walden CB10 1XL,
U.K.
Rebecca E. Jarvis − Charles River, Saffron Walden CB10
1XL, U.K.
Wesley Blackaby − Charles River, Saffron Walden CB10 1XL,
U.K.
Maria Eznarriaga − Charles River, Saffron Walden CB10
1XL, U.K.
(total plasma concentration − free concentration)
Fu = 100 −
total plasma concentration
Ovadia Lazari − Charles River, Saffron Walden CB10 1XL,
U.K.
× 100
Dawn Yates − Charles River, Saffron Walden CB10 1XL, U.K.
Mark Rose − CHDI Management/CHDI Foundation, Los
Angeles, California 90045, United States
Sung-Wook Jang − CHDI Management/CHDI Foundation,
Los Angeles, California 90045, United States
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00114.
■
sı
Ignacio Munoz-Sanjuan − CHDI Management/CHDI
̃
Additional synthetic methods, HPLC chromatograms,
biological data, mouse PK parameters for compound 55,
and Vps34 ATM surrogate crystal analysis details (PDF)
SMILES molecular formula strings (CSV)
Foundation, Los Angeles, California 90045, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00114
AUTHOR INFORMATION
Corresponding Author
Celia Dominguez − CHDI Management/CHDI Foundation,
Los Angeles, California 90045, United States;
Email: celia.dominguez@chdifoundation.org
Author Contributions
■
§A.V.D.P., L.T.S., and P.B. are equal contributors to this
publication.
Notes
The authors declare no competing financial interest.
Coordinates for the crystal structure of compound 44 have
been deposited in the Protein Data Bank with a pdb code 6ykg.
Authors will release the atomic coordinates and experimental
data upon article publication.
Authors
Amanda Van de Poël − Charles River, Saffron Walden CB10
1XL, U.K.
Leticia Toledo-Sherman − CHDI Management/CHDI
Foundation, Los Angeles, California 90045, United States
Perla Breccia − Charles River, Saffron Walden CB10 1XL,
U.K.; orcid.org/0000-0002-8552-9012
Roger Cachope − CHDI Management/CHDI Foundation,
Los Angeles, California 90045, United States
Jennifer R. Bate − Charles River, Saffron Walden CB10 1XL,
U.K.
Ivan Angulo-Herrera − Charles River, Saffron Walden CB10
1XL, U.K.
Grant Wishart − Charles River, Saffron Walden CB10 1XL,
U.K.
Kim L. Matthews − Charles River, Saffron Walden CB10
1XL, U.K.
Sarah L. Martin − Charles River, Saffron Walden CB10 1XL,
U.K.
Marcus Peacock − Charles River, Saffron Walden CB10 1XL,
U.K.
Amy Barnard − Charles River, Saffron Walden CB10 1XL,
U.K.
Helen C. Cox − Charles River, Saffron Walden CB10 1XL,
U.K.
ABBREVIATIONS USED
■
ATM, ataxia telangiectasia-mutated; ATR, ataxia- and rad3-
related; AUC, area under the curve; AUCnorm, normalized area
under the curve; BINAP, 2,2′-bis(diphenylphosphino)-1,1′-
binaphthyl; biochem, biochemical; Clint, intrinsic clearance;
CLp, plasma clearance; DIPEA, diisopropylethylamine; DNA-
PK, DNA-dependent protein kinase; dtbpf, 1,1′-bis(di-tert-
butylphosphino)ferrocene; EER, effective efflux ratio; H2AX,
histone 2AX; HD, Huntington’s disease; HTT, huntingtin
gene; KAP1, KRAB-associated protein 1; Kp, partition
coefficient; Kpuu, unbound partition coefficient; MDCK,
Madin−Darby canine kidney; mHTT, mutant huntingtin;
MLM, mouse liver microsomes; mTOR, mammalian target of
rapamycin; n.d., not determined; Papp, apparent permeability
coefficient; PIKK, phosphatidyl inositol-3-kinase-like kinase;
PoC, proof of concept; RuPhos, 2-dicyclohexylphosphino-
2′,6′-diisopropoxybiphenyl; RuPhos Pd G1, palladium(II)
phenethylamine chloride (1:1 MTBE solvate), chloro-(2-
dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2-
aminoethyl)phenyl]palladium(II)-methyl-t-butyl ether adduct;
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barrier-penetrating ATM inhibitor (AZ32) radiosensitizes intracranial
gliomas in mice. Mol. Cancer Ther. 2018, 17, 1637−1647.
SFC, supercritical fluid chromatography; SNAr, nucleophilic
aromatic substitution; TCR, transcription-coupled DNA
repair; TPSA, topological polar surface area; Vdss, volume of
distribution at steady state; Vps34, vacuolar protein sorting 34
(9) (a) Toledo-Sherman, L.; Breccia, P.; Cachope, R.; Bate, J. R.;
Angulo-Herrera, I.; Wishart, G.; Matthews, K. L.; Martin, S. L.; Cox,
H. C.; McAllister, G.; Penrose, S. D.; Vater, H.; Esmieu, W.; Van de
Poël, A.; Van de Bospoort, R.; Strijbosch, A.; Lamers, M.; Leonard, P.;
Jarvis, R. E.; Blackaby, W.; Barnes, K.; Eznarriaga, M.; Dowler, S.;
Smith, G. D.; Fischer, D. F.; Lazari, O.; Yates, D.; Rose, M.; Jang, S.-
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