Discovery of 4-{4-[(3R)-3-methylmorpholin-4-yl]-6-[1-(methylsulfonyl) cyclopropyl]pyrimidin-2-yl}-1H-indole (AZ20): A potent and selective inhibitor of ATR protein kinase with monotherapy in vivo antitumor activity

Article abstract of DOI:10.1021/jm301859s

ATR is an attractive new anticancer drug target whose inhibitors have potential as chemo- or radiation sensitizers or as monotherapy in tumors addicted to particular DNA-repair pathways. We describe the discovery and synthesis of a series of sulfonylmorpholinopyrimidines that show potent and selective ATR inhibition. Optimization from a high quality screening hit within tight SAR space led to compound 6 (AZ20) which inhibits ATR immunoprecipitated from HeLa nuclear extracts with an IC₅₀ of 5 nM and ATR mediated phosphorylation of Chk1 in HT29 colorectal adenocarcinoma tumor cells with an IC₅₀ of 50 nM. Compound 6 potently inhibits the growth of LoVo colorectal adenocarcinoma tumor cells in vitro and has high free exposure in mouse following moderate oral doses. At well tolerated doses 6 leads to significant growth inhibition of LoVo xenografts grown in nude mice. Compound 6 is a useful compound to explore ATR pharmacology in vivo.

Full text of DOI:10.1021/jm301859s

Article
pubs.acs.org/jmc
Discovery of 4‑{4-[(3R)‑3-Methylmorpholin-4-yl]-6-[1-
(methylsulfonyl)cyclopropyl]pyrimidin-2-yl}‑1H‑indole (AZ20): A
Potent and Selective Inhibitor of ATR Protein Kinase with
Monotherapy in Vivo Antitumor Activity
Kevin M. Foote,*,† Kevin Blades,† Anna Cronin,† Shaun Fillery,† Sylvie S. Guichard,† Lorraine Hassall,†
Ian Hickson,‡ Xavier Jacq,‡ Philip J. Jewsbury,† Thomas M. McGuire,† J. Willem M. Nissink,†
Rajesh Odedra,† Ken Page,† Paula Perkins,† Abid Suleman,† Kin Tam,† Pia Thommes,†
Rebecca Broadhurst,† and Christine Wood†
†AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K.
‡KuDOS Pharmaceuticals, Cambridge Science Park, Milton Road, Cambridge, U.K.
S
* Supporting Information
ABSTRACT: ATR is an attractive new anticancer drug target
whose inhibitors have potential as chemo- or radiation
sensitizers or as monotherapy in tumors addicted to particular
DNA-repair pathways. We describe the discovery and synthesis
of a series of sulfonylmorpholinopyrimidines that show potent
and selective ATR inhibition. Optimization from a high quality
screening hit within tight SAR space led to compound 6
(AZ20) which inhibits ATR immunoprecipitated from HeLa
nuclear extracts with an IC50 of 5 nM and ATR mediated
phosphorylation of Chk1 in HT29 colorectal adenocarcinoma
tumor cells with an IC50 of 50 nM. Compound 6 potently inhibits the growth of LoVo colorectal adenocarcinoma tumor cells in
vitro and has high free exposure in mouse following moderate oral doses. At well tolerated doses 6 leads to significant growth
inhibition of LoVo xenografts grown in nude mice. Compound 6 is a useful compound to explore ATR pharmacology in vivo.
for broad utility in cancer patients as monotherapy or in
combination with chemo- or radiotherapy.
INTRODUCTION
■
Ataxia telangiectasia mutated and RAD3-related (ATR) is a
serine/threonine protein kinase that, together with ATM and
DNA-PK, forms part of the DNA-damage response (DDR)
coordinating the cellular response to DNA damage, stress, and
cell-cycle perturbation.1 ATR is essential to the viability of
replicating cells responding to accumulation of single strand
breaks (SSB) in DNA such as stalled replication forks and to
bulky DNA damage lesions such as those formed by
chemotherapeutics and ultraviolet (UV) radiation.2−4 Sensitiza-
tion of tumor cells to chemotherapeutic agents has been
demonstrated following genetic modulation of ATR activity,5
with weak ATR inhibitors such as caffeine,6,7 and more recently
with potent and selective ATR inhibitors such as VE-821.8,9
These studies suggest that combination of ATR inhibitors with
some cytotoxic agents may be therapeutically beneficial. A
further compelling utility is aligned with the concept of
synthetic lethality.10 Tumor cells that are deficient in G1
checkpoint controls, in particular p-53, or with other mutations
that promote replicative stress, are hypothesized to be more
reliant on ATR for survival.11 In such circumstances, specific
inhibition of ATR may lead to enhanced antitumor activity
while minimizing normal tissue toxicity. In summary, ATR is an
important new drug target whose inhibitors have the potential
ATR, ATM, and DNA-PK together with three other proteins
mTOR, hSMG-1, and TRRAP are members of an atypical class
of protein kinases known collectively as the phosphatidylino-
sitol 3-kinase related kinase (PIKK) family. These proteins are
structurally different from classical protein kinases and are more
closely related to the PI3-kinase family of phospholipid
kinases.12,13 A number of compounds are known to inhibit
the PIKK family, which have recently been reviewed else-
where.13 One of the first potent PIKK-family inhibitors to be
described was 1 (LY294002, Figure 1). Compound 1 inhibits
PI3K, mTOR, and DNA-PK and was shown, through X-ray
crystallography in complex with PI3Kγ, to make a crucial
hydrogen bond interaction to the ATP-binding domain through
the morpholine oxygen.14 Identification of the morpholine
hinge binding motif has led to a number of other inhibitors of
the PIKK family including ATM, DNA-PK, PI3K, and mTOR,
some of which have reached human trials.15 One such
compound is the pan-PI3K inhibitor 2 (GDC-0941), which is
currently in phase II trials for the treatment of non-small-cell
Received: December 17, 2012
Published: February 11, 2013
© 2013 American Chemical Society
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Figure 1. Example PIKK family and ATR inhibitors.
lung cancer.15a However, there are few reports describing
potent and selective inhibitors of ATR. Compound 3 (NVP-
BEZ235), originally described as a dual PI3K and mTOR
inhibitor,16 has also been shown to inhibit DNA-PK, ATM, and
ATR.17,18 Recently, however, scientists at Vertex have described
a series 3-amino-6-arylpyrazines such as 4 (VE-821).8,9
Compound 4 is a potent and selective inhibitor of ATR
which potentiates the in vitro cytotoxicity of ionizing radiation
(IR) and cisplatin in HCT116 colorectal cancer cells.
Scheme 1
a
Despite the recently described advances in the identification
of potent ATR inhibitors such as 4, a need exists to identify
inhibitors suitable for exploring the complex biology of ATR-
mediated DNA repair in vivo and of high enough quality to
enter human clinical trials. In this report, we describe our early
discovery efforts starting from the mTOR-derived screening hit
5,15d which led to the discovery of compound 6 (AZ20).19
Compound 6 shows monotherapy in vivo antitumor efficacy in
LoVo colorectal xenografts in nude mice and is a useful
compound for further studies.
CHEMISTRY
■
Compounds were prepared as shown in Schemes 1−4. Test
compounds were synthesized via Suzuki cross-coupling either
from 2-chloropyrimidine intermediates (Scheme 2, compounds
62 and 66−78) or from 2-thiomethyl substituted pyrimidines
(Scheme 3, compounds 84 and 85) with the appropriate
arylboronic acid or ester.20 The aryl boronate coupling partners
were either commercially available or made from the
corresponding aryl bromide or triflate using standard literature
methods.21 The iodomethyl compounds 50−52 (Scheme 1)
proved to be versatile intermediates allowing variation of the
side chain sulfone group and the aryl substituent at position 2
of the pyrimidine ring. Compounds 50−52 were prepared
starting with a careful substitution of the 4-chloro leaving group
in 43 with the appropriate morpholine followed by full
reduction of the ester to give the alcohols 47−49 and finally
iodination. The methylene sulfone unit was elaborated either by
direct reaction of the iodomethyl group in 50−52 with a
sulfinic acid to give 56−59 or via the thioethers 64 and 65
followed by oxidation to sulfones 60 and 61, respectively
(Scheme 2). Alternatively, the methylene sulfone moiety can be
a
Reagents: (a) Et3N, DCM, 0 °C → rt; (b) LiBH4, THF, 0 °C → rt;
(c) MeSO2Cl, Et3N, DCM, 0 °C → rt; LiI, acetone, rt or dioxane, 100
°C.
incorporated before the morpholine; compounds 54 and 55
were made starting from the pyrimidinedione 53 by displace-
ment of the chloro leaving group either directly with
methanesulfinate to give 54 or first with isopropanethiol
followed by full chlorination and then oxidation of sulfide to
sulfone to give 55. Again, careful reaction of the 4-chloro
leaving group with morpholine led to 62 and 63. Alkylation of
the methylene unit in intermediates 56−63 was carried out
using methods such as sodium tert-butoxide and MeI in DMF
to give 66 or by phase transfer catalysis using TBAB and
concentrated NaOH solution in toluene with the appropriate
alkylating agent to give the cyclic precursors 67, 68, and 71−78
(Scheme 2); standard functional group manipulation of the
benzyl protecting group in 68 gave 69 and 70. The 2-SMe
substituted pyrimidine precursors 84 and 85 were made from
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a
Scheme 2
a
Reagents: (a) compound 54: NaOS(O)Me, DMF, 125 °C, POCl3, reflux; compound 55: isopropanethiol, DBU, MeCN, rt, POCl3, reflux, mCPBA,
DCM, rt; (b) Et3N, DCM, morpholine, 0 °C → rt; (c) compounds 56 and 57: NaOS(O)Me, DMF, rt; compound 58: NaOS(O)Ph, MeCN, 80 °C;
compound 59: sodium 1-Z-piperidin-4-ylsulfinate, DMF, rt; (d) N,N-Diisopropylethylamine, R2SH, DMF or MeCN, rt; (e) mCPBA, DCM, rt; (f)
compound 66: MeI, NaO-t-Bu, DMF, 0 °C → rt; compound 67: 1-bromo-2-(2-bromoethoxy)ethane, 50% aq NaOH, TBAB, DCM, rt; compound
68: N-benzyl-2-chloro-N-(2-chloroethyl)ethanamine hydrochloride, NaH, TBAB, NMP, rt → 80 °C; compounds 71−78: 50% aq NaOH, 1,2-
dibromoethane, TBAB, toluene, 60 °C or DCM, 30 °C; (g) 1-Chloroethylcarbonochloridate, DCM, rt → reflux, MeOH, di-tert-butyl dicarbonate,
N,N-diisopropylethylamine, rt; (h) 1-chloroethylcarbonochloridate, DCM, rt → reflux; NaBH(OAc)3, aq HCHO, MeCO2H, DCM, rt; (i)
Pd2(Ph3P)2Cl2, aq Na2CO3, indole boronic acid/ester, 18% DMF in 7:3:2 DME/water/EtOH or 4:1 DME/water, microwave/Δ, 90−110 °C;
Pd2(dba)3, tricyclohexylphosphine, KOAc, bis(pinacolato)diboron, bromoindole, dioxane, 100 °C followed by substrate, Pd(Ph3P)4, aq Na2CO3, 100
°C; 1,1′-bis(diphenylphosphine)ferrocenePdCl2, indole triflate, bis(pinacolato)diboron, KOAc, dioxane, 90 °C followed by substrate, Pd(Ph3P)4, aq
Na2CO3, 90 °C.
an analogous cyclopropanation procedure from the methylene
sulfones 82 and 83 (Scheme 3). Intermediates 82 and 83 were
constructed either from 8115d or by reaction of symmetrical
dichloropyrimidine 79 with methyl 2-(methylsulfonyl)acetate
to give 80 followed by morpholine displacement and
decarboxylation. Finally, compounds 31, 32, 34−36 were
prepared by standard functional group manipulation starting
from the esters 86 and 88 (Scheme 4).
Modest ATR potency was observed across various structural
classes of compounds, but a single compound stood out in
terms of both potency and selectivity for ATR. Compound 5 is
a sulfonylmorpholinopyrimidine from a previously described
series inhibiting mammalian target of rapamycin (mTOR) from
AstraZeneca15d and exhibited high ATR enzyme potency and
ligand efficiency (LE) (Table 1). Compound 5 is moderately
lipophilic (log D7.4 = 1.7) and shows high selectivity against
other PIKK and PI3K enzymes. Moreover, compound 5
inhibited ATR-driven phosphorylation of Chk1 at serine-345, a
downstream substrate of ATR, following addition of 4-
nitroquinoline 1-oxide (4NQO mimics DNA damage caused
by UV radiation).22 ATR cellular IC50 was established in an
ArrayScan format assay in HT29 colorectal adenocarcinoma
cells. The IC50 of 5 was 1.1 μM in this assay, which compared
favorably with inhibition of mTOR/PI3K-driven phosphor-
ylation of AKT at serine-473 measured in MDA-MB-468 cells
(Table 1). Given this profile, compound 5 was an excellent start
point from which to develop more potent ATR inhibitors.
On the basis of knowledge from known PIKK inhibitors such
as 1, compound 5 was likely to bind to ATR through the weak
morpholine oxygen hydrogen bond acceptor.14,15 This
RESULTS AND DISCUSSION
■
A directed subset of the AstraZeneca compound collection was
screened against ATR in single-shot format at fixed
concentrations of 1, 10, and/or 30 μM. As follow-up, the set
of actives were filtered stringently based on consensus of
inhibition data at the different concentrations and prioritized
further using ligand efficiency and, where available, PIKK-family
selectivity information. Both screening and dose response
follow-up were performed in an ELISA format utilizing ATR
immunoprecipitated from HeLa nuclear extracts. A further
selection of actives from the screen was assessed for cellular
inhibition of ATR kinase activity.
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a
Scheme 3
Table 1. PIKK-Family Enzyme and Cell Inhibitory Potency
for Screening Hit 5
ATR
mTOR DNA-PK
PI3Kα
ATR cell
mTOR
IC
ATR
b
IC50
IC
IC50
IC50
cell IC
a5e0
,
50a
ac
,
50a
ac
,
ad
,
(μM)
LE
(μM)
(μM)
(μM)
(μM)
(μM)
0.030
0.29
0.33
2.7
3.8
1.1
5.9
a
Standard error of the mean (SEM) pIC50 measurement is ≤0.13 from
b
at least two repeat measurements per test. Ligand efficiency (LE):
ATR enzyme pIC50/heavy (non-hydrogen) atom count. IC50 derived
c
following acoustic dispensing of compound 5 using a Labcyte Echo
d
550. Inhibition of pChk1 Ser-345 in HT29 cells exposed to 3 μM
e
4NQO in the presence of compound 5 for 1 h. Inhibition of pAKT
Ser-473 in MDA-MB-468 cells.
activity. The modeled binding mode of compound 5 in an ATR
protein homology model based on the PI3Kγ-isoform offered a
tentative explanation of the selectivity for ATR, although a full
explanation of selectivity across the PI3K and PIKKs remains
elusive (Figure 2). The indole N−H group in 5 likely forms a
a
Reagents: (a) methyl 2-(methylsulfonyl)acetate, NaH, DMF, rt; (b)
morpholine, N,N-diisopropylethylamine, DMF, 80 °C; (c) NaOH,
MeOH, H2O, 60 °C; 2 M HCl; (d) morpholine, N,N-diisopropylethyl-
amine, DCM, rt; (e) NaH, 1,2-dibromoethane, DMF, rt or 50% aq
NaOH, 1,2-dibromoethane, TBAB, toluene, 60 °C; (f) Pd2(dba)3 or
Pd(Ph3P)4, 1H-indol-4-ylboronic acid, trifuran-2-ylphosphine, (thio-
phene-2-carbonyloxy)copper, THF or dioxane, 80 °C.
Figure 2. ATR homology model built from an in-house PI3Kγ
structure. The binding mode of compound 5 was modeled starting
from the morpholinopyrimidine placement observed for compound 2
(PDB structure 3DBS, PI3Kγ).
a
water-mediated hydrogen bond to Asp2335. Although it is
positioned in the binding pocket in a largely similar fashion to
PI3K and PIKK targets, we hypothesize that ATR inhibitors
from this sulfonylmorpholinopyrimidine series bind slightly
deeper into the back pocket. Modeling of the P-loop of the
ATR protein suggests further that this region differs from PI3K
isoforms, and this may explain in part the selectivity of ATR
inhibitors with regard to these targets. Inspection of the model
highlighted space for substitution around the methylsulfone
region to explore by further compound synthesis, though it was
less clear what the impact of such changes would be on
selectivity.
Scheme 4
Both the presence and position of the indole N−H
hydrogen-bond donor group were found to be critical for
inhibition of ATR. We were able to test some of the other
positional isomers of the indole in 5 with compounds from the
AstraZeneca compound collection (Table 2, compounds 7 and
8). In contrast to mTOR, which favors the 5-indole isomer,15d
ATR has a clear preference for the 4-substituted indole
compared with either of the 5- or 6-indole isomers. We also
synthesized the 7-indole analogue 9, which we found to be of
intermediary potency presumably by virtue of being able to
adopt a similar conformation and have shape complementarity
a
Reagents: (a) LiOH.H2O, THF, H2O, rt → 70 °C; (b) HATU, N,N-
diisopropylethylamine, DMF, rt; NH4Cl, rt; (c) trifluoroacetic
anhydride, pyridine, dioxane, 0 °C → rt; (d) HATU, N,N-
diisopropylethylamine, DMF, rt; amine, rt.
hypothesis was supported by attempts to substitute the
morpholine for other saturated six-membered-ring heterocyclic
groups lacking the hydrogen-bond acceptor oxygen, e.g.,
piperidine, which resulted in the loss of kinase inhibitory
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Table 2. Structures and ATR Inhibitory Potency of
Compounds 7−10 Compared with 5
Table 3. Structures and ATR Inhibitory Potency of
Compounds 11−15 Compared with 5
a
Uncertainty (95% confidence) for pIC50 measurements is 0.38 (2.4-
fold) based on an average of two repeat measurements per compound;
b
compound 7, N = 1. Uncertainty (95% confidence) for pIC50
measurement is 0.45 (2.8-fold) based on an average of two repeat
occasions per compound.
a
Uncertainty (95% confidence) for pIC50 measurements is 0.38 (2.4-
fold) based on an average of two repeat occasions per compound.
b
Uncertainty (95% confidence) for pIC50 measurement is 0.45 (2.8-
with 5 but able only to present a C−H group in place of an N−
H hydrogen-bond donor contact. N-Methylation of the indole
N−H in 5 further confirmed the importance of this group and
resulted in near-ablation of ATR potency (compound 10).
Alkylation of the methylene unit in the sulfone side chain led
to an increase in ATR potency (Table 3). Acyclic groups such
as the gem-dimethyl 11 and three- to six-membered rings (e.g.,
cyclopropyl 12, tetrahydropyran 13, and piperidine 14) were
constructed, and in each case the increase in ATR enzyme
potency was found to mirror an increase in cellular potency.
The gem-dimethyl, cyclopropyl, and tetrahydropyran linkers
increase potency with a simultaneous increase in lipophilicity
compared with 5. We favor ligand efficiency (LE), calculated as
enzyme pIC50/number of heavy (non-hydrogen) atoms, and
ligand lipophilicity efficiency (LLE), calculated as cellular pIC50
− log D7.4, indices to guide quality in lead discovery and
optimization.23−26 Both indices have the advantage of being
simple to calculate, but of greater significance is LLE being a
balance of two of the most fundamental parameters to drug
designers, namely, cellular potency and lipophilicity. Com-
pounds 11−14 maintain high LE; however, the gem-dimethyl
linker in 11 results in a decreased LLE compared to 5 (3.9 vs
4.3), whereas its cyclopropyl and tetrahydropyran counterparts
12 and 13 have LLE values identical to that of 5. The charged
piperidine 14 (pKa = 8.8) leads to a significant reduction in
log D7.4 at the same time as retaining potency in the ATR
enzyme and cell assay, thus leading to a significant increase in
LLE compared to 5 (5.7 vs 4.3). Compounds 11−14 have
excellent selectivity over PI3K but retain activity against mTOR
(Table 4). These compounds have low micromolar activity in
the mTOR cell assay, comparing favorably with <1 μM ATR-
fold) based on an average of two repeat occasions per compound.
c
Measured using shake-flask methodology with a buffer/octanol
volume ratio of 100:1. The concentration of compound in the
aqueous phase before and after partitioning with octanol was
determined by generic HPLC−UV analysis. Lipophilicity ligand
efficiency (LLE): ATR cell pIC50 − log D7.4.
d
driven cellular potency. The moderate lipophilicity of the
neutral alkylated sulfonylmorpholinopyrimidine series leads to
high free drug in rat plasma, and the compounds show low
inhibition of the hERG ion channel. As such, these compounds
were deemed of sufficient quality to explore their properties in
vivo.
Compounds 11, 12, and 13 have low crystalline aqueous
solubility, with the solubility of 11 being unmeasurable at
pH7.4, but high permeability in Caco-2 cells and reasonable
stability to rat hepatocytes in vitro. In contrast, the charged
piperidine group in 14 leads to much higher aqueous solubility
and unbound fraction all without negatively impacting hERG
potency. However, the high basicity of the unsubstituted
piperidine group in 14 was a concern for permeability;
therefore, we explored substitution on the nitrogen atom.
One such compound made, the N-methylated derivative 15, is
shown in Table 3. Unfortunately compound 15 together with
several other groups we explored resulted in a reduction in
potency compared with the unsubstituted piperidine 14.
Compounds 11−14 were formulated as solutions in propylene
glycol, to minimize effects of solubility, and dosed orally in
Han−Wistar rats. Despite its low aqueous solubility, compound
12 leads to a high AUC (Table 4); however, significantly lower
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Table 4. Selectivity and Property Data for Compounds 11−14
mTOR IC50
PI3Kα IC
mTOR cell IC50
% free
b
hERG IC
solubility, pH 7.4
Caco-2 A−B
rat
Clint
rat AUC
g
a
a 50
a
c 50
d
e
f
compound
(μM)
(μM)
(μM)
(rat)
(μM)
(μM)
(pH 6.5)
(μM·h)
11
12
13
14
0.19
0.18
nd
1.6
2.7
3.2
3.3
1.5
20
23
25
40
>33
>100
>33
>33
<0.4
4.4
16
21
28
0.2
41
<3
23
9.4
0.76
3.1
0.94
1.4
1.8
0.32
0.04
h
0.23
2.7
230
a
b
Standard error of the mean (SEM) pIC50 measurement is ≤0.42 from at least two repeat measurements per test. nd = not determined. Run in 10%
c
plasma; % free calculated for 100% plasma assuming a single-site binding model. Activity against the human ether-a-go-go-related gene (hERG)
encoded potassium channel was determined using automated whole-cell electrophysiology.28 Solid material agitated in 0.1 M pH 7.4 phosphate
d
buffer for 24 h, double centrifuged, and the supernatant analyzed for compound concentration by LC−UV−MS. Crystallinity assessed by polarized
e
f
light microscopy of remaining solid. Median A to B Papp (1 × 10−6 cm/s), 10 μM compound concentration. Median Clint: intrinsic clearance from
g
rat hepatocytes (μL/min per 1 × 106 cells, 1 μM compound concentration). Compounds were dosed orally to male Han−Wistar rats at 4 μmol/kg
formulated in propylene glycol. Semicrystalline sample.
h
AUCs were observed with the other linker groups. In particular
the poorer in vitro permeability for piperidine 14 is borne out
with the lowest exposure seen of all the compounds examined.
In a rat PK study, compound 12 has a moderate volume of
distribution (2.1 L/kg), moderate plasma clearance (15 mL
min−1 kg−1) and excellent bioavailability (70%). A much higher
exposure, and therefore higher dose, of 12 was required in
order to probe ATR inhibition in vivo because of moderate
cellular potency. When the dose of compound 12 was increased
to 200 mg/kg, formulated as a suspension, increased exposure
was accompanied by increased variability. Therefore, com-
pound 12 was not considered a suitable probe molecule for
further studies to investigate ATR pharmacology in vivo.
The reasonable LLE, selectivity for ATR over other PIKK-
family kinases, and promising PK profile of the cyclo-
propylsulfone 12, despite low solubility, made this compound
an ideal start point for an early lead optimization campaign. In
the next phase we fixed the cyclopropylsulfone linker and
explored optimization of the rest of the molecule with a goal to
improve solubility and cellular potency while maintaining high
permeability and low clearance. A crystalline sample of 12
showed a sharp melt at 221 °C indicative of efficient crystal
packing, thus leading to low solubility despite moderate
lipophilicity. A single-crystal X-ray structure of 12 was obtained
in order to understand crystal packing (Figure 3, CCDC
916869). Compound 12 occupies a P-1 space group, and its
solid-state structure shows a centrosymmetric methylsulfone to
methylsulfone contact as well as ring−ring stacking and
hydrogen bonds between indole N−H and sulfone oxygen in
the crystal lattice. From an analysis of small-molecule structures
in the Cambridge Structural Database (CSD),27 we conclude
that such methylsulfone contacts are a preferred motif in
crystals of methylsulfone compounds, and the motif seems to
be associated with low solubility and high melting points. We
were intent on retaining the sulfone moiety in order to
maintain potency and selectivity. However, making a significant
impact on solubility was thought likely to be a challenge given
the efficient crystal packing shown in Figure 3. In order to
improve solubility, we were left options including disrupting
crystal packing, reducing lipophilicity significantly, or introduc-
ing charged groups without negatively impacting potency or
permeability.
Figure 3. Single crystal X-ray structure of compound 12 (CCDC
916869). Inset picture: methylsulfone−methylsulfone contact.
(compounds 19 and 20) all retained potency but with
decreased LE relative to 12. Neutral groups that had similar
or lower lipophilicity did not improve solubility. Where a
charged group was employed, high solubility could be achieved,
but in all cases in this position of the molecule, cellular potency
was disappointing. We concluded that while this part of the
molecule tolerated a broad range of structural variation and
offered a way to introduce polar functionality, it was again an
unproductive compromise because effects on properties came
at the expense of LE; a simple methylsulfone seemed optimal to
balance potency and properties.
The SAR of the pyrimidinyl 2-substituent was similarly
uncompromising. Replacing the 4-indole with a phenyl group
(Table 5, compound 21) resulted in a loss of >30-fold of
potency. We attempted to reintroduce an N−H hydrogen bond
in a fashion similar to that achieved with the indole, bearing in
mind the limited ways of achieving this without adversely
affecting conformation, balance of N−H hydrogen bond
strength, lipophilic environment, and shape complementarity.
Addition of an amino group to the phenyl ring in 21 to give
compound 22 resulted in a reduction in potency. In the mTOR
series the indole could be effectively replaced with 4-
phenylamides and ureas.15d For ATR, however, we thought it
A selection of the compounds made to explore substitution
of the terminal methyl group of the sulfone is shown in Table 5
(compounds 16−20). The methylsulfone in 12 can be
substituted for larger groups but with a loss of LE. Higher
alkyls such as isopropyl (compound 16), saturated cyclo-
heteroalkyls (compounds 17 and 18), and aromatic groups
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result with benzimidazole 25, which results in a similarly large
reduction in ATR potency. The most likely conformation of
this compound is to present an acceptor in place of the indole
N−H while the tautomeric N−H forms an internal hydrogen
bond with the pyrimidine nitrogen. We concluded that the 4-
indole group in compound 12 is a preferred moiety that
balances torsional preference and fits into ATR. With this in
mind we next set about to examine the opportunity to add
additional groups onto the indole ring. A “methyl scan” of
compound 12 (Table 6, compounds 26−30) indicated that
Table 5. Structures and ATR Inhibitory Potency of
Compounds 16−25
Table 6. Structures and ATR Inhibitory Potency of
Compounds 26−39
ATR IC
ATR cell IC50
a50
b
compound
R
(μM)
(μM)
LE
26
27
28
29
30
31
32
33
34
35
36
37
38
39
2-Me
3-Me
5-Me
6-Me
7-Me
0.028
14
2.5
0.26
0.17
>30
>30
0.49
6.3
nd
0.008
0.22
1.1
0.28
0.23
0.19
0.22
0.24
0.19
0.21
0.24
0.27
0.27
0.25
2-CONH2
2-CN
>30
7.2
0.30
0.066
0.72
0.14
0.041
0.015
0.016
0.023
6-CN
1.5
6-CONMe2
6-CONHMe
6-CONH2
6-F
24
15
9.2
0.44
0.72
0.80
6-Cl
6-OMe
a
Uncertainty (95% confidence) for pIC50 measurements is 0.38 (2.4-
fold) based on an average of two repeat occasions per compound. nd =
b
not determined. Uncertainty (95% confidence) for pIC50 measure-
ment is 0.45 (2.8-fold) based on an average of two repeat occasions
per compound; compound 29, N = 1.
a
Uncertainty (95% confidence) for pIC50 measurements is 0.38 (2.4-
fold) based on an average of two repeat occasions per compound.
b
Uncertainty (95% confidence) for pIC50 measurement is 0.45 (2.8-
fold) based on an average of two repeat occasions per compound;
compound 25, N = 1.
positions 2 and, in particular, 6 of the indole were capable of
tolerating substituents with the 3- and 5-Me analogues
(compounds 27 and 28), causing a large reduction in potency.
The results for the latter substituents are most likely driven by
increasing the torsion between indole and pyrimidine rings.
Increasing this torsion, however, led to an increase in crystalline
solubility (e.g., for compound 28, pH 7.4, aqueous solubility of
96 μM) and reiterated the difficulty of reducing packing
interactions in order to improve solubility while in tandem
maintaining potency. A wider set of substituents in positions 2
and 6 of the indole ring were investigated (Table 6, compounds
31−39). The compounds made were a result of the desire to
explore a wide enough range of groups in order to affect
lipophilicity and make additional binding interactions or affect
those interactions in place already, in combination with
synthetic expedience. Compounds were made from either
commercially available indole boronic acids or a direct
precursor to them such as the appropriate 4-bromoindoles.
While facile availability of such building blocks is limited,
sufficient diversity was available to explore the majority of
unlikely that simple amide and urea derivatives of the
aminophenyl 22 would lead to potent inhibitors because it is
unlikely that the preferred conformation of such groups would
place the N−H bond in a vector similar to that of the 4-indole
N−H. However, cyclic amides such as the indolone 23
theoretically are able to place an N−H bond in a similar
position. Disappointingly, compound 23 showed only weak
ATR enzyme potency, potentially a result of a clash of the
indolone oxygen with the protein and/or resulting in a
nonplanar torsion relative to pyrimidine and thus leading to
less optimal positioning in the binding site. We anticipated
being able to replace the indole with other 6,5-heteroaryl
groups, in particular with an indazole group as found in the
PI3K inhibitor 2. Much to our disappointment, the indazole 24
was significantly less active than the corresponding indole
despite being able to present a similarly strong N−H hydrogen
bond to the protein backbone. Perhaps less surprising was the
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substituents of interest. Substituents in the 2-position, such as
the carboxamido and cyano groups (compounds 31 and 32),
led to a large reduction in ATR potency compared with methyl.
As indicated by the methyl scan, a broader range of substituents
were indeed tolerated in the 6-position (compounds 33−39).
However, only simple substituents such as 6-F (compound 37),
6-Cl (compound 38), and 6-OMe (compound 39) resulted in
broad retention of LE and cellular potency and none of these
groups were predicted nor found to improve solubility.
Although an exhaustive set of substituents has not been
described, our goal of substantially moving potency and
physical properties had not been realized although certain
substituents on the indole ring, such as fluoro, might have
additional utility such as blocking specific sites of metabolism.
We next turned our attention to the morpholine group in
compound 12. Interestingly the 3(S)-methylmorpholine 40
(Table 7) led to a tandem reduction of ATR enzyme and
binder were subsequently described by Zask et al. for a series
of mTOR inhibitors.15b A crystal structure of compound 6 (not
shown, CCDC 915814) shows similar methylsulfone stacking,
but 6 displays a different symmetry in the solid state from that
seen with 12. The morpholine 3(R)-methyl prefers to occupy
an axial position, as the aromatic linked nitrogen adopts an sp2
geometry, thus forcing the methyl to be axial. On the basis of
this structure, we hoped for an improvement in solubility. The
crystalline melting point of compound 6, albeit still relatively
high, is reduced to an extent compared with compound 12
(204 °C vs 221 °C) which translated into a small but consistent
increase in aqueous solubility (compound 6, pH 7.4, aqueous
solubility of 10 μM). We also explored larger substituents on
the morpholine. When the methyl group was replaced with an
ethyl group with the same configuration (compound 41), a
significant reduction in potency was observed. Finally, the
bridged morpholine, compound 42, was prepared and found to
be equipotent to morpholine albeit with reduced LLE.
Table 7. Structures and ATR Inhibitory Potency of
Compounds 40, 6, 41, and 42
Compound 6 seemed to represent a step forward in terms of
a probe compound compared with 12. Compound 6 has
improved LLE and excellent free drug levels and does not
significantly inhibit the hERG ion channel when measured in a
whole cell assay (Table 8).28 Compound 6 was assessed for
drug−drug interaction (DDI) potential specifically from
inhibition of cytochrome P450 enzymes.29 Interestingly,
compound 6 shows no significant reversible inhibition of any
of the five major cytochrome P450 isoforms, but when tested
for time-dependent inhibitory (TDI) activity following
incubation with human liver microsomes, 6 was found to
inhibit the cytochrome 3A4-mediated metabolism of mid-
azolam by 50% at 10 μM.30 Inhibition of the CYP3A family of
enzymes is of particular concern for DDI given that they are the
major metabolizing enzymes involved in the human metabolism
of many drug molecules. In addition to DDI, covalent
modification associated with TDI can result in toxicological
consequences especially in the liver. In a recent study of 400
registered drugs, 16 (4%) were found positive for 3A4 TDI
while the proportion was much greater (20%) when lead-
optimization chemistry was tested.31 In addition, although TDI
is often coupled with reversible inhibition, 35% of the drugs
that were tested TDI positive showed no (>10 μM) reversible
inhibition, suggesting that TDI testing is important to fully
evaluate DDI risk in the selection of candidate drug
molecules.31 A number of mechanisms for TDI have been
proposed including formation of a tight-binding inhibitory
metabolite complex or by covalent adduct formation to heme
or protein.29,31 Specific structural features have been linked to
cytochrome P450 3A4 metabolism and inactivation,29,31,32 but a
full structural understanding of 3A4 TDI is not available. In the
case of compound 6 the precise structural feature(s)
responsible for the observed 3A4 TDI remains unknown. In-
silico classification models of cytochrome P450 3A4-mediated
TDI have been described and are likely to become of increasing
importance combined with in vitro screening approaches to
allow drug designers to efficiently identify compounds devoid
of this activity.32 Compound 6 has high permeability combined
with good stability to rat hepatocytes and, despite the lack of
progress in achieving markedly higher solubility, has respectable
bioavailability in a low dose rat PK study. Moreover, plasma
exposure at moderate oral doses in the mouse was high, relative
to in vitro ATR cellular potency, without undue variability. The
unbound concentration achieved at 50 mg/kg is below the
a
Uncertainty (95% confidence) for pIC50 measurements is 0.38 (2.4-
fold) based on an average of two repeat occasions per compound.
b
Uncertainty (95% confidence) for pIC50 measurement is 0.45 (2.8-
fold) based on an average of two repeat occasions per compound;
compound 41, N = 1.
cellular potency despite being a preferred group for affinity in
mTOR.15d We also made the corresponding 3(R)-methyl-
morpholine, compound 6, and to our surprise we discovered a
clear improvement in ATR enzyme and cell potency compared
to morpholine. An analysis involving 15 exact matched pairs
revealed an average increase in ATR cell potency of ∼5-fold
when morpholine is replaced with a 3(R)-methylmorpholine
group (Figure 4). The effect on potency is even more striking
when the two stereoisomers are compared directly; the R-
isomer 6 is nearly 100-fold more potent in cells than the
corresponding S-isomer 40. Moreover the 3(R)-methyl group
produces interesting effects on selectivity in addition to modest
improvements of physical properties. We discovered that while
potency against recombinant mTOR enzyme increases
compared with 12, compound 6 has improved PI3K selectivity
and notably the difference between ATR cellular and the
mTOR (pAKT) cell potency clearly increases in 6 compared
with 12 (Figure 5). Similar PI3K selectivity results for
compounds containing the 3(R)-methylmorpholine hinge
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Figure 4. ATR cell potency compared for matched-pair analogues of morpholine and 3(R)-Me morpholine hinge binders. Lower left: Each marker
represents an individual compound matching the substructure shown. Lines connect exact structural matches (∗ = position of variation). Lower
right: Each marker represents a matched pair; mean pIC50 difference = 0.765 (SE = 0.09, N = 15).
growth in cell lines with high baseline levels of replication stress
such as LoVo colorectal adenocarcinoma cells.34 The
combination of potent growth inhibitory effects in vitro and
high exposure led to significant antitumor effects in vivo.
Female nude mice bearing LoVo tumors were treated with
compound 6 orally at a dose of 25 mg/kg twice daily or 50 mg/
kg once daily for 13 days. Both schedules led to significant
tumor growth inhibition at the end of study compared with
vehicle treated controls (Figure 7). Body weight loss was in an
acceptable range throughout the study, and compound 6 was
generally well tolerated. Compound 6 is the first reported
inhibitor of ATR protein kinase demonstrating tumor growth
inhibition in vivo and is therefore a useful probe molecule to
aid further investigation of ATR tumor biology.
Figure 5. PIKK selectivity compared for compounds 12 and 6.
CONCLUSION
■
Compound 6 was discovered from a lead discovery and early
optimization campaign from an mTOR inhibitor screening hit.
The sulfonylmorpholinopyrimidine series has good potency
and selectivity for ATR, attractive LE and LLE, but low aqueous
solubility. Optimization within the confines of the described
SAR led to 6. Compound 6 has 20-fold improved cellular
potency compared to the screening hit 5, retains some residual
recombinant mTOR enzyme inhibitory potency, but shows
otherwise excellent kinase selectivity. Compound 6 is a poorly
soluble compound and is a time-dependent inhibitor of
cytochrome 3A4. However, 6 has high mouse free drug
exposure at moderate doses despite its low solubility and shows
tumor growth inhibition in LoVo xenografts in vivo at well
mTOR cell (pAKT) IC50 while achieving >12 h of exposure
over the ATR cell IC50 (Figure 6).
Compound 6 is a potent and selective inhibitor of ATR
(Table 9). Likewise, with the earlier compounds described in
this series, compound 6 inhibits recombinant mTOR enzyme
activity, but this leads to only weak potency in the mTOR
(pAKT) cell assay. Compound 6 shows good selectivity against
all of the PI3K isoforms together with ATM and DNA-PK, and
when tested in a large panel of kinases, 6 shows very high
general kinase selectivity. Out of a panel of 442 kinases only
two, mTOR and PI3Kα, had <50% residual activity when
incubated with 6 at 1 μM, representing just 0.5% of the panel
tested.33 Compound 6 leads to potent in vitro inhibition of cell
Table 8. Property and PK Data for Compound 6
% free
(rat)
hERG IC50
(μM)
3A4 IC50
a
3A4 TDI % inhibition, 10
solubility, pH 7.4
Caco-2 A−B (pH 6.5)
(1 × 10−6 cm/s)
rat
Clint
rat bioavailability
b
c
(μM)
μM
(μM)
(%)
15
50
>10
50
10
23
9.5
62
a
b
>10 μM vs 1A2, 2C19, 2C9, and 2D6. Mean value (N = 4). Compound 6 was preincubated 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 of incubation with 10 μM midazolam. Analysis of 1-hydroxymidazolam
was performed using liquid chromatography−tandem mass spectrometry.30 No activity was detected vs control for 1A2, 2C19, 2C9, and 2D6.
c
Compound 6 was administered orally to male Han−Wistar rats at 4 μmol/kg formulated as a solution in propylene glycol.
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Figure 6. Plasma concentration data in female nude mice dosed with 6 as a solution in 10% DMSO/40% propylene glycol/50% water orally at 25
○
(×) and 50 mg/kg ( ). Data shown are from individual animals. For 50 mg/kg: 2 h, N = 10; 8 h, N = 9; 24 h, N = 2. For 25 mg/kg: 8 h, N = 4. Solid
line is drawn through mean 50 mg/kg values. Hashed line is unbound concentration. Lower solid line is ATR cell IC50 (μM). Upper dotted line is
mTOR cell IC50 (μM).
Table 9. ATR, PIKK Selectivity, and Growth Inhibitory Potency for Compound 6
ATR IC50
(μM)
ATR cell
IC50 (μM)
mTOR IC50
mTOR cell
a
PI3Kα IC
PI3Kα cell IC50 ATM cell IC50
DNA-PK cell
d
selectivity
score S(50)
LoVo GI
af50
,
a
a 50
b
c
e
(μM)
IC50 (μM)
(μM)
(μM)
(μM)
IC50 (μM)
(μM)
0.005
0.050
0.038
2.4
13
>30
>30
>30
0.005
0.20
a
b
Standard error of mean (SEM) pIC50 measurement is ≤0.22 from at least two repeat measurements per test. Inhibition of pAKT T308 in BT-474
c
d
e
cells. Inhibition of pATM Ser-1981 in HT29 cells. Inhibition of pDNA-PK Ser-2056 in HT29 cells. The fraction of 442 kinases having <50%
residual activity in the presence of 1 μM compound 6. MTS assay with 72 h of continuous exposure to compound 6.
f
MeCN, 3:1. Intermediates were not necessarily fully purified, but their
structures and purity were assessed by TLC, NMR, HPLC, and mass
spectral techniques and are consistent with the proposed structures.
The purities of compounds for biological testing were assessed by
NMR, HPLC, and mass spectral techniques and are consistent with
the proposed structures; purity was ≥95% except for compound 17
(90%), compound 28 (90%), compound 30 (85%), compound 33
(92%), and compound 37 (89%). Electrospray mass spectral data were
obtained using a Waters ZMD or Waters ZQ LC/mass spectrometer
acquiring both positive and negative ion data, and generally, only ions
relating to the parent structure are reported. Unless otherwise stated,
1H NMR spectra were obtained using a Bruker DRX400 operating at
400 MHz in DMSO-d6 or CDCl3. Chemical shifts are reported as δ
values (ppm) downfield from internal TMS in appropriate organic
solutions. Peak multiplicities are expressed as follows: s, singlet; d,
doublet; dd, doublet of doublet; t, triplet; br s, broad singlet; m,
multiplet. Analytical HPLC was performed on C18 reverse-phase
silica, on a Phenomenex “Gemini” preparative reversed-phase column
(5 μm silica, 110A, 2 mm diameter, 50 mm length) using decreasingly
polar mixtures as eluent, for example, decreasingly polar mixtures of
water (containing 0.1% formic acid or 0.1% ammonia) as solvent A
and acetonitrile as solvent B or MeOH/MeCN, 3:1, with a flow rate of
about 1 mL/min, and detection was by electrospray mass spectrometry
and by UV absorbance at a wavelength of 254 nm. Accurate mass
spectra were recorded on a Themo LTQ-FT in +ve ion mode with a
Thermo Accela pump and Surveyor PDA+ with a CTC autosampler,
and the results agreed with the theoretical values to within 4 ppm.
Combustion analyses (C, H, N) were performed with a Carlo Erba
tolerated doses. Compound 6 is the first reported ATR
inhibitor with the required potency, selectivity, and properties
to explore ATR pharmacology in vivo.
MATERIALS AND METHODS
■
General Methods. All experiments were carried out under an inert
atmosphere and at room temperature unless otherwise stated.
Microwave reactions were performed using one of the following
reactors: Biotage initiator, Personal Chemistry Emrys optimizer,
Personal Chemistry Smithcreator, or CEM Explorer. Workup
procedures were carried out using traditional phase separating
techniques or by using strong cation exchange (SCX) chromatography
using Isolute SPE flash SCX-2 column (International Sorbent
Technology Limited, Mid Glamorgan, U.K.). When necessary, organic
solutions were dried over anhydrous MgSO4 or Na2SO4. Flash column
chromatography (FCC) was performed on Merck Kieselgel silica
(article 9385) or on Silicycle cartridges (40−63 μm silica, 4−330 g
weight) or on GraceResolv cartridges (4−120 g) either manually or
automated using an Isco Combi Flash Companion system. Preparative
reverse phase HPLC (RP HPLC) was performed on C18 reversed-
phase silica, for example, on a Waters “Xterra” or “XBridge”
preparative reversed-phase column (5 μm silica, 19 mm diameter,
100 mm length) or on a Phenomenex “Gemini” or “AXIA” preparative
reversed-phase column (5 μm silica, 110A, 21.1 mm diameter, 100 mm
length) using decreasingly polar mixtures as eluent, for example,
containing 1−5% formic acid or 1−5% aqueous ammonium hydroxide
(d = 0.88) as solvent A and acetonitrile as solvent B or MeOH/
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Figure 7. In vivo tumor growth inhibition (TGI) for compound 6. Female nude mice bearing established human LoVo colorectal adenocarcinoma
□
●
xenografts were dosed orally with either vehicle ( ) or 6 at 25 mg/kg twice daily ( , day 21 TGI = 78%, p < 0.0005) and 50 mg/kg once daily (×
̵
,
day 21 TGI = 77%, p < 0.0005) from day 9 to day 21.
EA1108 analyzer, and the results agreed with the theoretical values to
within 0.5%. Water was measured by the Karl Fischer method using
a Mettler DL 18.
4-{4-[1-(Methylsulfonyl)cyclopropyl]-6-morpholin-4-ylpyri-
midin-2-yl}-1H-indole (12). Bis(triphenylphosphine)palladium(II)
dichloride (82 mg, 0.12 mmol), 71 (371 mg, 1.17 mmol), 2 M
aqueous Na2CO3 (3.50 mL, 7.00 mmol), and 1H-indol-4-ylboronic
acid (225 mg, 1.40 mmol) were suspended in 18% DMF in 7:3:2
DME/water/EtOH (8 mL) and sealed into a microwave tube. The
mixture was heated to 110 °C for 1 h in a microwave reactor and then
allowed to cool to room temperature. The mixture was purified by
preparative HPLC using decreasingly polar mixtures of water
(containing 1% NH3) and MeCN as eluents. Fractions containing
the desired compound were combined and then evaporated. The
residue was purified by preparative HPLC using decreasingly polar
mixtures of water (containing 0.1% formic acid) and MeCN as eluents.
Fractions containing the desired compound were combined and then
evaporated to afford 12 (264 mg, 57%). 1H NMR (DMSO-d6): 1.60−
1.63 (2H, m), 1.71−1.74 (2H, m), 3.10 (3H, s), 3.74−3.78 (8H, m),
6.89 (1H, s), 7.21 (1H, t), 7.31 (1H, d), 7.46 (1H, t), 7.55 (1H, d),
8.04−8.06 (1H, m), 11.25 (1H, s). 13C NMR (DMSO-d6): 12.2, 40.3,
44.0, 46.1, 65.8, 100.5, 102.9, 113.7, 120.3, 120.5, 126.2, 129.3, 136.9,
161.5, 162.5, 164.4. HRMS-ESI m/z: 399.148 16 [MH]+ C20H22N4O3S
requires 399.148 54.
4-{4-[(3R)-3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)-
cyclopropyl]pyrimidin-2-yl}-1H-indole (6). Bis-
(triphenylphosphine)palladium chloride (1.692 g, 2.41 mmol),
compound 77 (8.00 g, 24.1 mmol), 1H-indol-4-ylboronic acid (4.27
g, 26.5 mmol), and 2 M aqueous Na2CO3 (36.2 mL, 72.3 mmol) were
suspended in 4:1 DME/water (170 mL) and heated to 90 °C
overnight. The DME was removed and the reaction mixture diluted
with EtOAc (100 mL). The mixture was washed with water (2 × 100
mL). The organics were separated, filtered through a pad of Celite, and
concentrated in vacuo onto silica. The residue was purified by
chromatography on silica with an elution gradient of 0−10% EtOAc in
DCM. Fractions containing product were combined and evaporated.
The residue was purified by chromatography on silica, eluting with a
gradient of 0−25% EtOAc in DCM. Fractions containing product were
combined and evaporated onto reverse phase C18 silica. The crude
product was purified by reverse phase using a 415g HP C18 column
using decreasingly polar mixtures of water (containing 1% NH3) and
MeCN as eluents. Fractions containing product were combined and
evaporated. The residue was taken up in dry MeOH and dried over
MgSO4. The mixture was filtered and the solvent evaporated, leaving a
gum. The gum was dissolved in DCM (500 mL), filtered and the
solvent removed under reduced pressure. The residue was dissolved in
MeOH (50 mL) and allowed to stir at room temperature overnight.
The resultant precipitate was collected by filtration to afford 6 (5.10 g,
51%). 1H NMR (DMSO-d6): 1.29 (3H, d), 1.57−1.64 (2H, m), 1.68−
1.78 (2H, m), 3.24−3.31 (1H, td), 3.29 (3H, s), 3.51 (1H, td), 3.67
(1H, dd), 3.80 (1H, d), 4.01 (1H, dd), 4.21 (1H, d), 4.61 (1H, m),
6.85 (1H, s), 7.21 (1H, t), 7.32 (1H, t), 7.46 (1H, t), 7.56 (1H, d),
8.06 (1H, dd), 11.25 (1H, s). 13C NMR (DMSO-d6): 12.2, 13.3, 38.9,
40.3, 46.1, 46.4, 66.0, 70.2, 100.5, 102.8, 113.7, 120.3, 120.5, 126.0,
126.2, 129.4, 137.0, 161.4, 161.9, 164.4. HRMS-ESI m/z: 413.163 97
[MH]+ C21H24N4O3S requires 413.164 19. Chiral HPLC: (HP1100
system 4, 5 μm Chiralpak AS-H (250 mm × 4.6 mm) column, eluting
with isohexane/EtOH/TEA 60/40/0.1) Rf = 8.815, >99%. Anal.
Found (% w/w): C, 60.65; H, 5.83; N, 13.4; S, 7.65; H2O, <0.3.
C21H24N4O3S requires C, 61.15; H, 5.86; N, 13.58; S, 7.77.
(R)-Methyl 2-Chloro-6-(3-methylmorpholino)pyrimidine-4-
carboxylate (45). (R)-3-Methylmorpholine (7.18 g, 71.0 mmol)
and triethylamine (12.87 mL, 92.31 mmol) were added to 43 (14.70 g,
71.01 mmol) in DCM (100 mL). The resulting mixture was stirred at
room temperature for 18 h. Water (100 mL) was added. The layers
were separated and extracted with DCM (5 mL). The combined
organics were dried over MgSO4, concentrated in vacuo and the
residue was triturated with Et2O to afford 45 (14.77 g, 77%). 1H NMR
(CDCl3): 1.33−1.37 (3H, d), 3.31−3.38 (1H, m), 3.52−3.59 (1H, m),
3.68−3.72 (1H, m), 3.79−3.83 (1H, m), 3.98 (3H, s), 4.02−4.05 (1H,
m), 4.12 (1H, m), 4.37 (1H, m), 7.16 (1H, s). MS-ESI m/z 272.43
[MH+]. The liquors were concentrated onto silica and purified by
chromatography on silica, eluting with a gradient of 20−40% EtOAc in
isohexane. Fractions containing product were combined and
1
evaporated to afford 45 (1.659 g, 9%). H NMR (CDCl3): 1.33−
1.37 (3H, d), 3.31−3.38 (1H, m), 3.52−3.59 (1H, m), 3.68−3.72 (1H,
m), 3.79−3.83 (1H, m), 3.98 (3H, s), 4.02−4.05 (1H, m), 4.12 (1H,
m), 4.37 (1H, m), 7.16 (1H, s). MS-ESI m/z 272.43 [MH+].
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1
(R)-(2-Chloro-6-(3-methylmorpholino)pyrimidin-4-yl)-
methanol (48). Lithium borohydride, 2 M in THF (18 mL, 36
mmol), was added dropwise to 45 (16.28 g, 59.92 mmol) in THF
(200 mL) at 0 °C over a period of 20 min under nitrogen. The
resulting solution was stirred at 0 °C for 30 min and then allowed to
warm to room temperature and stirred for a further 18 h. Water (200
mL) was added and the THF evaporated. The aqueous layer was
extracted with EtOAc (2 × 100 mL) and the organic phases were
combined, dried over MgSO4, and then evaporated to afford 48 (14.54
a solid which was dried under vacuum to afford 56 (7.3 g, 63%). H
NMR (DMSO-d6): 1.21 (3H, d), 3.12 (3H, s), 3.24 (1H, t), 3.42−3.49
(1H, td), 3.58−3.62 (1H, m), 3.74 (1H, d), 3.93−4.40 (3H, m), 4.47
(2H, s), 6.94 (1H, s). MS-ESI m/z 306.05 [MH+]. The mother liquors
were purified by chromatography on silica with an elution gradient of
40−90% EtOAc in isohexane. Fractions containing product were
combined and evaporated to afford a further crop of 56 (2.0 g, 17%).
MS-ESI m/z 306.12 [MH+].
2-Chloro-4-(methylsulfonylmethyl)-6-morpholin-4-ylpyrimi-
dine (62). Triethylamine (6.78 mL) was added to a cooled (−5 °C)
suspension of 54 (10.56 g, 43.8 mmol) in DCM (230 mL). A solution
of morpholine (3.85 mL) in DCM (30 mL) was then added dropwise
while keeping the temperature below −5 °C. The mixture was then
stirred at room temperature for 1 h. The solution was washed with
water (300 mL), dried (MgSO4), and concentrated in vacuo. The
residue was purified by chromatography on silica, eluting with 1:1
EtOAc/DCM to afford compound 62 (6.81g, 53%). 1H NMR
(DMSO-d6): 3.12 (3H, s), 3.63 (4H, s), 3.68−3.70 (4H, m), 4.45 (2H,
s), 6.96 (1H, s). MS-ESI m/z 292 [MH+].
4-(2-Chloro-6-(1-(methylsulfonyl)cyclopropyl)pyrimidin-4-
yl)morpholine (71). NaOH solution (9.60 mL, 96.0 mmol) was
added to a mixture of 62 (2.80 g, 9.60 mmol), 1,2-dibromoethane
(1.654 mL, 19.19 mmol), and TBAB (0.619 g, 1.92 mmol) in toluene
(120 mL). The resulting solution was then heated to 60 °C for 3 h.
The mixture was concentrated in vacuo to provide a residue which was
dissolved in EtOAc (200 mL). The solution was washed with water
(200 mL) and then saturated brine (100 mL). The solution was then
dried (MgSO4) and concentrated in vacuo. The residue was purified
by chromatography on silica, eluting with a gradient of 0−2.5% MeOH
in DCM to afford 71 (2.88 g, 94%). 1H NMR (DMSO-d6): 1.49−1.51
(2H, m), 1.62−1.65 (2H, m), 3.19 (3H, s), 3.66−3.69 (8H, m), 6.96
(1H, s). MS-ESI m/z 318 [MH+].
(R)-4-(2-Chloro-6-(1-(methylsulfonyl)cyclopropyl)pyrimidin-
4-yl)-3-methylmorpholine (77). A 50% aqueous solution of NaOH
(42 mL) was added to 56 (12.11 g, 39.60 mmol), 1,2-dibromoethane
(3.41 mL, 39.6 mmol), and TBAB (1.277 g, 3.96 mmol) in toluene
(120 mL). The resulting slurry was stirred at 60 °C for 3 h, and then
an additional portion of 1,2-dibromoethane (1 mL) was added and the
mixture stirred for a further 1 h. EtOAc (200 mL) was added and the
mixture washed with water (100 mL) and brine (100 mL). The
reaction mixture was dried over MgSO4 and concentrated in vacuo.
The residue was triturated with MeOH to give a solid which was
collected by filtration and dried under vacuum to afford 77 (9.65 g,
73%). 1H NMR (DMSO-d6): 1.21 (3H, d), 1.48−1.54 (2H, m), 1.61−
1.67 (2H, m), 3.20 (1H, td), 3.19 (3H, s), 3.44 (1H, td), 3.58 (1H,
dd), 3.72 (1H, d), 3.93 (1H, dd), 3.98−4.10 (1H, m), 4.41 (1H, m),
6.93 (1H, s). MS-ESI m/z 332.44 [MH+]. The MeOH mother liquors
were reduced in vacuo, and the residue was purified by
chromatography on silica with an elution gradient of 10−50%
EtOAc in DCM. Fractions containing product were combined and
1
g, 100%) which was used in the next step without purification. H
NMR (CDCl3): 1.32 (3H, d), 2.65 (1H, br s), 3.25−3.32 (1H, m),
3.51−3.57 (1H, m), 3.67−3.70 (1H, m), 3.78 (1H, d), 3.98−4.09 (2H,
m), 4.32 (1H, m), 4.59 (2H, s), 6.44 (1H, s). MS-ESI m/z 244.40
[MH+].
(R)-4-(2-Chloro-6-(iodomethyl)pyrimidin-4-yl)-3-methylmor-
pholine (51). (a) Methanesulfonyl chloride (4.62 mL, 59.7 mmol)
was added dropwise to 48 (14.54 g, 59.67 mmol) and triethylamine
(8.32 mL, 59.7 mmol) in DCM (250 mL) at 25 °C over a period of 5
min. The resulting solution was stirred at 25 °C for 90 min. The
reaction mixture was quenched with water (100 mL) and extracted
with DCM (2 × 100 mL). The organic phases were combined, dried
over MgSO4, filtered, and evaporated to afford (R)-(2-chloro-6-(3-
methylmorpholino)pyrimidin-4-yl)methyl methanesulfonate (20.14 g,
105%) which was used in the next step without further purification. 1H
NMR (CDCl3): 1.33 (3H, d), 3.13 (3H, s), 3.27−3.34 (1H, m), 3.51−
3.57 (1H, m), 3.66−3.70 (1H, m), 3.79 (1H, d), 3.99−4.03 (2H, m),
4.34 (1H, m), 5.09 (2H, d), 6.52 (1H, s). MS-ESI m/z 322.39 [MH+].
(b) Lithium iodide (17.57 g, 131.27 mmol) was added to (R)-(2-
chloro-6-(3-methylmorpholino)pyrimidin-4-yl)methyl methanesulfo-
nate (19.2 g, 59.67 mmol) in dioxane (300 mL) and heated to 100
°C for 2 h under nitrogen. The reaction mixture was quenched with
water (200 mL) and extracted with EtOAc (3 × 200 mL). The organic
layers were combined and washed with 2 M sodium bisulfite solution
(400 mL), water (400 mL), brine (400 mL), dried over MgSO4, and
then evaporated. The residue was triturated with Et2O to afford 51
(13.89 g, 66%). 1H NMR (CDCl3): 1.32 (3H, d), 3.24−3.32 (1H, m),
3.51−3.58 (1H, m), 3.67−3.71 (1H, m), 3.78 (1H, d), 3.98−4.02 (2H,
m), 4.21 (2H, s), 4.29 (1H, m), 6.41 (1H, s). MS-ESI m/z 354.31
[MH+]. The mother liquors were concentrated and triturated with
Et2O to afford a further crop of 51 (2.46 g, 12%). 1H NMR (CDCl3):
1.32 (3H, d), 3.24−3.32 (1H, m), 3.51−3.58 (1H, m), 3.67−3.71 (1H,
m), 3.78 (1H, d), 3.98−4.02 (2H, m), 4.21 (2H, s), 4.29 (1H, m), 6.41
(1H, s). MS-ESI m/z 354.31 [MH+].
2,4-Dichloro-6-(methylsulfonylmethyl)pyrimidine (54). (a)
Compound 53 (175 g, 1.09 mol) was dissolved in DMF (2 L), and
then sodium methanesulfinate (133.5 g, 1.31 mol) was added. The
mixture was heated to 125 °C for 2 h. After cooling to room
temperature, the mixture was filtered and the filtrate was concentrated
in vacuo. The crude material was washed with water, filtered, then
triturated with toluene. The solid was filtered and then triturated with
isohexane to provide 6-(methylsulfonylmethyl)-1H-pyrimidine-2,4-
dione (250 g), which was used without further purification. (b) 6-
(Methylsulfonylmethyl)-1H-pyrimidine-2,4-dione (132 g, 0.65 mol)
was added to POCl3 (1.2 L), and the mixture was heated to reflux for
16 h. The excess POCl3 was removed in vacuo and the residue
azeotroped with toluene (2 × 500 mL) and then dissolved in DCM.
This solution was poured slowly onto ice (4 L) (CARE: risk of
exotherm), and the mixture was stirred for 20 min. The mixture was
extracted with DCM (3 × 1 L) (insoluble black material was filtered
off and discarded) and EtOAc (2 × 1 L). The organic extracts were
combined, dried, and concentrated in vacuo to give compound 54 (51
g) which was used without further purification. 1H NMR (DMSO-d6):
3.13 (3H, s), 4.79 (2H, s), 7.87 (1H, s). MS-ESI m/z 239 [MH+].
(R)-4-(2-Chloro-6-(methylsulfonylmethyl)pyrimidin-4-yl)-3-
methylmorpholine (56). Sodium methanesulfinate (4.64 g, 45.5
mmol) was added in one portion to 51 (13.4 g, 37.9 mmol) in DMF
(100 mL). The resulting mixture was stirred at 25 °C for 2 h. The
reaction mixture was diluted with DCM and washed with water (2 ×
100 mL), aqueous sodium thiosulfate (50 mL), dried over MgSO4, and
concentrated in vacuo. The residue was triturated with MeOH to give
1
evaporated to afford 77 (2.16 g, 16%). H NMR (DMSO-d6): 1.21
(3H, d), 1.49−1.54 (2H, m), 1.59−1.68 (2H, m), 3.19 (3H, s), 3.20
(1H, td), 3.44 (1H, td), 3.58 (1H, dd), 3.71 (1H, d), 3.93 (1H, dd),
4.04 (1H, m), 4.41 (1H, m), 6.93 (1H, s). MS-ESI m/z 332.44 [MH+].
Biological Evaluation. IC50 values reported are geometric mean
values of at least two independent measurements unless otherwise
stated.
ATR Kinase Assay. ATR for use in the in vitro enzyme assay was
obtained from HeLa nuclear extract (CIL Biotech, Mons, Belgium) by
immunoprecipitation with rabbit polyclonal antiserum raised to amino
acids 400−480 of ATR (Tibbetts, R. S.; Brumbaugh, K. M.; Williams,
J. M.; Sarkaria, J. N.; Cliby, W. A.; Shieh, S. Y.; Taya, Y.; Prives, C.;
Abraham, R. T. A role for ATR in the DNA damage-induced
phosphorylation of p53. Genes Dev. 1999, 13, 152−157) contained in
the following buffer: 25 mM HEPES (pH 7.4), 2 mM MgCl2, 250 mM
NaCl, 0.5 mM EDTA, 0.1 mM Na3VO4, 10% v/v glycerol, and 0.01%
v/v Tween 20. ATR-antibody complexes were isolated from nuclear
extract by incubating with protein A-Sepharose beads (Sigma, no.
P3476) for 1 h and then through centrifugation to recover the beads.
In the well of a 96-well plate, 10 μL ATR-containing Sepharose beads
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Journal of Medicinal Chemistry
Article
were incubated with 1 μg of substrate glutathione S-transferase−
p53N66 (NH2-terminal 66 amino acids of p53 fused to glutathione S-
transferase were expressed in E. coli) in ATR assay buffer (50 mM
HEPES (pH 7.4), 150 mM NaCl, 6 mM MgCl2, 4 mM MnCl2, 0.1
mM Na3VO4, 0.1 mM DTT, and 10% (v/v) glycerol) at 37 °C in the
presence or absence of inhibitor. After 10 min with gentle shaking,
ATP was added to a final concentration of 3 μM and the reaction
continued at 37 °C for an additional 1 h. The reaction was stopped by
addition of 100 μL of PBS, and the reaction was transferred to a white
opaque glutathione coated 96-well plate (NUNC no. 436033) and
incubated overnight at 4 °C. This plate was then washed with PBS/
0.05% (v/v) Tween 20, blotted dry, and analyzed by a standard ELISA
(enzyme-linked immunosorbent assay) technique with a phosphoser-
ine 15 p53 (16G78) antibody (Cell Signaling Technology, no. 9286).
The detection of phosphorylated glutathione S-transferase−p53N66
substrate was performed in combination with a goat anti-mouse
horseradish peroxidase-conjugated secondary antibody (Pierce, no.
31430). Enhanced chemiluminescence solution (NEN, Boston, MA)
was used to produce a signal, and chemiluminescent detection was
carried out via a TopCount (Packard, Meriden, CT) plate reader. The
resulting calculated % enzyme activity (activity base, IDBS) was then
used to determine the IC50 values for the compounds (IC50 taken as
the concentration at which 50% of the enzyme activity is inhibited).
ATR Cell (pChk1) Assay in HT29 Tumor Cells. Compound dose
ranges were created by diluting in 100% DMSO and then further into
assay medium (EMEM, 10% FCS, 1% glutamine) using a Labcyte
Echo acoustic dispensing instrument. Cells were plated in 384-well
Costar plates at 9 × 104 cells per mL in 40 μL of EMEM, 10% FCS,
1% glutamine and grown for 24 h. Following addition of compound
the cells were incubated for 60 min. A final concentration of 3 μM
4NQO (prepared in 100% DMSO) was then added using the Labcyte
Echo, and the cells were incubated for a further 60 min. The cells were
fixed by adding 40 μL of 3.7% v/v formaldehyde solution for 20 min.
After removal of fix, cells were washed with PBS and permeabilized in
40 μL of PBS containing 0.1% Triton X-100. The cells were then
washed, and 15 μL primary antibody solution (pChk1 Ser345) was
added. The plates were incubated at 4 °C overnight. The primary
antibody was then washed off, and 20 μL of secondary antibody
solution (goat anti-rabbit Alexa Fluor 488, Invitrogen) and 1 μM
Hoechst 33258 (Invitrogen) added for 90 min at room temperature.
The plates were washed and left in 40 μL of PBS. Plates were then
read on an ArrayScan VTI instrument to determine staining
intensities, and dose responses were obtained and used to determine
the IC50 values for the compounds.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge the excellent technical expertise of the
scientists at KuDOS and AstraZeneca, in particular Lisa
Smith, Victoria Pearson, Aaron Cranston, and Elaine Brown
for biological evaluation, Paul Davey for help with mass spectra
analysis, and Drs. Matt Wood, Chris Jones, Robin Smith, Cliff
Jones, and Jason Kettle for helpful discussions in preparation of
this manuscript.
ABBREVIATIONS USED
■
ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia
mutated and RAD3-related; DNA-PK, DNA-activated protein
kinase; DDR, DNA-damage response; SSB, single strand break;
mTOR, mammalian target of rapamycin; PIKK, phosphatidy-
linositol 3-kinase related kinase; 4NQO, 4-nitroquinoline 1-
oxide; LLE, ligand lipophilicity efficiency; hERG, human ether-
a-go-go-related gene; Clint, intrinsic clearance; CSD, Cambridge
Structural Database; CCDC, Cambridge Crystallographic Data
Centre; TDI, time-dependent inhibition; TGI, tumor growth
inhibition; SCX, strong cation exchange; TEA, triethylamine;
MeCN, acetonitrile; HATU, O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and data for compounds 7−11, 13−42,
44, 46, 47, 49, 50, 52, 55, 57−61, 63−70, 72−76, 78, 80, 82−
89. This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*Telephone: +44(0)1625 518629. E-mail: kevin.foote@
astrazeneca.com.
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