Discovery of 3-Oxabicyclo[4.1.0]heptane, a non-nitrogen containing morpholine isostere, and its application in novel inhibitors of the PI3K-AKT-mTOR Pathway

Article abstract of DOI:10.1021/acs.jmedchem.9b00348

4-(Pyrimidin-4-yl)morpholines are privileged pharmacophores for PI3K and PIKKs inhibition by virtue of the morpholine oxygen, both forming the key hydrogen bonding interaction and conveying selectivity over the broader kinome. Key to the morpholine utility as a kinase hinge binder is its ability to adopt a coplanar conformation with an adjacent aromatic core favored by the morpholine nitrogen nonbonding pair of electrons interacting with the electron deficient pyrimidine Π-system. Few selective morpholine replacements have been identified to date. Herein we describe the discovery of a potent non-nitrogen containing morpholine isostere with the ability to mimic this conformation and its application in a potent selective dual inhibitor of mTORC1 and mTORC2 (29b).

Full text of DOI:10.1021/acs.jmedchem.9b00348

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Article
Discovery of 3-oxabicyclo[4.1.0]heptane, a non-nitrogen
containing morpholine isostere, and its application
in novel inhibitors of the PI3K-AKT-mTOR pathway.
Heather Hobbs, Gianpaolo Bravi, Ian B. Campbell, Maire A. Convery, Hannah Davies,
Graham Inglis, Sandeep Pal, Simon Peace, Joanna Redmond, and Declan Summers
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00348 • Publication Date (Web): 08 Jul 2019
Downloaded from http://pubs.acs.org on July 8, 2019
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Discovery of 3-oxabicyclo[4.1.0]heptane, a non-nitrogen containing
morpholine isostere, and its application in novel inhibitors of the PI3K-
AKT-mTOR pathway.
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Heather Hobbs*, Gianpaolo Bravi†, Ian Campbell, Maire Convery, Hannah Davies, Graham Inglis,
Sandeep Pal, Simon Peace, Joanna Redmond & Declan Summersψ.
GlaxoSmithKline R&D, Gunnels Wood Road, Stevenage, SG1 2NY, U.K.
†Current affiliations: †Dotmatics, The Old Monastery, Windhill, Bishops Stortford, CM23 2ND.
ψCostello Medical Consulting Ltd, Jupiter House 10, Cambridge, CB1 2JD.
Abstract
4-(Pyrimidin-4-yl)morpholines are privileged pharmacophores for PI3K and PIKKs inhibition by virtue
of the morpholine oxygen both forming the key hydrogen bonding interaction and conveying selectivity
over the broader kinome. Key to the morpholine utility as a kinase hinge binder is its ability to adopt a
co-planar conformation with an adjacent aromatic core favoured by the morpholine nitrogen non-
bonding pair of electrons interacting with the electron deficient pyrimidine π-system. Few selective
morpholine replacements have been identified to date. Herein we describe the discovery of a potent
non-nitrogen containing morpholine isostere, with the ability to mimic this conformation and its
application in a potent selective dual inhibitor of mTORC1 and mTORC2 [29b].
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Introduction
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The phospoinositide 3-kinase kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway
has attracted much attention from the scientific community as a key regulator of diverse cellular
processes including: growth; proliferation; metabolism; differentiation; autophagy; survival and
cytoskeleton rearrangement1. The pathway is one of the most commonly dysregulated in cancer2-4 and
is also implicated in metabolic disorders and neurodegeneration5-8. Several ATP competitive and non-
competitive pathway inhibitors are already in the clinic9-11 and it is likely that much of the
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pharmaceutical industry is still active in this field12-17
.
O
N
OH
OH
O
HO
O
O
O
N
O
N
N
HO
OH
N
OH
O
LY294002 [1]
Quercetin [2]
PI-103 [3]
Figure 1: Structures of LY294002 1, Quercetin 2, PI-103 3
Morpholine acts as the hinge binder in the very first reported synthetic inhibitor of the PI3Ks, LY294002
[1]18, 19 (Figure 1). The X-ray co-crystal structures of 1, and structurally related Quercetin [2], with
porcine PI3Kγ were published in 2000 and highlight the key hydrogen bonding interaction between the
morpholine oxygen of 1 and Val882 which mimics the N1 interaction of the natural substrate, ATP20.
Subsequent crystal structures, including dual PI3K-mTOR inhibitor PI-103 3 bound to mTOR21 further
support the key morpholine-hinge hydrogen bond hypothesis and highlight the co-planarity of the
morpholine and adjacent ring systems when binding to the respective active sites.
4-(Pyrimidin-4-yl)morpholine [A] is a privileged pharmacophore for the PI3K-AKT-mTOR pathway22-
24
and is widely conserved across selective PI3K and PIKK inhibitors. Minimization of the dihedral
angle CNcn (where C and N atoms belong to the aryl ring and the c and n atoms belonging to the
aromatic ring) of 4-(Pyrimidin-4-yl)morpholine [A] using Density Functional Theory (DFT) and
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6.31G** basis set in Jaguar25 shows that the morpholine ring and the aryl ring are co-planar. This is due
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to stabilising electronic overlap between the nitrogen lone pair and the aryl ring (Figure 2).
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0
50 100 150 200 250 300 350
Angle/ °
Figure 2: (a) Energy minimised structure of 4-(Pyrimidin-4-yl)morpholine [A]
using DFT. (b) Energy scanning plot of CNcn dihedral bond angle of A
Kinase inhibitors described to date possessing a morpholine hinge binder have shown exclusive
selectivity for the Lipid Kinases26 and the adjacent electron deficient pyrimidine ring induces co-
planarity, orientating the morpholine oxygen loan pair in the optimal vector for formation of the key
hydrogen bonding interaction. Replacements for the morpholine ring are rare and limited largely to
heteroaromatic rings or dihydropyran27, both of which conserve the co-planar geometry. However the
combination of multiple aromatic rings has significant implications for ‘drug like’ properties28, 29 and
simple, planar structures are often associated with poor physicochemical profiles and toxicity whilst
increasing carbon bond saturation (Fsp3), and structural complexity is correlated with improved
solubility and success of compounds progressing into the clinic30, 31. The dihydropyran (DHP) hinge
binder retains cross-kinome selectivity but has not been widely utilised in the literature, presumably
due to its reduced Fsp3 character, poor solubility profile and potential reactivity. Limited examples of
the fully saturated tetrahydropyran (THP) exist in the literature27 but despite retaining the key oxygen,
the inability of the THP to adopt a low energy co-planar conformation means the THP analogues are
invariably less active than their unsaturated or morpholine analogues due to the conformational energy
penalty to form the key hydrogen bonding interaction.
Herein we report the discovery of a novel, stable, carbon linked saturated cyclic system with increased
Fsp3 and differentiated physicochemical profile with the potential to mimic the binding mode of
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morpholine. Full comparative matched molecular pairs were prepared and described to give clear and
complete understanding of changing factors.
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Results and Discussion
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Figure 3: Dihydropyran pyrimidine [B], Tetrahydropyran pyrimidine [C] & CPP pyrimidine [D]
Energy minimised structures using MMFF94X forcefield in MOE
A brainstorm approach to novel alternative carbon-linked morpholine isosteres with improved Fsp3 lead
us to consider the potential for 3-oxabicyclo[4.1.0]heptane (cyclopropyl pyran or CPP) to adopt a co-
planar conformation with a pyrimidine core given that cyclopropyl carbon-carbon bonds are known to
form stabilising interactions with adjacent π-systems32-34. Energy minimization using molecular
mechanics using MMFF94X force field in MOE35 showed good structural overlap between morpholine
pyrimidine [A] and dihydropyran (DHP) pyrimidine [B], however tetrahydropyran (THP) pyrimidine
[C] and 3-oxabicyclo[4.1.0]heptane pyrimidine [D] were both predicted to adopt preferred orthogonal
geometries (Figure 3).
Similarly, investigation of the Cambridge crystallographic database also suggested that most
cyclopropyl-aromatic combinations afford orthogonal geometry [e.g. CSD code ULIXIX]; however,
the structures exemplified in the crystallography database are predominantly cyclopropyl-phenyl
combinations. Prior to discarding this option, rigorous quantum mechanical calculations using Density
Functional Theory (DFT) and 6.31G** basis set in Jaguar25 were undertaken which predicted a co-
planar conformation for CPP (D). (Figure 4).
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Figure 4: Dihedral scanning plots for various pyrimidine fragments
using DFT/6-31G**
The data in Table 1 supports the hypothesis documented in the literature27 that the dihydropyran [9] is
a reasonable replacement for the morpholine [5] and 3-S-methylmorpholine [6] across a range of
common lipid and lipid-like kinases with only a slight loss in affinity. The tetrahydropyran [11] loses
significant affinity, in line with the hypothesis that is can only form the key oxygen hydrogen bound
by adopting a less energetically favourable conformation.
The CPP [12] is a chiral molecule and exists as two isomers [12a] (3-(4-((1R,6S)-1-methyl-3-
oxabicyclo[4.1.0]heptan-6-yl)pyrimidin-2-yl)phenol)
oxabicyclo[4.1.0]heptan-6-yl)pyrimidin-2-yl)phenol).
and
[12b]
(3-(4-((1S,6R)-1-methyl-3-
The absolute stereochemistry of 12a and 12b are inferred from the small molecule crystal structure of
12b (Figure 5b, see supporting information)36 which has negative optical rotation. All subsequent.
cyclopropyl pyrans reported here are assigned relative to 12b by their optical rotation. Compounds with
negative optical rotation are assumed 1S,6R
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O
N
O
N
O
O
O
Hinge =
O
A
~
~
~
~
~
R
Hinge
Morpholine
Me
DHP
THP
CPP
Morpholine
~
O
O
~
~
N
OH
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O
R =
N
R
N
N
H
H
~
1R,6S CPP 1S,6R CPP
m-Phenol
Et Urea
Hinge
R
PI3Kα PI3Kδ mTOR pAKT
Fsp3 Chrom Solubility
a
a
a
a
pIC50
pIC50
pIC50
pIC50
LogPb
(µg/mL)c
5
6
Morpholine
6.3
6.6
5.9
5.7
0.29
0.33
0.2
2.7
107
Me
Morpholine
DHP
5.8
5.5
4.8
5.2
5.2
5.6
4.9
5.2
4.7
4.9
<4.5
4.6
6.0
5.9
5.1
5.4
5.5
5.9
5.0
5.3
4.8
5.3
4.6
4.6
5.7
5.7
4.5
4.8
<3d
5.1
6.5
6.1
6
5.4
5. 5
4.7
5.1
5.0
5.1
5.4
5.9
5.4
5.5
5.5
5.7
3.3
3.6
3.3
4.1
4.0
4.0
2. 7
3.4
3.6
4
119
113
100
102
123
141
109
136
24
9
11
12
THP
CPP
m-phenol
0.33
0.38
0.38
0.38
0.35
0.39
0.28
0.42
0.42
0.42
12a 1R,6S CPP
12b 1S,6R CPP
7
8
Morpholine
Me
Morpholine
DHP
10
13
Et urea
CPP
5.8
5.5
6.1
111
127
125
13a 1R,6S CPP
13b 1S,6R CPP
3.9
3.9
Table 1: Biochemical assay data for tool compounds 5-13
aMean value of at least two repeat measurements per data point, (see supporting information for details).
bChrom LogP is the lipophilicity of the compound in its neutral state measured using reversed phase
column chromatography (50 x 2 mm 3 µM Gemini NX C18, Phenomenex, UK) with fast acetonitrile
gradient at starting mobile phase of pHs 2, 7.4 and 10.5. Chrom LogP values are derived directly from
c
the gradient retention times by using a calibration line obtained for standard compounds. kinetic
solubility assay: 5µl of 10mM DMSO stock solution diluted to 100ul with pH7.4 phosphate buffered
saline, equilibrated for 1 hour at room temperature and filtered through Millipore MultiscreenHTS-PCF
filter plates and quantified by Charged Aerosol Detector. dData from high concentration screen (1 mmol
initial concentration).
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The PI3K and mTOR kinase affinities and cellular efficacy (pAKT) of the more potent 1S,6R CPP
isomer [12b] are broadly similar to the dihydropyran [9] although less potent and with lower lipophilic
ligand efficiency, due to its increased lipophilicity, than the 3S-methylmorpholine [6], supporting the
hypothesis that the 3-oxabicyclo[4.1.0]heptane motif can not only adopt a co-planar geometry with the
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5a
5c
5b
Figure 5a: QM predicted structure
using DFT/6-31G**
5b: small molecule crystal structure of
12b (CCDC 1864315 -See
supplementary information)
5c: A potential binding mode of 12b
which is consistent with a low
occupancy co-crystal structure in
PI3Kδ (not deposited).
adjacent pyrimidine ring but also is an active bioisostere for morpholine. The small molecule crystal
structure of 12b confirms co-planarity in the solid phase and the visible electron density in a low
occupancy co-crystal structure of 12b in PI3Kδ (Figure 5c, not deposited in PDB due to low occupancy,
see supporting information) is consistent with a favourable low energy co-planar binding conformation
and the ability of the CPP pyrimidine motif to sit close to the hinge in the active site.
The emerging utility of mTOR inhibitors in diseases including a variety of cancers, thrombosis, fibrosis
and diabetes37-43, along with a marked prevalence for morpholine pyrimidines amongst the selective
inhibitors in the literature lead us to apply our discovery to the development of potent selective 3-
oxabicyclo[4.1.0]heptane (CPP) mTOR inhibitors. Compounds with a classical mTOR ethyl urea back
pocket, such as 7, 8 and 9 illustrate the 10-30-fold increased affinity of such molecules for the mTOR
protein and improved selectivity over the closely structurally related PI3K isoforms. It is postulated that
this increased affinity is due to the additional hydrogen bonding interactions between the urea moiety
and the protein back pocket region. Gratifyingly this increased affinity for mTOR is conserved in the
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CPP molecules, with 13b demonstrating a full log unit of increased affinity over 12b and similar
potency to the morpholine [7], 3S-methylmorpholine [8] and dihydropyran [10] (with similar caveats
to 12b with respect to ligand efficiency and lipophilicity). Furthermore, the selectivity of the 1S,6R
analogue [12b] was improved by virtue of its reduced affinity for the class I PI3Ks. The CPP isomers
[13a and 13b], with considerably increased lipophilicities over the morpholines [7] and [8], might be
expected to exhibit a dramatic drop in aqueous solubility. Whilst the data shown are kinetic solubility
values, a significant drop in solubility is not apparent.
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PI3KK research in the recent literature that caught our attention are the publications by AstraZeneca
highlighting development of AZD3147 a dual mTORC1 mTORC2 inhibitor44 and the closely related
AZ20, a selective ATR inhibitor45. The authors postulate that increasing the ‘non-planarity’ in the hinge
region by incorporation of the 3(S)-methylmorpholine has a positive impact on cellular efficacy and
solubility due to disruption of hydrogen bonding networks, disruption of the crystal packing and an
increased twist between the morpholine and pyrimidine rings. Disrupting planarity by alkylation of the
methylene linker also had a positive impact on cellular potency but results in a significant increase
lipophilicity. We were interested in the impact of the CPP in this series and the interplay between
potency, lipophilicity, hydrogen bonding and solubility. To this end compounds 14, 15, 21, 22a, 22b, ,
23a and 23b were synthesised (Scheme 2 and 4) as benchmarks for us to assess the impact of our CPP
replacements in the sulphone series and are not included in our subsequent selectivity analysis as they
incorporate a classical non-selective kinase back pocket, however these compounds confirm the general
utility of the CPP replacement.
Whilst 1S,6R CPP 16b shows a significant 5-fold drop in affinity over the 3S-methylmorpholine [14],
their cellular efficacies (as determined by pAKT pIC50s: 5.5 vs 5.6) are almost identical, supporting
hypothesis of a positive impact of increasing non-planarity (or Fsp3). It should be noted that the CPP
isomers are also more lipophilic than the 3S-methylmorpholine (3.66 vs 3.33 respectively) and this
increased lipophilicity will also have a positive effect on permeability (also true for the 3S-
methylmorpholine vs morpholine matched pairs), however the increased lipophilicity should logically
have a negative impact on aqueous solubility
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O
O
O
~
O
~
O
~
Hinge =
N
~
N
~
O
A
R
Hinge
Morpholine
Me
Morpholine
DHP 1R,6S CPP 1S,6R CPP
O O
S
N
~
~
~
OH
O
O
H
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N
R2
R2 =
N
R
1 R1
N
H
N
N
H
H
m-Phenol
Et Urea
Glycinamide
mTOR pAKT PI3Kα PI3Kδ
Chrom Solubility
Hinge
R1
R2
Fsp3
a
a
a
a
pIC50
pIC50
pIC50
pIC50
LogPb
(µg/mL)c
14
21
Me
Morpholine
6.7
5.6
6.0
6.0
0.41
0.29
0.44
0.44
0.47
0.5
3.3
113
DHP
6.4
5.9
5.8
5.7
6.1
5.3
5.5
5.9
5.3
5.9
5.9
6.5
5.8
6.3
7.3
6.8
7.2
6.3
5.9
6.1
7.3
5.2
5.1
5.8
5.4
4.8
5.5
5.1
5.0
4.7
4.7
4.6
5.1
5.1
4.8
4.6
4.9
4.8
<4.5
5.8
3.3
3.7
3.7
4.4
4.8
4.8
2.7
3.2
3.6
3.6
4.4
4.7
4.7
2.6
3.1
3.1
4.1
27
145
75
H
22a 1R,6S CPP
22b 1S,6R CPP
m-
4.7
Phenol
15
Me
Morpholine
23a 1R,6S CPP
23b 1S,6R CPP
5.5
121
148
135
84
<5.4d
5.5
7.3
7,1
6.1
7
5.0
Me
4.8
0.5
18
5.4
0.42
0.45
0.48
0.48
0.5
Morpholine
16
Me
Morpholine
24a 1R,6S CPP
24b 1S,6R CPP
4.8
212
77
H
<4.5
<4.5
5.1
Et Urea
96
17
Me
Morpholine
25a 1R,6S CPP Me
25b 1S,6R CPP
7.3
6.6
6.9
7.7
7.1
7.4
8.0
130
78
4.6
0.52
0.52
0.45
0.48
0.48
0.5
<4.5
4.6
74
19
Me
Morpholine
26a 1R,6S CPP
90
H
<4.5
<4.5
4.8
167
158
198
26b 1S,6R CPP
glycin-
amide
20
Me
Morpholine
27a 1R,6S CPP
Me
7.3
6.8
4.6
5.9
0.52
4.3
257
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27b 1S,6R CPP
7.6
7.0
<4.5
4.9
0.52
4.3
217
5
6
Table 2: Biochemical assay data for tool compounds 14-27
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9
aMean value of at least two repeat measurements per data point. bChrom LogP is the lipophilicity of the
compound in its neutral state measured using reversed phase column chromatography (50 x 2 mm 3 µM
Gemini NX C18, Phenomenex, UK) with fast acetonitrile gradient at starting mobile phase of pHs 2,
7.4 and 10.5. Chrom LogP values are derived directly from the gradient retention times by using a
calibration line obtained for standard compounds. ckinetic solubility assay: 5µl of 10mM DMSO stock
solution diluted to 100ul with pH7.4 phosphate buffered saline, equilibrated for 1 hour at room
temperature and filtered through Millipore MultiscreenHTS-PCF filter plates and quantified by Charged
Aerosol Detector. Compound 23a tested inactive on two test occasions and positive on two test
occasions (pIC50 7.0 & 7.2), considering the other matched pair data we suspect that the active data
points are due to a technical error.
10
11
12
13
14
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16
17
18
19
20
21
22
23
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25
26
27
28
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d
which is not consistently apparent in our data. On average the CPP substituent solubility is broadly
similar to the 3S-methylmorpholine which in AstraZeneca’s hands were routinely 3-fold more soluble
than the parent morpholines.
Compounds 16 to 20 and 24 to 27 incorporating mTOR selective back pockets show the 1S,6R CPPs
[24b 25b, 26b and 27b] and are broadly equipotent with their morpholine [18] and 3S-
methylmorpholine analogues [16, 17, 19 and 20] but marginally more selective against the PI3K
isoforms (Figure 6a). Whereas the parent morpholine [18] shows limited cellular efficacy, the CPP
analogues [24b and 25b] are equiefficacious with the 3S-methylmorpholines [16 and 17]. It should be
noted that compounds 22a and 22b do not appear to follow the general trend of the 1S,6R isomer
[22b] being the more potent. In this matched pair the isomers are broadly equipotent. Our only
explanation for this is a subtle change in binding pose facilitated by the small and non-selective
phenol back pocket.
In line with AstraZeneca’s findings, increasing the lipophilicity sulphone linker region with the
dimethylated analogues resulted in increased cellular efficacy relative to the parent methylenes [17 vs
16 and 20 vs 19], which is evident in the 1S,6R CCP methylmorpholine matched pairs as a lower average
drop off from mTOR to pAKT pIC50’s. A preliminary analysis of the effect of the CPP on aqueous
solubility suggests a variable effect that is broadly positive in the polar m-phenol and glycinamide back
pockets whereas with the highly planar urea back pocket the negative effect of increased lipophilicity
dominates.
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Figure 6: MMP comparisons of morpholine, 3S-methylmorpholine and 1S,6R CPP analogues
Finally, we synthesised one of the more potent analogues from the AstraZeneca paper37 alongside the
two CPP analogues (Scheme 6). Compound 28 was synthesised as described in the literature using the
modified palladium cross coupling conditions used as standard in our labs.
1-(2-hydroxyethyl)-3-(4-(4-((1S,6R)-1-methyl-3-oxabicyclo[4.1.0]heptan-6-yl)-6-(2-(methyl-
sulfonyl)propan-2-yl)pyrimidin-2-yl)phenyl)thiourea 29b is a single-digit nanomolar, selective mTOR
inhibitor with excellent selectivity over closely related kinases. 29b compares favourably to the
literature compound 28 with respect to mTOR affinity, efficacy, PI3K isoform selectivity and aqueous
solubility.
To examine the potential of the CPP morpholine replacement to have an impact in a clinical setting we
further profiled compounds 8, 13a, 13b, 28 & 29b.
O
N
O
O
O
O
O
O
O
O
N
N
N
S
S
S
N
N
S
N
S
S
29a
29b
OH
OH
OH
28
N
N
N
N
N
N
H
H
H
H
H
H
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mTOR
pAKT
PI3Kα
PI3Kδ
Fsp3
Chrom
LogPb
Solubility
(µg/mL)c
a
a
a
a
pIC50
pIC50
pIC50
pIC50
6
7
8
9
28
8.4
8.2
8.6
7.4
7.1
7.5
5.5
5.1
4.6
4.9
4.6
4.5
0.5
3.6
3.9
3.9
151
145
173
29a
29b
0.52
0.52
10
11
12
13
14
15
16
17
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19
20
21
22
23
24
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27
28
29
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41
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60
Table 3: Biochemical assay data for nanomolar mTOR inhibitors 28-29
aMean value of at least two repeat measurements per data point. bChrom LogP is the lipophilicity of the
compound in its neutral state measured using reversed phase column chromatography (50 x 2 mm 3 µM
Gemini NX C18, Phenomenex, UK) with fast acetonitrile gradient at starting mobile phase of pHs 2,
7.4 and 10.5. Chrom LogP values are derived directly from the gradient retention times by using a
calibration line obtained for standard compounds. ckinetic solubility assay: 5µl of 10mM DMSO stock
solution diluted to 100ul with pH7.4 phosphate buffered saline, equilibrated for 1 hour at room
temperature and filtered through Millipore MultiscreenHTS-PCF filter plates and quantified by Charged
Aerosol Detector
The metabolic stability of matched pair compounds were assessed in human and rat microsomes to
determine the relative stability of the CPP with the m-phenol [13a & 13b] and the urea back pockets
[29b] compared to the 3S-methylmorpholine [8 and 28]. All three m-phenol compounds showed
moderate clearance profiles in human whereas in rat the CPP containing compounds were readily turned
over. Compounds 28 and 29b were assessed in both human and rat microsomes and hepatocytes.
Compound 28 was found to be a relatively low clearance compound, although it is moderately cleared
in rat hepatocytes. Compound 29b however is rapidly eliminated in both species in line with its
increased lipophilicity. This data suggests no measurable difference between the two CPP isomers and
but a specific metabolic liability with the cyclopropyl pyran substituent in rats.
As an exemplar of the CPP series, compound 27a was profiled across a range of 17 kinases closely
related to mTOR (ATM, ATR, CSNK2A1, CSNK2A2, PIK3C2A, PIK3C2B, PIK3C3, PIK3CA,
PIK3CB, PIK3CD, PIK3CG, PIK3R4, PI4KA, PI4KB, PI4K2B, PRKDC [DNAPK], RIOK2) in a
chemoproteomics kinobead screening assay and showed greater than x100 fold selectivity against each
with the exception of PIK3CB (x55 fold selectivity). Other cyclopropylpyrans, not reported here,
showed marked ATM and ATR potency.
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9
Hinge
Back Pocket
rat Clint
mL/min/g
human Clint
mL/min/g
rat Clint
mL/min/g
human Clint
mL/min/g
microsomes microsomes hepatocytes hepatocytes
8
Me
Morpholine
13a 1R,6S CPP
2.0
16.7
13.6
0.8
1.2
1.4
1.5
0.9
3.1
~
~
OH
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22
23
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28
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13b 1S,6R CPP
28
Me
Morpholine
2.8
0.9
5.1
O
N
N
29b
1.4
13.0
1S,6R CPP
H
H
Table 4: metabolic stability data for morpholine and CPP matched molecular pairs
Chemistry
Compounds 5 – 8 were synthesised by precedented chemistry44 according to the route outlined in
Scheme 1a. The reaction of morpholine or 3S-methylmorpholine with 2,4-dichloropyrimidine and
Hunig’s base proceeds selectively at room temperature to give intermediates A and B in good yield.
Installation of the non-selective m-phenol and mTOR selective back pockets via Suzuki-Miyaura cross
coupling46, 47 with the relevant pinacol boronic esters afforded tool compounds 5, 6, 7 and 8 in good
yields.
Similarly, Palladium mediated cross coupling furnished dihydropyran compounds intermediate C and
subsequently 9 and 10 in good yields. Compound 9 was reduced with hydrogen over a palladium on
carbon catalyst to afford the tetrahydropyran 11. The synthesis of 12 and 13 were affected by a Johnson-
Corey-Chaykovski cyclopropanation of 9 and 10 respectively in moderate yields. Interestingly
Simmons-Smith cyclopropanation afforded none of the desired products. Simmons-Smith
cyclopropanation is effective with electron rich systems, compounds 9 and 10 are both electron deficient
by virtue of the neighbouring pyrimidine hence favouring the Johnson-Corey-Chaykovski
cyclopropanation. Compounds 12 and 13 were subsequently separated into their single enantiomers
12a, 12b, 13a and 13b by chiral SFC.
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Scheme 1a:
5 6 7
8
and
General Synthesis of Parent Morpholines
,
,
O
N
O
O
6
7
8
9
R
Cl
N
O
N
N
R
a)
b)
N
R
+
&
N
N
N
R
N
H
OH
N
O
N
Cl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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N
Cl
N
N
H
H
A:
B:
5:
6:
7:
8:
R = H
R = Me
R = H
R = Me
R = H
R = Me
Scheme 1b:
9
10
11
12 13
and
General Synthesis of Dihydropyrans
&
, Terahydropyran
O
and Cyclopropylpyrans
O
O
O
N
Cl
N
b)
b)
+
&
N
B
N
O
O
N
N
Cl
OH
N
O
N
9
Cl
C
10
N
H
N
H
d)
O
c)
d)
O
O
N
N
N
N
OH
OH
N
O
N
N
H
N
H
11
12
13
13a, 13b
*
*
12a, 12b
Reagents: a) (S)-3-methylmorpholine, DIPEA rt. b) Borolane, PdCl2(dppf), K2CO3, isopropanol:water 5:1, 110C,
2h. c) Pd/C 5%, CH3CO2H, HCl/Zn 50C, 4h. * Chiral SFC d) Me3SO, NaH, DMSO, 65C, 4h.
The synthesis of 3-[4-(methanesulfonylmethyl)- and 3-[4-(2-methanesulfonylpropan-2-yl)- 6-[(3S)-3-
methylmorpholin-4-yl]pyrimidines is highlighted in Scheme 2 utilising literature intermediates D and
E44 and palladium cross coupling conditions favoured in-house to furnish the m-phenols 14 and 15, p-
phenyl ethyl ureas 16 and 17 and p-phenyl glycinamides 19 and 20. 1-ethyl-3-(4-(4-
((methylsulfonyl)methyl)-6-morpholinopyrimidin-2-yl)phenyl)urea (18) was synthesised as described
as previously44 using the modified palladium cross coupling conditions used as standard in our labs.
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Scheme 2:
14 20
to
General Synthesis of Compounds
~
~
OH
14:
15:
R1 = Me, R2 = H, R3 =
R1 = Me, R2 = Me, R3 =
O
O
N
N
N
R1
Cl
R1
R3
O
16:
17:
18:
R1 = Me, R2 = H, R3 =
R1 = Me, R2 = Me, R3 =
R1 = H, R2 = H, R3 =
a)
N
H
N
O
O
N
O
O
N
H
S
S
~
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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N
O
H
19:
20:
R1 = Me, R2 = H, R3 =
R1 = Me, R2 = Me, R3 =
D
R=H
N
R2
R2 R2
R2
N
H
E
: R=Me
Reagents: a) Borolane, PdCl2(dppf), K2CO3, isopropanol:water 5:1, 110C, 2h. .
The synthesis of 3-oxabicyclo[4.1.0]heptanes 22, 23, 24, 25, 26 and 27 was improved over the two-step
synthesis of tool compounds 12 and 13 by the initial synthesis of the 3-oxabicyclo[4.1.0]heptane pinacol
boronic ester, intermediate F and subsequent activation by conversion to the potassium 3-
oxabicyclo[4.1.0]heptan-6-yltrifluoroborate intermediate G (Scheme 3). Palladium mediated cross
couplings with intermediate G produced consistently better yields than coupling directly with
intermediate F.
Scheme 3:
Intermediate G
Synthesis of
O
O
O
b)
a)
K+
F
B
B
B-
F
F
O
O
O
O
F
G
Reagents: a) Et2Zn, CH2ClI, fluorobenzene, -5C, multiple additions, rt 16h.
b) KF/H2O, MeCN/MeOH, 2(2R,3R)-2,3-dihydroxysuccinic acid, THF rt.
The synthesis of 6-((methylsulfonyl)methyl)pyrimidine and 6-(2-(methylsulfonyl)propan-2-
yl)pyrimidine compounds 21 to 27, highlighted in Scheme 4, proceeds via the synthesis of known
intermediate H44. Suzuki-Miyaura cross-coupling chemistry, already described, furnished intermediate
I and conditions optimised specifically for the coupling of intermediate G were utilised to deliver
intermediate J in a single coupling step from the aryl chloride. Intermediate I was coupled with 3-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol to afford compound 21.
Intermediate J was reacted with methyl iodide in the presence of sodium tert-butoxide to give the
dimethylated intermediate K. Having established that the separate enantiomers were stable to the
palladium mediated cross-coupling conditions intermediates J and K were separated by chiral SFC to
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afford intermediates L, M, N and O prior to palladium-cross coupling to furnish compounds 22, 23, 24,
25, 26 and 27.
7
8
9
Scheme 4:
21 to 27
General Synthesis of Compounds
O
~
~
OH
21, 22, 23:
&
R2 =
: R2 =
: R2 =
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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44
45
46
47
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49
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O
15
O
24 25
&
a)
N
H
N
H
O
O
O
O
N
~
N
O
a)
26 27
H
&
S
S
Cl
N
N
N
O
R2
N
O
Cl
N
H
I
O
O
N
O
S
22
24
26
Cl
b)
H
a)
*
O
O
N
O
O
O
O
N
N
S
S
S
N
O
R2
N
L&M
R2
N
Cl
J
c)
O
O
23
25
27
a)
*
O
O
N
O
O
O
O
N
N
S
S
S
N
R2
N
N&O
R2
N
Cl
K

Intermediate G
Reagents: a) Borolane, PdCl2(dppf), K2CO3, isopropanol:water 5:1, 110 C, 2h. b)
Cs2CO3, toluene:water 10:1, 110C, 15h. * Chiral separation
, PdCl2,
Scheme 5
29a
29b
and
Synthesis of Compounds
O
O
O
O
O
N
O
O
N
O
O
N
S
S
N
S
S
N
N
N
Cl
OH
29a
O
P
N
H
N
H
NH2
a)
b)
O
O
O
O
N
O
O
N
O
O
N
S
S
N
S
S
N
N
O
Cl
OH
29b
Q
N
H
N
H
NH2
Reagents: a) Borolane, PdCl2(dppf), K2CO3, isopropanol:water 5:1, 110C, 2h. b) 1,1'-thiocarbonyldiimidazole,
DCM rt 4h then ethanolamine 0C to rt 18h.
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The synthesis of sub nanomolar mTOR inhibitors 29a and 29b is described in Scheme 5. Intermediates
N and O were coupled using our standard palladium cross-coupling conditions to afford intermediates
P and Q in moderate yields. Conversion of the aniline intermediates to the thioureas proceeded via a
two-step, one pot process involving initial formation of the thioisocyanates with di(imidazol-1-
yl)methanethione followed by addition of ethanolamine to afford 29a and 29b in excellent yields.
10
11
12
13
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19
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21
22
23
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Conclusion
Herein we have outlined the discovery of 3-oxabicyclo[4.1.0]heptane and demonstrated the use of in-
depth quantum mechanical calculations to predict the low energy co-planar conformation of the
cyclopropyl pyran attached to a pyrimidine ring. The anticipated co-planar geometry was confirmed
through a small molecule crystal structure of compound 12b and further supported by maintenance of
potency compared with morpholine analogues. A preliminary co-crystal structure of 12b in PI3Kδ
supported our hypothesis of co-planarity in the active site and is consistent with maintenance of the key
hydrogen bonding interaction, found in PI-103, that is common to all known morpholine based PI3K
and PIKK inhibitors. We have performed a very thorough matched molecular pair analysis and provided
a substantial data set illustrating that the incorporation of this novel moiety has increased FSp3 character
whilst removing an embedded aniline and therefore reducing the risk of genotoxic metabolites being
formed in vivo without a detrimental effect on solubility.
Following clear selectivity signals from our dataset we subsequently applied our discovery to a
published series of selective mTOR inhibitors and shown that the cyclopropyl pyran analogue 29b
retains mTOR affinity and cellular efficacy with an improved selectivity over the class I PI3K isoforms
and similar solubility profile when compared to the published 3S-methylmorpholine analogue 28.
The tractability of the single enantiomers of the CPP remains a synthetic challenge and efforts towards
a homochiral synthesis in house have, to date, proved unsuccessful with all single enantiomers described
herein being isolated by chiral chromatography. In addition, the disconnect between the metabolic
profile of the CPPs in rat and human has the potential to cause significant problems in obtaining rodent
safety cover and predicting human PK and dose using traditional allometric scaling techniques;
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however, such problems could be overcome using modern techniques such as PB/PK modelling. The
CPP matched molecular pairs have failed to address the metabolic clearance typically associated with
the 3S-methylmorpholine with consistently elevated clearance profiles, in good agreement with the
increased lipophilicity of the CPP substituent.
10
11
12
13
14
15
16
17
18
19
20
21
22
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The replacement of N-aryl saturated heterocycles with fused cyclopropanes extends well beyond the
lipid kinase field and, in principle, provides an alternative approach to control conformation besides the
use of an aryl nitrogen. A full investigation into the steric and electronic requirements to obtain co-
planar geometry has been undertaken and will be reported in due course36.
Experimental Section
General.
All solvents and reagents, unless otherwise stated, were commercially available from regular suppliers
such as Sigma-Aldrich and Fluorochem and were used as purchased without further purification. When
necessary organic phases were filtered through ISOLUTE® phase separator hydrophobic frits, which
contain a membrane which is selectively permeable to organic solutions to remove traces of water.
Intermediates were purified by flash column chromatography on Redisep® Rf flash columns using an
Isco Combi Flash Rf+ companion or similar eluting with increasing percentages of ethyl acetate in
cyclohexane. Compounds for biological testing were purified either by Mass Directed HPLC on C18
5um 30x150mM XSelect CSH columns using decreasingly polar mixtures of acetonitrile in water with
either a formic acid or ammonium carbonate modifier or by preparative HPLC on a Reveleris® prep
using C18 5um 30x100mM Waters Xbridge columns using decreasingly polar mixtures of acetonitrile
in water with an ammonium carbonate modifier or C18 5um 30x100mM Waters Sunfire columns using
decreasingly polar mixtures of acetonitrile in water with a formic acid or modifier. The purity of samples
was assessed by NMR and HPLC and mass spectroscopy and are consistent with the proposed
structures. The purity of compounds for biological testing were assessed by NMR, HPLC and mass
spectroscopy techniques and were ≥ 95% unless otherwise quoted. LCMS analysis was carried out on
a Waters Acquity UPLC instrument equipped with a BEH column (50 mm x 2.1 mm, 1.7 µm packing
diameter) and Waters Micromass ZQ MS using alternate-scan positive and negative electrospray.
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Analytes were detected as a summed UV wavelength of 210–350 nm. Samples were eluted with using
decreasingly polar mixtures of acetonitrile in water with either a formic acid or ammonium carbonate
modifier. Electrospray mass spectral data was obtained using a Waters ZQ/LC/mass spectrometer.
Proton and carbon nuclear magnetic resonance (1H NMR and 13C NMR) spectra were recorded in
deuterated solvents at ambient temperature on a Bruker AVI (400 MHz), or Bruker AVII+ (600 MHz)
spectrometer (with cryoprobe). Chemical shifts δ are reported in parts per million (ppm) relative to
tetramethylsilane and are internally referenced to the residual solvent peak. Coupling constants (J) are
given in hertz to the nearest 0.1 Hz. The following abbreviations have been used; s, singlet; d, doublet;
t, triplet; q quartet; br, broad; and m, multiplet. High-resolution mass spectra were recorded on a Waters
XEVO G2-XS quadrupole time-of-flight mass spectrometer, with analytes separated on an Acquity
UPLC CSH C18 column (100mm x 2.1mm, 1.7μm packing diameter). LC conditions were 0.8 mL/min
flow rate, 50 °C, injection volume 0.2 µL, using a gradient elution with (A) H2O containing 0.1% (v/v)
formic acid and (B) acetonitrile containing 0.1% (v/v) formic acid. Gradient conditions were initially
3% B, increasing linearly to 100% B over 8.5 min, remaining at 100% B for 0.5 min then decreasing
linearly to 3% B over 0.5 min followed by an equilibration period of 0.5 min prior to the next injection.
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General Method A
The appropriate chloropyrimidine or dichloropyrimidine (1 equiv.) was treated with the appropriate
4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.8 - 1.2 equiv.), PdCl2(dppf) (0.08 equiv.) and potassium
carbonate (2 equiv.) and the mixture suspended in degassed Isopropanol : Water 5 : 1. The reaction
mixture was bubbled through with nitrogen for 1 minute prior to heating at 110 °C for 2 h. The reaction
mixture was filtered through celite and eluted with EtOAc. The eluent was washed with water, filtered
through a hydrophobic frit and concentrated by blow down. Intermediate compounds were purified by
normal phase column chromatography, final compounds were purified by mass directed HPLC.
General Method B
Relevant 2-chloro-pyrimidine (1 equiv.) was treated with tert-butyl methyl(2-oxo-2-((4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)amino)ethyl)carbamate (1.2 equiv.), PdCl2(dppf) (0.08
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equiv.) and potassium carbonate (2 equiv.) and suspended in degassed Isopropanol : Water 5 : 1. The
reaction mixture was bubbled through with nitrogen for 1 minute prior to heating at 110 °C for 2 h. The
reaction mixture was filtered through celite and eluted with EtOAc. The eluent was washed with water,
filtered through a hydrophobic frit and concentrated by blow down. The sample was resuspended in
DCM and treated with TFA (~10 equiv.) and allowed to stand for 1 h prior to concentration by low
down and purification by mass directed HPLC.
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General Method C
Sodium hydride (2 equiv.) was treated with trimethyloxosulfonium chloride (2 equiv.) under a nitrogen
atmosphere. Anhydrous DMSO (2 mL/mmol) was added dropwise and the reaction mixture stirred for
20 min. A solution of relevant dihydro-2H-pyran-4-yl)pyrimidine (1 equiv.) in anhydrous DMSO (2
mL/mmol) was added and the reaction mixture stirred at 65 °C for 4 h. The reaction mixture was
partitioned between EtOAc and water and the aqueous phase extracted three times with EtOAc. The
combined organic phase was concentrated in vacuo and purified by column chromatography and
recrystallisation from toluene.
General Method D
A solution of relevant aniline (1 equiv.) in dichloromethane at 0 °C was treated portion wise with 1,1'-
thiocarbonyldiimidazole (2 equiv.) and the mixture was stirred at ambient temperature for 4 h. The
mixture was re-cooled to 0 °C and ethanolamine (10 equiv.) was added. The mixture was stirred at
ambient temperature for 18 h. The reaction mixture was concentrated in vacuo and the residue purified
by Mass Directed HPLC.
4-(2-Chloropyrimidin-4-yl)morpholine (Intermediate A)
To a clear, colourless solution of morpholine (0.871 mL, 10.00 mmol) and DIPEA (3.49 mL, 20.00
mmol) in anhydrous N,N-dimethylformamide (DMF) (13 mL), was added 2,4-dichloropyrimidine
(1.490 g, 10 mmol) dropwise and the resulting colourless solution was stirred at 20 °C for 1 h prior to
concentration in vacuo. The crude product was purified by column chromatography to afford 4-(2-
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chloropyrimidin-4-yl)morpholine (1.401 g, 7.02 mmol, 70% yield) as an amorphous white solid. H
NMR (400 MHz, CDCl3): δ ppm 8.07 (d, J = 6.06 Hz, 1H), 6.39 (d, J = 6.06 Hz, 1H), 3.74-3.82 (m,
4H), 3.62- 3.67 (m, 4H); ESI (m/z): [M+H]+ = 200
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(3S)-4-(2-Chloropyrimidin-4-yl)-3-methylmorpholine (Intermediate B)
A suspension of 2,4-dichloropyrimidine (500 mg, 3.36 mmol) in acetonitrile (6.7 mL) in a microwave
vial was treated with DIPEA (1759 μl, 10.07 mmol) and (S)-3-methylmorpholine (381 μl, 3.36 mmol).
The vial was sealed and stirred at ambient temperature for 19.5 h prior to addition of further (S)-3-
methylmorpholine (38.1 μl, 0.336 mmol). The reaction mixture was stirred at ambient temperature for
a further 2 h. The reaction mixture was partitioned between EtOAc (50 mL) and water (50 mL). The
aqueous phase was extracted with further EtOAc (2 x 20 mL) and the combined organic phase was
filtered through a hydrophobic frit and concentrated in vacuo. The residue was purified by column
chromatography to afford (3S)-4-(2-chloropyrimidin-4-yl)-3-methylmorpholine (483 mg, 67% yield).
1H NMR (600 MHz, DMSO-d6): δ ppm 1.19 (d, J = 6.8 Hz, 3 H) 3.16 (td, J = 12.9, 3.9 Hz, 1 H) 3.42
(td, J = 11.9, 3.1 Hz, 1 H) 3.57 (dd, J = 11.6, 3.2 Hz, 1 H) 3.70 (d, J = 11.6 Hz, 1 H) 3.92 (dd, J = 11.6,
3.9 Hz, 1 H) 3.98 (br d, J = 13.0Hz, 1 H) 4.33 (br s, 1 H) 6.79 (d, J = 6.2 Hz, 1 H) 8.10 (d, J = 6.2 Hz,
1 H); ESI (m/z): [M+H]+ = 214
2-Chloro-4-(3,6-dihydro-2H-pyran-4-yl)pyrimidine (Intermediate C)
From General Method A
Yield 6.4%. 1H NMR (400 MHz, CDCl3) δ ppm 8.57 (d, J = 5.14 Hz, 1H), 7.28 (d, J = 5.14 Hz, 1H),
7.26 (d, J = 5.14 Hz, 1H), 7.10 (m, 1H), 4.42 (q, J = 2.93 Hz, 2H), 3.95 (t, J = 5.38 Hz, 2H), 2.59 (m,
2H) ; ESI (m/z): [M+H]+ = 197
4,4,5,5-Tetramethyl-2-{3-oxabicyclo[4.1.0]heptan-6-yl}-1,3,2-dioxaborolane (Intermediate F)
From General Method A
A solution of 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1050 mg, 5
mmol) in fluorobenzene (10 mL) was cooled to -5 °C and was treated slowly with diethylzinc in hexanes
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(25.00 mL, 25.00 mmol) over 2 mins. The mixture was stirred at -5 °C for 5mins prior to treating with
a solution of chloroiodomethane (8819 mg, 50.0 mmol) in fluorobenzene (5 ml) dropwise over 5min.
The mixture was stirred at -5 °C for 10 min prior to three subsequent additions of diethylzinc and
chloroiodomethane in the same manner. The reaction mixture was allowed to warm to room temperature
and stirred for 16 h. The suspension was partitioned between aqueous NH4Cl (75 ml) and diethyl ether
(2 x 50 ml) and the dried over MgSO4. The extract was evaporated onto florisil and purified by column
chromatography to give 2-(3-oxabicyclo[4.1.0]heptan-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(485 mg, 2.164 mmol, 43.3% yield).
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1H NMR (400 MHz, CDCl3): δ ppm 4.02 (d, J = 11.25 Hz, 1H), 3.85 (dd, J = 3.42, 11.25 Hz, 1H), 3.61
(ddd, J = 2.45, 6.36, 11.25 Hz, 1H), 3.17 (dt, J = 4.89, 11.25 Hz, 1H), 2.00-2.08 (m, 1H), 1.62 -1.73
(m, 1H), 1.24 (s, 6H), 1.08 (br dd, J = 3.18, 5.62 Hz, 1H), 0.96 (dd, J = 2.93, 8.31 Hz, 1H), 0.66 (dd, J
= 3.18, 5.62 Hz, 1H)
Potassium trifluoro({3-oxabicyclo[4.1.0]heptan-6-yl})boranuide (Intermediate G)
2-(3-oxabicyclo[4.1.0]heptan-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (5 g, 22.31 mmol) was
dissolved in anhydrous acetonitrile (55 mL) and anhydrous MeOH (55 mL) under a nitrogen
atmosphere. Aqueous potassium fluoride (5.2 g, 90 mmol) in water (20 mL) was added and the
suspension was stirred at room temperature under nitrogen for 10 minutes. (2R,3R)-2,3-
dihydroxysuccinic acid (6.70 g, 44.6 mmol) was added followed by THF (2.5 mL). The reaction mixture
was stirred for 1.25 h and left standing over night after which the precipitate filtered off and washed
with acetonitrile. The filtrate was concentrated in vacuo and azeotroped with toluene (3 x 50 mL) and
triturated with diethyl ether (3 x 30 mL) to give potassium trifluoro({3-oxabicyclo[4.1.0]heptan-6-
yl})boranuide 4.83 g (106% yield).
1H NMR (400 MHz, CDCl3): δ ppm 4.02 (d, J = 11.25 Hz, 1H), 3.85 (dd, J = 3.42, 11.25 Hz, 1H), 3.61
(ddd, J = 2.45, 6.36, 11.25 Hz, 1H), 3.17 (dt, J = 4.89, 11.25 Hz, 1H), 1.86-2.11 (m, 1H), 1.58-1.85 (m,
1H), 1.20-1.29 (m, 9H), 1.03-1.16 (m, 1H), 0.96 (dd, J = 2.93, 8.31 Hz, 1H), 0.66 (dd, J = 3.18, 5.62
Hz, 1H)
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2-Chloro-4-(3,6-dihydro-2H-pyran-4-yl)-6-(methanesulfonylmethyl)pyrimidine (Intermediate I)
From General Method A
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Isolated as a mixture of 2,4-bis(3,6-dihydro-2H-pyran-4-yl)6((methylsulfonyl)methyl) pyrimidine with
2-chloro-4-(3,6-dihydro-2H-pyran-4-yl)-6-((methylsulfonyl)methyl)pyrimidine and used crude in the
synthesis of compound 21.
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2-Chloro-4-(methanesulfonylmethyl)-6-{3-oxabicyclo[4.1.0]heptan-6-yl}pyrimidine
(Intermediate J)
2,4-dichloro-6-((methylsulfonyl)methyl)pyrimidine
(200
mg,
0.830
mmol),
(3-
oxabicyclo[4.1.0]heptan-6-yl)trifluoro-l4-borane, potassium salt (203 mg, 0.995 mmol), PdCl2(dppf)
(60.7 mg, 0.083 mmol) and caesium carbonate (541 mg, 1.659 mmol) were suspended in toluene (2000
µl) : water (200 µl) and the resulting mixture was degassed under a flow of nitrogen, sealed, and heated
to 110 °C with stirring for 15 h. The reaction mixture was cooled to room temperature and treated with
EtOAc (5 mL), the mixture was filtered through Celite and eluted with EtOAc (3 x 5 mL). The filtrate
was evaporated in vacuo and purified by column chromatography (98.8 mg, 0.326 mmol, 39.3% yield).
1H NMR (400MHz, CDCl3): δ ppm 7.29 (s, 1H), 4.32 (s, 2H), 3.91 - 4.03 (m, 2H), 3.69 (ddd, 1H, J =
11.6 Hz, J = 5.7 Hz, J = 4.4 Hz), 3.42 (ddd, 1H, J = 11.7 Hz, J = 9.3 Hz, J = 5.1 Hz), 3.03 (s, 3H), 2.56
(dt, 1H, J = 13.9 Hz, J = 4.6 Hz), 2.09 (ddd, 1H, J = 14.2 Hz, J = 9.0 Hz, J = 5.9 Hz), 1.87 - 2.01 (m,
1H), 1.53 (dd, 1H, J = 9.2 Hz, J = 4.0 Hz), 1.33 (dd, 1H, J = 6.7 Hz, J = 4.3 Hz). ESI (m/z): [M+H]+ =
303
Intermediate J was purified by chiral SFC for chiral separation to afford:
2-Chloro-4-(methanesulfonylmethyl)-6-[(1S, 6R)-3-oxabicyclo[4.1.0]heptan-6-yl]pyrimidine
(Intermediate K)
1H NMR (400 MHz, DMSO-d6): δ ppm 7.61 (s, 1H), 4.68 (s, 2H), 3.78-3.97 (m, 2H), 3.55-3.62 (m,
1H), 3.32-3.41 (m, 1H), 3.11 (s, 3H), 2.44-2.57 (m, 1H), 1.96 (ddd, J = 5.70, 8.82, 14.20 Hz, 1H), 1.73-
1.86 (m, 1H), 1.42 (dd, J = 3.95, 9.21 Hz, 1H), 1.25 (dd, J = 4.17, 6.80 Hz, 1H); ESI (m/z): [M+H]+ =
303; [α]D20 = -67
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2-Chloro-4-(methanesulfonylmethyl)-6-[(1R,6S)-3-oxabicyclo[4.1.0]heptan-6-yl]pyrimidine
(Intermediate L)
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1H NMR (400 MHz, DMSO-d6): δ ppm 7.61 (s, 1H), 4.68 (s, 2H), 3.80-3.96 (m, 2H), 3.52-3.63 (m,
1H), 3.31-3.40 (m, 1H), 3.11 (s, 3H), 2.45-2.55 (m, 1H), 1.96 (ddd, J = 5.70, 8.82, 14.20 Hz, 1H), 1.73-
1.86 (m, 1H), 1.42 (dd, J = 4.06, 9.32 Hz, 1H), 1.25 (dd, J = 4.17, 6.80 Hz, 1H); ESI (m/z): [M+H]+ =
303; [α]D20 72
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2-Chloro-4-(2-methanesulfonylpropan-2-yl)-6-{3-oxabicyclo[4.1.0]heptan-6-yl}pyrimidine
(Intermediate M)
To a solution of 4-(3-oxabicyclo[4.1.0]heptan-6-yl)-2-chloro-6-((methylsulfonyl)methyl)pyrimidine
(300 mg, 0.991 mmol) in anhydrous tetrahydrofuran (THF, 4 mL) was added sodium 2-methylpropan-
2-olate 2M in THF (1.486 mL, 2.97 mmol) followed by dropwise addition of iodomethane (0.155 mL,
2.477 mmol). The resulting mixture was stirred at ambient temperature for 5 h. The reaction mixture
was quenched with saturated ammonium chloride (10 mL) and extracted with DCM (2 x 10 mL). The
organic phase was concentrated in vacuo and purified by column chromatography to afford 2-chloro-4-
(2-methanesulfonylpropan-2-yl)-6-{3-oxabicyclo[4.1.0]heptan-6-yl}pyrimidine (328 mg, 0.985 mmol,
99% yield).
1H NMR (400 MHz, CDCl3): δ ppm 7.42 (s, 1H), 3.99 (d, J = 2.93 Hz, 2H), 3.70 (ddd, J = 4.40, 5.75,
11.62 Hz, 1H), 3.44 (ddd, J = 5.26, 9.23, 11.68 Hz, 1H), 2.91 (s, 3H), 2.56 (td, J = 4.86, 14.00 Hz, 1H),
2.05-2.19 (m, 2H), 1.92-1.99 (m, 1H), 1.84-1.89 (m, 7H), 1.48-1.55 (m, 1H), 1.25-1.36 (m, 2H); ESI
(m/z): [M+H]+ = 328
Intermediate M was purified by chiral SFC for chiral separation to afford:
2-Chloro-4-(2-methanesulfonylpropan-2-yl)-6-[(1R,6S)-3-oxabicyclo[4.1.0]heptan-6-
yl]pyrimidine (Intermediate N)
1H NMR (400 MHz, CDCl3): δ ppm 7.42 (s, 1H), 3.99 (d, J = 2.69 Hz, 2H), 3.70 (ddd, J = 4.40, 5.87,
11.74 Hz, 1H), 3.44 (ddd, J = 5.14, 9.29, 11.74 Hz, 1H), 2.91 (s, 3H), 2.56 (td, J = 4.71, 14.06 Hz, 1H),
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2.12 (ddd, J = 5.87, 8.93, 14.31 Hz, 1H), 1.95 (br d, J = 2.69 Hz, 1H), 1.87 (s, 6H), 1.52 (dd, J = 4.16,
9.29 Hz, 1H), 1.32 (dd, J = 4.28, 6.72 Hz, 1H); ESI (m/z): [M+H]+ = 331; [α]D20 = -72
and
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2-Chloro-4-(2-methanesulfonylpropan-2-yl)-6-[(1S,6R)-3-oxabicyclo[4.1.0]heptan-6-
yl]pyrimidine (Intermediate O)
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Yield 66%. 1H NMR (400 MHz, CDCl3): δ ppm 7.42 (s, 1H), 3.99 (d, J = 2.69 Hz, 2H), 3.70 (ddd, J =
4.40, 5.87, 11.74 Hz, 1H), 3.44 (ddd, J = 5.26, 9.23, 11.68 Hz, 1H), 2.91 (s, 3H), 2.56 (td, J = 4.77,
13.94 Hz, 1H), 2.12 (ddd, J = 5.87, 8.99, 14.24 Hz, 1H), 1.95 (br d, J = 2.69 Hz, 1H), 1.87 (s, 6H), 1.52
(dd, J = 4.16, 9.29 Hz, 1H), 1.32 (dd, J = 4.28, 6.72 Hz, 1H). ESI (m/z): [M+H]+ = 331. [α]D20 = 78
4-[4-(2-Methanesulfonylpropan-2-yl)-6-[(1R,6S)-3-oxabicyclo[4.1.0]heptan-6-yl]pyrimidin-2-
yl]aniline (Intermediate P)
From Intermediate N, General Method A
Yield 43%. 1H NMR (400 MHz, DMSO-d6): δ ppm 7.99-8.20 (d, J = 8.80 Hz, 2H), 7.25 (s, 1H), 6.63
(d, J = 8.80 Hz, 2H), 5.65 (s, 2H), 3.79-4.04 (m, 2H), 3.51-3.71 (m, 1H), 3.38 (ddd, J = 5.26, 8.77,
11.62 Hz, 1H), 2.97 (s, 3H), 2.61-2.77 (dt, J = 5.12, 14.09 Hz,1H), 2.00 (ddd, J = 5.59, 8.60, 14.09 Hz,
1H), 1.88-1.95 (m, 1H), 1.45 (dd, J = 3.73, 9.21 Hz, 1H), 1.15 (dd, J = 3.84, 6.47 Hz, 1H); ESI (m/z):
[M+H]+ = 388; [α]D20 = -78
4-[4-(2-Methanesulfonylpropan-2-yl)-6-[(1S,6R)-3-oxabicyclo[4.1.0]heptan-6-yl]pyrimidin-2-
yl]aniline (Intermediate Q)
From Intermediate O, General Method A
Yield 47%. 1H NMR (400 MHz, DMSO-d6): δ ppm 8.04-8.18 (d, J = 8.80 Hz,, 2H), 7.25 (s, 1H), 6.63
(d, J = 8.80 Hz, 2H), 5.65 (s, 2H), 3.78-4.01 (m, 2H), 3.53-3.65 (m, 1H), 3.38 (ddd, J = 5.15, 8.66,
11.62 Hz, 1H), 2.97 (s, 3H), 2.66 (td, J = 4.96, 13.98 Hz, 1H), 2.00 (ddd, J = 5.59, 8.66, 14.03 Hz, 1H),
1.87-1.95 (m, 1H), 1.45 (dd, J = 3.73, 9.21 Hz, 1H), 1.15 (dd, J = 3.84, 6.47 Hz, 1H); ESI (m/z): [M+H]+
= 388; [α]D20 = 67
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3-[4-(3,6-Dihydro-2H-pyran-4-yl)pyrimidin-2-yl]phenol (9)
5
6
7
From Intermediate C, General method A
Yield 67%. 1H NMR (CDCl3, 400 MHz): δ ppm 8.74 (d, J = 5.4 Hz, 1H), 8.07 (dt, J = 7.8, 1.2 Hz, 1H),
8.00 (dd, J = 2.4, 1.5 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.22 (d, J = 5.4 Hz, 1H), 7.08 (dt, J = 2.9, 1.5
Hz, 1H), 7.00 (ddd, J = 8.1, 2.6, 0.9 Hz, 1H), 5.95-6.59 (m, 1H), 4.46 (q, J = 2.7 Hz, 2H), 4.00 (t, J =
5.5 Hz, 2H), 2.67-2.74 (m, 2H); 13C NMR (101 MHz, CDCl3): δ ppm 163.6, 163.3, 157.4, 156.2, 139.3,
133.1, 130.4, 129.9, 120.6, 118.0, 115.0, 113.2, 65.8, 64.3, 25.1; HRMS-ESI (m/z): calcd for
C15H15N2O2, 255.1133; found [M+H]+ 255.1121
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3-(4-{3-Oxabicyclo[4.1.0]heptan-6-yl}pyrimidin-2-yl)phenol (12)
From Compound 7, General Method C
Yield 17%. 1H NMR (400 MHz, CDCl3): δ ppm 8.66 (d, J = 5.56 Hz, 1H), 8.03 (d, J = 7.83 Hz, 1H),
7.90-7.96 (m, 1H), 7.37 (t, J = 7.96 Hz, 1H), 7.11 (d, J = 5.56 Hz, 1H), 6.98 (dd, J = 2.27, 7.58 Hz, 1H),
4.05 (d, J = 2.78 Hz, 2H), 3.67-3.81 (m, 1H), 3.50 (ddd, J = 5.05, 9.09, 11.62 Hz, 1H), 2.71 (td, J =
4.80, 13.89 Hz, 1H), 2.15 (ddd, J = 5.68, 8.91, 14.21 Hz, 1H), 1.99 (tdd, J = 2.91, 6.22, 9.19 Hz, 1H),
1.56 (dd, J = 4.04, 9.35 Hz, 1H), 1.26 (dd, J = 4.04, 6.57 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ ppm
173.2, 163.3, 156.6, 156.0, 139.4, 129.8, 120.6, 117.8, 114.8, 114.2, 65.6, 64.0, 25.2, 23.2, 23.1, 22;
HRMS-ESI (m/z): calcd for C16H17N2O2, 269.1290; found [M+H]+ 269.1276
The two enantiomers were separated by chiral HPLC on Chiralpak IC 250x20mm eluting with
Heptane:EtOH: Isopropylamine 90:10:0.2 to give 3-{4-[(1R,6S)-3-oxabicyclo[4.1.0] heptan-6-
20
yl]pyrimidin-2-yl}phenol (12a) [α]D = -100 and 3-{4-[(1S,6R)-3-oxabicyclo[4.1.0] heptan-6-
yl]pyrimidin-2-yl}phenol (12b) [α]D20 = 70
3-(2-Hydroxyethyl)-1-{4-[4-(2-methanesulfonylpropan-2-yl)-6-[(1S,6R)-3-oxabicyclo[4.1.0]
heptan-6-yl]pyrimidin-2-yl]phenylthiourea (29b)
From Intermediate P, General Method D
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Yield 73%. 1H NMR (600 MHz, DMSO-d6): δ ppm 9.75-9.97 (m, 1H), 8.35 (d, J = 8.80 Hz, 2H), 7.91
(br s, 1H), 7.69 (d, J = 8.80 Hz, 2H), 4.82 (br s, 1H), 3.92-3.99 (m, 1H), 3.84-3.91 (m, 1H), 3.54-3.67
(m, 4H), 3.41 (ddd, J = 5.32, 8.71, 11.65 Hz, 1H), 2.99 (s, 3H), 2.71 (td, J = 4.95, 13.94 Hz, 1H), 1.93-
2.09 (m, 2H), 1.84 (s, 6H), 1.52 (dd, J = 4.03, 9.17 Hz, 1H), 1.21 (dd, J = 4.03, 6.24 Hz, 1H); 13C NMR
(151 MHz, DMSO-d6): δ ppm 180.7, 174.3, 166.1, 162.0, 142.6, 132.6, 128.8, 122.2, 122.1, 113.1, 67.0,
65.1, 63.5, 59.6, 47.0, 36.5, 25.1, 23.5, 23.4, 22.8, 21.0; HRMS-ESI (m/z): calcd for C23H31N4O4S2,
491.1787; found [M+H]+ 491.1787; [α]D20 = 42
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Acknowledgements
The authors would like to thank Alan Kennedy, Department of Pure and Applied Chemistry, University
of Strathclyde, 295 Cathedral Street Glasgow, G1 1XL, U.K. for the small molecule crystal structure of
12b.
Abbreviations used
mTOR, mammalian target of rapamycin; mTORC1, mTOR complex1; mTORC2, mTOR complex 2;
PI3K, Phosphoinositide 3-kinase; PIKK, Phosphatidylinositol 3-kinase-related kinases; AKT, Protein
Kinase B; MOE, Molecular Operating Environment; TFA, trifluoracetic acid; EtOAc, ethyl acetate;
THF, tetrahydrofuran; DIPEA, diisopropylethylamine; DCM, dichloromethane; MeOH, Methanol;
DMSO, dimethylsulphoxide; MS, mass spectroscopy; HPLC, high pressure liquid chromatography.
Ancillary Information
Included in the supporting information are:
Molecular formular strings; data for the small molecule crystal structure of 9b; a low occupancy co-
crystal structure of 12b; protocols for the PI3K, mTOR and Akt assays and experimental data for the
remaining novel compounds 5 to 29 inclusive.
Contact emails: heather.x.hobbs@gsk.com; Gianpaolo.Bravi@dotmatics.com; ianb22@talktalk.net;
maire.a.convery@gsk.com;
hannah.r.davies@gsk.com;
graham.g.inglis@gsk.com;
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sandeep.8.pal@gsk.com;
simon.2.peace@gsk.com;
joanna.m.redmond@gsk.com
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