A Conformational Restriction Strategy for the Identification of a Highly Selective Pyrimido-pyrrolo-oxazine mTOR Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.9b00972

The mechanistic target of rapamycin (mTOR) plays a pivotal role in growth and tumor progression and is an attractive target for cancer treatment. ATP-competitive mTOR kinase inhibitors (TORKi) have the potential to overcome limitations of rapamycin derivatives in a wide range of malignancies. Herein, we exploit a conformational restriction approach to explore a novel chemical space for the generation of TORKi. Structure-activity relationship (SAR) studies led to the identification of compound 12b with a ～450-fold selectivity for mTOR over class I PI3K isoforms. Pharmacokinetic studies in male Sprague Dawley rats highlighted a good exposure after oral dosing and a minimum brain penetration. CYP450 reactive phenotyping pointed out the high metabolic stability of 12b. These results identify the tricyclic pyrimido-pyrrolo-oxazine moiety as a novel scaffold for the development of highly selective mTOR inhibitors for cancer treatment.

Full text of DOI:10.1021/acs.jmedchem.9b00972

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Article
A Conformational Restriction Strategy for the Identification of
a Highly Selective Pyrimido-Pyrrolo-Oxazine mTOR Inhibitor
Chiara Borsari, Denise Rageot, Alix Dall’Asen, Thomas Bohnacker, Anna Melone,
Alexander Markus Sele, Eileen Jackson, Jean-Baptiste Langlois, Florent Beaufils,
Paul Hebeisen, Doriano Fabbro, Petra Hillmann, and Matthias P. Wymann
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00972 • Publication Date (Web): 29 Aug 2019
Downloaded from pubs.acs.org on August 29, 2019
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A Conformational Restriction Strategy for the Identification of a Highly
Selective Pyrimido-Pyrrolo-Oxazine mTOR Inhibitor
†
†
‡
†
†
Chiara Borsari, Denise Rageot, Alix Dall’Asen, Thomas Bohnacker, Anna Melone, Alexander
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M. Sele, Eileen Jackson, Jean-Baptiste Langlois, Florent Beaufils, Paul Hebeisen, Doriano Fabbro,
‡
†*
Petra Hillmann, Matthias P. Wymann
†Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
‡PIQUR Therapeutics AG, Hochbergerstrasse 60, 4057 Basel, Switzerland
*
Correspondence to: matthias.wymann@unibas.ch, Dept. Biomedicine, University of Basel,
Mattenstrasse 28, 4058 Basel, Switzerland; Tel. +41 61 207 5046; Fax. +41 61 207 3566
KEYWORDS
Conformational restriction strategy; tricyclic pyrimido-pyrrolo-oxazines; mammalian or mechanistic
target of rapamycin (mTOR); mTOR kinase inhibitors; ATP-competitive; pharmacokinetic studies;
cancer.
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ABSTRACT
The mechanistic target of rapamycin (mTOR) plays a pivotal role in growth and tumor progression
and is an attractive target for cancer treatment. ATP-competitive mTOR kinase inhibitors (TORKi) have
the potential to overcome limitations of rapamycin-derivatives in a wide range of malignancies. Herein,
we exploit a conformational restriction approach to explore a novel chemical space for the generation
of TORKi. Structure Activity Relationship (SAR) studies led to the identification of compound 12b with
a ∼450-fold selectivity for mTOR over class I PI3K isoforms. Pharmacokinetic studies in male Sprague
Dawley rats highlighted a good exposure after oral dosing and a minimum brain penetration. CYP450
reactive phenotyping pointed out the high metabolic stability of 12b. These results identify the tricyclic
pyrimido-pyrrolo-oxazine moiety as a novel scaffold for the development of highly selective mTOR
inhibitors for cancer treatment.
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INTRODUCTION
The mechanistic target of rapamycin (mTOR, also mammalian TOR) is a key signaling node in the
phosphoinositide 3-kinase (PI3K)/mTOR signaling pathway and operates downstream of PI3K. mTOR
is a serine/threonine protein kinase and is part of two functionally distinct multiprotein complexes,
mammalian target of rapamycin complex 1 (mTORC1) and mammalian target of rapamycin complex 2
(mTORC2).1,2 TORC1 integrates inputs from cell surface receptors, cellular energy, stress levels,
availability of oxygen and amino acids. Cell surface receptors activate PI3K to produce PtdIns(3,4,5)P3,
which serves as a docking site for protein kinase B (PKB/Akt) and phosphoinositide-dependent protein
kinase 1 (PDK1). This results in phosphorylation of PKB/Akt at two regulatory sites, Thr308 by PDK1
and Ser473 by mTORC2, and other hydrophobic motif kinases.3-6
Through the activation of S6 kinase (S6K), TORC1 promotes phosphorylation of ribosomal protein
7
S6, which is a key regulator of protein and lipid synthesis. TORC1 also regulates lysosome function
and autophagy, while TORC2 promotes cytoskeletal rearrangement, cell survival, and cell cycle entry.8
The PI3K/mTOR axis is involved in many cellular processes including cell growth, proliferation and
metabolism, and it has been found to be dysregulated in fatal diseases, such as cancer, metabolic,
cardiovascular, and neurological disorders.5,9-12
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The pivotal role of mTOR in numerous human cancers has made this Ser/Thr -kinase a therapeutic
target for cancer treatment.
Rapamycin and its analogs (rapalogs) are allosteric inhibitors of TORC1 (Figure 1) and form a
TORC1/rapalog/FK506 binding protein 12 (FKBP12) complex.13 Rapalogs have been extensively
investigated in clinical trials in oncology14-17 and have been approved for a number of cancer treatments
such as lung, gastrointestinal neuroendocrine, noncancerous kidney, and advanced pancreatic tumors,
advanced ER+/HER2-negative breast and endometrial cancer, mantle cell lymphoma, tuberous sclerosis
complex (TSC), and pediatric subependymal giant cell astrocytoma (SEGA).16,18,19
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Their efficacy as single agents in major solid tumors, however, has turned out to be limited. This
might be explained by the selective allosteric inhibition of TORC1, which i) leaves mTORC2 intact;
and (ii) suppresses TORC1-dependent feedback loops.21-24 Small molecule ATP-competitive TORKi
target both mTORC1 and mTORC2. Hence, these inhibitors efficiently block two branches of PI3K
downstream signaling: i) one via PKB/Akt to tuberous sclerosis complex (TSC) and Ras homolog
enriched in brain (Rheb1) controlling TORC1, and ii) the PtdIns(3,4,5)P -medited Sin1-dependent
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TORC2 activation,25 explaining their robust antitumor efficacy.26 However, by targeting the ATP-
binding site of mTOR, TORKi potentially inhibit other protein and lipid kinases at elevated doses,
especially the structurally related PI3Ks. Thus, the development of highly selective TORKi is an
ongoing challenge. Structurally distinct mTOR-selective inhibitors have been reported and evaluated in
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a wide range of malignancies. Among the most noteworthy examples, CC-223 (55) , Sapanisertib
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(
INK128, MLN0128, 56) , and Vistusertib (AZD2014, 57) have been explored in clinical trials, and
were found superior to rapalogs in terms of cytostatic and cytotoxic potential (see Figure 1 for chemical
structures).30
Very recently, we have reported on PQR620 (58) (Figure 1), a highly selective and potent ATP-
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1
competitive mTOR inhibitor, targeting both mTORC1 and mTORC2 kinase activities. PQR620 (58)
had been discovered through chemical modification of PQR309 (1), a pan-PI3K and moderate mTOR
inhibitor currently in clinical trials for the treatment of lymphoma and solid tumors.32-35
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Allosteric Inhibitors (TORC1)
ATP-Competitive Inhibitors (TORC1 and TORC2)
RO
O
O
O
N
O
N
N
N
O
O
OH
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HO
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NH2
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CHF2
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H
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NH2
NH2
Rapamycin: R = H
Rapalogs: R  H
CC223 (55)
INK128 (56)
AZD2014 (57)
PQR620 (58)
Figure 1. Chemical structures of rapamycin and rapalogs, and a selection of ATP-competitive mTOR kinase
inhibitors (TORKi).
Here, we report the discovery of conformationally restricted compounds as a novel scaffold for the
development of highly selective TORKIs. The design of conformationally constrained analogs is often
used to minimize the entropic loss associated to target binding, to enhance the potency for the target and
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to increase the selectivity. This conformational restriction approach explores a novel chemical space
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and secured intellectual property. An extensive Structure Activity Relationship (SAR) study led us to
the identification of a highly selective and potent ATP-competitive inhibitor 12b. Compound 12b is
orally bioavailable, metabolically stable and has a minimal brain permeability, an advantage in the
treatment of systemic tumors. Our results disclose the tricyclic pyrimido-pyrrolo-oxazine moiety as an
innovative scaffold for selectively targeting mTOR kinase.
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RESULTS AND DISCUSSION
Rigidification Strategy.
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In our rigidification strategy, we started from PQR309 (1) and introduced a methylene bridge between
one of the morpholines and the aromatic core (Figure 2). This led to the conversion of the triazine ring
into a pyrimidine core. Moreover, we have removed the trifluoromethyl group from the 2-aminopyridine
moiety (Figure 2) to alter lipophilicity, increase solubility and change electronic and steric parameters.38
O
N
O
N
F
F
F
N
N
X
N
N
N
N
Y
O
O
N
NH2
N
NH2
PQR309 (1)
X = N; Y = C
or
X = C; Y = N
K mTOR = 93.4 nM
i
K p110 = 15.6 nM
i
Figure 2. Strategy for the development of mTOR selective inhibitors starting from PQR309 (1): rigidification
strategy (red dotted lines) and removal of trifluoromethyl group from the 2-aminopyridine moiety (blue).
For the designed compounds, two different regioisomers exist, each with two enantiomers (Table 1,
2
a/2b and 2c/2d). To evaluate the rigidification effect on compounds activity, compounds 2a-d were
evaluated for in vitro binding [K for mTOR and PI3Kα (p110α)] and for PI3K/mTOR signaling in
i
A2058 cells (IC for phosphorylated S6 to detect mTORC1 activity, and protein kinase B (PKB/Akt)
5
0
phosphorylation on Ser473 to detect mTORC2 activity). One regioisomer is more potent both in cells
and in vitro, as highlighted by comparing 2a with 2c and 2b with 2d. Compound 2a was >9-fold more
potent in cells and >11-fold more potent in vitro than 2c (Table 1).
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Table 1. Conformationally restricted regioisomers 2a/2b and 2c/2d.
O
N
O
N
O
N
O
N
(S)
N
(R)
N
N
N
N
N
Ar =
N
Ar
N
N
Ar
Ar
Ar
O
N
O
N
N
2b
(S)
2c
(R)
2d
N
NH2
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2a
O
O
Cellular Assays
in vitro Binding Assays
Selectivity
Ki(p110α
)/Ki(mTOR
)
Comp.
Name
a
b
IC50 [nM]
i
K [nM]
clogPc
pPKB
S473
pS6
S235/236
p110α
mTOR
PQR309 (1)
139
194
205
164
17
76
62
69
0.27
1.1
3.11
2.65
2.65
2.65
2.65
2
a
2b
118
96
432
867
541
24
18.3
1.1
2
c
1871
1560
1790
1857
812
1279
2
d
0.4
aPKB phosphorylation on Ser473 and ribosomal S6 phosphorylation on Ser235/236 were analyzed in A2058 cells exposed
to the indicated inhibitors and subsequent detection of phosphoproteins in an in-cell Western assay. Each experiment performed
b
n = 2. LogIC50s and Standard Errors are reported in Table S7 in the Supporting Information. Compounds were tested for the
in vitro binding to the ATP-binding site of p110α and mTOR using a commercially available time-resolved FRET (TR-FRET)
displacement assay (LanthaScreen). Each experiment performed n = 2. IC50s, LogIC50s and Standard Errors are reported in
c
Table S7 in the Supporting Information. Marvin/JChem 16.10.17 was used for calculation of logP (partition coefficient) values.
Elucidation of Binding Modes to PI3K and mTOR.
To explain activity differences of the regioisomers, we considered the possible interactions of the
3
3
inhibitors within the ATP-binding site of the kinases. As previously reported for PQR309 (1) and other
morpholino-substituted PI3K inhibitors,32,39 a crucial interaction for PI3K binding is the hydrogen bond
formation in the hinge region between the backbone amide of Val882 residue in PI3K (Val851 in
PI3Kα) and the morpholine oxygen atom. Herein, we started from the conformationally restricted
PQR309 (1) analogs (3a and 3b, Table 2) and replaced the morpholine with a piperidine (4a-4b and 5a-
5
b, Table 2) to investigate whether the methylene-bridged morpholine or the unrestricted morpholine
points towards and interacts with the hinge region Val of the ATP-binding pocket. The obtained Ki
values for PI3Kα suggested that the methylene-bridged morpholine locates to the solvent exposed
region, since only the compounds with a methylene-bridged piperidine (4a and 4b) displayed a strong
affinity for PI3Kα (K = 22 and 48 nM, respectively). The in vitro potency of 4a and 4b is comparable
i
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to that of the dimorpholine-substituted parental compounds (3a and 3b). On the contrary, replacement
of the unrestricted morpholine with a piperidine (5a and 5b) led to a drop in PI3K binding affinity.
Table 2. Binding mode: morpholine vs. piperidine in unrestricted and restricted positions.
1
1
1
1
1
1
1
1
1
1
2
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2
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2
2
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2
2
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0
1
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0
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0
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0
F
F
F
Ar =
Cellular Assays,
in vitro Binding
Assays, K [nM]
i
a
b
IC50 [nM]
N
NH2
pS6
S235/236
Name
Structure
pPKB S473
353
p110α
O
N
751
21
3
a
(
S)
N
N
O
N
Ar
O
N
3
3
55
21
524
1239
938
56
22
48
3
b
(R)
N
N
O
N
Ar
Ar
O
N
4b
(R)
N
N
N
O
N
N
407
4
5
a
a
(S)
N
N
N
N
N
N
Ar
Ar
Ar
N
N
2
1893
43317
2655
2835
138
(S)
O
O
N
N
2
075
5
b
(R)
aPKB phosphorylation on Ser473 and ribosomal S6 phosphorylation on Ser235/236 were analyzed in A2058 cells exposed
to the indicated inhibitors and subsequent detection of phosphoproteins in an in-cell Western assay. Each experiment performed
b
n = 2. LogIC50s and Standard Errors are reported in Table S8. Compounds were tested for the in vitro binding to the ATP-
binding site of p110α and mTOR using a commercially available time-resolved FRET (TR-FRET) displacement assay
(LanthaScreen). Each experiment performed n = 2. IC50s, LogIC50s and Standard Errors are reported in Table S8.
We thus concluded that the unrestricted morpholine binds to the hinge region and establishes a H-
bond with the Val backbone amide. We exploited the 2.48 Å resolution X-ray structure of PIKiN3 in
PI3Kγ (see Ref. 32; PDB code 5JHB), and substituted PIKiN3 for compound 3a to confirm its binding
mode. Energy minimization calculations of the resulting PI3Kγ-3a complex maintained important
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interactions such as H-bonds between the aminopyridine and Asp836/964 as well as the interaction of
the oxygen atom of the unrestricted morpholine and the backbone amide of Val882 (as present in the
parental structure with PIKiN3; Figure 3A). Moreover, the interaction of the core nitrogen of compound
3
a with the structured water molecule allows to stabilize the inhibitor binding through a H-bond
0
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network. In PI3K, this interaction had been proved to play a pivotal role in inhibitor binding (see Ref.
32; PDB code: 5JHB).
A
N951
Y867
B
D2357
C
D2357
V2240
M2345
V2240
M2345
D841
V882
D964
E2190
E2190
V851
D836
PI3Kγ
mTOR
mTOR
V851
D
E
D933
D933
D810
D810
Q859
Q859
D805
D805
PI3Kα
PI3Kα
Figure 3. A) Docking of compound 3a (plum) into PI3Kγ (gray) starting from PDB 5JHB (see Ref. 32).
Structural water molecules are shown in red and water-mediated H-bonds are depicted as dashed black lines. B)
Docking of compound 2a (gold) and C) compound 2b (green) into mTOR (turquoise) starting from PDB 4JT6.
The important features for mTOR selectivity are depicted in a ball and stick representation. D) Docking of
compound 2a (gold) and E) compound 2b (green) into PI3Kα (gray) starting from PDB 3ZIM. The exit vector
from the restricted morpholine oxygen is shown as a black arrow.
Given the high homology of the ATP-binding pocket of class I PI3Ks and mTOR, a similar binding
mode and orientation in mTOR and all PI3K isoforms can be assumed. Computational modeling studies
were performed to elucidate the binding mode of the conformationally restricted compounds 2a and 2b
in mTOR starting from the PI103-mTOR complex (PDB code: 4JT6). In analogy to PI3K, the higher
mTOR potency of the compounds bearing the unrestricted morpholine in the 4-position of the
pyrimidine core (2a and 2b, Scheme 1B) could be derived from the interaction of a pyrimidine core
nitrogen with a putative structured water molecule, even though the 3.6 Å structural resolution of mTOR
did not reveal such structured water molecules. Thus, the position of N-1 of the pyrimidine core (see
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Scheme 1 for numbering) in the ATP-binding pocket is an essential parameter for mTOR affinity. The
binding affinity data of compounds 2a-d suggests a similar structured water network in mTOR, which
is only maintained by binding of regioisomer 2a and 2b, but not with 2c and 2d (K 2a, 2b = 69, 24 nM
i
Vs K 2c, 2d = 812, 1279 nM). An additional stabilization of 2a/2b-mTOR complex is provided by
i
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hydrophobic interactions between the methylene bridge and Met2345 (Figure 3B and 3C). The
important features for mTOR selectivity are depicted in Figure 3B and 3C in a ball and stick
representation.
Within one regioisomer pair (2a and 2b), the (R)-enantiomer 2b displayed a stronger affinity for
mTOR, together with an 18-fold selectivity for mTOR kinase over PI3Kα (K for mTOR = 24 nM and
i
K for PI3Kα = 432 nM). In contrast, the (S)-enantiomer 2a is a dual mTOR/PI3Kα inhibitor (Table 1).
i
The 6-fold difference in PI3K inhibitory activity between compound 2a and 2b (K for PI3Kα = 76 and
i
4
32 nM, respectively) might be related to the different orientation of the morpholine due to scaffold
rigidification. In the (S)-configured compound 2a, the oxygen of the restricted morpholine points
towards the amide side-chain of Gln859 (4 Å) and a weak H-bond could stabilize the binding of 2a in
PI3Kα (see exit vector in Figure 3D). In the (R)-tricyclic pyrimido-pyrrolo-oxazine 2b the morpholine
oxygen is rotated by 1 Å with respect to the oxygen atom of 2a and the exit vector does not point towards
Gln859 (see Figure 3E). Therefore, no H-bond can be established.
Chemistry.
Procedures for library synthesis are depicted in Scheme 1. First, sulfamidates 33a and 33b were
prepared (Scheme 1A). Typically, sulfamidates are made from chiral amino alcohols in several steps.40
The 3-hydroxymethyl morpholines (34a and 34b) were synthesized by a chiral synthesis starting from
serine enantiomers. As for 34a, the amino group of (R)-serine was protected with a benzyl group
(compound 37b) since the ring closure has been described to proceed in higher yields when a protecting
group is present.41 N-Benzyl-(R)-serine (37b) underwent ring closure in presence of chloroacetyl
chloride to give (R)-4-benzyl-5-oxomorpholine-3-carboxylic acid (36b) in 71% yield. Reduction of 36b
using borane-dimethyl sulphide complex in tetrahydrofuran (THF) gave intermediate 35a with an 82%
yield. Hydrogenolysis of N-benzyl derivative (35a) over palladium on charcoal (Pd/C, H ) yielded (S)-
2
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2
2
2
2
2
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3
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5
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6
morpholin-3-ylmethanol (34a). Subsequent two-step cyclic sulfamidate formation was performed by
treating the amino alcohol (34a) with thionyl chloride, to give the corresponding cyclic sulfamidite,
followed by oxidation using NaIO and catalytic amounts of ruthenium(IV) oxide hydrate to give
4
sulfamidate 33b in 52% yield. The (S)-enantiomer (33a) was synthesized from (S)-serine following the
procedure described for 33b (Scheme 1A). The dichloropyrimidines (26-31) were prepared according
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to Scheme 1B starting from the commercially available 2,4,6-trichloropyrimidine. Morpholines M -M
0
5
were introduced as M substituents by nucleophilic aromatic substitution reaction. Following this
n
procedure, both regioisomers were generated and separated by column chromatography. The desired
regioisomers (27-31) were isolated in moderate to high yields (46-86%). The (S)-tricyclic intermediates
(20a-25a) were synthesized by sequential alkylation and nucleophilic aromatic substitution reactions of
dichloropyrimidines (26-31) with (R)-sulfamidate 33b using n-BuLi in THF at -78 °C and a catalytic
amount of copper(I) iodide followed by acidic hydrolysis of the intermediary sulfaminic acid and ring
4
2
closure mediated by base treatment. The same procedure was followed for the preparation of (R)-
tricyclic intermediates (20b-25b), starting from (S)-sulfamidate 33a. A subsequent palladium catalyzed
Suzuki coupling between choloropyrimidine intermediates (20a-25a and 20b-25b) and protected 2-
aminopyridine-5-boronic acid pinacol ester (38, see Supporting Information) afforded compounds 2a,
6a-10a and 2b, 6b-10b in moderate to high yields (49-88%; Scheme 1B). In addition, the chlorine of
intermediate 22b was displaced by heteroaryl moieties using Suzuki cross-coupling reaction with
boronic acid pinacol esters to give compounds 11b-17b (Scheme 1B). For not commercially available
boronic acid pinacol esters or corresponding bromo derivatives, the synthesis is reported in Scheme S1
and S2 in Supporting Information. The regioisomer bearing the unrestricted morpholine in the 2-position
of the pyrimidine core (enantiomers 2c and 2d) was synthesized starting from dichloropyrimidine 32
(Scheme 1C) following the procedure described for 2a and 2b. The synthesis of the conformationally
restricted PQR309 (1) analog 3a and 3b (Table 2) is depicted in Scheme S3A in Supporting Information
and the synthetic procedure is analogous to that described for 2a and 2b. The procedure for the
preparation of the compounds bearing a piperidine instead of a morpholine (4a-4b and 5a-5b, Table 2)
is reported in Scheme S3B and S3C in Supporting Information.
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O
A O
HO
O
OH
Bn
OH
Bn
OH
NH
O
S
O
S
*
iii)
*
iv)
*
v)
*
vi)
*
i
)
ii)
*
HO
*
OH
Bn
OH
O
N
O
O
N
O
O
N
O
N
O
O
NH2
HN
O
(
(
R)-Serine
S)-Serine
(R)-37b ((R)-Serine, 55%) (R)-36b (34b, 71%)
(S)-35a (33b, 82%)
(R)-35b (33a, 81%)
(S)-34a (32a, 99%)
(R)-34b (32b, 96%)
(R)-33b (31a, 52%)
(S)-33a (31b, 64%)
(S)-37a ((S)-Serine, 63%)
(S)-36a (34a, 51%)
M0 =
M1 =
B
O
O
O
O
O
N
Ar1 =
Cl
N
Mn
Mn
N
Mn
4
N
vii)
viii)
ix)
5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N3
*
*
(S)
(R)
N
N
NH2
NH2
O
*
N
N
N
N
Cl
N
Cl
Cl
N
Cl
O
N
Cl
O
N
N
2
Ar2 =
O
S
N
O
1
O
N
NH2
(
R)-33b
(S)-33a
M2 =
N
Mn
6a M0
Mn (from, yld)
Mn (from, yld)
2
(S)-20a M0 (33b, 66%)
(R)-20b M0 (33a, 73%)
(S)-2a M0 (20a, 65%)
(R)-2b M0 (20b, 76%)
(S)-6a M1 (21a, 76%)
(R)-6b M1 (21b, 64%)
(S)-7a M2 (22a, 74%)
(R)-7b M2 (22b, 49%)
(S)-8a M3 (23a, 67%)
(R)-8b M3 (23b, 86%)
(S)-9a M4 (24a, 69%)
(R)-9b M4 (24b, 88%)
(S)-10a M5 (25a, 70%)
(R)-10b M5 (25b, 88%)
Ar3 =
Ar4 =
2
2
2
3
3
7
8
9
0
1
M1 (72%)
M2 (71%)
M3 (86%)
M4 (80%)
M5 (46%)
M3 =
M4 =
(
(
S)-21a M1 (33b, 18%)
R)-21b M1 (33a, 24%)
N
NH2
NH2
(S)-22a M2 (33b, 73%)
(
(
(
(
R)-22b M2 (33a, 55%)
S)-23a M3 (33b, 71%)
R)-23b M3 (33a, 40%)
S)-24a M4 (33b, 59%)
N
O
M5 =
(R)-24b M4 (33a, 26%)
Ar5 =
O
N
(
(
S)-25a M5 (33b, 61%)
R)-25b M5 (33a, 66%)
N
NH2
C
Ar
11b Ar1 (84%)
2b Ar2 (68%)
3b Ar3 (31%)
14b Ar4 (66%)
15b Ar5 (44%)
16b Ar6 (76%)
17b Ar7 (76%)
O
O
O
O
O
O
N
) x)
xi) xii)
xiii)
O
ix
(R)
Ar6 =
Ar7 =
1
1
N
N
N
viii)
ix)
2
3
N
N
NH2
NH2
N
N
N
*
N
1N
N
(R)
N
N
O
4
O
N
*
N
Cl
Cl
Cl
O
Ar
O
N
S
O
N
N
5
32a,b
O
(
(
R)-33b
S)-33a
O
O
*
N
NH2
Mn (from, yld)
S)-20c M0 (33b, 76%)
R)-20d M0 (33a, 24%)
Mn (from, yld)
(S)-2c M0 (20c, 47%)
(R)-2d M0 (20d, 57%)
(
(
Scheme 1. A) Reagents and conditions: (i) 1) benzaldehyde, 2M NaOH, r.t, 30 min; 2) NaBH
hr; (ii) 1) chloroacetyl chloride, K CO , THF / H O, 0 °C, 1 hr; 2) NaOH, 5 °C, 2 hrs; (iii) borane-dimethylsulfide
complex, Et N, THF, 0 °C → 65 °C, 5 hrs; (iv) Pd/C, H , 2.8 bar, 48 hrs; (v) thionyl chloride, imidazole, DCM, -
5 °C → r.t. → 0 °C, 2 hrs; (vi) ruthenium(IV) oxide hydrate, NaIO , r.t., o/n. B) Reagents and conditions: (vii)
morpholine derivative (M -H), DIPEA, DCM, 0 °C → rt, o/n; (viii) 1) n-BuLi, CuI, -78 °C → r.t., o/n; 2) HCl
conc., MeOH, 45 °C, 4-6 hrs; 3) NaOH, H O, r.t., 1-16 hrs; (ix) 1) boronic ester 38 or 41, XPhosPdG2 (cat.),
PO , dioxane / H O, 95 °C, 2-16 hrs; 2) HCl, dioxane / H O, 60 °C, 3-16 hrs (for 2a, 2b, 2c, 2d, 6a, 6b, 7a, 7b,
a, 8b, 9a, 9b, 10a, 10b and 14b); (x) 2-aminopyridine-5-boronic acid pinacol ester, XPhosPdG2 (cat.), K PO
dioxane / H O, 95 °C (for 11b), o/n; (xi) boronic ester 39, Pd(dppf)Cl (cat.), CsCO , THF, Δ, o/n (for 12b); (xii)
) Boronic ester generated in situ, XPhosPdG2 (cat.), K PO , dioxane / H O, 95 °C, 3-3.5 hrs; 2) HCl, 80 °C, o/n
(for 13b and 15b); (xiii) 1) Boronic ester generated in situ, XPhosPdG2 (cat.), K PO , dioxane / H O, 95 °C, 2-16
hrs (isolated intermediates: 18b and 19b); 2) 18b or 19b, TFA, DCM, 0 °C → r.t., 1-3 hrs (for 16b and 17b).
4
, 5 °C → r.t., 1
2
3
2
3
2
4
n
2
K
3
4
2
2
8
3
4
,
2
2
3
1
3
4
2
3
4
2
a
b
prepared according to Ref. 32. prepared according to procedure (vii). After the reaction, the two regioisomers
26 and 32) were separated by column chromatography.
(
1
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6
Determination of Cellular Potency and PI3K vs mTOR Kinase Activities.
In the search for novel mTOR inhibitors, we selected the regioisomer bearing the unrestricted
morpholine in the 4-position of the pyrimidine core (Scheme 1B), and built up a Structure Activity
Relationship (SAR) study to investigate (i) the influence of the stereogenic center on mTOR selectivity
and (ii) the effect of substitution on the unrestricted morpholine. We prepared a series of compounds
introducing different morpholine derivatives (Mn substituents, Scheme 1B), such as 3-
methylmorpholine [M : (S) and M : (R)], 8-oxa-3-azabicyclo[3.2.1]octane (M ), 3-oxa-8-
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
azabicyclo[3.2.1]octane (M ) and 3,3-dimethylmorpholine (M ). Morpholines with increased steric
4
5
demand had already been shown to increase selectivity for mTOR over PI3K.31,43 For each
conformationally restricted compound both (S)- (2a, 6a-10a) and (R)-enantiomers (2b, 6b-10b) were
synthesized and tested in kinase binding and cellular assays. For all the regioisomer pairs, the (R)-
enantiomer had higher affinity for mTOR than the (S)-configured compound (Table 3), confirming the
results of compounds 2a and 2b. Also the cellular potency of the (R)-configured compounds was 2-7
fold higher than that of their corresponding enantiomers (see Table 3). The introduction of sterically
hindered morpholines in the hinge region of (R)-configured tricyclic pyrimido-pyrrolo-oxazines (6b-
1
0b) did not affect the potency for mTOR, but led to a significant improvement in mTOR selectivity.
With the exception of compound 10b, the selectivity of (R)-configured tricyclic compounds for mTOR
versus PI3Kα was higher than 35-fold [K (p110α)/K (mTOR) 6b = 43; 7b = 165; 8b = 53 and 9b = 36].
i
i
The (S)-3-methylmorpholine (M ) only slightly increased the compound potency and selectivity for
1
mTOR if compared with the morpholine (M ) [K for mTOR 2b = 24 nM; 6b = 9.1 nM; Selectivity
0
i
K (p110α)/K (mTOR) for 2b was 18-fold, and for 6b 43-fold]. On the contrary, the presence of a (R)-3-
i
i
methylmorpholine (M ) on the (R)-configured tricyclic pyrimido-pyrrolo-oxazine scaffold yielded the
2
highest selectivity of the series [K (p110α)/K (mTOR) for 7b was 165-fold]. In cells, compound 7b
i
i
displayed good activity (IC for phosphorylated PKB/Akt = 133 nM and for phosphorylated S6 = 61
5
0
nM) and was therefore selected for further optimization.
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Table 3. SAR study on the unrestricted morpholine of tricyclic pyrimido-pyrrolo-oxazines.
M
in vitro Binding
N
Cellular Assays
*
Assays
Selectivity
K (p110α)/
i
N
a
O
N
IC [nM]
5
0
b
clogPc
K
i
[nM]
N
NH2
K
i
(mTOR)
pPKB
S473
pS6
S235/236
164
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Name
Conf.
M
M
n
0
p110α
mTOR
69
1
1
4
94
16
16
76
2
a
(S)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
1.1
18
2.65
2.65
3.06
3.06
3.06
3.06
3.11
3.11
3.11
3.11
3.14
3.14
96
432
149
230
61
432
98
24
104
9.1
31
2b
6
a
0.9
43
M
M
M
M
M
1
2
3
4
5
194
363
386
6
b
923
7
a
30
1
4
2
33
03
07
2147
1219
1412
3230
1108
7187
376
13
7
b
165
13
245
160
354
132
991
136
96
8a
27
8
b
53
389
52
329
09
60
9
a
54
1
31
9
b
36
1
121
24
10a
10b
59
4
16
a
0 5
Chemical structures of M -M are depicted in Scheme 1. PKB phosphorylation on Ser473 and ribosomal S6
phosphorylation on Ser235/236 were analyzed in A2058 cells exposed to the indicated inhibitors and subsequent detection of
phosphoproteins in an in-cell Western assay. Each experiment performed n = 2. LogIC50s and Standard Errors are reported in
b
Table S7 in the Supporting Information. Compounds were tested for the in vitro binding to the ATP-binding site of p110α and
mTOR using a commercially available time-resolved FRET (TR-FRET) displacement assay (LanthaScreen). Each experiment
c
performed n = 2. IC50s, LogIC50s and Standard Errors are reported in Table S7 in the Supporting Information. Marvin/JChem
16.10.17 was used for calculation of logP (partition coefficient) values.
To further improve the potency and selectivity for mTOR, and to modulate the physico-chemical
properties of compound 7b, we investigated the role of the aryl moiety (Table 4). First, we replaced the
pyridine ring with a pyrimidine (11b) and a pyrazine (12b) to assess the effect of the polarity of the
heteroaromatic ring. Afterwards, we introduced a methyl (13b and 14b), a methoxy (15b) or a
dimethoxymethyl substituent (16b and 17b) on the 2-amino-substituted aryl group. The presence of a
pyrimidine or a pyrazine ring slightly increased mTOR potency, selectivity and cellular activity (see
comparison between 11b/12b and 7b, Table 4). Compound 13b, bearing a methyl substituent in position
4
of the 2-aminopyridine ring, had a moderate activity towards mTOR (K = 85 nM), and was poorly
i
active in cells (IC for phosphorylated PKB/Akt and for phosphorylated S6 > 800 nM, Table 4). In
5
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2
2
2
2
2
2
2
2
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3
3
3
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3
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4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
contrast, compound 14b, with a 3-methyl-substituted aminopyridine, maintained a moderate activity in
cells (IC50 for phosphorylated PKB/Akt was 488 nM and 289 nM for phosphorylated S6). The
introduction of a methoxy (15b) and a dimethoxymethyl substituent (16b) on the pyridine ring reduced
cellular potency and binding to both PI3Kα and mTOR. Compound 17b, bearing a dimethoxymethyl
substituent on the pyrimidine ring, is a dual PI3Kα/mTOR inhibitor (K for p110α = 66 nM; K for mTOR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
i
i
=
34 nM), but showed a limited potency in cells (IC pPKB > 500 nM and pS6 > 300 nM, Table 4).
50
With respect to compound 7b, the introduction of an additional nitrogen on the heteroaromatic ring
decreased the clogP value (compound 11b = 2.22 and 12b = 2.15), leading to compounds with predicted
optimal physicochemical and ADME properties for oral drugs.44 Moreover, the Polar Surface Area
(PSA) > 90 Å of compounds 11b and 12b is indicative that they might not cross the Blood Brain Barrier
(BBB). Limited brain uptake of mTOR inhibitors, normally used to treat systemic tumors, is an
advantage because inhibition of the mTOR pathway in the brain could lead to neurological side effects.
Table 4. SAR study on the aryl moiety of tricyclic pyrimido-pyrrolo-oxazines.
O
(
R)
in vitro Binding
Selectivity
(p110α/
N
Cellular Assays
clogPc
PSAc
Assays
K
i
a
(R)
N
IC [nM]
5
0
b
K
i
[nM]
K
i
(mTOR)
N
O
N
Ar
pPKB
S473
pS6
S235/236
Name
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
n
0
1
2
3
4
5
6
7
p110α
mTOR
13
7
b
133
61
2147
536
165
214
212
33
3.06
2.22
2.15
3.53
3.53
2.81
2.97
2.43
3.06
89.6
1
1
1b
2b
77
55
2.5
102.5
102.5
89.63
89.63
98.86
108.1
121.0
102.5
94
60
1695
2759
9627
15970
1122
66
8.0
13b
14b
15b
16b
17b
1544
488
3755
6522
534
190
826
289
2520
2425
377
85.2
85
56
172
77
208
300
34
3.7
2.0
389
PQR620 (58)*
Fig. 1
4203
10.8
a
0 7
Chemical structures of Ar -Ar are depicted in Scheme 1. *PQR620 data are reprinted from Ref. 31 for comparison. PKB
phosphorylation on Ser473 and ribosomal S6 phosphorylation on Ser235/236 were analyzed in A2058 cells exposed to the
indicated inhibitors and subsequent detection of phosphoproteins in an in-cell Western assay. Each experiment performed n =
b
2
. LogIC50s and Standard Errors are reported in Table S7 in the Supporting Information. Compounds were tested for the in
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vitro binding to the ATP-binding site of p110α and mTOR using a commercially available time-resolved FRET (TR-FRET)
displacement assay (LanthaScreen). Each experiment performed n = 2. IC50s, LogIC50s and Standard Errors are reported in
c
Table S7 in the Supporting Information. Marvin/JChem 16.10.17 was used for calculation of logP (partition coefficient) values.
1
1
1
1
1
1
1
1
1
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2
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Pharmacological Parameters.
To assess the oral availability and to determine BBB permeability, pharmacokinetic studies (PK)
were carried out for compounds 7b and 12b. Compounds 7b and 12b were administered orally (5 mg/kg
p.o.) in male Sprague Dawley rats, and compound concentrations in plasma and brain were determined
after a single dose (Figure 4A and 4B, respectively. See also Table S1-S4 in Supporting Information).
Both compounds showed plasma exposure sufficient to efficiently inhibit mTOR kinase, and brain levels
remined minimal. After 8 h, the total exposure AUC0−8 was 5730 ng·h/mL in plasma and 104 ng·h/g in
brain for compound 12b.
A
B
Compound 7b
Compound 12b
3000
2000
Plasma
Brain
1000
0
0
4
8
0
4
8
time, hours
time, hours
C
D
1
25
00
75
1
Dogs
5
0
5
0
Mice
Rats
Humans
2
0
50
100 150 200
0
50
100 150 200
time, minutes
time, minutes
Figure 4. Plasma and brain concentration of A) compound 7b and B) compound 12b after p.o. dosing at 5
mg/kg in male Sprague Dawley rats. Stability of compound 11b (5 µM) with primary hepatocytes from C) mice
(green) and rats (turquoise) D) dogs (red) and humans (black) (n = 2). All values mean ± SEM. Error bars not
shown when smaller than the symbols.
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To predict the metabolic stability of our restricted scaffold, in vitro assays were performed for
compound 11b, using hepatocyte cultures of different origin (CD-1 mice, Sprague Dawley rats, Beagle
dogs and humans). Compound 11b was only minimally metabolized when incubated with mouse, rat,
dog and human hepatocytes, as indicated by 81.2%, 93.6%, 93.0% and 93.2% remaining compound
after 3 hours of incubation (Figure 3C, Figure 3D and Table S5 in Supporting Information). As
compound 11b was highly stable across species, neither half-lives nor intrinsic clearance could be
determined under the experimental conditions.
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CYP450 Reactive Phenotyping for Compounds 7b, 11b and 12b.
CYP450 reactive phenotyping was performed to evaluate the involvement of different human hepatic
CYP isoenzymes (CYP1A1 and CYP1A2) in the metabolism of compounds 7b, 11b and 12b. All three
compounds turned out to be moderate CYP1A1 substrates, as indicated by < 77% parental compound
remaining after 60 minutes of incubation (Table 5). Compounds 7b and 11b were partially stable
towards CYP1A1 (44% and 51% compound remaining, respectively), while the lowest turnover was
observed with compound 12b (77% remaining). A low metabolization by CYP1A2 was observed with
1
1b (73% remaining). On the other hand, compounds 7b and 12b remained stable (Table 5), indicating
that they are no substrates of CYP1A2.
Table 5. CYP1A1 and CYP1A2 Reactive Phenotyping of 7b, 11b and 12b (1 µM).
%
remaining in corresponding
remaining test item w/ cofactors
Mean (%)
corr*
Test item
negative control w/o cofactors
mean %
SD
mean
SD
CYP1A1
7b
44
51
77
2.0
0.3
109
93
3.7
2.9
4.5
35
58
81
1
1
1b
2b
0.08
96
CYP1A2
7
b
98
73
92
0.6
0.4
0.2
94
91
91
0.1
2.4
0.8
104
83
1
1b
12b
101
Compounds remaining after 60 min of incubation with SupersomesTM at 25 pmol/ml (n=2), calculation based on absolute
amounts (nM). *Mean (%)corr = (100-% remaining negative control)+% remaining sample.
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Based on these results, compound 11b was further investigated to identify metabolites formed in
vitro by CYP1A1 (Figure 5A) and CYP1A2 (Figure 5B). Upon incubation of 11b with human
recombinant CYP1A1, the highest peak areas were observed for metabolite M2 (9.3% of total peak
areas, hydrolysis of the 3R-methyl-morpholinyl residue and oxidation to corresponding aldehyde) and
its secondary products M5 (7.5%, introduction of a C-C double bond (desaturation) at the tricyclic
backbone of M2; Figure 5C). Other minor metabolites were observed, highlighting that the preferred
site of oxidative metabolism of compound 11b was the 3R-methyl-substituted morpholinyl moiety
(Table S6 and Figure S1-S5 in Supporting Information). In CYP1A2 incubates, M2 was the only
metabolite (0.3%) suggesting that compound 11b is not a substrate of CYP1A2.
1
1
1
1
1
1
1
1
1
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2
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2
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A
B
1
1b: 73.3%
11b: 99.7%
M1: 1.2%
M2: 9.3%
M3: 2.9%
M4: 4.9%
M5: 7.5%
M6: 0.51%
M7: 0.34%
M8: 0.07%
M2: 0.3%
CYP1A1
CYP1A2
C
O
HO
O
HO
O
(R)
N
N
N
(R)
N
(R)
N
(R)
N
N
N
N
O
N
N
O
N
N
O
N
N
N
NH2
N
NH2
N
NH2
11b
M2
M5
Figure 5. Pie chart showing the percentage of compound 11b and its metabolites after 60 min of incubation
with human recombinant A) CYP1A1 and B) CYP1A2. C) Major metabolites observed upon incubation of 11b
with CYP1A1.
Enzymatic Profiling and Determination of Selectivity.
According to our SAR studies, compounds 7b, 11b and 12b were the most potent and selective
mTOR inhibitors showing high potency in cellular assays and good pharmacokinetic profile. Therefore,
7b, 11b and 12b were chosen for further characterization using the KINOMEScan platform of
DiscoverX (Table 6). DiscoverX KdELECT assays confirmed the excellent mTOR selectivity of
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compound 12b over class I and III PI3K and PI4K: 12b was ∼450-fold more potent for mTOR than for
class I PI3K isoforms. The selectivity of 12b for mTOR over PI3K exceeds competitor compounds such
as CC223 (55)45 (∼80×), INK128 (56) (∼40×) and AZD2104 (57) (∼230×) (Table 6). Moreover,
compound 12b showed negligible off-target effects when screened against a DiscoverX scanMAX
kinase assay panel containing a set of 468 protein and lipid kinases (Supporting Information, Figure S6
an Table S10). At a concentration of 10 μM, compound 12b and PQR620 (58) reached outstanding
selectivity scores [S(10) was 0.005 for both compounds; see Supporting Information, Table S9], while
the same value for INK128 (56) reached 0.14 (corresponding to 55 hits, Table S9).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. mTOR and lipid kinase binding constants of 7b, 11b, 12b and reference compounds.
*
Most
a
Inhibitor binding constants K
d
, [nM]
sensitive
PI3K/mTOR
Fold
Kinase

mTOR
34
PI3K PI3K
PI3K
PI3K
PI4K VPS34
selectivity
7
b
610
120
1600
4100
1400
7500
6200
1900
12000
8700
1800
11000
>30000
>30000
>30000
2100
980
3200
17.9
8.6
457
1
1
1b
2b
14
3.5
CC223
2
8
2300
18000
81
6200
30
7100
39
2500
8200
>80x
>40x
b
(55)
INK128
0
.092
15
3.7
n.d.
b
(56)
AZD2014
0
.14
.27
33
3300
1500
8400
>30000
>30000
23000
2750
>230x
b
(57)
PQR620
(58)
0
1000
22000
23000
18000
>3700x
aDissociation constants (K
) were determined using ScanMax technology (DiscoveRx) with 11 point 3-fold serial dilutions
d
of the indicated compounds. K
d
is the mean value from experiments performed in duplicate and were calculated from standard
b
dose response curves using the Hill equation. n.d. = not determined. Dissociation constants (K
d
) of CC223 (55), INK128 (56),
of most
AZD2014 (57) and PQR620 (58) are reprinted from Ref. 31. n.d. = not determined. *Fold selectivity: ratio of K
d
d
sensitive class I PI3K isoform (displayed in bold type) over K for mTOR.
CONCLUSION
In summary, we have exploited a conformational restriction strategy to enlarge the chemical space
around PQR309 (1), a pan-PI3K and moderate mTOR inhibitor. Our rigidification strategy allowed to
enhance potency and selectivity for mTOR. Molecular modeling elucidated interactions of the tricyclic
pyrimido-pyrrolo-oxazine scaffold in the ATP-binding pocket of PI3Ks and mTOR, and explained the
difference in potency between the regioisomers. An extensive SAR study led to the discovery of
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compound 12b, with a selectivity for mTOR higher than competitor compounds such as CC223 (55),
INK128 (56) and AZD2104 (57). The selectivity of PQR620 (58) for mTOR over PI3Ks exceeds that
of 12b, however 12b explores a novel chemical space and paves the way for additional chemical
modifications. A lead optimization process of the tricyclic pyrimido-pyrrolo-oxazine scaffold could lead
to further improve the mTOR potency and selectivity of 12b. Compound 12b efficiently inhibited
mTOR signaling in cells and showed plasma drug exposure after oral dosing that is expected to fully
inhibit mTOR in vivo. The minimal brain permeability of compounds 7b and 12b suggests the tricyclic
pyrimido-pyrrolo-oxazine as a novel scaffold for the development of highly selective and potent ATP-
competitive mTOR inhibitors to be exploited for the treatment of systemic tumors. PQR620 (58) and
the tricyclic pyrimido-pyrrolo-oxazine derivatives span along the different therapeutic applications of
mTOR inhibitors. PQR620 (58) showed an excellent brain penetration and could be exploited in the
treatment of neurological disorders. On the contrary, the conformationally restricted compounds have a
limited brain uptake and could find an optimal application in the treatment of systemic cancers, with the
advantage of avoiding neurological side effects. Our novel scaffold was also characterized for CYP450
related metabolism to assess the substrate specificity towards CYP1A1 and CYP1A2. The tricyclic
pyrimido-pyrrolo-oxazines 7b, 11b and 12b were metabolically stable, with compound 12b showing
the lowest turnover in CYP450 reactive phenotyping. On the basis of its remarkable mTOR
potency/selectivity, and favorable pharmacokinetic profile, compound 12b represents a promising
starting point for the development of a novel mTOR candidate in oncology.
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EXPERIMENTAL SECTION
General Information.
Reagents were purchased at the highest commercial quality from Acros, Sigma-Aldrich or
Fluorochem and used without further purification. Solvents were purchased from Acros Organics in
0
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®
AcroSeal bottles over molecular sieves. Cross coupling reactions were carried out under nitrogen
atmosphere in anhydrous solvents, and glassware was oven dried prior to use. Thin layer
chromatography (TLC) plates were purchased from Merck KGaA (Polygram SIL / UV254, 0.2 mm
silica with fluorescence indicator) and UV light (254 nm) was used to visualize the compounds.
Column chromatographic purifications were performed on Merck KGaA silica gel (pore size 60 Å,
2
30-400 mesh particle size). Alternatively, flash chromatography was performed with Isco
CombiFlash Companion systems using prepacked silica gel columns (40−60 μm particle size
1
19
13
RediSep). H, F and C NMR spectra were recorded on a Bruker Avance 400 spectrometer. NMR
spectra were obtained in deuterated solvents, such as CDCl , (CD ) SO or CD OD. The chemical shift
3
3 2
3
1
(δ values) are reported in ppm and corrected to the signal of the deuterated solvents (7.26 ppm ( H
13
1
13
NMR) and 77.16 ppm ( C NMR) for CDCl ; 2.50 ppm ( H NMR) and 39.52 ppm ( C NMR) for
3
1
13
19
(
CD ) SO; and 3.31 ppm ( H NMR) and 49.00 ppm ( C NMR) for CD OD). F NMR spectra are
3
2
3
calibrated relative to CFCl (δ = 0 ppm) as external standard. When peak multiplicities are reported,
3
the following abbreviations are used: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), td
(triplet of doublets), q (quartet), m (multiplet), br (broadened). Coupling constants, when given, are
reported in Hertz (Hz). High resolution mass spectra (HRMS) were recorded on a Bruker maxis 4G,
high resolution ESI-QTOF. All analysis were carried out in positive ion mode and in MeOH + 0.1 %
formic acid as solvent. Sodium formate was used as calibration standard. MALDI-ToF mass spectra
were obtained on a Voyager-DeTM Pro measured in m/z. The chromatographic purity of final
compounds was determined by high performance liquid chromatography (HPLC) analyses on an
Ultimate 3000SD System from ThermoFisher with LPG-3400SD pump system, ACC-3000
autosampler and column oven, and DAD-3000 diode array detector. An Acclaim-120 C18 reversed-
phase column from ThermoFisher was used as stationary phase. Gradient elution (5:95 for 0.2 min,
5
:95 → 100:0 over 10 min, 100:0 for 3 min) of the mobile phase consisting of CH CN /
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MeOH:H O
was used at a flow rate of 0.5 mL/min at 40 °C. The purity of all final compounds
(10:90)
2
23
was > 95%. Optical Rotations ([훼] ) were measured on a Perkin Elmer Polarimeter 341 (in a cuvette
퐷
(l = 1 dm)) at 23 °C at 589 nm.
1
1
1
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0
General procedure 1.
A flask was charged with the corresponding boronic acid pinacol ester (1 eq.), N,N-
dimethylformamide dimethyl acetal (DMF-DMA, 1.3-1.5 eq.) and THF (5 mL). The mixture was stirred
at 70 °C overnight. After completion of the reaction monitored by NMR, the solvents were evaporated
and the compound was dried under vacuum.
General Procedure 2.
To a solution of the desired morpholine (1.05 eq.) in DCM (approx. 1 ml / 0.6 mmol) at 0 °C, N,N-
diisopropylethylamine (2.1 eq.) was added, followed by 2,4,6-trichloropyrimidine (1.0 eq.). The reaction
mixture was allowed to warm up to r.t. and was stirred overnight. The reaction mixture was washed with
aqueous saturated NaHSO -solution (2 x). The aqueous layer was extracted with DCM and the combined
4
organic layers were dried over Na SO , filtered and concentrated under reduced pressure. The crude was
2
4
purified by column chromatography on silica gel.
Using this procedure, both regioisomers were generated and separated by column chromatography.
Only the characterization of the desired regioisomer is reported.
General procedure 3.
Under nitrogen atmosphere, a 1.6 M n-BuLi solution (1.0 mL, 1.52 mmol, 1.2 eq.) in THF (approx.
1
ml / 1.5 mmol) was cooled down to -78 °C and a solution of the corresponding morpholine (1 eq.) in
THF (approx. 1 ml / 0.4 mmol) was added dropwise. The mixture was stirred at -78 °C for 35 min. CuI
0.05 eq) and a solution of the corresponding sulfamidate (1-1.2 eq.) in THF (approx. 1 ml / 0.5 mmol)
(
were added. The mixture was stirred at -78 °C for 15 min, then allowed to warm to r.t. and stirred
overnight. The reaction was quenched by addition of water. Conc. HCl and methanol were added, and
the mixture was heated at 45 °C for 4-6 hrs. The reaction mixture was cooled down to 0 °C and a 6M
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NaOH-solution was added until pH 10. The reaction mixture was stirred at r.t. for 1-16 hrs, then
transferred to a separatory funnel. The layers were separated and the aqueous layer was extracted with
EtOAc (2 x). The combined organic layers were dried over Na SO , filtered and concentrated under
2
4
reduced pressure. The crude product was purified by column chromatography on silica gel.
0
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2
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7
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9
0
General procedure 4.
Chloropyrimidine derivative (1.0 eq.) and boronic acid pinacol ester (1.0 eq.) were charged in a flask.
Under nitrogen atmosphere, 1,4-dioxane (approx. 1 ml / 0.2 mmol) was added, followed by an aqueous
K PO -solution (2 eq.) and chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-
3
4
amino-1,1'-biphenyl)]-palladium(II) (XPhos Pd G2, 0.05 eq.). The resulting mixture was stirred at 95
C for 2-16 hrs. After completion of the reaction monitored by TLC, a 3 M aqueous HCl-solution (10
°
eq.) was added and the mixture was stirred at 60 °C for 3-16 hrs. A 2 M aqueous NaOH-solution was
added until pH 9-10. The aqueous layer was extracted with EtOAc (3 x). The combined organic layers
were dried over anhydrous Na SO , filtered and the solvent was evaporated under reduced pressure. The
2
4
crude product was purified by column chromatography on silica gel.
General procedure 5.
Step 1. The corresponding di-Boc protected bromo derivative 44 or 49 (1.0 eq.),
bis(pinacolato)diboron (1.5 eq.) and KOAc (3.0 eq.) were charged into a flask. Under nitrogen
atmosphere, 1,4-dioxane (approx. 1 ml / 0.2 mmol) was added, followed by [1,1′-
bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl , 0.1 eq.). The reaction mixture
2
was stirred at 100 °C for 2 hrs. Step 2. After completion of the reaction monitored by TLC, the
chloropyrimidine derivative 22b (200 mg, 0.65 mmol, 1 eq.) and K PO (3.0 eq.) in water (approx. 1 ml
3
4
/
0.3 mmol) and chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-
biphenyl)]-palladium(II) (XPhos Pd G2, 0.05 eq.) were added. The reaction mixture was stirred at 95
C for 2-16 hrs. A 15% NH Cl-solution was added and the aqueous layer was extracted with EtOAc (3
°
4
x). The combined organic layers were dried over anhydrous Na SO , filtered and the solvent was
2
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evaporated under reduced pressure. The crude product was purified by column chromatography on silica
gel.
General procedure 6.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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6
7
8
9
0
To a cold solution (0 °C) of the corresponding Boc-protected compound (1 eq.) in DCM (approx. 1
ml / 0.1 mmol), trifluoroacetic acid (TFA, 40 eq.) was added dropwise. The reaction mixture was
allowed to warm up to room temperature and was stirred for 1-3 hr. After completion of the reaction
monitored by TLC, a saturated NaHCO -solution was slowly added. The aqueous layer was extracted
3
with DCM (3 x). The combined organic layers were dried over anhydrous Na SO , filtered and
2
4
concentrated under reduced pressure. The crude product was purified by column chromatography on
silica gel.
(S)-5-(4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazin-2-
yl)pyridin-2-amine (2a) was prepared according to general procedure 4 from (S)-2-chloro-4-
morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazine 20a (131 mg, 0.44
mmol, 1 eq) and (E)-N,N-dimethyl-N'-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-
yl)formimidamide 38 (121 mg, 0.44 mmol, 1 eq.). Purification by column chromatography on silica gel
(cyclohexane / ethyl acetate 1:0 → 0:1) gave compound 2a as a colorless solid (102 mg, 0.29 mmol,
1
6
5%). H NMR (400 MHz, DMSO-d ):  8.83 (d, J = 2.3 Hz, 1 H), 8.18 (dd, J = 8.7, 2.3 Hz, 1 H), 6.45
6
(d, J = 8.6 Hz, 1 H), 6.27 (br s, 2 H), 4.04 (dd, J = 13.5, 2.6 Hz, 1 H), 3.87 (ddt, J = 10.5, 8.8, 4.3 Hz, 1
H), 3.79-3.71 (m, 3 H), 3.68-3.54 (m, 7 H), 3.35-3.28 (m, 1 H), 3.22-3.10 (m, 3 H), 2.63 (dd, J = 15.3,
1
3
1
4.7 Hz, 1 H). C{ H} NMR (101 MHz, DMSO-d ):  167.71 (s, 1 C), 161.20 (s, 1 C), 160.90 (s, 1 C),
6
1
58.28 (s, 1 C), 148.91 (s, 1 C), 136.73 (s, 1 C), 122.60 (s, 1 C), 107.34 (s, 1 C), 93.55 (s, 1 C), 70.07
(s, 1 C), 66.64 (s, 2 C), 66.17 (s, 1 C), 56.91 (s, 1 C), 45.90 (s, 2 C), 42.07 (s, 1 C), 29.14 (s, 1 C). HRMS
+
(
m/z): [M + H] calc. for C H N O 355.1877; found: 355.1880. HPLC (ACN with 0.1% TFA): t =
1
8
23
6
2
R
4
.98 min (98.7% purity).
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1
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
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3
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6
(
R)-5-(4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazin-2-
yl)pyridin-2-amine (2b) was prepared as described for its (S)-enantiomer 2a starting from (R)-2-chloro-
-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazine 20b (150 mg, 0.51
4
mmol, 1 eq) and boronic acid pinacol ester 38 in a 76% yield. The spectroscopic data are in agreement
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
with those reported for the (S)-enantiomer. HRMS (m/z): [M + H] calc. for C H N O 355.1877;
1
8
23
6
2
found: 355.1883. HPLC (ACN with 0.1% TFA): t = 4.95 min (97.4% purity).
R
(S)-5-(2-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazin-4-
yl)pyridin-2-amine (2c) was prepared according to general procedure 4 from (S)-4-chloro-2-
morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazine 20c (80 mg, 0.27
mmol, 1 eq) and (E)-N,N-dimethyl-N'-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-
yl)formimidamide 38 (81.5 mg, 0.30 mmol, 1.1 eq.). Purification by column chromatography on silica
gel (cyclohexane / ethyl acetate 1:0 → 0:1) gave compound 2c as a colorless solid (45 mg, 0.13 mmol,
1
47%). H NMR (400 MHz, DMSO-d ):  8.46 (d, J = 2.4 Hz, 1 H), 7.93 (dd, J = 8.7, 2.4 Hz, 1 H), 6.50
6
(
d, J = 8.7 Hz, 1 H), 6.31 (br s, 2 H), 3.96-3.89 (m, 2 H), 3.81-3.75 (m, 2 H), 3.69-3.61 (m, 8 H), 3.34-
13
1
3
.27 (m, 1 H), 3.24-3.11 (m, 3 H), 2.62 (dd, J = 15.9, 4.5 Hz, 1 H). C{ H} NMR (101 MHz, DMSO-
d6):  166.88 (s, 1 C), 161.13 (s, 1 C), 160.00 (s, 1 C), 152.84 (s, 1 C), 147.95 (s, 1 C), 136.12 (s, 1 C),
1
1
22.01 (s, 1 C), 107.22 (s, 1 C), 102.42 (s, 1 C), 70.07 (s, 1 C), 66.11 (s, 2 C), 65.54 (s, 1 C), 56.92 (s,
+
C), 44.23 (s, 2 C), 41.31 (s, 1 C), 27.75 (s, 1 C). HRMS (m/z): [M + H] calc. for C H N O 355.1877;
18 23
6
2
found: 355.1881. HPLC (ACN with 0.1% TFA): t = 3.57 min (99.8% purity).
R
(
R)-5-(2-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazin-4-
yl)pyridin-2-amine (2d) was prepared as described for its (S)-enantiomer 2c starting from (R)-4-chloro-
-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazine 20d (100 mg, 0.34
2
mmol, 1 eq) and boronic acid pinacol ester 38 in a 57% yield. The spectroscopic data are in agreement
+
with those reported for the (S)-enantiomer. HRMS (m/z): [M + H] calc. for C H N O 355.1877;
1
8
23
6
2
found: 355.1880. HPLC (ACN with 0.1% TFA): t = 3.61 min (99.2% purity).
R
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(
S)-5-(4-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazin-2-
yl)-4-(trifluoromethyl)pyridin-2-amine (3a) was prepared according to general procedure 4 from (S)-
-chloro-4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazine 20a (544
2
mg, 1.83 mmol, 1.0 eq.) and boronic acid pinacol ester 50 (818 mg, 2.38 mmol, 1.3 eq.) Purification by
column chromatography on silica gel (CH Cl / methanol 1:0 → 20:1) gave compound 3a as a colorless
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
1
solid (503 mg, 1.19 mmol, 65%). H NMR (400 MHz, CDCl ):  8.62 (s, 1 H), 6.77 (s, 1 H), 4.75 (br
3
s, 2 H), 4.10 (dd, J = 14.0, 2.5 Hz, 1 H), 4.01-3.93 (m, 1 H), 3.86-3.70 (m, 6 H), 3.70-3.58 (m, 4 H),
3.47 (td, J = 12.0, 2.8 Hz, 1 H), 3.35 (t, J = 11 Hz, 1 H), 3.27-3.17 (m, 2 H), 2.62 (dd, J = 15.0, 4.8 Hz,
19
1
13
1
1
H). F{ H} NMR (376 MHz, CDCl ):  − 60.2 (s, 3 F). C{ H} NMR (101 MHz, CDCl ):  167.70
3
3
(
s, 1 C), 161.65 (s, 1 C), 158.91 (s, 1 C), 158.33 (s, 1 C), 152.29 (s, 1 C), 138.0 (q, J = 32 Hz, 1 C),
1
1
23.91-123.81 (m, 1 C), 123.1 (q, J = 274 Hz, 1 C), 105.2 (q, J = 5.6 Hz, 1 C), 93.59 (s, 1 C), 70.44 (s,
C), 66.96 (s, 2 C), 66.59 (s, 1 C), 56.97 (s, 1 C), 45.94 (s, 2 C), 42.05 (s, 1 C), 29.66 (s, 1 C). HRMS
+
(
m/z): [M + H] calc. for C H F N O 423.1751; found: 423.1743. HPLC: t = 6.74 min (97.5%
19 22
3
6
2
R
2
0
purity). (S)-enantiomer: [α] = – 13.2 (c = 2.0, CHCl3).
퐷
(R)-5-(4-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazin-2-
yl)-4-(trifluoromethyl)pyridin-2-amine (3b) was prepared as described for its (S)-enantiomer 50a
starting
from
(R)-2-chloro-4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-
c][1,4]oxazine 20b (552 mg, 1.86 mmol, 1.0 eq.) and boronic acid pinacol ester 50 (83 mg, 2.42 mmol,
1
.3 eq.) in a 62% yield. The spectroscopic data are in agreement with those reported for the (S)-
+
enantiomer. HRMS (m/z): [M + H] calc. for C H F N O 423.1751; found: 423.1744. HPLC: t =
1
9
22
3
6
2
R
2
0
6.73 min (98.9% purity). (R)-enantiomer: [α] 퐷 = + 13.5 (c = 1.6, CHCl3).
(S)-5-(4-Morpholino-5,5a,6,7,8,9-hexahydropyrimido[5,4-b]indolizin-2-yl)-4-
(trifluoromethyl)pyridin-2-amine (4a) was prepared according to general procedure 4 from
intermediate 51a (150 mg, 0.51 mmol, 1.0 eq.) and boronic acid pinacol ester 50 (175 mg, 0.51 mmol,
1
.0 eq.). Purification by column chromatography on silica gel (cyclohexane / ethyl acetate 1:0 → 3:2)
1
gave compound 4a as a yellowish solid (90 mg, 0.21 mmol, 42%). H NMR (400 MHz, DMSO):  8.42
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
(
s, 1 H), 6.76 (s, 1 H), 6.67 (br s, 2 H), 4.05 (dd, J = 13, 4.2 Hz, 1 H), 3.67-3.49 (m, 9 H), 3.25 (dd, J =
1
1

5.0, 9.1 Hz, 1 H), 2.78-2.64 (m, 2 H), 1.83-1.67 (m, 2 H), 1.63-1.55 (m, 1 H), 1.51-1.37 (m, 1 H), 1.37-
1
9
1
13
1
.20 (m, 2 H). F{ H} NMR (376 MHz, DMSO):  − 59.1 (s, 3 F). C{ H} NMR (101 MHz, DMSO):
167.2 (s, 1 C), 160.7 (s, 1 C), 160.2 (s, 1 C), 157.5 (s, 1 C), 151.8 (s, 1 C), 135.7 (q, J = 31 Hz, 1 C),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
123.3 (q, J = 274 Hz, 1 C), 121.2-121.1 (m, 1 C), 104.1 (q, J = 5.9 Hz, 1 C), 99.3 (s, 1 C), 66.1 (s, 2 C),
58.9 (s, 1 C), 45.5 (s, 2 C), 41.3 (s, 1 C), 33.6 (s, 1 C), 31.5 (s, 1 C), 24.4 (s, 1 C), 23.6 (s, 1 C). HRMS
+
(m/z): [M + H] calc. for C H F N O 421.1958; found: 421.1962. HPLC: t = 8.44 min (97.8% purity).
2
0
24
3
6
R
(R)-5-(4-Morpholino-5,5a,6,7,8,9-hexahydropyrimido[5,4-b]indolizin-2-yl)-4-
(trifluoromethyl)pyridin-2-amine (4b) was prepared as described for its (S)-enantiomer 4a starting
from 51b (150 mg, 0.51 mmol, 1.0 eq.) and boronic acid pinacol ester 50 (175 mg, 0.51 mmol, 1.0 eq.)
in a 47% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer.
+
HRMS (m/z): [M + H] calc. for C H F N O 421.1958; found: 421.1966. HPLC: t = 8.45 min (98.7%
2
0
24
3
6
R
purity).
(S)-5-(4-(piperidin-1-yl)-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazin-
2-yl)-4-(trifluoromethyl)pyridin-2-amine (5a) was prepared as described for its (R)-enantiomer 5b
starting from 52a (100 mg, 0.34 mmol, 1.0 eq.) and boronic acid pinacol ester 50 (128 mg, 0.37 mmol,
1
.1 eq.) in a 75% yield. The spectroscopic data agree with those reported for the (R)-enantiomer. HRMS
+
(m/z): [M + H] calc. for C H F N O 421.1958; found: 421.1966. HPLC: t = 8.55 min (98.3% purity).
2
0
24
3
6
R
(R)-5-(4-(Piperidin-1-yl)-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-c][1,4]oxazin-
2-yl)-4-(trifluoromethyl)pyridin-2-amine (5b) was prepared according to general procedure 4 from
intermediate 52b (100 mg, 0.34 mmol, 1.0 eq.) and boronic acid pinacol ester 50 (128 mg, 0.38 mmol,
1
.1 eq.). Purification by column chromatography on silica gel (cyclohexane / ethyl acetate 1:0 → 3:2)
1
gave compound 5b as a colorless solid (114 mg, 0.27 mmol, 80%). H NMR (400 MHz, DMSO):  8.43
(s, 1 H), 6.76 (s, 1 H), 6.69 (br s, 2 H), 3.95-3.80 (m, 2 H), 3.77-3.67 (m, 2 H), 3.63-3.50 (m, 4 H), 3.29
(td, J = 12.0, 2.4 Hz, 1 H), 3.23-3.14 (m, 2 H), 3.08 (td, J = 13.0, 3.6 Hz, 1 H), 2.61 (dd, J = 15.0, 4.6
1
9
1
Hz, 1 H), 1.64-1.57 (m,. 2 H), 1.53-1.44 (m, 4 H). F{ H} NMR (376 MHz, DMSO):  − 58.9 (s, 3 F).
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1
C{ H} NMR (101 MHz, DMSO):  167.10 (s, 1 C), 160.88 (s, 1 C), 160.18 (s, 1 C), 157.47 (s, 1 C),
1
5
2
51.81 (s, 1 C), 135.70 (q, J = 31 Hz, 1 C), 123.30 (q, J = 274 Hz, 1 C), 121.16 (s, 1 C), 104.12 (q, J =
.7 Hz, 1 C), 92.47 (s, 1 C), 69.51 (s, 1 C), 65.62 (s, 1 C), 56.31 (s, 1 C), 45.88 (s, 2 C), 41.61 (s, 1 C),
+
9.11 (s, 1 C), 25.57 (s, 2 C), 24.43 (s, 1 C). HRMS (m/z): [M + H] calc. for C H F N O 421.1958;
2
0
24
3
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
found: 421.1963. HPLC: t = 8.55 min (98.9% purity).
R
5
-((S)-4-((S)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-
c][1,4]oxazin-2-yl)pyridin-2-amine (6a) was prepared according to general procedure 4 from (S)-2-
chloro-4-((S)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-
c][1,4]oxazine 21a (84 mg, 0.27 mmol, 1 eq) and (E)-N,N-dimethyl-N'-(5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)pyridin-2-yl)formimidamide 38 (275 mg, 0.27 mmol, 1 eq.). Purification by column
chromatography on silica gel (cyclohexane / ethyl acetate 1:0 → 0:1) gave compound 6a as a colorless
1
solid (76 mg, 0.21 mmol, 76%). H NMR (400 MHz, DMSO-d ):  8.82 (d, J = 2.2 Hz, 1 H), 8.17 (dd,
6
J = 8.7, 2.3 Hz, 1 H), 6.45 (dd, J = 8.7, 0.8 Hz, 1 H), 6.28 (br s, 2 H), 4.36-4.30 (m, 1 H), 4.09-4.02 (m,
2
3
H), 3.93-3.84 (m, 2 H), 3.79-3.63 (m, 4 H), 3.48 (td, J = 11.7, 2.9 Hz, 1 H), 3.35-3.29 (m, 1 H), 3.26-
1
3
1
.10 (m, 4 H), 2.65 (dd, J = 15.3, 4.7 Hz, 1 H), 1.18 (d, J = 6.7 Hz, 3 H). C{ H} NMR (101 MHz,
DMSO-d ):  167.63 (s, 1 C), 161.19 (s, 1 C), 160.88 (s, 1 C), 157.80 (s, 1 C), 148.91 (s, 1 C), 136.70
6
(s, 1 C), 122.69 (s, 1 C), 107.33 (s, 1 C), 93.14 (s, 1 C), 70.86 (s, 1 C), 70.10 (s, 1 C), 66.98 (s, 1 C),
66.17 (s, 1 C), 56.77 (s, 1 C), 47.58 (s, 1 C), 42.02 (s, 1 C), 40.11 (s, 1 C), 29.18 (s, 1 C), 14.23 (s, 1 C).
+
HRMS (m/z): [M + H] calc. for C H N O 369.2034; found: 369.2033. HPLC (ACN with 0.1%
1
9
25
6
2
TFA): t = 5.39 min (99.2% purity).
R
5
-((R)-4-((S)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-
c][1,4]oxazin-2-yl)pyridin-2-amine (6b) was prepared as described for its (S,R)-enantiomer 7a starting
from (R)-2-chloro-4-((S)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-
c][1,4]oxazine 21b (120 mg, 0.39 mmol, 1 eq) and boronic acid pinacol ester 38 in a 64% yield. The
spectroscopic data are in agreement with those reported for the (S,R)-enantiomer. HRMS (m/z): [M +
2
7
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Journal of Medicinal Chemistry
Page 28 of 65
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2
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+
H] calc. for C H N O 369.2034; found: 369.2033. HPLC (ACN with 0.1% TFA): t = 5.37 min
1
9
25
6
2
R
(
98.9% purity).
5-((S)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
0
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8
9
0
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7
8
9
0
1
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7
8
9
0
c][1,4]oxazin-2-yl)pyridin-2-amine (7a) was prepared according to general procedure 4 from (S)-2-
chloro-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-
c][1,4]oxazine 22a (84 mg, 0.27 mmol, 1 eq) and (E)-N,N-dimethyl-N'-(5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)pyridin-2-yl)formimidamide 38 (75 mg, 0.27 mmol, 1 eq.). Purification by column
chromatography on silica gel (cyclohexane / ethyl acetate 1:0 → 0:1) gave compound 7a as a colorless
1
solid (72 mg, 0.20 mmol, 74%). H NMR (400 MHz, DMSO-d ):  8.82 (d, J = 2.4 Hz, 1 H), 8.17 (dd,
6
J = 8.7, 2.3 Hz, 1 H), 6.45 (d, J = 8.7 Hz, 1 H), 6.27 (br s, 2 H), 4.35-4.30 (m, 1 H), 4.13-4.02 (m, 2 H),
3.92-3.81 (m, 2 H), 3.79-3.72 (m, 2 H), 3.69-3.61 (m, 2 H), 3.48 (td, J = 11.8, 2.9 Hz, 1 H), 3.36-3.29
1
3
1
(m, 1 H), 3.25-3.09 (m, 4 H), 2.57 (dd, J = 15.3, 4.7 Hz, 1 H), 1.21 (d, J = 6.7 Hz, 3 H). C{ H} NMR
(
101 MHz, DMSO-d ):  167.25 (s, 1 C), 160.73 (s, 1 C), 160.40 (s, 1 C), 157.20 (s, 1 C), 148.43 (s, 1
6
C), 136.23 (s, 1 C), 122.22 (s, 1 C), 106.87 (s, 1 C), 92.50 (s, 1 C), 70.41 (s, 1 C), 69.53 (s, 1 C), 66.53
(
s, 1 C), 65.68 (s, 1 C), 56.38 (s, 1 C), 47.12 (s, 1 C), 41.64 (s, 1 C), 28.84 (s, 1 C), 13.88 (s, 1 C). HRMS
+
(
m/z): [M + H] calc. for C H N O 369.2034; found: 369.2034. HPLC (ACN with 0.1% TFA): t =
1
9
25
6
2
R
5
.42 min (99.3% purity).
5
-((R)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-
c][1,4]oxazin-2-yl)pyridin-2-amine (7b) was prepared as described for its (S,S)-enantiomer 6a starting
from (R)-2-chloro-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5',4':4,5]pyrrolo[2,1-
c][1,4]oxazine 22b (150 mg, 0.48 mmol, 1 eq) and boronic acid pinacol ester 38 in a 49% yield. The
spectroscopic data are in agreement with those reported for the (S,S)-enantiomer. HRMS (m/z): [M +
+
H] calc. for C H N O 369.2034; found: 369.2035. HPLC (ACN with 0.1% TFA): t = 5.35 min
19 25
6
2
R
(99.6% purity).
2
8
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Page 29 of 65
Journal of Medicinal Chemistry
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2 7
5
-[(9S)-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0 , ]trideca-
2
(7),3,5-trien-4-yl]pyridin-2-amine (8a) was prepared according to general procedure 4 from (9S)-4-
2
7
chloro-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0 , ]trideca-2(7),3,5-
triene 23a (150 mg, 0.46 mmol, 1 eq) and (E)-N,N-dimethyl-N'-(5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)pyridin-2-yl)formimidamide 38 (128 mg, 0.46 mmol, 1 eq.). Purification by column
1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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0
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7
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9
0
chromatography on silica gel (cyclohexane / ethyl acetate 1:0 → 0:1) gave compound 8a as a colorless
1
solid (118 mg, 0.31 mmol, 67%). H NMR (400 MHz, DMSO-d ):  8.81 (dd, J = 2.3, 0.8 Hz, 1 H),
6
8.15 (dd, J = 8.6, 2.3 Hz, 1 H), 6.44 (dd, J = 8.7, 0.8 Hz, 1 H), 6.26 (br s, 2 H), 4.38-4.34 (m, 2 H), 4.09-
3
.95 (m, 3 H), 3.87-3.80 (m, 1 H), 3.77-3.69 (m, 2 H), 3.34-3.27 (m, 1 H), 3.22-3.05 (m, 5 H), 2.63 (dd,
13
1
J = 15.3, 4.8 Hz, 1 H), 1.84-1.74 (m, 4 H). C{ H} NMR (101 MHz, DMSO-d ):  167.09 (s, 1 C),
6
1
60.72 (s, 1 C), 160.25 (s, 1 C), 158.61 (s, 1 C), 148.43 (s, 1 C), 136.26 (s, 1 C), 122.22 (s, 1 C), 106.86
(
s, 1 C), 92.56 (s, 1 C), 73.16 (s, 1 C), 73.15 (s, 1 C), 69.60 (s, 1 C), 65.70 (s, 1 C), 56.38 (s, 1 C), 50.70
(
s, 1 C), 50.48 (s, 1 C), 41.59 (s, 1 C), 28.84 (s, 1 C), 27.56 (s, 1 C), 27.52 (s, 1 C). HRMS (m/z): [M +
+
H] calc. for C H N O 381.2034; found: 381.2033. HPLC (ACN with 0.1% TFA): t = 5.34 min
2
0
25
6
2
R
(98.2% purity).
2
7
5
-[(9R)-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0 , ]trideca-
(7),3,5-trien-4-yl]pyridin-2-amine (8b) was prepared as described for its (S)-enantiomer 8a starting
from (9R)-4-chloro-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-
2
2
7
triazatricyclo[7.4.0.0 , ]trideca-2(7),3,5-triene 23b (200 mg, 0.62 mmol, 1 eq) and boronic acid pinacol
ester 38 in a 86% yield. The spectroscopic data agree with those reported for the (S)-enantiomer. HRMS
+
(
m/z): [M + H] calc. for C H N O 381.2034; found: 381.2036. HPLC (ACN with 0.1% TFA): t =
2
0
25
6
2
R
5
.17 min (96.4% purity).
5
-[(9S)-6-{3-oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0²,⁷]trideca-
2(7),3,5-trien-4-yl]pyridin-2-amine (9a) was prepared according to general procedure 4 from (9S)-4-
chloro-6-{3-oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0²,⁷]trideca-2(7),3,5-
triene 24a (150 mg, 0.46 mmol, 1 eq) and (E)-N,N-dimethyl-N'-(5-(4,4,5,5-tetramethyl-1,3,2-
2
9
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