(S)-4-(Difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530), a Potent, Orally Bioavailable, and Brain-Penetrable Dual Inhibitor of Class i PI3K and mTOR Kinase

Article abstract of DOI:10.1021/acs.jmedchem.9b00525

The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is frequently overactivated in cancer, and drives cell growth, proliferation, survival, and metastasis. Here, we report a structure-activity relationship study, which led to the discovery of a drug-like adenosine 5′-triphosphate-site PI3K/mTOR kinase inhibitor: (S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530, compound 6), which qualifies as a clinical candidate due to its potency and specificity for PI3K and mTOR kinases, and its pharmacokinetic properties, including brain penetration. Compound 6 showed excellent selectivity over a wide panel of kinases and an excellent selectivity against unrelated receptor enzymes and ion channels. Moreover, compound 6 prevented cell growth in a cancer cell line panel. The preclinical in vivo characterization of compound 6 in an OVCAR-3 xenograft model demonstrated good oral bioavailability, excellent brain penetration, and efficacy. Initial toxicity studies in rats and dogs qualify 6 for further development as a therapeutic agent in oncology.

Full text of DOI:10.1021/acs.jmedchem.9b00525

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Article
(S)-4-(Difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-
triazin-2-yl)pyridin-2-amine (PQR530), a Potent, Orally Bioavailable
and Brain Penetrable Dual Inhibitor of Class I PI3K and mTOR Kinase
Denise Rageot, Thomas Bohnacker, Erhan Keles, Jacob A. McPhail, Reece M. Hoffmann,
Anna Melone, Chiara Borsari, Rohitha Sriramaratnam, Alexander Markus Sele, Florent Beaufils,
Paul Hebeisen, Doriano Fabbro, Petra Hillmann, John E. Burke, and Matthias P. Wymann
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00525 • Publication Date (Web): 07 Jun 2019
Downloaded from http://pubs.acs.org on June 8, 2019
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course of their duties.

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(S)-4-(Difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-
2-yl)pyridin-2-amine (PQR530), a Potent, Orally Bioavailable and Brain
Penetrable Dual Inhibitor of Class I PI3K and mTOR Kinase
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Denise Rageot,†$ Thomas Bohnacker,†$ Erhan Keles,† Jacob A. McPhail,§ Reece M. Hoffmann,§ Anna
Melone,† Chiara Borsari,† Rohitha Sriramaratnam,† Alexander M. Sele,† Florent Beaufils,† Paul
Hebeisen,‡ Doriano Fabbro,‡ Petra Hillmann,‡ John E. Burke,§ Matthias P. Wymann†,*
†Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland;
§Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia
BC V8W 2Y2, Canada
‡PIQUR Therapeutics AG, Hochbergerstrasse 60C, 4057 Basel, Switzerland;
$Equal contribution: these authors have contributed equally.
*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
Phosphoinositide 3-kinase (PI3K), mammalian or mechanistic target of rapamycin (mTOR); PI3K
inhibitor, mTOR kinase inhibitor; brain penetrable; cancer; ATP-competitive; rapamycin, rapalogs,
pharmacology, pharmacodynamics, clinical candidate
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ABSTRACT
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The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is frequently
overactivated in cancer, and drives cell growth, proliferation, survival and metastasis. Here, we report
a SAR-study which led to the discovery of a drug-like ATP-site PI3K/mTOR kinase inhibitor: (S)-4-
(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine
(PQR530, compound 6), which qualifies as a clinical candidate due to its potency and specificity for
PI3K and mTOR kinases, and its pharmacokinetic properties, including brain penetration. Compound
6 showed excellent selectivity over a wide panel of kinases and an excellent selectivity against unrelated
receptor enzymes and ion channels. Moreover, compound 6 prevented cell growth in a cancer cell line
panel. The preclinical in vivo characterization of compound 6 in an OVCAR-3 xenograft model
demonstrated good oral bioavailability, excellent brain penetration, and efficacy. Initial toxicity studies
in rats and dogs qualify 6 for further development as a therapeutic agent in oncology.
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INTRODUCTION
The phosphoinositide 3-kinase (PI3K) – mechanistic target of rapamycin (mTOR) signaling pathway
plays a fundamental role in cell proliferation, growth, and survival. Aberrant activation of this signaling
pathway has been shown to drive the progression of malignant tumors, which can be triggered by
mutated growth factor receptors, PI3K, loss of PTEN (phosphatase and tensin homolog on chromosome
10) and effector proteins such as Ras.1–3 The resulting increase of cellular PtdIns(3,4,5)P3 subsequently
recruits protein kinase B (PKB/Akt), which is fully activated by phosphorylation by phosphoinositide-
dependent kinase 1 (PDK1) and the TOR complex 2 (TORC2) kinase at the plasma membrane.4 By the
phosphorylation of tuberous sclerosis 2 (TSC2, tuberin), PKB/Akt initiates the activation and assembly
of TORC1 on endomembranes. TORC1 then augments cellular protein and lipid production via
phosphorylation of S6 kinase (S6K). While TORC1 affects endomembrane dynamics and autophagy,
TORC2 impacts on metabolic, cell cycle and cytoskeletal changes.3
Drugs targeting the pathway at multiple nodes, such as the first dual PI3K/mTOR inhibitor BEZ235
(dactolisib),5,6 have provided promising preclinical results attenuating tumor growth driven specifically
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by overactivated PI3K,7 but also block growth of cancer cells with multiple activated pathways, such
as in melanoma.5 Although subsequent clinical studies of BEZ235 were plagued by adverse effects,8
dual PI3K/mTOR inhibitors are still considered a valuable asset in cancer therapy, and are pursued in
clinical trials.2
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Recently, we presented PQR309 (bimiralisib, 5)9,10 as a novel, brain-penetrant pan-PI3K inhibitor,
which also moderately targets mTOR kinase activity (Figure 1). First clinical results for 5 have been
released,15 and the compound is currently in phase II clinical trials. To maximize potency, we focused
on the development of a brain permeant follow-up compound with a major enhancement in mTOR
kinase inhibition.
We analyzed the activity profiles of compounds with a similar structure and binding mode as for 5,
and assessed their in vitro dissociation constants (Kd) for class I PI3K isoforms (, , , and ) and
mTOR. A selection of relevant clinical compounds is depicted in Figure 1 (for data see Table S1 in
Supporting Information), and affinities for individual PI3K isoforms and mTOR provided a lead for
novel compounds. Compound 1 (alpelisib, BYL719) has been reported as a specific PI3K inhibitor12,
but displayed in our hands only 7-fold selectivity over PI3K, while sparing PI3K, PI3K and mTOR
(> 250-fold). Pictilisib (GDC0941, 4)13 is a potent pan class I inhibitor that strongly binds to PI3K, ,
and δ, with less affinity to PI3K however, its affinity to mTOR is >50-fold reduced compared to
p110. All other compounds are to various degrees dual class I PI3K [PI3K isoforms (, , , and )]
and mTOR kinase inhibitors: 2 (buparlisib, BKM120),9,14 5,9–11,15 and 3 (apitolisib, GDC0980)13, which
all target class I as well as mTOR. Compound 6 emerging from the study presented here is a sub-
nanomolar inhibitor of PI3K and mTOR, and targets other class I PI3Ks at around 10 nM (Table S1).
Here, we report the lead optimization process using 5 as a starting template to generate 6, a potent and
brain-penetrant clinical candidate compound.
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Figure 1. Affinity graph and chemical formulae of selected ATP-competitive inhibitors for class I PI3K isoforms
and mTOR kinase. A) Kd values for each compound and kinase are presented as a radar diagram with reciprocal and
logarithmic scaled axes. Data for compounds 2, 3, 4 and 5 are from Ref. 10. Dissociation constants (Kd) were
determined using the ScanMax platform from DiscoverX,16 see Supporting Information, Table S1. B) Chemical
structure of clinically relevant selected compounds.
RESULTS AND DISCUSSION
Chemistry
To identify potent and dual PI3K/mTOR inhibitors with increased potency, selectivity and brain
distribution profile, a library of pyrimidine and triazine compounds was produced (Scheme 1). All
triazine core compounds were prepared from cyanuric chloride (7), either via di-substitution by the
same morpholine moiety (Mn, route A for symmetric compounds) or by subsequent substitution with
two different morpholines (route B for asymmetric compounds). Depending on the morpholine
substituent, this reaction yielded the intermediates with good to excellent yields (74-87%). The
heteroaryl moieties were then introduced via Suzuki cross-coupling using the corresponding boronic
acid pinacol ester. Boronic acid pinacol esters were either protected at their 2-amino group, and isolated
or generated in situ (see experimental section and Supporting Information). The desired triazine core
derivatives (5, 6, 16-22 and 31-39) were obtained in moderate to good yields (21-87%). The synthetic
procedure for the preparation of pyrimidine core compounds (44-47) is depicted in route C. Similar to
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the synthesis of triazine derivatives, this synthesis involved a nucleophilic aromatic substitution
followed by a Suzuki cross-coupling reaction. The nucleophilic aromatic substitution of 2,4,6-
trichloropyrimidine by an unsubstituted morpholine to yield compounds 40 and 41 has been described
previously.9
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Scheme 1. Synthesis of tri-substituted triazine and pyrimidine core compounds.a
Mn
M0
M1
=
=
5
16
17
18
19
20
21
22
M0
A
O
O
N
M1 (64%)
M2 (35%)
M3 (87%)
M4 (77%)
M5 (44%)
M6 (57%)
M7 (35%)
Cl
Mn
N
Mn
N
F
F
F
i
ii
N
N
N
N
N
N
(R) N
Cl
N
Cl
Mn
Cl
Mn
N
NH2
7
8
15
-
(74 - 95 %)
M2
M3
M4
=
=
=
O
O
(S) N
B
Mn
M0
M0
M0
N
31
32
33
34
35
36
37
M1 (32%)
M2 (74%)
M3 (46%)
M4 (57%)
M5 (21%)
M6 (57%)
M7 (45%)
F
F
F
iii or iv
ii
N
N
N
N
N
N
N
Cl
N
Cl
Mn
N
Cl
Mn
N
Z
NH2
23
24 30
-
O
N
(74 - 87 %)
M0
N
F
F
Z, W
v, vi or vii
N
N
M5
M6
M7
=
=
=
(R)
6
38
39
H, CH (41%)
F, N (62%)
H, N (60%)
O
O
O
N
(S)
M2
W
N
NH2
C
(S)
M0
Y
M0
M0
Y
N
(S)
X, Y,
W
F
F
viii
ii or vii
N
X
N
X
N
X
44
45
46
47
CH, N, CH (57%)
N, CH, CH (74%)
CH, N, N (53%)
N, CH, N (63%)
Cl
Cl
M2
Y
Cl
M2
W
N
NH2
(R)
40
41
: X = CH, Y = N
: X = N, Y = CH
42
43
: X = CH, Y = N (54%)
: X = N, Y = CH (70%)
N
(R)
aReagents and conditions: (i) morpholine derivative (Mn-H), CH2Cl2, 0 °C → rt, o/n or as reported in literature17;
(ii) 1) boronic acid pinacol ester (RBpin) 48 or 49, XPhosPdG2 (cat.), K3PO4, dioxane / H2O, 95 °C, 2 - 15 h; 2)
HCl, H2O, 60 °C, 3 -15 h; (iii) Mn-H, DIPEA, EtOH, 0 °C → rt, o/n; (iv) Mn-H, DIPEA, dioxane, 70 °C, o/n; (v)
1) Pd(OAc) / PPh3 (cat.), RBpin 49, K2CO3, THF / H2O, 55 °C, 2 H; 2 ) HCl, H2O, 55 °C, o/n; (vi) RBpin,
XPhosPdG2 (cat.), K3PO4, dioxane / H2O, 95 °C, 2 h; (vii) RBpin generated in situ (see Supporting Information),
XPhosPdG2 (cat.), K3PO4, dioxane / H2O, 95 °C, 3 -15 h; (viii) Mn-H, DIPEA, 130 °C, o/n.
Determination of Cellular Potency and Dual PI3K and mTOR Kinase Activity
The SAR study focused initially on the exploration of substitutions on the morpholine moiety and its
influence on PI3K vs. mTOR inhibition. Therefore, two series of compounds were tested (Table 1): i)
symmetrical compounds (5, 16-22), substituted with two identical morpholines (M1-7), and ii)
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asymmetric compounds (31-37), with one unsubstituted morpholine (M0) and one substituted
morpholine (M1-7). Among the symmetric compounds, compound 20 (substituted with cis-3,5-
dimethylmorpholine) had the highest affinity for both mTOR (Ki = 40 nM) and p110 (Ki = 31 nM).
Overall, symmetric compounds with substituted morpholines showed increased concentrations for half-
maximal inhibition (IC50) of PKB/Akt and ribosomal protein S6 phosphorylation in cells. An exception
was compound 17, substituted with (S)-3-methylmorpholine (M2; IC50 of 248 nM for pPKB and of 283
nM for pS6). With the exception of compound 31 (Ki for mTOR = 109 nM), asymmetric compounds
showed a high in vitro potency for mTOR (Ki < 65 nM). Among this series, the most potent compounds
towards PI3K and mTOR were 32 with one (S)-3-methylmorpholine (M2) and 35 with cis-3,5-
dimethylmorpholine (M5; Table 1). The asymmetric compound 32 was 5-fold more potent on mTOR
than its symmetric analog bearing two (S)-3-methylmorpholine moieties (17) (Ki for mTOR = 25.5 and
137 nM, respectively). Overall, compounds with only one substituted morpholine were more potently
inhibiting cellular PI3K/mTOR signaling. As compound 32 showed a 2-fold increase in affinity for both
PI3K and mTOR with respect to PQR309 (5), it was selected for further optimization.
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Table 1. SAR-study of 4-(trifluoromethyl)-5-(1,3,5-triazin-2-yl)pyridine-2-amines.
5
6
7
8
9
M0
=
M1
=
M2
=
M3
=
O
N
O
O
(R) N
O
O
(S) N
O
O
N
N
R1
N
F
F
F
N
N
R2
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N
NH2
M5
=
M6
=
M7
=
(R)
(S)
(R)
(R)
M4
=
N
N
O
N
(S)
(S)
Cellular IC50, [nM]a
pPKB
Ki, [nM]b
p110α
17
Ki(p110α)/
Ki(mTOR)
Cpd.
R1
R2
clogPc
pS6
205
1122
283
299
691
437
531
14522
410
89.8
156
212
135
126
457
mTOR
62
PQR309 (5)
M0 M0 139
M1 M1 1702
M2 M2 248
M3 M3 755
M4 M4 947
M5 M5 567
M6 M6 610
M7 M7 >20000
M0 M1 373
M0 M2 196
M0 M3 420
M0 M4 274
M0 M5 122
M0 M6 100
M0 M7 435
0.27
4.01
0.05
60.5
4.12
0.79
0.14
32.7
0.49
0.33
0.46
0.65
1.42
0.26
1.38
3.11
3.94
3.94
4.04
4.04
4.76
4.76
4.76
3.52
3.52
3.58
3.58
3.94
3.94
3.94
16
17
18
19
20
21
22
31
32
33
34
35
36
37
684
171
6.8
137
4643
731
76.8
177
31.4
37.0
>20000
53.7
8.3
39.6
265
1892
109
25.5
43.1
47.8
19.3
59.2
64.5
19.9
31.0
27.4
15.2
88.9
aCellular phosphorylation of PKB/Akt on Ser473 and ribosomal S6 on Ser235/236 were analyzed in inhibitor
treated A2058 melanoma cells using in-cell western detection. Each experiment performed in triplicate or as a
multiple of n = 3. bCompounds were tested in a time-resolved FRET (TR)-assay (LanthaScreen), and inhibitor Kis
were calculated for PI3Kα and mTOR as described in methods. The PI3Kα/mTOR column depicts the ratio of
compound-specific PI3Kα Ki over the Ki for mTOR. Each experiment was performed at least twice or as a multiple
of n = 2. cMarvin/JChem 16.10.17 was used for calculation of logP (partition coefficient) and PSA (polar surface
area) values. For all compounds shown in this table the calculated PSA are equal to 102.5.
The results above, together with the SAR study that led to the development of a specific mTOR
inhibitor,
5-[4,6-bis({3-oxa-8-azabicyclo[3.2.1]octan-8-yl})-1,3,5-triazin-2-yl]-4-
(difluoromethyl)pyridin-2-amine (PQR620,17,18 67; for chemical structure see Appendix S1 in
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Supporting Information), encouraged the preparation of a compound series with i) a (S)-3-
methylmorpholine and ii) a difluoro- or trifluoromethyl group on the heteroaryl moiety (Table 2). With
the exception of compound 45, both triazine (6, 32, 38-39) and pyrimidine (44, 46 and 47) core
compounds showed very good potencies in cells. Moreover, these data confirmed our previous
observation that 4-difluoromethyl-substituted heteroaryls yielded increased affinity for mTOR, as
compared to their respective 4-trifluoromethyl analogs.17 The introduction of an additional N-atom in
the heteroaryl moiety moderately improved the affinity for PI3K (see Ki of 1.9 nM for 39 vs. 11 nM
for 6; and Ki of 2.0 nM for 47 vs. 14 nM for 45), but reduced mTOR binding (Ki of 192 nM for 47 vs
42 nM for 45), allowing a fine tuning of PI3K/mTOR inhibition ratios. Although the pyrimidine core
compound 44 showed a balanced PI3K/mTOR profile in vitro, compound 6 was selected (Ki for PI3K
of ~ 11 nM and for mTOR of ~ 7.4 nM) for further characterization due to its superior activity in cells.
Moreover, the triazine core yields symmetric compounds, and therefore eases large scale synthetic
access.
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Table 2. SAR of pyrimidine and triazine cores substituted with morpholine and (S)-3-
methylmorpholine.
O
7
8
9
N
Z
F
F
N
X
(S)
N
Y
W
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11
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O
N
NH2
Ki p110α/
Ki mTOR
Cellular IC50, [nM]a Ki, [nM]b
Cpd.
X
Y
Z
W
clogPc PSAc
pPKB
pS6
p110α mTOR
32
6
N
N
F
CH 195.7 89.8
8.3
25.5
7.4
0.33
1.50
0.11
0.18
1.16
0.32
0.27
0.01
3.52
2.80
3.08
2.26
2.40
2.40
1.85
1.85
102.52
102.52
115.41
115.41
89.63
N
N
H
F
CH 62.2
61.9
164
63.9
125
766
146
387
11.1
4.0
38
39
44
45
46
47
N
N
N
N
91.0
32.4
37.9
10.6
10.0
42.3
15.3
192
N
N
H
H
H
H
H
1.9
CH
N
N
CH 146
CH 614
11.6
13.7
4.1
CH
N
89.63
CH
N
N
N
77.3
99.6
102.52
102.52
CH
2.0
aCellular phosphorylation of PKB/Akt on Ser473 and ribosomal S6 on Ser235/236 were analyzed in inhibitor
treated A2058 melanoma cells using in-cell western detection. Each experiment performed in triplicate or as a
multiple of n = 3. bCompounds were tested in a time-resolved FRET (TR)-assay (LanthaScreen), and inhibitor Kis
were calculated for PI3Kα and mTOR as described in methods. The PI3Kα/mTOR column depicts the ratio of
compound-specific PI3Kα Ki over the Ki for mTOR. Each experiment was performed at least twice or as a multiple
of n = 2. cMarvin/JChem 16.10.17 was used for calculation of logP (partition coefficient) and PSA (polar surface
area) values.
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Binding Mode to PI3K and mTOR
5
6
7
8
The binding mode and interactions of PQR309 (5)10 and BKM120 (2)9 with the ATP-binding site of
PI3K have been elucidated. In detail, the orientation of the morpholine moiety in the hinge region is
well understood, and an identical binding mode for all class I PI3K isoforms can be assumed: the
morpholine O-atom forms a hydrogen bond interaction with the hinge Val882 residue in PI3K (Val851
in PI3K).
9
10
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16
17
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53
54
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58
59
60
In 4-trifluoromethyl compounds with two equally substituted morpholines, morpholines M1 (16), M3
(18), M4 (19) and M7 (22) severely attenuated binding to PI3K (Table 1, M5 (20) and M6 (21) had a
minor effect, while M (17) allowed to lower the Ki for p110 to 7 nM. These results suggest that the
(S)-enantiomer of methyl-morpholine (M2) is well accommodated when pointing towards the hinge
region (PI3K: Val882, PI3K: Val851), while the (M1) substitution abrogates high affinity
interactions. Specifically, 17 with two (S)-methyl-morpholines (2x M2) was 100-fold more potent on
PI3K than compound 16 with two (R)-methyl-morpholines (2x M1; Table 1). This tendency was
reproduced by compounds with a 4-difluoromethyl substituted heteroaromatic group, where (S)-
methyl-morpholine substitutions (for example 2x M1 in 60) augmented the affinity for PI3K by 53-
fold as compared to 61 (with 2x M2; see Table S2 in Supporting Information). The orientation of the
substituted morpholines towards the hinge Val was finally confirmed with a single-sided exchange of
a substituted morpholine with piperidine – thus forcing the substituted morpholine to form a H-bond
with the hinge Val (compounds 63 to 65 in Table S2).
To better understand the structural basis for the different affinity between compounds with (R)- and
(S)-methyl-morpholines, computational modeling studies were carried out for compound 6 and its (R)-
enantiomer (66): inhibitor-PI3K complexes were modelled using the PQR309 (5)/PI3K complex
crystal structure (PDB ID: 5OQ4) as a template to dock compounds with C3-substituted morpholines.
As depicted in Figure 2, molecular modelling suggests a preferred placement of the (S)-methyl group
pointing towards Tyr867 (Tyr836 in PI3K), while a steric clash is flagged at Ile881. The (R)-methyl
morpholine cannot be accommodated in PI3K, generating overlaps with the side chains of Ile879,
Glu880 and Met953. To validate this, a compound 6-p110 complex structure was resolved by X-ray
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crystallography at a resolution of 3.15 Å (PDB ID 6OAC; Figure S1). The core structure of compound
6 could be defined in the electron density, but distinct density for the methyl groups was not apparent
(Figure S1). Fitting compound 6 to the obtained electron density map, the inhibitor was best
accommodated with the methyl group in one of the two hydrophobic interfaces formed by side chains
of Tyr836, Ile848, Ile932 or Trp780, Ile800, and Val850 (numbering referring to PI3K). Val850 in
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59
60
PI3K corresponds to Ile881 in PI3K, and the predicted steric clash at this position in PI3K could not
be detected in PI3K - which might explain in part a preferred binding of compound 6 to PI3K vs.
PI3K (see Table S1, Kd for PI3K = 0.84; Kd for PI3K = 10 nM). The lack of a distinct signal for the
methyl group in the X-ray structure also suggests that compound 6 can bind to PI3Ks in multiple ways,
either with the substituted morpholine pointing towards the hinge region (Figure 2 and S1), or the
solvent exposed space.
A
B
Glu880
Glu880
Ile881
Ile881
Ile879
Ile879
H
H
H
CH3
N
N
(S)
(S)
O
O
CH3
H
H
H3C
Met953
Met953
C
Glu880
Glu880
Ile881
Ile881
Ile879
Ile879
H3C
H
H
H
(R)
N
N
(R)
O
O
CH3
H
H
H
Met953
Met953
Figure 2. Model of PI3K with docked compound 6 [(S)-enantiomer)] and its (R)-enantiomer 66. A) The PI3K
surface is depicted as mesh, and selected amino acids are shown as sticks (grey). Two space filling
conformations of the methyl-morpholino groups are shown for both 6 (green and yellow) and its enantiomer
66 (enantiomer, pink and cyan), oxygen atom pointing towards the hinge region Val882. The model is based
on a PQR309 (5)-PI3K complex (PDB ID: 5OQ4). Steric clashes conserved for class I PI3Ks are represented
as red lines, a steric clash detected PI3K is indicated in blue. B, C) Quadrant models for each possible position
of the methyl-substituent in the hinge region of PI3K are depicted for B) compound 6 and C) compound 66.
In vitro PI3K binding affinities for 6 are 11.1 nM (Ki) and 20.8 nM (Ki) for 66 (see Table S2). For the p110-
compound 6 complex see Figure S1 (deposited as PDB ID 6OAC).
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6
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9
A similar computational modelling analysis of the hinge region environment in mTOR showed that
the ATP-binding pocket is wider: here M1 and M2 [(R)- and (S)-3-methylmorpholine] can be
accommodated (Figure S2). These findings are supported by comparison of compound 16 to 17 (Table
1), 59 to 61 and 63 to 65 (Table S2), where mTOR affinities were very similar for M1 and M2 substituted
compounds.
10
11
12
13
14
15
16
17
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19
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51
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53
54
55
56
57
58
59
60
On- and Off-Target Activities
Compound 6 was selected for further elucidation of its activity profile. Binding of compound 6 to the
ATP-binding site of PI3K was confirmed by an in vitro Wortmannin19-competition assay, where 6
prevented covalent binding of wortmannin to Lys802 in PI3K20 (Figure S3). The selectivity for PI3K
and mTOR over a wide range of protein and lipid kinases was validated in a KINOMEScan panel. Here,
the selectivity of compound 6 was compared with data generated using the dual PI3K/mTOR inhibitor
GDC0980 (3) and the structurally related pan-PI3K inhibitor 510 (Figure S4 and Table S4). Even at 10
M, compound 6 achieved excellent selectivity scores of S(35) = 0.052 and S(10) = 0.03, exceeding
that of the dual PI3K/mTOR inhibitor 3 [S(35) = 0.22, S(10) = 0.09; exact calculation see Experimental
Section; simplified: S(35): fraction of total kinases with activities reduced to <35%].
Despite these differences in in vitro selectivity, the sensitivities of 44 tumor cell lines to 6 and 3
showed a good overall correlation across the cell panel (Figure 3 and Table S5). Similarly, half-maximal
growth inhibition (GI50) of 6 (0.43 µM in a 66 tumor cell line panel) was comparable to the mean GI50
of 44 cell lines 3 determined earlier (GI50 = 0.35 µM).9 As observed before for other PI3K/mTOR
inhibitors, compound 6 acted cytostatic, and minor cytotoxicity below 10 µM was only observed in
~10% of cell lines at concentrations surpassing >>10x the GI50 (Table S5, individual growth curves are
depicted in Figure S5).
Earlier reports have claimed that dual PI3K/mTOR inhibitors such as BEZ2355,6,21 excel over PI3K
inhibitors in the suppression of cancer cell proliferation. However, these reports usually compared
inhibitors that were, however, structurally and pharmacologically very different. Indeed, the cell-
specific sensitivity profile of 43 cell lines of BEZ235-mediated growth inhibition did not correlate
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significantly with the well aligned profiles of 6 and 3 (Figure 3, for BEZ235 see Figure S6). A
comparison of the impact of compound 6, the structurally related PI3K inhibitor 5 and the mTOR kinase
inhibitor PQR620 (67) on cell proliferation and PI3K/mTOR signaling confirmed an increased potency
of dual PI3K/mTOR inhibition by 6 as compared to 5, but was closely matched by 67 (Figure 4).
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60
Figure 3. A) Attenuation of cell proliferation in response to PQR530 (6) and GDC0980 (3) represented as a
waterfall plot. Concentrations of half-maximal growth inhibition (GI50) were obtained from dose-response
growth curves in 44 tumor cell lines. Log-transformed individual GI50 of a cell line was related to the log-
transformed mean GI50 of all cell lines, and cell lines were sorted by lowest to highest sensitivity for PQR530
(6) from left to right. Individual cell lines and GI50-values are given in Table S5. B) Correlation of 3 and 6
sensitivity of individual cell lines shown in A). The comparative dataset of 3 is from Ref. 9. Further
comparisons of cellular inhibition profiles for BEZ235, compound 3, and 5 are shown in Figures S6 and S7.
In line, compound 6 showed an increased potency in growth inhibition as compared to 5 (average over
44 cell lines: 2.5 to 3 fold lower GI50 and IC50). The very good correlation of the cellular inhibition
profiles of 5 and 6 is compatible with a similar mode of action (Figure S7).
Interestingly, half maximal growth inhibition is only achieved if phosphorylation of PKB/Akt is
suppressed by ca. 90%. In SKOV3 cells, a constitutively activated p110 mutant (H1047R) drives the
PI3K/mTOR pathway, while A2058 cells are devoid of the phosphoinositide 3-phosphatase PTEN, and
the B-Raf V600E mutation drives the activation of the MAPK (mitogen-activated protein kinase)
pathway, which contributes to an increased resistance to PI3K and mTOR inhibitors. In both tumor cell
lines, increased inhibition of mTOR by 6 enhanced its ability to suppress growth proliferation over that
of 5. While 67 demonstrated similar potency in these cells, in certain oncogenic contexts a selective
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TORC1 inhibitor may not be desirable due to a loss of feedback inhibition (via S6K and IRS-1) resulting
in reactivation of PI3K and PKB/Akt signaling [see Ref. 17 for a comparison of rapamycin and 67].
Thus, an inhibitor with high affinity towards both mTOR kinase and PI3K can be of advantage. The
analysis of PI3K and mTOR downstream targets (PKB/Akt; GSK3, S6K, S6) confirmed that 6 has an
enhanced potential to block the pathway as compared to selective pan-PI3K or mTOR inhibitors.
10
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51
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56
57
58
59
60
PQR309 (5)
PQR530 (6)
PQR620 (67)
SKOV3
Figure 4. Impact of PI3K and
mTOR inhibitors on cell
proliferation and PI3K/mTOR
downstream signaling. A)
A2058 and B) SKOV3 cells
were exposed to the indicated
inhibitors for 72h; and resulting
cell numbers are shown as % of
DMSO controls (n = 2, mean ±
SD). Dotted lines connect 50%
A
B
100
50
0
100
50
0
A2058
-8
-7
-6
-5
-8
-7
-6
-5
SKOV3
C
D
A2058
100
50
0
100
50
0
growth
inhibition
with
phosphorylation of PKB/Akt on
Ser473, analyzed in C) A2058
and D) SKOV3 cells after
exposure to the indicated
-8
-7
-6
-5
-8
-7
-6
-5
lg Inhibitor, [M]
lg Inhibitor, [M]
E
F
inhibitors
for
1h.
A2058
100
50
0
100
50
0
Phosphoprotein levels were
determined by an in-cell
Western assay (mean ± SD, n =
3). Phosphorylation of E)
PKB/Akt, F) GSK3, G) p70S6K
and H) pS6 in A2058 lysates are
A2058
-8
-9
-8
-7
-6
-5
-4
-9
-7
-6
-5
-4
G
H
shown as
%
of controls,
A2058
A2058
100
50
0
100
50
0
detected using the Mesoscale
Discovery platform (mean ±
SD, n = 2).
-9
-8
-7
-6
-5
-4
-9
-8
-7
-6
-5
-4
lg Inhibitor, [M]
lg Inhibitor, [M]
Off-target effects of compound 6 were tested in a CEREP BioPrint (P22-p) panel at a concentration of
10 M. Only very weak competitions with radioligands for cell surface and nuclear receptors,
membrane channels, transporters (Table S6) and enzyme activities of kinases, proteases, and
phosphodiesterases (Table S7) were detected, suggesting that 6 is a safe compound.
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Pharmacological Parameters
5
6
7
8
Metabolic stability of compound 6 was assessed in vitro prior to in vivo pharmacokinetic (PK)
properties. When evaluated in human, rat, dog and mouse liver microsomes, compound 6 showed little
clearance after 30 minutes of exposure (Table 3A). In hepatocyte cultures, compound 6 showed low
clearance in all species, dog, rat, mouse and human. The half-life of compound 6 was ~ 4.2 hours in
humans and, similarly, ~ 3.2-2.6 hours in the other species (Table 3B).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3A. Stability in liver microsomes of compound PQR530 (6).
% remaining in liver microsomesa
Cpd. \ species
6
Human
Rat
Dog
Mouse
81.5
7.45
54.8
106
28.5
2.9
97.4
1.8
73.2
1.5
7-ECb
Propranololb
30.1
6.8
b
aPercentage of compound remaining after 30 minutes (mean, n=2); Assay reference compounds: 7-EC, 7-
Ethoxycoumarin.
Table 3B. Stability of compound PQR530 (6) in hepatocyte cultures.
Cpd. \ species
Human
Rat
Dog
Mouse
aCLint
6.0
7.0
10
11
6
t1/2 [min]
CLint
250
20.0
70.0
65.5
21.1
192
30
142
163.3
8.49
138
10.2
142
74
7-ECb
7-HCb
t1/2 [min]
CLint
46.2
124
11.1
18.7
116
11.9
t1/2 [min]
aCLint [μl/min/106 cells]; t1/2, [min]; bAssay reference compounds: 7-EC, 7-Ethoxycoumarin; 7-HC, 7-
Hydroxycoumarin; Results are expressed as mean, n=3.
Subsequently, PK parameters of compound 6 were assessed in male C57BL/6J mice using a single
oral dose of 50 mg/kg (Table 4 and Figure 5). Drug levels were determined by LC-MS/MS in extracts
of plasma, brain and thigh muscle. Half-life was determined to be approximately 3.5 - 4.8 hours.
Concentration of compound 6 in the brain was higher than in plasma (Cmax of ~ 7799 ng/mL in plasma
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and of ~ 12627 ng/mL in brain), demonstrating that compound 6 passes the blood-brain barrier, showing
a ca. 1:1.67 distribution between plasma and brain. The maximal concentration was reached after 30
minutes in plasma, brain and thigh muscles.
9
The observed brain/plasma ratio corresponded with parallel artificial membrane permeability assay
(PAMPA, Table S8A) and MDCK permeability assays, which documented a fast and passive diffusion.
The lack of effect of the addition of the P-glycoprotein P-gp (P-glycoprotein 1, MDR1) inhibitor
cyclosporine A to the MDCK assay demonstrates, that  is not a substrate for P-gp transport (see Table
S8B), which predicts excellent brain penetration. Finally, a brain tissue binding assay revealed a fu
(fraction unbound) value of ~8%, which closely matched the levels of diazepam (fu ~9%, Table S8C)
with a proven brain activity. Altogether, the assembled parameters characterize 6 as a compound with
high on-target potency in brain tissue.
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. PK analysis of PQR530 (6) after oral application in male C57BL/6J mice (50 mg/kg p.o.).
Plasma
7799
0.5
Brain
12627
0.5
Thigh muscle
11946
0.5
Cmax [ng/mL]
Tmax [h]
t1/2 [h]
4.7
4.8
3.5
AUC0 - 8h [h*ng/mL]
Cl [mL/h/kg]
27910
1257
46483
729
39248
1003
p.o.: per os; Cmax: maximal concentration, Tmax: time of maximal concentration in hours; t1/2: half-life elimination
in hours; AUC: area under the curve, Cl: clearance; (n=3, mean for each time-point).
The physiological effect of compound 6 administration on plasma glucose and insulin levels in male
C57BL/6J mice is shown in Figure 5: vehicle treatment did not affect plasma insulin or glucose levels,
while compound 6 triggered an increase in plasma glucose and insulin levels. Ten minutes after
treatment with compound 6, plasma glucose levels rose significantly as compared to vehicle, and
reached maximal concentrations one hour after dosing. Plasma insulin levels reached maxima two hours
post-dosing. The rise of plasma glucose and insulin levels are typical for PI3K/mTOR pathway
inhibitors22 and have also been observed with pan-PI3K inhibitors, such as 5, in preclinical10 and phase
I clinical studies.15
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6
7
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9
A
B
105
104
103
102
101
2
1
PQR530 in
Muscle
Brain/Plasma
6 8
10
11
12
13
14
15
16
17
18
19
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21
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50
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52
53
54
55
56
57
58
59
60
Brain
Plasma
0
0
2
4
6
8
0
2
4
Time after administration [h]
Time after administration [h]
PQR530 Vehicle
C
D
PQR530 Vehicle
1000
100
10
1000
1
0.1
100
0
2
4
6
8
0
2
4
6
8
Time after administration [h]
Time after administration [h]
Figure 5. PK/PD assessment of PQR530 (6) in vivo. Compound 6 was administered to male C57BL/6J mice
as single dose per os (p.o., 50 mg/kg). A) Levels of 6 in C57BL/6J mouse tissues (n=3). Compound 6 was
extracted from each tissue at the indicated time-points, and was subsequently quantified using LC-MS. B)
Tissue distribution ratios of 6. Data of 6 in (B) was compared to rapamycin and everolimus in Ref. 18. C)
Glucose concentration after oral application in male C57BL/6J mice. Plasma glucose was quantified using the
Glucose Calorimetric Assay Kit (cat. No. Cay10009582-192, Biomol, Germany). D) Plasma insulin levels in
male C57BL/6J mice were quantified using a commercially available colorimetric immunoassay kit
(Rat/Mouse Insulin ELISA, cat. No. EZRMI-13K, Merck Millipore, Germany). All values mean ± SEM. Error
bars not shown when smaller than the symbols. A comparison of PK and brain/plasma distribution values of 6
with PQR309 (5) is shown in Figure S8.
In vitro and in vivo toxicokinetic parameters were determined in rats and dogs and are summarized
in Table S9. The side effects of compound 6 were consistent with its mechanism of action. In Wistar
rats, the no-observed adverse effect level (NOAEL) of compound 6 was considered to be 1 mg/kg/day
for males and 0.5 mg/kg for females (corresponding to steady state AUC0-8 levels of 335 h∙ng/mL and
599 h∙ng/mL, respectively). In Beagle dogs, the no-observed adverse effect level (NOAEL) of
compound 6 was 1 mg/kg/day for males (corresponding to steady state AUClast levels of 127 ± 45
h∙ng/mL) and 3 mg/kg/day for females (corresponding to steady state AUClast levels of 1070 ± 596
h∙ng/mL).
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In vivo Efficacy of PQR530 (6)
5
6
7
8
In order to establish the antiproliferative effect of compound 6 in vivo, an OVCAR-3 xenograft model
in BALB/c nude mice was used (Figure 6). To initiate tumor development, mice were inoculated
subcutaneously (region of right flank) with OVCAR-3 cells at day 0. From day 17, the control group
received vehicle once a day (QD), while the treated group received 25 mg/kg of compound 6 p.o. QD.
Compared to vehicle treated mice, compound 6 significantly inhibited tumor growth. No significant
body weight loss was observed.
9
10
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A
B
C
25
23
21
1000
500
0
1
0.5
0
$
$
$
$
$
$
$
$
*
$
$
**
*
$
$
$
$
20
30
40
20
30
40
Time after tumor inoculation, [days]
Time after tumor inoculation, [days]
Compounds, see below
PQR620 (67), 50 mg/kg
Vehicle PQR309 (5), 50 mg/kg
PQR530 (6), 25 mg/kg
Figure 6. OVCAR-3 human ovarian cancer xenograft model in BALB/c nude mice: tumor cells were
subcutaneously inoculated at day 0, and daily oral application of the indicated agents was started at day 17 (28
x QD). A) body weight was determined at the depicted time points. B) The tumor size was measured and
calculated as described in the experimental section. C) The tumor weight was determined at day 45. Statistics:
$
(*p<0.05; **p<0.001; p<0.0001; n=8; (A,B) Two-Way ANOVA with Bonferroni or Dunnett's correction for
multi-comparisons, mean ± SEM; (C) mean ± SEM; One-Way ANOVA with Tukey's correction for multi-
comparisons. PQR620 (67) and vehicle data were shown in Ref. 17.
CONCLUSION
An extensive SAR study led to the identification of 6, a potent ATP competitive dual pan-PI3K and
mTORC1/2 inhibitor with a sub-nanomolar Kd towards PI3K and mTOR (0.84 and 0.33 nM,
respectively). Compound 6 shows improved potency against mTOR kinase as compared to other
advanced candidates with dual pan-class I PI3K and mTOR inhibitory activity. Molecular modeling
and X-ray crystallography elucidated interactions of 6 in the ATP-binding pocket of PI3Ks and mTOR.
The selectivity of 6 for PI3Ks and mTOR over a wide range of protein and lipid kinases, a lack of
detectable off-target effects, in vitro and in vivo toxicity studies highlight the safety of 6. Compound 6
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displayed potency in inhibiting cancer cell line proliferation, and pharmacokinetic studies of 6 showed
good bioavailability and excellent brain penetration, culminating in a good in vivo efficacy in an
OVCAR-3 xenograft model. Altogether, the present results encourage the further development of the
compound as a clinical candidate in oncology.
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EXPERIMENTAL SECTION
5
6
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General Information
8
9
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
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, 230-400
mesh particle size). Alternatively, flash chromatography was performed with Isco CombiFlash
Companion systems using prepacked silica gel columns (40 - 60 μm particle size RediSep). 1H, 19F and
13C NMR spectra were recorded on a Bruker Avance 400 spectrometer. NMR spectra were obtained in
deuterated solvents, such as CDCl3 or (CD3)2SO. The chemical shift (δ values) are reported in ppm and
corrected to the signal of the deuterated solvents (7.26 ppm (1H NMR) and 77.16 ppm (13C NMR) for
CDCl3; and 2.50 ppm (1H NMR) and 39.52 ppm (13C NMR) for (CD3)2SO. 19F NMR spectra are
calibrated relative to CFCl3 (δ = 0 ppm) as external standard. When peak multiplicities are reported, 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 Thermo Fisher Scientific LTQ
Orbitrap XL (nanoESI-MS) spectrometer. 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 CH3CN / MeOH:H2O(10:90) was used at a flow rate of 0.5 mL / min at 40
°C. The purity of all final compounds was higher than 95%.
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General procedure 1.
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Step 1. Monochloro-triazine or -pyrimidine derivative (1.0 equiv), boronic acid pinacol ester 48 or
49 (1.0 - 1.1 equiv), K3PO4 (2.0-3.0 equiv) and chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-
1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (XPhos Pd G2, 0.05 equiv) were charged in a
flask. Under nitrogen atmosphere, absolute 1,4-dioxane (approx. 1 mL / 0.2 mmol) and deionized H2O
(approx. 1 mL / 0.4 mmol) were added and the resulting mixture was placed into an oil bath pre-heated
at 95 °C and stirred at this temperature for 3-15 hours. After completion of the reaction, the mixture
was allowed to cool down to room temperature. Step 2. A 3 M aqueous HCl-solution (> 10 equiv) was
added and the resulting mixture was heated at 60 °C for 3-15 hours. Reaction completion was monitored
by TLC. The mixture was allowed to cool down to room temperature, the pH was adjusted to 8-9 by
addition of a 2 M aqueous NaOH-solution and the aqueous layer was extracted with ethyl acetate (3 x).
The combined organic layers were dried over anhydrous Na2SO4, filtered and the solvent was
evaporated under reduced pressure. The crude product was purified by column chromatography on
silica gel.
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General procedure 2.
To a solution of 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine (23, 1.0 equiv) in 1,4-dioxane (approx.
1 mL / 0.3 mmol), the respective morpholine derivative (1.0 - 1.2 equiv) and N,N-diisopropylethylamine
(4.5 - 4.7 equiv) were added. The resulting mixture was stirred at 70 °C overnight. The solvent was then
removed under reduced pressure and the residue was purified by column chromatography on silica gel.
General procedure 3.
Step 1. Bis(pinacolato)diboron (1.5 equiv), potassium acetate (3.0 equiv), [1,1′-
bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl2, 0.10 equiv) and the respective
bromo derivative (1.0 equiv) were dissolved in absolute 1,4-dioxane (approx. 1 mL / 0.2 mmol) under
nitrogen atmosphere. The resulting mixture was heated at 95 °C for 6-8 hours. Step 2. Then, the mixture
was allowed to cool down to room temperature. Monochloro-triazine or -pyrimidine derivative (1.1
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equiv),
chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-
5
6
7
8
biphenyl)]palladium(II) (XPhos Pd G2, 0.05 equiv), K3PO4 (2.0 equiv) and deionized H2O (approx. 1
mL / 0.4 mmol) were added. The resulting reaction mixture was placed in a preheated oil bath at 95 °C
and stirred for 3-15 hours. After completion of the reaction, the mixture was allowed to cool down to
room temperature, deionized H2O was added and the mixture was extracted with dichloromethane (3
x). The combined organic layers were washed with brine (2 x), dried over anhydrous Na2SO4, filtered
and reduced to dryness under reduced pressure. Step 3. The above residue was dissolved in 1,4-dioxane
(approx. 1 mL / 0.1 mmol) and an aqueous solution of HCl (3 M, 10-15 equiv) was added. The reaction
mixture was stirred at 60 °C for 2 hours. The mixture was diluted with deionized H2O and washed with
ethyl acetate (1 x). The aqueous layer was basified to pH = 10 and then extracted with ethyl acetate (3
x). The combined organic layers were dried over anhydrous Na2SO4, filtered and reduced to dryness
under reduced pressure. The crude product was purified by column chromatography on silica gel.
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General procedure 4.
To a solution of the desired dichloropyrimidine (40 or 41, 1.0 equiv) in N,N-dimethylformamide
(approx. 1 mL / 0.3 mmol), the respective morpholine derivative (1.5 equiv) and N,N-
diisopropylethylamine (3.0 equiv) were added. The resulting mixture was stirred at 130°C overnight.
The solvent was then removed under reduced pressure and the residue was dissolved in
dichloromethane. The organic layer was washed with aqueous saturated NaHSO4-solution, dried over
anhydrous Na2SO4, filtered and the solvent was evaporated under reduced pressure. The crude product
was purified by column chromatography on silica gel.
O
N
F
F
F
N
N
N
N
O
N
NH2
5-[4,6-Bis(morpholin-4-yl)-1,3,5-triazin-2-yl]-4-(trifluoromethyl)pyridin-2-amine (PQR309, 5).
Compound 5 was prepared according to the literature.9
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O
N
F
F
6
N
N
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8
N
N
O
N
NH2
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4-(Difluoromethyl)-5-{4-[(3S)-3-methylmorpholin-4-yl]-6-(morpholin-4-yl)-1,3,5-triazin-2-
yl}pyridin-2-amine (PQR530, 6). Pd(OAc)2 (538 mg, 2.40 mmol, 0.04 equiv) and PPh3 (1.89 g, 7.21
mmol, 0.12 equiv) were dissolved in tetrahydrofuran (50 mL) and stirred at room temperature for 1
hour. This solution was added to a mixture of boronic acid pinacol ester 49 (19.5 g, 60.0 mmol, 1.0
equiv), intermediate 25 (18.0 g, 60.0 mmol, 1.0 equiv) and aqueous K2CO3 (2.4 M, 81.0 mL, 194 mmol,
3.2 equiv) in tetrahydrofuran (200 mL). Then, the reaction mixture was heated at 55 °C for 2 hours.
After this time, the reaction mixture was allowed to cool down to room temperature and an aqueous
HCl-solution (5 M, 123 mL, 615 mmol, 10 equiv) was slowly added. The resulting reaction mixture
was heated at 55 °C overnight. Upon completion of the reaction, the mixture was allowed to cool down
to room temperature and tetrahydrofuran was removed under reduced pressure. The pH was adjusted to
9 by addition of NaOH pellets. The aqueous layer was extracted with 2-methyltetrahydrofuran (2 x 200
mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated to
dryness under reduced pressure. The residue was purified by column chromatography on silica gel
(cyclohexane / ethyl acetate 1:0 → 7:3) to afford compound 6 as a colorless solid (10.2 g, 25.0 mmol,
41%). 1H NMR (400 MHz, CDCl3):  9.03 (s, 1 H), 7.67 (t, 1JH,F = 55 Hz, 1 H), 6.84 (s, 1 H), 4.84 (br
s, 2 H), 4.78-4.69 (m, 1 H), 4.45-4.34 (m, 1 H), 3.97 (dd, JH,H = 11, 3.7 Hz, 1 H), 3.89-3.71 (m, 9 H),
3.68 (dd, JH,H = 12, 3.2 Hz, 1 H), 3.52 (td, JH,H = 12, 3.0 Hz, 1 H), 3.27 (td, JH,H = 13, 3.8 Hz, 1 H), 1.32
(d, 3JH,H = 6.8 Hz, 3 H). 19F{1H} NMR (376 MHz, CDCl3):  − 115.9 (s, 2 F). 13C{1H} NMR (101 MHz,
CDCl3):  169.4 (s, 1 C), 164.9 (s, 1 C), 164.5 (s, 1 C), 160.2 (s, 1 C), 152.5 (s, 1 C), 143.7 (t, 2JC,F
=
22 Hz, 1 C), 121.6 (t, 3JC,F = 4.6 Hz, 1 C), 111.5 (t, 1JC,F = 239 Hz, 1 C), 104.1 (t, 3JC,F = 7.9 Hz, 1 C),
71.2 (s, 1 C), 67.1 (s, 1 C), 66.9 (br s, 2 C), 46.5 (s, 1 C), 43.8 (br s, 2 C), 38.7 (s, 1 C), 14.4 (s, 1 C).
NSI-HRMS (m/z): [M + H]+ calc. for C18H24F2N7O2, 408.1954; found: 408.1940. HPLC: tR = 7.73 min
(99.1% purity).
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8
O
N
N
N
N
N
Cl
O
9
2-Chloro-4,6-bis(morpholin-4-yl)-1,3,5-triazine (8). Compound 8 was prepared according to the
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literature.9
O
N
N
N
N
N
Cl
O
2‐Chloro‐4,6‐bis[(3R)‐3‐methylmorpholin‐4‐yl]‐1,3,5‐triazine (9). Compound 9 was prepared
according to the literature.17
O
N
N
N
N
N
Cl
O
2‐Chloro‐4,6‐bis[(3S)‐3‐methylmorpholin‐4‐yl]‐1,3,5‐triazine (10). To a solution of cyanuric
chloride (868 mg, 4.70 mmol, 1.0 equiv) in dichloromethane (20 mL) at 0 °C, a solution of (S)-3-
methylmorpholine (2.00 g, 19.8 mmol, 4.2 equiv) in dichloromethane (20 mL) was slowly added. The
resulting reaction mixture was stirred overnight, while it was allowed to warm up to room temperature.
Additional dichloromethane (50 mL) was added and the organic layer was washed with an aqueous
saturated NaHSO4-solution (2 x). The organic layer was dried over anhydrous Na2SO4, filtered and the
solvent was evaporated under reduced pressure. The crude product was purified by column
chromatography on silica gel (cyclohexane / ethyl acetate 1:0 → 3:1) to afford compound 10 as a
colorless solid (1.40 g, 4.46 mmol, 95%). 1H NMR (400 MHz, CDCl3):  4.76-4.53 (m, 2 H), 4.39-4.22
(m, 2 H), 3.93 (dd, JH,H = 11, 3.7 Hz, 2 H), 3.72 (d, JH,H = 11 Hz, 2 H), 3.63 (dd, JH,H = 11, 3.2 Hz, 2
H), 3.48 (td, JH,H = 12, 3.0 Hz, 2 H), 3.29-3.19 (m, 2 H), 1.30 (d, 3JH,H = 6.9 Hz, 6 H). 13C{1H} NMR
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(101 MHz, CDCl3):  169.7 (s, 1 C), 164.3 (s, 2 C), 71.0 (s, 2 C), 67.0 (s, 2 C), 46.8 (s, 2 C), 38.9 (s, 2
C), 14.6 (br s, 2 C). MALDI-MS: m/z = 314.4 ([M + H]+).
7
8
9
O
N
10
11
12
13
14
15
16
17
18
19
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N
N
N
N
Cl
O
8-(4-Chloro-6-{3-oxa-8-azabicyclo[3.2.1]octan-8-yl}-1,3,5-triazin-2-yl)-3-oxa-8-
azabicyclo[3.2.1]octane (11). Compound 11 was prepared according to the literature.9
O
N
N
N
N
N
Cl
O
3-(4-Chloro-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-1,3,5-triazin-2-yl)-8-oxa-3-
azabicyclo[3.2.1]octane (12). Compound 12 was prepared according to the literature.17
O
N
N
N
N
N
Cl
O
2-Chloro-4,6-bis[(3R,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazine (13). Compound 13 was
prepared according to the literature.17
O
N
N
N
N
N
Cl
O
2-Chloro-4,6-bis[(3S,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazine (14). Compound 14 was
prepared according to the literature.17
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5
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7
O
N
N
N
8
9
N
N
Cl
10
11
12
13
14
15
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17
18
19
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O
2-Chloro-4,6-bis[(3R,5R)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazine (15). Compound 15 was
prepared according to the literature.17
O
N
F
F
F
N
N
N
N
O
N
NH2
5-{4,6-Bis[(3R)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl}-4-(trifluoromethyl)pyridin-2-
amine (16). Compound 16 was prepared according to general procedure 1 from intermediate 9 (80.0
mg, 255 µmol, 1.0 equiv) and boronic acid pinacol ester 48 (87.1 mg, 254 μmol, 1.0 equiv). Purification
by column chromatography on silica gel (cyclohexane / ethyl acetate 1:0 →4:6) gave compound 16 as
a colorless solid (71.0 mg, 162 μmol, 64%). 1H NMR (400 MHz, CDCl3):  8.72 (s, 1 H), 6.78 (s, 1 H),
4.91 (br s, 2 H), 4.85-4.70 (m, 2 H), 4.51-4.33 (m, 2 H), 3.95 (dd, 2JH,H = 11 Hz, 3JH,H = 3.6 Hz, 2 H),
3.74 (d, 2JH,H = 11 Hz, 2 H), 3.66 (dd, 2JH,H = 11 Hz, 3JH,H = 3.2 Hz, 2 H), 3.51 (td, JH,H = 12, 3.0 Hz, 2
H), 3.31-3.19 (m, 2 H), 1.32 (d, 3JH,H = 6.8 Hz, 6 H). 19F{1H} NMR (376 MHz, CDCl3):  − 60.2 (s, 3
F). 13C{1H} NMR (101 MHz, CDCl3):  169.9 (s, 1 C), 164.5 (s, 2 C), 159.6 (s, 1 C), 152.9 (s, 1 C),
138.5 (q, 2JC,F = 33 Hz, 1 C), 123.0 (q, 1JC,F = 274 Hz, 1 C), 122.6 (s, 1 C), 105.7-105.2 (s, 1 C), 71.2
(s, 2 C), 67.2 (s, 2 C), 46.4 (s, 2 C), 38.6 (s, 2 C), 14.4 (s, 2 C). NSI-HRMS (m/z): [M + H]+ calc. for
C19H25F3N7O2, 440.2016; found: 440.2007. HPLC: tR = 8.50 min (98.1% purity).
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5
6
7
O
N
F
F
F
8
N
N
9
N
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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O
N
NH2
5-{4,6-Bis[(3S)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl}-4-(trifluoromethyl)pyridin-2-
amine (17). Compound 17 was prepared according to general procedure 1 from intermediate 10 (700
mg, 2.23 mmol, 1.0 equiv) and boronic acid pinacol ester 48 (770 mg, 2.24 mmol, 1.0 equiv).
Purification by column chromatography on silica gel (cyclohexane / ethyl acetate 1:2 → 1:3) gave
compound 17 as a colorless solid (344 mg, 782 μmol, 35%). 1H NMR (400 MHz, CDCl3):  8.70 (s, 1
H), 6.76 (s, 1 H), 4.84 (br s, 2 H), 4.81-4.69 (m, 2 H), 4.48-4.30 (m, 2 H), 3.93 (dd, 2JH,H = 11 Hz, 3JH,H
= 3.7 Hz, 2 H), 3.73 (d, 2JH,H = 11 Hz, 2 H), 3.64 (dd, 3JH,H = 12 Hz, 2JH,H = 3.2 Hz, 2 H), 3.55-3.45 (m,
2 H), 3.28-3.18 (m, 2 H), 1.30 (d, 3JH,H = 6.8 Hz, 6 H). 19F{1H} NMR (376 MHz, CDCl3):  − 60.2 (s,
3 F). 13C{1H} NMR (101 MHz, CDCl3):  169.9 (s, 1 C), 164.5 (s, 2 C), 159.6 (s, 1 C), 152.8 (s, 1 C),
138.5 (q, 2JC,F = 33 Hz, 1 C), 123.0 (q, 1JC,F = 274 Hz, 1 C), 122.5 (s, 1 C), 105.7-105.1 (s, 1 C), 71.2
(s, 2 C), 67.1 (s, 2 C), 46.4 (s, 2 C), 38.5 (s, 2 C), 14.4 (s, 2 C). NSI-HRMS (m/z): [M + H]+ calc. for
C19H25F3N7O2, 440.2016; found: 440.2006. HPLC: tR = 8.49 min (99.0% purity).
O
N
F
F
F
N
N
N
N
O
N
NH2
5-[4,6-Bis({3-oxa-8-azabicyclo[3.2.1]octan-8-yl})-1,3,5-triazin-2-yl]-4-
(trifluoromethyl)pyridin-2-amine (18). Compound 18 was prepared according to general procedure
1 from intermediate 11 (100 mg, 296 µmol, 1.0 equiv) and boronic acid pinacol ester 48 (112 mg, 325
µmol, 1.1 equiv). Purification by column chromatography on silica gel (cyclohexane / ethyl acetate 1:0
→ 3:7) gave compound 18 as a colorless solid (118 mg, 255 μmol, 87%). 1H NMR (400 MHz, CDCl3):
 8.71 (s, 1 H), 6.77 (s, 1H), 4.90 (br s, 2 H), 4.80-4.59 (m, 4 H), 3.83-3.73 (m, 4 H), 3.67-3.58 (m, 4
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H), 2.13-1.92 (m, 8 H). 19F{1H} NMR (376 MHz, CDCl3):  − 59.9 (s, 3 F). 13C{1H} NMR (101 MHz,
5
6
2
CDCl3):  170.4 (s, 1 C), 163.4 (s, 2 C), 159.6 (s, 1 C), 152.8 (s, 1 C), 138.5 (q, JC,F = 33 Hz, 1 C),
7
8
9
123.0 (q, 1JC,F = 274 Hz, 1 C), 122.5 (s, 1 C), 105.5 (q, 3JC,F = 6.0 Hz, 1 C), 72.1 (s, 2 C), 71.7 (s, 2 C),
54.9 (s, 2 C), 54.5 (s, 2 C), 27.0 (s, 4 C). NSI-HRMS (m/z): [M + H]+ calc. for C21H25F3N7O2, 464.2016;
found: 464.1999. HPLC: tR = 8.33 min (97.1% purity).
10
11
12
13
14
15
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19
20
21
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O
N
F
F
F
N
N
N
N
O
N
NH2
5-[4,6-Bis({8-oxa-3-azabicyclo[3.2.1]octan-3-yl})-1,3,5-triazin-2-yl]-4-
(trifluoromethyl)pyridin-2-amine (19). Compound 19 was prepared according to general procedure
1 from intermediate 12 (100 mg, 296 µmol, 1.0 equiv) and boronic acid pinacol ester 48 (112 mg, 325
µmol, 1.1 equiv). Purification by column chromatography on silica gel (cyclohexane / ethyl acetate 1:0
→ 3:7) gave compound 19 as a colorless solid (105 mg, 227 μmol, 77%). 1H NMR (400 MHz, CDCl3):
 8.69 (s, 1 H), 6.77 (s, 1 H), 4.89 (br s, 2 H), 4.48-4.37 (m, 6 H), 4.35-4.25 (m, 2 H), 3.21-3.12 (m, 4
H), 1.97-1.87 (m, 4 H), 1.83-1.73 (m, 4 H). 19F{1H} NMR (376 MHz, CDCl3):  − 59.8 (s, 3 F). 13C{1H}
NMR (101 MHz, CDCl3):  169.6 (s, 1 C), 165.7 (s, 2 C), 159.6 (s, 1 C), 152.8 (s, 1 C), 138.5 (q, 2JC,F
= 33 Hz, 1 C), 123.0 (q, 1JC,F = 274 Hz, 1 C), 122.7 (s, 1 C), 105.4 (q, 3JC,F = 5.7 Hz, 1 C), 74.2 (s, 2
C), 74.0 (s, 2 C), 49.4 (s, 2 C), 49.1 (s, 2 C), 27.9 (s, 2 C), 27.7 (s, 2 C). NSI-HRMS (m/z): [M + H]+
calc. for C21H25F3N7O2, 464.2016; found: 464.1998. HPLC: tR = 8.07 min (97.1% purity).
O
N
F
F
F
N
N
N
N
O
N
NH2
5-{4,6-Bis[(3R,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl}-4-(trifluoromethyl)pyridin-
2-amine (20). Compound 20 was prepared according to general procedure 1 from intermediate 13 (48.0
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Journal of Medicinal Chemistry
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mg, 140 µmol, 1.0 equiv) and boronic acid pinacol ester 48 (53.0 mg, 154 µmol, 1.1 equiv). Purification
by column chromatography on silica gel (cyclohexane / ethyl acetate 1:0 → 1:1) gave compound 20 as
a colorless solid (29.3 mg, 62.7 μmol, 44%). 1H NMR (400 MHz, CDCl3):  8.78 (s, 1 H), 6.79 (s, 1
H), 4.82 (br s, 2 H), 4.71-4.50 (m, 4 H 3.82 (d, 2JH,H = 11 Hz, 4 H), 3.64 (dd, 2JH,H = 11 Hz, 3JH,H = 3.4
Hz, 4 H), 1.37 (d, 3JH,H = 6.9 Hz, 12 H). 19F{1H} NMR (376 MHz, CDCl3):  − 60.4 (s, 3 F). 13C{1H}
10
11
12
13
14
15
16
17
18
19
20
21
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NMR (101 MHz, CDCl3):  169.6 (s, 1 C), 164.2 (s, 2 C), 159.4 (s, 1 C), 153.1 (s, 1 C), 138.5 (q, 2JC,F
= 33 Hz, 1 C), 123.0 (q, 1JC,F = 274 Hz, 1 C), 122.9 (s, 1 C), 105.4 (q, 3JC,F = 5.7 Hz, 1 C), 71.6 (s, 4
C), 45.7 (s, 4 C), 19.3 (br s, 4 C). NSI-HRMS (m/z): [M + H]+ calc. for C21H29F3N7O2, 468.2329; found:
468.2311. HPLC: tR = 9.72 min (97.2% purity).
O
N
F
F
F
N
N
N
N
O
N
NH2
5-{4,6-Bis[(3S,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl}-4-(trifluoromethyl)pyridin-
2-amine (21). Compound 21 was prepared according to general procedure 1 from intermediate 14 (185
mg, 541 µmol, 1.0 equiv) and boronic acid pinacol ester 48 (205 mg, 597 µmol, 1.1 equiv). Purification
by column chromatography on silica gel (cyclohexane / ethyl acetate 1:0 → 7:3) gave compound 21 as
a colorless solid (143 mg, 306 μmol, 57%). 1H NMR (400 MHz, CDCl3):  8.77 (s, 1 H), 6.79 (s, 1 H),
4.82 (br s, 2 H), 4.45-4.34 (m, 4 H), 4.23 (dd, 2JH,H = 11 Hz, 3JH,H = 3.3 Hz, 4 H), 3.74 (dd, 2JH,H = 11
Hz, 3JH,H = 2.3 Hz, 4 H), 1.46 (d, 3JH,H = 6.6 Hz, 12 H). 19F{1H} NMR (376 MHz, CDCl3):  − 60.4 (s,
3 F). 13C{1H} NMR (101 MHz, CDCl3):  169.4 (s, 1 C), 164.1 (s, 2 C), 159.4 (s, 1 C), 153.1 (s, 1 C),
138.3 (q, 2JC,F = 33 Hz, 1 C), 123.0 (q, 1JC,F = 274 Hz, 1 C), 122.8 (s, 1 C), 105.5-105.1 (m, 1 C), 68.0
(s, 4 C), 48.0 (s, 4 C), 19.8 (s, 4 C). NSI-HRMS (m/z): [M + H]+ calc. for C21H29F3N7O2, 468.2329;
found: 468.2321. HPLC: tR = 9.30 min (98.6% purity).
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