5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a Potent, Brain-Penetrant, Orally Bioavailable, Pan-Class i PI3K/mTOR Inhibitor as Clinical Candidate in Oncology

Article abstract of DOI:10.1021/acs.jmedchem.7b00930

Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, buparlisib). Compound 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compound's safety profile, identifies 1 as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.

Full text of DOI:10.1021/acs.jmedchem.7b00930

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES
Article
5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-
amine (PQR309): a Potent, Brain-Penetrant, Orally Bioavailable,
pan-Class I PI3K/mTOR Inhibitor as Clinical Candidate in Oncology
Florent Beaufils, Natasa Cmiljanovic, Vladimir Cmiljanovic, Thomas Bohnacker, Anna
Melone, Romina Marone, Eileen Jackson, Xuxiao Zhang, Alexander Sele, Chiara
Borsari, Jürgen Mestan, Paul Hebeisen, Petra Hillmann, Bernd Giese, Marketa
Zvelebil, Doriano Fabbro, Roger L. Williams, Denise Rageot, and Matthias P. Wymann
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00930 • Publication Date (Web): 22 Aug 2017
Downloaded from http://pubs.acs.org on August 22, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

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5ꢀ(4,6ꢀDimorpholinoꢀ1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ4ꢀ(trifluoromethyl)pyridinꢀ2ꢀamine
(PQR309): a Potent, BrainꢀPenetrant, Orally Bioavailable, panꢀClass I
PI3K/mTOR Inhibitor as Clinical Candidate in Oncology
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Florent Beaufils1,†,$, Natasa Cmiljanovic1,†,$, Vladimir Cmiljanovic1,†,$, Thomas Bohnacker1, Anna Melone1,
Romina Marone1, Eileen Jackson1, Xuxiao Zhang2, Alexander Sele1, Chiara Borsari1, Jürgen Mestan3, Paul
Hebeisen3, Petra Hillmann3, Bernd Giese4,††, Marketa Zvelebil1, Doriano Fabbro3, Roger L. Williams2, Denise
Rageot1,*, Matthias P. Wymann1,*
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1Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland;
2MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK;
3PIQUR Therapeutics AG, Hochbergerstrasse 60C, 4057 Basel, Switzerland;
4Department of Chemistry, University of Basel, St. JohannsꢀRing 19, 4056 Basel, Switzerland.
Current address:
†PIQUR Therapeutics AG, Basel, Switzerland;
††Department of Chemistry, University of Fribourg, Fribourg, Switzerland.
$These authors contributed equally to this work.
*Correspondence to:
Matthias.Wymann@UniBas.CH and Denise.Rageot@UniBas.CH
Dept. Biomedicine, University of Basel
Mattenstrasse 28
4058 Basel, Switzerland
Tel. +41 61 207 5046; Fax. +41 61 207 3566
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ABSTRACT
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Phosphoinositideꢀ3ꢀkinase (PI3K) is deregulated in a wide variety of human tumors, and triggers activation of
protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical
characterization of compound 1 (PQR309, Bimiralisib), a potent 4,6ꢀdimorpholinoꢀ1,3,5ꢀtriazineꢀbased panꢀclass
I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No offꢀtarget
interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover,
1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, Buparlisib). Compound 1 is
orally available, crosses the bloodꢀbrain barrier, and displayed favorable pharmacokinetic parameters in mice,
rats, and dogs. Compound 1 demonstrated efficiency inhibiting proliferation in tumor cell lines, and a rat
xenograft model. This, together with the compound's safety profile identifies 1 as a clinical candidate with a
broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is
currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.
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INTRODUCTION
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Phosphoinositide 3ꢀkinases (PI3Ks) are lipid kinases that play a central role in the control of cancer cell
growth, proliferation and metastasis.1–3 The PI3K family is divided into three classes according to sequence
homology and substrate specificity: soꢀcalled class I PI3Ks are activated downstream of cell surface receptors,
including receptor protein tyrosine kinases (RTKs), immunoglobulin receptors and Gꢀprotein coupled receptors
(GPCRs). Class IA PI3Ks consist of the catalytic subunits p110α, p110β, and p110δ. These interact with
dedicated phosphorylated tyrosine motifs on growth factor receptors or their substrates via two srcꢀhomology 2
(SH2) domains present in their tightly associated p85 regulatory subunits. The class IB PI3Kγ operates
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downstream of GPCRs, and is composed of a catalytic subunit p110γ and an adapter subunit, which is either p84
or p101.4
Class
I PI3Ks phosphorylate phosphatidylinositol(4,5)ꢀbisphosphate [PtdIns(4,5)P2] to yield
phosphatidylinositol(3,4,5)ꢀtrisphosphate [PtdIns(3,4,5)P3]. PtdIns(3,4,5)P3, produced at the inner leaflet of the
plasma membrane, provides a docking site for effector proteins with Pleckstrinꢀhomology (PH) domains. The
serine/threonine kinases phosphoinositideꢀdependent kinase 1 (PDKꢀ1) and protein kinase B (PKB/Akt) contain
a PH domain, and are thus activated by PI3K, and relay the activation of the mammalian target of rapamycin
complex 1 (mTORC1). In tumor cells, hyperꢀactivation of the PI3K/mTOR pathway can occur on multiple
levels, including mutation or overexpression of cell surface receptors such as HER25, oncogenes, the protein
tyrosine phosphatase nonꢀreceptor 12 (PTPN12)6, or the presence of activating hotspot mutations in PIK3CA
(encoding the catalytic PI3Kα subunit p110α).7 Other key regulators of the PI3K pathway include the lipid
phosphatase PTEN hydrolyzing PtdIns(3,4,5)P3 back to PtdIns(4,5)P2,2,8 and the inositol polyphosphate 4ꢀ
phosphatase type II (INPP4B)9 degrading PtdIns(3,4)P2. Loss of either lipid phosphatase activities or gain of
function mutations at the level of PI3K, and aberrations upꢀ and downstream in the pathway can thus lead to the
constitutive activation of PI3K, PKB/Akt and mTOR.
Besides promotion of tumor cell growth and angiogenesis, an overꢀactivated PI3K/mTOR pathway has
been associated with resistance to other cancer treatments, e.g. receptor tyrosine kinase inhibitors. PI3K
inactivating agents are currently in various clinical trials as either monotherapy or in combination with other
drugs.2,3,10
In our search for the development of a dual panꢀPI3K/mTOR inhibitor, we focused on diꢀmorpholino
substituted compounds with a triazine core, as their symmetry supports rapid access to a compound library with
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various aryl substituents. Furthermore, our goal was to develop brainꢀpenetrable compounds, to allow for the
treatment of brain metastasis and brain tumors. Here we describe a SAR study around the aryl moiety at the C2ꢀ
position of bismorpholinoꢀtriazines, which led to the identification of 1 (PQR309, Bimiralisib)11, a clinical
candidate and brain penetrable panꢀPI3K/mTOR inhibitor.
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RESULTS AND DISCUSSION
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Compound 1 evolved as the lead compound of a series of dimorpholinoꢀtriazine based compounds, and
is currently tested in Phase II clinical trials (clinicaltrials.gov). Initially inspired by 2ꢀ(2ꢀ
difluoromethylbenzimidazolꢀ1ꢀyl)ꢀ4,6ꢀdimorpholinoꢀ1,3,5ꢀtriazine (ZSTK474)12, a pan PI3K inhibitor with a
triazine core, development of 1 aimed to i) maximize compound solubility and bioavailability, ii) achieve blood
brain barrier penetration, iii) avoid microtubule interactions as observed for 4 (BKM120, Buparlisib)11,13, and iv)
introduce moderate mTOR inhibition. The rational for PI3K/mTOR dual activity was based on the observation
that persistent mTOR signaling was reported to confer resistance to PI3K inhibition14, while selective targeting
of the mTOR complex 1 (TORC1) initiates positive feedback leading to PI3K hyperꢀactivation15. To avoid this,
we selected for compounds with a higher potency for PI3K than mTOR kinase. Moreover, drugs targeting both
PI3K and mTOR have been shown early on to be more effective in tumors with multiple pathway activation
such as melanoma16,17, and to potentially support drug combinations18.
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A number of clinically advanced PI3K inhibitors with a pyrimidine, triazine or fused pyrimidine core
contain at least one morpholine moiety. This morpholine was demonstrated to be a crucial feature for PI3K
binding, as the morpholine oxygen atom forms a hydrogen bond interaction with the hinge region backbone NH
of the amino acid residue Val851 in PI3Kα (Val882 in PI3Kγ, and Val2240 in mTOR). Structurally related
compounds, which display distinct PI3K and mTOR kinase inhibitory profiles (Table 1) are shown in Figure 1:
1, 419 and 6 (GDCꢀ0980, Apitolisib / RG7422)20 target PI3K and mTOR kinase with some preference for PI3Kα,
while 3 (PIKꢀ587, Gedatolisib/ PFꢀ05212384)21 is a potent dual PI3K/mTOR inhibitor, and 5 (GDCꢀ0941,
Pictilisib)22 targets PI3K.
O
N
O
N
O
N
4
5 N
N 3 CF3
2
N
N
CF3
N
N
O
6
N
1
N
N
N
N
N
O
N
N
O
O
O
N
NH2
O
N
N
N
N
NH2
H
H
1
3
2
O
HO
O
N
O
N
N
N
N
N
S
N
NH
N
CF3
N
N
S
N
N
N
N
N
N
O
NH2
N
N
NH2
O
4
5
6
S
O
Figure 1. Structure of triazine core PI3K inhibitors 1, reference compound 2, and reported clinical candidate 3; and
pyrimidine or fused pyrimidine core containing molecules 4, 5, and 6.
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The above compounds are substituted with various aryl moieties (e.g. indazole, pyrimidine, pyridine,
benzoimidazole or aniline), which are situated in the PI3K's ATPꢀbinding site towards the affinity binding
pocket. The primary amine of 411,19 and 6,20 interacts via hydrogen bonding with the carboxyl groups of the
amino acid residues Asp841 and Asp836 in PI3Kγ. For 3 it has been proposed that the ureaꢀNH groups form
hydrogen bonds with Asp810 and the urea oxygen engages the wortmanninꢀreactive Lys80223,24 in PI3Kα.25
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Finally, 5 exploits its indazole NꢀNH atoms to form hydrogen bonds with Tyr867 and Asp841 in PI3Kγ. As
illustrated by the exchange of the aryl moiety from 4ꢀ(trifluoromethyl)pyridinꢀ2ꢀamine in 1 to 4ꢀ
(trifluoromethyl)pyrimidinꢀ2ꢀamine in compound 2, the aryl substitution can be used to fine tune PI3K vs.
mTOR activity.
Table 1. Affinity of triazine and pyrimidine core-containing molecules to PI3K and PI3K-related Kinases
mTOR/
PI3Kα
Binding constants Kd, [nM]a
Kinaseꢀ
PI3Kα PI3Kβ PI3Kγ PI3Kδ mTOR
PI3KC2β
VPS34
230
PI4KCβ DNAPK
40000 1600
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2
3
4
5
6
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11
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3.5
36
25
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25
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12
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820
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51
1.2
540
130
27000 >30000
>30000 >30000
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24
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0.94
0.46
2100
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200
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63
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130
260
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48
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40000
1800
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9800
0.76
0.62
2.1
3.8
7.7
2.6
3.3
3.3
n.d.
3.3
580
250
>30000
5.3
IC50, kinase assay, [nM]b
PI3Kα PI3Kβ PI3Kγ PI3Kδ mTOR
cα542Κ/545Κ/1047R
63 / 136 / 36
VPS34 DNAPK
1
4
33
45
661
607
708
778
451
110
89
94
8486
2581
8567
6917
2.6
13
111 / 216 / 87
aDissociation constants (Kd) were determined using ScanMax technology (DiscoveRx)26 with 11 point 3ꢀfold serial dilutions
of the indicated compounds. Kd is the mean value from experiments performed in duplicate, and were calculated from
standard dose response curves using the Hill equation depicted in Methods (n≥2). A TreeSpot representation of a full kinase
panel and kinase residual binding values are shown in Supporting Information, Figure S1 and Table S2 for all above
compounds except compound 2. bProkinase activity assays of the indicated enzymes and PI3Kα mutants. cInhibition of
PI3Kα E542K, E545K, and H2047R mutant activities are indicated. For these values, the wild type PI3Kα reference IC50s
were 122 nM for 1 and 142 nM for 4.
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Chemistry
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To achieve a balanced PI3K and mTOR inhibition, and to adjust physicochemical properties, a library
of 4,6ꢀdimorpholinoꢀ1,3,5ꢀtriazines substituted with various Cꢀ2ꢀaryl moieties was prepared from 4,4'ꢀ(6ꢀchloroꢀ
1,3,5ꢀtriazineꢀ2,4ꢀdiyl)dimorpholine (7, Scheme 1). The chlorine was displaced by aniline or heteroaryl moieties
using Suzuki crossꢀcoupling reactions with boronic acid pinacol esters. The desired products were obtained in
moderate to good yield (11ꢀ86%) depending on the used boronic acid pinacol ester derivative. Moreover,
compound 1 was transformed by acetylation, diazotization followed by bromination and Sandmeyer reaction in
order to obtain derivatives bearing different substituents at the Cꢀ2 pyridine (17ꢀ21). All final compounds were
analyzed for purity by analytical HPLC (> 95%).
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We have recently reported an optimized synthetic route to compound 111. This strategy allowed for
rapid access to large quantities of 1 starting from cyanuric chloride (81% yield) for preꢀ and clinical
development.
Scheme 1. Synthesis of C-2-aryl-substituted 4,6-dimorpholino-1,3,5-triazine analogsa
O
N
O
N
N
16 (34%)
b1 (86%)
2 (52%)
9 (45%)
10 (69%)
11 (17%)
13 (26%)
N
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NH2
NH2
NH2
NH2
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N
N
NH2
CF3
i, ii or iii
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N
N
N
14 (62%)
15 (75%)
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NH2
CF3
NH2
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N
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N
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CF3
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N
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N
12 (11%)
7: X = Cl; 8: X = I
N
NH2
NH2
O
N
O
N
17: R = NHAc
18: R = OH
19: R = Cl
(60%)
(33%)
(47%)
iv, v or vi
N
N
CF3
N
N
N
CF3
N
N
N
N
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20: R = NHOCH3 (15%)
21: R = Br (79%)
O
O
NH2
N
R
1
aReagents and conditions: (i) ArBpin, XPhosPdG2 (cat.), K3PO4, dioxane / H2O, 95 °C, 2ꢀ16 h, then HCl (aq.), 60 °C, 2ꢀ8 h;
(ii) ArBpin, Pd(dppf)Cl2 (cat.), Na2CO3, 1,2ꢀdimethoxyethane / H2O, 90 °C, 16 h; (iii) ArBpin, XPhosPdG2 (cat.), K3PO4,
dioxane / H2O, 95 °C, 2 h; (iv) AcCl, K2CO3, CH2Cl2, 0 °C → rt, 3.5 h; (v) HCl (conc.), NaNO2, 0 °C → rt, 1 h, then CuCl,
rt, 1 h; (vi) isopentyl nitrite, CH2Br2, rt, 5 min then (CH3)3SiBr, rt, 1 h; (vii) CH3ONH2·HCl, NaHCO3, DMSO, 100 °C, 6 h.
bCompound 1 was prepared according to the procedure recently reported11. ArBpin: boronic acid pinacol ester (the amino
group was protected in certain cases).
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Determination of cellular potency and PI3K vs. mTOR kinase activities
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In the following studies, the role of the aryl moiety was investigated systematically for enzymatic
activity and selectivity for class IA PI3K versus mTOR (Table 2). A first series of compounds including
variations of nitrogen atoms number and position was prepared to study the effect of the polarity of the aryl
group (9ꢀ12). Interestingly, 10 and 11 substituted with 2ꢀaminopyridine and 2ꢀaminopyrimidine, respectively,
were both found to be active in cellular and enzymatic assays. The calculated PSA (polar surface area)ꢀvalue for
the pyridineꢀsubstituted 10 (PSA=103) was slightly lower as compared to pyrimidine 11 (PSA=115), predicting
a better brain penetration for 10. Therefore, pyridines were substituted with lipophilic substituents such as
methyl and trifluoromethyl at pyridine C3ꢀand C4ꢀpositions to generate compounds (13-15). Introduction of a
C4ꢀtrifluoromethyl group significantly increased cellular potency and enzymatic targeting: compared with 13, 1
showed excellent cellular activities, good potency in vitro (Ki for PI3Kα of 17 nM), targeted mutated forms of
the catalytic PI3Kα subunit, and displayed the desired selectivity profile targeting PI3Kα>mTOR (Ki ratio of
mTOR/p110α ~ 3.6 [~ 8.0 from KINOMEscan™, see Table 1]). On the contrary, introduction of a lipophilic
substituent at the C3ꢀposition (methyl in 14 and trifluoromethyl in 15) diminished binding to PI3Kα as well as to
mTOR.
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While compound 11, bearing an unsubstituted 2ꢀaminopyrimidine, turned out to be a balanced inhibitor
for PI3Kα and for mTOR in vitro, (Ki mTOR/Ki PI3Kα ~2.3), a substitution at the pyrimidine C4ꢀposition with
methyl (16) or trifluoromethyl (2) enhanced cellular and in vitro activity towards PI3Kα, and diminished affinity
for mTOR, rising the mTOR/PI3Kα Ki ratio to ~16 to 25. Modifications at the C2ꢀpyridine position of the most
potent compound 1 were also investigated (17ꢀ21). These changes alter both the steric and the electronic
properties of the amine group, and are likely to affect hydrogen bond interactions with the aspartate carboxyꢀ
groups in the affinity binding pocket (Asp841 and Asp836 in PI3Kγ). Hydroxylamine and amide moieties
replacing primary amine functionality in 1 impaired binding to mTOR (Ki > 1 µM), and replacing the primary
amine moiety by a hydroxylamine, amide, hydroxy or halide decreased cellular potency considerably.
Compound 17 retained its affinity for PI3Kα in spite of an amide extension, likely due to a hydrogen bond of the
amide carbonyl with Lys802 (Lys833 in PI3Kγ; in analogy to compound 26 in Ref. 27 [see also PDB ID 3IBE]).
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Table 2. Direction of PI3K/mTOR selectivity with aromatic substituents for dimorpholino-triazine
4
5
Cellular IC50 [nM]a,b
in vitro Ki [nM]c
6
7
Cmpd
Growth Growth
mTOR/PI3Kα clogPd PSAd
pPKB/Akt
pS6
PI3Kα mTOR
8
9
A2058 SKOV3
12630
9
1744
367
1282
8504 1513
396
110
106
185
0.26
0.89
2.34
0.20
2.45
2.23
1.39
1.31
89.6
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10
11
12
415
415
817
3819
2161
9083
742
270
123
46
103
330
115
115
1283
4428
911
13
1
401
139
939
205
9113
2333
1504
237
61
17
609
10.00
3.60
2.69
3.11
102
102
62
14
15
1620
2370
1105
1638
14210
6221 1205
241
0.20
2.69
3.11
102
102
>20000
9213
342
4.3e6 284
6.5eꢀ5
16
2
169
85
831
312
2934
1636
13
224
203
17.60
25.10
1.56
2.67
115
115
73
8.1
20
17
3303
208
5588
729
19200
4545
689
2539
3.70
3.09
3.22
97.8
14480 11500
9.7 1213
125.00
105
18
19
21
1.3e5
51971
1.5e5
1.7e7 >20000 >20000 2299
n.d.
1.1e5
6.6e5
–
3.61
4.08
4.38
96.7
1.1e6
n.d.
4347 14050 32833
3.22
–
76.5
76.5
3033
3654
n.d.
aCellular phosphorylation of PKB/Akt on Ser473 and ribosomal S6 on Ser235/236 were analyzed in inhibitor treated A2058
b
melanoma cells using inꢀcell western detection. Each experiment performed in triplicate or as a multiple of n = 3. IC50s of
c
A2058 or SKOV3 cell proliferation were determined from 10ꢀpoint 2ꢀfold serial dilutions (n=3). Compounds were tested in
a timeꢀresolved FRET (TR)ꢀassay (LanthaScreen), and inhibitor Kis were calculated for PI3Kα and mTOR as described in
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methods. The mTOR/PI3Kα column depicts the ratio of compoundꢀspecific mTOR Ki over the Ki for PI3Kα. Each
4
5
d
experiment performed at least twice or as a multiple of n = 2. Marvin/JChem 16.10.17 was used for calculation of logP
6
7
(partition coefficient) and PSA (polar surface area) values.
8
9
The SARꢀstudy depicted above led to the identification of two potent compounds 1 and 2, both
substituted with a trifluoromethyl group on their aryl moiety. Both of them showed excellent potency for PI3Kα,
cellular activity profiles, and a calculated logPꢀvalues of ~3. Compound 1 was investigated in more detail, as it
displayed a mTOR/PI3Kα Ki selectivity ratio in the range of 3ꢀ8, while 2 had reduced impact on mTOR (Tables
1,2). An additional point in favor of compound 1 was a lower calculated PSA value of 102 as compared to 115
for 2, predicting a better brain penetration for 1.
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Binding mode of compound 1 to PI3K and mTOR kinase
Binding of compound 1 to the PI3Kγ catalytic subunit p110γ ATPꢀbinding site was confirmed by a
2.7 Å complex crystal structure, and identified key interactions of 1 with residues of p110γ. As reported for other
morpholinoꢀsubstituted PI3K inhibitors including compound 411,19, one morpholine of 1 forms a hydrogen bridge
with the backbone amide of Val882, a wellꢀknown and crucial interaction28. The compound 1/PI3Kγ complex
crystal structure (PDB ID code: 5OQ4; Table S1) also showed that the amino group of the 2ꢀaminoꢀ4ꢀ
(trifluoromethyl)pyridine moiety forms Hꢀbonds with Asp836, Asp841 and Asp964 (Figure 2A). Given the high
homology of class I PI3K catalytic pockets, and the nanomolar in vitro dissociation constants of 1 for all class I
PI3Ks (Table 1), an identical binding mode for all PI3K isoforms can be assumed. Besides the high affinity for
class I PI3K isoforms (highest affinity for PI3Kα), compound 1’s affinity for mTOR kinase is comparable to
PI3Kβ, PI3Kγ and PI3Kδ (Table 1).
Figure 2. Interactions of 1 with the ATPꢀbinding site of PI3Kγ catalytic subunit p110γ and mTOR kinase. A) Crystal
structure of 1 in PI3Kγ with a resolution of 2.7Å (PDB ID code: 5OQ4; Table S1). Compound 1 interacts with the hinge
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valine V882 in PI3Kγ, and its heteroaryl amine function forms hydrogen bonds with the carboxyl groups of aspartates D836,
4
5
6
7
8
9
D841, and D964. B) Overlay of the compound 1/PI3Kγ complex coordinates with the compound 4/PI3Kγ data from PDB
accession code 3SD5.19 The pyrimidine core nitrogen atom positions are corrected for the orientation as proposed in Ref. 11
(* denotes the position of CꢀH in compound 4); the two molecules are oriented exactly as in A). C) The coordinates of a
mTOR kinase/PI103 complex resolved to 3.6 Å (PDB code: 4JT6; docked, yellow) and mTOR at a resolution of 3.2 Å (PDB
code: 4JSN; relaxed, turquoise) were used to dock 1 into the ATPꢀbinding site of mTOR kinase. The yellow structure
represents the results of a manual preꢀdocking process (docked) where PI103 was replaced by 1 with subsequent energy
minimization; the turquoise structure represents the best fit of dynamic docking procedures to 4JSN (relaxed starting
conditions). Dotted lines indicate proposed Hꢀbond interactions.
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The current compound 1/PI3Kγ Xꢀray crystal structure combined with our previous structural studies on
4 and an asymmetrically substituted triazine (PIKiN3, PDB ID 5JHB),11 underlines the importance of Asp836,
Asp841 and Asp964, and associated structured water molecules, for the binding of this compound class.
Changes in the basicity of the aminoꢀheteroaryl (2ꢀaminoꢀ4ꢀ(trifluoromethyl)pyridine in 1 are therefore expected
to affect the affinity for PI3K, as it was the case for 2ꢀaminoꢀpyrimidine 2. The situation in the mTOR catalytic
pocket is less well defined: i) modelling compound 1/mTOR interactions starting with a preꢀdocked inhibitor
generate an interaction pattern similar to PI3K, where carboxyl groups of Asp2357, Glu2190, and Asp2195 are
at a distance of 3.5 to 4 Å from the heteroꢀaryl amino group and could execute hydrogen bonding; ii) starting
with less constraints, the inhibitor is slightly displaced by docking procedures, and the sole residue in hydrogen
bonding distance remains Glu2190. Although less conventional, the latter structure offers some explanation for
the differential effects of heteroꢀaryl modifications for PI3K and mTOR affinity shown in Table 2.
On and off target activities
In line with its activities against PI3K and mTOR, compound 1 induced a G1 cell cycle arrest in A2058
melanoma cells (mutated BꢀRaf, loss of PTEN) and SKOV3 cells (PI3Kα mutated at H1047R), which have both
constitutively activated PI3K/mTOR signaling pathways. In contrast, compound 4 triggered a mitotic arrest and
cells accumulated in G2/M or subG1 (Figure 2), which is in agreement with the reported binding of 4 to the
colchicineꢀbinding site of tubulin.11 In spite of compound 4's offꢀtarget effect, the compound attenuated PI3K
and mTOR signaling as determined by protein kinase B (PKB/Akt) phosphorylation on Ser473 and Thr308, and
S6 kinase (S6K, Thr389) and S6 phosphorylation on Ser235 and Ser236. Here, 4 closely matched the action of 1,
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5, 6, 3 and 2, while rapamycin by its specific action on TORC1 triggered an upregulation of PI3K activity, which
is in agreement with earlier observations.29
6
7
8
9
To validate the specificity of 1, the compound was tested in a KINOMEScan™ panel for interactions
with a wide range of protein (>400) and lipid kinases in parallel with 4, 5, 6 and 3. At 10 µM concentration 1, 4,
5 and 3 did show negligible interference with protein kinase activities (Figure S1, Table S2), while the dual
PI3K/mTOR kinase inhibitor 6 displayed multiple hits. Compound 1 reached excellent selectivity scores of
S(35) =0.025, S(10)= 0.018, as calculated according to Ref. 30.
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A
B
A2058
DMSO
Cmpd. 1
80
Cmpd. 4
SKOV3
DMSO
Cmpd. 1
80
Cmpd. 4
1000
800
600
400
200
0
250
200
150
100
50
****
****
60
40
20
60
40
20
**
**
ns
**
****
****
****
****
****
ns
**
****
****
**
0
0
0
Propidium Iodide
A2058
G1
S
G2 subG1
Propidium Iodide
A2058
G1
S
G2 subG1
C
D
SKOV3
pT308 pS473
SKOV3
3.0
2.0
1.0
0.0
pT308
pS473
3.0
2.0
1.0
0.0
pS6 S235/236
pS6K T389
pS6 S235/236
pS6K T389
1.0
0.5
0.0
1.0
0.5
0.0
pPKB T308
pPKB S473
PKB
pS6 S235/236
pS6K T389
S6
αꢀTubulin
S6K
Figure 3. Action of indicated compounds on cell cycle and PI3K and mTOR signaling. Cell cycle distribution of A2058 (A)
and SKOV3 (B) cells after 24 h exposure to 5 ꢀM (A) or 2 ꢀM (B) of indicated drugs (1, green; 4, red) or DMSO. After drug
exposure cells were collected, fixed, permeabilized and the DNA stained using propidium iodide. Cell cycle profiles were
acquired by fluorescence activated cell sorting. Left panels: Examples of cell cycle histograms. Right panels: Quantification
of cells in cell cycle phases G1, S, G2/M and sub G1 (as % of total, n=9, mean ± SD, one way ANOVA test with Dunnett’s
multiple comparison test for each cell cycle phase related to DMSO; ns: not significant, ** p>0.0021, **** p> 0.0001). C, D)
A2058 or SKOV3 cells were exposed to 1 ꢀM drug (Rapamycin 100 nM) for 1h, subsequently lysed and the denatured
lysates subjected to SDSꢀPAGE, Western blotting and immuneꢀdetection. C) Detection of total and phosphorylated (pT308
and pS473) PKB/Akt, and αꢀtubulin (left: A2058, right: SKOV3). Top: Quantification of phosphoꢀPKB/Akt levels related to
DMSO control (blue: pT308, red: pS473, n=3, mean ± SEM). D) Detection of total and phosphorylated (pS235/236)
ribosomal protein S6, and of total and phosphorylated (S6K T389) S6 Kinase (left: A2058, right: SKOV3). Top:
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Quantification of phosphoꢀprotein levels, related to DMSO control (blue: phospho S6, red: phospho S6K, n=3, mean ±
SEM).
6
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8
9
The impact of compound 1 on cell proliferation was analyzed in four independent human tumor cell
line collections of different tissue origin (NCI60;31 NTRC, Netherlands; Horizon Discoveries, UK; Clovis, UK;
135 different cell lines in total). Combined across the four cell panels the mean halfꢀmaximal growth inhibition
(GI50) was reached at ca. 0.7 µM (median: ca. 0.5 µM) of 1 as depicted in Figure 4.
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We have previously compared IC50ꢀindependent parameters such as Hill slopes of proliferation
inhibition curves and doseꢀdependent induction of nuclear condensation and histone H3 phosphorylation in a 44
cell line panel treated with either 1, 4, 5, or 6, which showed a coꢀclustering of the PI3K inhibitors 1, 5, and 6,
and grouped 4 with inhibitors of mitosis such as colchicine, nocodazole and MTD147 – a microtubuleꢀbinder
derived from 4,11 corroborating that 1 is free of the microtubuleꢀbinding offꢀtarget action of 4.
Figure 4. Cell proliferation in
response to 1 represented as a
1
waterfall blot. Concentrations of halfꢀ
maximal growth inhibition (GI50) for
1 were obtained from doseꢀresponse
growth curves derived from 4
0
ꢀ1
different tumor cell line panels (a
total of 135 cell lines; if a cell line
was contained in several panels its
GI50 was averaged). Individual GI50
of a cell line was related to the mean
GI50 of all cells lines, and cell lines were sorted by lowest to highest sensitivity for 1 from left to right. Individual cell lines
and values for the four cell panels are given in Table S3.
To evaluate other offꢀtarget effects, compound 1 was tested at 10 µM in a CEREP BioPrint panel,
where no or only very weak competition with radio ligands for cell surface and nuclear receptors, membrane
channels, transporters (Table S4) and enzyme activities of kinases, proteases and phosphodiesterases (Table S5)
were detected. Altogether, 1 qualifies as a specific panꢀclass I PI3K inhibitor without predicted offꢀtarget effects.
Pharmacological parameters of compound 1
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Before translation of the in vitro and cellular activities to in vivo tumor models, pharmacological
properties and metabolic stability of 1 were assessed. Compound 1 showed little clearance when exposed to rat,
dog and human liver microsomes, with a quicker turnover of 1 in mouse liver microsomes, where 40% of the
compound was eliminated within 30 min (Table 3A). Stability in microsome assays were matched with
clearance and halfꢀlive measurements in human, cynomolgus monkey, rat, dog and mouse hepatocyte cultures,
where 1 showed a low clearance in all species except for mouse. In the presence of human hepatocytes, the halfꢀ
life of compound 1 was 9.4 h, and ca. 45 min in mouse hepatocytes (Table 3B).
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Table 3A: Stability in liver microsomes of compound 1
% remaining in liver microsomesa
Cmpd \ species
1
Human
84.6 ± 2.2
7.45 ± 0.8
54.8 ± 6.7
Rat
99.9 ± 0.5
28.5 ± 3.1
2.9 ± 0.1
Dog
92.4 ± 1.6
1.8 ± 1.0
30.1 ± 2.6
Mouse
60.7 ± 4.6
1.5 ± 0.03
6.8 ± 0.9
7-ECb
Propranololb
apercentage of compound remaining after 30 min (mean ± SD, n=2); bAssay reference compounds: 7ꢀEC, 7ꢀ
Ethoxycoumarin.
Table 3B: Stability of compound 1 in hepatocyte cultures
Cmpd \ species
1
Human
2.5 ± 0.4
Monkey
3.5 ± 0.1
Rat
1.9 ± 0.1
Dog
Mouse
30.3 ± 0.1
45.7 ± 0.2
180.3 ± 0.1
7.7 ± 0.1
aCLint
2.4 ± 0.1
562.6 ± 95.5
37.8 ± 0.8
36.7 ± 0.8
98.2 ± 5.1
14.1 ± 0.7
396.4 ± 16.0
61.1 ± 3.5
22.7 ± 1.3
92.5 ± 3.5
15.0 ± 0.6
731.7 ± 54.5
16.0 ± 0.8
86.8 ± 4.6
192.8 ± 14.4
7.2 ± 0.5
577.6 ± 0.1
151.4 ± 2.8
9.2 ± 0.2
t1/2 [min]
CLint
7-ECb
7-HCb
t1/2 [min]
CLint
178.3 ± 0.7
7.8 ± 0.1
188.1 ± 8.6
7.4 ± 0.3
t1/2 [min]
aCLint [ꢁl/min/106 cells]; t1/2, [min]; bAssay reference compounds: 7ꢀEC, 7ꢀEthoxycoumarin; 7ꢀHC, 7ꢀHydroxycoumarin;
Results are expressed as Mean ± SD, n=2.
Subsequently, in vivo pharmacological parameters were determined in female CDꢀ1 mice, female
SpragueꢀDawley rats and male Beagle dogs to assess optimal dosing schedules for efficacy studies. Compound 1
was delivered either by a single intravenous (5 mg/kg) bolus or a single oral application (10 mg/kg). Drug levels
in extracts of plasma, brain and liver were determined by LCꢀMS/MS (Figure 5; PK parameters are summarized
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in Table 4). In female mice, plasma concentrations of 1 depended on the drug administration route, resulting in
halfꢀlives of ca. 13ꢀ36 min for p.o. administration vs. 9ꢀ10 min for i.v. administration. The fact that oral
administration yielded similar concentrations of 1 in brain and plasma samples, illustrates that compound 1
readily passes the blood brain barrier. In mice, both p.o. and i.v. application routes showed a rapid drop below
200 ng/ml (ca. 0.5 µM, Figure 5A) of 1 within <1 h (i.v.) to <2 h (p.o.) post administration, which reflects the
time point when the drug reaches the median GI50 determined in tumor cell lines.
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Figure 5. PK/PD assessment of 1 in
CD1 mice
A
4
3
2
4
3
2
1
0
mice, rats and dogs: time course of 1
abundance in vivo. Compound 1 was
administered in all species as single dose
either per os (p.o., 10 mg/kg) or via
intravenous injection (i.v., 5 mg/kg). At
indicated timeꢀpoints 1 was extracted
from tissue and its abundance determined
using HPLC/MSꢀMS. A) Levels of 1 in
CD1ꢀmice tissue. Right graph: Zoomed
view on early timeꢀpoints, indicated in
left graph by dashed square (n=3, mean±
SD). B) Concentrations of 1 in tissues
from SpragueꢀDawly rats. Right graph:
Zoomed view on early timeꢀpoints,
indicated in left graph by dashed square
(n=3, mean± SD). C) Plasma levels of 1
in male Beagle dogs. Panels on the right:
zoomed in to dotted line boxes to
visualize early timeꢀpoints (n=3, mean±
SD).
i.v. Plasma
p.o. Plasma
p.o. Brain
0
0
0
10
Hours
20
0
2
2
2
4
6
6
6
8
8
8
Hours
B
SpragueꢀDawly Rats
4
4
3
2
1
0
3
i.v.Plasma
i.v. Brain
p.o. Plasma
p.o. Brain
2
10
Hours
20
0
4
Hours
C
Beagle Dog
4
3
2
4
3
2
1
0
i.v. Plasma
p.o. Plasma
10
Hours
20
0
4
Hours
In female rats a single oral dose (10 mg/kg) achieved similar drug levels as a single intravenous
injection (5 mg/kg) with regard to Cmax (see Table 4). The halfꢀlife of 5 to 8 h, and an AUC0.25ꢀ12 of around
14'000 hr.ng/ml contributed to an excellent oral bioavailability of 1 (>50%). Twentyꢀfour hours post p.o.
administration, plasma levels of 1 were still > 2 µM (800ꢀ1000 ng/ml). Moreover, after 1 to 2 h exposure to 1,
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drug levels in rat brain samples were comparable to plasma levels (Fig. 5B), confirming rapid access of 1 to the
brain.
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Male Beagle dogs, exposed to 1 at 10 mg/kg p.o., showed maximal drug plasma concentrations Cmax of
583 ng/ml (approx. 1.5 µM) after 60 to 90 min and a halfꢀlife of >7 h, which results in drug levels of
approximately 0.38 µM (150 ng/ml) after 24 h. Intravenous delivery of 5 mg/kg of 1 resulted in high immediate
concentrations (>7000 ng/ml after 1 min.), and plasma levels decreased within 8 hours to similar concentrations
as observed for oral dosing (Fig. 4C). The oral bioavailability in male Beagle dogs was estimated to be 23%.
In vitro and in vivo toxicokinetic parameters were assessed and are summarized in Table S6. In rodents
and beagle dogs, compound 1 showed moderate but reversible target organ toxicity, which is expected for this
drug class and mechanism of action. Altogether the PK studies in the three models show rapid absorption of 1
and good oral bioavailability. Except for mice with a rapid clearance of 1, the late phase concentrations of 1 in
rats and dogs are in the expected range of predicted efficacy, independent of the administration route, and are
already reached after a single dose. The collected parameters are compatible with oral dosing in humans once
daily (QD).
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Altogether, compound 1 displays a superior solubility (Table S7) as compared with compound 2, is
orally bioavailable and brain penetrable, and does not display a microtubuleꢀbinding offꢀtarget effect like
compound 4.11
Table 4: PK analysis of 1 in plasma of mice, rats and dogs
Female CD-1 Mouse
Female Sprague-Dawley Rats
Male Beagle Dog
Route
i.v.
p.o.
i.v.
p.o.
i.v.
p.o.
Dose [mg/kg]
Cmax [ng/ml]
Tmax [min]
t1/2 [h]
5
10
5
10
5
10
3084 ± 379
5
1514 ± 334
15
3047 ± 557
5
4028 ± 1179
15
8760
5
583
120ꢀ240
8.6
0.15ꢀ0.33
0.22ꢀ0.60
2151
5.6ꢀ8.2
13974
5.6
7.6
AUC [h*ng/ml] 1583
AUC0 ꢀ 8h
3180
15313
12500
AUC0.25ꢀ 24h
411
6395
AUC0ꢀ 8h
AUC0.25ꢀ 12h
266
AUC0.25ꢀ 12h
AUC0.017ꢀ 24h
Cl [ml/h/kg]
BA [%]
68
55
23
i.v. intravenous (5 mg/kg); p.o.: per os (10 mg/kg); Cmax: maximal concentration, Tmax: time of maximal concentration in
hours; t1/2: halfꢀlife elimination in hours; AUC: Area under curve, Cl: clearance; BA: bioavailability; (n=3, mean ± SD for
each timeꢀpoint, species and route)
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Efficacy models
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Due to the short halfꢀlive of compound 1 in mice, a PC3 xenograft model in nude rats was used to
assess the antiꢀproliferative effects of 1 in vivo. Rats were injected with 2 x 107 human PC3 prostate cancer cells
into one flank and randomized after 16 days. From day 17 the control group received vehicle once daily.
Compound 1 was orally administered at 5 mg/kg, 10 mg/kg (both daily, QD1) or 15 mg/kg [5 consecutive days,
2 days off drug (QDx5, 2 days off)] for 28 days to match the timelines of regulatory toxicology studies. A
vinorelbineꢀtreated group (2.5 mg/kg i.v. weekly) was included as a “standard of care” control, with best treated–
toꢀcontrol ratios (T/C) of 17%.
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Treatment with 1 led to significant tumor size reductions: tumor growth was inhibited doseꢀdependently
(best T/C of 31ꢀ 12%, Fig. 6A). Compound 1 was best tolerated at 5 mg/kg without significant body weight
changes (Fig. 6B). At 10 mg/kg 1 caused a reduction of body weight, which accumulated to a reduction of 15%
after 28 days of treatment. Similarly, 15 mg/kg of 1 led to body weight loss after 5 days of treatment, which was
reversible during the recovery period. After 28 days of drug exposure (day 44 of the experiment), animals with
body weight loss fully recovered within a treatmentꢀfree period (day 45ꢀ 50) without overt signs of tumor cell
proliferation. In a subsequent treatment period tumor growth remained inhibited and body weight loss was only
observed in the 15 mg/kg group.
As shown in Figure 6C, Cmax of compound 1 is reached 15 min after a p.o. single dose in PC3ꢀtumor
bearing nude rats, and triggered a rapid and doseꢀdependent rise in plasma insulin and glucose levels (Figure 6Dꢀ
F), which are reliable markers for the drugs onꢀtarget action, as reported for other PI3K inhibitors targeting the
PI3Kα isoform.32
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Figure 6. Efficacy model for antiꢀtumor
activity of 1 in PC3 xenografts in nude
rats. A) Tumor volume: indicated
amounts of 1 (p.o., daily) or Vinorelbine
(i.v., weekly) were given from day 17ꢀ44
and from day 51ꢀ58 after tumor
inoculation (see materials and methods; n
= 4ꢀ8, mean± SEM). B) Body weight
changes in nude rats (treated as in (A), n
= 4ꢀ8, mean ± SEM). C) Time course of
1 abundance in nude rats. Compound 1
was administered as single dose per os
(p.o., 15 mg/kg). At indicated timeꢀpoints
1 was extracted from tissue and its
abundance determined using HPLC/MSꢀ
MS (n=3, mean± SD). D) Time course of
blood glucose and insulin levels tumor
bearing rats after a single dose of 1 (15
mg/kg p.o.; n=3, mean ± SEM; except
A
C
E
B
Control
cmpd 1 5mg/kg p.o.
cmpd 1 10mg/kg p.o.
cmpd 1 15mg/kg p.o.
Vinorelbine 2.5mg/kg IV
300
250
200
150
100
6
4
2
0
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0
20
Days
40
60
0
20
Days
40
60
D
cmpd 1, p.o., 15 mg/kg
Glucose [mM]
25 cmpd 1, p.o., 15 mg/kg
Insulin [ng/ml]
104
20
15
10
5
103
102
101
Plasma
Brain
Tumor
Liver
0
0
10
20
0
10
Hours
20
Hours
F
G
cmpd 1
p.o., 15 mg/kg
i.v., 10 mg/kg
1.5
1.0
0.5
0.0
25
15
10
5
***
***
***
20
15
10
5
***
0
0
10
Hours
20
cmpd 1,
[mg/kg]
0 5 1015
0 5 1015
for timeꢀpoints with values above detection limit). E) Plasma insulin and F) Plasma glucose concentrations on day 57 of the
efficacy model, 4 h after the last administration of 1 at indicated doses (n = 6ꢀ7, mean ± SEM, for insulin levels below
detection level – values were set constant to 0.04 [lowest measured value], for glucose levels above quantification limit,
levels were set 20 [upper quantification limit of kit]). G) Time course of phosphorylation of Ser473 in PKB/Akt in PC3
tumor xenograft lysates after a single dose of 1 (15 mg/kg p.o. or 10 mg/kg i.v.). Lysate were subjected to SDSꢀPAGE and
western blotting. Phosphorylated Ser473 levels were corrected for total PKB/Akt levels and compared to vehicleꢀtreated
xenografts (n=3, mean± SEM).
To further corroborate onꢀtarget activity of compound 1 in vivo, phosphorylation of pPKB/AKT was
measured in tumorꢀderived cell lysates. A single dose of 1 (15 mg/kg p.o. or 10 mg/kg i.v.) rapidly reduced
pPKB/AKT levels as compared to untreated control mice up to 12 h (Figure 6G), which matches high levels of 1
in the xenograft tumors at early time points (Figure 6C). After 24 h, PI3K signaling fully recovered, correlating
with a decrease of compound 1's concentration in tissue and tumors. Pharmacokinetics in plasma, brain and
tumor samples were very similar, suggesting that 1 distributes rapidly, and targets PI3K and mTOR in tumor and
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tissues. Proportionally elevated levels of 1 were detected in the liver, without signs of liver toxicity. The fast
reversibility of PI3K inhibition might be of advantage in patients, where mechanismꢀbased adverse effects are
expected to resolve with falling drug levels. Moreover, it has been observed that tumor cells reduce cell size in
response to PI3K inhibitors, while normal hepatocytes do not show mass reduction.17 This, and the observation
that interruption of drug administration (see 15 mg/kg schedule above) leads to body weight recovery but not
tumor regrowth, suggests that PI3K inhibition triggers latency in tumor growth that allows for the development
of intermittent scheduling protocols. To validate this treatment scheme, PC3 tumorꢀbearing nude rats were treated
every second, third and fourth day with 15 mg/kg p.o., which yielded a comparable efficacy, but substantially
alleviated the loss of body weight (Figure S2).
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CONCLUSION
The preclinical results presented here document that compound 1 is a highly selective panꢀPI3K
inhibitor with a balanced targeting of mTOR kinase. Compound 1's potent antiꢀproliferative action in vitro and
antiꢀtumor activity in vivo, paired with mild and reversible, mostly mechanismꢀbased toxicities have validated
the molecule as a clinical candidate. Compound 1 passed phase I studies and is now in phase II studies in
relapsed and refractory lymphoma and advanced solid tumors (clinicaltrials.gov).
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Experimental Section
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General Information.
8
9
Reagents were purchased at the highest commercial quality from Acros, SigmaꢀAldrich or Fluorochem
and used without further purification. Solvents were purchased from Acros Organics in AcroSeal® bottles over
molecular sieves. Column chromatographic purifications were performed on Merck KGaA silica gel (pore size
60 Å, 230ꢀ400 mesh particle size). Cross coupling reactions were carried out under nitrogen atmosphere in
anhydrous solvents and glassware was oven dried prior to use. TLC plates were obtained from Merck KGaA
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(Polygram SIL / UV254, 0.2 mm silica with fluorescence indicator) and UV light (254 nm) was used to visualize
13
1H, 19F and C NMR spectra were
the respective compounds.
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
were 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), 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 (ESIꢀMS) spectrometer. MALDIꢀToF mass spectra
were obtained on a VoyagerꢀDeTM Pro measured in m/z. The chromatographic purities of the final compounds
were determined by high performance liquid chromatography (HPLC) analyses on an Ultimate 3000SD System
from ThermoFisher with LPGꢀ3400SD pump system, ACCꢀ3000 autosampler and column oven, and DADꢀ3000
diode array detector. An Acclaimꢀ120 C18 reversedꢀphase column from ThermoFisher was used as stationary.
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 > 95% pure.
General procedure 1.
4,4'ꢀ(6ꢀChloroꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiyl)dimorpholine (7, 1.0 eq.), the respective boronic acid pinacol
ester (1.0ꢀ1.5 eq.), potassium phosphate tribasic (3.0 eq.) and chloro(2ꢀdicyclohexylphosphinoꢀ2',4',6'ꢀ
triisopropylꢀ1,1'ꢀbiphenyl)[2ꢀ(2'ꢀaminoꢀ1,1'ꢀbiphenyl)]palladium(II) (XPhos Pd G2, 0.05ꢀ0.10 eq.) were charged
in a flask. Under nitrogen atmosphere, 1,4ꢀdioxane (approx. 1 ml / 0.2 mmol) and deionized H2O (approx.
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1 ml / 0.4 mmol) were added and the resulting mixture was placed into an oil bath preꢀheated to 95 °C and
stirred at this temperature for 2ꢀ16 hours. After completion of the reaction, the mixture was allowed to cool to
room temperature, before addition of a 3 M aqueous HClꢀsolution and then heated at 60 °C for 2ꢀ8 hours. Then
the mixture was allowed to cool to room temperature, the pH was adjusted to 8ꢀ9 by addition of a 2 M aqueous
NaOHꢀsolution 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 the respective boronic acid pinacol ester (4.0 eq.), [1,1'ꢀ
(0.05 eq.) and 4,4'ꢀ(6ꢀchloroꢀ1,3,5ꢀtriazineꢀ2,4ꢀ
bis(diphenylphosphino)ferrocene]dichloropalladium(II)
diyl)dimorpholine (7, 1.0 eq.) in 1,2ꢀdimethoxyethane (approx. 1 ml / 0.2 mmol) under nitrogen atmosphere, a
2 M aqueous Na2CO3ꢀsolution (3.0 eq.) was added. The resulting reaction mixture was heated at 90°C for 16 h.
Then the mixture was allowed to cool to room temperature, poured onto an aqueous saturated NH4Clꢀsolution
and the aqueous layer was extracted with ethyl acetate (3x). The combined organic layers were washed with
deionized H2O, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated to dryness under reduced
pressure. The crude product was purified by column chromatography on silica gel.
General procedure 3.
N,NꢀDimethylformamide dimethyl acetal (1.5 eq.) and the respective amine (1.0 eq.) were dissolved in
tetrahydrofuran (approx. 1 ml / 0.3 mmol) and the resulting reaction mixture was stirred at 70 °C overnight. The
solvent was evaporated under reduced pressure and the residue was triturated with acetonitrile (3x) before it was
dried under vacuum.
5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (1) was prepared according to
the reported procedure in Ref. 11.
5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyrimidin-2-amine (2) was prepared according to
general procedure 1 from 4,4'ꢀ(6ꢀchloroꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiyl)dimorpholine (7, 300 mg, 1.05 mmol, 1.0 eq.)
and 5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ4ꢀ(trifluoromethyl)pyrimidinꢀ2ꢀamine (455 mg, 1.58 mmol,
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1.5 eq.). Purification by column chromatography on silica gel (cyclohexane / ethyl acetate 2:3 → 3:2) gave
3
4
5
6
7
product 2 as a colorless solid (225 mg, 546 ꢁmol, 52%). 1H NMR (400 MHz, CDCl3):
2 H), 3.39ꢀ3.78 (m, 8 H), 3.76ꢀ3.69 (m, 8 H); 19F{1H} NMR (376 MHz, (CD3)2SO):
δ
8.94 (s, 1 H), 5.65 (br s,
δ
− 63.8 (s, 3 F);
8
9
13C{1H} NMR (101 MHz, CDCl3):
δ
168.5 (s, 1 C), 164.7 (s, 2 C), 163.1 (s, 1 C), 162.7 (s, 1 C), 154.6 (q,
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1
2JC,F = 35 Hz, 1 C), 121.1 (s, 1 C), 120.8 (q, JC,F = 276 Hz, 1 C), 66.9 (s, 4 C), 43.7 (s, 4 C); ESI-HRMS (m/z):
[M + H]+ calc. for C16H20F3N8O2, 413.1656; found: 413.1662; HPLC: tR = 7.39 min (> 99.9% purity).
4,4'-(6-Chloro-1,3,5-triazine-2,4-diyl)dimorpholine (7) was prepared according to Ref. 11.
4,4'-(6-Iodo-1,3,5-triazine-2,4-diyl)dimorpholine (8). To an aqueous hydriodic acid solution (57 wt. %,
2.00 ml, 15.2 mmol, excess) diluted with deionized H2O (2.00 ml), 4,4'ꢀ(6ꢀchloroꢀ1,3,5ꢀtriazineꢀ2,4ꢀ
diyl)dimorpholine (7, 500 mg, 1.75 mmol, 1.0 eq.) was added at 0 °C. The reaction mixture was allowed to
warm to room temperature overnight and then poured onto a 1 M aqueous NaOHꢀsolution. The product 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 residue was purified by column chromatography on
silica gel (cyclohexane / ethyl acetate 1:0 → 1:1) to afford product 8 as a colorless solid (409 mg, 1.08 mmol,
1
61%). H NMR (400 MHz, CDCl3):
δ δ 162.1 (s, 2 C),
3.87ꢀ3.63 (m, 16 H); 13C{1H} NMR (101 MHz, CDCl3):
140.6 (s, 1 C), 66.8 (s, 4 C), 44.0 (s, 4 C); MALDI-MS: m/z = 378.1 ([M + H]+).
4-(4,6-Dimorpholino-1,3,5-triazin-2-yl)aniline (9) was prepared according to general procedure 1 from 4,4'ꢀ(6ꢀ
chloroꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiyl)dimorpholine (7, 150 mg, 525 ꢁmol, 1.0 eq.) and tertꢀbutyl (4ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)carbamate (22, 250 mg, 783 ꢁmol, 1.5 eq.). Purification by column
chromatography on silica gel (cyclohexane / ethyl acetate 1:0 → 3:2) gave product 9 as a colorless solid
1
3
3
(80.9 mg, 236 ꢁmol, 45%). H NMR (400 MHz, CDCl3):
δ 8.22 (d, JH,H = 8.7 Hz, 2 H), 6.69 (d, JH,H = 8.7 Hz,
2 H), 3.99ꢀ3.81 (m, 10 H), 3.79ꢀ3.72 (m, 8 H); 13C{1H} NMR (101 MHz, CDCl3):
δ
170.3 (s, 1 C), 165.3 (s,
2 C), 149.7 (s, 1 C), 130.1 (s, 2 C), 127.5 (s, 1 C), 114.2 (s, 2 C), 67.0 (s, 4 C), 43.7 (br s, 4 C); MALDI-MS:
m/z = 343.2 ([M + H]+); HPLC: tR = 7.27 min (> 99.9% purity). The spectroscopic data are consistent with
previous literature reports.34
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5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)pyridin-2-amine (10) was prepared according to general procedure 2
from 4,4'ꢀ(6ꢀchloroꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiyl)dimorpholine (7, 200 mg, 702 ꢁmol, 1.0 eq.) and 2ꢀaminopyridineꢀ5ꢀ
boronic acid pinacol ester (616 mg, 2.80 mmol, 4.0 eq.). Purification by column chromatography on silica gel
(dichloromethane / methanol 9:1) gave compound 10 as a colorless solid (166 mg, 483 ꢁmol, 69%).
1H NMR (400 MHz, CDCl3):
δ
9.07 (d, 4JH,H = 2.3 Hz, 1 H), 8.37 (dd, 3JH,H = 8.7 Hz, 4JH,H = 2.3 Hz, 1 H),
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3
6.50 (d, JH,H = 8.6 Hz, 1 H), 4.71 (br s, 2 H), 3.88 (br s, 8 H), 3.79ꢀ3.71 (m, 8 H); 13C{1H} NMR (101 MHz,
CDCl3):
δ 169.2 (s, 1 C), 165.1 (s, 2 C), 160.4 (s, 1 C), 150.0 (s, 1 C), 137.9 (s, 1 C), 123.6 (s, 1 C), 107.5 (s,
1 C), 67.0 (s, 4 C), 43.7 (s, 4 C); ESI-HRMS (m/z): [M + H]+ calc. for C16H22N7O2, 344.1829; found: 344.1832;
HPLC: tR = 8.59 min (96.2% purity).
5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2-amine (11) was prepared according to general
procedure 2 from 4,4'ꢀ(6ꢀchloroꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiyl)dimorpholine (7, 50.0 mg, 170 ꢁmol, 1.0 eq.) and 2ꢀ
aminopyrimidineꢀ5ꢀboronic acid pinacol ester (155 mg, 700 ꢁmol, 4.0 eq.). Purification by column
chromatography on silica gel (ethyl acetate / methanol 1:0 → 20:1) gave compound 11 as a colorless solid
(10.0 mg, 29.0 ꢁmol, 17%). 1H NMR (400 MHz, (CD3)2SO):
8 H), 3.64 (br s, 8 H); 13C{1H} NMR (101 MHz, (CD3)2SO):
δ
9.05 (s, 2 H), 7.26 (br s, 2 H), 3.94ꢀ3.62 (m,
δ
167.2 (s, 1 C), 164.8 (s, 1 C), 164.2 (s, 2 C),
158.6 (s, 2 C), 118.5 (s, 1 C), 66.0 (s, 4 C), 43.2 (br s, 4 C); ESI-HRMS (m/z): [M + H]+ calc. for C15H21N8O2,
345.1782; found: 345.1784; HPLC: tR = 6.06 min (95.4% purity).
5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)pyrazin-2-amine (12).
4,4'ꢀ(6ꢀiodoꢀ1,3,5ꢀtriazineꢀ2,4ꢀ
diyl)dimorpholine (8, 200 mg, 530 ꢁmol, 1.0 eq.), 5ꢀaminopyrazinꢀ2ꢀylꢀboronic acid pinacol ester (350 mg,
1.58 mmol, 3.0 eq.), potassium phosphate tribasic (339 mg, 1.60 mmol, 3.0 eq.) and chloro(2ꢀ
dicyclohexylphosphinoꢀ2',4',6'ꢀtriisopropylꢀ1,1'ꢀbiphenyl)[2ꢀ(2'ꢀaminoꢀ1,1'ꢀbiphenyl)]palladium(II)
(41.7 mg,
53.0 mmol, 0.10 eq.) were charged in a flask. Under nitrogen atmosphere, 1,4ꢀdioxane (5 ml) and deionized H2O
(1.5 ml) were added and the resulting mixture was placed into an oil bath preꢀheated and allowed to stir for
2 hours at 95 °C. After completion of the reaction, the mixture was allowed to cool to room temperature.
Dichloromethane and a 2 M aqueous NaOHꢀsolution were added. The aqueous layer was separated and extracted
with dichloromethane (3 x). The combined organic layers were then dried over anhydrous Na2SO4, filtered and
the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography
on silica gel (dichloromethane / methanol 1:0 → 9:1) to obtain compound 12 as a colorless solid (20.1 mg,
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58.4 ꢁmol, 11%). 1H NMR (400 MHz, CDCl3):
δ
9.08 (br s, 1 H), 8.10 (br s, 1 H), 4.86 (br s, 2 H), 4.08ꢀ
4
5
3.81 (m, 8 H), 3.81ꢀ3.71 (m, 8 H); 13C{1H} NMR (101 MHz, CDCl3):
δ 168.8 (s, 1 C), 165.2 (s, 2 C), 155.2 (s,
6
7
8
9
1 C), 144.5 (s, 1 C), 140.1 (s, 1 C), 131.6 (s, 1 C), 67.0 (s, 4 C), 43.8 (br s, 4 C); ESI-HRMS (m/z): [M + H]+
calc. for C15H21N8O2, 345.1782; found: 345.1780; HPLC: tR = 5.37 min (95.2% purity).
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5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-methylpyridin-2-amine (13) was prepared according to general
procedure 2 from 4,4'ꢀ(6ꢀchloroꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiyl)dimorpholine (7, 40.0 mg, 140 ꢁmol, 1.0 eq.) and 2ꢀ
aminoꢀ4ꢀmethylꢀpyridineꢀ5ꢀboronic acid pinacol ester (132 mg, 560 ꢁmol, 4.0 eq.). Purification by column
chromatography on silica gel (ethyl acetate / methanol 1:0 → 10:1) gave product 13 as a colorless solid
1
(13.1 mg, 36.7 ꢁmol, 26%). H NMR (400 MHz, CDCl3):
δ
8.78 (s, 1 H), 6.33 (s, 1 H), 4.54 (br s, 2 H), 3.90ꢀ
171.4 (s, 1 C), 165.0 (s,
3.80 (m, 8 H), 3.77ꢀ3.72 (m, 8 H), 2.60 (s, 3 H); 13C{1H} NMR (101 MHz, CDCl3):
δ
2 C), 159.3 (s, 1 C), 151.4 (s, 1 C), 149.4 (s, 1 C), 124.1 (s, 1 C), 109.8 (s, 1 C), 66.8 (s, 4 C), 43.7 (br s, 4 C),
22.3 (s, 1 C); ESI-HRMS (m/z): [M + H]+ calc. for C17H24N7O2, 358.1986; found: 358.1983; HPLC:
tR = 6.57 min (98.5% purity).
5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-3-methylpyridin-2-amine (14) was prepared according to general
procedure 1 from 4,4'ꢀ(6ꢀchloroꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiyl)dimorpholine (7, 150 mg, 525 ꢁmol, 1.0 eq.) and boronic
acid pinacol ester 23 (180 mg, 622 ꢁmol, 1.2 eq.). Purification by column chromatography on silica gel
(cyclohexane / ethyl acetate 1:0 → 4:1) gave product 14 as a colorless solid (116 mg, 32.5 ꢁmol, 62%).
4
1H NMR (400 MHz, CDCl3):
δ
8.89 (d, JH,H = 2.2 Hz, 1 H), 8.22 (br s, 1 H), 5.25 (br s, 2 H), 4.02ꢀ3.68 (m,
16 H), 2.19 (s, 3 H); 13C{1H} NMR (101 MHz, CDCl3):
δ 168.9 (s, 1 C), 165.0 (s, 2 C), 158.9 (s, 1 C), 145.2 (s,
1 C), 138.1 (s, 1 C), 123.5 (s, 1 C), 116.3 (s, 1 C), 67.0 (s, 4 C), 43.7 (br s, 4 C), 17.0 (s, 1 C); ESI-HRMS (m/z):
[M + H]+ calc. for C17H24N7O2, 358.1986; found: 358.1981; HPLC: tR = 6.91 min (99.3% purity).
5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-3-(trifluoromethyl)pyridin-2-amine (15) was prepared according to
general procedure 1 from 4,4'ꢀ(6ꢀchloroꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiyl)dimorpholine (7, 83.3 mg, 292 ꢁmol, 1.0 eq.) and
boronic acid pinacol ester 24 (100 mg, 292 ꢁmol, 1.0 eq.). Purification by column chromatography on silica gel
(cyclohexane / ethyl acetate 1:0 → 1:1) gave product 15 as a colorless solid (89.5 mg, 218 ꢁmol, 75%).
4
1H NMR (400 MHz, CDCl3):
δ
9.19 (s, 1 H), 8.63 (d, JH,F = 2.2 Hz, 1 H), 5.24 (br s, 2 H), 4.01ꢀ3.80 (m, 8 H),
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3.80ꢀ3.71 (m, 8 H); 19F{1H} NMR (376 MHz, (CD3)2SO):
δ
− 63.1 (s, 3 F); 13C{1H} NMR (101 MHz,
4
5
(CD3)2SO):
δ
167.3 (s, 1 C), 164.2 (s, 2 C), 157.3 (s, 1 C), 153.1 (s, 1 C), 134.5 (br s, 1 C), 124.1 (q,
6
7
8
9
1JC,F = 271 Hz, 1 C), 119.9 (s, 1 C), 105.1 (q, 2JC,F = 31 Hz, 1 C), 66.0 (s, 4 C), 43.2 (br s, 4 C); ESI-
HRMS (m/z): [M + H]+ calc. for C17H21F3N7O2, 412.1703; found: 412.1703; HPLC: tR = 8.40 min (> 99.9%
purity).
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5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-methylpyrimidin-2-amine (16) was prepared according to general
procedure 2 from 4,4'ꢀ(6ꢀchloroꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiyl)dimorpholine (7, 40.0 mg, 140 ꢁmol, 1.0 eq.) and 4ꢀ
methylꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)pyrimidinꢀ2ꢀamine (132 mg,
560 ꢁmol,
4.0 eq.).
Purification by column chromatography on silica gel (ethyl acetate 100%) gave product 16 as a colorless solid
(17.6 mg, 49.2 ꢁmol, 34%). 1H NMR (400 MHz, (CD3)2SO):
8 H), 3.66ꢀ3.59 (m, 8 H), 2.63 (s, 3 H); 13C{1H} NMR (101 MHz, CDCl3):
δ
8.90 (s, 1 H), 7.02 (br s, 2 H), 3.81ꢀ3.69 (m,
δ
170.1 (s, 1 C), 168.9 (s, 1 C),
164.6 (s, 2 C), 162.6 (s, 1 C), 160.9 (s, 1 C), 121.3 (s, 1 C), 66.9 (s, 4 C), 43.7 (br s, 4 C), 25.2 (s, 1 C); ESI-
HRMS (m/z): [M + H]+ calc. for C16H23N8O2, 359.1938; found: 359.1939; HPLC: tR = 6.22 min (97.3% purity).
N-(5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-yl)acetamide (17).
5ꢀ(4,6ꢀ
Dimorpholinoꢀ1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ4ꢀ(trifluoromethyl)pyridinꢀ2ꢀamine (1, 200 mg, 480 ꢁmol, 1.0 eq.) and
K2CO3 (134 mg, 972 mmol, 2.0 eq.) were mixed in dichloromethane (4 ml) and stirred at room temperature for 5
minutes. Then, the reaction mixture was cooled to 0 °C and acetyl chloride (41.4 ꢁl, 583 mmol, 1.2 eq.) was
added dropwise. The resulting mixture was stirred for 3.5 hours and allowed to warm up to room temperature.
Then, deionized H2O was added, the layers were separated and the aqueous layer was extracted with
dichloromethane (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
(cyclohexane / ethyl acetate 1:1 → 1:3) to afford compound 17 (120 mg, 265 mmol, 60%) as a colorless solid.
1H NMR (400 MHz, CDCl3):
2.26 (s, 3 H); 19F{1H} NMR (376 MHz, CDCl3):
δ 8.81 (s, 1 H), 8.61 (s, 1 H), 8.34 (s, 1 H), 3.85 (br s, 8 H), 3.78ꢀ3.67 (m, 8 H),
δ
− 59.5 (s, 3 F); 13C{1H} NMR (101 MHz, CDCl3):
δ 169.3 (s,
2
1 C), 168.7 (s, 1 C), 164.5 (s, 2 C), 152.6 (s, 1 C), 151.0 (s, 1 C), 138.8 (q, JC,F = 33 Hz, 1 C), 128.4 (s, 1 C),
1
3
122.5 (q, JC,F = 275 Hz, 1 C), 110.5 (q, JC,F = 5.1 Hz, 1 C), 66.7 (s, 4 C), 43.5 (br s, 4 C), 24.7 (s, 1 C); ESI-
HRMS (m/z): [M + H]+ calc. for C19H23F3N7O3, 454.1809; found: 454.1797; HPLC: tR = 7.94 min (> 99.9%
purity).
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5-(4,6-dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-ol (18)
and
4,4'-(6-(6-Chloro-4-
6
7
8
9
(trifluoromethyl)pyridin-3-yl)-1,3,5-triazine-2,4-diyl)dimorpholine (19). To a conc. HClꢀsolution (36%,
5.12 ml) at 0 °C, 5ꢀ(4,6ꢀdimorpholinoꢀ1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ4ꢀ(trifluoromethyl)pyridinꢀ2ꢀamine (1, 500 mg,
1.21 mmol, 1.0 eq.) was added dropwise followed by NaNO2 (1.70 g, 24.6 mmol, 20 eq.). The resulting mixture
was then allowed to warm to room temperature over 1 hour and CuCl (4.10 g, 41.4 mmol, 34 eq.) was added.
After 1 hour, the reaction mixture was quenched with a 2 M aqueous NaOHꢀsolution and 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 products were separated by column chromatography on silica gel
(cyclohexane / ethyl acetate 1:0 → 1:1). Compound 19 (247 mg, 573 ꢁmol, 47%) and compound 18 (166 mg,
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403 ꢁmol, 33%) were obtained in two separate fractions and isolated as colorless solids. Compound 19:
1H NMR (400 MHz, CDCl3):
19F{1H} NMR (376 MHz, CDCl3):
δ
8.91 (s, 1 H), 7.66 (s, 1 H), 3.89ꢀ3.78 (m, 8 H), 3.78ꢀ3.68 (m, 8 H);
− 59.7 (s, 3 F); 13C{1H} NMR (101 MHz, CDCl3):
δ
δ 169.0 (s, 1 C),
164.6 (s, 2 C), 153.2 (s, 1 C), 153.0 (s, 1 C), 154.1 (q, 2JC,F = 34 Hz, 1 C), 131.5 (s, 1 C), 122.1 (q,
3
1JC,F = 275 Hz, 1 C), 121.4 (q, JC,F = 5.4 Hz, 1 C), 66.9 (s, 4 C), 43.8 (br s, 4 C); ESI-HRMS (m/z): [M + H]+
calc. for C17H19F3ClN6O2, 431.1205; found: 431.1199; HPLC: tR = 9.91 min (> 99.9% purity). Compound 18:
1H NMR (400 MHz, (CD3)2SO):
3.55 (m, 8 H); 19F{1H} NMR (376 MHz, (CD3)2SO):
168.0 (s, 1 C), 164.0 (s, 2 C), 161.2 (s, 1 C), 141.4 (br s, 1 C), 138.9 (q, 2JC,F = 32 Hz, 1 C), 122.3 (q,
δ
12.6 (br s, 1 H), 8.15 (s, 1 H), 6.80 (s, 1 H), 3.79ꢀ3.67 (m, 8 H), 3.66ꢀ
− 59.4 (s, 3 F); 13C{1H} NMR (101 MHz, (CD3)2SO):
δ
δ
1JC,F = 275 Hz, 1 C), 119.2 (br s, 1 C), 113.3 (br s, 1 C), 65.9 (s, 4 C), 43.2 (s, 4 C); ESI-HRMS (m/z): [M + H]+
calc. for C17H20F3N6O3, 413.1543; found: 413.1535; HPLC: tR = 6.79 min (99.6% purity).
N-(5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-yl)-O-methylhydroxylamine (20).
4,4'ꢀ(6ꢀ(6ꢀChloroꢀ4ꢀ(trifluoromethyl)pyridinꢀ3ꢀyl)ꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiyl)dimorpholine (19, 200 mg, 464 ꢁmol,
1.0 eq.), methoxyamine hydrochloride (77.5 mg, 928 ꢁmol, 2.0 eq.) and NaHCO3 (79.0 mg, 940 ꢁmol, 2.0 eq.)
were mixed in dimethyl sulfoxide (3.0 ml). The resulting reaction mixture was stirred at 100 °C for 6 hours. The
solvent was partially evaporated under reduced pressure. Then, deionized H2O and dichloromethane were added.
The aqueous layer was separated and extracted with dichloromethane (3 x). The combined organic layers were
dried over anhydrous Na2SO4, filtered and the solvent was evaporated to dryness under reduced pressure. The
crude product was purified by column chromatography on silica gel (cyclohexane / ethyl acetate 1:1 → 0:1) to
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afford compound 20 (30.0 mg, 34.0 mmol, 15%) as a colorless solid. H NMR (400 MHz, CDCl3):
δ 8.77 (s,
4
5
1 H), 7.77 (br s, 1 H), 7.23 (s, 1 H), 3.90ꢀ3.79 (m, 11 H), 3.77ꢀ3.70 (m, 8 H); 19F{1H} NMR (376 MHz, CDCl3):
6
7
δ
− 59.6 (s, 3 F); 13C{1H} NMR (101 MHz, CDCl3):
δ 169.6 (s, 1 C), 164.8 (s, 2 C), 156.0 (s, 1 C), 151.8 (s,
8
9
2
1
3
1 C), 138.6 (q, JC,F = 274 Hz, 1 C), 127.5 (s, 1 C), 122.6 (q, JC,F = 275 Hz, 1 C), 110.1 (q, JC,F = 6.2 Hz, 1 C),
66.9 (s, 4 C), 63.1 (s, 1 C), 43.8 (br s, 4 C); ESI-HRMS (m/z): [M + H]+ calc. for C18H23F3N7O3, 442.1809;
found: 442.1799; HPLC: tR = 8.33 min (98.9% purity).
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4,4'-(6-(6-Bromo-4-(trifluoromethyl)pyridin-3-yl)-1,3,5-triazine-2,4-diyl)dimorpholine (21).
Compound
1
(1.00 g, 2.43 mmol, 1.0 eq.) was dissolved in dibromomethane (10 ml) and
isopentyl nitrite (392 ꢁl, 2.92 mmol, 1.2 eq.) was added. After 5 minutes, a solution of bromotrimethylsilane
(401 ꢁl, 3.04 mmol, 1.3 eq.) in dibromomethane (1.5 ml) was added and the resulting reaction mixture was
allowed to stir at room temperature for 1 hour. After this time, an aqueous saturated NaHCO3ꢀsolution was
added, the layers were separated and the aqueous layer was extracted with dichloromethane (3 x). The combined
organic layers were dried over anhydrous Na2SO4, filtered and the solvent was evaporated under reduced
pressure. Purification by column chromatography on silica gel (cyclohexane / ethyl acetate 1:0 → 1:1) gave
1
compound 21 (909 mg, 1.91 mmol, 79% yield) as colorless solid. H NMR (400 MHz, CDCl3):
δ
8.88 (s, 1 H),
7.81 (s, 1 H), 3.88ꢀ3.78 (m, 8 H), 3.78ꢀ3.68 (m, 8 H); 19F{1H} NMR (376 MHz, CDCl3):
13C{1H} NMR (101 MHz, CDCl3):
δ − 59.6 (s, 3 F);
δ
169.0 (s, 1 C), 164.6 (s, 2 C), 153.2 (s, 1 C), 143.7 (s, 1 C), 138.5 (q,
2JC,F = 34 Hz, 1 C), 131.8ꢀ131.7 (m, 1 C), 125.1 (q, JC,F = 5.3 Hz, 1 C), 122.0 (q, JC,F = 275 Hz, 1 C); 66.8 (s,
4 C), 43.7 (br s, 4 C); ESI-HRMS (m/z): [M + H]+ calc. for C17H19BrF3N6O2, 475.0699; found: 475.0710;
HPLC: tR = 10.11 min (97.8% purity).
3
1
tert-Butyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate (22) was prepared according to
the literature.33
N,N-Dimethyl-N'-(3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-
yl)formimidamide (23) was prepared according to general procedure 3 from 3ꢀmethylꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ
1,3,2ꢀdioxaborolanꢀ2ꢀyl)pyridinꢀ2ꢀamine (200 mg, 854 ꢁmol, 1.0 eq.). The desired product 23 was obtained in
quantitative yield as a brownish solid and was used in the next step without further purification.
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1H NMR (400 MHz, (CD3)2SO):
δ
8.50 (s, 1 H), 8.25 (s, 1 H), 7.61 (s, 1 H), 3.10 (s, 3 H), 3.02 (s, 3 H), 2.19 (s,
4
5
3 H), 1.27 (s, 12 H).
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8
9
N,N-Dimethyl-N'-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)pyridin-2-
yl)formimidamide (24) was prepared according to general procedure 3 from 5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
dioxaborolanꢀ2ꢀyl)ꢀ3ꢀ(trifluoromethyl)pyridinꢀ2ꢀamine (300 mg, 1.04 mmol, 1.0eq.). The desired product 24 was
obtained in quantitative yield as a yellowish solid and was used in the next step without further purification.
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1H NMR (400 MHz, (CD3)2SO):
δ 8.70 (s, 1 H), 8.54 (d, J = 1.8 Hz, 1 H), 7.96 (d, J = 1.9 Hz, 1 H), 3.16 (s,
3 H), 3.06 (s, 3 H), 1.29 (s, 12 H); 19F{1H} NMR (376 MHz, (CD3)2SO):
δ − 61.6 (s, 3 F).
Protein production and purification.
The ꢂABDp110γ was expressed in Sf9 cells using a baculovirus expression system as has been
described previously.24,35 Briefly, human ꢂABDp110γ was expressed with a Cꢀterminal His6ꢀtag and purified by
immobilized metalꢀaffinity chromatography, heparin chromatography and gel filtration. The protein in gel
filtration buffer (20 mM TrisꢀHCl 0.5 mM (NH4)2SO4, 5 mM DTT, 1% ethylene glycol and 0.02% CHAPS) was
concentrated to 8 mg/ml then frozen in liquid nitrogen and stored at ꢀ80 °C.
Crystallization of PI3Kγ; soaking with compound 1.
Drops containing 1 ꢁl of ꢂABDp110γ protein at 4 mg/ml was and 1 ꢁl of crystallization buffer (16%
PEG4K, 250 mM (NH4)2SO4, 0.1 M TrisꢀHCl pH 7.5 at RT) were hair seeded with crushed ꢂABDp110γ crystals
and incubated at 17°C. The crystals reached their maximum size (0.2 mm ×0.1mm × 0.1mm) in about 10
days. The inhibitor 1 was prepared in DMSO and diluted in cryoꢀprotectant containing 25% PEG 4000, 15%
glycerol, 250mM (NH4)2SO4 and 100mM Tris pH 7.5 to a final concentration of 1 mM. The inhibitor in cryoꢀ
protectant solution was added to the drop containing the crystals in a stepwise manner. The crystals were soaked
in the final 1 mM inhibitor solution for 4 hours. Crystals were then transferred to fresh inhibitor in cryoꢀ
protectant solution for 30 s and flash frozen in liquid nitrogen.
Data collection and structure determination.
Diffraction datasets were collected at the ESRF beamline ID23ꢀ1 on an ADSC Quantum Q315r
detector, using 1.00720 Å wavelength radiation. Images were processed using Mosflm36 or XDS37 and scaled
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with SCALA38. The crystal structure of the ꢂABDp110γ/compound 1 complex was solved by molecular
replacement using Phaser,39 with the previously published human ꢂABDp110γ as the search model (PDB entry
1E8Y). Coot was used to manually build inhibitors in the unaccounted FoꢀFc difference electron density within
the active site of the map.40 Initial models of the inhibitors were generated from SMILES strings using the
ELBOW module of PHENIX.41 Refmac refinement42 was iterated with manual rebuilding using Coot until the
structure converged. In the final model for the ꢂABDp110γ/compound 1 complex, 89.2% of residues were in the
core regions of the Ramachandran plot, with one residue in a disallowed region.
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Cellular PI3K and mTOR signaling
Downstream signals of PI3K and mTOR were determined in InꢀCell Western assays detecting
phosphorylation of Ser473 of PKB/Akt (pPKB/Akt) and Ser235/236 on the ribosomal protein S6 (pS6),
respectively. Briefly, 2x104 A2058 cells/well in 96ꢀwell plates (Cell Carrier, Perkin Elmer) were grown for 24 h,
before inhibitors or DMSO were added (1 h; 37°C, 5%CO2). Cells were subsequently fixed (4% PFA in PBS for
30 min at RT), and then blocked (1% BSA/0.1% Triton Xꢀ100/5% goat serum in PBS for 30 min, RT).
pPKB/Akt was detected with a rabbit polyclonal antibody (Cell Signaling Technology, #4058), and pS6 with a
rabbit monoclonal antibody (Cell Signaling Technology, #4856). Tubulin staining (mouse antiꢀαꢀtubulin, Sigma
#T9026) was assessed as internal standard. As readout, IRDye680ꢀconjugated goat antiꢀmouse, and IRDye800ꢀ
conjugated goat antiꢀrabbit antibodies (LICOR # 926ꢀ68070 and # 926ꢀ32211) were used, and fluorescence was
measured on an Odyssey CLx infrared imaging scanner (LICOR). Percentage of remaining phosphoꢀsubstrate
signals were calculated in relation to cellular tubulin. Further details and calculations are explained in Ref. 11.
Determination of inhibitor dissociation constants
Dissociation constants of compounds (Ki) for p110α and mTOR were determined by LanthaScreen
technology (Life Technologies) as described in detail in 11. Briefly, the AlexaFluor647ꢀlabeled Kinase Tracer314
(#PV6087) was used for p110α with a determined Kd of 2.2 nM at 20 nM, and for mTOR with a Kd of 19 nM
used at a final concentration of 10 nM. While recombinant p110α was Nꢀterminally (His)6ꢀtagged, and combined
with a biotinylated antiꢀ(His)6ꢀtag antibody (2 nM, #PV6089) and LanthaScreen EuꢀSteptavidin (2 nM,
#PV5899); truncated mTOR (amino acids 1360ꢀ2549; #PR8683B) fused to the Cꢀterminus of GST was detected
with a LanthaScreen Euꢀlabelled antiꢀGST antibody (2 nM, #PV5594). The p110α assay buffer was composed
of 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, and 0.01% (v/v) Brijꢀ35, and the mTOR assay buffer
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