The discovery and optimisation of pyrido[2,3-d]pyrimidine-2,4-diamines as potent and selective inhibitors of mTOR kinase

Article abstract of DOI:10.1016/j.bmcl.2009.08.038

We describe a novel series of potent inhibitors of the kinase activity of mTOR. The compounds display good selectivity relative to other PI3K-related kinase family members and, in cellular assays, inhibit both mTORC1 and mTORC2 complexes and exhibit good antiproliferative activity.

Full text of DOI:10.1016/j.bmcl.2009.08.038

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5950–5953
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery and optimisation of pyrido[2,3-d]pyrimidine-2,4-diamines
as potent and selective inhibitors of mTOR kinase
a
a
a
a
a
a
Karine Malagu , Heather Duggan , Keith Menear , Marc Hummersone , Sylvie Gomez , Christine Bailey ,
a
a
a
a
a
Peter Edwards , Jan Drzewiecki , Frédéric Leroux , Mar Jimenez Quesada , Gesine Hermann ,
a
a
a
a
a
a
Stephanie Maine , Carrie-Anne Molyneaux , Armelle Le Gall , James Pullen , Ian Hickson , Lisa Smith ,
Sharon Maguire , Niall Martin , Graeme Smith , Martin Pass
KuDOS Pharmaceuticals Ltd, 410 Cambridge Science Park, Milton Road, Cambridge CB4 0WG, UK
AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
a
a
a,b
b,
*
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe a novel series of potent inhibitors of the kinase activity of mTOR. The compounds display
good selectivity relative to other PI3K-related kinase family members and, in cellular assays, inhibit both
mTORC1 and mTORC2 complexes and exhibit good antiproliferative activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 6 July 2009
Revised 6 August 2009
Accepted 9 August 2009
Available online 13 August 2009
Keywords:
Antitumour
mTOR
Serine/threonine kinase
The mammalian target of rapamycin (mTOR) is a key target in
the development of antitumour therapies.1 This central regulator
of cell growth and proliferation is activated by growth factor/mito-
genic stimulation of the phosphatidylinositol 3-kinase (PI3K)/Akt
signalling pathway; one of the most frequently dysregulated path-
Therefore, it is proposed that direct targeting of the kinase do-
main of mTOR would inhibit the signalling through both mTORC1
and mTORC2 and that such a compound would exhibit a different
spectrum of pharmacology compared with rapamycin. As such a
number of groups including our own have sought to identify selec-
2
11,12
ways in cancer. mTOR is a mammalian serine/threonine kinase of
tive inhibitors of mTORC1 and mTORC2.
approximately 289 kDa in size and a member of the evolutionary
conserved eukaryotic PI3K like kinase (PIKK) family of proteins,
for example, DNA-PKcs (DNA dependent protein kinase), ATM
Herein we describe the discovery and optimisation of a series of
substituted pyrido[2,3-d]pyrimidine-2,4-diamines as potent and
selective inhibitors of mTOR kinase.
Our programme to develop small molecule mTOR inhibitors be-
gan with a high throughput screen which identified the racemate
(1), which inhibited the kinase activity of mTOR with an IC50 of
(
Ataxia-telangiectasia mutated).3–5
The known mTOR inhibitor rapamycin and its analogues
RAD001, CCI-779, AP23573) bind to the FKBP12/rapamycin com-
(
plex binding domain (FRB), resulting in suppression of signalling
1.0 lM in an ELISA based assay using immunoprecipitated full-
length mTOR with a 4E-BP1 substrate.
to the downstream targets p70S6K and 4E-BP1 (Fig. 1).6
,7
13
The potent but non-specific inhibitors of PI3K, LY294002 and
wortmannin, have also been shown to inhibit the kinase function
of mTOR but in this case by targeting the catalytic domain of the
O
N
N
8
protein.
Recently, it has been shown that mTOR exists in two complexes,
mTORC1, a rapamycin sensitive complex signalling to p70S6K and
N
N
N
4
E-BP1 and mTORC2 a rapamycin insensitive complex that signals
9
to Akt, which provides an explanation for the differences observed
in the earlier work of Brunn et al. and Edinger et al.
(
1)
8
10
Our initial exploration focused on the role of the pyridine nitrogen
of the bicyclic core. Synthesis of two structural isomers and the ben-
zenoid analogue of (1) was undertaken.
*
Corresponding author.
E-mail address: martin.pass@astrazeneca.com (M. Pass).
1
4
0
960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.038

K. Malagu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5950–5953
5951
Table 1
Growth factor/mitogen
Testing the importance of the pyridine nitrogen
O
N
PI3K IRS-1
PIP2 PIP3
N
A
PDK1
N
N
PTEN
Thr308
Compound
A ring
mTOR inhibition
Ser473 Akt
IC50
, lM
1
2
3
4
1.0
TSC2
TSC1
Rapamycin/FKBP12
mTORC1
N
N
<30% at 10
<30% at 10
<30% at 10
l
l
l
M
N
mTORC2
GTP
Rictor mTOR
Rheb
mTOR Raptor
M
M
4
E-BP1
p70S6K
S6
elF4E
directly in a key hydrogen-bonding interaction in the ATP-binding
1
8
site of PI3K
c
.
By analogy we reasoned that the C4 morpholine
Translation
Figure 1. PI3K-Akt-mTOR pathway.
ether oxygen of our mTOR inhibitors may be involved in a hydro-
gen bond interaction with residue Val 133 in the hinge region of
the mTOR kinase domain. In order to test this argument we com-
bined a preferred C2 substituent with a small number of changes
to the C4 amino substituent.
Replacement of the ether oxygen by a carbon, compound (16),
gave a significant reduction in activity. The slight change of geom-
etry and increase in bulk associated with the change from morpho-
line to homomorpholine, as in compound (17), did not result in a
complete loss of activity but did cause a significant reduction in
potency. Both findings are consistent with our view of the involve-
ment of the C4 morpholine oxygen in a key hydrogen binding
interaction.
The results, summarized in Table 1, demonstrate that the opti-
mal placement of the pyridine nitrogen is at the 8-position with
the other two isomers and the benzenoid analogue possessing
much reduced mTOR inhibitory activity.
At this point we decided to determine the preferred absolute
stereochemistry of the methyl substituent on the piperidine and
investigate the influence of C-2 and C-4 substituents.
Compounds exemplified in Table 2 were made according to the
method shown in Scheme 1.
Docking studies using an homology model of mTOR built from
The nicotinic acid (5) was subject to a fusion reaction with
potassium cyanate and ammonium chloride to give the dione (6).
This was chlorinated in the presence of phosphorus oxychloride
and diisopropylethylamine to give the dichloro species (7). The
cyclic amine, for example, morpholine, was selectively introduced
to the C4 position (8) allowing the final chlorine displacement with
selected amines to give compounds of the general structure (9).
The S-enantiomer (10) of compound (1) showed marginally im-
proved potency relative to the R-enantiomer (11) and the race-
mate, and gave us our first sub-micromolar mTOR inhibitor.
Moving the methyl group to the 3-position of the piperidine, com-
pound (12), results in a modest loss of activity. In general substitu-
tion on the piperidine was quite well tolerated (data not shown)
without significant loss of potency. Of particular interest were
the analogues (14) and (15) where the piperidine has been re-
placed by morpholine, as they offered the possibility of reducing
overall lipophilicity of the molecule. For example, Clog P com-
an in house crystal structure of PI3Kc suggested that substitution
of the pyridopyrimidine C7 position could access additional bind-
ing pockets within the ATP binding site. For these investigations
we elected to use the dimethyl-substituted morpholine substituent
at C2 as this gave a good balance of potency and lipophilicity.
Accordingly, the 7-chloro-substituted intermediate (18) was pre-
pared by an analogous route to that described in Scheme 1.
O
N
N
Cl
N
N
N
O
(18)
15
pound (11) is 2.39 and 1.00 for compound (14). The importance
of the methyl substituent is evidenced by the much-reduced activ-
ity of unsubstituted analogue (13).
This compound was then subject to either displacement by nitrogen
1
9
20
nucleophiles or Buchwald or Suzuki coupling reactions to intro-
duce aromatic substituents. The results for key analogues are sum-
marized in Table 3.
We next embarked on an exercise to characterize the require-
ments of the C4 substituent. The morpholine is a well precedented
1
6,17
structural motif in inhibitors of PI3K and PI3K-like kinases
and,
The introduction of aromatic amino substituents, as in com-
pounds (19) and (20), resulted in potency improvements as did
the introduction of directly bonded aromatic and heteroaromatic
at least for the case of the prototypical PI3K inhibitor LY294002,
the morpholine ether oxygen has been shown to participate

5
952
K. Malagu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5950–5953
Table 2
Table 3
Modifications to C2 and C4 substituent
Structure–activity of 7-substituted analogues
4
R
O
N
N
2
R
N
N
N
Compound
R2
R4
mTOR inhibition
IC50
7
R
N
N
N
, lM
O
N
N
N
N
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
4-Morpholino
4-Morpholino
4-Morpholino
4-Morpholino
4-Morpholino
4-Morpholino
0.69
1.2
Compound
R7
mTOR inhibition
IC50
, lM
1
9
0
0.12
N
H
3.0
N
2
0.087
0.12
N
H
<30% at 10
1.8
lM
O
21
O
S
N
N
O
O
2
2
2
2
3
4
0.033
0.22
1.3
N
N
N
1-Piperidino
<30% at 10
4.8
lM
0.062
0.045
0.039
O
N
25
O
2
2
2
6
7
8
substituents, as in compounds (21), (22), (23) and (24). An investi-
gation of substituted analogues of the phenyl compound (24) re-
vealed that electron-donating substituents in the para position,
as in compounds (25) and (26), were well tolerated. However, a
different picture was apparent for meta substituents. Here elec-
tron-donating character did not seem to be beneficial. Rather the
presence of a hydrogen bond donor either as a phenol compound
HO
O
0.49
(
28) or as a primary alcohol as in compound (29) was preferable.
HO
0.053
0.093
0.037
Cl
N
O
O
HO
HO
+
29
30
(
a)
(b)
N
OH
NH2
N K
O
N
Cl
N
N
N
H
(
5)
(6)
(7)
O
N
O
N
HO
OH
(
d)
(
c)
N
N
31
0.016
1
2
N
N
Cl
N
N
NR R
O
(
8)
(9)
Scheme 1. Reagents and conditions: (a) KCNO, NH
Hünig’s base, toluene, 100 °C, 3 h, 20%; (c) morpholine, Hünig’s base, CH
d) selected amine, DMA, 80 °C.
4
Cl, 200 °C, 76%; (b) POCl
3
,
Combining the electron donor at the para position with the hydro-
gen bond donor at the meta position resulted in the still more po-
2
Cl , rt, 40%;
2
(

K. Malagu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5950–5953
5953
Table 4
PI3K-Akt-mTOR pathway in disease and assess their potential as
therapeutic agents.
Activity of compound (31), KU-63794 against PI3K family enzymes
Enzyme
IC50, lM
References and notes
mTOR (4E-BP1 substrate)
0.016
0.003
8.9
>30
>30
>5.3
>10
>10
a
mTOR (p70S6K peptide substrate)
b
1. Huang, S.; Houghton, P. J. Curr. Opin. Pharmacol. 2003, 3, 371.
PI3K
PI3Kb
PI3K
a
2.
3.
4.
Yap, T. A.; Garrett, M. D.; Walton, M. I.; Raynaud, F.; de Bono, J. S.; Workman, P.
Curr. Opin. Pharmacol. 2008, 8, 393.
Brown, E. J.; Albers, M. W.; Shin, T. B.; Ichikawa, K.; Keith, C. T.; Lane, W. S.;
Schreiber, S. L. Nature 1994, 369, 756.
c
PI3Kd
ATR
DNA-PK
Sabatini, D. M.; Erdjument-Bromage, H.; Lui, M.; Tempst, P.; Snyder, S. H. Cell
1994, 78, 35.
a
5. Abraham, R. T. Curr. Opin. Immunol. 1996, 8, 412.
Inhibition of mTOR mediated phosphorylation was determined using truncated
6
7
.
.
Fingar, D. C.; Salama, S.; Tsou, C.; Harlow, E.; Blenis, J. Genes Dev. 2002, 16, 1472.
Fingar, D. C.; Richardson, C. J.; Tee, A. R.; Cheatham, L.; Tsou, C.; Blenis, J. Mol.
Cell Biol. 2004, 24, 200.
mTOR expressed in HEK293 and a biotinylated p70S6K peptide substrate. Ref. 21.
b
PI3K isoform inhibition was measured using PIP2 phosphorylation in an
alphascreen competition assay (biotinylated PIP3). Ref. 22.
8.
Brunn, G. J.; Williams, J.; Sabers, C.; Wiederrecht, G.; Lawrence, J. C., Jr.;
Abraham, R. T. EMBO J. 1996, 15, 5256.
9.
Sarbassov, D. D.; Ali, S. M.; Sabatini, D. M. Curr. Opin. Cell Biol. 2005, 17, 596.
1
0. Edinger, A. L.; Linardic, C. M.; Chiang, G. G.; Thompson, C. B.; Abraham, R. T.
tent analogue (31). These subtle structure–activity relationships at
the C7 substituent were not readily explained by the homology
model.
Further characterization of compound (31), KU-63794, suggests
a good degree of selectivity for mTOR relative to other PIKK family
members has been achieved (Table 4). In addition compound (31)
showed no significant activity when tested against a range of 200
Cancer Res. 2003, 63, 8451.
11. Thoreen, C. C.; Kang, S. A.; Chang, J. W.; Liu, Q.; Zhang, J.; Gao, Y.; Reichling, L. J.;
Sim, T.; Sabatini, D. M.; Gray, N. S. J. Biol. Chem. 2009, 284, 8023.
2. Feldman, M. E.; Apsel, B.; Uotila, A.; Loewith, R.; Knight, Z.; Ruggero, D.; Shokat,
K. M. PLoS Biol. 2009, 7, 371.
3. Inhibition of mTOR kinase was assayed using full-length mTOR (extracted from
HeLa cytoplasmic extract by immunoprecipitation) with a recombinant 4E-BP1
1
1
protein substrate and an ATP concentration of 50
lM. Detection of
phosphorylated product through an ELISA format was essentially as
described in Ref. 8. Figures quoted are the mean of at least 3 determinations
and variability around the mean was <50%.
non-PI3K related kinases at a concentration of 10 lM.
To determine if the compounds inhibited both mTORC1 and
mTORC2 complexes in cellular environment U87MG glioblastoma
cells were treated with compound (31) for 2 h and phosphorylation
of the indirect mTORC1 downstream effector S6 ribosomal protein
1
4. Compounds (2), (3) and (4) were prepared according to the same methods
described in Scheme 1 from 3-amino-isonicotinic acid, 3-aminopicolinic acid
and anthranilic acid, respectively.
15. BioByte www.biobyte.com CLog P Version 4.3.
16. Hickson, I.; Zhao, Y.; Richardson, C. J.; Green, S. J.; Martin, N. M.; Orr, A. I.;
(
Ser235/236) and the direct mTORC2 substrate Akt (Ser473) were
measured by Western blot. Both endpoints were inhibited with
IC50s of 0.10 and 0.15 M, respectively. Moreover inhibition of pro-
Reaper, P. M.; Jackson, S. P.; Curtin, N. J.; Smith, G. C. Cancer Res. 2004, 64,
9152.
l
17. Griffin, R. J.; Fontana, G.; Golding, B. T.; Guiard, S.; Hardcastle, I. R.; Leahy, J. J. J.
Med. Chem. 2005, 48, 569.
liferation of a range of cell types was demonstrated. For example,
the T47D breast cancer cell line was inhibited with a GI50 of
18. Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann,
M. P.; Williams, R. L. Mol Cell 2000, 6, 909.
0
.35
l
M using a sulforhodamine B endpoint.
19. Typical Buchwald conditions: (18), relevant amine, tris(dibenzylideneacetone)
dipalladium(0), Xantphos, cesium carbonate, dioxane, 150 °C, 40 min.
In conclusion we have discovered a series of highly selective,
2
2
2
0. Typical Suzuki conditions: (18), relevant boronate ester, tetrakis(PPh
3 4
) Pd,
sub-micromolar potency mTOR kinase inhibitors. The activity of
these inhibitors has been confirmed in a cellular environment
where they inhibit both mTOR complexes. Thus the compounds
warrant further characterization to understand the role of the
K
2
CO , CH CN, H O, 95 °C, 2 h.
3
3
2
1. Hummersone, M. G.; Gomez, S.; Menear, K. A.; Smith, G. C. M.; Malagu, K.;
Duggan, H. M. E.; Cockcroft, X.-L. F.; Hermann, G. J. WO2007060404, 2007.
2. Bailey, J. P.; Giles, M. B.; Pass, M. WO2006005914, 2006.