4-Methylpteridinones as orally active and selective PI3K/mTOR dual inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.08.045

Pteridinones were designed based on a non-selective kinase template. Because of the uniqueness of the PI3K and mTOR binding pockets, a methyl group was introduced to C-4 position of the peteridinone core to give compounds with excellent selectivity for PI3K and mTOR. This series of compounds were further optimized to improve their potency against PI3Kα and mTOR. Finally, orally active compounds with improved solubility and robust in vivo efficacy in tumor growth inhibition were identified as well.

Full text of DOI:10.1016/j.bmcl.2010.08.045

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6096–6099
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Methylpteridinones as orally active and selective PI3K/mTOR dual inhibitors
⇑
Kevin K.-C. Liu , Shubha Bagrodia, Simon Bailey, Hengmiao Cheng, Hui Chen, Lisa Gao, Samantha Greasley,
Jacqui E. Hoffman, Qiyue Hu, Ted O. Johnson, Dan Knighton, Zhengyu Liu, Matthew A. Marx,
Mitchell D. Nambu, Sacha Ninkovic, Bernadette Pascual, Kristina Rafidi, Caroline M.-L. Rodgers,
Graham L. Smith, Shaoxian Sun, Haitao Wang, Anle Yang, Jing Yuan, Aihua Zou
Pfizer Global Research and Development, Chemistry Department, 10770 Science Center Drive, La Jolla, CA 92120, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Pteridinones were designed based on a non-selective kinase template. Because of the uniqueness of the
PI3K and mTOR binding pockets, a methyl group was introduced to C-4 position of the peteridinone core
to give compounds with excellent selectivity for PI3K and mTOR. This series of compounds were further
Received 2 June 2010
Revised 6 August 2010
Accepted 10 August 2010
Available online 14 August 2010
optimized to improve their potency against PI3K
a and mTOR. Finally, orally active compounds with
improved solubility and robust in vivo efficacy in tumor growth inhibition were identified as well.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
PI3K
mTOR
Kinase inhibitor
Cancer
The phosphatidylinositol-3-kinase (PI3K)/mammalian target of
rapamycin (mTOR) signaling pathway plays a central role in driv-
ing tumor cell proliferation, survival, angiogenesis and metastasis
by activating mutation, deletion or amplification on one or several
of its components.1 PI3Ks are lipid kinases that regulate cellular
growth and metabolism by phosphorylation of the 3-hydroxy of
phosphatidylinositol to generate phosphatidylinositol triphos-
phate (PIP3). One of the pivotal downstream effectors, Akt, is acti-
vated by PIP3 via membrane recruitment and phosphorylation at
Thr-308 by the 3-phosphoinositide-dependent protein kinase-1
(PDK-1). The PIP3 formation is also responsible for facilitating the
second phosphorylation at Ser473 of Akt by mTOR–rictor complex
ferent complexes,6 mTORC1, a rapamycin sensitive complex signal-
ing to S6K1 and 4E-BP1 and mTORC2, an rapamycin insensitive
complex signals to Akt as mentioned earlier. The existence of this
rapamycin insensitive component of the mTOR signaling pathway
thus provides new opportunities to inhibit mTOR.
A small molecule inhibitor that targets mTOR kinase should
prevent signaling through both mTORC1 and mTORC2 to have a
CH3
4
Ar
O
N
Ar
N
N
6
(mTORC2).2 Among different subtype of PI3Ks, PI3K
a is the pri-
2R1RN
N
N
R3
8
2R1RN
N
N
O
mary target for the treatment of cancers since functional loss of
PTEN (phosphatase and tensin homolog protein is encoded by
PTEN gene, the most commonly mutated tumor-suppressor gene
in cancer after p53), oncogenic mutations in the PIK3CA gene
R3
2
1
selective PI3K/mTOR inhibitors
non-selective PI3K/mTOR inhibitors
OMe
encoding PI3Ka, amplification of the PIK3CA gene and over-expres-
CH3
sion of Akt have been established in many human cancers.3
mTOR is a mammalian serine/threonine kinase and a member
of PI3K like kinase (PIKK) family of proteins. Rapamycin and its
analogs (e.g., RAD001 (everolimus), CCI-779 (temsirolimus), and
AP23573 (deforolimus)) have demonstrated anti-tumor activity
in clinical trials.4 Temsirolimus was approved in May 2007 for
the treatment of advanced kidney cancer.5 mTOR exists in two dif-
OMe
N
OMe
N
N
N
H2N
N
N
O
H2N
N
N
O
1a
1b
PI3Ka Ki = 38 nM
PI3Ka Ki = 840 nM
mTOR Ki > 4000 nM
mTOR Ki = 5 nM
Selective PI3K/mTOR inhibitor
⇑
Corresponding author.
E-mail address: kevin.k.liu@pfizer.com (K.K.-C. Liu).
Scheme 1. From non-selective to selective PI3K/mTOR inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.045

K. K.-C. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6096–6099
6097
Asp 841
H
N
CH3
N
N
N
N
N
H2N
O
Val 882
2a
PI3Kα Ki = 7 nM
mTOR Ki = 9 nM
Scheme 3. Co-crystal structure of compound 2a and PI3Kc.
we were looking for. From molecular modeling and docking stud-
ies, a small methyl group at C-4 position of compound 1 such as
compound 1a will orient the C-4 methyl group to fill this unique
PI3K/mTOR binding pocket as shown in the top graph of Scheme 2.
Compound 1a7 was made to test this model and indeed this com-
pound shows excellent selectivity against other kinases in our ki-
nase screening panels which covers more than sixty different
kinases. The compound also has good and moderate binding affin-
ity on PI3Ka and mTOR, respectively. This unique binding pocket
for PI3K/mTOR turns out to be very small as we had predicted ear-
lier since compound 1b which has 4-methoxy instead of 4-mehtyl
at C-4 position does not bind to mTOR at >4
weaker binding affinity for PI3K as well.
lM and has much
a
Scheme 2. Top: the 4-methyl analog of compounds 1, 1a, was docking to PI3K
mTOR (PI3K surface: light blue, mTOR surface: light gray); bottom: PI3K
binding site (purple color) vs protein kinases (green color: CDK2, CHK1 and PAK6K).
c
a
and
ATP
To further increase structural diversity from 1, we decided to re-
place the pyridopyrimidinone with pteridinone as the core struc-
ture (compound 2 in Scheme 1) while keeping the critical C-4
methyl group for selectivity. Based on earlier SAR learning from
compound 1,7 we jump started to make compound 2a (Scheme 3)
with the pteridinone core and hoped the SAR transfer well between
these two series. Indeed, compound 2a shows good potency on
broad and advantageous spectrum of pharmacology over mTORC1
specific rapamycin. Given the pivotal role of the PI3K
pathway in the biology of human cancers, we believe a dual
PI3K /mTOR inhibitor can provide a great understanding in cancer
treatment.
Finding leads for this PI3K/mTOR dual inhibitor project, we
a/Akt/mTOR
a
PI3Ka and mTOR with great selectivity against other kinases as
compound 1a. We then focused on this 4-methyl-pteridinone ser-
ies to improve both PI3K and mTOR potency with acceptable ADME
properties.
started with promiscuous kinase hits with a general structure as
compound 1 in Scheme 1. To improve compound selectivity
against other kinases, PI3K ATP binding site was analyzed and
compared with other protein kinase ATP binding sites. It is inter-
esting to find out that there is an additional small binding pocket
in the PI3Ks and it is not available in regular protein kinases as
shown in Scheme 2. This additional binding pocket should be able
to accommodate a larger molecule and give us the selectivity that
To improve PI3Ka
and mTOR potency, small alkyl groups at R1
and R2 are preferred. From the co-crystal structure of compound
2a and PI3Kc8 (Scheme 3, PDB: 3OAW), it is consistent with our
docking model and indicated that the C-2 amino and N-3 interact
with Val 882 in the hinge region. Additionally, the pyrazole group
at C-6 position picks up an addition interaction with Asp 841
(Scheme 3). Both Val 882 and Asp 841 are conserved in PI3K
a as
Table 1
Representative compounds 2. 2b–g on the C-6 aryl modifications
N
N
HN
O
HN
N
N
N
N
N
N
N
N
N
N
OMe
H2N
N
O
H2N
N
N
H2N
N
O
2c
2b
2d
PI3K Ki = 20 nM
mTOR Ki =6 nM
PI3K Ki = 60 nM
PI3K Ki = 82 nM
mTOR Ki =113 nM
mTOR Ki = 3940 nM
OMe
N
OMe
N
N
N
N
N
N
N
N
OMe
N
N
N
H2N
N
N
O
H2N
N
O
H2N
N
O
2g
2e
PI3K Ki = 6 nM
mTOR Ki = 30 nM
2f
PI3K Ki = 27 nM
PI3K Ki = 2 nM
mTOR Ki = 974 nM
mTOR Ki =18 nM

6098
K. K.-C. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6096–6099
Table 2
Representative compounds 2. 2h–l
N
H
2i
N
N
2h
PI3K Ki: 46 nM
mTOR Ki: 0.8 nM
S473 pAKT IC50: 68 nM
N
N
N
N
PI3K Ki: 2.75 nM
N
N
mTOR Ki: 5.11 nM
S473 pAKT IC50: 5.74 nM
HLM ER <0.3
RLM ER <0.3
DLM ER <0.3
RRCK = 21
H2N
N
O
HLM ER<0.2
RLM ER = 0.5
DLM ER = 0.2
H2N
N
O
O
O
HO
N
O
N
2j
2k
PI3K Ki: 2.8 nM
mTOR Ki: 6.8 nM
S473 pAKT IC50: 14.3 nM
PI3K Ki: 13 nM
N
N
N
N
mTOR Ki: 5 nM
S473 pAKT IC50: 25 nM
HLM ER <0.2
RLM ER = 0.3
DLM ER = 0.1
N
N
HLM ER < 0.2
DLM ER = 0.2
RRCK = 32
H2N
N
O
H2N
N
O
O
OH
2l
N
PI3K Ki: 2.8 nM
mTOR Ki: 0.85 nM
S473 pAKT IC50: 5.7 nM
HLM ER < 0.2
RRCK = 32
N
N
N
H2N
N
O
OH
Antitumor activity of Compound 2j in U87 Xenograft model
Val 851 and Asp 810. However, there is some subtle SAR on mTOR
and PI3K potency in the C-6 binding pocket. Small changes in the
C-6 aryl group will have profound effects on either PI3K or mTOR
potency such as examples shown in Table 1.
Nevertheless, all the compounds are still selective against most
of other protein kinases.
One ADME drawback in this series is solubility, and fortunately
the R3 (N-8 position) points to the solvent exposed surface area
according to co-crystal structures. Therefore, we can modify R3 to
modulate ADME properties of pteridinones to improve compounds’
solubility.
TGI on Day10
11%(P>0.05)
Vehicle
Compound 2j 3mg/kg
1500
1250
1000
750
500
250
0
Compound 2j 10mg/kg 58%(P<0.05)
Compound 2j 20mg/kg 75%(P<0.01)
After mixing and matching, compounds 2h–l were made with
solvating groups at R3 for better solubility, and with promising het-
n=9-12
9
eroaryl groups at C-6 to interact with Asp 810 residue in PI3Ka for
better potency. All the compounds 2h–l still keep their excellent
selective against other kinases as 1a. Compounds 2h–l are potent
0
1
2
3
4
5
6
7
8
10 11
Study Day (post Dosing)
binders for both PI3Ka and mTOR with Ki <15 nM, and all the com-
Effect on body weight of Compound 2j
in U87 xenograft model
pounds are potent in our cellular functional assay, phosphorylation
of S473 of AKT in BT20 cell line. In addition, these compounds have
improved solubility and excellent microsomal stability across dif-
ferent species as shown in Table 2.
Compound 2j was picked for our in vivo tumor growth inhibi-
tion (TGI) studies because of favorable human PK predictions. A
shown in Scheme 4, a nice dose–response curve was observed in
the TGI studies when 2j was orally dosed in U87 (glioma cell line)
mouse xenograft model. Additionally, there was no effect on body
weight in the same studies.
General synthesis of compound 2 is described below and as
shown on Scheme 5. Compounds of general structure 4 are pre-
pared from 2,4-dichloro-6-methyl-5-nitropyrimidine (3) with
ammonia or optionally substituted amines in THF and triethyl-
amine as the base to yield compound 4. Compound 4 without puri-
fication are then treated with ammonia or optionally substituted
amines under the same condition as the first N-alkylation with
or without triethylamine base to generate compound 5 in 40–
5
0
-5
-10
-15
-20
n=9-12
9 10 11
-25
1
2
3
4
5
6
7
8
Study Day (Post Dosing)
Scheme 4. TGI and body weight studies of compound 2j.
80% yield in two steps. The nitro group in compound 5 is reduced
with Pd/C under hydrogenation in MeOH to give compound 6 with

K. K.-C. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6096–6099
6099
CH3
CH3
CH3
NO2
NO2
Cl
NO2
R3 NH2
R1R2NH
N
N
N
2R1RN
NHR3
N
Cl
N
Cl
N
NH
R3
THF or MeOH, (TEA)
40-80%
THF, TEA
5
3
4
O
O
CH3
CH3
N
EtO
N
NH2
N
N
Pd/C, H2
H
2R1RN
O
2R1RN
NHR3
N
N
AcOH, EtOH
refluxed
R3
MeOH
70-99%
20-77%
6
7
CH3
CH3
N
YO
B
Ar
N
Ar
O
N
Br
O
N
N
NBS
YO
2R1RN
N
N
2R1RN
N
DMF
PdCl2(PPh3)2, K2CO3
DMF/H2O = 2:1
35-90%
R3
R3
55-85%
8
2
Scheme 5. Synthesis of compounds 2.
free amino group in 70–99% yield. Compound 6 are cyclized with
ethyl glyoxalate and acetic acid in refluxing ethanol to give com-
pound 7 in 20–77% yield which are subsequently treated with
NBS in DMF to yield brominated compound 8 in 55–85% yield. Fi-
nally, compound 6 are reacted with an appropriate aryl boronic
acid or ester under the Suzuki–Miyaura coupling9 condition with
catalytic amount of PdCl2 (PPh3)2 and 2 equiv of K2CO3 in the
DMF/H2O = 2:1 mixed solvent system at 100 °C to generate com-
pound 2 in 35–90% yield.
In summary, from a non-selective kinase template, 2-aminopyr-
idopyrimidinone (1), 4-methylpteridinones were designed based
on a small special pocket within PI3K and mTOR binding site to
give us a series of selective PI3K/mTOR dual inhibitors. After opti-
mization in compound potency and solubility, Compounds with
good cellular potency were found. In addition, several orally active
compounds were identified with robust efficacy in our tumor
growth inhibition models as well.
References and notes
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8. We cannot get PI3K
a co-crystal structures so we used PI3Kc enzyme instead of
PI3K enzyme for our dual inhibitor program.
a
9. A review on Suzuki–Miyaura reaction: Miyaura, N.; Suzuki, A. Chem. Rev. 1995,
95, 2457.