Discovery and biological profiling of potent and selective mTOR inhibitor GDC-0349

Article abstract of DOI:10.1021/ml3003132

Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.

Full text of DOI:10.1021/ml3003132

Letter
pubs.acs.org/acsmedchemlett
Discovery and Biological Profiling of Potent and Selective mTOR
Inhibitor GDC-0349
Zhonghua Pei,*,† Elizabeth Blackwood,‡ Lichuan Liu,§ Shiva Malek,∥ Marcia Belvin,‡
Michael F. T. Koehler,† Daniel F. Ortwine,† Huifen Chen,† Frederick Cohen,† Jane R. Kenny,§
Philippe Bergeron,† Kevin Lau,† Cuong Ly,† Xianrui Zhao,† Anthony A. Estrada,† Tom Truong,‡
Jennifer A. Epler,‡ Jim Nonomiya,∥ Lan Trinh,∥ Steve Sideris,∥ John Lesnick,∥ Linda Bao,⊥
Ulka Vijapurkar,‡ Sophie Mukadam,§ Suzanne Tay,§ Gauri Deshmukh,§ Yung-Hsiang Chen,§ Xiao Ding,§
Lori S. Friedman,‡ and Joseph P. Lyssikatos*,†
‡
§
∥
†Departments of Discovery Chemistry, Translational Oncology, DMPK, Biochemical and Cellular Pharmacology, and
⊥Pharmaceutics, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been
observed in human tumors and tumor cell lines, indicating that these protein kinases may be
attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated
in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective
ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and
dose-dependent efficacy in mouse xenograft cancer models.
KEYWORDS: Mammalian target of rapamycin, mTOR, TDI, urea bioisostere
Scheme 1
he mammalian target of rapamycin (mTOR) is an
Timportant component of the PI3K-Akt signaling pathway
that regulates cell growth and proliferation.1 Since aberrant
activation of this pathway is found in many types of cancer,
inhibition of PI3K, Akt, and mTOR has become an attractive
strategy for treating cancer.2,3 mTOR is a serine/threonine
kinase belonging to the phosphoinositide kinase-related kinase
(PIKK) family. A mTORC1 complex is formed when mTOR
associates with RAPTOR and mLST8. A mTORC2 complex is
formed by mTOR associating with RICTOR, mLST8, PPR5,
and SIN1. The natural product rapamycin and its analogs
(rapalogs) only inhibit the mTORC1 complex and are
efficacious in reducing tumor cell growth, leading to two
rapalogs being approved for oncology indications.4 However,
the mTORC1 complex negatively affects upstream insulin
signaling through IRS1/2 via activation of the phosphorylated
substrate S6K.5 This negative feedback loop can activate a
growth factor signaling cascade that includes mTORC2 and
may limit the efficacy of rapalogs. Small molecule mTOR kinase
inhibitors that inhibit both mTORC1 and mTORC2 may be
more efficacious than the rapalogs.6−10
(THQ) class of inhibitors required for potency: (a) the
morpholine oxygen atom binds to the hinge of the mTOR
kinase domain; (b) the two N−H and carbonyl groups of the
urea moiety hydrogen-bond with both Asp2195 and Lys2187,
respectively; (c) the ethyl group of the urea imparts selectivity
over PI3K; and (d) the (S)-methyl on the morpholine
enhances the selectivity of 1b by occupying a hydrophobic
cavity formed by Trp2239 that is absent in PI3K (Supporting
Information Figure S1). However, compound 1 exhibited
potent time-dependent inhibition (TDI) of cytochrome P450
(CYPs), especially CYP3A4, and it possessed relatively high
free plasma clearance in animals. As TDI can lead to drug−drug
We recently reported the discovery of potent, selective, ATP-
competitive mTOR inhibitors exemplified by compound 1
(Scheme 1).11,12 Examination of the structure−activity relation-
ship (SAR) and docking into a homology model of mTOR
revealed key structural features of this tetrahydroquinazoline
Received: October 4, 2012
Accepted: November 29, 2012
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
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interactions and potential toxicity,13,14 we decided to identify
compounds that are devoid of TDI. Here we report our efforts
to improve the above properties while maintaining/improving
the potency, leading to the discovery of a drug development
candidate.
vitro liver microsome metID study (data not shown).
Introduction of an electron-withdrawing fluorine atom at the
end of the ethyl group led to compound 5a, which retained
potency and selectivity over PI3K-α (Table 1). A pyrimidine
a
Synthesis of the 7-aza tetrahydroquinazolines (7-aza-THQ)
with diversified R2 groups is outlined in Scheme 2. Treatment
Table 1. SAR of the Right-Hand Moiety of 7-aza-THQ
a
Scheme 2
a
Reagents and conditions: For 5a, phosgene, TEA, then 2-fluoroethyl-
amine; For 5b: cat. Pd(OAc)2, 2-chloropyrimidine, Cs2CO3, dioxane,
160 °C, microwave. For synthesis of 5c- 5f, see supporting material.
of aniline 4 with phosgene followed by 2-fluoroethylamine gave
5a. Palladium-mediated cross-coupling with 2-chloropyrimidine
gave 5b. Synthesis of the 7-aza-THQ with diversified R3 groups
is outlined in Scheme 3. After the 4-chlorine atom in dichloride
a
Scheme 3
a
Reagents and conditions: (a) morpholine or (S)-3-morpholine,
DIPEA, DMF; (b) 4-(EtNHCONH)Ph boronic ester, Pd(PPh3)4,
base, 120 °C; (c) TFA, DCM; (d) when R3 = heteroaromatic: R3Cl,
DMF, 80-115 °C; (e) when R3 = alkyl: alkyl ketone, then
NaBH(OAc)3, 1,2-dichloroethane.
a
b
Potencies in nanomolar. Ratio of Ki values of PI3Kα over mTOR.
c
ND: not determined.
group replacement led to significantly reduced mTOR potency
(5b), presumably due to the loss of the hydrogen-bond
donating group of the urea NH. Triazolone 5c, designed to
mimic the urea group, retained substantial mTOR potency and
selectivity over PI3K-α compared to 1b. Introduction of a
squaramide led to compound 5d with ∼9-fold potency
reduction. Pyridone 5e has a Ki of 6.6 nM, but the
antiproliferative activity is reduced, likely caused by higher
plasma protein binding. Pyrimidone 5f exhibited a Ki of 0.4 nM
and 630-fold selectivity over PI3K-α. It also demonstrated high
antiproliferative potency (25 nM) in PC3 cells. However, none
of the compounds in Table 1 is superior to 1b in terms of
overall profile (e.g., 5f displayed high in vivo clearance of 78
mL/(min·kg) in mouse and no oral bioavailability) and,
therefore, was not profiled further.
6 was selectively displaced with a morpholine or (S)-3-
methylmorpholine, the 2-chlorine atom participated in Suzuki
coupling with a boronic ester to give biphenyl 7. The Boc
protecting group was removed and the R3 group was
introduced via either a nucleophilic replacement or a reductive
amination to afford final compounds 8. Final compounds were
tested in both mTOR and PI3K-α enzymatic assays, and
inhibitory potency is reported as Ki values.11,12 Anti-
proliferative activities (reported as EC50 values) were measured
in a PC3 prostate cancer cell line, where the PI3K-AKt-mTOR
pathway is activated via the loss of the tumor suppressor PTEN.
Our first effort was to modify or replace the ethyl urea group
with the hope to improve the metabolic stability, as deethylated
product of 1a was observed as one of the metabolites in the in
B
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a
Although we were unable to experimentally identify the
reactive metabolites, we hypothesized that one of the
possibilities is the formation of diaza quinone iminium15 M1
via CYP-mediated oxidation of the aminopyrimidine moiety,
which would lead to TDI (Scheme 4).
Table 2. SAR of the N-Substitution of 7-aza-THQ
Scheme 4
This led our attention to modifying the left-hand side of the
molecule. Analogs with a fluorine atom at the C5 position of
pyrimidine (8a), regioisomers of pyrimidine (8b and 8c), or an
imidazole ring (8d) were designed to block the formation of
the diaza quinone iminium species (Table 2). These four
analogs maintained potency and selectivity. Replacing the
pyrimidine ring by an isopropyl group (8e) or a cyclopentyl
group (8f) decreased potency relative to 1b. The nitrogen atom
with alkyl groups attached is quite basic, which could bring
potential liabilities, such as blockade of the hERG channel.16
Indeed, compound 8e, with a pKa of 7.6, has an hERG IC50 of
8.5 μM (patch clamp assay). Compounds 8g and 8h, with
reduced basicity of the nitrogen atom, maintained most of the
potency and selectivity of 1b. Introduction of an oxetane group
reduced the basicity of 8h (pKa = 5.0), which translated into
diminished hERG liability (IC50 > 100 μM).
Compounds 8a−h were tested in a TDI shift assay in human
liver microsomes in the presence of cofactor NADPH with
midazolam as a CYP3A4 substrate.17 When a reactive
metabolite is formed and reacts with CYP during a 30-min
preincubation with the compound, a left shift of the CYP
activity curve is observed compared with no pre-incubation and
the compound is designated as TDI positive. TDI IC50 is
calculated from this shifted curve. When there is no shift with
30-min preincubation, the compound is designated as TDI
negative. Of the three compounds with modified pyrimidines in
Table 2, two (8a and 8b) had a similar level of TDI compared
to 1b and one compound (8c) had significantly less TDI
(Table 2). Compounds with alkyl groups (8e and 8h) or a
sulfonamide (8g) had decreased TDI potency compared to 1b,
consistent with the hypothesis that minimizing the formation of
diaza quinone iminium could decrease TDI.
Compound 8h is a remarkably selective mTOR inhibitor,
with less than 25% inhibition of 266 kinases, including all
isoforms of PI3K when tested at Invitrogen at 1 μM
(Supporting Information Table S1). Compound 8h has ∼10-
fold reduced free plasma clearance in both mice (100 mL/min/
kg) and rats (171 mL/min/kg in rat) compared to 1b (1818
mL/min/kg in mice, 1538 mL/min/kg in rats).
a
b
Potency and EC50 in nM. Ratio of Ki values of PI3Kα over mTOR.
c
d
IC50 value (in μM) is with imidazolam as the probe. ND: not
determined.
(Figure 1). Body weight change was less than 10% up to the
highest dose. Compound 8h was also efficacious in other
xenograft models, including PC3 (PTEN null) and 786-0 (VHL
mutant) (Supporting Information Figure S3). Similar levels of
tumor growth inhibition were achieved when 8h was
administered once every three days at higher doses compared
to once every day (Supporting Information Figure S4). While
the single agent mTOR inhibitor (GDC-0349), PI3K inhibitor
(GDC-0941),18 or MEK (GDC-0973)19 inhibitor showed only
modest tumor growth delay in the A549 mouse xenograft
model (KRas mutant, LKB1 deficient), combination of mTOR
and MEK inhibitors achieved the same level of tumor stasis as
the combination of PI3K and MEK inhibitors (Figure 2). All
the combinations were well tolerated and caused no significant
change in body weight.
Compound 8h inhibited downstream markers of mTOR,
including phospho-4EBP1 and phospho-Akt(s473) in an in
vivo PK/PD study in mouse (Supporting Information Figure
S2), consistent with an inhibition of both mTORC1 and
mTORC2 complexes.
When dosed orally once daily in athymic mice in a MCF7-
neo/Her2 tumor xenograft model (PI3K mutation), compound
8h inhibited tumor growth in a dose-dependent manner,
achieving stasis (99% TGI) at the maximum tolerated dose
In summary, we report here further optimization of our
advanced lead compound 1. Urea bioisosteric replacements
have been identified that possess equal or better mTOR
potency, as exemplified by 5e and 5f. SAR efforts culminated in
the discovery of selective and potent mTOR kinase inhibitors,
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ACKNOWLEDGMENTS
■
We acknowledge the analytical group for purification and
characterization support and Wuxi chemists for synthesizing
some of the analogs.
ABBREVIATIONS
■
mTOR, mammalian target of rapamycin; PI3K, phosphatidy-
linositol-3-kinase; TDI, time-dependent inhibition; THQ,
tetrahydroquinazoline
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and characterization data, kinase
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additional in vivo efficacy studies. This material is available free
of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Z.P.: phone, 650-467-1542; e-mail, pei.zhonghua@gene.com.
J.P.L.: phone, 650-467-6671; e-mail, lyssikatos.joseph@gene.
com.
■
Notes
The authors declare no competing financial interest.
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