Identification of GNE-477, a potent and efficacious dual PI3K/mTOR inhibitor

Article abstract of DOI:10.1016/j.bmcl.2010.03.046

Efforts to identify potent small molecule inhibitors of PI3 kinase and mTOR led to the discovery of the exceptionally potent 6-aryl morpholino thienopyrimidine 6. In an effort to reduce the melting point in analogs of 6, the thienopyrimidine was modified by the addition of a methyl group to disrupt planarity. This modification resulted in a general improvement in in vivo clearance. This discovery led to the identification of GNE-477 (8), a potent and efficacious dual PI3K/mTOR inhibitor.

Full text of DOI:10.1016/j.bmcl.2010.03.046

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2408–2411
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Bioorganic & Medicinal Chemistry Letters
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Identification of GNE-477, a potent and efficacious dual PI3K/mTOR inhibitor
b
a
c
d
Timothy P. Heffron a, , Megan Berry , Georgette Castanedo , Christine Chang , Irina Chuckowree ,
Jennafer Dotson a, Adrian Folkes d, Janet Gunzner a, John D. Lesnick c, Cristina Lewis c, Simon Mathieu a,
Jim Nonomiya c, Alan Olivero a, Jodie Pang e, David Peterson c, Laurent Salphati e, Deepak Sampath b,
Steve Sideris c, Daniel P. Sutherlin a, Vickie Tsui a, Nan Chi Wan d, Shumei Wang a, Susan Wong e,
Bing-yan Zhu a
*
a Discovery Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA
b Small Molecule Translational Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA
c Biochemical Pharmacology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA
d Piramed Pharma, 957 Buckingham Avenue, Slough, Berkshire SL1 4NL, UK
e Drug Metabolism and Pharmacokinetics,, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Efforts to identify potent small molecule inhibitors of PI3 kinase and mTOR led to the discovery of the
exceptionally potent 6-aryl morpholino thienopyrimidine 6. In an effort to reduce the melting point in
analogs of 6, the thienopyrimidine was modified by the addition of a methyl group to disrupt planarity.
This modification resulted in a general improvement in in vivo clearance. This discovery led to the iden-
tification of GNE-477 (8), a potent and efficacious dual PI3K/mTOR inhibitor.
Received 2 February 2010
Revised 8 March 2010
Accepted 9 March 2010
Available online 12 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
PI3K
mTOR
Kinase inhibitors
Owing to its common association with oncogenic malignancies,
the PI3K/AKT/mTOR signaling pathway is an attractive area of re-
search for the identification of oral small molecule inhibitors.1
We have previously reported the discovery of the PI3K inhibitor
GDC-0941 and have continued our efforts to identify additional
molecules that inhibit PI3K or both PI3K and mTOR.2
proliferation assay using MCF7.1 cells. In fact, compound 2, which
lacks any substitution at the 6-position, still maintains reasonable
potency, demonstrating that the potency is largely driven by the
interactions of the morpholine and aminopyrimidine with the en-
zyme.5 Compounds 3–5 are of comparable potency in each assay,
demonstrating that a wide range of substituents is tolerated at
the 6-position of the thiophene.
During our investigations, using GDC-0941 as a starting point,
we discovered that the inhibition of PI3K-a was maintained
when the indazole moiety was replaced by 2-aminopyrimidine
(1, Table 1). Furthermore, compound 1 was found to be a potent
inhibitor of mTOR kinase activity and had improved potency in
the MCF7.1 cell proliferation assay.3 This replacement group
proved to be an entry into the fruitful identification of many attrac-
tive dual PI3K/mTOR inhibitors.4
By holding constant the morpholino thienopyrimidine core
along with the 2-aminopyrimidine, we were able to consistently
generate molecules with attractive inhibitory activities against
Table 1
Comparison of relevant potency data for GDC-0941 and 1
O
N
O
N
S
S
N
N
NH
N
N
N
N
N
N
N
NH2
N
O
N
O
PI3K-a and mTOR. A representative set of compounds from our
Me
S
O
Me
S
O
GDC-0941
1
SAR development at the 6-position of the thienopyrimidine core
is presented in Table 2. Compound 1 has excellent potency against
both PI3K-
a
and mTOR and exhibits good cell-based activity in a
Compd
PI3K-
(nM)
a
IC50
mTOR Kiapp
(nM)
MCF7.1 Proliferation
EC50 (nM)
GDC-0941
1
3
2
570
29
720
130
* Corresponding author. Tel.: +1 650 467 3214; fax: +1 650 225 2061.
E-mail address: theffron@gene.com (T.P. Heffron).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.046

T. P. Heffron et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2408–2411
2409
Table 2
Table 3
SAR of 6-position substitution on the thienopyrimidine core
Rat pharmacokinetic data and key physicochemical properties for 6 and 7
O
O
N
N
S
S
6
N
N
6
R
N
N
N
N
7
7
MeO2S
R
N
NH2
N
NH2
Compd
R
P13K-
a
mTOR
MCF7.1
Thermodynamic
Compd R1
Rat LM, Clhep
(mL/min/kg)
Rat Cl
(mL/min/
kg)
F
TD Sol, pH
Melting
pointa
(°C)
IC50 (nM) Kiapp (nM) proliferation solubility, pH
(%) 6.5 ( g/mL)
l
EC50 (nM)
6.5 (lg/mL)
6b
7c
H
7
56
13
3
4
1
3
276
234
N
Me 12
1
2
29
0.13
62
a
MeO2SN
MeO2S N
Melting points obtained using crystalline material using the same lot used for
rat PK studies.
Male rats were dosed with the TFA salt as a solution intraveinously (1 mg/kg) in
7% DMSO/5% cremophor and dosed orally as a solution in 5% DMSO/76% PEG (5 mg/
kg).
2
3
5
1
42
10
0.43
0.17
170
15
b
Me
c
Male rats were dosed with the HCl salt as a solution intraveinously (1 mg/kg) in
10% DMSO/60% PEG and dosed orally as a solution in 10% DMSO/80% PEG (5 mg/kg).
O
4
5
N
5
7
50
59
0.19
0.29
770
Me
Me
1100
H2N
with reduced clearance, would have limited oral bioavailability
due to solubility-limited absorption.
6
<1
5
0.04
1
The aryl group at the 6-position resulted in superior potency
and we sought to improve solubility while maintaining this motif.
In doing so, we sought to improve oral bioavailability of further
analogs of compound 6 by increased rate of dissolution. The ther-
modynamic solubility of small molecules is often inversely propor-
tional to the compound melting point.6 We believed that the high
melting point of 6 was due to efficient crystal packing of the highly
planar molecule and so developed a strategy to disrupt the co-pla-
narity of the aryl group and the thienopyrimidine core.7 To this
end, we modified the core by introducing a methyl group at the
7-position (Table 3). Quantum mechanics calculations predicted
that the methylated thiophene (as in 7) preferred to be 45° out
of plane with the aryl sulfone, with an energy difference of
2.14 kcal/mol relative to the planar conformation.8
MeO2S
While the first five examples in Table 2 each have PI3K and
mTOR potencies similar to each other (1–7 nM, and 29–59 nM,
respectively) resulting in similar cell proliferation activities, an
exception in this series was the aryl analog 6 which was signifi-
cantly more potent in each of the biochemical and cell-based
assays.
Given the superior potency of 6 in both the biochemical and
cell-based assays we evaluated the pharmacokinetic profile of this
compound. Despite moderate stability in liver microsomes, com-
pound 6 had high clearance in vivo as well as low oral bioavailabil-
ity (Table 3). While the low oral bioavailability could be attributed
to first-pass metabolism, because the thermodynamic solubility of
6 was so low we remained concerned that related analogs, even
Compound 7, the direct analog of 6, was first prepared to assess
the promise of this approach. The melting point of 7 was reduced by
more than 40 °C relative to compound 6 resulting in a modest
Table 4
Comparison of analogs with and without methyl at R2
O
N
N
S
N
R1
N
R2
N
NH2
Compd
R1
R2
P13K-
(nM)
a
IC50
mTOR Kiapp
(nM)
MCF7.1 proliferation
EC50 (nM)
Rat LM Clhep
(mL/min/kg)
Rat Cl
(mL/min/kg)
Rat PPB
(%)
Rat Clu
(mL/min/kg)
1
8
H
Me
2
4
29
21
130
143
16
15
24
13
52
75
50
52
N
MeO2SN
O
N
4
9
H
Me
5
3
50
30
187
164
2
20
50
21
47
51
94
43
Me
Me
6
7
H
Me
<1
<1
5
4
33
68
7
12
56
13
93
97
800
433
MeO2S
10
11
H
Me
1
2
10
4
78
65
20
38
52
15
ND
ND
ND
ND
N
MeN
O

2410
T. P. Heffron et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2408–2411
solubility improvement. This reduction in melting point, however,
did not result in an improvement in oral bioavailability. The poor
oral bioavailability observed for 7 suggested that solubility was still
insufficient despite the reduction in melting point. Furthermore,
in vivo clearance and passive cell permeability were favorable
and hence did not appear to be compromising factors (Table 3).9
While the introduction of the methyl group to the 7-position
of the thiophene core did not lead to the desired improvement
in oral bioavailability, we observed that 7 had reduced rat clear-
ance in vivo relative to the des-methyl analog 6. This reduction
in clearance occurred despite in vitro studies suggesting that
the des-methyl analog was of comparable metabolic stability (Ta-
ble 3). In addition, it did not appear that methyl substitution was
blocking a site of metabolism as numerous in vitro metabolite
identification studies did not indicate significant metabolic activa-
tion of the thiophene ring. Encouraged that the remarkable
potency of 6 was unchanged on the modified core of 7, we ex-
tended our studies to determine whether the desirable impact
on clearance with maintained potency was a general trend. To
this end, we synthesized several pairs of analogs both with and
without the methyl group at the 7-position of the thienopyrimi-
dine core. A representative summary of these efforts is contained
in Table 4.
Each of the 6-aryl compounds was also synthesized by a general
route shown in Scheme 1. From the common intermediate 13, the
6-iodo compound 16 was prepared by lithiation and iodine
quench. Iterative regioselective Suzuki couplings were then used
Table 5
Comparison of in vivo pharmacokinetics for GNE-477 (8) and 1
O
N
O
N
S
S
N
N
N
N
N
N
N
N
Me
N
NH2
N
O
N
NH2
N
O
Me
S
O
Me
S
O
1
GNE-477 (8)
Compd
Species
Mousea
Cl (mL/min/kg)
F (%)
1
49
15
24
13
22
5
81
98
75
52
35
90
GNE-477
1
GNE-477
1
Ratb
Dogc
GNE-477
In each pair, the potency of the molecules does remain compa-
rable in each assay. Also in each case, the tetrasubstituted thieno-
pyrimidine core has reduced in vivo clearance in rats relative to the
trisubstituted analog despite being consistently more labile in rat
liver microsomes. The general improvement in in vivo clearance
of the tetrasubstituted core is likely a combination of multiple con-
tributing factors. One significant contribution to the reduced clear-
ance is the increase in plasma protein binding observed for the
analogs on the core with the methyl group (Table 4). Nevertheless,
it does not account for the magnitude of the difference in each case
as in two pairs the modified core has reduced unbound clearance
(4/9, and 6/7). Evidently, the additional thiophene substitution re-
duces metabolism of the molecule not represented in liver micro-
somes. The other factors contributing to reduced clearance are not
well understood. Regardless, the improvement in in vivo clearance
of this core was general and while 7 exhibited poor oral bioavail-
ability, other analogs with improved physicochemical properties
were identified. These maintained very attractive potency and re-
duced plasma protein binding. Of these, compound 8 (GNE-477)
was selected for further evaluation.
a
Female nu/nu mice were dosed with the HCl salt as a solution intraveinously
(1 mg/kg) in 5% DMSO/5% cremophor and dosed orally as a solution in 80% PEG
(5 mg/kg).
b
Male rats were dosed with the TFA salt as a solution intraveinously (1 mg/kg) in
5% DMSO/5% cremophor and dosed orally as a solution in 80% PEG (5 mg/kg).
c
Male beagle dogs were dosed with the HCl salt as a solution intraveinously
(1 mg/kg) in 10% HP-b-CD and dosed orally as a suspension in MCT (2 mg/kg).
A direct comparison of 8 with its des-methyl analog (1) reveals
that the trend of reduced in vivo clearance in rats is also observed
in mice and dogs (Table 5). The improvement in clearance was par-
ticularly significant in dogs where 1 was cleared at two-thirds the
rate of hepatic blood flow whereas 8 has low clearance.
The appealing pharmacokinetic profile of inhibitor 8 justified
further evaluation of this compound. In an experiment evaluating
the tumor growth inhibition of a PC3 tumor xenograft10 over
14 days, stasis was achieved at a 20 mg/kg QD dose and significant
inhibition was observed with doses as low as 1 mg/kg QD. The
administered drug was generally well tolerated during this study
as demonstrated by acceptable levels of weight loss comparable
to that observed with the animals in the vehicle cohort (Fig. 1).11
The synthetic route to GNE-477, as well as to other analogs de-
picted within, is described in Scheme 1.12 Commencing with
aminoester 12, chloropyrimidine intermediate 13 was formed by
reaction with molten urea followed by POCl3 mediated chlorina-
tion and subsequent nucleophilic aromatic substitution by mor-
pholine. Next, an aldehyde intermediate useful in targeting
compounds 1–3, 9, 14 and GNE-477 was produced by lithiation
of the thiophene core followed by reaction with DMF. Finally,
reductive amination with N-sulfonylpiperazine followed by Suzuki
coupling afforded GNE-477.
Figure 1. Efficacy study of GNE-477 (8) in the PC3-NCI tumor xenograft model. The
plot shows tumor volumes over time including vehicle cohort. Vehi-
a
cle = MCT = 0.5% methylcellulose/0.2% Tween-80; QD = every day. a% TGI = percent
of tumor growth inhibition at the end of study (day 14) compared with the vehicle
control group. bPR = partial response (reduction of >50% but <100% in tumor
volume, compared with the starting tumor volume, observed on any day of the
study. cCR = complete response (reduction of 100% in tumor volume, compared with
the initial tumor volume, observed on any day of the study. dTI = tumor incidence
(ratio of number of animals with measurable tumors remaining in each group at the
end of the study (day 14) compared to the number of animals with measurable
*
tumors at the beginning of the study. p-Values comparing treated groups with the
vehicle group were calculated at day 14 using the Students t-test.

T. P. Heffron et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2408–2411
2411
SO2Me
SO2Me
N
N
O
O
O
N
O
N
S
S
N
S
N
N
d, e
a, b, c
f
S
OMe
NH2
N
N
N
N
R
R
N
N
12
R
R
N
Cl
Cl
14
13
N
R = Me or H
NH2
GNE-477
R = Me :
j
R = H : 1
g, h
O
O
O
O
N
N
S
S
O
Ar
N
N
S
H2N
S
k, f
i, f
N
N
I
H2N
N
N
N
N
R
N
N
R
5
N
N
Cl
Cl
16
15
N
N
NH2
NH2
Scheme 1. Reagents and conditions: (a) Urea, 200 °C, 4 h; (b) POCl3, CH3CN, reflux, 24 h; (c) morpholine, MeOH, 1 h; (d) nBuLi, THF, À78 °C, DMF; (e) N-sulfonylpiperazine,
1,2-DCE, HC(OCH3)3, Na(OAc)3BH; (f) 2-aminopyrimidine-5-boronic acid pinacol ester, PdCl2(PPh3)2, 1 M aq KOAc, CH3CN, microwave, 150 °C, 15 min; (g) nBuLi, THF, À78 °C,
CO2; (h) HOAT, HATU, iPr2NEt, DMF, NH4Cl; (i) ZrCl4, THF, CH3MgBr, À10 °C to rt; (j) nBuLi, THF, À78 °C, I2; (k) aryl boronate, PdCl2(PPh3)2, 1 M aq Na2CO3, CH3CN, microwave,
100 °C, 10 min.
Patel, S.; Pergl-Wilson, G. H.; Raynaud, F. I.; Robson, A.; Saghir, N.; Salphati, L.;
Sohal, S.; Ultsch, M. H.; Valenti, M.; Wallweber, H. J. A.; Wan, N. C.; Wiesmann,
C.; Workman, P.; Zhyvoloup, P.; Zvelebil, M. J.; Shuttleworth, S. J. J. Med. Chem.
2008, 51, 5522.
to install the remaining aromatic rings. The carbinamine 4 was
produced via primary amide 15 using ZrCl4 and excess methyl
Grignard followed by a Suzuki coupling.13
3. MCF7.1 is a HER2 transfected clone of the original MCF7 cell line (ATCC,
In summary, an effort to improve rate of dissolution of high
melting point compounds resulted in an improved central core
Manassas, VA) that expresses elevated levels of HER2 and contains the PI3K
E545K activating mutation found in the parental line.
for morpholino thienopyrimidine PI3K inhibitors. The tetrasubsti-
tuted thiophene generally has identical potency to compounds of
the des-methyl core but benefits from improved clearance. This
observation led to the identification of GNE-477, a potent dual
PI3K/mTOR inhibitor that displays desirable pharmacokinetic
properties in each of three species studied.14 GNE-477 also exhib-
ited stasis in a PC3 tumor growth inhibition study. Our efforts with
these and additional PI3K inhibitors is ongoing.
4. Sutherlin, D. P.; Sampath, D.; Berry, M.; Castanedo, G.; Chang, Z.; Chuckowree,
I.; Dotson, J.; Folkes, A.; Friedman, L.; Goldsmith, R.; Heffron, T.; Lee, L.; Lesnick,
J.; Lewis, C.; Mathieu, S.; Nonomiya, J.; Olivero, A.; Pang, J.; Prior, W. W.;
Salphati, L.; Sideris, S.; Tian, Q.; Tsui, V.; Wan, N. C.; Wang, S.; Wiesmann, C.;
Wong, S.; Zhu, B.-Y. J. Med. Chem. 2010, 53, 1086.
5. The interactions with the enzyme gained by the aminopyrimidine are
significant. The PI3K-
a IC50 for the analog of 2 in which the aminopyrimidine
is replaced by the 4-indazole present in GDC-0941 is 40 nM. For a description
of these inhibitors and their key interactions within the binding site, and
detailed methods, see Ref. 4.
6. Yalkowsky, S. H.; Valvani, S. C. J. Pharm. Sci. 1980, 69, 912.
7. Quantum mechanics calculations suggest that the lowest energy conformation
Acknowledgments
of
6 positions the aryl sulfone 30° out of plane, but with the co-planar
conformation accessible at just 0.4 kcal/mol penalty.
8. Quantum mechanical energies were computed using Jaguar version 7.5 by
The authors wish to thank Mengling Wong, Martin Struble, and
Wen Chiu for compound purification and determination of purity
by HPLC, mass spectroscopy, and 1H NMR. We thank Krista K. Bow-
man, Alberto Estevez, Kyle Mortara, and Jiansheng Wu for technical
assistance of protein expression and purification. We thank Emil
Plise for plasma protein binding data.
**
Schrodinger (Schrodinger, Inc.), at the 6-31G +/B3LYP level of theory. The
‘Relaxed Coordinate Scan’ method was used to vary the dihedral angle between
the thiophene/methylthiophene and the aryl ring between 0° and 90° at 30°
intervals.
9. For
7
Papp A À B = 14 Â 10À6 cm/s and Papp B À A = 23 Â 10À6 cm/s in
a
permeability assay using MDCK cells.
10. PC3 is a cancer cell line that is PTEN(À). Cell proliferation EC50 using this cell
line was 174 nM.
11. Each group, including vehicle, exhibited similar and acceptable weight loss of
less than 10% relative to starting mass.
References and notes
12. (a) Bayliss, T.; Chuchowree, I.; Folkes, A.; Oxenford, S.; Wan, N. C.; Castanedo,
G.; Goldsmith, R.; Gunzner, J.; Heffron, T.; Mathieu, S.; Olivero, A.; Staben, S.;
Sutherlin, D. P.; Zhu, B.-Y. WO2008/070740 A1.; (b) Castanedo, G.; Dotson, J.;
Goldsmith, R.; Gunzner, J.; Heffron, T.; Mathieu, S.; Olivero, A.; Staben, S.;
Sutherlin, D. P.; Tsui, V.; Wang, S.; Zhu, B.-Y.; Bayliss, T.; Chuckowree, I.; Folkes,
A.; Wan, N. C. WO2008/073785 A2.
1. (a) Vivanco, I.; Sawyers, C. L. Nat. Rev. Cancer 2002, 2, 489; (b) Cantley, L. C.
Science 2002, 296, 1655; (c) Guertin, D. A.; Sabatini, D. M. Cancer Cell 2007, 12,
9; (d) Fasolo, A.; Sessa, C. Expert Opin. Invest. Drugs 2008, 17, 1717; (e) Bellacosa,
A.; Kumar, C. C.; Di Cristofano, A., et al Adv. Cancer Res. 2005, 94, 29; (f) Ihle, N.
T.; Powis, G. Mol. Cancer Ther. 2009, 8, 1; (g) Maira, S.-M.; Stauffer, F.; Schnell,
C.; García-Echeverría, C. Biochem. Soc. Trans. 2009, 37, 265.
2. Folkes, A. J.; Ahmadi, K.; Alderton, W. K.; Alix, S.; Baker, S. J.; Box, G.;
Chuckowree, I. S.; Clarke, P. A.; Depledge, P.; Eccles, S. A.; Friedman, L. S.; Hayes,
A.; Hancox, T. C.; Kugendradas, A.; Lensun, L.; Moore, P.; Olivero, A. G.; Pang, J.;
13. Denton, S. M.; Wood, A. Synlett 1999, 55.
14. GNE-477 (8) inhibits PI3K-a, b, d, and c with IC50’s of 4, 86, 6, and 15 nM,
respectively.