Discovery and optimization of potent and selective benzonaphthyridinone analogs as small molecule mTOR inhibitors with improved mouse microsome stability

Article abstract of DOI:10.1016/j.bmcl.2011.04.129

Starting from small molecule mTOR inhibitor Torin1, replacement of the piperazine ring with a phenyl ring resulted in a new series of mTOR inhibitors (as exemplified by 10) that showed superior potency and selectivity for mTOR, along with significantly improved mouse liver microsome stability and a longer in vivo half-life.

Full text of DOI:10.1016/j.bmcl.2011.04.129

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4036–4040
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of potent and selective benzonaphthyridinone
analogs as small molecule mTOR inhibitors with improved mouse
microsome stability
Qingsong Liu a,b, Jinhua Wang a,b, Seong A. Kang c,e, Carson C. Thoreen a,b, Wooyoung Hur a,b
,
Hwan Geun Choi a,b, David L. Waller a,b, Taebo Sim a,b, David M. Sabatini c,d,e, Nathanael S. Gray a,b,
⇑
a Department of Cancer Biology, Dana Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA
b Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, USA
c Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
d Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
e Koch Center for Integrative Cancer Research at MIT, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting from small molecule mTOR inhibitor Torin1, replacement of the piperazine ring with a phenyl
ring resulted in a new series of mTOR inhibitors (as exemplified by 10) that showed superior potency
and selectivity for mTOR, along with significantly improved mouse liver microsome stability and a longer
in vivo half-life.
Received 5 February 2011
Revised 26 April 2011
Accepted 27 April 2011
Available online 7 May 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
mTOR
PI3K
Torin1
Mammalian target of rapamycin (mTOR) is a key node of the
PI3K/Akt/mTOR signal transduction pathway. mTOR pathway has
been found to be deregulated in a variety of cancers and has been
extensively studied as an oncology drug discovery target.1–4 Upon
activation by external or internal stimuli such as growth factors,
nutrients, stress, and energy, mTOR regulates cell growth, prolifer-
ation, metabolism, and autophagy through the mTORC1 and
mTORC2 complexes by phosphorylation of downstream targets
S6K, 4EBP1, and Akt.5 The successful clinical application of rapa-
mycin in renal cell carcinoma has validated mTOR as an anti-can-
cer drug discovery target. However, a lack of activity against
mTORC2, incomplete inhibition of mTORC1 function and negative
reactivation of Akt through the S6K/IRS1 pathway may explain
the limited clinical efficacy observed with rapamycin related com-
pounds (rapalogs).6–8 Currently, there is significant interest in the
development and characterization of ATP-competitive mTOR
inhibitors, which would result in complete inhibition of both
mTORC1 and mTORC2.
cers, and are currently being evaluated as an anti-cancer drug tar-
get in the clinic.1 The structural similarity between PI3Ks and
mTOR endowed many early PI3K or mTOR inhibitors, like PI-103
and BEZ-235, with dual activity against both targets.10,11 Because
PI3Ks are upstream regulators of Akt, a central hub of many critical
cellular processes, it is believed that mTOR-selective inhibitors
may demonstrate improved toxicology relative to dual PI3K/mTOR
inhibitors. Many of the recently disclosed mTOR inhibitors, includ-
ing Torin1, have demonstrated that it is possible to selectively in-
hibit mTOR versus PI3Ks.12–22
We recently prepared a series of tricyclic benzonaphthyridinon-
es, exemplified by Torin1, as highly potent and selective mTOR
inhibitors. Torin1 has more than 800-fold selectivity for mTOR rel-
ative to PI3Ks in cellular assays and has no other significant protein
kinase off-targets among the 450 kinases profiled by KINOME-
scanTM 12
Torin1 exhibited anti-tumor activity in a U87-MG mouse
.
xenograft that correlated with target inhibition. However, Torin1
exhibits poor mouse microsome stability and a short in vivo half-
life which limit its utility as a pharmacological agent in vivo. In this
Letter, we describe our efforts to improve the pharmacokinetic
properties of Torin1 by replacing the metabolically labile phenyl-
piperazine moiety with a biphenyl system to yield compounds
such as 10 (Fig. 1).
mTOR is a member of the PI3K kinase family, which consists of
the PI3Ks, DNA-PK, ATR, ATM, and SMG-1.9 PI3Ks, especially PI3K
a,
have been found to be hyperactivated in a wide spectrum of can-
The preparation of compound 10 and analogs is illustrated in
Scheme 1. Ethyl-4,6-dichloroquinoline-3-carboxylate (1) was sub-
⇑
Corresponding author. Tel.: +1 617 582 8590; fax: +1 617 582 8615.
E-mail address: Nathanael_gray@dfci.harvard.edu (N.S. Gray).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.129

Q. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4036–4040
4037
Based on the Torin1 series, we first retained the quinoline moi-
ety at the R1 position, which was presumably responsible for the
selectivity and potency against mTOR. Replacing the phenylpiper-
azine ring with a biphenyl system provided compounds that dem-
onstrated excellent potency against mTOR and selectivity relative
to PI3K. Methyl amide 11, THP-protected benzyl alcohol 12, benzyl
alcohol 13, carboxylic acid 14, and primary amide 16 all demon-
strated the same level of potency against mTOR (EC50 values be-
tween 15–30 nM) and the same selectivity over PI3K (EC50 over
300 nM). The methyl ester 15 lost a significant amount of activity
against mTOR, suggesting that the active members of this series
of compounds possess hydrogen bond donors through either an –
OH, –COOH or –CONH2 functionality. N-methyl-piperidin-4-amine
amide 10 exhibited the best potency against mTOR (EC50 = 5 nM,
the same as Torin1) and 200-fold selectivity over PI3K. The com-
pounds derived from replacing the quinoline moiety with amino-
pyrimidine (17) or pyrazole (18) systems were slightly less
potent against mTOR, indicating that the inner hydrophobic pocket
required a larger substituent. In order to reduce the molecular
weight and obtain better solubility, while the R2 fragment was kept
as methyl-piperidin-4-amine, the CF3 group was replaced with a
CH3 group and the R1 side chain was varied to include a pyrazole
(20), methylpyrazole (21), 7-azaindole (22), hydrogen (23) and
aminopyridine (24). However, all of these analogs failed to produce
the same level of potency against mTOR, although the selectivity
over PI3Ks was retained. Compound 19, which bears quinoline side
chain at the R1 position, retained the same potency and selectivity
against mTOR as Torin1 and compound 10. This reaffirmed that
quinoline was the best side chain to occupy the inner hydrophobic
pocket, and that the hydrogen bond provided by the –OH or –NH2
was critical to maintain potency.
Figure 1. Structures of Torin1 and 10.
jected to nucleophilic substitution with 4-bromo-3-trifluorometh-
aneaniline (2) to afford compound 3. Reduction of ethyl ester 3
with NaBH4 generated benzyl alcohol 4, which was subjected to
benzylic oxidation with MnO2 and olefination/cyclization in situ
to furnish general intermediate 5. Pinacol boronic ester 6 was ob-
tained from 4-bromo-benzonic acid (8) via Suzuki coupling and
amide formation. A bromine-selective Suzuki coupling was used
to install the benzoate side chain (or other substituent in the ana-
logs) to afford the intermediate 7. Finally, the quinoline side chain
(or other substituent in the analogs) was attached at the chlorine
position using more vigorous Suzuki coupling conditions to afford
the final product illustrated by compound 10.
Following this general synthetic strategy, a focused library of
compounds was generated by varying the benzoate side chain,
quinoline side chain and CF3/CH3 moiety. The selected compounds
were evaluated in parallel in biochemical assays with mTORC1
complex in cellular assays using a mouse embryonic fibroblast
(MEF) cell line by examining the phosphorylation status of mTOR
downstream targets such as S6K (T389), and for PI3K activity with
the Akt S473D PC-3 cell line by examining the phosphorylation sta-
tus of AktT308. The results are summarized in Table 1.
With the best activity and selectivity, compounds 10 and 19 were
chosen for mouse stability study, single point CYP450 inhibition test
Scheme 1. Reagents and conditions: (a) 1,4-dioxane, 85 °C, 4 h, 80% yield; (b) NaBH4, EtOH, rt, 4 h, 50% yield; (c) MnO2, CH2Cl2, rt, 2 h, then triethylphosphonoacetate, K2CO3,
EtOH, 100 °C, 12 h, 60% yield; (d) 6, PdCl2(Ph3P)2, Na2CO3, 1,4-dioxane, 80 °C, 2 h, 60% yield; (e) quinoline-3-boronic acid, PdCl2(Ph3P)2, t-Bu-Xphos, Na2CO3, 1,4-dioxane,
100 °C, 4 h, 50% yield; (f) PdCl2(dppf), bis(pinacolato)diboron, KOAc, 1,4-dioxane, 80 °C, 12 h; crude material was used without purification; (g) HATU, THF, 1-methyl-
piperidin-4-amine, diisopropylethylamine, rt, 50% over two steps.

4038
Q. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4036–4040
Table 1
Data from biochemical and cellular assaysa
Entry
10
R1
R2
R3
mTORC1 biochemical IC50 (nM)
mTOR cellular EC50 (nM)
PI3K cellular EC50 (nM)
CF3
21.7
5
1000
11
12
CF3
CF3
10.2
188
15
25
>300
>300
13
14
CF3
CF3
17.3
22.9
30
20
>300
>300
15
16
CF3
CF3
299
200
25
>300
>300
56.3
17
18
19
20
21
CF3
CF3
CH3
CH3
CH3
43.2
14.1
14.5
38.1
283
10
1000
1000
1000
>1250
>1250
10
1
150
150

Q. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4036–4040
4039
Table 1 (continued)
Entry
R1
R2
R3
mTORC1 biochemical IC50 (nM)
mTOR cellular EC50 (nM)
PI3K cellular EC50 (nM)
22
CH3
18.9
25
1000
23
24
H
CH3
>3000
39.8
>1000
>1250
>1250
CH3
50
a
IC50 determinations are the mean of two of independent measurements with a standard error of <20%.
Table 2
Table 4
Ambit and Invitrogen profiles of compound 10 against PIKKs
Mouse microsome stability and CYP450 inhibition results
Compound Mouse
microsome
NADPH-
dependent inhibition
CYP3A4 %
CYP2D6 %
inhibition
(10 lM)
Kinase symbol
PI4K
PI4Kb
PI3K-C2a
PI3K-C2b
hVPS34
Invitrogen IC50 (nM)
Ambit score (%)
a
>10000
3440
1110
437
170
922
1170
1170
3.01
ND
ND
ND
0.65
ND
0.1
2
stability (min)
(10
lM)
10
19
Torin1
46
42
1.3
Y
Y
N
61
34
ND
51
24
ND
P110
P110d/P85
P110
mTOR
a
/P85
a
a
c
0.05
0
(Table 2), and in vivo mouse pharmacokinetic analysis (Table 3). In
comparison to Torin1, compounds 10 and 19 demonstrated signifi-
cant improvements in stability in the mouse microsome assay (46
and 42 min, respectively), where both were subjected to NADPH-
dependent metabolism. In the single point CYP450 metabolic en-
zyme inhibitory assay, compound 10 showed more than 60% and
were also superior to those of Torin1. The slower Tmax of compound
10 (4 h) compared to compound 19 (1 h) and Torin1 (0.5 h) was
indicative of poor solubility and/or slow absorption.
50% inhibition at 10 lM against the major metabolic enzymes
To evaluate the kinase selectivity of compound 10, it was sub-
jected to the Ambit kinome-wide screen using KINOMEscanTM
technology. The assay showed that compound 10 was very selec-
tive and did not strongly hit any other protein kinases among the
353 kianses tested, except for several PI3K family lipid kinases
(Table 4).
Compound 10 was evaluated in an in vivo pharmacodynamic
study, where it exhibited significant inhibitory activity against
the downstream targets of mTOR, S6K, and Akt, and blocked 80–
90% phosphorylation of S6K (T389) and pAkt (S473) in liver and
lung tissues even after 6 h at a dosage of 20 mg/kg.
In summary, starting from Torin1, replacement of the metabol-
ically labile 4-amino-phenylpiperazine moiety with a biphenyl
system provided a new series of inhibitors that were exemplified
by compound 10, which demonstrated significant improvements
in mouse microsome stability and in vivo pharmacokinetic proper-
ties. Compound 10 is a potent and selective mTOR inhibitor suit-
able for use in cell culture and in vivo. Further elaboration of this
CYP3A4 and CYP2D6, respectively, while compound 19 demon-
strated weaker inhibition (34% and 24%, respectively). Further
investigations of the ability of these compounds to inhibit metabo-
lism are warranted prior to performing combination studies.
The improved microsome stability profile of compounds 10 and
19 encouraged us to evaluate their in vivo pharmacokinetic prop-
erties. Upon intravenous (7.5% NMP and 40% PEG400 in water)
and oral (0.1% v/v Tween-80, 0.5% w/v NaCMC in water) adminis-
tration, compound 10 demonstrated superior pharmacokinetic
properties relative to compound 19, although both were signifi-
cantly better than Torin1.12 The half-life was improved to 3.6 h
(10) and 1.8 h (19) from that of Torin1 (0.5 h). The bioavailability
of compound 10 (10.1%) was double that of Torin1 (5.5%), while
the bioavailability of compound 19 was 5.4%. Compound 10 also
demonstrated much better exposure using both IV and PO delivery
routes compared to Torin1 (1388/1411 vs 720/396 hrꢀng/mL).
Other pharmacokinetic properties such as clearance rate (11.9 vs
23.0 mL/min/Kg) and volume of distribution (1.95 vs 0.59 L/Kg)
Table 3
In vivo mouse pharmacokinetic data
Compound Cmax (ng/mL) i.v./P.O. Tmax (h) i.v./P.O AUC(0–1) (hrꢀng/ml) i.v./P.O. T1/2 (h) i.v./P.O. CL (mL/min/kg) i.v./P.O. Vss (L/kg) i.v./P.O. F (%) i.v./P.O.
10
19
Torin1
1408/191
1046/115
2757/223
–/4.0
-/1
–/0.25
1388/1411
737/391
720/396
3.61/–
1.85/–
0.5/0.79
11.9/–
22.6/–
23.0/–
1.95/–
1.82/–
0.59/–
–/10.1
–/5.4
–/5.49

4040
Q. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4036–4040
12. Liu, Q.; Chang, J. W.; Wang, J.; Kang, S. A.; Thoreen, C. C.; Markhard, A.; Hur, W.;
Zhang, J.; Sim, T.; Sabatini, D. M.; Gray, N. S. J. Med. Chem. 2010, 53, 7146.
13. Feldman, M. E.; Apsel, B.; Uotila, A.; Loewith, R.; Knight, Z. A.; Ruggero, D.;
Shokat, K. M. PLoS Biol. 2009, 7, e38.
scaffold class to improve the drug-like properties will be reported
in due course.
14. Garcia-Martinez, J. M.; Moran, J.; Clarke, R. G.; Gray, A.; Cosulich, S. C.; Chresta,
C. M.; Alessi, D. R. Biochem. J. 2009, 421, 29.
Acknowledgments
15. Yu, K.; Toral-Barza, L.; Shi, C.; Zhang, W. G.; Lucas, J.; Shor, B.; Kim, J.; Verheijen,
J.; Curran, K.; Malwitz, D. J.; Cole, D. C.; Ellingboe, J.; Ayral-Kaloustian, S.;
Mansour, T. S.; Gibbons, J. J.; Abraham, R. T.; Nowak, P.; Zask, A. Cancer Res.
2009, 69, 6232.
16. Barr, S.; Russo, S.; Buck, E.; Epstein, D.; Miglarese, M. AACR 101st Annual
meeting, Washington DC, April, 2010, 1632.
17. Chresta, C. M.; Davies, B. R.; Hickson, I.; Harding, T.; Cosulich, S.; Critchlow, S.
E.; Vincent, J. P.; Ellston, R.; Jones, D.; Sini, P.; James, D.; Howard, Z.; Dudley, P.;
Hughes, G.; Smith, L.; Maguire, S.; Hummersone, M.; Malagu, K.; Menear, K.;
Jenkins, R.; Jacobsen, M.; Smith, G. C.; Guichard, S.; Pass, M. Cancer Res. 2010,
70, 288.
The authors thank Dr. Michael Cameron (Scripps Florida) for the
mouse microsome stability studies. The authors also thank the Life
Technologies Corporation (Invitrogen) SelectScreenÒ Kinase profil-
ing service for performing enzymatic biochemical kinase profiling
and Ambit Biosciences for performing KINOMEscanTM profiling.
References and notes
1. Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Nat. Rev. Drug Disc. 2009, 8, 627.
2. Shor, B.; Gibbons, J. J.; Abraham, R. T.; Yu, K. Cell Cycle 2009, 8, 3831.
3. Meric-Bernstam, F.; Gonzalez-Angulo, A. M. J. Clin. Oncol. 2009, 27, 2278.
4. Liu, Q.; Thoreen, C. C.; Wang, J.; Sabatini, D. M.; Gray, N. S. Drug Discovery Today:
Ther. Strateg. 2009, 6, 47.
5. Guertin, D. A.; Sabatini, D. M. Cancer Cell 2007, 12, 9.
6. Sarbassov, D. D.; Ali, S. M.; Sengupta, S.; Sheen, J. H.; Hsu, P. P.; Bagley, A. F.;
Markhard, A. L.; Sabatini, D. M. Mol. Cell 2006, 22, 159.
18. Menear, K. A.; Gomez, S.; Malagu, K.; Bailey, C.; Blackburn, K.; Cockcroft, X. L.;
Ewen, S.; Fundo, A.; Le Gall, A.; Hermann, G.; Sebastian, L.; Sunose, M.; Presnot,
T.; Torode, E.; Hickson, I.; Martin, N. M.; Smith, G. C.; Pike, K. G. Bioorg. Med.
Chem. Lett. 2009, 19, 5898.
19. Richard, D. J.; Verheijen, J. C.; Curran, K.; Kaplan, J.; Toral-Barza, L.; Hollander,
I.; Lucas, J.; Yu, K.; Zask, A. Bioorg. Med. Chem. Lett. 2009, 19, 6830.
20. Yu, K.; Shi, C.; Toral-Barza, L.; Lucas, J.; Shor, B.; Kim, J. E.; Zhang, W. G.;
Mahoney, R.; Gaydos, C.; Tardio, L.; Kim, S. K.; Conant, R.; Curran, K.; Kaplan, J.;
Verheijen, J.; Ayral-Kaloustian, S.; Mansour, T. S.; Abraham, R. T.; Zask, A.;
Gibbons, J. J. Cancer Res. 2010, 70, 621.
7. 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.
8. Carracedo, A.; Pandolfi, P. P. Oncogene 2008, 27, 5527.
21. Verheijen, J. C.; Yu, K.; Toral-Barza, L.; Hollander, I.; Zask, A. Bioorg. Med. Chem.
Lett. 2010, 20, 375.
9. Abraham, R. T. DNA Repair 2004, 3, 883.
10. Park, S.; Chapuis, N.; Bardet, V.; Tamburini, J.; Gallay, N.; Willems, L.; Knight, Z.
A.; Shokat, K. M.; Azar, N.; Viguie, F.; Ifrah, N.; Dreyfus, F.; Mayeux, P.; Lacombe,
C.; Bouscary, D. Leukemia 2008, 22, 1698.
11. Maira, S. M.; Stauffer, F.; Brueggen, J.; Furet, P.; Schnell, C.; Fritsch, C.;
Brachmann, S.; Chene, P.; De Pover, A.; Schoemaker, K.; Fabbro, D.; Gabriel, D.;
Simonen, M.; Murphy, L.; Finan, P.; Sellers, W.; Garcia-Echeverria, C. Mol.
Cancer Ther. 2008, 7, 1851.
22. Malagu, K.; Duggan, H.; Menear, K.; Hummersone, M.; Gomez, S.; Bailey, C.;
Edwards, P.; Drzewiecki, J.; Leroux, F.; Quesada, M. J.; Hermann, G.; Maine, S.;
Molyneaux, C. A.; Le Gall, A.; Pullen, J.; Hickson, I.; Smith, L.; Maguire, S.;
Martin, N.; Smith, G.; Pass, M. Bioorg. Med. Chem. Lett. 2009, 19, 5950.