Quinazolines with intra-molecular hydrogen bonding scaffold (iMHBS) as PI3K/mTOR dual inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.12.026

Intra-molecular hydrogen bonding was introduced to the quinazoline motif to form a pseudo ring (intra-molecular H-bond scaffold, iMHBS) to mimic our previous published core structures, pyrido[2.3-D]pyrimidin-7-one and pteridinone, as PI3K/mTOR dual inhibitors. This design results in potent PI3K/mTOR dual inhibitors and the purposed intra-molecular hydrogen bonding structure is well supported by co-crystal structure in PI3Kγ enzyme. In addition, a novel synthetic route was developed for these analogs.

Full text of DOI:10.1016/j.bmcl.2010.12.026

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1270–1274
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Quinazolines with intra-molecular hydrogen bonding scaffold (iMHBS)
as PI3K/mTOR dual inhibitors
Kevin K. C. Liu a, , Xiaojun Huang a, , Shubha Bagrodia a, Jeffrey H. Chen a, Samantha Greasley a,
⇑
Hengmiao Cheng a, Shaoxian Sun a, Dan Knighton a, Caroline Rodgers a, Kristina Rafidi a,
Aihua Zou a, Jiezhan Xiao b, Shengyong Yan b
a Pfizer Global Research and Development, Chemistry Department, 10770 Science Center Drive, La Jolla, CA 92120, USA
b WuXi ApTec Co. Ltd #288 FuTe ZhongLu, Shanghai 200131, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Intra-molecular hydrogen bonding was introduced to the quinazoline motif to form a pseudo ring (intra-
molecular H-bond scaffold, iMHBS) to mimic our previous published core structures, pyrido[2.3-D]pyrim-
idin-7-one and pteridinone, as PI3K/mTOR dual inhibitors. This design results in potent PI3K/mTOR dual
inhibitors and the purposed intra-molecular hydrogen bonding structure is well supported by co-crystal
Received 22 September 2010
Revised 1 December 2010
Accepted 6 December 2010
Available online 10 December 2010
structure in PI3K
c
enzyme. In addition, a novel synthetic route was developed for these analogs.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
PI3K
mTOR
Kinase inhibitor
iMHBS
CH3
CH3
H
2
Ar
The phosphatidylinositol-3-kinase (PI3K)/mammalian target of
rapamycin (mTOR) signaling pathway plays a central role in driving
tumor cell proliferation, survival, angiogenesis and metastasis by
activating mutation, deletion or amplification on one or several of
its components.1 mTOR is a mammalian serine/threonine kinase
and a member of PI3K like kinase (PIKK) family of proteins. Rapamy-
cin analogs (rapalogues) such as RAD001 (everolimus), CCI-779
(temsirolimus), and AP23573 (deforolimus)) have demonstrated
anti-tumor activity in clinical trials.2 mTOR exists in two different
complexes,3 mTORC1, a rapamycin sensitive complex signaling to
S6K1 and 4E-BP1, and mTORC2, an rapamycin insensitive complex
signals to Akt. Studies have shown that rapalogues are protein–pro-
tein inhibitors of mTOR through mTORC1 but not mTORC2.4 Exis-
tence of this rapamycin insensitive component of the mTOR
signaling pathwaythus provides newopportunitiesto inhibitmTOR.
A small molecule inhibitor that targets mTOR kinase should prevent
signaling through both mTORC1 and mTORC2 to have a broad and
advantageous spectrum of pharmacology over rapamycin. There
are also reports that rapalogues led to PI3K/AKT activation and
attenuated the agents’ antitumor activities.5 This complexity results
from the negative regulation of the insuline/IGF-1 receptor signaling
X
Ar
N
N
N
N
6
4
1
8
N
R1
O
H2N
4
H2N
O
IMHBS
N
1
8
R2
1 X = C
2 X = N
3
Figure 1. New quinazoline scaffold, 3 as IMHBS for templates 1 and 2.
by mTORC1.6 Recently, several PI3K inhibitors and rapalogues com-
bination studies indicate the rapalogue-induced AKT activation was
prevented and resulted in a synergistic inhibition of tumor growth.
Furthermore, AKT activation induced by rapalogues has been as-
cribed to drug resistance.7 All these data suggest a PI3K and mTOR
dual inhibitor will be a useful tool to understand the resistance in
the clinic and as a promising agent for the treatment of cancers.
In the process to find other novel leads for our PI3K/mTOR dual
inhibitor program and to strengthen chemical leads’ IP position,
we decided to rationally re-design our templates from the previ-
ously reported series, pyrido[2.3-D]pyrimidin-7-one (1) and pterid-
inone(2) cores.8,9 Weemployedade-construction approachtobreak
the bi-cyclic ring in template 1 and 2 to a new quinazoline ring tem-
plate 3 in Figure 1. A secondary amide side chain was introduced to
the C-8 position to form a pseudo-ring through the intra-molecular
⇑
Corresponding author.
E-mail addresses: Kevin.K.Liu@pfizer.com (K.K.C. Liu).
Present address: Roche, 3431 Hill view Ave., Palo Alto, CA94304, USA.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.026

K. K. C. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1270–1274
1271
N
O
HN
N
N
HN
O
HN
N
N
N
N
HN
N
N
HN
N
N
HN
8
N
H2N
O
H2N
H2N
O
H2N
O
7
6
3b
3b
3a
OMe
PI3Ka Ki = 26 nM
LE = 0.49
mTOR Ki = 13 nM
clogP = 1.22
PI3Ka Ki = 18 nM
LE = 0.44
mTOR Ki = 416 nM
clogP = 2.46
1a
Figure 4. PI3Kg co-crystal structure overlay between compound 3b (code: 3PRZ)
and 1a (code: 3PRE).
Figure 2. The first round of quinazoline IMHBS, compound 3a and 3b.
2, respectively. Compounds 3a and 3b were submitted for co-
crystallization with the PI3Kc enzyme under a soaking condition
hydrogen bonding between the quinazoline core N-1 nitrogen and
the C-8 amide hydrogen atom to lock the quinazoline ring and to mi-
mic the original bi-cyclic ring space in templates 1 and 2. In addition,
the C-8 amide side chain nicely overlaps with the C-6 aryl groups in 1
or 2. For kinase selectivity reason, we engineered the important
methyl group to the C-2 of the quinazoline template which is corre-
sponding to the C-4 methyl group in 1 or 2. This methyl group plays a
crucial role on dual PI3K/mTOR inhibitors selectivity over regular
protein kinases as was reported recently.8c,9 We named this type de-
signed scaffold as iMHBS (intra-molecular hydrogen bonding scaf-
fold). This type of design has been used in small molecule drug
discovery10 or in mimicking biologically active protein conforma-
tion.11 This approach should allow us a quick entry to this newly de-
signed iMHBS by leveraging the SAR with our knowledge on series 1
and 2, since these templates share common chemistry vectors/han-
dles and a similar conformation.
to confirm the iMHBS design. Much to out delight, the co-crystal
structures of compounds 3a and 3b confirmed our design
hypothesis as shown in Figure 3. These X-ray structures showed
compound conformations which are favorable in making the
intra-molecular H-bonding; the N–N distances between N-1
nitrogen and C-8 amide nitrogen are 2.4(3a)/2.5(3b) Å, with
N-1 and amide free hydrogen distances 1.5(3a)/1.7(3b) Å and
bond angels of 134°(3a)/132°(3b) (Fig. 3). These observed geom-
eters are within the range of well-established H-bond geome-
tries.13 As was observed in series 1 and 2 earlier,8c,9the N-3
nitrogen and C-4 free NH2 are making critical interaction with
the hinge Val-882 in PI3K
picks up an extra interaction with Lys-833 in PI3K
882 and Lys-833 are conserved in PI3K
c
. In addition, the C-8 carbonyl group
. Both Val-
as the corresponding
c
a
Val-851 and Lys-802, respectively.
When we superimposed the co-crystal structures of iMHBS, 3b
and compound 1a from series 1 together as shown in Figure 4, it
became evident and once again supported our modeling that mod-
ifications on C-6 and C-7 of iMHBS 3 should provide us with com-
pounds that mimicking the N-8 R1 groups in series 1 or 2.
To implement this iMHBS strategy, we decided to make com-
pounds 3a and 3b which represent an amalgamation of these ideas.
These two compounds have R2 = H for the ease of synthesis to al-
low rapid validation of our hypothesis. Indeed, both compounds
3a and 3b have good to moderate binding potency for either PI3K
or mTOR and with excellent ligand efficacy12 around 0.45 based on
PI3K Ki as shown in Figure 2.
a
In general and as shown in Table 1, the PI3Ka binding potency
improves with C-6 methyl substitution and deteriorates badly with
C-7 methyl substitution when compared with their corresponding
parent compounds, R1 = H such as 3d, 3c versus 3a; 3f, 3e versus
3b; 3n, 3m versus 3l and 3q, 3p versus 3o. When we tried to fur-
ther elaborate the C-6 methyl group by bringing in solvating
a
Encouraged by this result, we then focused our efforts on
potency improvement by expanding the SAR efforts on the
C-6/C-7 and C-8 of iMHBS 3 which correspond to the important
pharmacophores of C-6 aryl and N-8 R1 groups in series 1 and
Figure 3. Co-crystal structures of compound 3a (left, code:3PS6) and 3b (right, code: 3PRZ) in PI3Kc.

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K. K. C. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1270–1274
Table 1
SAR of IMBHS 3 and highlighted compounds with in vitro PI3K
a
and mTOR binding potency
CH3
2
H
Ar
N
N
N
1
O
H2N
4
8
R1
3
Compound
R1
Ar
PI3K
a
mTOR
Ki (nM)
Ki (nM)
OMe
OMe
8%@
100 nM
15%@
1 lM
3c
3d
3e
3f
7-CH3
6-CH3
7-CH3
N
N
0.5
4550
7
265
984
3
N
N
N
N
HN
HN
HN
HN
6-CH3
6-CH2
N
O
3g
3h
15
13
6-CH2NMe2
188
1330
CH3
3i
H
0.5
31
N
CH3
CH3
3j
7-CH3
6-CH3
4550
0.5
3250
N
N
3k
66
CH3
57%@
1 lM
N
N
N
3l
H
54
N
N
N
CH3
CH3
0%@
100 nM
3m
3n
7-CH3
6-CH3
ND
CH3
CH3
12
1
162
5
CH3
NH
NH
NH
3o
3p
3q
3r
H
N
N
N
7-CH3
6-CH3
H
631
0.3
6
216
17
H
N
21
N
N
N
H
N
3s
3t
7-CH3
6-CH3
6%@ 100 nM
5
ND
84
H
N
ND = not determined; please see Ref. 8a for assay condition.
groups, it resulted in compounds with lower binding affinity such
as 3g, 3h versus 3f. From this exercise, we did find potent PI3K
mTOR dual inhibitors such as 3f, 3g and 3o. Interestingly, potent
PI3K
as well. These compounds have binding selectivity against mTOR
from 60- to 440-fold. One thing to note is that in our PI3K
a selective compounds such as 3d, 3k and 3i were identified
a/
a

K. K. C. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1270–1274
1273
enzymatic assay, 0.5 nM was our lowest detection limit. Therefore,
these compounds could be more potent in PI3K binding and more
selective against mTOR. In addition, iMHBS 3 has great selectivity
against regular protein kinases because of the C-2 methyl group for
the reason that we mentioned earlier.
The synthesis of IMHBS, 3, turned out to be more challenging
than we originally expected, and a novel synthetic route was
developed to realize our designs. The synthesis is depicted in
Scheme 1 by using compound 3a as an example.
a
2-Bromoaniline (4) in a hot HCl solution was treated with
trichloroacetaldehyde monohydrate in water in the presence
hydroxylamine hydrochloride and sodium sulfate to give isonitros-
oacetanilide 5. The intermediate 5 was treated with concentrated
H2SO4 to give the known cyclized compound 6 in 44% yield in
two steps according to the literature procedure.14 Compound 6
was then dissolved in methanol, and treated with hydrogen perox-
ide and sodium methoxide to give methyl ester compound 7 in 90%
yield. Compound 7 was hydrolyzed with lithium hydroxide to give
free acid 8 in 87% yield. The amino acid 8 was treated with acetic
anhydride at 120 °C to give cyclized product 9 in 90% yield. The
oxazin-4-one 9 was then treated with ammonium acetate at
140 °C to give quinazolin-4-ol 10 in 91% yield. Compound 10 in tol-
uene was chlorinated with POCl3 and N,N-dimethylaniline to give
chloroquinazoline 11 in 93% yield which was subsequently treated
with ammonium hydroxide in refluxed THF to give compound 12
in 56% yield. Compound 12 was subjected to palladium catalyzed
carbonylation in methanol to give methyl ester 13 which was
hydrolyzed to free carboxylic acid 14 with lithium hydroxide in
excellent yield. Compound 14 was then coupled with amine 15
to give our final target, compound 3a.
In addition to good binding, compounds with good cellular po-
tency were also observed as shown in Table 2. The table lists com-
pounds with cellular potency less than 120 nM in BT20 cell line for
the phosphorylation of S473 residue of AKT.
Table 2
Selective IMBHS 3 with cellular potency in BT20 cell line of phosphorylation of AKT
s473
Compound
R1
Ar
IC50 (nM)
pAKT-s473 (BT20)
N
HN
3f
6-CH3
35
CH3
CH3
3i
H
112
88
N
N
3k
6-CH3
NH
3q
3t
6-CH3
6-CH3
43
24
N
N
H
N
In summary, an iMHBS was designed based on quinazoline tem-
plate to inhibit both PI3K and mTOR. Co-crystal structures of 3a
and 3b confirmed our iMHBS design. In addition to developing a
novel synthetic route to these molecules, a series of potent and
selective dual PI3K/mTOR inhibitors were discovered.
Please see Ref. 8a for assay condition.
O
Br
O
NH2
NH2
Br
H
N
N
OH
Br
HN
O
O
NH2-OH/HCl
H2O2
H2SO4
47%
Br
reflux
rt
Cl3CCHO
O
CH3OH/CH3ONa
(2 steps)
90%
6
7
4
5
O
NH2
O
N
N
N
Br
NH4OAc
N
N
POCl3
HO
Br
Br
O
Br
120 o
C
Cl
140 oC
91%
THF
HO
80o
C
toluene
93%
Ac2O
90%
rt
LiOH
87%
8
9
10
11
N
N
O
N
N
aq. LiOH
N
O
N
CO
NH3
Br
H2N
O
H2N
H2N
OH
THF
93%
Et3N, Pd(PPh3)4
MeOH
THF
56%
70%
13
14
12
O
N
O
H2N
15
O
N
N
N
N
H
H2N
TPTU, DIPEA
15%
3a
Scheme 1. Synthesis of compound 3a.

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K. K. C. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1270–1274
A. D.; Nambu, M. D.; Ninkovic, S.; Pascual, B.; Rafidi, K.; Rodgers C., M-L.; Smith,
References and notes
G. L.; Sun, S.; Wang, H.; Yang, A.; Yuan, J.; Zhang, E.; Zou, A. Bioorg. Med. Chem.
Lett. 2010, 20, 6096.
1. Engelman, J. A.; Luo, J.; Cantley, L. C. Nat. Rev. Genet. 2006, 7, 606.
2. Huang, S.; Houghton, P. J. Curr. Opin. Pharmacol. 2003, 3, 371.
3. Sarbassov, D. D.; Ali, S. M.; Sabatini, D. M. Curr. Opin. Cell Biol. 2005, 17, 596.
4. Guertin, D. A.; Sabatini, D. M. Sci. Signal. 2009, 2, 24.
5. Sun, S.-Y.; Rosenberg, L. M.; Wang, X.; Zhou, Z.; Yue, P.; Fu, H.; Khuri, F. R.
Cancer Res. 2005, 65, 7052.
10. (a) Furet, P.; Bold, G.; Hofmann, F.; Manley, P.; Meyer, T.; Altmann, K.-H. Bioorg.
Med. Chem. Lett. 2003, 13, 2967–2971; (b) Honma, T.; Hayashi, K.; Aoyama, T.;
Hashimoto, N.; Machida, T.; Fukasawa, K.; Iwama, T.; Ikeura, C.; Ikuta, M.;
Suzuki-Takahashi, I.; Iwasawa, Y.; Hayama, T.; Nishimura, S.; Morishima, H. J.
Med. Chem. 2001, 44, 4615; (c) Dumas, J.; Smith, R. A.; Lowinger, T. B. Curr. Opin.
Drug Discov. Devel. 2004, 7, 600; (d) Wan, Z.; Boehm, J. C.; Bower, M. J.; Kassis,
S.; Lee, J. C.; Zhao, B.; Adams, J. L. Bioorg. Med. Chem. Lett. 2003, 13, 1191; (e)
Furet, P.; Caravatti, G.; Guagnano, V.; Lang, M.; Meyer, T.; Schoepfer, J. Bioorg.
Med. Chem. Lett. 2008, 18, 897.
11. Patgiri, A.; Jochim, A. L.; Arora, P. S. Acc. Chem. Res. 2008, 41, 1289.
12. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
13. Steiner, T. Angew. Chem., Int. Ed. 2002, 41, 48.
14. Beauchard, A.; Laborie, H.; Rouillard, H.; Lozach, O.; Ferandin, Y.; Le Guevel, R.;
Guguen-Guillouzo, C.; Meijer, L.; Besson, T.; Thiery, V. Bioorg. Med. Chem. 2009,
17, 6257.
6. Carracedo, A.; Pandolfi, P. P. Oncogene 2008, 27, 5527.
7. O’Reilly, K. E.; Rojo, F.; She, Q.-B.; Solit, D.; Mills, G. B.; Smith, D.; Lane, H.;
Hoffmann, F.; Hicklin, D. J.; Ludwig, D. L.; Baselga, J.; Rosen, N. Cancer Res. 2006,
66, 1500.
8. (a) Cheng, H.; Bhumralkar, D.; Dress, K. R.; Hoffman, J. E.; Johnson, M. C.; Kania,
R. S.; Le, P. T. Q.; Nambu, M. D.; Pairish, M. A.; Plewe, M. B.; Tran, K. T. WO
2008032162, 2008.; (b) Bruendl, M. M.; Gogliotti, R. D.; Goodman, A. P.;
Reichard, G. WO 2005105801, 2005.; (c) Cheng, H.; Bagrodia, S.; Bailey, S.;
Edwards, M.; Hoffman, J.; Hu, Q.; Kania, R.; Knighton, D. R.; Marx, M. A.;
Ninkovic, S.; Sun, S.; Zhang, E. MedChemComm 2010, 1, 139.
9. Liu, K. K.-C.; Bagrodia, S.; Bailey, S.; Cheng, H.; Chen, J.; Guo, L.; Hemkens, M.;
Hoffman, J. E.; Hu, J.; Johnson, T. O.; Knighton, D.; Liu, Z.; Marx, M. A.; McHarg,