Structure-based optimization of pyrazolo-pyrimidine and -pyridine inhibitors of PI3-kinase

Article abstract of DOI:10.1016/j.bmcl.2010.08.067

Starting from HTS hit 1a, X-ray co-crystallization and molecular modeling were used to design potent and selective inhibitors of PI3-kinase. Bioavailablity in this series was improved through careful modulation of physicochemical properties. Compound 12 d

Full text of DOI:10.1016/j.bmcl.2010.08.067

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6048–6051
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based optimization of pyrazolo-pyrimidine and -pyridine
inhibitors of PI3-kinase
Steven T. Staben a, , Timothy P. Heffron a, Daniel P. Sutherlin a, Seema R. Bhat a, Georgette M. Castanedo a,
⇑
Irina S. Chuckowree d, Jenna Dotson a, Adrian J. Folkes d, Lori S. Friedman b, Leslie Lee b, John Lesnick c,
Cristina Lewis c, Jeremy M. Murray f, Jim Nonomiya c, Alan G. Olivero a, Emile Plise e, Jodie Pang d,
Wei Wei Prior b, Laurent Salphati d, Lionel Rouge f, Deepak Sampath b, Vickie Tsui a, Nan Chi Wan d,
Shumei Wang a, Christian Weismann f, Ping Wu f, 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 Medicinal Chemistry Department, 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
f Structural Biology, 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:
Starting from HTS hit 1a, X-ray co-crystallization and molecular modeling were used to design potent and
selective inhibitors of PI3-kinase. Bioavailablity in this series was improved through careful modulation
of physicochemical properties. Compound 12 displayed in vivo knockdown of PI3K pharmacodynamic
markers such as pAKT, pPRAS40, and pS6RP in a PC3 prostate cancer xenograft model.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 27 April 2010
Revised 12 August 2010
Accepted 12 August 2010
Available online 19 August 2010
Keywords:
PI3-kinase
Oncology
Structure-based drug design
Mutation and over-expression of PI3-kinase
tion of its negative regulator PTEN, in a variety of cancers underline
a
, as well as dele-
tion between hinge-residue Val882 and the pyrazole nitrogen of
the pyrazolo-pyrimidine core (Fig. 1B). The increased potency of
1d relative to the others in this series likely results from an inter-
action between one of the sulfone oxygens with Lys833.5 Analysis
of the structure encouraged design of 6-substituents to interact
with Tyr867 and Asp841, a strategy successful in the development
of GDC-0941.3a
the importance of PI3Ka in signal transduction of cell growth and
proliferation.1 The inhibition of PI3K as a means to modulate cer-
tain cancer types has indeed been an active area of research and
the subject of several recent review articles.2
Screening of our internal library of small molecules identified
1a as a sub-micromolar PI3K
a
inhibitor (Fig. 1A). The prevalence
Table 1 summarizes modifications of the 2-pyrimidyl substitu-
ent with the goal of gaining additional polar interactions to in-
crease potency. Hydrogen-bond accepting substituents on the
meta-position of 6-phenyl pyrazolopyrimidines brought modest
improvements in potency (2a–2c). However, this effect was sensi-
tive to steric size or orientation of the hydrogen-bond acceptor (2d
and 2e). Larger improvements in biochemical potency were
achieved with heterocyclic substitution (3 and 4). To our delight,
4-indazolyl substitution at the 6-position of the pyrazolo-pyrimi-
dine core, as in compound 5a, resulted in single-digit nanomolar
of PI3-kinase inhibitors with a morpholine hinge-binding substruc-
ture3 led us to first modify the 6-substituent to extend (1b and 1d)
or hinder (1c) the hydrogen-bond acceptor’s expected interaction
to the hinge residue (Val882 in PI3Kc). To our surprise, these mod-
ifications had little effect on the biochemical potency of these par-
ticular inhibitors. Even with removal of the acceptor, as in
piperidine 1f, sub-micromolar inhibition was maintained. From
this initial SAR, we concluded the morpholine of 1a was likely
not acting as a hinge-binding motif.
Co-crystallization of the most potent molecule from this series
inhibition of PI3Ka. The proximity of the H-bond accepting inda-
(1d) with PI3K
c
confirmed an alternate mode of binding: interac-
zole nitrogen to back pocket Tyr867 in an X-ray co-crystal struc-
ture of 6 explained the increased biochemical potency (Fig. 2). In
contrast to the co-crystal structure of GDC-0941, the indazole
N–H of 6 does not appear to be in H-bond proximity to Asp841.
⇑
Corresponding author. Tel.: +1 6504673103.
E-mail address: stevents@gene.com (S.T. Staben).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.067

S. T. Staben et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6048–6051
6049
R
A
B
N
O
N
OH
6
N
N
1b, 0.430 µM
4
1a, 0.162 µM
Me
Me
N
NH
N
R =
O
N
S
O
N
1f, 0.359 µM
N
O
OMe
Me
1 a-e
1d, 0.075 µM
1c, 0.371 µM
Figure 2. Co-crystal structure overlay of 6 (gray, PDB id: 3NZU) and GDC-0941
(cyan, PDB id: 3DBS) in PI3Kc. Potential H-bonding interactions between hinge
residue Val882 and back pocket residue Tyr867 are indicated by dashed lines.
Table 2
4-Pyrazolo-pyrimidine substitution
N
Me
N
Figure 1. (A) PI3K
a IC50 values with changing 2-pyrimidyl substitution. (B) Co-
crystal structure of 1d in PI3Kc (PDB id: 3NZS). Potential H-bonding interactions
between Val882 and Lys833 are indicated by dashed lines.4
R
N
N
N
NH
Table 1
SAR for 6-pyrazolo-pyrimidine substitution
Entry Compound
R
p110a IC50 (lM) Prolif Rat Clp
a
N
EC50
(mL/
Me
N
MeO
(lM) min/kg)
N
HN
N
H
SO2Me
OH
1
2
6
7
0.0014
0.0011
0.403 139
N
R
N
H
ND
103
176
Me Me
N
Entry
Compound
R
PI3Ka IC50 (lM)
H
N
1
2
2a
2b
R3 = OMe
0.134
0.056
0.072
3.64
R3
3
4
8
9
0.0083
0.0014
1.50
R
3 = SO2Me
Me
3
4
5
2c
2d
2e
R3 = CONH2
R3 = CONHMe
R3 = NHAc
H
N
0.386
0.087
20
N
N
SO2Me
6
7
3
4
0.029
0.016
a
Proliferation EC50 determined in an MCF7 cell line. ND = not determined.
N
NH2
Y
8
9
10
5a
X = NH, Y = H
X = NH, Y = NH2
X = O, Y = NH2
0.0014
0.011
0.0017
permeability. This anticipation was supported by relatively high
TPSA7 (118 Å2) in combination with two hydrogen-bond donors.
Indeed 9 exhibited a low apparent permeability in a MDCK perme-
ability assay (Papp A–B = 0.37 Â 10À6 cm/s, Table 3). Interestingly,
the molecule also exhibited pronounced efflux (B ? A/
A ? B = 6.7) which might have affected the bioavailability at low
doses.8,9 This effect was accentuated when tested in both MDR
and BCRP transfected MDCK cell lines.10 We hypothesized that
decreasing the TPSA and H-bond donor count might both increase
the passive permeability of these molecules as well as alleviate
efflux.11
Replacement of the NH with an oxygen atom in aryl ether 10
improves apparent permeability ꢀsevenfold and reduces efflux in
the wild-type, MDR1-transfected and Bcrp1-transfected MDCKI/II
cells. Bioavailability is improved substantially. Removal of the lin-
ker between the pyrazolo-pyrimidine core and the phenylsulfonyl
moiety (11) reduces the H-bond donor count and TPSA and pro-
vides the most dramatic increase in passive permeability, although
at the cost of solubility. It is likely that this extreme increase in per-
meability also results from a decrease in the number of rotatable
bonds in 11. We sought to further decrease the TPSA of 10 by
5ba
N
X
5ea
a
2,3-Dimethoxyaniline in replace of 3-methoxyaniline.
In support of this finding, a 3-aminobenzisoxazole was also a well-
tolerated replacement for the indazole, suggesting the indazole N–
H is superfluous (compare 5a–5c).
Potency was maintained when 4-anilino substitution was re-
placed with more polar alkylamines directed toward the solvent
exposed region of the binding site (Table 2). However, pyrazolo-
pyrimidines bearing 4-alkylamino substitution typically had clear-
ance higher than hepatic blood flow in rodents (6–8). In contrast,
4-anilino substitution typically resulted in molecules with moder-
ate-to-low clearance as exemplified by 9.
Although 9 displayed moderate clearance in rat (20 mL/min/kg),
it was not bioavailable at a 5 mg/kg solution dose (Table 3).6 Albeit
only modestly soluble (7.1
lack of bioavailability of a solution dose was a result of poor
lg/mL at pH 6.5), we anticipated the

6050
S. T. Staben et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6048–6051
Table 3
Relationship between physicochemical properties and permeability, solubility, and bioavailability
MeO2S
N
Me
N
X
N
Y
N
NH
 10À6 cm/s
B–A/A–Bc
Solubility pH 6.5
g/mL)
Rat Clp
(mL/min/kg)
F%
a
Compound
X
Y
PI3K
M)
a
IC50
Donor
count
TPSA
118
Papp
A–B
(l
(cell line)b
(l
9
NH
N
0.0014
2
1
1
1
0.37 (A)
0.13 (B)
0.03 (C)
6.7
79
311
7.1
20
0
10
11
12
O
N
0.003
115
106
102
2.5 (A)
0.8 (B)
0.05 (C)
0.7
4.2–8.8
55.1
16
<1
15
2.0
ND
8.4
29
ND
Bond
O
N
0.0049
0.0068
31.5 (A)
7.6 (B)
3.8 (C)
0.5
3.2
4.8
CH
13.9 (A)
1.2 (B)
0.8
9.3
107
a
b
c
Papp A–B: apparent permeability in the apical to basolateral direction.
Cell lines: (A) MDCKI; (B) MDR1-MDCKI; (C) Bcrp1-MDCKII.
B–A/A–B = Papp B–A/Papp A–B. ND = not determined. Permeability assays run at 10 lM compound concentration.
Figure 3. (A) Compound 12 displays significant inhibition of PI3K PD markers pAKT (S473), pPRAS40, and pS6 in the PC3 prostate cancer xenograft mouse model (50 mg/kg
po, single dose). (B) Plasma and tumor drug levels of compound 12 from 1 to 6 h post dose. (C) Potency and PK profile for 12.

S. T. Staben et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6048–6051
6051
Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.; Szabo, S.; Yan, H.; Gazdar, A.; Powell,
S. M.; Riggins, G. J.; Willson, J. K.; Markowitz, S.; Kinzler, K. W.; Vogelstein, B.;
Velculescu, V. E. Science 2004, 30, 554; (c) Parsons, D. W.; Wang, T. L.; Samuels,
Y.; Bardelli, A.; Cummins, J. M.; DeLong, L.; Silliman, N.; Ptak, J.; Szabo, S.;
Willson, J. K.; Markowitz, S.; Kinzler, K. W.; Vogelstein, B.; Lengauer, C.;
Velculescu, V. E. Nature 2005, 436, 792.
Me
Me
Me
H2N
R
Cl
N
Cl
N
N
N
N
e-g
a,b
N
N
N
N
R = NH2
R = OEt
Cl
14
O
Cl
15
13a R = NH2
2. (a) Hennessy, B. T.; Smith, D. L.; Ram, P. T.; Lu, Y.; Mills, G. B. Nat. Rev. Drug Disc.
2005, 4, 98; (b) Wee, S.; Lengauer, C.; Wiederschain, D. Curr. Opin. Oncol. 2008,
20, 77; (c) Chalhoub, N.; Baker, S. J. Annu. Rev. Pathol. Mech. Dis. 2009, 4, 127; (d)
Marone, R.; Cmiljanovic, V.; Giese, B.; Wymann, M. P. Biochim. Biophys. Acta
2008, 159; (e) Yap, T. A.; Garrett, M. D.; Walton, M. I.; Raynaud, F.; de Bono, J. S.;
Workman, P. Curr. Opin. Pharmacol. 2008, 8, 393; (f) Crabbe, T.; Welham, M. J.;
Ward, S. G. Trends Biochem. Sci. 2007, 32, 460.
13b
R = OEt
Me
c,d
N
h,i
N
HN
N
N
X
3. For select examples see: (a) Folkes, A. J.; Ahmadi, K.; Alderton, W. K.; Aliz, 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.; Patel, S.; Pergl-Wilson, G.; 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, A.; Zvelebil, M. J.;
Shuttleworth, S. J. J. Med. Chem. 2008, 51, 5522; (b) Vlahos, C. J.; Matter, W. F.;
Hui, K. Y.; Brown, R. F. J. Biol. Chem. 1994, 269, 5241; (c) Hayakawa, M.;
Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi, T.; Yamano, M.; Okada, M.;
Ohta, M.; Tsukamoto, S.-I.; Raynaud, F. I.; Workman, P.; Waterfield, M. D.;
Parker, P. Bioorg. Med. Chem. Lett. 2007, 17, 2438; (d) Hayakawa, M.; Kaizawa,
H.; Moritomo, H.; Koizumi, T.; Ohishi, T.; Okada, M.; Ohta, M.; Tsukamoto, S.-I.;
Parker, P.; Workman, P.; Waterfield, M. Bioorg. Med. Chem. 2006, 14, 6847; (e)
Rikki, A.; Ahrani, B.; Batchelor, M.; Brookings, D.; Crepy, K.; Crabbe, T.; Deltent,
M.-F.; Driessens, F.; Gill, A.; Harris, S.; Hutchinson, G.; Kulisa, C.; Merriman, M.;
Mistry, P.; Parton, T.; Turner, J.; Whitcombe, I.; Wright, S. Bioorg. Med. Chem.
Lett. 2008, 18, 4316; (f) Dehnhardt, C. M.; Venkatesan, A. M.; Delos Santos, E.;
Chen, Z.; Santos, O.; Ayral-Kaloustian, S.; Brooijmns, N.; Mallon, R.; Hollander,
I.; Feldberg, L.; Lucas, J.; Chudhary, I.; Yu, K.; Gibbons, J.; Abraham, R.; Mansour,
T. S. J. Med. Chem. 2010, 53, 798; (g) Zask, A.; Kaplan, J.; Verheijen, J. C.; Richard,
D. J.; Curran, K.; Brooijmans, N.; Bennett, E. M.; Toral-Barza, L.; Hollander, I.;
Ayral-Kaloustian, S.; Yu, K. J. Med. Chem. 2009, 52, 7942; (h) Perry, B.;
Alexander, R.; Bennett, G.; Buckley, G.; Ceska, T.; Crabbe, T.; Dale, V.; Gowers,
L.; Horsley, H.; James, L.; Jenkins, K.; Crepy, K.; Kulisa, C.; Lightfoot, H.; Lock, C.;
Mack, S.; Morgan, T.; Nicolas, A.-L.; Pitt, W.; Sabin, V.; Wright, S. Bioorg. Med.
Chem. Lett. 2008, 18, 4700; (i) 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; (j) Heffron, T. P.; Berry, M.; Castanedo, G.; Chang, C.; Chuckowree, I.;
Dotson, J.; Folkes, A.; Gunzner, J.; Lesnick, J. D.; Lewis, C.; Mathieu, S.;
Nonomiya, J.; Olivero, A.; Pang, J.; Peterson, D.; Salphati, L.; Sampath, D.;
Sideris, S.; Sutherlin, D. P.; Tsui, V.; Wan, N. C.; Wang, S. M.; Wong, S.; Zhu, B. Y.
Bioorg. Med. Chem. Lett. 2010, 20, 2408.
O
X = N, 10
12
X = CH,
SO2Me
Scheme 1. Synthesis of 10 and 12. Reagent and conditions: (a) urea, 180 °C, 84%;
(b) PCl5, POCl3, 150 °C, 64%; (c) 4-(methylsulfonyl)phenol, NaH, THF; (d) 5 mol %
(PPh3)2PdCl2, Na2CO3 (3 equiv), MeCN/H2O, 80 °C microwave; (e) dimethylmalo-
nate, NaOEt, EtOH, 100 °C; then (f) 15% NaOH, 100 °C, 60% over (e and f); (g)
PhP(O)Cl2, 170 °C, 75%; (h) 5 mol % (PPh3)2PdCl2, NaHCO3 (3 equiv), MeCN/H2O,
150 °C microwave, 66%; (i) 4-(methylsulfonyl)phenol, K2CO3, DMF, 155 °C micro-
wave, 40%.
removal of a core-nitrogen atom. Through this modification, we
anticipated an increase in passive permeability and solubility
(esp. at low pH from increased pKa of core nitrogen). Pyrazolopyr-
idine 12 has a substantially higher apparent permeability, without
reduction in solubility, and thus is >100% bioavailable at a 5 mg/kg
solution dose.
Importantly, 12 displayed no significant inhibition in a 59-
member kinase panel at 1
PI3-kinases over other PIKK family members (i.e., DNA-PK
IC50 = 1.02 M, mTOR Ki,app >10 M). In addition, 12 was not a po-
tent inhibitor of human CYP isozymes (IC50 >20 M) and was very
l
M12 and also was selective for class I
l
l
l
stable in hepatocyte incubations (human 94% remaining,
mouse ꢀ100% remaining, 3 h).
Compound 12 was dosed at 50 mg/kg po in athymic nude mice
implanted with PC3-cell xenografts to evaluate its effect on down-
stream markers of PI3K. Suppression of pAkt, pPRAS40, and pS6RP
was observed through the 6 h post dose time period when plasma
drug levels remain elevated (Fig. 3).13
4. X-ray structures of 6 and 1d have been deposited in the PDB (id: 3NZS and
3NZU).
5. Lys833 is not well defined in the crystal structure density. This interaction
could be more important in PI3Ka.
6. No detectable concentration after oral dosing at 5 mg/kg in 80% PEG/EtOH
vehicle.
Synthesis of 10 and 12 is shown in Scheme 1.14 By slight mod-
ification of a literature procedure,15 heating 13a in molten urea,
followed by POCl3-mediated chlorination provided dichloropyraz-
olopyrimidine 14. A two-pot procedure was developed for the syn-
thesis of dichloropyrazolopyridine 15. Both 14 and 15 were
converted to the titled compounds by standard SNAr and Suzuki
coupling procedures.
7. TPSA (topological polar surface area) calculation similar to that as described in:
Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.
8. (a) Kwon, H.; Lionberger, R. A.; Yu, L. X. Mol. Pharm. 2004, 1, 455; (b) Lin, J.;
Chiba, M.; Baillie, T. Pharmacol. Rev. 1999, 1, 135; (c) Collett, A.; Tanianis-
Hughes, J.; Hallifax, D.; Warhurst, G. Pharm. Res. 2004, 21, 819.
9. Consistent with this statement, co-administration of Gefitinib (a known PgP
and BCRP inhibitor) with compound 9 did result in improved bioavailablity of
9.
10. This ratio was decreased when co-administered with known inhibitors of PgP
(GF120918, Elacridar) and BCRP (Fumitremorgin C). For example: Papp
A–B = 0.63 Â 10À6 cm/s, B–A/A–B = 17.60 for 9 in cell line C (Bcrp1-MDCKII) in
the presence of Fumitremorgin C.
11. For examples, see (a) Roberts, L. R.; Bryans, J.; Conlon, K.; McMurray, G.; Stobie,
A.; Whitlock, G. A. Bioorg. Med. Chem. Lett. 2008, 18, 6437; (b) Raub, T. J. Mol.
Pharm. 2005, 3, 3.
12. Study performed at Invitrogen; most significant was 27% inhibition of FLT 3.
13. The attenuated pathway knockdown for 12 can be attributed to its modest
cellular pAKT activity as well as low exposure of free drug in this experiment
(mouse plasma protein binding = 98.8%). Compound 12 was not tested in a
mouse xenograft model for tumor growth inhibition. Determination of a MTD
was not attempted.
In conclusion, co-crystallization and molecular modeling were
used to design analogs of HTS hit 1a with improved potency. Care-
ful analysis of physicochemical properties led to the identification
of specific linker and core modifications that improved passive per-
meability and minimized efflux leading to dramatically improved
bioavailability. Compound 12 displayed a pronounced pharmaco-
dynamic effect on PI3K markers in an in vivo human prostate can-
cer xenograft model.
Acknowledgments
14. (a) For experimental data and characterization see: Dotson, J.; Heffron, T.;
Olivero, A. O.; Sutherlin, D. P.; Staben, S. T.; Wang, S.; Zhu, B-Y.; Chuckowree, I.
S.; Folkes, A. J.; Wan, N. C. WO/2010/059788; PCT/US2009/065085.; (b) Dotson,
J.; Heffron, T.; Olivero, A. O.; Sutherlin, D. P.; Wang, S.; Zhu, B-Y.; Chuckowree, I.
S. Folkes, A. J.; Wan, N. C. WO/2009/097446; PCT/US2009/032459.
15. Ferroni, R.; Simoni, D.; Orlandini, P.; Bardi, A.; Franze, G. P.; Guarneri, M.
Arzneimittelforschung 1990, 40, 1328.
The authors thank Mengling Wong, Chris Hamman, Mike Hayes,
and Baiwei Lin for analytical/purification support.
References and notes
1. (a) Shayesteh, L.; Kuo, W. L.; Baldocchi, R.; Godfrey, T.; Collins, C.; Pinkel, D.;
Powell, B.; Mills, G. B.; Gray, J. W. Nat. Genet. 1999, 21, 99; (b) Samuels, Y.;