Pyrazolo[3,4-d]pyrimidines as potent inhibitors of the insulin-like growth factor receptor (IGF-IR)

Article abstract of DOI:10.1016/j.bmcl.2007.07.037

A high throughput screen of Abbott's compound repository revealed that the pyrazolo[3,4-d]pyrimidine class of kinase inhibitors possessed moderate potency for IGF-IR, a promising target for cancer chemotherapy. The synthesis and subsequent optimization of this class of compounds led to the discovery of 14, a compound that possesses in vivo IGF-IR inhibitory activity.

Full text of DOI:10.1016/j.bmcl.2007.07.037

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5406–5409
Pyrazolo[3,4-d]pyrimidines as potent inhibitors of the
insulin-like growth factor receptor (IGF-IR)
Robert D. Hubbard,* Nwe Y. Bamaung, Fabio Palazzo, Qian Zhang, Peter Kovar,
Donald J. Osterling, Xiaoming Hu, Julie L. Wilsbacher, Eric F. Johnson, Jennifer Bouska,
Jieyi Wang, Randy L. Bell, Steven K. Davidsen and George S. Sheppard
Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA
Received 2 July 2007; revised 11 July 2007; accepted 12 July 2007
Available online 25 July 2007
Abstract—A high throughput screen of Abbott’s compound repository revealed that the pyrazolo[3,4-d]pyrimidine class of kinase
inhibitors possessed moderate potency for IGF-IR, a promising target for cancer chemotherapy. The synthesis and subsequent opti-
mization of this class of compounds led to the discovery of 14, a compound that possesses in vivo IGF-IR inhibitory activity.
Ó 2007 Elsevier Ltd. All rights reserved.
Receptor tyrosine kinases (RTKs) have recently become
validated targets for cancer chemotherapy. The clinical
successes of agents that inhibit RTKs, such as Bcr-Abl
(GleevecÒ), EGFR (TarcevaÒ), and EGFR/ErbB-2
(TykerbÒ), have revealed that a variety of RTKs can
be successfully exploited for cancer treatment.1 There-
fore, the search for additional RTKs has led to the pro-
posal that small-molecule inhibitors of the insulin-like
growth factor receptor (IGF-IR) might yield effective
anti-cancer agents.2 IGF-IR is a member of a complex
system of growth factors and receptors that includes
the following: insulin receptor (IR), insulin-like growth
factor I/II (IGF-I/IGF-II), and six insulin-like growth
factor binding proteins.3 The complexity of the signaling
family is further increased due to the presence of hybrid
receptors, which are composed of both IGF-IR and IR.4
In normal tissue, IGF-IR is essential for growth, devel-
opment, and the suppression of apoptosis. This is a con-
sequence of the ability of IGF-IR to simultaneously
activate the anti-apoptotic phosphoinositide-3-kinase/
Akt pathway, and the mitogenic extracellular signal reg-
ulated kinase (ERK)/mitogen-activated protein kinase
pathway.5 In malignant cells, the ability of IGF-IR to
simultaneously control both the pro-survival and the
proliferative aspects of the cellular machinery allows
these cells to avoid apoptosis, induce the production
of key angiogenic factors, and promote tumor cell
invasion.6
A variety of approaches have been or are currently being
employed to target IGF-IR, which include antibodies to
the extracellular domain of the receptor, dominant-neg-
ative receptor proteins, antisense RNA, siRNA, and
small-molecule inhibitors.7 The latter agents can be di-
vided into roughly two categories, substrate inhibitors
of IGF-IR8 and ATP-competitive inhibitors of IGF-
IR.9 One of the first examples of an ATP-competitive
inhibitor of IGF-IR in both enzymatic and cellular as-
says was recently disclosed (1, NVP-ADW742,
Fig. 1).10 Prior to the completion of our SAR efforts,
compound 2 was reported to also possess IGF-IR enzy-
matic and cellular activity.11 During a high throughput
screen of our compound repository, we noted that the
pyrazolopyrimidine class of kinase inhibitors, such as
3 possessed moderate potency for IGF-IR.12 The
remainder of this communication will focus on the syn-
thesis and IGF-IR inhibitory properties of this promis-
ing class of compounds.
The synthesis of the pyrazolopyrimidines begins with
boronation of the appropriate bromide 5 with diborane
reagent 4 to afford 6 in 85–99% yield (Scheme 1). The
known ketone 7 was treated with morpholine and for-
mic acid, which after recrystallization from DMF/IPA
afforded the desired trans-diastereomer,13 which was re-
acted with the aforementioned boronates 6 under Suzuki
Keywords: Insulin-like growth factor receptor (IGF-IR); Kinase; Inhi-
bitor; Pyrazolo[3,4-d]pyrimidine.
*
Corresponding author. Tel.: +1 847 936 9242; fax: +1 847 935
5165; e-mail: robert.d.hubbard@abbott.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.037

R. D. Hubbard et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5406–5409
5407
R1
O
N
NH2
N
NH2
N
NH2
N
N
N
N
N
N
N
N
N
N
N
O
N
NVP-ADW742 (1)
2
3
Figure 1. Structures of reported IGF-IR kinase inhibitors (1,2) and generic structure for the pyrazolo[3,4-d]pyrimidines 3.
R1 R2
NH
ylation of N1 of the benzimidazole did not affect the en-
zyme or cell activity; however, the C7-methyl-substituted
benzimidazole was completely void of enzyme activity
(13 vs 12).
R1 R2
NH
O
O
a
+
B
O
B
O
NO2
2
Br
NO2
4
5
6
The data provided in Table 1 indicated that the benzyl-
substituted benzimidazole analog 10 provided an
acceptable balance of cell and enzyme activity. There-
fore, we focused our attention toward further defining
the optimal substituent(s) on the aryl ring. As shown
in Table 2, a variety of substituents were tolerated at
the ortho-position, such as, –F, Cl, –Me, –OMe, and
even –Br, yielding analogs 14, 15, 16, 17, and 18, respec-
tively. All the aforementioned analogs provided potent
enzyme inhibitors, however, the cellular activity suf-
fered, with 14 providing the only analog with improved
cell and enzyme activity relative to 10.
R2
R2
N
R3
NH
R1
NH2
R1
NO2
N
NH2
N
I
NH2
N
N
b, c
d
N
N
N
N
N
N
N
N
N
O
N
N
O
O
7
8
9-37
Table 1. Enzyme and cellular activities of substituted benzimidazole
pyrazolopyrimidines
Scheme 1. Reagents and conditions: (a) PdCl2 Æ dppf, KOAc, DMF,
100 °C, 85–99%; (b) morpholine, HCO2H, NMP, 100 °C, 25% yield of
trans-diastereomer; (c) 6, (Ph3P)2PdCl2, 2 M aq Na2CO3, DME/H2O 2/
1 (v/v), 80 °C, 40–87%; (d) R3-CHO, 1 M Na2S2O4, EtOH, 130 °C,
20 min, microwave heating, 35–55%.
R2
N
R3
R1
N
NH2
N
N
reaction conditions to afford 8, in 40–87% yield. The ni-
tro-aniline was treated with the appropriate aldehyde in
N
N
14
the presence of 1 M Na2S2O4 in a microwave reactor
at 130 °C for 20 min to afford analogs 9–37, in 35–
55% yield.
N
O
The analogs, described herein, were synthesized to probe
the IGF-IR inhibitory properties of differentially substi-
tuted benzimidazoles, in the presence of the fixed mor-
pholino-cyclohexane moiety.15 Therefore, analogs 9–
37, in addition to 1, and 2, were tested for intrinsic po-
tency versus IGF-IR enzyme,16 and their ability to inhi-
bit intracellular phosphorylation (p-IGF-IR) of the
receptor in MiaPaCa-2 cells, a human pancreatic tumor
cell line.17 The initial set of analogs tested revealed that
the C2-phenyl substituted benzimidazole provided a po-
tent inhibitor of the enzyme (9, Table 1). Insertion of a
methylene unit affording 10 further increased enzyme
activity, while providing equipotent cell activity. Meth-
Analog
Benzimidazole
IGF-IR Cellular
enzyme phosphorylation
R1
R2
R3
(IC50
nM)
,
(p-IGF-IR,
IC50, nM)
1
2
—
—
—
—
—
—
78
37
323
58
9
–H
–H
–H
–H
–H
–H
–Ph
–CH2Ph
176
64
787
94
10
11
12
13
–CH2CH2Ph
–CH2Ph
328
4334
35
487
2794
132
–Me –H
–H
–Me –CH2Ph

5408
R. D. Hubbard et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5406–5409
Table 2. Enzyme and cellular activities of substituted benzyl benz-
imidazole pyrazolopyrimidines
usually decreased. A series of disubstituted analogs were
prepared affording compounds 25–32. The preferred
analogs possessed 2,6-disubstitution patterns, for exam-
ple, the di-fluoro analog 25 possessed enzyme and cellu-
lar activity comparable to the 2-F, 6-Cl-analog 26,
which was analogous to the di-chloro-analog 30. A sur-
vey of putative heterocyclic replacements for the aryl
ring in 10 indicated that the thiophene-derived analogs
(33, or 34) provided improved cellular activity, with
comparable enzymatic activity. However, other rings
systems like pyridyl-analog 35, the thiazole-analog 37,
and the larger naphthyl-ring-analog 36 were all found
to lack relevant enzymatic and cellular activity.
R
HN
N
NH2
N
N
N
N
N
O
From the data in Table 2, we decided to evaluate the
murine pharmacokinetic (PK) properties of three of
the more promising analogs (10, 14, 33, Table 3). Com-
parison of key PK parameters dose normalized AUC
(DNAUC), clearance (CLp), and half-lives (t1/2) indi-
cated that the compounds were characterized as possess-
ing short half-lives and moderate to high clearance.
Although the isosteric replacement of a phenyl with a
thiophene (10 ! 33) was tolerated at the enzyme and
cellular level, the aforementioned substitution yielded
much lower oral and intravenous (IV) murine exposure.
Fortunately, the 2-chloro-substituent yielded an analog
(14) that exhibited increased oral and IV exposure, as
well as decreased clearance. As a result of the promising
enzyme and cellular profile of 14, in combination with
improved pharmacokinetic properties, this compound
was chosen for further evaluation in a pharmacody-
namic model for in vivo IGF-IR activity (Fig. 2). Com-
pound 14 given orally at 12.5 mg/kg produced drug
plasma concentrations of 2.4–3.7 lM at a 4 h time point
after dosing, and resulted in nearly complete inhibition
of IGF-IR kinase activity (receptor autophosphoryla-
tion) in mouse lung tissue (92–99%).18 This confirms
that the in vitro IGF-IR inhibitory activity observed
for this class of compounds does indeed translate to
in vivo activity.
Analog
R
IGF-IR
enzyme
(IC50, nM)
Cellular
phosphorylation
(p-IGF-IR,
IC50, nM)
10
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
–Ph
64
37
94
90
–2-Cl–Ph
–2-Me–Ph
–2-F–Ph
38
51
117
168
142
65
–2-OMe–Ph
–2-Br–Ph
3-Cl–Ph
81
71
123
186
58
372
267
140
637
257
1250
104
113
183
197
819
252
429
NDa
–3-Me–Ph
–3-F–Ph
–4-Cl–Ph
642
70
4-Me–Ph
4-F–Ph
924
79
2,6-di-F–Ph
2-F, 6-Cl–Ph
3,5-di-F–Ph
2,3-di-F–Ph
3,4-di-F–Ph
2,6-di-Cl–Ph
3,4-di-Cl–Ph
2,3-di-Cl–Ph
90
152
173
674
125
488
2322
33
34
23
32
56
84
S
Table 3. Comparison of the murine oral and intravenous pharmaco-
kinetic properties of 10, 14, and 33
S
N
Compound PO
IV
CLp
(L/h/kg)
t1/2
F (%)
DNAUCa DNAUCa
(h)
35
36
2337
1309
2646
2335
10
14
33
0.16
0.43
0.039
1.1
1.5
2.3
1.2
4.6
1
15
28
9
1.2
0.60
0.42
a Units of DNAUC: lmol h/L/mg/kg.
S
N
37
3310
1542
IGF-I, IV 0.5 mg/kg
a ND, not determined.
No
Vehicle, PO
14, PO
treatment
12.5 mg/kg
< p-IGF-IR
The SAR indicated that for the simple mono-substituted
analogs, the preferred position was the ortho-position,
while the substituents at the meta- and para-position
were tolerated, the enzymatic and cellular activity was
< total IGF-IR
Figure 2. Inhibition of IGF-IR in vivo by 14.

R. D. Hubbard et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5406–5409
5409
Steinig, A. G.; Sun, Y.; Weng, Q.; Werner, D. S.; Wyle, M.
J.; Zhang, T. PCT Int. Appl. 2005, WO 200597800.
12. For a discussion of related pyrazolo[3,4-d]pyrimidines as
lck inhibitors, see: Burchat, A.; Borhani, D. W.; Calder-
wood, D. J.; Hirst, G. C.; Li, B.; Stachlewitz, R. F. Bioorg.
Med. Chem. Lett. 2006, 16, 118, and references cited
therein.
13. Wishart, N.; Friedman, M.; Arnold, L. D.; Yang, B.; Fix-
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Holms, J. H.; Steinman, D. H.; Tian, Z.; Wittenberger, S.
J. PCT Int. Appl. 2005, WO 2005074603.
In conclusion, we have disclosed a novel scaffold for
inhibiting the insulin-like growth factor receptor, the
pyrazolo[3,4-d]pyrimidine. Appropriate substitution of
the scaffold afforded 14, which possessed potent enzyme
and cellular activity (<100 nM), while simultaneously
displaying promising murine pharmacokinetics and
activity versus an in vivo pharmacodynamic model. Fur-
ther in vitro and in vivo characterization of 14, as well as
further SAR exploration of the pyrazolo[3,4-d]pyrimi-
dines, will be disclosed in due course.
14. Yang, D.; Fokas, D.; Li, J.; Yu, L.; Baldino, C. M.
Synthesis 2005, 1, 47.
References and notes
15. The SAR of changing the morpholine ring in related
pyrazolo[3,4-d]pyrimidine-based analogs to other ring
systems will be the focus of future publications.
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16. IGF-IR kinase activity was assayed by a homogeneous
time-resolved fluorescence in vitro kinase assay. Specifi-
cally, 10 lL of C-terminal GST-tagged, recombinant,
human IGF-IR, amino acids 954–1367 expressed by
baculovirus in Sf21 cells (Cell Signaling Technology) was
mixed with 10 lL of an inhibitor (various concentrations,
2% final DMSO) and 10 lL of ATP (50 lM final
concentration) in reaction buffer (50 mM HEPES, pH
7.5, 10 mM MgCl2, 2 mM MnCl2, 0.1% BSA, and 1 mM
DTT, 40 lL final volume).
17. MiaPaCa-2 cells were treated with serial dilutions (0.0003–
30 lM) of compounds for 6 h in growth media containing
10% fetal bovine serum before stimulation with IGF-I for
10 min. Cells were then lysed and phosphorylation of IGF-
IR was determined using the human p-IGF-IR DuoSet IC
ELISA kit from R&D Systems (Minneapolis, MN).
18. A pharmacodynamic model to determine the activity of
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´
IGF-IR kinase inhibitor in mice was adapted from Garcıa-
Echeverrıa, C. Pearson, M. A.; Marti, A.; Meyer, T.;
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tors, see: Hubbard, R. D.; Wilsbacher, J. L. Chem Med
Chem. 2007, 2, 41.
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orally at 12.5 mg/kg in C57BL6 mice; 4 h later IGF-I at
0.5 mg/kg was injected intravenously, and the lung tissue
was collected 10 min thereafter. p-IGF-IR and total IGF-
IR in lung tissue lysates were determined by Western Blot
using specific antibodies (Biosource, Camarillo, CA and
Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
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