Design of potent IGF1-R inhibitors related to bis-azaindoles

Article abstract of DOI:10.1111/j.1747-0285.2010.00991.x

From an azaindole lead, identified in high throughput screen, a series of potent bis-azaindole inhibitors of IGF1-R have been synthesized using rational drug design and SAR based on a in silico binding mode hypothesis. Although the resulting compounds produced the expected improved potency, the model was not validated by the co-crystallization experiments with IGF1-R.

Full text of DOI:10.1111/j.1747-0285.2010.00991.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.00991.x
Chem Biol Drug Des 2010; 76: 100–106
Research Article
Design of Potent IGF1-R Inhibitors Related to
Bis-azaindoles
Conception Nemecek1, William A. Metz2,
very potent hit 1 belonging to an azaindole chemical series from a
Syk kinase inhibitor program (8).
Sylvie Wentzler1, Fa-Xiang Ding2, Corinne
Venot3, Catherine Souaille4, Anne
OMe
4
4
´
Dagallier , Sebastien Maignan , Jean-
Pierre Guilloteau4, Franc¸ois Bernard1,
Alain Henry1, Sandrine Grapinet1 and
Dominique Lesuisse5,*
OMe
N
N
N
Me
H
1Medicinal Chemistry, Sanofi-aventis, 13 Quai Jules Guesde, 94300
Vitry-sur-Seine, France
1
IC50 = 55 nM
2Medicinal Chemistry, Sanofi-aventis, Route 202-206, Bridgewater,
NJ 08807, USA
As 1 was already quite potent on the target, we decided to evalu-
ate it in an extensive panel of biological, Absorption, Distribution,
Metabolism, Elimination (ADME), drug metabolism and Pharmacoki-
netics assays to anticipate potential liabilities and set the objec-
tives of our optimization program.
3Oncology Department, Sanofi-aventis, 13 Quai Jules Guesde,
94300 Vitry-sur-Seine, France
4Structural Biology and Drug Design, Sanofi-aventis, 13 Quai Jules
Guesde, 94300 Vitry-sur-Seine, France
5Sanofi-aventis, 1 av Pierre Brossolette, 91385 Chilly-Mazarin, France
*Corresponding author: Dominique Lesuisse,
dominique.lesuisse@sanofi-aventis.com
We first evaluated 1 in a series of secondary assays related to IGF
function. The potency in an ELISA IGF1-R autophosphorylation assay
was comparable to the one obtained in the HTRF assay. Compound
1 was also able to inhibit the IGF1-induced proliferation in two
different cell lines, a Mouse Embryonic Fibroblasts (MEF) cell line
engineered to over express IGF1-R and an MCF7 breast tumor cell
line, with IC50 's of 1.6 and 4.3 lM, respectively (Table 1).
From an azaindole lead, identified in high through-
put screen, a series of potent bis-azaindole inhibi-
tors of IGF1-R have been synthesized using
rational drug design and SAR based on a in silico
binding mode hypothesis. Although the resulting
compounds produced the expected improved
potency, the model was not validated by the co-
crystallization experiments with IGF1-R.
Selectivity against a panel of kinases was also evaluated. As
expected because of its origin, 1 was a potent Syk inhibitor
(Table 2). As expected also because of its high homology, there
was no selectivity versus insulin receptor kinase (IRK). Inhibitors of
IRK would be expected to induce insulin resistance (hyperglycemia).
It was hypothesized that for a short treatment period (about
4 months), there should not be any major metabolic disturbances
(compared with the physiological insulin resistance during late state
of pregnancy). In addition, some dual inhibitors of IGF1-R and IR-A
such as 1H-Benzoimidazol-2-yl)-1H-pyridin-2-one BMS-536924 (9) are
undergoing preclinical studies and could be advantageous for the
treatment of tumours expressing both IGF1-R and IR-A (10). Finally,
some inhibitors in development, such as the pyrrolo[2,3-d]pyrimidine
NVP-AEW541 (11) displayed good selectivity for IGF1-R versus IR in
intact cells in spite of no enzymatic selectivity for the kinases.
Owing to the little experience at that time with non-selective IGF-R
inhibitors, it was decided to assess the possible drawbacks of an
unselective inhibitor later in the development. From this initial
kinase panel, only KDR and GSK3b were inhibited albeit at higher
concentrations (3.6 and 5.5 lM, respectively).
Key words: drug discovery, enzyme structure, kinase, phosphatase,
structure-based drug design
Received 5 February 2010, revised 10 April 2010 and accepted for publi-
cation 18 April 2010
Insulin-like growth factor (IGF) promotes growth and mediates meta-
bolic signals (1). There is evidence that links IGF to cancer or tumori-
genesis, and examination of the literature (2–5) from the last decade
suggests that agents capable of inhibiting IGF receptor would have
potential in the treatment of such malignancies (6). To date, the first
results from clinical trials on antibodies or small molecules targeting
IGF-1 receptor are starting to be reported and confirm these hypoth-
eses (7). We now wish to report our own findings in this area.
We initiated a high throughput screen (HTS) on IGF1-R using an
Homogeneous Time-resolved Fluorescence (HTRF) assay relying on
the receptor autophosphorylation. This led to the identification of a
Solubility, transport, and human metabolism of 1 were acceptable,
and there was no PGP efflux. A preliminary single PK in rat showed
100

Design of Potent IGF1-R Inhibitors
Table 1: Evaluation of 1 in IGF1-related assaysa
IGF1-R (HTRF) (lM)
IGF1-R (ELISA) (lM)
IGF1-induced proliferation (MEF) (lM)
1.6
IGF1-induced proliferation (MCF7) (lM)
1
0.055
0.145
4.3
aBriefly, the activities were determined as follows: IGF1R–HTRF: Inhibition of autophosphorylation activity was determined using a time-resolved fluorescent
assay. The cytoplasmic domain of human IGF1R has been cloned as glutathione S-transferase (GST) fusion into the pFastBac–GST-tagged baculovirus expression
vector. The protein has been expressed in SF21 cells and purified to about 80% homogeneity. Kinase activity was determined in 50 mM Hepes pH 7.5 contain-
ing 5 mM MnCl2, 50 mM NaCl, 3% Glycerol, 0.025% Tween 20, 120 lM adenosine triphosphate. Enzyme reactions were terminated by the addition of 100 mM
Hepes buffer pH 7.0, containing 0.4 M KF, 133 mM EDTA, BSA 0.1% containing an anti-GST antibody labeled with XL665, and an anti-phosphotyrosine antibody
conjugated to a europium cryptate (Eu-K). Features of the two fluorophores, XL-665 and Eu-K, are given in G.Mathis et al., Anticancer Research, 1997, 17,
pages 3011-3014. The specific long time signal of XL-665, produced only when the IGF1R enzyme is autophosphorylated, was measured on a Victor analyser
(Perkin-elmer Life and Analytical Sciences, Courtaboeuf, France). Inhibition of IGF1R kinase activity with compounds of the invention was expressed as percen-
tage inhibition of control activity exhibited in the absence of test compounds.
IGF1R–ELISA: Inhibition of autophosphorylation in MCF7 cell line after IGF1 stimulation was evaluated by ELISA technique. MCF-7 cells were seeded at 600 000
cells per well in 6-multiwell plates, left over night in 10% serum and then serum-starved for 24 h. Compounds are added to medium 1 h before IGF1 stimula-
tion. After 10 min of IGF1 stimulation, cells are lysed with Hepes 50 mM pH7.6, Triton X100 1%, Orthovanadate 2 mM, proteases inhibitors. Cell lysates are
incubated on 96-multiwell plates precoated with anti-IGF1R antibody, followed by incubation with an anti-phosphotyrosine antibody coupled to peroxidase
enzyme. Peroxidase activity level (measured by OD with a luminescent substrate) reflects receptor phosphorylation status.
IGF1-Induced MCF7 cell proliferation: The antiproliferative effect of the molecules on MCF-7 cells was evaluated by [14C]-thymidine uptake 72 h after IGF1-
induced cell proliferation. MCF-7 cells were seeded at 25 000 cells per well in Cytostar 96-multiwell plates (Amersham Saclay, France) at 37 ꢀC, 5% CO2, at
day 1, left overnight in EMEM medium supplemented with 10% of FCS to allowed cell attachment. At day 2, the medium culture was changed for EMEM ⁄
HamF12, 50 ⁄ 50 in order to deprive the cells for 24 h. On day 3, cell medium was replaced by fresh EMEM with 1 % of sodium pyruvate, penicillin, streptomy-
cin, and 50 ng ⁄ mL final concentration of IGF1. Then, 0.1 lCi of [14C]-Thymidine and 3 lL of compounds were added in 213 lL final volume. Cells were incu-
bated at 37 ꢀC, 5% CO2 for 72 h. [14C]-Thymidine uptake was quantified by counting the radioactivity 72 h after IGF1-induced proliferation (Microbeta trilux
counter, Perkin-elmer). IC50 determination was performed in duplicate with 10 increasing concentrations.
moiety were interacting with the hinge portion of the kinase (12).
Indeed, removing N7 in the bis-indole 2 resulted in total loss of
the IGF1-R inhibiting activity. On the other hand, moving this nitro-
gen atom around the aromatic ring from position 7 to position 4 of
the azaindole (data not shown) also led to loss of activity. The fact
that methylation on the N1-position of the azaindole showed com-
plete loss of activity, further strengthened this binding hypothesis.
Table 2: Evaluation of 1 in a panel of kinases
Kinase
IC50 (lM)
ATP (lM)a
Kinase
IC50 (lM)
ATP (lM)
SYK
IRK
IGF1-R
0.055
0.086
0.055
0.280
>10
>10
5.480
>10
>10
>10
>10
>10
2
250b
120c
40
40
20
16
1
80
30
PLK1
bARK
CK1
>10
>10
>10
40
6
40
AURORA2
KDR
GSK3b
FAK
Tie2
>10
>10
>10
>10
>10
>10
>10
>10
>10
20
40
30
8
30
160
20
32
20
Rock2
PKCd
CK2
Cdk2
Src
PKA
PDK4
MEKK1
O
O
4
AKT
5
CDK4
JNK3
P38
N
6
6
N
1
X
300
1.7
7
R
PAK3
>10
1 X = N R = H 55 nM
2 X = C R = H > 10 µM
3 X = N R = Me >10 µM
IRK, insulin receptor kinase
aInternal kinase profiling; [ATP] = 2 Km.
bCoupled PK-LDH assay.
cHTRF assay.
Even though this hit arose from a series that had already been
thoroughly investigated in the Syk program, we could gather rela-
tively little information about the SAR for IGF1-R because the
5¢-methoxy group was essential to the activity. Table 4 shows the
subsequent loss of activity with removal of the methoxy groups on
the indole ring. We therefore designed a program of chemical modi-
fications keeping these groups in place.
acceptable exposure and bioavailability in spite of a high clearance
and short half-life (Table 3). Cytochrome P450 (CYP) inhibition did
not appear to be an issue (human recombinant CYP 3A4, 1A2, 2D6,
3C9, 2C19 enzymes, data not shown).
The goals of our optimization program were therefore set by this
initial analysis: optimize potency, in particular cellular potency and
selectivity versus Syk and other closely related kinases and optimize
metabolic stability to achieve a half-life of around 5 h in mice. This
article deals with optimization of the potency.
(OMe)n
5'
6'
4'
4
7'
3
5
6
N
N
N
2'
H
Even though we had no experimental information on the binding
mode of 1 in IGF1-R, the SAR data we had supported the hypoth-
esis that the two nitrogen atoms N1 and N7 of the azaindole
Substitution on positions 3 and 2¢ on the azaindole and indole
respectively also proved to be detrimental to the activity. On the
Chem Biol Drug Des 2010; 76: 100–106
101

Nemecek et al.
Table 3: Solubility, ADME and PK profile of 1
Sol (mg ⁄ mL)
In vivo PKd
AUC po (h lg ⁄ mL)
5.32
CaCo2
P total
Efflux
ratio
pH 7.4
0.010
pH 2.7
24.4
Met Staba,b
Met Staba,c
Fe (%)
52
Clf (L h ⁄ kg)
t1 ⁄ 2 po (h)
70
26
156
1.36
4.9
1.5
aPercent of compound remaining after 15 min of S9 fraction exposure.
bHuman S9 hepatic fraction.
cMouse S9 hepatic fraction.
dRat; Vehicle 5%PS80. 4%HCl 0.1 N in Gluco5%; 50 mpk.
eBioavailability.
fClearance.
Table 4: Activity as a function of methoxy position
Table 5: Activity as a function of methoxy position
Cpd
Position of MeO
IGF1-R (HTRF) (lM)
Cpd
R and position
IGF1-R (HTRF) (lM)
1
5¢, 6¢
5¢
5¢, 7¢
6¢
0.055
0.65
1.6
>10
>10
8
4-Cl
0.043
0.062
0.104
0.019
0.168
0.406
3
4 (8)
5
9
4-CN
4-Me
5-F
4-CH2OH
5-Cl
4-NH2
6-Cl
6-NH2
10
11
12
13
14
15
16
6
7
4¢, 5¢
>10
>10
other hand, the 6¢-position was more permissive as was also the
case for the 1¢-position. These positions were heavily explored with
production of libraries of various alkoxy, alkyl, and alkyl-substituted
side chains, but even if some derivatives were able to, at best,
keep the activity of the starting prototype, no substantial gain of
affinity was seen (data not shown) and we kept some of these in
mind for final modulation of the physicochemical properties like
solubility (see later).
phosphorylated forms but we had no in-house experimental
co-structure of an azaindole with IGF1-R. A 3D homology model
was built using the ATP-bound structure of a 3-phosphorylated form
of IGF1-R (PDB code 1K3A). Figure 1A shows a ribbon representa-
tion of the ATP-binding site, highlighting the hinge portion which
contains the hydrogen bond partners of ATP and the solvent region.
In terms of structure-based drug design, our knowledge of this che-
mical series was enriched by experimental 3D structures solved in-
house of various azaindole analogs complexed with Jnk3 and Aur-
ora2 kinase domains. The position of the azaindole scaffold is the
same, in all structures, with the azaindole nitrogen atoms hydro-
gen-bonded to the hinge backbone and the indole substituent lying
along the backbone at the entry of the ATP-binding site. Docking
calculations allowed proposing two binding modes. The first one,
showed in Figure 1B, is similar to the one observed in Jnk3 and
Aurora2. The second one places also the indazole moiety in front of
the hinge region, but the indole ring is flipped 180ꢀ around the C-C
bond between the two bicycles, as shown in Figure 1C. This ligand
conformation is allowed in IGF1-R because of a small hydrophobic
pocket which can accommodate the 5¢-methoxy group, with a Leu-
cine residue in the hinge region (Leu1051), where a Tyrosine and a
Leucine residue are found in Aurora2 and Jnk3, respectively. Our
SAR data in particular in position 5¢ along with the selectivity of
the compounds versus Aurora2 and Jnk3 supported the hypothesis
of the alternative binding mode.
Position 4, 5, and 6 on the azaindole were substituted with halo-
gens and a few other groups (Table 5) showing that small hydro-
phobic substituents on position 4 like chlorine (8), nitrile (9), and
alkyls (10 and 12) are well tolerated (IC50's 43–168 nM) while polar
substituents like amine (14, IC50 3 lM) and larger substituents
resulted, most of the time, in loss of affinity. Position 5 only
accepted limited changes with fluorine (11) even displaying activity
enhancement (IC50 19 nM) while activity dropped rapidly with chlor-
ine (13) displaying an IC50 of 400 nM. Disappointingly, the introduc-
tion of a primary amine in position 6 as in 16, to improve the
affinity by adding a hydrogen bond with the hinge, resulted in loss
of activity.
O
O
4
R
5
N
6
N
N
H
The crystallization of IGF1-R is complicated by the fact that the
receptor exists under various stable conformations depending on its
phosphorylation state. At the time we initiated this work, X-Ray
data were publicly available for the 0- (13), 2- (14), and 3- (15)
To assess this binding hypothesis, we introduced a nitrogen atom
on position 4¢ of the indole to stabilize this conformation through
an internal hydrogen bond and increase the inhibitory activity of the
compound. Quantum mechanics calculations favour this form by
102
Chem Biol Drug Des 2010; 76: 100–106

Design of Potent IGF1-R Inhibitors
A
Hinge
Solvent
Met1052
B
Figure 2: X-Ray of 25 with IGF1-R 2P form (resolution 3 ꢀ).
O
Glu1050
O
H
H
N
N
Hinge
N
H
N
H
Leu1O051
O
Thr1053
H
N
N
O
O
N
O
O
4'
N
H
H
H
3
3
4'
O
O
N
N
N
N
N
N
H
H
Figure 3: Active conformations of 1 (left) and 17 (right) bound
to IGF1-R.
C
Secondary
binding
pocket
Met1052
O
H
N
O
Glu1050
H
N
N
Hinge
N
H
H
Table 6: Activity as a function of azaindole substitution
O
O
Leu1O051
Thr1053
O
IGF1-R
(HTRF)
(nM)
IGF1-induced
proliferation
(MEF) (nM)
H
N
N
Cpd
X
R'1
R4
R5
N
1
18
8
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
Me
H
H
Cl
Cl
Cl
H
H
H
Cl
H
H
H
H
H
H
H
F
F
F
F
H
H
H
H
F
54
58
43
27
15
19
22
23
5.5
6.4
4.8
2
1600
1100
305
749
301
117
215
169
26
198
11
184
1.1
CH2CH2Morpholine
Me
CH2CH2Morpholine
CH2CH2PiperazineNMe
Me
CH2CH2Morpholine
CH2CH2PiperazineNMe
CH2CH2Morpholine
Me
CH2CH2Morpholine
Me
19
20
11
21
22
23
17
24
25
26
27
28
Figure 1: (A) Ribbon representation of a schematic kinase ATP-
binding site; (B) azaindazole-binding mode in IGF1-R similar to Jnk3
and Aurora2; (C) alternative binding mode in IGF1-R (same orienta-
tion of ATP site in all figures).
Cl
Cl
H
CH2CH2Morpholine
CH2CH2Morpholine
CH2CH2Morpholine
1.5
2.6
22
3–4 kcal ⁄ mol,1 a value compatible with a hydrogen bond. Indeed,
the addition of this nitrogen as in 17 resulted in substantial activity
improvement with an IC50 of 6 nM for IGF1-R inhibition.
8
32
Cl
F
Chem Biol Drug Des 2010; 76: 100–106
103

Nemecek et al.
Table 7: Activity of 26 on a panel of cell lines
graphic structures with azaindole analogs complexed to IGF1-R
showed the same binding conformation (data not shown).
Tissue
Cell line
IC50 (lM)
NSCLCa
NSCLCa
NSCLCa
Colon
A549
H460
H1299
HCT116
HT29
HeLa
1.49
1.49
1.18
3.1
0.14
2.12
7.1
Thus quantum mechanics energy calculations on the unbound 17
did not explain the observed improvement of activity. A hypothesis
for the observed activity enhancement of the bis-azaindole series is
that the presence of the 4¢-nitrogen atom favors the active flat con-
formation of the two bicycles observed in the crystallographic struc-
ture by removing the steric clash between the 3- and 4¢-hydrogens
in 1. There might even be a small additional stabilization through
an internal hydrogen-bond between 4¢-N and azaindole 1-H atoms
even though this type of H-bond is not very documented in the lit-
erature (Figure 3).
Colon
Uterus
Prostate
Prostate
PC3
DU145
6.44
aNon-small cell lung cancer.
N
We kept this new bis-azaindole scaffold in our final optimization.
The best compounds resulting from our chemistry effort are sum-
marized in Table 6. Overall, the 10-fold activity enhancement was
observed for all compounds with a nitrogen atom at position 4¢ of
the indole moiety (17 and 24–28) compared to equivalent analogs
with a carbon atom (1,8,11,19–23). This modification has also
resulted in a striking impact on the cellular activity, the most potent
compound (26) displaying a potency of 1 nM in the MEF IGF-
induced proliferation assay. Also, quite interesting was the effect of
the aminoethyl side chains at position 1¢ of the azaindole on the
cellular potency (compare for instance 17 with 24 and 25 with
26). This effect was much less pronounced on the initial carbon
N
H
N
N
4'
O
O
17 (6 nM)
We finally succeeded in obtaining crystal structures of IGF1-R com-
plexed to azaindazole analogs. Compound 25 was co-crystallized
with the bisphosphorylated form of IGF1-R2 demonstrating that the
bound conformation is the same as the one observed in Aurora2
and Jnk3, displayed in Figure 1B (Figure 2). Subsequent crystallo-
B(OH)2
I
O
O
O
b
a
N
N
R3
N
R3
R3
H
boc
R2
boc
R2
R2
A
B
C
R4
R4
SiMe3
R4
R4
R5
R5
R5
R6
f,g
R5
R6
e
c,d
I
N
R6
N
N
R6
N
N
NH2
N
NH2
H
Tosyl
D
E
F
G
O
O
R3
R3
R4
R4
N
R5
h
R2
R5
R6
R2
i or j,k
G
C
+
N
N
N
R6
N
H
N
R1
Tosyl
H
H
I
Scheme 1: Reagents and conditions : a. KOH, I2, DMAP, MeI, DMF or KOH, I2, DMAP, (Boc)2O, DMF; b. nBuLi, B(OMe)3; c. NIS,AcOH; d.
TMS-Ethynyl, CuI, LiCl, TEA, PdCl2(dppf), DMF; e. t-BuOK, NMP, 70 ꢀC; f. TsCl, BuN4NHSO4, NaOH, H2O, toluene; g. BuLi, THF, I2; h. Pd(PPh3)4,
NaHCO3 aq, DMF, 110 ꢀC; i. NaH, DMF, MeI; j. K2CO3, DMF, ClCH2CH2morpholino ⁄ HCl; k. KOH, MeOH reflux.
104
Chem Biol Drug Des 2010; 76: 100–106

Design of Potent IGF1-R Inhibitors
series. Compound 26 proved a tight binding IGF1-R inhibitor with a
Ki of 1.6 nM (data not shown). It was kept for further profiling.
(17,18) group, and cyclization under basic conditions. The resulting
azaindoles F were protected on the nitrogen by a tosyl group and
iodinated under basic conditions to afford the 2-iodo derivatives G.
These two building blocks C and G underwent palladium-catalyzed
coupling to afford the bis-bicyclic derivatives H. Alkylation on the
indole under basic conditions followed by deprotection of the tosyl
group afforded the final compounds I. From these, functional modi-
fications led to the azaindoles 9, 10, 12, and 141.
O
O
R4
X
R5
N
N
H
N
R'1
The general synthesis of the 2-[3-(4-azaindolyl)]-7-azaindoles 17,
24–28 (compounds R) is outlined in Scheme 2 (19). The 4-azain-
dole-building block N was obtained via a quite original synthesis:
Compound 26 was profiled in a panel of proliferating cell lines.
The huge improvement of potency in the MEF cell line did not
translate into the same improvement versus these additional cell
lines. The best responding cell line turned out to be HT29, a colon
cell line, with an IC50 of 140 nM (Table 7).
the 3-hydroxy-2-bromo-pyridine
J was converted to the 2,3-
dimethoxy-6-iodo-pyridine K after iodination (20) in position 6
followed by methylation of the phenol and substitution of the
2-bromine by sodium methoxide. Metal lithium exchange with nBuLi
followed by formylation with Dimethylformamide (DMF) afforded the
6-formyl derivative which was nitrated at position 5 with cupric
nitrate trihydrate (21). Condensation with nitro methane under
classic Henry conditions (22,23) followed by a modified procedure
reported by Novellino (24) gave the hydroxylated nitrostyrene which
upon heating with sodium acetate in acetic anhydride gave the
nitrostyrene intermediate L as a mixture of isomers. Silica gel-
assisted reductive cyclization (25) of this unstable intermediate,
The general synthesis of the 2-(3-indolyl)-7-azaindoles 4, 5, 8 11,
13, and 18–22 (compounds I) is outlined in Scheme 1 (16). The
substituted 1-Boc-3-boronic acids 5-methoxy-indoles
C were
obtained from 5-methoxyindole A after iodination in position 3
under basic conditions followed by iodine lithium exchange and
trapping with trimethylborate. The substituted 7-azaindoles F were
synthesized from the corresponding 2-aminopyridines D after 3-iodi-
nation, displacement of the 3-iodine by a trimethylsilylethynyl
O
N+
Br
N
O
O
N
O
O
N
I
a-d
e-g
O
O
N+
O
HO
J
K
L
h
R4
Br
O
O
N
R5
O
N
i,j
SnBu3
+
N
H
N
N
N
O
Tosyl
BOC
P
N
M
m
k,l
O
O
O
O
n, o
or
n, p, q, r
R4
R4
R4
N
N
N
R5
R5
R5
N
N
N
N
N
N
N
Boc
R1
H
Tosyl
H
O
Q
R
Scheme 2: Reagents and conditions : a. K2CO3, I2, H2O; b. MeI, K2CO3, DMF; c.NaOMe, DMF, 100 ꢀC; d. nBuLi, ⁄ THF, )78 ꢀC, DMF; e.
.
Ac2O,Cu(NO3)2 3H2O,; f. NaOEt, MeNO2,THF ⁄ EtOH, HCl.; g. NaOAc, Ac2O, reflux; h. Fe, SiO2, AcOH, tol.uene, reflux; i. Br2,, DMF, )15 ꢀC; j.
Boc2O, DMAP, TEA, DMF; k. ClSO2Ph-Me, NaOH, (nBu4)NHSO4; l. nBuLi, (nBu3)SnCl, THF, )75 ꢀC; m. Pd(PPh3)4, CuI, toluene, reflux; n. TFA,
AcOH, CH2Cl2; o. NaH, DMF, MeI; p. Cl(CH2)2Cl, Bu4NBr, KOH, Na2CO3, 50 ꢀC; q. NaI, MEC; r. morpholine, K2CO3, CH3CN, reflux.
Chem Biol Drug Des 2010; 76: 100–106
105

Nemecek et al.
with iron in acetic acid, provided the desired 4-azaindole M. This
compound was further brominated in position 3 and protected
with Boc-carbonate to afford N. On the other hand, the 7-azain-
doles O were protected with a tosyl group which then allowed
the specific introduction of the tributyltin moiety in position 2 to
afford P. Compounds P and N were coupled using standard Stille
conditions and after subsequent deprotection of the Boc and tosyl
groups, followed by N-alkylations the desired compounds R (cpds
17, 24–28) were obtained (Scheme 2).
11. Garcıa-Echeverrıa C., Pearson M.A., Marti A., Meyer T., Mestan
J., Zimmermann J., Gao J. et al. (2004) In vivo antitumor activ-
ity of NVP-AEW541 as a novel, potent, and selective inhibitor of
the IGF-IR kinase. Cancer Cell;5:231.
12. Ghose A.K., Herbertz T., Pippin D.A., Salvino J.M., Mallamo J.P.
(2008) Knowledge Based Prediction of Ligand Binding Modes
and Rational Inhibitor Design for Kinase Drug Discovery. J Med
Chem;51:5149.
13. Munshi S., Kornienko M., Hall D.L., Reid J.C., Waxman L., Stirdi-
vant S.M., Darke P.L., Kuo L.C (2002) Crystal structure of the
apo, unactivated insulin-like growth factor-1 receptor kinase.
Implication for inhibitor specificity. J Biol Chem;277:38797.
14. Pautsch A., Zoephel A., Ahorn H., Spevak W., Hauptmann R.,
Nar H. (2001) Crystal structure of bisphosphorylated IGF-1
receptor kinase. Insight into domain movements upon kinase
activation. Structure (Cambridge, MA, United States);9:955.
15. Favelyukis S., Till J.H., Hubbard S.R., Miller W.T. (2001) Structure
and autoregulation of the insulin-like growth factor 1 receptor
kinase. Nat Struct Biol;8:1058.
A lead from HTS was optimized for the inhibition of IGF1-R using
rational drug design. This led to the discovery of a very potent ser-
ies of bis-azaindoles, although the subsequent crystallographic
experimental data did not confirm the binding mode proposed by
the in silico calculations.
Acknowledgments
16. Wentzler S., El Ahmad Y., Filoche Romme B., Nemecek C., Venot
C. (2005) Fr. Demande 217 pp.; WO2006 ⁄ 037875.
17. Kumar V., Dority J.A., Bacon E.R., Singh B., Lesher G.Y. (1992)
Synthesis of 7-azaindole and 7-azaoxindole derivatives through a
palladium-catalyzed cross-coupling reaction. J Org Chem;57:
6997.
We are grateful to Antonio Guerreiro and Catherine Souaille for
helpful discussions and to the analytical department for compound
analyses.
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Notes
1Systematic conformational analysis with the Merck molecular force
field. (Thomas A. Halgren, I. Basis, form, scope, parameterization,
and performance of MMFF94, J. Comp. Chem.; 1996; 490–519) then
geometry optimisation using Density Functional Theory (B3LYP ⁄
6-31G*), no statistical thermodynamics corrections were applied.
Calculation done with Spartan08 (Wavefunction).
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2Crystal structure of 25 with IGF1-R have been deposited in the
PDB with accession code 3LVP.
106
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