Discovery of the first non-ATP competitive IGF-1R kinase inhibitors: Advantages in comparison with competitive inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.03.003

A new series of IGF-1R inhibitors related to hydantoins were identified from a lead originating from HTS. Their noncompetitive property as well as their slow binding characteristics provided a series of compounds with unique selectivity and excellent cellular activities.

Full text of DOI:10.1016/j.bmcl.2011.03.003

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2224–2228
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of the first non-ATP competitive IGF-1R kinase inhibitors:
Advantages in comparison with competitive inhibitors
Dominique Lesuisse b, , Jacques Mauger a, Conception Nemecek b, Sébastien Maignan e, Janine Boiziau d,
⇑
Greg Harlow a, Augustin Hittinger b, Swen Ruf c, Hartmut Strobel c, Anil Nair a, Kurt Ritter c,
Jean-Luc Malleron b, Anne Dagallier e, Youssef El-Ahmad b, Jean-Pierre Guilloteau e, Houlfa Guizani b,
Hervé Bouchard b, Corinne Venot d
a Highthroughput Medicinal Chemistry, Sanofi-aventis, 2090 East Innovation Park Drive, Oro Valle, AZ 85755-1965, USA
b Medicinal Chemistry, Sanofi-aventis, 13 Quai Jules Guesde, 94300 Vitry-sur-Seine, France
c Medicinal Chemistry, Sanofi-aventis, Industriepark Hoechst, Frankfurt am Main 65926, Germany
d Oncology Therapeutic Domain, Sanofi-aventis, 13 Quai Jules Guesde, 94300 Vitry-sur-Seine, France
e Structural Biology and Molecular Modelling, Sanofi-aventis, 13 Quai Jules Guesde, 94300 Vitry-sur-Seine, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of IGF-1R inhibitors related to hydantoins were identified from a lead originating from HTS.
Their noncompetitive property as well as their slow binding characteristics provided a series of com-
pounds with unique selectivity and excellent cellular activities.
Received 3 January 2011
Revised 28 February 2011
Accepted 1 March 2011
Available online 4 March 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
IGF-1R
Kinase inhibitors
ATP
Noncompetitive
Slow binding inhibitor
The IGF-1R (Insulin like Growth Factor 1 Receptor) tyrosine ki-
nase is the main receptor involved in the transduction of growth
metabolic signals1 of IGF-1 (Insulin Growth Factor 1). Several lines
of evidence link this growth factor and receptor to the emergence
and proliferation of various tumors.2 During the course of a pro-
gramme aimed at finding inhibitors of IGF-1R3 we initiated a high
throughput screening (HTS) for compounds with affinity for this
receptor. The initial lead identified was an azaindole 1 that was
optimized and previously described.4
The present communication describes a chemically unrelated
and new series that originated from HTS and was expeditiously
optimized by our combinatorial chemistry group.
IGF-1R is widely expressed in cells that have undergone onco-
genic transformation. IGF-1, the canonical growth factor of IGF-
1R, induces receptor autophosphorylation on Tyr1131, Tyr1135
and Tyr1136 in the activation loop of the cytoplasmic domain.5
IGF-1R inhibitors were identified in an autophosphorylation-HTRF
OMe
OMe
F
F
F
O
O
S
N
N
N
N
Me
1
IC50 = 55 nM
O
O
N
N
⇑
Corresponding author. Present Address: Sanofi-Aventis, 1 av Pierre Brossolette
91385 Chilly Mazarin, France. Tel.: +33 1 60496857; fax: +33 1 60497492.
2
IC50 = 0.5 µM (Table 1)
E-mail address: dominique.lesuisse@sanofi-aventis.com (D. Lesuisse).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.003

D. Lesuisse et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2224–2228
2225
assay6 with
a nonphosphorylated recombinant GST-IGF-1R
Table 3
Evaluation of 1 in IGF-1-related assays
(aa882-1367) protein. This led to the identification of a hit 2
belonging to a hydantoin chemical series.
IGF-1R (HTRF)
M)
IGF-1R (ELISA)
M)
IGF-1-induced proliferation
(lM)
(
l
(l
As 2 was generated in a full combinatorial library, hundreds of ana-
logues were available to establish the initial SAR. The key struc-
tural features are as follows: The oxidation state of the sulfur
was not essential to the activity as the sulfone 2, displayed the
same activity as the thioether 4, both only about 5-fold more active
than the corresponding sulfoxide 3. On the other hand, the activity
was totally lost when replacing the trifluoromethyl group by a
methyl group as in 5. The methyl on the hydantoin was important
for the activity as shown by the loss of activity of unsubstituted 6
but both enantiomers 2 and 7 displayed about the same level of
activity, as the analog incorporating two methyl groups 8 (Table 1).
Larger alkyl or polar groups in this position all resulted in loss of
activity (data not shown).
1
2
0.055
0.5
0.145
0.6
1.6
0.3
On the other hand, there was very little flexibility on the 4-qui-
nolyl group in the southern part of the molecule (Table 2). It could
be replaced by a 4-pyridyl group with only a 3-fold loss in activity.
All other replacements like 3- and 2-pyridyls (compounds 10 and
11), 3-quinolyl (12), 4-imidazolyl (13), 4-piperidyl (14), phenyl
Table 1
SAR around 2: variations of R1, R2, R3 and n
R1
S(O)n
N
O
O
N
R2
R3
N
Figure 1. (a) Inhibition of IGF-1R by 2 as a function of ATP concentration; (b)
Dixon-plot analysis of the above curves.
Compd
R1
R2
R3
Chirality
n
HTRF (
lM)
2
3
4
5
6
7
8
CF3
CF3
CF3
CH3
CF3
CF3
CF3
Me
Me
Me
Me
H
H
H
H
H
H
H
S
S
S
S
—
R
—
2
1
0
2
2
2
2
0,5
2,52
0,41
>100
5,4
(15) were much less active as well as the oxides of the 4-pyridyl
and 4-quinolyl derivatives (16 and 17).
Compound 2 was evaluated against a panel of 23 kinases
(a combination of Serine/Threonine and Tyrosine proteins)7 and
found inactive on all of them except the highly homologous IRK
Me
Me
0,3
0,46
Me
(Insulin Receptor Kinase) with an IC50 of 0.3 lM.
As 2 showed high permeability in a model of intestinal absorp-
tion (TC7 cells)8 along with good metabolic stability in hepatic
mouse and human microsomes (83% and 82% compound remain-
ing after 15 min, respectively), it was engaged in a PK experiment.
The absolute bioavailability following oral administration to the fe-
male Balb/c mouse at a dose level of 10 mg/kg was found to be in
excess of 100% (i.e., dose 1 mg/kg). Systemic exposure to 2 was
good after oral dosage at 10 mg/kg (AUC0–1 = 13234 h ng/ml and
Cpmax = 1491 ng/ml) with evident levels up to 24 h. The compound
Table 2
SAR around 2: replacement of the 4-quinolyl group
F
F
F
O
O
S
N
O
O
was not an inhibitor of CYP3A4, 1A2, 2D6 and 2C19 (IC50’s >15
and a slight inhibitor of CYP2C9 (IC50 = 0.7 M). The solubility of 2
was rather low (9.9–13 g/ml). These properties prompted us to
lM)
N
l
l
R
engage in an optimization programme with the objectives of
improving the solubility and optimizing the potency on the target
to low nanomolar cellular potency.
Compound 2 was evaluated in a cellular IGF-1 phosphorylation
assay (ELISA) and in an IGF-1-dependant cell proliferation assay
(MEF).9 The antiproliferative activity of 2 was in fact quite striking
compared to that of our initial azaindole lead 1.4 While 1 displayed
a 30- and 80-fold lower potency than enzymatic activity, 2 was
more potent in the antiproliferative assay than in the enzymatic
N
N
N
N
N
N
2
0.5
9
1.6
10
79.7
11
12
l
M
l
M
l
M
>100
lM
>100
lM
N
N
N
N
O
O
13
M
14
M
15
16
17
>100
l
>100
l
>100
lM
>100
lM
>100 lM
assay (0.3 vs 0.5 lM) (Table 3).

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D. Lesuisse et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2224–2228
F
F
F
F
F
F
S
N
S
N
O
O
O
O
N
N
N
N
18
19
As hypothesized the compounds possessing an NH in 2-position of
the hinge binding nitrogen, like the amino pyridine 43 and its acetyl
derivative 42 indeed displayed an improved activity, whereas bulk-
ier groups in this position tended to impede the hinge contact (com-
pounds 20–23).
The small amino substituents of 40, 42, 43 also led to marked
increase in solubility (Table 4). An additional amine in 6-position
of the pyridine to further enhance the hinge binding did not lead
to improved activity (data not shown).
Figure 2. Overlay of IGF-1R kinase domain 3P (green) and 0P (cyan), respectively, in
complex with AMPPNP (nonhydrolysable form of ATP) (grey) and 2 (orange). The
activation loop conformations are different. It is DFG-out for the complex with 2.
The best compounds 18, 40–43 were evaluated in the cellular
IGF-1 phosphorylation assay and IGF-1-dependant cell prolifera-
tion assay.7 The results are outlined in Table 5. 42 turned out to
be extremely potent in the proliferation assay, once again much
more potent than anticipated from its inhibitory potency on IGF-1R.
The X-ray structure of 2 bound to IGF-1R along with its non-
competitive behavior strongly suggested that this series of inhib-
itors was stabilizing an inactive form of the kinase, therefore it
was anticipated that they should behave as slow binding inhib-
itors.12 An experiment was set up to verify this assumption. The
apparent inhibitory potency of the hydantoin 42, the azaindole 1
and staurosporine (a pan-kinase inhibitor) was monitored as a
function of preincubation time between rec-IGF-1R and com-
pound (max. of 2 h for enzyme stability). Only for the hydantoin
42 did we observe that the apparent IC50 value was clearly
dependent on the preincubation time with an almost 300-fold
decrease over an hour of time. This behavior was later shown
to be common to all compounds belonging to this series albeit
the extent of IC50 lowering differed from one compound to the
other (data not shown). This time-dependent kinetic was clearly
correlated to a better cellular activity (Table 6).
The compounds from combinatorial libraries (compounds 2–17)
were synthesized on solid phase as shown in Scheme 1.13 Fmoc pro-
tected Rink resin A was deprotected in a mixture of 20% piperidine/
DMF and capped by a Fmoc protected amino acid in the presence of
diisopropylaminecarbodiimide and hydroxybenzotriazole. The
immobilized Fmoc amino acid B was then deprotected and reacted
with aldehydes in a mixture of THF/triethyl orthoformate to afford
the corresponding Schiff base that upon reduction, using sodium
cyanoborohydride, provided the amines C. Isocyanates (commer-
cially available or prepared from the corresponding amines by reac-
tion with phosgene) were condensed with the resulting amine to
give the respective ureas D. Final cleavage from the solid support
(95% TFA/water) directly provided the hydantoins E as their salts.
The compounds from the optimization program (18–43) were
obtained as depicted in Schemes 2–5.14 The synthesis of a first
set of hydantoins was performed analogously as on solid phase:
the unsubstituted hydantoin 44 was obtained starting from 4-tri-
This result, along with the high selectivity of this compound
motivated us to elucidate the mechanism of action of 2 with IGF-
1R versus ATP. The reactions were done using the same HTRF pro-
tocol as before and using three concentrations of ATP (80, 120, and
160 lM). Compound 2 showed mixed uncompetitive/noncompeti-
tive type kinetics with respect to ATP (Fig. 1a and b).
The noncompetitive kinetics might explain the good selectivity
of this compound. These observations also suggested a novel bind-
ing mode with the kinase. We had hypothesized from the impor-
tance of nitrogen position on the activity (see Table 2) that the
quinolyl nitrogen was involved in the hinge binding with the ki-
nase, but we had no structural information. Several compounds
from our azaindole series had been co-crystallized in the 2P (bis-
phosphorylated form) of IGF-1R. However, this form as well as
the 3P phosphorylated active form of IGF-1R exposed to 2 (co-crys-
tallization and soaking) did not allow us to obtain measurable crys-
tals. The success came from the nonphosphorylated (inactive form)
of IGF-1R. (Fig. 2).
This structure confirmed that the quinolyl nitrogen was inter-
acting with the hinge binding region of the kinase.10 In addition,
it also pointed to a DFG-out binding with the kinase.11 This confor-
mation of the protein is characterized by a large change of position
and orientation of the DFG motif found at the beginning of the acti-
vation loop in most kinases. Whereas the phenylalanine side chain
in the DFG-in conformation occupies a volume outside of the active
site, in the DFG-out conformation it lies close to the location of the
ribose pocket. In the process it allows access to a secondary pocket
at the back of the active site. Part of 2 binds in that secondary
pocket.
One rational way to enhance affinity of 2 for the kinase was to
reinforce the hinge interaction by adding an H-bond donor in the
close vicinity of the H-bond acceptor as to be able to obtain the
classical two-pronged interaction. One of our objectives being also
the optimization of the solubility, it appeared to us that this prop-
erties could also be favourably impacted by the addition of polar
groups on this aromatic ring.
From the previous SAR studies, it was decided to start from
compound 18 incorporating the simplest features required for
fluoromethylthiophenyl isocyanate and methyl
a-aminoisobuty-
the activity, namely
a trifluoromethyl thioether, a nonchiral
rate. From there, condensation with various 2-substituted
4-bromomethylpyridines15 afforded compounds 18, 24, 29, 30,
35 and 38 (Scheme 2).
dimethylhydantoin backbone and a pyridine as hinge interaction
motif (this was also in turn more potent than the corresponding
quinoline 19 on this particular scaffold).

D. Lesuisse et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2224–2228
2227
Table 4
SAR from pyridine substitution
O
H
N
NH
a,b
a,c
O
N
H
O
O
R1
O
F
R2
F
F
S
N
B
A
Fmoc protected Rink resin
H
R4
N
O
N
O
R4
O
O
O
H
e
d
F
F
N
O
O
F
N
R3
N
R1
N
N
H
R1 R2R3
R2
H
N
R2
R1
HO
O
R3
E
D
C
N
R
Scheme 1. Reagents and conditions: (a) 20% piperidine, DMF; (b) FmocAA(OH)
(AA = S-Ala (compounds 2–5 and 9–17), R-Ala (compd 7), Gly (compd 6) or Me-Ala
(compd 8), Diisopropylcarbodiimide (DIC), HOBt, DMF; (c) R3CHO, HC(OEt)3, THF;
NaBH3CN; (d) R4-N@C@O, DCM; (e) TFA, H2O.
Compd
R
Sol (l
g/ml)a
IGF-1R HTRF (nM)
18
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
H
<0.02
264
>10000
>10000
>10000
>10000
>10000
>10000
4706
4501
2950
2897
1851
1241
1089
1061
1052
995
CF3
O
1-Piperidyl
4-Morpholinyl
1-(4-Me-Piperaziny
1-Piperazinyl
CN
COOH
NHSO2Me
NMe2
CONH2
F
OEt
NH-cyclohexyl
NHCH2PhOMe
NHPh
NHBn
Cl
NHCOOtBu
CONHMe
Et
NHiPr
NHMe
NHEt
NHAc
<0.024
0.037
0.159
<0.024
<0.021
0.142
<0.025
<0.022
ND
S
O
CF3
(a)
NH
H2N
S
N
O
+
O
44
NCO
+
R
O
R
(b)
CF3
N
0.032
ND
N
S
N
N
Br
O
<0.025
<0.027
<0.025
<0.026
<0.022
ND
<0.023
<0.022
<0.023
0.075
<0.022
0.035
0.113
18 R = H; 24 R = CN; 29 R = F; 30 R = OEt; 35 R = Cl; 38 R = Et
Scheme 2. Reagents and conditions: (a) toluene/TEA; (b) NaH/DMF.
784
703
574
453
202
154
98
92
O
NHR
O
Cl
CF3
N
CF3
N
S
N
N
S
N
N
O
O
NH2
20-23, 27, 31, 33-34, 39-41
35
a
Nephelometric solubility.
Scheme 3. Reagents and conditions: RNH2/microwave, NMP or DMF.
Table 5
Evaluation of 18 and 40–43 in IGF-1-related assays
OMe
Compd
IGF-1R (HTRF)
10 min (nM)
IGF-1R (ELISA)
(nM)
IGF-1-induced
proliferation (nM)
O
O
NH
NH2
CF3
CF3
(a)
N
N
S
N
18
40
41
42
43
264
202
154
98
117
43
47
7
282
312
104
8
S
N
N
N
O
O
43
32
92
34
97
(b)
O
NHR
CF3
N
S
N
N
Table 6
Activity as a function of preincubation time
O
26, 36, 42
Preincubation
IC50’s (nM)
time (min)
Staurosporine
1
42
Scheme 4. Reagents and conditions. (a) TFA/DCM; (b) 26 MeSO2Cl/Py; 36 (BOC)2O/
tBuOH/rt; 42 Ac2O/80 °C.
IGF-1R in vitro (HTRF, nM)
0
5
1132
1325
1701
1533
1646
—
39
34
34
33
35
1975
175
87
28
7
10
30
60
60
—
From the 2-chloropyridine hydantoin 35 a series of amines were
prepared by direct displacement (Scheme 3).
Trifloracetic acid deprotection of the 2-(p-methoxybenzylami-
nopyridine hydantoin 32 afforded the free amine which could then
IGF-1R in cell (ELISA, nM)
IGF-1R-dep proliferation
(nM)
180
439
7
8
—

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D. Lesuisse et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2224–2228
O
fluorescent assay. The cytoplasmic domain of human IGF-1R has been cloned
O
CN
COOH
CF3
CF3
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 containing 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 labelled with XL665 and an
(a)
N
N
S
N
N
S
N
N
O
O
24
25
(b)
(c)
anti-phosphotyrosine antibody conjugated to
a europium cryptate (Eu-K).
Features of the two fluorophores, XL-665 and Eu-K are given in Mathis et al.,
Anticancer Res. 1997, 17, 3011. The specific long time signal of XL-665,
produced only when the IGF1R enzyme is autophosphorylated, was measured
on a Victor analyser (Perkin–Elmer). Inhibition of IGF1R kinase activity with
compounds of the invention was expressed as percentage inhibition of control
activity exhibited in the absence of test compounds.
O
CONH2
O
CONHMe
CF3
CF3
N
N
S
N
N
S
N
N
O
O
37
7. Internal panel of kinases: Akt, Fak, p38, Tie2, Jnk3, Plk1, Pak3, Aurora2, GSK3b,
Cdk4; CEREP panel of kinases: Abl, CAMKII, CKII, Cdk1, Cdk2, EGF, IRK, MAP,
MEK1, PKCa, Src, ZAP70.
8. Turco, L.; Catone, T.; Caloni, F.; i Consiglio, E. D.; Testai, E.; Stammati, A. Toxicol.
in Vitro 2011, 25, 13.
28
Scheme 5. Reagents and conditions: (a) HCl 5 N, reflux; (b) MeNH2, EDCI, HOBT,
DCM; (c) H2SO4, 40 °C.
9. Briefly, the activities were determined as follows: IGF-1R–ELISA: Inhibition of
autophosphorylation in MCF7 cell line after IGF-1 stimulation was evaluated by
ELISA technique. MCF-7 cells were seeded at 6,00,000 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 IGF-1 stimulation. After 10 min of
IGF1 stimulation, cells are lysed with Hepes 50 mM pH 7.6, Triton X100 1%,
orthovanadate 2 mM, protease inhibitors. Cell lysates are incubated on 96-
multiwell plates pre-coated with anti-IGF-1R antibody, followed by incubation
with an anti-phosphotyrosine antibody coupled to peroxydase enzyme.
be acylated or sulfonylated to afford compounds 26, 36 and 42
(Scheme 4).
Finally a series of hydantoins could be obtained from the 2-cya-
nopyridine hydantoin derivative 24 after hydrolysis to the corre-
sponding carboxylic acid 25 followed by amide formation with
methylamine to afford 37. Alternatively it could be directly hydro-
lyzed to the primary amide 28 (Scheme 5).
Peroxidase activity level (measured by OD with
reflects receptor phosphorylation status.
a luminescent substrate)
A lead from HTS was optimized and characterized as a noncom-
petitive slow binding inhibitor of IGF-1R. To our knowledge, this is
the first reported IGF-1R DFG-out kinase inhibitor. The potential
advantages of these types of inhibitors both in terms of selectiv-
ity16 and sustained effects17 (in vitro and in vivo) could be quite
considerable. In addition slow kinetics often leads to extended tar-
get occupancy and improved therapeutic responses. The optimiza-
tion of this series bears promises for the treatment of IGF-related
malignancies.
IGF-1-Induced MEF cell proliferation: MEF (Mouse Embryo Fibroblast 3T3) Tet off
cells (BD Bioscience) were stably transfected with IGF-1R tetracycline-
regulatable expression plasmid. MEF-IGF-1R cells were seeded in a 96-well
Cytostar microplates (Amersham) at 5000 cells/well under 0.2 ml of EMEM
culture medium and incubated at 37 °C for 18 h. After this period of time, cells
were washed twice with serum-free EMEM and left in this culture medium for
24 h. Compounds were then added in the presence of rhIGF1 (100 ng/ml; RD
Systems) and 0.1 l
Ci of Thymidine [methyl-14C]. After an incubation time of
72 h in the presence of the compounds, at 37 °C under 5% CO2, the 14C-
thymidine incorporation was measured on a Micro-beta counter (Perkin–
Elmer).
10. The crystal structure of 2 with IGF-1R has been deposited in the PDB with
accession code 3O23.
11. For precedents of kinase DFG-out inhibitors see for instance: Liu, Y.; Grey, N. S.
Nat. Chem. Biol. 2006, 2, 358; Liao, J. J.-L. J. Med. Chem. 2007, 50, 409.
12. For general reviews on slow binding inhibitors see for instance: Morrison, J. F.;
Walsh, C. T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988, 61, 201; Schloss, J. V. Acc.
Chem. Res. 1988, 21, 348.
13. Patek, M.; Nair, A.; Hittinger, A.; Nemecek, C.; Bond, D.; Harlow, G.; Bouchard,
H.; Mauger, J.; Malleron, J.-L.; Palermo, M.; Al_Obeidi, F.; Strobel, H.; Faitg, T.;
Strobel, H.; Ruf, S.; Ritter, K.; El-Ahmad, Y.; Lesuisse, D.; Benard, D. PCT Patent
Publication N WO2004/070050 A2, August 19th 2004.
Acknowledgements
We are grateful to Bill Metz and Carlos Garcia-Echeverria for
their help on reviewing the manuscript and to the analytical
departments for compound analyses.
References and notes
14. Patek, M.; Nahir, A.; Hittinger,A.; Nemecek, C.; Bond, D.; Harlow, G.; Bouchard,
H.; Mauger, J.; Malleron, J.-L.; Palermo, M.; Al-Obeidi, F.; Faitg, T.; Strobel, H.;
Ruf, S.; Ritter, K.; El-Ahmad, Y.; Lesuisse, D. US2004/248884 A1.
15. The starting bromomethylpyridine precursors of compounds 18 and 24 were
commercially available; the precursors of compounds 30, 35 and 38 were
prepared from the corresponding methyl ester after sodium borohydride
reduction and dibromotriphenylphosphine bromination; finally the 2-fluoro-
4-bromomethyl-pyridine was prepared from the 4-methyl analog after
benzoylperoxide-catalyzed NBS bromination.
16. See for instance: Liu, H.; Kuhn, C.; Feru, F.; Jacques, S. L.; Deshmukh, G. D.; Ye,
P.; Rennie, G. R.; Johnson, T.; Kazmirski, S.; Low, S.; Coli, R.; Ding, Y.-H.; Cheng,
A. C.; Tecle, H.; English, J. M.; Stanton, R.; Wu, J. C. Bioorg. Med. Chem. Lett. 2010,
20, 4885.
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6. Briefly, the activities were determined as follows: IGF-1R–HTRF: Inhibition of
autophosphorylation activity was determined using
a
time-resolved