The discovery of substituted 4-(3-hydroxyanilino)-quinolines as potent RET kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.07.104

Substituted 4-(3-hydroxyanilino)-quinoline compounds, initially identified as small-molecule inhibitors of src family kinases, have been evaluated as potential inhibitors of RET kinase. Three compounds, 38, 31, and 40, had K_i's of 3, 25, and 50

Full text of DOI:10.1016/j.bmcl.2007.07.104

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5886–5893
The discovery of substituted 4-(3-hydroxyanilino)-quinolines
as potent RET kinase inhibitors
R. Graham Robinett,a Alex J. Freemerman,b Michael A. Skinner,b,
Lisa Shewchuka and Karen Lackeya,*
aGlaxoSmithKline, Molecular Discovery Research Chemistry, 5 Moore Drive, Research Triangle Park, NC 27709, USA
bDuke University Medical Center, Department of Surgery, Durham, NC 27710, USA
Received 22 June 2007; revised 26 July 2007; accepted 27 July 2007
Available online 25 August 2007
Abstract—Substituted 4-(3-hydroxyanilino)-quinoline compounds, initially identified as small-molecule inhibitors of src family
kinases, have been evaluated as potential inhibitors of RET kinase. Three compounds, 38, 31, and 40, had Ki’s of 3, 25, and
50 nM in an in vitro kinase assay; while a cell based kinase assay showed Ki’s of 300, 100, and 45 nM, respectively. These compounds
represent potential new leads for the treatment of medullary and papillary thyroid cancer.
ꢀ 2007 Elsevier Ltd. All rights reserved.
RET (Rearranged in Transcription) is a receptor tyro-
sine kinase expressed in the thyroid gland, elements of
the peripheral and central nervous systems, the kidney,
and adrenal glands.1 Gain-of-function mutations in
RET can be sporadic or inherited and can lead to certain
types of cancer.2 Inherited point mutations in RET are
associated with multiple endocrine neoplasia 2A
(MEN2A), MEN2B, and familial medullary thyroid car-
cinoma (FMTC).2 Individuals with these cancer syn-
dromes develop various types of tumors, but the most
virulent type of tumor, and common to all three syn-
dromes, is medullary thyroid cancer (MTC).3 A hall-
mark of MTC is a constitutively activated RET and
we hypothesize that it is this RET signaling that renders
these tumors generally unresponsive to chemotherapy
and radiation through a mechanism of apoptosis
inhibition.3
inhibition, and all have some drawbacks for use in che-
motherapy. In an in vitro RET kinase assay, PP1 and
PP2 have reported IC50 values of 80 and 100 nM, respec-
tively.6,9 However, PP1 and PP2 also inhibit numerous
other kinases at similar or lower concentrations than
their activity in a RET kinase assay.9 ZD6474, a
targeted VEGFR2 inhibitor, has a reported IC50 value
of 100 nM for RET and is in clinical trials as an
anti-angiogenesis agent used to treat metastatic lung,
breast, and colon cancer, and more recently to treat
MTC.7 BAY 43-9006 (Sorafenib) was developed as a
Raf/VEGFR2 dual kinase inhibitor, but it also inhibits
many other kinases, and has recently also been shown
to inhibit RET.8 However, like ZD6474, clinical trials
with Sorafenib have shown some significant side effects
including hand–foot skin reactions, fatigue, and
hypertension.10
Recent reports have examined the ability of well-known
kinase inhibitors to inhibit RET activity as an added
activity over their intended target.4 For example, Pyrazol-
o-pyrimidines, PP15 and PP2,6 ZD6474 (Vandetanib),7
and BAY 43-9006 (Sorafenib)8 demonstrated RET kinase
Based on the evidence of RET kinase inhibition as a via-
ble approach, we believe the design of a small-molecule
inhibitor of RET would be an ideal targeted therapeutic
agent with the potential for a complementary inhibition
profile with reduced side effects. The strategy for identi-
fying a novel RET kinase inhibitor is summarized in
Figure 1. We started with a small set of known kinase
inhibitors with a broad range of potent activity across
a panel of >35 kinase assays. The idea was to use the
active compounds identified from this generic set to
provide a template to search the GlaxoSmithKline
compound collection for more novel, selective, and
Keywords: c-src; RET; Kinase inhibitors; 4-Anilinoquinolines.
*
Corresponding author. Tel.: +1 919 483 6240; e-mail:
karen.e.lackey@gsk.com
Present address: UT Southwestern Medical Center, Children’s
Medical Center of Dallas, Room B03-250, 1935 Motor Street, Dallas,
TX 75235, USA.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.104

R. Graham Robinett et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5886–5893
5887
Table 1. KisCollab set
Quinoline Series
KisCollab
27 compounds
32 compounds
Structure
Compound
Substructure
Searching based
On active compounds
Testing in Enzyme &
provided SAR
Cellular Assays
OH
O
111
211
3
HO
O
Quinoline Leads
4 compounds
With desired cellular activity
Scale-up &
In Vivo Studies
2 compounds
In silico methods
& PK data used to
Evaluated best
OH
OH
O
O
Ret kinase inhibitors
Figure 1. Strategy for rapid identification of a ret kinase inhibitor.
HO
F
Cl
N
potent RET kinase inhibitors. The next iteration of test-
ing would be done to establish SAR, and the top com-
pounds were selected based on cellular activity. In this
report, we will describe the compound selection, synthe-
sis, and SAR that led to the identification of potent RET
kinase inhibitors.
N
N
S
H
N
S
N
N
N
O
412
512
6
The initial set of 27 compounds, named KisCollab
shown in Table 1, was chosen based on their potent ki-
nase inhibitory activity. The molecules listed in Table 1
were prepared according to published accounts, where
the unreferenced compounds are available commer-
cially.11–18 Compounds were selected as a set intending
to show activity in every kinase in the assay panel of
>35 kinases, while individually, providing selective and
potent coverage of a few panel members. For example,
compound 25 inhibits 2 of 18 kinases tested at a
pIC50 > 7.0. Also compound 13 inhibits 5 of 14 kinases
tested at a pIC50 > 7.0, both sets providing mutually
exclusive inhibitory coverage of different kinases in the
internal assay panel. The KisCollab set was tested at a
single concentration in the RET kinase enzyme assay
in triplicate to determine active compounds.19 The data
are shown in Table 2 for only the active compounds.
This initial screen provided four potential series for fur-
ther evaluation. Using the substitution patterns on the
active compounds and selecting the quinoline as the core
scaffold, the GlaxoSmithKline compound collection was
searched for more novel and potent RET kinase inhibi-
tors. We chose the quinoline scaffold because there was
evidence that we would select drug-like molecules.20
S
O
NH2
O
N
H
H
N
N
N
O
S
O
O
NH2
N
N
N
S
N
N
O
N
N
N
712
812
912
1013
N
S
O
N
O
O
N
Br
Cl
N
O
N
N
N
N
N
S
O
O
O
Syntheses to generate the compounds in the quinoline
series listed in Tables 3–5 are summarized in Schemes
1 and 2. The 6-bromo-4-chloroquinoline starting mate-
rial shown in Scheme 1 can be obtained from commer-
cial vendors. The introduction of a 6-aryl group is well
precedented by using standard Suzuki conditions using
1,4-dioxane and palladium dichloroditriphenyl phos-
phine on the functionalized core quinoline.21 The result-
N
N
Br
O
N
H
N
ing 6-aryl-4-chloroquinoline can then undergo
a
N
1114
displacement reaction with a variety of anilines in sol-
vents such as DMSO and acetonitrile to yield the 4-ani-
lino-6-aryl substituted quinolines. 7-Position substituted
quinolines were generated using commercially available
4-chloro-7-iodoquinoline using the same methodology
as the 6-position analogs.
O
N
O
O
S
H
1215
S
N
HN
N
N
N
S
O
To synthesize the 6- and 7-position disubstituted com-
pounds, an exemplary synthetic scheme for generating 30
is summarized in Scheme 2. Amine 61 was synthesized
O
(continued on next page)

5888
R. Graham Robinett et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5886–5893
Table 1 (continued)
Table 1 (continued)
Structure
Compound
Structure
Compound
F
O
Cl
N
N
O
N
N
1316
2418
O
S
O
O
O
OH
O
S
Cl
N
O
O
F
2518
N
HN
1415
HN
N
Cl
O
O
N
N
O
N
N
N
H
N
Br
HN
S
26
O
N
1512
1612
1712
1812
1912
2012
O
O
S
O
O
N
H
N
H
N
N
S
S
N
N
N
N
N
NH
O
2712
S
S
O
O
O
O
O
S
N
O
N
H
N
H
N
OH
NH
O
Table 2. Compounds with activity <1500 cpm tested at 5000 nMa
O
N
H
Compound
cpm
H
N
O
S
6
7
950 ( 142)
1208 ( 130)
1312 ( 41)
428 ( 23)
355 ( 44)
298 ( 7)
S
N
O
O
15
18
23
25
13
N
H
N
O
N
N
O
O
460 ( 55)
S
N
O
a Values of counts per minute (cpm) are means of three experiments,
standard deviation is given in parentheses.19
N
N
O
N
O
Table 3. Trisubstituted 4-anilinoquinoline
S
NH2
H2N
S
O
R1
O
O
HN
N
R3
R2
Cl
N
O
2117
Compound R1
R2
R3
IC50 (nM)
100
Cl
Cl
S
O
7 –
6 –
28
3-Cl
N
O
O
N
N
Br
OH
Br
29
2-CF3
>1000
2217
O
O
S
N
N
O
N
from commercially available 2-bromo-1-fluoro-4-nitro-
benzene. The fluoro substituent of the nitrobenzene
was displaced using sodium sulfoxide followed by reduc-
tion of the nitro group using iron/acetic acid. The key
intermediate, 6-methylsulfonyl-4-chloro-7-bromoquino-
line, was synthesized using well-established procedures
from the functionalized aniline.22–24 Anilines can be
Br
OH
Br
O
O
S
2313
H2
N
O
N
N

R. Graham Robinett et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5886–5893
Table 4. 6- or 7-substituted 4-anilinoquinoline
5889
R1
HN
N
R2
R3
Compound
R1
R2
R3
IC50 (nM)
O
F
Cl
30
H
H
H
H
H
75
25
25
75
6
O
N
N
N
31
32
33
34
35
36
37
38
39
S
O
O
O
Cl
F
N
O
S
N
O
O
O
O
O
O
N
H
80
95
90
3
N
O
N
H
H
H
6 –
N
O
S
6 –
O
O
S
O
Br
O
H
85
O
introduced in the 4-position with the aforementioned
displacement reaction, followed by a Pd coupling reac-
tion to install the heterocycle in the 7-position. A variety
of amines can be used to probe the SAR by generating a
diverse set via a reductive amination step.
Generally speaking, there appeared to be considerable
tolerance for substitution in the 6- and 7-position. Based
on experience with other kinase inhibitor drug discovery
programs, we believed we could use these as attachment
points to examine improved cellular activity.26 Table 4
includes representative examples of the active quinoline
compounds tested where either the 6- or 7-position has a
substituent. All compounds tested with 7-position sub-
stitution contain the key 3-phenol pharmacophore in
the 4-anilino substituent. Additional substitution on
the 4-anilino portion is well tolerated as seen in exam-
ples 30, 31, and 32. Compounds 33 (IC50 value 75 nM)
and 32 (IC50 value 6 nM) differ by a single methyl sub-
stituent in the 2-position of the 4-anilino portion of
All compounds tested with a substituent in the 2-posi-
tion of the 4-anilinoquinoline series showed no activity
at the concentrations tested. The 3-position substituents
retained activity, but did not offer any advantages in
binding properties. Two representative examples are
shown in Table 3. In an era of emphasis on ligand effi-
ciency,25 we opted to keep 2- and 3-position as unsubsti-
tuted in our pursuit of a RET kinase inhibitor.

5890
R. Graham Robinett et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5886–5893
Table 5. 4-Anilino-6,7-disubstituted quinoline
added potency advantage when compared with the hal-
ogen substituent.
R1
HN
R2
A model of 40 bound into the ATP binding pocket of
RET kinase is shown in Figure 2 and compares favor-
ably with the structures published by Phillip P. Knowles
and co-workers.27 The sulfone appears to be within
hydrogen bonding distance of the backbone NH and
side chain hydroxyl of Ser811 as well as the side chain
of Arg878. A sulfonamide at the analogous position sig-
nificantly increased the potency of CDK2 inhibitors, so
it is reasonable to conclude that this interaction is
important in boosting affinity.12 The meta-substituted
4-aniline group likely interacts with the side chains of
Asp892 and Lys758 as observed in other quinoline struc-
tures.28 Potency is often achieved by kinase inhibitors
that bind in the ATP binding pocket by interacting with
the side chain located on the first turn of the helix at the
solvent interface. In RET, this residue is a glycine, mak-
ing the potential interactions minimal. Longer substitu-
ents, like the morpholinomethylthiazole, at the
7-position could potentially interact with Glu818, while
shorter groups, like the thiophene carbaldehyde, may
interact directly with the backbone of the hinge. How-
ever, since these positions are very solvent exposed, it
is difficult to predict the strength of these types of inter-
actions. There are no cysteine or serine residues close to
the aldehyde that could form a covalent bond (Ser411 is
R3
N
Compound R1
R2
R3
IC50
(nM)
O
O
40
N
34
S
N
O
F
O
S
O
O
N
41
Cl
>1000
O
Cl
the molecule and show a log difference in potency.
Changes in the 6-position, 35, 36, and 37, show no sig-
nificant difference in potency, all with IC50 determina-
tions in the range of ꢀ85 nM. However, the methyl
sulfone in the 6-position, as in 38, results in a log in-
crease in potency compared to compounds 35, 36, and
37.
Finally, Table 5 summarizes examples of compounds
where both the 6- and 7-position are simultaneously
substituted. While disubstitution of this ring is tolerated,
it appears that the methyl sulfone confers a specific
˚
ꢀ4 to 5 A). Substitutions in the 2-position are not
accommodated since the quinoline ring packs tightly
against the back of the pocket. Small substituents in
Cl
Ar
Cl
HN
Br
1,4-Dioxan
Ar
Y
R
+
Ar
N
PdCl2(PPh3)2
N
N
Y = SnBu3, B(OH)2
R = aryl
Scheme 1. Reagents and conditions for 4,6-substituted quinolines.
O
S
O
S
F
Br
b
c
a
O
O
NO2
Br
NO2
Br
NH2
Cl
N
O
S
OH
HN
N
O
S
d
OH
HN
O
S
e
O
O
N
O
O
Br
Br
N
H
S
OH
HN
O
S
O
f
O
N
N
N
S
30
Scheme 2. Reagents and conditions: (a) MeSO2Na, DMA; (b) Fe/ACOH; (c) Na2CO3/H2O; (d) 3-amino-4-methylphenol, AcCN, D;
(e) 4-(tributylstannanyl)-1,3-thiazole-2-carbaldehyde, Pd(PPh3)2Cl2, Ag2O, dioxane; (f) Morpholine, Mol. Sieves, AcOH, NaBH(AcO)3, NaHCO3.

R. Graham Robinett et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5886–5893
5891
Panel A: kinase selectivity for 38
10
9
8
7
6
*
*
*
*
*
*
*
* * * *
*
*
*
*
5
*
*
4
assay name
Panel B: kinase selectivity for 31
10
9
8
7
6
5
4
*
* *
* *
* *
*
*
* * * *
*
Figure 2. Model of 40 bound in the active site of RET based on the
protein coordinates of PDB2IVV. The figure was generated with
PYMOL.
assay name
Panel C: kinase selectivity for 40
10
9
nM of 38
nM of 31
nM of 40
8
7
-pRET
6
*
*
*
*
* *
* *
* *
*
-RET
5
*
*
*
-Actin
4
assay type
Figure 3. Western blot showing dose–response of RET inhibition by
compounds.
Figure 4. Selectivity profile of representative assays for 38 (panel A),
31 (panel B) and 40 (panel C). Denotes maximum concentration
*
tested not pIC50 value determined.
the 3-position are tolerated due to the presence of the
small gatekeeper residue, valine.
mutation, whereas MZ-CRC-1 cells are human MTC
where RET kinase is constitutively activated by the
MEN2B mutation. Western blots of TT and MZ-
CRC-1 cell lysates treated with 0.5 lM kinase inhibitor
for increasing amounts of time from 30 min to 24 h
are included in the Supplementary data. The data dem-
onstrate the inhibition of mutant RET phosphorylation
in both cell lines compared to a non-treatment control
(Supplementary data).
Three compounds (31, 38, 40) were selected for further
evaluation in a ret kinase cellular assay. The data are
shown in Figure 3 as a Western blot with increasing con-
centration of each inhibitor and using antibody detec-
tion.29 The assay uses SK-N-MC cells that express a
chimeric EGFR/RET protein (EGFR is extracellular,
RET is intracellular). The cells are incubated for 1 h
with compound before activating RET by adding
100 ng/mL of EGF for 15 min. The actin levels were
used to demonstrate equivalent loading of total protein.
The phosphorylated levels of a chimeric EGFR-RET
protein were measured to determine the effectiveness of
each RET kinase inhibitor’s ability to block a signaling
event. In a dose responsive manner, each of these three
compounds demonstrated potent activity with approxi-
mate ED50 values in the 45–300 nM range. The protein
levels (RET) were compared with the inhibition of phos-
phorylation (pRET) to be certain that only the signaling
was inhibited, as opposed to cytotoxicity or other mech-
anism that might reduce the phosphorylation levels. It is
noteworthy that these compounds were also effective in
cellular systems that contained the MEN2A and
MEN2B mutations. TT cells are human MTC where
RET kinase is constitutively activated by the MEN2A
Another important factor to examine was the overall ki-
nase inhibition profile for these effective ret kinase inhib-
itors. A summary bar graph of a representative set of
kinase assays is shown in Figure 4. The inhibition pat-
tern across the different kinase families suggests that
these three compounds are quite similar. The other tar-
gets inhibited include c-src,30 lyn,
31
VEGFR2,32 and
TIE232 which are also potentially important anticancer
targets.
We used a strategy to identify a ret kinase inhibitor that
included a small kinase probe set of 27 compounds cre-
ated by selecting compounds whose inhibition profiles
covered a panel of >35 kinases. The seven active com-
pounds found from screening this generic set allowed

5892
R. Graham Robinett et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5886–5893
W. J.; Rusnak, D.; Shewchuk, L.; Veal, J. M.; Walker, D.
H.; Kuyper, L. F. J. Med. Chem. 2001, 44, 4339.
13. Cheung, M.; Hunter, R. N., III; Peel, M. R.; Lackey, K.
E. Heterocycles 2001, 55, 1583.
14. Mologni, L.; Sala, E.; Cazzaniga, S.; Rostagno, R.;
Kuoni, T.; Puttini, M.; Bain, J.; Cleris, L.; Redaelli, S.;
Riva, B.; Formelli, F.; Scapozza, L.; Gambacorti-Passe-
rini, C. J. Mol. Endocrinol. 2006, 37, 199.
15. Brignola, P. S.; Lackey, K.; Kadwell, S. H.; Hoffman, C.;
Horne, E.; Carter, H. L.; Stuart, J. D.; Blackburn, K.;
Moyer, M. B.; Alligood, K. J.; Knight, W. B.; Wood, E.
R. J. Biol. Chem. 2002, 277, 1576.
us to rapidly identify the quinoline series with the appro-
priate substitutions that confer potent RET kinase
inhibition. Several compounds, 31, 38, and 40, demon-
strated potent inhibition of intracellular autophosphory-
lation in a ligand stimulated chimeric assay system.
Finally, these compounds had similar kinase inhibition
profiles that included important anticancer targets such
as c-src, VEGFR2, TIE2, and lyn. They will be evalu-
ated in more extensive models of efficacy and mechanis-
tic information in tumors driven by RET activity.33
16. Synthesis is included in this paper, and in a pending patent
application publication.
Acknowledgments
17. Lackey, K.; Cory, M.; Davis, R.; Frye, S. V.; Harris, P.
A.; Hunter, R. N.; Jung, D. K.; McDonald, O. B.;
McNutt, R. W.; Peel, M. R.; Rutkowske, R. D.; Veal, J.
M.; Wood, E. R. Bioorg. Med. Chem. Lett. 2000, 10, 223.
18. Hudson, A. T.; Vile, S.; Barraclough, P.; Franzmann, K.
W.; McKeown, S. C.; Page, M. J. PCT Int. Appl. 1996,
WO9609294 A1.
Kinase selectivity data were supplied by the Molecular
Discovery Chemistry, GlaxoSmithKline enzyme screen-
ing department. Kinase inhibition data for Table 1 are
included in Supplementary data.
19. The ret kinase assay used the intracellular portion of ret
(amino acids 713–1072 of the short isoform) produced in
SF9 insect cells and purified on a nickel column. The assay
Supplementary data
was performed in
a 45 lL volume with increasing
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.07.104.
concentrations of tyrosine kinase inhibitor (TKI) in
triplicate in a 96-well plate format as follows: 4.5 lL of
500 mM morpholinopropanesulfonic acid (500 mM), pH
7.4; 4.5 lL of 100 lM adenosine triphosphate 4.5lL of
250 mM MgCl2, 1.0 lL c-ATP33 (1 lCi) (3000 Ci/mmol);
2.0 lL (5 mg/mL) poly Glutamic acid-Tyrosine (Sigma (P-
0275)); 23 lL H20; 1.0 lL recombinant ret (100 ng); 4.5 lL
of 10· Tyrosine kinase inhibitor. The components were
incubated in triplicate for 30 min at room temperature
within a 96-well plate in triplicate. The reaction was
stopped by adding 45 lL of 0.5% H3PO4 to each well.
Seventy-five microliters of the reaction mixture was
spotted onto phosphocellulose paper (Millipore, cat #
MAPHN0B50) and was washed three times with 200 lL
of 0.5% H3PO4. Each phosphocellulose filter was trans-
ferred into a separate scintillation vial, 1 mL cytoscint
fluid was added, and the sample counted on a Packard
1900TR liquid scintillation analyzer for 1 min.
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acids 658–1072]). The human neuroblastoma cell line,

R. Graham Robinett et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5886–5893
5893
SK-N-MC, was utilized to establish cell lines expressing
the EGFR/RET construct and an empty vector control.
The assay was performed by pre-incubating EGFR/RET-
expressing cells for 30 min in serum-free MEM with
increasing concentrations of GW569998X, GW585550A,
or GW559768X and then activating RET by the addition
of human epidermal growth factor (EGF) (100 ng/mL) for
10 min. Experimental incubations were terminated by
collecting and directly lysing cells in 1· Laemmli (1%
SDS, 10% glycerol, 100 mM dithiothreitol, and 50 mM
Tris, pH 6.8) for Western blots. Western blots were
sequentially probed for phospho-RET (Tyr-1062-R),
RET9 (C-19) (Santa Cruz Biotechnology, Santa Cruz,
CA), and actin (MAB1501) (Chemicon International,
Temecula, CA). Yeatman, T. J. Nat. Rev. Cancer 2004,
4, 470–480.
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33. More indepth biological evaluation of these ret kinase
inhibitors is included in a submitted manuscript by
Michael A. Skinner, Jeffrey F. Moley, Karen E. Lackey,
and Alex J. Freemerman, titled RET oncogene
activation raises the apoptotic threshold in SK-N-MC
cells.