Design, synthesis, and biological activity of urea derivatives as anaplastic lymphoma kinase inhibitors

Article abstract of DOI:10.1002/cmdc.201100168

In anaplastic large-cell lymphomas, chromosomal translocations involving the kinase domain of anaplastic lymphoma kinase (ALK), generally fused to the 5' part of the nucleophosmin gene, produce highly oncogenic ALK fusion proteins that deregulate cell cycle, apoptosis, and differentiation in these cells. Other fusion oncoproteins involving ALK, such as echinoderm microtubule-associated protein-like 4-ALK, were recently found in patients with non-small-cell lung, breast, and colorectal cancers. Recent research has focused on the development of inhibitors for targeted therapy of these ALK-positive tumors. Because kinase inhibitors that target the inactive conformation are thought to be more specific than ATP-targeted inhibitors, we investigated the possibility of using two known inhibitors, doramapimod and sorafenib, which target inactive kinases, to design new urea derivatives as ALK inhibitors. We generated a homology model of ALK in its inactive conformation complexed with doramapimod or sorafenib in its active site. The results elucidated why doramapimod is a weak inhibitor and why sorafenib does not inhibit ALK. Virtual screening of commercially available compounds using the homology model of ALK yielded candidate inhibitors, which were tested using biochemical assays. Herein we present the design, synthesis, biological activity, and structure-activity relationships of a novel series of urea compounds as potent ALK inhibitors. Some compounds showed inhibition of purified ALK in the high nanomolar range and selective antiproliferative activity on ALK-positive cells.

Full text of DOI:10.1002/cmdc.201100168

MED
DOI: 10.1002/cmdc.201100168
Design, Synthesis, and Biological Activity of Urea
Derivatives as Anaplastic Lymphoma Kinase Inhibitors
Gustav Boije af Gennꢀs,[a] Luca Mologni,[b] Shaheen Ahmed,[c] Mohanathas Rajaratnam,[a]
Oriano Marin,[d] Niko Lindholm,[a] Michela Viltadi,[b] Carlo Gambacorti-Passerini,*[b]
Leonardo Scapozza,*[c] and Jari Yli-Kauhaluoma*[a]
In anaplastic large-cell lymphomas, chromosomal transloca-
tions involving the kinase domain of anaplastic lymphoma
kinase (ALK), generally fused to the 5’ part of the nucleophos-
min gene, produce highly oncogenic ALK fusion proteins that
deregulate cell cycle, apoptosis, and differentiation in these
cells. Other fusion oncoproteins involving ALK, such as echino-
derm microtubule-associated protein-like 4-ALK, were recently
found in patients with non-small-cell lung, breast, and colorec-
tal cancers. Recent research has focused on the development
of inhibitors for targeted therapy of these ALK-positive tumors.
Because kinase inhibitors that target the inactive conformation
are thought to be more specific than ATP-targeted inhibitors,
we investigated the possibility of using two known inhibitors,
doramapimod and sorafenib, which target inactive kinases, to
design new urea derivatives as ALK inhibitors. We generated a
homology model of ALK in its inactive conformation com-
plexed with doramapimod or sorafenib in its active site. The
results elucidated why doramapimod is a weak inhibitor and
why sorafenib does not inhibit ALK. Virtual screening of com-
mercially available compounds using the homology model of
ALK yielded candidate inhibitors, which were tested using bio-
chemical assays. Herein we present the design, synthesis, bio-
logical activity, and structure–activity relationships of a novel
series of urea compounds as potent ALK inhibitors. Some com-
pounds showed inhibition of purified ALK in the high nanomo-
lar range and selective antiproliferative activity on ALK-positive
cells.
Introduction
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase
(RTK) that is composed of an extracellular ligand binding
domain, a putative transmembrane domain, and a cytoplasmic
kinase domain.[1] Among the RTKs, the extracellular domain of
ALK is most similar to that of leukocyte tyrosine kinase (LTK),[2]
which is a member of the superfamily of insulin receptors. ALK
plays an important role in the development of the central and
peripheral nervous system.[3] However, ALK can be aberrantly
activated as a result of the (2;5)(p23;q35) chromosomal trans-
location in anaplastic large-cell lymphomas (ALCLs).[4,5] In most
cases, the translocation causes fusion of the intracellular cata-
lytic domain of ALK with the oligomerization domain of nucle-
ophosmin (NPM). The resulting NPM–ALK fusion product has
constitutive kinase activity and is highly oncogenic.[6] Several
additional fusion partners that have dimerization domains
have been described.[7] Oncogenic mutants or fusion variants
of ALK have also been identified in neuroblastomas, inflamma-
tory myofibroblastic tumors and diffuse large B-cell lympho-
ma.[1] Furthermore, the echinoderm microtubule-associated
protein-like 4 (EML4)–ALK fusion gene was recently discovered
to be expressed in a subset of non-small-cell lung cancers
(NSCLCs), breast and colorectal cancers.[8,9]
lymphomas (NHL) in this age group. Although ALK-positive
ALCL patients are responsive to cytotoxic drugs, relapses occur
frequently and bear a dismal prognosis.
There are several reasons for the increase in ALK research for
the treatment of ALCLs. Importantly, ALK expression is restrict-
ed to rare scattered neural cells.[11] Despite the role of ALK in
[a] G. Boije af Gennꢀs,+ Dr. M. Rajaratnam, N. Lindholm,
Prof. J. Yli-Kauhaluoma
Division of Pharmaceutical Chemistry
Faculty of Pharmacy, University of Helsinki
P.O. Box 56 (Viikinkaari 5E), 00014 Helsinki (Finland)
Fax: (+358)9-191-59556
E-mail: jari.yli-kauhaluoma@helsinki.fi
[b] Dr. L. Mologni,+ M. Viltadi, Prof. C. Gambacorti-Passerini
Department of Clinical Medicine and Prevention
University of Milano-Bicocca, Via Cadore 48, 20052 Monza (Italy)
Fax: (+39)039-233-3539
E-mail: carlo.gambacorti@unimib.it
[c] Dr. S. Ahmed,+ Prof. L. Scapozza
Group of Pharmaceutical Biochemistry
School of Pharmaceutical Sciences, University of Geneva
Quai Ernest-Ansermet 30, 1211 Genꢁve 4 (Switzerland)
Fax: (+41)22-379-3360
E-mail: leonardo.scapozza@unige.ch
[d] Prof. O. Marin
Sixty to eighty percent of ALCLs are ALK-positive, and 70–
80% of ALK-positive ALCLs express the NPM–ALK fusion pro-
tein.[10] ALK-positive ALCL tends to affect children and favor
males. Primary systemic ALCL has a peak incidence in children
or young adults, accounting for ~20–30% of non-Hodgkin
Peptide Facility, CRIBI-Biotechnology Centre
University of Padova, Viale Colombo 3, 35131 Padova (Italy)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100168.
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Urea Derivatives as ALK Inhibitors
development and expression patterns, ALK-deficient mice
appear normal and display no visible tissue abnormalities.[12]
Therefore, minimal side effects result from ALK inhibition. In
addition, children show more ALK-positive ALCLs than adults,
encouraging the search for safe treatments that will be effec-
tive for long-term use. Another important reason for research
of ALK is that immunological responses elicited against ALK
will probably not produce a relevant autoimmune disease. Fi-
nally, new inhibitors are needed to combat mutations in the
oncogene. For instance, the c-Met/ALK inhibitor crizotinib is
the first agent in phase III clinical trials that selectively targets
EML4–ALK in NSCLC.[13]
Sorafenib, a multi-kinase inhibitor that targets VEGFR,
PDGFR, KIT, FLT-3 and RET receptor tyrosine kinases, MAP kinas-
es, and Raf kinases, was approved in 2006 for the treatment of
advanced renal cell carcinoma (primary kidney cancer). Dora-
The majority of RTK inhibitors target the highly conserved
ATP binding pocket[14] and have a high risk of producing side
effects.[15] Therefore, to obtain kinase specificity, inhibitors that
also bind to less conserved residues outside the ATP binding
pocket are desirable.[16] In addition, inhibitors that target the
inactive conformation of kinases would have increased specif-
icity.[17,18]
mapimod (BIRB-796) is a potent inhibitor of MAP kinases with
an IC50 value of 63 nm. It also inhibits other kinases, such as
Abl kinase, with considerable potency. This compound was
tested in phase II clinical trials for patients of rheumatic arthri-
tis, Crohn’s disease, and psoriasis,[25] but was withdrawn owing
to liver toxicity.[26] In this study, a novel series of urea com-
pounds, which were derived from sorafenib and doramapimod
as well as hits from virtual screening of the ZINC database,
were designed, synthesized, and tested for ALK inhibition.
Doramapimod is a weak inhibitor of ALK (IC50 =45 mm),
whereas sorafenib does not inhibit ALK and only weakly inhib-
its Abl (IC50 =25 mm), but is a potent inhibitor of RET (IC50 =
6 nm).[17] Doramapimod and sorafenib bind the inactive confor-
mation (DFG-Asp-out) of p38 MAP kinase (Figure 1A and Fig-
ure 2B).[27,28] Sorafenib also binds the inactive conformation of
B-Raf.[29] To elucidate the activity of these inhibitors toward
ALK, doramapimod and sorafenib were docked into the active
site of a homology model of ALK in its inactive (DFG-Asp-out)
conformation. Superposition of the model of inactive ALK with
the crystal structures of ALK containing NVP-TAE684 (PDB ID:
2XB7)[22] and missing the activation loop yielded an overall
root mean square deviation (RMSD) of 0.68 ꢂ, indicating very
good agreement between the model and the recently solved
crystal structures. The primary difference between the active
site of the model and that of the crystal structure is the confor-
mation of the DFG motif, which is in the DFG-Asp-in conforma-
tion (active) in the X-ray structure and in the inactive DFG-Asp-
out conformation in the model. Furthermore, the model and
structure differ in the P-loop, which adopts a different confor-
mation depending on the inhibitor bound.[19,20] Figure 1B
shows the docking pose of doramapimod bound to inactive
ALK superimposed on the X-ray structure of the compound
bound to p38 MAP kinase (PDB ID: 1KV2). There are some sig-
nificant differences in these two binding modes, which may
explain why doramapimod is a weak inhibitor of ALK. The
bulky gatekeeper residue in ALK (leucine), in contrast to that
of p38 MAP kinase (threonine), prevents the naphthyl ring of
doramapimod from adopting the same conformation as ob-
served when bound to p38 MAP kinase. Thus, molecular dock-
ALK is a clinically relevant target, and there is a need for
new therapeutics for the treatment of ALK-positive tumors.
Drug resistance, nonspecific binding by ATP-competitive inhibi-
tors, and poor life expectancy for the patients involved are the
primary reasons we sought to develop novel ALK-targeted in-
hibitors. Herein we present a novel series of urea compounds
as potent ALK inhibitors, some of which show activity in both
purified ALK and cellular assays.
Results and Discussion
Design of urea compounds as potent NPM–ALK inhibitors
A model of ALK corresponding to the inactive conformation of
the catalytic site was generated using mouse c-Abl (PDB IDs:
1IEP, 1OPJ; 34% identity with ALK amino acid sequence) and
human insulin receptor (PDB ID: 1IRK; 40% identity with ALK)
as templates. The model was built before X-ray crystal struc-
tures of ALK were available; however, the overall structure is
very close to the published one, the only difference being that
the X-ray structure shows a DFG-Asp-in conformation, typical
of an active conformation.[19,20]
The ZINC database,[21] a free database of commercially avail-
able compounds, was virtually screened by using our homolo-
gy model of ALK (see Experimental Section for details). This ini-
tial screen yielded a series of potential ALK inhibitors. Among
the top-ranked hits, several urea-derived molecules were iden-
tified, and they were chosen as starting points for the develop-
ment of derivatives with increased potency and specificity, as
there is structural data available on how related compounds
from this class (such as sorafenib and doramapimod) bind to
other kinases (MAP kinase in particular). Our docking protocol
yielded a very similar binding mode for the ureas in the ALK
inactive model, as compared with available X-ray data with
protein kinase–urea inhibitor complexes. Urea-based com-
pounds have previously been described as potent kinase inhib-
itors.[22–24]
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sponding naphthyl moiety in doramapimod when docked in
ALK (Figure 2C) relative to p38 MAP kinase. As for doramapi-
mod, a disrupted hydrogen bonding pattern relative to that of
sorafenib in ALK has been observed. This finding suggests an
unfavorable strain in the docked poses of doramapimod and
sorafenib in ALK. Because sorafenib does not inhibit ALK, we
hypothesize that ALK is not able to adopt the specific hinge
conformation for binding sorafenib. Furthermore, recently
solved ALK structures reveal a hinge region conformation very
similar to our model (figure S1 in Supporting Information).[19,20]
Based on this comparative analysis, the model of the ALK inac-
tive DFG-Asp-out conformation seems to be able to discrimi-
nate, at least to some extent, between urea-based binders
(doramapimod) and non-binders (sorafenib).
Figure 1. A) Binding mode of doramapimod in the active site of p38 MAP
kinase (PDB ID: 1KV2). Doramapimod is shown in sticks with green carbon
atoms. Residues E71 of the a-helix C and D186 of the DFG motif as well as
M109 of the hinge region are shown as sticks and are colored by atom type.
Hydrogen bonds (donor–acceptor distance: 2.8–3.2 ꢂ, angle: 140–1808) are
depicted with black dotted lines. B) Doramapimod (sticks, cyan carbon
atoms) docked in the active site of inactive ALK showing the necessary flip
of the naphthyl moiety. The position of doramapimod in the crystal struc-
ture of p38 MAP kinase is superimposed (sticks, green carbon atoms). The
residues M259 of the hinge, E227 of the a-helix C and D330 of the DFG
motif of ALK are shown as sticks with blue carbon atoms. Hydrogen bonds
are depicted with black dotted lines. The 3D structure of p38 MAP kinase (A)
and the ALK kinase domain (B) are shown as ribbons with their secondary
structures colored as follows: b-strands: magenta, helices: cyan, loops: pink.
For the sake of clarity, the gatekeeper residue (L256 for ALK and T106 for
p38 MAP kinase) situated behind the naphthyl moiety of the inhibitor is not
shown.
Virtual hits from the in silico screen were further assayed in
vitro for inhibition of recombinant ALK and ALK-dependent
cell proliferation. Biochemical assays revealed six experimental
hits I–VI as shown in Figure 3. For a direct comparison, ALK in-
hibitor NVP-TAE684[18] inhibited the isolated enzyme with an
IC50 value of 60 nm and showed selective anti-NPM–ALK cell
proliferation (BaF3-NPM–ALK: IC50 =110 nm; BaF3 parental:
IC50 =980 nm) when measured under the same conditions.
These candidates were selected as a backbone for the devel-
opment of novel urea compounds with improved potency and
selectivity toward ALK. Whereas some of the candidate inhibi-
tors I–VI showed good inhibitory potency with sub-micromolar
IC50 values in cell-free assays, no inhibitors showed activity at
solubility limits in cells. Thus, in addition to improving inhibi-
tion of the purified enzyme, one of the major challenges was
to gain inhibitor activity and selectivity in cells. From the virtu-
al screening hits, it was apparent that the additional amide
bonds of IV relative to III are not involved in hydrogen bond-
ing to the protein, as these two compounds have very similar
IC50 values. Therefore, we investigated the possibility of form-
ing hydrogen bonds to the hinge region by changing the sub-
stituents on rings 1 and 2 of candidates I–IV (Figure 3). In addi-
tion, we wanted to explore the possibility of forming interac-
tions to the gatekeeper (L256) by adding a hydrophobic sub-
stituent at ring 2 in I or III. Moreover, we designed analogues
of candidates I and II to compare the influence of various sub-
stituents on the pyrazolyl ring toward cellular and enzymatic
activity. To investigate the aforementioned interactions and the
influence of structural modifications on enzymatic and cellular
activity, we selected the 1-phenyl-3-(pyrazol-5-yl)urea and 1,3-
diphenylurea backbones of candidates I–VI to design three
series of compounds (Tables 1–3).
ing predicts an approximate 608 tilt of the naphthyl moiety.
This tilt causes loss of the hydrogen bond interaction between
the morpholino moiety and the hinge region. Furthermore, a
slight shift is observed in the urea portion of the molecule,
where strong hydrogen bonding that involves the side chain
of E71 from a-helix C and the backbone of D168 within the
DFG motif is observed in the crystal structure of p38 MAP
kinase. These observations may explain why doramapimod is
only a weak inhibitor of ALK.
The crystal structure of p38 MAP kinase complexed with sor-
afenib (PDB ID: 3GCS, Figure 2B) reveals a marked conforma-
tional change at the hinge region relative to both the p38
MAP kinase–doramapimod complex (1KV2) and our ALK inac-
tive model (Figure 2A). This conformational change is thought
to be essential for the tight hydrophobic interactions of sorafe-
nib with residues V30 and V38 in the p38 MAP kinase active
site. However, the modified conformation of the hinge region
adopted by p38 MAP kinase in complex with sorafenib would
not allow binding of doramapimod because of a clash be-
tween the morpholino moiety and the hinge (Figure 2A). The
conformation of the ALK hinge region was modeled closer to
that of p38 MAP kinase in complex with doramapimod (1KV2)
because the conformational change observed in the p38 MAP
kinase–sorafenib complex has been rarely observed and might
not be possible in ALK due to the different length and amino
acid sequence of the hinge region (MAGGD [residues 259–263]
for ALK and MGAD [residues 109–112] for p38 MAP kinase; see
alignment in Figure 2D). Furthermore, the phenyl ring of sora-
fenib is in close proximity to the bulky gatekeeper residue of
ALK and shows a similar conformational change to the corre-
Synthesis
Candidates I and II were used in the design of the two first
series of compounds (Figure 3, Table 1, and Table 2). Com-
pounds 1, 7, 8, 13, and 15 were each prepared in a single step
with the respective commercially available isocyanates 25–28
and pyrazoles 29–31 (Scheme 1). For the synthesis of com-
pounds 11 and 12, pyrazoles 29 and 30, respectively, were first
treated with phosgene, and the resulting isocyanates 32 and
33 were allowed to react with 4-(2-morpholin-4-ylethoxy)phe-
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Urea Derivatives as ALK Inhibitors
Figure 2. A) Superposition of the ALK inactive conformation (cyan), p38 MAP kinase complexed with doramapimod (magenta, PDB ID: 1KV2) and p38 MAP
kinase complexed with sorafenib (green, PDB ID: 3GCS). The crystallographic binding position of sorafenib in the active site of p38 MAP kinase is depicted as
sticks with green carbon atoms. The view is rotated by 608 along the y-axis relative to Figure 1A to emphasize the hinge region. The black arrow indicates
the various conformations of the hinge region and the clash between doramapimod (sticks, violet carbon atoms) and the hinge region of the sorafenib p38
conformation (green). B) Sorafenib (sticks, green carbon atoms) in the active site of p38 MAP kinase (PDB ID: 3GCS). Residues involved in hydrogen bonding
(E71 and D186) and M109 within the hinge region are shown as sticks and colored by atom type. Hydrogen bonds are depicted with black dotted lines.
C) Sorafenib (sticks, cyan carbon atoms) docked in the active site of inactive ALK. A crystal structure of sorafenib bound to p38 MAP kinase is superimposed
(sticks, green carbons). Hydrogen bonds are depicted with black dotted lines. The same residues as in Figure 1B (E227, M259, and D330) are shown as sticks.
The protein in Figure 2B,C is shown in Figure 1A. For the sake of clarity, the gatekeeper is not shown. D) Structural alignment of ALK and p38 MAP kinase do-
mains. Identical and similar residues are indicated with a violet background with yellow and white letters, respectively. The hinge region is denoted.
as the 2,2,2-trichloroethyl carbamates 34 and 35 and subse-
quent microwave irradiation in the presence of respective
amines in DMF gave the final products 4, 5, 9, 10, and 14 in
19–86% yield (Scheme 3). Amine 37, used in the final step of
the syntheses of ureas 9 and 10, was prepared by starting
from nicotinoyl chloride hydrochloride and 4-nitroaniline in the
presence of pyridine (py). Subsequently, the nitro compound
36 was reduced by catalytic hydrogenation (Pd/C) to yield the
desired aniline 37 (Scheme 4).
The pyrazole starting materials 38–41 for the preparation of
Scheme 1. Reagents and conditions: a) THF, RT, 12–15 h, 65–90% (1, 7, 8, 13)
compounds 2, 3, 6, and 16 were conveniently synthesized
from the respective hydrazines 42 and 43 and benzoylacetoni-
triles 44–47 in methanol using microwave irradiation
or THF, RT!708C, 17 h, 48% (15).
nylamine in THF (Scheme 2). The reactions with phosgene
were not clean and required extensive purification. Therefore,
we explored additional methods for the formation of com-
pounds 4, 5, 9, 10, and 14. Indeed, activation of the pyrazoles
(Scheme 5). Pyrazoles 38–41 were obtained in 44–71% yields.
The pyrazoles were subsequently allowed to react with 4-(ben-
zyloxy)phenyl isocyanate 25 to give ureas 2, 3, 6, and 16 in
48–66% yields. Similarly, urea 17 was synthesized by starting
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Urea formation of the third series of compounds
18–20 and 23 and 24 (Table 3) was performed by
using isocyanates 25, 26 and 28 and amines 37 and
49–51 (Scheme 6). Synthesis of derivative 21 required
aniline 52 to be prepared in two steps from nicotinic
acid (Scheme 7). Conventional procedures for amida-
tion (acyl chloride, amine, and 4-(dimethylamino)pyri-
dine (DMAP) or pyridine or triethylamine) gave traces
of the desired amide (53). The use of other bases
such as sodium hydroxide and sodium hydride[30] was
also explored, but all the attempted reactions failed
to give satisfactory yields of the amide. Therefore, we
tried amide coupling reagents, and indeed, 1,1’-car-
bonyldiimidazole (CDI) worked well, giving 53 in
38% yield. Catalytic hydrogenation (Pd/C) of the
nitro compound 53 under reflux gave a modest yield
(14%) of the aniline 52. However, with palladium-cat-
alyzed silicon hydride reduction of the nitro group
[poly(methylhydrosiloxane) PMHS, Pd(OAc)2, KF (aq,
1m) in THF],[31] aniline 52 was obtained in 33% yield.
This aniline was subsequently treated with 4-(benzy-
Figure 3. Candidate inhibitors I–VI from virtual screening. The IC50 values derived from
inhibition of recombinant ALK and data from cell proliferation inhibition assays are
shown.
from pyrazole 48 and isocyanate 25, with a 40% yield (Table 2,
Scheme 5).
loxy)phenyl isocyanate 25 to give the urea 21 in 51% yield.
Benzamide 54, for preparation of urea 22, was synthesized in
two steps by starting from ani-
line and 4-nitrobenzoyl chloride
in the presence of DMAP as a
Table 1. IC50 values of the synthesized pyrazolyl urea derivatives determined in cell-free assays using purified
nucleophilic catalyst to yield
compound 55 (Scheme 8). The
nitro amide 55 was catalytically
hydrogenated (Pd/C) to the ani-
line, which was subsequently
treated with 4-(benzyloxy)phenyl
isocyanate 25 to give the urea
product 22 in 85% yield.
recombinant ALK, and in cell proliferation assays.
IC50 [mm][a]
BaF3 Parental
Compd
R1
R2
R3
R4
ALK
BaF3-NPM–ALK
Biological activity and struc-
ture–activity relationships of
synthesized urea compounds
1
2
3
4
5
6
7
8
BnO
BnO
BnO
BnO
4-Cl-BnO
BnO
H
H
H
Cl
H
H
H
H
H
H
H
H
H
Cl
H
H
H
Me
OMe
Cl
Cl
H
2.3ꢀ0.3
4.3ꢀ1.9
4.5ꢀ1.2
2.6ꢀ0.2
0.79ꢀ0.07
0.82ꢀ0.03
2.5ꢀ0.05
4.1ꢀ2.1
74ꢀ4
>100
4.1ꢀ0.1
3.1ꢀ1.4
>100
49ꢀ3
>100
4.6ꢀ1.1
1.8ꢀ1.4
>100
The synthesized compounds had
a purity of at least 97% and
were submitted to two in vitro
tests: an inhibition assay using
the isolated enzyme and a prolif-
eration inhibition assay using
NPM–ALK-transfected BaF3 cells.
Non-transfected BaF3 cell lines
were used as a control. The bio-
logical data are summarized in
Tables 1–3. In a first step, the vir-
tual screening hit I (IC50 =0.9 mm,
no activity on cells) was derivat-
ized (Figure 3 and Table 1).
3.0ꢀ0.6
16ꢀ7
1.4ꢀ0.1
5ꢀ2
PhO
PhCO
Cl
Cl
0.6ꢀ0.04
2.6ꢀ0.4
9
H
H
Cl
15ꢀ2
0.14ꢀ0.05
3.5ꢀ0.2
0.38ꢀ0.04
7.1ꢀ1.1
10
H
H
H
H
H
H
>100
11
12
>100
22ꢀ7
27ꢀ7
H
H
H
Cl
Cl
41ꢀ2
5.0ꢀ1.0
3.2ꢀ1.3
13
14[b]
H
Bn
2.3ꢀ0.8
1.5ꢀ0.2
25ꢀ3
>100
32ꢀ2
>100
In compound 1 of this series,
the para-chloro function of
ring 4 was removed to deter-
mine the contribution of this
(5-(4-Cl-Ph)-2-Me-2H-pyrazol-3-yl)2CO
[a] IC50 values were determined as described in the Experimental Section and represent the mean ꢀSEM (n=
3). [b] The structure of compound 14 is also shown in Scheme 3 and in the Supporting Information.
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Urea Derivatives as ALK Inhibitors
halogen to the activity of I (Figure 3, Table 1). Remov-
al of the chlorine decreased the inhibitory activity
toward the purified enzyme by a factor of ~2–3, indi-
cating that a chlorine substituent at this position of
the molecule is favorable. Compound 1 was used to
verify the mechanism of action for this series of in-
hibitors. Double reciprocal plots of compound 1
versus ATP show that these molecules behave as
mixed-type inhibitors for ALK (figure S2 in Supporting
Information). The activity of compound 2, in which
Scheme 2. Reagents and conditions: a) COCl2 (20% in PhMe), NaHCO3 (aq, satd)/CH2Cl2
(1:1), 08C, 15 min; b) 4-(2-morpholin-4-ylethoxy)phenylamine, THF, RT, 24 h, 13–18%.
Table 2. IC50 values of the synthesized pyrazolyl urea derivatives deter-
mined in vitro on recombinant ALK and in cell proliferation.
IC50 [mm][a]
BaF3 Parental BaF3-NPM–ALK
Compd R1 R2 R3
R4
ALK
15
16
17
H
Ph
Ph Cl
H
H
H
H
H
OMe 1.8ꢀ0.5
>100 >100
Cl
H
1.7ꢀ0.1
8.4ꢀ2.2 1.4ꢀ0.4
5.4ꢀ1.5 0.70ꢀ0.22
Scheme 3. Reagents and conditions: a) Cl3CCH2OCOCl, NaOH (aq, 2m)/EtOAc
(1:1), 08C!RT, 3.5 h, 71–81%; b) amine, DMF, mw, 100–1308C, 30–90 min,
19–86%.
0.61ꢀ0.28
[a] IC50 values were determined as described in the Experimental Section
and represent the mean ꢀSEM (n=3).
the para-chlorine of ring 4 of I is
replaced by a para-methyl sub-
stituent is even slightly worse
than compound 1, bearing only
a hydrogen at this position, indi-
Scheme 4. Reagents and conditions: a) py, CH2Cl2, 08C!RT, 17 h, 33%; b) H2, Pd/C (10%), EtOH/THF (2:1), RT, 1.5 h,
cating that the para-chlorine is
probably involved in halogen
bonding to a water molecule.
Furthermore, the lack of cellular
72%.
activity for this compound suggests that purely hy-
drophobic substituents at the para position of ring 4
are unfavorable for cell penetration. If the methyl
group of compound 2 is replaced by a methoxy
group (compound 3), cellular activity is improved by
a factor of 18 relative to compound 1. As compounds
1–3 in comparison with I have shown that the chlor-
ine of I at R4 is most favorable in terms of enzymatic
activity, we decided to modify groups R1 and R2 of
the scaffold in our endeavor to ameliorate cellular ac-
tivity. In compound 4 a chlorine was added as the R2
group which indeed shows good cellular activity,
with enzymatic activity similar to that of compound
1. According to modeling, ring 2 is in close proximity
to the gatekeeper residue L256. The slightly lower ac-
tivity of compound 4 relative to I could be explained
that in order to accommodate the chlorine, ring 2
needs to tilt slightly, or small conformational changes
Scheme 5. Reagents and conditions: a) MeOH, 1208C, mw, 40 min, 44–71%; b) 4-BnO-
(C6H4)NCO (25), THF, RT, 15–22 h, 62–66% (2, 3, 6) or 4-BnO(C6H4)NCO (25), 1,4-dioxane
or THF, mw, 708C, 60–120 min!RT, 16–21 h, 40–48% (16, 17).
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in the protein around the gate-
Table 3. IC50 values of the synthesized phenyl urea derivatives determined using purified ALK kinase domain,
keeper residue are required.
When adding a chlorine at the
para position of ring 1 of I (com-
pound 5) instead of the R2 posi-
tion, the potency toward the pu-
rified enzyme is slightly im-
proved, but the compound is
not active on cells. In contrast,
moving the chlorine from the
para to the meta position of
ring 4 not only yields favorable
activity on the enzyme, but also
shows remarkably improved cel-
lular activity (compound 6).
and the effects of these compounds on cell proliferation.
IC50 [mm][a]
BaF3 Parental
Compd
R1
R2
R3
R4
ALK
BaF3-NPM–ALK
18
19
BnO
PhO
Cl
H
H
Cl
H
H
1.7ꢀ0.2
5.0ꢀ0.2
44ꢀ6
10ꢀ5
50ꢀ8
1.5ꢀ0.9
20
H
H
H
0.80ꢀ0.12
61ꢀ13
42ꢀ5
To investigate whether cellular
activity can be improved, the
linker and ring 1 of I was altered
(Figure 3). Shortening the linker
by one carbon atom (compound
7), thereby making it more rigid,
slightly decreased potency, but
was again beneficial for cell per-
meability. Furthermore, this com-
pound showed the best ratio
obtained thus far between BaF3
parental cells and NPM–ALK-pos-
itive cells. For compound 8, the
oxygen linker of compound 7
21
22
H
H
H
H
3.6ꢀ0.7
>100
>100
86ꢀ1
>100
H
0.39ꢀ0.1
23
24
H
H
H
H
8.9ꢀ3.2
2.0ꢀ0.4
>100
>100
11ꢀ1
H
Bn
Cl
7.1ꢀ0.2
[a] IC50 values were determined as described in the Experimental Section and represent the mean ꢀSEM (n=
3).
was replaced by
a carbonyl
group, making the substituent
even more rigid. Whereas activi-
ty toward the enzyme slightly
worsened again, which is proba-
bly due to the fact that it cannot
adopt an optimal position in the
active site of ALK, compound 8
was very potent toward cells.
The four last compounds (9–12)
of this series were synthesized in
an attempt to establish an addi-
tional hydrogen bond with a
backbone atom of the hinge
region to improve potency. In
this line we extended the linker
of I by keeping it rigid; an amide
bond was therefore introduced
(compound 9). In addition, a pyr-
idine ring was introduced in-
stead of the benzene ring in I, as
modeling suggested that in this
way an additional hydrogen
bond with the backbone nitro-
gen of M256 at the hinge region
could be established. Unfortu-
nately this was not the case, as
compound 9 showed worse ac-
Scheme 6. Reagents and conditions: a) CH2Cl2 or THF, RT, 12–30 h, 41–87%.
Scheme 7. Reagents and conditions: a) 1. CDI, DMF, RT, 1 h, 2. 5-nitroindoline, RT!608C, 46 h, 38%; b) 1. Pd(OAc)2,
KF (aq, 1m), THF, 2. PMHS, RT!608C, 20 h, 33%; c) 4-BnO(C6H4)NCO (25), CH2Cl2, RT, 18 h, 51%.
Scheme 8. Reagents and conditions: a) 4-nitrobenzoyl chloride, DMAP, THF, RT, 6 h, 71%; b) H2, Pd/C (10%), MeOH/
EtOAc (2:1), RT, 12 h, 59%; c) 4-BnO(C6H4)NCO (25), THF, RT, 12 h, 85%.
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Urea Derivatives as ALK Inhibitors
tivity toward the enzyme, as was the case for compound 8.
This is most likely due to the nitrogen atom of the pyridine
ring coming to rest in an unfavorable hydrophobic area in the
active site, and due to the rigidity of the linker the substituent
cannot accommodate. However, with this compound the cellu-
lar activity could be improved more than fourfold. Removing
the chlorine atom on ring 4 (compound 10) was an attempt to
create a bit more space for the compound so that it could
adapt better to the active site of ALK, but this resulted in both
a decrease in enzymatic and cellular activity. The fact that the
compound was inactive up to 100 mm, and showed only mod-
erate cellular activity, illustrates the immanent role of the para-
chlorine at ring 4 which is thought to interact with amino
acids F305, I306, I230, and F234, forming a favorable hydro-
phobic environment (Figure 4). In a further effort to establish a
hydrogen bond at the hinge region, compounds 11 and 12
(compound 11 without the para-chlorine at ring 4) were syn-
thesized. The linker was made more flexible and was extended
by one carbon atom, and the pyridine ring of compound 9
was replaced by a morpholino group, analogous to the inter-
action between doramapimod and p38 MAP kinase. Similar ob-
servations could be made as described for compounds 9 and
10. Compound 13, in which the substituent at R1 was removed
and a benzyloxy group at R2 was introduced instead, shows
that similar enzymatic activity could be obtained by either sub-
stituting R1 or R2. Whether equally favorable cellular activity
can be obtained remains to be determined in follow-up com-
pounds of this series currently in preparation. Finally, com-
pound 14, the last compound of this series, although inactive
at the cellular level, shows the symmetric nature of the active
site and the resulting challenge to predict the correct binding
mode of urea derivatives. In conclusion, with this series of 1-
methylpyrazolyl urea derivatives we were able to improve cel-
lular activity from inactive to active at the sub-micromolar
level. Furthermore, we show for the first time that urea deriva-
tives are a promising class of compounds to inhibit ALK, for
which only few compounds have so far been described as in-
hibitors. Unfortunately, no additional hydrogen bond interac-
tions with the protein could be established, most probably
due to a difference in conformation of the hinge region of ALK
relative to that of MAP kinase as a result of the difference in
length of this segment between the two proteins. Therefore,
further refinements of the model are required for generating a
more potent compound from this series in a second round of
synthesis, where the lessons learned from the compounds de-
scribed above will be of great importance.
Compounds 15–17 (Table 2) were prepared in order to de-
termine the importance of the methyl group at the pyrazole
ring of I. In compound 15, the methyl group at the pyrazole
ring was removed relative to compound 3, leading to a polar
NH group at this position. Although this modification was well
tolerated regarding enzymatic activity compared with 3 (activi-
ty slightly improved from 4.5 to 1.8 mm), the compound is
completely inactive on cells. This observation suggests that a
polar group at this position is detrimental for cellular activity.
Further support for this fact comes from compounds 16 and
17, in which the methyl group of I is substituted by a phenyl
ring and where cellular activity is restored. In addition, com-
pound 17, with a chlorine atom at the ortho position of ring 4,
shows a remarkable 10-fold selectivity in favor of BaF3 NPM–
ALK-positive cells over BaF3 parental cells.
To better characterize the activity of compound 17 in NPM–
ALK-positive cells, proliferation and apoptosis assays were car-
ried out using the human ALCL-derived cell line SUDHL-1. As
shown in Figure 5A, compound 17 inhibited SUDHL-1 cell
growth with an IC50 value of 0.8 mm, similar to the case with
BaF3-NPM–ALK cells. The compound was less toxic toward
NPM–ALK-negative human leukemic cells U937 (IC50 =3.2 mm).
Compound 17 induced cell death in SUDHL-1 cells, as indicat-
ed by annexin V staining (Figure 5B). These effects correlated
with a specific block of NPM–ALK autophosphorylation at Y664
(Figure 5C), which is a marker of kinase activation.[32] These
data open interesting new perspectives for further improve-
ments in second-round derivatives.
Figure 4. Binding mode of representative urea derivatives as depicted by docking into the inactive ALK model. M259 of the hinge region, E227 of a-helix C
and D330 of the DFG motif are shown as sticks with carbon atoms in cyan. Hydrogen bonds are shown as black dotted lines. For the sake of clarity, the gate-
keeper L256 is not displayed. A) Compound 1 representing the 1-methylpyrazolyl urea derivatives. B) Compound 17 representing the 1-phenylpyrazolyl urea
derivatives. C) Compound 19 representing the phenyl urea derivatives.
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Figure 5. Activity of compound 17 in SUDHL-1 cells. A) Inhibition of cellular growth was determined with increasing doses of inhibitor 17 by [3H]thymidine in-
corporation assay and values are reported as a percentage of vehicle-treated control. B) SUDHL-1 cells were treated with vehicle or 17 at the indicated con-
centrations for 24 h; apoptosis is shown as a percent of annexin V-positive cells in the cell culture. C) NPM–ALK autophosphorylation was monitored by anti-
pY664 antibody; equal gel loading is verified by actin staining; *p<0.05 versus control; **p<0.01 versus control.
To explore the potential of the central 1,3-diphenylurea scaf-
Experimental Section
fold of the screening hits III–V, a series of derivatives was pre-
pared (Table 3). Compound 18, a derivative of III with an addi-
tional chlorine at position R2, which, according to the model-
Materials and general procedures
All reagents were commercially available and were acquired from
ing, is expected to make favorable nonpolar interactions with
the gatekeeper residue, exhibits only low activity in cell-based
assays, but is rather potent on the enzymatic level. Compound
19, with a phenoxy substituent at R1 and a chlorine at ring 3, is
selective against NPM–ALK-positive cells (greater than seven-
fold), indicating that the position of the chlorine offers an addi-
tional possibility for the synthesis of new selective compounds.
It has the best cellular activity obtained for this series. Com-
pounds 20–23 were synthesized in an attempt to establish an
additional hydrogen bond with the hinge region (similar to
compounds 9–12, which were not successful for the same rea-
sons already indicated for the 1-methylpyrazolyl urea deriva-
tives). In addition, compounds 20–23 are only moderately
active or inactive toward cells. Finally, for compound 24 a simi-
lar approach was used as with compound 16 to exemplify that
nonpolar substituents at either R1 or R2 lead to similar enzy-
matic activities. The scaffold explored in Table 3 seems to be
less favorable in terms of cell permeability, but considering the
small number of derivatives, more compounds probably need
to be synthesized in order to draw a final conclusion. No signif-
icant improvement of the enzymatic activity relative to the un-
derlying screening hits was observed. The positive feature
about the core scaffold of compounds listed in Table 3 is that
it is more permissive than those shown in Tables 1 and 2. How-
ever, its symmetric nature makes binding mode elucidations
more difficult.
ABCR (Karlsruhe, Germany), Asdi (Newark, NJ, USA), BDH (Poole,
UK), Fluka (Buchs, Switzerland), Matrix Scientific (Columbia, SC,
USA), Maybridge (Cambridge, UK) and Sigma–Aldrich (Schnelldorf,
Germany). THF and Et2O were distilled from sodium/benzophenone
ketyl. CHCl3 was distilled from CaH2. Anhydrous DMF was from
Fluka (Buchs, Switzerland) and was stored over molecular sieves
(4 ꢂ) under an inert atmosphere of dry argon. All reactions in anhy-
drous solvents were performed in flame-dried glassware under an
inert atmosphere of dry argon. The progress of chemical reactions
was monitored by thin-layer chromatography (TLC) on silica gel
60 F254 plates (Merck; Darmstadt, Germany) using phosphomolyb-
dic acid stain (10% by weight in EtOH) or ninhydrin stain (1.5% by
weight in EtOH). Flash SiO2 column chromatography was per-
formed with Merck silica gel 60 (230–400 mesh) or with a Biotage
high-performance flash chromatography Sp4 system (Uppsala,
Sweden) using a 0.1 mm pathlength flow cell UV detector/recorder
module (fixed wavelength: 254 nm), 12 mm or 25 mm flash car-
tridges, and the indicated mobile phase.
1H NMR, 13C NMR, and DEPT spectra were recorded on a Varian
Mercury 300 MHz or a Varian Unity 500 MHz spectrometer (Varian,
Palo Alto, CA, USA) as solutions in CDCl3, [D6]DMSO, or CD3OD.
Deuterated solvents were purchased from Sigma. Chemical shifts
(d) are given ppm relative to the NMR solvent signals (CDCl3: 7.26
and 77.21 ppm, [D6]DMSO: 2.50 and 39.52 ppm, CD3OD: 3.31 and
1
49.00 ppm for H and 13C NMR, respectively). Multiplicities are indi-
cated by brs (broad singlet), s (singlet), d (doublet), dt (doublet of
triplets), ddd (doublet of doublet of doublet), m (multiplet), and t
(triplet).
HPLC–MS analysis of the synthesized compounds was performed
to determine the purity of each compound using methods A, B, or
C. For method A, an Agilent 1100 series HPLC instrument (Agilent
Technologies, Palo Alto, CA) with a UV detector (l 210 nm) and an
Esquire-LC quadrupole ion trap mass spectrometer equipped with
an ESI interface (Bruker Daltonics, Bremen, Germany) and LC–MSD
Trap software, version 5.2 (Bruker Daltonics) was used. Signal sepa-
ration was performed using a Phenomenex Luna C8(2) column
(2.0ꢃ150 mm, 5.0 mm). The column was operated at a constant
temperature of 408C. The eluent consisted of H2O (plus 0.1%
formic acid, solvent A) and 2-propanol (plus 0.1% formic acid, sol-
vent B) and a gradient run was performed (95:5!0:100 over
Conclusions
A series of new inhibitors targeted to the catalytic site of ALK
were designed, synthesized, and screened for biological activi-
ty. These novel ALK inhibitors have IC50 values as low as
390 nm. More importantly, three derivatives (compounds 16,
17, and 19) preferentially inhibited the growth of NPM–ALK-
transfected cells. These results support the development of
urea-based compounds as potent, selective, and cell-permea-
ble ALK inhibitors.
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Urea Derivatives as ALK Inhibitors
35 min and 0:100 for 10 min). For method B, an Agilent 1100 series
Virtual screening: The virtual screen was performed on a subset
of the ZINC database consisting of ~40000 compounds of a May-
bridge (part of Thermo Fisher ScientificTM) lead-like compound li-
brary complying with Lipinski’s rule of five. Initial binding poses in
the inactive model of ALK were obtained using the program Dock
(v. 4.0), which is particularly suited to dock large compound data-
bases with a good balance between accuracy and speed. The
active site of ALK for the virtual screening was defined as outlined
above in the Molecular docking section, and no further constraints
were applied. The obtained binding poses were subjected to con-
sensus scoring as implemented in Sybyl 6.3 (Tripos). After visual in-
spection (to exclude binding poses that do not occupy most parts
of the active site) the best 1000 poses were chosen for a second
docking round using FlexX. The resulting poses were again consen-
sus scored, and the 20 top-ranked compounds were ordered and
tested experimentally for inhibitory activity against ALK. The urea
derivatives among these hits were finally used as starting points
for the synthesis of new derivatives described in the current study.
HPLC instrument (Agilent Technologies) with
a UV detector
(l 210 nm) and a PerkinElmer Sciex API3000 triple-quadrupole LC–
MS–MS mass spectrometer (Applied Biosystems/MDS Sciex, Con-
cord, ON, Canada) with a turbo ESI source was used. Signal separa-
tion was carried out by an XTerra MS RP18 column (4.6ꢃ30 mm,
2.5 mm). The column was operated at a constant temperature of
358C. The eluent consisted of H2O (plus 0.1% formic acid, solvent
A) and 2-propanol (plus 0.1% formic acid, solvent B) and a gradient
run was performed (95:5 for 1 min, 95:5!0:100 over 24 min, and
0:100 for 4 min). Method C was otherwise the same as method B
except that the column temperature was kept at 408C, and the
eluent consisted of H2O (plus 0.1% formic acid, solvent A) and ace-
tonitrile (solvent B). An isocratic run was performed (95:5 for
40 min).
Fourier transform infrared (FTIR) spectra were recorded using a
Vertex 70 spectrometer (Bruker Optik GmbH, Ettlingen, Germany)
and MIRacleꢄ ATR accessory (Pike Technologies, Madison, WI, USA)
or from a KBr tablet. Melting points were measured with an Elec-
trothermal IA 9100 apparatus and are uncorrected. Purity of all
tested compounds was >97%.
Synthesis of compounds
One representative synthesis of each procedure used to prepare
the intermediates and products is presented here (urea derivatives
1, 9, 12, 16, 17, 20–22, and intermediates 35–37, 41, and 52–55).
Procedures for the synthesis and analysis of the remaining com-
pounds are available in the Supporting Information.
Molecular modeling
Homology model: A model of ALK corresponding to a closed (in-
active) conformation of the catalytic site was generated using the
following protocol. The sequences of human ALK were retrieved
from the SwissProt database[33] (accession code Q9M73). The tyro-
sine kinase sequence of human ALK, spanning 278 residues, was
taken as a query to search the RCSB Protein Data Bank (PDB) using
PSI-Blast[34] to find putative templates for ALK homology modeling.
Further filtering criteria were applied, including preferences for
high-resolution X-ray structures, gapless backbones, and high se-
quence identity/similarity. Finally, the following templates were
chosen to generate model of ALK: mouse c-Abl (PDB IDs: 1IEP,
1OPJ) and human insulin receptor (INSR) mutant (PDB ID: 1IRK).
ALK shares 34% identity and 48% similarity with Abl, and 40%
identity and 56% similarity with INSR. Reliable pairwise sequence–
structure alignments, which were necessary for accurate homology
modeling, were obtained using the Verta algorithm of the Super-
imposer plug-in in the program Bodil.[35] These pairwise alignments
were merged to multiple alignments in the program malign[36]
using the STRMAT110 substitution matrix.[37] Homology modeling
was performed with the program Modeller6v2[38] using default pa-
rameters, including a round of minimization. For each conforma-
tion, 100 models were generated, and their overall geometrical
quality was assessed by Procheck.[39] The models showing geomet-
rical qualities similar to the templates were chosen for docking
studies. For binding mode evaluation of urea derivatives, models
of the active and inactive conformation of ALK were used. Docking
of the urea compounds into the active model did not result in any
plausible binding orientations that could support the experimental
data.
1-[4-(Benzyloxy)phenyl]-3-(1-methyl-3-phenyl-1H-pyrazol-5-
yl)urea (1): A solution of 4-(benzyloxy)phenyl isocyanate (0.25 g,
1.1 mmol, 1.1 equiv) and 5-amino-1-methyl-3-phenylpyrazole
(0.17 g, 1.0 mmol) in THF (10 mL) was stirred at RT for 12 h. The
solvent was evaporated in vacuo, and the solid residue was
washed with a mixture of cold n-hexane and THF (1:1, 2ꢃ5 mL)
and filtered. The crude product was recrystallized from a mixture
of n-hexane and THF (2:1) to give a white solid (compound 1;
1
0.36 g, 90% yield). H NMR (300 MHz, [D6]DMSO): d=8.74 (s, 1H),
8.58 (s, 1H), 7.79–7.71 (m, 2H), 7.48–7.22 (m, 10H), 7.00–6.91 (m,
2H), 6.61 (s, 1H), 5.07 (s, 2H), 3.72 ppm (s, 3H); 13C NMR (75 MHz,
[D6]DMSO): d=153.7, 151.9, 148.0, 138.6, 137.6, 133.6, 132.6, 128.6,
128.4, 127.7, 127.6, 127.3, 124.7, 120.1, 115.0, 94.6, 69.4, 35.4 ppm;
LC–MS: (398.17) m/z 399.2 [M+H]+, 412.2 [M+Na]+; tR =28.1 min,
purity 99% (method A).
N-[4-[3-[3-(4-Chlorophenyl)-1-methyl-1H-pyrazol-5-yl]ureido]phe-
nyl]nicotinamide (9):
A solution of compounds 35 (0.13 g,
0.33 mmol) and 37 (0.10 g, 0.47 mmol, 1.3 equiv) in DMF (3 mL)
was irradiated under mw at 1008C for 30 min. H2O (20 mL) was
added to the reaction mixture, and the precipitate was filtered.
The solid was collected and purified by flash SiO2 column chroma-
tography (CHCl3/MeOH, 1:0!10:1) to give a pink solid (compound
9; 0.05 g, 32% yield); mp: 2338C (dec); 1H NMR (300 MHz,
[D6]DMSO): d=10.34 (s, 1H), 9.07 (s, 1H), 8.90 (s, 1H), 8.72 (d, 1H,
J=4.7 Hz), 8.66 (s, 1H), 8.25 (d, 1H, J=7.7 Hz), 7.74 (d, 2H, J=
8.3 Hz), 7.66 (d, 2H, J=8.4 Hz), 7.53 (dd, 1H, J=7.6, 5.1 Hz), 7.48–
7.37 (m, 4H), 6.63 (s, 1H), 3.70 ppm (s, 3H); 13C NMR (75 MHz,
[D6]DMSO): d=163.7, 152.0, 151.8, 148.6, 146.9, 138.8, 135.5, 135.4,
133.4, 132.5, 131.7, 130.6, 128.6, 126.4, 123.4, 121.1, 118.6, 94.7,
Molecular docking: All dockings were performed using the pro-
grams Gold[40] and FlexX.[41] The active site of the ALK model was
defined by superimposing it onto the crystal structure of PDB ID:
1IEP and taking into account all residues of the ALK model corre-
sponding to the amino acids of Abl positioned 6.5 ꢂ around imati-
nib of 1IEP. Thirty docking solutions were generated per docking
run with FlexX and ten per docking run with Gold. A general con-
sensus in the docking poses has been observed between FlexX
and Gold.
35.5 ppm; IR (KBr): n˜ =3288, 3151, 3054, 1686, 1646, 1565 cmꢁ1
;
LC–MS (446.13): m/z 447.1 [M+H]+, tR =14.4 min, purity >99%
(method B).
1-[3-(4-Chlorophenyl)-1-methyl-1H-pyrazol-5-yl]-3-[4-(2-morpho-
linoethoxy)phenyl]urea (12): A mixture of 3-(4-chlorophenyl)-1-
methyl-1H-pyrazol-5-ylamine (0.21 g, 1.0 mmol), CH2Cl2 (14 mL) and
a saturated solution of NaHCO3 (14 mL) was cooled to 08C. Phos-
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gene (2.0 mL, 20% in toluene, 4 equiv) was added, and the reac-
tion mixture was stirred at 08C for 15 min. The CH2Cl2 phase was
separated, dried (Na2SO4) and evaporated in vacuo. CH2Cl2 (5 mL)
and 4-(2-morpholino-4-ylethoxy)phenylamine (0.20 g, 0.89 mmol,
0.89 equiv) was added to the solid, and the resulting reaction mix-
ture was stirred at RT for 24 h. The reaction mixture was filtered,
and the crude product was purified by flash SiO2 column chroma-
tography (Biotage MPLC, CHCl3/MeOH, 4:1) to give a white solid
MS (438.17): m/z 439.2 [M+H]+, 461.1 [M+Na]+, tR =24.2 min,
purity >99%. (method A).
1-[4-(Benzyloxy)phenyl]-3-(1-nicotinoylindolin-5-yl)urea (21):
A
solution of 4-(benzyloxy)phenyl isocyanate (0.059 g, 0.26 mmol)
and compound 52 (0.063 g, 0.26 mmol) in CH2Cl2 (6 mL) was stirred
at RT for 18 h. The reaction mixture was filtered, and the solid was
washed with a small amount of CH2Cl2. The crude product was pu-
rified by flash SiO2 column chromatography (Biotage MPLC, CHCl3/
MeOH, 1:0!10:1) to give a pale-yellow solid (compound 21;
1
(compound 12; 0.08 g, 18% yield). H NMR (300 MHz, [D6]DMSO):
d=8.75 (s, 1H), 8.63 (s, 1H), 7.77 (d, 1H, J=8.4 Hz), 7.43 (d, 2H, J=
8.3 Hz), 7.36 (d, 2H, J=8.9 Hz), 6.88 (d, 2H, J=8.9 Hz), 6.63 (s, 1H),
4.04 (t, 2H, J=5.8 Hz), 3.72 (s, 3H), 3.57 (t, 4H, J=4.6 Hz), 2.66 (t,
2H, J=5.7 Hz), 2.46 ppm (t, 4H, J=4.6 Hz); 13C NMR (75 MHz,
[D6]DMSO): d=153.8, 151.9, 146.9, 138.9, 132.5, 132.4, 131.7, 128.6,
126.4, 120.1, 114.7, 94.7, 66.2, 65.5, 57.1, 53.6, 35.5 ppm; LC–MS
(455.17): m/z 456.2 [M+H]+, 478.2 [M+Na]+; tR =18.9 min, purity
>99% (method A).
1
0.062 g, 51% yield), H NMR (300 MHz, [D6]DMSO): d=8.79 (s, 1H),
8.70 (dd, 1H, J=4.7, 1.4 Hz), 8.59 (s, 1H), 8.45 (s, 1H), 8.02 (s, 2H),
7.58–7.27 (m, 9H), 7.19 (s, 1H), 7.00–6.86 (m, 2H), 5.06 (s, 2H), 4.02
(t, 2H, J=7.8 Hz), 3.08 ppm (t, 2H, J=8.2 Hz); 13C NMR (75 MHz,
[D6]DMSO): d=165.2, 153.4, 152.7, 150.7, 147.7, 137.3, 136.9, 136.4,
134.7, 133.3, 133.0, 128.4, 127.7, 127.6, 123.5, 119.9, 117.0, 116.5,
115.0, 69.4, 50.5, 28.1 ppm; LC–MS (464.18): m/z 465.2 [M+H]+;
tR =15.2 min, purity >99% (method B).
1-[4-(Benzyloxy)phenyl]-3-[3-(4-chlorophenyl)-1-phenyl-1H-pyra-
zol-5-yl]urea (16):
A
solution of compound 41 (0.083 g,
4-[3-[4-(Benzyloxy)phenyl]ureido]-N-phenylbenzamide (22): A so-
lution of 4-(benzyloxy)phenyl isocyanate (0.25 g, 1.1 mmol,
1.1 equiv) and compound 54 (0.21 g, 1.0 mmol) in THF (10 mL) was
stirred at RT for 12 h. The solvent was evaporated in vacuo, and
the solid residue was washed with a mixture of cold n-hexane and
THF (1:1, 2ꢃ5 mL) and filtered. The crude product was recrystal-
lized from a mixture of n-hexane and THF (2:1) to give a white
solid (compound 22; 0.37 g, 85% yield). 1H NMR (300 MHz,
[D6]DMSO): d=10.1 (brs, 1H), 8.95 (brs, 1H), 8.61(brs, 1H), 7.94–
6.92 (m, 18H), 5.1 ppm (s, 2H); 13C NMR (75 MHz, [D6]DMSO): d=
165.0, 153.7, 152.5, 143.1, 139.4, 137.3, 132.6, 128.7, 128.5, 128.4,
127.7, 127.6, 123.3, 120.3, 120.2, 119.8, 117.0, 116.7, 115.0,
69.4 ppm; LC–MS (437.17) m/z 438.3 [M+H]+, 460.2 [M+Na]+;
tR =27.7 min, purity 97% (method A).
0.31 mmol) and 4-(benzyloxy)phenyl isocyanate (0.069 g,
0.31 mmol) in 1,4-dioxane (3 mL) was irradiated under mw at 708C
for 120 min and stirred at RT for 16 h. The reaction mixture was
evaporated in vacuo, and the crude product was purified by flash
SiO2 column chromatography (Biotage MPLC, n-hexane/EtOAc,
4:1!1:1) to give a white solid (compound 16; 0.073 g, 48% yield).
1H NMR (300 MHz, [D6]DMSO); H NMR (300 MHz, [D6]DMSO): d=
1
8.88 (brs, 1H), 8.49 (brs, 1H), 7.87 (d, 2H, J=8.7 Hz), 7.77 (d, 2H,
J=8.7 Hz), 7.68–7.27 (m, 12H), 6.97–6.88 (m, 3H), 5.05 ppm (s, 2H);
13C NMR (75 MHz, [D6]DMSO): d=153.7, 151.7, 149.0, 138.9, 137.3,
132.5, 132.3, 132.0, 129.4, 128.7, 128.4, 128.0, 127.7, 127.6, 126.8,
124.7, 120.0, 115.0, 96.1, 69.4 ppm; LC–MS (494.15): m/z 495.1 [M+
H]+, tR =19.4 min, purity 98% (method B).
1-[4-(Benzyloxy)phenyl]-3-[3-(2-chlorophenyl)-1-phenyl-1H-pyra-
zol-5-yl]urea (17): Following the procedure for compound 16,
urea derivative 17 was synthesized from a solution of 3-(2-chloro-
phenyl)-1-phenyl-1H-pyrazol-5-ylamine (0.10 g, 0.37 mmol) and 4-
(benzyloxy)phenyl isocyanate (0.084 g, 0.37 mmol) in THF (4 mL),
which was irradiated under mw at 708C for 60 min and stirred at
RT for 21 h. The crude product was recrystallized from THF and
washed with n-hexane to give a white solid (compound 17;
0.074 g, 40% yield). 1H NMR (300 MHz, [D6]DMSO): d=8.91 (brs,
1H), 8.52 (brs, 1H), 7.88–7.84 (m, 1H), 7.67–7.29 (m, 15H), 6.98 (s,
1H), 6.93 (d, 2H, J=9.0 Hz), 5.05 ppm (s, 2H); 13C NMR (75 MHz,
[D6]DMSO): d=153.7, 151.6, 147.9, 138.1, 138.0, 137.2, 132.5, 131.8,
131.1, 130.3, 129.4, 128.4, 128.0, 127.7, 127.6, 127.3, 124.6, 120.0,
115.0, 99.3, 69.4 ppm; LC–MS (494.15): m/z 495.1 [M+H]+; tR =
18.8 min, purity 98% (method B).
[5-(4-Chlorophenyl)-2-methyl-2H-pyrazol-3-yl]carbamic
acid
2,2,2-trichloroethyl ester (35): A 2m solution of NaOH in H2O
(8.0 mL, 16 mmol, 7.5 equiv) was added to a solution of 3-(4-chlor-
ophenyl)-1-methyl-1H-pyrazol-5-ylamine (0.48 g, 2.1 mmol) in
EtOAc (8 mL) at 08C, and the resulting mixture was stirred at 08C
for 30 min. 2,2,2-Trichloroethyl chloroformate (0.54 mL, 3.8 mmol,
1.8 equiv) was added portionwise at 08C over a period of 1 h to
this mixture, and the resulting mixture was stirred at RT for 2 h.
The aqueous layer was extracted with EtOAc (2ꢃ20 mL). The com-
bined organic layers were washed with brine (2ꢃ20 mL), dried
(Na2SO4), filtered and evaporated in vacuo. The crude product was
purified by flash SiO2 column chromatography (n-hexane/EtOAc,
1:0!2:1) to give a white solid (compound 35; 0.66 g, 81% yield);
1
mp: 1368C; H NMR (300 MHz, CDCl3): d=7.67 (2H, d, J=8.4 Hz),
7.34 (2H, d, J=6.9 Hz), 6.86 (1H, s), 6.49 (1H, s), 4.85 (2H, s),
3.81 ppm (3H, s); 13C NMR (75 MHz, CDCl3): d=149.2, 135.5, 133.8,
131.8, 129.0, 126.8, 97.8, 95.0, 75.3, 35.9, 29.9 ppm; IR (KBr): n˜ =
3293, 3214, 2955, 1749, 1714 cmꢁ1
.
N-[4-[3-[4-(Benzyloxy)phenyl]ureido]phenyl]nicotinamide
(20):
Urea derivative 20 was synthesized from a solution of 37 (0.10 g,
0.49 mmol, 1.1 equiv) and 4-(benzyloxy)phenyl isocyanate (0.10 g,
0.44 mmol) in THF (11 mL), which was stirred at RT for 18 h. The re-
action mixture was filtered, and the crude product was purified by
flash SiO2 column chromatography (Biotage MPLC, CHCl3/MeOH,
1:0!10:1) to give a white solid (compound 20; 0.080 g, 41%
yield). 1H NMR (300 MHz, [D6]DMSO): d=10.34 (s, 1H), 9.10 (dd,
1H, J=2.2, 0.7 Hz), 8.75 (dd, 1H, J=4.8, 1.6 Hz), 8.59 (brs, 1H), 8.48
(brs, 1H), 8.28 (dt, 1H, J=8.0, 2.2 Hz), 7.66 (d, 2H, J=9.0 Hz), 7.56
(ddd, 1H, J=8.0, 4.9, 0.8 Hz), 7.47–7.29 (m, 9H), 6.94 (d, 2H, J=
9.1 Hz), 5.06 ppm (s, 2H); 13C NMR (75 MHz, [D6]DMSO): d=163.6,
153.4, 152.7, 152.0, 148.6, 137.3, 136.1, 135.3, 133.0, 132.9, 130.6,
128.4, 127.7, 127.6, 123.5, 121.1, 119.9, 118.3, 115.0, 69.4 ppm; LC–
N-(4-Nitrophenyl)nicotinamide (36): A mixture of nicotinoyl chlo-
ride hydrochloride (1.9 g, 11 mmol, 1.5 equiv), pyridine (1.3 mL,
16 mmol, 2.3 equiv) and CH2Cl2 (20 mL) was stirred at 08C for
30 min. 4-Nitroaniline (1.0 g, 7.2 mmol) was added to the mixture,
which was slowly warmed to RT and then stirred at RT for 16 h.
The solvents were removed, and the crude product was successive-
ly washed with warm H2O and recrystallized from MeOH to give a
1
yellow solid (compound 36; 0.59 g, 33% yield). H NMR (300 MHz,
[D6]DMSO): d=11.09 (s, 1H), 9.19 (s, 1H), 8.91–8.80 (m, 1H), 8.43
(d, J=8.0 Hz, 1H), 8.29 (d, J=9.2 Hz, 2H), 8.07 (d, J=9.3 Hz, 2H),
7.68 ppm (dd, J=7.9, 5.0 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO):
1690
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1680 – 1692

Urea Derivatives as ALK Inhibitors
d=164.5, 151.6, 148.0, 145.0, 142.7, 136.9, 130.4, 124.9, 124.0,
120.0 ppm.
0.50 equiv) were added to the reaction mixture. After stirring at
608C for 24 h, the reaction mixture was quenched with ice H2O
(30 mL). The resulting mixture was extracted with EtOAc (3ꢃ
60 mL), washed with a saturated NaHCO3 solution (2ꢃ25 mL), H2O
(2ꢃ50 mL) and brine (2ꢃ50 mL), dried (Na2SO4), filtered and
evaporated in vacuo. The crude product was purified by SiO2 flash
column chromatography (CHCl3/MeOH, 10:1!4:1) to give a yellow
solid (compound 53; 0.72 g, 38% yield); mp: 2218C; 1H NMR
(300 MHz, [D6]DMSO): d=8.85 (dd, 1H, J=2.2, 0.8 Hz), 8.74 (dd,
1H, J=4.9, 1.7 Hz), 8.22–8.16 (m, 2H), 8.11–8.07 (m, 2H, J=7.9, 2.1,
1.8 Hz), 7.56 (ddd, 1H, J=7.9, 4.9, 0.9 Hz), 4.15 (t, 2H, J=8.5 Hz),
3.21 ppm (t, 2H, J=8.3 Hz); 13C NMR (75 MHz, [D6]DMSO): d=
167.0, 151.3, 148.3, 147.8, 143.2, 134.8, 132.1, 124.1, 123.5, 120.6,
116.0, 51.2, 27.2 ppm; IR (KBr): n˜ =3262, 3060, 2940, 1642,
N-(4-Aminophenyl)nicotinamide (37): A mixture of compound 36
(0.21 g, 0.86 mmol) and Pd/C (10%, 0.020 g) in EtOH (40 mL) and
THF (20 mL) was hydrogenated at RT for 1.5 h. The reaction mix-
ture was filtered through a pad of Celite 545 and washed with
EtOH. The filtrate was evaporated in vacuo, and the crude product
was purified by flash SiO2 column chromatography (Biotage MPLC,
CHCl3/MeOH, 1:0!10:1) to give a brown solid (compound 37;
1
0.13 g, 72% yield). H NMR (300 MHz, [D6]DMSO): d=10.06 (s, 1H),
9.06 (s, 1H), 8.72 (d, 1H, J=4.8 Hz), 8.25 (d, 1H, J=7.9 Hz), 7.53
(dd, 1H, J=8.0, 4.8 Hz), 7.37 (d, 2H, J=8.3 Hz), 6.55 (d, 2H, J=
8.3 Hz), 4.96 ppm (s, 2H); 13C NMR (75 MHz, [D6]DMSO): d=163.1,
151.7, 148.5, 145.5, 135.2, 130.9, 127.7, 123.4, 122.2, 113.7 ppm;
LC–MS (213.09): m/z 214.2 [M+H]+, tR =1.6 min, purity >99%
(method C).
1564 cmꢁ1
.
4-Amino-N-phenylbenzamide (54): A mixture of compound 55
(0.40 g, 1.7 mmol) and Pd/C (10%, 0.08 g) in MeOH and EtOAc (2:1,
15 mL) was hydrogenated and stirred at RT overnight. The reaction
mixture was filtered through Celite 545 and washed with MeOH.
The filtrate was dried (Na2SO4), filtered and evaporated in vacuo to
give a yellowish–brown crude solid. This was recrystallized from a
mixture of n-hexane and THF (2:1) to give a brown solid (com-
pound 54; 0.13 g, 59% yield). 1H NMR (300 MHz, [D6]DMSO): d=
9.75 (brs, 1H), 7.76–7.70 (m, 4H), 7.30 (t, 2H, J=7.9 Hz), 7.03 (t,
1H, J=7.4 Hz), 6.60 (d, 2H, J=8.6 Hz), 5.75 (brs, 2H); 13C NMR
(75 MHz, [D6]DMSO): d=165.3 152.1, 139.8, 129.3, 128.4, 122.9,
121.1, 120.1, 112.5 ppm; LC–MS (212.09) m/z 213.0 [M+H]+, 235.0
[M+Na]+; tR =14.5 min, purity 95% (method A).
3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-5-ylamine (41): A mix-
ture of phenylhydrazine hydrochloride (0.250 g, 1.73 mmol) and 4-
chlorobenzoylacetonitrile (0.318 g, 1.78 mmol, 1.03 equiv) in MeOH
(3 mL) was irradiated under mw at 1208C for 40 min. The reaction
mixture was cooled to RT, and the solvent was evaporated in va-
cuo. A 2m solution of NaOH in H2O (20 mL) was added to the
crude product, and the resulting mixture was extracted with
CH2Cl2 (2ꢃ20 mL). The combined organic phases were washed
with brine (2ꢃ20 mL) and evaporated in vacuo. The crude product
was recrystallized from a mixture of n-hexane and THF (1:1) to give
1
a white solid (compound 41; 0.21 g, 44% yield). H NMR (300 MHz,
CDCl3): d=7.75 (d, 2H, J=8.4 Hz), 7.63 (d, 2H, J=8.4 Hz), 7.55–7.48
(m, 2H, J=7.7 Hz), 7.40–7.34 (m, 3H), 5.94 (s, 1H), 3.86 ppm (brs,
2H); 13C NMR (75 MHz, CDCl3): d=150.5, 146.1, 138.6, 133.7, 132.1,
129.8, 128.9, 127.9, 127.1, 124.4, 88.2 ppm.
4-Nitro-N-phenylbenzamide (55): 4-Nitrobenzoyl chloride (0.20 g,
1.1 mmol, 1.1 equiv) was slowly added to a stirred solution of ani-
line (0.093 g, 1.0 mmol) and DMAP (6.4 mg, 0.05 mmol, 0.05 equiv)
in THF (10 mL) at 08C. The resulting solution was allowed to warm
to RT and stirred at RT for 6 h. A saturated NaHCO3 solution (3 mL)
was added to the reaction mixture, and the resulting mixture was
extracted with EtOAc (3ꢃ10 mL). The combined organic layers
were dried (Na2SO4), filtered and evaporated in vacuo to give the
crude product as a yellowish brown solid. The solid was recrystal-
lized from a mixture of n-hexane and EtOAc (2:1) to give a pale-
(5-Amino-2,3-dihydroindol-1-yl)pyridin-3-ylmethanone (52):
A
mixture of compound 53 (0.20 g, 0.74 mmol) and palladium(II) ace-
tate (0.0080 g, 0.037 mmol, 0.05 equiv) in THF (5 mL) was stirred at
RT for 10 min. A 1m solution of KF in H2O (1.5 mL, 2.0 equiv) was
added to the mixture and the resulting mixture was stirred at RT
for 5 min. Poly(methylhydrosiloxane) (PMHS) (0.18 mL, 2.0 mmol,
4.0 equiv) was added dropwise at RT over 3 min, and the resulting
black solution was stirred at RT for 20 h. PMHS (0.060 mL,
0.67 mmol, 1.3 equiv) was then added dropwise. The reaction mix-
ture was heated at 608C for 20 min, and a 1m solution of KF in
H2O (0.50 mL, 0.66 equiv) was added. Et2O (20 mL) was added to
the mixture and stirred for 5 min. The aqueous layer was then ex-
tracted with Et2O (2ꢃ10 mL), the combined organic phases were
filtered through Celite 545, and the cake was washed with EtOAc
(40 mL). The filtrate was washed with brine (2ꢃ30 mL), dried
(Na2SO4), filtered and evaporated in vacuo. The crude product was
purified by SiO2 flash column chromatography (EtOAc/MeOH,
1:0!0:1) to give a brown solid (compound 52; 0.058 g, 33%
1
yellow solid (compound 55; 0.58 g, 71% yield). H NMR (300 MHz,
[D6]DMSO): d=10.56 (brs, 1H), 8.45–8.26 (m, 2H), 8.23–7.97 (m,
2H), 7.78 (d, J=8.4 Hz, 2H), 7.38 (t, J=7.9 Hz, 2H), 7.14 ppm (ddd,
J=7.1, 2.2, 1.1 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO): d=163.9,
156.7, 140.5, 139.8, 129.3, 128.7, 124.2, 123.5, 120.5 ppm; LC–MS
(242.07) m/z 243.1 [M+H]+, 265.0 [M+Na]+; tR =21.7 min, purity
96% (method A).
Biochemical and cellular assays
1
Recombinant ALK catalytic domain was expressed in the Baculovi-
rus system, purified by affinity chromatography and used in an
ELISA-based kinase assay as described previously.[42] Dose–response
curves were generated by plotting normalized kinase activity
versus inhibitor concentration. IC50 values represent the concentra-
tion that causes 50% inhibition relative to the vehicle-treated con-
trol samples. Cell growth assays were performed according to the
[3H]thymidine incorporation method. Murine IL3-dependent BaF3
cells (control) and NPM–ALK-transfected BaF3 cells[43] were seeded
in 96-well plates and incubated with inhibitors for 72 h. An eight-
hour [3H]thymidine pulse before harvesting allowed direct measure
of the proliferation rate by liquid scintillation. Dose–response
curves were obtained, and IC50 values were calculated. Every inhibi-
yield). H NMR (300 MHz, [D6]DMSO): d=8.76 (1H, s), 8.65 (1H, d,
J=3.3 Hz), 7.99 (1H, d, J=8.1 Hz), 7.81 (1H, d, J=8.7 Hz), 7.49 (1H,
ddd, J=7.8, 4.9, 0.9 Hz), 6.49 (1H, d, J=2.1 Hz), 6.42 (1H, d, J=
8.4 Hz), 4.98 (2H, brs), 3.93 (2H, t, J=1.8 Hz), 2.95 ppm (2H,
1.8 Hz); 13C NMR (75 MHz, [D6]DMSO): d=164.2, 150.4, 147.7,
145.9, 134.6, 134.5, 132.5, 123.4, 117.6, 111.7, 110.3, 50.3, 28.1 ppm.
(5-Nitro-2,3-dihydroindol-1-yl)pyridin-3-ylmethanone (53): A so-
lution of nicotinic acid (1.0 g, 8.1 mmol) and 1,1’-carbonyldiimida-
zole (1.4 g, 8.0 mmol, 0.99 equiv) in DMF (5 mL) was stirred at RT
for 1 h. 5-Nitroindoline (1.2 g, 7.1 mmol, 0.87 equiv) was added to
the solution and, after 15 min of mixing, the solution was heated
at 608C for 21 h. DMF (15 mL) and CDI (0.73 g, 4.0 mmol,
ChemMedChem 2011, 6, 1680 – 1692
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1691

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We thank Matti Wahlsten and Miikka Olin for technical support.
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Revised: May 22, 2011
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