Discovery of novel fibroblast growth factor receptor 1 kinase inhibitors by structure-based virtual screening

Article abstract of DOI:10.1021/jm901386e

Fibroblast growth factors (FGFs) play important roles in embryonic development, angiogenesis, wound healing, and cell, proliferation and differentiation. In search, of inhibitors of FGFRl kinase, 2.2 million compounds were docked into the ATP binding site of the protein. A co-crystal structure, which shows two alternative conformations for the nucleotide binding loop, is reported. Docking was performed on both conformations and, ultimately, 23 diverse compounds were purchased and assayed. Following hit validation, two compounds 10 and, 16, a benzylidene derivative of pseudothiohydantoin and a thienopyrimidinone derivative, respectively, were discovered that, inhibit FGFRl kinase with IC₅₀ values of 23 and 50 μM. Initial optimization of 16 led to the more unsaturated, 40, which has significantly enhanced potency, 1.9 μM., The core structures represent new structural motifs for FGFRl, kinase inhibitors. The study also illustrates complexities associated with the choice of protein structures for docking, possible use of multiple kinase structures to seek, selectivity, and hit identification.

Full text of DOI:10.1021/jm901386e

1662 J. Med. Chem. 2010, 53, 1662–1672
DOI: 10.1021/jm901386e
Discovery of Novel Fibroblast Growth Factor Receptor 1 Kinase Inhibitors by Structure-Based
Virtual Screening†
Krishna P. Ravindranathan,‡ Valsan Mandiyan,§ Anil R. Ekkati,‡ Jae H. Bae,§ Joseph Schlessinger,§ and
William L. Jorgensen*,‡
‡Department of Chemistry, Yale University, New Haven, Connecticut 06520, and §Department of Pharmacology, Yale University School of
Medicine, New Haven, Connecticut 06520
Received September 17, 2009
Fibroblast growth factors (FGFs) play important roles in embryonic development, angiogenesis,
wound healing, and cell proliferation and differentiation. In search of inhibitors of FGFR1 kinase,
2.2 million compounds were docked into the ATP binding site of the protein. A co-crystal structure,
which shows two alternative conformations for the nucleotide binding loop, is reported. Docking was
performed on both conformations and, ultimately, 23 diverse compounds were purchased and assayed.
Following hit validation, two compounds 10 and 16, a benzylidene derivative of pseudothiohydantoin
and a thienopyrimidinone derivative, respectively, were discovered that inhibit FGFR1 kinase with IC50
values of 23 and 50 μM. Initial optimization of 16 led to the more unsaturated 40, which has significantly
enhanced potency, 1.9 μM. The core structures represent new structural motifs for FGFR1 kinase
inhibitors. The study also illustrates complexities associated with the choice of protein structures for
docking, possible use of multiple kinase structures to seek selectivity, and hit identification.
Introduction
addition, activating mutations in FGFR genes have been
associated with various human skeletal disorders such as
Crouzon syndrome,24,25 achondroplasia,26-29 and thana-
tophoric dysplasia.29-31 Therefore, discovery of inhibitors
of FGFR kinases has substantial potential therapeutic
value.32,33
Protein tyrosine kinases play an important role in the
signaling pathways that control cell proliferation and diffe-
rentiation. Enhanced protein kinase activity due to activating
mutations or overexpression has been implicated in many
cancers.1-3 The fibroblast growth factor receptors (FGFR1a
through FGFR4)4 play an important role in embryonic
development, angiogenesis, wound healing, and malignant
transformation.5 In response to growth factor stimulation,
these transmembrane receptors undergo ligand dependent
dimerization, which activates their intracellular tyrosine
kinase domains, resulting in autophosphorylation and sub-
sequent interaction with and recruitment of downstream
cellular target proteins.6 Inappropriate activation of FGF
receptors have been implicated in several angiogenic patho-
logies including diabetic retinopathy, rheumatoid arthritis,
atherosclerosis, and tumor neovascularization.7-9 Aberrant
FGFR kinase activity has been implicated in different cancers
including breast cancer,10-15 human pancreatic cancer,16
astrocytomas,17,18 salivary gland adenosarcoma,19 Kaposi’s
sarcoma,20 ovarian cancer,21 and prostate cancer.22,23 In
Kinase inhibition can be achieved by competition with the
substrate, with ATP, or by locking the kinase into an inactive
state.34,35 The human genome encodes at least 518 protein
kinases.36 All protein kinases share common sequences and
structural homology in their ATP binding sites, making
selectivity an issue in the development of kinase inhibitors.
However, the less well conserved areas of the ATP binding site
can be exploited to increase selectivity, if desired. Important
classes of FGFR1 inhibitors presently known include in-
dolinones32 such as SU4984 (1) and SU5402 (2) in Figure 1,
substituted pyrido[2,3-d]pyrimidines such as PD173074
(3),33,37 and the closely related 3-aryl-1,6-napthyridine-2,7-
diamines.38 These compounds show varying kinase inhibitory
strengths and selectivities. 1 inhibits the kinase activities of
FGFR1, PDGFR, and insulin receptor (InsR), but it does not
inhibit the kinase activity of EGFR.32 2 is more selective. It
inhibits the tyrosine kinase activity of FGFR1, it is a weak
inhibitor of PDGFR, and it does not inhibit the activity of
InsR and EGFR.32 1 and 2 inhibit the activity of FGFR1
kinase with IC50 values of 10-20 μM.32 However, 3 shows
high selectivity for FGFR1, inhibiting its activity with nano-
molar potency while inhibiting Src, InsR, EGFR, PDGFR,
and several other kinases with 1000-fold or higher IC50
values.33 Several FGFR kinase inhibitors, particularly in the
indolinone and 1H-quinolin-2-one classes, are currently in
clinical trials. Like the highly successful indolinone sunitinib,
they are multikinase inhibitors.39
†PDB accession code for the crystal structure of FGFR1 kinase with 4
is 3JS2.
*To whom correspondence should be addressed. Phone: 203-432-
6278. Fax: 203-432-6299. E-mail: William.Jorgensen@yale.edu.
a Abbreviations: ATP, adenosine triphosphate; EDTA, ethylenedia-
minetetraacetic acid; EGFR, epidermal growth factor receptor; FEP,
free energy perturbation; FGFR, fibroblast growth factor receptor; GB/
SA, generalized Born/surface area; InsR, insulin receptor; MC, Monte
Carlo; MEK, mitogen-activated extracellular signal-regulated kinase;
MES, 2-(N-morpholino)ethanesulfonic acid; OPLS-AA, optimized po-
tentials for liquid simulations-all-atom; PDGFR, platelet-derived
growth factor receptor; SP, standard precision; VEGFR, vascular
endothelial growth factor receptor; XP, extra precision.
r
pubs.acs.org/jmc
Published on Web 02/02/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1663
Figure 1. Examples of inhibitors of FGFR1 kinase with available
crystal structures: indolinones 1 and 2, the pyridopyrimidine 3 and 4.
Figure 3. Crystal structures of FGFR1 kinase with 4 illustrating
the two conformations for the nucleotide binding loop: extended or
up (blue) and down (brown).
nicotinic acid, 4 (Figure 1), at a resolution of 2.2 A.40 This
small inhibitor resides in the ATP binding site and forms a
hydrogen bond with the nitrogen of Ala564. Notably, the
crystal structure also reveals two conformations in the same
crystallographic unit, one with the nucleotide binding loop
down (brown in Figure 3) and the other with the loop
extended, forming part of a β-strand, and pointing upward
(blue in Figure 3). An issue with structure-based inhibitor
design for FGFR1 kinase is, thus, the flexibility of the protein
in this region.
Consequently, in view of the potential therapeutic impor-
tance and available crystallographic data, FGFR1 kinase is a
compelling target for structure-based inhibitor design. How-
ever, no prospective virtual screening studies have been
reported previously for discovery of FGFR kinase inhibitors.
Thus, the present study was carried out (a) to seek novel leads
as inhibitors of FGFR1 kinase by virtual screening and (b) to
test the viability of current docking methodology for this
purpose. High potency and selectivity were not expected at
this stage; rather, a foundation for subsequent lead optimiza-
tion was sought. The study illustrates complexities associated
with the choice of protein structures for docking, possible use
of multiple kinase structures to seek selectivity, practical
restrictions on numbers of compounds that can be purchased
and assayed, and hit identification. In particular, we have
docked a large database of molecules into the ATP binding
site of FGFR1 kinase using both conformations for the
nucleotide binding loop in the cocrystal structure for 4.
Compound selection was also influenced by docking results
for five additional kinases. Subsequent in vitro assaying
followed by validation of active compounds and initial opti-
mization did lead to the discovery of several compounds with
new core structures that exhibit low-micromolar inhibition of
FGFR1 kinase.
Figure 2. Indolinone 1 bound in the ATP binding site of FGFR1
kinase. Hydrogen bonds formed with Glu562 and Ala564, which are
in the hinge region, are highlighted.
FGFR1 kinase consists of two subdomains enclosing the
ATP binding cleft.32,33 This cavity, which is occupied by
adenine of ATP or the core of the inhibitors, is lined by
hydrophobic residues. When bound, 1 forms two hydrogen
bonds with the backbone carbonyl oxygen of Glu562 and
nitrogen of Ala564, which belong to the hinge region con-
necting the two lobes (Figure 2). Crystal structures of the
inhibitors 1, 2, and 3 bound to FGFR1 kinase32,33 indicate
that they reside in the ATP binding siteand have at least one of
the two hydrogen bonds with the hinge region.
The crystal structure of FGFR1 kinase bound to 132 shows
the nucleotide binding loop in a disordered conformation,
whereas the loop is in an extended conformation in the crystal
structures of FGFR1 kinase complexed with 232 and 3.33 At
the outset of the present work, we determined a crystal stru-
cture of FGFR1 kinase with the 5-thiophen-2-yl derivative of
Methods
Virtual Screening. The ZINC database41 was docked into the
two conformations from the FGFR1-4 structure40 using Glide

1664 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Ravindranathan et al.
4.0.42,43 The ZINC collection contains about 2.2 million small
molecules that have been filtered to be reasonable starting points
for drug design and that are commercially available. The
molecules are created, as appropriate, with multiple protonation
and tautomeric states. The FGFR1 kinase conformations were
prepared using standard Glide protocols.42 This includes addi-
tion of hydrogens, restrained energy-minimizations of the pro-
tein structure with the OPLS-AA force field,44 and finally setting
up the Glide grids using the Protein and Ligand Preparation
Module. Crystal structures of kinases with inhibitors bound in
the ATP site reveal that the inhibitors form at least one hydro-
gen bond with backbone amide or carbonyl groups in the hinge
region. Thus, imposition of pharmacophore constraints with the
hinge region has been routine in docking of molecules into the
ATP sites of kinases to improve the chances of success in finding
active compounds.45-47 This practice was adopted here such
that any acceptable protein-ligand complex was required to
have at least one hydrogen bond with Glu562 or Ala564
(Figure 2). All 2.2 million compounds were first docked and
ranked using standard precision (SP) Glide.42 The resultant top
40000 compounds were then docked using the more accurate
and computationally intensive extra-precision (XP) mode.43
The 41 compounds48 known to exhibit activity against FGFR1
kinase including 15 indolinones such as 1 and 2, 15 pyrido-
[2,3-d]pyrimidines such as 3, and 10 napthyridines, were also
included. The processing of the known active compounds was
intended to help gauge the effectiveness of the screening stra-
tegy. The top 1000 compounds for both conformations from the
XP docking were also docked into five additional kinase struc-
tures, EGFR,49 InsR,50 VEGFR2,51 Src,52 and MEK,53 in order
to see if the current scoring is accurate enough to provide some
kinase selectivity. Compounds that were eventually purchased
ranked in the top 100 for FGFR1 but not in the top 100 for any
of the other five kinases.
Docking with Glide effectively performs a systematic search
of the conformational, orientational, and positional space for
the docked ligand in the binding site of the protein. This is
followed by torsionally flexible energy optimization on an
OPLS-AA44 potential energy grid to arrive at a few final
candidate poses. The lowest energy poses are further refined
by Monte Carlo sampling. The scoring function used in (SP)
Glide includes terms for ligand-protein interaction energies,
hydrophobic interactions, hydrogen bonds, internal energies,
and desolvation.42 The more sophisticated XP scoring function
also employs terms that account for ligand-protein structural
motifs that lead to enhanced binding affinity.43 This includes
hydrophobic enclosure, where lipophilic ligand atoms are en-
closed on opposite faces by lipophilic protein atoms, and single
or correlated hydrogen bonds in hydrophobic environments,
which are relevant for kinase inhibitors in the ATP site. Thus,
XP Glide is expected to be well suited for the study of
ligand-kinase complexation.
were built starting from the crystal structure for the complex
with 4 and included the 170 amino acid residues nearest the
ligands. Short conjugate-gradient minimizations were carried
out on the initial structures for all complexes to relieve any
unfavorable contacts. Coordinates for the unbound ligands
were obtained by extraction from the complexes. The unbound
ligands and complexes were solvated in 25 A caps with ca. 2000
and 1250 water molecules. The FEP calculations utilized 11
windows of simple overlap sampling. Each window covered
10-15 million (M) configurations of equilibration and 20-30 M
configurations of averaging. The ligand and side chains with any
atom within ca. 10 A of the ligand were fully flexible, while the
protein backbone was kept fixed during the MC simulations.
The energetics were evaluated with the OPLS-AA force field for
the protein, OPLS/CM1A for the ligands, and TIP4P for
water.44
Experimental. Purchased compounds were initially assayed
as received. Structures of active and new compounds were
validated through 1H and 13C NMR and high-resolution mass
spectrometry, as detailed in the Supporting Information. The
purity of all active and new compounds was demonstrated to be
>95% by high-performance liquid chromatography. Synthetic
schemes for new compounds are provided below, while the
synthetic details are provided in the Supporting Information.
Biological assaying was performed using ALPHAScreen
(Amplified Luminescent Proximity Homogeneous Assay) from
Perkin-Elmer. FGFR1 kinase, the biotinylated peptide, ATP,
and the potential inhibitor were added to each of 10 wells with
inhibitor concentrations typically ranging from 0.01 to 250 μM.
Phosphorylation of the peptide was allowed to proceed for
30 min, and then the reaction was stopped by adding EDTA.
The resultant differential phosphorylation of the peptide by
FGFR1 kinase in each well depends on the effectiveness of the
inhibitor. Acceptor beads coated with antiphosphotyrosine
antibodies and donor beads coated with streptavidin conjugates
are then introduced into each well. Biotinylated peptide is
efficiently captured by the donor bead. The FGFR1 kinase
domain was obtained and purified as previously described.55
Other kinases used in this study, EGFR, InsR, and Src, were
obtained from Millipore Corp.
Purified FGFR1 kinase domain (20 mg/mL) was cocrystal-
lized with 2-thienyl nicotinic acid, 4 (1 mM), by vapor diffusion
in 0.1 M MES-NaOH (pH 6.5), 14% PEG 4000, 0.2 M
(NH4)2SO4, and 5% glycerol at 4 °C. The crystals were flash
frozen in 0.1 M MES (pH 6.5), 25% PEG 4000, 0.2 M
(NH4)2SO4, and 10% glycerol. Diffraction data were collected
on beamline X29 at the National Synchrotron Light Source.
They were processed by using HKL2000, and a molecular
replacement solution for the complex was found by using the
structure of inactive FGFR1 (PDB ID: 1FGK).56 The resultant
structure has been deposited in the Protein Data Bank (PDB ID:
3JS2).40
The resultant most desirable compounds were also evaluated
for conformational strain in the bound conformation. One
measure of this strain is the root-mean-square deviation
between a molecule’s docked structure and its structure after
energy minimization in vacuum. Another measure that was
applied is the difference in energy after minimization and the
lowest energy conformation resulting from a 200 conformation
Monte Carlo search with GB/SA hydration using the program
BOSS.54 These measures revealed that most compounds of
interest were in reasonably favorable conformations in the
protein binding site. Visual inspection of poses was also essential
in arriving at the final selections, which were then purchased
from commercial vendors.
Results
Docking Known Inhibitors. The top 40000 compounds
emerging from the SP Glide calculations plus the 41 com-
pounds possessing known activity were processed with XP
Glide. For both loop conformations, eight of the known
active compounds ended up in the top 1000. There were four
active compounds in common, and thus 12 unique active
compounds were retrieved. Assuming equal distribution of
the 41 compounds in 40000, there is roughly 1 in every 1000.
Thus, the finding of eight in the top 1000 reflects significant
enrichment. If the top 10000 compounds are considered, 17
and 11 of the known active compounds were identified using
the protein conformation with the binding loop down and
up, respectively. The performance of SP Glide alone can also
be noted. When the 41 known active compounds are added
MC/FEP Simulations. Some Monte Carlo/free-energy per-
turbation calculations were executed to compute relative free
energies of binding in order to guide initial modifications of
active compounds. The calculations were performed following
standard protocols with the program MCPRO.54 The models

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1665
to the 2.2 million library compounds, 9 and 10 are ranked in
the top 10000 using the protein conformations with the
binding loop down and up, respectively, and 12 and 11
compounds are among the top 50000. The random result
would be retrieval of roughly 1 known active compound per
50000. Thus, docking with Glide in both SP and XP modes
demonstrated success in identifying known FGFR1 kinase
inhibitors. Additional benefits of the XP mode over SP were
not clearly apparent in this exercise. As detailed below, the
performance of XP Glide for the known inhibitors was also
class specific.
Superposition of the crystal structures with the poses from
Glide reveals good correspondence for the positioning of 1
and 2 (Figure 4). In general, among the known active
compounds, ones with the indolinone core show the correct
orientation when compared to the crystal structures of the
complexes for 1 and 2.32 There is also very good overlap of
the positions of 4 in the crystal structure40 and in the docked
complexes for both conformations of the nucleotide binding
loop. However, the docked structure for FGFR1 kinase with
the nucleotide binding loop up complexed with 3 has in-
correct orientation of the ligand (Figure 4). Superposition of
structures of complexes with compounds having the pyrido-
[2,3-d]pyrimidine or napthyridine cores from the XP docking
and the crystal structure of 3 reveal a 180° flip in the binding
site (Figure 4). The crystal structures used for the docking
were the ones for 4, as this was the most complete structure
with no missing residues. However, most of the pyridopyri-
midine inhibitors have large side chains, which clash steri-
cally with the side chain of Lys514 when the inhibitor’s core
is properly oriented. Thus, incorrect docking of the pyrido-
[2,3-d]pyrimidine class could be improved by screening
against additional conformations of FGFR1 kinase. This
would hopefully lead to additional compounds with very low
docking scores that could be further scrutinized. Alterna-
tively, the conformational problem argues in favor of frag-
ment screening to discover viable core structures without the
need to accommodate large substituents. Owing to these
considerations, 8 of the 12 unique active compounds in the
top 1000 from the XP docking with the nucleotide binding
loop in either orientation belong to the indolinone class. The
correct docking of these compounds presumably accompa-
nies their good rankings. The compounds in the pyrido-
[2,3-d]pyrimidine and the napthyridine classes are in-
correctly docked and rank poorly.
The distribution of XP scores for the 40000 compounds
from the ZINC library, when docked into either FGFR1
kinase conformer, covers a 16 kcal/mol range (Figure 5).
Many library compounds have scores as low as those for the
best-ranked known actives (in red in Figure 5). Although it is
encouraging that many compounds from ZINC yield such
favorable scores, many known active compounds have poor
scores in view of the incorrect docking.
Compound Selection and Activity. The 2000 library com-
pounds, 1000 for each FGFR1 conformer, with the best XP
scores, were processed further. For both conformers, to
favor compounds that could selectively inhibit FGFR1
kinase, the top 1000 compounds were docked into the ATP
binding sites for the five other kinases, EGFR, InsR,
VEGFR2, Src, and MEK. While the kinome has roughly
500 members,36 this subset was chosen based on potential
availability of the protein for assaying and, of course, avail-
ability of a cocrystal structure of the kinase with a ligand in
the ATP site. Compounds ranked within the top 100 for
FGFR1 kinase, but not in the top 100 for any of the other 5
kinases, were retained for further consideration. Their
docked protein-ligand complexes received extensive visual
inspection. Ligands that possessed inappropriate geometries
such as twisted amides or esters were rejected.57 The com-
pounds were also checked for excessive conformational
strain in the bound conformation, as described above.
At this point, for the set of compounds from the screening
of the conformation with the binding loop up, a total of
31 desirable compounds remained. To promote structural
diversity, this set was further partitioned into 14 chemical
classes. Eventually, 11 compounds, which were mostly the
Figure 4. Overlays of the crystal structure (gray) with the XP
GLIDE pose (green) for 1, 2, 3, and 4. Hydrogen bonds with
Glu562 and Ala564 are highlighted.
Figure 5. XP Glide score frequency for 40000 compounds from the ZINC collection in black and known active compounds in red (scaled by
300) for the conformation with (A) the nucleotide binding loop up and (B) with the nucleotide binding loop down.

1666 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Ravindranathan et al.
Figure 6. Structures of the purchased compounds obtained by docking into the conformation of FGFR1 kinase with the nucleotide binding
loop up. Although 7 was ordered, it turned out to be the isomer 10. Samples of both 7 and 10 were synthesized.
top-ranked in their class, were purchased. These are shown in
Figure 6. In one case, 7, structural ambiguity arose, and it
was subsequently determined that the purchased compound
was actually the isomer 10. As described below, this was
demonstrated through synthesis and assaying of both 7 and
10. The docked structures and scores for 7 and 10 are
essentially identical, as the structural difference is in the edge
that is solvent-exposed in the complexes. Thus, the structures
of the two isomers, 7 and 10, and the 10 other purchased
compounds are shown in Figure 6. This set has a good
representation of five- and six-membered heterocycles in-
cluding six with fused polycyclic ring systems. Not surpris-
ingly, in the docked structures, the polycyclic cores overlap
the position of adenine of ATP when bound. All computed
structures of the complexes feature hydrogen bonds in the
hinge region and extensive overlap with the positions of
known inhibitors in crystal structures for complexes with
FGFR1 kinase.
FGFR1 kinase inhibitors were sought; indolinones, naph-
thyridines, and compounds with 2-aminopyrinidine frag-
ments were avoided. A total of 23 compounds was purchased
and tested in vitro for inhibition of FGFR1 kinase using the
AlphaScreen assaying system. The results of the assaying
along with Glide scores and ranks are shown in Table 1. Two
of the compounds were found to inhibit the activity of
FGFR1 kinase. Although follow-up of these hits is enough
to consume our available resources, purchase and assaying
of at least all 68 of the compounds deemed most desirable
would likely have generated additional alternatives. It is also
expected that many of the core structures for the inactive
compounds among 5-28, in fact, provide viable platforms
for discovery of active inhibitors; small changes are often all
that is needed to turn an inactive compound into an active
one.58,59
7, 10, and Analogues. One of the purchased compounds
from ChemBridge Corp., presumed to be 7, had an IC50
value of 23 μM. For initial follow-up, 7 and several analo-
gues, 29-34, were synthesized as summarized in Scheme 1.
The intention was exploration of modifications to the methoxy
group in 7 and a chlorine scan for the terminal phenyl
ring. MC/FEP results indicated that chlorine and methyl
substitution should be most favorable at the meta positions
In the same manner, the top 1000 compounds from the
docking calculations with the FGFR1 binding loop in the
down conformation were narrowed to 37 compounds, which
were assigned to 15 classes. Among these, the 12 high-
ranking ones in Figure 7 were ultimately purchased. In both
cases, compounds with cores not previously reported as

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1667
Figure 7. Structures of the purchased compounds obtained by docking into the conformation of FGFR1 kinase with the nucleotide binding
loop down. Compounds with stereocenters are racemic mixtures.
in this ring. The benzylidene derivatives were prepared from
either commercially available p-hydroxybenzaldehyde or
3-alkoxy-4-hydroxybenzaldehydes. In the case of 29, first
p-hydroxybenzaldehyde (41) was converted to 3- methoxy-
methyl-4- hydroxybenzaldehyde (42c). The aldehydes 43a-c
resulted from O-alkylation of 42a-c with 1,3-dibrompro-
pane. The O-alkylation of alkyl bromides 43a-c with phe-
nols 44a-d then provided aldehydes 45a-g, and
Knoevenagel condensation of 45a-g with thiohydantoin
afforded 7 and 29-34.
The compounds were assayed (Table 2), and it was sur-
prising to find that 7 was now inactive and that only 29 and
30 showed weak activity. A small molecule crystal structure
was then obtained for 31, which confirmed the structures in
this series to be as shown in Table 2, notably with the
thiohydantoin substructure and Z-olefin geometry. Thus,
the purchased compound displaying the 23 μM activity was
not 7. Scrutiny of the NMR spectra for the purchased
compound and the synthesized 7 led to the expectation that
the purchased compound might be the pseudothiohydantoin
isomer 10. This compound was then prepared by Knoeve-
nagel condensation of aldehyde 45a with pseudothiohydan-
toin (Scheme 1). The AlphaScreen assay demonstrated that
10 was the active compound (Table 1), and the spectral data
confirmed that 10 and the purchased compound were the
same. Compound 10 was subsequently docked into FGFR1
kinase. The predicted pose is almost identical to that for 7,
and it was found to rank well (Table 1). 10 also showed poor
ranking against the other 5 kinases, thus meeting the com-
putational selectivity criterion. In summary, extensive vali-
dation was needed to confirm that 10 rather than 7 was the
active compound found in the screening.
16 and Analogues. Thienopyrimidinone 16 is the other
purchased compound that was found to inhibit the activity of
FGFR1 kinase. However, the IC50 value of 50 μM was
modest, so some initial lead optimization was pursued to
see if a simple analogue in the low μM range could be
obtained. To examine the role of the carboxylic acid side
chain of 16, which was predicted to be largely solvent
exposed in the complex with FGFR1 kinase, compounds
35 and 36 were synthesized, as shown in Scheme 2. Relatives
with the tricyclic core fully unsaturated such as 37-40
were also pursued. MC/FEP calculations were performed
and indicated that replacement of H by methyl at R1 and R2
should improve the free energy of binding by 2.2-2.6 kcal/mol.
Introduction of a methyl group at the open position adjacent

1668 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Ravindranathan et al.
to R1 was computed to be a little less favorable, while a
methyl group at the remaining site adjacent to R2 is highly
unfavorable owing to steric clashes with the backbone of
Glu562. Finally, given the experience with 7 and 10, 16 too
was synthesized.
Preparation of thiouracil derivatives 16 and 35-40 com-
menced with 3- or 4-substituted cyclohexanones. Gewald
reaction of cyclohexanones 46a-e with 2-cyanoacetamide
provided 2-aminothiophenes 48a-e. Condensation of 48a
with aldehyde 52 provided ester 49, which was hydrolyzed to
afford 16. The spectra for the purchased 16 and synthesized 16
were the same. The reaction of 2- aminothiophene 48a with
3,4-dimethoxybenzaldehyde and 3-methoxybenzaldehyde
resulted in the formation of 35 and 36. For the synthesis of
benzothiophene derivatives 37-40, first, the 2-aminothio-
phenes 48a-e were converted to 2-aminobenzothiophenes
50a-e. Acid-catalyzed condensation of 50a-e with aldehyde
52 resulted in esters 51a-e, which were hydrolyzed to yield
37-40.
As summarized in Table 3, the decarboxylated 35 and 36
do not inhibit FGFR1 kinase. However, the unsaturated
analogues, 37 and 40, provided significant advance (Table 4).
Conversion of the cyclohexyl ring to phenyl in going from 16
to 37 lowered the IC50 from 50 to 4 μM. This outcome was
not obvious owing to the trade-off between greater hydro-
phobicity with 16 and greater rigidity with 37. Replacing the
methyl substituent by ethyl in proceeding from 37 to 38
yielded little changeinactivity, while introduction of trifluoro-
methyl at R1 in 39 eliminated activity. Consistent with the
MC/FEP results, a methyl group was found to have similar
effect at R1 and R2, with 40 being the most active analogue
with an IC50 of 1.9 μM. Overall, the unsaturated analogues
37, 38, and 40 provide a novel core structure for FGFR1
kinase inhibition and a much improved starting point for full
lead optimization.
Table 1. Experimental IC50 Values for Inhibition of FGFR1 Kinase
and Glide Scores and Rankings
compd
XP rank
XP score
SP rank
IC50 (μM)a
5
1
7
-16.18
-14.84
-14.29
-14.20
-13.98
-13.79
-13.71
-13.66
-13.64
-13.49
-13.48
-13.45
-18.53
-17.38
-17.01
-16.96
-16.93
-16.89
-16.84
-16.84
-16.78
-16.75
-16.62
-16.60
112
11628
33929
22167
550
na
na
na
na
na
23
na
na
na
na
na
50
na
na
na
na
na
na
na
na
na
na
na
na
6
7
16
19
25
38
46
51
52
74
77
84
2
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
61627
8409
Table 2. Experimental IC50 Values for Inhibition of FGFR1 Kinase by
Analogues of 7
13883
6377
36663
396
20052
429
10
29
37
41
45
55
56
64
72
93
97
10443
12782
7676
IC50
(μM)
14137
19473
9711
compd
R1
OMe
OEt
R2
Cl
R3
R4
R5
7
Me
Me
Me
Me
Me
Me
na
29
30
31
32
33
34
250
3300
na
15905
6525
4238
CH2OMe
OMe
OMe
Cl
na
na
27217
22135
OMe
OMe
Cl
Me
na
a na indicates not active in the assay.
Scheme 1a
a Reagents and conditions: (a) (1) HCHO, HCl, 50 °C, 3 h; (2) MeOH, reflux, 3 h, 57% two steps. (b) 1,3-Dibromopropane, K2CO3, DMF, 80 °C, 1 h
(43-60%). (c) K2CO3, DMF, 80 °C, 1 h (44-91%). (d) Thiohydantoin, pipiridine, MeOH, ref lux (54-74%). (e) pseudothiohydantoin, AcONa, AcOH,
170 °C, 15 min, MW, 53%.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1669
Scheme 2a
a Reagents and conditions: (a) S, piperidine, EtOH, 50 °C, 4 h (23-60%); (b) p-chloranil, 1,4-dioxane, 90 °C, 5 h; (c) 3,4-dimethoxybenzaldehyde or
3-methoxybenzaldehyde, conc HCl, n-BuOH, 80 °C, 8H (48-67%); (d) ethyl 2-(4-formyl-2-methoxyphenoxy)acetate (52), conc HCl, n-BuOH, reflux,
2 h (30-62%); (e) 5N NaOH, EtOH, reflux, 2 h (30%-quant).
Table 3. Inhibitory Activities of FGFR1 Kinase by Analogues of 16
Table 5. Some Properties of the Lead Compounds Predicted Using
QikProp 3.0
N
compd MWa QP log Pb QP log Sc QP PCaco2d metabolitese
10
40
412.5
396.4
4.49
3.26
-6.1
-5.6
673
36
6
5
a Molecular weight. b Log of the octanol/water partition coefficient.
c Log of the aqueous solubility S (mol/L). d Caco2 cell permeability in
nm/s. e Number of primary metabolites.
compd
R3
IC50 (μM)
16
35
36
OCH2CO2H
OMe
H
50
na
na
derivative 40 with an IC50 of 1.9 μM. To our knowledge, no
compounds with these core structures have been demon-
strated previously to be FGFR1 kinase inhibitors. Both
compounds evolved from the docking calculations that used
the conformation of FGFR1 kinase with the nucleotide
binding loop in the extended conformation (Figure 3). The
chosen compounds that came from the docking calculations
with the nucleotide binding loop down (Figure 7) were all
inactive. This should not be overinterpreted in view of the
modest success in finding only one true active compound, 16,
among the 23 compounds, which were purchased. More
favorable XP scores were obtained for the conformation
with the binding loop down (Table 1); however, a possible
reorganization penalty for achieving this conformation is not
included in the calculations.
Table 4. Inhibitory Activities of FGFR1 Kinase by Tetradehydro
Analogues of 16
compd
R1
R2
IC50 (μM)
To provide an initial sense of expected pharmacological
properties, the program QikProp60 was used to make the
predictions in Table 5. The selected properties are expected
to influence bioavailability through dissolution, cell permea-
tion, and metabolism. When QikProp is run for a set of 1700
oral drugs, 95% are predicted to have molecular weights
between 130 and 500, log P values between -2 and 6, log S
values between -6.0 and 0.5, PCaco2 values greater than
25 nm/s, and seven or fewer primary metabolites.61 The
predicted properties of the two key compounds compare
37
38
39
40
Me
Et
H
4.0
5.5
na
H
CF3
H
H
Me
1.9
Computed Properties, Structures, and Selectivity for 10 and
40. Thus, the screening and subsequent synthetic efforts deli-
vered two principal compounds, which are suitable for fur-
ther lead optimization, the pseudothiohydantoin derivative
10 with an IC50 of 23 μM and the benzothienopyrimidinone

1670 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Ravindranathan et al.
Figure 8. Computed structures for the complexes of FGFR1 kinase with 10 (A) and 40 (B). Selected backbone and side-chain atoms of the
kinase are shown; carbon atoms of the inhibitors are colored green. Hydrogen bonds are highlighted with black lines.
favorably with these ranges, although poorer solubility needs
to be avoided during further lead optimization. For 10, the
predicted primary metabolites arise from ether cleavages,
benzylic methyl oxidations, and possible sulfur oxidation.
For 40, the predicted metabolic processes are for oxidation
of the sulfur atom and the three side chains leading to
possible catechol formation.
Table 6. Inhibitory Activities for Four Kinases
IC50 (μM)
compd
FGFR1
EGFR
Src
InsR
10
40
23
1.9
56
2.4
10
1.9
47
na
toward Lys514 and Phe489 and truncation at the other end
of the inhibitor.
The structures from the Glide XP docking for 10 and 40
are illustrated in Figure 8. Both ligands are predicted to
bind in the hinge region and both feature two hydrogen
bonds with Ala564 via the amido fragments (OdC-NH) in
the pseudothiohydantoin and pyrimidinone rings. There is
considerable overlap with the observed positioning of the
indolinones, e.g., in Figure 2.32 However, the hydrogen-
bonding motif is interestingly different because, for the
indolinones, the order of the amido fragments (HN-CdO)
is reversed and the complementarity is with the backbone
carbonyl oxygen of Glu562 and the NH of Ala564. The
bound 10 also extends more to the right toward Phe489
than for the indolinones. In addition, the complex for 10
has a hydrogen bond between the methoxy group on the
ligand’s central ring and the side-chain ammonium group
of Lys514. The assay results for 29 in Table 2 indicate that
the methoxy to ethoxy change is beneficial for binding,
perhaps owing to favorable additional hydrophobic inter-
actions in the Val492-Lys514 area, while change to methoxy-
methyl (30) is less productive. Another notable motif is
the sandwiching of the dimethylphenyl ring of 10 between
the side chains of Phe489 and Lys514, forming presumably
constructive π-π and cation-π interactions. This does
impose conformational restrictions on the 1,3-dioxypropyl
linker. Returning to the 7 versus 10 conundrum, the
computed structures do not provide an obvious reason
for the inactivity of 7 because the SdC-NH and HN=C-S
edges are predicted to be solvent exposed. Presumably,
there is sensitivity of the critical hydrogen bonding with
Ala564 to the geometrical and electronic differences
between the isomeric rings or there are subtleties in
their hydration; further computational investigation is
warranted.
Finally, testing of compounds 10 and 40 for activity
against other kinases was performed using EGFR, InsR,
and Src (Table 6). Compound 10 is an inhibitor of all four
kinases with IC50 values of 10-56 μM, while 40 does not
inhibit InsR, but it is a 2 μM inhibitor for FGFR1, EGFR,
and Src. Thus, the limited computational selectivity filter
was not effective; however, fundamentally, the diverse re-
sults in Figure 5 for known inhibitors of FGFR1 kinase need
to be noted along with the fact that the two active com-
pounds from the present screening had essentially the two
worst scores in Table 1. Under the circumstances, the current
docking and scoring methodology does not appear to be
accurate enough to guide economically viable compound
acquisition in the absence of substantial human postproces-
sing.62 It seems even less likely that the current methodology
is accurate enough to successfully address kinase selectivity,
which requires reliability of the results for multiple targets.
The issue is complicated by the recognition of potential
clinical benefits for compounds with multikinase activity.39
Nevertheless, there is clear value in the docking, as it did
provide a key component of a compound selection protocol
that enabled the discovery of two new series of FGFR1
kinase inhibitors. Selectivity is typically expected to be
addressed during lead optimization through combination
of more computational modeling, synthesis, assaying, and
crystallography.59
Conclusions
The ZINC database of 2.2 million compounds was screened
using two conformations of FGFR1 kinase. On docking with
XPGlide, 8 of 41knownactivecompounds emergedin the top
1000 of 40000 compounds, which were the best ranked ones
using SP Glide. The indolinone class of inhibitors and nico-
tinic acid derivative 4 were handled well, while the docked
structures and scoring for the pyrido[2,3-d]pyrimidine and
napthyridine classes were inaccurate. The conformation of
Lys514 in the utilized structure led to steric incompatibilities
with many of the known inhibitors. The virtual screening
progressed to the purchase of 23 structurally diverse com-
pounds. Two compounds were initially found to be active.
However, much effort was needed to demonstrate that the
structure for one was incorrectly assigned and that the isomer
For 40, additional hydrogen bonding is indicated via salt
bridge formation between the ligand’s carboxylate group
and the ammonium terminus of Lys482. These groups can
also be fully solvent exposed. Thus, the energetic benefit of
the salt bridge is not clear; however, the results in Table 3
indicate that the carboxylate group is making a positive
contribution to the activity. The beneficial methyl groups
at R1 and R2 in 37, 38, and 40 (Table 4) are inserted into the
hydrophobic region near Val492. Comparison of the com-
puted structures for the complexes of 10 and 40 suggests that
lead optimization for 40 has opportunities in expansion

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1671
10 was the true active compound; synthesis of both isomers
was required. 10, a benzylidene derivative of pseudothio-
hydantoin, and 16, a thienopyrimidinone derivative, were found
to show inhibitory activity toward FGFR1 kinase with IC50
values of 23 and 50 μM. Modifications of 16 led to the more
unsaturated 40, which showed a much improved IC50 value of
1.9 μM. Thepredictedstructuresfor the complexes ofFGFR1
kinase with 10 and 40 appear reasonable in comparison to
known crystal structures, and they are generally consistent
with the initial structure-activity results presented here. Both
compounds are expected to form two hydrogen bonds with
the oxygen and amide NH of Ala564; aryl-aryl, cation-π,
and salt bridge interactions are also represented. Finally, the
selectivity of 10 and 40 for FGFR1 kinase received some
analysis through assaying with three additional kinases,
EGFR, InsR, and Src. Little selectivity was found, except
that 40 shows no inhibition of InsR kinase. Although the
applied computational selectivity filter could be made more
restrictive, it is unlikely that the accuracy of the current
docking and scoring methodology is sufficient to provide a
solid basis for this purpose. Optimization of the two new
series of FGFR1 kinase inhibitors for both potency and
selectivity is being pursued using a combined approach
featuring free-energy perturbation calculations, organic
synthesis, biological assaying, and protein crystallography.
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Supporting Information Available: Synthetic details and
spectral data for compounds 7, 10, 16, and 29-40. This material
is available free of charge via the Internet at http://pubs.acs.org.
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