Aryl extensions of thienopyrimidinones as fibroblast growth factor receptor 1 kinase inhibitors

Article abstract of DOI:10.1016/j.tetlet.2010.12.081

Optimization of thienopyrimidinone derivatives as FGFR1 kinase inhibitors is being pursued. The present results confirm predictions of computational modeling that an aryl substituent can be introduced at the 2-position in structure 3. The substituent is anticipated to project deeper into the binding site and provide opportunities for enhanced activity and selectivity. The most potent analog reported herein, 13, has a 4-hydroxyphenyl substituent and yields an IC₅₀ of 6 μM for inhibition of phosphorylation by FGFR1 kinase. It was also found that the western anisole-containing substituent in 3 can be replaced by a propionic acid group with no loss in potency and with potentially significant gains in pharmacologically relevant properties.

Full text of DOI:10.1016/j.tetlet.2010.12.081

Tetrahedron Letters 52 (2011) 2228–2231
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Aryl extensions of thienopyrimidinones as fibroblast growth factor receptor 1
kinase inhibitors
Anil R. Ekkati a, Valsan Mandiyan b, Krishna P. Ravindranathan a, Jae H. Bae b, Joseph Schlessinger b,
William L. Jorgensen a,
⇑
a Department of Chemistry, Yale University, New Haven, CT 06520, United States
b Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization of thienopyrimidinone derivatives as FGFR1 kinase inhibitors is being pursued. The present
results confirm predictions of computational modeling that an aryl substituent can be introduced at the
2-position in structure 3. The substituent is anticipated to project deeper into the binding site and pro-
vide opportunities for enhanced activity and selectivity. The most potent analog reported herein, 13, has a
Received 14 October 2010
Revised 11 December 2010
Accepted 19 December 2010
Available online 24 December 2010
4-hydroxyphenyl substituent and yields an IC50 of 6 lM for inhibition of phosphorylation by FGFR1
kinase. It was also found that the western anisole-containing substituent in 3 can be replaced by a
propionic acid group with no loss in potency and with potentially significant gains in pharmacologically
relevant properties.
This paper is dedicated with great affection
to Prof. Harry H. Wasserman, friend and
Yale colleague, on the occasion of his 90th
birthday
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
FGFR1 kinase inhibitors
Structure-based inhibitor design
Thienopyrimidinones
ATP-competitive small molecule inhibitors
Fibroblast growth factors (FGF) play an important role in many
biological processes including cell proliferation, migration, differ-
entiation, survival, morphogenesis, and angiogenesis.1 The diverse
biological effects of FGFs are mediated by cell surface receptors
(FGFR1-4) with intrinsic protein tyrosine kinase activity.2 FGFR
is activated by binding of FGF to its extracellular domain, result-
ing in receptor dimerization and autophosphorylation of specific
tyrosine residues in the cytoplasmic domain. The activated FGFR
interacts with downstream intracellular proteins to initiate signal
transduction, such as in the MAPK and PKB/Akt pathways.3
Abnormal FGF signaling either through gain or loss of function
by mutations in FGF or FGFR has been implicated in cancer and
other disorders, including skeletal, cardiovascular, immunological,
and neurological diseases.1,4 Therefore, targeted inhibition of
FGFR kinases with ATP-competitive small molecule inhibitors
has become an attractive therapeutic strategy. Several classes of
such inhibitors have been reported, including indolinones,5 pyr-
ido[2,3-d]pyrimidines,6 napthyridines,7 triazines,8 indenes,9 quin-
olines,10 and thioindoles.11 However, only a limited number of
FGFR inhibitors have entered clinical trials.12–17 Furthermore,
since the human genome encodes at least 518 protein kinases
and nearly all kinase inhibitors target the well-conserved ATP
binding site,18 development of selective kinase inhibitors contin-
ues to be a significant challenge.
In the course of studies to identify new FGFR1 kinase inhibitors,
we discovered two inhibitors 1 and 2, a benzylidene derivative of
pseudothiohydantoin and a thienopyrimidinone derivative, by
structure-based virtual screening followed by initial lead optimiza-
tion.19 1 and 2 inhibit phosphorylation by FGFR1 kinase with IC50
values of 23 and 1.9
1 was also found to be an inhibitor of EGFR, Src, and InsR kinases
with IC50 values of 10–56 M. The thienopyrimidinone 2 was
found to inhibit EGFR and Src kinases with IC50 values of 2.4 and
1.9 M, while it did not inhibit InsR. Thus, little selectivity was
lM, respectively. The pseudothiohydantoin
l
l
observed with these compounds. In an effort to develop more
potent and selective FGFR1 kinase inhibitors, the thienopyrimidi-
nones were chosen for further optimization.
A structure from docking calculations19 for the 21-
lM inhibitor
3 complexed with FGFR1 kinase is illustrated in Figure 2A. Forma-
tion of two hydrogen bonds between the amide fragment in the
pyrimidinone ring of 3 and Ala564 in the hinge region is antici-
pated. The tricyclic core of thienopyrimidinone 3 is spatially
limited to the ATP binding site, and the western end of the inhib-
itor is solvent exposed. Exploration eastward, deeper into the
active site, is expected to be potentially more fruitful, especially
⇑
Corresponding author. Tel.: +1 203 432 6278; fax: +1 203 432 6299.
E-mail address: William.Jorgensen@yale.edu (W.L. Jorgensen).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.12.081

A. R. Ekkati et al. / Tetrahedron Letters 52 (2011) 2228–2231
2229
Me
3
4
O
N
Me
O
N
O
2
OMe
3 O
HN
HN
HN
HN
1
S
S
S
Me
HO2C
O
HO2C
O
O
1
2
3
OMe
OMe
Figure 1. Chemical structure of 1, 2, and 3.
Figure 2. Computed structures of FGFR1 kinase with 3 (A) and 4 (B). Selected residues of FGFR1 kinase are shown; carbon atoms of the inhibitors are colored green. Hydrogen
bonds are highlighted with black lines.
O
O
O
HO
O
MgX
R
c
b
a
O
O
O
R
15a-h
16a-h
14
R
R
17a-h
18a-i
d
O
O
NH2
NH2
NH2
NH2
O
h
e
HN
R
S
MeO
S
S
N
20a-i
19a-i
22d-h
O
R
R
f
O
N
O
O
N
HN
R
R
S
S
N
23d-h
21a-c, 21i
EtO2C
O
OMe
g
g
O
N
O
HN
HN
R
HO
R
S
S
N
O
7-12
4-6, 13
HO2C
O
OMe
Scheme 1. Reagents and conditions: (a) RMgX, THF, 10 °C–rt, 3 h, 70–95%; (b) TFA, DCM, rt, 5 h, 84%-quant; (c) 10% Pd/C, EtOAc, rt, 16 h, 73–83%; (d) 2-Cyanoacetamide, S,
piperidine, EtOH, 50 °C, 4 h, 42–50%; (e) p-chloranil, 1,4-dioxane, 90 °C, 5 h, 56–65%; (f) ethyl 2-(4-formyl-2-methoxyphenoxy)acetate (24), concd HCl, n-BuOH, reflux, 2 h,
57–80%; (g) 5 N NaOH, EtOH, reflux, 2 h, 60%-quant; (h) methyl-4-oxobutanone (25), concd HCl, n-BuOH, 80 °C, 8 h, 48–67%.
for selectivity owing to variation among kinases in this area. In the
eastern end of 3 (see Figs. 1 and 2A), position 1 is solvent exposed
and substitutions here should have limited effect on modulating
activity. However, the de novo ligand-design program BOMB20,21
(Biomolecular and Organic Molecule Builder) was used to consider
introduction of substituents at positions 2–4 on the ligand in the
binding site; the utilized protein coordinates came from the struc-
ture of the complex with a nicotinic acid derivative, which we
previously reported (PDB ID: 3JS2).19,22 Substituents larger than
methyl cannot be introduced at positions 3 and 4 as they would

2230
A. R. Ekkati et al. / Tetrahedron Letters 52 (2011) 2228–2231
clash with the protein backbone and likely cause disruption of the
critical hydrogen bonds between the tricyclic core and the hinge
region. However, extension at the 2-position with monocyclic
and possibly fused bicyclic aryl substituents emerged as feasible
from the model building. The computed structure of the complex
with 4 (Fig. 2B) shows that it appears possible to insert a phenyl
group between Lys514 and Ile545 with potential benefits of cat-
benefits of the change to the smaller propionic acid side chain are a
predicted 100-fold enhancement in aqueous solubility in going
from 6 to 7 and lowering of the computed log Po/w for 6 of 4.7 to
3.4 for 7.24 Mono substitution in the meta position was again found
to lead to inactivity for the methyl and chloro analogs 9 and 12.
The one case with an ortho substituent, 10, showed roughly a
two-fold gain in activity over 4. However, the methyl-scan repre-
sented by 8–10 revealed that para-substitution of the phenyl ring
ion–p interactions with Lys514 and hydrophobic contacts with
Ile545, Val561, and Ala640. Importantly, this is achieved while
retaining the position of the tricyclic core and the hydrogen bonds
with the hinge region. The present Letter summarizes initial exper-
imental results that have been obtained to test this prediction.
Successful introduction of a phenyl group would open up explora-
tion of a wide range of substituted analogs and other aryl
substituents.
may be the most fruitful with the IC50 for 8 (26
three-fold improvement over the unsubstituted 4. Additional gain
was found for the para-chloro analog 11 at 18 M, and finally the
para-hydroxy analog 13 yielded an additional three-fold boost to
M. Further examination of analogs with ortho and/or para sub-
lM) showing a
l
6
l
stituents is indicated.
In summary, as predicted by the computational modeling, aryl
extension at the 2-position on the tricyclic core of theinopyrimid-
inone 3 was shown to be possible. It was also found that the ani-
sole-based western substituent could be replaced by the smaller
propionic acid alternative with no loss of activity and with con-
comitant expected enhancement of pharmacologically relevant
properties. The most potent analog reported herein, the hydroxy-
To pursue this notion, syntheses of compounds 4–13 with a phe-
nyl group at the 2-position on the core of 3 were performed as sum-
marized in Scheme 1. On the western end, 4–6 and 13 retain the
substituted anisole ring as previously reported, for example, in
2.19 However, 7–12 incorporate a smaller alternative, propionic
acid, that was found to not diminish activity (vide infra). Prepara-
tion of 5–12 commenced with ketal 14 and the syntheses of 4 and
13 began with commercially available cyclohexanones 18a and
18i. Grignard reaction of ketal 14 with phenyl magnesium halides
15a–h produced hydroxy ketals 16a–h. Simultaneous removal of
the protecting group and dehydration with TFA provided 17a–h,
which after hydrogenation and Gewald reaction with 2-cyanoacet-
amide yielded 2-aminothiophenes 19a–i. p-Choranil oxidation of
19a–i delivered the benzothiophenes 20a–i. Condensation of 20a–
c and 20i with aldehyde 24 (see Scheme 1) resulted in esters 21a–
c and 21i, which were hydrolyzed to give 4–6, and 13. Condensation
of 20d–h with aldehyde 25 afforded 23d–h via intermediate forma-
tion of 22d–h. Hydrolysis of esters 23d–h produced 7–12. The struc-
tures of 4–13 were validated through NMR and high-resolution
mass spectrometry,23 and their purities were typically >95% by
HPLC. The ALPHAScreen assay was performed using purified FGFR1
kinase domain and a biotinylated peptide substrate, as described
previously.19 The assay results are summarized in Table 1.
phenyl analog 13, yields inhibitory activity of 6
kinase. Though the activity is similar to that of 2 (1.9
l
M for FGFR1
M), the
l
present results provide a platform for further elaboration of
analogs to probe beyond the ATP binding site in search of enhanced
interactions and selectivity. Such studies are underway with
continued emphasis on the synergies between computational
modeling, synthesis, and crystallography.
Acknowledgments
Gratitude is expressed to the National Institutes of Health
(GM032136, AR051448, AR051886, P50-AR054086) and to Yale
University (YSM0061AM) for support.
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Table 1
Inhibitory activities of FGFR1 kinase by 4–13
R4
O
O
R =
or
R3
OH
O
CO2H
HN
OMe
R2
a
b
R1
S
R
N
a
Compd
R
R1
R2
R3
R4
IC50
(
lM)
4
5
6
7
8
a
a
a
b
b
b
b
b
b
a
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
Me
H
H
Cl
H
OH
H
H
OMe
OMe
H
H
H
H
H
75
na
28
21
26
na
43
18
na
6
OMe
OMe
OMe
H
Me
H
H
Cl
H
9
10
11
12
13
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a
na indicates not active in the assay.

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2231
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acetic acid (6) 1H NMR (500 MHz, DMSO-d6) d 12.94 (s, 1H), 8.50 (d, J = 8.3, 1H),
8.42 (d, J = 1.4 Hz, 1H), 7.90–7.82 (m, 3H), 7.03 (d, J = 9.3 Hz, 1H), 6.92 (d,
J = 2.2 Hz, 2H), 6.53 (t, J = 2.2 Hz, 1H), 4.81 (s, 2H), 3.92 (s, 3H), 3.84 (s, 6H); 13
C
NMR (126 MHz, DMSO-d6) d 169.8, 168.3, 166.9, 160.9, 158.5, 154.4, 150.4,
148.6, 141.7, 137.8, 135.5, 133.0, 124.8, 124.1, 123.8, 121.4, 120.9, 115.1, 112.5,
111.3, 104.9, 99.7, 64.7, 55.7, 55.3; HRMS (ESI-TOF) calcd for C27H23N2O7S
[M+H]+: 519.1226, found: 519.1221. 3-(7-(3,5-dimethoxyphenyl)-4-oxo-3,4-
dihydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)propanoic acid (7) 1H NMR
(500 MHz, DMSO-d6) d 12.82 (s, 1H), 12.29 (s, 1H), 8.46 (d, J = 8.3 Hz, 1H),
8.40 (d, J = 1.4 Hz, 1H), 7.85 (dd, J = 1.7, 8.4 Hz, 1H), 6.91 (d, J = 2.2 Hz, 2H), 6.52
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(t, J = 2.2 Hz, 1H), 3.83 (s, 6H), 2.96 (t, J = 6.9 Hz, 2H), 2.78 (t, J = 7.0 Hz, 2H); 13
C
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23. Spectral data for representative compounds: 2-(4-(7-(3,5-dimethoxyphenyl)-4-
oxo-3,4-dihydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)-2-methoxyphenoxy)
NMR (126 MHz, DMSO-d6) d 173.4, 166.6, 160.9, 159.7, 158.0, 141.7, 137.7,
135.2, 132.9, 124.8, 123.7, 120.9, 115.3, 104.9, 99.7, 55.3, 29.8, 28.9; HRMS
(ESI-TOF) calcd for C21H19N2O5S [M+H]+: 411.1015, found: 411.1014.
24. Results from QikProp v. 3.0, Schrödinger Inc., New York, NY, 2006.