Thienopyrimidine ureas as novel and potent multitargeted receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1021/jm050458h

A series of novel thienopyrimidine-based receptor tyrosine kinase inhibitors has been discovered. Investigation of structure-activity relationships at the 5- and 6-positions of the thienopyrimidine nucleus led to a series of N,N′-diaryl ureas that potentl

Full text of DOI:10.1021/jm050458h

6066
J. Med. Chem. 2005, 48, 6066-6083
Thienopyrimidine Ureas as Novel and Potent Multitargeted Receptor Tyrosine
Kinase Inhibitors
Yujia Dai,*,† Yan Guo,† Robin R. Frey,† Zhiqin Ji,† Michael L. Curtin,† Asma A. Ahmed,‡ Daniel H. Albert,†
Lee Arnold,‡ Shannon S. Arries,† Teresa Barlozzari,‡ Joy L. Bauch,† Jennifer J. Bouska,† Peter F. Bousquet,‡
George A. Cunha,‡ Keith B. Glaser,† Jun Guo,† Junling Li,† Patrick A. Marcotte,† Kennan C. Marsh,†
Maria D. Moskey,‡ Lori J. Pease,† Kent D. Stewart,† Vincent S. Stoll,† Paul Tapang,† Neil Wishart,‡
Steven K. Davidsen,† and Michael R. Michaelides†
Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park,
Illinois 60064-6100, and Abbott Bioresearch Center, 100 Research Drive, Worcester, Massachusetts 01605-5314
Received May 13, 2005
A series of novel thienopyrimidine-based receptor tyrosine kinase inhibitors has been discovered.
Investigation of structure-activity relationships at the 5- and 6-positions of the thienopyri-
midine nucleus led to a series of N,N′-diaryl ureas that potently inhibit all of the vascular
endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine
kinases. A kinase insert domain-containing receptor (KDR) homology model suggests that these
compounds bind to the “inactive conformation” of the enzyme with the urea portion extending
into the back hydrophobic pocket adjacent to the adenosine 5′-triphosphate (ATP) binding site.
A number of compounds have been identified as displaying excellent in vivo potency. In
particular, compounds 28 and 76 possess favorable pharmacokinetic (PK) profiles and
demonstrate potent antitumor efficacy against the HT1080 human fibrosarcoma xenograft
tumor growth model (tumor growth inhibition (TGI) ) 75% at 25 mg/kg‚day, per os (po)).
Introduction
and survival and ultimately leads to new vessel forma-
tion and stabilization. Similar to KDR, Tie2 is also an
endothelium-specific receptor tyrosine kinase and pro-
motes tumor angiogenesis through interaction with
angiopoietin ligands.8 Platelet-derived growth factor
receptor (PDGFR) tyrosine kinases are another struc-
turally related subfamily of RTKs, consisting of PDG-
FRâ, cKit, CSF1R (colony-stimulating factor 1 receptor),
and FLT3. PDGFR kinases are believed to not only
indirectly promote tumor angiogenesis but also contrib-
ute directly to tumor growth.9-13 Due to the vital role
of RTK signaling in tumor progression, inhibition of
RTK signaling pathways has emerged as one of the most
compelling targets for therapeutic intervention in can-
cer. Enormous effort has been allocated toward this area
in the past two decades.14-18 In particular, development
of antiangiogenesis-based agents by the interruption of
KDR signaling has been intensively pursued.19-22 The
recent approval by the Federal Drug Administration
(FDA) of Avastin, a VEGF antibody for treating colorec-
tal cancer, has promoted even greater interest in this
field.
The first generation of therapeutic agents based on
RTK signaling interruption primarily targeted selective
RTK inhibition. However, the focus has shifted in recent
years toward multitargeted inhibitors. While selective
RTK inhibitors should be less likely to affect normal
cells and, thus, should produce fewer side effects, broad-
acting and multitargeted RTK inhibitors may be re-
quired to overcome redundancies in signaling pathways
and, thus, effectively inhibit tumor growth.23,24 This is
evidenced by the significant preclinical antitumor ef-
ficacy of the RTK inhibitor SU11248 (Sutent)25 and the
RAF/RTK inhibitor BAY 43-9006 (Sorafenib).26 Both
Receptor tyrosine kinases (RTKs) are a family of
transmembrane proteins possessing extracellular ligand-
binding domains and intracellular kinase domains.
Mitogenic signaling, mediated by a variety of RTKs,
represents a major type of signal transduction pathway
in the communication among eukaryotic cells and plays
a central role in the regulation of diverse biological
processes.1,2 Upon binding to their specific extracellular
growth factors, receptor tyrosine kinases undergo dimer-
ization and autophosphorylation, initiating a cascade of
downstream signaling events. RTK activity in normal
cells is tightly regulated. Abnormal activation of RTKs
has been linked to the development and progression of
a variety of human cancers.
A principal subfamily of RTKs is vascular endothelial
growth factor receptor (VEGFR) tyrosine kinases which
are specifically expressed in vascular endothelial cells
and include FLT1 (Fms-like tyrosine kinase 1; VEG-
FR1), KDR (kinase insert domain-containing receptor
tyrosine kinase; VEGFR2), and FLT4 (VEGFR3). Acti-
vation of VEGFR family RTKs and, in particular, of
KDR by vascular endothelial growth factors (VEGFs)
plays a primary role in tumor angiogenesis.3,4 It has
been shown that angiogenesis is a rate-limiting step in
tumor development.5 Without blood supply, a solid
tumor is limited to a maximum size of 1-2 mm.6,7
VEGF-mediated KDR signaling induces a series of
endothelial responses such as proliferation, migration,
* To whom correspondence should be addressed at R47J/AP10,
Cancer Research, 100 Abbott Park Road, Abbott Park, IL 60064. Tel
(847) 937-8977; fax (847) 935-5165; e-mail yujia.dai@abbott.com.
† Abbott Laboratories.
‡ Abbott Bioresearch Center.
10.1021/jm050458h CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/26/2005

Thienopyrimidine Ureas as Multitargeted RTK Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6067
triethylamine and then reacting with the corresponding
amines.
By substituting various p-nitrophenyl ketones for
p-nitropropiophenone (2) in Scheme 1, the alternatively
C6-substituted thienopyrimidine analogues, including
ureas 74, 76, 79-90, and 93, were synthesized in a
similar fashion. The commercially unavailable p-nitro-
phenyl ketones 34a and b, 36a and b, 39, and 41a-d
were prepared as illustrated in Scheme 2.
Aldol condensation of p-nitropropiophenone with 3-
and 4-pyridinecarboxaldehyde yielded unsaturated
ketones 33a and 33b, respectively, which underwent
palladium-catalyzed conjugation reduction29 to give rise
to 34a and 34b, respectively. Ketone 36a was synthe-
sized using a palladium-catalyzed and Zn-Cu couple-
mediated coupling reaction30 between 4-nitrobenzoyl
chloride and iodide 35a. The same method was also used
to prepare ketone 36b from 4-nitrobenzoyl chloride and
iodide 35b. Ketone 36a also served as the intermediate
for preparation of ketone 39, which was synthesized
from 36a via the sequences of the desilylation, mesy-
lation, and reaction with dimethylamine. Ketones 41a-d
were conveniently prepared through the reaction of the
corresponding acid chlorides 40a-d with dimethyl
malonate and subsequent decarboxylation of the inter-
mediate 2-acylmalonates.
Figure 1. Initial KDR screening hit (I) and the design of
thienopyrimidine kinase inhibitors (II).
compounds are multitargeted kinase inhibitors cur-
rently in phase III clinical trials.
To avoid tedious preparation of the required p-
nitrophenyl ketones in some cases, the synthesis out-
lined in Scheme 1 could be started with the correspond-
ing phenyl ketones instead, and the required nitro group
could be introduced later by nitration. This approach is
represented by the synthesis of amide ureas 51-53
shown in Scheme 3. The nitro group in 49a-c was
incorporated through the nitration of 44 and 48a-b
using nitric acid in concentrated sulfuric acid. Interest-
ingly, heating amino nitrile 44 with formamide at 155
°C not only led to the formation of a pyrimidine ring
but also converted the carboxylic acid functionality into
its amide (45).
An alternative method to introduce diversifying sub-
stituents to the 6-position of the thienopyrimidines is
to functionalize the 6-methyl group in 5. This is dem-
onstrated by the synthesis of ureas 59-61 via bromide
55 shown in Scheme 4. Prior to bromination of the
6-methyl group, the amino group in 5 was protected
with a tert-BOC group. Without protecting the amino
group, direct bromination of 5 with 2,2′-azobisisobuty-
ronitrile (AIBN) and N-bromosuccinimide (NBS) in
benzene led to a complex mixture. However, bromina-
tion of 54 often failed to reach completion, and the
separation of 55 from unreacted 54 was difficult. Thus,
the bromination mixture was usually treated directly
with the corresponding amines, and the unreacted 54
was then removed from 56a-c using flash column
chromatography. Removal of the tert-BOC group fol-
lowed by reduction of the nitro group and reaction of
the resulting anilines with m-tolyl isocyanate provided
59-61.
Part of our program toward counterscreening of
compounds prepared as small molecule lymphoid T-cell
protein-tyrosine kinase (LCK) inhibitors led to the
pyrazolopyrimidine urea I, which was found to moder-
ately inhibit KDR (IC50 ) 0.26 µΜ).27 On the basis of
this result, we investigated the replacement of the
pyrazolopyrimidine template with alternate five-mem-
bered heterocycle-fused pyrimidines. In this paper, we
report the synthesis and characterization of a series of
potent VEGFR and PDGFR kinase inhibitors based on
a thienopyrimidine scaffold (II) (Figure 1).28
Chemistry
A general synthesis of the C5- and C6-substituted
aminothienopyrimidines is exemplified by the synthesis
of the 6-methyl-substituted analogues in Scheme 1.
Knoevenagal condensation of ketone 2 with malono-
nitrile gave rise to compound 3, which, through treat-
ment with elemental sulfur in the presence of an organic
base such as diethylamine, was converted to thiophene
analogue 4 (Gewald reaction). Heating of 4 with forma-
mide at 155 °C generated thienopyrimidine 5. Aniline
6, obtained via reduction of 5, served as a key interme-
diate for the preparation of various 6-methyl-substituted
thienopyrimidine target compounds. For instance, cou-
pling aniline 6 with m-toluic acid afforded amide 7,
whereas reaction of 6 with benzenesulfonyl chloride
produced sulfonamide 8. Ureas 9-32 were synthesized
either by directly reacting 6 with commercially available
isocyanates or in a stepwise fashion by first treating 6
with p-nitrophenyl chloroformate in the presence of
As described in Scheme 5, amide 64 (cf. 7) was
prepared from bromide 62, which was synthesized from
p-bromopropiophenone following a sequence similar to
that of the synthesis of 5 shown in Scheme 1. Acid 63
was then smoothly generated by the treatment of 62
with n-butyllithium, followed by the reaction with

6068 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Dai et al.
Scheme 1. Synthesis of 6-Methyl Thienopyrimidine RTK Inhibitorsa
a Conditions: (a) EtMgBr, ZnCl2, Pd(PPh3)4, THF; (b) NCCH2CN, NH4OAc, benzene, reflux; (c) S8, Et2NH, EtOH; (d) HCONH2, 155 °C;
(e) Fe, NH4Cl, EtOH, THF, H2O; (f) m-toluic acid, HOBt, EDC, NMM, DMF, room temperature (rt); (g) PhSO2Cl, pyridine, CH2Cl2, 0 °C
f rt; (h) RNCO, CH2Cl2, THF, 0 °C f rt or (i) p-O2NC6H4OCOCl, Et3N, THF, 0 °C; (ii) RNH2, rt.
Scheme 2. Syntheses of p-Nitrophenyl Ketonesa
a Conditions: (a) NaOH, EtOH, H2O, rt; (b) Bu3SnH, Pd(PPh3)4, THF, rt; (c) Zn-Cu, Pd(PPh3)4, p-NO2C6H4COCl, benzene, DMF; (d)
TBAF, THF; (e) MsCl, Et3N, CH2Cl2; (f) Me2NH, DMF; (g) (i) dimethyl malonate, MgCl2, Et3N, toluene, rt; (ii) DMSO, H2O, reflux.
carbon dioxide. Finally, 1-hydroxybenzotriazole-medi-
ated coupling of 63 with m-toluidine yielded amide
64.
Aniline 6 was also used to prepare N-cyanoguanidine
67 and aminooxazole 69 as shown in Scheme 6. Heating
aniline 6 with diphenyl cyanocarbonimidate in dimeth-
ylformamide (DMF) afforded 66, which was then con-
verted to 67 by reaction with m-toluidine under micro-
wave conditions. Synthesis of 69 was achieved via the
sequential treatment of 6 with 1,1-thiocarbonyldiimi-
dazole, 2-aminophenol, and 1-[3-(dimethylamino)pro-
pyl]-3-ethylcarbodiimide hydrochloride (EDC). The syn-
thesis of compound 72, a urea N-methylation derivative
of 11, is also described in Scheme 6. N-Methylaniline

Thienopyrimidine Ureas as Multitargeted RTK Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6069
Scheme 3. Synthesis of Thienopyrimidine Amide Ureas 51-53a
a Conditions: (a) NCCH2CN, NH4OAc, benzene, reflux; (b) S8, Et2NH, EtOH, rt; (c) HCONH2, 155 °C; (d) HNR1R2, HOBT, EDC, NMM,
DMF; (e) concentrated HNO3, concentrated H2SO4, 0 °C f rt; (f) Fe, NH4Cl, EtOH, THF, H2O, 80 °C; (g) m-tolyl isocyanate, DMF, 0 °C
f rt.
Scheme 4. Synthesis of C6-Substituted Thienopyrimidinesa
a Conditions: (a) NaH, DMF, (BOC)2O, 0 °C; (b) AIBN, NBS, benzene, 80 °C; (c) amine, DMF, rt, overnight; (d) TFA, CH2Cl2, rt; (e) Fe,
NH4Cl, EtOH, THF, H2O; (f) m-tolyl isocyanate, DMF, rt.
71 was obtained from an N-formylation and reduction
(1.0 mM, a physiologically relevant concentration31).
Gratifyingly, thienopyrimidene-based urea 9 displayed
potent inhibition of KDR (IC50 ) 36 nM, see Table 1),
representing a 9-fold boost in activity relative to that
of the initial pyrazolopyrimidine hit (I). A further 6-fold
boost was obtained upon introduction of an m-methyl
group (11, IC50 ) 6 nM). The urea moiety turned out to
be optimal for KDR inhibition; all attempts to replace
the urea link in this series were detrimental to the KDR
sequence.
Results and Discussion
Our initial studies of structure-activity relationships
(SARs) were focused on optimizing activity against
KDR, as this kinase plays the primary role in tumor
angiogenesis. Activity was measured in the presence of
a high concentration of adenosine 5′-triphosphate (ATP)

6070 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Scheme 5. Synthesis of Amide 64a
Dai et al.
in its interaction with the hinge region of KDR. Hydro-
gen bonds are formed between (1) the exocyclic amino
group of 11 and the backbone carbonyl of Glu 917 and
(2) the proximal ring nitrogen and the backbone N-H
of Cys 919. Our earlier modeling exercise of an isoin-
dolinone urea series33 suggested that the urea unit of
those KDR inhibitors accesses the back hydrophobic
pocket adjacent to the ATP binding site that is unavail-
able when KDR is in an “active” kinase conformation.
This also appears to be true for this series of compounds.
The urea unit of 11 is nicely bound to the region with
the carbonyl oxygen within H-bonding distance of the
backbone N-H of Asp 1046, while both urea N-H
groups of 11 project toward the side chain carboxylate
of Glu 885, with the external N-H forming a more
optimal H-bond interaction. This binding motif has also
been reported by others as a general recognition mode
for urea moieties.28b,34 The importance of the interaction
between the urea unit and the hydrophobic region is
clearly reflected by the dramatic increase of the KDR
inhibitory activity of 11 compared to that of 6. This is
also consistent with the deterioration of KDR activity
when the urea link was replaced (Table 1). The sugges-
tion that the external N-H of the urea has a better
hydrogen bond interaction with the protein (the car-
boxylate side chain of Glu 885) is supported by the KDR
potency of compound 72 versus 73. While the methy-
lation of the internal urea nitrogen (72) resulted in a
9-fold decrease in activity when compared to that of 11,
a 310-fold loss was observed for 73, in which the
terminal urea nitrogen was methylated.
a Conditions: (a) n-BuLi (2.5 equiv), THF, -78 °C; then CO2,
-78 °C f rt; (b) m-toluidine, HOBt, EDC, NMM, DMF, rt.
inhibitory activity. For instance, significant reduction
in KDR activity was observed for sulfonamide 8, thio-
urea 65, cyanoguanidine 67, 2-indole amide 68, amide
7, and its close analogue 64. A less dramatic deteriora-
tion of KDR activity was seen with 69, a cyclized version
of urea. The much weaker activity shown by 70 and 73
in comparison to that of 11 shows the importance of the
external urea NH toward KDR inhibition in this series.
In contrast to the manipulation of the external urea
NH unit (70 and 73), introduction of a methyl group on
the internal urea nitrogen is fairly well tolerated, as
only a slight drop in activity was measured for 72. The
necessity of the terminal aryl group in retaining high
potency is evidenced by the fact that substituting an
aliphatic residue, including benzyl (31), cyclohexanyl-
methyl (32), cyclohexanyl (75), n-butyl (77), and isobutyl
(78), led to a significant decrease of KDR activity.
To help understand the SARs observed at this point
in the project and guide further SAR studies, a homology
model of inhibitor 11 bound to the ATP binding site of
KDR kinase was created using a recently published
crystal structure of homologue Kit kinase (51% identity
to KDR kinase within the catalytic domain) in the
inactive conformation (Figure 2).32
The modeling also suggested that the terminal m-tolyl
group in 11 projects into a hydrophobic region, which
is comprised of the side chains of Ile 892, Ile 888, Leu
889, Val 898, and Leu 1019. This is consistent with the
observed preference for an aromatic residue over an
alkyl group in the urea terminus. The decrease of
activity caused by the replacement of the terminal aryl
group with alkyl residues is probably due to the
combined effect of an inferior interaction of the alkyl
groups with the hydrophobic region and the increased
entropy of the flexible alkyl groups.
In the modeling process, it was assumed that the
amino thienopyrimidine core mimics the adenine of ATP
Concluding that an N,N′-diaryl urea moiety is optimal
for achieving potent KDR activity, we examined the
Scheme 6. Synthesis of Compounds 67, 69, and 72a
a Conditions: (a) (PhO)2CNCN, DMF, 90 °C; (b) m-toluidine, DMF, microwave, 180 °C; (c) (i) 1,1-thiocarbonyldiimidazole, pyridine, 0
°C; (ii) 2-aminophenol, rt; (iii) EDC, 50 f 60 °C; (d) (i) CH3CO2CHO, THF, -15 °C; (ii) Red-Al, benzene, rt f reflux; (e) m-tolyl isocyanate,
CH2Cl2, 0 °C f rt.

Thienopyrimidine Ureas as Multitargeted RTK Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6071
Table 1. KDR Inhibitory Activity of C5- and C6-Substituted Thienopyrimidines
a Each IC50 determination was performed with seven concentrations, and each assay point was determined in duplicate.
effect of substitution on the terminal phenyl (ring B;
rings of the diaryl urea are labeled for clarity in the
figure of Table 2) of the urea. The results are sum-
marized in Table 2. To illustrate the inhibitory activity
of this series of compounds against other subfamilies
of RTKs, Table 2 also includes the potency of these
compounds against cKit (PDGFR-family) and Tie2 (Tie-
family). Substituting a small lipophilic group into the
meta position of ring B is generally well tolerated. In
fact, m-methyl substitution delivered the best KDR
enzymatic potency. In contrast, substitution at the ortho
position is unfavorable and resulted in diminished
potency. Even compound 16, which bears an o-fluoro
group, is approximately 3-fold less potent than the
parent compound 9. Substitution at the para position
is also detrimental to the activity in most cases, but the
drop in activity is generally less striking than that with
ortho substitution. Disubstitution of ring B with small
lipophilic groups also appears to be tolerated. In addi-
tion to their potent activity against KDR, the compounds
in Table 2 also displayed comparable potency against
cKit with similar SAR trends but were generally less
potent against Tie2 with the exception of inhibitor 28,
which exhibited an IC50 value of 17 nM against Tie2. A
similar enhancement of Tie2 potency by the combination
of a 2-F and a 5-CF3 on the urea terminal phenyl was
also reported recently for furanopyrimidine urea Tie2/
VEGFR2 inhibitors.28b
Investigation of C6-substitution of the thienopyrim-
idines was also conducted (Table 3). The results are

6072 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Dai et al.
Table 3. Substitutions at the 6-Position of the
Thienopyrimidines
IC50a (nM)
compd
X
KDR
cKit
Tie2
11
51
52
53
59
Me
6
17
330
210
210
9
160
400
930
580
110
120
470
140
430
(CH2)2CONH2
(CH2)2CONHMe
(CH2)2CONMe2
Figure 2. Model of 11 bound to KDR kinase. Hydrogen bonds
in black are shown between the urea NHs and Glu 885
carboxylate, between the exocyclic amine and Glu 917 back-
bone carbonyl, and between the ring nitrogen and Cys 919
N-H. Also, in thick bonds are residues Asp 1046 and Phe 1047
of the DFG motif in the “inactive” conformation (“DFG-out”).
60
590
800
630
61
74
76
79
80
81
82
CH2NMe2
Et
180
26
3
26
130
280
34
28
6
82
340
720
290
580
880
250
H
n-Pr
i-Pr
Bn
Table 2. Substitutions at the Terminal Benzene Ring (Ring B)
of N,N′-Diaryl Ureas
790
960
83
53
270
84
85
86
87
CH2CH2OH
(CH2)2OMe
(CH2)3OH
19
64
37
360
26
260
220
110
190
15
IC50a (nM)
(CH2)2NMe2
230
810
compd
R
KDR
cKit
Tie2
a Each IC50 determination was performed with seven concentra-
tions, and each assay point was determined in duplicate.
9
H
36
260
6
53
110
34
69
110
54
52
340
35
96
46
120
52
290
12
32
68
9
83
50
19
15
1200
1300
110
890
920
360
1600
1000
400
850
900
210
1200
130
1100
100
510
170
360
17
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
o-CH3
m-CH3
p-CH3
o-CH3O
m-CH3O
p-CH3O
o-F
methyl (61), and dimethylaminoethyl (87) groups. A
large decrease in activity (about 70-fold compared to
that of 76) was also seen for benzyl groups (81);
however, significantly better activity was measured for
compounds with a 3- or 4-pyridylmethyl group (82, IC50
) 34 nm; 83, IC50 ) 53 nM). In contrast to a bulky
residue, groups attached to the 6-position through a two-
or three-carbon link appear to be well tolerated. Both
compounds 84 and 86, as well as the methyl ether
analogue 85, exhibited low double-digit nanomolar KDR
inhibitory activity. The flexibility of the tethered groups
in these molecules may help them avoid an unfavorable
interaction with Phe 1047. The tolerance of some C6-
substitutions may provide potential opportunities for
tuning the physical properties and improving the phar-
macokinetic profiles of this series of inhibitors. The large
difference in potency between primary amide 51 and
N-methyl analogue 52 or N-dimethyl amide 53 is also
noteworthy. As for activity against cKit, substitutions
at the 6-position of the thienopyrimidines result in
effects very similar to those for KDR inhibitory activity.
No significant selectivity for cKit versus KDR was
observed for most compounds except 82 and 83, which
are 23- and 18-fold less potent against cKit than against
KDR, respectively. In contrast to the inhibitory activity
against KDR and cKit, the inhibitory activity against
Tie2 is generally less susceptible to changes in C6-
substitution, as all the compounds in Table 3 exhibit
submicromolar potency against Tie2.
m-F
27
33
p-F
o-Cl
110
22
m-Cl
p-Cl
29
m-Br
50
p-Br
48
m-CF3
m-CO2Et
3,5-di-CH3
3,5-di-Cl
2-F-5-CF3
140
43
19
175
182
160
45
a Each IC50 determination was performed with seven concentra-
tions, and each assay point was determined in duplicate.
consistent with the modeling, which suggested that the
methyl group at the 6-position of the thienopyrimidine
nucleus of KDR-bound 11 lies near the hydrophobic Phe
1047 of the “DFG (Asp-Phe-Gly)” motif. Consequently,
a sterically demanding group was predicted to be less
favorable. Although the 6-methyl group in 11 has little
impact on the KDR potency (11 versus 76 in Table 3),
introduction of larger groups at the 6-position of 76 led
to deterioration of the potency, with the degree of loss
correlating with the steric size of the introduced groups.
For example, an 8-fold drop in potency was observed
for both ethyl (74) and n-propyl (79) groups and a more
than 40-fold drop for isopropyl (80). An even more severe
drop in potency was recorded for 4-morpholinomethyl
(59), 4-methylpiperazinomethyl (60), dimethylamino-
A brief SAR investigation on the substitution at the
linking phenyl (ring A) was also conducted (Table 4).
The modeling studies suggested that there is only
limited space available for substitutions on ring A in

Thienopyrimidine Ureas as Multitargeted RTK Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6073
Table 4. Substitutions at the Linking Phenyl (Ring A) of
Thienopyrimidine Ureas
Table 5. KDR Enzymatic and Cellular Inhibitory Activity and
in Vivo Oral Uterine Edema Inhibitory Activity of Selected
Thienopyrimidine Ureas
IC50a (nM)
KDR KDR (cell)
compd
Y
KDR
cKit
FLT3
(IC50
,
(IC50
,
UE
76
88
89
90
91
92
93
94
H
3
11
6
27
200
180
38
54
28
3
10
59
140
67
700
130
36
compd
X
Y
R
nM)a
nM)b
(ED50, mg/kg)
1′-F
9
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
36
6
35
96
52
10
<10
28
1% @ 50c,d
1′-Cl
1′-MeO
2′-F
2′-Cl
2′-MeO
100
100
19
11
H
H
H
H
H
H
H
H
m-Me
m-Cl
p-Cl
43% @ 50c,d
20
19% @ 60c
21
15% @ 50c
83
24
m-CF3
10e
66
19
5
13
28
2-F-5-CF3 45
150
29
p-CF3
2-F-5-Me
m-Me
61
34
3
590
<10e
1
12% @ 100c
30
22
5
76
91
H
2′-F m-Me
H
H
19
50
3
2
44% @ 3c
95
Et
m-CF3
m-Cl
23
19
19
9
96
H
9
SU11248
18
22
a Each IC50 determination was performed with seven concentra-
tions, and each assay point was determined in duplicate. b Each
cellular IC50 determination was performed with five concentra-
tions, and each assay point was determined in duplicate. c Percent
inhibition @ mg/kg. d Intraperitoneal dosing. e Determined by
Western blot analysis.
a Each IC50 determination was performed with seven concentra-
tions, and each assay point was determined in duplicate.
part due to the presence of the “gatekeeper” residue in
its proximity (Val in both cKit and KDR, and Phe in
FLT3). Given that the gatekeeper residue in FLT3 is a
sterically more demanding group, it was predicted that
the FLT3 inhibitory activity would be even more sus-
ceptible to substitution of this ring, especially at the 2′-
position (see the numbering on the structure of Table
4). The results of SAR studies were consistent with
these predictions (Table 4). The introduction of a meth-
oxy or chloro group at either of the positions of ring A
in 76 resulted in deterioration of inhibitory activity
against KDR, cKit, and FLT3. However, the most
dramatic drop in potency (over 230-fold) was seen for
the FLT3 enzyme in the case of 92 (IC50 ) 700 nM)
bearing a 2′-chloro substituent. Compound 94, an an-
nulation analogue formed by connecting the 6-position
of the thienopyrimidine nucleus of 76 and the 1′-position
of ring A with an ethylene linker, displayed a much
weaker activity (50-fold) against KDR than 76. This may
be due to sterics akin to the substitution at the 1′-
position or due to significant reduction of the torsion
angle between the thienopyrimidine ring system and
ring A of the diaryl urea unit; this angle reduction could
result in severe warping of the thienopyrimidine nucleus
to accommodate the NH2 group and ring A interaction
and consequently lead to less optimized interactions of
the aminopyrimidine moiety with the hinge region and
of the urea unit with the back hydrophobic pocket. A
torsion angle of approximately 68° was modeled for
compound 11 bound to KDR; for compound 94, however,
the angle was calculated at only about 21°.
edema model (UE) of VEGF activity. Increased vascular
permeability is a hallmark of VEGF-induced responses.
It has been shown that VEGF mRNA and protein are
up-regulated in a temporal fashion following a bolus
injection of estradiol in rat uterus, resulting in increased
vascular permeability and edema.35 The protocol was
modified for use in mice. With tested compounds
administered orally, the mouse uterine edema enabled
rapid screening of KDR inhibitors’ in vivo efficacy and
systemic oral exposure. The cellular KDR potency and
in vivo uterine edema activity of selected compounds
are shown in Table 5. In general, compounds that
possess a small lipophilic meta substituent such as
m-Me, m-CF3, and m-Cl on the urea terminal phenyl
(ring B) and no substituent or a small alkyl group at
the 6-position of the theinopyrimidine core delivered
both potent KDR cellular activity and outstanding oral
activity of uterine edema inhibition. For instance,
outstanding oral activity was observed for compounds
24, 28, 30, 76, 95, and 96. In particular, an ED50 value
of 5 mg/kg was recorded for both 28 and 76, which are
more potent than SU11248 (ED50 ) 11 mg/kg) in the
same assay.
On the basis of their outstanding uterine edema
activity, the pharmacokinetic profiles and the antitumor
activity of compounds 28 and 76 were evaluated. As
shown in Table 6, both 28 and 76 possess a reasonable
mouse PK profile. A moderate volume of distribution
(Vd) value was observed in CD-1 mouse for both com-
pounds after intravenous dosing. With lower plasma
clearance, 28 displayed a longer plasma elimination
half-life than 76 while compound 76 had a much higher
oral bioavailability (65%) than 28 (33%), presumably
due to better absorption. Compound 76 was further
studied in beagle dog and cynomolgus monkey for its
pharmacokinetics. A similar profile was observed for 76
in both species, characterized by low plasma clearance
Compounds with significant KDR inhibitory activity
(<100 nM) were further evaluated utilizing an enzyme-
linked immunosorbent assay (ELISA) based screen for
their inhibition of cellular KDR phosphorylation, which
was induced by VEGF in 3T3 murine fibroblasts engi-
neered to express human KDR (Table 5). Compounds
with significant activity in the KDR cellular assay were
then tested in an estradiol-induced mouse uterine

6074 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Dai et al.
Table 6. Physical Properties and Pharmacokinetic Profiles of Thienopyrimidine Ureas 28 and 76
compd
MW
c log P
PSA (Å2)
species
T1/2 (h)
Vd (L/kg)
Cl (mL/min‚kg)
F (%)
AUC (po, µg‚h/mL)
28
76
461
375
5.1
4.2
93
93
mousea
mousea
dogb
2.1
0.7
3.2
1.9
1.55
1.30
0.5
8.7
20
1.8
1.7
33
65
40
42
4.8
4.9
11.7
15.9
monkeyb
0.3
a Dosed intravenously at 3 mg/kg and orally at 10 mg/kg. b Dosed both intravenously and orally at 5 mg/kg.
Table 7. Kinase Inhibition Profiles of Thienopyrimidine Ureas 28 and 76
IC50 (nM)
compd
KDR
FLT1
FLT4
FLT3
cKit
CSF1R
PDGFRa
Tie2
FGFR
EGFR
LCK
cMET
28
76
45
3
170
2
87
13
27
2
180
6
57
3
60
48
17
730
1500
4200
>50 000
>50 000
4 500
20 000
>50 000
>50 000
a IC50 values of cellular phosphorylation by ELISA assay.
(Cl e 1.8 mL/min‚kg) and low volumes of distribution
(Vd e 0.5 L/kg). Consequently, compound 76 displayed
a longer half-life in both dog and monkey than in mouse.
The oral bioavailability in both species averaged at
about 40%. While both 28 and 76 possess favorable
animal PK profiles, poor aqueous solubility was mea-
sured for both compounds (0.02 µg/mL for 28 and 3.1
µg/mL for 76 in phosphate buffered saline (PBS)).
The antitumor activity of 28 and 76 was evaluated
in an HT1080 fibrosarcoma mouse flank xenograft
model. As shown in Figure 3, both 28 and 76 demon-
strated excellent antitumor efficacy in a dose-dependent
fashion. When orally dosed twice a day with a total daily
dose of 50 mg/kg, both compounds inhibited tumor
growth by at least 80% compared to the control, in which
only the vehicle was administered. Approximately 75%
inhibition was observed for both compounds at the 25
mg/kg daily dose. Both compounds compare favorably
to SU11248 in the model, as a daily dose of 65 mg/kg
was needed for SU11248 to achieve 75% inhibition in
the same model.
The significant antitumor efficacy of 28 and 76 may
be ascribed to their multitargeted kinase inhibiton.
Table 7 shows the inhibitory potency of 28 and 76
against a panel of kinases. These data clearly demon-
strate that 28 and 76 are multitargeted receptor ty-
rosine kinase inhibitors with potent activity against all
the kinases of both the VEGFR and PDGFR families.
However, they are much less active against those
kinases structurally less homologous to KDR such as
fibroblast growth factor receptor tyrosine kinase (FGFR),
epidermal growth factor receptor tyrosine kinase (EGFR),
LCK, and cMET.
Figure 3. Effects of inhibitors 28 and 76 on the growth of
HT1080 human xenografts. Dosing (BID) started on day seven.
Tumor volumes are expressed as means ( SEM; n ) 10 per
group. Significant differences (p < 0.05 vs control) in mean
tumor volume were observed for all treatment groups by day
20. Dose (mg/kg‚day), and the percent inhibition of control (2
g) for each dosage, is indicated in the legend.
Conclusion
A series of thienopyrimidine-based receptor tyrosine
kinase inhibitors has been discovered through a rational
design based on an initial KDR screening hit. Extensive
SAR studies led to the conclusion that an N,N′-diaryl
urea moiety at the 5-position of the thienopyrimidine
nucleus is optimal for KDR inhibition. A KDR homology
model suggested that these compounds bind to the ATP
binding site of KDR in an inactive conformation, with
the urea portion interacting with the back hydrophobic
pocket. These compounds are multitargeted receptor
tyrosine kinase inhibitors, displaying potent activity
against other structurally related kinases of the VEGFR
and PDGFR families. Optimization of substituents at
both the urea terminal phenyl ring and the 6-position
of the thienopyrimidine nucleus generated a series of
compounds with excellent enzymatic and cellular activ-
ity. Utilizing a mouse uterine edema model, a number
of compounds have been identified to possess superior
in vivo activity. In particular, compounds 28 and 76
have demonstrated significant oral efficacy in tumor
growth inhibition and displayed favorable pharmaco-
kinetic profiles.
Experimental Section
Chemistry. 1H NMR spectra were recorded on a 300 MHz
spectrometer (Nicolet QE-300 or General Electric GN-300) if

Thienopyrimidine Ureas as Multitargeted RTK Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6075
not otherwise indicated, and chemical shifts are reported in
parts per million (ppm, δ) relative to tetramethylsilane as an
internal standard. Mass spectra were obtained on a Finnigan
MAT SSQ700 instrument. The above spectral data were
obtained through the Department of Structural Chemistry,
Abbott Laboratories. Elemental analysis was performed by
Robertson Microlit Laboratories, Inc., Madison, NJ, and the
results indicated by elemental symbols are within (0.4% of
theoretical values. Silica gel 60 (E. Merck, 230-400 mesh) was
used for preparative column chromatography.
1-(4-Nitrophenyl)propan-1-one (2). A solution of 2 M
ethylmagnesium chloride in THF (15 mL, 30 mmol) was added
to a solution of 0.5 M ZnCl2 in THF (60 mL, 30 mmol) at 0 °C.
After being stirring first at 0 °C for 10 min and then at room
temperature for 20 min, the reaction mixture was cooled to 0
°C and treated sequentially with Pd(PPh3)4 (1.73 g, 1.5 mmol)
and a solution of 4-nitrobenzoyl chloride (6.12 g, 33 mmol) in
THF (20 mL). The mixture was stirred at 0 °C for 40 min,
diluted with water, adjusted to pH 1 with a 2 N aqueous HCl
solution, and extracted with ethyl acetate three times. The
combined extracts were washed sequentially with saturated
aqueous Na2CO3, water, and brine, dried (MgSO4), filtered, and
concentrated. The crude product was purified by flash column
chromatography on silica gel eluting with 6:1 hexanes/ethyl
acetate to provide the title compound (2.17 g, 40%) as a yellow
solid. 1H NMR (DMSO-d6) δ 1.10 (t, J ) 7.12 Hz, 3H), 3.14 (q,
J ) 7.12 Hz, 2H), 8.18 (d, J ) 8.82 Hz, 2H), 8.34 (d, J ) 8.82
Hz, 2H).
2-[1-(4-Nitrophenyl)propylidene]malononitrile (3). A
mixture of 2 (3.40 g, 19 mmol), malononitrile (1.25 g, 19 mmol),
ammonium acetate (1.40 g, 26 mmol), and acetic acid (2 mL)
in benzene (50 mL) was heated to reflux in a round-bottomed
flask fitted with a Dean-Stark trap for 14 h. Additional
ammonium acetate (1.40 g, 26 mmol) and acetic acid (2 mL)
were added. The reaction mixture was heated to reflux for an
additional 4 h, cooled to room temperature, and partitioned
between water and ethyl acetate. The aqueous layer was
extracted with ethyl acetate two times, and the combined
extracts were washed with water and brine, dried (MgSO4),
filtered, and concentrated. The crude product was purified by
flash column chromatography on silica gel eluting with 3:1
hexanes/ethyl acetate to provide the title compound (4.01 g,
93%) as a yellow solid. 1H NMR (DMSO-d6) δ 0.97 (t, J ) 7.63
Hz, 3H), 3.00 (q, J ) 7.57 Hz, 2H), 7.89 (d, J ) 9.15 Hz, 2H),
8.40 (d, J ) 9.15 Hz, 2H).
1H), 7.01 (d, J ) 8.4 Hz, 2H), 6.70 (d, J ) 8.4 Hz, 2H), 5.39 (s,
2H), 2.27 (s, 3H); MS (CI) m/z 257 (M + H)+. Anal. (C13H12N4S‚
0.2EtOAc‚0.2H2O) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-3-methylbenzamide (7). A mixture of 6 (80 mg, 0.31
mmol), m-toluic acid (42 mg, 0.31 mmol), HOBT (46 mg, 0.34
mmol), EDC (66 mg, 0.34 mmol), and 4-methylmorpholine
(NMM) (86 µL, 0.78 mmol) in DMF (3 mL) was stirred at room
temperature for 14 h and then partitioned between water and
ethyl acetate. The aqueous layer was extracted with ethyl
acetate two times, and the combined extracts were washed
with water and brine, dried (MgSO4), filtered, and concen-
trated. The crude product was purified by flash column
chromatography on silica gel eluting with ethyl acetate to
provide the title compound (39 mg, 35%). 1H NMR (DMSO-
d6) δ 10.42 (s, 1H), 8.28 (s, 1H), 7.98 (d, J ) 8.4 Hz, 2H), 7.80-
7.75 (m, 2H), 7.45-7.36 (m, 4H), 2.42 (s, 3H), 2.31 (s, 3H); MS
(CI) m/z 375 (M + H)+. Anal. (C12H18N4OS‚0.2EtOAc) C, H,
N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]benzenesulfonamide (8). A solution of 6 (100 mg,
0.39 mmol) in dichloromethane (4 mL) was treated at 0 °C
with pyridine (38 µL, 0.47 mmol) and benzenesulfonyl chloride
(50 µL, 0.40 mmol) and stirred at 0 °C for 1 h and then at
room temperature overnight. The reaction mixture was diluted
with water and extracted with ethyl acetate three times. The
combined extracts were washed with brine, dried (MgSO4),
filtered, and concentrated. The concentrate was triturated from
ethyl acetate/hexanes to provide the title compound (91 mg,
1
59%). H NMR (DMSO-d6) δ 10.49 (s, 1H), 8.25 (s, 1H), 7.77
(m, 2H), 7.65-7.55 (m, 3H), 7.24 (m, 4H), 1.99 (s, 3H); MS
(ESI) m/z 397 (M + H)+. Anal. (C19H16N4O2S2‚0.3EtOAc) C,
H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-phenylurea (9). A solution of 6 (80 mg, 0.3 mmol)
in dichloromethane (4 mL) at 0 °C was treated with phenyl
isocyanate (36 µL, 0.33 mmol) and stirred overnight as the
temperature slowly rose to room temperature. To the resulting
suspension, hexane was added to yield more precipitate. The
solid material was collected by filtration to provide the title
compound (103 mg, 87%). 1H NMR (DMSO-d6) δ 8.89 (s, 1H),
8.75 (s, 1H), 8.26 (s, 1H), 7.63 (d, J ) 8.7 Hz, 2H), 7.47 (d, J
) 8.7 Hz, 2H), 7.33-7.26 (m, 4H), 6.99 (t, J ) 7.5 Hz, 1H),
2.30 (s, 3H); MS (CI) m/z 376 (M + H)+. Anal. (C20H17N5OS‚
0.1CH2Cl2) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(2-methylphenyl)urea (10). Compound 10 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 2-tolyl isocyanate for phenyl
2-Amino-5-methyl-4-(4-nitrophenyl)thiophene-3-car-
bonitrile (4). Diethylamine (0.6 mL, 5.75 mmol) was added
to a suspension of 3 (1.31 g, 5.75 mmol) and sulfur (0.18 g,
5.75 mmol) in ethanol (20 mL). The mixture was heated at 70
°C for 1.5 h, cooled to room temperature, and concentrated.
The crude product was purified by flash column chromatog-
raphy on silica gel eluting with 3:2 hexanes/ethyl acetate to
provide the title compound (1.27 g, 85%). 1H NMR (DMSO-d6)
δ 2.18 (s, 3 H), 7.63 (d, J ) 8.82 Hz, 2H), 8.30 (d, J ) 8.81 Hz,
2H); MS (CI) m/z 277 (M + NH4)+.
6-Methyl-5-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-
amine (5). A suspension of 4 (4.03 g, 15.5 mmol) in formamide
(60 mL) was stirred at 155 °C for 17 h, cooled to room
temperature, diluted with water, and filtered. The filter cake
was dried to provide the title compound (4.13 g, 93%). 1H NMR
(DMSO-d6) δ 8.36 (d, J ) 9.0 Hz, 2H), 8.30 (s, 1H), 7.68 (d, J
) 9.0 Hz, 2H), 2.32 (s, 3H); MS (CI) m/z 287 (M + H)+.
1
isocyanate. H NMR (DMSO-d6) δ 9.24 (s, 1H), 8.27 (s, 1H),
8.01 (s, 1H), 7.82 (d, J ) 7.5 Hz, 1H), 7.64 (d, J ) 8.1 Hz, 2H),
7.31 (d, J ) 8.1 Hz, 2H), 7.21-7.13 (m, 2H), 6.97 (t, J ) 7.5
Hz, 1H), 2.30 (s, 3H), 2.27 (s, 3H); MS (ESI) m/z 390 (M +
H)+. Anal. (C21H19N5OS‚0.7H2O) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(3-methylphenyl)urea (11). Compound 11 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 3-tolyl isocyanate for phenyl
1
isocyanate. H NMR (DMSO-d6) δ 8.87 (s, 1H), 8.67 (s, 1H),
8.26 (s, 1H), 7.63 (d, J ) 8.1 Hz, 2H), 7.32-7.23 (m, 4H), 7.17
(t, J ) 7.8 Hz, 1H), 6.81 (d, J ) 7.8 Hz, 1H), 2.30 (s, 3H), 2.29
(s, 3H); MS (ESI) m/z 388 (M - H)-. Anal. (C21H19N5OS‚
0.5H2O) C, H, N.
5-(4-Aminophenyl)-6-methylthieno[2,3-d]pyrimidin-4-
amine (6). A suspension of 5 (1.01 g, 3.53 mmol) in ethanol
(60 mL), THF (20 mL), and water (10 mL) was treated with
ammonium chloride (0.19 g, 3.53 mmol) and iron powder (1.18
g, 21.2 mmol). After being stirred at 80 °C for 2 h, the mixture
was diluted with ethanol (40 mL) and filtered through a pad
of diatomaceous earth (Celite) while still hot. The pad was
washed with ethanol, and the filtrate was concentrated. The
concentrate was diluted with water and extracted with ethyl
acetate three times. The combined extracts were washed with
brine, dried (MgSO4), filtered, and concentrated to provide the
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(4-methylphenyl)urea (12). Compound 12 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 4-tolyl isocyanate for phenyl
1
isocyanate. H NMR (DMSO-d6) δ 8.84 (s, 1H), 8.63 (s, 1H),
8.26 (s, 1H), 7.62 (d, J ) 8.4 Hz, 2H), 7.35 (d, J ) 8.4 Hz, 2H),
7.30 (d, J ) 8.4 Hz, 2H), 7.10 (d, J ) 8.4 Hz, 2H), 2.29 (s, 3H),
2.25 (s, 3H); MS (ESI) m/z 390 (M + H)+. Anal. (C21H19N5OS‚
0.5H2O‚0.1C6H12) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(2-methoxyphenyl)urea (13). Compound 13 was
1
title compound (0.89 g, 98%). H NMR (DMSO-d6) δ 8.23 (s,

6076 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Dai et al.
prepared using the same procedure as described for the
synthesis of 9 by substituting 2-methoxyphenyl isocyanate for
phenyl isocyanate. 1H NMR (DMSO-d6) δ 9.55 (s, 1H), 8.31 (s,
1H), 8.27 (s, 1H), 8.15 (dd, J ) 7.8, 2.1 Hz, 1H), 7.63 (d, J )
8.7 Hz, 2H), 7.31 (d, J ) 8.7 Hz, 2H), 7.04 (dd, J ) 8.1, 1.8 Hz,
1H), 6.70-6.87 (m, 2H), 3.90 (s, 3H), 2.30 (s, 3H); MS (ESI)
m/z 406 (M + H)+. Anal. (C21H19N5O2S‚0.25H2O) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(3-methoxyphenyl)urea (14). Compound 14 was
prepared using the same procedure as described for the
synthesis 9 by substituting 3-methoxyphenyl isocyanate for
phenyl isocyanate.1H NMR (DMSO-d6) δ 8.88 (s, 1H), 8.76 (s,
1H), 8.26 (s, 1H), 7.63 (d, J ) 8.7 Hz, 2H), 7.31 (d, J ) 8.7 Hz,
2H), 7.22-7.17 (m, 2H), 6.95 (m, 1H), 6.57 (m, 1H), 3.74 (s,
3H), 2.30 (s, 1H); MS (ESI) m/z 406 (M + H)+. Anal.
(C21H19N5O2S‚0.3H2O) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(4-methoxyphenyl)urea (15). Compound 15 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 4-methoxyphenyl isocyanate for
phenyl isocyanate. 1H NMR (DMSO-d6) δ 8.81 (s, 1H), 8.56 (s,
1H), 8.27 (s, 1H), 7.62 (d, J ) 8.1 Hz, 2H), 7.38 (d, J ) 9.0 Hz,
2H), 7.30 (d, J ) 8.1 Hz, 2H), 6.89 (d, J ) 9.0 Hz, 2H), 3.73 (s,
3H), 2.30 (s, 3H); MS (ESI) m/z 406 (M + H)+. Anal.
(C21H19N5O2S‚0.2C6H14‚0.3H2O) C, H, N.
1H), 8.26 (s, 1H), 7.63 (d, J ) 8.7 Hz, 2H), 7.51 (s, J ) 9.0 Hz,
2H), 3.37-7.29 (m, 4H), 2.29 (s, 3H); MS (ESI) m/z 410 (M +
H)+.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(3-bromophenyl)urea (22). Compound 22 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 3-bromophenyl isocyanate for
phenyl isocyanate. 1H NMR (DMSO-d6) δ 8.97 (s, 1H), 8.96 (s,
1H), 8.27 (s, 1H), 7.88 (t, J ) 1.8 Hz, 1H), 7.64 (d, J ) 9.0 Hz,
2H), 7.36-7.29 (m, 3H), 7.25 (t, J ) 7.8 Hz, 1H), 7.19-7.14
(m, 1H), 2.30 (s, 3H); MS (ESI) m/z 454, 456 (M + H)+. Anal.
(C20H16BrN5OS) C, H, N
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(4-bromophenyl)urea (23). Compound 23 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 4-bromophenyl isocyanate for
phenyl isocyanate. 1H NMR (DMSO-d6) δ 8.93 (s, 1H), 8.90 (s,
1H), 8.26 (s, 1H), 7.63 (d, J ) 8.7 Hz, 2H), 7.46 (s, 4H), 7.32
(d, J ) 8.7 Hz, 2H), 2.29 (s, 3H); MS (ESI) m/z 454, 456 (M +
H)+. Anal. (C20H16BrN5OS‚0.4H2O‚0.2C8H18) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-[3-(trifluoromethyl)phenyl]urea (24). Com-
pound 24 was prepared using the same procedure as described
for the synthesis of 9 by substituting 3-trifluoromethylphenyl
1
isocyanate for phenyl isocyanate. H NMR (DMSO-d6) δ 9.12
(s, 1H), 9.01 (s, 1H), 8.27 (s, 1H), 8.03 (s, 1H), 7.67-7.59 (m,
3H), 7.53 (t, J ) 7.8 Hz, 1H), 7.33 (m, 3H), 2.30 (s, 1H); MS
(ESI) m/z 444 (M + H)+. Anal. (C21H16F3N5OS) C, H, N.
Methyl 3-[({[4-(4-Amino-6-methylthieno[2,3-d]pyrimi-
din-5-yl)phenyl]amino}carbonyl)amino]benzoate (25).
Compound 25 was prepared using the same procedure as
described for the synthesis of 9 by substituting methyl 3-iso-
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(2-fluorophenyl)urea (16). Compound 16 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 2-fluorophenyl isocyanate for
phenyl isocyanate 1H NMR (DMSO-d6) δ 9.30 (s, 1H), 8.63 (d,
J ) 2.4 Hz, 1H), 8.27 (s, 1H), 8.17 (td, J ) 8.1, 1.5 Hz, 1H),
7.64 (d, J ) 8.4 Hz, 2H), 7.33 (d, J ) 8.4 Hz, 2H), 7.26 (ddd,
J ) 12.0, 8.1, 1.2 Hz, 1H), 7.16 (t, J ) 7.8 Hz, 1H), 7.05-6.99
(m, 1H), 2.30 (s, 3H); MS (ESI) m/z 394 (M + H)+. Anal.
(C20H16FN5OS‚0.2C8H18) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(3-fluorophenyl)urea (17). Compound 17 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 3-fluorophenyl isocyanate for
phenyl isocyanate. 1H NMR (DMSO-d6) δ 9.00 (s, 1H), 8.97 (s,
1H), 8.27 (s, 1H), 7.64 (d, J ) 8.4 Hz, 2H), 7.51 (dt, J ) 12.0,
2.1 Hz, 1H), 7.36-7.28 (m, 3H), 7.15 (d, J ) 8.1 Hz, 1H), 6.80
(td, J ) 8.1 Hz, 2.4 Hz), 2.30 (s, 3H); MS (ESI) m/z 394 (M +
H)+. Anal. (C20H16FN5OS‚0.3H2O) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(4-fluorophenyl)urea (18). Compound 18 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 4-fluorophenyl isocyanate for
phenyl isocyanate. 1H NMR (DMSO-d6) δ 8.89 (s, 1H), 8.79 (s,
1H), 8.26 (s, 1H), 7.63 (d, J ) 8.4 Hz, 2H), 7.51-7.46 (m, 2H),
7.31 (d, J ) 8.4 Hz, 2H), 7.14 (t, J ) 9.0 Hz, 2H), 2.29 (s, 3H);
MS (ESI) m/z 394 (M + H)+.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(2-chlorophenyl)urea (19). Compound 19 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 2-chlorophenyl isocyanate for
phenyl isocyanate. 1H NMR (DMSO-d6) δ 9.64 (s, 1H), 8.40 (s,
1H), 8.27 (s, 1H), 8.17 (dd, J ) 8.4, 1.8 Hz, 1H), 7.65 (d, J )
8.4 Hz, 2H), 7.48 (dd, J ) 7.8, 1.8 Hz, 1H), 7.36-7.28 (m, 3H),
7.05 (td, J ) 8.4, 1.8 Hz, 1H), 2.30 (s, 1H); MS (ESI) m/z 410,
412 (M + H)+.
1
cyanatobenzoate for phenyl isocyanate. H NMR (DMSO-d6)
δ 9.04 (s, 1H), 8.94 (s, 1H), 8.27 (s, 1H), 8.22 (t, 1H), 7.65 (d,
3H), 7.59 (dt, 1H), 7.44 (t, 1H), 7.32 (d, 2H), 3.86 (s, 3H), 2.30
(s, 3H); MS (ESI) m/z 434 (M + H)+. Anal. (C22H19N5O3S‚
0.5H2O) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(3,5-dimethylphenyl)urea (26). Compound 26
was prepared using the same procedure as described for the
synthesis of 9 by substituting 3,5-dimethylphenyl isocyanate
for phenyl isocyanate. 1H NMR (DMSO-d6) δ 8.85 (s, 1H), 8.59
(s, 1H), 8.26 (s, 1H), 7.62 (d, J ) 8.4 Hz, 2H), 7.30 (d, J ) 8.4
Hz, 2H), 7.09 (s, 2H), 6.63 (s, 1H), 2.30 (s, 3H), 2.24 (s, 6H);
MS (ESI) m/z 404 (M + H)+. Anal. (C22H21N5OS) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(3,5-dichlorophenyl)urea (27). Compound 27
was prepared using the same procedure as described for the
synthesis of 9 by substituting 3,5-dichlorophenyl isocyanate
for phenyl isocyanate. 1H NMR (DMSO-d6) δ 9.14 (s, 1H), 9.11
(s, 1H), 8.27 (s, 1H), 7.64 (d, J ) 8.4 Hz, 2H), 7.56 (d, J ) 1.8
Hz, 2H), 7.33 (d, J ) 8.4 Hz, 2H), 7.19 (t, J ) 1.8 Hz, 1H),
2.29 (s, 3H); MS (ESI) m/z 444, 446 (M + H)+.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-[2-fluoro-5-(trifluoromethyl)phenyl]urea (28).
Compound 28 was prepared using the same procedure as
described for the synthesis of 9 by substituting 2-fluoro-5-
1
(trifluoromethyl)phenyl isocyanate for phenyl isocyanate. H
NMR (DMSO-d6) δ 9.39 (s, 1H), 8.98 (d, J ) 2.7 Hz, 1H), 8.64
(dd, J ) 7.2, 1.8 Hz, 1H), 8.27 (s, 1H), 7.65 (d, J ) 8.4 Hz,
2H), 7.52 (t, J ) 9.0 Hz, 1H), 7.44-7.37 (m, 1H), 7.34 (d, J )
8.4 Hz, 2H), 2.30 (s, 3H); MS (ESI) m/z 462 (M + H)+. Anal.
(C21H15F4N5OS) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(3-chlorophenyl)urea (20). Compound 20 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 3-chlorophenyl isocyanate for
phenyl isocyanate. 1H NMR (DMSO-d6) δ 8.97 (s, 2H), 8.27 (s,
1H), 7.73 (m, 1H), 7.64 (d, J ) 8.7 Hz, 2H), 7.34-7.29 (m, 4H),
7.06-7.02 (m, 1H), 2.30 (s, 1H); MS (ESI) m/z 410 (M + H)+.
Anal. (C20H16ClN5OS‚0.2H2O) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(4-chlorophenyl)urea (21). Compound 21 was
prepared using the same procedure as described for the
synthesis of 9 by substituting 4-chlorophenyl isocyanate for
phenyl isocyanate. 1H NMR (DMSO-d6) δ 8.92 (s, 1H), 8.89 (s,
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-[4-(trifluoromethyl)phenyl]urea (29). Com-
pound 29 was prepared using the same procedure as described
for the synthesis of 9 by substituting 4-(trifluoromethyl)phenyl
1
isocyanate for phenyl isocyanate. H NMR (DMSO-d6) δ 9.29
(s, 1H), 9.02 (s, 1H), 8.27 (s, 1H), 7.71-7.63 (m, 6H), 7.33 (d,
J ) 8.7 Hz, 2H), 2.30 (s, 3H); MS (ESI) m/z 444 (M + H)+.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(2-fluoro-5-methylphenyl)urea (30). Compound
30 was prepared using the same procedure as described for
the synthesis of 9 by substituting 2-fluoro-5-methylphenyl

Thienopyrimidine Ureas as Multitargeted RTK Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6077
1
isocyanate for phenyl isocyanate. H NMR (DMSO-d6) δ 9.28
mmol) and a solution of 4-nitrobenzoyl chloride (3.4 g, 18.3
mmol) in benzene (36 mL) and stirred at room temperature
for 1 h. The mixture was filtered through diatomaceous earth
(Celite) and partitioned between saturated aqueous am-
monium chloride solution and ethyl acetate. The aqueous
phase was extracted with ethyl acetate two times, and the
combined extracts were washed with water and brine, dried
over Na2SO4, filtered, and concentrated. The crude product was
purified by flash column chromatography on silica gel eluting
with 5% ethyl acetate/hexanes to provide the title compound
(2.81 g, 47%). 1H NMR (DMSO-d6) δ 0.00 (s, 6H), 0.83 (s, 9H),
1.83 (m, 2H), 3.12 (t, J ) 6.95 Hz, 2H), 3.64 (t, J ) 6.27 Hz,
2H), 8.16 (d, J ) 8.82 Hz, 2H), 8.33 (d, J ) 9.16 Hz, 2H); MS
(ESI) m/z 322 (M - H)-.
(s, 1H), 8.56 (d, J ) 2.7 Hz, 1H), 8.27 (s, 1H), 8.00 (dd, J )
8.1, 2.1 Hz, 1H), 7.63 (d, J ) 9.0 Hz, 2H), 7.32 (d, J ) 9.0 Hz,
2H), 7.12 (dd, J ) 11.4, 8.1 Hz, 1H), 6.82 (m, 1H), 2.30 (s, 3H),
2.28 (s, 3H); MS (ESI) m/z 408 (M + H)+.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-benzylurea (31). Compound 31 was prepared
using the same procedure as described for the synthesis of 9
by substituting benzyl isocyanate for phenyl isocyanate. 1H
NMR (DMSO-d6) δ 8.81 (s, 1H), 8.26 (s, 1H), 7.59 (d, J ) 8.7
Hz, 2H), 8.38-7.28 (m, 4H), 7.27-7.22 (m, 3H), 6.71 (t, J )
6.0 Hz, 1H), 4.33 (d, J ) 6.0 Hz, 2H), 2.28 (s, 3H); MS (ESI)
m/z 390 (M + H)+.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-cyclohexylmethylurea (32). To a solution of 6
(159 mg, 0.62 mmol) in THF (9 mL) at 0 °C was added
triethylamine (95 µL, 0.68 mmol) and 4-nitrophenyl chloro-
formate (137 mg, 0.68 mmol). After being stirred at 0 °C for 1
h, cyclohexanylmethylamine (81 µL, 0.62 mmol) and trieth-
ylamine (95 µL, 0.68 mmol) were added. The reaction mixture
was stirred overnight while the temperature slowly rose to
room temperature. The reaction mixture was partitioned
between ethyl acetate and water, extracted with ethyl acetate,
dried over MgSO4, filtered, and concentrated. The crude
product was purified by flash column chromatography on silica
gel eluting with ethyl acetate to provide the title compound
(24 mg, 10%). 1H NMR (500 MHz, DMSO-d6) δ 0.85-0.97 (m,
2H), 1.07-1.27 (m, 3H), 1.36-1.46 (m, 1H), 1.63 (d, J ) 11.29
Hz, 1H), 1.66-1.75 (m, 4H), 2.28 (s, 3H), 2.96 (t, J ) 6.41 Hz,
2H), 6.25 (t, J ) 5.80 Hz, 1H), 7.23 (d, J ) 8.54 Hz, 2H), 7.55
(d, J ) 8.54 Hz, 2H), 8.26 (s, 1H), 8.61 (s, 1H); MS (ESI) m/z
396 (M + H)+. Anal. (C21H25N5OS‚0.2EtOAc) C, H, N.
1-(4-Nitrophenyl)-3-pyridin-3-ylpropenone (33a). A sus-
pension of 3′-nitroacetophenone (5 g, 30.3 mmol) and 4-
pyridinecarboxaldehyde (2.89 mL, 30.3 mmol) in water (45 mL)
at room temperature was treated with 6% NaOH in H2O/
ethanol (2:1, 0.60 mL), stirred overnight, and filtered. The filter
cake was washed with water and a small amount of ethanol
and dried to provide the title compound (7.6 g, 98%). 1H NMR
(DMSO-d6) δ 7.52 (dd, J ) 7.80, 4.75 Hz, 1H), 7.84 (d, J )
15.93 Hz, 1H), 8.11 (d, J ) 15.60 Hz, 1H), 8.36-8.44 (m, 5H),
8.65 (dd, J ) 4.75, 1.36 Hz, 1H), 9.06 (d, J ) 2.03 Hz, 1H); MS
(ESI) m/z 255 (M + H)+.
1-(4-Nitrophenyl)-3-pyridin-3-ylpropan-1-one (34a).
Tributyltin hydride (0.36 mL, 1.34 mmol) was slowly added
using a syringe pump at room temperature to a mixture of
33a (200 mg, 0.78 mmol) and Pd(PPh3)4 (27 mg, 0.023 mmol)
in THF (28 mL). After the addition was complete (1.3 h), the
mixture was stirred at room temperature overnight, then
diluted with water, and extracted with ethyl acetate three
times. The combined extracts were washed with brine, dried
over Na2SO4, filtered, and concentrated. The crude product was
purified by flash column chromatography on silica gel eluting
with 80% ethyl acetate/hexanes to provide the title compound
(113 mg, 50%). 1H NMR (DMSO-d6) δ 2.98 (t, J ) 7.46 Hz,
2H), 3.53 (t, J ) 7.29 Hz, 2H), 7.31 (dd, J ) 7.80, 4.75 Hz,
1H), 7.72 (dt, J ) 7.80, 2.03 Hz, 1 H), 8.22 (d, J ) 9.16 Hz,
2H), 8.34 (d, J ) 8.82 Hz, 2H), 8.40 (dd, J ) 4.75, 1.70 Hz,
1H), 8.53 (d, J ) 2.37 Hz, 1H); MS (ESI) m/z 257 (M + H)+.
5-(tert-Butyldimethylsilanyloxy)-1-(4-nitrophenyl)-pen-
tan-1-one (36b). Compound 36b was prepared using the same
procedure as described for the synthesis of 36a by substituting
1
35b for 35a. H NMR (DMSO-d6) δ 0.00 (s, 6H), 0.83 (s, 9H),
1.44-1.56 (m, 2H), 1.58-1.71 (m, 2H), 3.11 (t, J ) 7.12 Hz,
2H), 3.59 (t, J ) 6.27 Hz, 2H), 8.16 (d, J ) 9.15 Hz, 2H), 8.31
(d, J ) 8.82 Hz, 2H); MS (ESI) m/z 336 (M - H)-.
4-Hydroxy-1-(4-nitrophenyl)butan-1-one (37). To a so-
lution of 36a (1.5 g, 4.60 mmol) in THF (20 mL) at 0 °C was
dropwise added a 1.0 M tetrabutylammonium fluoride solution
in THF (6.9 mL, 6.9 mmol). The reaction mixture was stirred
overnight while the temperature rose slowly to room temper-
ature, diluted with water, extracted with ethyl acetate, washed
with brine, dried over Na2SO4, filtered, and concentrated. The
crude product was purified by flash column chromatography
on silica gel with 80% ethyl acetate/hexanes to provide the title
compound (0.66 g, 74%). 1H NMR (DMSO-d6) δ 1.72-1.85 (m,
2H), 3.13 (t, J ) 7.29 Hz, 2H), 3.46 (m, 2H), 4.52 (t, 1H), 8.18
(d, J ) 8.81 Hz, 2H), 8.34 (d, J ) 9.15 Hz, 2H).
Methanesulfonic Acid 4-(4-Nitrophenyl)-4-oxobutyl
Ester (38). A solution of 37 (5.68 g, 29.4 mmol) in dichloro-
methane (60 mL) at 0 °C was treated with triethylamine (4.9
mL, 35 mmol) and methylsulfonyl chloride (2.7 mL, 35 mmol).
After being stirred at 0 °C for 3 h, the reaction mixture was
poured into cold water and extracted with dichloromethane
three times. The combined extracts were washed with brine,
dried over Na2SO4, filtered, and concentrated. The crude
product was purified by silica gel chromatography eluting with
50% ethyl acetate/hexanes to provide the title compound (6.42
g, 76%). 1H NMR (DMSO-d6) δ 1.95-2.16 (m, 2H), 3.18 (s, 3H),
3.25 (t, J ) 6.95 Hz, 2H), 4.29 (t, J ) 6.44 Hz, 2H), 8.20 (d, J
) 8.81 Hz, 2H), 8.35 (d, J ) 8.81 Hz, 2H).
4-(Dimethylamino)-1-(4-nitrophenyl)-1-butanone (39).
A mixture of 38 (3.0 g, 10.5 mmol), a 2.0 M dimethylamine
solution in THF (21 mL, 42.0 mmol), and triethylamine (2.9
mL, 21.0 mmol) in DMF (25 mL) was heated at 85-90 °C for
1.5 h and then cooled to room temperature, diluted with water,
and extracted with ethyl acetate. The extract was washed with
brine, dried over Na2SO4, filtered, and concentrated. The crude
product was purified by silica gel chromatography eluting with
10% methanol/dichloromethane to provide the title compound
1
(1.27 g, 51%). H NMR (DMSO-d6) δ 1.70-1.85 (m, 2H), 2.08
(s, 6H), 2.26 (t, J ) 6.95 Hz, 2H), 3.08 (t, J ) 6.95 Hz, 2H),
8.16 (d, J ) 8.82 Hz, 2H), 8.33 (d, J ) 8.82 Hz, 2H); MS (ESI)
m/z 237 (M + H)+.
1-(4-Nitrophenyl)-3-pyridin-3-ylpropan-1-one (34b).
Compound 34b was prepared using the same sequence as
described for the synthesis of 34a by substituting 4′-nitro-
acetophenone for 3′-nitroacetophenone. 1H NMR (DMSO-d6)
δ 2.98 (t, J ) 7.29 Hz, 2H), 3.54 (t, J ) 7.46 Hz, 2H), 7.33 (d,
J ) 6.10 Hz, 2H), 8.22 (d, J ) 8.82 Hz, 2H), 8.34 (d, J ) 9.16
Hz, 2H), 8.46 (d, J ) 6.10 Hz, 2H); MS (ESI) m/z 257 (M +
H)+.
4-{[tert-Butyl(dimethyl)silyl]oxy}-1-(4-nitrophenyl)-
butan-1-one (36a). A mixture of Zn-Cu couple (2.68 g, 41.3
mmol) and tert-butyl(3-iodopropoxy)dimethylsilane (35a) (8.26
g, 27.5 mmol) in benzene (55 mL) and DMF (3.6 mL) was
stirred vigorously at room temperature for 1 h and, then,
heated at 60 °C for 4 h. After being cooled to room temperature,
the reaction mixture was treated with (Ph3P)4Pd (0.85 g, 0.73
1-(3-Methoxy-4-nitrophenyl)ethanone (41d). A suspen-
sion of MgCl2 (932 mg, 9.8 mmol) in toluene (13 mL) was
treated with triethylamine (4.7 mL, 33.4 mmol) and dimethyl
malonate (1.9 mL, 16.6 mmol). After being stirred at room
temperature for 1.5 h, the reaction mixture was treated
portionwise over 30 min with 3-methoxy-4-nitrobenzoyl chlo-
ride (40d) (3 g, 13.9 mmol). The stirring was continued for an
additional 45 min, and then, the mixture was treated with
concentrated HCl (4 mL). The organic layer was separated,
dried over Na2SO4, filtered, and concentrated. The residue was
dissolved in DMSO (11.5 mL) and water (0.5 mL), heated to
reflux overnight, and then cooled to room temperature and
partitioned between water and ethyl acetate. The organic
phase was washed sequentially with aqueous saturated NaH-
CO3, water, and brine, dried over MgSO4, filtered, and

6078 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Dai et al.
1
concentrated to provide the title compound (1.63 g, 60%). H
chromatography (HPLC) on a Waters Symmetry C8 column
(25 mm × 100 mm, 7 µm particle size) using a gradient of 10-
100% acetonitrile:0.1% aqueous trifluoroacetic acid (TFA) over
8 min (10 min run time) at a flow rate of 40 mL/min to provide
the title compound as the trifluoroacetate salt (35 mg, 41%).
1H NMR (DMSO-d6) δ 2.29 (s, 3H), 2.36 (t, J ) 7.63 Hz, 2H),
2.85 (t, J ) 7.46 Hz, 2H), 6.81 (d, J ) 7.46 Hz, 1H), 6.85 (s, br,
1H), 7.17 (t, J ) 7.80 Hz, 1H), 7.26 (d, J ) 9.00 Hz, 1H), 7.32
(m, 4H), 7.64 (d, J ) 8.81 Hz, 2H), 8.33 (s, 1H), 8.71 (s, 1H),
8.93 (s, 1H); MS (ESI) m/z 447 (M + H)+. Anal. (C23H22N6O2S‚
1.0CF3CO2H) C, H, N.
NMR (DMSO-d6) δ 2.66 (s, 3H), 4.00 (s, 3H), 7.65-7.72 (m,
1H), 7.75 (d, J ) 1.70 Hz, 1H), 8.00 (d, J ) 8.48 Hz, 1H); MS
(ESI) m/z 194 (M - H)-.
1-(2-Fluoro-4-nitrophenyl)ethanone (41a). Compound
41a was prepared using the same procedure as described for
the synthesis of 41d by substituting 40a for 40d. 1H NMR
(DMSO-d6) δ 2.65 (d, J ) 3.73 Hz, 3H), 8.01-8.09 (m, 1H),
8.12-8.20 (m, 1H), 8.26 (dd, J ) 10.51, 2.03 Hz, 1H); MS (ESI)
m/z 182 (M - H)-.
1-(2-Chloro-4-nitrophenyl)ethanone (41b). Compound
41b was prepared using the same procedure as described for
the synthesis of 41d by substituting 40b for 40d. 1H NMR
(DMSO-d6) δ 2.63 (s, 3H), 7.94 (d, J ) 8.14 Hz, 1H), 8.28 (dd,
J ) 8.48, 2.03 Hz, 1H), 8.37 (d, J ) 2.71 Hz, 1H); MS (ESI)
m/z 197 (M - H)-.
1-(2-Methoxy-4-nitrophenyl)ethanone (41c). Compound
41c was prepared using the same procedure as described for
the synthesis of 41d by substituting 40c for 40d. 1H NMR
(DMSO-d6) δ 2.57 (s, 3H), 4.01 (s, 3H), 7.74 (d, J ) 7.80 Hz,
1H), 7.84-7.89 (m, 1H), 7.91 (d, J ) 2.03 Hz, 1H); MS (ESI)
m/z 194 (M - H)-.
6,6-Dicyano-5-phenylhex-5-enoic Acid Methylamide
(46a). A mixture of 43 (5.0 g, 20.81 mmol), methylamine
hydrochloride (1.41 g, 20.81 mmol), HOBT (3.09 g, 22.89
mmol), EDC (4.39 g, 22.89 mmol), and NMM (5.7 mL, 52.03
mmol) in DMF was stirred at room temperature overnight and
then partitioned between ethyl acetate and water. The aqueous
phase was extracted with ethyl acetate, and the combined
extracts were washed with brine, dried over MgSO4, filtered,
and concentrated. The crude material was purified by flash
column chromatography on silica gel eluting with ethyl acetate
to provide the title compound (3.94 g, 75%). 1H NMR (DMSO-
d6) δ 1.42-1.60 (m, 2H), 2.05 (t, J ) 7.46 Hz, 2H), 2.54 (d, J
) 4.41 Hz, 3H), 2.98 (t, J ) 7.50 Hz, 2H), 7.50-7.69 (m, 5H),
7.75 (s, 1H); MS (ESI) m/z 271 (M + NH4)+.
6,6-Dicyano-5-phenylhex-5-enoic Acid (43). Compound
43 was prepared using the same procedure as described for
the synthesis of 3 by substituting 5-oxo-5-phenylvaleric acid
1
3-(5-Amino-4-cyano-3-phenylthiophen-2-yl)-N-methyl-
propionamide (47a). Compound 47a was prepared using the
same procedure as described for the synthesis of 4 by substi-
(42) for 1-(4-nitrophenyl)propan-1-one (2). H NMR (DMSO-
d6) δ 1.45-1.59 (m, 2H), 2.25 (t, J ) 7.29 Hz, 2H), 3.03 (t, J )
9.00 Hz, 2H), 7.53-7.62 (m, 3H), 7.62-7.69 (m, 2H), 12.16 (s,
br, 1H); MS (ESI) m/z 239 (M - H)-.
1
tuting 46a for 3. H NMR (DMSO-d6) δ 2.26 (t, J ) 7.46 Hz,
2H), 2.54 (d, J ) 4.41 Hz, 3H), 2.70 (t, J ) 7.46 Hz, 2H), 7.07
(s, 2H), 7.28-7.51 (m, 5 H), 7.77 (s, br, 1H); MS (ESI) m/z 286
(M + H)+.
3-(5-Amino-4-cyano-3-phenylthiophen-2-yl)propionic
Acid (44). Compound 44 was prepared using the same
procedure described for the synthesis of 4 by substituting 43
for 3 and using two molar equivalents of diethylamine. 1H
NMR (DMSO-d6) δ 2.41 (t, J ) 7.29 Hz, 2H), 2.72 (t, J ) 7.46
Hz, 2H), 7.09 (s, br, 2H), 7.29-7.35 (m, 2H), 7.37-7.50 (m,
3H), 12.23 (s, br, 1H).
3-(4-Amino-5-phenylthieno[2,3-d]pyrimidin-6-yl)propi-
onamide (45). Compound 45 was prepared using the same
procedure as described for the synthesis of 5 by substituting
44 for 4. In addition to the formation of the pyrimidine ring,
the acid group in 44 was converted into the amide in the
reaction. 1H NMR (DMSO-d6) δ 2.35 (t, J ) 7.63 Hz, 2H), 2.82
(t, J ) 7.63 Hz, 2H), 6.83 (s, br, 2H), 7.33 (s, br, 1H), 7.39-
7.46 (m, 2H), 7.51-7.59 (m, 3H), 8.27 (s, 1H); MS (ESI) m/z
299 (M + H)+.
3-(4-Amino-5-phenylthieno[2,3-d]pyrimidin-6-yl)-N-me-
thylpropionamide (48a). Compound 48a was prepared using
the same procedure as described for the synthesis of 5 by
substituting 47a for 4. 1H NMR (DMSO-d6) δ 2.36 (t, J ) 7.63
Hz, 2H), 2.54 (d, J ) 4.41 Hz, 3H), 2.83 (t, J ) 7.46 Hz, 2H),
7.38-7.45 (m, 2H), 7.51-7.62 (m, 3H), 7.79 (s, br, 1H), 8.27
(s, 1H); MS (ESI) m/z 313 (M + H)+.
3-{4-Amino-5-[4-({[(3-methylphenyl)amino]carbonyl}-
amino)phenyl]thieno[2,3-d]pyrimidin-6-yl}-N-methyl-
propanamide (52). Compound 52 was prepared as the
trifluoroacetate salt using the same sequence as described for
the synthesis of 51 by substituting 48a for 45. 1H NMR
(DMSO-d6) δ 2.29 (s, 3H), 2.36 (t, J ) 7.46 Hz, 2H), 2.54 (d, J
) 4.75 Hz, 3H), 2.86 (t, J ) 7.29 Hz, 2H), 6.81 (d, J ) 7.46
Hz, 1H), 7.17 (t, J ) 7.80 Hz, 1H), 7.26 (m, 1H), 7.29-7.34
(m, 3H), 7.64 (d, J ) 8.81 Hz, 2H), 7.80 (m, 1H), 8.33 (s, 1H),
8.71 (s, 1H), 8.93 (s, 1H); MS (ESI) m/z 461 (M + H)+.
3-[4-Amino-5-(4-nitrophenyl)thieno[2,3-d]pyrimidin-6-
yl]propionamide (49a). To a suspension of 45 (2.50 g, 7.66
mmol) in concentrated sulfuric acid (25 mL) at 0 °C was added
dropwise a solution of concentrated HNO3 (0.53 mL) in
concentrated sulfuric acid (8 mL). The mixture was stirred at
0 °C for 1 h and, then, at room temperature for 45 min, poured
into ice, and neutralized with solid Na2CO3. The yellow solid
was filtered, washed with water, dried over MgSO4, and
3-{4-Amino-5-[4-(3-m-tolylureido)phenyl]thieno[2,3-d]-
pyrimidin-6-yl}-N,N-dimethylpropionamide (53). Com-
pound 53 was prepared as the trifluoroacetate salt using the
same sequence as described for the synthesis of 51 by
1
1
concentrated to provide the title compound (2.13 g, 81%). H
substituting 48b for 45. H NMR (DMSO-d6) δ 2.29 (s, 3H),
NMR (DMSO-d6) δ 2.37 (t, J ) 7.46 Hz, 2H), 2.84 (t, J ) 7.46
Hz, 2H), 6.84 (s, 1H), 7.33 (s, br, 1H), 7.69 (d, J ) 8.82 Hz,
2H), 8.30 (s, br, 1H), 8.36 (d, J ) 8.48 Hz, 2H); MS (ESI) m/z
344 (M + H)+.
2.60 (t, J ) 7.12 Hz, 2H), 2.80 (s, 3H), 2.83-2.92 (m, 5H), 6.81
(d, J ) 7.12 Hz, 1H), 7.17 (t, J ) 7.50 Hz, 1H), 7.26 (d, J )
7.50 Hz, 1H), 7.29-7.37 (m, 3H), 7.64 (d, J ) 8.48 Hz, 2H),
8.36 (s, 1H), 8.73 (s, 1H), 8.94 (s, 1H); MS (ESI) m/z 475 (M +
H)+.
3-[4-Amino-5-(4-aminophenyl)thieno[2,3-d]pyrimidin-
6-yl]propionamide (50a). Compound 50a was prepared
using the same procedure as described for the synthesis of 6
by substituting 49a for 5 and using i-PrOH/CHCl3 (1:3) for
the extraction instead of ethyl acetate during the aqueous
workup. 1H NMR (DMSO-d6) δ 2.34 (dd, J ) 8.48, 6.44 Hz,
2H), 2.82 (dd, J ) 8.65, 6.61 Hz, 2H), 5.42 (s, br, 2H), 6.70 (d,
J ) 8.48 Hz, 2H), 6.83 (s, br, 1H), 7.01 (d, J ) 8.48 Hz, 2H),
7.32 (s, br, 1H), 8.23 (s, 1H); MS (ESI) m/z 314 (M + H)+.
3-{4-Amino-5-[4-({[(3-methylphenyl)amino]carbonyl}-
amino)phenyl]thieno[2,3-d]pyrimidin-6-yl}propanamide
(51). To a solution of 50a (60 mg, 0.19 mmol) in DMF (3 mL)
at 0 °C was added 3-tolyl isocyanate (24 µL, 0.19 mmol). The
mixture was stirred overnight as the temperature rose slowly
to room temperature. After DMF was removed, the crude
material was purified by preparative high-performance liquid
[6-Methyl-5-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4-
yl]carbamic Acid tert-Butyl Ester (54). To a solution of 5
(1.50 g, 5.24 mmol) in THF (40 mL) at 0 °C was added sodium
hydride (60% dispersion in mineral oil, 524 mg, 13.10 mmol)
in portions. After the mixture was stirred at 0 °C for 10 min,
a solution of di-tert-butyl dicarbonate (1.26 g, 5.76 mmol) in
THF (10 mL) was dropwise added. The mixture was stirred
at 0 °C for 2 h, quenched with saturated aqueous ammonium
chloride solution, and extracted with ethyl acetate three times.
The extracts were washed with brine, dried over MgSO4,
filtered, and concentrated. The solid residue was triturated
with ethyl acetate to provide the title compound (1.02 g, 63%).
1H NMR (DMSO-d6) δ ppm 1.06 (s, 9H), 2.50 (s, 3H), 7.63 (d,
J ) 8.82 Hz, 2H), 8.27 (d, J ) 8.82 Hz, 2H), 8.83 (s, 1H), 9.73
(s, 1H); MS (CI) m/z 387 (M + H)+.

Thienopyrimidine Ureas as Multitargeted RTK Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6079
[6-Morpholin-4-ylmethyl-5-(4-nitrophenyl)thieno[2,3-
d]pyrimidin-4-yl]carbamic Acid tert-Butyl Ester (56a).
To a suspension of 54 (1.0 g, 2.59 mmol) in benzene (100 mL)
was added N-bromosuccinimide (NBS) (0.51 g, 2.85 mmol) and
2,2′-azobisisobutyronitrile (AIBN) (100 mg). After being stirred
at 80 °C for 3 h, the mixture was cooled, absorbed on silica
gel, and purified by flash column chromatography eluting with
hexanes/ethyl acetate (3:2) to provide a mixture of bromide
55 and the unreacted 54 (730 mg) with a ratio of 3:1.
The above mixture was dissolved in DMF (15 mL) and
treated with morpholine (994 µL, 11.4 mmol) at room temper-
ature. After being stirred at room temperature overnight, the
reaction mixture was poured into a saturated sodium bicar-
bonate solution, extracted with ethyl acetate, washed with
water and brine, dried with MgSO4, filtered, and concentrated.
The crude product was purified by flash column chromatog-
raphy on silica gel eluting with ethyl acetate/hexanes (2:1) to
provide the title compound (476 mg, 39% from 54). MS (ESI)
m/z 472 (M + H)+.
[6-Morpholin-4-ylmethyl-5-(4-nitrophenyl)thieno[2,3-
d]pyrimidin-4-yl]carbamic Acid tert-Butyl Ester (57a).
To a solution of 56a (220 mg, 0.47 mmol) in methylene chloride
(4 mL) at 0 °C was added trifluoroacetic acid (4 mL). After
being stirred at 0 °C for 1.5 h, the mixture was concentrated.
The residue was neutralized with saturated aqueous sodium
bicarbonate solution, extracted with chloroform/2-propanol
(3:1), dried over MgSO4, filtered, and concentrated to provide
the title compound (170 mg, 98%). 1H NMR (DMSO-d6) δ 2.35
(t, J ) 4.41 Hz, 4H), 3.51 (s, 2H), 3.55 (t, J ) 4.41 Hz, 4H),
7.67 (d, J ) 8.82 Hz, 2H), 8.31 (s, 1H), 8.36 (d, J ) 8.81 Hz,
2H).
5-(4-Aminophenyl)-6-morpholin-4-ylmethylthieno[2,3-
d]pyrimidin-4-ylamine (58a). Compound 58a was prepared
using the same procedure as described for the synthesis of 6
by substituting 57a for 5. 1H NMR (DMSO-d6) δ 2.35 (t, br, J
) 3.73 Hz, 4H), 3.50 (s, 2H), 3.55 (t, J ) 4.41 Hz, 4H), 5.41 (s,
2H), 6.68 (d, J ) 8.48 Hz, 2H), 6.99 (d, J ) 8.48 Hz, 2H), 8.24
(s, 1 H); MS (ESI) m/z 342 (M + H)+.
N-{4-[4-Amino-6-(morpholin-4-ylmethyl)thieno[2,3-d]-
pyrimidin-5-yl]phenyl}-N′-(3-methylphenyl)urea (59).
Compound 59 was prepared using the same procedure as
described for the synthesis of 11 by substituting 58a for 6. 1H
NMR (DMSO-d6) δ 8.90 (s, 1H), 8.69 (s, 1H), 8.28 (s, 1H), 7.63
(d, J ) 8.7 Hz, 2H), 7.33-7.23 (m, 4H), 7.17 (t, J ) 7.5 Hz,
1H), 6.81 (d, J ) 7.5 Hz, 1H), 3.56 (s, br, 4H), 3.51 (s, 2H),
2.36 (s, br, 4H), 2.29 (s, 3H); MS (ESI) m/z 475 (M + H)+. Anal.
(C25H26N6O2S‚0.5H2O) C, H, N.
°C for 30 min, the mixture was treated with an excess of dry
ice and then stirred at -78 °C for an additional 30 min,
warmed to room temperature, diluted with water, and adjusted
to pH 3 with aqueous 2 N HCl. The solid material was filtered
to provide the title compound (686 mg, 51%). 1H NMR (DMSO-
d6) δ 13.13 (s, br, 1H), 8.29 (s, 1H), 8.09 (d, J ) 8.4 Hz, 2H),
7.53 (d, J ) 8.4 Hz, 2H), 2.28 (s, 3H); MS (CI) m/z 285 (M+).
4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-N-
phenylbenzamide (64). A suspension of 63 (89 mg, 0.31
mmol) and 1-hydroxybenzotriazole (46 mg, 0.35 mmol) in DMF
(4 mL) at room temperature was treated with m-toluidine (29
µL, 0.31 mmmol), 4-methylmorpholine (86 µL, 0.78 mmol), and
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochlo-
ride (66 mg, 0.34 mmol). After being stirred at room temper-
ature overnight, the reaction mixture was partitioned between
water and ethyl acetate. The aqueous phase was extracted
with ethyl acetate three times, and the combined organic
extracts were washed with water and brine, dried over Na2-
SO4, filtered, and concentrated. The residue was triturated
with hexanes/ethyl acetate to provide the title compound (84
1
mg, 75%). H NMR (DMSO-d6) δ 10.40 (s, 1H), 8.30 (s, 1H),
8.12 (d, J ) 8.4 Hz, 2H), 7.80 (d, J ) 7.5 Hz, 2H), 7.58 (d, J )
8.4 Hz, 2H), 7.37 (t, J ) 7.5 Hz, 2H), 7.12 (t, J ) 7.5 Hz, 1H),
2.32 (s, 3H); MS (ESI) m/z 361 (M + H)+. Anal. (C20H16N4OS‚
0.1EtOAc) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(3-methylphenyl)thiourea (65). Compound 65
was prepared using the same procedure as described for the
synthesis of 11 by substituting 3-tolyl thioisocyanate for 3-tolyl
isocyanate. 1H NMR (DMSO-d6) δ ppm 2.30 (s, 6H), 6.97 (d, J
) 6.78 Hz, 1H), 7.17-7.30 (m, 3H), 7.34 (d, J ) 8.14 Hz, 2H),
7.64 (d, J ) 8.48 Hz, 2H), 8.27 (s, 1H), 9.84 (s, 1H), 9.95 (s,
1H); MS (ESI) m/z 406 (M + H)+.
Phenyl N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-
5-yl)phenyl]-N′-cyanoimidocarbamate (66). A mixture of
6 (0.40 g, 1.56 mmol) and diphenyl cyanocarbonimidate (0.37
g, 1.56 mmol) in DMF (10 mL) was heated at 90 °C for 2 days.
After being cooled to room temperature, the reaction mixture
was poured into water, and the solid material was filtered.
The filter cake was suspended in hot ethanol and filtered. The
filtrate was concentrated and purified by flash column chro-
matography on silica gel eluting with 5-8% methanol in
dichloromethane to provide the title compound (150 mg, 24%).
1H NMR (DMSO-d6) δ 11.06 (s, 1H), 8.27 (s, 1H), 7.67 (d, J )
8.4 Hz, 2H), 7.50-7.43 (m, 4H), 7.36-7.29 (m, 3H), 2.29 (s,
3H); MS (ESI) m/z 401 (M + H)+.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′′-cyano-N′-(3-methylphenyl)guanidine (67). A
solution of 66 (40 mg, 0.01 mmol) and 3-methylaniline (12 µL,
0.01 mmol) in DMF (1 mL) was heated in a Smith Synthesizer
microwave at 180 °C for 20 min. After being cooled, the
reaction mixture was partitioned between water and ethyl
acetate. The aqueous phase was extracted with ethyl acetate
three times, and the combined extracts were washed with
water and brine, dried over Na2SO4, filtered, and concentrated.
The crude material was purified by flash column chromatog-
raphy on silica gel eluting with 8% methanol in dichlo-
romethane to provide the title compound (15 mg, 38%). 1H
NMR (DMSO-d6) δ 9.65 (s, 1H), 9.48 (s, 1H), 8.26 (s, 1H), 7.45
(d, J ) 8.4 Hz, 2H), 7.35 (d, J ) 8.4 Hz, 2H), 7.23 (t, J ) 7.8
Hz, 1H), 7.15-7.10 (m, 2H), 6.96 (d, J ) 7.8 Hz, 1H), 2.29 (s,
6H); MS (ESI) m/z 414 (M + H)+.
N-(4-{4-Amino-6-[(4-methylpiperazin-1-yl)methyl]-
thieno[2,3-d]pyrimidin-5-yl}phenyl)-N′-(3-methylphen-
yl)urea (60). Compound 60 was prepared using the same
sequence as described for the synthesis of 59 by substituting
N-methylpiperazine for morpholine in the case of 56a. 1H NMR
(DMSO-d6) δ 8.88 (s, 1H), 8.68 (s, 1H), 8.27 (s, 1H), 7.62 (d, J
) 9.0 Hz, 2H), 7.32-7.23 (m, 4H), 7.17 (t, J ) 7.5 Hz, 1H),
6.81 (d, J ) 7.5 Hz, 1H), 3.50 (s, 2H), 2.45-2.20 (s, br, 8H),
2.29 (s, 3H), 2.14 (s, 3H).
N-(4-{4-Amino-6-[(dimethylamino)methyl]thieno[2,3-
d]pyrimidin-5-yl}phenyl)-N′-(3-methylphenyl)urea (61).
Compound 61 was prepared using the same sequence as
described for the synthesis of 59 by substituting dimeth-
1
ylamine for morpholine in the case of 56a. H NMR (DMSO-
d6) δ 8.92 (s, 1H), 8.71 (s, 1H), 8.28 (s, 1H), 7.32-7.22 (m, 4H),
7.17 (t, J ) 7.8 Hz, 1H), 6.81 (d, J ) 7.8 Hz, 1H), 3.45 (s, br,
2H), 2.29 (s, 3H), 2.17 (s, 6H); MS (ESI) m/z 433 (M + H)+.
5-(4-Bromophenyl)-6-methylthieno[2,3-d]pyrimidin-4-
amine (62). Compound 62 was prepared using the same
sequence as described for the synthesis of 5 by substituting
4-bromophenyl ethyl ketone for 2. 1H NMR (DMSO-d6) δ 8.27
(s, 1H), 7.74 (d, J ) 8.1 Hz, 2H), 7.36 (d, J ) 8.1 Hz, 2H), 2.28
(s, 3H); MS (ESI) m/z 320, 322 (M + H)+.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-1H-indole-2-carboxamide (68). Compound 68 was
prepared using the same procedure as described for the
synthesis of 7 by substituting indole-2-carboxylic acid for
1
m-toluic acid. H NMR (DMSO-d6) δ 11.80 (s, 1H), 10.40 (s,
1H), 8.28 (s, 1H), 8.01 (d, J ) 8.7 Hz, 2H), 7.70 (d, J ) 7.5 Hz,
1H), 7.48 (d, J ) 7.5 Hz, 2H), 7.42 (d, J ) 8.7 Hz, 2H), 7.24 (t,
J ) 7.5 Hz, 1H), 7.08 (t, J ) 7.5 Hz, 1H), 2.32 (s, 3H); MS
(ESI) m/z 400 (M + H)+.
4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)ben-
zoic Acid (63). To a solution of 62 (1.5 g, 4.68 mmol) in THF
(50 mL) at -78 °C was dropwise added 2.5 M n-butyllithium
in hexanes (4.7 mL, 11.71 mmol). After being stirred at -78
5-[4-(1,3-Benzoxazol-2-ylamino)phenyl]-6-methylthieno-
[2,3-d]pyrimidin-4-amine (69). A solution of 6 (100 mg, 0.39
mmol) in pyridine (3 mL) was added dropwise over 5 min to a

6080 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Dai et al.
solution of 1,1-thiocarbonyldiimidazole (77 mg, 0.39 mmol) in
pyridine (3 mL) at 0 °C. After being stirred at 0 °C for 1.5 h,
the mixture was treated with 2-aminophenol (43 mg, 0.39
mmol) and stirred at room temperature overnight. The reac-
tion mixture was then treated with 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (90 mg, 0.47 mmol),
heated at 50 °C for 20 h, and then concentrated. The residue
was partitioned between ethyl acetate and water. The aqueous
phase was extracted with ethyl acetate, and the combined
extracts were washed with brine, dried over MgSO4, filtered,
and concentrated. The crude product was purified by flash
column chromatography on silica gel eluting with ethyl acetate
to provide the title compound (28 mg, 20%). 1H NMR (DMSO-
d6) δ 10.89 (s, 1H), 8.27 (s, 1H), 7.94 (d, J ) 8.4 Hz, 2H), 7.51
(m, 2H), 7.42 (d, J ) 8.4 Hz, 2H), 7.25 (td, J ) 7.5, 1.5 Hz,
1H), 7.16 (td, J ) 7.5, 1.5 Hz, 1H), 3.10 (s, 3H); MS (CI) m/z
374 (M + H)+. Anal. (C20H15N5OS‚0.2EtOAc‚0.2H2O) C, H, N.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-2-(3-methylphenyl)acetamide (70). Compound 70
was prepared using the same procedure as described for the
synthesis of 7 by substituting m-tolylacetic acid for m-toluic
acid. 1H NMR (DMSO-d6) δ 10.37 (s, 1H), 8.26 (s, 1H), 7.78 (d,
J ) 8.4 Hz, 2H), 7.32 (d, J ) 8.4 Hz, 2H), 7.26-7.13 (m, 3H),
7.07 (d, J ) 7.5 Hz, 1H), 3.64 (s, 2H), 2.31 (s, 3H), 2.27 (s,
3H); MS (ESI) 389 (M + H)+. Anal. (C22H20N4OS) C, H, N.
6-Methyl-5-[4-(methylamino)phenyl]thieno[2,3-d]pyri-
midin-4-amine (71). A suspension of 6 (400 mg, 1.56 mmol)
in dichloromethane (10 mL) and THF (10 mL) at -20 °C was
treated with formic acetic anhydride (135 µL, 1.7 mmol). After
being stirred at -20 °C for 1 h, the reaction mixture was
concentrated. The concentrate was then suspended in benzene
(50 mL), treated with 65% Red-Al in toluene (2.4 mL, 7.8
mmol), and stirred first at room temperature for 20 min and
then at reflux for 6 h. The mixture was cooled to room
temperature and partitioned between aqueous Rochelle’s salt
solution and ethyl acetate. The aqueous phase was extracted
with ethyl acetate three times. The combined extracts were
washed with water and brine, dried over Na2SO4, filtered, and
concentrated. The crude material was purified by flash column
chromatography on silica gel eluting with 7% methanol in
dichloromethane to provide the title compound (89 mg, 21%).
1H NMR (DMSO-d6) δ 2.27 (s, 3H), 2.73 (d, J ) 5.09 Hz, 3H),
5.99 (q, J ) 4.97 Hz, 1H), 6.67 (d, J ) 8.48 Hz, 2H), 7.08 (d, J
) 8.48 Hz, 2H), 8.23 (s, 1H); MS (ESI) m/z 271 (M + H)+.
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N-methyl-N′-(3-methylphenyl)urea (72). Com-
pound 72 was prepared using the same procedure as described
for the synthesis of 9 by substituting 71 for 6. 1H NMR (DMSO-
d6) δ 8.32 (s, 1H), 8.28 (s, 1H), 7.47 (d, J ) 8.4 Hz, 2H), 7.40
(d, J ) 8.4 Hz, 2H), 7.29-7.23 (m, 2H), 7.12 (t, J ) 7.8 Hz,
1H), 6.78 (d, J ) 7.8 Hz, 1H), 3.33 (s, 3H), 2.33 (s, 3H), 2.25
(s, 3H); MS (ESI) m/z 404 (M + H)+.
N-[4-(4-Amino-6-ethylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-cyclohexylurea (75). Compound 75 was pre-
pared using the same procedure as described for the synthesis
of 9 by substituting isocyanatocyclohexane for phenyl iso-
cyanate. 1H NMR (DMSO-d6) δ 1.00-1.90 (m, 14H), 2.63 (q, J
) 7.69 Hz, 2H), 6.17 (d, J ) 7.80 Hz, 1H), 7.23 (d, J ) 8.48
Hz, 2H), 7.54 (d, J ) 8.48 Hz, 2H), 8.27 (s, 1H), 8.54 (s, 1H);
MS (ESI) m/z 396 (M + H)+. Anal. (C21H25N5OS‚0.3H2O) C,
H, N.
N-[4-(4-Aminothieno[2,3-d]pyrimidin-5-yl)phenyl]-N′-
(3-methylphenyl)urea (76). Compound 76 was prepared
following the same sequence as described for the synthesis of
11 by replacing 2 with 4′-nitroacetophenone. 1H NMR (DMSO-
d6) δ 8.88 (s, 1H), 8.67 (s, 1H), 8.34 (s, 1H), 7.61 (d, J ) 8.7
Hz, 2H), 7.43 (s, 1H), 7.39 (d, J ) 8.7 Hz, 2H), 7.31 (s, 1H),
7.25 (d, J ) 7.5 Hz, 1H), 7.17 (t, J ) 7.5 Hz, 1H), 6.81 (d, J )
7.5 Hz, 1H); MS (ESI) m/z 376 (M + H)+. Anal. (C20H17N5OS)
C, H, N.
N-[4-(4-Aminothieno[2,3-d]pyrimidin-5-yl)phenyl]-N′-
butylurea (77). Compound 77 was prepared following the
same sequence as described for the synthesis of 32 by replacing
2 with 4′-nitroacetophenone in the case of 3 and n-butylamine
for cyclohexanylamine in the case of 32, respectively. 1H NMR
(DMSO-d6) δ 0.90 (t, J ) 7.1 Hz, 3H), 1.26-1.47 (m, 4H), 3.10
(q, J ) 6.4 Hz, 2H), 6.20 (t, J ) 5.3 Hz, 1H), 7.32 (d, J ) 8.8
Hz, 2H), 7.43 (s, 1H), 7.54 (d, J ) 8.8 Hz, 2H), 8.36 (s, 1H),
8.61 (s, 1H); MS (ESI) m/z 342 (M + H)+. Anal. (C17H19N5OS‚
0.5CF3CO2H) C, H, N.
N-[4-(4-Aminothieno[2,3-d]pyrimidin-5-yl)phenyl]-N′-
isobutylurea (78). Compound 78 was prepared using the
same procedure as described for the synthesis of 77 by
substituting 2-methylpropylamine for n-butylamine. 1H NMR
(DMSO-d6) δ 0.88 (d, J ) 6.4 Hz, 6H), 1.66-1.75 (m, 1H), 2.94
(apparent t, J ) 6.3 Hz, 2H), 6.26 (t, J ) 5.9 Hz, 1H), 7.33 (d,
J ) 8.5 Hz, 2H), 7.44 (s, 1H), 7.54 (d, J ) 8.5 Hz, 2H), 8.38 (s,
1H), 8.62 (s, 1H); MS (ESI) m/z 342 (M + H)+. Anal. (C17H19N5-
OS‚0.6CF3CO2H) C, H, N.
N-[4-(4-Amino-6-propylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(3-methylphenyl)urea (79). Compound 79 was
prepared following the same sequence as described for the
synthesis of 11 by replacing 2 with 1-(4-nitrophenyl)pentan-
1-one, which was prepared using the same procedure as
described for the synthesis of ketone 36a by substituting
n-butyl iodide for 35a. 1H NMR (DMSO-d6) δ 0.85 (t, J ) 7.29
Hz, 3H), 1.50-1.68 (m, 2H), 2.29 (s, 3H), 2.61 (t, J ) 7.63 Hz,
2H), 6.81 (d, J ) 7.80 Hz, 1H), 7.24 (m, 5H), 7.62 (d, J ) 8.48
Hz, 2H), 8.26 (s, 1H), 8.67 (s, 1H), 8.87 (s, 1H); MS (ESI) m/z
418 (M + H)+. Anal. (C23H23N5OS‚0.2H2O) C, H, N.
N-[4-(4-Amino-6-isopropylthieno[2,3-d]pyrimidin-5-
yl)phenyl]-N′-(3-methylphenyl)urea (80). Compound 80
was prepared following the same sequence as described for
the synthesis of 11 by replacing 2 with 1-(4-nitrophenyl)-3-
methylbutan-1-one, which was prepared using the same
procedure as described for the synthesis of ketone 36a by
substituting isobutyl iodide for 35a. 1H NMR (DMSO-d6) δ
1.20-1.23 (d, J ) 6.9 Hz, 6H), 2.29 (s, 3H), 3.00-3.09 (m, 1H),
6.79-6.82 (d, J ) 7.8 Hz, 1H), 7.14-7.19 (t, J ) 7.5 Hz, 1H),
7.24-7.27 (d, J ) 8.1 Hz, 1H), 7.30-7.33 (m, 3H), 7.61-7.64
(d, J ) 9.0 Hz, 2H), 8.26 (s, 1H), 8.67 (s, 1H), 8.88 (s, 1H); MS
(ESI) m/z 418 (M + H)+. Anal. (C23H23N5OS) C, H, N.
N-[4-(4-Amino-6-benzylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(3-methylphenyl)urea (81). Compound 81 was
prepared following the same sequence as described for the
synthesis of 11 by replacing 2 with 1-(4-nitrophenyl)-3-
phenylpropan-1-one, which was prepared using the same
procedure as described for the synthesis of ketone 36a by
N-[4-(4-Amino-6-methylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-methyl-N′-(3-methylphenyl)urea (73). Com-
pound 73 was prepared using the same procedure as described
for the synthesis of 32 by substituting N-methyl-m-toluidine
1
for cyclohexanylmethylamine. H NMR (DMSO-d6) δ 2.28 (s,
3H), 2.34 (s, 3H), 3.28 (s, 3H), 7.09 (d, J ) 7.32 Hz, 1H), 7.14
(d, J ) 7.93 Hz, 1H), 7.19 (s, 1H), 7.24 (d, J ) 8.54 Hz, 2H),
7.31 (t, J ) 7.78 Hz, 1H), 7.62 (d, J ) 8.54 Hz, 2H), 8.26 (s,
1H), 8.30 (s, 1H); MS (ESI) m/z 404 (M + H)+. Anal. (C22H21N5-
OS‚0.2EtOAc) C, H, N.
N-[4-(4-Amino-6-ethylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-(3-methylphenyl)urea (74). Compound 74 was
prepared following the same sequence as described for the
synthesis of 11 by replacing 2 with 1-(4-nitrophenyl)butan-1-
one, which was prepared using the same procedure as de-
scribed for the synthesis of ketone 36a by substituting n-propyl
iodide for tert-butyl(3-iodopropoxy)dimethylsilane (35a). 1H
NMR (DMSO-d6) δ 8.84 (s, 1H), 8.64 (s, 1H), 8.24 (s, 1H), 7.62
(d, J ) 8.5 Hz, 2H), 7.10-7.35 (m, 5H), 6.80 (d, J ) 7.2 Hz,
1H), 2.62 (q, J ) 7.5 Hz, 2H), 2.24 (s, 3H), 1.18 (t, J ) 7.5 Hz,
3H); MS (ESI) m/z 404 (M + H)+. Anal. (C22H21N5OS‚
0.15EtOAc) C, H, N.
1
substituting (2-iodoethyl)benzene for 35a. H NMR (DMSO-
d6) δ 2.29 (s, 3H), 3.99 (s, 2H), 6.79-6.82 (d, J ) 7.5 Hz, 1H),
7.14-7.32 (m, 8H), 7.35-7.38 (d, J ) 8.7 Hz, 2H), 7.63-7.66
(d, J ) 8.4 Hz, 2H), 8.26 (s, 1H), 8.67 (s, 1H), 8.89 (s, 1H); MS
(ESI) m/z 466 (M + H)+. Anal. (C27H23N5OS‚0.75H2O) C, H,
N.
N-{4-[4-Amino-6-(pyridin-3-ylmethyl)thieno[2,3-d]-
pyrimidin-5-yl]phenyl}-N′-(3-methylphenyl)urea (82).

Thienopyrimidine Ureas as Multitargeted RTK Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6081
Compound 82 was prepared following the same sequence as
N-[4-(4-Aminothieno[2,3-d]pyrimidin-5-yl)-3-chloro-
phenyl]-N′-(3-methylphenyl)urea (89). Compound 89 was
prepared following the same sequence as described for the
1
described for the synthesis of 11 by replacing 2 with 34a. H
NMR (DMSO-d6) δ 2.29 (s, 3H), 4.04 (s, 2H), 6.79-6.82 (d, J
) 7.5 Hz, 1H), 7.14-7.19 (t, J ) 7.8 Hz, 1H), 7.24-7.33 (m,
3H), 7.35-7.37 (d, J ) 8.4 Hz, 2H), 7.53-7.56 (td, J ) 2.1, 7.8
Hz, 1H), 7.63-7.66 (d, J ) 8.4 Hz, 2H), 8.27 (s, 1H), 8.34-
8.35 (d, J ) 1.8 Hz, 1H), 8.42-8.44 (dd, J ) 1.5, 4.8 Hz, 1H),
8.67 (s, 1H), 8.89 (s, 1H); MS (ESI) m/z 467 (M + H)+. Anal.
(C26H22N6OS‚0.2H2O) C, H, N.
1
synthesis of 11 by substituting 41b for 2. H NMR (DMSO-
d6) δ 2.29 (s, 3H), 6.82 (d, J ) 7.5 Hz, 1H), 7.18 (t, J ) 7.5 Hz,
1H), 7.23-7.26 (m, 1H), 7.31-7.34 (s, br, 1H), 7.41-7.42 (m,
2H), 7.49 (s, 1H), 7.91 (s, br, 1H), 8.33 (s, 1H), 8.75 (s, 1H),
9.05 (s, 1H); MS (ESI) m/z 409.9, 411.9 (M + H)+. Anal. (C20H16-
ClN5O‚0.5H2O) C, H, N.
N-{4-[4-Amino-6-(pyridin-4-ylmethyl)thieno[2,3-d]-
pyrimidin-5-yl]phenyl}-N′-(3-methylphenyl)urea (83).
Compound 83 was prepared following the same sequence as
N-[4-(4-Aminothieno[2,3-d]pyrimidin-5-yl)-3-meth-
oxyphenyl]-N′-(3-methylphenyl)urea (90). Compound 90
was prepared following the same sequence as described for
11 by substituting 41c for 2. The crude material was purified
by preparative HPLC using the conditions described for the
purification of 51 to provide the title compound as the
1
described for the synthesis of 11 by replacing 2 with 34b. H
NMR (DMSO-d6) δ 2.28 (s, 3H), 3.32 (s, 2H), 6.79-6.82 (d, J
) 7.5 Hz, 1H), 7.13-7.30 (m, 5H), 7.32-7.35 (d, J ) 8.4 Hz,
2H), 7.61-7.64 (d, J ) 8.4 Hz, 2H), 8.28 (s, 1H), 8.45-8.47
(dd, J ) 4.2, 1.5 Hz, 2H), 8.67 (s, 1H), 8.88 (s, 1H); MS (ESI)
m/z 467 (M + H)+; HRMS (FAB) Calcd for C26H23N6OS
467.1654, found 467.1649.
1
trifluoroacetate salt. H NMR (DMSO-d6) δ 2.29 (s, 3H), 3.72
(s, 3H), 6.81 (d, J ) 7.1 Hz, 1H), 7.05 (dd, J ) 8.1, 2.0 Hz,
1H), 7.14-7.25 (m, 3H), 7.34 (s, 1H), 7.38 (s, 1H), 7.50 (d, J )
2.0 Hz, 1H), 8.36 (s, 1H), 8.67 (s, 1H), 8.93 (s, 1H); MS (ESI)
m/z 406.0 (M + H)+. Anal. (C21H19N5O2S‚1.0CF3CO2H‚0.2H2O)
C, H, N.
N-[4-(4-Aminothieno[2,3-d]pyrimidin-5-yl)-2-fluoro-
phenyl]-N′-(3-methylphenyl)urea (91). Compound 91 was
prepared following the same sequence as described for the
synthesis of 51 by replacing 42 with 3′-fluoroacetophenone.
1H NMR (DMSO-d6) δ 2.30 (s, 3H), 6.83 (d, J ) 7.1 Hz, 1H),
7.18 (t, J ) 7.6 Hz, 1H), 7.23-7.27 (m, 2H), 7.32 (s, br, 1H),
7.39 (dd, J ) 12.0, 1.9 Hz, 1H), 7.49 (s, 1H), 8.30 (d, J ) 8.5
Hz, 1H), 8.34 (s, 1H), 8.70 (d, J ) 2.7 Hz, 1H), 9.06 (s, 1H);
MS (ESI) m/z 394 (M + H)+. Anal. (C20H16FN5OS) C, H, N.
N-[4-(4-Aminothieno[2,3-d]pyrimidin-5-yl)-2-chloro-
phenyl]-N′-(3-methylphenyl)urea (92). Compound 92 was
prepared following the same sequence as described for the
synthesis of 51 by replacing 42 with 3′-chloroacetophenone.
1H NMR (DMSO-d6) δ 2.30 (s, 3H), 6.83 (d, J ) 7.1 Hz, 1H),
7.19 (apparent t, J ) 7.6 Hz, 1H), 7.26 (d, J ) 8.1 Hz, 1H),
7.33 (s, 1H), 7.39 (dd, J ) 8.7, 2.2 Hz, 1H), 7.52 (s, 1H), 7.58
(d, J ) 2.0 Hz, 1H), 8.34 (d, J ) 8.8 Hz, 1H), 8.34 (s, 1H), 8.44
(s, 1H), 9.43 (s, 1H); MS (ESI) m/z 410 (M + H)+. Anal. (C20H16-
ClN5OS‚0.7H2O) C, H, N.
N-[4-(4-Aminothieno[2,3-d]pyrimidin-5-yl)-2-meth-
oxyphenyl]-N′-(3-methylphenyl)urea (93). Compound 93
was prepared following the same sequence as described for
the synthesis of 11 by substituting 41d for 2. 1H NMR (DMSO-
d6) δ 2.29 (s, 3H), 3.94 (s, 3H), 6.80 (d, J ) 7.5 Hz, 1H), 7.01
(dd, J ) 8.3, 1.7 Hz, 1H), 7.12 (d, J ) 1.7 Hz, 1H), 7.17 (t, J )
7.8 Hz, 1H), 7.24 (d, J ) 8.5 Hz, 1H), 7.32 (s, 1H), 7.46 (s,
1H), 8.29 (d, J ) 8.5 Hz, 1H), 8.34 (s, 1H), 8.38 (s, 1H), 9.31
(s, 1H); MS (ESI) m/z 406 (M + H)+. Anal. (C21H19N5O2S‚
0.7H2O) C, H, N.
N-(11-Amino-5,6-dihydro-7-thia-8,10-diazabenzo[c]-
fluoren-3-yl)-N′-(3-methylphenyl)urea (94). Compound 94
was prepared following the same sequence as described for
the synthesis of 51 by substituting 1-tetralone for 42. 1H NMR
(DMSO-d6) δ 2.28 (s, 3H), 2.91 (m, 4H), 6.6-7.0 (s, br, 2 H),
6.79 (d, J ) 7.1 Hz, 1H), 7.16 (m, 1H), 7.23 (d, J ) 8.1 Hz,
1H), 7.32 (s, 1H), 7.40 (m, 2H), 7.54 (d, J ) 1.7 Hz, 1H), 8.30
(s, 1H), 8.61 (s, 1H), 8.72 (s, 1H); MS (ESI) m/z 402 (M + H)+.
Anal. (C22H19N5OS‚0.7H2O) C, H, N.
N-[4-(4-Amino-6-ethylthieno[2,3-d]pyrimidin-5-yl)-
phenyl]-N′-[3-(trifluoromethyl)phenyl]urea (95). Com-
pound 95 was prepared using the same procedure as described
for the synthesis of 74 by substituting 3-trifluoromethylphenyl
isocyanate for m-tolyl isocyanate. 1H NMR (DMSO-d6) δ 9.18
(s, 1H), 9.06 (s, 1H), 8.32 (s, 1H), 8.02 (s, 1H), 7.50-7.70 (m,
4H), 7.32 (d, J ) 8.4 Hz, 3H), 2.62 (q, J ) 7.5 Hz, 2H), 1.98 (t,
J ) 7.5 Hz, 3H). Anal. (C22H18F3N5OS‚0.9CH2Cl2) C, H, N.
N-{4-[4-Amino-6-(2-hydroxyethyl)thieno[2,3-d]pyrimi-
din-5-yl]phenyl}-N′-(3-methylphenyl)urea (84). Compound
84 was prepared following the same sequence as described for
the synthesis of 11 by replacing 2 with 36a, except that the
reaction of the corresponding amino nitrile with formamide
was conducted in a Smith Synthesizer microwave (70 min at
180 °C) to form the pyrimidine ring and that the tert-
butyldimethylsilyl group was removed with tetrabutylammo-
nium fluoride (TBAF) (in THF at room temperature) in the
1
last step. H NMR (DMSO-d6) δ 2.29 (s, 3H), 2.75-2.80 (t, J
) 6.6 Hz, 2H), 3.54-3.60 (m, 2H), 4.85-4.89 (t, J ) 5.7 Hz,
1H), 6.79-6.82 (d, J ) 7.5 Hz, 2H), 7.14-7.19 (t, J ) 7.5 Hz,
1H), 7.24-7.32 (m, 4H), 7.61-7.63 (d, J ) 8.4 Hz, 2H), 8.26
(s, 1H), 8.67 (s, 1H), 8.87 (s, 1H); MS (ESI) m/z 420 (M + H)+.
Anal. (C22H21N5O2S‚0.4H2O) C, H, N.
N-{4-[4-Amino-6-(2-methoxyethyl)thieno[2,3-d]pyrimi-
din-5-yl]phenyl}-N′-(3-methylphenyl)urea (85). Compound
85 was prepared following the same sequence as described for
11 by replacing 2 with 4-methoxy-1-(4-nitrophenyl)butan-1-
one, which was prepared using the same procedure as de-
scribed for the synthesis of ketone 36a by substituting
3-methoxypropyl iodide for 35a. 1H NMR (DMSO-d6) δ 2.29
(s, 3H), 2.83-2.87 (t, J ) 6.6 Hz, 2H), 3.22 (s, 3H), 3.47-3.52
(t, J ) 6.6 Hz, 2H), 6.79-6.82 (d, J ) 7.5 Hz, 2H), 7.14-7.19
(t, J ) 7.5 Hz, 1H), 7.24-7.32 (m, 4H), 7.61-7.64 (d, J ) 9.0
Hz, 2H), 8.27 (s, 1H), 8.67 (s, 1H), 8.87 (s, 1H); MS (ESI) m/z
434 (M + H)+.
N-{4-[4-Amino-6-(3-hydroxypropyl)thieno[2,3-d]pyri-
midin-5-yl]phenyl}-N′-(3-methylphenyl)urea (86). Com-
pound 86 was prepared following the same sequence as
described for the synthesis of 84 by substituting 36b for 36a.
1H NMR (DMSO-d6) δ 1.70 (m, 2H), 2.29 (s, 3H), 2.68 (m, J )
6.27 Hz, 2H), 3.37 (t, J ) 6.27 Hz, 2H), 6.81 (d, J ) 7.80 Hz,
1H), 7.17 (t, J ) 7.63 Hz, 1H), 7.23-7.33 (m, 4H), 7.62 (d, J )
8.81 Hz, 2H), 8.27 (s, 1H), 8.69 (s, 1H), 8.90 (s, 1H); MS (ESI)
m/z 434 (M + H)+.
N-(4-{4-Amino-6-[2-(dimethylamino)ethyl]thieno[2,3-
d]pyrimidin-5-yl}phenyl)-N′-(3-methylphenyl)urea (87).
Compound 87 was prepared following the same sequence as
1
described for the synthesis of 11 by substituting 39 for 2. H
NMR (DMSO-d6) δ 2.11 (s, 6H), 2.29 (s, 3H), 2.42-2.46 (t, J )
7.2 Hz, 2H), 2.72-2.77 (t, J ) 6.0 Hz, 2H), 6.79-6.82 (d, J )
7.5 Hz, 1H), 7.14-7.19 (t, J ) 7.5 Hz, 1H), 7.24-7.32 (m, 4H),
7.61-7.64 (d, J ) 6.6 Hz, 2H), 8.26 (s, 1H), 8.67 (s, 1H), 8.87
(s, 1H); MS (ESI) m/z 447 (M + H)+.
N-[4-(4-Aminothieno[2,3-d]pyrimidin-5-yl)-3-fluoro-
phenyl]-N′-(3-methylphenyl)urea (88). Compound 88 was
prepared following the same sequence as described for the
synthesis of 11 by substituting 41a for 2. 1H NMR (500 MHz,
DMSO-d6) δ 2.29 (s, 3H), 6.82 (d, J ) 7.5 Hz, 1H), 7.18 (t, J )
7.8 Hz, 1H), 7.25 (d, J ) 10.0 Hz, 1H), 7.27 (dd, J ) 8.6, 2.0
Hz, 1H), 7.32 (s, 1H), 7.37 (t, J ) 8.4 Hz, 1H), 7.52 (s, 1H),
7.66 (dd, J ) 12.6, 2.0 Hz, 1H), 8.34 (s, 1H), 8.75 (s, 1H), 9.09
(s, 1H); MS (ESI) m/z 394.0 (M + H)+. Anal. (C20H16FN5OS‚
0.2H2O) C, H, N.
N-[4-(4-Aminothieno[2,3-d]pyrimidin-5-yl)phenyl]-N′-
(3-chlorophenyl)urea (96). Compound 96 was prepared
using the same procedure as described for the synthesis of 76
by substituting 3-chlorophenyl isocyanate for m-tolyl isocyan-
1
ate. H NMR (DMSO-d6) δ 8.96 (s, 1H), 8.34 (s, 1H), 7.73 (s,
1H), 7.61 (d, J ) 8.4 Hz, 2H), 7.44 (s, 1H), 7.40 (d, J ) 8.4 Hz,

6082 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Dai et al.
2H), 7.32-7.28 (m, 2H), 7.03 (dt, J ) 2.1 Hz, 1H); MS (ESI)
m/z 396 (M + H)+.
containing 0.1% Tween 20 (PBST) before addition of the cell
lysates. Cell lysates were incubated in the KDR ELISA plate
with constant shaking on a microtiter plate shaker for 2 h at
room temperature. The cell lysate was then removed, and the
plate was washed five times with PBST. Detection of phospho-
KDR was performed using a 1:2000 dilution of biotinylated
4G10 anti-phosphotyrosine (UBI, Lake Placid, NY). The wells
were incubated with constant shaking for 1.5 h at room
temperature and washed five times with PBST, and for
detection, a 1:2000 dilution of streptavidin-HRP (UBI, Lake
Placid, NY) was added and incubated with constant shaking
for 1 h at room temperature. The wells were then washed five
times with PBST, and K-Blue HRP ELISA substrate (Neogen)
was added to each well. The development time was monitored
at 650 nm in a SpectraMax Plus plate reader until 0.4-0.5
absorbance units were obtained (approximately 10 min) in the
VEGF only wells. Phosphoric acid (1 M) was added to stop the
reaction, and the plate was read at 450 nm. The percent
inhibition was calculated using the VEGF only wells as 100%
controls and wells containing 5 µM pan-kinase inhibitor as 0%
controls (no VEGF wells were used to monitor the endogenous
phosphorylation state of the cells). IC50 values were calculated
by nonlinear regression analysis of the concentration response
curve. Each IC50 determination was performed with five
concentrations, and each assay point was determined in
duplicate.
KDR Phosphorylation Determined by Western Blot
Analysis. NIH3T3 cells stably transfected with full length
human KDR (VEGFR2) were cultured in T75 or T150 flasks;
compounds were added in the serum-free growth medium for
20 min prior to stimulation for 3 min with VEGF (50 ng/mL).
The cells were washed one time with PBS and lysed with 1
mL/flask of RIPA buffer containing protease inhibitors, NaF,
and Na3VO4 for 5 min on ice. Cells were scraped, and the lysate
was incubated for an additional 20 min on ice with occasional
mixing in a 2 mL microcentrifuge tube. The lysate was
centrifuged at 20000g for 20 min at 4 °C, and the supernatant
was used to immunoprecipitate KDR. The lysate, 500 µg, was
precleared with Protein-G agarose beads (Calbiochem) for 30
min at 4 °C while rotating. The lysate was centrifuged (20000g
for 1 min), and the supernatant was immunoprecipitated with
4 µg of an anti-VEGFR2 antibody (R&D Systems) overnight
at 4 °C while rotating. After incubation, 50 µL of Protein-G
agaraose was added and incubated for 1 h at 4 °C while
rotating. Beads were washed five times with PBS containing
Na3VO4 (1 mM) and protease inhibitors (Sigma). The washed
beads were resuspended in 50 µL of sample buffer, electro-
phoresed on a 8-16% Tris-glycine gel (Invitrogen), transferred
to a nitrocellulose membrane, blocked with 5% milk in PBST,
and probed with an anti-KDR antibody (sc-315, Santa Cruz).
KDR was visualized with enhanced chemoluminescence detec-
tion, and IC50 values were determined by comparison of the
treated samples to the vehicle (DMSO) treated samples.
Computational Analysis. The homology model of KDR
kinase in the inactive conformation was built (Insight II
software, HOMOLOGY module, Accelrys, San Diego, CA)
using the homologous Kit kinase (51% identity to KDR kinase
within the catalytic domain, inactive conformation, PDB entry
1T46).32 Manual docking of 11 into the homology model of KDR
was accomplished by creating two canonical hinge hydrogen
bonds between (1) the exocyclic amino group of 11 and the
backbone carbonyl of Glu 917 and (2) the ring nitrogen and
the backbone N-H of Cys 919. The torsions of the diaryl urea
unit of 11 were then manually adjusted to follow the general
path of the inhibitor STI-571, an inhibitor targeting the
inactive conformations of Kit and c-Abl kinases, as observed
in PDB entry 1T46.32 Energy minimization using the CFF force
field (25 iterations in a static active site, followed by 50
iterations of full ligand-protein minimization) provided the
final model shown in Figure 2. No solvation was included.
Homogeneous Time-Resolved Fluorescence (HTRF)
Assays of Receptor Tyrosine Kinases. Assays were per-
formed in a total of 40 µL in 96-well Costar black half-volume
plates using HTRF technology.36 A peptide substrate (Biotin-
Ahx-AEEEYFFLFA-amide) at 4 µM, 1 mM ATP, enzyme, and
inhibitors were incubated for 1 h at ambient temperature in
50 mM Hepes/NaOH (pH 7.5), 10 mM MgCl2, 2 mM MnCl2,
2.5 mM DTT, 0.1 mM orthovanadate, and 0.01% bovine serum
albumin (BSA). Inhibitors were added to the wells at a final
concentration of 3.2 nM to 50 µM with 5% DMSO added as a
cosolvent. The reactions were stopped with 10 µL/well of 0.5
M EDTA, and then, 75 µL of buffer containing streptavidin-
allophycocyanin (Prozyme) (1.1 µg/mL) and PT66 antibody
europium cryptate (Cis-Bio) (0.1 µg/mL) was added to each
well. The plates were read from 1 to 4 h after addition of the
detection reagents, and the time-resolved fluorescence (665:
615 ratio) was measured using a Packard Discovery instru-
ment. The amount of each tyrosine kinase added to the wells
was calibrated to give a control (no inhibitor) to background
(prequenched with ethylenediaminetetraacetic acid (EDTA))
ratio of 10-15, and it was shown to be in the low nanomolar
concentration range for each kinase. The inhibition of each
well was calculated using the control and background readings
for that plate. KDR, CSF1R, cKit, FLT1, and FLT3 were
assayed using the above protocol. Tie2 does not phosphorylate
this peptide substrate; therefore, 10 ng/well of biotinylated
poly(Glu-Tyr) (Cis-Bio) was used, and the assay was quenched
with EDTA after 10 min. The HTRF detection was done in
the same way as that for the other kinases. Each IC50
determination was performed with seven concentrations, and
each assay point was determined in duplicate.
Cellular KDR Phosphorylation Assay. KDR Phos-
phorylation Determined by ELISA. NIH3T3 cells stably
transfected with full length human KDR (VEGFR2) were
maintained in a Dulbecco’s modified Eagle’s medium (DMEM)
with 10% fetal bovine serum and 500 µg/mL Geneticin. KDR
cells were plated at 20 000 cells per well into duplicate 96-
well tissue culture plates and cultured overnight in an
incubator at 37 °C with 5% CO2 and 80% humidity. The growth
medium was replaced with a serum-free growth medium for
2 h prior to compound addition. Compounds in DMSO were
diluted in the serum-free growth medium (final DMSO con-
centration 1%) and added to cells for 20 min prior to stimula-
tion for 10 min with VEGF (50 ng/mL). Cells were lysed by
addition of RIPA buffer (50 mM Tris-HCl (pH 7.4), 1%
IGEPAL, 150 mM NaCl, 1 mM EDTA, and 0.25% sodium
deoxycholate) containing protease inhibitors (Sigma cocktail),
NaF (1 mM), and Na3VO4 (1 mM) and placed on a microtiter
plate shaker for 10 min. The lysates from duplicate wells were
combined, and 170 µL of the combined lysate was added to
the KDR ELISA plate. The KDR ELISA plate was prepared
by adding an anti-VEGFR2 antibody (1 µg/well, R&D Systems)
to an unblocked plate and incubated overnight at 4 °C. The
plate was then blocked for at least 1 h with 200 µL/well of 5%
dry milk in PBS. The plate was washed two times with PBS
Estradiol-Induced Murine Uterine Edema Assay.
Twelve week old balb/c female mice (Taconic, Germantown,
NY) were pretreated with 10 units of pregnant mare’s serum
gonadotropin (PMSG) (Calbiochem) intraperitoneally (ip) ad-
ministered 72 and 24 h prior to estradiol. Mice were random-
ized the day of the experiment. Test compounds were formu-
lated in a variety of vehicles and administered po 30 min prior
to stimulation with an intraperitoneal injection of water
soluble 17â-estradiol (20-25 µg/mouse). Animals were sacri-
ficed and uteri removed 2.5 h following estradiol stimulation
by cutting just proximal to the cervix and at the fallopian
tubes. After the removal of fat and connective tissue, uteri were
weighed, squeezed between filter paper to remove fluid, and
weighed again. The difference between wet and blotted weights
represented the fluid content of the uterus. Compound treated
groups were compared to vehicle treated groups of animals
after subtracting the background water content of unstimu-
lated uteri. The experimental group size was 5 or 6.
HT1080 Tumor Growth Inhibition Model. The 1080
human fibrosarcoma cells were obtained from the American
Type Tissue Culture Collection and maintained in DMEM

Thienopyrimidine Ureas as Multitargeted RTK Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6083
Stokes, E. S. E.; Curry, B.; Richmond, G. H. P.; Wadsworth, P.
F.; Bigley, A. L.; Hennequin, L. F. ZD6474 Inhibits Vascular
Endothelial Growth Factor Signaling, Angiogenesis, and Tumor
Growth Following Oral Administration. Cancer Res. 2002, 62,
4645-4655.
supplemented with 10% fetal bovine serum and antibiotics.
For tumor xenograft studies, cells were suspended in PBS,
mixed with an equal volume of matrigel (phenol red free) to a
final concentration of 2 million cells/mL, and inoculated (0.25
mL) into the flank of SCID-beige mice. One week after
inoculation, tumor-bearing animals were divided into groups
(n ) 10), and administration of vehicle (2% EtOH, 5% Tween
80, 20% PEG 400, and 73% saline) or inhibitor at the indicted
dose was initiated. Tumor growth was assessed every 2-3 days
by measuring tumor size and calculating tumor volume using
the formula [length × width2]/2.
(22) Fraley, M. E.; Hoffman, W. F.; Arrington, K. L.; Hungate, R.
W.; Hartman, G. D.; McFall, R. C.; Coll, K. E.; Rickert, K.;
Thomas, K. A.; McGaughey, G. B. Curr. Med. Chem. 2004, 11,
709-719
(23) Adams, J., Huang, P.; Patrick, D. A Strategy for the Design of
Multiplex Inhibitors for Kinase-mediated Signaling in Angio-
genesis. Curr. Opin. Chem. Biol. 2002, 6, 486-492.
(24) Arteaga, C. L. Molecular Therapeutics: Is One Promiscuous
Drug against Multiple Targets Better than Combinations of
Molecule-specific Drugs? Clin. Cancer Res. 2003, 9, 1231-1232.
(25) Mandel, D. B.; Laird, A. D.; Xin, X.; Louie, S. G.; Christensen,
J. G.; Li, G.; Schreck, R. E.; Abrams, T. J.; Ngai, T. J.; Lee, L.
B.; Murray, L. J.; Carver, J.; Chan, E.; Moss, K. G.; Naznedar,
J. O.; Sukbuntherng, J.; Blake, R. A.; Sun, L.; Tang, C.; Miller,
T.; Shirazian, S.; McMahon, G.; Cherrington, J. M. In vivo
References
(1) Ullrich, A.; Schlessinger, J. Signal Transduction by Receptors
with Tyrosine Kinase Activity. Cell 1990, 61, 203-212.
(2) Hubbard, S. R.; Till, J. H. Protein Tyrosine Kinases: Structure
and Function. Annu. Rev. Biochem. 2000, 69, 373-398.
(3) Yancopoulos, G. D.; Davis, S.; Gale, N. W.; Rudge, J. S.; Wiegand,
S. J.; Holash, J. Vascular-specific Growth Factors and Blood
Vessel Formation. Nature 2000, 407, 242-248.
(4) Bilodeau, M. T.; Fraley, M. E., Hartman, G. D. Kinase Insert
Domain-containing Receptor Kinase Inhibitors as Antiangiogenic
Agents. Expert Opin. Invest. Drugs 2002, 11 (6), 737.
(5) Carmeliet, P.; Jain, R. K. Angiogenesis in Cancer and Other
Diseases. Nature 2000, 407, 249-257.
(6) Holmgren, L.; O’Reilly, M. S.; Folkman, J. Dormancy of Mi-
crometastases: Balanced Proliferation and Apoptosis in the
Presence of Angiogenesis Suppression. Nat. Med. 1995, 1, 149-
153.
(7) Zetter, B. R. Angiogenesis and Tumor Metastasis. Annu. Rev.
Med. 1998, 49, 407-424.
Antitumor Activity of SU11248,
A Novel Tyrosine Kinase
Inhibitor Targeting Vascular Endothelial Growth Factor and
Platelet-derived Growth Factor Receptors: Determination of a
Phamarcokinetic/pharmacodynamic Relationship. Clin. Cancer
Res. 2003, 9, 327-337.
(26) Wilhelm, S. M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.;
Rong, H.; Chen, C.; Zhang X.; Vincent, P.; McHugh, M.; Cao,
Y.; Shujath, J.; Gawlak, S.; Eveleigh, D.; Rowley, B.; Liu, L.;
Adnane, L.; Lynch, M.; Auclair, D.; Taylor, I.; Gedrich, R.;
Voznesensky, A.; Riedl, B.; Post, L. E.; Bollag, G.; Trail P. A.
BAY 43-9006 Exhibits Broad Spectrum Oral Antitumor Activity
and Targets the RAF/MEK/ERK Pathway and Receptor Tyrosine
Kinases Involved in Tumor Progression and Angiogenesis.
Cancer Res. 2004, 64, 7099-7109.
(27) Burchat, A. F.; Calderwood, D. J.; Friedman, M. M.; Hirst, G.
C.; Li, B.; Rafferty, P.; Ritter, K.; Skinner, B. S. Bioorg. Med.
Chem. Lett. 2002, 12, 1687-1690.
(28) Scientists at GlaxoSmithKline have independently discovered
thienopyrimidine and furanopyrimidine ureas as kinase inhibi-
tors: (a) WO 03/022852 A2. (b) Miyazaki, Y.; Matsungaga, S.;
Tang, J.; Maeda, Y.; Nakano, M.; Philippe, R. J.; Shibahara, M.;
Liu, W.; Sato, H.; Wang, L.; Nolte, R. T. Novel 4-Amino-furo-
[2,3-d]pyrimidines as Tie-2 and VEGFR2 Dual Inhibitors. Bioorg.
Med. Chem. Lett. 2005, 15, 2203-2207.
(29) Keinan, E.; Gleize, P. A. Organo Tin Nucleophiles IV. Palladium
Catalyzed Conjugation Reduction with Tin Hydride. Tetrahedron
Lett. 1982, 23, 477-480.
(30) Tamaru, Y.; Ochiai, H.; Sanda, F.; Yoshida, Z. A Convenient and
Efficient Unsymmetrical Ketone Synthesis from Acid Chlorides
and Alkyl Iodides Catalyzed by Palladium. Tetrahedron Lett.
1985, 26, 5529-5532.
(31) Gribbles, F. M.; Loussouarn, G.; Tucker, S. J.; Zhao, C.; Nichols,
C. G.; Ashcroft, F. M. A Novel Method for Measurement of
Submembrane ATP Concentration. J. Biol. Chem. 2000, 275,
30046-30049.
(32) Mol, C. D.; Dougan, D. R.; Schneider, T. R.; Skene, R. J.; Kraus,
M. L.; Scheibe, D. N.; Snell, G. P.; Zou, H.; Sang, B.-C.; Wilson,
K. P. Structural Basis for the Autoinhibition and STI-571
Inhibition of c-Kit Tyrosine Kinase. J. Biol. Chem. 2004, 279,
31655-31663.
(33) Curtin, M. L.; Frey, R. R.; Heyman, H. R.; Sarries, K. A.;
Bousquet, P. F.; Cunha, G. A.; Moskey, M. D.; Ahmed, A. A.;
Pease, L. J.; Glaser, K. B.; Stewart, K. D.; Davidsen, S. D.;
Michaelides, M. R. Isoindolinone Ureas: A Novel Class of KDR
Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 4505-
4509.
(34) Dumas, J.; Smith, R. A.; Lowinger, T. B. Recent Developments
in the Discovery of Protein Kinase Inhibitors from the Urea
Class. Curr. Opin. Drug Discovery Dev. 2004, 7, 600-616. (b)
Dumas, J. Protein Kinase Inhibitors from the Urea Class. Curr.
Opin. Drug Discovery Dev. 2002, 5, 718-727.
(35) Ma, W.; Tan, J.; Matsumoto, H.; Robert, B.; Abrahamson, D. R.;
Das, S. K.; Dey, S. K. Adult Tissue Angiogenesis: Evidence for
Negative Regulation by Estrogen in the Uterus. Mol. Endocrinol.
2001, 15, 1983-1992.
(36) Kolb, A. J.; Kaplita, P. V.; Hayes, D. J.; Park, Y.-W.; Pernell,
C.; Major, J. S.; Mathis, G. Tyrosine Kinase Assays Adapted to
Homegeneous Time-resolved Fluorescence. Drug Discovery To-
day 1998, 3, 333-342.
(8) Kim, I.; Kim, H. G.; Moon, S. O.; Chae, S. W.; So, J.-N.; Koh, K.
N.; Ahn, B. C.; Koh, G. Y. Angiopoietin-1 Induces Endothelial
Cell Sprouting through the Activation of Focal Adhesion Kinase
and Plasmin Secretion. Circ. Res. 2000, 86, 952-959.
(9) Skobe, M.; Fusenig, N. E. Tumorigenic Conversion of Immortal
Human Keratinocytes through Stromal Cell Activation. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 1050-1055.
(10) Lindahl, P.; Johansson, B. R.; Leveen, P.; Betsholtz, C. Pericyte
Loss and Microaneurysm Formation in PDGF-B-deficient Mice.
Science 1997, 277, 242-245.
(11) Demetri, G. D. Targeting c-Kit Mutations in Solid Tumors:
Scientific Rationale and Novel Therapeutic Options. Semin.
Oncol. 2001, 28, 19-26.
(12) Lin, E. Y.; Nguyen, A. V.; Russell, R. G.; Pollard, J. W. Colony-
stimulating Factor 1 Promotes Progression of Mammary Tumors
to Malignancy. J. Exp. Med. 2001, 193, 727-740.
(13) Stirewalt, D. L.; Radich, J. P. The Role of FLT3 in Haemato-
poietic Malignancies. Nat. Rev. Cancer 2003, 3, 650-665.
(14) Gschwind, A.; Fischer, O. M.; Ullrich, A. The Discovery of
Receptor Tyrosine Kinases: Targets for Cancer Therapy. Nat.
Rev. 2004, 4, 361-370.
(15) Dancey, J.; Sausville, E. A. Issues and Progress with Protein
Kinase Inhibitors for Cancer Treatment. Nat. Rev. Drug Dis-
covery 2003, 2, 296-313.
(16) Renhowe, P. A. Inhibitors of Growth Factor Receptor Kinase-
dependent Signaling Pathways in Anticancer Chemotherapy -
Clinical Progress. Curr. Opin. Drug Discovery Dev. 2002, 5, 214-
224.
(17) Bergsland, E. K. Update on Clinical Trials Targeting Vascular
Endothelial Growth Factor in Cancer. Am. J. Health-Syst.
Pharm. 2004, 61 (Suppl. 5), S12-S20.
(18) Bridges, A. J. Chemical Inhibitors of Protein Kinases. Chem.
Rev. 2001, 101, 2541-2571.
(19) Weng, D. E.; Usman, N. Angiozyme: A Novel Angiogenesis
Inhibitor. Curr. Oncol. Rep. 2001, 3, 141-146.
(20) Wood, J. M.; Bold, G.; Buchdunger, E.; Cozens, R.; Ferrari, S.;
Frei, J.; Hofmann, F.; Mestan, J.; Mett, H.; O′Reilly, T.; Persohn,
E.; Rosel, J.; Schnell, C.; Stover, D.; Theuer, A.; Towbin, H.;
Wenger, F.; Woods-Cook, K.; Menrad, A.; Siemeister, G.; Schirn-
er, M.; Thierauch, K.-H.; Schneider, M. R.; Drevs, J.; Martiny-
Baron, G.; Totzke, F.; Marme, D. PTK787/ZK222584, A Novel
and Potent Inhibitor of Vascular Endothelial Growth Factor
Receptor Tyrosine Kinases, Impairs Vascular Endothelial Growth
Factor-induced Responses and Tumor Growth after Oral Ad-
ministration. Cancer Res. 2000, 60, 2178-2189.
(21) Wedge, S. R.; Ogilvie, D. J.; Dukes, M.; Kendrew, J.; Chester,
R.; Jackson, J. A.; Boffey, S. J.; Valentine, P. J.; Curwen, J. O.;
Musgrove, H. L.; Graham, G. A.; Hughes, G. D.; Thomas, A. P.;
JM050458H