Structure-activity study leading to identification of a highly active thienopyrimidine based EGFR inhibitor

Article abstract of DOI:10.1016/j.ejmech.2014.01.042

Based on the thieno[2,3-d]pyrimidine scaffold, a series of new 4-amino-6-aryl thienopyrimidines have been prepared and evaluated as EGFR tyrosine kinase inhibitors. The in vitro activity was found to depend strongly on the substitution pattern in the 6-aryl ring, the stereochemistry, and the basicity at the secondary 4-amino group. A stepwise optimization by combination of active fragments led to the discovery of three structures with EGFR IC ₅₀ < 1 nM. The most potent drug candidate had an IC₅₀ of 0.3 nM towards EGFR and its mutants L858R and L861Q. Studies using human cancer cell lines and an EGFR-L858R reporter cell system revealed good cellular potency, verifying the identified thienopyrimidines as promising lead structures.

Full text of DOI:10.1016/j.ejmech.2014.01.042

European Journal of Medicinal Chemistry 75 (2014) 354e374
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity study leading to identification of a highly active
thienopyrimidine based EGFR inhibitor
,
c
Steffen Bugge a, Svein Jacob Kaspersen a, Synne Larsen a, Unni Nonstad b, Geir Bjørkøy b
,
,
Eirik Sundby d, Bård Helge Hoff a
*
a Department of Chemistry, Norwegian University of Science and Technology, Høgskoleringen 5, NO-7491 Trondheim, Norway
b Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Prinsesse Kristinas Gate 1, NO-7491 Trondheim, Norway
c Sør-Trøndelag University College, Erling Skjalgssons Gate 1, NO-7004 Trondheim, Norway
d Sør-Trøndelag University College, E.C. Dahls Gate 2, NO-7004 Trondheim, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the thieno[2,3-d]pyrimidine scaffold, a series of new 4-amino-6-aryl thienopyrimidines have
been prepared and evaluated as EGFR tyrosine kinase inhibitors. The in vitro activity was found to depend
strongly on the substitution pattern in the 6-aryl ring, the stereochemistry, and the basicity at the
secondary 4-amino group. A stepwise optimization by combination of active fragments led to the dis-
covery of three structures with EGFR IC50 < 1 nM. The most potent drug candidate had an IC50 of 0.3 nM
towards EGFR and its mutants L858R and L861Q. Studies using human cancer cell lines and an EGFR-
L858R reporter cell system revealed good cellular potency, verifying the identified thienopyrimidines
as promising lead structures.
Received 28 November 2013
Received in revised form
16 January 2014
Accepted 19 January 2014
Available online 31 January 2014
Keywords:
Thienopyrimidine
Benzylamine
Suzuki-coupling
EGFR-TK
Ó 2014 Elsevier Masson SAS. All rights reserved.
SAR
Erlotinib
1. Introduction
responses [1,6]. In contrast, properly designed low molecular
weight compounds are attractive as inhibitors of the intracellular
Epidermal growth factor receptor tyrosine kinase (EGFR-TK)
represents a major target in small molecular cancer therapy [1e3].
This transmembrane receptor contains an extracellular binding site
for epidermal growth factors (EGF), and an intracellular tyrosine
kinase domain. Binding of EGF induces dimerization and thereby
activation of the kinase domain [2]. Monoclonal antibodies such as
Cetuximab and Panitumumab interfere with ligand binding and/or
activation of the extracellular kinase domain [4,5], and some
monoclonal antibodies might in addition trigger immune
kinase domain, since they also can inhibit ligand independent
mutant receptors, and for their more moderate cost profile. Ex-
amples of ATP competitive inhibitors on the marked include Gefi-
tinib, Erlotinib, Lapatinib and Vandetanib, which all are based on a
central quinazoline core, see Fig. 1. Both intra- and extracellular
inhibitors have the effect of down regulating downstream signal-
ling events and thereby the growth and survival of the tumour [7].
X-ray structural data is available for EGFR-TK co-crystallized
with quinazoline based inhibitors [8e11], and also the pyrrolo-
pyrimidine AEE-788 [10]. The ATP binding site is in a cleft formed
by the C- and N terminal loops of the kinase. The X-ray structures
indicate that the inhibitors affinity for the binding site originates
from hydrogen bonding between N-1 of the pyrimidine to the main
chain NH of Met793 [10]. For AEE-788 a hydrogen bond interaction
was observed between N-3 and Thr854 via a bridged water mole-
cule [10].
Abbreviations: ATP, adenosine triphosphate; BEI, binding efficiency; cGMP, cy-
clic guanosine monophosphate; DFG, Asp-Phe-Gly; EGF, epidermal growth factor;
EGFR-TK, epidermal growth factor receptor tyrosine kinase; HER, human epidermal
growth factor receptor; LE, ligand efficacy; LLE, lipophilic ligand efficiency; LELP,
lipophilicity corrected ligand efficiency; MIDA, N-methyliminodiacetic acid; SEI,
surface efficiency index; IL-3, interleukin-3; TP53, tumour protein p53.
* Corresponding author. Tel.: þ47 73593973; fax: þ47 73544256.
The 4-amino group is directed in a small hydrophobic pocked
allowing for stabilization by dispersion forces. None of the X-ray
studies have indicated that the amine function at C-4 is involved in
binding. The substituents present at the benzo, pyrrolo or thieno
E-mail addresses: Steffen.Bugge@chem.ntnu.no (S. Bugge), Svein.Jacob.
Kaspersen@chem.ntnu.no (S.J. Kaspersen), synnelar@gmail.com (S. Larsen), unni.
nonstad@ntnu.no (U. Nonstad), geir.bjorkoy@hist.no (G. Bjørkøy), eirik.sundby@
hist.no (E. Sundby), bard.helge.hoff@chem.ntnu.no, bard.hoff@online.no (B.H. Hoff).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.042

S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
355
1
O
O
N
O
O
N
N
N
N
2
O
O
N3
N
N
O
N
4
N
O
S
O
O
Cl
F
N
H
H
H
H
F
O
Cl
Gefitinib
Erlotinib
Lapatinib
H
N
N
O
O
N
N
N
N
N
N
N
N
N
H
N
HN
S
N
H
O
S
N
N
S
O
O
O
H3C
N
N
H
H
Cl
F
H
F
F
Br
O
I
Cl
II
AEE-788
Vandetanib
Fig. 1. Structure of some potent EGFR inhibitors.
fragment of the heterocycle, often called “solubilizing tail”, are
directed towards the entrance of the ATP binding site.
but the X-ray structure of AEE-788 (Fig. 1) bond to EGFR did not
indicate the pyrrole nitrogen as important in binding [10]. Also, the
longer SeC bond in the thiophene fragment as compared to the
pyrrole could affect EGFR-ligand binding, and therefore could
require a different substitution pattern. Herein, the 6-aryl group has
been used as a scaffold in scanning for favourable/unfavourable
lipophilic, dipolar and hydrogen bond interactions. Besides the
more direct binding interactions, long range electronic effects to
the pyrimidine nitrogens might also play a role. Moreover, the re-
ported X-ray studies have not indicated any clear role for the C-4
amino group in binding. We have also addressed this issue by
evaluating the performance of designed model compounds.
Our laboratory has recently investigated 4-benzylamine
substituted pyrrolopyrimidines as EGFR tyrosine kinase ATP
competitive inhibitors [12]. Herein we report an extension of this
research by employing thieno[2,3-d]pyrimidine as the scaffold.
Thienopyrimidines in general have become an interesting structural
element in development of pharmaceutical compounds [13,14], and
have among others been evaluated as cGMP phosphodiesterase in-
hibitors [15], anti-viral agents [16], but also as kinase inhibitors and
potential anti-cancer agents [17e20]. Research on thienopyrimidine
analogues of Lapatinib such as I, and compound II both revealed low
nanomolar potency towards EGFR [21,22]. In addition, irreversible
thienopyrimidine based EGFR inhibitors have been developed [23].
Based on an efficient synthetic route we have synthesized a series of
new chiral thieno[2,3-d]pyrimidine derivatives and evaluated their
efficiency as EGFR-TK inhibitors.
2.2. Synthesis
To investigate the effect of structural variations in Fragment A
(Fig. 2) on EGFR inhibitory potency, a series of target molecules
were constructed as shown in Scheme 1.
2. Result and discussion
6-Bromo-4-chlorothieno[2,3-d]pyrimidine (1), synthesized in
house by a four step procedure in multi-gram scale [25], was reacted
with (R)-1-phenylethylamine ((R)-2) giving (R)-3. The following
Suzuki couplings using Pd(PPh3)4 gave the derivatives 5, mostly in
>70% yield. As compared to our previous study [25], tuning of the
reaction conditions by lowering the reaction temperature and the
amount of catalyst, gave easier purifications. Loss in yield in these
reactions was mainly due to purification issues rather than low
conversions, as some cross-coupling of the boronic acids occurred.
2.1. Design of the inhibitors
Our intention was to use the thienopyrimidine as a substitute for
quinazoline or pyrrolpyrimidine for construction of EGFR-TK in-
hibitors (Fig. 2), a strategy known as scaffold hopping. As compared
to the previously reported active pyrrolopyrimidines [12,24], the
thienopyrimidines lack the NeH unit as presented by the pyrrole,
R1
1
H
N
R1'
R1
N
2 N
N
S
Fragment A
R1
6
N
3 N
N
5
4
HN
N
NH
R3
R3
R4
R2'
Fragment B
R2
R2
Pyrrolopyrimidines
R2
Thienopyrimidines
Quinazolines
Fig. 2. Structure of benzo-, pyrrolo- and the investigated thienopyrimidines.

356
S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
Scheme 1. Synthesis of the thienopyrimidines (R)-5aev.
We were also able to couple the sometimes demanding 2-
aminophenylboronic acid in 84% yield. In selected cases the corre-
sponding N-methyliminodiacetic acid (MIDA) boronate esters were
used, with Pd(OAc)2 and SPhos as the catalyst system, with some-
what lower yields. During optimization of EGFR-TK activity, com-
pound (R)-5s was synthesized, which by sodium borohydride
reduction gave access to the disubstituted derivative (R)-5t.
In order to investigate the effect of structural variation in Frag-
ment B on EGFR-TK inhibitory potency, a series of related derivatives
25e33 were prepared using similar chemistry, Scheme 2. The ami-
nation reactions proceeded as for (R)-3 in every case except when
using the monofluoroamine, (rac)-12, which was unstable at higher
temperatures. Thus, the reaction was performed at 50 ꢀC for 72 h.
The following Suzuki coupling on 15e23 gave complete conversion
in every case, but fine tuning of the work-up and purification was
needed to fully purify the compounds. Selected derivatives were
also prepared by an alternative route via 24a [25] and 24c. However,
due to a moderate selectivity in the Suzuki coupling the yields were
somewhat low. The building blocks 24a and c were then trans-
formed to compounds (S)-5a, (R)-5c and (R)-25c, through standard
thermal amination chemistry. The analogue ether (rac)-36n was
synthesized from 1 and racemic 1-phenylethanol (34) with sodium
hydride as base forming the ether (rac)-35, which was converted to
compound (rac)-36n in the usual manner.
H
N
N
N
N
N
S
N
N
CH3
CH3
H
H
IC50= 2 nM
IC50= 58 nM
(R)
III
-5a
Fig. 3. Scaffold hopping by heterocyclic replacement used for discovery of new EGFR-
TK inhibitors.
2.3.1. Structural variations in Fragment A
The initial plan to improve potency was to “scan” for possible
hydrogen bonding interactions and lipophilic contacts. Sub-
stituents varied in the 6-aryl group (Fragment A) were OH and
CH2OH with the idea of mimicking the hydrogen bonding ability
of the pyrrole; methyl groups to search for possible lipophilic
contacts and as a control of any conformational effects; and
methoxy substituents as possible hydrogen bond acceptors. Later,
additional compounds were included to refine the structureeac-
tivity study. The results in terms of IC50 for the mono substituted
derivatives 5aer, and the trisubstituted compounds 5u, v are
shown in Fig. 4.
As compared to the starting point, (R)-5a, the potency was
increased from 58 nM to 7 and 9 nM by introducing a para
hydroxymethyl group (comp. (R)-5e) or an ortho methoxy group
(comp. (R)-5p). Compound (R)-5l having a meta hydroxymethyl
group also showed promising activity (9 nM).
The purity of all compounds was evaluated by HPLC. To verify
that the enantiomeric excess of the products were not affected by
the chemistry, a chiral analysis was developed for 5a using a Lux 5u
Cellulose HPLC column. Both (R)- and (S)-5a were measured to have
an ee of 99%, indicating no racemization.
2.3. EGFR inhibitory potency
As compared to the corresponding pyrrolopyrimidine III [12],
analysed by the same assay (Supporting information Table S1), a
significant drop in EGFR-TK potency was noticed for the structurally
related thienopyrimidine (R)-5a, see Fig. 3.
We therefore took on a stepwise approach where the effects of
the 6-aryl group (Fragment A) and the 4-amino group (Fragment B)
on EGFR-TK inhibitory potency were separately investigated and
optimized.
The major substituent effects observed are visualized in Fig. 5.
Methyl substitution decreased the potency in any position. For
compounds having the phenolic- or the methoxy group, the activity
increase in the order para < meta < ortho. This indicated that a
hydrogen bond acceptor is beneficial in the ortho position. Further
testing of the ortho aniline, (R)-5o, resulted in an IC50 value of
123 nM. All these groups are regarded as weak hydrogen bond
acceptors [26], and the activity trend OMe > OH > NH2 might

S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
357
Scheme 2. Synthesis and structures of the thienopyrimidines (S)-5a, 25e33 and (rac)-36n.
therefore be due to difference in desolvation energies, leading to
the highest potency for the least solvated compound (R)-5p.
For the hydroxymethyl group the potency increased in the order
ortho < meta < para, strongly indicating that a hydrogen donor/
acceptor group at this position promotes binding. Possibly, this
interaction can be reached also from the meta position, explaining
the similar activity of (R)-5l and (R)-5e.
To investigate the effect of substituent size and possible long
range electronic effects on N-1 and N-3, the additional compounds
(R)-5gei were made and tested. The aldehyde derivative (R)-5i,
being small and electron withdrawing had a decent potency
(11 nM), whereas the bulky tert-butyl and trifluoromethyl com-
pounds having opposite electronic character both were not toler-
ated in the para position. This shows that bulky para substituents
reduce activity. The 13C NMR spectroscopic shift changes at C-2 and
C-4 of compounds (R)-5aer followed normal substituent effects
where electron donating groups increase shielding (see Supporting
information). However, no correlation of the 13C chemical shifts
with the IC50 values was seen. Thus, the data do not indicate that
long range electronic effects have any major influence on EGFR
inhibitory potency. A low activity was also noticed for the 2,4,6-
trisubstituted derivatives, (R)-5u, v.
2.3.2. Structural variations in Fragment B
To clarify the role of the 4-amino group (Fragment B, Fig. 2) in
protein-ligand binding, the size and electronic properties of R3 have
been varied and R4 blocked as methyl. Moreover, modification of R3
might reveal favourable interactions and size limitations in this

358
S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
OMe
Me
CH2OH
OH
OH
OH
O
CHO
F
CF3
para
113 nM
(R)-5f
58 nM
(R)-5a
>1000 nM
(R)-5g
11 nM
>1000
(R)-5h
74 nM
54 nM
7 nM
(R)-5e
92 nM
(R)-5b
(R)-
-
5i
(R)
5c
(R)-
5d
OH
OH
O
N
N
S
meta
ortho
N
H
CH3
46 nM
(R)-5j
37 nM
78 nM
9 nM
(R)-5l
(R)-
(R)-
5k
5m
OH
OH
O
H2N
35 nM
153 nM
(R)-
123 nM
(R)-
38 nM
(R)-5q
9 nM
(R)-
(R)-
>1000 nM >1000 nM
(R)-5u
(R)-5v
5n
5r
5o
5p
Fig. 4. IC50 values of compounds 5aer and 5u, v towards EGFR-TK. (Single point data and standard deviations of the IC50 are compiled in Supporting information).
part of the binding pocket. The results of the testing are summa-
rized in Fig. 6.
as EGFR reversible inhibitor with an IC50 value of 720 nM, which is
36 times less potent than Gefitinib in the assay used [23]. The same
group later discovered that the hydroxymethyl side chain is
hydrogen bonded to Asp in the Asp-Phe-Gly motif [29].
Conservative modifications at R3 as exemplified with the ben-
zylamine based 26n and 1-phenyl-1-propanamine derivative (R)-
27n had similar activity to that of (R)-5n. The racemic monofluoro
and isopropyl analogues (rac)-31n and (rac)-28n showed IC50
values of 67 and 55 nM, respectively, indicating that the enantio-
merically pure analogues also would have a decent EGFR inhibitory
potency. The trifluoromethyl compound (rac)-30n on the other
hand had low activity. As the trifluoromethyl group has a size
similar to an isopropyl [27,28], the low potency could be due to the
inductive effect reducing amine basicity, although conformational
effects cannot be ruled out.
A low activity was also the case when removing the hydrogen
donor ability of the 4-amino group by methylation, as in compound
(R)-29n, and when using oxygen as heteroatom (compound (rac)-
36n). This strongly suggests that a secondary 4-amino group is
crucial for binding interactions with EGFR-TK.
Testing of compound (S)-5a (IC50 > 1000) verified that the ab-
solute stereochemistry is of uttermost importance. This was also
confirmed in the case of (S)-5n, (R)-32n and (R)-33n. Note that
although the relative stereochemistry is the same, the R/S
nomenclature in case of derivatives 32 and 33 changes as a result of
the higher priority of the CH2Oe as compared to the phenyl group.
Based on this we conclude that trifluoromethyl as R3, the
hydrogen bond donor ability of the 4-amino group, and wrong
stereochemistry affects potency negatively. The steric bulk of the R3
group is of less importance for potency, while a hot spot interaction
can be reached with
stereocentre.
a hydroxymethyl substituent at the
2.3.3. Fluoro insertion in the aromatic part of Fragment B
In the search for hydrogen bonding interactions and strength-
ened lipophilic contacts the R3 side chain was also varied with a
methoxymethyl and a hydroxymethyl group. To our delight com-
pound (S)-33n (IC50 3 nM) was found to be 10 times more potent
than the reference compound (R)-5n. A decent activity was also
seen for the methylated analogue (S)-32n (IC50 14 nM). In the late
phase of our work we discovered that a related thienopyrimidine,
containing the same hydroxylmethyl side chain, has been identified
Previous work on pyrrolopyrimidines indicated that only
limited structural variation was tolerated in the aromatic part of
Fragment
B [12], however para-fluoro substituted analogues
showed promising activity. As blocking of metabolic labile sites by
introduction of fluoro atoms is an efficient strategy in drug design
[30,31], selected para-fluoro derivatives were prepared and tested.
The obtained EGFR-TK IC50 values are compared with those for the
corresponding nonfluorinated derivatives in Fig. 7.
The para-fluoro substituted derivatives 25 showed consistently
lower potency as compared to the non-fluorinated analogues 5.
This systematic trend strongly suggests that all ligands, indepen-
dent of the 6-aryl substitution pattern, bind in a similar way. The
lower activity observed on fluoro insertion did not inspire more
elaborate structureeactivity studies in this region of the molecule.
2.3.4. Increasing potency by combining active fragments
To investigate if more active compounds could be developed,
the favourable substitution patterns identified were combined into
new compounds. (S)-2-Amino-2-phenylethan-1-ol ((S)-14) and
(R)-1-phenylethylamine ((R)-2) were used as building blocks in
Fragment B, while the substructures in Fragment A included 2-
Fig. 5. Effect of 6-aryl substitution pattern on EGFR potency.

S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
359
N
N
H
H
HO
O
OH
N
N
S
N
3 nM
(S)
14 nM
(S)
-33n
32n
-
N
N
N
N
H
H
H
H
H
F
35 nM
(R)
29 nM
26n
33 nM
67 nM
(rac)-31n (rac)-28n
55 nM
(R)
-
-
5n
27n
N
N
N
N
O
N
CF3
H
H
H
H
O
OH
>1000 nM
(R)
>1000 nM >1000 nM >1000 nM >1000 nM >1000 nM
(R) (S) (R)
33n
-
32n
-
5n
-29n
(rac)-36n (rac)-30n
-
Fig. 6. EGFR inhibitory potency represented by IC50 values upon variation of Fragment B. (Single point data and standard deviations of the IC50 are compiled in Supporting
information).
methoxyphenyl, 4-hydroxymethylphenyl and a combination of
these. The compounds made, and the test results in terms of IC50
values are shown in Fig. 8.
Three compounds were identified as highly potent (<1 nM), of
which the most active, compound (S)-33t, showed a 200 fold
improvement in IC50 as compared to the starting point (R)-5a.
Moreover, in every case the combination strategy was successful for
improving the potency.
initially indicated several binding modes. Herein, we have assumed
that binding should involve the N-1 to Met793 interaction as
shown in the co-crystal structure of AEE-788 and EGFR [10].
Upon variations in Fragment B, an increase in EGFR potency was
seen when having hydroxymethyl or methyl ether in the R3 side
chain. Modelling indicates these groups to engage in a hydrogen
bonding network involving Thr854 and Asp855 in the DFG motif.
The same type of interaction has been seen in a related 5,6-
diarylfuropyrimidine [29].
Further, a secondary amine function is needed at C-4, as also
seen in other classes of EGFR inhibitors [32]. Moreover, the elec-
tronic nature of the R3 substituent, influencing the basicity of this
amine, and the stereochemistry are of outmost importance. Overall,
this points to the fact that the secondary NH group is involved in
2.3.5. Structureeactivity relationships and molecular modelling
The major conclusions regarding the identified structureeac-
tivity relationships are summarized in Fig. 9.
Also, molecular docking was undertaken using crystal data from
a protein co-crystallized with AEE-788 (2J6M) (Fig. 11), which
N
N
S
N
27 nM
27 nM
35 nM
73 nM
134 nM
197 nM
256 nM
25c
143 nM
25a
CH3
H
(R)-25e
(R)-25p
(R)-25l
(R)-25n
(R)-25q
(R)-25j
(R)-
(R)-
F
OH
OH
O
HO
HO
OH
F
N
N
S
N
7 nM
9 nM
9 nM
35 nM
38 nM
46 nM
74 nM
58 nM
CH3
H
5e
(R)-5p
(R)-5l
(R)-5n
(R)-5q
(R)-5j
(R)-5c
(R)-
(R)-5a
Fig. 7. Comparison of EGFR-TK inhibitory activity for fluorinated and nonfluorinated analogues. (Single point data and standard deviations of the IC50 are compiled in Supporting
information).

360
S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
N
N
S
N
0.3 nM
0.8 nM
1.5 nM
2 nM
3 nM
H
OH
(S)
-
(S)
(S)
-
(S)
-
33n
-
-33t
(S) 33s
33p
33e
O
O
O
HO
O
OH
OH
N
N
S
N
0.7 nM
3 nM
9 nM
7 nM
35 nM
H
(R)
(R)
(R)
-
5n
(R)
-
5e
-
5t
(R)
5s
-
-5p
Fig. 8. EGFR-TK inhibitory activity (IC50 nM) by combining active fragments. (Single point data and standard deviations of the IC50 are compiled in Supporting information).
binding interactions. Two water molecules were seen in close
proximity (2.6e2.8 A) to the C-4 amino group, whereas one of these
higher activity in the pyrrolopyrimidine III as compared to (R)-5a, is
due to the slight change in orientation of the 6-aryl ring (Fig. 10).
The 6-aryl group (Fragment A) is directed towards the bulk
solvent, and an indication of hydrogen bonding is seen from para
hydroxymethyl to Leu718. Further, it is postulated that this site also
could be reached from the meta position with the same group.
Finally, a weak hydrogen bond acceptor (OMe) was preferred in
the ortho position, and oriented towards NH of Gly796, which
might be a hydrogen bond donor, although the O$$HN distance was
ꢀ
appears to donate a hydrogen bond to N-3 as seen from Fig. 11.
Possibly, the secondary amino group forms part of a hydrogen
bonding network involving these water molecules as indicated in
the crystallization and modelling work of Park et al. [11].
The naked phenyl ring (Fragment B) is located in a hydrophobic
pocket as observed for AEE-788. This model explains the reduced
activity of the para-fluoro containing derivatives 25 to be due to
repulsive interactions between the fluoro substituent and espe-
cially Thr790. Other residues in close proximity include Leu777,
Leu788 and Ile789. As compared to the analogues pyrrolopyr-
imidines [12], a higher sensitivity to fluoro insertion was seen.
Thus, a slightly distorted binding of the ligands as compared to the
pyrrolopyrimidines is indicated.
ꢀ
a long as 3.1 A. Gly796 has previously been postulated to be
engaged in a van der Waals interaction with the methoxy group of
Gefitinib [3], which offers an alternative explanation of the effect
exerted by the methoxy group.
The pyrimidine nitrogens have been proposed as crucial for
EGFR ligand binding. The 13C NMR spectroscopic data (Supporting
information) indicates that a long range electronic effect from the
6-aryl group does affect the electron density of the pyrimidine.
However, in the activity range studied other variables, mainly polar
interactions of the side chains seem to be of higher importance.
It was initially speculated that the pyrrole nitrogen could be
involved in hydrogen bonding as a donor. This is an interaction
which is not satisfied by the thienopyrimidine based molecules.
Placing hydrogen bond donor groups as R1 (OH, NH2 or CH2OH) did
not have a huge impact on potency, indicating that the intrinsic
2.3.6. Further profiling of selected compounds
Compounds (S)-33t, (R)-5t, (S)-33p and Erlotinib, were further
evaluated at 10 and 100 nM ATP concentrations towards EGFR-TK,
Fig. 10. Overlaid structures of III (dark grey) and (R)-5a (light grey). Replacement of
ꢀ
the heteroatom leads to a shift of 0.7 A of the carbon in para position of the 6-aryl ring
Fig. 9. Structureeactivity relationships identified in this study.
(Fragment A).

S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
361
(data not shown) [3]. Overall, compound (S)-33t compares
favourably with Erlotinib.
Off-target activity is often a challenge in development of kinase
inhibitors. The selectivity of compounds (S)-33t and (R)-5t as ki-
nase inhibitors were therefore evaluated towards a panel of 47
other kinases at a test concentration of 500 nM, see Table 2.
Compounds (R)-5t and (S)-33t have very similar structures, and
this is also reflected in the selectivity screen. For the majority of the
48 kinases tested, the two compounds have approximately the
same potency. The selectivity profiles of the two compounds are
compared with that of Erlotinib by a Gini plot (Fig. 12), where the
cumulative fraction of kinases are plotted towards the cumulative
fraction of total inhibition [33].
As seen (S)-33t (Gini coefficient: 0.60) and Erlotinib (Gini coef-
ficient: 0.58) have comparable kinase selectivity profile, whereas
compound (R)-5t (Gini coefficient: 0.68) is a somewhat more se-
lective EGFR kinase inhibitor.
Although a low activity was seen towards most of the kinases, a
mediocre inhibition efficiency was observed towards HER2, and the
kinases LYN A and LYN B. Whereas HER2 is directly involved in
EGFR signalling as a dimerization partner, the LYN kinases are
overexpressed in myelodysplastic syndrome and leukaemia [34e
36], and also affect EGFRvIII activity [37]. Thus, their inhibition in
cancerous diseases might be beneficial [36,37]. The inhibition of
some kinases like Aurora kinases, BRAF, PLK’s, PDGER’s, VEGFR’s,
KIT, ABL, JAK2, PTK2 (FAK) and PIM have been indicated as risk
factors for cardiac toxicity [38]. Compounds (R)-5t and (S)-33t
showed a moderate 22% and 20% inhibition of ABL1 and BRAF at
500 nM test concentration, whereas the others of this selection of
kinases showed inhibition of less than 9%.
Fig. 11. Docked structure of (S)-33t in EGFR. Polar interactions and hydrogen bonds
towards Leu718, Met793, Thr854 and Gly796 are highlighted.
and the EGFR mutants L858R, L861Q and T790M. The results are
shown in Table 1.
As the ATP concentration equal to Km is close to 10
testing at 10 M of ATP (Table 1, entry 2) was only useful for veri-
fying the high activity. By increasing the ATP concentration to
100 M the IC50 values were increased, confirming that these are
mM, the
m
m
reversible inhibitors. In addition, very similar high potencies were
also seen towards the EGFR L858R and L861Q mutants (entries 4, 5).
As expected for reversible inhibitors, these compounds were not
active in the nanomolar range towards the EGFR T790M mutants
Table 2
Inhibition of various kinases of compound (S)-33t and (R)-5t (500 nM), mean % of
two measurements with [ATP] ¼ Km.
Kinase
Inhibition (%)
Kinase
Inhibition (%)
Table 1
(S)-33t
(R)-5t
(S)-33t
(R)-5t
Inhibitory potency of (S)-33t, (R)-5t, (S)-33p and Erlotinib towards EGFR at different
ATP concentrations and towards the mutants L858R, L861Q and T790M. Unless
otherwise stated reported IC50 values are the mean of two titration curves (a total of
20 data points).
ABL1
22
5
10
20
7
<1
<1
1
LCK
LYN A
LYN B
MAP2K1
(MEK1)a
MAPK1
6
11
61
57
4
AURKA (Aurora A)
AURKB (Aurora B)
BRAFa
40
56
18
CHEK1 (CHK1)
CFS1R (FMS)
15
3
<1
7
<1
(ERK2)
2
MAPK14
(p38 alpha)
MAPK8 (JNK1)a
MAPKAPK2
MET (cMet)
NEK1
PDGFRA
(PDGFR alpha)
PDGFRB
(PDGFR beta)
PIM2
PLK1
PRKACA (PKA)
PRKCB1
(PKC beta I)
PRKCQ
(PKC theta)
PTK2 (FAK)
RET
13
<1a
CSK
EGFR (ErbB1)
EPHA1
EPHB1
ERBB2 (HER2)
5
100
<1
4
7
98
<1
<1
57
14
8
3
2
<1
15
13
<1
<1
<1
50
ERBB4 (HER4)
63
30
<1
3
FER
FGFR1
FGR
FLT1 (VEGFR1)
8
4
58
<1
10
<1
40
4
1
<1
<1
2
<1
ndb
4
Entry Kinase
ATP conc. Erlotinib (S)-33t
(R)-5t
(S)-33p
<1
1
2
3
4
5
IC50 (nM) EGFR Km app
0.4 ꢁ 0.1 0.3 ꢁ 0.1 0.7 ꢁ 0.2 1.5 ꢁ 1.0
0.3 ꢁ 0.0 0.4 ꢁ 0.0 0.6 ꢁ 0.1 2.6 ꢁ 0.6
1.7 ꢁ 0.1 1.0 ꢁ 0.0 2.0 ꢁ 0.3 10.2 ꢁ 0.1
0.6 ꢁ 0.1 0.3 ꢁ 0.1 1.0 ꢁ 0.1 1.3 ꢁ 0.2
0.5 ꢁ 0.0 0.3 ꢁ 0.0 0.8 ꢁ 0.1 1.0 ꢁ 0.5
(n ¼ 6)a
FLT4 (VEGFR3)
<1
3
<1
<1
IC50 (nM) EGFR 10
(n ¼ 2)
mM
FYN
12
8
<1
7
<1
9
6
5
IC50 (nM) EGFR 100 mM
GSK3B (GSK3 beta)
HCK
IKBKB (IKK beta)
JAK2
KDR (VEGFR2)
KIT
(n ¼ 2)
20
1
9
5
10
7
SRC
9
8
10
8
IC50 (nM) EGFR- Km app
L858R (n ¼ 2)
STK22B (TSSK2)
SYK
TEK (Tie2)
YES1
10
13
37
8
<1
12
IC50 (nM) EGFR- Km app
L861Q (n ¼ 2)
12
<1
12
7
a
a
Reported IC50 values are the mean of six titration curves (a total of 60 data
[ATP] ¼ 100
mM.
b
points).
Not determined.

362
S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
Fig. 13. Effect of (S)-33t, (R)-5t, (R)-5n and Erlotinib on Ba/F3 EGFR-L858R cellular
growth.
marrow-derived cell line which is dependent on the growth factor
interleukin-3 (IL-3), but is rendered IL-3 independent by stable
expression of different tyrosine kinase oncogenes. Upon expression
of activated EGFR mutant proteins the Ba/F3 cells display growth
and survival independent of IL-3, but dependent on the constitutive
EGFR signal. Using a Ba/F3 EGFR cellular system, novel inhibitors can
be tested for potency against EGFR induced cell growth. On target
cellular EGFR activity can thus be confirmed by comparing survival
in Ba/F3 cells expressing other activated kinases.
Fig. 12. Gini plot for comparing kinase selectivity of compounds (R)-5t, (S)-33t and
Erlotinib. The solid line represents a non-selective compound.
The Ba/F3 proliferation study was done using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-assay (MTT).
Compounds (S)-33t and (R)-5t inhibited Ba/F3 EGFR-L858R in a dose
dependent manner in the nanomolar range, see Fig. 13. The Ba/F3
experiments reflected trends from the enzymatic IC50 values. Deriva-
tive (S)-33t (IC50 26 nM) was almost equipotent to Erlotinib (IC50
22 nM), while (R)-5t (IC50 97 nM) was less potent. The ortho hydroxy
analogue (R)-5n, included as a reference for an intermediate potent
EGFR inhibitor, showed considerably less activity. None of the com-
pounds affected proliferation of the Ba/F3 EGFR-T790M cells (data not
shown). Thus, this shows that the tested compounds affect cell pro-
liferation mainly by interfering with the EGFR function.
The IC50 values were used to estimate the relative druglike
properties of the candidate inhibitors [39e43], see Table 3. Ligand
efficacy (LE) and binding efficiency (BEI) are measures of binding
efficiency per heavy atom and molecular weight, respectively. The
LE and BEI numbers should be as high as possible. Surface efficiency
index (SEI) describes how dependent activity is of polar in-
teractions. A SEI value of 18 has been suggested as a guideline value
for good oral bioavailability. Lipophilic ligand efficiency (LLE) de-
scribes how dependent binding is of lipophilicity and values in the
range of 5e7 are preferable. Lipophilicity corrected ligand effi-
ciency (LELP), is derived from calculated log P, and LELP values
below 10 has been suggested as optimal.
Further testing of (S)-33t and Erlotinib was performed with A-
431 cells. These cells express abnormally high levels of EGFR
[45,46], and contains no functional tumour suppressor protein
TP53 [47]. As in the Ba/F3 EGFR-L858R cell system, Erlotinib was
The binding efficiency as measured pr. heavy atom (LE) or by
molecular weight (BEI) of (S)-33t, (R)-5t and (S)-33p are comparable
to that of Erlotinib. The surface efficiency index (SEI) is a measure of
activity per polar surface area. The suboptimal SEI value of (S)-33t
indicate that the compound might be too polar. On the other hand
the hydrophilic/lipophilic balance of (S)-33t as defined by LLE and
LELP indicate favourable properties as compared to Erlotinib.
Compounds (R)-5t and (S)-33p are suggested to be too lipophilic.
To reveal if the new lead compounds (S)-33t and (R)-5t also had
on-target cellular potency towards EGFR mutants, they were
compared with Erlotinib using a Ba/F3 EGFR-L858R and Ba/F3 EGFR-
T790M reporter cells [44]. These systems consist of a murine bone
more potent than (S)-33t, with IC50 values of 0.4 mM and 1.1 mM,
respectively (Table 4). Finally, the compounds were evaluated using
human cancer cell lines with different oncogenic profiles, Table 4.
EGFR has been implicated as important for cell signalling in
cervical cancers [48]. However, C-33A, a cervix carcinoma cell line,
has low EGFR expression [49e52], and no inhibition in cell growth
was previously detected using Cetuximab [52]. C-33A was therefore
regarded as a challenging cell model. Interestingly, Erlotinib and
(S)-33t both inhibited cell viability with IC50 of 0.9 mM and 1.6 mM,
respectively. This could indicate that targets other than EGFR are of
importance in the inhibitory action of both Erlotinib and (S)-33t.
Table 3
Estimated LE, BEI, SEI, LLE and LELP for (R)-5t, (S)-33p (S)-33t and Erlotinib based on
IC50 values (For details of calculation see Supporting information).
Parameter
Ref. value
(S)-33t
(R)-5t
(S)-33p
Erlotinib
Table 4
Cell proliferation study using human cancer cell lines.a
IC50 (nM)
MW (Da)
clog Pa
PSAb
LE
BEI
SEI
LLE
LELP
0.3
407.5
3.5
87.5
0.33
23.4
10.9
6.0
0.7
391.5
4.5
67.3
0.33
23.4
13.6
4.7
1.5
377.5
4.2
67.3
0.33
23.4
13.1
4.7
0.4
393.4
4.0
74.7
0.32
23.9
12.6
5.4
200e500
2e5
Cell line EGFR profile Erlotinib IC50
(m
M) (S)-33t IC50
(
m
M) (R)-5t IC50 (mM)
A-431
C-33A
CAL-27 EGFR
FaDu EGFR
AU-565 Low EGFR
NCI-H82 Low EGFR
High EGFR
Low EGFR
0.4 ꢁ 0.0
0.9 ꢁ 0.0
1.3 ꢁ 0.6
18 ꢁ 6
1.1 ꢁ 0.3
1.6 ꢁ 0.1
2.8 ꢁ 1.4
43 ꢁ 6
Ndb
4.3 ꢁ 0.8
5.2 ꢁ 1.0
Ndb
0.36
27
18
5e7
<10
3.3 ꢁ 0.6
>20
6.3 ꢁ 0.4
10.9 ꢁ 0.2
15 ꢁ 1
10.8
13.8
12.8
12.3
>20
a
a
Calculated log P.
Calculated polar surface area (A ).
The data points are shown in Supporting information.
Not determined.
b
2
ꢀ
b

S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
363
The head and neck cell lines CAL-27 (tongue carcinoma) and
FaDu (hypopharnyx carcinoma) also contain multiple genetic de-
fects [53]. These cells were selected as they are known to possess
EGFR activity [54,55] and also respond to Erlotinib [56]. In our
assay, CAL-27 was considerable more sensitive towards the in-
hibitors than the FaDu cells, and in both cases Erlotinib showed
stronger inhibition of cell proliferation than (S)-33t.
AU-565, a breast adenocarcinoma cell line has overexpressed
HER2, HER3, HER4, and are classified as EGFR negative [57e59].
Although the compounds tested have a decent HER2 and HER4
inhibitory activity in the enzymatic profiling, the cellular IC50
amine ((R)-13), (S)- and (R)-2-amino-2-phenylethan-1-ol ((S)-
14, (R)-14), K2CO3, trifluoroacetophenone, acetophenone, 2-methyl-
1-phenylpropan-1-one, NaBH4, NaBH3CN, NaH, Pd(PPh3)4, Pd(OAc)2,
Pd2(dba)3,
2-dicyclohexylphosphino-20,60-dimethoxybiphenyl
(SPhos) and the arylboronic acids, were from Sigma Aldrich. (2-
Hydroxyphenyl)boronic acid (4n) and (R)-1-(4-fluorophenyl)ethan-
amine ((R)-6) were from Alfa Aesar. (4-Formyl-2-methoxyphenyl)
boronic acid (4s) was from Combi-Blocks. Compound 1 [25], 2-fluoro-
1-phenylethan-1-amine (rac-12) [63], were prepared as described
previously, while 4-chloro-6-phenylthieno[2,3-d]pyrimidine (24a)
[25], (rac)-35 and (rac)-36n [64], were prepared and characterised in
other studies from our laboratory.
values obtained were only mediocre (3.3e15 mM). This indicates
that even higher pan-HER inhibitory profile is needed to effectively
block cell growth in this system.
Silica-gel column chromatography was performed using silica
gel 60A from Fluka, pore size 40e63 mm. Celite 545 from Fluka was
NCI-H82, a small cell lung carcinoma cell line, contains multiple
oncogens [60,61], and also possesses low level of EGFR and HER2
[62,62], however some potency towards Gefitinib has been
observed [62]. Unfortunately, neither of the compounds tested had
any considerable activity towards the NCI-H82 cell line.
Overall, the cellular potency of the developed EGFR inhibitor,
(S)-33t, is generally lower than Erlotinib. Based on the comparable
EGFR enzymatic inhibitory profile, and the Ba/F3 EGFR-L858R cell
model assay, this is most likely due to other cytotoxic effects caused
by Erlotinib, rather than differences in druglike properties.
also used.
4.2. Analyses
1H and 13C NMR spectra were recorded with Bruker Avance 400
spectrometer operating at 400 MHz and 100 MHz, respectively. 19
F
NMR was performed on a Bruker Avance 500 operating at 564 MHz.
For 1H and 13C NMR chemical shifts are in ppm rel. toTMS or DMSO-
d6, while for 19F NMR the shift values are relative to hexa-
fluorobenzene. Compounds isolated as their HCl salts were free-
based prior to NMR spectroscopic analysis. Coupling constants
are in hertz. HPLC (Agilent 110-Series) with a G1379A degasser,
G1311A Quatpump, G1313A ALS autosampler and a G1315D Agilent
detector (254 nm) was used to determine the purity of the syn-
thesised compounds. All compounds evaluated for EGFR inhibitory
potency had a purity of ꢂ95%. Conditions: Method A: Poroshell C18
(100 ꢃ 4.6 mm) column, flow rate 0.8 mL/min, elution starting with
water/CH3CN (90/10), 5 min isocratic elution, then linear gradient
elution for 35 min ending at CH3CN/water (100/0). Method B:
Poroshell C18 (100 ꢃ 4.6 mm) column, flow rate 1.0 mL/min, elution
starting with water þ 0.1% TFA/CH3CN (95/5), linear gradient
elution for 25 min ending at CH3CN/water þ 0.1% TFA (100/0), then
20 min isocratic elution. Method C: Poroshell C18 (100 ꢃ 4.6 mm)
column, flow rate 1.0 mL/min, elution starting with water þ 0.1%
TFA/CH3CN (98/2), linear gradient elution for 25 min ending at
CH3CN/water þ 0.1% TFA (90/10), then 20 min isocratic elution. The
software used with the HPLC was Agilent ChemStation. Chiral HPLC
was conducted on a Lux 5u Cellulose-1 4.6 ꢃ 250 mm chiral column
(part no. 00G-4459-E0). Eluting with hexane (cont. 0.2% diethyl
amine)/i-PrOH, 96/4, flow rate: 2 mL/min, detection at 254 nm. The
retention times were: (R)-5: 5.7 min and (S)-5: 7.8 min, both with
99% ee.
3. Conclusion
A series of 4-N-substituted 6-aryl-thienopyrimidines synthe-
sized using Suzuki coupling as the key step, have been evaluated
and optimized as EGFR-TK inhibitors.
A stepwise development by combination of favourable substi-
tution patterns led to the discovery of three structures with EGFR
IC50 < 1 nM. The most potent drug candidate identified was equi-
potent to Erlotinib in enzymatic inhibition studies. Testing towards
a panel of kinases revealed a selectivity profile similar to Erlotinib
with moderate activity towards HER2, HER4, FGR and LYN A and B
kinases. Experiments using a Ba/F3 EGFR-L858R reporter cell-
system and human cancer cell lines showed that the candidate
compounds had promising cell potency.
The structureeactivity relationships study showed that the po-
tency was dependent on the substitution pattern in the 6-aryl group
(Fragment A). The activity increased when having a hydroxymethyl
substituent in para or meta position or when placing a methoxy
group in the ortho position. Both experimental data and modelling
indicate that these are polar directional interactions. On the other
side a drastic negative effect on potency was seen on introduction of
lipophilic and bulky groups in the para position. For Fragment B,
compounds containing the secondary amine function derived from
(R)-configured 1-phenylethylamine, 1-phenylpropylamine and
benzylamine were all potent inhibitors. Even more active inhibitors
were derived from (S)-configured 2-amino-2-phenylethan-1-ol. In
contrast, using the opposite enantiomers, or when less basic groups
were introduced at the stereocentre, a drastic drop in activity was
seen. The importance of the secondary amine function was further
proved by the low potency of selected model compounds. No evi-
dence suggests that long range electronic effects, originating from
the 6-aryl ring, influence potency.
Accurate mass determination (ESI) was performed on an Agilent
G1969 TOF MS instrument equipped with a dual electrospray ion
source, or EI (70 eV) using a Finnigan MAT 95 XL. Accurate mass
determination in positive and negative mode was performed on a
“Synapt G2-S” Q-TOF instrument from Waters. Samples were
ionized by the use of an ASAP probe, no chromatography separation
was used before the mass analysis. FTIR spectra were recorded on a
Thermo Nicolet Avatar 330 infrared spectrophotometer. All melting
points are uncorrected and measured by a Stuart automatic melting
point SMP40 apparatus.
4.3. Synthesis of primary amines
4. Experimental
4.3.1. (rac)-2-Methyl-1-phenylpropan-1-amine ((rac)-9) [65]
2-Methyl-1-phenylpropan-1-one (3.05 g, 20.6 mmol) was
mixed with ammonium acetate (15.8 g, 206 mmol) in dry MeOH
(50 mL). Sodium cyanoborohydride (1.31 g, 20.6 mmol) was added,
and the reaction was stirred at 50 ꢀC for 18 h. After cooling to rt, the
reaction mixture was concentrated in vacuo and CH2Cl2 (50 mL)
4.1. General
Compounds (S)- and (R)-1-phenylethanamine ((S)-2, (R)-2), ben-
zylamine (7), (R)-1-(phenyl)-1-propanamine ((R)-8), (R)-N-methyl-1-
phenylethan-1-amine ((R)-10), (R)-2-methoxy-1-phenylethan-1-

364
S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
was added. The mixture was washed with water (3 ꢃ 50 mL), and
the water phases were back extracted with CH2Cl2 (2 ꢃ 50 mL). The
combined organic phases were washed with saturated aq NaHCO3
solution (50 mL), dried over anhydrous Na2SO4, filtrated and
concentrated. This gave 3.96 g (18.9 mmol, 92%) of (rac)-9 as a clear
(2 ꢃ 50 mL) or EtOAc (2 ꢃ 50 mL). The combined organic phases
were washed with saturated aq NaCl solution (50 mL), dried over
anhydrous Na2SO4, filtered and concentrated in vacuo, before the
crude oil was dried under reduced pressure to constant weight to
remove excess benzylamine. The compounds were purified by
silica-gel column chromatography or crystallized as specified for
each individual compound.
oil. 1H NMR (300 MHz, CDCl3)
d: 7.33e7.20 (m, 5H), 3.58 (d, J ¼ 7.3,
1H), 1.90e1.79 (m, 1H), 1.57 (br s, 2H), 0.97 (d, J ¼ 6.7, 3H), 0.76 (d,
J ¼ 6.8, 3H). The shifts were ca 0.04 ppm up-field shifted as
compared to that reported [65].
4.4.2. (R)-6-Bromo-N-(1-phenylethyl)thieno[2,3-d]pyrimidin-4-
amine hydrochloride ((R)-3 HCl) [25]
4.3.2. (rac)-2,2,2-Trifluoro-1-phenylethanamine ((rac)-11) [66]
Compound 11 was made in two steps as reported previously by
Scopes et al. [66]. Trifluoroacetophenone (30.0 g, 172 mmol), hy-
drochloric hydroxylamine (23.9 g, 344 mmol) and sodium acetate
(32.5 g, 396 mmol) were dissolved in water (100 mL) and EtOH
(50 mL). The mixture was heated to 75 ꢀC and stirred for 24 h. EtOH
was removed in vacuo and the mixture was left standing at 4 ꢀC for
72 h. The precipitate formed was isolated by filtration, washed with
cold water (3 ꢃ 50 mL) and dried under reduced pressure to give
30.7 g (162 mmol, 94%) of 2,2,2-trifluoro-1-phenylethanone oxime
Compound 1 (5.00 g, 20.04 mmol) was mixed with (R)-1-
phenylethanamine ((R)-2) (7.7 mL, 0.60 mol) and 1-butanol
(16.5 mL) and agitated at 145 ꢀC for 18 h. Then the mixture was
cooled to rt, diluted with water (300 mL) and diethyl ether
(300 mL). After phase separation, the water phase was extracted
with more diethyl ether (2 ꢃ 150 mL). The combined organic phases
were washed with saturated aq NaCl solution (150 mL), dried over
anhydrous Na2SO4, filtered and concentrated in vacuo, before the
crude oil was dried under reduced pressure to constant weight to
remove excess amine. The product was dissolved in diethyl ether
(150 mL) and precipitated upon addition of HCl in diethyl ether
(8 mL). The solid formed was isolated by filtration and washed with
diethyl ether (2 ꢃ 100 mL). Drying gave 6.89 g (18.6 mmol, 94%) of
as white crystals, mp 62e64 ꢀC; 1H NMR (300 MHz, CDCl3)
d: 8.48
(br s, 1H), 7.53e7.44 (m, 5H). 2,2,2-Trifluoro-1-phenylethanone
oxime (30.0 g, 159 mmol) was mixed with Pd/C (10%, 1.13 g) in
THF (200 mL) and acetic acid (11.4 g,10.9 mL,190 mmol). In a closed
reactor the mixture was flushed with hydrogen at 5.5 bar for 23 h,
filtered over celite and washed with CH2Cl2 (200 mL). The solvent
was removed in vacuo and 5 M HCl was added to reach pH ¼ 1. The
mixture was extracted with diethyl ether (3 ꢃ 80 mL). The water
phase was added sodium hydroxide until the solution reached
pH ¼ 14. Extraction with diethyl ether (3 ꢃ 80 mL), removal of the
solvent in vacuo and distillation (50 mbar, 77 ꢀC) gave 20.6 g
(117.3 mmol, 74%) of (rac)-11 as a colourless liquid. 1H NMR
(R)-3$HCl as a white solid, mp 186e195 ꢀC; HPLC purity: 99%
20
(method A), tR ¼ 27.3 min; [
a
]
¼ ꢄ182.0 (c 0.70, DMSO); 1H NMR
D
(400 MHz, DMSO-d6)
d
: 8.27 (s, 1H), 8.23 (d, J ¼ 7.8, 1H), 7.99 (s, 1H),
7.42e7.37 (m, 2H), 7.34e7.28 (m, 2H), 7.24e7.19 (m, 1H), 5.51e5.42
(m, 1H), 1.53 (d, J ¼ 7.0, 3H). The spectroscopic properties corre-
sponds well with that reported previously [25].
4.4.3. (S)-6-Bromo-N-(1-phenylethyl)thieno[2,3-d]pyrimidin-4-
amine ((S)-3)
(300 MHz, CDCl3)
d
: 7.35e7.28 (m, 5H), 4.28 (q, J ¼ 7.4, 1H), 1.68 (br
The synthesis was performed as described in Section 4.4.1
starting with compound 1 (501 mg, 2.01 mmol) and (S)-1-
phenylethanamine ((S)-2) (730 mg, 6.03 mmol). The reaction
time was 19 h. The crude product was purified by silica-gel column
chromatography (EtOAc/n-pentane, 4/1), Rf ¼ 0.69. Isolation and
drying gave 545 mg (1.64 mmol, 81%) of (S)-3 as a white solid, mp
s, 2H). 1H NMR corresponds well with that reported previously at
500 MHz [67].
4.3.3. (S)-2-Methoxy-1-phenylethan-1-amine ((S)-13) [68]
Sodium hydride (700 mg, 29.2 mmol) was dissolved in dry THF
(10 mL) under an N2 atmosphere. Then adding (S)-2-amino-2-
phenylethan-1-ol ((S)-14) (2.21 g, 14.58 mmol) dissolved in dry
THF (20 mL) was added drop wise and stirred for 1 h. The solution
was cooled to 0 ꢀC before 2 M MeI in tert-butyl methyl ether
(7.7 mL, 15.4 mmol) was added drop wise over 30 min using a sy-
ringe pump. The reaction was heated at reflux for 3 h. Then the
mixture was cooled to rt, diluted with water (50 mL) and diethyl
ether (150 mL). After phase separation, the water phase was
extracted with more diethyl ether (2 ꢃ 60 mL). The combined
organic phases were washed with saturated aq NaCl solution
(50 mL), dried over anhydrous Na2SO4, filtered and concentrated.
The crude product was purified by silica-gel column chromatog-
raphy (CH2Cl2/MeOH, 19/1), Rf ¼ 0.59. Isolation and drying gave
1.79 g (11.8 mmol, 81%) of (S)-13 as a colourless oil. 1H NMR
121e124 ꢀC; HPLC purity: 98% (method A), tR ¼ 28.5 min;
20
[a
]
¼ 205.1 (c 0.55, DMSO); 1H NMR (400 MHz, DMSO-d6)
d: 8.26
D
(s, 1H), 8.23 (d, J ¼ 7.7, 1H), 7.99 (s, 1H), 7.41e7.38 (m, 2H), 7.36e7.28
(m, 2H), 7.23e7.19 (m, 1H), 5.51e5.42 (m, 1H), 1.53 (d, J ¼ 7.1, 3H);
79
HRMS (APCI/ASAP, m/z): 334.0009 (calcd. C
[M þ H]þ).
H BrN3S, 334.0014,
14 13
4.4.4. (R)-6-Bromo-N-(1-(4-fluorophenyl)ethyl)thieno[2,3-d]
pyrimidin-4-amine hydrochloride ((R)-15$HCl)
The synthesis was performed as described in Section 4.4.1
starting with compound 1 (1.50 g, 6.00 mmol) and (R)-1-(4-
fluorophenyl)-ethylamine ((R)-6) (2.66 g, 19.2 mmol). The reac-
tion time was 24 h. The product was purified by silica-gel column
chromatography (EtOAc), Rf ¼ 0.67. The product was precipitated by
dissolving in diethyl ether (30 mL), and then addition of HCl in
diethyl ether (0.5 mL) followed by crystallization at 4 ꢀC for 2 h. The
solid was isolated by filtration. This gave 2.06 g (5.30 mmol, 89%) of
(300 MHz, CDCl3)
d
: 7.41e7.25 (m, 5H), 4.21 (ddAB, J ¼ 8.8, 3.8, 1H),
3.51 (ddAB, J ¼ 8.7, 3.9, 1H), 3.32e3.39 (m, 4H), 1.74 (s, 2H). The 1H
NMR spectroscopic shifts correspond to that reported [68].
(R)-15$HCl as a white solid, mp 127e129 ꢀC; HPLC purity: 99%
20
4.4. Synthesis of 6-bromo-thieno[2,3-d]pyrimidin-4-amines
(method C), tR ¼ 18.2 min; [
a
]
¼ ꢄ200.3 (c 1.01, DMSO); 1H NMR
D
(400 MHz, DMSO-d6)
d
: 8.27 (s, 1H), 8.23 (d, J ¼ 7.9, 1H), 7.97 (s, 1H),
4.4.1. General procedure for thermal amination of 4-chloro-thieno
[2,3-d]pyrimidins
7.46e7.40 (m, 2H), 7.17e7.10 (m, 2H), 5.50e5.40 (m, 1H), 1.52 (d,
J ¼ 7.1, 3H); 13C NMR (100 MHz, DMSO-d6)
d: 166.5, 162.2 (d,
Compound 1 (1.00 g, 4.01 mmol) was mixed with the benzyl-
amine (12.03 mmol) and 1-butanol (3.5 mL) and agitated at 145 ꢀC
for 18e24 h. Then the mixture was cooled to rt, diluted with water
(50 mL) and diethyl ether (150 mL) or EtOAc (150 mL). After phase
separation, the water phase was extracted with more diethyl ether
J ¼ 240.7), 154.7, 154.1, 140.6 (d, J ¼ 3.0), 128.0 (d, J ¼ 8.0, 2C), 122.8,
116.8, 115.1 (d, J ¼ 21.1, 2C), 109.7, 48.5, 22.5; 19F NMR (564 MHz,
DMSO-d6)
d
: ꢄ118.6 (s); IR (neat, cmꢄ1): 3222, 3026, 2358, 1600,
1501, 790, 555; HRMS (EI, 70 eV, m/z): 350.9835 (calcd.
C
14H11N739BrFS, 350.9836, [M]þ).

S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
365
4.4.5. N-Benzyl-6-bromothieno[2,3-d]pyrimidin-4-amine (16)
4.4.9. (rac)-6-Bromo-N-(2,2,2-trifluoro-1-phenylethyl)thieno[2,3-
d]pyrimidin-4-amine ((rac)-20)
The synthesis was performed as described in Section 4.4.1
starting with (1.72 g, 6.91 mmol) and 2,2,2-trifluoro-1-
The synthesis was performed as described in Section 4.4.1
starting with compound 1 (1.00 g, 4.01 mmol) and benzylamine
(7) (1.30 mL,1.28 g, 11.9 mmol). The reaction time was 19 h. Workup
and drying gave 1.19 g (3.33 mmol, 83%) of 16 as a light yellow solid,
mp 188e190 ꢀC; HPLC purity: 99% (method B), tR ¼ 15.1 min; 1H
1
phenylethanamine ((rac)-11) (3.63 g, 20.7 mmol) and reacting for
20 h. After drying over anhydrous Na2SO4 the concentrated black
oil was dried under reduced pressure for 48 h. The obtained ma-
terial was purified by silica-gel column chromatography (n-
pentane/diethyl ether, 4/1), Rf ¼ 0.21. Isolation and drying gave
1.60 g (4.12 mmol, 60%) of (rac)-20 as a white solid, mp 204 ꢀC
(dec.); HPLC purity 99% (method A), tR ¼ 29.5 min; 1H NMR
NMR (400 MHz, DMSO-d6)
d
: 8.51 (t, J ¼ 5.9,1H), 8.33 (s,1H), 7.85 (s,
1H), 7.37e7.29 (m, 4H), 7.27e7.21 (m, 1H), 4.73 (d, J ¼ 5.9, 2H); 13
C
NMR (100 MHz, DMSO-d6) d: 166.5, 155.6, 154.2, 139.1, 128.4 (2C),
127.3 (2C), 126.9, 122.6, 116.9, 109.7, 43.3; IR (neat, cmꢄ1): 3235,
3024, 2922, 1594, 1448, 1309, 1084, 848, 774, 746, 694; HRMS (ESI,
79
m/z): 319.9851 (calcd. C
H
BrN3S, 319.9852, [M þ H]þ).
(400 MHz, CDCl3) d: 8.49 (s, 1H), 7.48 (m, 2H), 7.43e7.38 (m, 3H),
13 11
7.28 (s, 1H), 6.38e6.32 (m, 1H), 5.72 (d, J ¼ 9.2, 1H); 13C NMR
(100 MHz, CDCl3) d: 168.1, 154.2,153.6,133.0,129.4,129.1 (2C),127.9
4.4.6. (R)-6-Bromo-N-(1-phenylpropyl)thieno[2,3-d]pyrimidin-4-
amine ((R)-17)
(2C), 124.7 (q, J ¼ 211.3), 119.3, 117.3, 113.8, 54.9 (q, J ¼ 31.3); 19F
NMR (564 MHz, DMSO-d6)
d
: ꢄ74.1 (s); IR (neat, cmꢄ1): 2996, 2359,
The synthesis was performed as described in Section 4.4.1
starting with compound 1 (200 mg, 0.80 mmol) and (R)-1-
phenylpropan-1-amine ((R)-8) (329 mg, 2.43 mmol). The reaction
time was 20 h. The crude product was absorbed onto silica and
purified by silica-gel column chromatography (n-pentane/EtOAc, 4/
1), Rf ¼ 0.35. Drying gave 205 mg (0.53 mmol, 67%) of (R)-17 as a
2341, 2159, 2029, 564; HRMS (EI, 70 eV, m/z): 387.9487, (calcd.
79
C
14
H
BrF3N3S, 387.9487, [M]þ).
9
4.4.10. (rac)-6-Bromo-N-(2-fluoro-1-phenylethyl)thieno[2,3-d]
pyrimidin-4-amine ((rac)-21)
Compound 1 (182 mg, 0.73 mmol) was mixed with 2-fluoro-1-
phenylethan-1-amine ((rac)-12) (304 mg, 2.19 mmol) and 1-
butanol (3 mL), and agitated at 50 ꢀC for 72 h. Then the mixture
was cooled to 25 ꢀC, diluted with diethyl ether (20 mL) and washed
with water (3 ꢃ 20 mL) and saturated aq NaCl solution (10 mL).
After drying over Na2SO4 and concentration in vacuo, the obtained
material was purified by silica-gel column chromatography (n-
white solid, mp 142e147 ꢀC; HPLC purity: 99% (method B),
20
tR ¼ 18.2 min; [
a
]
¼ ꢄ166.8 (c 0.98, DMSO); 1H NMR (400 MHz,
D
DMSO-d6)
d
: 8.26 (s, 1H), 8.17 (d, J ¼ 8.2, 1H), 8.02 (s, 1H), 7.42e7.37
(m, 2H), 7.33e7.24 (m, 2H), 7.23e7.17 (m, 1H), 5.27e5.18 (m, 1H),
1.96e1.78 (m, 2H), 0.92 (t, J ¼ 7.3, 3H); 13C NMR (100 MHz, DMSO-
d6) d: 166.5, 155.3, 154.1, 143.6, 128.3 (2C), 126.8, 126.5 (2C), 122.8,
116.8, 109.6, 55.5, 29.3, 11.4; IR (neat, cmꢄ1): 3048, 2963, 2872,
pentane/diethyl ether, 4/1), Rf
¼
0.24. This gave 205 mg
1599, 1474, 1363, 844, 756, 697; HRMS (ESI, m/z): 348.0165 (calcd.
79
(0.584 mmol, 80%) of (rac)-21 as white solid, mp 135e137 ꢀC (dec.);
HPLC purity 96% (method A), tR ¼ 25.8 min; 1H NMR (400 MHz,
C
H
BrN3S, 348.0165, [M þ H]þ).
15 15
DMSO-d6) d: 8.49e8.47 (m, 1H), 8.31 (s, 1H), 8.04 (s, 1H), 7.49e7.47
4.4.7. (rac)-6-Bromo-N-(2-methyl-1-phenylpropyl)thieno[2,3-d]
pyrimidin-4-amine ((rac)-18)
(m, 2H), 7.38e7.34 (m, 2H), 7.30e7.27 (m, 1H), 5.81e5.72 (m, 1H),
4.81e4.75 (m, 1H), 4.69e4.64 (m, 1H); 13C NMR (100 MHz, DMSO-
The synthesis was performed as described in Section 4.4.1
starting with compound 1 (538 mg, 2.16 mmol) and 2-methyl-1-
phenylpropan-1-amine ((rac)-9) (965 mg, 6.47 mmol). The reac-
tion time was 18 h. The crude product was purified by silica-gel
column chromatography (n-pentane/EtOAc, 1/1), Rf ¼ 0.70. Drying
gave 677 mg (1.81 mmol, 84%) of (rac)-18 as an off white solid, mp
158e161 ꢀC; HPLC purity: 97% (method A), tR ¼ 29.9 min; 1H NMR
d6)
d
: 167.2, 155.7, 154.4, 138.5 (d, J ¼ 6.2), 128.9 (2C), 128.1, 127.6
(2C),123.2,117.4, 110.5, 84.7 (d, J ¼ 174.7), 54.2 (d, J ¼ 19.9); 19F NMR
(564 MHz, DMSO-d6)
3009, 1585, 1540, 1445, 1017, 693; HRMS (EI, 70 eV, m/z): 350.9831,
(calcd. C
d
: ꢄ220.1 (s); IR (neat, cmꢄ1): 3216, 3073,
79
H
BrFN3S, 350.9831, [M]þ).
14 11
4.4.11. (S)-6-Bromo-N-(2-methoxy-1-phenylethyl)thieno[2,3-d]
pyrimidin-4-amine ((S)-22)
(400 MHz, DMSO-d6) d: 8.27 (s, 1H), 8.11e8.09 (m, 1H), 8.05 (s, 1H),
7.43e7.41 (m, 2H), 7.33e7.29 (m, 2H), 7.22e7.19 (m, 1H), 5.06e5.02
(m, 1H), 2.22e2.10 (m, 1H), 1.03 (d, J ¼ 6.6, 3H), 0.75 (d, J ¼ 6.7, 3H);
The synthesis was performed as described in Section 4.4.1
starting with compound 1 (213 mg, 0.854 mmol) and (S)-2-
methoxy-1-phenylethanamine ((S)-13) (387 mg, 2.56 mmol). The
reaction time was 22 h. The crude product was purified by silica-gel
column chromatography (n-pentane/EtOAc, 1/1), Rf ¼ 0.63. Isola-
tion and drying gave 238 mg (0.653 mmol, 77%) of (S)-22 as an off-
13C NMR (100 MHz, DMSO-d6)
d: 166.9, 155.7, 154.5, 143.1, 128.6
(2C), 127.8 (2C), 127.2, 123.3, 117.2, 109.9, 60.7, 33.3, 20.4, 20.3; IR
(neat, cmꢄ1): 3230, 2958, 1581, 1514, 699, 579; HRMS (APCI/ASAP,
79
m/z): 362.0327 (calcd. C
H
BrN3S, 362.0327, [M þ H]þ).
16 17
white solid, mp 158e160 ꢀC; HPLC purity: 99% (method A),
¼ ꢄ246.9 (c 0.79, DMSO); 1H NMR (400 MHz,
20
4.4.8. (R)-6-Bromo-N-methyl-N-(1-phenylethyl)thieno[2,3-d]
pyrimidin-4-amine ((R)-19)
tR ¼ 25.0 min; [
a
]
D
DMSO-d6)
d
: 8.31 (br s, 1H), 8.28 (s, 1H), 8.05 (s, 1H), 7.44e7.42 (m,
The synthesis was performed as described in Section 4.4.1
starting with compound 1 (522 mg, 2.09 mmol) and (R)-N-
methyl-1-phenylethanamine ((R)-10) (849 mg, 6.28 mmol). The
reaction time was 18 h. The crude product was purified by silica-gel
column chromatography (EtOAc/n-pentane, 4/1), Rf ¼ 0.57. Isola-
tion and drying gave 660 mg (1.90 mmol, 91%) of (R)-19 as white
2H), 7.35e7.31 (m, 2H), 7.26e7.23 (m, 1H), 5.64e5.59 (m, 1H), 3.72
(ddAB, J ¼ 10.1, 8.8, 1H), 3.62 (ddAB, J ¼ 10.1, 5.1, 1H), 3.30 (s, 3H); 13
C
NMR (100 MHz, DMSO-d6) d: 167.0, 155.7, 154.5, 140.7, 128.8 (2C),
127.6, 127.4 (2C), 123.3, 117.3, 110.2, 75.1, 58.5, 53.7; IR (neat, cmꢄ1):
3363, 2888, 1575, 1536, 1494, 1096, 702; HRMS (APCI/ASAP, m/z):
79
364.0119 (calcd. C
H
BrN3OS, 364.0119, [M þ H]þ).
15 15
solid, mp 249 ꢀC (dec.); HPLC purity: 99% (method A), tR ¼ 30.7 min;
20
[
a]
¼ ꢄ23.4 (c 1.04, DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 8.37
4.4.12. (R)-6-Bromo-N-(2-methoxy-1-phenylethyl)thieno[2,3-d]
pyrimidin-4-amine ((R)-22)
D
(s, 1H), 7.78 (s, 1H), 7.38e7.27 (m, 5H), 6.40e6.35 (m, 1H), 3.03 (s,
3H), 1.59 (d, J ¼ 7.0, 3H); 13C NMR (100 MHz, DMSO-d6)
d
: 170.1,
The synthesis was performed as described in Section 4.4.11
starting with compound 1 (535 mg, 2.15 mmol) and (R)-2-
methoxy-1-phenylethanamine ((R)-13) (973 mg, 6.44 mmol). The
reaction time was 21 h. This gave 696 mg (1.85 mmol, 85%) of (R)-22
as an off-white solid, mp 157e160 ꢀC; HPLC purity: 98% (method A),
157.0, 153.4, 141.1, 129.1 (2C), 127.6, 127.4 (2C), 125.9, 116.5, 108.8,
53.7, 33.5, 16.5; IR (neat, cmꢄ1): 2977, 1590, 1566, 1390, 760, 694;
79
HRMS (APCI/ASAP, m/z): 348.0170 (calcd. C
H BrN3S, 348.0170,
15 15
[M þ H]þ).

366
S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
20
tR ¼ 24.9 min; [
a
]
D
¼ 199.4 (c 0.68, DMSO); 1H NMR (400 MHz,
4.6.1. General procedure for Suzuki coupling on 6-bromothieno[2,3-
d]pyrimidines
DMSO-d6)
d
: 8.30 (br s, 1H), 8.28 (s, 1H), 8.04 (s, 1H), 7.44e7.42 (m,
2H), 7.35e7.31 (m, 2H), 7.26e7.23 (m,1H), 5.64e5.58 (m,1H), 3.75e
Compound (R)-3$HCl (201 mg, 0.54 mmol) was mixed with the
selected boronic acid (4) (0.81 mmol), fine powdered K2CO3
(298 mg, 2.16 mmol), Pd(PPh3)4 (6 mg, 0.005 mmol), 1,4-dioxane
(2 mL) and water (2 mL). The reaction was then stirred at 80 ꢀC
for 2e3 h under N2 atmosphere. The solvent was removed and the
product was diluted with water (20 mL) and extracted with diethyl
ether or EtOAc (25 mL), the water phase was extracted with more
diethyl ether or EtOAc (3 ꢃ 10 mL). The combined organic phases
were washed with saturated aq NaCl solution (10 mL), dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. Purification
was performed as specified for each individual compound.
3.62 (m, 2H), 3.30 (s, 3H); HRMS (APCI/ASAP, m/z): 364.0120 (calcd.
79
C
H
BrN3OS, 364.0119, [M þ H]þ). The spectroscopic properties
15 15
confirmed with that reported for (S)-22.
4.4.13. (R)-2-((6-Bromothieno[2,3-d]pyrimidin-4-yl)amino)-2-
phenylethanol ((R)-23)
The synthesis was performed as described in Section 4.4.1
starting with compound 1 (300 mg, 1.20 mmol) and (R)-2-amino-
2-phenylethan-1-ol ((R)-14) (495 mg, 3.61 mmol). The reaction
time was 24 h. The crude product was purified by silica-gel column
chromatography (EtOAc), Rf ¼ 0.71. Isolation and drying gave
346 mg (0.988 mmol, 82%) of (R)-23 as a white solid, mp 171e
4.6.2. (S)-6-Phenyl-N-(1-phenylethyl)thieno[2,3-d]pyrimidin-4-
amine ((S)-5a)
20
173 ꢀC; HPLC purity: 96% (method A), tR ¼ 20.6 min; [
a
]
D
¼ 196.4 (c
0.59, DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 8.26 (s, 1H), 8.20e
4-Chloro-6-phenylthieno[2,3-d]pyrimidine (24a) [25] (30 mg,
0.122 mmol) and (S)-phenylethanamine ((S)-2) (45 mg,
0.371 mmol) were added to 1-butanol (1 mL) under N2 atmosphere
and heated at 145 ꢀC for 24 h. After cooling to rt, 1-butanol was
evaporated in vacuo, and water (10 mL) was added. The mixture
was extracted with EtOAc (3 ꢃ 10 mL). The combined organic
phases were washed with saturated aq NaCl solution (10 mL) and
dried over anhydrous Na2SO4, filtrated and evaporated. The ob-
tained material was purified by silica-gel column chromatography
(n-pentane/EtOAc, 1/1), Rf ¼ 0.76. This gave 32 mg (0.097 mmol,
79%) of (S)-5a as a white solid, mp 171e173 ꢀC; HPLC purity: 99%
8.18 (m, 1H), 8.04 (s, 1H), 7.42e7.40 (m, 2H), 7.33e7.29 (m, 2H),
7.24e7.21 (m, 1H), 5.44e5.38 (m, 1H), 5.03e5.00 (m, 1H), 3.78e3.68
(m, 2H); 13C NMR (100 MHz, DMSO-d6)
d: 166.9, 155.9, 154.5, 141.3,
128.7 (2C), 127.4 (3C), 123.4, 117.4, 109.9, 65.1, 56.9; IR (neat, cmꢄ1):
3421, 3042, 1602, 1477, 1357, 698; HRMS (APCI/ASAP, m/z):
79
349.9966 (calcd. C
H
BrN3OS, 349.9963, [M þ H]þ).
14 13
4.4.14. (S)-2-((6-Bromothieno[2,3-d]pyrimidin-4-yl)amino)-2-
phenylethanol ((S)-23)
The synthesis was performed as described in Section 4.4.13
starting with compound 1 (300 mg, 1.20 mmol) and (S)-2-amino-2-
phenylethan-1-ol ((S)-14) (495 mg, 3.61 mmol). The reaction time
was 24 h. The crude product was purified by silica-gel column chro-
matography (n-pentane/EtOAc, 2/8), Rf ¼ 0.58. This gave 360 mg
(method A), tR ¼ 32.1 min, ee: 99% tR ¼ 7.8 min (Lux 5u Cellulose-1);
20
[a
]
¼ 272.1 (c 0.72, DMSO); 1H NMR (400 MHz, DMSO-d6)
d: 8.28
D
(s,1H), 8.24 (d, J ¼ 7.9, 1H), 8.21 (s,1H), 7.71e7.69 (m, 2H), 7.54e7.48
(m, 2H), 7.46e7.37 (m, 3H), 7.35e7.30 (m, 2H), 7.25e7.20 (m, 1H),
5.56e5.47 (m, 1H), 1.56 (d, J ¼ 7.1, 3H). HRMS (APCI/ASAP, m/z):
331.1142 (calcd. C20H18N3S, 331.1143, [M þ H]þ). Other spectro-
scopic properties were identical to that of the (R)-5a reported
previously [25].
(1.03 mmol, 86%) of (S)-23 as a white solid, mp 169e172 ꢀC; HPLC
20
purity: 98% (method A), tR ¼ 20.6 min; [
a
]
¼ ꢄ210.4 (c 0.73, DMSO);
D
1H NMR (400 MHz, DMSO-d6)
d
: 8.26 (s, 1H), 8.20e8.18 (m, 1H), 8.04
(s,1H), 7.42e7.40 (m, 2H), 7.33e7.29 (m, 2H), 7.24e7.21 (m,1H), 5.44e
5.38 (m, 1H), 5.03e5.00 (m, 1H), 3.78e3.68 (m, 2H); HRMS (APCI/
79
ASAP, m/z): 349.9966 (calcd. C
H
14 13
BrN3OS, 349.9963, [M þ H]þ). The
4.6.3. (R)-6-(4-Fluorophenyl)-N-(1-phenylethyl)thieno[2,3-d]
pyrimidin-4-amine hydrochloride ((R)-5c$HCl)
spectroscopic properties confirmed with that reported for (R)-23.
Compound 24c (99 mg, 0.374 mmol) was mixed with (R)-1-
phenylethanamine ((R)-2) (0.15 mL, 143 mg, 1.18 mmol) and 1-
butanol (2 mL), and agitated at 145 ꢀC for 21 h. Then the mixture
was cooled to rt, diluted with EtOAc (40 mL) and washed with
water (3 ꢃ 15 mL). The combined organic phases was dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The crude
product was absorbed onto silica and purified with silica-gel col-
umn chromatography (n-pentane/EtOAc, 3/1), Rf ¼ 0.20. The
product was precipitated from diethyl ether (50 mL)/HCl in diethyl
ether (1.5 mL) at ꢄ18 ꢀC for 24 h. The solid formed was isolated by
filtration and washed with diethyl ether (2 ꢃ 25 mL). Drying gave
4.5. 4-Chloro-6-(4-fluorophenyl)thieno[2,3-d]pyrimidine (24c) [69]
Compound
1 (1.00 g, 4.02 mmol) was mixed with (4-
fluorophenyl)boronic acid (4c) (844 mg, 6.03 mmol), fine
powdered K2CO3 (1.68 g,12.1 mmol), Pd2(dba)3 (186 mg, 0.20 mmol)
and 1,4-dioxane (20 mL). The reaction was then stirred at 110 ꢀC for
26 h under N2 atmosphere. The solvent was removed and the
product was dissolved in diethyl ether (150 mL) and washed with
water (3 ꢃ 150 mL). The organic phase was dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The crude product was
absorbed onto silica and purified by silica-gel column chromatog-
raphy (n-pentane/acetone, 19/1), Rf ¼ 0.28. Drying gave 415 mg
(1.57 mmol, 39%) of 24c as a pale white solid, mp 142e143 ꢀC; HPLC
purity: 91% (method B), tR ¼ 23.5 min; 1H NMR (400 MHz, DMSO-d6)
57 mg (0.148 mmol, 40%) of (R)-5c$HCl as a white solid, mp 182e
20
187 ꢀC; HPLC purity: 99% (method B), tR ¼ 18.6 min; [
a
]
¼ ꢄ280.7
D
(c 0.97, DMSO); 1H NMR (400 MHz, DMSO-d6)
d: 8.28 (s, 1H), 8.25
(d, J ¼ 7.9, 1H), 8.15 (s, 1H), 7.80e7.68 (m, 2H), 7.47e7.40 (m, 2H),
d
: 8.93 (s, 1H), 8.04e7.98 (m, 3H), 7.43e7.36 (m, 2H); 13C NMR
7.40e7.28 (m, 4H), 7.27e7.19 (m, 1H), 5.56e5.47 (m, 1H), 1.56 (d,
(100 MHz, DMSO-d6)
d
: 167.6, 163.0 (d, J ¼ 248.6), 153.3, 152.8, 144.1,
J ¼ 7.0, 3H); 13C NMR (100 MHz, DMSO-d6)
d: 165.2, 162.1 (d,
130.8, 129.2 (d, J ¼ 8.7, 2C), 128.5 (d, J ¼ 3.1), 116.5 (d, J ¼ 22.0, 2C),
J ¼ 246.1), 155.8, 153.9, 144.6, 136.9, 129.9 (d, J ¼ 3.0), 128.3 (2C),
127.8 (d, J ¼ 8.5, 2C), 126.7, 126.1 (2C), 117.4, 116.4 (d, J ¼ 22.0, 2C),
115.1; 19F NMR (564 MHz, DMSO-d6)
d
: ꢄ115.3 (s); IR (neat, cmꢄ1):
3063, 1884, 1505, 1429, 1228, 1165, 813, 759, 670; HRMS (ESI, m/z):
115.7, 49.1, 22.5; 19F NMR (564 MHz, DMSO-d6)
d: ꢄ115.4 (s); IR
35
264.9995 (calcd. C
H
12
ClFN2S, 264.9997, [M þ H]þ).
(neat, cmꢄ1): 3059, 2978, 1604, 1491, 1233, 832, 766, 699; HRMS
7
(ESI, m/z): 350.1122 (calcd. C20H17FN3S, 350.1122, [M þ H]þ).
4.6. Synthesis of potential EGFR inhibitors
4.6.4. (R)-(4-(4-((1-Phenylethyl)amino)thieno[2,3-d]pyrimidin-6-
yl)phenyl)methanol ((R)-5e)
Compound (R)-3$HCl (201 mg, 0.542 mmol) was mixed with the
MIDA boronate ester of 4-(hydroxymethyl)phenylboronic acid (4e)
The synthesis and spectral properties of the compounds (R)-5a,
(R)-5b, (R)-5d, (R)-5h, (R)-5j, (R)-5n, (R)-5u and (R)-5v have been
described earlier [25].

S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
367
(215 mg, 0.817 mmol), SPhos (22 mg, 0.054 mmol), Pd(OAc)2
(8.5 mg, 0.038 mmol) and 1,4-dioxane (5 mL). The mixture was
stirred for 10 min, 3 M K3PO4 solution (1.5 mL) was added and the
temperature increased to 80 ꢀC for 3 h under an N2 atmosphere. The
reaction mixture was cooled to rt and added 1 M NaOH solution
(5 mL) and diethyl ether (25 mL). The water phase was extracted
with diethyl ether (2 ꢃ 10 mL). The combined organic phases were
dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The crude product was absorbed onto silica and purified with silica-
gel column chromatography (n-pentane/EtOAc, 11/9), Rf ¼ 0.17.
Drying gave 95 mg (0.262 mmol, 48%) of (R)-5e as a white solid, mp
was absorbed onto silica and purified by silica-gel column chro-
matography (n-pentane/EtOAc, 3/2), Rf ¼ 0.28. The purified product
was recrystallized from acetonitrile. Filtration and drying gave
15 mg (0.042 mmol, 30%) of (R)-5i as a yellow solid, mp 210e218 ꢀC;
20
HPLC purity: 99% (method A), tR ¼ 28.0 min; [
a
]
¼ ꢄ506.2 (c 0.52,
D
DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 10.04 (s, 1H), 8.42 (s, 1H),
8.32 (s, 1H), 8.36 (d, J ¼ 7.9, 1H), 8.05e8.03 (m, 2H), 7.92e7.90 (m,
2H), 7.45e7.43 (m, 2H), 7.35e7.31 (m, 2H), 7.25e7.21 (m, 1H), 5.56e
5.49 (m, 1H), 1.58 (d, J ¼ 7.0, 3H); 13C NMR (100 MHz, DMSO-d6)
d:
192.3, 165.9, 156.0, 154.5, 144.4, 138.7, 136.4, 135.5, 130.6 (2C), 128.3
(2C), 126.8, 126.1 (2C), 126.0 (2C), 118.2, 117.4, 49.1, 22.5; IR (neat,
cmꢄ1): 3369, 2973, 1683, 1575, 1491, 1116, 816, 775, 698, 553; HRMS
(APCI/ASAP, m/z): 360.1169 (calcd. C21H18N3OS, 360.1171, [M þ H]þ).
215e217 ꢀC; HPLC purity: 99% (method B), tR ¼ 21.9 min;
20
[
a]
¼ ꢄ255.2 (c 0.97, DMSO); 1H NMR (400 MHz, DMSO-d6)
d:
D
8.28 (s, 1H), 8.22 (d, J ¼ 7.9, 1H), 8.18 (s, 1H), 7.68e7.64 (m, 2H),
7.47e7.41 (m, 4H), 7.36e7.30 (m, 2H), 7.25e7.19 (m, 1H), 5.56e5.47
(m, 1H), 5.29 (t, J ¼ 5.8, 1H), 4.55 (d, J ¼ 5.7, 2H), 1.57 (d, J ¼ 7.1, 3H);
4.6.8. (R)-6-(3-Methoxyphenyl)-N-(1-phenylethyl)thieno[2,3-d]
pyrimidin-4-amine ((R)-5k)
13C NMR (100 MHz, DMSO-d6)
d
: 165.0, 155.7, 153.8, 144.6, 143.2,
Compound (R)-5k was prepared as described in Section 4.6.4,
but starting with the MIDA boronate ester of (3-methoxyphenyl)
boronic acid (4k) (287 mg, 1.09 mmol). The reaction time was 3 h.
The crude product was absorbed onto silica and purified by silica-
gel column chromatography (n-pentane/EtOAc, 7/3), Rf ¼ 0.26.
Drying gave 114 mg (0.315 mmol, 58%) of (R)-5k as a pale yellow
138.2,131.6,128.3 (2C),127.3 (2C),126.7,126.1 (2C),125.4 (2C),117.5,
115.1, 62.5, 49.0, 22.5; IR (neat, cmꢄ1): 3246, 3142, 2860, 1578, 1493,
1313, 1102, 777, 694; HRMS (EI, 70 eV, m/z): 361.1242 (calcd.
C
21H19N3OS, 361.1243, [M]þ).
4.6.5. (R)-N-(1-Phenylethyl)-6-(p-tolyl)thieno[2,3-d]pyrimidin-4-
amine ((R)-5f)
solid, mp 135e137 ꢀC; HPLC purity: 99% (method B), tR ¼ 26.9 min;
20
[a
]
¼ ꢄ336.4 (c 1.01, DMSO); 1H NMR (400 MHz, DMSO-d6)
d: 8.29
D
Compound (R)-5f was prepared as described in Section 4.6.1, but
starting with p-tolylboronic acid (4f) (110 mg, 0.816 mmol). The
reaction time was 2 h. The crude product was purified by silica-gel
column chromatography (n-pentane/EtOAc, 3/1), Rf ¼ 0.29. Drying
gave 162 mg (0.467 mmol, 86%) of (R)-5f as a pale yellow solid, mp
(s,1H), 8.22 (d, J ¼ 7.9,1H), 8.19 (s,1H), 7.46e7.39 (m, 3H), 7.36e7.30
(m, 2H), 7.29e7.20 (m, 3H), 7.01e6.97 (m, 1H), 5.57e5.48 (m, 1H),
3.85 (s, 3H), 1.57 (d, J ¼ 7.1, 3H); 13C NMR (100 MHz, DMSO-d6)
d:
165.1, 159.9, 155.8, 154.0, 144.6, 137.9, 134.6, 130.5, 128.3 (2C), 126.7,
126.1 (2C), 118.2, 117.3, 115.8, 114.2, 110.9, 55.3, 49.0, 22.5; IR (neat,
cmꢄ1): 3230, 1577, 1514, 1491, 1289, 780, 699; HRMS (EI, 70 eV, m/
z): 361.1241 (calcd. C21H19N3OS, 361.1243, [M]þ).
78e81 ꢀC; HPLC purity: 99% (method B), tR
¼
27.7 min;
20
[
a]
¼ ꢄ413.1 (c 1.02, DMSO); 1H NMR (400 MHz, DMSO-d6)
d: 8.27
D
(s, 1H), 8.20 (d, J ¼ 7.9, 1H), 8.14 (s, 1H), 7.61e7.57 (m, 2H), 7.45e7.41
(m, 2H), 7.35e7.29 (m, 4H), 7.25e7.19 (m, 1H), 5.56e5.47 (m, 1H),
4.6.9. (R)-(3-(4-((1-Phenylethyl)amino)thieno[2,3-d]pyrimidin-6-
yl)phenyl)methanol ((R)-5l)
2.35 (s, 3H), 1.57 (s, J ¼ 7.0, 3H); 13C NMR (100 MHz, DMSO-d6)
d:
164.9, 155.7, 153.7, 144.6, 138.3, 138.2, 130.5, 129.9 (2C), 128.3 (2C),
126.7, 126.1 (2C), 125.5 (2C), 117.5, 114.8, 49.0, 22.5, 20.8; IR (neat,
cmꢄ1): 3259, 2973, 1576, 1493, 1351, 810, 775, 697; HRMS (EI, 70 eV,
m/z): 345.1292 (calcd. C21H19N3S, 345.1294, [M]þ).
Compound (R)-5l was prepared as described in Section 4.6.1,
but starting with (3-(hydroxymethyl)phenyl)boronic acid (4l)
(99 mg, 0.646 mmol). The reaction time was 3 h. The crude product
was absorbed onto silica and purified with silica-gel column
chromatography (n-pentane/EtOAc, 7/3), Rf ¼ 0.67 (EtOAc). The
resulting product was dissolved in diethyl ether (2 mL), and slowly
added to n-pentane (30 mL) to give precipitation. Isolation and
drying gave 140 mg (0.387 mmol, 90%) of (R)-5l as a white solid,
4.6.6. (R)-6-(4-(tert-Butyl)phenyl)-N-(1-phenylethyl)thieno[2,3-d]
pyrimidin-4-amine ((R)-5g)
Compound (R)-5g was prepared as described in Section 4.6.1,
but starting with (4-(tert-butyl)phenyl)boronic acid (4g) (123 mg,
0.691 mmol). The reaction time was 3 h at 60 ꢀC. The crude product
was absorbed onto silica and purified by silica-gel column chro-
matography (n-pentane/EtOAc, 3/1), Rf ¼ 0.24. The purified product
was dissolved in diethyl ether (100 mL), n-pentane was added to
the mixture until the product precipitated. Filtration and drying
mp 173e176 ꢀC; HPLC purity: 98% (method A), tR ¼ 27.0 min;
20
[a
]
¼ ꢄ302.7 (c 0.83, DMSO); 1H NMR (400 MHz, DMSO-d6)
d:
D
8.29e8.27 (m, 2H), 8.23 (s, 1H), 7.72 (s, 1H), 7.56e7.54 (m, 1H),
7.47e7.43 (m, 3H), 7.35e7.31 (m, 3H), 7.24e7.21 (m, 1H), 5.55e5.48
(m, 1H), 5.37e5.34 (m, 1H), 4.60e4.58 (m, 2H), 1.57 (d, J ¼ 7.0, 3H);
13C NMR (100 MHz, DMSO-d6)
d: 165.5, 155.7, 153.9, 145.1, 144.3,
gave 145 mg (0.374 mmol, 69%) of (R)-5g as a white solid, mp 165e
138.8, 133.5, 129.6, 128.8 (2C), 127.2, 127.0, 126.5 (2C), 124.5, 123.7,
117.9, 115.9, 63.0, 49.5, 23.0; IR (neat, cmꢄ1): 3309, 1584, 998, 759,
20
166 ꢀC; HPLC purity: 98% (method A), tR ¼ 35.9 min; [
a
]
¼ ꢄ305.1
D
(c 0.37, DMSO); 1H NMR (300 MHz, DMSO-d6)
d
: 8.27 (s, 1H), 8.24
702; HRMS (APCI/ASAP, m/z): 362.1324 (calcd.
362.1325, [M þ H]þ).
C21H20N3OS,
(d, J ¼ 7.9, 1H), 8.14 (s, 1H), 7.64e7.62 (m, 2H), 7.53e7.51 (m, 2H),
7.44e7.42 (m, 2H), 7.35e7.31 (m, 2H), 7.24e7.20 (m, 1H), 5.55e5.48
(m, 1H), 1.57 (d, J ¼ 7.0, 3H), 1.32 (s, 9H); 13C NMR (100 MHz, DMSO-
4.6.10. (R)-N-(1-Phenylethyl)-6-(m-tolyl)thieno[2,3-d]pyrimidin-4-
amine ((R)-5m)
d6) d: 164.9, 155.7, 153.8, 151.3, 144.6, 138.2, 130.5, 128.3 (2C), 126.7,
126.2 (2C), 126.1 (2C), 125.4 (2C), 117.5, 114.8, 49.0, 34.5, 31.0 (3C),
22.5; IR (neat, cmꢄ1): 3265, 2960, 1575, 1521, 1493, 1351, 1306, 1104,
827, 776, 697, 554; HRMS (APCI/ASAP, m/z): 388.1843 (calcd.
Compound (R)-5m was prepared as described in Section 4.6.4,
but starting with the MIDA boronate ester of m-tolylboronic acid
(4m) (205 mg, 0.831 mmol). The reaction time was 3 h. The crude
product was purified by silica-gel column chromatography (n-
pentane/EtOAc, 3/1), Rf ¼ 0.46. Drying gave 127 mg (0.368 mmol,
C
24H26N3S, 388.1847, [M þ H]þ).
4.6.7. (R)-4-(4-((1-phenylethyl)amino)thieno[2,3-d]pyrimidin-6-
yl)benzaldehyde ((R)-5i)
Compound (R)-5i was prepared as described in Section 4.6.1, but
starting with (4-formylphenyl)boronic acid (4i) (163 mg,
1.09 mmol). The reaction time was 19 h at 60 ꢀC. The crude product
68%) of (R)-5m as a pale yellow solid, mp 82e85 ꢀC; HPLC purity:
20
99% (method B), tR ¼ 27.8 min; [
a]
¼ ꢄ443.5 (c 1.03, DMSO); 1H
D
NMR (400 MHz, DMSO-d6)
d
: 8.25 (s, 1H), 8.23 (d, J ¼ 7.9, 1H), 8.19
(s, 1H), 7.54e7.52 (m, 1H), 7.50e7.46 (m, 1H), 7.45e7.41 (m, 2H),
7.41e7.36 (m, 1H), 7.35e7.30 (m, 2H), 7.25e7.19 (m, 2H), 5.56e5.47

368
S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
(m, 1H), 2.39 (s, 3H), 1.57 (d, J ¼ 7.1, 3H); 13C NMR (100 MHz, DMSO-
4.6.14. (R)-(2-(4-((1-Phenylethyl)amino)thieno[2,3-d]pyrimidin-6-
yl)phenyl)methanol ((R)-5q)
d6) d: 165.0, 155.7, 153.9, 144.6, 138.6, 138.2, 133.1, 129.27, 129.25,
128.3 (2C), 126.7, 126.1 (3C), 122.9, 117.4, 115.4, 49.0, 22.5, 21.0; IR
(neat, cmꢄ1): 3259, 2973, 1577, 1513, 1350, 772, 697; HRMS (EI,
70 eV, m/z): 345.1291 (calcd. C21H19N3S, 345.1294, [M]þ).
Compound (R)-5q was prepared as described in Section 4.6.1,
but starting with compound (2-(hydroxymethyl)phenyl)boronic
acid (4q) (94 mg, 0.618 mmol), and reacting for 5 h. The crude
product was purified by silica-gel column chromatography
(EtOAc/n-pentane, 7/3), Rf ¼ 0.60. The purified product was then
dissolved in diethyl ether (2 mL) and slowly added to n-pentane
(30 mL) to give precipitation. Isolation and drying gave 141 mg
4.6.11. (S)-2-(4-((1-Phenylethyl)amino)thieno[2,3-d]pyrimidin-6-
yl)phenol ((S)-5n)
Compound (S)-5n was prepared as described in Section 4.6.1,
but starting with (S)-3 (150 mg, 0.449 mmol) and (2-
hydroxyphenyl)boronic acid (4n) (93 mg, 0.673 mmol), and react-
ing for 4 h. The crude product was purified by silica-gel column
chromatography (CH2Cl2/MeOH, 19/1), Rf ¼ 0.45. The purified
product was dissolved in diethyl ether (2 mL), slowly added to n-
pentane (30 mL) to give precipitation. This gave 136 mg
(0.390 mmol, 95%) of (R)-5q as a white solid, mp 137e139 ꢀC;
20
HPLC purity: 97% (method A), tR ¼ 27.2 min; [
a
]
¼ ꢄ294.2 (c
D
0.67, DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 8.31 (s, 1H), 8.27e
8.25 (m, 1H), 7.81 (s, 1H), 7.49e7.31 (m, 9H), 5.58e5.50 (m, 1H),
5.34 (s, br, 1H), 4.71 (s, 2H), 1.57 (d, J ¼ 7.0, 3H); 13C NMR
(100 MHz, DMSO-d6) d: 165.6, 155.8, 153.7, 144.6, 140.2, 136.4,
131.5, 130.5, 130.0, 128.5, 128.3 (2C), 127.8, 127.1, 126.7, 126.0 (2C),
116.8, 60.9, 49.0, 22.4; IR (neat, cmꢄ1): 3313, 2974, 1581, 1494,
1352, 996, 761; HRMS (APCI/ASAP, m/z): 362.1326 (calcd.
(0.393 mmol, 87%) of (S)-5n as a pale white solid, mp 227e230 ꢀC;
20
HPLC purity: 96% (method A), tR ¼ 28.0 min; [
a]
¼ 287.2 (c 0.80,
D
DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 10.41 (s, 1H), 8.62 (s, 1H),
C
21H20N3OS, 362.1325, [M þ H]þ).
8.23 (s, 1H), 8.22 (d, J ¼ 8.0, 1H), 7.69e7.65 (m, 1H), 7.44e7.41 (m,
2H), 7.34e7.29 (m, 2H), 7.24e7.18 (m, 2H), 7.03e6.99 (m, 1H), 6.97e
6.91 (m, 1H), 5.57e5.51 (m, 1H), 1.56 (d, J ¼ 7.1, 3H); HRMS (APCI/
ASAP, m/z): 348.1167 (calcd. C20H18N3OS, 348.1171, [M þ H]þ). Other
spectroscopic properties were identical to that of the (R)-5n re-
ported previously [25].
4.6.15. (R)-N-(1-Phenylethyl)-6-(o-tolyl)thieno[2,3-d]pyrimidin-4-
amine ((R)-5r)
The MIDA boronate ester of o-tolylboronic acid (4r) (240 mg,
0.971 mmol) was dissolved in THF (10 mL). 1 M NaOH solution
(3 mL) was added and the mixture was stirred for 25 min. The
mixture was diluted with 0.5 M pH 7 sodium phosphate buffer
(10 mL) and diethyl ether (10 mL). The aqueous phase was extracted
with THF/diethyl ether 1/1 (2 ꢃ 10 mL). The combined organic
phases were dried over anhydrous Na2SO4, filtered and concen-
trated in vacuo. Drying gave 108 mg (0.797 mmol, 82%) of 4r as a
white solid. The freshly prepared o-tolylboronic acid (4r) (108 mg,
0.797 mmol) was used in a cross-coupling reaction as described in
Section 4.6.1. The reaction time was 17 h. The crude product was
absorbed onto silica and purified by silica-gel column chromatog-
raphy (n-pentane/EtOAc, 3/1), Rf ¼ 0.29. Drying gave 151 mg
4.6.12. (R)-6-(2-Aminophenyl)-N-(1-phenylethyl)thieno[2,3-d]
pyrimidin-4-amine ((R)-5o)
Compound (R)-5o was prepared as described in Section 4.6.1,
but starting with (2-aminophenyl)boronic acid hydrochloride
(4o$HCl) (140 mg, 0.805 mmol). The reaction time was 17 h. The
crude product was purified by silica-gel column chromatography
(n-pentane/EtOAc, 1/1), Rf
¼
0.37. Drying gave 158 mg
(0.455 mmol, 84%) of (R)-5o as a dark orange solid, mp 68e72 ꢀC;
20
HPLC purity: 97% (method B), tR ¼ 15.1 min; [
a]
¼ ꢄ336.2 (c 0.53,
D
DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 8.27 (s, 1H), 8.20 (d,
(0.438 mmol, 81%) of (R)-5r as a pale yellow solid, mp 79e83 ꢀC;
J ¼ 7.9, 1H), 7.91 (s, 1H), 7.45e7.41 (m, 2H), 7.35e7.29 (m, 2H),
7.25e7.19 (m, 2H), 7.13e7.08 (m, 1H), 6.86e6.82 (m, 1H), 6.68e6.63
(m, 1H), 5.57e5.47 (m, 1H), 5.32 (s, 2H), 1.56 (d, J ¼ 7.0, 3H); 13C
20
HPLC purity: 99% (method B), tR ¼ 26.8 min; [
a]
¼ ꢄ309.8 (c 1.06,
D
DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 8.30 (s,1H), 8.24 (d, J ¼ 7.9,
1H), 7.86 (s, 1H), 7.50e7.46 (m, 1H), 7.45e7.41 (m, 2H), 7.40e7.29
(m, 5H), 7.25e7.19 (m, 1H), 5.57e5.50 (m, 1H), 2.49 (s, 3H), 1.57 (d,
NMR (100 MHz, DMSO-d6) d: 164.9, 155.6, 153.4, 145.8, 144.8, 136.7,
130.2, 129.4, 128.3 (2C), 126.6, 126.1 (2C), 117.5, 117.2, 117.1, 116.7,
116.0, 49.0, 22.5; IR (neat, cmꢄ1): 3311, 3025, 2972, 1575, 1490,
1349, 1302, 747, 698; HRMS (EI, 70 eV, m/z): 346.1243 (calcd.
J ¼ 7.0, 3H); 13C NMR (100 MHz, DMSO-d6)
d: 165.5, 155.9, 153.7,
144.6, 137.5, 135.5, 133.0, 131.2, 130.1, 128.6, 128.3 (2C), 126.7, 126.4,
126.1 (2C), 118.8, 116.8, 49.0, 22.4, 21.0; IR (neat, cmꢄ1): 3255, 2972,
1576, 1513, 1348, 755, 697; HRMS (EI, 70 eV, m/z): 345.1294 (calcd.
C
20H18N4S, 346.1247, [M]þ).
C
21H19N3S, 345.1294, [M]þ).
4.6.13. (R)-6-(2-Methoxyphenyl)-N-(1-phenylethyl)thieno[2,3-d]
pyrimidin-4-amine ((R)-5p)
Compound (R)-5p was prepared as described in Section 4.6.1,
but starting with (2-methoxyphenyl)boronic acid (4p) (93 mg,
0.605 mmol). The reaction time was 3 h. The crude product was
absorbed onto silica and purified by silica-gel column chroma-
tography (n-pentane/EtOAc, 4/1), Rf ¼ 0.53. The resulting product
was dissolved in diethyl ether (2 mL) and slowly added to n-
pentane (30 mL) to give precipitation. Isolation and drying gave
122 mg (0.387 mmol, 84%) of (R)-5p as a white solid, mp 158e
4.6.16. (R)-3-Methoxy-4-(4-((1-phenylethyl)amino)thieno[2,3-d]
pyrimidin-6-yl)benzaldehyde ((R)-5s)
Compound (R)-5s was prepared as described in Section 4.6.1, but
starting with (4-formyl-2-methoxyphenyl)boronic acid (4s)
(584 mg, 3.230 mmol). The reaction time was 9 h. Purification was
by silica-gel column chromatography (n-pentane/Et2O, 3/7),
Rf ¼ 0.30. This gave 0.891 mg (2.287 mmol, 84%) of (R)-5s as a bright
yellow solid, mp 155e158 ꢀC; HPLC purity: 99% (method A),
20
161 ꢀC; HPLC purity: 98% (method A), tR
¼
23.7 min;
tR ¼ 30.5 min; [
a
]
¼ ꢄ486.3 (c 0.98, DMSO); 1H NMR (400 MHz,
D
20
[
a]
¼ ꢄ300.3 (c 1.03, DMSO); 1H NMR (400 MHz, DMSO-d6)
d:
DMSO-d6)
d
: 10.03 (s, 1H), 8.47 (s, 1H), 8.34 (d, J ¼ 7.9, 1H), 8.31 (s,
D
8.32 (s, 1H), 8.29 (s, 1H), 8.25 (d, J ¼ 8.0, 1H), 7.81e7.78 (m, 1H),
7.49e7.46 (m, 2H), 7.45e7.40 (m, 1H), 7.38e7.34 (m, 2H), 7.28e7.22
(m, 2H), 7.17e7.12 (m, 1H), 5.58e5.48 (m, 1H), 3.99 (s, 3H), 1.62 (d,
1H), 8.01e7.97 (m, 1H), 7.70e7.66 (m, 2H), 7.46e7.42 (m, 2H), 7.36e
7.30 (m, 2H), 7.25e7.20 (m, 1H), 5.59e5.50 (m, 1H), 4.06 (s, 3H), 1.58
(d, J ¼ 7.1, 3H); 13C NMR (100 MHz, DMSO-d6)
d: 192.2, 166.4, 155.8,
J ¼ 7.2, 3H); 13C NMR (100 MHz, DMSO-d6)
d
: 166.1, 156.1, 155.9,
155.7, 154.2, 144.6, 136.6, 132.3, 128.3 (2C), 128.2, 127.6, 126.7, 126.1
(2C), 122.8, 119.4, 116.1, 112.2, 56.2, 49.0, 22.4; IR (neat, cmꢄ1): 3277,
3100, 2935, 2810, 1687, 1579, 1499, 1254, 1028, 735, 696, 559; HRMS
154.2, 145.2, 134.4, 130.2, 128.8 (2C), 128.4, 127.1, 126.6 (2C), 122.3,
121.5, 117.5, 116.6, 113.0, 56.3, 49.4, 22.9; IR (neat, cmꢄ1): 2359,
1609, 751, 699; HRMS (EI, 70 eV, m/z): 361.1241 (calcd. C21H19ON3S,
361.1243, [M]þ).
(APCI/ASAP, m/z): 390.1274 (calcd.
C22H20N3O2S, 390.1276,
[M þ H]þ).

S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
369
4.6.17. (R)-(3-Methoxy-4-(4-((1-phenylethyl)amino)thieno[2,3-d]
pyrimidin-6-yl)phenyl)methanol ((R)-5t)
5.54e5.45 (m, 1H), 1.55 (d, J ¼ 7.0, 3H); 13C NMR (100 MHz, DMSO-
d6)
d: 165.2, 162.2 (d, J ¼ 246.3), 161.1 (d, J ¼ 242.1), 155.7, 153.9,
Compound (R)-5s (285 mg, 0.733 mmol) was dissolved in MeOH
(25 mL) and added NaBH4 (30 mg, 0.793 mmol). The mixture was
stirred for 10 min at 0 ꢀC, before another portion of NaBH4 (30 mg,
0.793 mmol) was added. The stirring was continued at rt for 2 h.
The reaction mixture was then concentrated in vacuo, diluted with
water (100 mL) and EtOAc (50 mL) and the water phase was
extracted with EtOAc (2 ꢃ 50 mL). The combined organic phases
were washed with saturated aq NaCl solution (30 mL), dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The crude
product was absorbed onto silica and purified by silica-gel column
chromatography (Et2O/EtOAc, 3/1), Rf ¼ 0.21. Drying gave 194 mg
140.8 (d, J ¼ 2.9), 137.1, 129.9 (d, J ¼ 3.3), 128.0 (d, J ¼ 8.1, 2C), 127.8
(d, J ¼ 8.3, 2C), 117.5, 116.4 (d, J ¼ 22.0, 2C), 115.7, 115.0 (d, J ¼ 21.2,
2C), 48.5, 22.6; 19F NMR (564 MHz, DMSO-d6)
d
: ꢄ115.3 (s), ꢄ118.6
(s); IR (neat, cmꢄ1): 3057, 2966, 2874, 1603, 1491, 1234, 832, 699;
HRMS (ESI, m/z): 368.1029 (calcd.
C20H16F2N3S, 368.1028,
[M þ H]þ).
4.6.20. (R)-(4-(4-((1-(4-Fluorophenyl)ethyl)amino)thieno[2,3-d]
pyrimidin-6-yl)phenyl)methanol ((R)-25e)
The preparation of the 4-(hydroxymethyl)phenylboronic acid
(4e) (140 mg, 0.920 mmol) and the cross-coupling reaction with the
fluorinated compound (R)-15$HCl (200 mg, 0.515 mmol) were
performed as described in Section 4.6.4. The reaction time was
2.5 h. The crude product was absorbed onto celite and purified by
silica-gel column chromatography (EtOAc/n-pentane, 7/3),
Rf ¼ 0.26. Drying gave 77 mg (0.203 mmol, 39%) of (R)-25e as a
(0.497 mmol, 68%) of (R)-5t as a white solid, mp 177e179 ꢀC; HPLC
20
purity: 98% (method A), tR ¼ 26.6 min; [
a
]
¼ ꢄ377.3 (c 1.04,
D
DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 8.26 (s, 1H), 8.22 (s, 1H),
8.19 (d, J ¼ 8.0, 1H), 7.72e7.69 (m, 1H), 7.46e7.41 (m, 2H), 7.35e7.29
(m, 2H), 7.24e7.19 (m, 1H), 7.16e7.14 (m, 1H), 7.08e7.04 (m, 1H),
5.58e5.49 (m, 1H), 5.31 (t, J ¼ 5.8, 1H), 4.56 (d, J ¼ 5.7, 2H), 3.95 (s,
white solid, mp 203e205 ꢀC; HPLC purity: 98% (method A),
20
3H), 1.57 (d, J ¼ 7.1, 3H); 13C NMR (100 MHz, DMSO-d6)
d
: 165.5,
tR ¼ 27.2 min; [
a
]
D
¼ ꢄ265.7 (c 1.17, DMSO); 1H NMR (400 MHz,
155.5, 155.4, 153.5, 144.8, 144.7, 134.1, 128.3 (2C), 127.6, 126.7, 126.1
(2C), 120.1, 118.9, 116.5, 116.2, 110.2, 62.6, 55.8, 48.9, 22.5; IR (neat,
cmꢄ1): 3281, 2971, 2933, 2361, 1588, 1499, 1019, 767, 698; HRMS
DMSO-d6)
d
: 8.28 (s, 1H), 8.22 (d, J ¼ 7.8, 1H), 8.14 (s, 1H), 7.68e7.63
(m, 2H), 7.50e7.42 (m, 4H), 7.18e7.11 (m, 2H), 5.55e5.46 (m, 1H),
5.29 (t, J ¼ 5.7, 1H), 4.55 (d, J ¼ 5.7, 2H), 1.56 (d, J ¼ 7.0, 3H); 13C NMR
(APCI/ASAP, m/z): 392.1426 (calcd.
C
22H22N3O2S, 392.1433,
(100 MHz, DMSO-d6)
d: 165.4, 161.5 (d, J ¼ 241.7), 156.1, 154.2, 143.7,
[M þ H]þ).
141.3 (d, J ¼ 3.1), 138.8, 132.1, 128.5 (d, J ¼ 8.1, 2C), 127.8 (2C), 125.9
(2C), 117.9, 115.50, 115.46 (d, J ¼ 21.2, 2C), 62.9, 48.9, 23.0; 19F NMR
4.6.18. (R)-N-(1-(4-Fluorophenyl)ethyl)-6-phenylthieno[2,3-d]
pyrimidin-4-amine hydrochloride ((R)-25a$HCl)
(564 MHz, DMSO-d6)
d
: ꢄ118.7 (s); IR (neat, cmꢄ1): 3242, 3131,
2924, 2854, 1578, 1510, 1312, 1227, 1099, 831, 801, 777; HRMS (APCI/
Compound 24a (100 mg, 0.405 mmol) was mixed with (R)-1-(4-
fluorophenyl)ethanamine ((R)-6) (0.16 mL, 168 mg, 1.21 mmol) and
1-butanol (2 mL), and agitated at 145 ꢀC for 21 h. Then the mixture
was cooled to rt, diluted with EtOAc (50 mL) and washed with
water (3 ꢃ 15 mL). The combined organic phases were dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The crude
product was purified by silica-gel column chromatography (EtOAc),
Rf ¼ 0.60. The product was further purified by precipitation from
diethyl ether (50 mL) by addition of HCl in diethyl ether (1.5 mL)
and storage at ꢄ18 ꢀC for 24 h. The solid was isolated by filtration
and washed with diethyl ether (2 ꢃ 25 mL). Drying gave 108 mg
ASAP, m/z): 380.1226 (calcd. C21H19FN3OS, 380.1233, [M þ H]þ).
4.6.21. (R)-3-(4-(1-(4-Fluorophenyl)ethylamino)thieno[2,3-d]
pyrimidin-6-yl)phenol hydrochloride ((R)-25j$HCl)
Compound (R)-25j$HCl was prepared as described in Section
4.6.1, but starting with (3-hydroxyphenyl)boronic acid (4j) (115 mg,
0.834 mmol) and the fluorinated compound (R)-15$HCl (215 mg,
0.553 mmol). The reaction time was 3.5 h. The crude product was
purified by silica-gel column chromatography (EtOAc), Rf ¼ 0.59.
The product was precipitated from n-pentane (100 mL) by addition
of HCl in diethyl ether (2 mL) and storage at 4 ꢀC for 4.5 h. Drying
gave 213 mg (0.530 mmol, 96%) of (R)-25j$HCl as a white solid; mp
(0.280 mmol, 69%) of (R)-25a$HCl as a white solid, mp 147e151 ꢀC;
20
HPLC purity: 95% (method B), tR ¼ 18.6 min; [
a
]
D
¼ ꢄ268.0 (c 1.02,
180e184 ꢀC; HPLC purity: 98% (method C), tR ¼ 15.7 min;
20
DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 8.29 (s,1H), 8.24 (d, J ¼ 7.8,
[a
]
¼ ꢄ282.7 (c 1.00, DMSO); 1H NMR (400 MHz, DMSO-d6)
d:
D
1H), 8.18 (s, 1H), 7.73e7.67 (m, 2H), 7.54e7.44 (m, 4H), 7.43e7.37
(m, 1H), 7.18e7.10 (m, 2H), 5.56e5.46 (m, 1H), 1.56 (d, J ¼ 7.0, 3H);
9.73 (s, 1H), 8.28 (s, 1H), 8.22 (d, J ¼ 7.8, 1H), 8.12 (s, 1H), 7.48e7.44
(m, 2H), 7.31e7.27 (m, 1H), 7.17e7.12 (m, 3H), 7.09e7.08 (m, 1H),
6.82e6.79 (m, 1H), 5.54e5.45 (m, 1H), 1.55 (d, J ¼ 7.0, 3H); 13C NMR
13C NMR (100 MHz, DMSO-d6)
d
: 165.1, 161.0 (d, J ¼ 242.2), 155.7,
153.9, 140.8 (d, J ¼ 3.4), 138.2, 133.2, 129.4 (2C), 128.6, 128.0 (d,
J ¼ 8.1, 2C), 125.7 (2C), 117.4, 115.5, 115.0 (d, J ¼ 21.1, 2C), 48.5, 22.5;
(100 MHz, DMSO-d6)
d
: 165.0, 162.2 (d, J ¼ 240.8), 158.0, 155.7,
153.8, 140.8 (d, J ¼ 3.0), 138.4, 134.4, 130.5, 128.0 (d, J ¼ 8.0, 2C),
19F NMR (564 MHz, DMSO-d6)
d
: ꢄ118.6 (s); IR (neat, cmꢄ1): 3058,
117.4, 116.5, 115.7, 115.3, 115.1 (d, J ¼ 21.1, 2C), 112.4, 48.5, 22.5; 19F
2977, 1607, 1509, 1223, 838, 753, 688; HRMS (ESI, m/z): 350.1123
NMR (564 MHz, DMSO-d6)
d
: ꢄ118.7 (s); IR (neat, cmꢄ1): 3059,
(calcd. C20H17FN3S, 350.1122, [M þ H]þ).
2737, 2348, 1594, 1511, 1444, 1220, 763, 670, 571; HRMS (EI, 70 eV,
m/z): 365.0995 (calcd. C20H16ON3FS, 365.0993, [M]þ).
4.6.19. (R)-6-(4-Fluorophenyl)-N-(1-(4-fluorophenyl)ethyl)thieno
[2,3-d]pyrimidin-4-amine hydrochloride ((R)-25c$HCl)
4.6.22. (R)-(3-(4-(1-(4-Fluorophenyl)ethylamino)thieno[2,3-d]
pyrimidin-6-yl)phenyl)methanol ((R)-25l)
Compound (R)-25c$HCl was prepared as described in Section
4.6.18, but starting with compound 24c (100 mg, 0.378 mmol). The
crude product was purified by silica-gel column chromatography
(n-pentane/acetone, 7/3), Rf ¼ 0.38. The product was further puri-
fied by precipitation from diethyl ether (50 mL) by addition of HCl
in diethyl ether (1.5 mL) and storage at ꢄ18 ꢀC for 24 h. The solid
was isolated by filtration and washed with diethyl ether
(2 ꢃ 25 mL). Drying gave 106 mg (0.263 mmol, 70%) of (R)-25c$HCl
Compound (R)-25l was prepared as described in Section 4.6.1,
but starting with (3-(hydroxymethyl)phenyl) boronic acid (4l)
(149 mg, 0.981 mmol) and the fluorinated compound (R)-15$HCl
(210 mg, 0.540 mmol). The crude product was purified by silica-gel
column chromatography (EtOAc), Rf ¼ 0.52. Further purification
was done by crystallization from MeOH (1 mL) using water as anti-
solvent (1 mL). This gave 143 mg (0.377 mmol, 70%) of (R)-25l as a
as a white solid, mp 166e174 ꢀC; HPLC purity: 97% (method B),
slightly yellowish solid, mp 182e185 ꢀC; HPLC purity: 98% (method
20
20
tR ¼ 19.4 min; [
a
]
D
¼ ꢄ244.8 (c 0.92, DMSO); 1H NMR (400 MHz,
C), tR ¼ 14.9 min; [
a
]
¼ ꢄ324.9 (c 0.93, DMSO); 1H NMR (400 MHz,
D
DMSO-d6)
d
: 8.29 (s, 1H), 8.25 (d, J ¼ 7.8, 1H), 8.12 (s, 1H), 7.76e7.61
DMSO-d6)
d
: 8.29 (s, 1H), 8.26 (d, J ¼ 7.8, 1H), 8.20 (s, 1H), 7.72 (s,
(m, 2H), 7.50e7.43 (m, 2H), 7.40e7.32 (m, 2H), 7.18e7.10 (m, 2H),
1H), 7.56e7.54 (m, 1H), 7.49e7.43 (m, 3H), 7.33e7.31 (m, 1H), 7.17e

370
S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
20
7.12 (m, 2H), 5.55e5.46 (m, 1H), 5.34 (t, J ¼ 5.7, 1H), 4.59 (d, J ¼ 5.7,
161 ꢀC; HPLC purity 99% (method A), tR ¼ 24.4 min; [
a
]
¼ ꢄ273.0
D
2H), 1.56 (d, J ¼ 7.0, 3H); 13C NMR (100 MHz, DMSO-d6)
d
: 165.0,
(c 1.01, DMSO); 1H NMR (400 MHz, DMSO-d6)
d: 8.31 (s,1H), 8.25 (d,
162.2 (d, J ¼ 240.8), 155.7,153.9,143.8,140.8 (d, J ¼ 3.0),138.4,133.0,
129.1, 128.0 (d, J ¼ 8.1, 2C), 126.5, 124.0, 123.3, 117.5, 115.4, 115.1 (d,
J ¼ 21.1, 2C), 62.5, 48.5, 22.5; 19F NMR (564 MHz, DMSO-d6)
J ¼ 7.8, 1H), 7.77 (s, 1H), 7.68e7.66 (m, 1H), 7.49e7.44 (m, 4H) 7.40e
7.36 (m, 1H), 7.17e7.12 (m, 2H), 5.57e5.48 (m, 1H), 5.34 (t, J ¼ 5.4,
1H), 4.68 (d, J ¼ 5.4, 2H), 1.55 (d, J ¼ 7.0, 3H); 13C NMR (100 MHz,
d
: ꢄ118.7 (s); IR (neat, cmꢄ1): 3272, 2976, 2867, 1589, 1500, 1220,
DMSO-d6)
d
: 166.1,162.2 (d, J ¼ 240.8),155.8,153.7,140.8 (d, J ¼ 3.0),
1096, 774, 680, 566; HRMS (EI, 70 eV, m/z): 379.1149 (calcd.
C
140.2, 136.5, 131.5, 130.0, 128.6, 128.0 (d, J ¼ 8.0, 2C), 127.8, 127.2,
21H18FN3OS, 379.1149, [M]þ).
119.0, 116.8, 115.1 (d, J ¼ 21.1, 2C), 60.9, 48.4, 22.5; 19F NMR
(564MHz, DMSO-d6)
d
: ꢄ118.7 (s); IR (neat, cmꢄ1): 3324, 2971,
4.6.23. (R)-2-(4-(1-(4-Fluorophenyl)ethylamino)thieno[2,3-d]
pyrimidin-6-yl)phenol ((R)-25n)
2883, 1574, 1506, 1225, 1127, 763, 581; HRMS (EI, 70 eV, m/z):
379.1153 (calcd. C21H18ON3FS, 379.1149, [M]þ).
Compound (R)-25n was prepared as described in Section 4.6.1,
but starting with (2-hydroxyphenyl)boronic acid (4n) (115 mg,
0.834 mmol) and the fluorinated compound (R)-15$HCl (212 mg,
0.545 mmol). The reaction time was 3 h. The crude product was
purified by silica-gel column chromatography (EtOAc), Rf ¼ 0.52.
The product was precipitated from n-pentane (100 mL) by addition
of HCl in diethyl ether (1 mL). This gave 156 mg (0.388 mmol, 71%)
of a white powder with purity of 96% (HPLC). For further purifica-
tion a fraction (82 mg, 0.204 mmol) was free-based and crystallized
from MeOH (2 mL) using water as anti-solvent. This gave 55 mg
4.6.26. 2-(4-(Benzylamino)thieno[2,3-d]pyrimidin-6-yl)phenol
(26n)
Compound 26n was prepared as described in Section 4.6.1, but
starting with (2-hydroxyphenyl)boronic acid (4n) (90 mg,
0.653 mmol) and compound 16 (202 mg, 0.630 mmol). The reaction
time was 2.5 h. The crude product was purified by silica-gel column
chromatography (CH2Cl2/MeOH, 24/1), Rf ¼ 0.15. Drying gave 43 mg
(0.128 mmol, 20%) of 26n as a white solid, mp 177e180 ꢀC; HPLC
purity: 97% (method A), tR ¼ 24.2 min; 1H NMR (400 MHz, DMSO-
(0.151 mmol, 67%) of (R)-25n as a white powder, mp 167e174 ꢀC;
d6)
d
: 10.42 (s, 1H), 8.50 (t, J ¼ 5.9, 1H), 8.32 (s, 1H), 8.14 (s, 1H),
20
HPLC purity: 98% (method C), tR ¼ 15.0 min; [
a
]
¼ ꢄ347.3 (c 0.62,
7.64e7.60 (m, 1H), 7.41e7.30 (m, 4H), 7.27e7.17 (m, 2H), 7.03e6.98
D
DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 10.42 (s, 1H), 8.26 (s, 1H),
(m, 1H), 6.95e6.90 (m, 1H), 4.76 (d, J ¼ 5.9, 2H); 13C NMR (100 MHz,
8.24e8.19 (m, 2H), 7.67e7.65 (m, 1H), 7.48e7.45 (m, 2H), 7.23e7.19
(m, 1H), 7.16e7.12 (m, 2H), 7.02e7.00 (m, 1H), 6.96e6.92 (m, 1H),
5.56e5.47 (m, 1H), 1.56 (d, J ¼ 7.0, 3H); 13C NMR (100 MHz, DMSO-
DMSO-d6) d: 165.2, 156.3, 154.0, 153.5, 139.6, 135.1, 129.3,128.3 (2C),
127.8, 127.4 (2C), 126.8, 120.0, 119.7, 116.51, 116.46, 116.2, 43.3; IR
(neat, cmꢄ1): 3422, 2922, 2852, 1578, 1454, 1348, 1107, 745; HRMS
d6)
d
: 165.4, 162.2 (d, J ¼ 240.5), 155.5, 154.0, 153.3, 141.0 (d, J ¼ 3.0),
(APCI/ASAP, m/z): 334.1013 (calcd.
[M þ H]þ).
C19H16N3OS, 334.1014,
135.0, 129.4, 128.0 (d, J ¼ 8.0, 2C), 127.8, 120.1, 119.7, 116.5, 116.3,
116.2, 115.1 (d, J ¼ 21.1, 2C), 48.4, 22.5; 19F NMR (564 MHz, DMSO-
d6)
d
: ꢄ118.8 (s); IR (neat, cmꢄ1): 2919, 1574, 1501, 1201, 737, 550;
4.6.27. (R)-2-(4-((1-Phenylpropyl)amino)thieno[2,3-d]pyrimidin-
6-yl)phenol ((R)-27n)
HRMS (EI, 70 eV, m/z): 365.0994 (calcd. C20H16ON3FS, 365.0993,
[M]þ).
Compound (R)-27n was prepared as described in Section 4.6.1,
but starting with (2-hydroxyphenyl)boronic acid (4n) (21 mg,
0.149 mmol) and compound (R)-17 (37 mg, 0.096 mmol). The re-
action time was 20 h. The crude product was absorbed onto celite
and purified by silica-gel column chromatography (EtOAc/n-
pentane, 3/2), Rf ¼ 0.37. This gave 19 mg (0.053 mmol, 55%) of (R)-
4.6.24. (R)-N-(1-(4-Fluorophenyl)ethyl)-6-(2-methoxyphenyl)
thieno[2,3-d]pyrimidin-4-amine ((R)-25p)
Compound (R)-25p was prepared as described in Section 4.6.1,
but starting with (2-methoxyphenyl)boronic acid (4p) (119 mg,
0.785 mmol) and the fluorinated compound (R)-15$HCl (201 mg,
0.516 mmol). The reaction time was 3.5 h. The crude product was
absorbed onto celite and purified by silica-gel column chromatog-
raphy (n-pentane/EtOAc, 1/1), Rf ¼ 0.43. Drying gave 163 mg
27n as a light brown solid, mp 167e171 ꢀC; HPLC purity: 95%
20
(method A), tR ¼ 29.3 min; [
a]
¼ ꢄ256.0 (c 0.42, DMSO-d6); 1H
D
NMR (400 MHz, DMSO-d6)
d
: 10.24 (s, 1H), 8.26e8.24 (m, 2H), 8.15
(d, J ¼ 8.3, 1H), 7.70e7.66 (m, 1H), 7.46e7.42 (m, 2H), 7.35e7.29 (m,
2H), 7.24e7.18 (m, 2H), 7.03e6.99 (m, 1H), 6.97e6.95 (m, 1H), 5.33e
5.25 (m, 1H), 1.98e1.82 (m, 2H), 0.95 (t, J ¼ 7.3, 3H); 13C NMR
(0.431 mmol, 83%) of (R)-25p as a white solid, mp 98e102 ꢀC; HPLC
20
purity: 98% (method A), tR ¼ 31.8 min; [
a
]
D
¼ ꢄ296.0 (c 1.03,
DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 8.28 (s, 1H), 8.24e8.20 (m,
(100 MHz, DMSO-d6) d: 165.4,156.0,153.9,153.3,143.9,134.9,129.3,
2H), 7.77e7.72 (m, 1H), 7.50e7.43 (m, 2H), 7.42e7.36 (m, 1H), 7.23e
7.18 (m, 1H), 7.18e7.07 (m, 3H), 5.57e5.48 (m, 1H), 3.95 (s, 3H), 1.56
128.2 (2C), 127.8, 126.7, 126.6 (2C), 120.1, 119.6, 116.5, 116.3, 116.2,
55.3, 29.3, 11.5; IR (neat, cmꢄ1): 2923, 2853, 1578, 1451, 1354, 1294,
1105, 745, 697; HRMS (APCI/ASAP, m/z): 362.1324 (calcd.
(d, J ¼ 7.0, 3H); 13C NMR (100 MHz, DMSO-d6)
d: 165.6, 161.0 (d,
J ¼ 242.1), 155.53, 155.47, 153.6, 140.9 (d, J ¼ 2.9), 134.0, 129.8, 128.0
C
21H20N3OS, 362.1327, [M þ H]þ).
(d, J ¼ 8.2, 2C), 127.9, 121.8, 121.1, 117.0, 116.2, 115.0 (d, J ¼ 21.2, 2C),
112.5, 55.9, 48.4, 22.5; 19F NMR (564 MHz, DMSO-d6)
d
: ꢄ118.7 (s);
4.6.28. (rac)-(4-((2-Methyl-1-phenylpropyl)amino)thieno[2,3-d]
pyrimidin-6-yl)phenol ((rac)-28n)
IR (neat, cmꢄ1): 3242, 2971, 2836, 1577, 1506, 1221, 1115, 832, 747;
HRMS (APCI/ASAP, m/z): 380.1230 (calcd. C21H19FN3OS, 380.1233,
[M þ H]þ).
Compound (rac)-28n was prepared as described in Section 4.6.1,
but starting with compound (rac)-18 (150 mg, 0.414 mmol) and (2-
hydroxyphenyl)boronic acid (4n) (86 mg, 0.621 mmol) and reacting
for 4 h. The crude product was purified with silica-gel column
chromatography (CH2Cl2/MeOH, 49/1), Rf ¼ 0.40, and then precip-
itated from diethyl ether (2 mL) and n-pentane (30 mL). Evapora-
tion and drying gave 127 mg (0.339 mmol, 82%) of (rac)-28n as a
white solid, mp 159e162 ꢀC; HPLC purity: 96% (method A),
4.6.25. (R)-(2-(4-(1-(4-Fluorophenyl)ethylamino)thieno[2,3-d]
pyrimidin-6-yl)phenyl)methanol ((R)-25q)
Compound (R)-25q was prepared as described in Section 4.6.1,
but starting with (2-(hydroxymethyl)phenyl)boronic acid (4q)
(85 mg, 0.559 mmol) and the fluorinated compound (R)-15$HCl
(141 mg, 0.363 mmol). The reaction time was 4.5 h at 90 ꢀC. The
crude product was purified by silica-gel column chromatography
(EtOAc), Rf ¼ 0.46. This was followed by crystallization from MeOH
(1.5 mL) using water as anti-solvent, which gave 108 mg
(0.285 mmol, 80%) of (R)-25q as a white fluffy powder, mp 157e
tR ¼ 28.2 min; 1H NMR (400 MHz, DMSO-d6)
d: 10.41 (s, 1H), 8.25e
8.24 (m, 2H), 8.11e8.07 (m, 1H), 7.71e7.67 (m, 1H), 7.47e7.45 (m,
2H), 7.34e7.31 (m, 2H), 7.23e7.19 (m, 2H), 7.01e6.93 (m, 2H), 5.12e
5.07 (m, 1H), 2.27e2.15 (m, 1H), 1.07 (d, J ¼ 6.6, 3H), 0.77 (d, J ¼ 6.7,
3H); 13C NMR (100 MHz, DMSO-d6)
d: 165.4, 156.1, 153.9, 153.3,

S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
371
143.0, 134.8, 129.3, 128.1 (2C), 127.8, 127.4 (2C), 126.7, 120.1, 119.6,
116.5, 116.1, 83.2, 60.2, 32.8, 20.1, 19.9; IR (neat, cmꢄ1): 3035, 2961,
1606, 1367, 758, 701; HRMS (APCI/ASAP, m/z): 376.1484 (calcd.
116.50, 116.0, 84.3 (d, J ¼ 174.9), 53.7 (d, J ¼ 20.2); 19F NMR
(564 MHz, DMSO-d6)
d
: ꢄ219.7 (s); IR (neat, cmꢄ1): 3029, 2696,
1738, 1580, 1451, 1217, 749, 693; HRMS (APCI/ASAP, m/z): 366.1075
(calcd. C20H17FN3OS, 366.1075, [M þ H]þ).
C
22H22N3OS, 376.1484, [M þ H]þ).
4.6.29. (R)-2-(4-(Methyl(1-phenylethyl)amino)thieno[2,3-d]
pyrimidin-6-yl)phenol ((R)-29n)
4.6.32. (S)-2-(4-((2-Methoxy-1-phenylethyl)amino)thieno[2,3-d]
pyrimidin-6-yl)phenol ((S)-32n)
Compound (R)-29n was prepared as described in Section 4.6.1,
but starting with compound (R)-19 (150 mg, 0.428 mmol) and (2-
hydroxyphenyl)boronic acid (4n) (89 mg, 0.646 mmol), and react-
ing for 3.5 h. The crude product was purified with silica-gel column
chromatography (n-pentane/EtOAc, 1/1), Rf ¼ 0.43. The purified
product was dissolved in diethyl ether (2 mL) and slowly added to
n-pentane (30 mL) to give precipitation. Evaporation and drying
gave 136 mg (0.378 mmol, 88%) of (R)-29n as a white solid, mp
Compound (S)-32n was prepared as described in Section 4.6.1,
but starting with compound (S)-22 (130 mg, 0.357 mmol) and (2-
hydroxyphenyl)boronic acid (4n) (74 mg, 0.537 mmol), and react-
ing for 5 h. The crude product was purified with silica-gel column
chromatography (n-pentane/EtOAc, 1/1), Rf ¼ 0.48. The purified
product was dissolved in diethyl ether (2 mL) and slowly added to
n-pentane (30 mL) to give precipitation. Evaporation and drying
gave 113 mg (0.299 mmol, 84%) of (S)-32n as a white solid, mp
227e230 ꢀC; HPLC purity: 96% (method A), tR ¼ 28.1 min;
221e224 ꢀC; HPLC purity (method A): 97%, tR ¼ 26.8 min;
¼ ꢄ15.7 (c 0.80, DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 10.41
[a
]
¼ ꢄ282.3 (c 0.67, DMSO); 1H NMR (400 MHz, DMSO-d6)
d:
20
20
[
a]
D
D
(s, 1H), 8.38 (s, 1H), 8.01 (s, 1H), 7.71e7.69 (m, 1H), 7.39e7.35 (m,
4H), 7.31e7.27 (m, 1H), 7.20e7.16 (m, 1H), 6.97e6.95 (m, 1H), 6.90e
10.44 (s, 1H), 8.27e8.25 (m, 3H), 7.69e7.67 (m, 1H), 7.48e7.46 (m,
2H), 7.35e7.32 (m, 2H), 7.26e7.20 (m, 2H), 7.02e7.00 (m, 1H), 6.96e
6.93 (m, 1H), 5.71e5.65 (m, 1H), 3.78 (ddAB, J ¼ 10.1, 8.8, 1H), 3.65
(ddAB, J ¼ 10.1, 5.2, 1H), 3.32 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
6.86 (m, 1H), 6.45e6.40 (m, 1H), 3.13 (s, 3H), 1.63 (d, J ¼ 7.0, 3H); 13
C
NMR (100 MHz, DMSO-d6) d: 168.3, 157.4, 154.1, 152.3, 141.1, 133.3,
129.3, 128.5 (2C), 128.2, 127.1, 126.9 (2C), 119.9, 119.6, 118.9, 116.3,
115.6, 53.4, 33.1, 16.3; IR (neat, cmꢄ1): 3052, 1589, 1380, 753, 699;
HRMS (APCI/ASAP, m/z): 362.1322 (calcd. C21H20N3OS, 362.1327,
[M þ H]þ).
d: 165.9, 156.4, 154.4, 153.8, 141.1, 135.5, 129.8, 128.7 (2C), 128.3,
127.6, 127.5 (2C), 120.5, 120.1, 117.0, 116.9, 116.7, 75.2, 58.5, 53.5; IR
(neat, cmꢄ1): 3439, 2887, 1580, 1106, 745, 689; HRMS (APCI/ASAP,
m/z): 378.1276 (calcd. C21H20N3O2S, 378.1276, [M þ H]þ).
4.6.30. (rac)-2-(4-((2,2,2-Trifluoro-1-phenylethyl)amino)thieno
[2,3-d]pyrimidin-6-yl)phenol ((rac)-30n)
4.6.33. (R)-2-(4-((2-Methoxy-1-phenylethyl)amino)thieno[2,3-d]
pyrimidin-6-yl)phenol ((R)-32n)
Compound (rac)-30n was prepared as described in Section 4.6.1,
but starting with compound (rac)-20$HCl (221 mg, 0.521 mmol)
and (2-hydroxyphenyl)boronic acid (4n) (108 mg, 0.781 mmol),
and reacting for 3.5 h. The crude product was purified with silica-
gel column chromatography (diethyl ether), Rf ¼ 0.48. The puri-
fied product was dissolved in diethyl ether (2 mL), slowly added to
n-pentane (30 mL) to give precipitation. Isolation and drying gave
162 mg (0.403 mmol, 85%) of (rac)-30n as a white solid, mp 236 ꢀC
(dec.); HPLC purity: 97% (method A), tR ¼ 30.5 min; 1H NMR
Compound (R)-32n was prepared as described in Section 4.6.1,
but starting with compound (R)-22$HCl (150 mg, 0.374 mmol) and
(2-hydroxyphenyl)boronic acid (4n) (77 mg, 0.558 mmol), and
reacting for 4 h. The crude product was purified by silica-gel col-
umn chromatography (n-pentane/EtOAc, 1/1), Rf ¼ 0.46. The puri-
fied product was dissolved in diethyl ether (2 mL) and slowly added
to n-pentane (30 mL) to give precipitation. Evaporation and drying
gave 130 mg (0.344 mmol, 92%) of (R)-32n as a white solid, mp
219e222 ꢀC; HPLC purity: 96% (method A), tR ¼ 26.7 min;
20
(400 MHz, DMSO-d6)
d
: 10.59 (br s, 1H), 8.85 (d, J ¼ 9.5, 1H), 8.47e
[a
]
¼ 263.3 (c 0.76, DMSO); 1H NMR (400 MHz, DMSO-d6)
d: 10.45
D
8.45 (m, 2H), 7.75e7.71 (m, 3H), 7.49e7.40 (m, 3H), 7.26e7.22 (m,
(s, 1H), 8.27e8.24 (m, 3H), 7.69e7.67 (m, 1H), 7.48e7.46 (m, 2H),
7.35e7.31 (m, 2H), 7.26e7.21 (m, 2H), 7.02e7.00 (m, 1H), 6.96e6.93
(m, 1H), 5.71e5.65 (m, 1H), 3.81e3.76 (m, 1H), 3.67e3.63 (m, 1H),
3.32 (s, 3H); HRMS (APCI/ASAP, m/z): 378.1277 (calcd. C21H20N3O2S,
378.1276, [M þ H]þ). The spectroscopic properties confirmed with
that reported for (S)-32n.
1H), 7.05e7.03 (m, 1H), 6.98e6.94 (m, 1H), 6.63e6.54 (m, 1H); 13C
NMR (100 MHz, DMSO-d6) d: 166.9, 155.7, 154.5, 153.2, 136.5 (2C),
133.9, 130.1, 129.5, 129.1 (2C), 129.0 (2C), 128.3, 125.6 (q, J ¼ 282.7),
120.3, 120.1, 117.0, 116.5, 54.5 (q, J ¼ 22.9); 19F NMR (564 MHz,
DMSO-d6)
d
: ꢄ74.1 (s); IR (neat, cmꢄ1): 3313, 2983, 1546, 1072, 654;
HRMS (EI, 70 eV, m/z): 401.0805, (calcd. C20H14F3N3OS, 401.0804,
[M]þ).
4.6.34. (S)-2-((6-(4-(Hydroxymethyl)phenyl)thieno[2,3-d]
pyrimidin-4-yl)amino)-2-phenylethan-1-ol ((S)-33e)
4.6.31. (rac)-2-(4-((2–Fluoro-1-phenylethyl)amino)thieno[2,3-d]
pyrimidin-6-yl)phenol ((rac)-31n)
Compound (S)-33e was prepared as described in Section 4.6.1,
but starting with compound (S)-23$HCl (150 mg, 0.388 mmol) and
(4-(hydroxymethyl)phenyl)boronic acid (4e) (88 mg, 0.579 mmol),
and reacting for 4 h. The crude product was purified by silica-gel
column chromatography (EtOAc), Rf ¼ 0.18. The purified product
was dissolved in diethyl ether (2 mL) and slowly added to n-
pentane (30 mL) to give precipitation. Evaporation and drying gave
Compound (rac)-31n was prepared as described in Section 4.6.1,
but starting with compound (rac)-21 (150 mg, 0.42 mmol) and (2-
hydroxyphenyl)boronic acid (4n) (88 mg, 0.639 mmol). The reac-
tion was then stirred at 50 ꢀC for 72 h under N2 atmosphere. The
crude product was purified with silica-gel column chromatography
(n-pentane/EtOAc, 1/1), Rf ¼ 0.43. The purified product was dis-
solved in diethyl ether (2 mL) and slowly added to n-pentane
(30 mL) to give precipitation. Evaporation and drying gave 79 mg
(0.216 mmol, 51%) of (rac)-31n as a white solid, mp 176e179 ꢀC;
HPLC purity: 96% (method A), tR ¼ 24.2 min; 1H NMR (400 MHz,
123 mg (0.326 mmol, 84%) of (S)-33e as a white solid, mp 206e
20
209 ꢀC; HPLC purity: 96% (method A), tR ¼ 19.3 min; [
a]
¼ ꢄ309.3
D
(c 0.63, DMSO); 1H NMR (400 MHz, DMSO-d6)
d: 8.27 (s,1H), 8.24 (s,
1H), 8.19e8.17 (m, 1H), 7.69e7.67 (m, 2H), 7.46e7.44 (m, 4H), 7.34e
7.31 (m, 2H), 7.26e7.21 (m, 1H), 5.48e5.43 (m, 1H), 5.30e5.28 (m,
DMSO-d6)
d
: 10.49 (s, 1H), 8.46e8.44 (m, 1H), 8.29e8.27 (m, 2H),
1H), 5.06e5.03 (m, 1H), 4.56e4.55 (m, 2H), 3.82e3.72 (m, 2H); 13
C
7.69e7.67 (m, 1H), 7.52e7.50 (m, 2H), 7.39e7.35 (m, 2H), 7.31e7.27
(m, 1H), 7.24e7.20 (m, 1H), 7.03e7.01 (m, 1H), 6.96e6.93 (m, 1H),
5.87e5.78 (m, 1H), 4.87e4.80 (m, 1H), 4.75e4.69 (m, 1H); 13C NMR
NMR (100 MHz, DMSO-d6) d: 165.4, 156.9, 154.2, 143.6, 141.5, 138.6,
134.1, 128.6 (2C), 127.8 (2C), 127.5 (2C), 127.4, 125.8 (2C), 118.1, 115.7,
65.2, 62.9, 56.8; IR (neat, cmꢄ1): 3326, 3261, 1595, 1316, 1041, 699;
HRMS (APCI/ASAP, m/z): 378.1276 (calcd. C21H20N3O2S, 378.1276,
[M þ H]þ).
(100 MHz, DMSO-d6)
d
: 165.5, 155.9, 154.1, 153.2, 138.4 (d, J ¼ 6.0),
135.4, 129.4, 128.4 (2C), 127.9, 127.5, 127.2 (2C), 120.0, 119.6, 116.54,

372
S. Bugge et al. / European Journal of Medicinal Chemistry 75 (2014) 354e374
4.6.35. (S)-2-(4-((2-Hydroxy-1-phenylethyl)amino)thieno[2,3-d]
pyrimidin-6-yl)phenol ((S)-33n)
(168 mg, 0.931 mmol) and the building block (S)-23$HCl (307 mg,
0.778 mmol). The reaction time was 3 h. The crude product was
purified by silica gel column chromatography (EtOAc/n-pentane, 9/
1), Rf ¼ 0.27. This gave 225 mg (0.554 mmol, 71%) of (S)-33s as a
Compound (S)-33n was prepared as described in Section 4.6.1,
but starting with compound (S)-23$HCl (150 mg, 0.388 mmol) and
(2-hydroxyphenyl)boronic acid (4n) (80 mg, 0.580 mmol), and
reacting for 3 h. The crude product was purified with silica-gel
column chromatography (EtOAc), Rf ¼ 0.30. The purified product
was dissolved in diethyl ether (2 mL) and slowly added to n-
pentane (30 mL) to give precipitation. Evaporation and drying gave
bright yellow solid, mp 203e206 ꢀC; HPLC purity: 96% (method A),
20
tR ¼ 22.6 min; [
a
]
¼ ꢄ249.8 (c 0.92, DMSO); 1H NMR (400 MHz,
D
DMSO-d6)
d
: 10.04 (s, 1H), 8.52 (s, 1H), 8.33e8.27 (m, 2H), 8.04e
7.99 (m, 1H), 7.71e7.66 (m, 2H), 7.48e7.42 (m, 2H), 7.37e7.29 (m,
2H), 7.27e7.20 (m, 1H), 5.53e5.43 (m, 1H), 5.06 (t, J ¼ 5.6, 1H), 4.07
116 mg (0.319 mmol, 82%) of (S)-33n as a white solid, mp 137e
(s, 3H), 3.83e3.73 (m, 2H); 13C NMR (100 MHz, DMSO-d6)
d: 192.2,
20
139 ꢀC; HPLC purity: 97% (method A), tR ¼ 23.5 min; [
a
]
¼ ꢄ293.3
166.3, 156.5, 155.7, 154.2, 141.0, 136.6, 132.3, 128.20 (2C), 128.18,
127.6, 127.0 (2C), 126.9, 122.8, 119.5, 116.2, 112.2, 64.6, 56.4, 56.2; IR
(neat, cmꢄ1): 3270, 3064, 2837, 1693, 1592, 1509, 1266, 1154, 1030,
773, 696, 562; HRMS (APCI/ASAP, m/z): 406.1222 (calcd.
D
(c 0.84, DMSO); 1H NMR (400 MHz, DMSO-d6)
d: 10.43 (s, 1H), 8.28
(s, 1H), 8.24 (s, 1H), 8.18e8.16 (m, 1H), 7.70e7.68 (m, 1H), 7.45e7.43
(m, 2H), 7.34e7.30 (m, 2H), 7.24e7.19 (m, 2H), 7.02e7.00 (m, 1H),
6.96e6.93 (m, 1H), 5.49e5.42 (m, 1H), 5.04e5.01 (m, 1H), 3.83e
C
22H20N3O3S, 406.1225, [M þ H]þ).
3.72 (m, 2H); 13C NMR (100 MHz, DMSO-d6)
d: 165.8, 156.6, 154.4,
153.8, 141.7, 135.3, 129.8, 128.6 (2C), 128.3, 127.5 (2C), 127.3, 120.6,
120.1, 117.0, 116.9, 116.8, 65.2, 60.2; IR (neat, cmꢄ1): 3057, 1578,
1450, 747, 698; HRMS (APCI/ASAP, m/z): 364.118 (calcd.
4.6.39. (S)-2-((6-(4-(Hydroxymethyl)-2-methoxyphenyl)thieno
[2,3-d]pyrimidin-4-yl)amino)-2-phenylethanol ((S)-33t)
Compound (S)-33t was prepared as described in Section 4.6.17,
but starting with compound (S)-33s (71 mg, 0.174 mmol). The crude
product was purified by silica-gel column chromatography (EtOAc),
Rf ¼ 0.20. Drying gave 33 mg (0.081 mmol, 47%) of (S)-33t as a pale
C
20H18N3O2S, 364.1120, [M þ H]þ).
4.6.36. (R)-2-(4-((2-Hydroxy-1-phenylethyl)amino)thieno[2,3-d]
pyrimidin-6-yl)phenol ((R)-33n)
yellow solid, mp 183e185 ꢀC; HPLC purity: 96% (method A):
20
Compound (R)-33n was prepared as described in Section 4.6.1,
but starting with compound (R)-23 (150 mg, 0.428 mmol) and (2-
hydroxyphenyl)boronic acid (4n) (89 mg, 0.645 mmol), and react-
ing for 6 h. The crude product was purified by silica-gel column
chromatography (EtOAc), Rf ¼ 0.32. The purified product was dis-
solved in diethyl ether (2 mL) and slowly added to n-pentane
(30 mL) to give precipitation. Evaporation and drying gave 124 mg
tR ¼ 18.8 min; [
a]
¼ ꢄ332.0 (c 1.00, DMSO); 1H NMR (400 MHz,
D
DMSO-d6)
d
: 8.27e8.24 (m, 2H), 8.15 (d, J ¼ 8.0, 1H), 7.75e7.71 (m,
1H), 7.46e7.42 (m, 2H), 7.35e7.29 (m, 2H), 7.26e7.20 (m, 1H), 7.17e
7.15 (m, 1H), 7.09e7.04 (m, 1H), 5.50e5.43 (m, 1H), 5.32 (t, J ¼ 5.7,
1H), 5.03 (t, J ¼ 5.7, 1H), 4.56 (d, J ¼ 5.7, 2H), 3.95 (s, 3H), 3.83e3.72
(m, 2H); 13C NMR (100 MHz, DMSO-d6)
d: 165.5, 156.2, 155.4, 153.5,
144.8, 141.2, 134.0, 128.2 (2C), 127.6, 127.0 (2C), 126.9, 120.2, 118.9,
116.5, 116.3, 110.2, 64.7, 62.6, 56.3, 55.8; IR (neat, cmꢄ1): 3267, 3027,
2834, 1595, 1509, 1049, 772, 692, 559; HRMS (APCI/ASAP, m/z):
408.1380 (calcd. C22H22N3O3S, 408.1382, [M þ H]þ).
(0.341 mmol, 80%) of (R)-33n as a white solid, mp 136e139 ꢀC;
20
HPLC purity: 97% (method A), tR ¼ 23.5 min; [
a]
¼ 275.2 (c 0.58,
D
DMSO); 1H NMR (400 MHz, DMSO-d6)
d
: 10.44 (s, 1H), 8.28 (s, 1H),
8.24 (s, 1H), 8.18e8.16 (m, 1H), 7.70e7.68 (m, 1H), 7.45e7.43 (m,
2H), 7.34e7.30 (m, 2H), 7.24e7.19 (m, 2H), 7.02e7.00 (m, 1H), 6.96e
6.93 (m, 1H), 5.49e5.42 (m, 1H), 5.04e5.01 (m, 1H), 3.83e3.72 (m,
Author contributions
2H); HRMS (APCI/ASAP, m/z): 364.1119 (calcd.
C20H18N3O2S,
Cellular experiments were performed by Unni Nonstad and Geir
Bjørkøy. The synthetic work was performed by Steffen Bugge, Svein
Jacob Kaspersen and Synne Larsen. Docking experiments were
performed by Eirik Sundby.
364.1120, [M þ H]þ). The spectroscopic properties confirmed with
that reported for (S)-33n.
4.6.37. (S)-2-((6-(2-Methoxyphenyl)thieno[2,3-d]pyrimidin-4-yl)
amino)-2-phenylethan-1-ol ((S)-33p)
Acknowledgements
Compound (S)-33p was prepared as described in Section 4.6.1,
but with starting compound (S)-23$HCl (150 mg, 0.388 mmol) and
(2-methoxyphenyl)boronic acid (4p) (88 mg, 0.579 mmol), reacting
for 4 h. The crude product was purified by silica-gel column chro-
matography (n-pentane/EtOAc, 2/8), Rf ¼ 0.29. The purified product
was dissolved in diethyl ether (2 mL) and slowly added to n-
pentane (30 mL) to give precipitation. Evaporation and drying gave
Susana Villa Gonzalez is acknowledged for the HRMS experi-
ments. Roger Aarvik, Jin Han and Ellen M. Skjønsfjell are
acknowledged for their contributions. Prof. Dr. Justus Duyster and
Dr. Nikolas von Bubnoff, Technical University of Munich, Munich,
Germany kindly provided EGFR Ba/F3 cells. Anders Jahres Foun-
dation and Technology Transfer Office (TTO-NTNU Trondheim) are
thanked for financial support.
130 mg (0.344 mmol, 89%) of (S)-33p as a white solid, mp 186e
20
189 ꢀC; HPLC purity: 98% (method A), tR ¼ 23.7 min; [
a
]
¼ ꢄ307.8
D
(c 0.70, DMSO); 1H NMR (400 MHz, DMSO-d6)
d: 8.28e8.26 (m, 2H),
Appendix A. Supporting information
8.18e8.16 (m, 1H), 7.78e7.76 (m, 1H), 7.45e7.37 (m, 3H), 7.34e7.30
(m, 2H), 7.25e7.19 (m, 2H), 7.13e7.09 (m, 1H), 5.50e5.45 (m, 1H),
5.06e5.03 (m, 1H), 3.96 (s, 3H), 3.83e3.73 (m, 2H); 13C NMR
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.042.
(100 MHz, DMSO-d6) d: 166.1, 156.7, 155.9, 154.0, 141.6, 134.3, 130.2,
128.6 (2C), 128.4, 127.5 (2C), 127.3, 122.3, 121.6, 117.5, 116.8, 113.0,
65.1, 56.8, 56.3; IR (neat, cmꢄ1): 3274, 3085, 2836, 1582, 1509, 1024,
747, 697; HRMS (APCI/ASAP, m/z): 378.1274 (calcd. C21H20N3O2S,
378.1276, [M þ H]þ).
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