Exploring the functionalisation of the thieno[2,3-d]pyrimidinedione core: Late stage access to highly substituted 5-carboxamide-6-aryl scaffolds

Article abstract of DOI:10.1016/j.tet.2018.06.019

The thieno[2,3-d]pyrimidinedione core is found as a component in a range of pharmaceutically active compounds, however, synthetic approaches to these scaffolds rely on access to functionalised, highly substituted thiophenes. Here we describe a new approach for the preparation of 5-carboxamide-6-aryl analogues that involves a two-step synthesis of the thieno[2,3-d]pyrimidinedione core from a readily available mercaptouracil derivative. Thio-alkylation with ethyl 3-bromopyruvate, followed by cyclisation and dehydration mediated by polyphosphoric acid allowed the scalable synthesis of the thieno[2,3-d]pyrimidinedione unit. The late-stage functionalisation of this core motif via bromination of the thiophene ring and a subsequent Suzuki-Miyaura reaction as the key steps permitted access to a novel library of 5-carboxamide-6-aryl analogues. The physicochemical properties of these compounds were determined, generating an insight into the potential bioavailability of these scaffolds. Based on these results, a selection of the novel 5-carboxamide-6-aryl analogues were tested as lactate uptake inhibitors of monocarboxylate transporters 1, 2 and 4 in Xenopus oocytes.

Full text of DOI:10.1016/j.tet.2018.06.019

Accepted Manuscript
Exploring the functionalisation of the thieno[2,3-d]pyrimidinedione core: Late stage
access to highly substituted 5-carboxamide-6-aryl scaffolds
Kerry M. O'Rourke, Erin S. Johnstone, Holger M. Becker, Sally L. Pimlott, Andrew
Sutherland
PII:
S0040-4020(18)30690-2
10.1016/j.tet.2018.06.019
TET 29615
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 18 April 2018
Revised Date: 5 June 2018
Accepted Date: 8 June 2018
Please cite this article as: O'Rourke KM, Johnstone ES, Becker HM, Pimlott SL, Sutherland A, Exploring
the functionalisation of the thieno[2,3-d]pyrimidinedione core: Late stage access to highly substituted 5-
carboxamide-6-aryl scaffolds, Tetrahedron (2018), doi: 10.1016/j.tet.2018.06.019.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Exploring the functionalisation of the
Leave this area blank for abstract info.
thieno[2,3-d]pyrimidinedione core: late stage
access to highly substituted 5-carboxamide-
6
-aryl scaffolds
a
a
b
c
a
Kerry M. O’Rourke, Erin S. Johnstone, Holger M. Becker, Sally L. Pimlott and Andrew Sutherland*
a
WestCHEM, School of Chemistry, The Joseph Black Building, University of Glasgow, Glasgow, G12 8QQ, UK.
b
Institute of Physiological Chemistry, University of Veterinary Medicine Hannover, D-30559 Hannover,
c
Germany. West of Scotland PET Centre, Greater Glasgow and Clyde NHS Trust, Glasgow, G12 0YN, UK.

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Exploring the functionalisation of the thieno[2,3-d]pyrimidinedione core: late stage
access to highly substituted 5-carboxamide-6-aryl scaffolds
a
a
b
c
a
Kerry M. O’Rourke, Erin S. Johnstone, Holger M. Becker, Sally L. Pimlott and Andrew Sutherland*
a
b
WestCHEM, School of Chemistry, The Joseph Black Building, University of Glasgow, Glasgow, G12 8QQ, UK. Institute of Physiological Chemistry,
c
University of Veterinary Medicine Hannover, D-30559 Hannover, Germany. West of Scotland PET Centre, Greater Glasgow and Clyde NHS Trust, Glasgow,
G12 0YN, UK.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The thieno[2,3-d]pyrimidinedione core is found as a component in a range of pharmaceutically
active compounds, however, synthetic approaches to these scaffolds rely on access to
functionalised, highly substituted thiophenes. Here we describe a new approach for the
preparation of 5-carboxamide-6-aryl analogues that involves a two-step synthesis of the
thieno[2,3-d]pyrimidinedione core from a readily available mercaptouracil derivative. Thio-
alkylation with ethyl 3-bromopyruvate, followed by cyclisation and dehydration mediated by
polyphosphoric acid allowed the scalable synthesis of the thieno[2,3-d]pyrimidinedione unit.
The late-stage functionalisation of this core motif via bromination of the thiophene ring and a
subsequent Suzuki-Miyaura reaction as the key steps permitted access to a novel library of 5-
carboxamide-6-aryl analogues. The physicochemical properties of these compounds were
determined, generating an insight into the potential bioavailability of these scaffolds. Based on
these results, a selection of the novel 5-carboxamide-6-aryl analogues were tested as lactate
uptake inhibitors of monocarboxylate transporters 1, 2 and 4 in Xenopus oocytes.
Available online
Keywords:
Thienopyrimidinedione
Thiophene synthesis
Halogenation
Suzuki-Miyaura reaction
Amide coupling
2
009 Elsevier Ltd. All rights reserved
.
2,5
1
. Introduction
aminothiophenes with isocyanates
or the alkylation of
mercaptouracil derivatives with α-halocarbonyl compounds,
Due to their similarity in structure to nucleic acid bases,
followed by cyclisation in the presence of Lewis acids such as
pyrrolo[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine scaffolds
3c,8,9
titanium tetrachloride.
1
display a wide range of biological activities. Within this
structural class, the thieno[2,3-d]pyrimidinediones have been of
particular interest, with pharmaceutical activity against a wide
range of disease states. For example, thieno[2,3-
d]pyrimidinediones bearing a N-piperazinylethyl moiety such as
1
(Fig. 1) are potent oral antihypertensive agents that can reduce
2
systolic blood pressure. In a programme to discover new
inhibitors of the monocarboxylate transporter 1, AstraZeneca
showed that thieno[2,3-d]pyrimidinediones with 5-carboxamide-
3
6
-alkyl substituents had potent immunomodulatory activity.
More recently, compounds from this series (e.g. AZD3965 2)
4
have been shown to kill tumour cells reliant on glycolysis. Other
examples include thieno[2,3-d]pyrimidinediones with a 6-(p-
5
methoxyureidophenyl) substituent, such as relugolix 3. These
compounds are selective antagonists of the gonadotropin-
releasing hormone receptor and are in phase 3 clinical trials for
6,7
the treatment of endometrosis and prostrate cancer.
Despite the importance of thieno[2,3-d]pyrimidinediones,
there are relatively few synthetic approaches that allow both the
efficient preparation of the bicyclic core and further
functionalisation of either ring. Common approaches to access
thieno[2,3-d]pyrimidinediones include the reaction of 2-
Fig 1. Pharmaceutically active thieno[2,3-d]pyrimidinediones.

2
Tetrahedron
edSC
Building on our research programme that
A
se
C
ek
C
s t
E
o
P
di
T
sc
E
ov
D
er MAut
N
ilis
U
a
R
tw
I
o-stage process involving S-alkylation with 2-
P
T
1
0
novel biologically active polycyclic scaffolds,
we were
chlorocarbonyl compounds followed by cyclisation and
3a,c
interested in developing a scalable synthesis of a thieno[2,3-
d]pyrimidinedione core that could then be further functionalised
allowing late stage access to a diverse series of highly substituted
analogues. We now report a three-stage synthetic approach for
the rapid preparation of a small library of novel thieno[2,3-
d]pyrimidinedione-5-carboxamide-6-aryl analogues. We have
determined various physicochemical properties of these
compounds, highlighting the potential of this scaffold for
medicinal chemistry applications. The ability of these novel
thieno[2,3-d]pyrimidinedione-5-carboxamide-6-aryl analogues to
inhibit lactate uptake of monocarboxylate transporters 1, 2 and 4
in Xenopus oocytes is also presented.
dehydration with titanium tetrachloride.
approaches generally result in low overall yields (18–37%).
We attempted a similar transformation with 7 and ethyl 3-
bromopyruvate. Although the S-alkylated intermediate 8 was
formed cleanly (by H NMR spectroscopy), cyclisation and
dehydration with titanium tetrachloride gave only a 24% overall
yield of thieno[2,3-d]pyrimidinedione 5 (Table 1, entry 1). In
addition, it was difficult to reproduce these results using this two-
stage process. As a consequence of the poor yields and
reproducibility of using titanium tetrachloride to form the
thiophene ring, other methods were investigated. Thieno[2,3-
d]pyrimidinediones have been prepared efficiently from
mercaptopyrimidinedione 7, by deprotonation with sodium
However, these
3a,c
1
1
1
acetate, followed by reaction with α-halocarbonyl compounds.
However, application of this method with 7 and ethyl 3-
bromopyruvate gave only a 10% yield of 5, as well as a number
of side-products (entry 2).
2
. Results and discussion
12
As outlined in Scheme 1, a three-stage approach was proposed
for the preparation of the target thieno[2,3-d]pyrimidinedione-5-
carboxamide-6-aryl analogues. The first-stage involved the
development of a reproducible, scalable synthesis of thieno[2,3-
d]pyrimidinedione 5 from commercially available 6-chloro-3-
methyluracil (4). The aim was then to investigate the reactivity of
this scaffold for the introduction of aryl groups at the 6-position.
Table 1. Optimisation of the synthesis of thieno[2,3-d]pyrimidinedione 5.
Finally, as thieno[2,3-d]pyrimidinediones bearing
a
5-
1
b,3
carboxamide moiety are often biologically active,
strategies
for rapid conversion of the thiophene-carboxylate ester to various
carboxamide groups were then explored.
a
Entry
1
Step 1
Step 2
Yield (%) of 5
K
2
CO
3
, DMF,
TiCl
4
2 2
, CH Cl , 0 ºC
24%
rt, 16 h
to rt, 16 h
NaOAc, H
2
O,
2
60 ºC, 48 h
10%
rt, 5 h
3
4
5
EtOH, rt, 1 h
EtOH, rt, 1 h
EtOH, rt, 1 h
PPA, 100 ºC, 12 h
PPA, 130 ºC, 6 h
PPA, 145 ºC, 3 h
35%
41%
54%
Scheme 1. Proposed approach to novel thieno[2,3-d]pyrimidinedione-5-
carboxamide-6-aryl analogues from 6-chloro-3-methyluracil (4).
a
Isolated yield over the two steps.
To investigate various thiophene-forming reactions, 6-
mercaptopyrimidinedione 7 was chosen as the key substrate. This
was prepared in two steps from 6-chloro-3-methyluracil (4), by
N-alkylation with isobutyl bromide under standard base-mediated
conditions, followed by an efficient substitution reaction with
sodium hydrosulfide (Scheme 2).
Ogura
and
co-workers
demonstrated
that
mercaptopyrimidinediones could undergo S-alkylation with
simple α-halocarbonyl compounds such as bromoacetone under
neutral conditions. Following isolation and purification of the S-
9
alkylated mercaptopyrimidinediones, subsequent cyclisation with
polyphosphoric acid (PPA) gave the corresponding thieno[2,3-
d]pyrimidinediones in good yields. Using this approach as a
starting
mercaptopyrimidinedione 7 with the highly electrophilic ethyl 3-
bromopyruvate to form ester-derived thieno[2,3-
d]pyrimidinedione 5 were investigated. Reaction of 7 with ethyl
point,
conditions
for
the
reaction
of
3
8
-bromopyruvate under neutral conditions did form intermediate
cleanly (Table 1, entry 3). However, unlike the alkyl and aryl
Scheme 2. Two-step synthesis of 6-mercaptopyrimidinedione 7.
12
Previous reactions using mercaptopyrimidinedione 7 for the
preparation of analogues of thieno[2,3-d]pyrimidinedione 5 have
derived intermediates from the Ogura study, S-alkylated
mercaptopyrimidinedione 8 could not be isolated and purified

3
due to significant decomposition. Therefore, in
directly converted to ester-derived thieno[2,3-d]pyrimidinedione
using the PPA-mediated cyclisation reaction. Initially, the
A
te
C
rm
C
ed
E
ia
P
te
T
8
E
w
D
as MAhy
N
dr
U
oly
S
si
C
s
R
ofIP
ethyl ester 10 was achieved using sodium
T
hydroxide in ethanol. Coupling of the resulting carboxylic acid
with morpholine was then attempted using various standard
coupling reagents (EDCI, HBTU and EDCI/HOBt), however, all
of these reactions gave low yields (9–32%) of 14. It was
proposed that the low yields were due to the steric hinderance
associated with the highly substituted thiophene ring and the
subsequent slow reaction with the bulky coupling agents. It was
believed that this could be overcome by using a smaller and more
reactive acid chloride intermediate. Therefore, the carboxylic
acid was converted to the acid chloride under mild conditions
using oxalyl chloride and DMF. Without purification, this was
treated with morpholine, resulting in the isolation of carboxamide
5
cyclisation step was found to proceed at 100 ºC, generating
thieno[2,3-d]pyrimidinedione 5 in modest yield (entry 3).
Optimisation of this step included increasing the reaction
temperature, resulting in shorter reaction times. At an optimal
temperature of 145 ºC, this gave 5 in 54% overall yield from the
13
one-pot, two step procedure (entry 5). More importantly, this
approach was found to be readily reproducible and could be used
for the multigram synthesis of 5.
The next stage of this research programme involved aryl
substitution of the 6-position of thieno[2,3-d]pyrimidinedione 5.
A two-step strategy was proposed involving bromination of the
thiophene ring, followed by a Suzuki-Miyaura reaction (Scheme
1
4 in 48% yield over the three steps (Scheme 4). Following the
development of a straightforward approach to access the
thieno[2,3-d]pyrimidinedione-5-carboxamide-6-aryl scaffold, the
scope of this three step transformation was explored with
structurally different amines, for the formation of carboxamides
bearing cyclic and acyclic groups. As Weinreb amides are
commonly biologically active due to an ability to hydrogen bond
to biological targets, a series of these were also formed. Overall,
all four Suzuki-Miyaura products 10–13 were easily converted
via the three step sequence to the corresponding carboxamides.
Yields were generally good for preparation of the morpholine
14
3
). Bromination of 5 under standard conditions with N-
bromosuccinimide (NBS), in the presence of acetic acid gave the
corresponding bromide 9 in 84% yield. Suzuki-Miyaura reaction
of 9 was then performed with various boronic acids using
Pd(PPh ) as the catalyst. To explore the electronic and steric
3
4
3
limitations of this process, both electron-rich and electron-
deficient phenylboronic acids bearing either ortho- or para-
substituents were investigated. Despite the variations,
consistently high yields were observed for all four analogues
formed from this reaction.
carboxamides 14
yields were observed over the three steps for the diethyl
carboxamide analogues 18 21. In comparison to the other
–17 and the Weinreb amides 22–25. Lower
–
amines, this is likely due to the less rigid and less reactive nature
of diethylamine.
Scheme 3. Bromination and Suzuki-Miyaura reaction of thieno[2,3-
d]pyrimidinedione-5-carboxylate ester 5.
Having demonstrated efficient functionalisation of the C-6
position of the thieno[2,3-d]pyrimidinedione core through
incorporation of various aryl groups, the final stage required
preparation of the C-5 carboxamide. Initial studies began with the
synthesis of morpholine carboxamide 14 (Scheme 4). Rapid
Scheme 4. Preparation of the thieno[2,3-d]pyrimidinedione 5-carboxamide-6-
a
aryl library. Isolated yields of 14–25 over the three steps are shown. Hünig’s
base was also used for the amidation step.

4
Tetrahedron
ACCEPTED MAas
N
co
U
nt
S
ro
C
l (
R
bla
I
nk) experiments, AR-C155858 (AR-C in Fig. 2), a
P
T
commercially available inhibitor of MCTs was also tested as a
With the successful synthesis of a library of novel thieno[2,3-
3c
standard. While none of the compounds showed any reduction
d]pyrimidinedione-5-carboxamides, we were interested in
evaluating how the incorporation of aryl groups at the C-6
position might affect the physicochemical properties. For
compounds that might find application in binding to neurological
receptors by penetrating the blood brain barrier, permeability
across the plasma membrane is important. Similarly, for
compounds that transport across cell membranes through passive
diffusion, evaluation of the membrane partition coefficient is
crucial. Therefore, the partition coefficient (log P), permeability
in lactate uptake against MCT1, morpholine carboxamide 15
showed significant activity against MCT2 (p≤0.01). For several
of the compounds, a tendency to inhibit MCT2 was also
observed, however the reduction in transport activity did not
significantly differ from the control cells. The morpholine
carboxamides also showed a slight inhibitory effect on MCT4,
with compounds 14 and 16 showing ~10% reduction in uptake.
Although these compounds are not significantly active in
inhibiting lactate uptake (each compound was tested at 1 µM),
this study has demonstrated the structure activity relationship of
this series and identified thieno[2,3-d]pyrimidinedione
morpholine carboxamide derivatives as potential scaffolds for
further development as lactate uptake inhibitors of MCTs.
(
P ), the membrane partition coefficient (K ) and the percentage
m
m
of plasma protein binding (%PPB) of all twelve thieno[2,3-
d]pyrimidinedione-5-carboxamides were evaluated using
established HPLC methods (Table 2). Previous work by Tavares
et al., of ten biologically active compounds, established the limits
of each of these parameters (log P < 4, P < 0.5, Km < 250,
m
15
%
PPB < 95%). Based on these criteria, the physicochemical
properties of the majority of these compounds were found to be
excellent and well within the acceptable limits. Despite the
incorporation of an aryl moiety and the resulting increase in
lipophilicity, these compounds possess properties that should
allow effective transport through cell membranes. Due to the
relatively lipophilic diethyl carboxamide group, compounds 18
and 19 were found to have the highest plasma protein binding
(%PPB). While these values are above the acceptable limit, there
are medicinally important compounds with similar plasma
13
protein binding that still demonstrate good bioavailability.
Therefore, all of these structural classes are of interest for further
development.
Table 2. Physicochemical values of compounds 14–25.
a
b
b
c
Entry
Compound
Log P
3.02
2.97
2.94
2.87
3.67
3.63
3.69
4.22
3.38
3.32
3.52
3.53
P
m
K
m
%PPB
87.8
93.2
90.5
92.4
95.8
96.4
90.5
93.4
87.8
88.2
88.4
86.3
1
2
3
4
5
6
7
8
9
14
15
16
17
18
19
20
21
22
23
24
0.12
0.17
0.18
0.16
0.44
0.42
0.25
0.33
0.23
0.17
0.20
0.18
51.74
77.39
82.00
75.01
189.16
181.54
111.51
147.24
96.05
69.68
88.19
76.75
1
1
1
0
1
2
25
a
b
Determined using: C18 column; immobilised artificial membrane (IAM)
c
column; human serum albumin (HSA) coated column.
As highlighted above, thieno[2,3-d]pyrimidinediones have
significant
biological
activity,
particularly
against
Fig 2. Inhibition of lactate uptake in Xenopus oocytes, expressing (A) MCT1,
(B) MCT2 + GP70 and (C) MCT4. Each compound was tested at a
concentration of 1 µM. The lactate uptake for each compound shown is the
mean ± SEM of eight independent experiments (n=8). The asterisks refer to
the values of control cells (white bars). * p≤ 0.05, *** p≤ 0.001.
monocarboxylate transporters (MCT), with certain compounds
showing potent immunomodulatory activity or the ability to kill
3,4
tumour cells that rely on glycolysis. For these reasons, a
selection of nine of the thieno[2,3-d]pyrimidinediones prepared
in this current study were tested as inhibitors of lactate uptake of
16
MCT1, MCT2 or MCT4 in Xenopus oocytes (Fig. 2). As well

5
3
. Conclusions
Isobutyl bromide (8.90 mL, 82.2 mmol) was added to a
ACCEPTED MANUSCRIPT
solution of 6-chloro-3-methyluracil (4) (12.0 g, 74.7 mmol) and
potassium carbonate (12.4 g, 89.7 mmol) in DMSO (120 mL).
The mixture was then heated to 60 °C and stirred for 48 h. The
mixture was then cooled to room temperature and diluted with
water (40 mL) and brine (40 mL). The product was then
extracted using diethyl ether (3 × 40 mL). The organic layers
In summary, a flexible and concise approach for the
preparation of novel thieno[2,3-d]pyrimidinedione scaffolds has
been developed. In particular, a reliable and scalable route for the
synthesis of a thieno[2,3-d]pyrimidinedione core bearing a C-5
ester moiety has been achieved from a mercaptouracil derivative
by an alkylation reaction with ethyl 3-bromopyruvate followed
by an acid-mediated cyclisation. The product of this process has
served as a key intermediate to explore the introduction of aryl
groups at the C-6 position via bromination and a Suzuki-Miyaura
reaction. Despite the highly substituted nature of the thiophene
ring, transformation of the C-6 substituted esters to the
corresponding amides was readily achieved via the formation of
acid chlorides under mild conditions. Determination of several
important physicochemical properties of this small library of
thieno[2,3-d]pyrimidinedione-5-carboxamide-6-aryl analogues
showed that despite the introduction of aryl units, the majority of
these compounds still retain the potential ability to transport
through cell membranes. Finally, a selection of compounds
prepared in this study was tested for the ability to inhibit lactate
uptake against various monocarboxylate transporters, with the
morpholine carboxamide analogues showing most activity. Work
is currently underway to further develop this class of thieno[2,3-
d]pyrimidinediones for a range of biological applications, as well
as explore further synthetic applications of this functionally rich
bicyclic core.
were combined, dried (MgSO ), filtered and concentrated in
4
vacuo to provide title compound 6 (13.7 g, 84%) as an orange oil.
This was used directly in the next step without further
purification. Spectroscopic data were consistent with the
17
1
literature. H NMR (400 MHz, CDCl ) δ 0.96 (6H, d, J = 6.8
3
Hz, CH(CH ) ), 2.11–2.22 (1H, m, 2’-H), 3.34 (3H, s, 3-CH ),
3
2
3
13
3
.90 (2H, d, J = 7.6 Hz, 1’-H ), 5.92 (1H, s, 5-H); C NMR (101
MHz, CDCl ) δ 19.7 (2 × CH ), 28.1 (CH ), 28.3 (CH), 53.5
CH ), 101.9 (CH), 146.0 (C), 151.2 (C), 160.9 (C); MS (CI) m/z
2
3
3
3
(
2
2
+
17 (M+H , 95%), 183 (17), 69 (10).
4
.2.2. 6-Mercapto-3-methyl-1-(2’-methylpropyl)-2,4-(1H,3H)-
3a
pyrimidinedione (7)
Sodium hydrosulfide monohydrate (1.28 g, 22.7 mmol) was
added to stirred solution of 6-chloro-3-methyl-1-(2’-
a
methylpropyl)-2,4-(1H,3H)-pyrimidinedione (6) (4.10 g, 18.9
mmol) in ethanol (60 mL). The mixture was stirred at room
temperature for 24 h before further sodium hydrosulfide
monohydrate (1.28 g, 22.7 mmol) was added and stirring
continued at room temperature for a further 16 h under an
atmosphere of argon. The solution was concentrated in vacuo and
the resulting oil diluted with water (30 mL) and washed with
ethyl acetate (30 mL). The aqueous layer was then acidified with
an aqueous solution of 1 M hydrochloric acid and then extracted
using ethyl acetate (3 × 30 mL). The combined organic layers
4
4
. Experimental section
.1 General methods
All reactions were performed under an atmosphere of air
were then dried (MgSO
give 6-mercapto-3-methyl-1-(2’-methylpropyl)-2,4-(1H,3H)-
pyrimidinedione (7) as a pale yellow solid (3.69 g, 90%).
4
), filtered and concentrated in vacuo to
unless otherwise stated. All reagents and starting materials were
obtained from commercial sources unless otherwise stated. Brine
refers to a saturated solution of sodium chloride. All dry solvents
were purified using a PureSolv 500 MD solvent purification
system. Flash column chromatography was carried out using
Merck Feduran Si 60 (40–63 µm). Macherey-Nagel aluminium-
backed plates pre-coated with silica gel 60 were used for thin
3
a
Spectroscopic data were consistent with the literature. Mp 111–
1
113 °C; H NMR (400 MHz, CDCl
CH(CH ), 2.24–2.31 (1H, m, 2’-H), 3.29 (3H, s, 3-CH
(2H, s, 5-H
MHz, CDCl
(CH ), 54.5 (CH
237 (M+Na , 60%), 159 (20), 307 (5), 449 (2).
) δ 0.92 (6H, d, J = 6.8 Hz,
), 4.14
); C NMR (101
), 28.6 (CH), 49.2
), 150.3 (C), 164.8 (C), 197.2 (C); MS (ESI) m/z
2
3
)
2
3
3
13
), 4.27 (2H, d, J = 7.4 Hz, 1’-H
) δ 20.1 (2 × CH ), 26.6 (CH
2
2
3
3
3
1
layer chromatography and were visualised under a UV lamp. H
2
13
+
NMR and C NMR spectra were recorded on a Bruker DPX 400
spectrometer or Bruker 500 spectrometer with chemical shift
values in ppm relative to trimethylsilane or residual chloroform
4
.2.3. Ethyl 1-isobutyl-3-methyl-2,4-dioxo-1,2,3,4-
3a
1
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (5)
as standard. J values are reported in Hz. The assignment of H
13
NMR spectra is based on COSY and HSQC experiments and
C
Ethyl bromopyruvate (1.70 mL, 13.3 mmol) was added to a
NMR spectra is based on DEPT experiments. Infrared spectra
were recorded using a Shimadzu FTIR-84005 spectrometer
directly as either a solid or liquid and mass spectra were obtained
using a JEOL JMS-700 spectrometer or a Bruker MicroTOFq
high-resolution mass spectrometer. Melting points were
determined on a Gallenkamp melting point apparatus. All
physicochemical analyses were performed using a Dionex
Ultimate 3000 series, and data acquisition and processing
solution
of
6-mercapto-1-isobutyl-3-methyl-2,4-(1H,3H)-
pyrimidinedione (7) (1.90 g, 8.87 mmol) in ethanol (40 mL) and
stirred at room temperature for 1 h. The reaction mixture was
concentrated in vacuo to give a red oil. A mixture of the crude
intermediate and polyphosphoric acid (10.0 g) were heated to 145
°C for 3 h, and then cooled to room temperature. A saturated
aqueous solution of sodium carbonate (15 mL) was added and the
mixture extracted with ethyl acetate (3 × 25 mL). The combined
14
performed using Chromeleon 6.8 Chromatography software.
organic layers were dried (MgSO ), filtered and concentrated in
4
Standard and test compounds were dissolved in 1:1
organic/aqueous phases, and prepared to a concentration of 0.5
mg/mL. The HPLC system was set to 25 °C, and UV detection
achieved using a diode array detector (190–800 nm). Analysis
was performed using 5 µL sample injections.
vacuo. Purification by flash column chromatography eluting with
20% ethyl acetate in petroleum ether gave ethyl 1-isobutyl-3-
methyl-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-
carboxylate (5) as a dark pink solid (1.46 g, 54%). Mp 62–66 °C.
3
a 1
Spectroscopic data were consistent with the literature. H NMR
(
400 MHz, CDCl ) δ 1.00 (6H, d, J = 6.9 Hz, CH(CH ) ), 1.40
3
3
2
4
.2 Experimental procedures and compound characterisation
.2.1. 6-Chloro-3-methyl-1-(2’-methylpropyl)-2,4-(1H,3H)-
(3H, t, J = 7.4 Hz, OCH CH ), 2.26–2.38 (1H, m, 2’-H), 3.42
2 3
4
(3H, s, 3-CH
3
), 3.81 (2H, d, J = 7.6 Hz, 1’-H
2
), 4.41 (2H, q, J =
), 7.29 (1H, s, 6-H); C NMR (101 MHz,
CDCl ) δ 14.2 (CH ), 20.0 (2 × CH ), 27.0 (CH), 28.5 (CH ),
17
13
pyrimidinedione (6)
7.4 Hz, OCH CH
2 3
3
3
3
3
5
6.2 (CH ), 61.9 (CH ), 112.6 (C), 119.4 (CH), 132.3 (C), 150.8
2 2

6
Tetrahedron
(
C), 154.2 (C), 157.0 (C), 162.9 (C); M
A
S
C
(E
C
SI
E
)
P
m/z
T
E
3
D
33 MAIR
NU
(n
ea
S
t)
C
29
R
6
I
3
P
(C
T
H), 1732 (CO), 1707 (CO), 1661 (CO), 1487,
+
−1 1
(M+Na , 100%).
1223, 1018, 758 cm ; H NMR (400 MHz, CDCl ) δ 1.01 (6H,
3
d, J = 7.3 Hz, CH(CH ) ), 1.26 (3H, t, J = 7.1 Hz, OCH CH ),
3
2
2
3
4
.2.4. Ethyl 6-bromo-1-isobutyl-3-methyl-2,4-dioxo-1,2,3,4-
2
7
.30–2.40 (1H, m, 2’-H), 3.42 (3H, s, 3-CH ), 3.82 (2H, d, J =
3
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (9)
.5 Hz, 1’-CH ), 4.35 (2H, q, J = 7.1 Hz, OCH CH ), 7.13–7.18
2
2
3
N-Bromosuccinimide (0.598 g, 3.36 mmol) was added to a
stirred solution of ethyl 1-isobutyl-3-methyl-2,4-dioxo-1,2,3,4-
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (5) (0.870 g,
(1H, m, 3”-H), 7.20 (1H, td, J = 7.8, 1.2 Hz, 5”-H), 7.36–7.42
(1H, m, 6”-H), 7.51 (1H, td, J = 7.8, 1.6 Hz, 4”-H); C NMR
13
(101 MHz, CDCl
28.3 (CH), 56.3 (CH
21.7 Hz, CH), 118.9 (d, JCF = 15.1 Hz, C), 124.5 (d, JCF = 3.9
) δ 14.0 (CH
), 62.1 (CH
2
), 20.1 (2 × CH
), 113.4 (C), 116.1 (d, JCF
), 27.0 (CH
),
=
3
3
3
3
2
2
.80 mmol) in dichloromethane (15 mL) and acetic acid (15 mL)
2
2
4
and stirred at room temperature for 5 h. The reaction mixture was
azeotroped under vacuum in the presence of toluene (3 × 30 mL).
Purification by flash column chromatography eluting with 20%
ethyl acetate in petroleum ether gave ethyl 6-bromo-1-isobutyl-3-
methyl-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-
carboxylate (9) as a white solid (0.911 g, 84%). Mp 110–114 °C;
IR (neat) 2961 (CH), 1736 (CO), 1707 (CO), 1661 (CO), 1481,
3
Hz, CH), 126.2 (C), 129.7 (C), 131.1 (d, JCF = 8.0 Hz, CH),
3
131.1 (d, JCF = 9.8 Hz, CH), 150.7 (C), 153.4 (C), 157.4 (C),
1
159.5 (d, JCF = 251.9 Hz, C), 164.1 (C); MS (ESI) m/z 427
+
(M+Na , 100%); HRMS (ESI) calcd for C20
H21FN
NaO
S
2
4
+
(M+Na ), 427.1098, found 427.1087.
−
1
1
4.2.7.
dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate
12)
Ethyl
1-isobutyl-6-(4”-methoxyphenyl)-3-methyl-2,4-
1
6
2
1
283, 1238 cm ; H NMR (400 MHz, CDCl ) δ 0.99 (6H, d, J =
3
.7 Hz, CH(CH ) ), 1.42 (3H, t, J = 7.1 Hz, OCH CH ), 2.24–
.33 (1H, m, 2’-H), 3.40 (3H, s, 3-CH ), 3.72 (2H, d, J = 7.8 Hz,
’-H ), 4.48 (2H, q, J = 7.1 Hz, OCH CH ); C NMR (101 MHz,
3
2
2
3
(
3
13
The reaction was carried out according to the previously
2
2
3
CDCl ) δ 14.1 (CH ), 20.0 (2 × CH ), 27.0 (CH), 28.4 (CH ),
described procedure for compound 10 using ethyl 6-bromo-1-
isobutyl-3-methyl-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-
d]pyrimidine-5-carboxylate (9) (0.20 g, 0.51 mmol) and 4-
methoxyphenylboronic acid (0.10 g, 0.67 mmol). This gave title
compound 12 as a white solid (0.18 g, 85%). Mp 129–131 °C; IR
3
3
3
3
5
6.5 (CH ), 62.6 (CH ), 103.5 (C), 113.1 (C), 132.5 (C), 150.5
2 2
+
(C), 153.2 (C), 156.4 (C), 162.7 (C); MS (ESI) m/z 411 (M+Na ,
79
+
1
4
00%); HRMS (ESI) calcd for C H BrN NaO S (M+Na ),
14 17 2 4
10.9985, found 410.9972.
(
1
neat) 2961 (CH), 1730 (CO), 1705 (CO), 1659 (CO), 1537,
483, 1294, 1252, 1204, 1020, 831, 729 cm ; H NMR (400
4
1
.2.5. Ethyl 6-(4”-fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (10)
−1
1
MHz, CDCl ) δ 1.01 (6H, d, J = 6.7 Hz, CH(CH ) ), 1.31 (3H, t,
3
3 2
Potassium phosphate (0.18 g, 0.78 mmol) was suspended in a
J = 7.3 Hz, OCH
2
CH
3
), 2.30–2.40 (1H, m, 2’-H), 3.41 (3H, s, 3-
solution of ethyl 6-bromo-1-isobutyl-3-methyl-2,4-dioxo-1,2,3,4-
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (9) (0.15 g, 0.39
mmol) and 4-fluorophenylboronic acid (0.070 g, 0.50 mmol) in
N,N’-dimethylformamide (10 mL) and degassed for 1 h.
Tetrakis(triphenylphosphine)palladium(0) (0.045 g, 0.039 mmol)
was added and the reaction heated under reflux for 22 h. The
reaction mixture was cooled to room temperature, extracted into
ethyl acetate (30 mL) and washed with an aqueous solution of
CH
4.39 (2H, q, J = 7.3 Hz, OCH
H and 5”-H), 7.43 (2H, d, J = 8.8 Hz, 2”-H and 6”-H); C NMR
(101 MHz, CDCl ) δ 14.0 (CH ), 20.0 (2 × CH ), 27.1 (CH ),
28.3 (CH), 55.4 (CH ), 56.3 (CH ), 62.2 (CH ), 114.0 (C), 114.4
3
), 3.80 (2H, d, J = 7.7 Hz, 1’-CH
2
), 3.84 (3H, s, 4”-OCH ),
3
CH ), 6.92 (2H, d, J = 8.8 Hz, 3”-
2
3
1
3
3
3
3
3
3
2
2
(2 × CH), 123.5 (C), 126.2 (C), 129.5 (2 × CH), 132.9 (C), 150.8
(C), 151.8 (C), 157.6 (C), 160.3 (C), 165.0 (C); MS (ESI) m/z
+
439 (M+Na , 100%); HRMS (ESI) calcd for C21
H
24
N
2
NaO
S
5
+
5
% lithium chloride (4 × 25 mL) and brine (2 × 20 mL). The
(M+Na ), 439.1298, found 439.1283.
organic layers were combined, dried (MgSO ), filtered and
4
4
.2.8.
dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate
13)
Ethyl
1-isobutyl-6-(2”-methoxyphenyl)-3-methyl-2,4-
concentrated in vacuo. Purification by flash column
chromatography eluting with 20% ethyl acetate in petroleum
ether gave ethyl 6-(4’’-fluorophenyl)-1-isobutyl-3-methyl-2,4-
dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate
(
The reaction was carried out according to the previously
(
10) as a white solid (0.13 g, 81%). Mp 143–145 °C; IR (neat)
described procedure for compound 10 using ethyl 6-bromo-1-
isobutyl-3-methyl-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-
2
1
7
2
1
2
963 (CH), 1732 (CO), 1707 (CO), 1663 (CO), 1537, 1483,
−1 1
294, 1206 cm ; H NMR (400 MHz, CDCl ) δ 1.02 (6H, d, J =
d]pyrimidine-5-carboxylate (9) (0.20 g, 0.51 mmol) and 2-
methoxyphenylboronic acid (0.10 g, 0.67 mmol). This gave title
compound 13 as a yellow solid (0.18 g, 86%). Mp 119–123 °C;
IR (neat) 2963 (CH), 1730 (CO), 1705 (CO), 1659 (CO), 1489,
3
.0 Hz, CH(CH ) ), 1.30 (3H, t, J = 7.3 Hz, OCH CH ), 2.30–
3
2
2
3
.40 (1H, m, 2’-H), 3.42 (3H, s, 3-CH ), 3.81 (2H, d, J = 7.7 Hz,
3
’-CH ), 4.38 (2H, q, J = 7.3 Hz, OCH CH ), 7.01–7.13 (2H, m,
2
2
3
13
−1 1
”-H and 6”-H), 7.46–7.51 (2H, m, 3”-H and 5”-H); C NMR
1479, 1244, 1200, 1020, 729 cm ; H NMR (400 MHz, CDCl ) δ
3
(
2
2
101 MHz, CDCl ) δ 14.0 (CH ), 20.0 (2 × CH ), 27.1 (CH ),
1.01 (6H, d, J = 6.3 Hz, CH(CH ) ), 1.23 (3H, t, J = 7.3 Hz,
3
3
3
3
3 2
2
8.3 (CH), 56.4 (CH ), 62.3 (CH ), 114.0 (C), 116.2 (d, J
=
OCH CH ), 2.31–2.41 (1H, m, 2’-H), 3.42 (3H, s, 3-CH ), 3.81
2
2
CF
2
3
3
4
2.3 Hz, 2 × CH), 127.2 (d, J = 3.8 Hz, C), 128.1 (C), 129.0
(2H, d, J = 7.8 Hz, 1’-CH ), 3.84 (3H, s, 2”-OCH ), 4.32 (2H, q,
CF
2
3
3
(
(
(
(
C), 130.2 (d, J = 9.8 Hz, 2 × CH), 150.7 (C), 152.3 (C), 157.5
C), 162.8 (d, J = 250.7 Hz, C), 167.7 (C); MS (ESI) m/z 427
M+Na , 100%); HRMS (ESI) calcd for C H FN NaO S
M+Na ), 427.1098, found 427.1093.
J = 7.3 Hz, OCH CH ), 6.95 (1H, br d, J = 7.9 Hz, 3”-H), 6.98
2 3
CF
1
(1H, td, J = 7.9, 1.4 Hz, 5”-H), 7.36 (1H, td, J = 7.9, 1.4 Hz, 4”-
CF
+
13
H), 7.43 (1H, dd, J = 7.9, 1.4 Hz, 6”-H); C NMR (101 MHz,
20
21
2
4
+
CDCl ) δ 13.9 (CH ), 20.0 (2 × CH ), 27.0 (CH ), 28.4 (CH),
3
3
3
3
5
5.5 (CH ), 56.1 (CH ), 61.8 (CH ), 111.2 (CH), 113.0 (C), 119.9
3 2 2
4
1
.2.6. Ethyl 6-(2”-fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (11)
(C), 120.8 (CH), 128.7 (C), 129.8 (C), 130.6 (CH), 130.9 (CH),
1
50.9 (C), 153.2 (C), 156.4 (C), 157.5 (C), 164.6 (C); MS (ESI)
+
The reaction was carried out according to the previously
m/z 439 (M+Na , 100%); HRMS (ESI) calcd for C21
(M+Na ), 439.1298, found 439.1281.
H
24
N
2
NaO
5
S
+
described procedure for compound 10 using ethyl 6-bromo-1-
isobutyl-3-methyl-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-
d]pyrimidine-5-carboxylate (9) (0.20 g, 0.51 mmol) and 2-
fluorophenylboronic acid (0.11 g, 0.67 mmol). This gave title
compound 11 as a light pink solid (0.18 g, 86%). Mp 94–98 °C;
4
1
.2.9.
6-(4”-Fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-N-
morpholinecarboxamide (14)

7
2
Ethyl
6-(4”-fluorophenyl)-1-isobutyl-3-
A
me
C
th
C
yl-
E
2,
P
4-
T
di
E
ox
(10)
D
o- MA(C
N
H
U
),
S
66
C
.2
R
(CIP
H ), 66.3 (CH ), 113.1 (C), 116.0 (d, J = 22.7
2 2 CF
2 4
T
2
1
(
(
2
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate
0.18 g, 0.44 mmol) was dissolved in ethanol (4 mL) and water
4 mL) prior to the addition of 4 M sodium hydroxide (1.5 mL,
Hz, CH), 118.7 (d, J = 14.5 Hz, C), 123.2 (C), 124.9 (d, J
=
CF
CF
3
3.8 Hz, CH), 131.2 (d, J = 8.3 Hz, CH), 131.5 (C), 131.5 (d,
CF
3
1
JCF = 9.3 Hz, CH), 150.8 (C), 154.2 (C), 157.6 (C), 159.4 (d, J
CF
+
.0 mmol) and stirred at 80 °C for 1 h. The reaction mixture was
= 252.8 Hz, C), 163.6 (C); MS (EI) m/z 445 (M , 62%), 402 (39),
diluted with water (10 mL) and washed with diethyl ether (2 × 10
mL). The aqueous layer was acidified with 1 M hydrochloric acid
and extracted with ethyl acetate (3 × 10 mL). The combined
359 (100), 303 (52), 276 (22), 246 (21), 190 (16), 86 (63);
+
HRMS (EI) calcd for C H FN O S (M ), 445.1472, found
22
24
3
4
445.1490.
organic layers were dried (MgSO ), filtered and concentrated in
4
4
1
.2.11.
1-Isobutyl-6-(4”-methoxyphenyl)-3-methyl-2,4-dioxo-
vacuo. The resulting yellow solid was dissolved in
dichloromethane (5 mL) and N,N’-dimethylformamide (2 drops)
and cooled to 0 °C prior to the addition of oxalyl chloride (0.045
mL, 0.53 mmol). The reaction mixture was warmed to 40 °C and
stirred for 3 h. After being cooled to room temperature, the
reaction mixture was concentrated in vacuo to yield the acid
chloride, which was used without further purification. The crude
acid chloride was dissolved in dichloromethane (5 mL) and
cooled to 0 °C. Morpholine (0.19 mL, 2.2 mmol) was added
dropwise to the stirring acid chloride solution. The reaction
mixture was stirred at 40 °C for 18 h under argon. The reaction
mixture was cooled to room temperature, diluted in water (10
mL) and extracted with dichloromethane (3 × 10 mL). The
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-N-
morpholinecarboxamide (16)
The reaction was carried out according to the previously
described procedure for compound 14 using ethyl 6-(4”-
methoxyphenyl)-1-isobutyl-3-methyl-2,4-dioxo-1,2,3,4-
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (12) (0.20 g,
0.48 mmol) and morpholine (0.21 mL, 2.4 mmol). This gave title
compound 16 (0.11 g, 48%) as a colourless oil. IR (neat) 2961
(CH), 1738 (CO), 1703 (CO), 1659 (CO), 1535 (C=C), 1485,
1252, 1113, 729 cm ; H NMR (400 MHz, CDCl ) δ 1.02 (6H,
d, J = 6.8 Hz, CH(CH ) ), 2.29–2.41 (1H, m, 2’-H), 3.00 (1H,
ddd, J = 10.8, 7.2, 2.8 Hz, NCHH), 3.12 (1H, ddd, J = 13.2, 5.2,
2.8 Hz, NCHH), 3.26 (1H, ddd, J = 13.2, 7.6, 3.2 Hz, NCHH),
−1 1
3
3
2
organic layers were combined, dried (MgSO ), filtered and
4
concentrated in vacuo. Purification by flash column
chromatography eluting with 60% ethyl acetate in petroleum
ether gave 6-(4”-fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-
3.40 (3H, s, 3-CH ), 3.47–3.54 (2H, m, NCHH and OCHH), 3.67
3
(1H, dd, J = 14.2, 7.6 Hz, 1’-HH), 3.72–3.90 (6H, m, OCHH,
OCH and 4”-OCH ), 3.94 (1H, dd, J = 14.2, 7.6 Hz, 1’-HH),
2
3
1
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-N-
6.93 (2H, d, J = 8.8 Hz, 3”-H and 5”-H), 7.46 (2H, d, J = 8.8 Hz,
13
morpholinecarboxamide (14) (0.094 g, 48%) as a colourless oil.
2”-H and 6”-H); C NMR (101 MHz, CDCl ) δ 20.0 (CH ), 20.1
3 3
IR (neat) 2970 (CH), 1740 (CO), 1724 (CO), 1366, 1229, 1217
(CH ), 27.1 (CH ), 28.3 (CH), 42.2 (CH ), 47.0 (CH ), 55.4
(CH ), 56.4 (CH ), 66.2 (CH ), 66.3 (CH ), 113.9 (C), 114.6 (2 ×
3 2 2 2
CH), 123.6 (C), 127.6 (C), 129.2 (2 × CH), 130.5 (C), 150.7 (C),
3 3 2 2
−1
1
cm ; H NMR (400 MHz, CDCl ) δ 1.03 (6H, d, J = 5.6 Hz,
3
CH(CH ) ), 2.31–2.40 (1H, m, 2’-H), 3.05 (1H, ddd, J = 8.5, 5.8,
3
2
2
3
.4 Hz, NCHH), 3.10 (1H, ddd, J = 10.6, 4.6, 2.4 Hz, NCHH),
.26 (1H, ddd, J = 10.6, 5.8, 2.4 Hz, NCHH), 3.41 (3H, s, 3-
152.4 (C), 157.6 (C), 160.3 (C), 164.3 (C); MS (ESI) m/z 480
+
(M+Na , 100%); HRMS (ESI) calcd for C H N NaO S
23
27
3
5
+
CH ), 3.47–3.57 (2H, m, NCHH and OCHH), 3.68 (1H, dd, J =
(M+Na ), 480.1564, found 480.1546.
3
1
1.4, 6.0 Hz, 1’-HH), 3.74–3.86 (3H, m, OCHH and OCH ), 3.97
2
4
.2.12. 1-Isobutyl-6-(2”-Methoxyphenyl)-3-methyl-2,4-dioxo-
(1H, dd, J = 11.4, 6.4 Hz, 1’-HH), 7.10–7.14 (2H, m, 2”-H and
13
1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-N-
morpholinecarboxamide (17)
6
”-H), 7.51–7.54 (2H, m, 3”-H and 5”-H); C NMR (101 MHz,
CDCl ) δ 20.0 (CH ), 20.1 (CH ), 27.1 (CH ), 28.3 (CH), 42.3
3
3
3
3
(
CH ), 47.0 (CH ), 56.5 (CH ), 66.2 (CH ), 66.3 (CH ), 113.9
2 2 2 2 2
2 4
The reaction was carried out according to the previously
described procedure for compound 14 using ethyl 1-isobutyl-6-
(2’’-methoxyphenyl)-3-methyl-2,4-dioxo-1,2,3,4-
(C), 116.4 (d, J = 22.3 Hz, 2 × CH), 127.4 (d, J = 3.8 Hz,
CF
CF
3
C), 128.9 (C), 129.1 (C), 129.8 (d, J = 8.2 Hz, 2 × CH), 150.7
CF
1
(C), 152.9 (C), 157.7 (C), 163.1 (d, J = 250.7 Hz, C), 164.0
CF
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (13) (0.18 g,
+
(C); MS (ESI) m/z 468 (M+Na , 100%); HRMS (ESI) calcd for
0
.44 mmol) and morpholine (0.19 mL, 2.2 mmol). This gave title
+
C H FN NaO S (M+Na ), 468.1364, found 468.1350.
22
24
3
4
compound 17 (0.082 g, 41%) as a colourless oil. IR (neat) 2961
(
7
CH), 1705 (CO), 1661 (CO), 1532 (C=C), 1489, 1256, 1113,
4
1
.2.10.
6-(2”-Fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-
−1 1
31 cm ; H NMR (400 MHz, CDCl ) δ 1.02 (3H, d, J = 6.7 Hz,
3
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-N-
CH(CH ) ), 2.28–2.42 (1H, m, 2’-H), 2.84 (1H, ddd, J = 10.4,
7
3
2
morpholinecarboxamide (15)
.6, 2.8 Hz, NCHH), 3.15 (1H, ddd, J = 13.2, 5.2, 3.2 Hz,
The reaction was carried out according to the previously
described procedure for compound 14 using ethyl 6-(2”-
fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-1,2,3,4-
NCHH), 3.26 (1H, ddd, J = 13.2, 6.4, 2.8 Hz, NCHH), 3.36 (1H,
ddd, J = 10.4, 7.6, 2.8 Hz, NCHH), 3.40 (3H, s, 3-CH ), 3.48
3
(1H, ddd, J = 11.4, 7.6, 2.8 Hz, OCHH), 3.59–3.67 (2H, m,
OCHH and 1’-HH), 3.76 (1H, ddd, J = 11.4, 6.4, 3.2 Hz,
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (11) (0.178 g,
0
.440 mmol) and morpholine (0.193 mL, 2.21 mmol). This gave
OCHH), 3.81–3.87 (4H, m, OCHH and 2”-OCH ), 4.01 (1H, dd,
3
title compound 15 as a clear oil (0.105 g, 54%). IR (neat) 2961
J = 14.0, 8.0 Hz, 1’-HH), 6.97 (1H, br d, J = 7.9 Hz, 3”-H), 7.01
(1H, td, J = 7.9, 1.4 Hz, 5”-H), 7.38 (1H, td, J = 7.9, 1.4 Hz, 4”-
−1
1
(CH), 1707 (CO), 1663 (CO), 1489, 1113, 1001, 762 cm ; H
13
NMR (400 MHz, CDCl ) δ 1.02 (3H, d, J = 5.6 Hz, CH(CH )),
H), 7.58 (1H, dd, J = 7.9, 1.4 Hz, 6”-H); C NMR (101 MHz,
CDCl ) δ 20.0 (CH ), 20.2 (CH ), 27.1 (CH ), 28.2 (CH), 42.2
3
3
1
3
1
.02 (3H, d, J = 5.2 Hz, CH(CH )), 2.30–2.40 (1H, m, 2’-H),
.04 (1H, ddd, J = 8.8, 6.0, 2.4 Hz, NCHH), 3.16 (1H, ddd, J =
0.6, 4.4, 2.4 Hz, NCHH), 3.29 (1H, ddd, J = 10.6, 6.0, 2.4 Hz,
3
3
3
3
3
(CH ), 47.0 (CH ), 55.6 (CH ), 56.1 (CH ), 66.3 (CH ), 66.3
2 2 3 2 2
(CH ), 111.0 (CH), 112.7 (C), 119.7 (C), 121.3 (CH), 126.7 (C),
2
NCHH), 3.42 (3H, s, 3-CH ), 3.43–3.47 (1H, m, OCHH), 3.56
130.1 (C), 130.8 (CH), 131.4 (CH), 150.9 (C), 154.1 (C), 156.2
3
+
(1H, ddd, J = 8.8, 4.4, 2.4 Hz, NCHH), 3.65–3.74 (2H, m, OCHH
(C), 157.7 (C), 164.1 (C); MS (ESI) m/z 480 (M+Na , 100%);
+
and 1’-HH), 3.77–3.84 (2H, m, OCH ), 3.97 (1H, dd, J = 11.2,
HRMS (ESI) calcd for C H N NaO S (M+Na ), 480.1564,
2
23 27
3
5
6
1
.4 Hz, 1’-HH), 7.15–7.20 (1H, m, 6”-H), 7.22 (1H, td, J = 7.7,
.3 Hz, 4”-H), 7.38–7.42 (1H, m, 3”-H), 7.65 (1H, td, J = 7.7, 1.3
found 480.1544.
13
Hz, 5”-H); C NMR (101 MHz, CDCl ) δ 20.0 (CH ), 20.1
3
3
(
CH ), 27.1 (CH ), 28.3 (CH), 42.2 (CH ), 47.0 (CH ), 56.4
3 3 2 2

8
Tetrahedron
4
.2.13.
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-N-
diethylcarboxamide (18)
6-(4”-Fluorophenyl)-1-isobutyl-3-
A
m
C
eth
C
yl
E
-2
P
,4-
T
di
E
ox
D
o- MA7.
N
2 H
U
z
S
, N
C
C
R
H
I
C
P
H
T
3), 2.32–2.39 (1H, m, 2’-H), 3.02–3.14 (2H, m,
2
1
NCH CH ), 3.40 (3H, s, 3-CH ), 3.48 (1H, dt, J = 13.6, 7.2 Hz,
2
3
3
NCHHCH ), 3.61–3.70 (2H, m, NCHHCH and 1’-HH), 3.82
3
3
(
3H, s, 4”-OCH ), 3.96 (1H, dd, J = 14.0, 8.0 Hz, 1’-HH), 6.89
3
The reaction was carried out according to the previously
described procedure for compound 14 using ethyl 6-(4”-
fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-1,2,3,4-
(2H, d, J = 8.9 Hz, 3”-H and 5”-H), 7.49 (2H, d, J = 8.9 Hz, 2”-H
13
and 6”-H); C NMR (101 MHz, CDCl ) δ 12.3 (CH ), 13.5
3
3
(CH ), 20.1 (CH ), 20.2 (CH ), 27.1 (CH ), 28.3 (CH), 39.2
3 3 3 3
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (10) (0.12 g,
(CH ), 43.0 (CH ), 55.3 (CH ), 56.3 (CH ), 114.0 (C), 114.4 (2 ×
2
2
3
2
0
.30 mmol) and diethylamine (0.16 mL, 1.5 mmol). This gave
CH), 124.1 (C), 129.0 (C), 129.2 (2 × CH), 129.7 (C), 150.9 (C),
52.3 (C), 157.6 (C), 160.0 (C), 165.0 (C); MS (ESI) m/z 466
title compound 18 (0.040 g, 31%) as a colourless oil. IR (neat)
1
2
1
963 (CH), 1707 (CO), 1665 (CO), 1636 (CO), 1533 (C=C),
+
−1
1
(M+Na , 100%); HRMS (ESI) calcd for C23
H
29
N
3
NaO
4
S
487, 1294, 1238, 841 cm ; H NMR (400 MHz, CDCl ) δ 0.80
+
3
(M+Na ), 466.1771, found 466.1764.
(
3H, t, J = 7.2 Hz, NCH CH ), 1.02 (3H, d, J = 6.4 Hz,
2 3
CH(CH )), 1.03 (3H, d, J = 6.8 Hz, CH(CH )), 1.21 (3H, t, J =
4.2.16.
1-Isobutyl-6-(2”-methoxyphenyl)-3-methyl-2,4-dioxo-
3
3
7
.2 Hz, NCH CH ), 2.30–2.41 (1H, m, 2’-H), 3.03–3.14 (2H, m,
1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-N-
2
3
NCH CH ) 3.40 (3H, s, 3-CH ), 3.43–3.52 (1H, m, NCHHCH ),
diethylcarboxamide (21)
2
3
3
3
3
1
7
1
.60–3.71 (2H, m, NCHHCH and 1’-HH), 3.96 (1H, dd, J =
3
The reaction was carried out according to the previously
described procedure for compound 14 using ethyl 1-isobutyl-6-
2”-methoxyphenyl)-3-methyl-2,4-dioxo-1,2,3,4-
4.0, 7.6 Hz, 1’-HH), 7.06–7.10 (2H, m, 2”-H and 6”-H), 7.54–
13
.58 (2H, m, 3”-H and 5”-H); C NMR (101 MHz, CDCl ) δ
3
(
2.2 (CH ), 13.5 (CH ), 20.0 (CH ), 20.1 (CH ), 27.1 (CH ), 28.3
3
3
3
3
3
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (13) (0.182 g,
0.440 mmol) and diethylamine (0.227 mL, 2.20 mmol). This
gave title compound 21 as a colourless oil (0.0680 g, 32%). IR
(neat) 2963 (CH), 1705 (CO), 1661 (CO), 1634 (CO), 1487,
(
CH), 39.2 (CH ), 43.0 (CH ) 56.4 (CH ), 114.0 (C), 116.1 (d,
2 2 2
4
2
JCF = 21.6 Hz, 2 × CH), 127.8 (d, J = 3.8 Hz, C), 128.4 (C),
CF
3
1
1
4
29.8 (d, J = 9.8 Hz, 2 × CH), 130.3 (C), 150.8 (C), 152.7 (C),
57.6 (C), 162.8 (d, J = 250.7 Hz, C), 164.7 (C); MS (ESI) m/z
54 (M+Na , 100%); HRMS (ESI) calcd for C H FN NaO S
CF
1
CF
−1 1
+
1251, 1020, 750 cm ; H NMR (400 MHz, CDCl
J = 7.2 Hz, NCH CH ), 1.01 (3H, d, J = 6.8 Hz, CH(CH
3H, d, J = 6.8 Hz, CH(CH )), 1.11 (3H, t, J = 7.2 Hz,
3
) δ 0.78 (3H, t,
22
26
3
3
+
2
3
3
)), 1.02
(M+Na ), 454.1571, found 454.1560.
(
3
4
1
.2.14.
6-(2”-Fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-
NCH CH ), 2.30–2.41 (1H, m, 2’-H), 3.00–3.19 (2H, m,
2 3
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-N-
NCH CH ), 3.31–3.43 (4H, m, 3-CH and NCHHCH ), 3.59–
2
3
3
3
diethylcarboxamide (19)
3.68 (2H, m, NCHHCH and 1’-HH), 3.88 (3H, s, 2”-OCH ),
3
3
4
.03 (1H, dd, J = 14.0, 8.0 Hz, 1’-HH), 6.92–6.99 (2H, m, 3”-H
The reaction was carried out according to the previously
described procedure for compound 14 using ethyl 6-(2”-
fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-1,2,3,4-
and 5”-H), 7.33 (1H, td, J = 7.8, 1.6 Hz, 4”-H), 7.68 (1H, dd, J =
7
1
13
.8, 1.6 Hz, 6”-H); C NMR (101 MHz, CDCl ) δ 12.2 (CH ),
3 3
3.4 (CH ), 20.0 (CH ), 20.2 (CH ) 27.1 (CH ), 28.2 (CH), 39.0
3
3
3
3
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (11) (0.178 g,
(CH ), 42.9 (CH ) 55.6 (CH ), 56.1 (CH ), 110.8 (CH), 112.7
2 3 2 2
0
.440 mmol) and diethylamine (0.228 mL, 2.20 mmol). This
(C), 120.1 (C), 121.1 (CH), 125.7 (C), 130.3 (CH), 131.5 (CH
gave title compound 19 (0.0710 g, 38%) as a colourless oil. IR
and C), 151.0 (C), 154.0 (C), 156.0 (C), 157.7 (C), 165.0 (C); MS
(EI) m/z 443 (M , 75%), 371 (28), 344 (8), 315 (19), 230 (9), 155
(
neat) 2961 (CH), 1707 (CO), 1663 (CO), 1643 (CO), 1533
+
−1
1
(C=C), 1485, 1227, 1113, 839 cm ; H NMR (400 MHz, CDCl )
3
(7), 117 (9), 105 (12), 84 (82), 72 (100); HRMS (EI) calcd for
δ 0.82 (3H, t, J = 7.2 Hz, NCH CH ), 1.02 (3H, d, J = 6.8 Hz,
+
2
3
C H N O S (M ), 443.1879, found 443.1867.
23
29
3
4
CH(CH )), 1.03 (3H, d, J = 6.4 Hz, CH(CH )), 1.14 (3H, t, J =
3
3
7
.2 Hz, NCH CH ), 2.32–2.40 (1H, m, 2’-H), 3.03–3.17 (2H, m,
4.2.17.
6-(4”-Fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-
2
3
NCH CH ), 3.34–3.43 (4H, m, 3-CH and NCHHCH ), 3.59–
1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-(N-methoxy-N-
2
3
3
3
3
.70 (2H, m, NCHHCH and 1’-HH), 4.00 (1H, dd, J = 14.0, 8.0
methyl)carboxamide (22)
3
Hz, 1’-HH), 7.11–7.20 (2H, m, 4”-H and 6”-H), 7.33–7.39 (1H,
13
The reaction was carried out according to the previously
described procedure for compound 14 using ethyl 6-(4”-
fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-1,2,3,4-
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (10) (0.080 g,
0.20 mmol) and a combination of N,O-dimethylhydroxylamine
m, 3”-H), 7.75 (1H, td, J = 7.6, 1.6 Hz, 5”-H); C NMR (101
MHz, CDCl ) δ 12.2 (CH ), 13.4 (CH ), 20.0 (CH ), 20.2 (CH ),
3
3
3
3
3
2
7.0 (CH ), 28.3 (CH), 39.0 (CH ), 43.0 (CH ), 56.3 (CH ), 113.1
3 2 2 2
2 2
(C), 115.7 (d, J = 21.7 Hz, CH), 119.1 (d, J = 15.1 Hz, C),
CF
CF
4
3
1
22.3 (C), 124.7 (d, J = 3.9 Hz, CH), 130.7 (d, J = 8.4 Hz,
CF CF
3
hydrochloride
(0.064
g,
0.66
mmol)
and
N,N’-
CH), 131.7 (d, J = 9.8 Hz, CH), 132.7 (C), 150.9 (C), 154.1
CF
1
diisopropylethylamine (0.23 mL, 1.3 mmol). This gave title
compound 22 as a viscous yellow oil (0.040 g, 38%). IR (neat)
(C), 157.6 (C), 158.9 (d, J = 251.9 Hz, C), 164.5 (C); MS
CF
+
(ESI) m/z 454 (M+Na , 100%); HRMS (ESI) calcd for
+
2959 (CH), 1707 (CO), 1662 (CO), 1535 (C=C), 1486, 1390,
C H FN NaO S (M+Na ), 454.1571, found 454.1558.
−1
22
26
3
3
9
91, 773 cm ; The compound exists as a 3:1 mixture of
1
4
1
.2.15.
1-Isobutyl-6-(4”-methoxyphenyl)-3-methyl-2,4-dioxo-
rotamers. Data for the major rotamer: H NMR (400 MHz,
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-N-
CDCl ) δ 1.02 (6H, d, J = 6.8 Hz, CH(CH ) ), 2.31–2.42 (1H, m,
3
3 2
diethylcarboxamide (20)
2’-H), 3.35 (3H, s, NCH ), 3.36 (3H, s, OCH ), 3.41 (3H, s, 3-
3
3
CH ), 3.68 (1H, dd, J = 14.1, 7.7 Hz, 1’-HH), 3.95 (1H, dd, J =
3
The reaction was carried out according to the previously
described procedure for compound 14 using ethyl 1-isobutyl-6-
1
7
4.1, 7.7 Hz, 1’-HH), 7.10 (2H, t, J = 8.8 Hz, 2”-H and 6”-H),
.57 (2H, dd, J = 8.8, 5.2 Hz, 3”-H and 5”-H); C NMR (101
13
(
4”-methoxyphenyl)-3-methyl-2,4-dioxo-1,2,3,4-
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (12) (0.083 g,
.20 mmol). This gave title compound 20 (0.026 g, 34%) as a
green oil. IR (neat) 2963 (CH), 1778 (CO), 1705 (CO), 1665
MHz, CDCl ) δ 20.0 (CH ), 20.1 (CH ), 27.1 (CH ), 28.3 (CH),
3
3
3
3
2
3
2
3.1 (CH ), 56.5 (CH ), 61.5 (CH ), 114.4 (C), 116.3 (d, J
2.2 Hz, 2 × CH), 127.7 (d, J = 3.1 Hz, C), 128.7 (C), 129.2
(C), 129.8 (d, JCF = 8.4 Hz, 2 × CH), 150.8 (C), 152.1 (C), 157.8
C), 162.9 (d, J = 248.9 Hz, C), 166.4 (C); MS (ESI) m/z 442
M+Na , 100%); HRMS (ESI) calcd for C H FN NaO S
M+Na ), 442.1207, found 442.1191.
=
3
2
3
CF
0
4
CF
3
−1
1
(
CO), 1439, 1294, 1254, 1182 cm ; H NMR (400 MHz, CDCl )
1
3
(
(
(
CF
δ 0.81 (3H, t, J = 7.2 Hz, NCH CH ), 1.01 (3H, d, J = 6.8 Hz,
+
2
3
20
22
3
4
CH(CH )), 1.02 (3H, d, J = 6.8 Hz, CH(CH )), 1.20 (3H, t, J =
+
3
3

9
4
1
.2.18.
6-(2”-Fluorophenyl)-1-isobutyl-3
A
-m
C
eth
C
yl
E
-2
P
,4-
T
di
E
ox
D
o- MA(C
N
H
U
), 1
S
7
C
03
R
(CIP
O), 1657 (CO), 1533 (C=C), 1489, 1252, 1123,
T
−1
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-(N-methoxy-N-
991, 729 cm ; The compound exists as a 3:1 mixture of
rotamers. Data for the major rotamer: H NMR (400 MHz,
1
methyl)carboxamide (23)
CDCl ) δ 1.01 (6H, d, J = 6.8 Hz, CH(CH ) ), 2.29–2.41 (1H, m,
3
3 2
The reaction was carried out according to the previously
described procedure for compound 14 using ethyl 6-(2”-
fluorophenyl)-1-isobutyl-3-methyl-2,4-dioxo-1,2,3,4-
2
’-H), 3.31 (3H, s, NCH ), 3.34 (3H, s, OCH ), 3.41 (3H, s, 3-
3
3
CH ), 3.64 (1H, dd, J = 14.1, 7.7 Hz, 1’-HH), 3.87 (3H, s, 2”-
3
OCH ), 4.00 (1H, dd, J = 14.1, 7.7 Hz, 1’-HH), 6.95 (1H, d, J =
3
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (11) (0.070 g,
7
7
.8 Hz, 3”-H), 7.00 (1H, t, J = 7.8 Hz, 5”-H), 7.34 (1H, td, J =
0
.17 mmol) and a combination of N,O-dimethylhydroxylamine
13
.8, 1.6 Hz, 4”-H), 7.66 (1H, dd, J = 7.8, 1.6 Hz, 6”-H);
C
hydrochloride (0.017 g, 0.18 mmol) and N,N’-
diisopropylethylamine (0.080 mL, 0.48 mmol). This gave title
compound 23 as a viscous yellow oil (0.038 g, 57%). IR (neat)
NMR (101 MHz, CDCl ) δ 20.0 (CH ), 20.2 (CH ), 27.1 (CH ),
3
3
3
3
2
8.2 (CH), 33.1 (CH ), 55.6 (CH ), 56.1 (CH ), 61.3 (CH ), 111.1
3 2 3 3
(CH), 113.0 (C), 120.3 (C), 121.2 (CH), 128.1 (C), 129.9 (C),
2
1
970 (CH), 1740 (CO), 1728 (CO), 1663 (CO), 1533 (C=C),
−1
130.3 (CH), 131.2 (CH), 151.0 (C), 153.3 (C), 156.1 (C), 157.8
366, 1217, 1119, 991 cm ; The compound exists as a 3:1
+
1
(C), 166.8 (C); MS (ESI) m/z 454 (M+Na , 100%); HRMS (ESI)
mixture of rotamers. Data for the major rotamer: H NMR (400
+
calcd for C H N NaO S (M+Na ), 454.1407, found 454.1389.
21
25
3
5
MHz, CDCl ) δ 1.02 (6H, d, J = 6.7 Hz, CH(CH ) ), 2.31–2.41
3
3 2
(
1H, m, 2’-H), 3.32 (3H, s, NCH ), 3.37 (3H, s, OCH ), 3.42 (3H,
4.3 Biological assay for the inhibition of lactate uptake in
3
3
s, 3-CH ), 3.68 (1H, dd, J = 14.1, 7.5 Hz, 1’-HH), 3.97 (1H, dd, J
Xenopus oocytes
3
=
14.1, 7.5 Hz, 1’-HH), 7.13–7.22 (2H, m, 4”-H and 6”-H), 7.33–
13
Oocytes were injected with 5 ng of cRNA coding for MCT1,
MCT2+GP70 and MCT4, respectively. Experiments were carried
out 5–7 days after injection. For each experiment, a batch of 8
oocytes was incubated in 1 mL of uptake solution (82.5 mM
7
.41 (1H, m, 3”-H), 7.72 (1H, td, J = 8.0, 1.6 Hz, 5”-H);
C
NMR (101 MHz, CDCl ) δ 20.0 (CH ), 20.1 (CH ), 27.0 (CH ),
3
3
3
3
2
8.3 (CH), 33.1 (CH ), 56.3 (CH ), 61.4 (CH ), 113.5 (C), 115.9
3 2 3
2 2
(d, J = 21.8 Hz, CH), 119.2 (d, J = 14.0 Hz, C), 123.2 (C),
CF
CF
3
3
NaCl, 2.5 mM KCl, 1 mM CaCl , 1mM MgCl , 1 mM Na HPO ,
2 2 2 4
1
24.8 (d, J = 3.5 Hz, CH), 130.8 (d, J = 9.0 Hz, CH), 131.1
CF CF
2
5 mM HEPES, pH7), containing 1 µM of inhibitor for 1 h. After
incubation, the solution was replaced with 100 µL of uptake
(
(
(
(
C), 131.5 (d, J = 2.8 Hz, CH), 150.7 (C), 153.4 (C), 157.7
C), 159.3 (d, J = 250.4 Hz, C), 166.1 (C); MS (ESI) m/z 442
M+Na , 100%); HRMS (ESI) calcd for C H FN NaO S
M+Na ), 442.1207, found 442.1191.
CF
1
CF
14
+
solution, containing 0.5 mM of C-labelled lactate (0.2 µCi).
20
22
3
4
+
Oocytes were incubated for 2.5 minutes. Uptake was stopped by
washing the oocytes three times with ice-cold uptake solution.
Each cell was placed into a scintillation vial and lysed with 200
µL of 10% sodium dodecyl sulfate (SDS). After lysis, 3 mL of
4
1
.2.19.
1-Isobutyl-6-(4”-methoxyphenyl)-3-methyl-2,4-dioxo-
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-(N-methoxy-N-
®
methyl)carboxamide (24)
scintillation fluid (Rotiszint eco plus scintillation cocktail, Carl
Roth) was added and the radioactivity determined by liquid
scintillation counting using a Tri-Carb 2810TR scintillation
counter (Perkin Elmer). To determine MCT-mediated fluxes,
values from native oocytes were subtracted from the values
measured in MCT-expressing cells. Statistical values are
presented as means ±standard error of the mean. For calculation
of significance in difference one way analysis of variance
The reaction was carried out according to the previously
described procedure for compound 14 using ethyl 1-isobutyl-6-
4”-methoxyphenyl)-3-methyl-2,4-dioxo-1,2,3,4-
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (12) (0.296 g,
.710 mmol) and a combination of N,O-dimethylhydroxylamine
hydrochloride (0.227 g, 2.32 mmol) and N,N’-
(
0
diisopropylethylamine (0.930 mL, 4.64 mmol). This gave title
compound 24 as a viscous yellow oil (0.102 g, 51%). IR (neat)
(ANOVA) was carried out, followed by means comparison using
either Scheffé or Bonferroni test, depending on whether datasets
show homogeneity of variance or not. Homogeneity of variance
was assessed using Levene’s test. All statistical tests were carried
out with OriginPro 8.6. In the figures shown, a significance level
of p ≤ 0.05 is marked with *, p ≤ 0.01 with **, and p ≤ 0.001
with ***.
2
1
961 (CH), 1705 (CO), 1657 (CO), 1537 (C=C), 1485, 1254,
−1
180, 733 cm ; The compound exists as a 3:1 mixture of
1
rotamers. Data for the major rotamer: H NMR (400 MHz,
CDCl ) δ 1.02 (3H, d, J = 6.0 Hz, CH(CH ) ), 2.29–2.42 (1H, m,
3
3 2
2
’-H), 3.35 (3H, s, NCH ), 3.37 (3H, s, OCH ), 3.41 (3H, s, 3-
3
3
CH ), 3.68 (1H, dd, J = 14.1, 7.5 Hz, 1’-HH), 3.83 (3H, s, 4”-
3
OCH ), 3.95 (1H, dd, J = 14.1, 7.5 Hz, 1’-HH), 6.92 (2H, d, J =
3
Acknowledgements
8
.8 Hz, 3”-H and 5”-H), 7.52 (2H, d, J = 8.8 Hz, 2”-H and 6”-H);
C NMR (101 MHz, CDCl ) δ 20.0 (CH ), 20.1 (CH ), 27.1
1
3
3
3
3
The authors are grateful to EPSRC (studentship to K.M.O.,
EP/K503058/1), the Deutsche Forschungsgemeinschaft (BE
(
CH ), 28.3 (CH), 33.2 (CH ), 55.4 (CH ), 56.4 (CH ), 61.5
3 3 3 2
(
CH ), 114.4 (C), 114.6 (2 × CH), 124.0 (C), 127.5 (C), 129.2 (2
3
4
310/6-1), the University of Glasgow and Scottish Health
×
CH), 130.5 (C), 150.8 (C), 151.7 (C), 157.9 (C), 160.0 (C),
+
Innovations Ltd for financial support.
1
66.8 (C); MS (ESI) m/z 454 (M+Na , 100%); HRMS (ESI)
+
calcd for C H N NaO S (M+Na ), 454.1407, found 454.1393.
21
25
3
5
4
1
.2.20.
1-Isobutyl-6-(2”-methoxyphenyl)-3-methyl-2,4-dioxo-
References and notes
,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-(N-methoxy-N-
methyl)carboxamide (25)
1
.
(a) Gupta, P. K.; Daunert, S.; Nassiri, M. R.; Wotring, L. L.;
Drach, J. C.; Townsend, L. B. J. Med. Chem. 1989, 32, 402. (b)
Devinyak, O. T.; Slivka, M. V.; Slivka, M. V.; Vais, V. M.;
Lendel, V. G. Med. Chem. Res. 2012, 21, 2263. (c) De Coen, L.
M.; Heugebaert, T. S. A.; García, D.; Stevens, C. V. Chem. Rev.
2016, 116, 80.
The reaction was carried out according to the previously
described procedure for compound 14 using ethyl 1-isobutyl-6-
(
2”-methoxyphenyl)-3-methyl-2,4-dioxo-1,2,3,4-
tetrahydrothieno[2,3-d]pyrimidine-5-carboxylate (13) (0.119 g,
.29 mmol) and a combination of N,O-dimethylhydroxylamine
0
2. Russell, R. K.; Press, J. B.; Rampulla, R. A.; McNally, J. J.;
Falotico, R.; Keiser, J. A.; Bright, D. A.; Tobia, A. J. Med. Chem.
hydrochloride (0.0933 g, 0.960 mmol) and N,N’-
diisopropylethylamine (0.334 mL, 1.92 mmol). This gave title
compound 25 as a viscous oil (0.070 g, 56%). IR (neat) 2961
1
988, 31, 1786.
3
.
(a) Murray, C. M.; Hutchinson, R.; Bantick, J. R.; Belfield, G. P.;
Benjamin, A. D.; Brazma, D.; Bundick, R. V.; Cook, I. D.;

10
Tetrahedron
Craggs, R. I.; Edwards, S.; Evans, L. R.; Harrison, R.; Holness, E.;
Dewar, D.; Pimlott, S. L.; Sutherland, A. Chem. Sci. 2015, 6,
4772. (d) Sloan, N. L.; Luthra, S. K.; McRobbie, G.; Pimlott, S.
L.; Sutherland, A. Chem. Commun. 2017, 53, 11008.
ACCEPTED MANUSCRIPT
Jackson, A. P.; Jackson, C. G.; Kingston, L. P.; Perry, M. W. D.;
Ross, A. R. J.; Rugman, P. A.; Sidhu, S. S.; Sulliven, M.; Taylor-
Fishwick, D. A.; Walker, P. C.; Whitehead, Y. M.; Wilkinson, D.
J.; Wright, A.; Donald, D. K. Nat. Chem. Biol. 2005, 1, 371. (b)
Guile, S. D.; Bantick, J. R.; Cheshire, D. R.; Cooper, M. E.; Davis,
A. M.; Donald, D. K.; Evans, R.; Eyssade, C.; Ferguson, D. D.;
Hill, S.; Hutchinson, R.; Ingall, A. H.; Kingston, L. P.; Martin, I.;
Martin, B. P.; Mohamed, R. T.; Murray, C.; Perry, M. W. D.;
Reynolds, R. H.; Thorne, P. V.; Wilkinson, D. J.; Withnall, J.
Bioorg. Med. Chem. Lett. 2006, 16, 2260. (c) Guile, S. D.;
Bantick, J. R.; Cooper, M. E.; Donald, D. K.; Eyssade, C.; Ingall,
A. H.; Lewis, R. J.; Martin, B. P.; Mohammed, R. T.; Potter, T. J.;
Reynolds, R. H.; St-Gallay, S. A.; Wright, A. D. J. Med. Chem.
11. Cheshire, D.; Cooke, A.; Cooper, M.; Donald, D.; Furber, M.;
Perry, M.; Thorne, P. U.S. Patent 6,180,635, 2001.
12. The side-products from this reaction were not fully characterised
due to decomposition during column chromatography. However,
1
from H NMR spectroscopy of the crude reaction mixture, these
appear to be compounds derived from both S- and C-5 alkylation
of 7 by ethyl 3-bromopyruvate.
13. A quantity (~10–15%) of the carboxylic acid of 5 is also generated
from the two-step transformation. This is separated from the
product by extraction from sodium carbonate during the work-up.
14. (a) Miyaura, N.; Yamada, K.; Suzuki, A. Tetrahedron Lett. 1979,
20, 3437. (b) Miyaura, N.; Yanagi, T.; Suzuki, A. Synth. Commun.
1981, 11, 513. (c) Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95,
2457.
2
007, 50, 254.
4
5
.
.
Polański, R.; Hodgkinson, C. L.; Fusi, A.; Nonaka, D.; Priest, L.;
Kelly, P.; Trapani, F.; Bishop, P. W.; White, A.; Critchlow, S. E.;
Smith, P. D.; Blackhall, F.; Dive, C.; Morrow, C. J. Clin. Cancer
Res. 2014, 20, 926.
15. Tavares, A. A. S.; Lewsey, J.; Dewar, D.; Pimlott, S. L. Nucl.
Med. Biol. 2012, 39, 127.
(a) Sasaki, S.; Cho, N.; Nara, Y.; Harada, M.; Endo, S.; Suzuki,
N.; Furuya, S.; Fujino, M. J. Med. Chem. 2003, 46, 113. (b) Miwa,
K.; Hitaka, T.; Imada, T.; Sasaki, S.; Yoshimatsu, M.; Kusaka, M.;
Tanaka, A.; Nakata, D.; Furuya, S.; Endo, S.; Hamamura, K.;
Kitazaki, T. J. Med. Chem. 2011, 54, 4998.
16. Becker, H. M.; Bröer, S.; Deitmer, J. W. Biophys. J. 2004, 86,
235.
17. Wang, H.; Yang, C.; Doherty, J. R.; Roush, W. R.; Cleveland, J.
L.; Bannister, T. D. J. Med. Chem. 2014, 57, 7317.
6
7
.
.
Streuli, I.; de Ziegler, D.; Borghese, B.; Santulli, P.; Batteux, F.;
Chapron, C. Expert Opin. Emerging Drugs 2012, 17, 83.
Elancheran, R.; Maruthanila, V. L.; Ramanathan, M.; Kabilan, S.;
Devi, R.; Kunnumakara, A.; Kotoky, J. Med. Chem. Commun.
Supplementary material
2
015, 6, 746.
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.tet.XXXXX
8
9
.
.
Roth, B. J. Med. Chem. 1969, 12, 227.
Ogura, H.; Sakaguchi, M.; Takeda, K. Chem. Pharm. Bull. 1972,
2
0, 404.
1
0. (a) Calder, E. D. D.; McGonagle, F. I.; Harkiss, A. H.;
McGonagle, G. A.; Sutherland, A. J. Org. Chem. 2014, 79, 7633.
(b) Calder, E. D. D.; Sharif, S. A. I.; McGonagle, F. I.; Sutherland
A. J. Org. Chem. 2015, 80, 4683. (c) Blair, A.; Zmuda, F.;
Malviya, G.; Tavares, A. A. S.; Tamagnan, G. D.; Chalmers, A. J.;