Synthesis and biological evaluation of thieno[3,2-d]- pyrimidinones, thieno[3,2-d]pyrimidines and quinazolinones: Conformationally restricted 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2) inhibitors

Article abstract of DOI:10.3390/molecules18044487

In this study, a series of conformationally restricted thieno[3,2-d] pyrimidinones, thieno[3,2-d]pyrimidines and quinazolinones was designed and synthesized with the goal of improving the biological activity as 17β-hydroxysteroid dehydrogenase type 2 inhi

Full text of DOI:10.3390/molecules18044487

Molecules 2013, 18, 4487-4509; doi:10.3390/molecules18044487
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of Thieno[3,2-d]-
pyrimidinones, Thieno[3,2-d]pyrimidines and Quinazolinones:
Conformationally Restricted 17-Hydroxysteroid
Dehydrogenase Type 2 (17-HSD2) Inhibitors
Enrico Perspicace 1, Sandrine Marchais-Oberwinkler 1 and Rolf W. Hartmann 1,2,
*
1
Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C23, D-66123 Saarbrücken,
Germany; E-Mails: e.perspicace@mx.uni-saarland.de (E.P.); s.marchais@mx.uni-saarland.de (S.M.-O.)
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C23,
D-66123 Saarbrücken, Germany
2
* Author to whom correspondence should be addressed; E-Mail: rwh@mx.uni-saarland.de;
Tel.: +49-681-302-70300; Fax: +49-681-302-70308.
Received: 22 March 2013; in revised form: 8 April 2013 / Accepted: 10 April 2013 /
Published: 16 April 2013
Abstract: In this study, a series of conformationally restricted thieno[3,2-d]pyrimidinones,
thieno[3,2-d]pyrimidines and quinazolinones was designed and synthesized with the goal
of improving the biological activity as 17-hydroxysteroid dehydrogenase type 2 inhibitors
of the corresponding amidothiophene derivatives. Two moderately active compounds were
discovered and this allowed the identification of the biologically active open conformer as
well as the extension of the enzyme binding site characterisation.
Keywords: thieno[3,2-d]pyrimidinones; thieno[3,2-d]pyrimidines; quinazolinones;
conformationally restricted analogues; enzyme inhibitors; 17-hydroxysteroid
dehydrogenase type 2; osteoporosis
1. Introduction
Osteoporosis is a systemic skeletal disease characterised by reduction of bone mineral density and
deterioration of the bone microarchitecture leading to an increase in bone fragility and thus elevated
bone fracture risk [1]. The decrease in active steroid levels like estradiol (E2) and testosterone (T) is

Molecules 2013, 18
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often associated with the development of osteoporosis (e.g., in women at menopause [2]). As this
disease affects elderly men and especially women and the population is getting older, it has become a
serious public health issue and new therapeutic alternatives have to be developed.
17-Hydroxysteroid dehydrogenase type 2 (17-HSD2, (EC1.1.1.51) [3]) belongs to the short-chain
steroid dehydrogenase/reductase protein family (SDR). This enzyme catalyses the NAD+-dependent
oxidation of the potent estradiol (E2) into estrone (E1) as well as testosterone (T) into androstenedione
(4-dione). 17-HSD2 is a membrane-associated protein located in the endoplasmic reticulum and
contains 387 amino acids. This enzyme is highly expressed in a wide variety of tissues such as
placenta, liver, small intestine, endometrium, urinary tract and to a lesser extend also in kidney,
pancreas, colon, uterus, breast, prostate and bone. The 3D-structure of this enzyme remains unknown.
Few 17-HSD2 inhibitors have been described in the literature [4,5]. The concept of improving
bone quality by inhibiting 17-HSD2 has been validated by Bagi et al. in a monkey osteoporosis
model [2] using a cis-pyrrolidinone derivative as 17-HSD2 inhibitor [6–8]. However, the treatment
with this compound led to few effects on bones. Therefore, it is necessary to develop new potent
17-HSD2 inhibitors with better in vivo efficacy, which are selective toward 17-HSD1, the
isoenzyme catalysing the reverse reaction, the reduction of E1 into E2 (Figure 1).
Figure 1. Interconversion of Estradiol (E2) to Estrone (E1) by 17-HSD2 and 17-HSD1.
Our group has been working for 20 years on the development of inhibitors of steroidogenic enzymes
(e.g., 5-reductase [9], aromatase [10,11], CYP17 [12,13], CYP11B2 [14–17], CYP11B1 [18,19]) and in
the last years a lot of experience was gained in the design of potent and selective inhibitors of 17-
HSD1 [20–25], and 17-HSD2 [26–32]. Recently, we described [27] N-(3-methoxybenzyl)-5-(3-
methoxyphenyl)-N-methylthiophene-2-carboxamide (compound A, Table 1) and N-(3-hydroxybenzyl)-
5-(3-hydroxyphenyl)-N-methylthiophene-2-carboxamide (compound B, Table 1) as 17-HSD2
inhibitors (63% and 70% 17-HSD2 inhibition at a concentration of 1 µM, for A and B, respectively). Both
compounds show a good selectivity toward 17-HSD1 (0% and 21% inhibition at 1 µM, respectively).
Compounds A and B consist of a central thiophene moiety connected to a substituted phenyl ring on one
side and to a substituted benzyl on the other side linked to the thiophene via a methylated carboxamide
bond. A and B show some flexibility at this amide bond, allowing for several conformations.
1
In the H-NMR spectra of A or B it can be seen that the peaks corresponding to N-Me group
(3.15/3.16 ppm) and to the CH2 of the benzyl group (4.76/4.79 ppm) appear as very broad singlets,
indicating that in solution at room temperature A and B exist as a mixture of several conformers in
equilibrium. However, when the compounds bind to the enzyme binding site, only one conformer is
expected to be present and therefore only one conformer is biologically active.

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Table 1. In vitro 17-HSD2 and 17-HSD1 inhibitory potencies of thiophene-2-
carboxamide derivatives A and B.
Percentage of inhibition at 1µM a
Cmpd
R1
R2
17-HSD2 b
63%
17-HSD1 c
A
B
OMe
OH
OMe
OH
0%
70%
21%
a
b
Mean value of three determinations, standard deviation less than 15%. Human placental, microsomal
c
fraction, substrate E2 [500 nM], cofactor NAD+ [1500 nM]. Human placental, cytosolic fraction, substrate
E1 [500 nM], cofactor NADH [500 nM].
Rigidification is an often applied method to increase the potency of active molecules. In addition to
freezing a compound in one conformation when several exist, it is also a good strategy to identify the
active conformation of a compound when it binds in the active site of a protein. Rigidification can be a
useful tool for structural optimisation and also to characterize the binding site in the protein.
The goal of this study was the structural optimization of the 17-HSD2 inhibitors A and B, using
the rigidification strategy as well as the identification of the active conformation taken by A and B in
the protein. In this study the design and the synthesis of new rigidified 17-HSD2 inhibitors,
mimicking the different geometries which can be adopted by the conformers derived from A and B as
well as the biological evaluation of the synthesized compounds (17-HSD2 potency as well as
selectivity toward 17-HSD1) are reported.
2. Results and Discussion
Compounds A and B show the highest flexibility at the amide bond, involving two sigma bonds
(σ1 between the thiophene and the carbonyl moiety and σ2 between the carbonyl and the amino group,
Figure 2). Since σ bonds are freely rotatable, rotation about the two sigma bonds (σ1 and σ2) of the
amide group leads to four different conformers I-IV as depicted in Figure 2. The conformational
freezing of A and B can be achieved by cyclisation leading to thiophene- or phenyl-fused derivatives:
the thieno[3,2-d]pyrimidin-4-one analogues 1a–b and 3a–d, locked conformers I and III, respectively
and quinazolin-4-one derivatives 2a–b and 4a–b,locked conformers II and IV, respectively (Figure 3).
As restrained conformers II and IV are not possible with the thiophene core (S is bivalent), the
heterocycle was replaced by the bioisosteric phenyl ring (Figure 3). In order to investigate the
necessity of the benzyl group and its position, compounds 5a–d were designed (conformer I or III),
and synthesized (Figure 3).

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Figure 2. Four major conformers of thiophene-2-carboxamide derivatives A and B.
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Figure 3. Thieno[3,2-d]pyrimidin-4-ones 1a–b, 3a–d and thieno[3,2-d]pyrimidine 5a–d as
conformationally restrained analogues of conformer I and III, and quinazolin-4-one 2a–b,
4a–b as conformationally restrained analogues of conformer II and IV, respectively.

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Compounds 1a–b were synthesized by a two- or a three-step procedure, respectively, as depicted on
Scheme 1, starting from compound 6. The starting materials methyl 3-amino-5-(3-methoxyphenyl)-
thiophene-2-carboxylate (6) and 3-amino-5-(3-methoxyphenyl)thiophene-2-carboxamide (11a) were
synthesized using the multi-step procedure well described in the literature [33]. The condensation of
molecule 6 with N,N-dimethylformamide dimethyl acetal (DMF-DMA) gave methyl 3-dimethyl-
aminomethylidene-amino-5-(3-methoxyphenyl)thiophene-2-carboxylate (7) in very good yield [34]
which was used directly in the next step without purification to afford compound 1a. The condensation of
6-(3-methoxyphenyl)-4H-thieno[3,2-d][1,3]oxazin-4-one (6a) with 3-methoxybenzyl amine as an
alternative route to produce 1a failed. After O-demethylation using boron trifluoride methyl sulfide
complex BF3.SMe2, the thieno[3,2-d]pyrimidin-4(3H)-one 1b was obtained (Scheme 1).
Scheme 1. Synthesis of the thieno[3,2-d]pyrimidin-4-ones derivatives 1a–b.
Reagents and conditions: (a) DMF-DMA, EtOH, microwave irradiation (100 °C, 80 W, 15 min),
98%; (b) DMF, microwave irradiation (100 °C, 80 W, 15 min), 31%; (c) BF3.SMe2, CH2Cl2, rt,
82%; (d) MeOH, reflux.
Synthesis of derivatives 2a–b was achieved by a three- or a four-step reaction pathway starting
from the 2-amino-3-bromobenzoic acid (8) as shown on Scheme 2. The starting material 8 was
prepared as described in the literature [35]. It reacted with triethyl orthoformate [CH(OEt)3] to give the
cyclized benzoxazin-4-one 9, which was condensed with 3-methoxybenzylamine to afford the quinazolin-
4-one 10 (Scheme 2) [36]. Suzuki-Miyaura cross coupling led to the methoxy derivative 2a in very
good yield. Ether cleavage using the above described procedure afforded the hydroxylated quinazolin-4-

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one 2b. It is worth mentioning, that the condensation of methyl 2-bromo-6-{[(1E)-
(dimethylamino)methylidene]amino}benzoate (9a) with the 3-methoxybenzyl amine, which was
successfully applied in the synthesis of the thiophene fused 1a, failed to react to the quinazolin-4-one 10.
Scheme 2. Synthesis of the quinazolin-4-ones derivatives 2a–b.
Reagents and conditions: (a) CH(OEt)3, microwave irradiation (160 °C, 80 W, 10 min), 61%; (b) dry
toluene, reflux, 66%; (c) Pd(PPh3)4, Cs2CO3, DME/EtOH/H2O (1:1:1), microwave irradiation
(150 °C, 150 W, 15 min), 98%; (d) BF3.SMe2, CH2Cl2, rt, 97%.
Synthesis of 3a–d was performed starting from the 3-amino-5-arylthiophene (11a) as depicted in
Schemes 3 and 4 following a two- or a three-step procedure. Compound 11a was condensed with
3-methoxybenzaldehyde in presence of concentrated hydrochloric acid to give first the hydrogenated
thienopyrimidinone which was then autoxidized to the corresponding thienopyrimidinone 12 on heating in
acidic medium [37]. Then, N-methylation was performed as previously described to afford 3a. Ether
cleavage led to the thieno[3,2-d]pyrimidin-4(3H)-one 3b (Scheme 3).
3-Amino-5-arylthiophene (11a) was also condensed with 3-methoxyphenylacetyl chloride in
presence of triethylamine. The cyclization step was realized with sodium hydroxide to obtain 13.
N-methylation was performed using methyliodide in presence of potassium carbonate as base to give
3c and ether cleavage was accomplished using the same procedure as described above providing 3d in
very good yield (Scheme 4).

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Scheme 3. Synthesis of the thieno[3,2-d]pyrimidin-4-ones derivatives 3a–b.
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Reagents and conditions: (a) HCl 37%, MeOH, reflux, 38%; (b) MeI, K2CO3, CH3CN, reflux,
48%; (c) BF3.SMe2, CH2Cl2, rt, 57%.
Scheme 4. Synthesis of the thieno[3,2-d]pyrimidin-4-ones derivatives 3c–d.
Reagents and conditions: (a) Et3N, dry THF, rt; (b) NaOH 2N, few drops of DMF, reflux, 49%
(over two steps); (c) MeI, K2CO3, CH3CN, reflux, 74%; (d) BF3.SMe2, CH2Cl2, rt, 98%.
The 2-substituted quinazolin-4-one 4b was synthesized starting from 2-amino-3-bromobenzoic acid
methyl ester (14) (Scheme 5), which was reacted with 3-methoxybenzoyl chloride to afford the amide
15. This intermediate was condensed with formamide to afford the 2-substituted quinazolin-4-one 16 [38].
Compound 16 could not be obtained from condensation of 2-amino-6-bromobenzamide (15a) with
3-methoxybenzaldehyde as described for the synthesis of the thiophene fused derivative 13, even using
different catalysts (p-toluenesulfonic acid or sodium metabisulfite). This indicates a different reactivity
of the amino group depending on its location on a thiophene or a phenyl ring. Subsequently, the

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Suzuki-Miyaura cross coupling led to compound 17, which was then N-methylated at the quinazolin-4-
one into 4a. Treatment with BF3.SMe2 was used to cleave the methoxy groups affording compound 4b.
Scheme 5. Synthesis of the quinazolin-4-ones derivatives 4a–b.
Reagents and conditions: (a) Et3N, dry CH2Cl2, rt, 15%; (b) H2NCHO, 180 °C, 81%; (c) Pd(PPh3)4,
Cs2CO3, DME/EtOH/H2O (1:1:1), microwave irradiation (150 °C, 150 W, 15 min), 77%;
(d) MeI, K2CO3, CH3CN, reflux, 64%; (e) BF3.SMe2, CH2Cl2, rt, 98%.
The synthesis of fused-compounds 5a–b and 5c–d was performed following two different synthetic
routes depicted in Scheme 6. The 3-amino-5-arylthiophene amides 11a,b were condensed with formic
acid under microwave irradiation to afford the corresponding thieno[3,2-d]pyrimidin-4-one 18a,b as
described in the literature [39]. The conversion of these analogues 18a,b to thieno[3,2-d]pyrimidines
19a,b was performed using phosphorus oxychloride. The nucleophilic aromatic substitution of 19a,
19b was accomplished with sodium methylate and gave the desired 4-methoxy-thieno[3,2-
d]pyrimidines 5a and 5b (Scheme 6). Compound 19a was reacted with the corresponding sodium
methoxyphenolates providing the substituted 4-phenoxythieno[3,2-d]pyrimidines 5c,d, respectively
(Scheme 6).
The synthesized compounds were tested for their ability to inhibit 17-HSD2 and 17-HSD1 using
placental enzymes as previously described (compound concentration: 1 µM) [40]. Results are reported
in Table 2 as percentage of inhibition. Compounds showing an inhibitory activity below 10% were
considered as inactive.

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Scheme 6. Synthesis of the thieno[3,2-d]pyrimidin-4-ones derivatives 5a–d.
4495
Reagents and conditions: (a) HCOOH, H2SO4, 50 °C overnight, 18a: 76%, 18b: 98%; (b) POCl3,
microwave irradiation (95 °C, 80 W, 20 min), 19a: 98%, 19b: 99%; (c) MeONa in MeOH, DMF,
rt, 2 h, 5a: 91%, 5b: 40%; (d) sodium 3-methoxyphenolate or sodium 4-methoxyphenolate, DMF
dry, rt, 2 h, 5c: 88%, 5d: 87%.
Table 2. In vitro inhibitory potencies toward 17-HSD2 and 17-HSD1 of compounds 1a–b,
2a–b, 3a–d, 4a–b and 5a–d, 12 and 13.
Percentage of inhibition at 1 µM a
Compound
17-HSD2 b
n.i.
17-HSD1 c
n.i.
1a
1b
2a
2b
3a
3b
3c
3d
4a
4b
5a
5b
5c
5d
13
12
n.i.
n.i.
n.i.
n.i.
11%
n.i.
n.i.
n.i.
36%
n.i.
50%
n.i.
25%
n.i.
48%
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
a
b
Mean value of three determinations, standard deviation less than 15%. Human placental, microsomal
c
fraction, substrate E2 [500 nM], cofactor NAD+ [1500 nM]. Human placental, cytosolic fraction, substrate
E1 [500 nM], cofactor NADH [500 nM]. d n.i.: no inhibition or inhibition below 10%.
The most active compounds 3b and 3d show moderate 17-HSD2 inhibition (36% and 25%,
respectively, Table 2). These two compounds are fused thiophenes and bear two hydroxyphenyl
moieties. The corresponding methoxy derivatives are inactive. Compounds 3b/3d both result from the
freezing of conformer III and differ only in the presence of a phenyl or a benzyl group in position 2 of the

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pyrimidinone. As they are the only active compounds identified in this study, this indicates that
conformation III is likely to reflect the favourite geometry adapted by compounds A and B in the active
site of the enzyme, where it is expected that the compounds are present as only one conformer. However, it
is striking that compounds 3b/3d and B present such a difference in activity (3b: 36% @ 1 M, 3d: 25%
@ 1 M vs B: 70% @ 1 M). It should be noticed that the corresponding methoxy derivatives 3a and
3c are inactive, while methoxy compound A has a similar biological activity as the hydroxy compound
B (A: 63% @ 1 M). One explanation for this finding it that A and B might have a different binding
mode in the enzyme binding site.
In order to better understand the biological activities, manual superimposition of 3b and 3d with B
were made using MOE 2010.10 (considering the lowest energy for each compound, Figure 4). The
thiophene moiety of 3b/3d was superimposed on the one of B as well as the corresponding carbonyl
and N-amide moieties. In the superimposition pictures it can be seen that the compounds do not
overlap exactly with each other (however, they show a better hit than the other conformers). The
hydroxyphenyl in the 5 position of the fused thiophenes 3b/3d superimposes well on B. The main
differences concern the second hydroxyphenyl/benzyl which is in a completely different position and
the presence of the nitrogen N1 of the thienopyrimidinone (absent in A/B). It might be that this polar N
with hydrogen bond acceptor activity is located in a lipophilic pocket or that there is no space in this
area to accept the closed thienopyrimidinone ring. These differences might explain the contrasting
biological activities observed for 3b/3d and B.
Figure 4. Superimposition between compounds 3b and B (Figure 4-I) and between
compounds 3d and B (Figure 4-II).
I
II
In Figure 4, it can also be seen that the planarity induced by the rigidified compounds is comparable
to the planarity of the amide and therefore does not seem to have an impact on the potency of the
compounds. In this series of compounds the planar triazole derivative C (Figure 5), a cyclised
bioisostere of the amide function, was synthesized and was also a moderate 17-HSD2 inhibitor with
inhibitory activity in the same range as 3b/3d (C: 42% @ 1 M, [31]).

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Figure 5. Described analogues of 3b/3d with rigidified amide bond moiety.
4497
In order to investigate the role of the anisole moiety linked to the thienopyrimidinone, compounds
5a,c, without OMe-Ph and with OMe-Ph on the O next to the thiophene, respectively, both derived
from rigidification of conformer I or III, were synthesized. Derivative 5a, without OMe-Ph was
inactive, indicating the importance of this group, which certainly in case of A is able to interact with
the active site. Compound 5c, with the OMe-Ph at the O next to the thiophene, was also inactive. This
anisole moiety, which covers a different area in the active site, might induce a steric clash, preventing
the binding of the compound in the enzyme binding site. It might indicate that the binding cavity is
elongated and cannot accommodate globular compounds. This could also explain the inactivity of
compounds 4a/4b, which have a different geometry compared to 3b/3d.
The thienopyrimidinones 3b/3d show a structural similarity to compound D [41], previously
described as 17-HSD1 inhibitor (17-HSD1: 95% inh @ 1 M, 17-HSD2: 77% inh @ 1 M).
D differs from 3b/3d in the geometry of the pyrimidinone and the OH-Ph fused to the thiophene. As
observed for D, 3b/3d also shows a slightly better 17-HSD1 inhibition profile.
The synthesis of the different rigidified conformers highlights a difference in the reactivity of the
amino group depending on its position on the phenyl ring or on the thiophene moiety. All experiments
show that the amino group on the thiophene is more reactive than the amino group at the phenyl in
spite of the fact that phenyl and thiophene are considered to have similar chemical properties.
3. Experimental
Chemical names follow IUPAC nomenclature. Starting materials were purchased from Aldrich,
Acros, Combi-Blocks or Fluka and were used without purification. Flash column chromatography
(FC) was performed on silica gel (70–200 μm), and reaction progress was monitored by TLC on
Alugram SIL G/UV254 (Macherey-Nagel). Visualization was accomplished with UV light.
1H-NMR and 13C-NMR spectra were measured on a Bruker AM500 spectrometer (at 500 MHz and
125 MHz, respectively) at 300 K in acetone-d6 or DMSO-d6. Chemical shifts are reported in δ (parts
per million: ppm), by reference to the hydrogenated residues of deuteriated solvent as internal standard:
2.05 ppm (1H-NMR) and 29.8 and 206.3 ppm (13C-NMR) for acetone-d6, 2.50 ppm (1H-NMR) and
39.5 ppm (13C-NMR) for DMSO-d6 and 7.26 ppm (1H-NMR) and 77.2 ppm (13C-NMR) for CDCl3.
Signals are described as br (broad), s (singlet), d (doublet), t (triplet), dd (doublet of doublets), ddd (doublet
of doublet of doublets), dt (doublet of triplets) and m (multiplet). All coupling constants (J) are given in
Hertz (Hz). Melting points (mp) were measured in open capillaries on a Stuart Scientific SMP3
apparatus and are uncorrected.

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Mass spectrometry was performed on a TSQ® Quantum (ThermoFisher, Dreieich, Germany). The
triple quadrupole mass spectrometer was equipped with an electrospray interface (ESI). The purity of
the compounds was assessed by LC/MS. The Surveyor®-LC-system consisted of a pump, an auto
sampler, and a PDA detector. The system was operated by the standard software Xcalibur®. A RP C18
NUCLEODUR® 100-5 (3 mm) column (Macherey-Nagel GmbH, Dühren, Germany) was used as
stationary phase. All solvents were HPLC grade. In a gradient run the percentage of acetonitrile
(containing 0.1% trifluoroacetic acid) was increased from an initial concentration of 0% at 0 min to
100% at 15 min and kept at 100% for 5 min. The injection volume was 15 µL and flow rate was set to
800 µL/min. MS analysis was carried out at a needle voltage of 3,000 V and a capillary temperature of
350 °C. Mass spectra were acquired in positive mode from 100 to 1,000 m/z and UV spectra
were recorded at the wave length of 254 nm and in some cases at 360 nm. All tested compounds
exhibited 95% chemical purity. Compounds 6 [20], 8 [22], 11a–b [20] were prepared according to
previously described procedures.
3.1. General Procedure for Suzuki — Miyaura Coupling (2a, 17) — Method A
A solution of 2a or 17 (1 eq.), 3-methoxyphenyl boronic acid (1.5 eq.), cesium carbonate (3 eq.) and
tetrakis(triphenylphosphine)palladium(0) (0.02 eq.) in oxygen free DME/EtOH/H2O solution (3 mL,
1:1:1) was heated under microwave irradiation at 150 °C/150 W for 15 min. After cooling to room
temperature, the mixture water/EtOAc (2 mL, 1:1) was added to quench the reaction. The solution was
extracted three times by EtOAc (3 × 10 mL). The organic layer was dried over MgSO4, filtered and the
solution was concentrated under reduced pressure. The residue was purified by silica gel column
chromatography using n-hexane and EtOAc as eluent.
3.2. General Procedure for Cleavage of Ethers 1b–4b — Method B
To a solution of 1a–4a (1 eq.) in CH2Cl2 (10 mL) was added dropwise boron fluoride-dimethyl
sulfide complex BF3.SMe2 (3 eq./each methoxy group) at 0 °C. After 1 h at 0 °C, the ice bath was
removed and the reaction mixture was stirred overnight at room temperature. The solution was
quenched by addition of 10 mL MeOH. After 30 min, the solvent were concentrated under reduced
pressure at 35 °C and the residue was triturated with cold water. The aqueous layer was extracted three
times with EtOAc (3 × 15 mL), the organic layer was dried over MgSO4, filtered and the solution
concentrated under reduced pressure. The residue was purified by silica gel column chromatography
using n-hexane and EtOAc as eluent or triturated in a mixture of diethylether/petroleum ether and
filtered off.
Methyl 3-Amino-5-(3-methoxyphenyl)thiophene-2-Carboxylate (6). To a solution of 3-chloro-3-(3-
methoxyphenyl)prop-2-enenitrile (3.56 g, 18.40 mmol) and dry potassium carbonate (3.05 g, 22.08 mmol)
in dry NMP (20 mL) was added dropwise under N2 atmosphere methyl thioglycolate (2.01 mL,
22.08 mmol). The reaction mixture was heated at 60 °C overnight. After cooling, the reaction mixture
was poured into ice water solution and stirred. The aqueous layer was extracted three times with
EtOAc (3 × 30 mL). The organic layer was washed once with water, dried over MgSO4, filtered and
the solution was concentrated under reduced pressure. The residue was purified by silica gel column

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chromatography (n-hexane/EtOAc 70:30) to afford 6 as a yellow solid. Yield: 50%. Mp: 72–74 °C.
1H-NMR (CDCl3)  3.84 (s, 3H), 3.85 (s, 3H), 5.47 (br, 2H), 6.76 (s, 1H), 6.90 (dd, J = 1.9, 8.2 Hz, 1H),
7.10 (t, J = 1.9 Hz, 1H), 7.16–7.19 (m, 1H), 7.30 (t, J = 8.0 Hz, 1H); 13C-NMR (CDCl3)  51.5, 55.6, 111.8,
114.8, 115.9, 118.8, 130.2, 134.9, 149.2, 154.4, 160.2, 165.2. LC-MS (ESI): [M+H]+ = 264.29.
Methyl 3-{[(1E)-(dimethylamino)methylidene]amino}-5-(3-methoxyphenyl)thiophene-2-carboxylate (7).
To a solution of methyl 3-amino-5-(3-methoxyphenyl)thiophene-2-carboxylate 6 (250 mg, 0.95 mmol) in
EtOH (10 mL) was added N-dimethoxymethyl-N,N-dimethylamine (189 µL, 1.42 mmol). The reaction
mixture was heated under microwave irradiation at 100 °C/80 W for 30 min. The solution was
concentrated under reduced pressure to give 7 as an orange oil. Yield: 99%. The title compound was
used for the next step without further purification.
3-(3-Methoxybenzyl)-6-(3-methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (1a). A solution of 7
(302 mg, 0.95 mmol) and 3-methoxybenzylamine (146 µL, 1.14 mmol) in DMF (4 mL) was heated under
microwave irradiation at 100 °C/80 W for 30 min. Water was added to the solution to quench the
reaction. The aqueous layer was extracted three times with EtOAc (3 × 15 mL). The organic layer was
washed once with brine and once with water, dried over MgSO4, filtered and the solution was
concentrated under reduced pressure. The residue was purified by silica gel column chromatography using
n-hexane and EtOAc as eluent (n-hexane/EtOAc 60:40) to afford 1a as a pale yellow solid. Yield: 31%.
Mp: 144–146 °C. 1H-NMR (CDCl3)  3.78 (s, 3H), 3.90 (s, 3H), 5.29 (s, 2H), 6.88 (dd, J = 2.3, 8.2 Hz,
1H), 6.99–7.05 (m, 3H), 7.28 (t, J = 8.0 Hz, 1H), 7.36–7.44 (m, 3H), 7.68 (s, 1H), 8.48 (s, 1H); 13C-NMR
(CDCl3)  49.6, 55.4, 55.8, 112.5, 114.1, 114.7, 116.2, 119.5, 120.9, 122.1, 123.2, 130.7, 131.3, 135.3,
139.4, 150.1, 152.6, 157.4, 158.9, 161.0, 161.4. LC-MS (ESI): [M+H]+ = 379.31.
3-(3-Hydroxybenzyl)-6-(3-hydroxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (1b). Synthesized according
to Method B using 1a (52 mg, 0.14 mmol) and BF3.SMe2 (88 µL, 0.84 mmol). The residue was triturated
in a mixture of diethyl ether/petroleum ether to afford 1b as a pale brown solid. Yield: 82%. Mp:
1
270–272 °C. H-NMR (DMSO-d6)  5.15 (s, 2H), 6.67 (ddd, J = 0.7, 2.4, 8.0 Hz, 1H), 6.71 (t,
J = 1.7 Hz, 1H), 6.77 (d, J = 7.7 Hz, 1H), 6.87 (dt, J = 2.0, 7.2 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H),
13
7.17–7.19 (m, 1H), 7.27–7.32 (m, 2H), 7.76 (s, 1H), 8.60 (s, 1H), 9.44 (s, 1H), 9.78 (s, 1H); C-NMR
(DMSO-d6)  48.4, 112.8, 114.2, 114.7, 116.8, 117.0, 118.0, 120.9, 121.4, 129.7, 130.5, 133.6, 138.1,
149.8, 151.5, 156.2, 157.6, 157.7, 158.0. LC-MS (ESI): [M]+ = 350.84.
8-Bromo-4H-3,1-benzoxazin-4-one (9). A solution of 2-amino-3-bromobenzoic acid (250 mg, 1.16 mmol)
and triethyl orthoformate (2 mL) was heated under microwave irradiation at 160 °C/80 W for 15 min.
After cooling, the precipitate formed was collected by filtration and washed with petroleum ether to afford
9 as colorless needles. Yield: 61%. Mp: 314–316 °C. 1H-NMR (acetone-d6)  7.57 (t, J = 7.9 Hz, 1H), 8.14
(s, 1H), 8.17 (dd, J = 1.5, 7.9 Hz, 1H), 8.20 (dd, J = 1.5, 8.0 Hz, 1H); 13C-NMR (acetone-d6)  121.7,
122.2, 128.6, 130.8, 140.9, 144.6, 152.1, 158.4.

Molecules 2013, 18
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8-Bromo-3-(3-methoxybenzyl)quinazolin-4(3H)-one (10). A solution of 9 (100 mg, 0.44 mmol) and
3-methoxybenzylamine (73 mg, 0.53 mmol) in 2 mL dry toluene was heated at reflux overnight. After
cooling, the solvent was removed under reduced pressure. The residue was triturated with diethyl ether
1
and filtered off to give 10 as a colorless solid. Yield: 66%. Mp: 139–140 °C. H-NMR (acetone-d6) 
3.78 (s, 3H), 5.27 (s, 2H), 6.88 (ddd, J = 0.6, 2.6, 8.4 Hz, 1H), 6.99–7.02 (m, 1H), 7.04 (t, J = 2.1 Hz, 1H),
7.28 (t, J = 8.0 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 8.11 (dd, J = 1.5 Hz, 7.8 Hz, 1H), 8.23 (dd, J = 1.5 Hz,
8.0 Hz, 1H), 8.53 (s, 1H); 13C-NMR (acetone-d6)  50.1, 55.5, 114.2, 114.7, 120.9, 123.1, 124.8, 127.1,
128.7, 130.7, 138.7, 139.1, 146.9, 149.1, 160.8, 161.0. LC-MS (ESI): [M]+ = 345.46, 347.40.
3-(3-Methoxybenzyl)-8-(3-methoxyphenyl)quinazolin-4(3H)-one (2a). Synthesized according to
Method A using 10 (100 mg, 0.29 mmol) and 3-methoxyphenylboronic acid (66 mg, 0.43 mmol). The
residue was purified by silica gel column chromatography (n-hexane/EtOAc 70:30) to afford 2a as
1
colorless oil. Yield: 98%. H-NMR (acetone-d6)  3.75 (s, 3H), 3.81 (s, 3H), 5.23 (s, 2H), 6.85 (ddd,
J = 0.8, 2.6, 8.3 Hz, 1H), 6.94 (ddd, J = 1.0, 2.7, 8.3 Hz, 1H), 6.98–7.01 (m, 1H), 7.04 (t, J = 2.1 Hz,
1H), 7.16 (ddd, J = 1.0, 1.5, 7.6 Hz, 1H), 7.21 (dd, J = 1.6, 2.5 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.33
(t, J = 8.0 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.81 (dd, J = 1.6, 7.4 Hz, 1H), 8.27 (dd, J = 1.6, 8.0 Hz,
13
1H), 8.36 (s, 1H); C-NMR (acetone-d6)  49.9, 55.56, 55.58, 113.5, 114.1, 114.8, 117.4, 120.9,
123.7, 123.9, 126.8, 127.7, 129.6, 130.7, 135.8, 139.4, 140.3, 140.9, 146.4, 147.5, 160.1, 161.0, 161.5.
LC-MS (ESI): [M]+ = 372.77.
3-(3-Hydroxybenzyl)-8-(3-hydroxyphenyl)quinazolin-4(3H)-one (2b). Synthesized according to
Method B using 2a (125 mg, 0.31 mmol) and BF3.SMe2 (196 µL, 1.86 mmol). The residue was
1
triturated in diethyl ether to afford 2b as a colorless solid. Yield: 97%. Mp: 155–158 °C. H-NMR
(acetone-d6)  5.43 (s, 2H), 6.84 (ddd, J = 0.8, 2.4, 8.1 Hz, 1H), 6.99–7.04 (m, 5H), 7.20–7.23 (m, 1H),
7.36–7.40 (m, 1H), 7.90 (t, J = 7.7 Hz, 1H), 8.01 (dd, J = 1.5, 7.5 Hz, 1H), 8.40 (dd, J = 1.5, 8.1 Hz,
1H), 8.43 (br, 1H), 8.67 (br, 1H), 9.48 (s, 1H); 13C-NMR (acetone-d6)  52.6, 116.5, 117.2, 117.4,
120.7, 121.6, 121.8, 127.9, 130.5, 130.9, 131.6, 135.2, 136.3, 136.5, 136.7, 137.9, 152.1, 158.7, 158.9,
159.3. LC-MS (ESI): [M]+ = 344.81.
3-Amino-5-(3-methoxyphenyl)thiophene-2-carboxamide (11a). A solution of sodium sulfide
nonahydrate Na2S.9H2O (24 g, 100 mmol) in DMF (100 mL) was heated at 40 °C for 30 min. To this
solution 3-chloro-3-(3-methoxyphenyl)prop-2-enenitrile (9.68 g, 50 mmol) in DMF (20 mL) was
added. After 2 hours at 50 °C, 2-chloroacetamide (8.42 g, 100 mmol) in DMF (20 mL) was added to
the reaction mixture. The solution was stirred overnight at 50 °C. Freshly prepared EtONa in EtOH
(2.30 g of sodium in 30 mL of absolute EtOH) was added to the reaction mixture, which was stirred
and kept at 50 °C for 2 h. After cooling to room temperature, the mixture was poured into ice water
solution under stirring. The aqueous layer was extracted three times with EtOAc (3 × 50 mL). The
organic layer was washed once with water, dried over MgSO4, filtered and the solution was
concentrated under reduced pressure. The residue was purified by silica gel column chromatography
(EtOAc 100%) to afford 11a as a pale brown solid. Yield: 21%. Mp: 104–106 °C. 1H-NMR (acetone-d6) 
3.85 (s, 3H), 6.28 (br, 2H), 6.67 (br, 2H), 6.93 (s, 1H), 6.95 (ddd, J = 0.9, 2.6, 8.3 Hz, 1H), 7.16 (t,
J = 2.1 Hz, 1H), 7.22 (ddd, J = 0.9, 1.6, 7.7 Hz, 1H), 7.34 (t, J = 8.1 Hz, 1H); 13C-NMR (acetone-d6) 

Molecules 2013, 18
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60.0, 111.1, 116.3, 119.37, 119.43, 123.2, 135.3, 140.4, 154.2, 157.0, 165.4, 166.2. LC-MS (ESI):
[M]+ = 248.94.
2,6-Bis(3-methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (12). A solution of 3-amino-5-(3-
methoxyphenyl)thiophene-2-carboxamide 11a (200 mg, 0.80 mmol), 4-methoxybenzaldehyde
(117 µL, 0.96 mmol) and concentrated hydrochloric acid 37% (250 µL) was refluxed for 2 hours. After
cooling, the precipitate was collected by filtration, rinsed with cold MeOH and washed twice with
1
diethyl ether to give 12 as a pale brown solid. Yield: 61%. Mp: 241–243 °C. H-NMR (DMSO-d6) 
3.86 (s, 3H), 3.88 (s, 3H), 7.03 (ddd, J = 1.3, 2.5, 7.9 Hz, 1H), 7.14 (ddd, J = 0.9, 2.7, 8.3 Hz, 1H), 7.36 (t,
J = 1.9 Hz, 1H), 7.37–7.47 (m, 3H), 7.71 (t, J = 1.8 Hz, 1H), 7.74 (ddd, J = 0.9, 1.5, 7.7 Hz, 1H), 7.81
(s, 1H); 13C-NMR (DMSO-d6)  56.0, 56.1, 112.3, 113.5, 116.0, 118.2, 119.2, 120.7, 121.1, 122.0,
130.2, 131.0, 134.4, 134.5, 151.5, 155.1, 158.5, 158.7, 160.1, 160.6. LC-MS (ESI): [M+H]+ = 365.26.
2,6-Bis(3-methoxyphenyl)-3-methylthieno[3,2-d]pyrimidin-4(3H)-one (3a). Methyl iodide (20 µL,
0.32 mmol) was added to a solution of 12 (75 mg, 0.21 mmol) and potassium carbonate (58 mg,
0.42 mmol) in dry CH3CN (2 mL). The reaction mixture was refluxed for 3 h. After cooling to room
temperature, the reaction was stopped by adding water. The aqueous layer was extracted three times
with EtOAc (3 × 5 mL). The organic layer was dried over MgSO4, filtered and the solution was
concentrated under reduced pressure. The residue was purified by silica TLC using n-hexane and
EtOAc as eluent (n-hexane/EtOAc 60:40) to afford 3a as a colorless solid. Yield: 48%. Mp:
1
148–150 °C. H-NMR (CDCl3)  3.54 (s, 3H), 3.871 (s, 3H), 3.873 (s, 3H), 6.96 (ddd, J = 0.8, 2.5,
8.2 Hz, 1H), 7.05–7.08 (m, 2H), 7.11 (dt, J = 1.2, 7.8 Hz, 1H), 7.23 (t, J = 2.0 Hz, 1H), 7.30 (ddd,
J = 1.0, 1.4, 7.7 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.51 (s, 1H). 13C-NMR
(CDCl3)  34.2, 55.4, 55.5, 112.1, 113.5, 114.9, 115.9, 119.0, 120.2, 120.8, 121.2, 130.1, 130.3, 134.5,
136.3, 152.5, 156.5, 157.8, 158.5, 159.9, 160.1. LC-MS (ESI): [M+H]+ = 379.29.
2,6-Bis(3-hydroxyphenyl)-3-methylthieno[3,2-d]pyrimidin-4(3H)-one (3b). Synthesized according to
Method B using 3a (27 mg, 0.08 mmol) and BF3.SMe2 (51 µL, 0.48 mmol). The residue was triturated
in a mixture of diethyl ether/petroleum ether to afford 3b as a pale pink solid. Yield: 57%. Mp:
303–304 °C. 1H-NMR (DMSO-d6)  3.38 (s, 3H), 6.87 (dt, J = 2.0, 7.2 Hz, 1H), 6.94 (ddd, J = 0.8, 2.5,
8.1 Hz, 1H), 7.00 (t, J = 1.7 Hz, 1H), 7.04–7.06 (m, 1H), 7.19–7.20 (m, 1H), 7.27–7.36 (m, 3H), 7.74
(s, 1H), 9.78 (br, 2H); 13C-NMR (DMSO-d6)  33.8, 112.7, 115.1, 116.79, 116.81, 117.0, 118.7, 119.8, 120.8,
129.6, 130.5, 133.7, 136.2, 151.2, 156.4, 157.3, 157.6, 157.99, 158.02. LC-MS (ESI): [M]+ = 350.85.
2-(4-Methoxybenzyl)-6-(3-methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (13). To a solution of
3-amino-5-(3-methoxyphenyl)thiophene-2-carboxamide 11a (100 mg, 0.40 mmol) in dry THF (3 mL)
was added Et3N (67 µL, 0.48 mmol) followed by 4-methoxyphenylacetyl chloride (73 µL, 0.48 mmol).
The reaction mixture was stirred at room temperature for 1 h. The solvent was removed under reduced
pressure before NaOH 2N (1.5 mL) and DMF (1.5 mL) were added to the residue. The solution was
refluxed for 30 min, cooled to room temperature and poured into cold water. The precipitate formed
was filtered off, washed with water and diethyl ether to give 13 as a colorless solid. Yield: 49%. Mp:
258–261 °C. 1H-NMR (DMSO-d6)  3.71 (s, 3H), 3.83 (s, 3H), 3.89 (s, 2H), 6.87 (d, J = 8.6 Hz, 2H),
7.00–7.03 (m, 1H), 7.28 (d, J = 8.6 Hz, 2H), 7.33–7.42 (m, 2H), 7.79 (s, 1H), 12.59 (br, 1H);13C-NMR

Molecules 2013, 18
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(DMSO-d6)  50.0, 60.3, 60.6, 114.6, 116.5, 118.3, 118.6, 119.2, 120.6, 123.7, 125.0, 133.6, 135.2,
135.8, 139.1, 155.8, 163.4, 163.9, 165.0. LC-MS (ESI): [M+H]+ = 379.22.
2-(4-Methoxybenzyl)-6-(3-methoxyphenyl)-3-methylthieno[3,2-d]pyrimidin-4(3H)-one (3c). Methyl
iodide (11 µL, 0.18 mmol) was added to a solution of 13 (46 mg, 0.12 mmol) and potassium carbonate
(66 mg, 0.24 mmol) in dry CH3CN (2 mL). The reaction mixture was refluxed for 3 hours. After
cooling to room temperature, the reaction was stopped by adding water. The aqueous layer was
extracted three times with EtOAc (3 × 5 mL). The organic layer was dried over MgSO4, filtered and
the solution was concentrated under reduced pressure. The residue was purified by preparative TLC
using n-hexane and EtOAc as eluent (n-hexane/EtOAc 60:40) to afford 3c as a pale yellow solid.
1
Yield: 74%. Mp: 133–134 °C. H-NMR (CDCl3)  3.61 (s, 3H), 3.87 (s, 3H), 3.96 (s, 3H), 4.27 (s,
2H), 6.95 (d, J = 8.7 Hz, 2H), 7.02–7.05 (m, 1H), 7.25 (d, J = 8.7 Hz, 2H), 7.30–7.32 (m, 1H),
7.34–7.40 (m, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.57 (s, 1H); 13C-NMR (CDCl3)  30.9, 42.0, 55.5, 55.6,
112.3, 114.7, 115.2, 119.2, 120.8, 121.1, 126.8, 129.6, 130.5, 134.8, 152.5, 156.7, 157.9, 158.7, 159.1,
160.3. LC-MS (ESI): [M+H]+ = 393.28.
2-(4-Hydroxybenzyl)-6-(3-hydroxyphenyl)-3-methylthieno[3,2-d]pyrimidin-4(3H)-one (3d). Synthesized
according to Method B using 3c (35 mg, 0.01 mmol) and BF3.SMe2 (56 µL, 0.54 mmol). The residue
was triturated in a mixture diethyl ether/petroleum ether to afford 3d as a pale brown solid. Yield:
1
98%. Mp: 308–310 °C. H-NMR (DMSO-d6)  3.46 (s, 3H), 4.16 (s, 2H), 6.72 (d, J = 7.1 Hz, 2H),
6.83–6.88 (m, 1H), 7.07 (d, J = 7.1 Hz, 2H), 7.17 (br, 1H), 7.26–7.33 (m, 2H), 7.72 (s, 1H), 9.35
(s, 1H), 9.79 (s, 1H); 13C-NMR (DMSO-d6)  30.7, 75.4, 111.8, 113.2, 116.0, 117.2, 117.5, 119.7, 121.3,
125.8, 130.1, 130.9, 134.2, 151.5, 156.7, 157.0, 158.1, 158.5, 159.3. LC-MS (ESI): [M+H]+ = 364.89.
Methyl 2-amino-3-bromobenzoate (14). A solution of 2-amino-3-bromobenzoic acid 8 (1.033 g,
4.78 mmol) and thionyl chloride (30 mL) was heated at reflux for 4 hours. After cooling, the solvent
was reduced under reduced pressure. The residue was poured into MeOH at 0 °C. Excess solvent was
removed under reduced pressure. The precipitate was filtered and purified by recrystallization in a
1
MeOH/water mixture to afford 14 as a yellow solid. Yield: 89%. Mp: 39–40 °C. H-NMR
(Acetone-d6)  3.87 (s, 3H), 6.57 (t, J = 7.8 Hz, 1H), 6.63 (br, 2H), 7.64 (dd, J = 1.6, 7.9 Hz, 1H), 7.85
(dd, J = 1.5, 8.1 Hz, 1H); 13C-NMR (Acetone-d6)  52.3, 110.8, 112.4, 117.0, 131.6, 138.2, 148.8, 168.5.
Methyl 3-bromo-2-{[(3-methoxyphenyl)carbonyl]amino}benzoate (15). To a solution of 14 (983 mg,
4.27 mmol) in dry CH2Cl2 (5 mL) was added Et3N (653 µL, 4.70 mmol) followed by
3-methoxyphenylacetyl chloride (600 µL, 4.27 mmol). The reaction mixture was stirred at room
temperature overnight. Water was added to the solution to stop the reaction. The aqueous layer was
extracted three times with CH2Cl2 (3 × 20 mL). The organic layer was dried over MgSO4, filtered and
the solution was concentrated under reduced pressure. The residue was purified by silica gel column
chromatography (n-hexane/EtOAc 95: 5 to 70:30) to afford 15 as a white viscous oil. Yield: 15%. 1H-NMR
(acetone-d6)  3.78 (s, 3H), 3.89 (s, 3H), 7.18 (ddd, J = 1.0, 2.6, 8.3 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H),
7.46 (t, J = 7.7 Hz, 1H), 7.59 (dd, J = 1.6, 2.6 Hz, 1H), 7.64 (ddd, J = 1.0, 1.5, 7.6 Hz, 1H), 7.90 (dd,
J = 1.5, 7.8 Hz, 1H), 7.93 (dd, J = 1.5, 8.0 Hz, 1H), 9.48 (br, 1H); 13C-NMR (acetone-d6)  52.7, 55.8,
113.7, 118.6, 120.6, 123.8, 128.4, 130.57, 130.59, 131.0, 136.9, 137.2, 137.4, 160.9, 166.1, 166.8.

Molecules 2013, 18
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8-Bromo-2-(3-methoxyphenyl)quinazolin-4(3H)-one (16). A solution of 15 in formamide (5 mL) was
heated at 180 °C for 6 hours. After cooling, the precipitate formed was dispersed with cold water and
filtered off. The solid was washed twice with water to give 16 as a pale brown solid. The title
compound was used for the next step without further purification. Yield: 81%. Mp: 236–239 °C. 1H-NMR
(DMSO-d6)  3.87 (s, 3H), 7.18 (ddd, J = 1.0, 2.5, 8.2 Hz, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.49 (t, J = 8.0
Hz, 1H), 7.83 (t, J = 1.8 Hz, 1H), 7.88 (ddd, J = 0.9, 1.4, 7.9 Hz, 1H), 8.14 (dd, J = 1.3, 5.4 Hz, 1H),
8.16 (dd, J = 1.4, 5.3 Hz, 1H), 12.75 (br, 1H); 13C-NMR (DMSO-d6)  55.4, 112.9, 117.8, 120.3,
122.2, 122.6, 125.7, 127.4, 129.8, 133.6, 137.9, 146.0, 152.6, 159.3, 161.9. LC-MS (ESI): [M]+ = 331.54,
333.35.
2,8-Bis(3-methoxyphenyl)quinazolin-4(3H)-one (17). Synthesized according to Method A using 16
(144 mg, 0.43 mmol) and 3-methoxyphenylboronic acid (98 mg, 0.65 mmol). The residue was purified
by silica gel column chromatography (n-hexane/EtOAc 70:30) to afford 17 as a colorless solid. Yield:
1
77%. Mp: 198–201 °C. H-NMR (DMSO-d6)  3.81 (s, 3H), 3.82 (s, 3H), 6.99 (dd, J = 2.0, 8.2 Hz,
1H), 7.09–7.13 (m, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.33 (t, J = 2.0 Hz, 1H), 7.39–7.44 (m, 2H), 7.59 (t,
J = 7.7 Hz, 1H), 7.74–7.77 (m, 2H), 7.90 (dd, J = 1.4, 7.4 Hz, 1H), 8.19 (dd, J = 1.4, 7.9 Hz, 1H), 12.61
(br, 1H); 13C-NMR (DMSO-d6)  55.0, 55.1, 112.3, 112.9, 116.2, 117.8, 119.8, 121.7, 122.9, 125.4, 126.5,
128.7, 129.7, 134.1, 135.1, 138.4, 139.6, 145.5, 150.8, 158.7, 159.3, 162.4. LC-MS (ESI): [M]+ = 358.76.
2,8-Bis(3-methoxyphenyl)-3-methylquinazolin-4(3H)-one (4a). Methyl iodide (29 µL, 0.47 mmol) was
added to a solution of 17 (112 mg, 0.31 mmol) and potassium carbonate (86 mg, 0.62 mmol) in dry
CH3CN (3 mL). The reaction mixture was refluxed for 3 h. After cooling to room temperature, the
solvent was removed under reduced pressure and water was added to the residue to dissolve the
inorganic salt. The aqueous layer was extracted three times with EtOAc (3 × 5 mL). The organic layer
was dried over MgSO4, filtered and the solution was concentrated under reduced pressure. The residue
was purified by silica gel column chromatography using n-hexane and EtOAc as eluent
1
(n-hexane/EtOAc 70:30) to afford 4a as a colorless solid. Yield: 64%. Mp: 241–244 °C. H-NMR
(acetone-d6)  3.49 (s, 3H), 3.81 (s, 3H), 3.86 (s, 3H), 6.88 (ddd, J = 1.0, 2.6, 8.2 Hz, 1H), 7.07 (ddd,
J = 0.9, 2.6, 8.3 Hz, 1H), 7.23 (dt, J = 1.2, 7.7 Hz, 1H), 7.25 (dt, J = 1.1, 7.6 Hz, 1H), 7.28–7.29 (m,
1H), 7.30 (t, J = 7.8 Hz, 1H), 7.35 (dd, J = 1.7, 2.5 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.59 (t, J = 7.7
Hz, 1H), 7.87 (dd, J = 1.6, 7.5 Hz, 1H), 8.26 (dd, J = 1.6, 7.9 Hz, 1H); 13C-NMR (acetone-d6)  34.4,
55.5, 55.8, 113.8, 114.8, 116.3, 117.3, 121.3, 122.4, 123.5, 126.7, 127.3, 129.5, 130.4, 135.6, 138.3,
139.9, 140.9, 145.5, 156.2, 160.0, 160.6, 163.0. LC-MS (ESI): [M]+ = 372.71.
2,8-Bis(3-hydroxyphenyl)-3-methylquinazolin-4(3H)-one (4b). Synthesized according to Method B
using 4a (65 mg, 0.17 mmol) and BF3.SMe2 (107 µL, 1.02 mmol). The residue was triturated in diethyl
1
ether to afford 4b as a colorless solid. Yield: 98%. Mp: 241–244 °C. H-NMR (acetone-d6)  3.48 (s,
3H), 6.80 (ddd, J = 1.0, 2.6, 8.2 Hz, 1H), 6.98 (ddd, J = 1.0, 2.4, 8.2 Hz, 1H), 7.11–7.15 (m, 3H), 7.18–7.19
(m, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.33 (t, J = 8.1 Hz, 1H), 7.56–7.59 (m, 1H), 7.81 (dd, J = 1.7, 7.5 Hz,
13
1H), 8.25 (dd, J = 1.6, 8.0 Hz, 1H), 8.35 (s, 1H), 8.75 (s, 1H); C-NMR (acetone-d6)  34.4, 115.0,
116.3, 117.6, 118.5, 120.4, 122.3, 122.7, 126.6, 127.3, 129.5, 130.4, 135.6, 138.2, 140.2, 141.0, 145.6,
156.3, 157.7, 158.3, 163.1. LC-MS (ESI): [M]+ = 344.77.

Molecules 2013, 18
4504
3.3. General Procedure for the Preparation of Compounds 18a–b — Method C
A solution of 3-amino-5-(3-methoxyphenyl)thiophene-2-carboxamide 11a (4 mmol) or 3-amino-5-
(4-methoxyphenyl)thiophene-2-carboxamide 11b (4 mmol) in formic acid (10 mL) and with catalytic
amounts of concentrated sulfuric acid (30 drops) was heated at 50 °C overnight. After cooling, the
reaction was quenched with cold water and the solid formed was collected by filtration. The residue
was triturated in diethyl ether and filtered off.
6-(3-Methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (18a). Synthesized according to Method C
using 11a (993 mg, 4 mmol) and HCOOH (10 mL). The residue was triturated in diethyl ether to
1
afford 18a as a pale green solid. Yield: 76%. Mp: 237–245 °C. H-NMR (DMSO-d6)  3.85 (s, 3H),
13
7.03 (dt, J = 2.4, 6.9, 1H), 7.38–7.43 (m, 3H), 7.87 (s, 1H), 8.17 (s, 1H), 12.55 (br, 1H); C-NMR
(DMSO-d6)  55.4, 111.5, 115.4, 118.5, 121.5, 122.2, 130.5, 133.8, 147.1, 150.4, 157.0, 158.4, 159.8.
LC-MS (ESI): [M+H]+ = 259.18.
6-(4-Methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (18b). Synthesized according to Method C
using 11b (993 mg, 4 mmol) and HCOOH (10 mL). The residue was triturated in diethyl ether to
1
afford 18b as a pale green solid. Yield: 98%. Mp: 296–299 °C. H-NMR (DMSO-d6)  3.82 (s, 3H),
13
7.04 (d, J = 8.9, 2H), 7.69 (s, 1H), 7.78 (d, J = 8.9, 2H), 8.14 (s, 1H), 12.45 (br, 1H); C-NMR
(DMSO-d6)  55.4, 114.7, 119.7, 121.2, 125.1, 127.6, 146.9, 150.8, 156.9, 158.6, 160.4. LC-MS (ESI):
[M+H]+ = 259.19.
3.4. General Procedure for the Preparation of Compounds 19a–b — Method D
A solution of 6-(3-methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (18a, 0.58 mmol) or 6-(4-
methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (18b, 0.58 mmol) in phosphorus oxychloride
(5 mL) was heated under microwave irradiation at 95 °C/80 W for 20 min. Excess of phosphorus
oxychloride was removed under reduced pressure. Ice was added to the residue and the precipitate
formed was collected by filtration. The compounds were pure enough and could be used in the next
step without further purification.
4-Chloro-6-(3-methoxyphenyl)thieno[3,2-d]pyrimidine (19a). Synthesized according to Method D
using 18a (150 mg, 0.58 mmol) and POCl3 (5 mL) afforded 19a as a brown solid. Yield: 98%. Mp:
153–156 °C. 1H-NMR (DMSO-d6)  3.87 (s, 3H), 7.10–7.14 (m, 1H), 7.47 (t, J = 8.2, 1H), 7.51–7.54
13
(m, 2H), 8.28 (s, 1H), 9.03 (s, 1H); C-NMR (DMSO-d6) 55.5, 111.9, 116.8, 119.2, 120.8, 129.4,
130.7, 132.9, 153.0, 154.3, 154.8, 159.9, 162.5. LC-MS (ESI): [M+H]+ = 277.17.
4-Chloro-6-(4-methoxyphenyl)thieno[3,2-d]pyrimidine (19b). Synthesized according to Method D using
18b (150 mg, 0.58 mmol) and POCl3 (5 mL) afforded 19b as a brown solid. Yield: 99%. Mp: 153–156 °C.
1H-NMR (DMSO-d6)  3.84 (s, 3H), 7.09 (d, J = 8.7, 2H), 7.92 (d, J = 8.9, 2H), 8.07 (s, 1H), 8.97 (s,
1H); 13C-NMR (DMSO-d6)  55.5, 114.9, 118.7, 124.1, 128.5, 128.8, 152.6, 154.6, 154.7, 161.4,
162.9. LC-MS (ESI): [M+H]+ = 277.16.

Molecules 2013, 18
4505
3.5. General Procedure for the Preparation of Compounds 5a–b – Method E
To a solution of 4-chloro-6-(3-methoxyphenyl)thieno[3,2-d]pyrimidine 19a (0.51 mmol) or
4-chloro-6-(4-methoxyphenyl)thieno[3,2-d]pyrimidine 19b (0.51 mmol) in dry DMF (2 mL) was
added sodium methanolate in methanol (0.51 mmol) and the reaction mixture was stirred at room
temperature for 2 hours. Water was added to quench the reaction. The precipitate formed was collected
by filtration and washed once with water and once with petroleum ether.
4-Methoxy-6-(3-methoxyphenyl)thieno[3,2-d]pyrimidine (5a). Synthesized according to Method E
using 19a (140 mg, 0.51 mmol) and NaOMe (0.51 mmol in 2 mL MeOH). The residue was triturated
in petroleum ether to afford 5a as a brown solid. Yield: 91%. Mp: 159–162 °C. 1H-NMR (DMSO-d6) 
3.86 (s, 3H), 4.13 (s, 3H), 7.07 (dt, J = 2.3, 7.4 Hz, 1H), 7.42–7.46 (m, 3H), 8.08 (s, 1H), 8.77 (s, 1H);
13C-NMR (DMSO-d6)  54.2, 55.4, 111.8, 115.9, 116.1, 118.8, 120.5, 130.6, 133.6, 151.3, 154.7,
159.9, 162.4, 163.5. LC-MS (ESI): [M+H]+ = 273.22.
4-Methoxy-6-(4-methoxyphenyl)thieno[3,2-d]pyrimidine (5b). Synthesized according to Method E
using 19b (140 mg, 0.51 mmol) and NaOMe (0.51 mmol in 2 mL MeOH). The residue was triturated
in petroleum ether to afford 5b as a brown solid. Yield: 40%. Mp: 184–186 °C. 1H-NMR (DMSO-d6) 
3.83 (s, 3H), 4.12 (s, 3H), 7.08 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H), 7.91 (s, 1H), 8.74 (s, 1H);
13C-NMR (DMSO-d6)  54.1, 55.4, 114.8, 115.4, 118.6, 124.8, 128.0, 151.6, 154.7, 160.8, 162.8,
163.3. LC-MS (ESI): [M+H]+ = 273.21.
3.6. General Procedure for the Preparation of Compounds 5c–d – Method F
To a solution of 4-chloro-6-(3-methoxyphenyl)thieno[3,2-d]pyrimidine (19a, 1 eq.) in dry DMF (2 mL)
was added freshly prepared dry sodium 3-methoxyphenolate or sodium 4-methoxyphenolate (1.2 eq.)
The reaction mixture was stirred at room temperature for 2 h. Water was added to stop the reaction.
Organic layer was washed once with NaOH 2N and once with brine, dried over MgSO4, filtered and
the solution was concentrated under reduced pressure. The precipitate formed was triturated with a
mixture of petroleum ether / diethyl ether and filtered off.
4-(3-Methoxyphenoxy)-6-(3-methoxyphenyl)thieno[3,2-d]pyrimidine (5c). Synthesized according to
Method F using 19a (100 mg, 0.36 mmol) and sodium 3-methoxyphenolate (56 mg, 0.43 mmol). The
residue was triturated in petroleum ether to afford 5c as a pale brown solid. Yield: 88%. Mp:
129–132 °C. 1H-NMR (DMSO-d6)  3.78 (s, 3H), 3.87 (s, 3H), 6.92 (dd, J = 2.4, 8.2 Hz, 2H), 6.97 (t,
J = 2.3 Hz, 1H), 7.09 (ddd, J = 1.1, 1.3, 8.0 Hz, 1H), 7.39 (t, J = 8.2 Hz, 1H), 7.46 (t, J = 8.2 Hz, 1H),
13
7.49–7.51 (m, 2H), 8.16 (s, 1H), 8.70 (s, 1H); C-NMR (DMSO-d6) 54.4, 55.4, 108.0, 111.86,
111.87, 114.0, 116.1, 116.3, 118.9, 120.5, 130.2, 130.6, 133.4, 152.3, 152.7, 154.6, 159.9, 160.4,
163.1, 163.6. LC-MS (ESI): [M+H]+ = 365.26.
4-(4-Methoxyphenoxy)-6-(3-methoxyphenyl)thieno[3,2-d]pyrimidine (5d). Synthesized according to
Method F using 19a (30 mg, 0.11 mmol) and sodium 4-methoxyphenolate (17 mg, 0.13 mmol). The
residue was triturated in petroleum ether to afford 5d as a pale brown solid. Yield: 87%. Mp: 139–146 °C.
1H-NMR (DMSO-d6)  3.85 (s, 3H), 3.90 (s, 3H), 6.98–7.01 (m, 3H), 7.15–7.17 (m, 1H), 7.20 (t,

Molecules 2013, 18
4506
13
J = 7.9 Hz, 1H), 7.29 (t, J = 2.0 Hz, 1H), 7.36–7.42 (m, 2H), 7.70 (s, 1H), 8.71 (s, 1H); C-NMR
(DMSO-d6) 55.4, 55.6, 112.5, 114.8, 115.4, 117.4, 119.3, 120.0, 122.7, 130.4, 134.3, 145.4, 153.2,
154.8, 157.5, 160.2, 163.6, 164.0. LC-MS (ESI): [M+H]+ = 365.25.
4. Conclusions
In this study, a series of six thieno[3,2-d]pyrimidin-4-ones, four thieno[3,2-d]pyrimidines and four
quinazolin-4-ones was synthesized and tested for 17-HSD2 and 17-HSD1 inhibition. Two
compounds 3b and 3d were identified as moderate 17-HSD2 inhibitors, 3b being the best one with an
inhibition of 36% at 1 µM. These compounds resulted from the rigidification of conformer III, which
can be considered as the active conformer in the enzyme binding site. The inactive globular
compounds like 4a/4b or 5c/5d are indicative of the enzyme binding site being rather elongated and
are tools to further characterize the 17-HSD2 active site. Compounds 3b/3d need further optimization
focussing on conformer III and exchanging the geometry of the pyrimidinone or replacing the polar
nitrogen of the pyrimidinone moiety by a carbon or reducing the size of the ring fused to the thiophene.
Acknowledgments
We thank Josef Zapp for the NMR measurements, and Isabella Mang, Laura Schrobildgen, and
Nina Hanke for performing the biological tests. We acknowledge the Deutsche Forschungsgemeinschaft
(DFG) for financial support (Grants HA1315/12-1).
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Sample Availability: Samples of the compounds 1a–b, 2a–b, 3a–d, 4a–b, 5a–d are available from
the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).