Synthesis and biological evaluation of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) inhibitors based on a thieno[2,3-d] pyrimidin-4(3H)-one core

Article abstract of DOI:10.1021/jm900928k

Many breast tumors are hormone-dependent, and estrogens, especially estradiol (E2), have a pivotal role in their growth and development. 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is a key enzyme in the biosynthesis of female sex steroids, catalyzing the NADPH-dependent reduction of estrone into biologically active estradiol. In this study, a library of fused (di)cycloalkeno thieno[2,3-d]pyrimidin-4(3H)-one based compounds was synthesized, and the biological activities against 17β-HSD1 in a cell-free and in a cell-based assay were evaluated. Several thieno[2,3-d]pyrimidin-4(3H)-one based compounds, at 0.1 and 1 μM test concentrations, were found to be potent 17β-HSD1 inhibitors. For example, 4-(3-hydroxyphenylthio)-1,2,7,8,9,10,11,13-octahydro-13-oxo-[1] benzothieno[2′,3′:4,5]-pyrimido[1,2-a]azepine-3-carboxaldehyde (7f) is one of the most potent nonsteroidal 17β-HSD1 inhibitors reported to date with 94% inhibition of the recombinant enzyme at 0.1 μM test concentration. Importantly, the majority of these compounds exhibited excellent selectivity over the oxidative isoform 17β-HSD2 and lacked estrogenic effects in an estrogen receptor (ER) binding assay.

Full text of DOI:10.1021/jm900928k

6660 J. Med. Chem. 2009, 52, 6660–6671
DOI: 10.1021/jm900928k
Synthesis and Biological Evaluation of 17β-Hydroxysteroid Dehydrogenase Type 1 (17β-HSD1)
Inhibitors Based on a Thieno[2,3-d]pyrimidin-4(3H)-one Core
Annamaria Lilienkampf,† Sampo Karkola,† Sari Alho-Richmond,† Pasi Koskimies,‡ Nina Johansson,‡ Kaisa Huhtinen,‡,§
€ € €
,†
Kimmo Vihko,‡,§ and Kristiina Wahala*
†Laboratory of Organic Chemistry, Department of Chemistry, P.O. Box 55, FIN-00014 University of Helsinki, Finland, and ‡Hormos Medical
Ltd., PharmaCity, FIN-20520 Turku, Finland. §Current address: Department of Physiology, Institute of Biomedicine, FIN-20520 University of
Turku, Finland.
Received June 23, 2009
Many breast tumors are hormone-dependent, and estrogens, especially estradiol (E2), have a pivotal
role in their growth and development. 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is a key
enzyme in the biosynthesis of female sex steroids, catalyzing the NADPH-dependent reduction of
estrone into biologically active estradiol. In this study, a library of fused (di)cycloalkeno thieno[2,3-
d]pyrimidin-4(3H)-one based compounds was synthesized, and the biological activities against 17β-
HSD1 in a cell-free and in a cell-based assay were evaluated. Several thieno[2,3-d]pyrimidin-4(3H)-one
based compounds, at 0.1 and 1 μM test concentrations, were found to be potent 17β-HSD1 inhibitors.
For example, 4-(3-hydroxyphenylthio)-1,2,7,8,9,10,11,13-octahydro-13-oxo-[1]benzothieno[20,30:4,5]-
pyrimido[1,2-a]azepine-3-carboxaldehyde (7f) is one of the most potent nonsteroidal 17β-HSD1
inhibitors reported to date with 94% inhibition of the recombinant enzyme at 0.1 μM test concentration.
Importantly, the majority of these compounds exhibited excellent selectivity over the oxidative isoform
17β-HSD2 and lacked estrogenic effects in an estrogen receptor (ER) binding assay.
Introduction
superfamily.6 17β-HSD type 1 (17β-HSD1) is a key enzyme in
female hormonal regulation, converting the less potent estro-
gen estrone (E1) into the biologically active E2 (Figure 1), and
is thus an attractive target for inhibitor development for the
prevention and control of breast tumor growth. On the other
hand, the reverse inactivating enzymatic reaction is catalyzed
by the 17β-HSD2 isoform. 17β-HSD1 is essential for both
gonadal and peripheral E2 synthesis and is mainly expressed
in female breast tissue, ovaries, and placenta. High 17β-HSD1
expression has been found in malignant breast tissue7 and
immunohistochemical studies suggest that 17β-HSD1 has an
important role in the in situ E2 production in hormone-
dependent breast cancer tumors.8 17β-HSD1 has been shown
to be an independent prognostic factor in breast cancer.9
Recently, in vivo efficacy of 17β-HSD1 inhibition has been
demonstrated. Specific 17β-HSD1 inhibitors were able to
reduce hormone-dependent tumor growth in immunodefi-
cient mice inoculated with MCF-7 cells expressing the human
recombinant 17β-HSD1 enzyme.10
Breast cancer is the most common cancer in women in the
Western countries. More than 180 000 new cases and over
40000 deaths are expected to occur in the United States in
2008.1 The majority of breast cancers are initially hormone-
dependent (estrogen receptor (ERa) positive), and estrogens,
especially estradiol (E2), have a crucial role in their develop-
ment and progression. Lowering the levels of both circulating
and tissue E2 by inhibiting the biosynthesis of active sex
steroids has emerged as an attractive approach for treating
hormone-dependent types of breast cancer.2 Inhibitors of
CYP19 aromatase, the enzyme converting androgens to
estrogens, are already used in breast cancer treatment.3
17β-Hydroxysteroid dehydrogenase (17β-HSD) enzymes
play a crucial role in the synthesis of active female and male
sex steroids.4 They catalyze the NAD(P)(H)-dependent oxi-
dation and reduction of hydroxy or keto groups at the C17
position of androgens and estrogens. At present, 14 mamma-
lian 17β-HSDs have been identified,5 most of them belonging
to the short-chain alcohol dehydrogenase reductase (SDR)
The human 17β-HSD1 is active as a soluble cytosolic
homodimer, both monomeric subunits having 327 residues
and a molecular mass of 34.9 KDa.11 17β-HSD1 has been
crystallized with estrogenic12 and androgenic13 ligands as well
as in the native form14 and with a known steroid-based
inhibitor.15 In addition, the active site has been studied by
molecular modeling16 and site-directed mutagenesis17 experi-
ments. As a member of the SDR superfamily,6 17β-HSD1 has
the highly conserved and catalytically crucial Tyr-Xaa-Xaa-
Xaa-Lys sequence and a generally conserved serine in the
activesite. Theligand-bindingcavityof17β-HSD1 is a narrow
*To whom correspondence should be addressed. Mailing address:
Laboratory of Organic Chemistry, Department of Chemistry, Faculty of
Science, P.O. Box 55, FIN-00014, University of Helsinki, Finland. Tel:
þ358-9-19150356. Fax: þ358-9-19150357. E-mail: kristiina.wahala@
helsinki.fi.
a Abbreviations: E1, estrone; E2, estradiol; ER, estrogen receptor;
HSD, hydroxysteroid dehydrogenase; MDS, molecular dynamics simu-
lation; SDR, short-chain alcohol dehydrogenase reductase; 17β-HSD,
17β-hydroxysteroid dehydrogenase; NAD, nicotinamide adenine dinu-
cleotide; NADP, nicotinamide adenine dinucleotide phosphate;
NADH/NADPH, reduced forms of NAD and NADP, respectively;
DDQ, 2,3-dichloro-5,6-dicyanobenzoquinone.
2
˚
tunnel (340 A ) where carbon atoms contribute 72% of the
r
pubs.acs.org/jmc
Published on Web 10/13/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6661
total surface.12b Polar regions are located at opposite ends of
the otherwise hydrophobic ligand-binding cavity. Superim-
position of available crystal structures has revealed variable
orientations of several active site residues.18
tones29 are known to show a modest activity against 17β-
HSD1. Better activity, however, has been obtained with small
biphenolic molecules, for example, with quinoline and
naphthalene derivatives30 and with certain bisphenyl-
(tri)azoles and bisphenyloxazoles.31 Recently, subsequent to
our findings on thieno[2,3-d]pyrimidin-4(3H)-one based in-
hibitors,32 a series of benzothienopyrimidinones has been
reported to inhibit 17β-HSD1.18,33
In our search for therapeutic agents for hormone-sensitive
breast cancer, we have investigated nonsteroidal small mole-
cules as inhibitors of E2 biosynthesis. Based on molecular
modeling studies on the active site of 17β-HSD1,16 a small
commercial library of compounds, which were likely to fit and
be complementary to the hydrophobic nature of the active
site, were chosen for biological screening in order to identify
potential nonsteroidal 17β-HSD1 inhibitors. The tetracyclic
dicycloalkeno thieno[2,3-d]pyrimidin-4(3H)-one derivative
1,34 bearing an aldehyde and an aromatic thioether moiety
in the ring A, was identified as a lead compound (Figure 2).
Herein, we report the chemical synthesis and biological
activity of 17β-HSD1 inhibitors based on a thieno[2,3-d]pyr-
imidin-4(3H)-one core. Furthermore, the selectivity between
the 17β-HSD1 and 17β-HSD2 isoforms and possible estro-
genicity of these compounds will be discussed.
Most of the known inhibitors of 17β-HSD1 are based on
modifications of steroidal structures.19 For example, E2
derivatives substituted at C1620 or at C1521 have shown
promising 17β-HSD1 inhibitory activity, along with estra-
1,3,5(10)-trien-17-ones22 and D-homoestra-1,3,5(10)-trien-17-
ones23 bearing small substituents at the C2 position. In
addition, E2-adenosine hybrid inhibitors, aimed at binding
both the ligand- and cofactor-binding domains of 17β-HSD1,
have been developed.15,24 Much less work has been published
regarding nonsteroidal molecules as 17β-HSD1 inhibitors.
These compounds could benefit from synthetic accessibility,
drug-likeness, selectivity, and nonestrogenicity when com-
pared with some of the steroid-based inhibitors. Various
phytoestrogens and related compounds have been reported
to inhibit multiple isoforms of 17β-HSD,25 but the lack of
selectivity and the potential estrogenic nature of these natural
compounds have prohibited their use as such.26 Substituted
2-benzyltetral-1-ones,27 benzopyranones,28 and phenyl ke-
Chemistry
Homologation of the tetracyclic skeleton and variation of
the thioether moiety in ring A were chosen for the first
Table 1. Yield (%) of the Oxidation of Thiophene R-Position with
PCC-Celite36 or K2S2O8-CuSO4
Figure 1. The primary enzymatic reactions catalyzed by 17β-HSD
type 1 and type 2.
oxidant
PCC-Celite
K2S2O8-CuSO4
KMnO4
20
4a
4b
52
51
54
56
24
43
49
75
77
85
78
65
68
70
80
4c
4d
4e
16a
16b
27
Figure 2. The lead compound 1,2,7,8,9,10,11,13-octahydro-13-
oxo-4-(phenylthio)-[1]benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-
3-carboxaldehyde (1, CAS name) and the labeling of the fused ring system.
Scheme 1. The Synthetic Route for the Lead Compound 1 and Its Analoguesa
a Reagents and conditions: (a) POCl3, lactam, 1,2-dichloroethane, reflux, 76-91%; (b) K2S2O8, CuSO4 5H2O, MeCN-H2O, reflux, 65-85%;
3
(c) POCl3-DMF, CH2Cl2, 32-83%; (d) RSH, NaOH, THF, 65-93%.

6662 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Lilienkampf et al.
alterations of the lead compound 1. Although 1 was commer-
cially available in milligram quantities, it was an unknown
compound in the chemical literature, and thus an efficient
synthetic route needed to be developed for 1 and its deriva-
tives. The dicycloalkeno thieno[2,3-d]pyrimidin-4(3H)-ones,
bearing thioether and aldehyde functionalities at the ring A,
were synthesized in four consecutive steps from commercially
available starting materials (Scheme 1). First, POCl3-cata-
lyzed cyclization of ethyl 2-amino-4,5,6,7-tetrahydrobenzo-
[b]thiophene-3-carboxylate (2a) or ethyl 2-aminocyclohepta-
[b]thiophene-3-carboxylate (2b) with five- or eight-membered
lactams produced the fused tetracyclic dicycloalkeno thieno-
[2,3-d]pyrimidin-4(3H)-ones 3a-3e in 76-91% yield. The
oxidation of the thiophene R-position in 3a-3e with
K2S2O8-CuSO435 in MeCN-H2O (1:1) proceeded with high
selectivity and significantly shortened the reaction times and
improved the yields of 4a-4e when compared with the pre-
viously reported PCC-Celite oxidation (Table 1).36 In the
next step, Vilsmeier haloformylation37 of the ketones was
employed to produce the corresponding β-chlorovinylalde-
hydes 5a-5d. The Vilsmeier reaction proceeds mainly via a
chloroalkene intermediate38 (colorless and less polar component
thieno[2,3-d]pyrimidin-4(3H)-one derivatives 15-18 were
synthesized (Scheme 3). The same four-step synthetic route
was employed as for the tetracyclic compounds with the
exception that 2a was reacted with acetamide or N-methyla-
cetamide. As described earlier for 4a and 4b, the amidine R-
position in the dimethyl derivative 16b is highly activated,36
and Vilsmeier haloformylation is unfavored, whereas 16a
gave the corresponding β-chlorovinylaldehyde 17 in excellent
yield.
Next, modifications in the ring A were investigated. Aromati-
zation of the unsaturated ring A with DDQ in compounds 1
and 6b produced the corresponding benzothieno[2,3-
d]pyrimidin-4(3H)-one derivatives 19a and 19b in 84-91%
yield (Scheme 4). The subsequent reduction of the aldehyde
functionality with NaBH4 in EtOH gave the benzylic alcohols
20a and 20b. The aldehyde moiety in compounds 1 and 6b,
with a nonaromatic ring A, could similarly be reduced with
NaBH4, but the corresponding allylic alcohols proved to be
relativelyunstableandwerenot taken further. Theconjugated
oxime derivative 21 was prepared by treating 6b with
NH2OH HCl under basic conditions. The aldehyde functionality
3
in ring A was also modified in the β-chloro compound 5b to
give corresponding oxime 22, alcohol 23, and carboxylic acid
24 derivatives (Scheme 5). The best yield and ease of purifica-
tion of 24 was obtained with sodium chlorite oxidation.
Thioether formation combined with aldol condensation to
givethe conjugated ketone25 couldbeperformedasa one-pot
reaction.
1
on TLC, in H NMR spectra the alkene proton apparent
triplet at ∼5.9 ppm) instead of the direct haloformylation by
way of an enaminoketone. With 4a and 4b, which have a five- or
six-membered ring fused to the pyrimidinone moiety, the
amidine R-position (in ring D) is more activated than the
carbonyl R-position,36 and thus the Vilsmeier haloformylation
becomes unpropitious as aminomethylenation, chloroalkene
formation, and direct haloformylation intervene as competing
reactions. For example, with 4a a moderate 32% yield of
β-chlorovinylaldehyde 5a was isolated when the reaction was
performed using DMF as a solvent. In the final step, the
introduction of various aliphatic and aromatic thioether side
chains to give compounds 1, 6a-6i, and 7a-7j (Table 2) was
done with an addition-elimination reaction of corresponding
thiols under basic conditions. The reaction time depended
heavily on the nucleophile used, varying from 15 min up to 2
days with the most hindered t-butyl group.
All the thiols used were commercially available except for
4-(mercaptoacetyl)morpholine (13), which was used in the
synthesis of 6i. Compound 13 was synthesized in three steps
starting from morpholine (Scheme 2).39 The reaction of
morpholine with chloroacetyl chloride gave 4-(chloroacetyl)-
morpholine (11) in 94% yield. For compound 11, two differ-
ent sets of 1H NMR data have been previously reported.39,40
We found our 1H NMR data to be in accordance with those
reported by Pielichowski and Popielarz,40 and we further
verified the structure of 11 with 13C NMR and mass spectros-
copy. The reactionof 11withpotassiumxanthateproduced 12
as an intermediate, which was subsequently hydrolyzed with-
out purification. The hydrolysis was first attempted with the
published procedure39 by treating 12 with ammonia (aq) at
room temperature. In our hands, compound 13 could not
be isolated after the hydrolysis, but the pyrolysis product41
4-[(ethylthio)acetyl]morpholine (14) was obtained instead,
whereas hydrolysis of 12 with 1,3-diaminopropane at 0 °C
gave the desired 13 in 67% yield. Also for 13 our 1H NMR
data varied from the data reported previously.39 Again,
further analysis of 13 with 13C NMR and mass spectroscopy
verified our structure to be correct. In addition, the inter-
mediate 12 was isolated and analyzed.
In addition to the thieno[2,3-d]pyrimidin-4(3H)-one based
inhibitors, bicyclic thiophene derivatives 26-29 were pre-
pared employing a similar synthetic strategy as for the thieno-
[2,3-d]pyrimidin-4(3H)-one compounds (Scheme 6).
Results and Discussion
The lead compound 1 had already shown auspicious bio-
logical activity in a cell-based assay with 41% and 99%
inhibition of 17β-HSD1 at 1 and 10 μM concentration,
respectively. Nine derivatives of 1 with an aliphatic thioether
side chain (6a-6i) and ten derivatives with an aromatic
thioether side chain (7a-7j) were synthesized, and their
biological activity against 17β-HSD1 and 17β-HSD2 was
evaluated (Table 2). Most of these compounds, at 0.1 and
1 μM test concentration, showed good to excellent activity
against 17β-HSD1 in a cell-free (recombinant enzyme) assay.
However, charged substituents like -COOH in 6h and 7c, as
well as the 2-(4-morpholinyl)-2-oxoethylthio group in 6i,
seemed not to be tolerated, and the inhibitory activity was
lost. The most potent compounds found in this study were 7f,
7d, 7b, and7g with 94%-86% inhibition of 17β-HSD1 at 0.1 μM
concentration.
The cyclized intermediates 3a-3e and 4a-4e did not show
significant activity against 17β-HSD1 or 17β-HSD2. The
activity was somewhat improved with the β-chlorovinylalde-
hyde intermediates 5a-5d. However, the best inhibitory
activity was obtained with compounds bearing a thioether
moiety in ring A. Homologation of the dicycloalkeno thieno-
[2,3-d]pyrimidin-4(3H)-one core of potent inhibitor 6b gave
compounds 8-10 and 18 with decreased biological activity
(Table 3); hence the original thieno[2,3-d]pyrimidin-4(3H)-
one core with a six-membered ring A and a seven-membered
ring D was chosen for further modifications. Aromatic ring A
did not significantly improve the inhibitory activity of deri-
vatives 19a and 19b. Similar inhibitory activitywas found with
With the aim of investigating the significance of the alipha-
tic ring D for the 17β-HSD1 inhibitory activity, tricyclic

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6663
Scheme 5a
Scheme 2a
a Reagents and conditions: (a) ClCOCH2Cl, Et2O, 94%; (b) EtOC-
(S)SK, DMF; (c) 1,3-diaminopropane, dry THF, 0 °C, 67%; (d) NH3-
(aq), rt, overnight.
Scheme 3a
a Reagents and conditions: (a) NH2OH HCl, AcONa, EtOH, 75%;
(b) NaBH4, THF, 78%; (c) NaClO2, NaH2PO4 H2O, 2-methyl-2-
3
3
butene, t-BuOH-H2O, 72%; (d) PrSH, NaOH, acetone, 65%.
Scheme 6a
a Reagents and conditions: (a) acetamide or N-methylacetamide,
POCl3, 1,2-dichloroethane, reflux, 74-85%; (b) K2S2O8, CuSO4 5H2O,
3
MeCN-H2O, reflux, 68-70%; (c) POCl3-DMF, CH2Cl2, rt, over-
night, 89%; (d) PrSH, NaOH, THF, 91%.
Scheme 4a
a Reagents and conditions: (a) pivaloyl chloride, Et3N, CH2Cl2, 92%;
(b) K2S2O8, CuSO4 5H2O, MeCN-H2O, reflux, 80%; (c) POCl3-DMF,
CH2Cl2, 92%; (d) PrSH, NaOH, THF, 73%.
3
of the aldehyde functionality in ring A included an introduc-
tion of an oxime and R,β-unsaturated ketone (compounds 21
and 25) to compound 6b, but neither of these substituents
was beneficial (Table 3). The ring A aldehyde functionality
was also modified in the β-chlorovinylaldehyde derivative 5b
by introducing oxime, methylenehydroxyl, and carboxy
moieties (compounds 22, 23 and 24, respectively). These
compounds failed to show good inhibitory activity suggesting
unfavorable stereoelectronic effects, also emphasizing the
importance of the thioether moiety for the biological activity.
The bicyclic benzothiophene derivatives 26-29 did not show
any activity against 17β-HSD1 and 17β-HSD2 in a cell-free
assay.
a Reagents and conditions: (a) NH2OH HCl, AcONa, EtOH, 83%;
3
(b) DDQ, benzene, reflux, 84-91%; (c) NaBH4, THF, 89-92%.
The nature of the thioether moiety at ring A seemed quite
strongly to affect the cell permeability of these (di)cycloalkeno
thieno[2,3-d]pyrimidin-4(3H)-one based 17β-HSD1 inhibi-
tors. With few exceptions, the inhibitory activity was retained
to a satisfactory level in MCF-7 cells expressing the human
recombinant 17β-HSD1 enzyme (Tables 2 and 3), especially at
the higher 10 μM test concentration. Compounds 6a-6d,
having a simple aliphatic thioether side chain, seemed to best
the benzylic alcohols 20a and 20b, proving that the potentially
labile aldehyde moiety is not essential for the inhibitory
activity. The reduction of the aldehyde functionality in 1
and 6b produced the corresponding allylic alcohols with
retained biological activity (data not shown). However, the
ring A allylic alcohols were chemically unstable and were
rejected as potential drug candidates. Further modifications

6664 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Lilienkampf et al.
Table 2. The Influence of the Thioether Substitution on the Biological Activity
a The dashed line indicates the point of attachment. b No inhibition. c Not determined. d Human recombinant enzyme. e MCF-7 cells stably transfected
with 17β-HSD1.
retain their biological activity in a cell-based assay. With
compound 7b, bearing a C40 fluoro substituent in the aromatic
ring, the activity decreased significantly compared with the
cell-free assay. The aromatization of the ring A (compounds
19 and 20) seemed to somewhat reduce the inhibitory activity
in the cell-based assay, whereas in the cell-free assay the
activity was retained. The results obtained from the cell-based
assay raise an interesting point for future investigations con-
cerning the bioavailability or metabolism of these com-
pounds.
In general, all of the 17β-HSD1 inhibitors based on the
thieno[2,3-d]pyrimidin-4(3H)-one core showed good to excel-
lent selectivity over the oxidative isoform 17β-HSD2 (Tables 2
and 3). Compounds 6a-6g, with an aliphatic thioether side
chain, failed to show any inhibition of 17β-HSD2 at 0.1 μM.
In addition, representative inhibitor structures were chosen,
and their binding to estrogen receptors was evaluated. Com-
pounds 1, 5d, 6b, 6e-6g, 7g, 10, and 19b did not show any
undesired estrogenicity in an ERR or ERβ binding assay, with
EC50 values over 1 μM (Supporting Information).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6665
Table 3. The Effect of Homologation of the Dicycloalkeno Thieno[2,3-d]pyrimidin-4(3H)-one Core and Ring A Modifications on the Biological
Activity
inhibition (%) 17β-HSD1 cell-free assayc
inhibition (%) 17β-HSD1 cell-based assayd
inhibition (%) 17β-HSD2 cell-free assayc
compd
0.1 μM
1 μM
1 μM
10 μM
0.1 μM
1 μM
3c
8.0
a
23.2
3.8
10.0
6.3
45.9
35.8
38.6
78.0
b
b
b
b
b
4b
5a
5b
8
3.3
26.1
78.6
67.2
87.0
74.7
55.8
90.6
88.4
85.9
87.5
2.9
19.7
27.9
b
b
b
32.4
16.7
35.2
63.3
24.3
83.0
26.9
69.0
80.5
0.1
2.5
b
1.9
b
9
43.9
15.6
19.5
28.5
15.1
35.6
30.5
b
100.0
57.0
48.2
86.4
87.9
78.6
46.9
b
a
a
10
18
19a
19b
20a
20b
21
22
23
25
31.4
12.9
3.8
a
36.9
17.4
12.7
a
13.6
30.8
19.7
b
20.6
37.3
41.1
b
4.1
17.9
39.9
13.9
14.0
25.9
8.4
15.1
36.2
8.9
14.5
1.1
b
b
b
b
a No inhibition. b Not determined. c Human recombinant enzyme. d MCF-7 cells stably transfected with 17β-HSD1.
Figure 3. The active site surface around the lead compound 1
(orange). The bulkythioether side chainisfacingthecofactorNADPþ
and is accommodated by a hydrophobic pocket. The aliphatic seven-
membered ring protrudes partially out of the active site.
Figure 5. A comparison of the binding modes of the best inhibitor
7f (green) and E2 (gray). Inhibitor 7f is docked into the representa-
tive structure of the MDS trajectory with the lead compound 1, and
the orientation of E2 is from the relaxed crystal structure of 17β-
HSD1. Hydrogen bonds from inhibitor 7f and E2 are shown as red
and green dashed lines, respectively. For clarity, NADPþ and
selected active site residues are displayed as thin sticks and hydro-
gens are omitted. The enzyme structures are superimposed based on
the backbone.
We have recently reported the plausible binding mode of
17β-HSD1 inhibitors based on a thieno[2,3-d]pyrimidin-
4(3H)-one core.42 A crystal structure of 17β-HSD1 (pdb code
1FDT)12a was first subjected to molecular dynamics simula-
tion (MDS) to obtain a relaxed enzyme structure with the
reduced product E2. The lead compound 1 was docked into
the active site of the relaxed enzyme structure and simulated
under physiological conditions. The MDS was done in order
to mimic the dynamic process of inhibitor binding and to
reveal plausible interactions between the active site and 1.
Next, a set of inhibitors, including 6a-6i, 7a-7j, 18, 19a, 19b,
20a, 20b, 21-25, and 29, were docked into the representative
structure of the simulation trajectory, resulting in a good
alignment. The binding mode of 1 shows an orientation where
the ring A aldehyde group is hydrogen bonded to Asn152 and
Tyr218 (Figures 3 and 4). Moreover, the pyrimidinone carbo-
nyl forms a hydrogen bond to Arg258. The bulky aromatic
Figure 4. The lead compound 1 (orange) and inhibitor 20b (gray)
docked into the representative structure of the 17β-HSD1 MDS
trajectory. Hydrogen bonds from 1 and the inhibitor 20b are shown
as red and green dashed lines, respectively. For clarity, NADPþ and
selected active site residues are displayed as thin sticks and hydro-
gens are omitted.

6666 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Lilienkampf et al.
thioether side chain is accommodated by a hydrophobic
pocket, formed during the MDS. Aromatization of ring A
and reduction of the aldehyde group of 1 results in a more
potent inhibitor 20b in which the hydroxy group serves as an
acceptor in the hydrogen bond from Tyr218 and as a donor in
the hydrogen bond to Ser222 (Figure 4). Inhibitors 6a-6i,
with an aliphatic side chain, adopt a comparable binding
mode with 1. The hydrophobicity of the pocket around
the thioether side chain explains the lack of activity of
inhibitors 6h and 7c, bearing a carboxylic acid substituent.
One of the most potent inhibitors in the series, 7f, has the same
interactions as 1, and an additional hydrogen bond from the
phenolic hydroxy group at C30 of the thiophenyl moiety to the
backbone carbonyl at Gly144 (Figure 5), which explains the
increase in activity compared with 1.
Recombinant 17β-HSD1 assay. Recombinant enzyme homo-
genate (0.1 μg/mL) was incubated in 20 mM KH2PO4, pH 7.4,
reaction buffer (final volume 0.2 mL), including protease in-
hibitors (Complete Protease Inhibitor Cocktail tablet, Roche
Diagnostics, 1697498), 30 nM E1 (Sigma, E9750) as a substrate,
800 000 cpm/mL [3H]-E1 (PerkinElmer, NET319001MC) as a
tracer substrate, and 1 mM β-NADPH (Sigma, N1630) as a
cofactor for 30 min at room temperature, in the presence of
potential inhibitors at concentrations of 0.1 or 1 μM. Inhibitor
stock solutions were prepared in DMSO. The enzyme reaction
was stopped by the addition of trichloroacetic acid (1% final
concentration). Samples were centrifuged in a microtiter plate at
4000 rpm for 10 min. Supernatants were applied to reverse-
phase HPLC on a Waters Symmetry C18 column, equipped with
a Waters Sentry Guard column. Isocratic HPLC runs were
performed at room temperature at a flow rate of 1 mL/min of
CH3CN-H2O 48:52 as an eluent. Radioactivity was monitored
in the eluate by a Packard Flow scintillation analyzer. Total
radioactivity for E1 and E2 were determined in each sample, and
percent inhibition of E2 conversion by inhibitor was calculated
according to the following formula: inhibition %=[(E2 con-
version % of control - E2 conversion % of sample)/E2 con-
version % of control]ꢀ100.
Conclusions
17β-HSD1 is an attractive biological target for inhibitor
development for treating and preventing hormone-dependent
disorders, for example, breast cancer. In this study, several
thieno[2,3-d]pyrimidin-4(3H)-one based compounds were
found to be potent and selective 17β-HSD1 inhibitors as
well as to lack any undesired estrogenic effects. An efficient
synthetic route was developed for the lead compound and
subsequently utilized in the synthesis of a molecule library
based on a cycloalkeno thieno[2,3-d]pyrimidin-4(3H)-one
core. K2S2O8-CuSO4 was shown to be an expedient reagent
for the oxidation of the thiophene R-position in these ring
structures.
This work shows that good inhibitory activity can be
obtained with nonsteroidal and nonphenolic small molecules.
For example, 4-(3-hydroxyphenylthio)-1,2,7,8,9,10,11,13-oc-
tahydro-13-oxo-[1]benzothieno[20,30:4,5]-pyrimido[1,2-a]aze-
pine-3-carboxaldehyde (7f) is one of the most potent 17β-
HSD1 inhibitors reported to date with 94% inhibition of the
recombinant enzyme at 0.1 μM test concentration (cell-free
assay), and also several other compounds were found to have
>80% inhibition. A plausible binding mode, consistent with
the structure-activity relationships for the series, was identi-
fied for the inhibitors by MDS and docking studies. Most of
the compounds showed good inhibitory activity also in a cell-
based 17β-HSD1 assay, where the thioether moiety in ring A
was shown to influence the cell permeability. Especially
compounds 6a-6e, bearing an aliphatic thioether moiety,
showed promising activity in the cell-based assay. In general,
these compounds did not show notable inhibition of the
oxidative isoform 17β-HSD2. The fused dicycloalkeno
thieno[2,3-d]pyrimidin-4(3H)-ones described in this study
offer a new template for the design and future development
of potent and selective 17β-HSD1 inhibitors.
Recombinant 17β-HSD2 Assay. The assay was performed as
described for 17β-HSD1 but with following modifications: 6 μg/
mL of recombinant enzyme homogenate, 50 nM E2 (Sigma,
E8875) as a substrate, 800 000 cpm/mL [3H]-E2 (PerkinElmer,
NET317) as a tracer substrate and 1 mM β-NAD (Sigma
N7004) as a cofactor were used.
Inhibition of 17β-HSD1 in a Cell-Based Assay.33 Briefly, the
cell-based assays make use of human MCF-7 breast cancer cells
stably transfected with cDNA coding for human 17β-HSD1.33
These assays were used to evaluate the activity and selectivity of
test compounds in a cellular environment. In this assay, the
transfected cells were incubated with E1 as a substrate and [3H]-
E1 as a tracer substrate in the presence of potential inhibitors at
concentrations of 1 and 10 μM for 1 h at 37 °C. The assays were
performed in a 96-well plate format with three parallel samples.
The analysis of substrate and the end product and percent
inhibition of conversion by inhibitor were done as described
above in recombinant 17β-HSD1 assay.
Chemistry. The NMR spectra were measured with a Varian
Unity INOVA 500 or Varian MercuryPlus 300 spectrometer at
€
27 °C. The melting points were measured on a Buchi B-545
melting point apparatus and are corrected. The HRMS were
measured with Bruker MicroTofLC (ESI) in positive mode using
Agilent ESI Tunemix as a calibration solution or with JEOL
JMS-SX102 (EI) operating at 70 eV. Silica gel (0.040-0.063 mm)
was purchased from Merck. 1,2-Dichloroethane was dried with
anhydrous CaCl2 and distilled. DMF and CH2Cl2 were distilled
from CaH2. Compounds 2a and 2b were purchased from Acros.
The purity of the target compounds was g95% by analytical
HPLC.
General Procedure for the Synthesis of 4a-4e, 16a, 16b, and
27. Compound 3c (4.0 g, 13.9 mmol), K2S2O8 (11.3 g, 41.6
mmol), and CuSO4 5H2O (10.4 g, 41.6 mmol) in 250 mL of
3
CH3CN-H2O (1:1) were heated to reflux for 20 min. The
reaction was quenched with ice and extracted with CH2Cl2 (3ꢀ
50 mL). The organic layer was washed with 10% sodium
thiosulfate (30 mL) and brine (30 mL), and dried over Na2SO4.
After filtration, the solvent was evaporated. Flash chromatog-
raphy (CH2Cl2-EtOAc 5:1 as an eluent) afforded 4c as a white
powder in 85% yield. The analytical data of compounds 4a-4d
and 16b corresponded to previously published data.36
3,4,9,10,11,12-Hexahydro-1H-cyclohepta[40,50]thieno[20,30:4,5]-
pyrimido[1,2-a]azepine-5,14(2H,8H)-dione (4e). Yield 65%,
(white powder); mp 198 °C (EtOH); 1H NMR (500 MHz, CDCl3)
δ 1.79-2.02 (m, 8H), 2.83 (m, 2H), 3.06 (m, 2H), 3.56 (m, 2H),
4.36 (m, 2H); 13C NMR (500 MHz, CDCl3) δ 21.4, 25.1, 25.6,
27.7, 27.9, 29.6, 37.8, 41.9, 42.5, 120.8, 137.3, 147.2, 159.4,
Experimental Section
Inhibition of 17β-HSD1 and 17β-HSD2 in Cell-Free Assay.
17β-HSD1 and 17-HSD2 recombinant enzyme assays were
performed as described previously.21a Briefly, 17β-HSD1 and
17β-HSD2 homogenates were prepared as follows: Recombi-
nant baculovirus of 17β-HSD1 and 17β-HSD2 were generated
by the Bac to Bac Expression System (Invitrogen). Recombinant
bacmid DNAs were transfected to Sf9 insect cells using Cellfec-
tin Reagent (Invitrogen). Cells were harvested 60 h later; the
soluble fraction of cell lysates for 17β-HSD117 and microsomal
fraction for 17β-HSD2 were isolated. Aliquots were stored
frozen until determination of enzymatic activity.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6667
162.8, 166.8, 196.6; HRMS (ESI) calculated for C16H19N2O2S
[M þ H]þ 303.1162, found 303.1158.
(1 equiv) in THF was added dropwise. The reaction mixture
was allowed to reach room temperature and stirred until no
starting material was detected on TLC (CH2Cl2-EtOAc 9:1 as
an eluent). Reaction mixture was poured into a large excess of
water (∼700 mL/mmol), neutralized with 1 M HCl, and stirred
vigorously for 1 h. The precipitated crude product was filtrated
followed by purification by recrystallization from a suitable
solvent to afford the product. If a clear precipitate is not formed
upon the addition of water and 1 M HCl, the product can be
alternatively isolated by extraction with CH2Cl2 or EtOAc. For
compounds 6h, 7c, and 7e-7g, 3.0 equiv of NaOH was used.
1,2,7,8,9,10,11,13-Octahydro-13-oxo-4-(phenylthio)-[1]benz-
othieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxaldehyde (1).
Yield 87%, yellow crystals; mp 154 °C (dec.) (EtOH); 1H
NMR (500 MHz, CDCl3) δ 1.75-1.83 (m, 6H), 2.86 (m, 2H),
2.99 (m, 2H), 3.28 (m, 2H), 4.32 (m, 2H), 7.19-7.33 (m, 5H),
10.47 (s, 1H); 13C NMR (500 MHz, CDCl3) δ 22.26, 22.36, 25.0,
27.6, 29.5, 37.6, 42.3, 120.2, 127.4, 129.4, 129.5, 133.8, 134.4,
138.7, 139.2, 141.3, 158.5, 161.3, 165.5, 190.7; HRMS (ESI)
calculated for C22H21N2O2S2 [M þ H]þ 409.1039, found
423.1045.
6,7-Dihydro-3-methyl-[1]benzothieno[2,3-d]pyrimidine-4,8(3H,5H)-
1
dione (16a). Yield 68% (white powder); mp 194 °C (EtOH); H
NMR (500 MHz, CDCl3) δ2.25(m,2H),2.68(m,2H),3.30(m,2H),
3.59 (s, 3H), 8.08 (s, 1H); 13C NMR (500 MHz, CDCl3) δ 24.0, 25.9,
34.0, 38.3, 122.5, 133.5, 149.1, 149.7, 158.5, 168.5; HRMS (ESI)
calculated for C11H11N2O2S [M þ H]þ 235.0536, found 235.0540.
General Procedure for the Synthesis of 5a-5d, 17, and 28.
POCl3 (72.8 mmol, 6.8 mL) was added dropwise into anhydrous
DMF (74.9 mmol, 5.8 mL) in anhydrous CH2Cl2 (20 mL) at 0 °C
under argon. After 30 min, 4c (3.0 g, 10.4 mmol) in anhydrous
CH2Cl2 (15 mL) was added dropwise. The reaction mixture was
allowed to reach room temperature and stirred for 4 days. The
reaction was quenched with saturated NaOAc (150 mL) in ice
bath (caution! exothermic), extracted with CH2Cl2 (3ꢀ50 mL),
washed with brine (30 mL) and water (30 mL), and dried with
Na2SO4. After filtration, the solvent was evaporated. Flash
chromatography (CH2Cl2-EtOAc 9:1 as an eluent) afforded
5b as yellow powder in 75% yield.
6-Chloro-1,2,3,8,9-hexahydro-10-oxo-[1]benzothieno[2,3-d]-
pyrrolo[1,2-a]pyrimidine-7-carboxaldehyde (5a). Compound 5a
was synthesized from 4a using DMF as a solvent. Yield 32%,
yellow crystals; mp 225 °C (dec.) (EtOH); 1H NMR (300 MHz,
CDCl3) δ 2.32 (m, 2H), 2.78 (m, 2H), 3.19 (apparent t, 2H, J=
7.9 Hz), 3.25 (m, 2H), 4.18 (m, 2H), 10.18 (s, 1H); 13C NMR
(300 MHz, CDCl3) δ 19.6, 21.8, 22.3, 32.5, 46.6, 120.8, 128.5,
131.0, 139.6, 139.8, 157.6, 161.6, 167.5, 188.6; HRMS (ESI) cal-
culated for C14H12ClO2N2S [MþH]þ 307.0303, found 307.0290.
4-Chloro-1,2,7,8,9,10,11,13-octahydro-13-oxo-[1]benzothieno-
[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxaldehyde (5b). Yield
75%, yellow powder; mp 218 °C (dec.) (EtOH); 1H NMR
(300 MHz, CDCl3) δ 1.78-1.88 (m, 6H), 2.79 (m, 2H), 3.06
(m, 2H), 3.26 (m, 2H), 4.36 (m, 2H), 10.19 (s, 1H); 13C NMR
(200 MHz, CDCl3) δ 21.8, 22.3, 25.0, 27.5, 29.5, 37.6, 42.4,
120.4, 128.5, 131.1, 139.9, 140.2, 158.5, 161.8, 165.6, 188.6;
HRMS (EI) calculated for C16H15ClO2N2S [M]þ 334.0543,
found 334.0531.
4-(Ethylthio)-1,2,7,8,9,10,11,13-octahydro-13-oxo-[1]benzot-
hieno[20,30:4,5]-pyrimido[1,2-a]azepine-3-carboxaldehyde (6a).
Yield 87%; mp 175 °C (dec.) (EtOH-petroleum ether); 1H
NMR (500 MHz, CDCl3) δ 1.27 (t, 3H, J=7.3 Hz), 1.83 (m, 6H),
2.77 (m, 2H), 2.91 (q, 2H, J=7.3 Hz), 3.06 (m, 2H), 3.22 (m, 2H),
4.36 (m, 2H), 10.49 (s, 1H); HRMS (ESI) calculated for
C18H21N2O2S2 [M þ H]þ 361.1039, found 361.1048.
1,2,7,8,9,10,11,13-Octahydro-13-oxo-4-(propylthio)-[1]benzo-
thieno[20,30:4,5]-pyrimido[1,2-a]azepine-3-carboxaldehyde (6b).
Yield 92%; mp 193 °C (dec.) (EtOH); 1H NMR (500 MHz,
CDCl3) δ 0.99 (t, 3H, J=7.3 Hz), 1.63 (q, 2H, J=7.3 Hz), 1.79
(broad s, 2H), 1.87 (m, 4H), 2.76 (m, 2H), 2.86 (t, 2H, J=7.3 Hz),
3.07 (m, 2H), 3.22 (m, 2H), 4.36 (m, 2H), 10.49 (s, 1H); HRMS
(ESI) calculated for C19H23N2O2S2 [M þ H]þ 375.1195, found
375.1184.
4-(Butylthio)-1,2,7,8,9,10,11,13-octahydro-13-oxo-[1]benzot-
hieno[20,30:4,5]-pyrimido[1,2-a]azepine-3-carboxaldehyde (6c).
Yield 89% (EtOAc); 1H NMR (500 MHz, CDCl3) δ 0.89
(t, 3H, J=7.3 Hz), 2.81 (m, 2H), 1.59 (m, 2H), 1.79 (broad s,
2H), 1.87 (m, 4H), 2.76 (m, 2H), 2.89 (t, 2H, J=7.3 Hz), 3.07 (m,
2H), 3.22 (m, 2H), 4.37 (m, 2H), 10.48 (s, 1H); HRMS (ESI)
calculated for C20H25N2O2S2 [M þ H]þ 389.1352, found
389.13656.
4-[(1-Methylethyl)thio)]-1,2,7,8,9,10,11,13-octahydro-13-
oxo-[1]benzothieno-[20,30:4,5]pyrimido-[1,2-a]azepine-
3-carboxaldehyde (6d). Yield 68%, yellow crystals; mp 177 °C
(dec.) (EtOAc); 1H NMR (300 MHz, CDCl3) δ 1.32 (d, 6H, J=
6.7 Hz), 1.73-1.91 (m, 6H), 2.78 (m, 2H), 3.06 (m, 2H), 3.22 (m,
2H), 3.41 (sept, 1H, J=6.7 Hz), 4.36 (m, 2H), 10.47 (s, 1H);
HRMS (ESI) calculated for C19H23N2O2S2 [M þ H]þ 375.1195,
found 375.1212.
4-Chloro-1,2,7,9,10,11,12,14-octahydro-14-oxo-8H-[1]benzo-
thieno[20,30:4,5]pyrimido[1,2-a]azocine-3-carboxaldehyde (5c).
From 4d, yield 60%; mp 229 °C (dec.) (EtOH); 1H NMR (300
MHz, CDCl3) δ 1.44 (m, 2H), 1.60 (m, 2H), 1.85-2.20 (m, 4H),
2.81 (m, 2H), 3.02 (m, 2H), 3.28 (m, 2H), 4.30 (broad s, 2H),
10.21 (s, 1H); 13C NMR (300 MHz, CDCl3) δ 21.9, 22.3, 24.2,
26.1, 28.7, 30.6, 35.8, 42.9, 120.6, 128.5, 131.0, 140.0, 140.1,
158.4, 161.4, 166.1, 188.6; HRMS (ESI) calculated for
C17H18ClO2N2S [M þ H]þ 349.0772, found 349.0698.
5-Chloro-2,3,8,9,10,11,12,14-octahydro-14-oxo-1H-cyclohe-
pta-[40,50]thieno[20,30:4,5]pyrimido[1,2-a]azepine-4-carboxaldeh-
yde (5d). From 4e, yield 83%; yellow powder; mp 112 °C
(EtOH); 1H NMR (300 MHz, CDCl3) δ 1.81 (m, 6H), 2.14 (m,
2H), 2.52 (m, 2H), 3.05 (m, 2H), 3.32 (m, 2H), 4.35 (m, 2H),
10.26 (s, 1H); 13C NMR (300 MHz, CDCl3) δ 25.1, 25.5, 27.6,
29.6, 30.02, 30.04, 37.7, 42.5, 121.6, 133.0, 137.3, 141.9, 144.6,
159.0, 162.4, 165.4, 190.3; HRMS (EI) calculated for
C17H17ClO2N2S2 [M]þ 348.0699, found 348.0706.
4-[(1,1-Dimethylethyl)thio)]-1,2,7,8,9,10,11,13-octahydro-13-
oxo-[1]benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxal-
dehyde (6e). Purified by flash chromatography using CH2-
Cl2-EtOAc 1:1 as an eluent. Yield 65%, yellow crystals; mp
151 °C (MeCN); 1H NMR (500 MHz, CDCl3) δ 1.37 (m, 9H),
1.79 (m, 2H), 1.86 (m, 4H), 2.80 (m, 2H), 3.06 (m, 2H), 3.24 (m,
2H), 4.36 (m, 2H), 10.42 (s, 1H); HRMS (ESI) calculated for
C20H25N2O2S2 [M þ H]þ 389.1353, found 389.1362.
8-Chloro-3,4,5,6-tetrahydro-3-methyl-4-oxo-[1]benzothieno-
[2,3-d]pyrimidine-7-carboxaldehyde (17). Compound 17 was
synthesized from 16a as described above except the crude
product was purified by recrystallization from EtOH. Yield
89%; yellow crystals; mp 218 °C (EtOH); 1H NMR (300 MHz,
CDCl3) δ 2.82 (m, 2H), 3.30 (m, 2H), 3.59 (s, 3H), 8.02 (s, 1H),
10.22 (s, 1H); 13C NMR (300 MHz, CDCl3) δ 22.2, 22.6, 34.3,
123.1, 129.4, 132.9, 139.75, 139.79, 148.0; HRMS (ESI) calcu-
lated for C12H10ClN2O2S [M þ H]þ 281.0146, found 281.0143.
General Procedure for the Synthesis of 1, 6a-6i, 7a-7j, 8-10,
18, and 29. NaOH (1 M aq solution, 1.5 equiv) was added
dropwise to the appropriate thiol (1.5 equiv) in distilled THF
(∼6 mL/mmol) at 0 °C. The solution was stirred for 5 min, after
which the appropriate β-chlorovinylaldehyde intermediate
4-(Cyclopentylthio)-1,2,7,8,9,10,11,13-octahydro-13-oxo-[1]-
benzothieno[20,30:4,5]-pyrimido[1,2-a]azepine-3-carboxaldehyde
(6f). Yield 80%, yellow crystals; mp 194 °C (petroleum
ether-EtOH, 3:7); 1H NMR (300 MHz, CDCl3) δ 1.73 (m, 14H),
2.76 (m, 2H), 3.06 (m, 2H), 3.22 (m, 2H), 3.61 (m, 1H), 4.37 (m, 2H),
10.46 (s, 1H); HRMS (EI) calculated for C21H24O2N2S2
400.1279, found 400.1284.
4-(Cyclohexylthio)-1,2,7,8,9,10,11,13-octahydro-13-oxo-[1]-
benzothieno[20,30:4,5]-pyrimido[1,2-a]azepine-3-carboxaldehyde
(6g). Yield 77%, yellow crystals; mp 209 °C (petroleum

6668 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Lilienkampf et al.
1
ether-EtOH, 3:7); H NMR (300 MHz, CDCl3) δ 1.25-1.99
214 °C (dec.) (AcOH-H2O); 1H NMR (300 MHz, d6-DMSO) δ
1.65 (m, 6H), 2.72 (m, 2H), 2.99 (m, 2H), 3.17 (m, 2H), 4.27 (m,
2H), 6.73 (dt, 1H, J=1.2, 7.6 Hz), 6.85 (dd, 1H, J=1.2, 7.9 Hz),
7.10 (m, 2H), 10.27 (s, 1H), 10.38 (s, 1H); HRMS (ESI)
calculated for C22H21N2O3S2 [M þ H]þ 425.0988, found
425.0991.
1,2,7,8,9,10,11,13-Octahydro-13-oxo-4-(4-pyridylthio)-[1]-
benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxaldehyde
(7h). Yield 80%, yellow crystals; mp 207 °C (EtOAc); 1H NMR
(300 MHz, CDCl3) δ 1.81 (m, 6H), 2.91 (m, 2H), 3.01 (m, 2H),
3.35 (m, 2H), 4.35 (m, 2H), 7.12 (m, 2H), 8.42 (d, 2H, J=5.9 Hz),
10.39 (s, 1H);HRMS(ESI) calculatedfor C21H20N3O2S2 [M þ H]þ
410.0991, found 410.1009.
(m, 16H), 2.77 (m, 2H), 3.07 (m, 2H), 3.14 (m, 1H), 3.22 (m, 2H),
4.37 (m, 2H), 10.47 (s, 1H); HRMS (EI) calculated for C22H26-
O2N2S2 414.1436 found 414.1456.
4-[(3-Formyl-1,2,7,8,9,10,11,13-octahydro-13-oxo-[1]benzot-
hieno[20,30:4,5]pyrimido[1,2-a]azepin-4-yl)thio]propionic acid (6h).
Compound 6h was synthesized as described above except that 3
equiv of NaOH was used. Yield 73%; 1H NMR (500 MHz,
CDCl3) δ 1.75-1.88 (m, 6H), 2.68 (t, 2H, J = 7.0 Hz), 2.76
(m, 2H), 3.07 (m, 2H), 3.16 (t, 2H, J = 7.0 Hz), 3.22 (m, 2H), 4.36
(m, 2H), 10.45 (s, 1H), -OH exchanged; HRMS (ESI) calculated
for C19H21N2O4S2 [M þ H]þ 405.0937, found 405.0954.
4-[2-(4-Morpholinyl)-2-oxoethylthio]-1,2,7,8,9,10,11,13-octa-
hydro-13-oxo-[1]benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-
carboxaldehyde (6i). Purified by flash chromatogaphy using 5%
1,2,7,8,9,10,11,13-Octahydro-13-oxo-4-(phenylmethylt-
hio)-[1]benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxal-
dehyde (7i). Yield 75%, yellow crystals; mp 182 °C (dec.)
1
MeOH-CH2Cl2 as an eluent. Yellow powder, yield 87%; H
1
(EtOAc); H NMR (300 MHz, CDCl3) δ 1.85 (m, 6H), 2.61
NMR (300 MHz, CDCl3) δ 1.75-1.86 (m, 6H), 2.78 (m, 2H),
3.06 (m, 2H), 3.23 (m, 2H), 3.47 (m, 2H), 3.57 (m, 2H), 3.68 (m,
4H), 3.70 (s, 2H), 10.44 (s, 1H); HRMS (ESI) calculated for
C22H26N3O4S2 [M þ H]þ 460.1359, found 460.1349.
(m, 2H), 3.10 (m, 4H), 4.03 (s, 2H), 4.38 (m, 2H), 7.04 (m, 2H),
7.23 (m, 3H), 9.91 (s, 1H); HRMS (ESI) calculated for
C23H23N2O2S2 [M þ H]þ 423.1195, found 423.1217.
1,2,7,8,9,10,11,13-Octahydro-4-(2-naphtalenylthio)-13-
oxo-[1]benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxa-
ldehyde (7j). Yield 81%, yellow crystals; mp 228 °C (dec.)
4-(4-Methylphenylthio)-1,2,7,8,9,10,11,13-octahydro-13-
oxo-[1]benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxal-
dehyde (7a). Yield 75%, yellow crystals; mp 211 °C (dec.)
(EtOH-petroleum ether 7:3); 1H NMR (CDCl3) δ 1.79 (m,
6H), 2.28 (s, 3H), 2.84 (m, 2H), 2.99 (m, 2H), 3.26 (m, 2H), 4.32
(m, 2H), 7.07 (d, 2H, J=7.9 Hz), 7.24 (m, 2H), 10.52 (s, 1H);
HRMS (ESI) calculated for C23H23N2O2S2 [M þ H]þ 423.1195,
found 423.1180.
1
(EtOAc); H NMR (300 MHz, CDCl3) δ 1.78 (m, 6H), 2.91
(m, 4H), 3.21 (m, 2H), 4.30 (m, 2H), 7.38 (dd, 1H, J=8.8 Hz, 1.8
Hz)), 7.47 (m, 2H), 7.72 (m, 3H), 7.82 (d, 1H, J=1.8 Hz), 10.57
(s, 1H); HRMS (ESI) calculated for C26H23N2O2S2 [M þ H]þ
459.1195, found 459.1210.
1,2,3,8,9,10-Hexahydro-10-oxo-6-(propylthio)-[1]benzothi-
eno[2,3-d]pyrrolo[1,2-a]pyrimidine-7-carboxaldehyde (8). Puri-
fied by flash chromatography using EtOAc-CH2Cl2 (1:5) as
an eluent. Yield 54%, yellow powder; 1H NMR (300 MHz,
CDCl3) δ 0.99 (t, 3H, J=7.3 Hz), 1.63 (m, 2H), 2.32 (m, 2H),
2.76 (m, 2H), 2.87 (t, 2H, J=7.3 Hz), 3.21 (m, 4H), 4.19 (m, 2H),
10.49 (s, 1H); 13C NMR (300 MHz, CDCl3) δ 13.3, 19.7, 22.2,
22.4, 23.2, 32.6, 38.8, 46.6, 121.3, 135.3, 137.8, 138.4, 143.3,
157.8, 161.1, 167.3, 191.0; HRMS (ESI) calculated for
C17H19N2O2S2 [M þ H]þ 347.0882, found 347.0880.
4-(4-Fluorophenylthio)-1,2,7,8,9,10,11,13-octahydro-13-
oxo-[1]benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxal-
dehyde (7b). Yield 86%; mp 230-231 °C (EtOAc); H NMR
(CDCl3) δ 1.79 (m, 6H), 2.85 (m, 2H), 3.00 (m, 2H), 3.26 (m,
2H), 4.33 (m, 2H), 6.98 (m, 2H), 7.34 (m, 2H), 10.53 (s, 1H);
HRMS (ESI) calculated for C22H20FN2O2S2 [M þ H]þ
427.0945, found 427.0949.
1
4-[(3-Formyl-1,2,7,8,9,10,11,13-octahydro-13-oxo-[1]benzot-
hieno[20,30:4,5]pyrimido[1,2-a]azepin-4-yl)thio]benzoic acid (7c).
Compound 7c was synthesized as described above except that 3
equiv of NaOH was used. Yield 82%, yellow crystals; 1H NMR
(300 MHz, d6-DMSO) δ 1.69 (m, 6H), 2.78 (m, 2H), 2.98 (m,
2H), 3.23 (m, 2H), 4.26 (m, 2H), 7.46 (m, 1H), 7.85 (m, 1H),
10.31 (s, 1H), 12.99 (broad s, 1H); HRMS (ESI) calculated for
C23H21N2O4S2 [M þ H]þ 453.0937, found 453.0915.
2,3,8,9,10,11,12,14-Octatahydro-14-oxo-5-(propylthio)-1H-
cyclohepta-[40,50]thieno[20,30:4,5]pyrimido[1,2-a]azepine-4-car-
boxaldehyde (9). Purified by flash chromatography using
EtOAc-CH2Cl2 (1:5) as an eluent. Yield 69%, yellow powder;
1H NMR (500 MHz, CDCl3) δ 0.94 (t, 3H, J=7.3 Hz), 1.53
(m, 2H), 1.81 (broad s, 2H), 1.88 (m, 4H), 2.34 (m, 4H), 2.70
(t, 2H, J=7.1 Hz), 3.08 (m, 2H), 3.11 (m, 2H), 4.39 (m, 2H),
10.45 (s, 1H); 13C NMR (500 MHz, CDCl3) δ 13.2, 23.5, 25.1,
25.3, 26.2, 27.6, 29.6, 37.1, 37.2, 37.6, 42.4, 121.1, 135.1, 144.1,
145.2, 148.6, 158.7, 161.2, 165.5, 189.7; HRMS (ESI) calculated
for C20H25N2O2S2 [M þ H]þ 389.1352, found 389.1336.
1,2,7,9,10,11,12,14-Octahydro-14-oxo-4-(propylthio)-8H-[1]-
benzothieno[20,30:4,5]pyrimido[1,2-a]azocine-3-carboxaldehyde (10).
Yield 76%, yellow crystals; mp 172 °C (EtOH-petroleum ether,
7:3); 1H NMR (300 MHz, CDCl3) δ 0.99 (t, 3H, J=7.3 Hz), 1.44
(m, 2H), 1.62 (m, 4H), 1.93 (m, 4H), 2.76 (m, 2H), 2.87 (t, 2H, J=
7.3 Hz), 3.02 (m, 2H), 3.22 (m, 2H), 4.30 (m, 2H), 10.49 (s, 1H);
13C NMR (300 MHz, CDCl3) δ 13.2, 22.1, 22.3, 23.1, 24.2, 26.1,
28.7, 30.6, 35.8, 38.5, 42.8, 121.0, 135.2, 138.2, 138.3, 143.3,
158.5, 160.8, 165.8, 191.0; HRMS (EI) calculated for
C20H24O2N2S2 [M]þ 388.1279, found 388.1266.
4-[4-Methoxyphenylthio]-1,2,7,8,9,10,11,13-octahydro-13-
oxo-[1]benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxal-
dehyde (7d). Yield 86%, yellow crystals; mp 189 °C (dec.)
(EtOH); 1H NMR (300 MHz, CDCl3) δ 1.79 (m, 6H), 2.82 (m,
2H), 3.0 (m, 2H), 3.24 (m, 2H), 3.76 (s, 3H), 4.32 (m, 2H), 6.81
(m, 2H), 7.32 (m, 2H), 10.56 (s, 1H); HRMS (ESI) calculated for
C23H23N2O3S2 [M þ H]þ 439.1145, found 439.1144.
1,2,7,8,9,10,11,13-Octahydro-4-(4-hydroxyphenylthio)-13-
oxo-[1]benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxal-
dehyde (7e). Yield 78%, yellow crystals; mp 235 °C (dec.)
1
(benzene-EtOH); H NMR (300 MHz, d6-DMSO) δ 1.67 (m,
6H), 2.69 (m, 2H), 2.99 (m, 2H), 3.13 (m, 2H), 4.26 (m, 2H), 6.75
(m, 2H), 7.28 (m, 2H), 9.77 (s, 1H), 10.41 (s, 1H); HRMS (ESI)
calculated for C22H21N2O3S2 [M þ H]þ 425.0988, found 425.1010.
1,2,7,8,9,10,11,13-Octahydro-4-(3-hydroxyphenylthio)-13-
oxo-[1]benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxal-
dehyde (7f). Purified by flash chromatography 10%
MeOH-CH2Cl2 as an eluent. Yield 70%, yellow crystals; mp
3,4,5,6-Tetrahydro-3-methyl-4-oxo-8-(propylthio)-[1]benzoth-
ieno[2,3-d]pyrimidine-7-carboxaldehyde (18). Purified by flash
chromatography with 10% MeOH-CH2Cl2 as an eluent. Yield
81%, yellow powder; 1H NMR (500 MHz, CDCl3) δ 1.00 (t, 3H,
J=7.3 Hz), 1.64 (m, 2H), 2.78 (m, 2H), 2.87 (t, 2H, J=7.1 Hz), 3.24
(m, 2H), 3.58 (s, 3H), 8.00 (s, 1H), 10.50 (s, 1H); 13C NMR (500
MHz, CDCl3) δ 13.2, 22.1, 22.3, 23.2, 33.9, 38.7, 123.1, 136.9,
137.7, 138.9, 142.8, 147.2, 158.2, 165.6, 191.0; HRMS (ESI)
calculated for C15H17N2O2S2 [M þ H]þ 321.0726, found 321.0740.
General Procedure for the Synthesis of 19a and 19b. Com-
pound 1 (510 mg, 1.25 mmol) and DDQ (370 mg, 1.62 mmol)
1
206 °C (dec.) (EtOH); H NMR (d6-DMSO) δ 1.59-1.78 (m,
6H), 2.72 (m, 2H), 2.97 (m, 2H), 3.17 (m, 2H), 4.25 (m, 2H), 6.63
(m, 1H), 6.70 (m, 1H), 7.11 (t, 1H, J=7.9 Hz), 9.65 (s, 1H), 10.3
(s, 1H); HRMS (ESI) calculated for C22H21N2O3S2 [M þ H]þ
425.0988, found 425.0991.
1,2,7,8,9,10,11,13-Octahydro-4-(2-hydroxyphenylthio)-13-
oxo-[1]benzothieno-[20,30:4,5]pyrimido[1,2-a]azepine-3-carboxal-
dehyde (7g). Purified by flash chromatography using 10%
MeOH-CH2Cl2 as an eluent. Yield 77%, yellow crystals; mp

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6669
were refluxed overnight in dry benzene (45 mL). The reaction
mixture was cooled to room temperature and filtered through a
short column of silica gel. The silical gel was washed with
CH2Cl2-EtOAc (9:1) solution, and the combined organic solu-
tions were evaporated. The crude product was purified by
recrystallization to give the product.
7,8,9,10,11,13-Hexahydro-13-oxo-4-(propylthio)-[1]benzothi-
eno[20,30:4,5]pyrimido-[1,2-a]azepine-3-carboxaldehyde (19a).
Compound 19a was prepared as described above except that 6b
was used as a starting material. White crystals, yield 84%; mp
186 °C (EtOH); 1H NMR (300 MHz, CDCl3) δ 0.99 (t, 3H, J=
7.2 Hz), 1.59 (m, 2H), 1.85 (m, 6H), 2.93 (t, 2H, J=7.2 Hz), 3.15
(m, 2H), 4.48 (m, 2H), 8.08 (d, 1H, J=8.2 Hz), 8.66 (dd, 1H, J=
0.6 Hz, J=8.3 Hz), 10.84 (d, 1H, J=0.9 Hz); 13C NMR (300
MHz, CDCl3) δ 13.6, 23.7, 25.2, 27.8, 29.9, 38.1, 40.1, 42.9,
116.5, 125.2, 126.1, 133.6, 135.3, 138.9, 145.1, 158.3, 164.0,
167.7, 192.1; HRMS (ESI) calculated for C19H21N2O2S2 [M þ
H]þ 373.1039, found 373.1048.
8-10, and 18, HPLC purity determinations for the target
compounds, and ERR and ERβ binding analysis for com-
pounds 1, 5d, 6b, 6e-6g, 7g, 10, and 19b.This material is
available free of charge via the Internet at http://pubs.acs.org.
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1
Yield 92%, white crystals; mp 162 °C (EtOH); H NMR (300
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6H), 2.32 (t, -OH, J = 6.4 Hz), 2.89 (m, 2H), 3.13 (m, 2H), 4.48
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8,9,10,11-Tetrahydro-3-(hydroxymethyl)-4-(phenylthio)-[1]-
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(EtOH); 1H NMR (300 MHz, CDCl3) δ 1,87 (m, 6H), 2.14
(broad s, -OH) 3.11 (m, 2H), 4.47 (m, 2H), 4.96 (s, 2H), 7.13 (m,
5H), 7.73 (d, 1H, J=8.2 Hz), 8.66 (d, 1H, J=8.2 Hz); 13C NMR
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123.7, 125.8, 126.3, 126.8, 127.4, 129.3, 134.31, 135.2, 142.7,
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