Synthesis and biological evaluation of spiro-δ-lactones as inhibitors of 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2)

Article abstract of DOI:10.2174/157018011795514230

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyzes the oxidation of the potent estradiol (E2) to the less active estrogen estrone (E1). Inhibitors of this enzyme should maintain the local level of E2 in bone tissue when the E2 concentration in t

Full text of DOI:10.2174/157018011795514230

406
Letters in Drug Design & Discovery, 2011, 8, 406-421
Synthesis and Biological Evaluation of Spiro-ꢀ-lactones as Inhibitors of
17ꢁ-Hydroxysteroid Dehydrogenase Type 2 (17ꢁ-HSD2)
Kuiying Xu1, Marie Wetzel1, Rolf W. Hartmann*,1,2 and Sandrine Marchais-Oberwinkler*,1
1Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C23, D-66123 Saarbrücken, Germany
2Helmholtz Institute for Pharmaceutical Sciences Saarland (HIPS), Saarland University, Campus C23, D-66123
Saarbrücken, Germany
Received September 21, 2010: Revised February 17, 2011: Accepted February 25, 2011
Abstract: 17ꢀ-Hydroxysteroid dehydrogenase type 2 (17ꢀ-HSD2) catalyzes the oxidation of the potent estradiol (E2) to
the less active estrogen estrone (E1). Inhibitors of this enzyme should maintain the local level of E2 in bone tissue when
the E2 concentration in the circulation drops and therefore might be useful for the treatment of osteoporosis. In this work,
novel non-steroidal spiro-ꢀ-lactone compounds designed as 17ꢀ-HSD2 inhibitors were synthesized and their
physicochemical and biological properties were investigated. These new spiro-ꢀ-lactones are not sufficiently stable for
further development and show low inhibition of the enzyme.
Keywords: 17ꢀ-hydroxysteroid dehydrogenase type
2
inhibitors, Drug design, Osteoporosis, Spiro-ꢁ-lactones,
Steroidomimetics.
INTRODUCTION
replacement therapy is efficient against bone loss and
osteoporotic fractures [9-11] but it can not be administered
to patients because of adverse effects (breast, endometrial
and ovarian cancer, stroke, thromboembolism). There are
also substantial evidences that active androgens like T (Fig.
1) and dihydrotestosterone (DHT) may as well be involved
in bone formation and may protect the bones against
osteoporosis [12, 13]. Controlled increase of active E2 and T
in bones of osteoporotic patients will certainly reduce
osteoporotic fractures and slow down bone loss by lowering
bone resorption and by raising bone formation.
Augmentation in E2 and T in bones might be achieved by
inhibition of 17ꢀ-HSD2 via an intracrine approach [14],
where the transformation of E2 and T to inactive precursors
will be blocked dominantly in the bone cells.
17ꢀ-Hydroxysteroid dehydrogenases (17ꢀ-HSDs) [1] are
a class of enzymes that catalyze the conversion of 17ꢀ-
hydroxy estrogens/androgens to 17ꢀ-keto analogs and vice
versa. These enzymes play a key role in vivo, controlling the
biological activities of the sex hormones in a tissue selective
manner [1-5]. Up to now, at least fourteen 17ꢀ-HSDs have
been identified. All of them belong to the short-chain
dehydrogenases/reductases (SDRs) except 17ꢀ-HSD5, which
is an aldo-keto reductase (AKR) [6].
17ꢀ-HSD2 catalyzes the transformation of both
estrogenic and androgenic substrates, showing oxidative as
well as reductive activity in vitro, depending on the presence
of the cofactor (NADP+ or NADPH, respectively). However,
it has a predominant oxidative activity in vivo converting the
highly active 17ꢀ-hydroxysteroids such as 17ꢀ-estradiol (E2)
and testosterone (T) to the less active keto analogs estrone
(E1) and 4-androstene-3,17-dione (A-dione), respectively,
using the cofactor NAD+ (Fig. 1). 17ꢀ-HSD2 is therefore
able to modulate the level in active E2 and T in the target
cells. 17ꢀ-HSD2 is also able to catalyze the interconversion
of 20ꢂ-dihydroprogesterone (20ꢂ-DHP) to progesterone.
This enzyme has not been crystallized yet, no 3D-structure is
available.
Thus, inhibition of 17ꢀ-HSD2 could help to maintain the
local level of E2 and T and therefore provides a novel
approach for the treatment of osteoporosis.
As
biological
counter-parts,
17ꢀ-hydroxysteroid
dehydrogenase type 1 (17ꢀ-HSD1) which reduces E1 into E2
(Fig. 1) and 17ꢀ-hydroxysteroid dehydrogenase type 3 (17ꢀ-
HSD3) which transforms A-dione into T (Fig. 1), can
increase the level of E2 and T, respectively, in the bone cells.
These enzymes should not be inhibited [15-16].
Only few potent 17ꢀ-HSD2 inhibitors have been reported
[1, 17-19]. The group of Poirier described a series of
steroidal spirolactone derivatives [20-23], the most potent
one is the C17-spiro-ꢁ-lactone 1 (Fig. 2, 65% inhibition at an
inhibitor concentration of 1 μM [21]). A novel class of
pyrrolidinones [24-26] was also reported as active and
selective non-steroidal 17ꢀ-HSD2 inhibitors, with 2 (Fig. 2)
being the most active compound (IC50 = 10 nM). A
derivative of the pyrrolidinone 2 was evaluated in an
osteoporosis model using ovariectomized cynomolgus
Estrogens appear to be essential hormones in bone
remodeling: their osteoprotective role is known [7]. Estrogen
deficiency or down regulation in the bone cells is believed to
be an important risk factor for osteoporosis e.g. after
menopause or after treatment with aromatase inhibitors [8],
which radically prevent the estrogen biosynthesis. Estrogen
*Address correspondence to these authors at the Pharmaceutical and
Medicinal Chemistry, Saarland University, Campus C23, D-66123
Saarbrücken, Germany; Tel: +49 681 302 70318; Fax: +49 681 302 70308;
E-mail: s.marchais@mx.uni-saarland.de
monkeys [27].
A decrease of bone resorption and
maintenance of bone formation were observed. Despite high
Tel: +49 681 302 70300; Fax: +49 681 302 70308;
E-mail: rwh@mx.uni-saarland.de
1570-1808/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

Spiro-ꢀ-Lactones as Inhibitors of 17ꢁ-HSD2
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5 407
O
OH
17 17ꢀ-HSD2, NAD+
17
17ꢀ-HSD1, NADPH
HO
3
HO
3
E1
E2
O
OH
17 17ꢀ-HSD2, NAD+
17
17ꢀ-HSD3, NADPH
O
3
O
3
A-dione
T
Fig. (1). 17ꢀ-HSD1, 2 and 3 in sex steroid metabolism.
O
OH
N
O
O
S
17
F
3
HO
1
2
Fig. (2). Described 17ꢀ-HSD2 inhibitors.
variation and the small effects observed, this in vivo
experiment validates this approach.
membered ring). A hydrophobic scaffold should be present
between these two polar groups to mimic the B/C ring of E2
or T. Based on these observations, a pharmacophore model
was proposed (Fig. 3).
As Bydal [18] describes the spirolactone moiety essential
for 17ꢀ-HSD2 activity and taking advantage of our
experience designing steroidomimetics for different targets
(like e.g. CYP19 [28-30], 5ꢀ-reductase [31-32], CYP17 [33-
34], CYP11B2 [35-39], CYP11B1 [40-41], 17ꢁ-HSD1 [42-
54] and 17ꢁ-HSD2 [19]), the aim of the work described in
the following is the design, synthesis and biological
evaluation of new non-steroidal spiro-ꢁ-lactone inhibitors of
17ꢀ-HSD2.
O
10 -12 Å
O
hydrophobic
scaffold
INHIBITOR DESIGN
3
In the design process, we decided that steroidal structures
should be avoided to limit the side effects due to interaction
with steroid hormone receptors. However, the compounds
should bind in the substrate binding site of 17ꢀ-HSD2 and be
capable of mimicking E2 or T: they must have two polar
moieties, mimicking positions C3 and C17 of the steroids. In
a SAR study [20] based on the steroidal spirolactone 1,
several features were identified, which are important for
17ꢀ-HSD2 inhibition. The OH-phenyl moiety is necessary to
mimic the 3-OH group of the steroid, protection as methoxy
is detrimental for activity. The spirolactone moiety is
essential for activity in position C17: a complete loss of
activity is observed when the carbonyl group is omitted. The
size of the lactone is optimum in case of a ꢁ-lactone (6-
HO
4
Fig. (3). Simple pharmacophore model.
Phenyl indanes and phenyl tetrahydronaphthalenes have
often been used as steroidomimetics [33, 35] and will be
used as scaffold for the inhibitors. Compounds 3-7 (Fig. 4),
bearing the spiro-ꢁ-lactone moiety, combine the features
described in the pharmacophore model and a hydrophobic
core structure. As the distance between the phenyl-OH and
the lactone should be similar to the corresponding two
moieties in E2 (10-12 Å), the hydroxy group on the phenyl
of 3-7 should be either in para (4) or in meta (3) position.

408 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5
Xu et al.
O
O
O
O
O
O
O
O
(CH2)n
3
4
3
3
3
4
4
4
R
R
R
R
3a: n = 1, R = 4-OH
3b: n = 1, R = 3-OH
3c: n = 1, R = 4-OMe
4a: n = 2, R = 4-OH
4b: n = 2, R = 3-OH
7a: R = 4-OH
7b: R = 3-OH
6a: R = 4-OH
6b: R = 3-OH
5a: R = 4-OH
5b: R = 3-OH
Fig. (4). Designed structures.
RESULTS AND DISCUSSION
Chemistry
amount tetrakis triphenylphosphine palladium and two
equivalents sodium carbonate 2N afforded 9a-c and 10a,b in
good yields following the described procedures (Method A)
[55, 56]. In a second step, nucleophilic addition of the
commercially available Grignard reagent of 4-bromo-1-
butene to the carbonyl function of 9a-c and 10a,b at 0°C
provided the racemic mixture 11a-c, 12a,b (Method C).
Then, the obtained terminal alkene functions were oxidized
with 9-BBN and basic hydrogen peroxide at 0°C (Method D)
and gave the primary alcohols 13a-c and 14a,b. In a last
step, selective oxidation of the primary hydroxy moiety
using Ley’s conditions with tetrapropylammonium
Synthesis of hydroxyphenyl indanes and tetrahydro-
naphthalenes spirolactone derivatives (3a-c and 4a,b)
Compounds 3a-c and 4a,b were prepared in a four steps
procedure starting from the 5-bromo-1-indanone (8a) and
from 6-trifluoromethanesulfonate-1-tetralone (8b) (Scheme
1). First, Suzuki-cross coupling between the bromo (8a) or
trifluoromethanesulfonate derivatives (8b) and the
corresponding boronic acids, in presence of a catalytic
O
O
(CH2)2CH=CH2
(CH2)n
HO
(i)
(ii)
(CH2)n
(CH2)n
R
R
R
11a: n = 1, R = 4-OH
11b: n = 1, R = 3-OH
11c: n = 1, R = 4-OMe
12a: n = 2, R = 4-OH
12b: n = 2, R = 3-OH
9a: n = 1, R = 4-OH
8a: n = 1, R = Br
9b: n = 1, R = 3-OH
8b: n = 2, R = OTf
9c: n = 1, R = 4-OMe
10a: n = 2, R = 4-OH
10b: n = 2, R = 3-OH
(iii)
O
(CH2)4OH
(CH2)n
HO
O
(iv)
(CH2)n
R
R
13a: n = 1, R = 4-OH
13b: n = 1, R = 3-OH
13c: n = 1, R = 4-OMe
14a: n = 2, R = 4-OH
14b: n = 2, R = 3-OH
3a: n = 1, R = 4-OH
3b: n = 1, R = 3-OH
3c: n = 1, R = 4-OMe
4a: n = 2, R = 4-OH
4b: n = 2, R = 3-OH
Scheme 1. Synthesis of compounds 3a-c and 4a,b.
Reagents and conditions: (i): p- or m-OH(or OMe)PhB(OH)2, Pd(PPh3)4, 2N Na2CO3, PhMe or DME, 80°C, Method A; (ii):
CH2=CH(CH2)2MgBr, THF, 0°C, Method C; (iii): 9-BBN, 60°C and H2O, NaOH (3M), H2O2, 0°C, Method D; (iv): TPAP, NMO, 4 Å
molecular sieves, CH2Cl2, rt, Method E.

Spiro-ꢀ-Lactones as Inhibitors of 17ꢁ-HSD2
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5 409
perruthenate (Method E) [57] followed by spontaneous
Stability Study
cyclisation afforded the desired lactones (3a-c, 4a,b).
As the spirolactones 3-7 and precursors were found
difficult to isolate with high yields, it was hypothesized that
they might have a limited chemical stability. The half-live
values of the spirolactones 3a-c, 4a,b and 7a,b, in aqueous
buffer solution (20% glycerol, 50 mM KH2PO4, 1 mM
EDTA disodium salt, pH 7.4) were determined UV-
spectrophotometrically and are listed in Table 1. The
analyzed compounds are rather unstable and are quickly
decomposed at room temperature. The presence of the
methoxy on the phenyl increases slightly the stability
compared to the hydroxy analog (3c vs 3a,b).
Synthesis of biphenyl ethyl spirolactone (5a,b),
hydroxyphenyl
fluorene
spirolactone
(6a,b)
and
hydroxyphenyl dimethyl indane spirolactone (7a,b)
derivatives
The synthesis of the spirolactones 5-7 is depicted on
Scheme 2 and occurred in a five steps reaction. First,
methoxylated intermediates 16, 21 and 26 were obtained by
Suzuki-cross coupling reaction of the appropriate brominated
ketone (15, 20, 25) with methoxyphenyl boronic acids using
the standard conditions (catalytic amount of tetrakis
triphenylphosphine palladium, 2 equivalents of 2N aqueous
sodium carbonate at reflux – method A). Demethylation was
performed with boron trifluoride dimethyl sulfide complex
(Method B) and led to the hydroxylated ketones 17, 22 and
27. Other demethylation reagents like boron tribromide or
aluminium chloride were also tried but appeared to be
inefficient or led to degradation. The formation of the final
spirolactones (5-7) was achieved following the synthetic
pathway described for 3a-c and 4a,b after nucleophilic
addition of the Grignard reagent (Method C), hydroboration-
oxydation with 9-BBN and H2O2 (Method D) and oxidation
Derivatives 5a,b, whose half lives could not be
determined by this method, were observed to be rather labile.
We suspect that the spirolactone ring opens and a water
molecule is eliminated, as we could isolate and characterize
the eliminated products 30 and 31 (Scheme 3). The hydroxy
group at 1-position of the indane group is very easily
eliminated as the product is conjugated to an aromatic
system. The reaction takes place spontaneously at room
temperature.
The presence of the adjacent annulated phenyl group is
certainly responsible for the instability of the newly
synthesized compounds compared to the steroidal analog 1
with tetrapropylammonium perruthenate (Method
Scheme 1).
E
-
O
O
HO
(CH2)2CH=CH2
(iv)
HO
(CH2)4OH
(v)
(i)
(iii)
5a,b
Br
HO
R
HO
15
16a,b: R = OMe
17a,b: R = OH
18a,b
19a,b
(ii)
H2C=HC(H2C)2
HO
HO(H2C)4
HO
O
O
(i)
(iii)
(iv)
(v)
Br
6a,b
HO
R
HO
20
21a,b: R = OMe
22a,b: R = OH
23a,b
24a,b
(ii)
HO
HO
(CH2)2CH=CH2
(iv)
O
(CH2)4OH
(v)
O
(i)
(iii)
7a,b
Br
HO
HO
R
25
26a,b: R = OMe
27a,b: R = OH
28a,b
29a,b
(ii)
a: 4-OMe or OH; b: 3-OMe or OH
Scheme 2. Synthesis of compounds 5a,b; 6a,b and 7a,b.
Reagents and conditions: (i): p- or m-MeOPhB(OH)2, 2N Na2CO3, Pd(PPh3)4, PhMe, 80°C, Method A; (ii): BF3·S(Me)2, CH2Cl2, rt, Method
B; (iii): CH2=CH(CH2)2MgBr, THF, 0°C, Method C; (iv): 9-BBN, 60°C and H2O, NaOH (3M), H2O2, 0°C, Method D; (v): TPAP, NMO, 4
Å molecular sieve, CH2Cl2, rt, Method E.

410 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5
Xu et al.
(CH2)2CH=CH2
HO
(CH2)2CH=CH2
OH
OH
30
11b
HO
(CH2)4OH
(CH2)4OH
H3CO
H3CO
31
13c
Scheme 3. Spontaneous conversion of compound 11b and 13c.
as the compounds formed like 30 and 31 are
thermodynamically more stable.
related to the low stability of the compounds as the duration
of the assay is longer (20 min incubation) than the half-life
of the compounds (a short half-life is also expected for 5a,b).
For the spirolactones 6a,b, which are chemically stable, as
well as for 7a,b, steric hindrance could play a role in the loss
of activity as these compounds are much larger than E2.
Compound 4b and 6b are the two most potent 17ꢀ-HSD2
inhibitors identified in this study (25 and 24% inhibition at 1
ꢁM, respectively), with 4b having the best selectivity profile
towards 17ꢀ-HSD1.
Compounds 6a,b and 7a,b, lacking a proton in ꢀ position
to the spirolactone ring, were synthesized with the goal to
stabilize the compounds and to minimize risk of water
elimination. The dimethyl derivatives 7a,b turned out to be
slightly more stable than the unsubstituted analogs without
dimethyl moiety (half-life of 7a,b: 28, 33 min vs. 3a,b: 5, 7
min) but still too labile for further drug development.
In opposite, the fluorene spirolactones 6a,b turned out to
present a reasonable chemical stability (half-life > 120 min).
Table 1. Half-Life of Compounds 3, 4, 7a
EXPERIMENTAL SECTION
Chemical Methods
Chemical names follow IUPAC nomenclature. Starting
materials were purchased from Aldrich, Acros, Lancaster,
Roth, Merck or Fluka and were used without purification.
Compound
Half-life (min)
Wavelength (nm)
3a
3b
3c
4a
4b
7a
7b
5
255
256
260
257
256
316
316
7
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). IR
spectra were recorded on a Bruker Vector 33FT-infrared
spectrometer (neat sample). Half-lives were measured with a
Cary-50 UV/Vis spectrophotometer (Varian, Australia) and
all values were measured more than twice and they were
repeatable.
1H NMR and 13C NMR spectra were measured on a
Bruker AM500 spectrometer (500 MHz) at 300 K. Chemical
shifts are reported in ꢀ (parts per million: ppm), by reference
to the hydrogenated residues of deuteriated solvent as
internal standard (CDCl3: ꢀ 7.26 ppm (1H NMR) and 77 ppm
(13C NMR); CD3COCD3: 2.05 ppm (1H NMR) and 29.84 and
206.3 ppm (13C NMR); CD3SOCD3: 2.50 ppm (1H NMR)
and 39.5 ppm (13C NMR). Signals are described as s
(singlet), d (doublet), t (triplet), dd (doublet of doublets), ddd
(double, double doublet), dt (doublet of triplets), br (broad)
and m (multiplet). All coupling constants (J) are given in
Hertz (Hz).
57
13
17
28
33
aDetermined in aqueous buffer (20% glycerol, 50 mM KH2PO4, 1 mM EDTA disodium
salt, pH = 7.4).
BIOLOGICAL RESULTS: 17ꢀ-HSD2 AND 17ꢀ-HSD1
INHIBITORY ACTIVITY
Human placental microsomal 17ꢁ-HSD2 and cytosolic
17ꢁ-HSD1 enzymes were isolated following a described
procedure [42, 47]. For determination of 17ꢀ-HSD2
inhibition, tritiated E2 was incubated with 17ꢀ-HSD2, NAD+
and the inhibitor (concentration: 1 ꢁM). The amount of
formed E1 was quantified by HPLC. Selectivity against 17ꢀ-
HSD1 was also determined by incubation of tritiated E1,
17ꢀ-HSD1, NADH and the inhibitor (concentration: 1 ꢁM).
The results are shown in Table 2. None of the synthesized
spiro-ꢂ-lactones 3-7 have a comparable 17ꢀ-HSD2 inhibitory
activity as the steroidal derivative 1 (65% inhibition at 1
ꢁM). In case of 3a,b and 4a,b, the loss in activity is certainly
Mass spectra were measured on a TSQ Quantum
(Thermo fischer) instrument. No mass peak was detected for
the synthesized compounds as the compounds did not

Spiro-ꢀ-Lactones as Inhibitors of 17ꢁ-HSD2
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5 411
Table 2. In Vitro Binding Potencies in 17ꢀ-HSD2 and 17ꢀ-HSD1 for the Synthesized Inhibitorsa
Compound
Inhibition of 17ꢀ-HSD2 –microsomal fraction (%) at 1ꢁMb
Inhibition of 17ꢀ-HSD1 – cytosolic fraction (%) at 1ꢁMc
1
65
0
8
0
3a
3b
3c
4a
4b
5a
5b
6a
6b
7a
7b
0
0
11
0
0
0
25
9
8
4
4
0
5
6
24
14
5
23
7
5
aMean value of three determinations, standard deviation less than 10%.
bHuman placental, microsomal fraction, substrate E2, [500 nM], cofactor NAD+, [1500 ꢁM].
cHuman placental, cytosolic fraction, substrate E1, [500 nM], cofactor NADH, [500 ꢁM].
ionized in electro-spray mode. All compounds showed more
than 95% purity (HPLC determination).
THF (5 ml/mmol) and 4-bromobutene (8 eq) under N2. The
reaction mixture was stirred at room temperature for 1 h and
the reaction was quenched by addition of 5% NaHCO3. The
mixture was extracted with ethyl acetate and the combined
organic layers were dried over magnesium sulfate,
evaporated to dryness under reduced pressure.
The following compounds were prepared according to
previously
described
procedures:
5-oxo-5,6,7,8-
(8b)
tetrahydronaphthalen-2-yltrifluoromethanesulfonate
[56], 5-(4-hydroxyphenyl)-2,3-dihydro-1H-inden-1-one (9a)
[55, 58], 5-(3-hydroxyphenyl)-2,3-dihydro-1H-inden-1-one
(9b) [58], 5-(4-methoxyphenyl)-2,3-dihydro-1H-inden-1-one
General Procedure for Hydroboration-Oxidation of the
Terminal Alkene - Method D
(9c)
1(2H)-one
[55], 6-(4-hydroxyphenyl)-3,4-dihydronaphthalen-
(10a) [55], 6-(3-hydroxyphenyl)-3,4-
9-BBN (6 eq, 0.5 M solution in THF) was added
dropwise to a solution of 3-butenyl derivative (1 eq) in THF
(3 ml/mmol) under nitrogen at 60°C. The reaction mixture
was stirred for 1 h at 60°C and cooled to 0°C. Water (2 ml)
was added and the mixture was stirred for 5 min at 0°C.
Sodium hydroxide (2 ml/mmol, 3M) was added and after
additional 5 min, 2 ml of hydrogen peroxide (30%) was
added dropwise. After 30 min the reaction was quenched by
addition of NaHCO3 (5% aqueous) and the mixture was
extracted with diethyl ether. The organic layers were dried
over sodium sulfate and evaporated under reduced pressure.
dihydronaphthalen-1(2H)-one (10b) [56], 2-bromo-fluoren-
9-one (20) [59].
General Procedure for Suzuki Coupling - Method A
A mixture of arylbromide (1 eq), methoxyphenyl boronic
acid (1.2 eq), sodium carbonate (2 eq) and
tetrakis(triphenylphosphine) palladium (0.1 eq) in an oxygen
free toluene/water (1:1) solution was stirred at 80°C for 4-16
h under nitrogen. The reaction mixture was cooled to room
temperature. The aqueous layer was extracted with
dichloromethane. The combined organic layers were washed
with brine, dried over sodium sulfate, filtered and
concentrated to dryness.
General Procedure for Formation of the Spirolactones -
Method E
To a solution of the diol derivative (1 eq) in anhydrous
General Procedure for Ether Cleavage - Method B
dichloromethane
(4
ml/mmol)
was
added
N-
methylmorpholine N-oxide (3.9 eq), tetrapropyl ammonium
per ruthenate (0.17 eq) and 4 Å molecular sieves (310
mg/mmol) at room temperature. The reaction mixture was
stirred for 2-30 min at room temperature, monitored by TLC.
The reaction mixture was diluted with dichloromethane (4
ml/mmol) and directly purified.
To a solution of bis(methoxyphenyl) derivative (1 eq) in
dry dichloromethane, borontrifluoride dimethyl sulfide
complex (6 eq) was added dropwise at 0°C. The reaction
mixture was stirred for 3-14 h at room temperature. Water
was added to quench the reaction, and the aqueous layer was
extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over sodium sulfate,
evaporated to dryness under reduced pressure.
1-But-3-en-1-yl-5-(4-hydroxyphenyl)indan-1-ol (RS, 11a)
Compound 11a was prepared by reaction of 5-(4-
hydroxyphenyl)-2,3-dihydro-1H-inden-1-one (9a, 70 mg,
0.31 mmol) with a Grignard reagent in THF prepared from 4-
bromobutene (0.25 ml, 2.5 mmol) and magnesium (48 mg,
2.0 mmol) according to the general procedure C. The
analytically pure product was obtained after purification by
FC (hexane/ethyl acetate 10:1ꢀ6:1) as colorless solid (75
General Procedure for Introducing the but-3-en-1-yl
Group - Method C
To the Grignard reagent in dry THF prepared from 4-
bromobutene (8 eq) and magnesium (6.6 eq) was added
dropwise a solution of the ketone derivative (1 eq) in dry

412 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5
Xu et al.
mg, 86%). 1H NMR (CD3COCD3) ꢀ 1.77-1.83 (m, 1H), 1.93-
1.99 (m, 1H), 2.10-2.20 (m, 2H), 2.22-2.31 (m, 2H), 2.80-
2.83 (m, 1H), 2.96-3.02 (m, 1H), 3.92 (br s, 1H), 4.88-4.91
(m, 1H), 5.83-5.91 (m, 1H), 6.89-6.93 (m, 2H), 7.34 (d, J =
7.9 Hz, 1H), 7.37-7.42 (m, 2H), 7.46-7.49 (m, 2H), 8.39 (br
s, 1H). 13C NMR (CD3COCD3) ꢀ 40.8, 83.0, 114.3, 116.5,
123.4, 124.3, 125.56, 128.9, 133.6, 140.1, 141.6, 144.4,
147.7, 157.8.
1-But-3-en-1-yl-6-(3-hydroxyphenyl)-1,2,3,4-
tetrahydronaphthalen-1-ol (RS, 12b)
Compound 12a was prepared by reaction of 6-(3-
hydroxyphenyl)-3,4-dihydronaphthalen-1(2H)-one (10b
[41], 150 mg, 0.63 mmol) with a Grignard reagent in THF
prepared from 4-bromobutene (0.51 ml, 5.0 mmol) and
magnesium (99 mg, 4.1 mmol) according to the general
procedure C. The analytically pure product was obtained
after purification by FC (hexane/ethyl acetate 10:1ꢁ6:1) as
1-But-3-en-1-yl-5-(3-hydroxyphenyl)indan-1-ol (RS, 11b)
1
colorless solid (98 mg, 53%). H NMR (CDCl3) ꢀ 1.80-2.10
Compound 11b was prepared by reaction of 5-(3-
hydroxyphenyl)-2,3-dihydro-1H-inden-1-one (9b [44], 60
mg, 0.27 mmol) with a Grignard reagent in THF prepared
from 4-bromobutene (0.22 ml, 2.2 mmol) and magnesium
(43 mg, 1.8 mmol) according to the general procedure C.
The analytically pure product was obtained after purification
by FC (hexane/ethyl acetate 10:1ꢁ6:1) as colorless solid (62
mg, 83%). 1H NMR (CDCl3) ꢀ 1.86-1.92 (m, 1H), 1.95 (br s,
1H), 2.03-2.09 (m, 1H), 2.14-2.21 (m, 2H), 2.23-2.31 (m,
1H), 2.33-2.39 (m, 1H), 2.85-2.91 (m, 1H), 3.03-3.09 (m,
1H), 4.96-4.99 (m, 1H), 5.06 (ddt, J = 17.3 Hz, 1.9 Hz, 1.6
Hz, 1H), 5.36 (br s, 1H), 5.84-5.92 (m, 1H), 6.80 (ddd, J =
8.2 Hz, 2.5 Hz, 0.9 Hz, 1H), 6.99 (dd, J = 2.2 Hz, 1.5 Hz,
1H), 7.13 (ddd, J = 7.6 Hz, 1.6 Hz, 0.6 Hz, 1H), 7.28 (t, J =
7.9 Hz, 1H), 7.35-7.37 (m, 1H), 7.40-7.43 (m, 2H). 13C NMR
(CDCl3) ꢀ 28.7, 29.5, 39.3, 40.1, 83.6, 114.1, 114.2, 114.6,
119.7, 123.1, 123.7, 126.0, 129.9, 138.6, 141.2, 143.0, 143.7,
146.4, 156.0.
(m, 9H), 2.15-2.22 (m, 1H), 2.74-2.87 (m, 2H), 4.94 (ddt, J =
10.4 Hz, 1.9 Hz, 1.3 Hz, 1H), 5.02 (ddt, J = 17.6 Hz, 1.9 Hz,
1.6 Hz, 1H), 5.64 (br s, 1H), 5.80-5.88 (m, 1H), 6.79 (ddd, J
= 8.0 Hz, 2.5 Hz, 0.6 Hz, 1H), 7.00 (dd, J = 2.5 Hz, 1.6 Hz,
1H), 7.11, (ddd, J = 7.6 Hz, 0.9 Hz, 0.6 Hz, 1H), 7.24-7.28
(m, 1H), 7.37 (dd, J = 8.2 Hz, 1.9 Hz, 1H), 7.56 (d, J = 8.2
Hz, 1H). 13C NMR (CDCl3) ꢀ 19.7, 28.6, 30.0, 36.0, 41.3,
72.6, 114.0, 114.3, 114.5, 119.4, 125.1, 126.7, 127.5, 129.9,
137.1, 138.6, 139.6, 141.1, 142.5, 156.0.
1-(4-Hydroxybutyl)-5-(4-hydroxyphenyl)indan-1-ol
13a)
(RS,
Compound 13a was prepared by reaction of 1-but-3-en-
1-yl-5-(4-hydroxyphenyl)indan-1-ol (11a, 140 mg, 0.5
mmol) with 9-BBN (6 ml, 0.5 M in THF, 3 mmol) and H2O2
(1 ml, 30% in H2O) according to method D. Purification by
FC (dichloromethane/methanol 30:1) afforded the desired
product as colorless solid (90 mg, 60%). 1H NMR
(CD3COCD3) ꢀ 1.40-1.47 (m, 1H), 1.50-1.59 (m, 3H), 1.72-
1.78 (m, 1H), 1.82-1.92 (m, 1H), 2.08-2.14 (m, 1H), 2.25-
2.30 (m, 1H), 2.79-2.83 (m, 1H), 2.95-3.01 (m, 1H), 3.40 (t,
J = 5.4 Hz, 1H), 3.54 (dt, J = 6.3 Hz, 5.3 Hz, 2H), 3.84 (s,
1H), 6.89-6.92 (m, 2H), 7.33 (dd, J = 7.6 Hz, 0.9 Hz, 1H),
7.38-7.41 (m, 2H), 7.46-7.49 (m, 2H), 8.38 (br s, 1H). 13C
NMR (CD3COCD3) ꢀ 21.6, 34.4, 40.8, 41.6, 62.5, 83.3,
116.5, 123.4, 124.3, 125.5, 128.8, 133.7, 141.5, 144.5, 148.0,
157.8.
1-But-3-en-1-yl-5-(4-methoxyphenyl)indan-1-ol (RS, 11c)
Compound 11c was prepared by reaction of 5-(4-
methoxyphenyl)-2,3-dihydro-1H-inden-1-one (9c, 70 mg,
0.29 mmol) with a Grignard reagent in THF prepared from 4-
bromobutene (0.23 ml, 2.3 mmol) and magnesium (46 mg,
1.9 mmol) according to the general procedure C. The
analytically pure product was obtained after purification by
FC (hexane/ethyl acetate 20:1ꢁ10:1) as colorless solid (76
mg, 88%). 1H NMR (CDCl3) ꢀ 1.79 (br s, 1H), 1.85-1.91 (m,
1H), 2.02-2.08 (m, 1H), 2.12-2.23 (m, 2H), 2.24-2.31 (m,
1H), 2.33-2.39 (m, 1H), 2.85-2.91 (m, 1H), 3.03-3.09 (m,
1H), 3.85 (s, 3H), 4.96-5.08 (m, 1H), 5.04-5.08 (m, 1H),
5.85-5.93 (m, 1H), 6.95-6.99 (m, 2H), 7.37 (d, J = 7.9 Hz,
1H), 7.40-7.44 (m, 2H), 7.51 (d, J = 8.8 Hz, 2H). 13C NMR
(CDCl3) ꢀ 28.7, 29.5, 39.4, 40.3, 55.3, 83.4, 114.2, 114.5,
123.0, 123.3, 125.6, 128.2, 133.8, 138.7, 141.2, 143.7, 145.8,
159.1.
1-(4-Hydroxybutyl)-5-(3-hydroxyphenyl)indan-1-ol
13b)
(RS,
Compound 13b was prepared by reaction of 1-but-3-en-
1-yl-5-(3-hydroxyphenyl)indan-1-ol (11b, 65 mg, 0.23
mmol) with 9-BBN (2.76 ml, 0.5 M in THF, 1.38 mmol) and
H2O2 (0.45 ml, 30% in H2O) according to method D.
Purification by FC (dichloromethane/methanol 30:1)
afforded the desired product as colorless solid (30 mg, 43%).
1H NMR (CD3COCD3) ꢀ 1.41-1.48 (m, 1H), 1.50-1.59 (m,
3H), 1.72-1.78 (m, 1H), 1.87-1.93 (m, 1H), 2.09-2.15 (m,
1H), 2.26-2.31 (m, 1H), 2.80-2.87 (m, 1H), 2.96-3.03 (m,
1H), 3.43 (t, J = 5.2 Hz, 1H), 3.52-3.57 (m, 2H), 3.90 (s,
1H), 6.82 (ddd, J = 8.0 Hz, 2.5 Hz, 0.9 Hz, 1H), 7.08-7.10
(m, 2H), 7.25 (t, J = 8.2 Hz, 1H), 7.36 (dd, J = 7.4 Hz, 0.9
Hz, 1H), 7.41-7.44 (m, 2H), 8.38 (br s, 1H). 13C NMR
(CD3COCD3) ꢀ 21.6, 30.1, 34.3, 40.7, 41.5, 62.5, 83.3,
114.7, 114.9, 119.0, 123.9, 124.3, 126.0, 130.6, 141.5, 143.8,
144.5, 148.9, 158.7.
1-But-3-en-1-yl-6-(4-hydroxyphenyl)-1,2,3,4-
tetrahydronaphthalen-1-ol (RS, 12a)
Compound 12a was prepared by reaction of 6-(4-
hydroxyphenyl)-3,4-dihydronaphthalen-1(2H)-one (10a [40],
150 mg, 0.63 mmol) with a Grignard reagent in THF prepared
from 4-bromobutene (0.51 ml, 5.0 mmol) and magnesium
(99 mg, 4.1 mmol) according to the general procedure C.
The analytically pure product was obtained after purification
by FC (dichloromethane/methanol 100:1) as colorless solid
(120 mg, 65%). 1H NMR (CD3COCD3) ꢀ 1.86-1.90 (m, 4H),
2.08-2.24 (m, 2H), 2.58-2.62 (m, 1H), 2.76-2.83 (m, 2H),
3.04 (t, J = 6.1 Hz, 1H), 3.76 (br s, 1H), 4.87-4.91 (m, 1H),
5.00 (ddt, J = 17.4 Hz, 3.6 Hz, 1.8 Hz, 1H), 5.81-5.89 (m,
1H), 6.89-6.93 (m, 2H), 6.94-6.97 (m, 1H), 7-25-7.28 (m,
1H), 7.38 (dd, J = 8.2 Hz, 2.1 Hz, 1H), 7.46-7.50 (m, 2H),
7.55-7.60 (m, 2H), 8.36 (br s, 1H).
1-(4-Hydroxybutyl)-5-(4-methoxyphenyl)indan-1-ol
13c)
(RS,
Compound 13c was prepared by reaction of 1-but-3-en-1-
yl-5-(4-methoxyphenyl)indan-1-ol (11c, 140 mg, 0.48 mmol)

Spiro-ꢀ-Lactones as Inhibitors of 17ꢁ-HSD2
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5 413
(br s, 1H). 13C NMR (CD3COCD3) ꢀ 18.1, 32.4, 39.9, 44.3,
50.8, 65.8, 93.1, 116.6, 123.7, 124.5, 126.1, 129.0, 133.2,
142.9, 143.7, 144. 8, 170.4.
with 9-BBN (5 ml, 0.5 M in THF, 2.5 mmol) and H2O2 (0.9
ml, 30% in H2O) according to the method D. Purification by
FC (dichloromethane/methanol 30:1) afforded the desired
1
product as colorless solid (120 mg, 80%). H NMR (CDCl3)
5-(3-Hydroxyphenyl)-2,3,4',5'-tetrahydrospiro[indene-1,2'-
pyran]-6'(3'H)-one (RS, 3b)
ꢀ 1.43-1.51 (m, 1H), 1.56-1.63 (m, 3H), 1.77-1.83 (m, 1H),
1.94-1.99 (m, 1H), 2.10-2.16 (m, 1H), 2.32-2.37 (m, 1H),
2.84-2.90 (m, 1H), 3.02-3.08 (m, 1H), 3.66 (t, J = 6.3 Hz,
2H), 3.85 (s, 3H), 6.95-6.98 (m, 2H), 7.35 (d, J = 7.9 Hz,
1H), 7.39-7.43 (m, 2H), 7.48-7.52 (m, 2H). 13C NMR
(CDCl3) ꢀ 20.5, 29.5, 33.1, 40.0, 40.2, 55.3, 62.7, 83.5,
114.2, 123.0, 123.3, 125.5, 128.2, 133.8, 141.2, 143.7, 146.0,
159.1.
Compound 3b was prepared by reaction of 1-(4-
hydroxybutyl)-5-(3-hydroxyphenyl)indan-1-ol (13b, 20 mg,
0.07 mmol) with N-methylmorpholine N-oxide (31 mg, 0.26
mmol) and tetrapropyl ammonium per ruthenate (10 mg,
0.01 mmol) according to method E. Purification by FC
(dichloromethane/ethyl acetate 50:1ꢁ30:1) afforded the
desired product as colorless solid (4 mg, 30%). IR (ꢂ, cm-1):
1-(4-Hydroxybutyl)-6-(4-hydroxyphenyl)-1,2,3,4-
tetrahydronaphthalen-1-ol (RS, 14a)
1
1696. H NMR (CDCl3) ꢀ 1.91-2.01 (m, 1H), 2.03-2.08 (m,
2H), 2.15-2.20 (m, 1H), 2.31-2.35 (m, 1H), 2.42-2.48 (m,
1H), 2.62-2.76 (m, 2H), 2.87-2.93 (m, 1H), 3.10-3.16 (m,
1H), 5.66 (br s, 1H), 6.83 (ddd, J = 8.1 Hz, 2.5 Hz, 0.9 Hz,
1H), 7.03 (dd, J = 2.2 Hz, 1.6 Hz, 1H), 7.10 (ddd, J = 7.9 Hz,
1.6 Hz, 0.9 Hz, 1H), 7.27 (t, J = 7.9 Hz, 1H), 7.32 (dd, J =
7.6 Hz, 1.3 Hz, 1H), 7.39-7.43 (m, 2H). 13C NMR (CDCl3) ꢀ
17.2, 29.3, 29.6, 31.8, 39.3, 93.1, 114.3, 114.5, 119.6, 123.5,
123.8, 126.1, 129.9, 142.2, 142.6, 142.7, 143.7, 156.2, 171.5.
Compound 14a was prepared by reaction of 1-but-3-en-
1-yl-6-(4-hydroxyphenyl)-1,2,3,4-tetrahydronaphthalen-1-ol
(12a, 100 mg, 0.34 mmol) with 9-BBN (4 ml, 0.5 M in THF,
2 mmol) and H2O2 (0.65 ml, 30% in H2O) according to the
method D. Purification by FC (dichloromethane/methanol
50:1ꢁ30:1) afforded the desired product as colorless solid
1
(71 mg, 67%). H NMR (CD3COCD3) ꢀ 1.35-1.40 (m, 2H),
1.43-1.56 (m, 3H), 1.79-1.91 (m, 5H), 2.70-2.79 (m, 2H),
3.41 (t, J = 5.4 Hz, 1H), 3.51 (dt, J = 6.2 Hz, 5.4 Hz, 2H),
3.67 (s, 1H), 6.87 (d, J = 8.8 Hz, 2H), 7.22 (s, 1H), 7.34 (dd,
J = 8.2 Hz, 2.1 Hz, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.55 (d, J =
8.2 Hz, 1H), 8.38 (s, 1H). 13C NMR (CD3COCD3) ꢀ 21.1,
21.4, 34.4, 36.8, 37.3, 43.6, 62.5, 71.6, 116.5, 124.6, 127.0,
128.0, 128.6, 128.7, 133.2, 137.6, 142.9, 157.8.
5-(4-Methoxyphenyl)-2,3,4',5'-tetrahydrospiro[indene-1,2'-
pyran]-6'(3'H)-one (RS, 3c)
Compound 3c was prepared by reaction of 1-(4-
hydroxybutyl)-5-(4-methoxyphenyl)indan-1-ol (13c, 30 mg,
0.10 mmol) with N-methylmorpholine N-oxide (44 mg, 0.37
mmol) and tetrapropyl ammonium per ruthenate (6 mg, 0.02
mmol) according to method E. Purification by FC
(dichloromethane/ethyl acetate 50:1) afforded the desired
product as colorless solid (15 mg, 50%). IR (ꢂ, cm-1): 2932,
1-(4-Hydroxybutyl)-6-(3-hydroxyphenyl)-1,2,3,4-
tetrahydronaphthalen-1-ol (RS, 14b)
Compound 14b was prepared by reaction of 1-but-3-en-
1-yl-6-(3-hydroxyphenyl)-1,2,3,4-tetrahydronaphthalen-1-ol
(12b, 100 mg, 0.34 mmol) with 9-BBN (4 ml, 0.5 M in THF,
2 mmol) and H2O2 (0.65 ml, 30% in H2O) according to the
method D. Purification by FC (dichloromethane/methanol
50:1ꢁ30:1) afforded the desired product as colorless solid
1
1704. H NMR (CDCl3) ꢀ 1.94-2.09 (m, 3H), 2.16-2.22 (m,
1H), 2.32-2.36 (m, 1H), 2.44-2.50 (m, 1H), 2.62-2.71 (m,
2H), 2.89-2.95 (m, 1H), 3.14-3.20 (m, 1H), 3.85 (s, 3H),
6.97 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 8.0 Hz, 1H), 7.26-7.45
(m, 2H), 7.51 (d, J = 8.8 Hz, 2H). 13C NMR (CDCl3) ꢀ 17.3,
29.4, 29.7, 31.9, 39.3, 55.3, 92.9, 114.2, 123.3, 123.5, 125.7,
128.3, 133.5, 142.1, 143.7, 159.3, 170.8.
1
(56 mg, 53%). H NMR (CD3COCD3) ꢀ 1.32-1.42 (m, 2H),
1.48-1.59 (m, 3H), 1.80-1.86 (m, 5H), 1.90-1.96 (m, 1H),
2.79-2.83 (m, 1H), 3.37 (t, J = 5.4 Hz, 1H), 3.52-3.56 (m,
2H), 3.71 (s, 1H), 6.81 (ddd, J = 8.2 Hz, 2.2 Hz, 0.9 Hz, 1H),
7.08-7.10 (m, 2H), 7.27-7.29 (m, 1H), 7.28 (s, 1H), 7.40 (dd,
J = 8.2 Hz, 1.9 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H), 8.33 (s,
1H). ). 13C NMR (CD3COCD3) ꢀ 21.1, 21.4, 30.7; 34.4, 36.7,
37.4, 43.6, 62.5, 72.1, 114.5, 115.0, 118.9, 125.1, 127.6,
128.0, 130.6, 137.7, 143.4, 143.9, 158.7.
6-(4-Hydroxyphenyl)-3,4,4',5'-tetrahydro-2H-
spiro[naphthalene-1,2'-pyran]-6'(3'H)-one (RS, 4a)
Compound 5a was prepared by reaction of 1-(4-
hydroxybutyl)-6-(4-hydroxyphenyl)-1,2,3,4-
tetrahydronaphthalen-1-ol (14a, 50 mg, 0.16 mmol) with N-
methylmorpholine N-oxide (73 mg, 0.62 mmol) and
tetrapropyl ammonium per ruthenate (10 mg, 0.03 mmol)
5-(4-Hydroxyphenyl)-2,3,4',5'-tetrahydrospiro[indene-1,2'-
pyran]-6'(3'H)-one (RS, 3a)
according
to
method
E.
Purification
by
FC
(dichloromethane/ethyl acetate 50:1ꢁ30:1) afforded the
desired product (20 mg, 40%) as colorless solid. IR (ꢂ, cm-1):
1696. 1H NMR (CD3COCD3) ꢀ 1.86-1.98 (m, 4H), 2.07-2.17
(m, 5H), 2.60-2.64 (m, 2H), 2.90-2.94 (m, 1H), 6.90-6.93
(m, 2H), 7.31-7.34 (m, 1H), 7.42-7.46 (m, 2H), 7.48-7.52
(m, 2H), 8.52 (br s, 1H).
Compound 3a was prepared by reaction of 1-(4-
hydroxybutyl)-5-(4-hydroxyphenyl)indan-1-ol (13a, 50 mg,
0.17 mmol) with N-methylmorpholine N-oxide (76 mg, 0.65
mmol) and tetrapropyl ammonium per ruthenate (10 mg,
0.03 mmol) according to method E, Purification by FC
(dichloromethane/ethyl acetate 50:1ꢁ30:1) afforded the
desired product as colorless solid (15 mg, 30%). IR (ꢂ, cm-1):
6-(3-Hydroxyphenyl)-3,4,4',5'-tetrahydro-2H-
spiro[naphthalene-1,2'-pyran]-6'(3'H)-one (RS, 4b)
1
2926, 1694. H NMR (CD3COCD3) ꢀ 1.98-2.03 (m, 1H),
2.07-2.13 (m, 1H), 2.21-2.26 (m, 1H), 2.38-2.42 (m, 2H),
2.45-2.47 (m, 1H), 2.59-2.62 (m, 2H), 2.91-2.97 (m, 1H),
3.06-3.12 (m, 1H), 6.92 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 8.5
Hz, 1H), 7.46-7.49 (m, 2H), 7.50 (d, J = 8.8 Hz, 2H), 8.42
Compound 4b was prepared by reaction of 1-(4-
hydroxybutyl)-6-(3-hydroxyphenyl)-1,2,3,4-tetrahydronaph-
thalen-1-ol (14b, 26 mg, 0.08 mmol) with N-methylmor-
pholine N-oxide (37 mg, 0.31 mmol) and tetrapropyl

414 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5
Xu et al.
4'-(1-Hydroxy-1-methylpent-4-en-1-yl)biphenyl-4-ol (RS,
18a)
ammonium per ruthenate (5 mg, 0.01 mmol) according to
method E. Purification by FC (dichloromethane/ethyl acetate
50:1ꢀ30:1) afforded the desired product (10 mg, 38%) as
Compound 18a was prepared by reaction of 1-(4'-
hydroxybiphenyl-4-yl)ethanone (17a, 132 mg, 0.62 mmol)
1
colorless solid. IR (ꢁ, cm-1): 1684. H NMR (CD3SOCD3) ꢂ
1.78-1.86 (m, 3H), 1.93-2.15 (m, 5H), 2.54-2.67 (m, 2H),
2.77-2.89 (m, 2H), 6.76 (ddd, J = 8.2 Hz, 2.4 Hz, 0.9 Hz,
1H), 7.00 (t, J = 1.9 Hz, 1H), 7.05 (dt, J = 8.2 Hz, 0.9 Hz,
1H), 7.24 (t, J = 7.9 Hz, 1H), 7.34 (s, 1H), 7.43-7.47 (m,
2H), 9.50 (s, 1H). 13C NMR (CD3SOCD3) ꢂ 16.2, 19.2, 28.6,
29.1, 33.1. 34.2, 83.0, 113.3, 114.4, 117.3, 124.3, 126.6,
127.5, 129. 8, 136.9, 138.5, 139.4, 141.1, 157.7, 170.4.
with
a Grignard reagent in THF prepared from 4-
bromobutene (0.51 ml, 4.98 mmol) and magnesium (100 mg,
4.1 mmol) according to method C. The analytically pure
product was obtained after purification by FC (hexane/ethyl
1
acetate 10:1ꢀ6:1) as colorless solid (120 mg, 72%). H
NMR (CDCl3) ꢂ 1.61 (s, 3H), 1.93-2.00 (m, 4H), 2.06-2.10
(m, 1H), 4.93-5.01 (m, 2H), 5.27 (br s, 1H), 5.78-5.82 (m,
1H), 6.90 (d, J = 8.8 Hz, 2H), 7.46-7.49 (m, 4H), 7.52 (d, J =
8.2 Hz, 2H).13C NMR (CDCl3) ꢂ 28.5, 30.3, 43.0, 74.9,
114.6, 115.7, 125.2, 126.4, 128.2, 133.5, 138.7, 139.0, 146.0,
155.2.
1-(4'-Methoxybiphenyl-4-yl)ethanone (16a)
Compound 16a was prepared by reaction of 1-(4-
bromophenyl)ethanone (15, 591 mg, 3.0 mmol) and 4-
methoxyphenyl boronic acid (547 mg, 3.6 mmol) with
tetrakis(triphenylphosphine)palladium (347 mg, 0.3 mmol)
as catalyst according to method A. Purification by FC
(hexane/ethyl acetate 10:1) afforded the desired product (650
4'-(1-Hydroxy-1-methylpent-4-en-1-yl)biphenyl-3-ol (RS, 18b)
Compound 18b was prepared by reaction of 1-(3'-
hydroxybiphenyl-4-yl)ethanone (17b, 100 mg, 0.47 mmol)
1
mg, 96%) as colorless solid. H NMR (CDCl3) ꢂ 2.62 (s,
with
a Grignard reagent in THF prepared from 4-
3H), 3.86 (s, 3H), 7.00 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8
Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H), 8.00 (d, J = 8.5 Hz, 2H).
13C NMR (CDCl3) ꢂ 26.5, 55.3, 114.3, 126.6, 128.3, 128.8,
132.2, 135.3, 145.3, 159.8, 197.6.
bromobutene (0.38 ml, 3.77 mmol) and magnesium (74 mg,
3.10 mmol) according to method C. The analytically pure
product was obtained after purification by FC (hexane/ethyl
acetate 10:1ꢀ6:1) as colorless solid (80 mg, 63%). 1H NMR
(CD3COCD3) ꢂ 1.57 (s, 3H), 1.86-1.96 (m, 3H), 2.11-2.19
(m, 1H), 3.96 (s, 1H), 4.85 (d, J = 10.4 Hz, 1H), 4.94 (d, J =
17.4 Hz, 1H), 5.76-5.84 (m, 1H), 6.82 (ddd, J = 8.0 Hz, 2.2
Hz, 0.9 Hz, 1H), 7.11-7.13 (m, 2H), 7.26 (t, J = 8.2 Hz, 1H),
7.57 (s, 4H), 8.37 (s, 1H). 13C NMR (CD3COCD3) ꢂ 29.2,
30.9, 44.4, 74.0, 114.2 114.5, 115.0, 118.9, 126.4, 127.1,
130.7, 139.5, 140.0, 143.2, 149.0, 158.7.
1-(3'-Methoxybiphenyl-4-yl)ethanone (16b)
Compound 16b was prepared by reaction of 1-(4-
bromophenyl)ethanone (15, 591 mg, 3.0 mmol) and 3-
methoxyphenyl boronic acid (547 mg, 3.6 mmol) with
tetrakis(triphenylphosphine)palladium (347 mg, 0.3 mmol)
as catalyst according to method A. Purification by FC
(hexane/ethyl acetate 10:1) afforded the desired product (655
1
mg, 97%) as colorless oil. H NMR (CDCl3) ꢂ 2.64 (s, 3H),
5-(4'-Hydroxybiphenyl-4-yl)hexane-1,5-diol (RS, 19a)
3.88 (s, 3H), 6.95 (ddd, J = 8.2 Hz, 2.5 Hz, 0.6 Hz, 1H), 7.15
(t, J = 2.2 Hz, 1H), 7.21 (ddd, J = 7.6 Hz, 1.6 Hz, 0.9 Hz,
1H), 7.39 (t, J = 8.0 Hz, 1H), 7.66-7.69 (m, 2H), 8.01-8.04
(m, 2H). 13C NMR (CDCl3) ꢂ 26.6, 55.3, 113.1, 113.5, 119.7,
127.3, 128.8, 130.0, 136.0, 141.4, 145.6, 160.0, 197.7.
Compound 19a was prepared by reaction of 4'-(1-
hydroxy-1-methylpent-4-en-1-yl)biphenyl-4-ol (18a, 110
mg, 0.41 mmol) with 9-BBN (4.92 ml, 0.5 M in THF, 2.46
mmol) and H2O2 (0.80 ml, 30% in H2O) according to the
method D. Purification by FC (dichloromethane/methanol
50:1ꢀ30:1) afforded the desired product as colorless solid
1-(4'-Hydroxybiphenyl-4-yl)ethanone (17a)
1
Compound 17a was prepared by reaction of 1-(4'-
methoxybiphenyl-4-yl)ethanone (16a, 226 mg, 1 mmol) with
borontrifluoride dimethyl sulfide complex (0.63 ml, 6 mmol)
according to method B. Purification by FC (hexane/ethyl
acetate 10:1) afforded the desired product as colorless solid
(170 mg, 80%). 1H NMR (CD3COCD3) ꢂ 2.59 (s, 3H), 6.96-
6.98 (m, 2H), 7.60-7.62 (m, 2H), 7.71-7.74 (m, 2H), 8.01-
8.04 (m, 2H), 8.62 (s, 1H). 13C NMR (CD3COCD3) ꢂ 26.6,
116.7, 127.0, 129.2, 129.7, 131.7, 136.2, 146.0, 158.8, 197.3.
(73 mg, 62%). H NMR (CD3COCD3) ꢂ 1.17-1.25 (m, 1H),
1.38-1.48 (m, 3H), 1.53 (s, 3H), 1.77-1.88 (m, 2H), 3.40-
3.44 (m, 1H), 3.47-3.50 (m, 2H), 3.89 (s, 1H), 6.92 (d, J =
8.8 Hz, 2H), 7.50 (d, J = 8.8 Hz, 2H), 7.52 (s, 4H), 8.43 (br
s, 1H). 13C NMR (CD3COCD3) ꢂ 21.3, 30.9, 34.2, 45.2, 62.5,
74.2, 116.5, 126.4, 128.6, 133.1, 139.4, 148.3, 157.8.
5-(3'-Hydroxybiphenyl-4-yl)hexane-1,5-diol (RS, 19b)
Compound 19b was prepared by reaction of 4'-(1-
hydroxy-1-methylpent-4-en-1-yl)biphenyl-3-ol (18b, 78 mg,
0.29 mmol) with 9-BBN (3.48 ml, 0.5 M in THF, 1.74
mmol) and H2O2 (0.70 ml, 30% in H2O) according to the
method D. Purification by FC (dichloromethane/methanol
50:1ꢀ30:1) afforded the desired product as colorless solid
1-(3'-Hydroxybiphenyl-4-yl)ethanone (17b)
Compound 17b was prepared by reaction of 1-(3'-
methoxybiphenyl-4-yl)ethanone (16b, 375 mg, 1.66 mmol)
with borontrifluoride dimethyl sulfide complex (1.05 ml,
9.94 mmol) according to method B. Purification by FC
(hexane/ethyl acetate 10:1) afforded the desired product as
colorless solid (288 mg, 82%). 1H NMR (CD3COCD3) ꢂ 2.61
(s, 3H), 6.89-6.92 (m, 1H), 7.17-7.20 (m, 2H), 7.32 (t, J =
8.2 Hz, 1H), 7.75 (d, J = 8.5 Hz, 2H), 8.05 (d, J = 8.5 Hz,
2H), 8.50 (s, 1H). 13C NMR (CD3COCD3) ꢂ 26.7, 114.8,
116.1, 119.2, 127.8, 129.7, 131.0, 137.1, 142.1, 146.2, 158.9,
197.5.
1
(75 mg, 90%). H NMR (CD3COCD3) ꢂ 1.18-1.25 (m, 1H),
1.41-1.49 (m, 3H), 1.54 (s, 3H), 1.77-1.89 (m, 2H), 3.35 (t, J
= 5.2 Hz, 1H), 3.46-3.50 (m, 2H), 3.88 (s, 1H), 6.82 (ddd, J
= 8.2 Hz, 2.5 Hz, 0.9, Hz, 1H), 7.11-7.13 (m, 2H), 7.26 (t, J
= 8.0 Hz, 1H), 7.55 (s, 4H), 8.38 (s, 1H). 13C NMR
(CD3COCD3) ꢂ 21.3, 30.9, 34.2, 45.2, 62.5, 74.2, 114.5,
114.9, 118.9, 126.4, 127.0, 130.7, 139.4, 143.3, 149.4, 158.7.

Spiro-ꢀ-Lactones as Inhibitors of 17ꢁ-HSD2
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5 415
6-(4'-Hydroxybiphenyl-4-yl)-6-methyltetrahydro-2H-pyran-
2-one (RS, 5a)
1H), 7.53-7.57 (m, 2H), 7.64-7.66 (m, 1H), 7.71 (dd, J = 7.6
Hz, 1.8 Hz, 1H), 7.89 (d, J = 1.5 Hz, 1H). 13C NMR (CDCl3)
ꢂ 55.4, 112.3, 113.5, 119.3, 120.4, 120.6, 123.0, 124.4,
129.0, 129.9, 133.2, 134.5, 134.8, 141.3, 142.1, 143.3, 144.2,
160.1, 193.8.
Compound 5a was prepared by reaction of 5-(4'-
hydroxybiphenyl-4-yl)hexane-1,5-diol (19a, 42 mg, 0.15
mmol) with N-methylmorpholine N-oxide (67 mg, 0.57
mmol) and tetrapropyl ammonium per ruthenate (9 mg, 0.03
mmol) according to method E. Purification by FC
(dichloromethane/Ethyl acetate 50:1ꢀ30:1) afforded the
desired product (12 mg, 28%) as colorless solid. IR (ꢁ, cm-1):
2-(4-Hydroxyphenyl)-fluoren-9-one (22a)
Compound 22a was prepared by reaction of 2-(4-
methoxyphenyl)-fluoren-9-one (21a, 286 mg, 1mmol) with
borontrifluoride dimethyl sulfide complex (0.63 ml, 6 mmol)
1
2922, 1695. H NMR (CD3COCD3) ꢂ 1.56-1.62 (m, 1H),
according
to
method
B.
Purification
by
FC
1.66 (s, 3H), 1.81-1.88 (m, 1H), 2.08-2.12 (m, 1H), 2.35-
2.41 (m, 2H), 2.45-2.52 (m, 1H), 6.93 (d, J = 8.5 Hz, 2H),
7.43 (d, J = 8.5 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.61 (d, J
= 8.5 Hz, 2H), 8.50 (s, 1H). 13C NMR (CD3COCD3) ꢂ 17.6,
31.3, 34.9, 85.4, 116.6, 125.9, 127.2, 128.8, 132.5, 140.6,
144.6, 158.2, 170.9.
(dichloromethane/CH3OH 100:1) afforded the desired
product (250 mg, 92%) as yellow solid. 1H NMR
(CD3COCD3) ꢂ 6.97 (dt, J = 8.5 Hz, 2.1 Hz, 2H), 7.37 (dt, J
= 7.3 Hz, 0.9 Hz, 1H), 7.57-7.63 (m, 4H), 7.73-7.81 (m, 4H),
8.57 (s, 1H). 13C NMR (CD3COCD3) ꢂ 116.7, 121.6, 122.1,
122.3, 124,6, 128.8, 129.9, 131.8, 133.4, 135.1, 135.6, 135.9,
143.1, 143.2, 145.2, 158.6, 193.8.
6-(3'-Hydroxybiphenyl-4-yl)-6-methyltetrahydro-2H-pyran-
2-one (RS, 5b)
2-(3-Hydroxyphenyl)-fluoren-9-one (22b)
Compound 5b was prepared by reaction of 5-(3'-
hydroxybiphenyl-4-yl)hexane-1,5-diol (19b, 50 mg, 0.17
mmol) with N-methylmorpholine N-oxide (79 mg, 0.67
mmol) and tetrapropyl ammonium per ruthenate (10 mg,
0.03 mmol) according to method E. Purification by FC
(CH2Cl2/Ethyl acetate 50:1ꢀ30:1) afforded the desired
product (15 mg, 30%) as colorless solid. IR (ꢁ, cm-1): 2945,
Compound 22b was prepared by reaction of 2-(3-
methoxyphenyl)-fluoren-9-one (21b, 300 mg, 1.05 mmol)
with borontrifluoride dimethyl sulfide complex (0.66 ml, 6.3
mmol) according to method B. Purification by FC
(dichloromethane/CH3OH 100:1) afforded the desired
product (260 mg, 91%) as yellow solid. 1H NMR
(CD3COCD3) ꢂ 6.90 (d, J = 7.9 Hz, 1H), 7.17-7.22 (m, 2H),
7.32 (t, J = 7.8 Hz, 1H), 7.39 (t, J = 7.3 Hz, 1H), 7.59-7.65
(m, 2H), 7.76-7.85 (m, 4H), 8.50 (s, 1H). 13C NMR
(CD3COCD3) ꢂ 114.4, 115.9, 118.8, 121.7, 122.1, 122.9,
124.7, 130.1, 131.0, 134.1, 135.1, 135.6, 136.0, 142.1, 143.1,
144.1, 145.1, 158.9, 193.6.
1
1727. H NMR (CD3COCD3) ꢂ 1.50-1.64 (m, 1H), 1.67 (s,
3H), 1.82-1.89 (m, 1H), 2.07-2.13 (m, 1H), 2.36-2.42 (m,
2H), 2.45-2.52 (m, 1H), 6.85 (ddd, J = 7.9 Hz, 2.2 Hz, 0.9
Hz, 1H), 7.12-7.14 (m, 2H), 7.28 (t, J = 8.0 Hz, 1H), 7.47
(dt, J = 8.9 Hz, 1.9 Hz, 2H), 7.65 (dt, J = 8.8 Hz, 2.2 Hz,
2H), 8.44 (s, 1H). 13C NMR (CD3COCD3) ꢂ 17.6, 29.8, 31.2,
34.9, 85.4, 114.6, 115.3, 118.94, 125.9, 127.8, 130.8, 140.7,
142.7, 145.6, 158.8, 170.8.
9-But-3-en-1-yl-2-(4-hydroxyphenyl)-9H-fluoren-9-ol (RS,
23a)
2-(4-Methoxyphenyl)-fluoren-9-one (21a)
Compound 23a was prepared by reaction of 2-(4-
hydroxyphenyl)-fluoren-9-one (22a, 100 mg, 0.36 mmol)
Compound 21a was prepared by reaction of 2-bromo-
fluoren-9-one [46] (20, 540 mg, 2.08 mmol) and 4-
methoxyphenyl boronic acid (380 mg, 2.50 mmol) with
tetrakis(triphenylphosphine)palladium (243 mg, 0.21 mmol)
as catalyst according to method A. Purification by FC
(hexane/dichloromethane 5:1) afforded the desired product
with
a Grignard reagent in THF prepared from 4-
bromobutene (0.30 ml, 2.94 mmol) and magnesium (58 mg,
2.43 mmol) according to method C. The analytically pure
product was obtained after purification by FC (hexane/ethyl
acetate 10:1ꢀ6:1) as colorless solid (50 mg, 41%). 1H NMR
(CD3COCD3) ꢂ 1.68-1.74 (m, 2H), 2.21-2.27 (m, 2H), 4.56
(s, 1H), 4.76-4.80 (m, 1H), 4.81-4.86 (m, 1H), 5.65-5.72 (m,
1H), 6.95 (dt, J = 8.8 Hz, 2.1 Hz, 2H), 7.31 (dt, J = 7.3 Hz,
1.2 Hz, 1H), 7.36 (dt, J = 7.3 Hz, 1.2 Hz, 1H), 7.53-7.56 (m,
1H), 7.56-7.60 (m, 3H), 7.70-7.73 (m, 1H), 7.74 (dd, J = 7.9
Hz, 0.6 Hz, 1H), 7.77 (dd, J = 1.5 Hz, 0.6 Hz, 1H), 8.47 (s,
1H). 13C NMR (CD3COCD3) ꢂ 40.6, 82.5, 114.4, 116.6,
120.6, 121.0, 122.4, 124.5, 127.5, 128.3, 128.8, 129.4, 133.2,
138.9, 139.3, 140.4, 141.5, 150.6, 151.2, 158.1.
1
(500 mg, 84%) as yellow solid. H NMR (CDCl3) ꢂ 3.86 (s,
3H), 6.98 (dt, J = 8.8 Hz, 2.2 Hz, 2H), 7.28 (dt, J = 7.3 Hz,
1.3 Hz, 1H), 7.48 (dt, J = 7.3 Hz, 1.3 Hz, 1H), 7.50-7.53 (m,
2H), 7.55 (dt, J = 8.8 Hz, 2.2 Hz, 2H), 7.64-7.67 (m, 2H),
7.85 (d, J = 1.6 Hz, 1H). 13C NMR (CDCl3) ꢂ 55.3, 114.3,
120.2, 120.6, 122.4, 124.3, 127.8, 128.8, 132.3, 132.5, 134.4,
134.7, 134.9, 141.9, 142.5, 144.4, 159.6, 193.9.
2-(3-Methoxyphenyl)-fluoren-9-one (21b)
Compound 21b was prepared from 2-bromo-fluoren-9-
one (20, 540 mg, 2.08 mmol) and 3-methoxyphenyl boronic
acid (380 mg, 2.50 mmol) with tetrakis(triphenylphos-
phine)palladium (243 mg, 0.21 mmol) as catalyst according
to method A. Purification by FC (hexane/dichloromethane
5:1) afforded the desired product (520 mg, 87%) as yellow
9-But-3-en-1-yl-2-(3-hydroxyphenyl)-9H-fluoren-9-ol (RS,
23b)
Compound 23b was prepared by reaction of 2-(3-
hydroxyphenyl)-fluoren-9-one (22b, 136 mg, 0.50 mmol)
with
a Grignard reagent in THF prepared from 4-
1
solid. H NMR (CDCl3) ꢂ 3.88 (s, 3H), 6.93 (ddd, J = 8.2
bromobutene (0.41 ml, 4.00 mmol) and magnesium (79 mg,
2.43 mmol) according to method C. The analytically pure
product was obtained after purification by FC (hexane/ethyl
acetate 10:1ꢀ6:1) as colorless solid (60 mg, 37%). 1H NMR
Hz, 2.7 Hz, 0.9 Hz, 1H), 7.14, (t, J = 2.0 Hz, 1H), 7.20 (ddd,
J = 7.6 Hz, 1.5 Hz, 0.9 Hz, 1H), 7.30 (dt, J = 7.3 Hz, 0.9 Hz,
1H), 7.37 (t, J = 8.0 Hz, 1H), 7.49 (dt, J = 7.3 Hz, 0.9 Hz,

416 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5
Xu et al.
(CD3COCD3) ꢀ 1.69-1.75 (m, 2H), 2.21-2.32 (m, 2H), 4.58
(s, 1H), 4.76-4.80 (m, 1H), 4.81-4.86 (m, 1H), 5.65-5.73 (m,
1H), 6.86 (ddd, J = 8.2 Hz, 2.5 Hz, 0.9 Hz, 1H), 7.18-7.20
(m, 2H), 7.30 (t, J = 8.0 Hz, 1H), 7.33 (dt, J = 7.4 Hz, 1.3
Hz, 1H), 7.37 (dt, J = 7.4 Hz, 1.3 Hz, 1H), 7.55-7.57 (m,
1H), 7.63 (dd, J = 7.9 Hz, 1.9 Hz, 1H), 7.74 (ddd, J = 7.3 Hz,
1.3 Hz, 0.6 Hz, 1H), 7.76-7.79 (m, 1H), 7.79 (dd, J = 1.6 Hz,
0.6 Hz, 1H), 8.39 (s, 1H). 13C NMR (CD3COCD3) ꢀ 40.6,
82.5, 114.45, 114.6, 115.2, 119.0, 120.7, 121.0, 123.0, 124.5,
128.1, 128.60, 129.4, 130.8, 139.3, 139.9, 140.2, 141.5,
143.4, 150.8, 151.2, 158.8.
7.81-7.83 (m, 2H), 7.86 (dd, J = 1.6 Hz, 0.6 Hz, 1H), 8.48
(br s, 1H). 13C NMR (CD3COCD3) ꢀ 18.4, 33.0, 42.8, 90.2,
116.6, 118.6, 121.1, 121.5, 123.0, 125.1, 128.6, 128.7, 129.0,
130.5, 132.8, 138.2, 139.9, 142.1, 148.7, 158.3, 170.5.
2-(3-Hydroxyphenyl)-4',5'-dihydrospiro[fluorene-9,2'-
pyran]-6'(3'H)-one (RS, 6b)
Compound 6b was prepared by reaction of 9-(4-
hydroxybutyl)-2-(3-hydroxyphenyl)-9H-fluoren-9-ol (24b,
30 mg, 0.09 mmol) with N-methylmorpholine N-oxide (40
mg, 0.34 mmol) and tetrapropyl ammonium per ruthenate (5
mg, 0.015 mmol) according to method E. Purification by FC
(dichloromethane/ethyl acetate 50:1ꢁ30:1) afforded the
desired product (10 mg, 30%) as colorless solid. IR (ꢂ, cm-1):
1700. 1H NMR (CD3COCD3) ꢀ 2.07-2.13 (m, 2H), 2.17-2.25
(m, 1H), 2.35-2.39 (m, 3H), 6.86 (ddd, J = 7.9 Hz, 2.2 Hz,
0.9 Hz, 1H), 7.16-7.19 (m, 2H), 7.29 (dt, J = 7.6 Hz, 0.6 Hz,
1H), 7.37, (dt, J = 7.6 Hz, 0.9 Hz, 1H), 7.47 (dt, J = 7.6 Hz,
1.3 Hz, 1H), 7.68 (dt, J = 7.6 Hz, 0.9 Hz, 1H), 7.70 (dd, J =
7.9 Hz, 1.6 Hz, 1H), 7.82 (ddd, J = 7.6 Hz, 1.3 Hz, 0.6 Hz,
1H), 7.85 (dd, J = 7.9 Hz, 0.6 Hz, 1H), 7.88 (dd, J = 1.6 Hz,
0.6 Hz, 1H), 8.46 (s, 1H). 13C NMR (CD3COCD3) ꢀ 18.4,
22.7, 33.0, 42.8, 90.2, 114.8, 115.5, 118.9, 119.1, 121.3,
121.6, 123.6, 125.1, 128.9, 129.3, 130.6, 130.8, 139.2, 139.7,
143.0, 148.1, 148.6, 170.5.
9-(4-Hydroxybutyl)-2-(4-hydroxyphenyl)-9H-fluoren-9-ol
(RS, 24a)
Compound 24a was prepared by reaction of 9-but-3-en-
1-yl-2-(4-hydroxyphenyl)-9H-fluoren-9-ol (23a, 50 mg, 0.15
mmol) with 9-BBN (1.83 ml, 0.5 M in THF, 0.91 mmol) and
H2O2 (0.32 ml, 30% in H2O) according to the method D.
Purification by FC (dichloromethane/methanol 50:1ꢁ30:1)
afforded the desired product as colorless solid (30 mg, 57%).
1H NMR (CD3COCD3) ꢀ 0.99-1.04 (m, 2H), 1.35-1.41 (m,
2H), 2.15-2.25 (m, 2H), 3.33-3.37 (m, 3H), 4.50 (br s, 1H),
6.93-6.97 (m, 2H), 7.29 (dt, J = 7.3 Hz, 1.2 Hz, 1 H), 7.34
(dt, J = 7.3 Hz, 1.2 Hz, 1H), 7.52-7.55 (m, 1H), 7.56-7.59
(m, 3H), 7.68-7.71 (m, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.75 (d,
J = 1.5 Hz, 1H), 8.50 (br s, 1H). 13C NMR (CD3COCD3) ꢀ
21.5, 34.0, 41.3, 62.3, 82.7, 116.6, 120.5, 120.9, 122.3,
124.4, 127.4, 128.3, 128.8, 129.2, 133.3, 139.0, 140.4, 141.4,
150.9, 151.5, 158.0.
5-(4-Methoxyphenyl)-2,2-dimethylindan-1-one (26a)
Compound 26a was prepared by reaction of 5-bromo-
2,2-dimethyl-2,3-dihydro-1H-inden-1-one [45] (25, 200 mg,
0.84 mmol) and 4-methoxyphenyl boronic acid (152 mg,
0.88 mmol) with tetrakis(triphenylphosphine)palladium (97
mg, 0.08 mmol) as catalyst according to method A.
Purification by FC (hexane/dichloromethane 15:1) afforded
9-(4-Hydroxybutyl)-2-(3-hydroxyphenyl)-9H-fluoren-9-ol
(RS, 24b)
Compound 24b was prepared by reaction of 9-but-3-en-
1-yl-2-(3-hydroxyphenyl)-9H-fluoren-9-ol (23b, 60 mg, 0.18
mmol) with 9-BBN (2.20 ml, 0.5 M in THF, 1.10 mmol) and
H2O2 (0.36 ml, 30% in H2O) according to the method D.
Purification by FC (dichloromethane/methanol 50:1ꢁ30:1)
afforded the desired product as colorless solid (30 mg, 47%).
1H NMR (CD3COCD3) ꢀ 0.99-1.06 (m, 2H), 1.35-1.42 (m,
2H), 2.18-2.23 (m, 2H), 3.29-3.39 (m, 2H), 3.29 (t, J = 5.3
Hz, 1H), 3.35-3.39 (m, 2H), 4.51 (br s, 1H), 6.85 (ddd, J =
8.0 Hz, 2.5 Hz, 1.2 Hz, 1H), 7.17-7.19 (m, 2H), 7.29 (t, J =
8.2 Hz, 1H), 7.31 (dd, J = 7.3 Hz, 1.2 Hz, 1H), 7.36 (dt, J =
7.3 Hz, 1.3 Hz, 1H), 7.52-7.55 (m, 1H), 7.61 (dd, J = 7.9 Hz,
1.6 Hz, 1H), 7.68-7.71 (m, 1H), 7.72-7.74 (m, 2H). 13C NMR
(CD3COCD3) ꢀ 21.5, 34.1, 41.3, 62.2, 82.7, 114.6, 115.2,
118.9, 120.6, 120.9, 122.9, 123.9, 124.5, 128.0, 128.5, 129.3,
130.8, 139.9, 140.3, 141.4, 143.5, 151.1, 151.6, 158.8.
1
the desired product (210 mg, 94%) as colorless solid. H
NMR (CDCl3) ꢀ 1.26 (s, 6H), 3.03 (s, 2H), 3.87 (s, 3H), 7.00
(d, J = 8.8 Hz, 2H), 7.56-7.58 (m, 4H), 7.80 (d, J = 7.9 Hz,
1H). 13C NMR (CDCl3) ꢀ 25.4, 42.9, 45.8, 55.4, 114.4,
124.4, 124.8, 126.4, 128.6, 132.7, 133.7, 147.5, 152.9, 160.0.
210.9.
5-(3-Methoxyphenyl)-2,2-dimethylindan-1-one (26b)
Compound 26b was prepared by reaction of 5-bromo-
2,2-dimethyl-2,3-dihydro-1H-inden-1-one [45] (25, 270 mg,
1.13 mmol) and 3-methoxyphenyl boronic acid (206 mg,
1.36 mmol) with tetrakis(triphenylphosphine)palladium (130
mg, 0.11 mmol) as catalyst according to method A.
Purification by FC (hexane/dichloromethane 20:1) afforded
1
the desired product (230 mg, 77%) as colorless solid. H
2-(4-Hydroxyphenyl)-4',5'-dihydrospiro[fluorene-9,2'-
pyran]-6'(3'H)-one (RS, 6a)
NMR (CDCl3) ꢀ 1.25 (s, 6H), 3.03 (s, 2H), 3.86 (s, 3H), 6.96
(ddd, J = 8.2 Hz, 2.5 Hz, 0.9 Hz, 1H), 7.15 (dd, J = 2.5 Hz,
1.6 Hz, 1H), 7.21 (ddd, J = 7.6 Hz, 1.9 Hz, 0.9 Hz, 1H), 7.39
(t, J = 8.2 Hz, 1H), 7.58-7.61 (m, 2H), 7.82 (d, J = 7.9 Hz,
1H). 13C NMR (CDCl3) ꢀ 25.3, 42.9, 45.8, 55.4, 113.2,
113.7, 119.9, 124.8, 125.1, 127.0, 130.0, 134.4, 141.8, 147.8,
152.8, 160.0, 210.9.
Compound 6a was prepared by reaction of 9-(4-
hydroxybutyl)-2-(4-hydroxyphenyl)-9H-fluoren-9-ol (24a,
30 mg, 0.09 mmol) with N-methylmorpholine N-oxide (40
mg, 0.34 mmol) and tetrapropyl ammonium per ruthenate (5
mg, 0.015 mmol) according to method E. Purification by FC
(dichloromethane/ethyl acetate 50:1ꢁ30:1) afforded the
desired product (8 mg, 27%) as colorless solid. IR (ꢂ, cm-1):
5-(4-Hydroxyphenyl)-2,2-dimethylindan-1-one (27a)
1
2923, 1713. H NMR (CD3COCD3) ꢀ 2.16-2.22 (m, 1H),
Compound 27a was prepared by reaction of 5-(4-
methoxyphenyl)-2,2-dimethylindan-1-one (26a, 105 mg, 0.4
mmol) with borontrifluoride dimethyl sulfide complex (0.25
ml, 2.4 mmol) according to method B. Purification by FC
2.33-2.40 (m, 3H), 2.88-2.91 (m, 2H), 6.94 (d, J = 8.8 Hz,
2H), 7.35 (dt, J = 7.6 Hz, 1.3 Hz, 1H), 7.46 (dt, J = 7.6 Hz,
1.3 Hz, 1H), 7.58 (d, J = 8.7 Hz, 2H), 7.65-7.69 (m, 2H),

Spiro-ꢀ-Lactones as Inhibitors of 17ꢁ-HSD2
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5 417
1-(4-Hydroxybutyl)-5-(4-hydroxyphenyl)-2,2-
dimethylindan-1-ol (RS, 29a)
(hexane/ethyl acetate 10:1) afforded the desired product (95
mg, 95%) as colorless solid. H NMR (CD3COCD3) ꢀ 1.20
1
(s, 6H), 3.06 (s, 2H), 6.97 (d, J = 8.8 Hz, 2H), 7.61 (d, J =
8.5 Hz, 2H), 7.64-7.70 (m, 3H), 8.65 (s, 1H). ). 13C NMR
(CD3COCD3) ꢀ 25.5, 43.3, 46.1, 116.8, 125.0, 125.1, 126.8,
129.5, 132.1, 134.3, 148.4, 153.9, 159.0, 210.2.
Compound 29a was prepared by reaction of 1-but-3-en-
1-yl-5-(4-hydroxyphenyl)-2,2-dimethylindan-1-ol (28a, 60
mg, 0.19 mmol) with 9-BBN (2.33 ml, 0.5 M in THF, 1.17
mmol) and H2O2 (0.38 ml, 30% in H2O) according to the
method D. Purification by FC (dichloromethane/methanol
50:1ꢁ30:1) afforded the desired product as colorless solid
(30 mg, 47%). 1H NMR (CD3COCD3) ꢀ 1.00 (s, 3H), 1.14 (s,
3H), 1.38-1.55 (m, 3H), 1.65-1.72 (m, 3H), 2.67-2.76 (m,
2H), 3.41 (t, J = 5.4 Hz, 1H), 3.50-3.58 (m, 2H), 3.58 (s,
1H), 6.90 (d, J = 8.8 Hz, 2H), 7.31-7.37 (m, 3H), 7.47 (d, J =
8.8 Hz, 2H), 8.40 (br s, 1H). 13C NMR (CD3COCD3) ꢀ 21.2,
22.8, 24.9, 34.6, 37.3, 46.0, 48.6, 62.56, 84.9, 116.5, 123.5,
124.8, 125.7, 128.9, 133.7, 140.9, 142.3, 147.4, 157.8.
5-(3-Hydroxyphenyl)-2,2-dimethylindan-1-one (27b)
Compound 27b was prepared by reaction of 5-(3-
methoxyphenyl)-2,2-dimethylindan-1-one (26b, 210 mg, 0.8
mmol) with borontrifluoride dimethyl sulfide complex (0.50
ml, 4.74 mmol) according to method B. Purification by FC
(hexane/ethyl acetate 8:1) afforded the desired product (180
1
mg, 90%) as colorless solid. H NMR (CD3COCD3) ꢀ 1.20
(s, 6H), 3.08 (s, 2H), 6.91 (ddd, J = 7.9 Hz, 2.2 Hz, 0.9 Hz,
1H), 7.17-7.19 (m, 2H), 7.31 (t, J = 8.2 Hz, 1H), 7.65-7.67
(m, 1H), 7.71-7.73 (m, 2H), 8.80 (br s, 1H). 13C NMR
(CD3COCD3) ꢀ 25.5, 43.2, 46.1, 115.0, 116.2, 119.3, 125.0,
126.0, 127.5, 130.9, 135.1, 142.4, 148.5, 153.9, 158.9, 210.5.
1-(4-Hydroxybutyl)-5-(3-hydroxyphenyl)-2,2-
dimethylindan-1-ol (RS, 29b)
Compound 29b was prepared by reaction of 1-but-3-en-
1-yl-5-(3-hydroxyphenyl)-2,2-dimethylindan-1-ol (28b, 131
mg, 0.42 mmol) with 9-BBN (5.10 ml, 0.5 M in THF, 2.55
mmol) and H2O2 (0.84 ml, 30% in H2O) according to the
method D. Purification by FC (dichloromethane/methanol
50:1ꢁ30:1) afforded the desired product as colorless solid
(58 mg, 43%). 1H NMR (CD3COCD3) ꢀ 1.00 (s, 3H), 1.14 (s,
3H), 1.38-1.57 (m, 3H), 1.65-1.73 (m, 3H), 2.68-2.78 (m,
2H), 3.39 (t, J = 5.4 Hz, 1H), 3.50-3.54 (m, 2H), 3.62 (s,
1H), 6.81 (ddd, J = 8.2 Hz, 2.2 Hz, 1.3 Hz, 1H), 7.08-7.10
(m, 2H), 7.25 (t, J = 8.2 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H),
7.38-7.42 (m, 2H), 8.38 (s, 1H). 13C NMR (CD3COCD3) ꢀ
21.2, 22.7, 24.8, 34.6, 37.2, 45.9, 48.6, 62.6, 84.9, 114.6,
114.9, 119.0, 124.0, 125.3, 125.8, 130.6, 141.0, 142.4, 143.8,
148.4, 158.7.
1-But-3-en-1-yl-5-(4-hydroxyphenyl)-2,2-dimethylindan-1-
ol (RS, 28a)
Compound 28a was prepared by reaction of 5-(4-
hydroxyphenyl)-2,2-dimethylindan-1-one (27a, 80 mg, 0.32
mmol) with a Grignard reagent in THF prepared from 4-
bromobutene (0.26 ml, 2.54 mmol) and magnesium (50 mg,
2.09 mmol) according to method C. The analytically pure
product was obtained after purification by FC (hexane/ethyl
acetate 10:1ꢁ6:1) as colorless solid (75 mg, 77%). 1H NMR
(CD3COCD3) ꢀ 1.01 (s, 3H), 1.15 (s, 3H), 1.69 (dt, J = 12.9
Hz, 4.4 Hz, 1H), 1.79 (ddd, J =13.9 Hz, 12.0 Hz, 4.4 Hz,
1H), 2.01-2.09 (m, 1H), 2.37-2.45 (m, 1H), 2.70 (d, J = 15.1
Hz, 1H), 2.77 (d, J = 15.1 Hz, 1H), 3.65 (d, J = 0.6 Hz, 1H),
4.85-4.89 (m, 1H), 4.94-4.9 (m, 1H), 5.79-5.86 (m, 1H),
6.89-6.91 (m, 2H), 7.33 (d, J = 7.9 Hz, 1H), 7.34-7.40 (m,
2H), 7.48-7.50 (m, 2H), 8.36 (s, 1H). 13C NMR
(CD3COCD3) ꢀ 22.7, 24.8, 29.3, 36.6, 45.9, 48.6, 84.7,
114.3, 116.5, 123.6, 124.8, 125.7, 128.8, 133.6, 140.4, 141.1,
142.3, 147.0, 157.8.
5-(4-Hydroxyphenyl)-2,2-dimethyl-2,3,4',5'-
tetrahydrospiro[indene-1,2'-pyran]-6'(3'H)-one (RS, 7a)
Compound 7a was prepared by reaction of 1-(4-
hydroxybutyl)-5-(4-hydroxyphenyl)-2,2-dimethylindan-1-ol
(29a, 30 mg, 0.09 mmol) with N-methylmorpholine N-oxide
(42 mg, 0.36 mmol) and tetrapropyl ammonium per
ruthenate (5 mg, 0.015 mmol) according to method E.
Purification by FC (dichloromethane/ethyl acetate
50:1ꢁ30:1) afforded the desired product (10 mg, 34%) as
colorless solid. IR (ꢂ, cm-1): 2923, 1695. 1H NMR
(CD3COCD3) ꢀ 1.11 (s, 3H), 1.13 (s, 3H), 1.99-2.02 (m, 1H),
2.10-2.14 (m, 3H), 2.50-2.57 (m, 1H), 2.64-2.69 (m, 1H),
2.75 (d, J = 15.1 Hz, 1H), 2.95 (d, J = 15.4 Hz, 1H), 6.92 (d,
J = 8.8 Hz, 2H), 7.35-7.44 (m, 3H), 7.50 (d, J = 8.8 Hz, 2H),
8.46 (br s, 1H). 13C NMR (CD3COCD3) ꢀ 17.9, 23.1, 23.9,
27.3, 45.9, 49.3, 90.8, 116.6, 124.0, 125.0, 125.4, 128.9,
133.1, 140.4, 143.6, 148.3, 158.7, 170.6.
1-But-3-en-1-yl-5-(3-hydroxyphenyl)-2,2-dimethylindan-1-
ol (RS, 28b)
Compound 28b was prepared by reaction of 5-(3-
hydroxyphenyl)-2,2-dimethylindan-1-one (27b, 170 mg,
0.67 mmol) with a Grignard reagent in THF prepared from 4-
bromobutene (0.55 ml, 5.39 mmol) and magnesium (107 mg,
4.45 mmol) according to method C. The analytically pure
product was obtained after purification by FC (hexane/ethyl
1
acetate 10:1ꢁ6:1) as colorless solid (160 mg, 77%). H
NMR (CD3COCD3) ꢀ 1.01 (s, 3H), 1.16 (s, 3H), 1.69 (dt, J =
12.9 Hz, 4.6 Hz, 1H), 1.80 (ddd, J = 13.6 Hz, 12.3 Hz, 4.4
Hz, 1H), 2.01-2.09 (m, 1H), 2.38-2.46 (m, 1H), 2.70 (d, J =
15.1 Hz, 1H), 2.79 (d, J = 15.1 Hz, 1H), 3.69 (d, J = 0.6 Hz,
1H), 4.85-4.89 (m, 1H), 4.95-4.99 (m, 1H), 5.78-5.87 (m,
1H), 6.82 (ddd, J = 7.8 Hz, 2.2 Hz, 0.9 Hz, 1H), 7.09-7.11
(m, 2H), 7.25 (t, J = 8.2 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H),
5-(3-Hydroxyphenyl)-2,2-dimethyl-2,3,4',5'-
tetrahydrospiro[indene-1,2'-pyran]-6'(3'H)-one (RS, 7b)
Compound 7b was prepared by reaction of 1-(4-
hydroxybutyl)-5-(3-hydroxyphenyl)-2,2-dimethylindan-1-ol
(29b, 40 mg, 0.12 mmol) with N-methylmorpholine N-oxide
(56 mg, 0.48 mmol) and tetrapropyl ammonium per
ruthenate (7 mg, 0.02 mmol) according to method E.
Purification by FC (dichloromethane/ethyl acetate
50:1ꢁ30:1) afforded the desired product (14 mg, 35%) as
colorless solid. IR (ꢂ, cm-1): 2924, 1690. 1H NMR
7.39-7.41 (m, 1H), 7.43 (d, J = 7.9 Hz, 1H), 8.35 (s, 1H). 13
C
NMR (CD3COCD3) ꢀ 22.6, 24.8, 29.2, 36.6, 45.9, 48.7, 84.7,
114.3, 114.7, 114.9, 119.0, 124.2, 125.3, 125.8, 130.6, 140.4,
141.1, 142.4, 143.8, 148.0, 158.7.

418 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5
Xu et al.
(CD3COCD3) ꢀ 1.11 (s, 3H), 1.13 (s, 3H), 1.98-2.03 (m, 1H),
2.08-2.14 (m, 3H), 2.51-2.58 (m, 1H), 2.65-2.71 (m, 1H),
2.77 (d, J = 15.4 Hz, 1H), 2.96 (d, J = 15.4 Hz, 1H), 6.84
(ddd, J = 8.2 Hz, 2.5 Hz, 0.9 Hz, 1H), 7.10-7.12 (m, 2H),
7.27 (t, J = 8.0 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H), 7.46-7.50
(m, 2H), 8.42 (br s, 1H). 13C NMR (CD3COCD3) ꢀ 17.9,
23.1, 23.8, 27.3, 30.6, 45.9, 49.3, 94.7, 114.8, 115.3, 119.1,
124.6, 125.0, 126.0, 130.7, 142.5, 143.3, 143.6, 144.8, 158.8,
170.6.
adjusted to 1% in all samples. The enzymatic reaction was
started by addition of a mixture of unlabeled- and [3H]- E2
(final concentration: 500 nM, 0.11 ꢁCi). After 20 min, the
incubation was stopped with HgCl2 and the mixture was
extracted with ether. After evaporation, the steroids were
dissolved in acetonitrile. E1 and E2 were separated using
acetonitrile/water (45:55) as mobile phase in a C18 RP
chromatography column (Nucleodur C18, 3ꢁm, Macherey-
Nagel, Düren) connected to a HPLC-system (Agilent 1100
Series, Agilent Technologies, Waldbronn). Detection and
quantification of the steroids were performed using a
radioflow detector (Berthold Technologies, Bad Wildbad).
The conversion rate was calculated according to the
%E1
3-(3-(But-3-enyl)-1H-inden-6-yl)phenol (30)
Compound 30 was obtained after the spontaneous
1
conversion of compound 11b at room temperature. H NMR
(CDCl3) ꢀ 2.46-2.51 (m, 2H), 2.66-2.70 (m, 2H), 3.40 (d, J =
1.9 Hz, 2H), 4.92 (d, J = 3.8 Hz, 1H), 5.03 (dd, J = 10.1 Hz,
1.6 Hz, 1H), 5.12 (dd, J = 17.0 Hz, 1.6 Hz, 1H), 5.92-6.00
(m, 1H), 6.28 (t, J = 1.6 Hz, 1H), 6.81 (ddd, J = 7.9 Hz, 2.5
Hz, 0.9 Hz, 1H), 7.10 (t, J = 2.0 Hz, 1H), 7.21 (dt, J = 7.6
Hz, 1.3 Hz, 1H), 7.31 (t, J = 7.9 Hz, 1H), 7.42 (d, J = 7.9 Hz,
1H), 7.53 (dd, J = 7.9, 1.6 Hz, 1H), 7.67 (br s, 1H). 13C NMR
(CD3COCD3) ꢀ 27.2, 32.1, 37.8, 113.7, 114.1, 114.8, 119.0,
119.8, 122.6, 125.2, 128.6, 129.9, 137.3, 138.4, 143.6, 143.7,
144.9, 145.1, 155.8.
following equation:
.
%conversion =
•100
%E1+ %E2
Each value was calculated from at least three independent
experiments.
Inhibition of 17ꢀ-HSD1 in Cell-Free Assay
The 17ꢂ-HSD1 inhibition assay was performed similarly
to the 17ꢂ-HSD2 test. The cytosolic fraction was incubated
with NADH [500 ꢁM], test compound and a mixture of
unlabeled- and [3H]-E1 (final concentration: 500 nM, 0.15
ꢁCi) for 10 min at 37°C. Further treatment of the samples
and HPLC separation were carried out as mentioned above
for 17ꢂ-HSD2.
4-(6-(4-Methoxyphenyl)-1H-inden-3-yl)butan-1-ol (31)
Compound 30 was obtained after the spontaneous
1
conversion of compound 13c at room temperature. H NMR
(CDCl3) ꢀ 1.30-1.35 (m, 2H), 1.68-1.73 (m, 2H), 1.77-1.83
(m, 2H), 2.59-2.63 (m, 1H), 3.39 (d, J = 1.6 Hz, 2H), 3.71 (t,
J = 6.5 Hz, 2H), 3.86 (s, 3H), 6.22-6.24 (m, 1H), 6.96-7.00
(m, 2H), 7.40 (d, J = 7.9 Hz, 1H), 7.48-7.51 (m, 1H), 7.54-
7.57 (m, 2H), 7.65 (br s, 1H). 13C NMR (CD3COCD3) ꢀ
24.2, 27.5, 32.7, 37.8, 55.3, 62.9, 114.2, 119.1, 122.3, 124.8,
128.1, 128.7, 134.4, 137.5, 144.0, 144.1, 158.9.
CONCLUSION
In this study, we applied a ligand based approach to
design new non-steroidal 17ꢂ-HSD2 inhibitors bearing a
spiro-ꢀ-lactone moiety, which is believed to be responsible
for the 17ꢂ-HSD2 inhibitory activity of the steroidal
compound (1) [20]. Eleven spiro-ꢀ-lactone steroidomimetics
were synthesized and tested for 17ꢂ-HSD2 inhibitory
activity. None of the compounds could reach the activity of
the steroidal spiro-ꢀ-lactone 1, they were either inactive or
showed a low activity (25% for 4b and 24% for 6b) when
tested at a concentration of 1 ꢁM.
Biological Methods
[2,4,6,7-3H]-E1 and [2,4,6,7-3H]-E2 were purchased from
Perkin Elmer, Boston. Quickszint Flow 302 scintillator fluid
was bought from Zinsser Analytic, Frankfurt. Other
chemicals were purchased from Sigma, Roth or Merck.
Several problems, met during the synthesis, obliged us to
investigate a new synthesis pathway for the formation of the
spiro-ꢀ-lactone moiety, different to the one described [20]
for the synthesis of 1. The keto derivatives 9a-c were first
alkylated with lithium acetylenide resulting from the reaction
of tetrahydro-(butynyl)-2H pyrane and n-BuLi. The triple
bound was then hydrogenated in EtOH with palladium on
activated charcoal. The tetrahydropyranyl ether moiety of the
side chain was hydrolyzed by treatment with p-TSA but did
not lead to the expected dihydroxy derivative 13a-c: only the
primary OH function was identified in the molecule. The
synthetic pathway was optimized by introduction of the
alkene chain on the tetralone 9a-c and oxidized with 9-BBN
and H2O2 to lead to 13a-c as described on Scheme 1.
17ꢀ-HSD1 and 17ꢀ-HSD2 Enzyme Preparation from
Human Placental Enzyme
17ꢀ-HSD1 and 17ꢀ-HSD2 were obtained from human
placenta according to previously described procedures [47,
60]. Fresh human placenta was homogenized and the
enzymes were separated by fractional centrifugation at 1000
g, 10000 g and 150000 g. For the purification of 17ꢀ-HSD1,
the cytosolic fraction was precipitated with ammonium
sulfate. 17ꢀ-HSD2 was obtained from the microsomal
fraction. Aliquots containing 17ꢀ-HSD1 or 17ꢀ-HSD2 were
stored frozen.
Inhibition of 17ꢀ-HSD2 in Cell-Free Assay
Inhibitory activities were evaluated by an established
method with minor modifications [22, 47, 60]. Briefly, the
microsomal enzyme preparation was incubated with NAD+
[1500 ꢁM] in the presence of potential inhibitors at 37°C in a
phosphate buffer (50 mM) supplemented with 20% of
glycerol and EDTA 1mM. Inhibitor stock solutions were
prepared in DMSO. Final concentration of DMSO was
Despite the fact that compound 1 was stable, it appeared
that the new derivatives decomposed and were very difficult
to purify. This chemical instability was confirmed and
quantified by measurement of the half-life in aqueous buffer.
It might be due to the fact that the C ring of the steroidal 1 is
not aromatic, while the phenyl ring, present next to the
spirolactone in the synthesized tetrahydronaphathlene or

Spiro-ꢀ-Lactones as Inhibitors of 17ꢁ-HSD2
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5 419
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[11]
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ACKNOWLEDGEMENTS
We thank Dr. Josef Zapp for the NMR measurements.
We are grateful to Professor Donald Poirier for the kind gift
of compound 1. We thank the Alexander-von-Humboldt
Foundation for the research fellowship to Kuiying Xu. We
acknowledge Jannine Ludwig and Jeannine Jung for their
help in performing the enzyme inhibition tests (17ꢁ-HSD1,
17ꢁ-HSD2). We are grateful to Enrico Perspicace for careful
reading of the manuscript and fruitful discussion.
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