Estrogen receptor β-subtype selective tetrahydrofluorenones: Use of a fused pyrazole as a phenol bioisostere

Article abstract of DOI:10.1016/j.bmcl.2006.05.036

Synthesis of a series of fused pyrazole tetrahydrofluorenone analogs which are potent, ERβ subtype selective ligands is described. Analogs possessing subnanomolar ERβ binding, greater than 100-fold ERβ-selectivity, and oral bioavailability are reported.

Full text of DOI:10.1016/j.bmcl.2006.05.036

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3896–3901
Estrogen receptor b-subtype selective tetrahydrofluorenones:
Use of a fused pyrazole as a phenol bioisostere
R. R. Wilkening,a,* R. W. Ratcliffe,a A. K. Fried,a D. Meng,a W. Sun,a L. Colwell,a
S. Lambert,a M. Greenlee,a S. Nilsson,c A. Thorsell,c M. Mojena,d C. Tudela,d
K. Frisch,b W. Chan,b E. T. Birzin,b S. P. Rohrerb and M. L. Hammonda
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, 800-B116 Rahway, NJ 07065, USA
bDepartment of Endocrinology, Merck Research Laboratories, PO Box 2000, 800-B116 Rahway, NJ 07065, USA
cKaro Bio AB, Novum, S-14157 Huddinge, Sweden
dMerck, Sharp and Dohme Research Laboratories-CIBE, Madrid, Spain
Received 30 March 2006; accepted 12 May 2006
Available online 30 May 2006
Abstract—Synthesis of a series of fused pyrazole tetrahydrofluorenone analogs which are potent, ERb subtype selective ligands is
described. Analogs possessing subnanomolar ERb binding, greater than 100-fold ERb-selectivity, and oral bioavailability are
reported.
Ó 2006 Elsevier Ltd. All rights reserved.
The physiological effects of estrogen are mediated
through the estrogen receptor subtypes ERa1,2 and
ERb.3,4 While our understanding of the complex role
that these receptors play in the body is incomplete, it
is clear that decreasing levels of 17b-estradiol during
peri-menopause are associated with bone loss, cognitive
impairment, and hot flashes.5 Hormone replacement
therapy (HRT) provides relief from these symptoms
but does so with an increased risk of breast and uterine
cancer as well as an increase in the incidence of coronary
heart disease and stroke.6
effects of 17b-estradiol in tissues, such as the breast
and uterus, which predominately express ERa.
The identification of an ER subtype selective ligand is
further complicated by the structural similarity of
these receptors. In particular, the binding pockets of
ERa and ERb were observed to differ by only two
amino acid residues.4 In the ERb binding pocket,
Met336 was replaced by Leu384 and Ile373 was
replaced by Met421. In spite of the subtle changes
in receptor isoforms, selective ERa7,9,10 and ERb11–18
ligands have been described in the literature. We
recently reported a new class of potent, ERb selective
phenolic tetrahydrofluorenone analogs which are
exemplified by analog 1.19 In this paper, we will
describe modifications to 1 aimed at improving the
ERb binding, b-selectivity, and pharmacokinetic
properties (PK) of these compounds.
The observation that 17b-estradiol binds ERa and ERb
receptors with equal affinity, coupled with the differen-
tial distribution of these receptors throughout the
body,7,8 provides an intriguing possibility that tissues
can be targeted with receptor selective ligands. For
example, ERb is expressed in the lung, prostate, and
brain but is not the predominant receptor in the uterus
or breast tissues. Consequently, a b-selective ligand
could specifically target tissues which express ERb while
minimizing the risks associated with the proliferative
O
O
Br
Br
4
6
9a
HO
HN
8
Keywords: Estrogen; Selective; Biosostere; Tetrahydrofluorenone.
*
N
1
15
Corresponding author. Tel.: +1 732 594 5729; fax: +1 732 594
9556; e-mail: robert_wilkening@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.036

R. R. Wilkening et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3896–3901
3897
Analog 1 displayed poor oral bioavailability and high
O
H
plasma clearance rates in rats. The poor PK was, pre-
sumably, the result of first-pass glucuronidation of the
phenol group followed by rapid elimination from the
rat. Several approaches aimed at improving the PK of
these analogs were investigated. They included the syn-
thesis of a series of 6- and 8-substituted analogs in an at-
tempt to sterically minimize glucuronidation of the
phenol, the synthesis of prodrug derivatives of the phe-
nol, and replacement of the phenol by a fused pyrazole
ring.20 The results from the first approach, that substitu-
tion at the 8-position was found to be preferable to sub-
stitution at the 6-position, helped define regioisomer21
15 as the target for the third approach.22 The syntheses
of the pyrazole analogs 2–37 follow the procedures de-
scribed in Schemes 1–4.
O
c, d
O
O
R1
R1
N
N
H
41
42
H
Me
Me
a, b
R2
e
f
R2
O
O
O
R1
R1
H2N
N
H
43
Me
44
Me
g
f, g
R2
R2
O
O
Preparation of the requisite 2-substituted indanone 41 is
described in Scheme 1. Reductive alkylation of 38 pro-
vided the monoalkylated product 39 in high yield.23 Bro-
mination of 39 with NBS in dimethylformamide gave an
approximately 85:15 mixture of the 4- and 6-regioisom-
ers from which the 4-isomer 40 was obtained by crystal-
lization from acetonitrile. Conversion of 40 into the
4-methyl indanone 41 was accomplished under Stille
reaction conditions.
R1
R1
HN
N
HN
N
2-10
11-17
Scheme 2. Reagents and conditions: (a) EVK or PVK, NaOMe,
MeOH; (b) HOAc, 6 N HCl; (c) MVK, NaOMe, MeOH; (d)
pyrrolidine, HOAc, toluene; (e) NCS, DMF or Br2, NaHCO3, CH2Cl2;
(f) NaOMe, MeOH; (g) NOBF4, KOAc, dibenzo-18-crown-6, CH2Cl2.
Scheme 2 describes the Robinson annulation of 41 with
alkyl vinyl ketones and cyclization under acidic condi-
tions to provide the racemic tetrahydrofluorenone plat-
form. Michael reaction of 41 with methyl vinyl ketone
and cyclization of the addition product with pyrroli-
dine/acetic acid gave analog 42. The halogenation of
the enone function of 42 provided the chloro and bromo
derivatives 44. The addition product of 41 and ethyl
vinyl ketone or propyl vinyl ketone was cyclized in a
mixture of acetic acid/6 N HCl, with loss of the acetyl
group, to provide aniline 43.
O
O
Br
R2
a, f, g (18-20)
b, d, e, g (21, 22)
c, e, g (23)
O
R1
R1
N
HN
H
44
18-23
Me
N
Scheme 3. Reagents and conditions: (a) FSO2CF2CO2CH3, CuI,
DMF; (b) Me3Sn(vinyl), PdCl2(PPh3)2, toluene; (c) C6H5B(OH)2,
PdCl2(PPh3)2, (d) CH2N2, Et2O; (e) NaOMe, MeOH; (f) HCl, EtOH;
(g) NOBF4, KOAc, dibenzo-18-crown-6, CH2Cl2.
A one-pot nitrosation and concomitant cyclization24 of
anilines 43, and 44 after basic hydrolysis of the acetyl
group, with nitrosonium tetrafluoroborate, potassium
acetate, and dibenzo-18-crown-6 provided the racemic
pyrazole analogs 2–17. Chiral resolution25 of selected
racemic pyrazole products gave enantiomers A and B.
Derivatization of the bromo enone 44 is described in
Scheme 3. Introduction of a trifluoromethylgroup was
accomplished using copper(I) mediated chemistry.26
Phenyl and vinyl groups were introduced using Stille
chemistry. The vinyl group was then converted into a
cyclopropyl ring by reaction with excess diazomethane
in ether. Removal of the acetyl group was followed by
pyrazole ring formation and chiral resolution to afford
18–23.
O
O
O
O
O
O
O
O
a
c
Solvolysis of the trifluoromethyl group 45 under basic
conditions gave the esters 24 and 25 (see Scheme 4).
The reaction apparently proceeds via deprotonation of
the pyrazole nitrogen followed by conjugative elimina-
tion of fluoride from the trifluoromethyl group. Metha-
nol or water then adds to the vinyl fluoride intermediate
and the process is repeated until the ester is formed.
Interestingly, there is no evidence for the formation of
the acid product which would be expected from the
addition of two moles of water.
R1
N
H
N
H
38
41
39
b
R1
R1
N
H
N
H
40
Me
Br
Scheme 1. Reagents and conditions: (a) H2, RCHO, KOH or NaOH,
10% Pd/C, EtOH; (b) NBS, DMF; (c) SnMe4, PdCl2(PPh3)2, PPh3,
LiCl, DMF.
Halogenation of the pyrazolo tetrahydrofluorenone 45
was examined under a variety of reaction conditions

3898
R. R. Wilkening et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3896–3901
analogs.19 In both series, alkyl substitution of R1 with
Me
R2
O
O
ethyl, propyl, and butyl groups imparted the most favor-
able ERb binding and b-selectivity. The large difference
between the ERb binding values of the phenolic enanti-
omers was also seen in the pyrazole series, where the
inactive pyrazole enantiomer 6 exhibited less than 1%
of the ER binding of the active enantiomer 5.
Et
R1
HN
N
HN
N
31,32
R3
O
26-30
b
a
d
R2
R2
O
O
O
In general, an increase in the steric size of the R2 substi-
tuent produced an increase in ERb potency. When R1
was substituted with an ethyl group, the bromo 13
(2.2 nM), trifluoromethyl 19 (2.8 nM), and phenyl 23
(1.4 nM) analogs showed the most favorable ERb bind-
ing affinities, while the methyl 5 (190-fold), bromo 13
(113-fold), and trifluoromethyl 19 (130-fold) analogs
displayed the greatest ERb selectivity. When R1 was
substituted with a butyl group, the ethyl 10 (1.1 nM)
and trifluoromethyl 20 (0.8 nM) analogs possessed the
most potent ERb binding, while the trifluoromethyl 20
(220-fold) and methyl ester 25 (400-fold) analogs dis-
played the greatest ERb selectivity.
c
R1
R1
Cl Cl
46
HN
N
HN
N
45
e
R2
f
O
MeO2C
R1
R1
HN
N
HN
24,25
33-37
N
F
Scheme 4. Reagents and conditions: (a) NCS or Br2, NaOH, EtOH;
(b) (R2 = Cl or Br), K2S2O8, MeCN, H2O; (c) (R2 = CF3 or CH3),
NCS, CH2Cl2; (d) silica gel or HCl, EtOH; (e) (R2 = CF3), NaOH,
MeOH; (f) XeF2,MeCN.
In analogs where R1 was substituted with a cyclopropyl
group, the cyclopropylethyl analog 17 (1.6 nM) showed
the most potent and ERb selective binding affinity,
although, it was found to be somewhat less selective
than analog 15. Analogs 21 and 22, where R2 was substi-
tuted with a cyclopropyl group, also displayed slightly
reduced ERb potencies when compared with analogs 5
and 8.
(see Scheme 4). Chlorination and bromination of 45 un-
der basic conditions produced the anticipated pyrazole
ring halogenated products 31 and 32. However, when
the chlorination was performed in a non-polar solvent
and R2 was a trifluoromethyl group, the reaction pro-
duced the benzylic dichloro analog 46. This reaction
proceeded rapidly at room temperature without the
addition of a free radical initiator. The isolated dichloro
product 46 was found to slowly hydrolyze on silica gel
during purification or in dilute acid to give the ketones
29 and 30. Extension of this reaction to analogs 45 in
which R2 was a methyl, chloro or bromo group gave a
low yielding conversion to the dichloro intermediate,
in the case of the methyl analog, and a complex mixture
of products with the chloro and bromo derivatives.
Alternatively, oxidation of the chloro and bromo ana-
logs 45 with potassium persulfate in aqueous acetonitrile
provided the ketones 27 and 28.
Oxidation of the benzylic position to give ketones 26–30
had essentially no effect on ERb binding when com-
pared with the parent analogs 5, 11, 13, 19, and 20.
However, a small but significant decrease in ERb-selec-
tivity was observed for these compounds.
A comparison of the analogs, in which the pyrazole ring
was substituted with a halogen 31–37, showed that
decreasing steric size produced an increase in ERb
potency (F > Cl > Br). The fluorinated analogs 33–37
showed a 1–9-fold improvement over the parent analogs
in ERb binding and with the exception of 37, essentially
unchanged ERb selectivity. Analog 35 (0.3 nM) showed
an ERb potency which was 4-fold better than 17b-estra-
diol but with greatly improved ERb selectivity.
Few examples of pyrazole ring fluorination have been
described in the chemical literature and this target pre-
sented a synthetic challenge. After surveying a variety
of fluorinating reagents, it was found that reaction of
45 with xenon difluoride provided a low but useful yield
of analogs 33–37. This procedure produced a complex
mixture of products from which the desired product
and a small amount of a mono-fluorinated benzylic
product were isolated.
Compounds were also evaluated in a cell-based trans-
activation assay28 utilizing HEK 293 cells which were
stably cotransfected with human ERa or ERb and
the alkaline phosphatase reporter gene. The transcrip-
tional activity of the tetrahydrofluorenone analogs
was determined and compared as a percent response
of 17b-estradiol. These data are summarized in
Table 2.
The estrogen receptor binding affinities27 of racemic (A/B)
and resolved (A or B) tetrahydrofluorenone analogs
1–37 are summarized in Table 1. Comparison of the
ER binding SAR for the pyrazole and phenolic tetrahy-
drofluorenone analogs showed that, after correcting for
enantiomeric purity, the pyrazole analogs displayed
similar or slightly improved ERb binding affinity and
selectivity in comparison with the corresponding phenol
Overall, the transactivation data tracked reasonably well
with the ER binding data from Table 1. Several analogs
displayed subnanomolar ERb transactivation values
with the fluoropyrazole 35 exhibiting the greatest ERb
potency (0.2 nM). The potent and selective EC50 values
demonstrated that these analogs behaved as functional
ERb agonists.

R. R. Wilkening et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3896–3901
Table 1. Human estrogen receptor binding affinities
3899
R2
R2
O
O
9a
9a
R1
R1
HN
N
HN
N
R4 R5
R4 R5
R3
R3
A
B
Compound
R1
R2
R3
R4 = R5
9a stereochemistry
ERa IC50 (nM)
ERb IC50 (nM)
ERa/b
1.1
76
Estradiol
1
—
Bu
—
Br
—
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Br
F
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
H
H
H
H
H
H
H
—
1.3
141
1.2
1.8
618
283
69
A/B
A/B
A/B
A/B
A
2
Et
Bu
H
>10,000
>10,000
4410
1360
>10,000
976
>16
>35
64
3
H
Me
4
Me
Et
5
Me
Me
7.2
4460
12.6
12
190
>2
77
6
Et
Pr
B
7
Me
Me
A/B
A/B
A/B
A
8
Bu
Et
1050
223
85
56
9
Et
Et
4.0
1.1
7.7
26
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Bu
Et
93
470
83
61
Cl
Br
A
Me
Et
A/B
A
1990
249
77
Br
Br
2.2
5.4
4.9
4.0
1.6
8.0
2.8
0.8
7.7
7.0
1.4
207
5.2
31.8
4.1
8.0
1.5
0.9
42.5
103
1.1
1.6
0.3
0.3
1.0
113
123
288
58
Pr
Bu
A/B
A/B
A
663
Br
Br
1410
230
Cyclopropylmethyl
Cyclopropylethyl
Br
A
229
145
185
130
220
61
Me
Et
Bu
Et
Bu
Et
Et
Bu
Et
Et
Et
Et
Bu
Et
Et
Et
Et
Et
Bu
Bu
CF3
CF3
CF3
Cyclopropyl
Cyclopropyl
Phenyl
CO2Me
CO2Me
Me
A
1478
360
A
A
176
470
A
A/B
A
422
46
60
37
A/B
A
>10,000
2080
624
>48
400
20
A/B
A
Cl
Br
139
352
34
44
A/B
A
CF3
CF3
Me
58
40
39
43
A
A/B
A/B
A
2420
5880
82
57
57
Me
Cl
72
Br
F
A/B
A
197
124
170
87
CF3
Et
F
50.9
23.0
49.2
F
A
CF3
F
A
49
As was the case in the phenolic series,19 an increase
in the steric size of the R2 substituent produced a de-
crease in the ERb agonist response. Analogs that
showed a moderate percent agonist response were
then tested for their ability to antagonize estradiol’s
transcriptional effects. The cyclopropyl 21 and phenyl
23 analogs were observed to weakly antagonize the
effects of estradiol (526 and 428 nM, respectively)
and these analogs were identified as partial agonists/
antagonists.
with the inactive enantiomer 6 displaying a slight
advantage in AUC and plasma clearance rates over
the active enantiomer 5. In the halogenated analogs,
the trifluoromethyl analog 19 displayed improved PK
over the chloro 11 and bromo 13 analogs. A com-
parison of the trifluoromethyl analogs 19 and 20
showed ethyl substitution to be preferable to butyl
substitution.
In addition to minimizing glucuronidation using a fused
pyrazole group, an attempt was made to improve PK by
minimizing the potential for oxidative metabolism.
Potential metabolic sites on the tetrahydrofluorenone
were protected from oxidative metabolism with the
introduction of a fluoro, cyclopropyl, or keto group.
Unfortunately, the PK data for analogs containing a
A summary of rat pharmacokinetic (PK) data29 is
listed in Table 3. In contrast with phenolic analog
1, all pyrazole derivatives showed oral bioavailability
in the rat (21–74%). In the ethyl series, the pair of
resolved enantiomers showed relatively good PK

3900
R. R. Wilkening et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3896–3901
Table 2. Transactivation in ERa and ERb cotransfected HEK 293 cells
Example
ERa EC50 (nM)
% Estradiol agonism
ERb EC50 (nM)
% Estradiol agonism
2
>1000
104
1100
59
37
78
7
76
2.1
935
1.7
1.5
0.7
0.9
0.6
5.8
0.8
84
91
49
77
70
74
76
79
69
54
5
6
9
67
42
50
70
66
13
37
11
13
17
19
21
23
152
65
61
50
>1000
72
24
28
1170
49
33
65
210
1.5
61
85
29
34
35
10.4
33.1
8.3
90
71
75
0.4
0.8
0.2
86
81
67
Table 3. Pharmacokinetics in female Sprague–Dawley rats at an
intravenous dosing of 1 mg/kg and oral dosing of 2 mg/kg
the phenol tetrahydrofluorenones. Several analogs,
such as 19, 29, and 35, have shown a promising com-
bination of ERb potency, selectivity, and rat pharma-
cokinetic properties. Further modification of the
tetrahydrofluorenone class of ERb agonists will be
the subject of forthcoming publications from these
laboratories.
Example IV-AUC (lM h) Clp (mL/min/kg) T1/2 (h) F%
1
0.5
1.5
2.0
0.9
0.7
1.5
102
44
31
72
74
36
1.5
1.1
0.8
0.5
0.7
0.7
0
5
51
44
47
64
74
6
11
13
19
17
21
0.8
1.0
55
58
1.2
0.9
51
55
References and notes
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29
35
3.2
1.2
15
42
1.2
1.9
62
27
20
30
0.9
0.7
64
69
0.9
1.8
25
21
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cyclopropyl or a fluoro group were not improved
relative to related unsubstituted derivatives. For in-
stance, the rat PK of the cyclopropyl analogs 17 and
21 were not significantly different from that of the meth-
yl 5 or halogenated analogs 11 and 13. Likewise, the flu-
oro analog 35 displayed similar rat PK to that of the
parent analog 19.
In contrast, the ethyl substituted ketone 29 showed a
greater than 2-fold improvement in AUC and plasma
clearance over the parent compound 19. This analog dis-
played the best PK in this class of compounds and sug-
gested that the benzylic position was a site of oxidative
metabolism. The butyl substituted ketone 30, however,
showed a similar PK profile to the parent analog 20
and reinforced the conclusion that substitution of the
R1 group with an ethyl group was preferable to substitu-
tion with a butyl group.
In summary, we have shown that a fused pyrazole is
a suitable phenol group replacement in the previously
described tetrahydrofluorenone class of ERb agonists.
The pyrazole analogs were selective ERb agonists in
a
marked improvement in their PK properties over
cell-based transactivation assay and showed
a
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R. R. Wilkening et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3896–3901
3901
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of a series of fused triazole tetrahydrofluorenone analogs
will be the subject of forthcoming publications from these
laboratories.
23. All aldehydes in this step were commercially available with
the exception of cyclopropylmethylcarboxaldehyde which
was prepared from cyclopropylethanol using a TPAP/
NMO oxidation.
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and heptanes as the mobile phase. The stereochemistry of
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by analogy with the phenol tetrahydrofluorenones (ref.
19). This assignment was confirmed in three cases (5, 10,
and 19) by X-ray crystallography of ERb complexed with
the ligands (P. Fitzgerald unpublished results).
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27. The IC50 values were generated in an estrogen receptor
ligand binding assay. This scintillation proximity assay
was conducted in NEN Basic Flash plates using tritiated
estradiol and full-length recombinant human ERa and
ERb proteins with incubation times of 3–23 h. In our
experience, this assay provides IC50 values that are
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moieties have been reported. Wright, J.; Tracy, G.;
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21. The 6,7-fused regioisomer was prepared in a related
triazole series and was found to be significantly less
potent than the analogous 15 isomer. R. Wilkening,
unpublished results.
reproducible to within
a factor of 2–3. With the
exception of 17b-estradiol and analog 1, the majority of
analogs were tested once in the ER binding assay. The
analogs were tested in duplicate in the transactivation
assay (Table 2).
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29. Female Sprague–Dawley rats were dosed at 1 mpk IV and
2 mpk PO using a 1.0 mg/mL solution of compound
dissolved in EtOH/PEG400/H2O (2:3:5). Plasma samples
were mixed with acetonitrile, centrifuged, and analyzed by
LC–MS/MS on an Applied Biosystems MDS SCIEX API
4000 tandem mass spectrometer/HPLC system.