Synthesis and evaluation of 4-(substituted styryl/alkenyl)-3,5-bis(4- hydroxyphenyl)-isoxazoles as ligands for the estrogen receptor

Article abstract of DOI:10.1016/j.bmcl.2012.06.097

A series of 3,5-bis (4-hydroxyphenyl) isoxazoles bearing a styryl/alkyl vinyl group at the 4-position were prepared and evaluated as ligands for the estrogen receptor-alpha (ERα). The target compounds were prepared using the Suzuki reaction to couple an iodo-isoxazole intermediate with a series of styryl/alkenyl boronic acids, followed by O-demethylation. The products were evaluated for their estrogen receptor-α ligand binding domain (ERα-LBD) binding affinity using a competitive binding assay. The 4-(4-hydroxystyryl) derivative 4 h displays binding properties similar to those of the previously described pyrazole class of ER ligands, indicating that the ERα-LBD tolerates the presence of the added vinyl group at the 4-position of the isoxazole ring.

Full text of DOI:10.1016/j.bmcl.2012.06.097

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5999–6003
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 4-(substituted
styryl/alkenyl)-3,5-bis(4-hydroxyphenyl)-isoxazoles as ligands
for the estrogen receptor
Terra Haddad a, Rachel Gershman a, Robert Dilis a, David Labaree b, Richard B. Hochberg b,
Robert N. Hanson a,
⇑
a Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, United States
b Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3,5-bis (4-hydroxyphenyl) isoxazoles bearing a styryl/alkyl vinyl group at the 4-position were
Received 14 February 2012
Revised 25 June 2012
Accepted 27 June 2012
Available online 15 July 2012
prepared and evaluated as ligands for the estrogen receptor-alpha (ERa). The target compounds were
prepared using the Suzuki reaction to couple an iodo-isoxazole intermediate with a series of styryl/alke-
nyl boronic acids, followed by O-demethylation. The products were evaluated for their estrogen receptor-
a
ligand binding domain (ER
a-LBD) binding affinity using a competitive binding assay. The 4-(4-hydroxy-
styryl) derivative 4 h displays binding properties similar to those of the previously described pyrazole
Keywords:
Estrogen receptors
Isoxazoles
class of ER ligands, indicating that the ER
4-position of the isoxazole ring.
a
-LBD tolerates the presence of the added vinyl group at the
Ó 2012 Elsevier Ltd. All rights reserved.
Synthesis
Suzuki coupling reaction
Receptor binding
Conformations
Introduction
efficacy2–18 ( Fig. 1). Relative binding affinities (RBA) for these
derivatives ranged from 0.01% to 140% (compared to estradiol—
100%), depending both upon the nature of the heteroaryl group
and the R1/R2 substituents.
As part of our program to identify new structural motifs that
elicit the desired ER response, we also focused on the preparation
of new di- and tri-aryl heteroarenes. Although 3,5-diaryl isoxazoles
are electronically similar to the 3,5-diaryl pyrazoles, isoxazole
analogs had not been as extensively evaluated for estrogen
ligand development as the corresponding pyrazoles.10,18 We noted
that incorporation of a CH2–, C@O or CH2O-linker between the
The estrogen receptor displays the capacity to bind a wide vari-
ety of nonsteroidal ligands and to generate diverse biological re-
sponses as a consequence of that binding interaction.1 A number
of investigators have undertaken efforts to systematically prepare
and evaluate nonsteroidal ligand cores. The strategy, exemplified
by the work of Katzenellenbogen and others, typically involved
the preparation of a bis(4-hydroxyphenyl) heteroarene to mimic
the estradiol skeleton and the incorporation of additional func-
tional groups (R1/R2) that would enhance ER affinity and influence
OH
R1
Heteroaryl = pyrazole, imidazole, pyrrole, furan
thiophene, pyrimidine, pyridine,
Heteroaryl
scaffold
isoxazole, oxazoles, thiazoles
1 = substituted aryl,alkyl
R2
R
HO
R2 = alkyl
Figure 1. Nonsteroidal ER ligands based on bis(4-hydroxyphenyl) heteroarene scaffold and incorporating additional R1 and R2 substituents.
⇑
Corresponding author. Tel.: +1 617 373 3313; fax: +1 617 373 8795.
E-mail address: r.hanson@neu.edu (R.N. Hanson).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.097

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T. Haddad et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5999–6003
OH
OH
R = Substituted aryl
Substituted alkyl
R
R
N
N
O
O
HO
HO
Figure 2. Transformation of 4-substituted-3,5-bis(4-hydroxyphenyl) isoxazoles to corresponding 4-styryl/alkylvinyl-3,5-bis(4-hydroxyphenyl) isoxazoles.
isoxazole ring and the aryl group gave products with significant ER
affinity, however, none of the derivatives had employed a vinyl
group to link the isoxazole core with the terminal (substituted
phenyl) moiety. We hypothesized that an appropriate styryl/alkyl-
vinyl group would yield derivatives that retain significant affinity
satisfactory (77%), the yields for the subsequent Suzuki reactions
to give the 4-(substituted styryl)-3,5-bis(4-methoxyphenyl) isox-
azoles 3 were low (20–40%). Therefore we developed the second
approach, (Scheme 2) in which we coupled the 4-iodo intermediate
1 with a series of commercially available substituted styryl and
alkylvinyl boronic acids. In this case, the yields for the Suzuki reac-
tion were very good (56–98%). Subsequent O-demethylation of the
intermediates 3a–m, using boron tribromide or aluminum trichlo-
ride-ethyl mercaptan gave the initial series of target compounds
4a–m in good yields (53–98%).
Although the second approach was synthetically more success-
ful, it is limited by the availability of coupling partners, that is,
arylvinyl boronic acids are not as readily available as aryl halides.
As a basis for chemical library design, we demonstrated that other
arylvinyl boronic acids could be readily accessed for use in this
synthetic strategy. Conversion of aryl halides to the corresponding
aryl acetylenes using the Sonogashira reaction has been well de-
scribed. For our study, we used three such alkynes and converted
them to the corresponding aryl vinyl boronic acids21 and subse-
quently to the isoxazole derivatives (Scheme 3). The yields for
the catechol hydroboration, hydrolysis (43–61%) and coupling
steps for the representative compounds 5n–p22–24 were very satis-
factory (43–61%). Conversion to the isoxazole derivatives 3n–p
also proceeded in good yields (73–93%).
for the ERa-LBD (Fig. 2). In addition, the synthetic approach should
contain elements that would make it amenable to combinatorial
chemistry, thereby permitting rapid exploitation of the chemical
space. This paper describes the synthesis and receptor binding
results with this series of new 4-(styryl/alkenyl)-isoxazole
derivatives.
Results and discussion
Our synthetic objective involved the development of a direct,
reliable synthetic route to the preparation of the target com-
pounds. We examined two approaches, based upon a 4-iodo-3,5-
bis(4-methoxyphenyl)-isoxazole intermediate, either of which
could be expanded to combinatorial/parallel synthesis. In the first
approach (Scheme 1) we converted the 4-iodo intermediate 118 to
the 4-vinyl boronic acid derivative 2 using the Sonogashira
reaction,19,20 followed by catechol hydroboration and hydrolysis.
Subsequent Suzuki coupling with substituted aryl halides gave 4-
(substituted styryl)-3,5-bis(4-methoxyphenyl) isoxazoles 3. The
Sonogashira reaction, however, proceeded in low yield (ꢀ30%)
and was accompanied by side products that required careful
separation. Although the yield for the hydroboration step was
The initial series of 4-(substituted styryl/alkenyl)-3,5-bis(4-
hydroxyphenyl)isoxazoles 4a–4m were evaluated as ligands
for ERa
-LBD using a competitive binding assay.25,26 The relative
O
CH3
R
O
CH3
O
CH3
OH
B
HO
I
i,ii,iii
N
O
iv
N
N
O
O
H3C
H3C
H3C
O
O
O
2
3
1
(25% overall 3 steps)
23-41%
Scheme 1. Synthesis of 4-styryl-3,5-bis(4-methoxyphenyl) isoxazoles 3 via the 4-vinylboronic acid intermediate. Reagents and conditions: (i) Trimethylsilyl acetylene, TEA,
Cu(I)I, [P(C6H5)3]4Pd(0); (ii) TBAF, THF; (iii) catechol borane, reflux; (iv) aryl iodide, Pd(P(C6H5)3)2Cl2, DME, 90 °C,16 h.
O
CH3
OH
O
CH3
i
R
R
I
ii/iii
H3C
N
N
N
O
O
O
O
H3C
O
HO
4a-m
50-98%
1
3a-m
55-98%
Scheme 2. Synthesis of 4-styryl-3,5-bis(4-hydroxyphenyl) isoxazoles 4a–4m via the 4-iodo intermediate and commercially available styryl/alkylvinyl boronic acids.
Reagents and conditions: (i) Ar/R–CH@CH–B(OH)2—1.5 equiv, Pd(P(C6H5)3)2Cl2, NaHCO3, H2O–DME (ii) BBR3, CH2Cl2 (iii) AlCl3 (5.8 equiv), C2H5SH (4.9 equiv), CH2Cl2.

T. Haddad et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5999–6003
6001
O
CH3
R
HO
iii
i,ii
B OH
R
H
N
O
R
H3C
O
5 n-p
3n-p
F
R =
n
o
p
Scheme 3. Synthesis of novel arylvinyl boronic acids 5n–p and conversion to 4-(substituted styryl)-3,5-bis(4-methoxyphenyl) isoxazole derivatives 3n–p. Reagents and
conditions: (i) Catechol borane; (ii) H2O; (iii) 1, Pd(P(C6H5)3)2Cl2, NaHCO3, H2O–DME.
Table 1
patent18 reported a relative binding affinity (RBA) of 30% for
3,4,5-tris(4-hydroxyphenyl) isoxazole. In our case, introduction of
the vinyl group between the isoxazole ring and the aryl group at
Relative binding affinities (RBA) of isoxazole derivatives for the estrogen receptor-
ligand binding domain (ER -LBD)
a
a
the 4-position appears to be well tolerated in terms of ERa-LBD
OH
binding. The binding of the new compounds also compares favor-
ably with the other heterocyclic analogs. Most of the tris(4-
hydroxyphenyl) substituted pyrazoles reported by Katzenellenbo-
gen also have RBA values in the 5–25% range.3,5,8,10,11 Figure 3 pre-
sents the RBA value for 4h compared to two of the more potent
isoxazole and pyrazole compounds.
R
N
O
HO
The RBA values for the 4- and 3-hydroxyphenyl derivatives 4h
and 4i are also comparable to the highest values observed for the
tri- and tetrasubstituted pyrrole ER ligands recently reported by
Gust.14,15 It should be noted that pyrazoles and furans which have
higher reported RBA values than 4h may achieve the enhanced
affinity through the additional fourth substituent. This is not pos-
sible with the isoxazole scaffold. By comparison, the substituted
isoxazoles described in this study generally were better than the
previously reported imidazoles, oxazoles and thiazole which had
very low RBA (<1%) values for ER.10
4a-m
Compound
R
RBA S.D
4a
4b
4c
4d
4e
4f
4g
4h
4i
E-C6H5
Z-C6H5
3.5 1.0%
8.7 1.2
0.6 0.2%
3.1 0.7%
0.8 0.2%
5.3 1.2
1.5 0.7
16.4 3.8
8.7 1.5
1.8 1.2
2.5 0.7
1.3 0.6
4.3 3.2
4-CH3–C6H5
4-F-C6H5
4-Cl-C6H5
4-CF3–C6H5
3-CF3–C6H5
4-HO–C6H5
3-HO–C6H5
n-C7H15
Based prior studies of the interaction between nonsteroidal li-
gands and ER
gands that provides
a
-LBD, we proposed a binding mode for the new li-
rationale for the structure–activity
4j
4k
4l
n-C8H17
n-C9H19
t-C4H9
a
relationship. We illustrated this mode using compound 4d whose
RBA value is less than that of the highest affinity agent 4h but
somewhat more than the less active alkyl vinyl derivatives.27 As
with most nonsteroidal ligands of this type, the 3,5-bis(4-hydroxy-
phenyl)-isoxazole scaffold mimics the internal steroidal core of
estradiol. The vinyl moiety at the 4-position experiences torsional
strain due to the neighboring (4-fluorophenyl) groups and adopts a
non-coplanar conformation that will reduce the steric strain. The
more distal vinyl-aryl bond can also undergo rotation to achieve
4m
RBA = 100 Â [E]/[C] where [E] is the concentration of unlabeled estradiol necessary
to reduce the specific binding of [3H]-estradiol to the ER
-LBD by 50% and [C] is the
concentration of the competitive ligand necessary to reduce specific binding by 50%.
The RBA of estradiol is 100% at 25 °C. Curves for ligand and estradiol had correlation
coefficients >95%.
a
binding affinity (RBA) values for the new compounds are shown in
Table 1, where estradiol has a value of 100%. All of the compounds
demonstrated significant affinity for the ER
a
-LBD, with values
the most effective ligand–ERa-LBD contacts and further lower
ranging from <1% to 16% that of estradiol. The highest affinity
was observed for the 4-hydroxystyryl derivative 4h (16.4%), with
the 3-hydroxy isomer 4i (8.7%) slightly lower. Substitution of the
ring at the 4-position with alkyl or halogenated groups (4c–g) re-
duced binding affinity (RBA <1–5.3%) and as did replacement of
the aromatic ring by alkyl groups (4j–m, RBA = 1.3–4.3%). The Z-
the energy of the complex. Our study (Fig. 4) suggests that the ter-
minal 4-fluoro-group interacts with residues Leu-540(helix-12)
and Trp-383(helix5), as well as Thr-347 (helix-3), which is not
see in this depiction. Hydrogen bonding by either the 4-hydroxyl
(4h) or 3-hydroxyl (4i) group provides additional stabilization
within this pocket. Hydrophobic substituents such as choro- (4e),
methyl (4c) or trifluoromethyl (4f,4g) are tolerated but not fa-
vored. The alkyl vinl derivatives 4j–4m are also accommodated
within this hydrophobic pocket, but provide no additional interac-
tions that would strengthen binding. Other modeling studies have
identified Thr-347 as the residue in the 11ß-pocket most likely to
establish hydrogen bonding with the distal phenolic hydroxyl.4,5
This would appear to be the case as the two ligands with the high-
est RBA values were the 4- and 3-hydroxystyryl isoxazoles 4h
and 4i. Substitution with non-hydrogen bonding or hydrophobic
styryl isomer 4b appeared to be a slightly better ligand for ERa-
LBD than the E-isomer 4a (8.7% vs 4%). Because substitution on
the phenyl ring with groups other than hydroxyl appeared to re-
duce the binding affinity, we did not convert isoxazole derivatives
3n–3p to the corresponding bis(4-hydroxyphenyl) analogs 4n–4p
for evaluation.
The binding properties of this series of tri-substituted isoxaz-
oles compares favorably with previous studies on tris(4-hydroxy-
phenyl) five-membered heterocycles. For example, the Chiron

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T. Haddad et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5999–6003
OH
OH
OH
HO
HO
N
N
N
O
O
N
OH
HO
HO
RBA = 30% (ref 18)
HO
4h
RBA = 16%
RBA = 36% (ref 5)
Figure 3. Comparison of compound 4h with similar tris(4-hydroxyphenyl) heteroarenes.
Figure 4. Docking study with isoxazole 4d and ER
a-LBD showing interaction between 4-(4-fluorophenyl)-group and Leu-540 and Trp-383 residues.
groups reduces the binding interaction, but conformation mobility
may result in alternative binding modes that generate stable com-
plexes. One interesting observation in this series was the difference
between the 4-E- and 4-Z-styryl isoxazoles where the Z-isomer 4b
had a slightly higher affinity than the E-isomer 4a (8.7% vs 4%). It is
possible that the terminal phenyl group of the Z-derivative can ac-
cess a slightly different and/or more favorable binding pocket than
the E-counterpart.
adopt conformations that can access complementary residues,
such as Thr-347, Trp-383, and Leu-540 in the 11ß-binding pocket.
The results provide the basis for future studies that evaluate the
relationship between the 4-substituent and estrogenic potency.
Studies in which the 4-styryl group is substituted with dialkylami-
noethoxy groups are in progress as well.
Acknowledgments
Conclusions
The authors acknowledge support for this research through
Grants from the National Institutes of Health [PHS 1RO1
CA81049 (R.N.H.), PHS 1R01 CA37799 and R21MH082252
(R.B.H.)], and the U.S. Army Breast Cancer Research Program
[DAMD 17-99-1- 9333 and 17-00-1-00384 (R.N.H.)]. The substi-
tuted vinyl boronic acids were provided by Frontier Scientific for
which the authors are particularly grateful.
In this study we developed an approach to the synthesis of 4-
(substituted styryl)-3,5-bis(4-hydroxyphenyl) isoxazoles that is
efficient and can be extended to combinatorial expansion.
Although limited by the availability of the substituted styryl boro-
nic acids, such reagents can be prepared from commercially avail-
able starting materials. The resultant products demonstrate
significant binding affinity for the ER
a-LBD that is comparable to
Supplementary data
previously described bis- and tris-(4-hydroxyphenyl) isoxazole,
pyrazole and furan derivatives. The presence of the vinyl group
at the 4-position is well tolerated and may permit the distal 3-/
4-hydroxyphenyl moiety, present in compounds 4h and 4i, to
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
06.097.

T. Haddad et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5999–6003
15. Schaefer, A.; Wellner, A.; Gust, R. ChemMedChem 2011, 6, 794.
6003
References and notes
16. Zimmermann, J.; Liebl, R.; von Angerer, E. Steroid Biochem. Molec. Biol. 2005, 94,
57.
1. Gao, H.; Katzenellenbogen, J. A.; Garg, R.; Hansch, C. Chem. Rev. 1999, 99, 723.
2. Ghosh, U.; Ganessunker, D.; Sattigeri, V. J.; Carlson, K. E.; Mortensen, D. S.;
Katzenellenbogen, B. S.; Katzenellenbogen, J. A. Bioorg. Med. Chem. Lett. 2003,
11, 629.
3. Mortensen, D. S.; Rodriguez, A. L.; Carlson, K. E.; Sun, J.; Katzenellenbogen, B. S.;
Katzenellenbogen, J. A. J. Med. Chem. 2001, 44, 3838.
4. Mortensen, D. S.; Rodriguez, A. L.; Sun, J.; Katzenellenbogen, B. S.;
Katzenellenbogen, J. A. Bioorg. Med. Chem. Lett. 2001, 11, 2521.
5. Stauffer, S. R.; Coletta, C. J.; Tedesco, R.; Nishiguchi, G.; Carlson, K. E.; Sun, J.;
Katzenellenbogen, B. S.; Katzenellenbogen, J. A. J. Med. Chem. 2000, 43, 4934.
6. Nishiguchi, G. A.; Rodriguez, A. L.; Katzenellenbogen, J. A. Bioorg. Med. Chem.
Lett. 2002, 12, 947.
7. Huang, Y. R.; Katzenellenbogen, J. A. Org. Lett. 2000, 2, 2833.
8. Stauffer, S. R.; Katzenellenbogen, J. Comb. Chem. 2000, 2, 318.
9. Katzenellenbogen, J. A.; Katzenellenbogen, B. S.; Fink, B. E.; Stauffer, S. R.;
Mortensen, D. S.; Sattigeri, V. J.; Huang, Y. WO 2000019994 A1, 2000.
10. Fink, B. E.; Mortensen, D. E.; Stauffer, S. R.; Aron, Z. D.; Katzenellenbogen, J. A.
Chem. Biol. 1999, 6, 205.
17. Zimmermann, J.; Von Angerer, E. J. Steroid Biochem. Mol. Biol. 2007, 96, 259.
18. Huebner, V. D.; Lin, X.; James, I.; Chen, L.; Desai, M.; Moore, J. C.; Krywult, B.;
Navaratnam, T.; Singh, R.; Trainor, R.; Wang, L. WO 0008001 A1, 200002017.
19. Schnurch, M.; Flasik, R.; Khan, A. F.; Spina, M.; Mihovilovic, M. D.; Stanetty, P.
Eur. J. Org. Chem. 2006, 3283.
20. Chinchilla, R.; Najera, C. Chem. Soc. Rev. 2011, 40, 5084.
21. Churches, Q. I.; Stewart, H. E.; Cohen, S. B.; Shroder, A.; Turner, P.; Hutton, C. A.
Pure Appl. Chem. 2008, 80, 687.
22. Minkkila, A.; Saario, S. M.; Kasnanen, H.; Leppanen, J.; Poso, A.; Nevalainen, T. J.
Med. Chem. 2008, 51, 7057.
23. Peng, C.; Zhang, W.; Yan, G.; Wang, J. Org. Lett. 2009, 11, 1667.
24. Angibaud, P.; Van Emelen, K.; Decrane, L.; van Brandt, S.; ten Holte, P.; Pilatte,
I.; Roux, B.; Poncelet, V.; Speybrouck, D.; Queguiner, L.; Gaurrand, S.; Marien,
A.; Floren, W.; Janssen, L.; Verdonck, M.; van Dun, J.; van Gompel, J.; Gilissen,
R.; Mackie, C.; Du Jardin, M.; Peeters, J.; Noppe, M.; Van Hijfte, L.; Freyne, E.;
Page, M.; Janicot, M.; Arts, J. Bioorg. Med. Chem. Lett. 2010, 20, 294.
25. Labaree, D. C.; Shang, J.; Harris, H. A.; O’Connor, C.; Reynolds, T. Y.; Hochberg, R.
B. J. Med. Chem. 1886, 2003, 46.
11. Stauffer, S. R.; Huang, Y.; Coletta, C. J.; Tedesco, R.; Katzenellenbogen, J. A.
Bioorg. Med. Chem. 2001, 9, 141.
12. Gust, R.; Keilitz, R.; Schmidt, K.; von Rauch, M. Arch. Pharm. 2002, 335, 463.
13. Gust, R.; Busch, S.; Keilitz, R.; Schmidt, K.; von Rauch, M. Arch. Pharm. 2003,
336, 456.
26. Green, S.; Walter, P.; Kumar, V.; Krust, A.; Bonert, J. M.; Argos, P.; Chambon, P.
Nature 1986, 320, 134.
27. Insight II, 97.2/ Discover 2.9.7 (MSI Inc. San Diego) 1997.
14. Schaefer, A.; Wellner, A.; Strauss, M.; Wolber, G.; Gust, R. ChemMedChem 2011,
6, 2055.