Biphenyl C-cyclopropylalkylamides: New scaffolds for targeting estrogen receptor β

Article abstract of DOI:10.1016/j.bmcl.2009.03.075

The C-cyclopropylalkylamide scaffold was previously identified as a new structural framework for antiestrogens. A second generation library provided three compounds that bind estrogen receptor (ER)α. Further screening of this library identified an ERβ hit

Full text of DOI:10.1016/j.bmcl.2009.03.075

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2404–2408
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biphenyl C-cyclopropylalkylamides: New scaffolds for targeting estrogen
receptor b
Miranda J. Sarachine a, Jelena M. Janjic b, Peter Wipf b,c, Billy W. Day b,c,
*
a Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA
b Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261, USA
c Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The C-cyclopropylalkylamide scaffold was previously identified as a new structural framework for anti-
Received 19 January 2009
Revised 14 March 2009
Accepted 20 March 2009
Available online 25 March 2009
estrogens. A second generation library provided three compounds that bind estrogen receptor (ER)a. Fur-
ther screening of this library identified an ERb hit and inspired another round of SAR. A new focused
library was tested for binding to the ERs, and for effects on the growth of breast cancer cell lines and pro-
tein levels of common cell cycle regulators.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Estrogen receptor
Chemical synthesis
Receptor binding
Antiproliferative
The estrogen receptors (ERs) are ligand activated transcription
factors. ER
and ERb demonstrate ꢀ56% sequence homology in
their ligand binding domains, but their ligand-binding pockets dif-
fer by only two amino acids: ER
Leu384 versus ERb Met336, and
ER
Met421 versus ERb ILe373 1 ERb’s pocket is also about 100 Å3
smaller than that of ERa 2
Additionally, the tissue distributions of
Previously, through screening of a library of homoallylic
a
amides, allylic amides and C-cyclopropylalkylamides, we identified
a new structural scaffold for antiestrogens.16 The synthesis of a
second generation library provided three additional agents that
a
a
.
were found to bind tightly to ERa 17
. In the present analysis, we
.
screened this second generation library with a fluorescent E2
derivative (ES2) for competitive binding to ERb in a fluorescence
polarization (FP) assay.18 The percent displacement of the E2 deriv-
ative by the test compound was calculated from the difference in
measured FP values upon incubation with and without the test
sample. The efficiency of displacement demonstrated for ERb by
the initial screening library was significantly lower than that for
these receptors are distinct.3 There is much data demonstrating
the benefits of selectively targeting the ERb. ERb expression inhib-
its estradiol (E2)-induced proliferation in T47D and MCF-7 breast
cancer cells and leads to a reduction in xenograft volume.4–6 ERb
has a growth inhibitory mechanism in medullary thyroid carci-
noma, prostate cancer, and ovarian cancer.7–10 Also, ERb-selective
agonists demonstrate potential as anti-inflammatory agents.11
Several ERb-selective agents have been identified (Fig. 1). The
plant-derived ligand genistein (GEN (1)) has also been shown to in-
duce DNA strand breaks and cause chromosomal aberrations.3,12
The Katzenellenbogen group developed
a diarylpropionitrile
(DPN, 2) that is a 100-fold selective ERb agonist.13 Recently, biphe-
nyl derivatives have been investigated for ERb-selectivity. Simple
4-OH-biphenyls demonstrated 20–70-fold selectivity for ERb over
ERa 14
In order to attempt to mimic the C-ring of GEN, an oxime
.
moiety was incorporated, and altering the substitutions on the
biphenyl core provided various levels of selectivity: oximes 3 and
4 were 11-fold and 43-fold selective, respectively (Fig. 1).15
* Corresponding author. Tel.: +1 412 648 9706; fax: +1 412 383 5298.
E-mail address: bday@pitt.edu (B.W. Day).
Figure 1. Structures of several known ERb-selective ligands: GEN (1), DPN (2), and
biphenyl carbaldehyde oximes 3 and 4.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.075

M. J. Sarachine et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2404–2408
2405
chosen based upon published SAR for biphenyl-containing
agents.15 The phenylamide was changed into a smaller acetyla-
mide, or completely removed. Six new compounds, analogues of
5, were synthesized, 10a, 11b–d, 12, and 13 (Scheme 1). The palla-
dium catalyst trans-(Cy2NH)2Pd(OAc)2 (DAPCy) was used to per-
form Suzuki couplings in dioxane because it could be used at
room temperature, thereby suggesting functional group tolerance,
and due to its past use in similar couplings.19,20 This catalyst was
easily prepared in yellow crystalline form from palladium(II)ace-
tate and dicyclohexylamine.19 The cross-coupling of commercially
available benzyl- or methyl-O-protected 4-hydroxybenzeneboron-
ic acid to 4-bromobenzaldehyde using the DAPCy catalyst provided
biphenyl aldehydes 6a–c in good to excellent yields (65–88%).
Imine formation using titanium(IV) chloride as the Lewis acid ini-
tially gave phosphinoylimine 8a in poor to moderate yields (30–
47%).17 Changing the workup procedure to a fast filtration over a
pad of silica gel and washing with ethyl acetate significantly im-
proved the yields (80%) of the phosphinoylimines 8b and c. One-
pot hydrozirconation-transmetallation-aldimine addition in
Figure 2. Lead compound 5, and subunits A, B and C used in the SAR.
ER
a
. In fact, only a single compound (5; Fig. 2) demonstrated a
concentration-dependent displacement over the concentration
range tested and close to 50% displacement at the highest concen-
tration. The displacement values were: 0.2
lM, 26%; 1 lM, 31%;
and 5 M, 46%. Compound 5 was chosen as the lead structure for
l
efforts to improve ERb-selectivity. Because 5 did not quite reach
50% displacement on ERb, the selection criteria were modified to
include structural features previously known to promote ERb affin-
ity.15 Screening results on ER
and 5 was not a hit on ERa 17
a were used as co-selection criteria,
.
Figure 2 demonstrates the three regions in 5 that were further
evaluated in the next round of our SAR. The biphenyl core A was
shown to be beneficial for selective targeting of ERb, in analogy
to Yang et al.15 who used GEN as a lead structure to search for
new ERb-selective ligands. The biphenyl moiety was synthetically
readily accessible through diaryl Suzuki cross-couplings, and a
large number of substituted phenyl precursors were commercially
available. Biphenyl carbaldehyde oxime derivatives were synthe-
sized with various substituents. Upon placement of a fluorine
and/or chlorine substituent on the biphenyl core while maintain-
ing the phenol group, agents selective for ERb were obtained that
demonstrated greater than 10-fold (3) and up to 40-fold (4) selec-
tivity.15 Among the second generation library compounds, only one
compound containing the biphenyl system satisfied the criteria for
hit selection in the screen on ERa; however, this compound failed
to inhibit estradiol-stimulated growth of MCF-7 ER positive breast
cancer cells.17 Replacement of the biphenyl group with a smaller
arene, that is, p-chlorobenzene, preserved the displacement activ-
ity on ER
effect indicates that the sterically more demanding biphenyl core
is less tolerated by ER if combined with larger substituents on
a and recovered the cell-based activity. This substituent
a
the nitrogen atom in the benzylic position. When the biphenyl
group in 5 was replaced with a phenyl ring while keeping the
remaining structure constant, the activity at ERb dropped to
ꢀ10% and activity at ER
a was retained. Alterations to region B in
5 also led to detrimental consequences in the ability of the com-
pound to displace ES2 from ERb. Compound 5 demonstrated that
the best-tolerated function in sector B was a phenylamide group,
while other rings were better tolerated for ERa. When the phenyla-
mide was replaced with a biphenylamide, activity at ERb was abol-
ished. Therefore, we concluded that only one biphenyl core was
necessary and that increasing the size of the ligand would be det-
rimental to activity on ERb. As far as the alkyl chain in region C was
concerned, an increase in its size to a cyclohexyl group while keep-
ing the remainder of the molecule constant abolished activity at
both receptors. Therefore, region C was not altered in subsequent
synthetic efforts. Previous results demonstrated that the cyclopro-
pane ring was important for cellular activity.16 When compounds
containing the biphenyl core yet lacking the cyclopropane ring
were tested in the FP assay, activity at both receptors was lost. Re-
gions A and B were chosen for further elaboration and the struc-
tures were assembled with the biphenyl core, the cyclopropane
ring and the amide linkage preserved. The biphenyl group was
substituted with a key 40-OH group expected to promote hydrogen
bonding with ERb Glu305 and ERb Arg346. A fluorine substituent was
Scheme 1. Overview of the synthesis of six new biphenyl substituted C-cyclo-
propylalkylamides. Reagents and conditions: (a) DAPCy (2 mol %), Cs2CO3, dioxane,
rt; (b) Ph2P(O)NH2, TiCl4, Et3N, rt; (c) (i) Cp2Zr(H)Cl, 1-hexyne, CH2Cl2, (ii) Me2Zn,
toluene, ꢁ78 to 0 °C, (iii) mW 100 °C, 300 W, 10 min, (iv) CH2I2, rt; (d) (1) HCl,
MeOH, (2) R3COCl, DMAP, iPr2EtN, rt; (e) H2, Pd(OH)2/C, THF, MeOH, rt; (f) H2,
Pd(OH)2/C, THF, MeOH, rt; (g) HCl, MeOH. Percent yields: 6a, 70; 6b, 88; 6c, 50; 8a,
47; 8b, 67; 8c, 40; 9a, 30; 9b, 68; 9c, 40; 10a, 61; 10b, 57; 10c, 22; 10d, 75; 11b, 69;
11c, 92; 11d, 94; 12, 80; 13, 43. R1 = Me, R2 = F (6a, 8a, 9a); R1 = Bn, R2 = F (6b, 8b,
9b); R1 = Bn, R2 = H (6c, 8c, 9c); R1 = Me, R2 = F, R3 = Ph (10a); R1 = H, R2 = F, R3 = Ph
(10b, 11b); R1 = H, R2 = H, R3 = Ph (10c, 11c); R1 = H, R2 = F, R3 = Me (10d, 11d); R2 = F
(12, 13).

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M. J. Sarachine et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2404–2408
dichloromethane using a microwave (100 °C, 300 W, 10 min)
immediately followed by cyclopropanation in the presence of ex-
cess diiodomethane at room temperature (16–20 h) gave the phos-
phinoyl-protected biphenyl C-cyclopropylalkylamides 9a–c in
moderate overall yields (43–66%). Acidic deprotection followed
by acylation gave the final product 10a (61%) or benzyl-protected
compounds 10b–d (22–75%). Benzyl ethers were easily removed
by hydrogenolysis at 1 atm in the presence of palladium hydroxide
on carbon to give the final products 11b–d and 12. Deprotections
and acylations were performed in parallel using a Bradley Green-
house reactor, and final compounds were purified individually by
column chromatography. The deacetylated primary amine 13
was purified as its HCl salt.
This synthetic scheme allowed for diversification in two ways.
Substituents on the biphenyl core were varied by changing the
substitution on the arylboronic acid and aryl bromide coupling
components. The amide linkage also allowed for diversification
by altering the acylating agents, analogously to the synthesis of
the second generation library. After the acidic deprotection step,
the amine salt could be coupled without further purification with
diverse acyl chlorides, carbamoyl chlorides, and sulfonyl chlorides
in the presence of DMAP.17
3-Fluoro-40-hydroxy-1,10-biphenyl-4-carbaldehyde oxime
3
served as a positive control. The past synthesis of 3 was modified
as shown in Scheme 2.15 Suzuki cross-coupling using DAPCy gave
the desired biphenyl aldehyde 6a in 70% yield. The aryl methyl
ether was then removed with BBr3 at low temperature, and, after
simple basic aqueous work up, the resulting deprotected aldehyde
was used without further purification. Biphenyl carbaldehyde
oxime 3 was formed in two steps by treating the aldehyde 7 with
hydroxyloxime hydrochloride in methanol at room temperature.
The six new compounds 10a, 11b–d, 12 and 13 and the control
compound, oxime 3, were first evaluated for their ability to com-
pete with ES2 for binding to both ERs using the FP assay as de-
scribed (Fig. 3).18
Only two compounds, 11b and 13 (Fig. 4), significantly com-
peted with ES2 as shown in Figure 3. The curves were constructed
from one-site competition best-fit curves of polarization (mP) ver-
sus concentration using GraphPad Prism 4 software. The IC50 val-
ues determined from this fit are listed in Table 1. Biphenyl
carbaldehyde oxime 3 and E2 were used as positive controls, the
former giving an IC50 comparable to the previously determined va-
lue in a radioligand binding assay.15
Figure 3. Competition FP assay for 11b and 13 with ES2 for binding to ER
E2 and 3 were used as controls.
a
or ERb.
O
NH2•HCl
HN
F
Ph
F
11b
13
HO
HO
Figure 4. Lead compounds 11b and 13.
Table 1
IC50 values for competition at both ERs from the FP assay
a‘
a
Compounds
ERa
IC50 (nM)
ERb IC50 (nM)
The compounds were next evaluated for their ability to inhibit
E2-induced proliferation of MCF-7 human breast cancer cells and
E2
3
0.627
795
0.316
32.3
11b
13
>100,000
na
2048
15,030
a
Values are means of three experiments, (na = not active).
demonstrate their potential as anti-estrogens. This was done using
two variants of the MCF-7 cell line. The later passage ATCC variant
(MCF7) that expresses only ER
a, and an earlier passage (MCF-7)
that expresses both ER and ERb. The western blot demonstrating
a
this expression is in the Supplementary data. Figure 5 shows the
growth inhibition curves generated for compounds determined
to be selective for ERb using the MTS assay.21 Raloxifene (RAL),
an antiestrogen known to inhibit the proliferation of MCF-7 cells,
was included as a control.22 GraphPad Prism 4 was used to esti-
mate GI50 values using a non-linear best curve fit. In the ER
ERb expressing MCF-7 cells, compounds 11b and 13 gave GI50 val-
ues of 4.1 and 1.2 M, respectively.
In the ER expressing ATCC MCF7 cells, compound 11b did not
demonstrate inhibition, while compound 13 gave a GI50 value of
51.7 M. In these cells, all 3 compounds tested were toxic at a con-
a and
l
a
Scheme 2. Synthesis of positive control agent, 3-fluoro-40-hydroxy-1,10-biphenyl-
4-carbaldehyde oxime 3. Reagents and conditions: (a) DAPCy (2 mol %), Cs2CO3,
dioxane, rt, 70%; (b) BBr3; (c) NH2OHꢂHCl, 42%.
l

M. J. Sarachine et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2404–2408
2407
Table 2
Relative fold changes of protein levels induced by the indicated treatment in MCF-7
cells compared to DMSO vehicle treatment
10 nM E2
1
lM PPT
1
lM DPN
1
l
M 11b
1 lM 13
cMyca
p21a
3.84
1.15
1.76
2.17
4.05
2.47
2.67
3.47
3.32
0.77
3.64
3.90
0.59
1.10
1.51
p27a
a
ImageJ was used to determine band intensity, and levels were standardized to
actin.
the protein levels of c-Myc, p21, and p27. Western blot analyses
(Fig. 6) from lysates of MCF-7 cells treated with the indicated com-
pounds and densitometric analyses (Table 2) show compounds 11b
and 13 hold promise because of their ability to increase the levels
of cyclin-dependent kinase inhibitors and decrease the levels of c-
myc, likely inhibiting cell cycle progression and proliferation. PPT,
a 410-fold ERa-selective agonist, and DPN, a 70-fold ERb-selective
agonist, were included as controls.27,13
In conclusion, compounds 11b and 13 demonstrate selectivity
for ERb as well as promising antiproliferative effects in breast can-
cer cells. The compounds are likely acting as agonists for ERb based
on what has been shown about ERb’s activity in breast cancer,4–6
and this encourages future investigation. Compared to the lead
compounds 3 and 5, 11b and 13 show no affinity for ERa and mod-
erate affinity for ERb. This initial investigation into the biphenyl C-
cyclopropylalkylamide pharmacophore demonstrates the potential
of this scaffold to deliver ER-selective antiestrogens. Furthermore,
the preliminary SAR provides clues for improving selectivity for
ERb. A subtype-selective agent will help to understand the com-
plex biology of the ERs and their interplay. The arrival of new ER
ligands with differential subtype selectivity ratios enables the tai-
loring of antiestrogenic or estrogenic therapy according to the con-
dition and level of receptor isoforms present in patients.
Figure 5. Inhibition of E2-stimulated growth of ATCC MCF7 (top) or MCF-7 (bottom)
cells by biphenyl C-cyclopropylalkylamides 11b and 13. RAL was used as a positive
control. Data represents % growth inhibition (mean SD, N = 3).
centration of 25 lM. Additionally, the growth inhibition of ER-neg-
ative MDA-MB231 cells was also assayed.23,24 11b, 13, and RAL did
not demonstrate significant growth inhibitory activity against this
cell line but the compounds again demonstrated toxicity at 25 lM.
This can also be seen in the Supplementary data. Therefore, this
provides support that the compounds are exerting their action
through ERb, because in breast cancer cells expressing both ERb
Acknowledegments
and ER
demonstrated compared to breast cancer cells expressing only
ER or no ERs (Fig. 5).
c-Myc is known to play a major role in cell proliferation and
a a significantly more potent anti-proliferative effect was
Supported by grants from the Department of Defense
(W81XWH-04-1-0413 and W81XWH-08-1-0290) and the National
Institutes of Health (GM067082).
a
malignant transformation in breast cancer.25 E2 has been shown
to cause a rapid increase in the level of c-Myc expressed by human
breast cancer cells.26 Induction of the expression of ERb inhibits c-
Myc at both the mRNA and protein level, while increasing the lev-
els of its regulators, the cyclin-dependent kinase inhibitors, p21
(Cip1) and p27(Kip1).5 Because of these known links to breast can-
cer and E2, we chose to investigate how our compounds affected
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.03.075.
References and notes
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Figure 6. Western blot analysis of the levels of c-Myc, p21, p27, and actin in lysates
of MCF-7 cells treated with 10 nM E2, 1 lM PPT, 1 lM 11b and 1 lM 13 for 12 h.

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M. J. Sarachine et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2404–2408
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(concentrations ranging from 40 nM to 25 lM) along with 1 nM E2, and cells
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490 nm using an M5 plate reader (Perkin Elmer, Boston, MA) and the
background absorbance at 630 nm was subtracted after 2 h incubation with
the reagents. A plate of control cells (16 wells) was measured at day 0 to
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18. The ER competitor assays were performed according to the manufacturer’s
(Invitrogen, Carlsbad, CA) recommendations with some modifications. Human
recombinant ER was used at the recommended concentration of 15 nM and the
FluormoneTM ES2 was used at a concentration of 1 nM. Aliquots (20
lL) of the
mixture of ER and FluormoneTM ES2 were distributed in 384-well, black flat-
bottom plates and serial dilutions of test compounds in DMSO were added.
Each compound was tested in duplicate, and 1
l
M E2 was used as a positive
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experiment. After 2 h, the fluorescence polarization (FP) was measured using
an AnalystTM AD & HT Assay Detection Systems reader (Molecular Devices,
Sunnyvale, CA) equipped with 485 nm excitation and 530 nm emission filters
with the appropriate FL505 dichroic mirror. Data was then analyzed by linear
regression using GraphPad Prism’s one-site competition method. The fit was
constrained by the high polarization control, which was the ER/ES2 complex
with no competition, and the low polarization control, which was the ER/ES2
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and allowed to adhere for 72 h in phenol red-free RPMI-1640 containing 10%
FBS. Test agents were added (concentrations ranging from 3.2 nM to 50 lM)
and cells were incubated for 72 h. Cell density was determined using the MTS
assay as described above.
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