Tethered indoles as functionalizable ligands for the estrogen receptor

Article abstract of DOI:10.1016/j.bmcl.2008.11.043

To create ligands for the estrogen receptor that contain pendant groups for tethering to a poly(amido)amine (PAMAM) dendrimer, we have explored a class of N-substituted 2-phenyl indoles. Attachment of tethers of different length and chemical nature to thi

Full text of DOI:10.1016/j.bmcl.2008.11.043

Bioorganic & Medicinal Chemistry Letters 19 (2009) 485–488
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tethered indoles as functionalizable ligands for the estrogen receptor
*
Bridget G. Trogden , Sung Hoon Kim, Shuyi Lee, John A. Katzenellenbogen
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
To create ligands for the estrogen receptor that contain pendant groups for tethering to a poly(amido)-
amine (PAMAM) dendrimer, we have explored a class of N-substituted 2-phenyl indoles. Attachment
of tethers of different length and chemical nature to this non-steroidal indole scaffold gave high affinity
ligand-tether conjugates that can be easily functionalized. To further explore the utility of this system, an
indole-conjugated dendrimer was prepared and evaluated as an estrogen receptor ligand.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 18 September 2008
Revised 10 November 2008
Accepted 12 November 2008
Available online 18 November 2008
Keywords:
Estrogen receptor
Indole
PAMAM dendrimer
The estrogen receptor (ER) is a member of the nuclear hormone
receptor superfamily of ligand-regulated transcription factors. The
ER is regulated primarily by the endogenous estrogen, estradiol
(E2, Fig. 1), but it is also the target of pharmaceutical agents,
including estrogen agonists, antagonists, and selective estrogen
ents onto which ligands can be covalently attached. Generation-6
PAMAM has a nominal molecular weight of 58 kDa, similar to bo-
vine serum albumin, which has been used as an estradiol carrier6;
the dendrimer also has nominally 256 surface primary amines. A
distinct advantage of a PAMAM over a protein as a macromolecule
for hormone conjugation is that the PAMAM can withstand organic
solvents and extraction protocols needed to remove any remaining
free ligand, which can confound biological experiments.4–6
Production of ER ligand-macromolecule conjugates involves
four design steps: (I) identifying a good ligand scaffold with a posi-
tion for covalent attachment of the tether, (II) determining an opti-
mal tether length and chemical nature, (III) locating an appropriate
linker type for synthetic ease in attachment to the dendrimer, and
(IV) attachment of the dendrimer.
receptor modulators (SERMs) that activate the subtypes ER
a and
ERb.1 The binding of E2 or other agonists to ER results in conforma-
tional changes that affect its ability to recruit coactivators or core-
pressors and controls ER interaction with DNA-regulatory
sequences, termed estrogen response elements.2
In addition to its nuclear role to regulate gene transcription, ER
can also have non-genomic actions by directly activating kinase
cascades. These non-genomic effects are more rapid than the tran-
scriptional ones and are mediated through ER from membrane or
other extranuclear sites, where it acts as a classical activator of sig-
nal transduction.3 Extranuclear ER is most likely the same protein
as nuclear ER, yet it represents only a few percent of total cellular
ER, so rigorous characterization of membrane ER has been difficult.
Estrogens conjugated to poly(amido)amine (PAMAM) dendri-
mers have been used to study the non-genomic, extranuclear ef-
fects of the estrogen receptor.4,5 We have found that when an
estrogen is conjugated to the highly charged, abiotic PAMAM mac-
romolecule, this estrogen–dendrimer conjugate remains outside of
the nucleus, allowing the ligand to activate signaling of only non-
nuclear, non-genomic pathways.
Regarding the first design step, adding a tether to various posi-
tions on E2 generally reduces its affinity, although substitution at
7a, 11b, or 17
a
is typically well tolerated.7 Estradiol-macromolec-
ular conjugates attached through 7
a
or 17a that we prepared bind
well to ER and were useful as biological probes.4,5 Although these
steroidal estrogen conjugates were useful, many interesting non-
steroidal ligands for ER are known. While originally of interest
because of their ease of synthesis, non-steroidal ligands have
PAMAMs are available in a number of generations that corre-
spond to progressively increasing molecular weights and surface
functionalities. PAMAMs contain multiple surface amine substitu-
* Corresponding author.
E-mail address: jkatzene@uiuc.edu (J.A. Katzenellenbogen).
Present address: Department of Chemistry, Mercer University, 1400 Coleman Ave,
Macon, GA 31207, USA.
Figure 1. Estradiol (E2) and 2-phenylindole estrogens.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.043

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B. G. Trogden et al. / Bioorg. Med. Chem. Lett. 19 (2009) 485–488
noteworthy biological profiles and have provided ER subtype-selec-
tive ligands and supplied new therapeutic agents.2,8,9 Thus, we were
intrigued at the possibility of building functional ER-non-steroidal
ligand conjugates, particularly those with macromolecules.
To investigate the tolerance of ER to macromolecule conjugates
of non-steroidal ligands, we chose a series of 2-phenylindole li-
gands (Fig. 1). Many members of this class have pharmacological
activity,10–18 and attachment of alkyl groups to the indole nitrogen
generally provided favorable ER binding.10,17 The 2-phenylindole
estrogens have been tested as inhibitors of mammary tumor
growth15 and have been successfully attached to pendant chemo-
therapy agents to target ER+ tumors.11
Deprotection of 1 with BBr3 gave a dihydroxy indole intermediate,
which was reprotected as the di-isopropyl ether 2. This was neces-
sary to avoid incompatibility during eventual removal of protect-
ing groups from the indole and substituents placed on the indole
nitrogen.
The protected indoles 1 and 2 were used to prepare tether-con-
taining indoles to determine which attachment gives the highest
affinity ER ligand (Table 1). Indoles 1 or 2 were treated with NaH
followed by the appropriate electrophile to introduce N-benzyl or
N-alkyl groups. The isopropyl groups were selectively cleaved with
AlCl3 to give products 3–4 and 6–7, whereas treatment of the tri-
methoxy indoles with BBr3 gave 5 and 8.
Recent reports show that the second design step can often be
best achieved with short tethers.4,5,19 Addition of long or moder-
ate-length tethers to ER ligands can sometimes decrease their
accessibility to the receptor. Particularly when the ligand is conju-
gated to a macromolecule, shorter, more rigid tethers result in bet-
ter exposure of the ligand to the receptor, by projecting the ligand
outward, away from the conjugated molecule.5 Thus, ligands with
shorter tethers often have affinity similar to that of the analogous
ligands without these tethers.7 Thus, we prepared a small group of
N-substituted 3-methyl-2-phenylindoles with tethers of various
lengths and determined their binding affinities.
The affinity of these modified indoles for ERa and ERb was mea-
sured using a radiometric binding assay with [3H]estradiol as tra-
cer.20 The binding is expressed as relative binding affinity (RBA)
values, which represent a percent of the binding affinity of the
standard, E2 = 100% (Table 1). The affinities of these compounds
are quite promising. Especially noteworthy are N-alkyl indole 7
and N-benzyl indole 4, with ERa RBAs of 41% and 27%, respectively.
These two compounds show that the ER is able to tolerate flexible
tethers of some length on the N-1 position; thus, such compounds
are viable candidates for further study. The ERa binding affinity of
the other N-substituted indoles is also high, but in all cases, their
Using the Fischer indole synthesis (Scheme 1), 4-methoxyphen-
ylhydrazine hydrochloride was reacted with 4-methoxypropiophe-
none to obtain dimethoxy protected 3-methyl-2-phenylindole 1.
ERb affinity is less.
Some indoles were assayed for their agonistic and antagonistic
character as regulators of transcription by co-transfection reporter
Scheme 1. Reagents and conditions: (a) HCl, EtOH, 80 °C, 6 h, 84%; (b) BBr3, CH2Cl2, À78 °C to rt, 18 h, 82%; (c) 2-bromopropane, TBAH, acetone, 60 °C, 24 h, 46%.
Table 1
Synthesis and evaluation of estrogen receptor ligands
Reagents and conditions (a) NaH, electrophile, THF, 0 ˚C to r.t., 16-44%; (b) AlCl3, CH2Cl2, r.t., 25-33%; (c) BBr3, CH2Cl2, 0 ˚C to r.t., quant.
Compound
SM/deprotection scheme
2/(b)
R2
ERa
, ERb relative binding affinity (RBA)*
OCH3
3
11, 6.5
27, 1.2
10, 1.0
O(CH2)3CH3
OH
4
5
2/(b)
1/(c)
6
7
2/(b)
2/(b)
1/(c)
11, 2.0
41, 3.0
13, 1.4
(CH2)6OCH3
(CH2)6O(CH2)3CH3
8
(CH2)6OH
*
Binding affinities are expressed as relative binding affinity (RBA) values, where the affinity of the tracer and native ligand estradiol is set at 100. The Kd of estradiol is 0.2 nM
and 0.5 nM for ERb.
for ER
a

B. G. Trogden et al. / Bioorg. Med. Chem. Lett. 19 (2009) 485–488
487
Table 2
gene assays in human endometrial cancer cells, at ligand concen-
trations from 10À10 to 10À6 M. Agonist activity was measured with
the ligand alone, antagonistic activity with 10À9 M estradiol. None
of the compounds showed full agonist activity; all were mixed ago-
nist/antagonists of modest potency, consistent with the behavior of
other N-substituted-2-phenylindoles.10–17 They also showed only
RBA values for tethered indole ligands
Compound
Structure
ERa, ERb
relative
binding
affinity
(RBA)
limited selectivities for ERa and ERor b (data not shown).
10
11
13
15
See Scheme 2
See Scheme 2
See Scheme 2
See Scheme 2
0.188, 0.084
2.17, 0.24
0.314, 0.046
0.226, 0.048
As this class of tether-containing indoles was promising, we
prepared another group to address the third design challenge of
locating an appropriate chemical linker for attachment to the N-1
position. We utilized either ether-based phenolic or amine-based
aniline linkages. These molecules ended in an amine, which can
be used to anchor the PAMAM dendrimer.
For the ether linkage 9 (Scheme 2), we used 4-(3-iodoprop-
oxy)benzaldehyde to add a three-carbon linker to 1. Compound 9
was deprotected with EtSH to give 10, modifying the aldehyde as
well. This compound had low ER affinity (Table 2). From interme-
diate 9 we made 11, using reductive amination to attach a linker
that mimics the attachment site to the PAMAM dendrimer. Com-
19
0.5 0.1,
0.1 0.1
20
See Scheme 3
1.3 0.2a,
<0.007a
a
RBA values for the dendrimer conjugate are based on the concentration of
ligand, not the dendrimer conjugate. Based on the dendrimer, RBA values would be
25-fold higher.
pound 11 (Table 2) again shows a preference for the ERa.
For the amide linkage, we added 4-iodobutylphthalimide to 1 or
to a benzyl-protected indole. Deprotection of 12 and treatment
with hydrazine gave 13. An N-hydroxysuccinimide (NHS) ester
(16) was added to amine 14, and the product was deprotected to
mimics the functionality of last unit of a G-6 PAMAM, or with G-
6 PAMAM itself to form the dendrimer conjugate 20.
give 15. Compounds 13 and 15 also show selectivity for ER
a
. Thus,
To synthesize the indole conjugates, methyl-protected indole 1
was treated with NaH followed by 4-iodobutylphthalimide. Depro-
tection of the methyl ethers with AlCl3 and EtSH followed by repro-
tection with TBDMS gave indole 17. Hydrazinolysis gave the
primary amine, and a protected benzaldehyde was obtained by
treatment with NHS ester 21. The acetal protecting group was re-
moved with acid to give intermediate 18. Reductive amination
with N-acetyl ethylenediamine followed by cleavage of the silyl
protecting groups gave reference indole 19.
we determined that ether or amine linkages would be acceptable
for further design of an indole-PAMAM conjugate. For synthetic
ease, we choose a 4-carbon linker to a substituted benzamide (like
15) because it would provide the most direct route to the desired
conjugate.
For the fourth and final design step, we created an indole com-
pound 18 having an aldehyde-substituted phenyl ring through
which it could be conjugated to a PAMAM dendrimer (Scheme
3). Compound 18 was to be reacted with N-acetyl ethylenediamine
to afford conjugate 19, a reference compound whose structure
Protecting group cleavage of 18 followed by reductive amina-
tion was also performed with the G-6 PAMAM dendrimer to pro-
Scheme 2. Reagents and conditions: (a) NaH, 4-(3-iodopropoxy)benzaldehyde, DMF, 79%; (b) EtSH, AlCl3, rt, 89%-quant; (c) i—NH2(CH2)2NHAc, MeOH, 65 °C; ii—NaBH4,
MeOH; (d) NaH, 4-iodobutylphthalimide, rt, 62–83%; (e) H2NNH2, CH2Cl2CHCl2, MeOH, rt, quant; (f) i—16, CH2Cl2, quant; ii—Pd/C, MeOH, quant.

488
B. G. Trogden et al. / Bioorg. Med. Chem. Lett. 19 (2009) 485–488
Scheme 3. Reagents and conditions: (a) NaH, 4-iodobutylphthalimide, DMF, 0 °C to rt, 83%; (b) EtSH, AlCl3, rt, 85%; (c) TBDMSCl, imidazole, DMF, 91%; (d) H2NNH2, CH2Cl2,
MeOH, rt, 80%; (e) 21, CH2Cl2, THF, rt, 80%; (f) H+, acetone, rt, 72%; (g) i—NH2CH2CH2NHAc, CH2Cl2, ii—NaBH4; (h) 1N HCl/MeOH, rt, quant; (i) i—G-6 PAMAM, MeOH, rt; ii—
NaBH4, quant.
vide the indole-PAMAM conjugate 20, after removal of unreacted
indole by membrane filtration, as we have previously described.4
Because the reductive amination reaction is highly efficient, the
degree of dendrimer substitution by the indole ligand directly re-
flects the indole-to-PAMAM stoichiometric ratio.4,5 The uniformity
of substitution was determined by MALDI MS, which showed an
average of 25 indole substituents per PAMAM molecule, leaving
approximately 230 free amines.21
References and notes
1. Katzenellenbogen, B. S.; Katzenellenbogen, J. A. Science 2002, 295, 2380.
2. Lin, X.; Huebner, V. Curr. Opin. Drug Discov. Dev. 2000, 3, 383.
3. Levin, E. R. Steroids 2002, 67, 471.
4. Kim, S. H.; Katzenellenbogen, J. A. Angew. Chem. Int. Ed. 2006, 45, 7243.
5. Harrington, W. R.; Kim, S. H.; Funk, C. C.; Madak-Erdogan, Z.; Schiff, R.;
Katzenellenbogen, J. A.; Katzenellenbogen, B. S. Mol. Endocrinol. 2006, 20,
491.
6. Stevis, P. E.; Deecher, D. C.; Suhadolnik, L.; Mallis, L. M.; Frail, D. E.
Endocrinology 1999, 140, 5455.
7. Anstead, G. M.; Carlson, K. E.; Katzenellenbogen, J. A. Steroids 1997, 62, 268.
8. Veeneman, G. H. Curr. Med. Chem. 2005, 12, 1077.
The binding affinity analyses of 19 and 20 are given in Table 2.
As an ER ligand, the reference compound 19 had relatively low
affinity for both ER
jugate 20 showed respectable binding to ER
ity preference (ca. 200-fold) for ER over ERb. Notably, this is the
first ligand–dendrimer conjugate that shows such a large ER
a
and ERb. By contrast, the indole-PAMAM con-
9. Buijsman, R. C.; Hermkens, P. H.; van Rijn, R. D.; Stock, H. T.; Teerhuis, N. M.
Curr. Med. Chem. 2005, 12, 1017.
a, with very high affin-
10. von Angerer, E.; Prekajac, J.; Strohmeier, J. J. Med. Chem. 1984, 27, 1439.
11. Rink, S. M.; Yarema, K. J.; Solomon, M. S.; Paige, L. A.; Tadayoni-Rebek, B. M.;
Essigmann, J. M.; Croy, R. G. Proc. Natl. Acad. Sci. USA 1996, 93, 15063.
12. Greenberger, L. M.; Annable, T.; Collins, K. L.; Komm, B. S.; Lyttle, C. R.; Miller, C.
P.; Satyaswaroop, P. G.; Zhang, Y.; Frost, P. Clin. Cancer Res. 2001, 7, 3166.
13. Miller, C. P.; Collini, M. D.; Tran, B. D.; Harris, H. A.; Kharode, Y. P.; Marzolf, J. T.;
Moran, R. A.; Henderson, R. A.; Bender, R. H. W.; Unwalla, R. J.; Greenberger, L.
M.; Yardley, J. P.; Abou-Gharbia, M. A.; Lyttle, C. R.; Komm, B. S. J. Med. Chem.
2001, 44, 1654.
14. Biberger, C.; von Angerer, E. J. Steroid Biochem. Mol. Bio. 1998, 64, 277.
15. Biberger, C.; von Angerer, E. J. Steroid Biochem. Mol. Bio. 1996, 58, 31.
16. Golob, T.; Biberger, C.; Walter, G.; von Angerer, E. Arch. Pharm. 2000, 333,
305.
17. von Angerer, E.; Strohmeier, J. J. Med. Chem. 1987, 30, 131.
18. von Angerer, E.; Knebel, N.; Kager, M.; Ganss, B. J. Med. Chem. 1990, 33, 2635.
19. Ahmed, N.; Dubuc, C.; Rousseau, J.; Benard, F.; van Lier, J. E. Bioorg. Med. Chem.
Lett. 2007, 17, 3212.
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Biochemistry 1997, 36, 14897.
a
a
-
selectivity, a finding that supports further study within this com-
pound class.
In summary, we have prepared a series of N-substituted indoles
having good binding affinities for the estrogen receptor and show
the high tolerance of the ER for tethers of various lengths on N-1
of the 2-phenylindole. A dendrimer-conjugated indole was pro-
duced that had good affinity and selectivity for ERa. This novel
compound can be used in further studies to characterize the bio-
logical functions of extranuclear ER.
Acknowledgments
21. MALDI-TOF (DHB) Mn 57117, Mw 58260, PI 1.02 (reference G6 PAMAM
dendrimer, Mn 46825, Mw 47573, PI 1.02: theoretical MW 58047). 1H NMR
(500 MHz, D2O) 1.65 (indole, CH2), 2.26 (G6), 2.42 (G6), 2.62 (G6), 2.85 (G6),
3.10 (G6), 3.23 (G6), 6.40–7.40 (m, indole).
This research was supported by a Grant from the NIH (PHS 5R37
DK15556). We thank Drs. F. Stossi and B. Katzenellenbogen for per-
forming co-transfection assays and Kathryn Carlson for the binding
assays.