Blocking estrogen signaling after the hormone: Pyrimidine-core inhibitors of estrogen receptor-coactivator binding

Article abstract of DOI:10.1021/jm800698b

As an alternative approach to blocking estrogen action, we have developed small molecules that directly disrupt the key estrogen receptor (ER)/coactivator interaction necessary for gene activation. The more direct, protein-protein nature of this disruption might be effective even in hormone-refractory breast cancer. We have synthesized a pyrimidine-core library of moderate size, members of which act as α-helix mimics to block the ERα/coactivator interaction. Structure-activity relationships have been explored with various C-, N-, O-, and S-substituents on the pyrimidine core. Time-resolved fluorescence resonance energy transfer and cell-based reporter gene assays show that the most active members inhibit the ERα/steroid receptor coactivator interaction with K_i's in the low micromolar range. Through these studies, we have obtained a refined pharmacophore model for activity in this pyrimidine series. Furthermore, the favorable activities of several of these compounds support the feasibility that this coactivator binding inhibition mechanism for blocking estrogen action might provide a potential alternative approach to endocrine therapy.

Full text of DOI:10.1021/jm800698b

6512
J. Med. Chem. 2008, 51, 6512–6530
Blocking Estrogen Signaling After the Hormone: Pyrimidine-Core Inhibitors of Estrogen
Receptor-Coactivator Binding
Alexander A. Parent, Jillian R. Gunther, and John A. Katzenellenbogen*
Department of Chemistry, UniVersity of Illinois at Urbana-Champaign, 600 South Mathews AVenue, Urbana, Illinois 61801
ReceiVed June 9, 2008
As an alternative approach to blocking estrogen action, we have developed small molecules that directly
disrupt the key estrogen receptor (ER)/coactivator interaction necessary for gene activation. The more direct,
protein-protein nature of this disruption might be effective even in hormone-refractory breast cancer. We
have synthesized a pyrimidine-core library of moderate size, members of which act as R-helix mimics to
block the ERR/coactivator interaction. Structure-activity relationships have been explored with various C-,
N-, O-, and S-substituents on the pyrimidine core. Time-resolved fluorescence resonance energy transfer
and cell-based reporter gene assays show that the most active members inhibit the ERR/steroid receptor
coactivator interaction with Ki′s in the low micromolar range. Through these studies, we have obtained a
refined pharmacophore model for activity in this pyrimidine series. Furthermore, the favorable activities of
several of these compounds support the feasibility that this coactivator binding inhibition mechanism for
blocking estrogen action might provide a potential alternative approach to endocrine therapy.
Introduction
While in many cases antiestrogen therapy in hormone-
responsive breast cancer can stop or reverse tumor progression,
resistance develops with time so that typically after ca. 5 years,
tumor growth resumes despite continued treatment with
antiestrogens.18,19 In fact, in laboratory models of breast cancer
and even in the clinic, it has been shown that antiestrogens can
even become stimulatory in hormone-resistant breast cancer.20
While a molecular-level understanding of the mechanism of
resistance to endocrine therapy is still incomplete, it is clear
that breast cancer cells can undergo adaptations such that
antiestrogens can no longer block estrogen signaling. Neverthe-
less, tumor growth in this hormone-refractory state still appears
to rely on the ER-SRC interaction.21
Although historically considered female reproductive hor-
mones, estrogens are now recognized to be major regulators of
physiological functions in both reproductive and nonreproductive
tissues in both men and women.1,2 While estrogen activity is
required or is beneficial in many cases, such as for fertility,3,4
during pregnancy, and for bone health and metabolic function,5-9
the pro-proliferative effect of estrogens in many target tissues
can be pathological.10,11 Such is the case with breast cancer,
where tumor growth is driven by estrogen in ca. one-third of
tumors.12 In such patients, good therapeutic responses can often
be achieved by blocking estrogen action with antiestrogens
(estrogen antagonists).13
Such considerations motivated us to search for molecules that
might inhibit this key ER-SRC interaction directly. Such small
molecules, termed coactivator binding inhibitors (CBIs), would
be designed to bind in the hydrophobic groove of the ER-agonist
complex, thereby blocking the interaction between ER and SRC
(Figure 1C).22 Because the CBIs would be blocking this key
protein-protein interaction in a direct manner, rather than
indirectly as do conventional estrogen antagonists, they might
not be subject to the resistance mechanisms that compromise
endocrine therapy with antiestrogens. The distinction between
inhibition of estrogen signaling through ER by antiestrogens,
which act in the ligand binding pocket, and CBIs, which act in
the coactivator groove, is illustrated in Figure 1.
Both estrogens and antiestrogens work through the estrogen
receptors, of which there are two subtypes, ERR and ERꢀ.2,14
The ERsa are ligand-modulated transcription factors that act
through cis-regulatory gene sequences in chromatin to regulate
patterns of gene expression in target cells. Estrogen agonists
bind to the ERs and stabilize conformations that recruit key
coregulatory proteins, in particular, members of the p160 steroid
receptor coactivator family (SRCs) (Figure 1A).15 The ER-SRC
interaction involves protein-protein contact between signature
LXXLL sequences in the SRC and a hydrophobic groove on
the surface of the ligand-binding domain of the ER-agonist
complex with which the three leucine residues of the helix
interact. The coactivators that are recruited by the ER-agonist
complex are responsible for altering chromatin architecture,
loosening nucleosome structure, and activating RNA polymerase
II (polII), as required to up-regulate gene transcription.16
Estrogen antagonists occupy the same ligand binding site as
agonists, but they stabilize different ER conformations, ones
that preclude or reduce the binding of the SRC coactivators,
thereby indirectly interrupting estrogen signaling (Figure 1B).17
In designing CBIs, we have used a structure-guided approach,
seeking to find simple cyclic systems that could be assembled
in a modular fashion and would display three hydrophobic
substituents that mimic the three key leucine residues in the
LXXLL interaction sequence. In our earlier work, in which we
explored a number of different approaches and core heterocyclic
systems, we found that pyrimidine-core molecules appeared to
be promising structures as based on their CBI activity and ease
of preparation.22 Others have also reported on compounds with
ER-CBI activity. Our initial publication was closely followed
by a study from Shao et al., which identified “guanylhydrazone”
ER CBIs23 and compounds based on bicyclo[2.2.2]octane24 and
pyridylpyridone25 scaffolds, have also been reported to have
* To whom correspondence should be addressed. Phone: 217-333-6310.
Fax: 217-333-7325. E-mail: jkatzene@uiuc.edu.
a Abbreviations: ER, estrogen receptor; LBD, ligand binding domain,
SRC, steroid receptor coactivator; CBI, coactivator binding inhibitor; TR-
FRET, time-resolved fluorescence resonance energy transfer; RBA, relative
binding affinity.
10.1021/jm800698b CCC: $40.75
2008 American Chemical Society
Published on Web 09/12/2008

Pyrimidine CoactiVator Binding Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6513
Figure 1. Cartoon representation of classical vs CBI antagonism of ER. (A) Conformation of agonist-bound ER with helix 12 forming part of the
steroid receptor coactivator (SRC) binding site. (B) Conformation of antagonist-bound ER in which helix 12 occupies the SRC binding site, disrupting
the ER-SRC interaction indirectly. (C) Conformation of agonist-bound ER in which a CBI occupies the SRC binding site, disrupting the ER-SRC
interaction directly.
similar activity. Most recently, our laboratory has also published
promising results utilizing the amphipathic nature of alternately
subsititued trialkyl/triamino benzenes as disruptors of the ER/
SRC interaction.26
In this study, we have explored a wide variety of trisubstituted
pyrimidine-core systems as potential CBIs and we have
characterized their potency as inhibitors of the interaction of
ERR and ERꢀ with SRC-3 using a time-resolved fluorescence
resonance energy transfer assay (TR-FRET) as well as in cell-
based assays of estrogen transcriptional activity. Through this
study, we have developed a refined pharmacophore model for
CBIs of this pyrimidine class and we have obtained a number
of compounds that have low micromolar potency as CBIs in
blocking the ER-coactivator interaction in vitro. These com-
pounds are also effective in blocking estrogen-stimulated
transcriptional activity in breast cancer cells in a manner that is
insurmountable by increased estrogen, a hallmark that critically
distinguishes CBIs from conventional estrogen antagonists.
Nearly all of these CBIs also have high selectivity for ERR
over ERꢀ. The results of this study suggest that blocking
estrogen action using CBIs might provide a viable alternative
approach to endocrine therapies.
Results and Discussion
Figure 2. Structure-based design of pyrimidine core molecules based
on the ER-SRC-2 interaction (3erd). (A) Rendering of SRC-2 peptide
from 3erd crystal structure (the internal H691 and R692 residues are
On the basis of our initial success in developing ER-CBIs
using 2,4-diamino-6-alkyl pyrimidines,22 we wanted to inves-
tigate this pharmacophore type further in hopes of increasing
the affinity of the CBI. As shown in Figure 2, the design of the
initial pyrimidine library was based on the roughly triangular
orientation of the L690, L693, and L694 residues of an ER-
bound LXXLL-containing peptide from SRC-2 (also known as
glucocorticoid receptor interacting protein 1, GRIP-1).27 Al-
though we first reported that the 6-alkyl-2,4-diaminopyrimidines
bound with Ki′s in the mid micromolar range (29-49 µM as
measured by a fluorescence polarization assay), when they were
tested in a more sensitive TR-FRET assay, the best of these,
2,4-diisobutylamino-6-isoamylpyrimidine (Figure 2b), was found
to bind with a Ki approaching submicromolar concentrations.24
The high affinity of these compounds, as well as the relative
ease with which substituted pyrimidine heterocycles can be
synthesized, provided a fruitful starting point for the preparation
of an expanded ER-CBI library.
deleted for clarity). (B) Minimized structure of CBI 2,4-diisobuty-
lamino-6-isopentylpyrimidine. (C) Overlay of the SRC-2 peptide and
CBI. (D) Side view of SRC-2 peptide and CBI overlay in coactivator
groove.
involving the preformed heterocycle, finally settling upon 2,4,6-
trichloropyrimidine, which is a cheap, easily modified starting
material. As detailed below, we eventually applied a wide range
of reactions, including aminations, alkylations, alkoxylations,
and sulfide formation, on a variety of tri-, di-, and monochlo-
ropyrimidines, all of which proceeded in moderate to good
yields. Additionally, alkylations and aminations could be applied
to this precursor in a site-selective manner.28-31
We designed our pyrimidine-based library to incorporate the
leucine and phenylalanine-mimicking substituents of the previ-
ously synthesized compounds, as well as tryptophan-mimicking
naphthyl groups. In addition to the N- and C-side-arms
previously described, we also incorporated other heteroatom-
containing substituents (O, S, and SO2) into the pyrimidine core
to probe, more deeply, the nature of the binding mode of the
CBI in the coactivator groove. Triamino, trimercapto, and
trialkoxy pyrimidines were also synthesized. The overall goal
of this approach was to make a thorough exploration of the
Library Design and Synthesis. In our initial attempts at
expanding the pyrimidine library, we followed the synthetic
route previously described.22 Although this path can be used to
produce the desired substituted pyrimidines, it is laborious (due
to sparsely soluble intermediates) and prohibitively low-yielding.
Consequently, we quickly turned our attention to synthetic routes

6514 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Parent et al.
Scheme 1. Synthesis of “Symmetric” and Unsymmetric 2,4-Diamino-6-phenethylpyrimidines
structure-activity relationships of the 2-, 4-, and 6-positions
of the pyrimidine ring with respect to substituent size, polarity,
and hydrogen-bond donor/acceptor capability. In practice, the
library design was an iterative activity, evolving with the project
as progressive binding results were obtained.
displaced using an NaH-deprotonated tert-butyl carbamate to
form the 2-chloro-6-styryl-pyrimidin-4-yl carbamates 7a-c.30
The pyrimidinyl carbamates then undergo addition of a second
amine to form the SNAr products 8a-f. At this point, either
the double bond of the styryl group is reduced under mild
hydrogenation conditions (10% Pd/C, 1 atm H2) to give
phenethyl derivatives 9a-d, or the Boc group is removed in
TFA/CH2Cl2, followed by hydrogenation of the alkene, produc-
ing the unsymmetrical 2,4-diamino-6-phenethylpyrimidines
11a-f. This synthetic approach allowed for all four of the final
unsymmetrical product types, stemming from and including the
aminopyrimidinyl carbamates 8a-f, to be prepared and submit-
ted for CBI assays.
Isoamyl and Naphthethylpyrimidines. Because of the
limited accessibility of the associated boronic acids or esters,
we turned to the direct coupling of sp3-hybridized alkanes with
the pyrimidinyl trichloride to form the 6-isoamyl and 6-naph-
thethylpyrimidine series. This was effectively accomplished
through the use of a site-selective Fe(acac)3-catalyzed Kumada-
type coupling involving alkyl Grignard reagents, as developed
by the Fu¨rstner laboratory.28,34,35 Following their procedure, we
obtained the desired 6-alkyl pyrimidines 12a-c in moderate
yields. The major byproduct is the “symmetric”, disubstituted
4,6-dialkylpyrimidine, with both the 2-naphthethylpyrimidine
and the asymmetric, 2,6-dinaphthethylpyrimidine also isolated
as minor products. The 2,4-dichloro-6-alkylpyrimidines 12a-c
were then reacted with O-, N-, and S- nucleophiles to
produce the “symmetrically” substituted pyrimidines 13a-k.
Additionally, the dithioethers 13g and 13h were oxidized with
mCBPA in CHCl3 to give the respective disulfonylpyrimidines
14a and 14b (top pathway in Scheme 2).
Phenethyl and Styryl Pyrimidines. The first step toward
formation of the CBI library involves the site-selective
Suzuki-Miyaura cross-coupling reaction of trans-2-phenylvi-
nylboronic acid with 2,4,6-trichloropyrimidine, which gives the
2,4-dichloro-6-styryl-pyrimidine 1 as the major isomer (73%
yield), together with small amounts of the symmetric dicoupled
2-chloro-4,6-distyryl-pyrimidine as the only significant byprod-
uct.29 From this point, the synthesis splits into three routes. The
first route (top pathway in Scheme 1) involves the double
addition of an amine or alkoxy nucleophile to 1 to produce
“symmetrically” substituted pyrimidines 2a-d, followed by
hydrogenation of the styryl double bond over 10% Pd on carbon
at atmospheric pressure of H2 to give the final pyrimidine
products 3a-c. The second route (middle pathway in Scheme
1) involves the formation of intermediates that are labile under
hydrogenation conditions (most notably thioethers). As such, it
is necessary to first reduce the styryl double bond of 1 before
addition of any nucleophile. The reduced 2,4-dichloro-6-
phenethylpyrimidine (4) is subsequently reacted under SNAr
conditions with either an excess of alkoxide or thiolate to form
the “symmetrically” substituted pyrimidines 5a-c. The disul-
fanyl compounds 5b and 5c are then oxidized with mCPBA to
form the disulfonyl pyrimidines 6a and 6b (for complete
structural information, see Tables 1 and 2 and Supporting
Information).32,33
The third pathway of phenethylpyrimidine synthesis involves
the formation of unsymmetrically substituted 2,4-diaminopyri-
midines, as shown in the bottom pathway of Scheme 1. Starting
from 1, the chloride at the 4-pyrimidinyl position is selectively
Unfortunately, it proved more difficult to create unsymmetri-
cally substituted diamines from the alkylpyrimidines 12b and
12c than from the styryl-substituted pyrimidine 1. When 12b

Pyrimidine CoactiVator Binding Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6515
Table 1. Inhibitory Activity of 6-Alkyl-2,6-Diaminopyrimidines on the ERR and ERꢀ/Coactivator Interaction as Measured in TR-FRET and Luciferase
Reporter Gene Assays
TR-FRET Ki (µM)
ERR ERꢀ
0.066
reporter Gene IC50 (µM)
ERR
compd
R1
R2
R3
control
2a
2b
3a
3b
8b
9a
9b
SRC-1 BoxII
0.065
7.5
-styryl
-NHCH2CH(CH3)2
-NHCH2Ph
-NHCH2CH(CH3)2
-NHCH2Ph
-NHCH2CH(CH3)2
-NHCH2Ph
-NHCH2CH(CH3)2
-NHCH2Ph
-NHCH2CH(CH3)2
-NHCH2Ph
-NHCH2CH(CH3)2
-NHCH2Ph
-NHCH2-1-Nap
-NHCH2CH(CH3)2
-NHCH2-1-Nap
-NHCH2-1-Nap
-NHCH2Ph
-NHCH2-1-Nap
-NHCH2CH(CH3)2
-NHCH2-1-Nap
-NHCH2Ph
-NHCH2-1-Nap
-NH(CH2)2CH3
-NHCH2CH(CH3)2
-NHCH2Ph
-NHCH2-1-Nap
-NHCH2CH(CH3)2
-NHCH2Ph
242
>1000
>1000
>1000
132
6.9
>1000
2.1
-styryl
>1000
5.8
>1000
>1000
>1000
>1000
>1000
>1000
16
-CH2CH2Ph
-CH2CH2Ph
>1000
-styryl
-N(Boc)CH2Ph
-N(Boc)CH2CH(CH3)2
-N(Boc)CH2Ph
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2Ph
-CH2CH2Ph
-CH2CH2Ph
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
10a
10b
10c
10d
10g
11a
11b
11c
11d
11f
11g
13a
13b
13c
13d
13i
13j
13k
18a
18b
18c
18d
18e
18f
18g
18h
18j
18l
-styryl
12
>1000
9.8
-styryl
-styryl
-styryl
-NHCH2Ph
>1000
>1000
7.6
>1000
1.7
-styryl
-NHCH2-1-Nap
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2Ph
-CH2CH2Ph
4.5
>1000
1.9
-CH2CH2Ph
-CH2CH2Ph
-CH2CH2Ph
-NHCH2Ph
>1000
>1000
>1000
16
-CH2CH2Ph
-NHCH2-1-Nap
-NHCH2-1-Nap
-NH(CH2)2CH3
-NHCH2CH(CH3)2
-NHCH2Ph
>1000
>1000
13
-CH2CH2Ph
-CH2(CH2)2CH3
-CH2CH2CH(CH3)2
-CH2CH2CH(CH3)2
-CH2CH2CH(CH3)2
-CH2CH2-1-Nap
-CH2CH2-1-Nap
-CH2CH2-1-Nap
-CH2CH3
2.8
4.1
>1000
2.5
3.1
8.3
-NHCH2-1-Nap
-NHCH2CH(CH3)2
-NHCH2Ph
>1000
2.1
>1000
>1000
-NHCH2-1-Nap
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2Ph
-NHCH2-1-Nap
-NHCH2CH(CH3)2
-NHCH2Ph
>1000
7.3
10
7.9
7.2
5.8
-CH2CH2CH(CH3)2
-CH2CH2CH(CH3)2
-CH2CH2CH(CH3)2
-CH2CH2CH(CH3)2
-CH2CH2CH(CH3)2
-CH2CH2CH(CH3)2
-CH2CH2-1-Nap
-CH2CH2-1-Nap
-CH2CH2-1-Nap
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
1.8
4.1
5.8
-NHCH2-1-Nap
-NHCH2CH(CH3)2
-NHCH2-1-Nap
-NHCH2CH(CH3)2
-NHCH2Ph
-NHCH2Ph
>1000
2.4
>1000
>1000
-NHCH2-1-Nap
-NHCH2-1-Nap
-NHCH2CH(CH3)2
-NHCH2Ph
6.7
>1000
-NHCH2Ph
>1000
>1000
>1000
>1000
>1000
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2-1-Nap
-Cl
26
27d
-H
was submitted to the same SNAr conditions used to form 7a,
an unsymmetric dimer of the starting material was isolated as
the major product instead of the desired carbamate. This reaction
presumably proceeds through deprotonation of the isoamyl
R-carbon of one pyrimidine, followed by displacement of the
aromatic chloride on a second molecule. This problem is easily
avoided by first introducing the carbamate site-selectively into
2,4,6-trichloropyrimidine at the 4-position (compounds 15a-c).
This is followed by the Fe(acac)3-catalyzed coupling of the alkyl
Grignard to produce the 6-alkyl-pyrimidin-4-yl carbamates
16a-g. Amination under the same conditions used for the
chlorostyrylpyrimidines leads to the aminoalkylpyrimidinyl
carbamates 17a-m. The Boc group was subsequently removed
with TFA in CH2Cl2 to produce the final 2,4-diamino-6-
alkylpyrimidines 18a-m in modest overall yield (bottom
pathway in Scheme 2a).
of the Boc group to produce the 4,6-diamino-2-isoamylpyrim-
idines 21a-d, as shown in Scheme 2b.
To explore the SAR space of the pyrimidine library further,
4,6-dialkyl-2-aminopyrimidines were prepared from the 4,6-
dialkyl-2-chloropyrimidines 22a and 22b, which were obtained
by either Suzuki-Miyaura coupling of two equivalents of trans-
2-phenylvinylboronic acid to 2,4,6-trichloropyrimidine or as the
major byproduct from the reaction forming 12a. The final
monoamine products were obtained by amination of 22a and
22b, followed by reduction of the styryl groups of compounds
23d-f to provide the 2-amino-4,6-diisopropylpyrimidines
23a-c and 2-amino-4,6-diphenethylpyrimidines 24a-c (Scheme
3).
In addition to the alkyldiaminopyrimidines and dialkylami-
nopyrimidines, we also wanted to probe the effect of three
electronegative substituents on the binding of the pyrimidine
core molecules. Thus, we formed the “symmetric” trisubstituted
pyrimidines 25a-f with three heteroatom-containing substituents
by the reaction of 2,4,6-trichloropyrimidine with O, N, and S
nucleophiles (Scheme 4). In addition to the “symmetrically”
substituted triisobutylaminopyrimidine 25a, at lower temperature
and shorter reaction time, the unsymmetrical diamination
It should be noted that for both of the site-selective steps
described in the paragraph above, a small amount of the
undesired isomer is also formed. To explore the effect of the
exchange of the 6-alkyl and the 4-amino group on the pyrimidine
CBIs, four of these minor isomers, 19a-d, were subjected to
SNAr conditions with isobutylamine, with subsequent removal

6516 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Parent et al.
Table 2. Inhibitory Activity of Various Pyrimidines on the ERR and ERꢀ/Coactivator Interaction as Measured in TR-FRET and Luciferase Reporter
Gene Assays
TR-FRET Ki (µM)
ERR
Reporter Gene IC50 (µM)
ERR
compd
R1
R2
R3
ERꢀ
0.066
control SRC-1 BoxII
3c
5b
6a
0.065
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
5.7
-CH2CH2Ph
-CH2CH2Ph
-CH2CH2Ph
-CH2CH2CH(CH3)2 -OCH2CH(CH3)2
-CH2CH2CH(CH3)2 -SCH2CH(CH3)2
-CH2CH2CH(CH3)2 -SO2CH2CH(CH3)2
-OCH2CH(CH3)2
-SCH2CH(CH3)2
-SO2CH2CH(CH3)2
-OCH2CH(CH3)2
-SCH2CH(CH3)2
-SO2CH2CH(CH3)2
-OCH2CH(CH3)2
-SCH2CH(CH3)2
-SO2CH2CH(CH3)2
-CH2CH2CH(CH3)2
-CH2CH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2Ph
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
299
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
13e
13g
14a
21a
21d
23a
23b
23c
24a
25a
25c
25e
27a
27b
27c
28
29a
29c
30
31a
31c
32
34a
34c
35
37a
37b
39
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2-1-Nap
>1000
>1000
-CH2CH2CH(CH3)2 -CH2CH2CH(CH3)2
-CH2CH2CH(CH3)2 -CH2CH2CH(CH3)2
-CH2CH2CH(CH3)2 -CH2CH2CH(CH3)2
-NHCH2-1-Nap
-CH2CH2Ph
-CH2CH2Ph
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-OCH2CH(CH3)2
-SCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
>1000
2.4
>1000
>1000
9.5
-NHCH2CH(CH3)2
-OCH2CH(CH3)2
-SCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-OCH2CH(CH3)2
-SCH2CH(CH3)2
-NHCH2Ph
>1000
120
7.3
-OCH2CH(CH3)2
-SCH2CH(CH3)2
-SO2CH2CH(CH3)2
>1000
>1000
>1000
>1000
>1000
>1000
7.2
>1000
>1000
>1000
2.4
-CH2CH2CH(CH3)2 -N(CH3)CH2CH(CH3)2 -N(CH3)CH2CH(CH3)2
-CH2CH2-1-Nap
-CH2CH2Ph
-N(CH3)CH2CH(CH3)2 -N(CH3)CH2CH(CH3)2
-N(CH3)CH2CH(CH3)2 -N(CH3)CH2CH(CH3)2
-CH2CH2CH(CH3)2 -NHCH2CH(CH3)2
-CH2CH2-1-Nap
-CH2CH2Ph
-CH2CH2CH(CH3)2 -N(CH3)CH2CH(CH3)2 -NHCH2CH(CH3)2
-CH2CH2-1-Nap
-CH2CH2Ph
-CH2CH2CH(CH3)2 -NHCH2CH(CH3)2
-CH2CH2CH(CH3)2 -NHCH2CH(CH3)2
-CH2CH2CH(CH3)2 -NHCH2CH(CH3)2
-N(CH3)CH2CH(CH3)2
-N(CH3)CH2CH(CH3)2
-N(CH3)CH2CH(CH3)2
9.7
-NHCH2CH(CH3)2
-NHCH2CH(CH3)2
-N(CH3)CH2CH(CH3)2 -NHCH2CH(CH3)2
-N(CH3)CH2CH(CH3)2 -NHCH2CH(CH3)2
4.4
5.5
6.9
8.4
-OCH2CH(CH3)2
-SCH2CH(CH3)2
-SO2CH2CH(CH3)2
>1000
>1000
>1000
byproduct, 2,4-diisobutylamino-6-chloropyrimidine, 26, was also
produced in modest yield from the reaction of isobutylamine
with 2,4,6-trichloropyrimidine. Compound 26 was subsequently
substituted with benzyl amine, isobutylalcohol, or isobutylm-
ercaptan to form the unsymmetric triheteroatom substitutued
pyrimidines 27a-c. Reductive dechlorination of 26 gave the
6-proteopyrimidine 27d, and the isobutylsulfanyl pyrimidine 27c
was oxidized with mCPBA in CHCl3 to form the diaminosulfone
28.
The N4-methylated compounds could be accessed through a
K2CO3-catalyzed site-selective addition of N-methylisobuty-
lamine to 12b,c or as a byproduct from the formation of 29a,
followed by amination with isobutylamine and reduction of the
styryl moieties when present (bottom pathway of Scheme 5).
The binding results for the N-methylated 6-isoamylpyrim-
idines 29a, 31a, and 34a show that there is a strong preference
for a free N-H at the 4-position of the isoamyl-substituted
pyrimidine ring (see Table 2). As H-bonding at the 2-position
seemed less important to CBI binding, the O, S, and SO2-
containing 4-isobutylamino-6-isoamylpyrimidines 37a,b and 39
were also synthesized according to the methods described above
and as shown in Scheme 6. Based purely on polarity, these
compounds should show CBI activity similar to the diaminopy-
rimidines 13b and 31a. On the other hand, if H-bond donation
or overall planarity of the CBI is important, decreased affinity
would be expected.
In Vitro Time-Resolved FRET Assay of the Inhibition of
Coactivator Binding to ERr and ERꢀ by Pyrimidine-Core
CBIs. The inhibitory activity of members of our pyrimidine CBI
library for coactivator binding to both ERR and ERꢀ systems
was measured using a TR-FRET assay (Figure 3. This assay
employs a site-specifically labeled terbium/streptavidin-biotin/
ER-LBD construct and a fluorescein-labeled nuclear receptor
domain of the steroid receptor coactivator 3 (SRC-3-NRD). In
the presence of agonist (17ꢀ-estradiol) and the absence of a
CBI, the SRC-3-NRD binds to the ER-LBD, allowing transfer
of fluorescence resonance energy from the Tb donor (D) to the
Methylated Aminopyrimidines. During the course of this
study, it became clear that there was an important relationship
between the activity of the CBI and the nature of the heteroatom
substituents, suggesting that simple insertion of an electroneg-
ative atom was not sufficient to produce high-affinity compounds
(see Table 2). The apparent requirement for a nitrogen-
containing substituent at the 2- and/or 4-position of the
pyrimidine suggested that H-bond donation might be playing a
role in supporting the binding of the diaminopyrimidines. To
probe this hypothesis, we synthesized N2-methyl, N4-methyl,
and N2,N4-dimethyl diaminopyrimidines, as shown in Scheme
5. Methylation at these sites should obliterate the H-bond donor
capability of the secondary amine while retaining the polarity
and planarity induced by the aryl nitrogen.
Both the N2-methylated and the “symmetrically” substituted
N2,N4-dimethylated pyrimidines were easily synthesized by the
nonselective amination with N-methylisobutylamine and sub-
sequent reduction of compounds 1 and 12b,c and 7a and 16b,e
(see the middle and top pathways of Scheme 5, respectively).

Pyrimidine CoactiVator Binding Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6517
Scheme 2. Synthesis of Alkylpyrimidines: (A) Synthesis of 6-Isoamyl and 6-Naphthethyl Pyrimidines; (B) Synthesis of 2-Isoamyl and
2-Naphthethyl Pyrimidines
Scheme 3. Synthesis of 4,6-Dialkyl-2-aminopyrimidines
to 3a (converted by hydrogenation of the styryl double bond)
and compound 8b to 10c (involving simple removal/addition
of a Boc group from the 4-aminopyrimidinyl position). These
two factors seem to be important in increasing ERꢀ affinity,
while also decreasing affinity for ERR (compare 3a to 2a, 11a
to 10a, and 11c to 10c). It is likely that the combination of the
styryl functionality and the Boc-protecting group gives 8b its
ERꢀ selectivity, and this suggests that the ERꢀ binding groove
prefers compounds with greater rigidity coupled with increased
hydrophobicity. Selectivity of small-molecules for ERR/ERꢀ
CBI activity has been mentioned only briefly before23 and seems
surprising because an examination of the coactivator groove of
ERR and ERꢀ bound to LXXLL-containing peptides shows very
little dissimilarity. The homology of the binding groove is again
supported by assay of the control SRC-1 Box II peptide (LXXLL
motif), which has approximately equivalent affinity for both
receptors. Nonetheless, the preference of other peptide-based
CBIs for ERR over ERꢀ has been reported,36-38 and this
selectivity is significant in that it may increase the ultimate
therapeutic utility of these compounds.39-41
Table 1 contains the binding data for the “symmetrically”
and “unsymmetrically” substituted 2,4-diamino-6-alkylpyri-
midines and 2,4-diamino-6-styrylpyrimidines. Because these
compounds were the initial synthetic targets of this study,
as well as the compounds most closely related to those
reported in our first ER-CBI paper,22 we were hopeful that
these alternative and “unsymmetrical” additions would
provide increased CBI activity. While somewhat disappointed
by the lack of significant increase in affinity, we were
nonetheless pleased that this approach produced many
compounds with CBI activity having comparable or slightly
higher affinity for ERR.
fluorescein acceptor (A). With increasing concentrations of CBI,
the ER/SRC-3 complex is disrupted and fluorescence resonance
energy transfer decreases. This provides a dose-dependent
inhibition curve, typically plotted on an A/D × 1000 scale, from
which Ki values for the various compounds can be calculated.
An unlabeled peptide containing the NR Box II (LXXLL motif)
of SRC-1, a natural coactivator of ER, is used as a positive
control. The results from these binding studies are summarized
in Tables 1 and 2 (additional binding data can be found in
Supporting Information).
Initially, what is most striking about the data is the almost
universal selectivity of the pyrimidine core CBIs for ERR over
ERꢀ coactivator binding inhibition. With the exception of three
compounds that show only very modest affinity for ERꢀ (2a,
8b, and 25a), all compounds assayed show binding only to ERR.
(These results have been echoed in preliminary studies in cell-
based reporter gene systems, which have also confirmed the
ERR selectivity of pyrimidine compounds 3a, 13b, and 27a,
which show no mechanism-based inhibition of ERꢀ.) Of these
three, 2a and 25a are still over 30-fold ERR selective, while
only the Boc-protected compound, 8b, shows complete selectiv-
ity for ERꢀ with no activity for ERR. Loss of ERꢀ activity
occurs readily, as observed in the relationship of compound 2a
Included in Table 1 are compounds 3b, 13a, and 13b, which
represent the high affinity pyrimidine CBIs reported previ-

6518 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Scheme 4. Synthesis of Tri-Heteroatom-Substituted Pyrimidines
Parent et al.
Scheme 5. Synthesis of N-Methylated Pyrimidines
Scheme 6. Synthesis 2-Alkoxy, 2-Sulfanyl, and 2-Sulfonylpyrimidines
ously.22 Interestingly, while 13a and 13b show lower Ki values
in our more sensitive TR-FRET assay than those we reported
using our earlier fluorescence polarization assay, compound 3b
shows no measurable binding to either ERR or ERꢀ by TR-
FRET (Ki ) 49, 32, and 29 µM by FP vs >1000, 16, and 2.8
µM by TR-FRET, respectively, for 3b, 13a, and 13b). We
believe that the lower affinity value that we obtain for 3b by
the TR-FRET assay is the correct one because it is consistent
with a trend we have observed throughout this series of
compounds, namely the lack of affinity of the ERR coactivator
groove for extremely bulky ligands. This is apparent in the
general exclusion of pyrimidines that contain a naphthyl group,
exceptions being those pyrimidines (13i, 18c, and 18f) that
contain only two small isobutyl/isoamyl side-arms in addition
to the larger aromatic group. Additionally, while the coactivator
groove readily tolerates CBIs with two phenyl groups and a
single isoamyl/isobuyl group (11a, 11c, and 13c), addition of a
third aromatic substituent (3b) results in complete loss of
binding. In general, it appears that those compounds that bind
best to the coactivator groove are those that fill it snugly, without
exceeding certain steric limitations, as in compounds 11c, 13i,
and 18f.

Pyrimidine CoactiVator Binding Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6519
which have potencies similar to that of their alkyl-diamino
analogues 13b and 18d (Table 1), with Ki values of 5.7 and
7.3, and 2.8 and 5.8, respectively. Nonetheless, there is some
selectivity for atom type at the 6-position, as is evidenced by
the lack of activity of the 2,4-diisobutylaminopyrimidines 27b,c,
and 28, which contain O-, S-, and SO2-linked substituents at
that site (Table 2).
In addition to the amino pyrimidines already described, a
number of other heteroatom-containing pyrimidines were also
analyzed for CBI activity. Incorporation of the alkoxy, sulfanyl,
and sulfonyl pyrimidines into the library (Table 2 and Supporting
Information) allowed for further evaluation of the role of the
amino substituents in CBI binding. Although we had anticipated
that any electronegative atom or group would provide affinity
similar to the previously assayed aminopyrimidines, we found
that none of the pyrimidines incorporating two or three O or S
substituents exhibited any inhibition of coactivator binding. (The
lone exception to this is the triisobutylsulfanylpyrimidine 25e,
which bound ERR with the relatively high Ki of 120 µM.) Even
oxidation of the thioether function to the highly polar sulfone
failed to elicit activity.
Figure 3. In vitro coactivator binding inhibition assay. TR-FRET assay
shows displacement of SRC-3-NRD-Fl by control peptide and pyri-
midine CBIs.
Comparison of the pairs of compounds 11a and 11c, 18b
and 18d, and 18c and 18f in Table 1 yields another general
trend in CBI affinity. For each of these pairs, affinity is increased
1.5- to 3-fold when the amino substituents at the 2- and
4-positions are situated so that the larger moiety is in the
4-position. This effect becomes more dramatic as the size
difference in the groups increases (18c and 18f vs 18b and 18d).
Conversely, it appears that the size of the substituent on the
alkyl group at the 6-position has little effect on binding, as both
the 2,4-diisobutylaminopyrimidines with the large naphthethyl
group (13i) and the isoamyl group (13b) bind with comparable
affinity. Nevertheless, if the size of the group at the 6-position
is reduced drastically, binding is negatively affected, as in the
6-ethylpyrimidine 18a (Ki )10 µM), and substitution with a
chlorine or hydrogen atom at the same position results in
complete loss of activity (26 and 27d). As mentioned above,
introduction of a double bond at the 6-position decreases or, in
extreme cases, obliterates ERR affinity and, in all cases,
pyrimidines with Boc-protection at the N4-amine fail to bind
ERR (see Supporting Information for further examples of these
compounds).
To better understand the effect of substituent type and
placement on the pyrimidine ring, we next investigated the
binding of 4,6-dialkyl-2-amino- and 2-alkyl-4,6-diaminopyri-
midines, as shown in Table 2. Despite the overall hydrophobic
nature of the coactivator groove and the distinct structural
homology of the 4,6-dialkylpyrimidines 23a-24c to the high
affinity 2,4-diamino compounds, we failed to observe binding
with any members of this pyrimidine derivative class. Possibly
more surprising is the complete lack of binding exhibited by
the 2-alkyl-4,6-diaminopyrimidines 21a-21d. Although they
are simple isomers of compounds 13b, 13i, 18b, 18c, 18d, and
18f, which all have Ki values below 10 µM, the compounds
with a methylene group replacing the amino group at the
2-position suffer complete loss of CBI binding activity. To-
gether, these results suggest that at least two electronegative
substituents on the pyrimidine ring are required for CBI activity.
Moreover, it is not sufficient for these substituents to have a
general 1,3-relationship; rather, binding requires specific sub-
stitution at the 2- and 4-positions. Along with the two nitrogens
of the pyrimidine core, these two substituent nitrogen atoms
form a tetrad of electronegative atoms, which appears to provide
a unique and essential binding motif.
At this point, it became evident that there is a necessary
relationship between the presence of nitrogen-bearing substit-
uents at a specific position on the pyrimidine and the potency
of the CBI. We rationalized that the importance of the N
heteroatom was most likely based on either its ability to act as
a hydrogen-bond donor or its planarity due to resonance
interaction between the nitrogen lone pair and the aryl π-system.
If H-bond donation was required for CBI binding, it was likely
that it would be position specific. To probe these possibilities,
we synthesized and assayed the N2-methyl, N4-methyl, and N2,
N4-dimethyl diaminopyrimidine compounds, 29a-35.
As shown in Table 2, when both the N2- and N4-nitrogens
are methylated, affinity for ERR is completely lost (29a-d).
Interestingly, some activity is regained in the selectively
monomethylated compounds: when the N4 of the 6-isoamylpy-
rimidine is methylated, no binding is observed (34a), but activity
is nearly fully restored when the methyl group is switched to
N2 (31a). In the phenethyl and naphthethyl compounds, the
opposite is observed, and the compounds with a free N-H at
the N2-position bind (34c and 35), whereas the N4 isomers are
inactive (31c and 32).
This restoration of activity associated with monomethylation
suggests that N-H H-bond donation is indeed important to
binding. That this binding is specific to either the 2- or 4-position
of the aromatic ring, depending on the nature and/or size of the
C-6 substituent, implies that more subtle electronic effects are
also playing a role in orientation of the CBI in the coactivator
groove: sterically, the 2- and 4-positions of the pyrimidine
should be practically equivalent, but the dipole moment of the
heterocycle relative to the methyl-substituted amine is position-
dependent and the differential ability of the aromatic nitrogen
atoms to act as hydrogen bond acceptors must also be
considered. The preference for the free N-H to switch from
the 4- to 2-position as the steric bulk of the alkyl group increases
from isoamyl to phenethyl may indicate that a different binding
mode (i.e., 120° rotation) is involved in inhibition with these
larger molecules. The manner in which these two somewhat
contradictory observations might be understood has been
analyzed using computation docking experiments and is dis-
cussed below.
In contrast to the importance of the presence of the amino
substituents at the 2- and 4-positions of the pyrimidine ring,
the nature of the C-6 substituent seems to be of minimal
importance, as the alkyl group can be replaced with a third
amino substituent with minimal loss in binding affinity.
Examples of this are the triamino CBIs 25a and 27a (Table 2),
After showing the selective importance of at least one free
amine N-H at either the 2- or 4-position of the pyrimidine, we
wanted to probe the ability of other electronegative atoms to

6520 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Parent et al.
concentrations of CBI. After incubation for 24 h, cells were
lysed and assayed for luciferase reporter gene and ꢀ-galactosi-
dase activity.
Those CBIs that were active showed not only a concentration-
dependent reduction in reporter gene activity but they also gave
IC50 values that were unaffected by changing the estradiol
concentration. This inhibition, insurmountable by 100-fold
excess estradiol, indicates that the CBIs are not competing
against the ligand estradiol as a conventional antagonist would;
rather, they are acting through the CBI mechanism to block
coactivator binding. Examples of the estrogen insurmountable
reporter gene inhibitory activity of two representative CBIs (3a
and 13b) are shown in Figure 5a (TR-FRET curves for the same
compounds are shown in Figure 3).
Figure 4. Summary of the structure-activity relationships of the
pyrimidine-core CBIs.
offer the same binding properties as the non-H-bonding N-
methyl amine. To accomplish this, we prepared the 6-isoamyl-
4-isobutylpyrimidines 37a,b and 39. None of these O- or
S-containing pyrimidines showed any inhibition of the receptor/
coactivator complex. Although not conclusive, this gives strong
support for the need of two amino substituents at both the N2-
and N4-positions of the pyrimidine ring. Although it is possible
that subtle electronic changes are the basis of this marked affinity
differential, it is more likely that the planarity and associated
rigidity of the N-aryl bond creates an overall CBI core structure
that is more conducive to binding. A summary of the
structure-activity relationships of the pyrimidine-core CBIs is
shown in Figure 4.
In Vitro Radiometric Assay of CBI Binding Affinity
for the Ligand Binding Pocket of ERr and ERꢀ. To further
ensure that the inhibition we observe with these compounds in
the TR-FRET CBI assay is not due to the compound binding
as an antagonist at the ligand binding pocket, we conducted a
competitive radiometric ligand binding assay using tritium-
labeled estradiol as a tracer and full-length purified human ERR
and ERꢀ. The binding affinities of the CBIs relative to the tracer
and standard, estradiol, are expressed as relative binding affinity
(RBA) values. The pyrimidine CBIs generally have RBAs for
ERR of less than 0.005% (estradiol ) 100%), and while there
are a few significant exceptions, even the CBI with the highest
affinity for the ligand binding pocket (11a) binds with only
0.051% the affinity of estradiol.
To properly interpret these results, it must be remembered
that estradiol has a Kd for ERR of 0.2 nM; therefore, at the
concentration of estradiol used for the TR-FRET CBI assay (1
µM), a compound with an RBA of even 0.1% would show an
IC50 value of minimally 1 mM in the assay if it were acting as
a conventional antagonist (i.e., competing with estradiol) rather
than as a CBI (i.e., competing with SRC). Thus, we conclude
that the inhibitions we are observing in our TR-FRET assay
could not be due to the competition of these pyrimidines for
the natural ligand estradiol with concomitant induction of an
antagonist, coactivator non-binding conformation of the ER.
Rather, the active pyrimidine CBIs reported in this library are
disrupting the ER-SRC interaction by direct binding to the
protein surface at the coactivator binding groove, not to the
traditional, internal ligand binding site. (See Supporting Infor-
mation for specific RBAs.)
Cell-Based Assay of the Inhibition of Estrogen-Induced
Reporter Gene Transcriptional Activity by the CBIs. To
investigate the cellular activity of our pyrimidine core CBIs,
we utilized a estrogen-responsive luciferase reporter gene assay
performed in human endometrial cancer (HEC-1) cells lacking
endogenous estrogen receptor but transfected with a full-length
ERR expression vector, an estrogen-responsive luciferase,
reporter gene plasmid (2ERE Luc), and pCMV ꢀ-galactosidase
(ꢀ-gal; internal control). The cells were incubated with two
concentrations of estradiol (1 and 100 nM) and titrated
ERI-5, a previously published CBI inhibitor of the ERR-SRC
interaction reported to have an IC50 of 5.5 µM in a COS-7 cell
mammalian 2 hybrid assay,23 was used as a positive control in
these reporter gene assays; in our hands, it showed an IC50 value
of 1.4 µM. Because of its cellular impermeability, the SRC-1
Box II control peptide used for the in vitro TR-FRET work
could not be used in the cell-based assay. Measurement of
ꢀ-galactosidase activity simultaneously with CBI testing serves
as an internal control to confirm that decreased luciferase activity
corresponds to a CBI blockade of ERR–coactivator binding
rather than to a generalized mechanism of cellular toxicity. The
activity of the internal ꢀ-galactosidase control was affected by
only the highest concentration of CBI used in the assay, 20 µM,
which decreased the activity of the control to approximately
70% of the expected maximal value for only the most potent
pyrimidine compounds. A functionally different type of viability
assay, probing cellular mitochondrial function, was also em-
ployed to confirm that the decrease in luciferase activity we
observed was truly due to compound activity. This assay
(CellTiter 96 AQueous One Solution Cell Proliferation Assay,
Promega) was run using compounds that showed the greatest
discrepancy in IC50 values between in vitro and cellular assays
(i.e., 3a, 10a, 18b, 25a, and 37a) and provided results in
agreement with the ꢀ-galactosidase reporter gene assay controls,
also showing that viable cell counts decreased significantly only
at the highest doses of compound (20 µM). Because the IC50
values we see for these compounds are significantly lower than
20 µM where cell toxicity first becomes apparent, we have
concluded that the inhibition of estrogen-stimulated reporter
gene activity by the CBIs was a transcription-based response
rather than the result of general cell death.
In all instances where activity was observed in the TR-FRET
protein-binding assay, comparable potencies (or, in a number
of cases, increased potencies) were observed in the luciferase
reporter gene assay (Tables 1 and 2). A comparison of potencies
in the cell-based activity assay vs the in vitro binding assay is
illustrated in Figure 5b. The points below the dotted line
represent cases where higher potency was evident in cell-based
assay. While we can only speculate at this point, enhanced
potency in the cell-based assays could be the result of decreased
available concentrations of CBI compound in vitro because of
sequestration by the small amount of detergent that needs to be
present in the buffer of the TR-FRET assay. It could also be
due to cell-based mechanisms that increase the local concentra-
tions of the CBI by increased cellular uptake. In either case,
we are encouraged by the increased potencies of these com-
pounds when tested in a more physiologically relevant cell-
based assay: not only do these results confirm the CBI activity
of the library compounds observed in the TR-FRET assays but
they also provide evidence that these compounds are soluble

Pyrimidine CoactiVator Binding Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6521
Figure 5. Coactivator binding inhibition assays. (A) Dose-response of CBI inhibition of ER-mediated transcription in cell-based reporter gene
assay run at two concentrations of estradiol. (B) Plot of CBI Ki (µM) as calculated from in vitro TR-FRET assay vs CBI IC50 as determined by
cell-based reporter gene assay.
and cell-permeable at concentrations necessary for inhibition.
In a very low percentage of compounds, we have seen activity
in the cell-based assays that was not discovered using the TR-
FRET assay (10a and 37a). It is possible that these results are
due to an alternative mechanism of inhibition involving the AF-1
and/or DNA-binding domains of ER, which are present in the
full-length protein used in the reporter gene assays but not in
the ligand-binding domain of ER employed in the TR-FRET
assay. Together, these data provide support that these pyrimidine
CBI compounds could be effective as novel inhibitors of
ERR-SRC interactions and, thus, ER-mediated transcription.
Modeling of CBI Docking in ERr. To better understand
the binding mode(s) involved with the pyrimidine-core CBIs,
we have performed extensive docking experiments using, as a
starting point, the structure of ER-LBD cocrystallized with the
agonist diethylstilbestrol and a SRC-2 peptide (see http://
www.rcsb.org entry 3erd). After workup of the crystal structure,
the coactivator peptide was removed and the CBIs were docked
into the resulting empty binding groove using the FlexiDock
module of SYBYL 7.3 from Tripos.
Figure 6. Docking of pyrimidine-core CBIs into the coactivator groove
of ERR-LBD. (A) electrostatic rendering of 13b bound to ERR-LBD.
(B) Cut-away showing the interaction of Glu542 and Lys362 with 13b.
(C) electrostatic rendering of 13i bound to ERR-LBD. (D) Cut-away
showing the interaction of Glu542 and Lys362 with 13i.
As has been noted previously,22 these experiments confirm
that the coactivator binding groove of ER is flexible in nature
and modeling that is representative of reality is often compli-
cated by the apparent accommodation of a variety of orientations
of a single ligand. This is especially problematic when docking
quasisymmetrical compounds such as 13b. Fortunately, while
analyzing the various docking results, we have been able to
apply the empirical binding and activity data described above
to produce ER/CBI-binding models that are satisfying chemi-
cally, computationally, and experimentally (see Figure 6).
We have been especially interested in comparing the binding
orientation of the isopentyl pyrimidine 13b and the analogous
naphthylethyl pyrimidine 13i, as these compounds show es-
sentially equivalent binding affinity in the TR-FRET assay yet
exhibit opposite selectivity for their corresponding monomethy-
lated aromatic amines. Additionally, the pre-existing knowledge
of which N-methylated CBI (N2 vs N4) binds to the surface of
the LBD, greatly aids in determination of the binding mode of
the unmethylated compound.
in Figure 6A,B, 13b binds to the ER coactivator groove in such
a manner as to optimize its interaction with, not only the three
hydrophobic pockets but also both the carboxyl and amine
groups of the charge clamp. The isopentyl alkyl group fills the
small pocket on the right, pushing the amine in the 4-position
into the medium sized pocket on the left and toward Glu 542,
allowing for a positive interaction between the partially posi-
tively charged N-H of the amine and the negatively charged
carboxyl group. The second isobutylamine substituent occupies
the coactivator shelf so that there is minimal steric interaction
between the alkyl group and the charged amine of Lys362. This
allows for positive electrostatic interaction between Lys362 and
the lone pair of the pyrimidinyl ring nitrogen. Additionally,
docking of the associated monomethylated products 31a and
34a in this orientation allows for rationalization of the binding
selectivity observed: when the amine at the 2-position is
methylated (31a), there is very little rearrangement of either
the ligand or the protein and the interaction between the Lys362
and the pyrimidine nitrogen is maintained. Methylation at the
N4-position of the pyrimidine (34a) completely disrupts the
interaction between the N-H and Glu542, and, consequently,
affinity for the receptor is lost.
In general, the coactivator groove consists of three major
binding sites: a small pocket (shown on the right of the binding
site in Figure 6), a medium-size pocket (on the left), and a large
shelf (bottom center). Another important feature of the binding
site is the “charge clamp” composed of Glu542 and Lys362 of
the ERR-LBD, which, in the presence of a coactivator, interact
in a productive manner with the inherent dipole associated with
R-helical peptide backbone of the LXXLL sequence. As shown
In contrast to the quasisymmetric 13b, the large naphthyl
group of 13i constrains the CBI to only two possible orientations,
both of which place the bicyclic aromatic on the large nonpolar

6522 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Parent et al.
shelf. Of these two orientations, only that shown in Figure 6C,D
is situated so that monomethylation at the 4-position is tolerated
while methylation at the 2-position causes disruption of the ER/
CBI complex. As seen in the electrostatic rendering of the
complex (Figure 6C), this orientation also correctly matches
the negatively and positively charged residues of the “charge
clamp” with the electron-deficient N-H and the electron-rich
pyrimidine N. The cut-away rendering in Figure 6D more
explicitly shows this orientation. Although not shown, Figure
6D also provides an illustration of the effects of mono-N-
methylation of 13i: while there is ample room for methylation
at N4 (this in effect pushes the CBI to the right, deeper into the
small pocket), methylation at N2 disrupts the pyrimidine
nitrogen/Lys362 interaction, causing the CBI to be bumped out
of the binding pocket and the lysine residue to be pushed back
and to the right.
of this study suggest that blocking estrogen action using CBIs
might provide a viable alternative approach to endocrine
therapies.
Experimental Section
General Synthetic Methods. All reagents were used as pur-
chased except where noted. THF, ether, CH2Cl2, and DMF used in
reactions were dried using a solvent delivery system (neutral
alumina column). Solvents used for extraction and flash chroma-
tography were reagent or ultima grade purchased from either Aldrich
or Fisher Scientific. All reactions were run under dry N2 atmosphere
except where noted. Flash column chromatography was performed
on Silica P Flash silica gel (40-64 µM, 60 Å) from SiliCycle.
1HNMR and 13CNMR spectra were obtained on 500 MHz Varian
FT-NMR spectrometers. Except where noted, both low and high
resolution mass spectra were obtained using electrospray ionization
on either a Micromass Q-Tof Ultima or Waters Quattro instrument.
HPLC analysis was performed using a Waters 1525 binary HPLC
pump equipped with a Waters in-line degasser AF, Waters 2487
Dual λ absorbance detector, and a Waters Symmetry C18 5 µM,
4.6 mm × 150 mm column. Microanalysis was performed with a
CE 440 CHN analyzer.
Conclusions
In this report, we describe the progressive optimization of
the structure of 2,4,6-trisubstituted pyrimidines as inhibitors of
the interaction between agonist-liganded estrogen receptor (ER)
and a key coactivator protein, steroid receptor coactivator 3
(SRC-3). By inhibiting the interaction of ER with this important
mediator of ER transcriptional activity, these coactivator binding
inhibitors (CBIs) are able to block ER activity at a point after
ligand binding and we have demonstrated in cell-based reporter
gene assays that this inhibition is insurmountable by increasing
estrogen concentration. In this sense, inhibition of ER activity
by CBIs is direct and distinct from that of conventional estrogen
antagonists, which compete for agonist binding and interfere
with SRC binding indirectly by altering the topology of the ER
from an agonist conformation that recruits SRCs to an antagonist
conformation that does not.
We have achieved this optimization by developing an
expedited synthesis of 2,4,6-trisubstituted pyrimidines, which
has enabled us to systematically generate a series of focused
pyrimidine libraries having alkyl and aralkyl substituents of
various size and positioning, linked to the heterocyclic core
through carbon and heteroatoms. Additionally, we have probed
for the role of potential hydrogen bonding involvement of NH-
linking atoms.
Through these studies, we have developed a rather complete
structure-activity profile outlining a pharmacophore for the CBI
activity of these trisubstituted pyrimidines. This pharmacophore
specifies the following:
1. The need for N-linked side chains at the 2- and 4-positions
of the pyrimidine ring, with a preference for a smaller group
(isobutyl) at the 2-position.
2. Ready accommodation of either a C- or N-bound moiety
at the 6-position of the pyrimidine.
3. A strict size limit to the CBI binding pocket, with no more
than two medium (phenyl) groups or one large (naphthyl) group
tolerated at one time on the tridentate ligand.
Representative Syntheses (For Complete Synthetic Details
and HPLC Analysis of Active Compounds Please see Supporting
Information). 2,4-Dichloro-6-styryl-pyrimidine (1). On the basis
of the coupling described by Tan et al.,29 trans-2-phenylvinylbo-
ronic acid (2.546 g, 17.2 mmol), K3PO4 (7.307 g, 34.4 mmol), and
PdCl2(PPh3)2 (0.362 g, 0.52 mmol) were dissolved in 100 mL THF.
To this mixture, 2,4,6-trichloropyrimidine (3.156 g, 17.2 mmol)
dissolved in 20 mL THF was added, producing a cloudy yellow
suspension. H2O (15 mL) was added, and the now clear solution
was heated at reflux for 7 h. Approximately 100 mL of H2O were
added, and the biphasic mixture was extracted three times with
ether. The combined organic layers were washed with brine, dried
over anhydrous MgSO4, and the solvent removed with a rotary
evaporator. The product was purified by column chromatography
(gradient elution 5-10% EtOAc in hexanes) to provide 1 (3.1613
1
g, 73%). H NMR (500 MHz, CDCl3) δ ppm 6.95 (d, J ) 15.87
Hz, 1 H), 7.22 (s, 1 H), 7.41 (m, 3 H), 7.59 (dd, J ) 7.45, 2.08 Hz,
2 H), 7.96 (d, J ) 15.87 Hz, 1 H). 13C NMR (500 MHz, CDCl3)
δ ppm 117.1, 123.0, 128.2, 129.2, 130.6, 134.8, 140.9, 160.7, 162.8,
166.6. HRMS (ESI+) m/z calcd for C12H9N2Cl2+, 251.0143; found,
251.0101.
N,N′-Diisobutyl-(6-styryl-pyrimidin-2,4-yl)-diamine (2a). Com-
pound 1 (0.111 g, 0.44 mmol) was dissolved in 4 mL of
isobutylamine and heated with stirring in a sealed high-pressure
tube at 115 °C for 43 h. The reaction was allowed to cool to room
temperature and the excess amine removed in vacuo. The residue
was extracted from H2O three times with ether. The combined
organic layers were washed twice with brine, dried over MgSO4,
and the solvent removed by rotary evaporator. Purification was
accomplished with column chromatography (50% EtOAc in hex-
1
anes) to give 2a (67 mg, 47%). H NMR (500 MHz, CDCl3) δ
ppm 0.97 (d, J ) 6.65 Hz, 6 H), 0.98 (d, J ) 6.65 Hz, 6 H), 1.88
(sept, J ) 6.65 Hz, 1 H), 1.89 (sept, J ) 6.65 Hz, 1 H), 3.12 (bs,
2 H), 3.25 (t, J ) 6.32 Hz, 2 H), 4.78 (bs, 1 H), 4.90 (bs, 1 H),
5.74 (s, 1 H), 6.83 (d, J ) 15.87 Hz, 1 H), 7.28 (t, J ) 7.29 Hz,
1 H), 7.35 (t, J ) 7.50 Hz, 2 H), 7.55 (d, J ) 7.50 Hz, 2 H), 7.66
(d, J ) 15.86 Hz, 1 H). 13C NMR (500 MHz, CDCl3) δ ppm 20.5,
20.6, 28.7, 28.8, 49.3, 127.5, 127.7, 128.6, 128.9, 134.0, 136.8,
4. Electrostatic effects and/or hydrogen bonding that require
at least one free aryl N-H, the preferred position of which being
dependent on the size of the alkyl group at the 6-position of
the pyrimidine ring.
The best compounds we have obtained have Ki values (as
inhibitors of coactivator binding in vitro) and IC50 values (for
inhibition of reporter gene activity in cells) in the low micro-
molar region, and the vast majority of these compounds show
strong selectivity for ERR over ERꢀ. The most promising of
these compounds are being evaluated in further cell-based and
in other more biologically advanced activity assays. The results
+
162.8, 164.4. HRMS (ESI+) m/z calcd for C20H29N4 325.2392,
found 325.2393. Anal. (C20H28N4) H. C: calcd, 74.03; found, 73.44.
N: calcd, 17.27; found, 16.82.
2,4-Diisobutoxy-6-styryl-pyrimidine (2c). Clean Na0 (82 mg,
1.4 mmol) was dissolved in 2 mL of isobutyl alcohol while heating
at 70 °C with stirring (∼2 h). To the resulting slightly cloudy
solution, 150 mg (0.60 mmol) of 1 were added with stirring, forming
an opaque pale orange mixture. One more mL of isobutyl alcohol
was added, and the reaction was stirred at 70 °C for 4 h. The
reaction was allowed to cool to room temperature and extracted

Pyrimidine CoactiVator Binding Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6523
from H2O three times with ether. The combined organic layers were
washed with brine, dried over anhydrous MgSO4, and the solvent
and excess alcohol removed with a rotary evaporator. Purification
was accomplished with column chromatography (10% EtOAc in
6 H), 1.07 (d, J ) 6.65 Hz, 6 H), 1.95 (sept, J ) 6.65 Hz, 1 H),
2.01 (sept, J ) 6.65 Hz, 1 H), 2.86 (distorted dd, J ) 10.29, 9.22
Hz, 2 H), 3.00 (distorted dd, J ) 10.51, 8.79 Hz, 2 H), 3.07 (d, J
) 6.65 Hz, 4 H), 6.60 (s, 1 H), 7.20 (m, 3 H), 7.28 (t, J ) 7.40
Hz, 2 H). 13C NMR (500 MHz, CDCl3) δ ppm 22.1, 22.2, 28.6,
1
hexanes) to give 2c (0.172 g, 88%). H NMR (500 MHz, CDCl3)
δ ppm 1.01 (d, J ) 6.59 Hz, 6 H), 1.06 (d, J ) 6.59 Hz, 6 H), 2.08
(sept, J ) 6.84 Hz, 1 H), 2.19 (sept, J ) 6.84 Hz, 1 H), 4.14 (d,
J ) 6.59 Hz, 2 H), 4.18 (d, J ) 6.84 Hz, 2 H), 6.33 (s, 1 H), 6.93
(d, J ) 15.87 Hz, 1 H), 7.32 (t, J ) 7.20 Hz, 1 H), 7.37 (t, J )
7.32 Hz, 2 H), 7.57 (d, J ) 7.57 Hz, 2 H), 7.82 (d, J ) 15.87 Hz,
1 H). 13C NMR (500 MHz, CDCl3) δ ppm 19.4, 19.6, 28.0, 28.1,
72.9, 73.9, 99.6, 126.0, 127.7, 128.9, 129.1, 136.0, 136.1, 164.3,
165.3, 172.4. HRMS (ESI+) m/z calcd for C20H27N2O2+, 327.2073;
found, 327.2085. Anal. (C20H26N2O2) C, H, N.
N,N′-Dibenzyl-(6-phenethyl-pyrimidin-2,4-yl)-diamine (3b).
Compound 2b (80 mg, 0.20 mmol) and 10% Pd/C (22 mg, 0.020
mmol) were dissolved in 16 mL of MeOH. The resulting suspension
was stirred under 1 atm H2 for 1 h. The solvent was removed in
vacuo and the residue purified with column chromatography (25%
EtOAc in hexanes) to give 3b (42 mg, 52%). 1H NMR (500 MHz,
CDCl3) δ ppm 2.72 (distorted dd, J ) 10.51, 8.36 Hz, 2 H), 2.95
(distorted dd, J ) 10.51, 8.58 Hz, 2 H), 4.48 (d, J ) 4.07 Hz, 2
H), 4.61 (d, J ) 6.00 Hz, 2 H), 4.90 (bs, 1 H), 5.24 (bs, 1 H), 5.56
(s, 1 H), 7.16-7.37 (m, 15 H). 13C NMR (500 MHz, CDCl3) δ
ppm 34.9, 39.7, 45.6, 50.4, 126.1, 127.1, 127.6, 127.7, 127.8, 128.5,
128.6, 128.7, 128.9, 140.3, 141.9, 162.4. HRMS (ESI+) m/z calcd
for C26H27N4+, 395.2236; found, 395.2245.
28.7, 34.7, 37.6, 39.2, 39.5, 113.2, 126.3, 128.59, 128.64, 141.2,
+
167.3, 170.1, 171.5. HRMS (ESI+) m/z calcd for C20H29N2S2
,
361.1772; found, 361.1781.
2,4-Bis-(2-methyl-propane-1-sulfonyl)-6-phenethyl-pyrimidine (6a).
From the procedure described by Hurst,32,33 mCPBA (77%, 53 mg,
0.24 mmol) and 5b (18 mg, 0.050 mmol) were dissolved in 1.1
mL of CHCl3 with stirring. The solution was then allowed to stand
at room temperature for 36 h. CHCl3 (5 mL) was added, and the
solution was washed 2 times with saturated NaHCO3 solution. The
aqueous layers were extracted once with CHCl3 and the combined
organic layers were washed with saturated NaCl solution and dried
over MgSO4. The solvent was removed in vacuo, and chromatog-
raphy of the residue (30% EtOAc in hexanes) gave 6a in
1
quantitative yield. H NMR (500 MHz, CDCl3) δ ppm 1.10 (d, J
) 6.86 Hz, 6 H), 1.13 (d, J ) 6.86 Hz, 6 H), 2.29 (sept, J ) 6.65
Hz, 1 H), 2.40 (sept, J ) 6.65 Hz, 1 H), 3.16 (t, J ) 7.72 Hz, 2 H),
3.37 (m, 4 H), 3.42 (d, J ) 6.43 Hz, 2 H), 7.16 (d, J ) 7.08 Hz,
2 H), 7.22 (t, J ) 7.40 Hz, 1 H), 7.29 (t, J ) 7.40 Hz, 2 H), 7.91
(s, 1 H). 13C NMR (500 MHz, CDCl3) δ ppm 22.8, 22.9, 24.0,
24.1, 34.4, 40.0, 58.6, 58.7, 118.8, 127.0, 128.6, 129.0, 139.2, 166.4,
167.2, 177.2. HRMS (ESI+) m/z calcd for C20H29N2O4S2+, 425.1569;
found, 425.1588.
2,4-Dichloro-6-phenethyl-pyrimidine (4). Compound 1 (1.00
g, 4.0 mmol) and 10% Pd/C (118 mg, 0.11 mmol) were dissolved
in 40 mL of MeOH. The resulting suspension was stirred under 1
atm H2 for 1 h. The solvent was removed in vacuo and the residue
purified with column chromatography (10% EtOAc in hexanes) to
give 4 (0.521 mg, 52%). 1H NMR (500 MHz, CDCl3) δ ppm 3.06
(s, 4 H), 7.05 (s, 1 H), 7.17 (d, J ) 6.86 Hz, 2 H), 7.22 (t, J ) 7.29
Hz, 1 H), 7.29 (t, J ) 7.40 Hz, 2 H). 1H NMR (500 MHz, acetone-
d6) δ ppm 3.08 (AA′BB′, 2 H) 3.13 (AA′BB′, 2 H), 7.20 (t, J )
6.96 Hz, 1 H), 7.28 (m, 4 H), 7.53 (s, 1 H). 13C NMR (500 MHz,
CDCl3) δ ppm 34.4, 39.2, 119.4, 126.7, 128.5, 128.8, 139.9, 160.7,
162.5, 174.6. HRMS (ESI+) m/z calcd for C12H11N2Cl2+, 253.0299;
found, 253.0301.
(2-Chloro-6-styryl-pyrimidin-4-yl)-naphthalen-1-ylmethyl-car-
bamic Acid tert-Butyl Ester (7c). From the procedure described by
Zanda, et al.,30 compound 1 (0.700 g, 2.8 mmol) and naphthalen-
1-ylmethyl-carbamic acid tert-butyl ester (0.745 g, 3.06 mmol) were
dissolved in 20 mL DMF. To this solution, NaH (60% dispersion
in mineral oil, 0.134 g, 3.3 mmol) was added under nitrogen,
causing the solution to become a dark turquoise. The reaction was
stirred at room temperature for 1 h. The DMF was removed in
vacuo with heating and the residue extracted from brine twice with
ether and once with CH2Cl2. The combined organic layers were
washed with saturated NaCl solution and dried over MgSO4. The
solvent was removed in vacuo, and chromatography of the residue
(1:1 CH2Cl2:hexanes to CH2Cl2) gave 7c (573 mg, 44%). 1H NMR
(500 MHz, CDCl3) δ ppm 1.33 (s, 9 H), 5.79 (s, 2 H), 7.05 (d, J
) 15.87 Hz, 1 H), 7.21 (d, J ) 7.32 Hz, 1 H), 7.38 (m, 4 H),
7.50-7.62 (m, 4 H), 7.76 (d, J ) 8.06 Hz, 1 H), 7.91 (m, 2 H),
8.09 (d, J ) 8.30 Hz, 1 H), 8.10 (s, 1 H). 13C NMR (500 MHz,
CDCl3) δ ppm 28.0, 46.3, 83.5, 109.0, 122.99, 123.03, 125.2, 125.5,
125.8, 126.3, 127.6, 128.0, 128.99, 129.05, 129.7, 131.0, 133.6,
133.8, 135.7, 138.1, 153.4, 160.0, 162.7, 165.5. HRMS (ESI+) m/z
calcd for C28H27ClN3O2+, 472.1792; found, 472.1790.
2,4-Bis-benzyloxy-6-phenethyl-pyrimidine (5a). Clean Na0 (54
mg, 2.3 mmol) was dissolved in 2 mL of benzyl alcohol while
heating at 70 °C with stirring (∼2 h). To the resulting pale-yellow
solution, 100 mg (0.40 mmol) of 4 were added and the reaction
was stirred at 70 °C for 16 h. The reaction was allowed to cool to
room temperature and extracted from H2O three times with ether.
The combined organic layers were washed with brine, dried over
anhydrous MgSO4, and the solvent and excess alcohol removed in
vacuo (removal of the alcohol required heating to 80 °C).
Purification was accomplished with column chromatography (10%
EtOAc in hexanes) to give 5a (0.130 g, 83%). 1H NMR (500 MHz,
CDCl3) δ ppm 2.93 (distorted dd, J ) 9.77, 9.03 Hz, 2 H), 3.03
(distorted dd, J ) 10.25, 8.79 Hz, 2 H), 5.40 (s, 2 H), 5.45 (s, 2
H), 6.25 (s, 1 H), 7.20 (m, 3 H), 7.26-7.43 (m, 10 H), 7.51 (d, J
) 7.08 Hz, 2 H). 13C NMR (500 MHz, CDCl3) δ ppm 34.6, 39.3,
68.3, 69.2, 100.6, 126.2, 128.1, 128.2, 128.4, 128.56, 128.6, 128.7,
136.4, 137.0, 141.3, 164.8, 171.5, 172.3. HRMS (ESI+) m/z calcd
for C26H25N2O2+, 397.1916; found, 397.1916. Anal. (C26H24N2O2)
H, N. C: calcd, 78.76; found, 78.22.
(2-Benzylamino-6-styryl-pyrimidin-4-yl)-isobutyl-carbamic Acid
tert-Butyl Ester (8a). Compound 7a (0.059 g, 0.15 mmol) and
benzylamine (50 µL, 0.45 mmol) were dissolved in 3 mL of DMSO
and heated with stirring in a sealed high pressure tube at 90 °C for
16 h. As the reaction had not yet progressed to completion (as
shown by TLC), at this point the temperature was raised to 110 °C
and the solution was heated an additional 22 h. The DMSO was
removed in vacuo with heating, and the resulting residue was
extracted from brine three times with ether. The combined organic
layers were washed with brine and dried over MgSO4. The solvent
was removed in vacuo, and chromatography of the residue (5%
EtOAc in hexanes) produced 8a (49 mg, 70%). 1H NMR (500 MHz,
CDCl3) δ ppm 0.80 (d, J ) 6.59 Hz, 6 H), 1.55 (s, 9 H), 1.98
(sept, J ) 6.84 Hz, 1 H), 3.79 (d, J ) 7.32 Hz, 2 H), 4.65 (d, J )
5.86 Hz, 2 H), 5.35 (bs, 1 H), 6.94 (d, J ) 16.11 Hz, 1 H), 7.25 (t,
J ) 7.20 Hz, 1 H), 7.28-7.39 (m, 8 H), 7.55 (d, J ) 7.57 Hz, 2
H), 7.72 (d, J ) 15.87 Hz, 1 H). 13C NMR (500 MHz, CDCl3) δ
ppm 20.4, 28.2, 28.4, 45.8, 52.2, 81.9, 127.2, 127.49, 127.51, 127.6,
128.7, 128.86, 128.91, 135.0, 136.5, 140.0, 154.3, 161.8, 162.4,
163.6. HRMS (ESI+) m/z calcd for C28H35N4O2+, 459.2760; found,
459.2760.
2,4-Bis-isobutylsulfanyl-6-phenethyl-pyrimidine (5b). To a
suspension of 48 mg (1.2 mmol) NaH (60% dispersion in mineral
oil) in 5 mL of THF, 2-methyl-1-propanethiol (127 µL, 1.2 mmol)
was added and the resulting mixture was stirred for 30 min. To
this compound, 4 (0.100 g, 0.40 mmol) in 1 mL THF was added
and the suspension became a cloudy pink. The reaction was stirred
16 h at room temperature and then extracted from H2O three times
with CH2Cl2. The combined organic layers were washed with brine,
dried over anhydrous MgSO4, and the solvent removed with a rotary
evaporator. Purification was accomplished with column chroma-
tography (50% to 75% CH2Cl2 in hexanes) to give 5b (0.131 g,
1
92%). H NMR (500 MHz, CDCl3) δ ppm 1.04 (d, J ) 6.65 Hz,

6524 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Parent et al.
+
(2-Benzylamino-6-phenethyl-pyrimidin-4-yl)-isobutyl-carbam-
ic Acid tert-Butyl Ester (9a). Compound 8a(6 mg, 0.013 mmol)
and 10% Pd/C (2 mg, 0.002 mmol) were dissolved in 2 mL of
MeOH and 2 mL of CH2Cl2. The resulting suspension was stirred
under 1 atm H2 for 1 h. The solvent was removed in vacuo and the
residue purified with column chromatography (25% EtOAc in
hexanes) to give 9a in quantitative yield. 1H NMR (500 MHz,
CDCl3) δ ppm 0.78 (d, J ) 6.84, 6 H), 1.51 (s, 9 H), 1.95 (sept, J
) 6.84 Hz, 1 H), 2.84 (m, 2 H), 2.99 (distorted dd, J ) 11.23,
8.79 Hz, 2 H), 3.76 (d, J ) 7.32 Hz, 2 H), 4.60 (d, J ) 5.86 Hz,
2 H), 5.32 (bs, 1 H), 7.08 (s, 1 H), 7.16-7.35 (m, 10 H). 13C NMR
(500 MHz, CDCl3) δ ppm 20.4, 28.2, 28.4, 35.2, 40.2, 45.7, 52.2,
160.5, 162.4, 176.4. HRMS (ESI+) m/z calcd for C9H13N2Cl2
219.0456; found, 219.0462.
,
2,4-Dibenzylamino-6-isopentylpyrimidine (13c). Compound
12b (0.071 g, 0.32 mmol) was dissolved in benzylamine (1.5 mL)
and heated sequentially with stirring in a sealed high-pressure tube
at 110 °C for 5 h, 140 °C for 6 h, and finally 155 °C for 13 h. The
solution was cooled and extracted from saturated NaHCO3 solution
once with CHCl3 and twice with CH2Cl2. The combined organic
layers were washed with saturated NaHCO3 solution, H2O, and
brine, dried over anhydrous MgSO4, and the solvent was removed
with a rotary evaporator. Purification was accomplished with
column chromatography (50% EtOAc in hexanes) to give 13c in
1
81.8, 102.6, 126.1, 127.2, 127.4, 128.57, 128.65, 128.7, 133.6,
quantitative yield. H NMR (500 MHz, CDCl3) δ ppm 0.91 (d, J
+
135.0, 140.0, 154.2. HRMS (ESI+) m/z calcd for C28H37N4O2
,
) 6.65 Hz, 6 H), 1.51 (m, 2 H), 1.59 (sept, J ) 6.65 Hz, 1 H),
2.42 (t, J ) 8.15 Hz, 2 H), 4.49 (bs.d, J ) 5.15 Hz, 2 H), 4.59 (d,
J ) 6.00 Hz, 2 H), 4.98 (bs, 1 H), 5.20 (bs, 1 H), 5.61 (s, 1 H),
7.21-7.38 (m, 10 H). 13C NMR (500 MHz, CDCl3) δ ppm 22.7,
28.1, 36.0, 37.9, 45.6, 53.3, 105.5, 127.0, 127.1, 127.5, 127.71,
127.74, 128.4, 128.57, 128.6, 128.8, 140.3, 162.4, 163.8, 170.4.
HRMS (ESI+) m/z calcd for C23H29N4+, 361.2392; found, 361.2386.
Anal. (C23H28N4) H, N. C, calcd, 76.63; found, 77.36.
461.2917; found, 461.2920.
N2-Benzyl-N4-isobutyl-6-styryl-pyrimidine-2,4-diamine(10a).Com-
pound 8a (16 mg, 0.035 mmol) was dissolved in 1 mL of CH2Cl2,
and to this solution, 1 mL of TFA was added with stirring, causing
the solution to become bright yellow. The reaction was stirred at
room temperature for 30 min, and the solvent was removed in
vacuo. The resulting residue was extracted from H2O three times
with ether. The combined organic layers were washed with brine,
dried over MgSO4, and the solvent removed by rotary evaporator.
Column chromatography of the residue (40% EtOAc in hexanes)
gave 10a (12 mg, 98%). 1H NMR (500 MHz, CDCl3) δ ppm 0.96
(d, J ) 6.59 Hz, 6 H), 1.86 (sept, J ) 6.59 Hz, 1 H), 3.12 (bs, 2
H), 4.66 (d, J ) 5.86 Hz, 2 H), 4.74 (bs, 1 H), 5.12 (bs, 1 H), 5.78
(s, 1 H), 6.83 (d, J ) 15.87 Hz, 1 H), 7.23-7.42 (m, 8 H), 7.54 (d,
J ) 7.57 Hz, 2 H), 7.67 (d, J ) 15.87 Hz, 1 H). 13C NMR (500
MHz, CDCl3) δ ppm 20.5, 28.7, 45.8, 127.1, 127.5, 127.6, 127.8,
128.6, 128.9, 134.1, 136.8, 140.4, 162.5, 164.5. HRMS (ESI+) m/z
calcd for C23H27N4+, 359.2236; found, 359.2254.
2,4-Diisobutoxy-6-(3-methyl-butyl)-pyrimidine (13e). Clean
Na0 (63 mg, 2.7 mmol) was dissolved in 2 mL of isobutyl alcohol
while heating at 70 °C. To this solution, 12b (0.100 g, 0.46 mmol)
dissolved in 0.5 mL of isobutyl alcohol was added and the solution
became a cloudy pale orange. The reaction was stirred at 70 °C
for 12 h and allowed to cool to room temperature. The solution
was extracted from H2O three times with ether. The combined
organic layers were washed with brine, dried over anhydrous
MgSO4, and the solvent removed with a rotary evaporator.
Purification was accomplished with column chromatography (10%
EtOAc in hexanes) to give 13e (0.102 g, 76%). 1H NMR (500 MHz,
CDCl3) δ ppm 0.90 (d, J ) 6.22 Hz, 6 H), 0.97 (d, J ) 6.65 Hz,
6 H), 1.00 (d, J ) 6.65 Hz, 6 H), 1.49-1.64 (m, 3 H), 2.03 (sept,
J ) 6.86 Hz, 1 H), 2.10 (sept, J ) 6.65 Hz, 1 H), 2.55 (t, J ) 7.72
Hz, 2 H), 4.06 (d, J ) 6.65 Hz, 2 H), 4.07 (d, J ) 6.65 Hz, 2 H),
6.16 (s, 1 H). 13C NMR (500 MHz, CDCl3) δ ppm 19.4, 19.6,
22.6, 27.97, 28.04, 28.06, 35.70, 35.75, 72.6, 73.7, 99.4, 165.3,
171.9, 173.6. HRMS (ESI+) m/z calcd for C17H31N2O2+, 295.2386;
found, 295.2392. Anal. (C17H30N2O2) H, N. C: calcd, 69.35; found,
69.86.
N2-Benzyl-N4-isobutyl-6-phenethyl-pyrimidine-2,4-diamine (11a).
Compound 10a (9 mg, 0.025 mmol) and 10% Pd/C (12 mg, 0.011
mmol) were dissolved in 4 mL MeOH. The resulting suspension
was stirred under 1 atm H2 for 1 h. The solvent was removed in
vacuo and the residue purified with column chromatography (50%
EtOAc in hexanes) to give 11a in quantitative yield. 1H NMR (500
MHz, CDCl3) δ ppm 0.92 (d, J ) 6.59 Hz, 6 H), 1.80 (sept, J )
6.84 Hz, 1 H), 2.73 (distorted dd, J ) 10.25, 8.30 Hz, 2 H), 2.97
(distorted dd, J ) 10.50, 8.55 Hz, 2 H), 3.03 (bs, 2 H), 4.61 (d, J
) 5.86 Hz, 2 H), 4.63 (bs, 1 H), 5.08 (bs, 1 H), 5.52 (s, 1 H),
7.16-7.34 (m, 8 H), 7.37 (d, J ) 6.84 Hz, 2 H). 13C NMR (500
MHz, CDCl3) δ ppm 20.5, 28.6, 35.0, 39.8, 45.7, 126.0, 127.1,
127.8, 128.5, 128.6, 128.7, 142.0, 162.5, 164.1. HRMS (ESI+) m/z
calcd for C23H29N4+, 361.2392; found, 361.2398.
2,4-Bis-isobutylsulfanyl-6-(3-methyl-butyl)-pyrimidine (13g). To
a suspension of 41 mg (1.03 mmol) NaH in 4 mL of THF, 2-methyl-
1-propanethiol (93 mg, 1.03 mmol) was added and the resulting
mixture was stirred for 20 min. To this, 12b (0.078 g, 0.35 mmol)
in 3 mL of THF was added and the suspension became a cloudy
yellow. The reaction was stirred 16 h at room temperature and then
extracted from H2O three times with ether. The combined organic
layers were washed with brine, dried over anhydrous MgSO4, and
the solvent removed with a rotary evaporator. Purification was
accomplished with column chromatography (50% CH2Cl2 in
hexanes) to give 13g (0.114 g, 98%). 1H NMR (500 MHz, CDCl3)
δ ppm 0.91 (d, J ) 6.35 Hz, 6 H), 1.03 (d, J ) 6.59 Hz, 6 H), 1.04
(d, J ) 6.59 Hz, 6 H), 1.49-1.63 (m, 3 H), 1.89-2.03 (m, 2 H),
2.54 (t, J ) 7.81 Hz, 2 H), 3.05 (d, J ) 6.84 Hz, 2 H), 3.08 (d, J
) 6.84 Hz, 2 H), 6.64 (s, 1 H). 13C NMR (500 MHz, CDCl3) δ
2,4-Dichloro-6-(3-methyl-butyl)-pyrimidine (12b). On the basis
of methodology described by Fu¨rstner et al.,28,34,35 isopentylmag-
nesium bromide was formed by slow addition without stirring of
isoamylbromide (5.0 mL, 41.7 mmol) to the bottom of a three-
neck flask fitted with a condenser that contained magnesium metal
(1.116 g, 45.9 mmol) and 1,2-dibromoethane (500 µL, 5.8 µmol)
in ∼100 mL of diethyl ether. Once bubbles began to form, the
solution was stirred until evolution ceased (1.5 h). Before addition
to the following reaction mixture, the Grignard was titrated using
menthol and 1,10-phenanthroline dissolved in THF. In a separate
flask, 2,4,6-trichloropyrimidine (2.20 g,. 11.99 mmol), Fe(acac)3
(0.217 g, 0.614 mmol), and N-methyl-2-pyrolidinone (6.5 mL) were
combined in dry THF (85 mL), producing an orange solution. To
this solution, the isopentylmagnesium bromide (66 mL, 0.181 M
in ether, 11.05 mmol) was added dropwise over the course of 20
min, immediately producing a cloudy dark-orange precipitate. The
reaction was stirred for 1 h at room temperature and then quenched
with 1 M aqueous HCl. The mixture was extracted three times with
ether, and the combined organic layers were washed with saturated
NaHCO3 solution and brine, dried over MgSO4, and the solvent
removed in vacuo. Purification was accomplished by column
chromatography (CH2Cl2) to give 12b as the major product (1.377
ppm 22.15, 22.22, 22.6, 28.0, 28.7, 35.6, 37.6, 37.8, 39.5, 112.9,
+
168.9, 169.8, 171.3. HRMS (ESI+) m/z calcd for C17H31N2S2
327.1929; found, 327.1941.
,
4-(3-Methyl-butyl)-2,6-bis-(2-methyl-propane-1-sulfonyl)-pyri-
midine (14a). From the procedure described by Hurst,32,33 mCPBA
(77%, 142 mg, 0.63 mmol) in 1.5 mL of CHCl3 was added to a
solution of 13g (43 mg, 0.13 mmol) in 1 mL of CHCl3 with stirring.
The solution was then allowed to stand at room temperature for
36 h. CHCl3 (10 mL) was added, and the solution was washed two
times with saturated NaHCO3 solution. The aqueous layers were
extracted once with CHCl3, and the combined organic layers were
washed with saturated NaCl solution and dried over MgSO4. The
solvent was removed in vacuo, and chromatography of the residue
(25% EtOAc in hexanes) gave 14a (0.026 g, 50%). 1H NMR (500
MHz, CDCl3) δ ppm 0.97 (d, J ) 6.43 Hz, 6 H), 1.13 (d, J ) 6.86
1
g, 52%). H NMR (500 MHz, CDCl3) δ ppm 0.91 (d, J ) 6.43
Hz, 6 H), 1.58 (m, 3 H), 2.71 (AA′XX′, 2 H), 7.14 (s, 1 H). 13C
NMR (500 MHz, CDCl3) δ ppm 22.4, 28.0, 35.7, 37.69, 118.9,

Pyrimidine CoactiVator Binding Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6525
Hz, 6 H), 1.14 (d, J ) 6.86 Hz, 6 H), 1.62-1.72 (m, 3 H), 2.36
(sept, J ) 6.86 Hz, 1 H), 2.41 (sept, J ) 6.86 Hz, 1 H), 3.03 (m,
2 H), 3.39 (d, J ) 6.65 Hz, 2 H), 3.46 (d, J ) 6.65 Hz, 2 H), 8.02
(s, 1 H). 13C NMR (500 MHz, CDCl3) δ ppm 22.5, 22.87, 22.92,
24.0, 24.2, 28.2, 36.7, 37.7, 58.5, 58.7, 118.4, 166.3, 167.2, 179.0.
HRMS (ESI+) m/z calcd for C17H31N2O4S2+, 391.1725; found,
391.1732.
N4-Benzyl-N2-isobutyl-6-(3-methyl-butyl)-pyrimidine-2,4-di-
amine (18d). Compound 17c (0.030 g, 0.071 mmol) was dissolved
in 1.5 mL of CH2Cl2 followed by addition of 1.5 mL of TFA with
stirring. The reaction was stirred at room temperature for 1.5 h,
and the solvent and excess TFA were removed by rotary evaporator.
Direct purification of the residue was accomplished with column
chromatography (20% EtOAc and 2% TEA in hexanes) to give
18d in quantitative yield. 1H NMR (500 MHz, CDCl3) δ ppm 0.90
(d, J ) 6.65 Hz, 6 H), 0.93 (d, J ) 6.86 Hz, 6 H), 1.49 (m, 2 H),
1.58 (sept, J ) 6.65 Hz, 1 H), 1.82 (sept, J ) 6.65 Hz, 1 H), 2.39
(m, 2 H), 3.18 (t, J ) 6.32 Hz, 2 H), 4.50 (d, J ) 5.15 Hz, 2 H),
4.84 (bs, 1 H), 4.88 (bs, 1 H), 5.56 (s, 1 H), 7.30 (m, 5 H). 13C
NMR (500 MHz, CDCl3) δ ppm 20.5, 22.7, 28.2, 28.8, 36.1, 38.0,
45.5, 49.2, 91.9, 127.5, 127.7, 128.8, 162.8, 163.8. HRMS (ESI+)
m/z calcd for C20H31N4+, 327.2549; found, 327.2553.
(2,6-Dichloro-pyrimidin-4-yl)-isobutyl-carbamic Acid tert-Bu-
tyl Ester (15a). On the basis of the method described by Zanda et
al.,30 2,4,6-trichloropyrimidine (0.500 g, 2.73 mmol) and N-isobutyl-
tert-butyl carbamate (0.473 g, 2.73 mmol) were dissolved in 15
mL of DMF. NaH (60% dispersion in mineral oil, 0.163 g, 4.07
mmol) was added, and the solution turned yellow. The reaction
was stirred at room temperature overnight, quenched with saturated
NH4Cl solution and then extracted three times with ether. The
combined organic phases were washed with saturated NaCl solution
and dried over MgSO4. The solvent was removed in vacuo, and
column chromatography (1:1 CH2Cl2:hexanes) of the residue
produced 15a (0.642 g, 74%). 1H NMR (500 MHz, CDCl3) δ ppm
0.89 (d, J ) 6.86 Hz, 6 H), 1.54 (s, 9 H), 2.03 (sept, J ) 6.86 Hz,
1 H), 3.87 (d, J ) 7.29 Hz, 2 H), 8.08 (s, 1 H). 13C NMR (500
[6-Chloro-2-(3-methyl-butyl)-pyrimidin-4-yl]-isobutyl-carbam-
ic Acid tert-Butyl Ester (19a). Formed as a byproduct from the
1
synthesis of 16b (30 mg, 12%). H NMR (500 MHz, CDCl3) δ
ppm 0.89 (d, J ) 6.84 Hz, 6 H), 0.93 (d, J ) 6.35 Hz, 6 H), 1.55
(s, 9 H), 1.60 (sept, J ) 6.59 Hz, 1 H), 1.66 (m, 2 H), 2.02 (sept,
J ) 6.84 Hz, 1 H), 2.82 (distorted t, J ) 8.06 Hz, 2 H), 3.92 (d, J
) 7.08 Hz, 2 H), 7.88 (s, 1 H). 13C NMR (500 MHz, CDCl3) δ
ppm 20.4, 22.7, 27.9, 28.2, 28.3, 37.26, 37.32, 52.5, 82.9, 109.7,
153.8, 160.9, 161.6, 171.1. HRMS (ESI+) m/z calcd for
C18H31N3O2Cl+, 356.2105; found, 356.2099.
MHz, CDCl3) δ ppm 20.3, 28.0, 28.2, 52.7, 83.8, 110.4, 153.3,
+
158.9, 162.0, 163.0. HRMS (ESI+) m/z calcd for C13H20N3O2Cl2
,
320.0933; found, 320.0939.
[2-Chloro-6-(3-methyl-butyl)-pyrimidin-4-yl]-isobutyl-carbam-
ic Acid tert-Butyl Ester (16b). On the basis of the methodology
described by Fu¨rstner et al.,28,34,35 isopentylmagnesium bromide
was formed by slow addition without stirring of isoamylbromide
(0.700 mL, 5.84 mmol) to the bottom of a three-neck flask fitted
with a condenser that contained magnesium metal (0.50 g, 20.6
mmol) and 1,2-dibromoethane (75 µL, 0.87 µmol) in ∼20 mL of
diethyl ether. Once bubbles began to form, the solution was stirred
until evolution ceased (1.5 h). Before addition to the following
reaction mixture, the Grignard was titrated using menthol and 1,10-
phenanthroline dissolved in THF. Compound 15a (0.220 g,. 0.69
mmol), Fe(acac)3 (0.017 g, 0.048 mmol), and N-methyl-2-pyroli-
dinone (0.4 mL) were combined in dry THF (5.0 mL). To this
solution, isopentylmagnesium bromide (0.29 M in ether, 3.55 mL,
1.03 mmol) was added dropwise, causing a progression from the
starting brown solution to a cloudy orange, followed by a dark-
brown and finally a clear solution containing solid salts. The reaction
was stirred for 4 h at room temperature and then quenched with 1
M aqueous HCl. The mixture was extracted three times with ether,
and the combined organic layers were washed with brine, dried
over MgSO4, and the solvent removed in vacuo. Purification was
accomplished by column chromatography (CH2Cl2 followed by 3%
EtOAc in CH2Cl2) to give 16b (128 mg, 52%). 1H NMR (500 MHz,
CDCl3) δ ppm 0.88 (d, J ) 6.59 Hz, 6 H), 0.92 (d, J ) 6.35 Hz,
6 H), 1.52-1.67 (m, 12 H), 2.03 (sept, J ) 6.84 Hz, 1 H), 2.66
(m, 2 H), 3.87 (d, J ) 7.08 Hz, 2 H), 7.78 (s, 1 H). 13C NMR (500
MHz, CDCl3) δ ppm 20.3, 22.6, 28.2, 28.3, 36.3, 37.2, 38.2, 52.5,
82.9, 109.8, 153.7, 159.3, 162.6, 174.4. HRMS (ESI+) m/z calcd
for C18H31N3O2Cl+, 356.2105; found, 356.2114.
[2-Benzylamino-6-(3-methyl-butyl)-pyrimidin-4-yl]-isobutyl-car-
bamic Acid tert-Butyl Ester (17a). Compound 16b (0.020 g, 0.056
mmol) was dissolved in benzylamine (1 mL) and heated with
stirring in a sealed high-pressure tube at 100 °C for 24 h. The
solution was cooled and the excess amine removed in vacuo with
heating. The residue was extracted from saturated NaHCO3 solution
three times with CH2Cl2. The combined organic layers were washed
with H2O and brine, dried over anhydrous MgSO4, and the solvent
removed with a rotary evaporator. Purification was accomplished
with column chromatography (15% EtOAc in hexanes) to give 17a
in quantitative yield. 1H NMR (500 MHz, CDCl3) δ ppm 0.78 (d,
J ) 6.86 Hz, 6 H), 0.92 (d, J ) 6.43 Hz, 6 H), 1.50-1.66 (m, 12
H), 1.95 (sept, J ) 6.86 Hz, 1 H), 2.53 (m, 2 H), 3.75 (d, J ) 7.29
Hz, 2 H), 4.58 (d, J ) 6.00 Hz, 2 H), 5.31 (bs, 1 H), 7.06 (s, 1 H),
7.21-7.34 (m, 5 H). 13C NMR (500 MHz, CDCl3) δ ppm 20.3,
22.7, 28.2, 28.3, 28.4, 36.5, 38.3, 45.6, 52.2, 81.7, 127.1, 127.4,
128.7, 140.0, 145.3, 154.3, 161.7, 161.8, 172.6. HRMS (ESI+) m/z
calcd for C25H39N4O2+, 427.3073; found, 427.3068.
Isobutyl-[6-Isobutylamino-2-(3-methyl-butyl)-pyrimidin-4-yl]-
carbamic Acid tert-Butyl Ester (20a). Compound 19a (0.019 g,
0.053 mmol) was dissolved in isobutylamine (2 mL) and heated
with stirring in a sealed high-pressure tube at 100 °C for 16 h. The
solution was cooled and the excess amine removed in vacuo. Direct
purification of the residue was accomplished with column chro-
matography (15% EtOAc in hexanes) to give 20a (18 mg, 88%).
1H NMR (500 MHz, CDCl3) δ ppm 0.86 (d, J ) 6.84 Hz, 6 H),
0.92 (d, J ) 6.35 Hz, 6 H), 0.98 (d, J ) 6.84 Hz, 6 H), 1.52 (s, 9
H), 1.62 (m, 3 H), 1.86 (sept, J ) 6.59 Hz, 1 H), 1.95 (m, J )
6.84 Hz, 1 H), 2.63 (distorted t, J ) 8.06 Hz, 2 H), 3.06 (t, J )
5.74 Hz, 2 H), 3.87 (d, J ) 7.32 Hz, 2 H), 4.89 (bs, 1 H), 6.68 (s,
1 H). 13C NMR (500 MHz, CDCl3) δ ppm 20.4, 20.6, 22.8, 28.0,
28.4, 28.51, 28.54, 37.4, 49.5, 52.4, 81.4, 110.0, 157.3, 164.1.
N,N′-Diisobutyl-2-(3-methyl-butyl)-pyrimidine-4,6-diamine (21a).
Compound 20a (0.018 g, 0.047 mmol) was dissolved in 2 mL of
CH2Cl2 followed by addition of 2 mL TFA with stirring. The
reaction was stirred at room temperature for 1 h, and the solvent
and excess TFA were removed by rotary evaporator. Direct
purification of the residue was accomplished with column chro-
matography (25% EtOAc and 2% TEA in hexanes) to give 21a (6
1
mg, 47%). H NMR (500 MHz, CDCl3) δ ppm 0.92 (d, J ) 6.35
Hz, 6 H), 0.99 (d, J ) 6.84 Hz, 12 H), 1.61 (m, 3 H), 1.87 (sept,
J ) 6.84 Hz, 2 H), 2.51 (m, 2 H), 2.98 (t, J ) 6.10 Hz, 4 H), 4.83
(bs, 2 H), 5.05 (s, 1 H). 13C NMR (500 MHz, CDCl3) δ ppm 20.6,
22.8, 28.48, 28.54, 37.7, 37.9, 49.8, 110.0, 163.6. HRMS (ESI+)
m/z calcd for C17H33N4+, 293.2705; found, 293.2696.
2-Chloro-4,6-distyryl-pyrimidine (22a). On the basis of the
coupling described by Tan et al.,29 trans-2-phenylvinylboronic acid
(0.148 g, 1.00 mmol), K3PO4 (0.430 g, 2.03 mmol), and
PdCl2(PPh3)2 (0.010 g, 0.015 mmol) were combined in a 25 mL
round-bottom flask. To this mixture, 2,4,6-trichloropyrimidine
(0.093 g, 0.50 mmol) dissolved in THF (1.5 mL) was added,
producing a cloudy suspension. More THF (4.5 mL) and H2O (0.11
mL) were added, producing a white cloudy suspension, which was
heated at reflux for 19 h. Then 0.5 mL more of H2O was added,
eventually causing the precipitate to dissolve and the solution to
turn dark orange. After five more hours at reflux, the solution was
allowed to cool to room temperature. Then 35 mL of H2O were
added and the biphasic mixture was extracted three times with ether.
The combined organic layers were washed with brine, dried over
anhydrous MgSO4, and the solvent removed with a rotary evapora-
tor. The product was recrystallized from 5% EtOAc/hexanes and
the filtrate purified by column chromatography (5% EtOAc in
hexanes) to provide 22a (0.084 g, 52%). 1H NMR (500 MHz,
CDCl3) δ ppm 7.04 (d, J ) 15.87 Hz, 2 H), 7.20 (s, 1 H), 7.36-7.45

6526 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Parent et al.
(m, 6 H), 7.62 (d, J ) 6.84 Hz, 4 H), 7.95 (d, J ) 15.87 Hz, 2 H).
13C NMR (500 MHz, CDCl3) δ ppm 114.9, 124.6, 128.1, 129.2,
130.0, 135.5, 139.0, 161.7, 165.9. HRMS (ESI+) m/z calcd for
C20H16N2Cl+, 319.1002; found, 319.1005.
2-Chloro-4,6-bis-(3-methyl-butyl)-pyrimidine (22b). Formed
as a byproduct from the synthesis of 12b (279 mg, 9%). 1H NMR
(500 MHz, CDCl3) δ ppm 0.91 (d, J ) 6.22 Hz, 12 H), 1.53-1.63
(m, 6 H), 2.67 (distorted t, J ) 8.36 Hz, 4 H), 6.93 (s, 1 H). 13C
NMR (500 MHz, CDCl3) δ ppm 22.5, 28.2, 35.8, 38.0, 117.5, 161.0,
174.6. HRMS (ESI+) m/z calcd for C14H24N2Cl+, 255.1628; found,
255.1620.
with ether and once with CH2Cl2. The combined organic layers
were washed with brine, dried over anhydrous MgSO4, and the
solvent and excess alcohol removed with a rotary evaporator.
Purification was accomplished with column chromatography (5%
EtOAc in hexanes) to give 25c (0.151 g, 90%). 1H NMR (500 MHz,
CDCl3) δ ppm 0.96 (d, J ) 6.65 Hz, 12 H), 0.99 (d, J ) 6.65 Hz,
6 H), 2.03 (sept, J ) 6.65 Hz, 2 H), 2.11 (sept, J ) 6.65 Hz, 1 H),
4.02 (d, J ) 6.86 Hz, 4 H), 4.05 (d, J ) 6.86 Hz, 2 H), 5.66 (s, 1
H). 13C NMR (500 MHz, CDCl3) δ ppm 19.4, 19.5, 28.0, 28.1,
73.1, 73.9, 83.4, 164.9, 172.8. HRMS (ESI+) m/z calcd for
C16H29N2O3+, 297.2178; found, 297.2179. Anal. (C16H28N2O3) H,
N. C: calcd, 64.83; found, 65.26.
[4,6-Bis-(3-methyl-butyl)-pyrimidin-2-yl]-isobutyl-amine (23a).
Compound 22b (0.050 g, 0.20 mmol) was added to 1.5 mL of
isobutylamine, immediately forming a bright-yellow solution. With
stirring at room temperature (∼5 min), the solution became a light
brown. The reaction mixture was heated in a sealed high-pressure
tube at 95 °C for 16 h. The reaction was cooled, and the excess
isobutylamine was removed in vacuo. The resulting residue was
extracted from H2O three times with ether. The combined organic
layers were washed with brine, dried over anhydrous MgSO4, and
the solvent removed with a rotary evaporator. Purification was
accomplished with column chromatography (10% EtOAc in hex-
anes) to give 23a in quantitative yield. 1H NMR (500 MHz, CDCl3)
δ ppm 0.92 (d, J ) 6.35 Hz, 12 H), 0.95 (d, J ) 6.84 Hz, 6 H),
1.50-1.64 (m, 6 H), 1.85 (sept, J ) 6.59 Hz, 1 H), 2.50 (m, 4 H),
3.24 (dd, J ) 6.71, 5.98 Hz, 2 H), 4.99 (bt, J ) 5.37 Hz, 1 H),
6.24 (s, 1 H). 13C NMR (500 MHz, CDCl3) δ ppm 20.5, 22.7,
28.1, 28.8, 36.0, 38.0, 49.2, 108.1, 162.8, 171.7. HRMS (ESI+)
m/z calcd for C18H34N3+, 292.2753; found, 292.2755.
(4,6-Diphenethyl-pyrimidin-2-yl)-isobutyl-amine (24a). Com-
pound 23d (0.089 g, 0.25 mmol) was dissolved in a suspension of
13 mg (0.012 mmol) of 10% Pd/C in 20 mL of MeOH, forming a
bright-yellow mixture. The suspension was stirred under 1 atm of
H2 for 45 min, producing a clear mixture. The MeOH was removed
in vacuo and the residue purified with column chromatography (25%
EtOAc in hexanes) to give 24a (57 mg, 64%). 1H NMR (500 MHz,
CDCl3) δ ppm 1.00 (d, J ) 6.65 Hz, 6 H), 1.90 (sept, J ) 6.65
Hz, 1 H), 2.82 (distorted dd, J ) 10.29, 8.79 Hz, 4 H), 2.98
(distorted dd, J ) 10.29, 8.79 Hz, 4 H), 3.30 (t, J ) 6.22 Hz, 2 H),
5.10 (t, J ) 5.15 Hz, 1 H), 6.19 (s, 1 H), 7.20 (m, 6 H), 7.29 (m,
4 H). 13C NMR (500 MHz, CDCl3) δ ppm 20.5, 28.8, 34.9, 39.6,
49.2, 108.7, 126.2, 128.56, 128.62, 141.7, 162.9, 170.3. HRMS
(ESI+) m/z calcd for C24H30N3+, 360.2440; found, 360.2435. Anal.
(C24H29N3) C, H, N.
2,4,6-Tris-isobutylsulfanyl-pyrimidine (25e). To a suspension
of 147 mg NaH (3.67 mmol, 60% dispersion in mineral oil) in 15
mL of THF, 2-methyl-propane-1-thiol (0.35 mL, 3.3 mmol) was
added dropwise and the resulting mixture was stirred for 20 min.
To this, 2,4,6-trichloropyrimidine (0.150 g, 0.82 mmol) was added.
The reaction was stirred 16 h at room temperature, quenched with
H2O, and extracted three times with ether. The combined organic
layers were washed with brine, dried over anhydrous MgSO4, and
the solvent removed with a rotary evaporator. Purification was
accomplished with column chromatography (50% CH2Cl2 in
hexanes) to give 25e (0.226 g, 94%). 1H NMR (500 MHz, CDCl3)
δ ppm 1.01 (d, J ) 6.65 Hz, 12 H), 1.04 (d, J ) 6.65 Hz, 6 H),
1.92 (sept, J ) 6.65 Hz, 2 H), 1.98 (sept, J ) 6.65 Hz, 1 H), 3.02
(d, J ) 6.86 Hz, 2 H), 3.03 (d, J ) 6.86 Hz, 4 H), 6.64 (s, 1 H).
13C NMR (500 MHz, CDCl3) δ ppm 22.1, 22.2, 28.6, 28.7, 37.8,
+
39.5, 110.8, 168.0, 170.9. HRMS (ESI+) m/z calcd for C16H29N2S3
,
345.1493; found, 345.1479. Anal. (C16H28N2S3) C, H, N.
2,4-Diisobutylamino-6-chloropyrimidine (26). Isobutylamine
(1 mL) was added dropwise to 0.337 g (1.8 mmol) of 2,4,6-
trichloropyrimidine, producing a violent reaction accompanied by
the evolution of white smoke. The resulting mixture was heated in
a sealed pressure tube at 75 °C for 1 h. The reaction was cooled to
room temperature and extracted three times from saturated NaHCO3
solution with CH2Cl2. The combined organic layers were washed
with brine, dried over anhydrous MgSO4, and the solvent removed
with a rotary evaporator. Purification was accomplished with
column chromatography (5% EtOAc in hexanes) to give 26 (0.216
1
g, 45%). H NMR (500 MHz, CDCl3) δ ppm 0.90 (d, J ) 6.59
Hz, 6 H), 0.92 (d, J ) 6.59 Hz, 6 H), 1.76-1.88 (m, 2 H), 3.03
(bs, 2 H), 3.15 (t, J ) 6.35 Hz, 2 H), 4.98 (bs, 1 H), 5.16 (bs, 1 H),
5.66 (s, 1 H). 13C NMR (500 MHz, CDCl3) δ ppm 20.4, 28.5,
28.6, 49.0, 162.1, 164.4. HRMS (ESI+) m/z calcd for C12H22N4Cl+,
257.1533; found, 257.1522.
N,N′,N′′-Triisobutyl-pyrimidine-2,4,6-triamine (25a). 2,4,6-
Trichloropyrimidine (0.182 g, 0.11 mmol) was added to 4 mL of
isobutylamine, producing a violent reaction accompanied by the
evolution of light smoke. The reaction mixture was heated with
stirring in a sealed high-pressure tube at 115 °C for 43 h. The
reaction was removed from the heat, allowed to cool to room
temperature, and the excess amine was removed in vacuo. The
resulting residue was extracted from H2O three times with CH2Cl2.
The combined organic layers were washed with saturated NaHCO3
and brine and dried over anhydrous MgSO4. The solvent was
removed with a rotary evaporator and purification of the residue
was accomplished with column chromatography (100% EtOAc) to
give 25a (175 mg, 60%). 1H NMR (500 MHz, CDCl3) δ ppm 0.91
(d, J ) 6.84 Hz, 6 H), 0.94 (d, J ) 6.59 Hz, 12 H), 1.81 (m, 3 H),
2.96 (t, J ) 6.35 Hz, 4 H), 3.12 (t, J ) 6.59 Hz, 2 H), 4.52 (m, 3
H), 4.74 (s, 1 H). 13C NMR (500 MHz, CDCl3) δ ppm 20.5, 20.6,
28.6, 28.8, 49.1, 49.6, 71.6, 162.4, 164.5. HRMS (ESI+) m/z calcd
for C16H32N5+, 294.2658; found, 294.2656. Anal. (C16H31N5) C,
H, N.
2,4-Diisobutylamino-pyrimidine (27d). Compound 26 (0.035
g, 0.14 mmol) was dissolved in a suspension of 3 mL of MeOH,
23 µL of Et3N, and 24 mg (0.022 mmol) of 10% Pd/C. The
suspension was stirred under 1 atm of H2 for 24 h. The reaction
was filtered using a 2 µM syringe filter and the solvent removed
using a rotary evaporator. The residue was extracted three times
from H2O with CH2Cl2. The combined organic layers were washed
with brine, dried over anhydrous MgSO4, and the solvent removed
in vacuo. Purification was accomplished with column chromatog-
raphy (5% EtOAc and 1% TEA in hexanes) to give 27d (19 mg,
1
63%). H NMR (500 MHz, CDCl3) δ ppm 0.95 (d, J ) 6.84 Hz,
6 H), 0.95 (d, J ) 6.59 Hz, 6 H), 1.80-1.90 (m, 2 H), 3.07 (bs, 2
H), 3.17 (dd, J ) 6.71, 5.98 Hz, 2 H), 4.73 (bs, 1 H), 4.86 (s, 1 H),
5.66 (d, J ) 5.86 Hz, 1 H), 7.81 (bs, 1 H). 13C NMR (500 MHz,
CDCl3) δ ppm 20.47, 20.51, 28.6, 28.7, 49.1, 156.3, 162.6, 163.4.
HRMS (ESI+) m/z calcd for C12H23N4+, 223.1923; found, 223.1915.
N2,N4-Dimethyl-N2,N4-diisobutyl-(6-isopentyl-pyrimidin-2,4-yl)-
diamine (29a). N-Methylisobutylamine (1 mL) was added dropwise
to 12b (0.055 g, 0.22 mmol), causing the immediate formation of
a precipitate. The reaction mixture was stirred in a sealed high-
pressure tube at 80 °C for 16 h, allowed to cool to room temperature,
and extracted three times from aq NaHCO3 with CH2Cl2. The
combined organic layers were washed with brine, dried over
anhydrous MgSO4, and the solvent removed in vacuo. Purification
was accomplished with column chromatography (10% EtOAc in
2,4,6-Triisobutoxy-pyrimidine (25c). Clean Na0 (83 mg, 3.6
mmol) was dissolved in 4 mL of isobutylalcohol while heating at
70 °C. The resulting solution was cooled to room temperature and
104 mg (0.57 mmol) 2,4,6-trichloropyrimidine dissolved in 0.5 mL
of isobutyl alcohol were added with stirring, causing the solution
to immediately become cloudy. The reaction was stirred at room
temperature for 1 h and then at 60 °C for 24 h. The reaction was
allowed to cool to room temperature, extracted from H2O twice
1
hexanes) to give 29a (25 mg, 31%). H NMR (500 MHz, CDCl3)

Pyrimidine CoactiVator Binding Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6527
δ ppm 0.89 (d, J ) 6.65 Hz, 12 H), 0.92 (d, J ) 6.43 Hz, 6 H),
1.52-1.66 (m, 3 H), 2.06 (nonet, J ) 6.86 Hz, 2 H), 2.45 (t, J )
7.93 Hz, 2 H), 2.99 (s, 3 H), 3.12 (s, 3 H), 3.28 (d, J ) 6.22 Hz,
2 H), 3.39 (d, J ) 7.29 Hz, 2 H), 5.57 (s, 1 H). 13C NMR (500
MHz, CDCl3) δ ppm 20.58, 20.63, 22.8, 27.7, 27.8, 28.0, 36.2,
36.5, 36.7, 38.1, 57.0, 57.2, 89.6, 162.2, 163.2, 169.7. HRMS (ESI+)
m/z calcd for C19H37N4+, 321.3018; found, 321.3014.
7.80 (d, J ) 15.87 Hz, 1 H). 13C NMR (500 MHz, CDCl3) δ ppm
20.3, 27.5, 37.1, 125.6, 127.7, 129.0, 129.2, 136.1, 136.3 164.1.
HRMS (ESI+) m/z calcd for C17H21N3Cl+, 302.1424; found,
302.1421.
N4-Methyl-N2,N4-diisobutyl-(6-styryl-pyrimidin-2,4-yl)-di-
amine (34b). Compound 33b (0.050 g, 0.17 mmol) was dissolved
in 2.5 mL of isobutylamine and heated with stirring in a sealed
high-pressure tube at 100 °C for 24 h. The reaction was allowed to
cool to room temperature and the excess amine removed in vacuo.
Purification was accomplished with column chromatography (5%
Isobutyl-[2-(isobutyl-methyl-amino)-6-styryl-pyrimidin-4-yl]-car-
bamic Acid tert-butyl Ester (31b0). Compound 7a (0.063 g, 0.16
mmol) was dissolved in 1.5 mL of N-methylisobutylamine and
heated with stirring in a sealed high-pressure tube at 100 °C for
16 h. The reaction was allowed to cool to room temperature and
the excess amine removed in vacuo. The resulting residue was
extracted three times from H2O with ether. The combined organic
layers were washed with brine, dried over anhydrous MgSO4, and
the solvent removed by rotary evaporator. Purification was ac-
complished with column chromatography (CH2Cl2 followed by 10%
EtOAc in hexanes) to give 31b0 (32 mg, 45%). 1H NMR (500
MHz, CDCl3) δ ppm 0.90 (d, J ) 6.84 Hz, 6 H), 0.93 (d, J ) 6.84
Hz, 6 H), 1.56 (s, 9 H), 2.11 (m, 2 H), 3.21 (s, 3 H), 3.45 (d, J )
7.08 Hz, 2 H), 3.87 (d, J ) 7.32 Hz, 2 H), 6.95 (d, J ) 15.63 Hz,
1 H), 7.17 (s, 1 H), 7.29 (t, J ) 7.32 Hz, 1 H), 7.37 (t, J ) 7.45
Hz, 2 H), 7.56 (d, J ) 7.57 Hz, 2 H), 7.76 (d, J ) 15.87 Hz, 1 H).
13C NMR (500 MHz, CDCl3) δ ppm 20.5, 20.6, 27.6, 28.2, 28.5,
36.4, 52.3, 57.2, 81.7, 100.9, 127.6, 128.3, 128.6, 128.9, 134.4,
136.8, 154.4, 161.7, 161.8, 162.9. HRMS (ESI+) m/z calcd for
C26H39N4O2+, 439.3073; found, 439.3076.
1
EtOAc and 1% TEA in hexanes) to give 34b (27 mg, 47%). H
NMR (500 MHz, CDCl3) δ ppm 1H 0.92 (d, J ) 6.59 Hz, 6 H),
0.98 (d, J ) 6.59 Hz, 6 H), 1.91 (sept, J ) 6.59 Hz, 1 H), 2.08
(sept, J ) 6.84 Hz, 1 H), 3.06 (s, 3 H), 3.25 (t, J ) 6.35 Hz, 2 H),
3.33 (bs, 2 H), 4.93 (bs, 1 H), 5.83 (s, 1 H), 6.85 (d, J ) 15.87 Hz,
1 H), 7.28 (t, J ) 7.32 Hz, 1 H), 7.35 (t, J ) 7.57 Hz, 2 H), 7.55
(d, J ) 7.32 Hz, 2 H), 7.66 (d, J ) 15.87 Hz, 1 H). 13C NMR (500
MHz, CDCl3) δ ppm 20.5, 20.6, 27.8, 28.9, 36.8, 49.4, 57.3, 92.2,
127.4, 128.1, 128.4, 128.8, 133.5, 136.9, 162.5, 163.8. HRMS
(ESI+) m/z calcd for C21H31N4+, 339.2549; found, 339.2562.
N4-Methyl-N2,N4-diisobutyl-6-[(2-naphthalen-1-yl-ethyl)-pyrimi-
din-2,4-yl]-diamine (34c). Compound 33c (0.012 g, 0.03 mmol) was
dissolved in 4 mL of isobutylamine and heated with stirring in a
sealed high-pressure tube at 95 °C for 20 h. The reaction was
allowed to cool to room temperature and the excess amine removed
in vacuo. The resulting residue was extracted three times from H2O
with CH2Cl2. The combined organic layers were washed with brine,
dried over anhydrous MgSO4, and the solvent removed by rotary
evaporator. Purification was accomplished with column chroma-
tography (10% EtOAc and 1% TEA in hexanes) to give 34c (3
N2-Methyl-N2,N4-diisobutyl-(6-styryl-pyrimidin-2,4-yl)-di-
amine (31b). Compound 31b0 (0.032 g, 0.073 mmol) was dissolved
in 1.5 mL of CH2Cl2 and to this solution 1.5 mL of TFA was added
dropwise with stirring. The reaction was allowed to stir at room
temperature for 2.5 h. The solvent was removed in vacuo, and the
resulting residue was directly purified by column chromatography
(5% EtOAc and 1% TEA in hexanes) to give 31b in quantitative
yield. 1H NMR (500 MHz, CDCl3) δ ppm 0.93 (d, J ) 6.59 Hz, 6
H), 0.97 (d, J ) 6.84 Hz, 6 H), 1.90 (sept, J ) 6.84 Hz, 1 H), 2.12
(sept, J ) 6.59 Hz, 1 H), 3.15 (bs, 2 H), 3.19 (s, 3 H), 3.45 (d, J
) 7.32 Hz, 2 H), 4.64 (bs, 1 H), 5.67 (s, 1 H), 6.83 (d, J ) 15.87
Hz, 1 H), 7.28 (t, J ) 7.32 Hz, 1 H), 7.35 (t, J ) 7.57 Hz, 2 H),
7.55 (d, J ) 7.32 Hz, 2 H), 7.71 (d, J ) 15.87 Hz, 1 H). 13C NMR
(500 MHz, CDCl3) δ ppm 20.57, 20.64, 27.7, 28.8, 36.3, 49.2, 57.1,
127.5, 128.37, 128.43, 128.8, 133.5, 162.4. HRMS (ESI+) m/z calcd
for C21H31N4+, 339.2549; found, 339.2542.
1
mg, 21%). H NMR (500 MHz, CDCl3) δ ppm 0.86 (d, J ) 6.59
Hz, 6 H), 0.98 (d, J ) 6.84 Hz, 6 H), 1.87-2.03 (m, 2 H), 2.86
(m, 2 H), 2.97 (s, 3 H), 3.24 (t, J ) 6.35 Hz, 4 H), 3.44 (distorted
t, J ) 8.30 Hz, 2 H), 5.03 (bs, 1 H), 5.52 (s, 1 H), 7.37 (m, 2 H),
7.45-7.54 (m, 2 H), 7.71 (d, J ) 7.57 Hz, 1 H), 7.85 (dd, J )
7.93, 1.34 Hz, 1 H), 8.15 (d, J ) 8.30 Hz, 1 H). HRMS (ESI+)
m/z calcd for C25H35N4+, 391.2862; found, 391.2863.
N4-Dimethyl-N2,N4-diisobutyl-(6-phenethyl-pyrimidin-2,4-yl)-di-
amine (35). Compound 34b (0.016 g, 0.05 mmol) was dissolved in
a suspension of 10 mg (0.009 mmol) of 10% Pd/C in 2 mL MeOH.
The reaction was stirred under 1 atm of H2 for 45 min. The solvent
was removed in vacuo and residue was purified directly by column
chromatography (10% EtOAc and 1% TEA in hexanes) to give 35
N2-Methyl-N2,N4-diisobutyl-(6-phenethyl-pyrimidin-2,4-yl)-di-
amine (32). Compound 31b (0.022 g, 0.064 mmol) was dissolved
in a suspension of 16 mg (0.015 mmol) of 10% Pd/C in 4 mL of
MeOH. The reaction was stirred under 1 atm of H2 for 45 min.
The solvent was removed in vacuo and residue was purified directly
by column chromatography (10% EtOAc in hexanes) to give 32
1
(14 mg, 85%). H NMR (500 MHz, CDCl3) δ ppm 0.88 (d, J )
6.59 Hz, 6 H), 0.96 (d, J ) 6.59 Hz, 6 H), 1.88 (sept, J ) 6.84 Hz,
1 H), 2.00 (sept, J ) 6.59 Hz, 1 H), 2.72 (m, 2 H), 2.97 (m, 5 H),
3.21 (t, J ) 6.47 Hz, 2 H), 3.26 (bs, 2 H), 4.92 (bs, 1 H), 5.55 (s,
1 H), 7.18 (t, J ) 7.20 Hz, 1 H), 7.22 (d, J ) 6.84 Hz, 2 H), 7.27
(t, J ) 7.57 Hz, 2 H). 13C NMR (500 MHz, CDCl3) δ ppm 20.5,
1
(16 mg, 73%). H NMR (500 MHz, CDCl3) δ ppm 0.90 (d, J )
20.6, 27.7, 28.8, 35.4, 36.7, 40.0, 49.4, 57.2, 91.3, 126.0, 128.5,
6.59 Hz, 6 H), 0.94 (d, J ) 6.59 Hz, 6 H), 1.85 (sept, J ) 6.59 Hz,
1 H), 2.07 (sept, J ) 6.59 Hz, 1 H), 2.74 (bt, J ) 6.84 Hz, 2 H),
3.00 (dd, J ) 9.28, 6.84 Hz, 2 H), 3.06 (bs, 2 H), 3.12 (s, 3 H),
3.42 (d, J ) 7.32 Hz, 2 H), 4.52 (bs, 1 H), 5.44 (s, 1 H), 7.17 (t,
J ) 7.20 Hz, 1 H), 7.22 (distorted d, J ) 6.84 Hz, 2 H), 7.27 (t, J
) 7.57 Hz, 2 H). HRMS (ESI+) m/z calcd for C21H33N4+, 341.2705;
found, 341.2705.
+
128.7, 142.1, 162.3, 163.4. HRMS (ESI+) m/z calcd for C21H33N4
,
341.2705; found, 341.2713.
Isobutyl-[2-isobutylsulfanyl-6-(3-methyl-butyl)-pyrimidin-4-yl]-
carbamic Acid tert-Butyl Ester (36b). To a suspension of 45 mg
(1.1 mmol) of NaH (60% dispersion in mineral oil) in 3 mL of
THF, 2-methyl-propane-1-thiol (122 µL, 1.1 mmol) was added
dropwise and the resulting mixture was stirred for 15 min. To this
mixture, 16b (0.040 g, 0.11 mmol) was added dissolved in 1 mL
THF. The reaction was stirred 16 h at room temperature, quenched
with H2O, and extracted three times with ether. The combined
organic layers were washed with brine, dried over anhydrous
MgSO4, and the solvent removed with a rotary evaporator.
Purification was accomplished with column chromatography (10%
EtOAc in hexanes) to give 36b (0.036 g, 78%). 1H NMR (500
MHz, CDCl3) δ ppm 0.88 (d, J ) 6.84 Hz, 6 H), 0.92 (d, J ) 5.86
Hz, 6 H), 1.04 (d, J ) 6.59 Hz, 6 H), 1.54 (s, 9 H), 1.58 (bs, 3 H),
1.95 (sept, J ) 6.84 Hz, 1 H), 2.04 (sept, J ) 6.84 Hz, 1 H), 2.62
(bt, J ) 7.32 Hz, 2 H), 3.00 (d, J ) 6.35 Hz, 2 H), 3.89 (d, J )
7.57 Hz, 2 H), 7.44 (s, 1 H). 13C NMR (500 MHz, CDCl3) δ ppm
20.3, 22.3, 22.7, 28.1, 28.3, 28.4, 28.7, 36.3, 38.1, 39.5, 52.2, 82.3,
(2-Chloro-6-styryl-pyrimidin-4-yl)-isobutyl-methyl-amine (33b).
Compound 1 (50 mg, 0.20 mmol) and K2CO3 (82 mg, 0.60 mmol)
were mixed in 2.5 mL of DMF. To this suspsension, 24 µL (0.20
mmol) of N-methylisobutylamine were added with stirring at room
temperature. Consumption of starting material was observed by TLC
after 3 h. The reaction mixture was diluted with H2O and extracted
three times with ether. The combined organic layers were washed
with brine, dried over MgSO4, and the solvent removed by rotary
evaporator. Purification of the residue by column chromatography
(CH2Cl2 followed by 10% EtOAc in hexanes) afforded 33b (50
1
mg, 84%). H NMR (500 MHz, CDCl3) δ ppm 0.94 (d, J ) 6.35
Hz, 6 H), 2.08 (sept, J ) 6.84 Hz, 1 H), 3.11 (bs, 3 H), 3.42 (bs,
2 H), 6.25 (s, 1 H), 6.88 (d, J ) 15.87 Hz, 1 H), 7.32 (t, J ) 7.20
Hz, 1 H), 7.37 (t, J ) 7.32 Hz, 2 H), 7.56 (d, J ) 7.32 Hz, 2 H),

6528 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Parent et al.
107.4, 154.0, 160.7, 170.3, 171.8. HRMS (ESI+) m/z calcd for
Hz, 2 H), 5.55 (bs, 1 H), 6.23 (s, 1 H). HRMS (ESI+) m/z calcd
for C17H32N3O2S+, 342.2215; found, 342.2209.
C16H29N2S3+, 345.1493; found, 345.1479.
[2-Isobutoxy-6-(3-methyl-butyl)-pyrimidin-4-yl]-isobutyl-
amine (37a). NaH (60% dispersion in mineral oil, 34 mg, 0.85
mmol) was added to 3 mL of isobutanol and stirred for 30 min at
room temperature. To this solution, 28 mg (0.078 mmol) of 16b
dissolved in 3 mL of isobutanol were added. The resulting solution
was heated with stirring in a sealed high-pressure tube at 100 °C
for 4 h. The solution was allowed to cool to room temperature and
stirred an additional 16 h. Extraction of the reaction mixture was
performed three times from H2O with ether, and the combined
organic layers were washed with brine, dried over MgSO4, and the
solvent removed by rotary evaporator. The resulting residue was
dissolved in 2 mL of CH2Cl2 and 2 mL of TFA were added with
stirring. The reaction was stirred for 1.5 h at room temperature,
and the solvent was removed in vacuo. Purification of the residue
was accomplished with column chromatography (10% EtOAc and
Biological Methods
TR-FRET CBI Assay. Purified ERR-417, labeled site-
specifically with maleimide-biotin (Quanta BioDesign) and
SRC-3-NRD, labeled nonspecifically through cysteine residues
with iodoacetamide-fluorescein (Invitrogen), were used in the
time-resolved FRET assays.42 A 5 µL volume of a stock solution
mix of ERR-417 (8 nM), estradiol (4 µM), and LanthaScreen
streptavidin-terbium (Invitrogen) (2 nM) in TR-FRET buffer
(20 mM Tris, pH 7.5, 0.01% NP40, 50 mM NaCl) were placed
in separate wells of a black 96-well Molecular Devices HE high
efficiency microplate (Molecular Devices, Inc., Sunnyvale, CA).
In a second 96-well Nunc polypropylene plate (Nalge Nunc
International, Rochester, NY), a 0.02 M solution of each
coactivator binding inhibitor was serially diluted in a 1:10
fashion into DMF. Each concentration of coactivator binding
inhibitor or vehicle was then diluted 1:10 into TR-FRET buffer,
and 10 µL of this solution was added to the stock ERR solution
in the 96-well plate. After a 20 min incubation, 5 µL of 200
nM fluorescein-SRC-3-NRD was added to each well. This
mixture was allowed to incubate for 1 h at room temperature
in the dark. TR-FRET was measured using an excitation filter
at 340/10 nm and emission filters for terbium and fluorescein
at 495/20 and 520/25 nm, respectively. The final concentrations
of the reagents were as follows: ERR-417 (2 nM), streptavidin-
terbium (0.5 nM), estradiol (1 µM), coactivator binding inhibitor
(0-1 mM), SRC-3-NRD (50 nM).
Luciferase Reporter Gene Assay. Human endometrial
cancer (HEC-1) cells were maintained in culture and transfected
in 24 well plates as previously described.43 HBSS (50 µL/well),
Holo-transferrin (Sigma T1408) (20 µL/well), and lipofectin
(Invitrogen no. 18292-011) (5 µL/well) were incubated together
at room temperature for 5 min. A DNA mixture containing 200
ng of pCMVꢀ-galactosidase as an internal control, 500 ng of
the estrogen responsive reporter gene plasmid 2ERE Luc, and
100 ng of full-length ER alpha expression vector with 75 µL
of HBSS per well was added to the first mixture and allowed
to incubate for 20 min at room temperature. After changing the
cell media to Opti-MEM (350 µL/well), 150 µL of the
transfection mixture was added to each well. The cells were
incubated at 37 °C in a 5% CO2 containing incubator for 6 h
before the medium was replaced with fresh medium containing
5% charcoal-dextran-treated calf serum and the desired con-
centrations of ligands. Luciferase reporter gene activity was
assayed 24 h after ligand addition as described.43
1
2% TEA in hexanes) to give 37a (22 mg, 98%). H NMR (500
MHz, CDCl3) δ ppm 0.92 (d, J ) 6.35 Hz, 6 H), 0.96 (d, J ) 6.84
Hz, 6 H), 1.00 (d, J ) 6.59 Hz, 6 H), 1.51-1.64 (m, 3 H), 1.86
(sept, J ) 6.84 Hz, 1 H), 2.08 (sept, J ) 6.59 Hz, 1 H), 2.50
(distorted t, J ) 8.06 Hz, 2 H), 3.09 (bs, 2 H), 4.02 (d, J ) 6.84
Hz, 2 H), 4.90 (bs, 1 H), 5.81 (s, 1 H). 13C NMR (500 MHz, CDCl3)
δ ppm 19.6, 20.5, 22.7, 28.16, 28.18, 28.6, 37.9, 73.3, 165.0. HRMS
(ESI+) m/z calcd for C17H32N3O+, 294.2545; found, 294.2538.
Isobutyl-[2-isobutylsulfanyl-6-(3-methyl-butyl)-pyrimidin-4-yl]-
amine (37b). Compound 36b (18 mg, 0.043 mmol) was dissolved
in 2 mL of CH2Cl2 to which 2 mL of TFA were added with stirring.
The reaction was stirred for 1.5 h at room temperature, and the
solvent was removed in vacuo. Purification of the residue was
accomplished with column chromatography (10% EtOAc and 2%
1
TEA in hexanes) to give 37b in quantitative yield. H NMR (500
MHz, CDCl3) δ ppm 0.89 (d, J ) 6.35 Hz, 6 H), 0.95 (d, J ) 6.59
Hz, 6 H), 1.02 (d, J ) 6.59 Hz, 6 H), 1.47-1.61 (m, 3 H),
1.86-2.01 (m, 2 H), 2.52 (distorted t, J ) 7.81 Hz, 2 H), 3.01 (d,
J ) 6.59 Hz, 2 H), 3.22 (bs, 2 H), 6.09 (bs, 1 H). HRMS (ESI+)
m/z calcd for C17H32N3S+, 310.2317; found, 310.2309.
Isobutyl-[6-(3-methyl-butyl)-2-(2-methyl-propane-1-sulfonyl)-py-
rimidin-4-yl]-carbamic Acid tert-Butyl Ester (38). From the pro-
cedure described by Hurst, et al.,32,33 mCPBA (77%, 27 mg, 0.12
mmol) and 20 mg of 36b were dissolved in 2 mL of CHCl3 and
mixed well. The solution was then allowed to stand at room
temperature for 16 h. Then 20 mL of CHCl3 was added and the
solution was washed three times with saturated NaHCO3 solution.
The aqueous layers were extracted twice with CHCl3, and the
combined organic layers were washed with saturated NaCl solution
and dried over MgSO4. The solvent was removed in vacuo, and
chromatography of the residue (10% EtOAc in hexanes) gave 38
1
(9 mg, 40%). H NMR (500 MHz, CDCl3) δ ppm 0.91 (d, J )
Intheinitialscreen,compoundswereassayedinadose-response
format at concentrations ranging from 20 µM to 0.6 µM; their
inhibitory potential was determined by performing the assay in
the presence of 10-9 M estradiol (E2). Upon validation of
antagonistic activity, mechanism of action was examined by
repeating the compound titration in the presence of both 10-7
and 10-9 M E2 with an expectation that changing the concentra-
tion of E2 100-fold would not change the IC50 of true coactivator
binding inhibitors.
Estrogen Receptor-Relative Binding Affinity Assay. Rela-
tive binding affinities were determined using a modification of
a competitive radiometric binding assay as previously described,
using 2 nM [3H]estradiol as tracer ([2,4,6,7-3H]estra-1,3,510-
triene-3,17ꢀ-diol, 84-85 Ci/mmol, Amersham/GE Health Care,
Piscataway, NJ); purified full-length human ERR and ERꢀ were
purchased from PanVera/Invitrogen (Carlsbad, CA). Incubations
were conducted for 18-24 h at 0 °C. Hydroxyapatite (BioRad,
6.59 Hz, 6 H), 0.94 (d, J ) 6.59 Hz, 6 H), 1.10 (d, J ) 6.84 Hz,
6 H), 1.57 (s, 9 H), 1.59-1.67 (m, 3 H), 2.07 (sept, J ) 6.84 Hz,
1 H), 2.37 (sept, J ) 6.59 Hz, 1 H), 2.80 (m, 2 H), 3.39 (d, J )
6.59 Hz, 2 H), 3.94 (d, J ) 7.32 Hz, 2 H), 8.05 (s, 1 H). 13C NMR
(500 MHz, CDCl3) δ ppm 20.4, 22.6, 23.0, 24.1, 28.18, 28.24, 28.3,
36.3, 38.1, 52.8, 58.4, 83.4, 113.3, 153.7, 161.8, 164.5, 173.6.
Isobutyl-[6-(3-methyl-butyl)-2-(2-methyl-propane-1-sulfonyl)-py-
rimidin-4-yl]-amine (39). Compound 38 (9 mg, 0.019 mmol) was
dissolved in 2 mL of CH2Cl2, to which 2 mL of TFA were added
with stirring. The reaction was stirred for 2.5 h at room temperature
and the solvent was removed in vacuo. Purification of the residue
was accomplished with column chromatography (25% EtOAc and
2% TEA in hexanes) to give 39 in quantitative yield. 1H NMR
(500 MHz, CDCl3) δ ppm 0.93 (d, J ) 6.35 Hz, 6 H), 0.99 (d, J
) 6.59 Hz, 6 H), 1.10 (d, J ) 6.59 Hz, 6 H), 1.54-1.65 (m, 3 H),
1.89 (sept, J ) 6.59 Hz, 1 H), 2.37 (sept, J ) 6.59 Hz, 1 H), 2.66
(distorted t, J ) 8.06 Hz, 2 H), 3.06 (bs, 2 H), 3.37 (d, J ) 6.35

Pyrimidine CoactiVator Binding Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6529
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Hercules, CA) was used to absorb the receptor-ligand com-
plexes, and free ligand was washed away. The binding affinities
are expressed as relative binding affinity (RBA) values with
the RBA of estradiol set to 100%. Estradiol binds to ERR with
a Kd of 0.2 nM and to ERꢀ with a Kd of 0.5 nM.44,45
Molecular Modeling of the CBI/ER Interaction. All
modeling of the ER-LBD was performed using the Sybyl 7.3
module from Tripos. The crystal structure of ER-LBD cocrys-
tallized with diethylstilbestrol and an NR-Box II (LXXLL motif)
containing peptide from the steroid receptor coactivator 2 (SRC-
2) was used as the basis for coactivator groove modeling (http://
www.rcsb.org ID 3erd). The crystal structure was prepared for
docking experiments by addition of the missing internal amino
acids of the LBD using information contained in the RCSB web
site and the loop search (PRODAT database) feature of Sybyl.
Hydrogens, missing side chains, and charged end groups were
added to the protein and peptide, and the ER/SRC-2 structure
was minimized using MMFF94 calculated charges and an
MMFF94s force field in three steps (inserted loops, water
molecules, entire protein) with appropriate aggregates applied
for each. CBI structures were minimized using the Tripos
forcefield to termination gradient of 0.05 kcal/(mol·A). Initial
placement of the CBI was performed using least-squares
multiple atom fitting of the alkyl groups on the CBI with the
ER-interacting leucine residues of the SRC-2 peptide. Subse-
quent CBI orientations used for docking were manipulated into
the coactivator groove by hand. Before docking, the SRC-2
peptide and all water molecules were removed from the ER-
LBD structure. Docking experiments were performed using the
Sybyl FlexiDock module, with all bonds in the CBI and all side
chains within 10 Å of the coactivator groove designated as
rotatable. H-bond donor and acceptor sites were designated for
the appropriate atoms in the CBI as well as for Glu542 and
Lys362. Docking runs were begun from a random seed and
allowed to proceed for 100000 iterations. In most instances,
duplicate runs were performed to ensure validity of the docking
results. The lowest energy conformation of CBI/receptor thus
produced was then further minimized with the MMFF94s force
field to a termination gradient of 0.10 kcal/(mol·A), following
a four-step procedure to produce the final structures [(1) 10 Å
surrounding CBI with CBI and backbone static, (2) 10 Å
surrounding CBI with backbone static, (3) 10 Å surrounding
CBI with CBI static, and (4) 10 Å surrounding CBI)].
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coactivators: an update. Endocrinology 2002, 143, 2461–2465.
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tamoxifen therapy for breast cancer patients with negative lymph nodes
and estrogen receptor-positive tumors. J. Nat. Cancer Inst. 1996, 88,
1529–1542.
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A.; Giordano, A.; Perrone, F. Mechanisms of endocrine resistance and
novel therapeutic strategies in breast cancer. Endocr. Rel. Cancer 2005,
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function and its implication for endocrine therapy resistance in breast
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Design, Synthesis, and in Vitro Biological Evaluation of Small
Molecule Inhibitors of Estrogen Receptor a Coactivator Binding.
J. Med. Chem. 2004, 47, 600–611.
Acknowledgment. We greatly acknowledge support of this
researchfromtheNationalInstitutesofHealth(PHS5R37DK15556).
J.R.G. was supported by a David Robertson Fellowship and an
NIH Fellowship NRSA 1 F30 ES016484-01 and training grant
NRSA 5 T32 GM070421. A.A.P. received support from an
Illinois Distinguished Fellowship and a Robert D. Doolen
Fellowship. We are grateful for the helpful suggestions of Dr.
Sung Hoon Kim.
(23) Shao, D.; Berrodin, T. J.; Manas, E.; Hauze, D.; Powers, R.; Bapat,
A.; Gonder, D.; Winneker, R. C.; Frail, D. E. Identification of novel
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Supporting Information Available: See for complete synthetic
details, HPLC and CHN analyses, and more extensive biological
data, including RBA values. This material is available free of charge
via the Internet at http://pubs.acs.org.
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