Potent, orally bioavailable diazabicyclic small-molecule mimetics of second mitochondria-derived activator of caspases

Article abstract of DOI:10.1021/jm801254r

A series of small-molecule Smac mimetics containing a diazabicyclic core structure have been designed, synthesized, and evaluated. The most potent compound (6) binds to XIAP, cIAP-1, and cIAP-2 with K_i values of 8.4, 1.5, and 4.2 nM, respective

Full text of DOI:10.1021/jm801254r

8158
J. Med. Chem. 2008, 51, 8158–8162
Brief Articles
Potent, Orally Bioavailable Diazabicyclic Small-Molecule Mimetics of Second
Mitochondria-Derived Activator of Caspases
Yuefeng Peng,† Haiying Sun,† Zaneta Nikolovska-Coleska,† Su Qiu,† Chao-Yie Yang,† Jianfeng Lu,† Qian Cai,† Han Yi,†
Sanmao Kang,‡ Dajun Yang,‡ and Shaomeng Wang*,‡
ComprehensiVe Cancer Center and Departments of Internal Medicine, Pharmacology and Medicinal Chemistry, UniVersity of Michigan, 1500 E.
Medical Center DriVe, Ann Arbor, Michigan 48109, Ascenta Therapeutics, Inc., 101 Lindenwood DriVe, Suite 405, MalVern, PennsylVania 19355
ReceiVed August 14, 2008
A series of small-molecule Smac mimetics containing a diazabicyclic core structure have been designed,
synthesized, and evaluated. The most potent compound (6) binds to XIAP, cIAP-1, and cIAP-2 with Ki
values of 8.4, 1.5, and 4.2 nM, respectively, directly antagonizes XIAP in a cell-free functional assay and
induces cIAP-1 degradation in cancer cells. It inhibits cell growth with an IC50 value of 31 nM, effectively
induces apoptosis in the MDA-MB-231 cancer cell line, and has a good oral bioavailability.
Introduction
Inhibitor of apoptosis proteins (IAPs)a are key apoptosis
regulators characterized by the presence of one to three regions
known as baculoviral IAP repeat (BIR) domains.1,2 Among these
IAP proteins, cellular IAP-1 (cIAP-1) and cIAP-2 play critical
roles in regulation of tumor necrosis factor receptor-mediated
apoptosis,3,4 and X-linked IAP (XIAP) is a central regulator of
both death-receptor-mediated and mitochondria-mediated apo-
ptosis pathways.5 XIAP and cIAP-1 play a role in apoptosis
resistance of cancer cells to a variety of anticancer drugs and
are considered to be promising cancer therapeutic targets.5,6
Second mitochondria-derived activator of caspases (Smac),
a potent pro-apoptotic protein, is an endogenous antagonist of
IAP proteins.7,8 Previous studies have established that Smac
Figure 1. Chemical structures of designed Smac mimetics.
interacts with XIAP and cIAP-1/2 proteins via its AVPI
cells.19 However, because of their lower molecular weights,
tetrapeptide motif.2,9-12 In the past few years, intense research
properly designed monovalent Smac mimetics can possess major
efforts have been devoted to the design and development of
advantages over bivalent Smac mimetics for purposes of drug
Smac mimetics, small molecules which mimic the AVPI binding
design.
motif and function as antagonists of IAP proteins.13-22 Smac
Our laboratory has previously reported the structure-based
mimetics are considered to have the great potential to be
design, synthesis, and evaluation of a series of conformationally
developed as a new class of anticancer drugs by promoting
apoptosis in cancer cells. Two types of Smac mimetics,
monovalent and bivalent, have been reported. The monovalent
compounds are designed to mimic the binding of a single AVPI
constrained monovalent Smac mimetics.14,15,17,19 Compound 1
(Figure 1) binds to the XIAP BIR3 protein with a Ki value of
26 nM.19 Compound 1 directly antagonizes XIAP in a cell-free
functional assay and induces apoptosis in cancer cells.19 It also
binding motif to IAP proteins,14-18 and the bivalent compounds
binds to cIAP-1 and cIAP-2 with Ki values of 1.0 and 1.8 nM
contain two AVPI binding motif mimetics tethered together
(Table 1), respectively, and is thus a potent monovalent Smac
through a linker.13,19-22 We have shown that the bivalent Smac
mimetic.
mimetics can achieve much higher affinities to XIAP and are
To examine if compound 1 can be potentially a drug
1-2 orders of magnitude more potent than the corresponding
candidate, we evaluated its pharmacokinetics (PK) in rats.
monovalent Smac mimetic in induction of apoptosis in tumor
Compound 1 achieved a maximum concentration (Cmax) of 545
( 43 nM, an elimination half-life (T1/2) of 1.7 ( 0.3 h and
AUC(0-∞) (area-under-the-curve) of 482 ( 92 µg/L h at 25 mg/
kg administered orally and an oral bioavailability of 14%. Hence,
compound 1 has a modest AUC and oral bioavailability, making
it less than an ideal drug candidate for further development. To
improve the PK profile, we have decided to modify the core
structure of 1, which led to a new class of Smac mimetics. The
most promising new compound (6) binds to XIAP and cIAP-
1/2 with very high affinities, is more potent than 1 in cell-based
* To whom correspondence should be addressed. Phone: (734)615-0362.
Fax: (734)6479647. E-mail: shaomeng@umich.edu.
†
Comprehensive Cancer Center and Departments of Internal Medicine,
Pharmacology and Medicinal Chemistry, University of Michigan.
‡
Ascenta Therapeutics, Inc.
a Abbreviations: IAP, inhibitor of apoptosis protein; XIAP, X-linked IAP;
cIAP-1/-2, cellular IAP-1/2; Smac, second mitochondria-derived activator
of caspases; BIR, baculoviral IAP repeats (BIR) domain; BIR2, the second
BIR domain; BIR3, the third BIR domain; FP, fluorescence polarization;
PK, pharmacokinetics; Cmax, maximum concentration; T1/2, elimination half-
life; AUC, area-under-the-curve; PARP, poly(ADP-ribose) polymerase.
10.1021/jm801254r CCC: $40.75
2008 American Chemical Society
Published on Web 12/02/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8159
Scheme 2. Synthesis of Designed Smac Mimeticsa
Table 1. Binding Affinities of Smac Mimetics to XIAP, cIAP-1, and
cIAP-2, as Determined in Competitive, Fluorescence-Polarization Based
Assays (Assays Details Provided in Supporting Information)a
Ki ( SD (nM)
a Reagents and conditions: (a) 4 N HCl in 1,4-dioxane, methanol, rt,
overnight, 95%. (b) (i) Formaldehyde (37% in water), NaBH3CN, HOAc,
methanol, rt, overnight; (ii) 4 N HCl in 1,4-dioxane, methanol, rt, overnight,
71% over two steps. (c) (i) BnBr, NaHCO3, 1,4-dioxane, rt, overnight; (ii)
4 N HCl in 1,4-dioxane, methanol, rt, overnight 77% over two steps. (d)
(i) Ac2O or BnCOCl, N,N-diisopropylethylamine, CH2Cl2, rt, overnight;
(ii) 4 N HCl in 1,4-dioxane, methanol, rt, overnight, 85% for 5 and 87%
for 6 over two steps.
compd
XIAP
cIAP-1
cIAP2
1
2
3
4
5
6
26 ( 4
1.0 ( 0.3
1.2 ( 0.1
3.3 ( 1.3
3.7 ( 1.4
1.3 ( 0.3
1.5 ( 0.5
1.8 ( 0.6
4.6 ( 1.5
17.5 ( 4.3
9.8 ( 4.1
1.9 ( 0.8
4.2 ( 0.6
20.0 ( 14.5
341 ( 65.9
91.8 ( 30.4
5.4 ( 3.0
8.4 ( 1.6
a Data were obtained using 3-5 independent experiments.
Figure 2. Predicted binding models of compounds 1 and 6 to XIAP
BIR3 domain in superposition with Smac AVPI peptide. Compounds
1 and 6 are colored in green and the AVPI peptide in yellow. Binding
pockets are shown in transparent surface. Oxygen, nitrogen, and sulfur
atoms are colored in red, blue, and yellow, respectively. Hydrogen
bonds are depicted in dash lines.
Figure 3. Inhibition of caspase-3/-7 activity by XIAP and antagonism
of Smac mimetics to XIAP to recover the activity of caspase-3/-7 in a
cell-free functional assay. (A) Caspase-3/-7 was activated by addition
of cytochrome c and dATP into cell lysates and recombinant XIAP
BIR3 protein at 0.5 µM completely inhibited caspase activation.
Compound 6 dose-dependently recovered the caspase-3/-7 activity. (B).
Dose-dependent recovery of caspase-3/-7 activity by 1, 4, and 6 to the
maximum activation. Caspase-3/-7 activity at 30 min time point was
used.
Scheme 1. Synthesis of the Key Intermediate 10a
benzyl group on the nitrogen resulted in 3 and 4, and
introduction of an acetyl group, or a phenylacetyl group on the
nitrogen atom, yielded 5 and 6, respectively.
Compounds 3 and 4 have Ki values of 341 and 91.8 nM to
XIAP BIR3, respectively, 4-17 times weaker than compound
2. Although both compounds bind to cIAP-1 with high affinities,
they are 3 times weaker than that of 2. Similarly, compounds 3
and 4 bind to cIAP-2 with affinities 2-3 times weaker than
that of 2.
In binding to XIAP BIR3, both 5 and 6 have higher binding
affinities than 2, with Ki values of 5.4 and 8.4 nM, respectively.
These compounds, with Ki values of 1.3 and 1.5 nM, respec-
tively, are as potent as 2 to cIAP-1. Compound 6 is as potent
and compound 5 is twice as potent as 2 in binding to cIAP-2.
These binding data for compounds 2-6 showed that modifica-
tions at this site can significantly impact the binding affinities
of these Smac mimetics to XIAP, cIAP-1, and cIAP-2 proteins.
a Reagents and conditions: (a) (i) CbzCl, NaHCO3, 1,4-dioxane, rt,
overnight; (ii) (Boc)2O, NaHCO3, 1,4-dioxane, rt, overnight, 65% over three
steps. (b) (i) 2 N LiOH, 1,4-dioxane, 3 h, then 1 N HCl; (ii) aminodiphe-
nylmethane, EDC, HOBt, N,N-diisopropylethylamine, rt, overnight, 82%
over two steps. (c) (i) 4 N HCl in 1,4-dioxane, methanol, rt, overnight; (ii)
L-N-Boc-N-methyl-alanine, EDC, HOBt, N,N-diisopropylethylamine, rt,
overnight; (iii) H2, 10% Pd-C, methanol, overnight, 77% over three steps.
assays, and has improved PK parameters and oral bioavailability
as compared to 1.
Results and Discussion
Our model of 1 complexed with XIAP BIR3 showed that
while the 8-membered ring has van der Waals contacts with
Trp323 in XIAP BIR3, the middle portion of this 8-membered
ring is largely exposed to solvent (Figure 2). On the basis of
this model, we predicted that replacement of one of the carbons
in the 8-membered ring atoms by a nitrogen may not be
detrimental to the binding to XIAP.
To test this idea, we designed and synthesized compound 2
(Figure 1, Schemes 1 and 2), which has a diazabicyclic core
structure. This compound binds to XIAP BIR3 with a Ki value
of 20 nM in our fluorescence polarization-based (FP-based)
XIAP binding assay23 and is thus as potent as 1. It also binds
to cIAP-1 and cIAP-2 with Ki values of 1.2 and 4.6 nM,
respectively, determined in FP-based competitive binding assays
for these two proteins. Hence, 2 is a potent Smac mimetic and
a promising compound for further optimization.
We next evaluated if compounds 2-6 behave as antagonists
of XIAP in cell-free functional assays. The results for three
representative compounds, 1, 4, and 6, are shown in Figure 3.
The XIAP BIR3 protein effectively inhibits the activity of
caspase-3/-7, and these Smac mimetics dose-dependently an-
tagonize XIAP and restore caspase activity (Figure 3A and
Supporting Information). Consistent with the binding affinities,
compound 6 has twice the potency of 1 and 10 times that of 4
in antagonizing XIAP in this functional assay.
Previous studies showed that Smac mimetics can effectively
inhibit cell growth and induce apoptosis in the MDA-MB-231
human breast cancer cell line.16,17 We have evaluated these new
Smac mimetics for cell growth inhibition in the MDA-MB-231
cancer cell line (Figure 4) and found that, while all these
compounds are effective, 5 and 6, with IC50 values of 69 and
31 nM, respectively, are the two most potent; 6 is 8 times more
potent than 1 in this cell growth assay. Compounds 3 and 4 are
Modifications of the secondary amino nitrogen in the
8-membered ring of 2 were conducted to explore structure-
activity relationships at this site. Introduction of a methyl or

8160 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Brief Articles
3, two biochemical markers of apoptosis, within 24 h and is
more effective than 1, 2, or 4.
We next evaluated the pharmacokinetics in rats of 6 dosed
orally and found that it achieves a Cmax of 532 ( 249 nM, a
T1/2 of 4.7 ( 2.2 h, and AUC(0-∞) of 1985 ( 614 µg/L h at 30
mg/kg. The Cmax value for 6 at 30 mg/kg is 17 times of its IC50
value in the cell growth assay in the MDA-MB-231 cancer cell
line and it has an oral bioavailability of 24% at doses of 30
mg/kg in rats. In direct comparison, 6 has a longer T1/2 and a
larger AUC value than 1 and a better oral bioavailability. Hence,
compound 6 has a significantly better PK profile than 1.
The synthesis of compounds 2-6 is shown in Schemes 1
and 2. Compound 7 (Scheme 1) was synthesized using a
published method.24 Protection of the amino group in 7 with
carbobenzoxy gave a carbamate that was treated with SOCl2 in
MeOH to transform the tert-butyl ester to a methyl ester. The
primary amino group was reprotected with t-Boc to afford 8,
hydrolysis of whose methyl ester followed by condensation of
the resulting acid with aminodiphenylmethane yielded 9.
Removal of the t-Boc protecting group in 9 and subsequent
condensation of the resulting amine with L-N-t-Boc-N-methyl
alanine furnished an amide. Cleavage by hydrogenation of the
Cbz protecting group in this amide afforded the common key
intermediate 10.
Figure 4. Inhibition of cell growth by Smac mimetics in the MDA-
MB-231 cancer cell line. Cells were treated with Smac mimetics for 4
days and cell growth was determined using a WST-based assay.
The synthesis of compounds 2-6 is shown in Scheme 2.
Methylation or benzylation of 10 group furnished the alkylated
amines and removal of the t-Boc protecting group in these
compounds provided 3 and 4, respectively. Condensation of 10
with acetic anhydride or phenylacetyl chloride afforded the
respective amides and subsequent removal of the t-Boc protect-
ing group gave 5 and 6, respectively.
Figure 5. Induction of apoptisis by compound 6 in the MDA-MB-
231 cell line. Cells were treated with different concentrations of 6 for
24 or 48 h. Apoptosis was analyzed using Annexin V and propidium
iodide double staining by flow cytometry. Percentage of early apoptotic
(Annexin V+/PI-), late apoptotic (Annexin V+/PI+), and dead (Annexin
V-/PI+) cells are shown, respectively.
Summary
A new class of Smac mimetics has been designed and
synthesized through modifications of the [8,5] bicyclic core
structure in our initial compound 1. Several highly potent and
cell-permeable Smac mimetics were obtained. The most potent
compound, 6, binds to XIAP, cIAP-1, and cIAP-2 with Ki values
of 8.4, 1.5, and 4.2 nM, respectively. Compound 6 potently
inhibit cell growth in the MDA-MB-231 cell line with an IC50
value of 31 nM and effectively induces apoptosis at 100 nM
within 24 h of treatment. Significantly, compound 6 is orally
bioavailable and has excellent aqueous solubility. These data
reveal that compound 6 is a promising lead compound for further
evaluation and optimization. Extensive optimization and in vitro
and in vivo testing of 6 and its analogues are under way and
will be reported in due course.
Figure 6. Induction of cIAP-1 degradation, cleavage of PARP, and
processing of caspase-3 by compounds 1, 2, 4, and 6 in the MDA-
MB-231 cell line. Cells were treated with different concentrations of
Smac mimetics for 24 h and levels of cIAP-1, cleaved PAPR (CL
PARP), and cleaved caspase-3 (CL C3) were probed by Western blot.
the two least potent, with IC50 values of 1300 and 1000 nM,
respectively.
We evaluated the most potent compound 6 for its ability to
induce apoptosis in the MDA-MB-231 cancer cell line. Com-
pound 6 effectively induces apoptosis in this cancer cell line in
a dose- and time-dependent manner (Figure 5). For example,
compound 6 at 100 nM for 24- and 48-h treatment induces 31%
and 47% of the MDA-MB-231 breast cancer cells to undergo
apoptosis, respectively.
Experimental Section
Chemistry. General Methods. NMR spectra were measured at
1
300 MHz. H chemical shifts are reported relative to DHO (4.79
ppm) as internal standard. Final products were purified by C18
reverse phase semipreparative HPLC column with solvent A (0.1%
of TFA in water) and solvent B (0.1% of TFA in CH3CN) as
eluents.
Several recent studies have shown that Smac mimetics induce
cIAP-1 degradation in cancer cells, mediating apoptosis induc-
tion, and cIAP-1 is a direct target of Smac mimetics.20-22 We
have evaluated our Smac mimetics for their ability to induce
cIAP-1 degradation and cleavage of caspase-3 and poly(ADP-
ribose) polymerase (PARP) in the MDA-MB-231 cell line. We
found that 6 at concentrations as low as 10 nM effectively
induces the degradation of cIAP-1 and is more potent than 1,
2, and 4 (Figure 6). Consistent with the potent activity in the
apoptosis assay (Figure 5), at concentrations as low as 100 nM,
6 induces robust cleavage of PARP and processing of caspase-
(5S,8S,10aR)-N-Benzhydryl-5-((S)-2-(methylamino) propana-
mido)-6-oxodeca-hydropyrrolo[1,2-a][1,5]diazocine-8-carbox-
amide (2). HCl solution (4 N in 1,4-dioxane, 1 mL) was added to
a solution of 10 (35 mg, 0.06 mmol) in methanol (5 mL). The
solution was stirred at room temperature overnight and then
concentrated to give crude 2, which was purified by reverse phase
semipreparative HPLC to give pure 2 (40 mg, 95%). The gradient
ran from 80% of solvent A and 20% of solvent B to 60% of solvent
1
A and 40% of solvent B in 40 min. H NMR (300 MHz, D2O) δ
7.42-7.29 (m, 10H), 6.03 (d, J ) 6.8 Hz, 1H), 5.36 (m, 1H), 4.75
(m, 1H), 4.61 (m, 1H), 3.99 (dd, J ) 12.0, 5.4 Hz, 1H), 3.68 (m,

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8161
1H), 3.55 (m, 1H), 3.43 (m, 1H), 3.25 (m, 1H), 2.69 (s, 3H), 2.49
(m, 1H), 2.30-1.82 (m, 5H), 1.52 (d, J ) 7.1 Hz, 3H). ESI MS:
m/z 500.4 (M + Na)+. Anal. (C27H35N5O3 ·2.7CF3COOH): C, H,
N.
mg, 0.08 mmol) in CH2Cl2 (10 mL). The mixture was stirred at
room temperature overnight, concentrated, and then partitioned
between EtOAc (20 mL) and brine (5 mL). The organic layer was
dried over Na2SO4 then concentrated, and the residue was purified
by chromatography to give an amide. HCl (4N in 1,4-dioxane, 1
mL) was added to a solution of this amide in MeOH. The solution
was stirred at room temperature overnight and then concentrated
to give crude 6, which was purified by reverse phase semiprepara-
tive HPLC to give pure product (44 mg, 87% over two steps). The
gradient ran from 70% of solvent A and 30% of solvent B to 50%
of solvent A and 50% of solvent B in 40 min. 1H NMR (300 MHz,
D2O) δ 7.27-6.90 (m, 15H), 5.95 (brs, 1H), 4.65 (m, 1H), 4.38
(m, 1H), 4.06 (m, 1H), 3.85 (m, 1H), 3.78-3.30 (m, 6H), 2.55
(brs, 3H), 2.08 (m, 1H), 1.98-1.30 (m, 8H). ESI MS: m/z 596.3
(M + H)+. Anal. (C35H41N5O4 ·1.0HCl·1.2CF3COOH): C, H, N.
(5S,8S,10aR)-N-Benzhydryl-3-methyl-5-((S)-2-(methylamino)-
propanamido)-6-oxodeca-hydropyrrolo[1,2-a][1,5]diazocine-8-
carbox-amide (3). Aqueous formaldehyde solution (37%, 0.2 mL)
and HOAc (0.2 mL) were added to a solution of 10 (44 mg, 0.076
mmol) in MeOH (5 mL). After addition of NaBH3CN (50 mg, 0.79
mmol) at 0 °C, the solution was warmed to room temperature and
stirred for 3 h. The mixture was concentrated and then partitioned
between EtOAc (20 mL) and brine (5 mL). The organic layer was
dried over Na2SO4 and concentrated, and the residue was purified
by chromatography to give the methylated amine. HCl solution (4
N in 1,4-dioxane, 1 mL) was added to a solution of this amine in
MeOH (5 mL). The solution was stirred at room temperature
overnight and then concentrated to give crude 3, which was purified
by reverse phase semipreparative HPLC to give pure 3 (38 mg,
71% over two steps). The gradient ran from 75% of solvent A and
25% of solvent B to 60% of solvent A and 40% of solvent B in 40
min. 1H NMR (300 MHz, D2O) δ 7.42-7.25 (m, 10H), 6.07 (d, J )
6.8 Hz, 1H), 5.35 (m, 1H), 4.70 (m, 1H), 4.61 (m, 1H), 3.99 (dd, J )
14.0, 7.0 Hz, 1H), 3.86 (m, 1H), 3.65 (m, 1H), 3.56 (m, 1H), 3.30 (m,
1H), 3.00 (s, 3H), 2.68 (s, 3H), 2.49 (m, 1H), 2.32-1.82 (m, 5H),
1.51 (d, J ) 7.1 Hz, 3H). ESI MS: m/z 492.3 (M + H)+. Anal.
(C28H37N5O3 ·3.1CF3COOH): C, H, N.
(5S,8S,10aR)-N-Benzhydryl-3-benzyl-5-((S)-2-(methylamino)-
propanamido)-6-oxodeca-hydropyrrolo[1,2-a][1,5]diazocine-8-
carbox-amide (4). Benzyl bromide (0.1 mL) and NaHCO3 (0.3 g)
were added a solution of 10 (48 mg, 0.083 mmol) in DMF (5 mL).
The mixture was stirred at room temperature overnight and then
concentrated before being partitioned between EtOAc (20 mL) and
brine (5 mL). The organic layer was dried over Na2SO4 then
concentrated, and the residue was purified by chromatography to give
a benzylated amine. To a solution of this amine in methanol (5 mL)
was added HCl solution (4 N in 1,4-dioxane, 1 mL). The solution
was stirred at room temperature overnight and then concentrated to
give crude product, which was purified by reverse phase semiprepara-
tive HPLC to give pure 4 (51 mg, 77% over two steps). The gradient
ran from 75% of solvent A and 25% of solvent B to 60% of solvent
A and 40% of solvent B in 40 min. 1H NMR (300 MHz, D2O) δ 7.34
(m, 2H), 7.28-7.02 (m, 13H), 6.05 (d, J ) 6.9 Hz, 1H), 5.38 (m,
1H), 4.72 (m, 1H), 4.52 (m, 1H), 4.25 (ABq, J ) 8.4 Hz, 2H), 3.97
(dd, J ) 13.5, 6.8 Hz, 1H), 3.82-3.56 (m, 2H), 3.49 (m, 1H), 3.18
(m, 1H), 2.67 (s, 3H), 2.35 (m, 1H), 2.08 (m, 1H), 1.75-1.52 (m,
4H), 1.47 (d, J ) 7.0 Hz, 3H). ESI MS: m/z 568.3 (M + H)+. Anal.
(C34H41N5O3 ·2.9CF3COOH): C, H, N.
(5S,8S,10aR)-3-Acetyl-N-benzhydryl-5-((S)-2-(methylamino)-
propanamido)-6-oxodeca-hydro pyrrolo[1,2-a][1,5]diazocine-8-
carbox-amide (5). Acetic anhydride (0.1 mL) and N,N-diisopro-
pylethylamine (0.3 mL) were added to a solution of 10 (46 mg,
0.08 mmol) in CH2Cl2 (5 mL) at 0 °C. The mixture was stirred at
room temperature overnight, concentrated, and then partitioned
between EtOAc (20 mL) and brine (5 mL). The organic layer was
dried over Na2SO4 and then concentrated. The residue was purified
by chromatography to give an amide. HCl solution (4N in 1,4-
dioxane, 1 mL) was added to a solution of this residue in MeOH
(5 mL). The solution was stirred at room temperature overnight
and then concentrated to give crude 5, which was purified by reverse
phase semipreparative HPLC to give pure product (38 mg, 85%
over two steps). The gradient ran from 70% of solvent A and 30%
of solvent B to 50% of solvent A and 50% of solvent B in 40 min.
1H NMR (300 MHz, D2O) δ 7.38-7.19 (m, 10H), 5.95 (brs, 1H),
4.96 (m, 1H), 4.40 (m, 1H), 4.25 (m, 1H), 3.94 (m, 1H), 3.66 (m,
1H), 3.60-3.35 (m, 3H), 2.63 (s, 3H), 2.25 (m, 1H), 2.15-1.65
(m, 8H), 1.47 (d, J ) 7.1 Hz, 3H). ESI MS: m/z 520.3 (M+H)+.
Anal. (C29H37N5O4 ·1.0HCl·1.5CF3COOH): C, H, N.
Acknowledgment. We are grateful for financial support from
the National Cancer Institute, National Institutes of Health
(R01CA109025 to S.W.), the Breast Cancer Research Founda-
tion (S.W.), the Prostate Cancer Foundation (S.W.), Ascenta
Therapeutics Inc. (S.W.), the Susan G. Komen Foundation
(H.S.), and the University of Michigan Cancer Center Core grant
(P30CA046592).
Supporting Information Available: An experimental section
including details of the synthesis and chemical data of intermediates,
biochemical and cellular assays, molecular modeling, and pharma-
cokinetics. This material is available free of charge via the Internet
at http://pubs.acs.org.
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