Orally bioavailable antagonists of inhibitor of apoptosis proteins based on an azabicyclooctane scaffold

Article abstract of DOI:10.1021/jm801450c

A series of IAP antagonists based on an azabicyclooctane scaffold was designed and synthesized. The most potent of these compounds, 14b, binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domain of c-IAP1 with K_i values of 14

Full text of DOI:10.1021/jm801450c

J. Med. Chem. 2009, 52, 1723–1730
1723
Orally Bioavailable Antagonists of Inhibitor of Apoptosis Proteins Based on an
Azabicyclooctane Scaffold∞
Frederick Cohen,*,† Bruno Alicke,‡ Linda O. Elliott,# John A. Flygare,† Tatiana Goncharov,| Stephen F. Keteltas,†
Matthew C. Franklin,| Stacy Frankovitz, Jean-Philippe Stephan, Vickie Tsui,† Domagoj Vucic,| Harvey Wong,§ and
Wayne J. Fairbrother|
Departments of DiscoVery Chemistry, Translational Oncology, Biochemical Pharmacology, Protein Engineering, Assay and Automation
Technology, and Drug Metabolism and Pharmacokinetics, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080
ReceiVed NoVember 17, 2008
A series of IAP antagonists based on an azabicyclooctane scaffold was designed and synthesized. The most
potent of these compounds, 14b, binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the
BIR3 domain of c-IAP1 with Ki values of 140, 38, and 33 nM, respectively. These compounds promote
degradation of c-IAP1, activate caspases, and lead to decreased viability of breast cancer cells without affecting
normal mammary epithelial cells. Finally, compound 14b inhibits tumor growth when dosed orally in a
breast cancer xenograft model.
Introduction
parts of the peptide, the software program CAVEAT was
utilized.19 This program takes as input the structure of a peptide
or small molecule bound in the target receptor site. Selected
bond vectors are searched against prebuilt databases of three-
dimensional compounds to find ring systems that could connect
to and maintain the vectors in their input spatial arrangement.
We used the 2.3 Å resolution crystal structure of peptide Ala-
Val-Pro-2,2-diphenethylamine (1) bound to the ML-IAP BIR
domain as the input structure (Figure 1A).20 One vector was
defined from the proline nitrogen atom to the valine carbonyl
carbon atom, while a second vector was defined from the proline
carbonyl carbon atom to the diphenethylamine nitrogen (Figure
2A). CAVEAT databases of minimized compounds from
commercially available small molecules were constructed and
searched. Ring systems that overlapped with atoms of the ML-
IAP BIR domain were not considered in subsequent analyses.
The conformations and qualitative geometric fit of all unique
hits were examined visually. In particular, some fused-ring
motifs (Figure 2B) looked especially promising from a geometric
perspective. However, upon further inspection, these were
deemed either synthetically infeasible within our desired time
frame or chemically unstable. These bicyclic motifs provided
the inspiration to manually search the literature for similar
bicyclic templates that would be more synthetically accessible;
from this search emerged an azabicyclooctane (Figure 2C). A
further conceptual jump was to realize that use of this scaffold
would also require reversing the direction of the P3-P4 amide
bond for both synthetic accessibility and geometric fit.
Programmed cell death, or apoptosis, is a crucial mechanism
for maintaining homeostasis and removal of damaged or
malignant cells. This pathway is tightly controlled by a number
of positive and negative regulatory elements. The inhibitor of
apoptosis (IAPa) proteins negatively regulate this process
through a variety of mechanisms including direct inhibition of
effector caspase enzymes or modulation of TNF receptor-
mediated signaling pathways.1-6 Members of this family are
up-regulated in various cancers and promote resistance to
chemotherapy. Thus, inhibition of these proteins may be a new
therapeutic mechanism for treating cancer.7,8
Each of the IAP proteins contains at least one BIR domain,
some of which bind to the N-termini of their targets through
interactions with a shallow cleft. Although the peptide-binding
pocket of the BIR domains is shallow, several groups have
reported peptidomimetics9-12 or other small molecules13-16 that
bind to this pocket with high affinity, disrupt IAP-caspase or
IAP-SMAC binding, and show in vivo efficacy in a mouse
xenograft model of cancer.17,18
In this communication we report our initial efforts toward
developing a molecule with reduced peptide character that would
possess the appropriate potency, permeability, and pharmaco-
kinetic properties to be a useful therapeutic agent. To generate
ideas for small molecule scaffolds that could potentially displace
∞
Coordinates and structure factors have been deposited in the RCSB
Protein Data Bank. Access codes are as follows: 1, 3F7G; 14a, 3F7H; 14b,
3F7I.
When the azabicyclooctane scaffold was computationally
inserted into the rest of the peptide, using a reversed amide to
link the diphenyl group to the scaffold (Figure 2D), it was found
to fit well within the peptide binding site. Using Glide, a
minimized conformation of this molecule was docked to the
ML-IAP BIR domain peptide-binding site.21 The conformations
that emerged were similar to the conformations of 1 both in
the crystal structure and in the docked model (Figure 1B). The
docking score of the azabicyclooctane compound (-9.5) is
comparable to that of 1 (-9.3). The main difference between
the crystal structure and docked models relates to which of the
prochiral phenyl groups is buried in the P4 pocket. In the crystal
structure of 1, the pro-S phenyl ring of the 2,2-diphenylethy-
lamine moiety packs into the hydrophobic P4 pocket where it
* To whom correspondences should be addressed. Phone: 650-225-2976.
Fax: 650-467-8922. E-mail: fcohen@gene.com.
†
Department of Discovery Chemistry.
Department of Translational Oncology.
Department of Biochemical Pharmacology.
Department of Protein Engineering.
‡
#
|
Department of Assay and Automation Technology.
Department of Drug Metabolism and Pharmacokinetics.
§
a Abbreviations: IAP, inhibitor of apoptosis; XIAP, X-chromosome-
linked inhibitor of apoptosis; BIR, baculoviral inhibitor of apoptosis repeat;
ML-IAP, melanoma inhibitor of apoptosis; SMAC, second mitochondrial
activator of caspase; Teoc, (trimethylsilyl)ethyl carbamate; TFA, trifluo-
roacetic acid; TMS, trimethylsilyl; EDC, N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride; HATU, 2-(7-aza-1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluroniumhexafluorophosphate;TASF,tris(dimethylamino)sulfonium
difluorotrimethylsilicate; HMEC, human mammary epithelial cells.
10.1021/jm801450c CCC: $40.75
2009 American Chemical Society
Published on Web 02/19/2009

1724 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Cohen et al.
Scheme 1. Synthesis of Amide Analogues 14a-ta
Figure 1. (A) Crystal structure of the ML-IAP BIR domain in complex
with 1. The key P1 and P4 binding pockets are labeled. (B) Overlay of
the highest-scoring docked Ala-Val-Pro-2,2-diphenethylamine confor-
mation (gray) and the highest-scoring docked azabicyclooctane com-
pound (Figure 2D) (cyan) in the ML-IAP peptide-binding site.
a Reagents and conditions: (a) I2, CH2Cl2. (b) 4 and 5: NH4OH, THF,
HPLC separation, 29%, two steps. (c) 6 and 7: NaN3, acetone, water; SiO2
separation 35%, two steps; (d) 10% Pd ·C, 1 atm of H2, EtOAc, 94%; (e)
TMSCH2CH2OC(O)OC6H4NO2, NaHCO3, CH2Cl2 55%; (f) H2, 20%
Pd(OH)2 ·C, MeOH, quant; (g) Cbz-t-Leu-OH, HATU, DIPEA, DMF; (h)
H2, 20% Pd(OH)2 ·C, MeOH, 72% two steps; (i) Boc-N-Me-Ala-OH, EDC,
DMAP, MeCN; (j) TASF, MeCN, 50 °C, 88-94%, two steps; (k) R1COCl
or R1CO2H, EDC, DMAP, MeCN; (l) TFA, CH2Cl2, HPLC purification,
16-79% two steps.
with I2 in CH2Cl2 provided bicyclic amino iodide 3 (Scheme
1). Subjection of this crude mixture to NH4OH and THF led to
displacement of the iodide by ammonia, with retention of
configuration, providing diastereomeric diamines 4 and 5. These
compounds were separated by HPLC on a C18 column, eluting
with 0-10% MeCN/H2O, 0.1% trifluoroacetic acid (TFA). The
desired diastereomer 5 elutes first under these conditions.
Assignment of the stereochemistry of 4 and 5 was made initially
by carrying both isomers forward to IAP antagonists and
comparing potency in the fluorescence polarization assay. Our
assignment was subsequently verified by X-ray analysis of a
cocrystal of an antagonist with an IAP BIR domain (vida infra).
While acceptable for making exploratory quantities of these
compounds, the requirement for separating diastereomers by
HPLC limited our ability to make larger amounts of material
and led us to investigate another nitrogen source. Thus, treatment
of iodide 3 with sodium azide in a mixture of acetone-water
cleanly led to a mixture of azides 6 and 7, which were separable
on silica gel, greatly improving throughput. Hydrogenation under
mild conditions provided the primary amine 5, with no trace of
benzyl deprotection.
Figure 2. Evolution of azabicyclooctane proline mimetic: (A) input
vectors for CAVEAT search; (B) bicyclic hits from CAVEAT search;
(C) azabicyclooctane inspired by search hits; (D) elaborated construct
used to compare scaffold design to 1.
makes extensive contacts with protein residues Thr116, Lys121-
Arg123, and Gly130-Gln132 (Figure 1A). The second (pro-R)
phenyl ring of the 2,2-diphenylethylamine moiety packs more
on the surface of the protein with one edge making contact with
the hydrophobic portion of the side chain of Lys121. By contrast,
both models predicted that the pro-R phenyl group would insert
into the P4 pocket and the pro-S phenyl group would be more
exposed (Figure 1B).
Synthesis
The synthesis of this scaffold is based on the work of Corey22
and Kawabata.23 We began with amine 2, which when treated

Antagonists Based on Azabicyclooctane
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1725
Table 1. Structure and Potency of IAP Selected Antagonistsa
Figure 3. Crystal structures of 14a and 14b in complex with
MLXBIR3SG at 1.8 and 1.9 Å resolution, respectively.
summarized in Table 1. The potency of diphenylacetyl derivative
14a was within 2-fold of the potency of the peptide lead 1 (Ki
) 43 nM for MLXBIR3SG, a chimeric BIR domain that
preserves the native ML-IAP peptide-binding site,20 and Ki )
345 nM for XIAP BIR3). Most of the other moieties chosen
for the P4 position lost potency relative to the starting peptide.
However, a series of 1-napthyl derivatives 14b-e retained or
increased affinity for both MLXBIR3SG and XIAP BIR3. The
2-napthyl isomer 14f lost affinity for XIAP BIR3 and retained
affinity for MLXBIR3SG, making it the most selective com-
pound in the series. This series also shows that polar substituents
are not tolerated in the P4 pocket.
To gain a more detailed understanding of the interactions
between the azabicyclooctane-containing IAP antagonists and
the BIR domain of ML-IAP, the crystal structures of complexes
between MLXBIR3SG and compounds 14a and 14b were
determined to resolutions of 1.8 and 1.9 Å, respectively (Figure
3). X-ray data collection and refinement statistics are listed in
Supporting Information Table 1. Key contacts observed in the
structure of 1 in complex with the ML-IAP BIR domain (Figure
1A) are conserved in the complexes with 14a and 14b. In
particular, hydrogen bonds with the main chain of Gln132 and
the side chain carboxylate of Asp138 are maintained. In the
case of 14a, the phenyl rings of the diphenylacetyl moiety are
positioned very similarly to the phenyl rings of the 2,2-
diphenylethylamine moiety of 1. The 1-naphthyl group of 14b
also makes extensive contacts with the hydrophobic P4 pocket
of the protein, although in order to make these contacts, the
conformations of the azabicyclooctane and the intervening amide
group differ slightly from that observed for 14a.
a Ki determined as described in ref 10. b Average of two measurements.
After experimenting with several protecting group schemes,
we arrived at (trimethylsilyl)ethyl carbamate (Teoc) as the
optimal group for the primary amine. Thus, the primary amine
of 5 was acylated with Teoc-p-nitrophenyl carbonate under
biphasic conditions, and the benzyl group was removed by
hydrogenolysis with 20% Pd(OH)2 ·C, to provide amine 9 in
46% yield for the two steps. We chose to fix the P1 and P2
amino acids as N-Me-Ala and tert-Leu, respectively, for this
study, as SAR in the peptide series demonstrated that these
changes retained potency and afforded increased hydrolytic
stability (data not shown). Thus, coupling of Cbz-protected tert-
Leu was accomplished with N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDC) and the Cbz group
removed with H2 and 20% Pd(OH)2 ·C to afford amine 11 in
72% yield over two steps. The P1 amino acid was coupled in
Boc-protected form with EDC. Finally, the Teoc group was
removed with tris(dimethylamino)sulfonium difluorotrimethyl-
silicate (TASF) in warm MeCN.24 This route was readily
amenable to preparation of half-gram quantities of common
intermediate 13.
With common intermediate 13 in hand, we sought to explore
the SAR of the P4 position. Carboxylic acid derivatives were
selected by a combination of computational virtual reagent
selection and medicinal chemistry principles. These were
coupled through either the acid chloride or EDC dehydration.
The Boc group was removed with TFA, and the final product
was purified by HPLC. The structures of the most potent
antagonists 14a-f, made via this route, are shown in Table 1.
A complete list of the antagonists synthesized is in the
Supporting Information.
The azabicyclooctane scaffold both reverses the direction of
the P3-P4 amide bond and adds extra conformational constraint
relative to 1. We sought to dissect these two effects through
the synthesis of control molecules. Thus, the two peptides 15a
and 15b (Table 2) were synthesized using routine peptide
chemistry from S-2-(aminomethyl)-1-tert-butoxycarbonylpyr-
rolidine. These compounds lack the constraint of the bicyclic
scaffold and yet retain the reversal of the P3-P4 amide bond.
Compound 15a lost approximately 3- to 5-fold in binding
affinity relative to 14a, while 15b lost approximately 10- to
15-fold in binding affinity relative to 14b. These data show that
The Ki values binding to the BIR3 domain of XIAP and the
single BIR domain of ML-IAP were determined using a
fluorescence polarization competition assay.10 The results are

1726 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Table 2. Potency of Monocyclic Controlsa
Cohen et al.
a Ki determined as described in ref 10. b Average of two measurements.
Figure 4. Cell-killing in response to 3 day treatment with IAP
antagonists in MDA-MB-231 cells.
Figure 5. Caspase 3/7 activation in response to treatment with 14a
and 14b for 6 h in various cell lines.
Table 3. EC50 Values for Selected Compounds in 3-Day Cell Viability
Assay (Values in µM, Average of Three Determinations)
Table 4. EC50 for Caspase Activation in Cell Culture
EC50 (µM)
EC50 (µM)
compd
MDA-MB-231
BT-549
HMEC
compd
MDA-MB-231
HMEC
14a
14b
14c
14d
14f
3.59 ( 0.20
0.49 ( 0.01
4.45 ( 0.03
1.58 ( 0.11
1.32 ( 0.04
1.81 ( 0.45
0.47 ( 0.02
4.07 ( 0.11
1.45 ( 0.04
0.19 ( 0.01
>10
>10
>10
>10
>10
14a
14b
14c
14d
14f
9.64 ( 1.00
0.98 ( 0.14
10.2 ( 0.30
3.12 ( 0.69
3.29 ( 0.11
>10
>10
>10
>10
>10
the extra ring provided by the scaffold has a significant
contribution to the potency of these antagonists.
correlates well with the potency in the viability assay. Example
data for compounds 14a and 14b are shown in Figure 5, and
potencies are tabulated in Table 4. Consistent with the viability
assays, these compounds did not activate effector caspases in
normal human mammary epithelial cells (HMEC).
A selection of the more potent IAP antagonists were tested
in 3-day cell-viability assays (Figure 4, Table 3). These
compounds led to a decrease in cell viability in the MDA-MB-
231 breast cancer cell line. This is in contrast to starting peptide
1, which does not induce cell death in a 3-day viability assay.
These compounds did not cause cell death in normal human
mammary epithelial cells (HMEC).
To assess the mechanism of cell killing, these compounds
were tested for activation of the effector caspases 3 and 7 in
the breast cancer cell lines BT-549 and MDA-MB-231. These
compounds were found to activate caspases 3 and 7 in a dose
dependent manner. The potency in the caspase activation assay
The effects of the IAP antagonist compounds 14a and 14b
on the stability of c-IAP1 protein were also examined. Treatment
of MDA-MB-231 breast carcinoma cells with 14a for 1 h had
only a minor effect on the levels of c-IAP1, while similar
treatment with 14b led to almost complete loss of c-IAP1 protein
(Figure 6) in agreement with reported studies.2,4 These data
correlate with the measured potency of 14a and 14b against
the c-IAP1 BIR3 domain (14a Ki ) 129 nM; 14b Ki ) 33 nM).

Antagonists Based on Azabicyclooctane
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1727
Figure 6. Treatment with IAP antagonist compound 14b leads to
degradation of c-IAP1. MDA-MB-231 cells were treated for 1 h with
the indicated concentrations of 14b or 14a, and cell lysates were probed
with the indicated antibodies.
In preparation for efficacy studies, compounds 14a and 14b
were evaluated for oral exposure in mice. Thus, each was dosed
at 25 mg/kg as a solution in 15% aqueous hydroxypropyl-ꢀ-
cyclodextrin with 20 mM succinic acid. Both compounds were
found to have significant exposure upon oral dosing. Compound
14a was found to have a Cmax of 5.0 µM, AUC of 16 h ·µM,
and T1/2 of 1.5 h, while 14b was found to have a Cmax of 3.6
µM, AUC of 5.0 h ·µM, and T1/2 of 1.3 h.
Compounds 14a and 14b were further studied in a mouse
cancer xenograft model (Figure 7). To that effect, mice bearing
tumors (n ) 14 per group) derived from the MDA-MD-231
cell line were dosed orally with either vehicle or compounds at
50 (mg/kg)/day. Compound 14b potently inhibited tumor growth
in this model, with a median time to progression (defined as
time to 6-fold increase in tumor volume from day 0) of 25 days,
versus 14 days for the vehicle control, without affecting body
weight. Compound 14a had little effect on either tumor growth
or body weight.
Conclusion
We have designed and synthesized a novel azabicyclooctane
proline mimetic and incorporated it into IAP antagonists. Several
of these compounds were more potent than lead peptide 1, with
Ki values of <50 nM against select IAP BIR domains. X-ray
crystallographic analysis confirmed that these compounds bind
to the expected binding site on the ML-IAP BIR domain. These
compounds induce degradation of c-IAP1 and activate effector
caspases in cancer cell lines in preference to normal cell lines.
Compounds that potently activated caspases also lead to
decreased viability of cancer cell lines without affecting
“normal”, nontumor cells. This potency in cell culture assay
translates to significant potency in a xenograft model of breast
cancer where compound 14b nearly doubles the time to
progression when dosed orally. These studies further validate
the IAP proteins as promising targets for cancer treatment.
Figure 7. Efficacy of compounds 14a and 14b compared to vehicle
control. Compounds were dosed 50 (mg/kg)/day po, n ) 14 per group.
MeOH in CO2 flowing at 2.35 mL/min (SFCd method) or a Berger
pyridine column, 4.6 mm × 150 mm, with a 6 min gradient of
5-50% MeOH in CO2 flowing at 2.35 mL/min (SFCp). All final
compounds were purified to >95% purity. High-resolution mass
spectra were obtained at the University of California at Berkeley
on a ZAB2-EQ mass spectrometer (Micromass, Manchester, U.K.)
equipped with a FAB ion source. The source was operated at 8
kV; the Cs gun was operated at 20 kV.
(3aS,6S,6aR)-1-((R)-1-Phenylethyl)octahydrocyclopenta[b]-
pyrrol-6-ylamine (4) and (3aR,6S,6aS)-1-((R)-1-Phenylethyl)-
octahydrocyclopenta[b]pyrrol-6-ylamine (5). To a solution of
amine 2 (3.13 g, 14.5 mmol) in CH2Cl2 (60 mL) was added I2 (4.43
g, 17.4 mmol) portionwise over 30 min with vigorous stirring. After
2 h, LCMS indicated complete conversion to iodide 3. The mixture
was concentrated under reduced pressure to afford a dark-brown
foam.
The residue from above was dissolved in THF (50 mL) and
concentrated NH4OH (50 mL) and stirred at room temperature for
17 h. The THF was removed under reduced pressure and the residue
diluted with 1 N NaOH (50 mL) and extracted with CH2Cl2 (4 ×
50 mL). The combined organic phases were extracted with 1 N
HCl (4 × 50 mL). The combined aqueous phases were washed
Experimental Section
All chemicals were purchased from commercial suppliers (Al-
drich, VWR) and used as received. Flash chromatography was
carried out with RediSep prepacked SiO2 cartridges on an ISCO
Companion chromatography system. NMR spectra were recorded
on a Varian Inova 400 NMR spectrometer and referenced to
tetramethylsilane. For all compounds that contain secondary amides
the data for the major rotamers are reported. Preparative HPLC
was performed on a Polaris C18 5 µm column (50 mm × 21 mm).
Purity analysis of final compounds was performed by HPLC-MS
with a Chromasil C18 column (4.6 mm × 50 mm) with a gradient
of 0-90% acetonitrile (containing 0.038% TFA) in 0.1% aqueous
TFA (over 11 min, flow ) 2 mL/min). Low-resolution mass spectra
were recorded on a Sciex 15 mass spectrometer in ES+ mode.
Purity analysis of final compounds was also performed on a Berger
Instruments SFC system operating at 100 bar, 35 °C, column )
Berger diol, 4.6 mm × 150 mm, with a 6 min gradient of 20-60%

1728 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Cohen et al.
with CH2Cl2 (1 × 20 mL), then made basic (pH > 10) with solid
NaOH (∼10 g, with cooling). The aqueous phase was extracted
with CH2Cl2 (4 × 50 mL). The combined organic phases were dried
(K2CO3), filtered, and concentrated. The residue was purified by
reverse-phase HPLC (C18, MeCN-H2O, 0.1% TFA). Fractions
containing the product were concentrated under reduced pressure
until all of the MeCN was removed, made basic with 1 N NaOH
(pH > 11), and exhaustively extracted with CH2Cl2. The combined
organic phases were dried (Na2SO4), filtered, and concentrated to
residue was purified by flash chromatography (40 g of SiO2, 0-10%
ethyl acetate in hexanes with 2% triethylamine) to afford 397 mg
1
(65%) of 8 as a colorless oil: H NMR (400 MHz, CDCl3) δ ppm
7.46-7.00 (m, 5H), 4.42-4.18 (m, 1H), 4.18-3.97 (m, 2H),
3.97-3.80 (m, 1H), 3.77-3.43 (m, 1H), 3.10-2.82 (m, 1H),
2.72-2.62 (m, 1H), 2.56 (d, J ) 8.38 Hz, 1H), 2.36 (q, J ) 9.29
Hz, 1H), 2.01-1.88 (m, 1H), 1.84-1.79 (m, 1H), 1.76-1.61 (m,
1H), 1.42-1.39 (m, 1H), 1.37 (d, J ) 6.71 Hz, 3H), 1.28 (s, 1H),
0.92 (dd, J ) 9.57, 7.59 Hz, 2H), 0.02-0.01 (m, 9H); 13C NMR
(100 MHz, CDCl3) δ ppm 144.70, 128.00, 127.60, 126.60, 59.20,
49.15, 41.21, 31.48, 30.47, 30.21, 30.18, 17.71, -1.51; HRMS
(FAB) m/z 375.2474 (375.2468 calcd for C21H35N2O2Si M + H).
(3aR,6S,6aS)-(Octahydrocyclopenta[b]pyrrol-6-yl)carbamic
Acid 2-Trimethylsilanylethyl Ester (9). A mixture of 8 (397 mg,
1.06 mmol), 20% Pd(OH)2 ·C (120 mg), acetic acid (0.30 mL), and
MeOH (15 mL) was evacuated and refilled with H2 3× and then
stirred vigorously under 1 atm of H2 for 2 h. The mixture was
filtered through Celite, then concentrated. The residue was dissolved
in CH2Cl2 (50 mL) and washed with 1 N NaOH (3 × 20 mL). The
combined aqueous phases were extracted with CH2Cl2 (1 × 20 mL).
The combined organic phases were dried (K2CO3), filtered, and
concentrated to afford 265 mg (92%) of amine 9 as a colorless oil:
1H NMR (400 MHz, CDCl3) δ ppm 4.81-4.56 (m, 1H), 4.10 (m,
2H), 3.69-3.50 (m, 1H), 3.46-3.28 (m, 1H), 2.85 (t, J ) 6.28
Hz, 2H), 2.67-2.50 (m, 1H), 2.28 (s, 1H), 1.96 (dt, J ) 11.67,
5.67 Hz, 1H), 1.91-1.69 (m, 2H), 1.50-1.16 (m, 4H), 1.09-0.85
(m, 2H), 0.02-0.01 (m, 9H); 13C NMR (100 MHz, CDCl3) δ ppm
69.82, 62.82, 46.14, 40.87, 33.44, 31.48, 31.46, 30.35, 17.73, -1.53;
HRMS (FAB) m/z 271.1836 (271.1842 calcd for C13H27N2O2Si M
+ H).
1
provide amine 5 (720 mg, 28%) as a colorless oil: H NMR (400
MHz, CDCl3) δ ppm 7.46-7.18 (m, 5H), 3.45 (q, J ) 6.65 Hz,
1H), 3.16-2.98 (m, 1H), 2.65 (t, J ) 8.91 Hz, 3H), 2.30 (ddd, J )
11.48, 9.04, 5.47 Hz, 1H), 2.18 (d, J ) 2.48 Hz, 4H), 1.97-1.82
(m, 1H), 1.82-1.66 (m, 2H), 1.49-1.28 (m, 6H), 0.02-0.01 (m,
9H); 13C NMR (100 MHz, CDCl3) δ ppm 145.44, 128.12, 128.09,
127.15, 65.17, 58.90, 53.52, 42.37, 32.33, 31.16, 30.90, 29.56,
20.84; HRMS (FAB) m/z 231.1858 (231.1861 calcd for C15H23N2
M + H). Data for 4: 1H NMR (400 MHz, CDCl3) δ ppm 7.45-7.07
(m, 5H), 3.52 (q, J ) 6.75 Hz, 1H), 3.34-3.19 (m, 1H), 2.76-2.52
(m, 3H), 2.11 (dt, J ) 10.80, 5.63 Hz, 1H), 2.03-1.84 (m, 2H),
1.84-1.70 (m, 1H), 1.51-1.34 (m, 6H), 1.29 (m, 5H); 13C NMR
(100 MHz, CDCl3) δ ppm 145.06, 128.10, 127.45, 126.67, 76.24,
64.27, 58.56, 53.31, 42.04, 32.55, 31.40, 29.62, 23.45.
(3aS,6R,6aR)-6-Azido-1-((R)-1-phenylethyl)octahydrocyclo-
penta[b]pyrrole (6) and (3aR,6S,6aS)-6-Azido-1-((R)-1-phenyl-
ethyl)octahydrocyclopenta[b]pyrrole (7). To a solution of amine
2 (6.71 g, 31.2 mmol) in CH2Cl2 (70 mL) was added I2 (8.70 g,
34.3 mmol) portionwise over 20 min with vigorous stirring. After
2 h, LCMS indicated complete conversion to iodide 3. The mixture
was concentrated under reduced pressure to afford a dark-brown
foam.
[(3aR,6S,6aS)-1-((S)-2-Benzyloxycarbonylamino-3,3-dimeth-
ylbutyryl)octahydrocyclopenta[b]pyrrol-6-yl]carbamic Acid 2-T-
rimethylsilanylethyl Ester (10). A mixture of amine 9 (437 mg,
1.62 mmol), Cbz-N-tert-butylglycine (514 mg, 1.94 mmol), HATU
(1.23 g, 3.23 mmol), DIPEA (1.4 mL, 8.1 mmol), and DMF (10
mL) was maintained at room temperature 4 h. The mixture was
diluted with ethyl acetate (100 mL) and washed with 1 N HCl (3
× 20 mL). The combined aqueous phases were extracted with ethyl
acetate (1 × 10 mL). The combined organic phases were dried
(Na2SO4), filtered, and concentrated. The residue was purified by
flashchromatography(40gcolumnSiO2,0-40%ethylacetate-hexanes)
to afford 485 mg (58%) of the product as a colorless oil, which
was carried on directly. HRMS (FAB) m/z 569.3741 (569.3734
calcd for C28H53N4O6Si M + H).
[(3aR,6S,6aS)-1-((S)-2-Amino-3,3-dimethylbutyryl)octahydro-
cyclopenta[b]pyrrol-6-yl]carbamic Acid 2-Trimethylsilanylethyl
Ester (11). A mixture of the amide 10 (500 mg, 0.97 mmol), MeOH
(20 mL), AcOH (0.8 mL), and 20% Pd(OH)2 · C (200 mg) was
stirred vigorously under 1 atm of H2 for 3 h. The mixture was
filtered through Celite with MeOH. The MeOH was removed under
reduced pressure, and the residue was dissolved in CH2Cl2 (50 mL)
and washed with 0.5 N NaOH (3 × 10 mL), dried (Na2SO4),
filtered, and concentrated to afford 300 mg (81%) of amine 11 as
a colorless oil: 1H NMR (400 MHz, CDCl3) δ ppm 6.31-5.85 (m,
1H), 4.31 (dd, J ) 8.79, 6.99 Hz, 1H), 4.20-4.00 (m, 2H),
3.72-3.62 (m, 1H), 3.57-3.44 (m, 2H), 3.26 (s, 1H), 2.67 (d, J )
6.66 Hz, 1H), 2.41-2.24 (m, 1H), 2.01 (m, 2H), 1.43-1.30 (m,
1H), 1.06-0.86 (m, 11H), 1.75-1.44 (m, 5H), 0.02-0.01 (m, 9H);
13C NMR (100 MHz, CDCl3) δ ppm 173.89, 157.07, 156.11, 67.31,
62.60, 60.07, 59.80, 47.69, 39.30, 35.49, 33.26, 31.76, 28.95, 26.20,
17.61, -1.50, -1.53; HRMS (FAB) m/z 384.2690 (384.2682 calcd
for C19H38N3O3Si M + H).
The residue from above was dissolved in acetone (100 mL) and
water (40 mL). Sodium azide (10.1 g, 156 mmol) was added and
the mixture stirred at room temperature for 2 h. Aqueous 1 N NaOH
(50 mL) was added and the acetone removed under reduced
pressure. Water (100 mL) was added and extracted with CH2Cl2
(1 × 100 mL, 2 × 50 mL). The combined organic phases were
dried (K2CO3), filtered, and concentrated. The residue was purified
by chromatography on a 4 cm × 20 cm column of SiO2 with slow
elution of 2-4% ether-hexanes modified with 2% triethylamine
to afford 2.36 g (32%) of 6 and 1.97 g (27%) of 7 which was carried
1
on directly. Diagnostic data for 7: H NMR (400 MHz, CDCl3) δ
7.36-7.25 (m, 5H), 3.53 (q, J ) 6.7, 1H), 3.09 (d, J ) 4.0, 1H),
3.04-2.96 (m, 1H), 2.92 (d, J ) 8.5, 1H), 2.71-2.58 (m, 1H),
2.31 (ddd, J ) 11.5, 8.9, 5.6, 1H), 1.98-1.89 (m, 1H), 1.85-1.65
(m, 2H), 1.56 - 1.47 (m, 1H), 1.44-1.39 (m, 4H), 1.38 - 1.24
(m, 1H); 13C NMR (100 MHz, CDCl3) δ 144.83, 128.59, 128.13,
127.55, 74.28, 68.07, 52.76, 41.91, 31.61, 30.10, 29.19, 20.59.
1
Diagnostic data for 6: H NMR (400 MHz, CDCl3) δ 7.34-7.20
(m, 5H), 3.86 (d, J ) 4.8, 1H), 3.55 (q, J ) 6.8, 2H), 2.94 (d, J )
8.5, 1H), 2.70-2.60 (m, 3H), 2.08 (ddd, J ) 11.6, 9.0, 5.5, 1H),
2.00-1.94 (m, 2H), 1.85-1.74 (m, 3H), 1.57 (d, J ) 3.1, 1H),
1.52-1.46 (m, 2H), 1.44 (d, J ) 6.8, 3H), 1.34 - 1.21 (m, 2H).
Hydrogenation of 7 to 5. A mixture of azide 7 (446 mg, 1.74
mmol), ethyl acetate (20 mL), and 10% Pd ·C (200 mg) in a 100
mL round-bottom flask was evacuated and refilled 3× with H2 and
then maintained under H2 (1 atm) with vigorous stirring for 4 h.
The mixture was filtered through Celite and concentrated to afford
377 mg (94%) of amine 5 as a colorless oil. Spectral data were
identical to material prepared by aminolysis of iodide 3.
[(3aR,6S,6aS)-1-((R)-1-Phenylethyl)octahydrocyclopenta[b]-
pyrrol-6-yl]carbamic Acid 2-Trimethylsilanylethyl Ester (8). To
a mixture of amine 5 (377 mg, 1.64 mmol), CH2Cl2 (6 mL), and
saturated aqueous NaHCO3 (6 mL) was added 4-nitrophenyl
2-(trimethylsilyl)ethylcarbamate (510 mg, 1.80 mmol), and the
mixture was stirred vigorously at room temperature for 14 h. The
mixture was partitioned between 1 N NaOH (50 mL) and CH2Cl2
(50 mL). The phases were separated, and the organic phase was
washed with 1 N NaOH (3 × 20 mL). The combined aqueous
phases were extracted with CH2Cl2 (1 × 20 mL). The combined
organic phases were dried (K2CO3), filtered, and concentrated. The
((3aR,6S,6aS)-1-{(S)-2-[(S)-2-(tert-Butoxycarbonylmethylami-
no)propionylamino]-3,3-dimethylbutyryl}octahydrocyclopenta-
[b]pyrrol-6-yl)carbamic Acid 2-Trimethylsilanylethyl Ester (12).
To a solution of amine 11 (300 mg, 0.79 mmol) in acetonitrile
(3.0 mL) were added N-Boc-N-methylalanine (185 mg, 0.090
mmol), DMAP (catalytic), and EDC (172 mg, 0.09 mmol), and
the solution was stirred at room temperature for 1.5 h. The solution
was diluted with CH2Cl2 (50 mL) and then washed with 1 N HCl
(3 × 10 mL), 1 N NaOH (3 × 10 mL), brine (1 × 10 mL), dried

Antagonists Based on Azabicyclooctane
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1729
(Na2SO4), filtered, and concentrated to afford 426 mg (93%) of 12
as a colorless oil, which was used without further purification: H
Inc.; San Diego, CA) for 5-10 min. The detached cells were
washed with phosphate buffered saline (PBS) and resuspended in
assay media. Cells were placed in tissue culture-treated, white-wall,
clear-bottom, 96-well plates (Corning, Inc.; Corning, NY) at 1 ×
104 cells/well in a volume of 50 µL. The plates were incubated at
37 °C in 5% CO2 overnight.
The next day, the media were removed and compounds added
to the cells in assay media. Plates were incubated at 37 °C in 5%
CO2 for 3-4 days before cell viability was measured using the
CellTiter-Glo luminescent cell viability assay kit (Promega Corp.;
Madison, WI) according to the manufacturer’s instructions.
Caspase-3/7 Assays. Cells seeded in black-wall, clear-bottom
plates were incubated at 37 °C and 5% CO2 for 3-24 h before
caspase-3 and -7 activities were assessed using the Apo-ONE
homogeneous caspase-3/7 assay kit (Promega Corp.; Madison, WI)
according to the manufacturer’s instructions.
Western Blot Analysis. Western blot analysis was performed
as described previously25,26 using the following lysis buffer: 1%
Triton X-100, 150 mM NaCl, 50 mM Tris, pH 7.5, 5 mM EDTA,
protease inhibitory cocktail (Roche). The primary antibodies against
c-IAP1 were purchased from R&D (affinity-purified goat antibody)
and against Beta Tubulin from MP Biopharmaceuticals (mouse
monoclonal).
Inhibition of MDA-MB-231-X1.1 Xenograft Tumor Growth
by IAP Antagonists. All procedures involving animals were
performed in accordance with Genentech’s Institutional Animal
Care and Use Committee guidelines. To generate tumors, 100 µL
of a single-cell suspension containing 1 × 107 MDA-MB-231-X1.1
cells (in-house GFP-expressing variants of MDA-MB-231 cells
(ATCC, Manassas, VA)) were injected subcutaneously into the
upper right flanks of 8-week-old female C.B-17 SCID.bg mice
(Charles River Laboratories, Hollister, CA). Tumor volume was
calculated using the mean diameter measured with vernier calipers
using the formula V ) 0.5ab2, where a and b are the smallest and
largest perpendicular tumor diameters, respectively. Once-daily
administration of IAP antagonists by oral gavage was initiated once
tumors reached a size of ∼200 mm3 (∼1.5 weeks after inoculation).
IAP antagonists were formulated in 15% hydroxypropyl-ꢀ-cyclo-
detrin and 20 mM succinic acid and administered in a volume of
100 µL. Tumor volumes and body weights were measured twice
weekly until end of study.
1
NMR (400 MHz, CDCl3) δ ppm 7.40-7.26 (m, 5H), 5.85-5.67
(m, 1H), 5.61-5.55 (m, 1H), 5.20-5.00 (m, 3H), 4.35-4.21 (m,
2H), 4.20-4.05 (m, 3H), 3.90-3.80 (m, 1H), 3.60-3.45 (m, 2H),
2.35-2.20 (m, 1H), 2.05-1.85 (m, 3H), 1.85-1.45 (m 3H),
1.45-1.35 (m, 1H), 1.10-0.90 (m, 11H), 0.10--0.90 (m, 9H);
13C NMR (100 MHz, CDCl3) δ ppm 171.11, 170.33, 156.85,
156.14, 67.26, 62.59, 59.65, 56.33, 47.88, 39.38, 35.78, 35.52,
33.22, 31.62, 29.81, 29.63, 28.92, 28.29, 26.32, 17.56, -1.54;
HRMS (FAB) m/z 569.3741 (569.3734 calcd for C28H53N4O6Si M
+ H).
{(S)-1-[(S)-1-((3aR,6S,6aS)-6-Aminohexahydrocyclopenta[b]-
pyrrole-1-carbonyl)-2,2-dimethylpropylcarbamoyl]ethyl}meth-
ylcarbamic Acid tert-Butyl Ester (13). Protected amine 12 (420
mg) was treated with excess TAS-F in MeCN (10 mL) at 50 °C
for 18 h. The mixture was diluted with CH2Cl2 (50 mL) and washed
with 0.5 N NaOH (3 × 30 mL), dried (Na2SO4), filtered, and
concentrated to afford quantitative yield of amine 13 as a colorless
oil: 1H NMR (400 MHz, CDCl3) δ ppm 6.94-6.49 (m, 1H),
4.80-4.48 (m, 2H), 3.98-3.92 (m, 1H), 3.91-3.80 (m, 1H),
3.48-3.35 (m, 1H), 3.26-3.14 (m, 1H), 3.05-2.98 (m, 1H), 2.76
(s, 5H), 2.04-1.77 (m, 3H), 1.77-1.52 (m, 3H), 1.52-1.40 (m,
11H), 1.40-1.19 (m, 6H), 1.01-0.79 (m, 10H); 13C NMR (100
MHz, CDCl3) δ ppm 171.02, 170.94, 169.73, 169.71, 71.96, 60.62,
60.60, 56.51, 47.16, 41.30, 35.74, 34.77, 30.19, 29.82, 28.29, 26.41;
HRMS (FAB) m/z 425.3130 (425.3128 calcd for C22H41N4O4 M +
H).
Naphthalene-1-carboxylic Acid {(3aR,6S,6aS)-1-[(S)-3,3-
Dimethyl-2-((S)-2-methylaminopropionylamino)bu-
tyryl]octahydrocyclopenta[b]pyrrol-6-yl}amide (14b). To a solu-
tion of amine 13 (84 mg, 0.20 mmol) and MeCN (1 mL) were
added 1-naphthoic acid (42 mg, 0.24 mmol), DMAP (catalytic),
and EDC (46 mg, 0.24 mmol), and the mixture was stirred at room
temperature for 1 h. The mixture was diluted with CH2Cl2 (50 mL),
washed with 1 N HCl (1 × 50 mL), 1 N NaOH (1 × 50 mL),
brine (1 × 50 mL), dried (Na2SO4), filtered, and concentrated. The
residue was chromatographed (SiO2, 60% ethyl acetate-hexanes)
to afford 84 mg of the N-Boc-protected intermediate.
The residue from above was dissolved in CH2Cl2 (2 mL) and
TFA (1 mL) and maintained at room temperature for 2 h. The
solvents were removed under reduced pressure, and the residue was
purified by HPLC, and the solvents were removed by lyophylization
to afford 43 mg (28%) of 14b as a colorless powder: tR (C18) )
3.98 min; tR (SFCd) ) 3.47 min; MS m/z ) 479 (M + H+).
Benzeneacetamide, N-[(3aR,6S,6aS)-Octahydro-1-(N-methyl-
L-alanyl-3-methyl-L-valyl)cyclopenta[b]pyrrol-6-yl]-r-phenyl-
(14a). Yield, 71%; tR (C18) ) 4.59 min; tR (SFCd) ) 3.18 min;
MS m/z ) 519 (M + H+).
4-Fluoronaphthalene-1-carboxylic Acid {(3aR,6S,6aS)-1-[(S)-
3,3-Dimethyl-2-((S)-2-methylaminopropionylamino)bu-
tyryl]octahydrocyclopenta[b]pyrrol-6-yl}amide (14c). Yield, 33%;
tR (C18) ) 4.30 min; tR (SFCd) ) 3.29 min; MS m/z ) 497 (M +
H+).
4-Methylnaphthalene-1-carboxylic Acid {(3aR,6S,6aS)-1-[(S)-
3,3-Dimethyl-2-((S)-2-methylaminopropionylamino)bu-
tyryl]octahydrocyclopenta[b]pyrrol-6-yl}amide (14d). Yield, 39%;
tR (C18) ) 4.16 min; tR (SFCd) ) 3.44 min; MS m/z ) 493 (M +
H+).
2-Methoxy-naphthalene-1-carboxylic Acid {(3aR,6S,6aS)-1-
[(S)-3,3-Dimethyl-2-((S)-2-methylaminopropionylamino)bu-
tyryl]octahydrocyclopenta[b]pyrrol-6-yl}amide (14e). Yield, 52%;
tR (C18) ) 3.98 min; tR (SFCd) ) 3.56 min; MS m/z ) 509 (M +
H+).
Naphthalene-2-carboxylic Acid {(3aR,6S,6aS)-1-[(S)-3,3-
Dimethyl-2-((S)-2-methylaminopropionylamino)bu-
tyryl]octahydrocyclopenta[b]pyrrol-6-yl}amide (14f). Yield, 79%;
tR (C18) ) 4.24 min; tR (SFCd) ) 3.41 min; MS m/z ) 479 (M +
H+).
Acknowledgment. We thank members of the DMPK and
Purification groups within Genentech Small Molecule Drug
Discovery for analytical support. X-ray crystallographic data
were collected at The Advanced Light Source and Berkeley
Center for Structural Biology which are supported in part by
the U.S. Department of Energy and the National Institutes of
Health.
Supporting Information Available: Experimental procedures
for synthesis and characterization of additional IAP antagonists,
table of additional antagonists and potency in FP assay, and X-ray
data collection and refinement statistics. This material is available
free of charge via the Internet at http://pubs.acs.org.
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