Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152)

Article abstract of DOI:10.1021/jm300060k

A series of compounds were designed and synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP based on the N-terminus, AVPI, of mature Smac. Compound 1 (GDC-0152) has the best profile of these compounds; it binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domains of cIAP1 and cIAP2 with K_i values of 28, 14, 17, and 43 nM, respectively. These compounds promote degradation of cIAP1, induce activation of caspase-3/7, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Compound 1 inhibits tumor growth when dosed orally in the MDA-MB-231 breast cancer xenograft model. Compound 1 was advanced to human clinical trials, and it exhibited linear pharmacokinetics over the dose range (0.049 to 1.48 mg/kg) tested. Mean plasma clearance in humans was 9 ± 3 mL/min/kg, and the volume of distribution was 0.6 ± 0.2 L/kg.

Full text of DOI:10.1021/jm300060k

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pubs.acs.org/jmc
Discovery of a Potent Small-Molecule Antagonist of Inhibitor of
Apoptosis (IAP) Proteins and Clinical Candidate for the Treatment of
Cancer (GDC-0152)
John A. Flygare,*,† Maureen Beresini,‡ Nageshwar Budha,§ Helen Chan,∥ Iris T. Chan,⊥
Sravanthi Cheeti,§ Frederick Cohen,† Kurt Deshayes,# Karl Doerner,○ S. Gail Eckhardt,◆
Linda O. Elliott,‡ Bainian Feng,† Matthew C. Franklin,# Stacy Frankovitz Reisner,‡ Lewis Gazzard,†
Jason Halladay,∥ Sarah G. Hymowitz,¶ Hank La,∥ Patricia LoRusso,◊ Brigitte Maurer,¶ Lesley Murray,○
Emile Plise,∥ Clifford Quan,# Jean-Philippe Stephan,‡ Shin G. Young,∥ Jeffrey Tom,# Vickie Tsui,†
Joanne Um,† Eugene Varfolomeev,# Domagoj Vucic,# Andrew J. Wagner,⬡ Heidi J. A. Wallweber,¶
Lan Wang,† Joseph Ware,§ Zhaoyang Wen,† Harvey Wong,∥ Jonathan M. Wong,† Melisa Wong,†
Susan Wong,∥ Ron Yu,∇ Kerry Zobel,# and Wayne J. Fairbrother*,#
†
‡
§
∥
Departments of Discovery Chemistry, Biochemical and Cellular Pharmacology, Clinical Pharmacology, Drug Metabolism and
Pharmacokinetics, Oncology Exploratory Clinical Development, Early Discovery Biochemistry, ○Translational Oncology,
⊥
#
¶Structural Biology, and ∇Biostatistics, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
◆Division of Medical Oncology, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado 80045, United States
◊Department of Oncology, Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, United States
⬡Center for Sarcoma and Bone Oncology, Dana Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115,
United States
S
* Supporting Information
ABSTRACT: A series of compounds were designed and
synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP
based on the N-terminus, AVPI, of mature Smac. Compound 1
(GDC-0152) has the best profile of these compounds; it binds
to the XIAP BIR3 domain, the BIR domain of ML-IAP, and
the BIR3 domains of cIAP1 and cIAP2 with Ki values of 28, 14,
17, and 43 nM, respectively. These compounds promote
degradation of cIAP1, induce activation of caspase-3/7, and
lead to decreased viability of breast cancer cells without affect-
ing normal mammary epithelial cells. Compound 1 inhibits
tumor growth when dosed orally in the MDA-MB-231 breast cancer xenograft model. Compound 1 was advanced to human
clinical trials, and it exhibited linear pharmacokinetics over the dose range (0.049 to 1.48 mg/kg) tested. Mean plasma clearance
in humans was 9 3 mL/min/kg, and the volume of distribution was 0.6 0.2 L/kg.
function in the regulation of signal transduction pathways
associated with malignancy.14−25 In particular, the cIAP proteins
regulate TNFα-mediated NF-κB activation via their C-terminal
RING ubiquitin E3-ligase domains, which have been shown to
ubiquitinate receptor interacting protein (RIP)-1 and NF-κB
inducing kinase, NIK.26
Efforts to target the IAP proteins have focused on the design
of small molecules that mimic the binding of the endogenous
IAP antagonist second mitochondria-derived activator of caspases/
direct IAP-binding protein with low pI (Smac/DIABLO)27,28 to
a shallow groove on the surface of select IAP baculoviral IAP
repeat (BIR) domains.29 The IAP BIR domains are approximately
INTRODUCTION
■
Apoptosis is a physiological cell-death program that is critical
for the maintenance of tissue homeostasis. This process results
in the removal of unwanted cells such as those with potentially
harmful genomic mutations or alterations in cell-cycle control.
Cancer cells, unlike normal cells, are under stress and highly
dependent on aberrations in the apoptosis signaling pathways
to remain viable. Therefore, drugs that can restore apoptosis in
tumor cells might be effective for the treatment of cancer.
Members of the mammalian inhibitor of apoptosis (IAP)
family of proteins, including X chromosome-linked IAP (XIAP),
cellular IAP 1 (cIAP1), cellular IAP 2 (cIAP2), and melanoma
IAP (ML-IAP), are frequently overexpressed in cancer cells,1−5
where they confer protection against a variety of pro-apoptotic
stimuli.6−13 The IAP proteins have also been demonstrated to
Received: January 13, 2012
Published: March 13, 2012
© 2012 American Chemical Society
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80-amino acid zinc-binding domains that are necessary for the
antiapoptotic function of the IAP proteins. The third BIR
domain (BIR3) of XIAP is a specific inhibitor of caspase-9,30−33
while the BIR2 domain is necessary for potent inhibition of
caspases-3 and -7.34−38 Antagonism of XIAP-mediated
inhibition of these caspases is required for efficient caspase-
dependent cell death via both the extrinsic death receptor-
mediated and the intrinsic mitochondrial-mediated apoptosis
pathways.39
Scheme 1. Solid Phase Synthesis of P1, P2, and P3
Derivatives
a
The four amino-acid N-terminus of mature Smac (AVPI) is
capable of antagonizing XIAP with a binding affinity of approx-
imately 500 nM against the BIR3 domain.40 AVPI also binds
with high affinity to the BIR3 domains of cIAP1/2, the single
BIR domain of ML-IAP, and with lower affinity to the BIR2
domain of XIAP. In an effort to uncover lead matter capable of
mimicking these interactions, we undertook several large high-
throughput-screening campaigns. Screening greater than two
million compounds for binding to the ML-IAP BIR domain
failed to uncover any viable starting points; thus, we relied
solely on a peptidomimetic approach. Herein, we report the
design, synthesis, and evaluation of a series of Smac mimetics
that were based on the AVPI tetrapeptide. This peptide
sequence has served as the lead structure for several reports
detailing the evaluation of monovalent and bivalent small-
molecule Smac mimetics capable of antagonizing the IAP
proteins.41−46 We sought to evolve the peptide into a
compound with improved potency, pharmacokinetic properties,
and cell-killing characteristics that would allow us to evaluate
the effectiveness of IAP antagonism in human clinical trials. We
took a systematic approach to evaluate the contributions of
each substituent in the P1 through P4 positions using a
combination of structure-based design and targeted compound
library generation. The efforts culminated in the discovery of
compound 1 (GDC-0152), a potent antagonist of cIAP1/2,
ML-IAP, and XIAP and the first compound targeting this class
of proteins to enter clinic trials.
a
Reagents and conditions: (a) 2,2-diphenethylamine, NaCNBH3; 1%
AcOH/DMF. (b) FmocPro, HATU; DIPEA/DMF (X = −CH2−). (c)
Fmoc-Val(R2 = isopropyl), HBTU/HOBt, DIPEA/DMF. (d)
FmocAla, HBTU/HOBt, DIPEA/DMF. (e) Aldehyde, NaCNBH3;
1% AcOH/DMF. (f) TFA/H2O. (g) Bromopropionoic acid, DIC/
DMF. (h) Primary amine, DMF. (i) TFA/H2O.
a
Scheme 2. Synthesis of P4 Derivatives
SYNTHESIS
■
a
Reagents and conditions: (a) DIC, HOBt, CH2Cl2, 24 h. (b) TFA, CH2Cl2.
Compounds were prepared using either a solid-phase (Scheme 1)
or a solution-phase synthesis (Schemes 2 and 3). In the solid-
phase method, DFPE polystyrene resin was treated with 2, 2-
diphenethylamine, and sodium cyanoborohydride to provide
amine 2. Substituted proline (R1) and valine (R2) derivatives
were prepared using HATU/DIPEA and HBTU/HOBt,
DIPEA, respectively, to yield 3 and 4. N-Capped anilines
(6 or 7) were made using two procedures from intermediate 4.
In the first method, Fmoc-alanine was coupled to 4 followed by
a reductive amination with an aldehyde and sodium
cyanoborohydride to give enantiomerically pure compound 6.
Alternatively, using the peptoid synthesis method developed by
Zuckermann,47 4 was coupled with bromopropionoic acid
followed by displacement with a primary amine to yield racemic
compound 7. The solution synthesis of P4 derivatives (Scheme 2)
all utilized carboxylic acid 8. Primary amines with a saturated or
unsaturated two-carbon spacer and an aromatic ring (9) were
coupled to the carboxylic acid using EDC/HOBt to yield 10. The
synthesis of 1 is outlined in Scheme 3. Thiadiazole 11 was coupled
to Boc-proline using EDC/HOBt to yield 12. Boc-cyclo-
hexylglycine and Boc-methylalanine were sequentially coupled to
13 using BOP, which, after deprotection with hydrochloric acid,
yielded 1.
a
Scheme 3
a
Reagents and conditions: (a) EDC, HOBt, DIPEA, DMF, 24 h; (b)
HCl, dioxane; (c) TEA, CH2Cl2; (d) Boc-Chg, BOP, DIPEA, DMF,
3 h; (e) HCl, dioxane; (f) TEA, CH2Cl2; (g) Boc-MeAla, BOP,
DIPEA, DMF, 3 h; (h) HCl, dioxane.
RESULTS AND DISCUSSION
■
We used an analysis of our previously published 2.3-Å resolu-
tion crystal structure of Ala-Val-Pro-2,2-diphenethylamine
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a
Table 1. Structure and Potency of IAP Antagonists
a
b
c
Ki determined as described in ref 50. Average of two measurements. Single measurement
bound to the BIR domain of ML-IAP48 as our starting point for
the design of our compounds. From this structure, we
understood that the alanine methyl group in P1 would be
difficult to replace as the methyl group was buried within a
hydrophobic pocket formed by the side chains of Leu131,
Trp134, and Glu143. Additional evidence for the need to
maintain this methyl group came from the phage-display of
naive peptide libraries.49 In that work Ala was the only residue
observed at the N-terminus following three sorts of phage
libraries of linear peptides against both ML-IAP-BIR and XIAP-
BIR3. The other three positions (P2−P4) all tolerated some
level of substitution. The N-terminus of the peptide binds in an
acidic site on the BIR domains, with the amino group of Ala
interacting with the side-chain carboxylates of Asp138 and
Glu143 in ML-IAP, Glu314 in XIAP, and Asp320 and Glu325
in cIAP1. Several bond vectors from this nitrogen could be
extended to potentially pick up additional binding interactions
with the BIR domains. To explore these interactions, libraries of
nitrogen-capped derivatives were made, fixing diphenethyl-
amine in P4, proline in P3, and valine in P2 as detailed in
Scheme 1. Primary amines and aldehydes were selected for this
library by carefully choosing derivatives from randomly
generated diversity sets of these compounds that contained
functionality that could potentially interact with the BIR
domain. Several hundred compounds were made and indi-
vidually purified by reverse-phase HPLC prior to analysis. The
compounds derived from the primary amines (7) were assayed
as diastereomeric mixtures, and the compounds from the
aldehydes (6) were enantiomerically pure. The binding affini-
ties of these compounds for ML-IAP-BIR and XIAP-BIR3 were
determined using a fluorescence polarization-based competition
assay. No substituent in this position was more effective than a
methyl group. For example, as shown in Table 1, replacement
of the P1 methyl group with either a cyclopropylmethyl group
(6c) or a 2, 3-dihydroxypropyl group (6d) failed to achieve the
same level of binding affinity versus either protein.
For the analysis of the P2 position, we fixed methyl-Ala in P1
and proline in P3 utilizing the solid phase synthesis in Scheme 1.
The P2 position tolerated the most substitution in our prior
peptide-phage selection; thus, we anticipated that this area
would accommodate a wide range of functionality. The endo-
genous valine makes van der Waals contact with the β-methylene
of Ser133 in ML-IAP, but compared with the other side chains
in the sequence, this contribution is minimal. Consistent with this
observation, very little difference was noted in binding affinity
when the valine was replaced with a large hydrophobic sub-
stituent such as a cyclohexyl (10a) or t-butyl (data not shown)
group. The rotation of bulky P2 side chains is restricted by the
presence of a Pro at P3, and these interactions favor a peptide
conformation that optimizes the interactions between P2-Pro and
the BIR domains. Larger P2 side chains also improve the
proteolytic stability of the P2-Pro peptide bond (data not
shown).
The proline in P3 productively orients the P1 and P4
substituents. We have published previous efforts to modify this
region with a 7−5 bicyclic ring,50 an azabicyclooctane fuzed
ring,48 or a thiazoline.51 Substitution directly off the proline
ring allows for favorable interactions with Phe148 of ML-IAP or
Phe330 of cIAP1-BIR3 but can result in a steric clash with the
corresponding Tyr324 of XIAP-BIR3. We have exploited this
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difference to make compounds that have selectivity (>2,000-
fold) for cIAP1 over XIAP.52 Given our desire in this work to
create a pan-specific compound, we limited our exploration of
proline replacements to changes directly in the ring. As
illustrated in Table 1, substitution of a methylene with a sulfur
is tolerated without any loss of binding affinity (6b).
Having used a solid-phase method to rapidly scan P1−P3, we
turned to the solution-phase synthesis outlined in Scheme 2 to
assess the binding contributions of the P4 residue. MeAla,
ChGly, and Pro were fixed in the P1, P2, and P3 positions,
respectively, and primary amines were added to the terminal
carboxylic acid. The peptide-phage data revealed a preference
for an aromatic ring in the P4 position over the endogenous
isoleucine of Smac. Consistent with this, the crystal structure of
Ala-Val-Pro-2,2-diphenethylamine bound to the BIR domain of
ML-IAP showed one of the phenyl rings occupying a
hydrophobic pocket defined by Thr116, Gly130, Gln132, and
the aliphatic portions of Lys121 and Arg123. With this
structural information, we limited our initial exploration to
primary amines with a one- or two-carbon spacer connected to
an aromatic ring. Compounds were selected based on their
ability to add additional binding interactions in this well-defined
pocket. Of particular interest were the thiadiazole compounds 1
and 14, both of which showed improved binding affinity
relative to diphenethylamine. We initiated further studies with
compound 1.
To understand the specific interactions resulting in high-
affinity interactions, crystal structures were determined of 1
bound to XIAP chimeras of ML-IAP BIR (MLXBIR3SG) and
cIAP1 BIR3 (cXBIR3CS) domains (Figure 1). Key interactions
are consistent between the two structures including the critical
hydrogen-bond interaction with Asp138 in ML-IAP and the
corresponding Asp320 in cIAP1. This helps position the α-
methyl group of the methyl-Ala in the P1 cavity formed in both
complexes. In both examples, the thiadiazole heterocycle
positions the attached aromatic ring optimally within the
hydrophobic P4 pocket.
Figure 1. Solvent-accessible surface representation of the peptide-
binding site of (upper panel) the 1.79-Å resolution crystal structure of
the cIAP1/XIAP chimeric BIR3 domain (cXBIR3CS) (PDB ID
3UW4) and (lower panel) the 1.71-Å resolution crystal structure of
the ML-IAP/XIAP chimeric BIR domain (MLXBIR3SG) (PDB ID
3UW5) in complex with 1. The protein surfaces are color-coded
according to the electrostatic surface potentials: red is negatively
charged; blue is positively charged. This figure was produced using the
program PyMol (www.pymol.org).
As we desired a pan-selective antagonist, we evaluated the
thiadiazole (1) for its ability to bind to the BIR domain of
ML-IAP and the BIR2 and BIR3 domains of cIAP1, cIAP2, and
XIAP using a fluorescence polarization-based competition assay
(Figure 2 and Table 2). The ML-IAP/XIAP chimeric BIR
domain MLXBIR3SG, which retains the native ML-IAP-BIR
Smac-peptide-binding site,53 was used as a surrogate for the
ML-IAP BIR domain. Compound 1 binds to XIAP-BIR3,
cIAP1-BIR3, cIAP2-BIR3, and the single BIR domain of
ML-IAP with affinities in the low nanomolar range (Ki < 50 nM).
By contrast, the Ki values of 1 for cIAP1-BIR2 and cIAP2-BIR2
are approximately 10 μM.
The ability of 1 to block protein−protein interactions that
involve IAP proteins and pro-apoptotic molecules was evaluated
by immunoprecipitation and immunoblotting. Using transiently
transfected HEK293T cells, 1 was shown to disrupt XIAP
binding to partially processed caspase-9 (Figure 3A) and to
disrupt the association of ML-IAP, cIAP1, and cIAP2 with
Smac (Figure 3B). In melanoma SK-MEL28 cells, the endo-
genous association of ML-IAP and Smac was effectively also
abolished by 1 (Supporting Information).
To assess how these interactions with IAP proteins translated
into cell killing, compound 1 was tested in a three-day cell
viability assay. As shown in Figure 4A, 1 led to a decrease in cell
viability in the MDA-MB-231 breast cancer cell line while
having no effect on normal human mammary epithelial
Figure 2. Competitive binding curves for 1 binding to MLXBIR3SG
and select BIR domains of XIAP, cIAP1, and cIAP2. The Ki values
(Table 1) were calculated from IC50 values as described previously.63
cells (HMEC). This result was consistent with the moderate
cell permeability of 1 in MDCK cells (A and B; Papp = 1.8 ×
10−6 cm/s).
The effect of 1 on the activity of the effector caspases 3 and 7
in the MDA-MB-231 breast cancer cell line was measured to
determine the specific mechanism of action. Compound 1 was
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Table 2. Summary of Ki Values (μM) for Compound 1
IAP protein BIR domain
Ki(1)
a
b
MLXBIR3SG
XIAP-BIR3
XIAP-BIR2
cIAP1-BIR3
cIAP1-BIR2
cIAP2-BIR3
cIAP2-BIR2
0.014 0.006
0.028 0.011
c
0.112
c
0.017
c
14.5
c
0.043
c
9.6
a
b
c
Average of 5 measurements. Average of 4 measurements. These
values were taken from Figure 2.
Figure 4. (A) MDA-MB-231 breast carcinoma cells (solid symbols)
and HMECs (open symbols) were treated with the indicated
concentrations of 1. Cell death was assessed using the CellTiter-Glo
luminescent cell viability assay 72 h following the start of treatment.
(B) Effect of 1 (solid symbols) or its enantiomer (open symbols) on
caspase-3 and -7 activities in MDA-MB-231 cells assessed using the
Apo-ONE homogeneous caspase-3/7 assay.
Previous studies have demonstrated that Smac mimetics can
induce autoubiquitination activity and rapid proteasomal
degradation of cIAP1.54 Consistent with these studies, 1 was
shown to induce rapid degradation of cIAP1 in A2058
melanoma cells (Figure 5). Compound 1 effectively induces
Figure 3. (A) HEK293T cells were transiently transfected with
caspase-9 and Flag-tagged XIAP-BIR3 domain or empty vector. Cells
were lysed, and lysates were incubated with the previously published
IAP protein antagonist compound 14a,48 its non-IAP protein binding
stereoisomer, compound 14a* (a negative control),48 or 1.
Immunoprecipitation was performed with anti-Flag antibody, and
samples were immunoblotted with anticaspase-9 and anti-Flag
antibodies. Whole-cell lysates are shown in the right panels. Astericks
denote nonspecific bands from immunoprecipitations. (B) HEK293T
cells were transiently transfected with Smac and Myc-tagged cIAP1,
cIAP2, ML-IAP, or empty vector. Cells were lysed, and lysates were
incubated with the indicated concentrations of 1 and immunopreci-
pitated with anti-Myc antibody (left panels). Samples were then
immunoblotted with anti-Smac and anti-Myc antibodies. Whole-cell
lysates are shown in the right panel.
Figure 5. A2058 cells were treated with increasing amounts of 1 or
with 10 μM of its enantiomer for the indicated time periods. Cells
were lysed, and lysates were immunoblotted with anti-c-IAP1 and
antibody.
degradation of cIAP1 at concentrations as low as 10 nM,
consistent with its affinity for cIAP1, while its enantiomer,
which does not bind to cIAP1, has no effect following a 2-h
exposure at a concentration of 10 μM.
Pharmacokinetic optimization was performed in parallel with
the optimization of potency. We focused on the identification
of compounds that would enable both preclinical proof of
concept in an in vivo efficacy model and testing of IAP
antagonism in the clinic. The compounds were not dissolution
limited as all had favorable solubility properties. The kinetic
found to activate caspases 3 and 7 in a dose- and time-
dependent manner as shown in Figure 4B. To probe the
specificity of the observed effects, the enantiomer of 1 was
assessed at the 24-h time point and found to not activate the
caspases at concentrations up to ∼10 μM. The potency of the
caspase activation correlated well with the cell viability results
and provided strong evidence that the cell killing was mediated
through the desired apoptosis signaling culminating in caspase-
3/7 activation.
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Figure 6. Efficacy of 1 compared to vehicle control in human-tumor xenograft mouse models of MDA-MB-231 breast cancer. Compounds were
dosed as an oral solution at the indicated time intervals and doses. Compound 1 showed significant dose-dependent growth inhibition throughout
dosing (p-value < 0.001 for all doses).
solubility of compound 1 was 40 mg/mL at pH 5.5, 2.0 mg/mL
at pH 6.7, and 0.46 mg/mL at pH 7.7. Compound 1 had
moderate predicted hepatic clearance based on metabolic
stability assays conducted using human liver microsomes (data
not shown). Additional in vitro ADME evaluation also included
an assessment of plasma−protein binding and blood−plasma
partitioning. The plasma−protein binding of 1 was evaluated in
mouse, rat, rabbit, dog, monkey, and human plasma by
equilibrium dialysis using [14C]1. Plasma−protein binding of
1 was moderate and comparable among mice (88−91%), rats
(89−91%), dogs (81−90%), monkeys (76−85%), and humans
(75−83%) over the range of concentrations investigated (0.1−
100 μM); higher plasma−protein binding was observed in
rabbits (95−96%). Compound 1 did not preferentially
distribute to red blood cells with blood−plasma partition ratios
ranging from 0.6 to 1.1 in all species tested.
The in vivo pharmacokinetics for 1 were evaluated initially in
mice in order to enable efficacy studies in human-tumor mouse
xenograft models. The monotrifluoroacetic acid salt of 1 was
dosed orally in female mice at 100 mg/kg as a solution in
phosphate-buffered saline (PBS). Significant exposure was
achieved with a Cmax of 53.7 μM and AUC of 203.5 h·μM. The
in vivo efficacy studies of 1 were performed using human-tumor
xenograft mouse models of MDA-MB-231 breast cancer with a
variety of dosing schedules and regimens. These in vivo studies
demonstrated that 1 has robust antitumor activity as a single
agent as shown in Figure 6. Dosing once weekly with 10, 50,
or 100 mg/kg resulted in significant tumor volume reduction
(p-value < 0.001 for all doses). Similar results were obtained
when dosed at 10 mg/kg daily or every third day for three
weeks. The initial rate of regression was greatest with doses of
100 mg/kg given once a week, although by day 21 all groups
dosed with 1 had experienced between 85 and 95% inhibition
of tumor growth, with four to six complete responses, relative
to the vehicle-control group. In mice that experienced less than
a partial response (defined as >50% but <100% reduction in
tumor volume, compared with the starting tumor volume),
tumors resumed growth after the final doses of 1 were administered.
Notably, 1 was well tolerated by all groups, and by day 28, all
remaining mice had a mean gain in body weight of ≥5%. A
transient loss in body weight was observed for mice that were given
weekly doses of 100 mg/kg, although body weights were similar
among all groups on day 28.
The preclinical pharmacokinetics of 1 were investigated in
mouse, rat, dog, and monkeys following single-dose intravenous
(IV) administration. Following a low IV dose (1 mg/kg), 1 had
a high plasma clearance (>80% of hepatic blood flow) in nude
mice, Sprague−Dawley rats, and cynomolgus monkeys, and a
moderate plasma clearance (52% of hepatic blood flow) in
beagle dogs (Table 3). At high doses (100 mg/kg in mice;
20 mg/kg in rats, dogs, and monkeys), mechanisms of clearance
from plasma became saturated in mouse and rat but not in the
monkey and dog.
Human clearance was predicted using in vivo preclinical
pharmacokinetic data and allometry adjusted for maximum life
potential (Figure 7) or using in vitro−in vivo extrapolation of
hepatocyte stability data. Both methods resulted in similar
moderate human clearance predictions of 9.6 (allometry) and
10 mL/min/kg (in vitro−in vivo extrapolation). Human volume
of distribution was predicted to be moderate at 1.2 L/kg using
allometry (Figure 7).
On the basis of the human PK predictions, the antitumor
efficacy, and the favorable safety profile observed in preclinical
species, compound 1 was advanced for evaluation in humans. In
humans, 1 exhibited linear pharmacokinetics over the dose
range (0.049 to 1.48 mg/kg) tested (Figure 8). Mean plasma
clearance was 9
was 0.6 0.2 L/kg (Supporting Information, Table S2), in
good agreement with preclinical predictions.
3 mL/min/kg and volume of distribution
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mimic the four-amino acid N-terminus of mature Smac. From
these studies, 1 was found to be a potent antagonist of cIAP1,
cIAP2, ML-IAP, and XIAP with the ability to disrupt IAP
binding to pro-apoptotic proteins such as partially processed
caspase-9 and Smac. X-ray crystallographic analysis confirmed
that 1 binds to the Smac-binding site of the cIAP1 BIR3 and
ML-IAP BIR domains with a binding mode very similar to that
observed for Smac-derived peptides. As described previously for
Smac mimetics, 1 also induces autoubiquitination activity and
rapid proteasomal degradation of cIAP1. Cell-based assays
confirmed that 1 promoted the activation of caspase-3 and -7,
resulting in cell killing in a three-day viability assay.
Administration of 1 orally led to effective tumor reduction in
a human-tumor xenograft mouse model of MDA-MB-231
breast cancer when dosed as a single agent. Predictions of
human pharmacokinetics, using in vivo preclinical pharmaco-
kinetic data and allometry or in vitro−in vivo extrapolation of
hepatocyte stability data, suggested that 1 would exhibit
moderate clearance in humans with a moderate volume of
distribution. On the basis of the described properties as a
whole, 1 was selected as the first compound targeting IAP
proteins to enter clinic trials. Pharmacokinetics observed in
humans were well behaved and consistent with preclinical
predictions.
Table 3. Pharmacokinetics Parameters (mean SD) of 1 in
Mouse, Rat, Dog, and Monkey after IV Bolus
Administration
a
nude
Sprague−
cynomolgus
monkey
parameter
sex
mouse
Dawley rat
beagle dog
male
female
male
male
N
27
1
3
3
1
3
1
low dose (mg/kg)
CL (mL/min/kg)
Vss (L/kg)
1
72
1.6
0.4
1.4
0.5
67 10
2.0 0.3
0.43 0.05
1.1 0.2
0.51 0.08
16
3
47
4
1.0 0.2
0.9 0.1
3.8 0.1
2.1 0.4
1.60 0.04
0.44 0.05
1.46 0.04
0.71 0.06
t1/2 (h)
Cmax (μM)
AUC0‑∞ (h·μM)
male/
female
sex
female
male
male
N
27
3
2
3
high dose (mg/kg)
CL (mL/min/kg)
Vss (L/kg)
100
23
20
27
20
19
1.5
2.0
30
36
20
2
36
6
1.7
1.7 0.2
2.2 0.3
t1/2 (h)
2.9
0.84 0.06
0.77 0.01
Cmax (μM)
118.6
147
31
25
4
2
24
19
3
3
AUC0‑∞ (h·μM)
a
AUC0−∞ = area under the concentration−time curve from time zero to
infinity; CL = total plasma clearance; Cmax = highest observed plasma
concentration of 1; t1/2 = terminal half-life; Vss = volume of distribution at
steady state. Compound 1 hydrochloride salt in 10% hydroxypropyl-β-
cyclodextrin was used in the mouse studies; 1-TFA salt in phosphate-
buffered saline was used in the rat, monkey, and dog studies.
EXPERIMENTAL SECTION
■
All chemicals were purchased from commercial suppliers (Aldrich,
VWR). Flash chromatography was carried out with RediSep pre-
packed SiO2 cartridges on an ISCO Companion chromatography
system. NMR spectra were recorded on a Varian Inova 400 system,
and referenced to tetramethylsilane. Preparative HPLC was performed
on a Polaris C18 5 μm column (50 × 21 mm). Low-resolution mass
spectra were recorded on a Sciex 15 in ES+ mode. Final compounds
were also purified on a Berger Instruments SFC system operating at
100 bar, 35 °C; column = Berger Diol 4.6 × 150 mm; with a six−
minute gradient of 20−60% MeOH in CO2 flowing 2.35 mL/min
(SFCd method) or a Berger Pyridine column 4.6 × 150 mm; with a
six-minute gradient of 5−50% MeOH in CO2 flowing at 2.35 mL/min
(SFCp). Purity analysis of final compounds was performed by a
20 min HPLC-MS analysis with a Chromasil C18 column (4.6 × 50 mm)
with a gradient of 0−90% acetonitrile (containing 0.038% TFA) in 0.1%
aqueous TFA (over 17 min, flow = 2 mL/min). All final compounds
were purified to >95% purity.
(S)-tert-Butyl 2-(4-phenyl-1,2,3-thiadiazol-5-ylcarbamoyl)-
pyrrolidine-1-carboxylate (12). 4-Phenyl-1,2,3-thiadiazol-5-amine
(1.0 g, 5.64 mmol), Boc-L-proline (1.28 g, 5.94 mmol), EDC (1.14 g,
5.94 mmol), HOBt, (0.8 g, 5.94 mmol), and DIPEA (1.04 mL, 5.94
mmol) were dissolved in 5 mL of DMF and stirred for 24 h at room
temperature. EtOAc (100 mL) was added to the solution, and the
organic layer was washed with saturated aqueous NaHCO3 (20 mL)
and separated. The aqueous layer was extracted with two 100 mL
portions of EtOAc and the organic layers combined. The combined
organic layer was washed one time with saturated aqueous NaHCO3
(20 mL), washed twice with saturated brine (20 mL), dried over
MgSO4, and concentrated to a solid residue. The crude material was
purified by reverse phase HPLC to obtain compound 12 as a 2/1
1
mixture of rotomers (1.60 g, 76%). H NMR (400 MHz, DMSO) δ
ppm 11.90 (s, 0.66H), 11.85 (s, 0.33H), 7.68−7.75 (m 2H), 7.50−
7.63 (m, 3H), 4.69 (dd, J = 8.41, 3.42 Hz, 0.33H), 4.63 (dd, J = 8.13,
4.12 Hz, 0.66H), 3.32−3.51 (m, 2H), 2.11−2.26 (m, 1H), 1.82−1.97
(m, 3H), 1.42 (s, 3H), 1.21 (s, 6H). 13C NMR (100 MHz, DMSO) δ
ppm 23.30, 23.94, 27.75, 28.01, 29.76, 30.89, 46.52, 46.70, 58.60, 58.73,
78.77, 79.17, 128.68, 128.74, 128.85, 128.94, 130.57, 145.40, 147.68,
152.74, 152.78, 172.37, 173.06. MS (ESI): m/z [M + H]+ 375.1.
(S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propan-
amido)acetyl)-N- (4-phenyl-1,2,3-thiadiazol-5-yl)pyrrolidine-2-
carboxamide (1). Amide 12 (2.32 g, 6.2 mmol) was treated with 4 N
Figure 7. Prediction of human clearance and volume of distribution of
compound 1 using allometric methods for clearance (CL) and volume
of distribution (Vss).
CONCLUSIONS
■
Using a combination of structure-based design and solid-phase
library synthesis, we have made a series of compounds that
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Figure 8. Compound 1 pharmacokinetics in humans. (A) Cycle 1 mean plasma concentration versus time profiles of compound 1 by cohort. (B)
Plasma exposure (AUCinf) of 1 plotted versus dose.
HCl/dioxane (25 mL) for 30 min, and the solution was concentrated.
TEA/CH2Cl2 (10:90, 20 mL) was added, and the solution was stirred
for 5 min and concentrated to a solid residue. Boc-L-cyclohexylglycine
(1.6 g, 6.2 mmol), BOP (2.7 g, 6.2 mmol), and DIPEA (2.16 mL, 12.4
mmol) in 10 mL of DMF were added and stirred for 3 h at room
temperature. EtOAc (100 mL) was added to the solution, and the
organic layer was washed with saturated aqueous NaHCO3 (20 mL)
and separated. The aqueous layer was extracted with two 100 mL
portions of EtOAc and the organic layers combined. The combined
organic layer was washed one time with saturated aqueous NaHCO3
(20 mL), washed twice with saturated brine (20 mL), dried over
MgSO4, and concentrated to a solid residue. The crude reaction
mixture was treated with 25 mL of 4 N HCl/dioxane for 30 min. The
solution was concentrated, TEA/CH2Cl2 (10:90, 20 mL) was added
and stirred for 5 min, and the solution was concentrated to a solid
residue. Boc-N-methylalanine (1.26, 6.2 mmol), BOP (2.7 g, 6.2
mmol), and DIPEA (2.16 mL, 12.4 mmol) in DMF (10 mL) were
added, and the solution was stirred for 3 h at room temperature.
EtOAc (100 mL) was added to the solution, and the organic layer was
washed with saturated aqueous NaHCO3 (20 mL) and separated. The
aqueous layer was extracted with two 100 mL portions of EtOAc and
the organic layers combined. The combined organic layer was washed
one time with saturated aqueous NaHCO3 (20 mL), washed twice
with saturated brine (20 mL), dried over MgSO4, and concentrated to
a solid residue. The crude material was purified by reverse phase
HPLC. The purified material was treated with 25 mL of 4 N HCl/
dioxane for 30 min and the solution concentrated to a solid residue
4.91 (dd, J = 8.08, 1.92 Hz, 1H), 4.45 (d, J = 8.23 Hz, 1H), 3.87 (q, J =
8.56 Hz, 1H), 3.58 (q, J = 5.04 Hz, 1H), 3.06 (q, J = 6.92 Hz, 1H),
2.69 (d, J = 12.46 Hz, 1H), 2.45 (s, 3H), 2.03−2.19 (m, 2H), 1.87−
2.03 (m, 1H), 1.40−1.69 (m, 1H), 1.35−1.60 (m, 5H), 1.32 (d, J =
10.31 Hz, 3H), 0.79−1.02 (m, 5H). 13C NMR (100 MHz, CDCl3) δ
ppm 175.13, 173.84, 168.92, 148.34, 144.87, 130.52, 129.20, 129.23,
128.92, 128.28, 128.27, 60.34, 59.72, 54.58, 47.99, 40.35, 35.21,
29.65, 28.17, 25.87, 25.74, 25.65, 25.63, 25.11, 19.62. MS (ESI): m/z
[M + H]+ 499.7
Preparation of Compounds 6a−6d. 2,2-Diphenylethylamine
(5.3 g, 27 mmol) and NaCNBH3 (1.7 g, 27 mmol) were added to 2-
(3,5-dimethoxy-4-formylphenoxy)ethoxymethyl (DFPE) polystyrene
resin (5 g, 4.5 mmol) in 1% HOAc/DMF (200 mL), and nitrogen was
bubbled through the reaction mixture for 3 days at room temperature.
The resin was dried and washed with DMF (2 × 100 mL) and CH2Cl2
(2 × 100 mL) and dried. Fmoc-Pro (4.6 g, 13.5 mmol), HATU (5.1 g,
13.5 mmol) and DIPEA (4.7 mL, 27 mmol) in 1:1 DCM/DMF
(100 mL) were added to the resin, and nitrogen was bubbled through
the reaction mixture for 4 h. The resin was dried and washed with
DMF (2 × 100 mL) and CH2Cl2 (2 × 100 mL) and dried to yield
Resin 3. Resin 3 was treated with 20% piperidine/DMA (100 mL) for
30 min, and the resin was dried and washed with DMF (2 × 100 mL)
and CH2Cl2 (2 × 100 mL) and dried. Fmoc Val (4.6 g, 13.5 mmol),
HBTU (5.1 g, 13.5 mmol), and DIPEA (4.7 mL, 27 mmol) were
added to 1:1 DCM/DMF (100 mL), and nitrogen was bubbled
through the reaction mixture for 3 h. The resin was dried and washed
with DMF (2 × 100 mL) and CH2Cl2 (2 × 100 mL) and treated with
20% piperidine/DMA (100 mL) for 30 min. The resin was dried and
washed with DMF (2 × 100 mL) and CH2Cl2 (2 × 100 mL) and dried
to yield resin 5. The aldehyde (13.5 mmol) and NaCNBH3 (0.85 g,
1
and purified by reverse phase HPLC to obtain 1 (2.28 g, 74%) H
NMR (400 MHz, CDCl3) δ ppm 7.75 (d, J = 7.23 Hz, 2H), 7.62 (d,
J = 9.08 Hz, 1H), 7.59 (t, J = 7.69 Hz, 2H), 7.50 (t, J = 7.38 Hz, 1H),
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13.5 mmol) were added to resin 5 in 1% HOAc/DMF (100 mL) and
nitrogen was bubbled through the reaction mixture for 3 days at room
temperature. TFA and H2O (95:5, 100 mL) were added, and nitrogen
was bubbled through the solution for 1 h. The solution was
concentrated to a solid residue and purified by reverse phase HPLC
to yield the product.
(S)-N-(2,2-Diphenylethyl)-1-((S)-3-methyl-2-((S)-2-
(methylamino)propanamido)butanoyl)pyrrolidine-2-carboxamide
(6a). 1H NMR (400 MHz, CD3OD) δ 7.35−7.22 (m, 8H), 7.22−7.10
(m, 2H), 4.45 (t, J = 9.5 Hz, 1H), 4.28−4.19 (m, 2H), 4.05 (ddd, J =
13.4, 9.0, 7.0 Hz, 1H), 3.92−3.83 (m, 1H), 3.84−3.73 (m, 1H), 3.67−
3.53 (m, 2H), 2.64 (d, J = 3.1 Hz, 3H), 2.18−2.03 (m, 1H), 2.00−1.77
(m, 3H), 1.50 (t, J = 5.6 Hz, 1H), 1.45 (d, J = 7.0 Hz, 3H), 1.05 (d, J =
6.8 Hz, 3H), 0.99 (d, J = 6.7 Hz, 3H), 0.97−0.86 (m, 1H). 13C NMR
(75 MHz, CD3OD) δ 173.62, 172.64, 172.11, 170.80, 168.78, 168.18,
161.85, 161.40, 158.50, 156.69, 144.64, 142.38, 132.91, 128.70, 128.40,
128.34, 128.29, 128.06, 127.89, 127.47, 126.49, 126.44, 117.95, 83.38,
60.53, 60.43, 57.36, 57.17, 57.07, 56.76, 50.86, 50.58, 44.11, 43.80,
32.03, 31.89, 30.79, 30.43, 29.61, 24.80, 22.13, 18.75, 18.50, 17.54,
17.41, 15.49, 15.29. LC/MS (ESI+): m/z 480 [M + H].
(R)-3-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)-
acetyl)-N-(2,2-diphenylethyl)thiazolidine-4-carboxamide (6b). 1H
NMR (400 MHz, CDCl3) δ 7.34−7.23 (m, 8H), 7.20 (ddd, J =
10.1, 8.6, 6.1 Hz, 2H), 5.03 (d, J = 8.3 Hz, 1H), 4.60−4.51 (m, 1H),
4.49 (s, 2H), 4.27 (t, J = 7.9 Hz, 1H), 4.16 (dd, J = 14.0, 7.2 Hz, 1H),
3.90 (s, 1H), 3.73 (ddt, J = 8.8, 6.0, 4.3 Hz, 1H), 3.60 (d, J = 19.8 Hz,
1H), 2.91 (dd, J = 12.0, 7.2 Hz, 1H), 2.63 (d, J = 9.3 Hz, 1H), 2.55 (s,
3H), 1.66 (t, J = 24.0 Hz, 4H), 1.35 (d, J = 5.3 Hz, 3H), 1.28−1.07 (m,
4H), 1.08−0.85 (m, 3H). 13C NMR (101 MHz, CD3OD) δ 169.00,
168.60, 168.44, 142.63, 142.52, 128.33, 128.23, 127.85, 127.80, 126.29,
126.23, 61.73, 55.62, 55.49, 49.98, 49.42, 42.96, 33.00, 30.59, 28.41,
27.79, 25.66, 25.45, 25.34, 15.70, 15.56. LC/MS (ESI+): m/z 539
[M + H].
80% EtOAc/hexane) to yield the product. The compound was treated
with 1:1 TFA and CH2Cl2 at room temperature for 30 min. The
reaction mixture was diluted with CH2Cl2 (100 mL), washed with
water (2 × 20 mL), and the organic layer was dried, concentrated, and
purified by HPLC to yield the product. (48 mg, 85%).
1H NMR (500 MHz, DMSO) δ 7.84 (dt, J = 11.0, 5.5 Hz, 1H), 7.81
(d, J = 9.0 Hz, 1H), 7.36−7.30 (m, 1H), 7.38−7.23 (m, 9H), 7.23−
7.08 (m, 2H), 4.35 (dd, J = 16.9, 8.4 Hz, 1H), 4.20−4.12 (m, 2H),
3.93 (ddd, J = 22.1, 13.6, 7.5 Hz, 1H), 3.69−3.58 (m, 1H), 3.54−3.48
(m, 1H), 3.48−3.41 (m, 1H), 2.91 (q, J = 6.8 Hz, 1H), 2.13 (d, J =
10.5 Hz, 3H), 1.83−1.55 (m, 10H), 1.13 (dt, J = 11.3, 8.5 Hz, 2H),
1.06 (t, J = 9.3 Hz, 3H), 1.03−0.81 (m, 3H). 13C NMR (500 MHz,
DMSO) δ 173.4, 171.1, 169.3, 142.8, 129.1, 128.1, 126.3, 59.2, 54,
50.2, 46.9, 42.8, 34.2, 29.2, 28.9, 27.9, 25.9, 25.7, 25.5, 24.1, 19.1. LC/
MS (ESI+): m/z 534 [M + H].
(S)-N-(4-Chlorobenzyl)-1-((S)-2-cyclohexyl-2-((S)-2-
(methylamino)propanamido)acetyl)pyrrolidine-2-carboxamide
(10b). Boc-MeAla-Chg-Pro (8) (50 mg, 0.11 mmol) was diluted with
CH2Cl2 (10 mL), and treated with 4-chlorophenylmethanamine (24
mg, 0.171 mmol), DIC (0.03 mL, 0.171 mmol) and HOBT (28 mg,
0.171 mmol). The reaction was stirred at room temperature for 1 h,
and the reaction mixture was diluted with CH2Cl2 (100 mL) and
washed with water (2 × 20 mL), and the organic layer was dried,
concentrated, and purified by ISCO chromatograpy (50−80% EtOAc/
hexane) to yield the product. The compound was treated with 1:1
TFA and CH2Cl2 at room temperature for 30 min. The reaction
mixture was diluted with CH2Cl2 (100 mL) and washed with water (2
× 20 mL), and the organic layer was dried, concentrated, and purified
1
by HPLC to yield the product. (40 mg, 79%). H NMR (500 MHz,
DMSO) δ 8.46−8.32 (m, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.42−7.31
(m, 2H), 7.28 (t, J = 8.4 Hz, 2H), 4.41 (t, J = 8.1 Hz, 1H), 4.31 (dd, J
= 8.0, 5.2 Hz, 1H), 4.27 (t, J = 6.8 Hz, 1H), 4.25−4.18 (m, 1H), 3.73
(dt, J = 9.5, 6.6 Hz, 1H), 3.59 (dt, J = 9.5, 6.9 Hz, 1H), 3.01−2.88 (m,
1H), 2.17 (d, J = 3.3 Hz, 3H), 2.12−2.02 (m, 1H), 1.97 (dd, J = 12.5,
6.1 Hz, 1H), 1.89−1.75 (m, 2H), 1.66 (ddd, J = 44.7, 26.1, 13.0 Hz,
6H), 1.19−1.11 (m, 2H), 1.08 (dd, J = 10.2, 4.9 Hz, 3H), 1.06−0.84
(m, 3H). 13C NMR (500 MHz, DMSO) δ 174.1, 171.6, 169.8, 138.6,
131.1, 128.8, 128, 59.5, 59.2, 54.1, 47.1, 41.5, 34.3, 29.3, 28.9, 27.8,
25.9, 25.6, 25.5, 24.6, 19.1. LC/MS (ESI+): m/z 519 [M + H].
(S)-1-((S)-2-((S)-2-(Methylamino)propanamido)-2-phenylace-
tyl)-N-(4-phenyl-1,2,3-thiadiazol-5-yl)pyrrolidine-2-carboxamide
(14). Amide 12 (1.16 g, 3.1 mmol) was treated with 15 mL of 4 N
HCl/dioxane for 30 min, and the solution was concentrated. TEA/
CH2Cl2 (10:90, 15 mL) was added, and the solution was stirred for
5 min and concentrated to a solid residue. Boc-L-Phenylglycine (0.8 g,
3.1 mmol), 1.35 g (3.1 mmol) of BOP, and 1.08 mL (6.2 mmol) of
DIPEA in 10 mL of DMF were added and stirred for 3 h at room
temperature. One hundred milliliters of EtOAc was added to the
solution, and the organic layer was washed with saturated aqueous
NaHCO3 (20 mL) and separated. The aqueous layer was extracted
with two 100 mL portions of EtOAc and the organic layers combined.
The combined organic layer was washed one time with saturated
aqueous NaHCO3 (20 mL), washed twice with saturated brine (20 mL),
dried over MgSO4, and concentrated to a solid residue. The crude
reaction mixture was treated with 25 mL of 4 N HCl/dioxane for
30 min. The solution was concentrated, TEA/CH2Cl2 (10:90, 15 mL)
was added and stirred for 5 min, and the solution was concentrated to a
solid residue. Boc-N-methylalanine (0.63 g, 3.1 mmol), BOP (1.35 g,
3.1 mmol), and DIPEA (1.08 mL, 12.4 mmol) in 10 mL of DMF were
added, and the solution was stirred for 3 h at room temperature. EtOAc
(100 mL) was added to the solution, and the organic layer was washed
with saturated aqueous NaHCO3 (20 mL) and separated. The aqueous
layer was extracted with two 100 mL portions of EtOAc and the organic
layers combined. The combined organic layer was washed one time with
saturated aqueous NaHCO3 (20 mL), washed twice with saturated brine
(20 mL), dried over MgSO4, and concentrated to a solid residue. The
crude material was purified by reverse phase HPLC. The purified
material was treated with 25 mL of 4 N HCl/dioxane for 30 min, the
solution concentrated to a solid residue, and purified by reverse phase
HPLC to obtain 14 (1.19 g, 78%). 1H NMR (400 MHz, CDCl3)
(S)-1-((S)-2-((S)-2-(Cyclopropylamino)propanamido)-3-methyl-
1
butanoyl)-N-(2,2-diphenylethyl)pyrrolidine-2-carboxamide (6c). H
NMR (400 MHz, CD3OD) δ 7.37−7.23 (m, 8H), 7.23−7.09 (m, 2H),
4.44 (t, J = 6.7 Hz, 1H), 4.30−4.17 (m, 2H), 4.10−3.93 (m, 2H),
3.88−3.75 (m, 1H), 3.69−3.50 (m, 2H), 2.89 (dt, J = 14.4, 7.2 Hz,
1H), 2.76 (dt, J = 12.7, 7.7 Hz, 1H), 2.22−2.01 (m, 1H), 2.00−1.74
(m, 3H), 1.51 (t, J = 6.3 Hz, 1H), 1.47 (d, J = 7.0 Hz, 3H), 1.13−1.01
(m, 3H), 0.98 (d, J = 6.7 Hz, 3H), 0.92 (dd, J = 10.7, 6.8 Hz, 1H),
0.77−0.61 (m, 2H), 0.43−0.25 (m, 2H). 13C NMR (101 MHz,
CD3OD) δ 172.82, 172.74, 172.23, 170.88, 170.70, 168.93, 168.35,
142.56, 142.53, 142.41, 128.41, 128.36, 128.29, 128.06, 127.94, 127.90,
126.52, 126.49, 126.44, 60.34, 60.30, 56.96, 56.64, 55.47, 55.23, 51.14,
51.05, 50.41, 43.98, 43.75, 43.64, 31.86, 31.58, 30.19, 29.46, 24.63,
21.92, 18.56, 18.32, 17.37, 17.19, 15.63, 15.47. LC/MS (ESI+): m/z
538 [M + H].
(S)-1-((S)-2-((S)-2-((S)-2,3-Dihydroxypropylamino)propanamido)-
3-methylbutanoyl)-N-(2,2-diphenylethyl)pyrrolidine-2-carboxamide
(6d). 1H NMR (400 MHz, CD3OD) δ 7.36−7.24 (m, 8H), 7.23−7.12
(m, 2H), 4.49−4.40 (m, 1H), 4.32−4.17 (m, 1H), 4.09−4.00 (m, 2H),
3.92−3.86 (m, 1H), 3.79 (t, J = 14.5 Hz, 1H), 3.67−3.55 (m, 3H),
3.55−3.45 (m, 1H), 3.21−3.11 (m, 1H), 3.02−2.89 (m, 1H), 2.10 (dt,
J = 14.1, 4.7 Hz, 1H), 2.01−1.77 (m, 3H), 1.52−1.47 (m, 3H), 1.44 (t,
J = 6.1 Hz, 1H), 1.05 (dd, J = 6.8, 2.1 Hz, 3H), 1.00−0.96 (m, 3H),
0.93 (dd, J = 12.1, 6.8 Hz, 1H). 13C NMR (101 MHz, CD3OD) δ
172.84, 170.86, 169.77, 168.95, 161.54, 142.53, 128.35, 128.30, 128.07,
127.96, 127.90, 127.73, 126.50, 126.44, 67.28, 67.20, 63.94, 60.35,
57.00, 56.70, 55.84, 50.72, 50.44, 48.88, 43.75, 31.61, 30.31, 30.22,
29.46, 24.61, 21.90, 18.56, 18.32, 17.42, 17.39, 17.25, 16.56, 16.41,
15.50, 15.28. LC/MS (ESI+): m/z 540 [M + H].
(S)-1-((S)-2-cCyclohexyl-2-((S)-2-(methylamino)-
propanamido)acetyl)-N-(2,2-diphenylethyl)pyrrolidine-2-car-
boxamide (10a). Boc-MeAla-Chg-Pro (8) (50 mg, 0.11 mmol) was
diluted with CH2Cl2 (10 mL) and treated with 2, 2-diphenylethyl-
amine (33.67 mg, 0.171 mmol), DIC (0.03 mL, 0.171 mmol), and
HOBT (28 mg, 0.171 mmol). The reaction was stirred at room
temperature for 1 h, and the reaction mixture was diluted with CH2Cl2
(100 mL) and washed with water (2 × 20 mL), and the organic layer
was dried, concentrated, and purified by ISCO chromatograpy (50−
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δ 7.88−7.76 (m, 2H), 7.74−7.61 (m, 2H), 7.58−7.47 (m, 1H), 7.29−
7.21 (m, 1H), 7.22−7.12 (m, 2H), 7.04−6.92 (m, 2H), 5.54 (s, 1H),
5.02−4.89 (m, 1H), 3.78−3.58 (m, 1H), 3.24 (t, J = 7.6 Hz, 1H), 3.13
(q, J = 6.9 Hz, 1H), 2.60−2.51 (m, 1H), 2.47 (s, 3H), 2.12−1.80 (m,
3H), 1.25 (d, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 171.92,
168.96, 167.87, 167.62, 167.27, 157.71, 157.48, 138.10, 136.06, 128.62,
128.56, 128.24, 128.04, 127.88, 127.52, 56.23, 55.79, 54.67, 46.76, 46.66,
31.55, 31.15, 30.49, 29.16, 24.61, 21.83, 20.96, 16.09, 15.38. LC/MS
(ESI+): m/z 493 [M + H].
X-ray Crystallography. Compound 1 was soaked into crystals of
the chimeric BIR domain MLXBIR3SG in complex with the peptide
AVPW using protocols described previously.50 Data for the
MLXBIR3SG/1 complex were collected at beamline 9−1 of the
Stanford Synchrotron Radiation Lightsource. The starting models for
refinement of these structures were derived from a 1.3-Å resolution
structure of a different peptidomimetic complex (details to be published
elsewhere), which was stripped of the peptidomimetic antagonist
molecule and all water molecules within 10 Å of it. After one round of
refinement of the antagonist-free model, 1 was built into clear difference
electron density visible in Fo − Fc maps. New water molecules were
picked automatically using the program Arp/wArp,55 and the entire new
complex model was subjected to several rounds of positional, anisotropic
B-factor, and translation−libration−screw (TLS) refinement using
Refmac5.56 The structure has been deposited with the Protein Data
Bank and assigned the accession code 3UW5.
cIAP2-BIR2 in polarization buffer to 5 nM AVPFAK(5-FAM)K (Hid-
FAM). The reactions were read after an incubation time of 30 min at
room temperature with standard cutoff filters for the fluorescein
fluorophore (λex = 485 nm; λem = 530 nm) in 384-well black HE96
plates (Molecular Devices Corp.). Fluorescence polarization values
were plotted as a function of the protein concentration, and the EC50
values were obtained by fitting the data to a 4-parameter equation
using KaleidaGraph software (Synergy software, Reading, PA). The
apparent Kd values were determined from the EC50 values.
Inhibition constants (Ki) for the antagonists were determined by
the addition of the IAP protein constructs to wells containing serial
dilutions of the antagonists or the peptide AVPW, and the Hid-FAM
probe or AVP-diPhe-FAM probe, as appropriate, in the polarization
buffer. Samples were read after a 30-min incubation. Fluorescence
polarization values were plotted as a function of the antagonist
concentration, and the IC50 values were obtained by fitting the data to
a 4-parameter equation using KaleidaGraph software. Ki values for the
antagonists were determined from the IC50 values.57
Cells, Reagents, and Co-Immunoprecipatation. HEK293T,
melanoma SK-MEL28 and A2058, and breast cancer MDA-MB-231
cells were obtained from ATCC and maintained in the recommended
media. Antibodies against Myc (Upstate), Flag (SIGMA), c-IAP1 (R & D
Systems), caspase-9 (Cell Signaling), and Smac (ProSci) were purchased,
and ML-IAP antibody was described previously.58 zVAD was purchased
from Biomol.
DNA encoding for a chimeric human cIAP1 composed of cIAP1
residues 266−354 with the mutation C309S and residues 344−354
replaced by residues 336−348 of XIAP (hereafter referred to as
cXBIR3CS) was subcloned into the pET15b vector and expressed in
E. coli strain BL-21(DE3) grown in TB media supplemented with 1%
glycerol and 50 μM zinc acetate. Cultures were transformed and grown
at 37 °C until an OD600 of 2.0, induced with 0.5 mM IPTG, grown
overnight at 16 °C, harvested by centrifugation, and lysed in 50 mM
Tris-HCl at pH 8.3, 300 mM NaCl, 0.5 mM TCEP, and 5 mM
imidazole with 2 Complete Protease Inhibitor tablets (Roche). Soluble
cXBIR3CS was purified using Ni-NTA agarose (Qiagen) affinity
chromatography and eluted in 50 mM Tris-HCl at pH 8.3, 300 mM
NaCl, 0.5 mM TCEP, and 300 mM imidazole. Thrombin was added to
the cXBIR3CS-containing fractions followed by dialysis for 36 h at
4 °C against 20 mM Tris-HCl at pH 8.5, 200 mM NaCl, 0.2 mM
TCEP, 2 mM CaCl2 to remove the His-tag. Uncleaved material was
removed by repassage over the Ni-NTA agarose column. The flow
through was concentrated and loaded onto a S-75 sizing column pre-
equilibrated in 20 mM Tris-HCl at pH 8.3, 200 mM NaCl, and
0.2 mM TCEP. Purified cXBIR3CS was concentrated to 4 mg/mL in
50 mM HEPES, pH 7.2, 300 mM NaCl, and 0.2 mM TCEP. Com-
pound 1 (50 mM stock solution in DMSO) was added to a final
concentration of 1 mM. The cXBIR3CS−compound 1 complex was
crystallized by vapor diffusion from hanging drops containing equal
volumes of protein and reservoir solution (0.1 M Tris-HCl pH 8.6,
and 0.5 M magnesium formate). Crystals were cryo-protected in 0.1 M
Tris-HCl at pH 8.5, 0.5 M magnesium formate, and 3 M sodium
formate. A data set was measured at the Advanced Light Source
(Berkeley) beamline 5.0.2 and processed with the HKL suite of
programs. The structure was solved by molecular replacement with the
program Phaser using the BIR3 domain of cIAP2 (PDB code 2UVL)
as a search model and was refined with Refmac5. The structure has
been deposited with the Protein Data Bank and assigned the accession
code 3UW4.
HEK293T cells were transiently transfected with the indicated
constructs, and 40 h after transfection, the cells were lysed in NP40 lysis
buffer. Lysates were incubated with 0, 1, 10, or 100 mM concentrations
of the indicated test materials for 2 h, followed by immunoprecipitation
with anti-Flag or anti-Myc antibodies for 3 h, SDS−PAGE and immuno-
blotting with anti-Flag, anti-Myc, anti-SMAC, and anticaspase-9
antibodies.
SK-MEL28 cells were treated with Gemcitabine (0.5 mM) and 1
(0.5 mM) for 20 h in the presence of the caspase inhibitor, zVAD
(10 mM). At that time, the cells were lysed in NP40 lysis buffer,
immunoprecipitated with the anti-Smac antibody, resolved on SDS−
PAGE, and immuno-blotted with anti-ML-IAP and anti-Smac
antibodies.
Cell Viability and Caspase Activation Assays. Human breast
carcinoma MDA-MB-231 were obtained from ATCC. Normal human
mammary epithelial cells (HMECs) were obtained from Cambrex
Corp. Cells were dissociated from tissue culture flasks by incubation
with Accutase (Innovative Cell Technology Inc.) for 5−10 min.
Detached cells were washed with phosphate-buffered saline (PBS) and
were resuspended in assay media (MDA-MB-231 cells: RPMI1640
supplemented with 10% fetal bovine serum [Sigma-Aldrich] and
2 mM L-glutamine [GlutaMAX-1; Gibco/Invitrogen Corp.]) or culture
media (HMECs: MEBM with MEGM SingleQuots provided by
Cambrex Corp.). Cells were placed in tissue culture-treated, white-wall
(Corning, Inc.) or black-wall (PE Biosystems), clear-bottom, 96-well
plates at 1 × 104 cells/well in a volume of 50 μL. The plates were
incubated at 37 °C and 5% CO2 overnight, the media was removed,
and 1 or its enantiomer was added to the assay media. Cells cultured
in white-wall, clear-bottom plates were incubated at 37 °C and 5%
CO2 for 3 days before cell viability was measured using the CellTiter-
Glo luminescent cell viability assay kit (Promega Corp.) according to
the manufacturer’s instructions. 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.) according to
the manufacturer’s instructions.
Refinement statistics for the complex structures are in Table S1
(Supporting Information).
Binding Assays. Initial polarization experiments were performed
in order to determine dissociation constants (Kd) between IAP protein
BIR domains and fluorescent probes. Samples for fluorescence
polarization affinity measurements were prepared by the addition of
serial dilutions of MLXBIR3SG, XIAP-BIR3, XIAP-BIR2, cIAP1-BIR3,
cIAP2-BIR3 in polarization buffer (50 mM Tris [pH 7.2], 120 mM
NaCl, 1% bovine globulins, 5 mM DTT, and 0.05% octylglucoside) to
5 nM 5-carboxyflourescein (5-FAM)-conjugated AVP-diphenylala-
nine-AKK (AVP-diPhe-FAM) or serial dilutions of cIAP1-BIR2 and
Tumor Xenograft Study. All procedures involving animals were
performed in accordance with the guidelines of the Genentech
Institutional Animal Care and Use Committee. Human breast cancer
MDA-MB-231 cells were obtained from American Type Culture
Collection (Manassas, VA). Cells were resuspended in PBS, and the
cell suspension was mixed 1:1 with Matrigel (BD Biosciences). The
cells (1.5 × 107) were then implanted subcutaneously into the right
flank of 130 female nude mice (Charles River Laboratories, Hollister,
CA) aged 6−8 weeks. Tumor volumes were calculated using the mean
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diameter measured with vernier calipers using the formula v = 0.5 ×
a × b2, where a and b are the largest and smallest perpendicular tumor
diameters, respectively. Ten mice with the appropriate mean tumor
volume were assigned randomly to each of six groups. The mean
ASSOCIATED CONTENT
* Supporting Information
X-ray data collection and refinement statistics, summary of
clinical pharmacokinetics parameters for 1 from day 1 cycle 1,
and immunoprecipitation and immunoblotting of melanoma
SK-MEL28 cells. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
tumor volume
the standard error of the mean (SEM) for all six
groups was 168 3 mm3 at the initiation of treatment (day 0). Mice
were dosed with 1 or vehicle (PBS) by oral gavage with a dose volume
of 4.0 mL/kg, according to the schedule indicated in Figure 6. The
mice were observed on each day of the study, and tumor volumes and
body weights were measured twice each week. Percent tumor growth
inhibition was calculated using the formula % TGI = 100 × (1 −
tumor volumedose/tumor volumevehicle).
Accession Codes
Compound 1/cXBIR3CS, 3UW4; compound 1/MLXBIR3SG,
3UW5.
Pharmacokinetic Studies in Mouse, Rat, Dog, and Monkey.
Compound 1 was intravenously administered to Sprague−Dawley rats
(n = 3 per dose level), Beagle dogs (n = 3 at 1 mg/kg and n = 2 at
20 mg/kg), and cynomolgus monkeys (n = 3 per dose level) at doses
of 1 and 20 mg/kg. Serial plasma samples were collected following
dosing up to 24 h postdose. In nude mice (n = 27 per dose level), 1
was administered intravenously at doses of 1 and 100 mg/kg. Mice
plasma samples (n = 3 per time point) were collected for up to 24 h
postdose. Compound 1 plasma concentrations were determined using
LC-MS/MS. Pharmacokinetic parameters were determined using
noncomparmental analysis.59
Plasma Protein Binding. The plasma protein binding of 1 was
determined at 0.1, 1, 10, and 100 μM by equilibrium dialysis using a
96-well equilibrium dialysis block (HTDialysis LLC, Gales Ferry, CT,
USA). Compound 1 (mix of unlabeled + 14C-labeled) in pooled
plasma from female CD-1 mice, male rats, female rabbits, male dogs,
male monkeys, and male humans (n ≥ 3 for all species) was obtained.
The radioactivity of the postdialysis buffer and plasma samples were
quantified by liquid scintillation counter. The percent unbound
fraction in plasma was calculated by dividing the radiation counts in
the postdialysis buffer side by the radiation counts in the postdialysis
plasma side and multiplying by 100.
AUTHOR INFORMATION
Corresponding Author
*(J.A.F.) Phone: 650-225-6437. E-mail: jflygare@gene.com.
(W.J.F.) Phone: 650-225-6372. E-mail: fairbro@gene.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank members of the DMPK and Purification groups
within Genentech Small Molecule Drug Discovery for analytical
support. We also thank the staff at the Advanced Light Source
and Stanford Synchrotron Radiation Lightsource for their
assistance with sample handling and data collection. The
Advanced Light Source is supported by the Director, Office of
Science, Office of Basic Energy Sciences, of the U.S. Department
of Energy under Contract No. DE-AC02-05CH11231. The
Stanford Synchrotron Radiation Lightsource is a Directorate of
SLAC National Accelerator Laboratory and an Office of Science
User Facility operated for the U.S. Department of Energy Office
of Science by Stanford University. The SSRL Structural
Molecular Biology Program is supported by the DOE Office of
Biological and Environmental Research and by the National
Institutes of Health, National Center for Research Resources,
Biomedical Technology Program (P41RR001209).
BLOOD PLASMA PARTITIONING
■
Blood−plasma partitioning was assessed at total (unlabeled +
14C-labeled) 1 concentrations of 0.1, 1, 10, and 100 μM in
pooled blood from female CD-1 mice, male rats, female rabbits,
male dogs, male monkeys, and male humans (n ≥ 3 for all
species). The blood samples were incubated at 37 °C for 0.5 h.
Radioactivity in blood and plasma samples was quantified by
liquid scintillation counter.
ABBREVIATIONS USED
■
IAP, inhibitor of apoptosis; XIAP, X chromosome-linked
inhibitor of apoptosis; ML-IAP, melanoma inhibitor of
apoptosis; RIP, receptor interacting protein; Smac, second
mitochondria-derived activator of caspases; DIABLO, Direct
IAP binding protein with low pI; BIR, baculoviral IAP repeat
domain; HATU, 2-(1H-7-azabenzotriazol-1-yl)--1,1,3,3-tetra-
methyl uronium hexafluorophosphate methanaminium;
DIPEA, diisopropylethylamine; BOP, benzotriazol-1-yloxytris-
(dimethylamino)-phosphonium hexafluorophosphate; HBTU,
O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-
phosphate; HOBt, N-hydroxybenzotriazole; ADME, absorp-
tion, distribution, metabolism, and excretion; DIC, N,N′-
diisopropylcarbodiimide; EDC, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide; PBS, phosphate-buffered saline; IV, intra-
venous; PK, pharmacokinetic; AUC, area under the curve;
DFPE, 2-(3,5-dimethoxy-formylphenoxy)ethyl polystyrene;
HOBt, N-hydroxybenzotriazole; DMF, dimethylformamide
Hepatocyte Stability Assay and Hepatic Clearance
Prediction. Compound 1 (1 μM) was incubated with human
cryopreserved hepatocytes (0.5 × 106 cells/mL) at 37 °C for 0,
1, 2, and 3 h. Concentrations were assessed by LC/MS/MS.
Intrinsic clearance based upon hepatocyte stability data was
determined using a substrate depletion method and scaled to
hepatic clearance using the well-stirred model.60
Allometric Scaling. Allometric scaling based on maximum
life expectancy potential (MLP) and simple allometric scaling
were used to predict human plasma clearance and volume of
distribution, respectively.61
Human Pharmacokinetics. A phase Ia, first-in-human,
open-label, multicenter, standard dose-escalation study (3 + 3
design) of 1 was conducted in patients with locally advanced or
metastatic solid malignancies or non-Hodgkin’s lymphoma
without leukemic phase. Patients were given 1 doses of 0.049,
0.1, 0.2, 0.28, 0.39, 0.54, 0.76, 1.06, or 1.48 mg/kg as a 0.5 h
intravenous infusion every 14 days. Serial plasma samples were
collected at predetermined time points up to approximately
24 h postdose during the first two cycles. Compound 1 plasma
concentrations were measured using LC/MS/MS. Pharmaco-
kinetic parameters were calculated using noncompartmental
analysis.62
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