Fragment-based design of small molecule X-linked inhibitor of apoptosis protein inhibitors

Article abstract of DOI:10.1021/jm8006992

We report on a general structure- and NMR-based approach to derive druglike small molecule inhibitors of protein-protein interactions in a rapid and efficient manner. We demonstrate the utility of the approach by deriving novel and effective SMAC mimetics targeting the antiapoptotic protein X-linked inhibitor of apoptosis protein (XIAP). The XIAP baculovirus IAP repeat 3 (Bir3) domain binds directly to the N-terminal of caspase-9, thus inhibiting programmed cell death. It has been shown that in the cell this interaction can be displaced by the protein second mitochondrial activator of caspases (SMAC) and that its N-terminal tetrapeptide region (NH2-AVPI, Ala-Val-Pro-Ile) is responsible for this activity. However, because of their limited cell permeability, synthetic SMAC peptides are inefficient when tested in cultured cells, limiting their use as potential chemical tools or drug candidates against cancer cells. Hence, as an application, we report on the derivation of novel, selective, druglike, cell permeable SMAC mimics with cellular activity.

Full text of DOI:10.1021/jm8006992

J. Med. Chem. 2008, 51, 7111–7118
7111
Fragment-Based Design of Small Molecule X-Linked Inhibitor of Apoptosis Protein Inhibitors
Jui-Wen Huang, Ziming Zhang, Bainan Wu, Jason F. Cellitti, Xiyun Zhang, Russell Dahl, Chung-Wai Shiau, Kate Welsh,
Aras Emdadi, John L. Stebbins, John C. Reed, and Maurizio Pellecchia*
Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, California 92037
ReceiVed June 9, 2008
We report on a general structure- and NMR-based approach to derive druglike small molecule inhibitors of
protein-protein interactions in a rapid and efficient manner. We demonstrate the utility of the approach by
deriving novel and effective SMAC mimetics targeting the antiapoptotic protein X-linked inhibitor of apoptosis
protein (XIAP). The XIAP baculovirus IAP repeat 3 (Bir3) domain binds directly to the N-terminal of
caspase-9, thus inhibiting programmed cell death. It has been shown that in the cell this interaction can be
displaced by the protein second mitochondrial activator of caspases (SMAC) and that its N-terminal
tetrapeptide region (NH2-AVPI, Ala-Val-Pro-Ile) is responsible for this activity. However, because of their
limited cell permeability, synthetic SMAC peptides are inefficient when tested in cultured cells, limiting
their use as potential chemical tools or drug candidates against cancer cells. Hence, as an application, we
report on the derivation of novel, selective, druglike, cell permeable SMAC mimics with cellular activity.
Introduction
the desired potency is achieved, keeping molecular weight and
other druglike properties in check (Figure 1).
The X-linked inhibitor of apoptosis protein (XIAPa) bacu-
lovirus IAP repeat 3 (Bir3) domain binds directly to the
N-terminal of caspase-9, thus inhibiting programmed cell
death.1-3 It has been shown that in the cell this interaction can
be displaced by the protein SMAC (second mitochondrial
activator of caspases) and that its N-terminal tetrapeptide region
(AVPI) is responsible for the binding.3,4 However, the use of
synthetic SMAC-derived peptides as therapeutic compounds is
hindered because of their limited cell permeability, proteolytic
instability, and poor pharmacokinetics.5-8 A number of research
groups have reported the discovery of small-molecule Bir3
inhibitors by various methods,6-14 including peptidomimetic
approaches,8,10,14 virtual screening/structure-based design,6,11-13
or screening of natural-product or synthetic libraries.7,9 In this
study, we present a simple strategy in which individual amino
acids are replaced in an iterative manner with more druglike
scaffolds (Figure 1). By starting from the single most important
amino acid of the template peptide, alanine,8-11,15,16 a first
virtual library is obtained by coupling the selected amino acid
with low molecular weight, druglike, synthetically accessible
scaffolds. Subsequently, the library elements are docked against
the target in order to select those compounds with the best fit
for the binding site. Following chemical synthesis of top scoring
compounds, these are experimentally tested by NMR spectros-
copy techniques. The use of NMR is pivotal to the approach
given that at this stage only high-micromolar binders, at best,
are expected. Hit compounds are subsequently used for a second
round of in silico derivatizations followed by synthetic chemistry
of top scoring compounds. The approach can be repeated until
Results and Discussion
As an application we report the derivation of druglike, cell
permeable SMAC mimics. Following our strategy, we first
designed an initial virtual library of L-alanine derivatives, a
critical amino acid in SMAC peptides, by coupling it with 578
primary and 815 secondary commercially available, low mo-
lecular weight, druglike, amines. After molecular docking
studies, a series of 15 selected candidate compounds (structures
and Goldscore values are listed in Supporting Information
Figure 1A) were synthesized and tested experimentally by NMR
for their ability to bind to the Bir3 domain of XIAP (Supporting
Information Figure 1B). Through observation the differences
of chemical shift perturbations on Bir3 in the presence of the
selected putative SMAC mimics, compound 1 (BI-75A1, Figure
1) was identified as a weak binder (Kd ≈ 200 µM) for the Bir3
domain. Molecular docking studies support that 1 presents
several binding characteristics that overlap these observed with
the SMAC peptide, in particular mimicking the interactions
provided by the first three amino acids in AVPI (see also
Supporting Information Figure 1C). From the experimentally
derived structure of Bir3 in complex with AVPI, it appears clear
that the Ala and Val residues occupy the first of two subpockets
(P1 and P2 in Figure 2A) on the surface of the domain, while
the side chain of the Ile residue occupies the second (Figure
2A). Therefore, structure modifications of 1 at position 2 of
the 4-phenoxybenzene scaffold could be proposed in a second
iteration, which may result in the selection of an additional
scaffold mimicking the interactions provided by the isoleucine
residue of AVPI, into the P2 subpocket (Figure 2).
On the basis of this hypothesis, a second virtual library of
compound 1’s derivatives (about 900 compounds) was similarly
designed, compounds rank-ordered by using in silico docking,
and top scoring compounds, such as compound 2 (BI-75D1;
Goldscore is 63.0, Figure 1 and compounds listed in Supporting
Information Figure 2), were further synthesized and tested by
NMR. The molecular docking model of the analogue of 2,
compound 3 (BI-75D2) is shown in Figure 2A. As also
corroborated by NMR chemical shift mapping data with 15N
* To whom correspondence should be addressed. Phone: 858-646-3159.
Fax: 858-713-9925. E-mail: mpellecchia@burnham.org.
a Abbreviations: XIAP, X-linked inhibitor of apoptosis protein; Bir3,
baculovirus IAP repeat 3; SMAC, second mitochondrial activator of
caspases; IAPs, inhibitor of apoptosis proteins; FITC-labeled, fluorescein-
isothiocyanate-labeled; ITC, isothermal titration calorimetry; MEF, mouse
embryonic fibroblasts; TRAIL, tumor necrosis factor-related apoptosis-
inducing ligand; CI, combination index; AML, acute myeloid leukemia;
AVPI, alanine-valine-proline-isoleucine.
10.1021/jm8006992 CCC: $40.75
2008 American Chemical Society
Published on Web 10/29/2008

7112 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Huang et al.
Figure 1. Schematic representation of approach used to derive non-peptide SMAC mimics.
labeled Bir3 (Figure 2B and Supporting Information Figure 3A),
compound 3 docks on the Bir3 surface by occupying each of
the two subpockets occupied by the SMAC peptide AVPI.
Additional interactions between the domain and the bridging
proline residue in AVPI are mimicked by the peripheral phenyl
ring in compound 3 (Figure 2A).
From this second iteration and structure refinement by
N-methylation to improve druglikeness,10,14 compound 3 emerged
showing the tightest binding affinity, among the tested com-
pounds, for Bir3, with a Kd value of 1.2 µM (Figure 2C). When
tested in fluorescence polarization displacement assay with
FITC-labeled SMAC peptides, the compound appeared weaker
depending on experimental conditions (with inhibition constants
of ∼30 µM). This may be explained by possible nonspecific
interactions of the FITC peptide with the protein that cannot
be displaced by the compound. Moreover, the binding affinity
between Bir3 domain of XIAP and compound 3 was also
analyzed by isothermal titration calorimetry (ITC), indicating
a binding affinity (Ka) of (6.5 ( 3.3) × 105 (M-1) (Kd ≈ 2.5
µM) (Figure 2D).
Given the superior druglike characteristics, better human
plasma stability, longer drug metabolic stability, high perme-
ability, and good solubility of the compound (Table 1) compared
to the SMAC N-terminal peptides such as AVPI or AVPF (cell
activity with IC50 > 50 µM), compound 3 exhibited marked
cellular activity as an apoptotic inducer (IC50 ) 16.4 µM) in
MDA-MB-231 breast cancer cell lines (Figure 3A). The cell
line was chosen because it is one of the most sensitive human
cancer lines to the reported Bir3 inhibitors and has high inhibitor
of apoptosis proteins (IAPs) expression levels.10,17 In fact, the
IAP induced resistance in these cells resulted in no obvious
DNA fragmentation when treated with TRAIL (30 ng/mL) or
etoposide (5 µM).17 To confirm whether the proapoptotic activity
of compound 3 is mainly linked to its interaction with the Bir3
domain of XIAP with concomitant caspase-9 activation, we
treated XIAP knockout (-/-) and wild type mouse embryonic
fibroblasts (MEF) cells with similar amounts of compound 3.
Because the compounds are designed to induce apoptosis by
inhibition of the antiapoptotic protein XIAP, it is expected that
XIAP-/- cells would be indifferent to the compound. As
expected, compound 3 exhibited much less proapoptotic activity
in XIAP knockout (-/-) MEF cells rather than in wild type
cells (Figure 3B), further confirming that compound 3 down-
regulates the XIAP function in MEF cells. It is noteworthy that
compound 3 still killed XIAP knockout (-/-) MEF cells,
although at much higher concentrations. This may be caused
by off-target effects at higher concentrations and/or by the
possible interaction with of other Bir3 containing proteins.
Compound 3 also increases caspase-3 activation in MDA-MB-
231 cells (Figure 3C), further confirming that the proapoptotic
activity of compound 3 in cancer cells occurs through caspase
activation. Staurosporine was used as control to validate the
assay.
It has been reported that SMAC-mimic compounds sensitize
tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-
induced apoptosis in breast cancer cells;17 hence, we tested
whether compound 3 has synergistic effects with this agent.
MDA-MB-231 cells were treated with compound 3 (Figure 3D)
or TRAIL alone for 24 or 20 h, respectively. When used
together, compound 3 was added 4 h earlier than TRAIL. Cell
viability was determined by ATPLite assay. The synergy
combination index (CI) value18 of the combination of compound
3 and TRAIL is 0.48 at both IC50 values (CI < 1), indicating
that those agents induce apoptosis in MDA-MB-231 cells in a

Rational Design of XIAP Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7113
Figure 2. (A) Superposition between the X-ray structure of AVPI (magenta) in complex with the Bir3 domain of XIAP (surface representation)
and the docked conformation of compound 3. The Ala and Val residues occupy the first of two subpockets (P1 and P2) on the surface of the
domain, while the Ile occupies the second. (B) Chemical shift mapping data with 15N Bir3 and compound 3. The surface of the Bir3 domain of
XIAP is colored according to the shifts induced by compound 3: red > orange > yellow . white ) no shifts). (C) Fractional changes (p) of
chemical shifts in Bir3 as a function of ligand concentration. The experimental data were fitted to the nonlinear equation as described in the
methods, resulting in Kd ) 1.2 µM. (D) Calorimetric titration of Bir3 with compound 3. The top panel shows raw data in power versus time. The
bottom panel shows data after peak integration, subtraction of blank titration data, and concentration normalization. The solid line is the fit to a
single binding site model. Kd ≈ 2.5 µM.
synergistic manner. The synergistic effect is rational, since
TRAIL is believed to trigger apoptosis through regulation of
extrinsic death-receptor pathway, and compound 3, a Bir3
inhibitor, should activate caspase-9 to induce intrinsic mito-
chondrial pathway of apoptosis. TRAIL (Genentech, South San
Francisco, CA) has recently entered clinic trials but showed
resistances in certain cancers, such as acute myeloid leukemia
(AML).19-23 It has been reported that the resistances of TRAIL
can be overcome, at least in part, by down-regulation of
XIAP.19,24-27 On the other hand, TRAIL itself shows very little
effect on inducing tumor cell death at low concentrations (up
to 110 ng/mL, Figure 3D). Therefore, the combination of
compound 3 and TRAIL may provide a novel approach for
TRAIL-based therapeutic strategy, such as lower resistance and
lower dose of TRAIL.
the role of XIAP in tumorigenesis but also provides a viable
starting point for the development of novel anticancer agents.
Experimental Section
Protein Expression and Purification. The plasmid of His6-
tagged Bir3 were described previously.28 The protein was expressed
in the Escherichia coli strain BL21(DE3) and purified using Ni2+
affinity chromatography. The uniformly 15N-lableled Bir3 was
produced by growing the bacteria in M9 minimal media containing
15NH4Cl as the sole nitrogen source. The NMR samples were
dissolved in 20 or 100 µM sodium phosphate buffer (pH 7.0) for
1D or 2D NMR experiments containing 90%/10% (H2O/2H2O) or
2
99.5% H2O.
General Synthetic Procedures. All commercially available
starting materials were purchased from Sigma-Aldrich, Acros, or
Chembridge and were used without further purification. Column
chromatography was performed with silica gel (63-200 Å, Mach-
rey-Nagel GmbH & Co. KG) or RediSep flash column (ISCO Ltd.).
1H NMR and 13C NMR for QC analysis were acquired on a Varian
Inova 300 MHz or Bruker 600 MHz spectrometer. Chemical shifts
are reported in ppm from residual solvent peaks (δ 7.27 or 3.31
for CDCl3 or CD3OD for 1H NMR, respectively). High resolution
ESI-TOF mass spectra were acquired at the Center for Mass
Spectrometry, the Scripps Research Institute, La Jolla, CA.
Compounds were all found to be in excess of 95% pure as
established by LC-MS.
Conclusion
In conclusion, we propose a simple and effective strategy
that, combining in silico docking, fragment-based drug design,
and NMR spectroscopy, can be used to rapidly derive peptide
mimics with improved druglike properties. When applied to the
design of SMAC mimetics, the approach resulted in the design
of compound 3 with demonstrated activity in cell. This novel
class of compounds not only can be used to further investigate
HPLC analyses were performed using the following conditions:
Waters Symmetry C18 column (reverse phase, 4.6 mm × 150 mm);

7114 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Huang et al.
Table 1
(S)-5-(2-Aminopropanamido)-2-(4-methoxyphenoxy)-N-((2-
(thiophen-2-yl)thiazol-4-yl)methyl)benzamide (2). To a stirring
solution of 2-(4-methoxyphenoxy)-5-nitrobenzoyl chloride (0.5188
g, 1.69 mmol) in DMF (5 mL) at 0 °C was added 4-dimethylami-
nopyridine (DMAP, 0.2716 g, 2.22 mmol) in one potion. After 30
min, to the the stirring mixture was added (2-(2-thienyl)-1,3-thiazol-
4-yl)methylamine (0.3496 g, 1.78 mmol) in a small amount DMF
dropwise. The solution was then stirred overnight at room temper-
ature under nitrogen. The mixture was quenched by saturated
NH4Cl(aq), extracted with ethyl acetate, and dried over Na2SO4. The
resulting residue was purified by the CombiFlash Companion
machine (ISCO, Inc., Lincoln, NE) with 4 g RediSep normal-phase
flash columns with hexane and ethyl acetate solvent system to afford
2-(4-methoxyphenoxy)-5-nitro-N-((2-(thiophen-2-yl)thiazol-4-yl)m-
ethyl)benzamide (0.5926 g, 75%).
AVPI
compound 3
human plasma stabilitya,e 23 min
>120 min (93.9% ( 7.7)
drug metabolic stabilityb,e t1/2 ) 11 min t1/2 ) 14.5 min (>45 min);f
CLint ) 47.7
permeability (2%)c,e
solubilityd,e
low (5.2%)
>100 µg/mL >100 µg/mL
0.73 µM 2.5 µM
high (27%)
Kd from ITC
a The test compounds were incubated in human plasma at 37 °C for 15,
60, 120 min. Liquid chromatography/tandem mass spectrometry (LC/MS/
MS) has been applied in quantitative analysis of drug candidates in plasma.
Triple quadrupole mass spectrometer (API 3200 QTrap, Applied Biosystems/
MDS SCIEX) was used to conduct mass analysis in multiple reaction
monitoring (MRM) mode. The plasma stability expressed as the percentage
of the remaining compound to the initial concentration was calculated using
Analyst software (version 1.4.2) and Excel template. b To determine the
metabolic stability, the compounds in triplicate were incubated with S9
fraction in the presence of NADPH at 37 °C for 30 min. Liquid
chromatography/tandem mass spectrometry (LC/MS/MS) has been applied
in quantitative analysis of drug candidates in biological matrix. Triple
quadrupole mass spectrometry (API 3200 QTrap, Applied Biosystems/MDS
SCIEX) was used to conduct mass analysis in multiple-reaction-monitoring
(MRM) mode. The t1/2 and CLint of a test compound in the liver microsomes
were calculated according to “in vitro half-life” approach using Analyst
software (version 1.4.1) and Excel template. Intrinsic clearance (CLint) was
determined using the following equation based on the “well-stirred” model:
To hydrogenate the nitro group, the product from the last step
was dissolved in ethyl acetate (8 mL), and a catalytic amount of
palladium/C (10 wt %) and a balloon of hydrogen gas were added.
The mixture was stirred overnight at room temperature, filtered,
and concentrated under reduced pressure. The resulting residue was
used for the next step without further purification.
N-(tert-Butoxycarbonyl)-L-alanine (0.1334 g, 0.71 mmol) and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(WSC·HCl, 0.2022 g, 1.05 mmol) were mixed in 8 mL of THF in
a round-bottomed flask, and 5-amino-2-(4-methoxyphenoxy)-N-((2-
(thiophen-2-yl)thiazol-4-yl)methyl)benzamide (the hydrogenated
product, 0.2238 g, 0.5 mmol) in a small amount of THF was then
added dropwise into the flask. The reaction mixture was stirred
overnight at room temperature under nitrogen. The mixture was
extracted with ethyl acetate and saturated NaHCO3(aq) and dried
over Na2SO4. The crude product was purified by the CombiFlash
Companion machine (ISCO, Inc., Lincoln, NE) by 4 g RediSep
normal-phase flash columns with hexane and ethyl acetate solvent
system following the recommend procedures. (S)-tert-Butyl 1-(4-
(4-methoxyphenoxy)-3-((2-(thiophen-2-yl)thiazol-4-yl)methylcar-
bamoyl)phenylamino)-1-oxopropan-2-ylcarbamate was obtained
after drying the samples in an evaporator and high-pressure vacuum
system (0.2070 g, 68%).
CLint (µL/(min · mg))
)
k(min-1) · Vincubation(µL)/Pincubation(mg) where
Vincubation is the incubation volume in µL and Pincubation is the amount of
microsomal protein in the incubation volume. c The permeability was
determined by using the PAMPA method described in the literature.44 d The
apparent solubility was determined by using a nephelometry-based method
described in the literature.45 The compounds were tested at eight concentra-
tions in triplicate. The compound was dissolved in DMSO to 10 mM solution
on day 1. The assay was performed on day 1, and detection was performed
on day 2. e The assay was performed by ASINEX Ltd. (Winston-Salem,
NC). f The major metabolic product of compound 3 is its N-demethylation
product (compound 2), which is still active against Bir3.
a linear gradient from 5% acetonitrile and 95% water to 95%
acetonitrile and 5% water over 20 min; flow rate of 1 mL/min; UV
detection at 254 nm. The LC column was maintained at ambient
temperature.
To deprotect Boc groups, the purified (S)-tert-butyl 1-(4-(4-
methoxyphenoxy)-3-((2-(thiophen-2-yl)thiazol-4-yl)methylcarbam-
oyl)phenylamino)-1-oxopropan-2-ylcarbamate (0.2070 g) was dis-
solved in dichloromethane (CH2Cl2), and then 10 equiv of
trifloroacetic acid was added. The reaction mixture was stirred at
room temperature for 2-3 h after the starting material was all
consumed (checked by TLC). After solvent was removed, the
reactions were then quenched by saturated Na2CO3(aq) solution. The
product was extracted with ethyl acetate, and the combined organic
phase was adjusted to pH 2.0 with concentrated HCl. The final
product (salt form) was obtained after drying the samples in an
evaporator and high-pressure vacuum system (0.1435 g, 83%).
HPLC purity 98.11%, tR ) 12.0 min. 1H NMR (600 MHz, CD3OD)
δ 8.16 (s, 1 H), 7.72 (d, J ) 6.60 Hz, 1 H), 7.61 (s, 2 H), 7.25 (s,
1 H), 7.15 (s, 1 H), 7.01 (d, J ) 7.20 Hz, 2 H), 6.92 (d, J ) 7.80
Hz, 2H), 6.88 (d, J ) 8.40 Hz, 1 H), 4.71 (s, 2 H), 4.09 (m, 1 H),
3.86 (s, 3 H), 1.62 (d, J ) 6.00 Hz, 3 H). HRMS exact mass of (M
+ H)+, 509.1312 amu; observed mass of (M + H)+, 509.1326
amu.
(S)-2-Amino-N-(4-(2-chlorophenoxy)phenyl)propanamide (1). N-
(tert-Butoxycarbonyl)-L-alanine (0.1925 g, 1.02 mmol) and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSC ·HCl,
0.2341 g, 1.22 mmol) were mixed in 5 mL of THF in a round-
bottomed flask, and then 4-(2-chlorophenoxy)aniline (0.2116 g, 0.96
mmol) in small amount THF was added dropwise into the flask.
The reaction mixture was stirred overnight at room temperature
under nitrogen. The mixture was extracted with ethyl acetate and
saturated NaHCO3(aq) and dried over Na2SO4. The crude product
was purified by the CombiFlash Companion machine (ISCO, Inc.,
Lincoln, NE) by 4 g RediSep normal-phase flash columns with
hexane and ethyl acetate solvent system following the recommend
procedures. (S)-tert-Butyl 1-(4-(2-chlorophenoxy)phenylamino)-1-
oxopropan-2-ylcarbamate was obtained after drying the samples
in an evaporator and high-pressure vacuum system (0.2088 g, 73%).
To deprotect Boc groups, the purified (S)-tert-butyl 1-(4-(2-
chlorophenoxy)phenylamino)-1-oxopropan-2-ylcarbamate (0.2088
g) was dissolved in dichloromethane (CH2Cl2) and then 10 equiv
of trifloroacetic acid was added. The reaction mixture was stirred
at room temperature for 2-3 h after the starting material was all
consumed (checked by TLC). After solvent was removed, the
reactions were than quenched by saturated Na2CO3(aq) solution. The
product was extracted with ethyl acetate, and the combined organic
phase was adjusted to pH 2.0 with concentrated HCl. The final
product (salt form) was obtained after drying the samples in an
evaporator and high-pressure vacuum system (0.2341 g, 85%).
HPLC purity >99.9%, tR ) 11.26 min. 1H NMR (600 MHz, CDCl3)
δ 9.47 (s, 1 H, -NH), 7.95-8.32 (br, 2 H, -NH2), 7.49 (d, J )
5.40 Hz, 2 H), 7.32 (d, J ) 7.24 Hz, 2 H), 7.07 (m, 1 H), 6.98 (m,
1H), 6.79 (d, J ) 7.2 Hz, 1H), 6.68 (d, J ) 6.00 Hz, 2 H), 4.64
(m, 1 H) 1.87 (d, 3 H). HRMS exact mass of (M + H)+, 291.0895
amu; observed mass of (M + H)+, 291.0900 amu.
(S)-2-(4-Methoxyphenoxy)-5-(2-(methylamino)propanamido)-N-
((2-(thiophen-2-yl)thiazol-4-yl)methyl)benzamide (3). To a stirring
solution of 2-(4-methoxyphenoxy)-5-nitrobenzoyl chloride (1.4882
g, 4.84 mmol) in DMF (10 mL) at 0 °C was added 4-dimethy-
laminopyridine (DMAP, 0.7115 g, 5.85 mmol) in one potion. After
30 min, to the stirring mixture was added (2-(2-thienyl)-1,3-thiazol-
4-yl)methylamine (1.002 g, 5.11 mmol) in a small amount DMF
dropwise. The solution was then stirred overnight at room temper-
ature under nitrogen. The mixture was quenched by saturated
NH4Cl(aq), extracted with ethyl acetate, and dried over Na2SO4. The
resulting residue was purified by the CombiFlash Companion
machine (ISCO, Inc., Lincoln, NE) with 12 g RediSep normal-
phase flash columns with hexane and ethyl acetate solvent system

Rational Design of XIAP Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7115
Figure 3. (A) Cell based activity of compounds AVPI and compound 3. MDA-MB-231 breast cancer cells were cultured in 10% FBS DMEM
media in 96-well plates overnight and followed by dosage with AVPI, compound 3, or vehicle at varying concentrations in serum-free DMEM
media. After 48 h of treatment, the effects on cells proliferation were evaluated by the ATPLite assay. Cell survival was expressed as percentage
of control ( SEM, compared to DMSO control, from two independent experiments, each having four wells per drug concentration. (B) Similar
amounts of wild type and XIAP knockout (-/-) mouse embryonic fibroblasts (MEF) cells were cultured 24 h in 10% FBS RMPI-1640 media in
white 96-well plate before treatment by compound 3 or DMSO (control) at varying concentrations. After 24 h of treatment in 10% FBS RMPI-1640
media, the effects on cells proliferation were evaluated by the ATPLite assay. Cell survival was expressed as percentage of control ( SEM,
compared to DMSO control, from three independent experiments, each having four wells per drug concentration. (C) Caspase-3 activation assay.
After being cultured in 10% FBS DMEM media overnight, MDA-MB 231 breast cancer cells were treated by compound 3 at various concentrations,
DMSO vehicle, or staurosporine 100 nM. After 24 h of culture, the cells were lysed and caspase-3 activities of the lysates were detected by meso
scale discovery (MSD) caspase-3 assay kit detecting cleaved versus total caspase-3. Staurosporine was used to indicate the efficiency of the assay.
(D) Growth inhibition of MDAMB-231 cells by compound 3 alone or in combination with TRAIL. Cells were cultured in 10% FBS DMEM media
in white 96-well plates overnight and then treated with TRAIL or compound 3 at indicated concentrations alone for 20 and 24 h, respectively.
When used together, compound 3 was added 4 h before TRAIL (total incubation time, 24 h). Cell viability (%) was determined by ATPLite assay
and was expressed as percentage of control ( SEM, compared to DMSO control, from two independent experiments, each having four wells per
drug concentration.
to afford 2-(4-methoxyphenoxy)-5-nitro-N-((2-(thiophen-2-yl)thi-
azol-4-yl)methyl)benzamide (1.4708 g, 65%).
filtered, and concentrated under reduced pressure. The resulting
residue was used for the next step without further purification.
To hydrogenate the nitro group, the product from the last step
was dissolved in dichloromethane (CH2Cl2, 10 mL), and a catalytic
amount of palladium/C (10 wt %) and a balloon of hydrogen gas
were added. The mixture was stirred for 5 h at room temperature,
Boc-N-methyl-L-alanine (0.1222 g, 0.60 mmol) and 1-(3-dim-
ethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSC ·HCl,
0.1435 g, 0.75 mmol) were mixed in 8 mL of THF in a round-
bottomed flask, and 5-amino-2-(4-methoxyphenoxy)-N-((2-(thiophen-

7116 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Huang et al.
2-yl)thiazol-4-yl)methyl)benzamide (the hydrogenated product,
0.2238 g, 0.5 mmol) in a small amount of THF was then added
dropwise into the flask. The reaction mixture was stirred overnight
at room temperature under nitrogen. The mixture was extracted
with ethyl acetate and saturated NaHCO3(aq) and dried over Na2SO4.
The crude product was purified by the CombiFlash Companion
machine (ISCO, Inc., Lincoln, NE) by 4 g RediSep normal-phase
flash columns with hexane and ethyl acetate solvent system
following the recommend procedures. (S)-tert-Butyl 1-(4-(4-meth-
oxyphenoxy)-3-((2-(thiophen-2-yl)thiazol-4-yl)methylcarbamoyl)phe-
nylamino)-1-oxopropan-2-yl(methyl)carbamate was obtained after
drying the samples in an evaporator and high-pressure vacuum
system (0.2460 g, 79%).
Isothermal Titration Calorimetry (ITC). Purified Bir3 domain
of XIAP and the 3 compounds were dissolved in PBS buffer (pH
7.4) and then degassed for 10 min prior to sample loading. Titrations
were performed using a VP-ITC calorimeter from Microcal
(Northampton, MA). The solution containing Bir3 (100 µM) was
loaded into the sample cell, whereas the compound 3’s solution (1
mM) was loaded into the injection syringe. The calorimeter was
first equilibrated at 25 °C, and the baseline was monitored during
equilibration. The total observed heat effects were corrected for
these small contributions. All titration data were analyzed using
Microcal Origin software provided by the ITC manufacturer
(MicroCal, LLC). The association constant (Ka) was obtained
through the equation ∆G ) -RT ln Ka, where R is the gas constant
and T is the absolute temperature in kelvin. Equilibrium dissociation
constant (Kd) was calculated as the reciprocal of Ka.
Molecular Modeling. Some commercially available alanine-
containing compounds are downloaded from ZINC databases (total
2 062 906 druglike compounds from RYAN, CHEMBRIDGE,
CHEMDIV, SIGMA Aldrich, SPECS, COMGENEX, OTAVA,
ASINEX, NCI, TIMTEC, ENAMINE, MAYBRIDGE, AMBINT-
ER, INTERBIOSCREEN, LIFECHEMICALS, KEY ORGANICS,
MICROSOURCE, NANOSYN, PHARMEKS, PUBCHEM, IN-
TERCHIM databases).31 The libraries of commercial available
amines were modified by Combichem into a virtual alanine-
containing library with 1400 compounds.32-34 The 3D structures
of the 1400 compounds were built with CONCORD35 and energy-
minimized with SYBYL (Tripos, St. Louis, MO).36 The X-ray
coordinates of SMAC-XIAP-BIR3 are available (PDB ID 1G73,
2.0 Å resolution),37 and the compound was used as the docking
host of the virtual compounds. Docking calculations were performed
with GOLD, version 3.0 (Cambridge Crystallographic Data Centre,
Cambridge, U.K.).38,39 For each compound, 10 solutions were
generated and subsequently ranked according to their Goldscore.40
The top 200 compounds predicted by GOLD were further analyzed
using SYBYL. The goals are to visually inspect all their docking
poses and select compounds whose alanine moieties primarily bind
to BIR3 in the same mode as that of AVPI observed in the X-ray
structure. The figure was made with SYBYL, and the molecular
surface was generated with MOLCAD.41 The second virtual library
of compound 1’s derivatives (about 900 compounds) was similarly
built up by downloading druglike amines from Sigma-Aldrich,
Acros, and Chembridge and further modifying into alanine-
containing compounds by Combichem.
To deprotect Boc groups, the purified (S)-tert-butyl 1-(4-(4-
methoxyphenoxy)-3-((2-(thiophen-2-yl)thiazol-4-yl)methylcarbam-
oyl)phenylamino)-1-oxopropan-2-yl(methyl)carbamate was dis-
solved in dichloromethane (CH2Cl2), and then 10 equiv of
trifloroacetic acid was added. The reaction mixture was stirred at
room temperature for 2-3 h after the starting material was all
consumed (checked by TLC). After solvent was removed, the
reactions were then quenched by saturated Na2CO3(aq) solution. The
product was extracted with ethyl acetate, and the combined organic
phase was adjusted to pH 2.0 with concentrated HCl. The final
product (salt form) was obtained after drying the samples in an
evaporator and high-pressure vacuum system (0.1817 g, 88%, HCl
1
salt form). HPLC purity 99.14%, tR ) 12.28 min. H NMR (600
MHz, CD3OD) δ 8.19 (s, 1 H), 7.72 (d, J ) 7.20 Hz, 1 H), 7.59 (s,
2 H), 7.25 (s, 1 H), 7.14 (s, 1 H), 7.00 (d, J ) 7.20 Hz, 2 H), 6.91
(d, J ) 7.80 Hz, 2 H), 6.88 (d, J ) 8.40 Hz, 1 H), 4.61 (s, 2 H),
4.11 (m, 1 H), 3.90 (s, 3 H), 2.88 (s, 3 H), 1.74 (d, J ) 6.0 Hz, 3
H). HRMS exact mass of (M + H)+, 523.1468 amu; observed mass
of (M + H)+, 523.1485 amu.
NMR Spectroscopy. Spectra were acquired on a 600 MHz Bruker
Avance spectrometer equipped with TXI probe and z-shielded
gradient coils or on a 600 MHz Bruker Avance equipped with a
TCI cryoprobe.
Dissociation equilibrium constants (Kd) of ligands were deter-
mined by monitoring the protein chemical shift changes as a
function of ligand concentration. Data were collected for a set of
1
resolved Bir3 (concentration was 100 µM) H NMR resonances
and fitted to a single binding site model according to the following
equation:29
Kd ) [To](1 - p)([Lo] - [To]p) ⁄ ([To]p)
Cell Culture. ER-negative MDA-MB 231 breast cancer cells
were obtained from the American Type Culture Collection (Ma-
nassas, VA) and were maintained in DMEM medium supplemented
with 10% fetal bovine serum (FBS, Gibco) at 37 °C in a humidified
incubator containing 5% CO2. Wild type and XIAP knockout (-/
-) mouse embryonic fibroblasts (MEF)2,42 were cultured in RPMI-
1640 medium supplemented with 10% fetal bovine serum (FBS,
Gibco) at 37 °C in a humidified incubator containing 5% CO2.
ATPlite Cell Viability Assay. The effect of individual test agents
on cell viability was assessed by using the ATPlite one step
luminescence assay system (PerkinElmer, Waltham, MA)43 in four
replicates. For cell viability data of compound 3 and AVPI (Figure
3A), MDA-MB-231 cells (8000 cells/well) were seeded and
incubated in white 96-well, flat-bottomed plates in DMEM medium
with 10% FBS overnight. The cells were exposed to various
concentrations of test agents dissolved in DMSO (final DMSO
concentration, 0.1%) in serum-free DMEM medium for 48 h. To
compare the difference of cell growth inhibition of compound 3
between wild type and XIAP knockout (-/-) MEF cells (Figure
3B), the same amount of each type of cell (5000 cells/well) was
seeded and incubated in white 96-well, flat-bottomed plates in
RPMI-1640 medium with 10% FBS overnight. The cells were
exposed to various concentrations of compound 3 dissolved in
DMSO (final DMSO concentration, 0.1%) in 10% FBS RPMI-1640
medium for 24 h. For TRAIL combination experiment (Figure 3D),
MDA-MB-231 cells (8000 cells/well) were seeded and incubated
in white 96-well, flat-bottomed plates in DMEM medium with 10%
FBS overnight. The cells were exposed to various concentrations
[To] and [Lo] are total concentrations of target protein and ligand,
respectively. The parameter p represents the fractional population
of bound versus free species at equilibrium, which for fast
exchanging ligands is measured as
p ) (δobs - δf) ⁄ (δsat - δfree
)
δobs is the observed receptor chemical shift during the titration, and
δfree and δsat are the chemical shifts for the receptor in the unbound
and fully bound (saturated) states, respectively.
Compounds with estimated Kd values of 100 µM or less are
subsequently determined more accurately by a more complete NMR
titration. At least six data points were collected for different
concentrations of ligand, and Kd can then be determined by a
nonlinear least-squares fit of p versus [Lo].
p){([To] + [Lo] + Kd) -
(([T ] + [L ] + K )2 - 4[L ][T ])} ⁄ 2[T ]
√
o
o
d
o
o
o
All NMR data were processed and analyzed using TOPSPIN2.0
(Bruker BioSpin Corp., Billerica, MA) and SPARKY.30 For
chemical shift mapping the NMR samples contained 0.1 mM 15N-
labeled Bir3, 100 mM potassium phosphate buffer (pH 7.0), 5%
DMSO, and 7% D2O. 2D [15N, 1H]-HSQC experiments were
acquired using 32 scans with 2048 and 128 complex data points in
1
the H and 15N dimensions at 300 K.

Rational Design of XIAP Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7117
molecule inhibitors of XIAP. Bioorg. Med. Chem. Lett. 2005, 15, 771–
775.
of compound 3 alone or TRAIL alone or combined in 10% FBS
DMEM medium. The cells were treated by compound 3 for 24 h
and TRAIL for 20 h. When used together, compound 3 was added
into cells 4 h before TRAIL. Controls received DMSO vehicle at
a concentration equal to that of drug-treated cells. After the indicated
length of treatments, the cells in each well were treated with10 µL
of ATPlite substrate solution. The plate was adapted to the dark
for 5-10 min, and the luminescence was measured by a Wallac
1420 plate reader (PerkinElmer). The curves or graphs were
generated using Excel (Microsoft), and the IC50 was calculated by
PRISM (Graphpad). The combination index (CI) of 3 and TRAIL
were calculated by the CalcuSyn (version 2.1, Biosoft) software.
Caspase-3 Activation. MDA-MB-231 cells were cultured in 12
wells plates (100 000 cells/well) with 10% FBS DMEM media
overnight. The cells were treated compound 3 (varying concentra-
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another 24 h. The effect of individual test agents on caspase-3
activation was assessed by using the cleaved/total caspase-3 assay
(Meso Scale Discovery, Meso Scale Diagnostics, LLC, Gaithers-
burg, MD) following the recommend buffer and procedures in two
replicates. The number of relative caspase-3 activation is calculated
by following equation:
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number of relative caspase-3 activation )
[(signal of cleaved caspase-3 activity) ⁄
(signal of total caspase-3 activity)] × 100
Acknowledgment. This work was supported in part by NIH
Grants U19 CA113318 (to M.P. and J.C.R.), R01 AI059572
(to M.P.), and P01 CA102583 (to M.P.).
Supporting Information Available: General synthetic schemes,
details about molecular models, and NMR spectra of the derivatives
of compound 1; NMR Bir3 15N-HSQC spectra and the mapping
data of compound 3; statistical analysis of the synergy data reported
in Figure 3. This material is available free of charge via the Internet
at http://pubs.acs.org.
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