Screening multicomponent reactions for X-linked inhibitor of apoptosis-baculoviral inhibitor of apoptosis protein repeats domain binder

Article abstract of DOI:10.1021/jm101341m

We report a second example of a general reaction screening approach to discover low molecular weight inhibitors of protein protein interactions. On the basis of the known pharmacophore model of SMAC mimetics, we predicted several inhibitors based on four

Full text of DOI:10.1021/jm101341m

890 J. Med. Chem. 2011, 54, 890–900
DOI: 10.1021/jm101341m
Screening Multicomponent Reactions for X-Linked Inhibitor of Apoptosis-Baculoviral Inhibitor
of Apoptosis Protein Repeats Domain Binder
†
Ilaria Monfardini, Jui-Wen Huang, Barbara Beck, Jason F. Cellitti, Maurizio Pellecchia, and Alexander Domling*
‡
†
‡
‡
,†
€
†University of Pittsburgh, Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, Pennsylvania 15261, United States, and
‡Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, United States
Received November 3, 2010
We report a second example of a general reaction screening approach to discover low molecular weight
inhibitors of protein protein interactions. On the basis of the known pharmacophore model of SMAC
mimetics, we predicted several inhibitors based on four different multicomponent reactions. The
predicted inhibitors were subsequently synthesized, tested, and found to bind to the antiapoptotic
protein X-linked inhibitor of apoptosis protein (XIAP) and showed cellular activity. Also the
compounds are currently not highly potent. They could form a starting point for future medicinal
chemistry optimization.
Introduction
introduced a new complementary approach to discover protein-
protein interaction antagonists based on structural informa-
tion of the interface. Herein we propose to excavate the
most buried amino acid from the PPI interface and to rename
this amino acid side chain “ANCHOR”. Next we impose this
fragment on efficiently chemical-accessible scaffolds (multicom-
ponent reaction chemistry, MCR) and create virtual libraries
based on several MCR scaffolds. These compound libraries
are then docked into the PPI interface whereby the com-
pounds’ “anchors” align with the protein “anchor” as a
starting pose. Manual inspection of the docking results or
automatic scoring functions is then used to choose compounds
for synthesis and screening. This novel approach was recently
validated by the discovery of several new classes of HDM2
and the first HDM4 antagonists, as well as by dual-action
HDM2/4 antagonists with high potency and cell activity.18-20
Herein we describe our attempts to use the approach to
discover XIAP antagonists.
Analyzing the X-ray structures available between XIAP
and AVPI peptides, (e.g., PDB code 1TFQ) and the wealth of
biochemical data, we chose the N-terminus of AVPI including
the R-carbon and side chain of valine, not including the
residual peptide as an essential fragment serving as an “anchor”
endowed with initial affinity to XIAP (Figure 2).
The anchor residue then serves as a fragment for the
generation of a virtual library of compounds based on differ-
ent molecular scaffolds. These scaffolds were chosen from a
database of several hundred multicomponent reactions (MCR)
which provides high molecular diversity and fast and efficient
chemistry to test our binding hypothesis.21 To generate virtual
libraries based on different MCR scaffolds, we used REAC-
TOR from Chemaxon. This program supports “smart” reac-
tions (generic reaction equations combined with reaction rules)
generating chemically feasible products even in batch mode.22
Thus, the fragment was imprinted onto different MCR scaf-
folds and for each scaffold several representatives were gen-
erated based on available starting materials for the corre-
sponding MCR. Next these virtual libraries were docked into
Inhibitors of apoptosis proteins (IAPa) are key regulators
of apoptosis and are thus important new mechanism-based
cancer targets.1,2 IAPs negatively regulate caspases, the ex-
ecutioner of apoptosis by a selective protein protein interac-
tion (PPI). Smac (second mitochondria-derived activator of
caspases) is a naturally occurring antagonist of IAPs and can
effectively antagonize the inhibition of several initiator and
effector caspases. Smaccanrelease the specific bindingof both
the Bir3 domain of XIAP (X-linked inhibitor of apoptosis)
with caspase-9 and the linker region between Bir1 and Bir2
with caspase-3 and -7. The hotspot of this protein-protein
interaction is the tetrapeptide motif AVPI (Ala-Val-Pro-Ile).
Current strategies focus mostly on the design of AVPI mi-
metics that target the Bir3 domain;3 however, Bir2 domain
directed strategies have also been reported.4 The differential
importance of the two domains, however, is currently subject
to controversies.5 A major disadvantage of many currently
described AVPI mimetics is that they retain a very high peptide-
like character, thus reducing their propensity to be orally
bioavailable.6-11 In contrast, more druglike AVPI mimetics
tend to have a considerably reduced affinity for XIAP.7 Re-
cently, both Genentech and Novartis launched AVPI mimetics
into a phase I clinical trial; however, the chemical structure of the
compounds have not been disclosed (Figure 1).12-14 Typical
examples of published XIAP antagonists and their activities are
shown in parts a,15 b,16 c,14 d,7 e,4 and f17 of Figure 1.
Discovery/Design Strategy of AVPI Mimetics
Current AVPI mimetics have been designed and discovered
mostly by peptidomimetic approaches.3,6,12-15 We recently
*To whom correspondence should be addressed. Phone: 001-412-
651-8425. Fax: 001-412-383-5298. E-mail: asd30@pitt.edu.
a Abbreviations: XIAP, X-linked inhibitor-of-apoptosis protein;
IAP, inhibitor-of-apoptosis protein; BIR3, baculoviral inhibitor of
apoptosis protein repeat; HSQC, heteronuclear single quantum coherence;
FP, fluorescence polarization; ITC, isothermal titration calorimetry; AVPI,
Ala-Val-Pro-Ile.
r
pubs.acs.org/jmc
Published on Web 01/11/2011
2011 American Chemical Society

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 891
efficiently into a reasonable starting geometry and subse-
quently to optimize the compound. The target compounds for
synthesis were chosen by the overall shape complementarities
and the number and quality of proposed hydrogen bond
interactions. According tothisalgorithm, wepredicted several
MCR scaffolds that looked promising as candidates to bind
XIAP and were then chosen for further synthesis (Table 1).
Typical docking poses for such predicted MCR compounds
are shown in Figure 3.
Results and Discussion
Most currently known SMAC mimetics have a central
moiety with an angle of <180° represented by Pro in the
AVPI type peptides. This is an important feature, since it
allows connecting the molecule parts addressing the Ala and
Ile pockets in an efficient way. Our predicted scaffolds mimic
this bending feature and therefore are based on four different
MCRs and represent the heterocyclic backbones thiazolidine,
(homo)piperazine, thiazole, and tetrazole (Table 1).
The rather complex structures incorporating a thiazoline as
the proline mimetic can be convergent-synthesized by the
combination of two MCRs, the well-known Asinger and the
Ugi four-component condensation (Scheme 1).24 The cyclic
Schiff base thiazoline can be accessed in a great manifold by
the MCR of R-haloaldehydes, sodium sulfide, ammonia, and
oxo components, e.g., aldehydes or ketones.29 The particular
thiazolidine 5 was prepared from chloroacetaldehyde 1, sodium
hydrogen sulfide 2, acetone 3, and ammonia 4.30,31 The Asinger
reaction yields the thiazoline in 70% yield as described in the
literature. The subsequent Ugi reaction with diphenylmethyl
isocyanide 7 and N-Boc protected Ala-Val dipeptide 6 yields
after deprotection with TFA the target compound 9 in 22%
yield over two steps.24 Thus, a predicted XIAP binding molecule
was synthesized in only three steps from commercially avail-
able starting materials. This synthetic approach can be poten-
tially used to make derivatives in the central heterocyclic part,
e.g., thiazolines with different substitution patterns or oxazo-
lines or six-membered oxazines and thiazines.32-36 Inaddition
the isocyanide part and the dipeptide part should be amenable
to straightforward variations.
Figure 1. Representative XIAP antagonist classes.
The next scaffold contains a piperazine as a central cis-
constrained moiety. Corresponding compounds 14 and 15 are
quickly and efficiently accessible by a MCR involving mono-
N-substituted ethylenediamines 10 and 11, diphenyl isocya-
nide 7, chloroacetaldehyde 1, and a dipeptide derived car-
boxylic acid 6 (Scheme 2).25,26 This reaction involves a Ugi
reaction and an in situ nucleophilic substitution reaction of
the intermediates 12 and 13, respectively. Target compounds
14 and 15 were synthesized in only two steps according to this
procedure followed by TFA mediated deprotection in quanti-
tative and 56% yield, respectively.
Additionally we were interested in investigating the influence
of the central ring size on the affinity to XIAP. Fortunately,
simply by a switch to propylene diamines, the corresponding
seven-membered diazepine 18 is accessible analogously,
although in lower yield (Scheme 3).27
On the basis of these results, we predicted that compounds
with a central thiazolemoietywould also be of interest. Herein
the thiazole nitrogen could potentially substitute the valine
carbonyl and undergo similar hydrogen bonds. In addition
the thiazole substitution pattern geometry seems to induce a
steep enough angle to direct the carboxylic acid amide sub-
stituent into the hydrophobic subbinding site occupied by
Figure 2. Schematic interaction between the tetrapeptide AVPI
and XIAP (PDB code 1OWZ; AVPF bound to BIR3-XIAP).
Hydrogen bonds are shown in blue, charge-charge interaction is
shown in pink, and hydrophobic interactions are in yellow. The
importance of the charge-charge interaction and the adjacent
hydrogen bond network led us to define the Ala-Val dipeptide
excluding the valine carbonyl as our anchor (shown in red).
the structure under a zero distance constraint regarding the
fragment of the virtual library compounds and the Ala-Val
fragment in the X-ray structure. As the modeling and docking
suite, we used the software MOLOC.23 The zero distance
constraint was used to pose the compounds quickly and

892 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Monfardini et al.
Table 1. MCR Scaffolds with Predicted XIAP Bir3 Antagonistic Activity
isoleucine in the tetrapeptide AVPI (Figure 1e). The thiazole
MCR was introduced by us several years ago and proved to be
very reliable and broad in scope.27,37-41 It involves the reac-
tion of a thiocarboxylic acid, a primary amine, an oxo com-
primary assay. Initial screening of all compounds was carried
out at 100, 10, 1, and 0.1 μM. For active compounds, six to
seven point serial dilutions are made in duplicates. In order to
exclude compounds with highly reactive bonds, which are
likely to form covalent bonds, we look for changes in the IC50
in the presence of 1 mM Cys and/or 1 mM DTT. In addition,
preincubation of inhibitor with the Bak-BH3 peptide before
addition of Bcl protein and the resulting IC50 can give an
indication of a fast and reversible binder. The result is summar-
ized in Table 2. Clearly all of the predicted and tested com-
pounds showed inhibition at the highest concentration mea-
sured (100 μM). Four compounds, however, also showed
considerable activity at 1 μM or below. These are the piper-
azine14, the thiazoles 27 and 34, and the tetrazole39. TheIC50
of the best compound 14AB in the TR-FRET assay was
determined to be 4.9 μM (Figure 4).
ponent, and an isocyanide first described by Schollkopf.42 A
€
target thiazole can be synthesized according to Scheme 4 in
four steps from commercial starting materials. Boc-protected
thioalanine 20 is made from the alanine precursor 19.43 Next,
2,4-dimethoxybenzylamine 22 is used as an ammonia sub-
€
stitute together with isobutyric aldehyde 21 and Schollkopf’s
isocyanide 23 to yield thiazole 24 in 23% yield. We prepared
26 in 91% yield by a recently developed solventless direct
conversion of methyl esters to amides,44 which after TFA-
mediated deprotection yields the target thiazole 27.
Our modeling analysis suggests that a substitutent in the
5-position of the thiazole ring can potentially address the
hydrophobic binding site on top of the Pro ring (Figure 3a,e).
To further establish the physical binding of the compounds
to the Bir3 domain, we performed isothermal titration calo-
rimetry (ITC) and 1D and 2D NMR experiments (Figure 5).
Several compounds show considerably low micromolar affi-
nity to the Bir3 domain, including 9, 14, and 15.
€
We synthesized the required phenyl substituted Schollkopf
isocyanide according to known procedures.45 Analogous to
the above and according to Scheme 5, we were able to synthesize
thiazole target compound 34.
The fourth backbone that was predicted to be active in
binding to the AVPI site in XIAP was a hydroxymethylte-
trazole amenable to synthesis by a tetrazole Passerini variant.28
As a starting material, the Ala-Val dipeptide aldehyde 35 was
required which was synthesized according to known proce-
dures from dipeptide 6.46 Next, the Passerini reaction yielded
the tetrazole target 39 after deprotection of 38 as a mixture of
two diasteromers (Scheme 6). Interestingly, we were able to
separatethe twodiastereomersatthe stage of compound38by
silica gel chromatography. However, acid deprotection
yielded epimerization of the target compound 39.
Next the compounds were tested for their ability to induce
apoptosis in cancer cells. We treated MDA-MB-231 cells with
different compounds (Table 3). The MDA-MB-231 cell line
was chosen because of its reported high inhibitor of apoptosis
proteins (IAPs) expression levels and its sensitivity to Bir3
inhibitors.47,48 Compound 15 can induce apoptosis at IC50 of
31 μM (Figure 6).
Conclusion
In summary, we aimed to discover several unprecedented
XIAP binding AVPI mimicking scaffolds using the interplay
of several techniques, including computational docking of the
To test the binding activities of the predicted compounds to
the Bir3 domain, we performed a fluorescence polarization

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 893
Figure 3. (a, upper left) X-ray structure of small molecular weight AVPI mimetic bound to XIAP (PDB code 1TFQ). The surface of XIAP
is shown in gray. (a) The tetrapeptide mimetic is shown in pink rendered sticks. Hydrogen bond contacts are shown as yellow dotted lines.
(b, upper right) Thiazoline AVPI mimetic 9 docking pose. (c, middle left) Piperazine AVPI mimetic 15 docking pose. (d, middle right) Tetrazole
AVPI mimetic 38 docking pose. (e, lower left) Thiazole AVPI mimetic 32 docking pose. Pictures were rendered using PyMol.
Scheme 1. Synthesis of Thiazolidine Target 9
Scheme 2. Synthesis of Piperazine Targets 14 and 15
on different scaffolds, which can serve as a starting point for
further optimization. In fact several predicted and synthesized
compounds are shown to bind in the micromolar range by
several independent biophysical methods including NMR
(HSQC), FP, and ITC assays. Additionally, compound 15 is
cell-permeable and shows induction of apoptosis in the breast
Ala anchor incorporating scaffold libraries from a MCR
database, biophysical screening, and use of efficient and fast
MCR chemistry. The goal of our study was to investigate if
this method can deliver reasonable active compounds based

894 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Scheme 3. Synthesis of Homopiperazine Target 18
Monfardini et al.
Scheme 5. Synthesis of Thiazole Target 34
Scheme 4. Synthesis of Thiazole Target 27
Scheme 6. Synthesis of Tetrazole Target 39
cancer cell line MDA-MB-231. Binding of the compound
classes into the XIAP groove is rationalized using computa-
tional modeling. Further studies are needed to understand the
structure-activityrelationshipaswellasthe modeof action of
this lead series. Moreover, a wider variety of starting materials
should lead to compounds with improved biological and
pharmacokinetic activities.
Table 2. TR-FRET Assay Results
inhibition, %
concn
9
9A 9AB (1/4) 14 14A 15 18 27 34 39 AVPI
The major method currently used to discover protein-protein
interaction antagonist is high throughput screening (HTS).
Whereas HTS campaigns in protein-protein interaction pro-
jects often yield very poor hit rates, the herein described
process is more successful.49,50 Recently, we showed the
success of the method with p53/mdm2;18 herein we show the
method to work also with an unrelated protein-protein
interaction XIAP(Bir3)/caspase-9. p53/Hdm2 is an R-helix
mediated PPI with largely hydrophobic contacts.51 The hot
spot of XIAP/caspase-9, the tetrapeptide AVPI, however,
shows a more diverse energetic interaction including charge-
charge, hydrogen bonding, and hydrophobic interactions.
100 μM 83.2 97.1
10 μM 15.8 67.4
1 μM
500 nM
100 nM
83.6
16.7
96.9 23.5 79.9 82.5 23.4 97.2 20.7
77.2
23.6
3.8 15.6 19.9 58.0 15.0
11.1 12.3 10.8 75.0
55.4
25.5
3.6
9.1
Our designed compounds presented herein have to be com-
pared to the activities of the described compounds in Figure 1,
with activities ranging from 100 μM to 31 nM. These are the
results of a HTS campaign (Figure 1e) or extended optimiza-
tion studies (Figure 1a-d,f). Clearly our compounds are far
from being optimized and for all four predicted scaffolds only

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 895
[M þ Na]þ: (calcd) 619.2930; (found) 619.2936. HPLC-MS tR:
12.95 min. m/z [M þ H]þ: 597.2.
Diastereomer A. 1H NMR (CDCl3, 600 MHz): δ = 0.88-1.02
(7H, m), 1.11-1.18 (2H, m), 1.45 (9H, s), 1.81 (3H, s), 1.84
(3H, s), 1.89-1.99 (1H, m), 3.20 (1H, q, J = 5), 3.61 (1H, d, J =
12), 4.02-4.10 (1H, m), 4.15-4.24 (1H, m), 4.67 (1H, s), 4.86
(1H,d,J= 5), 6.27 (1H, d, J=7),6.76(1H,s),7.16-7.41 (10H, m),
8.12 (1H, s). 13C NMR (CDCl3, 600 MHz): δ = 17.3, 19.7, 27.8,
28.4, 28.8, 32.3, 52.4, 57.4, 58.2, 66.7, 74.5, 127.4, 127.8, 128.4,
128.5,141.1, 169.3, 172.3.
Diastereomer B. 1H NMR (CDCl3, 600 MHz): δ = 0.55 (3H,
d, J = 7) 0.72 (3H, d, J = 7), 1.32 (3H, d, J = 8), 1.42 (9H, s),
1.78-1.98 (7H, m), 3.16 (1H, d, J = 13), 3.43 (1H, q, J = 7),
4.12-4.18 (1H, m), 4.19-4.21 (1H, m), 4.93 (1H, s), 5.33 (1H, s),
6.23 (1H, d, J = 8), 6.62 (1H, s), 7.03-7.48 (11H, m). 13C NMR
(CDCl3, 600 MHz): δ = 17.3, 19.3, 27.3, 28.3, 32.8, 52.1,
57.6, 57.9, 66.7, 74.4, 127.0, 127.6, 128.7, 128.9, 141.3, 158.9,
169.7.
3-((S)-2-((S)-2-Aminopropanamido)-3-methylbutanoyl)-N-
benzhydryl-2,2-dimethylthiazolidine-4-carboxamide, Diaster-
eomer A (9). In a 5 mL round-bottom flask is placed tert-butyl
1-(1-(4-(benzhydrylcarbamoyl)-2,2-dimethylthiazolidin-3-yl)-
3-methyl-1-oxobutan-2-ylamino)-1-oxopropan-2-ylcarbamate 8
(0.03 mmol, 19 mg), and trifluoroacetic acid is added (100 μL). The
solution is stirred for 10 min, and the trifluoroacetic acid residue is
evaporated. A TLC plate DCM/MeOH 9:1 þ 1% Et3N on the
crude gives a yellow solid 9 (9 mg, 59%). C27H36N4O3S, MW:
496.66 g/mol. HRMS (ESI-TOF) m/z [M þ Na]þ: (calcd)
519.2406; (found) 519.2423. HPLC-MS tR: 9.28 min. m/z
[M þ H]þ: 497.0. 1 H NMR (CDCl 3, 600 MHz): δ = 0.98 (3H,
d, J = 7), 1.02 (3H, d, J = 7), 1.14 (3H, d, J = 7), 1.45-1.67
(2H, m), 1.83 (3H, s), 1.86 (3H, s), 1.95-2.04 (1H, m), 3.20
(1H, q, J = 6), 3.32 (1H, q, J = 6), 3.62-3.66 (1H, m),
4.14-4.21 (1H, m), 4.9 (1H, d, J = 5), 6.27 (1H, d, J = 8),
7.11-7.41 (10H, m), 7.71 (1H, s), 8.24 (1H, s).
Figure 4. FP determined IC50 = 4.9 μM of 14AB.
one or two derivatives were synthesized. The advantages of
the current method are that several scaffolds can be poten-
tially found as starting points for medicinal chemistry opti-
mization based on efficient MCR chemistry. Parallel optimi-
zation of several backbones potentially reduces the preclinical
attrition rate. In contrast to HTS, however, the current methods
are dependent on structural information that is only available
for a minority of PPIs.
Experimental Section
Chemistry. Standard syringe techniques were applied for
transfer of air sensitive reagents and dry solvents. Solvents were
purchased from Aldrich, Fisher Scientific, Acros Organics, and
Alfa Aesar and used as received. 1H and 13C NMR spectra were
recorded on Ultrashield Plus 600 and Bruker at 600 MHz and on
Oxford 400 Varian at 400 MHz, respectively. Chemical shift
values are in ppm relative to external TMS with high frequency
shifts signed positive. Abbreviations used are as follows: s =
singlet, d = doublet, m = multiplet, t = triplet, q= quartet. Data in
parentheses are given in the following order: number of protons,
multiplicity, and coupling constants in Hz. Flash chromatogra-
phy was performed with the indicated solvent (mixture) and
3-((S)-2-((S)-2-Aminopropanamido)-3-methylbutanoyl)-N-
benzhydryl-2,2-dimethylthiazolidine-4-carboxamide, Diaster-
eomer B (9). In a 5 mL round-bottom flask is placed tert-butyl
1-(1-(4-(benzhydrylcarbamoyl)-2,2-dimethylthiazolidin-3-yl)-
3-methyl-1-oxobutan-2-ylamino)-1-oxopropan-2-ylcarba-
mate 8 (0.05 mmol, 30 mg), and trifluoroacetic acid is added
(100 μL). The solution is stirred for 10 min, and the trifluor-
oacetic acid residue is evaporated. A TLC plate DCM/MeOH 9:1 þ
1% Et3N on the crude gives a yellow solid 9 (5.4 mg, 21%).
˚
silica gel, MP Silitech 32-63 D, 60 A, Bodman. Chromatotron
chromatography was performed on Harrison Research chro-
matotron, serial no. 65F, with the indicated solvent (mixture)
and silica gel, Merck, TLC grade 7749, with gypsium binder and
fluorescent indicator, Sigma Aldrich. Thin layer chromatogra-
phy was performed using Whatmann flexible-backed TLC plates on
aluminum with fluorescence indicator. Compounds on TLC
were visualized by UV or ninhydrin detection.
C
27H36N4O3S, MW: 496.66 g/mol. HRMS (ESI-TOF) m/z
[M þ Na]þ: (calcd) 519.2406; (found) 519.2423. HPLC-MS
tR: 9.35 min. m/z [M þ H]þ: 497.0. 1H NMR (CDCl3, 600
MHz): δ = 0.59 (3H, d, J = 7), 0.74 (3H, d, J = 7), 1.36 (3H,
d, J = 7), 1.53-1.74 (2H, m), 1.81-1.98 (7H, m), 3.18 (1H, d,
J = 13), 3.46 (1H, q, J = 7), 3.52-3.57 (1H, m), 4.11-4.19
(1H, m), 5.53 (1H, d, J = 7), 7.09 (1H, s), 7.21-7.44 (10H, m),
7.55 (1H, s).
Purity (g95%) of the compounds was determined by HPLC-
MS measurements with Shimadzu Prominence UFLC (HPLC)
and API 2000 LC/MS/MS systems and Applied Biosystems
MDS SCIEX (MS) using water with 0.1% acetic acid and
acetonitrile: gradient, 5-90% acetonitrile for 7 min; injection
volume, 5 μL; detection wavelength, 254 nm.
tert-Butyl 1-(1-(2-(Benzhydrylcarbamoyl)-4-ethyl piperazin-1-yl)-
3-methyl-1-oxobutan-2-ylamino)-1-oxopropan-2-ylcarbamate (12).
To a solution of N-ethylethylenediamine 10 (2 mmol, 210 μL)
and BocAlaValOH 6 (2.12 mmol, 611 mg) in 2 mL of MeOH
is added diphenyl isocyanide 7 (425 mg, 2.2 mmol) and aque-
ous chloroacetaldehyde solution 1 (4 mmol, 50% wt sol.,
516 μL). The reaction mixture is stirred for 48 h at room tem-
perature. The reaction mixture is diluted with EtOAc, washed
three times with saturated NaHCO3 solution and once with
brine, dried, and evaporated. The resulting crude is purified by
column chromatography on silica gel with EtOAc/MeOH 8:2 to
give a tan solid 12 (490 mg, 41%, diastereomeric ratio: 1:1).
C33H47N5O5, MW: 593.76 g/mol. HRMS (ESI-TOF) m/z [M þ
H]þ: (calcd) 594.3655; (found) 594.3608. HPLC-MS tR: 9.15 min.
m/z [M þ H]þ: 593.8.
tert-Butyl 1-(1-(4-(Benzhydrylcarbamoyl)-2,2-dimethylthiazo-
lidin-3-yl)-3-methyl-1-oxobutan-2-ylamino)-1-oxopropan-2-yl-
carbamate (8). 2,2-Dimethyl-2,5-dihydrothiazole 5 (1 mmol,
155 mg) and the dipeptide BocAlaValOH 6 (1 mmol, 288 mg)
are dissolved in 10 mL of MeOH. Diphenylmethyl isocyanide 7
(1 mmol, 193 mg) is added to the solution, and the mixture is
stirred for 24 h at room temperature. The reaction mixture is
concentrated under vacuum, diluted in EtOAc, and washed
three times with 5% citric acid, three times with NaHCO3, and
once with brine. The organic layer is dried over Na2SO4 and
evaporated under vacuum. The resulting crude is purified by
column chromatography on silica gel with CH2Cl2/CH3OH (99:1)
to give a tan solid 8 (220 mg, 37%, diasteromeric ratio: 1:1). A
subsequent column chromatography on silica gel with CH2Cl2/
CH3OH (99:1) affords separation between the two diastereo-
mers. C32H44N4O5S MW: 596.78 g/mol. HRMS (ESI-TOF) m/z
Diastereomer A. 1H NMR (CDCl3, 600 MHz): δ = 0.28-0.37
(3H, m), 0.95-1.05 (3H, m), 1.27-1.31 (3H, m), 1.36-1.53
(12H, m), 3.49-3.69 (1H, m), 3.69-3.95 (4H, m), 3.95-4.19

896 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Monfardini et al.
Figure 5. (a) Calorimetric titration of Bir3 with compounds 14 (a; Kd = 21.6 μM). The top panel shows raw data in power versus time. The
bottom panel shows data after peak integration, subtraction of blank titration data, concentration normalization, and the fit to a single binding
site model. (b) NMR titration of compound 14 to a 10 mM solution of Bir3. The experimental data were fitted to the nonlinear equation as
described in the methods section, resulting in Kd = 27.7 μM. (c) HSQC NMR of 100 μM Bir3 (red) in H2O phosphate buffer (50 mM, pH 7) and
in the presence of 500 μM 9 (green) and 500 μM 15 (blue).
Diastereomer B. 1H NMR (CDCl3, 600 MHz): δ = 0.80-0.89
Table 3. Cell Viability Assay with MDA-MB-231 Breast Cells and
Selected Compounds
(3H, m), 0.93-1.01 (3H, m), 1.07-1.18 (3H, m), 1.26-1.37 (12H,
m), 2.74-2.92 (1H, m), 3.06-3.19 (1H, m), 3.61-3.83 (3H, m),
3.83-4.06 (3H, m), 4.16-4.40 (1H, m), 4.58-4.72 (1H, m), 5.05-
IC50, μM
9
15 14A
9A 9AB (1/4)
>50
14
18
27
34
39
5.18 (1H, m), 5.80 (1H, s), 6.18 (1H, s), 7.10-7.66 (10H, m), 9.10
(1H, s), 9.96(1H, m). 13CNMR(CDCl3, 600 MHz): δ= 14.2, 16.2,
19.5, 28.3, 29.3, 43.1, 44.5, 47.5, 49.3, 53.1, 57.2, 59.5, 60.3, 79.5,
126.7, 127.1, 127.8, 128.6, 141.1, 141.9, 154.8, 166.5, 167.7, 174.4.
1-((S)-2-((S)-2-Aminopropanamido)-3-methylbutanoyl)-N-benz-
hydryl-4-ethylpiperazine-2-carboxamide 2,2,2-Trifluoroacetate (14).
In a 5 mL round-bottom flask is placed tert-butyl 1-(1-(2-(benz-
hydrylcarbamoyl)-4-ethylpiperazin-1-yl)-3-methyl-1-oxobutan-2-
ylamino)-1-oxopropan-2-ylcarbamate 12 (0.16 mmol, 97 mg), and
>50 31 >50 >50
>50 38 >50 >50 >50
(2H, m), 4.19-4.41 (2H, m), 4.41-4.66 (2H, m), 5.96-6.19
(2H, m), 7.13-7.24 (2H, m), 7.24-7.36 (4H, m), 7.36-7.46
(2H, m), 7.46-7.59 (2H, m), 9.70 (1H, s), 9.78 (1H, m). 13C
NMR (CDCl3, 600 MHz): δ = 13.7, 18.4, 18.7, 20.6, 28.3,s 43.0,
44.6, 47.6, 50.8, 52.7, 57.4, 59.6, 60.2, 80.12, 127.1, 127.2,
127.8, 128.6, 140.9, 142.2, 155.7, 167.0, 167.3, 171.1, 175.2.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 897
with EtOAc, washed three times with saturated NaHCO3 solu-
tion and once with brine, dried, and evaporated. The resulting
crude is purified by column chromatography on silica gel with
EtOAc/MeOH 8:2 to give a tan solid 17 (99 mg, 7%, diastere-
omeric ratio: 4/6). C38H55N5O5, MW: 661.87 g/mol. HRMS (ESI-
TOF) m/z [M þ H]þ: (calcd) 661.420320; (found) 661.421219.
1
HPLC-MS tR: 9.97 min. m/z [M þ H2O]þ: 680.3. H NMR
(CDCl 3, 600 MHz): δ = 0.50-0.97 (8H, m), 0.97-1.19 (5H, m),
1.19-1.28 (3H, m), 1.32 (4.5H, s), 1.33 (4.5H, s), 1.38-1.49
(2H, m), 1.49-1.62 (1H, m), 1.62-1.79(2H, m), 1.78-2.18 (4H, m),
2.67-3.00 (3H, m), 3.87-4.61 (4H, m), 5.19 (0.5H, s), 5.35
(0.5H, s), 6.16 (0.5H, d, J = 8), 6.20 (0.5H, d, J = 8), 7.10-7.30
13
(11H, m), 8.38 (0.5H, s), 8.48 (0.5H, s). C NMR (CDCl ,
3
600 MHz): δ = 17.8, 18.2, 19.1, 24.5, 24.8, 28.3, 29.0, 30.6, 43.2,
45.2, 50.0, 57.1, 57.6, 58.4, 64.3, 79.9, 127.4, 127.8, 128.6, 128.7,
141.4, 155.6, 171.2, 173.2.
1-((S)-2-((S)-2-Aminopropanamido)-3-methylbutanoyl)-N-
benzhydryl-4-cyclohexyl-1,4-diazepane-2-carboxamide 2,2,2-
Trifluoroacetate (18). In a 5 mL round-bottom flask is placed
tert-butyl 1-(1-(7-(benzhydrylcarbamoyl)-4-cyclohexyl-1,4-
diazepan-1-yl)-3-methyl-1-oxobutan-2-ylamino)-1-oxopro-
pan-2-ylcarbamate 17 (0.07 mmol, 47 mg), and trifluoroace-
tic acid is added (100 μL). The solution is stirred for 10 min
and the trifluoroacetic acid residue is evaporated to give a
tan solid (58 mg, quantitative yield). C35H48F3N5O5, MW:
675.78 g/mol. HRMS (ESI-TOF) m/z [M þ H2O]þ: (calcd)
580.3863; (found) 580.3815. HPLC-MS tR: 8.15 min. m/z
[M þ H2O]þ: 580.0.
Figure 6. Concentration dependent cell viability of MDA-MB-231
breast cancer cells of 15.
trifluoroacetic acid is added (200 μL). The solution is stirred for
10 min and the trifluoroacetic acid residue is evaporated to give a
tan solid 14 (122 mg, quantitative yield). C30H40F3N5O5, MW:
607.66 g/mol. HRMS (ESI-TOF) m/z [M þ H]þ: (calcd) 494.3131;
(found) 494.3119. HPLC-MS tR: 7.93 min. m/z [M þ H]þ: 494.2.
tert-Butyl 1-(1-(2-(Benzhydrylcarbamoyl)-4-benzyl piperazin-
1-yl)-3-methyl-1-oxobutan-2-ylamino)-1-oxopropan-2-ylcar-
bamate (13). To a solution of N-benzylethylenediamine 11 (1 mmol,
150 mg) and BocAlaValOH 6 (1.06 mmol, 305 mg) in 1.5 mL of
MeOH are added diphenyl isocyanide 7 (212 mg, 1.1 mmol) and
aqueous chloroacetaldehyde solution 1 (2 mmol, 50% wt sol.,
258 μL). The reaction mixture is stirred at room temperature for
48 h. The reaction mixture is diluted with EtOAc, washed three times
with saturated NaHCO3 solution and once with brine, dried, and
evaporated. The resulting crude is purified by column chromatog-
raphy on silica gel with CH2Cl2/CH3OH (95:5) to give a tan solid
(144 mg, 22%, diastereomeric ratio: 1:1). C38H49N5O5 MW: 655.83
BocAlaSH (20). To a solution of BocAlaOH 19 (5 g, 26 mmol)
in DMF (26 mL) is added carbonyldiimidazole (8.6 g, 53 mmol),
and the reaction mixture is stirred at room temperature for 4 h.
NaSH (5.8 g, 104 mmol) is then added, and stirring continued at
room temperature for 16 h. The reaction mixture is poured in
HCl, 2 N, and extracted with AcOEt. The organic layer is then
washed with brine, dried over Na2SO4, and evaporated. A white
solid 20 was obtained (5 g, 94%). C8H15NO3S, MW: 205.27 g/mol.
1H NMR (CDCl3, 600 MHz): δ = 1.44 (3H, d, J = 7), 1.46
(9H, s), 4.30 (0.4H, s), 4.52 (0.6H, s), 5.31 (0.7H, s), 5.48 (0.3H).
13C NMR (CDCl3, 600 MHz): δ = 18.07, 28.07, 57.22, 80.79,
154.94, 196.83.
g/mol. HRMS (ESI-TOF) m/z [M þ H]þ: (calcd) 656.3812; (found)
1
656.3829. HPLC-MS tR: 10.32 min. m/z [M þ H]þ: 656.2.
H
NMR (CDCl 3, 600 MHz): δ = 0.08 (1.5H, d, J = 7), 0.72-0.84
(2H, m), 0.87 (1.5 H, d, J = 7), 0.97-1.08 (1H, m), 1.12-1.53
(12H, m), 2.25-2.37 (0.5H, m), 2.70-2.84 (0.5H, m), 2.98-3.09
(0.5H, m), 3.29-3.46 (1H, m), 3.46-3.58 (1H, m), 3.58-3.72 (1H, m),
3.72-3.85 (1H, m), 3.85-3.92 (0.5H, m), 3.92-4.05 (1H, m),
4.05-4.18 (0.5H, m), 4.18-4.30 (0.5H, m), 14.18-4.45 (1.5H, m),
4.45-4.59 (0.5H, m), 4.59-4.81 (1.5H, m), 4.814.93 (0.5H, m), 5.79
(0.5H, s), 5.97(0.5H, s), 6.02 (0.5H, d, J = 10), 6.08 (0.5H, d, J =
11), 6.96-7.50 (15H, m), 9.25 (0.5H, s), 9.71 (0.5H, s), 9.79 (0.5H, s),
10.05 (0.5H, s). 13 C NMR (CDCl 3, 600 MHz): δ = 18.2, 18.7, 19.4,
20.6, 28.3, 28.6, 44.7, 48.2, 49.3, 51.1, 52.4, 52.5, 57.4, 59.7, 80.4,
127.1, 127.5, 128.6, 129.4, 140.8, 142.2, 155.9, 167.1, 175.6.
Methyl 2-(1-((S)-2-(tert-Butoxycarbonylamino)-N-(2,4-di-
methoxybenzyl)propanamido)-2-methylpropyl)thiazole-4-car-
boxylate (24). The isobutyraldehyde 21 (91 μL, 1 mmol) is added
under nitrogen at 0 °C to a solution of 2,4 dimethoxybenzyla-
mine 22 (152 μL, 1 mmol) in MeOH (0.5 mL) and treated with
MgSO4 (120 mg, 1 mmol). The Schiff base is precondensed for
1 h, and the solution is cooled to -10 °C. Then BocAlaSH 20
(205 mg, 1 mmol) and the methyl 3-(dimethylamino)-2-isocya-
noacrylate 23 (154 mg, 1 mmol) are added and the reaction
volume is increased to 1 mL. The reaction mixture is allowed to
warm to room temperature and stirred for 3 days. The solvent is
removed in vacuo, and the residue is treated with dichloro-
methane and water. The organic layer is separated and washed
several times with HCl (3%) and with saturated NaHCO3 solution,
dried over Na2SO4, and concentrated in vacuo. The resulting
crude is purified by silica gel column chromatography. The eluting
mixture petroleum ether/ethyl acetate 8:2 affords separation
between the two diastereomers 24 to give a white solid (125 mg,
23%, diastereomeric ratio: 1:1). C26H37N3O7S, MW: 535.65 g/mol.
1H NMR (CDCl3, 600 MHz):δ= 0.70 (1H, s), 0.77 (3H, d, J= 7),
0.92 (2H, d, J = 7), 1.23-1.35 (3H, m), 1.44 (4.5H, s), 1.45
(4.5H, s), 2.91-3.04 (1H, m), 3.64 (2H, s), 3.76 (3H, s), 3.78
(1H, s), 3.89 (2H, s), 3.92 (1H, s), 4.36-4.50 (1H, m), 4.58-4.76
(2H, m), 4.86-4.98 (1H, m), 5.42-5.48 (0.5H, m), 5.54-5.60
(0.5H, m), 6.20 (1H, s), 6.29-6.39 (2H, m), 6.89-6.98 (1H, m),
8.03 (0.5H, s), 8.05 (0.5H, s). 13C NMR (CDCl3, 600 MHz): δ =
19.01, 19.54, 20.41, 20.57, 28.35, 29.70, 30.33, 47.21, 52.23,
54.90, 55.24, 79.38, 98.11, 101.98, 103.57, 116.30, 129.05,
130.39, 144.82, 154.99, 158.40, 168.87, 161.82, 169.21, 174.16.
1-((S)-2-((S)-2-Aminopropanamido)-3-methylbutanoyl)-N-
benzhydryl-4-benzylpiperazine-2-carboxamide (15).In a 5 mL round-
bottom flask is placed tert-butyl 1-(1-(2-(benzhydrylcarbamoyl)-4-
benzylpiperazin-1-yl)-3-methyl-1-oxobutan-2-ylamino)-1-oxopropan-
2-ylcarbamate 13 (0.05 mmol, 33 mg), and trifluoroacetic acid is
added (100 μL). The solution is stirred for 10 min, and the tri-
fluoroacetic acid residue is evaporated. A TLC plate DCM/MeOH
9:1 þ 1% Et3N on the crude gives a yellow solid 15 (15.5 mg, 56%).
C
33H41N5O3, MW: 555.71 g/mol. HRMS (ESI-TOF) m/z
[M þ H]þ: (calcd) 556.3288; (found) 556.3251. HPLC-MS
tR: 8.71 min. m/z [M þ H]þ: 556.1.
tert-Butyl 1-(1-(7-(Benzhydrylcarbamoyl)-4-cyclohexyl-1,
4-diazepan-1-yl)-3-methyl-1-oxobutan-2-yl amino)-1-oxopropan-
2-ylcarbamate (17). To a solution of N-(3-aminopropyl)cyclo-
hexylamine 16 (2 mmol, 340 μL) and BocAlaValOH 6 (2.12 mmol,
611 mg) in 2 mL of MeOH is added diphenyl isocyanide 7
(425 mg, 2.2 mmol) and aqueous chloroacetaldehyde solution 1
(4 mmol, 50% wt sol., 516 μL). The reaction mixture is stirred at
room temperature for 5 days. The reaction mixture is diluted

898 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Monfardini et al.
tert-Butyl (2S)-1-((1-(4-(Benzylcarbamoyl)thiazol-2-yl)-2-
methylpropyl)-(2,4-dimethoxybenzyl)amino)-1-oxopropan-2-yl-
carbamate (26). To a stirred solution of methyl 2-(1-((S)-2-(tert-
butoxycarbonylamino)-N-(2,4-dimethoxybenzyl) propanamido)-
2-methylpropyl)thiazole-4-carboxylate 24 (54 mg, 0.1 mmol)
is added TBD (4 mg, 0.03 mmol) followed by benzylamine 25
(13 μL, 0.12 mmol) under nitrogen atmosphere. The reaction
mixture is slowly warmed to 75 °C and stirred for 12 h, allowed
to cool to room temperature, and concentrated in vacuo. The
residue obtained is chromatographed on silica gel using petro-
leum ether/ethyl acetate 6:4 as eluent to afford a white solid 25
(43.6 mg, 71%). C32H42N4O6S, MW: 610.76 g/mol. HRMS
(ESI-TOF) m/z [M þ Na]þ: (calcd) 633.2723; (found) 633.2722.
1H NMR (CDCl3, 600 MHz): δ = 0.79 (3H, d, J = 7), 0.83
(1H, s), 0.94 (2H, d, J = 7), 1.21-1.31 (3H, m), 1.41 (4.5H, s),
1.43 (4.5H, s), 2.75-2.89 (1H, m), 3.56 (1.5H, s), 3.58 (1.5H, s),
3.69 (1.5H, s), 3.70 (1.5H, s), 4.44-4.80 (6H, m), 5.32-5.39
(0.5H, m), 5.50-5.55 (0.5H, m), 6.15-6.26 (3H, m), 6.62 (1H, s),
7.21-7.40 (5H, m), 7.94 (0.5H, s), 7.96 (0.5H, s). 13C NMR
(CDCl3, 600 MHz): δ = 19.25, 19.32, 20.60, 21.06, 28.33, 30.13,
43.16, 46.86, 47.41, 53.43, 54.93, 79.40, 97.97, 98.34, 103.68,
116.21, 116.67, 124.25, 127.45, 127.78, 128.70, 138.34, 154.74,
155.05, 160.67, 161.03, 161.10, 168.22, 174.20.
(0.5H, m), 2.93-3.05 (0.5H, m), 3.49 (1H, s), 3.59 (1H, s),
3.65-3.89 (4H, m), 4.41-4.99 (6H, m), 5.33-5.42 (0.5H, m),
5.55-5.63 (0.5H, m), 6.21-6.31 (1H, m), 6.31-6.40 (1H, m),
6.40-6.53 (1H, m), 6.65-6.84 (0.5 H, m), 6.89-7.06 (0.5 H, m),
7.20-7.59 (10H, m). 13C NMR (CDCl3, 600 MHz): δ = 19.04,
19.31, 19.59, 20.62, 28.33, 29.71, 30.15, 43.00, 43.79, 46.87,
47.47, 50.85, 55.00, 55.28, 79.37, 98.27, 103.39, 116.03, 127.34,
127.70, 127.88, 127.90, 127.93, 128.07, 128.61, 128.64, 128.99,
129.74, 130.17, 138.58, 154.86, 158.50, 159.64, 160.78, 161.46,
162.60, 165.22, 173.76, 174.22.
2-(1-((S)-2-Aminopropanamido)-2-methylpropyl)-N-benzyl-5-
phenylthiazole-4-carboxamide (34). To the tert-butyl (2S)-1-
((1-(4-(benzylcarbamoyl)-5-phenylthiazol-2-yl)-2-methylpro-
pyl)-(2,4-dimethoxybenzyl)amino)-1-oxopropan-2-ylcarbamate
33 (20 mg, 0.03 mmol) is added TFA (200 μL), and the mixture is
refluxed overnight. TFA is removed in vacuo and the crude
residue is purified by silica gel column chromatography with the
eluting mixture ethyl acetate/methanol 9:1 to give a yellow solid
34 (6 mg, 46%). C24H28N4O2S, MW: 436.57 g/mol. HRMS
(ESI-TOF) m/z [M þ H]þ: (calcd) 437.2011; (found) 437.2007.
HPLC-MS tR: 10.85. m/z [M þ H]þ: 437.0. 1 H NMR (CDCl 3,
600 MHz): δ = 0.76-1.08 (6H, m), 1.31-1.58 (3H, m), 2.24-
2.40 (1H, m), 3.56-3.71 (1H, m), 4.29-4.55 (2H, m), 4.87-5.05
2-(1-((S)-2-Aminopropanamido)-2-methylpropyl)-N-benzyl-
thiazole-4-carboxamide (27). To the tert-butyl (2S)-1-((1-
(4-(benzylcarbamoyl)thiazol-2-yl)-2-methylpropyl)-(2,4-di-
methoxybenzyl)amino)-1-oxopropan-2-ylcarbamate 26 (44 mg,
0.07 mmol) is added TFA (700 μL), and the mixture is refluxed
overnight. TFA is removed in vacuo and the crude residue is
purified by silica gel column chromatography with a gradient
eluting mixture ethyl acetate/methanol and subsequently by
TLC plate with the same mixture to give a white solid 27 (5 mg,
20%). C18H24N4O2S, MW: 360.47 g/mol. HRMS (ESI-TOF)
(1H, m), 7.03-7.59 (10H, m), 8.17-8.56 (2H, m), 8.71-8.82
13
(1H, m), 8.92-9.03 (1H, m) . C NMR (CDCl , 600 MHz):
3
δ = 17.42, 17.90, 18.43, 19.34, 29.72, 33.14, 43.30, 49.45, 52.16,
55.39, 56.77, 127.38, 128.18, 128.25, 128.60, 128.74, 128.81,
129.00, 129.36, 129.86, 130.02, 139.35, 147.06, 162.99, 168.44,
169.63, 170.63.
tert-Butyl (2S)-1-((2S)-1-Hydroxy-3-methyl-1-(1-phenethyl-
1H-tetrazol-5-yl)butan-2-ylamino)-1-oxopropan-2-ylcarbamate
(38). To a solution of BocAlaValH 35 (170 mg, 0.6 mmol) and
phenethyl isocyanide 36 (79 mg, 0.6 mmol) in 1 mL of dichlor-
omethane is added TMSN3 37 (69 mg, 0.6 mmol), and the reaction
mixture is stirred at room temperature for 4 days. The solution is
concentrated and the crude residue is purified by silica gel
column chromatography with the eluting mixture dichloro-
methane/methanol 95:5 and subsequently by TLC plate with
the same mixture to give a white foam 38 (55 mg, 20%). C22H34-
N6O4, MW: 446.54 g/mol. HRMS (ESI-TOF) m/z [M þ Na]þ:
(calcd) 469.2539; (found) 469.2560. HPLC-MS tR: 10.46. m/z
[M þ H]þ: 447.1. 1 H NMR (CDCl 3, 600 MHz): δ = 0.55-1.03
(6H, m), 1.03-1.22 (3H, m), 1.22-1.63 (9H, m), 1.82-2.31
(1H, m), 3.14-3.38 (2H, m), 3.53-3.83(1H, m), 3.88-4.28 (1H, m),
m/z [M þ H]þ: (calcd) 361.1698; (found) 361.1688. HPLC-
1
MS tR: 8.43 min. m/z [M þ H]þ: 360.9. H NMR (CDCl ,
3
600 MHz): δ = 0.93-1.02 (6H, m), 1.35-1.44 (3H, m), 1.87
(2H, s), 2.36-2.46 (1H, m), 3.58-3.68 (1H, m), 4.62-4.72 (2H, m),
5.11-5.18 (1H, m), 7.27-7.34 (2H, m), 7.34-7.43 (3H, m),
7.66 (1H, s), 8.04-8.12 (2H, m).
Methyl 2-(1-((S)-2-(tert-Butoxycarbonylamino)-N-(2,4-di-
methoxybenzyl)propanamido)-2-methylpropyl)-5-phenylthiazole-
4-carboxylate (32). The isobutyraldehyde 21 (183 μL, 2 mmol),
the 2,4 dimethoxybenzylamine 22 (304 μL, 2 mmol), and MgSO4
(241 mg, 2 mmol) are stirred under inert and water-free condi-
tions in dry MeOH (2 mL) at 0 °C. The imine is precondensed for
2 h, and the solution is cooled to -10 °C. BocAlaSH 20 (411 mg,
2 mmol) and (Z)-methyl 3-bromo-2-isocyano-3-phenylacrylate
31 (532 mg, 2 mmol) are added, and the reaction volume is
increased to 4 mL. The mixture is allowed to warm to room tem-
perature and stirred for 48 h. Then the reaction mixture is
diluted with dichloromethane and the organic layer is washed
with saturated NaHCO3 solution, 3% HCl, and saturated NaCl
solution, dried over NaSO4, and concentrated in vacuo. The
resulting compound 32 could not be efficiently purified by silica gel
chromatography, so the crude compound was used directly for
the following reaction. C32H41N3O7S, MW: 611.75 g/mol.
tert-Butyl (2S)-1-((1-(4-(Benzylcarbamoyl)-5-phenylthiazol-2-
yl)-2-methylpropyl)-(2,4-dimethoxybenzyl)amino)-1-oxopropan-
2-ylcarbamate (33). To a stirred solution of the methyl 2-(1-((S)-
2-(tert-butoxycarbonylamino)-N-(2,4-dimethoxybenzyl)pro-
panamido)-2-methylpropyl)-5-phenylthiazole-4-carboxylate 32
(crude mixture: 396 mg, 0.65 mmol) is added TBD (27 mg,
0.195 mmol) followed by benzylamine 25 (85 μL, 0.78 mmol)
under nitrogen atmosphere. The reaction mixture is slowly
warmed to 75 °C and stirred for 12 h, allowed to cool to room
temperature, and concentrated in vacuo. The residue obtained is
chromatographed on silica gel using petroleum ether/ethyl
acetate as eluent to afford a yellow solid 33 (20 mg). C38H46N4O6S,
4.45-4.90 (3H, m), 5.08-5.44 (1H, m), 7.04-7.19(2H, m),
13
7.19-7.38 (3H, m). C NMR (CDCl , 600 MHz): δ = 18.16,
3
19.04, 19.48, 19.92, 27.64, 28.25, 35.96, 36.34, 39.22, 49.71,
50.64, 51.99, 58.93, 60.63, 62.40, 66.13, 80.15, 80.81, 127.32,
128.84, 136.73, 155.54, 174.29.
(2S)-2-Amino-N-((2S)-1-hydroxy-3-methyl-1-(1-phenethyl-1H-
tetrazol-5-yl)butan-2-yl)propanamide (39). In a 5 mL round-bottom
flask is placed tert-butyl (2S)-1-((2S)-1-hydroxy-3-methyl-
1-(1-phenethyl-1H-tetrazol-5-yl)butan-2-ylamino)-1-oxopro-
pan-2-ylcarbamate 38 (0.06 mmol, 28 mg), and trifluoroacetic
acid is added (100 μL). The solution is stirred for 1 h, and the
trifluoroacetic acid residue is evaporated. A silica gel column
chromatography with a gradient eluting mixture DCM/MeOH
on the crude gives a colorless solid 39 (10 mg, 50%). C17H26-
N6O2, MW: 346.43 g/mol. HRMS (ESI-TOF) m/z [M þ H]þ:
(calcd) 347.2195; (found) 347.2193. HPLC-MS tR: 8.20. m/z
[M þ H]þ: 347.0. 1 H NMR (CDCl 3, 600 MHz): δ = 0.53-1.15
(4H, m), 1.15-1.40 (1H, m), 1.40-1.79 (4H, m), 1.79-1.93 (0.5H,
m), 1.93-2.11 (0.5H, m), 3.06-3.42 (2H, m), 3.81-4.39 (2H, m),
4.49-5.02 (2H, m), 5.19-5.35 (0.5H, m), 5.35-5.51 (0.5H, m),
6.94-7.17 (2H, m), 7.17-7.36 (3H, m), 8.33 (0.3H,s), 8.42 (0.4H, s),
8.56 (0.3H, s).
Biology. 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 TCI cryoprobe. Dissociation equilibrium
1
MW: 686.86 g/mol. H NMR (CDCl3, 600 MHz): δ = 0.69-
1.13 (6H, m), 1.20-1.38 (3H, m), 1.38-1.50 (9H, m), 2.79-2.93

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 899
constants (Kd) of ligands were determined by monitoring the
protein chemical shift changes as a function of ligand concen-
tration. Data were collected for a set of resolved BIR3 (con-
assay was performed by two independent experiments and each
having two wells per drug concentration.
Cell Culture and ATPlite Cell Viability Assay. ER-negative
MDA-MB 231 breast cancer cells were obtained from the American
Type Culture Collection (Manassas, 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. The effect of individual test agents on cell viability was
assessed by using the ATPlite one step luminescence assay system
(PerkinElmer, Waltham, MA)55 in four replicates. For cell viability
data of compounds and AVPI, MDA-MB-231, cells (5000 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 72 h. After the indicated length of treat-
ments, the cells in each well were treated with 10 μL ATPlite
substrate solution. The plate was dark-adapted 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).
1
centration was 100 μM) H NMR resonances and fitted to a
single binding site model according to the following equation:52
Kd ¼ ½Toꢀð1 - pÞð½Loꢀ - ½ToꢀpÞ=ð½ToꢀpÞ
[To] and [Lo] are total concentration of target protein and ligand,
respectively. The parameter p represents the fractional popula-
tion of bound versus free species at equilibrium, which for fast
exchanging ligands is measured as
p ¼ ðδobs - δf Þ=ðδsat: - δfree
Þ
δ
obs 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. Com-
pounds with estimated Kd values of 100 μM or less are subse-
quently 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].
References
p ¼ fð½Toꢀ þ ½Loꢀ þ KdÞ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
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2
-
ðð½Toꢀþ½LoꢀþKdÞ - 4½Loꢀ½ToꢀÞg=ð2½ToꢀÞ
All NMR data were processed and analyzed using TOP-
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