Design and synthesis of a simplified inhibitor for XIAP-BIR3 domain

Article abstract of DOI:10.1016/j.bmcl.2009.09.058

Based on tetrapeptide AVPI, we were able to design and synthesize a new simplified scaffold to inhibit the BIR3 domain of the XIAP protein at low micromolar range. The uncomplicated synthesis and the binding activity of the molecule disclosed here represe

Full text of DOI:10.1016/j.bmcl.2009.09.058

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6413–6418
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of a simplified inhibitor for XIAP-BIR3 domain
Fernando R. Pinacho Crisóstomo, Yongmei Feng, Xuejun Zhu, Kate Welsh, Jing An, John C. Reed,
Ziwei Huang *
The Burnham Institute for Medical Research, 10901 N. Torrey Pines Rd., La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2009
Revised 4 September 2009
Accepted 14 September 2009
Available online 17 September 2009
Based on tetrapeptide AVPI, we were able to design and synthesize a new simplified scaffold to inhibit the
BIR3 domain of the XIAP protein at low micromolar range. The uncomplicated synthesis and the binding
activity of the molecule disclosed here represent an attractive alternative to develop new compounds tar-
geting the protein–protein interaction of XIAP/caspase9.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
XIAP-BIR3 domain
Molecular simplification
Small molecule
Apoptosis plays a crucial role in the homeostasis and develop-
ment of living organisms. Deregulation in this mechanism is asso-
ciated with many diseases including several types of cancer. In the
apoptosis pathway, the inhibitors of apoptosis proteins (IAPs) are
one of the mechanisms used by tumor cells to evade programmed
pounds based on the AVPI structure. As common features, the ana-
logs should contain: (1) an alanine residue or a N-methyl alanine
residue, (2) the presence of a rigid core (3) an aromatic residue
as a surrogate of the isoleucine, and (4) the molecules should adopt
a ‘U-conformation’ for a suitable interaction with the protein
(Fig. 2). Most of the compounds with biological activity in vitro
at nano molar range follow this pattern.
1
cell death.
The XIAP is the most potent caspase inhibitors among IAPs pro-
tein family. This protein interacts with initiator caspase 9 and exe-
cutioner caspase 3 and 7 through its BIR3 and BIR2 domains
Structural simplification represents an efficient drug design
strategy to shorten synthetic routes while keeping or enhancing
2
6
respectively. The search for new compounds able to disrupt the
the biological activity of complex compounds. Combining the
XIAP-caspase interaction has attracted the attention of scientific
community as a promising strategy for cancer treatment.
molecular simplification concept with the guidelines highlighted
previously, we report here a series of simplified compounds in-
spired by the Smac-AVPI tetrapeptide.
Preserving the alanine residue, we proposed a molecular simpli-
fication where the second and third amino acids were substituted
by thiazole ring fused to a carbocycle with different sizes as rigid
The natural inhibitor of XIAP is a protein (SMAC/DIABLO) re-
leased from the mitochondria into the cytosol in response to apop-
totic stimuli. SMAC removes XIAP inhibition of caspase 9 by
binding to the BIR3 domain of XIAP through AVPI tetrapeptide pres-
ent in the N-terminal part of SMAC. This interaction (AVPI/BIR3) has
3
been determined unequivocally by X-ray crystallography.
Using the AVPI structure, Fesik and co-workers have performed
a comprehensive study to determine which amino acids could be
substituted without compromising its binding affinity. The authors
have determined the essential amino acids residues to preserve the
activity of this tetrapeptide to be the alanine (first amino acid) and
4
proline (third amino acid) (Fig. 1).
5
Based on precedents in the literature, it is possible to rational-
ize about some structural features for peptidomimetic derivatives
and postulate general ‘structural guidelines’ to design new com-
*
Corresponding author. Current address: Department of Pharmacology, SUNY
Upstate Cancer Research Institute, State University of New York (SUNY), Upstate
Medical University, Syracuse, New York 13210, USA.
E-mail address: huangz@upstate.edu (Z. Huang).
Figure 1. Tetrapeptide from the N-terminal part of SMAC protein.
0
960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.058

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F. R. Pinacho Crisóstomo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6413–6418
central core. In this approach, we eliminated one chiral center
while at same time conferring more rigidity to the molecule. Final-
ly, different aromatic moieties linked through an amide bond to
the rigid portion provided analogs structurally less complex
(Fig. 3).
The retrosynthetic analysis of these molecules is depicted in the
Scheme 1. It is important to mention that the final compounds
were predicted to adopt the necessary ‘U-conformation’ for a suit-
able interaction with the protein based on molecular modeling
7
studies.
After docking analysis of the molecules containing different
sizes in the central core with the BIR3 domain of the XIAP protein
Figure 2. Common structural features found in most of XIAP-BIR3 domain
inhibitors.
(
RCSB PDB ID 2jk7), one of the suitable candidates to initiate the
synthesis was the compound containing the seven member ring
Figure 3. New synthetic scaffold using a thiazole ring fused to a carbocycle as peptide surrogate.
Scheme 1. Retrosynthesis and conformational analysis of the proposed compounds.

F. R. Pinacho Crisóstomo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6413–6418
6415
carbocycle.8 Furthermore, examples containing a seven member
ring fused to a five member ring have been reported in the litera-
ture with excellent biological activity.
deprotection of the Boc group present in the amino acid residue
was performed using a solution of 10%TFA (v/v) in CH
2 2
Cl provid-
5b,7
ing the first set of molecules. (Scheme 2).
The synthesis started with the bromination of the commercially
available compound methyl-2-oxo-1-cycloheptanecarboxilate
These compounds were synthesized to identify which amino
1
3
acid residue (R ) and aromatic moieties (R ) would provide conve-
nient substitution patterns for this scaffold. Their binding affinities
to XIAP-BIR3 domain were evaluated using competitive fluores-
cence polarization BIDING assay (FPA).
1
0
(1). The product 2 was used without purification in the thiazole
formation assisted by microwave irradiation affording the fused
bicyclic compound 3. The peptide coupling with the amino acids
was achieved using standard conditions (DIC/HOAt/CH
2
Cl
2
) lead-
Despite the preliminary docking result, which indicated the
thiazole ring fused with a seven member carbocycle as a promis-
ing candidates, these molecules did not exhibit relevant
ing to compound 4 in good yields (85–95%). The methyl ester 4
was hydrolyzed using LiOH and the product 5 was used in amide
formation using different commercial available amines. Finally,
1
1
activity.
Scheme 2. Synthesis of the thiazole ring fused to a seven member ring carbocycle.

6
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F. R. Pinacho Crisóstomo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6413–6418
Focusing our attention in the central part of the proposed scaf-
FPA assay of XIAP inhibitors
fold, we synthesized another group of molecules containing a five
member ring in the rigid portion. This modification was envisioned
to confer more rigidity to system and at same time probe any unfa-
vorable steric interactions in the binding pocket of the protein.
The synthesis of these compounds followed the same procedure
as described above although the starting material was the com-
mercially available compound cyclopetanone-2-carboxylic acid
methyl ester (Fig. 4).
100
80
6
4
2
0
0
0
0
The results obtained in the fluorescence polarization assay
(
FPA) experiment prompted us to make some considerations about
the structure–activity relationship (SAR) of this set of molecules
Fig. 5).
As shown in Figure 5, all the compounds with the N-methyl ala-
(
-
20
nine residue exhibited better activity than those containing the
alanine. On the other hand, the compounds with 1,2,3,4-tetrahy-
dronaphthalene moiety showed better results when compared
with others with different aromatic substitution patterns.
Bir-3+
Rh-
Smac
T5B
T5BH
T5D
T5DH
T5T
T5TH
1
2
Figure 5. FPA result obtained with the thiazole fused to a five member ring
i
The promising K = 37 lM obtained with the compound T5T
carbocycle derivatives.
has provided valuable information about this scaffold, which in-
cludes:(1) the amino acid residue must contain an N-methyl
group; (2) the aromatic moiety should be the 1,2,3,4-tetrahydro-
naphthalene; and (3) probably because of unfavorable steric inter-
actions, the thiazole ring in the central core should be fused with a
five member ring.
T5T exists in a diasteroisomeric mixture of four compounds
(Fig. 6).
To determine which diasteroisomer was responsible for the
inhibitory activity, we performed a docking study of these different
diasteroisomers. The result obtained indicated that the only diaste-
roisomer suitable for the interaction with the XIAP-BIR3 domain is
T5TR1 with the configuration S,R,R, highlighted in the Figure 6. The
docked conformation of this diasteroisomer is shown in Figure 7.
To verify experimentally that T5TR1 is indeed the diasteroisom-
er responsible for XIAP-BIR3 binding, we conducted synthesis of
the four diasteroisomers using the same conditions as depicted
in the Scheme 2. However, in the coupling with the aromatic moi-
ety, the commercially available enantiopure compounds (R) and
Another important aspect for the activity of the small molecules
targeting the BIR3 domain is the correct configuration of the chiral
centers present in the molecule. This is not unexpected, considering
that the binding pocket is a chiral environment and many examples
support the importance of the correct diasteroisomer.5
b,9,13
In our initial studies we wanted to determine the suitable sub-
stitution pattern and also the convenient central core for BIR3
binding activity. Nonetheless, the chiral aspect of the molecules
was not considered. As can be observed, only one of the three chiral
centers (the amino acid residue) present in the structure is defined
while the other two are in the racemic form. Thus, the compound
(S)-1,2,3,4-tetrahydronaphthalene were used. The diasteroisomeric
mixture from the coupling with (R)-1,2,3,4-tetrahydronaphthalene
Figure 4. Set of molecules containing thiazole ring fused to five member ring carbocycle.

F. R. Pinacho Crisóstomo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6413–6418
6417
Figure 6. Diasteroisomers present in the T5T mixture.
FPA assay of XIAP inhibitors
100
8
6
4
2
0
0
0
0
0
1uM 10uM 100uM 10uM 100uM 10uM 100uM 10uM 100uM 10uM 100uM
Bir3+ BV6
20
Rh-
T5T
T5TR1
T5TR2
T5TSM
R1+R2+SM
-
Smac
Figure 8. FPA result obtained with diasteroisomers of compound T5T.
that T5TR1 is indeed the active diasteroisomer responsible for
XIAP-BIR3 binding (Fig. 9).
i
Although the K value for the compound T5TR1 was approxi-
Figure 7. The docked conformation of the diasteroismer T5TR1 (shown in Fig. 6) in
XIAP-BIR3 domain (RCSB PDB ID 2jk7) as predicted by molecular docking
calculation using the program SYBYL 8.0.
mately seven times less potent compared to the control BV6, the
uncomplicated synthesis (six steps) and the structural simplicity
of our designed scaffold make it an interesting starting point to de-
velop new analogs.
was possible to separate in a regular (silica gel) chromatographic
column, affording the compounds T5TR1 and T5TR2. In the dock-
ing analysis, the isomers from the coupling with (S)-1,2,3,4-tetra-
hydronaphthalene (T5TS1 and T5TS2) did not exhibit interaction
with protein. We therefore tested these compounds as diasteroiso-
meric mixture (T5TSM) (Fig. 8).
Precedents in the literature have in their structures between 4
and 5 chiral centers (as in the case of the original AVPI peptide),
and in some examples the synthesis has up to 10 chemical steps
5
c,11
to achieve the final product.
In conclusion, we have designed a simplified analog using the
AVPI tetrapeptide as template. The expedient synthetic route (six
steps) combined with binding activity of this new compound
(T5TR1) represents an alternative to other more complex com-
pounds attacking the XIAP-BIR3 domain. This lead compound
presents the structural novelty of a thiazole ring fusioned to five
member ring carbocycle (2-amino-5,6-dihydro-4H-cyclopenta[d]-
As a control experiment we have used the known compound
BV6, which was determined to have a K
BIR3 domain in our FPA assay.14 The K
obtained for T5TR1 was
.58 M while the other disteroisomers did not exhibit significant
binding activity, supporting the docking result and demonstrating
i
value of 0.90 lM for
i
5
l

6
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F. R. Pinacho Crisóstomo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6413–6418
Oltersdorf, T.; Rosenberg, S.; Shoemaker, A.; Tomaselli, K.; Zou, H.; Fesik, S. W. J.
Competition of Rh-Smac binding by T5TR1
Med. Chem. 2004, 47, 4417.
1
00
5. (a) Park, C. M.; Sun, C.; Olejniczak, E. T.; Wilson, A.; Meadows, R.; Betz, S.;
Elmore, S.; Fesik, S. W. Bioorg. Med. Chem. Lett. 2005, 15, 771; (b) Sun, H.;
Coleska, Z. N.; Yang, C. Y.; Xu, L.; Liu, M.; Tomita, Y.; Pan, H.; Yoshika, Y.;
Krajewski, K.; Roller, P.; Wang, S. J. Am. Chem. Soc. 2004, 126, 16686; (c) Sun, H.;
Coleska, Z. N.; Yang, C. Y.; Lu, J.; Meagher, J. L.; Yang, C. Y.; Qiu, S.; Tomita, Y.;
Ueda, Y.; Jiang, S.; Krajewski, K.; Roller, P.; Stuckey, J.; Wang, S. J. Am. Chem. Soc.
8
0
0
0
0
2007, 129, 15279; (d) Wist, A. D.; Gu, L.; Riedl, S.; Shi, Y.; McLendon, G. L. Bioorg.
Med. Chem. 2007, 15, 2935.
6
4
2
6
7
8
.
.
.
Wender, P. A.; Verma, V. A.; Paxton, T. J.; Pillow, T. H. Acc. Chem. Res. 2008, 41,
40.
The structure was minimized using AM1 semi-empirical method in the
program HyperChem 7.5 for windows.
Docking result for the thiazole ring fused to a seven member ring carbocycle
using SYBYL 8.0 and the XIAP-BIR3 domain crystal structure with RCSB PDB ID
2jk7.
0
-
9
-8
-7
-6
-5
-4
-3
T5TR1 Log(M)
i
Figure 9. Titration curve to determine the K value of compound T5TR1.
thiazole-4-carboxylic acid) as a peptide surrogate. New analogs
inspired by this scaffold are undergoing evaluation in our laboratory.
Acknowledgment
9.
Zobel, K.; Wang, L.; Varfolomevv, E.; Franklin, M. C.; Elliott, L. O.; Wallweber, H.
J. A.; Okawa, D. C.; Flygare, J. A.; Vucic, D.; Fairbrother, W. J.; Deshayes, K. ACS
Chem. Biol. 2006, 8, 525.
This work was supported by the NIH PO1 grants CA055164,
U01-CA113318.
10. Hedberg, C.; Kallstrom, K.; Brandt, P.; Hansen, L. K.; Andersson, P. G. J. Am.
Chem. Soc. 2006, 128, 2995.
1
1
1. These compounds did not exhibit activity at concentration of 100
lM.
References and notes
2. The K value was calculated according to Nikolovska-Coleska, Z.; Wang, R.;
i
Fang, X.; Pan, H.; Tomita, Y.; Li, P.; Roller, P. P.; Krajewski, K.; Saito, N. G.;
Stuckey, J. A.; Wang, S. Anal. Biochem. 2004, 332, 261.
3. Sun, W.; Coleska, Z. N.; Qin, D.; Sun, H.; Yang, C. Y.; Bai, L.; Qiu, S.; Wang, Y.; Ma,
D.; Wang, S. J. Med. Chem. 2009, 52, 593.
4. Varfolomeev, E.; Blankenship, J. W.; Wayson, S. M.; Fedorova, A. V.; Kayagaki,
N.; Garg, P.; Zobel, K.; Elliot, L. O.; Wallweber, H. J. A.; Flygare, J. A.; Fairbrother,
W. J.; Deshayes, K.; Dixit, V. M.; Vucic, D. Cell 2007, 131, 669.
1
2
.
.
Reed, J. Nat. Rev. Drug Disc. 2002, 1, 111.
Shiozaki, E. N.; Chai, J.; Rigotti, D. J.; Riedl, S. J.; Li, P.; Srinivasula, S. M.; Alnemri,
E. S.; Fairman, R.; Shi, Y. Mol. Cell. 2003, 11, 519.
Liu, Z.; Sun, C.; Olejniczak, E. T.; Meadows, R. P.; Betz, S. F.; Oost, T.; Hermann,
J.; Wu, J. C.; Fesik, S. W. Nature 2000, 408, 1004.
Oost, T. K.; Sun, C.; Armstrong, R. C.; Assad, A.-S.; Betz, S.; Deckwerth, T. L.;
Ding, H.; Elmore, S. W.; Meadows, R. P.; Olejniczak, E. T.; Oleksijew, A.;
1
3
.
.
1
4