Design and synthesis of a potent biotinylated Smac mimetic

Article abstract of DOI:10.1016/j.tetlet.2005.08.055

A biotinylated Smac mimetic (2) was designed based upon our previously reported potent, conformationally constrained Smac mimetic (1). Smac mimetic (2) was synthesized and determined to bind to XIAP protein with a high-affinity (K_i value of 13

Full text of DOI:10.1016/j.tetlet.2005.08.055

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 7015–7018
Design and synthesis of a potent biotinylated Smac mimetic
Haiying Sun, Zaneta Nikolovska-Coleska, Chao-Yie Yang and Shaomeng Wang*
Departments of Internal Medicine and Medicinal Chemistry and Comprehensive Cancer Center,
University of Michigan, 1500 East Medical Center Drive, Ann Arbor, MI 48109, USA
Received 16 July 2005; revised 10 August 2005; accepted 11 August 2005
Abstract—A biotinylated Smac mimetic (2) was designed based upon our previously reported potent, conformationally constrained
Smac mimetic (1). Smac mimetic (2) was synthesized and determined to bind to XIAP protein with a high-affinity (Ki value of
13 nM) and is therefore a useful pharmacological tool for probing the intracellular targets of this class of potent Smac mimetics.
Ó 2005 Published by Elsevier Ltd.
Smac/DIABLO (Second Mitochondria-derived Activa-
tor of Caspase or Direct IAP Binding protein with
LOw pI) is a protein released from mitochondria in
response to apoptotic stimuli.1,2 Smac/DIABLO pro-
motes apoptosis in cells, at least in part by directly inter-
acting with the inhibitor of apoptosis proteins (IAPs)
and functions as a potent endogenous cellular antago-
nist of IAPs.1,2 Structural3,4 and biological5 studies have
demonstrated that Smac binds to a surface groove in the
third Birculovirus IAP repeat (BIR3) of X-linked IAP
(XIAP) through its exposed four N-terminal residues
(alanine–valine–proline–isoleucine or AVPI).6 Short
Smac peptides derived from the N-terminal residues of
Smac fused to a carrier peptide for intracellular delivery
have been shown to overcome resistance to apoptosis of
cancer cells possessing high levels of IAP proteins. Such
Smac peptides enhance the activity of anti-cancer drugs
in vitro and in vivo, and have little toxicity to normal
cells in vitro or to normal tissues in vivo.7,8 These studies
thus strongly suggest that Smac mimetics may have
great therapeutic potential to be developed as a new
class of anti-cancer drugs.
stability, and bioavailability, and we have recently
reported the design and synthesis of a series of conform-
ationally constrained non-peptidic Smac mimetics.9,10
Compound 1 (Fig. 1) is the most potent Smac mimetic
we have obtained from our previous studies,9,10 and
has a Ki value of 25 nM to XIAP as determined in
our fluorescence-polarization-based (FP-based) binding
assay.14 Toward understanding the precise molecular
mechanisms of action and probing the intracellular
molecular targets of our designed Smac mimetics, we
have designed and synthesized a biotinylated Smac
mimetic based upon the structure of compound 1.
In the design of a biotinylated ligand, it is critical to
identify a suitable position for biotinylation of the
ligand so that the biotin tag will not compromise the
binding of the ligand to its target protein(s). Based upon
our predicted binding model for compound 1 (Fig. 2),
the pro (S)-phenyl ring projects from the XIAP binding
groove with apparently no specific interaction with the
protein.10 In comparison, the pro (R)-phenyl ring buries
into a hydrophobic pocket in XIAP, so a biotin tag at
this phenyl ring would clash with the protein atoms
and decrease ligand binding to XIAP. Based upon this
analysis, the biotinylated compound 2 was designed in
which the 3-amino-propyl group was attached to the
para-position of the pro-(S)-phenyl ring of compound
1 (Figs. 1 and 2). The biotin tag is then linked to the
amino group by the formation of an amide bond
(Fig. 1).
Smac peptides have several intrinsic limitations such
as poor cell-permeability, poor in vivo stability and
bioavailability. Consequently, our laboratory9–11 and
others12,13 are interested in design of Smac peptido-
mimetics and non-peptidic mimetics with much
improved binding affinity, cell-permeability, in vivo
The synthesis of compound 2 is detailed in Scheme 1 and
2. First, a previously reported method was used to syn-
thesize the substituted (S)-diphenylmethylamine 9.15
Keywords: Smac mimetic; Biotinylated ligand; XIAP.
*
Corresponding author. Tel.: +1 734 615 0362; fax: +1 734 647
9647; e-mail: shaomeng@umich.edu
0040-4039/$ - see front matter Ó 2005 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2005.08.055

7016
H. Sun et al. / Tetrahedron Letters 46 (2005) 7015–7018
pro-(S)
O
N
N
NH
H
O
NH2
O
1
O
O
NH
N
O
(S)
HN
N
H
NH
H
N
NH2
O
S
O
2
Figure 1. Design of biotinylated Smac mimetic.
Briefly, reduction of the commercially available acid 3
with NaBH4–I2 gave an alcohol, and protection of the
hydroxyl group with TBS yielded a silyl ether. Treat-
ment of this silyl ether with BuLi followed by the addi-
tion of the resulted aryl lithium to DMF furnished the
aldehyde 4. Condensation of 4 with (S)-phenylglycinol
gave aldimine/oxazolidine 5a/5b. Addition of 4 equiv
of phenyl magnesium bromide to 5a/5b afforded the
aminoalcohol 6 as a single isomer. Cleavage of the aux-
iliary in 6 by Pb(OAc)4 oxidation and removal of the
TBS protecting group with 2N HCl gave an amine. Pro-
tection of the amino group with Boc afforded compound
7. Treatment of 7 with MsCl followed by reaction of the
resulted mesylate with NaN3 yielded azide 8. Reduction
of the azide with PPh3 in THF–H2O followed by protec-
tion of the resulted amine with carbobenzoxy chloride
Figure 2. Predicted binding model of compound 1 in complex with
XIAP BIR3. The red arrow indicates the labeling position for biotin.
Hydrogen bonds formed between 1 and XIAP BIR3 are shown in
dashed lines.
a
b
OHC
Br
(CH ) OTBS
2 3
(CH ) COOH
2 2
4
3
Ph
H
N
H
Ph
N
TBSO(H C)
TBSO(H C)
OH
2
3
2
3
O
5b
5a
Ph
OH
HN
NHBoc
Ph
d
c
e
.
HO(H C)
2
3
TBSO(H C)
2
3
7
6
HCl
NHBoc
Ph
NH
2
f
N (H C)
CbzHN(H C)
2 3
3
2
3
Ph
8
9
Scheme 1. Reagents and conditions: a. (i) NaBH4–I2, THF; (ii) TBSCl, Et3N, CH2Cl2; (iii) BuLi, À78 °C, then DMF, THF, 85% for three steps; b.
(S)-phenylglycinol (1.2equiv), toluene, reflux, 98%; c. PhMgBr (4 equiv), THF, reflux; 56%; d. (i) Pb(OAc) 4, MeOH, then 2N HCl; (ii) (Boc) 2O,
Et3N, CH2Cl2, 68% for two steps; e. (i) MsCl, Et3N, CH2Cl2; (ii) NaN3, DMF, 85% for two steps; f. (i) PPh3, THF–H2O; (ii) CbzCl, Et3N, CH2Cl2;
(iii) 4 M HCl in 1,4-dioxane, MeOH, 82% for three steps.

H. Sun et al. / Tetrahedron Letters 46 (2005) 7015–7018
7017
Ph
a
N
N
BocHN
BocHN
NH
OH
O
O
O
O
O
(CH2)3NHCbz
10
11
Ph
b
N
c
N
NH
H
O
NHBoc
O
(CH2)3NHCbz
HN
12
O
O
Ph
NH
N
N
NH
H
H
N
O
O
NH2
HCl
S
O
2
Scheme 2. Reagents and conditions: a. 9, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2, 95%; b. (i) 4 M HCl in 1,4-dioxane, MeOH; (ii) (S)-N-
Boc-2-aminobutyric acid, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2, 89% for two steps; c. (i) 10% Pd–C, H2, MeOH; (ii) (+)-biotin N-
hydroxy-succinimide ester, N,N-diisopropylethylamine, CH2Cl2; (iii) 4 M HCl in 1,4-dioxane, MeOH, 77% over three steps.
furnished a carbomate. Removal of the Boc protective
group in this carbomate gave our desired chiral amine
9 (Scheme 1).
References and notes
1. Du, C.; Fang, M.; Li, Y.; Wang, X. Cell 2000, 102, 33–42.
2. Verhagen, A. M.; Ekert, P. G.; Pakusch, M.; Silke, J.;
Connolly, L. M.; Reid, G. E.; Moritz, R. L.; Simpson, R.
J.; Vaux, D. L. Cell 2000, 102, 43–53.
3. Wu, G.; Chai, J.; Suber, T. L.; Wu, J. W.; Du, C.; Wang,
X.; Shi, Y. Nature 2000, 408, 1008–1012.
4. Liu, Z.; Sun, C.; Olejniczak, E. T.; Meadows, R. P.; Betz,
S. F.; Oost, T.; Herrmann, J.; Wu, J. C.; Fesik, S. W.
Nature 2000, 408, 1004–1008.
5. Srinivasula, S. M.; Hegde, R.; Saleh, A.; Datta, P.;
Shiozaki, E.; Chai, J.; Lee, R. A.; Robbins, P. D.;
Fernandes-Alnemri, T.; Shi, Y.; Alnemri, E. S. Nature
2001, 410, 112–116.
Condensation of acid 1010 and chiral amine 9 afforded
amide 11. Removal of the Boc protective group in 11
followed by condensation of the resulted ammonium
salt with (S)-N-Boc-2-aminobutyric acid furnished com-
pound 12. Removal of the Cbz protecting group in 12 by
hydrogenation over 10% Pd–C yielded an amine.
Condensation of this amine with (+)-biotin N-hydroxy-
succinimide ester furnished
a biotinylated amide.
Finally, removal of the Boc protective group from this
amide gave the designed biotinylated compound 2.16
6. Fulda, S.; Wick, W.; Weller, M.; Debatin, K. M. Nature
Med. 2002, 8, 808–815.
Compound 2 was tested for its binding affinity to XIAP
BIR3 using our established FP-based assay14 and was
shown to have a Ki value of 13 nM. The binding affinity
of 2 is comparable to that of parent compound 1
(Ki = 25 nM), hence confirming our design strategy. In
our preliminary experiments, we have used compound
2 to probe the intracellular molecular target(s) and
shown that compound 2 indeed binds to endogenous
XIAP protein in human prostate cancer PC-3 cells (data
not shown).
7. Arnt, C. R.; Chiorean, M. V.; Heldebrant, M. P.; Gores,
G. J.; Kaufmann, S. H. J. Biol. Chem. 2002, 277, 44236–
44243.
8. Yang, L.; Mashima, T.; Sato, S.; Mochizuki, M.; Saka-
moto, H.; Yamori, T.; Oh-Hara, T.; Tsuruo, T. Cancer
Res. 2003, 63, 831–837.
9. Sun, H.; Nikolovska-Coleska, Z.; Yang, C.-Y.; Xu, L.;
Tomita, Y.; Krajewski, K.; Roller, P. P.; Wang, S.; Wang,
S. J. Med. Chem. 2004, 47, 4147–4150.
10. Sun, H.; Nikolovska-Coleska, Z.; Yang, C.-Y.; Xu, L.;
Liu, M.; Tomita, Y.; Pan, H.; Yoshioka, Y.; Krajewski,
K.; Roller, P. P.; Wang, S. J. Am. Chem. Soc. 2004, 126,
16686–16687.
11. Sun, H.; Nikolovska-Coleska, Z.; Chen, J.; Yang, C.-Y.;
Tomita, Y.; Pan, H.; Yoshioka, Y.; Krajewski, K.; Roller,
P. P.; Wang, S. Bioorg. Med. Chem. Lett. 2005, 15, 793–
797.
12. Oost, T. K.; Sun, C.; Armstrong, R. C.; Al-Assaad, A. S.;
Betz, S. F.; Deckwerth, T. L.; Ding, H.; Elmore, S. W.;
Meadows, R. P.; Olejniczak, T. T.; Oleksijew, A.; Olters-
dorf, T.; Rosenberg, S. H.; Shoemaker, A. R.; Tomaselli,
K. J.; Zou, H.; Fesik, S. W. J. Med. Chem. 2004, 47, 4417–
4426.
In summary, we have designed and synthesized a biotin-
ylated Smac mimetic, which binds to XIAP with a high-
affinity (Ki = 13 nM). This potent biotinylated Smac
mimetic is being used as a pharmacological tool to prove
the cellular targets for this class of potent Smac mimetics
in our laboratory.
Acknowledgement
We are grateful for the financial support from the
National Cancer Institute, National Institutes of Health
(R01CA109025 to S.W.).
13. Li, L.; Thomas, R. M.; Suzuki, H.; De Brabander, J. K.;
Wang, X.; Harran, P. G. Science 2004, 305, 1471–1474.

7018
H. Sun et al. / Tetrahedron Letters 46 (2005) 7015–7018
14. Nikolovska-Coleska, Z.; Wang, R.; 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,
261–273.
15. Pridgen, L. N.; Mokhallalati, M. K.; Wu, M. J. J. Org.
Chem. 1992, 57, 1237–1241.
16. Selected chemical data of compound 2: Compound 2 was
purified by HPLC on reverse phase C18 column. The
gradient ran from 75% of solvent A (0.1% TFA in H2O)
and 25% of solvent B (0.1% TFA in CH3CN) to 60% of
solvent A and 40% solvent B in 30 min. The purity of 2
was confirmed by analytical HPLC to be over 98%. 1H
NMR was reported with DHO (4.79 ppm), 13C NMR was
reported with 1,4-dioxane (67.16 ppm). 1H NMR
(300 MHz, D2O) d 7.30–7.16 (m, 5H), 7.10–7.01 (m,
4H), 5.90 (s, 1H), 4.50–4.40 (m, 2H), 4.32–4.22 (m, 1H),
4.13–4.08 (m, 1H), 3.95–3.82 (m, 2H), 3.08–2.90 (m, 3H),
2.76–2.60 (m, 1H), 2.58–2.42 (m, 3H), 2.18–1.10 (m, 20H),
0.92(t, J = 7.5 Hz, 3H); 13C NMR (75 MHz, D2O, 1,4-
dioxane) d 177.03, 173.49, 172.76, 169.44, 165.79, 142.04,
141.63, 139.22, 129.44, 128.32, 127.79, 62.64, 62.56, 60.72,
60.15, 57.96, 55.97, 54.85, 54.31, 40.28, 39.49, 36.02, 33.12,
32.71, 30.36, 29.95, 28.45, 28.28, 27.58, 25.76, 25.01,
9.03.