Development of target protein-selective degradation inducer for protein knockdown

Article abstract of DOI:10.1016/j.bmc.2011.03.057

Our previous technique for inducing selective degradation of target proteins with ester-type SNIPER (Specific and Nongenetic Inhibitor-of-apoptosis- proteins (IAPs)-dependent Protein ERaser) degrades both the target proteins and IAPs. Here, we designed a

Full text of DOI:10.1016/j.bmc.2011.03.057

Bioorganic & Medicinal Chemistry 19 (2011) 3229–3241
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of target protein-selective degradation inducer for protein
knockdown
Yukihiro Itoh a, Minoru Ishikawa a, Risa Kitaguchi a, Shinichi Sato a, Mikihiko Naito b, Yuichi Hashimoto a,
⇑
a Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Our previous technique for inducing selective degradation of target proteins with ester-type SNIPER
(Specific and Nongenetic Inhibitor-of-apoptosis-proteins (IAPs)-dependent Protein ERaser) degrades both
the target proteins and IAPs. Here, we designed a small-molecular amide-type SNIPER to overcome this
issue. As proof of concept, we synthesized and biologically evaluated an amide-type SNIPER which
induces selective degradation of cellular retinoic acid binding protein II (CRABP-II), but not IAPs. Such
small-molecular, amide-type SNIPERs that induce target protein-selective degradation without affecting
IAPs should be effective tools to study the biological roles of target proteins in living cells.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 10 March 2011
Revised 22 March 2011
Accepted 24 March 2011
Available online 29 March 2011
Keywords:
Protein knockdown
Protein degradation inducer
IAPs
CRABP-II
1. Introduction
ligase (E3) activity.4,5 Under physiological conditions, caspase
degradation occurs via two steps: (i) IAPs bind to caspases and
Methods to down-regulate target proteins in cells or animals
are useful not only for biological studies and medical research,
but also for developing therapeutic strategies in cases where
expression of a target protein is involved in the pathogenesis of
disease. Thus far, deletion and suppression of proteins at the pre-
translational level by means of gene knockout and gene knock-
down have been widely used for ablating target proteins. Such
techniques have yielded insight into the biological functions of
numerous proteins in cells or animals. However, complicated and
time-consuming genetic manipulation is required for gene knock-
out. Gene knockdown using RNA interference is easy, but cannot
remove existing proteins and so is especially ineffective for pro-
teins with a long half-life. Therefore, the utility of these genetic
methods for medical applications is limited. Proteolysis-targeting
chimeric molecules (protacs) represent a pioneering approach in
the field of down-regulating proteins post-translationally.1 How-
ever, the membrane permeability or stability of peptide structures
is often inadequate for biological studies and therapeutic applica-
tions.2 Therefore, new general techniques, which overcome these
issues, are needed.
promote poly-ubiquitination, (ii) the polyubiquitinated caspases
are degraded by proteasome (Fig. 1a). Protein knockdown is
achieved by the use of small molecules designed as conjugates of
an IAP recognition moiety with a specific ligand for a target pro-
tein. Such a molecule, which we named SNIPER (Specific and
Nongenetic IAPs-dependent Protein ERaser), induces formation of
an artificial (non-physiological) complex of IAP and the target pro-
tein, thereby promoting poly-ubiquitination and proteasomal deg-
radation of the target protein (Fig. 1b). We adopted methyl bestatin
(MeBS, 2) (Fig. 1)6,7 as a ligand for cellular inhibitor of apoptosis
protein 1 (cIAP1), which is one of the IAPs,5,8 and all-trans retinoic
acid (ATRA, 3) (Fig. 2) as a specific ligand for cellular retinoic acid
binding protein II (CRABP-II),9 and synthesized SNIPER (4) (Fig. 3)3
as a tool for degradation of CRABP-II. However, this ester-type SNI-
PER (4) was found to promote auto-degradation of cIAP1, like MeBS
(2),3,6,7 and thus, the selectivity of ester-type SNIPER (4) for its tar-
get protein is poor. In addition, the ester group of 4 might be easily
hydrolyzed in cells. Therefore, we aimed to develop a new type of
SNIPER which would overcome these problems. Herein, we de-
scribe the design, synthesis and biological evaluation of an
amide-type SNIPER which induces selective degradation of
CRABP-II, without affecting IAPs.
Recently, we have reported protein knockdown as a technique
for inducing selective degradation of proteins post-translationally
by using small molecules which have sufficient membrane perme-
ability.3 This approach mimics physiological caspase degradation
by inhibitor of apoptosis proteins (IAPs), which show ubiquitin
2. Chemistry
The compounds prepared for this study are shown in Figure 3
(compounds 5–8) and Figure 6 (DanBE, 16). Syntheses were carried
⇑
Corresponding author. Tel.: +81 3 5841 7847; fax: +81 3 5841 8495.
E-mail address: hashimot@iam.u-tokyo.ac.jp (Y. Hashimoto).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.057

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Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 3229–3241
Figure 1. (a) Physiological protein degradation. (b) Artificial protein degradation (protein knockdown) induced by SNIPER.
with N-Boc bestatin (14) and subsequent deprotection of the Boc
group of compound 20 gave DanBE (16).
Compounds 6 and 8 were synthesized as outlined in Scheme 3.
Alcohol 2110 was converted to azide 23 by tosylation and subse-
quent treatment with sodium azide. Amine 24, obtained by reduc-
tion of azide 23, was allowed to react with compounds 14 and 253
to yield amides 26 and 27. Condensation of acid 283 with amines
29 and 30, derived by deprotection of the Boc group of compounds
26 and 27, afforded amides 31 and 32. Deprotection of the 2-cya-
noethyl groups of compounds 31 and 32 with tetrabutylammo-
nium fluoride (TBAF) gave acid 33 and compound 8.3,11 Removal
of the 9-fluorenylmethyloxycarbonyl (Fmoc) group of compound
33 with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)3,12 gave com-
pound 6.
Figure 2. Structures of bestatin (1), bestatin methyl ester (MeBS, 2), and ATRA (3).
out as outlined in Schemes 1–3. The route for synthesis of com-
pounds 5 and 7 is illustrated in Scheme 1. Condensation of amine
10 with acid 11 afforded amide 12. Hydrolysis of ester 12 under an
alkaline condition gave acid 14. Bestatin (1) was treated with di-
tert-butyl dicarbonate ((Boc)2O) to yield N-Boc bestatin (13). Con-
densation of acid 13 or 14 with methylamine afforded amides 15
and 7. Removal of the Boc group of 15 gave BE04 (5).
3. Results and discussion
3.1. Molecular design
DanBE (16) was synthesized from 17 and 18 via the route
shown in Scheme 2. Condensation of sulfonyl chloride 17 with
amine 18 gave sulfonamide 19. Alcohol 19 was allowed to react
We selected MeBS (2) as a ligand for cIAP1 in the previous study
because MeBS (2) activates auto-ubiquitination of cIAP1 and
Figure 3. Structures and molecular design of SNIPERs.

Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 3229–3241
3231
maintains the E3 activity of cIAP1. Therefore, we considered that
highly selective SNIPERs might be obtained by replacement of
MeBS (2) in the ester-type SNIPER (4) with a structure which binds
to cIAP1, but does not induce auto-degradation of cIAP1 and does
not inhibit the E3 activity of cIAP1. Therefore we attempted to
identify such a structure. Naito’s group investigated the cIAP1-
binding affinity and auto-degradation activity of MeBS analogs.6
The order of binding affinity evaluated by means of surface plas-
mon resonance (SPR) binding experiments indicated that the
binding affinity of BE04 (5) (Fig. 3) is higher than that of bestatin
(1), which induced degradation of cIAP1, but lower than that of
MeBS (2). On the other hand, the cIAP1-degradation activity of
BE04 (5) is apparently weaker than that of bestatin (1) or MeBS
(2).6 These results indicate that the two activities (binding activity
and auto-degradation activity) should be separable. We further
investigated the effect of BE04 (5) on cIAP1 levels by means of
Western blotting. Treatment of HT1080 cells expressing FLAG-
partially retained and the amide has no influence on E3 activity.
This substitution is also expected to be advantageous for prolong-
ing the duration of target protein destabilization, because amide
groups are generally more stable than ester groups in the intracel-
lular environment. Based on these findings, we designed and syn-
thesized SNIPER (6) as a selective degradation inducer of CRABP
(Fig. 3). We also prepared compound 8 as a negative control
(Fig. 3), based on compound 7,14 which does not bind to cIAP1
and does not induce auto-degradation of cIAP1 (Figs. 4a, c and 6).
3.2. Selectivity and time course of degradation of CRABP-II by
the synthesized compound
We initially evaluated the cIAP1/CRABP-II degradation selectiv-
ity of SNIPER (6) and compound 8. The CRABP-II degradation activ-
ity of 6 was similar to that of SNIPER (4) as evaluated by Western
blot analysis. As expected, 6 had no influence on cIAP1 levels even
tagged cIAP1 with even 1000
lM BE04 (5) had no influence on
at 30 lM (Fig. 7a and b). On the other hand, compound 8 did not
cIAP1 levels (Fig. 4a–c). Accordingly, it appears that BE04 (5) does
not induce auto-degradation of cIAP1. In addition, the results
shown in Figure 5 indicate that BE04 (5) has no influence on E3
activity, because ubiquitination of cIAP1 is mainly mediated by
the E3 activity of cIAP1 itself.13 Next, we investigated the binding
of BE04 (5) to cIAP1. The results of fluorescence polarization (FP)
assay7 indicated that BE04 (5) did bind to cIAP1 (Fig. 6) and
showed competitive binding with respect to DanBE (16),7 a FP
probe. Moreover, it was suggested that BE04 (5) and MeBS (2)
bound to cIAP1 in a mutually competitive manner by means of
Western blot analysis (Fig. 4b). Taken together, these results indi-
cate that BE04 (5) has the activity we expected. Therefore, it ap-
pears that substitution of the ester group of 4 to amide greatly
decreases cIAP1-degradation activity, but the binding affinity is
induce degradation of either cIAP1 or CRABP-II (Fig. 7a). Next we
examined whether 6 induced degradation of X-linked inhibitor of
apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein
2 (cIAP2), which (i) are members of the IAP family, (ii) have E3
activity, and (iii) induce auto-degradation.5,8 As shown in Figs.
4b, c, and 6 had no effect on the levels of XIAP and cIAP2, as was
the case for BE04 (5) (Fig. 7c and d). These results indicated that
amide-type SNIPER (6) had high specificity for degradation of
CRABP-II. In addition, the reduced CRABP-II level was maintained
for 12 h with 4, whereas it was maintained for 48 h with 6
(Fig. 7e). The replacement of the ester group with amide thus
greatly prolonged the duration of CRABP-II destabilization.
We next confirmed the mechanism of CRABP-II degradation by
6, using the previously reported methods,3 including pretreatment
Scheme 1. Reagents and conditions: (a) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole hydrate (HOBtꢀH2O), Et3N,
tetrahydrofuran (THF), rt, 100%; (b) LiOH, MeOH, H2O, rt, 93%; (c) di-tert-butyl dicarbonate, NaOH, H2O, acetone, rt, 61%; (d) methylamine hydrochloride, EDCI, HOBtꢀH2O,
iPr2NEt, dimethylformamide (DMF), CH2Cl2, rt, 88–99%; (e) HCl, 1,4-dioxane, rt, 99%.
Scheme 2. Reagents and conditions: (a) Et3N, CH2Cl2, rt, 68%; (b) N-Boc bestatin (13), EDCI, HOBtꢀH2O, iPr2NEt, CH2Cl2, rt, 24%; (c) HCl, 1,4-dioxane, CH2Cl2, rt, 100%.

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Scheme 3. Reagents and conditions: (a) p-toluenesulfonyl chloride, N,N-dimethylaminopyridine (DMAP), Et3N, CH2Cl2, rt; (b) NaN3, DMF, 60 °C, 98% (two steps); (c) Pd/C, H2,
EtOH, rt, quant.; (d) EDCI, HOBtꢀH2O, CH2Cl2, rt, 78–88%; (e) HCl, 1,4-dioxane, CH2Cl2, quant.; (f) EDCI, HOBtꢀH2O, THF, rt, 56–62%; (g) tetrabutylammonium fluoride (TBAF),
MeOH, THF, rt; (h) 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), n-C12H25SH, CH2Cl2, rt, 57% (two steps); (i) TBAF, MeOH, THF, rt, 70%.
Figure 4. (a) Western blot-detection of cIAP1 levels in HT1080 cells expressing FLAG-tagged cIAP1. The cells were treated with compounds for 6 h. (b) BE04 (5) or (c)
compound 7 was added to the culture 1 h prior to the addition of MeBS (2). BE04 (5) inhibited degradation of cIAP1 by MeBS (2) and compound 7 did not. These results
suggested that: (i) BE04 (5) and compound 7 do not induce degradation of cIAP1, (ii) BE04 (5) and MeBS (2) bind to cIAP1 in a mutually competitive manner in cells, but
compound 7 and MeBS (2) do not show competitive binding.

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Figure 5. Western blot-detection of cIAP1 levels in HT1080 cells expressing FLAG-tagged cIAP1 after (a) 15-min, (b) 30-min, (c) 60-min, and (d) 120-min treatment with
reagents. MG132 was added to the culture 30 min prior to the addition of MeBS (2) or BE04 (26). Lane 1, control; lane 2, 1000 M BE04 (26); lane 3, 30 M MeBS (2); lane 4,
10 M MG132; lane 5, mixture of 10 M MG132 and 1000 M BE04 (26). Proteasome inhibitor MG132 inhibited auto-degradation of cIAP1 and increased the accumulation of
l
l
l
l
l
cIAP1, but BE04 (5) had no influence on cIAP1 level. In addition, BE04 (5) had no influence on the accumulation of cIAP1 induced by MG132 (lanes 4 and 5). Given that
ubiquitination and degradation of cIAP1 are mediated by the ubiquitin ligase activity of cIAP1 itself,8 these results indicate that BE04 (5) did not inhibit the ubiquitin ligase
activity of cIAP1.
with an excess of MeBS (2) (Fig. 8a), GST pull-down assay (Fig. 8b),
combined use of proteasome inhibitors (Fig. 8c), combined use of
MeBS (2) or BE04 (5) and ATRA (3) (Fig. 9), RARa degradation assay
and RAR reporter gene assay (data not shown). In these mechanis-
tic analyses, all actions of 6 were similar to those of 4, except for
degradation activity towards cIAP1. Therefore, SNIPER (6) is sug-
gested to induce (i) formation of an artificial ternary complex with
cIAP1 and CRABP-II, (ii) ubiquitination of CRABP-II by cIAP1, and
(iii) degradation of ubiquitinated CRABP-II by proteasome.3
cIAP1, because it binds to cIAP1. Such inhibition would be unfavor-
able for investigation of the biological function of a target protein
by protein knockdown. Therefore, we investigated whether 6
inhibits the function of cIAP1. It is thought that inhibition of IAP
function activates caspase 3/7 and induces apoptosis.5,15 Indeed,
cIAP1 destabilization by MeBS (2) enhanced apoptosis of cancer
cells by chemotherapeutic drugs such as cisplastin and etoposide
(ETP).6 Therefore, we evaluated the effect on apoptosis in HT1080
cells of MeBS (2), ester-type SNIPER (4) and 6 in the presence or ab-
sence of ETP, using lactate dehydrogenase (LDH) assay (Fig. 10a). In
the absence of etoposide, MeBS (2), 4, and 6 hardly induced apop-
tosis. However, MeBS (2) and 4, which induce cIAP1 degradation,
enhanced apoptosis of HT1080 cells by ETP in a dose-dependent
fashion, while 6, which has no effect on cIAP1 levels, did not en-
hance apoptosis of HT1080 cells. Furthermore, we investigated
the effects of these compounds on caspase 3/7 activation. As
shown in Figure 10b, these compounds did not activate caspase
3/7 in the absence of ETP. In the presence of ETP, MeBS (2) and 4
activated caspase 3/7, whereas 6 had no effect on caspase 3/7. Col-
lectively, these results suggest that the binding of 6 to cIAP1 nei-
ther induces degradation of cIAP1 nor inhibits cIAP1 function. All
the results mentioned above indicated that amide-type SNIPER
(6) exhibits satisfactory selectivity and stability, overcoming the is-
sues found in the case of ester-type SNIPER.
3.3. Effect on cIAP1 inhibition at the level of function by the
synthesized compound
Although amide-type SNIPER (6) degraded CRABP-II without
affecting cIAP1 levels, 6 may inhibit the biological function of
3.4. Characterization of CRABP-II in neuroblastoma cells using
SNIPER
Finally, we attempted to demonstrate that amide-type SNIPER
is useful for investigation of the biological function of CRABP-II
by protein knockdown. It was reported that CRABP-II increases
expression of the oncogene MycN in neuroblastoma cells.16 In
addition, MycN inhibits caspase and the apoptotic pathway17 and
MycN knockdown inhibits proliferation of neuroblastoma cells.18
Therefore, we investigated the effects of amide-type SNIPER (6)
on MycN expression, caspase activation and proliferation of neuro-
blastoma cells. Consistent with the reported role of CRABP-II in
MycN expression, 6 decreased MycN levels (Fig. 11a) concomi-
tantly with a decrease of CRABP-II protein (Fig. 11b). Next, we
investigated the effects of MeBS (2), BE04 (5), and 6 on caspase
3/7 in IMR-32 cells (Fig. 11c). MeBS (2) and BE04 (5) hardly
activated caspase 3/7, whereas 6 significantly activated caspase
Figure 6. (a) Structure of DanBE (16), a fluorescence polarization (FP) probe. (b)
Binding assay. Competitive displacement studies were performed with MeBS (2),
BE04 (5), and compound 7 by means of FP assay. MeBS (2), BE04 (5) or compound 7
was incubated in the presence of DanBE (16) and GST-BIR-3 (GST-tagged BIR3
domain of cIAP1, to which MeBS (2) binds) for 5 min at room temperature in
phosphate-buffered saline (pH 7.4). After incubation, the fluorescence polarization
of DanBE (16) was measured.

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Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 3229–3241
Figure 7. (a) Western blot-detection of CRABP-II levels in HT1080 cells expressing FLAG-tagged cIAP1 after 6-h treatment with compounds 4, 6, and 8. (b) Western blot-
detection of CRABP-II and cIAP1 levels in HT1080 cells expressing FLAG-tagged cIAP1 after 6-h treatment with SNIPER (6). Compound 6 induced dose-dependent down-
regulation of CRABP-II, but not cIAP1. (c) Western blot-detection of XIAP levels in HT1080 cells expressing FLAG-tagged XIAP. The cells were treated with compounds for 6 h.
(d) Western blot-detection of cIAP2 levels in HT1080 cells expressing FLAG-tagged cIAP2. The cells were treated with compounds for 6 h. (e) Western blot-detection of cIAP1
levels and CRABP-II in HT1080 cells expressing FLAG-tagged cIAP1 treated with ester-type SNIPER (4) and amide-type SNIPER (6). With 4, the reduced CRABP-II level was
maintained for 12 h whereas with 6, it was maintained for 48 h.
Figure 8. (a) Influence of pretreatment of HT1080 cells expressing FLAG-tagged cIAP1 with MeBS (2) on induction of CRABP-II degradation. The cells were treated with 1
6 for 6 h. MeBS (2) (1000 M) was added to the culture 1 h prior to the addition of 6. Pretreatment with an excess amount of MeBS (2) led to the complete disappearance of
cIAP1 (lane 2). CRABP-II levels were down-regulated by treatment with amide-type SNIPER (6) [without pretreatment with MeBS (2)] as mentioned above (lane 3), whereas
amide-type SNIPER (6) scarcely decreased the CRABP-II levels in cells pretreated with 1000 M MeBS (2) (lane 4). These results indicate that the reduction of CRABP-II by
lM
l
l
amide-type SNIPER (6) is mediated by cIAP1. (b) Western blot-detection of GST-BIR3, GST, and CRABP-II levels of samples prepared by pull-down assay in vitro: lane 1,
CRABP-II levels; lane 2, mixture of GST-BIR3 and CRABP-II; lane 3, mixture of GST-BIR3, CRABP-II, and 4; lane 4, mixture of GST, CRABP-II, and 6; lane 5, mixture of GST-BIR3,
CRABP-II, and 6; lane 6, mixture of GST-BIR3, CRABP-II, and 8. GST-BIR3 pulled down CRABP-II in the presence of amide-type SNIPER (6) (lane 5) as well as ester-type SNIPER
(4) (lane 3), but not in the absence of amide-type SNIPER (6) (lane 2) or in the presence of compound 8 (lane 6). GST did not co-precipitate CRABP-II even in the presence of
amide-type SNIPER (6) (lane 4). This result indicates that CRABP-II is held in proximity to cIAP1 by amide-type SNIPER (6), but not by compound 8. (c) Influence of
pretreatment with proteasome inhibitors on induction of CRABP-II degradation. Western blot-detection of CRABP-II level in HT1080 cells expressing FLAG-tagged cIAP1. The
cells were treated with 1 lM 6 for 6 h. MG132 (10 lM) and lactacystin (10 lg/mL) were added to the culture 30 min prior to the addition of 6. The down-regulation of CRABP-
II by amide-type SNIPER (6) was cancelled by proteasome inhibitors, MG132 and lactacystin. This result suggested that the down-regulation of CRABP-II induced by amide-
type SNIPER (6) is mediated by proteasomal degradation.
3/7. Finally, we evaluated inhibition of cell proliferation by MeBS
(2), BE04 (5), and 6, using IMR-32 cells (Fig. 12). The results were
consistent with the results of caspase 3/7 assay. MeBS (2) and
BE04 (5) did not show strong inhibition of cell proliferation,
whereas 6 showed strong and dose-dependent inhibition. On the
MCF-7 cells, which express CRABP-II and cIAP1, but not MycN,
though it decreased CRABP-II in these cells (Fig. 13). These results
suggested that the cell proliferation inhibition by amide-type
SNIPER (6) is specific for neuroblastoma cells expressing MycN.
With regard to caspase activation and cell proliferation, ester-
type SNIPER (4), as well as the combination of amide-type SNIPER
other hand,
6 hardly inhibited proliferation of HT1080 and

Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 3229–3241
3235
4. Conclusion
In conclusion, the previously reported ester-type SNIPER in-
duces degradation of not only its target protein, but also cIAP1,
and therefore in the present work we designed amide-type SNIPER
to improve the target protein/cIAP1 degradation selectivity. We
synthesized the designed amide-type SNIPER, and confirmed that
it does indeed shows improved selectivity. This is an interesting
example illustrating that modification of just one atom of a bioac-
tive molecule can cause a major change of activity. We also con-
firmed that amide-type SNIPER is an effective chemical tool to
study the involvement of CRABP-II in MycN-mediated caspase acti-
vation in living neuroblastoma cells, finding that: (i) degradation of
CRABP-II induces caspase activation and inhibits proliferation of
neuroblastoma cells, (ii) degradation of both CRABP-II and cIAP1
is more effective than down-regulation of CRABP-II alone to inhibit
the cell proliferation. Amide-type SNIPER should be adaptable to
various other target proteins by using appropriate specific ligands,
and we are currently examining protein knockdown of other target
proteins with amide-type SNIPERs. We believe that highly selective
target protein knockdown with SNIPERs will provide new insights
into the chemical biology of various proteins and open up new
strategies for drug therapy.
Figure 9. (a) Influence of combination of MeBS (2) and ATRA (3). Western blot-
detection of CRABP-II and cIAP1 levels in HT1080 cells expressing FLAG-tagged
cIAP1. The cells were treated with 1 lM MeBS (2), 1 lM ATRA (3), or 1 lM 6 for 6 h.
(b) Influence of combination of BE04 (5) and ATRA (3). Western blot-detection of
CRABP-II and cIAP1 levels in HT1080 cells expressing FLAG-tagged cIAP1. The cells
were treated with 1 lM BE04 (2), 1 lM ATRA (3), or 1 lM 6 for 6 h. SNIPER (6)
5. Experimental section
5.1. Chemistry
induced a decrease of CRABP-II, whereas the mixture of MeBS (2) or BE04 (5) and
ATRA (3) did not. The treatments with MeBS (2) or BE04 (5), and the combination of
MeBS (2) or BE04 (5) and ATRA (3) did not affect the CRABP-II level in HT1080. ATRA
(3) did not cause any decrease of cIAP1 or CRABP-II. Thus, single treatment of
HT1080 cells expressing FLAG-tagged cIAP1 with BE04 (5) and ATRA (3), which are
partial structures of SNIPER (6), or their combination, did not induce degradation of
CRABP-II.
Proton nuclear magnetic resonance spectra (1H NMR) and car-
bon nuclear magnetic resonance spectra (13C NMR) were recorded
on a JEOL JNMGX500 (500 MHz) spectrometer in the indicated sol-
vent. Chemical shifts (d) are reported in parts per million relative to
the internal standard tetramethylsilane. High-resolution mass
spectra (HRMS) and fast atom bombardment (FAB) mass spectra
were recorded on a JEOL JMA-HX110 mass spectrometer. Bestatin
(1) and methyl bestatin (MeBS, 2) were provided by Nippon Kaya-
ku Co., Ltd (Tokyo, Japan). The other chemical reagents and sol-
vents were purchased from Aldrich, Merck, Tokyo Kasei Kogyo,
Wako Pure Chemical Industries, and Kanto Kagaku and used with-
out purification. Flash column chromatography was performed
(6) and MeBS (2), showed a stronger effect than amide-type SNIPER
(6) alone (Figs. 11c and 14), even though 4 and 6 equally decreased
CRABP-II and MycN (Fig. 11a and b). These results suggested that
down-regulation of both cIAP1 and CRABP-II is more effective than
down-regulation of CRABP-II alone to inhibit cell growth and
induce apoptosis of neuroblastoma cells. This discovery was made
possible by the development of CRABP-II-specific amide-type
SNIPER (6).
Figure 10. (a) LDH assay. HT1080 cells were treated with compounds in the presence or absence of 30
lg/mL etoposide (ETP) for 24 h. The data are the means of triplicate
determinations. (b) Caspase activity assay. HT1080 cells were treated with compounds in the presence or absence of 30
l
g/mL etoposide (ETP) for 24 h. The data are the
means of triplicate determinations. ⁄⁄P <0.01; ANOVA and Bonferroni-type multiple t test results indicated significant differences between ETP control and the combination of
ETP with MeBS (2), and between ETP control and the combination of ETP with 4.

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Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 3229–3241
(Boc)2O (1.40 g, 6.41 mmol) with cooling in an ice-bath. The result-
ing mixture was stirred at room temperature for 24 h, then concen-
trated in vacuo, and the residue was dissolved in AcOEt. The
organic solution was washed with 10% aqueous citric and brine,
and dried over MgSO4. Filtration, evaporation of the solvent in va-
cuo and purification of the residue by flash column chromatogra-
phy (AcOEt/n-hexane = 1:2 to AcOEt only) gave 770 mg (61%) of
13 as a colorless solid; 1H NMR (DMSO-d6, 500 MHz, d; ppm);
7.74 (1H, d, J = 8.5 Hz), 7.27–7.16 (5H, m), 6.12 (1H, d, J = 9.3 Hz),
6.02 (1H, m), 4.28 (1H, m), 3.90 (1H, m), 3.82 (1H, m), 2.78 (1H,
dd, J = 13.1, 7.0 Hz), 2.63 (1H, dd, J = 13.1, 7.9 Hz), 1.64–1.59 (2H,
m), 1.47 (1H, m), 1.27 (9H, s), 0.86 (3H, d, J = 6.0 Hz), 0.81 (3H, d,
J = 6.1 Hz); MS (FAB) m/z: 431 (MNa+), 409 (MH+).
5.1.3. (R)-2-Isobutyl-4-oxo-7-phenylheptanoic acid (14)
LiOHꢀH2O (823 mg, 19.6 mmol) was added to a solution of 12
(2.48 g, 8.51 mmol) in MeOH/H2O (60.0 mL/30.0 mL) and the
resulting mixture was stirred at room temperature for 15 h. Then
the reaction mixture was acidified with 2 N HCl and concentrated
in vacuo. The residue was extracted with AcOEt. The organic solu-
tion was washed with brine, and dried over Mg2SO4. Filtration and
evaporation of the solvent in vacuo gave a crude solid, which was
suspended in AcOEt. Insoluble material was collected by filtration
to give 2.20 g (93%) of 14 as a colorless solid; 1H NMR (DMSO-d6,
500 MHz, d; ppm): 12.43 (1H, br), 8.04 (1H, d, J = 7.9 Hz), 7.26
(2H, t, J = 7.9 Hz), 7.16 (3H, m), 4.21 (1H, m), 2.54 (2H, t,
J = 7.3 Hz), 2.12 (2H, d, J = 7.3 Hz), 1.75 (2H, quin, J = 7.3 Hz), 1.63
(1H, m), 1.49 (2H, m), 0.87 (3H, d, J = 6.7 Hz), 0.83 (3H, d,
J = 6.7 Hz); MS (FAB) m/z: 278 (MH+).
5.1.4. tert-Butyl (2R,3S)-3-hydroxy-4-[(S)-4-methyl-1-
(methylamino)-1-oxopentan-2-ylamino]-4-oxo-1-
phenylbutan-2-ylcarbamate (15)
Figure 11. (a) Western blotting detection of MycN in IMR-32 cells. Western blot-
detection of MycN levels in IMR-32 cells after 48-h treatment with MeBS (2), 4, and
6. (b) Western blot-detection of cIAP1 and CRABP-II levels in IMR-32 cells after 24-h
treatment with MeBS (2), SNIPER (4), and SNIPER (6). (c) Caspase activity assay.
IMR-32 cells were treated with compounds for 24 h. The data are the means of
triplicate determinations. ⁄⁄P <0.01; ANOVA and Bonferroni-type multiple t test
EDCI (72.0 mg, 0.376 mmol) was added to a solution of 14
(104 mg, 0.255 mmol), methylamine hydrochloride (75.0 mg,
1.11 mmol), HOBtꢀH2O (78.0 mg, 0.509 mmol) and iPr2NEt
(220 lL, 1.26 mmol) in DMF/CH2Cl2 (4.00 mL/3.00 mL) with cool-
results indicated significant differences from the control for 4,
combination of MeBS (2) with 6.
6 and the
ing in an ice-bath. The resulting mixture was stirred at room tem-
perature for 22 h, then quenched with water, and extracted with
AcOEt. The organic layer was washed with brine, and dried over
MgSO4 Filtration, evaporation of the solvent in vacuo and purifica-
tion of the residue by flash column chromatography (AcOEt only)
gave 94.0 mg (88%) of 15 as a colorless solid; 1H NMR (CDCl3,
500 MHz, d; ppm): 7.29 (2H, t, J = 6.7 Hz), 7.23 (3H, m), 6.97 (1H,
d, J = 8.5 Hz), 6.36 (1H, br), 5.44 (1H, d, J = 4.9 Hz), 4.98 (1H, d,
J = 7.9 Hz), 4.44 (1H, m), 4.11 (2H, dd, J = 7.3, 3.0 Hz), 4.00 (1H,
m), 3.11 (1H, m), 3.04 (1H, m), 2.78 (1.5H, s), 2.77 (1.5H, s), 1.65
(3H, m), 1.39 (9H, s), 0.93 (3H, d, J = 6.0 Hz), 0.89 (3H, d,
J = 6.1 Hz); MS (FAB) m/z: 444 (MNa+), 422 (MH), 322 (MH+ꢁBoc).
using Silica Gel 60 (particle size 0.060–0.210 mm) supplied by Kan-
to Kagaku.
5.1.1. (S)-Methyl 4-methyl-2-(4-phenylbutanamido)pentanoate
(12)
EDCI (2.61 g, 13.6 mmol) was added to a solution of leucine
methyl ester hydrochloride (10) (2.45 g, 13.5 mmol), 4-phenylbu-
tanoic acid (11) (1.50 g, 9.14 mmol), HOBtꢀH2O (2.86 g, 18.7 mmol)
and Et3N (5.60 mL, 40.4 mmol) in THF (150 mL) with cooling in an
ice-bath. The resulting mixture was stirred at room temperature
for 5 h, then concentrated, and the residue was extracted with
AcOEt. The organic solution was washed with 10% aqueous citric
acid, saturated NaHCO3 and brine, and dried over Mg2SO4. Filtra-
tion and evaporation of the solvent in vacuo gave 2.65 g (100%)
of 12 as a colorless oil; 1H NMR (CDCl3, 500 MHz, d; ppm): 7.30–
7.16 (5H, m), 5.74 (1H, br d, J = 7.3 Hz), 4.65 (1H, m), 3.73 (3H, s),
2.66 (2H, t, J = 7.3 Hz), 2.21 (2H, t, J = 7.3 Hz), 1.98 (2H, quin,
J = 7.3 Hz), 1.54 (3H, m), 0.94 (3H, d, J = 5.7 Hz), 0.93 (3H, d,
J = 5.7 Hz); MS (FAB) m/z: 292 (MH+).
5.1.5. (S)-2-[(2S,3R)-3-Amino-2-hydroxy-4-phenylbutanamido]-
N,4-dimethylpentanamide hydrochloride (BE04, 5)
4 N HCl in 1,4-dioxane (500 lL, 2.00 mmol) was added to 15
(63.2 mg, 0.150 mmol) in CH2Cl2 (1.00 mL) with cooling in an
ice-bath and the resulting mixture was stirred at room tempera-
ture for 2 h, then concentrated in vacuo to give 53.2 mg (99%) of
5 as a colorless solid; 1H NMR (DMSO-d6, 500 MHz, d; ppm):
8.04–7.97 (5H, m), 7.34–7.23 (5H, m), 6.66 (1H, d, J = 6.1 Hz),
4.22 (1H, m), 3.99 (1H, m), 3.53 (1H, br), 2.95–2.82 (2H, m), 2.54
(1.5H, s), 2.53 (1.5H, s), 1.52–1.42 (3H, m), 0.87 (3H, d,
J = 6.1 Hz), 0.84 (3H, d, J = 6.7 Hz); 13C NMR (CDCl3, 125 MHz, d;
ppm): 171.97, 170.64, 136.27, 129.44, 128.65, 126.94, 68.18,
54.20, 51.24, 40.92, 34.70, 25.55, 24.24, 22.85, 21.91; MS (FAB)
m/z: 322 (MH+); HRMS (FAB) calcd for C17H28N3O3þ, 322.2131;
found 322.2115.
5.1.2. Preparation of (S)-2-[(2S,3R)-3-(tert-butoxycarbonyl)-2-
hydroxy-4-phenylbutanamido]-4-methylpentanoic acid (N-Boc
bestatin, 13)
To a suspension of bestatin (1) (950 mg, 3.08 mmol) in acetone
(140 mL) was added 2 N aqueous NaOH (3.10 mL, 6.20 mmol) and

Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 3229–3241
3237
Figure 12. Viability of IMR-32 cells treated with (a) 4, (b) 6, (c) MeBS (2), and (d) BE04 (5).
5.1.6. (S)-N,4-Dimethyl-2-(4-phenylbutanamido)pentanamide (7)
Compound 7 (yield; 106 mg, 99%) was prepared from 14
(102 mg, 0.368 mmol), methylamine hydrochloride (134 mg,
1.98 mmol) using the procedure described for 15; yellow solid;
1H NMR (CDCl3, 500 MHz, d; ppm): 7.28 (2H, t, J = 7.3 Hz), 7.21
(1H, d, J = 7.3 Hz), 7.17 (2H, d, J = 7.3 Hz), 6.10 (1H, br), 5.83 (1H,
d, J = 8.5 Hz), 4.41 (1H, m), 2.80 (1.5H, s), 2.79 (1.5H, s), 2.63 (2H,
t, J = 7.6 Hz), 2.20 (2H, t, J = 7.3 Hz), 1.95 (2H, quin, J = 7.6 Hz),
1.66–1.49 (3H, m), 0.93 (3H, d, J = 5.4 Hz), 0.92 (3H, d, J = 6.0 Hz);
13C NMR (CDCl3, 125 MHz, d; ppm): 173.02, 172.75, 141.49,
128.68, 128.63, 126.29, 51.64, 41.24, 35.90, 35.32, 27.19, 26.49,
24.96, 23.04, 22.43; MS (FAB) m/z: 291 (MH+); HRMS (FAB) calcd
for C17H27N2O2þ, 291.2073; found 291.2076.
0.367 mmol) using the procedure described for 15; fluorescent so-
lid; 1H NMR (CDCl3, 500 MHz, d; ppm): 8.55 (1H, d, J = 8.5 Hz), 8.28
(1H, d, J = 8.5 Hz), 8.22 (1H, dd, J = 7.0, 1.2 Hz), 7.53 (1H, t,
J = 7.8 Hz), 7.50 (1H, t, J = 7.3 Hz), 7.35–7.15 (6H, m), 5.83 (1H,
m), 5.45 (1H, m), 5.17 (1H, br d, J = 6.7 Hz), 4.40 (1H, m), 4.20–
4.12 (2H, m), 4.11–4.05 (2H, m), 3.25–3.00 (4H, m), 2.89 (6H, s),
1.63–1.52 (3H, m), 1.37 (9H, s), 0.91 (3H, d, J = 6.1 Hz), 0.89 (3H,
d, J = 6.1 Hz); MS (FAB) m/z: 684 (M+), 585 (MH+ꢁBoc).
5.1.9. (S)-2-[1-(Dimethylamino)naphthalene-5-
sulfonamido]ethyl 2-[(2S,3R)-3-amino-2-hydroxy-4-
phenylbutanamido]-4-methylpentanoate dihydrochloride
(DanBEꢀ2HCl, 16ꢀ2HCl)
Compound 9ꢀ2HCl (yield; 42.6 mg, 100%) was prepared from 20
(44.6 mg, 0.0651 mmol) using the procedure described for 5; fluo-
rescent powder; 1H NMR (DMSO-d6, 500 MHz, d; ppm): 8.66 (1H,
br d, J = 8.5 Hz), 8.43 (1H, br d, J = 7.9 Hz), 8.31 (2H, m), 8.13 (1H,
d, J = 6.7 Hz), 8.04 (3H, br), 7.68 (1H, t, J = 7.9 Hz), 7.66 (1H, t,
J = 7.3 Hz), 7.54 (1H, br), 7.30–7.20 (5H, m), 4.22 (1H, m), 4.00
(1H, d, J = 3.6 Hz), 3.93 (2H, m), 3.49 (1H, m), 3.06–2.82 (10H, m),
1.65–1.40 (3H, m), 0.86 (3H, d, J = 6.1 Hz), 0.82 (3H, d, J = 6.1 Hz);
13C NMR (DMSO-d6, 125 MHz, d; ppm) 171.89, 171.12, 136.36,
136.21, 129.42, 128.88, 128.84, 128.58, 128.32, 128.09, 127.78,
126.86, 124.39, 68.26, 66.34, 63.34, 54.27, 50.39, 45.49, 41.12,
34.50, 24.16, 22.65, 21.54; MS (FAB) m/z: 585 (MH+); HRMS
(FAB) calcd for C30H41N4O6S+, 585.2747; found 585.2727.
5.1.7. 5-(Dimethylamino)-N-(2-hydroxyethyl)naphthalene-1-
sulfonamide (19)
2-Aminoethanol (18) (250
tion of 5-(dimethylamino)naphthalene-1-sulfonyl chloride (17)
(216 mg, 0.801 mmol) and Et3N (600 L, 4.33 mmol) in CH2Cl2,
lL, 4.14 mmol) was added to a solu-
l
(20.0 mL) with cooling by an ice-bath and the resulting mixture
was stirred at room temperature for 2 h, then poured into water.
The organic layer was washed with brine and dried over Mg2SO4.
Filtration, evaporation of the solvent in vacuo and purification of
the residue by flash column chromatography (CH2Cl2/
MeOH = 10:1) gave 160 mg (68%) of 19 as a fluorescent powder;
1H NMR (CDCl3, 500 MHz, d; ppm): 8.55 (1H, d, J = 8.5 Hz), 8.28
(1H, d, J = 8.5 Hz), 8.26 (1H, d, J = 7.3 Hz), 7.58 (1H, t, J = 8.2 Hz),
7.53 (1H, t, J = 7.3 Hz), 7.20 (1H, d, J = 7.3 Hz), 5.20 (1H, br d,
J = 7.3 Hz), 3.60 (2H, q, J = 4.8 Hz), 3.04 (2H, q, J = 5.9 Hz), 2.89
(6H, s), 1.89 (1H, br); MS (FAB) m/z: 294 (M+).
5.1.10. tert-Butyl 2-[2-(2-azidoethoxy)ethoxy]ethylcarbamate
(22)
A mixture of p-toluenesulfonyl chloride (583 mg, 3.06 mmol) in
CH2Cl2 (20.0 mL) was added to
1.52 mmol), Et3N (500 L, 3.61 mmol) and DMAP (37.0 mg,
a
solution of 211 (380 mg,
5.1.8. (S)-2-[1-(Dimethylamino)naphthalene-5-
l
sulfonamido]ethyl 2-[(2S,3R)-3-(tert-butoxycarbonyl)-2-
hydroxy-4-phenylbutanamido]-4-methylpentanoate (20)
Compound 20 (yield; 83.2 mg, 24%) was prepared from N-Boc
bestatin (13) (202 mg, 0.495 mmol) and alcohol 19 (108 mg,
0.303 mmol) CH2Cl2 (2.00 mL) in a dropwise fashion over a period
of 1 h. The resulting mixture was stirred at room temperature for
1 h, then poured into water. The organic layer was washed with
brine, and dried over MgSO4. Filtration, evaporation of the solvent

3238
Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 3229–3241
Figure 13. (a) Western blot-detection of cIAP1 and MycN levels in HT1080 cells, MCF-7 cells and IMR-32 cells. (b) Western blot-detection of CRABP-II levels in HT1080 cells after
6-h treatment with MeBS (2), ester-type SNIPER (4) and amide-type SNIPER (6). (c) Western blot-detection of CRABP-II levels in MCF-7 cells after 24-h treatment with MeBS (2),
ester-type SNIPER (4), and amide-type SNIPER (6). (d–i) Viability of HT1080 and MCF-7 cells treated with MeBS (2), ester-type SNIPER (4) and amide-type SNIPER (6).
in vacuo and purification of the residue by flash column chroma-
tography (AcOEt/n-hexane = 1:1 AcOEt only) gave 515 mg (84%)
of 22 as a colorless oil. A suspension of 22 (500 mg, 1.24 mmol)
and NaN3 (180 mg, 2.77 mmol) in DMF (5.00 mL) was stirred at
60 °C for 8 h. Then, the reaction mixture was poured into water
and extracted with AcOEt. The organic layer was washed with
brine, and dried over MgSO4. Filtration and evaporation of the sol-
vent in vacuo gave 335 mg (98%) of 23 as a colorless oil; 1H NMR
(CDCl3, 500 MHz, d; ppm): 5.01 (1H, br), 3.69 (6H, m), 3.56 (2H, t,
J = 4.9 Hz), 3.41 (2H, t, J = 7.5 Hz), 3.32 (2H, m); 1.44 (9H, s); MS
(FAB) m/z: 297 (MNa+), 275 (MH+), 175 (MH+ꢁBoc).
temperature under H2 for 13 h, the reaction mixture was filtered
though Celite. The solvent was removed by concentration in vacuo
to give crude 24 (191 mg) as a colorless oil, which was used in the
next reaction without further purification; MS (FAB) m/z: 249
(MH+).
5.1.12. (S)-2-{2-[2-(tert-Butoxycarbonyl)ethoxy]ethoxy}ethyl 2-
((2S,3R)-3-{[(9H-fluoren-9-yl)methoxy]carbonyl}-2-hydroxy-4-
phenyl-butanamido)-4-methylpentanamide (26)
Compound 26 (56.0 mg, 78%; from 25) was prepared from crude
amine (24) (140 mg) and 252 (50.1 mg, 0.0944 mmol) using the
procedure described for 15; colorless oil; 1H NMR (CDCl3,
500 MHz, d; ppm): 7.76 (2H, d, J = 7.3 Hz), 7.51 (2H, m), 7.38 (2H,
t, J = 7.6 Hz), 7.31–7.14 (7H, m), 6.70 (1H, br), 5.80 (1H, br d,
J = 6.1 Hz), 5.48 (1H, m), 5.43 (1H, m), 5.12 (1H, m), 4.48 (1H, m),
4.43–4.20 (5H, m), 3.54–3.40 (10H, m), 3.28 (2H, br q, J = 4.3 Hz),
5.1.11. tert-Butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate
(24)
10% Pd/C (20.2 mg) was added to a solution of 23 (211 mg,
0.769 mmol) in EtOH (5.00 mL). After having been stirred at room

Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 3229–3241
3239
J = 7.3 Hz), 7.34–7.22 (7H, m), 7.00 (1H, dd, J = 15.2, 11.0 Hz), 6.80
(1H, br), 6.66 (1H, m), 6.32–6.16 (4H, m), 5.78 (1H, s), 5.48 (1H,
d, J = 8.5 Hz), 5.36 (1H, d, J = 5.5 Hz), 4.55 (2H, s), 4.36 (1H, m),
4.34 (7H, m), 3.60 (10H, m), 3.37 (2H, m), 2.98 (2H, m), 2.70 (2H,
t, J = 6.1 Hz), 2.63 (2H, t, J = 6.4 Hz), 2.33 (3H, s), 2.01 (3H, s), 1.83
(3H, s), 1.67 (1H, m),1.58 (4H, m), 1.06 (6H, s), 0.84 (3H, d,
J = 6.0 Hz), 0.81 (3H, d, J = 6.1 Hz); MS (FAB) m/z: 1083 (M+).
5.1.17. (2E,4E,6E,8E)-2-Cyanoethyl 3,7-dimethyl-9-((E)-2,6,6-
trimethyl-3-{2-[2-(2-{2-[(S)-4-methyl-2-(4-
phenylbutanamido)pentanamido]ethoxy}ethoxy)ethylamino]-
2-oxoethoxyimino}cyclohex-1-enyl)nona-2,4,6,8-tetraenoate
(32)
Compound 32 (yield; 23.2 mg, 62% from 28) was prepared from
28 (20.0 mg, 0.454 mmol) and crude 30ꢀHCl (45.3 mg) using the
procedure described for 12; yellow solid; 1H NMR (CDCl3,
500 MHz, d; ppm): 7.27 (2H, t, J = 7.3 Hz), 7.19 (1H, t, J = 7.3 Hz),
7.16 (2H, d, J = 7.3 Hz), 7.03 (1H, dd, J = 14.6, 11.6 Hz), 6.68 (1H,
br), 6.57 (1H, br), 6.36–6.20 (4H, m), 5.82 (1H, s), 4.58 (2H, s),
4.45 (1H, m), 4.33 (2H, t, J = 6.1 Hz), 3.59–3.40 (12H, m), 2.73
(2H, t, J = 6.1 Hz), 2.66 (4H, m), 2.37 (3H, s), 2.21 (2H, t,
J = 7.3 Hz), 2.03 (3H, s), 1.95 (2H, quin, J = 7.9 Hz), 1.86 (3H, S),
1.68–1.54 (5H, m), 1.09 (6H, s), 0.93 (3H, d, J = 2.4 Hz), 0.92 (3H,
d, J = 2.4 Hz); MS (FAB) m/z: 830 (MH+).
Figure 14. Relative inhibition of proliferation of IMR-32 cells by treatment with
MeBS (2), SNIPER (4), SNIPER (6), and the combination of MeBS (2) and SNIPER (6).
The graph was drawn based on calculated IMR-32 cells viability. SNIPER (4) or the
combination of SNIPER (6) and MeBS (2) inhibited proliferation of IMR-32 cells
more strongly than did SNIPER (6). The activity of the combination of SNIPER (6)
and MeBS (2) was similar to that of SNIPER (4).
3.05 (2H, m), 1.62 (3H, m), 1.44 (9H, s), 0.93 (3H, d, J = 6.0 Hz), 0.89
(3H, d, J = 6.1 Hz); MS (FAB) m/z: 761 (MH+), 661 (MH+ꢁBoc).
5.1.18. (2E,4E,6E,8E)-9-[(E)-3-(2-{2-[2-(2-{(S)-2-[(2S,3R)-3-
Amino-2-hydroxy-4-phenylbutanamido]-4-
5.1.13. (S)-tert-Butyl 2-(2-{2-[4-methyl-2-(4-
phenylbutanamido)pentanamido]ethoxy}ethoxy)ethyl–
carbamate (27)
methylpentanamido}ethoxy)ethoxy]ethylamino}-2-
oxoethoxyimino)-2,6,6-trimethylcyclohex-1-enyl]-3,7-
dimethylnona-2,4,6,8-tetraenoic acid (amide-type SNIPER, 6)2–4
Compound 27 (yield; 47.5 mg, 88%) was prepared from 14
(29.5 mg, 0.106 mmol) and crude amine (24) (50.0 mg) using the
procedure described for 15; colorless oil; 1H NMR (CDCl3,
500 MHz, d; ppm): 7.27 (2H, t, J = 7.3 Hz), 7.18 (1H, t, J = 7.3 Hz),
7.17 (2H, d, J = 6.7 Hz), 6.65 (1H, m), 6.02 (1H, m), 5.20 (1H, m),
4.47 (1H, m), 3.76–3.44 (10H, m), 3.32 (2H, m), 2.63 (2H, t,
J = 7.6 Hz), 2.21 (2H, t, J = 7.3 Hz), 1.95 (2H, quin, J = 7.6 Hz), 1.62
(2H, m), 1.59 (1H, m), 1.43 (9H, s), 0.93 (3H, d, J = 6.1 Hz); MS
(FAB) m/z: 508 (MH+), 408 (MH+ꢁBoc).
1 N TBAF in THF (370
lL, 0.370 mmol) was added to a solution
of 31 (40.3 mg, 0.0372 mmol) and MeOH (30.0
lL, 0.741 mmol).
After having been stirred at room temperature for 1 h, the mixture
was purified by flash column chromatography (CHCl3/
MeOH = 20:1) to give 33 mg of crude acid as a yellow oil. DBU
(17.0
lL, 0.114 mmol) was added to a solution of the crude acid
and dodecyl mercaptan (13.0
lL, 0.0552 mmol) in CH2Cl2
(1.00 mL). After having been stirred at room temperature for 1 h,
the mixture was purified by flash column chromatography
5.1.14. (9H-Fluoren-9-yl)methyl (2R,3S)-4-((S)-1-{2-[2-(2-
aminoethoxy)ethoxy]ethylamino}-4-methyl-1-oxopentan-2-
ylamino)-3-hydroxy-4-oxo-1-phenylbutan-2-ylcarbamate
hydrochloride (29ꢀHCl)
(CHCl3/MeOH = 9:1)
and
PTLC
(CHCl3/MeOH/NH3
aque-
ous = 3:1:0.1) to give 17.2 mg (57%; two steps) of 6 as a yellow
oil; 1H NMR (CDCl3, 500 MHz, d; ppm): 7.87 (1H, d, J = 6.7 Hz),
7.30–7.24 (5H, m), 7.01 (1H, m), 6.97 (1H, dd, J = 15.2, 12.1 Hz),
6.73 (1H, s), 6.38–6.18 (4H, m), 5.82 (1H, s), 4.58 (2H, s), 4.44
(1H, m), 4.07 (1H, m), 3.72–3.50 (13H, m), 3.33 (2H, m), 3.02
(2H, m), 2.66 (2H, t, J = 6.7 Hz), 2.33 (3H, s), 2.02 (3H, s), 1.86
(3H, s), 1.67 (5H, m), 1.09 (6H, s), 0.93 (3H, d, J = 6.1 Hz), 0.92
(3H, d, J = 6.0 Hz); 13C NMR (CDCl3, 125 MHz, d; ppm): 173.34,
172.02, 170.67, 158.88, 150.34, 139.35, 138.38, 138.06, 136.72,
131.64, 129.34, 128.75, 126.74, 126.61, 124.79, 73.03, 70.32,
70.21, 69.92, 69.76, 54.64, 51.84, 40.63, 39.31, 38.80, 35.97,
4 N HCl in 1,4-dioxane (250 lL, 1 mmol) was added to 26
(56.1 mg, 0.0736 mmol) with cooling in an ice-bath and the result-
ing mixture was stirred at room temperature for 1 h. The reaction
mixture was concentrated in vacuo to give crude 29ꢀHCl (51.2 mg),
which was used in the next step without further purification; MS
(FAB) m/z: 661 (MH+ꢁHCl).
5.1.15. (S)-N-{2-[2-(2-Aminoethoxy)ethoxy]ethyl}-4-methyl-2-
(4-phenylbutanamido)pentanamide hydrochloride (30ꢀHCl)
Crude 30ꢀHCl was prepared from 27 (47.5 mg, 0.0936 mmol)
using the procedure described for 29ꢀHCl. Crude 30ꢀHCl (45.3 mg)
was used in next step without further purification.
34.88, 27.62, 24.88, 23.02, 21.73, 20.23, 14.91, 13.86, 12.82; MS
(FAB) m/z: 808 (MH+); HRMS (FAB) calcd for C44H66N5O9
,
þ
808.4861; found 808.4825.
5.1.19. (2E,4E,6E,8E)-3,7-Dimethyl-9-((E)-2,6,6-trimethyl-3-{2-
[2-(2-{2-[(S)-4-methyl-2-(4-
phenylbutanamido)pentanamido]ethoxy}ethoxy)ethylamino]-
2-oxoethoxyimino}cyclohex-1-enyl)nona-2,4,6,8-tetraenoic
acid (8)
5.1.16. (2E,4E,6E,8E)-2-Cyanoethyl 9-((E)-3-{2-[2-(2-{2-[(S)-2-
((2S,3R)-3-{[(9H-fluoren-9-yl)methoxy]carbonyl}-2-hydroxy-4-
phenyl butanamido)-4-
methylpentanamido]ethoxy}ethoxy)ethylamino]-2-
oxoethoxyimino}-2,6,6-trimethylcyclohex-1-enyl)-3,7-
dimethylnona-2,4,6,8-tetraenoate (31)
1 N TBAF in THF (280
of 32 (23.2 mg, 0.0279 mmol) and MeOH (22.0
THF (560 L). The mixture was stirred at room temperature for 2 h,
lL, 0.280 mmol) was added to a solution
l
L, 0.543 mmol) in
Compound 31 (40.3 mg, 56% from 28) was prepared from 282
(29.2 mg, 0.0662 mmol) and crude 29ꢀHCl (51.2 mg) using the pro-
cedure described for 12; yellow oil; 1H NMR (CDCl3, 500 MHz, d;
ppm): 7.72 (2H, d, J = 7.3 Hz), 7.46 (2H, d, J = 7.3 Hz), 7.35 (2H, t,
l
then purified by flash column chromatography (CHCl3 only to
CHCl3/MeOH = 20:1) and PTLC (CHCl3/MeOH = 20:1) to give
15.1 mg (70%) of 8 as a yellow oil; 1H NMR (CDCl3, 500 MHz, d;

3240
Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 3229–3241
ppm): 7.25 (2H, t, J = 7.3 Hz), 7.17 (1H, t, J = 7.3 Hz), 7.14 (2H, d,
J = 6.7 Hz), 7.01 (1H, dd, J = 15.2, 11.6 Hz), 6.68 (1H, br), 6.61 (1H,
br), 6.35–6.17 (4H, m), 5.82 (1H, s), 4.56 (2H, s), 4.45 (1H, m),
3.89–3.32 (12H, m), 2.65–2.58 (4H, m), 2.34 (3H, s), 2.20 (2H, t,
J = 6.7 Hz), 2.00 (3H, s), 1.93 (2H, quin, J = 7.3 Hz), 1.84 (3H, S),
1.59–1.47 (5H, m), 1.07 (6H, s), 0.93 (3H, d, J = 6.1 Hz), 0.92 (3H,
d, J = 6.1 Hz); 13C NMR (CDCl3, 125 MHz, d; ppm): 172.84, 172.43,
170.47, 169.97, 158.76, 154.31, 150.15, 141.40, 139.21, 138.90,
136.25, 131.44, 131.06, 128.47, 128.39, 126.89, 125.98, 124.92,
118.32, 73.08, 70.40, 70.21, 69.91, 69.69, 51.55, 41.51, 39.29,
38.76, 35.99, 35.18, 34.87, 27.62, 27.11, 24.80, 22.88, 22.15,
20.22, 14.87, 13.95, 12.85; MS (FAB) m/z: 777 (MH+); HRMS
(FAB) calcd for C44H65N4O8þ, 777.4802; found 777.4843.
fluorescence spectrometer (Jasco FP-6500; excitation at 335 nm,
emission at 550 nm). In competitive displacement studies, MeBS
(2), BE04 (5) or compound 7 was incubated with GST-BIR3 for
5 min before the addition of DanBE.
5.2.5. Pull down assay
Pull down assays were carried out according to the method re-
ported in Ref. 3.
5.2.6. Reporter gene assay
Reporter gene assays were carried out according to the method
reported in Ref. 3.
5.2.7. Cell death assay
5.2. Biology
Etoposide was purchased from Sigma–Aldrich Corporation.
CytoTox 96Ò Non-Radioactive Cytotoxicity Assay (Promega) was
used for cell death assay. HT1080 cells were plated in 96-well
5.2.1. Cell culture conditions
Human fibrosarcoma HT1080 cells were cultured in RPMI 1640
containing 10% heat-inactivated fetal bovine serum (FBS), penicil-
lin, and streptomycin mixture at 37 °C in a humidified atmosphere
of 5% CO2 in air. Human neuroblastoma IMR-32 cells were cultured
in Eagle’s minimal essential medium with non-essential amino
acids and 10% FBS at 37 °C in a humidified atmosphere of 5% CO2
in air. Human embryonic kidney (HEK) 293 cells and human mam-
mary tumor MCF-7 cells were cultured in D-MEM medium con-
taining 5% FBS and 10% FBS, respectively, at 37 °C in a humidified
atmosphere of 5% CO2 in air.
plates at the initial density of 2 ꢂ 104 cells/well (50
lL/well) and
incubated at 37 °C. After 24 h, cells were exposed to test com-
pounds by adding solutions (50
ious concentrations in medium at 37 °C under 5% CO2 in air for
24 h. Lysis buffer (15 L) was added to high control wells and
the plates were incubated at 37 °C for 45 min. Then 50 L aliquots
lL/well) of the compounds at var-
l
l
were taken from all wells using a multichannel pipette and trans-
ferred to fresh 96-well flat-bottomed (enzymatic assay) plates, and
50 lL of reconstituted Substrate Mix was added to each well of the
enzymatic assay plate containing samples transferred from the
cytotoxicity assay plate. The plates were incubated for 30 min at
5.2.2. FLAG-IAPs transfection
room temperature, then 50 lL of stop solution was added to each
The transfection experiments were carried out according to the
method reported in Ref. 6.
well and the absorbance at 490 nm in each well was measured
with an ARVO™ SX microplate reader. The percentage cell growth
was calculated from the absorbance readings.
5.2.3. Western blotting
HT1080, FLAG-IAPs HT1080, IMR-32 and MCF-7 (1 ꢂ 106) cells
were treated for the indicated period with MeBS (2) (Nippon Kaya-
ku Co., Ltd), ATRA (3) (Tokyo Kasei Kogyo), MG132 (Peptide Insti-
tute Inc.), lactacystin (Toronto Research Chemicals Inc.) and/or
synthetic compounds at the indicated concentrations in an appro-
priate cell culture medium supplemented with 10% FBS, then the
cells were collected and extracted with SDS buffer. Protein concen-
trations of the lysates were determined using a BCA protein assay.
Equivalent amounts of protein from each lysate were resolved in
10–20% SDS–polyacrylamide gels and transferred onto PVDF mem-
branes. After blocking with TBS containing 5% skim milk, the
transblotted membranes were probed with rabbit polyclonal
CRABP-II antibody (Novus Biologicals, Inc.) (1:1000 dilution), affin-
5.2.8. Caspase assay
Apo-ONEÒ Homogeneous Caspase-3/7 Assay (Promega) was
used for caspase assay. HT1080 and IMR-32 cells were plated in
96-well plates at the initial density of 2 ꢂ 104 cells/well (50
lL/
well) and incubated at 37 °C. After 24 h, cells were exposed to test
compounds by adding solutions (50
various concentrations in medium at 37 °C under 5% CO2 in air for
24 h. Then 100 L of caspase activity detection reagent was added
lL/well) of the compounds at
l
to each well. The plates were further incubated for 10 h (HT1080)
or 0.5 h (IMR-32) at room temperature, and the fluorescence in
each well was measured with an ARVO™ SX microplate reader
(excitation at 485 nm, emission at 535 nm). The fold change of cas-
pase activity was calculated from the fluorescence readings.
ity-purified goat cIAP1 antibody (R&D systems) (0.5
lg/mL), mouse
monoclonal RAR antibody (Perseus Proteomics Inc.) (1:1000 dilu-
a
5.2.9. Cell proliferation inhibition assay
tion), rabbit MycN antibody (Cell Signal Technology) (1:1000 dilu-
tion), anti-goat IgG-horseradish peroxidase conjugates (Kirkegaard
& Perry Laboratories, Inc.) (1:5000 dilution), anti-mouse IgG-horse-
radish peroxidase conjugates (Chemicon) (1:2000 dilution), goat
anti-rabbit IgG-horseradish peroxidase conjugates (Amersham)
(1:2000 dilution), FLAG antibody (Sigma) (1:500 dilution), b-actin
antibody (Santa Cruz Biotechnology, Inc.) (1:2000 dilution) in
can-get-signal solution (Toyobo). After probing, the membrane
was washed twice more with TBS-T. The immunoblots were visu-
alized by enhanced chemiluminescence with Immobilon™ Wes-
tern Chemiluminescent HRP Substrate (Millipore).
IMR-32, HT1080, and MCF-7 cells were plated in 96-well plates
lL/well) and incu-
bated at 37 °C. After 24 h, cells were exposed to test compounds
at the initial density of 2 ꢂ 104 cells/well (50
by adding solutions (50
centrations in medium at 37 °C under 5% CO2 in air for 0–72 h. The
mixtures were then treated with 10
L of AlamarBlueÒ (Invitro-
lL/well) of the compounds at various con-
l
gen), and incubation was continued at 37 °C for 3 h. The fluores-
cence in each well was measured with an ARVO™ SX microplate
reader (excitation at 540 nm, emission, at 590 nm). The percentage
cell growth was calculated from the fluorescence readings.
Acknowledgments
5.2.4. Binding assay
Recombinant GST-BIR3 and GST were produced and purified as
The work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of
Education, Culture, Sports, Science and Technology, Japan and the
Japan Society for the Promotion of Science. This work was also
supported financially by the Takeda Science Foundation and the
described previously.10 Serial dilutions of GST-BIR3 or GST were
incubated in the presence of DanBE (1 lM) at room temperature
for 5 min in phosphate-buffered saline (PBS, pH 7.4). After incuba-
tion, the fluorescence polarization of DanBE was measured on

Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 3229–3241
3241
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