Double protein knockdown of cIAP1 and CRABP-II using a hybrid molecule consisting of ATRA and IAPs antagonist

Article abstract of DOI:10.1016/j.bmcl.2012.04.134

Protein knockdown can be achieved by the use of a small molecule that possesses affinity for both the target protein and ubiquitin ligase. We have designed such a degradation-inducing molecule targeting cIAP1 and CRABP-II, which are involved in proliferation of several cancer cell lines and in neuroblastoma growth, respectively. As a CRABP-II-recognizing moiety, all-trans retinoic acid (ATRA, 3), a physiological ligand of CRABP, was chosen. As a cIAP1-recognizing moiety, MV1 (5), which is a cIAP1/cIAP2/XIAP pan-ligand, was chosen. Although cIAP1 itself possesses ubiquitin ligase activity, we expected that its decomposition would be efficiently mediated by related molecules, including cIAP2 and XIAP, which also possess ubiquitin ligase activity. The designed degradation inducer 6, in which ATRA (3) and MV1 (5) moieties are connected via a linker, was synthesized and confirmed to induce efficient degradation of both cIAP1 and CRABP-II. It showed potently inhibited the proliferation of IMR32 cells.

Full text of DOI:10.1016/j.bmcl.2012.04.134

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4453–4457
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Double protein knockdown of cIAP1 and CRABP-II using a hybrid
molecule consisting of ATRA and IAPs antagonist
Yukihiro Itoh a, Minoru Ishikawa a, Risa Kitaguchi a, Keiichiro Okuhira b, 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:
Protein knockdown can be achieved by the use of a small molecule that possesses affinity for both the
target protein and ubiquitin ligase. We have designed such a degradation-inducing molecule targeting
cIAP1 and CRABP-II, which are involved in proliferation of several cancer cell lines and in neuroblastoma
growth, respectively. As a CRABP-II-recognizing moiety, all-trans retinoic acid (ATRA, 3), a physiological
ligand of CRABP, was chosen. As a cIAP1-recognizing moiety, MV1 (5), which is a cIAP1/cIAP2/XIAP pan-
ligand, was chosen. Although cIAP1 itself possesses ubiquitin ligase activity, we expected that its decom-
position would be efficiently mediated by related molecules, including cIAP2 and XIAP, which also
possess ubiquitin ligase activity. The designed degradation inducer 6, in which ATRA (3) and MV1 (5)
moieties are connected via a linker, was synthesized and confirmed to induce efficient degradation of
both cIAP1 and CRABP-II. It showed potently inhibited the proliferation of IMR32 cells.
Received 10 April 2012
Revised 16 April 2012
Accepted 17 April 2012
Available online 23 May 2012
Keywords:
cIAP1
CRABP-II
Protein knockdown
Ó 2012 Elsevier Ltd. All rights reserved.
Inhibitor of apoptosis proteins (IAPs) are overexpressed in cer-
tain tumor cells and inhibit apoptosis induced by a variety of stim-
uli.1–4 X-linked inhibitor of apoptosis protein (XIAP) and cellular
inhibitor of apoptosis protein 1 and 2 (cIAP1 and cIAP2) show ubiq-
uitin ligase (E3) activity and they promote proteasomal degradation
of their substrate proteins, including caspase, which is an effector of
apoptosis.5,6 Therefore, inhibition of these IAPs is a potential strat-
egy for cancer treatment.7,8 The mitochondrial protein Smac (sec-
ond mitochondria-derived activator of caspases) is an endogenous
IAPs antagonist that binds to the BIR domain of IAP proteins. Potent
and cell-permeable small-molecular IAP antagonists, mimicking
Smac’s AVPI tetrapeptide sequence, have provided useful tools to
investigate the apoptosis-regulatory function of IAPs.9 It was also
reported recently that antagonism of both cIAPs and XIAP is re-
quired for efficient induction of cancer cell death by IAPs antago-
nists.10 On the other hand, Naito’s group reported that bestatin
methyl ester (MeBS: 4, Fig. 1) specifically binds to the BIR3 domain
of cIAP1 and promotes auto-ubiquitination and degradation of
cIAP1.11 Further, cIAP1 degradation induced by MeBS enhanced
apoptosis of cancer cells induced by chemotherapeutic agents such
as cisplastin and etoposide.11
(RARs).12–14 CRABP-II is expressed in several cancers, including
neuroblastoma and Wilms tumor.15,16 In particular, it is thought
to play a role in cancer development in MycN-amplified neuroblas-
toma IMR-32 cells.13 We reported that decrease of CRABP-II induces
down-regulation of MycN, activation of caspase and growth inhibi-
tion of MycN-amplified neuroblastoma IMR-32 cells.17
Several groups have developed methods to regulate protein lev-
els.18–22 We have reported protein knockdown, that is, degradation
of target proteins by small molecules in living cells by making use
of the E3 activity of cIAP1.17,23–25 This approach for targeted pro-
tein degradation involves three steps; (i) formation of an artificial
(non-physiological) complex of cIAP1 and a target protein via con-
jugation with a small molecule (named SNIPER), (ii) polyubiquiti-
nation of the target protein mediated by cIAP1’s E3 activity, (iii)
degradation of the polyubiquitinated protein by proteasome
(Scheme 1). We previously identified compounds 1 and 2 (Fig. 1)
as CRABP-II SNIPERs.17,24 Compound 2 is a hybrid small molecule
designed as a conjugate of ATRA (3) with MeBS (4). Therefore, com-
pound 2 induces degradation of both cIAP1 and CRABP-II. On the
other hand, the amide derivative of MeBS binds to cIAP1 but does
not induce auto-degradation of cIAP1, and therefore amide 1 in-
duces selective degradation of CRABP-II. The previous study also
showed that degradation of both CRABP-II and cIAP1 (with 2) re-
sults in more potent inhibition of neuroblastoma IMR32 cell prolif-
eration than down-regulation of CRABP-II alone (with 1). This
result indicates that 2 might be a candidate therapeutic agent for
MycN-amplified neuroblastoma. However, compound 2 has some
issues, that is, rather low activity and chemical instability due to
Cellular retinoic acid binding protein II (CRABP-II) resides in
cytoplasm and specifically binds to all-trans retinoic acid (ATRA:
3, Fig. 1), an endogenous ligand of retinoic acid receptors
⇑
Corresponding author. Tel./fax: +81 3 5841 7847.
E-mail address: hashimot@iam.u-tokyo.ac.jp (Y. Hashimoto).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.134

4454
Y. Itoh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4453–4457
O
OH
O
OH
N
O
O
O
NH2
H
N
X
Ph
ATRA (3)
O
N
H
2
O
OH
1; X = NH
2; X =O
O
H
N
N
O
MeO
O
NH2
O
MeO
Ph
Ph
Ph
O
N
H
HN
O
OH
NHMe
MeBS (4)
MV-1 (5)
O
OH
N
O
O
Ph
6
H
N
H
N
N
HN
O
O
O
Ph
O
O
HN
O
NHMe
Figure 1. Structures of compound (1, 2, 6), ATRA (3), MeBS (4), and MV1 (5).
Figure 2. Western blotting detection of cIAP1 levels in HT1080 cells after 6 h
treatment with compounds.
Scheme 1. Protein knockdown by SNIPERs.
SNIPER (i.e., 6) than 2 as a candidate for MycN-amplified neuro-
blastoma therapy. The linker was designed to be introduced at
the C-terminus of MV1 (5), because (i) X-ray crystal structure
determination of the complex of BIR3 domain of IAPs and AVPI tet-
rapeptide indicated that the C-terminus of the peptide is located
outside the binding site,27,28 and (ii) a dimer of MV1, linked at
the C-terminus, showed higher binding affinities for cIAP1 and
XIAP than did MV1 monomer (5).26
Synthesis of compound 6 was accomplished as shown in
Schemes 2 and 3. Amine 7,29,30 and acid 1031 were prepared by
using the reported procedure. Stepwise liquid-phase peptide syn-
thesis was performed to give tetrapeptide 12. Removal of the C-ter-
minal O-benzyl group by catalytic reduction gave acid 13 (Scheme
2). Two-step conversion of the N-Boc group of 13 to a N-Fmoc
group gave compound 14. Condensation of 14 with amine 1517 fol-
lowed by deprotection of the Boc group of the amide gave amine
17. The condensation product of amine 17 with acid 16, whose
Fmoc group and 2-cyanoethyl group were deprotected with tetra-
butylammonium fluoride (TBAF), gave compound 6.17,32,33
First, we examined the cIAP1 and CRABP-II degradation-induc-
the ester group. Therefore we attempted to identify new com-
pounds with potent activity to induce degradation of both cIAP1
and CRABP-II. Herein we describe the design, synthesis and biolog-
ical evaluation of a new type of SNIPER that induces potent degra-
dation of both cIAP1 and CRABP-II. We also investigated the effect
of this SNIPER on neuroblastoma IMR-32 cells.
We expected that simultaneous utilization of multiple ubiquitin
ligases would result in more efficient dual SNIPERs than 2, which
has a MeBS (4: a cIAP1-specific ligand) moiety. This consideration
led us replace the MeBS (4) moiety with a MV1 (5) moiety which is
a pan-antagonist for all of XIAP, cIAP1 and cIAP2.26 MV1 (5) has
been reported to induce cancer cell death, and is cell-permeable26
Indeed, our studies suggested that MV1 (5) induces degradation of
cIAP1 more effectively than does MeBS (4) (Fig. 2). Thus, compound
6 was designed (without an ester group; see Fig. 1). Considering
the previous finding that a cIAP1/cIAP2/XIAP antagonist induces
cancer cell death more potently than a cIAP1/cIAP2 antagonist,10
we hoped that IAPs antagonism by MV1-conjugated SNIPER 6
might result in potent induction of cancer cell death. In other
words, we expected that replacement of the MeBS (4) moiety of
compound 2 with a MV1 (5) moiety would yield a more effective
ing activity of compound
6 in HT1080 cells overexpressing

Y. Itoh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4453–4457
4455
HCl
NH2
O
O
H
N
a
b, c
N
HO
BnO
Ph
BnO
Ph
+
N
Boc
O
Boc
O
Ph
Ph
9
8
7
O
H
O
H
N
N
N
BnO
Ph
d, e
N
O
BnO
Ph
O
O
Ph
O
O
Ph
HN
NHBoc
11
NBoc
12
O
H
N
N
HO
Ph
f
O
Ph
O
O
HN
NBoc
13
Scheme 2. (a) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 1-hydroxybenzotriazole hydrate (HOBtÁH2O), i-Pr2NEt, N,N-dimethylformamide (DMF), rt, 22 h, 84%;
(b) HCl, 1,4-dioxane, rt, 4.5 h; (c) (S)-2-(tert-butoxycarbonylamino)-2-cyclohexylacetic acid (10), EDCI, HOBtÁH2O, i-Pr2NEt, DMF, rt, 13 h, 86% (2 steps); (d) HCl, 1,4-dioxane,
rt, 4.5 h; (e) (S)-2-[(tert-butoxycarbonyl)(methyl)amino]propanoic acid, EDCI, HOBtÁH2O, i-Pr2NEt, DMF, rt, 15 h, 80% (two steps); (f) Pd/C, H2, 1,4-dioxane, rt, 6.5 h, 100%.
O
CN
O
N
O
O
OH
H
N
16
N
HO
O
a, b
O
13
Ph
Ph
O
O
e, f
HN
+
6
NFmoc
O
H
14
H2N
N
c, d
N
O
N
H
+
O
O
Ph
Ph
O
O
BocHN
HN
O
NH2
O
NFmoc
17
15
Scheme 3. (a) TFA, CH2Cl2, rt, 3.5 h, 87%; (b) 9-fluorenylmethyloxycarbonyl chloride (FmocCl), K2CO3, THF, rt, 3 h, 56%; (c) EDCI, HOBtÁH2O, CH2Cl2, rt, 20 h; (d) HCl, 1,4-
dioxane, rt, 1 h; (e) EDCI, HOBtÁH2O, Et3N, CH2Cl2, rt, 20 h; (f) tetrabutylammonium fluoride (TBAF), MeOH, THF, rt, 0.5 h, 14% (four steps; from 16).
FLAG-cIAP1 by means of Western blotting analysis, as previously
reported.17 As expected, compound 6 induced down-regulation of
both cIAP1 and CRABP-II in a dose-dependent manner (Fig. 3).
Compound 6 is approximately 10 times more potent than com-
pound 2 (Fig. 3). We previously reported that compound 1 does
pected that 6 would not induce RARa degradation because both 6
and 2 possess same CRABP-recognition moiety. Next, we investi-
gated the mechanistic features of the cIAP1/CRABP-II decrease
induced by compound 6. To confirm the contribution the ubiqui-
tin-proteasome pathway, the incubation mixture was pretreated
with proteasome inhibitor MG132 (Fig. 4). As expected, the de-
crease in both cIAP1 and CRABP-II levels by compound 6 was
blocked by MG132. These results suggested that the reduction in
cIAP1 and CRABP-II levels by compound 6 was caused by proteaso-
mal degradation. In addition, we confirmed that the decrease in
CRABP-II was not caused by a partial structure of compound 6, or
by a mere mixture of MV1 (5) and ATRA (3) (Fig. 5). Compound 6
induced a decrease of CRABP-II, whereas the mixture of MV1 (5)
not bind to RARs and does not induce RARa
degradation.24 It is ex-
and ATRA (3) did not. Treatments with 10
combination of 10 M MV1 (5) and 10 M ATRA (3) decreased
the cIAP1 level, but did not affect the CRABP-II level in HT1080.
ATRA (3) at 10 M did not cause any decrease of cIAP1 or
CRABP-II. Thus, conjugation of MV1 (5) and ATRA (3) in a single
lM MV1 (5), and the
l
l
Figure 3. Down-regulation of cIAP1 and CRABP-II by treatment with compound 6.
Western blot detection of cIAP1 and CRABP-II levels in HT1080 cells expressing
FLAG-tagged cIAP1 after 6 h treatment.
l

4456
Y. Itoh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4453–4457
Figure 4. Influence of pretreatment with proteasome inhibitors on CRABP-II
degradation induction. Western blot detection of CRABP-II levels in HT1080 cells.
The cells were treated with 10
lM 6 for 6 h. MG132 (10 lM) was added to the
culture 30 min prior to the addition of 6.
Figure 7. Relative cell numbers of IMR-32 cells treated with 2 and 6.
Table 1
Effects of compounds on caspase activitya,b
Entry
Compound
Caspase activity (fold increase)
1
2
3
4
—
1
2
1.00
1.84
3.61
4.44
Figure 5. The influence of combination of MV-1 (5) and ATRA (3). Western blot
detection of CRABP-II and cIAP1 levels in HT1080 cells expressing FLAG-tagged
cIAP1 after 6-h treatment with each reagent.
6
a
Values are means of at least two experiments.
IMR-32 cells were treated with compounds for 24 h. The data are the means of
b
triplicate determinations.
molecule is mandatory for the cIAP1/CRABP-II dual degradation-
inducing activity. Taken together, these results indicate that com-
pound 6 induces cIAP1/CRABP-II dual degradation via the expected
mechanism (Scheme 1). In addition, our findings indicate that pro-
tein knockdown using MV1 (5)-bearing molecules might be avail-
able as a general strategy by replacing the ATRA (3) moiety with
other specific ligands of various target proteins.
and caspase-activating activities than 2 in the assay using MycN-
amplified neuroblastoma IMR-32 cells. These results suggested
that compound 6 might be a new therapeutic candidate/lead com-
pound for the treatment of neuroblastoma. Dual degradation of
cancer-related proteins, including IAPs, by treatment with IAPs
antagonist-bearing molecules seems likely to be of therapeutic va-
lue as a novel strategy for cancer therapy. A detailed mechanistic
analysis of cancer cell death induced by 6 is under way.
Finally, we investigated the effect of compound 6 on MycN-
amplified neuroblastoma IMR-32 cells. Down-regulation of
CRABP-II and cIAP1 levels in IMR32 cells by 6 is shown in Figure
6. The cIAP1 and CRABP-II degradation-inducing activity decreased
in the order of 6 > 2 > 1 and 6 >1 = 2, respectively (Fig. 6). We also
evaluated the cell proliferation-inhibiting activity of compounds 6
and 2. Compound 6 showed strong inhibition of cell proliferation,
and the activity was higher than that of compound 2 (Fig. 7). Then,
we tested caspase 3/7 activation by compound 6 (Table 1). Com-
pound 1, which is a weaker inhibitor of IMR-32 cell proliferation
than 2,17 enhanced caspase activity about 1.8-fold compared with
the controls. The caspase activity enhancement elicited by com-
pound 2 was estimated to be about 3.6-fold. Compound 6 showed
the most potent caspase activity enhancement among the tested
compounds, that is about 4.4-fold. These results are consistent
with the idea that 6 inhibits IMR32 cell proliferation by decreasing
CRABP-II and cIAP1, thereby resulting in activation of caspase 3/7.
In summary, we have prepared a novel SNIPER molecule, com-
pound 6, in which ATRA (3) and MV1 (5) moieties are conjugated
via a linker. Compound 6 induced efficient degradation of both
cIAP1 and CRABP-II. Mechanistic analysis indicated that cIAP1
and CRABP-II were degraded in a proteasome-dependent manner.
Compound 6 also showed stronger cell proliferation-inhibiting
Acknowledgments
The work described in this Letter was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of Educa-
tion, 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 Naito Foun-
dation. We are grateful to Nippon Kayaku Co., especially Dr. Keiko
Sekine, for providing MeBS (4), to HSRRB for providing IMR32 cells
(IFO50283), and to Dr. Yukihide Tomari for help with Western blot
detection.
References and notes
1. Kurokawa, M.; Kornbluth, S. Cell 2009, 138, 838.
2. Degterev, A.; Yuan, J. Nat. Rev. Mol. Cell Biol. 2008, 9, 378.
3. Salvesen, G. S.; Riedl, S. J. Adv. Exp. Med. Biol. 2008, 615, 13.
4. Taylor, R. C.; Cullen, S. P.; Martin, S. J. Nat. Rev. Mol. Cell Biol. 2008, 9, 231.
5. Riedl, S. J.; Renatus, M.; Schwarzenbacher, R.; Zhou, Q.; Sun, C.; Fesik, S. W.;
Liddington, R. C.; Salvesen, G. S. Cell 2001, 104, 791.
6. Roy, N.; Deveraux, Q. L.; Takahashi, R.; Salvesen, G. S.; Reed, J. C. EMBO J. 1997,
16, 6914.
7. Deveraux, Q. L.; Reed, J. C. Genes Dev. 1999, 13, 239.
8. Salvesen, G. S.; Duckett, C. S. Nat. Rev. Mol. Cell Biol. 2002, 3, 401.
9. Wang, S. Curr. Top. Microbiol. Immunol. 2011, 348, 89.
10. Ndubaku, C.; Varfolomeev, E.; Wang, L.; Zobel, K.; Lau, K.; Elliott, L. O.; Maurer,
B.; Fedorova, A. V.; Dynek, J. N.; Koehler, M.; Hymowitz, S. G.; Tsui, V.;
Deshayes, K.; Fairbrother, W. J.; Flygare, J. A.; Vucic, D. ACS Chem. Biol. 2009, 4,
557.
11. Sekine, K.; Takubo, K.; Kikuchi, R.; Nishimoto, M.; Kitagawa, M.; Abe, F.;
Nishikawa, K.; Tsuruo, T.; Naito, M. J. Biol. Chem. 2008, 283, 8961.
12. Donovan, M.; Olofsson, B.; Gustafson, A. L.; Dencker, L.; Eriksson, U. J. Steroid
Biochem. Mol. Biol. 1995, 53, 459.
Figure 6. Western blot detection of cIAP1 and CRABP-II levels in IMR32 cells after
24 h treatment.
13. Wolf, G. Nutr. Rev. 2000, 58, 151.

Y. Itoh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4453–4457
4457
14. Fogh, K.; Voorhees, J. J.; Aström, A. Arch. Biochem. Biophys. 1993, 300, 751.
15. Gupta, A.; Williams, B. R.; Hanash, S. M.; Rawwas, J. Cancer Res. 2006, 66, 8100.
16. Gupta, A.; Kessler, P.; Rawwas, J.; Williams, B. R. Exp. Cell Res. 2008, 314, 3663.
17. Itoh, Y.; Ishikawa, M.; Kitaguchi, R.; Sato, S.; Naito, M.; Hashimoto, Y. Bioorg.
Med. Chem. 2011, 19, 3229.
18. Sakamoto, K. M.; Kim, K. B.; Kumagai, A.; Mercurio, F.; Crews, C. M.; Deshaies,
R. J. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8554.
19. Schneekloth, J. S., Jr.; Fonseca, F. N.; Koldobskiy, M.; Mandal, A.; Deshaies, R.;
Sakamoto, K.; Crews, C. M. J. Am. Chem. Soc. 2004, 126, 3748.
20. Lee, H.; Puppala, D.; Choi, E. Y.; Swanson, H.; Kim, K. B. Chembiochem 2007, 8,
2058.
24. Itoh, Y.; Ishikawa, M.; Naito, M.; Hashimoto, Y. J. Am. Chem. Soc. 2010, 132, 5820.
25. Itoh, Y.; Kitaguchi, R.; Ishikawa, M.; Naito, M.; Hashimoto, Y. Bioorg. Med. Chem.
2011, 19, 6768.
26. Varfolomeev, E.; Blankenship, J. W.; Wayson, S. M.; Fedorova, A. V.; Kayagaki,
N.; Garg, P.; Zobel, K.; Dynek, J. N.; Elliott, L. O.; Wallweber, H. J.; Flygare, J. A.;
Fairbrother, W. J.; Deshayes, K.; Dixit, V. M.; Vucic, D. Cell 2007, 131, 669.
27. Kulathila, R.; Vash, B.; Sage, D.; Cornell-Kennon, S.; Wright, K.; Koehn, J.; Stams,
T.; Clark, K.; Price, A. Acta Crystallogr., Sect. D 2009, 65, 58.
28. 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.
29. Royo, S.; Jiménez, A. I.; Cativiela, C. Tetrahedron: Asymmetry 2006, 17, 2393.
30. Hsieh, K. H.; LaHann, T. R.; Speth, R. C. J. Med. Chem. 1989, 32, 898.
31. Moffat, D. F. C.; Pintat, S. PCT Int. Appl. WO 2008053158, 2008.
32. Kita, Y.; Maeda, H.; Takahashi, F.; Fujui, S.; Ogawa, T.; Hatayama, K. Chem.
Pharm. Bull. 1994, 42, 147.
21. Bargagna-Mohan, P.; Baek, S. H.; Lee, H.; Kim, K.; Mohan, R. Bioorg. Med. Chem.
Lett. 2005, 15, 2724.
22. Cyrus, K.; Wehenkel, M.; Choi, E. Y.; Han, H. J.; Lee, H.; Swanson, H.; Kim, K. B.
Mol. Biosyst. 2010, 7, 359.
23. Okuhira, K.; Ohoka, N.; Sai, K.; Nishimaki-Mogami, Y.; Itoh, Y.; Ishikawa, M.;
Hashimoto, Y.; Naito, M. FEBS Lett. 2011, 585, 1147.
33. Bernardes, G. J.; Grayson, E. J.; Thompson, S.; Chalker, J. M.; Errey, J. C.; El
Oualid, F.; Claridge, T. D.; Davis, B. G. Angew. Chem., Int. Ed. 2008, 47, 2244.