Solution-phase synthesis of ICG-001, a β-turn peptidomimetic molecule inhibitor of β-catenin-Tcf-mediated transcription

Article abstract of DOI:10.1016/j.tet.2007.10.048

The solution-phase synthesis of the β-turn peptidomimetic ICG-001, a selective inhibitor of Wnt/β-catenin signalling, which has been found to be important for both initiation and progression of cancers of different tissues and has been exploited as an ext

Full text of DOI:10.1016/j.tet.2007.10.048

Available online at www.sciencedirect.com
Tetrahedron 63 (2007) 12912–12916
Solution-phase synthesis of ICG-001, a b-turn peptidomimetic
molecule inhibitor of b-catenin–Tcf-mediated transcription
a
a
a
Alessandro Piergentili,a, Fabio Del Bello, Francesco Gentili, Mario Giannella,
*
Wilma Quaglia,a Cristian Vesprini,a Russell J. Thomasb and Graeme M. Robertsonb
aDipartimento di Scienze Chimiche, Universitaꢀ di Camerino, Via S. Agostino, 1, 62032 Camerino, Italy
bSiena Biotech S.p.A., Via Fiorentina, 1, 53100 Siena, Italy
Received 10 July 2007; revised 14 September 2007; accepted 11 October 2007
Available online 13 October 2007
Abstract—The solution-phase synthesis of the b-turn peptidomimetic ICG-001, a selective inhibitor of Wnt/b-catenin signalling, which has
been found to be important for both initiation and progression of cancers of different tissues and has been exploited as an extremely useful
chemogenomic tool, was developed. This route is particularly suitable for the multigram scale preparation of ICG-001.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitors of b-catenin–Tcf have been identified by high-
throughput screens of large synthetic compound collec-
tions.6
In a screen for regulators of b-catenin–T cell factor (Tcf)-
mediated transcription in the Wnt pathway, the compound
ICG-001 (Fig. 1), bearing the tetrahydro-1H-pyrazino[1,2-
a]pyrimidine-4,7(6H,8H)-dione skeleton, a b-turn bicyclic
peptidomimetic, proved to be one of the most potent
inhibitors.1
Among these, ICG-001 binds selectively cAMP response
element-binding protein (CBP), which is an essential tran-
scriptional co-activator protein, and prevents its interaction
with b-catenin–Tcf-responsive gene.7 ICG-001 selectively
induces apoptosis in transformed cells but not in normal
colonic epithelial cells by increasing caspase activity, re-
duces in vitro growth of colon carcinoma cells, and is effica-
cious in the Min mouse and nude mouse xenograft models of
coloncancer.7 Due to these positiveeffects, ICG-001 not only
has potential therapeutic use in colon cancer, but also repre-
sents a lead compound for the development of new agents.
Additionally, ICG-001 has been used as a chemogenomic
tool compound to help tease apart the mechanisms behind
survivin gene transcription,8 and ready access to this mole-
cule will undoubtedly facilitate similar studies in the future.
The Wnt signalling cascade has important functions in tissue
development, homeostasis and regeneration.2 Wnts bind to
members of the Frizzled family of cell-surface receptors
and are known to activate at least three separate signalling
pathways to effect gene expression.3 One of these is the
‘canonical’ pathway, which regulates the ability of the b-
catenin protein to drive activation of specific target genes.2
Deregulation of Wnt/b-catenin signalling is frequently
found in various human cancers and its activation has been
shown to be important for both initiation and progression
of cancers of different tissues, such as colorectal cancers, he-
patocellular carcinomas or gastric cancers.4 In the nucleus,
b-catenin regulates gene expression promoting cell prolifer-
ation, migration and invasiveness. In fact, it acts as a tran-
scriptional activator in conjunction with the DNA-binding
T cell factor/lymphoid enhancer factor (Tcf/Lef) proteins.5
Therefore, targeted inhibition of Wnt/b-catenin signalling
has been a rational and promising new approach for the ther-
apy of cancers of various origins and, though many efforts
have been made, at present only a few small molecule
The published synthesis of ICG-001 uses a solid-phase
synthetic protocol9 as is the case for many such peptido-
mimetics. Though very useful in terms of rapid analogue
NH
O
H
N
O
N
N
O
Keywords: Wnt pathway; Solution-phase synthesis; Inhibitor; Peptido-
mimetic; ICG-001.
* Corresponding author. Tel.: +390737402237; fax: +390737637345;
e-mail: alessandro.piergentili@unicam.it
OH
Figure 1. Structure of ICG-001.
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.10.048

A. Piergentili et al. / Tetrahedron 63 (2007) 12912–12916
12913
C2H5O
OC2H5
OC2H5
OC2H5
O
Fmoc NH
N
O
H
C2H5O
c
a
b
HN
+
NH2
OC2H5
OC(CH3)3
2
1
C2H5O
OC2H5
C2H5O
OC2H5
C2H5O
NH2
NH
OC2H5
Fmoc
NH
NH
H2N
N
O
N
d
N
f
e
O
O
O
O
OC(CH3)3
OC(CH3)3
OC(CH3)3
4
5
3
O
NH
C2H5O
OC2H5
H
N
O
NH NH
O
g
N
N
O
N
NH
O
O
OH
ICG-001
OC(CH3)3
6
Scheme 1. (a) NaBH4, EtOH, 85%; (b) Fmoc-L-Tyr(t-Bu)-OH, HATU, DIEA, DMF, 78%; (c) DEA, DCM, 83%; (d) Fmoc-b-Ala-OH, HATU, DIEA, DMF,
77%; (e) DEA, DCM, 82%; (f) C6H5CH2NCO, DCM, 76%; (g) HCOOH, 76%.
generation, this strategy is less efficient in terms of providing
larger quantities of ICG-001. The use of multiple equivalents
of costly raw materials required for this solid-phase route
is largely responsible for this efficiency shortfall. Moreover,
the solid-phase approach for small molecules can be limited
by unfavourable heterogeneous reaction kinetics, longer
development times and difficulties in analyzing resin-
bound intermediates.10 Therefore, the investigation of
alternative synthetic methodologies is a matter of particular
importance.
diethoxyethanamine, followed by the reduction of the Schiff
base intermediate (Scheme 1). The amine compound 1 was
coupled with Fmoc-L-Tyr(t-Bu)-OH. Fmoc was chosen as
protecting group11 as its cleavage is easily accomplished
with diethylamine (DEA).12 The reaction with triethylamine
and ethyl chlorocarbonate in CHCl3 gave compound 2 in
moderate yield (30%). The yield of this synthetic step
was improved to 78% by using O-(7-azabenzotriazol-1-
yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate
(HATU) and diisopropylethylamine (DIEA) as the base in
dimethylformamide (DMF). The removal of the Fmoc
group, to obtain compound 3 in 83% yield, was performed
in dichloromethane (DCM) with DEA instead of piperidine,
which was used in the solid-phase synthetic protocol.9 Since
the reaction workup foresaw the evaporation to dryness, the
advantage lay in the fact that DEA has a boiling point lower
than that of piperidine. The reaction of compound 3 with
Fmoc-b-Ala-OH in the presence of HATU and DIEA in
DMF afforded compound 4 in 77% yield. The cleavage of
the Fmoc protection (yield 82%) and subsequent reaction
with benzyl isocyanate gave compound 6 in 76% yield,
which, finally, was cyclized upon treatment with formic
acid for 12 h at rt to afford ICG-001 (76% yield). The final
cyclization step produced only a single diastereomer, in
which, as previously reported,13 the hydrogen at the ring
junction is trans to the hydrogen at the 6-position. In addi-
As for the preparation of ICG-001 the number of amino acid
units to be combined was small, in the present paper an effi-
cient method in solution-phase synthesis (Scheme 1), which
is a method of choice for producing short peptides, was de-
veloped and is the subject of this paper. Scale-up of a solu-
tion-phase synthesis is more predictable and less expensive
than a solid-phase synthesis and allows purification, isola-
tion and characterization of the reaction intermediates to
be possible after each reaction step.
2. Results and discussion
The synthetic approach to ICG-001 involved the solution-
phase sequential amino acid addition to (2,2-diethoxy-
ethyl)-naphthalen-1-ylmethyl-amine (1), prepared in 85%
yield by condensation of 1-naphthaldehyde with 2,2-
1
tion, on the basis of the H NMR spectra the structure of
the product here described is consistent with that previously
reported.9

12914
A. Piergentili et al. / Tetrahedron 63 (2007) 12912–12916
3. Conclusion
126.2, 126.1, 125.8, 125.6, 123.9, 102.4, 62.5, 52.3, 51.7,
15.6; MS (ESI): m/z¼274.1 (M+H)+. Anal. Calcd for
C17H23NO2: C, 74.69; H, 8.48; N, 5.12. Found: C, 74.99;
H, 8.41; N, 5.05.
In conclusion we have described the solution-phase synthe-
sis of ICG-001, a selective inhibitor of Wnt/b-catenin signal-
ling, which has been found to be important for both initiation
and progression of cancers of different tissues and is a very
useful tool for chemogenomic analysis of this, and related
pathways. Since in all steps good yields, ranging from
76% to 85%, were obtained, the new synthetic method has
proven suitable for the multigram scale preparation of
ICG-001.
4.1.2. {(S)-2-(4-tert-Butoxy-phenyl)-1-[(2,2-diethoxy-eth-
yl)-naphthalen-1-ylmethyl-carbamoyl]-ethyl}-carbamic
acid 9H-fluoren-9-ylmethyl ester (2). (a) DIEA (5.84 g,
45.2 mmol) and HATU (17.18 g, 45.2 mmol) were added
to a stirred solution of Fmoc-L-Tyr(t-Bu)-OH (18.84 g,
41.0 mmol) in DMF (150 mL) and the mixture was stirred
at rt for 30 min. Compound 1 (12.24 g, 45.2 mmol) was
then added and the reaction mixture was stirred at rt for
16 h. Removal of the solvent to dryness gave a residue,
which was purified by column chromatography, eluting
with cyclohexane/EtOAc (9:1) to give 2 as a white foam:
22.86 g (78% yield). Rf¼0.10; IR (neat) 3285, 2975, 1713,
4. Experimental
4.1. General
All commercially available reagents were used without fur-
ther purification or were purified by standard methods prior
to use. Melting points were taken in glass capillary tubes on
1634, 1236, 1044, 738 cmꢂ1 1H NMR (400 MHz,
;
CDCl3): d¼1.02–1.15 (m, 6H, CH3CH2), 1.15 and 1.40
(two s, 9H, C(CH3)3), 2.90 and 2.99 (two d, J¼7.0, 9.2 Hz,
4H, NCH2CH and Tyr-CH2), 3.45 (m, 4H, OCH2CH3),
4.10–4.79 (m, 5H, Fmoc-CH, Fmoc-CH2, Tyr-CH, CHO),
4.81–5.10 (m, 2H, Napht-CH2), 5.79 (br d, 1H, NH, ex-
changeable with D2O), 6.60–8.19 (m, 19H, ArH); 13C
NMR (100 MHz, CDCl3): d¼172.5, 155.7, 154.4, 144.1,
141.5, 134.1, 132.6, 132.0, 131.4, 130.0, 128.9, 128.2,
127.9, 127.3, 126.7, 126.2, 125.6, 125.4, 125.3, 124.4,
124.3, 120.1, 101.8, 78.5, 67.2, 63.7, 53.0, 50.0, 48.3,
47.2, 39.9, 28.9, 15.4; MS (ESI): m/z¼753.4 (M+K)+.
Anal. Calcd for C45H50N2O6: C, 75.60; H, 7.05; N, 3.92.
Found: C, 75.96; H, 7.14; N, 3.85.
1
€
a Buchi SMP-20 apparatus and are uncorrected. IR and H
NMR spectra were recorded on Perkin–Elmer Spectrum
100 and Varian Mercury 400 instruments, respectively.
Chemical shift values are reported in parts per million
(ppm) relative to tetramethylsilane (TMS), used as an inter-
nal reference standard and spin multiplicities are given as s
(singlet), br s (broad singlet), br t (broad triplet), d (doublet),
dd (double doublet), t (triplet), q (quartet), or m (multiplet).
1
Compounds 2–6 were observed as rotamers at 20 ꢀC in H
and 13C NMR: here only the major peaks are reported.
Mass spectra were obtained using a Hewlett Packard 1100
MSD instrument utilizing electron-spray ionization (ESI).
Elemental analyses were performed at the Microanalytical
Laboratory of our department. All reactions were monitored
by thin-layer chromatography (TLC) using silica gel plates
(60 F254; Merck), visualizing with ultraviolet light or iodine.
Chromatographic separations were performed on silica gel
columns (Kieselgel 40, 0.040–0.063 mm, Merck) by flash
chromatography. Compounds were named following IUPAC
rules as applied by Beilstein-Institut AutoNom, a software
for systematic names in organic chemistry.
(b) Ethyl chlorocarbonate (1.22 g, 11.3 mmol) was added
dropwise to a stirred and cooled (0 ꢀC) solution of
Fmoc-L-Tyr(t-Bu)-OH (5.19 g, 11.3 mmol) and Et3N
(1.14 g, 11.3 mmol) in CHCl3 (100 mL). After 30 min a solu-
tion of 1 (3.09 g, 11.3 mmol) in CHCl3 (60 mL) was added.
The resulting reaction mixture was stirred at rt overnight and
then washed with 2 N NaOH (3ꢁ15 mL) and water (15 mL).
Removal of solvents in vacuo gave an oil, which was purified
by column chromatography to give 2 as a white foam 2.42 g
(30% yield).
4.1.1. (2,2-Diethoxy-ethyl)-naphthalen-1-ylmethyl-amine
(1). A mixture of 1-naphthaldehyde (15.6 g, 0.1 mol) and
2,2-diethoxyethanamine (13.3 g, 0.1 mol) was heated on
a steam bath for 20 min, then cooled to rt and diluted with
EtOH (75 mL). Sodium borohydride (3.8 g, 0.1 mol) was
added in small portions and the mixture was stirred at rt
for 24 h. After evaporation of the solvent under reduced
pressure, the residue was partitioned between H2O
(50 mL) and EtOAc (200 mL). The organic layer was
washed with H2O (2ꢁ25 mL) and dried over Na2SO4. The
solvent was concentrated in vacuo to give a residue, which
was purified by column chromatography, eluting with petro-
leum ether/CHCl3/CH3OH/NH4OH (10:10:0.2:0.01) to af-
ford the product as an oil: 23.2 g (85% yield). Rf¼0.51; IR
4.1.3. (S)-2-Amino-3-(4-tert-butoxy-phenyl)-N-(2,2-di-
ethoxy-ethyl)-N-naphthalen-1-ylmethyl-propionamide
(3). DEA (122.5 mL) was added to a solution of 2 (17.5 g,
24.5 mmol) in DCM (250 mL) at rt. The reaction, moni-
tored by TLC, was complete in 3 h at rt. The solvent
and DEA were removed and the residue was purified by
column chromatography gradient eluent, eluting with cy-
clohexane/EtOAc (8:2) first and EtOAc next, to give 3
as an oil: 10.0 g (83% yield). Rf¼0.08 (EtOAc); IR
1H NMR (400 MHz, CDCl3): d¼1.02–1.21 (m, 6H,
CH3CH2), 1.25 and 1.35 (two s, 9H, C(CH3)3), 1.99 (br
s, 2H, NH2, exchangeable with D2O), 2.66–3.21 (m, 4H,
NCH2CH and Tyr-CH2), 3.45 (m, 4H, OCH2CH3), 3.71
(m, 1H, Tyr-CH), 3.91–4.72 (m, 1H, CHO), 4.81–5.22
(m, 2H, Napht-CH2), 6.76–8.11 (m, 11H, ArH); 13C
NMR (100 MHz, CDCl3): d¼176.5, 154.2, 134.0, 133.1,
132.4, 129.9, 129.2, 128.8, 128.0, 127.6, 126.7, 126.2,
125.4, 124.3, 122.2, 101.6, 78.4, 63.6, 53.5, 50.5, 48.8,
42.0, 29.1, 15.4; MS (ESI): m/z¼493.4 (M+H)+. Anal.
(neat) 3373, 2975, 1640, 1235, 1160, 1056, 751 cmꢂ1
;
1
(neat) 2974, 1597, 1217, 1120, 1057, 751 cmꢂ1; H NMR
(400 MHz, CDCl3): d¼1.19 (t, J¼7.0 Hz, 6H, CH3), 1.64
(br s, 1H, NH, exchangeable with D2O), 2.89 (d,
J¼5.0 Hz, 2H, CH2N), 3.52 and 3.66 (two q, J1¼7 Hz,
J2¼10 Hz, 4H, OCH2), 4.28 (s, 2H, CH2Ar), 4.62 (t,
J¼5.0 Hz, 1H, CH), 7.42–8.19 (m, 7H, ArH); 13C NMR
(100 MHz, CDCl3): d¼136.1, 134.1, 132.1, 128.9, 127.9,

A. Piergentili et al. / Tetrahedron 63 (2007) 12912–12916
12915
Calcd for C30H40N2O4: C, 73.14; H, 8.18; N, 5.69. Found:
C, 73.34; H, 8.19; N, 5.91.
was purified by column chromatography, eluting with
EtOAc, to yield 6 as a viscous oil: 7.9 g (76% yield).
Rf¼0.26; IR (neat) 3306, 2975, 1738, 1630, 1505, 1235,
4.1.4. (2-{(S)-2-(4-tert-Butoxy-phenyl)-1-[(2,2-diethoxy-
ethyl)-naphthalen-1-ylmethyl-carbamoyl]-ethylcarba-
moyl}-ethyl)-carbamic acid 9H-fluoren-9-ylmethyl ester
(4). DIEA (4.7 g, 36.0 mmol) and HATU (13.6 g,
36.0 mmol) were added to a stirred solution of Fmoc-
b-Ala-OH (10.2 g, 33.0 mmol) in DMF (120 mL) and the
mixture was left at rt for 30 min. After the addition of com-
pound 3 (18.0 g, 36.0 mmol), the reaction mixture was stirred
at rt for 16 h. Removal of the solvent to dryness gave a
residue, which was purified by column chromatography,
eluting with cyclohexane/EtOAc (1:1), to give 4 as a white
foam: 21.8 g (77% yield). Rf¼0.23; IR (neat) 3300, 2975,
1160, 1056, 697 cmꢂ1 1H NMR (400 MHz, CDCl3):
;
d¼1.06–1.12 (m, 6H, CH3CH2), 1.20 and 1.28 (two s, 9H,
C(CH3)3), 1.63 (br s, 1H, NH, exchangeable with D2O),
2.21–2.38 (m, 2H, CH2CO), 2.76–3.72 (m, 10H, NCH2CH,
Tyr-CH2, OCH2CH3, CH2CH2CO), 4.22–5.48 (m, 7H, Tyr-
CH, CHO, CH2Ar, Napht-CH2, NH, exchangeable with
D2O), 6.26 and 6.40 (br d, 1H, Tyr-NH, exchangeable with
D2O), 6.68–8.11 (m, 16H, ArH); 13C NMR (100 MHz,
CDCl3): d¼172.8, 171.7, 158.3, 154.4, 139.8, 134.1,
132.4, 131.9, 131.4, 130.0, 128.9, 128.6, 128.3, 127.6,
127.3, 126.7, 126.2, 125.6, 124.3, 124.0, 122.4, 101.7,
78.3, 63.9, 51.2, 49.9, 48.9, 47.9, 44.5, 38.8, 36.8, 28.9,
15.5; MS (ESI): m/z¼719.5 (M+Na)+. Anal. Calcd for
C41H52N4O6: C, 70.66; H, 7.52; N, 8.04. Found: C, 70.76;
H, 7.62; N, 8.20.
1
1721, 1631, 1236, 1056, 740 cmꢂ1; H NMR (400 MHz,
CDCl3): d¼1.03–1.21 (m, 6H, CH3CH2), 1.19 and 1.41
(two s, 9H, C(CH3)3), 2.30 and 2.40 (br s, 1H, Fmoc-NH,
exchangeable with D2O), 2.82–3.39 (m, 6H, NCH2CH,
Tyr-CH2, CH2CO), 3.52–3.79 (m, 8H, OCH2CH3,
Fmoc-CH2, CH2CH2NH), 4.21–5.52 (m, 5H, Fmoc-CH,
Tyr-CH, CHO, Napht-CH2), 6.21 and 6.41 (br d, 1H,
Tyr-NH, exchangeable with D2O), 6.62–8.15 (m, 19H,
ArH); 13C NMR (100 MHz, CDCl3): d¼172.5, 170.7,
156.6, 154.6, 144.2, 141.5, 134.1, 132.5, 132.0, 131.6,
129.9, 128.9, 128.3, 128.0, 127.2, 126.6, 126.2, 125.5,
125.4, 125.3, 124.4, 124.3, 120.1, 101.6, 78.3, 66.8,
63.7, 51.3, 50.0, 48.5, 47.5, 39.4, 37.4, 36.1, 28.9, 15.4;
MS (ESI): m/z¼786.9 (M+H)+. Anal. Calcd for
C48H55N3O7: C, 73.35; H, 7.05; N, 5.35. Found: C, 73.54;
H, 7.15; N, 5.42.
4.1.7. (6S,9aS)-6-(4-Hydroxy-benzyl)-8-naphthalen-1-
ylmethyl-4,7-dioxo-hexahydro-pyrazino[1,2-a]pyrimi-
dine-1-carboxylic acid benzylamide (ICG-001).
Compound 6 (6.0 g, 8.55 mmol) was stirred in formic acid
(60 mL) for 16 h at rt. Evaporation to dryness yielded
4.5 g of ICG-001 as a solid, which was recrystallized from
EtOAc: 3.6 g (76% yield); mp 133–134 ꢀC. Rf¼0.48
(EtOAc/CH3OH 9.5:0.5); IR (neat) 3321, 2928, 1627,
1
1515, 1260, 1101, 1056, 783 cmꢂ1; H NMR (400 MHz,
CDCl3): d¼2.21 (m, 1H, CH2CO), 2.35 (m, 1H, CH2CO),
2.90 (m, 1H, CH2N), 3.05 (m, 1H, Tyr-CH2), 3.11 (m, 1H,
Tyr-CH2), 3.28 (dd, J1¼5 Hz, J2¼14 Hz, 1H, CHCH2N),
3.40 (dd, J1¼5 Hz, J2¼14 Hz, 1H, CHCH2N), 3.82 (m,
1H, CH2N), 4.19 (dd, J1¼5 Hz, J2¼14 Hz, 1H, CH2NH),
4.31 (dd, J1¼5 Hz, J2¼14 Hz, 1H, CH2Ar), 4.56 (br t, 1H,
NH, exchangeable with D2O), 4.98 (d, J¼15 Hz, 1H,
Napht-CH2), 5.04 (dd, J1¼4 Hz, J2¼11 Hz, 1H, Tyr-CH),
5.23 (d, J¼15 Hz, 1H, Napht-CH2), 5.39 (t, J¼6 Hz, 1H,
NCHN), 6.58 (m, 2H, ArH), 7.05 (m, 2H, ArH), 7.13 (m,
2H, ArH), 7.30 (m, 4H, ArH), 7.37 (m, 1H, ArH), 7.55 (m,
2H, ArH), 7.83 (m, 1H, ArH), 7.88 (m, 1H, ArH), 8.08
4.1.5. (S)-2-(3-Amino-propionylamino)-3-(4-tert-butoxy-
phenyl)-N-(2,2-diethoxy-ethyl)-N-naphthalen-1-yl-
methyl-propionamide (5). DEA (125 mL) was added to
a solution of 4 (20.0 g, 25.0 mmol) in DCM (250 mL).
The reaction, monitored by TLC, was complete in 3 h at
rt. The solvent and DEA were removed and the residue
was purified by column chromatography gradient eluent,
eluting with cyclohexane/EtOAc (1:1) first and with
EtOAc/MeOH (8:2) next, to give 5 as an oil: 11.6 g (82%
yield). Rf¼0.20 (EtOAc/CH3OH/NH4OH 8:2:0.01); IR
1
(m, 1H, ArH); H NMR (400 MHz, DMSO-d6): d¼2.09
(m, 1H, CH2CO), 2.99–3.39 (m, 5H, CH2N, Tyr-CH2,
CHCH2N), 3.59 (t, J¼11 Hz, 1H, NH, exchangeable with
D2O), 3.92 (m, 1H, CH2N), 4.19 (dd, J1¼6 Hz, J2¼15 Hz,
1H, CH2Ar), 4.32 (dd, J1¼6 Hz, J2¼15 Hz, 1H, CH2Ar),
4.86 (d, J¼15 Hz, 1H, Napht-CH2), 5.18 (m, 1H, Tyr-
CH), 5.18 (d, J¼5 Hz, 1H, Napht-CH2), 5.79 (dd,
J1¼4 Hz, J2¼11 Hz, 1H, NCHN), 6.52 (d, J¼9 Hz, 2H,
ArH), 6.89 (d, J¼9 Hz, 2H, ArH), 7.19–8.18 (m, 12H,
ArH), 9.19 (br s, 1H, OH, exchangeable with D2O); 13C
NMR (100 MHz, CD3OD): d¼167.3, 167.1, 157.1, 156.4,
139.7, 134.3, 131.7, 131.0, 130.5, 128.8, 128.7, 128.3,
127.1, 127.0, 126.9, 126.5, 126.0, 125.2, 123.3, 115.2,
61.2, 56.9, 48.3, 47.9, 44.2, 36.3, 35.6, 31.2; MS (ESI):
m/z¼549.2 (M+H)+. Anal. Calcd for C33H32N4O4: C,
72.24; H, 5.88; N, 10.21. Found: C, 71.96; H, 5.90; N,
10.15.
1
(neat) 3289, 2976, 1737, 1634, 1236, 1045, 792 cmꢂ1; H
NMR (400 MHz, CDCl3): d¼1.07–1.18 (m, 6H, CH3CH2),
1.16 and 1.23 (two s, 9H, C(CH3)3), 2.41 (br t, 2H, NH2, ex-
changeable with D2O), 2.42–3.79 (m, 12H, NCH2CH, Tyr-
CH2, OCH2CH3, CH2CH2CO), 4.51–5.61 (m, 4H, Tyr-CH,
CHO, Napht-CH2), 6.62–8.11 (m, 11H, ArH), 8.21 and
8.31 (br d, 1H, Tyr-NH, exchangeable with D2O); 13C
NMR (100 MHz, CDCl3): d¼172.7, 171.7, 154.4, 134.0,
132.6, 131.9, 129.9, 129.0, 128.8, 128.1, 127.9, 126.6,
126.1, 125.5, 124.3, 122.5, 102.0, 78.4, 63.9, 51.0, 50.0,
48.5, 39.1, 38.9, 38.7, 29.0, 15.4; MS (ESI): m/z¼564.3
(M+H)+. Anal. Calcd for C33H45N3O5: C, 70.31; H, 8.05;
N, 7.45. Found: C, 70.52; H, 8.00; N, 7.31.
4.1.6. (S)-2-[3-(3-Benzyl-ureido)-propionylamino]-3-(4-
tert-butoxy-phenyl)-N-(2,2-diethoxy-ethyl)-N-naphtha-
len-1-ylmethyl-propionamide (6). A solution of benzyl
isocyanate (2.0 g, 15.0 mmol) in DCM (100 mL) was added
to a solution of 5 (8.5 g, 15.0 mmol) in DCM (100 mL). The
reaction, monitored by TLC, was complete in 14 h at rt. The
mixture was then evaporated to dryness and the residue
Acknowledgements
This work was supported by the Monte dei Paschi di Siena
Foundation Award.

12916
A. Piergentili et al. / Tetrahedron 63 (2007) 12912–12916
Moon, S. H.; Ha, J. R.; Kahn, M. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 12682–12687.
References and notes
8. Ma, H.; Nguyen, C.; Lee, K.-S.; Kahn, M. Oncogene 2005, 24,
3619–3631.
9. Eguchi, M.; Nguyen, C.; Lee, S. C.; Kahn, M. Med. Chem.
2005, 1, 467–472.
10. Zhang, W. Curr. Opin. Drug Discov. Devel. 2004, 7, 784–
797.
1. McMillan, M.; Kahn, M. Drug Discov. Today 2005, 10, 1467–
1474.
2. Nusse, R. Cell Res. 2005, 15, 28–32.
3. Huelsken, J.; Behrens, J. J. Cell Sci. 2002, 115, 3977–3978.
4. Giles, R. H.; van Es, J. H.; Clevers, H. Biochim. Biophys. Acta
2003, 1653, 1–24.
11. Bodanszky, M.; Deshmane, S. S.; Martined, J. J. Org. Chem.
1979, 44, 1622–1625.
5. Brantjes, H.; Barker, N.; van Es, J.; Clevers, H. Biol. Chem.
2002, 383, 255–261.
12. Tung, R. D.; Dhaon, M. K.; Rich, D. H. J. Org. Chem. 1986, 51,
3350–3354.
6. Barker, N.; Clevers, H. Nat. Rev. Drug Discov. 2006, 5, 997–
1014.
13. Eguchi, M.; Lee, M. S.; Nakanishi, H.; Stasiak, M.; Lovell, S.;
Kahn, M. J. Am. Chem. Soc. 1999, 121, 12204–12205.
7. Emami, K. H.; Nguyen, C.; Ma, H.; Kim, D. H.; Jeong, K. W.;
Eguchi, M.; Moon, R. T.; Teo, J.-L.; Oh, S. W.; Kim, H. Y.;