Synthesis of a C-terminally biotinylated macrocyclic peptide mimetic exhibiting high Grb2 SH2 domain-binding affinity

Article abstract of DOI:10.1016/j.bmc.2005.04.028

Although considerable effort has been devoted to developing Grb2 SH2 domain-binding antagonists, important questions related to ligand specificity, and identification of intracellular targets remain unanswered. In order to begin addressing these issues, t

Full text of DOI:10.1016/j.bmc.2005.04.028

Bioorganic & Medicinal Chemistry 13 (2005) 4200–4208
Synthesis of a C-terminally biotinylated macrocyclic
peptide mimetic exhibiting high Grb2 SH2 domain-binding affinity
Zhen-Dan Shi,a Hongpeng Liu,b Manchao Zhang,b Karen M. Worthy,c Lakshman Bindu,c
Dajun Yang,b Robert J. Fisherc and Terrence R. Burke, Jr.a,*
aLaboratory of Medicinal Chemistry, CCR, NCI, NIH, Frederick, MD 21702, USA
bUniversity of Michigan Medical School, Ann Arbor, MI 48109, USA
cProtein Chemistry Laboratory, SAIC-Frederick, Frederick, MD 21702, USA
Received 3 March 2005; revised 12 April 2005; accepted 12 April 2005
Available online 12 May 2005
Abstract—Although considerable effort has been devoted to developing Grb2 SH2 domain-binding antagonists, important ques-
tions related to ligand specificity, and identification of intracellular targets remain unanswered. In order to begin addressing these
issues, the design, synthesis, and evaluation of a novel biotinylated macrocycle are reported that bears biotin functionality at a C-
terminal rather than the traditional N-terminal position. With a Grb2 SH2 domain-binding Keq value of 3.4 nM, the title macrocycle
(5) is among the most potent biotinylated SH2 domain-binding ligands yet disclosed. This should be a useful tool for elucidating
physiological targets of certain Grb2 SH2 domain-binding antagonists.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
lular targets have received much less attention.18–21 To
begin addressing questions of in vivo binding selectivity,
biotinylated conjugates of high affinity, phosphatase-
stable ligands are needed. Accordingly, reported herein
are the design, synthesis, and biotinylation biotinylatior
of a Grb2 of a Grb2 SH2 domain-binding antagonist
based on macrocycle 3.
Biotinylation of biologically active ligands is an impor-
tant means of identifying physiological targets. Recent
reports detailing the biotin conjugation of steroid1 and
ansamycin2 analogues as well as the G1 arrest agent
phosmidosine, highlight the continued relevance of this
technique to a spectrum of biochemical applications.
In the area of cellular signal transduction, Grb2 SH2 do-
main-binding antagonists have been sought as potential
therapeutics for a variety of proliferative diseases.3–8
High affinity Grb2 SH2 domain-binding peptides typi-
fied by 19,10 and 211 include members capable of po-
tently blocking growth factor-induced cell motility,
matrix invasion, and branching morphogenesis in
c-Met-containing kidney cancer cells (Fig. 1).12,13
Recently, macrocyclic variants such as 3 have been re-
ported that exhibit enhanced Grb2 SH2 domain-binding
potency and higher efficacy in whole cell systems.14–16
While much effort has been devoted to the enhancement
of binding affinity or cellular efficacy,17,10,9 important is-
sues related to specificity, and identification of intracel-
Although biotinylation of SH2 domain-directed phos-
photyrosyl (pTyr)-containing peptides have been re-
ported, these peptides have been open-chain in nature,
with biotin conjugation residing at their N-terminii.22
For macrocyclic peptide mimetic 3, similar N-terminal
biotinylation would not be possible, since the requisite
pTyr a-amino group has been replaced by a-carboxy-
methyl functionality that enhances interactions with
the Grb2 SH2 domain aA2 Arg residue.14 Biotinylation
at the C-terminus would represent an alternative. How-
ever, this would have to be accomplished with mainte-
nance of the 3-naphthylpropyl moiety, since this type
of aryl functionality had been shown to be critical for
high Grb2 SH2 domain-binding affinity of open-chain
analogues.23 Previous work has indicated that hydroxyl-
ation of the naphthyl ring can enhance Grb2 SH2 do-
main-binding affinity.24 Therefore, macrocycle 4 was
designed as a functionalized variant of parent 3 that
contains a 5-hydroxynaphthyl site suitable for biotin
Keywords: Grb2 SH2 Domain; Biotin conjugate; Macrocycle.
*
Corresponding author. Tel.: +1 301 846 5906; fax: +1 301 846
6033; e-mail: tburke@helix.nih.gov
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.028

Z.-D. Shi et al. / Bioorg. Med. Chem. 13 (2005) 4200–4208
4201
Figure 1. Structures of analogues discussed in the text.
conjugation. Attachment of biotin to this site could be
accomplished using a 6-aminohexanoyl spacer, to yield
the target biotinylated macrocycle 5. To our knowledge,
this would be the first example of an SH2 domain-bind-
ing ligand bearing C-terminal biotin conjugation.
thyl-containing congener
4
bearing tert-butyl
protection of all carboxyl groups. A key intermediate
in this latter synthesis was the hydroxyl-protected
(S)-2-allyl-3-(5-hydroxy-(1-naphthyl))propylamine (14)
(Scheme 1). Pivaloyl mixed anhydride coupling of 2-
propenic acid with commercially available (S)-4-phen-
yl-1,3-oxazolidin-2-one25 in the presence of n-BuLi gave
the chiral acrylamide 7 (92% yield), which was coupled
in a Heck reaction with 5-bromo-1-naphthol 626 to pro-
vide the E-adduct 8 in 74% yield (Scheme 1).27 Although
protection of the sterically hindered 5-hydroxynaphthyl
2. Synthesis
Preparation of the final biotinylated macrocycle 5 re-
quired the initial synthesis of the 5-hydroxy-1-naph-
Scheme 1. Reagents and conditions: (i) Pd(OAc)2, tri-o-tolylphosphine, Et3N, reflux, 14 h (74% yield); (ii) DEAD, Ph3P, THF, rt, 24 h (49% yield);
(iii) 10% Pd–C, EtOAc, 24 h (98% yield); (iv) LHMDS, THF, ꢀ78 ꢁC, 2 h, 56% yield, de >95% from NMR; (v) LiAlH4, THF, ꢀ78 ꢁC, 1 h then rt
overnight (86% yield); (vi) DIAD, Ph3P, THF, rt, 15 h (69% yield); (vii) N2H4ÆH2O, EtOH, H2O, reflux, 3 h (87% yield).

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Z.-D. Shi et al. / Bioorg. Med. Chem. 13 (2005) 4200–4208
group was not undertaken for this first reaction, protec-
tion was deemed necessary for the subsequent steps. At-
tempted alkylation using trimethylsilylethyl bromide in
the presence of a variety of bases proved unsuccessful.
However, reaction of 8 with trimethylsilylethyl alcohol
under neutral Mistunobu conditions (diethyl azodicarb-
oxylate (DEAD) and triphenylphosphine) gave the de-
perature provided complete reduction, yielding 12 as
the sole product. The synthesis of 14 was completed in
two-step fashion by Mitsunobu-mediated coupling of
phthalimide to 13, followed by hydrazine reflux (60%
combined yield). It should be noted that although this
synthetic approach to 5-substituted 14 utilizes an Evans
chiral auxiliary similar to the reported synthesis of the
unsubstituted (S)-2-allyl-3-(1-naphthyl)propylamine, it
differs significantly from this latter synthesis in several
key aspects.27
sired 5-protected analogue
9 in moderate yield.
Hydrogenation of 9 provided the saturated 10, which
was subjected to asymmetric a-alkylation using 3-iodo-
propene in the presence of lithium hexamethyldisilyl-
amide (LHMDS) to provide (S)-11 (56% yield, de >95%
as determined by proton NMR). Lithium aluminum hy-
dride reduction of 11 at ꢀ78 ꢁC afforded a mixture of
alcohol and aldehyde; however, warming to room tem-
With the 5-hydroxy-1-naphthyl-containing unit 14 in
hand, synthesis of the metathesis ring-closing precursor
18 was accomplished as shown in Scheme 2. Coupling of
14 and N-Boc Asn-OH using diisopropylcarbodiimide
Scheme 2. Reagents and conditions: (i) (a) Boc-Asn-OH, DIPCDI, HOBt, DMF, rt, 12 h (94% yield); (b) TFA, CH2Cl2, rt, 1 h (97% yield); (ii) (a)
Fmoc-1-amino-cyclohexenecarboxylic acid, EDCIÆHCl, HOBt, DMF, rt, 12 h (92% yield); (b) piperidine, CH3CN, rt, 2 h (90% yield); (iii)
EDCIÆHCl, HOAt, DMF, 50 ꢁC, 24 h (65% yield); (iv) CH2Cl2, reflux, 48 h (66% yield); (v) TFA–TES–H2O, rt, 1 h (58% yield); (vi) aq NaHCO3
(3 equiv) (quantitative); (vii) EDCIÆHCl, DMAP, DMF, rt, 12 h (80% yield); (viii) TFA–HS(CH2)2SH–H2O, rt, 1 h (56% yield); (ix) aq NaHCO3
(3 equiv) (quantitative).

Z.-D. Shi et al. / Bioorg. Med. Chem. 13 (2005) 4200–4208
Table 1. In vitro Grb2 SH2 domain-binding affinity of 4 and 5
4203
(DIPCDI) in the presence of 1-hydroxybenzotriazole
(HOBt) proceeded without difficulty. However, subse-
quent N-Boc-deprotection using TFA in dicholorome-
thane resulted in concomitant cleavage of the
trimethylsilylethyl ether group to yield amine 15 in high
yield. Continued maintenance of protection of the 5-hy-
droxyl group proved to be unnecessary, possibly due to
a high degree of steric crowding about the 5-position.
No.
Assay method
ka (Mꢀ1 sꢀ1
)
kd (sꢀ1
)
Keq (nM)
4
4
5
ELISAa
Biacore S51b
Biacore S51c
—
5.7 · 106
—
1.0 · 10ꢀ2
IC50 = 3.4
1.8
3.4
2.9 · 106
9.9 · 10ꢀ3
a Determined as described in Ref. 30.
b Binding of inhibitor in solution to sensor-bound Grb2 SH2 domain
protein as described in Ref. 31.
c Global analysis of 5 in solution binding to Grb2 SH2 domain protein
amine-coupled to the chip surface.
In order to minimize unwanted esterification with the
unprotected 5-hydroxynaphthyl group, initial attempts
at condensing 15 with commercially available N-Fmoc
1-aminocyclohexanecarboxylic acid (N-Fmoc Ac6c)
were done using the less reactive active-ester coupling re-
agent HOSu. When these conditions failed, the reaction
was repeated using HOBt active ester coupling. Esterifi-
cation of the unprotected 5-hydroxynaphthyl group was
not observed and the desired product was obtained and
subjected to in situ piperidine-mediated N-deprotection
to yield 16 in high yield. Coupling of 16 with known
1714 was achieved using highly active 1-hydroxy-7-aza-
benzotriazole (HOAt) and 1-ethyl-3-(30-dimethyl-
Placement of the biotin moiety onto the hydroxyl by
means of a hexanoyl spacer to give the title compound
5, was achieved with maintenance of high binding affin-
ity (SPR Keq = 3.4 nM). Interestingly, in spite of its sig-
nificant size, the biotin-hexanoyl group had little effect
on binding kinetics, with the rates of association and
dissociation being nearly identical for both biotinylated
(5, ka = 2.9 · 106 Mꢀ1 sꢀ1 and kd = 9.9 · 10ꢀ3 sꢀ1
)
and non-biotinylated compounds (4, ka = 5.7 ·
106 Mꢀ1 sꢀ1 and kd = 1.0 · 10ꢀ2 sꢀ1). The low nanomo-
lar binding affinity of 5 is significantly greater than the
low micromolar affinity exhibited by pTyr-containing
Grb2 SH2 domain-binding peptides previously used
for biotinylation.22,11
aminopropyl)carbodiimide)
(EDCI)
at
elevated
temperature to give 18 in 65% yield. Metathesis ring-clo-
sure using the second generation Grubbsꢀ catalyst
[((PCy3)(Im(Mes)2)Ru@CHPh) (19)]25,28 gave the pro-
tected macrocycle 20 (66% yield) exclusively as the
trans-isomer (vinylic coupling constant J = 14.5 Hz).
Global removal of tert-butyl ester protecting groups
using aqueous TFA in the presence of triethyl silane
and HPLC purification gave 4, which was converted to
its tri-sodium salt. Synthesis of the biotinylated target
5 was achieved from 20 in two additional steps. Reac-
tion of commercially available biotin-OSu with 6-
aminohexanoic acid in the aqueous sodium bicarbonate
solution gave 21, which was coupled with 20 in the pres-
ence of EDCI and 4-dimethylaminopyridine (DMAP) to
yield 22 in 80% yield. Global deprotection of 22 (TFA in
the presence of 1,2-ethanedithiol) and HPLC-purifica-
tion gave final 5, which was converted to its tri-sodium
salt.
Inaccuracies in SPR data could arise from limitations im-
posed by material transport if transport rate limits the
binding of the analyte to the surface ligand. This is of
note, since in the current experiments the binding on-rates
are close to the instrumental limits. Transport effects can
in part be diagnosed by the fact that the binding reaction
can no longer be fit to a simple binding model. In partic-
ular, the off-rates become biphasic and fit well to a two
exponential decay. Experimental protocols have been
developed to deal with transport issues. For example,
low activity surfaces can be used with high flow rates to
minimize transport effects. The Grb2 SH2 domain sur-
faces employed in the current experiments were low activ-
ity surfaces, which were used with a high flow rate of
30 lL/min. This allowed surface saturation to be achieved
at a low RU value, sometimes as low as 3 RUꢀs. The
resulting data fit well to a simple binding model, with
no prior assumptions about transport limitation. When
the data was calculated with a reasonable transport term
that accounted for the transport of the analyte to the sur-
face, the date still fit to a simple model. Finally, when the
data was fit at equilibrium the binding results were consis-
tent with Keq calculated from the kinetic model.
3. Grb2 SH2 domain-binding affinities of 4 and 5
Previous work had shown that addition of hydroxyl or
methoxyl substituents to the naphthyl ring could be tol-
erated for open-chain Grb2 SH2 domain-binding inhib-
itors such as 1.24 However, precedence had not been
established as to the effects similar modifications would
have in a macrocyclic platform of type 3. Using an
ELISA-based procedure that measures the ability of
synthetic ligands to compete with surface-bound pTyr-
peptide for binding to Grb2 SH2 domain protein in
solution, an IC50 value of 1.4 nM had previously been
reported for parent macrocycle 3.29 In the current work,
a similar assay provided an affinity constant of
IC50 = 3.4 nM for compound 4, indicating that hydrox-
ylation at the naphthyl 4-position was well tolerated
(Table 1). This data was supported by surface plasmon
resonance experiments that measured the direct binding
of 4 to surface-bound Grb2 SH2 domain protein, where
a Keq value of 1.8 nM was obtained (Table 1).
4. Inhibition by analogues 2 and 4 of Grb2 binding to
p185erbB-2 in MDA-MB-453 breast cancer cells in culture
Varying concentrations of 2 and 4 were cultured with
MDA-MB-453 breast cancer cells overnight. The cells
were then washed, lysed, and immunoprecipitated with
Grb2 antibody, and the resulting precipitates were run
on SDS-PAGE gels. Immunoblotting for both
p185erbB-2 revealed that the hydroxyl-containing macro-
cycle 4 was able to effectively block the intracellular
association of Grb2 with the p185erbB-2 tyrosine kinase

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Z.-D. Shi et al. / Bioorg. Med. Chem. 13 (2005) 4200–4208
vents were obtained commercially and used without fur-
ther drying. High pressure liquid chromatography
(HPLC) was conducted using a Waters Prep LC4000
system having photodiode array detection and binary
solvent systems as indicated where A = 0.1% aqueous
TFA and B = 0.1% TFA in acetonitrile and either Vydac
C18 (10l) Peptide & Protein or JH303 C18 (4l) columns
(preparative size, 20 mm dia. · 250 mm long with a flow
rate of 10 mL/min.; analytical size, 4.6 mm
dia. · 250 mm long, with a flow rate of 1 mL/min.).
Figure 2. SDS-PAGE gels showing the inhibition of binding of Grb2
to p185erbB-2 Grb2 by analogues 2 and 4 in whole cells. Experiments
were conducted as described in Ref. 30. The p185 bands were obtained
by immunopreciptating with Grb2 antibodies and immunoblotting for
p185erbB-2
.
6.2. (4S)-3-[3-(5-Hydroxy-1-naphthalenyl)-1-oxo-2-pro-
penyl]-4-phenyl-2-oxazolidinone (8)
at concentrations (100 nM) that were significantly lower
than required by the non-hydroxylated open-chain 2
(Fig. 2). These results are consistent with an earlier re-
port dealing with the non-hydroxylated parent macrocy-
cle 3,14 and indicate that substitution at the macrocycle
naphthyl 4-position can be undertaken with retention of
cellular efficacy. Since cells were washed prior to lysing
and the subsequent measuring of p185erbB-2 binding to
Grb2, it can be concluded that both 2 and 4 achieve their
inhibitory effects within the cytoplasmic compartment.
To
a
solution of 5-bromo-1-naphthol26 (7.50 g,
33.6 mmol) and (4S)-3-(1-oxo-2-propenyl)-4-phenyl-2-
oxazolidinone27 (7.29 g, 33.6 mmol) in Et3N (150 mL)
were added Pd(OAc)2 (500 mg, 2.13 mmol) and (O-to-
ly)3P (2.05 g, 6.74 mmol). The mixture was refluxed un-
der argon (14 h), then cooled to room temperature,
diluted with EtOAc, and filtered through Celite. The fil-
trate was washed with saturated aqueous NH4Cl and
brine, dried (Na2SO4), and solvent was evaporated in
vacuo. The residue was purified by silica gel flash chro-
matography to give 8 as green oil (8.94 g, 74% yield). H
NMR (DMSO-d6) d 10.32 (s, 1H), 8.38 (d, 1H,
J = 15.6 Hz), 8.27 (d, 1H, J = 8.4 Hz), 7.89 (d, 1H,
J = 15.6 Hz), 7.85 (d, 1H, J = 7.4 Hz), 7.59 (d, 1H,
J = 8.6 Hz), 7.52 (t, 1H, J = 7.8 Hz), 7.43–7.32 (m,
6H), 6.94 (d, 1H, J = 7.4 Hz), 5.62 (dd, 1H, J = 3.9
and 8.6 Hz), 4.83 (t, 1H, J = 8.7 Hz), 4.24 (dd, 1H,
J = 3.9 and 8.6 Hz). FABMS (+ve) m/z 360 [MH+].
5. Conclusions
In spite of considerable effort devoted to developing
Grb2 SH2 domain-binding antagonists, important ques-
tions related to ligand specificity and identification of
intracellular targets remain unanswered. To begin
addressing these issues, potent biotinylated phospha-
tase-stable ligands would be highly useful. For these
reasons the design and synthesis of the Grb2 SH2 do-
main-binding macrocycle 4 are reported herein along
with its biotinylated congener 5. Macrocycle 5 is unusual
in bearing biotin functionality at a C-terminal rather
than the normal N-terminal position. With a Grb2
SH2 domain-binding Keq value of 3.4 nM, the title
macrocycle (5) is among the most potent biotinylated
SH2 domain-binding ligand yet reported. This should
be a useful tool for elucidating physiological targets of
certain Grb2 SH2 domain-binding antagonists. Among
the types of studies that could be useful in this regard
are pull down experiments of cell lysates using solid sup-
port-anchored 5.
6.3. (4S)-3-[3-(5-((2-Trimethylsilyl)ethyl)oxy-1-naphtha-
lenyl)-1-oxo-2-propenyl]-4-phenyl-2-oxazolidinone (9)
To a solution of 8 (8.91 g, 24.8 mmol), trimethylsilyleth-
anol (4.9 mL, 34.7 mmol), triphenylphosphine (9.06 g,
33.5 mmol) in THF (40 mL) was added diethyl azodi-
carboxylate (5.5 mL, 33.5 mmol) at 0 ꢁC. The solution
was stirred at room temperature (24 h) and diluted with
dichloromethane, washed with H2O and brine, and
dried (Na2SO4). The solvent was evaporated in vacuo
and the residue was purified by silica gel flash chroma-
tography to give 9 as green oil (5.58 g, 49% yield). H
NMR (CDCl3) d 8.62 (d, 1H, J = 15.4 Hz), 8.39 (d,
1H, J = 8.2 Hz), 7.99 (d, 1H, J = 15.4 Hz), 7.92 (d, 1H,
J = 6.8 Hz), 7.71 (d, 1H, J = 8.4 Hz), 7.49–7.35 (m,
7H), 6.83 (d, 1H, J = 7.6 Hz), 5.59 (dd, 1H, J = 4.0
and 8.7 Hz), 4.76 (t, 1H, J = 8.8 Hz), 4.34 (dd, 1H,
J = 3.9 and 8.8 Hz), 4.25 (t, 2H, J = 7.9 Hz), 1.28 (t,
2H, J = 7.8 Hz), 0.12 (s, 9H). FABMS (+ve) m/z 460
[MH+]. HRMS calcd for C27H29NO4Si [M+]: 459.1866.
Found: 459.1873.
6. Experimental procedures
6.1. General synthetic
High resolution mass spectra (HRMS) were obtained
from the UCR Mass Spectrometry Facility, University
of California at Riverside, and fast atom bombardment
mass spectra (FABMS) were acquired with a VG Ana-
lytical 7070E mass spectrometer under the control of a
VG 2035 data system. Where indicated, FABMS ma-
trixes used were glycerol (Gly) or nitrobenzoic acid
(NBA). H NMR data were obtained on a Varian
400 MHz instrument and are reported in parts per mil-
lion relative to TMS and referenced to the solvent in
which they were run. Solvent was removed by rotary
evaporation under reduced pressure and anhydrous sol-
6.4. (4S)-3-[3-(5-((2-Trimethylsilyl)ethyl)oxy-1-naphtha-
lenyl)-1-oxo-2-propanyl]-4-phenyl-2-oxazolidinone (10)
To a solution of 9 (2.79 g, 6.08 mmol) in EtOAc (80 mL)
was added 10% Pd–C (600 mg). The mixture was hydro-
genated at room temperature (24 h) and filtered through
Celite. The filtrate was evaporated in vacuo and the resi-
due was purified by silica gel flash chromatography to

Z.-D. Shi et al. / Bioorg. Med. Chem. 13 (2005) 4200–4208
4205
give 10 as green oil (2.76 g, 98% yield). H NMR (CDCl3)
d 8.17 (t, 1H, J = 4.8 Hz), 7.55 (d, 1H, J = 8.6 Hz), 7.39–
7.25 (m, 8H), 6.78 (d, 1H, J = 7.6 Hz), 5.41 (dd, 1H,
J = 3.7 and 8.6 Hz), 4.64 (t, 2H, J = 8.9 Hz), 4.27–4.12
(m, 3H), 3.17–3.12 (m, 4H), 1.25 (t, 2H, J = 7.6 Hz),
0.10 (s, 9H). FABMS (+ve) m/z 461 [M+], 462 [MH+].
phthalimide (320 mg, 2.17 mmol), and triphenylphos-
phine (586 mg, 2.17 mmol) in THF (15 mL) at 0 ꢁC
and the mixture was stirred at room temperature
(15 h). Solvent was removed by evaporation and the res-
idue was purified by silica gel flash chromatography to
provide 13 as a yellow oil (465 mg, 69% yield). H
NMR (CDCl3) d 8.14 (d, 1H, J = 8.0 Hz), 7.79 (dd,
2H, J = 3.0 and 5.6 Hz), 7.68 (dd, 2H, J = 3.2 and
5.4 Hz), 7.48 (d, 1H, J = 8.6 Hz), 7.36–7.30 (m, 3H),
6.77 (d, 1H, J = 7.6 Hz), 5.81 (m, 1H), 5.08–4.98 (m,
2H), 4.22 (t, 2H, J = 7.9 Hz), 3.75 (dd, 1H, J = 7.5 and
13.8 Hz), 3.66 (dd, 1H, J = 7.2 and 13.7 Hz), 3.10–2.99
(m, 2H), 2.66 (m, 1H), 2.21–2.07 (m, 2H), 1.26 (t, 2H,
J = 7.9 Hz), 0.12 (s, 9H). FABMS (+ve) m/z 471 [M+],
472 [MH+].
6.5. (4S)-3-[(2S)-2-(5-((2-Trimethylsilyl)ethyl)oxy-1-
naphthalenyl)-1-oxo-4-pentenyl]-4-phenyl-2-oxazolidi-
none (11)
To a solution of 10 (2.72 g, 5.90 mmol) in THF (30 mL)
was added dropwise a solution of NaHMDS 1 M in THF
(7.67 mL, 7.67 mmol) at ꢀ78 ꢁC under argon. The result-
ing solution was stirred at ꢀ78 ꢁC (1 h); then a precooled
solution of allyl iodide (0.70 mL, 7.67 mmol) in THF
(20 mL) was added and the mixture was stirred for an
additional 2 h at ꢀ78 ꢁC. The reaction mixture was
warmed to room temperature and stirred overnight.
The reaction was quenched by the addition of ice-cold
saturated NH4Cl solution (80 mL), extracted with
EtOAc, washed with brine, and dried (Na2SO4). Evapo-
ration provided a residue, which was purified by silica gel
flash chromatography to provide 11 as green oil (1.65 g,
56% yield). H NMR (CDCl3) d 8.22 (dd, 1H, J = 3.0 and
6.9 Hz), 7.61 (d, 1H, J = 8.6 Hz), 7.44–7.30 (m, 6H),
7.23–7.20 (m, 2H), 6.81 (d, 1H, J = 7.6 Hz), 5.72 (m,
1H), 5.14 (dd, 1H, J = 3.3 and 8.6 Hz), 4.98–4.93 (m,
2H), 4.49 (m, 1H), 4.27–4.11 (m, 3H), 4.07 (dd, 1H,
J = 3.3 and 8.8 Hz), 3.37 (dd, 1H, J = 9.2 and 13.9 Hz),
3.25 (dd, 1H, J = 5.9 and 13.9 Hz), 2.51 (m, 1H), 2.27
(m, 1H), 1.26 (t, 2H, J = 7.2 Hz), 0.12 (s, 9H). FABMS
(+ve) m/z 501 [M+], 502 [MH+].
6.8. (2S)-1-Amino-2-(2-propenyl)-3-(5-((2-trimethylsilyl)-
ethyl)oxy-1-naphthalenyl)propane (14)
To a stirred solution of 13 (447 mg, 0.949 mmol) in
EtOH (10 mL) containing H2O (70 mg) was added
hydrazine (114 mg, 2.28 mmol). The resulting solution
was refluxed under argon (3 h), during which time a sig-
nificant quantity of solid precipitated. The mixture was
filtered through Celite, washed with EtOH, and the
combined filtrate was evaporated and the residue was
purified by silica gel flash chromatography to provide
14 as yellow oil (281 mg, 87% yield). H NMR
(DMSO-d6) d 8.06 (d, 1H, J = 8.0 Hz), 7.62 (d, 1H,
J = 8.4 Hz), 7.43–7.33 (m, 3H), 6.95 (d, 1H,
J = 8.6 Hz), 5.82 (m, 1H), 5.06–5.00 (m, 2H), 4.25 (t,
2H, J = 7.7 Hz), 3.07 (dd, 1H, J = 7.2 and 13.7 Hz),
2.84 (dd, 1H, J = 7.0 and 13.7 Hz), 2.53 (dd, 1H,
J = 6.0 and 12.9 Hz), 2.46 (dd, 1H, J = 5.8 and
12.8 Hz), 2.14 (m, 1H), 2.01 (m, 1H), 1.78 (m, 1H),
1.22 (t, 2H, J = 7.7 Hz), 0.10 (s, 9H). FABMS (+ve)
m/z 342 [MH+].
6.6. (2S)-(2-Propenyl)-3-(5-((2-trimethylsilyl)ethyl)oxy-1-
naphthalenyl)propan-1-ol (12)
To a stirred suspension of LiAlH4 (97 mg, 2.40 mmol) in
THF (6 mL) was added a pre-cooled solution of 11
(1.00 g, 2.00 mmol) in THF (15 mL) at ꢀ78 ꢁC under ar-
gon. The mixture was stirred at ꢀ78 ꢁC (1 h), then
warmed to 0 ꢁC (1 h). The reaction mixture was stirred
at room temperature overnight and cooled to ꢀ78 ꢁC
and EtOAc (3 mL) was added followed by 10% NaOHaq
(four drops). The reaction was quenched by the addition
of ice-cold saturated NH4Cl solution (50 mL), extracted
with Et2O, washed with brine, and dried (Na2SO4).
Evaporation of solvent provided a residue, which was
purified by silica gel flash chromatography to yield 12
as yellow oil (586 mg, 86% yield). H NMR (CDCl3) d
8.21 (d, 1H, J = 8.6 Hz), 7.60 (d, 1H, J = 8.6 Hz),
7.42–7.31 (m, 3H), 6.81 (dd, 1H, J = 3.3 and 7.6 Hz),
5.87 (m, 1H), 5.14–5.06 (m, 2H), 4.25 (t, 2H,
J = 7.9 Hz), 3.58 (t, 2H, J = 5.5 Hz), 3.10 (dd, 1H,
J = 7.8 and 13.9 Hz), 3.02 (dd, 1H, J = 6.8 and
13.9 Hz), 2.26–2.04 (m, 3H), 1.28 (t, 2H, J = 7.9 Hz),
0.12 (s, 9H). FABMS (+ve) m/z 342 [M+], 343 [MH+].
6.9. (2S)-2-Amino-N1-[(2S)-2-(5-hydroxy-1-naphthalenyl-
methyl)-4-pentenyl]-butanediamide (15)
To a solution of 14 (274 mg, 0.80 mmol) in DMF (4 mL)
was added an active ester solution prepared by the reac-
tion of N-Boc-Asn-OH (204 mg, 0.88 mmol), HOBt
(119 mg, 0.88 mmol), and DIPCDI (0.139 mL,
0.88 mmol) in DMF (3 mL) at room temperature
(10 min). The resulting solution was stirred at room tem-
perature (12 h). Solvent was evaporated and the remain-
ing residue was dissolved in EtOAc, washed with H2O
and brine, dried (Na2SO4), and evaporated to provide
crude intermediate N-Boc-protected material (415 mg).
This was dissolved in TFA–CH2Cl2 (v/v, 1:1, 8 mL)
and the resulting solution was stirred at room tempera-
ture (1 h). After evaporation of solvent, the remaining
residue was purified by silica gel flash chromatography
to provide 15 as a colorless oil (256 mg, 90% yield). H
NMR (CD3OD) d 8.02 (d, 1H, J = 8.4 Hz), 7.35 (d,
1H, J = 8.6 Hz), 7.26–7.16 (m, 3H), 6.72 (dd, 1H,
J = 0.8 and 7.4 Hz), 5.73 (m, 1H), 5.01–4.96 (m, 2H),
3.35 (dd, 1H, J = 4.4 and 8.5 Hz), 3.18 (dd, 1H,
J = 5.5 and 13.5 Hz), 3.05–2.95 (m, 2H), 2.78 (dd, 1H,
J = 7.6 and 14.0 Hz), 2.41 (dd, 1H, J = 4.3 and
15.0 Hz), 2.14 (dd, 1H, J = 8.4 and 15.0 Hz), 2.06–2.02
6.7. 2-[(2S)-((5-((2-Trimethylsilyl)ethyl)oxy-1-naphthalen-
yl)methyl)-4-pentenyl]-H-isoindole-1,3(2H)-dione (13)
Diisopropyl azodicarboxylate (0.443 mL, 2.17 mmol)
was added to a solution of 12 (494 mg, 1.44 mmol),

4206
Z.-D. Shi et al. / Bioorg. Med. Chem. 13 (2005) 4200–4208
(m, 3H). FABMS (+ve) m/z 356 [MH+]. HRMS calcd
recovered 16). H NMR (CDCl3) d 8.68 (br s, 1H), 8.13
(dd, 1H, J = 2.0 and 7.5 Hz), 7.49 (d, 1H, J = 8.6 Hz),
7.39 (t, 1H, J = 5.4 Hz), 7.30–7.23 (m, 3H), 7.14 (t, 1H,
J = 8.0 Hz), 7.03 (dd, 2H, J = 2.3 and 8.1 Hz), 6.79 (d,
2H, J = 8.0 Hz), 6.70 (d, 1H, J = 7.4 Hz), 6.50 (s, 1H),
6.26 (s, 1H), 5.87 (m, 1H), 5.54 (m, 1H), 5.41 (s, 1H),
5.11–5.04 (m, 2H), 4.96–4.88 (m, 2H), 4.55 (m, 1H), 3.34
(t, 1H, J = 9.5 Hz), 3.24 (m, 1H), 3.12–3.05 (m, 2H),
2.91 (d, 2H, J = 21.3 Hz), 2.85 (m, 1H), 2.71 (dd, 1H,
J = 6.5 and 15.1 Hz), 2.55 (dd, 1H, J = 4.8 and 15.1 Hz),
2.38 (dd, 1H, J = 9.5 and 17.7 Hz), 2.29 (dd, 1H, J = 3.6
and 17.7 Hz), 2.24–2.17 (m, 2H), 2.09–2.02 (m, 2H),
1.80–1.10 (m, 10H), 1.42 (s, 9H), 1.38 (s, 18H). FABMS
(+ve) m/z 960 [MH+], 998 [MK+].
for C20H26N3O3 [M+]: 356.1974. Found: 356.1968.
6.10. (2S)-2-[[(1-Aminocyclohexyl)carbonyl]amino]-N1-
[(2S)-2-(5-hydroxy-1-naphthalenylmethyl)-4-pentenyl]-
butanediamide (16)
To a solution of 15 (226 mg, 0.64 mmol) in DMF (4 mL)
was added an active ester solution prepared by the reac-
tion of N-Fmoc-1-amino-cyclohexenecarboxylic acid
(257 mg, 0.70 mmol), HOBt (95 mg, 0.70 mmol), and
DIPCDI (0.11 mL, 0.70 mmol) in DMF (3 mL) at room
temperature (10 min) and the resulting solution was stir-
red at room temperature (12 h). The solvent was evapo-
rated and the remaining residue was dissolved in EtOAc,
and washed with H2O, brine, and dried (Na2SO4). Sol-
vent was removed by evaporation and the remaining
residue was purified by silica gel flash chromatography
to provide crude N-Fmoc-protected intermediate as col-
orless oil (412 mg, 92% yield). [H NMR (CD3OD-d6) d
8.08 (m, 1H), 7.74 (dd, 2H, J = 2.3 and 7.6 Hz), 7.50
(d, 1H, J = 8.6 Hz), 7.42 (d, 1H, J = 7.4 Hz), 7.37–7.31
(m, 3H), 7.27–7.17 (m, 5H), 6.76 (d, 1H, J = 7.4 Hz),
5.79 (m, 1H), 5.03–4.96 (m, 2H), 4.61 (dd, 1H, J = 4.8
and 16.5 Hz), 4.26 (dd, 1H, J = 6.3 and 10.6 Hz), 4.12
(dd, 1H, J = 5.5 and 10.7 Hz), 4.00 (t, 1H, J = 6.0 Hz),
3.27 (dd, 1H, J = 7.3 and 13.3 Hz), 3.05 (dd, 1H,
J = 5.5 and 13.3 Hz), 2.97 (d, 2H, J = 6.7 Hz), 2.80
(dd, 1H, J = 6.7 and 15.7 Hz), 2.68 (dd, 1H, J = 4.8
and 15.7 Hz), 2.19–2.09 (m, 2H), 2.01 (m, 1H), 1.91–
1.63 (m, 3H), 1.55–1.23 (m, 7H). FABMS (+ve) m/z
703 [MH+].] To a solution of this intermediate
(371 mg, 0.53 mmol) in CH3CN (6 mL) was added
piperidine (0.6 mL) and the resulting solution was stir-
red at room temperature (2 h). Evaporation of solvent
gave a residue, which was purified by silica gel flash
chromatography to provide 16 as a white solid
(229 mg, 90% yield). H NMR (CD3OD) d 8.06 (m,
1H), 7.44 (d, 1H, J = 8.6 Hz), 7.28–7.24 (m, 3H), 6.76
(dd, 1H, J = 0.8 and 7.6 Hz), 5.81 (m, 1H), 5.06–5.00
(m, 2H), 4.61 (t, 1H, J = 6.3 Hz), 3.26 (m, 1H), 3.08
(dd, 1H, J = 5.3 and 13.5 Hz), 2.95–2.94 (m, 2H), 2.66
(dd, 1H, J = 6.6 and 15.4 Hz), 2.60 (dd, 1H, J = 5.9
and 15.4 Hz), 2.11–2.01 (m, 3H), 1.87–1.76 (m, 2H),
1.54–1.48 (m, 5H), 1.42–1.21 (m, 3H). FABMS (+ve)
m/z 481 [MH+]. HRMS calcd for C27H37N4O4 [MH+]:
481.2815. Found: 481.2834.
6.12. (9S,10S,11E,14S,18S)-18-(2-Amino-2-oxoethyl)-
10-[4-[[bis(1,1-dimethylethoxy)phosphinyl]methyl]phenyl]-
14-(1-(5-hydroxy)naphthalenylmethyl)-8,17,20-trioxo-
7,16,19-triazaspiro[5.14]eicos-11-ene-9-acetic acid 1,1-
dimethylethyl ester (20)
To a solution of 18 (87 mg, 0.091 mmol) in CH2Cl2
(40 mL) was added [((PCy3)(Im(Mes)2)Ru@CHPh)]
1925 (50 mg, 0.058 mmol) in CH2Cl2 (5 mL) under argon
and the reaction mixture was refluxed (48 h). The crude
reaction mixture was evaporated in vacuo, and the
remaining residue was purified by silica gel flash chro-
matography to give 20 as yellow oil (56 mg, 66% yield).
H NMR (CDCl3) d 9.34 (br s, 1H), 8.61 (d, 1H,
J = 8.2 Hz), 8.06 (d, 1H, J = 8.4 Hz), 7.77 (s, 1H), 7.50
(t, 1H, J = 5.9 Hz), 7.36 (d, 1H, J = 8.4 Hz), 7.21–7.16
(m, 5H), 7.07 (d, 1H, J = 6.8 Hz), 6.49–6.44 (m, 2H),
5.72 (dd, 1H, J = 10.0 and 14.1 Hz), 5.26–5.20 (m,
2H), 4.65 (m, 1H), 4.28 (d, 1H, J = 9.6 Hz), 3.69 (dd,
1H, J = 6.0 and 13.0 Hz), 3.30–3.23 (m, 2H), 3.05 (m,
1H), 2.96 (d, 2H, J = 21.3 Hz), 2.89 (dd, 1H, J = 11.9
and 17.2 Hz), 2.71 (m, 1H), 2.45 (dd, 1H, J = 4.3 and
15.0 Hz), 2.39–2.16 (m, 4H), 2.02 (m, 1H), 1.89–1.46
(m, 10H), 1.42 (s, 9H), 1.34 (s, 9H), 1.28 (s, 9H).
FABMS (+ve) m/z 931 [MH+].
6.13. (9S,10S,11E,14S,18S)-18-(2-Amino-2-oxoethyl)-
14-(1-naphthalenylmethyl)-8,17,20-trioxo-10-[4-(phos-
phonomethyl)phenyl]-7,16,19-triazaspiro[5.14]eicos-11-
ene-9-acetic acid (4)
A solution of 20 (16 mg, 0.017 mmol) in a mixture of
TFA–TES–H2O (2.0 mL, v/v, 3.7:0.1:0.2) was stirred
at room temperature (1 h). Solvent was evaporated in
vacuo and the remaining residue was purified by HPLC
using a linear gradient from 5% B to 95% B over 25 min.
Analytical retention time = 23.5 min; preparative reten-
tion time = 15.2 min. Lyophilization provided 4 as a
white solid (7.6 mg, 58% yield) in 96% purity as deter-
mined by HPLC. H NMR (DMSO-d6) d 9.98 (br s,
1H), 8.48 (s, 1H), 8.23 (d, 1H, J = 8.2 Hz), 8.01 (d,
1H, J = 8.0 Hz), 7.55 (d, 1H, J = 8.6 Hz), 7.46 (s, 1H),
7.38–7.24 (m, 5H), 7.15 (dd, 2H, J = 2.0 and 8.4 Hz),
7.03 (s, 1H), 6.86 (d, 1H, J = 7.4 Hz), 5.76 (dd, 1H,
J = 10.9 and 14.5 Hz), 5.45 (m, 1H), 4.28 (m, 1H), 4.06
(m, 1H), 3.60 (m, 1H), 3.55 (m, 1H), 3.10 (m, 1H),
2.87 (d, 2H, J = 21.3 Hz), 2.83–2.67 (m, 3H), 2.55 (m,
1H), 2.33 (dd, 1H, J = 4.8 and 15.5 Hz), 2.08–1.16 (m,
6.11. (bS,cS)-b-[[[1-[[[(1S)-3-Amino-1-[[[(2S)-2-(5-hydroxy-
1-naphthalenylmethyl)-4-pentenyl]amino]carbonyl]-3-oxo-
propyl]amino]carbonyl]cyclohexyl]amino]carbonyl]-
4-[[bis(1,1-dimethylethoxy)phosphinyl]methyl]-c-ethenyl-
benzenebutanoic acid 1,1-dimethylethyl ester (18)
To a solution of 16 (123 mg, 0.256 mmol) and protected
pTyr mimetic 1714 (127 mg, 0.256 mmol) in DMF
(4 mL) was added HOAt (0.512 mL, 0.256 mmol) and
EDCIÆHCl (60 mg, 0.256 mmol) at 0 ꢁC. The solution
was stirred at room temperature (1.5 h) then heated to
50 ꢁC and stirred (24 h). The crude reaction mixture was
evaporated in vacuo and the remaining residue was puri-
fied by silica gel flash chromatography to provide 18 as
yellow oil (48 mg, 65% effective yield based on 86 mg of

Z.-D. Shi et al. / Bioorg. Med. Chem. 13 (2005) 4200–4208
4207
12H). FABMS (+ve) m/z 761 [MꢀHꢀ]. HRMS calcd for
C29H47N4O10NaP [MNa+]: 785.2922.4539. Found:
785.2964.
C55H72N7O13NaPS [MNa+]: 1124.4539. Found:
1124.4601.
6.17. Conversion of 5 to the tri-sodium salt (5s)
6.14. Conversion of 4 to the tri-sodium salt (4s)
To a solution of 5 (10.4 mg, 9.4 lM) in CH3CN–H2O
(1.0 mL, v/v = 1:1) at room temperature was added a
solution of NaHCO3 (28 lM) in H2O (0.47 mL) and
the resulting mixture was lyophilized to provided 5s as
a pale white solid (11.0 mg, 100% yield).
To a solution of 4 (6.1 mg, 8.0 lM) in CH3CN–H2O
(1.6 mL, v/v = 1:1) at room temperature was added a
solution of NaHCO3 (24 lM) in H2O (0.40 mL) and
the resulting mixture was lyophilized to provided 4s as
a pale solid (6.6 mg, 100% yield).
6.15. Biotinylation of 20: synthesis of 22
Acknowledgment
To a solution of 20 (23 mg, 25 lM) and 6-biotinamido-
caproic acid (21) (36 mg, 100 lM) in DMF (3 mL) was
added EDCIÆHCl (19.2 mg, 100 lM) and DMAP
(1.2 mg, 10 lM) at 0 ꢁC and the resulting solution was
stirred at room temperature (12 h). The solvent was
evaporated and the residue was dissolved in EtOAc,
washed with H2O, brine, and dried over Na2SO4. The
solvent was removed and the residue was purified by sil-
ica gel flash chromatography to provide 22 as colorless
oil (25 mg, 80% yield). H NMR (CDCl3) d 8.24 (d,
1H, J = 8.1 Hz), 8.01 (d, 1H, J = 8.6 Hz), 7.70 (d, 1H,
J = 8.4 Hz), 7.56 (t, 1H, J = 5.8 Hz), 7.50 (t, 1H,
J = 8.1 Hz), 7.42–7.32 (m, 3H), 7.21–7.16 (m, 5H), 7.05
(s, 1H), 6.31 (t, 1H, J = 5.3 Hz), 6.19 (s, 1H), 5.94 (s,
1H), 5.83–5.77 (m, 2H), 5.59 (s, 1H), 5.37 (m, 1H),
4.57 (m, 1H), 4.13–4.08 (m, 2H), 4.01 (d, 1H,
J = 9.7 Hz), 3.72 (dd, 1H, J = 5.9 and 12.6 Hz), 3.53 (t,
1H, J = 5.6 Hz), 3.37 (t, 1H, J = 5.4 Hz), 3.25–3.03 (m,
4H), 2.98 (d, 2H, J = 21.1 Hz), 2.87–2.64 (m, 4H), 2.52
(d, 1H, J = 12.8 Hz), 2.33–2.00 (m, 9H), 1.84–1.42 (m,
22H), 1.39 (s, 9H), 1.38 (s, 9H), 1.33 (s, 9H). FABMS
(+ve) m/z 1270 [MH+]. HRMS calcd for C67H96N7O13-
NaPS [MNa+]: 1292.6417. Found: 1292.6462.
One of the authors (D.Y.) expresses appreciation to the
Susan G. Komen Breast Cancer Foundation for finan-
cial support.
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ethanedithiol–H2O (2.0 mL, v/v, 3.8:0.1:0.1) was stirred
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7.48–7.45 (m, 3H), 7.32–7.25 (m, 4H), 7.15 (dd, 2H,
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7.6 Hz), 4.06 (d, 1H, J = 10.0 Hz), 3.52 (m, 1H), 3.30
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