Design and synthesis of amidine-type peptide bond isosteres: Application of nitrile oxide derivatives as active ester equivalents in peptide and peptidomimetics synthesis

Article abstract of DOI:10.1039/c0ob01193b

Amidine-type peptide bond isosteres were designed based on the substitution of the peptide bond carbonyl (CO) group with an imino (CNH) group. The positively-charged property of the isosteric part resembles a reduced amide-type peptidomimetic. The peptidy

Full text of DOI:10.1039/c0ob01193b

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PAPER
Design and synthesis of amidine-type peptide bond isosteres: application of
nitrile oxide derivatives as active ester equivalents in peptide and
peptidomimetics synthesis†
Eriko Inokuchi,a Ai Yamada,a Kentaro Hozumi,b Kenji Tomita,a Shinya Oishi,a Hiroaki Ohno,a
Motoyoshi Nomizub and Nobutaka Fujii*a
Received 17th December 2010, Accepted 16th February 2011
DOI: 10.1039/c0ob01193b
Amidine-type peptide bond isosteres were designed based on the substitution of the peptide bond
carbonyl (C O) group with an imino (C NH) group. The positively-charged property of the isosteric
part resembles a reduced amide-type peptidomimetic. The peptidyl amidine units were synthesized by
the reduction of a key amidoxime (N-hydroxyamidine) precursor, which was prepared from nitrile oxide
components as an aminoacyl or peptidyl equivalent. This nitrile oxide-mediated C–N bond formation
was also used for peptide macrocyclization, in which the amidoxime group was converted to peptide
bonds under mild acidic conditions. Syntheses of the cyclic RGD peptide and a peptidomimetic using
both approaches, and their inhibitory activity against integrin-mediated cell attachment, are presented.
Introduction
The backbone modification of amide bonds 1 in bioactive peptides
is one of the most promising approaches for improving their re-
sistance towards degradation by peptidases.1 A number of peptide
bond isosteres that reproduce their electrostatic properties and
secondary structure conformations have been reported.2 Reduced
amide bonds (–CH2–NH–) 2 with a positively-charged secondary
amine provide a flexible and hydrogen bond-donating substructure
(Fig. 1). The success of this substructure is exemplified by several
enzyme inhibitors of HIV-1 protease3 and neuronal nitric oxide
synthase.4 Alkene dipeptide isosteres (–CR CH–, R = H, F or
Me) 32,5 also represent steady-state peptide bond mimetics. This
motif has been employed for the preparation of functional probes
to identify indispensable peptide bonds. During the course of
our medicinal chemistry studies using these isosteres, it has been
demonstrated that a heavy atom corresponding to the carbonyl
Fig. 1 Structures of the peptide bond and mimetics.
oxygen in peptide bonds favorably modulates local and global
peptide conformations.6
amide structure. However, there are few reports on amidine-type
peptide bond isosteres 4,7,8 while acyclic amidines9 and cyclic
amidines10 have been utilized as equivalents of the basic guanidino
group for several bioactive molecules.
Whereas amidines have been synthesized directly by the Pinner
reaction7,8b or by the coupling of imidyl chlorides with amines,
these reactions are not applicable to peptidyl amidine synthe-
sis because of the harsh reaction conditions or the arduous
substrate preparation. We have postulated that amidoximes (N-
hydroxyamidines) 8 represent an appropriate key precursor for
peptidyl amidine synthesis, which are obtained by the coupling
of nitrile oxides 6 with nucleophilic amines 7 (Scheme 1).11
Reduction10 or hydrolysis under mild acidic conditions12 of the key
The uncharged form of amidines 4 resemble the peptide bond
structure 1, in which both imino and amino functional groups
share an sp2 carbon. Under physiological conditions, amidines
are protonated and the positive charge of the conjugated acid is
delocalized over two nitrogens. Characteristic substructure 4¢ can
be viewed as a modified motif of the peptide bond and/or reduced
aGraduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku,
Kyoto, 606-8501, Japan. E-mail: nfujii@pharm.kyoto-u.ac.jp; Fax: +81 75-
753-4570; Tel: +81 75-753-4551
bSchool of Pharmacy, Tokyo University of Pharmacy and Life Sciences,
1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c0ob01193b
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reactive nitrile oxide components for peptide and peptidomimetic
synthesis.
Initially, we optimized the coupling conditions of a-
aminoaldoxime 914 and a-amino ester 12. This consisted of a
two-step process including chlorination of aldoxime 9 and the
subsequent nucleophilic attack of amino ester 12 onto nitrile oxide
11, derived from 10, under basic conditions (Table 1). The major
isomer of N-Boc-valine aldoxime, 9a, reacted with an NaOCl
solution11 followed by work-up and treatment with amino ester 12
to give the desired amidoxime (N-hydroxyamidine) product, 13,
in 77% yield (Table 1, entry 1), while minor isomer 9b produced
a complex mixture of unidentified products with the same reagent
(Table 1, entry 2). This was presumably due to the concomitant
formation of unstable nitrile oxide 11 under the basic conditions
of the first chlorination step. Treatment of both aldoxime isomers
9a and 9b with N-chlorosuccinimide (NCS) in DMF without base
provided the same product, 13, in satisfactory yields (Table 1,
entries 3 and 4). Of note, the chlorination of 9a with NCS in CHCl3
did not work, resulting in the recovery of the starting material.
As such, a facile protocol to prepare amidoximes from the both
isomers of amino acid-derived aldoximes was established.
Conversion processes from amidoxime 13 were next investi-
Scheme 1 Synthetic scheme for amidine-type peptide bond isosteres 4 and
native peptide bonds 1 using nitrile oxides 6 as reactive acyl equivalents.
amidoximes 8 would provide the target peptidyl amidine 4 or the
parent peptide bond 1, respectively. It was also expected that highly
reactive nitrile oxides 6 derived from peptide aldoximes 5 could be
exploited as active ester equivalents for fragment condensation to
prepare various protected peptides and peptidomimetics. Herein,
we describe a novel approach for the synthesis of peptides and
amidine-type peptidomimetics via peptide amidoximes.
R
gated. The hydrogenation of 13 with RANEYꢀ Ni10 cleaved the
N–O bond to afford the expected amidine-type isosteric unit,
14, in 67% yield (Scheme 2). Alternatively, the hydrolysis of
13 under mild acidic conditions containing NaNO2 gave the
parent dipeptide unit, 15, in 62% yield.12 No epimerization of
the amidoximes occurred during the coupling process, which
was verified by comparing 15 with two authentic diastereomers
prepared by the standard protocol for peptide synthesis.
Results and discussion
Preparation of amino acid-derived nitrile oxides and their
application to the synthesis of peptide bond and amidine-type
peptidomimetics
Nitrile oxides are useful reactive species that can be formed
from aldoximes by treatment with a chlorinating agent and a
weak base.11 There have been a number of reports of 1,3-dipolar
cycloadditions of nitrile oxides with olefins to produce isoxazoline
derivatives,13 whereas examples utilizing nitrile oxides as active
ester equivalents are limited. We expected that a-aminoaldoximes
and peptide aldoximes would serve as useful precursors of
Scheme 2 Conversion of N-hydroxyamidine 13 to amidine 14 and peptide
bond 15.
Table 1 Optimization of the aldoxime–amino acid coupling conditions
Entry
Substratea
Step a
Step b
Yield (%)
1
2
3
4
9a
9b
9a
9b
NaOClb (3.0 equiv.), Et3N (3.0 equiv.)
NaOClb (3.0 equiv.), Et3N (3.0 equiv.)
NCS (1.4 equiv.)
Et3N (6.0 equiv.)/CH2Cl2
Et3N (6.0 equiv.)/CH2Cl2
Et3N (4.0 equiv.)/Et2O
Et3N (4.0 equiv.)/Et2O
77
Decomp.
71c
NCS (1.4 equiv.)
81
a Substrates 9 were prepared from Boc-valinal according to literature procedures.14 b 30% aqueous solution. c When CHCl3 was used as the reaction solvent
in step a, starting material 9a was recovered.
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Table 2 Preparation of aldoxime resin 19
(TIS)/CH2Cl2 (0.5/0.1/99.4) provided linear peptide aldoxime
21 in a quantitative yield.
Cyclization of acyclic peptide aldoxime 21 by treatment with
NCS, followed by Et3N, gave amidoxime-containing peptide
22 in a moderate yield (36%, Scheme 4). The yield of the
aldoxime-mediated cyclization was comparable with approaches
using the azide method or DPPA-mediated cyclizations (11–52%
cyclization yields for the RGD peptide 16 and the derivatives).15a
Subsequently, amidoxime 22 was converted smoothly to amide
23a and amidine 23b in 46 and 95% yields, respectively, by
Entry
Additive (1.2 equiv.)
Conditionsa
Loading (%)
1
2
3
4
5
—
rt, o/n
rt, o/n
83
56
90
99
99
Et3N
AcOH
AcOH
AcOH
rt, o/n
R
NaNO2-mediated acidic hydrolysis and RANEYꢀ Ni-mediated
60 ◦C, o/n
60 ◦C, 2 h
hydrogenation. The protecting groups for Arg and Asp were
cleaved off using a cocktail of 1 M TMSBr–thioanisole/TFA in the
presence of m-cresol and 1,2-ethanedithiol (EDT) in a short time,
providing the desired parent RGD peptide 16 and peptidomimetic
17 in 87 and 73% yields, respectively. It is of note that no hydrolyzed
product 16 was observed during the deprotection treatment of 23b
and subsequent HPLC purification process.
a Dichloroethane (0.15 M), HC(OMe)3 (0.2 M).
Solid-phase synthesis of peptide aldoximes and the application of
nitrile oxide-mediated coupling to cyclic peptide synthesis
The nitrile oxide-mediated synthesis of peptides and pep-
tidomimetics was applied by a solid-phase approach. We chose
cyclic RGD peptide 16, cyclo(–Arg–Gly–Asp–D-Phe–Val–),15
which is a highly potent integrin avb3 antagonist that in-
cludes two reactive side-chains (Arg and Asp), and mimetic 17
cyclo(–Arg–Gly–y[C( NH)–NH]–Asp–D-Phe–Val–) as target
peptides. We planned to synthesize RGD peptide 16 and pep-
tidomimetic 17 by a nitrile oxide-mediated cyclization, followed
by hydrolysis and hydrogenolysis, respectively. For application to
solid-phase synthesis, the preparation of aldoxime resin 19 was
investigated. The direct attachment of Fmoc-protected aminoal-
doximes such as Fmoc–NH–CH2–CH NH–OH onto the (2-
Cl)trityl chloride resin failed to afford the expected resin under any
conditions. In contrast, resin 18 was prepared from the (2-Cl)trityl
chloride resin and Fmoc-protected hydroxyamine (89% loading),
followed by piperidine treatment. The reaction of an Fmoc-
protected a-aminoaldehyde with aminooxy (2-Cl)trityl resin 18
gave the desired aldoxime resin, 19 (83% loading; Table 2, entry
1). An acidic additive improved the reactivity, and the reaction
proceeded smoothly at 60 ◦C within 2 h to give aldoxime resin 19
in 99% yield (Table 2, entry 5).16
Peptide elongation was performed by the standard
Fmoc-based solid-phase synthesis approach using N,N¢-
diisopropylcarbodiimide (DIC)/HOBt in DMF to give peptide
aldoxime resin 20 (Scheme 3). During the solid-phase process,
the oxime–ether linker was inert, even when treated with 20%
piperidine in DMF for Fmoc removal. For peptide cleavage
from the solid support, the standard conditions [30% 1,1,1,3,3,3-
hexafluoropropan-2-ol (HFIP) in CH2Cl2, rt,
2
h17] were
Scheme 4 Synthesis of cyclic RGD peptide 16 and amidine-type isosteric
congener 17.
ineffective for aldoxime resin 20, indicating that the oxime–ether
linkage is less acid-labile compared with peptide acids and peptide
alcohols. The treatment of resin 20 in TFA/triisopropylsilane
Biological activity of the cyclic RGD Peptide with an amidine-type
isosteric unit for the Gly-Asp dipeptide
The resulting cyclic RGD peptidomimetic 17 was evaluated for
its inhibitory effect of integrin-mediated cell attachment (Fig. 2).
Peptide 17, with an amidine moiety, showed moderate inhibitory
activity (IC50 = 4.77 mM) compared with original peptide 16
(peptide 16, IC50 = 0.157 mM). The X-ray crystal structure of
the avb3 integrin-cyclic RGD peptide complex indicated that the
Scheme 3 Preparation of peptide aldoxime 21.
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with CH2Cl2, and the extract was washed with H2O and dried over
Na2SO4. Concentration under reduced pressure followed by flash
chromatography over silica gel with n-hexane–EtOAc (3/1) gave
the title compounds 9a and 9b (3.44 g, 82% yield, 9a/9b = 58/42)
both as white solids.
◦
Compound 9a: mp 35–36 C; [a]2D6 +11.0 (c 0.58, CHCl3); dH
(500 MHz, DMSO, Me4Si) 0.82 (6H, dd, J = 13.7 and 6.9 Hz),
1.37 (9H, s), 1.75 (1H, td, J = 13.7 and 6.9 Hz), 3.72–3.78 (1H, m),
6.96 (1H, d, J = 8.8 Hz), 7.14 (1H, d, J = 7.3 Hz) and 10.63 (1H,
s); dC (125 MHz, DMSO-d6, Me4Si) 18.6, 18.8, 28.2 (3C), 30.9,
55.4, 77.7, 149.0 and 155.1. Anal. calc. for C10H20N2O3: C, 55.53;
H, 9.32; N, 12.95. Found: C, 55.29; H, 9.17; N, 12.81%.
Compound 9b: mp 114–115 ◦C; [a]D26 +50.0 (c 0.18, CHCl3); dH
(500 MHz, DMSO-d6, Me4Si) 0.81 (6H, t, J = 7.2 Hz), 1.37 (9H,
s), 1.76–1.85 (1H, m), 4.54 (1H, dd, J = 15.7 and 7.1 Hz), 6.51
(1H, d, J = 7.1 Hz), 6.95 (1H, d, J = 8.9 Hz) and 10.86 (1H, s); dC
(125 MHz, DMSO, Me4Si) 18.3, 18.7, 28.2 (3C), 30.7, 50.2, 77.7,
149.9 and 155.2. Anal. calc. for C10H20N2O3: C, 55.53; H, 9.32; N,
12.95. Found: C, 55.25; H, 9.32; N, 12.71%.
Fig. 2 The inhibitory effect of cyclic RGD peptides on HDF attachment
to vitronectin. HDFs were allowed to attach to human vitronectin in the
presence of various concentrations of cyclic RGD peptides. Peptides were
added to the cell suspension and the cells were plated. After a 30 min
incubation period, the attached cells were stained with crystal violet and
dissolved in a 1% SDS solution. The absorbance at 570 nm was measured.
Triplicate experiments gave similar results.
uncharged amide NH of Gly-Asp is located proximal to the
integrin residue Arg216, which is likely to be involved in the
interaction.18 These results suggest that substitution of the Gly-
Asp peptide bond with a positively-charged amidine unit partially
eliminated the highly potent binding affinity towards the avb3
integrin.
tert-Butyl (S)-2-{[(S)-2-tert-butoxycarbonylamino-N-hydroxy-
3-methylbutanimidoyl]amino}-3-phenylpropionate (13). To a so-
lution of aldoxime 9b (30.0 mg, 0.140 mmol) in DMF (0.6 cm3)
was added N-chlorosuccinimide (26.2 mg, 0.200 mmol) and the
mixture stirred at room temperature for 4 h. The reaction mixture
was extracted with EtOAc, the extract washed with a solution
of H2O/brine (1/1) and dried over Na2SO4. After concentration
under reduced pressure, the residue was dissolved in Et2O (5 cm3).
To the solution were added Et3N (77 mm3, 0.560 mmol) and H-
Phe-OtBu 12 (30.0 mg, 0.140 mmol), and the mixture stirred at
room temperature overnight. The reaction mixture was washed
with brine and dried over Na2SO4. Concentration under reduced
pressure followed by flash chromatography over silica gel with n-
hexane–EtOAc (3/1) gave title compound 13 (50.0 mg, 81% yield,
inseparable mixture of major/minor = 97/3) as a colorless oil:
[a]2D6 -19.2 (c 0.73, CHCl3); dH (500 MHz, DMSO-d6, Me4Si) 0.63
(3H, d, J = 6.6 Hz), 0.73 (3H, d, J = 6.6 Hz), 1.32 (9H, s), 1.37
(9H, s), 1.79 (1H, dt, J = 21.7 and 6.6 Hz), 2.84–2.94 (2H, m), 3.70
(1H, t, J = 9.0 Hz), 4.44–4.52 (1H, m), 5.38 (1H, d, J = 10.5 Hz),
6.66 (1H, d, J = 9.5 Hz), 7.19–7.29 (5H, m) and 10.86 (1H, s);
dC (125 MHz, DMSO-d6, Me4Si) 18.3, 19.8, 27.5 (3C), 28.2 (3C),
29.8, 55.0, 56.1, 77.8, 80.6, 126.5, 128.0 (3C), 129.5 (2C), 137.0,
150.5, 155.3 and 171.4; HRMS (FAB) m/z calc. for C23H38N3O5
([M + H]+) 436.2811, found 436.2808.
Conclusion
In conclusion, we have established a novel approach to synthesize
acyclic amidine and amide units via a key amidoxime (N-
hydroxyamidine) precursor, which was prepared from a nitrile
oxide component as an active ester equivalent. This method was
used for the Fmoc-based solid-phase synthesis of peptides and
peptidomimetics containing an amidine-type isostere. The peptide
aldoxime represented a functional precursor for a protected cyclic
peptide and peptidomimetic, suggesting that the nitrile oxide-
mediated coupling reaction could serve as an alternative method
for peptide macrocyclizations. Further studies on the scope and
limitations of this approach, as well as applications for structure–
activity relationship studies of bioactive peptides, are currently in
progress.
Experimental section
tert-Butyl
(S)-2-{[(S)-2-tert-butoxycarbonylamino-3-methyl-
Synthesis
butanimidoyl]amino}-3-phenylpropionate (14). To a solution of
tert-Butyl [(S)-1-(hydroxyiminomethyl)-2-methylpropyl]carba-
mate (9). To a solution of Boc-Val-NMe(OMe) (5.00 g,
19.2 mmol) in Et2O (60 cm3) was added dropwise a solution of
LiAlH4 (1.02 g, 27.0 mmol) in Et2O (20 cm3) at -40 ◦C and
the mixture was stirred for 40 min. The reaction was quenched
at -40 ◦C by the addition of an Na2SO4 solution. The reaction
mixture was washed with saturated aqueous NaHCO3 and brine,
and dried over Na2SO4. Concentration under reduced pressure
gave the Boc-valinal. To a solution of NH2OH·HCl (1.66 g,
23.9 mmol) and AcONa (1.96 g, 23.9 mmol) in EtOH (50 cm3)
was added the solution of the aldehyde in EtOH (15 cm3). The
reaction mixture was stirred at 80 ◦C for 15 min. The mixture was
concentrated under reduced pressure. The residue was extracted
amidoxime 13 (29.1 mg, 0.0670 mmol) in MeOH (1 cm3) and
R
AcOH (0.011 cm3) was added RANEYꢀ Ni (0.85 cm3, slurry in
H2O) and the mixture stirred under an atmosphere of hydrogen
at room temperature for 1 h. The mixture was filtered through
R
Celiteꢀ. Concentration under reduced pressure followed by flash
chromatography over silica gel with n-hexane–EtOAc (3/1) gave
title compound 14 (18.9 mg, 67% yield) as a yellow oil: [a]2D6 +7.53
(c 0.46, CHCl3); dH (500 MHz, DMSO-d6, Me4Si) 0.76 (6H, dd,
J = 13.5 and 6.7 Hz), 1.28 (9H, s), 1.38 (9H, s), 1.80–1.88 (1H, m),
2.86 (1H, br s), 2.92 (1H, dd, J = 13.5 and 6.9 Hz), 3.77 (1H, br s),
4.26 (1H, br s), 4.99 (1H, d, J = 9.5 Hz), 6.16 (1H, br s), 6.96 (1H,
d, J = 9.5 Hz) and 7.14–7.26 (5H, m); dC (125 MHz, DMSO-d6,
Me4Si) 18.0 (2C), 19.3, 27.5 (3C), 28.2 (3C), 31.0, 37.8, 59.5, 77.8,
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78.9, 126.1, 127.9 (2C), 127.9, 129.2 (2C), 138.1, 155.2 and 171.2;
HRMS (FAB) m/z calc. for C23H38N3O4 ([M + H]+) 420.2862,
found 420.2864.
Cyclo[–Arg(Pbf)–Gly–w[C( NOH)NH]–Asp(OtBu)–D-Phe–
Val–] (22). To a solution of peptide aldoxime 21 (74.0 mg)
in DMF (1 cm3) was added N-chlorosuccinimide (14.7 mg,
0.100 mmol). The solution was stirred at room temperature
overnight, and then DMF (40 cm3) and Et3N (0.4 cm3) added.
The mixture was stirred at room temperature overnight and then
concentrated under reduced pressure. The residue was extracted
with EtOAc and the extract washed with brine. The organic layer
was dried over Na2SO4 and concentrated under reduced pressure
to give a yellow oil, which was purified by column chromatography
over silica gel with CH2Cl2–MeOH (95/5) to give 22 (26.9 mg, 36%
yield, major/minor = 79/21) as a yellow solid: mp 168–169 ◦C;
[a]2D5 -52.7 (c 0.28, CHCl3); dH (500 MHz, DMSO-d6, Me4Si) 0.85
(major, 3H, d, J = 6.9 Hz), 0.68 (minor, 3H, t, J = 6.4 Hz), 0.73
(major, 3H, d, J = 6.7 Hz), 0.72–0.76 (minor, 3H, m), 1.25–1.50
(2H, m), 1.34 (minor, 9H, s), 1.37 (major, 9H, s), 1.36 (minor,
6H, s), 1.41 (major, 6H, s), 1.74–1.76 (2H, m), 2.00 (3H, s), 2.41
(3H, s), 2.47 (3H, s), 2.30–2.50 (2H, m), 2.59 (1H, dd, J = 15.9
and 5.9 Hz), 2.82–2.91 (2H, m), 2.96 (2H, s), 3.79 (major, 1H, t,
J = 7.0 Hz), 3.82–3.88 (minor, 1H, m), 3.98 (major, 1H, dd, J =
14.7 and 7.7 Hz), 4.02–4.08 (minor, 1H, m), 4.10–4.14 (minor, 1H,
m), 4.16–4.25 (major, 1H, m), 4.50 (major, 1H, dd, J = 14.6 and
8.4 Hz), 4.35–4.45 (minor, 1H, m), 4.54–4.65 (major, 1H, m), 4.60–
4.75 (minor, 1H, m), 5.17 (minor, 1H, d, J = 9.6 Hz), 5.27 (major,
1H, d, J = 10.6 Hz), 6.37 (major, 1H, br s), 6.70 (minor, 1H, br
s), 7.12–7.33 (5H, m), 7.42–7.53 (1H, m), 8.05–8.14 (2H, m), 8.32
(minor, 1H, d, J = 7.3 Hz), 8.45 (major, 1H, d, J = 5.9 Hz), 9.17
(minor, 1H, s) and 9.63 (major, 1H, s); dC (125 MHz, DMSO-d6,
Me4Si) 12.1, 12.3, 17.3 (minor), 17.6 (major), 17.8 (major, 2C),
17.9 (minor, 2C), 18.9, 19.0 (major), 19.1 (minor), 21.1, 27.7 (3C),
27.7, 28.3 (major, 2C), 28.8 (minor, 2C), 36.3, 42.5 (2C), 52.0, 52.6,
55.0, 59.8 (minor), 60.3 (major), 62.8, 79.7 (minor), 80.3 (major),
86.3, 116.3, 124.3, 126.5, 128.1 (minor, 2C), 128.2 (major, 2C),
129.1 (major, 2C), 129.3 (minor, 2C), 131.4, 134.2, 137.0, 137.3,
148.9, 156.0, 157.5, 169.3 (major), 169.5 (minor), 170.7, 171.2,
172.2 and 172.4; HRMS (FAB) m/z calc. for C43H64N9O10S ([M +
H]+) 898.4497, found 898.4502.
tert-Butyl (S)-2-[(S)-2-tert-butoxycarbonylamino-3-methylbu-
tyrylamino]-3-phenylpropionate (15). To a solution of amidoxime
13 (35.3 mg, 0.0810 mmol) in MeOH (0.8 cm3) and H2O (0.8 cm3)
were added AcOH (0.00800 cm3, 0.120 mmol) and NaNO2
(8.30 mg, 0.120 mmol). The mixture was stirred at room tem-
perature overnight. The mixture was concentrated under reduced
pressure. The residue was extracted with CH2Cl2, and the extract
was washed with H2O and dried over Na2SO4. Concentration
under reduced pressure followed by flash chromatography over
silica gel with n-hexane–AcOEt (3/1) gave title ◦compound 15
(21.0 mg, 62% yield) as a white solid: mp 115–116 C; [a]2D4 +60.0
(c 0.87, CHCl3); dH (500 MHz, DMSO-d6, Me4Si) 0.87 (3H, d,
J = 5.6 Hz), 0.93 (3H, d, J = 6.8 Hz), 1.38 (9H, s), 1.45 (9H, s),
2.04–2.14 (1H, m), 3.04–3.11 (2H, m), 3.91 (1H, t, J = 6.8 Hz), 4.74
(1H, dd, J = 13.8 and 6.2 Hz), 5.16 (1H, d, J = 6.6 Hz), 6.30 (1H,
d, J = 6.2 Hz) and 7.14–7.31 (5H, m); dC (125 MHz, DMSO-d6,
Me4Si) 17.7, 19.2, 27.9 (3C), 28.3 (3C), 38.2, 52.2, 53.6, 55.1, 82.2,
82.3, 126.9, 127.0 (2C), 128.4 (2C), 129.5, 136.0, 170.3 and 171.0;
HRMS (FAB) m/z calc. for C23H37N2O5 ([M + H]+) 421.2702,
found 421.2702.
H2N–O-(2-Cl)Trt resin (18). 2-Chlorotrityl resin chloride
(loading: 1.31 mmol g-1, 76.3 mg) was reacted with Fmoc–NHOH
(128 mg, 0.500 mmol) and pyridine (0.0810 cm3, 1.00 mmol) in
THF (0.8 cm3) at 60 ◦C for 6 h. The solution was removed by
decantation and the resulting resin washed with a solution of
DMF/(iPr)2NEt/MeOH (17/2/1). The Fmoc protecting group
was removed by treating the resin with a DMF/piperidine solution
(80/20, v/v). The loading was determined by measuring at 290 nm
the UV absorption of the piperidine-treated sample: 0.900 mmol
g-1, 89%.
H–Asp(OtBu)–D - Phe–Val–Arg(Pbf)–Gly–aldoxime - (2 - Cl)Trt
resin (20). Solid-supported hydroxyamine 18 (loading:
0.900 mmol g-1, 91.6 mg, 0.0820 mmol) was reacted with Fmoc-
glycinal (0.500 mmol) in dichloroethane (0.7 cm3), HC(OMe)3
(0.5 cm3) and AcOH (0.001 cm3) at 60 ◦C for 2 h. The solution was
removed by decantation and the resulting resin was washed with
DMF to afford resin 19. Peptide resin 20 was manually constructed
using an Fmoc-based solid-phase synthesis on resin 19. The
Fmoc protecting group was removed by treating the resin with
a DMF/piperidine solution (80/20, v/v). The Fmoc-protected
amino acid (0.500 mmol, 6.1 equiv.) was successively condensed
using 1,3-diisopropylcarbodiimide (0.0770 cm3, 0.500 mmol,
6.1 equiv.) in the presence of N-hydroxybenzotriazole (77 mg,
0.500 mmol, 6.1 equiv.) to give resin 20. The tBu ester for Asp and
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for Arg
were employed for side-chain protection.
Cyclo[–Arg(Pbf)–Gly–Asp(OtBu)–D-Phe–Val–] (23a). To a so-
lution of amidoxime 22 (20.0 mg, 0.0220 mmol) in MeOH
(0.5 cm3) and H2O (0.2 cm3) were added AcOH (0.00500 cm3)
and NaNO2 (4.60 mg, 0.0660 mmol). The mixture was stirred at
room temperature overnight. The mixture was concentrated under
reduced pressure. The residue was extracted with EtOAc, and the
extract washed with H2O and dried over MgSO4. Concentration
under reduced pressure followed by PTLC purification with
CH2Cl2–MeOH (95/5) gave title compound 23a (8.90 mg, 46%
yield) as a white solid: mp 247–248 ◦C; [a]D25 -32.3 (c 0.27, MeOH);
dH (500 MHz, DMSO-d6, Me4Si) 0.70 (6H, dd, J = 20.7 and
6.7 Hz), 1.18–1.50 (2H, m), 1.34 (9H, s), 1.41 (6H, s), 1.65–1.72
(1H, m), 1.80–1.88 (1H, m), 2.01 (3H, s), 2.36 (1H, dd, J = 15.7
and 8.9 Hz), 2.41 (3H, s), 2.46 (3H, s), 2.80 (1H, dd, J = 13.7 and
6.6 Hz), 2.91–3.06 (2H, m), 2.96 (2H, s), 3.28 (2H, s), 3.82 (1H,
t, J = 7.6 Hz), 4.00–4.10 (2H, m), 4.54–4.62 (2H, m), 6.35 (1H,
br s), 6.70–6.80 (1H, m), 7.13–7.28 (5H, m), 7.42–7.50 (5H, m),
7.74 (2H, dd, J = 11.5 and 8.3 Hz), 7.95 (1H, d, J = 8.3 Hz), 8.06
(1H, d, J = 7.6 Hz) and 8.36 (1H, dd, J = 7.3 and 4.4 Hz); dC
(125 MHz, DMSO-d6, Me4Si) 12.3, 17.6, 18.2, 18.9, 19.2, 25.8,
27.6 (3C), 28.3 (2C), 28.4, 29.7, 36.4, 37.1, 39.8, 42.5, 43.1, 48.9,
H–Asp(OtBu)–D-Phe–Val–Arg(Pbf)–Gly–aldoxime
(21).
Resin 20 was treated with TFA/TIS/CH2Cl2 (20 cm3,
0.5/0.1/99.4) at room temperature for 1.5 h. After removal
of the resin by filtration, the filtrate was concentrated under
reduced pressure to give crude peptide aldoxime 21 as a yellow
oil (74.0 mg, quant. from resin 18). The crude product was used
without further purification.
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Org. Biomol. Chem., 2011, 9, 3421–3427 | 3425
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synthesized using Fmoc-based solid-phase synthesis. [a]2D5 -21.6
(c 0.27, MeOH); dH (500 MHz, DMSO-d6, Me4Si) 0.68 (3H, d,
J = 6.7 Hz), 0.75 (3H, d, J = 6.7 Hz), 1.32–1.45 (2H, m), 1.45–1.55
(1H, m), 1.69–1.80 (1H, m), 1.80–1.90 (1H, m), 2.38 (1H, dd, J =
16.4 and 5.5 Hz), 2.72 (1H, dd, J = 16.4 and 8.9 Hz), 2.81 (1H,
dd, J = 13.5 and 6.1 Hz), 2.94 (1H, dd, J = 13.5 and 8.0 Hz),
3.05–3.14 (2H, m), 3.26 (1H, dd, J = 15.2 and 4.2 Hz), 3.82 (1H, t,
J = 7.4 Hz), 4.04 (1H, dd, J = 15.2 and 7.7 Hz), 4.08–4.16 (1H, m),
4.55 (1H, dd, J = 14.2 and 7.2 Hz), 4.60–4.68 (1H, m), 6.58–7.11
(1H, br s), 7.15–7.25 (5H, m), 7.58 (1H, t, J = 5.7 Hz), 7.78 (1H,
d, J = 7.4 Hz), 7.87 (1H, d, J = 8.0 Hz), 8.00 (1H, d, J = 7.4 Hz),
8.08 (1H, d, J = 8.6 Hz), 8.36 (1H, dd, J = 7.4 and 4.2 Hz) and
12.3 (1H, s); dC (125 MHz, DMSO-d6, Me4Si) 18.1, 19.1, 25.3,
28.2, 29.5, 34.8, 37.1, 40.2, 43.0, 48.8, 52.0, 53.9, 60.1, 126.1, 128.0
(2C), 129.0 (2C), 137.3, 156.6, 158.3, 169.4, 169.8, 170.6, 171.1
and 171.6; HRMS (FAB) m/z calc. for C26H39N8O7 ([M + H]+)
575.2942, found 575.2952.
52.2, 53.9, 60.1, 80.0, 86.3, 116.3, 119.7, 124.3, 126.2, 128.1 (2C),
129.0 (2C), 130.3, 131.4, 137.3, 156.0, 157.4, 169.1, 169.4, 169.9,
170.8, 171.0 and 171.1; HRMS (FAB) m/z calc. for C43H63N8O10S
([M + H]+) 883.4388, found 883.4397.
Cyclo[–Arg(Pbf)–Gly–w[C( NH)NH]-Asp(OtBu)–D-Phe–
Val–] (23b). To
a solution of amidoxime 22 (30.0 mg,
0.0330 mmol) in MeOH (0.6 cm3) and AcOH (0.006 cm3) was
R
added RANEYꢀ Ni (0.440 cm3, slurry in H2O), and the mixture
stirred under a H2 atmosphere at room temperature for 2 h.
R
The mixture was filtered through Celiteꢀ. Concentration under
reduced pressure followed by flash chromatography over silica gel
with CH2Cl2–MeOH (95/5) gave title compound 23b (27.4 mg,
95% yield) as a colorless oil: [a]2D6 -53.3 (c 0.14, CHCl3); dH
(500 MHz, CD3OD, Me4Si) 0.74 (6H, dd, J = 14.7 and 6.9 Hz),
1.43 (9H, s), 1.45 (6H, s), 1.45–1.52 (1H, m), 1.55–1.60 (1H, m),
1.82–1.89 (1H, m), 1.95–2.00 (1H, m), 2.07 (3H, s), 2.56 (3H, s),
2.59 (1H, d, J = 6.6 Hz), 2.77 (1H, dd, J = 16.5 and 6.9 Hz), 2.94
(1H, dd, J = 13.3 and 6.7 Hz), 2.99 (2H, s), 3.05 (1H, dd, J = 13.2
and 9.0 Hz), 3.11–3.18 (1H, m), 3.53 (1H, d, J = 15.2 Hz), 3.87
(1H, d, J = 6.9 Hz), 4.28 (1H, d, J = 15.2 Hz), 4.34–4.37 (1H, m),
4.39–4.45 (1H, m), 4.68 (1H, dd, J = 9.0 and 6.9 Hz) and 7.15–
7.29 (5H, m); dC (125 MHz, CD3OD, Me4Si) 12.5, 18.4, 18.7, 19.6,
19.7, 28.4, 28.4, 28.4 (3C), 29.6 (2C), 30.9, 37.9, 38.4, 44.0, 49.5,
49.7, 54.0, 56.4, 62.5, 82.6, 87.7, 118.5, 126.0, 127.9, 129.6 (2C),
130.4 (2C), 132.4, 133.5, 134.4, 138.0, 139.4, 158.1, 160.0, 172.0,
173.3, 173.6, 173.9, 174.2 and 174.3; HRMS (FAB) m/z calc. for
C43H62N9O9S ([M - H]-) 880.4397, found 880.4395.
Evaluation of inhibitory activity against integrin-mediated cell
attachment. Human dermal fibroblasts (HDFs; AGC Techno
Glass, Chiba, Japan) were maintained in DMEM containing
10% FBS, 100 U cm-3 penicillin and 100 mg cm-3 streptomycin
(Invitrogen, Carlsbad, CA, USA). Human plasma vitronectin
(0.1 mg in 0.050 cm3 well-1; EMD Chemicals Inc., Gibbstown, NJ,
USA) were added to 96-well plates (Nalge Nunc, Rochester, NY,
USA) and incubated for 1 h at 37 ◦C. The plates were washed and
blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich, St.
Louis, MO, USA) in DMEM. HDFs were incubated at room
temperature for 15 min in various concentrations of peptides
(0.001–200 mM in 1% DMSO). Then, 0.100 cm3 HDFs (2 ¥ 104
cells) in DMEM containing 0.1% BSA were added to each well
and incubated at 37 ◦C for 30 min in 5% CO2. The attached cells
were stained with a 0.2% crystal violet aqueous solution in 20%
MeOH (0.150 cm3) for 15 min. After washing with Milli-Q water,
the plates were dried overnight at room temperature and dissolved
in 0.150 cm3 of a 1% SDS solution. The absorbance at 570 nm
was measured. Each sample was assayed in triplicate, and cells
attached to the BSA were subtracted from all measurements. 1%
DMSO did not have any effect on HDF attachment to vitronectin.
Cyclo[–Arg–Gly–w[C( NH)NH]–Asp–D-Phe–Val–]
(17).
Protected amidine 23b (7.90 mg, 0.00900 mmol) was treated
with 1 M TMSBr–thioanisole in TFA (10 cm3) in the presence
of m-cresol (0.1 cm3) and 1,2-ethanedithiol (0.5 cm3) at 4 ◦C
for 15 min. The mixture was poured into ice-cold dry Et2O
(50 cm3). The resulting powder was collected by centrifugation
and washed three times with ice-cold dry Et2O. The crude product
was purified by preparative HPLC to afford expected peptide
17 as a white powder (5.30 mg, 0.00660 mmol, 73% yield): [a]D25
-129.2 (c 0.17, MeOH); dH (500 MHz, DMSO-d6, Me4Si) 0.70
(3H, d, J = 6.6 Hz), 0.74 (3H, d, J = 6.6 Hz), 1.32–1.60 (3H, m),
1.73–1.84 (1H, m), 1.88–1.98 (1H, m), 2.59 (1H, dd, J = 17.0 and
5.7 Hz), 2.78 (1H, dd, J = 13.5 and 6.5 Hz), 2.84 (1H, dd, J =
17.2 and 8.2 Hz), 3.00 (1H, dd, J = 13.0 and 8.4 Hz), 3.04–3.13
(2H, m), 3.72–3.78 (2H, m), 3.90–3.98 (1H, m), 4.23 (1H, dd, J =
13.5 and 8.2 Hz), 4.43 (1H, t, J = 16.2 and 7.0 Hz), 4.53–4.60
(1H, m), 4.62–4.68 (1H, m), 6.80–7.40 (2H, br s), 7.16–7.28 (5H,
m), 7.72 (1H, t, J = 5.7 Hz), 7.93 (1H, dd, J = 11.3 and 8.4 Hz),
8.12 (1H, d, J = 7.7 Hz), 8.28–8.32 (1H, m), 8.53 (1H, d, J =
7.7 Hz), 8.92–8.98 (1H, m), 9.10–9.20 (1H, m) and 9.64 (1H, s);
dC (125 MHz, DMSO-d6, Me4Si) 17.9, 25.3, 28.2, 29.6, 34.2, 37.0,
37.1, 40.2, 51.7, 51.9, 54.2, 59.9, 126.4, 128.2 (2C), 129.1 (2C),
137.2, 156.8, 158.4, 164.8, 166.8, 170.7, 171.2, 171.3 and 171.7;
HRMS (FAB) m/z calc. for C26H40N9O6 ([M + H]+) 574.3102,
found 574.3101.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research
and the Targeted Protein Research Program from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan. E.
I. and K. T. are grateful for Research Fellowships from the JSPS
for Young Scientists.
Notes and references
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63, 6088.
Cyclo(–Arg–Gly–Asp–D-Phe–Val–) (16). By an identical pro-
cedure to that described for the preparation of 17, 23a (8.00 mg,
0.00900 mmol) was converted into cyclic RGD peptide 16
(0.00790 mmol, 87% yield). All characterization data were in
agreement with the data for the control peptide, which was
3426 | Org. Biomol. Chem., 2011, 9, 3421–3427
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3 K. A. Newlander, J. F. Callahan, M. L. Moore, T. A. Tomaszek, Jr. and
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16 Racemization (20%) was observed when Fmoc–D-Phe–H was utilized
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5.
17 R. Bollhagen, M. Schmiedberger, K. Barlos and E. Grell, J. Chem. Soc.,
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The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 3421–3427 | 3427
©