Structure-activity relationship study of a CXC chemokine receptor type 4 antagonist, FC131, using a series of alkene dipeptide isosteres

Article abstract of DOI:10.1021/jm2016914

A structure-activity relationship study on a highly potent CXC chemokine receptor type 4 (CXCR4) antagonist, FC131 [cyclo(-d-Tyr¹-Arg ²-Arg³-Nal⁴-Gly⁵-)], was carried out using a series of alkene isosteres of the d-Tyr¹-l/d-Arg² dipeptide to investigate the binding mode of FC131 and its derivatives with CXCR4. The structure-activity relationships of isostere-containing FC131 analogues were similar to those of the parent FC131 and its derivatives, suggesting that a trans-conformer of the d-Tyr¹-Arg² peptide bond is the dominant contributor to the bioactive conformations of FC131. Although NMR analysis demonstrated that the two conformations of the peptidomimetic containing the d-Tyr¹-d-Arg² isostere are possible, binding-mode prediction indicated that the orientations of the alkene motif within d-Tyr¹-MeArg² peptidomimetics depend on the chirality of Arg² and the β-methyl group of the isostere unit, which makes the dominant contribution for binding to the receptor. The most potent FC122 [cyclo(-d-Tyr¹-d-MeArg²-Arg ³-Nal⁴-Gly⁵-)] bound with CXCR4 by a binding mode different from that of FC131.

Full text of DOI:10.1021/jm2016914

Article
pubs.acs.org/jmc
Structure−Activity Relationship Study of a CXC Chemokine Receptor
Type 4 Antagonist, FC131, Using a Series of Alkene Dipeptide
Isosteres
Kazuya Kobayashi,† Shinya Oishi,*,† Ryoko Hayashi,† Kenji Tomita,† Tatsuhiko Kubo,†
Noriko Tanahara,† Hiroaki Ohno,† Yasushi Yoshikawa,‡ Toshio Furuya,‡ Masaru Hoshino,†
and Nobutaka Fujii*,†
†Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
‡PharmaDesign Inc., Chuo-ku, Tokyo 104-0032, Japan
S
* Supporting Information
ABSTRACT: A structure−activity relationship study on a
highly potent CXC chemokine receptor type 4 (CXCR4)
antagonist, FC131 [cyclo(-D-Tyr1-Arg2-Arg3-Nal4-Gly5-)], was
carried out using a series of alkene isosteres of the D-Tyr1-L/D-
Arg2 dipeptide to investigate the binding mode of FC131 and
its derivatives with CXCR4. The structure−activity relation-
ships of isostere-containing FC131 analogues were similar to those of the parent FC131 and its derivatives, suggesting that a
trans-conformer of the D-Tyr1−Arg2 peptide bond is the dominant contributor to the bioactive conformations of FC131.
Although NMR analysis demonstrated that the two conformations of the peptidomimetic containing the D-Tyr1-D-Arg2 isostere
are possible, binding-mode prediction indicated that the orientations of the alkene motif within D-Tyr1-MeArg2 peptidomimetics
depend on the chirality of Arg2 and the β-methyl group of the isostere unit, which makes the dominant contribution for binding
to the receptor. The most potent FC122 [cyclo(-D-Tyr1-D-MeArg2-Arg3-Nal4-Gly5-)] bound with CXCR4 by a binding mode
different from that of FC131.
derived anti-human immunodeficiency virus (HIV) peptide
T140.20 Since this novel scaffold for CXCR4 antagonists was
identified, a series of cyclic peptides and peptidomimetics have
been designed for potential anti-HIV and antimetastatic
agents.21 For example, substitution of Arg2 in FC131 with D-
Arg and/or N-methylarginine (MeArg) provided interesting
insights into SARs:22 (1) the D-Arg2-substituted derivative
(FC092) showed slightly less potent activity than FC131 did,
(2) in the low-energy structures of FC092, the orientation of
the D-Tyr1−D-Arg2 peptide bond was flipped (Figure 1a,b), (3)
the MeArg2-substituted peptide (FC162) is less potent than
FC131 is, (4) the D-MeArg2-substituted peptide (FC122) is the
most potent, and approximately 30% of the N-methylamide
bonds in D-Tyr1-D-MeArg2 exist as the cis-conformer, and (5)
the local conformation around D-Tyr1-D-MeArg2 in FC122 is
similar to that of FC131 (Figure 1a,c). The D-Tyr1-Arg2
substructure in FC131 is therefore involved in direct or
indirect contributions to binding with CXCR4. The different
biological effects among the derivatives are likely to be derived
from two possible pseudo-1,3-allylic strains between the Arg2
side chain and the D-Tyr1 carbonyl group and between the D-
Tyr1 side chain and the N-methyl group of MeArg2; these can
affect the orientations of the peptide bond (Figure 1b,c). In this
study, we investigated the electrostatic and steric effects around
INTRODUCTION
■
Cyclic peptides provide versatile scaffolds for the development
of therapeutic agents in drug discovery from peptide ligands.1
The cyclic structure provides several advantages, including
preorganized conformations to improve the affinity for the
target molecule(s),2 protection from proteolytic degradation by
exopeptidases,3 and increased membrane permeability.4,5 The
restricted conformations can facilitate the identification of
preferred spatial distributions of functional groups necessary for
bioactivity. Cyclic peptides therefore offer promising lead
compounds for optimization of small-molecule ligands via
improvement of the bioactivity and/or selectivity in ligand-
based drug design.6 For example, Kessler and co-workers
developed a cyclic RGD pentapeptide for highly potent
antagonists of αvβ3 integrin.7 The subsequent structure−activity
relationship (SAR) studies identified a more potent and
selective cyclic peptide.8 Using information on the spatial
distributions of the pharmacophoric elements of cyclic RGD
peptides, small-molecule inhibitors with a variety of druglike
scaffolds have been developed.9−12 Endothelin receptor
antagonists,13−15 endomorphin-1 analogues,16,17 and somatos-
tatin analogues18,19 with cyclic peptide scaffolds have also been
exemplified.
Previously, we developed a highly potent CXC chemokine
receptor type 4 (CXCR4) antagonist, FC131 [cyclo(-D-Tyr1-
Arg2-Arg3-Nal4-Gly5-)], from a library of cyclic pentapeptides
consisting of pharmacophore residues of the polyphemusin-II-
Received: December 15, 2011
Published: February 21, 2012
© 2012 American Chemical Society
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Figure 1. Structures of cyclic pentapeptide CXCR4 antagonists and the bioactivity-relevant peptide bond orientations in D-Tyr-L-Arg in FC131 (a),
D-Tyr-D-Arg in FC092 (b), and D-Tyr-D-MeArg in FC122 (c). Nal = L-3-(2-naphthyl)alanine.
the D-Tyr1-Arg2 substructure using a series of alkene dipeptide
isosteres. Computational analysis was also performed to assess
the binding mode to CXCR4 of FC131 and its derivatives.
isosteres,26,27 a γ-methyl group serves as a substituent
corresponding to the carbonyl oxygen of a peptide bond.
Tetrasubstituted alkene isosteres mimic N-methylamide
bonds.28 Our expectation was that, using a series of alkene
isosteres, the steric effects of the carbonyl group and N-methyl
group of the D-Tyr−Arg peptide bond on the peptide
conformation and bioactivity could be understood. The
contributions of the D-Tyr−Arg peptide bond to the hydro-
gen-bonding interactions could also be revealed by replacement
with the isosteres because these substituted alkene motifs
cannot engage in dipole interactions (Figure 3).
RESULTS AND DISCUSSION
■
Design and Synthesis of a Series of FC131 Derivatives
Containing Alkene Dipeptide Isosteres. Peptide bonds
constitute the assembly units for secondary and tertiary
structures as well as the functional motifs for intermolecular
interactions with binding partners via hydrogen bond acceptor/
donor properties. Because replacement of peptide bonds with
isosteric mimetics is one of the usual practices in performing
SAR studies on bioactive and functional peptides, a number of
peptide bond isosteres have been developed and used in
medicinal chemistry. To identify the dominant conformations
that contribute to the bioactivity of FC131 derivatives, alkene
dipeptide isosteres were used for the SAR study (Figure 2). A
planar alkene motif can restrict the possible cis/trans isomer-
ization of peptide bonds.23−25 In the trisubstituted alkene
Figure 3. Design of alkene isostere-containing derivatives of cyclic
pentapeptide-based CXCR4 antagonists. Nal = L-3-(2-naphthyl)-
alanine.
For the coupling component in the solid-phase peptide
synthesis, a fully protected D-Tyr-Orn isostere, 10 (Orn = L-
ornithine), was designed in which the Orn δ-amino group can
be converted into an Arg guanidino group after peptide
synthesis.29,30 A synthetic method for disubstituted alkene
dipeptide isosteres has previously been reported.31 Initially, the
D-tyrosine derivative 1 was converted to syn-allyl alcohol 2 by a
one-pot reduction and vinylation (Scheme 1). After protection
of the hydroxyl group of 2 with an acetyl group, ozonolysis and
Figure 2. Structures of a series of alkene dipeptide isosteres. Xaa and
Yaa = amino acid side chains.
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a
a
Scheme 1
Scheme 2
a
Reagents and conditions: (a) DIBAL-H, CH2Cl2−toluene, −78 °C,
20 min, then H2CCHMgBr, ZnCl2, LiCl, THF, −78 °C, 3 h (31%);
(b) recrystallization; (c) Ac2O, pyridine, DMAP, CHCl3, 0 °C, 2 h
(quantitative); (d) (i) O3, EtOAc, −78 °C, then Me2S, 0 °C, 30 min;
(ii) (EtO)2P(O)CH2CO2-t-Bu, LiCl, (i-Pr)2NEt, MeCN, 0 °C, 3 h
(62%); (e) K2CO3, MeOH, rt, 2 h (96%); (f) MsCl, Et3N, CH2Cl2, 0
°C, 2 h (96%); (g) TBSO(CH2)3Li, CuCN, LiCl, THF−Et2O−n-
pentane, −78 °C, 30 min (94%); (h) (i) H2SiF6(aq), MeCN−H2O, rt,
2 h; (ii) NsNH(Cbz), DEAD, PPh3, THF−toluene, 0 °C, 3 h (86%);
(i) PhSH, K2CO3, DMF, rt, 3 h (96%); (j) (i) TFA, CH2Cl2, rt, 2 h;
(ii) FmocOSu, Et3N, MeCN−H2O, rt, 2 h (71%).
a subsequent Horner−Wadsworth−Emmons reaction (HWE
reaction) afforded an (E)-isomer of α,β-enoate 4. Alcoholysis of
the acetyl group followed by mesylation yielded γ-(mesyloxy)-
α,β-enoate 6, which is a key substrate for organocopper-
mediated SN2′ alkylations. Treatment of 6 with TBSO(CH2)3Li
in the presence of CuCN and LiCl gave an α-alkylated product,
7. The side chain silyl ether group in 7 was converted to a Cbz-
protected amino group via a Mitsunobu reaction using
NsNH(Cbz). Removal of Ns, Boc and t-Bu groups, followed
by N-Fmoc protection, provided the expected isostere
component 10.
Tri- and tetrasubstituted alkene isosteres for D-Tyr-Orn
dipeptide were synthesized according to the protocol
established in our previous studies.32,33 syn-Allyl alcohols
12a,b were obtained by sequential methylation and alkenylation
of Weinreb amide 11 using Grignard reagents (Scheme 2).
Cyclization of 12a,b under basic conditions followed by Boc
protection gave oxazolidinones 13a,b. 5-Vinyloxazolidinone
13a was converted to an α,β-unsaturated ester, 15a, by
ozonolysis and HWE reaction. In contrast, the same HWE
reaction of ketone 14, which was derived from 5-propenylox-
azolidinone 13b, did not proceed. The β-methylated congener
15b was provided via Wittig reaction of 14 using Ph3P
CHCO2-t-Bu. Organocopper-mediated alkylations of 15a,b
gave the anti-SN2′ products 16a,b with moderate (E)-selectivity.
Although the (E)- and (Z)-isomers of 16a were not separated
in this step, the (E)-isomer of alcohol 17a was isolated by
column chromatography. Alcohols 17a,b were converted to the
desired Fmoc-protected amino acids 20a,b using methods
identical to those described for the synthesis of 10.
a
Reagents and conditions: (a) (i) MeMgCl, THF, −78 °C, 1.5 h; (ii)
CH2CXMgBr, CeCl3, THF, 0 °C, 3 h; (iii) recrystallization (12a,
48%; 12b, 47%); (b) NaH, THF, reflux, 1 h, then (Boc)2O, rt, 2 h
(13a, quantitative; 13b, 87%); (c) (i) O3, EtOAc, −78 °C, then Me2S,
−78 °C, 30 min; (ii) (EtO)2P(O)CH2CO2-t-Bu, LiCl, (i-Pr)2NEt,
MeCN, 0 °C, 3.5 h (56%); (d) O3, EtOAc, −78 °C, then Me2S, −78
°C, 15 min (99%); (e) Ph3PCHCO2-t-Bu, toluene, reflux, 10 h
(quantitative); (f) TBSO(CH2)3Li, CuCN, LiCl, THF−Et2O−n-
pentane, −78 °C, 2 h (16a, 92%, E/Z = 80/20; 16b, quantitative,
E/Z = 79/21); (g) H2SiF6(aq), MeCN−H2O, rt, 13.5 h (17a, 51%;
17b, 91%); (h) NsNH(Cbz), DEAD, PPh3, THF−toluene, 0 °C,
overnight (18a, 61%; 18b, quantitative); (i) PhSH, K2CO3, DMF, rt,
overnight (19a, 81%; 19b, 84%); (j) (i) TFA, CH2Cl2, rt, 2 h; (ii)
FmocOSu, (i-Pr)2NEt, MeCN−H2O, rt, 14.5 h (20a, 57%; 20b, 90%).
The epimeric D-Tyr-D-Orn dipeptide isostere 26 was also
synthesized. Although a (Z)-selective HWE reaction34 or
modified Wittig reaction35 of ketone 14 failed, Peterson
olefination36,37 provided an E/Z mixture of α,β-unsaturated
esters in moderate yield (15b/21 = 3/2) (Scheme 3). The
mixture was converted to three α-alkylated products, namely,
22, (E)-16b, and (Z)-16b. After cleavage of the TBS group,
Mitsunobu reaction, and removal of the Ns group, a mixture of
25 and 19b was separated from the (Z)-product derived from
(Z)-16b. The desired D-Tyr-D-Orn isostere 26 was obtained via
TFA-mediated deprotection and N-Fmoc protection, followed
by separation from 20b by column chromatography.
A representative synthesis of the isostere-containing FC131
derivatives is shown in Scheme 4. The protected peptide resin
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a
using 1H-pyrazole-1-carboxamidine gave the expected peptide
31a with a D-Tyr-Arg isostere. The derivatives 31b−d were also
obtained by the same procedure. In addition, an FC131
analogue, 31e, with a D-Tyr-Arg ethylene isostere was prepared
by hydrogenation of 31a using Pd/BaSO4.
Scheme 3
Structure−Activity Relationships of FC131 Deriva-
tives for CXCR4 Binding. We assessed the receptor binding
of cyclic peptides 31a−e with CXCR4 for inhibitory potency
against [125I]stromal-cell-derived factor-1 (SDF-1) binding to
CXCR4 (Table 1). The biological activities of the disubstituted
Table 1. Inhibitory Activity of FC131 Derivatives against
SDF-1 Binding to CXCR4
a
peptide
sequence
IC50 (μM)
FC131 cyclo(-D-Tyr1-L-Arg2-L-Arg3-L-Nal4-Gly5-)
0.084 0.037
0.33 0.074
31a
31b
31c
31d
31e
cyclo(-D-Tyr1-ψ[(E)-CHCH]-L-Arg2-L-Arg3-
L-Nal4-Gly5-)
cyclo(-D-Tyr1-ψ[(E)-CMeCH]-L-Arg2-L-Arg3- 0.50 0.21
L-Nal4-Gly5-)
cyclo(-D-Tyr1-ψ[(E)-CMeCMe]-L-Arg2-L-
2.5 1.0
0.10 0.029
>10
Arg3-L-Nal4-Gly5-)
cyclo(-D-Tyr1-ψ[(E)-CMeCMe]-D-Arg2-L-
a
Reagents and conditions: (a) TMSCH2CO2-t-Bu, LDA, THF−n-
Arg3-L-Nal4-Gly5-)
hexane, −78 °C, 4 h (53%, 15b/21 = 3/2); (b) (TBS)O(CH2)3Li,
CuCN, LiCl, THF−Et2O−n-pentane, −78 °C, 2 h (89%); (c)
H2SiF6(aq), MeCN−H2O, rt, 13.5 h (90%); (d) NsNH(Cbz), DEAD,
PPh3, THF−toluene, 0 °C, overnight (95%); (e) PhSH, K2CO3, DMF,
rt, overnight (82%); (f) (i) TFA, CH2Cl2, rt, 2 h; (ii) FmocOSu, (i-
Pr)2NEt, MeCN−H2O, rt, 14.5 h (26, 39%; 20b, 54%).
cyclo(-D-Tyr1-ψ[CH2−CH2]-L-Arg2-L-Arg3-L-
Nal4-Gly5-)
FC162 cyclo(-D-Tyr1-L-MeArg2-L-Arg3-L-Nal4-Gly5-)
FC122 cyclo(-D-Tyr1-D-MeArg2-L-Arg3-L-Nal4-Gly5-)
0.29 0.12
0.063 0.032
a
IC50 values are the concentrations for 50% inhibition of the
125I]SDF-1α binding to CXCR4 (n = 3).
[
a
Scheme 4
alkene-containing peptide 31a and trisubstituted alkene-
containing peptide 31b were slightly less than that of FC131.
These results suggested that the hydrogen-bonding capability of
the D-Tyr1−L-Arg2 peptide bond in FC131 is not necessary, but
is partly effective. The nearly equipotent activities of 31a and
31b indicated that the steric effects of a γ-methyl group of the
alkene isostere in 31b, which corresponds to the D-Tyr1
carbonyl oxygen of FC131, are not critical to the antagonistic
activity. Peptide 31c containing a tetrasubstituted alkene
isostere for D-Tyr1-L-MeArg2 exhibited low activity, whereas
the D-MeArg2 congener 31d showed activity nearly equipotent
to that of FC131. The bioactivity profile of the isostere-
containing peptides was similar to that of a series of the parent
peptides, including FC162 and FC122, except that their
potencies were somewhat lower than those of the parents.
These observations suggested that the trans-isomer of the D-
Tyr1−L/D-Arg2 peptide bond contributes to the bioactivity of
FC131 and its derivatives and that tri- and tetrasubstituted
alkene isosteres closely mimic the D-Tyr1-L-Arg2 dipeptide and
N-methylated congeners (D-Tyr1-L-MeArg2 and D-Tyr1-D-
MeArg2), respectively. On the other hand, an ethylene
isostere-containing analogue, 31e, did not bind with CXCR4.
The increased flexibility of the peptide backbone as a result of
the ethylene substructure led to a higher entropy loss upon
receptor binding, indicating that the fixed planar structure of
the D-Tyr1−L-Arg2 peptide bond is indispensable for biological
activity.
a
Reagents and conditions: (a) Fmoc-based solid-phase peptide
synthesis; (b) HFIP, CH2Cl2, rt, 2 h; (c) DPPA, NaHCO3, DMF,
−40 °C to rt, 48 h; (d) (i) TMSOTf−thioanisole in TFA, 0 °C to rt,
3.5 h; (ii) 1H-pyrazole-1-carboxamidine hydrochloride, Et3N, DMF, rt,
2 days (22% from 27); (e) H2, Pd/BaSO4, MeOH, rt, 36 h (33%).
28a was prepared on a 2-chlorotrityl [(2-Cl)Trt] resin using
standard Fmoc-based solid-phase peptide synthesis. After
cleavage of 28a from the resin, the linear peptide 29a was
cyclized to 30a using diphenylphosphoryl azide (DPPA).
Removal of the side chain protecting groups in 30a followed
by conversion of the Orn δ-amino group to a guanidino group
Molecular Modeling Study of Cyclic Peptidomimetics
and Identification of an Alternative Binding Mode of
Cyclic Pentapeptide CXCR4 Antagonists. To investigate
the bioactive conformations of cyclic pentapeptide-based
1
CXCR4 antagonists, the H NMR spectra of 31a−d were
obtained. Peptides 31a−c showed nuclear Overhauser effect
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Figure 4. Superimposed low-energy structures of FC131 (gray) and the isostere-containing derivatives: (a) 31a (blue), (b) 31b (green), (c) 31c
(yellow), (d) 31d-A (orange), and (e) 31d-B (pink).
Figure 5. Binding modes of FC131 derivatives: (a) FC131, (b) 31a, and (c) 31b.
(NOE) patterns around the isostere similar to those seen with
FC131. Interestingly, the D-Tyr1-D-MeArg2 isostere-containing
31d existed as a 1:1 mixture of two conformers; one conformer,
31d-A, exhibited NOE patterns similar to those of FC131, and
the other conformer, 31d-B, showed different patterns. We also
calculated their low-energy conformations in solution using a
molecular dynamics simulation based on the NMR data. The
calculations were performed by MacroModel using the Merck
molecular force field (MMFFs). The backbone conformations
of peptides 31a−c and 31d-A were similar to that of FC131
(Figure 4a−d), but 31d-B exhibited a conformation different
from that of FC131 with respect to the orientation around the
isostere alkene substructure in the D-Tyr1-D-MeArg2 dipeptide
(Figure 4e).
Recently, we and others have reported pharmacophore
models and binding models of FC131.38−41 In our FC131−
CXCR4 complex model,41 using NMR-based calculated
conformations of FC13120,22 and an X-ray crystal structure of
CXCR442 (Figure 5a), a guanidino group of L-Arg2 interacts
with both Asp97 and Asp187 in CXCR4, and an amide proton
of L-Arg2 forms a hydrogen bond network with Glu288 via a
crystal water molecule (w1), which is also involved in an
OH−π interaction with an aromatic ring of D-Tyr1. The L-Arg3
guanidino group interacts with His113, Thr117, and Asp171 in
CXCR4. In addition, the carbonyl oxygen of L-Nal4 is involved
in a second hydrogen bond network including Tyr255 and
Glu288 side chains via another crystal water molecule, and
hydrogen bonds are present between D-Tyr1 phenol and Tyr45
phenol and between the carbonyl oxygen of Gly5 and the
Ser285 hydroxyl group (see the Supporting Information). Most
of these interactions were maintained in all the following
binding conformations of FC131 derivatives.
Using this FC131−CXCR4 complex model, we next carried
out a prediction of the binding mode for 31a−d with CXCR4.
Models for binding of 31a−d with CXCR4 were obtained by
energy minimization of the complex structure using the
MMFF94s force field in the Molecular Operating Environment
(MOE) software package43 (Figures 5−7). The global
conformation of FC131 was little altered by substitution of
the D-Tyr1-L-Arg2 substructure with a disubstituted [ψ[(E)-
CHCH]] alkene unit in 31a (Figure 5b). The interactions of
the L-Arg2 guanidino group and the OH−π interaction of the D-
Tyr1 phenol group with the water molecule (w1) were also
maintained. The slightly less potent binding of 31a may be
attributable to the loss of the hydrogen bond networks by the
D-Tyr1−L-Arg2 peptide bond. Peptide 31b with a ψ[(E)-
CMeCH] isosteric unit also exhibited a binding conforma-
tion similar to that of FC131 with the same interaction modes
as those of 31a with CXCR4 (Figure 5c). The similar biological
activities and binding modes of 31a and 31b suggested that the
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steric effect of the D-Tyr1 carbonyl oxygen in FC131 and the γ-
methyl group of the isostere unit in 31b did not cause any
favorable or unfavorable interactions with CXCR4. This may be
a result of the outward orientation of the D-Tyr1 carbonyl group
in FC131 from the receptor.
mode. A possible alternative binding mode of FC122 was
obtained on the basis of the characteristic binding mode of 31d,
which is the isosteric peptide corresponding to FC122 with a D-
Tyr1-D-MeArg2 substructure (Figure 7a). Although the binding
structure of FC122 was inconsistent with its NMR-based
structure with respect to the orientation of the D-Tyr1−D-
MeArg2 peptide bond,41 the local conformation and binding
mode around the D-Tyr1−D-MeArg2 peptide bond in FC122
were similar to those of 31d. In this binding mode, the N-
methyl group of D-MeArg2 in FC122 probably restricts the
peptide backbone conformation, as does the isostere β-methyl
group in 31d. The less potent bioactivity of 31d compared with
that of the isosteric FC122 accounted for these similar binding
modes. The D-Tyr1 carbonyl oxygen in FC122 formed a
hydrogen bond network via a water molecule (w1), but in 31d,
the corresponding hydrogen bond was missing because of the
isosteric tetrasubstituted alkene.
The whole backbone conformation of 31c was also
maintained (Figure 6b), whereas the water molecule (w1)
In this study, docking simulations of FC131 derivatives were
performed using the X-ray crystal structure of CXCR4 in a
complex with a 16-residue cyclic peptide, CVX15.42 The
binding modes of FC131 and its derivatives met the
requirements of the shared indispensable functional groups
between CVX15 and FC131: the L-Arg3 guanidino group and
L‑Nal4 naphthalene group in FC131 correspond to Arg2 and
Nal3 in CVX15, respectively. Although the backbone
conformations varied in the cases of FC122 and 31d as a
result of the substitution mode of the D-Tyr1−D-MeArg2
peptide bond, the binding modes of the side chain functional
groups were maintained.
Figure 6. Binding modes of FC131 derivatives: (a) FC162 and (b)
31c.
was not settled below the antagonist because of the isostere β-
methyl group. The lower potency of 31c than that of 31a,b
could be rationalized by the loss of the w1-mediated OH−π
interaction. Similarly, it was suggested that the lower receptor
binding of the parent FC162 compared with that of FC131 was
a result of the N-methyl group of L-MeArg2 in FC162
preventing the OH−π interaction (Figure 6a).
A calculation using the FC131-like conformer 31d-A as an
initial structure afforded a binding mode for 31d similar to that
of 31a−c; however, the binding mode failed to provide a
possible explanation for the improved bioactivity of 31d. In
contrast, an alternative reasonable binding mode of 31d with
CXCR4 was obtained when another conformation, 31d-B, with
the flipped alkene substructure was used for the calculation
(Figure 7b). In this model, peptide 31d was bound to CXCR4
CONCLUSIONS
■
To investigate bioactive conformations, four alkene isosteres for
D-Tyr1-L/D-Arg2 dipeptides in the selective CXCR4 antagonist
FC131 and its derivatives were synthesized. The bioactivity
profiles of a series of the cyclic peptidomimetics suggested that
the D-Tyr1−L-Arg2 and D-Tyr1−L/D-MeArg2 peptide bonds of
the FC131 derivatives existed as trans-conformers in the
bioactive conformations. In the SAR study, the tetrasubstituted
alkene dipeptide isosteres adequately mimicked the N-
methylamide bonds in D-Tyr1-L-MeArg2 and D-Tyr1-D-MeArg2
dipeptides. NMR studies indicated that the backbone structures
of all the isostere-containing derivatives in solution were similar
to that of FC131, except that a different orientation of the
isostere alkene substructure was observed in 31d-B, containing
a D-Tyr1-D-MeArg2 isostere. A comparative biological evaluation
and binding-mode prediction suggest that the L-Arg2 amide
hydrogen in FC131 is involved in indirect receptor binding via
water molecules. Although the addition of a methyl group on
the D-Tyr1−L-Arg2 peptide bond (FC162) or the corresponding
isostere β-position (31c) did not influence the peptide
conformation in a complex with CXCR4, the potential
hydrogen bond network via water molecules in FC131 was
eliminated. On the other hand, an alternative binding mode was
identified in the D-MeArg2 congener 31d, which is the best
among the isostere-containing peptides. On the basis of this
binding mode of 31d, the previously unknown binding mode of
the D-MeArg2-substituted peptide (FC122) was identified; this
was not calculated directly from the NMR-based conforma-
tions. On the basis of this binding mode for CXCR4, it is
concluded that the improved potency of FC122 may be derived
from a secondary conformation stabilized by both chirality and
the N-methyl group of D-MeArg2. These results suggest that
two distinct binding modes of cyclic pentapeptide-based
Figure 7. Binding modes of FC131 derivatives: (a) FC122 and (b)
31d.
at a slightly different position on CXCR4 with the w1-mediated
OH−π interaction maintained, and the D-MeArg2 guanidino
group of 31d bound only with Asp187 via bimodal interactions.
The isostere β-methyl group may possibly restrict the peptide
backbone structure to a low-energy conformation such as 31d-
B, observed in NMR analysis, which leads to decreased entropy
loss upon receptor binding.
The binding-mode analysis of the most potent FC122 using
NMR-based conformations afforded a binding structure similar
to that of FC131 (data not shown); however, the highly potent
activity of FC122 could not be fully rationalized by this binding
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Article
concentrated under reduced pressure and extracted with EtOAc. The
extract was washed successively with 1 N HCl, brine, 5% NaHCO3,
and brine and dried over MgSO4. Concentration under reduced
pressure followed by flash chromatography over silica gel with n-
hexane−EtOAc (4:1) gave the title compound 3 (418 mg, 1.20 mmol,
quantitative) as colorless crystals: mp 96−97 °C; [α]D26 +48.8 (c 1.21,
CXCR4 antagonists may provide new insights into the design
of more potent derivatives and small-molecule antagonists with
novel scaffolds.
EXPERIMENTAL SECTION
■
1
CHCl3); IR (neat) 3355 (NH), 1742 (CO), 1712 (CO); H
General Procedures. All moisture-sensitive reactions were
performed using syringe−septum cap techniques under an argon
atmosphere, and all glassware was dried in an oven at 80 °C for 2 h
prior to use. Melting points were measured by a hot stage melting
point apparatus and are uncorrected. Optical rotations were measured
with a JASCO P-1020 polarimeter. For flash chromatography,
Wakogel C-300E was employed. For analytical HPLC, a COSMOSIL
5C18-ARII column (4.6 × 250 mm, Nacalai Tesque Inc., Kyoto,
Japan) was employed with a linear gradient of MeCN containing 0.1%
(v/v) TFA at a flow rate of 1 mL/min on a Shimadzu LC-20ADvp
(Shimadzu Corp., Ltd., Kyoto, Japan). Preparative HPLC was
performed using a COSMOSIL 5C18-ARII column (20 × 250 mm,
Nacalai Tesque Inc.) with a linear gradient of MeCN containing 0.1%
(v/v) TFA at a flow rate of 8 mL/min on a Shimadzu LC-6AD
(Shimadzu Corp., Ltd.). 1H NMR spectra were recorded using a JEOL
ECA-500 spectrometer, and chemical shifts are reported in δ (ppm)
relative to TMS (in CDCl3) or DMSO (in DMSO-d6) as an internal
standard. 13C NMR spectra were recorded using a JEOL ECA-500
spectrometer and referenced to the residual CHCl3 or DMSO signal.
Chemical shifts were reported in parts per million with the residual
NMR (500 MHz, CDCl3) δ 1.39 (s, 9H), 2.12 (s, 3H), 2.72 (d, J = 6.9
Hz, 2H), 3.78 (s, 3H), 3.96−4.07 (m, 1H), 4.67 (d, J = 9.2 Hz, 1H),
5.18−5.30 (m, 3H), 5.74−5.84 (m, 1H), 6.82 (d, J = 8.6 Hz, 2H), 7.08
(d, J = 8.6 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 21.0, 28.3 (3C),
37.5, 54.3, 55.2, 74.4, 79.4, 113.9 (2C), 118.0, 129.3, 130.2 (2C),
133.7, 155.4, 158.3, 169.7; HRMS (FAB) m/z calcd for C19H28NO5
(MH+) 350.1962, found 350.1958.
tert-Butyl (4R,5R,2E)-4-Acetoxy-5-[N-(tert-butoxycarbonyl)-
amino]-6-(4-methoxyphenyl)hex-2-enoate (4). Ozone gas was
bubbled through a stirred solution of the acetate 3 (3.29 g, 9.50 mmol)
in EtOAc (95 mL) at −78 °C until a blue color persisted. Me2S (14.0
mL, 190 mmol) was added to the solution at −78 °C. After being
stirred for 30 min at 0 °C, the mixture was dried over MgSO4 and
concentrated under reduced pressure to give the crude aldehyde,
which was used for the next reaction without further purification. To a
stirred suspension of LiCl (805 mg, 19.0 mmol) in MeCN (35 mL)
was added tert-butyl diethylphosphonoacetate (4.79 g, 19.0 mmol) in
MeCN (30 mL) and (i-Pr)2NEt (3.31 mL, 19.0 mmol) at 0 °C under
argon. After 20 min, the above aldehyde in MeCN (30 mL) was added
to the mixture at 0 °C, and the stirring was continued for 3 h. The
reaction was quenched with saturated NH4Cl at 0 °C. The mixture was
concentrated under reduced pressure and extracted with EtOAc. The
extract was washed successively with saturated citric acid, brine, 5%
NaHCO3, and brine and dried over MgSO4. Concentration under
reduced pressure followed by flash chromatography over silica gel with
n-hexane−EtOAc (4:1) gave the title compound 4 (2.63 g, 5.85 mmol,
62% yield) as a colorless oil: [α]D23 +44.7 (c 1.24, CHCl3); IR (neat)
1
solvent peak used as an internal standard. H NMR spectral data are
given as follows: chemical shift, multiplicity (br = broad, s = singlet, d
= doublet, t = triplet, q = quartet, m = multiplet), number of protons,
and coupling constant(s). Exact mass (HRMS) spectra were recorded
on a JMS-HX/HX 110A mass spectrometer. Infrared (IR) spectra
were obtained on a JASCO FT/IR-4100 FT-IR spectrometer with a
JASCO ATR PRO410-S. The purity of the peptides for bioassay was
calculated as >95% by HPLC on a COSMOSIL 5C18-ARII analytical
column at 220 nm absorbance (see the Supporting Information).
(3R,4R)-4-[N-(tert-Butoxycarbonyl)amino]-5-(4-
methoxyphenyl)pent-1-en-3-ol (2). To a solution of LiCl (4.11 g,
96.9 mmol) and ZnCl2 (13.2 g, 96.9 mmol) in THF (50 mL) was
added dropwise a solution of vinylmagnesium bromide in THF (1.3
M, 75.0 mL, 96.9 mmol) at −78 °C under argon, and the mixture was
stirred at 0 °C for 30 min. To a solution of Boc-L-Tyr(Me)OMe (10.0
g, 32.3 mmol) in CH2Cl2 (40 mL) and toluene (80 mL) was added
dropwise a solution of DIBAL-H in toluene (0.99 M, 72.0 mL, 71.1
mmol) at −78 °C under argon, and the mixture was stirred at −78 °C
for 30 min. To this solution was added dropwise the above solution of
vinylzinc reagent at −78 °C, and the mixture was stirred for 3 h with
warming to 0 °C. The reaction was quenched with 0.5 M Rochelle salt
and saturated NH4Cl. The mixture was concentrated under reduced
pressure and extracted with EtOAc. The extract was washed with
saturated citric acid, brine, saturated NaHCO3, and brine and dried
over MgSO4. Concentration under reduced pressure followed by flash
chromatography over silica gel with n-hexane−EtOAc (4:1) gave the
title compound 2 (3.07 g, 9.99 mmol, 31% yield) as white solids: mp
91−92 °C; [α]D28 +27.9 (c 1.39, CHCl3); IR (neat) 3323 (OH and
1
1706 (CO); H NMR (500 MHz, CDCl3) δ 1.39 (s, 9H), 1.45 (s,
9H), 2.13 (s, 3H), 2.72 (d, J = 6.9 Hz, 2H), 3.77 (s, 3H), 3.97−4.15
(m, 1H), 4.50−4.70 (m, 1H), 5.38 (ddd, J = 5.2, 2.9, 1.1 Hz, 1H), 5.81
(dd, J = 15.5, 1.1 Hz, 1H), 6.69 (dd, J = 15.5, 5.2 Hz, 1H), 6.82 (d, J =
8.6 Hz, 2H), 7.06 (d, J = 8.6 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ
20.3, 27.6 (3C), 27.9 (3C), 36.9, 53.7, 54.7, 72.4, 79.0, 80.1, 113.6
(2C), 124.2, 128.7, 129.7 (2C), 141.4, 154.9, 158.1, 164.4, 169.0;
HRMS (FAB) m/z calcd for C24H34NO7 (MH−) 448.2341, found
448.2344.
tert-Butyl (4R,5R,2E)-5-[N-(tert-Butoxycarbonyl)amino]-4-hy-
droxy-6-(4-methoxyphenyl)hex-2-enoate (5). To a solution of
the acetate 4 (1.21 g, 2.70 mmol) in MeOH (27 mL) was added
K2CO3 (746 mg, 5.40 mmol) at 0 °C, and the mixture was stirred for 2
h at room temperature. After the mixture was filtered, the filtrate was
concentrated under reduced pressure and extracted with EtOAc. The
extract was washed with brine and dried over MgSO4. Concentration
under reduced pressure followed by flash chromatography over silica
gel with n-hexane−EtOAc (3:1) gave the title compound 5 (1.06 g,
2.60 mmol, 96% yield) as colorless solids: mp 98−99 °C; [α]D24 +60.3
1
(c 1.04, CHCl3); IR (neat) 3436 (OH and NH), 1698 (CO); H
1
NMR (500 MHz, CDCl3) δ 1.38 (s, 9H), 1.45 (s, 9H), 2.81 (dd, J =
13.7, 7.4 Hz, 1H), 2.90 (dd, J = 13.7, 7.4 Hz, 1H), 3.75 (s, 3H), 3.77−
3.85 (m, 1H), 4.23−4.27 (m, 1H), 4.96−5.05 (m, 1H), 5.98 (dd, J =
15.5, 1.7 Hz, 1H), 6.78−6.84 (m, 3H), 7.14 (d, J = 8.6 Hz, 2H); 13C
NMR (125 MHz, CDCl3) δ 28.0 (3C), 28.2 (3C), 36.8, 55.06, 55.09,
70.8, 79.5, 80.3, 114.0 (2C), 123.3, 130.07 (2C), 130.10, 146.8, 156.0,
158.3, 165.6. Anal. Calcd for C22H33NO6: C, 64.84; H, 8.16; N, 3.44.
Found: C, 64.67; H, 8.19; N, 3.55.
NH), 1696 (CO); H NMR (500 MHz, CDCl3) δ 1.39 (s, 9H),
2.29−2.39 (m, 1H), 2.75−2.85 (m, 1H), 2.88 (dd, J = 13.7, 7.4 Hz,
1H), 3.69−3.77 (m, 1H), 3.78 (s, 3H), 4.07−4.14 (m, 1H), 4.79 (d, J
= 6.3 Hz, 1H), 5.18 (d, J = 10.3 Hz, 1H), 5.27 (d, J = 17.2 Hz, 1H),
5.89 (ddd, J = 17.2, 10.3, 5.7 Hz, 1H), 6.83 (d, J = 8.6 Hz, 2H), 7.15
(d, J = 8.6 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 28.3 (3C), 37.0,
55.2, 56.1, 72.6, 79.4, 113.9 (2C), 116.0, 130.3 (3C), 138.4, 156.2,
158.2; HRMS (FAB) m/z calcd for C17H26NO4 (MH+) 308.1856,
found 308.1855.
tert-Butyl (4R,5R,2E)-5-[N-(tert-Butoxycarbonyl)amino]-4-
[(methylsulfonyl)oxy]-6-(4-methoxyphenyl)hex-2-enoate (6).
To a stirred solution of the alcohol 5 (897 mg, 2.20 mmol) in
CH2Cl2 (22 mL) were added Et3N (3.06 mL, 22.0 mmol) and
methanesulfonyl chloride (851 μL, 11.0 mmol) at 0 °C, and the
mixture was stirred for 2 h at the same temperature. After the reaction
was quenched with water, the mixture was concentrated under reduced
pressure, and the residue was extracted with EtOAc. The extract was
(3R,4R)-4-[N-(tert-Butoxycarbonyl)amino]-5-(4-
methoxyphenyl)pent-1-en-3-yl Acetate (3). To a solution of the
alcohol 2 (368.9 mg, 1.20 mmol) in CHCl3 (12 mL) were added
pyridine (1.94 mL, 24.0 mmol), Ac2O (1.13 mL, 12.0 mmol), and 4-
(dimethylamino)pyridine (DMAP; 14.7 mg, 0.12 mmol) at 0 °C, and
the mixture was stirred for 2 h at the same temperature. The reaction
was quenched with saturated NH4Cl at 0 °C. The mixture was
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Journal of Medicinal Chemistry
Article
washed with brine and dried over MgSO4. Concentration under
reduced pressure followed by recrystallization from n-hexane−EtOAc
(10:1) gave the title compound 6 (1.03 g, 2.12 mmol, 96% yield) as
colorless crystals: mp 145−146 °C; [α]D22 +42.3 (c 1.00, CHCl3); IR
tert-Butyl (2R,5R,3E)-2-{3-[N-[(Benzyloxy)carbonyl]amino]-
propyl}-5- [N- (tert -b ut ox ycarbonyl)amino]-6-(4-
methoxyphenyl)hex-3-enoate (9). To a stirred solution of enoate
8 (2.69 g, 3.50 mmol) in DMF (35 mL) were added thiophenol (715
μL, 7.00 mmol) and K2CO3 (1.45 g, 10.5 mmol) at room temperature,
and the mixture was stirred at the same temperature for 3 h. After
concentration under reduced pressure, the residue was extracted with
EtOAc, washed with saturated citric acid, brine, 5% NaHCO3, and
brine, and dried over MgSO4. Concentration under reduced pressure
followed by flash chromatography over silica gel with n-hexane−
EtOAc (3:1) gave the title compound 9 (1.95 g, 3.35 mmol, 96%
yield) as a colorless oil: [α]D22 −19.8 (c 1.09, CHCl3); IR (neat) 3342
1
(neat) 3372 (NH), 1707 (CO); H NMR (500 MHz, CDCl3) δ
1.38 (s, 9H), 1.47 (s, 9H), 2.74 (dd, J = 14.3, 8.0 Hz, 1H), 2.89 (dd, J
= 14.3, 6.9 Hz, 1H), 3.06 (s, 3H), 3.78 (s, 3H), 4.04−4.13 (m, 1H),
4.71 (d, J = 9.2 Hz, 1H), 5.18−5.24 (m, 1H), 6.02 (d, J = 15.5 Hz,
1H), 6.78 (dd, J = 15.5, 5.7 Hz, 1H), 6.84 (d, J = 8.6 Hz, 2H), 7.16 (d,
J = 8.6 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 28.0 (3C), 28.2
(3C), 36.6, 39.2, 54.9, 55.2, 80.0, 80.3, 81.2, 114.0 (2C), 126.9, 128.5,
130.2 (2C), 139.2, 155.1, 158.5, 164.3. Anal. Calcd for C23H35NO8S:
C, 56.89; H, 7.26; N, 2.88. Found: C, 56.96; H, 7.03; N, 2.85.
tert-Butyl (2R,5R,3E)-5-[N-(tert-Butoxycarbonyl)amino]-2-[3-
(tert-butyldimethylsiloxy)propyl]-6-(4-methoxyphenyl)hex-3-
enoate (7). To a suspension of CuCN (1.79 g, 20.0 mmol) and LiCl
(1.70 g, 40.0 mmol) in THF (40 mL) was added dropwise a solution
of TBSO(CH2)3Li in n-pentane−Et2O (0.5 M, 40.0 mL, 20.0 mmol)
at −78 °C under argon, and the mixture was stirred for 30 min at 0 °C.
To the above mixture was added dropwise a solution of the mesylate 6
(2.43 g, 5.0 mmol) in THF (20 mL) at −78 °C, and the mixture was
stirred for 30 min at −78 °C. The reaction was quenched at −78 °C by
the addition of a saturated NH4Cl/28% NH4OH solution (1:1, 50
mL), with additional stirring at room temperature for 3 h. After the
mixture was concentrated under reduced pressure, the residue was
extracted with Et2O. The extract was washed with water and brine and
dried over MgSO4. Concentration under reduced pressure followed by
flash chromatography over silica gel with n-hexane−EtOAc (6:1) gave
the title compound 7 (2.64 g, 4.68 mmol, 94% yield) as a colorless oil:
[α]D23 −14.9 (c 1.09, CHCl3); IR (neat) 3372 (NH), 1715 (CO);
1H NMR (500 MHz, CDCl3) δ 0.04 (s, 6H), 0.89 (s, 9H), 1.28−1.48
1
(NH), 1700 (CO); H NMR (500 MHz, CDCl3) δ 1.31−1.43 (m,
21H), 1.60−1.68 (m, 1H), 2.64−2.72 (m, 1H), 2.76−2.85 (m, 2H),
3.06−3.17 (m, 2H), 3.75 (s, 3H), 4.27−4.37 (m, 1H), 4.50−4.60 (m,
1H), 4.83−4.91 (m, 1H), 5.09 (s, 2H), 5.39−5.48 (m, 2H), 6.80 (d, J
= 8.0 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 7.27−7.37 (m, 5H); 13C
NMR (125 MHz, CDCl3) δ 27.2, 27.9 (3C), 28.3 (3C), 29.4, 40.6
(2C), 49.3, 52.9, 55.1, 66.5, 79.2, 80.6, 113.6 (2C), 127.96 (2C),
128.01, 128.4 (2C), 128.7, 129.4, 130.4 (2C), 132.4, 136.5, 155.0,
156.3, 158.1, 173.0; HRMS (FAB) m/z calcd for C33H45N2O7 (MH−)
581.3232, found 581.3239.
(2R,5R,3E)-2-{3-[N-[(Benzyloxy)carbonyl]amino]propyl}-5-{N-
[(9-fluorenylmethoxy)carbonyl]amino}-6-(4-methoxyphenyl)-
hex-3-enoic Acid (10). To a stirred solution of enoate 9 (1.11 g, 1.90
mmol) in CH2Cl2 (20 mL) was added trifluoroacetic acid (5 mL), and
the mixture was stirred for 2 h at room temperature. After
concentration under reduced pressure, the residue was dissolved in
water (10 mL). To this solution were added Et3N (792 μL, 5.70
mmol) and FmocOSu (641 mg, 1.90 mmol) in MeCN (10 mL) at 0
°C, and the mixture was stirred for 2 h at room temperature. The
reaction was quenched by addition of 1 N HCl. After concentration
under reduced pressure, the residue was extracted with EtOAc. The
extract was washed with 1 N HCl and brine and dried over MgSO4.
Concentration under reduced pressure followed by flash chromatog-
raphy over silica gel with n-hexane−EtOAc (1:1) containing 1% AcOH
gave the title compound 10 (875 mg, 1.35 mmol, 71% yield) as
colorless solids: mp 166−167 °C; [α]D22 +1.7 (c 1.09, DMSO); IR
(m, 21H), 1.66−1.75 (m, 1H), 2.62−2.87 (m, 3H), 3.56 (t, J = 6.3 Hz,
2H), 3.77 (s, 3H), 4.25−4.51 (m, 2H), 5.40−5.52 (m, 2H), 6.81 (d, J
= 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H); 13C NMR (125 MHz, CDCl3)
δ −5.4 (2C), 18.3, 25.9 (3C), 28.0 (3C), 28.3 (3C), 28.9, 30.0, 40.8,
49.5, 52.8, 55.1, 62.6, 79.2, 80.4, 113.7 (2C), 128.9, 129.4, 130.5 (2C),
132.1, 155.0, 158.2, 173.3; HRMS (FAB) m/z calcd for C31H54NO6Si
(MH+) 564.3715, found 564.3712.
1
(neat) 1705 (CO); H NMR (500 MHz, DMSO-d6) δ 1.26−1.46
tert-Butyl (2R,5R,3E)-2-{3-[N-[(Benzyloxy)carbonyl]-N-[(2-
n i t r o p h e n y l ) s u l f o n y l ] a m i n o ] p r o p y l } - 5 - [ N - ( t e r t -
butoxycarbonyl)amino]-6-(4-methoxyphenyl)hex-3-enoate
(8). To a solution of the TBS ether 7 (2.59 g, 4.60 mmol) in MeCN−
H2O (1:1, 46 mL) was added aqueous H2SiF6 (3.28 M, 701 μL, 2.30
mmol) at 0 °C, and the mixture was stirred at room temperature for 2
h. After the mixture was concentrated, the residue was extracted with
EtOAc. The extract was washed with 5% K2CO3 and brine and dried
over MgSO4. Concentration under reduced pressure gave the
corresponding alcohol, which was used in the next step without
further purification. To a solution of the alcohol, PPh3 (1.81 g, 6.90
mmol), and NsNH(Cbz) (1.70 g, 5.06 mmol) in THF (50 mL) was
added diethyl azodicarboxylate (DEAD) in toluene (2.2 M, 2.51 mL,
5.52 mmol) at 0 °C under argon, and the mixture was stirred at the
same temperature for 3 h. The reaction was quenched at 0 °C by the
addition of MeOH (10 mL), with additional stirring at the same
temperature for 30 min. Concentration under reduced pressure
followed by flash chromatography over silica gel with n-hexane−
EtOAc (3:1) gave the title compound 8 (3.03 g, 3.95 mmol, 86%
yield) as a colorless oil: [α]D22 −11.0 (c 1.10, CHCl3); IR (neat) 3411
(m, 3H), 1.52−1.69 (m, 1H), 2.70 (d, J = 6.9 Hz, 2H), 2.85−2.93 (m,
1H), 2.94−3.04 (m, 2H), 3.67 (s, 3H), 4.12−4.27 (m, 4H), 5.03 (s,
2H), 5.46 (dd, J = 15.5, 8.6 Hz, 1H), 5.57 (dd, J = 15.5, 5.7 Hz, 1H),
6.80 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 2H), 7.25−7.39 (m, 8H),
7.39−7.45 (m, 2H), 7.51 (d, J = 8.6 Hz, 1H), 7.63−7.71 (m, 2H), 7.88
(d, J = 7.4 Hz, 2H), 12.3 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ
27.0, 29.3, 40.0 (2C, overlapped with DMSO peaks), 46.7, 48.0, 54.2,
54.9, 65.2, 65.3, 113.5 (2C), 120.1 (2C), 125.3 (2C), 127.1 (2C),
127.6 (2C), 127.7 (2C), 127.7, 128.1, 128.4 (2C), 130.3 (2C), 130.4,
133.0 (2C), 137.3, 140.7 (2C), 143.9 (2C), 155.4, 156.2, 157.6, 175.0;
HRMS (FAB) m/z calcd for C39H41N2O7 (MH+) 649.2908, found
649.2921.
(3R,4R)-4-[N-(tert-Butoxycarbonyl)amino]-5-(4-methoxy-
phenyl)-3-methylpent-1-en-3-ol (12a). To a stirred solution of
Boc-D-Phe-NMe(OMe) 11 (1.43 g, 4.23 mmol) in THF (35 mL) was
added dropwise a solution of MeMgCl in THF (1.9 M, 6.7 mL, 12.7
mmol) at −78 °C under argon, and the mixture was stirred for 1.5 h at
−78 °C. The reaction was quenched with saturated citric acid at −78
°C, and the whole was extracted with EtOAc. The extract was washed
successively with saturated citric acid, brine, saturated NaHCO3, and
brine and dried over MgSO4. Concentration under reduced pressure
gave a crude ketone, which was used immediately in the next step
without further purification. To a stirred suspension of anhydrous
CeCl3 (3.13 g, 12.7 mmol) and the above ketone in THF (30 mL) was
added dropwise a solution of vinylmagnesium bromide in THF (1.3
M, 9.8 mL, 12.7 mmol) at 0 °C under argon. After 3 h, the reaction
was quenched with saturated citric acid at −78 °C. The mixture was
concentrated under reduced pressure and extracted with EtOAc. The
extract was washed successively with saturated citric acid, brine,
saturated NaHCO3, and brine and dried over Na2SO4. Concentration
under reduced pressure followed by flash chromatography over silica
1
(NH), 1720 (CO); H NMR (500 MHz, CDCl3) δ 1.41 (s, 9H),
1.42 (s, 9H), 1.46−1.56 (m, 1H), 1.65−1.78 (m, 3H), 2.70−2.83 (m,
2H), 2.84−2.91 (m, 1H), 3.76 (s, 3H), 3.83 (t, J = 7.4 Hz, 2H), 4.21−
4.61 (m, 2H), 5.10 (s, 2H), 5.43−5.53 (m, 2H), 6.82 (d, J = 8.6 Hz,
2H), 7.08 (d, J = 8.6 Hz, 2H), 7.18−7.23 (m, 2H), 7.29−7.36 (m,
3H), 7.41−7.47 (m, 1H), 7.62−7.71 (m, 2H), 8.09 (d, J = 8.0 Hz,
1H); 13C NMR (125 MHz, CDCl3) δ 27.4, 27.9 (3C), 28.3 (3C), 29.2,
40.6, 47.7, 49.2, 52.8, 55.1, 69.3, 79.1, 80.6, 113.6 (2C), 124.2, 128.4
(2C), 128.6 (2C), 128.7, 129.3, 130.0, 130.4, 130.5, 131.5, 132.4,
132.7, 134.0 (2C), 134.2, 147.6, 151.6, 155.0, 158.1, 172.8; HRMS
(FAB) m/z calcd for C39H48N3O11S (MH−) 766.3015, found
766.3011.
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Article
gel with n-hexane−AcOEt (4:1) and recrystallization with n-hexane−
AcOEt (10:1) gave the title compound 12a (650 mg, 2.02 mmol, 48%
yield) as white solids: mp 94−96 °C; [α]D22 +78.2 (c 0.98, CHCl3); IR
14 h. The reaction was quenched by addition of saturated NH4Cl.
After concentration under reduced pressure, the residue was extracted
with EtOAc. The extract was washed with saturated NH4Cl, brine,
saturated NaHCO3, and brine and dried over Na2SO4. Concentration
under reduced pressure followed by flash chromatography over silica
gel with n-hexane−EtOAc (1:1) gave the title compound 17a (21.7
mg, 0.047 mmol, 51% yield) as a colorless oil: [α]D25 −37.3 (c 1.03,
1
(neat) 3436 (NH), 1691 (CO); H NMR (500 MHz, CDCl3) δ
1.28−1.37 (m, 12H), 2.50−2.60 (m, 1H), 2.90 (s, 1H), 3.04 (dd, J =
14.3, 3.4 Hz, 1H), 3.64−3.71 (m, 1H), 3.77 (s, 3H), 4.52 (d, J = 8.0
Hz, 1H), 5.15 (d, J = 10.3 Hz, 1H), 5.34 (d, J = 17.2 Hz, 1H), 5.98
(dd, J = 17.2, 10.3 Hz, 1H), 6.81 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 8.6
Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 24.7, 28.2 (3C), 34.6, 55.3,
60.1, 75.7, 79.5, 113.3, 113.8 (2C), 130.0 (2C), 130.8, 142.8, 156.5,
158.1. Anal. Calcd for C18H27NO4: C, 67.26; H, 8.47; N, 4.36. Found:
C, 67.23; H, 8.49; N, 4.42.
1
CHCl3); IR (neat) 1699 (CO); H NMR (500 MHz, CDCl3) δ
1.28−1.34 (m, 3H), 1.37−1.41 (m, 10H), 1.42 (s, 9H), 1.62−1.70 (m,
4H), 2.76 (d, J = 6.9 Hz, 2H), 3.03−3.12 (m, 1H), 3.51 (t, J = 6.9 Hz,
2H), 3.77 (s, 3H), 4.09−4.31 (m, 1H), 4.64 (d, J = 8.0 Hz, 1H), 5.16
(d, J = 9.2 Hz, 1H), 6.80 (d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.6 Hz, 2H);
13C NMR (125 MHz, CDCl3) δ 14.3, 28.0 (3C), 28.3 (3C), 28.7, 29.9,
(4R,5R)-N-(tert-Butoxycarbonyl)-5-ethenyl-4-(4-methoxy-
benzyl)-5-methyl-1,3-oxazolidin-2-one (13a). To a stirred
suspension of NaH (1.64 g, 41.1 mmol) in THF (20 mL) was
added dropwise a solution of the known allyl alcohol 12a (3.30 g, 10.3
mmol) in THF (80 mL) at 0 °C under argon, and the mixture was
heated under reflux for 1 h and stirred for 30 min at room
temperature. (Boc)2O (4.50 g, 20.6 mmol) was added to the mixture
at 0 °C, and the mixture was stirred for 2 h with warming to room
temperature. The mixture was poured into water at 0 °C, and the
whole was extracted with EtOAc. The extract was washed successively
with water and brine and dried over Na2SO4. Concentration under
reduced pressure followed by flash chromatography over silica gel with
n-hexane−EtOAc (6:1) gave the title compound 13a (3.58 g, 10.3
mmol, quantitative) as white solids: mp 117−118 °C; [α]D22 +82.3 (c
1.02, CHCl3); IR (neat) 1798 (CO), 1714 (CO); 1H NMR (500
MHz, CDCl3) δ 1.40 (s, 3H), 1.43 (s, 9H), 2.92 (dd, J = 14.3, 8.6 Hz,
1H), 3.06 (dd, J = 14.3, 5.7 Hz, 1H), 3.79 (s, 3H), 4.31 (dd, J = 8.6,
5.7 Hz, 1H), 5.16 (d, J = 10.9 Hz, 1H), 5.34 (d, J = 17.2 Hz, 1H), 5.76
(dd, J = 17.2, 10.9 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 8.6
Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 20.6, 27.8 (3C), 35.1, 55.2,
63.2, 82.1, 83.6, 114.2 (2C), 114.5, 128.5, 130.1 (2C), 139.5, 149.3,
151.4, 158.5. Anal. Calcd for C19H25NO5: C, 65.69; H, 7.25; N, 4.03.
Found: C, 65.40; H, 7.37; N, 4.03.
tert-Butyl (E)-3-[(4R,5R)-N-(tert-Butoxycarbonyl)-4-(4-me-
thoxybenzyl)-5-methyl-1,3-oxazolidin-2-on-5-yl]prop-2-
enoate (15a). Ozone gas was bubbled into a stirred solution of the
oxazolidin-2-one 13a (211 mg, 0.61 mmol) in EtOAc (10 mL) at −78
°C until a blue color persisted. To the solution was added dimethyl
sulfide (890 μL, 12.2 mmol) at −78 °C, and the mixture was stirred for
0.5 h at −78 °C. The mixture was dried over Na2SO4 and concentrated
under reduced pressure to give the corresponding aldehyde, which was
used for the next reaction without further purification. To a stirred
suspension of LiCl (52.0 mg, 1.22 mmol) in MeCN (4.0 mL) were
added tert-butyl diethylphosphonoacetate (380 μL, 1.22 mmol) and (i-
Pr)2NEt (213 μL, 1.22 mmol) successively at 0 °C under argon. After
30 min, the above aldehyde in MeCN (2.0 mL) was added to the
mixture at 0 °C, and the stirring was continued for 3.5 h. The reaction
was quenched by addition of saturated NH4Cl. After concentration
under reduced pressure, the residue was extracted with EtOAc. The
extract was washed with saturated citric acid, brine, saturated
NaHCO3, and brine and dried over Na2SO4. Concentration under
reduced pressure followed by flash chromatography over silica gel with
n-hexane−EtOAc (4:1) gave the title compound 15a (152 mg, 0.34
mmol, 56% yield) as white solids: mp 140−141 °C; [α]D26 +92.8 (c
0.99, CHCl3); IR (neat) 1800 (CO), 1714 (CO); 1H NMR (500
MHz, CDCl3) δ 1.42 (s, 3H), 1.47 (s, 18H), 2.94 (dd, J = 14.3, 9.2 Hz,
1H), 3.12 (dd, J = 14.3, 4.6 Hz, 1H), 3.80 (s, 3H), 4.37 (dd, J = 9.2,
4.6 Hz, 1H), 6.00 (d, J = 16.0 Hz, 1H), 6.63 (d, J = 16.0 Hz, 1H), 6.86
(d, J = 8.0 Hz, 2H), 7.15 (d, J = 8.0 Hz, 2H); 13C NMR (125 MHz,
CDCl3) δ 20.5, 27.8 (3C), 28.0 (3C), 34.9, 55.2, 62.8, 81.2, 81.3, 84.2,
114.3 (2C), 122.6, 128.0, 130.0 (2C), 146.1, 149.0, 150.9, 158.6, 165.0.
Anal. Calcd for C24H33NO7: C, 64.41; H, 7.43; N, 3.13. Found: C,
64.48; H, 7.23; N, 3.10.
38.8, 45.1, 55.2, 58.2, 62.4, 79.3, 80.4, 113.7 (2C), 124.5, 129.8, 130.1
(2C), 136.9, 155.0, 158.1, 173.5; HRMS (FAB) m/z calcd for
C26H42NO6 (MH+) 464.3007, found 464.3010.
tert-Butyl (2R,5R,3E)-2-{3-[N-[(Benzyloxy)carbonyl]-N-[(2-
n i t r o p h e n y l ) s u l f o n y l ] a m i n o ] p r o p y l } - 5 - [ N - ( t e r t -
butoxycarbonyl)amino]-6-(4-methoxyphenyl)-4-methylhex-3-
enoate (18a). To a solution of the alcohol 17a (21.7 mg, 0.047
mmol), PPh3 (24.6 mg, 0.094 mmol), and NsNH(Cbz) (33.0 mg,
0.094 mmol) in THF (0.47 mL) was added DEAD in toluene (2.2 M,
43 μL, 0.094 mmol) at 0 °C under argon, and the mixture was stirred
at the same temperature overnight. Concentration under reduced
pressure followed by flash chromatography over silica gel with n-
hexane−EtOAc (3:1) gave the title compound 18a (22.4 mg, 0.029
mmol, 61% yield) as a yellow oil: [α]D25 −24.0 (c 1.02, CHCl3); IR
1
(neat) 1726 (CO); H NMR (500 MHz, CDCl3) δ 1.38 (s, 9H),
1.40−1.48 (m, 10H), 1.57−1.78 (m, 6H), 2.69−2.85 (m, 2H), 3.06−
3.18 (m, 1H), 3.77 (s, 3H), 3.80 (t, J = 7.4 Hz, 2H), 4.12−4.28 (m,
1H), 4.50−4.67 (m, 1H), 5.11 (s, 2H), 5.19 (d, J = 9.7 Hz, 1H), 6.80
(d, J = 8.6 Hz, 2H), 7.06 (d, J = 8.6 Hz, 2H), 7.17−7.25 (m, 2H),
7.29−7.40 (m, 3H), 7.41−7.51 (m, 1H), 7.61−7.76 (m, 2H), 8.10 (d, J
= 8.0 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 14.3, 27.4, 27.9 (3C),
28.3 (3C), 29.4, 39.0, 45.0, 47.9, 55.2, 58.0, 69.3, 79.1, 80.4, 113.7
(2C), 124.2, 124.3, 128.5 (2C), 128.6 (2C), 128.7, 129.8, 130.1 (2C),
131.5, 132.8, 134.09, 134.11, 134.2, 137.5, 147.7, 151.6, 155.0, 158.1,
173.0; HRMS (FAB) m/z calcd for C40H52N3O11S (MH+) 782.3317,
found 782.3319.
(4R,5S)-5-Acetyl-N-(tert-butoxycarbonyl)-4-(4-methoxyben-
zyl)-5-methyl-1,3-oxazolidin-2-one (14). Ozone gas was bubbled
into a stirred solution of the oxazolidin-2-one 13b (1.20 g, 3.32 mmol)
in EtOAc (40 mL) at −78 °C until a blue color persisted. To the
solution was added dimethyl sulfide (2.4 mL, 33.2 mmol) at −78 °C,
and the mixture was stirred for 15 min at −78 °C. Concentration
under reduced pressure followed by flash chromatography over silica
gel with n-hexane−EtOAc (2:1) gave the title compound 14 (1.19 g,
3.28 mmol, 99% yield) as colorless crystals: mp 91−92 °C; [α]D26 +48.0
1
(c 1.02, CHCl3); IR (neat) 1817 (CO), 1725 (CO); H NMR
(500 MHz, CDCl3) δ 1.35 (s, 3H), 1.44 (s, 9H), 2.28 (s, 3H), 2.93
(dd, J = 14.9, 8.6 Hz, 1H), 3.05 (dd, J = 14.3, 5.2 Hz, 1H), 3.79 (s,
3H), 4.86 (dd, J = 8.6, 5.2 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 7.16 (d, J
= 8.6 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 17.6, 24.8, 27.8 (3C),
34.8, 55.2, 60.0, 84.3, 86.5, 114.3 (2C), 127.7, 130.0 (2C), 148.4,
150.4, 158.6, 207.8. Anal. Calcd for C19H25NO6: C, 62.80; H, 6.93; N,
3.85. Found: C, 62.68; H, 6.80; N, 3.89.
tert-Butyl (E)-3-[(4R,5R)-N-(tert-Butoxycarbonyl)-4-(4-me-
thoxybenzyl)-5-methyl-1,3-oxazolidin-2-on-5-yl]but-2-enoate
(15b). The ketone 14 (490 mg, 1.35 mmol) and Ph3P=CHCO2-t-Bu
(1.11 g, 2.97 mmol) were dissolved in toluene (6.0 mL), and the
mixture was gently refluxed for 10 h. Concentration under reduced
pressure followed by flash chromatography over silica gel with n-
hexane−EtOAc (3:1) gave the title compound 15b (621 mg, 1.35
mmol, quantitative) as colorless crystals: mp 174−175 °C; [α]D26 +76.7
1
(c 1.00, CHCl3); IR (neat) 1813 (CO), 1714 (CO); H NMR
(500 MHz, CDCl3) δ 1.41 (s, 3H), 1.46 (s, 9H), 1.49 (s, 9H), 1.91 (d,
J = 1.2 Hz, 3H), 2.94 (dd, J = 14.3, 9.2 Hz, 1H), 3.12 (dd, J = 14.3, 4.6
Hz, 1H), 3.80 (s, 3H), 4.42 (dd, J = 9.2, 4.6 Hz, 1H), 5.95 (d, J = 1.2
Hz, 1H), 6.87 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 8.6 Hz, 2H); 13C NMR
(125 MHz, CDCl3) δ 14.7, 20.2, 27.9 (3C), 28.1 (3C), 35.3, 55.2,
tert-Butyl (2R,5R,3E)-5-[N-(tert-Butoxycarbonyl)amino]-2-(3-
hydroxypropyl)-6-(4-methoxyphenyl)-4-methylhex-3-enoate
(17a). To a solution of the TBS ether 16a (52.6 mg, 0.091 mmol) in
MeCN−H2O (1:1, 2.0 mL) was added aqueous H2SiF6 (3.28 M, 28
μL, 0.091 mmol) at room temperature, and the mixture was stirred for
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61.5, 80.5, 84.2, 84.4, 114.3 (2C), 117.1, 128.1, 130.1 (2C), 149.0,
150.6, 153.9, 158.6, 165.7. Anal. Calcd for C25H35NO7: C, 65.06; H,
7.64; N, 3.03. Found: C, 64.97; H, 7.71; N, 3.07.
4.32−4.41 (m, 1H), 4.53 (ddd, J = 7.6, 7.6, 6.9 Hz, 1H), 5.39 (ddd, J =
15.1, 8.9, 1.4 Hz, 1H), 5.55 (dd, J = 15.1, 4.1 Hz, 1H), 6.62 (d, J = 8.3
Hz, 2H), 6.93 (d, J = 8.3 Hz, 2H), 7.02 (d, J = 8.3 Hz, 1H), 7.30 (dd, J
= 8.3, 2.1 Hz, 1H), 7.42−7.50 (m, 2H), 7.50−7.57 (m, 2H), 7.61 (s,
1H), 7.76 (d, J = 8.9 Hz, 1H), 7.79−7.87 (m, 4H), 8.35 (dd, J = 6.9,
5.5 Hz, 1H), 9.15 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 25.3,
26.3, 27.9, 28.4, 29.0, 38.1, 40.1 (overlapped with DMSO peaks), 40.3,
43.5, 50.3, 51.5, 54.3, 54.5, 115.0 (2C), 125.5, 126.0, 127.4, 127.4,
127.5, 127.6, 127.66, 127.75, 128.0, 130.1 (2C), 131.9, 132.8, 132.9,
134.8, 155.6, 156.72, 156.75, 167.6, 170.6, 171.5, 172.2; HRMS (FAB)
m/z calcd for C37H49N10O5 (MH+) 713.3882, found 713.3881.
Cyclo(D-Tyr-ψ[-CH2-CH2]-Arg-Arg-Nal-Gly-)·2TFA (31e). The
cyclic pseudopeptide 31a (2.64 mg, 0.0281 mmol) was treated with
Pd/BaSO4 (59.5 mg, 0.0281 mmol) in MeOH (300 μL) under a H2
atmosphere at room temperature for 36 h. After filtration and
concentration under reduced pressure, purification by preparative
HPLC gave the bistrifluoroacetate of the title peptide 31e (0.874 mg,
0.00927 mmol, 33% yield) as a colorless freeze-dried powder: [α]D29
tert-Butyl (Z)-3-[(4R,5R)-N-(tert-Butoxycarbonyl)-4-(4-me-
thoxybenzyl)-5-methyl-1,3-oxazolidin-2-on-5-yl]but-2-enoate
(21). To a solution of diisopropylamine (41.0 μL, 0.29 mmol) in THF
(0.29 mL) at −78 °C was added dropwise n-BuLi in n-hexane (1.65 M,
0.18 mL, 0.29 mmol). After the mixture was stirred at 0 °C for 30 min,
a solution of TMSCH2CO2-t-Bu (66.0 μL, 0.30 mmol) in THF (0.19
mL) was added dropwise at −78 °C. After the mixture was stirred at
−78 °C for 2 h, a solution of ketone 14 (21.0 mg, 0.058 mmol) in
THF (0.19 mL) was added dropwise. The resulting mixture was stirred
at −78 °C for 4 h. The reaction was quenched by addition of saturated
NH4Cl at −78 °C. The whole mixture was stirred at room temperature
for 15 min, extracted with Et2O, and dried over MgSO4. Concentration
under reduced pressure followed by flash chromatography over silica
gel with n-hexane−EtOAc (3:1) gave an E/Z mixture of the title
compound 21 and 15b (14.2 mg, 0.031 mmol, 53% yield, 15b/21 = 3/
2) as a clear oil. Data for compound 21 (purified by preparative
HPLC): colorless oil; [α]D25 −115.7 (c 1.01, CHCl3); IR (neat) 1818
1
−31.7 (c 0.0933, DMSO); H NMR (500 MHz, DMSO-d6) δ 1.08−
1.53 (m, 10H), 1.61−1.79 (m, 2H), 1.91−2.04 (m, 1H), 2.35 (dd, J =
13.2, 7.4 Hz, 1H), 2.46−2.58 (m, 1H, overlapped with DMSO peak),
2.95−3.10 (m, 4H), 3.14 (dd, J = 13.7, 9.7 Hz, 1H), 3.24 (dd, J = 13.7,
4.6 Hz, 1H), 3.44−3.48 (m, 1H), 3.61−3.72 (m, 1H), 3.75−3.84 (m,
1H), 3.84−3.93 (m, 1H), 4.42−4.52 (m, 1H), 6.45 (d, J = 9.7 Hz,
1H), 6.62 (d, J = 8.0 Hz, 2H), 6.89 (d, J = 8.0 Hz, 2H), 7.41−7.56 (m,
5H), 7.76 (s, 1H), 7.82−7.90 (m, 3H), 8.10 (d, J = 7.4 Hz, 1H), 8.13−
8.21 (m, 1H), 8.26−8.36 (m, 1H), 9.14 (s, 1H); 13C NMR (125 MHz,
DMSO-d6) δ 25.4, 26.5, 27.0, 29.0, 29.3, 31.2, 36.4, 40.2, 40.4, 41.2,
42.5, 46.9, 50.4, 53.7, 56.1, 115.0 (2C), 125.5, 126.1, 127.2, 127.4,
127.47, 127.55, 127.8, 128.7, 129.9 (2C), 131.8, 133.0, 135.6, 155.5,
156.68, 156.70, 168.0, 170.7, 172.6, 174.8; HRMS (FAB) m/z calcd for
C37H51N10O5 (MH+) 715.4038, found 715.4046.
1
(CO), 1705 (CO); H NMR (500 MHz, DMSO-d6) δ 1.16 (s,
9H), 1.52 (s, 9H), 1.73 (s, 3H), 1.94 (d, J = 1.7 Hz, 3H), 2.66 (dd, J =
13.7, 10.3 Hz, 1H), 3.30 (dd, J = 13.7, 3.4 Hz, 1H), 3.77 (s, 3H), 4.76
(dd, J = 10.3, 3.4 Hz, 1H), 5.74 (d, J = 1.7 Hz, 1H), 6.82 (d, J = 8.6
Hz, 2H), 7.14 (d, J = 8.6 Hz, 2H); 13C NMR (125 MHz, DMSO-d6) δ
19.2, 22.7, 27.4 (3C), 28.1 (3C), 36.2, 55.2, 65.2, 80.7, 82.9, 85.9,
114.0 (2C), 119.0, 129.0, 130.7 (2C), 148.7, 151.6, 158.5, 160.8,
164.7; HRMS (FAB) m/z calcd for C25H36NO7 (MH+) 462.2486
found 462.2486.
Peptide Synthesis. The protected linear peptides 28a−c were
constructed by Fmoc-based solid-phase synthesis on H-Gly-(2-Cl)Trt
resin (0.66 mmol/g, 152 mg, 0.10 mmol). The Pbf group for Arg was
employed for side chain protection. Fmoc-protected amino acids (0.30
mmol) were coupled by using N,N′-diisopropylcarbodiimide (DIC)
(46.4 μL, 0.3 mmol) and HOBt·H2O (45.9 mg, 0.3 mmol) in DMF.
Coupling of dipeptide isostere 10, 20a, or 20b (0.30 mmol) was
carried out with DIC (46.4 μL, 0.3 mmol) and HOAt (40.8 mg, 0.30
mmol). Completion of each coupling reaction was ascertained using
the Kaiser ninhydrin test. The Fmoc protecting group was removed by
treating the resin with 20% piperidine in DMF.
[
125I]SDF-1 Binding and Displacement. Membrane extracts
were prepared from HEK293 cell lines expressing CXCR4. For ligand
binding, 50 μL of the inhibitor, 25 μL of [125I]SDF-1α (0.3 nM,
Perkin-Elmer Life Sciences), and 25 μL of the membrane/bead
mixture [7.5 μg of membrane/well, 0.5 mg of PVT WGA beads
(Amersham)/well] in assay buffer (25 mM HEPES, pH 7.4, 1 mM
CaCl2, 5 mM MgCl2, 140 mM NaCl, 250 mM sucrose, 0.5% BSA)
were incubated in the wells of an Optiplate plate (Perkin-Elmer Life
Sciences) at room temperature for 1 h. The bound radioactivity was
counted for 1 min/well in a TopCount (Packard). The inhibitory
activity of the test compounds was determined on the basis of the
inhibition of [125I]SDF-1 binding to the receptors (IC50, triplicate
experiments, Table 1).
By use of a procedure identical with that described for the
preparation of the protected linear peptide, 28d was obtained from H-
Gly-(2-Cl)Trt resin (0.80 mmol/g, 125 mg, 0.10 mmol) using
dipeptide isostere 26 (0.30 mmol).
Cyclo(D-Tyr-ψ[(E)-CHCH]-Arg-Arg-Nal-Gly-)·2TFA (31a).
The resulting protected peptide resin 28a (275 mg) was subjected
to hexafluoro-2-propanol (HFIP)−CH2Cl2 (2:8, 15 mL) treatment at
room temperature for 2 h. After filtration of the residual resin, the
filtrate was concentrated under reduced pressure to give a crude linear
peptide, 29a. To a mixture of the linear peptide and NaHCO3 (42.0
mg, 0.500 mmol) in DMF (40 mL) was added diphenylphosphoryl
azide (DPPA; 53.9 μL, 0.250 mmol) at −40 °C. The mixture was
stirred for 40 h with warming to room temperature and then filtered.
The filtrate was concentrated under reduced pressure, followed by
flash chromatography over silica gel with CHCl3−MeOH (90:10) to
give the protected cyclic peptide 30a. The peptide 30a was treated
with 1 M TMSOTf−thioanisole in TFA (3 mL) at room temperature
for 3 h. Concentration under reduced pressure gave an oily residue,
which was used immediately in the next step without purification. To a
solution of the crude mixture in DMF (2 mL) were added (i-Pr)2NEt
(261 μL, 1.50 mmol) and 1H-pyrazole-1-carboxamidine hydrochloride
(73.3 mg, 0.500 mmol), and the mixture was stirred at room
temperature for 2 days. After concentration under reduced pressure,
purification by preparative HPLC gave the bistrifluoroacetate of the
title cyclic peptide 31a (20.4 mg, 0.0217 mmol, 22% yield based on H-
Gly-(2-Cl)Trt resin) as a colorless freeze-dried powder: [α]D28 −43.4 (c
NMR Spectroscopy. The peptide sample was dissolved in DMSO-
d6 at 5 mM. 1H NMR spectra of the peptides were recorded at 300 K.
The assignment of the proton resonance was achieved by use of
1H−1H COSY spectra. COSY spectra were composed of 2048
complex points in the F2 dimension and 256 complex points, which
were zero-filled to yield a final data matrix of 2048 × 512 points.
3J(HN,Hα) coupling constants were measured from one-dimensional
spectra. The mixing time for NOESY experiments was set at 200 ms.
NOESY spectra were composed of 1024 complex points in the F2
dimension and 512 complex points, which were zero-filled to yield a
final data matrix of 1024 × 1024 points, with 32 scans per t1
increment. The cross-peak intensities were classified on the basis of
the number of contour lines.
Structural Calculations. The structure calculations were
performed by MacroModel using the MMFFs. Pseudoatoms were
defined for the CH3 protons on the alkene of 31b−d, methylene
protons of D-Tyr1, D/L-Arg2, L-Arg3, and L-Nal4, and aromatic protons
1
of D-Tyr1, the prochiralities of which were not identified from H
NMR data. The dihedral φ angle constraints were calculated on the
3
basis of the Karplus equation: J(HN,Hα) = 6.7 cos2 θ − 1.3 cos θ +
1.5. Lower and upper angle errors were set to 15°. The NOESY
spectrum with a mixing time of 200 ms was used for the estimation of
the distance restraints between protons. The NOE intensities were
classified into three categories (strong, medium, and weak) on the
basis of the number of contour lines in the cross-peaks to define the
1
0.133, DMSO); H NMR (500 MHz, DMSO-d6) δ 1.25−1.50 (m,
5H), 1.52−1.70 (m, 3H), 2.58 (d, J = 7.6 Hz, 2H), 2.74 (ddd, J = 8.2,
8.2, 7.6 Hz, 1H), 2.94−3.15 (m, 6H), 3.29 (dd, J = 15.8, 5.5 Hz, 1H),
3.66 (dd, J = 15.8, 6.9 Hz, 1H), 4.14 (ddd, J = 8.9, 8.2, 8.2 Hz, 1H),
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upper limit distance restraints (2.7, 3.5, and 5.0 Å, respectively). The
upper limit restraints were increased by 1.0 Å for the involved
pseudoatoms except the aromatic protons, for which the restraints
were increased by 2.0 Å. Lower bounds between nonbonded atoms
were set to their van der Waals radii (1.8 Å). A total of 100 000
random structures were generated by molecular dynamic simulation
starting with any initial structure in water; the structures matched with
the restraints from the NMR data were then selected. The structure in
the lowest potential energy was defined as the most stable structure in
solution.
Docking of Peptidomimetics to CXCR4. Initial structures of
31a−c, 31d-A, and 31d-B were built by energy minimization of NMR-
based structures described above. The resulting models were
incorporated into CXCR4, and the water molecules of the crystal
structure of CXCR4 bound to CVX15 (PDB code 3OE0) were
manually input as appropriate. After that, the structures of
peptidomimetics were minimized in the receptor structure in MOE
using MMFF94s and a distance-dependent dielectric constant of 1
with a 10 Å cutoff distance. The steepest descent algorithm was used
for the minimization, followed by the conjugate gradient method. The
maximum iterations of each run were set to 100 steps, and the root-
mean-square (rms) gradient value of 0.01 was set for the criteria of the
minimizations. In this calculation, the backbone atoms of the receptor
were fixed.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and characterization data for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(11) Nicolaou, K. C.; Trujillo, J. I.; Jandeleit, B.; Chibale, K.;
Rosenfeld, M.; Diefenbach, B.; Cheresh, D. A.; Goodman, S. L.
Design, synthesis and biological evaluation of nonpeptide integrin
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J.; DeGrado, W. F.; Mousa, S.; Jadhav, P. K. Rapid synthesis of RGD
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AUTHOR INFORMATION
Corresponding Author
■
*Phone: +81-75-753-4551. Fax: +81-75-753-4570. E-mail:
soishi@pharm.kyoto-u.ac.jp (S.O.); nfujii@pharm.kyoto-u.ac.jp
(N.F.).
(13) Ihara, M.; Noguchi, K.; Saeki, T.; Fukuroda, T.; Tsuchida, S.;
Kimura, S.; Fukami, T.; Ishikawa, K.; Nishikibe, M.; Yano, M.
Biological profiles of highly potent novel endothelin antagonists
selective for the ETA receptor. Life Sci. 1992, 50, 247−255.
(14) Kikuchi, T.; Ohtaki, T.; Kawata, A.; Imada, T.; Asami, T.;
Masuda, Y.; Sugo, T.; Kusumoto, K.; Kubo, K.; Watanabe, T.;
Wakimasu, M.; Fujino, M. Cyclic hexapeptide endothelin receptor
antagonists highly potent for both receptor subtypes ETA and ETB.
Biochem. Biophys. Res. Commun. 1994, 200, 1708−1712.
(15) Iqbal, J.; Sanghi, R.; Das, S. K. Endothelin receptor antagonists:
an overview of their synthesis and structure-activity relationship. Mini-
Rev. Med. Chem. 2005, 5, 381−408.
(16) Cardillo, G.; Gentilucci, L.; Tolomelli, A.; Spinosa, R.; Calienni,
M.; Qasem, A. R.; Spampinato, S. Synthesis and evaluation of the
affinity toward μ-opioid receptors of atypical, lipophilic ligands based
on the sequence c[-Tyr-Pro-Trp-Phe-Gly-]. J. Med. Chem. 2004, 47,
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(18) Veber, D. F.; Freidinger, R. M.; Perlow, D. S.; Paleveda, W. J.
Jr.; Holly, F. W.; Strachan, R. G.; Nutt, R. F.; Arison, B. H.; Homnick,
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Grants-in-Aid for Scientific
Research and the Targeted Protein Research Program from
MEXT and Health and Labor Science Research Grants
(Research on HIV/AIDS, Japan). K.K. and K.T. are grateful
for JSPS Research Fellowships for Young Scientists.
ABBREVIATIONS USED
■
CXCR4, CXC chemokine receptor type 4; HWE reaction,
Horner−Wadsworth−Emmons reaction; HIV, human immu-
nodeficiency virus; MMFFs, Merck molecular force field; MOE,
Molecular Operating Environment; Nal, 3-(2-naphthyl)alanine;
Orn, ornithine; SAR, structure−activity relationship; SDF-1,
stromal-cell-derived factor-1
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