Synthesis and biological evaluation of selective CXCR4 antagonists containing alkene dipeptide isosteres

Article abstract of DOI:10.1039/b917236j

A set of cyclic peptide analogues of a selective CXCR4 antagonist FC131 [cyclo(-d-Tyr-Arg-Arg-Nal-Gly-)] were synthesized and bioevaluated. Using (E)-alkene and (Z)-fluoroalkene dipeptide isosteres for Arg-Arg and Arg-Nal substructures, indispensable or the partial contribution of the two peptide bonds to the CXCR4 antagonism and anti-HIV activity was demonstrated. FC131 and the analogues were shown to selectively inhibit SDF-1 binding to CXCR4, whereas no inhibition of binding of SDF-1 to CXCR7 was observed.

Full text of DOI:10.1039/b917236j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of selective CXCR4 antagonists
containing alkene dipeptide isosteres†
Tetsuo Narumi,a,b Ryoko Hayashi,a Kenji Tomita,a Kazuya Kobayashi,a Noriko Tanahara,a Hiroaki Ohno,a
Takeshi Naito,a Eiichi Kodama,c Masao Matsuoka,c Shinya Oishi*a and Nobutaka Fujii*a
Received 21st August 2009, Accepted 31st October 2009
First published as an Advance Article on the web 4th December 2009
DOI: 10.1039/b917236j
A set of cyclic peptide analogues of a selective CXCR4 antagonist FC131
[cyclo(-D-Tyr-Arg-Arg-Nal-Gly-)] were synthesized and bioevaluated. Using (E)-alkene and
(Z)-fluoroalkene dipeptide isosteres for Arg-Arg and Arg-Nal substructures, indispensable or the
partial contribution of the two peptide bonds to the CXCR4 antagonism and anti-HIV activity was
demonstrated. FC131 and the analogues were shown to selectively inhibit SDF-1 binding to CXCR4,
whereas no inhibition of binding of SDF-1 to CXCR7 was observed.
Introduction
Chemokine receptor CXCR4 belongs to the G-protein coupled re-
ceptor family1 and plays important roles in physiological functions
including angiogenesis,2 chemotaxis,3 and neurogenesis.4 CXCR4
is associated with various pathological conditions including cancer
metastasis,5 HIV-1 infection6 and rheumatoid arthritis.7 The broad
spectrum of biological activities has led to extensive research
towards the development of specific inhibitors directed against
CXCR4.8,9
We have previously identified a highly potent CXCR4 antag-
onist, T140 1, which is a b-sheet-like 14-mer peptide with a
single disulfide bridge (Fig. 1).10 The indispensable residues for
bioactivity are four amino acids positioned across the disulfide
bridge: Arg2, L-3-(2-naphthyl)alanine3 (Nal3), Tyr5 and Arg14.
These residues were used for further molecular-size reductions.
Using these critical residues for a characteristic combination of
cyclic pentapeptide libraries, a potent CXCR4 antagonist FC131
Fig. 1 Structures of T140 (1), FC131 (2), and the (E)-alkene and
(Z)-fluoroalkene FC131 analogues. Bold residues of 1 are indispensable for
the potent CXCR4-antagonistic activity. Nal = L-3-(2-naphthyl)alanine.
2 was identified, which exerts comparable anti-HIV activity to
T140.11
Structure–activity relationship (SAR) studies of FC131 by
various modifications such as amino acid substitution,12 tuning of
the ring structure,13 andbackbone modifications,14,15 demonstrated
that the potent bioactivity of FC131 is attributed to the ideal
spatial dispositions of the side-chain functional groups. For
example, N-methylation of the peptide bonds of FC131 and
the epimeric congeners significantly altered the bioactivity.14 The
appropriate combination of sequence, chirality and auxiliary
groups on the cyclic pentapeptide backbone can accommodate
the bioactive conformations.
Replacement of the planar amide bond with a surrogate
alkene substructure, including unsubstituted,15,16 fluorinated,17
multi-substituted,18 and trifluoromethylated19 alkenes, represents
a promising approach to probe structural and electrostatic re-
quirements in bioactive peptides. In particular, fluorinated or
substituted alkene isosteres are considered to be more appropri-
ate peptide bond mimetics when compared with unsubstituted
alkene isosteres because of the favorable electrostatic and steric
properties.20 In this study, the contributions of the Arg2-Arg3
and Arg3-Nal4 peptide bonds to the bioactivity of FC131 were
investigated through the synthesis and bioevaluation of alkene
aGraduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-
ku, Kyoto 606-8501, Japan. E-mail: soishi@pharm.kyoto-u.ac.jp, nfujii@
pharm.kyoto-u.ac.jp; Fax: +81-75-753-4570; Tel: +81-75-753-4551
bInstitute of Biomaterials and Bioengineering, Tokyo Medical and Dental
University, Chiyoda-ku, Tokyo 101-0062, Japan
=
analogues of FC131, cyclo[(-D-Tyr-Arg-y[trans-CX CH]-Arg-
Nal-Gly-)] 3E/3F and cyclo[(-D-Tyr-Arg-Arg-y[(trans-CX CH]-
=
cLaboratory of Virus Control, Institute for Virus Research, Kyoto University,
Sakyo-ku, Kyoto 606-8507, Japan
Nal-Gly-)] 4E/4F (X = H or F). The comparative study using
unsubstituted and fluorinated isosteres aimed to reveal the electro-
static contributions of the amide carbonyl groups of these peptide
bonds to the bioactivity of FC131.
† Electronic supplementary information (ESI) available: Additional ex-
perimental procedures, NMR spectra and HPLC charts. See DOI:
10.1039/b917236j
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of 10 and the subsequent Mitsunobu reaction using CbzNHNs
to give a bis(sulfonamide) 12. The expected Fmoc-Orn(Cbz)-
Results and discussion
Synthesis of alkene dipeptide isosteres and the application to
FC131 analogues
=
y[(E)-CH CH]-Orn(Cbz)-OH 14 was obtained by sequential
manipulation of the protecting groups including cleavage of two
Ns groups in 12 and N-Fmoc protection and deprotection of the
t-Bu ester.
In our previous synthesis of the Arg-Nal type (E)-alkene dipeptide
isostere (EADI),15 a protected arginine was employed as the
starting material. However, the derivatives were not experimentally
tractable in the same synthetic process due to the presence of
the protected guanidino group. Consequently, the synthesis of
FC131 analogue 3E bearing Arg-Arg type EADI began with
Boc-Orn(Cbz)-OMe 5 (Orn = L-ornithine, Scheme 1). Ornithine
includes a 3-aminoprop-1-yl group that can be used as a precursor
of the arginine side-chain. Successive treatment of the ester 5 with
diisobutylaluminium hydride (DIBAL-H) and vinylzinc chloride,
gave a syn and anti-mixture of allylic alcohols 6 (syn : anti =
87 : 13). The syn-isomer 6a was obtained by recrystallization. Boc
cleavage of 6a with TFA followed by N-2-nitrobenzenesulfonyl
(Ns) protection produced a Ns-amide 7. The intramolecular
Mitsunobu reaction of 7 proceeded to provide 2,3-cis-aziridine
8 in high yield. Ozonolysis of 8 and the subsequent Horner–
Wadsworth–Emmons reaction predominantly afforded the (E)-
isomer of b-aziridinyl-a,b-enoate 9 in 57% yield. Organocopper-
mediated anti-SN2¢ type alkylation of 9 gave the a-alkylated prod-
uct 10 with a TBS-protected 3-hydroxyprop-1-yl group, that can be
modified to provide another Arg side-chain. Transformation to the
Orn side-chain was performed by TBAF-mediated deprotection
Diastereoselective synthesis of (Z)-fluoroalkene dipeptide
isosteres (FADI) has recently been accomplished.17e The key step
in this synthesis is the one-pot reaction involving organocopper-
mediated reduction/asymmetric alkylation via transmetalation to
establish the a-alkylated isostere with appropriate configuration.
According to the previous synthetic study of peptide3F bearing the
Arg-Arg type FADI,17i the preparation of the Orn-Nal type FADI
was carried out (Scheme 2). The one-pot reaction of g,g-difluoro-
a,b-enoyl sultam 1517i with 2-(bromomethyl)naphthalene yielded
the corresponding a-alkylated sultam 16. Cleavage of the TBS
group with aqueous H2SiF6 followed by the Mitsunobu reaction
with BocNHNs afforded the sulfonamide 17. The sulfonamide
17 was converted to the Fmoc-protected FADI 18 by a standard
deprotection/protection manipulation.
Scheme 2 Synthesis of the Orn-Nal-type (Z)-fluoroalkene dipeptide
isostere. Reagents and conditions: (a) (i) Me2CuLi·LiI·2LiBr, THF–Et2O,
-78 ◦C, 0.5 h; (ii) Hexamethylphosphoric triamide (HMPA), -78 ◦C, 0.5 h;
(iii) Ph3SnCl, THF, -40 ◦C, 10 min; (iv) 2-(bromomethyl)naphthalene,
-40 ◦C, 20 h (79%); (b) (i) H2SiF6 aq. MeCN–MeOH, 0 ◦C, 1 h; (ii)
BocNHNs, DEAD, PPh3, THF, rt, 12 h (98%); (c) (i) 1 N LiOH, H2O2,
THF–H2O, rt, 2 h; (ii) TFA, CH2Cl2, rt, 0.5 h; (iii) Fmoc-OSu, Et3N,
DMF–H2O–MeCN, rt, 12 h (85%).
The resulting isosteres 14 and 18 were incorporated into
the peptide-chain by standard Fmoc-based solid-phase peptide
synthesis (Scheme 3). Briefly, the protected peptides 21a,b were
cleaved off the resins 20a,b with 1,1,1,3,3,3-hexafluoroisopropanol
(HFIP). After diphenylphosphoryl azide (DPPA)-mediated cy-
clization, the Cbz- or Ns-groups on the ornithine d-amino
group(s) of 22a,b were deprotected by treatment with 1 M
TMSBr/thioanisole in TFA or with 95% aqueous TFA fol-
lowed by 2-mercaptoethanol/1,8-diazabicyclo[5,4,0]-7-undecene
(DBU), respectively. Subsequently, the amino group(s) of 23a,b
were modified using 1H-pyrazole-1-carboxamidine to provide the
expected peptidomimetics 3E and 4F with the Arg guanidino
group(s).
Scheme
1 Synthesis of the Orn-Orn-type (E)-alkene dipeptide
isostere. Reagents and conditions: (a) (i) Diisobutylaluminium hydride
(DIBAL-H), CH2Cl2–toluene, -78 C, 1 h; (ii) H2C CHMgCl, ZnCl2,
◦
=
LiCl, -78 ◦C, 3 h (42%, syn : anti = 87 : 13); (b) recrystallization;
(c) (i) TFA, CH2Cl2, 0 ◦C, 1 h; (ii) 2-nitrobenzenesulfonyl chloride
(NsCl), Et3N, CH2Cl2, rt, 1 h (74%); (d) diethyl azodicarboxylate
(DEAD), PPh3, THF, rt, 9 h (93%); (e) (i) O3, EtOAc, -78 ◦C, then
Me2S; (ii) (EtO)2P(O)CH2CO2t-Bu, LiCl, (i-Pr)2NEt, MeCN, 0 ◦C,
4 h (57%); (f) TBSO(CH2)3Li, CuCN, LiCl, THF–Et2O–n-pentane,
-78 ◦C, 2 h (66%); (g) tetrabutylammonium fluoride (TBAF), THF,
0
24
◦C, 14
(93%); (i) (i) PhSH, K2CO3, MeCN–DMSO, 50 ◦C,
2
h
(85%); (h) CbzNHNs, DEAD, PPh3, THF,
0
◦C,
h;
h
(ii) N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu), Et3N,
THF–H2O, 0 ◦C, 4 h (quant); (j) 4 N HCl–dioxane, rt, 8 h (65%).
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Table 1 Inhibitory activity of FC131 and the derivatives against [125I]-
SDF-1 binding to CXCR4 and CXCR7
IC50/mMc
Peptide
CXCR4
CXCR7
FC131 2
0.068
1.46
1.78
> 10
> 10
> 10
> 10
> 10
> 10
> 10
cyclo[(-D-Tyr-Arg-WE -Arg-Nal-Gly-)] 3Ea
cyclo[(-D-Tyr-Arg-WF -Arg-Nal-Gly-)] 3Fb
cyclo[(-D-Tyr-Arg-Arg-WE -Nal-Gly-)] 4Ea
cyclo[(-D-Tyr-Arg-Arg-WF -Nal-Gly-)] 4Fb
a The WE indicates the isosteric y[(E)-CH CH] substructure. b The WF
=
indicates the isosteric y[(Z)-CF CH] substructure. c IC50 values are the
=
concentrations for 50% inhibition of the [125I]-SDF-1a binding to CXCR4
or CXCR7 transfectants of CHO-K1 cells.
amide hydrogen of Arg3 and/or the conformational change by the
backbone modification. Comparison of the biological activities
of the two analogues 3E and 3F showed that the unsubstituted
alkene analogue 3E was essentially equipotent in inhibiting the
binding of SDF-1 to CXCR4 to the fluoroalkene analogue 3F.
This observation indicates that the presence of the fluorine atom
did not aid the appropriate mimicry of the steric and electrostatic
effects of the Arg2 carbonyl group.
Our previous studies on N-methylamino acid-scanning14 and
EADI replacement15 (4E) revealed that the modification of Arg3-
Nal4 peptide bond resulted in a significant loss of CXCR4-binding
inhibition activity. This is possibly due to the absence of the
amide hydrogen and/or the dissolution of the pseudo-1,3-allylic
strain between the Arg3 carbonyl group and the Nal4 side chain.
Although the possible mimicking ability of the fluorine atom
was expected,20 the introduction of the FADI into the Arg3-Nal4
dipeptide (4F) also led to the loss of CXCR4-binding activity again
[IC50(4F) > 10 mM]. This result indicates that the amide hydrogen
within the Arg3-Nal4 dipeptide of FC131 may contribute to a
critical interaction required for binding to CXCR4.
Furthermore, inhibitory activity of the peptides for CXCR7,
which is also a target receptor of SDF-1, was also examined;
however, no inhibition was observed even at 10 mM. This
observation showed that FC131 and the related analogues are
selective CXCR4 antagonists and show similar target specificity
as the T140 derivatives.21
Scheme 3 Synthesis of the alkene analogues of FC131. Reagents and
conditions: (a) Fmoc-based SPPS; (b) 1,1,1,3,3,3-hexafluoroisopropanol
(HFIP), CH2Cl2; (c) diphenylphosphoryl azide (DPPA), NaHCO3,
DMF, -40 ◦C to rt; (d) 23a: 1 M TMSBr/thioanisole in TFA,
m-cresol, 1,2-ethandithiol, 6 h; 23b: (i) TFA-H2O, 3 h; (ii) 2-mer-
captoethanol, 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), DMF, 50 ◦C,
2.5 h; (e) 3E: 1H-pyrazole-1-carboxamidine·HCl, (i-Pr)2NEt, DMF; 4F:
1H-pyrazole-1-carboxamidine·HCl, Et3N, DMF.
Anti-HIV activity based on the inhibition of HIV-1 entry
into the target cells was examined by the MAGI assay using
three strains including NL4-3, IIIB and Ba-L (Table 2). As
in the case of CXCR4-binding inhibition, moderate anti-HIV
activity against NL4-3 and IIIB strains was observed for peptides
3E/3F containing EADI and FADI for the Arg2-Arg3 dipeptide
Biological evaluation of FC131 analogues with EADI and FADI
The biological activities of cyclic pseudopeptides 3E/3F17i and
4E15/4F were comparatively evaluated, in which the Arg2-Arg3
and Arg3-Nal4 dipeptide sites were substituted with EADI or
FADI. The inhibitory potency against [125I]-SDF-1-binding to
CXCR4 or CXCR7 was measured (Table 1). Both EADI and
FADI analogues (3E and 3F) with substitution at the Arg2-Arg3
dipeptide moderately inhibited the SDF-1 binding to CXCR4
[IC50(3E) = 1.46 mM; IC50(3F) = 1.78 mM]. The potency was
approximately 20-fold lower than the original FC131 2 [IC50(2) =
0.068 mM], indicating the partial contribution of the amide bond
within the Arg2-Arg3 dipeptide to the bioactivity of FC131. This is
consistent with the bioactivity of the FC131 analogue containing
the Arg2-MeArg3 dipeptide substructure,14 suggesting that the
less potent activity may be attributed to the loss of the H-bonding
Table 2 Anti-HIV activities of FC131 and the derivatives
EC50/mMa
Peptide
NL4-3
IIIB
Ba-L
2
0.014 0.002
0.234 0.004
0.332 0.073
> 10
0.019 0.003
0.295 0.069
0.403 0.051
> 10
> 10
> 10
> 10
> 10
> 10
3E
3F
4E
4F
> 10
> 10
a EC50 is the concentration that blocks HIV-1 infection by 50%.
618 | Org. Biomol. Chem., 2010, 8, 616–621
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[IC50(3E) = 0.234 mM (NL4-3) and 0.295 mM (IIIB); IC50(3F) =
0.332 mM (NL4-3) and 0.403 mM (IIIB)]. The potency was
significantly less compared with the original FC131 2 [IC50(2) =
0.014 mM (NL4-3) and 0.019 mM (IIIB)]. Substitutions of Arg3-
Nal4 dipeptides with EADI and FADI resulted in the loss of the
anti-HIV activity [IC50(4E/4F) > 10 mM (NL4-3 and IIIB)], which
also correlates with the observation of no CXCR4 antagonistic
activity of these peptides. For the Ba-L strain, that utilizes CCR5
for entry, all peptides showed no inhibitory activity at 10 mM.
and 8.6), 5.42 (1 H, d, J 8.0), 7.23–7.31 (5 H, m), 7.61–7.65 (2 H,
m), 7.74–7.80 (1 H, m) and 7.99–8.06 (1 H, m); dC (125 MHz,
CDCl3, Me4Si) -5.4 (2 C), 18.2, 25.9 (3 C), 26.0, 27.9 (3 C), 28.7,
29.9, 33.0, 40.3, 49.0, 56.5, 62.5, 66.5, 80.6, 125.2, 128.0 (3 C),
128.4 (2 C), 130.9, 131.2, 132.8, 133.2, 133.3, 134.8, 136.5, 147.7,
156.4 and 172.6; HRMS (FAB), m/z calcd for C35H52N3O9SSi
([M - H]-) 718.3199, found 718.3190.
(2R,5S,3E)-8-[N -(Benzyloxycarbonyl)amino]-2-{3-[N -(ben-
zyloxycarbonyl) amino]prop - 1 - yl} - 5 - [N - (fluorenylmethoxycar -
bonyl)amino]oct-3-enoic acid (14). Compound 13 (610 mg,
0.790 mmol) was dissolved in 4 N HCl–dioxane (8 cm3) and
the mixture was stirred at room temperature for 8 h. After the
mixture was concentrated 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 chromatography over silica gel with
EtOAc–n-hexane–AcOH (1/1/0.02) gave the title compound 14
Conclusions
In conclusion, Orn-Orn type EADI 14 and Orn-Nal type FADI
18 were synthesized and incorporated into FC131 analogues.
Comparative bioevaluation of a set of peptides containing EADI
or FADI at Arg2-Arg3 and Arg3-Nal4 positions revealed the sig-
nificant contribution of these peptide bonds to FC131 bioactivity.
Although substitutions with alkene isosteres resulted in a decrease
in bioactivity, the structural and functional requirements of the
corresponding amide bonds to biological activity was shown. The
results will be useful for the development of cyclic pentapeptide-
based CXCR4 antagonists. Additionally, it was demonstrated that
FC131 and the analogous were selective CXCR4 antagonists,
which did not inhibit SDF-1 binding to CXCR7. Further studies
on the synthesis and biological evaluation of CXCR4 antagonists
with peptide bond mimetics are the subject of an ongoing
investigation.
◦
(367 mg, 65%) as a white solid: mp 162–163 C; [a]2D4 -16.6 (c
1.02, DMSO); dH (500 MHz, DMSO, Me4Si) 1.38–1.40 (7 H, m),
1.55–1.66 (1 H, m), 2.87 (1 H, m), 2.97 (4 H, m), 3.93 (1 H, m),
4.17–4.24 (1 H, m), 4.24–4.31 (1 H, m), 4.96–5.03 (5 H, m), 5.47
(2 H, m), 7.28–7.41 (17 H, m), 7.65–7.69 (2 H, m), 7.86–7.88 (2 H,
m) and 12.20 (1 H, s); dC (125 MHz, DMSO, Me4Si) 26.1, 27.0,
29.2, 30.9, 40.0 (2 C), 46.7, 47.8, 51.9, 65.1, 65.2 (2 C), 120.0 (2
C), 125.2 (2 C), 127.0 (2 C), 127.5 (2 C), 127.6 (3 C), 127.7 (3 C),
128.3 (4 C), 133.2, 137.2 (2 C), 140.7, 143.8 (2 C), 143.9 (2 C),
156.1 (3 C) and 174.8; HRMS (FAB), m/z calcd for C42H44N3O8
([M - H]-) 718.3134, found 718.3125.
Experimental
(2R,5S,3Z)-5-[(tert-Butoxycarbonyl)amino]-8-(tert-butyldi-
methylsiloxy)-4-fluoro-2-(naphthalen-2-ylmethyl)oct-3-enoyl (S)-
sultam (16). To a suspension of CuI (2.22 g, 11.6 mmol) in THF
Synthesis
◦
tert-Butyl
(2R,5S,3E)-8-[N-(benzyloxycarbonyl)amino]-2-
(250 cm3) at -78 C under argon was added dropwise a solution
[3-(tert-butyldimethylsiloxy)prop-1-yl]-5-[N -(o-nitrobenzenesul-
fonyl)amino]oct-3-enoate (10). 1.57 M t-BuLi in n-pentane
solution (28.7 cm3, 45 mmol) was added dropwise to a stirred
solution of I(CH2)3OTBS (6.78 g, 22.5 mmol) in dry Et2O
(10.6 cm3) under argon at -78 ◦C. Following stirring at -78 ◦C
for 30 min, the mixture was stirred at room temperature for
10 min. To a stirred solution of CuCN (1.26 g, 14.1 mmol) and
LiCl (1.19 g, 28.1 mmol) in dry THF (20 cm3) under argon at
-78 ◦C, the above 0.5 M TBSO(CH2)3Li in THF–Et2O–n-pentane
solution (28.2 cm3) was added dropwise, and the mixture was
further stirred at 0 ◦C for 10 min. To the above mixture, a solution
of the enoate 9 (1.92 g, 3.51 mmol) in dry THF (20 cm3) was
added dropwise at -78 ◦C, and the mixture was further stirred
for 2 h at -78 ◦C. The reaction was quenched by the addition
of a saturated NH4Cl/28% NH4OH solution (1/1, 30 cm3),
with additional stirring at room temperature for 1 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
EtOAc–n-hexane (1/5) gave the title compound 10 (1.68 g, 66%)
as a colorless oil: [a]D24 -89.8 (c 1.00, CHCl3); dH (500 MHz,
CDCl3, Me4Si) 0.00 (6 H, s), 0.85 (9 H, s), 1.22–1.26 (2 H, m), 1.34
(9 H, s), 1.46–1.51 (6 H, m), 2.59–2.64 (1 H, m), 3.12–3.14 (2 H,
m), 3.45–3.48 (2 H, m), 3.89–3.93 (1 H, m), 4.79–4.87 (1 H, m),
5.04 (2 H, s), 5.22 (1 H, dd, J 15.5 and 7.4), 5.34 (1H, dd, J 15.5
of MeLi·LiBr complex in Et2O (1.5 M, 15.5 cm3, 23.2 mmol), and
the mixture was stirred for 10 min at 0 ◦C. To the solution of
the above organocopper reagent at -78 ◦C was added dropwise
a solution of the N-enoyl sultam 15 (1.80 g, 2.90 mmol) in THF
(70 cm3). The mixture was stirred for 30 min at -78 ◦C and HMPA
(8.31 cm3, 46.4 mmol) was◦added dropwise to the mixture. After
stirring for 30 min at -78 C, a solution of triphenyltin chloride
(2.24 g, 5.80 mmol) in THF (20 cm3) was added dropwise, and
the mixture was subsequently stirred for 10 min at -40 ◦C. 2-
(Bromomethyl)naphthalene (5.13 g, 23.2 mmol) in THF (30 cm3)
was added dropwise and the mixture was stirred for 20 h at
-40 ◦C. The reaction was quenched at -40 ◦C by the addition
of a saturated NH4Cl/28% NH4OH solution (1/1, 50 cm3) and
the mixture was stirred at room temperature for an additional
30 min. The mixture was extracted with Et2O and the extract was
washed with brine and dried over MgSO4. Concentration under
reduced pressure followed by flash chromatography over silica gel
with EtOAc–n-hexane (1/3) gave the title compound 16 (1.71 g,
79%) as a colorless oil: [a]2D4 -74.3 (c 1.00, CHCl3); dH (500 MHz,
CDCl3, Me4Si) 0.02 (6 H, s), 0.30 (3 H, s), 0.76 (3 H, s), 0.88 (9 H,
s), 1.18–1.30 (2 H, m), 1.38–1.48 (11 H, m), 1.52–1.66 (4 H, m),
1.70–1.82 (2 H, m), 1.91 (1 H, dd, J 13.7 and 8.0), 2.97 (1 H, dd,
J 13.7 and 6.9), 3.24–3.36 (3 H, m), 3.46–3.56 (2 H, m), 3.64–3.79
(1 H, m), 4.08–4.21 (1 H, m), 4.48–4.60 (1 H, m), 4.67 (1 H, d, J
8.6), 5.06 (1 H, dd, J 36.1 and 9.2), 7.38–7.44 (3 H, m), 7.64 (1
H, s) and 7.71–7.78 (3 H, m); dC (125 MHz, CDCl3, Me4Si) -5.3
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(2 C), 18.3, 19.6, 19.8, 26.0 (3 C), 26.3, 28.3 (3 C), 28.6, 28.7, 32.8,
38.2, 40.6 (d, J 2.4), 43.0, 44.5, 47.3, 48.0, 51.7, 52.9, 62.5, 64.9,
79.6, 103.7 (d, J 13.1), 125.3, 125.7, 127.5, 127.6, 127.8, 127.9,
127.9, 132.4, 133.4, 135.1, 154.9, 158.8 (d, J 261.1) and 172.2; dF
(125 MHz, CDCl3, CFCl3) -119.5; HRMS (FAB), m/z calcd for
C40H58FN2O6SSi ([M - H]-) 741.3774, found: 741.3768.
resin by filtration, the filtrate solution was concentrated under
reduced pressure to give a crude protected peptide 21a. To a
mixture of 21a and NaHCO3 (21 mg, 0.25 mmol) in DMF (20 cm3)
was added DPPA (0.0270 cm3, 0.13 mmol) at -40 ◦C. The mixture
was stirred for 66 h with warming to room temperature and then
filtered. The filtrate was concentrated under reduced pressure to
give the protected cyclic peptide 22a. The peptide 22a was treated
with 1 M TMSBr/thioanisole in TFA (10 cm3) in the presence
of m-cresol and 1,2-ethandithiol (0.117 cm3) for 6 h at 0 ◦C. The
mixture was poured into ice-cold dry Et2O. The resulting powder
was collected and washed three times with ice-cold dry Et2O. To
a stirred solution of the precipitant 23a in DMF (1 cm3) were
added (i-Pr)2NEt (0.014 cm3, 0.08 mmol) and 1H-pyrazole-1-
carboxamidine·HCl (12 mg, 0.04 mmol), and the mixture was
stirred at room temperature for 60 h. After concentration under
reduced pressure, purification by preparative HPLC gave the bis-
trifluoroacetate salt of the title peptide 3E (1.9 mg, 9% yield based
on H–Gly–(2-Cl)Trt resin, >98% purity by HPLC analysis) as
a colorless freeze-dried powder: HRMS (FAB), m/z calcd for
C37H49N10O5 ([M+H]+) 713.3882, found 713.3886.
(2R,5S,3Z)-5-[N-(Fluorenylmethoxycarbonyl)amino]-4-fluoro-
2-(naphthalen-2-ylmethyl)-8-[N-(o-nitrobenzenesulfonyl)amino]oct-3-
enoic acid (18). To a solution of the sultam 17 (986 mg,
1.08 mmol) and aqueous 50% H2O2 (0.383 cm3, 5.62 mmol)
in THF–H2O (5/1, 15 cm3) at 0 ◦C was added aqueous 1 N
LiOH (2.16 cm3, 2.16 mmol). The mixture was stirred at room
temperature for 2 h. Following dilution with EtOAc (50 cm3),
the mixture was washed with 0.1 N HCl and dried over MgSO4.
Concentration under reduced pressure gave the corresponding
acid, which was used in the next step without purification. TFA
(5 cm3) was added to a solution of the acid in CH2Cl2 (5 cm3)
at 0 ◦C, and the mixture was stirred at room temperature for
30 min. Concentration under reduced pressure gave an oily
residue, which was dissolved in MeCN–DMF–H2O (10/9/1,
40 cm3). Fmoc-OSu (584 mg, 1.73 mmol) and Et3N (0.332 cm3,
2.38 mmol) were added to the mixture at 0 ◦C and the mixture
was stirred at room temperature for 12 h. After being diluted
with EtOAc (280 cm3), the reaction mixture was washed with
1 N HCl and dried over MgSO4. Concentration under reduced
pressure followed by flash chromatography over silica gel with
EtOAc–n-hexane–AcOH (1/1/0.02) gave the title compound 18
(673 mg, 85%) as a colorless semisolid: [a]2D5 -27.4 (c 1.00, CHCl3);
=
cyclo(–D–Tyr–Arg–Arg–W[(Z)-CF CH]–Nal–Gly–)·2TFA
(4F). Cyclic peptide 4F was synthesized by a procedure identical
with that described for the synthesis of 3E. The protected peptide
22b (32.0 mg, 0.0270 mmol) was treated with aqueous TFA/H2O
(95/5, 10 cm3) for 3 h. Concentration under reduced pressure gave
an oily residue. To a solution of the residue in DMF (8 cm3) were
added 2-mercaptoethanol (0.0191 cm3, 0.270 mmol) and◦DBU
(0.0809 cm3, 0.540 mmol), and the mixture was stirred at 50 C for
2.5 h. After concentration under reduced pressure, the residue
23b was treated with Et3N (0.112 cm3, 0.810 mmol) and 1H-
pyrazole-1-carboxamidine·HCl (39.6 mg, 0.270 mmol) in DMF
(2 cm3). After concentration under reduced pressure, purification
by preparative HPLC gave the bis-trifluoroacetate salt of the title
peptide 4F (3.6 mg, 6% yield based on H–Gly–(2-Cl)Trt resin,
89% purity by HPLC analysis): HRMS (FAB), m/z calcd for
C37H48FN10O5 ([M+H]+) 731.3788, found 731.3796.
d
H (500 MHz, CDCl3, Me4Si) 1.31–1.40 (2 H, m), 1.41–1.55 (2 H,
m), 2.93–2.99 (3 H, m), 3.28 (1 H, dd, J 13.7 and 6.3), 3.78–3.87
(1 H, m), 4.08–4.16 (2 H, m), 4.28 (1 H, dd, J 10.3 and 6.9), 4.40
(1 H, dd, J 10.3 and 6.9 Hz), 4.81 (1 H, d, J 9.2), 4.93 (1 H, dd,
J 36.1 and 9.7), 5.38 (1 H, t, J 5.7), 7.26–7.79 (18 H, m) and
8.02–8.07 (1 H, m); dC (125 MHz, CDCl3, Me4Si) 25.6, 28.9, 38.4,
42.9, 47.1, 51.6 (d, J 27.6), 62.3, 66.7, 104.7 (d, J 14.4), 120.0
(2 C), 124.4, 124.9, 125.0 (2 C), 125.2 (2 C), 125.5 (2 C), 125.9,
127.1, 127.5 (2 C), 127.7, 127.8, 130.9, 132.2, 132.7, 133.3, 133.5,
133.5, 135.6, 141.3 (2 C), 143.7, 143.8, 147.9, 158.0 (d, J 262.0),
163.0 and 177.0; dF (125 MHz, CDCl3, CFCl3) -120.8; HRMS
(FAB), m/z calcd for C40H35FN3O8S ([M - H]-) 736.2134, found:
736.2137.
[
125I]-SDF-1 binding and displacement
Membrane extracts were prepared from CHO-K1 cell lines ex-
pressing either CXCR4 or CXCR7. For ligand binding, 0.050 cm3
of the inhibitor, 0.025 cm3 of [125I]-SDF-1a (0.3 nM, Perkin-Elmer
Life Sciences) and 0.025 cm3 of the membrane/beads mixture
[CXCR4: 7.5 mg well-1 of membrane, 0.5 mg well-1 of PVT WGA
beads (Amersham); CXCR7: 3 mg well-1 of membrane, 0.25 mg
well-1 of PVT-PEI type A beads (Amersham)] 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 places (Perkin-Elmer Life Sciences) at room
temperature for 1 h. The bound radioactivity was counted for
1 min well-1 in a TopCount (Packard). Inhibitory activity of the
test compounds was determined based on the inhibition of [125I]-
SDF-1 binding to the receptors (IC50).
Peptide synthesis
The protected linear peptides 20a,b were constructed on H–
Gly–(2-Cl)Trt resin (0.8 mmol g-1, 38 mg, 0.03 mmol). t-Bu
was employed for Tyr side-chain protection. Fmoc-protected
amino acids (0.3 mmol) were coupled by using DIC (0.046 cm3,
0.3 mmol) and HOBt·H2O (46 mg, 0.3 mmol) in DMF. Coupling
of EADI 14 (33mg, 0.045 mmol) was carried out with HOAt
(6.3 mg, 0.045 mmol), HATU (17 mg, 0.045 mmol) and (i-
Pr)2NEt (0.009 cm3, 0.045 mmol). Completion of each coupling
reaction was ascertained using the Kaiser ninhydrin test. The
Fmoc-protecting group was removed by treating the resin with
a DMF/piperidine solution (80/20, v/v).
Determination of anti-HIV activity
The peptide sensitivity of three HIV-1 strains was determined by
the MAGI assay with some modifications.22 Briefly, the target
cells (HeLa-CD4/CCR5-LTR-b-gal; 104 cells well-1) were plated
in 96-well flat microtiter culture plates. On the following day,
=
cyclo(–D–Tyr–Arg–W[(E)-CH CH]–Arg–Nal–Gly–)·2TFA
(3E). The obtained resin 20a was treated with HFIP/CH2Cl2
(2/8, 15 cm3) at room temperature for 2 h. After removal of the
620 | Org. Biomol. Chem., 2010, 8, 616–621
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the cells were inoculated with the HIV-1 (60 MAGI U/well,
giving 60 blue cells after 48 h of incubation) and cultured in the
presence of various concentrations of the drugs in fresh medium.
Forty-eight hours after viral exposure, all the blue cells stained
with X-Gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside)
were counted in each well. The activity of test compounds was
determined as the concentration that blocked HIV-1 replication
by 50% (50% effective concentration [EC50]).
12 (a) H. Tamamura, A. Esaka, T. Ogawa, T. Araki, S. Ueda, Z. Wang,
J. O. Trent, H. Tsutsumi, H. Masuno, H. Nakashima, N. Yamamoto,
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4394; (b) T. Tanaka, H. Tsutsumi, W. Nomura, Y. Tanabe, N. Ohashi,
A. Esaka, C. Ochiai, J. Sato, K. Itotani, T. Murakami, K. Ohba, N.
Yamamoto, N. Fujii and H. Tamamura, Org. Biomol. Chem., 2008, 6,
4374–4377; (c) T. Tanaka, W. Nomura, T. Narumi, A. Esaka, S. Oishi,
N. Ohashi, K. Itotani, B. J. Evans, Z. Wang, S. C. Peiper, N. Fujii and
H. Tamamura, Org. Biomol. Chem., 2009, 7, 3805–3809.
13 H. Tamamura, T. Araki, S. Ueda, Z. Wang, S. Oishi, A. Esaka, J. O.
Trent, H. Nakashima, N. Yamamoto, S. C. Peiper, A. Otaka and N.
Fujii, J. Med. Chem., 2005, 48, 3280–3289.
14 S. Ueda, S. Oishi, Z. Wang, T. Araki, H. Tamamura, J. Cluzeau, H.
Ohno, S. Kusano, H. Nakashima, J. O. Trent, S. C. Peiper and N. Fujii,
J. Med. Chem., 2007, 50, 192–198.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research
from MEXT, Japan, and Health and Labour Science Research
Grants (Research on HIV/AIDS). T.N. and K.T. are grateful
for the JSPS Research Fellowships for Young Scientists. We are
grateful to Prof. Hirokazu Tamamura and Mr. Tomohiro Tanaka
for helpful discussions.
15 H. Tamamura, K. Hiramatsu, S. Ueda, Z.-X. Wang, S. Kusano, S.
Terakubo, J. O. Trent, S. C. Peiper, N. Yamamoto, H. Nakashima, A.
Otaka and N. Fujii, J. Med. Chem., 2005, 48, 380–391.
16 For some recent exapmles of unsubstituted alkene dipeptide isosteres,
see: (a) S. Oishi, H. Tamamura, M. Yamashita, N. Hamanaka, A.
Otaka and N. Fujii, J. Chem. Soc., Perkin Trans. 1, 2001, 2445–2451;
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17 For some recent exapmles of fluoroalkene dipeptide isosteres, see:
(a) A. Otaka, J. Watanabe, A. Yukimasa, Y. Sasaki, H. Watanabe,
T. Kinoshita, S. Oishi, H. Tamamura and N. Fujii, J. Org. Chem.,
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Meester, A.-M. Lambeir, M.-B. Maes, S. Scharpe`, A. Haemers and K.
Augustyns, J. Med. Chem., 2005, 48, 1768–1780; (c) Y. Nakamura, M.
Okada, A. Sato, H. Horikawa, M. Koura, A. Saito and T. Taguchi,
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Parrot-Lopez and P. Goekjian, Tetrahedron Lett., 2007, 48, 6177–6180;
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Ohno and N. Fujii, Tetrahedron, 2008, 64, 4332–4346; (j) Y. Yamaki,
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18 For some recent examples of tri- or tetrasubstituted alkene isosteres,
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Yamamoto, K. Ajito, H. Tamamura, A. Otaka and N. Fujii, J. Org.
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20 S. Couve-Bonnaire, D. Cahard and X. Pannecoucke, Org. Biomol.
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21 S. Oishi, R. Masuda, B. Evans, S. Ueda, Y. Goto, H. Ohno,
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