Identification of novel low molecular weight CXCR4 antagonists by structural tuning of cyclic tetrapeptide scaffolds

Article abstract of DOI:10.1021/jm050009h

A highly potent CXCR4 antagonist, compound 2, was previously found by using two orthogonal cyclic pentapeptide libraries involving conformation-based and sequence-based libraries based on the pharmacophore of a 14-mer peptidic antagonist, 1. Herein, cyclic tetrapeptides derived from replacements of the dipeptide unit (Nal-Gly) with a γ-amino acid and pseudopeptides cyclized by disulfide and olefin bridges were synthesized to find novel scaffold structures different from that of cyclic pentapeptides. These compounds contain a reduced number of peptide bonds compared to compound 2. Furthermore, several analogues with chemical modification of the side chain of Arg⁴ in 2 were also prepared. From these, several new leads possessing high to moderate CXCR4-antagonistic activity were characterized.

Full text of DOI:10.1021/jm050009h

3280
J. Med. Chem. 2005, 48, 3280-3289
Identification of Novel Low Molecular Weight CXCR4 Antagonists by Structural
Tuning of Cyclic Tetrapeptide Scaffolds
,#
Hirokazu Tamamura,*, Takanobu Araki, Satoshi Ueda, Zixuan Wang,§ Shinya Oishi, Ai Esaka,
John O. Trent,| Hideki Nakashima,† Naoki Yamamoto,‡ Stephen C. Peiper,§ Akira Otaka, and Nobutaka Fujii*,
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan; Institute of Biomaterials
and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku Tokyo 101-0062, Japan; Medical College of Georgia,
Augusta, Georgia 30912; James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40202;
St. Marianna University, School of Medicine, Miyamae-ku, Kawasaki 216-8511, Japan; and Tokyo Medical and Dental
University, School of Medicine, Bunkyo-ku, Tokyo 113-8519, Japan
Received January 5, 2005
A highly potent CXCR4 antagonist, compound 2, was previously found by using two orthogonal
cyclic pentapeptide libraries involving conformation-based and sequence-based libraries based
on the pharmacophore of a 14-mer peptidic antagonist, 1. Herein, cyclic tetrapeptides derived
from replacements of the dipeptide unit (Nal-Gly) with a γ-amino acid and pseudopeptides
cyclized by disulfide and olefin bridges were synthesized to find novel scaffold structures
different from that of cyclic pentapeptides. These compounds contain a reduced number of
peptide bonds compared to compound 2. Furthermore, several analogues with chemical
modification of the side chain of Arg4 in 2 were also prepared. From these, several new leads
possessing high to moderate CXCR4-antagonistic activity were characterized.
Introduction
The chemokine receptor, CXCR4, is a seven trans-
membrane (7TM) GPCR that transduces signals of its
endogenous ligand, stromal cell-derived factor-1 (SDF-
1).1-4 The SDF-1/CXCR4 system plays an important role
in the migration of progenitors during embryologic
development of the cardiovascular, hemopoietic, and
central nervous systems. Recently, this system has been
Figure 1. Reduction of the molecular size of a peptide 1 to
cyclic pentapeptides 2 and 3. Bold residues are the indispen-
shown to be involved in several diseases, including HIV
infection,5 cancer metastasis/progression,6-20 and rheu-
matoid arthritis (RA).21 CXCR4 was initially identified
as a coreceptor that is utilized in T cell line-tropic (X4-)
HIV-1 entry.5 Mu¨ller et al. reported that CXCR4 and
another chemokine receptor, CCR7, are highly ex-
pressed in human breast cancer cells, while SDF-1 and
a CCR7 ligand, CCL21, are highly expressed in lymph
nodes, bone marrow, lung, and liver, which represent
the primary metastatic destinations of breast cancer,
suggesting that the SDF-1/CXCR4 system might deter-
mine the metastatic destination of tumor cells.6 Re-
cently, this system has been recognized to be involved
in the metastasis of several types of cancers, such as
pancreatic cancer,7,8 melanoma,6,9 prostate cancer,10
kidney cancer,11 neuroblastoma,12 non-Hodgkin’s lym-
phoma,13 lung cancer,14 ovarian cancer,15,16 multiple
myeloma,17 chronic lymphocytic leukemia,18 acute lym-
phoblastic leukemia,19 and malignant brain tumor.20
Nanki et al. reported that the memory T cells highly
express CXCR4 and the SDF-1 concentration is ex-
tremely high in the synovium of RA patients, and that
sable residues of 1 for the expression of strong CXCR4-
antagonistic activity.
SDF-1 stimulates migration of the memory T cells and
inhibits T cell apoptosis, indicating that the SDF-
CXCR4 interaction plays a critical role in T cell ac-
cumulation in the RA synovium.21 Thus, CXCR4 is
thought to be an important therapeutic target for these
diseases. Compound 1 and its analogues, 14-mer pep-
tides, were previously found to be specific CXCR4
antagonists that were characterized as HIV-entry in-
hibitors,22 anticancer-metastatic agents8,23 and anti-RA
agents.24 Arg2, L-3-(2-naphthyl)alanine (Nal)3, Tyr5, and
Arg14 were proven to constitute the critical pharma-
cophores of 1 (Figure 1).25 The efficient utilization of two
orthogonal cyclic pentapeptide libraries consisting of
conformation-based and sequence-based libraries in-
volving the critical residues of 1 led to molecular-size
reduction of 1 and discovery of cyclic pentapeptides, 2
and 3, which have strong CXCR4-antagonistic activity,
comparable to that of 1.26 In this paper, we describe the
fine-tuning of ring structures based on cyclic pentapep-
tide templates,27-34 and chemical modifications of side
chains for an increase in potency and a reduction of the
peptide characteristics of 2.35
* Corresponding authors. Tel: +81 75 753 4551, Fax: +81 75 753
4570,
e-mail:
tamamura@pharm.kyoto-u.ac.jp
and
nfujii@
pharm.kyoto-u.ac.jp.
Kyoto University.
# Institute of Biomaterials and Bioengineering.
§ Medical College of Georgia.
| University of Louisville.
† St. Marianna University.
‡ School of Medicine.
Chemistry
γ-Amino Acid-Containing Cyclic Tetrapeptides
(11a-d). Requisite Nγ-Fmoc-γ-amino acids, (2E,4S)-Nγ-
10.1021/jm050009h CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/13/2005

Low Molecular Weight CXCR4 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3281
Scheme 1a
Scheme 3a
a Reagents: (i) DIBAL-H; (ii) (EtO)2P(O)CH2CO2But,LiCl,
DIPEA; (iii) 95% aqueous TFA; (iv) Fmoc-OSu, Et3N; (v) H2, Pd/
C.
Scheme 2a
a Reagents: (i) Fmoc-based SPPS; (ii) 1H-pyrazole-1-carbox-
amidine hydrochloride, DIPEA; (iii) EDT/H2O/TFA; (iv) aqueous
AcONH4 pH 8.
and NaHCO3, subsequent deprotection with TFA and
HPLC purification gave the desired cyclic peptide 11.26
Disulfide-Bridged Cyclic Peptides (15a-p and
16a-p). The protected peptide resin was constructed
by general Fmoc-based solid-phase synthesis on an NH2-
Rink amide resin (for 15a-p) or a tyramine-Wang
PEG resin (for 16a-p) 12 (Scheme 3). Fmoc-â-Ala-OH
was condensed to the N-terminus of the protected resin
as the final residue. After deprotection of the Fmoc
group, N-guanylation of the resulting free â-amino
group with 1H-pyrazole-1-carboxamidine hydrochloride
and DIPEA, followed by cleavage from the resin and
removal of the 2,2,4,6,7-pentamethyldihydrobenzo-
furan-5-sulfonyl (Pbf) and Trt groups with EDT/H2O/
TFA, gave the crude reduced peptide (2SH-peptide) 14.
Subsequent air-oxidation of the crude 2SH-peptide 14
and HPLC purification yielded the desired disulfide
peptide 15 or 16.
Disulfide-Bridged Cyclic Peptides (15q and 16q).
The protected peptide resin was constructed on an
Fmoc-NH-Rink amide resin (for 15q) or an Fmoc-
tyramine-Wang PEG resin (for 16q) in the same manner
as in the synthesis of 15e or 16e (Table 1). Reductive
amination of the N-terminal amino group of the pro-
tected resin with N-Fmoc-3-aminopropanal and NaBH-
(OAc)3,36 followed by removal of the Fmoc group and the
subsequent N-guanylation of the resulting free amino
group in the same manner as in the synthesis of 15e or
16e, obtained the protected pseudo-peptide resin. Cleav-
age from the resin and removal of the Pbf and Trt
groups, the subsequent air-oxidation, and HPLC puri-
fication were performed in the same manner as in the
a Reagents: (i) 6 or 8, DIPEA, DMF/CH2Cl2; (ii) Fmoc-based
SPPS; (iii) AcOH/TFE/CH2Cl2; (iv) DPPA, NaHCO3; (v) basic
alumina column; (vi) 95% aqueous TFA.
Fmoc-4-amino-5-naphthalen-2-yl-pent-2-enoic acid (γ-
(E)-Nal) 6 and (4R)-Nγ-Fmoc-4-amino-5-naphthalen-2-
yl-pentanoic acid (γ-Nal) 8, were synthesized according
to Scheme 1. Boc-Nal-NMe(OMe) 4 was treated with
DIBAL followed by modified Horner-Wadsworth-Em-
mons olefination to yield Boc-γ-(E)-Nal-OBut 5. NR-
Fmoc-protection after the cleavage of the NR-Boc and
But groups of 5 with TFA afforded a desired compound,
Fmoc-γ-(E)-Nal-OH 6. Hydrogenation of 5 obtained a
reduced compound, 7, which was similarly converted to
another desired compound, Fmoc-γ-Nal-OH 8. The
protected peptide resin 10 was constructed by general
Fmoc-based solid-phase synthesis on a (2-chloro)trityl
resin 9, in which the above γ-amino acid, 6 or 8, was
introduced as a C-terminal residue by DIPEA in DMF/
CH2Cl2 (Scheme 2). Cleavage of the liner peptide from
the resin with AcOH/TFE/CH2Cl2 (1:1:3 (v/v)) followed
by cyclization with diphenylphosphoryl azide (DPPA)

3282 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Tamamura et al.
Scheme 4a
Table 1. Inhibitory Activity of Disulfide/Olefin-Bridged Cyclic
Peptides against SDF-1 Binding to CXCR4
IC50 ( SD
compd
m
n
X
R1 R2
R3
NH2
(µM)a
15e
16e
15q
16q
15r
16r
20a
20b
20c
20d
2
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
2
2
1
S-S
S-S
S-S
S-S
S-S
S-S
O
H
H
H
H
0.69 ( 0.40
O
tyramine 0.53 ( 0.28
H2
H2
O
NH2
tyramine ca. 1
>10
Me tyramine >10
ca. 1
Me NH2
O
(E)-CHdCH
(E)-CHdCH
(E)-CHdCH
(E)-CHdCH
O
H
H
H
H
NH2
NH2
NH2
1-10
1-10
1-10
O
O
O
tyramine 1-10
0.0043 ( 0.0012
0.0084 ( 0.0038
3
a IC50 values are based on the inhibition of [125I]-SDF-1 binding
to CXCR4 transfectants of CHO cells. All data with standard
deviation (SD) are the mean values for at least three independent
experiments.
synthesis of 15e or 16e to yield the desired pseudo-
peptide, 15q or 16q.
a Reagents: (i) Fmoc-based SPPS; (ii) Grubbs catalyst 2nd
generation, (iii) piperdine; (iv) 1H-pyrazole-1-carboxamidine hy-
drochloride, DIPEA; (v) 1 M TMSBr-thioanisole/TFA.
Disulfide-bridged Cyclic Peptides (15r and 16r).
The protected peptide resin was constructed on an
Fmoc-NH-Rink amide resin (for 15r) or an Fmoc-
tyramine-Wang PEG resin (for 16r) (Table 1). NR-
Methylation of the Arg(Pbf) residue in the protected
resin was performed by the Fukuyama-Mitsunobu
reaction.37 NR-o-Nitrobenzenesulfonyl (Ns) protection of
H-Arg(Pbf)-Nal-D-Cys-NH-Rink amide (or -tyramine-
Wang PEG) resin and the subsequent NR-methylation
with MeOH, Ph3P, and diethyl azodicarboxylate (DEAD)
were followed by removal of the NR-Ns group with DBU
and 2-mercaptoethanol. The next residue Fmoc-D-Tyr-
(But)-OH was condensed to the secondary NR-amino
group of the MeArg(Pbf) residue on the protected resin
by using HATU, HOAt, and DIPEA. N-Guanylation,
cleavage, deprotection, air-oxidation, and HPLC puri-
fication were subjected to yield the desired peptide, 15r
or 16r.
Olefin-Bridged Cyclic Peptides (20a-d). The
protected peptide resin was constructed on an NH2-Rink
amide resin (for 20a-c) or an Fmoc-tyramine-Wang
PEG resin (for 20d) (Scheme 4) 12. D-2-Allylglycine38
(for 20a or 20d) or Fmoc-D-2-homoallylglycine39,40 (for
20b or 20c) was used as the C-terminal residue, while
Fmoc-L-2-allylglycine41 (for 20a, 20b, or 20d) or Fmoc-
L-2-homoallylglycine42 (for 20c) was used as the N-
terminal second residue. Fmoc-â-Ala-OH was condensed
to the N-terminus of the protected resin as the final
residue. The protected peptide resin 17 was subjected
to ring-closing olefin metathesis with Grubbs catalyst
second generation43 to give the cyclized peptide resin
18. After deprotection of the Fmoc group, N-guanylation
of the resulting free â-amino group, followed by treat-
ment with 1 M TMSBr-thioanisole/TFA and HPLC
purification, gave the desired peptide 20. The E geom-
etry of the olefin units in 20a-d was easily established
from the coupling constants (J ) 15.5, 15.6, 15.2, and
1
15.2 Hz, respectively) of the two olefinic protons by H
NMR analysis.
Analogues with Substitution for Arg4 (23a-p).
Each peptide was synthesized in a general manner
similar to that in the synthesis of 11a. 23d and 23f:
NR-Methylation and coupling reaction to the NR-meth-
ylated residue were performed in a manner similar to
that in the synthesis of 15r. 23j,k and 23o,p: After
cyclization and deprotection, N-guanylation of the re-
sulting free γ- or ꢀ-amino group of the L-2,4-diaminobu-
tyric acid (Dab)4 or Lys4 residue (for 23j or 23k,
respectively) was performed with 1H-pyrazole-1-car-
boxamidine hydrochloride and DIPEA (Scheme 5). N-
Guanylation of the resulting free γ-amino group of the
trans- or cis-4-aminoproline residue44 (for 23o or 23p,
respectively) was similarly performed.
Biological Results and Discussion
γ-Amino Acid-Containing Cyclic Tetrapeptides.
Cyclic pentapeptides, 2 and 3, have a Gly residue as a
spacer for cyclization. To reduce the ring size, the Nal-
Gly sequences of 2 and 3 were replaced by a γ-Nal or
γ-(E)-Nal unit (Scheme 2). Among these γ-amino acid-
containing cyclic tetrapeptides (11a-d), only 11a [sub-
stitution of γ-Nal for Nal-Gly of 3] showed high CXCR4-
antagonistic activity (IC50 ) 54 nM) (Figure 2), although

Low Molecular Weight CXCR4 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3283
Scheme 5a
µM. 15e and 16e have a common combination of
chiralities of composed amino acids, suggesting that
these compounds form similar conformations. It seems
that a phenol group of 16e is not disposed in the suitable
position, since 15e and 16e have almost the same
potencies. However, notably, L,L-chiralities of Arg-Nal
are critical for high activity, as shown in 2 and 3. The
synthetic Arg side chain involving an N-3-guanidino-
propanoyl moiety has an extra atom, compared to the
original Arg side chain. This might account for the lower
activity.
In addition, 15e/16e analogues, 15q and 16q, in
which the amide bond of N-3-guanidinopropanoyl-Cys
was replaced by a reduced amide bond, and 15r and 16r,
in which the R-amino group of Arg was N-methylated,
were prepared. Substitution of the reduced amide bond
for the amide bond of N-3-guanidinopropanoyl-Cys
brought about no significant difference in potency (IC50
of 15q and 16q ) c.a. 1 µM). This suggested that the
planar character of this amide bond and the carbonyl
group have little effect on potency. N-Methylation of Arg
caused remarkable decrease in potency (IC50 of 15r and
16r > 10 µM), indicating that conformation surrounded
by the Cys-Arg amide bond might be changed or the
Cys-Arg amide proton might be required for high
activity.
a Reagents: (i) Fmoc-based SPPS; (ii) AcOH/TFE/CH2Cl2 (1:1:3
(v/v)); (iii) DPPA, NaHCO3; (iv) basic alumina column; (v) 95%
aqueous TFA; (vi) 1H-pyrazole-1-carboxamidine hydrochloride,
DIPEA.
Olefin-Bridged Cyclic Peptides. Cyclic analogues
that were bridged by an olefin instead of a disulfide in
15e and 16e were synthesized. C-Terminal-amidated
compounds, 20a-c, which differ in bridge length, and a
C-terminal tyramide-type compound, 20d, showed lower
activity (IC50 ) 1-10 µM), compared to those of 15e and
16e (Table 1). It is thought that the constraint of the
olefin unit into the E-form is not an effective optimiza-
tion component.
Figure 2. The structure of 11a.
this activity is 6-fold lower than that of 3. This result
suggested that the Gly residue and the amide bond of
the Nal-Gly sequence are not necessary for high activity.
The reason for no significant activity of 11b [substitu-
tion of γ-Nal for Nal-Gly of 2] (IC50 > 1 µM) cannot be
well explained, however, the difference of chirality of
L/D-Arg3 in 2 and 3 might cause a global conformational
change of the ring. Both γ-(E)-Nal-substituted ana-
logues, 11c and 11d, showed no significant activity (IC50
> 1 µM), suggesting that constraint of the γ-amino acid
to the E-form might not be suitable.
Disulfide-Bridged Cyclic Peptides. To optimize
the ring structures of compound 2-derived compounds,
the use of scaffold templates different from that of cyclic
pentapeptides was investigated. Since the four requisite
residues of 1 are disposed in close vicinity each other
due to the disulfide bridge [Cys4-Cys13]45 and cyclic
peptides possessing the Arg-Arg-Nal sequence, such as
2 and 3, showed high CXCR4-antagonistic activity,
we designed and separately prepared disulfide-
bridged cyclic peptide libraries consisting of the N-3-
guanidinopropanoyl-L/D-Cys(S-)-L/D-Arg-L/D-Nal-L/D-Cys-
(S-)-NH2 (or -tyramine) sequence, which included 32
compounds (2 × 24 stereoisomers) (15a-p or 16a-p,
respectively, Scheme 3). Among these synthetic com-
pounds, 15e [N-3-guanidinopropanoyl-Cys(S-)-Arg-Nal-
D-Cys(S-)-NH2] and 16e [N-3-guanidinopropanoyl-Cys-
(S-)-Arg-Nal-D-Cys(S-)-tyramine] exhibited moderate
CXCR4-antagonistic activity (IC50 ) 690 and 530 nM,
respectively) (Table 1), although the other compounds
did not show any activity up to the concentration of 1
Analogues Constrained in the Peripheral Re-
gion of Arg4. The significant difference of activity
between 23e ([Ala4]-2, Ala-substitution for Arg4, IC50
) 63 nM) and 23c ([D-Ala4]-2 ) [D-Ala4]-3, D-Ala-
substitution for L/D-Arg4, IC50 ) 230 nM) indicates a
biological importance of the amide bond direction be-
tween D-Tyr and L/D-Arg. Thus, relationships between
the conformation surrounded by Arg4 of 2 and activity
were investigated. L/D-Pro-substitutions for Arg4 also
gave the difference of activity: An L-Pro-substituted
analogue, 23b, is 4-fold stronger than a D-Pro-substi-
tuted analogue, 23a (Table 2). N-Methylation of D-Ala4
in 23c (IC50 ) 230 nM) brought about a significant
increase in potency (IC50 of 23d ) 42 nM), which was
comparable to that of 23e ([Ala4]-2) (IC50 ) 63 nM),
while N-methylation of Ala4 in 23e caused a significant
decrease in potency (IC50 of 23f ) 490 nM). NMR and
simulated annealing molecular dynamics (SA-MD) analy-
sis showed that the backbone structure of 23d is very
similar to that of 23e, but different from that of 23c,
especially in the direction of the D-Tyr-L/D-Arg amide
bond (Figure 3A).46 N-Methylation of D-Ala4 in 23c
might cause an inversion of the D-Tyr3-D-Ala4 amide
bond (180° rotation of φ and ψ torsion angles) to reduce
the 1,3-pseudo-allylic strain between the side chain of
D-Tyr3 and the N-methyl group, resulting in a confor-
mation that is similar to that of 23e.
Ala-substitution for Arg4 in 2 (23e) did not bring
about a severe decrease in potency (IC50 ) 63 nM),

3284 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Tamamura et al.
Table 2. Inhibitory Activity of Cyclic Pentapeptides with Sub-
stitution for Arg4 in Compound 2 against SDF-1 Binding to
CXCR4
dino-Lys (g-Lys)-substituted analogues, 23j and 23k,
also showed higher activity (IC50 ) 24 and 33 nM,
respectively) than 23e. However, these analogues were
weaker than compound 2, suggesting that Arg is the
most suitable at position 4 among the Arg/Lys mimetics
used in this study. Asn/Gln/Glu-substituted analogues,
23l-n, showed a remarkable decrease in activity (IC50
) 270, 170, and 610 nM, respectively). This proved that
a basic functional group, such as an amino or guanidino
group, is necessary for strong activity in the side chain
of the amino acid at position 4, and that a hydrophilic
(not basic) or acidic group is not preferable to a methyl
group of Ala.
compd
sequencea
IC50 ( SD (µM)
23a
23b
23c
23d
23e
23f
23g
23h
23i
23j
23k
23l
23m
23n
23o
cyclo(-Nal-Gly-D-Tyr-D-Pro-Arg-)
cyclo(-Nal-Gly-D-Tyr-Pro-Arg-)
cyclo(-Nal-Gly-D-Tyr-D-Ala-Arg-)
cyclo(-Nal-Gly-D-Tyr-D-MeAla-Arg-)
cyclo(-Nal-Gly-D-Tyr-Ala-Arg-)
cyclo(-Nal-Gly-D-Tyr-MeAla-Arg-)
cyclo(-Nal-Gly-D-Tyr-Dab-Arg-)
cyclo(-Nal-Gly-D-Tyr-Orn-Arg-)
cyclo(-Nal-Gly-D-Tyr-Lys-Arg-)
cyclo(-Nal-Gly-D-Tyr-g-Dab-Arg-)
cyclo(-Nal-Gly-D-Tyr-g-Lys-Arg-)
cyclo(-Nal-Gly-D-Tyr-Asn-Arg-)
cyclo(-Nal-Gly-D-Tyr-Gln-Arg-)
cyclo(-Nal-Gly-D-Tyr-Glu-Arg-)
cyclo(-Nal-Gly-D-Tyr-trans-4-
guanidino-Pro-Arg-)
1.6 ( 0.1
0.42 ( 0.29
0.23 ( 0.0064
0.042 ( 0.0088
0.063 ( 0.013
0.49 ( 0.090
0.016 ( 0.010
0.019 ( 0.011
0.097 ( 0.0035
0.024 ( 0.0040
0.033 ( 0.0001
0.27
Conformationally constrained Arg mimetics, trans-4-
guanidino-Pro and cis-4-guanidino-Pro (Table 2), were
synthesized with the idea of fixing the backbone and
side chain of Arg, according to the reported procedure.44
These Arg mimetics were incorporated in compound 2
to determine the spatial disposition of guanidino group
and increase in potency. 23o ([trans-4-guanidino-Pro4]-
2, trans-4-guanidino-Pro-substitution for Arg4) and 23p
([cis-4-guanidino-Pro4]-2, cis-4-guanidino-Pro-substitu-
tion for Arg4) showed high CXCR4-antagonistic activi-
ties (IC50 ) 10 and 9.9 nM, respectively) that were twice
as strong as that of 23j ([g-Dab4]-2, g-Dab-substitution
for Arg4), having the same length of the linear-type side
chain of the amino acid at position 4 (IC50 ) 24 nM),
while 23b ([Pro4]-2, Pro-substitution for Arg4) did not
show high activity (IC50 ) 420 nM). In consideration of
the fact that the introduction of a pyrrolidinyl ring
caused a significant reduction of potency (23a and 23b),
it is thought that fixing the side chain effectively
increased potency. From these results, we have learned
that the constrained guanido group might efficiently
interact with CXCR4. In addition, NMR and SA-MD
analysis of 23o and 23p showed similar dispositions of
guanidino groups of trans/cis-4-guanidino-Pro residues
in space (Figure 3B),46 which might be the reason for
essentially no difference in potency between 23o and
23p.
0.17
0.61
0.010 ( 0.0015
23p
cyclo(-Nal-Gly-D-Tyr-cis-4-
0.0099 ( 0.0043
guanidino-Pro-Arg-)
a MeAla ) NR-Me-Ala.
whereas Ala-substitution for Nal1, D-Tyr3, and Arg5
completely deminished activity of the parent compound
(IC50 > 1 µM, data not shown). These results suggest
that the side chain of Arg4 in 2 has relatively little effect
on the expression of activity, and that the spatial
disposition of the δ-guanidino group of Arg4 might not
be optimized. Thus, several analogues, in which Arg4
was replaced by Arg/Lys mimetics with various lengths
of alkyl chains, were synthesized. Dab/L-ornithine (Orn)-
substituted analogues, 23g and 23h, showed higher
activity (IC50 ) 16 and 19 nM, respectively) than the
Ala-substituted analogue, 23e, whereas a Lys-substi-
tuted analogue, 23i, was slightly weaker (IC50 ) 97 nM)
than 23e (Table 2). γ-N-Amidino-Dab (g-Dab)/ꢀ-N-ami-
Figure 3. Superimpositions of low-energy structures of 23c (red), 23d (green), and 23e (blue) (A), and 23o and 23p (B).

Low Molecular Weight CXCR4 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3285
under reduced pressure. The residue was extracted with
EtOAc, and the extract was washed successively with satu-
rated aqueous citric acid, brine, saturated aqueous NaHCO3,
and brine and dried over MgSO4. Concentration under reduced
pressure followed by flash chromatography over silica gel with
EtOAc-n-hexane (2:3) and subsequent recrystalization with
Et2O-n-hexane gave 12.8 g (35.7 mmol, 77% yield from L-3-
(2-naphthyl)alanine) of 4 as colorless crystals. mp: 110-111
°C; [R]35D +29.41 (c 0.714, CHCl3); 1H NMR (400 MHz, CDCl3)
δ 7.73-7.82 (m, 3H), 7.62, (s, 1H), 7.39-7.48 (m, 2H), 7.31 (dd,
J ) 8.6, 1.5 Hz, 1H), 5.17-5.24 (br d, 1H), 5.06 (br, 1H), 3.66
(s, 3H), 3.01-3.25 (m, 4H), 1.36 (s, 9H); Anal. Calcd for
C20H26N2O6: C, 67.02; H, 7.31; N, 7.82. Found: C, 66.78; H,
7.47; N, 7.68.
Conclusion
The fine-tuning of the ring structure of compound 2
led to findings of CXCR4 antagonists involving scaffold
structures that are different from cyclic pentapeptide
structures: A cyclic tetrapeptide including a γ-amino
acid and pseudopeptides cyclized by disulfide and olefin
bridges, having a smaller number of peptide bonds
compared to compound 2, might be useful lead com-
pounds. Furthermore, we have learned from L/D-NR-Me-
Ala- and L/D-Pro-substitutions for Arg4 that the direction
of the carbonyl group in the D-Tyr3-L/D-Ala4 amide bonds
causes a remarkable effect on CXCR4-antagonistic
activity. It is also clear from this study that a basic
functional group, such as amino or guanidino group, in
the amino acid side-chain at position 4 is indispensable
for strong activity. In addition, analogues of compound
2, where a conformationally constrained Arg mimetic,
trans- or cis-4-guanidino-Pro, was incorporated at posi-
tion 4, showed high CXCR4-antagonistic activity, indi-
cating that spatial constraint of a guanido group is
important for an efficient interaction with CXCR4.
These results for modifications of compound 2 provide
useful insights for the future design of new low molec-
ular weight CXCR4 antagonists, considering in connec-
(2E,4S)-N-Boc-4-amino-5-(2-naphthyl)-2-pentenoic Acid
tert-Butyl Ester (5). To a stirred solution of 4 (7.00 g, 19.5
mmol) in CH2Cl2 (50 mL) was added dropwise a solution of
DIBAL-H in toluene (1.0 M, 38.6 mL, 38.6 mmol) at -78 °C
under argon, and the mixture was stirred at -78 °C for 2 h.
The reaction was quenched with saturated aqueous citric acid
(50 mL) at -78 °C, and the organic solvents were concentrated
under reduced pressure. The residue was extracted with
EtOAc, and the extract was washed successively with brine,
aq. 50% NaHCO3, and brine and dried over MgSO4. Concen-
tration under reduced pressure gave the crude aldehyde as a
white solid, which was used directly in the following step
without purification. To a stirred suspension of LiCl (1.65 g,
39.0 mmol) in CH3CN (30 mL) were added (EtO)2P(O)CH2CO2-
But (6.31 mL, 39.0 mmol) and DIPEA (6.79 mL, 39.0 mmol)
at 0 °C under argon, and the mixture was stirred at 0 °C for
1 h. The above aldehyde in CH3CN (30 mL) was added to the
mixture at 0 °C, and the mixture was allowed to warm to room
temperature and stirred overnight. The reaction mixture was
concentrated under reduced pressure. The residue was ex-
tracted with EtOAc, and the extract was washed successively
with saturated aqueous citric acid, brine, saturated aqueous
NaHCO3, and brine and dried over MgSO4. Concentration
under reduced pressure followed by flash chromatography over
silica gel with EtOAc-n-hexane (1:4) and subsequent recrys-
talization with Et2O-n-hexane gave 6.05 g (15.2 mmol, 78%
47-53
tion with other CXCR4 antagonists.
Experimental Section
General. 1H NMR spectra were recorded using a JEOL EX-
270, a JEOL AL-400, or a JNM-ECA600 spectrometer at 270,
400, or 600 MHz 1H frequency, respectively, in CDCl3 or
DMSO-d6. Chemical shifts are reported in parts per million
downfield from internal tetramethylsilane. Nominal (LRMS)
and exact mass (HRMS) spectra were recorded on a JEOL
JMS-01SG-2 or JMS-HX/HX 110A mass spectrometer. Ion-
spray (IS)-mass spectrum was obtained with a Sciex APIIIIE
triple quadrupole mass spectrometer (Toronto, Canada). Opti-
cal rotations were measured in CHCl3 or H2O with a JASCO
DIP-360 digital polarimeter (Tokyo, Japan) or a Horiba high-
sensitive polarimeter SEPA-200 (Kyoto, Japan). For flash
column chromatography, silica gel 60 H (silica gel for thin-
layer chromatography, Merck) and Wakogel C-200 (silica gel
for column chromatography, Wako Pure Chemical Industries,
Ltd., Osaka, Japan) were employed. HPLC solvents were H2O
and CH3CN, both containing 0.1% (v/v) TFA. For analytical
HPLC, a Cosmosil 5C18-AR column (4.6 × 250 mm, Nacalai
Tesque Inc., Kyoto, Japan) was eluted with a linear gradient
of CH3CN at a flow rate of 1 mL/min on a Waters model 600
(Nihon Millipore, Ltd., Tokyo, Japan). Preparative HPLC was
performed on a Waters Delta Prep 4000 equipped with a
Cosmosil 5C18-AR column (20 × 250 mm, Nacalai Tesque Inc.)
using an isocratic mode of CH3CN at a flow rate of 15 mL/
min.
from 4) of 5 as colorless crystals. mp: 135-137 °C; [R]35
D
1
-12.84 (c 0.467, CHCl3); H NMR (400 MHz, CDCl3) δ 7.72-
7.87 (m 3H), 7.62 (s, 1H), 7.39-7.51 (m, 2H), 7.31 (dd, J )
8.6, 1.7 Hz, 1H), 6.85 (dd, J ) 15.6, 5.1 Hz, 1H), 5.81 (dd, J )
15.6, 1.5 Hz, 1H), 4.69 (br, 1H), 4.57 (br, 1H), 2.94-3.12 (m,
2H), 1.46 (s, 9H), 1.36 (s, 9H); Anal. Calcd for C24H31NO4: C,
72.58; H, 7.86; N, 3.52. Found: C, 72.44; H, 7.86; N, 3.22.
(2E,4S)-N-Fmoc-4-amino-5-(2-naphthyl)-2-pentenoic
Acid (6). 5 (1.00 g, 2.51 mmol) was treated with aq. 95% TFA
(20 mL) at room temperature for 2.5 h. TFA was removed
under reduced pressure, and the residue was dissolved in
DMF-H2O-CH3CN (9:1:15 (v/v), 24 mL). To the stirred
solution were added DIPEA (876 µL, 5.03 mmol) and Fmoc-
OSu (891 mg, 2.64 mmol) at 0 °C, and the reaction mixture
was stirred at room temperature for 6 h. The mixture was
concentrated under reduced pressure and acidified with aq. 1
M HCl. The mixture was extracted with EtOAc, and the
extract was washed successively with aq. 0.1 M HCl (× 3) and
brine and dried over MgSO4. After removal of the solvent
under reduced pressure, the resulting crude product was
purified by flash chromatography on silica gel with CHCl3-
MeOH (20:1) and subsequent recrystalization with (CHCl3-
n-hexane-MeOH) to give 345.1 mg (0.753 mmol, 30% yield
[1-(N,N-Methoxy-methyl-carbamoyl)-2(S)-(2-naphthyl)-
ethyl]-carbamic Acid tert-Butyl Ester (Boc-Nal-NMe
(OMe)) (4). To a stirred solution of L-3-(2-naphthyl)alanine
(10.0 g, 46.4 mmol) in THF-H2O (1:1 (v/v), 200 mL) were
added triethylamine (12.5 mL, 92.9 mmol) and Boc2O (9.63 g,
44.1 mmol) at room temperature, and the mixture was stirred
at this temperature overnight. The reaction mixture was
concentrated under reduced pressure. The residue was ex-
tracted with EtOAc, and the extract was washed successively
with saturated aqueous citric acid, aq. 5% citric acid (× 3),
H2O (× 5), and brine and dried over MgSO4. Concentration
under reduced pressure gave the crude product as a white
powder, which was used directly in the following step without
purification. To a solution of the crude product in DMF (150
mL) were added N,O-dimethylhydroxylamine hydrochloride
(10.3 g, 106 mmol), triethylamine (14.3 mL, 106 mmol), HOBt
(8.11 g, 52.9 mmol), and DCC (11.8 g, 53.0 mmol) at 0 °C, and
the mixture was stirred at room temperature overnight. The
reaction mixture was filtered, and the filtrate was concentrated
from 5) of 6 as colorless crystals. mp: 189-190 °C; [R]34
D
-15.33 (c 0.326, CHCl3); 1H NMR (400 MHz, DMSO-d6) δ 12.3
(br, 1H), 7.67-7.95 (m, 7H), 7.51-7.62 (m, 2H) 7.40-7.51 (m,
3H), 7.30-7.41 (m, 2H) 7.08-7.30 (m, 2H), 6.86 (dd, J ) 15.6,
5.4 Hz, 1H), 5.81 (d, J ) 15.6 Hz,1H), 4.43-4.61 (m, 1H), 4.04-
4.26 (m, 3H), 3.00-3.14 (m, 1H), 2.83-3.00 (m, 1H); LRMS
(FAB), m/z 464 (MH+, base peak), 307, 179, 165, 154, 136, 141,
136, 121, 107, 91, 89, 77; HRMS (FAB), m/z calcd for C30H26-
NO4 (MH+): 464.1862, found: 464.1853.
N-Boc-4(R)-amino-5-(2-naphthyl)-pentanoic Acid tert-
Butyl Ester (7). To the solution of 5 (2.50 g, 6.29 mmol) in

3286 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Tamamura et al.
EtOAc (100 mL) was added 10% Pd/C (200 mg). The reaction
vessel was charged with atmosphere of H2 (balloon), and the
mixture was stirred at room temperature for 8 h. Completion
of the reaction was monitored by RP-HPLC. The reaction
mixture was filtered through a pad of Celite, rinsing with
EtOAc, and the filtrate was concentrated under reduced
pressure. The resulting crude product was purified by flash
chromatography on silica gel with EtOAc-n-hexane (1:5) and
subsequent recrystalization with Et2O-n-hexane to give 1.87
mL × 3), MeOH (20 mL × 3), CHCl3 (20 mL × 3) and Et2O
(20 mL × 3) and dried in vacuo. The loading rate of the resin
was estimated by Fmoc-quantification (0.126 mmol/g (41%)).
Representative Procedure for the Synthesis of Disul-
fide-Bridged Cyclic Peptides (16e). Protected peptide resin
was manually constructed by Fmoc-based solid-phase peptide
synthesis on Fmoc-tyramine-Wang PEG resin (0.126 mmol/g,
397 mg, 0.05 mmol). Fmoc-â-Ala-OH was condensed to the
N-terminal amino group of H-Cys-Arg(Pbf)-Nal-D-Cys-tyramine-
Wang PEG resin, and Fmoc group was deprotected by treat-
ment with 20% piperidine in DMF (20 min). To a mixture of
the resin and DIPEA (52,5 µL, 0.300 mmol) in DMF (5 mL)
was added 1H-pyrazole-1-carboxamidine hydrohloride (22.0
mg, 0.150 mmol) at room temperature. The reaction mixture
was stirred at room temperature for 12 h. This N-guanylation
procedure was repeated once again. The protected resin was
treated with EDT/H2O/TFA (2.5:2.5:95 (v/v), 6 mL) at 0 °C for
2 h. The reaction mixture was filtered, and the filtrate was
concentrated by bubbling with N2 gas. Cooled Et2O was added
to the residue, and the resulting precipitate was separated by
centrifugation. The precipitate was washed with Et2O three
times. The crude peptide was dissolved in H2O, and pH of this
solution was adjusted to approximately 8 with aq. 0.28% NH3.
Air oxidation for 1 d and purification by HPLC gave the cyclic
pseudopeptide 16e (6.5 mg, 16% yield from Fmoc-tyramine-
Wang PEG resin) as a freeze-dried powder.
Synthesis of 15q. Protected peptide resin was manually
constructed by Fmoc-based solid-phase peptide synthesis on
Fmoc-HN-Rink amide resin (0.36 mmol/g, 139 mg, 0.05 mmol).
To a suspension of H-Cys-Arg(Pbf)-Nal-D-Cys-HN-Rink amide
resinin DCM were added N-Fmoc-3-aminopropanal (44.0 mg,
0.15 mmol) and NaBH(OAc)3 (53.0 mg, 0.25 mmol) at room
temperature. The mixture was stirred at room-temperature
overnight. Deprotection of the Fmoc group, N-guanylation,
deprotection/cleavage from the resin, air oxidation, and HPLC
purification were performed by use of a procedure identical
with that described for the synthesis of 16e to yield the cyclic
pseudopeptide 15q (3.0 mg, 9% yield from Fmoc-HN-Rink
amide resin) as a freeze-dried powder.
g (4.65 mmol, 74%) of 7 as colorless crystals. mp: 90-91 °C;
1
[R]35 -20.83 (c 0.432, CHCl3); H NMR (400 MHz, CDCl3) δ
D
7.71-7.86 (m, 3H), 7.62 (s, 1H), 7.38-7.52 (m, 2H), 7.34 (d, J
) 8.1 Hz, 2H), 4.42-4.58 (br d, 1H), 2.94-3.09 (m, 1H), 2.79-
2.94 (m, 1H), 2.28 (t, 2H), 1.56-1.92 (m, 2H), 1.41 (s, 9H), 1.39
(s, 9H); Anal. Calcd for C24H33NO4: C, 72.15; H, 8.33; N, 3.51.
Found: C, 71.92; H, 8.49; N, 3.21.
N-Fmoc-4(R)-amino-5-(2-naphthyl)-pentanoic Acid (8).
7 (1.00 g, 2.50 mmol) was treated with aq. 95% TFA (20 mL)
at room temperature for 2.5 h. TFA was removed under
reduced pressure, and the residue was dissolved in DMF-
H2O-THF (9:1:10 (v/v), 20 mL). To the stirred solution were
added DIPEA (1.03 mL, 5.89 mmol) and Fmoc-OSu (1.29 g,
3.83 mmol) at 0 °C, and the reaction mixture was stirred at
room temperature overnight. The mixture was concentrated
under reduced pressure and acidified with aq. 1 M HCl. The
mixture was extracted with EtOAc, and the extract was
washed successively with aq. 0.1 M HCl (× 3) and brine and
dried over MgSO4. After removal of the solvent under reduced
pressure, the resulting crude product was purified by flash
chromatography on silica gel with CHCl3-MeOH (20:1) and
subsequent recrystalization with (CHCl3-n-hexane) to give
747.6 mg (1.60 mmol, 64% yield from 7) of 8 as colorless
1
crystals. mp: 167-169 °C; [R]34 -7.31 (c 0.410, CHCl3); H
D
NMR (400 MHz, DMSO-d6) δ 7.73-7.92 (m 5H), 7.67 (s, 1H),
7.56-7.62 (m, 2H) 7.41-7.48 (m, 2H), 7.33-7.41 (m, 3H),
7.18-7.33 (m, 3H), 4.06-4.24 (m, 3H), 3.76 (br, 1H), 2.85 (d,
J ) 6.6 Hz, 2H), 2.14-2.32 (m, 2H), 1.68-1.82 (m, 1H), 1.54-
1.68 (m, 1H); LRMS (FAB), m/z 466 (MH+, base peak), 179,
154, 136, 141, 136; HRMS (FAB), m/z calcd for C30H28NO4
(MH+): 466.2018, found: 464.2024.
Representative Procedure for the Synthesis of Olefin-
Bridged Cyclic Peptides (20a). Protected peptide resin was
manually constructed by Fmoc-based solid-phase peptide
synthesis on Fmoc-HN-Rink amide resin (0.29 mmol/g, 172 mg,
0.05 mmol). Fmoc-â-Ala-OH was condensed to the N-terminal
amino group of H-L-2-allylGly-Arg(Pbf)-Nal-D-2-allylGly-HN-
Rink amide resin. To a suspension of the protected resin in
dry DCM (5 mL) was added Grubbs catalyst second generation
(4.20 mg, 5.00 µmol) at room temperature under argon. The
reaction mixture was refluxed for 9 h. After removal of the
organic solvent, this reaction was repeated once again for 12
h in the presence of the catalyst (21 mg, 0.025 mmol) in dry
DCM (5 mL). The obtained resin was treated with 20%
piperidine in DMF (20 min). To a mixture of the resin and
DIPEA (52.5 µL, 0.300 mmol) in DMF was added 1H-pyrazole-
1-carboxamidine hydrohloride (22.0 mg, 0.15 mmol) at room
tempereture. The reaction mixture was stirred at room tem-
perature for 12 h. This N-guanylation procedure was repeated
once again. The protected resin was treated with thioanisole
(940 µL), TFA (7.2 mL), and TMSBr (1.32 mL) at 0 °C for 2 h.
The reaction mixture was filtered, and the filtrate was
concentrated by bubbling with N2 gas. Cooled Et2O was added
to the residue, and the resulting precipitate was separated by
centrifugation. The precipitate was washed with Et2O. The
crude peptide was purified by HPLC to give the cyclic
pseudopeptide 20a (3.3 mg, 10% yield from Fmoc-HN-Rink
amide resin) as a freeze-dried powder.
Representative Procedure for the Synthesis of γ-Ami-
no Acid-Containing Cyclic Tetrapeptides (11a). Cl-Trt-
(2-Cl) resin (1.25 mmol/g, 400 mg, 0.5 mmol) was treated with
Fmoc-γ-Nal-OH (256 mg, 0.55 mmol) and DIPEA (383 µL, 2.2
mmol) in CH2Cl2 (4 mL) at room temperature for 2 h to yield
Fmoc-γ-Nal-Trt(2-Cl) resin (0.79 mmol/g, 97%). Protected
peptide 11a resin was manually constructed by Fmoc-based
solid-phase peptide synthesis on Fmoc-γ-Nal-Trt(2-Cl) resin
(0.79 mmol/g, 127 mg, 0.1 mmol). But for Tyr and Pbf for Arg
were employed for side-chain protection. The protected peptide
11a resin was treated with 1,1,1,3,3,3-hexafluoro-2-propanol
(HFIP)-DCM (1:4 (v/v), 7 mL) at room temperature for 2 h.
After filtration, the filtrate was concentrated under reduced
pressure to give the crude protected linear peptide. To a stirred
mixture of the protected peptide and N-methylmorphorine
(54.9 µL, 0.5 mmol) in DMF (25 mL) was added diphenylphos-
phoryl azide (DPPA) (53.2 µL, 0.247 mmol) at -40 °C. The
reaction mixture was stirred for 24 h with warming up to room
temperature and concentrated under reduced pressure. The
residue was subjected to solid-phase extraction over basic
alumina in CHCl3-MeOH (9:1) to remove inorganic salts
derived from DPPA. The resulting cyclic protected peptide was
treated with aq. 95% TFA (10 mL) at room temperature for 2
h. Concentration under reduced pressure and purification by
HPLC gave the cyclic pseudopeptide 11a (42.9 mg, 61% yield
from Fmoc-γ-Nal-Trt(2-Cl) resin) as a freeze-dried powder.
Fmoc-tyramine-Wang PEG Resin. To a mixture of Wang
PEG resin (0.33 mmol/g, 1.82 g, 0.600 mmol), Fmoc-tyramine
(647 mg, 1.80 mmol), Ph3P (472 mg, 1.80 mmol), and N-
methylmorphorine (66.0 µL, 0.600 mmol) in CH2Cl2-THF (3:1
(v/v), 16 mL) was added diisopropyl azodicarboxylate (DIAD)
(40% solution in toluene, 886 µL, 1.80 mmol) at 0 °C. The
reaction mixture was stirred at room temperature for 2 d. The
obtained resin was washed with THF (20 mL × 3), DMF (20
Representative Procedure for the Synthesis of Cyclic
Pentapeptides (cyclo(-D-Tyr-Pro-Arg-Nal-Gly-) (23b)). Pro-
tected peptide resin was manually constructed by Fmoc-based
solid-phase peptide synthesis on Fmoc-Gly-Trt(2-Cl) resin
(0.741 mmol/g, 101 mg, 0.075 mmol). But for Tyr and Pbf for
Arg were employed for side-chain protection. Cleavage from
the resin, cyclization, deprotection, and HPLC purification
were performed by use of a procedure identical with that

Low Molecular Weight CXCR4 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3287
described for the synthesis of 11a to yield the cyclic peptide
23b (25.2 mg, 50% yield from Fmoc-Gly-Trt(2-Cl) resin) as a
freeze-dried powder.
4H), 3.93-4.36 (m, 5H), 3.60-3.75 (m, 1H), 3.05-3.21 (m, 1H),
2.32-2.58 (m, 1H), 1.67-1.90 (m, 1H), 1.44 (s, 9H); LRMS
(FAB), m/z 451 [(M - H)-], 229, 155, 153, 152 (base peak);
HRMS (FAB), m/z calcd for C25H27N2O6 [(M - H)-]: 451.1868,
found: 451.1883.
(2S,4R)-NR-Cbz-4-N-Boc-aminoproline Benzyl Ester. By
use of a procedure identical with that described for the
preparation of (2S,4S)-NR-Cbz-4-N-Boc-aminoproline benzyl
ester, (2S,4R)-NR-Cbz-4-aminoproline benzyl ester (5.56 g, 15.7
mmol) was converted into 6.24 g (13.8 mmol, 88% yield) of the
Representative Procedure for the Synthesis of N-
Methylated Cyclic Pentapeptides (cyclo(-D-Tyr-D-MeAla-
Arg-Nal-Gly-) (23d)). According to a procedure identical with
that described for the preparation of 23b, 23d was synthesized
except for N-methylation of the D-Ala residue. N-Methylation
was performed by the Fukuyama-Mitsunobu reaction:28 To a
mixture of H-D-Ala-Arg(Pbf)-Nal-Gly-Trt(2-Cl) resin (0.075
mmol) and o-nitrobenzenesulfonyl chloride (49.9 mg, 0.225
mmol) in CH2Cl2 (5 mL) was added 2,4,6-collidine (49.6 µL,
0.375 mmol) at room temperature. The reaction mixture was
stirred at room temperature for 2 h, and the resin was washed
successively with CH2Cl2 (10 mL × 3) and DMF (10 mL × 3).
To a mixture of the resin, Ph3P (98.4 mg, 0.375 mmol), and
MeOH (15.2 µL, 0.375 mmol) in dry THF (5 mL) was added
diethyl azodicarboxylate (DEAD) (40% solution in toluene, 170
µL, 0.375 mmol) at 0 °C. The reaction mixture was stirred at
room temperature for 2 h. The resin was washed successively
with THF (10 mL × 3) and DMF (10 mL × 3). To a mixture of
the resin and DBU (56.1 µL, 0.375 mmol) in DMF (5 mL) was
added 2-mercaptoethanol (51.4 µL, 0.750 mmol) at room
temperature, and the reaction mixture was stirred at room
temperature for 2 h. Subsequent condensation of the next
residue (D-Tyr) to the secondary amino group on the resin was
performed by treatment with Fmoc-D-Tyr(But)-OH (5 equiv),
HATU (4.9 equiv), HOAt (5 equiv), and DIPEA (10 equiv) in
DMF (5 mL) for 1 h (× 2). 23d (freeze-dried powder): 3.0 mg,
6.0% yield from Fmoc-Gly-Trt(2-Cl) resin.
title compound as colorless crystals. mp: 97-99 °C; [R]25
D
-47.61 (c, 0.714, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.15-
7.43 (m, 10H), 4.93-5.27 (m, 4H), 4.56-4.70 (br, 1H), 4.37-
4.54 (m, 1H), 4.21-4.35 (br, 1H), 3.78-3.89 (m, 1H), 3.30-
3.50 (m, 1H), 2.12-2.34 (m, 2H), 1.45 (s, 9H); LRMS (FAB),
m/z 477 (MNa+, base peak), 455 (MH+), 445, 399, 355, 263,
91; HRMS (FAB), m/z calcd for C25H31N2O6 (MH+): 455.2182,
found: 455.2176.
(2S,4R)-NR-Fmoc-4-N-Boc-aminoproline. By use of a
procedure identical with that described for the preparation of
(2S,4S)-NR-Fmoc-4-N-Boc-aminoproline, (2S,4R)-NR-Cbz-4-N-
Boc-aminoproline benzyl ester (2.75 g, 6.10 mmol) was con-
verted into 2.05 g (4.51 mmol, 74% yield) of the title compound
as colorless crystals. mp: 85-87 °C; [R]24 -36.92 (c 0.325,
D
CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.33 (s, 1H), 7.82-7.97
(m, 2H), 7.56-7.72 (m, 2H), 7.26-7.49 (m, 4H), 3.96-4.36 (m,
4H), 3.50-3.67 (m, 1H), 3.18-3.40 (m, 2H), 1.99-2.26 (m, 2H),
1.44 (s, 9H); LRMS (FAB), m/z 451 [(M - H)-], 229, 155, 153,
151 (base peak); HRMS (FAB), m/z calcd for C25H27N2O6 [(M
- H)-]: 451.1868, found: 451.1887.
(2S,4S)-NR-Cbz-4-N-Boc-aminoproline Benzyl Ester.
(2S,4S)-NR-Cbz-4-aminoproline benzyl ester (1.44 g, 3.93 mmol),
which was prepared from (2S,4S)-NR-Cbz-4-azidoproline benzyl
ester according to Tamaki’s procedure,35 was dissolved in THF
(20 mL), and Boc2O (1.38 g, 6.31 mmol) and triethylamine (1.10
mL, 7.89 mmol) were added to the solution at 0 °C. The
reaction mixture was stirred at room temperature for 7.5 h.
The mixture was concentrated under reduced pressure, and
the residue was extracted with Et2O. The extract was washed
successively with saturated aqueous citric acid (× 2), H2O (×
2), and brine and dried over MgSO4. After removal of the
solvent under reduced pressure, the resulting crude product
was purified by flash chromatography on silica gel with
EtOAc-n-hexane (1:4) to give 1.70 g (3.77 mmol, 96% yield)
of the title compound as a colorless oil. [R]29D -39.47 (c 0.304,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.18-7.45 (m, 10H),
4.95-5.40 (m, 5H), 4.28-4.50 (m, 2H), 3.68-3.82 (m, 1H),
3.44-3.60 (m, 1H), 2.39-2.57 (m, 1H), 1.89-2.05 (m, 1H), 1.45
(s, 9H); LRMS (FAB), m/z 477 (MNa+, base peak), 455 (MH+),
445, 399, 355, 91; HRMS (FAB), m/z calcd for C25H31N2O6
(MH+): 455.2182, found: 455.2186.
Representative Procedure for the Synthesis of Cyclic
Pentapeptides Containing Arginine Mimetic (cyclo(-D-
Tyr-trans-4-guanidino-Pro-Arg-Nal-Gly-) (23o)). Accord-
ing to a procedure identical with that described for the
preparation of 23b, the cyclic protected peptide was synthe-
sized. The obtained protected peptide (0.05 mmol) was treated
with aq. 95% TFA (5 mL) for 2 h at room temperature, and
concentration under reduced pressure gave the crude cyclic
peptide. To a mixture of the crude product and 1H-pyrazole-
1-carboxamidine hydrochloride (22.0 mg, 0.15 mmol) in DMF
(4 mL) was added DIPEA (52.3 µL, 0.300 mmol) at room
temperature. The reaction mixture was stirred at room tem-
perature for 12 h. This procedure (N-guanylation of the
γ-amino group of the trans-4-amino-Pro residue) was repeated
once again. Concentration under reduced pressure and puri-
fication by HPLC gave the cyclic peptide 23o (5.1 mg, 14%
yield from Fmoc-Gly-Trt(2-Cl) resin) as a freeze-dried powder.
[
125I]-SDF-1 Binding and Displacement. Stable CHO cell
transfectants expressing CXCR4 variants were prepared as
described previously.54 CHO transfectants were harvested by
treatment with trypsin-EDTA, allowed to recover in complete
growth medium (MEM-R, 100 µg/mL penicillin, 100 µg/mL
streptomycin, 0.25 µg/mL amphotericin B, 10% (v/v)) for four
to 5 h and then washed in cold binding buffer (PBS containing
2 mg/mL BSA). For ligand binding, the cells were resuspended
in binding buffer at 1 × 107 cells/mL, and 100 µL aliquots were
incubated with 0.1 nM of [125I]-SDF-1 (PerkinElmer Life
Sciences) for 2 h on ice under constant agitation. Free and
bound radioactivity were separated by centrifugation of the
cells through an oil cushion and bound radioactivity was
measured with a gamma-counter (Cobra, Packard, Downers
Grove, IL). Inhibitory activity of test compounds was deter-
mined based on the inhibition of [125I]-SDF-1-binding to
CXCR4 transfectants (IC50).
(2S,4S)-NR-Fmoc-4-N-Boc-aminoproline. (2S,4S)-NR-Cbz-
4-N-Boc-aminoproline benzyl ester (1.70 g, 3.77 mmol) was
dissolved in EtOH (80 mL), and 5% Pd/C (170 mg) was added
to the solution. The reaction vessel was charged with atmo-
sphere of H2 (balloon), and the mixture was stirred at room
temperature for 12 h. After removal of Pd/C by filtration, the
filtrate was concentrated under reduced pressure to give the
crude product as a colorless oil, which was used directly in
the following step without purification. To the crude product
in H2O-THF (4:3 (v/v), 70 mL) were added triethylamine (1.41
mL, 7.55 mmol) and Fmoc-OSu (2.54 g, 7.55 mmol) in CH3CN
(10 mL) at 0 °C, and the reaction mixture was stirred at room
temperature for 2 h. The mixture was acidified with saturated
aqueous citric acid and concentrated under reduced pressure.
The residue was extracted with EtOAc, and the extract was
washed successively with saturated aqueous citric acid, H2O
(× 3), and brine and dried over MgSO4. After removal of the
solvent under reduced pressure, the resulting crude product
was purified by flash chromatography on silica gel with
CHCl3-MeOH (20:1) to give 1.18 g (2.60 mmol, 69% yield) of
NMR Spectroscopy (23c-e, 23o, and 23p). The peptide
sample was dissolved in DMSO-d6 at a concentration of 5 mM.
1H NMR spectra of the peptides were recorded at 300 K. The
assignments of the proton resonances were achieved by use
of 1H-1H COSY spectra. 3J(HN,HR) coupling constants were
measured from one-dimensional spectra. The mixing time for
the NOESY experiments was set at 400 ms. NOESY spectra
were composed of 512 real points in the F2 dimension and 256
real points, which were zero-filled to 256 points in the F1
dimension, with 144 scans per t1 increment. The cross-peak
the title compound as colorless crystals. mp: 87-89 °C; [R]24
D
-33.81 (c 0.621, CHCl3); 1H NMR (400 MHz, DMSO-d6) δ 8.30
(s, 1H), 7.82-7.97 (m, 2H), 7.57-7.75 (m, 2H), 7.25-7.49 (m,

3288 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Tamamura et al.
(5) Feng, Y.; Broder, C. C.; Kennedy, P. E.; Berger, E. A. HIV-1 entry
co-factor: Functional cDNA cloning of a seven-transmembrane,
G protein-coupled receptor. Science 1996, 272, 872-877.
(6) Mu¨ller, A.; Homey, B.; Soto, H.; Ge, N.; Catron, D.; Buchanan,
M. E.; McClanahan, T.; Murphy, E.; Yuan, W.; Wagner, S. N.;
Barrera, J. L.; Mohar, A.; Verastegui, E.; Zlotnik, A. Involvement
of chemokine receptors in breast cancer metastasis. Nature 2001,
410, 50-56.
intensities were evaluated by relative build-up rates of the
cross-peaks. Temperature dependence of the chemical shifts
of all of the amide protons was investigated in 23c-e, 23o,
and 23p. In 23c, the temperature coefficients for all of the NH
protons were large. In 23d, the only temperature coefficient
for the NH of D-Tyr3 was small, but NOE was not observed
between the Nal1 CRH and D-Tyr3 NH. In 23e, 23o, and 23p,
the temperature coefficients for the NH of D-Tyr3 and Arg5
were small, but NOE was not observed between the Nal1 CRH
and D-Tyr3 NH or between the D-Tyr3 CRH and Arg5 NH. Thus,
no hydrogen bond restraints were used in the simulated
annealing calculations of 23c-e, 23o, and 23p.
(7) Koshiba, T.; Hosotani, R.; Miyamoto, Y.; Ida, J.; Tsuji, S.;
Nakamura, S.; Kawaguchi, M.; Kobayashi, H.; Doi, R.; Hori, T.;
Fujii, N.; Imamura, M. Expression of stromal cell-derived factor
1 and CXCR4 ligand receptor system in pancreatic cancer:
a
possible role for tumor progression. Clin. Cancer Res. 2000, 6,
3530-3535.
Calculation of Structures. The structure calculations
were performed on a Silicon Graphics Origin 2000 workstation
with the NMR-refine program within the Insight II/Discover
package using the consistent valence force field (CVFF).55
Pseudoatoms were defined for the methylene protons of Nal1,
D-Tyr3, Arg4, and Arg5 prochiralities of which were not identi-
fied by 1H NMR data. The restraints, in which the Gly2
R-methylene participated, were defined for the separate
protons without definition of the prochiralities. The dihedral
φ angle constraints were calculated based on the Karplus
equation: 3J(HN,HR) ) 6.7cos2(θ - 60) - 1.3cos(θ - 60) + 1.5.56
Lower and upper angle errors were set to 15°. The NOESY
spectrum with a mixing time of 400 ms was used for the
estimation of the distance restraints between protons. The
NOE intensities were classified into three categories (strong,
medium, and weak) based on the number of contour lines in
the cross-peaks to define the 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. Lower
bounds between nonbonded atoms were set to their van der
Waals radii (1.8 Å). These distance and dihedral angle
restraints were included with force constants of 25-100 kcal/
mol‚Å2 and 25-100 kcal/mol‚rad2, respectively. The 50 initial
structures generated by the NMR refine program randomly
were subjected to the simulated annealing calculations. The
final minimization stage was achieved until the maximum
derivative became less than 0.01 kcal/mol‚Å2 by the steepest
descents and conjugate gradients methods.
(8) Mori, T.; Doi, R.; Koizumi, K.; Toyoda, E.; Tulachan, S. S.; Ito,
D.; Kami, K.; Masui, T.; Fujimoto, K.; Tamamura, H.; Hira-
matsu, K.; Fujii, N.; Imamura, M. CXCR4 antagonist inhibits
stromal cell-derived factor 1-induced migration and invasion of
human pancreatic cancer. Mol. Cancer Ther. 2004, 3, 29-37.
(9) Robledo, M. M.; Bartolome, R. A.; Longo, N.; Miguel Rodriguez-
Frade, J.; Mellado, M.; Longo, I.; van Muijen, G. N. P.; Sanchez-
Mateos, P.; Teixido, J. Expression of functional chemokine
receptors CXCR3 and CXCR4 on human melanoma cells. J. Biol.
Chem. 2001, 276, 45098-45105.
(10) Taichman, R. S.; Cooper, C.; Keller, E. T.; Pienta, K. J.;
Taichman, N. S.; McCauley, L. K. Use of the stromal cell-derived
factor-1/CXCR4 pathway in prostate cancer matastasis to bone.
Cancer Res. 2002, 62, 1832-1837.
(11) Schrader, A. J.; Lechner, O.; Templin, M.; Dittmar, K. E. J.;
Machtens, S.; Mengel, M.; Probst-Kepper, M.; Franzke, A.;
Wollensak, T.; Gatzlaff, P.; Atzpodien, J.; Buer, J.; Lauber, J.
CXCR4/CXCL12 expression and signalling in kidney cancer. Br.
J. Cancer 2002, 86, 1250-1256.
(12) Geminder, H.; Sagi-Assif, O.; Goldberg, L.; Meshel, T.; Rechavi,
G.; Witz, I. P.; Ben-Baruch, A. A possible role for CXCR4 and
its ligand, the CXC chemokine stromal cell-derived factor-1, in
the development of bone marrow metastases in neuroblastoma.
J. Immunol. 2001, 167, 4747-4757.
(13) Bertolini, F.; Dell’Agnola, C.; Mancuso, P.; Rabascio, C.; Burlini,
A.; Monestiroli, S.; Gobbi, A.; Pruneri, G.; Martinelli, G. CXCR4
neutralization, a novel therapeutic approach for non-Hodgkin’s
lymphoma. Cancer Res. 2002, 62, 3106-3112.
(14) Kijima, T.; Maulik, G.; Ma, P. C.; Tibaldi, E. V.; Turner, R. E.;
Rollins, B.; Sattler, M.; Johnson, B. E.; Salgia, R. Regulation of
cellular proliferation, cytoskeletal function, and signal trans-
duction through CXCR4 and c-Kit in small cell lung cancer cells.
Cancer Res. 2002, 62, 6304-6311.
Acknowledgment. This work was supported in part
by a 21st Century COE Program “Knowledge Informa-
tion Infrastructure for Genome Science”, a Grant-in-Aid
for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology, Japan, and the
Japan Health Science Foundation. Computation time
was provided by the Supercomputer Laboratory, Insti-
tute for Chemical Research, Kyoto University. S.U. is
grateful for a Research Fellowship from the Japan
Society for the Promotion of Science for Young Scien-
tists.
(15) Scotton, C. J.; Wilson, J. L.; Milliken, D.; Stamp, G.; Balkwill,
F. R. Epithelial cancer cell migration: a role for chemokine
receptors? Cancer Res. 2001, 61, 4961-4965.
(16) Scotton, C. J.; Wilson, J. L.; Scott, K.; Stamp, G.; Wilbanks, G.
D.; Fricker, S.; Bridger, G.; Balkwill, F. R. Multiple actions of
the chemokine CXCL12 on epithelial tumor cells in human
ovarian cancer. Cancer Res. 2002, 62, 5930-5938.
(17) Sanz-Rodriguez, F.; Hidalgo, A.; Teixido, J. Chemokine stromal
cell-derived factor-1R modulates VLA-4 integrin-mediated mul-
tiple myeloma cell adhesion to CS-1/fibronectin and VCAM-1.
Blood 2001, 97, 346-351.
(18) Tsukada, N.; Burger, J. A.; Zvaifler, N. J.; Kipps, T. J. Distinctive
features of “nurselike” cells that differentiate in the context of
chronic lymphocytic leukemia. Blood 2002, 99, 1030-1037.
(19) Juarez, J.; Bradstock, K. F.; Gottlieb, D. J.; Bendall, L. J. Effects
of inhibitors of the chemokine receptor CXCR4 on acute lym-
phoblastic leukemia cells in vitro. Leukemia 2003, 17, 1294-
1300.
(20) Rubin, J. B.; Kung, A. L.; Klein, R. S.; Chan, J. A.; Sun, Y.-P.;
Schmidt, K.; Kieran, M. W.; Luster, A. D.; Segal, R. A. A small-
molecule antagonist of CXCR4 inhibits intracranial growth of
primary brain tumors. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
13513-13518.
(21) Nanki, T.; Hayashida, K.; El-Gabalawy, H. S.; Suson, S.; Shi,
K.; Girschick, H. J.; Yavuz, S.; Lipsky, P. E. Stromal cell-derived
factor-1-CXC chemokine receptor 4 interactions play a central
role in CD4+ T cell accumulation in rheumatoid arthritis
synovium. J. Immunol. 2000, 165, 6590-6598.
(22) Tamamura, H.; Xu, Y.; Hattori, T.; Zhang, X.; Arakaki, R.;
Kanbara, K.; Omagari, A.; Otaka, A.; Ibuka, T.; Yamamoto, N.;
Nakashima, H.; Fujii, N. A low molecular weight inhibitor
against the chemokine receptor CXCR4: a strong anti-HIV
peptide T140. Biochem. Biophys. Res. Commun. 1998, 253, 877-
882.
(23) Tamamura, H.; Hori, A.; Kanzaki, N.; Hiramatsu, K.; Mizumoto,
M.; Nakashima, H.; Yamamoto, N.; Otaka, A.; Fujii, N. T140
analogs as CXCR4 antagonists identified as anti-metastatic
agents in the treatment of breast cancer. FEBS Lett. 2003, 550,
79-83.
Supporting Information Available: Characterization
data of representative synthetic compounds and HPLC charts
of 11a, 11c, 15e, 16e, 15q, 16q, 20a, 20d, 23d, 23j, 23o, and
23p. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) Nagasawa, T.; Kikutani, H.; Kishimoto, T. Molecular cloning and
structure of a pre-B-cell growth-stimulating factor. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 2305-2309.
(2) Bleul, C. C.; Farzan, M.; Choe, H.; Parolin, C.; Clark-Lewis, I.;
Sodroski, J.; Springer, T. A. The lymphocyte chemoattractant
SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry.
Nature 1996, 382, 829-833.
(3) Oberlin, E.; Amara, A.; Bachelerie, F.; Bessia, C.; Virelizier, J.-
L.; Arenzana-Seisdedos, F.; Schwartz, O.; Heard, J.-M.; Clark-
Lewis, I.; Legler, D. F.; Loetscher, M.; Baggiolini, M.; Moser, B.
The CXC chemokine SDF-1 is the ligand for LESTR/fusin and
prevents infection by T-cell-line-adapted HIV-1. Nature 1996,
382, 833-835.
(4) Tashiro, K.; Tada, H.; Heilker, R.; Shirozu, M.; Nakano, T.;
Honjo, T. Signal sequence trap: a cloning strategy for secreted
proteins and type I membrane proteins. Science 1993, 261, 600-
603.

Low Molecular Weight CXCR4 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3289
(24) Tamamura, H.; Fujisawa, M.; Hiramatsu, K.; Mizumoto, M.;
Nakashima, H.; Yamamoto, N.; Otaka, A.; Fujii, N. Identification
of a CXCR4 antagonist, a T140 analog, as an anti-rheumatoid
arthritis agent. FEBS Lett. 2004, 569, 99-104.
(25) Tamamura, H.; Omagari, A.; Oishi, S.; Kanamoto, T.; Yamamoto,
N.; Peiper, S. C.; Nakashima, H.; Otaka, A.; Fujii, N. Pharma-
cophore identification of a specific CXCR4 inhibitor, T140, leads
to development of effective anti-HIV agents with very high
selectivity indexes. Bioorg. Med. Chem. Lett. 2000, 10, 2633-
2637.
(26) Fujii, N.; Oishi, S.; Hiramatsu, K.; Araki, T.; Ueda, S.; Tama-
mura, H.; Otaka, A.; Kusano, S.; Terakubo, S.; Nakashima, H.;
Broach, J. A.; Trent, J. O.; Wang, Z.; Peiper, S. C. Molecular-
size reduction of a potent CXCR4-chemokine antagonist using
orthogonal combination of conformation- and sequence-based
libraries. Angew. Chem., Int. Ed. 2003, 42, 3251-3253.
(27) Fukami, T.; Nagase, T.; Fujita, K.; Hayama, T.; Niiyama, K.;
Mase, T.; Nakajima, S.; Fukuroda, T.; Saeki, T.; Nishikibe, M.;
Ihara, M.; Yano, M.; Ishikawa, K. Structure-activity relation-
ships of cyclic pentapeptide endothelin A receptor antagonists.
J. Med. Chem. 1995, 38, 4309-4324.
(28) Haubner, R.; Gratias, R.; Diefenbach, B.; Goodman, S. L.;
Jonczyk, A.; Kessler, H. Structural and functional aspects of
RGD-containing cyclic pentapeptides as highly potent and
selective integrin RVâ3 antagonists. J. Am. Chem. Soc. 1996,
118, 7461-7472.
(29) Spatola, A. F.; Crozet, Y.; deWit, D.; Yanagisawa, M. Rediscover-
ing an endothelin antagonist (BQ-123): A self-deconvoluting
cyclic pentapeptide library. J. Med. Chem. 1996, 39, 3842-3846.
(30) Wermuth, J.; Goodman, S. L.; Jonczyk, A.; Kessler, H. Stereo-
isomerism and biological activity of the selective and superactive
RVâ3 integrin inhibitor cyclo(-RGDfV-) and its retro-inverso
peptide. J. Am. Chem. Soc. 1997, 119, 1328-1335.
(31) Haubner, R.; Finsinger, D.; Kessler, H. Stereoisomeric peptide
libraries and peptidomimetics for designing selective inhibitors
of the RVâ3 integrin for a new cancer therapy. Angew. Chem.,
Int. Ed. Engl. 1997, 36, 1374-1389.
(32) Porcelli, M.; Casu, M.; Lai, A.; Saba, G.; Pinori, M.; Cappelletti,
S.; Mascagni, P. Cyclic pentapeptides of chiral sequence DLDDL
as scaffold for antagonism of G-protein coupled receptors:
Synthesis, activity and conformational analysis by NMR and
molecular dynamics of ITF 1565 a substance P inhibitor.
Biopolymers 1999, 50, 211-219.
(33) Oishi, S.; Kamano, T.; Niida, A.; Odagaki, Y.; Hamanaka, N.;
Yamamoto, M.; Ajito, K.; Tamamura, H.; Otaka, A.; Fujii, N.
Diastereoselective synthesis of new ψ[[(E)-CHdCMe]- and ψ-
[[(Z)-CHdCMe]-type alkene dipeptide isosteres by organocopper
reagents and application to conformationally restricted cyclic
RGD peptidomimetics. J. Org. Chem. 2002, 67, 6162-6173.
(34) Nikiforovich, G. V.; Kover, K. E.; Zhang, W.-J.; Marshall, G. R.
Cyclopentapeptides as flexible conformational templates. J. Am.
Chem. Soc. 2000, 122, 3262-3273.
cally pure, Fmoc-protected R-amino acids. Helv. Chim. Acta
1992, 75, 2572-2582.
(42) Douat, C.; Heitz, A.; Martinez, J.; Fehrentz, J.-A. Stereoselective
synthesis of allyl- and homoallylglycines. Tetrahedron Lett. 2001,
42, 3319-3321.
(43) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Synthesis and
activity of a new generation of ruthenium-based olefin meta-
thesis catalysts coordinated with 1,3-dimesityl-4,5-dihydroimi-
dazol-2-ylidene ligands. Org. Lett. 1999, 1, 953-956.
(44) Tamaki, M.; Han, G.; Hruby, V. J. Practical and efficient
synthesis of orthogonally protected constrained 4-guanidinopro-
lines. J. Org. Chem. 2001, 66, 1038-1042.
(45) Tamamura, H.; Sugioka, M.; Odagaki, Y.; Omagari, A.; Kan, Y.;
Oishi, S.; Nakashima, H.; Yamamoto, N.; Peiper, S. C.; Ha-
manaka, N.; Otaka, A.; Fujii, N. Conformational study of a
highly specific CXCR4 inhibitor, T140, disclosing the close
proximity of its intrinsic pharmacophores associated with strong
anti-HIV activity. Bioorg. Med. Chem. Lett. 2001, 11, 359-362
and 2409.
(46) Intramolecular hydrogen bonds were not observed in the calcu-
lated structures of these cyclic pentapeptides. Thus, these
peptides do not seem to exist in a characteristic turn conforma-
tion such as a âII′γ turn as reported in several papers concerning
normal cyclic pentapeptides, see Weisshoff, H.; Prasang, C.;
Henklein, P.; Frommel, C.; Zschunke, A.; Mugge, C. Mimicry of
âII′-turns of proteins in cyclic pentapeptides with one and
without D-amino acids. Eur. J. Biochem. 1999, 259, 776-788.
(47) Murakami, T.; Nakajima, T.; Koyanagi, Y.; Tachibana, K.; Fujii,
N.; Tamamura, H.; Yoshida, N.; Waki, M.; Matsumoto, A.;
Yoshie, O.; Kishimoto, T.; Yamamoto, N.; Nagasawa, T. A small
molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1
infection. J. Exp. Med. 1997, 186, 1389-1393.
(48) Schols, D.; Struyf, S.; Van Damme, J.; Este, J. A.; Henson, G.;
De Clercq, E. Inhibition of T-tropic HIV strains by selective
antagonization of the chemokine receptor CXCR4. J. Exp. Med.
1997, 186, 1383-1388.
(49) Donzella, G. A.; Schols, D.; Lin, S. W.; Este J. A.; Nagashima,
K. A.; Maddon, P. J.; Allaway, G. P.; Sakmar, T. P.; Henson, G.;
De Clercq, E.; Moore, J. P. AMD3100, a small molecule inhibitor
of HIV-1 entry via the CXCR4 co-receptor. Nat. Med. 1998, 4,
72-77.
(50) Doranz, B. J.; Grovit-Ferbas, K.; Sharron, M. P.; Mao, S.-H.;
Bidwell Goetz, M.; Daar, E. S.; Doms, R. W.; O’Brien, W. A. A
small-molecule inhibitor directed against the chemokine receptor
CXCR4 prevents its use as an HIV-1 coreceptor. J. Exp. Med.
1997, 186, 1395-1400.
(51) Howard, O. M. Z.; Oppenheim, J. J.; Hollingshead, M. G.; Covey,
J. M.; Bigelow, J.; McCormack, J. J.; Buckheit, Jr., R. W.;
Clanton, D. J.; Turpin, J. A.; Rice, W. G. Inhibition of in vitro
and in vivo HIV replication by a distamycin analogue that
interferes with chemokine receptor function: a candidate for
chemotherapeutic and microbicidal application. J. Med. Chem.
1998, 41, 2184-2193.
(35) Tamamura, H.; Hiramatsu, K.; Ueda, S.; Wang, Z.; Kusano, S.;
Terakubo, S.; Trent, J. O.; Peiper, S. C.; Yamamoto, N.; Na-
kashima, H.; Otaka, A.; Fujii, N. Stereoselective synthesis of
[L-Arg-L/D-3-(2-naphthyl)alanine]-type (E)-alkene dipeptide iso-
steres and its application to the synthesis and biological evalu-
ation of pseudopeptide analogues of the CXCR4 antagonist
FC131. J. Med. Chem. 2005, 48, 380-391.
(36) Abdel-Magid, A. F.; Maryanoff, C. A.; Carson, K. G. Reductive
amination of aldehydes and ketones by using sodium triacetoxy-
borohydride. Tetrahedron Lett. 1990, 31, 5595-5598.
(37) Fukuyama, T.; Jow, C.-K.; Cheung, M. 2- and 4-Nitrobenzene-
sulfonamides: exceptionally versatile means for preparation of
secondary amines and protection of amines. Tetrahedron Lett.
1995, 36, 6373-6374.
(52) Fujii, N.; Nakashima, H.; Tamamura, H. The Therapeutic
Potential of CXCR4 Antagonists in the Treatment of HIV. Expert
Opin. Investig. Drugs 2003, 12, 185-195.
(53) Tamamura, H.; Fujii, N. Two Orthogonal Approaches to Over-
come Multi-Drug Resistant HIV-1s: Development of Protease
Inhibitors and Entry Inhibitors Based on CXCR4 Antagonists.
Curr. Drug Targets-Infect. Disord. 2004, 4, 103-110.
(54) Navenot, J. M.; Wang , Z. X.; Trent, J. O.; Murray, J. L.; Hu, Q.
X.; DeLeeuw, L.; Moore, P. S.; Chang, Y.; Peiper, S. C. Molecular
anatomy of CCR5 engagement by physiologic and viral chemo-
kines and HIV-1 envelope glycoproteins: Differences in primary
structural requirements for RANTES, MIP-1R, and vMIP-II
binding. J. Mol. Biol. 2001, 313, 1181-1193.
(55) Miyamoto, K.; Nakagawa, T.; Kuroda, Y. Solution structure of
the cytoplasmic linker between domain III-S6 and domain IV-
S1 (III-IV linker) of the rat brain sodium channel in SDS
micelles. Biopolymers 2001, 59, 380-393.
(38) Myers, A. G.; Gleason, J. L.; Yoon, T.; Kung, D. W. Highly
practical methodology for the synthesis of D- and L-R-amino
acids, N-protected R-amino acids, and N-methyl-R-amino acids.
J. Am. Chem. Soc. 1997, 119, 656-673.
(39) Travins, J. M.; Etzkorn, F. A. Facile synthesis of D-amino acids
from an L-serine-derived aziridine. Tetrahedron Lett. 1998, 39,
9389-9392.
(56) Ludvigsen, S.; Andersen, K. V.; Poulsen, F. M. Accurate mea-
surements of coupling-constants from 2-dimensional nuclear-
magnetic-resonance spectra of proteins and determination of
φ-angles J. Mol. Biol. 1991, 217, 731-736.
(40) Biagini, S. C. G.; Gibson, S. E.; Keen, S. P. Cross-metathesis of
unsaturated R-amino acid derivatives. J. Chem. Soc., Perkin.
Trans. 1 1998, 2485-2499.
(41) Oppolzer, W.; Lienard, P. Non-destructive cleavage of N-acyl-
sultams under neutral conditions: preparation of enantiomeri-
JM050009H