Structure-activity relationship study on artificial CXCR4 ligands possessing the cyclic pentapeptide scaffold: The exploration of amino acid residues of pentapeptides by substitutions of several aromatic amino acids

Article abstract of DOI:10.1039/b908286g

Previously, downsizing of a 14-residue peptidic CXCR4 antagonist 1 has led to the development of a highly potent CXCR4 antagonist 2 [cyclo(-d-Tyr ¹-Arg²-Arg³-Nal⁴-Gly⁵-)]. In the present study, cyclic

Full text of DOI:10.1039/b908286g

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Structure-activity relationship study on artificial CXCR4 ligands possessing
the cyclic pentapeptide scaffold: the exploration of amino acid residues of
pentapeptides by substitutions of several aromatic amino acids†
Tomohiro Tanaka,a Wataru Nomura,*a Tetsuo Narumi,a,b Ai Esaka,b Shinya Oishi,b Nami Ohashi,a
Kyoko Itotani,a Barry J. Evans,c Zi-xuan Wang,c,d Stephen C. Peiper,c Nobutaka Fujiib and
Hirokazu Tamamura*a
Received 30th April 2009, Accepted 9th June 2009
First published as an Advance Article on the web 20th July 2009
DOI: 10.1039/b908286g
Previously, downsizing of a 14-residue peptidic CXCR4 antagonist 1 has led to the development of a
highly potent CXCR4 antagonist 2 [cyclo(-D-Tyr1-Arg2-Arg3-Nal4-Gly5-)]. In the present study, cyclic
pentapeptide libraries that were designed by substitutions of several amino acids for D-Tyr1 and Arg2 in
peptide 2 were prepared and screened to evaluate binding activity for CXCR4. The above
structure-activity relationship study led to the finding of several potent CXCR4 ligands.
Introduction
The chemokine receptor CXCR4, which has an endogenous
ligand, stromal-cell derived factor-1a (SDF-1a)/CXCL12,1,2 be-
longs to the G-protein coupled receptor (GPCR) family.3,4 The
CXCR4-CXCL12 axis plays an important role in various physio-
logical functions: chemotaxis,5 angiogenesis6,7 and neurogenesis8,9
in embryonic stage. However, CXCR4 is also relevant to multi-
ple intractable diseases: AIDS,10,11 cancer metastasis,12 progress
of leukemia,13 and rheumatoid arthritis14 in adulthood. Thus,
CXCR4 is thought to be an attractive drug target against
these diseases, and CXCR4 antagonists would be useful for the
development of potent therapeutic agents.15–17 Various CXCR4
antagonists such as AMD310018,19 and KRH-163620 have been
reported to date. A 14-residue cyclic peptide CXCR4 antagonist
1 was previously found by structure optimization of an 18-residue
bicyclic peptide polyphemusin analogue (Fig. 1).21,22 Furthermore,
the downsizing of 1 using its pharmacophore residues [Arg ¥ 2,
L-3-(2-naphthyl)alanine (Nal), Tyr] brought the development of
cyclic pentapeptide 2 as a CXCR4 antagonist.23
Fig. 1 Development of a cyclic pentapeptide 2 based on the pharma-
cophore of a CXCR4 antagonistic peptide 1. Further conversion from
1 into a biostable derivative 3 and from 2 into new cyclic pentapep-
In addition, a biologically stable analogue 3 was derived from
tide leads 4 and 5. Cit = L-citrulline, Nal = L-3-(2-naphthyl)alanine,
Nal(1) = L-3-(1-naphthyl)alanine.
1 with the addition of a 4-fluorobenzoyl group as a new pharm-
acophore moiety at the N-terminus.24 We have studied structure-
activity-relationship (SAR) of 2 through various modifications
such as changes of the ring size and amino acid substitutions.25–27
Potent CXCR4 ligands contain aromatic and cationic groups,28
suggesting that these groups are involved in binding to CXCR4
mediated by hydrophobic and electrostatic interactions. In a
previous study, D-Tyr1 and Arg2 in peptide 2 were replaced by a
bicyclic aromatic amino acid and a cationic amino acid to identify
novel pharmacophores and to find new lead compounds. Com-
pounds 4, with replacement of D-Tyr1 by D-3-(1-naphthyl)alanine
(D-Nal(1)), and 5 with the sequence of Arg1-D-Nal2 based on
shuffling cationic and aromatic amino acids at positions 1 and
2 of compound 2 showed high CXCR4 binding activity.29 Thus,
in this study, the design of a cyclic pentapeptide library based on
substitutions of several aromatic amino acids at positions 1 and
2 led to the development of novel analogues of 2 to explore new
pharmacophore moieties.
aInstitute of Biomaterials and Bioengineering, Tokyo Medical and Den-
tal University, Chiyoda-ku, Tokyo, 101-0062, Japan. E-mail: nomura.
mr@tmd.ac.jp, tamamura.mr@tmd.ac.jp; Fax: +813 5280 8039; Tel: +813
5280 8036
bGraduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku,
Kyoto, 606-8501, Japan
cDepartment of Pathology, Anatomy and Cell Biology, Thomas Jefferson
University, Philadelphia, PA 19107, USA
dDepartment of Surgery, Thomas Jefferson University, Philadelphia, PA
19107, USA
† Electronic supplementary information (ESI) available: Characterization
data (MS and [a]D) and HPLC charts of novel synthetic compounds. See
DOI: 10.1039/b908286g
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Table 1 Inhibitory activity of the synthetic compounds 6–10 against
binding of [125I]-SDF-1a to CXCR4
Table 2 Inhibitory activity of the synthetic compounds 11–16 against
binding of [125I]-SDF-1a to CXCR4
Compd
cyclo(-Xaa1-Xaa2-Arg3-Nal4-Gly5-). Xaa1-Xaa2
IC50/mMa
Compd
cyclo(-Xaa1-Arg2-Arg3-Nal4-Gly5-). Xaa1
IC50/mMa
2
D-Tyr1-Arg2
0.0079
0.22
0.31
0.22
2.2
2
D-Tyr1
0.0079
1.2
2.3
0.059
0.088
1.1
6
D-Phe(4-F)1-Arg2
11
12
13
14
15
16
D-Phe(4-Cl)1
D-Phe(4-Br)1
D-Phe(2-F)1
D-Phe(3-F)1
L/D-Phg1
D-Thi1
7
D-Phe(4-F)1-D-Arg2
Phe(4-F)1-Arg2
8
9
Phe(4-F)1-D-Arg2
10
cyclo(-D-Tyr1-Arg2-Arg3-Nal4- Phe(4-F)5-)
4.4
0.056
a IC50 values are the concentrations for 50% inhibition of the [125I]-SDF-
1a binding to CXCR4 transfectants of CHO cells. All data are the mean
values for at least three experiments.
Biological results and discussion
SAR of analogues with L/D-Phe(4-F)1-L/D-Arg2
a IC50 values are the concentrations for 50% inhibition of the [125I]-SDF-
1a binding to CXCR4 transfectants of CHO cells. All data are the mean
values for at least three experiments.
Since compound 6, where D-Tyr1 of 2 was replaced by 4-fluoro-
D-phenylalanine [D-Phe(4-F)] relevant to the new pharmacophore
4-fluorobenzoyl group, showed relatively potent CXCR4-binding
activity as reported previously,26 initially, 6 and its three analogues
7–9, [L/D-Phe(4-F)1, L/D-Arg2]-2, were synthesized to evaluate the
configuration effects of amino acids at positions 1 and 2 (Table 1).
These analogues except for 9 showed high CXCR4 binding activity
(IC50 = 0.2–0.4 mM, Table 1), although the potencies were much
lower than that of 2. Compound 9 showed moderate potency
(IC50 = 2.2 mM), suggesting that the combination of Phe(4-F)1 and
D-Arg2 is not suitable. In addition, compound 10 was synthesized,
where Gly5 of 2 was replaced by Phe(4-F) with maintenance of
D-Tyr1, since both Phe(4-F) and D-Tyr are thought to be important
pharmacophore residues. However, compound 10 did not show
high potency (IC50 = 4.4 mM), probably due to a conformational
change.
for the side-chain of the amino acid at position 1. New leads, 13
and 16, having D-Phe(2-F)1 and D-Thi1, respectively, were found
although the potencies were approximately one-eighth of that of 2.
SAR of analogues with Arg1-aromatic D-amino acid2
Since 5, an analogue with the sequence of Arg1-D-Nal2 based
on shuffling cationic and aromatic amino acids at positions 1
and 2 of compound 2, showed high CXCR4 binding activity,29a
series of analogues with the sequence of Arg1-aromatic D-amino
acid2 (substitution for D-Nal2) were synthesized. Among halogen
substituents at position 4 on the phenyl ring of D-Phe2, fluorine is
the most suitable, whereas chlorine or bromine is not preferable
as shown in activity of 17,26 18 and 19 (IC50 = 0.035, 0.79 and
0.57 mM, respectively, Table 3). In addition, a 4-nitro group
is not suitable although this group is an electron-withdrawing
group, possibly due to steric hindrance (20, IC50 = 0.94 mM).
A 4-hydroxy group with electron-donating action and a 4-amino
group with strong electron-donating action is not favorable (21,
IC50 = 0.97 mM, 22, IC50 = 15 mM). As a result, fluorine is the most
suitable substituent at position 4 on D-Phe2 among these atoms
and groups. In the investigation of the preference of positions of
fluorine on the phenyl ring of D-Phe2, para-position is superior to
SAR of analogues with replacement of D-Phe(4-F)1 of 6 by an
aromatic D-amino acid
Based on the configuration of [D-Phe(4-F)1, L-Arg2] of 6, a series
of analogues with replacement of D-Phe(4-F)1 by several aromatic
amino acids were synthesized. The order of preference of halogen
atoms as a substituent of position 4 on D-Phe1 is fluorine, chlorine
and bromine as shown in activity of 6, 11 and 12 (IC50 = 0.22,
1.2 and 2.3 mM, respectively, Tables 1 & 2). It suggests that a
small or electron-withdrawing group is favorable for a substituent
of position 4 on D-Phe1. Next, preference of positions of fluorine
on the phenyl ring of D-Phe1 was investigated. As a result, the
order of preference is ortho, meta and para-positions, as shown
in activity of 13, 14 and 6 (IC50 = 0.059, 0.088 and 0.22 mM,
respectively). In the previous paper, a D-Nal(1)1-substituted ana-
logue 4 (IC50 = 0.043 mM) showed much higher CXCR4 binding
Table 3 Inhibitory activity of the synthetic compounds 17–24 against
binding of [125I]-SDF-1a to CXCR4
Compd
cyclo(-Xaa1- Xaa2-Arg3-Nal4-Gly5-). Xaa1-Xaa2
IC50/mMa
2
D-Tyr1-Arg2
0.0079
0.035
0.79
0.57
0.94
0.97
15
17
18
19
20
21
22
23
24
Arg1-D-Phe(4-F)2
Arg1-D-Phe(4-Cl)2
Arg1-D-Phe(4-Br)2
Arg1-D-Phe(4-NO2)2
Arg1-D-Tyr2
activity than a D-Nal1-substituted analogue, [D-Nal1]-2 (IC50
>
2.0 mM).29 Taken together, a para-substituent on the phenyl ring of
the D-amino acid residue at position 1 is not appropriate for high
potency, possibly due to the steric hindrance between the para-
substituent on the phenyl ring and CXCR4. In addition, two other
analogues were prepared. Compound 15, [L/D-Phg1]-2 (racemic),
did not show high CXCR4 binding activity. Compound 16, [b-
(2-thienyl)-D-alanine (D-Thi)1]-2, showed very potent CXCR4
binding activity, suggesting a thienyl group is relatively suitable
Arg1-D-Phe(4-NH2)2
Arg1-D-Phe(2-F)2
Arg1-D-Phe(3-F)2
7.1
6.1
a IC50 values are the concentrations for 50% inhibition of the [125I]-SDF-
1a binding to CXCR4 transfectants of CHO cells. All data are the mean
values for at least three experiments.
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Table 4 Inhibitory activity of the synthetic compounds 25–31 against
binding of [125I]-SDF-1a to CXCR4
Table 5 Inhibitory activity of the synthetic compounds 32–37 against
binding of [125I]-SDF-1a to CXCR4
Compd
cyclo(-D-Tyr 1-Xaa2-Xaa3-Nal4-Gly5-). Xaa2-Xaa3 IC50/mMa
Compd
cyclo(-Xaa1- Xaa2-Arg3-Nal4-Gly5-). Xaa1-Xaa2
IC50/mMa
2
Arg2-Arg3
Hph2-Arg3
D-Phg2-Arg3
Phg2-Arg3
His2-Arg3
D-His2-Arg3
Arg2-His3
His2-His2
0.0079
0.075
6.0
2
D-Tyr1-Arg2
Arg1-His2
0.0079
0.40
0.96
1.7
8.1
5.0
25
26
27
28
29
30
31
32
33
34
35
36
37
Arg1-D-His2
Arg1-D-Thi2
Arg1-D-Tpi2
Arg1-D-Hph2
Arg1-L/D-Phg2
0.17
0.037
0.035
5.0
5.9
12
a IC50 values are the concentrations for 50% inhibition of the [125I]-SDF-
1a binding to CXCR4 transfectants of CHO cells. All data are the mean
values for at least three experiments.
a IC50 values are the concentrations for 50% inhibition of the [125I]-SDF-
1a binding to CXCR4 transfectants of CHO cells. All data are the mean
values for at least three experiments.
ortho or meta as shown in the activity of 17, 23 and 24 (IC50 = 0.035,
7.1 and 6.1 mM, respectively). In the previous paper, a D-Nal2-
substituted analogue 5 (IC50 = 0.045 mM) showed much higher
CXCR4 binding activity than a D-Nal(1)2-substituted analogue,
[Arg1-D-Nal(1)2]-2 (IC50 > 2.0 mM).29 Taken together, a para-
substituent on the phenyl ring of the D-amino acid residue at
position 2 is suitable for high potency, possibly due to hydrophobic
or p interaction between the para-substituent on the phenyl ring
and the receptor CXCR4.
most potent compound, with almost the same CXCR4 binding
activity as 29 (D-His2-Arg3). Thus, 32 and 33, where D-Tyr1 of
28 and 29 was replaced by Arg, respectively, were synthesized.
However, 32 and 33 are more than 10 fold weaker than 28 and 29
(Table 5), indicating that Arg1 is not suitable in these analogues.
D-Thi, (3R)-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic acid
(D-Tpi), D-Hph and L/D-Phg (racemic)-substituted analogues did
not show high potency as shown in 34, 35, 36 and 37, respectively.
It suggests that these series of analogues with the sequence of
Arg1-aromatic-amino acid2 are not potent compounds, although
5 and 17 showed high potency.
SAR of analogues with replacement of Arg2 of 2 by an aromatic
amino acid
SAR of analogues with D-Phe(4-F)1-Arg/His2-Arg/His3 or
Arg/His1-D-Phe(4-F)2-Arg/His3
Since [D-Tyr1-Phe(4-F)2]-2 showed high CXCR4 binding activ-
ity in the previous paper,26 analogues having incorporation of
an aromatic amino acid into position 2 were synthesized. An
L-homophenylalanine (Hph)-substituted analogue 25 showed
potent CXCR4 binding activity (IC50 = 0.075 mM, Table 4),
whereas an L-phenylglycine (Phg)-substituted analogue 27 showed
lower CXCR4 binding activity (IC50 = 0.17 mM), although a
D-phenylglycine (D-Phg)-substituted analogue 26 showed even
lower CXCR4 binding activity (IC50 = 6.0 mM). Since His has
both basic and aromatic character, it would be a useful amino acid
substitution at position 2. Practically, L- and D-His-substituted
analogues 28 and 29 showed high potency (IC50 = 0.037 and
0.035 mM, respectively), indicating that the chirality of L/D-His
at position 2 does not affect CXCR4 binding. The potencies are
approximately one-fourth of that of 2. Next, we extended the His-
substitution to position 3 (Arg3). Analogues with the sequences of
Arg2-His3 and His2-His3 were synthesized (30 and 31, respectively).
However, these analogues did not show potent CXCR4 binding
activity (IC50 = 5.2 and 12 mM, respectively), suggesting that His-
substitution for Arg3 is not appropriate.
Since 6, [D-Phe(4-F)1]-2, showed moderate CXCR4 binding
activity,26 a series of analogues with the sequence of D-Phe(4-F)1-
Arg/His2-Arg/His3 were synthesized (38, 39 and 40). However,
significantly potent analogues could not be found (IC50 > 10 mM,
Table 6). Thus, to interchange the order of positions 1 and 2,
a series of analogues with the sequence of Arg/His1-D-Phe(4-
F)2-Arg/His3 were synthesized (41, 42 and 43). CXCR4 binding
Table 6 Inhibitory activity of the synthetic compounds 38–43 against
binding of [125I]-SDF-1a to CXCR4
cyclo(-Xaa1- Xaa2-Xaa3-Nal4-Gly5-).
Compd
Xaa1-Xaa2-Xaa3
IC50/mMa
2
D-Tyr1-Arg2-Arg3
0.0079
22
10
>100
>100
5.7
38
39
40
41
42
43
D-Phe(4-F)1-Arg2-His3
D-Phe(4-F)1-His2-Arg3
D-Phe(4-F)1-His2-His3
Arg1-D-Phe(4-F)2-His3
His1-D-Phe(4-F)2-Arg3
His1-D-Phe(4-F)2-His3
>100
SAR of analogues with Arg1-aromatic-amino acid2
a IC50 values are the concentrations for 50% inhibition of the [125I]-SDF-
1 binding to CXCR4 transfectants of CHO cells. All data are the mean
values for at least three experiments.
Among analogues with a combination of the sequences of
Arg/His2-Arg/His3, 28 having the sequence of His2-Arg3 is the
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activities of these analogues are relatively weak (IC50 > 5 mM),
indicating that incorporation of D-Phe(4-F) at position 1 and His-
substitution for Arg3 are not suitable.
temperature for 2 h. After filtration of the residual resin, the
filtrate was concentrated under reduced pressure to give a crude
protected peptide. To a stirred mixture of the protected peptide
and NaHCO3 (57.1 mg, 0.680 mmol) in DMF (41 mL) was added
diph◦enylphosphoryl azide (DPPA, 0.0879 mL, 0.408 mmol) at
-40 C. The mixture was stirred for 36 h with warming to room
temperature and filtered. The filtrate was concentrated under
reduced pressure to give an oily residue, which was subjected
to solid phase extraction over basic alumina in CHCl3-MeOH
(9 : 1 (v/v)) to remove inorganic salts derived from DPPA. The
resulting cyclic protected peptide was treated with 95% TFA
solution for 1.5 h at room temperature. Concentration under re-
duced pressure and purification by preparative HPLC gave a cyclic
peptide.
Conclusion
In this paper, SAR of several cyclic pentapeptides having CXCR4
binding activity was studied to discover useful lead compounds. (1)
Of the analogues with replacement of D-Tyr1 of 2 by an aromatic D-
amino acid, a D-Phe(2-F)1-substituted analogue, 13, and a D-Thi1-
substituted analogue, 16, have potent CXCR4 binding activity. A
para-substituent on the phenyl ring of the D-amino acid residue at
position 1 is not favorable for high potency. (2) Among a series of
analogues based on shuffling cationic and aromatic amino acids
at positions 1 and 2, an [Arg1-D-Phe(4-F)2]-containing analogue,
17, showed the most potent CXCR4 binding activity. A para-
substituent on the phenyl ring of the D-amino acid residue at
position 2 is suitable for high potency. (3) Analogues, where
Arg2 of 2 was replaced by L/D-His which have both basic and
aromatic characters, have high CXCR4 binding activity. (4) Arg1-
substituted analogues or His3-substituted analogues are not potent
leads. Taken together, in the present study several new leads were
found, and aromatic amino acid residues, Phe(2-F), Phe(4-F), Thi
and His, were identified to be new pharmacophore residues in
addition to Arg, Nal, Nal(1) and Tyr. The present data will be
important for the development of CXCR4 antagonists. In future,
the introduction of fluorophenyl, thienyl, imidazoyl groups, etc.
involving the combinational use of the above groups into the
cyclic pentapeptide templates and into low molecular weight
linear type scaffolds will bring us the development of new-type
leads of CXCR4 antagonists. The present data of preferences
of the target CXCR4 such as inclination of aromatic and basic
groups will be useful to disclose an unknown detail binding
mode of CXCR4 and cyclic pentapeptide-type ligands on the cell
membrane.
CXCR4 receptor binding assay30
Stable CHO cell transfectants expressing CXCR4 were prepared
as described previously.31 CHO transfectants were detached by
treatment with trypsin-EDTA, allowed to recover in complete
growth medium (MEM-R, 100 mg/mL penicillin, 100 mg/mL
streptomycin, 0.25 mg/mL amphotericin B, 10% FBS (v/v)), 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 cell/mL, and 100 mL aliquots were incubated with
0.1 nM of [125I]-SDF-1 (Perkin-Elmer Life Sciences) for 1 h on
ice under constant agitation. Free and bound radioligands 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, USA). Inhibitory activity
of test compounds was determined based on the inhibition of
[
125I]-SDF-1 binding to CXCR4 transfectants (IC50).
Acknowledgements
This work was supported by New Energy and Industrial Tech-
nology Development Organization (NEDO), Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan, and Health and Labour
Sciences Research Grants from Japanese Ministry of Health,
Labor, and Welfare.
Experimental
Chemistry
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