Low-Molecular-Weight CXCR4 Ligands with Variable Spacers

Article abstract of DOI:10.1002/cmdc.201200390

Low-molecular-weight CXCR4 ligands based on known lead compounds including the 14-mer peptide T140, the cyclic pentapeptide FC131, peptide mimetics, and dipicolylamine-containing compounds were designed and synthesized. Three types of aromatic spacers, 1,4-phenylenedimethanamine, naphthalene-2,6-diyldimethanamine, and [1,1′-biphenyl]-4,4′-diyldimethanamine, were used to build four pharmacophore groups. As pharmacophore groups, 2-pyridylmethyl and 1-naphthylmethyl are present in all of the compounds, and several aromatic groups and a cationic group from 1-propylguanidine and 1,1,3,3-tetramethyl-2-propylguanidine were also used. Several compounds showed significant CXCR4 binding affinity, and zinc(II) complexation of bis(pyridin-2-ylmethyl)amine moieties resulted in a remarkable increase in CXCR4 binding affinity.

Full text of DOI:10.1002/cmdc.201200390

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DOI: 10.1002/cmdc.201200390
Low-Molecular-Weight CXCR4 Ligands with Variable
Spacers
Tetsuo Narumi,[a] Haruo Aikawa,[a] Tomohiro Tanaka,[a] Chie Hashimoto,[a] Nami Ohashi,[a]
Wataru Nomura,[a] Takuya Kobayakawa,[a] Hikaru Takano,[a] Yuki Hirota,[a]
Tsutomu Murakami,[b] Naoki Yamamoto,[c] and Hirokazu Tamamura*[a]
Low-molecular-weight CXCR4 ligands based on known lead
compounds including the 14-mer peptide T140, the cyclic pen-
tapeptide FC131, peptide mimetics, and dipicolylamine-con-
taining compounds were designed and synthesized. Three
types of aromatic spacers, 1,4-phenylenedimethanamine, naph-
thalene-2,6-diyldimethanamine, and [1,1’-biphenyl]-4,4’-diyldi-
methanamine, were used to build four pharmacophore
groups. As pharmacophore groups, 2-pyridylmethyl and 1-
naphthylmethyl are present in all of the compounds, and sev-
eral aromatic groups and a cationic group from 1-propylguani-
dine and 1,1,3,3-tetramethyl-2-propylguanidine were also used.
Several compounds showed significant CXCR4 binding affinity,
and zinc(II) complexation of bis(pyridin-2-ylmethyl)amine moi-
eties resulted in a remarkable increase in CXCR4 binding affini-
ty.
Introduction
CXCR4 is a chemokine receptor that transduces sig-
nals of its endogenous ligand, CXCL12/stromal cell-
derived factor-1 (SDF-1).[1–4] This receptor is
a member of the seven-transmembrane GPCR family,
and has been reported to exist and function as an
oligomer,[5] which was elucidated by our molecular
ruler approach.[6] The CXCR4–CXCL12 axis plays
a physiological role in embryonic stages in chemotax-
is,[7] angiogenesis,[8,9] and neurogenesis.[10,11] CXCR4 is
associated with many disorders including cancer cell
metastasis,[12–14] leukemia cell progression,[15,16] HIV in-
fection/AIDS,[17,18] and rheumatoid arthritis;[19,20] it is
therefore a major target in the discovery of chemo-
therapeutic treatments for these diseases. To date,
many researchers, including ourselves, have devel-
oped potent CXCR4 antagonists. A 14-mer peptide,
T140, and a cyclic pentapeptide, FC131, have been
Figure 1. Reported low-molecular-weight CXCR4 antagonists.
found to be potent CXCR4 antagonists.[21–27] In addi-
tion, downsizing of these peptides has led to the de-
velopment of active small-molecular peptide mimetics.[28] An-
other peptide mimetic, KRH-1636,[29] and
a
bicyclam,
[a] Dr. T. Narumi, Dr. H. Aikawa, Dr. T. Tanaka, C. Hashimoto, Dr. N. Ohashi,
Dr. W. Nomura, T. Kobayakawa, H. Takano, Y. Hirota, Prof. H. Tamamura
Institute of Biomaterials and Bioengineering
AMD3100,[30,31] have also been reported. Furthermore, several
compounds based on monocyclams[32] and noncyclams[33,34]
have been reported. Other aza-macrocyclic compounds such
as the Dpa–Zn complex 1[35] and the Dpa–cyclam compound
2[36] have been developed as non-peptide leads (Figure 1).
These lead compounds have 1,4-phenylenedimethanamine
structures with amino groups presenting basic/aromatic moiet-
ies. We recently developed small-molecular peptide mimetics
containing benzyl and 2-pyridylmethyl amino groups, such as
compound 3[37] and cyclic pentapeptide FC131 derivatives con-
taining two naphthalene moieties (e.g., 4).[38] In the study pre-
sented herein, we tried to develop more effective small mole-
Tokyo Medical and Dental University
2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062 (Japan)
E-mail: tamamura.mr@tmd.ac.jp
[b] Dr. T. Murakami
AIDS Research Center, National Institute of Infectious Diseases
1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640 (Japan)
[c] Prof. N. Yamamoto
Department of Microbiology, Yong Loo Lin School of Medicine
National University of Singapore
Block MD4, 5 Science Drive 2, Singapore 117597 (Singapore)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200390.
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cules based on these lead compounds and to perform appro-
priate structure–activity relationship studies.
duced on a nitrogen atom of the above spacer templates, and
guanidino and tetramethylguanidino groups were used as sub-
stituents for Y3 in compounds 37a–42d.
Results and Discussion
Chemistry
Design
The synthesis of compounds 19a–c is shown in Scheme 1.
Condensation of N-Boc-3-aminopropylbromide (6) and N-Ns-
aminonaphthalen-1-yl-methane (9; Ns=2-nitrobenzenesulfon-
yl) followed by removal of the Ns group produced the amine
11. The N-Ns-4-aminomethylbenzoic acid derived Weinreb
amide 14 was treated with
We initially designed compounds that contain 1,4-phenylenedi-
methanamine, one amino group of which is linked to guani-
dine and naphthalene moieties, and the other to 2-pyridyl-
methyl and naphthalene analogues, as shown in Figure 2. The
DIBAL to afford the correspond-
ing aldehyde, the reductive ami-
nation of which was performed
by treatment with amine 11 to
afford the tertiary amine 15. In-
troduction of a 2-pyridinylmethyl
group into 15 by means of Mit-
sunobu reaction followed by re-
moval of the Ns group yielded
Figure 2. New compounds containing the 1,4-phenylenedimethanamine structure.
amine 17. Introduction of 2-
methylquinoline, 2-methylnaph-
thalene, and 2-methoxy-6-meth-
adoption of these functional moieties is based on structures of
compound 3, which contains 4-fluorobenzyl and 2-pyridyl-
methyl amino groups, and compound 4, which contains two
naphthalene moieties. Thus, 2-methylquinoline, 2-methylnaph-
thalene, 2-methoxy-6-methylnaphthalene, 2-bromo-6-methyl-
naphthalene, and 2-fluoro-6-methylnaphthalene (X-CH2) moiet-
ies were introduced on a nitrogen atom of the 1,4-phenylene-
dimethanamino group in compounds 19a–c and 23d,e. Fur-
thermore, compounds with 1,4-phenylenedimethanamine,
naphthalene-2,6-diyldimethanamine, and [1,1’-biphenyl]-4,4’-
diyldimethanamine structures as spacer templates (H2N-Y2-NH2)
were designed as shown in Figure 3 to refine the spacers.
Monocyclic aromatic groups, 4- or 2-pyridylmethyl, 4-fluoro-
benzyl, and 4-trifluoromethylbenzyl groups (Y1-CH2) were intro-
ylnaphthalene groups by reductive amination of 17 produced
amines 18a–c, respectively, and subsequent removal of the
Boc group followed by N-guanylation yielded the desired com-
pounds 19a–c.
As shown in Scheme 2, introduction of 2-bromo-6-methyl-
naphthalene and 2-fluoro-6-methylnaphthalene moieties into
15 by Mitsunobu reaction followed by removal of the Ns
group yielded amines 21d and 21e, respectively. Introduction
of a 2-pyridinylmethyl group by reductive amination of 21d
and 21e produced amines 22d and 22e, respectively, and sub-
sequent removal of the Boc group followed by N-guanylation
yielded the desired compounds 23d and 23e.
Scheme 3 shows the synthesis of 37a–39d and 40a–42d.
Introduction of 4-pyridylmethyl, 2-pyridylmethyl, or 4-fluoro-
benzyl and 4-trifluoromethylbenzyl groups into N-Ns-(pyridin-
2-ylmethyl)amide 25 by Mitsunobu reaction followed by re-
moval of the Ns group yielded amines 27a–d, respectively.
Treatment of 1,4-phenylenedimethane, naphthalene-2,6-diyldi-
methane, and [1,1’-biphenyl]-4,4’-diyldimethane-derived dibro-
mides 28–30 with amine 11 afforded the tertiary amines 31–
33, respectively. Subsequent treatment of 31–33 with amines
27a–d yielded amines 34a–36d. Subsequent removal of the
Boc group followed by N-guanylation and N-tetramethylguany-
lation yielded the desired compounds 37a–39d and 40a–42d,
respectively.
Biological studies
The CXCR4 binding affinity of the synthesized compounds was
assessed through inhibition of [125I]CXCL12 binding to Jurkat
cells, which express CXCR4.[38] The activity was evaluated for
compounds 19a–c containing 2-methylquinoline, 2-methyl-
naphthalene, 2-methoxy-6-methylnaphthalene, and 23d,e,
Figure 3. New compounds containing the 1,4-phenylenedimethanamine,
naphthalene-2,6-diyldimethanamine, and [1,1’-biphenyl]-4,4’-diyldimethana-
mine structures.
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thoxynaphthalene group, the
compounds showed significant
but very weak binding affinity.
With an electron-donating me-
thoxy group, the 2-methoxy-
naphthalene moiety is an elec-
tron-rich aromatic group. The
quinoline, 2-bromonaphthalene,
and 2-fluoronaphthalene moiet-
ies are electron-deficient aromat-
ic groups because of the elec-
tron-deficient pyridine ring and
electron-withdrawing
fluorine
and bromine atoms. It is sug-
gested that when X represents
bicyclic or electron-rich aromatic
groups, the compounds are un-
likely to be potent ligands.
Because some compounds
containing bicyclic or electron-
rich aromatic groups at the
group X position in Figure 2 do
not have high binding affinity
for CXCR4, compounds in
Figure 3 in which Y1 is a monocy-
clic and electron-deficient aro-
matic group were designed: 4-
pyridylmethyl, 2-pyridylmethyl,
4-fluorobenzyl, and 4-trifluoro-
methylbenzyl groups (Y1-CH2)
were introduced onto the nitro-
gen atom. In addition, as spacer
templates (H2N-Y2-NH2) 1,4-phe-
nylenedimethanamine, naphtha-
lene-2,6-diyldimethanamine, and
[1,1’-biphenyl]-4,4’-diyldimethan-
amine structures were intro-
duced to refine the spacer struc-
Scheme 1. Reagents and conditions: a) Boc2O, Et3N, MeOH/MeCN (1:1), 98%; b) LiAlH4, THF, 08C, 89%; c) NsCl, Et3N,
THF, 78%; d) K2CO3, DMF, 608C, 96%; e) PhSH, K2CO3, DMF, 95%; f) NsCl, Et3N, THF, 88%; g) EDCI·HCl, HOBt·H2O,
NHCH3(OCH3)·HCl, Et3N, DMF, 88%; h) DIBAL/n-hexane, CH2Cl2, À788C; i) NaBH(OAc)3, AcOH, amine 11, 1,2-di-
chloroethane, 43% (two steps); j) PPh3, DEAD, 2-pyridinemethanol, THF, 76%; k) PhSH, K2CO3, DMF, 87%; l) NaBH-
(OAc)3, AcOH, X-CHO, 1,2-dichloroethane, 70% (18a), 69% (18b), 49% (18c); m) 4m HCl/dioxane; n) N,N-diisopro-
pylethylamine, 1-amidinopyrazole·HCl, DMF, 28% (19a), 58% (19b), 46% (19c) (two steps). Ns=2-nitrobenzene-
sulfonyl.
with 2-bromo-6-methylnaphthalene and 2-fluoro-6-methyl-
naphthalene moieties, respectively (X-CH2), introduced on a ni-
trogen atom of the 1,4-phenylenedimethanamino group. The
percent inhibition data for all compounds at 10 mm are listed
in Table 1. With the exception of 19c, which contains a 2-me-
tures, and guanidino and tetramethylguanidino groups were
used as Y3 substituents. The CXCR4 binding affinities of com-
pounds 37a–42d were evaluated (Table 2). None of these
compounds showed more than 50% inhibition at 10 mm. In
general, 4-trifluoromethylbenzyl, [1,1’-biphenyl]-4,4’-diyldime-
thanamine, and tetramethylguanidino moieties seem to be
more suitable as candidates for Y1-CH2, H2N-Y2-NH2, and Y3, re-
spectively. Among these synthetic compounds, 40b, contain-
ing 2-pyridylmethyl, 1,4-phenylenedimethanamine and tetra-
methylguanidino groups, and 42d containing 4-trifluorome-
thylbenzyl, [1,1’-biphenyl]-4,4’-diyldimethanamine and tetrame-
thylguanidino groups, have the highest binding affinity for
CXCR4.
Table 1. CXCR4 binding affinities of compounds 19a–c and 23d,e.
Compd
X[a]
Inhibition [%][b]
19a
19b
19c
a
b
c
14.4Æ1.0
7.0Æ0.6
0
23d
23e
FC131
d
e
–
9.0Æ2.2
9.5Æ1.3
100
As described above in the Introduction, aza-macrocyclic
compounds such as the Dpa–Zn complex 1[35] and the Dpa–
cyclam compound 2[36] have high binding affinities toward
CXCR4. The zinc complex of 2 also has a higher CXCR4 binding
affinity. Thus, the CXCR4 binding affinities of the zinc com-
plexes of 19a, containing 2-pyridylmethyl and 2-methylquino-
[a] The structures of X (a–e) are shown in Figure 2. [b] CXCR4 binding af-
finity was assessed based on inhibition of [125I]CXCL12 binding to Jurkat
cells; percent inhibition values for all compounds at 10 mm were calculat-
ed relative to that of FC131 (100%).
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Figure 4. Zinc complexes of a) 19a and b) 37b, 38b, 39b, 40b, 41b, and
42b. The shaded circle represents the position of the zinc cation in the che-
late. The structures of Y2 and Y3 are shown in Figure 3 as A–C and i–ii, re-
spectively.
except 39b is observed if the inhibitory activities of the zinc
complexes at 5 mm (Table 3) are compared with those of the
corresponding metal-free compounds at 10 mm (Tables 1 and
2). The high activity of the zinc complexes is consistent with
results reported in our previous work,[35,36] and suggests that
the formation of chelates of the nitrogen atoms in the com-
pounds with the zinc(II) ion might enhance their interaction
with CXCR4. Fixation of the functional moieties by zinc(II) che-
lation, progression of electron
Scheme 2. Reagents and conditions: a) PPh3, DEAD, X-CH2OH, THF, RT, 97%
(20d), 59% (20e); b) PhSH, K2CO3, DMF, RT, 42% (21d), 64% (21e); c) NaBH-
(OAc)3, AcOH, 2-pyridinecarbaldehyde, 1,2-dichloroethane, RT, 78% (22d),
85% (22e); d) 4m HCl/dioxane, RT; e) DIPEA, 1-amidinopyrazole·HCl, DMF,
RT, 24% (23d), 18% (23e) (two steps).
deficiency of the aromatic moiet-
Table 2. CXCR4 binding affinities of compounds 37a–42d.
ies, interaction of the zinc(II) ion
with residues on CXCR4, etc.,
Compd
Y1[a]
Y2[b]
Y3[c]
Inhibition [%][d]
Compd
Y1[a]
Y2[b]
Y3[c]
Inhibition [%][d]
might be considered as reasons
for the enhanced CXCR4 binding
affinity of the zinc complexes.
According to previous re-
ports,[39,40] in the case of chela-
tion of the zinc complexes of
AMD3100, a divalent metal ion
such as zinc(II) in one of the bi-
cyclam rings increased this com-
pound’s affinity for CXCR4
37a
37b
37c
37d
38a
38b
38c
38d
39a
39b
39c
39d
a
b
c
d
a
b
c
d
a
b
c
A
A
A
A
B
B
B
B
C
C
C
C
i
i
i
i
i
i
i
i
i
i
i
i
9.6Æ1.9
21.4Æ2.8
8.5Æ1.8
22.3Æ1.4
0
4.7Æ1.3
4.2Æ6.0
4.1Æ4.1
8.1Æ1.1
18.0Æ1.1
26.0Æ3.0
27.9Æ5.2
40a
40b
40c
40d
41a
41b
41c
41d
42a
42b
42c
42d
a
b
c
d
a
b
c
d
a
b
c
A
A
A
A
B
B
B
B
C
C
C
C
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
0
41.5Æ4.8
12.7Æ4.0
23.8Æ6.0
3.2Æ2.2
21.6Æ2.6
13.2Æ1.5
18.4Æ1.2
8.8Æ1.0
0
26.6Æ4.4
d
d
45.0Æ3.0
through
a specific interaction
[a–c] The structures of Y1, Y2, and Y3 are shown in Figure 3 as a–d, A–C, and i–ii, respectively. [d] CXCR4 binding
affinity was assessed based on the inhibition of [125I]CXCL12 binding to Jurkat cells; percent inhibition values
for all compounds at 10 mm were calculated relative to that of FC131 (100%).
with the carboxylate of Asp262
of CXCR4. A similar phenomenon
could be occurring in the zinc
complexes of the present com-
pounds. The IC50 values of the
line groups, and 37b, 38b, 39b, 40b, 41b, and 42b, contain-
ing the Dpa group, were evaluated (Figure 4). ZnCl2 (10 equiv
relative to each compound) was added to phosphate-buffered
saline (PBS) solutions of these compounds to form zinc(II) com-
plexes. Chelation of the nitrogen atoms of 37b and 40b with
the zinc(II) ion has been demonstrated by changes in NMR
chemical shifts upon ZnCl2 titration as zinc chelates as de-
scribed in our previous studies.[35,36] The percent inhibition of
the zinc complexes at 5 mm is listed in Table 3. A remarkable in-
crease in CXCR4 binding affinity of all the zinc complexes
zinc complexes of 37b and 40b containing 1,4-phenylenedi-
methanamine were evaluated to be 2.1 mm. In comparing the
CXCR4 binding affinity of the zinc complexes of 37b, 38b,
39b, 40b, 41b, and 42b, 1,4-phenylenedimethanamine is the
most suitable spacer template (H2N-Y2-NH2), and naphthalene-
2,6-diyldimethanamine is the second most effective. As sub-
stituents for Y3, the tetramethylguanidino group is more ap-
propriate than guanidine. The reason for this property has not
been clarified yet; however, the tetramethyl group might stabi-
lize a positively charged nitrogen atom, or might enhance a hy-
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than
the
2-methylquinoline
group as X-CH2 or Y1-CH2 intro-
duced on the nitrogen atom.
Conclusions
New
low-molecular-weight
CXCR4 ligands were designed
and synthesized. The most
potent compounds are 37b and
40b, zinc complexes with a Dpa
group on the 1,4-phenylenedi-
methanamine spacer template.
The distances between all the
functional moieties of the com-
pounds linked by the 1,4-phenyl-
enedimethanamine spacer might
be appropriate for interaction
with CXCR4. These compounds
exhibited IC50 values at micro-
molar levels in CXCR4 binding
affinity. Zinc complexation of
Dpa-containing compounds re-
sulted in a remarkable increase
in CXCR4 binding affinity relative
to the corresponding zinc-free
compounds. The results reported
herein might provide useful in-
sight into the design of novel
CXCR4 ligands, complementing
information from other com-
pounds such as T140, FC131,
and KRH-1636. These com-
pounds will be useful for the de-
velopment of future therapeutic
strategies for CXCR4-relevant
diseases.
Experimental Section
Chemistry
Synthetic strategies of compounds
reported in the present study are
described in Results and Discus-
sion above, and details are provid-
ed in the Supporting Information.
ZnII complex formation was per-
formed by treatment of the com-
pounds with 10 equiv ZnCl2 in PBS.
The ZnII complexes were character-
ized by the chemical shifts of their
methylene protons in 1H NMR
spectroscopic analysis. The Dpa–
ZnII complex was characterized
previously.[35] Detailed data are provided in the Supporting Infor-
mation.
Scheme 3. Reagents and conditions: a) NsCl, Et3N, THF, 84%; b) Y1-CH2OH, DEAD, PPh3, THF, 53% (26a), 92% (26b),
70% (26c), 97% (26d); c) PhSH, K2CO3, DMF, 97% (27a), 74% (27b), 91% (27c), 91% (27d); d) KI, K2CO3, 11,
MeCN, 78% (31), 53% (32), 71% (33); e) KI, K2CO3, amine 27a–d, MeCN, 25% (34a), 78% (34b), 80% (34c), 90%
(34d), 38% (35a), 75% (35b), 67% (35c), 55% (35d), 23% (36a), 59% (36b), 80% (36c), 80% (36d); f) 4m HCl/
dioxane; g) DIPEA, 1-amidinopyrazole·HCl, DMF, 19% (37a), 49% (37b), 52% (37c), 30% (37d), 42% (38a), 56%
(38b), 62% (38c), 44% (38d), 39% (39a), 48% (39b), 87% (39c), 50% (39d) (two steps); h) 4m HCl/dioxane;
i) DIPEA, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, DMF, 24% (40a), 36% (40b),
31% (40c), 32% (40d), 31% (41a), 14% (41b), 47% (41c), 27% (41d), 37% (42a), 25% (42b), 27% (42c), 44%
(42d) (two steps).
drophobic interaction with residues on CXCR4. Comparison of
the CXCR4 binding affinity of the zinc complexes of 19a and
37b shows that the 2-pyridylmethyl group is more suitable
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Compd
Inhibition [%][a]
IC50 [nm][b]
19a
34.5Æ6.5
93.4Æ6.4
25.6Æ2.4
0
98.0Æ1.0
80.7Æ0.8
35.9Æ0.9
100
ND
2100
ND
ND
2100
ND
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39b
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41b
42b
FC131[c]
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[a] CXCR4 binding affinity was assessed based on the inhibition of
125I]CXCL12 binding to Jurkat cells; percent inhibition values for all zinc
complexes at 5 mm were calculated relative to that of FC131 (100%).
[b] IC50 zinc complex concentration required for 50% inhibition of
125I]CXCL12 binding to Jurkat cells; all data are the mean values from at
[
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