Development of a linear type of low molecular weight CXCR4 antagonists based on T140 analogs

Article abstract of DOI:10.1039/b603818b

A linear type of several low molecular weight CXCR4 antagonists were developed based on T140 analogs, which were previously found to be strong CXCR4 antagonists that block X4-HIV-1 entry and have inhibitory activities against cancer metastasis/progression and rheumatoid arthritis. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b603818b

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Development of a linear type of low molecular weight CXCR4 antagonists
based on T140 analogs†
Hirokazu Tamamura,*a Hiroshi Tsutsumi,a Hiroyuki Masuno,a Satoko Mizokami,b Kenichi Hiramatsu,b Zixuan
Wang,c John O. Trent,d Hideki Nakashima,e Naoki Yamamoto, f ,g Stephen C. Peiperc and Nobutaka Fujii*b
Received 14th March 2006, Accepted 24th April 2006
First published as an Advance Article on the web 12th May 2006
DOI: 10.1039/b603818b
A linear type of several low molecular weight CXCR4
antagonists were developed based on T140 analogs, which
were previously found to be strong CXCR4 antagonists that
block X4-HIV-1 entry and have inhibitory activities against
cancer metastasis/progression and rheumatoid arthritis.
Nanki et al. reported that the memory T cells highly express
CXCR4 and the concentration of CXCL12 is extremely high in the
synovium of RA patients, and that CXCL12 stimulates migration
of the memory T cells and inhibits T cell apoptosis followed by T
cell accumulation in the RA synovium.4a Taken together, CXCR4
is thought to represent an important therapeutic target.5 Thus,
several antagonists directed against CXCR4 have been developed.
We previously found a 14-mer peptide, T140, which specifically
antagonizes CXCR4,6 and that Arg2, L-3-(2-naphthyl)alanine
(Nal)3, Tyr5 and Arg14 constitute the biologically critical residues of
T140 (Fig. 1).7 Recently, its potent analogs, 4F-benzoyl-TN14003
and 4F-benzoyl-TE14011, possessing increased stability in serum
and liver homogenate, were developed by introduction of a p-
fluorobenzoyl group, which was defined as a new pharmacophore,
into the N-terminus.8 4F-benzoyl-TN14003 and 4F-benzoyl-
TE14011 showed strong anti-HIV activity in vitro, anti-metastatic
activity against breast cancer3b and melanoma3g and anti-RA
activity in experimental model mice.4b Furthermore, T140-related
analogs exhibited significant inhibition against CXCL12-induced
migration/activation/invasion of small-cell lung cancer cells,3h
acute lymphoblastic leukemia cells3e and pancreatic cancer cells3c,f
in vitro. Molecular-size reduction of T140 based on the above four
critical residues (Arg × 2, Nal and Tyr) led to discovery of a low
molecular weight CXCR4 antagonist with a cyclic pentapeptide
template, FC131.9 In this paper, identification of the enhanced
pharmacophore involving an electron-deficient aromatic ring
at the N-terminus of 4F-benzoyl-TN14003 and 4F-benzoyl-
TE14011, such as a p-fluorobenzoyl or p-trifluoromethylbenzoyl
moiety, prompted us to develop novel linear-type low molecular
weight CXCR4 antagonists. By combining substructure units of
Introduction
A system of a chemokine receptor, CXCR4, and its endoge-
nous ligand, stromal cell-derived factor-1 (SDF-1/CXCL12), has
multiple important functions in normal physiology involving the
migration of progenitors during embryologic development of
the cardiovascular, hemopoietic and central nervous systems.1
The CXCL12/CXCR4 system has been also recognized to be
involved in several pathologic conditions, such as HIV infection,2
cancer metastasis/progression3 and rheumatoid arthritis (RA).4
First, CXCR4 was identified as a co-receptor that is used in the
entry of T cell line-tropic (X4-) HIV-1 into T cells.2 Second, it
is found that the CXCL12/CXCR4 system is involved in the
metastasis of several types of cancers, including breast cancer,
pancreatic cancer, melanoma, prostate cancer, kidney cancer,
neuroblastoma, non-Hodgkin’s lymphoma, lung cancer, ovarian
cancer, multiple myeloma, chronic lymphocytic leukemia, acute
lymphoblastic leukemia and malignant brain tumor,3 and that
this system might determine the metastatic destination of tumor
cells. For instance, Mu¨ller et al. reported that CXCR4 is highly
expressed in human breast cancer cells, while CXCL12 is highly
expressed in lymph nodes, bone marrow, lung and liver, which
represent the primary metastatic destinations of breast cancer,
and that breast cancer metastasis can be significantly inhibited
by neutralization using anti-CXCR4 antibodies in mice.3a Third,
aInstitute of Biomaterials and Bioengineering, Tokyo Medical and Dental
University, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail: tamamura.mr@
tmd.ac.jp; Fax: 81 3 5280 8039; Tel: 81 3 5280 8036
bGraduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku,
Kyoto 606-8501, Japan. E-mail: nfujii@pharm.kyoto-u.ac.jp; Fax: 81 75
753 4570; Tel: 81 75 753 4551
cMedical College of Georgia Augusta, GA 30912, USA
dJames Graham Brown Cancer Center, University of Louisville, Louisville,
KY 40202, USA
eSt. Marianna University, School of Medicine, Miyamae-ku, Kawasaki 216-
8511, Japan
f
AIDS Research Center, National Institute of Infectious Diseases, Shinjuku-
ku, Tokyo 162-8640, Japan
gGraduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo
113-8519, Japan
Fig. 1 Development of bio-stable CXCR4 antagonists, 4F-benzoyl-
TN14003 and 4F-benzoyl-TE14011, and a downsized antagonist, FC131.
Nal = L-3-(2-naphthyl)alanine, Cit = L-citrulline.
† Electronic supplementary information (ESI) available: experimental and
characterization data (MS) of novel synthetic compounds. See DOI:
10.1039/b603818b
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the above four critical residues (Arg × 2, Nal and Tyr) that were
electron-deficient aromatic ring, several compounds were designed
and synthesized.
used in the development of FC131, in addition to the above
Biological results and discussion
Biological activities of the present synthetic compounds were
evaluated by two assays: the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT) assay based on the inhibition
of X4-HIV-1 (HIV-1IIIB)-induced cytopathogenicity in MT-4 cells
by test compounds (anti-HIV activity)10 and a blocking assay
based on displacement of CXCL12 binding to CXCR4 by
test compounds (binding affinity for CXCR4).11 Initially, three
tripeptide mimetics containing amide bonds and/or reduced
amide bonds, 1–3 were designed based on the sequence of Arg1–
Arg2–Nal3 in the N-terminal region of T140 (Fig. 1 and 2)
and synthesized using solution-phase techniques involving amide
bond-forming condensation and reductive amination reactions.
In this study, (S)-(−)-1-(1-naphthyl)ethylamide, which was used
in another CXCR4 antagonist KRH-1636,12 was introduced with
the view to enhancement of biostability. Compounds 2 and 3
showed significant anti-HIV activity, while compound 1 did not
exhibit activity until the 100 lM concentration, suggesting that
a reduced amide bond possessing the conformational flexibility
might be more suitable for the interaction of CXCR4 (Table 1).
Fig.
2 Development of tri- and tetrapeptide mimetics with
CXCR4-antagonistic activity.
Fig. 3 Design of tripeptide library containing three pharmacophores of the N-terminal region of 4F-benzoyl-TN14003 and 4F-benzoyl-TE14011
(aromatic ring, Arg2 and Nal3) and the development of new leads.
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Table 1 Cytotoxicity, anti-HIV activity and inhibitory activity against
lead compounds for anti-HIV agents, 11 and 14, although these
compounds did not show significant binding affinity for CXCR4
until the 1 lM concentration. Compound 8, which contains
Arg1 based on the original 4F-benzoyl-TN14003 and 4F-benzoyl-
TE14011 sequence, also exhibited moderate anti-HIV activity and
significant CXCR4-binding affinity. These results suggest that
Arg1 can be replaced by conformationally restricted units in terms
of anti-HIV activity. The other compounds that were contained
in library 1 did not show significant anti-HIV activity until the
100 lM concentration.
Next, in due consideration of an increase in biostability, focused
library of compounds with the C-terminal substituted amide was
constructed based on the structures of compounds 8, 11 and 14 by
solid-phase techniques using Kenner’s sulfonamide safety-catch
linker14 (Fig. 3, library 2): C-terminal Nal-amide of compounds
8, 11 and 14 was replaced by several amides possessing various
naphthalene units. The synthetic scheme for compound 39 is
shown as a representative in Scheme 1. Compounds 30 and
39 showed moderate and strong anti-HIV activity, respectively,
although each compound did not show significant CXCR4-
binding affinity until the 1 lM concentration. Anti-HIV potency
of compounds is not always in proportion to binding affinity
for CXCR4, especially in case of these small compounds, since
there is a significant difference between the interactive site of
HIV and the binding site of CXCL12 on CXCR4. There is
a great interest in this result: compound 39, possessing (R)-
(+)-1-(1-naphthyl)ethylamide in the C-terminus, is stronger than
compound 38, possessing (S)-(−)-1-(1-naphthyl)ethylamide in the
CXCL12 binding to CXCR4 of the synthetic compounds
Compound
CC50/lMa
EC50/lMb
IC50/lMc
1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
66
>100
52
46
22
26
11
1.7
45
7.7
0.32–1
0.32–1
0.32–1
0.090
0.30
0.32–1
>1
0.30
>1
>1
>1
2
3
4
5
6
7
8
11
14
6.0
61
7.4
30
39
>1
FC131
T140
AZT
>100
>10
>100
0.073
0.026
0.014
0.0032
0.0045
a CC50 values are based on the reduction of the viability of mock-infected
MT-4 cells. Since the cytotoxicity of T140 was previously evaluated as
CC50 > 40 lM, further estimation at high concentrations was omitted
in this study. b EC50 values are based on the inhibition of HIV-induced
cytopathogenicity in MT-4 cells. c IC50 values are based on the inhibition
of [125I]-CXCL12 binding to CXCR4 transfectants of CHO cells. All data
are the mean values for at least three independent experiments.
Thus, we synthesized two tetrapeptide mimetics, 4 and 5, where
a Tyr residue was added in the N-terminus of compounds 2 and
3, respectively, based on the sequence of the FC131 sequence.
Compounds 4 and 5 showed approximately twice stronger anti-
HIV activity than compounds 2 and 3, indicating that an N-
terminal addition of a Tyr residue is effective for an increase in anti-
HIV activity. Furthermore, compounds 4 and 5 exhibited stronger
binding affinity for CXCR4, compared to compounds 1–3. Next,
we synthesized p-fluorobenzoylated tripeptide mimetics, 6 and 7,
based on the N-terminal sequence of 4F-benzoyl-TN14003 and
4F-benzoyl-TE14011. As a result, p-fluorobenzoylation caused an
increase in anti-HIV activity. Compound 7 showed strong anti-
HIV activity, suggesting that introduction of a reduced amide
bond between two Arg residues is more suitable than that between
Arg and naphthalenylethylamine. However, binding affinity of
compound 7 for CXCR4 could not be exhibited until the 1 lM
concentration, and compound 6 is weaker than compounds 4 and 5
in terms of binding affinity for CXCR4, although anti-HIV activity
of compounds 6 and 7 is stronger than that of compounds 4 and
5. This discrepancy might be caused by the difference between
the interactive site of HIV and the binding site of CXCL12 on
CXCR4.13
Since hit compounds with significant anti-HIV activity were
found among several compounds that were synthesized using
solution-phase techniques, we attempted to prepare more com-
pounds by solid-phase synthesis: A tripeptide library containing
three pharmacophores of the N-terminal region of 4F-benzoyl-
TN14003 and 4F-benzoyl-TE14011 (aromatic ring, Arg2 and
Nal3) and the C-terminal carboxy amide was designed (Fig. 3).
Since Arg1 is not an indispensable residue for high activity,
it was replaced by several spacers involving conformationally
constrained units, such as 4-piperidinecarboxylic acid and 4-
(aminomethyl)benzoic acid. Use of this library involving 20
synthetic compounds, which was constructed by solid-phase
peptide synthesis (Fig. 3, library 1), led to the discovery of
Scheme 1 Reagents: (i) Fmoc-Arg(Pbf)–OH, DIPEA, PyBOP, CHCl3;
(ii) 20% (v/v) piperidine–DMF; (iii) Fmoc-(4-aminomethyl)benzoic acid,
DIPCDI, HOBt, DMF; (iv) 20% (v/v) piperidine–DMF; (v) 4-trifluoro-
methylbenzoic acid, DIPCDI, HOBt, DMF; (vi) TMSCHN2, hex-
ane, THF; (vii) (R)-(+)-1-(1-naphthyl)ethylamine, DMF, reflux; (viii)
thioanisole, TFA; Pbf = 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-
sulfonyl, DIPEA = N,N-diisopropylethylamine, PyBOP = benzotriazole-
1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, DIPCDI =
N,N-diisopropylcarbodiimide, HOBt = N-hydroxybenzotriazole.
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C-terminus, which is a common structure unit with KRH-1636.
Compound 39 is thought to be a useful lead possessing chemically
modified N- and C-terminal ends. The other 10 compounds that
were contained in library 2 did not show significant anti-HIV
activity until the 100 lM concentration.
In summary, several compounds that were synthesized based
on pharmacophores of T140 analogs showed significant anti-
HIV activity and binding affinity for CXCR4. According to these
results, two types of libraries based on the N-terminal region of 4F-
benzoyl-TN14003 and 4F-benzoyl-TE14011 were constructed to
find effective lead compounds. Linear-type low molecular weight
compounds obtained in this study are thought to be useful leads
for chemotherapy of AIDS, cancer and RA.
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Acknowledgements
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This work was supported in part by a 21st Century COE
Program “Knowledge Information Infrastructure for Genome
Science”, a Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology, Japan,
the Japan Health Science Foundation, The Mochida Memorial
Foundation for Medical and Pharmaceutical Research and Philip
Morris USA Inc. and Philip Morris International.
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