Synthesis and biological evaluation of peptide mimics derived from first extracellular loop of CCR5 toward HIV-1

Article abstract of DOI:10.1248/cpb.52.1479

Peptide mimics derived from the first extracellular loop of CCR5 bearing non-peptide spacers in place of Ala-Ala-Ala sequence in the peptide moiety were synthesized, and the effects of these compounds on the inhibition against HIV-1 were examined. Compoun

Full text of DOI:10.1248/cpb.52.1479

December 2004
Notes
Chem. Pharm. Bull. 52(12) 1479—1482 (2004)
1479
Synthesis and Biological Evaluation of Peptide Mimics Derived from First
Extracellular Loop of CCR5 toward HIV-1
*
Kiyoshi IKEDA, Yuko ISHII, Koji KONISHI, Masayuki SATO, and Kiyoshi TANAKA
School of Pharmaceutical Sciences, University of Shizuoka; 52–1 Yada, Shizuoka 422–8526, Japan.
Received June 28, 2004; accepted August 23, 2004
Peptide mimics derived from the first extracellular loop of CCR5 bearing non-peptide spacers in place of
Ala-Ala-Ala sequence in the peptide moiety were synthesized, and the effects of these compounds on the inhibi-
tion against HIV-1 were examined. Compound 2b having m-aminophenoxyacetic acid derivative as a non-peptide
spacer significantly inhibited HIV-1.
Key words HIV-1 inhibitor; first extracellular loop; CCR5 peptide mimic; chemokine receptor; non-peptide spacer
The chemokine receptor CCR5, a member of the seven- Ala-Ala sequence in the peptide moiety for prevention of
transmembrane G-protein coupled family of receptors,1) has HIV-1 infection based on a strategy of binding to gp120.
been identified as a primary co-receptor with CD4 by which
macrophage tropic human immunodeficiency virus type-1 Results and Discussion
(HIV-1) strains infect their host cells.2) Since the discovery of
In the amino acid sequence of the N-terminal domain of
CCR5 as a co-receptor with CD4 for HIV-1 cell entry, there CCR5, we chose the region of Tyr89-Gly97 including alanine
has been interest in discovering small molecule CCR5 antag- tripeptide. The peptide of natural type peptide 1 and its mim-
onists3—6) as well as CXCR4 antagonists7) and HIV-1 pro- icking peptides 2a—c consisting of unnatural-type amino
tease inhibitors8) as potential agents for the treatment of HIV- acids derived from aminophenol as the spacer unit were pre-
1 infection.
pared. First, the nona-peptide 1 was synthesized manually by
The entry of HIV-1 to cells involves the binding of the a stepwise liquid-phase procedure. The synthesis of 2a—c is
trimeric viral envelope glycoprotein gp120/gp41 to cell sur- shown in Chart 2. Aminophenoxyacetates 3a—c as spacers
face receptor CD4 and chemokine co-receptor CXCR4 or were prepared from the corresponding nitrophenols and tert-
CCR5, which triggers conformational changes in the enve- butyl bromoacetate.19,20) Coupling of compounds 3a—c with
lope proteins. Gp120 then dissociates from gp41, allowing 9-fluorenylmethoxycarbonyl (Fmoc)-Tyr(Bn) in the pres-
for the fusion peptides to be inserted into the target mem- ence of 1-benzotriazoyloxy-tris-dimethylaminophosphonium
brane and the pre-hairpin configuration of the ectodomain to hexafluorophosphate (BOP) and 1-hydroxy-1H-benzotriazole
form.9—11) Regulation of CCR5 number on cells is important (HOBt) in N,N-dimethylformamide (DMF) gave compounds
in determining the infection rate by HIV-1. Therefore, CCR5 4a—c in 72%, 73% and 86% yield, respectively. The depro-
is a highly valued target for the treatment of HIV-1 infection. tection of tert-butyl group of 4a—c with trifluoroacetic acid
It was demonstrated that the Gly-Gly-Gly sequence of the (TFA) afforded compounds 5a, 5b, and 5c in 71%, quant.
peptide moiety in lipopeptides could be replaced with a non- and 93% yield, respectively. Compounds 5a—c were coupled
peptide spacer.12) We designed compounds with non-peptide with penta-peptide 6 in the presence of 1-(3-dimethylamino-
spacers such as a benzene ring in place of the Ala-Ala-Ala propyl)-3-ethylcarbodiimide hydrochloride (EDCl) and
sequence of the peptide moiety of CCR5. As part of a HOBt in DMF to give compounds 7a—c in 78%, 81% and
program aimed at the development of new HIV-1 inhibit- 95% yield, respectively. Finally, hydrogenolysis of com-
ors,13—18) we would like to report the design and synthesis of pounds 7a—c catalyzed by 20% Pd(OH)2 in EtOH–AcOH
CCR5 peptide mimics derived from the first extracellular (6 : 1) at 45 °C, followed by treatment with TFA–
loop of CCR5 bearing non-peptide spacers in place of Ala- CH2Cl2–anisole–ethanedithiol (43 : 50 : 2 : 5), then 20% piper-
Chart 1. Amino Acid Sequence of First Extracellular Loop of CCR5
∗ To whom correspondence should be addressed. e-mail: ikeda@u-shizuoka-ken.ac.jp
© 2004 Pharmaceutical Society of Japan

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Vol. 52, No. 12
Chart 2. Synthesis of Peptide Mimics Derived from First Extracellular Loop of CCR5 toward HIV-1
Fig. 1. Mechanism of HIV-1 Fusion with Target Cell Membrane
idine-DMF afforded target compounds 2a—c in 75%, 67%
and 67% yields, respectively.
Table 1. Anti-HIV Activities of Peptide Mimics (2a—c) Derived from
First Extracellular Loop of CCR5a)
Drug concentration (mg/ml)b)
Cytotoxicity
(1000 mg/ml)
Biological Activity
Compd.
The anti-HIV-1 activities of 1 and 2a—c are shown in
Table 1. The anti-HIV-1 activity was tested by the syncytium-
formation assay method using MT-4 cells according to our
previously reported method.8) Among the synthesized com-
pounds, interestingly, 2b as m-aminophenoxyacetic acid type
showed the most potent inhibitory activity against HIV-1 and
had cytotoxicity at 1000 mg/ml. This finding is in distinct
contrast to the result that peptide 1 derived from the native
sequence of CCR5 had little antiviral activity against HIV-1.
However, 2a had a moderate activity and 2c was practically
inactive against HIV-1. The reason compound 2b showed
higher inhibitory activity is unclear in this study, but there is
the possibility that the configuration of aminophenoxyacetic
acid derivatives as a non-peptide spacer affects the expres-
sion of antiviral activity against HIV-1.
1000
100
10
1
ꢁꢂꢃ
ꢁ
n.d.c)
ꢁꢂꢃ
ꢃ
ꢁꢂꢃ
ꢁꢁ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢁꢂꢃ
ꢁꢁ
ꢃ
2a
2b
2c
a) C8166/GUN1WT (dual cell-tropic HIV strain) syncytium assay; b) inhibitory ac-
tivity: ꢃ, 0%; ꢁ, ꢄ10%; ꢁꢁ, 10—50%; c) not determined.
Experimental
All melting points are uncorrected. Optical rotations were measured with
a JASCO P-1030 polarimeter. IR spectra were recorded on a JASCO IR-810
spectrometer. 1H-NMR spectra were recorded with a JEOL JNM-EX 270
(270 MHz) spectrometer. 1H chemical shifts are given in ppm relative to
Me4Si (dꢀ0) in CDCl3 or CD3OD as internal standards at ambient tempera-
ture. Fast atom bombardment (FAB) mass spectra were obtained with a
JEOL JMS SX-102 mass spectrometer in the positive ion mode using NBA
or glycerol-thioglycerol matrix. Column chromatography was performed on
silica gel 60 (70—230 mesh, Merck) and Sephadex LH-20 (Pharmacia).
Precoated thin-layer chromatography (TLC) plates (silica gel 60-F254
(Merck)) were used to monitor the reaction and ascertain the purity of the
reaction products. The spots were detected by spraying the plates with 5%
In conclusion, it was demonstrated that the Ala-Ala-Ala
sequence of the peptide moiety could be replaced with a non-
peptide spacer. Synthesized peptide analogues are attractive
candidates as new lead compounds for the development of
chemokine receptor-directed anti-HIV-1 drugs.

December 2004
1481
aqueous sulfuric acid and 10% 12-molybdo(VI) phosphoric acid n-hydrate gel column chromatography (CH2Cl2–MeOH 10 : 1) to give 6 (0.70 g, 88%)
in EtOH solution, and then heating.
as powder. Positive FAB-MS m/z: 1074 MHꢁ.
L-Tyrosinyl-L-alanyl-L-alanyl-L-alanyl-L-glutaminyl-L-tryptophanyl-L-
aspartyl-L-phenylalanyl-glycine (1) A solution of Fmoc-Tyr(Bn)-Ala-
Ala-Ala-Gln(Trt)-Trp-Asp(Bn)-Phe-Gly-OBn (0.025 g, 0.014 mmol) in
EtOH–AcOH (6 : 1) (2.0 ml) was hydrogenated over 20% Pd(OH)2–C
(0.030 g) for 20 h at 45 °C, then filtered and concentrated. The residue was
treated with TFA–CH2Cl2–anisole–ethanedithiol (43 : 50 : 2 : 5) (1.5 ml) at
[Na-2-(Na-9-Fluorenylmethoxycarbonyl-O-benzyl-L-tyrosinamido]
phenoxy]acetyl-L-alanyl-L-alanyl-L-alanyl-Nw-trityl-L-glutaminyl-L-
tryptophanyl-L-aspartyl (b-benzyl ester)-L-phenylalanyl-glycine Benzyl
Ester (7a) To a solution of 5a (0.116 g, 0.18 mmol), peptide 6 (0.197 g,
0.18 mol) and HOBt (0.031 g, 0.20 mmol in DMF (2.0 ml) was added EDCl
(0.038 g, 0.20 mmol) at 0 °C and the mixture was stirred for 15 h at room
room temperature for 20 h to give nona-peptide derivative which was dis- temperature. The reaction mixture was washed with saturated aqueous
solved in 20% piperidine-DMF (1.0 ml) for 18 h. After evaporation of the
solvent, the residue was purified by silica gel column chromatography
(CH2Cl2–MeOH–H2O 60 : 60 : 10) and Sephadex LH-20 (CH2Cl2–MeOH–
NaHCO3, 10% aqueous citric acid and saturated aqueous brine, dried
(MgSO4) and concentrated. The residue was chromatographed on silica gel
(CH2Cl2–MeOH 10 : 1) to give 7a (0.240 g, 78%) as prisms, mp 129—
H2O 12 : 8 : 1) to give amorphous 1 (0.014 g, 97%). Positive FAB-MS m/z: 131 °C, [a]D ꢃ49° (cꢀ1.2, CHCl3). IR (KBr): 3303, 3059, 3032, 2932,
1028 MHꢁ.
tert-Butyl
1719, 1659, 1512, 745, 700 cmꢃ1. Positive FAB-MS m/z: 1699 MHꢁ.
7b (m-Form Compound) The reaction was carried out using 5b
(0.122 g, 0.19 mmol) and 6 (0.208 g, 0.19 mmol) in a manner similar to the
preparation of 7a to give 7b (0.260 g, 81%) as prisms, mp 127—129 °C,
[a]D ꢃ26° (cꢀ1.1, CHCl3). IR (KBr): 3297, 3059, 3032, 2930, 1719, 1663,
743, 700 cmꢃ1. Positive FAB-MS m/z: 1699 MHꢁ.
7c (p-Form Compound) The reaction was carried out using 5c
(0.135 g, 0.21 mmol) and 6 (0.224 g, 0.21 mmol) in a manner similar to the
preparation of 7a to give 7c (0.340 g, 95%) as prisms, mp 126—128 °C,
[2-(Na-9-Fluorenylmethoxycarbonyl-O-benzyl-L-tyrosin-
amido)phenoxy]acetate (4a) BOP (2.44 g, 4.7 mmol) was added to a solu-
tion of 3a (0.88 g, 3.9 mmol), Fmoc-L-Tyr(Bn) (1.93 g, 3.9 mmol) and HOBt
(0.63 g, 4.7 mmol) in DMF (15 ml) at 0 °C and the mixture was stirred for
1 h at 0 °C and 15 h at room temperature under an Ar atmosphere. After re-
moval of the solvent, the residue was dissolved in CH2Cl2 and washed with
saturated aqueous NaHCO3, 10% aqueous citric acid and saturated aqueous
NaCl, dried over anhydrous MgSO4 and concentrated. The residue was puri-
fied by silica gel column chromatography (CH2Cl2–acetone 10 : 1) to give 4a [a]D ꢃ42° (cꢀ2.5, CHCl3). IR (KBr): 3301, 3058, 2928, 1719, 1659, 741,
(0.92 g, 72%) as prisms, mp 65—67 °C, [a]D ꢃ8.6° (cꢀ1.2, CHCl3). IR 698 cmꢃ1. Positive FAB-MS m/z: 1699 MHꢁ.
(KBr): 3318, 2977, 1725, 1607, 758, 741, 696 cmꢃ1. H-NMR (CDCl3) d:
1.43 (s, 9H, tert-Bu), 3.01—3.17 (m, 2H, Tyr; CHCH2), 4.18—4.44 (m, 4H,
[Na-(2-L-Tyrosinamido]phenoxy]acetyl-L-alanyl-L-alanyl-L-alanyl-L-
1
glutaminyl-L-tryptophanyl-L-aspartyl-L-phenylalanyl-glycine
(2a) A
Tyr; CHCH2, Fmoc; CHCH2O), 4.47 (s, 2H, OCH2CO), 4.97 (s, 2H, Bn; solution of 7a (0.049 g, 0.029 mmol) in EtOH–AcOH (6 : 1) (3.0 ml) was hy-
CH2), 6.81—7.77 (m, 16H, aromatic-H). Positive FAB-MS m/z: 699 MHꢁ.
4b (m-Form Compound) The reaction was carried out using 3b
(0.94 g, 4.2 mmol) and Fmoc-L-Tyr(Bn) (2.07 g, 4.2 mmol) in a manner simi-
lar to the preparation of 4a to give 4b (2.25 g, 83%) as colorless prisms, mp
drogenated over 20% Pd(OH)2–C (0.070 g) for 20 h at 45 °C, then filtered
and concentrated. The residue was treated with TFA–CH2Cl2–anisole–
ethanedithiol (43 : 50 : 2 : 5) (1.5 ml) at room temperature for 20 h to give
Fmoc compound which was dissolved in 20% piperidine-DMF (1.5 ml) for
106—107 °C, [a]D ꢃ9.2° (cꢀ2.1, CHCl3). IR (KBr): 3299, 2977, 2930, 21 h. After evaporation of the solvent, the residue was purified by silica gel
1752, 1671, 1611, 843, 739, 696 cmꢃ1
tert-Bu), 3.05—3.10 (m, 2H, Tyr; CHCH2), 4.17—4.45 (m, 4H, Tyr; LH-20 (CH2Cl2–MeOH–H2O 12 : 8 : 1) to give amorphous 2a (0.021 g,
CHCH2, Fmoc; CHCH2O), 4.49 (s, 2H, OCH2CO), 5.00 (s, 2H, Bn; CH2), 75%), after lyophilization from a H2O suspension. Positive FAB-MS m/z:
.
1H-NMR (CDCl3) d: 1.48 (s, 9H, column chromatography (CH2Cl2–MeOH–H2O 60 : 60 : 10) and Sephadex
6.64—7.76 (m, 16H, aromatic-H). Positive FAB-MS m/z: 699 MHꢁ.
4c (p-Form Compound) The reaction was carried out using 3c
(0.847 g, 3.8 mmol) and Fmoc-L-Tyr(Bn) (1.88 g, 3.8 mmol) in a manner
similar to the preparation of 4a to give 4c (2.29 g, 86%) as colorless prisms,
mp 154—156 °C, [a]D ꢃ4.2° (cꢀ2.2, CHCl3). IR (KBr): 3306, 2978, 2938,
964 MHꢁ.
2b (m-Form Compound) The reaction was carried out using 7b
(0.052 g, 0.031 mmol) in a manner similar to the preparation of 2a to give
amorphous 2b (0.020 g, 67%). Positive FAB-MS m/z: 986 (M+Na)ꢁ.
2c (p-Form Compound) The reaction was carried out using 7c
(0.053 g, 0.031 mmol) in a manner similar to the preparation of 2a to give
1752, 1694, 1663, 1611, 826, 741, 696 cmꢃ1. H-NMR (CDCl3) d: 1.43 (s,
1
9H, tert-Bu), 2.93—2.99 (m, 2H, Tyr; CHCH2), 4.09—4.38 (m, 4H, Tyr; amorphous 2c (0.020 g, 67%). Positive FAB-MS m/z: 964 MHꢁ, 986
CHCH2, Fmoc; CHCH2O), 4.42 (s, 2H, OCH2CO), 4.92 (s, 2H, Bn; CH2), (MꢁNa)ꢁ.
6.76—7.69 (m, 16H, aromatic-H). Positive FAB-MS m/z: 699 MHꢁ.
[2-(Na-9-Fluorenylmethoxycarbonyl-O-benzyl-L-tyrosinamido)phe-
Acknowledgements We thank Professor H. Hoshino, Department of
Hygiene, Gunma University School of Medicine, for the biological data.
This work was partly supported by the Japan Health Science Foundation.
noxy]acetic Acid (5a)
A solution of 4a (0.87 g, 1.24 mmol) and
TFA–CH2Cl2 (1 : 3) (20 ml) was stirred for 7 h. After evaporation of the sol-
vent, the residue was chromatographed on silica gel (CH2Cl2–MeOH 10 : 1)
to give 5a (0.57 g, 71%) as prisms, mp 150—153 °C, [a]D ꢃ6.3° (cꢀ1.2, References
AcOH). IR (KBr): 3295, 3063, 3036, 1694, 1607, 741, 696 cmꢃ1. H-NMR
(CDCl3–CD3OD) d: 2.93—3.23 (m, 2H, Tyr; CHCH2), 4.15—4.37 (m, 4H,
Tyr; CHCH2, Fmoc; CHCH2O), 4.58 (s, 2H, OCH2CO), 4.95 (s, 2H, Bn;
CH2), 6.87—7.77 (m, 16H, aromatic-H). Positive FAB-MS m/z: 643 MHꢁ,
665 (MꢁNa)ꢁ.
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CH2), 6.77—7.68 (m, 16H, aromatic-H). Positive FAB-MS m/z: 643 MHꢁ,
665 (MꢁNa)ꢁ, 681 (MꢁK)ꢁ.
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Gly-OBn (0.96 g, 0.74 mmol) was dissolved in 20% piperidine-DMF (15 ml)
for 2 h. After evaporation of the solvent, the residue was purified by silica

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Vol. 52, No. 12
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