Pharmacophore-based small molecule CXCR4 ligands

Article abstract of DOI:10.1016/j.bmcl.2012.04.032

Low molecular weight CXCR4 ligands were developed based on the peptide T140, which has previously been identified as a potent CXCR4 antagonist. Some compounds with naphthyl, fluorobenzyl and pyridyl moieties as pharmacophore groups in the molecule showed significant CXCR4-binding activity and anti-HIV activity. Structure-activity relationships were studied and characteristics of each of these three moieties necessary for CXCR4 binding were defined. In this way, CXCR4 ligands with two types of recognition modes for CXCR4 have been found.

Full text of DOI:10.1016/j.bmcl.2012.04.032

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4169–4172
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pharmacophore-based small molecule CXCR4 ligands
Tetsuo Narumi a, Tomohiro Tanaka a, Chie Hashimoto a, Wataru Nomura a, Haruo Aikawa a, Akira Sohma a,
Kyoko Itotani a, Miyako Kawamata b, Tsutomu Murakami b, Naoki Yamamoto c, Hirokazu Tamamura a,
⇑
a Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan
b AIDS Research Center, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan
c Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
Low molecular weight CXCR4 ligands were developed based on the peptide T140, which has previously
been identified as a potent CXCR4 antagonist. Some compounds with naphthyl, fluorobenzyl and pyridyl
moieties as pharmacophore groups in the molecule showed significant CXCR4-binding activity and
anti-HIV activity. Structure–activity relationships were studied and characteristics of each of these three
moieties necessary for CXCR4 binding were defined. In this way, CXCR4 ligands with two types of
recognition modes for CXCR4 have been found.
Received 16 March 2012
Revised 4 April 2012
Accepted 7 April 2012
Available online 20 April 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
HIV entry inhibitors
Chemokine receptor
AIDS
Low molecular weight CXCR4 ligands
The chemokine receptor CXCR4 is classified into a family of G
protein-coupled receptors (GPCRs), and transduces signals of its
endogenous ligand, CXCL12/stromal cell-derived factor-1 (SDF-
1).1 The CXCR4-CXCL12 axis plays a physiological role in chemo-
taxis,2 angiogenesis3 and neurogenesis4 in embryonic stages. The
CXCR4 receptor is linked to many disorders including HIV infec-
tion/AIDS,5 metastasis of cancer cells,6 leukemia cell progression,7
rheumatoid arthritis.8 Since CXCR4 is an important drug target in
these diseases, it is thought that effective agents directed to this
receptor may be useful leads for therapeutic agents. To date, we
and others have developed several potent CXCR4 antagonists. A
highly potent antagonist, T140, a 14-mer peptide with a disulfide
bridge, and its downsized analogue, FC131, with a cyclic pentapep-
tide scaffold, and several other related compounds have been re-
ported.9 Based on T140 and FC131, small-sized linear anti-HIV
agents such as ST34 (1) have been developed (Fig. 1).10
AMD3100,11 KRH-1636,12 Dpa–Zn complex (2)13 and other azamac-
rocyclic compounds such as 3,14 which like 1, contain benzylamine
and electron-deficient aromatic groups, have also been reported as
nonpeptidic antagonists. Compound 1 possesses significant anti-
HIV activity but does not have high CXCR4 binding affinity. In the
present study, more effective linear CXCR4 antagonists derived
from compound 1 have been examined, and structure–activity rela-
tionship studies of these compounds have been performed.
Initially, three segments of compound 1 were selected for
structural modification to support the design of new synthetic
compounds: replacement of the 4-trifluoromethylbenzoyl group
(Fig. 2, R1), modification of the stereochemistry of the 1-naphthyl-
ethylamine moiety (R2) and introduction of pyridine moieties on
the nitrogen atom (R3). In a previous study of T140 analogues, 4-flu-
orobenzoyl was found to be superior to 4-trifluoromethylbenzoyl as
an N-terminal moiety. Thus, 4-fluorobenzyl, 4-fluorobenzoyl and
4-fluorophenylethyl groups were used as substitutes for the 4-tri-
fluoromethylbenzoyl group (R1) in 1. The (R)-1-naphthylethylamine
moiety in 1 is also present in KRH-1636 where it has the (S)-stereo-
chemistry and thus both the (R) and (S)-stereoisomers were investi-
gated in the present study. Several CXCR4 antagonists such as KRH-
1636,12 Dpa-Zn complex (2)13 and Dpa-cyclam compound (3),14
contain pyridyl rings. Thus, 2, 3, or 4-pyridylmethyl and 2, 3, or
4-pyridylethyl groups were introduced on the nitrogen atom of the
4-aminomethylbenzoyl group (R3). With these modifications, a total
of 3 Â 2 Â 6 = 36 compounds (12–47) were designed (Fig. 2).
The synthesis of the structural fragment, Unit 1 is shown in
Scheme 1. N-nosylation of 4-amino-methylbenzoic acid (4) with
2-nitrobenzenesulfonyl chloride and subsequent esterification
gave the t-butyl ester 5. Introduction of an R3 moiety by means
of a Mitsunobu reaction followed by removal of the Ns group
yielded amines 6A–F. Introduction of either 4-fluorobenzyl or
4-fluorophenylethyl groups by reductive amination of 6A–F
produced amines 7Ai–Fi or 7Aiii–Fiii, respectively. Conversion of
6A–F to the appropriate amide (7Aii-Fii), and subsequent depro-
tection of the tert-butyl group yielded Unit 1, 8Ai–Fiii.
⇑
Corresponding author.
E-mail address: tamamura.mr@tmd.ac.jp (H. Tamamura).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.032

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T. Narumi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4169–4172
O
O
O
H
N
H
N
H2N
j
i
Boc
OH
NH
Boc
N
H
N
H
NH
NH
Mts
Mts
Mts
HN
N
HN
N
NH
N
H
H
H
9
(R)-10; (S)-10
(R)-11; (S)-11
Scheme 2. Synthetic schemes of Unit 2, compounds (R/S)-11. Reagents and condi-
tions: (i) EDCIÁHCl, HOBtÁH2O, Et3N, (R/S)-(+/À)-1-(1-naphthyl)ethylamine, CH2Cl2,
83–97%; (j) TFA then 4 M HCl/EtOAc, quantitative; EDCIÁHCl = 1-ethyl-3-(3-dimeth-
ylaminopropyl)carbodiimide hydrochloride, HOBtÁH2O = 1-hydroxybenzotriazol
monohydrate, Mts = 2,4,6-trimethylphenylsulfonyl, Boc = tert-butoxycarbonyl.
R1
O
Figure 1. The structures of
compound (3).
1 (ST34), Dpa-Zn complex (2) and Dpa-cyclam
R1
k
N
O
H2N
H
N
N
R3
N
H
R3
OH
N
H
O
O
NH
8Ai-Fiii
NH
Mts
R4
HN
N
H
HN
N
H
11 11
(R)- ; (S)-
R
R
4 = Mts
4 = H
L1-L36
l
Scheme 3. Synthetic schemes of compounds 12–47. Reagents and conditions: (k)
EDCIÁHCl, HOBtÁH2O, Et3N, DMF, 36-95%; (l) TMSBr, m-cresol, 1,2-ethanedithiol,
thioanisole, TFA, 4–54%. The structures of R1 and R3 are shown in Figure 2 as i–iii
and A–F, respectively.
The CXCR4-binding activity of synthetic compounds was as-
sessed in terms of the inhibition of [125I]-CXCL12 binding to Jurkat
cells, which express CXCR4.16 The percent inhibition of all the com-
pounds at 10 lM is shown in Table 1. Several of the compounds
showed significant binding affinity. In general, compounds in which
the 1-naphthylethylamine moiety (R2) has the (S)-stereochemistry,
as in KRH-1636, are more potent than the (R)-stereoisomers. Ten
compounds (26–28, 30, 33, 36, 39, 44, 45 and 47, Table 1) were found
to induce at least 30% inhibition and compounds 26, 27 and 33,
which have a pyridyl group with a nitrogen atom at the b-position,
showed more than 60% inhibition. It is noteworthy that compounds
26 and 27 in which R2 is a (R)-1-naphthylethylamine moiety, are
both more potent than the corresponding (S)-stereoisomers 44
and 45. Compounds 26, 27 and 33, have a 4-fluorobenzyl or 4-fluor-
ophenylethyl group, which rather than an amide, is a reductive alkyl
type (R1). As can be seen from Table 1, there is a tendency for com-
pounds with a pyridyl group with a nitrogen atom at the b-position
(R3: C or D), to be more potent in terms of CXCR4-binding activity
than the corresponding compounds, which have a pyridyl group
Figure 2. The structures of substituents for three parts of compound 1 in the design
of new compounds.
H2N
HN
Ns
a, b
c, d
HN
R3
OtBu
OH
OtBu
O
O
6A-F
O
4
5
R1
R1
e, f or g
N
h
N
R3
OtBu
R3
OH
O
O
7Ai-Fiii
8Ai-Fiii
with a nitrogen atom at the a- or c
- position (R3: A, B, E or F), and
those with a reductive alkyl 4-fluorobenzyl or 4-fluorophenylethyl
group (R1: i or iii), to be more potent in CXCR4-binding activity than
the corresponding compounds, with a 4-fluorobenzoyl group (R1: ii).
Compounds were next evaluated for anti-HIV activity and
cytotoxicity. CXCR4 is the major co-receptor for the entry of T-cell
line-tropic (X4-) HIV-1.5 Accordingly, inhibitory activity against
X4-HIV-1 (NL4-3 strain)-induced cytopathogenicity in MT-4 cells
(anti-HIV activity), and reduction of the viability in MT-4 cells
(cytotoxicity) were assessed16 and are shown in Table 1. Com-
pounds 26 and 33–35 showed significant anti-HIV activity with
EC50 values in the micromolar range. Compounds 26 and 33
showed both potent CXCR4-binding activity (79% and 60% inhibi-
Scheme 1. The synthetic scheme of Unit 1, compounds 8Ai–Fiii. Reagents and
conditions and yields: (a) NsCl, Et3N, THF/H2O (1/1); (b) isobutene, THF/H2SO4 (10/
1), 39% (2 steps); (c) PPh3, DEAD, R3OH, THF; (d) PhSH, K2CO3, DMF, 42–92% (2
steps); (e) NaBH(OAc)3, 4-fluorobenzaldehyde, CH2Cl2; (f) NaBH(OAc)3, (4-fluoro-
phenyl)acetaldehyde, CH2Cl2; g) 4-fluorobenzoyl chloride, Et3N, CH2Cl2, 51-94%; (h)
TFA then 4 M HCl/EtOAc, quantitative; The structures of R1 and R3 are shown in
Fig. 2 as i–iii and A–F, respectively. Ns = 2-nitrobenzenesulfonyl, tBu = tert-butyl,
DEAD = diethyl azodicarboxylate.
The synthesis of Unit 2 is shown in Scheme 2. Condensation of
Boc-Arg(Mts)-OH (9) and (R)-1-naphthylethylamine or its (S) iso-
mer produced amides (R)-10 or (S)-10. Removal of the Boc group
gave Unit 2, (R)-11 and (S)-11.
Compounds 12–47 were synthesized by amide condensation of
Unit 1, 8Ai–Fiii, with Unit 2, (R)-11 and (S)-11, and subsequent
deprotection of the Mts group, as shown in Scheme 3.15 All the syn-
thetic compounds were purified by preparative reverse phase
HPLC. In cases where peaks derived from side products appeared
around the target peaks on the HPLC profile, the precise analysis
was accomplished, giving rise to lower yields (Scheme 3, l).
tion at 10
13 M, respectively), the two activities being highly correlated.
Compounds 34 and 35 have significant anti-HIV activity with
EC50 values of 8 and 10 M, respectively, which is higher than
CXCR4-binding activities, which are 16% and 20% inhibition at
10 M, respectively. Compound 27, which showed relatively high
CXCR4-binding activity (69% inhibition at 10 M), failed to show
lM, respectively) and anti-HIV activity (EC50 = 11 and
l
l
l
l

T. Narumi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4169–4172
4171
M)
Table 1
CXCR4-binding activity, anti-HIV activity and cytotoxicity of compounds 12–47
a
b
c
e
f
a
b
c
e
f
Compd no.
R1
R2
R3
Inhibitiond (%)
EC50
(
lM)
CC50
(lM)
Compd no.
R1
R2
R3
Inhibitiond (%)
EC50
(lM)
CC50
(l
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
i
i
i
i
i
i
ii
ii
ii
ii
ii
ii
iii
iii
iii
iii
iii
iii
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
A
B
C
D
E
F
A
B
C
D
E
F
A
B
C
D
E
F
0
1.7
0.7
>20
>4
35
23
37
n.d.
39
n.d.
38
41
45
n.d.
45
n.d.
n.d.
27
47
11
n.d.
n.d.
80
55
100
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
i
i
i
i
i
i
ii
ii
ii
ii
ii
ii
iii
iii
iii
iii
iii
iii
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
A
B
C
D
E
F
A
B
C
D
E
F
A
B
C
D
E
F
30 1.1
25 3.3
27 1.7
60 1.5
16 1.2
20 1.3
36 1.8
0
14 1.4
32 8.4
13 15
25 13
16 5.1
23 14
36 13
35 5.2
26 23
51 6.6
100
>4
11
24
41
65
44
44
37
43
57
n.d.
51
47
9.9
13
n.d.
n.d.
n.d.
n.d.
>10
66
4
6
>20
>20
13
8
10
>20
>20
>20
n.d.
>20
>20
>4
>20
n.d.
>20
n.d.
>20
>20
>20
n.d.
>20
n.d.
n.d.
>20
11
>11
n.d.
n.d.
0.33
0.062
0.058
24 1.7
12 3.0
16 2.2
0.9
3.9
11 0.8
22 4.1
3
6
6
2.7
12 1.9
15 2.1
13 0.6
79 14
69 5.0
44 5.4
0
>4
n.d.
n.d.
n.d.
n.d.
0.16
7.4
28
29
46
47
FC131
1 (ST34)
KRH-1636
AMD3100
AZT
100
n.d.
n.d.
n.d.
a,c
The structures of R1 and R3 are shown in Fig. 2 as i–iii and A–F, respectively.
b
The absolute configuration in stereochemistry of R2 shown in Fig. 2 is described.
d
CXCR4-binding activity was assessed based on the inhibition of the [125I]-CXCL12 binding to Jurkat cells. Inhibition percentages of all the compounds at 10
lM were
calculated relative to the inhibition percentage by T140 (100%).
e
EC50 values are the concentrations for 50% protection from X4-HIV-1 (NL4-3 strain)-induced cytopathogenicity in MT-4 cells.
CC50 values are the concentrations for 50% reduction of the viability of MT-4 cells. All data are the mean values from at least three independent experiments.
f
significant anti-HIV activity at concentrations below 11
cause of high cytotoxicity (CC50 = 11 M). With the exception of
27, 30, 42 and 43, the tested compounds showed no significant
cytotoxicity (CC50 >20 M, Table 1). On the other hand, compounds
26, 27, 33, 34 and 35 at concentrations below 100 M failed to
l
M be-
and Technology of Japan, Japan Human Science Foundation, and
Health and Labour Sciences Research Grants from Japanese Minis-
try of Health, Labor, and Welfare. T.T. and C.H. are grateful for the
JSPS Research Fellowships for Young Scientists.
l
l
l
show significant protective activity against macrophage-tropic
(R5-) HIV-1 (NL(AD8) strain)-induced cytopathogenicity in
PM-1/CCR5, whereas the EC50 of the CCR5 antagonist SCH-D17 in
References and notes
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c-position, in
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binding activity, but have a pyridyl group with a nitrogen atom
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and 33 compete with HIV-1 and CXCL12. Compound 27 does not
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Acknowledgements
This work was supported by Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science,

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T. Narumi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4169–4172
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(S)-11 (320 mg, 0.657 mmol, HCl salt) at 0 °C. The mixture was stirred at room
temperature for 43 h. The reaction mixture was diluted with CHCl3 and washed
with saturated citric acid, saturated NaHCO3 and brine, and dried over
MgSO4. Concentration under reduced pressure followed by flash column
chromatography over silica gel with CHCl3/MeOH (20/1) gave the
condensation product (175 mg, 0.208 mmol, 50% yield) as white powder. To
this compound were added m-cresol (75.0
(225 L, 2.68 mmol), thioanisole (225
bromotrimethylsilane (495 L, 3.82 mmol) with stirring at 0 °C, and the stirring
l
L, 0.714 mmol), 1,2-ethanedithiol
l
lL, 1.91 mmol), TFA (3 mL) and
l
was continued at room temperature for 3.5 h under N2. The reaction mixture
was concentrated under reduced pressure, followed by addition of Et2O to
precipitate the product. After washing with Et2O, the crude product was purified
by preparative HPLC and lyophilized to give the compound 30 (15.6 mg,
0.0236 mmol, 13%) as white powder. 1H NMR dH (400 MHz; DMSO-d6) 1.49 (m,
2H), 1.51 (d, J = 7.2 Hz, 3H), 1.80–1.62 (m, 2H), 3.07 (dd, J = 6.4, 12.8 Hz, 2H), 3.85
(s, 2H), 3.91 (s, 4H), 4.54 (m, 1H), 5.72 (m, 1H), 7.13 (t, J = 8.8 Hz, 2H), 7.40 (m,
1H), 7.60–7.45 (m, 10H), 7.75–7.95 (m, 5H), 8.10 (m, 1H), 8.40 (d, J = 8.0 Hz, 1H),
8.58 (m, 1H), 8.65 (d, J = 7.6 Hz, 1H); LRMS (ESI), m/z calcd for C39H42FN7O2
(MH)+ 660.34, found 660.31.
11. Schols, D.; Struyf, S.; Van Damme, J.; Este, J. A.; Henson, G.; DeClarcq, E. J. Exp.
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12. Ichiyama, K.; Yokoyama-Kumakura, S.; Tanaka, Y.; Tanaka, R.; Hirose, K.;
Bannai, K.; Edamatsu, T.; Yanaka, M.; Niitani, Y.; Miyano-Kurosaki, N.; Takaku,
H.; Koyanagi, Y.; Yamamoto, N. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4185.
13. Tamamura, H.; Ojida, A.; Ogawa, T.; Tsutsumi, H.; Masuno, H.; Nakashima, H.;
Yamamoto, N.; Hamachi, I.; Fujii, N. J. Med. Chem. 2006, 49, 3412.
14. Tanaka, T.; Narumi, T.; Ozaki, T.; Sohma, A.; Ohashi, N.; Hashimoto, C.; Itotani,
K.; Nomura, W.; Murakami, T.; Yamamoto, N.; Tamamura, H. ChemMedChem
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15. For example, the synthesis of compound 30: To a stirred solution of 8Ai (176 mg,
0.415 mmol, HCl salt) in DMF (4 mL) were added EDCIÁHCl (104 mg,