Design, synthesis and biological evaluation of low molecular weight CXCR4 ligands

Article abstract of DOI:10.1016/j.bmc.2019.02.013

The chemokine receptor CXCR4/stromal cell-derived factor-1 (SDF-1: CXCL12) signaling axis represents a crucial drug target due to its relevance to several diseases such as HIV-1 infection, cancer, leukemia, and rheumatoid arthritis. With the aim of enhancing the binding affinity and anti-HIV activity of a potent CXCR4 ligand as a lead, 23 low molecular weight compounds containing dipicolylamine (Dpa) and cyclam cationic moieties with varying spacers and spatial positioning were designed, synthesized and biologically evaluated. All of the synthesized compounds screened at 1.0 μM in the NanoBRET assay system exhibited >70% inhibition of the binding of a competitive probe TAMRA-Ac-TZ14011 (10 nM) to CXCR4 in the presence of zinc (II) ion. Furthermore, selected compounds 3, 8, 9, 19 and 21 with spatial distances between the next carbon to Dpa and the next carbon to cyclam within the range of 6.5–7.5 ? showed potent binding affinity selective for CXCR4 with IC₅₀ values of 1.6, 7.9, 5.7, 3.5 and 4.5 nM, respectively, with corresponding high anti-HIV activity with EC₅₀s of 28, 13, 21, 28 and 61 nM, respectively, in the presence of zinc (II) ion. Some compounds with remarkably more potent CXCR4-binding affinity than that of an initial lead were obtained. These compounds interact with different but overlapping amino acid residues of CXCR4. The present studies have developed new low molecular weight CXCR4 ligands with high CXCR4-binding and anti-HIV activities, which open avenue into the development of more potent CXCR4 ligands.

Full text of DOI:10.1016/j.bmc.2019.02.013

Accepted Manuscript
Design, synthesis and biological evaluation of low molecular weight CXCR4
ligands
Maxwell M. Sakyiamah, Takuya Kobayakawa, Masayuki Fujino, Makoto
Konno, Tetsuo Narumi, Tomohiro Tanaka, Wataru Nomura, Naoki Yamamoto,
Tsutomu Murakami, Hirokazu Tamamura
PII:
S0968-0896(18)32183-7
https://doi.org/10.1016/j.bmc.2019.02.013
BMC 14749
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 December 2018
1 February 2019
5 February 2019
Please cite this article as: Sakyiamah, M.M., Kobayakawa, T., Fujino, M., Konno, M., Narumi, T., Tanaka, T.,
Nomura, W., Yamamoto, N., Murakami, T., Tamamura, H., Design, synthesis and biological evaluation of low
molecular weight CXCR4 ligands, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.
2019.02.013
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Design, synthesis and biological evaluation of low molecular weight
CXCR4 ligands
Maxwell M. Sakyiamaha,b,#, Takuya Kobayakawaa,#,*, Masayuki Fujinoc, Makoto Konnoa, Tetsuo
Narumia,$, Tomohiro Tanakaa, Wataru Nomuraa, Naoki Yamamotod, Tsutomu Murakamic,*,
Hirokazu Tamamuraa,b,*
aInstitute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-
3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
bGraduate School of Medical and Dental Sciences, Tokyo Medical and Dental University
(TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8150, Japan
cAIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku,
Tokyo 162-8640, Japan
dYong Loo Lin School of Medicine, National University of Singapore, Singapore 117597,
Singapore
#equally contributed
$present address: Graduate School of Integrated Science and Technology, Shizuoka University, 3-5-1
Johoku, Hamamatsu, Shizuoka 432-8561, Japan
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if
required according to the journal that you are submitting your paper to)
*To whom correspondence should be addressed; tkobmr@tmd.ac.jp, phone; +81-3-5280-8037,
fax; +81-3-5280-8039, tmura@nih.go.jp, phone; +81-3-4582-2816, fax; +81-3-5285-5037, or
tamamura.mr@tmd.ac.jp, phone; +81-3-5280-8036, fax; +81-3-5280-8039
1

2

Abstract
The chemokine receptor CXCR4/stromal cell-derived factor-1 (SDF-1: CXCL12) signaling axis
represents a crucial drug target due to its relevance to several diseases such as HIV-1 infection, cancer,
leukemia, and rheumatoid arthritis. With the aim of enhancing the binding affinity and anti-HIV activity
of a potent CXCR4 ligand as a lead, 23 low molecular weight compounds containing dipicolylamine
(Dpa) and cyclam cationic moieties with varying spacers and spatial positioning were designed,
synthesized and biologically evaluated. All of the synthesized compounds screened at 1.0 M in the
NanoBRET assay system exhibited more than 70 % inhibition of the binding of a competitive probe
TAMRA-Ac-TZ14011 (10 nM) to CXCR4 in the presence of zinc (II) ion. Furthermore, selected
compounds 3, 8, 9, 19 and 21 with spatial distances between the next carbon to Dpa and the next carbon
to cyclam within the range of 6.5-7.5 Å showed potent binding affinity selective for CXCR4 with IC50
values of 1.6, 7.9, 5.7, 3.5 and 4.5 nM, respectively, with corresponding high anti-HIV activity with EC50s
of 28, 13, 21, 28 and 61 nM, respectively, in the presence of zinc (II) ion. Some compounds with
remarkably more potent CXCR4-binding affinity than that of an initial lead were obtained. These
compounds interact with different but overlapping amino acid residues of CXCR4. The present studies
have developed new low molecular weight CXCR4 ligands with high CXCR4-binding and anti-HIV
activities, which open avenue into the development of more potent CXCR4 ligands.
Keywords
anti-HIV
azamacrocyclic compound
CXCR4-binding
dipicolylamine
3

NanoBRET assay
4

1. Introduction
CXC chemokine receptor type 4 (CXCR4) is a seven transmembrane (7TM) G-protein coupled receptor
(GPCR), which is expressed mainly in immune cells and in hematopoietic stem cells. In response to its
natural ligand stromal cell-derived factor-1 (SDF-1: CXCL12), it transmits signals to enhance
development and chemotaxis of these cells.1 However, the CXCR4/CXCL12 signaling axis plays relevant
roles in a multiple of diseases such as HIV-1 infection, metastasis of various cancers and inflammatory
diseases including rheumatoid arthritis.2 Thus, this axis represents a prospective target for therapeutic
interventions. To this aim, drugs and their candidates ranging from peptides such as T140 and 4F-
benzoyl-TN14003 (LL-8040/BKT140), through to low molecular weight non-peptide compounds such as
AMD3100 (plerixafor) and its analogs have been developed.3 Our previous studies have developed a new
class of low molecular weight CXCR4 antagonists such as SKA028-p (Fig. 1)4 with bis(pyridin-2-
ylmethyl)amine (dipicolylamine: Dpa) and 1,4,8,11-tetraazacyclotetradecane (cyclam) moieties attached
to a p-xylene spacer, based on modification of the parent compounds, AMD3100 and Dpa-zinc(II)
complex.5 According to these studies, it is deduced that the positioning of the cationic moieties and the
length of the spacer could influence the binding affinity of the compound for CXCR4 as well as its anti-
HIV activity. In addition, the information might be useful that linker optimization was a key contributing
factor leading to the synthesis of AMD3100.6 Based on these backdrops, in this study we tried the design,
synthesis and structure-activity relationship studies of a series of compounds with emphasis on varying
spacers and positioning of the cationic moieties aimed at development of potent low molecular weight
CXCR4 ligands.
2. Results and Discussion
2.1. Design and synthesis of the desired compounds
Compounds 1-23 were designed and synthesized employing spacers which would increase the spatial
distance between the next carbon to Dpa and the next carbon to cyclam based on a lead compound
5

SKA028-p as shown in Fig. 1. In our previous study, SKA028-p, having the p-xylene spacer with a
length of 5.9 Å, showed remarkably higher anti-HIV activity than the derivative having the m-xylene
spacer with a length of 5.0 Å, in particular, in the presence of zinc (II) ion,4 suggesting that longer
distances between the cationic moieties could enhance binding affinity for CXCR4 and HIV-inhibitory
activity. In place of benzene, naphthalene was therefore introduced to achieve a longer spatial length with
a degree of angular restrain in compounds 1-3. To further increase the length, a biphenyl spacer was
utilized in compounds 4-12. The biphenyl spacer was modified by introducing crosslinks to hinder the
rotation around the single bond of the biphenyl spacer in compounds 15-18. From our previous studies,
the azamacrocyclic compounds showed enhanced activity in the presence of a bivalent cation, zinc (II).4,5
Therefore, we modified the biphenyl and napththalene structures by introducing nitrogen atoms to derive
bipyridine (compounds 13, 14), phenylpyridine (compounds 19-22) and quinoline (compound 23) (Fig. 1),
to possibly enhance the chelation effects of the resulting compounds. In addition to the difference in
spacer types, the positioning of the cationic moieties was varied with the aim of achieving the optimal
spatial orientation of the Dpa and cyclam moieties for potent CXCR4-binding affinity and anti-HIV
activity (Table 1).
6

Figure 1. Design of the desired compounds with Dpa-CH2 and cyclam-CH2 moieties via various
spacers based on our previous lead compound SKA028-p.
In the compound synthesis, depending on spacer types, synthetic schemes were changed.
Schemes 1-3 are representative of the synthetic schemes for compounds 1-23. Detail synthetic procedures
and characterization data of all of the compounds 1-23 are described in Supplementary data. In the
synthesis of a naphthalene spacer-containing compound 1, 2,7-bis(bromomethyl)naphthalene 1a, which
was obtained by treatment of 2,7-dimethylnaphthalene with N-bromosuccinimide (NBS) and benzoyl
peroxide (BPO), was reacted with tri-tert-butyl 1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate
((Boc)3-cyclam) in the presence of KI and K2CO3, and then reacted with bis(pyridin-2-ylmethyl)amine
7

(Dpa) in the presence of KI and K2CO3 to obtain a tri-N-Boc-protected amine 1c, followed by TFA
treatment to yield the desired compound 1 (Scheme 1).
Scheme 1. Synthesis of a naphthalene spacer-containing compound 1. Reagents and conditions: (a) N-
bromosuccinimide (NBS), benzoyl peroxide (BPO), CCl4, reflux, 67%; (b) tri-tert-butyl 1,4,8,11-
tetraazacyclotetradecane-1,4,8-tricarboxylate ((Boc)3-cyclam), KI, K2CO3, CH3CN, rt; (c) bis(pyridin-2-
ylmethyl)amine (Dpa), KI, K2CO3, CH3CN, 80 °C; (d) TFA/CHCl3/H2O (5 : 47.5 : 0.25) (v/v), 0 °C to rt,
11% from 1a.
In the synthesis of a crosslinked biphenyl spacer-containing compound 16, 5,5'-dimethyl-[1,1'-biphenyl]-
2,2'-diol 16a, which was obtained by treatment of p-cresol with copper(II) chloride and cyclohexylamine,
was reacted with bromochloromethane in the presence of sodium hydride to obtain the dioxepine
derivative 16b, followed by treatment with NBS and BPO to obtain the corresponding dibromide 16c.
The dibromide 16c was reacted with (Boc)3-cyclam in the presence of KI and K2CO3, and then reacted
with Dpa in the presence of KI and K2CO3 to obtain a tri-N-Boc-protected amine 16d, followed by TFA
treatment to yield the desired compound 16 (Scheme 2).
8

Scheme 2. Synthesis of a crosslinked biphenyl spacer-containing compound 16. Reagents and conditions:
(a) CuCl2•2H2O, cyclohexylamine, MeOH, 30 °C, 30%; (b) NaH, CH2BrCl, dry DMF, rt, 49%; (c) NBS,
BPO, CCl4, reflux; (d) (Boc)3-cyclam, KI, M K2CO3, CH3CN, rt; (e) Dpa, KI, K2CO3, CH3CN, 80 °C; (f)
TFA/CHCl3/H2O (5 : 47.5 : 0.25) (v/v), 0 °C to rt, 29 % from 16b.
In the synthesis of
a
2-phenylpyridine spacer-containing compound 22, 2-bromo-4-
(bromomethyl)pyridine 22a, which was obtained by treatment of 2-bromo-4-methylpyridine with NBS
and BPO, was reacted with Dpa in the presence of K2CO3 to yield 1-(2-bromopyridin-4-yl)-N,N-
bis(pyridin-2-ylmethyl)methanamine 22b, followed by treatment with (4-formylphenyl)boronic acid,
tetrakis(triphenylphosphine)palladium(0) and cesium carbonate to obtain the corresponding 4-
carbaldehyde 22c. The 4-carbaldehyde 22c was reacted with (Boc)3-cyclam and sodium
cyanoborohydride in the presence of acetic acid, and then treated with TFA to yield the desired compound
22 (Scheme 3).
9

Scheme 3. Synthesis of a 2-phenylpyridine spacer-containing compound 22. Reagents and conditions: (a)
NBS, BPO, CCl4, reflux, 31%; (b) Dpa, K2CO3 CH3CN, rt, 99%; (c) (4-formylphenyl)boronic acid, 4
mol% Pd(PPh3)4, Cs2CO3, MeOH, 150 °C, MW; (d) (Boc)3-cyclam, NaBH3CN, AcOH, MeOH, rt; (e)
TFA/CHCl3/H2O (47.5 : 47.5 : 0.25) 0 °C to rt, 9 % from 22b.
2.2. Evaluation of CXCR4-binding affinity of the synthesized compounds
The binding affinity of the synthesized compounds was evaluated using the NanoBRET assay system,
which was recently established by us,7 based on their ability to compete for the binding of the fluorescent
labeled ligand (TAMRA-Ac-TZ14011) in CHO cells stably expressing CXCR4 with Nanoluc tagged to
its N-terminus. The NanoBRET assay system, which has recently gained wide application, is a non-
radioactive assay system based on the transfer of energy from a bioluminescent donor (Nanoluc
luciferase) to a fluorescent acceptor (5(6)-carboxy-tetramethylrhodamine; TAMRA) in close proximity
(approximately 10 nm) upon binding of TAMRA-Ac-TZ14011 to CXCR4. We used the NanoBRET
assay system due to its enhanced sensitivity and usefulness in high throughput screening of GPCR
ligands.8
The present synthesized compounds, as well as the parent compounds AMD3100 and Dpa-Zn
complex, have the propensity to coordinate with bivalent cations such as zinc, copper and nickel resulting
10

in highly stable metal complexes with enhanced binding affinity for CXCR4.4,5,9 Both Dpa and cyclam
moieties have the propensity to coordinate with zinc (II) ions.4,5 Thus, the synthesized compounds 1-23
were evaluated at a predetermined concentration of 1.0 µM in the presence and absence of 10 equivalent
of zinc (II) ion,4 and the results were comparable to values obtained by radioisotope (RI)-based binding
assay using [125I]-CXCL12 as a competitive probe10 (Fig. 2, Supplementary data Table S1). All of the
compounds 1-23 screened showed more than 70 % inhibition of TAMRA-Ac-TZ14011 binding in the
presence of zinc (II) ion (Fig. 2) whereas in the absence of zinc (II) ion, compounds 1-3 with a
naphthalene spacer, 10 and 11 with a biphenyl spacer, 13 and 14 with a bipyridine spacer, 20 and 22 with
a 2-phenylpyridine spacer, and 23 with a quinoline spacer showed more than 70 % inhibition. This
observation corroborates with the aforementioned assertion that binding of the azamacrocyclic
compounds is generally improved in the presence of bivalent ions. In the presence of zinc (II) ion, these
compounds are predicted to assume more stable octahedral conformation upon interaction with the zinc
(II) ions through the nitrogen atoms, and this enhances their affinity for CXCR4.11
A
150
100
50
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23 SKAFC131
MKN Compound
11

B
150
100
50
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14
15 16 17 18 19 20 21
22 23 SKAFC131 Zn
MKN Compound
Figure 2. Single point percentage inhibition of the synthesized compounds using the NanoBRET assay
system. CHO cells stably expressing Nluc-CXCR4 (5 x104) were seeded 24 h prior to experiments. Cells
were incubated with 10 nM TAMRA-Ac-TZ14011 in the presence of 1.0 µM of the synthesized
compounds without (A) or with 10 µM ZnCl2 (B) for 1 h at 25°C. Values are mean ± SEM, n = 3.
Green: SKA (SKA028-p).4 Blue: CXCR4 antagonist, FC131.3h Red: 10 µM ZnCl2 without compounds.
2.3. Evaluation of anti-HIV activity of the synthesized compounds
The inhibitory activity of the synthesized compounds against T-cell-line tropic (X4-) HIV-1 (NL4-3
strain)-induced cytopathogenicity in MT-4 cells was evaluated. CXCR4 is a major co-receptor for the
entry of X4-HIV-1,1,2 therefore a potential target for anti-HIV drug development.
As a result, the compounds with 2-phenylpyridine as a spacer 19-22, in particular 19 and 20,
appeared to show higher anti-HIV activity which increased by the presence of zinc (II) ion (Table 1), and
19 with zinc (II) ion is the most potent (EC50 = 28 nM). A similar increase by the presence of zinc (II) ion
12

was observed in binding affinity of 19 and 21 for CXCR4 (Fig. 2). The Dpa/cyclam-CH2 moieties at the
3/4-positions on pyridine/phenyl rings of the 2-phenylpyridine spacer of 19-22 seem to favor high
CXCR4-binding affinity which possibly leads to high anti-HIV activity in the presence of zinc (II) ion.
The additional nitrogen atom in the 2-phenylpyrine spacer might contribute to the interaction with the
zinc (II) ion and thus, its increased affinity for CXCR4 in the presence of zinc (II) ion. However, contrary
to what was expected, 13 or 14 with Dpa/cyclam-CH2 moieties at the 5/5’ or 4/4’-position, respectively,
on two pyridine rings of the bipyridine spacer, did not show higher anti-HIV activity compared to 19or20
with the 2-phenylpyridine spacer. Furthermore, the presence of zinc (II) ion did not improve the CXCR4-
binding affinity of 13 or 14 in spite of existence of the two nitrogen atoms (Fig. 2), suggesting the
coordinative interaction with zinc (II) ion might cause some conformational change. Compounds 1-3 with
the naphthalene spacer showed high CXCR4-binding activity in the presence and absence of zinc (II) ion
(Fig. 2), and 3 showed potent anti-HIV activity in the presence of zinc (II) ion (EC50 = 28 nM). All of the
compounds with the biphenyl spacer 4-12, with the exception of 8 and 9, did not show high anti-HIV
activity in the presence or absence of zinc (II) ion whereas some showed potent binding affinity in the
presence of zinc (II) ion. Similarly, 15-18 having the crosslinked biphenyl spacer did not show high anti-
HIV activity in the presence or absence of zinc (II) ion although 15-18 exhibited enhanced binding
affinity for CXCR4 in the presence of zinc (II) ion (Fig. 2). This suggest that the introduced angular
restrain did not necessarily contribute to the anti-HIV activity of 15-18. Compound 23, with a mixture of
isomers 23AB, having the quinoline spacer did not show high anti-HIV activity in the presence or
absence of zinc (II) ion, and did not exhibit increased binding affinity for CXCR4 in the presence of zinc
(II) ion (Fig. 2). All 23 compounds, with the exception of 5, did not show significant cytotoxicity in the
absence of zinc (II) ion (CC50 > 10 µM, Table 1). Three selected compounds, 2, 13 and SKA028-p did not
show significant cytotoxicity in the presence of zinc (II) ion (CC50 > 10 µM, data not shown). All 23
compounds, with the exception of 9 in the presence of zinc (II) ion (58% inhibition at 10 M), did not
show significant anti-HIV activity against macrophage-tropic (R5-) HIV-1 (NL(AD8) strain) in the
13

presence or absence of zinc (II) ion (< 30% inhibition at 10 M) (data not shown), suggesting that these
compounds selectively bind for CXCR4.
Table 1. Anti-HIV and CXCR4-binding activities of the synthesized compounds.
compd. No. structure
spacer anti-HIV activity
cytotoxicity
CXCR4-binding
activity
length EC50 (µM)
EC50 (µM) CC50 (µM)
IC50 (nM)
(Å)
(- Zn(II))[a] (+ Zn(II))[a] (- Zn(II))[b]
(+ Zn(II))[c]
naphthalene
spacer-
containing compounds
1
7.5
8.1
0.79
0.47
1.1
>10
>10
2
3
0.24
10
6.7
0.22
0.028
>10
1.6
biphenyl spacer-containing
compounds
4
8.7
0.75
0.98
>10
5
6
10.2
6.8
0.78
2.6
1.0
4.8
0.89
>10
7
8
5.4
6.5
4.2
1.1
>10
>10
0.040
0.013
7.9
14

9
6.6
9.1
4.7
0.040
0.80
1.0
0.021
1.1
>10
>10
>10
5.7
10
11
5.8
12
9.1
3.1
1.0
>10
bipyridine
spacer-
containing compounds
13
10.0
8.8
0.68
0.92
0.90
0.20
>10
>10
14
crosslinked
spacer-containing
compounds
15
biphenyl
7.5
1.9
0.97
>10
16
17
7.2
7.8
0.80
0.58
0.87
0.30
>10
>10
18
7.3
0.67
0.28
>10
2-phenylpyridine spacer-
containing compounds
15

19
7.5
0.17
0.028
>10
3.5
4.5
20
21
10.1
6.7
0.59
0.11
0.13
>10
>10
0.061
22
9.1
0.14
0.045
>10
quinoline spacer-containing
compounds
23AB
8.0
5.9
>0.97
5.5
>10
>10
control
SKA028-p
0.0065
0.0027
32
[a]EC50 values are the concentrations of test compounds in the presence and absence of ZnCl2 (10 eq)
corresponding to 50% protection from X4-HIV-1 (NL4-3 strain)-induced cytopathogenicity in MT-4 cells.
[b]CC50 values are the concentrations of test compounds in the presence of ZnCl2 (10 eq) which
correspond to 50 % reduction in the viability of MT-4 cells. All data are mean values of at least three
independent experiments. [c]IC50 values are the concentrations of test compounds in the presence of ZnCl2
(10 eq) based on the inhibition of TAMRA-Ac-TZ14011 binding to CXCR4 in Chinese hamster ovary
(CHO) stable cells using the NanoBRET assay system. : Dpa-CH2 moiety : cyclam-CH2 moiety.
Spacer length: spatial distance between the next carbon to Dpa and the next carbon to cyclam as shown in
Fig. 1.
Contrary to our initial hypothesis, the synthesized compounds did not show the expected trend of
increasing CXCR4-binding and anti-HIV activities with increasing spatial distances between the next
carbon to Dpa and the next carbon to cyclam (Fig. 3), and rather an undulating pattern was observed.
16

Compounds with shorter spatial distances such as 11 with a spacer length of 4.7 Å exhibited weaker anti-
HIV activity (EC50 = 5.8 µM, Table 1) compared to the control SKA028-p (EC50 = 0.0027µM, Table1)
with a length of 5.9 Å, whereas compounds with longer spatial distances such as compounds 1, 4, 5 and
23 with lengths of 7.5 Å, 8.7 Å, 10.2 Å, and 8.0 Å, respectively, showed weaker anti-HIV activity than
SKA028-p, in spite of the presence or absence of zinc (II) ion. Compounds 19 and 22, however, exhibited
high anti-HIV activity despite having longer spatial lengths, 7.5 Å and 9.1 Å, respectively. Compounds 3,
8, 9 and 21 with spatial distances within the range of 6.5-6.7 Å showed higher anti-HIV activity in the
presence and absence of zinc (II) ion (Table 1). This implies that a spatial distance within the range of
6.5-6.7Å could be the most suitable length for optimal interaction between the synthetic compounds and
the corresponding amino acid residues in the binding pocket of CXCR4. However, the spatial orientations
and the presence of a basic group such as a nitrogen atom for the zinc (II) ion interaction could partly or
altogether complement the contribution of the spatial distance between the cationic moieties to enhance
the CXCR4-binding and anti-HIV activities of the compounds (Compounds 13,14 and 19-22, Fig. 2 and
Table 1).
In addition to binding affinity, the selectivity of candidates is imperative in the drug discovery
targeting GPCRs. The present control compound, SKA028-p-Zn(II) complex, as well as AMD3100, have
been reported in previous studies to show high selectivity for CXCR4 over CCR5.3j,10,12 Similarly, all of
the 23 compounds synthesized here, with the exception of 9 in the presence of zinc (II) ion did not show
significant anti-HIV activity against R5-HIV-1 (NL(AD8) strain) in the presence or absence of zinc (II)
ion (< 30% inhibition at 10 M). Compound 9 with zinc (II) ion exhibited quite slightly activity against
R5-HIV-1 (58% inhibition at 10 M). This suggests that the present compounds are also highly selective
for CXCR4 because R5-HIV-1 strains prefer CCR5 to CXCR4 as the main co-receptor for viral entry.
Taken together, the present compounds such as 3, 8, 9, 19 and 21 are potential CXCR4 ligands.
A
B
17

8
6
4
2
0
-Zn2+
150
100
50
-Zn2+
+Zn2+
+Zn2+
0
4
5
6
7
8
9
10
11
4
5
6
7
8
9
10
11
Length Å
Length Å
Figure 3. Correlation of CXCR4-binding affinity (competitive inhibition (%) of binding of TAMRA-Ac-
TZ14011 (10 nM) in CHO cells stably expressing CXCR4 by the synthesized compounds (1.0 M) in the
presence/absence of 10 M ZnCl2) (A) and anti-HIV activity (EC50 (M)) (B) of the synthesized
compounds with increasing spatial distances (length (Å)) between the next carbon to Dpa and the next
carbon to cyclam (Fig. 1).
2.4. Structure-activity relationship studies (SARs) of selected potent CXCR4 ligands
Based on the anti-HIV activity (Table 1) and the initial data for the CXCR4-binding affinity (Fig. 2) of
the synthesized compounds, the IC50 values of six selected compounds: 2 and 3 with the naphthalene
spacer; 8 and 9 with the biphenyl spacer; 19 and 21 with the 2-phenylpyrindine spacer, were evaluated
through a competitive binding assay using the NanoBRET assay system in the presence of zinc (II) ion.
All the six compounds showed high binding affinity for CXCR4 compared to the control SKA028-p, with
3 showing the highest activity (IC50 = 1.6 nM, Table 1, Supplementary data Fig. S2). This observation
could be attributed to the spacer distances and/or spatial positioning of the cationic moieties of these six
compounds, suggesting that positioning of Dpa and cyclam moieties orient these compounds to achieve
suitable spatial length for the interaction with CXCR4 leading to higher binding affinity. As indicated
above, a spacer within the range of 6.5-6.7 Å appear to enhance the binding characteristics of the
compounds to CXCR4.4,13 Compounds 3, 8, 9 and 21 possessing spatial lengths within the 6.5 - 6.7 Å
range (that is, 6.7Å, 6.5Å, 6.6Å, and 6.5Å, respectively) showed high CXCR4-binding affinity (IC50 = 1.6,
18

7.9, 5.7 and 4.5 nM, Table 1, Supplementary data Fig. S2). Compound 19 with a spatial distance of 7.5Å,
slightly over the 6.5-6.7 Å range, showed high affinity (IC50 = 3.5 nM) whereas 2 with 8.1Å, certainly
over the 6.5- 6.7 Å range, showed slightly weaker affinity (IC50 = 10 nM) than other five compounds.
Furthermore, among the three group of spacers and spatial positioning of cationic moieties, the rigidity of
the naphthalene spacer and the 1, 6-positioning of compound 3 probably contributed largely to the
stability of its zinc (II) ion complex resulting in the highest binding affinity of 3 for CXCR4.
There was, however, no defined correlation between the CXCR4-binding and ant-HIV activities
of the six compounds analyzed (Table 1). In other words, strong binding to CXCR4 did not necessarily
translate into high anti-HIV activity. Compound 3, for example, does not have the highest anti-HIV
activity in the presence of zinc (II) ion (EC50 = 28 nM) while it possesses the highest binding affinity for
CXCR4 (IC50 = 1.6 nM) among the six compounds analyzed. Similarly, compound 8 with the highest
anti-HIV activity (EC50 = 13 nM) exhibited lower binding affinity (IC50 = 7.9 nM) compared to
compound 3 in the presence of zinc (II) ion. These differences could be attributed to possible variations in
the CXCR4 amino acid residues responsible for TAMRA-Ac-TZ14011 and HIV-1 binding to CXCR4.
2.5. Analysis of binding mode to CXCR4
The plausible binding modes of compound 3 and SKA028-p to CXCR4 (PDB ID: 3ODU) were analyzed
using 2D Molecular Operating Environment (MOE) software in order to expatiate the SARs of the
synthesized compounds on binding to CXCR4 (Fig. 4). In this analysis zinc (II) ions cannot be included.
AMD3100 on binding to CXCR4 is predicted to interact with three main residues, Asp171
(transmembrane (TM) IV), Asp262 (TM VI), and Glu288 (TM VII) of CXCR4, whereby one of its two
cyclam moieties interacts with Asp171 while the other cyclam moiety is sandwiched by Asp262 and
Glu288.14 Similarly, in the present molecular docking simulation studies, SKA028-p and compound 3
showed interactions with Asp171, Asp262 and Glu288, whereby the cyclam moiety of SKA028-p and a
picolyamine moiety of compound 3 are sandwiched by Asp262 and Glu288. In addition, both SKA028-p
19

and compound 3 showed hydrogen bond interactions with Arg188 and Tyr116. In addition, compound 3
interacts with 1H-imidazole of His203 using a lone pair of a nitrogen atom of the cyclam moiety as an H-
bond acceptor, and is located close to Val197, Gln200, Ser263, Ile265, Leu266, Gln277 and Ile 284 with
non-bonded interactions. Furthermore, the naphthalene spacer of compound 3 was seated in the
hydrophobic pocket of CXCR4 interacting with Val196, F199 and Ile259 to enhance its stability.
SKA028-p, on the other hand, interacts with His281 (through H-bonding) and with Asp187 (through ionic
interaction), and is located close to Arg30, Trp94, Trp102, Val112, His113, Cys186 and Ile259 with non-
bonded interactions. Taken together, the variation in spacer lengths and/or orientation of cationic moieties
appear to influence the CXCR4 amino acid residues available for ligand-receptor interaction, and thus the
binding affinity. This observation could be the cause of the disparities in the binding affinities of the six
selected compounds and the inconsistency in the trends of the binding affinity and anti-HIV activity. For
instance, in addition to Asp262, HIV-1 entry involves Asp193 of the extracellular loop (ECL) 2, mostly
N-terminal residues of CXCR4 and the upper region of TM II such as Tyr 97,10,12,14 which are different
from the residues observed to the interaction of compound 3. SKA028-p was observed to interact with
Arg30 (N-terminal region) and Trp94 (TM II), which could account for the higher anti-HIV activity
observed despite the weaker CXCR4-binding affinity compared to compound 3.
20

A
B
Figure 4. Binding models of (A) SKA028-p and (B) compound 3 to CXCR4 (PDB: 3ODU). Green circle:
hydrophobic residues without Tyr, purple circle: hydrophilic residues including Tyr (blue outline: basic
21

residues, red outline: acidic residues), indigo shade: ligand exposure, cyano blue shadow: receptor
exposure.
3. Conclusion
In summary, a series of low molecular weight CXCR4 ligands have been designed and synthesized based
on a lead compound SKA028-p by introduction of spacers which provide longer lengths between the
cationic moieties, Dpa-CH2 and cyclam-CH2, with emphasis on the spatial positioning of these moieties.
All of the synthesized compounds at 1.0 M with zinc (II) ion exhibited more than 70% inhibition of the
binding of a competitive probe TAMRA-Ac-TZ14011 (10 nM) to CXCR4 in the NanoBRET assay.
Compounds 3, 8, 9, 19 and 21 with spacers within the range of 6.5 Å-7.5Å were identified to exhibit high
binding affinity selective for CXCR4 as well as high anti-HIV activity in the presence of zinc (II) ion.
The selected compounds interact with varying but overlapping amino acid residues of CXCR4 due to the
differences in the spacer lengths and/or orientation of the cationic moieties, accounting for disparities in
CXCR4-binding and anti-HIV activities. At least, concerning CXCR4-binding affinity compounds 3, 8, 9,
19 and 21 are remarkably more potent than an initial lead SKA028-p in the presence of zinc (II) ion
whereas the present study has not produced a higher anti-HIV compound compared to that of SKA028-p.
The present data provide a new window for exploration into the development of more potent CXCR4
ligands, especially low molecular weight compounds, which can be considered in the future drug
development for chemotherapy of diseases including cancer.
4. Experimental
4.1. Chemistry
Three representative compounds were synthesized as shown in Schemes 1-3. Detailed procedures and
characterization data are described in Supplementary data.
22

4.2. Biological assay methods
NanoBRET assay
The NanoBRET assay system for CXCR4 ligands was developed in our laboratory.7 Briefly, post
transfected CHO cells stably expressing Nluc-CXCR4 (5 x 104) were seeded into a white Thermo
Scientific Matrix 96 well microplate and incubated for 24 h at 37 °C under 5% CO2 prior to experiments.
The medium in each well was removed and replaced with OptiMEM (reduced serum). Single point
competitive binding assay was performed by incubating seeded cells with TAMRA-Ac-TZ14011 (10 nM)
in the presence of a test compound (1 µM) with and without ZnCl2 (10 eq). For IC50 value evaluation, the
seeded cells were incubated with TAMRA-Ac-TZ14011 (10 nM) in the presence of increasing
concentrations of a test compound with ZnCl2 (10 eq). The substrate, furimazine (Promega corperation,
USA), reconstituted in the LCS dilution buffer (provided by the manufacturer) was then added to each
well (25 µL) according to the manufacturer’s protocol and BRET was read immediately using the Wallac
1420 ARVO MX plate reader (fitted with 100 nm filter) at room temperature. The donor emission of Nluc
was measured at 460 nm (80 nm bandpass) and the acceptor emission of TAMRA was measured at >610
nm (longpass). Raw BRET ratio was given as;
cceptor emission
Raw BRET ratio
onor emission
The percentage inhibition was calculated with equation below;
Inhibition (%) =
Where E0, Et and E100 represent the BRET ratio without a test compound, BRET ratio in the presence of
a test compound, and BRET ratio with excess amount of Ac-TZ14011 as a competitor, respectively.
Anti-HIV assay
Anti-HIV-1 activity was determined based on the protection against X4-HIV-1(NL4-3 stain)-induced
cytopathogenicity in MT-4 cells. Various concentrations of a test compound were added to HIV-1-
infected MT-4 cells at multiplicity of infection (MOI) of 0.001 and placed in wells of a flat-bottomed
microtiter tray (2.0 × 104 cells/well). fter 5 days’ incubation at 37 °C in a CO2 incubator, the number of
23

viable cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) method.10
Data presentation and statistical analysis
The data were presented as mean of independent experiments conducted in triplicate and analyzed using
GraphPad Prism software (version 5.0). The IC50 and EC50 values were generated from non-linear
regression analysis.
4.3. Molecular modeling
Molecular modeling calculations were performed using compounds docked into CXCR4 (PDB: 3ODU)
with MOE (version 2018.0101, Chemical Computing Group Inc., Montreal, QC, Canada, 2018). In the
molecular modeling, global energy minimization of compounds docked into CXCR4 was searched.
Declaration of interest
Conflicts of interest: none.
Acknowledgments
This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan (JSPS KAKENHI Grant Number JP15H04652). It was
also supported in part by the Platform for Drug Discovery, Informatics, and Structural Life Science of
MEXT, Japan, and the Cooperative Research Project of Research Center for Biomedical Engineering.
M.S. was also supported by MEXT through the award of a scholarship. Our profound gratitude also goes
to Prof. Shoji Yamaoka for his immense support.
Supplementary data
Supplementary data (synthetic procedures and characterization data of compounds, an RI-based
binding assay method and CXCR4-binding assay data) associated with this article can be found, in the
online version, at doi: .bmc. .
24

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