The natural product CCR5 antagonist anibamine and its analogs as anti-prostate cancer agents

Article abstract of DOI:10.1016/j.bmcl.2011.07.058

Prostate cancer is a leading cause of death among males in the United States. As the chemokine receptor CCR5 is over-expressed in more aggressive forms of prostate cancer, and is also a critical receptor in inflammation, chemokine receptor CCR5 antagonist

Full text of DOI:10.1016/j.bmcl.2011.07.058

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5159–5163
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The natural product CCR5 antagonist anibamine and its analogs as anti-prostate
cancer agents
Kendra M. Haney a, Feng Zhang a, Christopher K. Arnatt a, Yunyun Yuan a, Guo Li a, Joy L. Ware b,d
,
David A. Gewirtz c,d, Yan Zhang a,d,
⇑
a Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298, USA
b Department of Pathology, Virginia Commonwealth University, Richmond, VA 23298, USA
c Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298, USA
d Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Prostate cancer is a leading cause of death among males in the United States. As the chemokine receptor
CCR5 is over-expressed in more aggressive forms of prostate cancer, and is also a critical receptor in
inflammation, chemokine receptor CCR5 antagonists could potentially act as anti-prostate cancer agents.
Anibamine, a natural product CCR5 antagonist, provides a unique molecular scaffold for the generation of
novel analogs with possible anti-prostate cancer activity. A series of analogs of anibamine were designed,
synthesized and tested against several prostate cancer cell lines. The analogs all acted as CCR5 antago-
nists at micromolar range affinity to the receptor while their anti-proliferative activity varied depending
on the cell line type and their chemical structural properties. Further basal cytotoxicity characterization
on these compounds indicated some of them may be suitable for in vivo studies.
Received 29 April 2011
Revised 14 July 2011
Accepted 15 July 2011
Available online 23 July 2011
Keywords:
CCR5
Antagonist
Anibamine
Prostate cancer
Published by Elsevier Ltd.
The CC chemokine receptor CCR5 belongs to the G-protein-
coupled seven-transmembrane receptor superfamily, and its
endogenous ligands include the chemokines CCL3 (MIP-1a), CCL4
between prostate cancer and chronic inflammation, several genes
and proteins of the inflammatory network were evaluated in pros-
tatic tissues of various disease states.23 It was found that in some
cases prostate cancer tissues express CCL5 and CCR5 mRNA.23 This
discovery prompted the investigation of the effect of a CCR5 antag-
onist, TAK-779, which significantly reduced the growth and inva-
siveness of prostate cancer cells in the presence of CCL5.24
Therefore, development of an appropriate chemokine receptor
CCR5 antagonist may provide a novel prostate cancer therapy.
Anibamine, a unique pyridine quaternary alkaloid recently iso-
lated from Aniba panurensis, has been found to effectively bind to
(MIP-1b), and CCL5 (RANTES).1,2 CCR5 has been identified as an
essential co-receptor for HIV virus entry to host cells3–6 and has
therefore become an attractive target for anti-HIV therapeutics
development.7–11 A number of small molecule CCR5 antagonists
identified through high-throughput screening efforts have shown
potent activities in blocking chemokine function and HIV entry,
such as Maraviroc,12 TAK-779,13 and Vicriviroc14 (Fig. 1).
Prostate cancer is a leading cause of death in American males
and there is no cure for this disease once it becomes androgen-
independent and/or metastatic.15 Historically, chronic inflamma-
tion has been believed to be at the root of several cancers, includ-
ing prostate cancer16 and increased expression of the members of
the chemokine network (an important component in inflammation
response) by tumor cells has been found to correlate with the pro-
gression of many types of cancers.17–20 For example, the chemo-
kine CCL5 was shown to induce breast cancer cell migration,
mediated by the chemokine receptor CCR5. It was then shown that
the expression of CCR5 and CCL5 correlated with breast cancer dis-
ease progression while a CCR5 antagonist inhibited breast tumor
growth in the presence of CCL5.21,22 Because of the possible link
the chemokine receptor CCR5 with an IC50 at 1 lM in competition
with 125I–gp120, a HIV viral envelop protein that has high binding
affinity to CCR5.25 As the first natural product CCR5 antagonist,
anibamine provides a structural skeleton, that is, remarkably dif-
ferent from all previously identified lead CCR5 antagonists
(Fig. 1). Our recent studies26 demonstrated that anibamine pro-
duced significant inhibition of prostate cancer cell proliferation at
micromolar to submicromolar concentrations as well as suppress-
ing adhesion and invasion of the highly metastatic M12 prostate
cancer cell line. Preliminary in vivo studies indicated that anib-
amine also inhibits prostate tumor growth in mice.26 These find-
ings indicate that anibamine may serve as a new lead compound
for the development of prostate cancer therapeutic agents.
Compared with other known CCR5 antagonists, anibamine’s
moderate binding affinity to CCR5 is likely due to its long aliphatic
side chains. It is believed that rational structural modification of
⇑
Corresponding author.
E-mail address: yzhang2@vcu.edu (Y. Zhang).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.07.058

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K. M. Haney et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5159–5163
F
N
N
N
O
F
N
+
N
HN
TFA
O
Anibamine
Maraviroc
H
N
N
N
N
N
N
O
N
O
O
F3C
TAK 779
Vicriviroc
Figure 1. Anibamine and some known CCR5 antagonists.
this natural product based on its novel structural skeleton could
lead to the development of new lead compounds that would act
as CCR5 antagonists and also express some unique pharmacologi-
cal profiles that would be useful for anti-cancer activity. Recently,
the total synthesis of anibamine was reported by our group.27
Modification of this synthetic pathway provides opportunities for
generating anibamine analogs whose structure–activity relation-
ship could be investigated as CCR5 antagonists. Here we report
the initial structural modification of anibamine and the biological
screening of these analogs against prostate tumor cell lines.
Primary structural modifications of anibamine were focused on
the size of the fused ring embodied pyridine quaternary ion and
the configuration of the double bond in each side chain, two major
structure features of the parent natural product. The central fused
ring system of anibamine largely determines the binding mode of
the whole molecule to the receptor28 as the central positively
charged nitrogen atom is believed to be essential for the binding
of anibamine to CCR5. Altering the size of the fused ring might
influence the binding affinity of the whole molecule to the receptor
and the anti-prostate cancer activity of the products. Isomerization
and saturation of the double bond were carried out to test the
influence on the anti-prostate cancer activity since, that is, another
important structural feature of anibamine. The ring-size-modified
anibamine analogs were prepared following previously reported
procedures (Scheme 1).27 The palladium-catalyzed Sonogashira
reaction between the bromide 1 and the appropriate PMB pro-
tected alcohol afforded the intermediate 2. The hydrogenation of
compound 2 using palladium on carbon as a catalyst at room tem-
perature gave the side chain saturated intermediate 3 in quantita-
tive yield. The aldehyde 4 was prepared by DIBAL-H reduction of 3
in toluene. Adopting LHMDS as the base, the Wittig reaction prod-
ucts 5 and 6 were obtained as major products. Without further sep-
aration of the isomers, the PMB group of compounds 5 and 6 was
removed under acidic conditions to give compounds 7 and 8,
respectively. The ring-closure reaction was achieved by treating
compounds 7 and 8 with methanesulfonylchloride and triethyl-
amine at room temperature except for the seven-membered ring
derivatives which required heating up to 50 °C. The crude products
9 and 10 were purified by preparative HPLC using the previously
reported condition.27 The saturated products 11 were obtained
by catalytic hydrogenation at room temperature. All the final com-
pounds were obtained with reasonable yields and characterized
NC
CN
Br
NC
CN
NC
CN
i
ii
iii
OPMB
( )
N
N
N
n
OPMB
( )
n
2
3
1
( )7
( )7
OHC
CHO
+
( )7
OPMB
( )7
iv
OPMB
OPMB
( )
n
( )
( )
n
N
N
N
n
5 (a,b,c)
6 (a,b,c)
4
( )7
( )7
+
( )7
v
( )7
vi
OH
OH
( )n
( )n
N
N
7 (a,b,c)
8 (a,b,c)
( )7
( )7
( )9
( )9
( )7
+
( )7
vii
n=1, compounds a series
n=2, compounds b series
n=3, compounds c series
N
N
N
+
-
-
+
-
+
TFA
TFA
11 (a,b,c)
TFA
9 (a,b,c)
( )n
( )n
( )n
10 (a,b,c)
Scheme 1. Reagents: (i) HC„C(CH2)nOPMB (n = 1, 2, and 3), CuI, PdCl2(PPh3)2, TEA; (ii) Pd/C; (iii) DIBAL-H, toluene; (iv) CH3(CH2)7CH@PPh3, LHMDS; (v) 1 N HCl, EtOH; (vi)
(a) MsCl, TEA; (b) preparative HPLC; (vii) Pd/C.

K. M. Haney et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5159–5163
5161
Table 2
with NMR, IR, MS, and HPLC (see Supplementary data). It should be
Inhibition of proliferation of prostate cancer cell lines
noted that two other minor products (isomers with double bond
configuration as (11E, 22Z) and (11Z, 22E), respectively) were ob-
tained as a mixture. The stereo-selective syntheses of these two
isomers have been pursued and will be reported in due course.
Inhibitory concentrations of CCR5 antagonists determined by
inhibition of chemokine induced calcium ion mobilization has
been demonstrated to correlate well with radioligand binding
inhibitory concentrations.29 Anibamine and its eight analogs along
with the established CCR5 antagonist TAK-779 (as a control), were
evaluated for their ability to inhibit CCL5 stimulated calcium mobi-
lization in MOLT-4 cells. This CCR5-expressing T-lymphoblastoid
cell line30 seemed to be the most sensitive one in our calcium
mobilization assay among all the cell lines we explored. The results
are shown in Table 1. The fact that the IC50 values of TAK-779
Compounds
ED50 SEa
DU-145
(lM)
M12
PC-3
9a (Anibamine)
3.26 0.35
2.18 0.34
1.92 0.44
3.24 0.46
0.43 0.13
1.93 0.47
2.57 0.07
4.20 2.99
2.67 0.03
1.02 0.17
0.84 0.02
1.31 0.15
0.36 0.10
0.46 0.07
0.97 0.01
0.76 0.12
0.60 0.15
1.41 0.18
1.13 0.01
0.64 0.06
0.64 0.08
0.28 0.07
0.19 0.04
0.55 0.19
0.31 0.05
0.24 0.08
0.43 0.19
10a
11a
9b
10b
11b
9c
10c
11c
a
Values shown were mean S.E. mean from at least three separate experiments
performed in triplicate. The ED50 (concentration to inhibit 50% of cell proliferation
compared with the control) values were calculated using Prism.
(7.9 2.5 nM) and anibamine (9a, 5.43 0.91
their CCL5 inhibitory binding affinities reported in literature
(TAK-779, 1.4 nM,13b and anibamine, 1.0 M25) provides a valida-
lM) correlated with
l
a given cell line, the analogs showed similar anti-proliferation
activity as indicated by their ED50 values, which is at least partially
in correlation to their comparable binding affinity to the receptor.
In general, the PC-3 cells appeared to be more sensitive to either
the M-12 or the DU-145 cells. This could be related to the different
receptor expression levels for each prostate cancer cell line.23,24,26
Among all the analogs, compound 10b appeared to be the most
potent agent against all three cancer cell lines. For example, it
showed about 10 times improvement in ED50 over the original lead
(anibamine) against M12 and PC-3 prostate cancer cell lines.
Therefore this compound was defined as the new lead.
tion of this experimental approach. For five- and six-membered
ring analogs of anibamine (9b, 9c, 10a, 10b, and 10c), their side
chain double bond configuration did not appear to influence bind-
ing affinity as significantly as for the seven-membered ring analogs
(11a, 11b, and 11c). In addition, it appeared that a seven-
membered ring was unfavorable for binding affinity. All of the
analogs were also tested for their capacity to stimulate calcium
release in the absence of CCL5, but none acted as agonists (data
not shown). Not surprisingly, most of the new ligands did not show
much improved receptor affinity in comparison to the parent
compound anibamine, likely due to the fact that at this stage the
structural modifications did not significantly alter the shape and
hydrophobicity of the molecule, and therefore no observable
change to the binding mode to the receptor either.
As indicated previously, the expression of CCL5 and CCR5 has
been observed in various prostate cancer cell lines, including
M12, DU145 and PC-3.26,29,31 Therefore, the anti-proliferative
activity of anibamine and its analogs was evaluated against these
prostate cancer cell lines. The first CCR5 antagonist, TAK-779, has
been reported to inhibit proliferation and invasion of cancer cells
pre-treated with CCL5 (10 ng/mL).24 Under such CCL5 stimulation
condition, TAK-779, at 500 nM, showed significant proliferation
inhibition effect (40–50%) for DU145 and LNCaP cell lines. A mod-
ified protocol without the CCL5 stimulation condition was adopted
here for anibamine and its analogs in an effort to avoid the poten-
tial non-specific stimulation effect of CCL5 interacting with other
chemokine receptors expressed in the cancer cells that it can bind
to20 and to better simulate a clinically applicable scenario for drug
development purpose. Results of these assays were summarized in
Table 2. Each compound showed dose-dependent anti-proliferative
activity in all three cell lines (data not shown). Across the board, for
Interestingly, in our assay TAK-779 showed lower anti-
proliferative activity (ED50 values, M-12, 20.40 1.10
lM;
DU-145, 57.27 9.47 M; and PC-3, 37.85 0.99 M, respectively)
l
l
than reported by others.24 This may be because the current studies
were performed in the absence of CCL5 stimulation. TAK-779
entered clinical trial based on its anti-HIV activity as a result of
optimization of a series of compounds by following their binding
affinity to the CCR5 receptor as well as their anti-HIV activities.13
Its mechanism of action against prostate cancer cell is not clear
and the involvement of the CCL5/CCR5 axis in prostate cancer
development is not yet defined. Studies to optimize lead com-
pounds based on their binding affinity to CCR5 as well as their
anti-cancer activities may help elucidate the role of CCL5/CCR5
pathway in prostate cancer progression. In this context, the fact
that anibamine and its analogs showed somewhat higher potency
than TAK-779 in the anti-proliferation assays suggests the possibil-
ity that these new ligands may also be affecting other target sites.
Because of the structure similarity of these ligands and their
limited ‘drug-like’ property based on Lipinski’s Rule of 5,32 more
extensive structural modification would be needed to further
understand their structure–activity relationship.
Table 1
Table 3
Inhibitory effects on CCL5 induced Ca2+ mobilization in MOLT-4/
CCR5 cell
Cytotoxicity results of anibamine and its analogs
Compounds
HC50 SEa
Sheep red blood cells NR/3T3
(l
M)
TC50 SEa
(l
M) TC50 SEa
WST-1/3T3
(lM)
Compounds
IC50 SEa
(lM)
9a (Anibamine)
5.43 0.91
6.53 1.79
7.80 1.38
10.01 0.38
4.60 1.60
15.24 7.87
8.40 0.94
9a (Anibamine) >100
22.65 7.38
2.52 0.75
2.79 1.11
19.00 1.65
6.81 1.77
1.98 0.47
7.66 0.91
0.83 0.35
1.86 0.18
23.47 2.36
4.35 0.57
5.08 2.40
29.08 0.60
17.53 4.56
3.76 0.47
10.09 1.21
4.76 0.58
11.66 1.48
10a
11a
9b
10b
11b
9c
10a
11a
9b
>100
77.1 6.7
>100
10b
11b
9c
10c
11c
58.1 12.5
96.2 6.5
63.0 11.4
>100
10c
11c
48.09 21.72
37.61 5.40
>100
a
a
Values shown were mean S.E. mean from at least three
Values shown were mean S.E. mean from at least three separate experiments
separate experiments performed in triplicate. The IC50 values
(concentration to inhibit 50% of calcium release induced by CCL5
compared with the control) were calculated using Prism.
performed in triplicate. The HC50 (concentration to induce 50% of hemolysis com-
pared with the control) values and TC50 (concentration to induce 50% of cell death
compared with the control) values were calculated using Prism.

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K. M. Haney et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5159–5163
Table 4
Therapeutic index (TI) for 9a (anibamine), 9b, and 10b
Compound
3T3 versus M12
3T3 versus DU145
3T3 versus PC3
NR
6.95
5.86
15.84
WST-1
NR
WST-1
NR
WST-1
9a (Anibamine)
9b
10b
7.20
8.98
40.77
22.21
52.78
14.80
23.01
80.78
38.11
20.04
67.86
35.84
20.77
103.86
92.26
To further verify whether the inhibitory effect of our lead com-
pound, anibamine, against prostate cancer cell line proliferation
was due to its inhibition of CCL5 binding on the chemokine recep-
tor CCR5, we retested its anti-proliferation activity against the
CCL5 (30 nM) treated M12 cell line. Compound 9, anibamine,
showed higher potency under this condition, as indicated by its
the MOLT-4/CCR5 cell line and TAK-779. We also thank Dr. Xianjun
Fang (Department of Biochemistry, VCU) for providing the NIH 3T3
cell line for the basal cytotoxicity screening.
Supplementary data
ED50 value at 0.84 0.08 lM. Such result at least partially
Supplementary data (chemical synthesis procedures and com-
pounds analysis data) associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2011.07.058.
supported our hypothesis that our lead compound may inhibit
the proliferation of prostate cancer cell M12 through its inhibition
of CCL5/CCR5 axial function.
Due to the reported hemolysis by anibamine,32 all the
compounds were further evaluated for their hemolytic toxicity at
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Acknowledgments
We are grateful to the funding support from US Army Prostate
Cancer Research Program PC073739, and A.D. Williams Multi-
school Research Award at VCU. The content is solely the responsi-
bility of the authors and does not necessarily represent the official
views of the US Army Prostate Cancer Research Program. We thank
NIH AIDS Research and Reference Reagent Program for providing

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