Structure activity relationship studies of natural product chemokine receptor CCR5 antagonist anibamine toward the development of novel anti prostate cancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.07.049

Recent studies have indicated that the CCR5 chemokine receptor may be a potential target for treating prostate cancer. Thus, development of CCR5 antagonists may provide novel prostate cancer therapy. Anibamine, a novel pyridine quaternary alkaloid isolated from Aniba sp., was found to effectively compete with ¹²⁵I-gp120 in binding to the chemokine receptor CCR5, with an IC₅₀ = 1 μM. Anibamine is the first natural product reported as a CCR5 antagonist, and thus provides a novel structural skeleton unique from other lead compounds that have generally been identified from high-throughput screening efforts. In order to refine the lead compound's structure and improve the therapeutic index of anibamine derivatives as potential anti prostate cancer agents, the approach of "deconstruction- reconstruction-elaboration" was applied in the structure-activity relationship studies of this work. Here, we report the design, syntheses and anti prostate cancer activities of anibamine and 17 analogues. The results from the in vitro and in vivo studies described here show that this class of compounds has potential to provide novel leads as anti prostate cancer agents.

Full text of DOI:10.1016/j.ejmech.2012.07.049

European Journal of Medicinal Chemistry 55 (2012) 395e408
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure activity relationship studies of natural product chemokine receptor
CCR5 antagonist anibamine toward the development of novel anti prostate cancer
agents
Feng Zhang a, Christopher K. Arnatt a, Kendra M. Haney a, Harrison C. Fang a, John E. Bajacan a,
,
,c,
*
Amanda C. Richardson b, Joy L. Ware b c, Yan Zhang a
a Department of Medicinal Chemistry, Virginia Commonwealth University, 800 E Leigh Street, Richmond, VA 23298-0540, USA
b Department of Pathology, Virginia Commonwealth University, Richmond, VA 23298-0662, USA
c Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298-0037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Recent studies have indicated that the CCR5 chemokine receptor may be a potential target for treating
prostate cancer. Thus, development of CCR5 antagonists may provide novel prostate cancer therapy.
Anibamine, a novel pyridine quaternary alkaloid isolated from Aniba sp., was found to effectively compete
Received 5 April 2012
Received in revised form
25 July 2012
Accepted 31 July 2012
Available online 7 August 2012
with 125I-gp120 in binding to the chemokine receptor CCR5, with an IC50 ¼ 1
mM. Anibamine is the first
natural product reported as a CCR5 antagonist, and thus provides a novel structural skeleton unique from
other lead compounds that have generally been identified from high-throughput screening efforts. In order
to refine the lead compound’s structure and improve the therapeutic index of anibamine derivatives as
potential anti prostate cancer agents, the approach of “deconstructionereconstructioneelaboration” was
applied in the structureeactivity relationship studies of this work. Here, we report the design, syntheses
and anti prostate cancer activities of anibamine and 17 analogues. The results from the in vitro and in vivo
studies described here show that this class of compounds has potential to provide novel leads as anti
prostate cancer agents.
Keywords:
Prostate cancer
Chemokine receptor CCR5
Antagonist
Anibamine
Published by Elsevier Masson SAS.
1. Introduction
there is a high need for treatments that will stop the metastasis and
invasion of prostate cancer cells.
Prostate cancer is the most common non-cutaneous solid cancer
occurring among men in the USA. The most recent estimates from
the American Cancer Society [1] showed that about 240,890 new
cases of prostate cancer would be diagnosed in 2011, with 33,720
deaths attributable to prostate cancer in the United States alone.
About one of six males in the U.S. may be afflicted with this cancer,
and the risk is increased drastically for older males. Moreover, the
risk of death due to metastatic prostate cancer is 1 in 36. Genetics,
age, race, diet, and family history, and even lifestyle may all
contribute to prostate cancer risk [2]. The treatment options for
prostate cancer are surgery, chemotherapy, cryotherapy, hormonal
therapy and/or radiation, but all are only beneficial at the early
stages, with no significant effects after metastasis [3]. Therefore,
Historically, chronic inflammation has been believed to play
a role in the development of many types of cancer in humans [4e7],
including prostate cancer [2,8]. During the past decade, genetic
studies have provided evidence that infection or inflammation of
the prostate does contribute to the development of prostate cancer.
Inflammation is thought to incite carcinogenesis by causing cell and
genome damage, promoting cellular turnover, and creating a tissue
microenvironment that can enhance cell replication, angiogenesis
and tissue repair [9]. It is usually self-limiting and thus does not
cause any harm to the body. However, sometimes the control is lost
and inflammation becomes chronic leading to various diseases
such as Crohn’s disease, Blau syndrome and cancer, among others
[10]. The inflammatory response begins when injured tissue cells
release chemical signals (inflammatory mediators or cytokines),
which then attract other immune cells, increase capillary perme-
ability, and cause fever. Chemokines are a family of small cytokines
that play a number of important roles in acute and chronic
inflammation and also have immunoregulatory functions. These
specialized cytokines attract monocytes, macrophages, neutrophils,
* Corresponding author. Department of Medicinal Chemistry, Virginia
Commonwealth University, 800 E Leigh Street, Richmond, VA 23298-0540, USA.
Tel.: þ1 804 828 0021.
E-mail address: yzhang2@vcu.edu (Y. Zhang).
0223-5234/$ e see front matter Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2012.07.049

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F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
and eosinophils, cells that are responsible for nonspecific inflam-
matory immune response [11]. Since chronic inflammation has
been linked to tumors and cancer, chemokines may also play
important roles in creating a microenvironment that favors or
enhances tumor survival. Chemokine receptors expressed by target
cells may promote numerous downstream events related to tumor
angiogenesis, growth, survival and metastasis through the binding
of these soluble protein ligands [4].
natural products contain more diverse skeletons with wider ranges of
shape and chemical features, often with specific biological activities
[33,34]. Thus, natural products are desirable and useful resources for
drug discovery and development.
Anibamine (1), a unique pyridine quaternary alkaloid recently
isolated from the Guyanese plant Aniba panurensis [35,36], has been
found to effectively compete with 125I-gp120 for binding with the
chemokine receptor CCR5 with an IC50 value of 1 mM. As the first re-
Expression of the inflammatory chemokine CCL5 (RANTES) by
tumor cells is thought to correlate with the progression of several
cancers [6,12e14]. For example, the chemokine CCL5 was shown to
induce breast cancer cell migration that was mediated by the che-
mokine receptor CCR5. It was also shown that the expression of CCR5
and CCL5 correlated with breast cancer disease progression while
a CCR5 antagonist inhibited breast tumor growth in the presence of
CCL5 [15,16]. A recent investigation also showed that CCR5 is over-
expressed in prostate cancer specimens [17]. A preclinical study of
the CCR5 antagonist TAK-779 [18] showed that TAK-779 blocked
RANTES-induced cell invasion and proliferation of prostate cancer
cells [14]. Thus, development of an appropriate chemokine receptor
CCR5 antagonist may provide a novel prostate cancer therapy.
CCR5, a G-protein-coupled receptor, has also been identified as an
essential co-receptor for HIV-1 entry to host cells [19,20] and has thus
become an attractive target for development of anti-HIV therapeutics
[21e23]. A number of small molecule CCR5 antagonists developed
based on leads identified through high-throughput screening, e.g.,
SCHC [24], aplaviroc [25], vicriviroc [26,27], maraviroc [28] and TAK-
779 [18,29] (Fig. 1), have shown potent activities in blocking che-
mokine function and HIV entry. However, SCHC caused a modest but
dose-dependent prolongation of the corrected cardiac QT interval
(QTc) [27]; the vicriviroc clinical trial was stopped because the drug
did not meet primary efficacy endpoints in late stage trials [30]; and
development of TAK-779 as an anti-HIV-1 agent was discontinued
due to its poor oral bioavailability [31]. The first CCR5 antagonist in
clinical trials, aplaviroc, showed initial potent antiviral activity but,
after severe hepatotoxicity occurred in several patients, its develop-
ment was stopped in October 2005 [25]. In fact, to date only one drug,
maraviroc, has been approved by the FDA (on August 6, 2007),
although concerns were raised that maraviroc could be associated
with increased risks of liver damage, lymphoma, infections and heart
attack [32]. It is clear that, in order to develop new CCR5 antagonists,
there is an urgent need to explore new chemical structures and
templates with a wider range of structural features. Compared to
high-throughput screening hits, lead compounds derived from
ported natural product that is a CCR5 antagonist, anibamine possesses
a structural skeleton that is remarkably different from all previous
lead compounds, all of which were discovered through high-
throughput screening studies. Recent studies also demonstrated
that anibamine produced significant inhibition of prostate cancer cell
proliferation at micromolar to submicromolar levels as well as sup-
pressing adhesion and invasion of the highly metastatic M12 prostate
cancer cell line [37]. These findings suggest that anibamine may serve
as a new lead for development of CCR5 antagonists with potential
applications in prostate cancer therapy. In this paper we explore and
define the structureeactivity relationships of anibamine and its
analogues as anti prostate cancer agents with application of the
“deconstructionereconstructioneelaboration” approach. The results
reported here will be the basis of future studies in our long-term goal
to discover an optimized CCR5 antagonist.
2. Results and discussion
2.1. Molecular design
The “deconstructionereconstructioneelaboration” approach
(Fig. 2) has been successfully applied to improve the pharmacolog-
ical activities of both synthetic agents and natural products [38e41].
In this case, each structural component of the lead was “removed”
one at a time to test the influence of that component on CCR5
antagonism and anti prostate cancer activity. Once the essential
structural components were defined, they were retained in the core
structure that was then further modified to improve these activities.
2.2. Chemistry
The total synthesis of anibamine was first reported by our
laboratory a few years ago [42]. Very recently, we accomplished an
improved synthetic methodology with high stereo- and regio-
selectivity [43]. These approaches were applied in the present work
for the syntheses of newly designed derivatives of anibamine to
O
N
O
CH3
N
O
O
OH
N
N
N
N
N
O
Br
F3C
NH
N
N
N
N
O
HOOC
O
O
Vicriviroc
Aplaviroc
SCHC
N
N
H
N
N
F
N
N
O
N
HN
Cl
F
O
TFA
O
Maraviroc
Anibamine
TAK-779
Fig. 1. Chemical structures of anibamine and some of the known CCR5 antagonists.

F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
397
Deconstruction:remove
Elaboration: different configuration: cis or
trans; or saturated side chain
this side chain to test
its influence on the
anti-cancer activity
H3C
( )5
Deconstruction:remove
this side chain to test
its influence on the
anti-cancer activity
CH3
( )5
TFA
N
Deconstruction: remove
the ring system to see its
influence on activity
Elaboration: different configuration: cis or
trans; or saturated side chain
Fig. 2. Initial structural modifications of anibamine.
identify the critical pharmacophore and, ideally, a next generation
lead compound for drug development.
Based on molecular modeling and docking studies of anibamine and
three other potent CCR5 antagonists [49], the positively charged
nitrogenous center of the core ring system appeared to be especially
important by interacting with Glu283 in the common antagonist
binding locus in the CCR5 receptor. The first ring deconstruction
derivative was analogue 18. In it, a methyl group was retained at
position 2 to support retention of the overall conformation, which
might have been lost by removal of the fused ring. The synthetic route
for 18 is shown in Scheme 3. Bromination of 2,4,6-trimethylpyridine
was conducted with excess NBS, with added TFA to boost the acidity
of the concentrated sulfuric acid medium [50]. The dibrominated
product 17 was generated quantitatively at 50 ꢀC. Subsequently,
under Suzuki cross-coupling conditions, 3,5-di((Z)-dec-1-enyl)-
2,4,6-trimethylpyridine (18) was obtained in excellent yield.
The second molecule, 21, in the ring deconstruction set was
designed to retain the count of carbon atoms (from the aliphatic
ring) to minimize the change in hydrophobicity. To facilitate the
synthesis, a methoxyl group was applied to cap the end of the new
side chain. The synthesis scheme for 21 (Scheme 4) is nearly iden-
tical to the total synthesis of anibamine we reported earlier [43]. In
order to regioselectively introduce 3-methoxypropyl at the 2-
position, the key intermediate 2,3,5-tribromo-4,6-dimethylpyridine
was coupled with methyl propargyl ether to afford compound 3,5-
dibromo-2-(3-methoxyprop-1-ynyl)-4,6-dimethylpyridine (19) in
94.6% yield. Catalytic hydrogenation of the triple bond, followed by
Suzuki coupling, yielded the final compound 21.
2.2.1. Side chain deconstruction
The first deconstruction efforts were to remove the side chains at
either position 3 or 5 on the core ring of the molecule. This tests the
role of each side chain with respect to the anticancer activity of
anibamine. The syntheses of analogues retaining the 5-position side
chain are illustrated in Scheme 1. Bromination of commercially
available 2-amino-4,6-dimethylpyridine in glacial acetic acid gave
the desired 2-amino-5-bromo-4,6-dimethylpyridine (1) [44], which
was diazotized under Br2/NaNO2 in 48% HBr to afford 2 [45]. The
regioselective synthesis of the key intermediate 2-alkynyl-5-bromo-
4,6-dimethylpyridine (3) was achieved by reacting 2 with a protected
terminal acetylene under palladium-catalyzed Sonogashira condi-
tions [46,47] with 76% yield. Then, following the similar scheme for
the total synthesis of anibamine [43], the corresponding analogue 7
was finished with yield of 79%. In order to prepare analogues
retaining the side chain at the 3-position (Scheme 2), we first
condensed cyanoacetamide and acetylacetone in
a potassium
carbonate solution of approximately pH 9 at room temperature
overnight [42]. The product 8 precipitates in 92% yield. The bromi-
nation of 8 with TBAB/P2O5 led to the 2-bromo-substituted inter-
mediate 9 [48]. After hydrolysis and Hofmann rearrangement [48],
the key intermediate 10 was afforded in 75% yield. Following
palladium-catalyzed Sonogashira coupling reactions, reduction,
diazotization and bromination, the intermediates 13 were afforded in
reasonable yields. Applying a similar scheme as used for compound 7
produced analogue 16 with a satisfactory yield of 78%.
2.2.3. Simultaneous deconstruction of core ring and side chains
In order to further elucidate the structureeactivity relationship
of the core ring and the side chain functionalities, analogues that
simultaneously deconstructed both the core ring and side chains
were also designed and synthesized. These compounds were
synthesized following procedures similar to those for 7 and 16.
Synthesis of analogue 24 (Scheme 5) began with intermediate 2,
2.2.2. Core ring deconstruction
By deconstructing the rings, we aimed to clarify the contributions
of anibamine’s fused five-member ring; the aliphatic portion of the
core ring system was thus removed in deconstruction derivatives.
Br
Br
Br
i
ii
iii
iv
N
N
2
Br
N
1
NH2
N
NH2
OPMB
3
6
Br
6
6
v
vi
N
vii
N
N
N
Cl
OH
OPMB
OPMB
5
7
6
4
Scheme 1. The synthesis of 5-position side chain analogues. Reagents and conditions: (i) Br2, CH3COOH, 68%; (ii) Br2, NaNO2, HBr (48%), 92%; (iii) HC^CCH2OPMB, CuI,
PdCl2(PPh3)2, Piperidine, 76%; (iv) H2, PtO2, TEA, EtOH, 86%; (v) diisopropyl (Z)-1-decenylboronate (1.5 equiv), Pd(OAc)2, PPh3, Na2CO3, 58%; (vi) 1 N HCl, EtOH, 74%; (vii) MsCl, TEA,
79%.

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F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
CN
OH
O
O
O
CN
Br
NH2
Br
N
ii
i
iii
iv
+
H2N
NH2
N
N
N
8
9
10
NH2
Br
v
vi
vii
OPMB
OPMB
N
N
N
OPMB
12
13
11
6
6
6
x
viii
viiii
N
N
N
Cl
16
OPMB
OH
14
15
Scheme 2. The synthesis of 3-position side chain analogues. Reagents and conditions: (i) K2CO3, H2O, 92%; (ii) TBAB, P2O5, 95%; (iii) H2SO4, 120 ꢀC, 75%; (iv) Br2, NaOH, 100%; (v)
HC^CCH2OPMB, CuI, PdCl2(PPh3)2, Piperidine, 69%; (vi) H2, PtO2, TEA, EtOH, 77%; (vii) CuBr2, BuNO2, CHBr3, 50%; (viii) diisopropyl (Z)-1-decenylboronate (1.5 equiv), Pd(OAc)2,
PPh3, Na2CO3, 84%; (viiii) 1N HCl, EtOH, 80.3%; (x) MsCl, TEA, 78%.
which was converted to 3-bromo-6-(3-methoxyprop-1-ynyl)-2,4-
dimethylpyridine (22) by the Sonogashira coupling reaction using
commercial available 3-methoxyprop-1-yne. Catalytic hydrogena-
tion of the triple bond, followed by Suzuki coupling, afforded the
final compound 24 in good yield.
Scheme 6 describes the synthesis of analogue 28. 2-Bromo-4,6-
dimethylpyridin-3-amine (10) was coupled with 3-methoxyprop-
1-yne under Sonogashira coupling conditions, followed by catalytic
hydrogenation, diazotization and bromination, and finally Suzuki
coupling, to yield 28. Similarly, compound 30 was prepared
(Scheme 7) to examine the influence of removal of both the entire
aliphatic ring (retaining only a methyl vestige as in 18) and one side
chain. The procedure was very similar to that of 18, except that the
bromination reaction was carried out at room temperature.
variants of anibamine. Compounds 39 and 41 tested a combination
of the deconstruction and elaboration illustrated above by removing
one side chain in anibamine while changing the stereochemistry on
the other. The chemical syntheses of compounds 39 and 41 are
shown in Scheme 9. Compounds 40 and 42 were prepared by simply
reducing the double bonds of 39 and 41, respectively.
2.2.6. Elaboration of ring size
Because the positively charged nitrogen atom of anibamine is
believed to be an essential feature for binding to the CCR5 receptor,
we wanted to investigate how changes in the size (and resulting
conformation) of the anibamine core might change its binding
affinity and, further, influence anticancer activity. The ring size-
modified anibamine analogues 37 and 38 (Fig. 3) were prepared
following previously reported procedures, and both final
compounds were obtained in good yields [51].
2.2.4. Elaboration of side chains
In designing the set of compounds that elaborate the side chain,
our goal was to elucidate the role of bond order and configuration
in the two side chains with respect to the observed anticancer
activity. In order to change the side chain alkene configuration from
cis to trans, (E)-dec-1-enylboronic acid was used instead of diiso-
propyl (Z)-1-decenylboronate (Scheme 8). Under the same Suzuki
cross-coupling reaction conditions as above, the coupled products
with E-configuration were afforded stereoselectively in excellent
yield. The unsaturated analogue, compound 34, was prepared by
reducing the double bond of 33. Furthermore, we recently reported
the regio- and stereo-selective syntheses of the (11E, 22Z) and (11Z,
22E) anibamine isomers (35 and 36, respectively, Fig. 3) [43].
In summary, a series of anibamine analogues were designed
following
the
“deconstructionereconstructioneelaboration”
approach to identify the critical pharmacophore of the natural
product lead compound. A total of seventeen novel ligands that are
anibamine derivates have been synthesized through multi-step
chemical reactions. They all have been fully characterized by NMR,
IR, MS, and HPLC (see Supporting information) prior to character-
ization of their biological activities, which will be described below.
2.3. Biological screening
We adopted a series of screening methods to assess the bio-
logical activity of the newly synthesized anibamine analogues as
anti prostate cancer agents. First, a Ca2þ mobilization assay was
developed to characterize their binding affinity and function
against the CCR5 receptor. Similar Ca2þ mobilization assays have
previously been applied in characterizing the binding affinity and
function of other GPCR ligands [52,53]. Second, since the expres-
sion of CCL5 and CCR5 has been observed in various prostate cancer
cell lines, including M12, DU145 and PC-3 [41,52,54], the anti-
proliferative activities of anibamine and its analogues were also
evaluated against these cell lines. Further, a basal cytotoxicity assay
was conducted to evaluate the therapeutic window for these
ligands to help us identify our “next generation” lead. Lastly,
a prostate tumor mouse model was applied to test in vivo our lead’s
anti prostate cancer activity.
2.2.5. Side chain deconstruction/stereochemical elaboration
Further deconstruction/elaboration products based on the anib-
amine main skeleton were designed to exhaust the structural
6
Br
Br
i
ii
6
N
N
N
17
18
Scheme 3. The synthesis of the 2-methyl analogue (18). Reagents and conditions: (i)
NBS, TFA, H2SO4, 50 ꢀC, 100%; (ii) diisopropyl (Z)-1-decenylboronate (4.0 equiv),
Pd(OAc)2, PPh3, Na2CO3, 82%.

F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
399
6
Br
Br
Br
Br
Br
Br
Br
iii
6
ii
i
OCH3
N
N
N
N
OCH3
19
20
OCH3
21
Scheme 4. The synthesis of the methoxypropyl analogue (21). Reagents and conditions: (i) HC^CCH2OCH3, CuI, PdCl2(PPh3)2, Piperidine, 95%; (ii) H2, PtO2, TEA, EtOH, 80%; (iii)
diisopropyl (Z)-1-decenylboronate (4.0 equiv), Pd(OAc)2, PPh3, Na2CO3, 80%.
2.3.1. Ca2þ mobilization assay
lines. We adopted a modified protocol (without CCL5 stimulation)
for anibamine and its analogues in an effort to simulate a more
clinically applicable scenario as well as to avoid the possibility of
non-specific stimulation arising from CCL5 interacting with other
chemokine receptors normally expressed in these cancer cells.
To assess the anti-proliferative effect of these compounds, three
prostate cancer cell lines were grown in the presence of anibamine
and its analogues for 72 h. At that time, the colorimetric reagent
WST-1 (Roche) was added and after 3 h of incubation, absorbance
values were read on a FlexStation-3 (Molecular Devices) microplate
reader. Most of the compounds showed dose-dependent anti-
proliferative activity in all three cell lines (data not shown). The
ED50 values of these compounds are listed in Tables 1 and 2.
For all cell lines, the ring-deconstructed derivatives (18, 21, 24,
28 and 30) were found to be, at best, weakly active up to the highest
tested concentrations, which is consistent with the calcium mobi-
lization assay results. On the other hand, compounds 7 and 16, each
carrying only one aliphatic side chain, showed relatively higher
activity than the parent. However, considering their low affinity to
CCR5 (i.e., calcium mobilization), this anti-proliferation effect on
prostate cancer cells may stem from interaction with an off-target
protein. The elaborated analogues (33, 34, 35 and 36, Table 1) e
containing the same core but different double bond configurations
The inhibitory effects of CCR5 antagonists in chemokine-
induced calcium ion mobilization have been demonstrated to
correlate well with their affinity in radioligand competition binding
assays [51e53]. In this study, anibamine and its analogues were
tested for their ability to inhibit chemokine CCL5-induced calcium
mobilization in MOLT-4/CCR5 cells as an indicator of their antag-
onism of CCR5 [51]. The results are shown in Tables 1 and 2. All of
the analogues were tested first for their capacity as agonists to
stimulate calcium release in the absence of CCL5. None of the
anibamine analogues acted as agonists of CCR5 (data not shown),
but most of the acted as antagonists at the low micromolar level.
However, the core ring deconstruction analogues, 18, 21, 24, 28 and
30, had no significant antagonism towards CCR5 (IC50 > 30 mM),
which suggests that the positively charged nitrogenous center is
critical for receptor binding. The side chain deconstruction prod-
ucts, 7 and 16, showed significantly decreased inhibition of
receptor function compared to the parent compound anibamine,
which further indicated that both side chains should be retained.
For the compounds we synthesized elaborating the two side chains
(33, 34, 35 and 36), double bond configuration (trans, cis, or satu-
rated) showed an insignificant difference overall in their functional
inhibition of the receptor as measured with this assay. Further-
more, both the six- and seven-member ring analogues of anib-
amine (37 and 38), showed relatively weaker activity compared to
the parent lead. For the dual modification analogues (compounds
39 through 42), similar to the other side chain deconstruction
analogues in Table 1 (7 and 16), much lower receptor inhibition
activity was observed compared to that of the parent natural
product.
on the two side chains
e showed similar or higher anti-
proliferation activities compared with anibamine. For the six- and
seven-member ring analogues (37 and 38), in line with their
antagonist activity, ring size did not appear to significantly influ-
ence anti-proliferation activity against the three cancer cell lines.
Overall, the deconstruction/elaboration modification analogues
(39e42) showed higher inhibition effects in this assay compared to
the natural product lead, which again suggests off-target interac-
tions since the Ca2þ mobilization assay indicates much lower
activities for these compounds.
2.3.2. Proliferation of prostate cancer cell lines without stimulation
of CCL5
Immortalized prostate cancer cell lines provide in vitro models
for studying this cancer. They allow the study of anti-proliferative,
anti-invasive and anti-metastasis factors. For this project, three
immortalized cell lines were used to study the effect of CCR5
antagonists on prostate cancer cell proliferation. The first reported
CCR5 antagonist, TAK-779, was reported to inhibit proliferation and
invasion of cancer cells that were pre-treated (10 ng/mL) with CCL5
[14]. Under this stimulation, TAK-779 at 500 nM showed significant
inhibition of proliferation (40%e50%) for the DU145 and LNCaP cell
2.3.3. Basal cytotoxicity
Since some hemolysis activity has been previously reported for
anibamine [36], we were interested whether the observed anti-
proliferative activity of its analogues might also be associated with
interactions at off-target proteins. Thus, these compounds were
further evaluated for their basal cytotoxicity by adopting a Neutral
Red protocol in NIH 3T3 cells [55e57]. The associated data are
presented in Tables 1 and 2. For all side chain-deconstructed
Br
6
Br
Br
i
ii
iii
N
N
N
N
Br
OCH3
OCH3
OCH3
2
22
23
24
Scheme 5. The synthesis of 3-((Z)-dec-1-enyl)-6-(3-methoxypropyl)-2,4-dimethylpyridine (24). Reagents and conditions: (i) HC^CCH2OCH3, CuI, PdCl2(PPh3)2, Piperidine, 96%; (iv)
H2, PtO2, TEA, EtOH, 57%; (v) diisopropyl (Z)-1-decenylboronate (1.5 equiv), Pd(OAc)2, PPh3, Na2CO3, 80%.

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F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
2.3.5. In vivo study of lead compounds on tumor growth
NH2
NH2
Br
NH2
Lastly, to assess the effect of anibamine and its analogue 38 on
prostate tumor growth in vivo, we injected mice subcutaneously with
M12 prostate cancer cells M12, followed by treatment with drug at
a dose of 0.3 mg/kg (or saline) once every four days for total of sixteen
days before data analysis. No apparent toxicity of the compounds to
the animal, e.g., significant weight loss, was observed during the
experiments. The experiment was terminated when the control
group mice were lost due to their fully developed tumors. The results,
in Fig. 5, show that anibamine reduced the subcutaneous growth of
M12 tumors inathymicnudemiceby nearly 50% atthisdose, while 38
reduced the growth of the tumors, even more, by over 70% (P
value < 0.05). The tumor growth reduction effects were also signifi-
cantly different between anibamine and 38 (P value < 0.05). These
results establish compound 38 as our new lead for future molecular
design and development of a novel anti prostate cancer agent.
i
ii
OCH3
OCH3
N
N
N
10
25
26
6
Br
iii
iv
N
OCH3
N
OCH3
27
28
Scheme 6. The synthesis of 3-((Z)-dec-1-enyl)-2-(3-methoxypropyl)-4,6-dimethylpyridine
(28). Reagents and conditions: (i) HC^CCH2OCH3, CuI, PdCl2(PPh3)2, Piperidine, 64%;
(ii) H2, PtO2, TEA, EtOH, 85%; (iii) CuBr2, BuNO2, CHBr3, 51%; (iv) diisopropyl (Z)-1-
decenylboronate (1.5 equiv), Pd(OAc)2, PPh3, Na2CO3, 72%.
3. Conclusions
analogues, including those with stereochemical elaboration (7, 16
and 39e42), significantly higher cytotoxicities towards 3T3 cells
were observed as compared with anibamine. Considering their
lower receptor effects (Ca2þ mobilization) compared to the parent
compound, clearly deconstruction of the side chain produces
analogues that are non-selective. For compounds 33e36, changes
in the double bond configuration seemed to significantly increase
their basal cytotoxicity as well, which was surprising since our
preliminary molecular modeling studies suggested that the overall
conformation of the molecule will not change dramatically with
this elaboration [43]. Compounds 18, 21, 24, 28, and 30, the ring
deconstruction analogues, were not tested due to their low activity
in both the calcium mobilization and anti-proliferation assays.
Lastly, both ring size-elaboration products showed relatively low
basal cytotoxicity with the six-member ring analogue 37 more
tolerated with a TC50 similar to that of anibamine.
There is no cure for metastatic prostate cancer. Anibamine and its
analogues may represent a new class of compounds that can disrupt
proliferation and metastasis of prostate cancer. A series of these
analogues was designed and synthesized based on the
“deconstructionereconstructioneelaboration”
concept.
All
compounds that retained the core fused ring system acted as CCR5
antagonists in calcium mobilization assays. These analogues were also
able to inhibit proliferation of prostate cancer cells in vitro. The core
ring system appeared to be far more important than the configuration
of double bonds in the side chains with respect to their overall anti-
proliferation activity and cytotoxicity. One of these analogues, 38,
inhibited growth of M12 prostate tumor cells significantly in an in vivo
model. Further structure activity relationship studies of anibamine
and the new lead may help identify the next generation of anti
prostate cancer agents as well as elucidate the potential role of CCL5/
CCR5 axial in prostate cancer development and progression.
2.3.4. Proliferation of prostate cancer cell line under stimulation of
CCL5
4. Experimental section
To further simulate experimental conditions reported in the
literature, the growth of the M12 prostate cancer cell line under the
stimulation of different concentrations of CCL5 was next tested. The
results are summarized in Fig. 4. It is apparent that the proliferation
of M12 cells show dose response to the stimulation of CCL5; under
CCL5 stimulation of 30 nM, this proliferation was significantly
higher than without CCL5. To verify whether the inhibitory effects
of our anibamine lead compound and the two ring size-elaborated
analogues (37 and 38) against prostate cancer cells proliferation
were due to inhibition of CCL5 binding on CCR5, we tested this
activity against CCL5 (10 and 30 nM) treated M12 cells. As shown in
Table 2, under these conditions, all three compounds showed
higher inhibition activity than without CCL5 stimulation. This
seems to support the hypothesis that their effect on proliferation of
M12 cells was related to their interfering in the function of the
CCL5/CCR5 axial in these cells. Most interestingly, compound 38
seemed to be more sensitive to the stimulated M12 cells than
compound 37.
4.1. Chemistry
All reagents were used directly as obtained commercially unless
otherwise noted. Melting points were determined on a Fisher-
Scientific melting point apparatus and are uncorrected. 1H NMR
and 13C NMR spectra were taken on a Bruker Avance III 400 MHz
spectrometer with tetramethylsilane as an internal standard. 1H
spectra were collected at 400 MHz and 13C spectra were collected at
100 MHz. Chemical shift values are expressed in ppm and coupling
constants (J) in Hertz. Infrared spectra were obtained on a Nicolet
5ZDX FT-IR spectrometer. LCeMS was performed on a Waters
Micromass QTOF-2 instrument using ESI ion source. Column chro-
matography was performed on silica gel (grade 60 mesh; Bodman
Industries, Aston, PA). The purity of the title compounds was
assessed by means of a Varian Prostar 210 HPLC instrument equip-
ped with Prostar 325 UV/vis as the detector and working at 254 nm.
A Microsorb 100-5 CN column (250 ꢁ 21.5 mm) chromatographic
column was used at room temperature, a flow rate of 4.0 mL/min and
run over 30 min. The eluent was acetonitrile (0.5% TFA)/water (60/
40). Using the analytical conditions reported above, the retention
times of the compounds range between 5.79 and 12.80 min. All the
synthesized compounds were generally more than 95% pure.
Br
6
i
ii
N
N
N
30
29
4.1.1. 2-Amino-5-bromo-4,6-dimethylpyridine (1)
A solution of 2-amino-4,6-dimethylpyridine (1.22 g, 10 mmol) in
10 mL of glacial acetic acid under N2 was treated with a solution of
1.6 g (10 mmol) Br2 in 2 mL of glacial acetic acid over 15 min with
Scheme 7. The synthesis of compound 30. Reagents and conditions: (i) NBS, TFA,
H2SO4, r.t., 90%; (ii) diisopropyl (Z)-1-decenylboronate (4.0 equiv), Pd(OAc)2, PPh3,
Na2CO3, 82%.

F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
401
6
6
6
Br
i
ii
iii
v
N
N
N
N
Br
6
6
6
Cl
33
Cl
OPMB
OPMB
34
31
32
Scheme 8. The synthesis of compounds 33 and 34. Reagents and conditions: (i) (E)-dec-1-enylboronic acid (4.0 equiv), Pd(OAc)2, PPh3, Na2CO3, 87%; (ii) 1 N HCl, EtOH, 91%; (iii)
MsCl, TEA, 71%; (v) H2, 10%Pd/C, 66%.
water bath cooling to keep the reaction temperature below 20 ꢀC.
The solution became a solid mass and was allowed to stand at room
temperature for 1 h. After cooling in an ice bath, the material was
made alkaline with 20% cold NaOH solution. Then the mixture was
extracted with CH2Cl2 three times, the combined organic layer was
dried over anhydrous Na2SO4, then filtered and concentrated. The
residue was purified on silica gel (HexaneeEtOAc, 2:1 to 1:1) to
afford the product (1.37 g, 68%) as a yellow solid. Mp 143e144 ꢀC.
(lit. [40] 143 ꢀC). 1H NMR (CDCl3) 6.24 (s, 1H), 4.32 (br, 2H), 2.50
(s, 3H), 2.28 (s, 3H).
4.1.4. 2-(3-(4-Methoxybenzyloxy)propyl)-5-bromo-4,6-
dimethylpyridine (4)
A solution of 3 (272 mg, 0.76 mmol) in 3 mL of ethanol and
0.06 mL of triethylamine was hydrogenated over 7 mg (0.03 mmol)
of PtO2 for 3 h. The reaction mixture was filtered and concentrated.
The residue was purified on silica gel (HexaneeEtOAc, 8:1) to afford
the product (237 mg, 86%) as a pale yellow oil. IR (KBr, cmꢂ1
)
nmax
:
2930, 2853, 1511, 1244, 1097, 1022, 818. 1H NMR (CDCl3)
d 7.25 (m,
2H), 6.87 (m, 2H), 6.82 (s, 1H), 4.43 (s, 2H), 3.80 (s, 3H), 3.48 (t,
J ¼ 6.4 Hz, 2H), 2.75 (m, 2H), 2.64 (s, 3H), 2.34 (s, 3H), 1.96e2.03 (m,
2H). 13C NMR (CDCl3)
d 159.14, 159.06, 156.6, 147.2, 130.7, 129.2,
4.1.2. 3,6-Dibromo-2,4-dimethylpyridine (2)
129.2, 122.7, 121.4, 113.8, 113.8, 72.5, 69.2, 55.3, 34.1, 29.8, 25.6, 23.2.
To a solution of 1 (201 mg, 1 mmol) in HBr (48%, 3 mL) at 0 ꢀC,
a solution of NaNO2 (172 mg, 2.5 mmol) in H2O (0.6 mL) was added
dropwise. Then the mixture was stirred at 0e5 ꢀC for 30 min, Br2
(448 mg, 2.8 mmol) was added, while keeping the temperature
below 5 ꢀC. After a further 30 min, the mixture was made alkaline
with 20% cold NaOH solution. Then the mixture was extracted with
EtOAc three times, the combined organic layer was dried over
anhydrous Na2SO4, filtered and concentrated. The residue was
purified on silica gel (HexaneeEtOAc, 10:1) to afford the product
(243 mg, 92%) as a pale yellow solid. Mp 62e63 ꢀC. (lit. [41] 63 ꢀC).
MS (ESI) m/z: 386.3 (M þ Na)þ.
4.1.5. 6-(3-(4-Methoxybenzyloxy)propyl)-3-((Z)-dec-1-enyl)-2,4-
dimethylpyridine (5)
Pd(OAc)2 (7 mg, 0.03 mmol), PPh3 (25 mg, 0.1 mmol), and 4
(196 mg, 0.54 mmol) were stirred in toluene (1.08 mL) and aq
Na2CO3 (0.54 mL, 2 M) under N2 for 0.5 h. To this solution was
added a solution of diisopropyl (Z)-1-decenylboronate (216 mg,
0.81 mmol) in ethanol (0.54 mL). The solution was refluxed over-
night, then diluted with EtOAc, filtered and concentrated, after
which the residue was purified on silica gel (HexaneeEtOAc, 8:1) to
1H NMR (CDCl3)
d 7.20 (s, 1H), 2.65 (s, 3H), 2.37 (s, 3H).
afford the product (132 mg, 58%) as a pale yellow oil. IR (KBr, cmꢂ1
nmax: 2999, 2923, 2852, 1590, 1512, 1245, 1098, 1036, 819, 723. 1H
NMR (CDCl3) 7.27 (m, 2H), 6.87 (m, 2H), 6.80 (s, 1H), 6.22 (d,
)
4.1.3. 2-(3-(4-Methoxybenzyloxy)prop-1-ynyl)-5-bromo-4,6-
dimethylpyridine (3)
d
A solution of 2 (263 mg, 1 mmol), 1-methoxy-4-((prop-2-
ynyloxy)methyl)benzene (264 mg, 1.5 mmol), CuI (19 mg,
0.1 mmol) and piperidine (255 mg, 3 mmol) in 6 mL THF was
purged by the passage of N2 through the solution, and 35 mg
(0.05 mmol) of PdCl2(PPh3)2 was added all at once. The reaction
mixture was stirred at room temperature for 3 h. Then the mixture
was diluted with EtOAc, washed with water, saturated brine and
dried (Na2SO4), filtered and concentrated. The residue was purified
on silica gel (HexaneeEtOAc, 8:1) to afford the product (272 mg,
J ¼ 11.2 Hz, 1H), 5.69 (m, 1H), 4.44 (s, 2H), 3.80 (s, 3H), 3.51 (m, 2H),
2.78 (m, 2H), 2.39 (s, 3H), 2.14 (s, 3H), 1.98e2.06 (m, 2H), 1.76e1.81
(m, 2H), 1.19e1.34 (m, 12H), 0.86 (t, J ¼ 6.8 Hz, 3H). 13C NMR (CDCl3)
d
159.1, 158.7, 155.3, 145.5, 134.7, 130.8, 129.4, 129.2, 129.2, 125.2,
121.2, 113.8, 113.8, 72.5, 69.6, 55.3, 34.6, 31.8, 30.0, 29.4, 29.3, 29.2,
29.0, 28.7, 23.1, 22.6, 19.8, 14.1. MS (ESI) m/z: 424.7 (M þ H)þ.
4.1.6. 3-(5-((Z)-Dec-1-enyl)-4,6-dimethylpyridin-2-yl)propan-1-ol
(6)
76%) as a yellow oil. IR (KBr, cmꢂ1
1020, 818. 1H NMR (CDCl3)
7.30 (m, 2H), 7.16 (s, 1H), 6.88 (m, 2H),
4.60 (s, 2H), 4.37 (s, 2H), 3.80 (s, 3H), 2.68 (s, 3H), 2.38 (s, 3H). 13C
NMR (CDCl3) 159.4, 158.0, 147.7, 139.9, 129.8, 129.8, 129.4, 126.8,
)
nmax: 2933, 2835, 1512, 1246,
To a solution of 5 (132 mg, 0.31 mmol) in 6 mL of EtOH was
added 1 N HCl (3 mL). Then the mixture was refluxed for 3 h. After
cooling, the mixture was concentrated to remove EtOH. The water
layer was extracted with CH2Cl2 (3 ꢁ 20 mL) and the combined
organic layers were washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated. The residue was purified on
d
d
124.2, 113.9, 113.9, 85.6, 85.0, 71.6, 57.5, 55.3, 25.8, 23.1. MS (ESI)
m/z: 360.5 (M þ H)þ.
6
6
6
6
6
6
6
N
N
N
N
6
Cl
Cl
Cl
35
Cl
36
37
38
Fig. 3. Isomers of anibamine and the ring size-modified anibamine analogues.

402
F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
Br
6
6
OPMB
OPMB
N
N
N
Cl
Cl
4
39
i
ii
iii
v
40
or
or
or
Br
6
6
N
N
N
Cl
Cl
13
41
42
Scheme 9. The syntheses of compounds 39e42. Reagents and conditions: (i) (E)-dec-1-enylboronic acid (4.0 equiv), Pd(OAc)2, PPh3, Na2CO3; (ii) 1 N HCl, EtOH; (iii) MsCl, TEA; (v)
H2, 10%Pd/C.
silica gel (DCMeMeOH, 30:1) to give 78 mg pale yellow oil in 74%
yield as the hydrochloride salt. IR (KBr, cmꢂ1
nmax: 3266, 3000,
2923, 2853, 1593, 1512, 1463, 1245, 1036, 817, 729. 1H NMR (CDCl3)
J ¼ 6.8 Hz, 3H). 13C NMR (CD3OD)
d 158.2, 157.5, 151.0, 139.9, 136.2,
)
123.4, 122.9, 58.4, 33.1, 33.0, 30.5, 30.4, 30.3, 30.1, 29.8, 23.7, 21.8,
21.5, 18.1, 14.4. MS (ESI) m/z: 286.2 (Mþ). HPLC: >95% pure
(tR ¼ 7.37 min).
d
6.85 (s, 1H), 6.20 (d, J ¼ 11.2 Hz, 1H), 5.80 (m, 1H), 3.74 (m, 2H),
2.91 (m, 2H), 2.39 (s, 3H), 2.16 (s, 3H), 1.93e1.99 (m, 2H), 1.75e1.81
(m, 2H), 1.19e1.33 (m, 12H), 0.86 (t, J ¼ 6.8 Hz, 3H). 13C NMR (CDCl3)
4.1.8. 2-Hydroxy-4,6-dimethylpyridine-3-carbonitrile (8)
d
158.0, 154.9, 146.4, 135.1, 129.7, 124.7, 121.8, 62.9, 35.8, 31.9, 31.1,
Cyanoacetamide (8.4 g, 100 mmol) was dissolved in a solution of
K2CO3 (4 g, 28.9 mmol) in water (200 mL). Then acetylacetone (10 g,
100 mmol) was added and the reaction stirred overnight at room
temperature. The precipitate was filtered and washed with hexane
to give the product (13.6 g, 92% yield) as a white powder. Mp
29.4, 29.3, 29.2, 29.0, 28.8, 22.7, 22.6, 19.9, 14.1. MS (ESI) m/z: 304.3
(M þ H)þ.
4.1.7. 6-Dec-1-(Z)-enyl-5,7-dimethyl-2,3-dihydro-1H-indolizinium
chloride (7)
188e189 ꢀC. (lit. [42] 188e189 ꢀC). 1H NMR (CDCl3)
d 12.76 (br, 1H),
Methanesulfonyl chloride (38 mg, 0.33 mmol) was added to an
ice-cooled solution of 6 (50 mg, 0.16 mmol) and triethylamine
(50 mg, 0.5 mmol) in 5 mL CH2Cl2. The resulting mixture was
allowed to warm to room temperature over 1 h. The mixture was
diluted with CH2Cl2 and washed with 1 N HCl twice, dried over
Na2SO4, filtered and concentrated. The residue was purified on
silica gel (DCMeMeOH, 10:1) to afford the product (42 mg, 79%) as
6.07 (s, 1H), 2.43 (s, 3H), 2.41 (s, 3H).
4.1.9. 2-Bromo-4,6-dimethylnicotinonitrile (9)
A solution of 8 (4.44 g, 30 mmol), TBAB (13.52 g, 42 mmol), and
P2O5 (11.08 g, 78 mmol) in toluene (200 mL) was refluxed overnight.
The toluene layer was decanted and washed with saturated solution
of NaHCO3 and then with water. To the oily residue left in the flask
was added water and then powered NaHCO3 portion-wise until
there was no further evolution of gas. The mixture was extracted
with CH2Cl2 and washed with brine. The toluene and CH2Cl2 solu-
tions were combined and dried over Na2SO4, filtered and concen-
trated, followed by filtration of the product through a short pad of
a pale yellow oil. IR (KBr, cmꢂ1
) nmax: 3384, 2956, 2923, 2853, 1626,
1434, 1247, 727. 1H NMR (CD3OD)
1H), 6.07 (dt, J ¼ 7.4, 11.3 Hz, 1H), 4.71 (t, J ¼ 7.6 Hz, 2H), 3.49 (t,
J ¼ 7.4 Hz, 2H), 2.64 (s, 3H), 2.44e2.52 (m, 2H), 2.44 (s, 3H),
1.83e1.85 (m, 2H), 1.38e1.41 (m, 2H), 1.24e1.31 (m, 10H), 0.88 (t,
d
7.70 (s, 1H), 6.31 (d, J ¼ 11.3 Hz,
Table 1
Biological screening results of anibamine and its analogues.
Compounds
IC50 ꢃ SE (
m
M)a
ED50 ꢃ SE (
m
M)a
TC50 ꢃ SE (
m
M)a
Ca2þ
M12
DU-145
PC-3
NR/3T3
Anibamine
7
5.4 ꢃ 0.9
16.3 ꢃ 4.2
9.2 ꢃ 0.4
>100
>30
>30
3.3 ꢃ 0.4
0.8 ꢃ 0.2
1.3 ꢃ 0.2
>30
18.7 ꢃ 3.8
6.6 ꢃ 1.0
31.7 ꢃ 0.1
>30
2.2 ꢃ 0.3
1.9 ꢃ 0.4
2.2 ꢃ 0.3
2.4 ꢃ 0.2
3.2 ꢃ 0.5
2.6 ꢃ 0.1
0.3 ꢃ 0.1
1.7 ꢃ 0.2
1.3 ꢃ 0.2
0.9 ꢃ 0.1
1.0 ꢃ 0.2
0.9 ꢃ 0.6
0.2 ꢃ 0.1
>30
>30
>30
1.1 ꢃ 0.1
0.5 ꢃ 0.1
0.2 ꢃ 0.1
>30
>30
>30
22.7 ꢃ 7.4
4.2 ꢃ 0.4
1.9 ꢃ 0.1
n.d.b
n.d
n.d.
16
18
21
24
28
30
33
34
35
36
37
38
39
40
41
42
>30
>30
>30
>30
>30
>30
n.d.
n.d.
6.5 ꢃ 1.8
7.8 ꢃ 1.4
10.1 ꢃ 3.9
9.2 ꢃ 0.6
10.0 ꢃ 0.4
8.4 ꢃ 0.9
13.7 ꢃ 2.3
16.4 ꢃ 3.6
9.2 ꢃ 0.4
10.5 ꢃ 2.4
0.8 ꢃ 0.1
1.3 ꢃ 0.2
0.2 ꢃ 0.1
0.8 ꢃ 0.1
0.7 ꢃ 0.1
0.8 ꢃ 0.1
0.8 ꢃ 0.5
1.6 ꢃ 0.2
0.6 ꢃ 0.1
0.5 ꢃ 0.1
0.6 ꢃ 0.1
0.6 ꢃ 0.1
0.5 ꢃ 0.1
0.7 ꢃ 0.1
0.3 ꢃ 0.1
0.3 ꢃ 0.1
0.2 ꢃ 0.1
1.5 ꢃ 0.4
0.6 ꢃ 0.1
0.9 ꢃ 0.1
2.5 ꢃ 0.8
2.8 ꢃ 1.1
3.7 ꢃ 2.3
3.2 ꢃ 1.5
19.0 ꢃ 1.7
7.7 ꢃ 0.9
4.3 ꢃ 0.9
7.0 ꢃ 1.3
1.9 ꢃ 0.1
3.2 ꢃ 0.3
a
Values shown were mean ꢃ S.E. mean from at least three separate experiments performed in triplicate. The IC50 (concentration of CCR5 antagonist for 50% inhibition of
chemokine-induced calcium ion mobilization), ED50 (concentration to inhibit 50 percent of cell proliferation compared with the control) and TC50 (toxic concentration for 50
percent of mouse embryonic fibroblast cells) values were calculated using Prism.

F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
403
Table 2
Anti tumor growth assay at 16 days
Anti-proliferation assay of lead compounds against M12 cell line under CCL5 stim-
ulation conditions.
400
300
200
100
0
*
Compound
ED50 ꢃ SE (
m
M)a
No CCL5
10 nM CCL5
30 nM CCL5
Anibamine
37
38
3.3 ꢃ 0.4
3.3 ꢃ 0.5
2.6 ꢃ 0.1
1.4 ꢃ 0.2
1.5 ꢃ 0.2
1.6 ꢃ 0.1
0.8 ꢃ 0.1
1.9 ꢃ 0.9
0.9 ꢃ 0.1
*
a
Values shown were mean ꢃ S.E. mean from at least three separate experiments
performed in triplicate. The ED50 (concentration to inhibit 50 percent of cell
proliferation compared with the control) values were calculated using Prism.
silica eluting with Hexane/EtOAc (2:1). The product was afforded
(6.0 g, 95% yield) as a white solid. Mp 116e117 ꢀC. (lit. [48]
116e117 ꢀC). 1H NMR: (CDCl3)
d 7.10 (s, 1H), 2.57 (s, 3H), 2.54 (s, 3H).
4.1.10. 2-Bromo-4,6-dimethylpyridin-3-amine (10)
A solution of 9 (420 mg, 2 mmol) in concentrated H2SO4 (2 mL)
was heated at 120 ꢀC for 2 h. Then the reaction mixture was cooled
to room temperature and poured into ice water. The solution was
neutralized with solid NaHCO3. The mixture was extracted with
CH2Cl2, washed with brine and dried over Na2SO4, then filtered.
Fig. 5. In vivo studies of prostate tumor growth inhibition of two lead compounds.
Statistical analysis was conducted by using t-test.
with EtOAc, washed with water, saturated brine and dried
(Na2SO4), filtered and concentrated. The residue was purified on
silica gel (hexaneeEtOAc, 1:1) to afford the product (410 mg, 69%)
Evaporation
of
the
solvent
afforded
2-bromo-4,6-
dimethylpyridine-3-carboxamide (343 mg, 75%) as a white solid.
Mp 179e180 ꢀC. (lit. [48] 179e180 ꢀC). Br2 (2.8 g, 17.5 mmol) was
added to a solution of NaOH (2.1 g, 52.5 mmol) in 21 mL of water
at ꢂ5 ꢀC. 2-bromo-4,6-dimethylpyridine-3-carboxamide (3.19 g,
14 mmol) was added in one portion. The reaction mixture was
stirred at 0 ꢀC for 1 h and then at 70 ꢀC for 1 h. The reaction mixture
was then cooled to room temperature and extracted with CH2Cl2;
the organic layer was washed with brine, dried (Na2SO4), filtered
and concentrated. The residue was purified on silica gel
(HexaneeEtOAc, 2:1) to afford the product (2.8 g, 100%) as a pale
yellow solid. Mp 61e62 ꢀC. (lit. [48] 61e62 ꢀC). 1H NMR (CDCl3)
as a yellow oil. IR (KBr, cmꢂ1
)
nmax: 3362, 2934, 2837, 1733, 1612,
1512, 1462, 1243, 1074, 1033, 818. 1H NMR (CD3Cl3)
7.30 (m, 2H),
6.88 (m, 2H), 6.84 (s, 1H), 4.61 (s, 2H), 4.45 (s, 2H), 4.04 (br s, 2H),
3.80 (s, 3H), 2.40 (s, 3H), 2.15 (s, 3H). 13C NMR (CD3Cl3)
159.4,
d
d
148.1, 141.3, 131.1, 129.7, 129.7, 129.5, 126.4, 125.2, 113.9, 113.9, 90.4,
82.8, 71.4, 57.7, 55.3, 23.4, 17.0. MS (ESI) m/z: 297.2 (M þ H)þ.
4.1.12. 2-(3-(4-Methoxybenzyloxy)propyl)-4,6-dimethylpyridin-3-
amine (12)
A solution of 11 (740 mg, 2.5 mmol) in 9 mL of ethanol and
0.18 mL of triethylamine was hydrogenated over 23 mg (0.1 mmol)
of PtO2 for 3 d. The reaction mixture was filtered and concentrated.
The residue was purified on silica gel (DCMeMeOH, 20:1) to afford
d
6.80 (s, 1H), 3.90 (br s, 2H), 2.39 (s, 3H), 2.18 (s, 3H).
4.1.11. 2-(3-(4-Methoxybenzyloxy)prop-1-ynyl)-4,6-
dimethylpyridin-3-amine (11)
the product (575 mg, 77%) as a brown oil. IR (KBr, cmꢂ1
)
nmax: 3359,
2923, 2855, 1611, 1511, 1463, 1244, 1094, 1031, 818. 1H NMR (CD3Cl3)
7.27 (m, 2H), 6.88 (m, 2H), 6.72 (s, 1H), 4.46 (s, 2H), 3.81 (s, 3H),
3.68 (br, 2H), 3.50 (t, J ¼ 5.9 Hz, 2H), 2.83 (t, J ¼ 7.3 Hz, 2H), 2.40 (s,
3H), 2.12 (s, 3H), 1.97e2.04 (m, 2H). 13C NMR (CD3Cl3)
159.2, 146.3,
A solution of 10 (400 mg, 2 mmol), 1-methoxy-4-((prop-2-
ynyloxy)methyl)benzene (422 mg, 2.4 mmol), CuI (19 mg,
0.1 mmol) and piperidine (510 mg, 6 mmol) in 10 mL THF was
purged by passing N2 through the solution, and 140 mg (0.2 mmol)
of PdCl2(PPh3)2 was added all at once. The reaction mixture was
stirred at room temperature for 2 d. Then, the mixture was diluted
d
d
144.8,136.9, 130.5,129.3,129.3,129.3, 123.1,113.8,113.82, 72.5, 69.0,
55.3, 29.6, 28.3, 23.0, 17.2. MS (ESI) m/z: 301.2 (M þ H)þ.
4.1.13. 2-(3-(4-Methoxybenzyloxy)propyl)-3-bromo-4,6-
dimethylpyridine (13)
To a solution of 12 (575 mg, 1.92 mmol) and CuBr2 (513 mg,
2.3 mmol) in bromoform (6 mL), was added dropwise BuNO2
(237 mg, 2.3 mmol) at room temperature and stirred for 2 h. Then
the solution was filtered and concentrated. The residue was purified
on silica gel (hexaneeEtOAc, 6:1) to afford the product (349 mg, 50%)
as a pale yellow oil. IR (KBr, cmꢂ1
)
nmax: 2930, 2853, 1511, 1244,1033,
1021, 818. 1H NMR (CD3Cl3)
d
7.27 (m, 2H), 6.87 (m, 2H), 6.85 (s, 1H),
4.46 (s, 2H), 3.80 (s, 3H), 3.55 (t, J ¼ 6.6 Hz, 2H), 3.00e3.04 (m, 2H),
2.42 (s, 3H), 2.35 (s, 3H), 2.03e2.08 (m, 2H). 13C NMR (CD3Cl3)
d
159.3, 159.1, 155.8, 147.4, 130.9, 129.2, 129.2, 123.3, 120.9, 113.7, 113.7,
72.4, 69.7, 55.3, 34.8, 28.6, 23.7, 23.3. MS (ESI) m/z: 386.3 (M þ Na)þ.
4.1.14. 2-(3-(4-Methoxybenzyloxy)propyl)-3-((Z)-dec-1-enyl)-4,6-
dimethylpyridine (14)
Pd(OAc)2 (4 mg, 0.02 mmol), PPh3 (12 mg, 0.05 mmol), and 13
(91 mg, 0.25 mmol) were stirred in toluene (0.5 mL) and aqueous
Na2CO3 (0.25 mL, 2 M) under N2 for 0.5 h. To this solution was
added a solution of diisopropyl (Z)-1-decenylboronate (100 mg,
Fig. 4. CCL5 stimulation of M12 prostate cancer cell proliferation.

404
F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
0.38 mmol) in ethanol (0.25 mL). The solution was refluxed 5 h,
then diluted with EtOAc, filtered and concentrated. The residue was
purified on silica gel (hexaneeEtOAc, 4:1) to afford the product
of diisopropyl (Z)-1-decenylboronate (214 mg, 0.8 mmol) in ethanol
(0.4 mL). The solution was refluxed 6 h, diluted with EtOAc, filtered
and concentrated; the residue was purified on silica gel
(hexaneeEtOAc, 10:1) to afford the product (65 mg, 82%) as a pale
(89 mg, 84%) as a pale yellow oil. IR (KBr, cmꢂ1
)
nmax: 3000, 2923,
2852, 1590, 1512, 1245, 1098, 1036, 819, 723. 1H NMR (CDCl3)
d
7.25
yellow oil. IR (KBr, cmꢂ1
)
nmax: 3000, 2955, 2922, 2853, 1559, 1440,
(m, 2H), 6.86 (m, 2H), 6.82 (s, 1H), 6.25 (d, J ¼ 11.2 Hz, 1H), 5.78 (dt,
J ¼ 7.2, 11.2 Hz, 1H), 4.42 (s, 2H), 3.80 (s, 3H), 3.48 (t, J ¼ 6.7 Hz, 2H),
2.75 (br, 2H), 2.46 (s, 3H), 2.14 (s, 3H), 1.90e1.98 (m, 2H), 1.75e1.81
(m, 2H), 1.19e1.33 (m, 12H), 0.86 (t, J ¼ 6.8 Hz, 3H). 13C NMR (CDCl3)
722. 1H NMR (CDCl3)
d
6.25 (d, J ¼ 11.2 Hz, 2H), 5.78 (dt, J ¼ 7.3,11.2 Hz,
2H), 2.40 (s, 6H), 2.05 (s, 3H), 1.75e1.81 (m, 4H), 1.18e1.33 (m, 24H),
0.86 (t, J ¼ 6.9 Hz, 6H). 13C NMR (CDCl3)
d 153.2, 153.2, 143.6, 134.5,
134.5, 129.2, 129.2, 125.8, 125.8, 31.9, 31.9, 29.4, 29.4, 29.3, 29.3, 29.2,
29.2, 28.9, 28.9, 28.7, 28.7, 23.1, 23.1, 22.6, 22.6,17.4,14.1,14.1. MS (ESI)
m/z: 398.5 (M þ H)þ. HPLC: >95% pure (tR ¼ 9.25 min).
d
159.0, 158.5, 155.4, 145.6, 134.9, 131.0, 129.1, 129.1, 128.7, 124.8,
121.9, 113.7, 113.7, 72.3, 70.0, 55.3, 32.6, 31.8, 29.4, 29.4, 29.2, 29.2,
29.0, 28.8, 24.1, 22.6, 19.8, 14.1. MS (ESI) m/z: 424.7 (M þ H)þ.
4.1.19. 3,5-Dibromo-2-(3-methoxyprop-1-ynyl)-4,6-
4.1.15. 3-(3-((Z)-Dec-1-enyl)-4,6-dimethylpyridin-2-yl)propan-1-
ol (15)
dimethylpyridine (19)
A solution of 2,3,5-tribromo-4,6-dimethylpyridine (682 mg,
2 mmol), methyl propargyl ether (168 mg, 2.4 mmol), CuI (19 mg,
0.1 mmol) and piperidine (510 mg, 6 mmol) in 10 mL THF was purged
by passing N2 through the solution, and 140 mg (0.2 mmol) of
PdCl2(PPh3)2 was added all at once. The reaction mixture was stirred
at room temperature for 1 h. Then the mixture was diluted with
EtOAc, washed with water, saturated brine and dried (Na2SO4),
filtered and concentrated. The residue was purified on silica gel
(hexaneeEtOAc, 10:1) to afford the product (626 mg, 95%) as a pale
To a solution of 14 (80 mg, 0.19 mmol) in 4 mL of EtOH was
added 1 N HCl (2 mL). Then the mixture was refluxed for 3 h. After
cooling, the mixture was concentrated to remove EtOH. The water
layer was extracted with CH2Cl2 (3 ꢁ 10 mL) and the combined
organic layers were washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated. The residue was purified on
silica gel (DCMeMeOH, 30:1) to give 51 mg pale yellow oil in 80%
yield as the hydrochloride salt. IR (KBr, cmꢂ1
)
nmax: 3266, 3002,
6.85 (s, 1H),
2922, 2853, 1593, 1455, 1059, 722. 1H NMR (CDCl3)
d
yellow solid. Mp 41e42 ꢀC. IR (KBr, cmꢂ1
2235,1539,1428,1360,1098,1046, 902.1H NMR (CDCl3)
3.50 (s, 3H), 2.64 (s, 3H), 2.63 (s, 3H). 13C NMR (CDCl3)
) nmax: 2978, 2930, 2820,
6.23 (d, J ¼ 11.2 Hz, 1H), 5.81 (dt, J ¼ 7.3, 11.2 Hz, 1H), 3.70 (t,
J ¼ 5.4 Hz, 2H), 2.92 (m, 2H), 2.46 (s, 3H), 2.16 (s, 3H), 1.87e1.93 (m,
2H), 1.74e1.80 (m, 2H), 1.19e1.33 (m, 12H), 0.86 (t, J ¼ 6.8 Hz, 2H).
d
4.40 (s, 2H),
156.5, 147.4,
d
140.7,123.8,123.0, 89.9, 84.7, 60.3, 58.0, 25.8, 24.8. MS(ESI)m/z: 332.2
13C NMR (CDCl3)
d 157.7, 154.8, 146.6, 135.3, 129.0, 124.6, 122.2, 63.0,
(M þ H)þ.
33.5, 31.8, 30.2, 29.4, 29.3, 29.2, 28.9, 28.8, 23.5, 22.6, 20.0, 14.1. MS
(ESI) m/z: 304.2 (M þ H)þ.
4.1.20. 3,5-Dibromo-2-(3-methoxypropyl)-4,6-dimethylpyridine(20)
A solution of 19 (104 mg, 0.31 mmol) in 5 mL of ethanol and
0.1 mL of triethylamine was hydrogenated over 3 mg (0.01 mmol) of
PtO2 for 1 h. The reaction mixture was filtered and concentrated.
The residue was purified on silica gel (HexaneeEtOAc, 20:1) to
afford the product (84 mg, 80%) as a pale yellow solid. Mp 45e46 ꢀC.
4.1.16. 8-Dec-1-(Z)-enyl-5,7-dimethyl-2,3-dihydro-1H-
indolizinium chloride (16)
Methanesulfonyl chloride (32 mg, 0.28 mmol) was added to an
ice-cooled solution of 15 (41 mg, 0.14 mmol) and triethylamine
(42 mg, 0.42 mmol) in 5 mL CH2Cl2. The resulting mixture was
allowed to warm to room temperature over 1 h. The mixture was
diluted with CH2Cl2 and washed with 1 N HCl twice, dried over
Na2SO4, filtered and concentrated. The residue was purified on
silica gel (DCMeMeOH, 10:1) to afford the product (34 mg, 78%) as
IR (KBr, cmꢂ1
NMR (CDCl3)
)
d
nmax: 2922, 2866, 1547, 1438, 1385, 1117, 1040, 974. 1H
3.46 (t, J ¼ 6.5 Hz, 2H), 3.35 (s, 3H), 2.98 (m, 2H), 2.62
(s, 3H), 2.61 (s, 3H), 1.96e2.03 (m, 2H). 13C NMR (CDCl3)
d 157.6,
155.1,146.6,121.4,121.0, 72.2, 58.5, 34.6, 28.2, 25.7, 24.8. MS (ESI) m/
z: 336.2 (M þ H)þ.
a pale yellow oil. IR (KBr, cmꢂ1
) nmax: 3374, 2956, 2925, 2854, 1628,
1459, 1326, 733. 1H NMR (CDCl3)
d
7.42 (s, 1H), 6.18 (d, J ¼ 11.4 Hz,
4.1.21. 3,5-Di((Z)-dec-1-enyl)-2-(3-methoxypropyl)-4,6-
dimethylpyridine (21)
1H), 6.02 (dt, J ¼ 7.4, 11.4 Hz, 1H), 4.98 (t, J ¼ 7.5 Hz, 2H), 3.33 (t,
J ¼ 7.4 Hz, 2H), 2.84 (s, 3H), 2.51e2.59 (m, 2H), 2.40 (s, 3H),
1.80e1.86 (m, 2H), 1.18e1.38 (m, 12H), 0.86 (t, J ¼ 6.8 Hz, 3H). 13C
Pd(OAc)2 (11 mg, 0.05 mmol), PPh3 (38 mg, 0.14 mmol), and 20
(134 mg, 0.4 mmol) were stirred in toluene (1.6 mL) and aq Na2CO3
(0.8 mL, 2 M) under N2 for 0.5 h. To this solution was added
a solution of diisopropyl (Z)-1-decenylboronate (427 mg, 1.6 mmol)
in ethanol (0.8 mL). The solution was refluxed 5 h, diluted with
EtOAc, filtered and concentrated; the residue was purified on silica
gel (hexaneeEtOAc, 8:1) to afford the product (146 mg, 80%) as
NMR (CDCl3) d 156.3,156.1,150.2,139.1,131.7,127.5,120.4, 57.2, 32.3,
31.8, 29.4, 29.3, 29.19, 29.15, 28.8, 22.6, 20.9, 20.3, 20.1, 14.1. MS (ESI)
m/z: 286.2 (Mþ). HPLC: >95% pure (tR ¼ 7.71 min).
4.1.17. 3,5-Dibromo-2,4,6-trimethylpyridine (17)
To a solution of 2,4,6-trimethylpyridine (605 mg, 5 mmol) in TFA
(3 mL) were added concentrated H2SO4 (4.1 mL) and NBS (5.34 g,
30 mmol) and the resultant reaction was stirred at 50 ꢀC for 2 d. The
contents were poured into crushed ice and the solution was made
alkaline with 10% NaOH solution. The suspension was extracted
with EtOAc twice. The organic layers were combined, washed with
brine, dried over Na2SO4, filtered and concentrated. The residue
was purified on silica gel (hexaneeEtOAc, 10:1) to afford the
product (988 mg, 71%) as a white crystal. Mp 81e82 ꢀC. (lit. [50]
a pale yellow oil. IR (KBr, cmꢂ1
) nmax: 3000, 2955, 2922, 2853, 1456,
1377, 1119, 722. 1H NMR (CDCl3)
d
6.29 (d, J ¼ 11.1 Hz, 1H), 6.25 (d,
J ¼ 11.2 Hz, 1H), 5.78 (m, 2H), 3.42 (t, J ¼ 6.7 Hz, 2H), 3.32 (s, 3H),
2.73 (br, 2H), 2.39 (s, 3H), 2.05 (s, 3H), 1.92 (m, 2H), 1.78 (m, 4H),
1.19e1.33 (m, 24H), 0.86 (t, J ¼ 6.8 Hz, 6H). 13C NMR (CDCl3)
d 156.3,
153.4, 143.5, 134.7, 134.4, 129.2, 128.8, 125.9, 125.4, 72.7, 58.4, 32.5,
31.9, 29.7, 29.6, 29.6, 29.4, 29.4, 29.3, 29.2, 29.0, 28.94, 28.92, 28.8,
28.7, 23.3, 22.7, 22.7, 17.5, 14.1, 14.1. MS (ESI) m/z: 456.5 (M þ H)þ.
HPLC: >95% pure (tR ¼ 11.01 min).
81.5 ꢀC). 1H NMR (CDCl3)
d 2.62 (s, 6H), 2.60 (s, 3H).
4.1.22. 3-Bromo-6-(3-methoxyprop-1-ynyl)-2,4-dimethylpyridine
4.1.18. 3,5-Di((Z)-dec-1-enyl)-2,4,6-trimethylpyridine (18)
(22)
Pd(OAc)2 (6 mg, 0.03 mmol), PPh3 (19 mg, 0.07 mmol), and 17
(55 mg, 0.2 mmol) were stirred in toluene (0.8 mL) and aq Na2CO3
(0.4 mL, 2 M) under N2 for 0.5 h. To this solutionwas added a solution
A solution of 3,6-dibromo-2,4-dimethylpyridine (316 mg,
1.2 mmol), methyl propargyl ether (101 mg, 1.4 mmol), CuI (11 mg,
0.06 mmol) and piperidine (306 mg, 3.6 mmol) in 8 mL THF was

F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
405
purged by the passage of N2 through the solution, and 84 mg
(0.12 mmol) of PdCl2(PPh3)2 was added all at once. The reaction
mixture was stirred at room temperature for 1 h. Then the mixture
was diluted with EtOAc, washed with water, saturated brine and
dried (Na2SO4), filtered and concentrated. The residue was purified
on silica gel (hexaneeEtOAc, 6:1) to afford the product (290 mg,
NMR (CDCl3) d 146.7, 145.1, 136.7, 130.9, 122.9, 71.7, 58.4, 29.8, 28.2,
23.3, 17.2. MS (ESI) m/z: 195.1 (M þ H)þ.
4.1.27. 3-Bromo-2-(3-methoxypropyl)-4,6-dimethylpyridine (27)
To a solution of 26 (219 mg, 1.13 mmol) and CuBr2 (303 mg,
1.36 mmol) in bromoform (4 mL), was added dropwise BuNO2
(140 mg, 1.36 mmol) at room temperature, followed by stirring for
2 h. Then the solution was filtered and concentrated. The residue
was purified on silica gel (hexaneeEtOAc, 6:1) to afford the product
96%) as a pale yellow solid. IR (KBr, cmꢂ1
1446, 1358, 1099, 1020, 907. 1H NMR (CDCl3)
2H), 3.45(s, 3H), 2.67 (s, 3H), 2.38 (s, 3H). 13C NMR (CDCl3)
)
nmax: 2926, 2821, 1575,
7.17(s, 1H), 4.32(s,
158.0,
d
d
147.7, 139.9, 126.7, 124.2, 85.3, 85.0, 60.3, 57.9, 25.7, 23.1. MS (ESI) m/
(148 mg, 51%) as a pale yellow oil. IR (KBr, cmꢂ1
1586, 1446, 1117, 1020. 1H NMR (CDCl3)
6.86 (s, 1H), 3.47 (t,
J ¼ 6.6 Hz, 2H), 3.35 (s, 3H), 3.01 (m, 2H), 2.43 (s, 3H), 2.35 (s, 3H),
) nmax: 2922, 2867,
z: 254.5 (M þ H)þ.
d
4.1.23. 3-Bromo-6-(3-methoxypropyl)-2,4-dimethylpyridine (23)
A solution of 22 (290 mg, 1.15 mmol) in 6 mL of ethanol and
0.12 mL of triethylamine was hydrogenated over 10 mg (0.04 mmol)
of PtO2 for 1.5 h. The reaction mixture was filtered and concentrated.
The residue was purified on silica gel (hexaneeEtOAc, 6:1) to afford
2.01 (m, 2H). 13C NMR (CDCl3)
d 159.3, 155.8, 147.5, 123.3, 120.9,
72.4, 58.5, 34.7, 28.5, 23.7, 23.3. MS (ESI) m/z: 258.5 (M þ H)þ.
4.1.28. 3-((Z)-Dec-1-enyl)-2-(3-methoxypropyl)-4,6-dimethylpyri-
dine (28)
the product (168 mg, 57%) as a pale yellow solid. IR (KBr, cmꢂ1
)
nmax
6.86 (s,
1H), 3.41 (t, J ¼ 6.4 Hz, 2H), 3.34 (s, 3H), 2.73 (m, 2H), 2.65 (s, 3H),
:
Pd(OAc)2 (4 mg, 0.02 mmol), PPh3 (12 mg, 0.05 mmol), and 27
(64 mg, 0.25 mmol) were stirred in toluene (0.5 mL) and aqueous
Na2CO3 (0.25 mL, 2 M) under N2 for 0.5 h. To this solution was
added a solution of diisopropyl (Z)-1-decenylboronate (100 mg,
0.38 mmol) in ethanol (0.25 mL). The solution was refluxed 5 h,
then diluted with EtOAc, filtered and concentrated. The residue was
purified on silica gel (hexaneeEtOAc, 4:1) to afford the product
2922, 2866, 1586, 1448, 1389, 1116, 1021. 1H NMR (CDCl3)
d
2.36 (s, 3H),1.96 (m, 2H). 13C NMR (CDCl3)
d 159.0,156.7,147.3,122.7,
121.5, 72.0, 58.6, 34.0, 29.7, 25.6, 23.2. MS (ESI) m/z: 258.5 (M þ H)þ.
4.1.24. 3-((Z)-Dec-1-enyl)-6-(3-methoxypropyl)-2,4-dimethylpyr-
idine (24)
Pd(OAc)2 (4 mg, 0.02 mmol), PPh3 (14 mg, 0.05 mmol), and 23
(77 mg, 0.3 mmol) were stirred in toluene (0.6 mL) and aqueous
Na2CO3 (0.3 mL, 2 M) under N2 for 0.5 h. To this solution was added
(57 mg, 72%) as a pale yellow oil. IR (KBr, cmꢂ1
)
nmax: 2955, 2922,
d 6.82 (s,1H), 6.26
2853,1590,1454,1373,1118, 723. 1H NMR (CDCl3)
(d, J ¼ 11.2 Hz, 1H), 5.79 (dt, J ¼ 7.2, 11.2 Hz, 1H), 3.40 (t, J ¼ 6.7 Hz,
2H), 3.32 (s, 3H), 2.74 (m, 2H), 2.47 (s, 3H), 2.14 (s, 3H), 1.91 (m, 2H),
1.79 (m, 2H), 1.20e1.34 (m, 12H), 0.86 (t, J ¼ 6.8 Hz, 3H). 13C NMR
a
solution of diisopropyl (Z)-1-decenylboronate (120 mg,
0.45 mmol) in ethanol (0.3 mL). The solutionwas refluxed overnight,
then diluted with EtOAc, filtered and concentrated, the residue was
purified on silica gel (HexaneeEtOAc, 8:1) to afford the product
(CDCl3) d 158.4, 155.4, 145.6, 134.9, 128.7, 124.7, 121.9, 72.6, 58.4,
32.4, 31.8, 29.4, 29.3, 29.2, 29.0, 28.9, 28.8, 24.1, 22.6, 19.8, 14.0. MS
(ESI) m/z: 318.1 (M þ H)þ. HPLC: >98% pure (tR ¼ 6.13 min).
(76 mg, 80%) as a pale yellow oil. IR (KBr, cmꢂ1
)
nmax: 2954, 2922,
6.83 (s,1H), 6.22
2853,1591, 1458,1383,1119, 723. 1H NMR (CDCl3)
d
4.1.29. 3-Bromo-2,4,6-trimethylpyridine (29)
(d, J ¼ 11.2 Hz,1H), 5.78 (dt, J ¼ 7.3,11.2 Hz,1H), 3.43 (t, J ¼ 6.5 Hz, 2H),
3.35 (s, 3H), 2.76 (t, J ¼ 7.3 Hz, 2H), 2.40 (s, 3H), 2.15 (s, 3H), 1.98 (m,
2H),1.29 (m, 2H),1.19e1.34 (m,12H), 0.86 (t, J ¼ 6.8 Hz, 3H).13C NMR
To a solution of 2,4,6-trimethylpyridine (1.21 g, 10 mmol) in TFA
(2 mL) were added conc. H2SO4 (2.7 mL) and NBS (5.34 g, 30 mmol)
and the resultant reaction was stirred at room temperature for 2 d.
The contents were poured into crushed ice and the solution was
made alkaline with 10% NaOH. The suspension was extracted with
EtOAc twice. The organic layers were combined, washed with brine,
dried with Na2SO4, filtered and concentrated. The residue was
purified on silica gel (hexaneeEtOAc, 4:1) to afford the product
(CDCl3) d 158.7,155.4,145.6,134.7,129.4,125.2,121.2, 72.3, 58.5, 34.5,
31.8, 29.9, 29.4, 29.3, 29.2, 29.0, 28.7, 23.1, 22.6,19.9,14.1. MS (ESI) m/
z: 318.1 (M þ H)þ. HPLC: >95% pure (tR ¼ 6.75 min).
4.1.25. 2-(3-Methoxyprop-1-ynyl)-4,6-dimethylpyridin-3-amine (25)
A solution of 10 (400 mg, 2 mmol), methyl propargyl ether
(168 mg, 2.4 mmol), CuI (19 mg, 0.1 mmol) and piperidine (510 mg,
6 mmol) in 10 mL THF was purged by passing N2 through the
solution, and 140 mg (0.2 mmol) of PdCl2(PPh3)2 was added all at
once. The reaction mixture was stirred at room temperature over-
night. Then the mixture was diluted with EtOAc, washed with
water, saturated brine and dried (Na2SO4), filtered and concen-
trated. The residue was purified on silica gel (HexaneeEtOAc, 1:2)
(1.79 g, 90%) as a pale yellow oil. 1H NMR (CDCl3)
(s, 3H), 2.44 (s, 3H), 2.35 (s, 3H). (lit. [50])
d 6.86 (s, 1H), 2.64
4.1.30. 3-((Z)-Dec-1-enyl)-2,4,6-trimethylpyridine (30)
Pd(OAc)2 (3 mg, 0.01 mmol), PPh3 (10 mg, 0.04 mmol), and 29
(40 mg, 0.2 mmol) were stirred in toluene (0.4 mL) and aqueous
Na2CO3 (0.2 mL, 2 M) under N2 for 0.5 h. To this solution was added
a solution of diisopropyl (Z)-1-decenylboronate (80 mg, 0.3 mmol)
in ethanol (0.2 mL). The solution was refluxed overnight, then
diluted with EtOAc, filtered and concentrated. The residue was
purified on silica gel (hexaneeEtOAc, 8:1) to afford the product
to afford the product (242 mg, 64%) as a yellow oil. IR (KBr, cmꢂ1
nmax: 3344, 3199, 2925, 2820, 1620, 1462, 1105, 1094. 1H NMR
(CDCl3) 6.83 (s, 1H), 4.40 (s, 2H), 4.05 (br, 2H), 3.46 (s, 3H), 2.40 (s,
)
d
3H), 2.15 (s, 3H). 13C NMR (CDCl3)
d
148.1, 141.2, 131.1, 126.3, 125.2,
(46 mg, 88%) as a pale yellow oil. IR (KBr, cmꢂ1
)
nmax: 3000, 2955,
6.82 (s, 1H),
90.0, 82.9, 60.5, 57.8, 23.4, 17.0. MS (ESI) m/z: 190.9 (M þ H)þ.
2922, 2853, 1592, 1456, 1381, 721. 1H NMR (CDCl3)
d
6.22 (d, J ¼ 11.2 Hz, 1H), 5.78 (d t, J ¼ 7.3, 11.2 Hz, 1H), 2.46 (s, 3H),
4.1.26. 2-(3-Methoxypropyl)-4,6-dimethylpyridin-3-amine (26)
A solution of 25 (300 mg, 1.58 mmol) in 7.5 mL of ethanol and
0.16 mL of triethylamine was hydrogenated over 14 mg (0.06 mmol)
of PtO2 overnight. The reaction mixture was filtered and concen-
trated. The residue was purified on silica gel (DCMeMeOH, 20:1) to
2.40 (s, 3H), 2.14 (s, 3H), 1.79 (m, 2H), 1.19e1.33 (m, 12H), 0.86 (t,
J ¼ 6.8 Hz, 3H). 13C NMR (CDCl3)
d 155.3, 145.6, 134.8, 129.1, 125.2,
121.8, 31.8, 29.4, 29.3, 29.2, 29.0, 28.7, 24.0, 23.1, 22.6, 19.8, 14.1. MS
(ESI) m/z: 260.2 (M þ H)þ. HPLC: >98% pure (tR ¼ 5.79 min).
afford the product (261 mg, 85%) as a pale yellow oil. IR (KBr, cmꢂ1
)
4.1.31. 6,8-Didecyl-5,7-dimethyl-2,3-dihydro-1H-indolizinium
chloride (34)
nmax: 3358, 2921, 2868, 1633, 1463, 1111. 1H NMR (CDCl3)
d 6.72 (s,
1H), 3.67 (br, 2H), 3.41 (t, J ¼ 5.92 Hz, 2H), 3.36 (s, 3H), 2.79 (t,
Hydrogenationwas conducted on 33 (15 mg, 0.027 mmol) and 10%
Pd/C (2 mg,10%by weight)inMeOH (2 mL) under 50 psiH2 for 6 h. The
J ¼ 7.24 Hz, 2H), 2.38 (s, 3H), 2.14 (s, 3H), 1.94e2.01 (m, 2H). 13C

406
F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
mixture was filtered through celite to remove Pd/C and concentrated
yellow oil. IR (KBr, cmꢂ1
1199,1122,1040.1H NMR (CD3CN)
2H), 2.74 (m, 2H), 2.62 (s, 3H), 2.51 (s, 3H), 2.38 (m, 2H),1.44 (m, 4 H),
1.28 (m, 12 H) 0.89 (m, 3H). 13C NMR (CD3CN)
155.9, 154.5, 149.8,
)
nmax: 2922, 2853, 2666, 2654, 1690, 1633,
7.53 (s,1H), 4.58 (m, 2H), 3.37 (m,
to remove MeOH to afford the product (10 mg, 66% yield) as a pale
d
yellow oil. 1H NMR (CD3CN)
d 4.57 (m, 2H), 2.75 (m, 2H), 2.72 (m, 2H),
2,58 (s, 3H), 2.40 (s, 3H), 2.35 (m, 4H),1.44 (m, 6H),1.28 (m, 26 H), 0.89
d
(m, 6H). (lit. [47]) HPLC: >95% pure (tR ¼ 12.80 min).
137.8, 122.3, 56.9, 48.5, 38.5, 31.4, 31.3, 29.0, 28.72, 28.67, 28.1, 27.9,
22.1, 20.2, 19.7, 16.1, 13.1. MS (ESI) m/z: 289.0 (Mþ).
4.1.32. 6-Dec-1-(E)-enyl-5,7-dimethyl-2,3-dihydro-1H-
indolizinium chloride (39)
4.1.34. 8-Dec-1-(E)-enyl-5,7-dimethyl-2,3-dihydro-1H-
Pd(OAc)2 (5 mg, 0.02 mmol), PPh3 (18 mg, 0.07 mmol), and 4
(138 mg, 0.38 mmol) were stirred in toluene (0.76 mL) and
aqueous Na2CO3 (0.38 mL, 2 M) under N2 for 0.5 h. To this solution
was added a solution of (E)-dec-1-enylboronic acid (105 mg,
0.57 mmol) in ethanol (0.38 mL). The solution was refluxed
overnight, then diluted with EtOAc, filtered and concentrated. The
residue was purified on silica gel (hexaneeEtOAc, 8:1) to afford
the product 6-(3-(4-methoxybenzyloxy)propyl)-3-((E)-dec-1-
enyl)-2,4-dimethylpyridine (116 mg, 72%) as a pale yellow oil. IR
indolizinium chloride (41)
Pd(OAc)2 (4 mg, 0.02 mmol), PPh3 (12 mg, 0.05 mmol), and 13
(91 mg, 0.25 mmol) were stirred in toluene (0.5 mL) and aqueous
Na2CO3 (0.25 mL, 2 M) under N2 for 0.5 h. To this solution was
added a solution of (E)-dec-1-enylboronic acid (69 mg, 0.38 mmol)
in ethanol (0.25 mL). The solution was refluxed overnight,
then diluted with EtOAc, filtered and concentrated; the residue
was purified on silica gel (hexaneeEtOAc, 4:1) to afford the
product
4,6-dimethylpyridine (91 mg, 86%) as a pale yellow oil. IR (KBr,
cmꢂ1
nmax: 2953, 2923, 2852, 1590, 1512, 1454, 1245, 1098, 1036,
969. 1H NMR (CDCl3)
7.25 (m, 2H), 6.86 (m, 2H), 6.80 (s, 1H), 6.29
2-(3-(4-methoxybenzyloxy)propyl)-3-((E)-dec-1-enyl)-
(KBr, cmꢂ1
(CDCl3)
)
nmax: 2923, 2852, 1511, 1245, 1099, 1035, 819. 1H NMR
7.26 (m, 2H), 6.87 (m, 2H), 6.79 (s, 1H), 6.26 (d,
d
)
J ¼ 16.1 Hz, 1H), 5.69 (dt, J ¼ 6.9, 16.1 Hz, 1H), 4.44 (s, 2H), 3.80 (s,
3H), 3.50 (t, J ¼ 6.5 Hz, 2H), 2.76 (t, J ¼ 6.9 Hz, 2H), 2.48 (s, 3H), 2.24
(s, 3H), 2.24 (m, 2H), 2.01 (m, 2H), 1.47 (m, 2H), 1.26e1.38 (m, 10H),
d
(d, J ¼ 16.1 Hz, 1H), 5.65 (dt, J ¼ 6.9, 16.1 Hz, 1H), 4.43 (s, 2H), 3.80 (s,
3H), 3.49 (t, J ¼ 6.7 Hz, 2H), 2.83 (m, 2H), 2.44 (s, 3H), 2.22 (s, 3H),
2.19 (m, 2H), 1.96 (m, 2H), 1.47 (m, 2H), 1.28e1.36 (m, 10H), 0.88 (t,
0.89 (t, J ¼ 6.6 Hz, 3H). 13C NMR (CDCl3)
d 159.1, 158.2, 155.3, 137.1,
131.6, 130.8, 130.4, 129.2, 129.2, 125.4, 121.9, 113.8, 113.8, 72.5, 69.6,
55.3, 34.5, 33.4, 31.9, 30.0, 29.5, 29.4, 29.3, 29.2, 23.6, 22.7, 20.4,
14.1. MS (ESI) m/z: 424.7 (M þ H)þ.
J ¼ 6.7 Hz, 3H). 13C NMR (CDCl3)
d 159.0, 158.5, 154.9, 145.3, 137.1,
130.9, 130.0, 129.1, 129.1, 125.1, 122.4, 113.7, 113.7, 72.3, 70.0, 55.3,
33.4, 32.6, 31.9, 29.6, 29.5, 29.4, 29.3, 29.3, 24.0, 22.7, 20.4, 14.1. MS
(ESI) m/z: 424.7 (M þ H)þ.
To a solution of 6-(3-(4-methoxybenzyloxy)propyl)-3-((E)-dec-
1-enyl)-2,4-dimethylpyridine (205 mg, 0.48 mmol) in 8 mL of EtOH
was added 1 N HCl (4 mL). Then the mixture was refluxed for 3 h.
After cooling, the mixture was concentrated to remove EtOH. The
water layer was extracted with CH2Cl2 (3 ꢁ 20 mL) and the
combined organic layers were washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated. The residue was
purified on silica gel (DCMeMeOH, 30:1) to give 3-(5-(E)-dec-1-
enyl)-4,6-dimethylpyridine as 156 mg pale yellow oil in 95% yield
To a solution of 2-(3-(4-methoxybenzyloxy)propyl)-3-((E)-dec-
1-enyl)-4,6-dimethylpyridine (80 mg, 0.19 mmol) in 6 mL of EtOH
was added 1 N HCl (3 mL). Then the mixture was refluxed for 3 h.
After cooling, the mixture was concentrated to remove EtOH. The
water layer was extracted with CH2Cl2 (3 ꢁ 20 mL) and the
combined organic layers were washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated. The residue was
purified on silica gel (DCMeMeOH, 30:1) to give 3-(3-((E)-dec-1-
enyl)-4,6-dimethylpyridin-2-yl)propan-1-ol as 55 mg pale yellow
as the hydrochloride salt. IR (KBr, cmꢂ1
)
nmax: 3261, 2954, 2923,
6.84 (s,
2853, 1626, 1486, 1336, 1233, 1040, 977. 1H NMR (4CDCl3)
d
oil in 85% yield as the hydrochloride salt. IR (KBr, cmꢂ1
) nmax: 3262,
1H), 6.24 (d, J ¼ 16.2 Hz, 1H), 5.70 (dt, J ¼ 6.9, 16.2 Hz, 1H), 3.73 (t,
J ¼ 5.6 Hz, 2H), 2.89 (t, J ¼ 6.0 Hz, 2H), 2.47 (s, 3H), 2.25 (s, 3H), 2.24
(m, 2H), 1.94 (m, 2H), 1.24e1.51 (m, 12H), 0.89 (t, J ¼ 6.5 Hz, 2H). 13C
2954, 2923, 2853, 1593, 1455, 1246, 1036, 970. 1H NMR (CDCl3)
d
6.83 (s, 1H), 6.26 (d, J ¼ 16.1 Hz, 1H), 5.65 (dt, J ¼ 6.9, 16.1 Hz, 1H),
3.69 (t, J ¼ 5.6 Hz, 2H), 2.99 (m, 2H), 2.44 (s, 3H), 2.23 (m, 2H), 2.23
(s, 3H), 1.92 (m, 2H), 1.47 (m, 2H), 1,29e1.38 (m, 10H), 0.89 (t,
NMR (CDCl3)
d 157.5, 154.8, 146.0, 137.5, 130.7, 125.0, 122.4, 62.8,
35.7, 33.4, 31.9, 31.2, 29.4, 29.3, 29.3, 29.2, 23.2, 22.7, 20.4, 14.1. MS
(ESI) m/z: 304.3 (M þ H)þ. Methanesulfonyl chloride (105 mg,
0.92 mmol) was added to an ice-cooled solution of 3-(5-((E)-dec-1-
enyl)-4,6-dimethylpyridine (156 mg, 0.46 mmol) and triethylamine
(139 mg, 1.38 mmol) in 8 mL CH2Cl2. The resulting mixture was
allowed to warm to room temperature over 1 h. The mixture was
diluted with CH2Cl2 and washed with 1 N HCl twice, dried over
Na2SO4, filtered and concentrated. The residue was purified on
silica gel (DCMeMeOH, 10:1) to afford the product (78 mg, 53%) as
J ¼ 6.6 Hz, 2H). 13C NMR (CDCl3)
d 157.7, 154.2, 146.2, 137.6, 130.4,
124.8, 122.6, 62.8, 33.4, 33.3, 31.9, 30.6, 29.4, 29.3, 29.3, 29.2, 23.4,
22.7, 20.5, 14.1. MS (ESI) m/z: 304.3 (M þ H)þ.
Methanesulfonyl chloride (34 mg, 0.15 mmol) was added to an
ice-cooled solution of 3-(3-((E)-dec-1-enyl)-4,6-dimethylpyridin-
2-yl)propan-1-ol (51 mg, 0.15 mmol) and triethylamine (45 mg,
0.45 mmol) in 6 mL CH2Cl2. The resulting mixture was allowed to
warm to room temperature over 1 h. The mixture was diluted with
CH2Cl2 and washed with 1 N HCl twice, dried over Na2SO4, filtered
and concentrated. The residue was purified on silica gel
(DCMeMeOH, 10:1) to afford the product (39 mg, 82%) as a pale
a pale yellow oil. IR (KBr, cmꢂ1
)
nmax: 3385, 2956, 2923, 2853, 1626,
7.57 (s, 1H), 6.21 (d,
1486, 1336, 1233, 1040, 977. 1H NMR (CDCl3)
d
J ¼ 16.2 Hz, 1H), 5.86 (dt, J ¼ 6.9, 16.2 Hz, 1H), 5.00 (t, J ¼ 7.5 Hz, 2H),
3.58 (t, J ¼ 7.9 Hz, 2H), 2.82 (s, 3H), 2.58 (m, 2H), 2.46 (s, 3H), 2.29
(m, 2H), 1.49 (m, 2H), 1.29e1.37 (m, 10H), 0.89 (t, J ¼ 6.6 Hz, 3H). 13C
yellow oil. IR (KBr, cmꢂ1
1464, 1340, 1039, 978. 1H NMR (CDCl3)
)
nmax: 3445, 2956, 2923, 2853, 1626, 1490,
7.41 (s, 1H), 6.25 (d,
d
J ¼ 16.2 Hz, 1H), 6.05 (dt, J ¼ 6.8, 16.2 Hz, 1H), 5.05 (t, J ¼ 7.7 Hz, 2H),
3.51 (t, J ¼ 7.8 Hz, 2H), 2.87 (s, 3H), 2.57 (m, 2H), 2.47 (s, 3H), 2.28
(m, 2H), 1.49 (m, 2H), 1.24e1.37 (m, 10H), 0.89 (t, J ¼ 6.8 Hz, 3H). 13C
NMR (CDCl3)
d 155.9, 155.2, 149.8, 142.2, 136.2, 122.5, 121.6, 57.9,
33.2, 32.4, 31.8, 29.3, 29.1, 29.1, 28.7, 22.6, 21.7, 21.0, 18.7, 14.0. MS
(ESI) m/z: 286.2 (Mþ). HPLC: >97% pure (tR ¼ 7.29 min).
NMR (CDCl3) d 155.5, 155.0, 149.5, 142.1, 132.3, 127.9, 121.2, 57.3,
33.5, 33.0, 31.8, 29.3, 29.21, 29.17, 28.8, 22.6, 21.4, 20.7, 20.3, 14.1. MS
4.1.33. 6-Decyl-5,7-dimethyl-2,3-dihydro-1H-indolizinium chloride
(40)
(ESI) m/z: 286.2 (Mþ). HPLC: >99% pure (tR ¼ 6.75 min).
Hydrogenationwas conducted on 39 (25 mg, 0.08 mmol) and Pd/C
(2.5 mg, 10% by weight) in MeOH (2 mL) under 50 psi H2 for 6 h. The
mixture was filtered through celite to remove Pd/C and concentrated
to remove MeOH to afford the product (18 mg, 72% yield) as a pale
4.1.35. 8-Decyl-5,7-dimethyl-2,3-dihydro-1H-indolizinium chloride
(42)
Hydrogenation was conducted on 41 (29 mg, 0.09 mmol) and
Pd/C (3 mg, 10% by weight) in MeOH (3 mL) under 50 psi H2 for 6 h.

F. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 395e408
407
The mixture was filtered through celite to remove Pd/C and
incubation, media was removed from the plates, each well was
washed with 200 L of Hank’s Buffered Salt Solution (HBSS, with
calcium and magnesium, Invitrogen) and the rinsing solution was
removed from the plates. To each well, 200 L of 25 g/mL of neutral
concentrated to remove MeOH to afford the product (21 mg, 72%
m
yield) as a pale yellow oil. IR (KBr, cmꢂ1
)
nmax: 2924, 2855, 2663,
2647, 1690, 1632, 1199, 1122, 1041. 1H NMR (CD3CN)
d
7.43 (s, 1H),
m
m
4.54 (m, 2H), 3.39 (m, 2H), 2.69 (m, 2H), 2.60 (s, 3H), 2.48 (s, 3H),
2.41 (m 2H), 1.49 (m, 2H), 1.40 (m, 2H) 1.29 (m, 12 H) 0.89 (m, 3H).
red (NR, 0.33% solution in DPBS; Sigma) in DMEM containing 5%
NBCS and 1% penicillin:streptomycin was added and plates were
incubated for 3 ꢃ 0.1 h. After incubation, NR media was removed
13C NMR (CD3CN)
d 156.0, 154.5, 149.8, 135.0, 127.4, 56.1, 31.3, 30.8,
29.0, 28.9, 28.71, 28.66, 28.5, 27.6, 22.6, 22.1, 20.0, 18.4,18.3, 13.1. MS
from the plates and each well was washed with 200
mL of HBSS. The
(ESI) m/z: 289.2 (Mþ).
washing solution was decanted from the plates and 100 mL of
a solution containing 50% ethanol, 49% H2O, and 1% glacial acetic acid
was added. Plates were shaken rapidly for 20 min while being pro-
tected from light. Once removed from the shaker, plates were
allowed to sit for 5 min and absorbance at 540 nM was measured by
a microplate reader (FlexStation-3). Absorbance values were ob-
tained using SoftMaxPro software and TC50 values were calculated
using non-linear regression curves on Prism.
4.2. Calcium mobilization assay
MOLT-4/CCR5 cells were plated in black 96-well plates with
transparent bottoms (Greiner Bio-one) at 100,000 cells per well in
50:1 HBSS:HEPES assay buffer. They were incubated for 1 h at 37 ꢀC
and 5% CO2 with control buffer or varying concentration of antag-
onist for a total volume of 130
bated with 50 L of Fluo-4-AM loading buffer (40
dye, 100 L 2.5 mM probenecid, in 5 mL assay buffer) for an addi-
tional hour. Then 20 L 200 nM RANTES solution in assay buffer or
assay buffer alone were added to the wells right before changes in
Ca2þ concentration were monitored by RFU for 90 s using a micro-
plate reader (FlexStation-3, Molecular Devices). Peak values were
obtained using SoftMaxPro software (Molecular Devices) and non-
linear regression curves were generated using Prism (GraphPad) to
calculate IC50 values.
m
L per well. Cells were then incu-
m
m
L 2 M Fluo-4
m
4.5. In vivo M12 tumor growth inhibition of two leads
m
m
Mice (each group of ten, either control group or drug group) were
injected subcutaneously with 2 ꢁ 106 M12 cells. When tumors
reached a measurable size of 33 mm3, each mouse was injected in
the tail vein with 0.3 mg/kg of the anibamine or compound 38 every
four days over a sixteen days period. The controls were injected with
saline. At the time of necropsy, tumor size was established by gross
tumor mass and estimated tumor volume and analyzed.
4.3. Anti-proliferation assay
Acknowledgments
All cell lines, PC-3, DU-145 and M12, were incubated at 37 ꢀC in
the presence of 5% CO2. RPMI 1640 serum free media (GIBCO
We are grateful for the funding support from US Army Prostate
Cancer Research Program PC073739. The content of this report is
solely the responsibility of the authors and does not necessarily
represent the official views of the US Army Prostate Cancer
Research Program. We appreciate Dr. Glen E. Kellogg for the help he
offered in editing the manuscript. We thank the NIH AIDS Research
and Reference Reagent Program for providing the MOLT-4/CCR5
cell line.
Invitrogen) containing 1%
L-glutamine, 0.1% ITS (insulin, 5
mg/mL;
transferrin, g/mL; and selenium, 5 m
5
m
g/mL; Collaborative
Research, Bedford) and 0.1% gentamicin was used to cultivate all
cells. M12 cells were first incubated in media with 5% fetal bovine
serum (FBS); after 24 h serum free media was added with 0.01%
epidermal growth factor (EGF). DU-145 and PC-3 cell lines were
incubated in media containing 10% FBS at all times. Prostate cancer
tumor cells (PC-3, M12, or DU-145) were plated into 96 well plates
(BD Falcon, VWR) at a concentration of 1000 cells per well. Each cell
line was plated in its respective serum-containing media for a total
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.07.049.
concentration of 100
of drugs in a 50 L PBS solution were added to the cells. Control
cells were given 50 L of PBS. 72 h after incubation with the drug,
the serum containing media was replaced with 100 L of a 9:1
mL per well. After 24 h, various concentrations
m
m
m
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obtained using the SoftMaxPro software and non-linear regression
curves were generated using Prism to calculate IC50 values.
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