Design, synthesis and cytotoxicity studies of dithiocarbamate ester derivatives of emetine in prostate cancer cell lines

Article abstract of DOI:10.1016/j.bmc.2015.06.072

A small library of emetine dithiocarbamate ester derivatives were synthesized in 25-86% yield via derivatization of the N2′- position of emetine. Anticancer evaluation of these compounds in androgen receptor positive LNCaP and androgen receptor negative P

Full text of DOI:10.1016/j.bmc.2015.06.072

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and cytotoxicity studies of dithiocarbamate ester
derivatives of emetine in prostate cancer cell lines
Emmanuel S. Akinboye a,e, Zebalda D. Bamji b, Bernard Kwabi-Addo b, David Ejeh c, , Robert L. Copeland d,
Samuel R. Denmeade e, Oladapo Bakare a,
⇑
a Department of Chemistry, Howard University, 525 College St NW, Washington, DC 20059, USA
b Department of Biochemistry and Cancer Center, Howard University, Washington, DC 20059, USA
c Archer Daniel Midland Company, James R Randall Research Center, Decatur, IL 62521, USA
d Department of Pharmacology, College of Medicine, Howard University, Adams Bldg., 520 W St NW, Washington, DC 20059, USA
e The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins School of Medicine, 1650 Orleans St, Baltimore, MD 21231, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A small library of emetine dithiocarbamate ester derivatives were synthesized in 25–86% yield via deriva-
tization of the N20- position of emetine. Anticancer evaluation of these compounds in androgen receptor
positive LNCaP and androgen receptor negative PC3 and DU145 prostate cancer cell lines revealed time
dependent and dose-dependent cytotoxicity. With the exception of compound 4c, all the dithiocarbamate
ester analogs in this study showed appreciable potency in all the prostate cancer cell lines (regardless of
whether it is androgen receptor positive or negative) with a cytotoxicity IC50 value ranging from
Received 29 April 2015
Revised 22 June 2015
Accepted 30 June 2015
Available online xxxx
Keywords:
Emetine
Dithiocarbamate ester
Anti-cancer activities
Prostate cancer
Emetine derivatives
1.312 0.032
salt 1, all the dithiocarbamate ester analogs (2 and 4a–4g) displayed lower cytotoxicity than compound
1 (PC3, IC50 = 0.087 0.005 M; DU145, IC50 = 0.079 0.003 M and LNCaP, IC50 = 0.079 0.003 M) on
lM to 5.201 0.125 lM by day 7 of treatment. Compared to the sodium dithiocarbamate
l
l
l
day 7 of treatment. Consequently, it appears that S-alkylation of compound 1 leads to a more stable
dithiocarbamate ester derivative that resulted in lower anticancer activity in the prostate cancer cell
lines.
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
bone-targeted denosumab, radiation therapy with radium-223
and immunotherapy vaccine, sipuleucel-T.2,3 All these agents only
Prostate cancer is the most common non-cutaneous malignancy
among American men. Patients diagnosed at stage III and IV of the
disease, are usually treated with androgen-deprivation therapy
(surgical or chemical castration) and virtually all patients progress
to the castration resistant stage and ultimately resulting in mortal-
ity once it escapes the confines of the gland. It was estimated that
more than 29,000 men in the United States would die from pros-
tate cancer in 2014.1 Recently, several new agents have been
approved for the treatment of castration resistant disease based
on a few months median survival improvement over placebo.
These include anti-androgen therapy abiraterone acetate and
enzalutamide, second-line chemotherapy with cabazitaxel,
extend patient life by several months and there remains an urgent
need to develop new therapies for castration resistant prostate
cancer.3 Currently, efforts are being directed along several frontli-
nes to develop more efficacious therapy with unique anticancer
mechanisms, improved survival benefit and relatively low sys-
temic toxicity to normal cells.3,4 Natural products continue to be
valuable source of compounds with tremendous biological and
medicinal importance including compounds with excellent anti-
cancer activities. Thus, natural products remain a vital source of
scaffolds and compounds for the development of useful anticancer
agents. In our efforts to develop clinically useful anticancer agents
based on natural products scaffold, we find emetine to be an
attractive scaffold for chemical transformation.
Akinboye and Bakare recently reviewed the biological activities
of emetine; this account shows the versatility of emetine as a bio-
active natural product.5 Among other biological activities, it is a
⇑
Corresponding author. Tel.: +1 202 806 6888; fax: +1 202 806 5442.
E-mail address: obakare@howard.edu (O. Bakare).
Currently at.
http://dx.doi.org/10.1016/j.bmc.2015.06.072
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Akinboye, E. S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.072

2
E. S. Akinboye et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
8
A
7
B
MeO
MeO
9
6
5
MeO
MeO
11bN
C
1
4
N
N
10
MeO
11
MeO
H
H
13
H
H
H
3
2
14
12
7'
H
H
H
S
H
H
1'
S
8'
H
2'
OMe
OMe
OMe
HN
3'
OMe
OMe
-
Na+
S
N
D
N
E
S
6'
OMe
5'
4'
Emetine
2
1
well known anti-cancer alkaloid from Psychotria ipecacuanha.6–8 Its
anticancer activity was evaluated in several clinical studies up to
Phase II for treating solid tumor, however narrow therapeutic
index and dose dependent side effects stopped further studies.9
Recently, some studies reported novel mechanisms of action for
the cytotoxicity of emetine. For instance, among other alkaloids
screened for the inhibition of the activation of Hypoxia-inducible
Factor-1 (HIF-1) in breast cancer, emetine demonstrated an
appreciable potency.6 It regulates the alternative splicing of Bcl-x
Pre-mRNA, producing an up-regulation of the levels of the pro-
apoptotic variant, Bcl-xS, and down-regulation of the levels of
the anti-apoptotic variant Bcl-xL in different cancer cell lines.10
In a separate study on leukemia cells, emetine itself induced apop-
tosis, and also improved drug-induced apoptosis when combined
with other chemotherapeutic agents.11 Smukste et al., in a study
on the use of small molecules to overcome drug resistance induced
by a viral oncogene, also reported emetine and other protein
synthesis inhibitors to potentiate the lethality of doxorubicin in
RKO-E6 cell.12 More interestingly, emetine has been found to be
a substrate of permeability glycoprotein (P-gp).13 The glycoprotein
(P-gp) is a membrane protein that pumps drug molecules out of
cells so that they cannot elicit their cytotoxic effects, therefore,
contributing significantly to multidrug resistance (MDR).
chemo-preventive activities.16–19 In the present paper, we have
carried out a time-dependent cytotoxicity study of compound 2
and seven other dithiocarbamate ester analogs in androgen recep-
tor positive LNCaP and androgen receptor negative PC3 and DU145
prostate cancer cell lines. We herein report the synthesis, charac-
terization and preliminary anti-prostate cancer activities of a small
library of emetine dithiocarbamate ester analogs.
2. Results and discussion
2.1. Chemistry
As we previously15 described for compound 2, the synthesis of
the dithiocarbamate analogs of emetine (4a–4g) commenced with
the conversion of emetine dihydrochloride salt to the dithiocarba-
mate salt 1 by treating a solution of emetine dihydrochloride salt
in ethanolic NaOH with carbon disulfide (Scheme 1). Compound
1 was obtained in almost quantitative yield. Subsequent reaction
of 1 with different alkylating agents 3a–3g (Fig. 1) in acetonitrile
furnished the dithiocarbamate ester analogs of emetine (4a–4g)
in 25% to 86% yield. The resulting crude product was subjected to
a short flash chromatography on silica gel column using different
ratios of methanol and ethyl acetate as eluent. The main impurity
is believed to result from decomposition of the dithiocarbamate
salt, which resulted in a more polar by-product than the dithiocar-
bamate ester analog as shown by thin-layer chromatography (TLC)
on silica. No attempt was made to further isolate and characterize
this by-product. All compounds were characterized by infrared, 1H
and 13C NMR spectroscopy as well as by electrospray ionization
mass spectrometry.
Consequently,
a small library of emetine homodimers was
designed and synthesized to probe the substrate binding sites of
P-gp.14 One of these homodimers was reported to reverse the
MDR phenotype of MCF-7/DX1 cells at a non-cytotoxic concentra-
tion when co-administered with cytotoxic agent such as doxoru-
bicin.14 Thus, it appears that modification of emetine could lead
to anticancer drug candidates with better therapeutic index.
In our approach to obtain emetine derivatives without acute
systemic toxicity, but still retaining anti-cancer potency, we
designed the derivatization of the N20- position of emetine into
biologically interesting compounds for bioactivity studies. We ini-
tially synthesized and evaluated the anti-prostate cancer activities
of a diverse library of hydrolysable analogs and prodrugs of
emetine involving a variety of functional moieties. Among the
compounds studied in a 7 day drug treatment, the emetine dithio-
carbamate salt 1 showed considerable potency in the prostate can-
2.2. Biology
In a seven-day exposure, we initially compared the cytotoxicity
of emetine, the dithiocarbamate salt 1 and a benzyl substituted
dithiocarbamate ester analog 2 of emetine in PC3 and LNCaP.15 In
this study, emetine was about three folds more potent than the
dithiocarbamate salt 1 while it was about 50 folds more potent
than the dithiocarbamate ester analog in PC3. We established in
this initial study that the dithiocarbamate salt 1 served as a pro-
drug of emetine and was hydrolyzed to emetine in the slightly
acidic cancer cell growth medium mimicking the cancerous tumor
microenvironment, whereas the dithiocarbamate ester 2 was more
stable as a result of S-alkylation of the dithiocarbamyl group. More
importantly, a preliminary evaluation of the safety of compound
1 in healthy mice compared to emetine suggested that the dithio-
carbamate salt 1 is safer than emetine in vivo. Specifically, mice
that received a single dose of emetine at 100 mg/kg body weight
all died within 48 h. On the other hand, 100 mg/kg body weight
of compound 1 did not produce observable lethality. Further, when
33 mg/kg body weight of emetine or compound 1 was adminis-
tered, mice that received emetine were lethargic and showed
about four times weight loss compared with control; while, mice
cer cell lines studied (PC3, IC50 = 0.087 0.005
IC50 = 0.079 0.003 M) compared to the rest of the analogs stud-
ied. Upon alkylation of 1 to form the benzyl dithiocarbamate ester
2, the cytotoxic activity was reduced (PC3, IC50 = 1.560 M; LNCaP,
IC50 = 1.970
M).15 In order to establish the effect of alkylation of 1
lM; LNCaP,
l
l
l
on the anticancer properties of the resulting dithiocarbamate ester
and establish structure activity relationship (SAR) studies that
would allow us to identify a lead emetine dithiocarbamate ester
derivative for further studies, we designed the synthesis and anti-
cancer studies of a small library of emetine dithiocarbamate ester
derivatives. In addition, incorporating the dithiocarbamate moiety
into a library of emetine analogs is of interest to us because the
presence of this moiety in a number of compounds has been
associated with anti-carcinogenic, anti-mutagenic, and cancer
Please cite this article in press as: Akinboye, E. S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.072

E. S. Akinboye et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
MeO
MeO
MeO
MeO
MeO
N
.
2HCl
H
N
N
MeO
H
R
X
CS2
H
H
H
H
(3a-3g)
H
H
NaOH/H2O/EtOH,
-5 to +5 oC
H
H
H
S
H
Acetonitrile,
S
OMe
OMe
OMe Room temp
HN
Na+
-
R
OMe
OMe
S
N
S
N
OMe
Emetine.2HCl
1
4a-4g
Scheme 1. General synthetic scheme for the synthesis of dithiocarbamate ester analogs of emetine. R–X represents the various alkylating agents employed in the SN2
reactions.
Table 1
O
Br
O
Br
Cytotoxicity of dithiocarbamate analogs of emetine in androgen receptor positive
(LNCaP) and negative (PC3 and DU145) on day 3 of exposure
H2N
Cl
O
3a
3b
3c
Compound
Cytotoxicity IC50
PC3
(lM) on day 3
LNCaP
DU145
O
H
N
H
N
1
2
4a
4b
4c
4d
4e
4f
0.565 0.012
1.966 0.088
3.659 0.044
4.785 0.137
9.217 0.709
5.280 0.191
5.721 0.092
4.667 0.132
3.221 0.117
0.037 0.001
0.442 0.036
2.535 0.099
5.978 0.168
8.264 0.006
>10.000
3.868 0.277
3.594 0.113
4.363 0.084
3.207 0.071
0.035 0.001
0.377 0.018
2.741 0.092
3.658 0.053
3.220 0.031
>10.000
5.654 0.319
3.170 0.158
5.157 0.101
3.809 0.051
0.033 0.001
Cl
Cl
Cl
O
O
O
Br
Cl
O
3d
3e
3f
O
Br
4g
Emetine
N
O
3g
Table 2
Figure 1. Set of alkylating agents (R–X) for the synthesis.
Cytotoxicity of dithiocarbamate analogs of emetine in androgen receptor positive
(LNCaP) and negative (PC3 and DU145) on day 5 of exposure
Compound
Cytotoxicity IC50
PC3
(lM) on day 5
that received compound 1 appeared healthy and did not show any
significant difference in weight compared with control.15 These
results encouraged us to pursue further anticancer evaluation of
derivatives of compound 1 similar to compound 2. We were inter-
ested in developing more stable analogs that would not necessarily
act as prodrugs, but would retain anticancer activities while mini-
mizing the toxicities associated with emetine. Such a compound
would be closely related to compound 1 that we have proven to
be safer than emetine in vivo. The availability of such compounds
from emetine would make available clinically useful compounds
that could be used in combination therapy with existing anticancer
drugs, if not as a stand-alone chemotherapeutic agent. This will be
beneficial for the treatment of metastatic castration-resistant
prostate cancer particularly when the cancer is not responding to
available treatment options as a result of drug resistance to cur-
rently used chemotherapeutic agents.
Consequently, we set out to design a small library of dithiocar-
bamate ester analogs of emetine with potential anticancer proper-
ties and investigate the effects of various types of ‘alkyl groups’ on
the potency of the dithiocarbamate ester analogs of emetine in the
androgen receptor positive LNCaP and negative PC3 and DU145
prostate cancer cell lines. Each alkylating agent we employed is
unique, but they all can be grouped into two categories: ‘aliphatic
(non-aromatic)’ which gave compounds 4a–4c and ‘aromatic’
which gave 4d–4g. Our goal is to incorporate diversity into this
small chemical library to investigate the effects of the structure
and functional groups of each group on the general anticancer
activity of the analogs and possibly identify a lead dithiocarbamate
ester derivative. The cytotoxicity assay was done with a maximum
LNCaP
DU145
1
2
4a
4b
4c
4d
4e
4f
0.088 0.003
1.890 0.092
1.613 0.066
2.308 0.174
4.732 0.352
2.189 0.075
2.312 0.023
2.717 0.135
2.801 0.099
0.027 0.003
0.091 0.006
2.021 0.049
3.21 0.117
4.855 0.153
6.562 0.913
2.768 0.146
2.700 0.133
2.706 0.192
2.770 0.060
0.027 0.002
0.080 0.001
2.070 0.103
3.189 0.078
3.148 0.007
>10.000
2.796 0.151
3. 197 0.193
2.898 0.137
3.575 0.418
0.025 0.001
4g
Emetine
Table 3
Cytotoxicity of dithiocarbamate analogs of emetine in androgen receptor positive
(LNCaP) and negative (PC3 and DU145) on day 7 of exposure
Compound
Cytotoxicity IC50
PC3
(lM) on day 7
LNCaP
DU145
1
2
4a
4b
4c
4d
4e
4f
0.079 0.003
1.312 0.032
1.592 0.014
1.763 0.039
3.390 0.339
1.698 0.187
1.951 0.068
1.656 0.564
2.303 0.121
0.037 0.001
0.087 0.003
1.560 0.290
3.027 0.160
4.355 0.091
5.201 0.125
1.507 0.896
2.692 0.145
1.902 0.134
2.378 0.240
0.029 0.003
0.079 0.003
1.980 0.213
2.300 0.067
2.253 0.084
>10.000
1.603 0.093
2.449 0.162
2.467 0.263
3.050 0.070
0.024 0.001
4g
Emetine
Looking at the effects of analogs 2 and 4a–4g, it was observed
that by day 3 all the compounds, except 4c, showed appreciable
potency in all the prostate cancer cell lines (regardless of whether
drug concentration of 10
greater than 10 M is categorized as inactive and such values are
not reported (Tables 1–3 and Fig. 2A–C).
lM; any drug with cytotoxicity IC50 value
l
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4
E. S. Akinboye et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
IC50 = 3.390 lM; PC3, IC50 = 5.201 lM) as shown in Figure 2.
(A)
12
10
8
Though generally more potent than 4c, compound 4b, with an
ester functional group at the terminal of the aliphatic chain, is rel-
atively less potent in the more aggressive PC3 than the other two
cell lines (Tables 1–3). On the contrary, 4a without any other func-
tionality on the hexyl chain shows consistent potency across the
three cell lines with appreciable anticancer activity observed
Day 3
Day 5
Day 7
6
within the first three days (LNCaP, IC50 = 3.659
lM; PC3,
4
IC50 = 5.978 M; DU145, IC50 = 3.658 M). Compound 4a appears
l
l
to be the most potent of the three alkyl-substituted analogs show-
ing time-dependent increase in cytotoxicity in all three cell lines by
2
day
5
(LNCaP, IC50 = 1.613
l
7
M; PC3, IC50 = 3.21
(LNCaP, IC50 = 1.592
M; DU145, IC50 = 2.300 M) (Tables 1–3 and
l
M; DU145,
0
IC50 = 3.189
IC50 = 3.027
Fig. 2A–C).
lM), and day
l
M; PC3,
4a
4b
4c
4d
4e
4f
4g
l
l
Dithiocarbamate ester analogs of emeꢀne
(B)
Compounds 2, 4d, 4e, 4f, and 4g have aromatic group in the
dithiocarbamate ester substituent. Unlike compounds 4a, 4b, and
4c, each compound in this category shows almost the same level
of potency across the three cell lines with compounds 2 (LNCaP,
9
8
Day 3
Day 5
Day 7
7
IC50 = 1.312
and 4d (LNCaP, IC50 = 1.698
IC50 = 1.603 M) showing the lowest IC50 values (less than 2
l
M; PC3, IC50 = 1.560
l
M; DU145, IC50 = 1.980
M; DU145,
M)
lM)
6
lM; PC3, IC50 = 1.507
l
5
l
l
4
in all three cell lines by day 7 (Tables 1–3 and Fig. 2A–C).
Further, all the analogs 2 and 4d–4g show a gradual increase in
potency between day 3 and day 7 of exposing the prostate cancer
cell lines to the analogs (Tables 1–3). The resulting dithiocarba-
mate esters in this study (2, 4a–4g) do not necessarily act as pro-
drugs, but rather as less toxic analogs of emetine. The slight
differences in cytotoxicity observed in this study is most likely
due to conformational differences resulting from the size and nat-
ure of each substituent group. As previously reported, replacement
of the secondary amine hydrogen of the N-20 position of emetine
with a bulkier group would eliminate an electronic effect as a
result of loss of possible H-bonding of the N–H with potential
receptors at the active site. In addition, any substituted group
would result in conformational changes which appear to affect
the space between the tricyclic and bicyclic ring system of emetine
and consequently affect the bioactivity of the resulting analog
including protein synthesis inhibitory and anticancer activi-
ties.5,15,20 Hence, an appropriate substituent in this position would
result in a clinically useful emetine prodrug, or an emetine deriva-
tive that does not have the same systemic toxicity as emetine. We
are currently studying a diverse library of emetine analogs in this
regard.
3
2
1
0
4a
4b
4c
4d
4e
4f
4g
-1
Dithiocarbamate ester analogs of emeꢀne
(C)
7
6
5
4
3
2
1
0
Day 3
Day 5
Day 7
4a
4b
4d
4e
4f
4g
Dithiocarbamate ester analogs of emeꢀne
In summary, all the analogs 2 and 4a–4g consistently displayed
lower cytotoxicity than compound 1, establishing the fact that
once compound 1 is stabilized by S-alkylation, the cytotoxic effect
of the resulting dithiocarbamate ester (2, 4a–4g) is decreased rel-
ative to 1. In addition, for all the compounds (2, 4a–4g) there
was a time dependent increase in cytotoxicity between day 3
and day 7 of the study in androgen-receptor positive LNCaP and
androgen receptor negative PC3. The only exception is seen in
the androgen-receptor negative DU145 where compound 4c con-
Figure 2. Variation of the cytotoxicity IC50 of the dithiocarbamate analogs of
emetine with time over the seven-day exposure. (A) Cytotoxicity IC50 values in
LNCaP on the 3rd, 5th and 7th day of exposure. (B) Cytotoxicity IC50 values in PC3 on
the 3rd, 5th and 7th day of exposure. (C) Cytotoxicity IC50 values in DU145 on the
3rd, 5th and 7th day of exposure.
it is androgen receptor positive or negative) with a cytotoxicity IC50
value less than 10 lM (Table 1). Compounds 4a, 4b and 4c are
designed with aliphatic (non-aromatic) alkyl group, and different
functionalities at the aliphatic chain terminal. Compound 4c has
an amide functional group terminal and has a shorter chain (about
three carbon shorter) than 4a and 4b. As such, its relatively low
potency within the first 3 days of exposure as seen in all the three
cell lines, could be due to a number of factors including chain
length, possible H-bonding from the terminal amide or may be
due to poor cellular uptake. In both LNCaP and PC3, its potency
increased as the exposure period increased to five days (LNCaP,
sistently showed IC50 value of over 10 lM between days 3 to 7.
The reason for this exception is not immediately apparent as the
same compound 4c showed some increase in potency (IC50 of less
than 10 lM by day 5 and 7) in the more aggressive androgen
receptor negative PC3 prostate cancer cell line (Tables 1–3 and
Fig. 2A–C). Also, it is important to note that nearly all the com-
pounds showed appreciable potency in all the prostate cancer cell
lines regardless of whether it is androgen receptor positive or
androgen receptor negative. This might suggest a mechanism of
action that is independent of the androgen receptor. Finally, of
IC50 = 4.732 lM; PC3, IC50 = 6.562 lM) and seven days (LNCaP,
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E. S. Akinboye et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
the eight compounds studied here, compounds 2 and 4d appear to
be the most consistently potent in all three cell lines and further
SAR around these two compounds could lead to more active ana-
logs that could be clinically useful in combination therapy with
existing anticancer drugs, if not as a stand-alone chemotherapeutic
agent in the treatment of metastatic castration-resistant prostate
cancer.
3.3. General procedure for the synthesis of the dithiocarbamate
ester derivatives of emetine
For the synthesis of each of compounds 4a–4g, the salt 1
(200 mg, 0.35 mmol) was dissolved in acetonitrile (15 mL) and to
this was added a solution of alkylating agents 3a–3g (0.27 mmol)
in acetonitrile. The reaction mixtures were allowed to stir for
24 h at room temperature. Solvent was evaporated in-vacuo.
Residue obtained was triturated with water (20 mL) to dissolve
any inorganic substances, and then filtered under suction. The
crude product was air-dried and then purified by flash chromatog-
raphy on silica gel using EtOAc/MeOH (10:1) as eluent.
3. Experimental section
3.1. Chemistry: general procedures
All solvents and reagents used were bought from commercial
sources and used without any further purification. Melting points
were determined in open capillary tubes on a Mel-Temp melting
point apparatus and are uncorrected. The 1H and 13C NMR spectra
were obtained on a Bruker Avance 400 MHz spectrometer in
deuterated chloroform (CDCl3) or deuterated methanol (CD3OD).
Chemical shifts are in d units (ppm) with TMS (0.00 ppm), CHCl3
(7.27 ppm), or CH3OH (3.34 ppm) as the internal standard for 1H
NMR, and CDCl3 (77.00 ppm) or CD3OD (49.90 ppm) for 13C NMR.
The samples were analyzed with a Waters quadrupole Time-of-
Flight (QTOF) mass spectrometer (micro model) equipped with
both Electrospray Ionization (ESI) and Atmospheric Pressure
Chemical Ionization (APCI). The instrument was operated in posi-
tive-ion (ESI+) single-stage mode. The MS was calibrated in the
3.3.1. 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2H-
pyrido[2,1-a]isoquinolin-2-yl-methyl)-6,7-dimethoxy-3,4-dihy-
dro-1H-isoquinoline-2-carbodithioic acid hexyl ester (4a)
Yield: 45.2%, mp = 78–80 °C 1H NMR (400 MHz, CDCl3) d 0.78–
0.94 (6H, m), 1.03–1.15 (1H, m), 1.16–1.32 (7H, m), 1.33–1.53
(4H, m), 1.55–1.75 (3H, m), 2.03 (1H, ds t, J = 10.0 Hz), 2.36–2.52
(2H, m), 2.60 (1H, d, J = 15.2 Hz), 2.77 (1H, d, J = 14.5 Hz), 2.90–
2.98 (1H, m), 3.00–3.19 (3H, m), 3.20–3.30 (1H, m), 3.31–3.39
(2H, m), 3.61–3.72 (1H, m), 3.83 (3H, s), 3.84 (6H, s), 3.93 (3H, s),
4.68 (1H, dd, J = 4.8, 13.9 Hz), 6.55 (1H, s), 6.56 (1H, s), 6.57 (1H,
s), 6.86 (1H, dd, J = = 4.1, 11.5 Hz), 6.92 (1H, s). 13C NMR
(100 MHz, CDCl3) d 198.0, 148.0, 147.8, 147.3, 147.2, 130.1,
126.2, 124.7, 111.6, 111.3, 111.2, 110.0, 108.8, 62.9, 61.4, 59.8,
56.3, 56.2, 56.1, 55.9, 52.4, 43.3, 41.2, 40.6, 38.9, 37.7, 37.6, 37.1,
31.4, 29.7, 28.8, 27.9, 23.8, 22.5, 14.0, 11.2. HRESIMS m/z
641.4342 ([C36H52N2O4S2+H]+ calcd 641.3447).
mass-to-charge (m/z) range = 100–1500 using
g/mL NaI and 0.05 g/mL CsI in isopropyl alcohol/H2O 1:1 (vol:-
vol) introduced into the instrument by direct infusion at a flow of
25 L/min. All the mass spectra showed signals corresponding to
a solution of
2
l
l
l
3.3.2. 5-[1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2H-
pyrido[2,1-a]isoquinolin-2-yl-methyl)-6,7-dimethoxy-3,4-di-
hydro-1H-isoquinoline-2-carbothioylsulfanyl]-pentanoic acid
methyl ester (4b)
the molecular ion plus sodium (Na). The sodium was picked up
from the conditions used to operate the instrument, as is common
in this type of mass spectrometry, and was not present in the
actual compounds themselves.
Yield: 86.2%, mp = 74–76 °C 1H NMR (400 MHz, CDCl3) d 0.87
(3H, t, J = 7.4 Hz), 1.01–1.15 (1H, m), 1.15–1.31 (3H, m), 1.32–
1.43 (1H, m), 1.43–1.54 (1H, m), 1.54–1.67 (1H, m),1.68–1.82(4H,
m), 2.03 (1H, t, J = 7.4 Hz), 2.24–2.35 (2H, m), 2.36–2.53 (2H, m),
2.59 (1H, d, J = 15.6 Hz), 2.68–2.83 (1H, m), 2.88–2.98 (1H, m),
2.98–3.26 (4H, m), 3.27–3.42 (2H, m), 3.62 (3H, s), 3.65–3.75
(1H, m), 3.81 (3H, s), 3.83 (6H, s), 3.92 (3H, s), 4.65 (1H, dd,
J = 4.3, 13.6 Hz), 6.54 (1H, s), 6.55 (1H, s), 6.57 (1H, s), 6.82 (1H,
dd, J = 3.7, 11.3 Hz), 6.70 (1H, s). 13C NMR (100 MHz, CDCl3) d
197.1, 174.2, 148.5, 148.3, 147.8, 147.7, 130.5, 130.4, 128.9,
126.7, 125.1, 111.8, 110.5, 109.3, 63.3, 62.9, 60.4, 56.8, 56.6, 56.3,
52.8, 46.0, 43.9, 41.8, 41.1, 39.4, 37.3, 36.7, 34.0, 30.2, 29.7, 28.9,
28.3, 24.7, 24.3, 11.8. HRESIMS m/z 671.4057 ([C36H50N2O6S2+H]+
calcd 671.3189).
3.2. Synthesis of sodium salt of dithiocarbamic acid of
emetine 1
3.2.1. Sodium 1-(3-ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahy-
dro-2H-pyrido[2,1-a]isoquinolin-2-ylmethyl)-6,7-dimethoxy-3,
4-dihydro-1H-isoquinoline-2-carbodithioate (1)
A solution of NaOH (240.0 mg) in water (1.00 mL) and ethanol
(20.00 mL) was added to a solution of emetine dihydrochloride
hydrate (1.11 g, 2.00 mmol) in ethanol (10.0 mL) at ꢀ8 °C. This
was stirred at this temperature for 15 min after which CS2
(0.30 mL, 4.97 mmol) was added. The resulting mixture was stirred
at ꢀ5 to 1 °C for 2 h and at room temperature for 30 min. The sol-
vent was removed in-vacuo and the residue triturated with ace-
tonitrile and then filtered. The filtrate was evaporated to dryness
and the residue dissolved in ethyl acetate (3 mL). To this was added
diethyl ether which afforded the precipitation of 1 as a white solid
(950.0 mg, 82.0%). 1H NMR (400 MHz, CD3OD) d 0.93 (3H, t,
J = 7.4 Hz), 1.10–1.23 (1H, m), 1.14–1.20 (2H, m), 1.33–1.41 (2H,
m), 1.66–1.72 (1H, m), 2.10 (1H, t, J = 11.0 Hz), 2.38 (1H, t,
J = 12.8 Hz), 2.50 (1H, tb, J = 4.1, 11.6 Hz), 2.63–2.70 (2H, m),
2.99–3.20 (5H, m), 3.35 (1H, s), 3.44–3.51 (1H, m) 3.80 (3H, s),
3.81 (6H, br s), 3.84 (3H, s), 5.95 (1H, dd, J = 5.9, 12.8 Hz), 6.66
(1H, s), 6.69 (1H, s), 6.72 (1H,s), 7.20 (1H,s), 7.25 (1H, dd, J = 3.6,
11.8 Hz). 13C NMR (100 MHz, CD3OD) d 216.5, 147.4, 147.3,
147.2, 147.1, 133.0, 131.1, 127.0, 126.5, 112.6, 112.0, 111.0,
110.3, 65.4, 63.3, 61.6, 58.0, 56.5, 56.2, 56.1, 56.0, 55.9, 52.5,
41.7, 41.3, 37.6, 29.4, 27.3, 23.7, 11.6.
3.3.3. 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2H-
pyrido[2,1-a]-isoquinolin-2-yl-methyl)-6,7-dimethoxy-3,4-di-
hydro-1H-isoquinoline-2-carbodithioic acid 2-carbamoyl-ethyl
ester (4c)
Yield: 23.4%, mp = 107–109 °C 1H NMR (400 MHz, CDCl3) d 0.88
(3H, t, J = 7.4 Hz), 1.05–1.13 (1H, m), 1.20–1.27 (3H, m), 1.34–1.44
(1H, m), 1.45–1.57 (1H, m), 1.58–1.69 (1H, m), 1.94–2.10 (1H, m),
2.35–2.51 (2H, m), 2.61 (1H, d, J = 15.9 Hz), 2.70 (2H, t, J = 6.6 Hz),
2.78 (1H, d, J = 14.8 Hz), 2.86–3.27 (5H, m), 3.54–3.76 (3H, m), 3.83
(3H, s), 3.84 (6H, s), 3.92 (3H, s), 4.63 (1H, dd, J = 3.9, 13.9 Hz),
5.36–5.72 (2H, m), 6.55 (1H, s), 6.56 (1H, s), 6.58 (1H, s), 6.81
(1H, dd, J = 3.8, 11.2 Hz), 6.90 (1H, s). 13C NMR (100 MHz, CDCl3)
d 197.1, 173.2, 148.3, 147.9, 147.7, 147.3, 130.1, 129.8, 126.4,
124.6, 111.5, 111.4, 109.9, 108.9, 62.8, 61.4, 59.5, 56.5, 56.4, 56.2,
Please cite this article in press as: Akinboye, E. S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.072

6
E. S. Akinboye et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
56.1, 56.0, 52.4, 41.4, 40.7, 40.5, 38.8, 37.8, 35.4, 29.2, 27.1, 23.8,
41.3, 40.7, 40.1, 39.0, 38.0, 29.7, 29.1, 23.8, 14.4, 11.2. HRESIMS
11.2. HRESIMS m/z 628.3776 ([C33H45N3O5S2+H]+ calcd 628.2879).
m/z 762.4441 ([C41H51N3O7S2+H]+ calcd 762.3247).
3.3.4. 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2H-
pyrido[2,1-a]isoquinolin-2-yl-methyl)-6,7-dimethoxy-3,4-dihy-
dro-1H-isoquinoline-2-carbodithioic acid (4-bromo-
3.3.7. 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2H-
pyrido[2,1-a]isoquinolin-2-yl-methyl)-6,7-dimethoxy-3,4-dihy-
dro-1H-isoquinoline-2-carbodithioic acid 2-(1,3-dioxo-1,3-di-
hydro-isoindol-2-yl)-ethyl ester (4g)
phenylcarbamoyl)-methyl ester (4d)
Yield: 40.5%, mp = 136–138 °C 1H NMR (400 MHz, CDCl3) d 0.87
(3H, t, J = 7.3 Hz), 0.99–1.29 (4H, m), 1.29–1.45 (1H, m), 1.45–1.70
(2H, m), 1.88 (1H, t, J = 10.8 Hz), 2.30 (1H, ds t, J = 9.6 Hz), 2.43 (1H,
t, J = 12.8 Hz), 2.61 (1H, d, J = 15.6 Hz), 2.71–2.96 (3H, m), 2.96–
3.21 (4H, m), 3.83 (3H, s), 3.86 (6H, s), 3.91 (3H, s), 4.14 (1H, d,
J = 14.6 Hz), 4.37 (1H, d, J = 14.6 Hz), 4.61 (1H, br d, J = 11.09 Hz),
6.59 (2H, s), 6.60 (1H, s), 6.76 (1H, dd, J = 4.0, 11.0 Hz), 6.83 (1H,
s), 7.16 (2H, d, J = 8.4 Hz), 7.26 (2H, d, J = 8.4 Hz), 9.06 (1H, s). 1H
NMR (400 MHz, CD3OD) d 0.87 (3H, t, J = 7.5 Hz), 0.92–1.13 (3H,
m) 1.14–1.24 (1H, m), 1.25–1.39 (1H, m), 1.39–1.63 (2H, m), 2.02
(1H, t, J = 11.2 Hz), 2.26–2.42 (2H, m), 2.51–2.64 (1H, m), 2.69–
2.88 (2H, m), 2.91–3.05 (4H, m), 3.13 (1H, d, J = 12.3 Hz), 3.64
(3H, s), 3.66 (3H, s), 3.71 (3H, s), 3.72 (3H, s), 3.96 (1H, d,
J = 15.8 Hz), 4.32 (1H, d, J = 15.8 Hz), 4.68 (1H, dd, J = 4.8,
13.7 Hz), 6.54 (1H, s), 6.65 (1H, m), 6.66 (2H, m), 6.75 (1H, dd,
J = 4.0, 11.5 Hz), 6.99 (2H, d, J = 8.8 Hz), 7.21 (2H, d, J = 8.8 Hz)
7.25 (1H, s). 13C NMR (100 MHz, CDCl3) d 195.9, 166.8, 148.6,
148.3, 147.2, 147.4, 147.2, 137.1, 136.8, 131.8 (2C), 129.1, 127.7,
126.6, 124.3, 116.8, 111.5, 111.3, 109.7, 108.6, 62.8, 61.4, 61.3,
56.2 (2C), 56.0, 55.9, 52.5, 44.5, 40.7, 40.4, 38.9, 37.9, 29.7, 28.1,
27.3, 23.8, 11.2. HRESIMS m/z 768.3202 ([C38H46BrN3O5S2+H]+
calcd 768.2140).
Yield: 27.4%, mp = 100–102 °C 1H NMR (400 MHz, CDCl3) d 0.88
(3H, t, J = 7.4 Hz), 1.04–1.36 (4H, m), 1.37–1.52 (2H, m), 1.52–1.69
(1H, m), 1.97–2.16 (1H, m), 2.34–2.52 (2H, m), 2.60 (1H, d,
J = 13.7 Hz), 2.67–2.80 (1H, m), 2.86–2.98 (1H, m), 2.98–3.27 (4H,
m), 3.62–3.73 (3H, m), 3.83 (3H, s), 3.84 (6H, s), 3.95 (3H, s),
3.98–4.17, (2H, m), 4.61 (1H, dd, J = 4.8, 13.6 Hz), 6.54 (1H, s),
6.55 (1H, s), 6.58 (1H, s), 6.80 (1H, dd, J = 3.8, 11.4 Hz), 6.89 (1H,
s), 7.62–7.69 (2H, m), 7.73–7.82 (2H, m). 13C NMR (100 MHz,
CDCl3) d 195.9, 168.0 (2C), 148.0, 147.8, 147.4, 147.2, 133.9 (2C),
133.7, 133.0, 132.0 (2C), 129.8 (2C), 124.6, 123.2, 111.3, 111.2,
110.0, 108.8, 62.6, 61.3, 59.5, 56.3, 56.1, 56.0, 55.9, 52.3, 43.6,
40.7, 39.0, 37.4, 36.8, 34.8, 29.7, 29.2, 28.0, 23.8, 11.3. HRESIMS
m/z 730.4174 ([C40H47N3O6S2+H]+ calcd 730.2985).
3.4. Biology
3.4.1. Cytotoxicity assay
3.4.1.1. General information.
The human androgen receptor
positive (LNCaP) and negative (PC3 and DU145) prostate cancer
cell lines were purchased from the American Type Culture
Collection (Manassas, VA). All cells were grown in cell culture
flasks in RPMI culture medium with phenol red (GIBCO) supple-
mented with only 10% fetal bovine serum, 1% L-glutamine and 1%
3.3.5. 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2H-
pyrido[2,1-a]isoquinolin-2-yl-methyl)-6,7-dimethoxy-3,4-dihy-
dro-1H-isoquinoline-2-carbodithioic acid 2-(4-chloro-phenyl)-
2-oxo-ethyl ester (4e)
Penicillin-Streptomycin. Cells were cultured in a humidified atmo-
sphere of 95% air and 5% carbon dioxide at 37 °C. To sub-culture
cells for experiments, cells growing as monolayer cultures were
released from the tissue culture flasks by treatment with 0.05%
trypson/EDTA. Cell population density was determined with aid
of coulter counter (Beckman) and/or hemocytometer. For all the
in-vitro experiments, cell-growth was maintained in the log phase
and the cells were used while still in this logarithmic growth
phase.
Yield: 75.3%, mp = 99–101 °C 1H NMR (400 MHz, CDCl3) d 0.88
(3H, t, J = 7.4 Hz), 0.99–1.15 (1H, m), 1.16–1.28 (2H, m), 1.29–
1.42 (1H, m), 1.42–1.55 (1H, m), 1.56–1.69 (1H, m), 2.03 (1H, t,
J = 11.2 Hz), 2.35–2.52 (2H, m), 2.59 (1H, d, J = 15.4 Hz), 2.70–
2.88 (2H, m), 2.90–3.25 (4H, m), 3.69 (3H, s), 3.83 (10H, s), 4.65–
4.73 (2H, m), 4.91 (1H, d, J = 16.6 Hz), 6.54 (2H, s), 6.59 (1H, s),
6.72 (1H, dd, J = 3.9, 11.2 Hz), 6.77 (1H, s), 7.38 (2H, d, J = 8.5 Hz),
7.94 (2H, d, J = 8.5 Hz). 13C NMR (100 MHz, CDCl3) d 195.5, 192.2,
148.1, 147.9, 147.2, 140.0, 134.5, 134.0, 130.0, 129.7, 129.0,
128.1, 126.3, 125.9, 124.5, 111.5, 111.2, 109.9, 109.7, 108.7, 62.7,
61.4, 60.2, 56.2, 56.1, 56.0, 55.9, 52.4, 44.8, 44.2, 41.4, 40.6, 38.9,
37.8, 29.7, 28.1, 23.8, 11.3. HRESIMS m/z 709.3492
([C38H45ClN2O5S2+H]+ calcd 709.2537).
3.4.1.2. Cytotoxicity assay of emetine derivatives in DU145, PC3
and LNCaP cell lines.
The fast growing androgen receptor
negative PC3 and DU145 were plated at a population density of
2.5 ꢁ 103 cells/well while relatively slow growing androgen
receptor positive LNCaP cell lines were plated at a density of
6 ꢁ 103 cells/well into 96 well tissue culture plates. The cells were
incubated for 24 h before treatment. Compounds were dissolved in
100% DMSO to give various concentrations which were serially
3.3.6. 4-{2-[1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahy-
dro-2H-pyrido[2,1-a]-isoquinolin-2-ylmethyl)-6,7-dimethoxy-
3,4-dihydro-1H-isoquinoline-2-carbothioylsulfanyl]-acetylami-
no}-benzoic acid ethyl ester (4f)
diluted (0.01 lM to 10 lM) with complete RPMI medium to obtain
a uniform DMSO concentration of 0.1% in the medium. Control cells
were treated with an equivalent concentration of DMSO. Cells were
exposed to the drugs for seven days and MTT (3-[4,5-dimethylthi-
azol-2-yl]-2,5-diphenyltetrazolium bromide) cell proliferation
assay was done to determine the population of viable cells on
the 3rd, 5th, and 7th, day of treatment to evaluate the cytotoxic
effects of drugs and also measure if the control cells were in the
logarithmic growth phase. All assays were done in six replicates
and repeated at least twice.
Yield: 51.4%, mp = 90–92 °C 1H NMR (400 MHz, CDCl3) d 0.86
(3H, t, J = 7.4 Hz), 0.99–1.11 (1H, m), 1.12–1.26 (3H, m), 1.36 (3H,
t, J = 7.1 Hz), 1.48–1.69 (2H, m), 1.89 (1H, t, J = 11.1 Hz), 2.30 (1H,
t, J = 9.8 Hz), 2.44 (1H, t, J = 12.8 Hz), 2.59 (1H, d, J = 11.8 Hz),
2.78–2.94 (3H, m), 2.94–3.20 (4H, m), 3.83 (3H, s), 3.84 (3H, s),
3.86 (3H, s), 3.87 (1H, br s), 3.91 (3H, s), 4.19 (1H, d, J = 14.6 Hz),
4.31 (2H, q, J = 7.1 Hz), 4.38 (1H, d, J = 14.6 Hz), 4.60 (1H, br d,
J = 11.6 Hz), 6.58 (1H, s), 6.59 (1H, s), 6.60 (1H, s), 6.75 (1H, dd,
J = 4.2, 11.0 Hz), 6.84 (1H, s), 7.44 (2H, d, J = 8.7 Hz), 7.77 (2H, d,
J = 8.7 Hz), 9.27 (1H, s). 13C NMR (100 MHz, CDCl3) d 195.9, 167.1,
166.0, 148.3, 148.1, 147.5, 147.2, 141.8, 130.6, 129.7, 129.0,
126.6, 125.9, 125.8, 125.7, 124.3, 118.8, 111.5, 111.4, 109.9,
108.6, 62.8, 61.4, 61.1, 60.8, 56.3, 56.2, 56.0, 55.9, 52.4, 52.0,
Acknowledgments
We gratefully acknowledge NIH grant number 5-U54-CA914-31
(Howard University/Johns Hopkins Cancer Center Partnership) and
MRI grant number CHE-1126533 from the National Science
Foundation.
Please cite this article in press as: Akinboye, E. S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.072

E. S. Akinboye et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
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Please cite this article in press as: Akinboye, E. S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.072