Design, synthesis, and evaluation of pH-dependent hydrolyzable emetine analogues as treatment for prostate cancer

Article abstract of DOI:10.1021/jm300426q

The N-2′ position of the natural product emetine has been derivatized to thiourea, urea, sulfonamide, dithiocarbamate, carbamate, and pH responsive hydrolyzable amide analogues. In vitro studies of these analogues in PC3 and LNCaP prostate cancer cell lines showed that the analogues are generally less cytotoxic (average IC₅₀ ranging from 0.079 to 10 μM) than emetine (IC₅₀ ranging from 0.0237 to 0.0329 μM). The pH sensitive sodium dithiocarbamate salt 13 and the amide analogues 21, 22, 26 (obtained from maleic and citraconic anhydrides) showed the most promise as acid-activatable prodrugs under mildly acidic conditions found in the cancer microenvironment. These prodrugs released 12-83% of emetine at pH 6.5 and 41-95% emetine at pH 5.5. Compounds 13 and 26 were further shown to exhibit increased cytotoxicity in PC3 cell culture medium that was already below pH 7.0 at the time of treatment.

Full text of DOI:10.1021/jm300426q

Article
pubs.acs.org/jmc
Design, Synthesis, and Evaluation of pH-Dependent Hydrolyzable
Emetine Analogues as Treatment for Prostate Cancer
Emmanuel S. Akinboye,† Marc D. Rosen,‡ Samuel R. Denmeade,‡ Bernard Kwabi-Addo,§
and Oladapo Bakare*,†
†Department of Chemistry, Howard University, 525 College Street NW, Washington, D.C. 20059, United States
‡The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21239, United States
§Department of Biochemistry and Cancer Center, Howard University, Washington, D.C. 20059, United States
S
* Supporting Information
ABSTRACT: The N-2′ position of the natural product
emetine has been derivatized to thiourea, urea, sulfonamide,
dithiocarbamate, carbamate, and pH responsive hydrolyzable
amide analogues. In vitro studies of these analogues in PC3 and
LNCaP prostate cancer cell lines showed that the analogues are
generally less cytotoxic (average IC50 ranging from 0.079 to 10
μM) than emetine (IC50 ranging from 0.0237 to 0.0329 μM).
The pH sensitive sodium dithiocarbamate salt 13 and the amide
analogues 21, 22, 26 (obtained from maleic and citraconic
anhydrides) showed the most promise as acid-activatable
prodrugs under mildly acidic conditions found in the cancer
microenvironment. These prodrugs released 12−83% of
emetine at pH 6.5 and 41−95% emetine at pH 5.5. Compounds 13 and 26 were further shown to exhibit increased
cytotoxicity in PC3 cell culture medium that was already below pH 7.0 at the time of treatment.
of choice for amoebiasis, amebic dysentery, and trypanoso-
miasis.6
INTRODUCTION
■
Prostate cancer is the most commonly diagnosed cancer in men
in the United States with approximately 240 000 cases
annually.1,2 About 33 000 American men die of prostate cancer
each year with African American men having the highest death
rate in the world.2,3 Androgen ablation therapy has remained
the gold standard first line treatment of metastastic prostate
cancer since its discovery in the 1940s. While producing
remarkable palliative benefit, androgen ablation is never
curative and all men progress to a castration resistant state.
Recently, several new agents have been approved for the
treatment of castration resistant disease based on a few months
of median survival improvement over placebo. While these
results are encouraging, there remains an urgent need to
develop new therapies for castration resistant prostate cancer.
One method for identifying new treatments is to look for
older therapies that may have unique mechanisms of action that
could be effective in cancer treatment. Emetine (1, Figure 1) is
one such older agent that is a natural product alkaloid found in
the root of Psychotria ipecacuanha.4 It is the active ingredient in
the ipecac root long used in traditional folk medicine as an
emetic and expectorant.5 Ipecac remains a staple in most
households where it is used to induce vomiting in the event of
accidental ingestion of toxic agents. Direct subcutaneous
injection of emetine was, for a long time, also the treatment
While emetine has been shown to inhibit a number of
important cell processes, its major mechanism of action appears
to be related to its ability to inhibit ribosomal protein synthesis
as first reported by Grollman5 while Lietman later reported the
same in the mitochondria.7 As a protein synthesis inhibitor, its
anticancer activities were investigated in several phase I−II
clinical trials in a number of solid tumors between 1969 and
1974.8−13 While clinical responses were observed in these
studies, emetine was reported to have a very narrow therapeutic
index, and dose dependent side effects such as muscle fatigue
and cardiac toxicity were observed. These were the same side
effects observed in the treatment of amoebiasis patients.14,15
On the basis of the toxicity observed in these clinical studies,
development of emetine as an anticancer drug ceased.16 Since
then, this natural product has been used as an important tool in
cell biology and pharmacology for in vitro studies that require
inhibition of protein biosynthesis. These more recent studies
have revealed the potency of emetine in modulating different
biological pathways associated with cancer growth.6 However,
to further develop emetine-based therapeutics, an analogue
must be developed that is only active upon reaching the tumor
Received: March 26, 2012
Published: August 6, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structure of emetine and some of its natural analogues.
microenvironment while relatively inactive in the general
circulation and in normal tissues. Thus, to achieve this goal,
it is important that we understand the effects of its structure
and functional moieties on its biological activities (Figure 2).
Figure 2. General chemical structure of proposed emetine analogues
with tunable handle.
An account of structure−activity relationship (SAR) studies
on emetine and its biological activities done by various research
groups has been recently reviewed.6 The available SAR data
demonstrate that a secondary amine in the N-2′ position of
emetine is critical for protein synthesis inhibition. A relatively
significant loss of protein synthesis inhibitory activity was
observed in both N-methylemetine (2) and O-methylpsycho-
trine (3); the hydrogen of the secondary amine might be
involved in a crucial hydrogen bonding interaction of emetine
to its targeted receptor.17 In addition, a more recent
conformational study reported the critical need for the region
between the tricyclic system (A, B, C rings) and the bicyclic
isoquinoline (D, E) system to be unoccupied for emetine and
its analogues to be active.18 Minimum energy conformational
studies revealed that this region is occupied in inactive
analogues while unoccupied in active analogues.18
Figure 3. Energy minimized diagram of compounds 1 and 4: (A)
emetine (1) and (B) an N2′ derived thiourea analogue of emetine with
benzyl substituent, compound 4.
These studies document that modification of the N-2′
position of emetine results in reduced biological activity
including protein synthesis inhibition and toxicity. Replacement
of the N-2′ secondary amine hydrogen with a bulkier group
may also lead to conformational changes that will affect the
region S (Figure 3) between the tricyclic and bicyclic systems.
Consequently, an appropriate substituent in this position that
could be selectively removed within the tumor microenviron-
ment but remain stable in normal tissue could result in an
emetine analogue with improved therapeutic index that could
be useful as cancer therapy. In our efforts to develop emetine
into clinically useful anticancer agent we have thus focused on
chemical modification of the N-2′ position to (1) generate a
functionally diverse library of emetine analogues with various
hydrolyzable moieties (such as sulfonamides, urea, thiourea,
carbamates, dithiocarbamate, and amide), which are also known
for incorporating bioactivities into small molecules,19−21 and
(2) generate a set of prodrugs that will only be activated to
release emetine in the cancer cells or tumor microenvironment.
The prodrugs could be activated either at the cancer
microenvironment by exploiting the lower extracellular pH
(pH ≈ 6.4−6.9) of some tumor cells compared to that of
normal tissue22−24 or the mild acidic conditions of the
intracellular vesicles such as endosomes (pH ≈ 5.5−6) and
lysosomes (pH ≈ 4.5−5).25
The present paper reports our initial studies on the synthesis
and anticancer activities of novel bioactive emetine derivatives
and acid-hydrolyzable prodrugs by chemical modification of the
N-2′ position. The cytotoxic activities of these compounds
were evaluated on androgen receptor positive LNCaP and
negative PC3 prostate cancer cell lines.
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4-dimethylaminopyridine (DMAP) (Scheme 1c). This reaction
progressed well with DMAP but not with triethylamine as base.
The preparation of the dithiocarbamate analogue 14 was
achieved in two steps. Emetine dihydrochloride was reacted
with carbon disulfide in ethanolic sodium hydroxide at −5 to
+5 °C to produce the dithiocarbamic acid salt 13 which on
treatment with benzyl bromide afforded 14 in 45% yield
(Scheme 1d).
The carbamate analogue 15 was synthesized in 46% yield by
reacting emetine dihydrochloride with benzyl chloroformate in
the presence of at least 1 molar equivalent of DMAP in excess
triethylamine using chloroform as solvent (Scheme 1e).
Attempts to conduct this reaction in CH2Cl2, THF, and a
number of other solvents were not very successful.
As stated earlier, we desire to target emetine directly to the
cancer region in the form of prodrugs that will be activated only
at the cancer microenvironment either intercellularly or
intracellularly. To achieve this goal, we also synthesized pH-
responsive amide analogues of emetine (20−27) from cyclic
anhydrides (Scheme 1f and Scheme 1g). These analogues were
synthesized by reacting emetine with appropriate anhydride in
CHCl3 in the presence of triethylamine to furnish the amide
derivatives. Compounds 20−22 were isolated in 89−94% yield.
To remove all the residual triethylamine from the cis-
aconitylamide analogue (23), this analogue was treated with
0.1 N aqueous NaOH. Thus, the dicarboxylic acid 23 was
converted to its disodium salt 27 in about 79% overall yield.
Similarly, compounds 20−22 were converted to the respective
sodium salts 24−26 with overall yield of 78−88% from
emetine. With the amide analogues obtained as the sodium
salts, 24−27, it was possible to do direct structure−activity
relationship studies on the bioactivity and acid responsiveness
of the four compounds.
Initial in Vitro Determination of the Antiprostate
Cancer Potency of the Emetine Analogues. First, to
determine the general effect of derivatizing emetine at the N-2′
on its cytotoxicity and to do structure−activity relationship
studies on these synthetic analogues, we began by assaying their
cytotoxicity in androgen receptor positive LNCaP and
androgen receptor negative PC3 human prostate cancer cell
lines. Six concentrations of each compound were tested on each
of the two cell lines over a period of 7 days. We utilized the
colorimetric MTT assay to determine the viable cells after the
third, fifth, and seventh day of exposing the prostate cancer cell
lines to the compounds. The anticancer potency of each
compound on the seventh day measured as IC50 value is
reported in Table 2.
RESULTS AND DISCUSSION
■
Evaluation of the Antiprostate Cancer Activity of
Emetine. Because of lack of data on the cytotoxicity of emetine
in prostate cancer cell lines, we began this study by first
evaluating its anticancer potency in a representative androgen
receptor positive LNCaP and androgen receptor negative
metastatic PC3 and DU145 prostate cancer cell lines. Cell
survival and proliferation were determined spectrophotometri-
cally using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-
lium bromide (MTT) colorimetric assay (Supporting Informa-
tion). For the biological studies, emetine was used as the
dihydrochloride salt. Time and concentration dependent
potency of emetine in this study revealed that emetine could
be a novel antineoplastic agent in prostate cancer drug
development (Table 1) if the therapeutic index is somehow
Table 1. Time and Concentration Dependent Cytotoxicity of
Emetine in Prostate Cancer Cell Lines
IC50 (nM)
exposure time
LNCaP
PC3
DU145
day 3
day 5
day 7
37.87 0.76
27.82 3.84
31.58 2.42
35.13 1.02
26.84 2.27
29.43 3.27
32.88 0.99
25.27 0.8
23.74 1.22
improved. Emetine is equally potent in both androgen receptor
positive and negative prostate cancer cell lines. As shown from
the IC50 values (concentration of drug that reduce the average
growth value of the cancer cell lines by 50% relative to the
average control growth value), cell death due to emetine
relative to control was significant within the first 3 days of
exposing the cells to emetine dihydrochloride hydrate with IC50
values in the nanomolar range. Encouraged by this initial
screening result with emetine in prostate cancer cell lines, we
designed and synthesized thiourea, urea, sulfonamide, carba-
mate, dithiocarbamate, and amide derivatives of emetine via
modification of the N-2′ position.
Synthesis of Emetine Analogues 4−15 and 20−27. As
depicted in Scheme 1a, efforts on the synthesis of thiourea
analogues commenced with commercially available amines.
Treatment of the appropriate amines with carbon disulfide in
tetrahydrofuran in the presence of Et3N resulted in an in situ
generation of dithiocarbamic acid salt which on subsequent
treatment with 4-toluenesulfonyl chloride as described by
Wong and Dolman26 gave the corresponding isothiocyantes in
74−93% yields. Reaction of the isothiocyanate with emetine
dihydrochloride in CH2Cl2 in the presence of pyridine
furnished the appropriate thiourea derivatives 4−6 in 47−
51% yield.
The urea analogues 7−9 were synthesized in two steps
(Scheme 1b). First, the requisite isocyanates were prepared by
treating an appropriate amine with trichloromethyl chlorofor-
mate in CH2Cl2 in the presence of non-nucleophilic amine base
1,8-bis(dimethylamino)naphthalene.27 A predetermined quan-
tity of emetine dihydrochloride was then added to each
isocyanate in CH2Cl2 in the presence of 4-dimethylaminopyr-
idine (DMAP) to produce compounds 7−9 in 74−85% yield.
Unlike the thiourea synthesis, the urea analogues were isolated
in a relatively high yield.
This study further confirms that derivatizing emetine at the
N-2′ position to produce a tertiary nitrogen results in reduction
of its cytotoxicity. The extent of reduction depends not only on
the functional groups bonded to this position but also on the
structural features of the substituents attached. Thus, the N-2′
position can serve as a tunable point where chemical
modification can be carried out with the goal of generating
emetine analogues and prodrugs that avoid the systemic toxicity
associated with the parent compound. As observed for emetine,
almost all the analogues have relatively potent cytotoxicity in
both androgen receptor positive LNCaP and negative PC3
prostate cancer cells. The IC50 values are below 10 μM except
for those of compounds 9 and 10 in PC3 cell lines. Among the
thiourea analogues (4−6), compound 6 with electron with-
drawing chlorine atom at the para position is the most active
(Table 2). However, a distinct preference for electron
The sulfonamide analogues 10−12 were obtained in 74−
86% yield by treating emetine dihydrochloride with the
appropriate sulfonyl chlorides in CH2Cl2 in the presence of
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a
Scheme 1. Synthesis of Emetine Analogues (4−15, 20−27)
a
(a) Synthesis of thiourea derivatives of emetine (4−6) from isothiocyanates. The requisite isothiocyanates were prepared from corresponding
amines. (b) Synthesis of urea derivatives of emetine (7−9) from isocyanates. The isocyanates were made from commercially available amines and
trichloromethyl chloroformate. (c) Synthesis of sulfonamide derivatives (10−12) by reacting emetine with sulfonyl chlorides. (d) Synthesis of
dithiocarbamate derivative of emetine (14) by the synthesis of dithiocarbamic acid salt (13) followed by SN2 reaction between the salt and benzyl
bromide. (e) Preparation of carbamate derivative (15) by reaction of emetine with benzyl chloroformate. (f, g) Preparation of amide derivatives
(20−27) by treating emetine with appropriate cyclic anhydride.
withdrawing group over electron donating group is not too
obvious, as the IC50 values for compound 5 with electron
donating methoxy group are slightly lower than that of
compound 4 in PC3. A consistent observation is that these
thiourea analogues have relatively lower IC50 values in LNCaP
than in PC3 cell lines.
Similarly the urea analogues 7 and 9 exhibit more potency in
LNCaP than PC3 as seen from the IC50 values; however, the
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easily hydrolyzable. Maleic anhydride has been used for the
reversible blocking or protection of amino groups of proteins
and peptides in a reversible pH sensitive reaction.28−30
Hydrolysis of the protecting maleic acid monoamide is
known to occur under mildly acidic conditions by intra-
molecular acid catalyzed hydrolysis which initially causes
reversion to the amino group and maleic anhydride.29−32 The
maleic anhydride from the deprotection can then undergo
further hydrolysis to maleic acid. A similar reversible blocking
of amino groups of protein was reported for citraconic
anhydride (a maleic anhydride derivative) and found to
undergo faster hydrolysis than blocking with maleic anhy-
dride.33 A number of studies have employed maleic acid amide
derivatives as acid-activatable linkers for daunomycin- and
doxorubicin-macromolecule conjugates in targeted drug deliv-
ery to tumor cells.34−36 The cis-aconitic monoamide derivative
of daunomycin has been reported as a pH activated prodrug
that releases daunomycin under acidic pH but more stable at
neutral pH.34,37 Hence, the proposed maleic acid monoamide
analogues (21, 22, 25−27) are expected to release emetine in
the acidic cancer environment while remaining stable at the
physiological pH range of 7.0−7.4.
Each amide with a carboxylic acid terminal (20−22) has a
slightly higher cytotoxic activity (lower IC50 value) than the
corresponding sodium salt, 24−26 (Table 2). The citraconyl
derivative 22 with a vinylic methyl substituent is the most active
in the acid category of the amides (20−22), and its
corresponding sodium salt 26 is the most active among the
sodium salts (24−27). Though not as active as the
dithiocarbamate salt 13, compounds 22 and 26 are significantly
more potent than emetine analogues 4−12 and 15. The
remaining amide analogues 20, 21, 24, 25, and 27 show similar
cytotoxicity levels as the other emetine analogues 4−12 and 15
in this study. As mentioned above, the citraconyl moiety in
compounds 22 and 26 had been previously employed in
reversible blocking of amino groups in proteins and gave
satisfactory results in the ease of deblocking by hydrolysis of the
amide bond in slightly acidic pH conditions. It is therefore
possible that the citraconyl group in compounds 22 and 26 was
hydrolyzed to emetine in the in vitro cancer microenvironment
of the tissue culture medium. In light of this possibility, we
further evaluated the time-dependent cytotoxicity of some of
these emetine analogues in prostate cancer cell lines and
studied their pH-dependent hydrolysis to emetine.
To obtain an understanding of the time-dependent
cytotoxicty of some of the compounds and use the information
to gain insight into a possible pH activated prodrug
development for emetine, we studied the progress of the in
vitro cytotoxicity of these emetine analogues over a 7-day
period by comparing the IC50 values obtained on the third, fifth,
and seventh day after exposing the cells to the drugs. We
wanted to know how long it took to reach the optimum
cytotoxic activity for each drug compared to that for emetine
and also to see if there is a progressive cytotoxicity common to
each group of compounds over the 7-day period. For each
compound, a similar progression in cytotoxicity is observed in
both LNCaP and PC3. So we present the data obtained in PC3
cell lines in Figure 4. On the basis of these data, it is clear that
the cytotoxicity of emetine reached an optimum by the third
day of exposing the prostate cancer cells to emetine
dihydrochloride. The fifth and seventh day studies gave results
very close to that of day 3 (Figure 4a). An almost similar
pattern was observed for the cytotoxic activity of thiourea
Table 2. In Vitro Cytotoxic Evaluation of Emetine Analogues
4−22 and 24−27 in LNCaP and PC3 Prostate Cancer Cell
Lines Presented as IC50 (in μM) after 7-Day Exposure
compd
LNCaP
PC3
4
2.057 0.438
3.388 0.139
1.590 0.151
2.115 0.576
7.839 0.315
5.411 0.212
2.263 0.798
2.692 0.055
3.153 0.099
0.079 0.003
1.970 0.088
3.241 0.175
2.409 0.053
6.781 0.283
0.368 0.024
2.451 0.122
8.901 0.079
0.458 0.012
5.465 0.039
6.916 0.071
5.002 0.307
2.864 0.066
7.613 0.681
3.669 0.796
10.000 0.512
>10.0
5
6
7
8
9
10
11
12
13
14
15
20
21
22
24
25
26
27
4.214 0.435
6.075 0.105
0.087 0.005
1.560 0.290
6.071 0.144
2.102 0.172
5.041 0.087
0.378 0.010
2.149 0.131
7.708 0.151
0.428 0.008
2.889 0.123
urea analogue 8 with the para electron donating methoxy group
exhibited distinctly better potency (IC50, 3.669 μM) in the
androgen receptor negative PC3 than androgen receptor
positive LNCaP (IC50, 7.839 μM). By comparison of the
thiourea and urea analogues, it appears that without any
substituent at the para position, replacement of the sulfur in
thiourea by oxygen atom in the urea analogue does not
significantly affect the cytotoxicity in either cell lines as shown
by the similar IC50 values obtained for thiourea derivative 4
(LNCap, 2.057 μM; PC3, 6.916 μM) and urea derivative 7
(LNCap, 2.115 μM; PC3, 7.613 μM).
All the sulfonamide analogues 10−12 also have higher
potency (lower IC50 values) in LNCaP than in PC3 but the
range is very narrow in LNCaP, making it difficult to tell if the
substituent at the para-position really affects the cytotoxicity.
Compound 10 with no substituent on the sulfonamidophenyl
ring is the least active in PC3 cell lines (IC50 > 10).
Interestingly, the dithiocarbamic acid salt 13 is significantly
more potent in both LNCaP and PC3 than the emetine
analogues 4−12 discussed above. The seventh day IC50 value is
only about 3-fold higher than what we obtained for emetine.
However, upon alkylation of 13 to the benzyl dithiocarbamate
ester 14, a significant reduction in potency measured by a
higher IC50 value of about 24-fold increase in LNCaP and 18-
fold increase in PC3 (Table 2) was observed. The significance
of this observation is further investigated in our prodrug
development (Figures 4a,d and 6). We needed to know if 13 is
acting by a different pharmacophore compared to emetine or if
it was hydrolyzed in vitro and served as a prodrug for emetine.
The carbamate ester 15 has a slightly lower potency (higher
IC50 value) relative to dithiocarbamate 14 in both cell lines
(Table 2).
With the possibility that the better cytotoxicity of the
dithiocarbamate salt 13 is due to its ease of hydrolysis, thus
serving as an emetine prodrug, the maleic acid monoamide
derivatives 21, 22, and 25−27 were specifically designed as
acid-activatable prodrugs of emetine while succinic acid
monoamide derivatives 20 and 24 are not expected to be as
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Figure 4. Variation of cytotoxicity (IC50 values) of each compound over 7 days in PC3 cells. PC3 cell lines were incubated with six different
concentrations of emetine dihydrochloride salt and its synthetic analogues for 7 days. MTT cell proliferation assay was carried out to determine the
viable cells on each of the third, fifth, and seventh day of exposing the cells to each drug. The variation of cytotoxicity IC50 values of emetine and its
synthetic analogues with time over a 7-day exposure is shown in parts a−f.
analogue 6 and urea analogues 7 and 9 (Figure 4b,c). For these
compounds, the IC50 values also appear to reach an optimum
by day 3 and the potency did not change much from day 3 to
day 7. However, for compounds 5 (a thiourea, Figure 4b), 12
(a sulfonamide, Figure 4e), 13 (dithiocarbamate salt, Figure
4a), 26 (amide derivative, Figure 4a), and 24 (amide derivative,
Figure 4f), an optimum IC50 value was obtained by day 5 after
exposure. The IC50 value for each of these compounds on day 7
is similar to that of day 5. For urea analogue 8 (Figure 4c),
dithiocarbamate ester 14 (Figure 4d), and carbamate ester 15
(Figure 4d), there appear to be a progressive decrease in IC50
values (increase in cytotoxicity) from day 3 to day 7. The
remaining analogues, 4, 11, 25, and 27, appear to show very
close IC50 values for days 3, 5, and 7.
No consistent pattern is observed in each class of
compounds, and we believe that in addition to functional
moieties, the conformation of each analogue most likely
affected its potency. A study on the effect of conformation of
these analogues, its implication on the size of space “S” between
the tricyclic ABC rings and bicyclic DE rings (Figure 3), and
correlation of these to anticancer activities in emetine analogues
is currently being planned. Of much interest in the study
presented here, however, is the big increase in cytotoxicity
observed for compound 13 between day 3 and day 5. In line
with our design of an emetine prodrug, we were interested in
understanding if this compound was acting as a prodrug and
thus not as potent by day 3. And if so, was compound 13
hydrolyzed to emetine by day 5 such that the potency observed
after day 5 resulted from emetine? To have an insight into this
possibility, we used HPLC to study the acid-catalyzed release of
emetine from this compound and some of the other potentially
pH-activatable analogues (21, 22, 25, 26, and 27). It is
important to mention at this point that all the synthetic
emetine analogues presented in Scheme 1 are hydrolyzable with
different degrees of responsiveness to pH change and the time
required. However, compounds 13, 21, 22, 25, 26, and 27 are
expected to be more easily hydrolyzable under mildly acidic
environment as discussed above. The results of the evaluation
of the pH-responsiveness of 13, 21, 22, 25, 26, and 27 by
HPLC are presented in the following section and in Figure 5.
Evaluation of pH-Responsiveness of Compounds 13,
21, 22, and 25−27 by HPLC. Relative rates of hydrolysis of
these compounds over a 48 h period were evaluated using the
percent emetine released from the acid catalyzed hydrolysis of
13, 21, 22, and 25−27. The compounds were incubated in
aqueous phosphate buffer at pH 5.5, 6.5, and 7.4 at 37 °C over
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Figure 5. Quantitative analysis of emetine released in the pH responsive hydrolysis of emetine analogues. (A) Hydrolysis of emetine analogues to
emetine at pH 5.5. Percent conversion indicates percent emetine in the sample mixture after pure analogues are incubated at pH 5.5 phosphate
buffer at 37 °C for 24−48 h. (B) Amount of emetine released at pH 6.5, with the same study as in (A) carried out at pH 6.5.
a 48 h period. High performance liquid chromatography
(HPLC) was employed to analyze the samples and quantify
how much emetine was released. The data are summarized in
Figure 5. The compounds whose hydrolysis to emetine is
shown in Figure 5 are all 100% stable at physiological pH 7.4
up to a 5-day exposure. All the compounds studied here (13,
21, 22, 25, 26, and 27) were activated to emetine at pH 5.5
over a 48 h period, although to different extents. Percent
hydrolysis to emetine was found to drop at the less acidic pH
6.5. These results are consistent with earlier reports of the
hydrolysis of the monoamides of maleic anhydride derivatives
outlined above.29−34,37 Hence, amide analogues synthesized
from maleic anhydride and its derivatives in this study showed
great promise as seen particularly in 21, 22, and 26 which
release about 50%, 80%, and 50% emetine, respectively, within
48 h at pH 6.5. In addition, the free acid analogues 21 and 22
appear to be more pH-responsive and sensitive than the
corresponding sodium carboxylate salts 25 and 26. The pH
responsiveness of the sodium dithiocarbamate salt 13 is higher
at pH 5.5 than at 6.5, producing about 94.5% hydrolysis within
48 h at pH 5.5 but only 13.2% over the same period at pH 6.5.
The hydrolysis of 13 appears to be slower at pH 6.5, and it is
therefore conceivable that there is an increase in the amount of
emetine released between day 3 and day 5 in the in vitro study,
thus making this compound show optimum activity by day 5. A
brief evaluation of the other emetine analogues 4−12, 14, and
15 shows that they are stable at these slightly acidic pH over the
time period studied. Taken together, these data demonstrate
that pH-responsiveness of 13, 22, and 26, resulting in release of
emetine, may be playing a significant role in their increased
cytotoxicity (lower IC50 values) observed after day 3 when the
pH of growth medium has probably dropped to between 6.0
and 7.0 (Figures 4a, 5, and Table 1). These results encouraged
further in vitro studies of the activation of two of these
potential prodrugs (13 and 26) in cell culture medium under
slightly acidic pH (6.0−6.9).
In Vitro Cytotoxic Studies of Compounds 13 and 26 in
PC3 Cell Line under Pre-Established Acidic Cancer Cell
Culture Medium (pH < 7.0). Upon establishing the release of
emetine from these compounds under mildly acidic condition,
we rationalized that we could increase the potency of the pH-
responsive analogues if we could adapt the cancer cell to an
acidic medium so that the pH of the cell culture is already
below 7 at day 0. To investigate this, we selected compounds
13 and 26, which are two of the three most cytotoxic pH-
responsive analogues.
To accomplish our objective here, we first needed to
establish the range of pH changes of the PC3 cell lines in our in
vitro studies over a 7-day period. Hence, we investigated the
effects of the metabolism of PC3 cell lines on change in pH of
growth medium without any drugs, and the result is presented
in Figure 6. This study showed that the metabolic activities of
the PC3 cancer cells caused a fall in the pH of the growth
medium from 7.4 on day 0 to 7.05 on day 3 and 6.66 by day 7
(Figure 6).
In our initial in vitro cytotoxic studies on these compounds, a
gradual reduction in the pH of the growth medium was
observed as the cancer cells metabolized over a 7-day period
(Figure 6). We therefore reasoned that it is possible to adapt
the prostate cancer cells to low pH between 6.5 and 7.0. Hence,
confluent PC3 cell lines were left incubated at 37 °C until the
growth medium attained a low pH of 6.7−7.0. The cells were
further passaged twice into RPMI-1640 medium (pH 6.7−7.0)
to allow them to adapt to this low pH environment. These
cancer cells were then suspended in growth medium buffered at
pH 6.8 using KH2PO4 and plated in a 96-well plate. Cells from
the same passage were also plated in normal RPMI-1640
growth medium of pH 7.4. The growth of PC3 cell lines under
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Table 3. In Vitro Activation of pH-Responsive
a
Representative Emetine Prodrugs 13 and 26
b
b
compd
IC50 fold at pH 7.4
IC50 fold at pH < 7.0
13
26
16.5
16.8
1.9
2.5
a
Measured by change in ratio of cytotoxic IC50 of each drug compared
to that of emetine on day 5 in PC3 cell lines under different pH
b
conditions. IC50 fold at each pH is determined as (IC50 of each
drug)/(IC50 of emetine).
is thus in agreement with their activation to emetine under
slightly acidic environment. Hence, it appears that emetine is
the major cytotoxic agent when these compounds are subject to
pH < 7.0. In addition, the results at pH 7.4 compared to pH <
7.0 indicate that these compounds are far more activatable in
the acidic cancer environment, thus establishing these
compounds as potential prodrugs of emetine. In order to
carry out an initial assessment of the relative toxicity of the
prodrugs 13 and 26 compared to emetine, we opted for in vivo
toxicity studies in healthy mice.
Figure 6. Reduction of average pH of growth medium over 7 days due
to the metabolism of PC3 prostate cancer cell lines. Cell suspension
was made at a density of 2000 cells per 100 μL and then plated in a 96-
well plate at a density of 2000 cells/well. Medium was drawn off the
wells on day 0, day 3, day 5, and day 7 of incubation. pH was measured
and average pH calculated.
In Vivo Toxicity Study To Establish the Safety of
Emetine, 13, and 26 in Mice. The dose dependent side
effects associated with the use of emetine usually culminates in
lethality. To establish the safety of the N-2′ derived analogues
developed in this study, we further evaluated the in vivo toxicity
of emetine and compounds 13 and 26 in healthy mice.
These compounds were administered to the mice intra-
venously (iv). A single dose of emetine at 100 mg/kg body
weight killed all three mice within 48 h. However, no sign of
toxicity was shown in the mice that received 100 mg/kg body
weight of compounds 13 and 26 daily for 5 days. To further
investigate the safety of these compounds at 100 mg/kg, we
determined the change in the body weight of these mice and
that of the mice in the control group (mice that received just
1% DMSO in saline) within 48 h (Figure 8). The percent
these two pH conditions was monitored over a 5-day
incubation period. They both gave a comparable growth
curve (Figure 7). Thus, this became our in vitro model for
performing a pH-responsive prodrug activation assay.
Figure 7. Comparison of growth of PC3 cell lines in growth medium
of pH 6.8−7.0 and pH 7.4. These cells were adapted to growth in low
pH medium and then plated in growth medium of pH 6.8−7.0 and pH
7.4 for comparison. MTT cell proliferation assay was done on each of
day 1, day 2, and day 5. Growth of cells under these two conditions is
comparable.
Figure 8. Percent weight change in mice that received 100 mg/kg
body weight of compounds 13 and 26 and the control group over a 5-
day daily dosing.
Prostate cancer cell lines already adapted to growth under
this slightly acidic pH were thus plated at a higher density of 15
000 to 20 000 cells/well in growth medium of pH 6.8. Cells
were incubated for 48 h to give room for metabolism that might
lead to further lowering of pH before drug treatment. Cells
were then treated with six different concentrations of each drug
(13 or 26) prepared in a growth medium of pH 6.8. Emetine
dihydrochloride was used as a positive control. Because of
cancer cell metabolism, the average pH gradually decreased
further to as low as 6.4 by the fifth day of this study. As
expected, the difference in the cytotoxicity of 13 and 26
compared to that of emetine reduced drastically (only about 2-
fold difference from emetine) at pH < 7.0. On the other hand,
there is more than 16-fold difference in the cytotoxicity of 13
and 26 relative to emetine at pH 7.4 (Table 3). The increased
cytotoxicity of these prodrugs (13 and 26) at pH less than 7.0
weight change observed within 48 h in the mice that received
analogues 13 and 26 was very similar to that obtained for the
control group (Figure 8). This indicates that this small weight
change is not due to the toxicity effect of these drugs. The mice
were further studied for 3 weeks, and no noticeable effect of
toxicity was observed in them. To further establish the safety of
one of the top pH-responsive prodrugs of emetine compared to
emetine, we needed a dose of emetine that is not immediately
lethal to the mice (i.e., does not result in death of the mice). To
this end, we found that a lower dose of 33 mg/kg body weight
of emetine did not kill the mice within the period of study.
Hence, each mice group was given 33 mg/kg of emetine and
prodrug 13. Though mice that received 33 mg/kg of emetine
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did not die within 48 h, these mice appeared slightly lethargic
and weight loss was observed (Figure 9). A weight loss of 4.5%,
(obtained from maleic and citraconic anhydrides) showed the
most promise as potential pH-sensitive prodrugs for activation
by the slightly acidic cancer microenvironment with significant
stability at physiological pH. The results obtained from the
compounds investigated showed that the N-2′ position is
“tunable” for controlled release of emetine. The in vitro model
of slightly acidic prostate cancer microenvironment was also
developed and employed in in vitro activation of potential
prodrug in this study. Further, the relative safety, in health
mice, was demonstrated in vivo for two of the prodrugs 13 and
26 compared to emetine. Our findings in this preliminary study
are a significant foundation for further in vivo efficacy and
pharmacokinetic studies, as well as further efforts toward the
design of new synthetic analogues of emetine in prodrug
development.
Figure 9. Percent change in weight of mice that received emetine and
compound 13 at a dose of 33 mg/kg body weight.
ASSOCIATED CONTENT
* Supporting Information
■
S
(1) Details of the analytical instrument used, (2) experimental
details of all the intermediates and analogues of emetine, (3)
experimental details of the biological assays, (4) further details
on the pH responsiveness of emetine analogues. This material
is available free of charge via the Internet at http://pubs.acs.org.
2.0%, and 1.7% were observed in mice that received emetine,
prodrug 13, and control (1% DMSO in saline), respectively,
within 24 h. Over a 48 h period, mice that received emetine at
33 mg/kg showed an average weight loss of about 3.6% and
appeared lethargic while those that received 33 mg/kg of 13
showed a weight loss of 0.67% but appeared healthy with no
noticeable signs of toxicity (Figure 9). Within 48 h of this daily
dosing, the percent weight loss in mice that received emetine
was about 4 times the weight loss in control mice, while there
was no significant difference in weight loss between mice
injected with prodrug 13 and the control group (Figure 9).
These results indicate that the toxicity associated with the use
of emetine has been significantly reduced if not removed in
these pH-responsive prodrugs. The drugs are therefore
considered safe for further in vivo efficacy studies.
In summary, these results suggest that an appropriately
designed emetine analogue could become a valuable cancer
chemotherapy. The vital role of the N-2′ secondary amine is
seen in the reduced cytotoxicity of all the N-2′ derived
analogues. These analogues can be hydrolyzed to emetine at
variable rates that depend on the substituent at the “tunable
handle”. It is also vital to note that these compounds are
relatively stable at pH 7.4, indicating that emetine will most
likely not be released in the blood or the environment of
normal tissue as demonstrated in the nontoxic nature of two of
these prodrugs relative to emetine in healthy mice.
AUTHOR INFORMATION
Corresponding Author
*Phone: 202-806-6888. Fax: 202-806-5442. E-mail: obakare@
howard.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by Grant 5-U54-CA914-31
(Howard University/Johns Hopkins Cancer Center Partner-
ship); in part by Grant 2 G12 RR003048 from the RCMI
Program, Division of Research Infrastructure, National Center
for Research Resources, NIH; and in part by MRI Grant CHE-
1126533 from the NSF.
ABBREVIATIONS USED
■
MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide; RPMI, Roswell Park Memorial Institute; LNCaP,
androgen-sensitive human prostate adenocarcinoma; PC3,
androgen-insensitive human prostate cancer cell lines;
DU145, androgen-insensitive human prostate cancer cell lines
CONCLUSION
■
Overall, in an attempt to improve the therapeutic index of the
natural product emetine, we have developed several N-2′
derived analogues of emetine and they were assayed in both
androgen receptor positive and negative prostate cancer cell
lines. The data obtained clearly confirmed that the secondary
amine of the N-2′ position of emetine plays a crucial role in its
cytotoxic activity. All chemical modifications of this position to
thiourea, urea, dithiocarbamate, carbamate, sulfonamides, and
amides resulted in various analogues that are almost 5−400
times less potent than emetine. This is obviously a starting
point for designing an emetine prodrug that will avert the
systemic toxicity associated with its therapeutic use and thus
improve its therapeutic index. The current study utilized
synthetic analogues of emetine that are sensitive to pH change
for their controlled activation. The water-soluble sodium
dithiocarbamate salt 13 and the amide analogues 21, 22, 26
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