Total synthesis and biological studies of TMC-205 and analogues as anticancer agents and activators of SV40 promoter

Article abstract of DOI:10.1021/ml500025p

TMC-205 is a natural fungal metabolite with antiproliferative activity against cancer cell lines. The light- and air-sensitivity prevented in-depth exploitation of this novel indole derivative. Herein, we report the first synthesis of TMC-205. On the basi

Full text of DOI:10.1021/ml500025p

Letter
pubs.acs.org/acsmedchemlett
Total Synthesis and Biological Studies of TMC-205 and Analogues as
Anticancer Agents and Activators of SV40 Promoter
Yang Gao,† Sami Osman,† and Kazunori Koide*
Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania 15260, United States
S
* Supporting Information
ABSTRACT: TMC-205 is a natural fungal metabolite with anti-
proliferative activity against cancer cell lines. The light- and air-
sensitivity prevented in-depth exploitation of this novel indole
derivative. Herein, we report the first synthesis of TMC-205. On the
basis of its reactivity with reactive oxygen species, we developed air-
stable analogues of TMC-205. These analogues are 2−8-fold more
cytotoxic than TMC-205 against HCT-116 colon cancer cell line.
Importantly, at noncytotoxic dose levels, these analogues activated the
transcription of luciferase reporter gene driven by simian virus 40
promoter (SV40). Further, these small molecules also inhibit firefly luciferase, presumably by direct interaction.
KEYWORDS: natural product, indole,, TMC-205, simian virus 40 promoter, firefly luciferase, gene activation
a
Scheme 1. Synthesis of TMC-205
mall molecule-based activators of gene transcription are
Spowerful chemical tools to study the functions of a
particular gene through a gain-of-function approach.1−3
Constitutive promoters, such as SV40 early promoter and
cytomegalovirus (CMV) immediate early promoter, are
frequently used for driving the expression of transgenes in
mammalian cells.4 Ectopic gene expression assays using an
SV40 promoter facilitated the discoveries of natural anticancer
agents including azelaic bishydroxamic acid,5 trichostatin A,6
romidepsin (FR901228),7 herboxidiene,8 and FR901464.9
FR901464 and herboxidiene are now known as spliceosome
inhibitors, while the other three compounds are known as
histone deacetylase inhibitors.10 Romidepsin was approved by
the US Food and Drug Administration for the treatment of
cutaneous T-cell lymphoma. FR901464 analogues are widely
distributed to study splicing and the implication of the splicing
factor 3b (SF3b) subcomplex of the spliceosome in diseases; it
is currently unclear whether/how this activation of SV40
promoter is related to splicing inhibition.
Using a cell-based luciferase assay, TMC-205 (Scheme 1)
was discovered from an unidentified fungal strain, TC 1630, as
an activator of the SV40 promoter.11 This indolebased natural
product showed antiproliferative activity against various human
cancer cell lines with GI50 values in the range of 52−203 μM.
The fairly compact structure suggested to us that the potency
could be improved through chemical synthesis of its analogues.
In addition, TMC-205 was found to be light sensitive, and only
3.3 mg of TMC-205 was isolated, necessitating better access to
this natural product to carry out structure−activity relationship
(SAR) and biological studies. Herein, we report the first
synthesis of TMC-205 and its light- and air-stable analogues
and subsequent biological studies.
a
Reagents and conditions: (a) (CF3CO)2O (1.1 equiv), DMF, 0 to 23
°C; (b) 4 M aq. NaOH, reflux, 97% (over 2 steps); (c) 8 (3.0 equiv),
Pd(PPh3)4 (10 mol %), Cs2CO3 (3.0 equiv), THF, 80 °C, <1%; (d) 4
(1.0 equiv), 9 (2.0 equiv), Pd(PPh3)4 (10 mol %), DMF, 10%; (e)
TMSCHN2 (4.1 equiv), MeOH, 65% (from 2); (f) 8 (2.9 equiv),
Pd(PPh3)4 (5 mol %), Cs2CO3 (3.0 equiv), THF/MeOH (4:1), 70 °C,
98%; (g) 4 M NaOH, MeOH, 80 °C; 3 M KHSO4, 88%; (h)
catecholborane (0.4 equiv), 80 °C; (i) iPr2NH (1.2 equiv), nBuLi (1.2
n
equiv), Bu3SnH (1.15 equiv), CuCN (2.05 equiv), THF, −40 °C,
58%.
was the C6−C9 bond formation via a crosscoupling reaction.
We first examined Stille coupling12 and Suzuki−Miyaura
coupling13 reactions (Scheme 1). Treatment of 6-bromoindole
(2) with (CF3CO)2O followed by aqueous NaOH afforded
carboxylic acid 4 in 97% yield over 2 steps.14 With the known
Received: January 20, 2014
Accepted: June 19, 2014
Published: June 23, 2014
We first set out to devise a concise synthesis of TMC-205 for
subsequent biological studies. An obvious convergent approach
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
sequence (6 total steps) from the commercially available
bromide 2 in 64% overall yield. The GI50 value of the synthetic
TMC-205 against HCT-116 tumor cells was 68 3 μM (Table
1), which is in excellent agreement with the literature.11
Synthetic TMC-205 was used to determine the cause of its
noticeable decomposition in less than 24 h under ambient light
and air. The decomposition was not solvent-dependent (Figure
S2, Supporting Information). The 1H NMR spectrum of TMC-
205 in CD3OD (Figure S3, Supporting Information) revealed
the presence of an enone group, possibly from enone I (Figure
1A) or its derivative produced by an oxidation of the indole
ring (carboxylic acid III) and a formyl group resulted from the
oxidation of the C9−C10 and/or the C2−C3 bond (carboxylic
acid II and III, aldehyde IV, or aldehyde V; Figure 1A). LC−
MS analyses revealed enone I and aldehyde V as the probable
products of TMC-205 when exposed to light and air, based on
the limited spectroscopic information obtained.
Table 1. Antiproliferative Activity of TMC-205 and Its
Analogues against HCT-116 Colon Cancer cells
Indole derivatives are well-known for their antioxidant
potential, and the indole substitution patterns influence the
reactivity toward ROS.18 We proceeded to determine which
reactive oxygen species (ROS) was responsible for the oxidized
byproducts of TMC-205. A qualitative assessment revealed the
primary reactivity of synthetic TMC-205 to singlet oxygen, but
not superoxide, hydrogen peroxide, or hydroxy radical.19 In the
presence of NaN3, a widely used singlet oxygen scavenger,20 the
singlet oxygen-mediated oxidation of TMC-205 into V was
minimized as determined by HPLC analysis (Figures 1B and
S4, Supporting Information). Of note, the existence of I in the
chromatograph indicated the involvement of another ROS in
the oxidation of the diene moiety. Ketone 12 was more stable
than TMC-205, which is consistent with the literature that had
shown that electron-deficient indoles are less reactive toward
ROS.20
A series of SAR studies were subsequently preformed to
TMC-205 and analogues. We first probed the importance of
the carboxyl group for the cytotoxicity of TMC-205. The 72 h
antiproliferative assays revealed that the decarboxylated
analogue 10 (Table 1) was ∼3 times less cytotoxic than
TMC-205 against HCT-116 colon cancer cells. Alcohol 11 was
more sensitive to light, air, and heat than TMC-205, but was
equipotent, indicating that the negative charge of the carboxyl
organostannane 9,15,16 but not boronic ester 8,17 a palladium-
catalyzed reaction converted bromide 4 to TMC-205. However,
this transformation provided the natural product only in 10%
yield, prompting us to seek an alternative approach.
Both of the cross-coupling reactions described above suffered
from the poor solubility of carboxylic acid 4 in various solvents
(THF, MeOH, DMSO, and 1,4-dioxane). Methyl ester 5 was
prepared in 65% overall yield from 2 (Scheme 1A) and was
more soluble in these organic solvents. This ester was subjected
to the Suzuki−Miyaura coupling conditions with boronic ester
8 to form methyl ester 6 in 98% yield. Finally, treatment of 6
with aqueous NaOH followed by acidification with KHSO4
afforded TMC-205 in 88% yield. Therefore, the total synthesis
of TMC-205 was accomplished in 5 steps in the longest linear
Figure 1. (A) Structures of possible byproducts of decomposed TMC-205. (B) Reaction of TMC-205 with singlet oxygen in the presence or absence
of NaN3.
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Figure 2. (A) pGL3-control and pGL3-promoter. (B) TMC-205
upregulates expression of luciferase gene in pGL3-control, without loss
of cell viability. Data represent mean values
SD (n = 4). (C)
Figure 3. (A) Absorption spectrum of analogue 12 in DMSO. (B,C)
Analogue 12 inhibited luciferase. The graph shows that inhibition of
luciferase was relieved when the concentrations of D-luciferin (C), but
not ATP (B), were increased. Data represent mean values SD (n =
2). (D) RTPCR of luciferase expressed in HeLa cells transiently
transfected with pGL3-promoter and subsequently treated with
DMSO, meayamycin B (MAMB), 12, and resveratrol (RSV).
Activation of luciferase gene from each treatment was presented as
the relative intensity of the luciferase fragment from compound-treated
compared with DMSO-treated. Data represent results from three
independent experiments.
Analogue 12 upregulates expression of luciferase gene in pGL3-
control, without loss of cell viability. (D) Upregulation of SV40
promoter-mediated luciferase expression by 14 and resveratrol. Data
represent mean values SD (n = 2).
group is not necessary. In order to improve the stability of
TMC-205, we introduced an electron-withdrawing 3-trifluor-
omethyl group to TMC-205, yielding ketone 12. This ketone
was twice as potent as TMC-205, with a GI50 value of 39 12
μM.
The oxidation of the diene moiety might occur in cellular
environment to yield an enone functionality.21 We hence
speculated that derivatives with this moiety might display
superior bioactivity, as the bona fide bioactive species in cells.
Indeed, enone 13 exhibited a GI50 value of 14 4 μM and was
3 and 5 times more potent than 12 and TMC-205, respectively.
Thus, we asked whether the electrophilicity was of any
significance for the activity of this enone. The secondary allylic
alcohol 14 was equipotent to enone 13, suggesting that 14
might be oxidized to 13, although this hypothesis warrants
further investigation. The tertiary allylic alcohol 15 was far less
potent, which implies that the additional methyl group might
be too bulky for a putative binding pocket or may prevent the
alcohol from being oxidized to the enone. The rigidity of 1,3-
diene moiety was crucial as evidenced by a complete loss of
activity of the alkane analogue 16. The N-methylated analogue
17 was less cytotoxic than 12, implying that the N−H group
may serve as a hydrogen bonding donor or that a putative
binding pocket for this functional group may not tolerate
additional steric bulk. Figure S5a (Supporting Information)
shows representative data demonstrating the improvement of
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ACS Medicinal Chemistry Letters
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antiproliferative activity against HCT-116 colon cancer cells
through the SAR studies.
In conclusion, the first total synthesis of TMC-205 has been
accomplished. Degradation studies revealed that TMC-205
reacts with singlet oxygen. More oxidation-resistant analogues
were up to 8-fold more potent than TMC-205. These
compounds bind the D-luciferin binding pocket of luciferase
and inhibit the enzymatic activity. Importantly, they activated
the expression of an SV40 promoter-driven gene at non-
cytotoxic dosage.
Previously, TMC-205 was found to activate a luciferase gene
driven by an SV40 promoter in an SV40 enhancer-dependent
manner using the pGL3-control (Promega). The activation of
the luciferase gene driven by the SV40 promoter without the
SV40 enhancer (pGL3-promoter, Promega) was negligible in
the presence of TMC-205 (Figure 2A).11 In this study, with 24
h exposure, TMC-205 and 12 activated SV40 promoter in the
presence of enhancer in HeLa cells stably transfected with
pGL3-control (Figure 2B,C). Interestingly, pGL3-promoter
could be activated by TMC-205 as well (Figure S6a,b,
Supporting Information), meaning the gene activation was
independent of SV40 enhancer. Analogues 12 and 14 also
activated the expression of SV40:luciferase without inhibiting
cell growth (Figure S6c,d, Supporting Information). It is
noteworthy that within the 24 h time frame, before cell growth
could be influenced, analogue 12 activated gene transcription in
a nanomolar range (Figure 2C and S6c, Supporting
Information), demonstrating a property as small-molecule
activators for gain-of-function studies in live organisms.
Firefly luciferase is the reporter enzyme in the vectors used
both in the original isolation study and our current SAR studies
for TMC-205 and analogues. Recently, it was discovered that
certain heterocyclic small molecules perturb luciferase-catalyzed
production of luminescence either through luciferase binding or
compound-specific luminescence absorbance or scattering.22 In
the case of direct binding, these small molecules inhibit
luciferase, competitively or noncompetitively, with D-luciferin
and/or ATP. Concerns arise when the tested compounds that
are active against luciferase may also be active against a target of
interest. For example, high throughput screenings for enzymes,
such as kinases that are also ATP dependent, would inevitably
be susceptible to luciferase inhibitory activity of the hits.23 For
the current study, direct attenuation of luminescence was not a
concern for two reasons: first, as shown in Figure 3A, analogue
12 absorbs UV light with a peak at 290 nm, with no absorption
in the range of visible light (400−700 nm); second, compound-
containing cell medium was thoroughly removed for the
luciferase reporter assay.
We decided to examine the interaction between recombinant
luciferase and analogue 12 as a representative analogue of
TMC-205. Resveratrol was used as a positive control for
noncompetitive luciferase inhibition.24 Substrate competition
assays were carried out by varying concentrations of ATP or D-
luciferin to acquire the dose−response curves of luciferase
inhibition by 12. As expected, varying the concentration of
either ligand did not significantly change the IC50 values of
resveratrol (Figure S5b,c, Supporting Information). Luciferase
inhibition with 12 was perturbed by D-luciferin but not by ATP
(Figure 3B,C), indicating that 12 and D-luciferin antagonize
each other toward luciferase.
On the basis of these data, we questioned whether the strong
inhibition of luciferase enzyme activity underestimated the
activation of luciferase expression by 12. In order to answer this
question, we examined the expression of luciferase at the
mRNA level in HeLa cells transfected with the pGL3-promoter
vector (Figure 3D); in a dose-dependent manner, 12 activated
the SV40 promoter, which in turn upregulated luciferase
mRNA. Interestingly, resveratrol also activated the SV40
promoter-driven gene (Figure 3D), which is unprecedented
and warrants further investigation in the future.
ASSOCIATED CONTENT
* Supporting Information
■
S
Compound characterization and methods for syntheses and
biological studies. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(K.K.) E-mail: koide@pitt.edu. Tel: (412) 624 8767.
■
Author Contributions
†Y.G. and S.O. contributed equally to this work. Y.G. and S.O.
performed experiments. Y.G., S.O., and K.K. interpreted the
results and prepared the manuscript.
Funding
This work was in part supported by the US National Cancer
Institute (R01 CA120792) and the US National Science
Foundation (CHE-0911092).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Dr. Joel Gillespie at the Materials
Characterization Laboratory, Dr. Damodaran and K. Achary at
the NMR facility, and Dr. Bhaskar Godugu at the Mass
Spectrometry facility, all at the University of Pittsburgh.
ABBREVIATIONS
■
CMV, cytomegalovirus; SV40, simian virus 40; SF3b, splicing
factor 3b; SAR, structure−activity relationship; GI50, 50%
growth inhibitory concentration; MAMB, meayamycin B; Me,
methyl; RSV, resveratrol; ROS, reactive oxygen species;
RTPCR, reverse transcription-polymerase chain reaction;
ATP, adenosine triphosphate; DMSO, dimethyl sulfoxide;
THF, tetrahydrofuran; HPLC, high-performance liquid chro-
matography; UV, ultraviolet; SD, standard deviation
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