RITA Mimics: Synthesis and Mechanistic Evaluation of Asymmetric Linked Trithiazoles

Article abstract of DOI:10.1021/acsmedchemlett.6b00488

The established cytotoxic agent RITA contains a thiophene-furan-thiophene backbone and two terminal alcohol groups. Herein we investigate the effect of using thiazoles as the backbone in RITA-like molecules and modifying the terminal groups of these trithiazoles, thereby generating 41 unique structures. Incorporating side chains with varied steric bulk allowed us to investigate how size and a stereocenter impacted biological activity. Subjecting compounds to growth inhibition assays on HCT-116 cells showed that the most potent compounds 7d, 7e, and 7h had GI₅₀ values of 4.4, 4.4, and 3.4 μM, respectively, versus RITA (GI₅₀ of 800 nM). Analysis of these compounds in apoptosis assays proved that 7d, 7e, and 7h were as effective as RITA at inducing apoptosis. Evaluating the impact of 7h on proteins targeted by RITA (p53, c-Myc, and Mcl-1) indicated that it acts via a different mechanism of action to that of RITA. RITA suppressed Mcl-1 protein via p53, whereas compound 7h suppressed Mcl-1 expression via an alternative mechanism independent of p53.

Full text of DOI:10.1021/acsmedchemlett.6b00488

Letter
pubs.acs.org/acsmedchemlett
RITA Mimics: Synthesis and Mechanistic Evaluation of Asymmetric
Linked Trithiazoles
Adrian L. Pietkiewicz,† Yuqi Zhang,† Marwa N. Rahimi, Michael Stramandinoli, Matthew Teusner,
and Shelli R. McAlpine*
School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia
S
* Supporting Information
ABSTRACT: The established cytotoxic agent RITA contains
a thiophene−furan−thiophene backbone and two terminal
alcohol groups. Herein we investigate the effect of using
thiazoles as the backbone in RITA-like molecules and
modifying the terminal groups of these trithiazoles, thereby
generating 41 unique structures. Incorporating side chains with
varied steric bulk allowed us to investigate how size and a
stereocenter impacted biological activity. Subjecting com-
pounds to growth inhibition assays on HCT-116 cells showed
that the most potent compounds 7d, 7e, and 7h had GI50
values of 4.4, 4.4, and 3.4 μM, respectively, versus RITA (GI50
of 800 nM). Analysis of these compounds in apoptosis assays
proved that 7d, 7e, and 7h were as effective as RITA at inducing apoptosis. Evaluating the impact of 7h on proteins targeted by
RITA (p53, c-Myc, and Mcl-1) indicated that it acts via a different mechanism of action to that of RITA. RITA suppressed Mcl-1
protein via p53, whereas compound 7h suppressed Mcl-1 expression via an alternative mechanism independent of p53.
KEYWORDS: Heterocycle, RITA, thiazole, antitumor, Mcl-1, p53, c-Myc
eterocycles comprise one of the largest compound classes
is also present in NSC 652287 (RITA) (Figure 1c),21 which
Hof all known molecules. Over 10 million heterocyclic contains a 2,4-linked thiophene−furan−thiophene backbone
and terminal alcohol groups.21 RITA is of significant biological
interest as a cytotoxic agent because it interferes with the p53-
HDM-2 interaction, a key oncogenic binding event.22 Inhibiting
binding between HDM-2 and p53 activates p53, which
subsequently induces apoptosis. RITA also suppresses ex-
pression of numerous oncogenic proteins including Mcl-1 and
c-Myc, which also promote apoptosis.23 Recent studies
involving CRISPR-Cas9-mediated gene disruption have also
shown that RITA is cytotoxic in cells deficient in p53.24 Cells
resistant to RITA could be made RITA-sensitive when FancD2
or mTor proteins were inhibited.24 These results indicate that
RITA is biologically active even in the absence of p53 protein.
Elegant work by Wipf and co-workers describes a series of
linked thiophenes and furans that had similar structures and
activity to RITA (4−5 Figure 1c).20 Research by Jiang and co-
workers also showed that a linked trithiophene 6 has cytotoxic
potency.25 The primary alcohols on RITA, and structures 4−6
were essential to the molecules’ biological activity. Jiang et al.25
showed that replacing one of the thiophene groups with a furan
to form a thiophene-difuran diol lead to a loss in cytotoxic
activity by almost 10-fold. The linked trifuran species lead to a
complete loss in cytotoxic activity of the diol, suggesting that
scaffolds have been identified since 2000.1 Linked azole
moieties are present in many bioactive compounds,2−5 and
substituting a thiazole into the molecular backbone can
improve the biological activity of a molecule over other
heterocyclic substitutions.6−8 Thiazole-containing compounds
have efficacy as antibacterial,9 anticonvulsive,10 antifungal,11
antiviral,12 and anticancer13,14 agents. One successful thiazole
drug, dasatinib (Sprycel), was FDA-approved as a chronic
myeloid leukemia (CML) chemotherapeutic (Figure 1a).15
LCL-161 (Figure 1b) is currently in Phase II clinical trials,
where it is being assessed to treat myelofibrosis.16
Thiazoles provide rigidity to the compound’s structure
compared to alkyl or peptide moieties from which they are
generated, which leads to lower entropic costs upon binding to
a target compared to molecules with the same atoms that are
not locked into a ring system. Thiazoles also offer π-stacking
opportunities with protein targets.17,18 In contrast to thiazoles,
similar structures containing oxazoles and triazoles can be
significantly less active, in some cases by greater than 10-fold
than those containing thiazoles.6−8,19,20
Molecules containing three 2,4-linked thiazoles and an ester
or amide functional group at one end (structures 1−3, Figure
1c) are cytotoxic against the human colorectal carcinoma cell
line HCT-116.6,7 Similar structures where thiazoles in 1−3
were replaced with oxazoles were completely inactive.6,7 The
2,4-linked heterocylic scaffold present in molecules 1, 2, and 3
Received: December 1, 2016
Accepted: March 6, 2017
Published: March 6, 2017
© XXXX American Chemical Society
A
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a
Scheme 1. Synthesis of Trithiazole Analogue Series 7−10
Figure 1. (a) Thiazole-containing chemotherapeutic drug approved by
the FDA. (b) Thiazole-containing compound in clinical trials. (c)
Trithiazoles 1−36,7,20 and RITA20 with their reported activity against
HCT-116 colon cancer cell line, 4−5 generated from Wipf lab,20 and 6
from Gao and Jiang lab against MCF-7 breast cancer line.25
a
Reagents and conditions: (a) BOP, DIPEA, NH4OH, CH2Cl2, N2, rt,
16 h. (b) Lawesson’s reagent, benzene/THF, 60 °C/rt, 6 h. (c)
KHCO3, ethyl bromopyruvate, DME, rt, 16 h. (d) Py, TFAA, TEA,
DME, 0 °C−rt, 7 h. (e) NH4OH, MeOH, rt, 14 h. (f) TFA, anisole,
CH2Cl2, rt, 1 h. (g) NaNO2, H2SO4, 1,4-dioxane, 0 °C−rt, 14 h. (h)
LiAlH4, THF, N2, 0 °C−rt, 2 h.
increase compound hydrophilicity via H-bonding with the
nitrogen atom in the ring.27
Structures 7a−i, which are similar to 1 in that they contain
an ester and amine with a tert-butyloxy carbonyl (Boc)
protecting group, were synthesized with methyl (7b, 7c),
isopropyl (7d, 7e), benzyl (7f, 7g), (S)-cyclohexylmethyl (7h),
and (S)-tetrahydroisoquinoline (7i) at position R. These side
chains were chosen to evaluate the effect of biological activity as
the steric bulk of the side chain increased. Removal of the Boc
group produced series 8a−i. Conversion of the amine in each
structure to an alcohol generated molecules 9a−h. Reduction of
the ester to the alcohol produced diol compounds labeled as
10a−h. Conversion of the ester 7 to the amide gave molecules
labeled as 11a−h. An (S)-tetrahydroisoquinoline analogue 12
was also synthesized, containing Boc and alcohol groups.
With the goal of understanding how thiazoles impact the
biological activity and the relevance of the alcohol group
compared to other moieties, we synthesized and mechanisti-
cally evaluated series 7−11 (Figure 2). These series were
hybrid structures of 1−3 and 4−6, thereby allowing us to
Figure 2. Structures of 7a−i, 8a−i, 9a−h, 10a−h, 11a−h, and 12.
the thiophene moieties, and particularly the sulfur group, are
important for cytotoxic activity. Computational studies have
shown that thiazoles have a larger aromatic character compared
to furans, and behave similarly to thiophenes,26 which may
explain Jiang’s results. By increasing the aromatic character in
the molecular backbone, there is increased potential for the
compound to undergo π-stacking with target proteins.
Incorporating thiazole moieties into the compound can also
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Syntheses of structures 7a−i were based on the synthetic
strategy developed by Islam and co-workers (Scheme 1).7
Starting from the appropriate Boc-protected amino acid 13,
thiazole 14 was produced via reaction with ammonia in the
presence of (benzotriazol-1-yloxy) tris (dimethylamino)-
phosphonium hexafluorophosphate (BOP), N,N-diisopropyle-
thylamine (DIPEA), and ammonium hydroxide to form the
corresponding amide. The amide was then reacted with
Lawesson’s reagent to yield the thioamide. The thioamide
was condensed with ethyl bromopyruvate and KHCO3 and
subsequently dehydrated using trifluoroacetic acid anhydride
(TFAA) in the presence of pyridine (Py) and quenched with
triethylamine (TEA), employing a modified Hantzsch synthesis
to form the monothiazole (34−88% yield). Series 14 were
converted to the dithiazoles 15 by converting the monothiazole
ester to the amide using ammonium hydroxide and then
repeating the thioamidation and modified Hantzsch synthesis
(yields 28−83% over 4 steps). Conversion of the dithiazole into
trithiazole structures 7a−i was accomplished by amidation,
thioamidation, and modified Hantzsch synthesis to yield
compounds 7a−i (12−72% yield).
Scheme 2. Syntheses of Trithiazole Analogues 11d−i and 12
The compound series 7a−i was reacted with trifluoroacetic
acid (TFA) to produce compounds 8a−i (yields 20−57%).
The series 9a−h was synthesized by reacting 8a−h with sodium
nitrite in concentrated sulfuric acid and 1,4-dioxane (23−65%
yield). The diol series 10a−h was synthesized by reducing
compounds 9a−h with LiAlH4 (3−40% yield).
Compound 11a was not generated, and compounds 11b−c
have been previously reported7 and have no biological activity.
Series 11d−i were produced by converting analogues 7d−i to
their corresponding amide via sonication in ammonium
investigate how the trithiazole and primary alcohol contributed
to the compound’s overall activity. Mechanistic studies
including cytotoxicity against HCT-116 colon cancer cell
lines, apoptosis induction, and impact on expression of p53,
Mcl-1, and c-Myc are also described for the three most active
molecules.
Figure 3. (a) Growth inhibition assay on trithiazole analogues at 10 μM of compound over 24 h against HCT-116 cells. (b) GI50 values of most
cytotoxic analogues (see Supporting Information for growth inhibition curves). (c) Apoptosis induction caused by RITA, 7d, 7e, and 7h at 24 h in
HCT116 cells. All values shown are the average SEM from at least three independent experiments.
C
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contain a heteroatom on the ring and the alkyl bond is absent.
This alkyl bond appears to play a key role in the cytotoxicity of
RITA given that there is a difference between RITA and 10a.
After the initial growth inhibition screening, the most potent
analogues, 7d, 7e, and 7h, were tested for their GI50: 7d (4.4
1.0 μM), 7e (4.4 0.2 μM), and 7h (3.4 0.2 μM) (Figure
3b). Thus, the trithiazole esters with aliphatic bulky side chains
were the most promising leads. Despite being similar in size to
the side chains on the active molecules the benzyl and
tetrahydroisoquinoline (TIC) analogues (7e, 7f, and 7i,
respectively) were not nearly as active as those with isopropyl
and cyclohexylmethyl side chains (7d, 7e, and 7h).
Apoptosis assays were evaluated on the three most active
compounds (7d, 7e, and 7h) and run based on their GI50 values
in HCT-116 cells (Figure 3c). An initial concentration of 5 μM
was chosen for compounds 7d, 7e, and 7h as this concentration
was just above each compound’s GI50 value and would be easy
for others to replicate. Comparing 1 μM RITA to 5 μM 7d, 7e,
and 7h showed that RITA induced apoptosis more effectively
than 7d, 7e, and 7h. However, treating cells with 10-fold over
the GI50 concentration (10 μM RITA versus 50 μM 7d, 7e, and
7h) showed that 7d and 7e were slightly more effective and that
7h was almost 2-fold more effective at inducing apoptosis than
RITA.
We investigated the mechanism by which 7h, our most
effective molecule, induced apoptosis and compared it to
RITA’s mechanism for impacting p53 protein levels in the cell.
It is established that RITA activates p53, inducing expression of
this antiapoptotic protein. The current belief is that through
activation of p53, RITA suppresses the expression of numerous
oncogenic proteins including Mcl-1 and c-Myc.21−23 Thus, we
evaluated the expression levels of p53, c-Myc, and Mcl-1 when
HCT116 cells were treated with RITA and 7h. The impact of
RITA on protein levels are usually monitored over time rather
than varying the concentration of the compound in order to
observe the initial increase and then decrease of p53 at a single
concentration of RITA. The levels of p53 are then correlated to
a decrease in c-Myc and Mcl-1. Thus, we investigated changes
in p53, c-Myc, and Mcl-1 over time but also over concentration
(Figure 4a−c).
Figure 4. (a) Expression level of p53 at 6, 12, and 24 h against HCT-
116 cells following treatment with DMSO (1%), RITA (1 μM, 10
μM), and 7h (5 μM, 50 μM). (b) Expression levels of c-Myc at 6, 12,
and 24 h against HCT-116 cells following treatment with DMSO
(1%), RITA (1 μM, 10 μM), and 7h (5 μM, 50 μM). (c) Expression
level of Mcl-1 at 6, 12, and 24 h against HCT-116 cells following
treatment with DMSO (1%), RITA (1 μM, 10 μM), and 7h (5 μM, 50
μM).
Treating HCT116 cells with 1 μM RITA produced an
increase in p53 at 6 h. A peak in p53 protein levels was seen at
12 h when treating with 10 μM (∼10-fold over the GI50). In
contrast, treating cells with 7h did not produce an increase in
p53, rather p53 decreased with treatment time. Evaluating c-
Myc after treatment with 1 μM RITA over time showed that
initially c-Myc protein decreased relative to control, but then
returned to normal levels. The 10 μM c-Myc treatment,
however, produced an intense decrease in c-Myc protein
expression. This trend is similar to data produced by others,
where c-Myc decreases as p53 levels are increased. Monitoring
c-Myc upon treatment of 7h showed an increase in the protein
after 5 μM treatment and 24 h. These data correlate to the
levels of p53, which decreased by 10-fold after 24 h of 5 μM
treatment. Treatment with 7h at 50 μM produces the same
trend as treatment at 5 μM, where there is a decrease in p53
levels, which likely produces an increase in c-Myc levels.
Examining Mcl-1 protein levels after treatment with RITA
and 7h showed that both concentrations of RITA and 7h
produced a drop in Mcl-1 protein over time (almost 10-fold).
Levels of Mcl-1 are known to drop when treated with RITA
because they are regulated by the increase in p53. However,
treatment with 7h does not increase p53; therefore, the
hydroxide (Scheme 2a, yields 10−60%). The (S)-tetrahydroi-
soquinoline analogue 12 was synthesized by LiAlH4 reduction
of 7i in THF (27% yield, Scheme 2b).
Upon completing the compound synthesis all molecules
were tested for their ability to inhibit growth when colon cancer
HCT-116 cell line was treated with compounds at a
concentration of 10 μM for 24 h (Figure 3a). DMSO was a
negative control, and RITA was a positive control. Of the ester
series, 7d, 7e, and 7h were found to be the most potent
analogues, showing between 50 and 100% growth inhibition.
Surprisingly, no analogues in series 8, 9, 10, or 11 showed
significant cytotoxic activity despite structural similarities to
RITA.
The lack of cytotoxic activity in series 10 was surprising, as
this series most closely resembled the lead structures,
suggesting that the thiophene ring in RITA is important for
biological activity. Comparing 10a and RITA, we see the sulfur
groups of 10a are in a different position than in RITA. The
thiophene ring in RITA also contains a hydrophobic HC−CH
bond opposite the heteroatoms, whereas the thiazoles in 10a
D
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ACS Medicinal Chemistry Letters
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expression levels of Mcl-1 do not correlate to p53 expression
levels. Thus, in contrast to RITA, where an increase in p53 is
linked to a decrease in Mcl-1, 7h produces a decrease in p53,
but also a decrease in Mcl-1. Treating cells with 7h produces
almost a 10-fold decrease in the Mcl-1 protein (Figure 4c, 24 h,
50 μM). This interesting phenomenon suggests that a protein
other than p53 is regulating the decrease of Mcl-1. In
conclusion, we have synthesized 41 compounds that were
based on the structure of RITA. These compounds were tested
for their ability to kill HCT-116 cells, induce apoptosis, and
regulate p53 and oncogenic proteins c-Myc and Mcl-1. As
reported by others, RITA induces p53 protein and decreases
the level of both oncogenic proteins. Our most potent
compound 7h has a slightly higher GI50 than RITA and
induces apoptosis at similar levels to RITA. Yet 7h acts via a
mechanism that is distinct from that of RITA. It does not
induce high levels of p53, yet it increases the protein levels of c-
Myc and decreases the protein levels of Mcl-1 in cancer cells.
This behavior is distinct from the p53 inducing compound
RITA and, as such, provides an interesting tool for suppressing
Mcl-1 protein levels via an alternative mechanism.
dride; Py, pyridine; TEA, triethylamine; TFA, trifluoroacetic
acid
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00488.
Experimental procedures and all spectra collected during
the synthesis (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: s.mcalpine@unsw.edu.au. Tel: +61 4 1672-8896.
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Shelli R. McAlpine: 0000-0003-1058-5252
Author Contributions
†These authors contributed equally to the manuscript. A.L.P
synthesized “series f, g, and h” and created the first draft of the
manuscript. Y.Z. synthesized “series b, c, e, i, and 12” and
collated the Supporting Information. M.N.R. ran the biological
assays. M.S. synthesized “series a”. M.T. synthesized “series d”.
S.R.M. conceived of the project, assisted in experimental design
and interpretation, and heavily edited the final version of the
manuscript.
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Funding
The University of New South Wales, School of Chemistry
supported this research.
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South Wales for providing funding for this project. A.L.P. and
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̀
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DME, 1,2-dimethoxyethane; TFAA, trifluoroacetic acid anhy-
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DOI: 10.1021/acsmedchemlett.6b00488
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