Studies toward novel peptidomimetic inhibitors of thioredoxin-thioredoxin reductase system

Article abstract of DOI:10.1021/jm201359d

Thioredoxins (Trx) are ubiquitous multifunctional low-molecular weight proteins that together with thioredoxin reductases (TrxR) participate in the maintenance of protein thiol homeostasis in NADPH-dependent reactions. An increasing number of data reveal that the Trx-TrxR system is an attractive target for anticancer therapies. In this work, we have elaborated a new and simple synthetic approach employing Ugi reaction to synthesize several new inhibitors of this system. The influence of various electrophilic fragments of this new class of compounds on the inhibition of the Trx-TrxR system was evaluated. As a result, a new compound 19a (SK053), which inhibits the activity of the Trx-TrxR system and exhibits antitumor activity, was obtained. Biologic analyses revealed that 19a inhibits induction of NF-κB and AP-1 and decreases H₂O₂ scavenging capacity in tumor cells. Altogether, we show that 19a is a novel potential antitumor peptidomimetic inhibitor that can be used as a starting compound for further optimization.

Full text of DOI:10.1021/jm201359d

Article
pubs.acs.org/jmc
Studies toward Novel Peptidomimetic Inhibitors of Thioredoxin−
Thioredoxin Reductase System
‡
‡
†
Szymon Kłossowski,†,§ Angelika Muchowicz,‡,§ Małgorzata Firczuk, Marta Swiech, Adam Redzej,
́
Jakub Golab,*,‡ and Ryszard Ostaszewski*,†
†Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
‡Department of Immunology, Center of Biostructure Research, Medical University of Warsaw, Banacha 1A, F building, 02-097
Warsaw, Poland
S
* Supporting Information
ABSTRACT: Thioredoxins (Trx) are ubiquitous multifunctional low-molecular weight
proteins that together with thioredoxin reductases (TrxR) participate in the maintenance of
protein thiol homeostasis in NADPH-dependent reactions. An increasing number of data
reveal that the Trx−TrxR system is an attractive target for anticancer therapies. In this work,
we have elaborated a new and simple synthetic approach employing Ugi reaction to
synthesize several new inhibitors of this system. The influence of various electrophilic
fragments of this new class of compounds on the inhibition of the Trx−TrxR system was
evaluated. As a result, a new compound 19a (SK053), which inhibits the activity of the
Trx−TrxR system and exhibits antitumor activity, was obtained. Biologic analyses revealed that 19a inhibits induction of NF-κB
and AP-1 and decreases H2O2 scavenging capacity in tumor cells. Altogether, we show that 19a is a novel potential antitumor
peptidomimetic inhibitor that can be used as a starting compound for further optimization.
of a substrate disulfide, resulting in the formation of the
transient intermolecular disulfide bond. Next, the second
nucleophilic attack of the Cys35 occurs, which generates a
dithiol in a proteinatious substrate and a disulfide in Trx. To
enable the next cycle of the reduction, the Trx has to be
enzymatically reduced by NADPH-dependent TrxR.7 Human
TrxR are homodimeric enzymes containing a seleno-cysteine
(Sec) residue at the protein C terminus. This residue belongs
to the conserved Gly-Cys-Sec-Gly- sequence motif and plays an
essential role in catalysis of Trx reduction.12 The Sec residue is
a strong nucleophile and thus has been shown to be a molecular
target for electrophilic compounds such as antitumor quinols.13
The Trx−TrxR system plays a distinct functional role among
cellular antioxidants mainly due to its capacity to reduce other
proteins by cysteine thiol-disulfide exchange. By reducing
peroxiredoxins or glutathione peroxidase 3 that regulate
hydrogen peroxide (H2O2) levels, Trx provide a link between
major antioxidant systems. Trx can also reduce methionine
sulfoxide reductase, quench singlet oxygen (1O2), and act as
hydroxyl radical (•OH) scavengers.13−15 Recent observations
indicate that Trx are the only cellular enzymes that can
participate in protein denitrosylation.16 Inhibition of the Trx−
TrxR system can therefore lead to a profound imbalance in the
redox state that can ultimately lead to apoptosis. Additionally,
Trx-1 can directly protect cells from apoptotic signaling
through binding to apoptosis signal-regulating kinase
(ASK1),17 a member of the mitogen-activated protein (MAP)
1. INTRODUCTION
Thioredoxins (Trx) are ubiquitous multifunctional low-
molecular weight proteins that together with thioredoxin
reductases (TrxR) participate in the maintenance of protein
thiol homeostasis in nicotinamide adenine dinucleotide
phosphate (NADPH)-dependent reactions.1 Trx provide
reducing equivalents to a number of target molecules including
ribonucleotide reductase, participate in scavenging reactive
oxygen species (ROS) primarily by reducing peroxiredoxins,
and regulate the activity of various transcription factors, such as
p53,2 HIF-1, nuclear factor κB (NF-κB), and activating protein-
1 (AP-1)3,4 as well as protein tyrosine phosphatases, such as
phosphatase and tensin homologue deleted on chromosome
ten (PTEN)5 or Cdc25.6 The influence of Trx on some
transcription factors is mediated by reduction of a redox factor,
which has a DNA repair endonuclease activity.4 Two similar
Trx systems exist in eukaryotic cells: a cytosolic one consisting
of a 12 kDa Trx-1 and homodimeric seleno-protein TrxR-1 as
well as mitochondrial, which includes Trx-2 and TrxR-2.7
Trx belong to oxidoreductases, a class of enzymes catalyzing
redox reactions. Trx perform reduction of disulfide bonds in
proteinatious substrates. X-ray crystallography,8,9 protein
nuclear magnetic resonance (NMR),10 and biochemical
investigations11 jointly contributed to the elucidation of the
mechanism of the reaction catalyzed by Trx. The three-
dimensional structure of Trx revealed a common folding unit
named the Trx fold and the active site formed by two Cys
residues, which belong to the conserved CXXC sequence motif.
In human Trx-1, the motif comprises Cys32-Pro-Gly-Cys35. It
is believed that the reaction catalyzed by Trx is initiated by a
direct nucleophilic attack of the Cys32 thiolate on a sulfur atom
Received: June 8, 2011
Published: December 1, 2011
© 2011 American Chemical Society
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Figure 1. Compound 3 and electrophilic inhibitors of the Trx−TrxR system.
kinase family, or via inhibition of procaspase 3 processing and
the presence of double nucleophilic center in Trx can be
targeted by compounds that have two electrophilic groups. On
the basis of this assumption, we also hypothesized that
inhibition of Trx by 3-bromo-2-bromomethylpropionic acid
methyl ester (1a)42 can be explained by the mechanism
presented in Figure 2.
activation.18
Increased levels and activity of Trx are found in many
different tumor types including solid tumors, leukemias, and
lymphomas19 and are associated with more aggressive tumor
progression and worse prognosis.20,21 Trx may facilitate tumor
progression through growth-promoting and antiapoptotic
functions.22 At later stages of tumor progression, Trx may
promote angiogenesis through increased activity of hypoxia-
inducible factor (HIF-1) and secretion of vascular endothelial
growth factor (VEGF) or metastasis by modulating the activity
of matrix metalloproteinases (MMP).23,24 Moreover, increased
Trx levels have been associated with tumor resistance to
chemotherapeutics, such as cisplatin, docetaxel, mitomycin C,
etoposide, or doxorubicin.25−28
Figure 2. Putative alkylation of cysteines in Trx by 1a.
All of these biological activities indicate that the Trx−TrxR
system is an attractive target for anticancer treatment. Indeed,
several inhibitors of Trx have been developed, and one, PX-12
(3),29 progressed to phase II clinical trials as an anticancer
agent. Compound 3 is a substituted 2-imidazolyl disulfide that
thioalkylates cysteine residues of Trx, especially Cys73, which is
thioalkylated irreversibly. When Cys73 is alkylated, Trx cannot
be reduced, which leads to its inactivation.29,30 Phase I clinical
trials revealed, however, some undesired features of disulfides
such as pneumonitis induction as well as pungent, unpleasant,
garlic-like odor of thiol methabolites.31 Other Trx−TrxR
system inhibitors include napthoquinone spiroketal compounds
[such as palmarumycin CP1, PX-916,32,33 gold compounds,34
the organotellerium analogue of vitamin E,35 or PMX464 (2,
previously AW464)].36 Moreover, histone deacetylase inhibitor,
suberoyl anilide hydroxamic acid (SAHA), which is approved
for the treatment of cutaneous T-cell lymphoma, up-regulates
the expression of an endogenous Trx inhibitor−Trx-binding
protein 2 (TBP-2).37
For the initial screening of the capacity of the intermediate
compounds to inhibit the Trx−TrxR system, we have employed
a coupled, Trx−TrxR-dependent insulin reduction assay.43
Tested compounds were incubated for 30 min at 37 °C
together with all other assay reagents, and the IC50 values were
designated (see the Experimental Section). As shown in Table
Table 1. Biologic Activities of 3 and Peptidomimetic
Analogues of 1a and 1b
b
LD50 (μM)
a
compd
IC50 (μM)
EMT6
Panc02
3
12.3 0.6
1.2 0.8
>50
11.6 0.6
>50
3.9 0.2
>50
1a
c
13
ND
ND
14a
14b
15
>50
ND
ND
>50
ND
ND
>50
ND
ND
1b
0.44 0.02
>50
>50
>50
Considering the features of described inhibitors of the Trx−
TrxR system, we designed novel compounds with a
peptidomimetic scaffold that could target these enzymes to
become potential anticancer therapeutics.
6b
ND
ND
16a
16b
16c
16d
16e
17
22.3 4.2
10.0 0.7
3.9 0.5
8.9 1.6
>50
>50
>50
>50
>50
>50
>50
>50
>50
2. RESULTS AND DISCUSSION
>50
>50
2.4 0.5
0.4 0.03
0.2 0.02
3.3 1.3
3.0 0.2
>25
>50
A characteristic feature of the majority of known inhibitors of
Trx system is the presence of an electrophilic fragment, which
can covalently interact with cysteines or Sec in the active site of
Trx or TrxR (Figure 1).38,39 Compound 2 has two electrophilic
centers, and one of the proposed mechanisms of its action is
18a
18b
19a
19b
>25
>25
>25
>25
5.2 0.1
5.5 0.2
3.8 0.3
3.0 0.4
a
based on a two-step thioalkylation of both cysteines of Trx
Activities in recombinant Trx−TrxR insulin reduction assay.
b
c
Cytostatic/cytotoxic effects in cellular assay. ND, not determined.
within the active site by a single inhibitor molecule leading to a
macrocyclic adduct.40 A similar mechanism is also described for
Mannich bases, which also exhibit high Trx system inhibiting
activity.41 Considering these mechanisms, we hypothesized that
1, 1a strongly inhibited the activity of recombinant Trx−TrxR
in the insulin reduction assay, with IC50 values nearly 50-fold
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Figure 3. New concept for the synthesis of Trx−TrxR system inhibitors.
a
lower as compared with 3, the Trx inhibitor currently in clinical
trials. Considering that many of the Trx substrates are proteins
and that peptidomimetic structures serve as efficient scaffolds
for drug design,44 we designed a target structure I (Figure 3),
which contains an electrophilic fragment as well as a peptide
mimicking backbone. The synthetic approaches, which give an
access to peptidomimetics with electrophilic fragment, have not
been described so far, and to the best of our knowledge, no
peptidomimetic-based inhibitors of Trx−TrxR system exist.
The synthesis of peptides and peptidomimetics with one or
more electrophilic fragment using classical synthesis method-
ology is cumbersome. Also, the use of protective groups
methodology is a multistep procedure, which is undesirable in
searching new active structures. Therefore, the multicomponent
Ugi reaction was used, which we have previously successfully
employed for the synthesis of peptidomimetic scaffolds.45 Apart
from its simplicity and one-pot nature, the carboxylic group of
acidic component acts as a nucleophile during Ugi reaction.
Therefore, the presence of multielectrophilic centers in
substrate structure is allowed.
The retrosynthetic analysis and a new synthetic methodology
for target compounds with a general structure I are presented in
Figure 3. The 1,3-dihalide I can be obtained via functionaliza-
tion of 1,3-diols II. An intermediate II, on the other hand, is a
potential product of Ugi reaction with substrates III−VI.
2.1. Synthesis of Peptidomimetics with 1,3-Dihalo-
genes. For the Ugi reaction, isovaleric aldehyde, benzyl
isocyanoacetate, and benzylamine were used. Isovaleric
aldehyde represents a leucine residue in a product, whereas
isocyanoacetate introduced glycine protected with benzyl
group. Benzyl ester was used because of its easy removal,
which could be useful in further functionalization. According to
a retrosynthetic analysis, the introduction of the precursor of an
electrophilic fragment into a peptidomimetic requires 3-
hydroxy-2-hydroxymethylpropionic acid (5). Synthetic path-
ways for 5 and corresponding isocyanide are presented in
Scheme 1.
Scheme 1. Synthesis of the Components for Ugi Reaction
a
Reagents and conditions: (a) Cyclohexanone, p-TsOH, 63%. (b)
KOH, EtOH, 50%. (c) Py, dioxane, 90 °C, 75%. (d) BnOH, p-TsCl, p-
TsOH, 80 °C. (e) EtOH, SOCl2, reflux, 12 h, 88%. (f) CHCOOEt,
TEA, reflux, 20 h, 66−69%. (g) POCl3, DIPEA, DCM, 0 °C, 65−85%.
step was deprotection of 1,3-diol with 80% aqueous acetic acid
(compound 12, 71% yield, Scheme 2).
Compound 12 was a key intermediate in the synthesis of 1,3-
halides as well as their analogues. Next, in the Appel reaction of
12, the desired 1,3-dibromide 13 was obtained with 60% yield.
To determine the influence of the leaving group on the Trx−
TrxR system inhibition, other analogues of 13 were
synthesized. Ditosylate 14a and dimesylate 14b were obtained
with 92 and 100% yield, respectively. Then, ditosylate 14a was
chosen for substitution with sodium iodide leading to the
compound 15. The substitution was accomplished in acetone
producing a compound 15 with 68% yield. This efficient
synthesis provided four analogues of 1a (13, 14a, 14b, and 15)
with peptidomimetic fragments, which were tested in the Trx−
TrxR insulin reduction assay. Table 1 presents the IC50 values.
Surprisingly, all of the obtained analogues were inactive.
Assuming that 1a acts through S-alkylation of selenocysteine
in TrxR or cysteine residues in Trx, there are three possible
mechanisms of enzyme inhibition. The first one is the
alkylation of a solvent-exposed selenocysteine (or cysteine)
residue via the nucleophilic substitution (Scheme 3, pathway I:
1a → A → C). Alternatively, basic enzyme residues can
promote elimination reaction of an adduct A to its α,β-
unsaturated methyl methacrylate derivative B by elimination,
which may be followed by the Michael addition resulting in
adduct C (Scheme 3, pathway II: 1a → A → B → C). Finally, it
is also possible that 1a may be eliminated in the assay
conditions to form a more active alkylation agent methyl
bromomethacrylate (1b), which can alkylate the cysteine
(Scheme 3, pathway III; 1a → 1b → B → C). Considering
the structures of described inhibitors, the Michael addition to a
double bond might be the overall rate-determining step. It
suggests that the formation of intermediate B is most likely.
To investigate this assumption, the activity of 1b was
measured, and it turned out to be more active than 1a in the
Acid 5 was made from 2,2-bis(hydroxymethyl)-
diethylmalonate as described before.46 Reaction of p-
toluenesulfonic acid and cyclohexanone with 2,2-
bis(hydroxymethyl)diethylmalonate gave an acetale 4 with a
63% yield. After basic hydrolysis (50% yield) and decarbox-
ylation in pyridine, acid 5 was obtained with 75% yield.
Isocyanides 9 and 10 were obtained from their formyl
derivatives using phosphorus oxychloride as a dehydratating
agent as described before.47
A further synthetic strategy of new inhibitors based on
retrosynthethic analysis is presented in Scheme 2. The Ugi
reaction was performed in ethanol at room temperature, and
peptidomimetic 11 was obtained with a 48% yield. The next
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a
Scheme 2
a
Reagents and conditions: (a) EtOH, 24 h, room temperature, 48%. (b) 80% AcOH, 24 h, room temperature, 71%. (c) PBr3, PPh3, CCl4, 60%. (d)
MsCl, TEA, DCM, 0 °C, 2 h, quant. (e) TsCl, DIPEA, DMAP cat, DCM, room temperature, 24 h, 92%. (f) NaI, acetone, reflux, 16 h, 68%.
Scheme 3. Possible Pathways of Alkylation of Cysteine
Residues by 3-Bromo-2-(bromomethyl)-propionic Acid
Methyl Ester
methanol (54% yield). Then, the hydroxyl groups were
transformed into p-toluenesulphonate derivative 6a in reaction
with p-toluenesulphonic chloride with 45% yield. Refluxing
compound 6a with lithium bromide leads to the formation of a
desired compound 6b with 55% yield (Scheme 4).
a
Table 1 presents the IC50 values for two model compounds.
In contrast to 1b, compound 6b was inactive. This lack of
activity is in accordance with the mechanism presented in
Scheme 3, where the elimination reaction to 1b or B is blocked.
This indicates that the reaction proceeds with the α,β-
unsaturated methyl methacrylate intermediate and consists of
two Michael addition reactions to protein. This feature
corresponds well with the structure of other known inhibitors
of the Trx−TrxR system. A similar mechanism was postulated
for inactivation of TrxR from Plasmodium falciparum.41 The
interaction of organic molecules with two Michael centers with
proteins was already discussed before, and acronym ETAC
(equilibrium transfer alkylating cross-link reagent) was
proposed.48
2.2. Synthesis of Peptidomimetics with β-Bromoacry-
lamide Fragments and Their Analogues. The results
presented above demonstrate that an efficient inhibitor should
be able to form adduct B. Therefore, the synthesis of
peptidomimetics with acrylamide fragment was designed
(Scheme 5), and the influence of a leaving group in allylic
position was studied. Compound 16a was obtained by
elimination of tosylate from 14a in the presence of a base.
Reaction with various amines such as di-isopropylethylamine
(DIPEA), dimethylaminepyridin, or triethylamine (TEA) was
performed; however, compound 16a was not isolated under
these conditions. Finally, reaction with sodium hydride as a
base leads to the product 16a with 71% yield. The substitution
of the remaining tosylate resulted in compounds 16b and 16c
at room temperature with yields of 95 and 93%, respectively
(Scheme 5).
a
Pathway I: 1a → A → C. Pathway II: 1a → A → B → C. Pathway III:
1a → 1b → B → C.
Trx−TrxR insulin reduction assay (Table 1), indicating that the
proposed pathways III and II might be involved in enzyme
inhibition. At this point, however, pathway I still could not be
excluded. Thus, we proposed the synthesis of a model
compound, which cannot undergo elimination neither to the
adduct B nor to 1b during the inhibition process. Such a
compound can act only via pathway I. For this purpose, the
synthesis of 2-bis(bromomethyl)propionic acid methyl ester
(3) was proposed employing strategy presented in Scheme 4.
a
Scheme 4. Synthesis of Compound 3 for Model Studies
a
Reagent and conditions: (a) SOCl2, methanol, reflux, 16 h, 54%. (b)
p-TsCl, DIPEA, DMAP (cat.), DCM, 16 h, 45%. (c) LiBr, acetone,
reflux, 24 h, 55%.
The inhibitory activity of compounds 16a−c was tested in
the Trx−TrxR insulin reduction assay and revealed that they
are relatively potent inhibitors of the Trx system, with the IC50
values in the range of 3. In addition, the obtained results
revealed a correlation between chemical reactivity of the
electrophilic groups and their biological activity. These results
2,2-Bis(bromomethyl)propionic acid methyl ester 6b was
obtained from 2,2-bis(hydroksymethyl)propionic acid, which
was converted into methyl ester using thionyl chloride in
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a
Scheme 5. Synthesis of Peptidomimetics with Acrylamide Fragments
a
Reagents and conditions: (a) NaH, THF, −67 °C to room temperature, 2 h, 71%. (b) NaI, acetone, room temperature, 15 min, 93%. (c) LiBr,
acetone, 1 h, 95%.
a
Scheme 6. New Synthetic Strategy for Compounds 16d, 16e, and 17
a
Reagents and conditions: (a) R1 = DMB; X = Br; 10, EtOH, 6 h, 0 °C, 61%. (b) R1 = Bn, X = H, 9, EtOH, 24 h, room temperature, 36%. (c) TFA,
DCM, 60 min, room temperature, 70%.
indicate that inhibition of TrxR−Trx system may be a result of
covalent alkylation of cysteine or Sec residues.
Moreover, compound 16e containing methacrylic fragment was
not active, indicating that the leaving group in allylic position is
indispensable for the inhibition of the Trx−TrxR system.
We turned our attention to the similar examples of
biologically active compoundsα-(acyloxymethyl)ketones.50
These compounds are efficient inhibitors of cathepsin B, a
cysteine protease. The proposed mechanism of action is that
carbonyl group in ketone acts as an electrophile, whereas
acyloxy moiety acts as a leaving group.
Inspired by this observation, we decided to use carboxylic
acid as a leaving group instead of bromine in compound 17.
Allylic esters are less reactive in substitution reaction.
Therefore, we proposed a new structure of the electrophilic
fragment in compounds 18a,b. To this end, 2,6-dichlorobenzoic
acid or 2,6-(trifluorometylo)benzoic acid was used. The
advantage of selected acids is the fact that the bulky 2,6-
substitued acids may decrease the reactivity of the vicinal
double bond, which should increase the stability and selectivity
of the compound. Additionally, they are not naturally occurring
compounds, so they may be more stable in the presence of
endogenous esterases.
After the optimization of esterification conditions, com-
pounds 18a,b were obtained as model compounds from methyl
bromomethacrylate (Scheme 7). Esterification of bromome-
thacrylate proceeds easily with appropriate cesium salts 20a,b in
acetone, resulting in compounds 18a,b with good yields (88−
97%). The compounds were tested in the Trx−TrxR insulin
reduction assay. Results demonstrated that the proposed
fragment was highly active, with IC50 = 0.4 μM for both.
Because the results with model compounds were promising, the
peptidomimetics 19a,b were synthesized. Similar conditions of
Despite promising activities in enzymatic assay, compounds
16a−c did not exert any significant cytostatic/cytotoxic activity
in tumor cell lines (Table 1). This fact can be explained by high
reactivity of allylic halides, which under cellular conditions may
be quickly transformed into inactive derivatives. Therefore, the
synthesis of less reactive 16e was performed to study the
importance of the leaving group in allylic position. An
alternative synthetic pathway was employed utilizing meth-
acrylic acid as a substrate for Ugi reaction (Scheme 6). This
resulted in compound 16e with a 36% yield.
Compounds 16a−c contain bulky and highly hydrophobic
benzyl groups, which may interfere with enzymes active site
residues. Therefore, we considered compound 17, without
benzyl groups. 2,4-Dimethoxybenzylamine (DMBA) has been
used before as an efficient ammonia equivalent in Ugi
reactions.45 Bromomethacrylic acid, on the other hand, has
been reported to be a substrate in Ugi reaction, but the yields
were poor.49 During our studies, it turned out that acceptable
yields can be achieved when the reaction is carried out at low
temperatures of 0 or −78 °C. After the optimization, the Ugi
reaction with bromomethacrylic acid, DMBA, isovaleric
aldehyde, and ethyl isocyanoacetate was performed in ethanol
(Scheme 6). Compound 16d was obtained with decent yield
(61%). Then, the dimethoxybenzyl group was removed in
acidic conditions using a 20% trifluoroacetic acid in dichloro-
methane (DCM). As a result, compound 17 was obtained with
70% yield, and its biological activity was evaluated.
The results presented in Table 1 reveal that the activities of
compounds 16b and 17 are similar, indicating that removal of
benzyl groups did not affect the activity in enzymatic assay.
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a
the downstream cellular effects due to Trx−TrxR inhibition are
evident (see the Results and Discussion).
Scheme 7. Esterification of 17
In further experiments, we focused on the influence of 19a
on the Trx−TrxR system in in vitro experiments. Mass
spectrometry analysis of the recombinant human Trx incubated
with 19a confirmed covalent interaction. Following incubation
of the reduced form of the enzyme with 19a, we have observed
additional mass peaks corresponding to Trx plus one or two
inhibitor molecules attached (data not shown). Considering
that human Trx molecule has five Cys residues, there seems to
be some preference for certain cysteines over the others,
presumably resulting from their nucleophilicity. Additional
studies addressing the selectivity issue are underway. To further
investigate the nature of inhibition and to determine the
sensitivity of individual enzymes to 19a, we measured the Trx−
TrxR system activity in two different, modified variants: (1)
preincubation of Trx with 19a followed by an addition of the
excess of TrxR to assess the inhibition of Trx and (2)
preincubation of TrxR with 19a followed by an addition of the
excess of Trx to assess the inhibition of TrxR (for details, see
the Experimental Section). Interestingly, we have observed a
potent, dose and time dependent inhibition of insulin reduction
in both variants (Figure 5).
a
Reagents and conditions: (a) Acetone, reflux 4 h, 88−97%. (b)
Compound 20a or 20b, acetone, reflux, 5 h, 59−87%.
substitution of 17 were employed resulting in compounds 19a
and 19b with 87 and 59% yield, respectively.
The results of inhibition studies in the Trx−TrxR insulin
reduction assay shown in Table 1 demonstrate that compounds
18a,b and 19a,b are promising inhibitors. However, com-
pounds 18a and 18b are unsuitable for further development
due to their chemical instability and tendency to polymer-
ization. Our experiments revealed that these compounds do not
exert cytostatic/cytotoxic effects against tumor cells even at 25
μM concentrations (Table 1). Compound 19b exhibits
significant activity in enzymatic assay; however, it is almost
insoluble in biological media. Therefore, in further experiments,
we focused on 19a (SK053), which is more soluble, exhibits
activity in enzymatic assay, and is active against tumor cells
(Table 1). The stability of 19a in phosphor-buffered saline
(PBS) buffer (pH 7.4) and in pure culture medium was
determined together with the reference compounds 3 and 2.
Compound 19a is stable in PBS buffer but undergoes
decomposition in culture medium (Figure 4). Surprisingly,
Figure 5. Time-dependent inhibition of Trx and TrxR activities. (A)
Trx was first incubated with TrxR and NADPH for 20 min.
Subsequently, 19a at concentrations of 2.5−20 μM was added for
preincubation before the addition of Trx substrates. To start the
enzymatic reaction Trx and 19a were 5-fold diluted with insulin and
additional NADPH and TrxR, and the activity was measured with the
insulin reduction assay. (B) TrxR was first incubated with NADPH for
10 min and subsequently preincubated with 19a followed by addition
of insulin and Trx. Compound 19a concentrations presented in the
figure correspond to final inhibitor concentrations after dilution. Data
points were measured in triplicate in individual experiments, and error
bars represent SD.
Moreover, incubation of Raji tumor cells with 19a resulted in
decreased Trx−TrxR system activity (Figure 6) and exhibited
significant cytostatic/cytotoxic effects against these cells.
Therefore, 19a was tested in a wider range of tumor cells
with 3 used as a reference. In many cases, the cytostatic/
cytotoxic effects of 19a were slightly better or very similar to
those of 3 (Table 2). Experiments with normal cells (murine
bone marrow cells (mBM), mouse embryonic fibroblasts
(MEF), and human peripheral blood mononuclear cells
(hPBMC) revealed that 19a exerts cytostatic/cytotoxic effects
toward rapidly proliferating MEFs but demonstrates 5−8 times
weaker activity toward nonproliferating cells as compared with
tumor cells (Table 2).
Figure 4. Stability of the investigated compounds. The kinetics of 19a
degradation in 0.01 M PBS buffer (pH = 7.4) at 37 °C and the kinetics
of 19a, 2, or 3 degradation in pure biological medium without serum at
37 °C (for details, see section 5.6).
19a drops to about 20% of its initial amount within 5 h and
remains at this level for an additional 4 days. We have no
rational explanation so far whether the activity against tumor
cells results from the remaining 20% of the original compound
or is rather exerted by its derivatives. Nonetheless, the
inhibition of the Trx−TrxR system in the insulin reduction
assay, the cytostatic/cytotoxic effects against tumor cells, and
To further investigate the biologic effects of Trx−TrxR
system inhibitors, Raji cells were incubated with 3 or 19a, and
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of cells with the combination of tumor necrosis factor (TNF)
and interferon (IFN)-γ resulted in activation of NF-κB
promoter activity that was inhibited by 19a (Figure 8A).
Also, ionomycin-induced AP-1 promoter activity was sup-
pressed by 19a (Figure 8B). Because Trx also has a weak H2O2
scavenging activity, the influence of 19a on the fluorescence of
CM-H2DCFDA, a cell-permeant indicator for ROS, was
measured in HeLa cells incubated with H2O2. The results of
these experiments revealed that 19a increases CM-H2DCFDA
fluorescence but only at the higher 10 μM concentration
(Figure 8C).
Therefore, a potential antitumor activity of 19a was evaluated
in an in vivo study. For these experiments, BALB/c mice were
subcutaneously inoculated with 3.5 × 105 of murine breast
carcinoma (EMT6) cells and were intraperitoneally treated
with two doses of 19a dissolved in dimethylsulfoxide (DMSO)
(40 and 80 mg/kg daily, for 7 consecutive days starting from
day 7 after inoculation of tumor cells). Control mice received
DMSO in the same treatment schedule. For the comparison of
antitumor activity, another group of BALB/c mice was treated
with 3 administered at dose of 14 mg/kg (equimolar to 40 mg/
kg of 19a). Unexpectedly, mice treated with 3 experienced a
significant weight loss and died within 5 days of the treatment.
In contrast, administration of 19a was well tolerated by all
animals (no significant weight loss and no mortality).
Importantly, in mice treated with 80 mg/kg of 19a, a
statistically significant retardation of tumor growth and
prolongation of the survival time was observed (Figure 9).
Figure 6. Incubation of tumor cells with 19a inhibits Trx activity. Raji
cells were incubated with either 3 or 19a for 4 h. Then, tumor cells
were washed three times with PBS and lysed, and the Trx activity was
measured with Trx−TrxR insulin reduction assay. Data points were
measured in triplicate in individual experiments (n = 3), and error bars
represent SD; *P < 0.05, Student's t test.
Table 2. Cytostatic/Cytotoxic Effects of 3 and 19a (LD50,
μM)
tumor cell line
3
19a
EMT6
PANC02
CT26.WT
B78
11.6 0.6
3.9 0.2
5.2 0.4
5.0 0.3
8.8 0.9
3.4 0.2
5.5 0.6
4.6 0.6
5.2 0.1
3.8 0.3
3.3 0.1
4.3 0.1
3.7 0.3
3.7 0.3
4.0 0.3
5.6 0.3
RAJI
Ramos
K562
3. CONCLUSIONS
T24
a
mBM
>12.5
2.7 0.4
>25
>12.5
2.9 0.5
>25
The Trx−TrxR system is a promising molecular target for
antitumor compounds. The literature study on potential
mechanisms of action of known inhibitors of this system
allowed us to design novel, potent compounds reactive with the
nucleophilic residues within the active sites of Trx and TrxR.
The new and relatively simple synthetic approach employing
the Ugi reaction with reactive bromomethacrylic acid as a
substrate allowed the synthesis of peptidomimetics with various
electrophilic fragments that were analogues of the known Trx
system inhibitor 1. During this study, various electrophilic
fragments in target compounds were tested as potential Trx
system inhibitors, and their cytostatic/cytotoxic activity against
tumor cells was measured. As a result, a simple, three-step
synthesis is described, which results in a new class of a wide
spectrum of β-acyloxyacrylamides. This new class, represented
a
MEF
a
hPBMC
a
Normal cells: MEF, mouse embryo fibroblasts; n ≥ 3.
the amounts of Trx at mRNA and protein levels were
measured. These studies revealed that both compounds induce
expression of Trx at mRNA and protein levels (Figure 7),
indicating that this might be a rescue response of cells adopting
to a condition of decreased enzymatic activity of Trx.
Trx has been shown to participate in activation of various
transcription factors, including NF-κB and AP-1.3,4 Therefore,
to investigate whether 19a can modulate their activity HeLa
cells were transiently cotransfected with p-NFκB-luc and p-AP-
1-luc as well as with Renilla reporter plasmids. A 6 h stimulation
Figure 7. Expression of Trx in tumor cells incubated with 3 or 19a. Following incubation of Raji cells with 3 or 19a, the levels of mRNA (A) or
protein (B) for Trx were measured in tumor cell lysates.
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Figure 8. Compound 19a modulates Trx target activities. Data points were measured in triplicate in individual experiments (n = 3), and error bars
represent SD; *P < 0.05 vs controls, **P < 0.05 vs TNF/IFN, or ionomycin group; Student's t test.
Figure 9. Antitumor effects of Trx inhibitors. Balb/c mice were inoculated with 3.5 × 105 of EMT6 cells. Tumor treatment started on day 7 after
inoculation of tumor cells. Compound 19a or 3 was administered daily ip for 7 consecutive days (administration of 3 was terminated after three
doses due to a significant weight loss death of the first animal). Control mice received DMSO. Graphs show the influence of the treatment on the
growth of tumors and survival of mice. *P < 0.05 (two way Student's t test) in comparison with all other groups.
as described before (NMR spectra was the same as described, and the
by 19a, turned out to effectively inhibit the Trx−TrxR system
purity was confirmed by HPLC as well as elemental analysis).51 All key
activity and to exert potent antiproliferative and/or cytotoxic
compounds were proven by HPLC method to show >95% purity.
effects against tumor cells in in vitro studies as well as
4.2. Synthesis of Peptidomimetics. 4.2.1. Synthesis of {2-
significant antitumor activity in an in vivo tumor model. It is
[Benzyl-(3-bromo-2-bromomethyl-propionyl)amino]-4-methyl-
noteworthy that cytotoxicity of 19a toward various tumor cell
lines correlates with the in vitro Trx−TrxR inhibition potency
(LD50 between 2.9 and 5.6 μM vs 3.3 μM IC50). Moreover, 19a
turned out to be less toxic in mice as compared with 3. The
exact mechanism of action of this inhibitor has not been
established; however, it will be the subject of our further
studies. The structure of 19a will be also further optimized,
especially in terms of its peptidomimetic fragment. Further-
more, we cannot exclude that the inhibition of targets other
than Trx−TrxR system can contribute to antitumor activity of
the presented compounds.
pentanoylamino}acetic Acid Benzyl Ester (13). Compound 12 (100
mg, 0.2 mmol) was dissolved in DCM (10 mL), and carbon
tetrachloride (148 mg, 0.4 mmol) and triphenylphosphine (123 mg,
0.5 mmol) were added. The mixture was stirred for 24 h. The solvent
was evaporated, and the residue was purified by flush chromatography-
(silica gel, Rf = 0.82, hexane:ethyl acetate, 5:5, v:v). A colorless oil was
obtained (76 mg, 60% yield). 1H NMR (200 MHz, CDCl3): δ 0.77 (d,
J = 6.4 Hz, 3H, CHCH3), 0.85 (d, J = 6.4 Hz, 2H, CHCH3), 1.20−
1.62 (m, 2H, CH2CH(CH3)2), 1.64−1.88 (m, 1H, CH(CH3)2), 3.21−
3.88 (m, 1H, CHCH2), 4.00−4.78 (m, 3H, ArCH2, CHCO), 4.97−
5.30 (m, 3H, ArCH2, NH), 7.08−7.40 (m, 10H, ArH). 13C NMR (50
MHz, CDCl3): δ 22.7, 23.1, 25.6, 30.0, 36.6, 41.4, 48.1, 56.0, 64.5,
68.0, 126.5, 126.9, 127.8,128.2, 128.8, 129.0, 169.5, 171.3, 173.2. HR-
MS (ESI, [M + Na+]) calcd for C26H32N2O4Br2Na, 617.0621; found,
617.0643.
4. EXPERIMENTAL SECTION
4.1. General. NMR spectra were measured with Varian 200
Gemini, Varian 400 Gemini, and Bruker AM 500 spectrometers, with
TMS used as an internal standard. TLCs were performed with silica
gel 60 (230−400 mesh or 70−230 mesh, Merck) and silica gel 60
PF254 (Merck). CHN analysis was performed on a Vario EL III
(Elementor) elemental analyzer. High-resolution mass spectrometry
(HR-MS) spectra were recorded on an Mariner (PerSeptive
Biosystems) apparatus. Methyl bromomethyl acrylate (1b), methyl
methacrylate (MMA), and all starting materials were purchased in
Sigma-Aldrich. HPLC experiments were carried out on a Kromasil C-
18 column, λ = 230 nm: method 1 [eluent: acetonitrile/water 55:45
(v/v), flow of 1 mL/min]; method 2 [eluent: acetonitrile/water 45:55
(v/v), flow of 0.8 mL/min]; and method 3 [eluent: acetonitrile/water
3:2 (v/v), flow of 1.0 mL/min]. Compound 3 synthesis was performed
4.2.2. Synthesis of (2-{Benzyl-[3-(toluene-4-sulfonyloxy)-2-(tol-
uene-4-sulfonyloxymethyl)propionyl]amino}-4-methyl-
pentanoylamino)acetic Acid Benzyl Ester (14a). To a solution of 12
(30 mg, 0.06 mmol) in DCM (2 mL) N,N-diethylaminepirydine (10
mg) and N,N-diisopropyl-N-ethylamine (0.027 mL, 2.5 mmol) were
added. The solution of p-tolunesulfonylchloride (27 mg, 0.14 mmol)
in DCM (3 mL) was then added. The reaction was carried out at room
temperature for 18 h. The solvent was evaporated, and the residue was
purified by flush chromatography (silica gel, Rf = 0.69, 5:5,
hexane:ethyl acetate, v/v). A 45.6 mg amount of colorless oil was
obtained (92% yield). 1H NMR (200 MHz, CDCl3): δ 0,85 (d, J = 6,4
Hz, 3H, CHCH3), 0,93 (d, J = 6,2 Hz, 3H, CHCH3), 1,35−1,63 (m,
2H, CH2CH(CH3)2), 1,76 (s, 1H, CH(CH3)2), 2,50 (s, 3H, ArCH3),
2,53 (s, 3H, ArCH3), 3,32−3,6 (m, 1H, CHCO), 4,03−4,27 (m, 6H,
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OCH2, ArCH2N), 4,53−4,76 (m, 2H, CH2CO), 5,24 (s, 2H,
ArCH2O), 6,75−6,80 (m, 1H, NH), 7,16−7,42 (m, 14H, ArH),
7,70−7,83 (m, 4H, ArH). 13C NMR (50 MHz, CDCl3): δ 20.0, 22.8,
25.4, 37.0, 41.6, 42.8, 48.8, 56.6, 67.3, 67.9, 68.2, 126.4, 128.0, 128.2,
128.6, 128.7, 128.9, 129.2, 130.3, 130.4, 132.4, 136.9, 145.5, 169.9,
170.8, 171.3. Anal. calcd for C40H46N2O10S2: C, 61.68; H, 5.95; N,
3.60; S, 8.23. Found: C, 61.76; H, 5.81; N, 3.55; S, 8.46%.
(s, 1H, HCC), 5.49 (s, 1H, HCC), 7.32−7.37 (m, ArH 10H).
13C NMR (125 MHz, CDCl3): δ 22.4, 23.0, 25.1, 26.9, 33.2, 37.3, 41.3,
41.5, 52.4, 67.1, 118.9, 127.5, 128.4, 128.5, 128.6, 135.2, 140.1, 169.9,
170.4, 171.9. Anal. calcd for C26H31BrN2O4: C, 60.59; H, 6.06; N, 5.43.
Found: C, 60.92; H, 6.11; N, 5.01. HR-MS (ESI, [M + Na+]); calcd
for C26H31N2O4BrNa, 537.1359; found, 537.1356.
4.2.7. Synthesis of {2-[Benzyl-(2-iodomethyl-acryloyl)amino]-4-
methyl-pentanoylamino}acetic Acid Benzyl Ester (16c). To a
solution of compound 16a (15 mg, 0.02 mmol) in acetone (1 mL),
sodium iodide was added (22.6 mg, 0.15 mmol). The mixture was
stirred for 1 h in room temperature. The solvent was evaporated, and
the residue was purified by flush chromatography (silica gel, Rf = 0.58,
7:3, hexane:ethyl acetate, v:v). A colorless oil was obtained. (8 mg,
93%). 1H NMR (400 MHz, CDCl3): δ 0.76−0.88 (m, 6H,
CH(CH3)2), 1.40−1.80 (m, 2H, CH2CH(CH3)2), 1.82−2.04 (m,
1H, CH2CH(CH3)2), 3.95 (s, 2H, ICH2), 4.12 (s, 2H, ArCH2N)
4.50−4.80 (m, 3H, NCHCO, NCH2CO), 5.18 (s, 2H, ArCH2O), 5.28
(s, 1H, HCC), 5.48 (s, 1H, HCC), 7.26−7.36 (m, 10H, ArH).
13C NMR (100 MHz, CDCl3): δ 5.75, 22.6, 23.1, 25.6, 41.7, 67.4,
4.2.3. Synthesis of {2-[Benzyl-(3-methanesulfonyloxy-2-metha-
n e s u l f o n y l o x y m e t h y l - p r o p i o n y l ) a m i n o ] - 4 - m e t h y l -
pentanoylamino}acetic Acid Benzyl Ester (14b). Compound 12 (35
mg, 0.07 mmol) was dissolved in anhydrous DCM (3 mL), and TEA
(25 μL, 0.18 mmol) was added. The mixture was cooled to 0 °C.
Methylsulfonyl chloride (15 μL, 0.17 mmol) was added dropwise. The
reaction was allowed to reach room temperature and was stirred for 24
h. The solvent was evaporated, and the residue was filtered through
1
silica gel giving 37 mg of colorless oil (quant. yield). H NMR (200
MHz, CDCl3): δ 0.80 (t, J = 6.8 Hz, 6H, CHCH3), 1.25−1.58 (m, 2H,
CH2CH(CH3)2), 1.64−2.05 (m, 1H, CH(CH3)2), 3.02−3.18 (m, 7H,
SCH3, CHCO), 3.72−3.92 (m, 1H, CHCO), 4.00−4.34 (m, 4H,
OCH2), 4.61 (s, 2H, ArCH2N), 5.16 (s, 2H, ArCH2O), 7.10−7.17 (m,
2H, ArH), 7.23−7.38 (m, 8H, ArH). 13C NMR (50 MHz, CDCl3): δ
22.6, 22.7, 22.8, 23.1, 25.5, 26.0, 27.7, 36.9, 38.3, 40.4, 41.4, 48.5, 55.9,
61.3, 61.6, 67.5, 98.4, 126.1, 127.9, 128.8, 128.9, 129.0, 129.2, 135.5,
137.4, 169.7, 171.3, 173.8. HR-MS (ESI, [M + Na+]) calcd for
C28H38N2O10S2Na, 649.1860; found, 649.1870.
118.1, 127.8, 128.7, 128.8, 128.9, 141.3, 169.6, 170.2, 171.3. Anal. calcd
for C26H31IN2O4 + H2O: C, 53.80; H, 5.73; N, 4.83. Found: C, 53.87;
H, 5.73; N, 4.65. HR-MS (ESI, [M + Na+]) calcd for C26H31N2O4I Na,
585.1221; found, 585.1233.
4.2.8. Synthesis of [2-(2-Bromomethyl-acryloylamino)-4-methyl-
pentanoylamino]acetic Acid Ethyl Ester (17). Compound 16d (117
mg, 0.23 mmol) dissolved in DCM (2 mL) and trifluoroacetic acid
(475 μL) was added. The mixture was stirred for 1 h at room
temperature. DCM was added (4 mL). A saturated solution of sodium
bicarbonate was added until the mixture become transparent. The
water phase was extracted with DCM (3 × 8 mL). The combined
organic layer was washed with brine, dried over anhydrous magnesium
sulfate, and filtered, and volatiles were evaporated. The residue was
purified by flush chromatography (silica gel, Rf = 0.25, 6:4,
hexane:ethyl acetate, v/v). A 58 mg amount of white solid (mp =
78−79 °C) was obtained (70% yield). 1H NMR (200 MHz, CDCl3): δ
0.87−0.94 (m, 6H, CH(CH3)2), 1.25 (t, J = 7.2 Hz, 3H, OCH2CH3),
1.38−1.70 (m, 3H, CH2CH(CH3)2), 3.88−4.12 (m, 2H, NHCH2),
4.12−4.22 (m, 4H, OCH2CH3; BrCH2C), 4.60−4.78 (m, 1H,
NHCH), 5.69 (s, 1H, CCHH), 5.85 (s, 1H, CCHH), 6.90 (d, J =
8.0 Hz, 1H, NH), 7.14 (m, 1H, NH). 13C NMR (50 MHz, CDCl3): δ
14.5, 22.4, 23.3, 25.1, 30.7, 41.4, 41.7, 52.1, 61.8, 123.0,141.3, 166.4,
170.0, 172.6. HR-MS (ESI, [M + Na+]) calcd for C14H23BrN2O4 Na,
385.0733; found, 385.0740.
4.2.4. Synthesis of {2-[Benzyl-(3-iodo-2-iodomethyl-propionyl)-
amino]-4-methyl-pentanoylamino}acetic Acid Benzyl Ester (15). To
a solution of 14a (46.4 mg, 0.06 mmol) in acetone (3 mL), sodium
iodide was added (54.5 mg, 0.36 mmol). The mixture was refluxed for
24 h. The solvent was evaporated, and the residue was purified by flush
chromatography (silica gel, Rf = 0.87, 7:3, hexan:ethyl acetate, v/v).
1
The product was obtained as colorless oil (27.8 mg, 68% yield). H
NMR (200 MHz, CDCl3): δ 0.85−0.90 (m, 6H, CH(CH3)2), 1.46−
1.70 (m, 2H, CH2CH(CH3)2), 1.86−1.96 (m, CHCH3, 1H), 3.18−
3.23 (m, 5H, ICH2, CHCO), 4.01−4.05 (m, 2H, NCH2Ar), 4.66 (d, J
= 6.4 Hz, 2H,, NCH2CO), 5,18 (s, 2H, ArCH2O), 7.24−7.37 (m,
10H, ArH). 13C NMR (50 MHz, CDCl3): δ 5.2, 5.6, 22.8, 23.1, 23.1,
25.5, 37.4, 41.6, 48.2, 49.4, 56.8, 67.5, 126.9, 128.2, 128.7, 128.9, 129.0,
129.4, 135.5, 137.1, 169.6, 170.9, 173.6. Anal. calcd for C26H32I2N2O4:
C, 45.24; H, 4.67; N, 4.06. Found: C, 45.34; H, 4.77; N, 4.05.
4.2.5. Synthesis of (2-{Benzyl-[2-(toluene-4-sulfonyloxymethyl)-
acryloyl]-amino}-4-methyl-pentanoylamino)acetic Acid Benzyl
Ester (16a). The solution of compound 14a (27 mg, 0.4 mmol) in
tetrahydrofuran (THF) was cooled to −67 °C under atmosphere of
nitrogen. Sodium hydride (1 mg, 0.4 mmol) was added. The mixture
was allowed to stand to room temperature and stirred for 1 h. The
solvent was evaporated, and the residue was purified by flush
chromatography (silica gel, Rf = 0.59, 5:5, hexane:ethyl acetate, v:v).
4.3. General Procedure II for Synthesis Compounds 19a,b.
Compound 17 (1 mmol) was dissolved in acetone. The carboxylic acid
cesium salt (5 mmol) was suspended in the mixture. The reaction was
refluxed for 4 h. The solvent was evaporated, and the residue was
suspended in ethyl acetate. The organic layer was washed with water
and brine and dried with magnesium sulfate. The solvent was
evaporated, and the residue was purified using flush chromatography.
4.3.1. Synthesis of 2,6-Bis-trifluoromethyl-benzoic Acid 2-[1-
(Ethoxycarbonylmethyl-carbamoyl)-3-methyl-butylcarbamoyl]allyl
Ester (19a). General Procedure II. Yield, 87%; white solid; mp = 114−
1
The product was obtained as a colorless oil (15 mg, 71% yield). H
NMR (200 MHz, CDCl3): δ 0.77−0.85 (m, 6H, CHCH3), 1.40−1.68
(m, 1H, CHCH3), 1.80−2.10 (m, 2H, CH2CHCH3), 2.44 (s, 3H,
ArCH3), 3.92 (d, J = 5.8 Hz, 2H, OCH2), 3.80−4.0 (m, 5H, ArCH2N,
NCH2CO, NCHCO), 5.17 (s, 2H, ArCH2O), 5.44 (s, 1H, CHC),
5.55 (s, 1H, CHC), 7.23−7.34 (m, 12H, ArH), 7.77 (d, J = 8.6 Hz,
2H, ArH). 13C NMR (50 MHz, CDCl3): δ 22.0, 22.9, 23.8, 25.3, 32.7,
41.6, 67.4, 69.1, 103.4,119.9, 127.9, 128.1, 128.4, 128.6, 128.7, 128.9,
130.3, 135.6, 137.9, 169.8, 170.7, 171.3. Anal. calcd for C27H36N2O6 +
0.5H2O: C, 64.37; H, 6.38; N, 4.55; S, 5.21. Found: C, 64.31; H, 6.33;
N, 4.41; S, 5.17.
1
115 °C; Rf = 0.61 (1:1, hexane:ethyl acetate, v/v). H NMR (200
MHz, CDCl3): δ 0.85−0.91 (m, 6H, CH(CH3)2), 1.24 (t, J = 7.2 Hz,
3H, OCH2CH3), 1.51−1.70 (m, 3H, CH2CH(CH3)2), 3.84−4.08 (m,
2H, NHCH2COO), 4.17 (q, J = 7.2 Hz, 2H, OCH2CH3), 4.51−4.67
(m, 1H, NCH), 5.10 (s, 2H, OCH2C), 5.79 (s, 1H, CCHH), 6.02 (s,
1H, CCHH) 6.74 (d, J = 8.2 Hz, 1H, NH), 6.98−7.03 (m, 1H, NH),
7.72 (m, 1H, ArH), 7.91 (d, J = 7.8 Hz, 2H, ArH). 13C NMR (50
MHz, CDCl3): δ 14.4, 22.4, 23.0, 25.0, 30.7, 41.2, 41.6, 51.9, 61.8,
65.6, 120.0, 124.3, 125.8, 129.0, 129.7, 130.2, 130.7 137.9, 164.8 166.4,
169.9, 172.6. Anal. cacld for C23H26F6N2O6: C, 51.11; H, 4.85; N, 5.18.
Found: C, 51.00; H, 5.07; N, 4.86.
4.2.6. Synthesis of {2-[Benzyl-(2-bromomethyl-acryloyl)amino]-4-
methyl-pentanoylamino}acetic Acid Benzyl Ester (16b). To a
solution of compound 16a (90 mg, 0.1 mmol) in acetone (3 mL)
lithium bromide was added (12 mg, 0.14 mmol). The mixture was
stirred for 1 h at room temperature. The solvent was evaporated, and
the residue was purified by flush chromatography (silica gel, Rf = 0.26,
7:3, hexane:ethyl acetate, v:v). The product was obtained as oil (80
4.3.2. Synthesis of 2,6-Dichloro-benzoic Acid 2-[1-(Ethoxycarbo-
nylmethyl-carbamoyl)-3-methyl-butylcarbamoyl]allyl Ester (19b).
General Procedure II. Yield, 59%; white solid; mp = 120−121 °C, Rf =
1
mg, 95% yield). H NMR (500 MHz, CDCl3): δ 0.82−0.87 (m, 6H,
1
CH(CH3)2), 1.56−1.82 (m, 2H, CH2CH(CH3)2), 1.96−2.01 (m, 1H,
CH2CH(CH3)2) 3.95 (m, 2H, BrCH2), 4.20−4.25 (m, 2H, ArCH2N),
4.60−4.81 (m, 3H, NCHCO, NCH2CO), 5.18 (s, 2H, ArCH2O), 5.37
0.34 (1:1, hexane:ethylacetate, v:v). H NMR (200 MHz, CDCl3) δ
0.83 (d, J = 6 Hz, 6H, CH(CH3)2), 1.20 (t, J = 7.0 Hz, 3H,
OCH2CH3), 1.47−1.69 (m, 3H, CH2CH(CH3)2), 3.81−4.07 (m, 2H,
63
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NHCH2COO), 4.12 (q, J = 7.0 Hz, 2H, OCH2CH3), 4.51−4.60 (m,
1H, NCH), 5.07 (d, J = 4.2 Hz, 2H, OCH2C), 5.78 (s, 1H, CCHH),
6.00 (s, 1H, CCHH), 6.56 (d, J = 8.2 Hz, 1H, NH), 6.77 (bs, 1H,
NH), 7.20−7.25 (m, 3H, ArH). 13C NMR (50 MHz, CDCl3): δ 14.5,
22.4, 23.2, 25.1, 41.2, 41.7, 51.9, 61.9, 65.1, 124.6, 128.2, 131.4, 132.2,
138.1, 166.4, 169.8, 172.28. HR-MS (ESI, [M + Na+]) calcd for
C21H26Cl2N2O6 Na, 495.1060; found, 495.1081.
subsequent 4 h, plates were centrifuged, and cells were lysed with
DMSO. Finally, the absorbance was measured at 570 nm. The XTT
assay was used to evaluate the cytostatic/cytotoxic effects in hPBMC
and mBM. After a 4 h incubation with XTT (0.3 mg/mL), the
absorbance was measured at 490 nm with the reference wavelength of
690 nm. The relative viability of cells (% of control cultures incubated
with medium and corresponding concentrations of inhibitors solvent
− DMSO, never exceeding 1%) was calculated as follows: relative
viability = [(Ae − Ab)/(Ac − Ab)] × 100, where Ab is the background
absorbance, Ae is the experimental absorbance, and Ac is the
absorbance of untreated controls.
4.5.2. Trx/TrxR Insulin Reduction Assays. To measure the activity
of investigated compounds, the Trx−TrxR insulin reduction kit
(IMCO Co., Stockholm, Sweden) was used. The procedure is based
on the reduction of insulin by Trx.43 The reduced form of Trx is
maintained with TrxR and NADPH. The activity was measured in a
final volume of 50 μL containing 50 mM Tris-HCl buffer (pH 7.6), 20
mM EDTA, 0.8 μM NADPH, 0.325 μM TrxR, 0.25 μM Trx, and 316
μM bovine insulin (Sigma Aldrich) and the indicated concentrations
of studied compounds. At least seven different concentrations of
studied compounds were used in individual experiments. After 30 min
of incubation at 37 °C, the reaction was terminated by the addition of
8 mM DTNB [5,5′-dithiobis-(2-nitrobenzoic acid] solution in 8 M
guanidine HCl (Sigma Aldrich), to determine the number of free
thiols from reduced insulin. The relative amount of yellow product
thionitrobenzoate was evaluated by absorbance measurement at 412
nm. The IC50 values were evaluated with Four Parameter Logistic
Standard Curves Analysis using a SigmaPlot software. Data points
were measured in triplicates in individual experiments, and IC50 values
are EC50 SD, n ≥ 3. To measure the Trx activity in cells, Raji tumor
cells were incubated with 3 or 19a for 4 h, washed three times with
PBS, and lysed in 0.1% NP40 buffer, supplemented with Complete
protease inhibitors cocktail (Roche Diagnostics). The protein
concentration was measured using BCA protein assay (BioRad,
United Kingdom). The Trx activity was evaluated in 10 μg of total
protein as described above. The reaction was performed for 20 min in
37 at 37 °C according to kit protocol (IMCO Co.).
4.5.3. Time-Dependent Inhibition of Trx and TrxR Activity. To
determine the nature of inhibition of individual enzymes in the Trx−
TrxR system by 19a, two modified variants of insulin reduction assay
were employed. Because of large enzyme amounts needed, we used
human recombinant Trx produced according to the procedure
described above (section 4.4). To assess predominantly Trx inhibition,
Trx was incubated with TrxR and NADPH for 20 min to obtain
reduced enzyme form and subsequently preincubated with 19a at
concentrations from 2.5 to 20 μM for 0, 10, 20, 30, 60, and 90 min at
37 °C. To start the insulin reduction, Trx and 19a were 5-fold diluted
with insulin, additional NADPH, and TrxR. The final concentrations
of reaction components were as follows: 0.25 μM Trx, 6.5 μM TrxR, 1
μM NADPH, and 0.5−4 μM 19a. To evaluate predominantly the
TrxR inhibition, the enzyme was incubated with NADPH for 10 min
and subsequently preincubated with 19a at concentrations from 2.5 to
20 μM for 0, 10, 20, 30, 60, and 90 min at 37 °C. The reaction of
insulin reduction was initiated by the addition of insulin and Trx. The
final concentrations were as follows: 3.25 nM TrxR, 10 μM Trx, 1 μM
NADPH, and 0.5−4 μM 19a. In all experiments, the reactions of
insulin reduction were performed for 30 min, at 37 °C, in Tris-HCl
EDTA buffer, pH 7.6, in a final volume of 100 μL. Reactions were
terminated by the addition of 8 mM DTNB solution in 8 M guanidine
HCl. The relative amount of yellow product thionitrobenzoate was
evaluated by absorbance measurement at 412 nm.
4.4. Cloning, Expression, and Purification of Recombinant
Human Trx. The gene-encoding human Trx-1 was amplified by PCR
on the cDNA obtained from human embryonic kidney cell line HEK-
293T and cloned into pET15-mod, a derivative of Novagen
pET15b(+) vector, containing six additional N-terminal histidine
codons.52 The fusion protein overexpression was performed in
Escherichia coli, strain BL21 Codon Plus RIL. Bacterial cells were
grown at 37 °C until they reached an OD600 = 0.7−0.9 and then after
induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG),
shifted to 30 °C for additional 16 h. For purification, cells were
disrupted in lysozyme-containing buffer by sonication and ultra-
centrifuged, and the soluble fraction was subjected to nickel affinity
chromatography and subsequently to gel filtration chromatography
(column Superdex 75 High Load 16/60, Amersham Pharmacia) in
reducing conditions [buffer: 10 mM Tris, pH 7.5, 100 mM NaCl, and
10 mM dithiotreitol (DTT)]. Pure protein was concentrated with
Vivaspin concentrator (10 kDa cutoff) to the concentration about 20
mg/mL and frozen at −20 °C after the addition of glycerol for
cryoprotection. Before use, Trx was dialyzed two times against 10 mM
Tris, pH 7.5, buffer to remove DTT, glycerol, and NaCl.
4.5. Cell Lines and Reagents. Murine mammary carcinoma
(EMT6), murine colon carcinoma (CT26.WT), human bladder
carcinoma (T24), and human Burkitt's lymphoma (Ramos, Raji) cell
lines were purchased from ATCC (Manassas, VA). The B78-H1
melanoma (herein named B78), a poorly immunogenic amelanotic
subclone of murine B16 melanoma cell line, was kindly provided by
Dr. Lloyd H. Graf (University of Chicago, Chicago, IL). Murine
pancreatic carcinoma cells (Panc 02) were kindly obtained from
Carsten Ziske (Rheinische Friedrich-Wilhelms-Universitat, Bonn,
̈
Germany). The human erythromyeloblastoid leukemia cell line
(K562) was purchased from DSMZ (Braunschweig, Germany). Cells
were cultured in RPMI-1640 (CT26.WT, Ramos, Raji, K562),
Dulbecco's modified Eagle's medium (EMT6, B78, Panc02) or
McCoy's medium (T24) supplemented with 10% heat-inactivated
fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotic/
antimycotic solution (Sigma, St. Louis, MO). mBM cells were
collected from Balb/c female mice and cultured in ISCOV medium
(Invitrogen) supplemented with 10% heat-inactivated fetal bovine
serum (Invitrogen), antibiotic/antimycotic solution (Sigma), and 100
U of IL-3 (Peprotech, NJ). Histopaque 1077 was used to isolate
hPBMC from healthy donors. After a 30 min centrifugation (700g, 25
°C), white blood cell rings were washed twice with PBS and
resuspended in RPMI. MEFs were isolated from Balb/c mouse fetuses
on embryonic day 13.5 (E 13.5) by trypsin (0.2%) digestion at 37 °C
for 10 min in PBS. MEFs were cultured in Dulbecco's modified Eagle's
medium. All synthesized compounds were dissolved in DMSO to
produce stock solution of 10 mM.
4.5.1. Cytostatic/Cytotoxic Assays. The cytostatic/cytotoxic effects
were measured using crystal violet staining (EMT6, CT26.WT, T24,
B78, and Panc 02) or 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-
zolium bromide (MTT) assay (Raji, Ramos, and K562), or 2,3-Bis(2-
methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-anilide
(XTT) assay (hPBMC, mBM). First, tumor cells were dispensed into
96-well plates (Sarstedt, Numbrecht, Germany). The following day,
the investigated compounds were added at indicated concentrations
for a 72 h incubation. Then, the adherent cells were rinsed with PBS
and stained with 0.5% crystal violet in 2% ethanol for 10 min at room
temperature. Next, plates were washed with tap water, and cells were
lysed with 2% SDS solution. The absorbance was measured at 595 nm
using an ELISA reader (SLT Labinstrument GmbH, Salzburg,
Austria). To evaluate cytostatic/cytotoxic effects in nonadherent
cells, after a 72 h of incubation with 19a or 3, MTT solution in a
concentration of 5 mg/mL was added to each well of plate. After
4.5.4. RT-PCR. RNA from Raji cells was isolated using a trizol
reagent (Invitrogen) after incubation with 3 or 19a. RT-PCR was
performed with AMV reverse transcriptase (Eurex, Poland) according
to manufacturer's protocol. Subsequently, PCR was done using
ColorOptiTaq DNA Polymerase (Eurex, Poland) using the following
pairs of primers amplifying human Trx1: 5′-GCCAAGATGGTGAAG-
CAGAT-3′ (forward) and 5′-TTGGCTCCAGAAAATTCACC-3′
(reverse); and human GAPDH: 5′-CCCTTCATTGACCTCAACTA-
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Journal of Medicinal Chemistry
Article
CATGG-3′ (forward) and 5′-CCTGCTTCACCACCTTCTT-
GATGTC-3′ (reverse).
culture medium or PBS preincubated in 37 °C. After vortexing, the
aliquots 100 μL were diluted with 200 μL of 96% ethanol, cooled for
15 min at 4 °C, centrifuged (10000g, 5 min), and stored at −20 °C
before the analysis. Residual solutions were incubated at 37 °C up to 7
days. All samples were analyzed using RP-HPLC (C-18 Kromasil
column, eluent: acetonitrile:water, PDA detector, method 1 for 19a, tR
= 10.2 min, and method 2 for 3, tR = 5.9 min, and 2, tR = 5.1 min).
Standard curves were determined with minimum of five dilutions of
each compound, prepared as described above. Data points were
measured in triplicate in individual experiments (n = 3).
4.5.5. Western Blotting. For Western blotting analysis, cells were
cultured with 19a and 3 for 4 and 8 h. After they were washed with
PBS, the cells were lysed in 0.1% NP40 buffer supplemented with
Complete protease inhibitors cocktail (Roche Diagnostics, Mannheim,
Germany). The protein concentration was measured using BCA
protein assay (Biorad). Equal amounts of proteins were separated on
15% SDS-polyacrylamide gel, transferred onto Protran nitrocellulose
membranes (Schleicher and Schuell BioScience Inc., Keene, NH), and
blocked with TBST [Tris-buffered saline (pH 7.4) and 0.05% Tween
20] with 5% BSA (Sigma Aldrich). The following antibodies were used
for the overnight incubation: antithioredoxin (rabbit monoclonal, Cell
Signaling) and antitubulin (mouse monoclonal, Sigma Aldrich). After
extensive washing with TBST, the membranes were incubated for 45
min in corresponding HRP-coupled secondary antibodies (Jackson
Immuno Research, West Grove, PA). The reaction was developed
using 0.2 mM cumaric acid (Sigma Aldrich) and 1.25 mM luminol
(Sigma Aldrich), diluted in 50 mM Tris-HCL (pH 7.8) and
supplemented with H2O2, and the luminescence was acquired using
Stella bioimager (Raytest, Germany).
4.5.6. Luciferase Reporter Assay. Luciferase reporter plasmids
pTA-NFκB-luc and pTA-AP-1-luc were purchased from Clontech.
Renilla control plasmid with a constitutive CMV promoter (pRL-
CMV-luc) was purchased from Promega. Transient cotransfection was
performed on HeLa cells with 2 μg of luciferase and Renilla reporter
plasmids using the Amaxa Cell Line Nucleofector Kit R and Amaxa
Nucleofector (Lonza). After nucleofection, cells were seeded into wells
of a 24-well culture plate for 48 h. Then, NF-κB was induced by
incubation of cells with TNF (10 ng/mL) and IFN-γ (100 U) for 6 h.
For the induction of AP-1, cells were washed with PBS and cultured in
medium without antibiotics and FBS for 4 h. Next, ionomycin (1 μM)
was added for 6 h of incubation. To evaluate the influence of 19a on
NF-κB, and AP-1 activity cells were incubated with TNF, IFN-γ, and
19a or with ionomycin and 19a. After 6 h of incubation, the cells were
harvested and lysed in PLB buffer (Promega). Both firefly and Renilla
luciferase activities were assayed with the dual luciferase assay kit
(Promega), and the firefly luciferase activities were normalized to
activities of Renilla enzyme in each sample. The light emission was
determined with GloMax luminometr (Promega). All experiments
were repeated at least three times.
4.5.7. ROS Generation Assay. To determine the influence of 19a
on H2O2 scavenging activity, HeLa cells were seeded into six-well
plates and incubated for 4 h with 10 or 20 μM 19a. Next, cells were
trypsinized, washed, and resuspended in 1 mL of PBS. H2O2 at 1 mM
final concentration was added to indicated probes for 10 min
incubation before staining. One microliter of CM-H2DCFDA
(Invitrogen, 5 mM DMSO solution) was added to each sample for
15 min of incubation at 37 °C. Then, cells were washed twice with
PBS and evaluated in flow cytometry using FACSCalibur (Becton
Dickinson). For single analysis, 1 × 104 cells were used. Data were
analyzed with CELLQuest 1.2 software (Becton Dickinson).
4.5.8. Mice, Tumor Treatment, and Monitoring. BALB/c mice, 8−
12 weeks of age, were used in the experiments. Breeding pairs were
obtained from the Animal House of the Polish Academy of Sciences,
Medical Research Center (Warsaw, Poland). Mice were inoculated
subcutaneously with 3.5 × 105 EMT6 cells into the depilated right
thigh. Local tumor growth was determined with calipers by the
formula: tumor volume (mm3) = (longer diameter) × (shorter
diameter)2/2. When tumors reached a longer diameter of 5−7 mm
(day 7 of the experiment), the intraperitoneal administration of Trx
inhibitors was started and continued for 6 consecutive days. Three
groups of mice received investigated compounds3 at a dose of 14
mg/kg, 19a at a dose of 40 mg/kg (equimolar to 14 mg/kg of 3), or
80 mg/kg. The control group received ip DMSO injection, which was
a solvent for all inhibitors. All in vivo experiments were performed in
accordance with the guidelines approved by the Ethical Committee of
the Medical University of Warsaw.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and spectral data for 6a,6, 11, 12,
16d,e, and 18a,b. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +48 22 5992199. E-mail: jakub.golab@wum.edu.pl (J.G.).
Tel: +48 22 343 2120. E-mail: rysza@icho.edu.pl (R.O.).
■
Author Contributions
§These authors contributed equally to this work.
ACKNOWLEDGMENTS
■
This work was supported by project “Biotransformations for
pharmaceutical and cosmetics industry” No. POIG.01.03.01-00-
158/09-07 part-financed by the European Union within the
European Regional Development Fund. The author's research
was supported by Ministry of Science and Higher education
grants N N405 3202 33 (J.G.), N N405 127640 (A.M.), and
IP2010 009170 (M.F.). J.G., A.M., and M.F. are members of
TEAM Programme cofinanced by the Foundation for Polish
Science and the EU European Regional Development Fund.
J.G. is a recipient of the Mistrz Award from the Foundation for
Polish Science. We thank professor Dieter Seebach from ETH
Zurich for valuable suggestions to the mechanism of Trx/TrxR
̈
inhibition.
DEDICATION
■
This paper is dedicated to Prof. Janusz Jurczak on the occasion
of his 70th birthday.
ABBREVIATIONS
■
AP-1, activating protein-1; ASK1, apoptosis signal-regulating
kinase 1; DCM, dichloromethane; DIPEA, di-isopropylethyl-
amine; DMAP, 4-(dimethylamino)pyridine; DMBA, 2,4-
dimethoxybenzylamine; DMSO, dimethylsulfoxide; DTT,
dithiotreitol; hPBMC, human peripheral blood mononuclear
cells; HR-MS, high-resolution mass spectrometry; IFN,
interferon; IPTG, isopropyl β-D-1-thiogalactopyranoside;
MAP, mitogen-activated protein; mBM, murine bone marrow;
MEF, mouse embryonic fibroblasts; MMP, matrix metal-
loproteinase; NADPH, nicotinamide adenine dinucleotide
phosphate; NF-κB, nuclear factor κB; NMR, nuclear magnetic
resonance; PBS, phosphor-buffered saline; PTEN, phosphatase
and tensin homologue deleted on chromosome ten; ROS,
reactive oxygen species; SAHA, suberoyl anilide hydroxamic
acid; TBP2, Trx-binding protein-2; TEA, triethylamine; THF,
tetrahydrofuran; TNF, tumor necrosis factor; Trx, thioredoxin;
TrxR, thioredoxin reductase; VEGF, vascular endothelial
growth factor; XTT, 2,3-Bis(2-methoxy-4-nitro-5-sulfophen-
yl)-2H-tetrazolium-5-carbox-anilide
4.6. Stability Studies. Stock solutions (30 μL, initial concen-
tration: 5 mg/mL) prepared in DMSO were diluted with 3.0 mL of
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