Discovery of Novel, Drug-Like Ferroptosis Inhibitors with in Vivo Efficacy

Article abstract of DOI:10.1021/acs.jmedchem.8b01299

Ferroptosis is an iron-catalyzed, nonapoptotic form of regulated necrosis that results in oxidative lipid damage in cell membranes that can be inhibited by the radical-trapping antioxidant Ferrostatin-1 (Fer-1). Novel inhibitors derived from the Fer-1 sca

Full text of DOI:10.1021/acs.jmedchem.8b01299

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Article
Discovery of Novel, Drug-like Ferroptosis Inhibitors with In Vivo Efficacy
Lars Devisscher, Samya Van Coillie, Sam Hofmans, Dries Van Rompaey,
Kenneth Goossens, Eline Meul, Louis Maes, Hans De Winter, Pieter Van
der Veken, Peter Vandenabeele, Tom Vanden Berghe, and Koen Augustyns
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01299 • Publication Date (Web): 25 Oct 2018
Downloaded from http://pubs.acs.org on October 25, 2018
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Discovery of Novel, Drug-like Ferroptosis Inhibitors with
In Vivo Efficacy
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Lars Devisscher # a, Samya Van Coillie # b,c, Sam Hofmans , Dries Van Rompaey , Kenneth
a
a
a
b,c
d
a
a
Goossens , Eline Meul , Louis Maes , Hans De Winter , Pieter Van Der Veken , Peter
Vandenabeele b,c,e, Tom Vanden Berghe * , Koen Augustyns *
#
b,c
#
a
a
Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, 2610
Antwerp, Belgium.
b
Molecular Signalling and Cell Death unit, VIB Center for Inflammation Research,
Technologiepark 927, 9052 Ghent, Belgium.
c
Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent, Belgium.
d
Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp,
Universiteitsplein 1, 2610 Antwerp, Belgium.
e
Methusalem Program, Ghent University, Ghent, Belgium.
#
These authors contributed equally.
Keywords: ferroptosis, regulated necrosis
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ABSTRACT
Ferroptosis is an iron-catalysed, non-apoptotic form of regulated necrosis that results in oxidative lipid
damage in cell membranes that can be inhibited by the radical-trapping antioxidant Ferrostatin-1 (Fer-1).
Novel inhibitors derived from the Fer-1 scaffold inhibited ferroptosis potently but suffered from solubility
issues. In this paper, we report the synthesis of a more stable and readily soluble series of Fer-1 analogues
that potently inhibit ferroptosis. The most promising compounds (37, 38 and 39) showed an improved
protection compared to Fer-1 against multi-organ injury in mice. No toxicity was observed in mice after
daily injection of 39 (UAMC-3203) for 4 weeks. UAMC-3203 inserts rapidly in a phospholipid bilayer in
silico, which aligns with the current understanding of the mechanism of action of these compounds.
Concludingly, these analogues have superior properties compared to Fer-1, show in vivo efficacy and
represent novel lead compounds with therapeutic potential in relevant ferroptosis-driven disease models.
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INTRODUCTION
The classical view that a cell can undergo cell death by either one of two distinct prototypic cell death
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pathways, apoptosis or necroptosis, has become invalid. While apoptosis is carried out following a strict
mechanism involving cellular proteases called caspases, necroptosis critically depends on the concerted
action of several enzymes such as the receptor interacting protein kinases (RIPK) and the mixed lineage
kinase domain like pseudokinase (MLKL).2–4 To date, several other forms of regulated necrosis each with
their distinctive pathways and effector molecules are described.5,6
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One molecule that is able to induce a form of regulated necrosis is erastin. Erastin interacts with
mitochondrial voltage-dependent anion channels (VDACs) which induces a RAS-RAF-MEK-dependent
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form of cell death due to the formation of reactive oxidative species (ROS). Subsequently, the Stockwell
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lab found that erastin also inhibits the membrane-bound cystine/glutamate antiporter System X , which
c
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accounts for the induction of a non-apoptotic form of cell death. This novel form of cell death was
dubbed ferroptosis, which is characterized by iron-dependent accumulation of lipid hydroperoxides that
disrupt membrane integrity leading to cell death. Ferroptosis may thus play a key role in the pathogenesis
of degenerative diseases in which lipid peroxidation has been implicated. Interestingly, the execution of
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this form of cell death could be inhibited by the small molecule ferrostatin-1 (Fer-1, 1). Even though
target identification of Fer-1 remains a challenge, the mechanism of action of Fer-1 was recently proposed
to derive from its reactivity as a radical-trapping antioxidant.10
Unravelling the molecular pathway of ferroptosis is currently a topic of intense study. Because erastin
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induces ferroptosis through the inhibition of System X , it can be classified as a class I ferroptosis inducer
c
(FIN). Blockage of this antiporter impairs the cellular uptake of cystine, an essential precursor in the
synthesis of the cellular antioxidant glutathione (GSH). The resulting intracellular deficit of GSH in turn
triggers the accumulation of ROS, which causes cells to die by excessive oxidation of the membrane lipids
1
1
(
Figure 1). In addition to class I FINs, ferroptosis can also be induced by molecules that directly target
and inactivate glutathione peroxidase 4 (GPX4) and are classified as class II FINs.12,13
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Seiler et al. showed that GPX4, a specific type of GSH-dependent selenoprotein, holds an essential role in
the antioxidant network of the membranes of a cell. Inducible GPX4-KO significantly increased cell death
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due to excessive lipid peroxidation, a hallmark feature of ferroptosis. GPX4 has the ability to reduce
organic hydroperoxides to the corresponding alcohols while consuming GSH as a reducing agent, further
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implying the importance of intracellular GSH levels in ferroptosis. Inactivation of GPX4 either directly
or indirectly resulted in the accumulation of ROS followed by lipid membrane oxidation which disrupted
cell membrane integrity. These findings further solidified GPX4 as a crucial protective enzyme that
supresses ferroptosis and it underlines the importance of the GSH-GPX4-axis considering redox
homeostasis within the cell membrane (Figure 1).
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Figure 1. Schematic representation of GPX4-dependent conversion of reactive lipid peroxides to the corresponding lipid
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alcohols. System X
c
facilitates the cellular uptake of cystine which is an important precursor in GSH synthesis. Cystine is
reduced by cystine reductase (CR) to cysteine. Glutamate cysteine ligase (GCL) attaches a glutamate molecule to cysteine in
order to generate γ-glutamyl cysteine (γGC). Addition of a glycine molecule by glutathione synthase (GS) results in the formation
of a new molecule of GSH. GSH servers as an important reducing cofactor for GPX4 by catalysing the reduction of lipid
peroxides to their corresponding alcohols while also forming glutathione disulfide (GSSG). Erastin inhibits the cellular uptake of
cystine and thus impairs intracellular synthesis of GSH. Depletion of GSH leads to the indirect inactivation of GPX4 and the
resulting accumulation of lipid peroxides disrupts membrane integrity resulting in ferroptosis. The class II FIN RSL3 acts by
directly inactivating GPX4 and does not interfere with the cellular uptake of cystine or intracellular GSH synthesis.6,16
17
The clinical relevance of ferroptosis has been implied in various pathological settings. Dixon et al. first
showed that Fer-1 was able to prevent glutamate-induced neurotoxicity in a model using organotypic
hippocampal slice cultures.11 Another study in particular showed that ferroptosis might contribute in a
whole array of diseases by showing that inhibition of ferroptosis significantly ameliorated the clinical
outcome in experimental models for Huntington’s disease, periventricular leukomalacia and kidney
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dysfunction.18 Since then multiple studies have implicated an important role of ferroptosis not only in
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various pathological conditions like ischemia-reperfusion injury , neuronal dysfunction
and
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Alzheimer’s disease , but also in several other biological processes such as hair follicle morphogenesis ,
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blood coagulation and in the maturation of photoreceptor cells.
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The design of novel and improved ferroptosis inhibitors is thus an interesting field of research. Fer-1 as a
lead molecule itself has some unfortunate shortcomings. The presence of a labile ester moiety results in
the rapid hydrolysis of Fer-1 into its inactive carboxylic acid. Multiple attempts have been made to
improve the potency of this type of molecule while also improving pharmacokinetic parameters of Fer-1
(
2 and 3, Figure 2).18,25 While Skouta et al. thoroughly explored the structure-activity relationship (SAR)
of the Fer-1 scaffold, we previously reported that the replacement of the labile ester moiety of Fer-1 by a
more stable sulfonamide group yielded promising results (4, UAMC-2418, Figure 2). This intervention
significantly improved the metabolic stability while maintaining and even improving potency in a cell-
based assay for ferroptosis. These molecules however displayed a poorly soluble character, which limited
their use in in vivo settings.16
In this study, we expand on these earlier findings and we report the synthesis of a novel generation of
ferroptosis inhibitors derived from Fer-1 with improved ADME properties and an increased solubility.
The pharmacokinetic properties of the most potent molecules were characterized and the in vivo efficacy
and toxicity of these molecules was investigated. Additionally, a molecular dynamics experiment was
conducted as an effort to investigate the binding properties of these molecules in a phospholipid bilayer.
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NH2
HN
H
H
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N
O
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O
O
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Ferrostatin-1
SRS11-92
N
H
N
HN
H
N
N
N
O
HN
S
O
O
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4
SRS16-86
UAMC-2418
Figure 2. The chemical structure of Ferrostatin-1 and other reported analogues.
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RESULTS AND DISCUSSION
Compound design
Our earlier published results involved a thorough exploration of the SAR which provided the following
results: (1) The replacement of the labile ester moiety with a sulfonamide greatly improved stability as
well as potency (2) The cyclohexyl moiety was deemed to be the most ideal substituent with regard to
both potency and lipophilicity. More bulky alkyl groups tend to have a negative effect on the solubility of
the compounds and smaller moieties impair the potency of the molecules. (3) The introduction of an
aromatic group on the 3-amino position greatly improved potency, but also further decreased the solubility
of the compounds.16
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The major drawback of our previously published molecules was their poor solubility. Designing more
soluble compounds is a challenging task since these molecules have to act in the highly lipophilic
environment of membranes. We identified that the terminal position of the aliphatic chain on the
sulfonamide moiety could be derivatized further in order to increase the solubility of these compounds. A
variety of solubility improving groups were introduced at this terminal R position. The cyclohexylamine
a
group was kept as in Fer-1 and the R position was further derivatized with benzylic and pyridinic
b
substituents in a similar fashion to the previously reported sulfonamide analogues (Figure 3).
Rb
NH2
HN
HN
H
H
H
N
N
N
HN
S
O
HN
S
O
Ra
O
O
O
O
Ferrostatin-1
UAMC-2418
Novel inhibitors
Figure 3. Structural comparison of Fer-1, our previously reported sulfonamide analogue of Fer-1 and the newly
synthesized series.
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Chemistry
The newly developed series of inhibitors were synthesized according to the general synthetic strategy
displayed in Scheme 1.
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NO2
NO2
NO2
H
Cl
Cl
N
Cl
HN
S
HN
S
S
R1
R1
a
b
O
O
O
O
O
O
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13-19
6-12
R2
NH2
HN
H
N
H
N
HN
S
HN
S
O
c
R1
R1
d
O
O
O
2
0-26
30-36
40-46
e
e
R2
NH2
HN
H
N
H
N
HN
S
HN
S
O
R3
R3
O
O
O
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7-29
37-39
7-49
4
R =
NH
Boc
N
N
O
N
N Boc
N
N
N
O
1
Boc
R =
NH2
NH
N
NH
3
Scheme 1. General overview of the synthetic route.
Reagents and conditions: (a) Aliphatic amine analogue, triethylamine, THF, 1 h, -40°C (b) cyclohexylamine, K
h, 60°C (c) Palladium hydroxide on carbon, H
CO , DMF. (e) HCl in dioxane, DCM; in case Boc protection was present.
2
CO
3
, DMSO, 18
2
, methanol, 18 h, rt (d) benzylbromide or 4-(bromomethyl)pyridine hydrobromide,
K
2
3
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All molecules were synthesized starting from 4-chloro-3-nitrobenzenesulfonyl chloride 5. Treatment of 5
with the appropriate amine analogue resulted in the formation of 6-12. Subsequently a nucleophilic
aromatic substitution of 6-12 with cyclohexylamine under basic conditions yielded 13-19. Palladium-
catalysed hydrogenation of the 3-nitro group to its corresponding amine resulted in the generation of
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molecules 20-26. Finally, benzylic and pyridinic substituents were introduced at the R -position to afford
2
3
0-36 and 40-46 respectively. If applicable, the protective Boc group was removed under acidic
conditions to yield compounds 27-29, 37-39 and 47-49.
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Inhibition of Erastin-Induced Ferroptosis in vitro
The final set of compounds was evaluated in an assay with IMR-32 neuroblastoma cells in order to assess
their potency for inhibiting erastin-induced ferroptosis. In addition to this screening, the kinetic solubility
of all newly synthesized molecules was also determined (Table 1) (Additional data such as standard error
of the means can be found in the Supporting Information).
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The introduction of a cyclic or aliphatic amine at R increased the kinetic solubility of this type of
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molecule significantly when compared to our earlier published results (Table 1). The introduction of a
benzylic moiety at R was well tolerated and improved potency in most cases. Compounds 35-39 for
2
example, all of which contain a benzyl moiety in R , displayed a more potent inhibition of erastin-induced
2
ferroptosis than their corresponding analogues which possess a primary amine or pyridinic moiety at this
position. This potentiating effect was not observed as significantly when a pyridinic group was introduced
at the same position. A similar trend was observed in our previously published work.16
For example, compound 35 has an IC -value of 24 nM combined with a solubility range of 50-100 µM.
5
0
Even more remarkable is the fact that molecules 37-39 exhibit even lower IC -values combined with an
5
0
excellent kinetic solubility (> 200 µM). This increase in solubility while preserving the improved potency
when compared to Fer-1 is a very promising feature of these compounds. The four most promising
molecules (35, 37-39) were thus selected for further assessment and their in vitro ADME-parameters were
defined.
a
Solubility (µM) b
> 200
Compound
(Fer-1)
R or R
R2
-H
-H
-H
-H
-H
IC (nM)
1
3
50
1
/
33
2
2
2
0
4
5
-N(Me)2
N-Methylpiperazine
Morpholine
160
236
448
492
> 200
> 200
> 200
26
-OCH3
> 200
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-NH(Me)
-NH2
-H
214
> 200
> 200
> 200
> 200
> 200
50-100
25-50
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
100-200
> 200
-H
306
296
554
1072
24
Piperazine
-N(Me)2
-H
0
1
2
3
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7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
30
34
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Pyridinyl
Pyridinyl
Pyridinyl
Pyridinyl
Pyridinyl
Pyridinyl
Pyridinyl
N-Methylpiperazine
Morpholine
-OCH3
3
3
3
3
5
6
7
8
22
-NH(Me)
-NH2
3
12
39
40
Piperazine
-N(Me)2
10
1560
1520
191
97
4
4
4
4
4
5
6
7
N-Methylpiperazine
Morpholine
-OCH3
-NH(Me)
-NH2
157
112
442
48
49
Piperazine
Table 1. Synthesized fer-1 analogues and their antiferroptotic activity in response to erastin-induced ferroptosis in IMR-
3
2 Neuroblastoma cells.
a
Reported IC50 values are calculated from measurements in triplicate. Additional information such as the standard error of the
means can be found in the Supporting Information.
b
Final test compound concentration range between 3.125 µM and 200 µM [4 µM DMSO solution in 196 µM buffer solution (10
mM PBS pH 7.4)].
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ADME Assays
The determination of both the microsomal and plasma stability of 35, 37-39 revealed a remarkable
improvement in stability when compared to Fer-1 which is unstable under nearly all conditions (Table 2
and Table 3). However, significant intraspecies differences between the four lead molecules were
observed. Compounds 38 and 39 more specifically, showed a microsomal half-life (t ) of multiple hours
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
/2
across all species. Compound 39 has an impressive t1/2 when incubated with both human and rat
microsomes (t1/2 = 20.5 h and t1/2 = 16.5 h respectively) but was found to be relatively less stable when
incubated with murine microsomes (t1/2 = 3.46 h). In contrast, compound 38 has a microsomal t1/2 greater
than 10 hours under both human (t1/2 = 17.7 h) and murine (t1/2 = 13 h) conditions but was microsomally
less stable when incubated with rat microsomes (t = 2.05 h).
1/2
Microsomal stability
Plasma stability
(% recovery after 6h)
a
b
(
t ) (h)
1
/2
Compound
Human
Rat
Mouse
Human
Rat
Mouse
1
0
.109 ± 0.003
0.98 ± 0.35
0
0
47.3
100
1.1
0
(Fer-1)
3
5
(
(
(
UAMC-
0.173 ± 0.007
0.35 ± 0.01
2.05 ± 0.21
16.48 ± 4.66
0.051 ± 0.002
100
100
3
240)
3
7
UAMC-
234)
13.21 ± 4.10
17.71 ± 2.04
20.53 ± 5.49
0.45 ± 0.02
13.00 ± 4.15
3.46 ± 1.37
90.3
100
65.8
100
100
100
100
3
3
8
UAMC-
3
206)
39
(UAMC-
3203)
84.2
85.8
Table 2. Microsomal stability and plasma stability of compounds 1, 35 , 37-39.
a
Metabolism by microsomes (CYP450 and other NADP-dependent enzymes) was monitored and expressed as half-life (h).
Percentage of remaining parent compound.
b
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1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
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Calculated Intrinsic Clearance in vitro
(
Cl ) (mL/min/kg)
int
Compound
Human
2.6 ± 8.1
Rat
Mouse
1223.2 ± 48.0
138.6 ± 6.2
4.8 ± 1.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
35
2
325.9 ± 13.2
161.1 ± 4.6
27.4 ± 2.7
3.4 ± 1.0
(
(
(
(
UAMC-3240)
37
1
.7 ± 0.5
1.3 ± 0.1
1.1 ± 0.3
UAMC-3234)
38
UAMC-3206)
39
18.0 ± 7.1
UAMC-3203)
Table 3. Calculated Intrinsic Clearance in vitro of compounds 35, 37-39.
The Clint is calculated using the measured microsomal t1/2 and takes into account several experimental variables such as the protein
concentration and the volume of incubation. The exact formula as well as the species-specific parameters can be found in the
Supporting Information.
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In vivo evaluation of efficacy of novel fer-1 analogues
We have recently shown that pure cellular iron overload suffices to induce ferroptosis in IMR-32
2
6
neuroblastoma cells, which is referred to as non-canonical ferroptosis induction. This new concept of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
4
4
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4
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4
4
5
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
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9
0
1
2
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0
iron-induced ferroptosis has recently been shown by others by using FeCl2 in organotypic hippocampal
27
28
slice cultures , BAY 11-7085 in breast cancer cell lines and glioblastoma and FINO2 in HT1080
fibrosarcoma cells. Both in humans as well as in rodents31–33, an acute overdose of iron in vivo typically
2
9
30
results in multiple organ failure and often death.34,35 First, we have tested the potency of our inhibitors for
(
NH ) Fe(SO ) -induced ferroptosis in vitro. Similar to erastin-induced ferroptosis, we found that our most
4 2 4 2
potent inhibitors show similar or lower IC -values than Fer-1 when inhibiting ferrous ammonium
5
0
sulphate-induced ferroptosis. (See Supporting Information) Therefore, the selected compounds were
assessed for their efficacy in an experimental in vivo ferroptosis model using acute iron poisoning.
Intravenous injection of compounds 37, 38 and 39 in mice 15 minutes prior to iron sulphate injection
significantly lowered the plasma levels of lactate dehydrogenase (LDH), a general biomarker for organ
injury which is drastically elevated in response to iron poisoning. All three compounds were significantly
more potent in lowering LDH levels than Fer-1 (Table 4), which illustrates the in vivo functionality of
these Fer-analogues.
Trigger
/
Treatment
Vehicle
Plasma LDH (U/L)
484 ± 72
Statistics
FeSO4
FeSO4
Vehicle
1
3572 ± 185
2898 ± 178
* vs. Veh
(Fer-1)
3
7
FeSO4
FeSO4
FeSO4
2093 ± 90
2293 ± 148
2346 ± 99
**** vs. Veh ** vs. Fer-1
**** vs. Veh; * vs. Fer-1
**** vs. Veh; * vs. Fer-1
(
(
(
UAMC-3234)
3
8
UAMC-3206)
3
9
UAMC-3203)
Table 4. Pretreatment with the compounds significantly decreases LDH levels after acute iron poisoning.
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Vehicle, Fer-1 (1), compound 37, 38 or 39 was injected intravenously (20 µmol/kg) 15 minutes before intraperitoneal injection
with 300 mg/kg iron sulfate. Two hours after IP injection, mice were sacrificed and blood was taken. Plasma levels of LDH are
4
shown. Vehicle = 2% DMSO in 0.9% NaCl. Error represents SEM, n=5 for twice treated vehicle group, n=11-12 for FeSO -
treated groups. * = 0.01<p< 0.05; ** = 0.001<p<0.01; *** = 0.0001<p<0.001; **** = p < 0.0001. Statistical analysis by ordinary
one-way ANOVA.
0
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0
In vivo pharmacokinetics
In order to estimate how the in vitro ADME parameters correlate to an in vivo setting, compound 39
(UAMC-3203) was selected for a single intravenous (iv) administration to three male Wistar rats at a dose
of 5 mg/kg. Blood samples were obtained at various time points (0.083, 0.25, 0.5, 1, 2, 4, and 24h). The
concentration of compound 39 was determined using UPLC analysis coupled with tandem quadrupole
mass spectrometry. (See Supporting Information) Interestingly, plasma concentrations of compound 39
remained below the detection limit (< 53.1 ng/mL) in nearly all samples. These findings suggest that
compound 39 is rapidly removed from the bloodstream and redistributed into various tissues, as can be
expected for lipophilic basic compounds.
To assess the extent of tissue distribution of compound 39, various organs (liver, kidney and lungs) of all
three rats were collected on ice at necropsy 24 hours after administration before being homogenized and
analysed (Table 5). Significant concentrations of compound 39 were found in all analysed tissues, which
further solidifies our earlier findings considering tissue redistribution. Since the tissue concentration of
compound 39 was determined 24 hours after iv dosage, these results also confirm the increase in stability
when compared to Fer-1 which is unstable in in vivo conditions.
Tissue distribution profile of compound 39 (UAMC-3203)
(mg/kg organ, after 24h)
Liver
Kidney
Lung
0.19a
Rat 1
0.23
0.20
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Rat 2
Rat 3
0.22
0.15
0.35
0.38
0.48
0.53
Table 5. Tissue distribution profile of compound 39 in various organs.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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5
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Each rat received an iv dose at 5 mg/kg and the internal organs were collected at necropsy at 24h. The organs were homogenized
and further processed by protein precipitation (n=3) and centrifugation. Analysis of the samples was conducted by LC-MS.
a
For this measurement only two samples of organ homogenate were processed (n=2).
In vivo assessment of toxicity of compound 39
Since ferroptosis plays a role in chronic diseases such as neurodegeneration, we evaluated the toxicity of
daily application of compound 39 to wild type mice. Therefore, classic biomarkers of toxicity were
measured in blood plasma of mice having received a daily injection of compound 39 over a period of 4
weeks. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), LDH, creatine kinase
(CK), creatinine, urea and troponin T levels were all comparable to background values detected in control
mice. In addition, no difference in body temperature or weight was observed (Table 6).
Plasma
Plasma
Plasma
LDH
Plasma CK
(U/L)
Plasma
Plasma
urea
(mg/dL) (mg/dL)
Plasma
troponin T
(µg/L)
Body
temperature
(°C)
Rel. gain in
Body
Weight (%)
Compound
Vehicle
ALT AST (U/L)
(U/L)
creatinine
(U/L)
< 25
110 ± 8 227 ± 18 1501 ± 196
< 0,2
< 0,2
42 ± 2 0,041 ± 0,005 37,0 ± 0,6 14,8 ± 3,4
43 ± 2 0,044 ± 0,004 37,0 ± 0,6 10,4 ± 3,3
3
9
(
UAMC-
< 25 123 ± 11 263 ± 20 1865 ± 308
3
203)
Table 6. No toxicity was observed in mice after repeated injections of compound 39.
Compound 39 (UAMC-3203) was injected intraperitoneally (20 µmol/kg) daily, over a period of 4 weeks. Body temperature and
weight were measured daily, blood parameters were assessed at the 28-day endpoint after sacrifice. Plasma levels of ALT, AST,
LDH, CK, creatinine, urea and troponin T are shown. Error represents SEM, n=9/group. Vehicle = 2% DMSO in 0.9% NaCl.
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Bilayer insertion of compound 39 occurs on the nanosecond scale
A molecular dynamics study was conceived to investigate the interactions of compound 39 with a model
membrane bilayer. The setup consisted of a 128 POPC bilayer in a waterbox. Compound 39 was placed
approximately in the centre of the waterbox and allowed to diffuse freely.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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A similar system with alpha-tocopherol was included as a reference. Three separate 300 ns simulations
were performed for each system. In each of our simulations, compound 39 inserted itself into the lipid
bilayer. The solubility-enhancing piperazine ring and the sulphonamide of compound 39 engaged in
hydrogen bonding interactions with the phospholipid head groups, while the lipophilic core and ring
substituents interacted with the lipid tails (Figure 4A). Comparing compound 39 to the reference
compound alpha-tocopherol, it is apparent that compound 39 binds in a more shallow manner than to
alpha-tocopherol, which inserts deeply into the membrane (Figure 4B).
The radical-scavenging hydroxyl group of alpha-tocopherol is frequently engaged in hydrogen-bonding
interactions to the acyl groups, rendering it unavailable for radical scavenging. By contrast, the aryl amine
groups, most prominently the cyclohexylamine group, are less frequently involved in hydrogen
bonding.10,36 This difference in availability may help explain the improved efficacy of compound 39 as a
potent ferroptosis inhibitor.
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Figure 4. Graphical overview of bilayer insertion of compound 39 and alpha-tocopherol. (A) Compound 39 binds in a relatively
shallow manner in the phospholipid bilayer. The solubility improving piperazine moiety engages in several hydrogen bonds with
the hydrophilic head groups of the phospholipids. (B) Alpha-tocopherol binds more deeply in the phospholipid bilayer, while the
phenol moiety of the chromanol ring engages in a hydrogen bond interaction with the head group which renders it less suitable for
radical scavenging.
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CONCLUSION
Fer-1 has traditionally been the most frequently used tool compound in cell death research to inhibit
ferroptotic cell death. The use of Fer-1 in multiple in vitro settings resulted in a potent dose-dependent
inhibition of ferroptosis. Earlier findings suggest that the practical use of Fer-1 in vivo is limited due to its
poor metabolic stability. Our previously reported research indicated that it was possible to synthesize
novel inhibitors of ferroptosis by introducing structural modifications to the Fer-1 scaffold. The
elucidation of the SAR of these compounds resulted in a series of sulfonamide derivatives of Fer-1 that
were able to inhibit ferroptosis in the single-digit nanomolar range with improved metabolic stability. The
major drawback of this series was their poor solubility, preventing them from being used efficiently in in
vivo settings. In this paper, we report a follow-up series of potent Fer-1 analogues. By introducing both a
solubility improving group and a sulfonamide moiety to the Fer-1 scaffold, we were able to synthesize
novel molecules that are more potent than Fer-1 while simultaneously improving solubility and stability.
Compounds 35, 37, 38 and 39 were able to inhibit erastin-induced ferroptotic cell death more potently
than Fer-1. These novel compounds were also found to have a significantly improved solubility over our
previously published molecules. Additionally, molecules 38 and 39 demonstrate great recovery from
plasma after 6h, while also possessing microsomal half-lives of multiple hours across three species.
Interestingly, our three most promising compounds (37, 38 and 39) were significantly more potent than
Fer-1 in protecting against acute iron poisoning induced injury in mice, which illustrate their efficacy in
vivo. Due to its long microsomal t1/2 in rats, compound 39 (UAMC-3203) was selected for single iv dose
administration (5 mg/kg) in an effort to estimate the in vivo pharmacokinetic properties of these types of
molecules. Plasma levels of compound 39 remained below the detection limit in nearly all samples, which
implied a rapid redistribution of compound 39 into various tissues. A significant amount of compound 39
was found in liver, kidney and lungs after 24 hours confirming the hypothesis of rapid tissue distribution.
No toxicity was observed in mice after daily injection of compound 39 for 4 weeks. Taken together, these
findings indicate that these analogues could be used as potent and readily soluble lead compounds to
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investigate the process of ferroptotic cell death in various in vivo settings or validate their therapeutic
value in experimental ferroptosis-driven diseases.
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EXPERIMENTAL SECTION
Unless otherwise stated, laboratory reagent grade solvents were used. Reagents were obtained from
various commercial sources and were used without any prior purification. Characterization of all
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compounds was done with H and C NMR and mass spectrometry of which the spectra were recorded
with a 400 MHz Bruker Avance III Nanobay spectrometer with Ultrashield. All obtained spectra were
analysed using MestReNova analytical chemistry software. Chemical shifts are displayed in ppm and
coupling constants are shown in hertz (Hz). ES mass spectra were obtained from an Esquire 3000plus Ion
Trap Mass Spectrometer from Bruker Daltonics. Purities were determined with a HPLC system based
either on mass determination or on UV detection. A Waters SQD ESI mass spectrometer was used in
combination with a Waters TUV detector. Waters Acquity UPLC BEH C18 1.7 μm, 2.1 mm × 50 mm
column was used. The eluens was composed of two different solvents nl. solvent A consisted of water
with 0.1% formic acid while solvent B consisted of acetonitrile with 0.1% formic acid. Two methods were
used interchangeably on the HPLC system. Method I involved the following parameters: 0.15 min 95% A,
5
% B, then in 1.85 min from 95% A, 5% B to 95% B, 5% A, then 0.25 min (0.350 mL/min), 95% B, 5%
A. The wavelength for UV detection was 254 nm. Method II involved the following: flow 0.4 mL/min,
.25 min 95% A, 5% B, then in 4.75 min to 95% B, 5% A, then 0.25 min 95% B, 5% A, followed by 0.75
0
min 95% A, 5% B. The wavelength for UV detection was 214 nm. During the chemical synthesis, flash
purification was performed when necessary on a Biotage ISOLERA One flash system equipped with an
internal variable dual wavelength diode array detector (200−400 nm). For normal phase purifications
SNAP cartridges (10−340 g, flow rate of 10−100 mL/min) were used, and reversed phase purifications
were done while using of KP-C18 containing cartridges. Dry sample loading was done by self-packing
samplet cartridges using silica and Celite® 545, respectively, for normal and reversed phase purifications.
Gradients used varied for each purification. During the pharmacokinetic characterization, mouse and rat
plasma were obtained from Innovative Research. Liver microsomes were purchased from Corning B.V.
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Life Sciences. The turbidity in the kinetic solubility experiments was measured using the UV/VIS
spectrophotometer Synergy MX, Biotek with Gen5.
The following section comprises the synthetic procedures and analytical data for all compounds reported
in this manuscript. Several synthetic procedures that were used in the preparation of intermediates and
final products are summarized here as “General Procedures”. The purities of all final products were found
to be > 95%, unless stated otherwise.
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General procedure A
A solution containing the appropriate aliphatic or cyclic amine substituent (1 equiv.) and triethylamine (2
equiv.) in THF was added dropwise to a solution containing 4-chloro-3-nitrobenzene-1-sulfonyl chloride 5
(1 equiv.) in THF that was cooled down to -40°C. After the addition was complete, the resulting mixture
was allowed to warm up to room temperature over the course of one hour. Subsequently the reaction
mixture was diluted with EtOAc and washed three times with water. The organic layer was dried using
anhydrous sodium sulphate before being concentrated in vacuo. The crude reaction products 6-12 were
used without any further purification.
General procedure B
Intermediates 6-12 (1 equiv.) were dissolved in DMSO before potassium carbonate (2 equiv.) and
cyclohexanamine (1.2 equiv.) were added. The resulting mixture was heated to a temperature of 60°C and
then stirred overnight. After cooling down to room temperature, the reaction mixture was diluted with
EtOAc and washed three times with water. The organic layer was dried using anhydrous sodium sulphate
before being concentrated in vacuo. If deemed necessary, further purification was conducted with flash
chromatography on silica gel using a gradient consisting of heptanes and EtOAc to obtain intermediates
1
3-19.
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General procedure C
Intermediates 13-19 (1 equiv.) were dissolved in dry MeOH and the solution was purged using argon gas.
Palladium(II) hydroxide was added under inert atmosphere while continuously stirring. After the addition
was complete, the resulting mixture was put under hydrogen atmosphere and left to stir overnight. The
reaction mixture was filtered through a patch of Celite® and then purified by flash chromatography on
silica gel using a gradient consisting of heptanes and EtOAc to yield products 20-26.
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General procedure D
Compounds 20-26 (1 equiv.) were dissolved in DMF followed by the addition of potassium carbonate (1 –
3 equiv.) and the corresponding benzyl- or pyridinylderivate (1-2 equiv.). This mixture was allowed to stir
at various temperatures and timespans, all of which are further specified at the corresponding compounds
reported below. The crude product was then purified by either normal phase or reversed phase flash
chromatography to yield compounds 30-36 and 40-46. The exact conditions of these purifications are
reported below for each compound individually.
General procedure E
Compounds 21-23, 31-33 and 41-43 (1 equiv.) were dissolved in dichloromethane followed by the
addition of a 4M solution of hydrochloric acid in dioxane (8 equiv.) The reaction mixture was stirred at
room temperature for 2 hours. After reaction diethylether was added to the reaction mixture in order to
precipitate compounds 27-29, 37-39 and 47-49. The obtained HCl-salts were subsequently washed with a
minimal amount of diethylether.
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-chloro-N-(2-(dimethylamino)ethyl)-3-nitrobenzenesulfonamide (6)
By following General Procedure A and using N,N-dimethylethane-1,2-diamine (1.38 g, 15.62 mmol) as
the corresponding amine, the formation of 4-chloro-N-(2-(dimethylamino)ethyl)-3-
nitrobenzenesulfonamide 6 (4.67 g, 15.16 mmol) was achieved. (Yield: 97%)
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H NMR (400 MHz, DMSO-d ) δ 2.07 (s, 6H), 2.25 - 2.30 (m, 2H), 2.97 (t, J = 6.5 Hz, 2H), 8.05 (d, J =
6
8
.5 Hz, 1H), 8.12 (dd, J = 8.5, 2.2 Hz, 1H), 8.50 (d, J = 2.1 Hz, 1H).
13
C NMR (101 MHz, DMSO-d ) δ 40.62, 44.83, 58.06, 123.88, 128.93, 131.23, 132.84, 141.20, 147.31.
6
tert-butyl (2-((4-chloro-3-nitrophenyl)sulfonamido)ethyl)(methyl)carbamate (7)
By following General Procedure A and using tert-butyl (2-aminoethyl)(methyl)carbamate (2.04 g, 11.72
mmol)
as
the
corresponding
amine,
the
formation
of
tert-butyl
(2-((4-chloro-3-
nitrophenyl)sulfonamido)ethyl)(methyl)carbamate 7 (4.58 g, 11.62 mmol) was achieved. (Yield: 99%)
1
H NMR (400 MHz, Acetone-d ) δ 1.42 (s, 9H), 2.83 (d, J = 14.4 Hz, 3H), 3.20 (t, J = 6.3 Hz, 2H), 3.31 -
6
3
1
.42 (m, 2H), 7.03 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 8.15 (dd, J = 8.5, 2.2 Hz, 1H), 8.45 (d, J = 2.2 Hz,
H).
1
3
C NMR (101 MHz, Acetone-d ) δ 27.64, 41.25, 47.95, 48.67, 78.86, 124.14, 124.22, 129.91, 131.38,
6
133.07, 141.36, 148.03.
tert-butyl (2-((4-chloro-3-nitrophenyl)sulfonamido)ethyl)carbamate (8)
By following General Procedure A and using tert-butyl (2-aminoethyl)carbamate (3.28 g, 20.5 mmol) as
the
correspoding
amine,
the
formation
of
tert-butyl
(2-((4-chloro-3-
nitrophenyl)sulfonamido)ethyl)carbamate 8 (6.2 g, 16.32 mmol) was achieved (Yield: 84%)
1
H NMR (400 MHz, Acetone-d ) δ 1.37 (s, 9H), 3.09 - 3.17 (m, 2H), 3.17 - 3.23 (m, 2H), 6.11 (t, J = 6.0
6
Hz, 1H), 7.02 (t, J = 5.7 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 8.14 (dd, J = 8.4, 2.2 Hz, 1H), 8.44 (d, J = 2.2
Hz, 1H).
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C NMR (101 MHz, Acetone-d ) δ 27.74, 40.11, 43.17, 78.29, 124.27, 130.01, 131.43, 133.08, 141.28,
6
1
47.95, 156.09.
tert-butyl 4-(2-((4-chloro-3-nitrophenyl)sulfonamido)ethyl)piperazine-1-carboxylate (9)
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By following General Procedure A and using 4-N-(2-Aminoethyl)-1-N-Boc-piperazine (3.76 g, 16.4
mmol) as the correspoding amine, the formation of tert-butyl 4-(2-((4-chloro-3-
nitrophenyl)sulfonamido)ethyl)piperazine-1-carboxylate 9 (6.7 g, 16.32 mmol) was achieved (Yield: 96%)
1
H NMR (400 MHz, DMSO-d ) δ 1.38 (s, 9H), 2.22 (t, J = 5.0 Hz, 4H), 2.32 (t, J = 6.5 Hz, 2H), 2.98 (q,
6
J = 5.3 Hz, 2H), 3.20 (dd, J = 6.3, 3.6 Hz, 4H), 7.98 - 8.05 (m, 2H), 8.08 (dd, J = 8.5, 2.1 Hz, 1H), 8.46 (d,
J = 2.1 Hz, 1H).
1
3
C NMR (101 MHz, DMSO-d ) δ 28.00, 43.05, 52.23, 56.69, 78.70, 123.82, 129.01, 131.24, 132.96,
6
141.12, 147.40, 153.72.
4-chloro-N-(2-(4-methylpiperazin-1-yl)ethyl)-3-nitrobenzenesulfonamide (10)
By following General Procedure A and using 2-(4-methylpiperazin-1-yl)ethanamine (2.24 g, 15.16
mmol) as the corresponding amine, the formation of 4-chloro-N-(2-(4-methylpiperazin-1-yl)ethyl)-3-
nitrobenzenesulfonamide 10 (5.13 g, 14.14 mmol) was achieved. (Yield: 91%)
1
H NMR (400 MHz, DMSO-d ) δ 2.09 (s, 3H), 2.12 - 2.26 (m, 8H), 2.29 (t, J = 6.5 Hz, 2H), 2.97 (t, J =
6
6
.5 Hz, 2H), 8.02 (d, J = 8.5 Hz, 1H), 8.08 (dd, J = 8.5, 2.1 Hz, 1H), 8.45 (d, J = 2.1 Hz, 1H).
1
3
C NMR (101 MHz, DMSO-d ) δ 40.03, 45.63, 52.40, 54.39, 56.86, 123.81, 128.96, 131.21, 132.91,
6
1
41.33, 147.38.
4
-chloro-N-(2-morpholinoethyl)-3-nitrobenzenesulfonamide (11)
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