O -Phenylenediamine: A privileged pharmacophore of ferrostatins for radical-trapping reactivity in blocking ferroptosis

Article abstract of DOI:10.1039/c8ob00546j

Ferroptosis is a non-apoptotic, iron dependent form of regulated cell death that is characterized by the accumulation of lipid hydroperoxides. It has drawn considerable attention owing to its putative involvement in diverse neurodegenerative diseases. Ferrostatins are the first identified inhibitors of ferroptosis and they inhibit ferroptosis by efficiently scavenging free radicals in lipid bilayers. However, their further medicinal application has been limited due to the deficient knowledge of the lipid peroxyl radical-trapping mechanism. In this study, experimental and theoretical methods were performed to illustrate the possible lipid hydroperoxide inhibition mechanism of ferrostatins. The results show that an ortho-amine (-NH) moiety from ferrostatins can simultaneously interact with lipid radicals, and then form a planar seven-membered ring in the transition state, and finally present greater reactivity. NBO analysis shows that the formed planar seven-membered ring forces ortho-amines into better alignment with the aromatic π-system. It significantly increases the magnitudes of amine conjugation and improves spin delocalization in the transition state. Additionally, a classical H-bond type interaction was discovered between a radical and an o-NH group as another transition state stabilizing effect. This type of radical-trapping mechanism is novel and has not been found in diphenylamine or traditional polyphenol antioxidants. It can be said that o-phenylenediamine is a privileged pharmacophore for the design and development of ferroptosis inhibitors.

Full text of DOI:10.1039/c8ob00546j

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DOI: 10.1039/C8OB00546J
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O-phenylenediamine: A Privileged Pharmacophore of Ferrostatins
for Radical-Trapping Reactivity in Blocking Ferroptosis
Xie-Huang Sheng†, Cheng-Cheng Cui†, Chao Shan†, Yu-Zhen Li†, Duo-Hong Sheng‡, Bin Sun†*, De-
Zhan Chen†*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Ferroptosis is a non-apoptotic, iron dependent form of regulated cell death that is characterized by accumulation of lipid
hydroperoxides. It has drawn considerable attention owning to its putative involvement in diverse neurodegenerative
diseases. Ferrostatins are the first identified inhibitors of ferroptosis by efficiently scavenging free radical in lipid bilayers.
However, their further medicinal application has been confined due to the deficient knowledge of lipid peroxyl radical-
trapping mechanism. In this study, experimental and theoretical methods were performed to illustrate the possible lipid
hydroperoxides inhibition mechanism of ferrostatins. The results show that ortho-amines (-NH) moiety from ferrostatins can
simultaneously interact with lipid radical, then form a planar seven-membered ring in transition-state, and finally present
greater reactivity. NBO analysis shows that the formed planar seven-membered ring forces ortho-amines into better
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alignment with the aromatic π-system. It significantly increases the magnitudes of amine conjugation and improves spin
∗
→
delocalization in transition state. Additionally, a classic H-bond type nO ꢀꢁꢂ interaction was discovered between radical and
o-NH group as another transition state stabilizing effect. This type of radical-trapping mechanism is novel which has not
been found in diphenylamine or traditional polyphenols antioxidants. It would be that o-phenylenediamine is a privileged
pharmacophore for ferroptosis inhibitors design and development.
therapeutic opportunity for these diseases previously thought to be
untreatable. 11
1 Introduction
Ferroptosis is a newly discovered form of programmed cell death
that is distinct from apoptosis, necrosis and autophagy.1 It is
characterized by the aberrant accumulation of membrane (phospho)
lipid hydroperoxides (LOOH)2 and has been implicated in various
H
NH2
H
N
NH
N
H
N
Cl
O
N
N
H
O
neurodegenerative disease, including Parkinson’s3, Huntington’s4,5
,
ferrostatin-1
4,4'-di-tert-butyldiphenylamine
liproxstatin-1
Alzheimer’s diseases6, and hemorrhagic brain injury7. The process of
ferroptosis was switched on by pharmacological pertubation of lipid
redox systems involving gultathione (GSH) depletion and inactivation
of the glutathione peroxidase 4 (GPX4).8 Subsequent lipidomics
showed that an increase of polyunsaturated fatty acids (PUFAs)
esterified into membrane phospholipids and underwent
Fig 1 Structure of potent inhibitors of ferroptosis (inside the brackets is the
o-phenylene-diamine moiety).
Ferrostatins, as the first highly potent inhibitors of ferroptosis, can
attenuate oxidative, iron-dependent cell death when treated with
small molecules such as erastin8. Ferrostatin-1 (Fer-1, Fig. 1) is a
representative compound in ferrostatins which could efficiently
inhibit ferroptotic cell death in models of Huntington’s disease4,
Parkinson’s disease12, periventricular leukomalacia4 , et al. It is
autoxidation, culminating in ferroptotic cell death9,10
. Recent
research indicated that cell death triggered exclusively by ferroptosis
could be suppressed by iron chelators, lipophilic antioxidants,
inhibitors of lipid peroxidation.1 Moreover, ferroptosis inhibitors can
cross the blood-brain barrier efficiently, hence it may provide a new
believed that by preventing oxidative damage to membrane lipids13
,
the cytoprotection of Fer-1 takes into effect. Experimental evidence
indicates that fer-1 is an excellent radical-trapping antioxidant (RTA)
in phospholipid bilayers with rate constants of 4.6 × 104 M–1 s–1,
15
which is similar to liproxstatin-114,
(Lip-1, another ferroptosis
inhibitor with kinh = 1.2 × 104 M–1 s–1, Fig 1) and 4,4'-di-tert-
butyldiphenylamine (kinh = 2.5 × 104 M–1 s–1, Fig 1), but more
significantly reactive than canonical lipophilic antioxidant, α-
tocopherol (kinh = 4.7 × 103 M–1 s–1)16.
†College of Chemistry, Chemical Engineering and Materials Science, Collaborative
Innovation Center of Functionalized Probes for Chemical Imaging in Universities of
Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education,
Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals,
Shandong Normal University, Jinan 250014, P. R. China
As is well known, the efficacy of RTAs relies heavily on the stability
to one-electron oxidation17. For example, the reactiviy of Lip-1 and
4,4'-di-tert-butyldiphenylamine depends mainly on the extensive
delocalization of unpaired electron in the arinyl radical for
E-mail: sunbin_1978@sdnu.edu.cn; dchen@sdnu.edu.cn
‡State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100,
P. R. China
§ Electronic Supplementary Information (ESI) available: Additional computational
results and Cartesian coordinates of all optimized structures involved in this study.
See DOI: 10.1039/x0xx00000x
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minimizing the entropic cost of N-H bond breaking15, 18. However,
Journal Name
In order to understand the specific effect of o-phenylenediamine
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DOI: 10.1039/C8OB00546J
this theory does not seem to be suitable for interpreting the high moiety, a series of compounds with single amine named 1-13 were
reactivity of fer-1, because the delocalizition of unpaired electron is synthesized for comparison. As for the ferroptotic reaction occurring
just restricted on benzene and an electron-withdrawing group in lipid bilayers4, all these molecules are lipophilic with a cLogP value
(COOR) located para to the reactive N−H even weakens the stability (Tab.S1) larger than 2.5. From the data shown in Tab.1, the inhibition
to one-electron oxidation. Thus, we infer that an unreported radical- activity of ferroptosis between ortho-diamines (series R2) and single
trapping mechanism exists which can be used to explain the amine (series R1) is significantly different from each other. The
reactivity of fer-1 properly. Herein we describe our efforts to molecules with ortho-diamines present a strong anti-ferroptotic
characterize this distinctive mechanism of ferrostatins by employing activity with EC50 at nanomole level. In contrast, there is not a bit of
experimental and computational methods. In addition, we also pay activity found for molecules with single amine under the
great attention to the function of o-phenylenediamine moiety, a concentration of maximum non-toxic dose (MNTD). For example, fer-
common sub-structure of ferrostatins. We believe that the discovery 1 owns only one more NH2 than compound 1 in structure, but when
of the novel radical-trapping mechanism could widen the structural co-incubated with ferroptosis inducer erastin (5 μM), fer-1 expresses
space for ferroptosis inhibitor development.
full protection from erastin induced ferroptotic cell death while
compound 1 does not show any activity at all. These results imply
that o-phenylenediamine is a prerequisite pharmacophore for the
reactivity of ferrostatins.
2. Results and Discussion
2.1 O-phenylenediamine is a prerequisite pharmacophore for
ferrostatins
bEC50
(nM)
dBDE
e ∆G
bEC50
(nM)
dBDE
e∆G
aseries R1
c series R2
kcal mol-1
kcal mol-1
kcal mol-1
kcal mol-1
>12500
>12500
>12500
>12500
88
87
88
88
18.80
18.11
19.85
21.32
f50/95
265
120
90
80
80
80
80
10.45
10.05
10.76
10.52
Fer-1
1
2
SRS8-71
SRS8-73
CH3- 3
4
5
SRS8-48
>12500
>6250
88
88
17.58
18.29
62
80
79
10.76
10.35
SRS13-10
130
6
SRS8-90
SRS8-70
>12500
>12500
>12500
88
89
88
17.74
18.39
19.85
80
80
79
80
80
10.02
10.93
10.51
7
8
SRS8-94
380
9
SRS8-80
SRS8-52
>12500
>12500
89
89
17.51
17.23
650
80
10.63
10
11
12
13
>12500
>12500
89
89
17.25
17.41
Tab .1 Anti-ferroptotic cell death activity and the theoretical properties of ferrostatins.
Tab.1 Anti-ferroptotic cell death activity and the theoretical properties of series R1 and series R2. a: compounds of series R1 synthesized in
our laboratory. b: data of EC50 value from new tests. c:data of EC50 value were derived from reference 4.d: the lowest BDE value among the N-
H groups. e: the smallest energy barrier value among the N-H groups. f: the EC50 value of Fer-1 tested in our lab is 50 nM.
2 | J. Name., 2017, 00, 1-3
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Bond dissociation enthalpy (BDE) of aromatic N–H bond has been
widely accepted as an theoretical indicator of antioxidant activity.19-
21 In particular, low value of BDE indicates that the antioxidant is able
to donate hydrogen atom in free radical scavenging mechanism.22
The BDE values of series R1 are higher than 87 kcal mol-1, while all
the values of Series R2 are smaller than 80 kcal mol-1. These data
conform to the fact that the low hydrogen donating ability of series
R1 is due to their higher BDE values. Correspondingly, the value of
the required energy barrier for the reaction between series R1 and
methylperoxyl radical is >17.23 kcal mol-1, which is also significantly
larger than that of series R2 (<10.93 kcal mol-1). These theoretical
data are in accordance with the experimental results shown in the
second line of each structure in Tab.1. We take Compound 1 and fer-
1 as examples to demonstrate the effect of o-phenylenediamine on
the activity more clearly. The BDE (N–H) value of compound 1 is 88
kcal mol-1. It would plummet to be 80 (H4′) and 80 kcal mol-1(H3′)
respectively when added a NH2 group at ortho site (Fig .2). That is
nearly 8 kcal mol-1 smaller than that of compound 1. It suggests that
the ortho-diamines make it easier for the H-atom to transfer from
arylamine moieties to lipid peroxyl radicals.
Fig.2 The BDE value of Compound 1 and fer-1
2.2 The synergistic characteristics of o-phenylenediamine
To describe the distinctive radical-trapping mechanism of
ferrostatins, the possible H-abstraction paths of fer-1 are calculated
with density functional theory (DFT) methods. Fer-1 owns three
candidate hydrogens from two –NH(2) groups that may represent
attack sites by lipid peroxide radical. However, we have just found
two possible H-abstraction paths occurring at position H3′ and H3′′,
respectively. The H3′ is significantly favorable based on the energy
barrier and BDE analysis (Fig S2). The differences between the two
reactions are discussed in the Supporting Information. Here, we
focus on the H-abstraction reaction between 3′-NH and
methylperoxyl radical, as a representative feature of the antioxidant
capacity of fer-1. The optimized geometries obtained from the
MPWB1K calculation are shown in Fig 3, and the selected
geometrical parameters are listed in Tab 2.
The above results clearly state that o-phenylenediamine moiety
remarkably enhances ferrostatins’ ability in trapping lipid peroxyl
radicals. The synergistic effect of o-phenylenediamine would
decrease the BDE (N-H) value and ultimately improve the antioxidant
reactivity. Therefore, we speculate that a distinctive radical-trapping
mechanism of ferrostatins might not be reported in the past.
Fig 3 Optimized stationary points geometries and their classical potential-energy values, relative to separated reactants, in kcal mol-1,
calculated is at the MPWB1K/6-31+G(d,p) level.
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It is worth noting that two hydrogens (H3′ and H4′) of ortho- fer-1 the high reactivity to counteract the extremely fast lipid peroxyl
diamines in fer-1 are in a face-to-face position. This is different from radical (LOO·) generation.
traditional antioxidants containing ortho-diphenolic group with two
Tab 2. Selected Geometrical Parameters of the Fer-1, Reactant
hydrogens at the same direction in a planar structure, such as
Complex (RC), Transition state (TS), and fer-1 radical product in the
quercetin.23 The dihedral angle (φ) between N3′-H3′ and aromatic ring
gas phase a.
is 57.7˚, whereas the dihedral angle (ω) between N4′-H4′ and aromatic
Fer-1
Fer-1
RC
TS
ring is 27.8˚. This indicates that the steric hindrance might be the
main reason why the H-abstraction reaction occurs at position 3′ for
a similar BDE value (Fig 3 & Fig 2). The most important geometric
property of transition state is that two hydrogens from ortho-
diamines moiety simultaneously interact with methylperoxyl radical
radical
2.14
N3′-H4′
N3′-H3′
H3′-OA
N4′-H4′
H4′-OA
H3′-H4′
α
β
φ
ω
2.28
1.01
-
1.01
-
2.22
-
-
-
-
1.02
2.21
1.01
3.51
2.21
176.2
101.7
61.8
23.3
1.05
1.57
1.04
1.71
1.85
170.7
170.4
28.6
11.1
-
-
1.02
-
-
-
-
-
CH3OO·. These strong interactions make
a
substantial
rearrangement of fer-1 and CH3OO· to generate an almost planar
seven-membered heterocyclic ring. Another notable geometric
property is the reactive H-atom closing to the hydrogen bond donor.
The distance of breaking bond (N3′-H3′) is 1.05 Å, significantly
shorter than the forming bonds (H3′-OA) with 1.57 Å. It indicates that
TS of fer-1 in H-abstraction pathway is reactant-like, consistent with
the Hammond’s postulate24 for exothermic reaction. However, this
is different from the geometry of some traditional polyphenols
antioxidants, e.g. Epicatechin25 + CH3OO· reaction, where the
hydrogen atom locates almost in the center of reactant and product.
According to the above analysis, we can conclude that it is the
synergistic effect of ortho-diamines (3′-NH and 4′-NH) that endows
57.7
27.8
1.84
a See Fig 3 for atomic labels. Angles are defined as α= N3′-H3′-OA and
β = N4′-H4′-OA. Torsional angles are defined as φ = H3′-N3′-C3′-C4′ and
ω = H4′-N4′-C4′-C3′. Bond lengths are in angstroms and angles are in
degrees.
Fig 4 (a) Theoretical properties of ferrostatins. (b) Optimized transition structure of fer-1 and its derivatives. Hydrogen atoms from C-H bond
are omitted for clarity. # Data of EC50 value is derived from reference 4.
moiety but N,N-dialkyl substituted substrates does not. From the
data shown in Fig 4, it is important to be aware that the H-atom
2.3 The synergistic effect lost for N,N-dialkyl substitution
To explore whether o-phenylenediamine is essential for the
abstraction reaction of N,N-dialkyl substituted substrates is
antioxidant activity of ferrostatins, mono-N-alkyl substituted
thermodynamically unfavorable. The required energy barriers for
substrates and N,N-dialkyl substituted substrates are calculated. The
transferring hydrogen from SRS9-01 and SRS12-12 to CH3OO· are
mono-N-alkyl substituted substrates have o-phenylenediamine
19.34 and 16.64 kcal mol-1, respectively. They are significantly higher
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2.4 Intramolecular H-bonds destroy the synergistic effect
than that of mono-N-alkyl substituted substrates (SRS9-14 (8.95 kcal
mol-1) and fer-1 (10.45 kcal mol-1)) (Fig 4a). Correspondingly, the BDE
(N-H) values of SRS9-01 and SRS12-12 are over 7 kcal mol-1, larger
than that of SRS9-14. These theoretical parameters are consistent
with experiment results that mono-N-alkyl substituted substrates
would be 34-173 times more potent than N,N-dialkyl substituted
substrates in trapping lipid radical (Fig 4a).4
The reactivity difference between these two types of substituted
substrates can be well explained by the geometry of transition-state
(Fig 4b). For mono-N-alkyl substituted substrates (SRS9-14, fer-1),
the interaction between o-phenylenediamine and CH3OO· can form
a stable seven-membered ring which makes the H-atom easily to
transfer. But for N,N-dialkyl substituted substrates (SRS9-01, SRS12-
12), only one -NH group interacts with the methylperoxyl radical,
which forms a linear transition structure. This structure, not so stable
as the mono-N-alkyl substituted substrates, makes the hydrogen
difficultly to transfer. In addition, higher reactivity of mono-N-alkyl
substituted substrates than that of N,N-dialkyl substituted substrates
provides direct evidence to explain the different mechanism
between Fer-1 and classical RTAs. Because NH2 (for Fer-1) , NHR (for
mono-N-alkyl substituted substrates) and NR2 (for N,N-dialkyl
substituted substrates) are all strong electron-donating groups26,
these molecules have similar capacity to stabilize one-electron
oxidation.
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In some cases, ortho-diamines can be sDuObsI:ti1t0u.1te0d39/bCy8OhyBd0r0o5g4e6nJ
bond acceptor (HBA), such as groups C=OR and C=OOR. Although
these molecules contain o-phenylenediamine moiety, the carbonyl
oxygen rotates toward the 4′-NH group and finally forms a strong
intramolecular H-bond aided by an optimized geometric
arrangement. Under this condition, the primary impact is that the
ortho-diamines in these molecules could not simultaneously interact
with peroxy radical. Another impact is that the intramolecular H-
bond substantially stabilizes the reactive 4′-NH group which causes
an increase in BDE. 4, 27 Taking compound SRS11-97 as an example,
the substituted group C=OOMe at position 3' interacts with 4'-NH
group and forms a strong intramolecular H-bond with the length of
1.90 Å (Fig 5b). It actually stabilizes the parent molecule, and makes
the BDE (4' N-H) value (90 kcal mol-1) remarkably increase.
Correspondingly, the energy barrier is 29.72 kcal mol-1, significantly
larger than that of fer-1 by 19.72 kcal mol-1. As for the -NH group at
position 3' of SRS11-97, its H-abstraction reaction mode is like
compound 1 that just have a single -NH group. The BDE (3' N-H) value
is 88 kcal mol-1 and the associated energy barrier is 21.30 kcal mol-1.
Both are considerably larger than that of Fer-1. These data are
consistent with experimental results of the weak reactivity for SRS11-
97 in trapping lipid radical (Fig 5a).
In general, intramolecular H-bonding involving the reactive -NH
group will destroy the synergistic effect of o-phenylenediamine and
decrease the radical-trapping reactivity completely if -NH acts as H-
bond donor.
According to the above computational and experimental results,
the radical-trapping reactivity of fer-1 derivatives is quite weak once
the syngeneic effect of o-phenylenediamine loses. In other words,
the pharmacophore of the o-phenylenediamine is the fundamental
substructure for ferrostatins to perform the potent antioxidant
activity.
Fig 5 (a) Theoretical properties of ferrostatins. (b) Optimized structure of SRS11-97 and its transition state at position 3, position 4.
Hydrogen atoms from C-H bond are omitted for clarity. # Data of EC50 value derive from reference 4.
ψ = 3.81º) forces both N-H bonds into better alignment with the
2.5 Understanding the Superiority of Fer-1 as a RTA
aromatic π-system, increasing the magnitude of the lone pair
The efficacy of RTAs relies on the stability to one-electron
donation to the conjugated benzene ring28. NBO analysis indicates
oxidation by peroxy radical. Thus, The NBO calculations were
that the energies of nN → π∗ interactions between two amines and
employed to predict the stability of the transition states in H-
aromatic system are 18.92 and 25.70 kcal mol-1, respectively. They
abstraction step. As the data shown in Tab.3, the simultaneous
are significantly larger than that in N,N-dialkyl substituted substrate
interaction between two ortho-amino groups with the peroxy radical
(SRS12-12). Accordingly, the stabilization of conjugative geometry
would be the crucial factor in stabilizing the structure of TS. Under
should improve spin delocalization in TS and radical product.
this condition, the formed planar seven-membered ring (ф = 9.01º,
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Amines group is one of the stereoelectronic chameleons that can mechanism is significantly different from that of diarylamine and
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interact with the peroxy radical in several ways29. In order to follow traditional polyphenols antioxidants. DuriDngOI:t1h0e.10p3r9o/cCe8sOs,B0o0r5t4h6oJ-
the H-abstraction coordinate, the breaking N-H (3′) should be diamines from ferrostatins simultaneously interact with the
interacted with the half-filled oxygen orbital. In this interaction, N-H methylperoxyl radical, and then form a compacted seven-membered
bond is an effective donor. At the same time, the ortho-NH (4′) acts ring in transition-state making the H-abstraction reaction easier to
as an acceptor that can interact with the orthogonal fully filled p-type happen. Correspondingly, the radical-trapping reactivity will
∗
→
orbital at the radical center via a classic H-bond type nO ꢀꢁꢂ dramatically decrease or even disappear once the structure of o-
interaction. This intermolecular interaction between the lone pair of phenylenediamine is destroyed by N,N-dialkyl substitutions or
an O-radical and a low energy σ* orbitals provides another stabilizing hydrogen bond acceptors substitutions. Thus, o-phenylenediamine
force that assists the transition state formation.30
moiety would be considered as a privileged pharmacophore for
In summary, the o-phenylenediamine moiety provides a specific radical-trapping reactivity. We believe that the novel radical-trapping
TS-stabilizing interaction to the N-H abstraction step.
mechanism could widen the path for new RTAs design and
development. On the other hand, it might be a good strategy to
integrate o-phenylenediamine pharmacophore into the approved
antipsychotic drugs. The new drug candidate would add the role of
ferroptosis inhibition on the original pharmacological effects, which
would provide promising therapeutic agents to treat pathological
conditions previously thought to be untreatable.
3 Conclusion
In summary, we have discovered that the synergistic effect of o-
phenylenediamine moiety is a mechanism by which ferrostatins
inhibit ferroptotic cell death. And this novel type of radical-trapping
Tab 3 the o-substituent affect the stability to one-electron oxidation in TS
Compound 1
SRS12-12
Fer-1
2
3
O
O
2
O
2
H2N
HN
H2N
3
3
O
O
O
C8H17
HN
4
5
4
5
N
4
5
C8H17
O
O
O
O
O
O
Ha
Hb
Ha
Hb
N1
N1
N2
N2
N2
C6H11
OA
OB
Hc
OA
OA
Hc
C8H17
C8H17
OB
C6H11
OB
ψ = 10.70˚
ф = 28.57˚, ψ = 32.36˚
nꢂ1 → ꢅ∗ = 11.55
nꢂ2 → ꢅ∗ = 12.54
ф = 9.01˚, ψ = 3.81˚
nN2 → ꢃꢄ+4 = 48.60
nꢂ2 → ꢅ∗ = 3.6
nꢂ1 → ꢅ∗ = 18.92
nꢂ2 → ꢅ∗ = 25.70
nꢆꢇ → ꢀꢁ∗ꢈꢂ2 = 9.03
ΔG = 18.41
ΔG = 16.64
ΔG = 10.45
ф = [(C2-C3-N1-Ha) + (C4-C3-N1-Hb)]/2; ψ = [(C3-C4-N2-Hc/C) + (C5-C4-N2-C)]/2, all energies are in kcal mol-1
4
Methods
vacuo. The crude product was purified by column chromatography
on silica gel using ethyl acetate/ petroleum ether as eluent to afford
compound 1-13 as a white solid.
4.1 Experiments
Reagents and Compounds synthesis
Fer-1 and erastin were obtained from Sigma-Aldrich (Sigma
Compound 1: ethyl 4-(cyclohexylamino)benzoate: white solid,
Chemicals St. Louis, MO, USA). All reagents were obtained from yield 50%; 1H NMR: 7.67-7.64(2H, m), 6.58-6.56(2H, m), 4.19(2H, q),
commercial sources and were used without further purification 3.27-3.22(1H, m), 1.89(2H, t), 1. 74-1.69(4H, m), 1.35-1.20(4H, m),
unless otherwise stated. Acetone was dried with Type 4A Linde 1.27(3H, t, J=6.0 Hz), 1.18-1.11(2H, m); 13C NMR(75MHz, D6-DMSO):
molecular sieves and then distilled. All reactions were monitored by δ 166.33, 152.37, 131.44, 115.90, 111.44, 59.88, 50.69, 25.89, 24.92,
thin-layer chromatography (TLC). 1H 13C NMR spectra were 14.84; HRMS(ESI) m/z: calcd. for C15H21NO2: [M+H] 248.1645; found:
measured on a Bruker 400 MHz spectrometer, using deuterated 248.1629.
dimethyl sulfoxide (DMSO-d6) as solvent and TMS as internal
Compound 2: ethyl 4-(cyclobutylamino)benzoate: white solid,
standard. Chemical shifts are expressed as δ ppm. High-resolution yield 61%; 1H NMR: 7.68-7.66(2H, m), 6.56(2H, m), 4.19(2H, q), 2.96-
mass spectra were obtained on a Bruker mass spectrometer with an 2.93(1H, m), 1.85-1.74 (1H, m), 1.26(3H, t, J=6.0 Hz), 1.25-1.04(2H,
ESI source.
m), 0.50-0.40(2H, m), 0.25-0.20(2H, m); 13C NMR(75MHz, D6-DMSO):
The general procedure for compound 1-13 synthesis: A mixture of δ 166.35, 152.07, 131.39, 116.29, 111.25, 59.93, 47.30, 30.53, 15.33,
benzocaine (20.0 mmol), propargyl bromide (20.0 mmol), anhydrous 14.79; HRMS(ESI) m/z: calcd. for C13H17NO2: [M+H] 220.1332;
(20.0 mmol) potassium iodide (1.0 mmol) in N,N-dimethylformamide found: 220.1319.
(35 mL) was vigorously stirred at room temperature. After
Compound 3: ethyl 4-(ethylamino)benzoate: white solid, yield 75%;
completion of the reaction, as indicated by TLC, the reaction mixture 1H NMR: 7.70-7.66(2H, m), 6.58-6.47(2H, m), 4.19(2H, q), 3.12-
was diluted with ethyl acetate (40 mL) and washed with brine (20 mL 3.06(2H, m), 1.27(2H, t, J=6.0 Hz), 1,14(3H, t); 13C NMR(75MHz, D6-
× 3). The organic phases were dried over Na2SO4 and concentrated in DMSO): δ 166.36, 153.07, 131.39, 116.49, 111.33, 59.93, 37.44,
6 | J. Name., 2017, 00, 1-3
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14.78, 14.50; HRMS(ESI) m/z: calcd. for C11H15NO2: [M+H] 194.1175; 6.0 Hz); 13C NMR(75MHz, D6-DMSO): δ 166.11, 160.52, 153.00,
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found: 194.1161.
134.45, 131.29, 128.96, 117.55, 115.89, 111D.9O3I,: 1600..11013,95/C38.5O4B,01045.4763J;
Compound 4: ethyl 4-(isopropylamino)benzoate: white solid, yield HRMS(ESI) m/z: calcd. for C16H16FNO2: [M+H] 273.1243; found:
6 %; 1H NMR: 7.68-7.65(2H, m), 6.57-6.55(2H, m), 4.19(2H, q）, 3.65- 273.1265.
3.57(1H, m), 1.58(3H, d), 1. 26(3H, t, J=6.0 Hz), 1.14(3H, d); 13C
NMR(75MHz, D6-DMSO): δ 166.12, 154.39, 131.42, 118.05, 111.51, solid, yield 26%; H NMR: 7.71-7.67(2H, m), 7.50-7.45(2H, m), 7.35-
60.17, 48.28, 25.3, 14.75; HRMS(ESI) m/z: calcd. for C12H17NO2: [M+H] 7.28(2H, m), 6.61-6.58(2H, m), 4.41(2H, s), 4.19(2H, q), 1.27(3H, t, J =
Compound 13: ethyl 4-(4-chlorobenzylamino)benzoate: white
1
208.1332; found: 208.1315.
6.0 Hz); 13C NMR(75MHz, D6-DMSO): δ 166.25, 152.81, 136.58,
Compound 5: ethyl 4-(butylamino)benzoate: white solid, yield 132.83, 131.43, 129.26, 127.67, 127.67, 117.20, 60.03, 41.21, 14.81;
87%;1H NMR: 7.68-7.65(2H, m), 6.58-6.48(2H, m), 4.20(2H, q), 3.08- HRMS(ESI) m/z: calcd. for C16H16NO2 Cl: [M+2] 290.0992; found:
3.03(1H, m), 1.56-1.49(4H, m), 1.27(3H, t, J=6.0 Hz), 0.91(3H, t); 13
C
291.0928.
NMR(75MHz, D6-DMSO): δ 166.35, 153.36, 131.37, 116.20, 111.14, Inhibition of Ferroptosis Induced by Erastin
59.88, 42.45, 31.06, 20.20, 14.80, 14.17; HRMS(ESI) m/z: calcd. for
C13H19NO2: [M+H] 222.1488; found: 222.1471.
HT-108031 cell line was purchased from the Cell Bank of the
Chinese Academy of Sciences (Shanghai, China). The cells were
Compound 6: ethyl 4-(pentylamino)benzoate: white solid, yield grown in Eagle’s Minimum Essential Medium with 10% fetal bovine
81 %; 1H NMR: 7.68-7.65(2H, m), 6.58-6.55(2H, m), 4.19(2H, q), serum, 2 mM L-glutamine, and 100 U/ml of penicillin/streptomycin
3.03(1H, t), 1.56-1.51(1H, m), 1.37-1.25(6H, m), 1.26(3H, t, J=6.0 Hz), in a humidified incubator with 5% CO2 and 95% air. Cell death
0.88(3H, t); 13C NMR(75MHz, D6-DMSO): δ 166.34, 153.30, 131.38, inhibition (EC50) values were determined by treating HT-1080
116.22, 111.21, 59.91, 42.76, 29.27, 28.62, 22.42, 14.83, 14.39; fibrosarcoma cells with a lethal concentration of erastin (5 μM) in the
HRMS(ESI) m/z: calcd. for C14H21NO2: [M+1] ： 236.1645; found: presence of each new synthesized compound in a 10-point, 2-fold
234.1626.
dilution series starting at 100 μM for 48 h. Cell viability was assessed
Compound 7: ethyl 4-(allylamino)benzoate: white solid, yield 47%; later using the MTT assay according to the manufacturer’s
1H NMR: 7.74-7.66(2H, m), 6.76-6.60(1H, m), 6.57(1H, t) 5.91- instructions. Each experiment was carried out in six analytical
5.83(1H, m), 5.23-5.19(2H, m), 5.19(2H, t), 4.17(2H, q), 1.27(3H, t, replicates per concentration and repeated independently at least
J=6.0 Hz); 13C NMR(75MHz, D6-DMSO): δ 166.34, 153. 30, 138.66, three times.
131.40, 116.26, 116.05, 111.35, 59.92, 42.18, 31.15, 28.09, 14.81; 4.2 Computations
HRMS(ESI) m/z: calcd. for C12H15NO2: [M+Na] 228.0995; found:
228.0977.
The geometries of reactants, products and transition states were
fully optimized at the (U)MPWB1K function32 with 6-31+G(d,p) basis
Compound 8: ethyl 4-(pent-4-enylamino)benzoate: white solid, set and characterized by the number of imaginary frequencies. We
yield 72%; 1H NMR: 7.69-7.66(2H, m), 6.58-6.52(2H, m), 5.07-4.97(2H, adopted the MPWB1K function because it is more accurate than the
m), 4.22(2H, q), 3.09-3.04 (2H, m), 2.11-2.09(1H, m), 1.64(2H, t), traditional B3LYP method in describing the energy barrier of H-
1.27(3H, t, J = 6.0 Hz); 13C NMR(75MHz, D6-DMSO): δ 166.32, 153. 11, transfer reaction.33 Transition state structures were further
135.65, 131.31, 116.675, 116.05, 111.55, 59.95, 32.07, 14.81; confirmed by intrinsic reaction coordinate (IRC) calculations to
HRMS(ESI) m/z: calcd. for C14H19NO2: [M+H] 234.1488; found: connect the corresponding reactants and products. For all cited
234.1472.
energies, ZPE corrections were taken into consideration. The
Compound 9: ethyl 4-(prop-2-ynylamino)benzoate: white solid, discussed energies in this paper are referred to relative Gibbs free
yield 53%; 1H NMR: 7.74-7.72(2H, m), 6.88-6.85(1H, m), 6.68-6.64(1H, energies (ΔG≠298K). Natural bond orbital (NBO) analyses were
m), 4.21(2H, q), 3.95-3.93(2H, m), 3.14(1H, s), 1.27(3H, t, J = 6.0 Hz); performed at the (U)MPWB1K /6-311++G(d,p) level using NBO6w.34
13C NMR(75MHz, D6-DMSO): δ 166.29, 152.25, 131.23, 117.62, In order to ensure that the lowest energy conformation of
112.13, 81.78, 73.81, 60.11, 32.07, 14.82; HRMS(ESI) m/z: calcd for intermediates and transition states was presented and discussed in
C12H13NO2: [M+H] 204.1019; found: 204.1004.
the text, extensive conformational searches were conducted.
Compound 10: ethyl 4-(benzylamino)benzoate: white solid, yield Cartesian coordinates of all optimized structures are given in
69%; 1H NMR: 7.67-7.64(2H, m), 7.35-7.27(4H, m), 7.26-7.22(1H, m), supporting information. Bond Dissociation Enthalpies (BDEs) of N-H
6.62-6.60(2H, m), 4.35(2H, s), 4.21(2H, q), 1.27(3H, t, J = 6.0 Hz); 13
C
bond was used as the energetic parameter to evaluate the feasibility
NMR(75MHz, D6-DMSO): δ 166.28, 153. 10, 139.79, 131.38, 128.83, of H-atom transfer procession because of its relationship to the
127.63, 127.28, 116.80, 111.71, 59.98, 46.32, 14.82; HRMS(ESI) m/z: hydrogen atom transfer (HAT) mechanism of free radical
calcd. for C16H17NO2: [M+H] 256.1332; found: 256.1300.
scavenging,35, 36 and relatively low N-H BDE facilitated the H-atom
Compound 11: ethyl 4-(4-methylbenzylamino)benzoate : white transfer reaction between antioxidant and radical to break radical
1
solid, yield 46%; H NMR: 7.66-7.64(2H, m), 7.23-7.21(2H, m), 7.14- chain reaction.37
7.07(2H, m), 6.60-6.58(2H, m), 4.29-4.27(2H, m), 4.18(2H, q), 1.27(3H,
BDE = Hꢉ + Hꢁ − Hꢊ
t, J = 6.0 Hz), 1.25(3H, t); 13C NMR(75MHz, D6-DMSO): δ 166.29, 153. Where HR is the enthalpy for radical generated after H-abstraction
11, 136.67, 136.32, 131.30, 129.39, 127.60, 116.71, 111.70, 59.97, reaction, HH is the enthalpy for hydrogen atom, and HP is the
46.07, 21.12, 14.83; HRMS(ESI) m/z: calcd. for C17H19NO2: [M+H] enthalpy for parent molecule of radical.
270.1488; found: 270.1461.
All calculations were performed with Gaussian 09 program
Compound 12: ethyl 4-(4-fluorobenzylamino)benzoate: white package.38 The 3D structures were prepared using CYLview.39
1
solid, yield 56%; H NMR: 7.71-7.68(2H, m), 7.29-7.25(2H, m), 7.20-
Acknowledgments
7.14(2H, m), 6.75-6.72(2H, m), 4.77(2H, s), 4.18(2H, q), 1.27(3H, t, J =
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This work is supported by grants from the China Postdoctoral Nat Cell Biol, 2014, 16, 1180-1191.
Science Foundation (NO. 2014M561958), the Primary Research & 15. X. Sheng, C. Shan, J. Liu, J. Yang, B. Sun and D. Chen, Physical
Development Plan of Shandong Province（NO. 2015GSF118175）, Chemistry Chemical Physics, 2017, 19, 13153-13159.
the Projects of Science and Technology Development Plan of 16. O. Zilka, R. Shah, B. Li, J. P. Friedmann Angeli, M. Griesser, M.
Shandong province (NO. J13M07) and the National Undergraduates’ Conrad and D. A. Pratt, ACS Central Science, 2017, 3, 232-243.
Innovation and Entrepreneurship Training Program (NO. 17. K. U. Ingold and D. A. Pratt, Chemical Reviews, 2014, 114, 9022-
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O-phenylenediamine: A Privileged Pharmacophore of Ferrostatins for Radical-Trapping
Activity in Blocking Ferroptosis
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