Novel Ferroptosis Inhibitors with Improved Potency and ADME Properties

Article abstract of DOI:10.1021/acs.jmedchem.5b01641

Ferroptosis is a nonapoptotic, iron-catalyzed form of regulated necrosis that is critically dependent on glutathione peroxidase 4 (GPX4). It has been shown to contribute to liver and kidney ischemia reperfusion injury in mice. A chemical inhibitor discovered by high-throughput screening displayed inhibition of ferroptosis with nanomolar activity and was dubbed ferrostatin-1 (fer-1). Ferrostatins inhibit oxidative lipid damage, but suffer from inherent stability problems due to the presence of an ester moiety. This limits the application of these molecules in vivo, due to rapid hydrolysis of the ester into the inactive carboxylic acid. Previous studies highlighted the importance of the ethyl ester and suggested steric modifications of the ester for generating improved molecules. In this study, we report the synthesis of novel ferroptosis inhibitors containing amide and sulfonamide moieties with improved stability, single digit nanomolar antiferroptotic activity, and good ADME properties suitable for application in in vivo disease models.

Full text of DOI:10.1021/acs.jmedchem.5b01641

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Article
Novel ferroptosis inhibitors with improved potency and ADME properties
Sam Hofmans, Tom Vanden Berghe, Lars Devisscher, Behrouz Hassannia, Sophie Lyssens,
Jurgen Joossens, Pieter Van der Veken, Peter Vandenabeele, and Koen Augustyns
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01641 • Publication Date (Web): 22 Dec 2015
Downloaded from http://pubs.acs.org on December 25, 2015
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Novel ferroptosis inhibitors with improved potency
and ADME properties
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a
b,c
a
b,c
Sam Hofmans , Tom Vanden Berghe , Lars Devisscher , Behrouz Hassannia , Sophie
a
a
a
b,c,d
Lyssens , Jurgen Joossens , Pieter Van Der Veken , Peter Vandenabeele
, Koen
a
Augustyns
a
Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, Bꢀ2610
Antwerp, Belgium
b
Molecular Signaling and Cell Death unit, VIB Inflammation Research Center, Ghent,
Belgium.
c
Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent, Belgium.
d
Methusalem Program, Ghent University, Ghent, Belgium.
Keywords: ferroptosis, regulated necrosis
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ABSTRACT
Ferroptosis is a nonꢀapoptotic, ironꢀcatalysed form of regulated necrosis that is critically dependent on
glutathione peroxidase 4 (GPX4). It has been shown to contribute to liver and kidney ischemia
reperfusion injury in mice. A chemical inhibitor discovered by highꢀthroughput screening displayed
inhibition of ferroptosis with nanomolar activity and was dubbed ferrostatinꢀ1 (ferꢀ1). Ferrostatins
inhibit oxidative lipid damage, but suffer from inherent stability problems due to the presence of an
ester moiety. This limits the application of these molecules in vivo, due to rapid hydrolysis of the ester
into the inactive carboxylic acid. Previous studies highlighted the importance of the ethyl ester and
suggested steric modifications of the ester for generating improved molecules. In this study, we report
the synthesis of novel ferroptosis inhibitors containing amide and sulfonamide moieties with improved
stability, single digit nanomolar antiꢀferroptotic activity and good ADME properties suitable for
application in in vivo disease models.
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INTRODUCTION
Cell death research was revitalized by the understanding that necrosis can occur in a highly regulated
and genetically controlled manner. Although necroptosis is the best understood form of regulated
1
2
necrosis , other important necrotic signalling pathways are emerging. A new type of regulated
necrosis induced by erastin, an oncogenic toxic small molecule, was recently described. Erastin acts, at
ꢀ
least partially, through inhibition of the System X
C
Cys/Glu antiporter (Figure 1). This type of erastinꢀ
induced cell death was defined as ferroptosis because it critically depends on intracellular iron
3
metabolism and gluthathione peroxidase 4 (GPX4) inactivity. Ferroptosis was recently found to be a
4
,5,6
detrimental process in kidney and liver ischemia reperfusion injury.
It is inhibited by iron chelators,
7
,8
lipophilic antiꢀoxidants and/or ferrostatinꢀ1 (ferꢀ1, 1, figure 2). GPX4 inhibits the accumulation of
ꢀ
lipid peroxides and depends on the functionality of System X
C
Cys/Glu antiporter in the plasma
membrane. Treatment with erastin blocks uptake of cystine, the oxidized form of Cys, which reduces
the levels of intracellular Cys required for gluthatione (GSH) synthesis. Subsequently, this drop in
8
9
GSH levels leads to the inactivation of GPX4, which uses GSH as a cofactor. In addition, the redoxꢀ
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active iron pools are capable of directly catalysing formation of damaging free radical via e.g. Fenton
reactions. The aberrant accumulation of iron, reactive oxygen species (ROS) or the combination of
both are linked to a staggering number of traumas and chronic degenerative conditions including
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,5,10
stroke, sepsis, ischemia reperfusion injury, traumatic brain injury and neurodegenerative diseases.
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For this reason, iron chelators have been implemented or proposed as treatments for diverse
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pathologies caused by iron and/or ROS accumulation.
Ferroptosis may be blocked in vitro by the small molecule inhibitor ferrostatinꢀ1 (ferꢀ1, 1, Figure 2).
6,7
Ferrostatins are believed to act by preventing oxidative damage to membrane lipids. Ferꢀ1, an
arylalkylamine with antiꢀoxidative properties, was identified as one of the first inhibitors of
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ferroptosis. Ferꢀ1 attenuates oxidative, ironꢀdependent cell death in cancer cells treated with small
molecules such as erastin, however the exact target of ferꢀ1 remains unclear. A study by Skouta et al.
observed that ferꢀ1 treatment did not significantly change the levels of cysteine or glutathione (system
ꢀ
C
X
), in contrast it did preserve normal levels of polyunsaturated fatty acids (PUFAs) and PUFAꢀ
derivatives. PUFAs and derivatives are susceptible to oxidation either through enzymatic (e.g.
1
1
lipoxygenaseꢀmediated) or nonꢀenzymatic (ironꢀdependent Fenton chemistryꢀmediated) processes.
Ferꢀ1 is able to prevent depletion of oxidized PUFAs and their fragments by inhibiting their oxidative
destruction. Thus ferꢀ1 acts as a lipid peroxide reductant to intercept and scavenge a lipid radical
6
through hydrogen atom transfer or direct reduction. Similar data was obtained by Dixon et al. This
study hypothesized as well that ferꢀ1 is a lipid ROS scavenger, implying that the Nꢀcyclohexyl moiety
7
on the central aromatic core serves as a lipophilic anchor within the biological membranes of the cell.
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ꢀ
c
Figure 1. Schematic overview of System Xcꢀ Cys/Glu antiporter inhibition by erastin. System X Cys/Glu
antiporter is an amino acid antiporter that typically mediates the exchange of extracellular cystine and
intracellular Glu across the cellular plasma membrane. The import of cystine through this transporter is critical
for the production of glutathione (GSH) and protection against oxidative stress. Cystine reductase (CR) rapidly
catalyzes cystine to Cys. Glutamate cysteine ligase (GCL) catalyzes the synthesis of γꢀglutamyl cysteine (γGC)
from Glu and Cys, and gluthathione synthase (GS) generates GSH by adding Gly. GSH reduces
hydrogenperoxide (H
2
O
2
) catalyzed by gluthatione peroxidase 4 (GPX4), and is thereby converted to GSH
ꢀ
disulfide (GSSG). Inhibition of System X
c
Cys/Glu antiporter by erastin results in a sequential drop of
intracellular cysteine, Cys and GSH levels. The consequent loss of GPX4 activity due to the lack of GSH,
2+
2 2
reduces the antiꢀoxidant capacity of the cell. This results in accumulation of H O that upon reaction with Fe
produces highly reaction hydroxyl radicals (Fenton reaction). This increase in free radicals can initiate a lipid
peroxidation chain reaction resulting in membrane damage, and consequent cell death (referred to as ferroptosis).
A chemical inhibitor discovered by highꢀthroughput screening displayed inhibition of ferroptosis with nanomolar
activity and was dubbed ferrostatinꢀ1 (ferꢀ1). Ferꢀ1 is believed to act by preventing oxidative damage to
3
,7,8,9
membrane lipids.
GCL: glutamate cysteine ligase; GS: gluthathione synthetase; CR: cysteine reductase
So far only a few analogues of ferꢀ1 have been reported to inhibit ferroptosis (Figure 2). An initial
3
structureꢀactivity relationship (SAR) was established by Stockwell et al. Substitution of the primary
aromatic amine of ferꢀ1 for a nitro group or elimination of the Nꢀcyclohexyl moiety abolished the
antioxidant capability of ferꢀ1 as well as its ability to prevent erastinꢀinduced death in HTꢀ1080 cells.
In addition ten other ferꢀ1 analogues were synthesised, in which the number of carbons in the Nꢀ
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substituted cycloalkyl moiety varied. A significant correlation between the predicted lipophilicity and
erastinꢀdeath suppressing ability of each molecule was observed. In general, compounds containing a
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large, lipophilic Nꢀsubstituted cycloalkyl moiety were more potent. A second SARꢀstudy was
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reported by Skouta et al. A library of 67 analogues of ferꢀ1 was synthesised, designing monoꢀ and
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disubstituted amines. Secondly, insertion of heteroatoms into the Nꢀcyclohexyl moiety decreased
potency, suggesting that hydrophobicity of this part in the scaffold is essential for anchoring with
lipophilic membrane environments. Also, the ethyl ester of ferꢀ1 was modified and it was observed
that elongation of the ester was well tolerated. However, replacement of the ester with an amide as
shown in SRS9ꢀ11 (2, figure 2, IC50 = 950 nM) resulted in a 10ꢀfold decrease in potency when
1
3
compared to the original ferꢀ1 (IC50 = 95 nM). It was also observed that tertiary amines resulted in a
significant decrease in potency when compared to primary and secondary amine analogues. It became
also clear that substitution of the aryl group at Nꢀ3 with different electron withdrawing or electron
donating groups and heteroatoms resulted in decreased potency. Finally, modification of the central
aromatic core of the parent compound resulted in additional ferꢀ1 analogues with a decrease in
potency. Conclusively, this study generated additional ferꢀ1 analogues with improved properties. For
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example, SRS11ꢀ92 (3, Figure 2, IC50 = 6 nM) was about 16ꢀfold more potent than the original ferꢀ1.
These two studies mainly focussed on increasing potency, rather than improving the poor plasma
stability due to the presence of an ethyl ester. Linkermann et al. recently published a study reporting
ferꢀ1 analogues with improved stability. This study disclosed that variants of ferꢀ1 should be ester
analogues. In an effort to enhance plasma stability the ethyl ester moiety was replaced by a tertꢀbutyl
ester. An imine moiety was additionally introduced to increase microsomal stability. The most
promising compound in that study, SRS16ꢀ86 (4, Figure 2), displayed a high plasma stability (t1/2 ≥
120 min) and a high metabolic stability towards mouse microsomes (intrinsic clearance (CLint),
5
mL/min per g liver < 0.5). However,
4 did show a decrease in potency (IC50 ~ 350 nM) when
1
2
compared to ferꢀ1.
It has become clear that the development of analogues with high potency and specificity for ferroptotic
cell death is beneficial for numerous therapies. In this study we describe the synthesis and evaluation
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of amide and sulfonamide analogues of ferꢀ1. Despite earlier reports on the negative effect of
replacing the ester with an amide, we will describe highly potent compounds with excellent plasma
and microsomal stability.
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Figure 2. Reported analogues of ferꢀ1.
RESULTS AND DISCUSSION
Compound design
In order to obtain novel inhibitors with increased stability and potency, three types of modifications
were introduced in this series: (1) replacement of the ester bond by an amide or sulfonamide, (2)
increase of lipophilicity by variation of the number of carbon atoms in the cycloalkyl moeity of R , (3)
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addition of benzylic and pyridinylic substituents to the amine of R .
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Figure 3. Structural comparison of ferrostatinꢀ1 and novel inhibitors with improved stability.
Chemistry
A total of 38 novel inhibitors were synthesized for this study. All compounds were prepared following
the general strategies in Schemes 1, 2 and 3.
Prior to the synthesis of the amide and sulfonamide analogues of ferꢀ1, the methyl ester and carboxylic
acid were synthesized as a reference to ferꢀ1. The molecules were synthesized from the commercially
available 4ꢀchloroꢀ3ꢀnitrobenzoic acid
5 and the methyl 4ꢀchloroꢀ3ꢀnitrobenzoate 6. Nucleophilic
aromatic substitution with cyclohexylamine in basic conditions provided molecules 7-8 respectively.
Palladiumꢀcatalyzed hydrogenation of the 3ꢀnitro group lead to the corresponding 3ꢀamino analogs 9-
10.
Scheme 1: Reagents and conditions: (a) cyclohexylamine, K
2
CO
3
, DMSO, 17h, 60°C; (b) Palladium hydroxide,
H gas, methanol, 17h, rt;
2
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The synthesis of ethylamide analogues of ferꢀ1 is outlined in Scheme 2. Starting from the
commercially available 4ꢀchloroꢀ3ꢀnitrobenzoyl chloride 11, Nꢀethylamide formation was realized by
treatment of the starting material with ethylamine in basic conditions. Nucleophilic aromatic
substitution of 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzamide 12 with cyclohexylamine or 2ꢀadamantylamine in
basic conditions provided molecules 13-14. Palladiumꢀcatalysed hydrogenation of the 3ꢀnitro group
lead to the corresponding 3ꢀamino analogs 15-16. Treatment of respectively 15-16 with benzyl
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bromide or 4ꢀ(bromomethyl)pyridine hydrobromide resulted in target compounds 17-20
.
Scheme 2: Reagents and conditions: (a) ethylamine HClꢀsalt, triethylamine, DCM, 4h, rt; (b) cyclohexylamine
7
or 2ꢀadamantylamine HClꢀsalt, K
2 3 2
CO , DMSO, 17h, 60°C; (c) Palladium hydroxide, H gas, methanol, 17h, rt;
1
3
(d) benzyl bromide or 4ꢀ(bromomethyl)pyridine hydrobromide, K
2
3
CO , DMF, 1ꢀ4h, 60°C
Scheme 3 shows the synthesis of sulfonamide analogues of ferrostatinꢀ1. The derivatives were
synthesized from 4ꢀchloroꢀ3ꢀnitrobenzenesulfonyl chloride 21 in a similar way as described in scheme
2. Reaction of the appropriate amine analogue with sulfonylchloride delivered 22-24. Nucleophilic
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aromatic substitution of 22-24 with different cycloalkylamines in basic conditions resulted in 25-34
.
Palladiumꢀcatalyzed hydrogenation of the 3ꢀnitro group resulted in 35-44 and coupling of the resulting
3ꢀamines with benzyl bromide or 4ꢀ(bromomethyl)pyridine hydrobromide resulted in target
compounds 45-64
.
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NO2
NO2
NO2
H
Cl
Cl
N
a
b
R2
O
O
O
S
S
S
Cl
NH
NH
O
O
O
R1
R1
2
1
22-24
25-34
R3
NH2
HN
H
H
N
N
R2
d
R2
c
O
O
S
O
S
NH
R1
NH
R1
O
3
5-44
45-64
R1
R2
=
=
=
R3
N
Scheme 3: Reagents and conditions: (a) ethylamine HClꢀsalt, triethylamine, THF, 1h, ꢀ40°C to rt; (b)
cyclohexylamine or 2ꢀadamantylamine HClꢀsalt, K
2
CO
3
, DMSO, 17h, 60°C; (c) Palladium hydroxide, H
2
gas,
7
13
methanol, 17h, rt; (d) benzyl bromide or 4ꢀ(bromomethyl)pyridine hydrobromide, K
2
CO
3
, DMF, 1ꢀ4h, 60°C
Inhibition of erastin-induced ferroptosis
Compounds 9-10, 15-20 and 35-64 were evaluated for prevention of erastinꢀinduced ferroptotic cell
death and IC50ꢀvalues are presented in table 1. The 95% confidence interval for all IC50ꢀvalues is
reported in the supplementary information (S2ꢀ3).
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a
Compound
R
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R
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R
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IC50
cLogP
(
nM)
18
1
(Fer-1)
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Ethyl
H
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
2ꢀAdamantyl
Cyclohexyl
Cyclohexyl
2ꢀAdamantyl
2ꢀAdamantyl
Cyclopropyl
Cyclopentyl
Cyclohexyl
Norbornyl
H
3.7
2.6
3.1
2.1
3.8
4.3
2.8
6.0
4.5
0.8
1.2
2.2
2.5
3.4
3.9
1.7
3.4
2.9
4.6
3.9
H
210
34
10
Methyl
Ethyl
H
15
16
17
18
19
20
H
88
Ethyl
H
14
Ethyl
Benzyl
21
Ethyl
Pyridinyl
26
Ethyl
Benzyl
6.5
3.4
76
Ethyl
Pyridinyl
35
36
37
38
39
40
41
42
43
44
45
Ethyl
H
H
Ethyl
82
Ethyl
H
67
Ethyl
H
28
Ethyl
Cyclooctyl
H
9.3
17
Ethyl
2ꢀAdamantyl
Cyclohexyl
2ꢀAdamantyl
Cyclohexyl
2ꢀAdamantyl
Cyclopropyl
H
Methyl
Methyl
tertꢀButyl
tertꢀButyl
Ethyl
H
76
H
11
H
27
H
6.0
61
Benzyl
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Ethyl
Cyclopropyl
Cyclopentyl
Cyclopentyl
Cyclohexyl
Cyclohexyl
Norbornyl
Pyridinyl
128
8.2
36
1.5
3.9
2.4
4.4
2.9
4.7
3.2
5.5
4.0
6.1
4.6
3.9
2.4
5.6
4.1
5.1
3.6
6.8
5.3
Ethyl
Ethyl
Benzyl
Pyridinyl
Benzyl
Ethyl
4.0
23
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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47
48
49
50
51
52
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57
58
59
60
Ethyl
Pyridinyl
Benzyl
Ethyl
3.0
9.4
2.4
22
Ethyl
Norbornyl
Pyridinyl
Benzyl
53
54
55
56
57
58
59
60
61
62
63
64
Ethyl
Cyclooctyl
Cyclooctyl
2ꢀAdamantyl
2ꢀAdamantyl
Cyclohexyl
Cyclohexyl
2ꢀAdamantyl
2ꢀAdamantyl
Cyclohexyl
Cyclohexyl
2ꢀAdamantyl
2ꢀAdamantyl
Ethyl
Pyridinyl
Benzyl
Ethyl
3.5
3.9
3.7
15
Ethyl
Pyridinyl
Benzyl
Methyl
Methyl
Methyl
Methyl
tertꢀButyl
tertꢀButyl
tertꢀButyl
tertꢀButyl
Pyridinyl
Benzyl
4.9
6.4
6.2
11
Pyridinyl
Benzyl
Pyridinyl
Benzyl
3.4
5.2
Pyridinyl
Table 1. Antiꢀferroptotic activity of synthesized ferꢀ1 analogs library in response to erastinꢀinduced ferroptosis
in IMRꢀ32 neuroblastoma cells. In this research IMRꢀ32 neuroblastoma cells were used instead of HTꢀ1080 cells
because IMRꢀ32 neuroblastoma were found to be more sensitive for erastinꢀinduced ferroptosis.
a
Calculated LogP using PerkinElmer Informatics ChemBioDraw software Version 14.0
Based on the antiꢀferroptotic activities of the synthesized ferꢀ1 analogues, we established a structureꢀ
activity relationship (SAR).
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1
1
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
The presence of a carboxylic acid (9) is clearly unfavourable, since it decreases activity 10ꢀfold. This
demonstrates that ester hydrolysis leads to a significant decrease in potency. Comparing ferꢀ1 to its
corresponding amide and sulfonamide analogue, 15 and 37 respectively, it can be concluded that
amide and sulfonamide moieties are tolerated in this position and are only slightly less potent than the
original ester derivate. Methyl, ethyl and tertꢀbutyl groups were coupled to the sulfonamide moiety in
R , resulting in similar IC ꢀvalues.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
9
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1
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3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
50
6
The importance of cycloalkyl groups at R
It was clear that the sulfonamide analogues containing small lipophilic substituents in R
cyclopropyl, were less active than compounds containing a large, bulky lipophilic group such as 2ꢀ
adamantyl. In general, increasing the number of carbon atoms in the cycloalkyl moiety in R increases
potency of the inhibitor as this can be viewed in 35-40 and 45-56
2
for ester analogues was already suggested by Skouta et al.
2
, such as
2
.
At the R ꢀposition, we observed that addition of a benzyl group resulted in compounds with a higher
3
potency than those containing a primary amine. Substitution with pyridinyl was also investigated in an
attempt to reduce lipophilicity of the compounds. In general it can be stated that compounds
containing a pyridinyl moiety are less potent than compounds containing a benzyl moiety (2 to 10ꢀ
2
fold) with the exception of compounds that are combined with 2ꢀadamantyl at R . This is exemplified
with the equipotent compounds pairs 19 and 20 55 and 56 59 and 60 and finally 63 and 64.
,
,
We observed a general trend between antiꢀferroptotic activity and lipophilicity (Figure 4). Compounds
with a high cLogP are more likely to display a low IC50ꢀvalue. This supports the earlier stated
hypothesis that the potency of these inhibitors is correlated with their ability to interact with cellular
lipid structures. Further increase in cLogP above 4 only yields marginal improvements to the IC50. We
thus propose that compounds with single digit nanomolar activity and a cLogP around 4 represent an
optimal balance between potency and lipophilicity. These observations will prove useful in the
development of compounds with good drugꢀlike properties, suitable for use in in vivo disease models.
In conclusion, we were able to generate amide and sulfonamide analogues of the ferrostatin esters with
single digit nanomolar antiꢀferroptotic activity.
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49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Comparison between IC50ꢀvalues and cLogP of all ethylsulfonamide analogues.
ADME assays
Five of the most potent amide and sulfonamide analogues were compared with ferꢀ1 for their in vitro
ADME properties. We determined kinetic solubility at pH 7.4, microsomal (human and mouse) and
plasma (human and mouse) stability (Table 2) (supplementary information S4ꢀ8). The original ester
(ferꢀ1) was rapidly metabolised by human and mouse microsomes with halfꢀlife’s of approximately 7
and 2 minutes respectively. As anticipated, ferꢀ1 was also extremely unstable in mouse plasma, with
complete degradation after 30 minutes. Surprisingly, ferꢀ1 was not degraded in human plasma after 6
hours. This initial result was confirmed in different plasma samples containing different anticoagulants
(
supplementary information S8ꢀ9) and by a third party (supplementary information S9). These data
demonstrate that ferꢀ1 is not a suitable tool compound to demonstrate the potential of antiꢀferroptotic
activity in mouse disease models.
Compared to ferꢀ1, a significant increase in microsomal and plasma stability of the amide and
sulfonamide analogues was observed. The amide analogue 20 showed a halfꢀlife of about 16 minutes
in human and around 30 min in mouse microsomes. The corresponding sulfonamide analogue 56
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1
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2
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2
2
2
2
2
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3
3
3
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5
5
5
5
5
5
5
5
5
6
showed a slower metabolism by human microsomes (4ꢀfold). The most stable compound of this subset
is the sulfonamide analogue 49, displaying a halfꢀlife of about 90 minutes in human and over 180
minutes in mouse microsomes, with complete stability in human and mouse plasma for up to 6 hours.
As we anticipated, the presence of lipophilic groups at the R
kinetic solubility in comparison to the original ferꢀ1.
1 2
and R positions caused a decrease in
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
In conclusion combining the results from both IC ꢀdetermination and ADME assays, a general SAR
50
can be reported and is shown in figure 5.
Plasma stability
% recovery
Microsomal stability
a
Solubility
b
Compound
Halfꢀlife (t1/2) (min)
c
(µM)
after 6h
Human
6.9 ± 0.2
16 ± 2
Mouse
1.9 ± 0 .6
30 ± 3
Human
100
Mouse
0
1 (Ferꢀ1)
> 200
25ꢀ50
20
37
49
55
56
100
100
86.2
98.6
99.4
100
12.5ꢀ25
12.5ꢀ25
12.5ꢀ25
12.5ꢀ25
37 ± 3
31 ± 5
81.9
100
88 ± 16
36 ± 11
64 ± 10
187 ± 41
31 ± 6
100
26 ± 5
100
Table 2: in vitro ADME of selected compounds
a
Final test compound concentration range of between 3.125 µM and 200 µM [4 µM DMSO solution in 196 µM
buffer solution (10 mM PBS pH 7.4)]
b
Metabolism by microsomes (CYP450 and other NADPH ꢀdependent enzymes) was monitored and expressed as
halfꢀlife (min)
c
Percentage of remaining parent compound
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49
50
51
52
53
54
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58
59
60
Figure 5. General structureꢀactivity relationship (SAR) from combining antiꢀferroptotic activity and the results
from the ADME assays.
CONCLUSION
In this paper we demonstrate that ferrostatinꢀ1, a frequently used compound to inhibit ferroptosis, is
not suitable for use in disease mouse models, because of its very low metabolic stability. For the first
time, we report on potent ferroptosis inhibitors in which the labile ester is replaced with amide and
sulfonamide moieties. Investigation of the SAR around 3 positions of this scaffold afforded single
digit nanomolar ferroptosis inhibitors with improved ADME properties. Analogues of ferꢀ1 containing
a sulfonamide show a significant increase in microsomal and plasma stability. Compound 49 inhibits
erastinꢀinduced ferroptotic cell death with an IC50 of 4 nM and has a halfꢀlife of 3 hours upon
incubation with mouse microsomes and is completely stable in mouse plasma up to 6 hours. These
excellent properties will allow the use of this compound in animal models to demonstrate the
therapeutic potential of ferroptosis inhibition. Further optimization of this compound series towards
improved solubility is ongoing and will be reported in due course.
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6
EXPERIMENTAL SECTION
Unless otherwise stated, laboratory reagent grade solvents were used. Reagents were obtained from
SigmaꢀAldrich, Acros Organics or Fluorochem and were used without further purification.
1
13
1
13
Characterization of all compounds was done with H and C NMR and mass spectrometry. H and C
NMR spectra were recorded on a 400 MHz Bruker Avance III Nanobay spectrometer with Ultrashield
and analysed by use of MestReNova analytical chemistry software. Chemical shifts are in ppm, and
coupling constants are in hertz (Hz). ES mass spectra were obtained from an Esquire 3000plus ion trap
mass spectrometer from Bruker Daltonics. Purities were determined with two diverse HPLC systems
based either on mass determination or on UV detection. A Waters acquity UPLC system coupled to a
Waters TQD ESI mass spectrometer (HPLC System A) or a Waters SQD ESI mass spectrometer
(HPLC system B) was used both in combination with a Waters TUV detector. The same methods on
both HPLC system A and B was used for compound detection and purity determination. Water (A)
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
3
and CH CN (B) were used as eluents. Waters Acquity UPLC BEH C18 1.7 ꢁm, 2.1 mm × 50 mm
column was used. Solvent A consisted of water with 0.1% formic acid. Solvent B consisted of
acetonitrile with 0.1% formic acid. Method I involved the following: 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, 0.25
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
min 95% A, 5% B. The wavelength for UV detection was 214 nm. Where necessary, flash purification
was performed on a Biotage ISOLERA One flash system equipped with an internal variable
dualwavelength 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
making use of KPꢀC18 containing cartridges. Dry sample loading was done by selfꢀpacking samplet
cartridges using silica and Celite 545, respectively, for normal and reversed phase purifications.
Gradients used varied for each purification. Mouse and rat plasma came from Innovative Research.
Liver microsomes were obtained from Corning B.V. Life Sciences. The turbidity in the kinetic
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solubility experiments was measured using the UV/vis spectrophotometer Synergy MX, Biotek with
1
4
Gen5.
The following section comprises the synthetic procedures and analytical data for all compounds
reported in this manuscript. Several synthesis 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.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
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59
60
General procedure A
To a solution of ethylamine HClꢀsalt (1 equiv.) and triethylamine (2 equiv.) in DCM was added 4ꢀ
chloroꢀ3ꢀnitrobenzoyl chloride 11 (1 equiv.) under an argon atmosphere. The resulting mixture was
washed with water and purified by flashꢀcolumn chromatography on silica gel (10% methanol in
DCM) to provide the desired 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzamide analog 12.
General procedure B
A solution of 4ꢀchloroꢀ3ꢀnitrobenzeneꢀ1ꢀsulfonyl chloride 21 (1 equiv.) in THF was cooled down to ꢀ
40°C before being treated with the appropriate amine analog (1 equiv.) followed by triethylamine (2
equiv.). The reaction mixture was stirred and allowed to reach room temperature over 1h. The reaction
mixture was diluted with ethyl acetate, washed twice with brine and dried. The residue was purified by
flashꢀcolumn chromatography on silica gel (20ꢀ40% ethyl acetate in heptane) to provide the desired
sulfonamide analogues 22-24.
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
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5
5
6
General procedure C
To the appropriate 4ꢀchloroꢀ3ꢀnitrobenzoic acid 5 (1 equiv.), methyl 4ꢀchloroꢀ3ꢀnitrobenzoate 6 (1
equiv.), 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzamide 12 (1 equiv.) or 4ꢀchloroꢀ3ꢀnitrobenzenesulfonamide
2 3
intermediate 22-24 (1 equiv.) in dry DMSO was added K CO (2ꢀ4 equiv.) and the appropriate
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
cycloalkylamine analog (1.2 equiv.). The mixture was stirred for 17h at 60°C. The solution was
poured in water, extracted with 3x ethyl acetate and dried with MgSO . The solvent was removed
4
under reduced pressure. The residue was purified by flashꢀcolumn chromatography on silica gel to
respectively provide the desired 4ꢀ(cyclohexyl)ꢀ3ꢀnitrobenzoic acid 7, methyl 4ꢀ(cyclohexyl)ꢀ3ꢀ
nitrobenzoate 8, 4ꢀ(cycloalkylamino)ꢀ Nꢀethylꢀ3ꢀnitrobenzamide analogues 13-14 and 4ꢀ
(
cycloalkylamino)ꢀ3ꢀnitrobenzenesulfonamide analogues 25-34.
General procedure D
The appropriate 4ꢀ(cyclohexyl)ꢀ3ꢀnitrobenzoic acid intermediate 7 (1 equiv.), methyl 4ꢀ
(
cyclohexylamino)ꢀ3ꢀnitrobenzoate intermediate
nitrobenzamide intermediate 13-14 (1 equiv.) or 4ꢀ(cycloalkylamino)ꢀ3ꢀnitrobenzenesulfonamide
intermediate 25-34 (1 equiv.) was dissolved in methanol, flushed with argon and hydrogenated (H
8
(1 equiv.), 4ꢀ(cycloalkylamino)ꢀNꢀethylꢀ3ꢀ
2
gas) over 10% Palladium hydroxide (1.5 equiv.) for 17 h at room temperature. The solution was
filtered through a pad of celite and volatiles were removed under reduced pressure. If necessary, the
residue was purified by flashꢀcolumn chromatography on silica gel to respectively provide the desired
3ꢀaminoꢀ4ꢀ(cyclohexylamino)benzoic acid 9, methyl 3ꢀaminoꢀ4ꢀ(cyclohexylamino)benzoate 10, 4ꢀ
(
cycloalkylamino)ꢀ 3ꢀaminobenzamide analogues 15-16 or 4ꢀ(cycloalkylamino)ꢀ 3ꢀ
aminobenzenesulfonamide analog 35-44.
General procedure E
To the 4ꢀ(cycloalkylamino)ꢀ 3ꢀaminobenzamide intermediate 15-16 (1 equiv.) or 4ꢀ(cycloalkylamino)ꢀ
3ꢀaminobenzenesulfonamide intermediate 35-44 (1 equiv.) in DMF was added the appropriate benzyl
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9
2 3
or pyridinylderivate (1ꢀ2 equiv.) and K CO (2ꢀ4 equiv.). The mixture was stirred at 60°C for 1ꢀ4
hours then poured in water. The organic layer was extracted with 3x ethyl acetate and the solvent was
removed under reduced pressure. The residue was purified by flashꢀcolumn chromatography on silica
gel to respectively provide target amide 17-20 and sulfonamide compounds 45-64.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
35
36
37
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45
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49
50
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4
-(cyclohexyl)-3-nitrobenzoic acid (7)
Following general procedure C, using 4ꢀchloroꢀ3ꢀnitrobenzoic acid 5 (2 g, 9.92 mmol) and
cyclohexylamine (1.362 ml, 11.91 mmol). The reaction was purified by flashꢀcolumn chromatography
on silica gel (5% methanol in DCM) to afford 4ꢀ(cyclohexyl)ꢀ3ꢀnitrobenzoic acid (1.4 g, 5.46 mmol).
(
Yield: 55%)
1
6
H NMR (400 MHz, DMSO-d ) δ 1.18 ꢀ 1.32 (m, 1H), 1.34 ꢀ 1.49 (m, 4H), 1.55 ꢀ 1.66 (m, 1H), 1.65
ꢀ
1.75 (m, 2H), 1.90 ꢀ 2.03 (m, 2H), 3.69 (td, J = 4.06, 8.73, 9.56 Hz, 1H), 7.18 (d, J = 9.24 Hz, 1H),
.94 (ddd, J = 0.68, 2.14, 8.99 Hz, 1H), 8.26 (d, J = 7.70 Hz, 1H), 8.60 (d, J = 2.09 Hz, 1H), 12.93 (s,
7
1
1
13
6
H). C NMR (101 MHz, DMSO-d ) δ 24.48, 25.44, 32.26, 51.07, 115.39, 117.50, 129.08, 130.64,
36.54, 146.82, 166.34.
MS (ESI) m/z 265 [M +H] (HPLC System B)
Methyl 4-(cyclohexylamino)-3-nitrobenzoate (8)
Following general procedure C, using methyl 4ꢀchloroꢀ3ꢀnitrobenzoate 6 (1.5 g, 6.96 mmol) and
cyclohexylamine (0.955 ml, 8.35 mmol). The reaction was purified by flashꢀcolumn chromatography
on silica gel (5% methanol in DCM) to afford methyl 4ꢀ(cyclohexylamino)ꢀ3ꢀnitrobenzoate (1.7 g,
6.11 mmol). (Yield: 88%).
1
H NMR (400 MHz, DMSO-d6) δ 1.19 ꢀ 1.29 (m, 1H), 1.33 ꢀ 1.50 (m, 4H), 1.54 ꢀ 1.65 (m, 1H), 1.68
ꢀ
1.76 (m, 2H), 1.88 ꢀ 2.02 (m, 2H), 3.69 (dt, J = 5.41, 9.32 Hz, 1H), 3.83 (s, 3H), 7.19 (d, J = 9.21 Hz,
13
1H), 7.94 (ddd, J = 0.72, 2.18, 9.14 Hz, 1H), 8.27 (d, J = 7.72 Hz, 1H), 8.60 (d, J = 2.12 Hz, 1H). C
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2
2
2
2
2
2
2
2
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4
4
4
4
4
4
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5
5
5
5
5
6
NMR (101 MHz, DMSO-d
6
) δ 24.48, 25.42, 32.23, 51.12, 52.48, 115.56, 116.22, 129.02, 130.68,
136.14, 146.95, 165.23.
MS (ESI) m/z 279 [M +H] (HPLC System A)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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5
6
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8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3-amino-4-(cyclohexylamino)benzoic acid (9)
Following general procedure D, using 4ꢀ(cyclohexylamino)ꢀ3ꢀnitrobenzoic acid 7 (0.500 g, 1.892
mmol) to afford the desired 3ꢀaminoꢀ4ꢀ(cyclohexylamino)benzoic acid (0.180 g, 0.768 mmol) as an
amorphous powder. (Yield: 41%)
1
H NMR (400 MHz, MeOD) δ 1.16 ꢀ 1.29 (m, 3H), 1.46 ꢀ 1.32 (m, 2H), 1.60 ꢀ 1.70 (m, 1H), 1.70 ꢀ
1
2
5
.84 (m, 2H), 2.00 ꢀ 2.09 (m, 2H), 3.25 ꢀ 3.39 (m, 1H), 6.56 (dd, J = 0.68, 8.44 Hz, 1H), 7.39 (d, J =
13
.03 Hz, 1H), 7.47 (dd, J = 2.04, 8.40 Hz, 1H). C NMR (101 MHz, MeOD) δ 24.82, 25.66, 32.75,
1.23, 108.79, 117.30, 117.48, 123.55, 132.14, 141.37, 169.94.
Remark: amine protons were exchanged with solvent.
R
t 1.86 min, MS (ESI) m/z 235 [M +H] (100%) (HPLC System A)
Methyl 3-amino-4-(cyclohexylamino)benzoate (10)
Following general procedure D, using methyl 4ꢀ(cyclohexylamino)ꢀ3ꢀnitrobenzoate 8 (1.7 g, 6,11
mmol) to afford the desired methyl 3ꢀaminoꢀ4ꢀ(cyclohexylamino)benzoate (0.7 g, 2.82 mmol) as an
amorphous powder. (Yield: 46%)
1
H NMR (400 MHz, DMSO-d
6
) δ 1.15 ꢀ 1.26 (m, 3H), 1.31 ꢀ 1.44 (m, 2H), 1.57 ꢀ 1.67 (m, 1H), 1.70
ꢀ
1.79 (m, 2H), 1.90 ꢀ 2.01 (m, 2H), 3.25 ꢀ 3.33 (m, 1H), 3.72 (s, 3H), 4.76 (s, 2H), 4.92 (d, J = 7.48
13
Hz, 1H), 6.46 (d, J = 8.34 Hz, 1H), 7.15 ꢀ 7.22 (m, 2H). C NMR (101 MHz, MeOD) δ 24.85, 25.70,
32.75, 50.73, 51.17, 108.76, 116.81, 117.04, 123.19, 132.54, 141.43, 168.34.
t
R
1.79 min, MS (ESI) m/z 249 [M +H] (100%) (HPLC System B)
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4-chloro-N-ethyl-3-nitrobenzamide (12)
Following general procedure B and ethylamine hydrochloride as the corresponding amine, the
reaction was purified by flashꢀcolumn chromatography on silica gel (10% methanol in DCM) to afford
the desired afford 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzamide (3 g, 13.12 mmol). (Yield: 58%)
10
11
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54
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56
57
58
59
60
1
H NMR (400 MHz, DMSO-d
8.40 Hz, 1H), 8.15 (dd, J = 2.13, 8.43 Hz, 1H), 8.50 (d, J = 2.11 Hz, 1H), 8.85 (s, 1H).
MS (ESI) m/z 229 [M +H] (HPLC System A)
6
) δ 1.14 (t, J = 7.23 Hz, 3H), 3.30 (td, J = 5.43, 7.24 Hz, 2H), 7.89 (d, J
=
4
-(cyclohexylamino)-N-ethyl-3-nitrobenzamide (13)
Following general procedure C, using 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzamide 12 (2 g, 8.75 mmol) and
cyclohexylamine (1.201 ml, 10.50 mmol). The reaction was purified by flashꢀcolumn chromatography
on silica gel (50% ethyl acetate in heptane) to afford the desired 4ꢀ(cyclohexyamino)ꢀNꢀethylꢀ3ꢀ
nitrobenzamide (1g, 3.43 mmol). (Yield: 78%)
1
3
H NMR (400 MHz, CDCl ) δ 1.27 (t, J = 7.26 Hz, 3H), 1.30 ꢀ 1.52 (m, 6H), 1.70 (ddd, J = 3.04,
5.44, 13.10 Hz, 1H), 1.79 ꢀ 1.88 (m, 2H), 1.99 ꢀ 2.13 (m, 2H), 3.50 (qd, J = 5.36, 7.28 Hz, 2H), 6.26 (s,
1H), 6.92 (d, J = 9.12 Hz, 1H), 7.96 (ddd, J = 0.67, 2.28, 8.99 Hz, 1H), 8.35 (d, J = 7.34 Hz, 1H), 8.55
13
3
(d, J = 2.23 Hz, 1H). C NMR (101 MHz, CDCl ) δ 8.63, 14.93, 24.47, 25.47, 32.61, 35.00, 45.84,
51.30, 76.73, 77.05, 77.25, 77.37, 114.37, 121.07, 125.46, 130.40, 135.10, 146.20, 165.43.
MS (ESI) m/z 292 [M +H] (HPLC System A)
4
-(adamantan-2-ylamino)-N-ethyl-3-nitrobenzamide (14)
Following general procedure C, using 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzamide 12 (2.05 g, 8.97 mmol)
and 2ꢀadamantylamine hydrochloride (3.37 g, 17.93 mmol). The reaction was purified by flashꢀ
column chromatography on silica gel (8% methanol in DCM) to afford the desired 4ꢀ(adamantanꢀ2ꢀ
ylamino)ꢀNꢀethylꢀ3ꢀnitrobenzamide (2.9 g, 8.44 mmol). (Yield: 94%).
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1
H NMR (400 MHz, CDCl
3
) δ 1.25 (td, J = 4.21, 7.26 Hz, 3H), 2.11 ꢀ 1.65 (m, 15H), 3.40 ꢀ 3.56 (m,
2H), 3.82 (dt, J = 2.96, 6.86 Hz, 1H), 6.62 (t, J = 5.54 Hz, 1H), 6.85 (d, J = 9.13 Hz, 1H), 8.57 (d, J =
13
2.23 Hz, 1H), 8.84 (d, J = 7.45 Hz, 1H). C NMR (101 MHz, CDCl ) δ 14.89, 27.00, 31.57, 35.00,
3
36.99, 37.33, 56.68, 114.37, 121.10, 125.69, 130.49, 135.02, 146.15, 165.56,
MS (ESI) m/z 344 [M +H] (HPLC System A)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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0
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8
9
0
3
-amino-4-(cyclohexylamino)-N-ethylbenzamide (15)
Following general procedure D, using 4ꢀ(cyclohexylamino)ꢀNꢀethylꢀ3ꢀnitrobenzamide 13 (1.6 g,
5.49 mmol) to afford the desired 3ꢀaminoꢀ4ꢀ(cyclohexylamino)ꢀNꢀethylbenzamide (0.600 g, 2.296
mmol) as an amorphous powder. (Yield: 42%)
1
H NMR (400 MHz, CDCl
3
)
δ 1.25 (td, J = 4.21, 7.26 Hz, 3H), 1.65 ꢀ 2.11 (m, 15H), 3.40 ꢀ 3.56 (m,
2
H), 3.82 (dt, J = 2.96, 6.86 Hz, 1H), 6.62 (t, J = 5.54 Hz, 1H), 6.85 (d, J = 9.13 Hz, 1H), 8.57 (d, J =
1
3
2.23 Hz, 1H), 8.84 (d, J = 7.45 Hz, 1H). C NMR (101 MHz, CDCl
3
) δ 14.89, 27.00, 31.57, 35.00, 36.99,
3
7.33, 56.68, 114.37, 121.10, 125.69, 130.49, 135.02, 146.15, 165.56.
t
R
1.66 min, MS (ESI) m/z 262 [M +H] (100%) (HPLC System A)
4
-(adamantan-2-ylamino)-3-amino-N-ethylbenzamide (16)
Following general procedure D, using 4ꢀ(adamantanꢀ2ꢀylamino)ꢀNꢀethylꢀ3ꢀnitrobenzamide 14 (2.9 g,
.44 mmol) to afford the desired 4ꢀ(adamantanꢀ2ꢀylamino)ꢀ3ꢀaminoꢀNꢀethylbenzamide (1.9 g, 6.06
8
mmol) as an amorphous powder. (Yield: 72%)
1
3
H NMR (400 MHz, CDCl ) δ 1.17 ꢀ 1.30 (m, 3H), 1.55 ꢀ 2.13 (m, 16H), 3.31 (br.s, 1H), 3.41 ꢀ 3.52
(m, 2H), 3.61 (t, J = 2.84 Hz, 1H), 6.02 (s, 1H), 6.52 ꢀ 6.59 (m, 1H), 7.18 ꢀ 7.24 (m, 1H), 7.25 ꢀ 7.30
1
3
(m, 1H). C NMR (101 MHz, CDCl ) δ 15.09, 27.31, 31.65, 34.70, 37.31, 37.65, 56.48, 109.82,
3
116.53, 120.12, 123.01, 132.75, 140.61, 167.52.
t
R
1.89 min, MS (ESI) m/z 314 [M +H] (95%) (HPLC System A)
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-(benzylamino)-4-(cyclohexylamino)-N-ethylbenzamide (17)
Following general procedure E, using 3ꢀaminoꢀ4ꢀ(cyclohexylamino)ꢀNꢀethylbenzamide 15 (0.150 g,
.574 mmol) and benzyl bromide (0.069 ml, 0.574 mmol). The residue was purified by flashꢀcolumn
chromatography (0 to 100% methanol in water) on Cꢀ18 to afford the 3ꢀ(benzylamino)ꢀ4ꢀ
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13
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15
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59
60
0
(
cyclohexylamino)ꢀNꢀethylbenzamide (0.0026 g, 7,40 µmol) as an amorphous powder. (Yield: 1.3%).
1
H NMR (400 MHz, CDCl
3
) δ 1.20 ꢀ 1.29 (m, 4H), 1.35 ꢀ 1.48 (m, 2H), 1.59 (s, 3H), 1.66 ꢀ 1.74 (m,
1
5
1
H), 1.80 (dt, J = 3.83, 13.31 Hz, 2H), 2.08 (d, J = 12.50 Hz, 2H), 3.26 – 3.37 (m, 1H), 3.49 (qd, J =
.52, 7.21 Hz, 3H), 4.34 (s, 2H), 5.92 (s, 1H), 6.65 (d, J = 8.24 Hz, 1H), 7.21 (dd, J = 2.05, 8.24 Hz,
H), 7.27 (d, J = 2.00 Hz, 1H), 7.31 – 7.47 (m, 5H).
MS (ESI) m/z 352 [M +H] (95%) (HPLC System A)
4-(cyclohexylamino)-N-ethyl-3-((pyridin-4-ylmethyl)amino)benzamide (18)
Following general procedure E, using 3ꢀaminoꢀ4ꢀ(cyclohexylamino)ꢀNꢀethylbenzamide 15 (0.150 g,
0.574 mmol) and 4ꢀ(bromomethyl)pyridine hydrobromide (0.128 g, 0.746 mmol). The residue was
purified by preparative HPLC to afford the 4ꢀ(cyclohexylamino)ꢀNꢀethylꢀ3ꢀ((pyridinꢀ4ꢀ
ylmethyl)amino)benzamide (0.0204 g, 0.058 mmol) as an amorphous powder. (Yield: 10%)
1
3
H NMR (400 MHz, CDCl ) δ 1.19 (t, J = 7.26 Hz, 3H), 1.22 ꢀ 1.32 (m, 3H), 1.34 ꢀ 1.48 (m, 2H),
1
.65 ꢀ 1.74 (m, 1H), 1.75 ꢀ 1.85 (m, 2H), 2.03 ꢀ 2.13 (m, 2H), 3.28 ꢀ 3.37 (m, 1H), 3.42 (qd, J = 5.46,
.25 Hz, 2H), 4.36 (s, 2H), 6.16 (t, J = 5.54 Hz, 1H), 6.30 (s, 1H), 6.64 (d, J = 8.27 Hz, 1H), 7.13 (d, J
7
=
2.03 Hz, 1H), 7.18 (dd, J = 1.99, 8.23 Hz, 1H), 7.33 ꢀ 7.37 (m, 2H), 8.24 (s, 1H), 8.52 ꢀ 8.54 (m,
1
3
2H). C NMR (101 MHz, CDCl
3
) δ 14.96, 24.96, 25.88, 33.34, 34.82, 47.51, 51.66, 110.30, 112.34,
119.27, 122.98, 123.49, 134.82, 140.13, 148.31, 150.24, 167.92.
t
R
1.38 min, MS (ESI) m/z 353 [M +H] (100%) (HPLC System A)
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4-(adamantan-2-ylamino)-3-(benzylamino)-N-ethylbenzamide (19)
Following general procedure E, using 4ꢀ(adamantanꢀ2ꢀylamino)ꢀ3ꢀaminoꢀNꢀethylbenzamide 16
(
0.200 g, 0.638 mmol) and benzyl bromide (0.076 ml, 0.638 mmol). The residue was purified by
preparative HPLC to afford the 4ꢀ(adamantanꢀ2ꢀylamino)ꢀ3ꢀ(benzylamino)ꢀNꢀethylbenzamide (0.011
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
g, 0.027 mmol) as an amorphous powder. (Yield: 4%)
1
H NMR (400 MHz, CDCl ) δ 1.24 (t, J = 7.26 Hz, 3H), 1.47 ꢀ 1.52 (m, 2H), 1.59 ꢀ 1.66 (m, 2H),
3
1
4
ꢀ
.78 ꢀ 1.95 (m, 10H), 2.05 ꢀ 2.09 (m, 2H), 3.47 (qd, J = 5.53, 7.26 Hz, 2H), 3.62 (t, J = 2.76 Hz, 1H),
.36 (s, 2H), 5.95 (t, J = 5.25 Hz, 1H), 6.60 (d, J = 8.26 Hz, 1H), 7.23 (dd, J = 2.04, 8.23 Hz, 1H), 7.28
13
7.46 (m, 6H). C NMR (101 MHz, CDCl ) δ 15.04, 27.30, 27.95, 31.65, 34.78, 37.30, 37.64, 49.42,
3
56.61, 109.89, 113.34, 119.76, 123.32, 127.41, 127.87, 128.69, 135.41, 139.21, 140.75, 167.99.
t
R
2.33 min, MS (ESI) m/z 404 [M +H] (100%) (HPLC System A)
4-(adamantan-2-ylamino)-N-ethyl-3-((pyridin-4-ylmethyl)amino)benzamide (20)
Following general procedure E, using 4ꢀ(adamantanꢀ2ꢀylamino)ꢀ3ꢀaminoꢀNꢀethylbenzamide 16
(
0.150 g, 0.479 mmol) and 4ꢀ(bromomethyl)pyridine hydrobromide (0.182 g, 0.718 mmol). The
residue was purified by by flashꢀcolumn chromatography on silica gel (20% methanol in ethyl acetate)
to afford the 4ꢀ(adamantanꢀ2ꢀylamino)ꢀNꢀethylꢀ3ꢀ((pyridinꢀ4ꢀylmethyl)amino)benzamide (0.049 g,
0.121 mmol) as an amorphous powder. (Yield: 25%)
1
3
H NMR (400 MHz, CDCl ) δ 1.18 (t, J = 7.25 Hz, 3H), 1.57 ꢀ 2.09 (m, 15H), 3.42 (qd, J = 5.51, 7.22
Hz, 2H), 3.61 (s, 1H), 4.24 (s, 1H), 4.30 ꢀ 4.39 (m, 2H), 6.09 ꢀ 6.18 (m, 1H), 6.54 ꢀ 6.62 (m, 1H), 7.16
13
ꢀ
7.25 (m, 2H), 7.26 ꢀ 7.32 (m, 2H), 8.51 ꢀ 8.58 (m, 2H). C NMR (101 MHz, CDCl
3
) δ 15.03,
2
7.29, 31.68, 34.72, 37.29, 47.82, 56.59, 110.02, 113.33, 119.74, 122.43, 123.47, 134.85, 140.63,
48.56, 149.96, 167.63.
1
t
R
1.62 min, MS (ESI) m/z 405 [M +H] (100%) (HPLC System A)
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4-chloro-N-methyl-3-nitrobenzenesulfonamide (22)
Following general procedure B and methylamine hydrochloride as the corresponding amine, the
reaction was purified by flashꢀcolumn chromatography on silica gel (50% ethyl acetate in heptane) to
afford the desired afford 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide (2.98 g, 11.99 mmol). (Yield:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
1%)
1
H NMR (400 MHz, DMSO-d6) δ 2.50 (s, 4H), 7.86 (s, 1H), 8.03 ꢀ 8.05 (m, 2H), 8.42 (dd, J = 0.89,
13
1.64 Hz, 1H). C NMR (101 MHz, DMSO-d
6
) δ 29.10, 124.55, 129.82, 131.95, 133.60, 140.05,
147.94.
MS (ESI) m/z 499 [M+] (HPLC System A)
4-chloro-N-ethyl-3-nitrobenzenesulfonamide (23)
Following general procedure B and ethylamine hydrochloride as the corresponding amine, the
reaction was purified by flashꢀcolumn chromatography on silica gel (10% methanol in DCM) to afford
the desired afford 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide (3.5 g, 13.22 mmol). (Yield: 72%)
1
3
H NMR (400 MHz, CDCl ) δ 1.19 (t, J = 7.24 Hz, 3H), 3.12 (qd, J = 5.71, 7.23 Hz, 2H), 4.71 (s,
1H), 7.75 (dd, J = 0.37, 8.39 Hz, 1H), 8.02 (dd, J = 2.16, 8.43 Hz, 1H), 8.38 (dd, J = 0.55, 2.02 Hz,
13
1H). C NMR (101 MHz, CDCl
3
) δ 15.20, 38.51, 124.40, 131.07, 131.55, 133.01, 140.53, 147.91.
MS (ESI) m/z 265 [M +H] (HPLC System A)
4-chloro-N-(tert-butyl)-3-nitrobenzenesulfonamide (24)
Following general procedure B and tertꢀbutylamine as the corresponding amine, the reaction was
purified by flashꢀcolumn chromatography on silica gel (50% ethyl acetate in heptane) to afford the
desired afford 4ꢀchloroꢀNꢀ(tertꢀbutyl)ꢀ3ꢀnitrobenzenesulfonamide (2.1 g, 7.17 mmol). (Yield: 92%)
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2
2
2
2
2
2
3
3
3
3
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3
3
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3
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4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
1
H NMR (400 MHz, DMSO-d
6
) δ 1.14 (s, 9H), 7.93 (s, 1H), 8.00 (d, J = 8.47 Hz, 1H), 8.10 (dd, J =
1
3
2.19, 8.45 Hz, 1H), 8.48 (d, J = 2.15 Hz, 1H). C NMR (101 MHz, DMSO-d
6
) δ 30.18, 54.45,
124.12, 129.19, 131.52, 133.47, 144.96, 147.78.
MS (ESI) m/z 291 [M+] (HPLC System A)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
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0
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4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
-(cyclopropylamino)-N-ethyl-3-nitrobenzenesulfonamide (25)
Following general procedure C, using 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide 23 (0.500 g,
1.889 mmol) and cyclopropylamine (0.159 ml, 2.267 mmol). The reaction was purified by flashꢀ
column chromatography on silica gel (35% ethyl acetate in heptane) to afford the desired 4ꢀ
(
cyclopropylamino)ꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide (0.437 g, 1.532 mmol). (Yield: 81%).
1
H NMR (400 MHz, DMSO-d
6
) δ 0.67 ꢀ 0.72 (m, 2H), 0.89 ꢀ 0.94 (m, 2H), 0.99 (t, J = 7.21 Hz, 3H),
2
9
1
.69 ꢀ 2.74 (m, 1H), 2.79 (td, J = 5.67, 7.23 Hz, 2H), 7.53 ꢀ 7.58 (m, 2H), 7.89 (ddd, J = 0.68, 2.29,
1
3
6
.09 Hz, 1H), 8.37 ꢀ 8.39 (m, 1H), 8.42 (d, J = 2.22 Hz, 1H). C NMR (101 MHz, DMSO-d ) δ 8.01,
5.14, 25.43, 37.98, 117.22, 125.95, 127.50, 130.70, 133.62, 148.14.
MS (ESI) m/z 286 [M +H] (HPLC System A)
4
-(cyclopentylamino)-N-ethyl-3-nitrobenzenesulfonamide (26):
Following general procedure C, using 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide 23 (1 g, 3.78
mmol) and cyclopentylamine (0.449 ml, 4.53 mmol). The reaction was purified by flashꢀcolumn
chromatography on silica gel (70% ethyl acetate in heptane) to afford the desired 4ꢀ
(
cyclopentylamino)ꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide (1.003 g, 3.20 mmol). (Yield: 85%).
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H NMR (400 MHz, DMSO-d
6
) δ 0.99 (t, J = 7.21 Hz, 3H), 1.54 ꢀ 1.66 (m, 4H), 1.70 ꢀ 1.78 (m, 2H),
.05 ꢀ 2.15 (m, 2H), 2.77 (q, J = 7.22 Hz, 2H), 4.12 (p, J = 6.04, 6.52 Hz, 1H), 7.29 (d, J = 9.19 Hz,
H), 7.54 (s, 1H), 7.81 (ddd, J = 0.67, 2.33, 9.19 Hz, 1H), 8.27 (d, J = 6.73 Hz, 1H), 8.43 (d, J = 2.24
2
1
1
3
Hz, 1H). C NMR (101 MHz, DMSO-d
6
) δ 15.13, 24.05, 33.07, 37.97, 54.42, 116.77, 126.34,
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26.68, 130.27, 133.75, 146.77.
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MS (ESI) m/z 314 [M +H] (HPLC System A)
4-(cyclohexylamino)-N-ethyl-3-nitrobenzenesulfonamide (27)
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Following general procedure C, using 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide 23 (1 g, 3.78
mmol) and cyclohexylamine (0.519 ml, 4.53 mmol). The reaction was purified by flashꢀcolumn
chromatography on silica gel (5% methanol in DCM) to afford the desired 4ꢀ(cyclohexylamino)ꢀNꢀ
ethylꢀ3ꢀnitrobenzenesulfonamide (0.95 g, 2.90 mmol). (Yield: 77%).
1
3
H NMR (400 MHz, CDCl ) δ 1.13 (t, J = 7.24 Hz, 3H), 1.29 ꢀ 1.52 (m, 5H), 1.63 ꢀ 1.72 (m, 1H),
1.77 ꢀ 1.88 (m, 2H), 2.00 ꢀ 2.13 (m, 2H), 3.01 (qd, J = 5.92, 7.21 Hz, 2H), 3.52 ꢀ 3.65 (m, 1H), 5.04 (t,
J = 5.98 Hz, 1H), 6.99 (d, J = 9.17 Hz, 1H), 7.83 (ddd, J = 0.74, 2.37, 9.22 Hz, 1H), 8.43 (d, J = 7.51
1
3
Hz, 1H), 8.67 (s, 1H). C NMR (101 MHz, CDCl
14.96, 125.61, 127.66, 130.43, 133.52, 146.47
MS (ESI) m/z 328 [M +H] (HPLC System A)
3
) δ 15.09, 24.40, 25.39, 32.51, 38.24, 51.52,
1
4-(bicyclo[2.2.1]heptan-2-ylamino)-N-ethyl-3-nitrobenzenesulfonamide (28)
Following general procedure C, using 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide 23 (1 g, 3.78
mmol) and bicyclo[2.2.1]heptꢀ2ꢀylamine (0.504 g, 4.53 mmol). The reaction was purified by flashꢀ
column chromatography on silica gel (50% ethyl acetate in heptane) to afford the desired 4ꢀ
(
bicyclo[2.2.1]heptanꢀ2ꢀylamino)ꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide (0.9 g, 2.65 mmol). (Yield:
70%).
1
6
H NMR (400 MHz, DMSO-d ) δ 0.99 (t, J = 7.22 Hz, 3H), 1.13 ꢀ 1.25 (m, 2H), 1.30 ꢀ 1.41 (m, 2H),
1
7
1
.44 ꢀ 1.60 (m, 3H), 1.92 (ddd, J = 2.33, 7.63, 12.85 Hz, 1H), 2.29 ꢀ 2.34 (m, 2H), 2.77 (qd, J = 5.63,
.21 Hz, 2H), 3.60 (ddd, J = 2.51, 5.54, 10.75 Hz, 1H), 7.19 (d, J = 9.19 Hz, 1H), 7.55 (t, J = 5.71 Hz,
13
H), 7.82 (ddd, J = 0.66, 2.26, 9.18 Hz, 1H), 8.13 (d, J = 6.12 Hz, 1H), 8.43 (d, J = 2.23 Hz, 1H). C
) δ 15.12, 26.17, 28.30, 35.81, 35.86, 37.96, 41.79, 56.05, 116.72, 126.34,
26.89, 130.44, 133.80, 146.17.
NMR (101 MHz, DMSO-d
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MS (ESI) m/z 340 [M +H] (HPLC System A)
4-(cyclooctylamino)-N-ethyl-3-nitrobenzenesulfonamide (29)
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Following general procedure C, using 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide 23 (1 g, 3.78
mmol) and cyclooctylamine (0.577 g, 4.53 mmol). The reaction was purified by flashꢀcolumn
chromatography on silica gel (40% ethyl acetate in heptane) to afford the desired 4ꢀ(cyclooctylamino)ꢀ
Nꢀethylꢀ3ꢀnitrobenzenesulfonamide (1.1 g, 3.09 mmol). (Yield: 82%).
1
6
H NMR (400 MHz, DMSO-d ) δ 0.99 (t, J = 7.21 Hz, 3H), 1.52 ꢀ 1.64 (m, 8H), 1.66 ꢀ 1.79 (m, 4H),
1.84 ꢀ 1.92 (m, 2H), 2.77 (qd, J = 5.59, 7.19 Hz, 2H), 3.87 ꢀ 3.94 (m, 1H), 7.22 (d, J = 9.26 Hz, 1H),
7.54 (t, J = 5.69 Hz, 1H), 7.81 (ddd, J = 0.66, 2.34, 9.11 Hz, 1H), 8.34 (d, J = 7.76 Hz, 1H), 8.43 (d, J
13
=
6
2.25 Hz, 1H). C NMR (101 MHz, DMSO-d ) δ 15.13, 23.48, 25.40, 27.05, 31.70, 37.96, 52.49,
116.54, 126.49, 126.56, 130.11, 133.90, 146.07.
MS (ESI) m/z 356 [M +H] (HPLC System A)
4-(adamantan-2-ylamino)-N-ethyl-3-nitrobenzenesulfonamide (30):
Following general procedure C, using 4ꢀchloroꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide 23 (2.380 g, 8.99
mmol) and 2ꢀadamantylamine hydrochloride (1.688 g, 8.99 mmol). The reaction was purified by flashꢀ
column chromatography on silica gel (10% methanol in DCM) to afford the desired 4ꢀ(adamantanꢀ2ꢀ
ylamino)ꢀNꢀethylꢀ3ꢀnitrobenzenesulfonamide (3.05 g, 8.04 mmol). (Yield: 89%).
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7
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H NMR (400 MHz, CDCl ) δ 1.04 (t, J = 7.23 Hz, 3H), 1.57 ꢀ 2.04 (m, 14H), 2.90 (qd, J = 5.77,
3
.20 Hz, 2H), 3.79 (dt, J = 2.89, 6.46 Hz, 1H), 5.68 (t, J = 5.91 Hz, 1H), 6.92 (d, J = 9.30 Hz, 1H),
13
.76 (ddd, J = 0.62, 2.31, 9.13 Hz, 1H), 8.58 (d, J = 2.26 Hz, 1H), 8.86 (d, J = 7.46 Hz, 1H). C NMR
(
101 MHz, CDCl ) δ 14.88, 26.96, 31.49, 36.80, 37.21, 38.16, 56.71, 115.24, 125.75, 127.34, 130.25,
3
133.61, 146.36.
MS (ESI) m/z 380 [M +H] (HPLC System A)
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-(cyclohexylamino)-N-methyl-3-nitrobenzenesulfonamide (31)
Following general procedure C, using 4ꢀchloroꢀNꢀmethylꢀ3ꢀnitrobenzenesulfonamide 22 (1 g, 3.99
mmol) and cyclohexylamine (0.548 ml, 4.79 mmol). The reaction was purified by flashꢀcolumn
chromatography on silica gel (30% ethyl acetate in heptane) to afford the desired 4ꢀ
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(
cyclohexylamino)ꢀNꢀmethylꢀ3ꢀnitrobenzenesulfonamide (0.600 g, 1.915 mmol). (Yield: 48%).
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H NMR (400 MHz, DMSO-d ) δ 1.20 ꢀ 1.32 (m, 1H), 1.37 ꢀ 1.47 (m, 4H), 1.56 ꢀ 1.66 (m, 1H), 1.68
ꢀ
(
1.76 (m, 2H), 1.92 ꢀ 2.01 (m, 2H), 2.41 (s, 3H), 3.62 ꢀ 3.80 (m, 1H), 7.33 (d, J = 9.27 Hz, 1H), 7.45
s, 1H), 7.77 (ddd, J = 0.68, 2.29, 9.15 Hz, 1H), 8.29 (d, J = 7.74 Hz, 1H), 8.42 (d, J = 2.24 Hz, 1H).
13
C NMR (101 MHz, DMSO-d
6
) δ 24.47, 25.42, 29.06, 32.22, 51.16, 116.50, 125.23, 126.78, 130.07,
133.88, 146.38.
MS (ESI) m/z 314 [M +H] (HPLC System A)
4-(adamantan-2-ylamino)-N-methyl-3-nitrobenzenesulfonamide (32)
Following general procedure C, using 4ꢀchloroꢀNꢀmethylꢀ3ꢀnitrobenzenesulfonamide 22 (1 g, 3.99
mmol) and 2ꢀadamantanylamine hydrochloride (0.749 ml, 3.99 mmol). The reaction was purified by
flashꢀcolumn chromatography on silica gel (30% ethyl acetate in heptane) to afford the desired 4ꢀ
(
adamantanꢀ2ꢀylamino)ꢀNꢀmethylꢀ3ꢀnitrobenzenesulfonamide (0.670 g, 1.833 mmol). (Yield: 46%).
1
H NMR (400 MHz, DMSO-d
6
) δ 1.63 ꢀ 1.71 (m, 2H), 1.73 ꢀ 1.77 (m, 2H), 1.83 ꢀ 1.94 (m, 8H), 2.01
ꢀ
2.07 (m, 2H), 2.41 (s, 3H), 3.96 ꢀ 4.07 (m, 1H), 7.30 (d, J = 9.27 Hz, 1H), 7.46 (s, 1H), 7.78 (dd, J =
13
2.26, 9.09 Hz, 1H), 8.44 (d, J = 2.25 Hz, 1H), 8.79 (d, J = 7.60 Hz, 1H). C NMR (101 MHz,
6
DMSO-d ) δ 26.95, 29.06, 31.39, 31.66, 36.58, 37.26, 56.00, 116.55, 125.44, 126.80, 130.28, 133.98,
146.37.
MS (ESI) m/z 329 [M +H] (HPLC System A)
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