Discovery of a Highly Potent, Selective, and Metabolically Stable Inhibitor of Receptor-Interacting Protein 1 (RIP1) for the Treatment of Systemic Inflammatory Response Syndrome

Article abstract of DOI:10.1021/acs.jmedchem.6b01196

On the basis of its essential role in driving inflammation and disease pathology, cell necrosis has gradually been verified as a promising therapeutic target for treating atherosclerosis, systemic inflammatory response syndrome (SIRS), and ischemia injury, among other diseases. Most necrosis inhibitors targeting receptor-interacting protein 1 (RIP1) still require further optimization because of weak potency or poor metabolic stability. We conducted a phenotypic screen and identified a micromolar hit with novel amide structure. Medicinal chemistry efforts yielded a highly potent, selective, and metabolically stable drug candidate, compound 56 (RIPA-56). Biochemical studies and molecular docking revealed that RIP1 is the direct target of this new series of type III kinase inhibitors. In the SIRS mice disease model, 56 efficiently reduced tumor necrosis factor alpha (TNFα)-induced mortality and multiorgan damage. Compared to known RIP1 inhibitors, 56 is potent in both human and murine cells, is much more stable in vivo, and is efficacious in animal model studies.

Full text of DOI:10.1021/acs.jmedchem.6b01196

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Article
Discovery of a highly potent, selective, and metabolically
stable inhibitor of receptor-interacting protein 1 (RIP1) for
the treatment of systemic inflammatory response syndrome
Yan Ren, Yaning Su, Liming Sun, Sudan He, Lingjun Meng, Daohong Liao, Xiao Liu, Yongfen
Ma, Chunyan Liu, Sisi Li, Hanying Ruan, Xiaoguang Lei, Xiaodong Wang, and Zhiyuan Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01196 • Publication Date (Web): 19 Dec 2016
Downloaded from http://pubs.acs.org on December 20, 2016
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Discovery of a highly potent, selective, and
metabolically stable inhibitor of receptorꢀinteracting
protein 1 (RIP1) for the treatment of systemic
inflammatory response syndrome
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‡
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Yan Ren , Yaning Su , Liming Sun , Sudan He , Lingjun Meng , Daohong Liao , Xiao Liu ,
*
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†ǁ
†
Yongfen Ma , Chunyan Liu , Sisi Li , Hanying Ruan , Xiaoguang Lei , Xiaodong Wang and
†§
*
Zhiyuan Zhang
†
National Institute of Biological Sciences, Beijing 102206, China
§
Collaborative Innovation Center for Cancer Medicine, Beijing, China
ABSTRACT. Based on its essential role in driving inflammation and disease pathology, cell
necrosis has gradually been verified as a promising therapeutic target for treating atherosclerosis,
systemic inflammatory response syndrome (SIRS), and ischemia injury, among other diseases.
Most necrosis inhibitors targeting receptorꢀinteracting protein 1 (RIP1) still require further
optimization because of weak potency or poor metabolic stability. We conducted a phenotypic
screen and identified a micromolar hit with novel amide structure. Medicinal chemistry efforts
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yielded a highlyꢀpotent, selective, and metabolically stable drug candidate — compound 56
(RIPAꢀ56). Biochemical studies and molecular docking revealed that RIP1 is the direct target of
this new series of type III kinase inhibitors. In the SIRS mice disease model, 56 efficiently
reduced tumor necrosis factor alpha (TNFα)ꢀinduced mortality and multiꢀorgan damage.
Compared to known RIP1 inhibitors, 56 is potent in both human and murine cells, is much more
stable in vivo, and is efficacious in animal model studies.
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INTRODUCTION
Over the past few decades, necrosis has gradually been verified as an important pathway of
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programmed cell death, playing essential roles in ovulation, innate immunity, and
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inflammation in metazoan animals. Programmed cell necrosis is associated with lethal diseases
such as ischemia brain/kidney damage, atherosclerosis, myocardial infarction, pancreatitis, and
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systemic inflammatory response syndrome (SIRS).
Thus, targeting necrosis pathway can offer
a new opportunity for treating these diseases. Three important signaling proteins, receptorꢀ
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interacting protein 1 (RIP1), receptorꢀinteracting protein 3 (RIP3), and mixed lineage kinase
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domainꢀlike protein (MLKL), have been recognized as critical components in the regulated
necrotic cell death pathway. The identification of the first RIP1 inhibitor — 1 (Necrostatinꢀ1,
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Necꢀ1, Figure 1)
has greatly promoted necrosis pathway research. However, the micromolar
potency and poor metabolic stability (t1/2 < 5 min) of 1 has limited its application in drug
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discovery efforts. The most advanced necrostatin reported to date is 2 (Necꢀ1s) (Figure 1).
This compound has better metabolic stability than 1, but is still only moderately potent, and is
difficult to improve structurally, owing to its narrow structureꢀactivity relationship (SAR) profile.
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The hybridization of a typeꢀII panꢀkinase inhibitor ponatinib with 2 gave compound 3 (PN10)
(
Figure 1), which had both increased activity and higher kinase selectivity compared to the two
compounds upon which it was based. However, the high molecular weight makes 3 unfavorable
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as a drug and limits its further optimization. The other RIP1 inhibitors listed in Figure 1 were
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mostly developed by GlaxoSmithKline. The selectivity of 4 (GSK'481) to primate RIP1, the
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poor pharmacokinetic (PK) stability of 5 (GSK'963), and the poor kinase selectivity profile of 6
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Cpd27) would hinder the development of these compounds as drug candidates. Other known
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necrosis inhibitors target RIP3 or MLKL (Figure 1). RIP3 inhibitor — 7 (GSK'872), is
unsuitable for use as an antiꢀinflammatory agent because it induces cell apoptosis; the
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development of 8 (Necrosulfonamide, NSA) as an MLKL inhibitor has been restricted by its
moderate antiꢀnecrosis potency and its primateꢀspecific selectivity. Considering the intricately
linked involvement of cell necrosis and human diseases, as well as the various limitations of
known necrosis inhibitors, the discovery of both novel inhibitors and new protein targets in the
necrosis signaling pathway promises to be of significance to both basic biological research and
drug discovery.
Necrosomes, which are made up of RIP1, RIP3, and MLKL, induce RIPꢀkinase activityꢀ
dependent necrosis. Treatment of the human colon adenocarcinoma cell line HT29 with a
combination of tumor necrosis factor alpha (TNFα), second mitochondrialꢀderived activator of
caspases (Smac) mimetics, and ZꢀValꢀAlaꢀAspꢀ(OMe)ꢀFluoromethyl Ketone (zꢀVADꢀFMK)
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results in the formation of necrosomes.
Many biological studies have used TNFα/Smac
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mimetic/zꢀVADꢀFMK (TSZ)ꢀinduced HT29 cell necrosis assays;
indeed the very discovery
of the critical role that MLKL plays in the necrosis pathway was based on a screening hit from
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such assays. Here, we report the discovery of a new series of inhibitors identified from their
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Figure 1. Chemical structures of known necrosis inhibitors targeting RIP1, RIP3, and MLKL.
ability to block necrosis in the TSZꢀHT29 assay. Our leadꢀoptimization improved the activity of
the initial hit compound 340ꢀfold (EC from 4.1 µM to 12 nM). We subsequently identified and
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biochemically characterized RIP1 as the cellular target of this new series of type III kinase
inhibitors. The highly potent and selective lead compound, NꢀbenzylꢀNꢀhydroxyꢀ2,2ꢀ
dimethylbutanamide 56 (RIPAꢀ56), showed an impressive PK profile in mice, with a 3.1 h halfꢀ
life and efficient protection of mice from TNFαꢀinduced mortality and multiꢀorgan damage in the
SIRS disease model. Compared to known RIP1 inhibitors, 56 is potent in both human and
murine cells, has the best PK profile, and is efficacious in animal model studies; thus 56 is a
favorable tool compound for studies in animal disease models, and is a potential drug candidate
for clinical trials of necrosisꢀrelated diseases.
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RESULTS and DISCUSSION
Discovery of novel antiꢀnecrosis small molecules. To identify new necrosis inhibitors, a robust
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TSZꢀinduced HTꢀ29 cell necrosis assay
00,000 compounds. Hit compound 9 (Figure 2A) was confirmed to block necrotic cell death
with an EC of 4.1 µM; albeit less potent than 1, but compound 9 still offered a novel core for
was conducted to screen a chemical library of
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structural optimization (Figure 2B).
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Compound conc. (nM)
Figure 2. Characterization of hit compound 9. A, Chemical structure of hit compound 9. B,
Protection of 1 and 9 against TSZꢀinduced HTꢀ29 cell necrosis.
From hit to lead optimization. According to the structural features of the initial hit compound 9,
we designed modifications of the compound at its various positions (Table 1) with the goal of
improving its potency. We first examined the influences of various substituents on the phenyl
ring and the nitrogen of the amide group of hit compound 9 on antiꢀnecrosis potency.
Compounds modified from 9 were synthesized, and their inhibition potency was measured in
vitro using a TSZꢀinduced HTꢀ29 cell necrosis assay. Introduction of a fluoro group to the 2ꢀ
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position of the phenyl ring resulted in an approximately 5ꢀfold improvement in antiꢀnecrosis
potency (Table 1, 10 vs 9), while a larger group (e.g., bromine) substituted at both the 2ꢀ and 3ꢀ
positions resulted in dramatically decreased antiꢀnecrosis potency (11 and 12). We next turned
our efforts to modifications at the nitrogen of the amide. Methylation (13 and 14) significantly
improved the potency by approximately 10~40ꢀfold, while ethylation (15) caused a complete
loss of potency, indicating a limited space for further modification at this position. Next, the
SAR around the phenyl group was explored by keeping the methyl group on the nitrogen
constant and substituting the phenyl ring. Replacement of the fluoro group in the 2ꢀposition of
the phenyl ring with a chloro group (16) caused complete loss of antiꢀnecrosis potency. The 3ꢀ
fluorine substituted compound 17 improved the potency (EC50 = 72 nM), however no
improvements were observed for the 3ꢀchlorine substituted compound 18 or the 3ꢀbromine
substituted compound 19 or the 4ꢀfluorine substituted compound 21, and a hydroxyl group at the
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3ꢀposition of the phenyl ring (20) resulted in a complete loss of potency. Encouraged by these
results, we further synthesized diꢀsubstituted compounds 22ꢀ24. While the EC values of 2,3ꢀdiꢀ
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F compound 22 and 2,5ꢀdiꢀF compound 24 were further reduced to 58 nM and 36 nM,
respectively, the EC50 value of 2,4ꢀdiꢀF compound 23 was reduced to 626 nM. These SAR
results indicated that substitution with fluorine at the 2ꢀ, 3ꢀ, or 5ꢀposition increased potency and
further suggested that larger substitutions at these positions were detrimental to potency. As
expected, the 2,3,5ꢀ trifluoro substituted derivative (25) had yet further improved potency (EC50
=
12 nM). In the contrary, the 2,4,6ꢀtriꢀF compound 26 exhibited much weaker antiꢀnecrosis
potency than 25. Our SAR results suggested that the phenyl ring likely interacts with a tigh
hydrophobic pocket of putative target protein.
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Table 1. SAR study of compounds 9–26 evaluated with a TSZꢀinduced HTꢀ29 cell necrosis
assay.
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a
a
Compd
R1
R2 EC (nM) Compd
R1
R2
EC (nM)
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50
9
phenyl
H
H
H
H
4100 ± 952
898 ± 48
18 3ꢀClꢀphenyl
Me 628 ± 94
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13
2ꢀFꢀphenyl
2ꢀBrꢀphenyl
3ꢀBrꢀphenyl
phenyl
19 3ꢀBrꢀphenyl
20 3ꢀOHꢀphenyl
21 4ꢀFꢀphenyl
Me 1233 ± 102
Me > 3000
Me 815 ± 32
Me 58 ± 7
8562 ± 869
> 10,000
Me 457 ± 53
Me 125 ± 4
Et > 10,000
22 2, 3ꢀdiꢀFꢀphenyl
23 2, 4ꢀdiꢀFꢀphenyl
24 2, 5ꢀdiꢀFꢀphenyl
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2ꢀFꢀphenyl
2ꢀFꢀphenyl
Me 627 ± 1
Me 36 ± 7
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16
17
2ꢀClꢀphenyl Me > 10,000
25 2, 3, 5ꢀtriꢀF ꢀphenyl Me 12 ± 2
3ꢀFꢀphenyl
Me 72 ± 9
26 2, 4, 6ꢀtriꢀFꢀphenyl Me 1254 ± 174
a
EC data of each compound were determined in 2~3 independent assays, and mean EC ±
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SD values are shown in the table. Compd: abbreviation of compound; SD: standard deviation.
These data formats and abbreviations are used in the tables that follow.
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In order to further explore SAR of hit compound 9, we investigated replacement of the phenyl
ring with heterocyclic analogs (Table 2). Substitution of this moiety with pyridine derivatives
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e.g. compounds 27 and 29) resulted in a complete loss of potency. Compound 28 also had
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greatly reduced potency in comparison with compound 13. In contrast, replacement of the phenyl
ring with a thiophene moiety increased activity 2ꢀfold (30 vs 13), while compound 32 had a
similar potency as 13. Compounds 31 and 33 displayed weaker antiꢀnecrosis potency with EC50
values in the range of 1 - 3 µM. Compound 34, which has a furan group in place of the phenyl
ring had slightly lower activity than compound 13, while derivatives carrying either 2ꢀ
methylthiazole (35) or 1ꢀmethylꢀ1Hꢀpyrazole (36) groups lost all antiꢀnecrosis potency.
Table 2. SAR of Nꢀheterocycle substitutedꢀN,2,2ꢀtrimethylbutanamide series 27–36
Compd
HET
EC (nM)
Compd HET
EC (nM)
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27
> 10,000
32
378 ± 4
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1735 ± 140
> 10,000
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36
1308 ± 81
628 ± 72
> 10,000
> 10,000
221 ± 26
2527 ± 227
We also investigated the SAR involving the acyl group of compound 9. First, the original acyl
group was substituted with different branched alkylacyl groups. As shown in Table 3, the
corresponding region of the binding pocket appeared sensitive to both the size (compounds 37
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and 40–44) and polarity (compounds 38 and 39), of the substituted acyl groups. Among these,
compound 37 showed an EC50 of 97 nM, approximately 8ꢀfold less potent than its parent,
compound 25. The introduction of hydroxyl and methoxyl groups, as in compounds 38 and 39,
caused complete loss of activity. Cyclic analogs of these compounds (40–44) all showed a
dramatic loss of activity. Replacement of the amide moiety with urea also impaired the antiꢀ
necrosis potency; compounds 45–48 showed greatly weak potency compared to the amide
compound 25. Overall, the 2,2ꢀdimethylbutanamide moiety was shown to be the most effective
acyl group within this class of compounds for high levels of antiꢀnecrosis potency.
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Table 3. SAR of acyl group replacement (compounds 37–48)
Compd
R3
EC (nM)
Compd
R3
EC (nM)
50
50
3
7
8
98 ± 10
> 10,000
> 3000
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1234 ± 200
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490 ± 86
> 3,000
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> 10,000
> 3000
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8237 ± 1180
> 3,000
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1526 ± 204
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We also examined the influence of R substitution on compound 9 (Table 4). Substitution of the
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R group with a methyl group, –(CH ) and –CH CH ꢀ group, as in compounds 49, 52, and 54,
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respectively, caused inactivation (Table 4). Addition of a methyl group to the amide nitrogen of
compounds 49 and 52, to produce compounds 50 and 55, respectively, did not improve the
inhibitory potency of either original compound. Introduction of a hydroxymethyl group to
compound 9 (compound 51) increased potency by ~4ꢀfold relative to the original. However,
methylation of compound 51 nitrogen atom, to form compound 53, did not increase potency. The
results suggested that there is no more room for further SAR study at this area.
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Table 4. SAR of compound 49–55.
Compd
R2
H
R4
EC (nM)
50
49
50
51
52
53
Me
Me
ꢀCH
> 10,000
> 10,000
1077 ± 124
> 10,000
> 10,000
> 5,000
Me
H
OH
2
H
ꢀ(CH )
3 2
Me
H
ꢀCH
ꢀCH
OH
2
54
55
2
CH ꢀ
2
Me
ꢀ(CH
)
2
> 10,000
3
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Using the results of our SAR studies, a 340ꢀfold increase in bioactivity from EC of 4.1 µM
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for compound 9 to 12 nM for compound 25, we expanded our optimization study to produce
derivatives with increased stability. We selected a subꢀset of potent compounds for in vitro
microsomal stability assays using human and mouse liver microsomes. The stability of most Nꢀ
methylated compounds was low in both types of liver microsomes (t1/2 less than 5 min, Table s1).
In contrast, compounds 9, 49 and 51 had higher microsomal stability (t1/2 > 200 min, Table s1) in
human liver microsomes. Although none of the three compounds had a high potency (Tables 1
and 4), the additional polar groups (OH or NH) possibly contributed to their stability. Following
these results, we synthesized a subsequent set of Nꢀhydroxylated compounds (56–62) (Table 5).
These compounds were tested for cell necrosis inhibition, yielding results comparable to the
SAR of Nꢀmethylated compounds. Notably, compound 56 had a halfꢀlife of 128 min in human
liver microsomal stability assays and an EC of 28 nM in TSZꢀinduced HTꢀ29 necrosis assay.
10
11
12
13
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15
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45
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49
50
51
52
53
54
55
56
57
58
59
60
5
0
Compound 56 also demonstrated potency in protection of murine L929 cells from TZꢀinduced
necrosis (EC50 = 27 nM, Figure s1). This result is ~100ꢀfold more potent than GSK
16
benzoxazepinone compound 4 (Figure 1), which has higher activity in primate, rather than nonꢀ
primate, RIP1. We conclude from these stability and activity data that compound 56 is wellꢀ
suited as a lead for further study in the mouse model.
Identification of RIP1 as the direct target of the amide series of necrosis inhibitors. As this
is a novel series of necrosis inhibitors, we wanted to know if they target RIP1, RIP3, MLKL or
an unknown target associated with this necrosis pathway. It is known RIP1 and RIP3 are
phosphorylated following TSZ treatment; this phosphorylation can be monitored as upꢀshifted
bands as detected via western blotting. The phosphorylation level of RIP3 is known to depend on
11
both its own kinase activity and the kinase activity of RIP1. According to the band shift results
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Table 5. SAR and liver metabolic stability of NꢀOH compounds 56–62.
0
1
2
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7
8
9
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Human Liver
Microsomes
Mouse Liver
Microsomes
Compd Fn
EC (nM)
50
t1/2
Clint
t1/2
Clint
(min)
(µL/min/mg)
(min)
(µL/min/mg)
56
ꢀ
28 ± 3
128
178
198
107
52.9
66.0
66.6
5.40
3.90
3.50
6.50
13.1
10.5
10.4
35.5
24.1
23.2
11.3
13.9
7.89
9.21
19.5
28.8
29.9
61.4
49.9
87.8
75.3
5
5
5
7
8
9
4ꢀF
64 ± 11
198 ± 36
56 ± 4
2, 4ꢀdiꢀF
3, 4ꢀdiꢀF
60
2, 3,4ꢀtriꢀF 262 ± 28
3, 4,5ꢀtriꢀF 49 ± 5
2, 3,5ꢀtriꢀF 74 ± 17
6
6
1
2
shown in Figure 3A, 56 can block the phosphorylation of both RIP1 and RIP3. The 56ꢀmediated
inhibition was confirmed using an antibody specific for phosphorylation on Serine227 of RIP3.
5
6 thus appears to inhibit the kinase activity of RIP1 and/or RIP3, or to somehow interfere with
their upstream activators. The kinase activities of RIP1 and RIP3 were next monitored in vitro
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using an ADPꢀGlo assay. Compound 56 showed efficient inhibition of RIP1 kinase activity,
15
with an IC of 13 nM (Figure 3B), 57ꢀfold more potent than 1 (IC = 760 nM). It showed no
50
50
inhibition of RIP3 kinase activity at a 10 µM concentration (Figure s2A). Several new
compounds were evaluated using this RIP1 kinase activity assay. A good correlation was noted
between kinase inhibition activity data and cell necrosis inhibition data (Figure s2B), indicating
that RIP1 is the direct target of these new amide series inhibitors.
10
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49
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52
53
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55
56
57
58
59
60
2
0
According to a reported complex structure of RIP1ꢀNec1, an allosteric backꢀpocket is
isolated from its ATP binding site and forms an Lꢀshaped hydrophobic pocket. This pocket is
divided into two parts by phenylalanine (Phe)ꢀ162. Our molecular docking simulation study
suggested that compound 56 fits tightly in this pocket, similar to 1 (Figure 3C). This binding
mode implies the existence of a key hydrogen bond that is formed between the carbonyl group
on the inhibitor molecule and the NH of aspartic acid (Asp)ꢀ156 of RIP1. The phenyl group
occupies the left side of the pocket formed by leucine (Leu)ꢀ70, Leuꢀ129, valine (Val)ꢀ134,
histidine (His)ꢀ136, isoleucine (Ile)ꢀ154, and serine (Ser)ꢀ161, and it seems likely that this
narrow space would not tolerate larger substituents on the phenyl ring or polar hetero aromatic
ring, which is consistent with our SAR results reported in Tables 1 and 2. On the right side of the
pocket, only the 2,2ꢀdimethylbutanamide moiety could effectively occupy the hydrophobic
pocket space; both the presence of one additional carbon and the absence of one of these carbons
resulted in a dramatic decrease in potency (Table 3). Furthermore, inefficient and unsuitable
occupancy in this area by various urea analogs from our SAR studies seems likely to be a major
factor contributing to activity loss. For the NꢀOH compounds, the hydroxyl group may also form
a hydrogen bond with valine (Val)ꢀ76, which could explain the slight SAR changes among the
phenyl substitutions shown in Table 5. Furthermore, this proposed binding mode for the
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9
0
Figure 3. Target validation and kinase panel screening of lead compound 56. A, compound 56
inhibits TSZꢀinduced phosphorylation of RIP1 and RIP3 in HTꢀ29 cells. B, Compound 56 is
more potent than 1 in inhibiting RIP1 kinase activity. C, Based on the molecular docking
simulation study, compound 56 could form tight hydrophobic interactions with RIP1 through
both the phenyl group and the 2,2ꢀdimethylbutyl group, and form two important hydrogen bonds
(one between the carbonyl oxygen of 56 and the backbone NH on Asp156 of RIP1, the other
occurs between the NꢀOH of 56 and Val76 of RIP1). D, compound 56 shows good selectivity in
kinase panel screening (conducted by Reaction Biology Corp.)
inhibitors suggests that the compounds are likely type III kinase inhibitors that lock the RIP1
kinase in its inactive form. This would be in line with its absolute selectivity according to the
kinase panel screening results (Figure 3D, Figure s2C). Compound 56 was also tested in IDO
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8, 21, 22
enzyme activity assays. Unlike 1,
56 did not inhibit IDO activity at a concentration of 200
µM, which represents an estimated 10,000ꢀfold selectivity window based on the RIP1 ADPꢀGlo
activity of 13 nM; 56 therefore avoids the problematic offꢀtarget (antiꢀIDO) effect of 1 (Figure
s3).
10
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49
50
51
52
53
54
55
56
57
58
59
60
Pharmacokinetics and pharmacological efficacy of 56 in vivo. We conducted metabolic
stability assays and noted that 56 had a marked improvement in liver microsomes (Table 5). 56
also had an impressive PK profile in mice (Table s2, Figure 4), with a 3.1 h halfꢀlife, 22% oral
bioavailability (PO), and 100% bioavailability from intraperitoneal injection (IP). Encouraged by
its high potency and high selectivity in biochemical and cellular studies, and good metabolic
stability in these in vivo PK studies, we were also interested to see whether 56 could confer any
therapeutic effect in mice models of inflammatory diseases.
1
0000
1000
100
10
PO 10 mg/kg BW
IP 10mg/kg BW
IV 2 mg/kg BW
1
0
4
8
12
16
20
24
Hours post compound dose
Figure 4. Pharmacokinetics of oral (PO), intraperitoneal (IP), and intravenous (IV)
administration of 56 in C57BL/6 mice (n = 3; BW: body weight).
TNFα administration can cause multiple organ dysfunction and death in mice, exhibiting many
similarities with the highlyꢀlethal human SIRS disorder that can be induced by infection, trauma,
4,6,8,15ꢀ17
etc.
To test whether 56 has any efficacy in protecting against TNFαꢀinduced lethality, 5.5
µg of mouse TNFα (mTNFα) (~0.25 mg/kg body weight (BW)) was intravenously injected to
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C57BL/6 mice (6ꢀ8 weeks, 20ꢀ25 g) and mice were either treated with multipleꢀdose of 56 (0.1,
1, 3 mg/kg BW, IP, 17 min before mTNFα injection and once every 12 h) or a single dose of 56
or 1 (6 mg/kg BW, IP, 17 min before mTNFα injection). As shown in Figure 5, both multipleꢀ
dose and singleꢀdose of 56 treatment dramatically increased the survival rate of TNFαꢀtreated
mice, showing a doseꢀdependent effect. Mice treated with multipleꢀdose of 56 (3 mg/kg) or a
single 6 mg/kg dose of 56 had a survival rate of 100%, much higher than the TNFα/1 group,
which had a survival rate of 60%. The TNFα/vehicleꢀtreated mice group had a 50% survival rate.
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a
100
mTNFα/3mg/kg 56
mTNFα/1mg/kg 56
a
a
8
0
mTNFα/0.1mg/kg 56
b
mTNFα/6mg/kg 56
b
mTNFα/6mg/kg 1
60
mTNFα/vehicle
40
0
20
40
60
Hours post mTNFα treatment
Figure 5. Both multiple dose of 56 (0.1, 1, 3 mg/kg BW, IP, once every 12 h) and a single dose
of 56 (6 mg/kg BW, IP, once) can protect mice from mTNFαꢀinduce death (n = 6~10 per group).
This protection was significantly better than that conferred by a single 6 mg/kg BW dose of 1.
a
b
multipleꢀdose; single dose.
To test the efficacy of 56 in protecting against TNFαꢀinduced multiꢀorgan damage, 4 µg
mouse TNFα (mTNFα) (~0.18 mg/kg BW) was intravenously injected into C57BL/6 mice. The
levels of the mouse proꢀinflammatory cytokines interleukinꢀ6 (mILꢀ6) and interleukinꢀ1ß (mILꢀ
1ß) increased significantly after TNFα induction, but mice treated with 56 had lower or even
normal levels of cytokines (Figure 6A). Note that mice treated with a single 6 mg/kg dose of 1
had partiallyꢀdecreased levels of mILꢀ6 and mILꢀ1ß (Figure s4), a finding consistant with the
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reported protection effects of 1 on mice viability. Histological examination of haematoxylinꢀ
eosin (H&E) stained spleens of TNFαꢀtreated mice showed that both the red pulp and the white
pulp were infiltrated with many immune cells and macrophages; this pattern was also seen in the
cortex and medulla of the thymus (Figure 6B).The thickness of the periarteral lymphatic sheath
(PALS) also increased dramatically in TNFαꢀtreated mice. In contrast, the spleen and thymus of
mice that were additinally treated with 56 were infiltrated with only a few immune cells and
macrophages, and otherwise appeared similar to those of nonꢀTNFα treated control mice. In
addition to the immune organs (spleen and thymus), we also examined TNFαꢀinduced damage to
other internal organs, including the myocardium, kidneys, and pancreas. For TNFαꢀtreated mice,
the level of representive biomarkers, including serum lactate dehydrogenase (LDH), creatine
kinase (CK), aspartate aminotransferase (AST), creatinine, blood urea nitrogen (BUN), and
amylase, increased by 2 to 10ꢀfold as compared with control mice. Organ damage could also be
clearly interpreted from histological staining results, which exhibited infiltration of inflammatory
cells, disordered tissues, and damaged cells. In contrast, the biomarker levels of TNFαꢀtreated
mice that were also given 56 were close to those of healthy animals, and the extent of organ
damage in 56ꢀtreated was limited compared to the TNFαꢀtreated mice (Figure 6).
10
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49
50
51
52
53
54
55
56
57
58
59
60
Assessment of 56 as a drug candidate for clinical trials. The drugꢀlike properties of 56 were
further evaluated. 56 was tested for its ability to inhibit cytochrome P450 enzymes; it had IC50
values higher than 50 µM against CYP1A2, 2C19, 2C9, and 2D6, and higher than 3.5 µM
against CYP3A4. In addition, a tissueꢀdistribution study indicated that 56 could be delivered
readily to most organs where diseases might occur (Figure s5). For example, 56 could be
detected in the brains of mice following intravenous injection, and exhibited strong permeability
6
in MDCKꢀMDR1 cells (P , AꢀB and BꢀA > 30 × 10 cm/sec), with an efflux ratio of 0.87
app
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Figure 6. 56 can efficiently protect mice from mTNFαꢀinduced multiꢀorgan damage. A, 56
administration (0.3, 1, 3 mg/kg BW, IP, once every 12 h) can decrease the secretion of proꢀ
inflammatory cytokines and level of tissue damage biomarkers. B, 56 shows efficient protection
against TNFαꢀinduced injury of different organs. (H&E staining, magnification: ×200). Each
*
**
***
image is representative of at least three mice. P < 0.05, P < 0.01, P < 0.001, each vs. TNFα
model group.
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(
Table s3), showing great ability in transporting across blood brain barrier. This is attractive
when considering the requirements of therapies against neurodegenerative disease and in
treatment of ischemic or traumatic brain injuries, in that 56 could be administrated by IV or IP
injection rather than through intracerebroventricular administration. Manual patch clamp
evaluation of 56 demonstrated that it did not inhibit the hERG channel, even at concentrations as
high as 30 µM (conducted by WuXi AppTec). Multipleꢀ, highꢀstrength dose of 56 (50 mg/kg,
once every 3 h, for 24 h) caused no injury to mice. These results from both in vitro and in vivo
studies all indicate that 56 seems to be a safe and potentially efficacious candidate drug for the
treatment of necrosisꢀrelated diseases.
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Chemistry. Compounds 10–15 were synthesized by a route as shown in Scheme 1. The
syntheses began with acylation reaction of corresponding halogen substituted phenylamines with
2
, 2ꢀdimethylbutanoyl chloride to obtain compound 10–13, and compound 14 and 15 were
obtained through alkylation of 10 with methyl iodide and bromoethane in good yield.
Scheme 1. Synthesis of compounds 10–15.
Reagent and conditions: (a) 2, 2ꢀdimethylbutanoyl chloride, TEA, DCM, rt; (b) NaH, RꢀBr, rt,
THF.
Compounds 16–48 were prepared according to Scheme 2. Reductive amination of aromatic
aldehydes with methylamine using NaBH afforded benzylmethylamine compounds, followed by
4
acylation with 2,2ꢀdimethylbutanoyl chloride and different alkyl acid to yield the corresponding
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5
5
5
6
compounds 16–36 and 37–44. The urea compounds 45–48 were prepared by treatment of the
benzylmethylamine intermediates with triphosgene and different alkylamines.
Scheme 2. Synthesis of compounds 16–48.
0
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7
8
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R group represented substituent aromatic rings such as benzene, pyridine, and thiophene rings,
1
etc. R group represented branched or cyclic alkyl group; R and R represted hydrogen atom or
2
3
4
alkyl group. Reagent and conditions: (a) CH NH , HCl, K CO , MeOH, rt, 1.5 h; (b) NaBH , rt,
3
2
2
3
4
1
6 h; (c) 2, 2ꢀdimethylbutanoyl chloride, DIEA, THF or DCM, rt, 2 h (for 16–36); (d) R COOH,
2
HATU, DIEA, DMF, rt, 16 h (for 37–44); (d) NR R , triphosgene, DCM (for 45–48).
3
4
The branched benzyl compounds 49–55 were synthesized by two routes (Scheme 3). Starting
from 1ꢀphenylethanꢀ1ꢀamine 63, 2ꢀphenylpropanꢀ2ꢀamine 65, and cyclopropyl(phenyl)
methanamine 66, acylation with 2,2ꢀdimethylbutanoyl chloride gave compounds 49, 52, and 54
respectively. Compound 49 and 52 were further methylated with methyl iodide in presence of
sodium hydride to give desired products 50 and 55 (Scheme 3A). Compound 51 was obtained by
selective amide formation by individually treating Nꢀbenzylhydroxylamine 64 with 2,2ꢀ
dimethylbutanoyl chloride in a mixture of aqueous NaHCO and tetrahydrofuran.
3
Compound 53 was synthesized in three steps as illustrated in Scheme 3B. A commercial
available material, 2ꢀaminoꢀ2ꢀphenylethanꢀ1ꢀol 64, was treated with methyl carbonochloridate to
give intermediate 67 that was converted to Nꢀmethyl compound 68 by a reduction using LiAlH4,
followed by a standard acylation with 2,2ꢀdimethylbutanoyl chloride afforded the desired
product 53.
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Scheme 3. Synthesis of compounds 49–55.
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49
50
51
52
53
54
55
56
57
58
59
60
Reagent and conditions: (a) TEA, DCM, rt. (for 49, 52 and 54); (b) 2,2ꢀdimethylbutanoyl
o
o
chloride, aq. NaHCO , THF/ H O, 0 C to rt, 16 h (for 51 and 53); (c) NaH, DMF, 0 C to rt, 16 h
3
2
o
(
for 50 and 55); (d) ethyl carbonochloridate, aq. NaHCO , THF/ H O, 0 C to rt, 16 h; (e) LiAH ,
3 2 4
THF, reflux.
Compounds 56–62 were synthesized as outlined in Scheme 4. Nucleophilic displacement of tertꢀ
butyl((tertꢀbutoxycarbonyl)oxy)carbamate with fluoroꢀsubstituted benzyl bromides (69–75)
afforded intermediate 76–82. Removal of Boc group from 76–82 by treatment with TFA in DCM
gave hydroxylamine derivatives 83–89, which were treated with 2,2ꢀdimethylbutanoyl chloride
to yield the final products 56–62.
Scheme 4. Synthesis of compounds 56–62.
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6
Reagent and conditions: (a) 1 N NaOH, TBAB, DCM; (b) TFA, DCM, rt, 4 h; (c) 2,2ꢀ
o
dimethylbutanoyl chloride, aq. NaHCO , THF, H O, 0 C to rt, 16 h.
3
2
CONCLUSION
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We have described the optimization of an antiꢀnecrosis screening hit into a promising lead,
which has novel amide structures and inhibits RIP1 by locking RIP1 kinase in an inactive form
as a type III kinase inhibitor. The lead compound — 56 — possesses favorable properties for
both a tool compound and a candidate drug: high potency in both human cells and murine cells,
high selectivity, high metabolic stability, high efficacy in mice SIRS model, as well as other
safety profiles. We anticipate that 56 will be a valuable tool in the further investigations of cell
death signaling pathway, and has great potential to be used in clinical trials of inflammatory
diseases. Further studies of 56 in different animal models of necrosisꢀrelated diseases are in
progress.
EXPERIMENTAL SECTION
1. Materials and Methods for Chemistry. All chemical reagents were used as supplied by
SigmaꢀAldrich, J&K and Alfa Aesar Chemicals. DCM and DMF were distilled from calcium
1
hydride; THF was distilled from sodium/benzophenone ketyl prior to use. H NMR spectra were
recorded on a Varian 400 MHz spectrometer at ambient temperature with CDCl as the solvent
3
1
3
unless otherwise stated. C NMR spectra were recorded on a Varian 100 MHz spectrometer
1
(
with complete proton decoupling) at ambient temperature. Data for H NMR are reported as
follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet), integration and coupling constants. Highꢀresolution mass spectra were obtained using
Agilent Technolodies 6540 UHD AccurateꢀMass QꢀTOF LC/MS. All of the synthesized
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compounds were analyzed by UPLC/MS on a Waters Auto Purification LC/MS system (3100
Mass Detector, 2545 Binary Gradient Module, 2767 Sample Manager, and 2998 Photodiode
Array (PDA) Detector). The system was equipped with a Waters C 5ꢁm SunFire separation
1
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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36
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49
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56
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column(150*4.6 mm), equilibrated with UPLC grade water (solvent A) and UPLC grade
acetonitrile (solvent B) with a flow rate of 0.3 mL/min. All of the tested compounds were
determined to be ≥ 95% pure by UPLC.
2. Synthesis procedure for compounds 10–62.
General synthesis procedure for compounds 10-13.
To a mixture of substituted
phenylmethanamine (1 eq, 100 mg) and triethylamine (2 eq) in dry DCM (4 mL) was added 2,2ꢀ
o
dimethylbutanoyl chloride (1.2 eq) at 0 C. The mixture was allowed to stir at room temperature
for 2 h. After the amine was consumed as judged by TLC, the mixture was quenched with iceꢀ
water, and then extracted with DCM. The organic phase was dried over Na SO , filtered and
2
4
concentrated. The residue was purified by silica gel chromatography to give the desired products.
Nꢀ(2ꢀfluorobenzyl)ꢀ2,2ꢀdimethylbutanamide (10). From (2ꢀfluorophenyl)methanamine and 2,2ꢀ
1
dimethylbutanoyl chloride. Colorless oil. Yield, 72.6% . H NMR (400 MHz，CDCl ) δ 7.34ꢀ
3
7.30 (m, 1H), 7.25ꢀ7.23 (m, 1H), 7.11ꢀ7.0 (m, 2H), 6.06 (br, 1H), 4.48 (d, J = 6.0 Hz, 2H), 1.55
1
3
(
q, J = 7.6 Hz, 2H), 1.16 (s, 6H), 0.81 (t, J=7.6 Hz, 3H). C NMR (100 MHz, CDCl ) δ 177.79,
3
162.30, 159.85, 130.25 (d, J = 4.4 Hz), 129.18 (d, J = 8.1 Hz), 125.68 (d, J = 14.6 Hz), 124.33
(d, J = 3.6 Hz), 115.48, 115.26, 42.50, 37.70, 37.67, 33.94, 24.98, 9.17. HRMS (ESI) [M+H]
+
calad for C H FNO, 224.1445; found, 224.1464. UPLC purity 98%.
1
3
19
Nꢀ(2ꢀbromobenzyl)ꢀ2,2ꢀdimethylbutanamide (11). From (2ꢀbromophenyl) methanamine and 2,2ꢀ
1
dimethylbutanoyl chloride. White solid. Yield, 74%. H NMR (400 MHz, CDCl ) δ 7.55 (dd, J =
3
8.0, 1.2 Hz, 1H), 7.38 (dd, J = 7.6, 1.7 Hz, 1H), 7.30 – 7.27 (m, 1H), 7.14 (td, J = 7.7, 1.7 Hz,
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2
2
2
2
2
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3
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1
H), 4.50 (d, J = 5.9 Hz, 2H), 1.55 (q, J = 7.5 Hz, 2H), 1.16 (s, 6H), 0.80 (t, J = 7.5 Hz, 3H). C
NMR (100 MHz, CDCl ) δ 177.75, 137.73, 132.90, 130.75, 129.26, 127.85, 123.87, 44.11,
3
+
+
4
2.63, 34.06, 25.07, 9.27. HRMS (ESI) [M+H] calad for C H BrNO , 284.0645; found,
13 19
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
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2
3
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7
8
9
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284.0645. UPLC purity 98%.
Nꢀ(3ꢀbromobenzyl)ꢀ2,2ꢀdimethylbutanamide (12). From (3ꢀbromophenyl) methanamine and 2,2ꢀ
1
dimethylbutanoyl chloride. Colorless oil. Yield, 76%. H NMR (400 MHz, CDCl ) δ 7.41 – 7.36
3
(
m, 2H), 7.21 – 7.15 (m, 2H), 5.90 (s, 1H), 4.40 (d, J = 5.8 Hz, 2H), 1.56 (q, J = 7.5 Hz, 2H),
1
3
1.16 (s, 6H), 0.84 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, CDCl ) δ 177.85, 141.27, 130.66,
3
+
1
30.49, 130.31, 126.26, 122.72, 42.95, 42.54, 33.93, 25.06, 9.30. HRMS (ESI) [M+H] calad for
+
C H BrNO , 284.0645; found, 284.0651. UPLC purity 98%.
13 19
o
NꢀbenzylꢀN,2,2ꢀtrimethylbutanamide (13). To cold (0 C) a solution of Nꢀbenzylꢀ2,2ꢀ
dimethylbutanamide (50 mg) in dry DMF (4 mL), sodium hydride (14.6 mg) was added under
o
N . After the mixture was stirred for 2 h at 0 C, iodomethane (52 mg) was added and the mixture
2
was allowed to warm to room temperature and stirred for 11 h. The mixture was quenched with
cold water and extracted with DCM, the combined organic layers was washed with water, brine,
dried over Na SO concentrated and the residue was purified by silica gel column
2
4,
1
chromatography to give the product as a colorless oil (18.8 mg, 35%). H NMR (400 MHz,
cdcl ) δ 7.40 – 7.21 (m, 5H), 4.65 (s, 2H), 3.00 (s, 3H), 1.70 (q, J = 7.5 Hz, 2H), 1.31 (s, 6H),
3
1
3
0.92 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, CDCl ) δ 177.14, 128.72, 127.71, 127.34, 53.35,
3
+
4
2
3.30, 35.99, 33.42, 26.76, 9.91. HRMS (ESI) [M+H] calad for C H NO, 220.1696; found,
1
4
22
20.1696. UPLC purity 97%.
Nꢀ(2ꢀfluorobenzyl)ꢀN,2,2ꢀtrimethylbutanamide
(14).
Nꢀ(2ꢀfluorobenzyl)ꢀ2,2ꢀ
dimethylbutanamide (40 mg) was dissolved in 4 mL of dry DMF, 8.61 mg of NaH was added at
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C under N and stirred for 2 h. Methyl iodide (51 mg) was added and the mixture was allowed
2
to warm to room temperature and stirred for 11 h. The mixture was quenched with cold water
and extracted with DCM, the combined organic layers was washed with water, brine, dried over
Na SO concentrated and the residue was purified by silica gel column chromatography to give
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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27
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29
30
31
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33
34
35
36
37
38
39
40
41
42
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48
49
50
51
52
53
54
55
56
57
58
59
60
2
4,
1
the product as a light yellow oil (21 mg, 51%). H NMR(400 MHz，CDCl ) δ 7.28ꢀ7.22 (m,
3
2
H), 7.12ꢀ7.01 (m, 2 H), 4.68 (s, 2H), 3.05(s, 3H), 1.65 (q, J = 7.6 Hz, 2H), 1.27(s, 6H), 0.89(t, J
1
3
= 7.6 Hz, 3H). C NMR (100 MHz, CDCl ) δ 177.13, 162.23, 159.79, 129.67, 128.84, 128.76,
3
1
24.78, 124.64, 124.35, 124.31, 46.75, 46.71, 43.17, 36.28, 33.24, 26.53, 9.40. HRMS (ESI)
+
[
M+H] calad for C H FNO, 238.1602; found, 238.1604. UPLC purity 96%.
1
4
21
NꢀethylꢀNꢀ(2ꢀfluorobenzyl)ꢀ2,2ꢀdimethylbutanamide (15). Compound 15 was prepared from Nꢀ
(
2ꢀfluorobenzyl)ꢀ2,2ꢀdimethylbutanamide, NaH and iodoethane using the method described in
1
the preparation of compound 14 as a colorless oil in 65% yield. H NMR (400 MHz, CDCl ): δ
3
7
.26ꢀ7.22 (m, 2H), 7.12ꢀ7.03 (m, 2H), 4.69 (s, 2H), 3.43 (d, J = 5.2 Hz, 2H), 1.67 (q, J = 7.6 Hz,
+
2
H), 1.26 (s, 6H), 1.17 (t, J =6.8 Hz, 3H), 0.89 (t, J =7.6 Hz, 3H). LCꢀMS (ESI) [M+H] calad
for C H FNO 252.18; found, 252.21. UPLC purity 96%.
15 24
General synthesis procedure for compounds 16–36. A mixture of K CO (207 mg, 1.5 mmol)
2
3
and methanamine hydrochloride (202 mg, 3.0 mmol) in 5 mL of MeOH was stirred at rt for 30
min. Then the aromatic aldehyde (248 mg, 2.0 mmol) was added to the mixture and stirred at rt
o
for 1 h. The mixture was cooled to 0 C, and NaBH (113.5 mg, 3.0 mmol) was added in portions.
4
o
The mixture was stirred at 0 C for 1 h and warmed to room temperature and stirred for 2 h. The
solid was filtered and washed with EtOAc. The filtrate was evaporated to dryness and the residue
was dissolved in EtOAc and was washed with water, brine, dried over Na SO , concentrated.
2
4
The residue was dissolved in a mixture of dry THF (10 mL) and DIEA (264 mg, 2.05 mmol),
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and then 2,2ꢀdimethylbutanoyl chloride (275 mg, 2.05 mmol) was added slowly to the mixture at
o
0
C under nitrogen, stirred at room temperature for 2 h. The reaction was quenched by adding
water (15 mL) and extracted with EtOAc (10 mL x 3). The combined organic was washed with 1
M HCl, brine, dried over Na SO , filtered and concentrated. The residue was purified by silica
0
1
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9
0
1
2
3
4
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8
9
0
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gel column chromatography (PE/EA) to give the corresponding product.
Nꢀ(2ꢀchlorobenzyl)ꢀN,2,2ꢀtrimethylbutanamide (16). From 2ꢀchlorobenzaldehyde (281 mg),
methanamine hydrochloride (202 mg) and 2,2ꢀdimethylbutanoyl chloride (275 mg). White solid.
1
Yield, 48%. H NMR (400 MHz, CDCl ) δ7.37ꢀ7.35 (m, 1 H), 7.25ꢀ7.16 (m, 3 H), 4.74 (s, 2 H),
3
13
3
.05 (s, 3 H), 1.70 (q, J = 7.6 Hz, 2H), 1.28 (s, 6 H), 0.91 (t, J = 7.6 Hz, 3 H). C NMR (100
MHz, CD OD) δ 177.82, 134.54, 129.29, 128.34, 127.72, 126.88, 50.73, 42.97, 35.99, 32.77,
3
+
25.53, 8.46. HRMS (ESI) [M+H] calad for C H ClNO, 254.1306; found, 254.1306. UPLC
1
4
21
purity 95%.
Nꢀ(3ꢀfluorobenzyl)ꢀN,2,2ꢀtrimethylbutanamide (17). From 3ꢀfluorobenzaldehyde (124 mg),
methanamine hydrochloride (101 mg) and 2,2ꢀdimethylbutanoyl chloride (140 mg). Colorless oil.
1
Yield, 65%. H NMR (400 MHz, CDCl ) δ 7.31ꢀ7.25 (m, 1 H), 7.01ꢀ6.91 (m, 3 H), 4.61 (s, 2 H),
3
1
3
3.01 (s, 3 H), 1.69 (q, J = 7.6 Hz，2 H), 1.29 (s, 6 H), 0.90 (t, J = 7.6 Hz，3 H). C NMR (100
MHz, CDCl ) δ 177.01, 164.24, 161.79, 140.48, 140.40, 130.08, 130.00, 123.09, 114.19, 113.98,
3
+
52.82, 43.12, 36.04, 33.19, 26.50, 9.41. HRMS (ESI) [M+H] calad for C H FNO, 238.1602;
14
21
found, 238.1606. UPLC purity 95%.
Nꢀ(3ꢀchlorobenzyl)ꢀN,2,2ꢀtrimethylbutanamide (18). From 3ꢀchlorobenzaldehyde (140 mg),
methanamine hydrochloride (101 mg) and 2,2ꢀdimethylbutanoyl chloride(140 mg). White solid.
1
Yield, 48%. H NMR (400 MHz, CDCl ) δ7.26ꢀ7.20 (m, 3 H), 7.12ꢀ7.10 (m, 1 H), 4.59 (s, 2 H),
3
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.01 (s, 3 H), 1.68 (q, J = 7.6 Hz，2H), 1.28 (s, 6 H), 0.90 (t, J = 7.6 Hz，3H). HRMS (ESI)
+
[M+H] calad for C H ClNO, 254.1306; found, 254.1302. UPLC purity 96%.
1
4
21
Nꢀ(3ꢀbromobenzyl)ꢀN,2,2ꢀtrimethylbutanamide (19). From 3ꢀbromobenzaldehyde (185 mg),
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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49
50
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53
54
55
56
57
58
59
60
methanamine hydrochloride (101mg) and 2,2ꢀdimethylbutanoyl chloride (140 mg). White solid.
1
Yield, 48%. H NMR (CDCl , 400 MHz): δ 7 7.36ꢀ7.40 (m, 2 H), 7.16ꢀ7.22 (m, 2 H), 4.59 (s, 2
3
13
H), 3.01 (s, 3 H), 1.69 (q, 2 H, J = 7.6 Hz), 1.29 (s, 6 H), 0.90 (t, 3 H, J = 7.6 Hz). C NMR (100
MHz, CD OD) δ 179.24, 141.66, 131.57, 131.45, 131.41, 127.41, 123.59, 53.74, 49.64, 49.43,
3
+
4
9.21, 49.00, 48.79, 48.57, 48.36, 44.32, 36.90, 34.06, 26.97, 9.78. HRMS (ESI) [M+H] calad
for C H BrNO , 298.0796; found, 298.0802, 300.0782. UPLC purity 96%.
1
4
21
2
Nꢀ(3ꢀhydroxybenzyl)ꢀN,2,2ꢀtrimethylbutanamide (20). From 3ꢀhydroxybenzaldehyde (122 mg),
methanamine hydrochloride (101 mg) and 2,2ꢀdimethylbutanoyl chloride (140 mg). White solid.
1
Yield, 33%. H NMR (CDCl , 400 MHz):δ7.17 (t, 1 H, J = 7.6 Hz), 6.71ꢀ6.79 (m, 3 H), 4.58 (s,
3
1
3
2
H), 2.98 (s, 3 H), 1.68 (q, 2 H, J = 7.6 Hz), 1.29 (s, 6 H), 0.90 (t, 3 H, J = 7.6 Hz). C NMR
(
100 MHz, CDCl ) δ 177.67, 157.44, 129.74, 114.90, 77.48, 77.16, 76.84, 53.42, 43.34, 36.18,
3
+
3
3.30, 26.72, 9.59. LCꢀMS (ESI) [M+H] calad for C H NO , 236.1640; found, 236.1641.
14 22
2
UPLC purity 96%.
Nꢀ(4ꢀfluorobenzyl)ꢀN,2,2ꢀtrimethylbutanamide (21). From 4ꢀfluorobenzaldehyde (248 mg),
methanamine hydrochloride (202 mg) and 2,2ꢀdimethylbutanoyl chloride (275 mg). Colorless oil.
1
Yield, 54%. H NMR (400 MHz, CDCl ): δ 7.21 – 7.12 (m, 2H), 6.98ꢀ6.93 (m, 2H), 4.54 (s, 2H),
3
1
3
2
.95 (s, 3H), 1.64 (q, J = 7.5 Hz, 2H), 1.24 (s, 6H), 0.84 (t, J = 7.5 Hz, 3H). C NMR (100 MHz,
CDCl ) δ 176.77, 163.12, 160.68, 133.44, 133.41, 129.22, 129.14, 115.33, 115.12, 77.32, 77.00,
3
+
7
6.68, 52.39, 42.97, 35.66, 33.08, 26.39, 9.26. LCꢀMS (ESI) [M+H] calad for: C H FNO,
1
4
21
238.1602; found, 238.1598. UPLC purity 96%.
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1
1
2
2
2
2
2
2
2
2
2
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Nꢀ(2,3ꢀdifluorobenzyl)ꢀN,2,2ꢀtrimethylbutanamide (22). From 2,3ꢀdifluorobenzaldehyde (284
mg), methanamine hydrochloride (202 mg) and 2,2ꢀdimethylbutanoyl chloride (275 mg).
1
Colorless oil. Yield, 56%. H NMR (400 MHz, CDCl ) δ 7.11 – 6.94 (m, 3H), 4.66 (s, 2H), 3.06
3
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
13
(
s, 3H), 1.66 (q, J = 7.5 Hz, 2H), 1.25 (s, 6H), 0.85 (t, J = 7.5 Hz, 3H). C NMR (100 MHz,
CDCl ) δ 177.07, 151.64, 151.51, 150.17, 150.04, 149.17, 149.04, 147.71, 147.58, 127.33,
3
127.21, 124.17, 124.12, 124.10, 124.06, 115.98, 115.81, 46.49, 43.07, 36.36, 33.08, 9.28. HRMS
+
(
ESI) [M+H] calad for: C H F NO, 256.1507; found, 256.1511. UPLC purity 97%.
14
20 2
Nꢀ(2,4ꢀdifluorobenzyl)ꢀN,2,2ꢀtrimethylbutanamide (23). From 2,4ꢀdifluorobenzaldehyde (284
mg), methanamine hydrochloride (202 mg) and 2,2ꢀdimethylbutanoyl chloride (275 mg).
1
Colorless oil. Yield, 50%. H NMR(400 MHz, CDCl ) δ 7.25ꢀ7.31 (m, 1H), 6.76ꢀ6.86 (m, 2H),
3
1
3
4
.60 (s, 2H), 3.06 (s, 3H), 1.65 (q, J= 7.6 Hz，2H), 1.26 (s, 6 H), 0.85 (t, J= 7.6 Hz，3H ). C
NMR (100 MHz, CDCl ) δ 177.23, 163.46, 163.35, 162.32,162.20, 161.00, 160.88, 159.85,
3
1
4
2
59.73, 131.10, 120.89, 120.86, 120.74, 120.71, 111.68, 11.50, 111.47,103.93, 103.68, 103.42,
+
6.43, 46.40, 43.23, 36.40, 33.23, 26.58, 9.46. HRMS (ESI) [M+H] calad for: C H F NO,
1
4
20 2
56.1507; found, 256.1522. UPLC purity 97%.
Nꢀ(2,5ꢀdifluorobenzyl)ꢀN,2,2ꢀtrimethylbutanamide (24). From 2,5ꢀ difluorobenzaldehyde (284
mg), methanamine hydrochloride (202 mg) and 2,2ꢀdimethylbutanoyl chloride (275 mg). White
1
solid. Yield, 59%. H NMR (400 MHz, CDCl ) δ7.02ꢀ6.89 (m, 3H), 4.63 (s, 2H), 3.08 (s, 3H),
3
13
1
.68 (q, J = 7.6 Hz，2H), 1.28 (s, 6 H), 0.88 (t, J = 7.6 Hz，3H). C NMR (100 MHz, CD OD)
3
δ 179.27, 161.46, 161.44, 159.53, 159.50, 159.07, 159.04, 127.99, 127.91, 127.81, 127.74,
117.83, 117.7, 117.58, 117.49, 116.55, 116.46, 116.31, 116.22, 48.18, 48.14, 44.33, 37.36, 34.00,
+
2
6.85, 9.69. HRMS (ESI) [M+H] calad for: C H F NO, 256.1507; found, 256.1509. UPLC
1
4
20 2
purity 96%.
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N,2,2ꢀtrimethylꢀNꢀ(2,3,5ꢀtrifluorobenzyl)butanamide (25). From 2,3,5ꢀtrifluorobenzaldehyde
(320 mg), methanamine hydrochloride (202 mg) and 2,2ꢀdimethylbutanoylchloride (275 mg).
1
Colorless oil. Yield, 50%. H NMR (400 MHz, CDCl ) δ 6.85ꢀ6.75 (m, 2 H), 4.64 (s, 2 H), 3.11
3
10
11
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14
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26
27
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32
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34
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60
+
(
s, 3 H), 1.68 (q, J = 7.6 Hz，2H), 1.28 (s, 6 H), 0.88 (t, J = 7.6 Hz，3H). LCꢀMS (ESI) [M+H]
1
3
calad for C H F NO, 274.14; found 274.16. C NMR (100 MHz, CDCl ) δ 177.23, 128.45,
1
4
19
3
3
1
28.36, 128.31, 128.23, 110.71, 110.48, 104.48, 104.21, 104.00, 46.66, 43.11, 36.63, 33.06,
+
+
26.34, 9.32. HRMS (ESI) [M H] calad for C H F NO, 274.1413; found, 274.1412. UPLC
1
4
19 3
purity 96%.
N,2,2ꢀtrimethylꢀNꢀ(2,4,6ꢀtrifluorobenzyl)butanamide (26). From 2,4,6ꢀtrifluorobenzaldehydhyde
(320 mg), methanamine hydrochloride (202 mg) and 2,2ꢀdimethylbutanoylchloride (275 mg).
1
Colorless oil. Yield, 62%. H NMR (400 MHz, CDCl ): δ 6.61ꢀ6.69 (m, 2 H), 4.64 (s, 2 H), 3.06
3
1
3
(
s, 3 H), 1.64 (q, 2 H, J = 7.6 Hz ), 1.24 (s, 6 H), 0.82 (t, 3 H, J = 7.6 Hz). C NMR (100 MHz,
CDCl ) δ 176.56, 163.46, 163.40,163.31, 163.29, 160.99, 160.91, 160.83, 160.80, 160.76,
3
1
60.65, 110.10, 110.06, 109.91, 109.87, 109.72, 109.68, 100.56, 100.54, 100.31, 100.28, 100.25,
+
+
1
00.02, 100.00, 43.28, 41.42, 36.08, 33.15, 26.53, 9.29. HRMS (ESI) [M H] calad
forC H F NO, 274.1413; found, 274.1416. UPLC purity 95%.
1
4
19 3
N,2,2ꢀtrimethylꢀNꢀ(pyridinꢀ4ꢀylmethyl)butanamide (27). From isonicotinaldehyde (214 mg),
methanamine hydrochloride (202mg) and 2,2ꢀdimethylbutanoyl chloride (289 mg). Light yellow
1
oil. Yield, 86%. H NMR (CDCl , 400 MHz): δ 8.55 (brs, 2 H), 7.13 (d, 2 H, J = 5.6 Hz), 4.59
3
1
3
(
s, 2 H), 3.05 (s, 3 H), 1.69 (q, 2 H, J = 7.6 Hz), 1.28 (s, 6 H), 0.89 (t, 3 H, J = 7.6 Hz). C NMR
(100 MHz, CDCl ) δ 177.26, 149.99, 147.14, 122.46, 52.64, 43.19, 36.60, 33.22, 26.49, 9.50.
3
+
HRMS (ESI) [M+H] calad for C H N O, 221.1648; found, 221.1642. UPLC purity 97%.
1
3
21
2
ACS Paragon Plus Environment
29