Structure-Activity Studies of Bis-O-Arylglycolamides: Inhibitors of the Integrated Stress Response

Article abstract of DOI:10.1002/cmdc.201500483

The integrated stress response comprises multiple signaling pathways for detecting and responding to cellular stress that converge at a single event - the phosphorylation of Ser51 on the α-subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylation of eIF2α (eIF2α-P) results in attenuation of global protein synthesis via the inhibitory effects of eIF2α-P on eIF2B, the guanine exchange factor (GEF) for eIF2. Herein we describe structure-activity relationship (SAR) studies of bis-O-arylglycolamides, first-in-class integrated stress response inhibitors (ISRIB). ISRIB analogues make cells insensitive to the effects of eIF2α-P by activating the GEF activity of eIF2B and allowing global protein synthesis to proceed with residual unphosphorylated eIF2α. The SAR studies described herein support the proposed pharmacology of ISRIB analogues as binding across a symmetrical protein-protein interface formed between protein subunits of the dimeric eIF2B heteropentamer. Stress management: Herein we describe structure-activity studies of bis-O-arylglycolamides, first-in-class Integrated Stress Response InhiBitors (ISRIB). ISRIB analogues make cells insensitive to the effects of eIF2α phosphorylation by activating eIF2B, the guanine exchange factor for eIF2. ISRIB analogues are thought to bind and stabilize a protein-protein interface in the dimeric form of the eIF2B heteropentameric protein complex.

Full text of DOI:10.1002/cmdc.201500483

DOI: 10.1002/cmdc.201500483
Full Papers
Structure–Activity Studies of Bis-O-Arylglycolamides:
Inhibitors of the Integrated Stress Response
Brian R. Hearn,[a] Priyadarshini Jaishankar,[a] Carmela Sidrauski,[b] Jordan C. Tsai,[b]
Punitha Vedantham,[a] Shaun D. Fontaine,[a] Peter Walter,*[b] and Adam R. Renslo*[a]
The integrated stress response comprises multiple signaling
pathways for detecting and responding to cellular stress that
converge at a single event—the phosphorylation of Ser51 on
the a-subunit of eukaryotic translation initiation factor 2
(eIF2a). Phosphorylation of eIF2a (eIF2a-P) results in attenua-
tion of global protein synthesis via the inhibitory effects of
eIF2a-P on eIF2B, the guanine exchange factor (GEF) for eIF2.
Herein we describe structure–activity relationship (SAR) studies
of bis-O-arylglycolamides, first-in-class integrated stress re-
sponse inhibitors (ISRIB). ISRIB analogues make cells insensitive
to the effects of eIF2a-P by activating the GEF activity of eIF2B
and allowing global protein synthesis to proceed with residual
unphosphorylated eIF2a. The SAR studies described herein
support the proposed pharmacology of ISRIB analogues as
binding across a symmetrical protein–protein interface formed
between protein subunits of the dimeric eIF2B heteropentam-
er.
Introduction
The accumulation of unfolded proteins in the endoplasmic re-
ticulum (ER) induces an adaptive response known as the un-
folded protein response (UPR).[1–3] Pharmacological modulation
of the UPR has emerged as a promising new therapeutic ap-
proach in cancer and neurodegenerative disease.[4,5] Three mo-
lecular sensors of unfolded proteins underlie the UPR
(Figure 1): inositol-requiring enzyme 1a (IRE1a), activating tran-
scription factor 6 (ATF6), and PKR-like ER kinase (PERK). Activa-
tion of PERK results in both the attenuation of global protein
synthesis and the de-repression of activating transcription
factor 4 (ATF4) mRNA translation via phosphorylation of Ser51
on the a-subunit of eukaryotic translation initiation factor 2
(eIF2a). In addition to PERK, eIF2a-Ser51 phosphorylation is
catalyzed by three other kinases: protein kinase double-strand-
ed RNA-dependent (PKR) in response to viral infection, general
control non-derepressible-2 (GCN2) in response to amino acid
starvation, and heme-regulated inhibitor (HRI) in response to
heme deficiency, oxidative stress, heat shock, or osmotic
shock.[6] These various stress-induced signaling pathways that
Figure 1. UPR and ISR sensors detect and respond to a variety of cellular
stresses such as unfolded proteins in the ER, viral infection, heme deficiency,
and amino acid starvation. The four ISR sensors (PERK, PKR, HRI, and GCN2)
are eIF2a kinases that become activated in response to cellular stress and
result in phosphorylation of eIF2a. This phosphorylated form of eIF2a
(eIF2a-P) inhibits the eIF2B-catalyzed guanine nucleotide exchange reaction
of eIF2 and results in both the attenuation of global protein synthesis and
de-repression of ATF4 mRNA translation.
converge on eIF2a-P are collectively known as the integrated
stress response (ISR) (Figure 1).
[a] Dr. B. R. Hearn, P. Jaishankar, Dr. P. Vedantham, Dr. S. D. Fontaine,
Prof. A. R. Renslo
A recent small-molecule phenotypic screen of the PERK
pathway using an ATF4-luciferase reporter identified a novel
class of small molecules that render cells insensitive to eIF2a
phosphorylation, effectively releasing the brake on global pro-
tein synthesis inhibition in stressed cells.[7] Interestingly, these
compounds were shown to act downstream of eIF2a phos-
phorylation and thus are effective antagonists of the ISR gener-
ally, not only when activated via the PERK pathway. According-
ly, this compound series was named ISRIB (for Integrated
Stress Response InhiBitors). The progenitor compound in this
series is a symmetrical bis-O-arylglycolamide (1, Figure 2). In
Department of Pharmaceutical Chemistry and
Small Molecule Discovery Center
University of California, San Francisco, CA 94158 (USA)
E-mail: adam.renslo@ucsf.edu
[b] Dr. C. Sidrauski, J. C. Tsai, Prof. P. Walter
Department of Biochemistry and Biophysics
Howard Hughes Medical Institute
University of California, San Francisco, CA 94158 (USA)
E-mail: peter@walterlab.ucsf.edu
This article is part of a Special Issue on Protein–Protein Interactions. To
view the complete issue, visit: http://onlinelibrary.wiley.com/doi/10.1002/
cmdc.v11.8/issuetoc.
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tion that produces a half-maximal response in the lumines-
cence signal following induction of ER stress.
ISRIB analogues 1–6, 15, 30–35, 44, and 45 were prepared
as previously described.[7,10] Detailed experimental procedures
for all new compounds described herein are provided in the
experimental section. For symmetric ISRIB analogues, a central
diamine core was elaborated in one or two steps to the de-
sired compounds, as illustrated in Scheme 1.
Figure 2. Structure of the prototypical ISR inhibitor 1.[7]
mice, 1 was shown to improve memory consolidation in ro-
dents[7] and conferred neuroprotection in a mouse model of
prion disease, while avoiding the pancreatic toxicity associated
with direct inhibition of PERK with kinase inhibitors.[8]
Using genetic, biochemical, and biophysical approaches, two
groups[9,10] independently identified the molecular target of
ISRIB as eIF2B—a multimeric protein complex that serves as
the guanine nucleotide exchange factor (GEF) for eIF2 and
which is inhibited by phosphorylated eIF2a. ISRIB analogues
bind to and enhance the GEF activity of eIF2B, allowing protein
synthesis to proceed with residual unphosphorylated eIF2a.[10]
Although the precise binding site remains unknown, the
weight of current evidence suggests that ISRIB analogues sta-
bilize the dimeric form[11,12] of the eIF2B pentamer by binding
across a protein–protein interface formed between eIF2Bd sub-
units or between eIF2Bb and eIF2Bd regulatory subunits of the
decamer (dimer of pentamers). This putative mechanism of
action—the stabilization of an existing protein–protein interac-
tion that may be important for GEF activity—also provides
a satisfying explanation for the exceptional cellular potency
and ligand efficiency of 1 and closely related analogues.
The notion of an ISRIB binding site that spans a symmetrical
protein–protein interface is consistent with the results of ex-
tensive structure–activity studies performed on the ISRIB series
to date, as initially described previously[7,10] and more fully de-
tailed here. Specifically, we find that both halves of the sym-
metrical structure are required for ISR antagonism and that the
spatial orientation and distance between the distal aryl ether
groups is strongly correlated with biological activity. Further-
more, the exquisite sensitivity of the aryl moieties to substitu-
ent effects indicates that these groups mediate key binding in-
teractions with the target and are important drivers of binding
affinity. By subtle optimization of ring substitution and elec-
tronics we identified new ISRIB analogues with potencies in
the picomolar range in a cell-based ATF4 translational reporter
assay.
Scheme 1. Synthetic approaches to symmetric ISRIB analogues. Reagents
and conditions: a) ClCH2C(O)Cl, DIEA, CH2Cl2; b) ArOH, K2CO3, acetone; c) Ar-
OCH2COOH, EDC, HOBt, DIEA, DMF or ArCH2OCOOH, HATU, DIEA, DMF;
d) triphosgene, ArOH, DIEA, CH2Cl2.
Pseudo-symmetric ISRIB analogues were prepared from par-
tially Boc-protected diamines as illustrated in Scheme 2. Acyla-
tion of the free amino group to install a single O-arylglycola-
mide side chain was followed by removal of the Boc group to
afford a key amine intermediate as a TFA salt. Various reactions
of this key intermediate were then used to introduce the
second O-arylglycolamide or related side chain. For example,
O-arylglycolamide or N-arylglycinamide side chains could be in-
troduced in one step by coupling the key intermediate to the
corresponding acids or acid chlorides (Scheme 2, step e). An
S-arylthioacetamide side chain was introduced in two steps via
Results and Discussion
All structure–activity studies were guided by the cell-based lu-
ciferase reporter assay described previously.[7] Briefly, this assay
employs stably transfected HEK293T cells harboring a retroviral
vector containing the ORF of firefly luciferase fused to the
5’UTR of ATF4 mRNA. Induction of ER stress with tunicamycin
in these cells leads to eIF2a phosphorylation, de-repression of
ATF4 translation and the induction of luciferase activity. The
EC50 values reported herein reflect the compound concentra-
Scheme 2. Synthetic approach to pseudo-symmetric ISRIB analogues. Re-
agents and conditions: a) 4-ClPhCH2C(O)Cl, DIEA, THF, 82%; b) TFA, Et3SiH,
CH2Cl2, H2O; c) ClCH2C(O)Cl, DIEA, CH2Cl2; d) ArOH, K2CO3, acetone or ArSH,
DIEA, CH2Cl2; e) ArXCH2COOH, EDC, HOBt, DIEA, DMF (X=O or NH) or Ar-
OCH2C(O)Cl, DIEA, THF; f) triphosgene, ArOH, DIEA, CH2Cl2; g) ArOCH2CHO,
NaBH(OAc)3, AcOH, CH2Cl2; h) ArOCH2C(NH)OEt, DIEA, EtOH.
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acylation with chloroacetyl chloride followed by reaction of
the resulting chloroacetamide intermediate with an arylthiol
(steps c and d). Carbamate-linked side chains were prepared
by reacting the key amine intermediate (where n=1) with tri-
phosgene and a substituted phenol (step f). A saturated amino
side chain could be prepared from the key amine intermediate
via reductive amination with the appropriate aldehyde build-
ing blocks (step g). Finally, an amidine-linked side chain was in-
troduced by reaction with the appropriate carboximidate re-
agent (step h).
a preference for an extended binding conformation in which
both side chains project equatorially, or nearly so, from the
plane of the central ring.
Next, we altered the nature of the glycolamide linkage be-
tween the central cyclohexane and distal aryl rings of ISRIB an-
alogues. Remarkably, we found that replacing just one of the
aryl ether bonds with analogous nitrogen, sulfur, or carbon
linkages resulted in a near or complete loss of effect in the re-
porter assay (Table 2). Of the analogues prepared, only the gly-
cinamide 10 retained measurable activity, albeit ~100-fold less
Our SAR studies began with a focus on the central ring that
joins the O-arylglycolamide side chains. The original screening
hit possessed a symmetric a 1,4-cyclohexane ring in this posi-
tion, but with undefined stereochemistry. The two possible dia-
stereomers were thus synthesized and evaluated in the report-
er assay, where the trans diastereomer 1 was found to be at
least 100-fold more potent than the cis diastereomer 2
(Table 1). Next, we explored ISRIB analogues bearing a smaller
Table 2. SAR of the glycolamide linker in ISRIB analogues.
Compd
X
Y
ATF4-luc EC50 [nm][a]
Table 1. SAR of the central core (C) of ISRIB analogues.
1
O
NH
S
S=O
SO2
CH2
CH2
O
O
O
O
O
5
700
10
11
12
13
14
15
>10000
>10000
>10000
>10000
>10000
O
CH2
[a] ATF4-luciferase reporter assay EC50 (single determinations).
Compd
C
ATF4-luc EC50 [nm][a]
1
2
3
4
5
6
7
8
9
trans-1,4-cyclohexyl
cis-1,4-cyclohexyl
trans-1,3-cyclobutyl
cis-1,3-cyclobutyl
1,4-n-butyl
1,3-n-propyl
1,4-but-2-ynyl
1,4-(Z)-but-2-enyl
1,4-phenyl
5
600
142
potent than 1. Changes in bond lengths and/or angles in
these analogues seemed insufficient to explain the dramatic ef-
fects and so we considered two alternate explanations for the
loss of activity in analogues 10–15. The first possibility was
that binding of ISRIB analogues to their target is exquisitely de-
pendent on the electronic nature of the distal aryl moieties
and that a 4-chlorophenyl ether as in 1 is optimal or nearly so.
A second possibility was that ISRIB analogues act by covalent
modification of the target, which could occur via reaction at
the a-carbon of the glycolamide, with 4-chlorophenoxide serv-
ing as the leaving group. Arguing against this possibility was
the lack of activity observed for analogue 11, in which putative
SN2 reaction should be more favorable given the presence of
a superior leaving group. To more conclusively distinguish be-
tween these possibilities, a more extensive SAR survey of the
glycolamide linker and aryl ring substitution was undertaken.
A series of ISRIB analogues bearing four-atom linkages of
various chemotypes were prepared and evaluated (Table 3).
The addition of a methyl group at the a-position of the glyco-
lamide linker (analogue 16) led to a significant loss of potency
(EC50 =210 nm vs. 5 nm for 1). Replacement of the carbonyl
function with methylene (compound 17) produced a similar
decrease in potency. Amidine 18 and carbamate 19 were the
most potent of the new analogues, with EC50 values in the
low-nanomolar range, but still less potent than 1. Carbamate
20 and amide 21, though less potent, demonstrated that at
least one carbon-linked aryl side chain can be tolerated in
ISRIB analogues. Replacing both O-arylglycolamide side chains
1000
306
>10000
>10000
200
53
[a] ATF4-luciferase reporter assay EC50 (single determinations); EC50 data
for 1–7 and 9 were reported previously.[10]
cyclobutane core (3 and 4) as well as those with acyclic linkers
of four (5) or three carbons (6). All of these analogues were
significantly less potent than 1, but the new SAR revealed
a preference for a cyclic central core over acyclic, and for four
carbon over three carbon spacing (cf. 1 vs. 3 and 5 vs. 6). This
implied that the central ring/spacer plays a crucial role in prop-
erly orienting the two side chains in space. Consistent with
this, we found that forcing the side chains further apart with
an alkynyl spacer as in analogue 7 led to a complete loss of
measureable activity. Reducing the alkyne to a (Z)-alkene as in
8 restored activity that was similar to or somewhat higher than
the saturated, unconstrained comparator 5. A simple aryl core
as in 9 turned out to be the best tolerated replacement for
trans-1,4-cyclohexane, but still tenfold less potent than 1. Thus,
the spatial orientation and distance separating the O-arylglyco-
lamide side chains is strongly correlated with the activity of
ISRIB analogues. Moreover, this preliminary SAR indicated
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Table 3. SAR of the glycolamide linker (L) in ISRIB analogues.
Table 4. SAR of aryl substitution in ISRIB analogues.
R1
R2
ATF4-luc EC50 [nm][a]
Compd
L1
L2
ATF4-luc EC50 [nm][a]
Compd
1
Cl
I
Cl
Cl
5
5
1
5
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
I
I
Cl
CF3
Cl
CCH
Cl
F
Cl
CH3
Cl
CN
Cl
51
10
13
14
158
48
270
95
CF3
CF3
CCH
CCH
F
16
17
18
210
170
60
F
CH3
CH3
CN
327
263
CN
>10000
250
>10000
>10000
>10000
OMe
OMe
S(O)Me
C(O)Me
19
20
21
22
23
30
122
OMe
Cl
Cl
[a] ATF4-luciferase reporter assay EC50 (single determinations); EC50 data
for 1 and 30–34 were reported previously.[10]
150
10 nm), alkynyl (28; 14 nm), and fluoro (30; 48 nm). Less-well-
tolerated substituents included methyl (32; 95 nm), cyano (34;
263 nm), and methoxy (36; 250 nm), while the introduction of
branched substituents such as methyl sulfoxide (38) or methyl
ketone (39) was not tolerated. These data suggested a general
preference for spheroid hydrophobes with moderate or strong-
ly electron-withdrawing character. Interestingly, the least-well-
tolerated substituents examined were those possessing a signif-
icant dipole moment (i.e., OMe, CN, C(O)Me, and S(O)Me). The
polarizability of the para substituent, by contrast, appears less
important, with both highly polarizable groups (iodo and al-
kynyl) and non-polarizable (fluoro and trifluoromethyl) groups
tolerated.
>10000
>10000
[a] ATF4-luciferase reporter assay EC50 (single determinations).
with the new (and inferior) side chains (analogues 22 and 23)
produced the expected additional loss of potency, consistent
with the putative interaction of these compounds with analo-
gous binding sites on either side of a protein–protein inter-
face.
The reasonable (mid-nanomolar) activity observed for ana-
logues such as 17, 20, and 21 argued against the possibility
that ISRIB analogues act as covalent cross-linkers, as the modi-
fied (L1) side chains in these analogues cannot serve as viable
electrophiles in biological settings. Rather, the superior poten-
cy of analogues 20 and 21 over that of 14 is most consistent
with the notion that subtle electronic effects of the distal aryl
rings drive binding and potency in ISRIB analogues. Because
none of the modified linkers examined in these studies proved
superior to the glycolamide linker, further optimization of the
ISRIB scaffold was carried out in the context of the bis-O-aryl-
glycolamide pharmacophore exemplified by 1.
The trends noted above were largely recapitulated in the
case of symmetric ISRIB analogues in which both side chains
were modified. Thus, the trend in potency for symmetrical
para-substituted analogues was in the order Cl (1; EC50 =5 nm)
> CF3 (27; 14 nm) > I (25; 51 nm) > CCH (29; 158 nm) > F (31;
270 nm) ~ CH3 (33; 327 nm) @ CN (35; >10000 nm) ~ OMe
(37; >10000 nm). In each case, modification of both side
chains produced an additive effect when compared with modi-
fication of a single side chain. While the magnitude of the ad-
ditive effect differed by substituent, the overall trend was con-
sistent and in line with the idea that each half of the molecule
engages in a similar binding interaction.
The electronics of the aryl moiety were explored systemati-
cally by replacement of one or both chlorine atoms in
1 (Table 4). Among pseudo-symmetric ISRIB analogues in
which one of the two aryl rings was altered, the best tolerated
substituents were iodo (24; EC50 =5 nm), trifluoromethyl (26;
Next we explored the introduction of multiple substituents
on the aryl ring, focusing on the groups CF3, I, F, Cl, CCH, and
CH3 that had proven reasonably well tolerated at the para po-
sition. Initial SAR studies involving multiple chloro substituents
indicated that meta/para substitution was strongly preferred
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Experimental Section
Table 5. SAR of aryl substitution in ISRIB analogues.
ATF4-luc reporter assay
HEK293T cells containing an ATF4 luciferase reporter as previously
described[7,10] were plated on polylysine-coated 96-well plates
(Greiner Bio-One, Monroe, NC, USA) at 30000 cells per well in Dul-
becco’s modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum (FBS), l-glutamine, and antibiotics (penicillin
and streptomycin). Cells were then treated the following day with
tunicamycin at 1 mgmLÀ1 along with various concentrations of
each compound for 7 h. Luminescence was measured using One
Glo (Promega, Madison, WI, USA) as specified by the manufacturer.
EC50 values were calculated by plotting log10 [mm] for each com-
pound as a function of the relative luminescence intensity or re-
sponse.
Compd
R3
R4
ATF4-luc EC50 [nm][a]
1
H
CF3
I
CH3
CCH
Cl
F
CH3
Cl
H
H
H
H
H
H
H
CH3
Cl
F
5
11
40
41
42
43
44
45
46
47
48
5.2
2.4
12
1.0
1.9
3.0
0.8
0.6
Chemistry
F
Synthesis. Unless otherwise noted, all reagents and solvents used
were commercially available. Compounds 1 and 2 were prepared
as described previously.[7] Compounds 3–6, 15, 30–35, 44, and 45
were prepared as described previously.[10] Compound 9 was pur-
chased from Specs (The Netherlands). Air- and/or moisture-sensi-
tive reactions were carried out under an argon atmosphere in
oven-dried glassware using anhydrous solvents from commercial
suppliers. Air- and/or moisture-sensitive reagents were transferred
via syringe or cannula and were introduced into reaction vessels
through rubber septa. Solvent removal was carried out with
[a] ATF4-luciferase reporter assay EC50 (single determinations); EC50 data
for 1, 45, 47, and 48 were reported previously.[10]
over ortho/para substitution and so further SAR was focused
on the meta/para substitution pattern (Table 5). Introduction of
a meta substituent in the context of para-chloro substitution
provided pseudo-symmetric ISRIB analogues with potencies
measurably superior to 1, including analogues 44 (R3 =Cl;
EC50 =1.0 nm), 45 (R3 =F; 1.9 nm), and 42 (R3 =Me; 2.4 nm).
Only slightly less potent were analogues 40, 41, and 43, bear-
ing respectively CF3, I, and CCH substituents at the meta posi-
tion (Table 5). As expected, introducing these more optimized
aryl rings in both side chains produced further enhancements
in potency, culminating in the symmetrical analogues 47 and
48 respectively, which remarkably exhibited cellular EC50 values
in the high picomolar range (EC50 =0.8 and 0.6 nm).
1
a rotary evaporator at ~10–50 Torr. H NMR spectra were recorded
on a Varian INOVA-400 400 MHz spectrometer and a Bruker Avan-
ceIII HD 400 MHz spectrometer. Chemical shifts (d) are reported in
ppm. NMR spectra were referenced relative to residual NMR sol-
vent peaks. Coupling constants (J) are reported in hertz (Hz). Micro-
wave reactions were carried out in a CEM Discover microwave re-
actor. Column chromatography was carried out using Biotage SP1
and Isolera Four flash chromatography system and SiliaSep silica
gel cartridges from Silicycle. Reversed-phase chromatography was
carried out on a Waters 2535 Separation Module with a Waters
2998 Photodiode Array Detector. Separations were carried out on
XBridge Preparative C18 19”50 mm columns at ambient tempera-
ture using a mobile phase of water/methanol containing a constant
amount (0.05%) of trifluoroacetic acid. LC–MS data were acquired
on a Waters Micromass ZQ mass spectrometer equipped with
a Waters 2795 Separation Module, a Waters 2424 Evaporative
Light-Scattering Detector, and a Waters 2996 Photodiode Array De-
tector. Separations were carried out with an XTerra MS C18 column
(5 mm, 4.6”50 mm) at ambient temperature (unregulated) using
a mobile phase of water/methanol containing a constant amount
(0.1%) of formic acid.
Conclusions
Targeting protein–protein interactions (PPI) with small mole-
cules remains a frontier of drug discovery,[13] despite the fact
that many examples of druggable PPI are now known.[14] Mole-
cules that bind at PPI interfaces often stretch the boundaries
of what might be considered “drug-like” or “beautiful”[15] mole-
cules. This is particularly true for PPI inhibitors, which typically
must bind a significant portion of the buried surface area com-
prising the targeted PPI interface, resulting in ungainly mole-
cules that bind with relatively modest ligand efficiency. In con-
trast, the stabilization of a pharmacologically relevant PPI
might well be achieved with smaller molecules binding at
higher ligand efficiencies. It would appear based on current
data that the compounds described herein fall into this latter
category. At the very least, the exceptional cellular potency of
1 and analogues like 44–48 is more easily reconciled with
a mechanism of PPI stabilization/activation than with PPI dis-
ruption/inhibition. Additional studies will be required however
to structurally define the eIF2B binding site of ISRIB analogues
and to better understand the effects of dimerization on the
GEF activity of eIF2B.
General Procedure A for amide coupling. To a solution of the car-
boxylic acid (2 equiv) in N,N-dimethylformamide were sequentially
added 1-hydroxybenzotriazole hydrate (2 equiv), 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (2 equiv), the diamine
(1.0 equiv), and N,N-diisopropylethylamine (6 equiv). The reaction
mixture was stirred at room temperature until determined com-
plete by LC–MS and then diluted with water. The precipitate
formed was washed with water and 10% diethyl ether in dichloro-
methane. The precipitate was dried in vacuo to obtain the prod-
uct.
General Procedure B for amide coupling. To a solution of the car-
boxylic acid (1 equiv) in N,N-dimethylformamide, were sequentially
added 1-hydroxybenzotriazole hydrate (1.2 equiv), 1-(3-dimethyla-
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minopropyl)-3-ethylcarbodiimide hydrochloride (1.2 equiv), 2-(4-
chlorophenoxy)-N-[(1R,4R)-4-aminocyclohexyl]acetamide trifluoro-
acetic acid (1.0 equiv), and N,N-diisopropylethylamine (2.0 equiv).
The reaction mixture was stirred at room temperature until deter-
mined complete by LC–MS and then diluted with water. The mix-
ture was vigorously vortexed, centrifuged, and the water layer was
decanted. This washing protocol was repeated with water and
then with diethyl ether. The wet solid was dissolved in dichlorome-
thane and dried over anhydrous magnesium sulfate. The solids
were removed by filtration, and the filtrate was concentrated by
rotary evaporation to obtain the product.
with diethyl ether (2”15 mL). After decanting the ether washes, re-
sidual solvent was removed under vacuum to afford 499 mg (96%)
of 2-(4-chlorophenoxy)-N-[(1R,4R)-4-aminocyclohexyl]acetamide tri-
fluoroacetic acid as a white solid. 1H NMR (400 MHz, [D6]DMSO):
d=7.95 (d, J=7.8 Hz, 1H), 7.77 (brs, 3H), 7.31 (d, J=9.0 Hz, 2H),
6.93 (d, J=9.0 Hz, 2H), 4.43 (s, 2H), 3.54 (m, 1H), 2.93 (brs, 1H),
1.90 (d, J=9.2 Hz, 2H), 1.77 (d, J=9.3 Hz, 2H), 1.31 ppm (sxt, J=
11.5 Hz, 4H); LC–MS: m/z=283 [M+H, 35Cl]+, 285 [M+H, 37Cl]+.
To a solution of 2-[(4-chlorophenyl)amino]acetic acid (9.3 mg,
0.05 mmol) in N,N-dimethylformamide (0.6 mL), were sequentially
added 1-hydroxybenzotriazole hydrate (9.2 mg, 0.06 mmol), 1-(3-di-
methylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.5 mg,
0.06 mmol), 2-(4-chlorophenoxy)-N-[(1R,4R)-4-aminocyclohexyl]ace-
tamide trifluoroacetic acid (40 mg, 0.1 mmol) and N,N-diisopropyle-
thylamine (0.035 mL, 0.2 mmol). The reaction mixture was stirred at
room temperature until determined complete by LC–MS. The reac-
tion mixture was then diluted with ethyl acetate and was washed
with 5% potassium hydrogen sulfate, water, saturated sodium bi-
carbonate solution and brine. The organic layer was dried over an-
hydrous magnesium sulfate, concentrated by rotary evaporation
and triturated with water and ether to obtain 8.4 mg (37%) of the
title compound. 1H NMR (400 MHz, [D6]DMSO): d=7.89 (d, J=
8.1 Hz, 1H), 7.73 (d, J=8.1 Hz, 1H), 7.31 (d, J=8.6 Hz, 2H), 7.06 (d,
J=8.6 Hz, 2H), 6.93 (d, J=8.8 Hz, 2H), 6.50 (d, J=8.8 Hz, 2H), 6.03
(t, J=5.8 Hz, 1H), 4.41 (s, 2H), 3.54–3.56 (m, 4H), 1.71 (d, J=
11.4 Hz, 3H), 1.13–1.34 ppm (m, 5H); LC–MS: m/z=450 [M+H,
35Cl]+, 452 [M+H, 37Cl]+.
2-(4-Chlorophenoxy)-N-{4-[2-(4-chlorophenoxy)acetamido]but-2-
yn-1-yl}acetamide (7). To a solution of but-2-yne-1,4-diamine dihy-
drochloride (0.05 g, 0.31 mmol) in a 1:1.5 mixture of tetrahydrofur-
an/water (2.5 mL) were sequentially added potassium carbonate
(0.27 g, 1.86 mmol) and 2-(4-chlorophenoxy)acetyl chloride
(0.094 mL, 0.6 mmol). The reaction mixture was stirred at room
temperature until determined complete by LC–MS. The reaction
mixture was then diluted with ethyl acetate, washed with 5% po-
tassium bisulfate, saturated sodium bicarbonate solution and
brine. The organic layer was dried over magnesium sulfate and
concentrated down to obtain a white solid. The white solid was
then triturated with diethyl ether twice to obtain 51 mg (39%) of
the title compound as an off-white solid. 1H NMR (400 MHz,
[D6]DMSO): d=8.54 (brs, 2H), 7.32 (d, J=8 Hz, 4H), 6.95 (d, J=
8 Hz, 4H), 4.47 (s, 4H), 3.91 ppm (d, J=8 Hz, 4H); LC–MS: m/z=
421 [M+H, 35Cl]+, 423 [M+H, 37Cl]+.
2-(4-Chlorophenoxy)-N-[(2Z)-4-[2-(4-chlorophenoxy)acetamido]-
2-(4-Chlorophenoxy)-N-[(1R,4R)-4-{2-[(4-chlorophenyl)sulfanyl]-
acetamido}cyclohexyl]acetamide (11). To a cooled (08C) solution
of 2-(4-chlorophenoxy)-N-[(1R,4R)-4-aminocyclohexyl]acetamide tri-
fluoroacetic acid (0.127 g, 0.32 mmol) in dichloromethane (3 mL)
were added sequentially N,N-diisopropylethylamine (0.174 mL,
1.0 mmol) and chloroacetyl chloride (0.04 mL, 0.5 mmol). The reac-
tion mixture was warmed up to room temperature and stirred for
four days. The reaction mixture was concentrated down to obtain
2-(4-chlorophenoxy)-N-[(1R,4R)-4-(2-chloroacetamido)cyclohexyl]a-
cetamide, which was used without further purification.
but-2-en-1-yl]acetamide (8). To
a solution of 7 (0.016 g,
0.037 mmol) in a 10:1 mixture of ethyl acetate/methanol (1.5 mL)
were added pyridine (0.15 mL) and Lindlar’s catalyst (0.016 mg).
The suspension was stirred under hydrogen at atmospheric pres-
sure and room temperature for 30 min. The reaction mixture was
filtered through Celite, concentrated and isolated by flash column
chromatography (5–50% acetone/dichloromethane) to obtain
1
5.8 mg (37%) of the title compound. H NMR (400 MHz, CDCl3): d=
7.32 (brs, 2H), 7.26 (d, J=8 Hz, 4H), 6.85 (d, J=8 Hz, 4H), 5.60 (d,
J=4.9 Hz, 2H), 4.44 (s, 4H), 4.04 ppm (t, J=5.6 Hz, 4H); LC–MS: m/
z=423 [M+H, 35Cl]+, 425 [M+H, 37Cl]+.
To a solution of 2-(4-chlorophenoxy)-N-[(1R,4R)-4-(2-chloroacetami-
do)cyclohexyl]acetamide (0.118 g, 0.32 mmol) in dichloromethane
(6.0 mL) were sequentially added N,N-diisopropylethylamine
(0.078 mL, 0.45 mmol) and 4-chlorothiophenol (0.043 g, 0.3 mmol).
The reaction mixture was stirred at room temperature for 1 h. The
reaction mixture was then concentrated down, triturated with di-
ethyl ether followed by 1:10 mixture of methanol/water and then
dried in vacuo to obtain 96 mg (64%) of the title compound as
2-[(4-Chlorophenyl)amino]-N-[(1R,4R)-4-[2-(4-chlorophenoxy)-
acetamido]cyclohexyl]acetamide (10). To a mixture of tert-butyl
N-[(1R,4R)-4-aminocyclohexyl]carbamate (750 mg, 3.5 mmol) in THF
(20 mL) were sequentially added N,N-diisopropylethylamine
(0.914 mL, 5.25 mmol) and 4-chlorophenoxyacetyl chloride
(0.573 mL, 3.78 mmol). The reaction mixture was vigorously stirred
at ambient temperature for 3 h then diluted with water (100 mL).
The precipitate was filtered, washed with water (2”20 mL),
washed with diethyl ether (3”20 mL) and dried under vacuum to
afford 1.1 g (82%) of tert-butyl N-[(1R,4R)-4-[2-(4-chlorophenoxy)a-
1
a light-brown solid. H NMR (400 MHz, [D6]DMSO): d=7.98 (d, J=
7.7 Hz, 1H), 7.90 (d, J=8.1 Hz, 1H), 7.29–7.32 (m, 6H), 6.93 (d, J=
9.0 Hz, 2H), 4.41 (s, 2H), 3.57 (s, 2H), 3.32 (brs, 2H), 1.70–1.72 (m,
3H), 1.16–1.32 ppm (m, 5H); LC–MS: m/z=467 [M+H, 35Cl]+, 469
[M+H, 37Cl]+.
1
cetamido]cyclohexyl]carbamate as a white solid. H NMR (400 MHz,
[D6]DMSO): d=7.88 (d, J=7.87 Hz, 1H), 7.25–7.37 (m, 2H), 6.93 (d,
J=8.97 Hz, 2H), 6.68 (d, J=7.69 Hz, 1H), 4.41 (s, 2H), 3.51 (m, 1H),
3.13 (brs, 1H), 1.72 (t, J=13.19 Hz, 4H), 1.34 (s, 9H), 1.09–1.30 ppm
(m, 4H); LC–MS: m/z=405 [M+Na, 35Cl]+, 407 [M+Na, 37Cl]+.
2-(4-Chlorophenoxy)-N-[(1R,4R)-4-[2-(4-chlorobenzenesulfinyl)-
acetamido]cyclohexyl]acetamide (12). To a solution of 11 (10 mg,
0.021 mmol) in dichloromethane (0.5 mL) was added 3-chloroper-
oxybenzoic acid (4.7 mg, 0.021 mmol). The reaction mixture was
stirred at room temperature for 1 h and then partitioned between
dichloromethane and saturated sodium bicarbonate solution. The
organic layer was then washed with brine, filtered and concentrat-
ed to obtain 4 mg (38%) of the title compound as a white solid.
1H NMR (400 MHz, CDCl3): d=7.52 (s, 2H), 7.25 (s, 2H), 6.84 (d, J=
9.0 Hz, 2H), 6.65 (d, J=7.9 Hz, 1H), 6.29 (d, J=8.2 Hz, 1H), 4.42 (s,
2H), 3.82–3.84 (m, 1H), 3.65–3.73 (m, 2H), 3.38 (d, J=15 Hz, 1H),
To a suspension of tert-butyl N-[(1R,4R)-4-[2-(4-chlorophenoxy)acet-
amido]cyclohexyl]carbamate (500 mg, 1.31 mmol) in dichlorome-
thane (9 mL) were sequentially added triethylsilane (0.300 mL,
1.88 mmol), water (0.200 mL, 11.1 mmol) and trifluoroacetic acid
(3.00 mL, 39.2 mmol). The reaction mixture was vigorously stirred
at ambient temperature for 30 min then the solvent was removed
by rotary evaporation. The resulting colorless oil was triturated
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2.02–2.06 (m, 3H), 1.82 (brs, 1H), 1.22–1.36 ppm (m, 4H); LC–MS:
m/z=483 [M+H, 35Cl]+, 485 [M+H, 37Cl]+.
ed by reversed-phase column chromatography to obtain 2 mg
(2%) of the title compound as a trifluoroacetate salt. LC–MS: m/z=
437 [M+H, 35Cl]+, 439 [M+H, 37Cl]+.
2-(4-Chlorophenoxy)-N-[(1R,4R)-4-[2-(4-chlorobenzenesulfonyl)-
acetamido]cyclohexyl]acetamide (13). To a solution of 11 (20 mg,
0.043 mmol) in dichloromethane (1.0 mL) was added 3-chloroper-
oxybenzoic acid (19.8 mg, 0.086 mmol). The reaction mixture was
stirred at room temperature overnight and then partitioned be-
tween dichloromethane and 10% aqueous sodium thiosulfate. The
organic layer was then washed with saturated sodium bicarbonate
solution, brine and concentrated to obtain 8 mg (37%) of the title
2-(4-Chlorophenoxy)-N-[(1R,4R)-4-[2-(4-chlorophenoxy)ethan-
imidamido]cyclohexyl]acetamide (18). To a solution of 4-chloro-
phenoxyacetonitrile (0.42 g, 2.49 mmol) in diethyl ether (2.0 mL)
were sequentially added 4m solution of hydrochloric acid in diox-
ane (0.59 mL, 2.36 mmol) and ethanol (0.145 mL, 2.49 mmol). The
reaction mixture was stirred at room temperature overnight and
concentrated to obtain 0.49 g of crude ethyl 2-(4-chlorophenoxy)-
ethanecarboximidate hydrochloride as white solid that was used
without further purification.
1
compound as a white solid. H NMR (400 MHz, [D6]DMSO): d=8.10
(d, J=8 Hz, 1H), 7.89 (d, J=8 Hz, 1H), 7.82 (d, J=8.6 Hz, 2H), 7.70
(d, J=8.4 Hz, 2H), 7.30 (d, J=8.8 Hz, 2H), 6.93 (d, J=8.8 Hz, 2H),
4.41 (s, 2H), 4.22 (s, 2H), 1.68–1.71 ppm (m, 2H); LC–MS: m/z=499
[M+H, 35Cl]+, 501 [M+H, 37Cl]+.
To a solution of 2-(4-chlorophenoxy)-N-[(1R,4R)-4-aminocyclohexyl]-
acetamide (0.100 g, 0.3 mmol) in ethanol (1.0 mL) were added N,N-
diisopropylethylamine (0.063 mL, 0.4 mmol) and ethyl 2-(4-chloro-
phenoxy)ethanecarboximidate (0.082 g, 0.4 mmol). The mixture
was stirred at room temperature for 24 h, concentrated and sus-
pended in diethyl ether (10.0 mL). The suspension was vortexed,
and the ether layer was decanted. The ether washes were repeated
twice to obtain a white gel that was suspended in methanol
(0.15 mL) and dichloromethane (5.0 mL). The suspension was
gently heated and cooled to room temperature. The suspension
was vortexed, the organic solvents were decanted and the solids
were dissolved in methanol (5.0 mL). To this methanolic mixture
was added Silicycle Si-TMA acetate resin (0.9 g) and stirred at room
temperature overnight. The mixture was filtered, filtrate concen-
trated and triturated vigorously with hot diethyl ether (10 mL)
twice. The resulting white solids were filtered and dried to obtain
3-(4-Chlorophenyl)-N-[(1R,4R)-4-[3-(4-chlorophenyl)propanami-
do]cyclohexyl]propanamide (14). To a solution of the 3-(4-chloro-
phenyl)propionic acid (0.018 g, 0.101 mmol) in N,N-dimethylforma-
mide (0.17 mL) were sequentially added 1-hydroxybenzotriazole
hydrate (0.094 g, 0.70 mmol), 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (0.014 g, 0.101 mmol), 2-(4-chlorophe-
noxy)-N-[(1R,4R)-4-aminocyclohexyl]acetamide trifluoroacetic acid
(0.04 g, 0.101 mmol) and N,N-diisopropylethylamine (0.027 mL,
0.15 mmol). The reaction mixture was stirred at room temperature
until determined complete by LC–MS and subjected to conditions
described in procedure A to obtain 9 mg (20%) of the title com-
pound. 1H NMR (400 MHz, [D6]DMSO): d=7.90 (d, J=7.7 Hz, 1H),
7.67 (d, J=7.5 Hz, 1H), 7.29 (d, J=9.0 Hz, 4H), 7.18 (d, J=7.7 Hz,
2H), 6.93 (d, J=8.2 Hz, 2H), 4.41 (s, 2H), 3.42–3.54 (m, 2H), 2.75 (t,
J=7.1 Hz, 2H), 2.29 (t, J=7.1 Hz, 2H), 1.70 (d, J=10.4 Hz, 4H),
1.11–1.32 ppm (m, 4H); LC–MS: m/z=447 [M+H, 35Cl]+, 449 [M+
H, 37Cl]+.
1
79 mg (61%) of the product as an acetate salt. H NMR (400 MHz,
[D6]DMSO): d=7.93 (d, J=8.1 Hz, 1H), 7.32 (dd, J=8.3, 6.6 Hz, 4H),
6.94 (dd, J=8.8, 6.6 Hz, 4H), 4.41 (s, 2H), 4.42 (s, 2H), 3.58 (brs,
2H), 1.72–1.75 (m, 3H), 1.17–1.36 ppm (m, 5H); LC–MS: m/z=450
[M+H, 35Cl]+, 452 [M+H, 37Cl]+.
2-(4-Chlorophenoxy)-N-[(1R,4R)-4-[2-(4-chlorophenoxy)acetami-
do]cyclohexyl]propanamide (16). To a solution of 2-(4-chlorophe-
noxy)propionic acid (10 mg, 0.05 mmol) in N,N-dimethylformamide
(1.0 mL), were sequentially added 1-hydroxybenzotriazole hydrate
(9 mg, 0.06 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (11 mg, 0.06 mmol), 2-(4-chlorophenoxy)-N-[(1R,4R)-
4-Chlorophenyl N-{[(1R,4R)-4-[2-(4-chlorophenoxy)acetamido]cy-
clohexyl]methyl}carbamate (19). To a suspension of tert-butyl N-
{[(1R,4R)-4-aminocyclohexyl]methyl}carbamate (0.068 g, 0.3 mmol)
was in tetrahydrofuran (2.0 mL) were added N,N-diisopropylethyla-
mine (0.058 g, 0.4 mmol) and 4-chlorophenoxyacetyl chloride
(0.046 mL, 0.3 mmol) and stirred at room temperature overnight.
The reaction mixture was partitioned in 1:1 mixture of ethyl ace-
tate/5% aqueous potassium hydrogen sulfate The organic layer
was washed with brine, dried over magnesium sulfate, concentrat-
ed, triturated with 2:1 mixture of hexanes/diethyl ether (2”10 mL)
and dried to obtain 80 mg (68%) of tert-butyl N-{[(1R,4R)-4-[2-(4-
chlorophenoxy)acetamido]cyclohexyl]methyl}carbamate as a white
solid. 1H NMR (400 MHz, [D6]DMSO): d=7.84 (d, J=8.2 Hz, 1H),
7.31 (d, J=9 Hz, 2H), 6.93 (d, J=9 Hz, 2H), 6.77 (brs, 1H), 4.40 (s,
2H), 3.51–3.53 (brs, 2H), 2.73 (t, J=6.3 Hz, 2H), 1.63–1.73 (m, 4H),
1.34 (s, 9H), 1.13–1.22 (m, 2H), 0.83–0.92 ppm (m, 2H); LC–MS: m/
z=397 [M+H, 35Cl]+, 399 [M+H, 37Cl]+.
4-aminocyclohexyl]acetamide
trifluoroacetic
acid
(20 mg,
0.05 mmol) and N,N-diisopropylethylamine (0.013 mL, 0.075 mmol).
The reaction mixture was stirred at room temperature until deter-
mined complete by LC–MS and subjected to conditions described
in procedure B to obtain 17 mg (73%) of the title compound as
1
a white solid. H NMR (400 MHz, [D6]DMSO): d=7.93 (d, J=8.1 Hz,
1H), 7.90 (d, J=8.2 Hz, 1H), 7.28–7.32 (m, 4H), 6.86–6.94 (m, 4H),
4.60 (q, J=6.4 Hz, 1H), 4.41 (s, 2H), 3.53 (brs, 2H), 1.62–1.72 (m,
4H), 1.37 (d, J=6.4 Hz, 3H), 1.20–1.31 ppm (m, 4H); LC–MS: m/z=
465 [M+H, 35Cl]+, 467 [M+H, 37Cl]+.
2-(4-Chlorophenoxy)-N-[(1R,4R)-4-{[2-(4-chlorophenoxy)ethyl]-
amino}cyclohexyl]acetamide (17). To a suspension of 2-(4-chloro-
To a solution of tert-butyl N-{[(1R,4R)-4-[2-(4-chlorophenoxy)aceta-
mido]cyclohexyl]methyl}carbamate (0.079 g, 0.2 mmol) in dichloro-
methane (4.0 mL) were sequentially added triethylsilane (0.1 mL,
0.6 mmol), water (0.1 mL) and trifluoroacetic acid (1.0 mL). The mix-
ture was stirred at room temperature for 30 min, concentrated, tri-
turated with diethyl ether (10.0 mL) and dried to obtain 76 mg
(93%) of 2-(4-chlorophenoxy)-N-[(1R,4R)-4-(aminomethyl)cyclo-
hexyl]acetamide as trifluoroacetic acid salt. LC–MS: m/z=297
[M+H, 35Cl]+, 299 [M+H, 37Cl]+.
phenoxy)-N-[(1R,4R)-4-aminocyclohexyl]acetamide
trifluoroacetic
acid (0.119 g, 0.3 mmol) in dichloromethane (2.0 mL) was added
N,N-diisopropylethylamine (0.07 mL, 0.4 mmol). After stirring at
room temperature for 10 min, 2-(4-chlorophenoxy)acetaldehyde
(0.034 g, 0.2 mmol) and acetic acid (0.4 mL) were sequentially
added, followed by addition of sodium triacetoxyborohydride
(0.042 g, 0.2 mmol) after 1 h. The reaction mixture was stirred at
room temperature overnight, diluted with 10:1 mixture of dichloro-
methane/methanol, washed with dilute ammonium hydroxide,
dilute potassium hydrogen sulfate solution, sodium bicarbonate
solution and brine. The organic layer was concentrated and isolat-
To a solution of 4-chlorophenyl chloroformate (0.009 mL, 0.1 mmol)
in tetrahydrofuran (1.0 mL) were added dropwise a mixture of 2-(4-
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Full Papers
chlorophenoxy)-N-[(1R,4R)-4-(aminomethyl)cyclohexyl]acetamide
trifluoroacetic acid (0.025 g, 0.1 mmol) and N,N-diisopropylethyla-
mine (0.026 mL, 0.2 mmol) in tetrahydrofuran (0.5 mL). The mixture
was stirred at room temperature for 2 h and then partitioned be-
tween a mixture of dichloromethane/5% aqueous potassium hy-
drogen sulfate. The organic layer was dried, concentrated and puri-
fied by flash column chromatography (0–50% acetone/dichlorome-
thane) to obtain 13 mg (47%) of the title compound as a white
solid. 1H NMR (400 MHz, [D6]DMSO): d=7.87 (d, J=8.1 Hz, 1H),
7.80 (t, J=5.8 Hz, 1H), 7.39 (d, J=8.8 Hz, 2H), 7.31 (d, J=8.8 Hz,
2H), 7.11 (d, J=8.8 Hz, 2H), 6.94 (d, J=9 Hz, 2H), 4.41 (s, 2H), 3.53
(brs, 1H), 2.89 (t, J=6.1 Hz, 2H), 1.73 (t, J=10.4 Hz, 4H), 1.17–1.26
(m, 2H), 0.91–1.00 ppm (m, 2H); LC–MS: m/z=451 [M+H, 35Cl]+,
453 [M+H, 37Cl]+.
propylethylamine (0.064 mL, 0.37 mmol) in dichloromethane
(1.0 mL) was added dropwise the pre-formed chloroformate solu-
tion and was stirred at room temperature overnight. To the reac-
tion mixture was added methanol (0.3 mL) to quench unreacted
triphosgene and concentrated to dryness. The residue was washed
with water (2”10 mL), diethyl ether (3”10 mL) and dried to obtain
1
25 mg (32%) of the title compound as an off-white solid. H NMR
(400 MHz, [D6]DMSO): d=7.32–7.41 (m, 8H), 7.19 (d, J=7.5 Hz,
2H), 4.95 (s, 4H), 3.18 (brs, 2H), 1.74–1.76 (m, 3H), 1.18–1.23 ppm
(m, 5H); LC–MS: m/z=449 [M+H]+.
4-Chlorophenyl
N-{[(1R,4R)-4-{[(4-chlorophenoxycarbonyl)ami-
no]methyl}cyclohexyl]methyl}carbamate (23). To a solution of 4-
chlorophenyl N-{[(1R,4R)-4-(aminomethyl)cyclohexyl]methyl}carba-
mate (0.03 g, 0.1 mmol) and N,N-diisopropylethylamine (0.032 mL,
0.2 mmol) in tetrahydrofuran (0.5 mL) was added dropwise 4-chlor-
ophenyl chloroformate (0.01 mL, 0.1 mmol). After stirring at room
temperature for 20 min, water (3 mL) was added to the reaction
mixture. The precipitate was washed with water (3”2 mL), diethyl
ether (4”2 mL) and dried to obtain 23 mg (70%) of the title com-
(4-Chlorophenyl)methyl N-[(1R,4R)-4-[2-(4-chlorophenoxy)aceta-
mido]cyclohexyl]carbamate (20). To a cooled solution (08C) of 4-
chlorobenzyl alcohol (0.022 g, 0.2 mmol) and triphosgene (0.015 g,
0.1 mmol) in dichloromethane (1.0 mL), was added dropwise N,N-
diisopropylethylamine (0.026 mL, 0.15 mmol). After warming up to
room temperature and stirring for 1 h, the reaction mixture was
added dropwise to a solution of 2-(4-chlorophenoxy)-N-[(1R,4R)-4-
aminocyclohexyl]acetamide (0.040 g, 0.1 mmol) and N,N-diisopro-
pylethylamine (0.052 mL, 0.3 mmol) in dichloromethane (1.0 mL).
After stirring the mixture at room temperature for 2 h, the reaction
was quenched with methanol (0.5 mL) and partitioned between
1:1 dichloromethane/5% aqueous potassium hydrogen sulfate. The
organic layer was concentrated and the resulting solid was washed
with diethyl ether thrice to obtain 29 mg (64%) of the title com-
1
pound as a white solid. H NMR (400 MHz, [D6]DMSO): d=7.79 (t,
J=5.7 Hz, 2H), 7.39 (d, J=8.8 Hz, 4H), 7.11 (d, J=8.8 Hz, 4H), 3.29
(brs, 2H), 2.89 (t, J=6.2 Hz, 4H), 1.73 (d, J=7 Hz, 4H), 0.85–
0.89 ppm (m, 4H); LC–MS: m/z=451 [M+H]+.
2-(4-Iodophenoxy)-N-[(1R,4R)-4-[2-(4-chlorophenoxy)acetamido]-
cyclohexyl]acetamide (24). To a suspension of 2-(4-chlorophe-
noxy)-N-[(1R,4R)-4-(2-chloroacetamido)cyclohexyl]acetamide
(0.085 g, 0.2 mmol,) in acetone (5.0 mL) were added potassium car-
bonate (0.070 g, 0.4 mmol) and 4-iodophenol (0.092 g, 0.4 mmol)
and heated at 1208C for 1 h in the microwave reactor. The reaction
mixture was diluted with water (6 mL) and resulting solids were
washed with water (5 mL) and diethyl ether (5 mL). The solids were
suspended in dichloromethane and concentrated by rotary evapo-
ration to obtain 9.5 mg (52%) of the title compound as a brown
solid. 1H NMR (400 MHz, [D6]DMSO): d=7.91 (d, J=7.9 Hz, 2H),
7.57 (d, J=9.0 Hz, 2H), 7.31 (d, J=9.0 Hz, 2H), 6.94 (d, J=9.0 Hz,
2H), 6.76 (d, J=9.0 Hz, 2H), 4.40–4.42 (m, 4H), 3.55 (brs, 2H), 1.74
(d, J=5.9 Hz, 4H), 1.25–1.35 ppm (m, 4H); LC–MS: m/z=543 [M+
H]+.
1
pound as a white solid. H NMR (400 MHz, [D6]DMSO): d=7.89 (d,
J=7.9 Hz, 1H), 7.40 (d, J=8 Hz, 1H), 7.33 (d, J=8.6 Hz, 1H), 7.31
(d, J=9.0 Hz, 2H), 7.20 (d, J=7.7 Hz, 1H), 6.93 (d, J=9.0 Hz, 2H),
4.96 (s, 2H), 4.41 (s, 2H), 3.51–3.53 (m, 1H), 3.20–3.23 (m, 1H),
1.70–1.79 (m, 4H), 1.18–1.33 ppm (m, 4H); LC–MS: m/z=451 [M+
H, 35Cl]+, 453 [M+H, 37Cl]+.
2-(4-Chlorophenoxy)-N-[(1R,4R)-4-{[2-(4-chlorophenyl)acetami-
do]methyl}cyclohexyl]acetamide (21). To a solution of 4-chloro-
phenylacetic acid (0.010 g, 0.1 mmol) in N,N-dimethylformamide
(0.5 mL) were added sequentially 1-hydroxybenzotriazole hydrate
(0.011 g, 0.1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (0.014 g, 0.1 mmol), 2-(4-chlorophenoxy)-N-[(1R,4R)-
4-(aminomethyl)cyclohexyl]acetamide (0.025 g, 0.1 mmol), and N,N-
diisopropylethylamine (0.026 mL, 0.2 mmol). The reaction mixture
was stirred at room temperature until determined complete by
LC–MS. The reaction mixture was then diluted with ethyl acetate
and was washed with 5% potassium hydrogen sulfate, water, satu-
rated sodium bicarbonate solution and brine. The organic layer
was dried over anhydrous magnesium sulfate, concentrated by
rotary evaporation and triturated with water and ether to obtain
9.3 mg (33%) of the title compound as a white solid. 1H NMR
(400 MHz, [D6]DMSO): d=7.99 (brs, 1H), 7.85 (d, J=8.1 Hz, 1H),
7.31 (dd, J=8.4, 4.2 Hz, 4H), 7.24 (d, J=8.0 Hz, 2H), 6.94 (d, J=
8.8 Hz, 2H), 4.41 (s, 2H), 3.53 (brs, 2H), 3.37 (s, 2H), 2.86 (t, J=
6.0 Hz, 2H), 1.64–1.73 (m, 4H), 1.13–1.19 (m, 2H), 0.89–0.95 ppm
(m, 2H); LC–MS: m/z=449 [M+H]+.
2-(4-Iodophenoxy)-N-[(1R,4R)-4-[2-(4-iodophenoxy)acetamido]cy-
clohexyl]acetamide (25). To a suspension of 2-chloro-N-[(1R,4R)-4-
(2-chloroacetamido)cyclohexyl]acetamide (0.2 g, 0.7 mmol) and po-
tassium carbonate (0.517 g, 3.7 mmol) in N,N-dimethylformamide
(0.058 mL), were added 4-iodophenol (0.659 g, 3.0 mmol) and
sodium iodide (0.006 g, 0.037 mmol), and the mixture was heated
in the microwave at 1008C for 30 min. The reaction mixture was di-
luted with water (20 mL). The mixture was centrifuged and the
water layer was decanted. This washing protocol was repeated
thrice and the resulting wet solid was concentrated down with tol-
uene (10 mL) in a rotary evaporator. The residual product was
washed with diethyl ether (10 mL) and concentrated using rotary
evaporation to afford 0.28 g (59%) of the title compound as
1
a brown solid. H NMR (400 MHz, [D6]DMSO): d=7.90 (d, J=8.1 Hz,
2H), 7.57 (d, J=8.8 Hz, 4H), 6.76 (d, J=8.8 Hz, 4H), 4.40 (s, 4H),
3.54 (brs, 2H), 1.72 (d, J=5.9 Hz, 4H), 1.27–1.32 ppm (m, 4H); LC–
MS: m/z=634 [M+H]+.
(4-Chlorophenyl)methyl
N-[(1R,4R)-4-({[(4-chlorophenyl)meth-
oxy]carbonyl}amino) cyclohexyl]carbamate (22). To a solution of
4-chlorobenzyl alcohol (0.052 g, 0.4 mmol) in dichloromethane
(3.0 mL) was added triphosgene (0.036 g, 0.1 mmol) and cooled to
08C. To this mixture was added dropwise a solution of N,N-diiso-
propylethylamine (0.064 mL, 0.37 mmol) in dichloromethane
(1.0 mL) and stirred at room temperature for 2 h. To a mixture of
(1R,4R)-cyclohexane-1,4-diamine (0.020 g, 0.2 mmol) and N,N-diiso-
N-[(1R,4R)-4-[2-(4-Chlorophenoxy)acetamido]cyclohexyl]-2-[4-(tri-
fluoromethyl)phenoxy]acetamide (26). To a solution of 2-[4-(tri-
fluoromethyl)phenoxy]acetic acid (0.011 g, 0.052 mmol) in N,N-di-
methylformamide (0.3 mL), were sequentially added 1-hydroxyben-
zotriazole hydrate (0.008 g, 0.055 mmol), 1-(3-dimethylaminoprop-
yl)-3-ethylcarbodiimide hydrochloride (0.011 g, 0.055 mmol), 2-(4-
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chlorophenoxy)-N-[(1R,4R)-4-aminocyclohexyl]acetamide trifluoro-
acetic acid (0.020 g, 0.05 mmol) and N,N-diisopropylethylamine
(0.021 mL, 0.12 mmol). The reaction mixture was stirred at room
temperature until determined complete by LC–MS and then sub-
jected to conditions described in procedure B to obtain 17.7 mg
(69%) of the title compound as a white solid. 1H NMR (400 MHz,
[D6]DMSO): d=7.98 (d, J=7.9 Hz, 1H), 7.91 (d, J=8.1 Hz, 1H), 7.64
(d, J=9.0 Hz, 2H), 7.31 (d, J=8.0 Hz, 2H), 7.09 (d, J=8.8 Hz, 2H),
6.94 (d, J=8.0 Hz, 2H), 4.53 (s, 2H), 4.42 (s, 2H), 3.56 (brs, 2H),
1.74 (d, J=7.9 Hz, 4H), 1.28–1.33 ppm (m, 4H); LC–MS: m/z=485
[M+H, 35Cl]+, 487 [M+H, 37Cl]+.
[(1R,4R)-4-[2-(4-iodophenoxy)acetamido]cyclohexyl]acetamide (25)
(0.2 g,
0.3 mmol),
dichlorobis(triphenylphosphine)palladium
(0.023 g, 0.03 mmol) and copper(I) Iodide (0.03 mg, 0.015 mmol) in
a degassed mixture of triethylamine/N,N-dimethylformamide (1:1,
2 mL), was added a solution of ethynyltrimethylsilane (0.178 mL,
1.5 mmol) in triethylamine/N,N-dimethylformamide (1:1, 2 mL) as
a single portion and the mixture was stirred at room temperature
for 4 h. The reaction mixture was then diluted with ethyl acetate
(25 mL) and washed with 5% aqueous potassium hydrogen sulfate
solution (20 mL). The emulsion formed was filtered through
a Celite plug and washed with dichloromethane (150 mL). The or-
ganic layers were dried over magnesium sulfate, filtered and con-
centrated down to a brown solid. Purification by flash column
chromatography (0–80% acetone/dichloromethane) afforded
58 mg (32%) of 2-{4-[2-(trimethylsilyl)ethynyl]phenoxy}-N-[4-(2-{4-
[2-(trimethylsilyl)ethynyl]phenoxy}acetamido)cyclohexyl]acetamide
as a brown solid. 1H NMR (400 MHz, [D6]DMSO): d=7.92 (d, J=
8.1 Hz, 2H), 7.36 (d, J=8.8 Hz, 4H), 6.89 (d, J=8.8 Hz, 4H), 4.44 (s,
4H), 1.73 (d, J=5.9 Hz, 4H), 1.29–1.32 (m, 4H), 0.18 ppm (s, 18H);
LC–MS: m/z=575 [M+H]+.
N-[(1R,4R)-4-{2-[4-(Trifluoromethyl)phenoxy]acetamido}cyclohex-
yl]-2-[4-(trifluoromethyl)phenoxy]acetamide (27). To a solution of
4-(trifluoromethy)phenoxyacetic acid (0.19 g, 1.76 mmol) in N,N-di-
methylformamide (1.75 mL) were sequentially added 1-hydroxy-
benzotriazole hydrate (0.12 g, 1.76 mmol), 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (0.175 g, 1.76 mmol),
(1R,4R)-cyclohexane-1,4-diamine (0.05 g, 0.87 mmol) and N,N-diiso-
propylethylamine (0.47 mL, 5.62 mmol). The reaction mixture was
subjected to conditions described in procedure A to obtain 0.15 g
(66%) of the title compound. 1H NMR (400 MHz, [D6]DMSO): d=
7.98 (d, J=7.5 Hz, 2H), 7.63 (d, J=8.2 Hz, 4H), 7.09 (d, J=8.1 Hz,
4H), 4.53 (s, 4H), 3.57 (brs, 2H), 1.75–1.76 (m, 4H), 1.29–1.31 ppm
(m, 4H); LC–MS: m/z=519 [M+H]+.
To a solution of (trimethylsilyl)ethynyl]phenoxy}-N-[4-(2-{4-[2-(trime-
thylsilyl)ethynyl]phenoxy}acetamido)cyclohexyl]acetamide (0.020 g,
0.035 mmol) in 1:1 mixture of methanol/tetrahydrofuran (2.0 mL),
was added potassium carbonate (0.014 g, 0.1 mmol). The reaction
mixture was stirred at ambient temperature for 20 min and then
concentrated down to dryness. The residue was triturated thrice
with water (5 mL) and the resulting wet solid was suspended in
toluene (5 mL) and concentrated down to dryness to obtain 12 mg
(80%) of the title compound as a tan solid. 1H NMR (400 MHz,
[D6]DMSO): d=7.92 (d, J=7.9 Hz, 2H), 7.38 (d, J=8.8 Hz, 4H), 6.91
(d, J=8.6 Hz, 4H), 4.45 (s, 4H), 4.00 (s, 2H), 1.74 (d, J=5.9 Hz, 4H),
1.28–1.33 ppm (m, 4H); LC–MS: m/z=431 [M+H]+.
2-(4-Ethynylphenoxy)-N-[(1R,4R)-4-[2-(4-chlorophenoxy)acetami-
do]cyclohexyl]acetamide (28). To a solution of 2-(4-iodophenoxy)-
N-[(1R,4R)-4-[2-(4-chlorophenoxy)acetamido] cyclohexyl]acetamide
(24) (0.02 g, 0.036 mmol), dichlorobis(triphenylphosphine)palladi-
um (0.003 g, 0.0036 mmol) and copper(I) Iodide (0.003 g,
0.016 mmol) in a degassed mixture of triethylamine/N,N-dimethyl-
formamide (1:1, 0.4 mL), was added a solution of ethynyltrimethyl-
silane (0.009 mL, 0.1 mmol) in triethylamine/N,N-dimethylforma-
mide (1:1, 0.4 mL) as a single portion and the mixture was stirred
at room temperature for 1 h. The reaction mixture was then dilut-
ed with ethyl acetate (25 mL) and washed with 5% aqueous potas-
sium hydrogen sulfate solution (20 mL) and brine (20 mL). The or-
ganic phase was dried over magnesium sulfate, filtered and con-
centrated to obtain a brown colored oil. Purification by flash
column chromatography (20–100% ethyl acetate/hexanes) afford-
ed 20 mg (105%) of N-{4-[2-(4-chlorophenoxy)acetamido]cyclohex-
yl}-2-{4-[2-(trimethylsilyl)ethynyl]phenoxy}acetamide (contaminated
2-(4-Methoxyphenoxy)-N-[(1R,4R)-4-[2-(4-methylphenoxy)aceta-
mido]cyclohexyl]acetamide (36). To a solution of 4-(methoxy)phe-
noxyacetic acid (0.014 g, 0.076 mmol) in N,N-dimethylformamide
(0.3 mL) were sequentially added 1-hydroxybenzotriazole hydrate
(0.01 g, 0.076 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide hydrochloride (0.015 g, 0.076 mmol), (1R,4R)-cyclohexane-1,4-
diamine (0.03 g, 0.076 mmol) and N,N-diisopropylethylamine
(0.04 mL, 0.23 mmol). The reaction mixture was then diluted with
5% methanol/dichloromethane and was washed with 5% potassi-
um hydrogen sulfate, water, saturated sodium bicarbonate solution
and brine. The organic layer was dried over anhydrous magnesium
sulfate, filtered through a silica plug and concentrated by rotary
1
with triphenylphosphine oxide) as a white solid. H NMR (400 MHz,
CDCl3): d=7.44 (d, J=8.6 Hz, 2H), 7.27–7.30 (m, 2H), 6.84–6.88 (m,
2H), 6.34 (d, J=7.9 Hz, 2H), 4.46 (s, 2H), 4.44 (s, 2H), 3.86 (brs,
2H), 2.05 (d, J=5.5 Hz, 4H), 1.32–1.34 (m, 4H), 0.25 ppm (s, 9H);
LC–MS: m/z=513 [M+H]+.
1
evaporation to obtain 34 mg (58%) of the title compound. H NMR
(400 MHz, CDCl3): d=7.23–7.27 (m, 2H), 6.78–6.83 (m, 6H), 6.42 (d,
J=8.2 Hz, 1H), 6.35 (d, J=8.1 Hz, 1H), 4.38–4.40 (m, 4H), 3.84 (brs,
2H), 2.02 (d, J=5.5 Hz, 4H), 1.27–1.38 ppm (m, 4H); LC–MS: m/z=
447 [M+H]+.
To a solution of N-{4-[2-(4-chlorophenoxy)acetamido]cyclohexyl}-2-
{4-[2-(trimethylsilyl)ethynyl]phenoxy}acetamide
(0.015 g,
0.03 mmol) in a 1:1 mixture of methanol/tetrahydrofuran (2.0 mL),
was added potassium carbonate (0.006 g, 0.04 mmol). The reaction
mixture was stirred at ambient temperature for 30 min and then
concentrated down to dryness. The residue was triturated thrice
with water (5.0 mL) and the resulting wet solid was suspended in
toluene (5.0 mL) and concentrated down to dryness to obtain
10 mg (78%) of the title compound as a white solid. 1H NMR
(400 MHz, CDCl3): d=7.45 (d, J=8.6 Hz, 2H), 7.25–7.28 (m, 2H),
6.85 (dd, J=8.6, 5.1 Hz, 4H), 6.32 (d, J=7.9 Hz, 2H), 4.45 (s, 2H),
4.42 (s, 2H), 3.85 (brs, 2H), 3.01 (s, 2H), 2.04 (d, J=6 Hz, 4H), 1.24–
1.38 ppm (m, 4H); LC–MS: m/z=441 [M+H]+.
2-(4-Methoxyphenoxy)-N-[(1R,4R)-4-[2-(4-methoxyphenoxy)acet-
amido]cyclohexyl]acetamide (37). To a solution of the 2-(4-me-
thoxyphenoxy)acetic acid (0.32 g, 1.75 mmol) in N,N-dimethylfor-
mamide (1 mL) were sequentially added 1-hydroxybenzotriazole
hydrate (0.236 g, 1.75 mmol), 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (0.345 g, 1.75 mmol), trans-1,4-diaminocy-
clohexane (0.1 g, 0.87 mmol) and N,N-diisopropylethylamine
(1.0 mL, 5.2 mmol). The reaction mixture was stirred at 528C until
determined complete by LC–MS and then subjected to conditions
described in procedure A to obtain 150 mg (62%) of the title com-
pound. 1H NMR (400 MHz, CDCl3): d=6.83 (s, 8H), 6.41 (d, J=
8.1 Hz, 2H), 4.39 (m, 4H), 3.83 (brs, 2H), 2.03 (d, J=6.4 Hz, 4H),
2-(4-Ethynylphenoxy)-N-{4-[2-(4-ethynylphenoxy)acetamido]cy-
clohexyl}acetamide (29). To a solution of 2-(4-iodophenoxy)-N-
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1.28–1.38 ppm (m, 4H); LC–MS: m/z=443 [M+H, 35Cl]+, 445 [M+
title compound as a tan solid. 1H NMR (400 MHz, [D6]DMSO): d=
7.93 (t, J=8.7 Hz, 2H), 7.43–7.48 (m, 2H), 7.31 (d, J=8.8 Hz, 2H),
6.93–6.99 (m, 3H), 4.40–4.43 (m, 4H), 3.55 (brs, 2H), 3.28 (s, 3H),
1.74 (d, J=5.5 Hz, 4H), 1.31 ppm (brs, 4H); LC–MS: m/z=577 [M+
H, 35Cl]+, 579 [M+H, 37Cl]+.
H, 37Cl]+.
2-(4-Methanesulfinylphenoxy)-N-[(1R,4R)-4-[2-(4-chlorophenoxy)-
acetamido]cyclohexyl]acetamide (38). To a solution of 2-(4-metha-
nesulfinylphenoxy)acetic acid (0.011 g, 0.052 mmol) in N,N-dime-
thylformamide (0.3 mL), were sequentially added 1-hydroxybenzo-
triazole hydrate (0.008 g, 0.055 mmol), 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (0.011 mg, 0.055 mmol), 2-(4-
chlorophenoxy)-N-[(1R,4R)-4-aminocyclohexyl]acetamide trifluoro-
acetic acid (20 mg, 0.05 mmol) and N,N-diisopropylethylamine
(0.021 mL, 0.12 mmol). The reaction mixture was stirred at room
temperature until determined complete by LC–MS and then sub-
jected to conditions described in procedure B to obtain 16 mg
(66%) of the title compound as an off-white solid. 1H NMR
(400 MHz, [D6]DMSO): d=7.96 (d, J=8.2 Hz, 1H), 7.91 (d, J=8.2 Hz,
1H), 7.60 (d, J=8.8 Hz, 2H), 7.31 (d, J=9.0 Hz, 2H), 7.10 (d, J=
8.6 Hz, 2H), 6.94 (d, J=9.0 Hz, 2H), 4.50 (s, 2H), 4.42 (s, 2H), 3.57
(brs, 2H), 2.67 (s, 3H), 1.75 (d, J=8.1 Hz, 4H), 1.21–1.34 ppm (m,
4H); LC–MS: m/z=479 [M+H, 35Cl]+, 481 [M+H, 37Cl]+.
2-(4-Chloro-3-methylphenoxy)-N-[(1R,4R)-4-[2-(4-chlorophen-
oxy)acetamido]cyclohexyl]acetamide (42). To a solution of 4-
chloro-3-methylphenoxyacetic acid (0.010 g, 0.05 mmol) in N,N-di-
methylformamide (1.0 mL), were sequentially added 1-hydroxyben-
zotriazole hydrate (0.009 g, 0.06 mmol), 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (0.012 g, 0.06 mmol), 2-(4-chlor-
ophenoxy)-N-[(1R,4R)-4-aminocyclohexyl]acetamide trifluoroacetic
acid (0.020 g, 0.05 mmol) and N,N-diisopropylethylamine (0.013 mL,
0.075 mmol). The reaction mixture was stirred at room temperature
until determined complete by LC–MS and then subjected to condi-
tions described in procedure B to obtain 20 mg (85%) of the title
compound as a white solid. 1H NMR (400 MHz, [D6]DMSO): d=
7.88–7.92 (m, 2H), 7.26–7.33 (m, 3H), 6.93–6.99 (m, 2H), 6.77 (dd,
J=8.7, 3 Hz, 2H), 4.40–4.42 (m, 4H), 3.56 (brs, 2H), 2.25 (s, 3H),
1.73 (d, J=6.0 Hz, 4H), 1.28–1.36 ppm (m, 4H); LC–MS: m/z=465
[M+H, 35Cl]+, 467 [M+H, 37Cl]+.
2-(4-Acetylphenoxy)-N-[(1R,4R)-4-[2-(4-chlorophenoxy)acetami-
do]cyclohexyl]acetamide (39). To a solution of 4-acetylphenoxy-
acetic acid (0.012 g, 0.063 mmol) in N,N-dimethylformamide
(0.3 mL), were sequentially added 1-hydroxybenzotriazole hydrate
(0.012 g, 0.076 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide hydrochloride (0.014 g, 0.076 mmol), 2-(4-chlorophenoxy)-N-
[(1R,4R)-4-aminocyclohexyl]acetamide trifluoroacetic acid (0.049 g,
0.1 mmol) and N,N-diisopropylethylamine (0.022 mL, 0.1 mmol).
The reaction mixture was stirred at room temperature until deter-
mined complete by LC–MS and then subjected to conditions de-
scribed in procedure B to obtain 21.5 mg (39%) of the title com-
2-(4-Chlorophenoxy)-N-[(1R,4R)-4-[2-(4-chloro-3-ethynylphen-
oxy)acetamido]cyclohexyl]acetamide (43). To a solution of 2-
(4-chloro-3-iodophenoxy)-N-[(1R,4R)-4-[2-(4-chlorophenoxy)acetami-
do]cyclohexyl]acetamide (41) (0.03 g, 0.05 mmol), dichlorobis(tri-
phenylphosphine)palladium (0.004 g, 0.005 mmol) and copper(I)
Iodide (0.002 g, 0.01 mmol) in a degassed mixture of triethylamine/
N,N-dimethylformamide (1:1, 0.4 mL), was added a solution of ethy-
nyltrimethylsilane (0.014 mL, 0.1 mmol) in triethylamine/N,N-dime-
thylformamide (1:1, 0.4 mL) as a single portion and the mixture
was stirred at room temperature for 20 min. The reaction mixture
was then diluted with ethyl acetate and washed with 5% aqueous
potassium hydrogen sulfate solution, saturated sodium thiosulfate
solution and brine. The organic phase was dried over magnesium
sulfate, filtered and concentrated to obtain the crude. Purification
by flash column chromatography (0–40% acetone/dichlorome-
thane) afforded 12 mg (42%) of 2-{4-chloro-3-[2-(trimethylsilyl)ethy-
nyl]phenoxy}-N-[(1R,4R)-4-[2-(4-chlorophenoxy)acetamido]cyclohex-
yl]acetamide as a light-brown solid. LC–MS: m/z=547 [M+H,
35Cl]+, 549 [M+H, 37Cl]+.
1
pound as a tan solid. H NMR (400 MHz, [D6]DMSO): d=7.97 (d, J=
8.1 Hz, 2H), 7.88–7.92 (m, 2H), 7.31 (d, J=8.0 Hz, 2H), 6.93–7.02
(m, 4H), 4.52 (s, 2H), 4.42 (s, 2H), 3.55 (brs, 2H), 3.28 (s, 3H), 1.74
(d, J=9.2 Hz, 4H), 1.28–1.33 ppm (m, 4H); LC–MS: m/z=459 [M+
H, 35Cl]+, 461 [M+H, 37Cl]+.
2-[4-Chloro-3-(trifluoromethyl)phenoxy]-N-[(1R,4R)-4-[2-(4-chlor-
ophenoxy)acetamido]cyclohexyl]acetamide (40). To a suspension
of 2-(4-chlorophenoxy)-N-[(1R,4R)-4-(2-chloroacetamido)cyclohexy-
l]acetamide (0.036 g, 0.1 mmol) in acetone (1.0 mL) were added
potassium carbonate (0.021 g, 0.2 mmol) and 2-chloro-5-hydroxy-
benzotrifluoride (0.027 g, 0.13 mmol) and heated at 1208C for
20 min in the microwave reactor. The reaction mixture was diluted
with water (6 mL) and resulting solids were washed with water
(5 mL) and diethyl ether (5 mL). The solids were suspended in di-
chloromethane and concentrated by rotary evaporation to obtain
28 mg (54%) of the title compound as a brown solid. 1H NMR
(400 MHz, [D6]DMSO): d=7.99 (d, J=8.1 Hz, 1H), 7.91 (d, J=8.1 Hz,
1H), 7.62 (d, J=8.8 Hz, 1H), 7.37 (d, J=2.9 Hz, 1H), 7.31 (d, J=
8.0 Hz, 2H), 7.23 (dd, J=8.9, 3 Hz, 1H), 6.94 (d, J=8.0 Hz, 2H), 4.55
(s, 2H), 4.42 (s, 2H), 3.56 (brs, 2H), 3.28 (s, 3H), 1.74 (d, J=5.9 Hz,
4H), 1.28–1.36 ppm (m, 4H); LC–MS: m/z=519 [M+H]+.
To a solution of 2-{4-chloro-3-[2-(trimethylsilyl)ethynyl]phenoxy}-N-
[(1R,4R)-4-[2-(4-chlorophenoxy)acetamido]cyclohexyl]acetamide
(0.01 g, 0.018 mmol) in a 1:1 mixture of methanol/tetrahydrofuran
(0.9 mL), was added potassium carbonate (0.004 g, 0.027 mmol).
The reaction mixture was stirred at ambient temperature for one
hour and then concentrated down to dryness. The residue was tri-
turated thrice with water (10 mL), diethyl ether (10 mL) and con-
centrated down to dryness to obtain 7 mg (81%) of the title com-
1
pound as a tan solid. H NMR (400 MHz, [D6]DMSO): d=7.92 (t, J=
7.5 Hz, 2H), 7.42 (d, J=9.0 Hz, 2H), 7.31 (d, J=8.0 Hz, 2H), 7.13 (d,
J=2.9 Hz, 1H), 7.00 (dd, J=9.0, 3.1 Hz, 1H), 6.94 (d, J=8.0 Hz, 2H),
4.54 (s, 1H), 4.46 (s, 2H), 4.42 (s, 2H), 3.55 (brs, 2H), 1.74 (d, J=
5.7 Hz, 4H), 1.28–1.33 ppm (m, 4H); LC–MS: m/z=475 [M+H,
35Cl]+, 477 [M+H, 37Cl]+.
2-(4-Chloro-3-iodophenoxy)-N-[(1R,4R)-4-[2-(4-chlorophenoxy)-
acetamido]cyclohexyl]acetamide (41). To
a suspension of 2-
(4-chlorophenoxy)-N-[(1R,4R)-4-(2-chloroacetamido)cyclohexyl]ace-
tamide (0.071 g, 0.2 mmol) in acetone (1.5 mL) were added potassi-
um carbonate (0.041 g, 0.3 mmol) and 4-chloro-3-iodophenol
(0.050 g, 0.2 mmol) and heated at 1208C for 30 min in the micro-
wave reactor. The reaction mixture was diluted with water (10 mL)
and resulting solids were washed with water (10 mL) and diethyl
ether (10 mL). The solids were suspended in dichloromethane and
concentrated by rotary evaporation to obtain 57 mg (50%) of the
2-(4-Chloro-3-methylphenoxy)-N-[(1R,4R)-4-[2-(4-chloro-3-meth-
ylphenoxy)acetamido]cyclohexyl]acetamide (46). To a solution of
(1R,4R)-cyclohexane-1,4-diamine (0.025 g, 0.2 mmol) in N,N-dime-
thylformamide (1 mL) were added 4-chloro-3-methylphenoxyacetic
acid (0.088 g, 0.4 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (0.175 g,
0.5 mmol) and N,N-diisopropylethylamine (0.153 mL, 0.9 mmol).
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The reaction mixture was vigorously stirred at room temperature
until determined complete by LC–MS. The reaction mixture was di-
luted with water (10 mL) and resulting solids were washed thrice
with water (10 mL). The wet solid was then concentrated down
with toluene (10 mL) in a rotary evaporator. The residual product
was washed with diethyl ether (10 mL) and dried to obtain 99 mg
(94%) of the title compound as a cream colored solid. 1H NMR
(400 MHz, CDCl3): d=6.80 (s, 2H), 6.68 (d, J=8.6 Hz, 2H), 6.32 (d,
J=8.1 Hz, 2H), 4.41 (s, 4H), 3.84 (brs, 2H), 2.04 (d, J=6 Hz, 4H),
1.33 ppm (t, J=9.8 Hz, 4H); 13C NMR (100 MHz, [D6]DMSO): d=
166.9, 157.0, 136.8, 129.9, 125.5, 118.0, 114.3, 67.6, 47.3, 31.3,
20.2 ppm; LC–MS: m/z=479 [M+H, 35Cl]+, 481 [M+H, 37Cl]+.
(0.122 mL, 0.7 mmol). The reaction mixture was vigorously stirred
at room temperature until determined complete by LC–MS. The re-
action mixture was diluted with water (10 mL) and resulting solids
were washed thrice with water (10 mL). The wet solid was then
concentrated down with toluene (10 mL) in a rotary evaporator.
The residual product was washed with diethyl ether (10 mL) and
dried to obtain 85 mg (99%) of the title compound as a white
solid. 1H NMR (400 MHz, CDCl3 with CD3OD as co-solvent): d=
7.23–7.28 (m, 4H), 6.61–6.73 (m, 4H), 4.36 (s, 4H), 3.56 (m, 2H),
1.95 (d, J=6.2 Hz, 4H), 1.28–1.33 ppm (m, 4H); 13C NMR (100 MHz,
[D6]DMSO): d=166.5, 159.1, 158.4, 158.3, 156.7, 131.1, 112.8,112.8,
111.6, 111.5, 104.6, 104.4, 67.8, 47.4, 31.3 ppm; LC–MS: m/z=487
[M+H, 35Cl]+, 489 [M+H, 37Cl]+.
2-(3,4-Dichlorophenoxy)-N-[(1R,4R)-4-[2-(3,4-dichlorophenoxy)-
acetamido]cyclohexyl]acetamide (47). To a solution of (1R,4R)-cy-
clohexane-1,4-diamine (0.025 g, 0.2 mmol) in N,N-dimethylforma-
mide (1 mL) were added 3,4-dichlorophenoxyacetic acid (0.097 g,
0.4 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxid hexafluorophosphate (0.175 g, 0.5 mmol) and
N,N-diisopropylethylamine (0.153 mL, 0.9 mmol). The reaction mix-
ture was vigorously stirred at ambient temperature for 2 h. The re-
action mixture was diluted with water (10 mL) and resulting solids
were washed thrice with water (10 mL). The wet solid was then
concentrated down with toluene (10 mL) in a rotary evaporator.
The residual product was washed with diethyl ether (10 mL) and
dried under vacuum to obtain 107 mg (94%) of the title com-
Acknowledgements
This work was funded in part by a Collaborative Innovation
Award from the Howard Hughes Medical Institute (HHMI). P.W. is
an Investigator of the HHMI.
Keywords: integrated stress response · ISRIB · protein–protein
interactions · structure–activity relationships · unfolded protein
response
1
pound as a cream colored solid. H NMR (400 MHz, CDCl3): d=7.37
(d, J=8.8 Hz, 2H), 7.04 (s, 2H), 6.78 (d, J=8.8 Hz, 4H), 6.26 (d, J=
8.1 Hz, 2H), 4.42 (s, 4H), 3.85 (brs, 2H), 2.05 (d, J=6 Hz, 4H), 1.31–
1.39 ppm (m, 4H); 13C NMR (100 MHz, [D6]DMSO): d=166.5, 157.7,
131.9, 131.4, 123.4, 117.3, 116.1, 67.7, 47.4, 31.3 ppm; LC–MS: m/
z=519 [M+H, 35Cl]+, 521 [M+H, 37Cl]+.
[1] A. Korennykh, P. Walter, Annu. Rev. Cell Dev. Biol. 2012, 28, 251–277.
[2] P. Walter, D. Ron, Science 2011, 334, 1081–1086.
[3] D. Ron, P. Walter, Nat. Rev. Mol. Cell Biol. 2007, 8, 519–529.
[4] C. Hetz, E. Chevet, H. P. Harding, Nat. Rev. Drug Discovery 2013, 12, 703–
719.
[5] M. Halliday, G. R. Mallucci, Neuropathol. Appl. Neurobiol. 2015, 41, 414–
427.
[6] N. Donnelly, A. M. Gorman, S. Gupta, A. Samali, Cell. Mol. Life Sci. 2013,
70, 3493–3511.
[7] C. Sidrauski, D. Acosta-Alvear, A. Khoutorsky, P. Vedantham, B. R. Hearn,
H. Li, K. Gamache, C. M. Gallagher, K. K.-H. Ang, C. Wilson, V. Okreglak,
A. Ashkenazi, B. Hann, K. Nader, M. R. Arkin, A. R. Renslo, N. Sonenberg,
P. Walter, Elife 2013, 2, e00498.
[8] M. Halliday, H. Radford, Y. Sekine, J. Moreno, N. Verity, J. le Quesne, C. A.
Ortori, D. A. Barrett, C. Fromont, P. M. Fischer, H. P. Harding, D. Ron, G. R.
Mallucci, Cell Death Dis. 2015, 6, e1672.
2-(4-Chloro-3-fluorophenoxy)-N-[(1R,4R)-4-[2-(4-chloro-3-fluoro-
phenoxy)acetamido]cyclohexyl]acetamide (48). To a solution 4-
chloro-3-fluorophenol (0.100 g, 0.7 mmol) in N,N-dimethylforma-
mide (2 mL), were added potassium carbonate (0.189 g, 1.4 mmol)
and tert-butyl bromoacetate (0.111 mL, 0.8 mmol) and stirred at
658C for 2 h. The reaction mixture was diluted with ethyl acetate,
washed with water and brine. The organic layer was dried over
magnesium sulfate and concentrated in a rotary evaporator to
obtain 177 mg of tert-butyl 2-(4-chloro-3-fluorophenoxy)acetate as
a colorless oil which was used without further purification.
[9] Y. Sekine, A. Zyryanova, A. Crespillo-Casado, P. M. Fischer, H. P. Harding,
D. Ron, Science 2015, 348, 1027–1030.
To
a solution of tert-butyl 2-(4-chloro-3-fluorophenoxy)acetate
[10] C. Sidrauski, J. C. Tsai, M. Kampmann, B. R. Hearn, P. Vedantham, P. Jaish-
ankar, M. Sokabe, A. S. Mendez, B. W. Newton, E. L. Tang, E. Verschueren,
J. R. Johnson, N. J. Krogan, C. S. Fraser, J. S. Weissman, A. R. Renslo, P.
Walter, Elife 2015, 4, e07314.
[11] Y. Gordiyenko, C. Schmidt, M. D. Jennings, D. Matak-Vinkovic, G. D.
Pavitt, C. V. Robinson, Nat. Commun. 2014, 5, 3902.
[12] N. C. Wortham, M. Martinez, Y. Gordiyenko, C. V. Robinson, C. G. Proud,
FASEB J. 2014, 28, 2225–2237.
[13] M. R. Arkin, Y. Tang, J. A. Wells, Chem. Biol. 2014, 21, 1102–1114.
[14] J. A. Wells, C. L. McClendon, Nature 2007, 450, 1001–1009.
[15] G. R. Bickerton, G. V. Paolini, J. Besnard, S. Muresan, A. L. Hopkins, Nat.
Chem. 2012, 4, 90–98.
(177 mg, 0.7 mmol) in methanol/water (4.5 mL, 2:1) was added
aqueous 5N NaOH solution (0.7 mL, 3.5 mmol) and stirred at ambi-
ent temperature for 1 h. The reaction mixture was concentrated in
a rotary evaporator to remove methanol, diluted with water (5 mL)
and extracted with ethyl acetate (5 mL). The aqueous layer was ad-
justed to about pH 2 with 1n HCl and extracted with ethyl acetate
(3”5 mL). The organic extract was washed with brine (5 mL), dried
over magnesium sulfate and concentrated to obtain 108 mg of 2-
(4-chloro-3-fluorophenoxy)acetic acid as a white solid.
To a solution of (1R,4R)-cyclohexane-1,4-diamine (0.02 g, 0.2 mmol)
in N,N-dimethylformamide (1 mL) were added 2-(4-chloro-3-fluoro-
phenoxy)acetic acid (0.072 g, 0.4 mmol), 1-[bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoro-
phosphate (0.14 g, 0.4 mmol) and N,N-diisopropylethylamine
Received: October 15, 2015
Revised: December 15, 2015
Published online on January 20, 2016
ChemMedChem 2016, 11, 870 – 880
www.chemmedchem.org
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