Virtual screening for novel Atg5-Atg16 complex inhibitors for autophagy modulation

Article abstract of DOI:10.1039/c4md00420e

Two hit compounds (14 and 62) were identified using virtual high throughput screening (vHTS) inhibiting the autophagy process in A2780 ovarian cancer cells. The expression levels of the LC3-II and p62 autophagy marker proteins were monitored using Western blotting. Preliminary structure activity relationship (SAR) study of close structural analogues revealed another active compound 38. The three active compounds were tested in the MCF-7 human breast cancer cells and severe reduction of autophagosomes formation was observed confirming the activity of the inhibitors. The docking scaffold used for the vHTS was a lipophilic cleft on the Atg5 protein, which is occupied by a phenylalanine residue in the Atg16 polypeptide. To the best of our knowledge this is the first report on inhibitors that specifically modulate autophagy by directly inhibiting autophagy specific proteins, which is significant due the role autophagy plays in a number of morbid diseases such as cancer. This journal is

Full text of DOI:10.1039/c4md00420e

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CONCISE ARTICLE
Virtual screening for novel Atg5–Atg16 complex
inhibitors for autophagy modulation†
Cite this: DOI: 10.1039/c4md00420e
a
b
c
Elizabeth Robinson, Euphemia Leung, Anna M. Matuszek, Niels Krogsgaard-
c
c
c
a
Larsen, Daniel P. Furkert, Margaret A. Brimble, Alan Richardson
and Johannes Reynisson*
c
´
Two hit compounds (14 and 62) were identified using virtual high throughput screening (vHTS) inhibiting the
autophagy process in A2780 ovarian cancer cells. The expression levels of the LC3-II and p62 autophagy
marker proteins were monitored using Western blotting. Preliminary structure activity relationship (SAR)
study of close structural analogues revealed another active compound 38. The three active compounds
were tested in the MCF-7 human breast cancer cells and severe reduction of autophagosomes
formation was observed confirming the activity of the inhibitors. The docking scaffold used for the vHTS
was a lipophilic cleft on the Atg5 protein, which is occupied by a phenylalanine residue in the Atg16
polypeptide. To the best of our knowledge this is the first report on inhibitors that specifically modulate
autophagy by directly inhibiting autophagy specific proteins, which is significant due the role autophagy
plays in a number of morbid diseases such as cancer.
Received 19th September 2014
Accepted 31st October 2014
DOI: 10.1039/c4md00420e
www.rsc.org/medchemcomm
In this study, the crystal structure of the Atg5–Atg16 complex
at the binding site of Phe-46 in Atg-16 was used as a docking
Introduction
Autophagy (self-eating) is a catabolic pathway that sequesters scaffold for a vHTS to nd small molecular inhibitors for the
undesired cellular material into autophagosomes for delivery to autophagy process in ovarian and breast cancer cell lines.
lysosomes for the bulk degradation and is conserved in most
1
eukaryotes. Autophagic dysfunction is now associated with a
number of diseases, including infection by pathogens, inam-
Results
Pilot screen
2–4
matory bowel disease, neurodegeneration and cancer.
A
number of small molecules are known to modulate autophagy
however there is a dearth of compounds that directly interact
4
1
ꢀ 10 molecular entities were downloaded from the Chem-
12
5
Bridge diversity collection. It was ltered to render the list
with the specic autophagy proteins. A key step in the pathway
13
Lipinski compliant, resulting in 8373 molecules. These were
screened against the cle in Atg5 which the phenyl moiety of
Phe-46 from the Atg16 polypeptide occupies. The binding
pocket at Arg-35 (see Fig. 1) was also considered but several
is the covalent conjugation of the ubiquitin-related protein Atg8
(autophagy) to phosphatidylethanolamine in autophagic
membranes by a complex consisting of Atg16 and the Atg12–
1
Atg5 conjugate. The crystal structure of the Atg5–Atg16
complex is known and in vitro analysis showed that the Arg-35
and Phe-46 amino acids of Atg-16 are crucial for this interaction
6,7
to take place. The binding pockets are shown in Fig. 1. This
protein complex therefore represents a new target for drug
8
development, in particular for a virtual high throughput
screening (vHTS) campaign, which is an effective method to
9
–11
generate inhibitors of bio-molecular targets.
a
Institute for Science and Technology in Medicine, Keele University, Guy Hilton
Research Centre, Stoke-on-Trent, UK
b
Auckland Cancer Society Research Centre, University of Auckland, New Zealand
c
School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland
1
+
†
1
142, New Zealand. E-mail: j.reynisson@auckland.ac.nz; Fax: +64-9-373-7422; Tel:
64-9-373-7599 ext. 83746
Fig. 1 The two binding pockets of interest of the Atg5–Atg16 complex
identified by mutation studies. The Atg5 protein surface is rendered
and the Atg16 polypeptide is green. The amino acids Arg35 and Phe46
are shown in the stick format.
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4md00420e
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amino acidic were missing from the crystal structure, which are
a part of a exible loop directly in this pocket. The proximity of
the loop makes the shape of the pocket uncertain and less
attractive as a docking scaffold than the cle for Phe-46 and it
14
was therefore not considered further. The GoldScore and
15,16
ChemScore
tness functions were used for the initial screen.
All ligands without predicted hydrogen bonding were elimi-
nated as well as those with low ChemScore (<10); no such low
values were predicted by GoldScore. This le 512 candidates,
which were screened again with high search efficiency using Fig. 2 Immunoblotting of p62 and LC3-II protein in ovarian cancer
A2780 cell line. Cells were incubated with DMSO as negative control
both scoring functions. The candidates were eliminated based
(C), or PBS for 3 h to induce autophagy without (+) or with pre-
on the GoldScore (#30) and ChemScore (#20); and the ability to
form hydrogen bonds based on both scoring functions resulting
in 33 candidates. The results were inspected visually for
incubation of compounds for 1 h. p62 and LC3-II were detected in
total cell extracts by western blotting analysis, using specific anti-
bodies. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was
consensus of the best predicted conguration of the ligands used as loading control. The quantitative results are given in Fig. S7 in
the ESI.†
between the scoring functions, that the ligands had plausible
congurations, i.e., not strained, for lipophilic moieties point-
ing into the water environment and, nally for undesirable
17
moieties linked to cell toxicity and chemical reactivity. The suppression of autophagy. Two set of experiments were con-
screening methodology used has been previously successfully ducted for each compound. The representative results are
applied to nd active ligands of the phosphoinositide specic- shown in Fig. 2 for compounds 10–18 and in Fig. S1† in the ESI
18
phospholipase C-g2 enzyme. Eighteen compounds were for compounds 1–9; quantitation of both sets of data is also
selected for experimental testing and their structures are shown provided in the ESI (Fig. S6 and S7†).
in Table 1. A detailed description of the vHTS is given in the
Methodology section.
The control prole of the cellular proteins as base line was
shown on the le lane in Fig. 2. Upon induction of autophagy,
the expected decrease in LC3-II and increase in p62 was
observed. Compounds 13 and 14 reduced accumulation of
LC3-II and depletion of p62. Dose response experiments were
Biological testing
Microtubule-associated protein light chain 3 (LC3-II) is the performed to verify this activity of these ligands (Fig. S2 and S3
mammalian ortholog of yeast Atg-8 and a robust marker of in the ESI section†). These experiments demonstrated the
19
autophagosomes.
LC3 binds the polyubiquitin-binding activity of compound 14 was dose dependent (Fig. 3), but this
protein p62, an adapter protein that is selectively degraded via was not apparent for ligand 13.
8
autophagy. The eighteen virtual hits were tested in A2780
It can be clearly seen in Fig. 3 that an increased concentra-
ovarian tumour cell line for efficacy. Autophagy was induced by tion of ligand 14 causes the re-emergence of p62 compared to
starvation (PBS buffer for 3 hours) and the effect of the ligands the second lane (+) of autophagy cells and the reduction of LC3-
was assessed by measuring accumulation of LC3-II and p62 by II, which is a strong indication that the ovarian cell are reverted
immunoblotting (see methodology for detailed description). A from their autophagy state.
decrease in LC3-II and increase in p62 is consistent with
Table 1 The structures of the eighteen compounds experimentally tested for autophagy activity. Compounds 12 and 17 were tested as the
racemates
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Table 2 The structures of three compounds with the important ortho/
para chlorine substitution on the benzamide moiety
Fig. 3 Dose response effect for ligand 14 using immunoblotting of
p62 and LC3-II protein in ovarian cancer A2780 cell line. Cells were
incubated with DMSO as negative control (C), or PBS for 3 h to induce
autophagy (+) without or with pre-incubation of indicated concen-
tration of compound 14 for 1 h. GAPDH was used as loading control.
The quantitative result for compound 14 is given in Fig. S8 in the ESI.†
Preliminary structural activity relationship (SAR)
In order to explore the chemical space around the hit
compound 14 ten close structural analogues were purchased
and tested. In addition two compounds (13 and 16) from the
pilot screen are similar to 14 resulting in thirteen molecules for
the SAR analysis (Scheme 1).
Various single and double chlorine substitutions of the
benzamide ring did not lead to active compounds suggesting
that the ortho/para substitution pattern is crucial for the efficacy
observed. Ortho and para uorine single substitutions (13 and
and thirdly the tolerance of oxygen substitution in the alkyl
chain.
Biological testing revealed that only compound 38 showed
activity, giving increased p62 and decreased LC3-II expression.
This was veried with a dose response experiment. The efficacy
of ligand 38 demonstrates that the substitution of the benza-
mide phenyl moiety with a-naphthyl is possible and that
introduction of an ether link into the alkyl chain is also toler-
ated. The length of the linker chain is clearly important, with
four units optimal, as seen for both hit compound 14 and its
derivative 38 compared to ligands 32–37, which have a shorter
chain length. Even though twenty ve compounds were tested
in this preliminary analysis, a clear SAR was not evident. Thus,
substantially more analogues of the hit compounds need to be
tested in order to produce a full SAR.
26) on the benzamide ring were not tolerated. Furthermore,
bromine single substitution (ortho (22), meta (23) and para (24))
gave inconsistent effects on the expression levels of LC3-II and
p62. There is not a clear indication of inhibition and it can be
concluded that single bromine substitution of the benzamide
ring is not favourable. Finally, one derivative (28) with a shorter
aliphatic chain (n ¼ 0) and no substitution on the ring systems
was not active. In conclusion, the ortho/para chlorine substitu-
tion pattern is vital for the activity. Three compounds, 29–31,
with the ortho/para chlorine substitution on the benzamide
were tested and their structures are shown in Table 2. None of
the ligands demonstrated consistent activity, which suggests
that the dichlorinated benzamide moiety is on its own insuffi-
cient to modulate autophagy.
Main screen
For the main screen, the complete diversity ChemBridge
4
12
diversity collection was used consisting of 5 ꢀ 10 molecules.
4
Aer making it Lipinski compliant 4.3 ꢀ 10 entities remained.
All four scoring functions available in the GOLD soware suite
14
14,15
20
21
(
GoldScore, ChemScore,
ChemPLP and ASP ) were used
in conjunction with the consensus concept, i.e., only ligands
Synthetic chemistry
In order to expand the preliminary SAR further nine more
analogues were synthesised of hit compound 14 (Scheme 2).
Three features were deemed important to investigate: rst, the
tolerance of the benzamide phenyl moiety for substitution with
other functional groups; secondly the length of the alkyl chain
Scheme 1 The structures of twelve close structural analogues of hit Scheme 2 The nine analogues of hit compound 14 synthesised for
compound 14, which were tested for their activity for SAR generation. SAR generation.
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that scored well with all the functions and showed hydrogen mediated proteolysis and causes autophagolysosomes accu-
2
2,23
24
bonding activity were taken forward.
sufficed to take only compounds with good hydrogen bonding dansylcadaverine (MDC) is
HB $ 1) forward resulting in 1470 candidates. For the second autophagolysosomes as it labels vacuoles formation in later
For the initial screen it mulation.
The
auto-uorescent
compound
mono-
a
widely used marker of
(
25–27
screen, using higher search efficiency and more docking runs, stages in the autophagy degradation process.
Incubation of
again only ligands with good hydrogen bonding were taken cells with 75 mM CQ caused a signicant increase in the number
forward as well as a good score (GoldFitness $ 45, ChemSocre $ of autophagolysosomes, visualized by MDC accumulation in
20, PLPScore $ 50 and ASP $ 25) leaving 200 candidates. These vacuoles (Fig. 4). The administration of the hits, 14, 38 and 62,
were visually inspected using the same criteria as for the pilot resulted in effective suppression of the autophagolysosome
screen. Twenty-two ligands were selected for experimental formation in line with the hypothesis of Atg5–Atg16 complex
testing and their structures are shown in Table 3.
inhibition. Finally, hit 62 was tested in the well-established
From the twenty-two virtual hits tested in the ovarian cancer National Cancer Institute's NCI60 panel of 60 human tumour
28
cells ligand 62 caused an increase in p62 and decrease in LC3-II cells lines. It had a marginal effect on the growth of the cell
consistent with inhibition of autophagy (see Fig. S4†). This was lines with a mean of 90.1% at 10 mM as compared to 100% of
also seen for ligand 58 to some extent but the effect was less untreated cells. These data suggest that the effect of compound
pronounced than for 62.
62 was not dependent on inhibition of cell growth. The result
from the NCI60 experiment is shown in Fig. S5 in the ESI.†
Hit verication
Modelling of hit compounds
In order to verify the activity of the three hit compounds 14, 38
and 62 in blocking autophagy cells back to the normal state they The binding to Atg5 of the three hit compounds was investi-
were tested in the MCF-7 human breast cancer cell line. The gated. The predicted binding modes of hit 14 are shown in
results are shown in Fig. 4.
Fig. 5.
The modelling of 14 resulted in two plausible binding
Chloroquine (CQ) is a lysosomotropic agent that prevents
autophagy protein degradation by inhibiting lysosome- modes to Atg5. GoldScore and ChemPLP predict the same
Table 3 The structures of the twenty-two compounds tested for autophagy effect identified from the main virtual screen. Compound 41 and 60
were tested as the racemates
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Fig. 4 The effect of pre-treating MCF-7 human breast cancer cells with hit compounds 14, 38 and 62 before incubation with 75 mM chloroquine
(
3
CQ). Autophagolysosomes were visualized by monodansylcadaverine (MDC) staining (see arrows). MDC was loaded into cells and incubated at
7 C for 10 min and visualized by fluorescent microscopy. The pre-incubation with the hit compounds abrogated the CQ induced autopha-
ꢁ
golysosomes accumulation.
29
activity. The implication is that inhibiting autophagy with
small molecules may increase the efficacy of chemotherapeutic
regimes.
The three hits found in this project are a good start in
developing effective inhibitors for autophagy. At this stage,
we can not rule out the possibility that the compounds
inhibit autophagy through some mechanism other than
interfering with Atg5–Atg16 complex formation. The devel-
Fig. 5 The docked configuration of 14 in the binding site of Atg5 using opment of a specic biochemical assay, isothermal calori-
GoldScore and ChemScore. (A) The protein surface is rendered. The metric
phenyl group of 14 occupies a lipophilic cleft overlapping with the
Phe46 amino acid in Atg16, which is shown as green sticks. Red depicts
a positive partial charge on the surface, blue depicts negative partial
charge and grey shows neutral/lipophilic areas. (B) Hydrogen bonds
titration
(ICT)
experiments
and/or
X-ray
crystallography will answer this vital question. In particular,
the results of ICT experiments can reveal whether the hits can
perturb the Atg5–Atg16 complex formation and we are
are shown as green lines between ligand 14 and the amino acids Arg36 currently evaluating this. Furthermore, the exploration of
for the GoldScore results and Asn14 for the ChemScore configuration. chemical space surrounding the hits by chemical synthesis
will produce a more complete SAR, identifying more potent
ligands and aiding in developing a better molecular model.
conformation with hydrogen bonding to the backbone The SAR study presented here can only be viewed as prelim-
carboxylic moiety of Arg36 but ChemScore predicts bonding inary. Nevertheless, having the three hit compounds identi-
to the backbone carboxylic moiety of Asn14 (Fig. 5B). In both ed in this work is the crucial start to give traction to develop
cases the phenyl moiety of 14 occupies the lipophilic cle in autophagy-specic inhibitors and hopefully leading to an
place of Phe46 of the Atg16 polypeptide and overlaps with it effective adjunctive therapeutic approach in the treatment of
in general (Fig. 5A). The ASP scoring function produced a cancer.
different binding mode to its counterparts but the lipophilic
cle was also occupied by the phenyl moiety. Hit compounds
3
8 and 62 gave less consistent results when modelled
Conclusion
but in all cases the lipophilic binding cle was lled, sug-
gesting that this may be necessary for the efficacy of the In this work two hit compounds 14 and 62 were identied using
compounds.
virtual high throughput screening inhibiting autophagy in
ovarian A2780 cancer cells. Preliminary SAR was developed for
hit 14 leading to the discovery of another active compound 38.
The three active compounds were tested in MCF-7 breast cancer
cells and the characteristic autophagy vacuole formation was
Discussion
It is becoming more apparent that the autophagy process plays diminished upon the administration of the inhibitors verifying
a pivotal role in diseases such as cancer, e.g., apoptotic cancer their efficacy. These three compounds can now be developed
cells induced by chemotherapeutics can not only survive but further with more elaborate experimental/biological testing and
recover from damage with the aid of heightened autophagy by substantially extending the SAR analysis.
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pH 7.4, 150 mM NaCl, 0.1% Tween 20, for 1 h at RTP. The
Methodology
Modelling and screening
membrane was then incubated in antibodies for ꢂ16 h (over-
ꢁ
night) at temperature of 4 C, where LC3-2G6 (Nanotools) at 1/
The compounds were docked to the crystal structure of Atg16– 2000 dilution was utilised as anti-LC3 antibody and AB56416
6
˚
(Abcam) at 1/1000 dilution was used as anti-SQSTM1/p62 anti-
Atg5 (PDB ID: 2DYO, resolution 1.97 A), which was obtained
30,31
from the Protein Data Bank (PDB).
The Scigress Ultra version body. The dilution step was then repeated in 5% milk in TBST
.7.0.47 program was used to prepare the crystal structure for as above, and subsequently in anti-GAPDH antibody MAB374
32
7
docking, i.e., hydrogen atoms were added, the co-crystallised (Millipore) at 1/5000 dilution for 1 h at RTP. Aer each primary
polypeptide Atg-16 was removed as well as crystallographic antibody, the membrane was washed three times for 5 min. The
water molecules. The centre of the binding pocket was dened membrane was then incubated in anti-mouse IgG antibody
as the position of the Atg-16's Phe-46 of the para-carbon of the linked to HRP (Cell Signalling Technology) at 1/2000 dilution in
˚
a room temperature for a period of 1 h. Membrane was then
phenyl ring (x ¼ 87.120, y ¼ 88.114, z ¼ 69.571) with 10 A radius.
For the initial screen 30% search efficiency was used (virtual washed in TBST ve times consecutively, before the detection of
screen) with ten runs per compound. For the second phase protein bands, which was conducted using UptiLight HRP
(
re-dock) 100% efficiency was used in conjunction with y chemiluminescent substrate (Uptima) and a FluorChem M
14
15,16
docking runs. The GoldScore (GS), ChemScore (CS),
Imager. To quantitate individual proteins, signal intensity was
20
21
ChemPLP and ASP scoring functions were implemented to integrated over the area of each band and corrected for back-
validate the predicted binding modes and relative energies of ground. Results were normalized to GAPDH measured in the
the ligands using the GOLD v5.2 soware suite. The virtual high same sample.
throughput screen was conducted with the ChemBridge diver-
4
12
sity collection of 5 ꢀ 10 entities. Substructure and Tanimoto MCF-7 breast cancer cells
33
similarity search methods were used to identify the structur-
ally related compounds using the Hit2Lead web based
compound libraries.
Human breast cancer cell line MCF-7 grown in 96 well plates
were not treated (control) or treated with chloroquine (75 mM)
alone or with 14, 38 and 62 at 25 mM. DMSO (0.2%) was incu-
bated for vehicle control. Aer 5 hours, cells were incubated
with 10 mM monodansylcadaverine (MDC, Sigma, 30 432) for 20
12
Western blotting
ꢁ
min at 37 C, then washed with PBS three times. Images were
The investigational compounds identied through screening
for autophagic activity were used in cell-based assays to deter-
mine their effects on autophagy in ovarian cancer cells. A2780
examined immediately by uorescence microscopy (FLoid Cell
Imaging Station, 40ꢀ magnication).
5
cells (5 ꢀ 10 cells per well) were plated in 6-well plates in 1 mL
NCI's 60-cell line panel growth inhibition assay
of growth medium and pre-incubated for 24 h. Following this,
The compounds obtained were submitted to the National
Cancer Institute's Developmental Therapeutic Program (DTP)
where they were screened against a panel of sixty human
the cells were incubated with 50–100 mM of each compound for
1
h before replacing the medium in each well with PBS sup-
plemented with the corresponding compound. DMSO was
added to the control wells to equal the concentration in drug-
treated wells (max. 0.5%). Aer 3 h of PBS-induced autophagy,
cells were lysed and autophagy-specic proteins detected and
autophagy-specic proteins (LC3-II and p62) detected using
western blotting with GAPDH used as loading control. Each
28
tumour cell lines (NCI60, for further information see ref. and
references therein). The full description of the assay is given in
the ESI.†
Chemistry
compound was tested in two or three independent experiments. Known compounds 32–34 were prepared by acylation of phe-
Further dose–response assays were performed with investiga- nethylamine hydrochloride using standard conditions. Experi-
tional compounds that demonstrated autophagy inhibition in mental procedures and characterisation data are provided in
the initial assay. Similar assays were performed with four the ESI.† Compounds 35–40 were prepared from either 2- or
concentrations (12.5 mM, 25 mM, 50 mM and 100 mM) of each 3-phenoxylethylamine, as described below.
compound. The concentration of DMSO was normalised across
all wells for each experiment.
2-Phenoxyethylamine. A solution of 2-iodo-N-Boc-ethylamine
(313 mg, 1.2 mmol), phenol (163 mg, 1.7 mmol, 1.5 equiv.) and
The buffer used for A2780 ovarian cancer cells lysis was potassium carbonate (280 mg, 2.0 mmol, 1.8 equiv.) in dime-
prepared consisting of 20 mM HEPES, 150 mM NaCl, 2 mM thylformamide (3 mL) was stirred at room temperature for 24
EDTA, 0.5% sodium deoxycholate, 1% NP40, 120 mM leupeptin, hours before being poured into ether (25 mL). The organic
1
mM pepstatin and 1 mM PMSF. The concentration of protein phase was washed with water, 2 M NaOH and brine (15 mL),
within the lysate was established using Sigma supplied bicin- dried over anhydrous magnesium sulfate, ltered and concen-
choninic acid kit. Nusep 4–20% polyacrylamide gradient gel was trated in vacuo. The crude oil (226 mg, 0.95 mmol) was dissolved
used, loading between 5–15 mg protein per sample. Aer protein in dichloromethane (1.5 mL) and triuoroacetic acid (250 mL,
separation on gel, the proteins were transferred onto the PVDF 3.3 mmol, 3.4 equiv.) added. The reaction mixture was stirred at
membrane, which was then blocked in 5% skimmed milk room temperature for 18 h then poured into a mixture of ethyl
powder dissolved in TBST buffer consisting of 50 mM Tris–HCl, acetate and 1 M NaOH (1 : 1, 20 mL). The aqueous phase was
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extracted with further ethyl acetate (10 mL) and the combined ethyl acetate and the organic phase washed with 1 M HCl and 1
organic phases washed with brine (10 mL), dried over anhy- M NaOH. The combined organic phases were then washed with
drous magnesium sulfate, ltered and concentrated in vacuo to brine, dried over anhydrous magnesium sulfate, ltered and
give as a brown oil (130 mg) that was used without further concentrated in vacuo to give a light brown oil. Column chro-
purication.
matography (EtOAc–hexanes, 1 : 1–2 : 1) afforded 37 (95 mg,
1
3-Phenoxypropylamine. Prepared according to the procedure 35%) as an off-white solid. H NMR (400 MHz, CDCl ): 7.29 (2H,
3
for 2-phenoxyethylamine above, beginning from 3-iodo-N-Boc- tm, J ¼ 8.0 Hz), 6.97 (1H, tm, J ¼ 8.0 Hz), 6.89 (2H, dm, J ¼ 8 Hz),
propylamine (328 mg, 1.2 mmol) to give crude 3-phenox- 5.89 (1H, bs), 4.04 (2H, t, J ¼ 5 Hz), 3.67 (2H, q, J ¼ 6 Hz), 2.17–
1
3
ypropylamine (216 mg) as a colourless oil that was used without 2.05 (3H, m), 0.95 (6H, d, J ¼ 6 Hz); C NMR (100 MHz, CDCl
3
):
further purication.
172.8, 158.6, 129.7, 121.3, 114.6, 67.0, 46.3, 39.0, 26.3, 22.6; IR
N-(2-Phenoxyethyl)-1-naphthamide (35). To a solution of 1- (cm ) 3290, 3073, 2956, 2931, 2871, 1643, 1600, 1588, 1545,
naphthoic acid (163 mg, 0.95 mmol) in dichloromethane– 1496, 1464, 1387, 1368, 1300, 1243, 1216 cm ; HRMS (ESI) m/z
ꢃ1
ꢃ1
+
dimethylformamide (3 : 1, 4 mL) were added DCC (206 mg, 1.00 calcd for C13
mmol, 1.05 equiv.) and 6-Cl-HOBt (32 mg, 0.19 mmol, 0.20 0.27 (EtOAc–hexanes, 1 : 1).
equiv.) and the mixture stirred at room temperature for 30 min. N-(3-Phenoxypropyl)-1-naphthamide (38). Prepared accord-
A solution of crude 2-phenoxyethylamine (130 mg, 0.95 mmol, ing to the method described for 35 above, from 1-naphthoic
.0 equiv.) in dichloromethane (2 mL) was added over 15 min acid (254 mg, 1.48 mmol, 1.03 equiv.) and 3-phenoxyethylamine
H
19NNaO
2
[M + Na] : 244.1308, found 244.1315; R
f
1
and the mixture stirred at rt for 18 h. The reaction mixture was (216 mg, 1.43 mmol, 1.0 equiv.). The crude oil was puried by
then ltered and the combined ltrate and washings (EtOAc) chromatography (EtOAc–hexane, 2 : 1) to yield 38 (161 mg, 46%)
1
washed with 1 M HCl, 1 M NaOH and brine, dried over anhy- as a colourless solid. H NMR (400 MHz, CDCl ): 8.33 (1H, m),
3
drous magnesium sulfate, ltered and concentrated in vacuo. 7.91 (1H, d, J ¼ 8 Hz), 7.87 (1H, m), 7.59 (1H, dd, J ¼ 7, 1 Hz),
Column chromatography (EtOAc–hexanes) afforded 35 (126 mg, 7.52 (2H, m), 7.44 (1H, dd, J ¼ 8, 7 Hz), 7.27 (2H, m), 6.95 (1H, tt,
1
3
8%) as a colourless solid. H NMR (400 MHz, CDCl
3
): 8.34 (1H, J ¼ 7, 1 Hz), 6.85 (2H, dm, J ¼ 8 Hz), 6.47 (1H, bs), 4.15 (2H, t, J ¼
13
m), 7.91 (1H, d, J ¼ 8 Hz), 7.86 (1H, m), 7.61 (1H, dd, J ¼ 7, 0.8 6 Hz), 3.77 (2H, q, J ¼ 6 Hz), 2.18 (2H, quintet, J ¼ 6 Hz);
C
Hz), 7.56–7.49 (2H, m), 7.45 (1H, dd, J ¼ 8, 7 Hz), 7.30 (2H, dd, J NMR (100 MHz, CDCl ): 169.6, 158.5, 134.7, 133.8, 130.6, 130.2,
3
¼
8, 7 Hz), 6.98 (1H, t, J ¼ 7 Hz), 6.93 (2H, d, J ¼ 8 Hz), 6.52 (1H, 129.6, 128.3, 127.1, 126.4, 125.5, 124.9, 124.7, 121.1, 114.4, 66.5,
13
bs), 4.22 (2H, t, J ¼ 5 Hz), 3.95 (2H, q, J ¼ 6 Hz); C NMR (100 38.2, 29.1; IR: 3413, 3279, 3056, 2939, 2876, 1639, 1592, 1534,
ꢃ1
MHz, CDCl ): 169.8, 158.6, 134.4, 133.8, 130.9, 130.2, 129.7, 1497, 1471, 1434, 1393, 1300, 1243, 1215 cm ; HRMS (ESI) m/z
3
+
1
28.5, 127.3, 126.6, 125.5, 125.3, 124.9, 121.4, 114.6, 66.8, 39.7; calcd for C20
H19NNaO
2
[M + Na] : 328.1308, found: 328.1299;
IR: 3413, 3274, 3053, 2935, 2876, 1641, 1597, 1527, 1496, 1463, TLC: R
f
0.29 (40% EtOAc–hexane).
ꢃ1
1
436, 1392, 1244 cm ; HRMS (ESI) m/z calcd for C19
H17NNaO
2
N-(3-Phenoxypropyl)-1H-indole-2-carboxamide (39). Prepared
+
[M + Na] : 314.1151, found: 314.1147; TLC: R
f
0.27 (40% EtOAc– according to the method described for 35 above, from indole-2-
hexanes).
carboxylic acid (250 mg, 1.55 mmol, 1.08 equiv.) and 3-phenoxy-
N-(2-Phenoxyethyl)-1H-indole-2-carboxamide (36). Prepared ethylamine (216 mg, 1.43 mmol, 1.0 equiv.). The crude oil was
according to the method described for 35 above, from indole-2- puried by chromatography (EtOAc–hexane, 2 : 1) to yield 39
1
carboxylic acid (153 mg, 0.95 mmol, 1.00 equiv.) and 2-phenoxy- (189 mg, 46%) as a aky, off-white solid. H NMR (400 MHz,
ethylamine (130 mg, 0.95 mmol, 1.0 equiv.). The crude oil was CDCl
3
): 11.55 (1H, s), 8.55 (1H, t, J ¼ 6 Hz), 7.60 (1H, dd, J ¼ 8, 0.6
puried by recrystallization (hexane–EtOAc, reux) to afford 36 Hz), 7.42 (1H, dd, J ¼ 8, 1 Hz), 7.28 (2H, m), 7.17 (1H, ddd, J ¼ 8,
1
(
8
8
125 mg, 39%) as brown crystals. H NMR (400 MHz, d
6
-DMSO): 7, 1 Hz), 7.11 (1H, dd, J ¼ 2, 1 Hz), 7.02 (1H, ddd, J ¼ 8, 7, 1 Hz),
.70 (1H, t, J ¼ 6 Hz), 7.61 (1H, dd, J ¼ 8, 1 Hz), 7.43 (1H, dd, J ¼ 6.96–6.89 (3H, m), 4.05 (2H, t, J ¼ 6 Hz), 3.46 (2H, q, J ¼ 6 Hz),
1
3
, 1 Hz), 7.32–7.25 (2H, m), 7.17 (1H, ddd, J ¼ 8, 7, 1 Hz), 7.14 2.01 (2H, quint., J ¼ 6 Hz); C NMR (100 MHz, CDCl
3
): 161.2,
(
1H, dd, J ¼ 2, 1 Hz), 7.03 (1H, ddd, J ¼ 8, 7, 1 Hz), 7.00–6.96 (2H, 158.6, 136.4, 131.8, 129.5, 127.1, 123.2, 121.4, 120.5, 119.6, 114.4,
m), 6.93 (1H, tt, J ¼ 7, 1 Hz), 4.13 (2H, t, J ¼ 6 Hz), 3.67 (2H, q, J ¼ 112.3, 102.3, 65.1, 35.9, 29.1; IR: 3244, 3075, 3056, 2989, 2944,
1
3
6
1
3
1
C
0
Hz); C NMR (100 MHz, d -DMSO): 161.4, 158.4, 136.4, 131.5, 1609, 1563, 1497, 1471, 1425, 1418, 1383, 1368, 1341, 1317, 1307,
6
ꢃ1
29.5, 127.1, 123.3, 121.5, 120.7, 119.7, 114.5, 112.3, 102.6, 66.0, 1271, 1240, 1213 cm ; HRMS (ESI) m/z calcd for C20
H
19NNaO
2
+
8.5; IR: 3401, 3287, 2927, 2852, 1625, 1600, 1548, 1496, 1465, [M + Na] : 317.1260, found: 317.1253; TLC: R
f
0.29 (40%
ꢃ1
418, 1384, 1341, 1309, 1248 cm ; HRMS (ESI) m/z calcd for EtOAc–hexane).
+
17
H
16
N
2
NaO
2
[M + Na] : 303.1104, found: 303.1100; TLC: R
f
3-Methyl-N-(2-phenoxyethyl)butanamide (40). Prepared
according to the method described for 37 above, from 3-phenoxy-
-Methyl-N-(2-phenoxyethyl)butanamide (37). To a solution ethylamine (216 mg, 1.43 mmol, 1.0 equiv.) and isovaleryl
.27 (40% EtOAc–hexane).
3
of 2-phenoxyethylamine (216 mg, 1.4 mmol) and triethylamine chloride (250 mL, 2.06 mmol, 1.44 equiv.). The crude product
ꢁ
(
350 mL, 2.5 mmol, 1.8 equiv.) in dichloromethane (6 mL) at 0 C was puried by chromatography (EtOAc–hexane, 1 : 1–2 : 1) to
1
under nitrogen was added isovaleryl chloride (250 mL, 2.1 mmol, yield 40 (95 mg, 35%) as a aky, off-white solid. H NMR (400
.44 equiv.) dropwise over 5 min. The reaction mixture was MHz, CDCl
): 7.29 (2H, m), 6.96 (1H, tm, J ¼ 8 Hz), 6.89 (2H, dm,
stirred at room temperature for 24 h then poured into ethyl J ¼ 8 Hz), 5.82 (1H, bs), 4.04 (2H, t, J ¼ 6 Hz), 3.47 (2H, q, J ¼ 6
1
3
1
3
acetate (10 mL) and the organic phase washed with 1 M HCl and Hz), 2.11 (1H, m), 2.05–1.95 (4H, m), 0.95 (6H, d, J ¼ 6 Hz);
C
1
M NaOH. The aqueous phases were further extracted with NMR (100 MHz, CDCl
3
): 172.7, 158.7, 129.7, 121.1, 114.6, 66.4,
This journal is © The Royal Society of Chemistry 2014
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Concise Article
4
1
1
6.4, 37.5, 29.2, 26.3, 22.6; IR: 3299, 3069, 2956, 2927, 2871, 15 M. D. Eldridge, C. Murray, T. R. Auton, G. V. Paolini and
632, 1600, 1589, 1547, 1499, 1473, 1451, 1384, 1364, 1303,
P. M. Mee, J. Comput.-Aided Mol. Des., 1997, 11, 425–445.
ꢃ1
292, 1249 cm ; HRMS (ESI) m/z calcd for C H NNaO [M + 16 M. L. Verdonk, J. C. Cole, M. J. Hartshorn, C. W. Murray and
1
4
21
2
+
Na] : 258.1465, found: 258.1464; TLC: R 0.24 (hexane–EtOAc,
R. D. Taylor, Proteins, 2003, 52, 609–623.
f
1
: 1).
17 P. Axerio-Cilies, I. P. Casta n˜ eda, A. Mirza and J. Reynisson,
Eur. J. Med. Chem., 2009, 44, 1128–1134.
1
8 J. Reynisson, W. Court, C. O'Neill, J. Day, L. Patterson,
E. McDonald, P. Workman, M. Katan and S. A. Eccles,
Bioorg. Med. Chem., 2009, 17, 3169–3176.
Acknowledgements
Funding for this work was obtained from the Maurice and
Phyllis Paykel trust, the McClelland Trust, Auckland Medical 19 K. B. Larsen, T. Lamark, A. Øvervatn, I. Harneshaug,
Research Foundation and Genesis Oncology Trust (GOT-1429-
T. Johansen and G. Bjørkøy, Autophagy, 2010, 6, 784–793.
PDA). This work is also supported by Auckland Cancer Society 20 O. Korb, T. St u¨ tzle and T. E. Exner, J. Chem. Inf. Model., 2009,
Research Centre.
49, 84–96.
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1 W. T. M. Mooij and M. L. Verdonk, Proteins, 2005, 61, 272–
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87.
2 R. Wang and S. Wang, J. Chem. Inf. Comput. Sci., 2001, 41,
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