Development of second-generation indole-based dynamin GTPase inhibitors

Article abstract of DOI:10.1021/jm300844m

Focused library development of our lead 2-cyano-3-(1-(3-(dimethylamino) propyl)-2-methyl-1H-indol-3-yl)-N-octylacrylamide (2) confirmed the tertiary dimethylamino-propyl moiety as critical for inhibition of dynamin GTPase. The cyanoamide moiety could be replaced with a thiazole-4(5H)-one isostere (19, IC_{50(dyn I)} = 7.7 μM), reduced under flow chemistry conditions (20, IC_{50(dyn I)} = 5.2 μM) or replaced by a simple amine. The latter provided a basis for a high yield library of compounds via a reductive amination by flow hydrogenation. Two compounds, 24 (IC_{50 (dyn I)} = 0.56 μM) and 25 (IC_{50(dyn I)} = 0.76 μM), stood out. Indole 24 is nontoxic and showed increased potency against dynamin I and II in vitro and in cells (IC_50(CME) = 1.9 μM). It also showed 4.4-fold selectivity for dynamin I. The indole 24 compound has improved isoform selectivity and is the most active in-cell inhibitor of clathrin-mediated endocytosis reported to date.

Full text of DOI:10.1021/jm300844m

Article
pubs.acs.org/jmc
Development of Second-Generation Indole-Based Dynamin GTPase
Inhibitors
Christopher P. Gordon,† Barbara Venn-Brown,† Mark J. Robertson,† Kelly A. Young,† Ngoc Chau,‡
Anna Mariana,‡ Ainslie Whiting,‡ Megan Chircop,‡ Phillip J. Robinson,‡ and Adam McCluskey*,†
†Chemistry, Centre for Chemical Biology, School of Environmental and Life Sciences, The University of Newcastle, University Drive,
Callaghan, NSW 2308, Australia
‡Cell Signaling Unit and Cell Cycle Unit, Children’s Medical Research Institute, The University of Sydney, 214 Hawkesbury Road,
Westmead, NSW 2145, Australia
S
* Supporting Information
ABSTRACT: Focused library development of our lead 2-cyano-3-(1-(3-(dimethylamino)-
propyl)-2-methyl-1H-indol-3-yl)-N-octylacrylamide (2) confirmed the tertiary dimethyla-
mino-propyl moiety as critical for inhibition of dynamin GTPase. The cyanoamide moiety
could be replaced with a thiazole-4(5H)-one isostere (19, IC50(dyn I) = 7.7 μM), reduced
under flow chemistry conditions (20, IC50(dyn I) = 5.2 μM) or replaced by a simple amine.
The latter provided a basis for a high yield library of compounds via a reductive amination
by flow hydrogenation. Two compounds, 24 (IC50 (dyn I) = 0.56 μM) and 25 (IC50(dyn I)
=
0.76 μM), stood out. Indole 24 is nontoxic and showed increased potency against dynamin I
and II in vitro and in cells (IC50(CME) = 1.9 μM). It also showed 4.4-fold selectivity for
dynamin I. The indole 24 compound has improved isoform selectivity and is the most active in-cell inhibitor of clathrin-mediated
endocytosis reported to date.
throughout the body and is the major scission protein for CME,
while dynamin III (dynIII) is limited primarily to the brain and
testes and at low levels in a few other tissues. The dynamin I
(dynI) gene product is only found in neuronal cells where it
coexists with the other two forms. DynI is expressed in these
cells at about 50-fold higher levels compared with either dynIII
or dynII. This high level of dynI was expected to be critical for
neuronal cell function, however knockout studies have shown
both dynII and especially dynIII are able to facilitate SVE with
limited success, hinting to a range of redundancies within the
endocytic network.11 A crucial role has thus been suggested for
dynI to allow SVE to occur across a broad range of neuronal
activities and has been implicated as a potential target for
epilepsy.1
Control of endocytic pathways presents immense therapeutic
potential with defects in such pathways being involved in
multiple disease states such as Alzheimer’s disease, Hunting-
ton’s disease, Stiff-person syndrome, Lewy body dementias, and
Niemann−Pick type C disease.12−15 Endocytic pathways are
also utilized by viruses, including HIV,16 and bacteria to gain
entry into cells.17 Links to genetic disease are also emerging
with mutations in dynII causing Charcot−Marie−Tooth
disease and centronuclear myopathy, and dynII mutations are
involved in T-cell precursor acute lymphoblastic leukemia.18−20
Recently attention has also turned toward nonendocytic roles
that dynamin plays in the body including those involving the
INTRODUCTION
■
Dynamin is a large GTPase known to play a crucial role in
membrane remodelling, notably during endocytosis.1 Three
dynamin genes exist, and while the expressed proteins have
over 80% homology, they show differential tissue distribution,
suggesting they may have distinct biological roles. Common to
all are four domains, a GTPase domain (required for vesicle
fission),2,3 a pleckstrin homology domain (targeting domain
and potentially a GTPase inhibitory module),4 a bundle
signaling element (which controls dynamin self-assembly into
rings),5,6 and a proline-rich domain (which interacts with
proteins containing an SH3 domain2,4 and is the site for
dynamin phosphorylation in vivo).7,8 Two crystal structures of
mammalian dynamin I have been solved.9,10 Each lack the PRD
domain but provided insights into the complex dynamics
involved in oligomerization and how dynamin may act as a
scission protein in cells.
Endocytic mechanisms serve a variety of cellular functions
including the uptake of cellular nutrients, regulation of cell-
surface receptor expression and signaling, antigen presentation,
and maintenance of synaptic transmission. Clathrin mediated
endocytosis (CME) is one such mechanism and in mammalian
cells its usual function is internalization of extracellular
materials. In neuronal cells, this is a specialized pathway
termed synaptic vesicle endocytosis (SVE) and its role is
internalization of membrane to allow vesicle recycling.2,4 SVE
and CME thus perform distinct functional roles in different cell
types but share the same underlying protein machinery.
The differences in SVE and CME mostly arise from which
dynamin gene product is present. Dynamin II (dynII) is found
Received: June 15, 2012
© XXXX American Chemical Society
A
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Figure 1. BIM I scaffold simplification for rational design of dynamin inhibitors which afforded indoles 1 and 2.
a
Scheme 1
a
Reagents and conditions: (i) NaH, THF, reflux, 72 h; (ii) MeOH, rt, 4 h; (iii) EtOH, microwave, 120 °C, 150 W, 20 min.
actin cytoskeleton, the sperm acrosome reaction, or cytokine-
sis.21−23
and 5 (Scheme 1). These were prepared via a three step
procedure with alkylation of an indole (a, b), concurrent
formation of the cyanoamide (3), followed by coupling via a
Knoevenagel condensation.
Additional dimethylamino tail modified analogues (6−17)
were synthesized utilizing the Knoevenagel condensation and a
suite of commercially available N-alkylated indole-3-carbalde-
hydes possessing a range functionalities including hydroxyls,
amides, morpholines, and aromatic moieties (Scheme 2).34−41
There is now a toolbox of dynamin inhibitors available,
including the MiTMAB25 Bis-T,26,27 RTIL,28 Iminiodyn,29
Pthaladyn,30 Dyngo,31 and Rhodadyn32 dynamin inhibitors.
Within this palette perhaps the series displaying the most
potential as therapeutic agents is a series of indole analogues
(Figure 1).33 The two most active of this series, indole 1 and
indole 2, display high cell permeability and efficacy with cellular
IC50 values less than an order of magnitude from the in vitro
activity.33 Indole 2 is nontoxic, with fibroblast cells remaining
viable after 7 days.23,33 Indole 2, like MiTMAB, is a new class of
antimitotic agent with a novel mechanism of action, blocking
cytokinesis specifically at the abscission stage. Our lead, 2,
induces cell death in cancer cells specifically following
cytokinesis failure in dividing cells while nontumorigenic
fibroblasts are much less sensitive.23
a
Scheme 2
The indole-based dynamin inhibitors evolved from a series of
bisindolylmaleimides (BIMs) via a focused compound library
approach (Figure 1). Analogues in these initial libraries were
decorated with relatively simple functional groups including
cyclic and acyclic aliphatic hydrocarbons in addition to a
number of benzyl moieties.33 Given that these relatively simple
alterations imparted a 100-fold increase in dynI inhibition
potency, we sought to further explore this compound class and
the potential to install more drug-like moieties in developing
this second generation of indole-based dynamin inhibitors.
a
Reagents and conditions; (i) EtOH, microwave, 120 °C, 150 W, 20
min.
These compounds were evaluated as potential dynI
inhibitors, and the outcomes of this screening are presented
in Table 1. The absolute requirement of the dimethylamino tail
was confirmed. Increased steric shielding of the amino nitrogen
reduced activity (4, IC50 172 μM), while biosiosteric
replacement (−N(CH3)2 to −CH(CH3)2) (5) afforded an
analogue devoid of dynI inhibition. Similarly, all amide
substituted (9−14) and aromatic moieties (15−17) were
inactive. The hydroxy substituted analogues (6−8) all displayed
good to modest levels of dynamin inhibition, with IC50 values
in the range 4−18 μM. Interestingly here, encapsulation of the
dimethylamino tail as a cyclopentyl amine had no effect on
activity (6 and 7 are equipotent with IC50 values of 4.8 0.6
and 4.4 0.5 μM, respectively). While the −OH moiety does
introduce a chiral center, we note that oxidation to the amide
RESULTS AND DISCUSSION
■
We have previously demonstrated that the dimethylamino
moiety was a crucial element of the parent indole scaffold with
shortening the tail from propyl to ethyl, resulting in a slight
activity reduction while removal of the tail abolished inhibitory
activity.33 Given these limits, a more extensive investigation was
proposed. Herein our initial focus was directed to the nitrogen
atom of the dimethylamino tail with removal and increased
shielding to be examined with the synthesis of compounds 4
B
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Table 1. Inhibition of Purified Sheep Brain dynI GTPase Activity by Indole Analogues 1, 2, and 4−17
a
IC50 determinations are the mean 95% confidence interval (CI) of a single experiment performed in triplicate.
removes all activity, suggesting a potential hydrogen bond
donor role.
Thiazol-4(5H)-one isosteric replacement of the cyanoamide
moiety afforded 19, a 7.7 0.1 μM potent dynI inhibitor
Satisfied that the N,N-dimethylaminopropyl was a crucial and
optimal component of the indole scaffold, attention turned to
the cyanoamide linker (Figure 1). Research in our group
suggested that the thiazol-4(5H)-one moiety could function as
a cyanoamide bioisostere (unpublished data), and analogue 19
was synthesized via a two-step procedure (Scheme 3).
(Table 2). This represents only a slight reduction of activity
relative to the lead 2 (IC50 = 1.3 0.3 μM). This suggests that
the cyanoamide linker was more tolerable of chemical
alterations than the dimethylamino tail, and consequently we
turned our attention to the development of linker-altered
analogues.
C
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a
Scheme 3
a
Reagents and conditions: (i) acetonitrile, DIEA, MgCl2, rt, 48 h; (ii) 1-(3-dimethylaminopropyl)-1H-indole-3-carboxaldehyde,31 EtOH, microwave,
120 °C, 150 W, 20 min.
Table 2. Inhibition of Purified Sheep Brain dynI GTPase Activity by Linker Modified Indole Analogues 19 and 20
a
IC50 determinations are the mean 95% confidence interval (CI) of a single experiment performed in triplicate.
a
Scheme 4
a
Reagents and conditions; (i) ThalesNano H-cube hydrogenation, EtOH (conc 0.05 M), 50 °C, 50 bar, 1.0 mL/min 10% Pd/C.
Although our current synthetic methodologies had afforded
numerous indole-based analogues in excellent yields and
relatively short timeframes, we were eager to explore method-
ologies to improve synthetic efficiencies. Having access to a
range of flow chemistry instrumentation, we explored its
potential to optimize the synthetic approaches to these
analogues, potentially leading to enhanced synthetic efficiency,
increased yields, reduced reaction times and byproduct
formation, potentially minimizing complications typically
associated with scale-up.42−52 It was envisaged that the
ThalesNano H-cube (H-cube), a continuous flow hydro-
genation reactor, would allow rapid access to a series of
linker-altered analogues with potential modifications including
saturation of the cyanoamide linker and simplification of the
linker to an amine. Initially, reduction of the cyanoamide linker
was investigated whereby a solution of 2 was hydrogenated at
50 °C, under 50 bar of H2 pressure, with a 10% Pd/C catalyst
(Scheme 4). Concentration of the eluate afforded the reduced
analogue 20 in near quantitative yields, and screening revealed
it to be a 5.2 0.6 μM potent dynamin inhibitor (Table 2)
Successful flow hydrogenation of 2 led us to examine the
possibility of applying flow chemistry approaches to the
installation of the propyl dimethylamino tail in the first step
of the synthesis of this lead compound. Our previous approach
relied on sodium hydride (NaH) to install the propyl
dimethylamino tail onto the commercially available indole-3-
carboxyaldehyde.33 This suffered from unreliable yields. NaH
was problematic in any proposed flow chemistry route to
aldehyde 21 with the possibility of pressure build up, but of
greater concern was the requirement to develop a NaH-based
packed column. As flow chemistry requires all materials to be
either soluble in the flow solvent or to be suitable for packing
into an in-line column reactor, we thus investigated soluble
bases. Using the SYRRIS FRX100 twin pump system and a 4
mL tube reactor, our initial investigations centered on the use
of NaOH/MeOH mixtures (Scheme 5). As can be seen from
the data presented in Table 3, even at relatively low
D
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a
Given the success of this initial hydrogenation and our ability
to replace the cyanoamide moiety with 19 and 20, we
questioned the absolute requirement for such a moiety. We
investigated the potential of synthesizing additional analogues
by replacing the cyanoamide with a simple amine by simple
reductive amination of indole carboxaldehyde (21) with a series
of amines in the H-cube. The imine was preformed by
microwave irradiation of a suspension of octylamine,
substituted indole-3-carboxaldehyde (21), and MgSO4 in
toluene for 10 min. After workup, the resultant solution was
diluted and subjected to flow hydrogenation (50 °C, 50 bar H2
pressure, 1 mL/min flow rate). Concentration of the eluate
afforded compound 22 in a 78% yield (Scheme 6).
Scheme 5
a
Reagents and conditions: NaOH, 0.5 M (for solvent, see Table 3), 1
mL/min.
Table 3. Flow Chemistry Optimization of the Addition of 3-
Chloro-N,N-propan-1-amine with 1H-Indole-3-
carbaldehyde
Both hydrogenated analogues displayed good dynI inhibitory
activity with the saturated analogue 20 displaying a modest
reduction in activity (IC50 = 5.2 0.6 μM; Table 2), while the
“linker-absent” analogue 22 (IC50 = 2.3 0.7 μM) (Table 4),
a
base
solvent
temp (°C)
yield (%)
time
NaH
THF
56
80
45
<20
20
72 h
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
MeOH
10 min
10 min
10 min
10 min
10 min
10 min
10 min
Table 4. Inhibition of Purified Sheep Brain dynI GTPase
Activity by Analogues 22−31
MeOH
90
MeOH
100
110
120
130
160
23
MeOH
30
MeOH
35
MeOH
55
EtOH/H2O (1:1)
75
a
Flow rate total of 1 mL/min, solutions were 0.5M in specified
solvent.
temperatures of 80 °C the desired product was detectable,
1
<20%, by H NMR after a 10 min reaction run at a combined
flow rate of 1 mL/min. Increasing the reaction temperature in
10 °C increments, under increased system pressure (FRX
pressurization module), saw a steady increase in the product
yield with the best conversion noted in MeOH at 130 °C of
55%. Solvent switching to EtOH/H2O (1:1) allowed the
reaction to be conducted at 160 °C with a 75% conversion
noted. This was a significant enhancement on our originally
reported synthesis using NaH/THF for 72 h to effect a 45%
yield.33 Encouraged by the ease of addition under flow
chemistry conditions, we re-examined our batch chemistry
approaches with milder bases (than NaH). Of the numerous
bases examined, cesium carbonate afforded the best outcome,
with yields increased to 88%, respectively, after aqueous
workup and chromatography. Thus choice of base is an
important consideration with the so-called “cesium effect”.53
Further optimization via the addition of potassium iodide in a
Finkelstein modification gave quantitative yields of the desired
product (21) on simple aqueous workup without the need for
further purification.
a
IC50 determinations are the mean 95% confidence interval (CI) of
a single experiment performed in triplicate.
a
Scheme 6
a
Reagents and conditions; (i) Cs2CO3, KI, ACN, reflux, 28 h; (ii) MeOH, microwave, 100 °C, 150 W, 10 min; (iii) H-cube hydrogenation, EtOH
(conc 0.05 M), 50 °C, 50 bar, 1.0 mL/min 10% Pd/C.
E
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a
Scheme 7
a
Conditions and reagents; (i) MeOH, microwave, 100 °C, 150 W, 10 min; (ii) H-cube hydrogenation, EtOH (conc 0.05 M), 50 °C, 50 bar, 1.0 mL/
min 10% Pd/C.
was near equipotent to the lead indole 2 (IC50 = 1.30 0.3
μM). Thus, utilizing microwave and flow hydrogenation
methodologies, the potent dynamin inhibitor 22 was prepared
in an excellent yield in a simple two-step synthesis. The flow
rates and concentration used correspond to a conversion of
∼100 mg of compound per hour. Subsequently, we found that
the initial microwave step in toluene/MgSO4 was not necessary
and simple irradiation and dilution in MeOH under the same
flow conditions afforded the desired compound in quantitative
yields. This is highly adaptable to large-scale synthesis being
only limited by the volume of reagent (at 0.05 M) and the
lifespan of the catalyst cartridge.
Buoyed by this result, we set about synthesizing an additional
library of compounds utilizing the microwave-hydrogenation
sequential protocol. Previously significant alterations in
dynamin inhibitory activity were observed with extensions
and contractions of the aliphatic tail, thus a small series of
analogues (23−31) were prepared to investigate optimum
chain length (Scheme 7).
Table 5. Clathrin-Mediated Endocytosis of Tfn Inhibition in
U2OS Cells by Second-Generation Indole-Based Dynamin
Inhibitors
a
b
compd
dynI IC50 (μM)
CME IC50 (μM)
cLogP
c
d
d
d
2
1.30 0.30
4.8 0.6
5.0 0.9
3.72
2.71
3.52
4.53
5.45
6.39
7.31
5.22
c
6
3.5 0.5
c
7
4.4 0.5
4.5 1.1
c
22
24
25
26
15
2.3 0.7
3.5 0.4
c
0.56 0.09
0.76 0.05
1.1 0.3
1.9 0.3
2.8
c
6.8 2.0
d
not active
not active
a
IC50 determinations are the mean 95% confidence interval (CI) of a
b
single experiment performed in triplicate. Calculated using the
OSIRIS online molecular property calculator. IC50 represents mean
SEM of pooled data from at least two independent experiments. The
c
d
OSIRIS molecular property calculator does not deal with cyanamide
moietes well, these values should be used with caution.
As observed with the first-generation indole-based analogues
extension of the aliphatic chain significantly altered dynamin
inhibitory activity (Table 4), with the hexyl (23) and octyl (22)
analogues possessing IC50 values of 9.7 and 2.3 μM,
respectively, while the decyl analogue (24) displayed an IC50
value of 0.56 μM, the first submicromolar potent indole-based
analogue. In the first-generation indole series, significant
potency drop off was noted on extending the alkyl chain
from octyl (1 IC50 = 3.30 0.3 μM) to decyl (IC50 = 10.1 μM)
especially possibly via accumulation in cell membranes or
other off-target effects. Such interactions would not preclude
the development of these compounds as therapeutic agents
with other membrane interacting compounds in current clinical
use.56 To ascertain that this second generation of indole-based
dynamin inhibitors remained relatively nontoxic to cells, a
comparison was completed involving our two new most potent
analogues (24 and 25) and our lead 2 (Figure 2). Using a
Trypan Blue exclusion assay 24 and 25, at 30 μM drug
concentration, displayed considerably less toxicity (10%) than
our first-generation analogues (2, 80%).
to dodecyl (IC50 = 32
2 μM).31 Herein, our second-
generation indole analogues readily accommodate longer alkyl
chains, viz decyl (24, IC50 = 0.56 0.09 μM), dodecyl (25,
IC50 = 0.76 0.05 μM), and tetradecyl (26, IC50 1.1 0.3
μM). We attribute this increased alkyl chain length tolerance to
the removal of the −CC(CN)-(CO)− linker moiety. The
obvious penalty is an increase cLogP from 4.72 (2) to 5.45
(24) (Table 5). These analogues have incorporated relatively
simple and non “drug-like” straight chain aliphatic moieties. To
rectify this, a number of more “drug-like” amines were
investigated as potential tail units (27−31). The changes
were reasonably tolerated, with the chains terminating in
aromatic or bulky hydrophobic units (compounds 27, 30, and
31, respectively), retaining activity with the latter two being
more potent (Table 4). Additionally, this small series of
compounds suggest that a number of heteroatoms including
cyclic groups may be incorporated within this region of the
indole scaffold. This may allow fine-tuning to generate
preferred physicochemical properties in the future.
Figure 2. Cytotoxicity (%) of 2 and the second-generation indole
analogues 24 and 25 in HeLa cells after 8 h treatment with the data
normalized to drug/media background.
Alkyl chain elongation in analogues such as 24 and 25 would
be expected to promote additional off-target concerns,
F
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A subset of the most dynamin GTPase-active compounds
were next selected to undergo in-cell testing for activity in
clathrin mediated endocytosis (CME). This is a whole-cell
assay wherein endocytosis is mediated by dynII. Six dynI-active
compounds (6, 7, 22, and 24−26) and one dynI inactive
compound (15) were tested. Five of the six dynI active
compounds were found to be potent inhibitors of CME and
more potent than 2, and the sixth analogue (26) was marginally
less potent with an IC50 of 6.8 2.0 μM. The most potent, 24,
with a CME IC50 = 1.9 μM, was 3-fold more active than 2
(Table 5); the remaining CME active analogues returned CME
IC50 values of 2.8−6.8 μM. Analogues 6 and 7, which contain
the additional −OH moiety, are essentially equipotent CME
and dynI inhibitors, with CME IC50 values of 3.5 0.5 and 4.5
1.1 and dynI IC50 values of 4.8 0.6 and 4.4 0.5 μM,
respectively. This may be due to cell permeability, with only 6
and 7 having cLogP values less than 4. This highlights the
importance of including physicochemical properties in the
design of the next generation dynole inhibitors. The dynamin
inactive analogue 15 was also inactive in cells against CME,
supporting the likelihood that there is on-target specificity
(Table 5).
Given the development of 24 (which we now call the Dynole
2−24 compound), we were keen to determine if it may have
any selectivity for dynI vs dynII and to compare it to our lead 2.
IC50 values are arbitrary numbers that primarily depend on the
enzyme concentration employed in the assay because the
analogues bind to the enzymes. It is therefore appropriate to
compare the two dynamins under normalized GTPase assay
conditions wherein the concentration of each dynamin was
maintained at the same 50 nM (Table 6). This change was
pyrrolidine, piperidine, and morpholine moieties retaining
activity. In the absence of the β-hydroxy substituent,
encapsulation of the N,N-dimethylaminopropyl moiety was
not tolerated and was a crucial and optimal component for
activity. The overall SAR of the cyanoamide moiety identified
three isosteric modifications that either retained or enhanced
activity: reduction to a β-methylamino amide or replacement
with a thiazole-4(5H)-one or a secondary amine. Significant
enhancements were observed with the introduction of the
secondary amine. Current ongoing investigations are focused
on surveying an additional suite of drug-like amines, and the
results of these investigations will be reported in due course.
The flow chemistry steps of the synthesis produced
important advances in the rapid optimization of the reaction
conditions, a significant yield enhancement, and the use of
more environmentally friendly reagents relative to the original
synthesis that required NaH/THF. The combination of
microwave and flow chemistry methodologies allowed the
identification of batch reaction conditions, yielding significant
quantities of these second-generation dynamin inhibitors to be
generated in high yields and with minimum reaction workup. In
particular, the Finkelstein modification using KI/CaCO3
afforded virtually quantitative yields of the N-alkylated
products. Therefore our work represents the development of
a vastly simplified route to these compounds.
The decyl amine analogue 24 (dynI IC50 = 0.56 0.09 μM)
is the first indole to elicit submicromolar activity against
dynamin GTPase activity and only the second class of dynamin
inhibitor to be submicromolar potent. A range of these second-
generation indole analogues exhibited potent in-cell inhibition
of CME, indicating that they retain on-target specificity.
Despite that our inclusion of straight alkyl chains has the
potential to promote membrane interactions off-target activity,
24 has a reduced toxicity profile. Indole 24 also displays
enhanced dynII inhibition as well as a small improvement in
dynI vs dynII selectivity relative to 2. The enhanced dynII
inhibition correlates with enhanced CME block in U2OS cells,
and this compound is therefore the most potent reported in-cell
inhibitor of dynamin mediated CME.
Table 6. dynI vs dynII Selectivity of 2 and 24 Using
a
Normalized Assay Conditions
compd FL-dynI IC50 (μM) FL-dynII IC50 (μM) dynI/dynII selectivity
2
6.9 1.0
1.7 0.3
16 10
2.3
4.4
24
5.4 7.5
a
GTPase assays for dynamin I and II were normalized so that IC50
values for the two enzymes can be directly compared. The assays
contained 50 nM dyn I or II (pre-incubated with 10 μg/mL PS for 15
min at RT), 300 μM GTP, 1 μg/mL leupeptin, and 0.1 μM AEBSF.
The reaction time for dynI and II assays were 10 and 90 min,
respectively.
EXPERIMENTAL SECTION
■
Biology. Materials. Phosphatidylserine (PS) and Tween 80 were
from Sigma-Aldrich (St Louis, CA). 4-(2-Aminoethyl)-benzenesulfon-
yl fluoride hydrochloride (AEBSF) was from Calbiochem (USA). GTP
was from Roche Applied Science (Germany), and leupeptin was from
Bachem (Bubendorf, Switzerland). Gel electrophoresis reagents,
equipment, and protein molecular weight markers were from Bio-
Rad (Hercules, CA). Paraformaldehyde (PFA) was from Merck Pty
Ltd. (Kilsyth, Australia). Phosphate buffered salts, fetal bovine serum
(FBS), and Dulbecco’s Minimal Essential Medium (DMEM) were
from Invitrogen (Mount Waverley, Victoria, Australia). Alexa-594
conjugated Tfn (Tfn−A594) and DAPI were from Molecular Probes
(Oregon, USA). All other reagents were of analytical reagent grade or
better.
Drugs. Drugs were made in-house. They were made up as stock
solutions (30 mM) in 100% DMSO and diluted in 50% v/v DMSO/
20 mM Tris/HCl pH 7.4 or cell media prior to further dilution in the
assay. The final DMSO concentration in the GTPase or endocytosis
assays was at most 3% or 1%, respectively. The GTPase assay for dynI
was unaffected by DMSO up to 3.3%. Drug stocks can be stored at
−20 °C for several months.
made because the PS-stimulated activity of dynII was
considerably lower than that of dynI, in part due to its very
high intrinsic basal activity,54 thus its GTPase activity was not
within the linear part of the Malachite Green assay unless
elevated enzyme concentrations were used. In the normalized
assay, all other assay conditions were the same except for the
reaction time. Therefore the IC50 values for dynI under these
normalized conditions (Table 6, 50 nM) are higher than those
observed in Table 4 (at 20 nM dynI). This revealed that 2 is 2-
fold dynI-selective, while 24 is 4.4-fold dynI-selective (Table 6).
CONCLUSION
■
Our work represents a significant enhancement on the dynamin
inhibition SAR of the indole-based dynamin inhibitor scaffold
and highlights a number of advancements in the path to drug
development for these compounds. SAR showed that
encapsulation of the N,N-dimethylaminopropyl moiety was
permitted if a β-hydroxy substituent was added, with terminal
Protein Production. Endogenous dynI (18 mg) was purified from a
sheep brain by extraction from the peripheral membrane fraction of
whole brain and affinity purification on GST−Amph2−SH3−
sepharose as described.23 Full length rat dynII (His-6-tagged) was
G
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expressed as a recombinant protein from a pIEx-6 vector in insect cells
(SF21) using polyethylenimine as the transfection reagent using a
DNA: polyethylenimine = 1:5 for 48 h and was purified by affinity
purification on GST−Amph2−SH3−sepharose as described previ-
ously.29
percentage of control cells (vehicle treated). The average number of
cells for each data point was ∼1200. IC50 values were calculated using
Graphpad Prism v5, and data was expressed as mean
95%
confidence interval (CI) for three wells and ∼1200 cells.
Cell Viability Assay. Cell viability and membrane integrity was
assessed by the Trypan Blue exclusion assay. Cells were seeded into a
a10 cm dish at a density of 1 × 105 cells per dish. On day 0 (24 h after
seeding), cells were treated in the presence and absence of compounds
or vehicle control (0.5% DMSO) for 7 days. Cells (floating and
adherent) were collected, and cell viability was measured by Vi-CELL
XR cell viability analyzer 2.03 (Beckman Coulter).
Malachite Green GTPase Assay: Standard Assay Conditions
(dynI). Malachite Green method was used for the sensitive
colorimetric detection of orthophosphate (Pi). The assay is based on
stimulation of native sheep brain-purified dynI by sonicated PS
liposomes.55 Although in our earlier studies we used 200 nM dynI, in
the present study we used 20 nM, requiring an optimization of assay
volumes and the Malachite Green reagent.24,26 Purified dynI (20 nM)
(diluted in 6 mM Tris/HCl, 20 mM NaCl, 0.01% Tween 80, pH 7.4)
was incubated in GTPase assay buffer (5 mM Tris/HCl, 10 mM NaCl,
2 mM Mg2+, 0.05% Tween 80, pH 7.4, 1 μg/mL leupeptin, and 0.1
mM AEBSF) and GTP 0.3 mM in the presence of test compound for
30 min at 37 °C. The final assay volume was 150 μL in round-
bottomed 96-well plates. Plate incubations were performed in a dry
heating block with shaking at 300 rpm (Eppendorf Thermomixer).
Dynamin I GTPase activity was maximally stimulated by addition of
different concentrations of phosphatidylserine liposomes. This assay
can also be conducted in the absence of Tween with little effect on the
IC50 values noted for the indoles listed in Tables 1, 2, and 4. The
reaction was terminated with 10 μL of 0.5 M EDTA pH 8.0, and the
samples were stable for several hours at room temperature. To each
well was added 40 μL of Malachite Green solution (2% w/v
ammonium molybdate tetrahydrate, 0.15% w/v malachite green, and 4
M HCl; the solution was passed through 0.45 μm filters and stored in
the dark for up to 2 months at room temperature). Color developed
for 5 min (and was stable up to 2 h), and the absorbance was
determined on a microplate spectrophotometer at 650 nm. Phosphate
release was quantified against a standard curve of sodium dihydrogen
orthophosphate monohydrate (baked dry at 110 °C overnight) which
was run in each experiment. GraphPad Prism 5 (GraphPad Software
Inc., San Diego, CA) was used for plotting data points and analysis of
enzyme kinetics using nonlinear regression.
Malachite Green GTPase Assay: Normalized Assay Conditions
(dynI and II). Normalized assay conditions were developed in order to
compare the IC50 values for dynamin I and II at the same enzyme
concentration: 50 nM dynI and II (diluted in 6 mM Tris/HCl, 20 mM
NaCl, 0.01% Tween 80, pH 7.4) were used, with 10 μg/mL PS being
used to stimulate both. DynI or II were preincubated with PS for 15
min at room temperature without shaking (in 6 mM Tris-HCl, 20 mM
NaCl, 0.01% Tween-80, pH 7,4), following which the mixture was
incubated in GTPase assay buffer (5 mM Tris-HCl, 10 mM NaCl, 2
mM Mg2+, 0.05% Tween-80, pH 7.4, 1 μg/mL leupeptin, and 0.1 mM
AEBSF) with 300 μM GTP in the presence of test compound at 37 °C
with constant shaking at 800 rpm. The reaction times were 10 and 90
min for dynI and II, respectively, in order to maintain activity within
the dynamic range of the assay. Final assay volume and the shaking
conditions were the same for both assays. The reactions were
terminated with 10 μL of 0.5 M EDTA, pH 8.0.
Chemistry. General Methods. All reagents were purchased from
Sigma-Aldrich, Matrix Scientific, or Lancaster Synthesis and were used
without purification. With the exception of THF (anhydrous >99%)
obtained from Sigma-Aldrich, all solvents were redistilled from glass
prior to use.
1H and 13C NMR spectra were recorded on a Bruker Avance AMX
300 MHz spectrometer at 300.1315 and 75.4762 MHz, respectively.
Chemical shifts (δ) are reported in parts per million (ppm) measured
relative to the internal standards, and coupling constants (J) are
expressed in hertz (Hz). Mass spectra were recorded on a Shimadzu
LCMS 2010 EV using a mobile phase of 1:1 acetonitrile:H2O with
0.1% formic acid.
Melting points were recorded on a Stuart Scientific melting point
apparatus (UK) and are uncorrected. Thin layer chromatography
(TLC) was performed on Merck silica gel 60 F254 precoated aluminum
plates with a thickness of 0.2 mm. Column chromatography was
performed under “flash” conditions on Merck silica gel 60 (230−400
mesh) or using the Biotage SP4 flash purification system with a 100 g
prepacked snap column.
All compounds were >95% pure as determined by combustion
analysis.
A CEM Discover BenchMate microwave (120 °C, 200 W, 1 h) was
used to perform several refluxes. Hydrogenations were performed
using the ThalesNano H-cube continuous-flow hydrogenation reactor
utilizing a palladium−carbon (CatCart) catalyst, a flow rate of 1 mL/
min, 40 bar of pressure, and column temperature 40 °C. Optimization
of the addition to indole-3-carboxaldehyde were conducted using the
SYRRIS FRX100 equipped with a 4 mL tube reactor and a FRX
pressurization module.
General Procedure 1. 1-(3-Piperidin-1-yl-propyl)-1H-indole-3-
carbaldehyde (5). To a stirred solution of indole-3-carbaldehyde
(0.99 g, 6.8 mmol) and anhydrous THF (30 mL) at 0 °C was added
sodium hydride (50% dispersion in oil, 2.0 g) in small portions over a
10 min period. The resulting pink solution was stirred at 0 °C for 30
min prior to the addition of 1-(3-chloropropyl)piperidine mono-
hydrochloride (1.44 g, 7.3 mmol). The reaction mixture was heated at
reflux for 72 h, quenched with water (50 mL), the organic material
extracted with EtOAc (3 × 40 mL), dried (MgSO4), and concentrated
in vacuo. The resulting brown solid was purified by flash
chromatography (1:1 MeOH/DCM) to afford 1-(3-piperidin-1-yl-
1
propyl)-1H-indole-3-carbaldehyde (0.12 g, 7%) as an orange oil. H
Texas Red-Tfn Uptake. U2OS cells were cultured in DMEM
supplemented with 10% FBS at 37 °C and in 5% CO2 in a humidified
incubator. Tfn uptake was analyzed based on methods previously
described.33 Briefly, cells were grown in fibronectin-coated (5 μg/mL)
96-well glass-bottomed plates. The cells were serum-starved overnight
(16 h) in DMEM minus FBS. Cells were then incubated with test
compounds (1−300 μM) or vehicle for 30 min prior to addition of 4
μg/mL Tfn-TxR for 8 min at 37 °C. Cell surface-bound Tfn was
removed by incubating the cells in an ice-cold acid wash solution (0.2
M acetic acid + 0.5 M NaCl, pH 2.8) for 10 min and washed with ice
cold PBS for 5 min. Cells were immediately fixed with 4% PFA for 10
min at 37 °C. Nuclei were stained using DAPI. Quantitative analysis of
the inhibition of Tfn-TxR endocytosis in U2OS cells was performed
on large numbers of cells by an automated acquisition and analysis
system (Image Xpress Micro, Molecular Devices, Sunnyvale, CA).
Nine images were collected from each well, averaging 40−50 cells per
image. The average integrated intensity of Tfn-TxR signal per cell was
calculated for each well using IXM system, and the data expressed as a
NMR (CDCl3): δ 10.00 (1H, s), 8.31 (1H, m), 7.81 (1H, s), 7.42 (1H,
m), 7.35 (2H, m), 4.31 (2H, t, J = 6.6 Hz), 2.45 (4H, m), 2.34 (2H, t, J
= 6.9 Hz), 2.17 (2H, quin, J = 6.9 Hz), 1.68 (4H, m), 1.49 (2H, m).
13C NMR (CDCl3): δ 184.0, 138.2, 136.8, 125.7, 124.1, 123.4, 122.4,
121.6, 109.5, 54.5, 53.8, 44.2, 25.7, 24.9, 23.5. MS (ES+): m/z 271 (M
+ 1, 100%).
2-Methyl-1-(4-methylpentyl)-1H-indole-3-carbaldehyde (4).
Compound 2 was synthesized using general procedure 1, 2-methyl-
1H-indole-3-carbaldehyde (3.0 g, 18.9 mmol), 5-chloro-2-methylpen-
tane (3.1 g, 18.9 mmol), sodium hydride (50% dispersion in oil, 2.0 g),
and THF (20 mL) to afford the title compound as a yellow oil (2.67 g,
58%). 1H NMR (CDCl3): δ 10.12 (1 H, s), 8.28 (1 H, td, J = 5.7, 2.4
Hz), 7.35−7.19 (3 H, m), 4.00 (2 H, t, J = 6.7 Hz), 2.61 (3 H, s),
1.85−1.66 (2 H, m), 1.56 (1 H, sep, J = 6.6, Hz) 1.24 (2 H, m), 0.89
(6 H, d, J = 6.6 Hz). 13C NMR (CDCl3): δ 183.6, 146.7, 135.7, 125.3,
122.4, 122.1, 120.3, 113.6, 109.0, 43.1, 35.4, 27.2, 26.9, 21.9, 9.9. MS
(ES+): m/z 244 (M + 1, 100%).
H
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2-Cyano-N-octylacetamide (3). A solution of methyl cyanoacetate
(2.16 g, 21.8 mmol), n-octylamine (2.83 g, 21.8 mmol), and MeOH
(10 mL) were stirred at room temperature for 4 h. The resulting solid
was collected by vacuum filtration and recrystallized from EtOH to
afford 2-cyano-N-octylacetamide (3.38 g, 79%) as a white flaky solid
quin, J = 6.6 Hz), 1.28 (10H, m), 0.86 (3H, t, J = 6.3 Hz). 13C NMR
(DMSO-d6): δ 162.2, 146.8, 144.3, 137.4, 124.4, 122.2, 121.0, 120.8,
118.4, 110.7, 107.4, 98.2, 68.2, 60.0, 54.2, 48.3, 39.8, 31.2, 29.0, 28.6,
28.5, 26.3, 23.1, 22.0, 13.9, 11.8. MS (ES+): m/z 465 (M + 1, 100%).
3-(1-(2-Hydroxy-3-morpholinopropyl)-1H-indol-3-yl)-2-cyano-N-
octylacrylamide (8). Compound 8 was synthesized utilizing general
procedure 2, 1-(2-hydroxy-3-morpholin-4-yl-propyl)-1H-indole-3-car-
baldehyde (0.18 g, 0.63 mmol), and 2-cyano-N-octylacetamide (0.14 g,
0.73 mmol). Recrystallization from EtOH afforded 3-(1-(2-hydroxy-3-
morpholinopropyl)-1H-indol-3-yl)-2-cyano-N-octylacrylamide (270
1
(mp 68−70 °C). H NMR (CDCl3): δ 6.48 (1H, bs), 3.37 (2H, s),
3.25 (2H, q, J = 6.6 Hz), 1.51 (2H, quin, J = 6.6 Hz), 1.28 (10 H, m),
0.86 (3H, t, J = 6.3 Hz). 13C NMR (CDCl3): δ 160.6, 114.4, 39.8, 31.2,
28.9, 28.6, 28.5, 26.2, 25.3, 22.0, 13.9. MS (ES+): m/z 197 (M + 1,
100%).
1
mg, 92%) as an orange oil. H NMR (CDCl3): δ 8.66 (1H, s), 8.46
General Procedure 2. 3-(1-(3-Piperidin-1-yl-propyl)-1H-indol-3-
yl)-2-cyano-N-octylacrylamide (4). A solution of 1-(3-piperidin-1-
ylpropyl)-1H-indole-3-carbaldehyde (0.09 g, 0.33 mmol), 2-cyano-N-
octylacetamide (0.07 g, 0.36 mmol), piperidine (0.1 mL), and EtOH
(5 mL) was irradiated with microwaves (120 °C, 150 W) for 20 min.
The reaction vessel was cooled and placed on ice, and the resulting
precipitate was recrystallized from ethanol to afford 3-(1-(3-piperidin-
1-yl-propyl)-1H-indol-3-yl)-2-cyano-N-octylacrylamide (46 mg, 31%)
as a yellow crystalline solid (mp 116−117 °C). 1H NMR (DMSO-d6):
δ 8.45 (1H, s), 8.40 (1H, s), 8.23 (1H, t, J = 5.7 Hz), 7.93 (1H, d, J =
7.2 Hz), 7.66 (1H, d, J = 7.8 Hz), 7.30 (2H, m), 4.38 (2H, t, J = 6.6
Hz), 3.20 (2H, q, J = 6.4 Hz), 2.24 (4H, m), 2.13 (2H, t, J = 6.3 Hz),
1.93 (2H, quin, J = 6.3 Hz), 1.49 (6H, m), 1.36 (2H, m), 1.27 (10H,
m), 0.86 (3H, t, J = 6.4 Hz). 13C NMR (DMSO-d6): δ 161.8, 141.1,
135.9, 132.8, 127.7, 123.2, 121.6, 118.5, 118.5, 111.1, 108.6, 97.4, 54.7,
53.8, 44.4, 39.8, 31.1, 28.9, 28.6, 28.5, 26.3, 26.1, 25.4, 24.0, 22.0, 13.8.
MS (ES+): m/z 449 (M + 1, 100%).
(1H, s), 7.87 (1H, dd, J = 6.9 Hz, 1.8 Hz), 7.47 (dd, J = 6.9 Hz, 1.8
Hz), 7.34 (2H, m), 4.34 (1H, dd, J = 14.4 Hz, 3.6 Hz), 4.23 (1H, dd, J
= 14.3 Hz, 6.0 Hz), 4.14 (1H, m), 3.70 (4H, m), 3.45 (2H, q, J = 6.6
Hz), 2.65 (2H, m), 2.40 (4H, m), 1.61 (2H, quin, J = 6.9 Hz), 1.35
(10H, m), 0.89 (3H, t, J = 6.9 Hz). 13C NMR (CDCl3): δ 161.7, 143.9,
136.8, 133.4, 128.3, 123.7, 122.4, 119.4, 118.9, 110.6, 110.4, 95.9, 66.9,
65.7, 61.4, 53.6, 50.7, 40.4, 31.8, 29.6, 29.3, 29.2, 26.9, 22.6, 14.1. MS
(ES+): m/z 467 (M + 1, 100%).
3-(1-(2-Cyclopentylamino-2-oxoethyl)-1H-indol-3-yl)-2-cyano-N-
octylacrylamide (9). Compound 9 was synthesized utilizing general
procedure 2, N-cyclopentyl-2-(3-formylindol-1-yl)acetamide (0.20 g,
0.75 mmol), and 2-cyano-N-octylacetamide (0.16 g, 0.79 mmol).
Recrystallization from EtOH afforded 3-(1-(2-cyclopentylamino-2-
oxoethyl)-1H-indol-3-yl)-2-cyano-N-octylacrylamide (157 mg, 47%)
as a yellow solid, mp 184−186 °C. 1H NMR (DMSO-d6): δ 8.44 (2H,
bs), 8.40 (1H, s), 8.26 (1H, t, J = 5.7 Hz), 7.93 (1H, dd, J = 7.6 Hz, 1.5
Hz), 7.48 (1H, dd, J = 7.5 Hz, 1.5 Hz), 7.30 (2H, m), 5.00 (2H, s),
4.00 (1H, m, J = 6.8 Hz), 3.21 (2H, q, J = 6.5 Hz), 1.81 (2H, m), 1.67
(2H, m), 1.51 (4H, m), 1.42 (2H, m), 1.28 (10H, m), 0.86 (3H, t, J =
6.6 Hz). 13C NMR (DMSO-d6): δ 165.6, 161.8, 141.1, 136.4, 133.8,
127.6, 123.4, 121.8, 118.5, 118.5, 110.9, 108.8, 97.8, 50.6, 49.3, 39.8,
32.3, 31.2, 29.0, 28.7, 28.6, 26.4, 23.4, 22.1, 14.0. MS (ES+): m/z 449
(M + 1, 100%).
2-Cyano-3-(2-methyl-1-(4-methylpentyl)-1H-indol-3-yl)-N-octyla-
crylamide (5). Compound 5 was synthesized using general procedure
2, 2-methyl-1-(4-methylpentyl)-1H-indole-3-carbaldehyde (2.30 g,
9.46 mmol), 2-cyano-N-octylacetamide (0.45 g, 2.30 mmol),
piperidine (0.1 mL), and EtOH (5 mL). Recrystallization from
EtOH afforded the title compound as a yellow solid (2.47 g, 62%), mp
1
96−98 °C. H NMR (CDCl3): δ 8.59 (1 H, s), 8.19−8.12 (1 H, m),
7.34−7.23 (3 H, m), 6.37 (1 H, t, J = 5.5 Hz), 4.07 (2 H, t, J = 7.6 Hz),
3.47 (2 H, q, J = 7.6 Hz), 3.46−3.38 (1 H, m), 2.56 (3 H, s), 1.86−
1.70 (2 H, m), 1.68−1.57 (2 H, m), 1.68−1.50 (2 H, m), 1.46−1.24 (8
H, m), 1.20 (2 H, t, J = 7.6 Hz), 0.89 (9 H, dd, J = 6.7, 3.3 Hz). 13C
NMR (CDCl3): δ 161.9, 145.5, 144.2, 136.6, 124.9, 122.4, 121.4,
121.2, 119.0, 109.2, 108.7, 96.043.7, 39.9, 35.4, 31.2, 29.0, 28.7, 28.6,
27.2, 26.9, 26.4, 22.0, 21.8, 13.5, 11.3. MS (ES+): m/z 422 (M + 1,
100%).
3-(1-(2-Oxo-2-pyrrolidin-1-yl-ethyl)-1H-indol-3-yl)-2-cyano-N-oc-
tylacrylamide (10). Compound 10 was synthesized utilizing general
procedure 2, 1-(2-oxo-2-pyrrolidin-1-yl-ethyl)-1H-indole-3-carbalde-
hyde (0.20 g, 0.77 mmol), and 2-cyano-N-octylacetamide (0.16 g,
0.84 mmol). Recrystallization from EtOH afforded 3-(1-(2-oxo-2-
pyrrolidin-1-yl-ethyl)-1H-indol-3-yl)-2-cyano-N-octylacrylamide (278
1
mg, 83%) as a yellow crystalline solid, mp 144−145 °C. H NMR
(DMSO-d6): δ 8.41 (1H, s), 8.40 (1H, s), 8.26 (1H, t, J = 5.4 Hz),
7.92 (1H, dd, J = 6.9 Hz, 2.4 Hz), 7.55 (1H, d, J = 8.6 Hz), 7.28 (2H,
m), 5.32 (2H, s), 3.61 (2H, t, J = 6.7 Hz), 3.30 (2H, t, J = 6.7 Hz),
3.21 (2H, q, J = 6.5 Hz), 1.96 (2H, m), 1.83 (2H, m), 1.51 (2H, m),
1.28 (10H, m), 0.85 (3H, t, J = 6.6 Hz). 13C NMR (DMSO-d6): δ
164.5, 161.8, 141.0, 136.9, 133.8, 127.4, 123.0, 121.5, 118.4, 118.2,
111.3, 108.9, 97.7, 48.5, 45.7, 44.9, 39.8, 31.1, 28.9, 28.6, 28.5, 26.3,
25.5, 23.6, 22.0, 13.8. MS (ES+): m/z 435 (M + 1, 100%).
3-(1-(3-Dimethylamino-2-hydroxy-propyl)-1H-indol-3-yl)-2-
cyano-N-octylacrylamide (6). Compound 6 was synthesized utilizing
general procedure 2, 1-(3-dimethylamino-2-hydroxy-propyl)-1H-in-
dole-3-carbaldehyde (0.20 g, 0.81 mmol), and 2-cyano-N-octylaceta-
mide (0.17 g, 0.89 mmol). Recrystallization from EtOH afforded 3-(1-
(3-dimethylamino-2-hydroxy-propyl)-1H-indol-3-yl)-2-cyano-N-octy-
1
lacrylamide (120 mg, 35%) as a yellow solid, mp 103−105 °C. H
NMR (DMSO-d6): δ 8.48 (1H, s), 8.40 (1H, s), 8.16 (1H, t, J = 5.6
Hz), 7.91 (1H, dd, J = 7.1 Hz, 1.3 Hz), 7.62 (1H, d, J = 7.6 Hz), 7.29
(2H, m), 5.02 (1H, bs), 4.46 (1H, dd, J = 11.1 Hz, 3.2 Hz), 4.20 (1H,
dd, J = 7.2 Hz, 6.9 Hz), 3.94 (1H, m), 3.21 (2H, q, J = 6.7 Hz), 2.20
(8H, m), 1.50 (2H, quin, J = 6.3 Hz), 1.27 (10H, m), 0.86 (3H, t, J =
6.3 Hz). 13C NMR (DMSO-d6): δ 161.8, 141.0, 136.5, 133.8, 127.5,
122.9, 121.4, 118.4, 118.2, 111.3, 108.4, 97.2, 67.1, 62.5, 50.8, 45.8,
39.8, 31.0, 28.9, 28.5, 28.4, 26.2, 21.9, 13.7. MS (ES+): m/z 425 (M +
1, 100%).
3-(2-Methyl-1-(2-oxo-2-piperidin-1-yl-ethyl)-1H-indol-3-yl)-2-
cyano-N-octylacrylamide (11). Compound 11 was synthesized
utilizing general procedure 2, 2-methyl-1-(2-oxo-2-piperidin-1-yl-
ethyl)-1H-indole-3-carbaldehyde (0.20 mg, 0.70 mmol), and 2-
cyano-N-octylacetamide (0.16 g, 0.79 mmol). Recrystallization from
EtOH afforded 3-(2-methyl-1-(2-oxo-2-piperidin-1-yl-ethyl)-1H-indol-
3-yl)-2-cyano-N-octylacrylamide (224 mg, 69%) as a yellow solid, mp
1
164−166 °C. H NMR (DMSO-d6): δ 8.33 (1H, s), 8.15 (1H, t, J =
5.7 Hz), 7.94 (1H, dd, J = 6.6 Hz, 2.4 Hz), 7.48 (1H, dd, J = 6.6 Hz,
2.4 Hz), 7.21 (2H, m), 5.30 (2H, s), 3.58 (2H, m), 3.43 (2H, m), 3.21
(2H, q, J = 6.6 Hz), 2.43 (3H, s), 1.65 (4H, m), 1.48 (4H, m), 1.28
(10H, m), 0.86 (3H, t, J = 6.6 Hz). 13C NMR (DMSO-d6): δ 164.2,
162.2, 146.5, 144.2, 137.7, 124.4, 122.2, 120.8, 118.3, 118.2, 110.4,
107.6, 98.9, 45.2, 44.7, 42.6, 39.8, 31.2, 28.9, 28.6, 28.5, 26.3, 26.0,
25.2, 23.8, 22.0, 13.9, 11.4. MS (ES+): m/z 463 (M + 1, 100%).
3-(2-Methyl-1-(2-morpholino-2-oxoethyl)-1H-indol-3-yl)-2-
cyano-N-octylacrylamide (12). Compound 12 was synthesized
utilizing general procedure 2, 2-methyl-1-(2-morpholin-4-yl-2-oxo-
ethyl)-1H-indole-3-carbaldehyde (0.2 g, 0.70 mmol), and 2-cyano-N-
octylacetamide (0.15 g, 0.76 mmol). Recrystallization from EtOH
afforded 3-(2-methyl-1-(2-morpholino-2-oxoethyl)-1H-indol-3-yl)-2-
3-(1-(2-Hydroxy-3-pyrrolidin-1-ylpropyl)-2-methyl-1H-indol-3-yl)-
2-cyano-N-octylacrylamide (7). Compound 7 was synthesized
utilizing general procedure 2, 1-(2-hydroxy-3-pyrrolidin-1-ylpropyl)-
2-methyl-1H-indole-3-carbaldehyde (0.19 g, 0.67 mmol), and 2-cyano-
N-octylacetamide (0.15 g, 0.76 mmol). Recrystallization from EtOH
afforded 3-(1-(2-hydroxy-3-pyrrolidin-1-ylpropyl)-2-methyl-1H-indol-
3-yl)-2-cyano-N-octylacrylamide (110 mg, 35%) as a yellow solid, mp
1
129−131 °C. H NMR (DMSO-d6): δ 8.33 (1H, s), 8.11, (1H, t, J =
5.6 Hz), 7.95 (1H, d, J = 7.2 Hz), 7.54 (1H, d, J = 7.6 Hz), 7.23 (2H,
m), 5.05 (1H, d, J = 4.8 Hz), 4.40 (2H, dd, J = 14.7 Hz, 2.9 Hz), 4.15
(2H, dd, J = 8.4 Hz, 6.3 Hz), 3.92 (1H, m), 3.20 (2H, q, J = 6.6 Hz),
2.60 (4H, m), 2.52 (2H, m), 2.45 (3H, s), 1.71 (4H, m), 1.51 (2H,
I
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cyano-N-octylacrylamide (140 mg, 43%) as a yellow crystalline solid,
mp 156−157 °C. 1H NMR (DMSO-d6): δ 8.34 (1H, s), 8.16 (1H, t, J
= 5.4 Hz), 7.94 (1H, d, J = 7.5 Hz), 7.50 (1H, d, J = 7.5 Hz), 7.23 (2H,
m), 5.33 (2H, s), 3.69 (4H, m), 3.61 (4H, m), 3.21 (2H, q, J = 6.6
Hz), 2.45 (3H, s), 1.51 (2H, quin, J = 5.7 Hz), 1.28 (10H, m), 0.86
(3H, t, J = 6.3 Hz). 13C NMR (DMSO-d6): δ 165.0, 162.3, 146.6,
144.3, 137.8, 124.5, 122.6, 122.3, 121.0, 118.4, 110.5, 107.7, 99.1, 66.1,
46.8, 44.8, 42.0, 31.3, 29.0, 28.7, 28.6, 26.4, 22.1, 14.0, 11.6. MS (ES+):
m/z 465 (M + 1, 100%).
dol-1-yl)acetamide (0.20 g, 0.61 mmol), and 2-cyano-N-octylaceta-
mide (0.12 g, 0.62 mmol). Recrystallization from EtOH afforded 3-(1-
(2-benzo[d][1,3]dioxol-5-yl-amino-2-oxoethyl)-1H-indol-3-yl)-2-
cyano-N-octylacrylamide (245 mg, 79%) as a yellow solid, mp 199−
1
200 °C. H NMR (DMSO-d6): δ 10.46 (1H, s), 8.51 (1H, s), 8.43
(1H, s), 8.29 (1H, t, J = 5.7 Hz), 7.95, (1H, dd, J = 7.2 Hz, 1.5 Hz),
7.57 (1H, dd, J = 7.2 Hz, 1.2 Hz), 7.30 (3H, m), 6.95 (1H, d, J = 8.4
Hz), 6.88 (1H, d, J = 8.4 Hz), 5.99 (2H, s), 5.28 (2H, s), 3.22 (2H, q, J
= 6.5 Hz), 1.51 (2H, quin, J = 6.0 Hz), 1.28 (10H, m), 0.86 (3H, t, J =
6.5 Hz). 13C NMR (DMSO-d6): δ 164.9, 161.9, 147.1, 143.2, 141.1,
136.6, 134.0, 132.9, 127.6, 123.5, 121.9, 118.5, 118.4, 112.1, 111.1,
109.0, 108.2, 101.3, 101.1, 98.1, 49.7, 39.8, 31.1, 29.1, 28.7, 28.6, 26.4,
22.1, 14.0. MS (ES+): m/z 501 (M + 1, 100%).
3-(2-Methyl-1-(2-(4-methylpiperidin-1-yl)-2-oxoethyl)-1H-indol-
3-yl)-2-cyano-N-octylacrylamide (13). Compound 13 was synthe-
sized utilizing general procedure 2, 2-methyl-1-(2-(4-methylpiperidin-
1-yl)-2-oxoethyl)-1H-indole-3-carbaldehyde (0.19 g, 0.63 mmol), and
2-cyano-N-octylacetamide (0.19 g, 0.97 mmol). Recrystallization from
EtOH afforded 3-(2-methyl-1-(2-(4-methylpiperidin-1-yl)-2-oxoeth-
yl)-1H-indol-3-yl)-2-cyano-N-octylacrylamide (188 mg, 63%) as a
2-(Octylamino)thiazol-4(5H)-one (18). To a stirred solution of 2-
thio-4-thiazolin-4-one (0.82 g, 6.2 mmol), 2-octylamine (1.59 g, 12.4
mmol), and acetonitrile (15 mL) was added N,N-diisopropylethyl-
amine (0.72, 5.5 mmol). The mixture was cooled to 0 °C prior to the
addition of MgCl2 (1.70 g, 6.2 mmol). The resulting solution was
stirred at room temperature for 48 h, filtered through Celite,
concentrated in vacuo, dried (MgSO4), and subjected to flash silica
column chromatography (1:1 hexanes:EtOAc) to afford 2-
(octylamino)thiazol-4(5H)-one (0.71 g, 51%) as a yellow solid, mp
134−135 °C. 1H NMR (MeOD): δ 5.74 (2 H, s), 4.34 (1 H, t, J = 7.1
Hz), 2.49 (2 H, qd, J = 17.7, 7.0 Hz), 2.16 (12 H, m), 1.76 (3 H, t, J =
6.6 Hz). 13C NMR (MeOD): δ 188.8, 181.4, 44.5, 37.8, 31.0, 28.4,
28.3, 28.0, 25.8, 21.7, 12.5. MS (ES+): m/z 229 (M + 1, 100%).
5-((1-(3-(Dimethylamino)propyl)-1H-indol-3-yl)methylene)-2-
(octylamino)thiazol-4(5H)-one (19). Compound 19 was synthesized
utilizing general procedure 2, 1-(3-dimethylaminopropyl)-1H-indole-
3-carbaldehyde (2.45 g, 11.0 mmol), 2-(octylamino)thiazol-4(5H)-one
(18) (2.50 g, 11.0 mmol), piperidine (0.1 mL), and EtOH (20 mL).
Recrystallization from EtOH afforded the title compound (3.13 g,
65%) as a yellow solid, mp 136−137 °C. 1H NMR (DMSO-d6): δ 9.44
(1 H, s), 7.81 (2 H, m), 7.59 (1 H, s), 7.55 (1 H, d, J = 8.1 Hz), 7.25
(1 H, t, J = 7.3 Hz), 7.17 (1 H, t, J = 7.3 Hz), 4.30 (2 H, t, J = 6.6 Hz),
3.46 (2 H, t, J = 6.6 Hz), 2.15 (8 H, m), 1.96−1.81 (2 H, m), 1.56 (2
H, m), 1.21 (10 H, m), 0.82 (3 H, t, J = 5.7 Hz). 13C NMR (DMSO-
d6): δ 179.8, 172.0, 136.0, 130.1, 129.8, 127.2, 122.7, 120.9, 120.7,
120.3, 118.5, 110.5, 110.3, 55.3, 44.8, 44.2, 43.7, 31.1, 28.5, 28.4, 27.0,
26.2, 22.0, 13.7. MS (ES+): m/z 441 (M + 1, 100%).
General Procedure 3. 2-Cyano-3-(1-(3-(dimethylamino)propyl)-
1H-indol-3-yl)-N-octylpropanamide (20). A solution of 2-cyano-3-(1-
(3-(dimethylamino)propyl)-1H-indol-3-yl)-N-octylacrylamide (2)
(0.114 g, 0.28 mmol) and EtOH (10 mL) was hydrogenated utilizing
the ThalesNano H-cube system (1.0 mL/min) loaded with a 33 mm
10% Pd/C CatCart column heated to 50 °C under 50 bar of H2
pressure. The eluate was concentrated in vacuo to afford 2-cyano-3-(1-
(3-(dimethylamino)propyl)-1H-indol-3-yl)-N-octylpropanamide (0.11
g, 92%) as a yellow oil. 1H NMR (CDCl3): δ 7.50 (1 H, d, J = 7.6 Hz),
7.30 (1 H, d, J = 8.1 Hz), 7.15 (1 H, t, J = 7.4 Hz), 7.06 (1 H, t, J = 7.4
Hz), 6.13 (1 H, t, J = 5.5 Hz), 4.12 (2 H, t, J = 7.2 Hz), 3.65 (2 H, td, J
= 8.1, 6.4 Hz), 3.40 (1 H, ddd, J = 22.3, 14.5, 6.8 Hz), 3.22−3.05 (2 H,
m), 2.43 (3 H, s), 2.26 (2 H, t, J = 6.9 Hz), 2.22 (6 H, s), 1.87 (2 H, p,
J = 7.0 Hz), 1.40−1.11 (14 H, m), 0.87 (3 H, t, J = 6.8 Hz). 13C NMR
(CDCl3): δ 164.1, 135.5, 134.4, 126.6, 120.5, 118.7, 118.1, 117.2,
108.62, 104.9, 57.6, 55.96, 44.86, 40.6, 39.9, 39.7, 30.8, 28.4, 27.5, 25.8,
25.5, 21.9, 17.9, 13.5, 9.9. MS (ES+): m/z 215 (M + 2, 100%), 411 (M
+ 1, 60%).
1
yellow solid, mp 171−172 °C. H NMR (DMSO-d6): δ 8.33 (1H, s),
8.15 (1H, t, J = 5.7 Hz), 7.94 (1H, m), 7.48 (1H, m), 7.20 (2H, m),
5.31 (2H, s), 4.25 (1H, d, J = 12.0 Hz), 4.02 (1H, d, J = 12.0 Hz), 3.21
(2H, q, J = 6.3 Hz), 3.14 (1H, m), 2.62 (1H, m), 2.42 (3H, s), 1.75
(1H, m), 1.64 (2H, m), 1.51 (2H, quin, J = 6.3 Hz), 1.28 (12H, m),
0.95 (3H, d, J = 6.0 Hz), 0.86 (3H, t, J = 6.9 Hz). 13C NMR (DMSO-
d6): δ 164.2, 162.2, 146.5, 144.2, 137.7, 124.4, 122.2, 120.8, 120.8,
118.3, 110.4, 107.6, 98.9, 44.7, 44.5, 41.9, 39.8, 34.1, 33.4, 31.2, 30.2,
28.9, 28.6, 28.5, 26.3, 22.0, 21.5, 13.9, 11.5. MS (ES+): m/z 477 (M +
1, 100%).
3-[1-(2-Diethylamino-2-oxoethyl)-1H-indol-3-yl]-2-cyano-N-octy-
lacrylamide (14). Compound 14 was synthesized utilizing general
procedure 2, N,N-diethyl-2-(3-formyl-indol-1-yl)acetamide (0.20 g,
0.76 mmol), and 2-cyano-N-octylacetamide (0.31 g, 1.58 mmol).
Recrystallization from EtOH afforded 3-(1-(2-diethylamino-2-oxoeth-
yl)-1H-indol-3-yl)-2-cyano-N-octylacrylamide (152 mg, 46%) as a
1
white solid, mp 96−97 °C. H NMR (DMSO-d6): δ 8.42 (1H, s,
ArH2) 8.41 (1H, s), 8.26 (1H, t, J = 5.4 Hz), 7.92 (1H, m), 7.44 (1H,
m), 7.29 (2H, m), 5.42 (2H, s), 3.47 (2H, q, J = 7.0 Hz), 3.30 (2H, q, J
= 6.6 Hz), 3.21 (2H, q, J = 6.6 Hz), 1.51 (2H, quin, J = 6.2 Hz), 1.28
(10H, m), 1.26 (3H, t, J = 7.2 Hz), 1.04 (3H, t, J = 7.2 Hz), 0.86 (3H,
t, J = 6.1 Hz). 13C NMR (DMSO-d6): δ 165.3, 161.8, 141.1, 136.8,
134.1, 127.5, 123.1, 121.5, 118.3, 118.2, 111.2, 108.7, 97.5, 47.7, 40.6,
40.2, 39.8, 31.2, 28.9, 28.6, 28.5, 26.3, 22.0, 14.0, 13.9, 12.9. MS (ES+):
m/z 451 (M + 1, 100%).
3-(1-Benzyl-1H-indol-3-yl)-2-cyano-N-octylacrylamide (15).
Compound 15 was synthesized utilizing general procedure 2, 1-
benzylindole-3-carbaldehyde (0.46 g, 2.0 mmol), and 2-cyano-N-
octylacetamide (0.42 g, 2.2 mmol). Recrystallization from EtOH
afforded 3-(1-benzyl-1H-indol-3-yl)-2-cyano-N-octylacrylamide (40
1
mg, 50%) as a yellow crystalline solid, mp 128−130 °C. H NMR
(DMSO-d6): δ 8.56 (1H, s), 8.40 (1H, s), 8.24 (1H, t, J = 5.4 Hz),
7.92 (1H, m), 7.60 (1H, m), 7.28 (7H, m), 5.62 (2H, s), 3.33 (2H, m),
1.49 (2H, t, J = 6.3 Hz), 1.26 (10H, m), 0.84 (3H, t, J = 6.9 Hz). 13C
NMR (DMSO-d6): δ 161.7, 141.0, 136.7, 135.9, 132.6, 128.7, 127.7,
127.1, 123.4, 121.8, 118.6, 118.3, 111.5, 109.1, 98.1, 49.8, 39.8, 31.1,
28.9, 28.6, 28.5, 26.3, 22.0, 13.9. MS (ES+): m/z 414 (M + 1, 100%).
3-(1-Phenylsulfonyl-1H-indol-3-yl)-2-cyano-N-octylacrylamide
(16). Compound 16 was synthesized utilizing general procedure 2, 1-
(phenylsulfonyl)indole-3-carbaldehyde (0.48 g, 1.7 mmol), 2-cyano-N-
octylacetamide (0.47 g, 2.4 mmol), piperidine (0.1 mL), and EtOH (5
mL). Recrystallization from EtOH afforded 3-(1-phenylsulfonyl-1H-
indol-3-yl)-2-cyano-N-octylacrylamide (30 mg, 39%) as a white solid,
mp 141−142 °C. 1H NMR (DMSO-d6): δ 8.68 (1H, s), 8.53 (1H, t, J
= 5.7 Hz), 8.33 (1H, s), 8.09 (2H, dd, J = 7.8 Hz, 1.5 Hz), 7.99 (1H, d,
J = 8.2 Hz), 7.96 (1H, d, J = 8.9 Hz), 7.76 (1H, t, J = 7.2 Hz), 7. 64
(2H, t, J = 7.8 Hz), 7.45 (2H, m), 3.23 (2H, q, J = 6.6 Hz), 1.51 (2H,
quin, J = 6.6 Hz), 1.27 (10H, m), 0.85 (3H, t, J = 6.6 Hz). 13C NMR
(DMSO-d6): δ 160.5, 139.2, 136.0, 135.3, 133.4, 130.1, 128.7, 128.0,
127.0, 126.2, 124.4, 119.9, 117.0, 115.0, 113.2, 106.2, 39.8, 31.1, 28.8,
28.6, 28.5, 26.3, 22.0, 13.9. MS (ES+): m/z 464 (M + 1, 100%).
3-(1-(2-Benzo[d][1,3]dioxol-5-yl-amino-2-oxoethyl)-1H-indol-3-
yl)-2-cyano-N-octylacrylamide (17). Compound 17 was synthesized
utilizing general procedure 2, N-benzo[1,3]dioxol-5-yl-2-(3-formylin-
1-(3-Dimethylaminopropyl)-1H-indole-3-carbaldehyde (21). To a
stirred solution of indole-3-carbaldehyde (1.00 g, 6.9 mmol) in ACN
(30 mL) at 0 °C was added cesium carbonate (11.2 g, 5 equiv) and
potassium iodide (2.3 g, 2 equiv) in small portions. After 10 min, 3-
dimethylamino-1-propyl chloride hydrochloride (2 g, 13.8 mmol, 2
equiv) was added to the resulting suspension. The reaction mixture
was heated at reflux for 18 h, quenched with water (50 mL), the
organic material extracted with EtOAc (3 × 40 mL), dried (MgSO4),
and concentrated in vacuo to yield the desired compound as a yellow
oil (1.58 g, 100%). 1H NMR (CDCl3): δ 9.98 (s, 1H), 8.34−8.25 (m,
1H), 7.75 (s, 1H), 7.46−7.36 (m, 1H), 7.33−7.29 (m,, 2H), 4.26 (t, J
= 6.8 Hz, 2H), 2.21 (t, J = 6.7 Hz, 2H), 2.21(s, 6H), 2.00 (tt, J = 6.7,
J
dx.doi.org/10.1021/jm300844m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
6.7 Hz, 2H). 13C NMR (CDCl3): δ 184.2, 138.9, 137.0, 125.1, 123.7,
122.6, 121.8, 117.8, 110.0, 55.5, 45.1, 44.4, 27.2. MS (ES+): m/z 231
(M + 1, 100%).
Hz), 1.52 (2H, quin, J = 6.9 Hz), 1.25 (22H, m), 0.87 (3H, t, J = 6.9
Hz). 13C NMR (CDCl3): 136.4, 127.5, 126.4, 121.5, 118.9, 118.8,
113.3, 109.5, 56.5, 49.6, 45.4, 44.6, 43.9, 31.9, 29.9, 29.7, 29.3, 28.3,
27.4, 22.7, 14.1. MS (ES+): m/z 214 (M + 2, 100%), 428 (M + 1,
70%).
General Procedure 4. N-((1-(3-(Dimethylamino)propyl)-1H-indol-
3-yl)methyl)octan-1-amine (22). A solution of 1-(3-dimethylamino-
propyl)-1H-indole-3-carbaldehyde (0.35 g, 1.52 mmol), octylamine
(0.25 mL, 1.52 mmol), toluene (5.0 mL), and MgSO4 (0.5 g) was
irradiated with microwaves (100 °C, 150 W) for 10 min. The resulting
crude material was filtered, diluted with EtOH (30 mL, 0.05 M), and
hydrogenated with the ThalesNano H-cube system (1.0 mL/min)
loaded with a 33 mm 10% Pd/C CatCart column heated to 50 °C
under 50 bar H2 pressure. The eluate was concentrated in vacuo to
afford N-(1-(3-dimethylaminopropyl)-1H-indol-3-ylmethyl) octyl-
1-Benzyl-N-((1-(3-(dimethylamino)propyl)-1H-indol-3-yl)methyl)-
piperidin-4-amine (27). Compound 27 was synthesized utilizing
general procedure 4, 1-(3-(dimethylamino)propyl)-2-methyl-1H-in-
dole-3-carbaldehyde (21) (0.73 g, 3.0 mmol), and 1-benzylpiperidin-4-
amine (0.80 mL, 3.0 mmol), affording 1-benzyl-N-((1-(3-
(dimethylamino)propyl)-2-methyl-1H-indol-3-yl)methyl)piperidin-4-
amine (0.62 g, 49%) as a yellow solid, mp 62−64 °C. 1H NMR
(CDCl3): δ 7.58 (1 H, d, J = 7.5 Hz), 7.38−7.29 (4 H, m), 7.27 (1 H,
dt, J = 6.9, 6.5, 4.0 Hz), 7.15 (1 H, dd, J = 11.0, 4.0 Hz), 7.13−7.06 (1
H, m), 4.14 (2 H, t, J = 7.3 Hz), 3.96 (2 H, s), 3.53 (2 H, s), 2.89 (2 H,
m), 2.69−2.54 (1 H, m), 2.44 (3 H, s), 2.30 (2 H, t, J = 6.9 Hz), 2.24
(6 H, s), 2.08 (2 H, dt, J = 11.5, 2.2 Hz), 1.90 (4 H, m), 1.55 (4 H, m).
13C NMR (CDCl3): δ 138.2, 135.6, 133.1, 128.5, 127.6, 127.2, 126.3,
1
amine (0.43 mg, 78%) as a yellow oil. H NMR (CDCl3): δ 7.62
(1H, d, J = 7.8 Hz), 7.34 (1H, d, J = 8.1 Hz), 7.21 (1H, m), 7.10 (2H,
m), 4.14 (2H, t, J = 6.9 Hz), 3.97 (2H, s), 2.68 (2H, t, J = 7.2 Hz),
2.23 (2H, t, J = 7.2 Hz), 2.20 (6H, s), 1.95 (2H, quin, J = 6.9 Hz), 1.53
(2H, quin, J = 6.9 Hz), 1.26 (10H, m), 0.86 (3H, t, J = 6.9 Hz). 13C
NMR (CDCl3): δ 136.4, 127.5, 126.6, 121.5, 118.9, 118.8, 112.9,
109.5, 56.5, 49.4, 45.4, 44.4, 43.9, 31.8, 29.8, 29.5, 29.2, 28.2, 27.4,
22.6, 14.1. MS (ES+): m/z 172 (M + 2, 100%), 344 (M + 1, 50%).
N-((1-(3-(Dimethylamino)propyl)-1H-indol-3-yl)methyl)hexan-1-
amine (23). Compound 23 was synthesized utilizing general
procedure 4, 1-(3-dimethylaminopropyl)-1H-indole-3-carbaldehyde
(21) (0.38 g, 1.67 mmol), and hexylamine (0.22 mL, 1.67 mmol),
affording N-(1-(3-dimethylaminopropyl)-1H-indol-3-ylmethyl)-
120.1, 118.4, 117.3, 109.7, 108.4, 62.6, 56.1, 54.0, 51.9, 44.9, 40.5, 40.3,
32.2, 27.7, 9.7. MS (ES+): m/z 210 (M + 2, 100%), 419 (M + 1, 70%).
N,N-Dimethyl-3-(3-((2-(piperidin-1-yl)ethylamino)methyl)-1H-
indol-1-yl)propan-1-amine (28). Compound 28 was synthesized
utilizing general procedure 4, 1-(3-dimethylaminopropyl)-1H-indole-
3-carbaldehyde (21) (0.16 g, 0.70 mmol), and 1-(2-aminoethyl)-
piperidine (0.1 mL, 0.70 mmol), affording N,N-dimethyl-3-(3-(2-
piperidine-1-ylethylaminomethyl)-1H-indol-1-yl)propylamine (209
1
1
hexylamine (0.49 g, 93%) as an orange oil. H NMR (CDCl3): δ
mg, 87%) as an orange oil. H NMR (CDCl3): δ 7.63 (1H, dd, J =
7.63 (1H, d, J = 7.8 Hz), 7.34 (1H, d, J = 8.1 Hz), 7.21 (1H, m), 7.10
(2H, m), 4.14 (2H, t, J = 6.9 Hz), 3.98 (2H, s), 2.69 (2H, t, J = 7.3
Hz), 2.24 (2H, t, J = 7.0 Hz), 2.20 (6H, s), 1.95 (2H, quin, J = 6.9 Hz),
1.52 (2H, quin, J = 7.1 Hz), 1.30 (6H, m), 0.87 (3H, t, J = 6.7 Hz). 13C
NMR (CDCl3): δ 136.6, 127.7, 126.8, 121.7, 119.1, 119.0, 113.1,
109.7, 56.7, 49.6, 45.6, 44.6, 44.1, 31.9, 29.9, 28.4, 27.2, 22.8, 14.2. MS
(ES+): m/z 158 (M + 2, 100%), 316 (M + 1, 70%).
7.8, 0.9 Hz), 7.34 (1H, d, J = 8.1 Hz), 7.20 (1H, dt, J = 7.2, 1.2 Hz),
7.10 (2H, m), 4.15 (2H, t, J = 6.9 Hz), 4.00 (2H, s), 2.81 (2H, t, J =
6.3 Hz), 2.48 (2H, t, J = 6.3 Hz), 2.35 (4H, m), 2.24 (2H, t, J = 6.9
Hz), 2.20 (6H, s), 1.96 (2H, quin, J = 6.9 Hz), 1.51 (4H, m), 1.41
(2H, m). 13C NMR (CDCl3): δ 136.5, 127.5, 126.5, 121.6, 119.0,
118.8, 112.9, 109.5, 58.1, 56.5, 54.6, 45.8, 45.4, 44.5, 43.9, 28.2, 25.9,
24.4. MS (ES+): m/z 172 (M + 2, 100%), 343 (M + 1, 30%).
N,N-Dimethyl-3-(3-((2-(pyrrolidin-1-yl)ethylamino)methyl)-1H-
indol-1-yl)propan-1-amine (29). Compound 29 was synthesized
utilizing general procedure 4, 1-(3-dimethylaminopropyl)-1H-indole-
3-carbaldehyde (21) (0.14 g, 0.61 mmol), and 1-(2-aminoethyl)-
pyrrolidine (0.08 mL, 0.63 mmol), affording N,N-dimethyl-3-(3-(2-
(pyrrolidin-1-yl)ethylaminomethyl)-1H-indol-1-yl)propylamine (183
N-((1-(3-(Dimethylamino)propyl)-1H-indol-3-yl)methyl)decan-1-
amine (24), Dynole 2−24. Compound 24, Dynole 2−24, was
synthesized utilizing general procedure 4, 1-(3-dimethylaminopropyl)-
1H-indole-3-carbaldehyde (21) (0.55 g, 2.30 mmol), and decylamine
(0.44 g, 2.78 mmol), affording N-(1-(3-dimethylaminopropyl)-1H-
1
indol-3-ylmethyl)decylamine (0.42 mg, 46%) as a pale-yellow oil. H
1
NMR (CDCl3): δ 7.63 (1H, d, J = 7.8 Hz), 7.33 (1H, d, J = 8.1 Hz),
7.18 (1H, m), 7.08 (2H, m), 4.13 (2H, t, J = 6.9 Hz), 3.98 (2H, s),
2.69 (2H, t, J = 7.2 Hz), 2.19 (2H, t, J = 6.9 Hz), 2.06 (6H, s), 1.94
(2H, quin, J = 6.9 Hz), 1.53 (2H, quin, J = 6.9 Hz), 1.25 (14H, m),
0.88 (3H, t, J = 6.9 Hz). 13C NMR (CDCl3): δ 136.4, 127.6, 126.4,
121.4, 118.9, 118.8, 113.2, 109.4, 56.5, 49.5, 45.3, 44.5, 43.8, 31.8, 29.8,
29.5, 29.2, 28.2, 27.3, 22.6, 14.0. MS (ES+): m/z 186 (M + 2, 100%),
372 (M + 1, 50%).
mg, 92%) as a yellow oil. H NMR (CDCl3): δ 7.63 (1H, dd, J =
7.8, 0.9 Hz), 7.34 (1H, d, J = 8.1 Hz), 7.20 (1H, dt, J = 6.9, 1.2 Hz),
7.09 (2H, m), 4.15 (2H, t, J = 6.9 Hz), 4.00 (2H, s), 2.83 (2H, t, J =
6.6 Hz), 2.64 (2H, t, J = 6.9 Hz), 2.48 (4H, m), 2.22 (8H, m), 1.96
(2H, quin, J = 6.9 Hz), 1.76 (4H, m). 13C NMR (CDCl3): δ 136.5,
127.6, 126.4, 121.5, 118.9, 113.4, 109.5, 56.5, 55.7, 54.1, 47.9, 45.4,
44.7, 43.9, 28.3, 23.4. MS (ES+): m/z 164 (M + 2, 100%), 329 (M + 1,
60%).
N-((1-(3-(Dimethylamino)propyl)-1H-indol-3-yl)methyl)dodecan-
1-amine (25). Compound 25 was synthesized utilizing general
procedure 4, 1-(3-dimethylaminopropyl)-1H-indole-3-carbaldehyde
(21) (0.36 g, 1.56 mmol), and dodecylamine (0.29 g, 1.56 mmol),
affording N-(1-(3-dimethylaminopropyl)-1H-indol-3-ylmethyl)-
N,N-Dimethyl-3-(3-((3-phenylpropylamino)methyl)-1H-indol-1-
yl)propan-1-amine (30). Compound 30 was synthesized utilizing
general procedure 4, 1-(3-dimethylaminopropyl)-1H-indole-3-carbal-
dehyde (21) (0.24 g, 1.06 mmol), and phenylpropylamine (0.16 mL,
1.09 mmol), affording N,N-dimethyl-3-(3-(3-phenylpropylaminometh-
1
1
dodecylamine (0.56 mg, 89%) as an orange oil. H NMR (CDCl3):
yl)-1H-indol-1-yl)propylamine (347 mg, 94%) as an orange oil. H
δ 7.62 (1H, d, J = 7.8 Hz), 7.34 (1H, d, J = 8.1 Hz), 7.20 (1H, m), 7.10
(2H, m), 4.14 (2H, t, J = 6.9 Hz), 3.97 (2H, s), 2.69 (2H, t, J = 7.2
Hz), 2.23 (2H, t, J = 7.2 Hz), 2.20 (6H, s), 1.95 (2H, quin, J = 6.9 Hz),
1.52 (2H, quin, J = 6.9 Hz), 1.24 (18H, m), 0.87 (3H, t, J = 6.9 Hz).
13C NMR (CDCl3): δ 135.9, 127.0, 125.9, 121.0, 118.4, 118.3, 112.7,
NMR (CDCl3): δ 7.63 (1H, d, J = 7.8 Hz), 7.35 (1H, d, J = 8.2 Hz),
7.18 (6H, m), 7.11 (2H, m), 4.14 (2H, t, J = 6.9 Hz), 3.99 (2H, s),
2.74 (2H, t, J = 6.8 Hz), 2.65 (2H, t, J = 7.4 Hz), 2.24 (2H, t, J = 7.0
Hz), 2.21 (6H, s), 1.95 (2H, quin, J = 7.0 Hz), 1.81 (2H, quin, J = 7.7
Hz). 13C NMR (CDCl3): δ 141.9, 136.4, 128.5, 128.3, 127.5, 126.7,
125.7, 121.6, 119.0, 118.8, 112.4, 109.5, 56.4, 48.6, 45.3, 44.2, 43.9,
33.5, 31.1, 28.2. MS (ES+): m/z 175 (M + 2, 100%), 350 (M + 1,
80%).
109.0, 56.0, 49.0, 44.9, 44.0, 43.4, 31.4, 29.4, 29.1, 28.8, 27.7, 26.9,
22.1, 13.6. MS (ES+): m/z 400 (M + 2, 100%), 200 (M + 1, 60%).
N-(1-(3-Dimethylaminopropyl)-1H-indol-3-ylmethyl)-
tetradecylamine (26). Compound 26 was synthesized utilizing general
procedure 4, 1-(3-dimethylaminopropyl)-1H-indole-3-carbaldehyde
(21) (0.39 g, 1.69 mmol), and tetradecylamine (0.36 g, 1.69 mmol),
affording N-(1-(3-dimethylaminopropyl)-1H-indol-3-ylmethyl)-
N1-(Cyclohexylmethyl)-N3-((1-(3-(dimethylamino)propyl)-1H-
indol-3-yl)methyl)propane-1,3-diamine (31). Compound 31 was
synthesized utilizing general procedure 4, 1-(3-dimethylaminoprop-
yl)-1H-indole-3-carbaldehyde (21) (0.18 g, 0.76 mmol), and N-
cyclohexyl-1,3-propanediamino-N1-(cyclohexylmethyl)-N3-((1-(3-di-
methylaminopropyl)-1H-indol-3-yl)methyl)propane-1,3-diamine (246
1
tetradecylamine (0.54 mg, 75%). H NMR (CDCl3): δ 7.62 (1H, d,
J = 7.8 Hz), 7.34 (1H, d, J = 8.4 Hz), 7.20 (1H, dt, J = 8.1, 1.2 Hz),
7.10 (2H, m), 4.14 (2H, t, J = 6.9 Hz), 3.97 (2H, s), 2.69 (2H, t, J =
7.2 Hz), 2.23 (2H, t, J = 7.2 Hz), 2.20 (6H, s), 1.95 (2H, quin, J = 6.9
1
mg, 84%) as a yellow oil. H NMR (CDCl3): δ 7.62 (1H, d, J = 7.7
Hz), 7.34 (1H, d, J = 7.8 Hz), 7.20 (1H, m), 7.09 (2H, m), 4.16 (2H, t,
K
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J = 7.0 Hz), 3.97 (2H, s), 2.77 (4H, m), 2.21 (8H, m), 1.96 (4H, m),
1.69 (7H, m), 1.16 (4H, m). 13C NMR (CDCl3): δ 136.5, 127.5,
126.7, 121.6, 119.0, 118.8, 112.8, 109.6, 56.9, 56.5, 48.4, 45.8, 45.5,
44.5, 43.9, 40.8, 33.2, 26.0, 25.0. MS (ES+): m/z 193 (M + 2, 100%),
385 (M + 1, 30%), 129 (M + 3, 20%).
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ASSOCIATED CONTENT
* Supporting Information
■
S
Data parameters for 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31. Dynole,
Rhodadyn, MiTMAB, Bis-T, Dyngo, and Iminodyn are
trademarks of Children’s Medical Research Institute and
Newcastle Innovation Ltd. Dynole 2−24 and most other
dynamin inhibitors described in this paper are available from
Abcam Biochemicals (Bristol, UK). This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +612 4921 6486. Fax: +61 249 215472. E-mail: Adam.
McCluskey@newcastle.edu.au.
■
Notes
The authors declare the following competing financial
interest(s): We have entered into a commercial agreement
with Abcam Biochemicals (Bristol, UK) for the supply of our
dynamin inhibitors. This includes the compounds listed in this
paper.
ACKNOWLEDGMENTS
■
(15) Sung, J. Y.; Kim, J.; Paik, S. R.; Park, J. H.; Ahn, Y. S.; Chung, K.
C. Induction of neuronal cell death by Rab5A-dependent endocytosis
of alpha-synuclein. J. Biol. Chem. 2001, 276, 27441−27448.
(16) Mercer, J.; Schelhaas, M.; Helenius, A. Virus Entry by
Endocytosis. Annu. Rev. Biochem. 2010, 79, 803−833.
(17) Veiga, E.; Guttman, J. A.; Bonazzi, M.; Boucrot, E.; Toledo-
Arana, A.; Lin, A. E.; Enninga, J.; Pizarro-Cerda, J.; Finlay, B. B.;
Kirchhausen, T.; Cossart, P. Invasive and Adherent Bacterial
Pathogens Co-Opt Host Clathrin for Infection. Cell Host Microbe
2007, 2, 340−351.
This work was supported by grants from the National Health
and Medical Research Council (Australia), The Australia
Research Council, The Australian Cancer Research Foundation,
The Ramaciotti Foundation, The Children’s Medical Research
Institute, and Newcastle Innovation.
ABBREVIATIONS USED
■
CME, clathrin-mediated endocytosis; SVE, synaptic vesicle
endocytosis; MiTMAB, myristoyl trimethyl ammonium bro-
mide; OcTMAB, octyl trimethyl ammonium bromide; Bis-T,
bis-tyrphostin; RTIL, room temperature ionic liquid; BIM,
bisindolylmaleimide; PKC, protein kinase C; PSA, polar surface
area; U2OS, human bone osteosarcoma epithelial cells; dynI,
dynamin I; dynII, dynamin II; dynIII, dynamin III; FL-dyn I,
full length dynamin I; FL-dyn II, full length dynamin II
(18) Zuechner, S.; Noureddine, M.; Kennerson, M.; Verhoeven, K.;
Claeys, K.; De Jonghe, P.; Merory, J.; Oliveira, S. A.; Speer, M. C.;
Stenger, J. E.; Walizada, G.; Zhu, D.; Pericak-Vance, M. A.; Nicholson,
G.; Timmerman, V.; Vance, J. M. Mutations in the pleckstrin
homology domain of dynamin 2 cause dominant intermediate
Charcot−Marie−Tooth disease. Nature Genet. 2005, 37, 289−294.
(19) Bitoun, M.; Maugenre, S.; Jeannet, P.-Y.; Lacene, E.; Ferrer, X.;
Laforet, P.; Martin, J.-J.; Laporte, J.; Lochmueller, H.; Beggs, A. H.;
Fardeau, M.; Eymard, B.; Romero, N. B.; Guicheney, P. Mutations in
dynamin 2 cause dominant centronuclear myopathy. Nature Genet.
2005, 37, 1207−1209.
(20) Zhang, J.; Ding, L.; Holmfeldt, L.; Wu, G.; Heatley, S. L.; Payne-
Turner, D.; Easton, J.; Chen, X.; Wang, J.; Rusch, M.; Lu, C.; Chen, S.
C.; Wei, L.; Collins-Underwood, J. R.; Ma, J.; Roberts, K. G.; Pounds,
S. B.; Ulyanov, A.; Becksfort, J.; Gupta, P.; Huether, R.; Kriwacki, R.
W.; Parker, M.; McGoldrick, D. J.; Zhao, D.; Alford, D.; Espy, S.;
Bobba, K. C.; Song, G.; Pei, D.; Cheng, C.; Roberts, S.; Barbato, M. I.;
Campana, D.; Coustan-Smith, E.; Shurtleff, S. A.; Raimondi, S. C.;
Kleppe, M.; Cools, J.; Shimano, K. A.; Hermiston, M. L.; Doulatov, S.;
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