Inhibition of dynamin mediated endocytosis by the Dynoles - Synthesis and functional activity of a family of indoles

Article abstract of DOI:10.1021/jm900036m

Screening identified two bisindolylmaleimides as 100 μM inhibitors of the GTPase activity of dynamin I. Focused library approaches allowed development of indole-based dynamin inhibitors called dynoles. 100-Fold in vitro enhancement of potency was noted with the best inhibitor, 2-cyano-3-(1-(2- (dimethylamino)ethyl)-1H-indol-3-yl)-N-octylacrylamide (dynole 34-2), a 1.3 ± 0.3 μM dynamin I inhibitor. Dynole 34-2 potently inhibited receptor mediated endocytosis (RME) internalization of Texas red-transferrin. The rank order of potency for a variety of dynole analogues on RME in U2OS cells matched their rank order for dynamin inhibition, suggesting that the mechanism of inhibition is via dynamin. Dynoles are the most active dynamin I inhibitors reported for in vitro or RME evaluations. Dynole 34-2 is 15-fold more active than dynasore against dynamin I and 6-fold more active against dynamin mediated RME (IC₅₀ ～15 μM; RME IC₅₀ ～80 μM). The dynoles represent a new series of tools to better probe endocytosis and dynamin-mediated trafficking events in a variety of cells.

Full text of DOI:10.1021/jm900036m

3762
J. Med. Chem. 2009, 52, 3762–3773
Inhibition of Dynamin Mediated Endocytosis by the DynolessSynthesis and Functional Activity
of a Family of Indoles
Timothy A. Hill,† Christopher P. Gordon,† Andrew B. McGeachie,‡ Barbara Venn-Brown,† Luke R. Odell,† Ngoc Chau,‡
Annie Quan,‡ Anna Mariana,‡ Jennette A. Sakoff,§ Megan Chircop (nee Fabbro),‡ Phillip J. Robinson,‡ and Adam McCluskey*,†
Chemistry, School of EnVironmental and Life Sciences, The UniVersity of Newcastle, UniVersity DriVe, Callaghan NSW 2308, Australia, Cell
Signaling Unit, Children’s Medical Research Institute, The UniVersity of Sydney, 214 Hawkesbury Road, Westmead NSW 2145, Australia,
Department of Medical Oncology, CalVary Mater Newcastle, Edith Street, Waratah NSW 2298, Australia
ReceiVed January 13, 2009
Screening identified two bisindolylmaleimides as 100 µM inhibitors of the GTPase activity of dynamin I.
Focused library approaches allowed development of indole-based dynamin inhibitors called dynoles. 100-
Fold in vitro enhancement of potency was noted with the best inhibitor, 2-cyano-3-(1-(2-(dimethylamino)-
ethyl)-1H-indol-3-yl)-N-octylacrylamide (dynole 34-2), a 1.3 ( 0.3 µM dynamin I inhibitor. Dynole 34-2
potently inhibited receptor mediated endocytosis (RME) internalization of Texas red-transferrin. The rank
order of potency for a variety of dynole analogues on RME in U2OS cells matched their rank order for
dynamin inhibition, suggesting that the mechanism of inhibition is via dynamin. Dynoles are the most active
dynamin I inhibitors reported for in vitro or RME evaluations. Dynole 34-2 is 15-fold more active than
dynasore against dynamin I and 6-fold more active against dynamin mediated RME (IC50 ∼15 µM; RME
IC50 ∼80 µM). The dynoles represent a new series of tools to better probe endocytosis and dynamin-mediated
trafficking events in a variety of cells.
Introduction
and is the site for dynamin I and III phosphorylation in vivo).11,12
All domains are potential sites for small molecule interventions
in dissecting endocytosis and potential drug targets.
Dynamin is a large GTPase that plays a crucial role in both
synaptic vesicle endocytosis and receptor mediated endocytosis,
SVEa and RME, respectively.1-5 It catalyzes hydrolysis of GTP
to GDP, causing assembled dynamin to undergo an expansion
(length) and contraction (width) that results in the fission of
clathrin coated vesicles from the membrane. This mechanism
has multiple biological roles and facilitates the recycling of
synaptic vesicles,2,5 the internalization of hormones and nutri-
ents, and it is also used as a cellular entry point for viruses,
toxins, and symbiotic organisms.6 Neurotoxins botulinum and
tetanus inhibit transmitter release from synapses, causing two
severe neuroparalytic diseases, tetanus and botulism. Their
action is dependent on their internalization via endocytosis into
nerve terminals.7
Dynamin comprises three major isoforms: dynamin I (neu-
rons), dynamin II (ubiquitous), and dynamin III (neurons and
testes).3,4 Common to all are four domains, a GTPase (required
for vesicle fission),3,8 pleckstrin homology (PH) (targeting
domain and potentially a GTPase inhibitory module),4 a GTPase
effector domain (GED, which controls dynamin self-assembly
into rings),9,10 and a proline-rich domain (PRD) (which interacts
with proteins containing an Src homology 3 (SH3) domain3,4
Dynamin inhibitors have attracted widespread attention and
have been used to study endocytosis and other aspects of
membrane dynamics in a variety of cellular systems.13-16 Small
molecule inhibitors offer many distinct advantages over tradi-
tional means of dynamin inhibition in cells by expression of
dynamin GTPase mutants or by small interfering RNA-mediated
dynamin knockdown, which cannot readily be used to study
rapid cellular effects. Small molecule, cell-permeable inhibitors
are able to rapidly block endocytosis in minutes and are
frequently reversible.17,18 There is now a small but expanding
“palette” of drugs available to rapidly and reversibly block
dynamin I by distinct mechanisms of action: the myristoyl
trimethyl ammonium bromide (MiTMAB),18,19 Bis-tyrphostin
(Bis-T),20 room temperature ionic liquid (RTIL),21 and dyna-
sore17 series of dynamin inhibitors, which are operating at
different stages in its cycle of GTPase activity. For example,
MiTMAB and octyl trimethyl ammonium bromide (OcTMAB)
block dynamin recruitment to the cell membrane.18
Results
From Bisindolylmaleimides to IndolessSimplification
Improves Dynamin GTPase Inhibition. A medicinal chemistry
program requires a viable lead compound. Subsequent lead
optimization should facilitate the development of a more
selective, potent, and less toxic analogue. The most difficult
task is frequently the initial lead discovery. Most leads are
derived from existing compound libraries, combinatorial librar-
ies, or natural products. In the present study, we needed to
identify a lead amenable to rapid synthetic development.
Screening a number of compound libraries (data not shown)
identified the structurally interesting bisindolylmaleimides (BIMs)
1 and 2 as ∼100 µM potent inhibitors of the GTPase activity
of dynamin I (Figure 1). Note that 1 and 2 are pseudosym-
metrical. Enzyme kinetic studies that showed BIMs 1 and 2
* To whom correspondence should be addressed. Phone: +61 249
216486. Fax: +61 249 215472. E-mail: Adam.McCluskey@
newcastle.edu.au.
†
Chemistry, School of Environmental and Life Sciences, The University
of Newcastle.
‡
Cell Signaling Unit, Children’s Medical Research Institute, The
University of Sydney.
§
Department of Medical Oncology, Calvary Mater Newcastle.
a Abbreviations: RME, receptor medicated endocytosis; SVE, synaptic
vesicle endocytosis; GED, GTPase effector domain; PRD, proline rich
domain; PH, Pleckstrin homology; SH3, Src Homology 3; MiTMAB,
myristoyl trimethyl ammonium bromide; OcTMAB, octyl trimethyl am-
monium 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.
10.1021/jm900036m CCC: $40.75
2009 American Chemical Society
Published on Web 05/21/2009

Inhibition of Dynamin Mediated Endocytosis by the Dynoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3763
Scheme 1a
Figure 1. Lead bisindolmaleimides displaying modest levels of
dynamin inhibition.
a Reagents and conditions: (i) NaH, THF; (ii) 2-chloro-N,N-dimethyl-
ethanamine or 3-chloro-N,N-dimethylpropanamine.
Table 1. Library 1: Inhibition of Dynamin I GTPase Activity by
Substituted Indoles 8-17
Figure 2. BIM scaffold simplification for rational design of dynamin
inhibitors.
were competitive with GTP, indicating they are competitive
inhibitors at the GTPase domain. BIMs of this nature are known
inhibitors of protein kinase C (PKC) and glycogen synthase
kinase 3.22,23
Access to a crystal or molecular modeled structure of the
target protein greatly facilitates rational inhibitor development.
In this particular case, while there is an apo structure of the rat
GTPase domain published, it is the lack of a ligand that renders
this structure unsuitable for modeling, as the GTPase domain
undergoes considerable structural change upon GTP binding and
GDP release.24 There are no reports of the full-length structure
of dynamin I.
There are a number of literature syntheses leading to
compounds such as 1, especially in regard to their reported
inhibition of PKC.25 However, because the goal for our study
was to produce rapid access to focused compound libraries
(typically 12-24 compounds per iteration), 1 was viewed as
too complex.25-30 Accordingly, we aimed to identify a minimal
pharmacophore that would allow rapid, focused analogue
development. Our approach to the desired compound libraries
is the application of a robust focused library approach in which
we placed access to compounds above synthetic elegance. Initial
effort commenced by targeting the bare indole while maintaining
the dimethylamino moiety (Figure 2), as this was strongly
indicated from previous dynamin inhibitors we developed.20,21
Library 1 analogues were synthesized in a one-step procedure
commencing with the treatment of five commercially available
indoles (3-7) with NaH, and addition of either 2-chloro-N,N-
dimethylethanamine or 3-chloro-N,N-dimethylpropanamine to
give substituted indoles (8-17) in good to excellent yields
(Scheme 1).
a IC50 determinations are the average of at least three separate experi-
ments, each in duplicate. b ∼50% Inhibition at 300 µM drug concentrations,
full IC50 determination not conducted. c “- ” No inhibition at 300 µM drug
concentration.
Six members of Library 1 were moderate (IC50 > 100 e 300
µM) dynamin I GTPase inhibitors (Table 1). Analogues 16 and
17 possessed additional C3-substituents and were the most active
of this group at 131 ( 5 and 143 ( 7 µM, respectively.
Compounds 8-11 were also active but with IC50 values of g300
µM. Only analogues containing an indole-N-alkyl-N(CH3)2
moiety inhibited dynamin. Based on this, and in keeping with
our focused library approach, two approaches for Library 2
analogues were considered (Table 2). Exploration of the
available chemical space at C3 either via simple peptide coupling
approaches (-CO2H, 14-17); or via Knovenangel approaches
(-CHO; 12 and 13). Given our previous Knovenangel
approaches,20,28-30 Library 2 was developed using previously
reported cyanoamides (18a-h) (Scheme 2).20,31-33
The introduction of simple chains had a marked effect on
potency with C6 alkyl chains substituted analogues 25 and 33

3764 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Hill et al.
Scheme 2. Library 2a
Table 2. Library 2: Inhibition of Dynamin I GTPase Activity by
Substituted Indoles 19-34
a Reagents and conditions: (i) piperidine (cat), EtOH reflux, 3 h.
than the original lead compounds. Replacement of the alkyl side
chain with a benzyl moiety causes concomitant decrease in
dynamin inhibition potency (27, IC50 ) 201 ( 15 µM),
presumably via a failure to engage a favorable hydrophobic
binding interaction.
Elongation of the alkyl side chains via synthesis of analogues
with C10, C12, and C14 alkyl chains (35-40) afforded no further
potency enhancements. Despite this, all C10-C14 analogues,
35-40, returned inhibitory values better than the original lead
compounds 1 and 2 (Table 3). This lack of improvement is not
surprising, as entropic effects increase the disorder of the
terminal chain. It is also possible that chain elongation begins
to encroach upon a charged binding pocket, which would
generate unfavorable hydrophobic-charged domain interactions
and in turn encourage chain folding, preventing additional
beneficial interactions and hence any increase in activity. It is
also of note that the N,N-dimethylaminopropyl-substituted C10,
C12, and C14 analogues (38-40) are uniformly less active than
the corresponding N,N-dimethylaminoethyl analogues (35-37),
which suggests there is finite space available to accommodate
both the indole nucleus and the long alkyl side chains and that
the ultimate binding is a compromise of accommodating the
pivotal N,N-dimethylamino moiety and the steric bulk of the
alkyl chains.
The importance of the terminal N,N-dimethylamino moiety
at N1 was examined by the synthesis of analogues shown
lacking this moiety (41-51) and analogue 52, which lacks only
the N,N-dimethylamino nitrogen atom of 34-1 (IC50 ) 3.33 (
0.75 µM) in Table 3. With these simple modifications, no
changes that removed the N,N-dimethylamino moiety were
tolerated, strongly suggesting that this is a key pharmacophoric
unit for the inhibition of dynamin I by indoles of this nature.
While analogue 34 displays an excellent level of dynamin I
inhibition, it is also highly lipophilic with a calculated CLogP
) 5.01, on the cusp of a Lipinski violation,34,35 suggesting good
cell penetration (see below) but poor aqueous solubility. With
the exception of the initial series of analogues, we did not
explore the role of the indole C2-CH3 substituent, but simple
CLogP and polar surface area (PSA) calculations suggest that
the removal of the C2-CH3 will install favorable properties
with LogP and PSA calculated to be 4.81 and 43.3 Å2,
respectively. Synthesis was conducted as described in Schemes
1 and 2 from indole 5, affording the desired analogue (Figure
3).
Removal of the C2 methyl group resulted (34-2, Figure 3)
in a 3-fold increase in potency for dynamin I inhibition (IC50
) 1.30 ( 0.30 µM), now representing the most potent dynamin
I inhibitor thus far, 2 times more potent than our existing Bis-T
analogues and 15× more potent than dynasore.17,20 This increase
in potency is most likely a result of reduced steric strain and
greater flexibility associated with the cyanoamide moiety and
its ability to remain in the same plane as the indole nucleus.
Removal of the C2-CH3 moiety decreases the dihedral angle
a IC50 determinations are the average of at least three separate experi-
ments, each in duplicate. b ∼50% Inhibition at 300 µM drug concentrations,
full IC50 determination not conducted. [c] “-” No inhibition at 300 µM
drug concentration.
returning IC50 values of 7.69 and 6.62 µM, respectively, and
C8 alkyl analogues 26 and 34 returned IC50 values of 9.05 and
3.33 µM, respectively, which were 10-30 times more potent

Inhibition of Dynamin Mediated Endocytosis by the Dynoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3765
Table 3. Library 3: Inhibition of Dynamin I GTPase Activity by
Substituted Indoles 35-52
Figure 3. Chemical structures and dynamin IC50 values for dynole 34
and the C2 demethyl dynole 34. To emphasize the structural similarity
with the parent indole 34, we have renamed these compounds dynole
34-1 and 34-2.
8 min of internalization under control conditions (DMSO
treatment), the cells showed considerable endocytosis of Tf-
TxR (Figure 4, top row) in the periphery and perinuclear region
of the cell, which is typical of Tf localization in early and
recycling endosomes.37,38 After a 30 min preincubation in the
presence of 30 µM of indoles 26, 33, 34-1, and 35 or 10 µM
of 34-2, RME was greatly reduced (Figure 4). It is clear from
Figure 4 that 26, 33, 34-1, 34-2, and 35 differ in their ability
to block RME, as evidenced by the very low, but observable,
levels of Tf-TxR apparent (red coloration at the periphery of
the DAPI (blue) stained nucleus) with 26, 33, and 35. With
34-1 and 34-2, we note the complete or near-complete lack
of color associated with Tf-TxR uptake, representing a complete
block of endocytosis. Tf-TxR internalization was completely
blocked at 100 µM by all five compounds (data not shown).
Importantly, cell morphology was unaffected by the drug
treatment, even after 30 min exposure (data not shown),
indicating that there was no membrane disruption.
To quantify the effect of 26, 33, 34-1, 34-2, and 35 in U2OS
cells, we used our previously reported automated quantitative
RME assay based on endocytosis of Tf-A594. The IC50 for
inhibition of RME by 26 in U2OS cells was 13.2 ( 2.6 µM
(Figure 5), which compares favorably with its IC50 for dynamin
I inhibition in vitro, 9.05 ( 1.68 µM. Identical trends were
observed for indoles 33, 34-1, 34-2, and 35, returning IC50(RME)
values of 14.3 ( 2.7, 10.8 ( 1.4, 5.0 ( 0.9, and 58.2 ( 11.1
µM versus IC50(in vitro)s 6.62 ( 1.52, 3.33 ( 0.75, 1.30 ( 0.30,
and 10.1 µM, respectively. This rank order of potency for RME
inhibition closely matches the rank order of potency for
inhibition of dynamin’s GTPase activity, suggesting that the
mechanism of inhibition is via dynamin.
Indole 34-2 represents the most potent inhibitor of dynamin
mediated RME yet reported and is 15-fold more potent than
dynasore in both dynamin in vitro and RME based assays.17
a IC50 determinations are the average of at least three separate experi-
ments, each in duplicate. b “-” No inhibition at 300 µM drug concentration.
To determine whether the dynoles are toxic to cells, we
assessed the viability of five fibroblast cell lines (hTert-
immortalized breast stromal fibroblast, two primary skin
fibroblast lines, a primary myoblast cell line, and NIH3T3
mouse fibroblasts) following 7 days of continuous exposure
to dynole 34-2 using the Trypan Blue exclusion assay.
Membrane integrity was not affected because >98% of cells,
in all five cell lines, remained viable in the presence of dynole
34-2 (10 µM).
associated with the cyanoamide moiety from 41.69° to 0.84°,
essentially coplanar with the indole nucleus.36
In Vitro Inhibition of Dynamin Leads to a Complete
Block in Receptor Mediated Endocytosis. Given that dynamin
is essential for endocytosis, we felt it appropriate to determine
the ability of our five most potent indoles, 26, 33, 34-1, 34-2,
and 35, to block RME in cells, and based on earlier efforts, we
focused on human bone osteosarcoma epithelial (U2OS) cells.
Using previously established approaches, we examined the effect
of 26, 33, 34-1, 34-2, and 35 on the internalization of Texas
Red-transferrin (Tf-TxR), which binds to the transferrin receptor
and is internalized by a dynamin II-dependent pathway.18 After
Mechanism of Dynamin in Vitro Inhibition. Our RME
studies clearly demonstrate that indoles 26, 33, 34-1, 34-2,
and 35 are efficient blockers of endocytosis, with the RME IC50
values obtained following an identical trend for the in vitro

3766 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Hill et al.
Figure 4. Dynoles 26, 33, 34-1, 34-2, and 35 block internalization of Tf-TxR in U2OS cells. U2OS cells were preincubated with vehicle only
or different dynoles for 30 min and then incubated with Tf-TxR (A1) for 8 min at 37 °C and acid washed and fixed, and internalized Tf-TxR was
detected by fluorescence microscopy (left column). Nuclei were stained blue with DAPI to show the position of the cells (middle column). The
combined image (right column) shows the position of the cell nucleus (blue) and the internalized Tf-TxR (red).
inhibition of dynamin. Using traditional enzyme kinetics (Figure
6 top, bottom), we found that indole 34-1 is uncompetitive
with GTP.
afforded new inhibitors of dynamin I displaying potency 100×
that of the original lead compounds that we call the dynole series
(Dynamin inhibition by indoles). Our data clearly demonstrates
efficient block of endocytosis (RME), and the rank order of
potency (of the dynoles) in blocking endocytosis correlates well
with their rank potency order for dynamin I inhibition. This
suggests that the observed endocytosis block is via the inhibition
of dynamin GTPase activity.
Importantly, this potency is not confined to the test tube with
a complete endocytic block in cells being observed with dynoles
34-1 and 34-2. These analogues display high cell permeability
and efficiency with cellular IC50s less than an order of magnitude
Conclusion
Synthetically simple approaches facilitated the rational design
of the most potent dynamin inhibitors reported thus far.
Commencing with the simplification of the bisindolylmaleimide
leads, 1 and 2, we designed and synthesized a series of small
highly focused libraries exploring the structural requirements
for inhibition of dynamin I GTPase. Modifications developed
herein were restricted to synthetically simple approaches and

Inhibition of Dynamin Mediated Endocytosis by the Dynoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3767
dynasore, suggesting a greater ease of cell penetration for the
dynoles.36 While not a pivotal consideration for the development
of biochemical probes, the dynoles lack dynasores’ catechol
moiety and have lower number of hydrogen bond donor and
acceptor groups, rendering this new family of dynamin inhibitors
amenable for therapeutic development.35
We thus believe that the dynoles, in particular dynole 34-2,
with the synthetic ease of its access and its high dynamin I in
vitro inhibition (IC50 ) 1.30 ( 0.30 µM) and excellent ability
to inhibit RME (IC50 ) 5.0 ( 0.9 µM) represent a significant
advance for the study of dynamin mediated endocytosis and
will be of considerable value to researchers in the field.
Figure 5. IC50 curves for endocytosis block in U2OS cells of dynoles
26, 33, 34-1, 34-2, and 35. U2OS cells were preincubated with vehicle
only or different dynoles for 30 min and then incubated with Tf-TxR
(A1) for 8 min. Data is mean fluorescence as a percent of control cells
(triplicate determinations on approximately 1200 cells each) ( SEM.
The results are representative of three independent experiments.
Experimental Section
Biology. Materials. Phosphatidylserine (PS), phenylmethylsul-
fonylfluoride (PMSF), and Tween 80 were from Sigma-Aldrich (St.
Louis, MO). GTP was from Roche Applied Science (Germany),
leupeptin was from Bachem (Bubendorf, Switzerland). Gel elec-
trophoresis reagents, equipment, and protein molecular weight
markers were from Bio-Rad (Hercules, CA). Collagenase was from
Roche. Paraformaldehyde (PFA) was from Merck Pty Ltd. (Kilsyth,
Australia). Coverslips were from Lomb Scientific (Sydney, Aus-
tralia). Penicillin/streptomycin, phosphate buffered salts, fetal calf
serum (FCS), and Dulbecco’s minimal essential medium (DMEM)
were from Invitrogen (Mount Waverley, Victoria, Australia). Alexa-
594 conjugated Tf (Tf-A594) and DAPI were from Molecular
Probes (Oregon). All other reagents were of analytical reagent grade
or better.
Drugs. Drugs were made in-house or were purchased from
Sigma-Aldrich. The drugs were made up as stock solutions 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
3.3% or 1%, respectively. The GTPase assay for dynamin I was
unaffected by DMSO up to 3.3%. Drugs were dissolved as 30 mM
stocks in 100% DMSO and were light yellow in color. Stocks were
stored at -20 °C for several months. They were diluted into
solutions of 50% DMSO made up in 20 mM Tris/HCl pH 7.4 and
then diluted again into the final assay.
Protein Production. Dynamin I was purified from sheep brain
by extraction from the peripheral membrane fraction of whole
brain39 and affinity purification on GST-Amph2-SH3-sepharose as
described,40 yielding 8-10 mg of protein from 250 g of sheep brain.
Malachite Green GTPase assay. The malachite green method
was used for the sensitive colorimetric detection of orthophosphate
(Pi). It is based on the formation of a phosphomolybdate complex
at low pH with basic dyes, causing a color change. The assay
procedure is based on stimulation of native sheep brain-purified
dynamin I by sonicated phosphatidylserine (PS) liposomes.41
However, in our earlier studies, we used 200 nM dynamin I, while
in the present study we used 20 nM, requiring a reformulation of
assay volumes and the malachite green reagent for the present
study.19,20,41 Purified dynamin I (20 nM) (diluted in 6 mM Tris/
HCl, 20 mM NaCl, 0.01% Tween 80, pH 7.4) was incubated in
Figure 6. Kinetic analysis of interactions of dynole 34-1 with dynamin
GTPase. Data shows this dynole is uncompetitive with GTP.
of the in vitro data. In the presence of the lead dynole 34-2,
fibroblast cells remained viable for 7 days in culture, indicating
that the drug is not toxic to these cells.
The mechanism of inhibition by dynole 34-1 was uncom-
petitive. Uncompetitive inhibition is relatively unusual and can
occur when an inhibitor only binds to the complex between the
enzyme (dynamin) and substrate (GTP). This indicates that the
dynole binds to the enzyme-substrate complex at a site other
than the active site, which might be an allosteric site within the
GTPase domain that becomes available upon GTP binding to
dynamin.
Currently, dynasore is the primary inhibitor of dynamin
mediated endocytosis in routine use.17 The dynoles clearly
display superior IC50 values and their lipophilic nature facilitates
membrane permeability, evidenced by the highly efficient block
of RME. These data correlate well with predicted physicochem-
ical properties of dynole 34-2 with CLogP ) 4.81, a polar
surface area (PSA) ) 41.4 Å2, and a dipole of 5.93 debye versus
CLogP ) 3.65, PSA ) 89.0 Å2, and a dipole ) 6.63 debye for
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 PMSF)
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 Figures 4-5. 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

3768 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Hill et al.
dark for up to 2 months at room temperature). Color was allowed
to develop for 5 min (and was stable up to 2 h), and the absorbance
of samples in each plate was determined on a microplate spectro-
photometer at 650 nm. Phosphate release was quantified by
comparison with a standard curve of sodium dihydrogen ortho-
phosphate 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. The curves were
generated using the Michaelis-Menten equation V ) Vmax[S]/(Km
+ [S]) where S ) PS activator or GTP substrate. After the Vmax
and Km values were determined, the data was transformed using
the Lineweaver-Burke eq 1/V ) 1/Vmax + (Km/Vmax)(1/[S]).
Texas Red-Tfn Uptake. U2OS cells were cultured in DMEM
supplemented with 10% FCS at 37 °C and in 5% CO2 in a
humidified incubator. Tf uptake was analyzed based on methods
previously described.41 Briefly, cells were grown in fibronectin-
coated (5 µg/mL) 96-well plates. The cells were serum-starved
overnight (16 h) in DMEM minus FCS. Cells were then incubated
with indoles 26, 33, 34-1, 34-2, and 35 (usually 30 µM) or vehicle
for 30 min prior to addition of 4 µg/mL of Tf-TxR for 8 min at 37
°C. Cell surface-bound Tf 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 was rinsed 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 Tf-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 Tf-TxR signal per
cell was calculated for each well using IXM system and the data
expressed as a 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 3 wells and ∼1200
cells.
desired fractions and concentration under reduced pressure gave
the desired product as a yellow oil, 3.50 g (70%). 1H NMR (CDCl3)
δ 7.71 (dd, J ) 8.5 Hz, 1.0 Hz, 1H), 7.43 (d, J ) 8.4 Hz, 1H),
7.30 (dt, J ) 7.0 Hz, 1.0 Hz, 1H), 7.17 (m, 2H), 6.55 (d, J ) 3.5
Hz, 1H), 4.25 (t, J ) 7.0 Hz, 2H), 2.64 (t, J ) 7.0 Hz, 2H), 2.26
(s, 6H). 13C NMR (CDCl3) δ 135.7, 128.0, 127.3, 121.0, 120.3,
119.1, 109.1, 100.4, 56.0, 44.9, 43.8.
(3-Indol-1-ylpropyl)dimethylamine (9). Compound 9 was
prepared in a similar manner to the synthesis of 8, from indole (3)
and 3-chloro-N,N-dimethylpropanamine, to give a yellow oil in a
68% yield. 1H NMR (CDCl3) δ 7.72 (dd, J ) 8.4 Hz, 0.6 Hz, 1H),
7.43 (d, J ) 8.4 Hz, 1H), 7.28 (dt, J ) 7.0 Hz, 1.1 Hz, 1H), 7.17
(m, 2H), 6.56 (d, J ) 3.1 Hz, 1H), 4.24 (t, J ) 6.9 Hz, 2H), 2.31
(t, J ) 7.0 Hz, 2H), 2.26 (s, 6H), 2.00 (quin, J ) 6.9 Hz, 2H). 13
C
NMR (CDCl3) δ 135.6, 128.1, 127.4, 120.9, 120.4, 118.7, 108.9,
100.5, 55.9, 44.9, 43.5, 27.7.
Dimethyl-[2-(2-methylindol-1-yl)ethyl]amine (10). Compound
10 was prepared in a similar manner to the synthesis of 8, from
2-methylindole (4) and 3-chloro-N,N-dimethylethanamine, to give
1
a yellow oil in a 75% yield. H NMR (CDCl3) δ 7.45 (d, J ) 7.8
Hz, 1H), 7.37 (d, J ) 8.2 Hz, 1H), 7.10 (m, 2H), 7.00 (dt, J ) 7.8
Hz, 0.9 Hz, 1H), 4.15 (t, J ) 6.7 Hz, 2H), 2.56 (t, J ) 6.7 Hz,
2H), 2.22 (s, 3H), 2.17 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 135.9,
128.1, 126.2, 120.8, 118.4, 118.0, 109.3, 108.6, 58.6, 45.1, 43.2,
9.3.
Dimethyl-[3-(2-methylindol-1-yl)propyl]amine (11). Com-
pound 11 was prepared in a similar manner to the synthesis of 8,
from 2-methylindole (4) and 3-chloro-N,N-dimethylpropanamine,
1
to give a yellow oil in a 73% yield. H NMR (CDCl3) δ 7.47 (d,
J ) 7.8 Hz, 1H), 7.35 (d, J ) 8.2, 1H), 7.10 (m, 2H), 7.00 (dt, J
) 7.8 Hz, 1.1 Hz, 1H), 4.10 (t, J ) 7.8 Hz, 2H), 3.35 (t, J ) 6.9
Hz, 2H), 2.37 (s, 3H), 2.13 (t, J ) 6.9 Hz, 2H), 2.09 (s, 6H), 1.80
(quin, J ) 6.9 Hz, 2H). 13C NMR (CDCl3) δ 135.9, 128.1, 126.0,
120.8, 118.4, 117.9, 110.2, 108.6, 55.6, 45.0, 42.7, 27.8, 9.3.
1-(2-Dimethylaminoethyl)-2-methyl-1H-indole-3-carbalde-
hyde (12). Compound 12 was prepared in a similar manner to the
synthesis of 8, from 2-methylindole-3-carboxaldehyde (5) and
2-chloro-N,N-dimethylethanamine, to give, after recrystallization
from ethanol, a white solid in a 78% yield; mp 50-52 °C. 1H NMR
(CDCl3) δ 10.25 (s, 1H), 8.14 (d, J ) 8.1 Hz, 1H), 7.43 (d, J )
7.5 Hz, 1H), 7.24 (m, 2H), 4.29 (t, J ) 7.2 Hz, 2H), 2.72 (s, 3H),
2.62 (t, J ) 7.2 Hz, 2H), 2.29 (s, 6H). 13C NMR (CDCl3) δ 184.4,
148.6, 136.0, 125.2, 122.4, 121.9, 119.9, 113.1 108.9, 55.7, 43.9,
40.4, 8.38.
Cell Viability Assay. Cell viability and membrane integrity was
assessed by the Trypan Blue exclusion assay. Cells were seeded
into 10 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 dynole 34-2 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).
1-(3-Dimethylaminopropyl)-2-methyl-1H-indole-3-carbalde-
hyde (13). Compound 13 was prepared in a similar manner to the
synthesis of 8, from 2-methylindole-3-carboxaldehyde (5) and
2-chloro-N,N-dimethylpropanamine, to give, after recrystallization
from ethanol, an off-white solid in a 76% yield; mp 62-64 °C. 1H
NMR (CDCl3) δ 10.08 (s, 1H), 8.10 (d, J ) 6.6 Hz, 1H), 7.5 (d,
J ) 7.2 Hz, 1H), 7.20 (m, 2H), 4.2 (t, J ) 7.2 Hz, 2H), 2.70 (s,
3H), 2.19 (t, J ) 6.6 Hz, 2H), 2.12 (s, 6H), 1.83 (quin, J ) 6.9 Hz,
2H). 13C NMR (CDCl3) δ 184.1, 148.7, 136.1, 125.1, 122.6, 122.1,
120.0, 113.4, 110.2, 55.6, 44.9, 40.6, 26.8, 9.72.
Chemistry. General Methods. THF was freshly distilled from
sodium-benzophenone. Flash chromatography was carried out
1
using silica gel 200-400 mesh (60 Å). H and 13C NMR were
recorded at 300 and 75 MHz, respectively, using a Bruker Avance
300 MHz spectrometer in CDCl3 and DMSO-d6. GCMS was
performed using a Shimadzu GCMS-QP2100. The instrument uses
a quadrupole mass spectrometer and detects samples via electron
impact ionization (EI). The University of Wollongong, Australia,
Biomolecular Mass Spectrometry Laboratory, analyzed samples for
HRMS. The spectra were run on the VG Autospec-oa-tof tandem
high resolution mass spectrometer using CI (chemical ionization),
with methane as the carrier gas and PFK (perfluorokerosene) as
the reference. Microanalysis was conducted at the Microanalyis Unit
at the Australian National University, Canberra, Australia. All
samples returned satisfactory analyses.
[1-(2-Dimethylaminoethyl)-1H-indol-3-yl]acetic Acid (14). Com-
pound 12 was prepared in a similar manner to the synthesis of 8,
from 2-methyl-1H-indole-3-carboxylic acid (6) and 2-chloro-N,N-
dimethylethanamine, to give, after recrystallization from ethanol,
1
a white solid in a 82% yield; mp 54-56 °C. H NMR (CDCl3) δ
8.75 (br, 1H), 7.61 (d, J ) 7.8 Hz, 1H) 7.30 (d, J ) 7.9 Hz, 1H),
7.11 (m, 3H), 4.18 (t, J ) 7.1 Hz, 2H), 3.83 (s, 2H), 2.38 (t, J )
7.1 Hz, 2H), 2.18 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 171.6,
135.5, 126.6, 122.5, 121.4, 119.0, 118.3, 110.7, 107.7, 62.6, 55.5,
44.7, 30.1.
(2-Indol-1-ylethyl)dimethylamine (8). To a cooled solution (0
°C) of indole (3) (3.0 g, 25.6 mmol) and THF (50 mL) was added
slowly NaH (50% dispersion in mineral oil) (2.0 g, 41.7 mmol).
This solution was allowed to stir for 30 min, then N,N-dimethy-
lamino-2-chloroethane (3.2 g, 29.75 mmol) was added and the
solution was heated at reflux for 70 h. Solution was allowed to
cool and water added (50 mL). The organic layer was removed,
and the aqueous layer extracted with ethyl acetate (2 × 50 mL).
Combined organic layers were dried (MgSO4) and evaporated under
reduced pressure, giving orange oil. This was purified via short
column chromatography DCM:MeOH (90:10). Combination of the
[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]acetic Acid (15).
Compound 15 was prepared in a similar manner to the synthesis
of 8, from 2-methyl-1H-indole-3-carboxylic acid (6) and 2-chloro-
N,N-dimethylpropanamine, to give, after recrystallization from
1
ethanol, a white solid in a 84% yield; mp 56-58 °C. H NMR
(300 MHz, CDCl3) δ 8.71 (br, 1H), 7.62 (d, J ) 7.6 Hz, 1H) 7.30
(d, J ) 7.9 Hz, 1H), 7.10 (m, 3H), 4.16 (t, J ) 6.4 Hz, 2H), 3.8 (s,

Inhibition of Dynamin Mediated Endocytosis by the Dynoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3769
1
2H), 2.28 (t, J ) 7.2 Hz, 2H), 2.18 (s, 6H), 1.80 (quin, J ) 6.5 Hz,
2H). 13C NMR (CDCl3) δ 171.7, 135.7, 126.7, 122.7, 121.5, 118.9,
118.3, 110.7, 107.7, 62.6, 55.5, 44.7, 30.9, 26.3.
°C. H NMR (DMSO-d6) δ 8.33 (s, 1H), 8.07 (br, 1H), 7.96 (d, J
) 7.2 Hz, 1H), 7.57 (d, J ) 7.5 Hz, 1H), 7.22 (m, 2H), 4.29 (t, J
) 7.2 Hz, 2H), 3.24 (quin, J ) 6.6 Hz, 2H), 2.72 (s, 3H), 2.62 (t,
J ) 7.2 Hz, 2H), 2.29 (s, 6H), 1.14 (t, J ) 7.3 Hz, 3H). 13C NMR
(DMSO-d6) δ 162.2, 145.5, 144.2, 136.8, 124.5, 122.4, 121.1, 120.9,
118.3, 110.4, 107.5, 98.9, 55.7, 34.7, 43.9, 40.4, 13.6, 10.4.
3-[1-(2-Dimethylaminoethyl)-2-methyl-1H-indol-3-yl]propi-
onic Acid (16). Compound 16 was prepared in a similar manner
to the synthesis of 8, from 3-(2-methyl-1H-indol-3-yl)propanoic
acid (7) and 2-chloro-N,N-dimethylethanamine, to give, after
recrystallization from ethanol, a white solid in a 54% yield; mp
2-Cyano-N-propyl-3-[1-(2-dimethylaminoethyl)-2-methyl-1H-
indol-3-yl]acrylamide (23). Compound 23 was prepared in a
similar manner to the synthesis of 19, substituting N-propyl-2-
cyanoacetamide (18e) for N-benzyl-2-cyanoacetamide (18a) to give,
after recrystallization from ethanol, a yellow solid (54%); mp
1
68-70 °C. H NMR (CDCl3) δ 10.60 (br, 1H), 7.48 (d, J ) 7.9
Hz, 1H), 7.31 (d, J ) 8.1 Hz, 1H), 7.08 (m, 2H), 6.96 (dt, J ) 7.0,
1.1 Hz, 1H), 4.01 (t, J ) 5.9 Hz, 2H), 2.69 (t, J ) 7.4 Hz, 2H),
2.43 (t, J ) 5.8 Hz, 2H), 2.32 (t, J ) 7.3 Hz, 2H), 2.14 (s, 6H),
2.07 (s, 3H) 1.88 (quin, J ) 7.4 Hz, 2H). 13C NMR (CDCl3) δ
172.7, 136.2, 127.0, 122.3, 120.7, 118.1, 118.0, 113.6, 111.2, 61.4,
57.2, 45.2, 33.2, 25.3, 23.9.
1
136-138 °C. H NMR (DMSO-d6) δ 8.08 (s, 1H), 7.85 (br, 1H),
7.74 (d, J ) 6.9 Hz, 1H), 7.37 (d, J ) 6.8 Hz, 1H), 7.07 (m, 2H),
4.49 (t, J ) 5.6 Hz, 2H), 3.49 (q, J ) 5.8 Hz, 2H), 2.94 (s, 3H),
2.85 (t, J ) 5.6 Hz, 2H), 2.60 (s, 6H), 1.99 (quin, J ) 6.4 Hz, 2H)
1.42 (t, J ) 6.5 Hz, 3H). 13C NMR (DMSO-d6) δ 162.2, 145.5,
144.2, 136.8, 124.5, 122.4, 121.1, 120.9, 118.3, 110.4, 107.5, 98.9,
57.9, 45.4, 41.7, 41.3, 22.2, 11.4, 11.3.
3-(1-(3-Dimethylaminopropyl)-2-methyl-1H-indol-3-yl)pro-
panoic Acid (17). Compound 17 was prepared in a similar manner
to the synthesis of 8 from 3-(2-methyl-1H-indol-3-yl)propanoic acid
(7) and 3-chloro-N,N-dimethylpropanamine, to give, after recrys-
tallization from ethanol, a white solid in a 60% yield; mp 74-76
°C. 1H NMR (CDCl3) δ 10.61 (br, 1H), 7.50 (d, J ) 7.9 Hz, 1H),
7.31 (d, J ) 8.1 Hz, 1H), 7.10 (m, 2H), 6.95 (dt, J ) 7.1, 1.5 Hz,
1H), 4.01 (t, J ) 5.9 Hz, 2H), 2.69 (t, J ) 7.4 Hz, 2H), 2.38 (t, J
) 5.8 Hz, 2H), 2.31 (t, J ) 7.3 Hz, 2H), 2.14 (s, 6H), 2.07 (s, 3H)
1.88 (quin, J ) 7.4 Hz, 2H), 1.80 (quin, J ) 5.8 Hz, 2H). 13C
NMR (CDCl3) δ 173.0, 137.2, 128.0, 122.7, 120.9, 118.3, 118.0,
113.9, 112.3, 61.5, 57.5, 46.1, 33.1, 25.2, 23.9, 22.8.
N-Benzyl-2-cyano-3-[1-(2-dimethylaminoethyl)-2-methyl-1H-
indol-3-yl]acrylamide (19). 1-(2-Dimethylaminoethyl)-2-methyl-
1H-indole-3-carbaldehyde (12) (0.2 g, 0.87 mmol), N-benzyl-2-
cyanoacetamide (18a) (0.15 g, 0.87 mmol), ethanol (5 mL), and
piperidine were refluxed for 3 h. After this time, water (30 mL)
was added to solution. This was then extracted with ethyl acetate
(2 × 50 mL). Organic layers were combined, dried over MgSO4,
and concentrated under reduced pressure, giving an orange solid.
This was recrystallized from MeOH giving an orange solid (82%);
mp 146-148 °C. 1H NMR (DMSO-d6) δ 8.68 (t, J ) 5.4 Hz, 1H),
8.38 (s, 1H), 7.97 (d, J ) 7.1 Hz, 1H), 7.55 (d, J ) 7.1 Hz, 1H),
7.12 (m, 8H), 4.42 (d, J ) 4.3 Hz, 2H), 4.32 (t, J ) 4.5 Hz, 2H),
2.58 (m, 5H), 2.19 (s, 6H). 13C NMR (DMSO-d6) δ 162.4, 146.1,
144.6, 139.4, 136.9, 128.2, 127.3, 126.7, 124.5, 122.4, 121.2, 121.0
118.3, 110.5, 107.6, 98.2, 57.9, 45.5, 41.1, 40.6, 11.2.
2-Cyano-N-butyl-3-[1-(2-dimethylaminoethyl)-2-methyl-1H-
indol-3-yl] acrylamide (24). Compound 24 was prepared in a
similar manner to the synthesis of 19, substituting N-butyl-2-
cyanoacetamide (18f) for N-benzyl-2-cyanoacetamide (18a) to give,
after recrystallization from ethyl acetate, a yellow solid (54%); mp
1
137-139 °C. H NMR (DMSO-d6) δ 8.33 (s, 1H), 8.07 (br, 1H),
7.96 (d, J ) 7.2 Hz, 1H), 7.57 (d, J ) 7.5 Hz, 1H), 7.22 (m, 2H),
4.29 (t, J ) 7.2 Hz, 2H), 3.22 (q, J ) 6.9 Hz, 2H) 2.72 (s, 3H),
2.62 (t, J ) 7.2 Hz, 2H), 2.29 (s, 6H), 1.48 (quin, J ) 7.2 Hz, 2H),
1.33 (sept, J ) 7.9 Hz, 2H), 0.89 (t, J ) 7.2 Hz, 3H). 13C NMR
(DMSO-d6) δ 162.2, 145.5, 144.2, 136.8, 124.5, 122.4, 121.1, 120.9,
118.3, 110.4, 107.5, 98.9, 55.7, 43.9, 40.4, 39.5, 25.4, 19.4, 13.0,
12.2.
2-Cyano-N-hexyl-3-[1(2-dimethylaminoethyl)-2-methyl-1H-in-
dol-3-yl]-acrylamide (25). Compound 25 was prepared in a similar
manner to the synthesis of 19, substituting N-hexyl-2-cyanoaceta-
mide (18g) for N-benzyl-2-cyanoacetamide (18a) to give, after
recrystallization from ethyl acetate, a yellow solid (67%); mp
119-121 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.25 (s, 1H), 8.10
(br, 1H), 8.00 (d, J ) 7.1 Hz, 1H), 7.62 (d, J ) 7.1 Hz, 1H), 7.20
(m, 2H), 4.32 (t, J ) 7.2 Hz, 2H), 3.22 (q, J ) 7.2 Hz, 2H), 2.70
(s, 3H), 2.59 (t, J ) 7.2 Hz, 2H), 2.30 (s, 6H), 1.46 (quin, J ) 7.3
Hz, 2H), 1.27 (m, 6H), 0.85 (t, J ) 6.5 Hz, 2H); 13C NMR (75
MHz, DMSO-d6) δ 163.1, 145.2, 144.2, 136.2, 125.0, 122.3, 120.9,
19.9, 118.2, 110.4, 106.5, 99.0, 55.8, 44.1, 40.2, 38.7, 28.6, 25.8,
25.2, 21.7, 11.3.
2-Cyano-N-(4-methoxybenzyl)-3-[1-(2-dimethylaminoethyl)-
2-methyl-1H-indol-3-yl]-acrylamide (20). Compound 20 was
prepared in a similar manner to the synthesis of 19, substituting
N-(4-methoxybenzyl)-2-cyanoacetamide (18b) for N-benzyl-2-cy-
anoacetamide (18a) to give, after recrystallization from methanol,
2-Cyano-N-octyl-3-[1-(2-dimethylaminoethyl)-2-methyl-1H-
indol-3-yl]-acyrlamide (26). Compound 26 was prepared in a
similar manner to the synthesis of 19, substituting N-octyl-2-
cyanoacetamide (18f) for N-benzyl-2-cyanoacetamide (18a) to give,
after recrystallization from ethyl acetate, a yellow solid (56%); mp
1
an orange solid (47%); mp 165-167 °C. H NMR (DMSO-d6) δ
8.33 (s, 1H), 8.07 (br, 1H), 7.96 (m, 3H), 7.57 (d, J ) 7.5 Hz,
1H), 7.22 (m, 2H), 7.12 (d, J ) 8.7 Hz, 2H), 4.29 (t, J ) 7.2 Hz,
2H), 3.84 (s, 3H), 2.72 (s, 3H), 2.62 (t, J ) 7.2 Hz, 2H), 2.29 (s,
6H). 13C NMR (75 MHz, DMSO-d6) δ 162.4, 150.5, 145.5, 144.3,
136.1, 132.8, 125.2, 124.8, 122.3, 121.1, 120.7, 118.7, 114.2, 111.6,
107.6, 97.6, 58.1, 55.7, 43.9, 40.4, 11.4, 8.38.
1
112-113 °C. H NMR (DMSO-d6) δ 8.30 (s, 1H), 8.10 (br, 1H),
7.99 (d, J ) 7.0 Hz, 1H), 7.59 (d, J ) 7.5 Hz, 1H), 7.20 (m, 2H),
4.28 (t, J ) 7.1 Hz, 2H), 3.27 (q, J ) 6.4 Hz, 2H), 2.74 (s, 3H),
2.62 (t, J ) 7.2 Hz, 2H), 2.30 (s, 6H), 1.50 (quin, J ) 6.7 Hz, 2H),
1.31 (m, 12H), 0.92 (t, J ) 6.3 Hz, 3H). 13C NMR (DMSO-d6) δ
162.1, 144.2, 143.6, 1363, 125.5, 123.3, 121.0, 120.4, 118.3, 110.2,
107.4, 99.0, 56.7, 44.3, 41.4, 40.2, 28.9, 28.5, 26.1, 25.4, 22.1, 12.4.
2-Cyano-N-cyclohexyl-3-[1-(2-dimethylaminoethyl)-2-methyl-
1H-indol-3-yl]acrylamide (21). Compound 21 was prepared in a
similar manner to the synthesis of 21, substituting N-cyclohexyl-
2-cyanoacetamide (18c) for N-benzyl-2-cyanoacetamide (18a) to
give, after recrystallization from methanol, a yellow solid (76%);
2-Cyano-N-benzyl-3-[1-(3-dimethylaminopropyl)-2methyl-
1H-indol-3-yl]-acrylamide (27). Compound 27 was prepared in a
similar manner to the synthesis of 19 from 1-(3-dimethylamino-
propyl)-2-methyl-1H-indole-3-carbaldehyde (13) and N-benzyl-2-
cyanoacetamide (18a) to give, after recrystallization from ethanol,
1
mp 150-152 °C. H NMR (DMSO-d6) δ 8.35 (s, 1H), 8.07 (br,
1H), 7.95 (d, J ) 7.3 Hz, 1H), 7.60 (d, J ) 7.3 Hz, 1H), 7.21 (m,
2H), 4.28 (t, J ) 7.2 Hz, 2H), 3.71 (m, 1H), 2.75 (s, 3H), 2.60 (t,
J ) 7.1 Hz, 2H), 2.31 (s, 6H), 1.91 (m, 2H), 1.35 (m, 2H),
1.20-1.15 (m, 4H). 13C NMR (75 MHz, DMSO-d6) δ 162.4, 145.6,
144.1, 137.5, 125.5, 122.1 121.1, 120.9, 118.1, 110.3, 108.2, 98.4,
55.6, 48.9, 43.8, 40.3, 25.5, 24.3, 24.1, 10.9.
1
an orange solid (64%); mp 165-168 °C. H NMR (DMSO-d6) δ
8.58 (t, J ) 5.4 Hz, 1H), 8.28 (s, 1H), 7.95 (d, J ) 7.0 Hz, 1H),
7.64 (d, J ) 7.1 Hz, 1H), 7.13-7.10 (m, 8H), 4.54 (d, J ) 4.4 Hz,
2H), 4.20 (t, J ) 7.0 Hz, 2H), 2.72 (s, 3H), 2.17 (t, J ) 6.9 Hz,
2H), 2.10 (s, 6H), 1.82 (quin, J ) 6.9 Hz, 2H). 13C NMR (DMSO-
d6) δ 163.2, 146.3, 145.0, 140.4, 137.3, 128.1, 126.9, 126.5, 124.5,
122.3, 121.2, 120.8 118.2, 110.4, 108.0, 98.1, 54.3, 44.6, 40.6, 26.8,
9.9.
2-Cyano-N-ethyl-3-[1(2-dimethylaminoethyl)-2-methyl-1H-in-
dol-3-yl]acrylamide (22). Compound 22 was prepared in a similar
manner to the synthesis of 19, substituting N-ethyl-2-cyanoaceta-
mide (18d) for N-benzyl-2-cyanoacetamide (18a) to give, after
recrystallization from ethanol, a yellow solid (58%); mp 135-137

3770 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Hill et al.
2-Cyano-N-(4-methoxybenzyl)-3-[1-(3-dimethylaminopropyl)-
2-methyl-1H-indol-3-yl]- acrylamide (28). Compound 28 was
prepared in a similar manner to the synthesis of 19 from 1-(3-
dimethylaminopropyl)-2-methyl-1H-indole-3-carbaldehyde (13) and
(4-methoxybenzyl)-2-cyanoacetamide (18b) to give, after recrys-
tallization from ethanol, a yellow solid (74%); mp 179-180 °C.
1H NMR (DMSO-d6) δ 8.38 (s, 1H), 8.11 (br, 1H), 7.94 (m, 3H),
7.55 (d, J ) 7.5 Hz, 1H), 7.22 (m, 2H), 7.11 (d, J ) 8.5 Hz, 2H),
4.25 (t, J ) 7.5 Hz, 2H), 2.73 (s, 3H), 2.17 (t, J ) 6.7 Hz, 2H),
2.15 (s, 6H), 1.85 (quin, J ) 6.9 Hz, 2H). 13C NMR (DMSO-d6) δ
162.5, 150.5, 146.5, 144.1, 138.1, 132.3, 127.2, 124.9, 122.3, 121.0,
120.7, 118.5, 114.1, 110.6, 108.3, 97.9, 58.0, 55.3, 44.9, 43.3, 40.2,
26.8, 11.0.
7.5 Hz, 1H), 7.21 (m, 2H), 4.25 (t, J ) 6.2 Hz, 2H), 3.22 (q, J )
7.1 Hz, 2H), 2.58 (s, 3H) 2.20 (t, J ) 6.6 Hz, 2H), 2.10 (s, 6H),
1.85 (quin, J ) 6.9 Hz, 2H) 1.52 (quin, J ) 7.3 Hz, 2H), 1.27 (m,
6H), 0.89 (t, J ) 6.4 Hz, 2H). 13C NMR (DMSO-d6) δ 164.4, 147.5,
146.3, 138.0, 127.5, 124.1, 123.1, 121.7, 119.5, 112.2, 108.4, 98.6,
56.3, 45.0, 41.5, 32.0, 28.5, 27.8, 26.8, 26.0, 22.9, 14.1, 11.2, 9.7.
2-Cyano-N-octyl-3-[1-(3-dimethylaminopropyl)-2-methyl-1H-
indol-3-yl]-acrylamide (34-1). Compound 34-1 was prepared in
a similar manner to the synthesis of 19 from 1-(3-dimethylamino-
propyl)-2-methyl-1H-indole-3-carbaldehyde (13) and N-octyl-2-
cyanoacetamide (18h) to give, after recrystallization from ethanol,
a yellow solid (52%); mp 99-101 °C. 1H NMR (DMSO-d6) δ 8.32
(s, 1H), 8.05 (br, 1H), 8.01 (d, J ) 7.5 Hz, 1H), 7.55 (d, J ) 7.5
Hz, 1H), 7.22 (m, 2H), 4.10 (t, J ) 6.3 Hz, 2H), 3.81 (s, 2H), 3.29
(q, J ) 6.3 Hz, 2H), 2.31 (t, J ) 7.2 Hz, 2H), 2.20 (s, 6H), 1.85
(quin, J ) 6.5 Hz, 2H) 1.50 (quin, J ) 6.5 Hz, 2H) 1.25 (m, 12H),
0.80 (t, J ) 6.3 Hz, 3H). 13C NMR (DMSO-d6) δ 163.4, 146.2,
145.1, 137.4, 126.3, 123.4, 122.4, 121.9, 119.7, 112.5, 108.4, 98.2,
56.1, 45.7, 40.6, 32.2, 28.9, 28.6, 26.9, 26.0 25.7, 25.3, 22.5, 13.5,
10.5.
2-Cyano-N-cyclohexyl-3-[1-(3-dimethylaminopropyl)-2-meth-
yl-1H-indol-3-yl]-acrylamide (29). Compound 29 was prepared
in a similar manner to the synthesis of 19 from 1-(3-dimethylami-
nopropyl)-2-methyl-1H-indole-3-carbaldehyde (13) and N-cyclo-
hexyl-2-cyanoacetamide (18c) to give, after recrystallization from
1
ethanol, a light-yellow solid (67%); mp 163-165 °C. H NMR
(DMSO-d6) δ 8.40 (s, 1H), 8.16 (br, 1H), 8.02 (d, J ) 7.4 Hz,
1H), 7.67 (d, J ) 7.5 Hz, 1H), 7.26 (m, 2H), 4.28 (t, J ) 7.0 Hz,
2H), 3.74 (m, 1H), 2.72 (s, 3H), 2.20 (t, J ) 7.0 Hz, 2H), 2.08 (s,
6H), 1.90 (m, 2H), 1.82 (quin, J ) 7.0 Hz, 2H), 1.32 (m, 2H),
1.20-1.15 (m, 4H). 13C NMR (DMSO-d6) δ 162.2, 145.3, 144.0,
136.1, 125.1, 121.9, 120.8, 120.2, 118.5, 111.0, 107.1, 97.2, 55.4,
48.5, 44.5, 40.2, 31.0, 26.5, 25.0, 24.5, 23.8, 10.0.
2-Cyano-N-octyl-3-(1-(3-dimethylaminopropyl)-1H-indol-3-
yl)-acrylamide (34-2). Compound 34-2 was prepared in a similar
manner to the synthesis of 19 from 1-(3-dimethylaminopropyl)-
1H-indole-3-carbaldehyde and N-octyl-2-cyanoacetamide (18h) to
give, after recrystallization from ethanol, a yellow solid (67%); mp
105-106 °C. 1H NMR (CDCl3) δ 8.64 (s, 1H), 8.40 (s, 1H), 7.85
(dd, J ) 6.8, 2.1 Hz, 1H), 7.44 (dd, J ) 7.1, 1.6 Hz, 1H), 7.37-7.23
(m, 2H), 6.19 (t, J ) 5.4 Hz, 1H), 4.30 (t, J ) 6.8 Hz, 2H), 3.41
(q, J ) 6.9 Hz, 2H), 2.22 (s, 6H), 2.07-1.94 (m, 2H), 1.59 (q, J
) 6.96 Hz, 2H), 1.30 (m, 12H), 0.87 (t, J ) 6.6 Hz, 3H). 13C NMR
(CDCl3) δ 161.3, 143.3, 135.3, 132.3, 127.9, 123.0, 121.7, 119.0,
118.3, 110.0, 109.5, 94.6, 55.2, 44.8, 44.4, 39.9, 31.3, 29.1, 29.0,
28.8, 27.1, 26.4, 22.1, 13.6.
2-Cyano-N-ethyl-3-[1-(3-dimethylaminopropyl)-2-methyl-1H-
indol-3-yl]-acrylamide (30). Compound 30 was prepared in a
similar manner to the synthesis of 19 from 1-(3-(dimethylamino)-
propyl)-2-methyl-1H-indole-3-carbaldehyde (13) and N-ethyl-2-
cyanoacetamide (18d) to give, after recrystallization from ethanol,
1
a yellow solid (57%); mp 136-138 °C. H NMR (DMSO-d6) δ
8.34 (s, 1H), 8.07 (br, 1H), 7.96 (d, J ) 7.1 Hz, 1H), 7.55 (d, J )
7.1 Hz, 1H), 7.23 (m, 2H), 4.28 (t, J ) 6.3 Hz, 2H), 3.20 (q, J )
6.2 Hz, 2H), 2.66 (s, 3H), 2.21 (m, 2H), 2.10 (s, 6H), 1.82 (t, J )
6.0 Hz, 2H), 1.01 (t, J ) 7.2 Hz, 3H). 13C NMR (DMSO-d6) δ
162.0, 145.1, 144.1, 136.7, 124.3, 122.0, 119.9, 119.1, 118.2, 110.5,
107.2, 98.5, 56.0, 44.4, 39.9, 28.0, 22.1, 11.1.
2-Cyano-N-decyl-3-[1-(2-dimethylaminoethyl)-2-methyl-1H-
indol-3-yl]-acrylamide (35). Compound 35 was prepared in a
similar manner to the synthesis of 19 from 1-(2-dimethylaminoet-
hyl)-2-methyl-1H-indole-3-carbaldehyde (12) and N-decyl-2-cy-
anoacetamide to give, after recrystallization from ethyl acetate, a
2-Cyano-N-propyl-3-[1-(3-dimethylaminopropy)-2-methyl-
1H-indol-3-yl]-acrylamide (31). Compound 31 was prepared in a
similar manner to the synthesis of 19 from 1-(3-dimethylamino-
propyl)-2-methyl-1H-indole-3-carbaldehyde (13) and N-propyl-2-
cyanoacetamide (18e) to give, after recrystallization from ethanol,
1
yellow solid (70%); mp 95-97 °C. H NMR (DMSO-d6) δ 8.19
(s, 1H), 7.93 (br, 1H), 7.85 (d, J ) 7.0 Hz, 1H), 7.44 (d, J ) 6.9
Hz, 1H), 7.10 (m, 2H), 4.32 (t, J ) 6.9 Hz, 2H), 3.30 (q, J ) 6.9
Hz, 2H), 2.73 (s, 3H), 2.65 (t, J ) 6.6 Hz, 2H), 2.10 (s, 6H), 1.52
(quin, J ) 6.8 Hz, 2H), 1.25 (m, 14H), 0.85 (t, J ) 6.5 Hz, 3H).
13C NMR (DMSO-d6) δ 165.2, 148.6, 145.1, 138.8, 126.5, 123.0,
121.4, 121.0, 119.1, 110.1, 106.3, 100.1, 58.3, 44.7, 40.1,39.9, 30.1,
29.0, 28.7, 28.6, 28.5, 26.2, 22.1, 13.8, 10.4.
1
a yellow solid (78%); mp 142-144 °C. H NMR (DMSO-d6) δ
8.33 (s, 1H), 8.07 (br, 1H), 7.96 (d, J ) 7.2 Hz, 1H), 7.57 (d, J )
7.5 Hz, 1H), 7.22 (m, 2H), 4.24 (t, J ) 6.1 Hz, 2H), 3.19 (q, J )
6.0 Hz, 2H), 2.56 (s, 3H), 2.20 (m, 2H), 2.12 (s, 6H), 1.83 (t, J )
6.0 Hz; 2H), 1.53 (q, J ) 7.1 Hz, 2H), 0.88 (t, J ) 7.2 Hz, 3H).
13C NMR (DMSO-d6) δ 162.1, 145.2, 144.2, 136.9, 124.4, 122.2,
121.1, 120.9, 118.4, 110.5, 107.2, 98.9, 56.0, 44.9, 40.1, 28.3, 22.3,
11.3.
2-Cyano-N-dodecyl-3-[1-(2-dimethylaminoethyl)-2-methyl-
1H-indol-3-yl]-acrylamide (36). Compound 36 was prepared in a
similar manner to the synthesis of 19 from 1-(2-dimethylaminoet-
hyl)-2-methyl-1H-indole-3-carbaldehyde (12) and N-dodecyl-2-
cyanoacetamide to give, after recrystallization from ethyl acetate,
a yellow solid (49%); mp 90-92 °C. 1H NMR (CDCl3) δ 8.33 (s,
1H), 7.99 (br, 1H), 7.83 (d, J ) 6.9 Hz, 1H), 7.40 (d, J ) 6.9 Hz,
1H), 7.00 (m, 2H), 4.30 (t, J ) 7.3 Hz, 2H), 3.30 (q, J ) 7.3 Hz,
2H), 2.72 (s, 3H), 2.65 (t, J ) 7.2 Hz, 2H), 2.32 (s, 6H), 1.50
(quin, J ) 7.0 Hz, 2H), 1.31 (m, 16H), 0.90 (t, J ) 6.3 Hz, 3H).
13C NMR (CDCl3) δ 162.0, 145.0, 144.0, 135.8, 124.2, 122.1, 120.5,
119.5, 118.6, 111.0, 106.5, 96.5, 55.4, 43.9, 40.3, 31.2, 29.0, 28.7,
28.6, 26.1, 25.5, 21.4, 13.6, 11.2, 8.3.
2-Cyano-N-butyl-3-[1-(3-dimethylaminopropyl)-2-methyl-1H-
indol-3-yl]-acrylamide (32). Compound 32 was prepared in a
similar manner to the synthesis of 19 from 1-(3-dimethylamino-
propyl)-2-methyl-1H-indole-3-carbaldehyde (13) and N-butyl-2-
cyanoacetamide (18f) to give, after recrystallization from ethanol,
1
a yellow solid (75%); mp 124-126 °C. H NMR (DMSO-d6) δ
8.32 (s, 1H), 8.05 (br, 1H), 7.95 (d, J ) 7.4 Hz, 1H), 7.57 (d, J )
7.5 Hz, 1H), 7.25 (m, 1H), 4.26 (t, J ) 6.4 Hz, 2H), 3.23 (q, J )
5.9 Hz, 2H), 2.57 (s, 3H), 2.20 (m, 2H), 2.13 (s, 6H), 1.83 (t, J )
6.2 Hz, 2H), 1.49 (quin, J ) 7.14 Hz, 2H), 1.32 (q, J ) 7.3 Hz,
2H), 0.90 (t, J ) 7.1 Hz, 3H). 13C NMR (DMSO-d6) δ 162.2, 145.3,
144.2, 136.8, 124.5, 122.3, 121.1, 120.9, 118.4, 110.4, 107.5, 98.8,
55.6, 44.95, 41.22, 41.0, 31.13, 26.9, 19.5, 13.6, 11.3.
2-Cyano-N-tetradecyl-3-[1-(2-dimethylaminoethyl)-2-methyl-
1H-indol-3-yl]-acrylamide (37). Compound 37 was prepared in a
similar manner to the synthesis of 19 from 1-(2-dimethylaminoet-
hyl)-2-methyl-1H-indole-3-carbaldehyde (12) and N-tetradecyl-2-
cyanoacetamide to give, after recrystallization from ethyl acetate,
a yellow solid (56%); mp 94-96 °C. 1H NMR (DMSO-d6) δ 8.33
(s, 1H), 8.07 (br, 1H), 7.95 (d, J ) 7.4 Hz, 1H), 7.55 (d, J ) 7.5
Hz, 1H), 7.22 (m, 2H), 4.25 (t, J ) 7.2 Hz, 2H), 3.19 (q, J ) 6.3
Hz, 2H), 2.70 (s, 3H), 2.60 (t, J ) 7.2 Hz, 2H), 2.26 (s, 6H), 1.53
(quin, J ) 6.8 Hz, 2H), 1.30 (m, 22H), 0.88 (t, J ) 6.3 Hz, 3H).
13C NMR (DMSO-d6) δ 165.4, 147.4, 146.4, 138.8, 126.3, 123.4,
2-Cyano-N-hexyl-3-[1-(3-dimethylaminopropyl)-2-methyl-1H-
indol-3-yl]-acrylamide (33). Compound 33 was prepared in a
similar manner to the synthesis of 19 from 1-(3-dimethylamino-
propyl)-2-methyl-1H-indole-3-carbaldehyde (13) and N-hexyl-2-
cyanoacetamide (18g) to give, after recrystallization from ethanol,
1
a yellow solid (46%); mp 100-102 °C. H NMR (DMSO-d6) δ
8.35 (s, 1H), 8.10 (br, 1H), 7.98 (d, J ) 7.1 Hz, 1H), 7.58 (d, J )

Inhibition of Dynamin Mediated Endocytosis by the Dynoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3771
122.8, 121.3, 120.0, 112.2, 109.3, 100.1, 58.3, 44.1, 42.4, 37.5,
29.8, 28.9, 28.7, 28.6, 26.2, 25.2, 21.1, 13.5, 8.5.
Hz), 7.20 (2H, m), 3.85 (1H, m), 2.67 (3H, s) 1.94 (2H, m), 1.54
(2H, m), 1.20-1.16 (4H, m). 13C NMR (DMSO-d6) δ 162.1, 145.3,
144.2 135.8, 124.7, 124.2, 121.0, 120.5, 118.5, 110.9, 106.5, 96.8,
48.9, 31.2, 25.6, 24.8, 24.2, 12.1.
2-Cyano-N-decyl-3-[1-(3-dimethylaminopropyl)-2-methyl-1H-
indol-3-yl]-acrylamide (38). Compound 38 was prepared in a
similar manner to the synthesis of 19 from 1-(3-dimethylamino-
propyl)-2-methyl-1H-indole-3-carbaldehyde (13) and N-decyl-2-
cyanoacetamide to give, after recrystallization from ethyl acetate,
2-Cyano-N-ethyl-3-(2-methyl-1H-indol-3-yl)-acrylamide (44).
Compound 44 was prepared in a similar manner to the synthesis
of 19 from 2-methyl-1H-indole-3-carbaldehyde (5) and N-ethyl-2-
cyanoacetamide (18d) to give, after recrystallization from ethanol,
1
a yellow solid (64%); mp 110-112 °C. H NMR (DMSO-d6) δ
1
8.30 (s, 1H), 8.02 (br, 1H), 7.95 (d, J ) 7.5 Hz, 1H), 7.60 (d, J )
7.5 Hz, 1H), 7.25 (m, 2H), 4.26 (t, J ) 7.2 Hz, 2H), 3.30 (q, J )
6.5 Hz, 2H), 2.70 (s, 3H), 2.20 (t, J ) 6.5 Hz, 2H), 2.15 (s, 6H),
1.85 (quin, J ) 6.5 Hz, 2H), 1.55 (quin, J ) 6.6 Hz, 2H), 1.25 (m,
14H), 0.83 (t, J ) 6.4 Hz, 3H). 13C NMR (DMSO-d6) δ 163.4,
146.5, 145.1, 137.0, 125.2, 123.1, 121.4, 120.9, 119.2, 112.3, 108.1,
98.5, 56.6, 45.9, 41.6, 32.0, 30.0, 29.0, 28.9, 28.8, 28.6, 26.8, 26.2,
22.0, 12.3, 11.2.
a yellow solid (66%) mp 242-244 °C. H NMR (CDCl3) δ 8.35
(1H, s), 8.02 (2H, m), 7.42 (2H, d, J ) 6.5 Hz), 7.16 (2H, m), 3.25
(2H, quin, J ) 6.6 Hz), 2.54 (3H, s), 1.15 (3H, t, J ) 7.2 Hz). 13
C
NMR (CDCl3) δ 164.2, 146.3, 145.1, 136.2, 125.2, 122.2, 121.4,
120.9, 119.9, 112.0, 103.2, 98.3, 26.2, 14.6, 11.5.
2-Cyano-N-propyl-3-(2-methyl-1H-indol-3-yl)-acrylamide (45).
Compound 45 was prepared in a similar manner to the synthesis
of 19 from 2-methyl-1H-indole-3-carbaldehyde (5) and N-propyl-
2-cyanoacetamide (18e) to give, after recrystallization from ethanol,
2-Cyano-N-dodecyl-3-[1-(3-dimethylaminopropyl)-2-methyl-
1H-indol-3-yl]-acrylamide (39). Compound 39 was prepared in a
similar manner to the synthesis of 19 from 1-(3-dimethylamino-
propyl)-2-methyl-1H-indole-3-carbaldehyde (13) and N-dodecyl-
2-cyanoacetamide to give, after recrystallization from ethyl acetate,
1
a yellow solid (75%); mp 245-247 °C. H NMR (CDCl3) δ 8.30
(s, 1H), 8.00 (m, 2H), 7.40 (d, J ) 6.8 Hz, 2H), 7.15 (m, 2H), 3.20
(q, J ) 6.5 Hz, 2H), 2.56 (s, 3H), 1.54 (sex, J ) 7.2 Hz, 2H), 0.87
(t, J ) 7.32 Hz, 3H). 13C NMR (CDCl3) δ 162.4, 145.5, 144.3,
136.1, 125.2, 122.3, 121.1, 120.7, 118.7, 111.6, 107.6, 97.6, 41.33,
22.3, 12.7, 11.20.
1
a yellow solid (64%); mp 103-105 °C. H NMR (DMSO-d6) δ
8.32 (s, 1H), 8.06 (br, 1H), 7.94 (d, J ) 7.3 Hz, 1H), 7.50 (d, J )
7.5 Hz, 1H,), 7.20 (m, 2H), 4.20 (t, J ) 7.2 Hz, 2H), 3.25 (q, J )
7.2 Hz, 2H,), 2.71 (s, 3H), 2.14 (t, J ) 6.5 Hz, 2H), 2.13 (s, 6H),
1.82 (quin, J ) 6.7 Hz, 2H), 1.50 (quin, J ) 6.8 Hz, 2H), 1.26 (m,
16H), 0.84 (t, J ) 6.4 Hz, 3H). 13C NMR (DMSO-d6) δ 163.2,
146.2, 143.2, 135.3, 124.2, 123.5, 122.2, 120.5, 119.6, 112.4, 105.3,
98.4, 54.2, 44.3, 42.6, 31.6, 28.9, 28.7, 28.6, 26.8,26.5, 25.3, 21.2,
13.0, 11.4.
2-Cyano-N-butyl-3-(2-methyl-1H-indol-3-yl)-acrylamide (46).
Compound 46 was prepared in a similar manner to the synthesis
of 19 from 2-methyl-1H-indole-3-carbaldehyde (5) and N-butyl-
2-cyanoacetamide (18f) to give, after recrystallization from
ethanol, a yellow solid (78%); mp 220-222 °C. 1H NMR
(DMSO-d6) δ 8.38 (s, 1H), 8.09 (m, 2H), 7.43 (d, J ) 7.1 Hz,
2H), 7.18 (m, 2H), 3.24 (q, J ) 7.0 Hz, 2H), 2.60 (s, 3H), 1.49
(quin, J ) 7.1 Hz, 2H), 1.35 (sept, J ) 7.7 Hz, 2H), 0.92 (t, J
) 7.1 Hz, 3H). 13C NMR (DMSO-d6) δ 164.5, 146.2, 145.1,
138.2, 127.1, 124.2, 122.3, 121.2, 119.2, 112.6, 108.9, 99.6, 33.2,
26.3, 20.4, 13.1, 10.1.
2-Cyano-N-tetradecyl-3-[1-(2-dimethylaminoethyl)-2-methyl-
1H-indol-3-yl]-acrylamide (40). Compound 40 was prepared in a
similar manner to the synthesis of 19 from 1-(3-dimethylamino-
propyl)-2-methyl-1H-indole-3-carbaldehyde (13) and N-tetradecyl-
2-cyanoacetamide to give, after recrystallization from ethyl acetate,
a yellow solid (55%); mp 99-101 °C. 1H NMR (DMSO-d6) δ 8.35
(s, 1H), 8.07 (br, 1H), 7.93 (d, J ) 7.2 Hz, 1H), 7.57 (d, J ) 7.5
Hz, 1H), 7.25 (m, 2H), 4.22 (t, J ) 7.2 Hz, 2H), 3.23 (q, J ) 6.1
Hz, 2H), 2.70 (s, 3H), 2.14 (t, J ) 6.6 Hz, 2H), 2.05 (s, 6H), 1.82
(quin, J ) 6.9 Hz, 2H), 1.54 (quin, J ) 6.8 Hz, 2H), 1.30 (m,
22H), 0.88 (t, J ) 6.4 Hz, 3H). 13C NMR (DMSO-d6) δ 164.4,
145.5, 144.3, 136.2, 125.2, 122.2, 121.1, 120.8, 118.7, 111.5, 107.4
97.5, 55.6, 44.9, 40.3, 31.3, 29.2, 28.9, 28.7, 28.6, 26.8, 26.2, 25.3,
21.4, 13.3, 11.2.
2-Cyano-N-hexyl-3-(2-methyl-1H-indol-3-yl)-acrylamide (47).
Compound 47 was prepared in a similar manner to the synthesis
of 19 from 2-methyl-1H-indole-3-carbaldehyde (5) and N-hexyl-
2-cyanoacetamide (18g) to give, after recrystallization from
1
acetonitrile, a yellow solid (64%); mp 186-188 °C. H NMR
(DMSO-d6) δ 8.32 (s, 1H), 8.02 (m, 2H), 7.45 (d, J ) 6.9 Hz,
2H), 7.22 (m, 2H), 3.23 (q, J ) 7.1 Hz, 2H), 2.60 (s, 3H), 1.48
(quin, J ) 7.5 Hz, 2H), 1.29 (m, 6H), 1.0 (t, J ) 6.7 Hz, 2H).
13C NMR (DMSO-d6) δ 163.2, 145.2, 146.2, 137.2, 127.4 124.2,
122.3, 121.4, 119.6, 112.5, 106.2, 98.2, 32.4, 29.5, 26.8, 25.3,
22.7, 14.2, 11.5.
2-Cyano-N-benzyl-3-(2-methyl-1H-indol-3-yl)-acrylamide (41).
Compound 41 was prepared in a similar manner to the synthesis
of 19 from 2-methyl-1H-indole-3-carbaldehyde (5) and N-benzyl-
2-cyanoacetamide (18a) to give, after recrystallization from ethyl
acetate, a yellow solid (52%); mp 238-240 °C. 1H NMR
(DMSO-d6) δ 8.55 (t, J ) 5.5 Hz, 1H), 8.40 (s, 1H), 8.08 (d, J
) 7.2 Hz, 1H), 7.75 (d, J ) 7.2 Hz, 1H), 7.10- 7.00 (m, 8H),
4.50 (d, J ) 5.4 Hz, 2H), 2.62 (s, 3H,). 13C NMR (DMSO-d6)
δ 163.4, 146.0, 144.8, 140.4, 137.8, 128.5, 127.2, 126.5, 124.6,
123.2, 121.0, 119.0 118.4, 111.2, 108.3, 98.2, 58.1, 9.9.
2-Cyano-N-(4′-methoxybenzyl)-3-(2-methyl-1H-indol-3-yl)-
acrylamide (42). Compound 42 was prepared in a similar manner
to the synthesis of 19 from 2-methyl-1H-indole-3-carbaldehyde
(5) and N-(4′-methoxybenzyl)-2-cyanoacetamide (18b) to give,
after recrystallization from ethyl acetate, a yellow solid (47%);
2-Cyano-N-octyl-3-(2-methyl-1H-indol-3-yl)-acrylamide (48).
Compound 48 was prepared in a similar manner to the synthesis
of 19 from 2-methyl-1H-indole-3-carbaldehyde (5) and N-octyl-2-
cyanoacetamide (18h) to give, after recrystallization from aceto-
nitrile, a yellow solid (52%); mp 174-176 °C. 1H NMR (DMSO-
d6) δ 8.28 (s, 1H), 7.88 (m, 2H), 7.32 (d, J ) 7.2 Hz, 2H), 7.10
(m, 2H), 3.30 (q, J ) 6.8 Hz, 2H), 2.51 (s, 3H), 1.49 (quin, J )
6.8 Hz, 2H), 1.24 (m, 12H), 0.82 (t, J ) 6.3 Hz, 3H). 13C NMR
(DMSO-d6) δ 161.4, 144.3, 143.2, 137.1, 124.2, 121.5, 120.1, 119.7,
117.7, 110.5, 106.6, 98.5, 30.2, 27.6, 27.5, 24.2, 24.0, 23.3, 21.0,
12.5, 9.8.
2-Cyano-N-decyl-3-(2-methyl-1H-indol-3-yl)-acrylamide (49).
Compound 49 was prepared in a similar manner to the synthesis
of 19 from 2-methyl-1H-indole-3-carbaldehyde (5) and N-decyl-
2-cyanoacetamide to give, after recrystallization from acetonitrile,
1
mp 252-254 °C. H NMR (DMSO-d6) δ 8.60 (t, J ) 5.4 Hz,
1
1H), 8.35 (s, 1H), 7.98 (m, 3H), 7.53 (d, J ) 7.2 Hz, 1H) 7.20
(m, 3H) 4.53 (d, J ) 4.1 Hz, 2H), 3.91 (s, 3H), 2.54 (s, 3H).
13C NMR (DMSO-d6) δ 163.4, 151.5, 146.5, 145.2, 138.2, 133.5,
126.1, 123.2, 122.2, 121.4, 120.4, 118.4, 115.4, 112.4, 108.5,
98.6, 59.2, 11.3.
a yellow solid (46%); mp 166-168 °C. H NMR (DMSO-d6) δ
8.31 (s, 1H), 7.99 (m, 2H), 7.38 (d, J ) 6.9 Hz, 2H), 7.13 (m, 2H),
3.25 (q, J ) 7.0 Hz, 2H), 2.54 (s, 3H), 1.52 (quin, J ) 6.9 Hz,
2H), 1.24 (m, 14H), 0.85 (t, J ) 6.5 Hz, 3H); 13C NMR (DMSO-
d6) δ 165.4, 147.4, 145.3, 138.1, 127.2, 123.3, 120.9, 120.2, 117.6,
110.4, 106.2, 96.6, 31.2, 28.2, 28.0, 27.6, 27.5, 27.2, 26.2, 21.1,
12.2, 10.2.
2-Cyano-N-cyclohexyl-3-(2-methyl-1H-indol-3-yl)-acryla-
mide (43). Compound 43 was prepared in a similar manner to the
synthesis of 19 from 2-methyl-1H-indole-3-carbaldehyde (5) and
N-cyclohexyl-2-cyanoacetamide (18c) to give, after recrystallization
2-Cyano-N-dodecyl-3-(2-methyl-1H-indol-3yl)-acrylamide (50).
Compound 50 was prepared in a similar manner to the synthesis
of 19 from 2-methyl-1H-indole-3-carbaldehyde (5) and N-dodecyl-
2-cyanoacetamide to give, after recrystallization from acetonitrile,
1
from ethanol, a yellow solid (86%); mp 236-238 °C. H NMR
(DMSO-d6) δ 8.30 (1H, s), 8.01 (2H, m), 7.42 (2H, d, J ) 6.8

3772 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Hill et al.
1
dynamin I partner in synaptic vesicle endocytosis. Nat. Neurosci.
2006, 9, 752–760.
(13) Hilliard, L. M.; Osicka, T. M.; Robinson, P. J.; Nikolic-Paterson,
D. J.; Comper, W. D. Characterisation of the urinary degradation
pathway in the isolated perfused rat kidney. J. Lab. Clin. Med. 2006,
147, 36–44.
(14) Newton, A. J.; Kirchhausen, T.; Murthy, V. N. Inhibition of
dynamin completely blocks compensatory synaptic vesicle endocy-
tosis. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17955–17960.
(15) Boucrot, E.; Kirchhausen, T. Endosomal recycling controls plasma
membrane area during mitosis. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 7939–7944.
(16) Xu, J.; McNeil, B.; Wu, W.; Nees, D.; Bai, L.; Wu, L. G. GTP-
independent rapid and slow endocytosis at a central synapse. Nat.
Neurosci. 2008, 11, 45–53.
(17) Macia, E.; Ehrlich, M.; Massol, R.; Boucrot, E.; Brunner, C.;
Kirchhausen, T. Dynasore, a cell-permeable inhibitor of dynamin.
DeV. Cell 2006, 10, 839–850.
(18) Quan, A. M.; McGeachie, A. B.; Keating, D. J.; van Dam, E. M.;
Rusak, J.; Chau, N.; Malladi, C. S.; Chen, C.; McCluskey, A.;
Cousin, M. A.; Robinson, P. J. MiTMAB is a potent dynamin
inhibitor that targets the PH domain and blocks two forms of
endocytosis. Mol. Pharmacol. 2007, 72, 1425–1439.
(19) Hill, T. A.; Odell, L. R.; Quan, A.; Ferguson, G.; Robinson, P. J.;
McCluskey, A. Long chain amines and long chain ammonium salts
as novel inhibitors of dynamin GTPase activity. Bioorg. Med. Chem.
Lett. 2004, 14, 3275–3278.
(20) Hill, T.; Odell, L. R.; Edwards, J. K.; Graham, M. E.; McGeachie,
A. B.; Rusak, J.; Quan, A.; Abagyan, R.; Scott, J. L.; Robinson,
P. J.; McCluskey, A. Small molecule inhibitors of dynamin I
GTPase activity: development of dimeric tyrphostins. J. Med. Chem.
2005, 48, 7781–7788.
(21) Zhang, J.; Lawrance, G. A.; Chau, N.; Robinson, P. J.; McCluskey,
A. From Spanish fly to room temperature ionic liquids (RTILs):
synthesis, thermal stability and inhibition of dynamin 1 GTPase
by a novel class of RTILs. New J. Chem. 2008, 32, 28–36.
(22) Toullec, D.; Pianetti, P.; Coste, H.; Bellevergue, P.; Grand-Perret,
T.; Ajakane, M.; Baudet, V.; Boissin, P.; Boursier, E.; Loriolle,
F.; Duhamel, L.; Charon, D.; Kirilovsky, J. The bisindolylmaleimide
GF 109203X is a potent and selective inhibitor of protein kinase
C. J. Biol. Chem. 1991, 266, 15771–15781.
(23) Hers, I.; Tavare, J. M.; Denton, R. M. The protein kinase C
inhibitors bisindolylmaleimide I (GF 109203x) and IX (Ro 31-
8220) are potent inhibitors of glycogen synthase kinase-3 activity.
FEBS Lett. 1999, 460, 433–436.
(24) Reubold, T. F.; Eschenburg, S.; Becker, A.; Leonard, M.; Schmid,
S. L.; Vallee, R. B.; Kull, F. J.; Manstein, D. J. Crystal structure
of the GTPase domain of rat dynamin 1. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 13093–13098.
(25) Bartlett, S. B.; Jackson, R. M.; Kayser, V.; Kilner, C.; Leach, A.;
Nelson, N.; Oledzki, P. R.; Reid, G. D.; Warriner, S. L. Comparison
of the ATP binding sites of protein kinases using conformationally
diverse bisindolylmaleimides. J. Am. Chem. Soc. 2005, 127, 11699–
11708.
a yellow solid (54%); mp 165-167 °C. H NMR (DMSO-d6) δ
8.27 (s, 1H), 7.96 (m, 2H), 7.35 (d, J ) 6.8 Hz, 2H), 7.11 (m, 2H),
3.24 (q, J ) 7.0 Hz, 2H), 2.52 (s, 3H), 1.48 (quin, J ) 6.6 Hz,
2H), 1.25 (m, 16H), 0.84 (t, J ) 6.0 Hz, 3H). 13C NMR (DMSO-
d6) δ 162.3, 145.2, 144.0, 135.8, 125.0, 122.1, 119.7, 119.1, 117.7,
110.5, 105.2, 95.2, 30.2, 27.9, 27.7, 27.4, 26.1, 25.3, 22.2, 12.6,
9.9.
2-Cyano-N-tetradecyl-3-(2-methyl-1H-indol-3-yl)-acrylamide (51).
Compound 51 was prepared in a similar manner to the synthesis
of 19 from 2-methyl-1H-indole-3-carbaldehyde (5) and N-tet-
radecyl-2-cyanoacetamide to give, after recrystallization from
1
acetonitrile, a yellow solid (45%; mp 160-162 °C. H NMR
(DMSO-d6) δ 8.42 (s, 1H), 8.12 (m, 2H), 7.55 (d, J ) 6.8 Hz,
2H), 7.34 (m, 2H), 3.45 (q, J ) 6.2 Hz, 2H), 2.77 (s, 3H), 1.67
(quin, J ) 6.9 Hz, 2H), 1.40 (m, 22H), 1.12 (t, J ) 6.4 Hz,
3H). 13C NMR (DMSO-d6) δ 165.6, 147.2, 145.2, 138.2, 127.2,
124.1, 122.1, 121.3, 119.4, 112.5, 106.5, 98.5, 32.4, 30.0, 29.9,
28.9, 28.7, 26.4, 25.6, 22.0, 14.2, 11.1.
2-Cyano-N-octyl-3-(2-methyl-1H-indol-3-yl)-acrylamide (52).
Compound 52 was prepared in a similar manner to the synthesis
of 19 from 2-methyl-1-(4-methylpentyl)-1H-indole-3-carbalde-
hyde and N-octyl-2-cyanoacetamide (18h) to give, after recrys-
tallization from ethanol, a yellow solid (86%); mp 236-238 °C.
1H NMR (DMSO-d6) δ 8.30 (s, 1H), 8.01 (m, 2H), 7.42 (d, J )
6.8 Hz, 2H), 7.20 (m, 2H), 3.85 (m, 1H), 2.67 (s, 3H) 1.94 (m,
2H), 1.54 (m, 2H), 1.20-1.16 (m, 4H); 13C NMR (DMSO-d6) δ
162.1, 145.3, 144.2 135.8, 124.7, 124.2, 121.0, 120.5, 118.5,
110.9, 106.5, 96.8, 48.9, 31.2, 25.6, 24.8, 24.2, 12.1.
Acknowledgment. We are grateful for financial support
from the NH & MRC (Australia), the Children’s Medical
Research Institute (CMRI), and the University of Newcastle
(UN). This project was also supported by the Epilepsy
Research Foundation through the generous support of Finding
A Cure For Epilepsy And Seizures (f.a.c.e.s.). Swetha
Gaddipati is thanked for technical assistance. T.H. and L.O.
gratefully acknowledge scholarship support from UN &
CMRI and UN, respectively.
References
(1) McLure, S. J.; Robinson, P. J. Dynamin, endocytosis and intra-
cellular signaling. Mol. Membr. Biol. 1996, 13, 189–215.
(2) Cousin, M. A. R.; Robinson, P. J. Mechanisms of synaptic vesicle
recycling illuminated by fluorescent dyes. J. Neurochem. 1999, 73,
2227–2239.
(3) Brodin, L. L. P.; Shupliakov, O. Sequential steps in clathrin-
mediated synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 2000,
10, 312–320.
(4) Hinshaw, J. E. Dynamin and its role in membrane fission. Annu.
ReV. Cell DeV. Biol. 2000, 16, 483–519.
(5) Cousin, M. A. R.; Robinson, P. J. The dephosphins: dephospho-
rylation by calcineurin triggers synaptic vesicle endocytosis. Trends
Neurosci. 2001, 24, 659–665.
(6) Mukherjee, S.; Ghosh, R. N.; Maxfield, F. R. Endocytosis. Physiol.
ReV. 1997, 77, 759–803.
(7) Humeau, Y. D.; Grant, N. J.; Poulain, B. How botulinum and tetanus
neurotoxins block neurotransmitter release. Biochimie 2000, 82, 427–
446.
(8) Marks, B.; Stowell, M. H.; Vallis, Y.; Mills, I. G.; Gibson, A.;
Hopkins, C. R.; McMahon, H. T. GTPase activity of dynamin and
resulting conformation change are essential for endocytosis. Nature
2001, 410, 231–235.
(9) Hinshaw, J. E.; Schmind, S. L. Dynamin self-assembles into rings
suggesting a mechanism for coated vesicle budding. Nature 1995,
374, 190–192.
(26) Wood, J. L.; Stoltz, B. M.; Dietrich, H.-J. Total synthesis of (+)-
and (-)-K252a. J. Am. Chem. Soc. 1995, 117, 10413–10414.
(27) Wood, J. L.; Stoltz, B. M.; Dietrich, H.-J.; Pflum, D. A.; Petsch,
D. T. Design and implementation of an efficient synthetic approach
to furanosylated indolocarbazoles: total synthesis of (+)- and
(-)-K252a. J. Am. Chem. Soc. 1997, 119, 9641–9651.
(28) Trost, B. M.; Krische, M. J.; Berl, V.; Grenzer, E. M. Chemo-,
regio-, and enantioselective Pd-catalyzed allylic alkylation of
indolocarbazole pro-aglycons. Org. Lett. 2002, 4, 2005–2008.
(29) Zhu, G. C.; Conner, S. E.; Zhou, X.; Shih, C.; Li, T.; Anderson, B. D.;
Brooks, H. B.; Campbell, R. M.; Considine, E.; Dempsey, J. A.; Faul,
M. M.; Ogg, C.; Patel, B.; Schultz, R. M.; Spencer, C. D.; Teicher, B.;
Watkins, S. A. Synthesis, structure-activity relationship, and biological
studies
of
indolocarbazoles
as
potent cyclin D1-CDK4 inhibitors. J. Med. Chem. 2003, 46, 2027–3030.
(30) Kuethe, J. T.; Wong, A.; Davies, I. W. Effective strategy for the
preparation of indolocarbazole aglycons and glycosides: Total
synthesis of tjipanazoles B, D, E, and I. Org. Lett. 2003, 5, 3721–
3723.
(31) McCluskey, A.; Robinson, P. J.; Hill, T.; Scott, J. L.; Edwards,
J. K. Green chemistry approaches to the Knoevenagel condensation:
comparison of ethanol, water and solvent free (dry grind) ap-
proaches. Tetrahedron Lett. 2002, 43, 3117–3120.
(32) Correa, W. H. E.; Edwards, J. K.; McCluskey, A.; McKinnon, I.;
Scott, J. L. A thermodynamic investigation of solvent-free reactions.
Green Chem. 2003, 5, 30–33.
(33) Hill, T. A.; Sakoff, J. A.; Robinson, P. J.; McCluskey, A. Parallel
solution-phase synthesis of targeted tyrphostin libraries with
anticancer activity. Aus. J. Chem. 2005, 58, 94–103.
(10) Stowell, M. H. B.; Marks, B.; Wigge, P.; McMahon, H. T.
Nucleotide-dependent conformational changes in dynamin: evidence
for a mechanochemical molecular spring. Nat. Cell Biol. 1999, 1,
27–32.
(11) Powell, K. A.; Valvoa, V. A.; Malladi, C. S.; Jensen, O. N.; Larsen,
M. R.; Robinson, P. J. Phosphorylation of dynamin I on Ser-795
by protein kinase C blocks its association with phospholipids.
J. Biol. Chem. 2000, 275, 11610–11617.
(12) Anggono, V.; Smillie, K. J.; Graham, M. E.; Valova, V. A.; Cousin,
M. A.; Robinson, P. J. Syndapin I is the phosphorylation-regulated

Inhibition of Dynamin Mediated Endocytosis by the Dynoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3773
(34) Calculated using the New and improVed ClogP Calculator from
Chemaxon http://www.chemaxon.com (home page); http://intro.bio.
umb.edu/111-112/OLLM/111F98/newclogp.html (Applet page).
(35) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. AdV. Drug
DeliVery ReV. 1997, 23, 3–25.
(39) Robinson, P. J.; Sontag, J.-M.; Liu, J. P.; Fykse, E. M.; Slaughter,
C.; McMahon, H. T.; Su¨dhof, T. C. Dynamin GTPase regulated
by protein kinase C phosphorylation in nerve terminals. Nature
1993, 365, 163–166.
(40) Quan, A.; Robinson, P. J. Rapid purification of native dynamin I
and colorimetric GTPase assay. Methods Enzymol. 2005, 404
(Chapter 49), 556–569.
(41) van der Bliek, A. M.; Redelmeier, T. E.; Damke, H.; Tisdale, E. J.;
Meyerowitz, E. M.; Schmid, S. L. Mutations in human dynamin
block an intermediate stage in coated vesicle formation. J. Cell
Biol. 1993, 122, 553–563.
(36) Dihedral angles were measured in Spartan 06 version 1.0.4 (for
Macintosh). Wavefunction Inc., Irvine, CA, (www.wavefun.com),
based on 6-31** energy minimized structures.
(37) Sheff, D.; Pelletier, L.; O’Connell, C. B.; Warren, G.; Mellman, I.
Transferrin receptor recycling in the absence of perinuclear
recycling endosomes. J. Cell Biol. 2002, 156, 797–804.
(38) Gaborik, Z.; Hunyady, L. Intracellular trafficking of hormone
receptors. Trends Endocrinol. Metab. 2004, 15, 286–293.
JM900036M