Small molecule inhibitors of dynamin I GTPase activity: Development of dimeric tyrphostins

Article abstract of DOI:10.1021/jm040208l

Dynamin I is a GTPase enzyme required for endocytosis and is an excellent target for the design of potential endocytosis inhibitors. Screening of a library of tyrphostins, in our laboratory, against the GTPase activity of dynamin I gave rise to a ìèpotent lead, 2-cyano-3-(3,4- dihydroxyphenyl)thioacrylamide (1, IC₅₀ 70 μM). Our initial investigations suggested that only the dimeric form of 1 displayed dynamin I GTPase inhibitory activity. Subsequent synthetic iterations were based on dimeric analogues and afforded a number of small molecules, low μM potent, inhibitors of dynamin I GTPase, in particular, symmetrical analogues with a minimum of two free phenolic -OHs: catechol-acrylamide (9) (IC₅₀ = 5.1 ± 0.6 μM), its 3,4,5-trihydroxy congener (10) (IC₅₀ = 1.7 ± 0.2 μM), and the corresponding 3-methyl ether (11) (IC ₅₀ = 9 ± 3 μM). Increasing the length of the central alkyl spacer from ethyl to propyl (22-24) afforded essentially identical activity with IC₅₀'s of 1.7 ± 0.2, 1.7 ± 0.2, and 5 ± 1 μM, respectively. No decrease in activity was noted until the introduction of a hexyl spacer. Our studies highlight the requirement for two free amido NHs with neither the mono-N-methyl (86) nor the bis-N-methyl (87) analogues inhibiting dynamin I GTPase. A similar effect was noted for the removal of the nitrile moieties. However, modest potency was observed with the corresponding ester analogues of 9-11: ethyl ester (90), propyl ester (91), and butyl ester (92), with IC₅₀'s of 42 ± 3, 38 ± 2, and 61 ± 2 μM, respectively. Our studies reveal the most potent and promising dynamin I GTPase inhibitor in this series as (22), which is also known as BisT.

Full text of DOI:10.1021/jm040208l

J. Med. Chem. 2005, 48, 7781-7788
7781
Small Molecule Inhibitors of Dynamin I GTPase Activity: Development of
Dimeric Tyrphostins
Timothy Hill,† Luke R. Odell,† Jennifer K. Edwards,† Mark E. Graham,‡ Andrew B. McGeachie,‡ Jenny Rusak,‡
Annie Quan,‡ Ruben Abagyan,§ Janet L. Scott,| Phillip J. Robinson,*,‡ and Adam McCluskey*,†
Discipline of Chemistry, Chemistry Building, School of Environmental and Life Sciences, The University of Newcastle,
Callaghan, NSW 2308, Australia, Children’s Medical Research Institute, 214 Hawkesbury Road,
Westmead NSW 2145, Australia, Department of Molecular Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, TCP-28, La Jolla, California 92037, and Centre for Green Chemistry, PO Box 23,
Monash University, Victoria 3800, Australia
Received December 1, 2004
Dynamin I is a GTPase enzyme required for endocytosis and is an excellent target for the
design of potential endocytosis inhibitors. Screening of a library of tyrphostins, in our laboratory,
against the GTPase activity of dynamin I gave rise to a µM potent lead, 2-cyano-3-(3,4-
dihydroxyphenyl)thioacrylamide (1, IC50 70 µM). Our initial investigations suggested that only
the dimeric form of 1 displayed dynamin I GTPase inhibitory activity. Subsequent synthetic
iterations were based on dimeric analogues and afforded a number of small molecules, low µM
potent, inhibitors of dynamin I GTPase, in particular, symmetrical analogues with a minimum
of two free phenolic -OHs: catechol-acrylamide (9) (IC50 ) 5.1 ( 0.6 µM), its 3,4,5-trihydroxy
congener (10) (IC50 ) 1.7 ( 0.2 µM), and the corresponding 3-methyl ether (11) (IC50 ) 9 ( 3
µM). Increasing the length of the central alkyl spacer from ethyl to propyl (22-24) afforded
essentially identical activity with IC50’s of 1.7 ( 0.2, 1.7 ( 0.2, and 5 ( 1 µM, respectively. No
decrease in activity was noted until the introduction of a hexyl spacer. Our studies highlight
the requirement for two free amido NHs with neither the mono-N-methyl (86) nor the bis-N-
methyl (87) analogues inhibiting dynamin I GTPase. A similar effect was noted for the removal
of the nitrile moieties. However, modest potency was observed with the corresponding ester
analogues of 9-11: ethyl ester (90), propyl ester (91), and butyl ester (92), with IC50’s of 42 (
3, 38 ( 2, and 61 ( 2 µM, respectively. Our studies reveal the most potent and promising
dynamin I GTPase inhibitor in this series as (22), which is also known as BisT.
Introduction
cadaverine,8 intracellular potassium depletion,9 intra-
cellularacidification,10 anddecreasingmediumtemperature
to 4 °C.6 Each of these methods has poor specificity and
limited utility and no specific biological target. Nonethe-
less, their use has contributed to a better understanding
of endocytosis. Some have been used to demonstrate
that blocking endocytosis might eventually have clinical
applications for humans.11
Mammalian cells take up extracellular material and
recycle their membranes by endocytosis, which is the
formation of numerous types of membrane vesicles at
the plasma membrane.1-3 Vesicles occur in different
sizes, ranging from large phagosomes, smaller clathrin-
coated vesicles to tiny synaptic vesicles (SV). Endocytic
mechanisms subserve many cellular functions including
the uptake of extracellular nutrients, regulation of cell-
surface receptor expression and signaling, antigen pre-
sentation, and maintenance of synaptic transmission.
Among the various endocytic pathways are two that are
biochemically well-characterized. The first is the rapid
synaptic vesicle endocytosis (SVE) that follows vesicle
exocytosis in nerve terminals. SVE is not specifically
linked to receptor activation, but serves to retrieve emp-
ty SVs for later refilling.2,3 It requires the enzyme dyna-
min I.4,5 The second is receptor-mediated endocytosis
(RME) which is initiated upon ligand binding to cell sur-
face receptors and occurs via clathrin-coated pits in all
cells, including nerve terminals. It requires dynamin II.4
RME provides the main entry point into cells for plasma
membrane components (such as the receptor-ligand com-
plexes and membrane lipids) or for extracellular fluid.
Multiple endocytosis inhibitors exist, e.g., chlorpro-
mazine,6 concanavalin A, phenylarsine oxide,7 dansyl-
Targeting the GTPase activity of dynamin is a more
attractive candidate for an endocytosis inhibitor than
any currently known targets. There are three dynamin
genes, with dynamin I in neurons, dynamin II being
ubiquitously expressed and dynamin III in neurons and
testes.4,5 All dynamins have four main domains which
are potential drug targets. (i) The GTPase domain has
an unusually low affinity for GTP (10-25 µM) and
extremely high turnover rates compared with other
GTPases. It is required for vesicle fission.4,12 The crystal
structure of this domain of dynamin from Dictyostelium
was recently solved.13 (ii) The pleckstrin homology (PH)
domain is both a targeting domain and potentially a
GTPase inhibitory module and is essential for endocy-
tosis.5 Dynamin interacts with lipids via this domain,
and dynamin binding to nanotubules containing phos-
phatidylinositol bisphosphate (PtdIns(4,5)P2) greatly
stimulates GTPase activity.14 The PH domain is not
needed for self-assembly or GTPase activity; simply
deleting it (delta-PH dynamin) maximally increases
intrinsic GTPase activity.15 (iii) The GTPase effector
domain (GED) controls dynamin-dynamin interactions
and dynamin assembly into its basic tetrameric config-
uration, tetramers being subunits of the rings. About
* To whom correspondence should be addressed. A.M.: e-mail,
Adam.McCluskey@newcastle.edu.au; phone, +61 249 216486; fax, +61
249215472.P.J.R.: e-mail,phrobins@usyd.edu.au;phone,+61 296 872800;
fax, +61 296 872120.
† The University of Newcastle.
‡ Children’s Medical Research Institute.
§ The Scripps Research Institute.
| Monash University.
10.1021/jm040208l CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/29/2005

7782 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
McCluskey et al.
no dynamin I GTPase inhibition was noted (see Table
S1, Supporting Information).23
Somewhat surprised by the complete lack of inhibi-
tion, we reexamined the activity of 1 and noted that it
increased as a function of time in solution. ES-MS
experiments showed rapid decomposition of 1 in the
presence of air and the appearance of two major product
peaks (m/z ) 366.95; 408.99; 444.97; 574.97 and 652.96),
but not in the presence of DTT, a known quencher of
disulfide formation. This is in keeping with reports by
Wells22 that the decomposition of 1 is not solely a result
of disulfide formation, but is more complex most likely
involving oxidation of the catechol moieties. Thus the
observed inhibitory action of 1 resides in a higher
molecular weight analogue (see Supporting Informa-
tion). Therefore, given the effect of DTT and synthetic
ease we redirected our efforts to the synthesis of a
second library of symmetrically substituted analogues
of 2 (Figure 1). Related dimeric tyrphostins have been
reported previously as potent inhibitors of EGF receptor
tyrosine kinase activity.24
Retrosynthetic analysis suggested that simple ap-
plication of Knoevenagel chemistry involving substi-
tuted benzaldehydes and a series of appropriate R,ω-
bisamines would rapidly afford the desired libraries
(Scheme 1). To expedite the development of a SAR
profile, we limited the choice of aromatic groups to
thirteen commercially available benzaldehydes. In all
cases good to excellent yields were obtained (see Ex-
perimental Section and Supporting Information).21
Utilization of this approach allowed the rapid genera-
tion of a number of discrete sublibraries, based upon
the length of the alkane spacer arm with n ) 1-4 and
n ) 10. Our library structure allowed variation in aro-
matic ring substitution facilitating the rapid examina-
tion of key features required such as spatial location/
orientation of groups, the distances between aromatic
rings, and the effect of electron donating and electron
withdrawing groups, and in conjunction with biological
screening, the development of a preliminary structure
activity relationship. Initial biological screens for dy-
namin I GTPase activity were conducted at 100 µM.
More promising analogues were then screened across a
range of concentrations to determine their IC50 values
(Table 1). Of the 68 analogues synthesized only com-
pounds 9-11, 22-24, 35-37, 48-50, and 61-63 showed
noteworthy inhibition (operationally defined as having
an IC50 < 100 µM).
Within sublibrary 1 (n ) 0), aromatic compounds
containing no substitutions (6) or single -OH (7, 8), -Cl
(12, 13), -OMe (14, 15), or -COOH (18) showed no
dynamin inhibition (Table 1). Introduction of a second
oxygen-bearing substituent had a pronounced effect
with the 3,4-di-OH (9) active (IC50 ) 5.1 ( 0.6 µM);
however, monomethylation of the C3 -OH (17) and bis-
methylation of C3 and C4 -OH (16) rendered the
compounds inactive. Reintroduction of an oxygen-bear-
ing substituent at C3 (or the equivalent C5-position) as
an -OH resulted in low µM potency once more (11).
Examination of the additional sublibraries (n ) 1, 2,
etc.) highlights a similar trend, i.e., there is a require-
ment for a minimum of two free -OH groups on each
of the aromatic rings, and this trend is consistent
through each of the subsequent sublibraries developed.
Figure 1. Library design for symmetrical dimeric analogues
based on 2.
28-32 tetramers cooperatively self-assemble as a single
ring16 or as a helix around PtdIns(4,5)P2-containing lipid
mixtures.14 GED accounts for tetramer self-association
by binding to the GTPase domain.17 GED acts like a
GTPase activator protein to stimulate GTPase activity.18
(iv) The proline-rich domain (PRD) at dynamin’s C-
terminus interacts with many SH3 domain-containing
proteins4,5 and calcineurin19 and is the site for in vivo
dynamin phosphorylation.20
Our group recently discovered a series of long chain
amine and ammonium salts that inhibit dynamin I
GTPase activity.21 The most potent of these was myris-
toyl trimethyl ammonium bromide (MTMAB IC50 ) 3.2
( 0.6 µM). These compounds inhibit dynamin by
competing with lipid binding to the PH domain (unpub-
lished observations). As part of these and ongoing
studies in our laboratories we screened a small library
of tyrphostins and discovered that 1 displayed good in
vitro inhibition of dynamin I GTPase activity, while a
large series of other tyrphostins were ineffective. Herein
we report the design and synthesis of dynamin I
inhibitors based on 1 and on the development of a
structure activity profile.
Results and Discussion
Our initial library screening in the search for inhibi-
tors of dynamin I GTPase activity identified (1) as a 70
µM potent lead.
Concerned about the known instability of 1,22 we
aimed to develop a series of new analogues, focusing
initial synthetic efforts of the corresponding amido
analogue 2-cyano-3-(3,4-dihydroxyphenyl)acrylamide (2)
(Figure 1). Our primary targeted library synthesis was
designed to (a) determine the role of the aromatic
substituents, both the type and positioning, and (b)
determine the effect of additional substituents at the
amide NH2. With the 100 or so analogues synthesized,

Small Molecule Inhibitors of Dynamin I GTPase Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7783
Scheme 1. Synthesis of a Dimeric Analogue of Library 2a
a Details of substituents R1-R5 and the alkane spacer n are given in Table 1.
For example, the 3,4,5-trisubstituted aromatic com-
pound 10 was equipotent with 23. The corresponding
compounds in each of the longer analogues 22-24, 35-
37, 48-50, and 61-63 all displayed some level of
dynamin inhibiting activity, although these latter ana-
logues are dropping off in potency.
Alkane spacer chain elongation had little effect on po-
tency until n > 3. For example, chain extended ana-
logues of 9 (n ) 0), i.e., 22 (n ) 1), 35 (n ) 2), 48 (n )
3), 61 (n ) 4), and 71 (n ) 10) display IC50 values of 5.1
( 0.6, 1.7 ( 0.2, 3.2 ( 1.0, 5.0 ( 1.4, 26 ( 2, and >100
µM, respectively. In contrast, while examining a raft of
similar compounds against EGF receptor tyrosine ki-
nase phosphorylation of the poly-GAT substrate, Gazit
et al. observed that inhibition was independent of chain
length.24-26
This contrasts with the effects of dimeric tyrphos-
tins on tyrosine kinase potency, which resides in
their extended configuration and thus allows them to
fit the dimeric intermediate of the EGFR tyrosine
kinases.24,25 Introduction of a more rigid linker such as
piperazine (74) resulted in complete loss of activity
(Table 2). Increasing the conformational flexibility (but
still more rigid than the original straight chain linkers)
with 1,2-bis-aminocyclohexane (75) and 1,3-bis-benzyl-
amine (76), we note the return of sub 100 µM potencies
of all these analogues, albeit markedly less potent than
9 (Table 2). We propose that the observed reduction in
potency associated with 75 and 76 is due to increased
conformational rigidity resulting in poor alignment of
pivotal binding modes. Indeed, our synthetic efforts
commencing with 1,3-bis-aminopropan-2-ol and applica-
tion of similar chemistries to those described in Scheme
1 have shown that limited modifications at this point
are permissible, and the introduction of an -OH with
82 is well tolerated (Table 2, Scheme 2). However the
introduction of propyl or benzyl ether renders these
analogues, 83 and 84, inactive (Table 2).
Further synthetic modifications designed to probe
requirements of the central linker were also explored.
Installation of a benzoyl amide (85) via the azide and
standard peptide coupling approaches27 (Supporting
Information) gave rise to the same outcome as that
evidenced with ethers 83 and 84, i.e., no inhibition of
dynamin I GTPase.
With analogues 83, 84, and 85 the lack of biologi-
cal activity suggests that there is a finite binding
pocket at this point and that only small groups are
tolerated.

7784 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
McCluskey et al.
Table 1. Symmetrically Substituted Analogues of 2: Effect of
Aromatic Ring Modifications Analogues on Dynamin I GTPase
Activity
compd
R1
R2
R3
R4
R5
n
IC50 (µM)a
6
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0
-
7
OH
H
H
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
10
10
10
-
8
9
H
-
H
OH
OH
OH
H
5.1 ( 0.6
al to inhibition (Table 2). This suggests that the amide
NH’s also play an important role in binding to dynamin
I, either by stabilization of the active conformation or
by H-bonding within dynamin I GTPases active site (or
both).
However, replacement of the amide linkage with the
corresponding esters (Scheme 3) maintained a moderate
level of inhibition, which again was dependent upon the
spacer length (Table 3, analogues 90-93). Presumably
the reduction in inhibition is a result of the less
favorable H-bonding characteristics of the ester “O”
compared with the amide “NH”.28
Having determined that the amide moiety was im-
portant for inhibitory activity, relative to the corre-
sponding esters, and that methylation was detrimental,
the final logical exploration in this preliminary study
was delineation of the role of the pendant nitriles. This
role was established via the synthesis of the correspond-
ing cyano free analogues (Scheme 4). Synthesis com-
menced with 3,4-dihydroxycinnamic acid (94) and pro-
tection of the catechols as the corresponding acetates
(95). Treatment with SOCl2 in CHCl3 with catalytic
quantities of DMF afforded the acyl chloride disposed
for addition of R,ω-bis-amines (3a-e) to yield the ni-
trile free acetates (97a-e), and acid cleavage (HCl)
of the acetates gave the desired nitrile free analogues
(98-102),27 with varying spacer chain sizes as had
been examined in our original sublibraries. Biological
evaluation revealed the nitrile free analogues to be
inactive.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
OH
OMe
H
1.7 ( 0.2
9.0 ( 3.0
-
H
H
H
Cl
-
OMe
H
H
-
OMe
OMe
OMe
COOH
H
H
-
H
OMe
OH
H
-
H
-
H
-
H
H
-
OH
H
H
H
-
OH
OH
OH
OH
H
H
-
H
OH
OH
OH
H
1.7 ( 0.2
OH
OMe
H
1.7 ( 0.2
5.0 ( 1.0
-
H
Cl
H
H
-
OMe
H
H
-
OMe
OMe
OMe
COOH
H
H
-
H
OMe
OH
H
-
H
-
H
-
H
H
-
OH
H
H
H
-
OH
OH
OH
OH
H
H
-
H
OH
OH
OH
H
3.2 ( 1.0
OH
OMe
H
2.1 ( 0.2
8.0 ( 0.2
-
H
H
H
Cl
-
OMe
H
H
-
OMe
OMe
OMe
COOH
H
H
-
H
OMe
OH
H
-
H
-
H
-
H
H
-
OH
H
H
H
-
OH
OH
OH
OH
H
H
-
H
OH
OH
OH
H
5.0 ( 1.4
OH
OMe
H
1.7 ( 0.4
8.0 ( 0.2
-
Conclusions
H
H
H
Cl
-
OMe
H
H
-
Herein we have reported the development of a pre-
liminary structure-activity relationship of dimeric tyr-
phostins against the GTPase activity of the enzyme
dynamin I. We believe that this represents a significant
step forward in the development of specific dynamin
GTPase inhibitors. From the data presented herein it
is clear that inhibitory potency, for this class of com-
pounds, requires a dimeric compound which contains
two aromatic rings with these rings bearing 2× -OH
groups in the 3,4-positions; the presence of two free
amide -NH’s or an ester -O atom in the linker arm;
and, also of considerable import is the presence of the
-CN moieties.
OMe
OMe
OMe
COOH
H
H
-
H
OMe
OH
H
-
H
-
H
-
H
H
-
OH
H
H
H
-
OH
OH
OH
OH
H
H
-
H
OH
OH
OH
H
26 ( 2
OH
OMe
H
6.0 ( 2.0
80 ( 4
-
-
-
-
-
-
-
-
-
-
H
H
H
Cl
OMe
H
H
OMe
OMe
OMe
COOH
OH
OH
OH
H
H
OMe
OH
H
H
H
As yet it is still unknown as to how these compounds
are able interact with dynamin, although we have
preliminary evidence that these analogues are not GTP
competitive (Robinson and McCluskey, unpublished
observations). This study shows the first steps in
developing a model for what is required for these
compounds to interact with this enzyme.
OH
OH
OMe
H
OH
OH
a IC50 determinations conducted in triplicate; (-) >100 µM.
Our SAR studies also indicate that N-methylation of
one, 86, or both, 87, of the amide NH’s of 9 is detriment-

Small Molecule Inhibitors of Dynamin I GTPase Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7785
Table 2. Effect of Linker Modifications on Dynamin I GTPase Activity
a IC50 determinations conducted in triplicate; (-) >100 µM.
Scheme 2. Introduction of -OH (82), -OCH2CH2CH3 (83), and -OCH2Ph (84) into the Linker Units

7786 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Scheme 3. Synthesis of Ester Based Linker Analogues of 9a
McCluskey et al.
a Cf. cyano acetate linkers of Scheme 1.
Table 3. Effect of Introduction of Ester Linkers on Dynamin I GTPase Activity
a IC50 determinations conducted in triplicate; (-) >100 µM.
Scheme 4. Synthesis of Cyano Free Analogues of 9

Small Molecule Inhibitors of Dynamin I GTPase Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7787
117.8, 110.5, 107.8, 98.7, 55.7, 39.4, 28.6, 22.5. Anal. (C27H28N4-
O8): C, H, N.
The most promising inhibitor compound in our study
was2-cyano-N-{3-[2-cyano-3-(3,4,5-trihydroxyphenyl)acryl-
oylamino]ethyl}-3-(3,4,5-trihydroxyphenyl)acrylamide (10)
(IC50 ) 1.7 ( 0.2 µM).24 It will be of importance for
subsequent studies to directly test the hypothesis that
22 may be an inhibitor of endocytosis in a cellular
context.
2-Cyano-N-{3-[2-cyano-3-(3,4,5-trihydroxyphenyl)acryl-
oylamino]hexyl}-3-(3,4,5-trihydroxyphenyl)acrylamide
(62): synthesized as per 9 from 2-cyano-N-[3-(2-cyanoacetyl-
amino)hexyl]acetamide (5e)24 and 3,4,5-trihydroxybenzalde-
hyde; afforded a yellow solid, 67%, mp >300 °C.
1H NMR (DMSO): 8.11 (2H, t, J ) 5.5 Hz), 7.76 (2H, s),
6.98 (4H, s), 3.16 (4H, br s), 1.47 (4H, quin, J ) 6.1 Hz), 1.28
(4H, br s). 13C NMR (DMSO): 161.8, 150.5, 146.0, 141.2, 120.7,
117.5, 109.9, 99.2, 39.7, 28.8, 26.0. Anal. (C26H26N4O8‚
2.5H2O): C, H, N.
Experimental Section
General. All starting materials were purchased from Ald-
rich Chemical Co. and Lancaster Synthesis. Solvents were
bulk, and distilled from glass prior to use. 1H and 13C spectra
were recorded on a Bruker Advance AMX 300 MHz spectrom-
eter at 300.13 and 75.48 MHz, respectively. Chemical shifts
are relative to TMS as internal standard. All compounds
returned satisfactory analyses. The Experimental Section
details synthesis of previously unreported active analogues (10,
11, 24, 35, 50, 62, 82, 90, 91, and 92) only; all other ex-
perimental detail is located in the Supporting Information.
Synthetic Methods. 2-Cyano-N-{3-[2-cyano-3-(3,4,5-tri-
hydroxyphenyl)acryloylamino]ethyl}-3-(3,4,5-trihydroxy-
phenyl)acrylamide (10): synthesized as per 9 from 2-cyano-
N-[3-(2-cyanoacetylamino)ethyl]acetamide (5a)24 and 3,4,5-
trihydroxybenzaldehyde; afforded an orange solid, 82%, mp
>300 °C.
2-Cyano-N-{3-[2-cyano-3-(3,4-dihydroxy-5-methoxy-
phenyl)acryloylamino]hexyl}-3-(3,4-dihydroxy-5-meth-
oxyphenyl)acrylamide (63): synthesized as per 9 from
2-cyano-N-[3-(2-cyanoacetylamino)hexyl]acetamide (5e)24 and
3,4-dihydroxy-5-methoxybenzaldehyde; afforded a yellow solid,
86%, mp 243 °C.
1H NMR (DMSO): 8.17 (2H, t, J ) 5.5 Hz), 7.89 (2H, s),
7.19 (2H, d, J ) 1.6 Hz), 7.13 (2H, d, J ) 1.6 Hz), 3.77 (6H, s),
3.17 (4H, br s), 1.48 (4H, quin, J ) 6.1 Hz), 1.29 (4H, br s).
13C NMR (DMSO): 161.8, 150.5, 146.1, 146.0, 141.3, 120.7,
117.5, 109.9, 99.1, 39.5, 28.6, 22.3. Anal. (C28H30N4O8‚
0.75H2O): C, H, N.
2-Cyano-N-{3-[2-cyano-3-(3,4-dihydroxyphenyl)acryl-
oylamino]-2-hydroxypropyl)-3-(3,4-dihydroxyphenyl)-
acrylamide (82): synthesized as per 9 from 2-cyano-N-[3-(2-
cyanoacetylamino)-2-hydroxypropyl]acetamide (5j)24 and 3,4-
dihydroxybenzaldehyde (0.37 g, 2.7 mmol); afforded a yellow
solid, 56%, mp 255 °C.
1H NMR (DMSO): 8.29 (2H, t, J ) 5.5 Hz), 7.79 (2H, s),
6.99 (4H, s), 3.32 (4H, br s). 13C NMR (DMSO): 162.2, 150.7,
145.9, 140.2, 121.3, 117.3, 110.0, 99.8, 39.4. Anal. (C22H18N4O8):
C, H, N.
2-Cyano-N-{3-[2-cyano-3-(3,4-dihydroxy-4-methoxy-
phenyl)acryloylamino]ethyl}-3-(3,4-dihydroxy-5-meth-
oxyphenyl)acrylamide (11): synthesized as per 9 from
2-cyano-N-[3-(2-cyanoacetylamino)ethyl]acetamide (5a)24 and
3,4-dihydroxy-5-methoxybenzaldehyde; afforded an orange
solid, 66%, mp 274 °C.
1H NMR (DMSO): 7.9 (2H, t br), 7.86 (2H, s), 7.48 (2H, d,
J ) 2.0 Hz), 7.21 (2H, dd, J ) 8.4 Hz, 2.3 Hz), 6.68 (2H, d, J
) 8.4 Hz), 5.1 (OH, br), 3.72 (1H, quin, J ) 5.8 Hz), 3.25 (4H,
m). 13C NMR (DMSO): 162.5, 156.1, 150.5, 146.6, 127.2, 120.3,
118.1, 115.7, 113.7, 96.2, 68.0, 44.0, 22.2. Anal. (C23H20N4O7):
C, H, N.
1H NMR (DMSO): 8.34 (2H, t, J ) 5.5 Hz), 7.93 (1H, s),
7.20 (2H, d, J ) 1.92 Hz), 7.13 (2H, d, J ) 1.92 Hz), 3.77 (6H,
s), 3.35 (4H, br s). 13C NMR (DMSO): 161.9, 150.8, 148.0,
145.8, 139.9, 121.8, 117.2, 111.1, 107.2, 100.8. Anal. (C24H22N4O8‚
0.5H2O): C, H, N.
2-Cyano-3-(3,4-dihydroxyphenyl)acrylic acid 2-[2-cy-
ano-3-(3,4-dihydroxyphenyl)acryloyloxy]ethyl ester (90):
synthesized as per 9 from cyanoacetic acid 2-(2-cyanoacetoxy)-
ethyl ester (89a)28 and 3,4-dihydroxybenzaldehyde; afforded
a yellow solid, 73%, mp 232 °C.
1H NMR (DMSO): 8.11 (2H, t br), 7.63 (2H, d, J ) 2.1 Hz),
7.35 (2H, dd, J ) 8.4 Hz, 2.2 Hz), 6.87 (2H, d, J ) 8.3 Hz),
4.53 (4H, t br). 13C NMR (DMSO): 162.7, 155.1, 152.7, 145.8,
127.2, 122.6, 116.3, 116.2, 115.9, 95.7, 63.3. Anal. (C22H16N4O8):
C, H, N.
2-Cyano-N-{3-[2-cyano-3-(3,4-dihydroxy-5-methoxy-
phenyl)acryloylamino]propyl}-3-(3,4-dihydroxy-5-meth-
oxyphenyl)acrylamide (24): synthesized as per 9 from
2-cyano-N-[3-(2-cyanoacetylamino)propyl]acetamide (5b)24 and
3,4-dihyroxy-4-methoxybenzaldehyde; afforded an orange solid,
42%, mp >300 °C.
2-Cyano-3-(3,4-dihydroxyphenyl)acrylic acid 2-[2-cy-
ano-3-(3,4-dihydroxyphenyl)acryloyloxy]propyl ester (91):
synthesized as per 9 from cyanoacetic acid 2-(2-cyanoacetoxy)-
propyl ester (89b)28 and 3,4-dihydroxybenzaldehyde; afforded
a yellow solid, 61%, mp 195 °C.
1H NMR (DMSO): 8.35 (2H, t, J ) 5.4 Hz), 7.95 (2H, s),
7.21 (2H, d, J ) 1.9 Hz), 7.12 (2H, d, J ) 1.9 Hz), 3.21 (4H, q,
J ) 6.8 Hz), 1.71 (2H, quin, J ) 6.8 Hz). 13C NMR (DMSO):
161.3, 150.6, 147.2, 145.3, 121.0, 117.6, 110.6, 107.6, 98.7, 38.4,
28.9. Anal. (C25H24N4O8‚2H2O): C, H, N.
1H NMR (DMSO): 8.08 (2H, t br), 7.61 (2H, d, J ) 2.0 Hz),
7.35 (2H, dd, J ) 8.4 Hz, 2.1 Hz), 6.82 (2H, d, J ) 8.3 Hz),
4.32 (4H, t br), 1.63 (2H, quin br). 13C NMR (DMSO): 162.6,
155.3, 152.2, 145.6, 127.3, 122.6, 116.3, 116.2, 115.9, 95.4, 65.3,
24.2. Anal. (C23H18N4O8): C, H, N.
2-Cyano-N-{3-[2-cyano-3-(3,4-dihydroxy-5-methoxy-
phenyl)acryloylamino]butyl}-3-(3,4-dihydroxy-5-meth-
oxyphenyl)acrylamide (37): synthesized as per 9 from
2-cyano-N-[3-(2-cyanoacetylamino)butyl]acetamide (5c)24 and
3,4-dihydroxy-5-methoxybenzaldehyde; afforded a yellow solid,
70%, mp >300 °C.
2-Cyano-3-(3,4-dihydroxyphenyl)acrylic acid 2-[2-cy-
ano-3-(3,4-dihydroxyphenyl)acryloyloxy]butyl ester (92):
synthesized as per 9 from cyanoacetic acid 2-(2-cyanoacetoxy)-
butyl ester (89c)28 and 3,4-dihydroxybenzaldehyde; afforded
a yellow solid, 48%, mp 252 °C.
1H NMR (DMSO): 8.09 (2H, t, J ) 5.5 Hz), 7.86 (2H, s),
7.18 (2H, d, J ) 1.9 Hz), 7.10 (2H, d, J ) 1.9 Hz), 3.75 (6H, s),
3.19 (4H, br s), 1.48 (4H, br s).
13C NMR (DMSO): 161.7, 150.2, 148.7, 146.2, 120.3, 117.5,
109.5, 106.8, 98.8, 55.8, 39.3, 26.6.
1H NMR (DMSO): 8.10 (2H, t br), 7.62 (2H, d, J ) 2.0 Hz),
7.35 (2H, dd, J ) 8.5 Hz, 2.0 Hz), 6.85 (2H, d, J ) 8.5 Hz),
4.28 (4H, t br), 1.79 (4H, quin br). 13C NMR (DMSO): 162.8,
154.7, 152.9, 145.9, 127.2, 122.4, 116.4, 116.0, 115.9, 95.7, 65.2,
24.6. Anal. (C24H20N4O8): C, H, N.
Anal. (C26H26N4O8): C, H, N.
2-Cyano-N-{3-[2-cyano-3-(3,4-dihydroxy-5-methoxy-
phenyl)acryloylamino]pentyl}-3-(3,4-dihydroxy-5-meth-
oxyphenyl)acrylamide (50): synthesized as per 9 from
2-cyano-N-[3-(2-cyanoacetylamino)pentyl]acetamide (5d)24 and
3,4-dihydroxy-5-methoxybenzaldehyde; afforded a yellow solid,
81%, mp 256 °C.
GTPase Assay. Dynamin I was purified from sheep brain
as previously described.6 Dynamin I GTPase activity was
determined by hydrolysis of GTP by a method modified from
that used previously.29 Purified dynamin I (0.5 µg/well; 200
nM) was incubated in GTPase buffer (10 mM Tris, 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 12 min at 30 °C. The final assay volume was 40
1H NMR (DMSO): 8.09 (2H, t, J ) 5.5 Hz), 7.86 (2H, s),
7.18 (2H, d, J ) 2 Hz), 7.10 (2H, d, J ) 2 Hz) 3.75 (6H, s),
3.17 (4H, br s), 1.50 (4H, quin, J ) 6.8 Hz), 1.28 (4H, quin, J
) 6.9 Hz). 13C NMR (DMSO): 161.8, 150.5, 148.0, 146.2, 120.1,

7788 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
McCluskey et al.
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Acknowledgment. We are grateful for financial
support from the National Health & Medical Research
Council (Australia), the Children’s Medical Research
Institute (CMRI), and the University of Newcastle (UN).
T.H., L.R.O., and J.K.E. gratefully acknowledge scholar-
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Centre for Green Chemistry, respectively.
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Supporting Information Available: Table of microanal-
ysis data, experimental details, effects of analogues of 2 on
dynamin I GTPase activity, and HPLC and MS study of
degradation of 1. This material is available free of charge via
the Internet at http://pubs.acs.org.
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