The pthaladyns: GTP competitive inhibitors of dynamin I and II GTPase derived from virtual screening

Article abstract of DOI:10.1021/jm100442u

We report the development of a homology model for the GTP binding domain of human dynamin I based on the corresponding crystal structure of Dictyostelium discoidum dynamin A. Virtual screening identified 2-[(2-biphenyl-2-yl-1,3-dioxo- 2,3-dihydro-1H-isoin

Full text of DOI:10.1021/jm100442u

J. Med. Chem. 2010, 53, 5267–5280 5267
DOI: 10.1021/jm100442u
The Pthaladyns: GTP Competitive Inhibitors of Dynamin I and II GTPase Derived
from Virtual Screening
Luke R. Odell,†, Dian Howan,† Christopher P. Gordon,† Mark J. Robertson,† Ngoc Chau,‡ Anna Mariana,‡
Ainslie E. Whiting,‡ Ruben Abagyan,§ James A. Daniel,‡ Nick N. Gorgani,‡ Phillip J. Robinson,‡ and Adam McCluskey*,†
†Chemistry, 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, and §Department of Molecular Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, TCP-28, La Jolla, California 92037. Current address: Organic Pharmaceutical
Chemistry, Department of Medicinal Chemistry, Uppsala Biomedical Centre, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden.
Received April 10, 2010
We report the development of a homology model for the GTP binding domain of human dynamin I
based on the corresponding crystal structure of Dictyostelium discoidum dynamin A. Virtual screening
identified 2-[(2-biphenyl-2-yl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl)amino]-4-chlorobenzoic
acid (1) as a ∼170 μM potent inhibitor. Homology modeling- and focused library-led synthesis resulted
in development of a series of active compounds (the “pthaladyns”) with 4-chloro-2-(2-(4-(hydro-
xymethyl)phenyl)-1,3-dioxoisoindoline-5-carboxamido)benzoic acid (29), a 4.58 ( 0.06 μM dynamin I
GTPase inhibitor. Pthaladyn-29 displays borderline selectivity for dynamin I relative to dynamin II (∼5-10
fold). Only pthaladyn-23 (dynamin I IC50 17.4 ( 5.8 μM) was an effective inhibitor of dynamin I mediated
synaptic vesicle endocytosis in brain synaptosomes with an IC50 of 12.9 ( 5.9 μM. This compound was also
competitive with respect to Mg2þ GTP. Thus the pthaladyns are the first GTP competitive inhibitors of
3
dynamin I and II GTPase and may be effective new tools for the study of neuronal endocytosis.
Introduction
potential drug targets within a molecule which has multiple
domains, and the lack of a published structure to guide such
efforts. No full dynamin crystal structure has been published,
but thereare now two knownstructuresof individualdomains
(below). The three classical dynamins, dynamins I, II, and III,
are large GTPases of approximately 100 kDa. All members
of the dynamin superfamily include a GTPase domain, a
GTPase effector domain (GED), and a middle domain, while
some include a transmembrane region. The classicaldynamins
differ from others by two additional structural domains; a
pleckstrin homology (PH) domain and a C-terminal proline-
rich domain (PRD). Each of these five primary domains has
distinct properties allowing dynamin to have multiple func-
tions and subcellular locations.
The PH domain binds lipids such as phosphoinositide bis-
phosphate (PtdIns(4,5)P2 or PIP2) and is essential for dyna-
min localization and can promote a massive increase in
GTPase activity by acting as a template for dynamin helical
self-assembly.10,11 Both X-ray and NMR approaches have
been used to solve the structure of this domain.12-16 The PH
domain of dynamin I comprises a seven-stranded β-sheet
sandwich, three variable loops, and a C-terminal R-helix. Site
directed mutagenesis highlights K535 in variable loop 1 and
variable loop 3 as essential for lipid binding and transfer-
rin endocytosis, and the K558E mutation in variable loop 3
causes the human neurodegenerative disease Charcot-Marie-
Tooth.17 Thus the variable loops in the PH domain play pivotal
roles in mediating dynamins lipid and membrane interactions.
The middle domain displays no homology with any known
structural motif. Little is known about its role or func-
tion, although recent evidence indicates that middle domain
Our laboratory has reported the identification and devel-
opment of a number of discrete families of small molecule
inhibitors of the large GTPase, dynamin I. The MiTMABs,a,1,2
the Bis-Ts,3,4 the RTILs,5 the dynoles,6 and most recently, the
iminodyns exemplify these inhibitor families.7 Other groups
have reported dynasore,8 and some selective serotonin reuptake
inhibitors as dynamin inhibitors.9 None of these compounds
bind to the active site (i.e., none are Mg2þ GTP competitive).
3
Without exception, all inhibitors have been discovered by
biological assay-directed screening of chemical libraries, and
in those instances where lead development has ensued, synth-
esis has been via focused library development.
To date, there has been no computer assisted molecular
modeling-led development of dynamin inhibitors. This is in
part due to the complex nature of dynamin, the multiple
*To whom correspondence should be addressed. Phone: þ61 249
216486. Fax: þ61 249 215472. E-mail: Adam.McCluskey@newcastle.
edu.au.
a Abbreviations: RME, receptor mediated endocytosis; SVE, synap-
tic vesicle endocytosis; CME, clathrin-mediated endocytosis; GED,
GTPase effector domain; PRD, proline rich domain; PH, Pleckstrin
homology; SH3, Src homology 3; CMT, Charcot-Marie-Tooth dis-
ease; CNM, centronuclear myopathy; MiTMAB, myristoyl trimethyl
ammonium bromide; OcTMAB, octyl trimethyl ammonium bromide;
Bis-T, Bis-tyrphostin; RTIL, room temperature ionic liquid; SAR,
structure-activity relationship; EGFR, epidermal growth factor recep-
tor; Tf-TxR, Texas Red-Tf; U2OS, human bone osteosarcoma epithelial
cells; PS, phosphatidylserine; PMSF, phenylmethylsulfonylfluoride;
PFA, paraformaldehyde; FCS, fetal calf serum; DMEM, Dulbecco’s
Modified Eagle Medium; DMSO, dimethylsulfoxide; IXM, Image
Xpress Micro; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane sulfonic
acid; FM 4-64, N-(3-triethylammoniumpropyl)-4-(4-diethylaminophe-
nylhexatrienyl)pyridinium dibromide.
r
2010 American Chemical Society
Published on Web 06/24/2010
pubs.acs.org/jmc

5268 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Odell et al.
mutations in dynamin II may play a role in dominant centro-
nuclear myopathy, a degenerative muscle wasting disease.18
The middledomainof dynaminIIhas also been shown tobind
γ-tubulin, suggesting a role in centrosome cohesion.19
The GTPase domain is the site of GTP binding and
hydrolysis and is the most highly conserved domain in the
superfamily. It is characterized by an unusually low affinity
for GTP (10-25 μM), high rates of hydrolysis (1-20 min-1),
and extremely high turnover rates relative to other families of
GTPases.20,21 Dynamin also has the unique ability (among
GTPases) to self-assemble into macromolecular entities that
stimulate basal GTPase activity up to 1000-fold.10 The dyna-
min superfamily GTPase domain structure grossly resembles
that of other GTPases, with an eight-stranded β-sheet with six
parallel and two antiparallel strands surrounded by nine
helices.22
virtual screening program to identify new dynamin inhibitors.
The model can then be used to guide the subsequent rational
design of more potent inhibitors. This is not a new approach,
with known crystal structures and cocrystals virtual library
screening becoming an increasingly important tool in the
discovery of novel and diverse lead structures.35,36 Virtual
library screening has recently successfully identified new lead
compounds for a host of different biological targets including:
protein kinase inhibitors,37 reverse transcriptase of human im-
munodeficiency virus type-1,38 glutamate receptor agonists,39
dengue virus methyltransferase,40 proteasome inhibitors,41 and
falcipain-2 inhibitors as potential antimalarial agents.42 We
sought to utilize a structure based screening approach in
the discovery of novel dynamin inhibitors that may target the
nucleotide binding pocket for the first time. Herein, we describe
the generation and validation of a homology model of the
human dynamin I GTPase domain and the subsequent library
development of novel dynamin inhibitors that we have called
the “pthaladyns”.
Our interest in dynamin stems from the need to understand
dynamin biology and recognition of the potential therapeutic
implications associated with inhibiting a protein that is essen-
tial to the endocytotic machinery.23-25 All cells endocytose, it
is the process whereby key cellular materials are taken up, the
process by which synaptic vesicles are recycled; it is crucial for
cell, and thus host survival.26 But the molecular machinery
deployed by cells to internalize plasma membrane varies
according to task, which can range from nutrient absorption
to cell signaling. Dynamin does not mediate all forms of endo-
cytosis, but its action includes clathrin-mediated endocytosis
(CME). Clathrin-mediated endocytosis is a dynamic biologi-
cal process requiring logistically complex assembly and utili-
zation of numerous specialized protein and lipid complexes.
Synaptic vesicle endocytosis (SVE) is a neuronal endocytic
specialization that facilitates the recovery of synaptic vesicles
after membrane fusion and neurotransmitter release (exo-
cytosis) and involves at least two dynamin-dependent modes,
CME and bulk endocytosis.27 Receptor mediated endocytosis
(RME) is initiated on activation of surface bound receptors
(via endogenous ligand binding) and is the primary vehicle for
cells to uptake membrane material.28 Of the known forms
of endocytosis, clathrin-induced membrane deformation to
form nascent vesicles is the most fully understood.29 Clathrin
typically recruits membrane proteins (multimeric adaptor
proteins, AP-2).30 This assists in the development of an almost
fully closed invaginated vesicle but requiring the protein
dynamin to detach the vesicle from the membrane. In this
final step, GTP hydrolysis by dynamin appears to supply the
energy to release the vesicle from the membrane. Dynamin is
involved atthis point by targeting to sites of endocytosis via its
PH domain and then stimulating its GTPase activity.31 There
are numerous potential avenues for clinical application of
dynamin inhibitors in the future. One possibility is their use as
potential antiepileptic drugs due to their ability to produce a
run-down in synaptic transmission as a result of inhibiting
SVE.32 Their ability to block the function of dynamin II in
cytokinesis raises the possibility of their application as new
antimitotic agents.33 Their ability to block HIV entry into
cells implies a potential future application in the treatment of
infectious diseases.34
Results and Discussion
It is well established that reasonable structural models can
be constructed when a 3D template and target protein share
greater than 30% sequence identity.43 A pairwise sequence
alignment between the GTPase domains of dynamin I and
dynamin A revealed a 64% sequence identity. Dynamin I, like
all GTPases, has four consensus elements (G1-G4) which are
involved in nucleotide binding. The sequence identity between
the two proteins reaches 89% when the 18 residues forming
the nucleotide-binding site are examined.44 Consequently, we
were confident that an accurate structural model for the
dynamin I GTPase domain that included the nucleotide
binding pocket could be constructed utilizing dynamin A as
a template. The G2 motif, a Mg2þ coordinating flexible loop,
was not resolved in the crystal structure of dynamin A and
represented a potential limitation in the construction of our
dynamin I homology model. Notwithstanding this, other
crucial residues were expected to allow excellent alignment
and hence we anticipated that a homology model of the
GTPase domain of dynamin I suitable for ligand database
screening would eventuate.
We constructed seven different models via ICM homology
(ICM v3.1) based on the GDP bound structure of the
dynamin A GTPase domain (PDB code 1JWY) (for details
see Experimental Section).45 To include ligand information in
our models, GTPwas docked onto a grid representationof the
nucleotide-binding site and the best solutions refined using a
full atom representation of the binding site. GTP and GDP
were then redocked onto a grid representation of the refined
nucleotide-binding sites, and their ICM scores were evaluated
(Table 1). The ICM-score takes into account the ligand-
receptor interaction energy, conformational strain energy of
the ligand, conformational entropy loss, and desolvation
effects.46 The score reflects the overall quality of the ligand-
receptor complex witha more negative scoreindicatingamore
favorable interaction (Table 1). Two of the proposed struc-
tures (hDyn1-3 and hDyn1-7) showed greater predicted
binding affinity than the other models (e.g., GTP -79.13
and -79.87, respectively, vs -68.76). In addition, their ICM
scores were lower for GDP than GTP (-75.55 and -71.51
vs -79.13 and -79.87, respectively), consistent with dyna-
min’s known lower affinity for GDP.47 Accordingly, thesetwo
structures were selected for further validation studies.
While we have discovered and developed a large “palette”
of small molecule inhibitors of dynamin that target distinct
domains,1-7 none yet display GTP competitive kinetics,
indicative of binding at the active site of dynamin. Therefore
our goal is to impart our dynamin modulators program with a
degree of rational drug design by development of a dynamin
GTPase domain homology model with a view to conducting a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5269
Table 1. Homology Model Representations and Their ICM Scores for
the Interaction with GDP and GTP
ICM score
model
GDP
GTP
hDyn1-1
hDyn1-2
hDyn1-3
hDyn1-4
hDyn1-5
hDyn1-6
hDyn1-7
-60.87
-64.85
-75.55
-67.60
-69.08
-65.32
-71.51
-29.75
-39.95
-79.13
-68.76
-73.39
-49.17
-79.87
Figure 2. Backbone structure of homology models hDyn1-3 and
R
˚
hDyn1-7 (red) superimposed (C rmsd 1.09 A) on the rat dynamin I
GTPase domain crystal structure (green, PDB code 2AKA).
Figure 1. Binding modes of GDP predicted using hDyn1-3 (red)
and hDyn1-7 (green) relative to the conformation found in the
dynamin A crystal structure (blue, PDB code 1JWY).
within a drug design program, essentially allowing a medicinal
chemist to custom build ligands, a significant limitation of
currently available molecular docking software is that the
enzyme effectively remains static throughout a simulation.
Given the dynamic ligand-enzyme binding interactions that
typically occur in vivo, an understanding of the potential
movements and flexibility of the target enzyme is extremely
valuable in the assessment and validation of docking results.
To assess potential flexibility of the dynamin, GTPase-GDP
cocrystallized structure (1JWY) and the ligand free structure
(1JX2) were compared utilizing the PROFLEX program.
This revealed that the conformation of the crystal structures
To validate our models, GDP was docked in the modeled
structures using the ICM flexible ligand-grid receptor algo-
rithm and the predicted ligand pose was compared to the
crystal structure conformation. Each of the models was able
to effectively reproduce the crystal structure conformation
˚
with rmsd’s of 0.69 and 0.77 A for hDyn1-3 and hDyn1-7,
respectively (rmsd <2 A is considered accurate), thus
48
˚
illustrating the accuracy of our predicted binding site repre-
sentations (Figure 1).
The stereochemical properties of the hDyn1-3 and
hDyn1-7 models were assessed and found to be within
acceptable limits (PROCHECK).49 The Ramachandran plot
showed that, in both models, 84.5% of the residues are located
within the core region, 13.2% in the allowed and 2.3% in the
generously allowed regions. Additionally, checks for bond
lengths, side chain torsions, amide torsions, and backbone
torsions showed no major deviations from the allowed values.
Only 5.2% of residues showed bond angles with higher than
expected values (>4.4 standard deviations), however, these
residues were not located within the nucleotide-binding site
and accordingly were deemed less relevant to this study. These
analyses show that our homology models are not only con-
sistent with current understanding of protein structure but are
also able to accommodate GTP in a native conformation.
During the completion of this work, the apo crystal struc-
ture of the GTPase domain of Rattus norvegicus dynamin I
was solved and this protein shares a 99.3% sequence identity
with the human dynamin I GTPase domain.50 Thus, our
modeled structures could be validated retrospectively by
comparison to the solved structure (Figure 2). hDyn1-3
˚
deviated significantly (>8 A) in two regions, specifically the
side-chains of residues 152-156 and 252-254 (see Supporting
Information). Consequently, docked conformations in close
proximity to either of these flexible regions would need to be
assessed with a degree of caution.
Flexible ligand and receptor docking is still far too compu-
tationally expensive to be considered viable for large database
screening, e.g. ∼800000 compounds (ChemBridge, San Diego,
CA, and ChemDiv, San Diego, CA). To achieve this goal in a
reasonable time frame and to account for some receptor
flexibility, we used both homology model representations
(hDyn1-3 and hDyn1-7) and the ICM flexible ligand-grid
receptor algorithm for virtual ligand screening. The flexible
ligands passing a relaxed Lipinksi filter (MW<600, HBA<12,
and HBD < 6) and containing less than seven rotatable bonds
were docked into grid receptor field and ranked according to
the scoring function implemented in ICM.51 The solutions from
each receptor were merged and the top ranked 1000 com-
pounds were considered potential inhibitors and clustered into
diverse sets according to chemical similarity. Each compound,
docked into the nucleotide-binding site, was visually inspected,
and those displaying unfavorable interactions within the bind-
ing site or significant interactions with the G2 consensus region
were discarded. To ensure diversity in the final compound
selection, only 1-2 compounds from each cluster were retained.
Finally, 300 compounds were requested from the vendors,
of which 175 were still available for biological evaluation. The
available compounds were submitted for initial biological
R
˚
and hDyn1-7 have rmsd’s of 1.09 A for C atoms and 1.74
˚
and 1.79 A for all heavy atoms, respectively, when compared
to the crystal structure. Furthermore, the GTP binding resi-
dues in hDyn1-3 and hDyn1-7 have heavy atom rmsd’s of
0.71 and 0.70 A, respectively, when compared to the same
˚
residues in the crystal structure. These results confirmed the
accuracy of our models and their relevance in further ligand
docking studies. Although these structures are invaluable

5270 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Odell et al.
evaluation against in vitro dynamin I GTPase activity at
two concentrations, 30 and 300 μM using methods des-
cribed previously.1 Compound 1 (Figure 3) was identified as
a ∼170 μM inhibitor of the GTPase activity of full length
endogenous sheep brain dynamin I.
Second, the 2-phenyl substituent is largely solvent exposed
and makes only minimal van der Waals interactions with
Leu209. Thus, its removal was not expected to significantly
reduce potency, allowing a straightforward examination of
the effect of additional aromatic ring substituents via a raft of
simple commercially available anilines. Moreover, the binding
site residues in this region (Asp208, Asn236, Arg237, Ser238,
and Gln239) contain a number of potential hydrogen and
ionic bond forming groups (CONH, OH, CONH2, NH2,
C(NH)2NH2, COOH), which may interact with any comple-
mentary groups (i.e., OH, NH2, COOH) introduced into our
analogues (Figure 4). Therefore, we decided to prepare two
discrete libraries of analogues to explore these factors, con-
current with the synthesis of 1.
The strategy used in the synthesis of Library A, which
explores the 1,2-amidobenzoic acid moiety is illustrated in
Scheme 1. Synthesis commenced with the treatment of an
excess of trimellitic anhydride chloride with the appropriately
substituted aniline at 0 °C, to minimize the competing anhy-
dride reaction.52 Compounds 5 and 7 could be isolated as the
desired acid chloride adducts via crystallization and basic
aqueous workup, respectively, however 4, 6, and 8 could not
be purified in this manner and decomposed (presumably via
anhydride opening) when flash column chromatography was
attempted. Consequently, these compounds were used in the
next step, without purification, as crude mixtures containing
Considering dynamins’ affinity for GTP is 10-25 μM the
level of potency displayed by compound 1 was encouraging.21
While only a moderate dynamin inhibitor, the structural
simplicity and ease of access to discrete libraries appealed,
soweset aboutthe development of1 asa potentialnew class of
dynamin 1 GTPase inhibitor. Retrosynthetic analysis of 1
revealed that preparation could occur via two straightforward
condensation reactions from commercially available precur-
sors (Figure 4). The inherent simplicity of this process struck
us as amenable to rapid analogue development and the
generation of SAR data. Moreover, the predicted binding
mode of 1 in the active site could be used to guide analogue
development.
Compound 1 fits wellintothe GTPbindingsiteofdynamin,
showing several hydrogen bonding, ionic, and van der Waals
interactions with the active site residues (Figure 5). The acid
group forms a salt bridge with the Lys44 side chain and also
accepts hydrogen bonds from the backbone amide hydrogens
of Ala42, Gly43, and Lys44. The amide carbonyl acts as a
hydrogen bondacceptor withthe Ser45 -OH, whilethe amide
hydrogen donates a hydrogen bond to the Ser45 oxygen. In
addition, the aromatic ring makes van der Waals contacts
with the Ile63 side chain. The imide carbonyls can accept
hydrogen bonds from the side chain hydrogens in Glu239 and
Ser46. The phenyl group adjacent to the imide appears to
make van der Waals interactions with the Lys206 and Val235
side chains. The additional 2-phenyl substituent appears to
make van der Waals contacts with the Leu209 side chain.
This fitting revealed two factors pertinent to analogue
development. First, the 1-amido-2-benzoic acid moiety makes
a number of significant hydrogen bonding and ionic interac-
tions within the binding site. These interactions appear to
mimic that of phosphate groups in GTP and GDP, suggesting
they may be crucial to the activity exhibited by compound 1.
Figure 5. Predicted binding pose of compound 1 within the binding
site of hDyn1-3. Color codes: oxygen, red; nitrogen, blue; hydro-
gen, gray; chlorine, green (for clarity only polar binding site hydrogen
atoms are shown). Carbon atoms are shown in white (hDyn1-3) and
yellow (1). Hydrogen bonds are shown as yellow dotted lines with atom
Figure 3. Chemical structure of pthaladyn-1 identified by virtual
screening of the Chembridge and ChemDiv screening databases.
˚
distances in A.
Figure 4. Retrosynthetic analysis of pthaladyn-1 showing that preparation can occur via two condensation reactions from commercially
available precursors.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5271
Scheme 1a
a Reagents and conditions: (i) THF, 0 °C, 3 h; (ii) 2-phenylaniline, DMF, toluene, TEA, 130 °C, 18 h.
Table 2. Inhibition of Dynamin I GTPase Activity by Pthaladyns 1,
and 9-12
Scheme 2a
a Reagents and conditions: (i) RNH2, DMF, toluene, TEA, 130 °C,
18 h.
that removal of the second phenyl ring would generate a
pthaladyn with reduced activity but with sufficient space for
later introduction of a range of substituents capable of better
engagement with the aforementioned residues while at the
same time removing the noted van der Waals interactions.
Closer examination of this cavity reveals the presence of
Asp208 that might be accessible with the appropriate choice
of aromatic substituent, as are Asn236 and Ser238 on the
cavity periphery.
Treatment of phthalic anhydride (7) with the appropriate
aniline (Scheme 2, and see Table 3 for details) in the presence
of TEA at 130 °C afforded the desired Library B constituents,
13-33 in low (33: 7%) to excellent (18: 84%) yields.
Synthesis and evaluation of the “naked” aniline analogue
(13) gave, as anticipated, reduced biological activity (Table 3).
With the compound skeleton in place, we rationalized that the
Asp208 carboxylate was in sufficiently close proximity to
interact with an amino moiety, thus we synthesized the 2-, 3-,
and 4-NH2 pthaladyn analogues 14-16, respectively. Intro-
duction of, in particular, the 3-NH2 (15) gave rise to a signi-
ficant increase in dynamin inhibition with an IC50 value of
94 μM (Table 3). Both the 2- and 4-NH2 analogues (14, 16)
returned significantly lower levels of dynamin inhibition,
returning IC50 values of 311 ( 28 and >400 μM, respectively.
On the basis of these findings, a series of simple bioisosteric
replacements were undertaken. The first resulted in the intro-
duction of an -OH moiety, which was expected to hydrogen
bond with Asp208. Gratifyingly, we note an increase in dyna-
min inhibition with the 2- and 4-OH analogues (17 and 19 with
IC50 values of 61 ( 30.0 and 40 ( 10.0 μM, respectively). This
increase in dynamin I inhibition was >10 fold relative to 1 and
can be attributed, in our model, to an additional hydrogen
bond interaction, with the Asp208 side chain carbonyl accept-
ing a hydrogen bond from the inhibitor OH group. Our
preliminary modeling analysis suggests that the Asp208 side
chain carbonyl accepts a hydrogen bond from 19’s 4-OH
group. “Methylation” of 18’s and 19’s phenolic OH gave 20
and 21, of which only the 4-OCH3 analogue (21) retained, albeit
at a reduced level, dynamin inhibition with an IC50 value of
no.
R1
R2
R3
IC50 (μM)
>400a
1
COOH
H
H
H
H
H
I
Cl
H
Cl
H
H
9
10
11
12
H
COOH
COOH
a
∼40% inhibition at 400 μM.
ca. 80-90% (by 1H NMR) of the desired acid chloride
adducts. Treatment of 4-8 with biphenyl-2-ylamine in the
presence of triethylamine (TEA) at 130 °C afforded the
desired 1, 9-12 in moderate (40-70%) yields. We call this
series the “pthaladyns” - phthalimide-based inhibitors of
dynamin.
Library A comprised of five analogues which were evaluated
for their ability to inhibit dynamin and this data is presented in
Table 2. The most striking feature of Table 2 is that our in-
house synthesized version of 1 displayed significantly weaker
inhibitory activity than the vendor supplied compound (IC50
∼ 170 vs >400 μM (40% inhibition at 400 μM)). This may be
the result of an error in the original assay or an error relating
to vendor supply. Since completion of this work, we have
obtained another sample of 1 from the vendor and its IC50
correlated well with our synthesized version (IC50 for both
>400 μM). While a poor lead, 1 was still predicted to have a
GTP competition mechanism of action against dynamin
(Figure 5), providing us with a compelling reason to continue
with molecular modelling-led focused library generation. To
date, no GTP competitive inhibitors of dynamin have been
reported. Additionally, the SAR is consistent with the pre-
dicted role of the carboxylic acid in mediating active site
binding and suggests that an electron-withdrawing group,
preferably para to the acid, is also beneficial for inhibition.
We next set about developing SAR associated with the biaryl
moiety via modeling guided synthesis of Library B. In our
initial modeling analysis, we noted that the biphenyl moiety of
1 is encapsulated within a cavity defined by Lys206, Val235,
and Leu209 (Figure 5). From these interactions, we believed

5272 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Odell et al.
Table 3. Inhibition of Dynamin I GTPase Activity by Pthaladyns
13-33
no.
R1
R2
R3
IC50 (μM)
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
H
H
H
H
a
NH2
H
H
311 ( 28
94
NH2
H
H
H
NH2
H
a
OH
H
H
61 ( 30
a
OH
H
H
H
OH
H
39.8 ( 10.0
a
Figure 6. (a) Overlay of the predicted binding poses of compounds
1, 26, and 29 in the hDyn1-3 model. The Asp208 side chain has
twisted and rotated to accommodate the bulkier acid and methanol
groups in 26 and 29. The original position is shown in green and the
new position in red. (b) Predicted binding interactions of the acid
group in compound 26. (c) Predicted binding interactions of the
CH2OH group in compound 29. Color codes: oxygen, red; nitrogen,
blue; hydrogen, gray (for clarity only polar binding site hydrogens
are shown). Carbon atoms are shown in white (hDyn1-3) and
yellow (inhibitors). Hydrogen bonds are shown as yellow dotted
H
OCH3
H
H
OCH3
SCH3
H
78 ( 10.0
a
H
H
H
NO2
H
17.4 ( 5.8
16.6 ( 7.0
184 ( 84
95
H
NO2
H
CO2H
H
H
H
CO2H
H
CH2OH
H
H
a
111
CH2OH
H
H
CH2OH
CO2Et
H
4.58 ( 0.06
b
˚
lines with atom distances in A.
H
H
H
H
CH2CO2H
CH2CH2CO2H
CO2H
95.5 ( 17.5
104
moiety would afford better alignment of the Asp208 -CO2H
to better hydrogen bond with 29’s 4-CH2OH. A significant
improvement in potency was observed, with 29 displaying an
IC50 value of 4.58( 0.06 μM, as predicted from our homology
model. The positional preference for substituents at the
4-position of the aniline moiety is apparent with the CH2OH
analogues, with the 2-CH2OH (27) inactive and 3-CH2OH
(28) significantly less active with an IC50 value of 111 μM. The
observed activity increase, by modeling analysis, is a result of
the engagement of Ser238’s -OH moiety by 29. Pthaladyn
29’s -OH group is also predicted to form two additional
hydrogen bonds within the binding site where the Asn236
backbone carbonyl acts as a hydrogen bond acceptor and the
Ser238 -OH acts as a hydrogen bond donor. Additional van
der Waals contacts are also observed between the Val235 side
chain and 29’s CH2 group (Figure 6C).
The ability to accommodate bulkier groups in close proxi-
mity to Asp208 is also evident, with essentially no reduction in
dynamin inhibition with the 4-CH2CO2H(31) and4-CH2CH2-
CO2H (32) analogues with IC50 values of 95.5 ( 17.5 μM and
104 μM, respectively. In this instance, 32’s predicted binding
pose is similar to that observed with 26 and 28 with efficient
engagement of the additional hydrogen bonds associated with
Ser238 and Asn236 (not shown). In contrast, the introduction
of a 4-CO2Et moiety removes all activity with 30, presumably
a consequence of additional steric bulk.
H
H
OH
H
267 ( 0.1
a No activity observed at 400 μM. b ∼50% inhibition at 300 μM.
78 ( 10.0 μM. This is consistent with the removal of the hydro-
gen bond donating capability of an -OH but retention of the
hydrogen bond accepting capability. The 4-SCH3 analogue
(22) is inactive.
Next, we turned our attention to the possibility of better
engaging additional residues on the periphery of the binding
cavity and rationalized that the introduction of a -NO2
moiety may allow hydrogen bond donation from Asp208
and also interactions with Asn236 and possibly Ser238. Both
the 3-NO2 (23), and 4-NO2 (24) returned the highest levels
of dynamin inhibition thus far observed with IC50 values of
17.4 ( 5.8 and 16.6 ( 7.0 μM, respectively. Interestingly, the
2-CO2H (25) and 4-CO2H (26) displayed disappointing levels
of dynamin inhibition, with IC50 values of 184 ( 84 and
95 μM, respectively. This is presumably due to the more
restricted matching conformation required to properly align
both the pthaladyn -CO2H and the Asp208 -CO2H moi-
eties. Indeed, re-examination of the binding pose of 26 clearly
shows that the Asp208 -CO2H twists so as to accommodate
26’s -CO2H moiety but in doing so the extent of favorable
overlap is reduced, leading to a concomitant reduction in
potency (Figure 6A,B). Pthaladyn 26’s -CO2H moiety is
predicted to accept hydrogen bonds from the Asn236 back-
bone amide proton and the Ser238 side chain OH (Figure 6B).
It is possible that these additional binding site contacts
counteract the impact of a reduction in interaction with
Asp208.
Validation of the homology model by examination of the
analogues predicted to be active should be strongly supported
by the bioevaluation of analogues predicted to have reduced
activity. Therefore, within Library C, we sought to explore the
effect of modifications that increased the steric bulk of the
pendent aromatic group. Given our findings relating to steric
bulk and the ability of the Asp208 residue to rotate to accom-
modate bulkier groups and the potential favorable van der
Waals contacts with Val235, we synthesized Library C in which
To better engage with Asp208 (Figure 6A), we sought to
extend the H-bonding moiety closer to this amino acid, as we
have seen that direct interaction results in a rotation of
Asp208, but believed that the increased bulk of the CH2

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5273
Table 4. Inhibition of Dynamin I GTPase Activity by Pthaladyns
34-42
of dynamin inhibition. Synthesis and subsequent screening
showed 42 to be an 84 ( 8.51 μM dynamin inhibitor.
These outcomes strongly suggest that our homology model
is valid and is a useful tool for the synthesis of more potent
dynamin inhibitors.
Given that dynamin GTPase activity is essential for en-
docytosis, we next determined the ability of four of our most
potent pthaladyns 17, 19, 23, and 29 to block in-cell endocy-
tosis. We first used our well established automated quantita-
tive RME assay based on endocytosis of Texas Red-Tf (Tf-
TxR) into U2OS human osteosarcoma cells.6,7 After a 30 min
preincubation in the presence of increasing concentrations of
pthaladyns, 17, 19, 23, and 29, RME was unaffected (Table 5).
This was surprising and is the first such incidence where we
have noted thatall the dynamin activeanalogues evaluatedfor
RME block were inactive. With the dynoles6 and iminodyns,7
we noted a considerable, compound dependent, degree of
RME block. It is conceivable that the pthaladyns fail to gain
entry to the cell, are rapidly degraded or effluxed, or that
they are displaying an exquisite degree of dynamin I specifi-
city relative to dynamin II. RME is a dynamin II mediated
process, and a lack of RME block may be an indication of
dynamin I versus dynamin II specificity. We therefore exam-
ined the effect of pthaladyns 17, 19, 23, and 29 directly on the
inhibition of dynamin II GTPase activity. While dynamin I in
this study was the endogenousformpurified from sheep brain,
dynamin II was produced as a recombinant protein expressed
in Sf9 insect cells. Our dynamin I and dynamin II assays
require different dynamin and lipid concentrations to ensure
an appropriate optical response in the malachite green assays.
This means that we consider the data presented in Table 5 as
indicative of specificity only, not as an absolute determination
of dynamin I or dynamin II specificity. Notwithstanding this
caveat, we obtained dynamin II IC50 curves for pthaladyns 17,
19, 23, and 29 (Table 5). Pthaladyn 17 returned a dynamin II
IC50 value of 71.0 μM, essentially indistinguishable from its
dynamin I IC50 value. The other pthaladyns displayed varying
levels of dynamin I selectivity ranging from 3-fold (19 and 23
with dynamin II IC50 values of 114 and 63.1 μM, respectively)
to 9-fold dynamin I selective (29 with dynamin II IC50 value of
40.0 μM), suggesting that these compounds should be con-
sidered as pan inhibitors of dynamin (i.e., inhibitors of both
dynamin I and II). These data also suggest that the lack of
observed RME block is not a consequence of dynamin iso-
form specificity but more likely is a result of poor cellular
uptake or rapid efflux in U2OS cells.
a No activity observed at 400 μM.
polar groups were removed and replaced by hydrophobic
groups or moved distal to the imide moiety. Synthesis of these
analogues was conducted as per Scheme 2 and afforded
analogues 34-42 (Table 4). The caveat in this series is that
for true engagement of Asp208, the introduction of a polar
moiety is required. Thus, our modeling predicted that the
introduction of bulky groups would reduce dynamin inhibi-
tion, but the combination of bulk and a polar moiety would
restore dynamin inhibition. With the exception of the 4-tert-
butyl substituted 34 (IC50 112 ( 3.4 μM), all simple aromatic
substituted pthaladyns were inactive (35-40). The retention
of activity with 34 is a consequence of predicted engagement
with Val235 offsetting the reduction in interaction with
Asp208, but this latter residue twists to accommodate the
bulky 4-tert-butyl moiety (not shown). The ability to accom-
modate changes in hydrophobic bulk is limited, with the
simple methylene insertion with the synthesis of 41 rendering
this 4-OCH3 analogue inactive (cf. 21; IC50 78 ( 10.0 μM). In
keeping with our modeling analysis thus far, docking pre-
dicted that the introduction of a methyl-2-pyridyl moiety
would return the resultant pthaladyn (42) to a modest level
We next determined the effect of pthaladyns 17, 19, 23, and
29 on SVE. This is a dynamin I mediated process in neurons
because the concentration of dynamin I in neurons is approxi-
mately 50-fold higher than that of dynamin II.53 These studies
were conducted using isolated rat brain nerve terminals
(synaptosomes), and the effects of the pthaladyns were deter-
mined by measuring a change in the uptake of the styryl dye
FM4-64, a well characterized endocytic marker in this
system.54 Only pthaladyn 23 displayed noteworthy inhibition
of SVE, returning an IC50 value of 12.9 ( 5.9 μM. The other
pthaladyns values were >100 μM. While pthaladyn-23 had
no activity for RME (Table 5), it is a highly effective SVE
blocker, acting at close to its dynamin I IC50 (dynamin I IC50
17.4 ( 5.8 and IC50(SVE) 12.9 ( 5.9 μM). This analogue is
therefore a highly efficient inhibitor of SVE.
Finally, to explore the mechanism of inhibition of the
pthaladyns on dynamin I, we conducted a series of Michaelis-
Menten kinetic experiments with pthaladyn-23 (14-19 μM)

5274 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Odell et al.
Table 5. Inhibition of Dynamin I or Dynamin II in Vitro GTPase Activity, and in-Cell Receptor Mediated Endocytosis (RME) or Synaptic Vesicle
Endocytosis (SVE) by Pthaladyns 17, 19, 23, and 29
a IC50 determinations are the average of at least two independent experiments, each in triplicate. b Mean ( 95% confidence interval (CI) of a single
experiment performed in triplicate. c Not active.
I (Figure 7). This finding confirms that the pthaladyns target
the active site, as predicted by the homology model, and they
are therefore the first reported active site directed dynamin
inhibitors.
Conclusions
Utilizing the high resolution X-ray of the dynamin A
GTPase domain, we constructed a number of homology
models for the human dynamin I GTPase domain. Two of
our modeled structures (hDyn1-3 and hDyn1-7) were able
to accommodate GDP in a near native pose and were also
consistent with the recently solved X-ray structure of the rat
dynamin I GTPase domain. While this work was under
review, the crystal structure of human dynamin’s GTPase
domain was reported.55 This is the first report of an active and
Figure 7. Competitive kinetics of pthaladyn-23 with respect to
Mg2þ GTP. The data depicts pthaladyn-23 concentration depen-
nucleotide-bound form of dynamin I. The structure within the
nucleotide binding pocket was highly similar to our homo-
logy model (see Supporting Information). Our homology
model was used to screen approximately 800000 compounds
in silico to identify potential dynamin I GTPase inhibitors.
Pthaladyn-1 was originally identified as a 170 μM dynamin
inhibitor and used as a lead compound in the development of
focused compound libraries, including an in-house synthe-
sized version of 1. Subsequent rescreening showed that 1
was only weakly active against dynamin (IC50 > 400 μM).
3
dent changes in double-reciprocal plot between substrate (GTP at
60-200 μM) and reaction velocity (V). The data corresponds to
pthaladyn-23 concentrations of 19 (9) 18 (0), 17 ([), 16 (]), 15 (2),
and 14 (Δ) μM. Error bars represent mean ( SEM of four
independent experiments each conducted in triplicate.
and varying concentrations of the Mg2þ GTP substrate
3
(60-200 μM). The Lineweaver-Burke plots revealed clear
competitive inhibition with respect to Mg2þ GTP for dynamin
3

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5275
Notwithstanding this, our focused and homology modeling-
led library synthesis has identified the pthaladyns as the first
class of GTP competitive inhibitors of dynamin. Two findings
strongly demonstrating that our homology models could be
used to successfully aid inhibitor design. First, the model
guided the development of 4-chloro-2-(2-(4-(hydroxymethyl)-
phenyl)-1,3-dioxoisoindoline-5-carboxamido)benzoic acid
(29), a 4.58 ( 0.06 μM potent dynamin inhibitor, a compound
approximately 400-fold more potent than the lead. Second,
the in-cell active analogue, pthaladyn-23, proved to be GTP
competitive; the first reported series to compete at the active
site of dynamin. Our homology model structures and identi-
fied inhibitors are currently being utilized in a number of
structure-based drug design projects within our laboratory.
Surprisingly, none of the pthaladyns evaluated (17, 19, 23,
and 29) blocked RME, which is contrary to our previous
findings with the dynoles6 and iminodyns,7 both of which
displayed efficient RME block. This lack of RME block was
not an artifact of dynamin I selectivity with, at best, modest
levelsofdynamin I(versusdynaminII) selectivitybeing noted.
Pthaladyn-29 displays borderline dynamin I specificity (ca.
9-fold). The lack of RME block is more likely attributed to
poor cellular uptake. In a similar vein, only pthaladyn-23 was
an effective inhibitor of dynamin I mediated SVE with an IC50
of 12.9( 5.9 μM but was an ineffective inhibitor of RME. The
basis of its in-cell selectivity is not yet understood.
The vast majority of drugs have one or more off-target
actions. In the protein kinase inhibitor field, it has been
suggested that a minimum of two structurally unrelated drugs
should be used for cell-based studies to minimize such off-
target drug actions.56 We strongly agree with this principle
and suggest that these additional stringent criteria are now
available for dynamin. With the advent of the pthaladyns
reported here, it is now possible to target dynamin in cell-
based studies with up to three chemically unrelated drugs that
target three unrelated binding sites in dynamin (GTP binding
site: pthaladyns; PH domain: MiTMABs, RTILs; allosteric
sites: iminodyns, dynoles, dynasore).1-9 The pthaladyns greatly
expand the ability to use the dynamin inhibitor palette to more
effectively understand dynamins cellular roles. Because of the
diversity of cellular systems regulated by the dynamins, these
compounds should be evaluated in the future for potential
clinical applications in a variety of disorders ranging from
cancer to pathogen infection.
of the defined binding site. The resulting conformation was then
optimized with flexible ligand and receptor side chains using the
refine and dock function within ICM v.3.1.
Virtual Ligand Screening. The ICM virtual library screening
function was used to dock ∼800000 (Chembridge, San Diego,
CA, and Chemical Diversity, San Diego, CA) flexible ligands,
which passed a relaxed Lipinski filter (MW <600, HBA < 12, and
HBD < 6) and contained less than seven rotatable bonds onto the
receptor grids generated by ICM. Each ligand was assigned a score
reflecting the quality of the ligand-receptor complex. Docking
was conducted three times in parallel and the lowest score for each
ligand was retained. The top ranked 1000 compounds were
clustered according to chemical similarity, visually inspected, and
compounds displaying significant interactions (1 or more H-bond)
with the G2 consensus region were discarded. The top 300
compounds were selected for biological evaluation, of which 175
were still available for purchase.
Biology. Our assay methods for GTPase assay (dynamin I
and dynamin II) and in-cell screens (transferrin endocytosis
inU2OS cells and FM4-64 uptake in rat brain synaptosomes)
have been previously described in detail6,7 and are available in
the Supporting Information.
Chemistry. General Methods. All solvents were bulk quality
and redistilled prior to use. Flash chromatography was carried
1
out using silica gel 200-400 mesh (60 A). H and 13CNMR 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 (perfluoro-
kerosene) as the reference. Microanalysis was conducted at the
Microanalyis Unit at the Australian National University, Canberra,
Australia. Compound purity was confirmed by a combination of
LC-MS (HPLC), micro, and/or high resolution mass spectrometry
and NMR analysis. All analogues are g95% purity.
General Synthetic Method, Synthesis of 2-Biphenyl-2-yl-1,3-
dioxo-2,3-dihydro-1H-isoindole-5-carboxylic Acid Phenylamide
(9). To a chilled (0 °C) solution of trimellitic anhydride chloride
(0.50 g, 2.4 mmol) in THF (20 mL) was added dropwise a
solution of aniline (0.196 g, 2.1 mmol) in 10 mL of the same
solvent. The reaction mixture was stirred at 0 °C for 3 h and
diluted with EtOAc (30 mL) and 1 M HCl (10 mL). The organic
layer was separated, washed with brine (2 ꢀ 20 mL), and dried
(MgSO4). The solvent was removed in vacuo to afford crude 1,3-
dioxo-N-phenyl-1,3-dihydroisobenzofuran-5-carboxamide (4),
which was used without purification in the next step.
Experimental Section
To a suspension of crude 4 (0.128 g, 0.48 mmol) and biphenyl-
2-ylamine (0.097 g, 0.56 mmol) in dry toluene (5 mL) was added
DMF (1 mL) and triethylamine (0.5 mL). The resulting solution
was heated at 130 °C for 18 h, cooled, and partitioned between
EtOAc (30 mL) and 1 M HCl (10 mL). The organic layer was
separated, washed with brine (2 ꢀ 20 mL), dried (MgSO4), and
the solvent removed in vacuo. The residue was purified by flash
column chromatography eluting with petrol/EtOAc (8:2), fol-
lowed by EtOAc to yield the desired compound as a tan solid
and then recrystallized from MeOH; Yield 54%; mp 264-265 °C.
1H NMR (DMSO-d6): δ 7.15 (t, J = 7.3 Hz, 1H), 7.26 (m,
5H), 7.34 (m, 2H), 7.55 (m, 4H), 7.76 (d, 7.4 Hz, 2H), 7.99 (d, J =
8.1 Hz, 1H), 8.35 (dd, J = 1.5, 8.0 Hz, 1H), 8.39 (d, J = 1.4 Hz,
1H), 10.51 (s, 1H).
Molecular Modeling Methods. Homology Model Construc-
˚
tion. The 2.3 A crystal structure of the GDP-bound GTPase
domain of Dictyostelium discoideum dynamin A (PDB code
1JWY) was used as a template to construct a predicted model
of the human dynamin I GTPase domain. The homology model
was generated using the build model function within ICM 3.1
(Molsoft, San Diego, CA) where the target sequence was aligned
on the 3D template (see Supporting Information), missing loops
predicted though PDB-database search and the energy of the
system minimized by a Monte Carlo simulation through a series
of random global moves, and gradient local minimization in the
internal coordinates space.57
Binding Site Identification. The nucleotide-binding site was
˚
defined as an 8 A sphere surrounding the crystal structure
13C NMR (DMSO-d6): δ 120.4, 122.2, 123.6, 124.1, 127.5,
127.6, 128.3, 128.4, 128.6, 129.2, 129.7, 130.0, 130.3, 131.4,
133.3, 134.4, 138.2, 138.6, 140.5, 140.9, 163.5, 166.4, 166.5.
2-[(2-Biphenyl-2-yl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl)-
amino]-4-chlorobenzoic Acid (1). Compound 1 was prepared in a
similar manner to the synthesis of 9 from 2-chloro-2-aminobenzoic
coordinates of dynamin A bound GDP (PDB code 1JWY).
Receptor-Ligand Docking. Initial docking was carried out
with a flexible ligand and 0.5 A grid potential representation of
˚
the receptor accounting for hydrophobicity, van der Waals
boundaries, hydrogen-bonding profile, and electrostatic potential

5276 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Odell et al.
acid. The residue was purified by flash column chromatography
eluting with petrol/EtOAc (8:2) followed by EtOAc and then recrys-
tallized from MeOH to give a tan solid in a 41% yield (two steps); mp
262-264 °C.
followed by EtOAc/MeOH (8:2) to yield the desired compound
as a brown solid; yield 68%; mp 299-300 °C.
1H NMR (DMSO-d6): δ 7.26 (dd, J = 1.8, 8.4 Hz, 1H), 7.47
(m, 3H), 7.53 (m, 2H), 8.01 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 7.8
Hz, 1H), 8.30 (s, 1H), 8.35 (d, J = 7.8 Hz, 1H), 8.67 (d, J = 2.0
Hz, 1H), 12.41 (s, 1H).
1H NMR (DMSO-d6): δ 7.01 (dd, J = 2.0, 8.3 Hz, 1H), 7.18
(m, 5H), 7.49 (m, 4H), 7.90 (d, J = 7.9 Hz, 1H), 8.05 (d, J = 8.3
Hz, 1H), 8.32 (s, 1H), 8.41 (d, J = 7.8 Hz, 1H), 8.71 (d, J = 2.1
Hz, 1H), 15.10 (s, 1H).
13C NMR (DMSO-d6): δ 115.8, 119.4, 121.1, 123.3, 124.0,
127.1, 128.1, 128.8, 131.7, 132.1, 132.7, 133.6, 134.2, 138.6,
139.3, 141.4, 162.9, 166.0, 169.3.
13C NMR (DMSO-d6): δ 118.3, 121.4, 121.6, 121.9, 123.6,
127.3, 127.5, 128.0, 128.1, 129.1, 129.4, 129.5, 130.3, 131.6,
132.6, 133.3, 133.6, 135.7, 138.1, 140.5, 140.9, 141.4, 162.3,
166.0, 166.1, 170.1.
C22H13ClN2O5 H2O: Calcd C 60.22, H 3.45, N 6.38. Anal. C
60.41, H 3.46, N 6.51.
3
2-{[2-(2-Aminophenyl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-
carbonyl]amino}-4-chlorobenzoic Acid (14). Synthesized using
the general procedure as for 13 with benzene-1,2-diamine to
yield the desired compound as a brown solid. Purified by
recrystallization from MeOH in a 49% yield; mp > 300 °C.
1H NMR (DMSO-d6): δ 6.56 (t, J = 7.6 Hz, 1H), 6.77 (d, J =
7.9 Hz, 1H), 7.01 (dd, J = 1.3, 7.8 Hz, 1H), 7.12 (m, 2H), 8.05 (d,
J = 7.7 Hz, 1H), 8.08 (d, J = 8.3 Hz, 1H), 8.40 (s, 1H), 8.45 (d,
J = 7.8 Hz, 1H), 8.74 (d, J = 2.1 Hz, 1H), 12.12 (s, 1H).
13C NMR (DMSO-d6): δ 115.2, 115.3, 115.7, 117.9, 121.0,
121.8, 123.4, 123.5, 129.7, 129.8, 132.8, 133.0, 133.1, 134.5,
134.8, 140.0, 141.7, 146.3, 162.8, 166.7, 166.8, 168.7.
C28H17ClN2O5 0.5H2O: Calcd C 65.31, H 3.72 N 5.44. Anal.
C 65.25, H 3.77, N 5.19.
3
2-Biphenyl-2-yl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carboxylic
Acid (3-Chlorophenyl)amide (10). Compound 10 was prepared in a
similar manner to the synthesis of 9 from 3-chloroaniline. The
residue was purified by flash column chromatography eluting with
petrol/EtOAc (8:2) followed by EtOAc and then recrystallized
from MeOH to give a tan solid in a 70% yield (two steps); mp
74-76 °C.
1H NMR (DMSO-d6): δ 7.16 (m, 7H), 7.40 (m, 4H), 7.62
(s, 1H), 7.80 (d, J = 7.4 Hz, 1H), 8.24 (m, 2H), 8.76 (s, 1H) 13.1
(s, 1H).
C22H14ClN3O5 3H2O: Calcd C 53.94, H 4.12 N 8.58. Anal. C
3
13C NMR (DMSO-d6): δ 118.1, 120.2, 121.4, 123.5, 124.5,
127.0, 127.4, 127.8, 128.0, 128.2, 128.5, 129.3, 129.4, 130.4,
131.3, 133.3, 133.8, 134.0, 137.9, 138.1, 139.7, 140.8, 163.2,
165.9, 166.4.
53.54, H 3.79, N 8.41.
2-{[2-(3-Aminophenyl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-
carbonyl]amino}-4-chlorobenzoic Acid (15). Synthesized using
the general procedure as for 13 with benzene-1,3-diamine; yield
57%; mp > 300 °C.
C27H17ClN2O3 3H2O: Calcd C 63.97, H 4.57 N 5.53. Anal. C
3
1H NMR (DMSO-d6): δ 6.55 (m, 3H), 7.08 (m, 2H), 8.06 (m,
2H), 8.40 (s, 1H), 8.46 (d, J = 7.8 Hz, 1H), 8.72 (d, J = 1.8 Hz,
1H), 12.04 (s, 1H).
64.00, H 3.71, N 5.47.
2-[(2-Biphenyl-2-yl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-car-
bonyl)amino] Benzoic Acid (11). Compound 11 was prepared in a
similar manner to the synthesis of 9 from 2-amino benzoic acid.
The residue was purified by flash column chromatography
eluting with petrol/EtOAc (8:2) followed by EtOAc and then
recrystallized from MeOH to give a tan solid in a 40% yield (two
steps); mp 251-53 °C.
13C NMR (DMSO-d6): δ 112.5, 113.6, 114.4, 117.9, 121.3,
121.9, 123.3, 123.7, 128.9, 132.1, 132.3, 132.8, 133.4, 133.8,
134.5, 140.4, 141.6, 149.2, 162.5, 166.2, 166.3, 168.8.
C22H14ClN3O5 4H2O: Calcd C 52.03, H 4.37, N 8.27. Anal. C
51.98, H 4.72, N 8.24.
3
1H NMR (DMSO-d6): δ 7.22 (m, 6H), 7.56 (m, 5H), 8.05 (d,
J = 7.8 Hz, 2H), 8.31 (s, 1H), 8.37 (dd, J = 1.5, 7.8, 1H), 8.59 (d,
J = 8.3 Hz, 1H), 12.5 (s, 1H).
2-{[2-(4-Aminophenyl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-
carbonyl]amino}-4-chlorobenzoic Acid (16). Synthesized using
the general procedure as for 13 with benzene-1,4-diamine; yield
55%; mp > 300 °C.
13C NMR (DMSO-d6):δ119.8, 120.1, 121.4, 123.1, 124.1, 127.5,
127.6, 128.3, 128.4, 129.2, 129.7, 129.8, 130.3, 131.1, 131.8, 132.8,
133.5, 133.7, 138.1, 140.3, 140.5, 140.9, 162.5, 166.2, 166.3, 169.9.
1H NMR (DMSO-d6): δ 6.63 (d, J = 8.5 Hz, 2H), 7.04 (d, J =
8.5 Hz, 2H), 7.11 (dd, J = 2.1, 8.4 Hz, 1H), 8.03 (d, J = 8.3 Hz,
1H), 8.05 (d, J = 7.8 Hz, 1H), 8.37 (s, 1H), 8.42 (d, J = 7.8 Hz,
1H), 8.71 (d, J = 2.1 Hz, 1H), 12.70 (s, 1H).
C28H18N2O5 2.25H2O: Calcd C 66.86, H 4.51, N 5.57. Anal.
3
C 66.57, H 4.32, N 5.33.
13C NMR (DMSO-d6): δ 113.4, 118.1, 119.4, 121.2, 122.0,
122.6, 123.6, 128.0, 132.2, 132.8, 133.4, 134.0, 134.9, 140.2,
141.6, 148.8, 162.7, 166.7, 166.8, 169.0.
2-[(2-Biphenyl-2-yl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-car-
bonyl)-amino]-5-iodo Benzoic Acid (12). Compound 12 was pre-
pared in a similar manner to the synthesis of 9 from 5-iodo-2-
amino benzoic acid. The residue was purified by flash column
chromatography eluting with petrol/EtOAc (8:2) followed by
EtOAc and then recrystallized from MeOH to give to give a tan
solid, yield 55% (two steps); mp > 300 °C.
C22H14ClN3O5 3.5H2O: Calcd C 52.97, H 4.24, N 8.42. Anal.
C 52.98, H 3.79, N 8.30.
3
4-Chloro-2-{[2-(2-hydroxyphenyl)-1,3-dioxo-2,3-dihydro-1H-
isoindole-5-carbonyl] amino}benzoic Acid (17). Synthesized using
the general procedure as for 13 with 2-hydroxyaniline. Purified
by recrystallization from MeOH; yield 73%; mp 290-294 °C.
1H NMR (DMSO-d6): δ 6.92 (dt, J = 1.2, 7.5 Hz, 1H), 7.00
(dd, J = 2.1, 8.2 Hz, 1H), 7.29 (m, 3H), 8.04 (d, J = 8.3 Hz, 1H),
8.12 (d, J = 7.7 Hz, 1H), 8.38 (s, 1H), 8.41 (dd, J = 1.5, 7.8 Hz,
1H), 8.73 (d, J = 2.1 Hz, 1H), 12.84 (s, 1H).
1H NMR (DMSO-d6): δ 7.26 (m, 5H), 7.56 (m, 4H), 7.97 (dd,
J = 1.7, 8.6 Hz, 1H), 8.03 (d, J = 7.5 Hz, 1H), 8.28 (m, 2H), 8.37
(m, 2H), 12.1 (s, 1H).
13C NMR (DMSO-d6): δ 87.0, 120.1, 121.3, 122.7, 124.1, 127.5,
127.6, 128.3, 128.4, 129.2, 129.7, 129.8, 130.1, 130.3, 131.8, 133.7,
138.1, 138.8, 139.7, 139.9, 140.9, 142.2, 162.8, 166.2, 166.3, 168.4.
C28H17IN2O5: Calcd C 57.16, H 2.91, N 4.76. Anal. C 57.12,
H 2.99, N 4.73.
13C NMR (DMSO-d6): δ 116.6, 116.9, 118.6, 119.1, 119.4,
121.1, 123.2, 124.0, 130.1, 130.4, 132.6, 132.8, 133.6, 134.6,
138.1, 139.5, 141.4, 153.9, 163.1, 166.2, 166.3, 169.2.
Synthesis of N-Substituted Pthaladyns 13-42. 4-Chloro-
2-[(1,3-dioxo-2-phenyl-2,3-dihydro-1H-isoindole-5-carbonyl)amino]-
benzoic Acid (13). To a suspension of 7 (0.15 g, 0.43 mmol) and
aniline (0.078 g, 0.65 mmol) in toluene (5 mL) was added DMF
(1 mL) and TEA (0.5 mL). The resulting solution was heated at 130
°C for 18 h, cooled, and neutralized with 10% HCl. The solution
was partitioned between EtOAc (30 mL) and water (10 mL) and the
organic layer separated, washed with brine (2 ꢀ 20 mL), dried
(MgSO4), and the solvent removed in vacuo. The residue was
purified by flash column chromatography eluting with EtOAc
C22H13ClN2O6 0.75H2O: Calcd C 57.34, H 3.43, N 6.08.
3
Anal. C 57.65, H 3.31, N 6.41.
4-Chloro-2-{[2-(3-hydroxyphenyl)-1,3-dioxo-2,3-dihydro-1H-
isoindole-5-carbonyl] amino}benzoic Acid (18). Synthesized using
the general procedure as for 13 with 3-hydroxyaniline. Purified
by recrystallization from MeOH; yield 84%; mp 290-291 °C.
1H NMR (DMSO-d6): δ 6.87 (m, 3H), 7.28 (m, 2H), 8.04 (d,
J = 8.4 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 8.36 (s, 1H), 8.40 (dd,
J = 1.2, 7.8 Hz, 1H), 8.70 (d, J = 2.1 Hz, 1H), 12.85 (s, 1H).

Article
13C NMR (DMSO-d6): δ 114.3, 115.2, 116.9, 117.7, 119.4,
121.2, 123.2, 124.0, 129.4, 132.2, 132.6, 132.8, 133.6, 134.3,
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5277
4-Chloro-2-{[2-(4-nitrophenyl)-1,3-dioxo-2,3-dihydro-1H-iso-
indole-5-carbonyl]-amino}-benzoic Acid (24). Synthesized using
the general procedure as for 13 with 4-nitroaniline. Purified by
recrystallization from MeOH; yield 47%; mp > 300 °C.
1H NMR (DMSO-d6): δ 7.30 (d, J = 7.5 Hz, 1H), 7.82 (m, 2H),
8.05 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 7.5 Hz, 1H), 8.39 (m, 4H),
8.71 (s, 1H), 12.54 (s, 1H).
138.0, 139.5, 141.4, 157.6, 163.1, 166.0, 166.1, 169.2.
C22H13ClN2O6 H2O: Calcd C 57.74, H 3.34, N 6.09. Anal. C
58.10, H 3.32, N 6.09.
3
4-Chloro-2-{[2-(4-hydroxyphenyl)-1,3-dioxo-2,3-dihydro-1H-
isoindole-5-carbonyl] amino}benzoic Acid (19). Synthesized using
the general procedure as for 13 with 4-hydroxyaniline. Purified
by recrystallization from MeOH; yield 48%; mp > 300 °C.
1H NMR (DMSO-d6): δ 6.88 (d, J = 8.7 Hz, 2H), 7.21 (d, J =
8.8 Hz, 2H), 7.31 (dd, J = 2.1, 8.4 Hz, 1H), 8.04 (d, J = 8.4 Hz,
1H), 8.11 (d, J = 7.7 Hz, 1H), 8.34 (s, 1H), 8.38 (dd, J = 1.2, 7.8
Hz, 1H), 8.70 (d, J = 2.0 Hz, 1H), 12.85 (s, 1H).
13C NMR (DMSO-d6): δ 119.7, 121.4, 123.4, 124.1, 124.3,
125.3, 132.2, 132.8, 133.9, 134.2, 137.5, 138.4, 139.8, 141.4,
146.3, 151.2, 163.1, 165.5, 165.6, 169.2.
C22H12ClN3O7 0.25H2O: Calcd C 56.18, H 2.68, N8.93.
3
Anal. C 56.29, H 2.66, N 8.72.
2-{[2-(2-Carboxyphenyl)-1,3-dioxo-2,3-dihydro-1H-isoindole-
5-carbonyl]amino}-4-chlorobenzoic Acid (25). Synthesized using
the general procedure as for 13 with 2-aminobenzoic acid.
Purified by recrystallization from MeOH; yield 60%; mp > 300 °C.
1H NMR (DMSO-d6): δ 7.31 (dd, J = 2.1, 8.6 Hz, 1H), 7.56
(dd, J = 1.1, 7.9 Hz, 1H), 7.49 (dt, J = 1.4, 7.6 Hz, 1H), 7.78 (dt,
J = 1.6, 7.7 Hz, 1H), 8.06 (m, 2H), 8.16 (d, J = 7.7 Hz, 1H), 8.40
(s, 1H), 8.45 (dd, J = 1.5, 7.8 Hz, 1H), 8.71 (d, J = 2.1 Hz, 1H),
12.40 (s, 1H).
13C NMR (DMSO-d6): δ 115.4, 116.1, 119.6, 121.0, 122.6,
123.4, 123.9, 128.6, 132.3, 132.8, 133.5, 134.4, 138.5, 139.6,
141.4, 157.3, 163.2, 166.4, 166.5, 169.2.
C22H13ClN2O6 2.5H2O: Calcd C 54.84, H 3.77, N 5.81. Anal.
3
C 54.51, H 3.46, N 5.61.
4-Chloro-2-{[2-(3-methoxy)-1,3-dioxo-2,3-dihydro-1H-isoindole-
5-carbonyl]-amino}-benzoic Acid (20). Synthesized using the
general procedure as for 13 with 3-methoxyaniline; yield 35%;
mp 242-246 °C.
13C NMR (DMSO-d6): δ 116.2, 119.7, 121.3, 123.4, 125.2,
129.0, 129.4, 130.5, 131.0, 131.2, 132.4, 132.8, 133.0, 133.9,
134.3, 138.5, 139.8, 141.4, 163.1, 165.9, 166.1, 166.2, 169.2.
1H NMR (DMSO-d6): δ 4.30 (s, 3H), 6.55 (d, J = 8.82 Hz,
1H), 7.61 (d, J = 8.81 Hz, 1H), 7.71 (dd, J = 7.94, 1.80 Hz, 2H),
7.84 (t, J = 6.49, 6.49 Hz, 1H), 7.94 (dd, J = 9.15, 2.66 Hz, 1H),
8.01 (s, 1H), 8.10 (d, J = 10.05 Hz, 1H), 8.20 (dd, J = 7.97, 0.62
Hz, 1H), 8.26 (s, 1H), 8.58 (d, J = 0.82 Hz, 1H), 10.71 (s, 1H).
13C NMR (DMSO-d6): δ 55.4, 98.5, 100.5, 102.6, 105.8, 107.9,
111.1, 116.9, 120.6, 122.8, 124.9, 128.4, 130.4, 132.5, 133.9,
140.5, 141.7, 149.8, 149.9, 160.2, 161.0, 166.3, 168.9.
C23H15ClN2O6: Calcd C 61.28, H 3.35 N 6.21 Anal. C 61.38,
H 3.65, N 6.45.
C23H15ClN2O7 0.25H2O: Calcd C 58.86, H 2.90, N 5.97.
3
Anal. C 58.90, H 3.12, N 6.32.
2-{[2-(4-Carboxyphenyl)-1,3-dioxo-2,3-dihydro-1H-isoindole-
5-carbonyl]amino}-4-chlorobenzoic Acid (26). Synthesized using
the general procedure as for 13 with 4-aminobenzoic acid.
Purified by recrystallization from MeOH; yield 36%; mp >
300 °C.
1H NMR (DMSO-d6): δ 7.27 (dd, J = 2.0, 8.6 Hz, 1H), 7.62
(d, J = 7.2 Hz, 2H), 8.02 (d, J = 8.5 Hz, 1H), 8.08 (d, J = 7.2 Hz,
2H), 8.14 (d, J = 7.7 Hz, 1H), 8.36 (s, 1H), 8.39 (d, J = 7.8 Hz,
1H), 8.68 (d, J = 2.0 Hz, 1H), 12.52 (s, 1H).
4-Chloro-2-[2-(4-methoxyphenyl)-1,3-dioxo-2,3-dihydro-1H-iso-
indole-5-carbonyl]aminobenzoic Acid (21). Synthesized using the
general procedure as for 13 with 4-methoxyaniline; yield 25%;
mp > 300 °C.
13C NMR (DMSO-d6): δ 116.2, 119.5, 121.3, 123.3, 124.2,
126.8, 129.8, 130.2, 132.2, 132.8, 133.7, 134.2, 135.5, 138.4,
139.6, 141.3, 163.0, 165.7, 165.8, 166.6, 169.2.
1H NMR (DMSO-d6): δ 3.83 (s, 3H), 7.09 (d, J = 8.91 Hz,
2H), 7.23 (m, 3H), 7.38 (d, J = 8.88, 1H), 8.09 (dd, J = 15.36,
8.12 Hz, 2H), 8.4 (s, 1H), 8.44 (dd, J = 7.80, 1.20 Hz, 1H), 8.74
(d, J = 2.12 Hz, 1H), 9.45 (s, 1H).
C23H15ClN2O7 0.5H2O: Calcd C 58.30, H 2.98, N 5.91. Anal.
3
C 57.97, H 3.40, N 6.26.
13C NMR (DMSO-d6): δ 55.3, 114.1, 114,5, 115.3, 118.3,
121.2, 121.6, 122,2, 123.8, 124.3, 128.6, 132.2, 132.7, 133.5,
134.0, 135.4, 140.2, 141.5, 159.0, 162.2, 162.7, 166.5, 169.0.
4-Chloro-2-{[2-(2-hydroxymethylphenyl)-1,3-dioxo-2,3-dihydro-
1H-isoindole-5-carbonyl]amino}benzoic Acid (27). Synthesized
using the general procedure as for 13 with (2-aminophenyl)-
methanol. Purified by recrystallization from MeOH; yield 22%;
mp 251-254 °C.
C23H15ClN2O6 2H2O: Calcd C 56.74, H 3.93, N 5.75. Anal. C
3
56.91, H 4.16, N 5.91.
1H NMR (DMSO-d6): δ 4.40 (s, 2H), 7.32 (dd, J = 2.0, 8.2
Hz, 1H), 7.39 (m, 2H), 7.49 (dt, J = 1.8, 7.6 Hz, 1H), 7.58 (d, J =
7.4 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 7.7 Hz, 1H),
8.40 (s, 1H), 8.41 (d, J = 7.8 Hz, 1H), 8.73 (d, J = 2.1 Hz, 1H),
12.44 (s, 1H).
4-Chloro-2-{[2-(4-methylsulfanylphenyl)-1,3-dioxo-2,3-dihy-
dro-1H-isoindole-5-carbonyl]-amino}-benzoic Acid (22). Synthe-
sized using the general procedure as for 13 with 4-methyl-
thioaniline; yield 14%; mp 180-182 °C.
1H NMR (DMSO-d6): δ 2.32 (s, 3H), 7.28 (dd, J = 8.52, 2.19
Hz, 2H), 7.4 (s, 1H), 8.03 (d, J = 8.54 Hz, 1H), 8.12 (d, J = 7.76
Hz, 2H), 8.34 (s, 1H), 8.40 (d, J = 1.00 Hz, 1H), 8.69 (d, J = 2.04
Hz, 1H), 12.5 (s, 1H).
13C NMR (DMSO-d6): δ 59.7, 116.3, 119.7, 121.3, 123.4,
124.2, 127.4, 127.7, 129.0, 129.1, 129.2, 132.5, 132.8, 133.7,
134.5, 138.5, 139.6, 140.4, 141.4, 163.2, 166.1, 166.2, 169.2.
13C NMR (DMSO-d6): δ 14.7, 115.4, 116.0, 119.5, 121.1,
123.2, 123.3, 124.0, 126.0, 127.6, 128.5, 130.4, 132.2, 132.7,
133.6, 134.3, 138.5, 138.6, 139.4, 141.4, 163.0, 168.1, 169.2.
C23H15ClN2O5S: Calcd C 59.17, H 3.24, N 6.00. Anal. C
59.51, H 3.39, N 6.51.
C23H15ClN2O6 0.5H2O: Calcd C 60.07, H 3.51, N 6.09. Anal.
3
C 60.22, H 3.68, N 6.38.
4-Chloro-2-{[2-(3-hydroxymethylphenyl)-1,3-dioxo-2,3-dihydro-
1H-isoindole-5-carbonyl]amino}benzoic Acid (28). Synthesized
using the general procedure as for 13 with (3-aminophenyl)-
methanol. Purified by recrystallization from MeOH; yield 47%;
mp 279-282 °C.
4-Chloro-2-{[2-(3-nitrophenyl)-1,3-dioxo-2,3-dihydro-1H-iso-
indole-5-carbonyl]-amino}-benzoic Acid (23). Synthesized using
the general procedure as for 13 with 3-nitroaniline. Purified by
recrystallization from MeOH; yield 56%; mp > 300 °C.
1H NMR (DMSO-d6): δ 7.26 (d, J = 8.2 Hz, 1H), 7.85 (m,
1H), 7.93 (m, 2H), 8.05 (d, J = 9.0 Hz, 1H), 8.21 (d, J = 8.1 Hz,
1H), 8.30 (m, 1H), 8.40 (m, 2H), 8.70 (s, 1H), 12.41 (s, 1H).
13C NMR (DMSO-d6): δ 116.1, 119.6, 121.4, 121.8, 122.8,
123.4, 124.3, 130.3, 132.2, 132.8, 133.5, 133.8, 134.2, 138.5,
139.7, 141.4, 147.8, 162.2, 163.1, 165.7, 165.8, 169.2.
C22H12ClN3O7: Calcd C 56.73, H 2.60, N 9.02. Anal. C 56.75,
H 2.40, N 8.75.
1H NMR (DMSO-d6): δ 4.56 (s, 2H), 7.09 (dd, J = 2.2, 8.3
Hz, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.41 (m, 2H), 7.49 (d, J = 7.7
Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 7.7 Hz, 1H), 8.43
(s, 1H), 8.48 (dd, J = 1.4, 7.8 Hz, 1H), 8.73 (d, J = 2.1 Hz, 1H),
12.41 (s, 1H).
13C NMR (DMSO-d6): δ 62.4, 115.9, 119.5, 121.1, 123.3,
124.0, 125.1, 125.5, 126.2, 128.5, 131.5, 132.2, 132.8, 133.6,
134.3, 138.6, 139.3, 141.4, 143.5, 163.0, 166.1, 166.2, 169.3.
C23H15ClN2O6 0.75H2O: Calcd C 59.49, H 3.58, N 6.03.
Anal. C 59.49, H 3.59, N 6.21.
3

5278 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Odell et al.
4-Chloro-2-{[2-(4-hydroxymethylphenyl)-1,3-dioxo-2,3-dihydro-
1H-isoindole-5-carbonyl]amino}benzoic Acid (29). Synthesized
using the general procedure as for 13 with (4-aminophenyl)-
methanol. Purified by recrystallization from MeOH; yield 28%;
mp 296-298 °C.
the general procedure as for 13 with 4-tert-butylaniline; yield
14%; mp > 300 °C.
1H NMR (DMSO-d6): δ 1.26 (m, 9H), 6.50 (d, J = 8.5 Hz,
1H), 7.02 (d, J = 8.5 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.34 (d,
J = 8.5 Hz, 1H), 7.59 (dd, J = 3.5, 8.5 Hz, 2H), 7.72 (d, J = 7.9
Hz, 1H), 8.06 (m, 1H) 8.17 (dd, J = 1.9, 8.0 Hz, 1H), 8.77(dd,
J = 2.0, 7.2 Hz, 1H), 10.34 (s, 1H).
1H NMR (DMSO-d6): δ 4.56 (s, 2H), 7.28 (dd, J = 2.2, 8.6
Hz, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.3 Hz, 2H), 8.04
(d, J = 8.5 Hz, 1H), 8.13 (d, J = 7.7 Hz, 1H), 8.35 (s, 1H), 8.38
(d, J = 7.8 Hz, 1H), 8.70 (d, J = 2.1 Hz, 1H), 12.41 (s, 1H).
13C NMR (DMSO-d6): δ 62.4, 116.0, 119.6, 121.1, 123.4,
124.0, 126.7, 126.9, 130.1, 132.3, 132.8, 133.6, 134.3, 138.5,
139.4, 141.4, 142.7, 163.1, 166.1, 166.2, 169.2.
13C NMR (DMSO-d6): δ 31.4, 33.9, 113.9, 118.7, 119.3, 122.5,
125.3, 126.4, 126.8, 127.8, 128.2, 128.5, 130.1, 130.6, 132.8,
135.0, 136.7, 139.1, 141.8, 145.4, 145.8, 163.4, 166.3, 166.9,
169.2.
HRMS: calcd for C26H21ClN2O5 476.1139; found (ESIþ)
(M þ H) 476.1140.
C23H15ClN2O6 0.75H2O: Calcd C 59.49, H 3.58, N 6.03.
Anal. C 59.49, H 3.59, N 6.21.
3
4-Chloro-2-{[2-(2,6-dimethylphenyl)-1,3-dioxo-2,3-dihydro-1H-
isoindole-5-carbonyl]-amino}benzoic Acid (35). Synthesized using
the general procedure as for 13 with 2,6-dimethylaniline; yield
47%; mp 224-226 °C.
4-Chloro-2-{[2-(4-ethoxycarbonylphenyl)-1,3-dioxo-2,3-dihydro-
1H-isoindole-5-carbonyl]-amino}benzoic Acid (30). Synthesized
using the general procedure as for 13 with ethyl 4-aminobenzoate;
yield 18%; mp > 300 °C.
1H NMR (DMSO-d6): δ 2.17 (s, 6H), 6.63 (t, J = 7.5 Hz, 1H),
6.93 (d, J = 7.5 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 7.07 (m, 2H),
7.20 (d, J = 7.4 Hz, 1H), 8.08 (d, J = 7.7 Hz, 1H), 8.13 (dd, J =
3.4, 8.4 Hz, 1H), 8.24 (dd, J = 1.7, 8.2 Hz, 1H), 8.40 (d, J = 8.2
Hz, 1H), 8.62 (s, 1H), 9.0 (s, 1H).
1H NMR (DMSO-d6): δ 1.04 (t, J = 7.0, 6.99 Hz, 3H), 2.06
(m, 2H), 6.58 (dd, J = 2.6, 9.8 Hz, 2H), 7.83 (d, J = 8.0 Hz, 1H),
7.91 (d, J = 9.2 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.46 (dd, J =
1.6, 8.0 Hz, 2H), 8.58 (d, J = 0.8 Hz, 1H), 11.0 (s, 1H).
13C NMR (DMSO-d6): δ 13.7, 61.2, 126.4, 126.9, 129.1, 129.2,
129.4, 129.8, 131.3, 131.7, 133.4, 133.8, 140.5, 140.6, 140.8,
140.9, 166.9, 167.3, 167.4, 167.8.
13C NMR (DMSO-d6): δ 12.6, 20.7, 113.4, 117.3, 118.8, 119.2,
120.8, 123.7, 124.9, 128.3, 129.0, 130.3, 131.4, 133.2, 134.6,
138.0, 139.3, 140.1, 142.8, 145.6, 161.2, 163.4, 165.8, 166.0.
HRMS: calcd for C24H17ClN2O5 448.0826; found (ESIþ)
(M þ H) 448.0829.
HRMS: calcd for C25H17ClN2O7 492.0724; found (ESIþ)
(M þ H) 492.0724.
2-{[2-(4-Carboxymethylphenyl)-1,3-dioxo-2,3-dihydro-1H-iso-
indole-5-carbonyl]-amino}-4-chlorobenzoic Acid (31). Synthesized
using the general procedure as for 13 with 2-(4-aminophenyl)-
acetic acid; yield 43%; mp > 272-275 °C.
4-Chloro-2-[(2-naphthalen-1-yl-1,3-dioxo-2,3-dihydro-1H-iso-
indole-5-carbonyl)-amino]benzoic Acid (36). Synthesized using
the general procedure as for 13 with 1-naphthylamine; yield 7%;
mp > 300 °C.
1H NMR (DMSO-d6): δ 3.7 (s, 2H), 7.28 (dd, J = 2.1, 8.5 Hz,
2H), 7.40 (m, 2H), 8.02 (d, J = 8.5 Hz, 2H), 8.12 (d, J = 7.8 Hz,
1H), 8.33 (s, 1H), 8.38 (d, J=7.8Hz, 2H), 8.68(d,J=1.9Hz, 1H),
12.4 (s, 1H).
1H NMR (DMSO-d6): δ 6.84 (s, 1H), 6.93 (d, J = 8.0 Hz, 1H),
7.07 (m, 2H), 7.26 (d, J = 7.0, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.6
(m, 2H), 7.82 (d, J = 7.7 Hz, 1H), 8.03 (d, J = 6.3 Hz, 1H), 8.15
(d, J = 7.3 Hz, 1H), 8.38 (m, 2H), 8.73 (s, 1H), 12.45 (s, 1H).
13C NMR (DMSO-d6): δ 106.3, 116.2, 118.4, 119.5, 120.9,
121.2, 123.1, 124.1, 125.9, 127.5, 128.9, 130.2, 132.8, 133.7,
134.9, 135.9, 136.2, 138.5, 139.5, 141.6, 145.9, 163.1, 168.2,
167.5, 169.4.
13C NMR (DMSO-d6): δ 45.4, 115.9, 119.5, 121.1, 123.4,
124.0, 127.0, 129.8, 130.1, 132.2, 132.8, 133.6, 134.3, 135.2,
138.6, 139.3, 141.4, 163.0, 166.1, 169.3, 172.3.
HRMS: calcd for C24H15ClN2O7 478.0568; found (ESIþ)
(M þ H) 478.0568.
HRMS: calcd for C26H15ClN2O5 470.0670; found (ESIþ)
(M þ H) 470.0667.
2-({2-[4-(2-Carboxyethyl)-phenyl]-1,3-dioxo-2,3-dihydro-1H-
isoindole-5-carbonyl}-amino)-4-chlorobenzoic Acid (32). Synthe-
sized using the general procedure as for 13 with 3-(4-aminophe-
nyl)propanoic; yield 16%; mp > 227-229 °C.
4-Chloro-2-{[1,3-dioxo-2-(1,2,3,4-tetrahydronaphthalen-1-yl)-
2,3-dihydro-1H-isoindole-5-carbonyl]-amino}benzoic Acid (37).
Synthesized using the general procedure as for 13 with 1-amino-
1,2,3,4-tetrahydronaphthalene; yield 11%; mp > 256-258 °C.
1H NMR (DMSO-d6): δ 1.9 (m, 6H), 4.45 (t, J = 5.2 Hz, 1H),
7.19 (m, 4H), 7.47 (d, J = 7.7 Hz, 1H), 8.07 (dd, J = 8.5 Hz, 2H),
8.16 (d, J = 8.2 Hz, 1H), 8.36 (m, 2H), 8.66 (d, J = 1.2 Hz, 1H),
8.77 (d, J = 2.0 Hz, 1H), 10.3 (s, 1H).
1H NMR (DMSO-d6): δ 2.81 (m, 4H), 7.13 (d, J = 8.2 Hz,
1H), 7.31 (dd, J = 1.6, 8.0 Hz, 1H), 7.37 (s, 1H), 7.45 (d, J = 8.0
Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 7.3 Hz, 1H), 8.37
(s, 1H), 8.41 (d, J = 7.4 Hz, 1H), 8.72 (d, J = 1.5 Hz, 1H), 9.74
(s, 1H), 12.46 (s, 1H).
13C NMR (DMSO-d6): δ 34.5, 35.0, 116.3, 119.1, 119.6, 121.2,
123.4, 124.1, 127.1, 128.3, 128.6, 129.7, 132.3, 132.8, 133.7,
134.3, 137.1, 138.4, 139.5, 141.4, 163.1, 166.2, 169.2, 170.1,
172.5.
13C NMR (DMSO-d6): δ 18.1, 27.4, 28.3, 47.9, 118.6, 118.7,
122.5, 122.6, 126.0, 128.1, 128.4, 128.9, 129.2, 130.2, 131.8,
132.4, 132.8, 134.6, 135.7, 136.9, 137.5, 141.8, 163.7, 167.3,
167.4, 169.3.
HRMS: calcd for C25H17ClN2O7 492.0724; found (ESIþ)
(M þ H) 492.0725.
HRMS: calcd for C26H19ClN2O5 474.0983; found (ESIþ)
(M þ H) 474.0984.
2-{[2-(3-Hydroxy-4-carboxyphenyl)-1,3-dioxo-2,3-dihydro-1H-
isoindole-5-carbonyl]-amino}-4-chlorobenzoic Acid (33). Synthe-
sized using the general procedure as for 13 with 4-amino-3-
hydroxybenzoic acid; yield 7%; mp > 242-244 °C.
1H NMR (DMSO-d6): δ 6.90 (s, 1H), 7.13 (d, J = 8.2 Hz, 1H),
7.30 (m, 2H), 7.81 (d, J = 8.1 Hz, 1H), 8.04 (s, 1H), 8.09 (d, J =
8.6 Hz, 1H), 8.4 (s, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.74 (d, J = 9.7
Hz, 1H), 10.4 (s, 1H).
2-[(2-Benzyl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl)-
amino]-4-chloro-benzoic Acid (38). Synthesized using the general
procedure as for 13 with benzylamine; yield 25%; mp 236-238 °C.
1H NMR (DMSO-d6): δ 4.80 (s, 2H), 7.08 (dd, J = 2.1, 8.3
Hz, 2H), 7.2 (m, 3H), 8.05 (d, J = 8.8 Hz, 1H), 8.35 (m, 3H), 8.42
(s, 1H), 8.56 (d, J = 7.5 Hz, 1H), 12.42 (s, 1H).
13C NMR (DMSO-d6): δ 45.8, 113.5, 117.8, 119.4, 120.7,
124.4, 125.2, 128.8, 129.5, 130.3, 131.2, 132.9, 133.6, 135.8,
137.3, 138.9, 141.5, 145.6, 150.0, 162.1, 165.5, 165.6, 167.8.
HRMS: calcd for C23H15ClN4O8 434.0670; found (ESIþ)
(M þ H) 434.0671.
13C NMR (DMSO-d6): δ 114.1, 114.7, 115.2, 117.7, 118,2,
121.3, 122.2, 123.9, 129.4, 130.0, 132.2, 132.6, 132.8, 133.5,
133.9, 135.2, 140.2, 141.5, 157.6, 162.6, 166.2, 169.1, 171.2.
HRMS: calcd for C23H13ClN2O8 480.03604; found (ESIþ)
(M þ H) 480.
4-Chloro-2-[1,3-dioxo-2-(2-phenethyl)-2,3-dihydro-1H-isoindole-
5-carbonyl]-amino}benzoic Acid (39). Synthesized using the general
procedure as for 13 with 2-phenylethanamine; yield 16%; mp
274-277 °C.
2-{[2-(4-tert-Butylphenyl)-1,3-dioxo-2,3-dihydro-1H-isoindole-
5-carbonyl]-amino}-4-chlorobenzoic Acid (34). Synthesized using

Article
1H NMR (DMSO-d6): δ 5.13 (m, 4H), 7.25 (m, 5H), 7.41 (d,
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5279
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J = 7.0 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 8.03 (dd, J = 3.0, 5.5
Hz, 1H), 8.11 (dd, J = 1.7, 7.9 Hz, 1H), 8.26 (s, 1H), 8.37 (d, J =
1.5 Hz, 1H), 8.75 (d, J = 2.0 Hz, 1H), 12.46 (s, 1H).
13C NMR (DMSO-d6): δ 42.0, 48.2, 115.7, 119.4, 121.0, 123.0,
126.1, 126.5, 127.8, 128.1, 128.7, 130.0, 130.8, 132.8, 133.4,
134.3, 138.5, 141.8, 142.1, 144.2, 163.1, 163.5, 166.9, 169.4.
HRMS: calcd for C24H17ClN2O5 448.0826; found (ESIþ)
(M þ H) 448.0827.
4-Chloro-2-{[1,3-dioxo-2-(4-phenylbutyl)-2,3-dihydro-1H-iso-
indole-5-carbonyl]-amino}benzoic Acid (40). Synthesized using
the general procedure as for 13 with 4-phenylbutanamine; yield
21%; mp 214-216 °C.
1H NMR (DMSO-d6): δ 1.15 (t, J = 7.1 Hz, 4H), 1.96 (d, J =
2.3 Hz, 2H), 2.5 (m, 2H), 3.59 (d, J = 6.1 Hz, 2H), 7.2 (m, 5H),
8.00 (dd, J = 5.5, 8.1 Hz, 1H), 8.22 (s, 1H), 8.28 (d, J = 1.3 Hz,
1H), 8.31 (d, J = 1.3 Hz, 1H), 8.66 (d, J = 2.1 Hz, 1H), 11.63 (s,
1H).
13C NMR (DMSO-d6): δ 27.4, 28.2, 34.5, 37.4, 116.3, 119.4,
120.8, 123.2, 123.6, 125.6, 128.2, 132.3, 132.8, 133.2, 134.3,
138.3, 139.1, 141.4, 141.8, 162.9, 166.9, 169.1, 169.2.
HRMS: calcd for C26H21ClN2O5 476.1139; found (ESIþ)
(M þ H) 476.1141.
4-Chloro-2-{[2-(4-methoxybenzyl)-1,3-dioxo-2,3-dihydro-1H-
isoindole-5-carbonyl]-amino}benzoic Acid (41). Synthesized
using the general procedure as for 13 with 4-methoxybenzyl
amine; yield 14%; mp 250-252 °C.
1H NMR (DMSO-d6): δ 3.98 (s, 3H), 4.71 (s, 2H), 6.86 (d, J =
8.7 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 7.14 (d, J = 2.2 Hz, 1H),
7.25 (d, J = 8.7 Hz 1H), 7.37 (d, J = 8.7 Hz, 1H), 8.04 (d, J = 8.3
Hz, 1H), 8.33 (s, 1H), 8.37 (dd, J = 1.3, 7.8 Hz, 1H), 8.71 (d, J =
2.2 Hz, 1H), 11.64 (s, 1H).
13C NMR (DMSO-d6): δ 15.1, 64.8, 106.8, 110.4, 124.6, 125.9,
127.2, 129.1, 129.4, 129.5, 131.3, 132.9, 134.0, 134.7, 135.3,
139.7, 140.5, 145.2, 145.9, 157.6, 162.7, 166.7, 167.1, 168.2.
HRMS: calcd for C24H17ClN2O6 464.0775; found (ESIþ)
(M þ H) 464.0777.
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4-Chloro-2-{[1,3-dioxo-2-pyridin-3-ylmethyl-2,3-dihydro-1H-
isoindole-5-carbonyl]-amino}benzoic Acid (42). Synthesized using
the general procedure as for 13 with 3-pyridylmethyl amine; yield
38%; mp > 300 °C.
(13) Ferguson, K. M.; Lemmon, M. A.; Schlessinger, J.; Sigler, P. B.
1H NMR (DMSO-d6): δ 4.83 (s, 2H), 7.11 (dd, J = 2.0, 8.4,
2.02 Hz, 1H), 7.33 (d, J = 5.4 Hz, 1H), 7.46 (d, J = 5.4 Hz, 1H),
8.05 (dd, J = 3.7, 8.0 Hz, 1H), 8.38 (s, 1H), 8.42 (d, J = 7.8 Hz,
1H), 8.50 (d, J = 5.5 Hz, 1H), 8.57 (d, J = 5.5 Hz, 1H), 8.72 (d,
J = 2.0 Hz, 1H), 8.76 (d, J = 1.9 Hz, 1H), 8.82 (d, J = 1.4 Hz,
1H), 10.41 (s, 1H).
˚
Crystal structure at 2.2 A resolution of the pleckstrin homology
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13C NMR (DMSO-d6): δ 41.1, 118.1, 121.3, 122.0, 123.1,
123.8, 128.4, 132.3, 132.7, 133.4, 133.9, 135.9, 137.7, 140.3,
141.5, 142.9, 145.1, 149.7, 162.6, 167.1, 167.6, 169.2.
HRMS: calcd for C22H14ClN3O5 435.0622; found (ESIþ)
(M þ H) 435.0624.
(17) Zuchner, S.; Noureddine, M.; Kennerson, M.; Verhoeven, K.;
Claeys, K.; Jonghe, P. D.; Merory, J.; Oliveira, S. A.; Speer, M. C.;
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Vance, J. M. Mutations in the pleckstrin homology domain of
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disease. Nat. Genet. 2005, 37, 289–294.
Acknowledgment. We are grateful for financial support
from the NH & MRC (Australia), the Children’s Medical
Research Institute (CMRI), and the University of Newcastle
(UoN). L.O. and D.H. gratefully acknowledge scholarship
support from UN & CMRI and AusAid respectively.
ꢀ
(18) Bitou, M.; Maugenre, S.; Jeannet, P. -Y.; Lacene, E.; Ferrer, X.;
^
€
Laforet, P.; Martin, J. -J.; Laporte, J.; Lochmuller, H.; Beggs,
A. H.; Fardeau, M.; Eymard, B.; Romero, N. B.; Guicheney, P.
Mutations in dynamin 2 cause dominant centronuclear myopathy.
Nat. Genet. 2005, 37, 1207–1209.
Supporting Information Available: Additional general synth-
esis information, details of screening methods, sequence align-
ment, and graphical output from Proflex. This material is
available free of charge via the Internet at http://pubs.acs.org.
(19) Salim, K.; Bottomley, M. J.; Querfurth, E.; Zvelebil, M. J.; Gout,
I.; Scaife, R.; Margolis, R. L.; Gigg, R.; Smith, C. I. E.; Driscoll,
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M. A. Dynamin 2 binds gamma-tubulin and participates in centro-
some cohesion. Nat. Cell Biol. 2004, 335–342.
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