Discovery of (2,4-Dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin-1- ylmethyl)-1,3-dihydroisoindol-2-yl]methanone (AT13387), a novel inhibitor of the molecular chaperone Hsp90 by fragment based drug design

Article abstract of DOI:10.1021/jm100060b

Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) are currently generating significant interest in clinical development as potential treatments for cancer. In a preceding publication (DOI: 10.1021/jm100059d) we describe Astex's approach to screening fragments against Hsp90 and the subsequent optimization of two hits into leads with inhibitory activities in the low nanomolar range. This paper describes the structure guided optimization of the 2,4-dihydroxybenzamide lead molecule 1 and details some of the drug discovery strategies employed in the identification of AT13387 (35), which has progressed through preclinical development and is currently being tested in man.

Full text of DOI:10.1021/jm100060b

5956 J. Med. Chem. 2010, 53, 5956–5969
DOI: 10.1021/jm100060b
Discovery of (2,4-Dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin-1-ylmethyl)-
1,3-dihydroisoindol-2-yl]methanone (AT13387), a Novel Inhibitor of the Molecular
Chaperone Hsp90 by Fragment Based Drug Design†
Andrew J. Woodhead,*,‡ Hayley Angove,§ Maria G. Carr,‡ Gianni Chessari, Miles Congreve,‡ Joseph E. Coyle,§ Jose Cosme,^
Brent Graham,# Philip J. Day,¥ Robert Downham,‡ Lynsey Fazal,^ Ruth Feltell,# Eva Figueroa,‡ Martyn Frederickson,‡
Jonathan Lewis,^ Rachel McMenamin,# Christopher W. Murray, M. Alistair O’Brien,‡ Lina Parra,^ Sahil Patel,¥
Theresa Phillips,‡ David C. Rees,‡ Sharna Rich,# Donna-Michelle Smith,^ Gary Trewartha,‡ Mladen Vinkovic,¥
Brian Williams,‡ and Alison J.-A. Woolford‡
§
#
‡Medicinal Chemistry, Biophysics, Computational Chemistry and Informatics, ^DMPK, Biology, and ¥Structural Biology,
Astex Therapeutics, Ltd., 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, U.K.
Received January 15, 2010
Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) are currently generating significant
interest in clinical development as potential treatments for cancer. In a preceding publication (DOI:
10.1021/jm100059d) we describe Astex’s approach to screening fragments against Hsp90 and the
subsequent optimization of two hits into leads with inhibitory activities in the low nanomolar range.
This paper describes the structure guided optimization of the 2,4-dihydroxybenzamide lead molecule 1
and details some of the drug discovery strategies employed in the identification of AT13387 (35), which
has progressed through preclinical development and is currently being tested in man.
Introduction
Because of its ubiquitous expression, concerns have
been expressed about the specificity of Hsp90 inhibitors
for tumor Hsp90 over that expressed in normal cells.7
However, in vitro experiments with the ansamycin antibio-
tic geldanamycin have shown that it possesses a higher
binding affinity for Hsp90 derived from tumor rather than
normal cell lines.8 Additionally geldanamycin shows more
potent cytotoxic activity in tumor cells than normal cells
and is sequestered at higher concentrations within tumors
in xenograft mouse models. This has subsequently been
confirmed by its clinical activity.9 Some evidence also exists
that the client protein bound to the Hsp90 cochaperone
complex is more susceptible to ubiquitination and target-
ing to the proteasome for degradation.10,11 These data when
taken together indicate the potential utility of Hsp90 in-
hibitors; consequently many biotechnology and pharma-
ceutical companies have active Hsp90 programs. At the
time of submission at least 10 Hsp90 inhibitors were in ac-
tive clinical development, according to the United States
National Institutes of Health Web site (www.clinicaltrials.
gov). These compounds range from natural products12-16 to
compounds derived from structure-based drug design.17-22
A number of clinical compounds have been disclosed in
recent publications, and there are now a number of reviews
describing Hsp90 inhibitors.23-26
Hsp90 is one of a family of molecular chaperones that are
produced in response to a range of cellular stresses.1 It is
involved in the activation and maintenance of a large number
of regulatory and signaling proteins by acting as a scaffold to
allow these proteins to fold into their correct functional states.
Aswellastheir rolesinnormalcellularfunction, manyofthese
client proteins are also directly implicated in cancer progres-
sion such that inhibition of Hsp90 may simultaneously target
many oncogenic pathways.2-4
The N-terminal domain of Hsp90 contains a nucleotide
binding region able to accommodate both adenosine tripho-
sphate (ATPa) and adenosine diphosphate (ADP), and this is
the target of the most advanced clinical compounds.5 The
binding and subsequent hydrolysis of ATP through its AT-
Pase activity is a key step in the activation process of Hsp90.6
†Coordinates of Hsp90 complexes with compounds 1, 2, 4, 35, and
ADP have been deposited in the Protein Data Bank under accession
codes 2xab, 2xjg, 2xjj, 2xjx, and 2xkt, respectively, together with the
corresponding structure factor files.
*To whom correspondence should be addressed. Phone: þ44 (0)1223
226287. Fax: þ44 (0)1223 226201. E-mail: a.woodhead@astex-therapeutics.
com.
aAbbreviations: ADP, adenosine diphosphate; aq, aqueous; ATP,
adenosine triphosphate; CDK, cyclin dependent kinase; DCE, 1,2-dichlor-
oethane; DMAW, dichloromethane, methanol, acetic acid, water; DMF,
N,N-dimethylformamide; EDC, 1-(3-dimethyaminopropyl)-3-ethylcarbo-
diimide hydrochloride; equiv, equivalents; hERG, human Ether-a-go-go
related gene; HPβCD, hydroxypropyl-β-cyclodextrin; HOBt, 1-hydroxy-
benzotriazole hydrate; HOAt, 1-hydroxy-7-azabenzotriazole; Hsp, heat
shock protein; ip, intraperitoneal; ITC, isothermal calorimetry; iv, intra-
venous; MMFF, Merck molecular force field; morph, morpholine; NMP,
1-methyl-2-pyrrolidinone; PD, pharmacodynamic; PK, pharmacokinetic;
T/C, mean tumor volume of treated animals divided by the mean control
tumor volume; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
In the preceding paper27 (DOI: 10.1021/jm100059d) we
describe the identification of a potent lead compound 1
(Table 1) (Kd = 0.54 nM; HCT116 cell IC50 = 31 nM) by
fragment screening and subsequent structure-based drug
design.27,28 In this publication we describe the biological
profile of 1 and how these data influenced our optimization
strategy, ultimately concluding in the discovery of the clinical
compound 35 (AT13387).
r
pubs.acs.org/jmc
Published on Web 07/28/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5957
Table 1. Structure-activity Relationships (SAR) for the 2,4-Dihydroxybenzamide Core
a Compounds were assayed two or more times. b Compounds were assayed n = 1. The cell assay was used as the primary screening assay. c Kd and cell
data for compound 36 were generated in house and compare well with values quoted in the literature.32
Results and Discussion
Analysis of this structure suggested that substitution at
the 4, 5, 6, and 7 positions of the isoindoline ought to be
well tolerated. The isopropyl group at the 5-position of the
central phenyl ring efficiently fills a proximal hydrophobic
cavity, bounded by the residues Leu107, Phe138, Val150,
and Val186, with the potential for further growth toward
where the ribose and phosphate groups of ADP bind.
Profile of Lead Compound 1. Compound 1 is an attractive
lead molecule, having a high binding affinity for the ATPase
domain of Hsp90 (Kd = 0.54 nM, as determined by isother-
mal calorimetry)31 and potent antiproliferative activity in
HCT116 (human colon cancer) cells of 31 nM (<100 nM
antiproliferative activity in a range of other cancer cell lines).
In HCT116 cells, 1 exhibits the expected cellular mode of
action for a specific Hsp90 inhibitor. This includes knock-
downofthe oncogenic clientprotein cyclin dependent kinase 4
(CDK4) (see preceding paper (DOI: 10.1021/jm100059d)27)32
and an increase in heat shock protein 70 (Hsp70) levels.
Increased expression of Hsp70 occurs as a cellular response
to Hsp90 inhibition and may result in the reduced effective-
ness of a Hsp90 inhibitors due to the cytoprotective nature
of Hsp70.55,56 However, the clinical efficacy displayed by
Description of the ATPase Site and Its Use in Structure-
Based Design. The apo structure of the N-terminus of Hsp90
is characterized by a network of highly ordered water mole-
cules at the base of the adenine binding pocket (Figure 1a).29
On binding of ADP, the endogenous ligand forms hydrogen
bonds with three of these conserved waters and one hydrogen
bond with the protein residue Asp93 (Figure 1b).30 Compound
1 is a potent inhibitor of Hsp90 which binds in the ATPase
binding site (Figure 1c). The 2-hydroxyl group forms hydrogen
bonds with the side chain carboxylate of Asp93 and a water
molecule (mediating to Asp93). The 4-hydroxyl group displaces
one of the conserved water molecules observed in the apo and
ATP bound structures and forms a hydrogen bond with one of
the remaining conserved water molecules. The tertiary amide
induces an important twist in the conformation of the molecule,
forcing the carbonyl out of the plane of the phenyl ring. This
allows the carbonyl to efficiently form a bidentate interaction to
a conserved water and the side chain hydroxyl of Thr184. The
low energy twisted conformation of the tertiary amide also
allows the isoindoline group to form positive hydrophobic
interactions with the backbones of Asp54 and Ala55.

5958 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Woodhead et al.
prototype Hsp90 inhibitors suggests this may not be
limiting.5,57,58
When administered to mice by the intravenous (iv) route, 1
exhibited a high plasma clearance of 115 (mL/min)/kg (Table 3),
above mouse liver blood flow (approximately 90 (mL/min)/kg),
and consequently 1 had a relatively short plasma half-life
(<1 h). We postulated that this was predominantly due to
phase II metabolism of the dihydroxyphenyl moiety.33 This
theory was tested by incubating 1 in the presence of mouse S9
fractions. The compound was shown to be rapidly eliminated
from reaction media, with LC-MS/MS analysis showing the
appearance of directly glucuronidated and sulfated species
over time. These species were also subsequently detected in
the in vivo plasma samples. Despite showing high plasma
clearance, 1 was dosed to mice bearing HCT116 tumor xeno-
grafts to determine the distribution of compound into tumor
tissue and whether a biomarker response could be observed.
A single dose of 1 was administered to mice by the intraper-
itoneal (ip) route at 50 mg/kg, and tumor samples were col-
lected for pharmacokinetic (PK) and pharmacodynamic
(PD) analysis up to 24 h postdose. Reasonably high levels of
compound were measured in all tumor samples such that the
concentration was around 1 μM at 24 h postdose (Table 3),
approximately 50-fold over the cellular IC50 in the HCT116
cell line. This corresponded to a markedly longer reten-
tion of compound 1 in tumor tissue compared with plasma,
where only trace levels were detected from 6 h postdose. Yet
despite reasonably high tumor levels a modest biomarker
response was observed. Increased levels of the molecular
chaperone protein Hsp70 were detected; however, no effect
was observed on levels of oncogenic client proteins such as
CDK4 and Raf-1. In an attempt to determine whether this
limited indication of a PK/PD response was a potential
indicator of in vivo efficacy, 1 was evaluated for antitumor
activity in a mouse xenograft model. 1 was dosed ip b.i.d.
(twice daily) at 10, 20, and 40 mg/kg to nude mice bearing
early stage HCT116 human colon carcinoma xenografts
for 10 days. All dose levels were well tolerated; however,
compound 1 had a modest effect on tumor volume at the
highest dose tested (see Supporting Information).60 This
modest effect in an efficacy model is perhaps consistent with
the PK/PD response and directed the project team toward
identifying compounds with a more significant effect on
oncogenic client protein levels.
Lead Optimization. 2,4-Dihydroxybenzamide Optimization.
From the outset of the lead optimization phase we felt that 1
had sufficient potency, given its good cell-based activity (when
compared to other clinical Hsp90 compounds)5 and that
optimization should focus on the physiocochemical and PK
properties of the molecule. Two separate strategies were
employed. The first involved directly blocking known sites of
metabolism by capping, steric crowding, or modification of the
electronics of the 2,4-dihydroxybenzamide.34,35,63 The second
was to alter the overall properties of the molecule in an attempt
to change its distribution characteristics.61,62 Optimization
of 1 initially focused on attempting to reduce the observed
secondary metabolism effects. Capping the 4-hydroxyl group
by methylation (2) in an attempt to block one site of
glucuronidation resulted in a 30-fold drop in enzyme acti-
vity (Kd=15 nM) but had a more significant impact on cell
activity, resulting in a 80-fold reduction (IC50=2.5 μM). The
Hsp90 X-ray cocrystal structure of 2 shows the molecule
maintaining a similar binding mode to 1; however, a highly
conserved water molecule has been displaced by the methyl
Figure 1. (a) Apo structure of the N-terminal 212 residues of
Hsp90. Of particular note is the network of highly ordered water
molecules at the base of the adenine binding pocket. (b, c) Hsp90
cocrystal structures of ADP (b) and compound 1 (c). Key: red
spheres, water molecules; purple dashed lines, protein-ligand and
water-ligand hydrogen bonds.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5959
group, causing a disruption in the network of stabilizing
hydrogen bonds (see Figure 2a and Figure 2b). In an attempt
to offset the penalty associated with this water displacement
the methyl group was tethered back into a cyclic ether. Dis-
appointingly, the 2,3-dihydrobenzofuran 3 was significantly
less active than 2 (Kd = 1.3 μM) and it proved difficult to
verify its binding mode because of the inability to obtain a
crystal structure. This suggested that the bicyclic ether was
unable to hydrogen-bond effectively to the conserved water
and that a hydrogen bond donor was required. The analo-
gous cyclic amine 4 was synthesized, and this recapitulates the
binding mode of 1, including forming seemingly favorable
interactions with both of the key waters in the active site. This
however did not translate into potent enzyme affinity (Kd =
0.31 μM).
It proved possible to replace the isopropyl group of 1 with
small aliphatic groups, such as ethyl and cyclopropyl (5, 6),
or bulkier groups including tert-butyl and sec-butyl (7, 8)
without losing significant enzyme affinity. None of these
compounds displayed improved cellular activity over 1, and
despite the introduction of steric crowding, no improvement
in PK properties was observed (data not shown).
An alternative optimization strategy for the dihydroxyben-
zamide core was to modify the electronics of the two hydroxyl
groups. Compound 1 has measured phenolic pKa values of
8.6 and 10.9. Introduction of a chlorine at the 5-position of
the benzamide to give 9 had little effect on enzyme affinity
(Kd=4 nM) but did reduce cellular potency (IC50=3.5 μM).
No effect on PK was observed. Despite lowering the calcu-
lated pKa of the 4-phenolic group by 2 log units, 9 had high
plasma clearance (data not shown). Structural data suggested
that there was little room for the introduction of further
substituents; however, it was possible that a small substituent
such as a fluorine may be tolerated. Fluorination of 1 resulted
in the readily separable mixture of compounds 10 and 11.
Compound 10 displayed improved PK properties with a low
intrinsic clearance of just 4 (μL/min)/mg protein in mouse
S9 fractions. While both compounds 10 and 11 showed
an indication of weak cellular activity (30 μM and 60% at
100 μM, respectively) we wereunable to determine the binding
affinities for Hsp90 by ITC or to obtain protein ligand crystal
structures presumably because they bind so weakly. There are
two possible explanations for this drop in affinity. The first
is the electronic impact that a fluorine atom has on the two
phenolic groups and the carbonyl of the tertiary amide, as all
of these groups form crucial hydrogen bonding interaction
either with the protein directly or through conserved water
molecules. The other possibility, particularly for compound
10, is a steric effect. If the compound bound in an analogous
fashion to 1 (Figure 1c), the electronegative fluorine atom
would be within van der Waals contact of the negatively
charged carboxylate of Asp93, an electronically unfavorable
interaction.
Modifications made to the dihydroxybenzamide core,
with the exception of compound 10 (which loses activity),
had little impact on PK. This region of the molecule is
important for providing high binding affinity, and as such,
conservative modifications reduce activity. As an alternative
strategy, optimization changed focus to the isoindoine ring
system.
Isoindoline Optimization. Further optimization of 1 turned
toward the bicyclic isoindoline ring. Substitution here might
provide two benefits. First introduction of a polar positively
charged group should alter the distribution properties of this
Figure 2. (a) Hsp90 cocrystal structure of 2. A conserved water
molecule at the base of the adenine pocket has been displaced
by the 4-methoxy group, which can be observed more readily
in (b) when overlaid with the cocrystal structures of 1. (c) Hsp90
cocrystal structure of 4. The water mediated H-bonding network
has been restored; however, a significant improvement in bind-
ing affinity is not observed. Key: red/cyan spheres, water mole-
cules; dashed lines, protein-ligand and water-ligand hydrogen
bonds.

5960 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Table 2. Isoindoline Structure-Activity Relationships
Woodhead et al.
Figure 3. Tumor profiles of compounds 1, 17 and 18 following ip
administration in mouse.
Table 3. Mouse PK for Compounds 1, 17, and 18
HCT116 tumor
concn 24 h after
iv dose
(mg/kg)
plasma clearance
((mL/min)/kg)
ip dose
(mg/kg)
compd
ip dose (μM)
1
1
115
130
170
50
1
17
18
10
10
40a
80a
6.5
11
a The doses of 17 and 18 used for tumor distribution experiments
represent efficacious doses.
simple modifications perhaps not surprisingly had little effect
on the in vitro PK of the series; for example, 12 and 15 had
high intrinsic clearance following incubation in mouse S9
fractions (62 and 77 (μL/min)/mg protein).
A basic moiety was introduced into the molecule to eva-
luate its effect on compound distribution in vivo.36,61,62 As
compound 14 indicates, substitution at the 5-position of the
isoindoline was well tolerated. We consequently synthesized
a range of groups at this position focusing on basic and other
solubilizing moieties. Introduction of morpholine was well
tolerated (16, Kd=2.7 nM); however, a 10-fold drop in cell
activity was observed compared to the parent compound 1.
Substitution with other solubilizing groups such as N-methyl-
piperazine (17), N,N-dimethylethanolamine (18), 2-methoxy-
ethoxy (19), and 4-morpholinylmethyl (20) resulted in both
good enzyme and cell activity. Compounds with basic func-
tionality such as 17 and 18 were observed to have increased
tumor retention when dosed in mice, compared to1 (Figure 3).
For compound 18 this resulted in tumor concentrations
of approximately 11 μM at 24 h (Table 3) following a
dose of 80 mg/kg by the ip route, which was approximately
500-fold greater than the cellular IC50. Encouragingly this
tumor concentration translated to a positive biomarker res-
ponse, including knockdown of the client proteins CDK4
and Raf-1 and an increase in levels of Hsp70 (see Figure 5
for the cellular mode of action for compound 18). Com-
pounds 17 and 18 were evaluated for their in vivo antitumor
activity in nude BALB/c mice bearing early stage HCT116
human colon carcinoma xenografts (mean starting volume
of approximately 100 mm3). In this study the hydrochloride
salts, dissolved in 0.9% saline were administered by the ip
route once daily for 8 consecutive days. Tumor growth
inhibition for the 80 mg/kg dose group at the end of the
experiment was 81% for 17 (% T/C=19, measured on day
10) and 63% for 18 (% T/C=37, measured on day 12), with
compound 17 showing a cytoreductive effect (see Support-
ing Information).60 This corresponded to a tumor growth
delay of 10.5 and 11.4 days for compounds 17 and 18,
respectively.59
a Compounds were assayed two or more times. b Compounds were
assayed n = 1. The cell assay was used as the primary screening assay.
c pKa values were measured by Pharmorphix, Ltd., Cambridge, U.K.
series of molecules. A common feature of basic drugs such as
the calcium channel blocker amlodipine is high tissue affi-
nity, as characterized by high apparent volumes of distribu-
tion.61,62 Second substitution of the isoindoline ring may
block potential sites of oxidative metabolism (although oxi-
dation of the isoindoline ring system had not previously been
observed for this series).36 A feature of benzylic systems is the
potential for cytochrome P450 mediated hydroxylation at
the paraposition; consequently blocking the 5 or 6 position of
the isoindoline ring should prevent this.63 Structural infor-
mation suggested that modification of the four unsubstituted
positions of the six-membered ring ought to be tolerated, with
growth vectors toward the solvent exposed surface of the
protein. Substitution at the 1 and 3 positions of the isoindo-
line, although possible, seemed less attractive fortwo reasons.
First, the protein architecture is very close, and second, a
chiral center would be introduced into the molecule, which
would complicate the synthesis. Simple modifications such as
introduction of halogens at the 4, 5, and 7 positions of the
isoindoline (compounds 12, 13, 14, and 15) were tolerated,
maintaining both enzyme and cell activity (Table 2). These

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5961
prepared. Properties that were evaluated during this phase
of the project included enzyme and cell potency, in vivo PK,
physicochemical properties (pKa, log D7.4, and aqueous solu-
bility), and off target effects such as P450 and hERG binding.
This program of optimization looked at varying a number of
parameters that included changing the point of substitution
to the isoindoline, altering the length and flexibility of linker
between the aromatic system and the basic group, sterically
crowding the basic group and modifying its pKa, and mod-
ifying the lipophilicity of the system. On the basis of the
properties of the initial compounds, attaching the correct
polar positively charged solubilizing group to the isoindoline
template could give us an excellent balance of properties for a
clinical development candidate.
Evaluating alternative isomers with essentially the same
physicochemical properties was an attractive starting point.
Consequently compounds substituted at the 4-position of the
isoindoline were synthesized, including the direct analogues of
17 and 18, compounds 22 and 24. These analogues had high
Hsp90 affinity (<10 nM), potent cell activity (<100 nM),
limited P450 binding (CYP3A4 < 50% inhibition at 10 μM),
and similar hERG affinity to 17 and 18. Replacing the ter-
tiary amine of previous compounds with a primary amine
gave compounds such as 25 and 26, with increased basicity
and reduced lipophilicity. This change was well tolerated
(Hsp90 Kd < 5 nM) and caused a reduction in hERG activity,
although these compounds had increased P450 binding (<10
nM against the 2C9 and 2C19 isoforms). Sterically crowding
compound 26 with bulky lipophilic groups (27 and 28),
although potent against the enzyme and in cells, led to in-
creased hERG inhibiton (97% and 92% at 30 μM, respec-
tively) compared to 17 and 18, possibly caused by a signifi-
cant increase in lipophilicity (cLogP > 4).
More rigid compounds such as piperazine amide 29 and
tertiary alcohol 30 showed potent activity in the cell based
assay (<100 nM), low P450 binding, and low activity in the
hERG patch clamp assay. These compounds are significantly
more polar than previous analogues (log D7.4 = 2.1 and 1.7,
respectively), indicating that side chain flexibility and polar-
ity are perhaps a contributing factor to hERG activity.
Replacing the amide linker of 29 with an amine (31) was
also well tolerated. This compound maintained polarity
(log D7.4=1.7) while introducing more conformational flexi-
bility. A similar P450 and hERG profile is observed to 17
and 18 (CYP3A4 34% inhibition at 10 μM; hERG = 72%
inhibition at 30 μM). The des-methyl analogue, cyclic sec-
ondary amine 32, is one of the most polar compounds we
synthesized in this series (log D7.4 =0.7), has good enzyme
and cell activity, very low activity against hERG (20% at
30 μM), and limited activity against CYP2C9 and 2C19
(approximately 1 μM against each). To test what effect the
introduction of other cyclic secondary amines would have,
the des-methyl analogue of 17 was prepared. Compound 33
had subnanomolar binding affinity to Hsp90 and potent
cellular activity and displayed similar hERG inhibitory acti-
vity to 17 (hERG=72% at 30 μM).
Figure 4. Computational analysis of side chain energy minima for
basic compounds 17, 18, 20, 21, 24, 25, 27-30, 32-35. A Monte
Carlo conformational search was performed on each molecule using
the MMFF force field as implemented in the Spartan ’04 modeling
package. The global minimum of each molecule was overlaid with
the others using only the resorcinol and the isoindolines atoms. The
colored spheres indicate where the basic centers of each of the
compounds 17, 18, 20, 21, 24, 25, 27-30, 32-35 is positioned. Key,
approximated hERG IC50: red spheres, <3 μM; orange spheres,
3-30 μM; green spheres, g30 μM.
Figure 5. Cellular biomarkers effects of compounds 18 and 35.
Immunoblot of HCT116 cell lysates treated for 18 h with the indi-
cated compound doses of 18 (a) or 35 (b). Client proteins: Raf-1 and
CDK4. Cochaperone: Hsp70. Loading control: glyceraldehyde
3-phosphate dehydrogenase (GAPDH).
Late Stage Optimization. Although compounds 17 and 18
were synthesized early in the project, they were promising
advanced lead molecules. Both compounds possessed good mec-
hanism based cell activity, potent in vivo efficacy, minimal
binding to the major cytochrome P450 isoforms, and selectivity
over a small in-house panel of enzymes. Compounds 17 and 18
both showed weak inhibition of hERG potassium channel
current at 3 μM (41% and 45%, respectively) in a patch clamp
assay. We felt it unlikely that this level of activity posed a
developability risk, particularly when considered alongside high
cellular potency and low plasma exposure at an efficacious dose.
As part of a preclinical candidate selection phase, a
number of closely related analogues of 17 and 18 were
Analogues of the methylene linked compound 20 were
also synthesized. These compounds were designed to in-
crease the pKa of the basic center and reduce lipophilicity.
As can be seen from Table 4, the N,N-dimethyl analogue 34
and the N-methylpiperazine analogue 35 possess good activ-
ity in the enzyme (<5 nM) and cell based assays (<100 nM),
reduced lipophilicity (log D7.4 = 2.1 and 2.7, respectively),
and increased basicity (pKa=9.2 and 7.7, respectively). The

5962 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Table 4. Substituted Isoindoline SAR
Woodhead et al.
Table 5. Mouse PK of Compound 35
half-life (h)
plasma
clearancea
((mL/min)/kg)
volume of
distributiona
(L/kg)
plasmaa bloodb muscleb tumorb
0.9-3.2 4.7 3.0 65
113-200
11-13
a The iv dose was 2-10 mg/kg. b Following an ip dose of 60 mg/kg.
P450 profiles for both these analogues were favorable. Com-
pound 34 caused approximately 50% inhibition at 10 μM
against 2C9 and 2C19, while 35 was <50% against all the
major P450 isoforms.
From this program of optimization we identified a number
of compounds including 29, 30, 31, 33, 34, and 35 possessing a
good balance of properties, including moderate to low human
plasma protein binding (ranging from 64% to 93%), accep-
table risk profiles against the major CYP450isoforms, and the
hERG ion channel, which were suitable candidates for more
detailed evaluation alongside the original compounds 17
and 18. Compounds 29, 30, 31, 33, 34, and 35 were profiled
initially for tolerabilityandthen efficacy innudeBALB/c mice
bearing early stage HCT116 human colon carcinoma xeno-
grafts (mean starting volume of approximately 100 mm3).
Despite having good in vitro properties including low plasma
protein binding (71% in mouse plasma), 29 produced a
reduced effect in an efficacy study, when compared to leads
17 and 18. Compounds 30, 31, 33, 34, and 35, however, de-
monstrated comparable efficacy at tolerated doses to 17 and
18, with a corresponding PK/PD response confirming an
Hsp90 driven mechanism of action. As previously described,
a common feature of this series of compounds was their high
plasma clearance (Tables 3 and 5). Compounds 17, 18, and 35
all showed glucuronide and sulfate conjugates when incu-
bated in S9 fractions (intrinsic clearance of 41, 34, and 46 (μL/
min)/mg of enzyme, respectively). This characteristic was
observed for all these late stage compounds, meaning that
they could not easily be differentiated using plasma kinetics.
Instead, these seven compounds were further assessed
for developability, with particular emphasis on dosing via
iv infusion. Key criteria that were considered during this
phase of the project included solubility of the free base and
hydrochloride salt in aqueous buffers, stability of a range of
pharmaceutically acceptable salts, and synthetic tractability
(see Supporting Information for details).
Compound 35 proved to have the best overall properties; the
hydrochloride, methanesulfonate, and L-lactate salts all dis-
played high solubility in a number of aqueous buffers which
spanned a range of pH values (solubility of >30 mg/mL in
0.1 M sodium acetate buffer at pH 5.5). Exposure to a range
of forcing conditions (including pH 1 and 14 at 40 ꢀC) during a
1 week stability study showed no degradation of 35. The
compound was readily scalable using the original lead optimi-
zation route. A number of other routes were also developed,
which will be detailed in a future publication.
We carried out an analysis of the hERG data for this series
of compounds, and there appear to be a number of subtle
factors that are interlinked and contribute to hERG affinity.
It is noted that the activity range is small and most of these
compounds have weak binding affinities. The data suggest
that important factors for reducing hERG affinity appear to
be the length of linker to the basic group, where two or five
atoms seemed to be optimal (34 and 35), and a minimum
amount of conformational flexibility (29 and 30). Figure 4
shows where the basic center is positioned for each minimized
a Compounds were assayed two or more times. b Compounds were
assayed n = 1. The cell assay was used as the primary screening assay.
c The log D and pKa values were measured by Pharmorphix, Ltd.,
Cambridge, U.K. d The hERG patch clamp assay was carried out by
Quiniltes, Ltd., U.K., using stably transfected HEK293 cells and
PatchXpress 7000A. Data are supplied as n = 1.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5963
structure. The compounds with the least hERG activity
appear to be clustered relatively closely together. Little
correlation could be drawn between the pKa values of active
and inactive compounds. The data for compounds 27 and 28
suggested that high lipophilicity gave rise to greater hERG
affinity, whereas compounds 26 and 32 with log D7.4 < 1
suggested that increased polarity helps to minimize hERG
affinity.37-41
Characterization of Compound 35. Cell-Based Activity.
Compound 35 is a potent inhibitor of HCT116 cell prolifera-
tion (used as a primary screen during lead optimization).42
Following 72 h of exposure, 35 potently inhibited the pro-
liferation of a panel of human tumor cell lines (well over 100
cell lines have been tested), showing IC50 < 100 nM against
more than 50 (data not shown).
The mechanism of action of 35 in cells was investigated
by monitoring the expression levels of a range of client
proteins, following treatment with 35 for 18 h. These studies
indicated that the expression of the oncogenic client pro-
teins Raf-1 and CDK4 is inhibited around 30 nM in the
HCT116 cell line, which is similar to the IC50 for prolifera-
tion (48 nM). The molecular chaperone Hsp70 is up-regu-
lated at 10 nM and is a more sensitive cellular response
to the inhibition of Hsp90. This effect is widely utilized as a
biomarker readout for Hsp90 inhibition in both cellular and
in vivo experiments.5,56
PK Study. As summarized in Table 5, the plasma clearance
of 35 in female BALB/c mice after iv dosing ranged from 113 to
200 (mL/min)/kg with a half-life of between 0.9 and 3.2 h.
Volume of distribution was 11-13 L/kg. Compound 35 was
found to partition preferentially into red blood cells, both in
vitro and in vivo (blood/plasma ratio up to 3:1), which may, at
least partially, account for the higher than liver blood flow
clearance measured in plasma. The tissue distribution of com-
pound 35 was investigated following an ip dose of 60 mg/kg
and was shown to have a half-life in HCT116 tumors of 65 h
and a tumor concentration of 9.3 μM after 24 h. This appeared
to be a selective effect for tumor, as the half-life in muscle
(taken as a surrogate normal tissue) was just 3 h, similar to
plasma. Encouragingly 35 showed some oral bioavailability
in mouse (31%) despite the high plasma clearance (113-
200 (mL/min)/kg). Protein binding in mouse plasma was 91%.
In Vivo Antitumor Activity. Compound 35 was evaluated
for its in vivo antitumor activity in nude BALB/c mice bearing
early stage HCT116 human colon carcinoma xenografts with
a mean starting volume of approximately 100 mm3 (Figure 6).
In this study the L-lactate salt of 35, dissolved in 17.5%
hydroxypropyl-β-cyclodextrin was administered by the ip
route using a repeated cycle of dosing of once per day for 3
days, no dose for 3 days, for four dosing cycles. Tumor
growth inhibition at the end of the dosing phase (day 21)
Figure 6. In vivo mouse efficacy study for 35 using HCT116,
human colon cancer xenografts (60 mg/kg ip 3 days on and 3 days
off for four cycles). Key: blue triangles, compound dosing time-
points; blue squares, 35 at 60 mg/kg; red diamonds, vehicle controls.
Scheme 1a
a Reagents and conditions: (a) substituted isoindoline, EDC, HOBt, DMF; (b) 10% Pd/C, EtOH, H2; (c) SnCl2 2H2O, EtOH; (d) EtNiPr2, nBu4NI,
NMP, [Cl(CH2)2]X; (e) (CH3)2SO4, K2CO3, CH3CN; (f) SelectFluor, CH3CN.
3

5964 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Woodhead et al.
Scheme 2a
a Reagents and conditions: (a) SOCl2, MeOH; (b) K2CO3, BrCH2C(CH3)dCH2, CH3CN; (c) nBu3SnCl, NaBH3CN, AIBN, C6H6; (d) NaOH,
MeOH, H2O; (e) (COCl)2, DMF, DCM; (f) isoindoline, NEt3, DCM; (g) LiOH, THF, MeOH, H2O; (h) isoindoline, EDC, HOBt, NEt3, THF, DCM;
(i) Cs2CO3, BrCH2C(CH3)dCH2, DMF; (j) Pd(OAc)2, HCO2Na, NEt3, nBu4NCl, DMF; (k) BBr3, DCM.
Scheme 3a
a Reagents and conditions: (a) K2CO3, BnBr, DMF; (b) KOH, MeOH, H2O; (c) isoindoline, EDC, HOBt, DCM; (d) boronate, Pd(dppf)Cl2, Cs2CO3,
THF, H2O; (e) 10% Pd/C, EtOH, H2; (f) BBr3, DCM.
was 78% for the 60 mg/kg dose level (% T/C = 22). Animals
were monitored for tumor regrowth after cessation of dosing,
and a tumor growth delay of 17 days was observed for this
dosing schedule.59,60 A more extensive biological character-
ization of compound 35 including additional in vivo efficacy
studies using Hsp90 sensitive cell lines and a more detailed
evaluation of PK/PD effects will be published separately.
Chemistry. Compound 1 was prepared by coupling the key
intermediate 2,4-bis-benzyloxy-5-isopropenylbenzoic acid
36 with isoindoline 37a using standard EDC/HOBt coupling
conditions (Scheme 1). Catalytic hydrogenation simulta-
neously removed the benzyl protecting groups and reduced
the isopropenyl group to the isopropyl, as described in the
preceding paper (DOI: 10.1021/jm100059d).27 Compounds
12-15, 20, 23, 24, 30, and 34 were prepared in an analogous
manner starting from the appropriate isoindoline. 2 was
prepared by direct methylation of 1 under basic condi-
tions, followed by chromatographic separation of the two
regioisomers. The bicyclic ether 3 was prepared by the tin
mediated free radical cyclization of the allylic ether 41
followed by saponification of the ester and coupling to
isoindoline (Scheme 2). The indoline analogue 4 was pre-
pared by a similar route starting from the aniline 43. Protec-
tion of the phenol was required, as the methyl ether and
cyclization were achieved using palladium catalysis. Com-
pounds 5, 6, and 7 were prepared by Suzuki cross coupling of
the bis-benzyl protected aryl bromide 49a with the appro-
priate activated boron species followed by catalytic hydro-
genation to remove the benzyl protecting groups (Scheme 3).
Compounds 8 and 9 were prepared in an analogous man-
ner to 1 starting from the appropriate 5-substituted benzoic
acid derivative (46b or 46c). Compounds 10 and 11 were
prepared directly from 1 by electrophilic aromatic fluorori-
nation using SelectFluor (Scheme 1). This produced a mix-
ture of the 3 and 6 fluorinated compounds which were readily
separable by flash column chromatography. 16 and 17 were

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5965
Scheme 4a
a Reagents and conditions: (a) K2CO3, R-X, DMF; (b) 10% Pd/C, EtOH, H2; (c) saturated HCl/EtOAc; (d) RR0CdO, NaBH(OAc)3, AcOH, DCE.
Scheme 5a
a Reagents and conditions: (a) K2CO3, BnBr, DMF; (b) 10% Pd/C, EtOH, H2; (c) NaOH, MeOH, H2O; (d) N-methylpiperazine or HN-
(OMe)Me HCl, EDC, HOBt, NEt3, DMF; (e) LiAlH4, THF; (f) N-methylpiperazine, NaBH(OAc)3, AcOH, DCE.
3
prepared by coupling 5-nitroisoindoline to 36 followed by
tin reduction to aniline 39. Microwave assisted cyclization
using the appropriate bis-alkylating agent under basic con-
ditions followed by catalytic hydrogenation gave the mor-
pholine 16 and N-methylpiperazine 17. Compounds 18 and
19 were prepared by alkylation of 50 with 2-dimethylami-
noethyl chloride and 2-chloroethyl methyl ether, respec-
tively, followed by removal of the benzyl protecting groups
(Scheme 4). Compounds 21, 25, and 26 were prepared in an
analogous manner, with 25 and 26 requiring a final BOC
deprotection under acidic conditions. 27 and 28 were pre-
pared directly from 25 by reductive amination, using sodium
triacetoxyborohydride and cyclopentanone or 3,3-dimethyl-
butyraldehyde, respectively. Compounds 22 and 31-33 were
prepared via palladium catalyzed Buchwald chemistry from
the relevant 4- or 5-bromoisoindoline intermediate (52 and
53) followed by deprotection (Scheme 5). Coupling of methyl
isoindoline-5-carboxylate to 36 gave intermediate 54. Sapo-
nification of the methyl ester followed by carbodiimide medi-
ated coupling with N-methylpiperazine, and debenzylation
gave compound 29. It proved difficult to convert 29 to 35,
and a number of conditions were attempted to reduce the
amide, but none of which were successful, even as the pro-
tected precursor. An alternative route from the Weinreb
amide 56 was used. Reduction of 56 to the aldehyde using
lithium aluminum hydride in THF followed by reductive
amination using N-methylpiperazine and sodium triacetoxy-
borohydride and final deprotection gave 35.
Conclusions
Using fragment screening and subsequent structure guided
design, we have identified a series of 2,4-dihydroxybenza-
mides with very high affinity for the ATPase site of Hsp90 and
potent Hsp90 driven cell-based activity. Two approaches were
taken in an effort to optimize compound 1. The first was
directed at blocking proposed sites of metabolism by capping,
sterically crowding, or modifying the electronics of the 2,4-
dihydroxybenzamide. This approach, while generally main-
taining activity against Hsp90, did not significantly modulate
the PK profile of 1. The second approach was to alter the
overall physical properties of the molecule in an attempt to
change its distribution characteristics. Substitution of the iso-
indoline ring system with a basic group gave compounds such
as 17 and 18, which, although still having high plasma clear-
ance, exhibited high tumor levels, potent biomarker modula-
tion, and efficacy in a mouse cancer model. A number of closely
related basic analogues of compounds 17 and 18 were synthe-
sized, and seven compounds were evaluated in a preclinical
candidate selection phase. Key parameters that were investi-
gated during this phase of the project were in vivo target
modulation, predicted human dose, selectivity/off-target ef-
fects, solubility, stability, formulation, and synthesis. Follow-
ing this process, compound 35 was selected as a preclinical
development candidate. 35 has potent affinity for Hsp90 (Kd =
0.5 nM) by binding in the ATPase site at the N-terminus of
Hsp90 (Figure 7). This enzyme affinity translates into good

5966 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Woodhead et al.
Agilent 1200SL-6140 LC/MS system using positive-negative
switching, both using a mobile phase of CH3CN/water with
0.1% formic acid. The purity is g95% unless otherwise expli-
citly stated.
2,4-Bis-benzyloxy-5-isopropenylbenzoic acid was prepared
as described previously in WO 2006/109075 A2. Experimental
details for noncommercially available starting materials (for
example, the substituted isoindolines) are available in the Sup-
porting Information.
(1,3-Dihydroisoindol-2-yl)-(2,4-dihydroxy-5-isopropylphenyl)-
methanone (1). A mixture of 2,4-bis-benzyloxy-5-isopropenyl-
benzoic acid 36 (187 mg, 0.5 mmol), EDC (116 mg, 1.2 equiv),
HOBt (81 mg, 1.2 equiv), and isoindoline (90 mg, 1.5 equiv) in
CH2Cl2 (10 mL) was stirred at room temperature overnight. The
organic layer was washed successively with 2 M HCl and 2 M
NaOH, then dried (MgSO4), filtered, and evaporated in vacuo.
The crude product 38a (220 mg) was dissolved in MeOH (8 mL),
treated with 10% Pd/C (20 mg), then subjected to atmospheric
hydrogenation for 16 h. The mixture was filtered through Celite
and washed with MeOH. The combined filtrates were concen-
trated in vacuo. The residue was triturated with Et2O, filtered,
and then dried under vacuum to give the title compound as an
off-white solid (50 mg, 33%). 1H NMR (DMSO-d6) δ 10.03 (s,
1H), 9.63 (s, 1H), 7.43-7.15 (m, 4H), 7.03 (s, 1H), 6.40 (s, 1H),
4.77 (br s, 4H), 3.15-3.09 (m, 1H), 1.14 (d, 6H). MS: [M þ H]þ
298. HPLC purity, >99%.
Figure 7. Protein-ligand crystal structure of compound 35 bound
in the ATPase site of Hsp90. The key binding features are identical
to those of lead compound 1 (Figure 1). The pendent N-methylpi-
perazine group binds in a highly solvent exposed region and
consequently has little impact on enzyme affinity. It does, however,
have a marked effect on the physicochemical and pharmacokinetic
properties of the molecule, resulting in elevated tumor levels,
biomarker modulation, and good efficacy in a mouse cancer model.
Methyl-2,3-dihydro-1H-isoindole-5-carboxylate Trifluoroace-
tate (37n). To an ice bath cooled solution of 3,4-dimethylbenzoic
acid (10 g, 67 mmol) in MeOH (100 mL) was added dropwise
thionyl chloride (5.84 mL, 81 mmol). The reaction mixture was
warmed to room temperature and then stirred for a further 24 h.
The reaction mixture was concentrated in vacuo and then
azeotroped in vacuo with toluene (ꢀ3) to give 3,4-dimethylben-
activity against a range of human tumor cell lines and statis-
tically significant cytostatic activity in a mouse anticancer
model. 35 has a clean profile against the major cytochrome
P450 isoforms (e40% inhibition at 10 μM for 1A2, 2D6, 3A4,
2C9, 2C19), low hERG affinity, and low protein binding (85%
in human plasma) and has high thermodynamic aqueous
solubility as the methanesulfonic, hydrochloride, or L-lactate
salt (>30 mg/mL in 0.1 M acetate buffer). Its synthetic tract-
ability makes it readily amenable to large scale synthesis. On
the basis of this and further data (to be published in a later
paper), compound 35 (AT13387) as the L-lactate salt was
selected as a clinical candidate and has subsequently entered
clinical development.
1
zoic acid methyl ester as a brown oil (10.9 g, 99%). H NMR
(DMSO-d6) δ 7.74 (d, 1H), 7.69 (dd, 1H), 7.28 (d, 1H), 3.82 (s,
3H), 2.29 (s, 3H), 2.28 (s, 3H).
A solution of methyl 3,4-dimethylbenzoate (10.9 g, 66 mmol),
N-bromosuccinimide (24.0 g, 133 mmol), and benzoyl peroxide
(500 mg, 2 mmol) in CCl4 (75 mL) was heated at reflux for 24 h.
Further N-bromosuccinimde (12 g, 67 mmol) and benzoyl
peroxide (250 mg, 1 mmol) were added, and the reaction mixture
was heated at reflux for a further 4 h. The reaction mixture was
filtered and then the mother liquour concentrated in vacuo to
give 3,4-bis-bromomethylbenzoic acid methyl ester as a pale-
orange oil (26.7 g, contains 20% of two trisubstituted bromo
compounds). The product was taken crude to the next step.
To a vigorously stirred solution of crude 3,4-bis-bromethylben-
zoic acid methyl ester (26.7 g, assume 66 mmol) and Na2CO3 (55.2
g, 521 mmol) in acetone (480 mL) and water (80 mL) was added
dropwise a solution of 4-methoxybenzylamine (10.4 mL, 80 mmol)
in acetone (80 mL). The reaction mixture was stirred vigorously for
a further 6 h. Acetone was evaporated in vacuo. The residue was
partitioned between EtOAc and HCl (2 N). The organic layer was
again washed with HCl (2 N). The aqueous layers were combined,
washed with EtOAc, and basified to ∼pH 10 with solid NaHCO3.
The product was extracted with CH2Cl2 (ꢀ2). The combined
organic layers were combined, dried (MgSO4), and evaporated in
vacuo. To the residue was added HCl in dioxane (4 M, 10 mL). The
material was concentrated in vacuo and then azeotroped twice with
toluene/MeOH (2:1) to give predominately 2-(4-methoxybenzyl)-
2,3-dihydro-1H-isoindole-5-carboxylic acid methyl ester as a light-
Experimental Section
Chemistry. Reagents and solvents were obtained from com-
mercial suppliers and used without further purification. Thin
layer chromatography (TLC) analytical separations were con-
ducted with E. Merck silica gel F-254 plates of 0.25 mm thick-
ness and were visualized with UV light (254 nM) and/or stained
with iodine, potassium permanganate, or phosphomolybdic
acid solutions followed by heating. Standard silica gel chroma-
tography was employed as a method of purification using the
indicated solvent mixtures. Compound purification was also
carried out using a Biotage SP4 system using prepacked dis-
posable SiO2 cartridges (4, 8, 19, 38, 40, and 90 g sizes) with
stepped or gradient elution at 5-40 mL/min of the indicated
solvent mixture. Proton nuclear magnetic resonance (1H NMR)
spectra were recorded in the deuterated solvents specified on
a Bruker Avance 400 spectrometer operating at 400 MHz.
Chemical shifts are reported in parts per million (δ) from the
tetramethylsilane internal standard. Data are reported as fol-
lows: chemical shift, multiplicity (br = broad, s = singlet, d =
doublet, t = triplet, sp = septet, m = multiplet), integration.
Compound purity and mass spectra were determined by a
Waters Fractionlynx/Micromass ZQ LC/MS platform using
the positive electrospray ionization technique (þES) or an
1
green solid hydrochloride salt (1.22 g, 6% over two steps). H
NMR (DMSO-d6) δ 11.74 (s, 1H), 8.01 (d, 1H), 7.96 (dd, 1H), 7.59
(dd, 2H), 7.55 (d, 1H), 7.04 (dd, 2H), 4.73-4.49 (m, 4H), 3.87 (s,
3H), 3.80 (s 3H).
A solution of 2-(4-methoxybenzyl)-2,3-dihydro-1H-isoindole-
5-carboxylic acid methyl ester (215 mg, 0.65 mmol) in anisole
(200 μL) and TFA (1 mL) was heated in a microwave (max 200 W)
at 140 ꢀC for 30 min. The reaction mixture was partitioned bet-
ween water and CH2Cl2. The aqueous layer was washed with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5967
CH2Cl2, evaporated in vacuo, and then azeotroped twice with a
mixture of MeOH and toluene (1:2) to give methyl-2,3-dihydro-
1H-isoindole-5-carboxylate trifluoroacetate as a pale-orange so-
lid (105 mg, 91%). 1H NMR (DMSO-d6) δ 9.70 (br. s, 2H), 8.03
(d, 1H), 7.97 (dd, 1H), 7.57 (d, 1H), 4.60 (d, 4H), 3.88 (s, 3H).
2-(2,4-Bis-benzyloxy-5-isopropylbenzoyl)-2,3-dihydro-1H-iso-
indole-5-carboxylic Acid Methyl Ester (54). A mixture of 2,4-bis-
benzyloxy-5-isopropenylbenzoic acid 36 (3.12 g, 8.35 mmol),
EDC (1.92 g, 1.2 equiv), HOBt (1.35 g, 1.2 equiv), methyl-2,3-
dihydro-1H-isoindole-5-carboxylate trifluoroacetate 37n (2.43 g,
8.35 mmol), and NEt3 (6 mL, 5 equiv) in DMF (20 mL) was
stirred at room temperature for 48 h and then evaporated. The
residue was partitioned between EtOAc and 5% citric acid solu-
tion. The organic layer was separated and washed successively
with saturated sodium bicarbonate solution and brine, then
dried (MgSO4), filtered, and evaporated in vacuo. The crude
material was purified by flash column chromatography, eluting
with 20%, 25%, then 33% EtOAc in 40-60 petroleum ether.
Product containing fractions were combined and evaporated in
vacuo to give the title compound as an off-white solid (3.2 mg,
72%). 1H NMR (DMSO-d6) δ 7.99-7.82 (s, 2H), 7.57-7.29 (m,
8H), 7.29-7.20 (m, 3H), 7.13 (s, 1H), 7.01 (s, 1H), 5.24 (s, 1H),
5.24 (s, 2H), 5.20 (s, 2H), 5.09 (d, 2H), 4.85 (s, 2H), 4.63 (s, 2H),
3.86 (d, 3H), 2.05 (s, 3H). MS: [M þ H]þ 534.
2H), 3.27-3.16 (m, 1H), 2.67-2.39 (m, 8H), 2.31 (s, 3H), 1.23 (d,
6H). MS: [M þ H]þ 410. HPLC purity, 95%.
Full Chemistry Experimental Procedures, in Vitro and in Vivo
Biological Assays, X-ray Crystallographic Data, and DMPK,
Solubility, and Stability Studies. Detailed experimental data can
be found in the Supporting Information.
Acknowledgment. The authors thank Rachel McMenamin
for bioassay support, Charlotte Cartwright for producing
Figures 3 and 6, and Anne Cleasby, Lyn Leaper, Neil
Thompson, John Lyons, Chris Johnson, and Harren Jhotti
for their useful comments on the manuscript and for provid-
ing support to the project.
Note Added after ASAP Publication. This paper was pub-
lished on the web on July 28, 2010 with a production error in
the Results and Discussion section. The revised version was
published on July 30, 2010.
Supporting Information Available: Experimental details of
syntheses, biological assays, and DMPK studies. This material
is available free of charge via the Internet at http://pubs.acs.org.
2-(2,4-Bis-benzyloxy-5-isopropylbenzoyl)-2,3-dihydro-1H-iso-
indole-5-carboxylic Acid Methoxymethylamide (56). A solution
of 2-(2,4-bis-benzyloxy-5-isopropylbenzoyl)-2,3-dihydro-1H-iso-
indole-5-carboxylic acid methyl ester 54 (390 mg) in MeOH
(10 mL) and 2 M NaOH (10 mL) was heated at 50 ꢀC for 48 h and
then evaporated in vacuo. The residue was acidified with 2 M
HCl. The solid was collected by filtration and washed with water
to give the corresponding acid as a colorless solid (255 mg). MS:
[M þ H]þ 520. A solution of this acid (1.76 g, 3.39 mmol), EDC
(0.78 g, 4.06 mmol), HOBt (0.55 g, 4.06 mmol), Et3N (1.0 mL,
6.78 mmol), and N,O-dimethylhydroxylamine hydrochloride
(0.36 g, 3.72 mmol) in DMF (20 mL) was stirred at room tem-
perature for 48 h, then evaporated under vacuum. The crude
material was dissolved in EtOAc and washed twice with saturated
NaHCO3, then brine, dried (MgSO4), filtered, then evaporated in
vacuo to give the title compound (1.84 g). MS: [M þ H]þ 563.
(2,4-Dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin-1-
ylmethyl)-1,3-dihydroisoindol-2-yl]methanone (35). A solution
of 56 (0.226 g, 0.4 mmol) in THF (5 mL) was cooled to 0 ꢀC under
N2 and treated with LiAlH4/THF (0.3 mL, 0.3 mmol, 1 M) and
stirred for 1 h. Further LiAlH4 (0.05 mL) was added, and the mix-
ture was stirred for 30 min. The reaction was quenched with satu-
rated KHSO4 solution, and the product extracted with EtOAc.
The organic layer was dried (MgSO4), filtered, and evaporated in
vacuo to give 2-(2,4-bis-benzyloxy-5-isopropylbenzoyl)-2,3-dihy-
dro-1H-isoindole-5-carbaldehyde (0.2 g). MS: [M þ H]þ 504.
To a solution of this aldehyde (0.316 g, 0.63 mmol) and N-
methylpiperazine (63 mg, 0.63 mmol) in CH2Cl2 (10 mL) were
added AcOH (38 mg, 0.63 mmol) and NaBH(OAc)3 (0.28 g, 1.33
mmol). Then the mixture was stirred at ambient temperature
for 5 h. The reaction was quenched with water, and the product
was extracted with CH2Cl2 (ꢀ3). The organics were combined,
washed with brine, dried (MgSO4), filtered, and evaporated in
vacuo to give (2,4-bis-benzyloxy-5-isopropylphenyl)-[5-(4-methyl-
piperazin-1-ylmethyl)-1,3-dihydroisoindol-2-yl]methanone (0.32 g).
MS: [M þ H]þ 588.
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Deprotection was carried out in a manner analogous to 1 with
the following modifications. K2CO3 (2 equiv) was added to the
reaction, and a MeOH/H2O (9:1) mixture was used as solvent.
Upon completion, the MeOH was evaporated in vacuo and the
mixture was diluted with water, neutralized using 1 M HCl, and
extracted with CH2Cl2 (ꢀ2). The combined organic layers were
dried (MgSO4), filtered, and evaporated under vacuum. The
product was purified by preparative HPLC to give the title
1
compound (21 mg, 9%). H NMR (Me-d3-OD) δ 7.37-7.23
(br s, 3H), 7.19 (s, 1H), 6.39 (s, 1H), 4.94-4.87 (br s, 4H), 3.57 (s,

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