Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90 as potent, selective and orally active antitumor agents

Article abstract of DOI:10.1021/jm900230j

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed from an unbiased screen to identify protein targets for a diverse compound library. These indol-4-one and indazol-4-one derived 2-aminobenzamides showed strong binding affinity to Hsp

Full text of DOI:10.1021/jm900230j

4
288 J. Med. Chem. 2009, 52, 4288–4305
DOI: 10.1021/jm900230j
Discovery of Novel 2-Aminobenzamide Inhibitors of Heat Shock Protein 90 as Potent, Selective and
Orally Active Antitumor Agents
Kenneth H. Huang,* James M. Veal, R. Patrick Fadden, John W. Rice, Jeron Eaves, Jon-Paul Strachan, Amy F. Barabasz,
Briana E. Foley, Thomas E. Barta, Wei Ma, Melanie A. Silinski, Mei Hu, Jeffrey M. Partridge, Anisa Scott, Laura G. DuBois,
Tiffany Freed, Paul M. Steed, Andy J. Ommen, Emilie D. Smith, Philip F. Hughes, Angela R. Woodward, Gunnar J. Hanson,
W. Stephen McCall, Christopher J. Markworth, Lindsay Hinkley, Matthew Jenks, Lifeng Geng, Meredith Lewis, James Otto,
Bert Pronk, Katleen Verleysen, and Steven E. Hall
Serenex Inc., 323 Foster Street, Durham, North Carolina 27701
Received February 21, 2009
A novel class of heat shock protein 90 (Hsp90) inhibitors was developed from an unbiased
screen to identify protein targets for a diverse compound library. These indol-4-one and indazol-4-one
derived 2-aminobenzamides showed strong binding affinity to Hsp90, and optimized analogues exhibited
nanomolar antiproliferative activity across multiple cancer cell lines. Heat shock protein 70 (Hsp70)
induction and specific client protein degradation in cells on treatment with the inhibitors supported Hsp90
inhibition as the mechanism of action. Computational chemistry and X-ray crystallographic analysis
of selected member compounds clearly defined the protein-inhibitor interaction and assisted the design of
analogues. 4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl]-2-[(trans-4-hy-
droxycyclohexyl)amino]benzamide (SNX-2112, 9) was identified as highly selective and potent (IC50
Her2 = 11 nM, HT-29 = 3 nM); its prodrug amino-acetic acid 4-[2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-
trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-phenylamino]-cyclohexyl ester methanesulfonate (SNX-
5422, 10) was orally bioavailable and efficacious in a broad range of xenograft tumor models (e.g. 67%
growth delay in a HT-29 model) and is now in multiple phase I clinical trials.
Introduction
redundant, oncogenic signaling pathways with a target-spe-
3
cific agent. The ability to affect multiple targets may also
diminish the potential for resistance to Hsp90 inhibitors.
a
Hsp90 has received significant recent attention as a ther-
6
1
apeutic target for cancer treatment. Hsp90 acts as a molec-
7
Hsp90 contains three relevant domains: (1) the C-terminal
domain which contains an ATP site capable of binding
2
ular chaperone that aids the folding, maturation, transport,
and maintenance of conformational stability of its client
8
the antibiotic novobiocin, (2) the charged middle linker
3
proteins. Many of these client proteins are involved in critical
9
region, and (3) the N-terminal domain, which also contains
an adenine binding pocket and is responsible for the protein’s
cellular functions that promote cell growth, proliferation, and
survival and are also themselves being pursued as anticancer
targets, for example, Her2, c-Met, and Cdk-4 as well as a wide
1
0
ATPase activity. Inhibition of this ATPase activity disrupts
an ongoing “folding” cycle, involving multiple cochaperone
proteins, and in turn leads to destabilization, ubiquitination,
4
range of mutated proteins. Additionally, Hsp90 is overex-
pressed in malignant cells and thus these cells may be more
6
5
dependent on theHsp90chaperoningfunction. Anattraction
in targeting Hsp90 is the ability to block multiple, possibly
and ultimately proteasomal degradation of client proteins.
Multiple research efforts have followed the initial dis-
covery that the natural product (Figure 1) geldanamycin
1
GA, 1) was a potent inhibitor of Hsp90, and these
1
(
*To whom correspondence should be addressed. Phone: 919-313-
657. Fax: 919-883-5095. E-mail: khuang@pamlicopharma.com. Cur-
efforts have led to the discovery of several compounds
now undergoing clinical testing. Owing to unacceptable
toxicity, GA was not pursued for clinical development,
but GA derivatives 17-allylamino-17-demethoxygelda-
9
rent address: Pamlico Pharmaceutical Inc., P.O. Box 110284, 7020 Kit
Creek Road, Suite 180, Research Triangle Park, NC 27709.
a
Abbreviations: Hsp90 and Hsp70, heat shock protein 90 and 70;
Her2, human epidermal growth factor receptor 2; HT-29, human colon
adenocarcinoma grade II cell line; GA, geldanamycin; 17-AAG, 17-
allylamino-17-demethoxygeldanamycin; 17-DMAG, 17-dimethylami-
no-ethylamino-17-demethoxygeldanamycin; SAR, structure-activity
relationship; HOAc, acetic acid; DMF, dimethylformamide; EtOH,
12
namycin (17-AAG, 2) and 17-dimethylamino-ethylamino-
13,15
1
7-demethoxygeldanamycin
(17-DMAG, 3) have been
investigated as less toxic analogues of GA. In particular,
7-AAG was the first Hsp90 inhibitor to undergo clinical
1
0
ethanol; DMSO, dimethyl sulfoxide; dppf or DPPF, 1,1 -bis(diphenyl-
trials and has displayed good tolerability and indications
of therapeutic efficacy in humans despite issues related to
phosphino)ferrocene; DMAP, 4-dimethylamino-pyridine; THF, tetra-
hydrofuran; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride; TFA, trifluoroacetic acid; MsOH, methanesulfonic acid;
PBS, phosphate-buffered saline; LCMS or LC-MS, liquid chromatog-
raphy-mass spectrometry; HPLC, high pressure liquid chromatogra-
phy; HRMS, high-resolution mass spectroscopy; TLC, thin layer
chromatography.
1
4
poor solubility and limited bioavailability. IPI-504 (4), a
7-AAG analogue containing a reduced hydroquinone moi-
ety, has improved water solubility properties, thereby facil-
1
15
itating formulation for parenteral administration. A second
r
pubs.acs.org/jmc
Published on Web 06/24/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4289
Figure 3. Purine based derivatives.
Figure 1. Natural product geldanamycin and its derivatives.
Figure 4. Novel class of 2-aminobenzamide derivatives.
Scheme 1. Hsp90 Screening hit 11; SAR and Synthetic
Strategies
Figure 2. Natural product radicicol and resorcinol derivatives.
1
natural product radicicol (5, Figure 2) was additionally
6
identified as a potent Hsp90 inhibitor in vitro but was largely
1
7
inactive in tumor xenograft models. However, this scaffold
has led to rationally designed resorcinol based pyrazole and
isoxazole inhibitors with improved solubility and in vivo
potencies. The most advanced clinical compound in this
1
8
class is VER-52296 (or NVP-AUY922, 6). A rational
choice for an Hsp90 inhibitor that targets ATPase activity
is purine based compounds. The first synthetic class of
such scaffolds was the PU series, represented by PU24-
FCl (7, Figure 3), which showed good in vitro potencies
of modifications to nearly all atoms around this scaffold
through robust synthetic chemistry, which would enable
rapid preparation of numerous diverse analogues for thor-
ough structure-activity relationship (SAR) exploration
and structural optimization (Scheme 1).
1
9
but low in vivo efficacy. Further exploration led to another
purine based scaffold, and one analogue, the orally bio-
available CNF-2024 (or BIIB021, 8), is also now in clinical
2
0
testing.
Computational models for binding of 11 were developed
to assist in the design of optimization strategies. The
benzamide moiety was considered likely to be a struc-
tural isostere for adenine, and so it was modeled to bind
consistent with the observed X-ray data for the binding
While the Hsp90 inhibitors evaluated to date have exhibited
promising activity, there remains a need for the identifica-
tion of small molecule inhibitors with enhanced potency
and improved pharmacokinetics. Herewereportour synthetic
and medicinal chemistry efforts in this area that have yielded
a new class of Hsp90 inhibitors unrelated to any previously
known scaffold (Figure 4). Our exploration has produced
a highly potent inhibitor, 9 (SNX-2112), and an orally bioa-
vailable and efficacious prodrug analogue, 10 (SNX-5422),
2
3
of ADP to N-terminal domain of Hsp90.
Specifi-
cally, hydrogen bonding, direct and water mediated, to
Asp 93 and Thr 184 was anticipated (Figure 5A). For
the indolone ring system, two alternative docking orienta-
tions were initially viewed as plausible. In one orienta-
tion (not shown), the ketone was mapped onto the quinone
of GA and reasonable alignment of the respective car-
bonyl groups from the two molecules was possible. An
alternate orientation relied on an induced binding pocket
resulting from movement of Leu 107, as was observed in
X-ray structures for PU3 and related analogues. In this
case, the carbonyl group of the indolone was modeled to
hydrogen bond to Tyr 139 with the gem-dimethyl being
buried into a hydrophobic region formed by Leu 107,
Phe 138, and Trp 162 (Figure 5A).
2
1
which is now in multiple phase I clinical trials.
Modeling and Medicinal Chemistry Strategies. Through
screening of a focused compound library against sets of ATP
binding proteins, we identified 4-(2,6,6-trimethyl-4-oxo-
2
2
4
,5,6,7-tetrahydro-1H-indol-1-yl)benzamide 11 (Scheme 1,
Figure 5) as a moderate inhibitor of Hsp90 (K =3.7 μM).
d
Although 11 was inactive in multiple cellular assays
(IC50 > 50 μM), this scaffold was prioritized over multiple
other internally identified hits for a number of carefully
considered reasons. More specifically, it showed high
selectivity for binding to Hsp90, possessed low molecular
weight and heteroatom count, and was a chemically novel
scaffold. Furthermore, our structural and retrosynthetic
analysis indicated it was feasible to make a broad range
Although both models were considered in detail, this
latter model was given preference due to the quality of the
potential hydrogen bonding and hydrophobic interac-
tions and the appeal of the gem-dimethyl group acting as a

4
290 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Huang et al.
Figure 5. Binding models for hit compound 11. (A) Docking of 11 into the N-terminal domain of Hsp90 (protein structure from PDB code
3
22
D0B). Putative hydrogen bonds are shown by cyan dots. (B) 2D schematic of the binding site.
a
a
Scheme 2. Preparation of Indol-4-ones
Scheme 3. Syntheses of Indolone Benzonitriles
a
Reagents and conditions: (a) 2-bromo-4-fluoro-benzonitrile, NaH,
DMF, rt.
reactions around the benzene ring and a convenient synthetic
precursor for the carboxamide.
Synthetic Chemistry. Chemical exploration of this scaffold
started with the syntheses of derivatives (15) of the indol-4-
one moiety. We utilized different routes to construct these
building blocks and alter the substitution pattern around
the carbon skeleton (Scheme 2). The known 2-methyl
indol-4-one (15a) was prepared as previously reported
a
Reagents and conditions: (a) glyoxal, Zn, HOAc, water, 75 °C;
(
b) Zn, HOAc, water, 75 °C, 5,5-dimethyl-cyclohexane-1,3-dione for
5c, 15d; or cyclohexane-1,3-dione for 15e.
1
2
4
leucine side chain isostere, thereby mimicking the displaced
Leu 107. The observations from this model were that sub-
stitution appeared quite reasonable ortho to the benzamide
with the expectation that these substitutions would orient to
the carbonyl side of the amide group (Figure 5B) because
the amino side was buried in the back of the Hsp90 binding
cleft. There was clear accessible space in this region with
the potential to additionally target both hydrophobic and
hydrogen bonding regions of Hsp90. Finally, substitution
of the pyrrole region appeared to be feasible. Subse-
quently, X-ray data were obtained for members of this
from dimedone and chloroacetone.
Another known
2
5
compound, 2,3-dihydrido indolone (15b), was prepared
through an improved one step treatment of 3-amino-5,5-
dimethyl-cyclohex-2-enone (16) with glyoxal and zinc pow-
der in a warm mixture of acetic acid (HOAc) and water.
2
6
27
28
3-Methyl indolone derivatives 15c, 15d, and 15e were
reported before; each was prepared here effectively in one
2
9
step via a modified Knorr procedure from a corresponding
available 2-oxime methyl ketone 17 and cyclohexane-1,3-
dione in the presence of Zn powder and HOAc. The coupling
reaction of 2-bromo-4-fluoro-benzonitrile with substituted
indolone derivatives 15a-e mediated by NaH in dimethyl-
formamide (DMF) gave the versatile 2-bromo-benzonitrile
intermediates, 18a-e (Scheme 3), which were subsequently
2
2
chemical scaffold that overall validated the binding
model for 11 and in particular confirmed the orientation
for the indolone ring.
3
0
Our overall synthetic and medicinal chemistry strategies
fully incorporated such binding analysis (Scheme 1). Analo-
gues of the scaffold (12) in general could be assembled from
top (13) and bottom (14) building blocks as shown, with each
bearing either preinstalled substitutions at designated posi-
tions or synthetic handles such as fluoride and bromide that
could be conveniently displaced later. The cyano group was
designed as both the activator for the sequential substitution
transformed into various analogues.
The screening hit 11 and its 3-methyl regioisomer 19
were prepared through a similar coupling reaction between
4-fluoro-benzonitrile and indolone 15a-b, respectively, fol-
lowed by H O catalyzed conversion of the resulting crude
2
2
indolone benzonitriles into benzamides at room temperature
in a mixture of EtOH and dimethyl sulfoxide (DMSO) (4:1
3
ratio) containing aqueous NaOH (Scheme 4). We found
1

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4291
a
a
Scheme 4. Preparation of Benzamide Analogues
Scheme 5. Synthesis of 2-Aminobenzamide Analogues
a
Reagents and conditions: (a) 4-fluoro-benzonitrile, NaH, DMF, rt;
rt; (c) pentylzinc bromide,
(dppf), THF, 85 °C microwave for 20a, or NaH, butanol, 100 °C
(
b) EtOH, NaOH, DMSO,
2 2
H O ,
a
2
Reagents and conditions: (a) Pd(OAc) , DPPF, NaOtBu, toluene,
PdCl
2
amine, or aniline, 110 °C microwave; (b) EtOH, NaOH, DMSO, H
rt; (c) Pd(OAc) , DPPF, NaOtBu, toluene, 3,4,5-trimethoxy-phenyla-
mine, 110 °C microwave.
2 2
O ,
microwave for 20b, or NaH, thiophenol, 100 °C microwave for 20c.
2
the addition of DMSO to our reaction systems consistently
improved the yield and accelerated the rate of this hydration
process, and many amide products would precipitate from
the reaction mixtures within 5-10 min. Derivatives from 11
carrying side chains linked to the phenyl ring through either
carries a 2-(trans-4-hydroxy-cyclohexylamino) side chain.
This method gave efficient entry into facile diversifica-
tion of the 2-amino side chains. Additional analogues
prepared through this method are reported in the Results
and Discussion and Experimental Sections.
a CH , O, or S ortho to the amide were prepared from
2
1
8a through nucleophilic replacements of the 2-bromide
and hydration of the cyano group. Thus, 2-pentyl analogue
0a was prepared from pentylzinc bromide and catalytic
An alternative synthesis for the indazolone analo-
gues is illustrated as method B (Scheme 7). The C-2 side
chain, such as trans-4-hydroxy-cyclohexylamino, was
introduced first to give 26 by treatment of 2,4-difluoroben-
zonitrile with trans-4-amino-cyclohexanol. Replacement
of the second fluorine with hydrazine under heat gave 27.
2
3
2
PdCl (dppf), 2-butoxy analogue 20b from butanol and
2
NaH, and 2-phenylsulfanyl analogue 20c from thiophenol
and NaH.
Treatment of 2-bromobenzonitriles 18 with amino nucleo-
3
3
35
36
philes under Buchwald-Hartwig conditions involving cat-
alytic Pd(OAc)2, 1,1 -bis(diphenylphosphino)ferrocene
Known triketones 28a, 28b, and novel 28c were conve-
niently prepared from the corresponding acid chloride
or anhydride and dimedone in warm with catalytic 4-di-
methylamino-pyridine (DMAP). Indazol-4-one formations
from 28a-c and 27 in hot mixtures of ethanol and acetic
acid, followed by hydration of the resulting benzonitriles
0
(
DPPF), and NaOtBu in toluene at 110 °C under microwave
irradiation introduced amino side chains to the scaffolds,
which were converted into a plethora of 2-aminobezamide
3
0
analogues (Scheme 5). Thus, 18a was converted into its
corresponding butylamine 21a, 2-methoxyethylamine 21b,
phenylamine 21c, and 4-methoxyphenylamine 21d deriva-
tives. Similarly, 2-(3,4,5-trimethoxy-phenylamino)-benza-
mide analogues 22a-c, 22e each was prepared from
corresponding 18, with methyl group(s) at varying positions
about of tetrahydro-indol-4-one moiety.
3
0
afforded analogues 29a-c. This method efficiently intro-
duced various substitutions at the C-3 position of the
indazolones.
A third route, or method C, was developed to prepare the
3-difluoromethyl and 3-trifluoromethyl substituted indazolone
2-aminobenzamide analogues (Scheme 8). Because of their poor
stability, the fluorinated methyl triketones of formula 28 could
not be obtained in meaningful yields, therefore the previous two
methods were not applicable. Instead, a known tosyl hydrazone
intermediate 30 was made from dimedone and tosyl hydrazide as
The tetrahydro-indol-4-one moieties of the scaffold
could be replaced by tetrahydro-indazol-4-ones, which
were prepared through a number of different routes. Method
A started by substituting the fluorine of 2-bromo-4-fluoro-
benzonitrile with hydrazine in tetrahydrofuran (THF) to
3
7
reported. It was subsequently converted into desired indazo-
lone 31a or 31b through treatment with trifluoro- or difluoro-
acetic anhydride in a warm mixture of tetrahydrofuran (THF)
3
4
give a known compound 23 (Scheme 6), followed by
reaction of 23 with readily available 2-acetyl-5,5-dimethyl-
cyclohexane-1,3-dione in a mixture of EtOH and HOAc
at 110 °C in a microwave reactor to afford 24a, a key 2-
bromo-benzoniltrile intermediate. 2-Aminobenzamide ana-
logues were prepared from 24a through Buchwald-Hartwig
coupling reactions with amines followed by hydration of
the resulting benzonitriles. Compound 25a, as an example,
30
and triethylamine, then NaOH in methanol and water.
Through similar conditions described above, coupling of 31a
and 31b with 2-bromo-4-fluoro-benzonitrile regioselectively af-
forded 32a, 32b, respectively, which were converted into the
desired 2-(trans-4-hydroxy-cyclohexylamino)-benzamide analo-
gues 9 and 33 under Buchwald-Hartwig conditions and nitrile

4
292 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Huang et al.
a
Scheme 6. Method A for Preparation of Indazolone 2-Aminobenzamide Analogues
a
Reagents and conditions: (a) NH
2
NH
2
2
, THF, rt; (b) 2-acetyl-5,5-dimethyl-cyclohexane-1,3-dione, EtOH, HOAc, 110 °C microwave; (c) Pd(OAc) ,
2 2
DPPF, NaOtBu, toluene, trans-4-amino-cyclohexanol, 110 °C microwave; (d) EtOH, NaOH, DMSO, H O , rt.
a
Scheme 7. Method B for Preparation of Indazolone 2-Aminobenzamide Analogues
a
Reagents and conditions: (a) trans-4-amino-cyclohexanol, THF, triethylamine, 110 °C microwave; (b) NH
2
NH
2 1
, THF, heat; (c) R COCl or
(R
1
CO)
2
O, pyridine, cat. DMAP, 70 °C; (d) 28, EtOH, HOAc, 110 °C microwave; (e) EtOH, NaOH, DMSO, H O
2 2
, rt.
a
Scheme 8. Method C for Preparation of Indazolone 2-Aminobenzamide Analogues
a
Reagents and conditions: (a) TsNHNH
2
, cat. p-TsOH, toluene, reflux; (b) (R
, DPPF, NaOtBu, toluene, trans-4-aminocyclohexanol, 110 °C microwave; (f) EtOH,
, rt; (g) N-(tert-butoxycarbonyl)glycine, EDC, cat. DMAP, CH Cl , rt; (h) TFA, CH Cl ; (i) methanesulfonic acid, CH Cl
1 2
CO) O, THF, triethylamine, 55 °C; (c) NaOH, MeOH, water, rt; (d) 2-
Bromo-4-fluoro-benzonitrile, NaH, DMSO, rt; (e) Pd(OAc)
NaOH, DMSO, H
2
2
O
2
2
2
2
2
2
2
.
hydration. Glycine ester mesylate salt 10 was prepared from
through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
Yet a fourth route, method D, was developed to prepare
the fluorinated 3-methylindazolone 2-aminobenzamide analo-
gues, especially for large scale production of the derivatives
(Scheme 9). The 4-fluoride of the 4-fluoro-2-aminobenzoni-
trile such as 26 was replaced by a fluoro 3-methyl indazol-4-
one, such as 31a in high yield with desired regioselectivity.
The nitrile hydration in the end, in this example, afforded
compound 9. Again, the first coupling step was not suitable
for nonfluorinated methyl indazolones due to poor regio-
selectivity.
9
hydrochloride (EDC), DMAP mediated coupling with
N-(tert-butoxycarbonyl)glycine, deprotection of the Boc group
using trifluoroacetic acid (TFA), and salt formation with
methanesulfonic acid (MsOH). This method was also highly
effective for the preparation of nonfluorinated indazolones
of formula 31 from 30 but gave poor regioselectivity (∼1:1)
for the C-N bond formation in the subsequent coupling
reactions with 2-bromo-4-fluoro-benzonitrile.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4293
Scheme 9. Method D for Preparation of Indazolone 2-Amino-
benzamide Analogues
Table 1. 2-Amino Substitutions Enhance both Hsp90 Affinity and
Client Effects
a
a
Reagents and conditions: (a) NaH, DMF, 100 °C microwave;
2 2
(b) EtOH, NaOH, DMSO, H O , rt.
a
Her-2 degradation Hsp90 affinity
b
compd
R
5
IC50 (μM)
d
K (μM)
Results and Discussion
1
7-AAG
0.003 ( 0.003
>100
>10
0.087
3.73
>50
1.96
1.06
0.39
0.39
0.37
0.69
0.22
We had envisioned the primary benzamide and indol-4-one
carbonyl groups as key structural features of this scaffold that
confer tight binding into the ATPase pocket of Hsp90, as
described above. This hypothesis was quickly validated by our
preliminary SAR explorations. Partial or total loss of binding
affinity to Hsp90 occurred when the primary benzamide was
replaced with amides with N-substitutions, a carboxylic acid
or its esters, imidamide, hydroxy imidamide, or its precursor
benzonitrile. Similar results were observed when the carbonyl
of the indol-4-one was converted into an oxime or other
1
2
2
2
1
H
0b
0a
0c
BuO
Pentyl
PhS
>10
>10
21a
21b
21c
BuNH
>10
>10
2-(MeO)EtNH
PhNH
4-(MeO)PhNH
6.4 ( 1.1
1.59 ( 0.20
0.49 ( 0.03
2
1d
2a
a
2
3
3,4,5-(MeO) PhNH
Values were determined from a Her2 imaging assay in AU565
3
8 b
3
0
cells.
assay.
Values were derived from an 8-pt nonenzymatic ATPase
1
moieties. Our follow-on strategy retained these structural
functionalities and focused optimization on the remaining
features around the scaffold.
2
One of our early strategies was to examine the impact of
side chains attached to the amido benzene ring of the scaffold
on Hsp90 binding affinity and cellular client protein degrada-
tion. We had expected such attachments might enhance
potencies because they would occupy the same space of
enzymepocket utilized by side chainsofPUseriesofinhibitors
as well as rings of 17-AAG (2) and radicicol (5). Our results
indicated there were indeed significant effects on binding
affinities, and they were highly dependent on the positions
of substitutions and the nature of the linker atoms. In general,
the C-3 (meta) substitutions on the benzene frequently led
to lower Hsp90 affinity, erratic SAR, or occasional binding
to off-target proteins that we did not fully characterize (data
not shown). The often reduced and erratic Hsp90 affinity
is believed to be due to steric interference with the Leu 107
residue of Hsp90, which is displaced, on ligand binding, to a
location proximal to the C-3 position. An additional point of
note is that the magnitude of the displacement of Leu 107 is
larger relative to the PU24FCl (7) and CNF-2024 (8) classes of
inhibitors due to the bulky gem-dimethyl group attached to
the indol-4-one moiety.
In sharp contrast, the C-2 (ortho) substitutions on the
benzene ring showed consistent SAR and tractable enhance-
ment of Hsp90 binding affinity (Table 1) for those substitu-
tions with an NH linker. Compounds containing an oxygen
linker, for example, compound 20b, did not exhibit measur-
able Hsp90 binding; this observation was consistent with the
binding model as electronic repulsion would be expected
between the oxygen linker and amide carbonyl oxygen.
Analogues containing methylene (20a) or sulfide (20c) linkers
improved only slightly the affinity for Hsp90 compared to
the parent hit (11), and neither had any measurable cellu-
lar potency. Conversely, amine linkers provided a signi-
ficant advance in optimization. Both alkyl and aryl groups
linked through NH at C-2 of the core scaffold improved
binding to Hsp90. Additionally, although linear alkyl chains
often conferred little or no cellular activity (21a, 21b), incor-
poration of cyclic aryl groups led to inhibitors able to induce
Her2 protein degradation in cellular assays (21c, 21d, 22a).
Oxygen containing rings (21d, 22a) appeared to confer more
potent cellular effects than the one without oxygen (21c). This
last trend was interesting because 17-AAG (2) also has an
oxygen located in thesamespace of theHsp90binding pocket.
Other types of C-2 substitutions without a NH linker were
also broadly explored and were generally disfavored (data not
shown). The data generated were generally consistent with the
binding model in which the NH linker formed a stabilizing
intramolecular interaction with the amide carbonyl group,
and the alkyl and aryl substitutions were involved in favorable
hydrophobic interactions with Hsp90 residues, especially Met
98. Additionally, Lys 58 was modeled to interact with the
oxygen containing substitutions, thereby affording an addi-
tional hydrogen bond. Compound 22a was the most potent
analogue we could achieve in this series by optimizing only the
2-amino group. Although potent Hsp90 binding affinity was
observed, modification of other parts of the scaffold became
necessary for improved cellular potencies.
Next we investigated the SAR of the indol-4-one moiety in
affectingbiologicalactivitiesby using 2-(3,4,5-trimethoxyphe-
nylamino)benzamide of 22a as a common and sensitive
template (Table 2). Deletion of either the 2-methyl (22b) or
the gem 6,6-dimethyl (22e) of indol-4-ones led to moderate
reduction of Hsp90 affinity but significant loss of the cellular
effect on Her2 degradation. Moving the C-2 indolone methyl
group of 22a to the C-3 position (22c) greatly improved the
cellular activity, leading to a compound showing nanomolar
potency. Compound 19, the C-3 methyl regioisomer of 11 and
the core scaffold component of 22c, was still active in cells
(Her2 IC50 = 8 μM, not shown in tables). While replacement
of the gem-dimethyl with a single ethyl was allowed, it created

4
294 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Huang et al.
Table 2. Critical Roles of Indolone Methyl Groups
Table 3. Versatile Indazolones and the Unique Aminocyclohexanol
a
Her-2 degradation Hsp90 affinity
b
a
Her-2 degradation Hsp90 affinity
b
compd
7-AAG
R
1
R
2
R
3
, R
4
IC50 (μM)
K
d
(μM)
compd
R
1
X
IC50 (nM)
K
d
(nM)
1
0.003 ( 0.003
0.49 ( 0.03
1.83 ( 0.12
1.61 ( 0.18
0.046 ( 0.011
0.087
0.22
0.59
0.94
0.39
17-AAG
34
3 ( 3
7 ( 3
30 ( 11
10 ( 0.8
9 ( 2
87
27
16
16
20
98
41
22a
H
H
Me
H
Me
Me
H
Me
Me
CH
N
22b
25a
9
2
2
2e
2c
a
Me
Me
H
CF
Et
3
N
H
Me
29a
33b
29c
N
CHF
2
N
16 ( 1
Values were determined from a Her2 imaging assay in AU565 cells.
Values were derived from an 8-pt nonenzymatic ATPase assay.
b
c-PrMe
i-Pr
Me
N
46 ( 14
203 ( 59
64 ( 9
2
9b
5
N
CMe
177
86
3
an unwanted chiral center, and larger groups were disfavored
atthatposition (bothdatanotshown). Similarly, 2,3-dimethyl
indolone analogues were also less potent, as in one example to
be shown later. These results clearly defined the importance of
the C-3 methyl and the gem-6,6-dimethyl groups of the
indolone moiety for potent Hsp90 inhibition and represented
thesecondcriticalSARtrendwediscovered withthis chemical
scaffold.
a
Values were determined from a Her2 imaging assay in AU565 cells.
Values were derived from an 8-pt nonenzymatic ATPase assay.
b
At this point in the discovery effort, compound 22c
represented the most advanced analogue and demonstrated
both strong Hsp90 binding and potent cellular activity
consistent with Hsp90 inhibition as the mechanism of action.
However, 22c was viewed as less than favorable with regards
to solubility and CLogP. In fact, this compound was neither
orally bioavailable nor effective in mouse xenograft tumor
models following ip dosing. Additionally, both the pyrrole
moiety and especially the electron rich trimethoxyaniline
ring were viewed as potential metabolic liabilities, with the
aniline having the potential to be metabolized to reactive
quinone species. To address these issues, additional modifica-
tions were pursued. Replacement of the trimethoxyaniline
ring with a trans-4-hydroxycyclohexylamino group afforded
compound 34 (Table 3), which was highly potent as an Hsp90
inhibitor. Additionally, this compound was shown to have
measurable oral bioavailability (ca. 10% in rat) and good in
vivo efficacy in mouse xenograft tumor models following ip
dosing (data not shown). Relative to 22c, the trans-4-hydro-
xycyclohexylamino substitution of 34 was attractive because
it was nonaromatic, while still maintaining achirality, and was
capable of forming similar favorable interactions with the
Hsp90 binding site, namely hydrophobic packing with Met 98
and hydrogen bonding to Lys 58. A binding model for
compound 34 overlaid onto 17-DMAG (3, Figure 6) illus-
trates the ability of the compound class to mimic, in an orally
available small molecule scaffold, multiple key pharmaco-
phore elements of the natural product ansamycins.
Figure 6. Overlay of binding model for 34 onto 17-DMAG (3, from
PDB code 1OSF). Green arrows show common pharmacophore
elements.
pyrazole provided increased compound polarity and perhaps
solubility and the opportunity to gain more structural diver-
sity via synthetic accessibility to the C-3 position of the
bottom moieties. These combined modifications significantly
lowered the ClogP values of the resulting indazolone analo-
gues listed in the table to within a well acceptable range
of 2.34-3.39. Close examination indicated that 3-methyl
indazolone 25a was less potent than its indolone equiva-
lent 34, but the trifluoromethyl version 9 was equally
potent as 34. Compound 9 would ultimately undergo
extensive evaluation (described below); its amorphous
form displayed good oral bioavailability (40%, mouse)
and in vivo antitumor efficacy in mouse xenograft models
following oral dosing.
We next replaced the seemingly dispensable 2-hydrido-
carbon (CH) of the indolone of 34 with nitrogen and ex-
plored substitutions at the C-3 position of the resulting
indazol-4-ones (Table 3). This new scaffold was designed
to lower the electron density and increase the stability
of the bottom moieties through the use of a pyrazole replace-
ment for the pyrrole of the original hit. Additionally, the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4295
Table 4. Broad Scope of the 2-Amino Substitutions
a
Her-2 degradation IC50 (nM)
b
Hsp90 affinity K
compd
R
1
X
R
6
d
(nM)
1
7-AAG
3 ( 3
87
43
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
10
a
Me
Me
CHF
Me
Me
Me
Me
Me
Me
Me
N
N
4-tetrahydropyranyl
cyclopent-3-enyl
50 ( 13
111 ( 19
35 ( 6
229 ( 116
184 ( 34
418 ( 59
>1000
15
11
2
N
4-tetrahydrothiopyranyl
cyclobutyl
N
114
51
N
cyclopropylmethyl
1-phenylethyl
furan-2-ylmethyl
CH
N
1020
216
63
N
2-(methylthio)ethyl
2-(methylsulfonyl)ethyl
1-methoxy-propan-2-yl
3-(2-oxopyrrolidin-1-yl)propyl
1,3-dimethoxypropan-2-yl
4-piperidinyl
226 ( 44
147 ( 32
115 ( 14
264 ( 103
181 ( 49
>7000
N
14
67
N
CF
CF
3
3
N
94
78
N
Me
Me
CH
CH
N
843
294
530
41
2-morpholinoethyl
4-cis-hydroxycyclohexyl
4-trans-(2-aminoacetoxy)cyclohexyl
b
>4000
248 ( 23
37 ( 9
CF
CF
3
3
N
Values were determined from a Her2 imaging assay in AU565 cells. Values were derived from an 8-pt nonenzymatic ATPase assay.
Table 5. Antiproliferative Activities for Selected Compounds
a
Table 6. Client Impacts for Selected Compounds
a
proliferation assays
17-AAG
34
9
10
client assays
17-AAG 34
9
10
A375 (melanoma)
LNCAP (prostate)
MCF-7 (breast)
HT-29 (colon)
1287 ( 212
82 ( 52
9 ( 2
4 ( 1
10 ( 5
7 ( 2
6 ( 1
7 ( 1
14 ( 5
10 ( 3
248 ( 72 18 ( 4
61 ( 5
3 ( 2
23 ( 5
3 ( 1
53 ( 46
3 ( 0.7
3 ( 0.5
6 ( 1
51 ( 6
31 ( 1
16 ( 6
32 ( 4
19 ( 6
25 ( 6
114 ( 14
38 ( 9
Hsp70 induction (A375 cells)
p-S6 degradation (A375 cells)
Her2 degradation (AU565 cells)
4 ( 3 14 ( 6 2 ( 0.9 13 ( 3
32 ( 19 7 ( 3 1 ( 0.6 61 ( 22
0.3 ( 0.3
0.1 ( 0.01
328 ( 20
134 ( 14
9 ( 4
194 ( 74
255 ( 61
1487 ( 212
126 ( 113
3 ( 3
7 ( 3 11 ( 5
5 ( 1
p-ERK degradation (AU565 cells) 8 ( 7 16 ( 3 41 ( 12 11 ( 3
SW620 (colon)
SK-MEL-5 (melanoma)
PC-3 (prostate)
a
Values are IC50 in nM and are the means of four experiments with
standard deviation shown.
38
17 ( 3
6 ( 1
MDA-MB-231 (breast)
NCI-H460 (NSCLC)
HCT-15 (colon)
K562 (erythroleukemia)
a
215 ( 95
52 ( 12 190 ( 18
6 ( 1 23 ( 4
Values are IC50 in nM and are the means of four experiments with
standard deviation shown.
38
A series of other analogues were prepared to further
evaluate the SAR (Table 3) of the C-2 and C-3 positions.
Ethyl 29a showed good potency, whereas difluoromethyl
analogue 33b, cyclopropylmethyl 29c, and isopropyl inda-
zolone 29b displayed reduced activity. The activity of the
2
,3-dimethyl indolone 35 was comparable but not superior
to34 or 22c. Ingeneral, larger or functional groupsintroduced
at available C-2 or C-3 or both positions of the indolone or
indazolone led to loss of potencies.
Figure 7. Exposure of compound 9 following iv and oral dosing
of compound 10 in female mice. Data are from an average of
four animals. Levels of prodrug were below the lower limit of
quantitation (LLOQ) at all time points.
Concurrently, we explored optimization of 2-amino sub-
stitutions in conjunction with the favored bottom moieties
bearing small C-3 groups and data for representative
compounds are summarized (Table 4). The overall SAR
trends were consistent with our earlier findings. The favored
side chains were aminocyclic structures that contained
oxygen (36) or a weak hydrogen bond acceptor such as
the less stable alkene (37) or sulfur (38). The less favored
side chains were either rings without such H-bond acceptors
(39) or rings having an extra linear spacer (40, 41, 42).
Open chain structures designed to improve flexibility and
solubility, were also less active (43, 44, 45, 46, 47). Particularly
disfavored side chains were those containing strongly
basic amines (48, 49). The comparator compound, epimeric

4
296 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Huang et al.
a
Table 7. Pharmacokinetic Data for Compound 10 in Female Mice
dose (mg/kg)
route
T
max (h)
C
max (ng/mL)
AUCInf (h ng/mL)
T
1/2 (h)
Vz_F_obs (mL/kg)
CL_F_obs (mL/h/kg)
3
1
1
2
5
0
0
5
0
iv
0.083
4455
747
4869
3968
2.6
2.5
2.9
3.3
7846
8928
9594
9547
2054
2520
2272
1988
po
po
po
1
1
2
2148
4015
11004
25153
a
Parameters were calculated using WinNonlin software.
4
-cis-hydroxycyclohexylamino (50), was 25- to 30-fold less
potent compared to the trans analogue 9. Compound 10, the
glycine ester prodrug of compound 9 that was made for
further evaluation for reasons described below, still main-
tained nanomolar level of potencies.
Overall, compounds 34 and 9 remained among the most
potent compounds. Their detailed cellular activity profiles,
along with those of prodrug 10 and 17-AAG (2), are pre-
sented in Tables 5 and 6. All compounds summarized
showed potent antiproliferative activity against a broad range
of cancer cell types (Table 5). Additionally, 17-AAG treat-
ment resulted in calculated IC50 values spanning a much
greater range (0.1-1487 nM) than 9 (3 nM to 53 nM). One
explanation for this observation is that the different cell
lines tested have differing abilities to reduce 17-AAG to its
more potent dihydroquinone form via DT-diaphorase
3
9
Figure 8. Example of in vivo activity of compound 10 following
oral dosing in an HT-29 colon tumor xenograft model. Compound
was dosed on a MWF schedule for 3 weeks.
(NQO1). Compounds 9, 10, and 34 all exhibited potent
effects on Her2 stability and caused expected up-regulation
of Hsp70 (Table 6). Additionally, treatment with the inhibi-
tors down regulated both the MAPK and AKT signaling
pathways as measured by the loss of p-S6 and p-ERK in
treated cells. These pathways are implicated in uncontrolled
cellular division and antiapoptotic signaling and have
been targeted for drug development. The results shown here
highlight the advantage of Hsp90 inhibition as a single
mechanism that is capable of modulating a number of known
pathways involved in cancer progression.
cleared from normal tissues to below the lower limit of
quantitation (LLOQ) by 24 h and exhibited good scaling with
oral dose (Table 7).
On the basis of the observed favorable pharmacokinetics,
compound 10 was evaluated in a range of xenograft models.
Shown in Figure 8 is the in vivo antitumor activity of 10 in
an HT-29 human colon tumor xenograft model. The com-
pound was orally administered to mice bearing subcutaneous
tumors 3 times a week for 3 weeks (qod ꢀ 3/2 ꢀ 3) at 5, 10, 25,
and 50 mg/kg. The 50 mg/kg dose was the most efficacious,
demonstrating a 67% growth delay over vehicle control.
Median time to end point (TTE) for the 50 mg/kg group
was 43.2 days compared to 25.9 days for vehicle control.
Two of 10 animals survived to the end of the study (61 days).
The 25 mg/kg dose showed tumor growth delay but at a much
lower percentage (14%). Median TTE was not statistically
significant over vehicle control. On the basis of body weight
measurements and clinical observations, compound 10 was
well tolerated at all dose levels tested. No treatment related
deaths were observed.
Lead compounds of this new class appeared highly selec-
tive for Hsp90. Routine screening through an internal affinity
21,40
chromatography assay,
designed to provide broad profiling
of purine binding proteins from either recombinant proteomes
or porcine tissues, showed strong Hsp90 binding and an
absence of off-target interactions. Furthermore, compound 9,
when screened through a panel of 75 enzymes and receptors at 1
μM, did not display any inhibitory activity greater than 20%,
nor did it exhibit significant inhibition against a panel of 9
cytochrome P450 isozymes (both provided by Cerep, Inc.,
Seattle, WA). This compound was also found sufficiently stable
to metabolism by human liver microsome (1 μM; 88% remain-
ing after 1 h in the S9 fractions).
Although amorphous 9 was orally bioavailable (F ∼ 0.4) and
had moderate solubility of 170 μM, its crystalline forms were
not, and aqueous solubility of these forms at physiological pH
Conclusion
A novel class of indol-4-one and indazol-4-one derived
2-aminobenzamides that potently inhibit Hsp90 has been
discovered. The overall synthetic schemes developed allowed
for facile preparation of the intermediates as well as diverse
analogues and were easily scalable. Computational chemistry
and X-ray crystallographic analysis of selected member
compounds clearly defined the protein-inhibitor interaction
and assisted the design of further analogues. Optimized
compounds of the new scaffolds exhibited low nanomolar
antiproliferation potencies across multiple cancer cell lines
with Hsp90 inhibition as a mechanism of action as demon-
strated through Hsp70 induction and specific client protein
degradation. Compound 10 displayed a superior profile of
oral bioavailability and efficacy and was selected as a clinical
(in phosphate-buffered saline or PBS, 7.4) was reduced 25-fold
to 6 μM. To improve bioavailability for oral dosing and also
provide additional solubility to allow the flexibility of parent-
eral administration, a simple amino ester prodrug strategy was
21
explored. One example was a β-analine analogue that showed
improved oral bioavailability and was highly water-soluble
(
∼10 mg/mL, pH 2-6); however, it was found to be too stable
to esterase hydrolysis both in vitro (60% conversion to 9, 1 h in
mouse plasma) and in vivo. The glycine ester 10 was crystalline,
stable, and reasonably soluble (∼1 mg/mL, pH 4-6). Conver-
sion of ester 10 to parent 9 was rapid and complete in vivo
following oral and intravenous dosing and resulted in excellent
exposure of the parent compound (Figure 7). Compound was

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4297
candidate; presently, it is being evaluated in multiple phase I
clinical trials.
tumors maintained by serial transplantation in athymic nude
mice. Each test mouse received a 1 mm HT-29 tumor fragment
3
implanted subcutaneously in the right flank, and
the growth of tumors was monitored as the average size ap-
Experimental Section
3
proached 80-120 mm . Fourteen days later, designated as
Day 1 of the study, individual tumor volumes ranged from
Determination of Compound Affinity for Hsp90. Test com-
pound affinity for Hsp90 was determined as previously de-
3
63 to 126 mm and the animals were placed into eight groups,
each consisting of 10 mice with group mean tumor volumes
21
scribed. Briefly, Hsp90 from porcine spleen extract was
isolated by affinity capture on a purine-affinity media. The
Hsp90 loaded media was then challenged with test compound
at a given concentration, ranging from 0.8 to 500 μM, and the
amount of Hsp90 liberated at each concentration was deter-
mined by Bradford protein assay. The resulting IC50 values were
corrected for the ATP ligand concentration and presented as
3
of 93.2-93.9 mm . Micronized 10 was preformulated in
1% microcrystalline cellulose/0.5% Tween80 in water. The
solutions were stored at 4 °C during the study and homoge-
nized just prior to dosing. Group 1 vehicle control mice
received D5W (5% dextrose) vehicle by oral gavage beginn-
ing on Day 1, every other day for three doses, followed by
two days without treatment, for three cycles ((qod ꢀ 3)/2 ꢀ 3
weeks, total of nine doses). Groups 2 to 5 animals received
10 at 5, 10, 25, or 50 mg/kg on the same schedule as
vehicle control group ((qod ꢀ 3)/2 ꢀ 3). Each treatment was
administered in a volume of 0.2 mL per 20 g of body weight
(10 mL/kg) and was scaled to the body weight of the animal.
Tumors were measured twice weekly using calipers.
d
apparent K values.
3
8
Determination of Cellular Activities of Compounds. All
assays were done in 96-well plates. All cell lines were purchased
from ATCC. Proliferation rates were measured by seeding
cells into 96-well plates, followed by compound addition 24 h
later. After addition of compound, cells were allowed to
grow for either an additional 72 or 144 h depending on rate
of growth. At harvest, media was removed and DNA content
for individual wells was determined using CyQuant DNA dye
Chemistry. General. Melting points were recorded using a
Stuart SMP3 and are uncorrected. Identity and purity confir-
mations were performed by LC/UV/MS using a Finnigan
Surveyor MSQ system (Thermo Fisher Scientific, Waltham,
MA). The diode array detector wavelength was 254 nm, and
the MS was operated in positive electrospray ionization mode.
The samples were maintained at rt in the autosampler, and an
aliquot (5 μL) was injected onto a Thermo BetaBasic-8 column,
50 mm ꢀ 4.6 mm, 5 μm (Thermo Fisher Scientific, Waltham,
MA) maintained at 35 °C. The samples were eluted at a flow rate
of 1 mL/min with a mobile phase system composed of solvent
A (water containing 0.1% formic acid) and B (acetonitrile
containing 0.08% formic acid) with a linear gradient from
10% B to 90% B in 4 min and then isocratic for 1 min with
90% B. The column was equilibrated back to the initial condi-
tions for 1 min before the next run. High-resolution mass
spectroscopic (HRMS) data were provided by Analytical In-
(
Invitrogen). Levels of Hsp90 client proteins and phosphoregu-
lated proteins in A375 and AU565 cells were measured by high
content analysis (HCA) using an ArrayScan 4.5 (Pittsburgh,
PA) instrument after 24 h of treatment with compound, fol-
lowed by methanol fixation. After fixation in 4% PBS-buffered
formalin and permeablization with 0.1% TX-100, cells were
probed with anti-Her2 (Millipore), antiphospho-S6 (pS6), anti-
pERK (Cell Signaling), and anti-Hsp70 (Assay Design) primary
antibodies, followed by TRITC or FITC conjugated secondary
antibodies. Nuclei where also stained with Hoechst DNA bind-
ing dye. For each well, 250-500 individual nuclei were identified
along with the average staining intensity for the client and
phosphoproteins for each cell. Average client staining intensities
were then calculated for each well.
Pharmacokinetic Studies. In-life work for the pharmacoki-
netic study was performed at Washington Biotechnology
1
strument Group, Inc. (Raleigh, NC). H NMR spectra were
(
Columbia, MD). Compound 10 was injected intravenously
recorded on a Varian Unity spectrometer at 400 or 300 MHz.
Elemental analyses were provided by Prevalere Life Sciences,
Inc. (Whitesboro, NY). Purities of all compounds reported here
were greater than 95%, by either the LC/UV method described,
or elemental analyses, or the two methods combined.
to one group of 40 female Swiss-Webster mice at 10 mg/kg.
Three other groups of 40 animals were dosed orally at 10, 25,
and 50 mg/kg. Blood samples were collected from four mice at
each time point: 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 8, 24, and 48 h
after dosing. Whole blood was collected into chilled tubes
containing EDTA and NaF, and the blood was converted to
plasma in a refrigerated centrifuge. The samples were stored
frozen until LC-MS/MS analysis. The concentrations of com-
pounds 9 and 10 in mouse plasma were determined by LC-MS/
MS following protein precipitation with acetonitrile. The cali-
bration curve ranges were 3.3-6000 ng/mL and 4.3-7800 ng/
mL for 10 and 9, respectively. The LC-MS/MS system consisted
of a Shimadzu Prominence HPLC (Shimadzu, Columbia,
MD) coupled to a hybrid triple quadrupole/linear ion trap mass
spectrometer with a Turbospray source (QTRAP, Applied
Biosystems, Foster City, CA). The samples were injected
onto a Synergy Hydro-RP column, 2.0 mm ꢀ 30 mm, 4 μm
2,6,6-Trimethyl-1,5,6,7-tetrahydroindol-4-one (15a). This com-
2
4
pound was prepared as reported previously from 5,5-dimethyl-
1,3-cyclohexanedione (20.0 g, 142.7 mmol) and chloroacetone
(11.36 mL, 142.7 mmol) over two steps (5 g, 20%) as a tan solid.
þ
1
LCMS m/z = 178.2 [M þ H] , t = 2.94 min. H NMR (400
R
MHz, DMSO-d ): δ 11.01 (bs, 1H, NH), 5.87 (s, 1H), 2.55 (s, 2H),
6
2.11 (s, 5H), 0.99 (s, 6H); mp = 189.6-191.1 °C. HRMS calcd for
þ
C H NO 178.1232 [Mþ H] , found 178.1231.
11
15
6,6-Dimethyl-1,5,6,7-tetrahydroindol-4-one (15b). 3-Amino-
5,5-dimethyl-cyclohex-2-enone (16, 1.0 g, 7.2 mmol) and glyoxal
(40% in H O, 1.04 mL, 7.2 mmol) were dissolved in AcOH and
2
H O (7:1, 5 mL) solution and warmed to 65 °C. When the
2
reaction temperature had reached 65 °C, Zn powder (923 mg,
14.4 mmol) was added portionwise. Once the addition was
complete, the reaction was heated to 90 °C and allowed to stir
overnight. After 18 h, the reaction was cooled. The reaction was
(
Phenomenex, Torrance, CA), and the analytes and internal
standard were eluted at a flow rate of 0.45 mL/min using a
water-methanol gradient in the presence of 0.08 to 0.1% formic
acid and 4 mM ammonium formate. The ESI mass spectrometer
was operated in positive ion mode using multiple reaction
monitoring.
diluted with H O and extracted with EtOAc (3 ꢀ 30 mL). The
2
organics were combined and washed with H O (2 ꢀ 20 mL),
2
once with saturated NaHCO solution (20 mL) and once with
3
HT-29 Xenograft. In-life work was performed at Pied-
mont Research Center (Morrisville, NC). Female nude
mice (nu/nu, Harlan) were 11 to 12 weeks old and had a body
weight range of 18.7-30.5 g on Day 1 of the study. Compliance
was observed with the recommendations of the Guide for
Care and Use of Laboratory Animals for veterinary care. Xeno-
grafts were initiated from HT-29 human colon carcinoma
2 4
brine (20 mL). The organic layer was dried over Na SO ,
filtered, and concentrated to give a yellow oil. The yellow oil
was loaded onto a Biotage column (25 mm) and eluted with
solvent gradient (5-65% EtOAc in hexanes). The pure fractions
were combined and concentrated to give 15b (352 mg, 30%)
þ
as yellowish solid. LCMS m/z = 164 [M þ H] , t = 3.64 min.
R
1
H NMR (300 MHz, DMSO-d ): δ 8.59 (bs, 1H), 6.69 (s, 1H),
6

4
298 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Huang et al.
6
1
.19 (s, 1H), 2.77 (m, 2H), 2.18 (m, 2H), 0.97 (s, 6H); mp =
71.2-172.1 °C. HRMS calcd for C10
225.4 °C. HRMS calcd for C18
found 357.0604.
2-Bromo-4-(2,3,6,6-tetramethyl-4-oxo-4,5,6,7-tetrahydro-indol-1-
yl)-benzonitrile (18d). Using the same procedure as described
for compound 18a, treatment of 2-bromo-4-fluorobenzonitrile
(0.275 g, 1.37 mmol) with 15d (0.314 g, 1.64 mmol) yielded 18d
H
17
N
2
OBr 357.0603 [M þ H]þ,
H13NO 164.1076 [M þ
þ
H] , found 164.1075.
3
,6,6-Trimethyl-1,5,6,7-tetrahydro-indol-4-one (15c). To a solu-
tion of anti-pyruvic aldehyde-1-oxime (10 g) and 5,5-dimethyl-1,3-
cyclohexanedione (16.1 g) in AcOH and H O (7:3, 200 mL) was
added zinc powder (14.95 g) slowly using a water bath to keep the
reaction mixture at rt. The mixture was then refluxed overnight,
concentrated to dryness, and partitioned between brine (300 mL)
and dichloromethane (300 mL). The pH was adjusted to ca. 6 with
saturated aqueous NaHCO
with dichloromethane (3 ꢀ 200 mL). The organic layers were
combined, dried over Na SO , filtered, and concentrated. The crude
product was purified by flash chromatography eluting with 5%
ethyl acetate in dichloromethane. The combined organic fractions
were concentrated, triturated in ether-hexanes (2:1) for 1 h, filtered,
and washed with hexanes to give 15c (9 g, 45%) as a solid. LCMS
2
þ
1
(0.23 g, 46%). LCMS m/z = 371 [M þ H] , t
R
= 4.06 min. H
NMR (400 MHz, DMSO-d ): δ 8.11 (d, J = 8.3 Hz, 1H), 8.01 (d,
6
J = 1.9 Hz, 1H), 7.60 (dd, J = 8.3 and 1.9 Hz, 1H), 2.44 (s, 2H), 2.20
(s, 2H), 2.15(s, 3H), 1.93(s, 3H), 0.95(s, 6H);mp=204.6-208.8 °C.
þ
3
, and then the mixture was extracted
HRMS calcd for C19
371.0760.
H
19
N
2
OBr 371.0759 [M þ H] , found
2-Bromo-4-(3-methyl-4-oxo-4,5,6,7-tetrahydro-indol-1-yl)-benzo-
nitrile (18e). Using the same procedure as described for com-
pound 18a, treatment of 2-bromo-4-fluorobenzonitrile (0.39 g,
1.95 mmol) with 15e (0.314 g, 1.64 mmol) afforded 18e (0.41 g,
2
4
þ
1
64%). LCMS m/z = 329 [M þ H] , t
= 3.64 min. H NMR
R
þ
1
m/z = 178.2 [M þ H] , t = 2.58 min. H NMR (400 MHz,
DMSO-d ): δ 10.68 (bs, 1H), 6.41 (s, 1H), 2.55 (s, 2H), 2.14 (s, 2H),
R
(300 MHz, DMSO-d ): δ 8.07 (d, J = 6 Hz, 1H), 8.01 (s, 1H),
6
7.66 (d, J= 6 Hz, 1H), 6.97 (s, 1H), 2.83 (s, 2H), 2.34 (s, 2H), 2.19 (s,
3H), 1.98 (s, 2H); mp = 190.2-198.2 °C. HRMS calcd for
6
2
C
.11 (s, 3H), 0.98 (s, 6H); mp = 161.3-162.9 °C.HRMS calcd for
þ
þ
11
H
15NO 178.1232 [M þ H] , found 178.1231.
C
16
H
13
N
2
OBr 329.0290 [M þ H] , found 329.0290.
2,3,6,6-Tetramethyl-1,5,6,7-tetrahydro-indol-4-one (15d). This
was prepared by the same procedure as for 15c. Treat-
ment of 2,3-butanedione monoxime (10 g, 99 mmol) with dimedone
(13.9 g, 99 mmol) in acetic acid (96 mL) and water (41 mL) with zinc
powder (12.7 g, 198 mmol) afforded 15d (5.6 g, 30%) as a yellow
General Procedure for Hydration of Benzonitriles into Benza-
mides. A crude or purified benzonitrile intermediate (3 mmol) was
dissolved in a mixture of EtOH and DMSO (4:1, 10-20 mL), 1 N
2 2
NaOH aq solution (1-3 mL), and H O (30%, 1-2 mL). The
reaction mixture was stirred at rt or higher temperature as indicated,
for 10 min to 3 h until the disappearance of the benzonitrile by thin
layer chromatography (TLC). The mixture was poured into satu-
þ
1
solid. LCMS m/z = 192.2 [M þ H] , t
R
= 2.81 min. H NMR
(300 MHz, DMSO-d ): δ 10.79 (bs, 1H), 2.55 (s, 2H), 2.10 (s, 2H),
6
2
.02 (s, 3H), 2.00 (s, 3H), 0.98 (s, 6H); mp = 227.0-228.8 °C.
rated NH
4
Cl aq solution (100 mL), extracted with EtOAc (3 ꢀ 50
þ
HRMS calcd for C12
H
17NO 192.1389 [M þ H] , found 192.1388.
mL), the combined organic layers dried over Na SO , filtered, and
2
4
concentrated to give a crude product. This was purified by silica gel
chromatography eluted with 10-50% EtOAc in hexanes followed
by removal of solvent to give the benzamide.
3-Methyl-1,5,6,7-tetrahydro-indol-4-one (15e). This was pre-
pared by the same procedure as for 15c. Treatment of anti-pyruvic
aldehyde-1-oxime (4 g, 46 mmol) and 1,3-cyclohexanedione (5.14 g,
46 mmol) in AcOH and H O (7:3, 85 mL) with zinc powder (5.9 g,
4-(2,6,6-Trimethyl-4-oxo-4,5,6,7-tetrahydro-indol-1-yl)-benza-
mide (11). 15a (600 mg, 3.4 mmol) and 4-fluorobenzonitrile
(410 mg, 3.4 mmol) were dissolved in anhydrous DMF (20 mL).
NaH (95%, 162 mg, 6.8 mmol) was added to this solution,
and the reaction mixture was stirred at 100 °C overnight.
The reaction mixture was cooled to rt and poured into
saturated NH Cl aq solution (250 mL), extracted with EtOAc
4
(3 ꢀ 150 mL), dried over Na
give crude 4-(2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-indol-1-
2
92 mmol) afforded 15e (1.9 g, 26%) as a solid. LCMS m/z = 150.1
þ
1
[
(
M þ H] , t
R
= 2.04 min. H NMR (400 MHz, DMSO-d ): δ 10.92
6
bs, 1H), 6.41 (s, 1H), 2.67 (t, J = 6.16 Hz, 2H), 2.23 (t, J = 6.96 Hz,
2
H), 2.11 (s, 3H), 1.99-1.88 (m, 2H); mp = 195.1-197.2 °C.
þ
HRMS calcd for C
9
H
11NO 150.0919 [M þ H] , found 150.0919.
2-Bromo-4-(2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-indol-1-yl)-
benzonitrile (18a). 15a (3.54 g, 20 mmol) and 2-bromo-4-fluoro-
benzonitrile (4 g, 20 mmol) were dissolved in anhydrous DMF
2 4
SO , filtered, and concentrated to
þ
yl)-benzonitrile (LCMS m/z = 279 [M þ H] ; t = 3.49 min).
(
50 mL). NaH (95%, 960 mg, 40 mmol) was added to the solution,
and the reaction mixture was stirred at 100 °C for 3 h. The reaction
mixture was cooled to rt and poured to saturated NH Cl solution
4
SO ,
R
Hydration of the benzonitrile afforded 11 (570 mg, 57% two
þ
1
steps). LCMS m/z = 297.2 [M þ H] , t
R
= 2.60 min. H NMR
4
(
(
(
6
400 MHz, DMSO-d ): δ 8.01 (d, J = 6 Hz, 2H), 7.52 (s, 1H), 7.45
(
250 mL), extracted by EtOAc (3 ꢀ 150 mL), dried over Na
2
d, J = 6.3 Hz, 2H), 2.41 (s, 2H), 2.22 (s, 2H), 2.01 (s, 3H), 0.96
s, 6H); mp = 199.0-203.0 °C. Anal. (C18 ) C, H, N.
4-(3,6,6-Trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-yl)benza-
filtered, concentrated, and purified using a Biotage column eluted
with 50% EtOAc in hexanes to give 18a (4.4 g, 62%). LCMS m/z =
20 2 2
H N O
þ
1
3
d
6
57, 359 [M þ H] , t
R
= 3.75 min. H NMR (400 MHz, DMSO-
): δ8.12 (d, J = 8.4 Hz, 1H), 8.05 (s, 1H), 7.64 (dd, J = 18 and 8.4
mide (19). Using the same procedures as described for compound 11,
treatment of 15c (177.3 mg, 1 mmol) with 4-fluorobenzonitrile
(121.1 mg, 1 mmol) afforded 19 (237.1 mg, 80% two steps) as a
white powder. LCMS m/z = 297.2 [M þ H] , t = 2.75 min. H
NMR (400 MHz DMSO-d ): δ 8.05 (s, 1H), 7.99 (d, J = 7.5 Hz,
Hz, 1H), 6.23 (s, 1H), 2.99 (s, 1H), 2.49 (s, 2H), 2.21 (s, 2H), 2.04
(
C
s, 3H), 0.96 (s, 6H); mp = 160- 164 °C. HRMS calcd for
þ
þ
1
18
H
2
17
N
2
OBr 357.0603 [M þ H] , found 357.0604.
R
-Bromo-4-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-indol-1-yl)-ben-
6
zonitrile (18b). Using the same procedure as described for com-
pound 18a, treatment of 15b (250 mg, 1.5 mmol) with NaH (61 mg,
2H), 7.50 (d, J = 8.7 Hz, 2H), 7.45 (s, 1H), 6.86 (s, 1H), 2.69 (s, 2H),
2.24 (s, 2H), 2.20 (s, 3H), 0.97 (s, 6H); mp = 246.8-248.0 °C.
þ
1
.5 mmol) and 2-bromo-4-fluorobenzonitrile (450 mg, 2.25 mmol)
HRMS calcd for C H N O 297.1604 [M þ H] , found 297.1603.
18
20
2 2
þ
1
afforded 18b.LCMSm/z= 343 [M þ H] ,t
300 MHz, DMSO-d ): δ8.10 (d, J= 6 Hz, 1H), 8.06 (s, 1H), 7.70 (d,
J = 5.1 Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), 6.53 (s, 1H), 2.78 (s, 2H),
.27 (s, 2H), 0.98 (s, 6H); mp =171.2-172.1 °C. HRMS calcd for
R
= 3.68 min. HNMR
2-(Pentyl)-4-(2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroindol-1-yl)-
benzamide (20a). 18a (200 mg, 0.56 mmol), pentylzinc bromide
solution (0.5 M in THF, 2.24 mL, 1.12 mmol), and PdCl (dppf)
(
6
2
2
C
(20.4 mg, 0.028 mmol) were placed in a microwave vial. The
reagents were stirred and the vessel purged with nitrogen. The
reaction vessel was sealed and heated to 85 °C for 600 s. Upon
cooling, the reaction was quenchedwith10%HCl, andwashedwith
EtOAc (3ꢀ). The organic layer was washed with saturated NaH-
CO (1ꢀ), brine (1ꢀ), and dried over MgSO . Solvent was removed
þ
17
H
2
15
N
2
OBr 343.0446 [M þ H] , found 343.0445.
-Bromo-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-indol-1-
yl)-benzonitrile (18c). Using the same procedure as described for
compound 18a, treatment of 2-bromo-4-fluorobenzonitrile
(
(
13.27 g, 66.4 mmol) with 15c (9.8 g, 55.3 mmol) afforded 18c
3
4
þ
1
= 3.95 min. H
16.5 g, 84%). LCMS m/z = 357.0 [M þ H] , t
R
in vacuo. The residue crude benzonitrile was hydrated at 50 °C for
2 h to afford 20a as a white powder (28 mg, 14%). LCMS m/z =
NMR (300 MHz, DMSO-d ): δ 8.07 (d, J = 8.4 Hz, 1H), 8.01 (d,
6
þ
1
J = 2.1 Hz, 1H), 7.66 (dd, J = 11.4 and 4.5 Hz, 1H), 6.98 (d, J =
.9 Hz, 1H), 2.24 (s, 2H), 2.20 (s, 3H), 0.98 (s, 6H); mp = 222.0-
367.2 [M þ H] , t = 3.49 min. H NMR (300 MHz, DMSO-d ):
R
6
0
δ 7.88 (bs, 1H), 7.47 (bs, 1H), 7.44 (s, 1H), 7.21-7.19 (m, 2H),

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4299
at 50 °C for 2 h to afford 21c as a yellow powder (55.6 mg, 47%).
6
2
0
.20 (d, J = 0.9 Hz, 1H), 2.78 (t, J = 7.5 Hz, 2H), 2.40 (s, 2H),
.21 (s, 2H), 2.01 (s, 3H), 1.59-1.54 (m, 2H), 1.28-1.22 (m, 4H),
.96 (s, 6H), 0.83 (t, J = 6.6 Hz, 3H); mp = 176.6-177.8 °C. Anal.
) C, H, N.
-(Butoxy)-4-(2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroindol-1-yl)-
þ
1
LCMS m/z=388.1 [M þ H] , t =3.48 min. H NMR (400 MHz,
R
DMSO-d ): δ 8.19 (bs, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.61 (bs, 1H),
6
(C
23
H
30
N O
2 2
7.34 (s, 1H), 7.30 (d, J = 7.5 Hz, 2H), 7.20 (d, J=7.2 Hz, 2H), 7.02
(t, J = 2.21 Hz, 1H), 6.90 (s, 1H), 6.72 (d, J=1.2 Hz, 1H), 6.15 (s,
1H), 2.43 (s, 2H), 2.19 (s, 2H), 1.96 (s, 3H), 0.95 (s, 6H);
2
benzamide (20b). 18a (71 mg, 0.2 mmol), butanol (0.5 mL),
and NaH (9.6 mg, 0.4 mmol) were placed in a microwave vial.
The reagents were stirred, and the vessel purged with nitrogen.
The reaction vessel was sealed and heated to 100 °C for 600 s.
Upon cooling, the reaction mixture was treated with 1 N KOH
mp = 224-226 °C. HRMS calcd for C24
25 3 2
H N O 388.2026 [M
þ
þ H] , found 388.2024.
2-(4-Methoxy-phenylamino)-4-(2,6,6-trimethyl-4-oxo-4,5,6,7-
tetrahydro-indol-1-yl)-benzamide (21d). 18a (71.5 mg, 0.2 mmol),
(
11.2 mg, 0.2 mmol) and H
2
O
2
(0.47 mL). After stirring this
2
p-anisidine (98.5 mg, 0.8 mmol), Pd(OAc) (6.7 mg, 5 mol %),
solution at 50 °C for 2 h, the mixture of nitrile and amide was
purified by column chromatography (silica, gradient EtOAc in
hexanes) to afford 20b as a white powder (36.4 mg, 49%). LCMS
DPPF (11.1 mg, 10 mol %), and NaOtBu (38.4 mg, 0.4 mmol)
were treated using the same procedure as for 21a followed by
hydration at 50 °C for 2 h to afford 21d as a yellow powder (43.9
þ
1
þ
1
m/z = 369.2 [M þ H] , t
R
= 3.49 min. H NMR (300 MHz,
): δ 7.86 (d, J = 8.1 Hz, 1H), 7.65 (bs, 1H), 7.59 (bs,
mg, 43%). LCMS m/z = 418.2 [M þ H] , t
R
= 3.40 min. H
DMSO-d
1
6
6
NMR (400 MHz, DMSO-d ): δ 8.19 (bs, 1H), 7.83 (d, J = 8.2
Hz, 1H), 7.58 (bs, 1H), 7.18 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 8.7
Hz, 2H), 6.63 (d, J = 8.2 Hz, 1H), 6.59 (s, 1H), 6.13 (s, 1H), 3.71
H), 7.12 (d, J = 2.1 Hz, 1H), 6.99 (dd, J = 6.3, 2.1 Hz, 1H), 6.20
d, J = 0.9 Hz, 1H), 4.13 (t, J = 6.6 Hz, 2H), 2.44 (s, 2H), 2.21
(
(
(
s, 2H), 2.04 (s, 3H), 1.76-1.69 (m, 2H), 1.46-1.38 (m, 2H), 0.97
(s, 3H), 2.40 (s, 2H), 2.18 (s, 2H), 2.00 (s, 3H), 0.94 (s, 6H); mp =
418.2133 [M þ H] ,
þ
s, 6H), 0.91 (t, J = 8.1 Hz, 3H); mp = 197.0-199.0 °C. HRMS
221-222 °C. HRMS calcd for C25
H
27
3
N O
3
þ
calcd for C H N O 369.2180 [M þ H] , found 369.2177.
found 418.2130.
22
28
2
3
2-Phenylsulfanyl-4-(2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-in-
2-[(3,4,5-Trimethoxyphenyl)amino]-4-(2,6,6-trimethyl-4-oxo-4,5,6,
7-tetrahydro-1H-indol-1-yl)benzamide (22a). Using the same meth-
ods as described for compound 21d, the title compound 22a (1.786 g)
dol-1-yl)-benzamide (20c). Using the same procedure as des-
cribed for 20b, treatment of 18a (100 mg, 0.28 mmol), thiophenol
þ
(
0.0286 mL, 0.28 mmol) with NaH (14.1 mg, 0.56 mmol) in DMF
was prepared from 18a. LCMS m/z = 478.2 [M þ H] , t
R
= 3.25
þ
1
(1 mL) gave 20c (23%). LCMS m/z = 405.1 [M þ H] , t = 3.23
min. H NMR (400 MHz, DMSO-d ): δ 8.16 (bs, 1H), 7.83 (d, J =
R
6
1
min. H NMR (300 MHz, DMSO-d ): δ 8.08 (bs, 1H), 7.69 (d, J =
8.2 Hz, 1H), 7.58 (bs, 1H), 6.84 (s, 1H), 6.68 (d, J=8.2Hz, 1H), 6.51
(s, 2H), 6.14 (s, 1H), 3.72 (s, 6H), 3.59 (s, 3H), 2.98 (s, 1H), 2.42 (s,
2H), 2.18 (s, 2H), 1.96 (s, 3H), 0.93 (s, 6H); mp = 246.0-247.0 °C.
Anal. (C H N O ) C, H, N.
6
7
.8 Hz, 1H), 7.63 (bs, 1H), 7.54-7.51 (m, 2H), 7.46-7.43 (m, 3H),
7.22 (dd, J = 6.3, 2.1 Hz, 1H), 6.58 (d, J = 1.8 Hz, 1H), 6.11 (d,
J = 0.3 Hz, 1H), 2.48 (s, 2H), 2.16 (s, 2H), 1.87 (s, 3H), 0.88 (s, 6H);
27
31
3 5
mp = 109.0-113.0 °C. HRMS calcd for C24
H
24
N
2
O
2
S 405.1638
2-[(3,4,5-Trimethoxyphenyl)amino]-4-(3,6,6-trimethyl-4-oxo-4,5,
6,7-tetrahydro-1H-indol-1-yl)benzamide (22c). Using the same
methods as described for compound 21d, the title compound 22c
(28.4 mg, 20%) was prepared from treatment of 18c (107.1 mg,
0.3 mmol) with 3,4,5-trimethoxyaniline (219.9 mg, 1.2 mmol), Pd
þ
[
M þ H] , found 405.1637.
2
-(Butylamino)-4-(2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-
indol-1-yl)benzamide (21a). 18a (200 mg, 558 μmol), Pd(OAc)
13 mg, 56 μmol), DPPF (31 mg, 56 μmol), and potassium
2
(
phosphate (355 mg, 1.7 mmol) were dissolved in dioxane (4 mL)
followed by n-butylamine (144 mg, 1.7 mmol) and the solution
bubbled with nitrogen for 1 h. More n-butylamine (144 mg,
(OAc) (3.4 mg, 5 mol %), DPPF (16.6 mg, 10 mol %), and
2
NaOtBu (57.7 mg, 0.6 mmol) followed by nitrile hydration, as a
þ
1
yellow powder. LCMS m/z = 478.2 [M þ H] , t
R
= 3.38 min. H
1
.7 mmol) was added and the mixture heated to 100 °C under
nitrogen for 16 h. The reaction mixture was treated with water
15 mL), and extracted with toluene (2 ꢀ 15 mL). The toluene
layers were added to a column and chromatographed (silica gel,
to 50% ethyl acetate in hexanes) to give the intermediate nitrile as
6
NMR (400 MHz, DMSO-d ): δ10.13 (s, 1H), 8.10 (bs, 1H), 7.81 (d,
J = 8.4 Hz, 1H), 7.50 (bs, 1H), 6.98 (d, J = 2.4 Hz, 1H), 6.77 (s,
1H), 6.76 (dd, J = 8.4 and 2.4 Hz, 1H), 6.54 (s, 1H), 3.82 (s, 6H),
3.61 (s, 3H), 2.55 (s, 2H), 2.14 (s, 2H), 2.11 (s, 3H), 0.98 (s, 6H);
mp = 125.6-128.2 °C. HRMS calcd for C H N O 478.2345
(
0
2
7
31
3 5
þ
a crystalline solid (120 mg, 61%). The benzonitrile (80 mg)
was hydrated to give 21a (46 mg, 54%) as a white powder. LCMS
[M þ H] , found 478.2340.
4-(6,6-Dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-yl)-2-[(3,4,
5-trimethoxyphenyl)amino]benzamide (22b). Using the same pro-
cedure as described for compound 21d, 18b (150 mg, 0.44 mmol)
was converted to 22b (0.103 g, 81%). LCMS m/z = 464.2 [M þ
þ
1
m/z = 368.2 [M þ H] , t
R
= 3.58 min. H NMR (400 MHz,
DMSO-d ): δ 8.29 (t, J = 5.2 Hz, 1H), 7.92 (bs, 1H), 7.72 (d, J =
6
8.3 Hz, 1H), 7.26 (bs, 1H), 6.51 (d, J = 1.8 Hz, 1H), 6.43 (dd, J =
þ
1
6.4, 1.8 Hz, 1H), 6.17 (s, 1H), 3.09 (m, 2H), 2.45 (s, 2H), 2.21
H] , t = 3.18 min. H NMR (300 MHz, DMSO-d ): δ 8.13 (bs,
R 6
(
s, 2H), 2.04 (s, 3H), 1.48-1.56 (m, 2H), 1.31-1.40 (m, 2H), 0.97
1H), 7.83 (d, J = 6 Hz, 1H), 7.57 (bs, 1H), 7.02 (s, 1H), 6.93 (s, 1H),
6.77 (d, J = 6.3 Hz, 1H), 6.55 (s, 1H), 6.44 (s, 1H), 3.73 (s, 6H), 3.61
(s, 3H), 2.64 (s, 2H), 2.24 (s, 2H), 0.94 (s, 6H); mp =145.2-
(
s, 6H), 0.89 (t, J = 7.3 Hz, 3H); mp = 253-255 °C. HRMS calcd
þ
for C22
H N O
29 3 2
368.2339 [M þ H] , found 368.2339.
þ
2-[(2-Methoxyethyl)amino]-4-(2,6,6-trimethyl-4-oxo-4,5,6,7-tet-
rahydro-1H-indol-1-yl)benzamide (21b). 18a (100 mg, 0.28 mmol),
146.7 °C. HRMS calcd for C H N O 464.2182 [M þ H] , found
26
29
3 5
464.2182.
2
5
0
-methoxyethylamine (84.1 mg, 1.12 mmol), Pd(OAc) (9.4 mg,
mol %), DPPF (15.5 mg, 10 mol %), and NaOtBu (53.8 mg,
.56 mmol) were treated using the same procedure as for 21a
2
4-(3-Methyl-4-oxo-4,5,6,7-tetrahydroindol-1-yl)-2-(3,4,5-tri-
methoxyphenylamino)benzamide (22e). Using the same proce-
dure as described for compound 21d, 18e (0.227 g, 0.7 mmol) was
þ
followed by hydration at 50 °C for 2 h to afford 21b as a yellow
converted to 22e (0.199 g, 66%). LCMS m/z = 450 [M þ H] ,
þ
1
= 3.15 min. H NMR (400 MHz, DMSO-d
powder (43.9 mg, 43%). LCMS m/z = 370.2 [M þ H] , t
R
= 2.93
t
R
6
): δ 10.13 (s, 1H),
1
min. H NMR (400 MHz, DMSO-d ): δ 8.34 (bs, 1H), 7.92 (bs,
8.10 (bs, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.50 (bs, 1H), 6.98 (s, 1H),
6.77 (s, 1H), 6.76 (dd, J = 8.4, 8.4 Hz, 1H), 6.54 (s, 2H), 3.62 (s,
3H), 2.76 (t, J = 6.0 Hz, 2H), 2.48 (s,6H), 2.31(s,2H), 2.16(s, 3H),
6
1H), 7.71 (d, J = 8.1 Hz, 1H), 7.26 (bs, 1H), 6.56 (s, 1H), 6.45 (d,
J = 1.5 Hz, 1H), 6.17 (s, 1H), 3.49 (t, J = 5.1 Hz, 2H), 3.32 (s, 5H),
2
2
3
.49 (s, 2H), 2.21 (s, 2H), 2.04 (s, 3H), 0.97 (s, 6H); mp = 221-
27 3 5
1.97-1.93 (m, 2H); mp = 134.3-137.0 °C. Anal. (C25H N O )
þ
22 °C. HRMS calcd for C H N O 370.2133 [M þ H] , found
C, H, N.
3-Bromo-4-cyanophenylhydrazine (23). In a clean, dry 250 mL
round-bottom flask, 2-bromo-4-fluorobenzonitrile (25.34 g)
21
27
3 3
70.2132.
2
-(Phenylamino)-4-(2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroin-
dol-1-yl)benzamide (21c). 18a (107.2 mg, 0.3 mmol), aniline
111.8 mg, 1.2 mmol), Pd(OAc) (10.1 mmol, 5 mol %), DPPF
2
was dissolved in THF (50 mL) under N . To this solution was
(
slowly added anhydrous hydrazine (50 mL). The solution color
changed from yellow to red-orange. The reaction was allowed
to stir at rt for 16 h. A yellow-white crystalline solid precipitated
2
(16.6 mg, 10 mol %), and NaOtBu (57.7 mg, 0.6 mmol) were
treated using the same procedure as for 21a followed by hydration

4
300 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Huang et al.
from the solution. The mixture was then diluted with THF
50 mL) to dissolve the solids. The organic layer was then washed
organic layer was dried by MgSO
(400 mg, 70%) as an oil. H NMR (400 MHz, DMSO-d ): δ
4
and concentrated to give 27
1
(
6
with saturated sodium bicarbonate solution until the pH of the
organic layer was approximately 8.5. The organic layer was
isolated, and the solvent was removed under reduced pressure
to give a white solid. This was place in a fritted glass funnel and
washed with water (1.5 L), followed by diethyl ether (ca. 200 mL).
The ether wash was then combined with the white solid and dried
under reduced pressure. Title compound 23 was isolated as a
fluffy, white or off-white solid (23.43 g, 87.2%). LCMS m/z =
10.54 (bs, 3H), 8.76 (bs, 1H), 7.37 (m, 1H), 6.42 (m, 1H), 6.26 (m,
1H), 5.39 (bs, 1H), 3.48 (m, 1H), 3.35 (m, 1H), 1.93 (m, 4H), 1.38
(m, 4H); mp = 216-127.5 °C. HRMS calcd for C13
18 4
H N O
þ
247.1559 [M þ H] , found 247.1559.
5,5-Dimethyl-2-propionyl-cyclohexane-1,3-dione (28a). 5,5-Di-
methyl-1,3-cyclohexanedione (14 g, 100 mmol) and DM-
AP (3.7 g, 30 mmol) were dissolved in methylene chloride
(100 mL) and treated with Hunig’s base (17.5 mL, 100 mmol).
A solution of propionyl chloride (9.25 g, 100 mmol) in
methylene chloride (25 mL) was added dropwise. The mix-
ture was heated to reflux. After 2 h, TLC showed mostly
product with a trace of the intermediate. The mixture was
concentrated then partitioned between 1 N HCl (50 mL) and
hexanes-EtOAc (2:1, 150 mL). The organic layer was washed
þ
1
2
12.1 [M þ H] , t
R 6
= 2.13 min. H NMR (400 MHz, DMSO-d ):
δ 8.02 (s, 1H), 7.45 (d, J = 8.7 Hz, 1H), 7.04 (d, J = 2 Hz, 1H),
6
.71 (dd, J = 8.7, 2 Hz, 1H), 4.38 (bs, 2H);mp= 143.4-146.5 °C.
-Bromo-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-indazol-
-yl)-benzonitrile (24a). Ina clean, dry 20mL microwave reaction
2
1
vial, 23 (2.49 g) was combined with 2-acetyl-5,5-dimethyl-1,3-
cyclohexanedione (2.14 g). The contents of the vial were dissolved
in ethanol and acetic acid (12 mL, 3:1). The vial was sealed and
agitated on a vortex. The vial was then placed in the microwave
reactor and heated to 150 °C for 15 min. The vial was then cooled
and placed in a refrigerator for 1 h. The cooled solution was then
diluted with water (8 mL) and poured onto a fritted glass funnel.
with brine (50 mL), dried over MgSO , concentrated, and
4
chromatographed using a Biotage column (40 mm) eluted
with EtOAc in hexanes (0 to 20%) to give 28a (17.56 g,
þ
1
89%). LCMS m/z = 197.2 [M þ H] , t
R
= 3.53 min. H
NMR (400 MHz, DMSO-d ): δ 2.95 (q, J = 7.2 Hz, 2H),
6
2.25-2.65 (bs, 4H), 1.01 (t, J = 7.2 Hz, 3H), 0.97 (s, 6H);
The orange solid was washed with H O (100 mL), followed by
2
mp = 37.7-38.5 °C. HRMS calcd for C11
H
16
O
3
197.1180
þ
ethanol(25mL). Thesolidwas thendriedunderreducedpressure.
Title compound 24a was obtained as a light-orange crystalline
[M þ H] , found 197.1177.
2-(Isobutyryl)-5,5-dimethylcyclohexane-1,3-dione (28b). 5,5-
Dimethyl-1,3-cyclohexanedione (1 g, 7.13 mmol) and DMAP
(260 mg, 2.14 mmol) were dissolved in methylene chlo-
ride (10 mL) and treated with Hunig’s base (920 mg, 7.13 mmol).
Isobutyric anhydride (1.13 g, 7.13 mmol) was added dropwise
and the mixture stirred. After 4 days, the solution was diluted
with dichloroethane (10 mL) and heated to 65 °C for 1 d. The
mixture was concentrated and partitioned between 1 N HCl
(15 mL) and hexanes (15 mL). The organic layer was added to
a column and the aqueous layer was extracted with more
hexanes (5 mL), which was also added to a column. The mixture
was chromatographed (silica gel, 0 to 20% EtOAc in hexanes)
to give 28b (195 mg, 14%) as an oil. LCMS m/z = 211.2 [M þ
þ
solid(3.75 g, 89%). LCMSm/z = 358.3[Mþ H] , t
R
= 3.75 min.
1
6
H NMR (400 MHz, DMSO-d ): δ 8.10 (d, J = 8.2 Hz, 1H), 8.08
(
(
d, J = 2 Hz, 1H), 7.79 (dd, J = 2, 8.2 Hz, 1H), 2.99 (s, 2H), 2.39
s, 3H), 2.32 (s, 2H), 1.01 (s, 6H); mp = 196.8-198.2 °C. HRMS
þ
calcd for C17
H
16
N
3
OBr 358.0555 [M þ H] , found 358.0556.
2-(trans-4-Hydroxy-cyclohexylamino)-4-(3,6,6-trimethyl-4-oxo-
,5,6,7-tetrahydro-indazol-1-yl)-benzamide (25a). A microwave
4
vial was charged with 24a (2 g, 5.58 mmol), trans-4-aminocyclo-
hexanol (1.29 g, 11.1 mmol), Pd(OAc) (64 mg, 5 mol %), DPPF
2
(312 mg, 10 mol %), and NaOtBu (1.08 g, 11.1 mmol). The
reagents were suspended in toluene (15 mL) and were heated with
microwave to 170 °C for 3 h. After allowing the reaction vessel to
cool, the suspension was filtered and the filtrate evaporated. The
residue was purified by flash chromatography, and the benzoni-
trile was hydrated to yield 25a (0.8 g, 67%) as a yellow powder.
þ
1
H] , t = 3.88 min. H NMR (400 MHz, DMSO-d ): δ 3.85
R
6
(sept, J = 6.8 Hz, 1H), 2.27-2.66 (bs, 4H), 1.03 (d, J = 6.8 Hz,
6H), 0.98 (s, 6H).
þ
1
LCMS m/z = 411.2 [M þ H] , t = 2.60 min. H NMR (300
2-(2-Cyclopropylacetyl)-5,5-dimethylcyclohexane-1,3-dione
(28c). To 5,5-dimethyl-1,3-cyclohexanedione (6.93 g, 49.4 mmol),
cyclopropylacetic acid (3.4 mL, 33 mmol), and DMAP (6.04 g,
49.4 mmol) in CH Cl (90 mL) at 0 °C was added dropwise
R
MHz, DMSO-d ): δ 8.35 (d, J = 7.8 Hz, 1H), 7.88 (bs, 1H), 7.71
6
(d, J = 8.6 Hz, 1H), 7.22 (bs, 1H), 6.75 (d, J = 1.8 Hz, 1H), 6.65
(dd, J = 8.6, 1.8 Hz, 1H), 3.46 (m, 1H), 2.92 (s, 2H), 2.38 (s, 3H),
2
2
2.32 (s, 2H), 1.97 (m, 2H), 1.82 (m, 2H), 1.27 (m, 4H), 0.99 (s, 6H);
a solution of N,N-dicyclohexylcarbodiimide (8.15 g, 39.5 mmol)
in CH Cl (90 mL). The solution was allowed to warm to 25 °C
mp = 133.8-136.1 °C. HRMS calcd for C23
30 4 3
H N O 411.2398
2
2
þ
[
M þ H] , found 411.2398.
and stirred for 14 h. The crude mixture was filtered through
celite and concentrated. It was taken up in EtOAc (200 mL) and
washed with 2 M aqueous HCl (2 ꢀ 200 mL). The aqueous layer
was back-extracted with EtOAc (200 mL). The combined organic
portions were washed with saturated aqueous NaCl (200 mL) and
4-Fluoro-2-(4-trans-hydroxy-cyclohexylamino)-benzonitrile (26).
,4-Difluorobenzonitrile (50.0 g, 0.359 mol), trans-4-aminocyclo-
2
hexanol (41.4 g, 0.359 mol), and diisopropylethylamine (62.6 mL,
.359 mol) were dissolved in DMSO (300 mL). The reaction vessel
0
was outfitted with a reflux condenser to avoid loss of diisopropy-
lethylamine. The reaction mixture was then placed in an oil bath
that had been preheated to 150 °C and was stirred at this
temperature for 20 min. The solution was then cooled, poured
2 4
dried over Na SO . Purification by gradient flash chromatogra-
phy, eluted with EtOAc in hexanes (0% to 25%) provided 28c
(6.2 g, 85%) as a pale yellow oil. LCMS m/z = 223.1 [M þ H] , t
þ
R
1
= 3.86 min. H NMR (400 MHz, DMSO-d ): δ 10.54 (bs, 3H),
6
into saturated aqueous NH Cl (750 mL), and extracted with
4
8.76 (bs, 1H), 7.37 (m, 1H), 6.42 (m, 1H), 6.26 (m, 1H), 5.39 (bs,
1H), 3.48 (m, 1H), 3.35 (m, 1H), 1.93 (m, 4H), 1.38 (m, 4H).
EtOAc (200 mL ꢀ 3). The combined organics were washed with
þ
brine (150 mL ꢀ 3), dried over Na SO , filtered, and concentrated
HRMS calcd for C H O 223.1336 [M þ H] , found 223.1334.
2
4
13 18 3
in vacuo. The residue was purified by column chromatography
(1:1 EtOAc in hexanes) to afford 26 (20.9 g, 25%) as a white
4-(3-Ethyl-6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-indazol-1-
yl)-2-(4-hydroxy-cyclohexylamino)-benzamide (29a). 28a (107
mg, 1 equiv) and 27 (140 mg, 1 equiv) were combined in AcOH
and EtOH (1:5, 20 mL) and microwaved at 120 °C for 10 min. The
reaction mixture was concentrated to dryness, and residue crude
benzonitrile hydrated to afford 29a (130 mg, 56%). LCMS m/z =
þ
1
powder. LCMS m/z = 235.1 [M þ H] , t
R
= 1.77 min. H NMR
(
400 MHz, DMSO-d ): δ 7.50 (m, 1H), 6.64 (m, 1H), 6.42 (m, 1H),
6
5
.76 (bs, 1H), 4.54 (m, 1H), 3.28-3.43 (m, 2H), 1.81 (m, 4H), 1.31
(m, 4H); mp = 126.2-128.9 °C. HRMS calcd for C13
H
15
N
2
OF
þ
þ
1
235.1247 [M þ H] , found 235.1246.
425.2 [M þ H] , t =2.83min. H NMR (400 MHz, DMSO-d ): δ
R
6
4
-Hydrazino-2-(4-trans-hydroxy-cyclohexylamino)-benzoni-
8.34 (d, J=7.6 Hz, 1H), 7.88 (b, 1H), 7.71 (d, J=8.5 Hz, 1H), 7.21
(b, 1H), 6.76 (d, J = 2.0 Hz, 1H), 6.66 (dd, J = 8.5, 2.0 Hz, 1H),
4.57 (d, J = 4.1 Hz, 1H), 3.51-3.42 (m, 1H), 2.92 (s, 2H), 2.8 (q, J
= 7.5 Hz, 2H), 2.32 (s, 2H), 2.00-1.96 (m, 2H), 1.83-1.79 (m, 2H),
1.36-1.21 (m, 4H), 1.81 (t, J = 7.5 Hz, 3H), 0.99 (s, 6H); mp =
trile (27). 26 (537 mg, 2.29 mmol) was dissolved in hydrazine
2 g, 64 mmol) and heated to 60 °C and stirred for 30 min. The
mixture was partially concentrated and then partitioned be-
tween EtOAc (25 mL) and half saturated NaHCO (25 mL). The
(
3

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4301
2
01.1-202.6 °C. HRMS calcd for C24
H
32
N
4
O
3
425.2555 [M þ
DMSO-d
6
): δ 2.78 (s, 2H); 2.35 (s, 2H), 1.01 (s, 6H); mp = 209-
þ
þ
H] , found 425.2552.
214 °C. HRMS calcd for C H N OF 233.0902 [M þ H] ,
1
0
11
2
3
2
-[(trans-4-Hydroxycyclohexyl)amino]-4-(3-isopropyl-6,6-di-
methyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide (29b).
8b (120 mg, 0.57 mmol), 27 (130 mg, 0.46 mmol), and sodium
found 233.0901.
3-Difluoromethyl-6,6-dimethyl-1,5,6,7-tetrahydro-indazol-
4-one (31b). The title compound (3.12 g, 50.6%) was prepared
using the same procedure as for 31a from 30 (8.86 g) and
difluoroacetic anhydride (5 g) in a mixture of THF (100 mL)
2
acetate (59 mg, 0.71 mmol) were combined in methanol (4 mL)
and stirred at rt for 16 h. The reaction mixture was concentrated
and the residue hydrated to give 29b (126 mg, 63%) as a pink
solid. Half of the material was recrystallized from methanol/water
þ
and triethylamine (30 mL). LCMS m/z = 215.2 [M þ H] ,
1
t = 2.51 min. H NMR (400 MHz, DMSO-d ): δ 7.04 (t, 1H,
R
6
þ
1
for analysis. LCMS m/z= 439 [M þ H] , t =3.13min. HNMR
J = 53 Hz), 2.76 (s, 2H); 2.32 (s, 2H), 1.01 (s, 6H); mp =
OF
R
(400 MHz, DMSO-d
6
): δ 8.43 (d, J = 7.7 Hz, 1H), 7.88 (bs, 1H),
145.1-146.8 °C. HRMS calcd for C10
H
12
N
2
2
215.0996
þ
7
6
3
1
0
4
.71 (d, J = 8.7 Hz, 1H), 7.20 (bs, 1H), 6.77 (d, J = 1.8 Hz, 1H),
[M þ H] , found 215.0997.
.67 (dd, J= 1.8, 8.7 Hz, 1 H), 4.56 (d, J= 4 Hz, 1H), 3.47 (m, 1H),
.29 (m, 1H) 2.92 (s, 2H), 2.33 (s, 2H), 1.94-2.03 (m, 2H),
.77-1.86 (m, 2H), 1.14-1.38 (m, 5H), 1.24 (d, J = 6.8 Hz, 6H),
.99 (s, 6H); mp 191-192 °C. HRMS calcd for C25
39.2711 [M þ H] , found 439.2709.
2-Bromo-4-(6,6-dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetra-
hydro-indazol-1-yl)-benzonitrile (32a). NaH (168 mg, 7.02 mmol)
was added to a solution of 31a (1.63 g, 7.02 mmol) in anhydrous
DMSO (35 mL). After 15 min, 2-bromo-4-fluorobenzonitrile
(2.25 g, 11.23 mmol) was added as a solid. The reaction mixture
was heated at 45 °C. After 23 h, the reactionmixturewas cooledto
34 4 3
H N O
þ
4-[3-(Cyclopropylmethyl)-6,6-dimethyl-4-oxo-4,5,6,7-tetrahy-
dro-1H-indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]ben-
zamide (29c). 28c (253 mg, 1.14 mmol) and 27 (281 mg, 1.14
mmol) in a mixture of EtOAc, EtOH, and AcOH (4:3:1, 2.2 mL)
was stirred at 150 °C for 16 h. The reaction mixture was diluted
with EtOAc (20 mL) and stirred at 25 °C for 16 h. The reaction
4
rt and quenched with saturated aqueous NH Cl (10 mL). The
mixture was diluted with water and extracted with EtOAc (4ꢀ).
The combined organic layers were washed with brine, dried over
sodium sulfate, filtered, and concentrated in vacuo. The residue
was purified on a Biotage column (SiO
afford 32a (1.83 g, 63%) as an off-white powder. LCMS m/z =
2
, hexanes-EtOAc) to
mixture was then washed with saturated NaHCO (1 ꢀ 50 mL),
3
þ
1
and the aqueous layer was back-extracted with EtOAc
(
412.0 [M þ H] , t = 4.06 min. H NMR (400 MHz, DMSO-d ):
R
6
1 ꢀ 20 mL). The reaction was concentrated and purified by
δ 8.19 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.88 (dd, J = 8.4 and
2.1 Hz, 1H), 3.03 (s, 2H), 2.43 (s, 2H), 1.02 (s, 6H); mp = 168.0-
column chromatography eluted with 75% EtOAc in hexanes
to provide 4-(3-cyclopropylmethyl-6,6-dimethyl-4-oxo-4,5,6,7-
tetrahydroindazol-1-yl)-2-(4-hydroxycyclohexylamino)-benzoni-
trile (220 mg, 45%) as an orange solid. The benzonitrile
was hydrated to provide 29c (215 mg, 94%) as a white solid.
þ
172.0 °C. HRMS calcd for C H N OBrF 412.0273 [M þ H] ,
1
7
13
3
3
found 412.0273.
2-Bromo-4-(6,6-dimethyl-4-oxo-3-difluoromethyl-4,5,6,7-tetrahy-
dro-indazol-1-yl)-benzonitrile (32b). Compound 32b was prepared
(2.82 g, 49.2%) as a white solid using the same procedure as for 32a
from 31b (3.12 g) and 2-bromo-4-fluorobenzonitrile (4.67 g).
þ
1
LCMS m/z = 451.2 [M þ H] , t = 3.10 min. H NMR (400
R
MHz, DMSO-d
6
): δ 8.18 (d, J = 7.6 Hz, 1H), 7.71 (bs, 1H), 7.54
d, J = 8.5 Hz, 1H), 7.04 (bs, 1H), 6.61 (d, J = 1.9 Hz, 1H), 6.50
dd, J = 8.5, 1.9 Hz, 1H), 4.38 (d, J = 4.1 Hz, 1H), 3.34-3.24 (m,
þ
1
(
(
1
2
LCMS m/z = 394 [M þ H] , t = 3.88 min. H NMR (300
R
MHz, DMSO-d ): δ 8.17 (m, 2H), 7.86 (dd, J = 8.4, 1.8 Hz, 1H),
6
H), 3.13-3.09 (m, 1H), 2.77 (s, 2H), 2.54 (d, J = 6.9 Hz, 2H),
7.22 (t, J= 54 Hz, 1H), 3.03 (s, 2H), 2.48 (s, 2H), 1.02 (s, 6H); mp =
.16 (s, 2H), 1.85-1.78 (m, 2H), 1.68-1.60 (m, 2H), 1.20-0.90
163.5-165.7 °C. HRMS calcd for C H N OBrF 394.0367 [M þ
17
14
3
2
þ
H] , found 394.0368.
(
(
m, 3H), 0.83 (s, 6H), 0.24-0.18 (m, 2H), 0.03-0.01
m, 2H); mp = 214.5-218.9 °C. Anal. (C H N O ) C, H, N.
2
6
34
4
3
4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-
indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]benzamide (9). A
microwave vial was charged with 32a (0.080 g, 0.19 mmol), trans-4-
2
aminocyclohexanol (0.044 g, 0.38 mmol), Pd(OAc) (2.5 mg, 5 mol
%), DPPF (11.1 mg, 10 mol %), and NaOtBu (0.038 g, 0.38 mmol).
The reagents were suspended in toluene (0.5 mL) and were heated
in a microwave to 170 °C for 3 h. After cooling, the suspension
was filtered and the filtrate evaporated. The residue was purified
by flash chromatography to afford 4-(6,6-dimethyl-4-oxo-3-triflu-
oromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-2-(4-hydroxy-cyclohex-
3,3-Dimethyl-5-(p-tolylsulfonylhydrazono)-cyclohexanone (30).
3
7
This compound was prepared as previously reported from 5,5-
dimethyl-1,3-cyclohexanedione (7.0 g, 49.9 mmol) and p-tolue-
nesulfonylhydrazide (9.3 g, 49.9 mmol) in heated toluene (300
mL)asa light-yellow solid (14.26 g, 93%):LCMSm/z = 309.1 [M
þ
1
þ H] , t = 2.68 min. H NMR (400 MHz, DMSO-d ): δ 9.74
R
6
(bs, 1H), 7.66 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 2.48
(s, 2H), 2.36 (s, 3H), 2.04 (s, 2H), 1.90 (s, 2H), 0.87 (s, 6H);
mp = 201.5-202.7 °C. HRMS calcd for C15
20 2 3
H N O S 309.1275
þ
þ
[
M þ H] , found 309.1272.
ylamino)-benzonitrile. LC/MS m/z = 447 [M þ H] . The benzo-
6
,6-Dimethyl-3-trifluoromethyl-1,5,6,7-tetrahydro-indazol-4-
nitrile (17.1 g, 38.3 mmol) was hydrated to afford 9(15.0 g, 84%) as a
= 3.11
þ
one (31a). To a suspension of 30 (4.0 g, 12.97 mmol) in a mix-
ture of THF (72 mL) and triethylamine (24 mL) was added
trifluoroacetic anhydride (1.8 mL, 12.97 mmol). The dark-red
reaction mixture was heated at 55 °C. After 15 min, the reac-
tion mixture was homogeneous. After 2 h, the reaction mix-
ture was cooled to rt. Methanol (16 mL) and a 1:1 solution of
water and 1 M aqueous sodium hydroxide (16 mL) were added.
After stirring for 3 h, the reaction mixture was diluted with
white amorphous owder. LCMS m/z = 465.2 [M þ H] , t
R
1
6
min. H NMR (400 MHz, DMSO-d ): δ 8.36 (d, J = 7.72 Hz, 1H),
7.94 (bs, 1H), 7.76 (d, J = 8.44 Hz, 1H), 7.29 (bs, 1H), 6.86 (s, 1H),
6.69 (dd, J = 8.4, 1.96 Hz, 1H), 3.51-3.41 (m, J = 4.24 Hz, 1H),
2.95 (s, 2H), 2.43 (s, 2H), 1.99-1.95 (m, 2H), 1.82-1.78 (m, 2H),
1.35-1.15 (m, 4H), 1.02 (s, 6H); mp = 265.2-266.3 °C. Anal.
27 4 3 3
(C23H N O F ) C, H, N.
4-[3-(Difluoromethyl)-6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-
indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]benzamide (33).
32b (0.394 g, 1 mmol), trans-4-aminocyclohexanol (0.288 g, 2.5
5
0 mL of saturated aqueous ammonium chloride and extracted
with ethyl acetate (3 ꢀ 50 mL). The combined organic layers
were washed with brine, dried over sodium sulfate, filtered, and
concentrated in vacuo. The residue was filtered through a plug
of silica gel and eluted with ethyl acetate. The filtrate was
concentrated in vacuo, and the residue was treated with ether.
The solids were collected by filtration and washed with ether.
The filtrate was concentrated in vacuo, and the resulting residue
was treated with ether. The solids were collected by filtration,
washed with ether, and combined with the initial solids to
provide 31a (1.24 g, 41%) as a reddish-orange solid. LCMS
mmol), Pd(OAc) (11.2 mg, 5 mol %), DPPF (55.4 mg, 10 mol %),
2
and NaOtBu (0.192 g, 2 mmol) were suspended in toluene (2 mL).
The reaction was microwaved at 120 °C for 20 min, cooled, and
concentrated. The residue was diluted with H O, extracted with
2
EtOAc (3ꢀ). The combined organic layers were dried over Na SO
2
4
and concentrated. The residue was purified by column chromatog-
raphy (CH Cl ) to afford 4-[3-(difluoromethyl)-6,6-dimethyl-4-
oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl]-2-[(trans-4-hydroxycyclo-
2
2
hexyl)amino]benzonitrile. The benzonitrile was hydrated to afford
33 (0.165 g, 37%). H NMR (300 MHz, DMSO-d ): δ 8.37 (d, J =
þ
1
1
m/z = 233.2 [M þ H] , t
R
= 2.92 min. H NMR (400 MHz,
6

4
302 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Huang et al.
7
6
.5 Hz, 1H), 7.94 (bs, 1H), 7.75 (d, J = 7.1 Hz, 1H), 7.28 (bs, 1H),
.82 (d, J = 2.1 Hz, 1H), 6.68 (dd, J = 7.1 and 1.8 Hz, 1H), 4.57 (d,
J = 4.2, 1H), 3.47-3.44 (m, 1H), 2.96 (s, 2H), 2.40 (s, 2H), 1.99-
.97 (m, 2H), 1.82-1.79 (m, 2H), 1.37-1.22 (m, 5H), 1.01 (s, 6H);
mp = 155.0-158.0 °C. Anal. (C23 ) C, H, N.
8.10 (d, J = 5.44 Hz, 1H), 7.44 (d, J = 6.64 Hz, 1H), 7.26 (s, 1H),
6.55 (d, J= 17.88 Hz, 2H), 6.44 (d, J= 6.64 Hz, 1H), 3.75 (bs, 1H),
2.66 (s, 2H), 2.15 (d, J = 8.76 Hz, 2H), 2.05 (bs, 4H), 1.59 (s, 3H),
1.49-1.37 (m, 5H), 1.26 (t, J = 5.52 Hz, 2H), 1.08 (s, 6H); mp =
1
H
28
N
4
O
3
F
2
31 3 3
170.7-173.4 °C. Anal. (C24H N O ) C, H, N.
Amino-acetic Acid 4-[2-Carbamoyl-5-(6,6-dimethyl-4-oxo-3-triflu-
oromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-phenylamino]-cyclohexyl
Ester Methanesulfonate (10). A mixture of 9 (44.8 g, 96.45 mmol),
2-[(trans-4-Hydroxycyclohexyl)amino]-4-(2,3,6,6-tetramethyl-4-
oxo-4,5,6,7-tetrahydro-1H-indol-1-yl)benzamide (35). 15d (1.91 g,
10 mmol) was dissolved in DMAC (60 mL). NaH (360 mg,
15 mmol) was added, and the reaction mixture was allowed to
stir at rt for 30 min. 26 (2.34 g, 10 mmol) was added, and the
reaction mixture was stirred at 150 °C for overnight. Upon
completion, the crude reaction mixture was poured into saturated
1
-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
37.0 g, 192.9 mmol), 4-dimethylaminopyridine (1.18 g, 9.645 mmol),
and N-(tert-butoxycarbonyl)glycine (33.8 g, 192.9 mmol) in CH Cl
1 L) was stirred at rt for overnight. The reaction mixture was poured
into H O (1 L), the organics were collected, and the aqueous layer
was extracted 2 times with CH Cl (500 mL). The organics were
(
2
2
(
2
NH
4
Cl aq solution, extracted with EtOAc (3 ꢀ 300 mL), dried
2
2
over Na SO , filtered, and concentrated to give 2-[(trans-4-hydro-
2
4
combined and washed one time each with saturated NaHCO , 1 N
3
xycyclohexyl)amino]-4-(2,3,6,6-tetramethyl-4-oxo-4,5,6,7-tetrahy-
dro-1H-indol-1-yl)benzonitrile. This was hydrated to give 35
HCl, and brine. The organics were then dried over Na SO and
2
4
þ
concentrated to a pale-yellow oil and then purified on a Biotage
column eluted with EtOAc in hexanes (30-80%). Pure fractions
were combined and concentrated to give tert-butoxycarbonylami-
no-acetic acid 4-[2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-trifluoro-
methyl-4,5,6,7-tetrahydro-indazol-1-yl)-phenylamino]-cyclohexyl
(500 mg, 12% for 2 steps). LCMS m/z = 424.20 [M þ H] ,
1
t
R
= 2.96 min; mp = 194.9-199.0 °C. H NMR (400 MHz,
DMSO-d ): δ 8.42 (d, J = 6.2 Hz, 1H), 8.24 (d, J = 5.7 Hz, 1H),
6
7.89 (bs, 1H), 7.69(d, J = 5.7 Hz, 1H), 7.23 (bs, 1H), 3.49-3.41 (m,
1H), 2.66 (s, 2H), 2.42 (s, 3H), 2.28 (s, 3H), 2.15 (m, 2H), 2.05 (bs,
4H), 1.49-1.37 (m, 5H), 1.16 (t, J = 5.1 Hz, 2H), 0.95 (s, 6H).
Anal. (C H N O ) C, H, N.
þ
ester (49.31 g). LCMS m/z = 622 [M þ H] . This compound
(
49.31 g, 79.32 mmol) was suspended in CH Cl , cooled to 0 °C,
2
2
25 33
3 3
and a mixtureof trifluoroacetic acid and CH Cl
2
2
(150 mL, 1:1) was
2-(Tetrahydro-pyran-4-ylamino)-4-(3,6,6-trimethyl-4-oxo-
4,5,6,-tetrahydro-indazol-1-yl)-benzamide (36). Using the same
procedure as described for compound 34, treatment of 24a
added dropwise over 10 min. The reaction was then stirred at 0 °C
for 10 min, and at rt for 90 min. To the resulting suspension was
added H O (1 L) and K CO (77 g, 555 mmol). This was stirred for
(1.26 mmol, 450 mg) with Pd(OAc) (0.06 mmol, 14 mg), 4-
2
2
2
3
1
0 min and then filtered to give amino-acetic acid 4-[2-carbamoyl-5-
aminotetrahydropyran (2.52 mmol, 255 mg), DPPF (0.13 mmol,
70 mg), and NaOtBu (2.52 mmol, 242 mg) followed by hydra-
tion of the benzonitrile yielded 36 (69%). LCMS m/z = 397.2
(6,6-dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-
yl)-phenylamino]-cyclohexyl ester (25.6 g). LCMS m/z = 522 [M þ
þ
þ
1
H] . This free amine (25.6 g, 49.1 mmol) was suspended in a mixture
[Mþ H] , t
R 6
= 2.83 min. H NMR(300 MHz, DMSO-d ):δ 8.48
of CH
3.18 mL, 49.1 mmol) was added. After stirred for 90 min, the
mixture was filtered under vacuum to afford 10 (27.5 g) as a white
2
Cl
2
and MeOH (700 mL, 4:1), and methanesulfonic acid
(d, J = 10.2 Hz, 1H), 7.86 (bs, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.23
(bs, 1H), 6.82 (d, J = 2.1 Hz, 1H), 6.67 (dd, J = 6.6, 1.8 Hz, 1H),
3.83 (m, 2H), 3.63 (m, 1H), 3.43 (m, 2H), 2.90 (s, 2H), 2.38 (s, 3H),
2.31 (s, 2H), 1.95 (m, 2H), 1.39 (m, 2H), 0.99 (s, 6H); mp =
(
þ
1
powder. LCMS m/z = 522.3 [M þ H] , t
300 MHz, DMSO-d ): δ8.17(s,3H), 7.99(bs, 1H), 7.78(d,J=8.44
Hz, 1H), 7.32 (bs, 1H), 6.88 (d, J = 1.92 Hz, 1H), 6.72 (dd, J = 8.4,
.96 Hz, 1H), 4.86-4.80 (m, 1H), 3.81-3.79 (m, 2H), 2.96 (s, 2H),
.43 (s, 2H), 2.30 (s, 3H), 2.05-2.02 (m, 2H), 1.95-1.91 (m, 2H),
.63-1.54 (m, 2H), 1.42-1.33 (m, 2H), 1.01 (s, 6H); mp = 297.7-
R
= 2.38 min. H NMR
(
6
28 4 3
171.8-174.6 °C. Anal. (C22H N O ) C, H, N.
2-(Cyclopent-3-enylamino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tet-
rahydro-indazol-1-yl)benzamide (37). Using the same procedure
as described for compound 34, treatment of 24a (2.00 g, 5.6 mmol)
with Pd(OAc)2 (64 mg, 5% mmol), 1-amino-3-cyclopentene
hydrochloride salt (1.34 g, 11.2 mmol), DPPF (328 mg, 10%
mmol), sodium hydroxide (0.5 M, 2.24 mL, 11.2 mmol), and
NaOtBu (1.13 g, 11.2 mmol) followed by hydration of the
benzonitrile yielded 37 (2.8 g, 63%). LCMS m/z = 379.2 [M þ
1
2
1
298.9 °C. Anal. (C H N O SF ) C, H, N.
26
34
5
7
3
4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-
H-indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]benza-
1
mide (9), Alternative Synthesis. NaH (225 mg, 9.39 mmol) was
added to a solution of 31a (1.98 g, 8.54 mmol) in N,N-dimethy-
lacetamide (17.1 mL) at rt. After gas evolution ceased, 26 (2.0 g,
þ
1
R 6
H] , t = 3.38 min. H NMR (300 MHz, DMSO-d ): δ 8.53 (d, J
= 6.6 Hz, 1H), 7.91 (bs, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.24 (bs,
1H), 6.74 (d, J = 1.8 Hz, 1H), 6.69 (dd, J = 8.1, 2.1 Hz, 1H), 5.75
(s, 2H), 4.17-4.11 (m, 1H), 2.93 (s, 2H), 2.84-2.76 (m, 2H), 2.52
(s, 2H), 2.38 (s, 3H), 2.31 (s, 2H), 2.17 (dd, J = 15 and 3.3 Hz, 2H),
1.00 (s, 6H); mp = 165.0-168.2 °C. HRMS calcd for
8
.54 mmol) was added and the flask was fitted with a condenser
and heated to 150 °C. After 6 h, the reaction mixture was cooled
to rt, poured into saturated aqueous NH Cl (200 mL), and
4
extracted with methyl t-butyl ether (100 mL ꢀ 3). The combined
þ
organic layers were washed with brine (100 mL ꢀ 2), dried over
C H N O 379.2135 [M þ H] , found 379.2133.
22
26
4 2
MgSO
purified by column chromatography (1:1 EtOAc in hexanes) to
4
, filtered, and concentrated in vacuo. The residue was
4-(3-Difluoromethyl-6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-
indazol-1-yl)-2-(tetrahydro-thiopyran-4-ylamino)-benzamide (38).
give 4-(6,6-dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahy-
dro-indazol-1-yl)-2-(4-hydroxy-cyclohexylamino)-benzonitrile
Using the same procedure as described for compound 34, treat-
ment of 32b (2.54 mmol, 1.00 g) with Pd(OAc) (0.13 mmol, 29 mg),
4-aminotetrahydrothiopyran (1.30 mmol, 423 mg), DPPF (0.27
mmol, 150 mg), and NaOtBu (5.37 mmol, 516 mg) followed by
hydration of the benzonitrile yielded 38 (77%). LCMS m/z= 449.1
2
þ
(
2.91 g, 76%). LCMS m/z = 447.5 [M þ H] . The benzonitrile
þ
was hydrated to 9 (2.7 g, 90%). LCMS m/z = 465.4 [M þ H] .
All analytical data were identical to those of the same compound
made by the previous method.
þ
1
[M þ H] , t = 3.58 min. H NMR (300 MHz, DMSO-d ): δ 8.54
R
6
2
-(trans-4-Hydroxy-cyclohexylamino)-4-(3,6,6-trimethyl-4-
(d, J = 8.1 Hz, 1H), 7.99 (bs, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.34
(bs, 1H), 6.83 (d, J = 2.1 Hz, 1H), 6.71 (dd, J = 6.3, 2.1 Hz, 1H),
3.53-3.50 (m, 1H), 2.95 (s, 2H), 2.76-2.61 (m, 4H), 2.39 (s, 2H),
2.21-2.19 (m, 2H), 1.97-1.96 (s, 1H), 1.59-1.52 (m, 2H), 1.01 (s,
6H); mp = 209.0-210.3 °C. Anal. (C H N O SF ) C, H, N.
2-(Cyclobutylamino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetra-
hydro-1H-indazol-1-yl)benzamide (39). Using the same proce-
dure as described for compound 34, treatment of 24a (1.00
oxo-4,5,6,7-tetrahydro-indol-1-yl)-benzamide (34). A microwave
vial was charged with 18c (1.072 g, 3 mmol), trans-4-aminocyclo-
hexanol (1.382 g, 12 mmol), Pd(OAc)
166.3 mg), and NaOtBu (576.7 mg, 6 mmol). The reagents were
suspended in toluene (20 mL) and were heated with a microwave to
2
(33.7 mg, 5 mol %), DPPF
(
2
2
26
4
2
2
1
15 °C for 12 min. After allowing the reaction vessel to cool, the
suspension was filtered and the filtrate evaporated. The residue was
purified by flash chromatography, and hydration of the benzoni-
trile yielded 34 (575 mg, 47%) as a yellow powder. LCMS m/z =
mmol, 357 mg) with Pd(OAc) (0.13 mmol, 30 mg), aminocy-
2
clobutane (3.0 mmol, 0.26 mL), DPPF (0.10 mmol, 56 mg), and
NaOtBu (2.0 mmol, 192 mg) followed by hydration of the
þ
1
410.2 [M þ H] , t
R
= 2.89. H NMR (400 MHz, DMSO-d
6
): δ

Article
benzonitrile yielded 39 (72%). LCMS m/z = 367.2 [M þ H] , t
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4303
þ
R
1.8 Hz, 1H), 6.76 (dd, J = 8.4 and 1.8 Hz, 1H), 3.63 (q, J = 6.4 Hz,
2H), 3.42 (t, J = 6.6 Hz, 2H), 3.02 (s, 3H), 2.95 (s, 2H), 2.39 (s, 3H),
2.31 (s, 2H), 1.00 (s, 6H); mp = 194.1-195.8 °C. Anal.
1
=
3.38 min. H NMR (300 MHz, DMSO-d ): δ 8.49 (d, J = 6
6
Hz, 1H), 7.86 (bs, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.24 (bs, 1H),
.70 (dd, J = 2.1 and 0.3 Hz, 1H), 6.60 (d, J = 2.1 Hz, 1H), 3.93
m, 1H), 2.90 (s, 2H), 2.38 (m, 5H), 2.31 (s, 2H), 1.78 (m, 4H),
6
(
26 4 4
(C20H N O S) C, H, N.
2-[(2-Methoxy-1-methylethyl)amino]-4-(3,6,6-trimethyl-4-oxo-4,
5,6,7-tetrahydro-1H-indazol-1-yl)benzamide (45). Using the same
procedure as described for compound 34, treatment of 24a (1.00
0
.99 (s, 6H); mp = 197.3-200.4 °C. Anal. (C H N O ) C, H, N.
21
26
4 2
2-[(Cyclopropylmethyl)amino]-4-(3,6,6-trimethyl-4-oxo-4,5,6,
-tetrahydro-1H-indazol-1-yl)benzamide (40). Using the same
7
procedure as described for compound 34, treatment of 24a (351.6
2
mmol, 357 mg) with Pd(OAc) (0.13 mmol, 30 mg), 2-amino-1-
methoxypropane (2.0 mmol, 0.21 mL), DPPF (0.10 mmol, 56 mg),
and NaOtBu (2.0 mmol, 192 mg) followed by hydration of the
mg, 0.96 mmol) with cyclopropanemethylamine (139.6 mg, 1.92
mmol), Pd(OAc) (11 mg, 0.05 mmol), DPPF (54.4 mg, 0.1 mmol),
þ
2
benzonitrile yielded 45 (77%). LCMS m/z= 385.2 [M þ H] , t
R
=
1
and NaOtBu (188.6 mg, 1.92 mmol) followed by hydration of the
benzonitrile afforded 40 (285 mg, 83% over 2 steps). LCMS m/z =
6
3.07 min. H NMR (300 MHz, DMSO-d ): δ 8.42 (d, J = 7.8 Hz,
1H), 7.89 (bs, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.22 (bs, 1H), 6.79 (d,
J = 2.4 Hz, 1H), 6.66 (dd, J = 6.6 and 1.8 Hz, 1H), 3.77-3.69 (m,
1H), 3.40-3.30 (m, 2H), 3.27 (s, 3H), 2.91 (s, 2H), 2.38 (s, 3H), 2.31
(s, 2H), 1.14 (d, J = 6.3 Hz, 3H), 1.00 (s, 3H), 0.99 (s, 3H); mp =
177.2-179.7 °C. Anal. (C H N O ) C, H, N.
þ
1
3
8
(
3
(
1
67.2 [M þ H] , t
.41 (t, J = 4.8 Hz, 1H), 7.91 (bs, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.24
bs, 1H), 6.73 (d, J = 1.8 Hz, 1H), 6.65 (dd, J = 8.4 and 1.8 Hz, 1H),
.09-2.98 (m, 2 H), 2.92 (s, 2H), 2.38 (s, 3H), 2.31 (s, 2H), 1.10-1.05
m, 1H), 0.99 (s, 6H), 0.53-0.44 (m, 2H), 0.26-0.21 (m, 2H); mp =
R 6
= 3.32 min. H NMR (300 MHz, DMSO-d ): δ
21
28
4 3
4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-
indazol-1-yl]-2-{[3-(2-oxopyrrolidin-1-yl)propyl]amino}benza-
mide (46). Using the same procedure as described for compound
84.0-187.0 °C. Anal. (C21
H
26
N
4
O
2
) C, H, N.
-{[(1S)-1-Phenylethyl]amino}-4-(3,6,6-trimethyl-4-oxo-4,5,6,
-tetrahydro-1H-indol-1-yl)benzamide (41). Using the same
2
7
2
34, treatment of 32a (0.41 g, 0.984 mmol) with Pd(OAc) (11 mg,
procedure as described for compound 34, treatment of 18c
(
(
0.049 mmol), 1-(3-aminopropyl)pyrrolidinone (0.28 mL, 1.97
mmol), DPPF (0.055 g, 0.098 mmol), and NaOtBu (0.1892 g,
1.97 mmol) followed by hydration of the benzonitrile yielded 46
420.8 mg) with (S)-methylbenzylamine (299.8 mL.), Pd(OAc)2
13.2 mg), DPPF (65.3 mg), and NaOtBu (226.4 mg) followed by
hydration of the benzonitrile afforded 41 (126 mg, 27%). LCMS
þ
1
(0.158 g, 33%). LCMS m/z = 492.2 [M þ H] , t
R
= 3.17 min. H
NMR (300 MHz, DMSO-d ): δ 8.38 (m, 1H), 7.98 (bs, 1H), 7.76
þ
1
m/z = 416.2 [M þ H] , t = 3.77 min. H NMR (300 MHz,
R
6
DMSO-d ): δ 8.91 (d, J = 6.6 Hz, 1H), 8.00-7.93 (m, 1H), 7.72
d, J = 8.7 Hz, 1H), 7.33 (s, 1H), 7.32 (m, 4H), 7.23-7.17 (m,
(d, J = 8.4 Hz, 1H), 7.35 (bs, 1H), 6.78 (d, 1H), 6.71 (dd, J = 8.4
and 2.1 Hz, 1H), 3.25 (m, 4H), 3.12 (m, 2H), 2.95 (s, 2H), 2.42 (s,
2H), 2.19 (m, 2H), 1.87 (m, 2H), 1.75 (m, 2H), 1.01 (s, 6H); mp =
92.1-93.8 °C. HRMS calcd for C H N O F 492.2224 [M þ
6
(
1
6
1
3
H), 6.58 (d, J = 0.9 Hz, 1H), 6.48 (dd, J = 8.3 and 2.0 Hz, 1H),
.30 (d, J = 2.1 Hz, 1H), 4.67-4.62 (m, 1H), 2.93 (s, 1H), 2.69 (s,
H), 2.11 (s, 2H), 1.43 (d, J = 6.6 Hz, 3H), 0.85 (s, 3H), 0.82 (s,
H); mp = 82.9-86.8 °C.
24
28
5 3 3
þ
H] , found 492.2222.
4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-
1H-indazol-1-yl]-2-{[2-methoxy-1-(methoxymethyl)ethyl]amino}
benzamide (47). 2,4-Difluorobenzonitrile (1.17 g, 8.39 mmol),
2-amino-1,3-dimethoxypropane (1 g, 8.39 mmol), and diiso-
propylethylamine (1.46 mL, 8.39 mmol) were dissolved in
DMSO (10 mL) and stirred at 150 °C for 30 min. The reac-
tion mixture was then cooled to rt and poured into satur-
2-[(2-Furylmethyl)amino]-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetra-
hydro-1H-indazol-1-yl)benzamide (42). Using the same procedure
as described for compound 34, treatment of 24a (396.6 mg, 1 equiv)
with furfurylamine (204.8 mL, 2 equiv), Pd(OAc) (12.4 mg, 5%
2
equiv), DPPF (61.4 mg, 10% equiv), and NaOtBu (212.8 mg,
2
equiv) followed by hydrolysis of the benzonitrile afforded 42
þ
1
= 3.43 min. H
(
105 mg, 25%). LCMS m/z = 393.2 [M þ H] , t
R
ated NH
(3ꢀ). The combined organic layers were washed with brine,
dried over MgSO , and concentrated. Purification of the
residue by column chromatography (SiO , 10% to 20% EtOAc
4
Cl. The aqueous phase was extracted with EtOAc
NMR (300 MHz, DMSO-d ): δ 8.73 (bs, 1H), 7.94 (bs, 1H), 7.75
6
(
6
bs, 1H), 7.59-7.58 (m, 1H), 7.32 (bs, 1H), 6.84 (bs, 1H), 6.78-
4
.70 (m, 1H), 6.40-6.38 (m, 1H), 6.32 (bs, 1H), 4.42 (bs, 2H), 2.81
2
(
°
3
s, 2H), 2.37 (s, 3H), 2.30 (s, 2H), 0.98 (s, 6H); mp = 172.6-176.4
in hexanes) afforded 4-fluoro-2-(2-methoxy-1-methoxymethyl-
ethylamino)-benzonitrile (0.376 g, 19%). LCMS m/z = 239
þ
C. HRMS calcd for C H N O 393.1929 [M þ H] , found
22
24
4 3
þ
93.1928.
2-{[2-(Methylthio)ethyl]amino}-4-(3,6,6-trimethyl-4-oxo-4,5,6,
-tetrahydro-1H-indazol-1-yl)benzamide (43). Using the same
[M þ H] .
4-Fluoro-2-(2-methoxy-1-methoxymethyl-ethylamino)-benzoni-
trile (0.376 g, 1.58 mmol), 31a (0.37 g, 1.58 mmol), and NaH (60%
in mineral oil, 0.076 g, 1.9 mmol) were suspended in
DMF (2 mL). The reaction mixture was microwaved at 200 °C
7
procedure as described for compound 34, treatment of 24a
(
(
1.81 g, 5.14 mmol) with Pd(OAc)2 (56 mg, 0.25 mmol), 2-
methylthio)ethylamine (0.95 mL, 10.28 mmol), DPPF (0.28 g,
for 10 min, cooled to rt, and treated with saturated NH
aqueous phase was extracted with EtOAc (3ꢀ). The combined
organic layers were washed with brine, dried over MgSO , and
concentrated. Purification of the residue by column chromatogra-
phy (SiO , 50% EtOAc in hexanes) afforded 4-(6,6-dimethyl-4-oxo-
4
Cl. The
0
hydration of the benzonitrile yielded 43 (1.48 g, 75%). LCMS
.51 mmol), and NaOtBu (0.99 g, 10.28 mmol) followed by
4
þ
1
m/z = 387.2 [M þ H] , t = 3.16 min. H NMR (300 MHz,
R
DMSO-d
6
): δ 8.50 (t, J = 5.7 Hz, 1H), 7.91 (bs, 1H), 7.73 (d, J =
2
8
8
2
1
.7 Hz, 1H), 7.26 (bs, 1H), 6.78 (d, J = 2.2 Hz, 1H), 6.69 (dd, J =
.7and 2.2 Hz, 1H), 3.36 (m, 2H), 2.92 (s, 2H), 2.73(t, J =6.8Hz,
H), 2.38 (s, 3H), 2.33 (s, 2H), 2.10 (s, 3H), 0.99 (s, 6H); mp =
3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-2-(2-methoxy-1-
methoxymethyl-ethylamino)-benzonitrile (0.515 g, 72%). LCMS
þ
m/z= 451 [M þ H] . The benzonitrile was hydrated at rt for 30 min
þ
04.8-106.8 °C. Anal. (C20
-{[2-(Methylsulfonyl)ethyl]amino}-4-(3,6,6-trimethyl-4-oxo-
,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide (44). To a solu-
26 4 2
H N O S) C, H, N.
to afford 47 (0.55 g, 100%). LCMS m/z = 469.2 [M þ H] , t
R
=
1
2
6
3.52 min. H NMR (300 MHz, DMSO-d ): δ 8.56 (d, J = 5.4 Hz,
4
1H), 7.98 (bs, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.30 (bs, 1H), 6.94 (d, J
= 2.1 Hz, 1H), 6.73 (dd, J= 8.1 and 1.8 Hz, 1H), 3.82 (m, 1H), 3.42
(d, J = 5.4 Hz, 4H), 3.28 (s, 6H), 2.96 (s, 2H), 2.43 (s, 2H), 1.02 (s,
tion of 43 (3.07 g, 7.94 mmol) in CHCl (45 mL) was added
3
MCPBA (2.74 g, 15.88 mmol), and the reaction mixture was stirred
at rt for 1 h. The solvent was evaporated under vacuum, and
NaHCO3 (saturated, 20 mL) was added to the residue. The
aqueous phase was extracted with EtOAc (3ꢀ). The combined
27 4 4 3
6H); mp = 85.3-88.7 °C. Anal. (C22H N O F ) C, H, N.
2-(Piperidin-4-ylamino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tet-
rahydro-indol-1-yl)-benzamide (48). Compound 18c (2.0 g,
organic layers were dried over Na
2
SO
4
, and concentrated. Purifi-
, EtOAc)
2
5.6 mmol), Pd(OAc) (63 mg, 0.3 mmol), 1-N-Boc-4-aminopi-
peridine (4.4 g, 22.4 mmol), DPPF (310 mg, 0.6 mmol), and
NaOtBu (1.08 g, 11.2 mmol) were suspended in toluene (20 mL)
cation of the residue by column chromatography (SiO
2
þ
afforded 44 (0.26 g, 8%). LCMS m/z = 419.1 [M þ H] , t = 2.55
R
1
min. H NMR (300 MHz, DMSO-d
6
): δ 8.50 (t, J = 6.1 Hz, 1H),
in a microwave vial and purged for 15 min with N
2
. The reaction
7.94 (bs, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.31 (bs, 1H), 6.81 (d, J =
vessel was placed in the microwave reactor for 15 min at 115 °C.

4
304 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Huang et al.
The mixture was filtered through celite and concentrated to give
-[2-cyano-5-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-in-
den-1-yl)-phenylamino]-piperidine-1-carboxylic acid tert-butyl
ester (1.7 g, 64%) as a yellow solid. This was dissolved in CH Cl
50 mL). Trifluoroacetic acid (5.5 mL) was added, and the
2001, 154, 267–274. (c) Picard, D. Heat-shock protein 90, a chaperone
for folding and regulation. Cell. Mol. Life Sci. 2002, 59, 1640–1648.
3) Pratt, W. B.; Toft, D. O. Regulation of Signaling Protein Function
and Trafficking by the hsp90/hsp70-Based Chaperone Machinery.
Exp. Biol. Med. 2003, 228, 111–133.
4) (a) Schulte, T. W.; Blagosklonny, M. V.; Romanova, L.;
Mushinsky, J. F.; Monia, B. P.; Johnston, J. F.; Nguyen, P.; Trepel,
J.; Neckers, L. M. Destabilization of Raf-1 by geldanamycin leads
to disruption of the Raf-1-MEK-Mitogen-Activated protein ki-
nase signalling pathway. Mol. Cell. Biol. 1996, 16, 5839–5845. (b)
Sato, S.; Fujita, N.; Tsuruo, T. Modulation of AKT kinase activity by
binding to Hsp90. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10832–
4
(
(
2
2
(
reaction mixture was stirred at rt. Once TLC analysis showed
complete consumption of starting material, the reaction mixture
was concentrated in vacuo to give a dark oil of the crude
benzonitrile, which was hydrated to afford 48. LCMS m/z =
þ
1
3
95.2 [M þ H] , t = 2.13. H NMR (300 MHz, DMSO-d ):
R
6
δ 8.49 (d, J = 5.7 Hz, 1H), 7.91 (bs, 1H), 7.70 (d, J = 5.7 Hz,
1
0837. (c) Richter, K.; Buchner, J. Hsp90: Chaperoning signal trans-
1
1
2
H), 7.25 (bs, 1H), 6.84 (s, 1H), 6.71 (s, 1H), 6.52 (d, J = 6.3 Hz,
H), 4.13 (m, 1H), 3.74 (m, 2H), 3.351 (m, 2H), 2.71 (s, 2H),
.52 (m, 2H), 2.23 (s, 2H), 2.20 (s, 3H), 1.97 (m, 2H), 0.98 (s, 6H);
duction. J. Cell. Physiol. 2001, 188, 281–290. (d) Kamal, A.; Boehm,
M. F.; Burrows, F. J. Therapeutic and diagnostic implications of Hsp90
activation. Trends Mol. Med. 2004, 10, 283–290.
(
5) Workman, P.; Powers, M. V. Chaperoning cell death: a critical dual
mp: 99.7-101.9 °C. HRMS calcd for C23
[
30 4 2
H N O 395.2448
þ
role for Hsp 90 in small-cell lung cancer. Nat. Chem. Biol. 2007, 3,
M þ H] , found 395.2448.
4
55–457.
2-[(2-Morpholin-4-ylethyl)amino]-4-(3,6,6-trimethyl-4-oxo-
,5,6,7-tetrahydro-1H-indol-1-yl)benzamide (49). Using the
(6) (a) Zhang, H.; Burrows, F. Targeting multiple signal transduction
pathways through inhibition of Hsp90. J. Mol. Med. 2004, 82, 488–
499. (b) Workman, P. Combinatorial attack on multistep oncogenesis by
inhibiting the Hsp90 molecular chaperone. Cancer Lett. 2004, 206,
4
same procedure as described for compound 34, treatment of
1
4
0
2
8c (178 mg, 0.5 mmol) with Pd(OAc) (5.6 mg, 0.03 mmol),
-(2-aminoethyl)morpholine (260 mg, 2.0 mmol), DPPF (28 mg,
.05 mmol), and NaOtBu (96 mg, 1.0 mmol) followed by
1
49–157. (c) Vilenchik, M.; Solit, D.; Basso, A.; Huezo, H.; Lucas, B.;
He, H.; Rosen, N.; Spampinato, C.; Modrich, P.; Chiosis, G. Targeting
wide-range oncogenic transformation via PU24FCl, a specific inhibitor
of tumor Hsp90. Chem Biol. 2004, 11, 787–797.
hydration of the benzonitrile yielded 49 (105 mg, 49% over
2
NMR (300 MHz, DMSO-d ): δ 8.42 (s, 1H), 8.10 (bs, 1H), 7.67
6
þ
1
= 2.02 min. H
steps). LCMS m/z = 425.2 [M þ H] , t
R
(7) (a) Pearl, L. H.; Prodromou, C. Structure, function, and mechan-
ism of the Hsp90 molecular chaperone. Adv. Protein Chem.
2
001, 59, 157–186. (b) Prodromou, C.; Peral, L. H. Structure and
functional relationship of the Hsp90. Curr. Cancer Drug Targets 2003,
, 301–323.
(
d, J = 7.8 Hz, 1H), 7.21 (bs, 1H), 6.83 (s, 1H), 6.58 (s, 1H), 6.51
(d, J = 7.8 Hz, 1H), 3.57 (s, 4H), 2.70 (s, 2H), 2.53 (s, 2H),
3
2
.40 (s, 4H), 2.23 (s, 2H), 2.19 (s, 3H), 0.97 (s, 6H); mp = 207.0-
(
(
8) Marcu, M. G.; Chadli, A.; Bouhouche, I.; Catelli, M.; Neckers,
L. M. The heat shock protein 90 antagonist novobiocin interacts
with a previously unrecognized ATP-binding domain in the car-
boxyl terminus of the chaperone. J. Biol. Chem. 2000, 275, 37181–
þ
210.0 °C. HRMS calcd for C24
H N O
32 4 3
425.2555 [M þ H] ,
found 425.2552.
-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-
H-indazol-1-yl]-2-[(cis-4-hydroxycyclohexyl)amino]benzamide
50). Using the same procedure as described for com-
pound 34, treatment of 32a (1.03 g, 2.5 mmol) with Pd(OAc)2
28 mg, 0.13 mmol), cis-4-aminocyclohexanol (862 mg, 7.5 mmol),
4
3
7186.
1
(
9) Lotz, G. P.; Lin, H.; Harst, A.; Obermann, W. M. Aha1 binds to
the middle domain of Hsp90, contributes to client protein activa-
tion, and stimulates the ATPase activity of the molecular chaper-
one. J. Biol. Chem. 2003, 278, 17228–17235.
(
DPPF (139 mg, 0.3 mmol), and NaOtBu (481 mg, 5.0 mmol)
followed by hydration of the benzonitrile yielded 50 (356 mg, 32%
(10) (a) Grenert, J. P.; Sullivan, W. P.; Fadden, P.; Haystad, T. A.;
Clark, J.; Mimmaugh, E.; Krutzsch, H.; Ochel, H. J.; Schulte,
T. W.; Sausville, E; Neckers, L. M.; Toft, D. O. The amino-
terminal domain of the heat shock protein 90 (hsp90) that binds
geldanamycin is an ATP/ADP switch domain that regulates hsp90
conformation. J. Biol. Chem. 1997, 272, 23843–23850. (b) Grenert,
J. P.; Johnson, B. D.; Toft, D. O. The importance of ATP binding and
hydrolysis by hsp90 in formation and function of protein heterocom-
plexes. J. Biol. Chem. 1999, 274, 17525–17533.
þ
1
over 2 steps). LCMS m/z = 465.2 [M þ H] , t = 3.27 min. H
R
6
NMR (300 MHz, DMSO-d ): δ 8.61 (d, J = 7.8 Hz, 1H), 7.98 (bs,
1
1
2
1
H), 7.77 (d, J = 8.4 Hz, 1H), 7.32 (bs, 1H), 6.83 (d, J = 1.5 Hz,
H), 6.68 (dd, J = 8.4 and 1.5 Hz, 1H), 3.62 (bs, 1H), 3.48 (bs, 1H),
.95 (s, 2H), 2.42 (s, 2H), 1.62-1.48 (m, 8H), 1.01 (s, 6H); mp =
27 4 3 3
54.0-156.0 °C. Anal. (C23H N O F ) C, H, N.
(
11) (a) Le Brazidec, J.-Y.; Kamal, A.; Busch, D.; Thao, L.; Zhang, L.;
Timony, G.; Grecko, R.; Trent, K.; Lough, R.; Salazar, T.; Khan,
S.; Burrows, F.; Boehm, M. F. Synthesis and biological evaluation
of a new class of geldanamycin derivatives as potent inhibitors of
Hsp90. J. Med. Chem. 2004, 47, 3865–3873. (b) Miyata, Y. Hsp90
inhibitor geldanamycin and its derivatives as novel cancer chemother-
apeutic agents. Curr. Pharm. Des. 2005, 11, 1131–1138. (c) Supko,
J. G.; Hickman, R. L.; Grever, M. R.; Malspeis, L. Preclinical pharma-
cologic evaluation of geldanamycin as an antitumor agent. Cancer
Chemother. Pharmacol. 1995, 36, 305–315.
Supporting Information Available: Additional analytical data
including elemental analyses and LC-UV spectra for com-
pounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(
1) (a) Maloney, A.; Workman, P. Hsp90 as a new therapeutic target
for cancer therapy: The story unfolds. Expert Opin. Biol. Ther.
(12) Schulte, T. W.; Neckers, L. M. Thebenzoquinoneansamycin-17-
allylamino-17-demethoxygeldanamycin binds to HSP90 and
shares important biologic activities with geldanamycin. Cancer
Chemother. Pharmacol. 1998, 42, 273–279.
(13) Kaur, G.; Belotti, D.; Burger, A. M.; Fisher-Nielson, K.; Borsotti,
P.; Riccardi, E.; Thillainathan, J.; Hollingshead, M.; Sausville, E.
A.; Giavazzi, R. Antiangiogenic properties of 17-(dimethylami-
noethylamino)-17-demethoxygeldanamycin: an orally bioavail-
able heat shock protein 90 modulator. Clin. Cancer Res. 2004, 10,
4813–4821.
2002, 2, 3–24. (b) Dymock, B. W.; Drysdale, M. J.; McDonald, E.;
Workman, P. Inhibitors of Hsp90 and other chaperones for the treatment
of cancer. Expert Opin. Ther. Patents 2004, 14, 837–847. (c) Isaacs,
J. S.; Wu, X.; Neckers, L. Heat shock protein 90 as a molecular target for
cancer therapeutics. Cancer Cell 2003, 3, 213–217. (d) Chiosis, G.;
Vilenchik, M.; Kim, J.; Solit, D. Hsp90: the vulnerable chaperone. Drug
Discovery Today 2004, 9, 881–888. (e) Whitesell, L.; Lindquist, S. L.
Hsp90 and the chaperoning of cancer. Nat. Rev. Cancer 2005, 5, 761–
7
72. (f) Calderwood, S. K.; Khaleque, M. A.; Sawyer, D. B.; Ciocca,
(14) (a) Glaze, E. R.; Smith, A. C.; Johnson, D. W.; McCormick, D. L.;
Brown, A. B.; Levin, B. S.; Krishnaraj, R.; Lyubimov, A.; Egorin,
M. J.; Tomaszewski, J. E. Dose range-finding toxicity studies of 17-
DMAG. Proc. Am. Assoc. Cancer. Res. 2003, 44, 162–163. (b)
Eiseman, J. L.; Lan, J.; Lagatutta, T. F.; Hamburger, D. R.; Joseph, E.;
Covey, J. M.; Egorin, M. J. Pharmacokinetics and pharmacodynamics
of 17-demethoxy 17-[[(2-dimethylamino)ethyl]amino]geldanamycin
(17DMAG, NSC 707545) in CB-17 SCID mice bearing MDA-MB-
231 human breast cancer xenografts. Cancer Chemother. Pharmacol.
2005, 55, 21–32.
D. R. Heat shock proteins in cancer: chaperones of tumorigenesis.
Trends Biochem. Sci. 2006, 31, 164–172. (g) Neckers, L. Heat shock
protein 90: the cancer chaperone. Bioscience 2007, 32, 517–530. (h)
Neckers, L.; Mimnaugh, E.; Schulte, T. W. Hsp90 as an anti-cancer
target. Drug Resist. Updates 2007, 2, 165–172. (i) Cortajarena, A.; Yi,
F.; Regan, L. Designed TPR Modules as Novel Anticancer Agents.
ACS Chem. Biol. 2008, 3, 161–166. (j) Loh, S. N. Disrupting Proteins
to Treat Cancer. ACS Chem. Biol. 2008, 3, 140–141.
(
2) (a) Buchner, J. Hsp90 & Co.;a holding for folding. Trends
Biochem. Sci. 1999, 24, 136–141. (b) Young, J.; Moarefi, I.; Hartl, F.
U. Hsp90: a specialized but essential protein folding tool. J. Cell. Biol.
(15) (a) Ge, J.; Normant, E.; Porter, J. R.; Ali, J. A.; Dembski, M. S.;
Gao, Y.; Georges, A. T.; Grenier, L.; Pak, R. H.; Patterson, J.;

Article
Sydor, J. R.; Tibbitts, T. T.; Tong, J. K.; Adams, J.; Palombella, V.
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4305
(23) Obermann, W. M.; Sondermann, H.; Russo, A. A.; Pavletich, N.
P.; Hartl, F. U. In Vivo Function of Hsp90 Is Dependent on ATP
Binding and ATP Hydrolysis. J. Cell. Biol. 1998, 143, 901–910.
(24) Ramadas, S. R.; Padmanabhan, S. A convenient method for the
syntheses of 4-oxo-4,5,6,7-tetrahydroindole and benzofuran deri-
vatives. Curr. Sci. 1979, 48, 52–53.
J. Design, synthesis, and biological evaluation of hydroquinone
derivatives of 17-amino-17-demethoxygeldanamycin as potent,
water-soluble inhibitors of Hsp90. J. Med. Chem. 2006, 49, 4606–
4
615. (b) Sydor, J. R.; Normant, E.; Pien, C. S.; Porter, J. R.; Ge, J.;
Grenier, L.; Pak, R. H.; Ali, J. A.; Dembski, M. S.; Hudak, J.; Patterson,
J.; Penders, C.; Pink, M.; Read, M. A.; Sang, J.; Woodward, C.; Zhang,
Y.; Grayzel, D. S.; Wright, J.; Barrett, J. A.; Palombella, V. J.; Adams, J.;
Tong, J. K. Development of 17-allylamino-17-demethoxygeldanamy-
cin hydroquinone hydrochloride (IPI-504), an anti-cancer agent direc-
ted against Hsp90. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17408–
(25) Bobbitt, J. M.; Dutta, C. P. An intramolecular aldehyde-enamine
condensation. Chem. Commun. 1968, 22, 1429.
(26) Nenitzescu, C. D.; Scortzbanu, V. Derivatives of Bz-tetrahydroin-
dole. II. Some derivatives of 4,5,6,7-tetrahydro-3,6,6-trimethyl-4-
ketoindole. Bull. Soc. Chim. Romania 1928, 10, 134–140.
(27) Bondietti, G.; Lions, F. Extension of Knorr’s pyrrole synthesis. J.
Proc. R. Soc. N.S.W. 1933, 66, 477–485.
1
7413.
(
(
(
16) Delmotte, P.; Delmotte-Plaque, J. A new antifungal substance of
fungal origin. Nature 1953, 171, 344–345.
(28) Calvo, L.; Gonzalez-Ortega, A.; Navarro, R.; Perez, M.; Sanudo,
17) Soga, S.; Shiotsu, Y.; Akinaga, S.; Sharma, S. V. Development of
radicicol analogues. Curr. Cancer Drug Targets 2003, 3, 359–369.
18) Brough, P. A.; Aherne, W.; Barril, X.; Borgognoni, J.; Boxall, K.;
Cansfield, J. E.; Cheung, K. J.; Collins, I.; Davies, N. G. M.;
Drysdale, M. J.; Dymock, B.; Eccles, S. A.; Finch, H.; Fink, A.;
Hayes, A.; Howes, R.; Hubbard, R. E.; James, K.; Jordan, A. M.;
Lockie, A.; Martins, V.; Massey, A.; Matthews, T. P.; McDonald,
E.; Northfield, C. J.; Pearl, L. H.; Prodromou, C.; Ray, S.;
Raynaud, F. I.; Roughley, S. D.; Sharp, S. Y.; Surgenor, A.;
Walmsley, D. L.; Webb, P.; Wood, M.; Workman, P.; Wrightt,
L. 4,5-Diarylisoxazole Hsp90 Chaperone Inhibitors: Potential
Therapeutic Agents for the Treatment of Cancer. J. Med. Chem.
M. C. Synthesis of pyrroles with fused carbocycles or heterocycles
from Weinreb N-vinyl-R -amino amides. Synthesis 2005, 18, 3152–
0
3158.
(29) Hauptmann, S.; Blume, H.; Hartmann, G.; Haendel, D.; Franke,
P. Hydroxyindoles. 4-oxo-4,5,6,7-Tetrahydroindoles from 1,3-cy-
clohexanedione and isonitroso carbonyl compounds. Z. Chem.
1966, 6, 107.
(30) Huang, K. H.; Eaves J.; Hanson, G.; Veal, J.; Barta, T.; Geng,
L.; Hinkley, L. Tetrahydroindolone and tetrahydroindazolone
derivatives. International Publication Number WO 06/091963,
2006.
(31) Sawaki, Y.; Ogata, Y. Mechanism of the Reaction of Nitriles with
Alkaline Hydrogen Peroxide. Reactivity of Peroxycarboximidic
Acid and Application to Superoxide Ion Reaction. Bull. Chem.
Soc. Jpn. 1981, 54, 793–799.
(32) Zhou, J.; Fu, G. C. Palladium-Catalyzed Negishi Cross-Coupling
Reactions of Unactivated Alkyl Iodides, Bromides, Chlorides, and
Tosylates. J. Am. Chem. Soc. 2003, 125, 12527–12530.
(33) Yang, B. H.; Buchwald, S. L. Palladium-catalyzed amination of
aryl halides and sulfonates. J. Organomet. Chem. 1999, 576, 125–
146.
2008, 51, 196–218.
(
19) (a) Chiosis, G.; Lucas, B.; Shtil, A.; Huezo, H.; Rosen, N. Devel-
opment of a purine-scaffold novel class of Hsp90 binders that
inhibit the proliferation of cancer cells and induce the degradation
of HER-2 tyrosine kinase. Bioorg. Med. Chem. 2002, 10, 3555–
3
564. (b) Chiosis, G.; Timaul, M. N.; Lucas, B.; Munster, P. N.; Zheng,
F. F.; Sepp-Lorenzino, L.; Rosen, N. A small molecule designed to bind
to the adenine nucleotide pocket of Hsp90 causes Her2 degradation and
the growth arrest and differentiation of breast cancer cells. Chem. Biol.
2
001, 8, 289–299.
(34) Voss, G.; Eichner, S. Simplified synthesis of the precursor for the
azo dye Chlorindazone DS. J. Prakt. Chem. 2000, 342, 201–204.
(35) Akhrem, A. A.; Lakhvich, F. A.; Budai, S. I.; Khlebnicova, T. S.;
Petrusevich, I. I. A new simple synthesis of 2-acylcyclohexane-1,3-
diones. Synthesis 1978, 12, 925–927.
(36) De Buyck, L.; De Pooter, H.; Verhe, R.; De Kimpe, N.; Schamp, N.
Synthesis of 5-alkyl-3,3-dimethyl-1,2,4-cyclopentanetriones, invol-
ving hydrogenation of 2-acyldimedones and polychlorination.
Bull. Soc. Chim. Belg. 1981, 90, 825–835.
(
20) (a) Kasibhatla, S. R.; Hong, K.; Biamonte, M. A.; Busch, D. J.;
Karjian, P. L.; Sensintaffar, J. L.; Kamal, A.; Lough, R. E.;
Brekken, J.; Lundgren, K.; Grecko, R.; Timony, G. A.; Ran, Y.;
Mansfield, R.; Fritz, L. C.; Ulm, E.; Burrows, F. J.; Boehm, M. F.
Rationally Designed High-Affinity 2-Amino-6-halopurine Heat
Shock Protein 90 Inhibitors That Exhibit Potent Antitumor Activ-
ity. J. Med. Chem. 2007, 50, 2767–2778.(b) Lundgren, K.; Zhang, H.;
Kamal, A.; Lough, R.; Timple, N.; Sensintaffar, J.; Busch, D.; Yang, C.;
Neely, L.; Khan, S.; Hong, K.; Kasibhatla, S.; Boehm, M.; Burrows, F.
BIIB021 is a Small Molecule Inhibitor of the Heat Shock Protein, Hsp90,
That Shows Potent Anti-tumor Activity in Preclinical Models. AACR-
NCI-EORTC International Conference on Molecular Targets and Cancer
Therapeutics, San Francisco, California, October 22-26, 2007.
(37) Hiegel, G. A.; Burk, P. Synthesis of cyclic 2-enones from cyclic 1,3-
diketones. J. Org. Chem. 1973, 38, 3637–3639.
(38) Barabasz, A.; Foley, B.; Otto, J. C.; Scott, A.; Rice, J. The Use of
High-Content Screening for the Discovery and Characterization of
Compounds That Modulate Mitotic Index and Cell Cycle Progres-
sion by Differing Mechanisms of Action. Assay Drug Dev. Technol.
2006, 4, 153–163.
(39) Guo, W.; Reigan, P.; Siegel, D.; Zirrolli, J.; Gustafson, D.; Ross, D.
Formation of 17-Allylamino-demethoxygeldanamycin (17-AAG)
Hydroquinone by NAD(P)H:Quinone Oxidoreductase 1: Role of
17-AAG Hydroquinone in Heat Shock Protein 90 Inhibition.
Cancer Res. 2005, 65, 10006–10015.
(40) Graves, P. R.; Kwiek, J. J.; Fadden, P.; Ray, R.; Hardeman, K.;
Coley, A. M.; Foley, M.; Haystead, T. A. Discovery of novel
targets of quinoline drugs in the human purine binding proteome.
Mol. Pharmacol. 2002, 62, 1364–1372.
(
21) Chandarlapaty, S.; Sawai, A.; Ye, Q.; Scott, A.; Silinski, M.;
Huang, K.; Fadden, P.; Partdrige, J.; Hall, S.; Steed, P.; Norton,
L.; Rosen, N.; Solit, D. B. SNX2112, a synthetic heat shock protein
90 inhibitor, has potent antitumor activity against HER kinase-
dependent cancers. Clin. Cancer Res. 2008, 14, 240–248.
22) Barta, T. E.; Veal, J. M.; Rice, J. W.; Partridge, J. M.; Fadden, R.
P.; Ma, W.; Jenks, M.; Geng, L.; Hanson, G. J.; Huang, K. H.;
Barabasz, A. F.; Foley, B. E.; Otto, J.; Hall, S. E. Discovery of
benzamide tetrahydro-4H-carbazol-4-ones as novel small mole-
cule inhibitors of Hsp90. Bioorg. Med. Chem. Lett. 2008, 18,
(
3517–3521.