Discovery of benzamide tetrahydro-4H-carbazol-4-ones as novel small molecule inhibitors of Hsp90

Article abstract of DOI:10.1016/j.bmcl.2008.05.023

Hsp90 maintains the conformational stability of multiple proteins implicated in oncogenesis and has emerged as a target for chemotherapy. We report here the discovery of a novel small molecule scaffold that inhibits Hsp90. X-ray data show that the scaffold binds competitively at the ATP site on Hsp90. Cellular proliferation and client assays demonstrate that members of the series are able to inhibit Hsp90 at nanomolar concentrations.

Full text of DOI:10.1016/j.bmcl.2008.05.023

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3517–3521
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of benzamide tetrahydro-4H-carbazol-4-ones as novel small
molecule inhibitors of Hsp90
*
Thomas E. Barta, James M. Veal , John W. Rice, Jeffrey M. Partridge, R. Patrick Fadden, Wei Ma,
Matthew Jenks , Lifeng Geng à, Gunnar J. Hanson §, Kenneth H. Huang, Amy F. Barabasz, Briana E. Foley,
James Otto –, Steven E. Hall
Serenex, Inc., 323 Foster St., Durham, NC 27701, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Hsp90 maintains the conformational stability of multiple proteins implicated in oncogenesis and has
emerged as a target for chemotherapy. We report here the discovery of a novel small molecule scaffold
that inhibits Hsp90. X-ray data show that the scaffold binds competitively at the ATP site on Hsp90. Cel-
lular proliferation and client assays demonstrate that members of the series are able to inhibit Hsp90 at
nanomolar concentrations.
Received 10 April 2008
Revised 2 May 2008
Accepted 6 May 2008
Available online 9 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Hsp90
Heat shock protein 90
Cancer therapeutic
Cancer inhibitor
X-ray structure
Her2
ATP
Carbazol-4-one
Benzamide
Hsp70
Heat shock protein 90 (Hsp90) has emerged as a promising tar-
get for the treatment of cancer and other diseases.1 The cellular
function of Hsp90 is to chaperone the folding and then maintain
the conformational integrity of multiple proteins. Hsp90’s pre-
ferred clients encompass a range of signaling proteins involved in
oncogenesis, including Her2, c-Kit, Met, Hif-1a, and androgen
receptor. Additionally, mutated proteins that are implicated in can-
cer are often sensitive clients and are generally more dependent on
Hsp90 chaperone function than their wild-type counterparts.2
Hsp90 is an ATPase and this function is essential to its chaperone
capability.3,4 Inhibition of the ATPase function results in client pro-
teins being ubiquinated and then degraded via proteosome path-
ways. The discoveries that the natural products geldanamycin
and radicicol could competitively inhibit ATP binding to Hsp905,6
have prompted significant research into the discovery of small
molecule inhibitors of Hsp90.7,8 We report here the discovery of
a novel class of small molecule carbazol-4-one benzamides that
are unrelated to published ansamycins, resorcinols, or purine-
based inhibitors. They potently inhibit Hsp90 and demonstrate
nanomolar cellular inhibition of the target.
Screening of a focused library, designed to inhibit proteins with
purine binding sites, yielded a novel benzamide hit for Hsp90 (Fig.
1). Synthetic and modeling analyses of this chemical scaffold
* Corresponding author. Tel.: +1 919 281 6038; fax: +1 919 281 6054.
E-mail address: jveal@serenex.com (J.M. Veal).
Present address: SCYNEXIS, Inc., 3501 C Tricenter Boulevard, Durham, NC 27713,
USA.
à
Present address: Philip Morris, 601 E. Jackson Street Richmond, VA 23261 USA.
Present address: Joyant Pharmaceuticals, 2600 N. Stemmons Frwy, Ste. 2620,
§
Dallas, TX 75207, USA.
Present address: Department of Pharmacology and Cancer Biology, Duke
–
Figure 1. Hsp90 benzamide screening hit and the 1,2,3,9-tetrahydro-4H-carbazol-
4-one analog 1.
University Medical Center, Durham, NC 27710, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.023

3518
T. E. Barta et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3517–3521
Table 1
prompted effort to combine the benzamide with a 1,2,3,9-tetrahy-
dro-4H-carbazol-4-one moiety. This carbazol-4-one functionality
has demonstrated favorable pharmacokinetic properties as it is
Structures and activities for benzamide tetrahydro-4H-carbazol-4-one analogs
incorporated into ondansetron,
medicine.
a well tolerated anti-emetic
The benzamides in this study were obtained in a straightfor-
ward manner and a representative synthesis is shown in Scheme
1. The 1,2,3,9-tetrahydro-4H-carbazol-4-one ring system was
established by means of combining 1,3-cyclohexanedione and phe-
nyl hydrazine via the Fischer indole synthesis in a Personal Chem-
istry microwave apparatus.9 Use of dimedone or the mono-methyl
reagent instead of 1,3-cyclohexanedione yielded the related ana-
logs described in the SAR. The purified tetrahydro-4H-carbazol-4-
one was then reacted with the desired 4-fluorobenzonitrile in the
presence of sodium hydride. For most analogs, the next step was
to incorporate the amine side chain of interest via a palladium cat-
alyzed aromatic amination utilizing Pd(OAc)2 and DPPF with
microwave irradiation.10 Hydrolysis of the nitrile to the carboxam-
ide gave the desired product.
The SAR for this series of compounds is reported in Table 1.
Shown are data for a non-enzymatic binding assay, previously de-
scribed,8 that allowed determination of binding affinity to mono-
meric Hsp90. An assay measuring effects on Her2 stability was
also established to evaluate cellular effects of the compound.11 It
is important to note that Hsp90 function is complex, involving
structural rearrangements as well as co-chaperone protein binding,
and so inhibition of monomeric Hsp90 is necessary but not suffi-
cient for cellular activity.4 Compound 1 showed promising sub-
micromolar Hsp90 binding although it lacked measurable cellular
activity. Investigation focused initially on the importance of the
gem-dimethyl group present in the hit. Removal of the methyl
groups was viewed as potentially desirable for solubility and
molecular weight reasons. Although compound 2 showed reduced
activity, it still retained moderate affinity. Addition of either a bro-
mo (3, 4) or cyano (5) group at the benzamide 2 position effectively
killed Hsp90 binding. The methoxy analog 6 was also inactive.
Compound 7 with an amino group showed reduced but still
measurable affinity, and binding models suggested room for addi-
tional substitution at this position. The affinities of cyclopropyl
analogs, 8, 9, and 10 were consistent with this observation, and
moreover, these compounds showed the first cellular activity ob-
served for the series. Compounds 9 and 10 demonstrated improved
binding relative to compound 1, and the gem-dimethyl analog 8
showed nanomolar cellular activity. Cylcopropyl methyl analogs
Compound
R1
H
R2
R3
R4
H
Hsp90a
0.75
Her2b
>50
1
Me
Me
2
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
1.8
>50
ND
>10
>50
>50
>10
0.61
7.0
3
Br
Br
Me
H
>10
>10
>10
>10
2.9
4
5
H
6
–OMe
–NH2
H
7
H
8
Me
Me
H
0.35
1.7
9
10
11
12
13
0.35
0.25
2.9
5.4
H
1.3
Me
H
H
H
6.9
0.27
1.3
14
Me
Me
H
>10
ND
15
16
17
18
H
H
H
H
H
H
H
H
H
H
F
1.0
2.1
8.9
4.1
5.3
0.29
0.78
0.35
Scheme 1. Preparation of 16: (a) phenylhydrazine (1.2 equiv), TFA, microwave/
140 °C/600 s; (b) NaH (2 equiv), DMF, 2-bromo-4-fluorobenzonitrile (1.3 equiv); (c)
methoxyethylamine (5 equiv), Pd(OAc)2 (0.01 equiv), DPPF (0.01 equiv), NaOt-Bu
(2 equiv), toluene, microwave/110 °C/1200 s; (d) DMSO (cat.), EtOH, KOH (ca. 10
equiv), H2O2 (32%, XS), 60 °C, 2.5 h.
H
(continued on next page)

T. E. Barta et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3517–3521
3519
Table 1 (continued)
O
NH2
R1
R2
H
R3
H
R4
F
Hsp90a
0.25
Her2b
5.4
H
N
Compound
O
19
N
20
21
22
23
24
25
26
27
28
29
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
0.31
>10
1.1
6.8
>10
6.5
5.0
>10
34
O
H
H
F
0.82
>10
1.2
F
1.1
2.8
31
H
F
1.9
0.27
0.90
0.41
1.6
1.2
0.56
H
H
M98
W162
L107
Y139
K58
30
I96
a
Values (lM) are estimated Kd’s derived from an 8 point non-enzymatic ATPase
T184
assay as described in text and Ref. 13.
b
Values (lM) are IC50 determinations from a Her2 ELISA in SKBR3 cells as
described in text and Ref. 11.
F138
12 and 13 were also well tolerated. An interesting observation was
that addition of a bulkier phenyl group, compounds 14 and 15, was
reasonably well tolerated when positions R2 and R3 were both
hydrogen but was completely inactive for the corresponding
gem-dimethyl substitution. The methyl ethyl ether analog, 16,
showed good Hsp90 binding, and at this point, X-ray crystallo-
graphic studies were undertaken for this compound to better
understand the Hsp90 binding mode for this series.12
Co-crystallization of 16 with the N-terminal binding domain of
Hsp90 proved successful, and a 1.74 Å high resolution structure
was obtained (Fig. 2). The ligand binding orientation was unambig-
uous from the Fo–Fc omit map. Previous models were in overall
good agreement with the structure, but the X-ray provided addi-
tional detailed information on the complex. As anticipated, the
benzamide acted as an adenine mimic with the amide group form-
ing hydrogen bonds with residues Asp 93, Thr 184, and a coordi-
nated water molecule. These residues were observed previously
to form a hydrogen bonding network in geldanamycin (GA) and
ADP X-ray structures.6,14 Sandwiching the benzamide ring were
residues Met 98 and Asn 51 with the interaction of the asparagine
involving pi stacking of the amide side chain moiety with the benz-
D93
N51
Figure 2. X-ray structure of compound 16 bound to the N-terminal domain of
Hsp90. Fo–Fc map at 3r is shown at top right; entire protein structure is shown in
middle; close-up view of active site region is shown at bottom. Some of the active
site water molecules are shown as purple spheres and ligand hydrogen bonds are
indicated by cyan dashes.
amide of 16. Multiple tightly bound structural waters were re-
solved in the binding site.
The tetrahydro-4H-carbazol-4-one group was observed to make
multiple hydrophobic interactions with Hsp90. More specifically, a
tight binding pocket was formed on three sides involving Leu 107,
Phe 138, and an edge to face interaction with Trp 162. Secondary
interactions involved Val 150 and Leu 103. Additionally, the car-
bonyl group made a water-mediated hydrogen bond with Tyr
109, and several additional water molecules were observed in

3520
T. E. Barta et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3517–3521
the region of Tyr 109. Overall, the protein conformation resembled
that previously seen for PU24FCl and related scaffolds in which Leu
107 is also seen to be displaced by ligand binding.15 The methyl
ethyl ether side chain of compound 16 was most proximal to the
side chain of Met 98, forming favorable hydrophobic contacts with
that residue as well as Ala 55 and Ile 96. The observed gauche con-
formation allowed the ether oxygen to form an intramolecular
hydrogen bond with the amino hydrogen. The tight packing of
the side chain with Met 98, coupled with the close fit of the tetra-
hydro-4H-carbazol-4-one into its region of Hsp90, indicated that
longer range allosteric effects could be possible and may explain
the divergent SAR for the gem-dimethyl analogs versus all-hydro-
gen analogs at position 2 of the carbazol-4-one. Superimposition of
the X-ray structures of compound 16 and GA6,14 bound to Hsp90
showed that the scaffold was able to effectively mimic key phar-
macophore elements. The benzamide of 16 tightly overlayed the
carbamate of GA while the methyl ethyl ether and indole compo-
nents of 16 closely mapped to GA macrocycle atoms 8–11 and 2–
5, respectively.
It was apparent that there was additional sterically accessible
space that could be targeted in the region off the 2 position of
the benzamide and that became the primary focus for additional
analogs. Compounds 17–20 further explored the potential to make
hydrophobic interactions with Met 98 and all showed good Hsp90
binding and moderate cellular potency. The introduction of a fluoro
group at the 6 position (17, 19) of the carbazol-4-one appeared to
be well tolerated and this was observed for later analogs as well. It
was anticipated that the fluoro would possibly interact favorably
with the hydroxyl of Tyr 109 while potentially improving both
metabolic and solubility properties of the series. Benzyl and ani-
sole analogs 21 and 24 were inactive. One strategy was to target
a proximal residue, Lys 58, for additional hydrogen bonding and
this strategy was attempted via the pyridyl analogs 22 and 23.
Although much better than their benzyl counterparts, they were
still not improved relative to earlier analogs. The incorporation of
solubilizing groups was also attempted for a set of analogs, 25–
27; all showed reasonable Hsp90 binding, but only the less basic
morpholino analog 26 had moderate cellular potency.
Figure 3. HCA images of AU565 cells showing effects of compound 30 inhibition at
10 lM on Her2 degradation and of A375 cells showing effects on pS6 and Hsp70
levels. Her2 and pS6 are colored red, Hsp70 is colored green, and DNA is colored
blue. pS6 and Hsp70 images are of the same cells for the individual treatments, but
pS6 is FITC exposure, Hsp70 is TRITC exposure. For each endpoint measured, length
of exposures was identical for direct comparison.
The final members of the series compounds, compounds 28, 29,
and 30 incorporated hydrophobic rings, coupled with hydrogen
bond acceptors, in an effort to target both Met 98 and Lys 58.
The improved potencies of these compounds, particularly com-
pound 30, supported this rationale. As a result, compound 30
was evaluated in multiple additional cellular assays.11 These assays
included cancer cell line proliferation as well as imaging assays for
signaling pathways impacted by Hsp90 inhibition. Also evaluated
was the induction of Hsp70 which is an expected result for
Hsp90 inhibition as a mechanism of action. The results of these as-
says are summarized in Table 2 and illustrated in Figure 3. As can
be seen, compound 30 potently inhibited cancer cell proliferation
with IC50’s below 400 nM in multiple cell lines. Potency was within
a factor of 5–10 of the geldanamycin analog, 17-AAG, used as a po-
sitive control (data not shown). Of note is that both MAPK and AKT
signaling pathways, as measured by phosphorylation of ERK and
S6, respectively, were also inhibited. Additionally, Hsp70 was in-
duced, fully supporting Hsp90 inhibition as a mechanism of action.
In conclusion, we have reported here a novel scaffold for com-
petitively inhibiting the ATP binding site of Hsp90. The binding
mode has been confirmed via X-ray crystallography. Syntheses of
multiple analogs allowed for discernment of key SAR trends and
led to potent Hsp90 inhibitors: 8, 13, 29, and 30. Detailed evalua-
tion of compound 30 showed it to be potent across a range of can-
cer cell lines and able to cause degradation of oncogenic Hsp90
clients. The promising data presented here have prompted further
evaluation of this scaffold and that work remains ongoing.
Table 2
Cellular potencies for compound 30
Acknowledgment
a
Assay
IC50 (lM)
The authors thank Bert Pronk for discussions regarding biolog-
ical assays and Active Sight (San Diego, CA) for crystallography
support.
A375 proliferation
HT29 proliferation
0.40 0.17
0.37 0.10
0.70 0.26
0.29 0.05
0.96 0.13
0.90 0.15
0.82 0.20
0.34 0.09
0.26 0.06
0.56 0.13
0.23 0.07
0.59 0.24
0.07 0.02
LNCAP proliferation
MCF-7 proliferation
MDAMB231 proliferation
NCI-H460 proliferation
PC-3 proliferation
SKMEL5 proliferation
SW620 proliferation
Her2 degradation, SKBR3b
Hsp70 induction, A375b
pErk inhibition, AU565b
pS6 inhibition, A375b
References and notes
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Values are means of four experiments with standard deviation shown.
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281 water molecules were located. Number reflections = 27945;
% completeness = 95; RCryst = 0.200; Rfree (5%) = 0.228; resolution (Å) = 1.74–
66.52; space group = I222; unit cell (Å): a = 66.85, b = 90.60, c = 98.22, a = 90°,
b = 90°, g = 90°. The PDB code is 3D0B.
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