Discovery of novel aminoquinazolin-7-yl 6,7-dihydro-indol-4-ones as potent, selective inhibitors of heat shock protein 90

Article abstract of DOI:10.1016/j.bmcl.2012.01.137

A novel class of Hsp90 inhibitors, structurally distinct from previously reported scaffolds, was developed from rational design and optimization of a compound library screen hit. These aminoquinazoline derivatives, represented by compound 15 (SNX-6833) or 1-(2-amino-4-methylquinazolin-7-yl)-3,6,6-trimethyl-6, 7-dihydro-1H-indol-4(5H)-one, selectively bind to Hsp90 and inhibit its cellular activities at concentrations as low as single digit nanomolar.

Full text of DOI:10.1016/j.bmcl.2012.01.137

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2550–2554
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel aminoquinazolin-7-yl 6,7-dihydro-indol-4-ones
as potent, selective inhibitors of heat shock protein 90
,
⇑
Kenneth H. Huang , Thomas E. Barta, John W. Rice, Emilie D. Smith, Andy J. Ommen, Wei Ma,
James M. Veal, R. Patrick Fadden, Amy F. Barabasz, Briana E. Foley, Philip F. Hughes, Gunnar J. Hanson,
Christopher J. Markworth, Melanie Silinski, Jeffrey M. Partridge, Paul M. Steed, Steven E. Hall
Serenex Inc., 323 Foster Street, 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:
A novel class of Hsp90 inhibitors, structurally distinct from previously reported scaffolds, was developed
from rational design and optimization of a compound library screen hit. These aminoquinazoline
derivatives, represented by compound 15 (SNX-6833) or 1-(2-amino-4-methylquinazolin-7-yl)-3,6,6-
trimethyl-6,7-dihydro-1H-indol-4(5H)-one, selectively bind to Hsp90 and inhibit its cellular activities
at concentrations as low as single digit nanomolar.
Received 8 August 2011
Revised 29 January 2012
Accepted 31 January 2012
Available online 15 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Hsp90
Chaperone
Inhibitors
Indolone
Aminoquinazoline
Quinazoline
Her2
Cancer
Heat shock protein 90 (Hsp90) has received significant atten-
tion as a therapeutic target for cancer treatment.1 Hsp90 acts as
a molecular chaperone that aids the folding,2 maturation, trans-
port, and maintenance of conformational stability of its client pro-
teins.3 Many of these client proteins are involved in critical cellular
functions that promote cell growth, proliferation and survival, and
are also themselves being pursued as anti-cancer targets, for exam-
ple, Her2, c-Met, and Cdk-4 as well as a wide range of mutated pro-
teins.4 Hsp90 is also over-expressed in malignant cells thus these
cells may be more dependent on the Hsp90 chaperoning function.5
Targeting Hsp90 blocks multiple oncogenic signaling pathways,3
thus diminish the potential for resistance to Hsp90 inhibitors.6
Inhibition of Hsp90 N-terminal domain ATPase activity7 disrupts
an ongoing ‘folding’ cycle, leads to destabilization, ubiquitination,
and ultimately proteasomal degradation of client proteins.6 The
natural products geldanamycin8 and radicicol9 are inhibitors of
Hsp90. More recently, additional classes of Hsp90 inhibitors have
been developed.10–14 Here we report our synthetic and medicinal
chemistry efforts in the discovery of a novel aminoquinazoline
scaffold that inhibits Hsp90.15
Through screen of a focused compound library against sets of
ATP binding proteins, we identified 4-(2,6,6-trimethyl-4-oxo-
4,5,6,7-tetrahydro-1H-indol-1-yl)benzamide (1, Fig. 1) as a moder-
ate hit for Hsp90 binding (Kd = 3.7
multiple cellular assays (IC50 >50
l
l
M), although it was inactive in
M).13 Structure-based design
led us to synthesize 1-(4-aminoquinazoline-7-yl)-2,6,6-trimethyl-
6,7-dihydro-1H-indol-4(5H)-one (2), which showed improved
activity in an Her2 degradation assay (SKBR3 cells; IC50 = 10
lM).
This scaffold appears not only small but also drug-like: both its
H2N
N
H2N
O
N
N
N
⇑
Corresponding author.
2
O
1
O
E-mail address: khuang@pamlicopharma.com (K.H. Huang).
Present address: Pamlico Pharmaceutical Inc., PO Box 110284, 7020 Kit Creek
Road Suite 180, Research Triangle Park, NC 27709, USA.
Figure 1. Two related Hsp90 inhibitors: an initial screen hit benzamide 1, and a
4-aminoquinazoline 2 with a variant cyclic binding head.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.137

K. H. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2550–2554
2551
Table 1
H2N
O
O
H2N
O
Structures and biological data for aminoquinazoline analogs
H
N
H
N
R4
N
R2
OH
OH
R5
N
N
N
N
R8
F3C
SNX-7081
SNX-2112
O
N
X
R9
Figure 2. Previous development of screen hit 1 has led to potent Hsp90 inhibitors:
benzamide SNX-7081 and SNX-2112.
O
R4
N
R2
Y
F
Compd R2
R8
R5
R4
NH2
NH2 Me CH
NH2 Me
NH2 Me CH
NH2 Me CH
NH2 Me CH
NH2 Me CH
NH2 Me CH
NH2 Me CH
NH2 Me CH
NH2 Me CH
NH2 Me CH
R9
H
X
Hsp90a Her2b
CMe 2.941
9.974d
R5
NH
F
a
R5
N
+
2
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
–SEt
R2
H2N
0.568c
5.813d
0.731c
1.898c
>20c
R8
3
H
0.245
0.628
0.628
2.549
>10
1.078
9.804
> 10
R8
4
H
N
5
6
7
Me
NH2
H
F
Ia Y = CN
II
III
Ib Y = C(=O)R4
+
8
H
F
0.665c
7.369c
7.752c
0.401c
3.655c
>20c
–OnBu
R4
R5
N
R2
H
9
H
10
11
12
13
14
15
16
17
18
19
20
21
H
H
H
H
NH2
NH2
H
H
H
–NHnBu
N
b
X
–SnBu
0.980
2.157
>10
N
–S-allyl
–SC2H4NH2
R9
R8
O
H
H
H
H
H
F
H
Me
Me CH
Me CH
0.157
0.098
0.705d
0.004d
0.498d
4.510d
16.96d
3.783d
16.84d
>20d
IV
N
X
NH2 Me CMe NDe
R9
NH2 Et
NH2 CF3
NH2 Me
N
N
N
NDe
>10
NDe
>10
>10
V
O
R5, R8 = F
H
NH2
H
c
R5, R8 = OBu, SBu, NHBu
H
H
NH2 Me CH
–OMe NH2 Me CH
Scheme 1. Preparations for the aminoquinazoline analogs. Reagents and condi-
tions: (a) NaH, DMF, 100 °C; (b) NaH, DMF, 50–150 °C; (c) NaH, DMF, BuOH or BuSH
or BuNH2, 120 °C microwave.
a
Values (lM) are estimated Kd’s derived from an 8 point binding assay as
described in ‘Text’ and in ‘References and Notes’.19
b
Values (lM) are IC50 determinations from a Her2 imaging assay in either SKBR3
or AU565 cells as described in ‘Text’ and ‘References and Notes’.20
c
SKBR3 cell line.
AU565 cell line.
Not determined.
d
e
top 4-aminoquinazoline and lower dihydro indolone moieties have
been utilized in marketed drugs, for example, the anti-cancer med-
icine Erlotinib and anti-schizophrenia agent Molindone, respec-
tively. Such favorable trend and features, combined with our
previous success on structurally related SNX-7081 and SNX-2112,
a clinical candidate (Fig. 2),13 have encouraged us to further
explore and optimize this novel scaffold also carrying an amino
[6,6] fused pyrimidine core that is shared less commonly by
reported Hsp90 inhibitors.16
The syntheses of 4-aminoquinazoline analogs started with an
available 2,4-difluorobenzonitrile (Ia, Scheme 1). Treating that
with formamidine, acetamidine, or guanidine (II) and sodium
hydride in DMF15,17 at 100 °C afforded 4-amino-7-fluoroquinazo-
line (III). This was then coupled with either a derivative of
6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-one or 6,6-dimethyl-
6,7-dihydro-1H-indazol-4(5H)-one (IV)13 in heated mixture of
NaH and DMF to afford a 4-amino regioisomer (V). Additional sub-
stituents could be optionally introduced later, for example, at the
C5 (R5) or C8 (R8) of the quinazoline ring, by displacement of exist-
ing extra fluorine(s) carried on the benzonitrile with nucleophiles
such as alkoxide, thio or amine in heated DMF. The corresponding
regioisomeric 2-aminoquinazolines were prepared similarly, from
guanidine and either a 2,4-difluoro benzaldehyde or acetophenone
(Ib).15,18
an imaging assay. In addition, the anti-proliferative activities of
these analogs were evaluated in multiple cancer cell lines.
One key initial improvement we made on analog 2 was to relo-
cate the 2-methyl of the lower indolone moiety to the nearby 3-po-
sition (3), based on our knowledge of SAR on a related SNX-7081
scaffold.13 This increased both the affinity to Hsp90 and cellular
potency for Her2 degradation by more than 10-fold. The parallel
3-methyl indazol-4-one analog (4), designed to improve solubility
and metabolic stability, lost affinity and potency significantly.
Additional modifications to the lower subunit did not provide more
improvement, evidenced by the 2,3-dimethyl indolone analog (16),
larger 3-ethyl (17) or 3-trifluoromethyl (18) indazolone analog.
Likewise, reduction of the indolone carbonyl to a hydroxyl or re-
moval of one to all three methyl(s) led to partial or total loss of
activities (data not shown). Thus the 3,6,6-trimethylindol-4-one
moiety appears optimized, and the steric hindrance imposed by
the methyl groups could enhance the metabolic stability of the
indolone and its analogs.22
Addition of a methyl group at the quinazoline C2 position (5)
caused a minor decrease in compound activities whereas an amino
group (6) was less favored. Introduction of a fluorine at C5 (7) was
not tolerated, but was somewhat tolerated at C8 of (8) and (19) and
we do not have a good explanation to these. Displacement of the
C8 fluoride with n-butanol (9) or n-butylamine (10) was disfa-
vored, whereas the n-butylthio (11) gave only slight improvement.
The biological data of these analogs are summarized (Table 1). A
binding assay, as previously described,21 was used to determine
the compound affinity to monomeric Hsp90. Hsp90 specific cellu-
lar effects on client proteins were also evaluated, including Her2
degradation as measured in either SKBR3 or AU565 cell lines using

2552
K. H. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2550–2554
Table 2
M98
W162
Cellular activities for compound 15, SNX-6833
I96
L107
Y139
K58
N
NH2
N
A111
V136
N
T184
F138
O
D93
I96
N51
D54
K58
Assay
IC50, l
Ma
M98
W162
A375 proliferation
HT29 proliferation
0.026 0.016
0.005 0.005
0.015 0.008
0.0007 0.0009
0.020 0.011
0.064 0.028
0.094 0.035
0.104 0.031
0.014 0.008
0.004 0.001
0.005 0.001
0.036 0.020
0.011 0.004
L107
Y139
LNCAP proliferation
MCF-7 proliferation
MDAMB231 proliferation
NCI-H460 proliferation
PC-3 proliferation
SKMEL5 proliferation
SW620 proliferation
Her2 degradation, AU565b
HSP70 induction, A375b
pErk inhibition, AU565b
pS6 inhibition, A375b
A111
V136
T184
D54
F138
D93
a
N51
Values are means of four experiments with standard deviation shown.
Both cellular readout and cell type are indicated.
b
of Hsp90. In particular the quinazoline ring was modeled to clo-
sely overlay the purine of ADP with the indolone moiety binding
into a hydrophobic pocket induced by displacement of Leu 107
( Fig. 3). The key relocation of the 2-methyl group for the
3-methyl (comparison of 2 to 3) results in the 3-methyl occupy-
ing a small hydrophobic pocket formed by residues Ala 111, Val
136, and Tyr 139. It is interesting to note that this region is in
part composed by residues of the ‘lid’ segment of Hsp90 which
plays a central role in the conformational rearrangements that
occur as part of the chaperoning mechanism. Modulation of
the lid conformation may offer a plausible rationalization for
the significantly improved cellular activity of compound 3. The
2-aminoquinazoline analog 15 was modeled to maintain overlap
of the N3 aromatic nitrogen and 2-amino group with N1 and N6
of ADP, thereby allowing the 2-amino group to engage in a well-
formed hydrogen bond with Asp 93. This overlap was accom-
plished through rotation about the biaryl torsion, which in turn
allowed the indolone to maintain a similar positioning relative to
compound 3. The quinazoline ring is also positioned to strongly
interact with Met 98.
Compound 15, or SNX-6833, potently inhibited cancer cell pro-
liferation across multiple cell lines at nanomolar concentrations
(Table 2). Importantly, it also promoted degradation of direct client
proteins such as Her2 and pERK, as well as induced Hsp70 synthe-
sis, at comparable concentrations. In addition to examining effects
on direct Hsp90 clients, markers for various signaling cascades
were studied. As expected for a compound that inhibited AKT
and MAPK pathways, compound 15 potently reduced levels of
pS6 (Fig. 4).
Figure 3. Binding models for compounds 3 (top) and 15 (middle). Protein structure
and active site water molecules are derived from protein databank entry 3D0B.24
Expected hydrogen bonds are indicated with dashed lines. Lower panel shows
protein based overlaps to ADP as taken from Hsp90—ADP structure.25
Other C8 attachments carrying additional functional groups such
as an alkene (12) or amine (13) did not improve potencies, nor
for a small ethylthio (20) or methoxyl (21) group placed at C5.
Another intriguing SAR feature of the scaffold was discovered,
when the 4-amino group of analog 6 was deleted, or the 4-amino
of 3 was relocated to C2. The resulted 2-amino analog 14 was
equally potent compared to its 4-amino regioisomer 3. In vitro
studies, however, indicated 14 to be not selective for Hsp90. Unlike
most analogs in this series, it also appeared to inhibit tubulin poly-
merization and active in cellular assays that measure progression
through M-phase23 (IC50 = 0.11
lM). Since C4 position of quinazo-
line is also known prone to metabolic oxidation, a blocking methyl
group was introduced there to lead to compound 15. Serendipi-
tously and unexpectedly, this modification greatly boosted both
Hsp90 affinity and cellular client activities to nanomolar range;
and surprisingly, this also eliminated the off-target activity against
tubulin (IC50 >10 lM, M block).
Computational models were used to assist in design and
understanding of the observed SAR for this quinazoline scaffold.
Development of initial models for the 4-aminoquinazoline series
was straightforward, based on X-ray data for binding of either a
related benzamide analog24 or ADP25 to the N-terminal domain
In conclusion, we have discovered a novel, selective, and drug-
like 2-amino-4-methylquinazolin-7-yl 6,7-dihydro-indol-4-one
scaffold that potently inhibits Hsp90 across multiple cellular
assays. The highly favorable features of this series of inhibitors
revealed here invite further investigations.

K. H. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2550–2554
2553
Figure 4. Merged images showing levels of pERK (green) and Her2 (red) in AU565 cells. Hsp70 (green, FITC) and pS6 (red, TRITC) were measured in A375 cells. Hsp70/pS6
images are of identical cells for each treatment condition (vehicle control or test compound), but were captured at different wavelengths. For all images, DNA was stained
with Hoescht (blue).
Mansfield, R.; Fritz, L. C.; Ulm, E.; Burrows, F. J.; Boehm, M. F. J. Med. Chem.
2007, 50, 2767.
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resulting IC50 values were corrected for the ATP ligand concentration and
presented as Kd values.
20. 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.
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72 or 144 h, depending on rate of growth. At harvest, media was removed and
DNA content for individual wells was determined using CyQuantÒ DNA dye
(Invitrogen). Her2 degradation in SKBR3 cells was measured by cellular ELISA
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using
a mouse anti-Her2 primary antibody (Millipore) and anti-mouse
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24 h with compound followed by methanol fixation. High content analysis
(HCA) was done using an ArrayScan 4.5 (Pittsburg, PA) or Becton Dickenson
435 (Rockville, MD) imager on cells treated 24 h with compound. After fixation
in 4% PBS-buffered formalin, 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.

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