Discovery of aminoquinolines as a new class of potent inhibitors of heat shock protein 90 (Hsp90): Synthesis, biology, and molecular modeling

Article abstract of DOI:10.1016/j.bmc.2008.05.047

The molecular chaperone Hsp90 plays important roles in maintaining malignant phenotypes. Recent studies suggest that Hsp90 exerts high-affinity interactions with multiple oncoproteins, which are essential for the growth of tumor cells. As a result, research has focused on finding Hsp90 probes as potential and selective anticancer agents. In a high-throughput screening exercise, we identified quinoline 7 as a moderate inhibitor of Hsp90. Further hit identification, SAR studies, and biological investigation revealed several synthetic analogs in this series with micromolar activities in both fluorescent polarization (FP) assay and a cell-based Western blot (WB) assay. These compounds represent a new class of Hsp90 inhibitors with simple chemical structures.

Full text of DOI:10.1016/j.bmc.2008.05.047

Bioorganic & Medicinal Chemistry 16 (2008) 6903–6910
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of aminoquinolines as a new class of potent inhibitors of heat
shock protein 90 (Hsp90): Synthesis, biology, and molecular modeling
Thota Ganesh a,b, Jaeki Min a,b, Pahk Thepchatri a, Yuhong Du a,c, Lian Li a, Iestyn Lewis a, Larry Wilson a,
Haian Fu a,c, Gabriela Chiosis d, Raymond Dingledine a,c, Dennis Liotta a,b, James P. Snyder a,b, Aiming Sun a,b,
*
a Chemical Biology Discovery Center, 1510 Clifton Road, Emory University, Atlanta, GA 30322, USA
b Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA
c Department of Pharmacology, Emory University, 1510 Clifton Road, Atlanta, GA 30322, USA
d Department of Medicine and Program in Molecular Pharmacology, and Chemistry, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The molecular chaperone Hsp90 plays important roles in maintaining malignant phenotypes. Recent
studies suggest that Hsp90 exerts high-affinity interactions with multiple oncoproteins, which are essen-
tial for the growth of tumor cells. As a result, research has focused on finding Hsp90 probes as potential
and selective anticancer agents. In a high-throughput screening exercise, we identified quinoline 7 as a
moderate inhibitor of Hsp90. Further hit identification, SAR studies, and biological investigation revealed
several synthetic analogs in this series with micromolar activities in both fluorescent polarization (FP)
assay and a cell-based Western blot (WB) assay. These compounds represent a new class of Hsp90 inhib-
itors with simple chemical structures.
Received 1 April 2008
Revised 8 May 2008
Accepted 22 May 2008
Available online 27 May 2008
Keywords:
Heat shock protein 90 (Hsp90)
High-throughput screening (HTS)
Aminoquinoline
Published by Elsevier Ltd.
1. Introduction
purine-scaffold was subsequently developed into the clinical
agents PU-H71 (5)8 and CNF2024 (6).9 Very recently, the pyra-
Heat shock protein 90 (Hsp90) has emerged as an important
biological target that modulates a variety of cellular processes
including cell maturation, stability, and conformational mainte-
nance of signature cancer proteins such as Akt, Raf-1, HER-2/ErbB2,
and p53.1 Reports indicate that Hsp90 from stress-induced cells
exhibits a higher affinity for small molecule inhibitors relative to
normal cells as a result of increased refolding requirements of its
mutated or altered clients.2,3 Thus, identification of selective tu-
mor-specific Hsp90 inhibitors could lead to the specific targeting
of cancer cells and circumvent systemic toxicities. The antibiotic
geldanamycin (1a) and its synthetic analogs (17-AAG, 17-DMAG,
1b, 1c)4 were the first small molecules found to bind competitively
to the ATP-binding pocket in the Hsp90 N-terminal domain and
thereby prevent the ATP-mediated conformational change needed
for protein–protein interactions of Hsp90 with its client polypep-
tides. While geldanamycin analogs are showing promising antican-
cer activity in clinical trials,5several other compounds either from
natural or synthetic sources have also been identified (Fig. 1).
Notable among these is the natural product radicicol (2),6 which
exhibits potent in vitro Hsp90 inhibitory activity, but lacks
in vivo efficacy. The first reported synthetic class of Hsp90 inhibi-
tors was the purine-scaffold series, represented by PU3 (4).7 The
zole/isoxazole class of compounds (3a and 3b) was discovered
from high-throughput screening.10 All these known analogs utilize
the same binding pocket as geldanamycin on Hsp90 (Fig. 1).8b,11,13
Two strategies have proven useful for identifying Hsp90 inhib-
itors. On one hand, structure-based design strategies were em-
ployed to develop potent analogs8,9,12 with X-ray templates of
Hsp90–ligand complexes.13 On the other hand, high-throughput
screening (HTS) methods are being investigated to find novel
chemical scaffolds.14 In the present study, we employed a fluores-
cence polarization (FP) assay15 to identify a new class of Hsp90
inhibitors by HTS. The aminoquinoline 7 was identified as a mar-
ginally active Hsp90 inhibitor hit. Structure validation, re-synthe-
sis, and structure–activity relationship (SAR) studies led to
several aminoquinoline analogs as low micromolar inhibitors of
Hsp90. The best compound 10 exhibits low micromolar activities
in both primary FP and cell-based Western blot (WB) assays
(Fig. 2). Hit identification, synthesis, SAR and bio-structural analy-
sis of the quinoline derivatives are described in detail below.
2. Results and discussion
Various bioassays have been developed to identify novel inhib-
itors of Hsp90.16 Most of these assays are amenable to HTS, since
they report the interaction of a small molecule with recombinant
* Corresponding author. Tel.: +1 404 727 6689; fax: +1 404 727 6689.
Hsp90-a or -b. These proteins are derived from yeast or human
E-mail address: asun2@emory.edu (A. Sun).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.05.047

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T. Ganesh et al. / Bioorg. Med. Chem. 16 (2008) 6903–6910
O
O
R
O
HO
HO
Cl
OH
Cl
O
Me
H
O
N
H
OMe
O
O
H
Me
MeO
MeO
HO
OH
H
N
X
N
OCONH2
O
2
3a
: X = NH (VER-49009); pyrazole
1a
1b
(Radicicol)
(geldanamycin): R = OCH3
(17AAG): R = NHAllyl
3b
: X = O (VER-50589); Isoxazole
1c (17DMAAG): R = NHCH2CH2NMe2
O
O
H2N
N
NH2
NH2
N
OMe
N
N
N
N
N
N
N
OMe
OMe
I
S
N
N
N
N
Cl
OMe
4 (PU3)
HN
6 (CNF-2024; BIIB021)
5 (PU-H71)
Figure 1. Structures of known Hsp90 inhibitors and purine analogs.
Table 1
O
HO
Bioactivity of aminoquinolines in FP and WB assays
a
b
N
ID
Compound
IC50
(l
M) (FP assay)
IC50 (lM) (Western blot)
N
NH
2HBr
NH
5
7
8a
8c
PU-H71
850375
TG2-193-3
TG2-205
0.043
5.8
40
0.06–0.13
5.0
NAc
NA
40
8
9
10
11
24724348
24724372
24724290
24724310
AS-222
4.9
29
1.6
6.0
H2N 2HCl
N
1.3 (0.73)d
2.4
1.0 (2–3)d
6.0
10
7 (SID: 850375)
(SID: 24724290)
18a
18d
19a
19b
19c
19d
19e
19f
19g
19h
19i
19j
19k
19l
19m
20a
20b
20c
20d
20f
20g
20h
20i
20j
20k
>50
>50
3.1
NA
NA
NA
NA
13
25
NA
NA
NA
25
NA
NA
25
NA
NA
NA
NA
NA
NA
20
20
30
NA
NA
AS-217
Figure 2. Hsp90 hit compound 7 and the most active analog 10.
24724364
24724377
24724374
JM1-156b
24724347
24724365
24724375
24724350
24724357
24724376
24724346
24724369
24724351
JM1-138b
JM1-152c
JM1-156c
JM1-135c
JM1-147d
24724309
24724292
24724291
JM1-128a
24724363
8.0
40.0
>50
>50
>50
14.4
44
>50
>50
2.1
14
>50
7.6
<1.6
<1.6
<1.6
<1.6
<1.0
0.8
normal cells, respectively, in which Hsp90 is found to be in a latent,
low-affinity form, compared to the high-affinity state present in
tumor cells.17 One way to determine the therapeutically relevant
state of Hsp90 is to develop an assay that makes use of human can-
cer cell derived lysates, instead of recombinant protein. The former
permits direct measurement of the interaction between small mol-
ecule and tumor-specific Hsp90. Such an assay is capable of leading
to identification of molecules that are specific for tumor cell Hsp90.
By employing a recently developed FP assay for HTS,15 we have
identified a highly promising compound, namely 7, for follow-up
by chemistry and molecular modeling.18
2.1. Hit identification
<1.6
1.8
1.1
In a high-throughput screening, it has become common for
molecules with known biological activities to emerge as modula-
tors of novel targets.19 One example in the present work is the
anti-malarial agent quinocide-dihydrochloride, namely 8-(4-amin-
opentylamino)-6-methoxyquinoline-dihydrochloride (7),20 which
appeared as a novel scaffold in the modulation of Hsp90 (Fig. 2).
This compound showed activities in the low micromolar range in
both FP and WB assays (Table 1). The FP assay measures the inter-
action of Cy3b-conjugated geldanamycin with Hsp90 in tumor cell
lysates for identifying ATP-binding inhibitors. Hsp90 uniquely sta-
bilizes the Her2/Hsp90 association.21 Addition of Hsp90 inhibitors
to cancer cells induces the proteasomal degradation of a small sub-
NA
a
Mean of two determinations.
Mean of three determinations.
NA, not active.
b
c
d
Values in parentheses are from re-synthesized 10.
set of proteins involved in signal transduction such as Raf1 kinase,
Akt, and certain transmembrane tyrosine kinases such as Her2.
Thus, Her2 degradation in cells is a functional read-out of Hsp90
inhibition. Correlation between Hsp90 binding and Her2 degrada-

T. Ganesh et al. / Bioorg. Med. Chem. 16 (2008) 6903–6910
6905
tion in cancer cells is indicative of a selective biological effect in
these cells via Hsp90. We used a WB-based assay to measure the
cellular level of Her2 protein in MCF-7 breast cancer cell lysates
collected after 24 h of compound treatment. The original hit com-
pound 7 was re-synthesized in both neutral and dihydrochloride
salt forms as previously reported.22 Surprisingly, both synthesized
namely, 8-alkylaminoalkyl-6-methoxy-quinolines and 8-alkylami-
noalkyl-6-hydroxyl-quinolines as shown in Figure 3. Table 1 sum-
marizes estimated in vitro binding affinities to Hsp90 as well as the
ability to prevent Hsp90 interaction with Her2 in the WB assay,
leading to the degradation of Her2.
forms of 7 showed only moderate biological activity (IC50: ꢀ30
lM
2.2. Chemistry and SAR-analyses of aminoquinolines
in FP assay) in both assays by comparison to the original sample
(SID 850375, collected from Discovery Partner International (DPI)
as part of MLSCN). As the purities of both synthetic and original
DPI sample for 7 were checked by LC/MS, the original DPI sample
showed about 60% purity, while the re-synthesized compound
was about 97% in purity. The biological activity of the original sam-
ple of 7 was believed to arise partially from degradation products
present in the original DPI library sample, which will be detailed
in a separate publication. Nevertheless, the structure of 7 serves
as a starting point for analog identification.
One of the advantages of compound 7 is that many analogs of
this scaffold (aminoquinolines) are available for SAR from the Na-
tional Cancer Institute (NCI) collection. To take advantage of this
collection, we performed 2D-tanimoto similarity searching on a
structural database consisting of 250,000 compounds available
from the NCI.23 2D descriptors were assigned to the NCI database
using the MDL public keys SciTegic’s Extended Connectivity Finger-
prints (ECFP_6) available in Pipeline Pilot.24 Threshold similarity
was set to 60% to introduce alternative scaffolds into the pool.
Thirty-five compounds were identified and ordered from NCI and
tested in both FP and WB assays. In addition, design and synthesis
for some aminoquinolines were also executed through Schemes 1
and 2. These compounds were segregated into 2-subclasses;
Compound 8 with a 2-carbon linker between the quinoline moi-
ety and diisobutyl amine is the only active compound in the series.
Replacing the isobutyl groups on the terminal amine of 8 with n-
dibutyl, diethyl, and diisopropyl units (8a–c) (Scheme 1) elimi-
nates the activities in both FP and WB assays. Likewise, 19a (with
sec-butyl group) and 19b (with one ethyl and one sec-pentyl
groups) show activity only in the FP assay, but fail to show a re-
sponse in WB assay, indicating the importance of diisobutyl groups
on the terminal nitrogen. Increasing the chain length to 3–4 car-
bons while swapping the alkyl groups on the terminal nitrogen
also eliminates activity (cf. 19c–d), suggesting the importance of
the 2-carbon linker. Likewise 9 with a 3-carbon linker, an extra hy-
droxyl group on the middle carbon and a methyl group at the quin-
oline C-2 position shows sixfold less activity in the FP assay and
threefold less in the WB assay compared to 8. Thus, longer the link-
ers appear to impair activity in this series. Likewise 19k with a 3-
carbon linker, but with additional appendages (anilino, PhN) at the
quinoline C-5 position shows mixed effects. Compound 19k shows
twofold increased activity in FP assay and threefold decreased
activity compared to 8, which suggests that a 2–3 carbon linker
is optimal for the 6-methoxy quinoline series. Supporting this
interpretation, compounds 19d–e, 19h–j with 4–5 carbon tethers
O
I
OH
N
N
NH
c
14
a
b
N
NH
12
13
8
N
O
O
N
N
NH
n
NH2
14
n
8a
8b
8c
8d
: n=1; R1, R2 = Bu
: n=1; R1, R2 = Et
i
: n=1; R1, R2 = Pr
N
R1
R2
i
: n=2; R1, R2 = Bu
Scheme 1. Synthesis of 6-methoxy-aminoquinoline derivatives. Reagents and conditions: (a) Bromoethanol, Et3N, THF, reflux; (b) Imidazole (3 equiv), PPh3 and I2 (1.5 equiv),
Et2O/MeCN; (c) excess Et3N, MeOH/DMF (2:1), 135 °C, 2 days.
HO
MeO
MeO
MeO
N
N
N
N
d
HN
a
c
HN
b
HN
HN
14
n
n
n
n
NC
NH2
N
R1
N
R1
R2
R2
15a:
n=1
18a
16a
17a
17b
17c
: n = 1, R1 = R2 = Et
: n = 2, R1= R2 = Et
: n = 1
: n = 1, R1 = R2 = Et
: n = 2, R1 = R2 = Et
: n = 2, R1 = H, R2 = iPr
15b: n=2
16b: n = 2
10
: n = 2, R1 = H, R2 = iPr
11
Scheme 2. Syntheses of 6-hydroxyl-aminoquinoline derivatives. Reagents and conditions: (a) Br(CH2)2(CH2)nCN, Et3N, EtOH, reflux, 3 days; (b) LAH, THF, 0 °C–rt;(c) CH3CHO,
NaBH(OAc)3; (d) 48% aq HBr, reflux.

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T. Ganesh et al. / Bioorg. Med. Chem. 16 (2008) 6903–6910
X
R1
N
HO
R1
O
R
N
N
R2
NH
NH
Y
n
R2
n
N
R3
R4
R3
R4
8: n=0, R = R1 = R2 = H; R3 = R4
=
=
s
s
-Bu; X = Y = H
-Bu, X = H, Y = OH
= s-Bu; X = Y = H
10:
n=3, R1 = R2 = H, R3 = R4 = Et
11: n=3, R1 = R2 = H, R3 = iPr, R4 = H
20a
9:
,
n=1 R = R1 = R2 = H; R3 = R4
19a: n = 0, R = R1 = R2 = H; R3 = H, R4
: n = 1, R1 = R2 = H, R3 = R4 = H
19b
: n = 0, R = R1 = R2 = H; R3 = Et, R4 = s-Pentyl; X = Y = H
19c: n = 1, R = R1 = R2 = H; R3 = H, R4 -pentyl; X = Y = H
19d
20b: n = 1, R1 = H, R2 = Et, R3 = R4 = H
20c: n = 2, R1 = R2 = R3 = R4 = H
=
3
: n = 2, R = R1 = R2 = H; R3 = R4 = H; X = Y = H
19e: n = 3, R = R1 = R2 = H; R3 = H, R4 -pentyl; X = Y = H
nPr, R4 = H; X = Y = H
19f
=
20d
: n = 2, R1 = H, R2 = Me, R3 = R4 = H
20e: n=2, R1 = R2 = H, R3 = R4 = Et
20f
=
t
: n = 2, R = R1 = Me, R2 = H, R3
: n = 3, R1 = H, R2 = Me, R3 = R4 = H
20g: n = 3, R1 = R = R3 = R4 = H
20h
19g: n = 2, R = Me, R1 = R2 = H, R3 = iPr, R4 = H; X = Y = H
: n = 2, R = Me, X = OMe, R1 = R2 = Y = H, R3 = iPr, R4 = H
: n = 2, R = Me, R1 = R2 = Y = H, X = p-Cl-PhO, R3 = R4 = H
19h
19i
: n =1, R1= Me, R2 = H, R3 = R4 = Me
20i: n = 2, R1 = Me, R2 = H, R3 = iPr, R4 = H
20j
19j: n=3, R = H, R1 = R2 = Y = H, X =
19k
p
-Meo-PhO, R3 =iPr, R4 = H
: n = 2, R1 = Me, R2 = R3 = R4 = H
20k: n = 2, R1 = Me, R2 = H, R3 = R4 = Et
: n = 1, R = R1 = R2 = H, X = PhN, Y = H, R3 = R4 = Et
19l: n =1, R1 = H, R2 = OMe, X = Y = H, R3 = R4 = Et
19m
: n =1, R1= Cl, R2 = H, X = Y = H, R3 = R4
= i-pr
Figure 3. Structure of quinoline analogs obtained from NCI collection25 and synthesis.
120
were found to be inactive or much less active compared to 8. Sub-
stitutions around the quinoline moiety also resulted in complete
loss of activity (cf. 19l–m, 19f–19j).
90
60
30
0
From the 6-hydroxyl-8-aminoquinilone series (Figs. 2 and 3 and
Scheme 2) only two compounds 10 and 11 show promising activity.
In this series, contrary to the methoxy quinoline series, the longer
linkers promote activity. Compound 10 exhibits nearly twice the
binding affinity of 11 in the FP assay and eightfold more active in
the WB assay. The difference between these two compounds is only
the alkyl groups on the terminal nitrogen. The former (compound
10) possesses two ethyl groups compared to the latter (compound
11), which incorporates only one isopropyl group. This result is con-
sistentwith the findingsfor the methoxyquinoline series and clearly
suggests that di- alkyl substituents on the terminal nitrogen are very
importantfor activity, especially in the WB assay. The latter observa-
tion also operates when 10 is compared with 20a–g, which do not
bear substituents on the terminal nitrogen and show no activities
in the WB assay, despite the fact 20a–g proved to be highly active
in the competitive-binding assay (Table 1).
To assure that the activities observed with the most active com-
pounds (8, 10, and 11) were truly due to the structures assigned by
NCI, the compounds were re-synthesized in our laboratory as
shown in Schemes 1 and 2. Compounds 8, 10, and 11 have very
similar structures to hit compound 7 with only slight changes in
the length of the linker, alkyl groups on the amine tail and the free
hydroxy group instead of methoxy group in some cases.
Compound 8 was prepared as shown in Scheme 1. Diisobutyl-
amine was reacted with bromoethanol to obtain 2-(N,N-diisobu-
tyl)-ethanol 12, which was treated with triphenyl phosphine and
iodine in the presence of imidazole to provide 2-(N,N-diisobutyl)-
ethyl iodide 13. Condensation of 13 with 6-methoxy-8-amino-
quinoline 14 in the presence of triethylamine provided compound
8. Similarly, derivatives 8a–c with different alkyl groups on the
amine nitrogen and a longer chain analog 8d were also obtained
in the same way.
The syntheses of 10 and 11 were carried out as shown in
Scheme 2. 6-Methoxy-8-amino-quinolin 14 was treated with ex-
cess bromoalkylnitrile in a mixed solvent of triethylamine and
methanol under refluxing for more than 48 h to obtain nitrile com-
pound 15 in low to moderate yields. Reduction of 15 with lithium
aluminum hydride (LAH) afforded the terminal amine 16, which
Compound 10
Re-synthesized 10
0.1
0.01
1
10
100
Compound (μM)
Figure 4. The Hsp90 inhibition activity of the resynthesized compound 10 was
confirmed in a FP assay. Increasing concentrations of compound 10 from the
compound library or resynthesized batch were added to the cy3B-geldanamycin/
MCF7 cell lysate (Hsp90) reaction and the FP readout was recorded. Results from a
representative of three independent experiments are shown.
was further alkylated by reductive-amination with either acetalde-
hyde or acetone to deliver 17a–c. Deprotection of methoxy analogs
17 delivered hit compounds 10, 11, and their analogs 18a.
As shown in Figure 4, the resynthesized 10 was highly active in
FP assay with an IC50 around 0.73 lM in a representative experi-
ment. Importantly, this compound appeared to be able to enter
cells and exerted a potent activity to induce Her2 degradation with
an average IC50 of 1.0
shown in Fig. 5).
lM (0.8 lM in a representative assay as
2.3. Discussion
An ideal small molecule probe should bind effectively to tumor
Hsp90, after which a conformational change will lead to disruption
of the Hsp90-client protein interaction and client degradation as
observed in Western blots. In the present study, Western blots
have been performed for Her2 degradation, to monitor client pro-
tein interaction with Hsp90. Her2 shows greatest sensitivity, and it
was selected as diagnostic in the current study. Compounds 20b–k
have shown potent activity in the FP assay with IC50 values in the
low micromolar range, but failed to demonstrate activity in the WB
assay.26 Poor membrane permeability might account for the differ-
ent behaviors in the cell-free and cell-based assay. (Table 1).

T. Ganesh et al. / Bioorg. Med. Chem. 16 (2008) 6903–6910
6907
100
A
B
80
60
Compound 10 (μM)
12.5 6.3 3.1 1.6 0.8 0.4 0.2
40
20
0
IC50: 0.8 μM
C
PU
Her2
Hsp90
0.1
1
10
Compound 10 (μM)
Figure 5. Compound 10-induced Her2 degradation. (A) Western blot assay. MCF7 cells were treated with test compounds for 24 h before being lysed for Western blot
analysis with antibodies to Her 2 and Hsp90. (B) The dose-curve of compound 10 in Her2 degradation assay. The density of each band in Western blot from (A) was quantified.
Data were normalized to total Hsp90 protein level and then normalized to the vehicle (DMSO) control well. Data shown are from one representative of 6 independent
experiments. Compound 5 was used as a positive control and was tested at 60–130 nM.
Among all the analogs, 8–11 obtained from NCI showed prom-
ising activity in both FP and WB assays. Compounds 8 and 9 both
feature a 6-methoxy group on the quinoline moiety, while 10
and 11 share a common scaffold with 6-hydroxyl substitution.
Compounds 8 and 9 were obtained as dihydrochloride salts and,
10 and 11 were available as dihydrobromide salts. These analogs
not only inhibited the binding of labeled geldanamycin to Hsp90
suggests that they bind competitively with the known N-terminal
inhibitor, geldanamycin. It is possible that the aminoquinolines are
able to influence the N-terminal ATP-binding site from an allosteric
location. However, no such site has been identified at this time,
though through space communication between the N-terminal
and C-terminal regions is being explored. The binding modes of
five N-terminal Hsp90 inhibitor classes have been solved by X-
ray crystallography and were used as a guide to model the amino-
quinolines into the N-terminal ATP-binding site.11–13,8b Despite the
wide conformational and functional diversity, they share three
common pharmacophore sites: hydrophobic cluster, hydrogen-
bonding acceptor, and hydrogen-bonding donor. Hsp90 inhibitors
interact with a hydrophobic cluster consisting of residues M84,
L93, F124, Y125, and V136 (Numbering based on Protein Databank
(PDB) code: 2BRE27). (Fig. 6). The two other common pharmaco-
phore sites involve an H-bond donor and an H-bond acceptor
(T171 and D79, respectively). Other hydrogen-bonding interac-
tions are observed as well, but these two appear most consistently
in analysis of all available co-crystals (Fig. 7).
with IC50 values in the range of 1–5
lM (except 9 which showed
IC50 = 29 M) but also induced degradation of Her2 with activities
l
in the low micromolar range. Resynthesised compound 10 exhibits
activity in both FP and WB assays, however, synthesized versions
of 8 and 11 had only marginal activities in either the FP or WB as-
says. It is difficult to pin down causes of the discrepancy, when
both samples showed similar analytic data. However, minor unde-
tectable impurities present in the NCI sample due to long-term
storage in DMSO could potentially account for the discrepancy.
Gratifyingly, resynthesized compound 10 shows consistent activi-
ties in both FP assay and cell-based WB assay with IC50 values in
low micromolar range (ꢀ1
lM).
The GLIDE protocol (Schrodinger Inc.)28 was used to dock 10
into the ATP-binding pocket of Hsp90. No constraints were imple-
mented during the docking calculation. Receptor-site coordinates
were derived from the Protein DataBank of 2BRE. Rendering for
all pictures was generated using PyMol (DeLano Scientific).29
2.4. Biostructural analysis
We have hypothesized that aminoquinolines bind to the N-ter-
minal region based on their activity in the in vitro FP assay, which
Figure 6. 3D representations of the hydrophobic and hydrogen-bonding interactions with the pyrazole (cyan) and purine (blue) inhibitors (PDB ID: 2BRE, 2FWZ,
respectively). Receptor represented as a molecular surface, where the hydrophobic region is highlighted in gray, the hydrogen-bonding region in red/blue. As shown in the
figure, the pyrazole interacts only with the surface of the hydrophobic cluster while the purine buries a benzodioxazole deep into the hydrophobic cluster.

6908
T. Ganesh et al. / Bioorg. Med. Chem. 16 (2008) 6903–6910
1H), 7.97 (dd, J = 8.2, 1.6 Hz, 1H), 7.30 (dd, J = 8.2, 4 Hz, 1H), 6.43
(d, J = 2.4 Hz, 1H), 6.25 (d, J = 2.4 Hz, 1H), 3.84 (s, OCH3, 3H), 3.21
(t, J = 6 Hz, 2H), 2.68 (t, J = 6 Hz, 2H). 2.15 (d, J = 6.8 Hz, 4H), 1.72
(m, 2H), 0.88 (d, J = 6.8 Hz, 12H). 13C NMR (100 MHz, CDCl3): d
159.6, 146.5, 144.7, 144.6, 135.9, 134.6, 129.8, 122.0, 96.7, 92.0,
64.3, 55.3, 54.2, 41.2, 26.9, 21.2, 21.1. HRMS calcd for C20H32N3O
[M+H]: 330.25399; found: 330.25355 (
20H31N3O: C, 72.91; H, 9.48; N, 12.75; observed: C, 73.19; H,
9.47; N, 12.75.
DÀ1.33). Anal. Calcd for
C
4.3. Synthesis of dihydrochloride salt of 8
A solution of 8 (10 mg) in dichloromethane (4 ml) was purged
with hydrochloride gas for 30 min. Evaporation of the solvent pro-
vided the title compound (100% yield). 1H NMR (400 MHz, MeOH-
d4): d 8.87 (dd, J = 8.4, 1.2 Hz, 1H), 8.80 (dd, J = 5.2, 1.2 Hz, 1H), 7.19
(dd, J = 8.6, 5.2 Hz, 1H), 7.05 (d, J = 2.4 Hz, 1H), 6.93 (d, J = 2 Hz, 1H),
3.97 (s, OCH3, 3H), 3.79 (t, J = 6.8 Hz, 2H), 3.67 (t, J = 6.8 Hz, 2H).
3.19 (d, J = 6.8 Hz, 4H), 2.20 (m, 2H), 1.07 (d, J = 6.8 Hz, 12H). Anal.
Calcd for C20H35Cl2N3O3 (2H2O): C, 54.79; H, 8.51; N, 9.58; ob-
served: C, 54.66; H, 7.98; N, 9.35.
Figure 7. Docking of the 10 (red) into the ATP N-terminal-binding site of Hsp90.
Docking results show that the compound makes contacts with key pharmacophoric
centers (gray—hydrophogic region, red/blue—hydrogen-bonding region observed in
Hsp90 crystal structures).
4.4. Compounds 8a–8d were synthesized by using the same
method as for synthesis of compound 8
3. Conclusions
4.4.1. N1,N1-Dibutyl-N2-(6-methoxyquinolin-8-yl)ethane-1,2-
diamine (8a)
In an HTS campaign, we identified compound 7 as an inhibitor
of Hsp90 with modest activity. SAR-study yielded 3 highly active
derivatives (8, 10, and 11) by FP assay, which all show IC50 values
in low micromolar range. These compounds also enhance degrada-
tion of the Hsp90 client proteins, for instance, Her2, as demon-
Yield 26%. 1H NMR (400 MHz, CDCl3): d 8.53 (dd, J = 4, 1.6 Hz, 1H),
7.89 (dd, J = 8, 1.6 Hz, 1H), 7.27 (dd, J = 8.2, 4 Hz, 1H), 6.45 (d, broad
triplet, J = 5.2 Hz, NH), 6.33 (d, J = 2.8 Hz, 1H), 6.27 (d, J = 2.4 Hz, 1H),
3.87 (s, OCH3, 3H), 3.30 (dd, J = 12, 6 Hz, 2H), 2.80 (t, J = 6.4 Hz, 2H).
2.49 (t, J = 7.2 Hz, 4H), 1.43 (m, 4H), 1.32 (m, 4H), 0.87 (t, J = 7.2 Hz,
6H). 13C NMR (100 MHz, CDCl3): d 159.6, 146.3, 144.7, 135.8,
134.7, 128.9, 122.0, 96.8, 92.1, 55.4, 54.2, 52.9, 41.2, 29.4, 20.8,
strated by
a cell-based Western blot assay. However, after
resynthesis and biological testing of synthesized materials only
one compound, namely 10, demonstrated consistent activities in
both FP assay and WB assay with low micromolar (1 lM) activities.
The use of compound 10 as a lead for further SAR exploration could
14.2. HRMS calcd for
330.25355 (
N, 12.75; observed: C, 72.67; H, 9.58; N, 11.55.
C20H32N3O [M+H]: 330.25399; found:
À1.33). Anal. Calcd for C20H31N3O: C, 72.91; H, 9.48;
D
yield optimized and therapeutically useful molecules.
4.4.2. N1,N1-Diethyl-N2-(6-methoxyquinolin-8-yl)ethane-1,2-
diamine (8b)
4. Experimental
Yield 25%. 1H NMR (400 MHz, CDCl3): d 8.53 (dd, J = 4.2, 2 Hz,
1H), 7.89 (dd, J = 8.4, 1.6 Hz, 1H), 7.28 (dd, J = 8.2, 4.4 Hz, 1H),
6.37 (t, J = 4.4 Hz, NH), 6.33 (d, J = 2.4 Hz, 1H), 6.28 (d, J = 2.4 Hz,
1H), 3.87 (s, OCH3, 3H), 3.34 (dd, J = 12.2, 6.8 Hz, 1H), 2.82 (t,
J = 6.8 Hz, 2H). 2.62 (q, J = 7.2 Hz, 4H), 1.06 (t, J = 7.2 Hz, 6H). 13C
NMR (100 MHz, CDCl3): d 159.6, 146.2, 144.7, 135.8, 134.8, 129.9,
122.0, 96.8, 92.2, 55.4, 51.7, 47.2, 41.2, 11.8. HRMS calcd for
4.1. 2-(Diisobutylamino)ethyl iodide (13)
To a solution of 2-(diisobutylamino)ethanol (12) (3 mmol) in
ether and acetonitrile (3:1, 12 ml) was sequentially added imidazole
(0.6 g, 9 mmol) followed by triphenyl phospine (1.2 g, 4.5 mmol)
and iodine (1.5 g, 4.5 mmol) at 0 °C. The resulting reaction mixture
was stirred for a further 30 min. The reaction was quenched with
Na2S2O3 solution and the reaction mixture portioned between ether
and water. The organic layer was separated and after the usual work-
up, the crude product was purified by silica gel chromatography to
give the title compound 13 (60% yield). 1H NMR: (400 MHz, CDCl3):
d 3.09 (t, J = 8.4 Hz, 2H), 2.74 (t, J = 8.0 Hz, 2H), 2.12 (d, J = 7.2 Hz, 4H),
1.64 (m, 2H), 0.86 (d, J = 6.4 Hz, 12H).
C
C
16H24N3O [M+H]: 274.19139; found: 274.10107. Anal. Calcd for
16H23N3O: C, 70.30; H, 8.48; N, 15.37; observed: C, 70.10; H,
8.23; N, 15.06 (
DÀ1.16).
4.4.3. N1,N1-Diisopropyl-N2-(6-methoxyquinolin-8-yl)ethane-
1,2-diamine (8c)
Yield 25%. 1H NMR (400 MHz, CDCl3): d 8.52 (dd, J = 4.2, 1.2 Hz,
1H), 7.89 (dd, J = 8.4, 1.6 Hz, 1H), 7.27 (dd, J = 8.4, 4.4 Hz, 1H), 6.48
(br s, NH), 6.32 (d, J = 2.4 Hz, 1H), 6.27 (d, J = 2.4 Hz, 1H), 3.87 (s,
OCH3, 3H), 3.21 (br s, 2H), 3.06 (t, J = 6 Hz, 2H). 2.82 (t, J = 6 Hz,
2H), 1.04 (d, J = 6 Hz, 6H). 13C NMR (100 MHz, CDCl3): d 159.6,
146.4, 144.7, 135.8, 134.8, 129.9, 121.9, 96.8, 92.1, 55.5, 48.6,
43.8, 43.2, 21.0. HRMS calcd for C18H28N3O [M+H]: 302.22269;
4.2. N1,N1-Diisobutyl-N2-(6-methoxyquinolin-8-yl)ethane-1,2-
diamine (8)
To a solution of 6-methoxy-8-amino quinoline (14) (106 mg,
0.6 mmol) and 13 (213 mg, 0.8 mmol) in methanol and dimethyl
formamide (2:1, 3 ml) was added triethylamine (0.254 ml,
1.8 mmol) and the reaction mixture heated at 135 °C for 48 h. Sat-
urated NaHCO3 was added to quench the reaction and the product
extracted with dichloromethane. The organic layer was subjected
to a usual work-up to provide the crude product. Silica gel chroma-
tography yielded the title compound 8 as a yellow liquid (35%
yield). 1H NMR (400 MHz, MeOH-d4): d 8.44 (dd, J = 4.2, 1.6 Hz,
found: 302.22235 (
D
À1.12).
4.4.4. N1,N1-Diisobutyl-N3-(6-methoxyquinolin-8-yl)propane-
1,3-diamine (8d)
Yield 35%. 1H NMR (400 MHz, CDCl3): d 8.52 (dd, J = 4.2, 2 Hz, 1H),
7.90 (dd, J = 8.4, 1.6 Hz, 1H), 7.28 (dd, J = 8.4, 4.4 Hz, 1H), 6.33 (d,

T. Ganesh et al. / Bioorg. Med. Chem. 16 (2008) 6903–6910
6909
J = 2.4 Hz, 1H), 6.30 (d, J = 2.8 Hz, 1H), 6.13 (t, J = 5.6 Hz, NH), 3.87 (s,
OCH3, 3H), 3.31 (dd, J = 7, 5.2 Hz, 2H), 2.46 (t, J = 6.8 Hz, 2H). 2.07 (d,
J = 2.4 Hz, 1H), 6.29 (d, J = 2.4 Hz, 1H), 6.13 (br, 1H), 3.89 (s, 3H),
2.48–2.58 (m, 6H), 1.77 (quintet, J = 8.0 Hz, 2H), 1.60–1.68 (m,
4H), 1.03 (t, J = 7.2 Hz, 6H). Compound 17c (5.3 mg, 27%). 1H
NMR (400 MHz, CDCl3): d 8.52 (dd, J = 1.6, 4.0 Hz, 1H), 7.91 (dd,
J = 1.6, 8.4 Hz, 1H), 7.28 (dd, J = 4.4, 8.4 Hz, 1H), 6.33 (d,
J = 2.8 Hz, 1H), 6.26 (d, J = 2.4 Hz, 1H), 6.07 (br, 1m), 3.88 (s, 3H),
3.26 (q, J = 6.8 Hz, 2H), 2.78 (septuplet, J = 6.4 Hz, 1H), 2.61 (t,
J = 6.8 Hz, 2H), 1.77 (quintet, J = 7.2 Hz, 2H), 1.46–1.59 (m, 4H),
1.03 (d, J = 6.0 Hz, 6H).
J = 7.2 Hz, 4H), 1.86(m, 2H), 1.70 (m, 2H), 0.88(d, J = 6.4 Hz, 12H). 13
C
NMR (100 MHz, CDCl3): d 159.7, 146.3, 144.5, 144.4, 135.6, 134.9,
129.9, 122.0, 96.7, 91.9, 64.2, 55.4, 53.2, 41.9, 27.2, 26.8, 21.2, 21.1.
HRMS calcd for C21H34N3O [M+H]: 344.26964; found: 344.26901
(
D
À1.83). Anal. Calcd for C21H33N3O: C, 73.43; H, 9.68; N, 12.01; ob-
served: C, 73.34; H, 9.88; N, 12.01.
4.4.5. 4-(6-Methoxyquinolin-8-ylamino)butanenitrile (15)
6-Methoxy-8-amino-quinoline (14) (1.5 mmol, 261.3 mg) and 4-
bromobutanenitrile (2.25 mmol, 333.0 mg) were refluxed in a mix-
ture of Et3N (2 ml) and MeOH (1 ml) for 24 h. More 4-bromobutane-
nitrile (2.25 mmol, 333.0 mg) was added and the mixture refluxed
for another 48 h. The mixture was concentrated to give a dark oily
residue which was purified by chromatography using hexane/EtOAc
(2:1) as eluent to afford compound 15a (148 mg, 40%). 1H NMR
(400 MHz, CDCl3): d 8.54 (dd, J = 1.6 Hz, 4.0 Hz, 1H), 7.95 (dd,
J=1.6 Hz, 8.4 Hz, 1H), 7.33 (dd, J = 8.0 Hz, 4.4 Hz, 1 H), 6.40 (d,
J = 2.8 Hz, 1H), 6.32 (d, J = 2.8 Hz, 1H), 6.20 (m, 1H), 3.90 (s, 3H),
3.49 (q, J = 6.4 Hz, 2H), 2.53 (t, J = 7.2 Hz, 2H), 2.11 (quintet,
J = 6.8 Hz, 2H).
4.6.2. 8-(5-(Diethylamino)pentylamino)quinolin-6-ol
dihydrobromide (10)
A solution of 17b (10 mg, 0.031 mmol) in HBr (1 ml, 48% aq)
was heated up to 120 °C in microwave initiator for 2.5 h. Cooled
down the reaction mixture, evaporated off the solvent by genevac
DD-4X evaporator. The resulting dark brown residue was purified
by silica gel chromatography to provide the title compound 10
(8 mg, 55%) as a pale brown solid. 1H NMR (400 MHz, DMSO-d6):
d 9.12 (br s, 1H), 8.60 (d, J = 3.9 Hz, 1H), 8.30 (d, J = 7.8 Hz, 1H),
7.56 (dd, J = 7.8, 4.6 Hz, 1H), 6.50 (s, 1H), 6.43 (s, 1H), 3.21 (t,
J = 7.0 Hz, 2H), 3.08 (quintet, J = 7.0 Hz, 4H), 3.03–2.98 (m, 2H),
1.74–1.62 (m, 4H), 1.47-1.39 (m, 2H), 1.16 (t, J = 7.0 Hz, 6H). 13C
NMR (100 MHz, DMSO-d6): d 158.5, 143.9, 142.0, 133.0, 132.9
130.7, 122.5, 100.4, 92.4, 54.5, 51.3, 46.8, 28.1, 24.4, 23.5, 9.2,
9.1. EI, m/z (C18H27N3O): calcd, 301.4; found: 302.3 (M+H). Anal.
Calcd for C18H29Br2N3OÁH2O: C, 44.92; H, 6.49; N, 8.73; observed:
C, 45.50; H, 6.26; N, 8.59.
4.5. Similar procedure was employed to make 15b
4.5.1. Compound 15b
Yield 41% . 1H NMR (400 MHz, CDCl3): d 8.54 (dd, J = 1.6 Hz,
4.4 Hz, 1H), 7.94 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 7.32 (dd, J = 4.0 Hz,
8.4 Hz, 1H), 6.37 (d, J = 2.8 Hz, 1H), 6.29 (d, J = 2.8 Hz, 1H), 6.12
(m, 1H), 3.90 (s, 3H), 3.35 (q, J = 6.0 Hz, 2H), 2.42 (t, J = 7.2 Hz,
2H), 1.84-1.95 (m, 4H). Anal. Calcd for C15H17N3O: C, 70.56; H,
6.71; N, 16.46; Found: C, 70.63; H, 6.75; N, 16.48.
4.7. Similar procedure was employed to get 11 and 18a
4.7.1. 8-(5-(Isopropylamino)pentylamino)quinolin-6-ol (11)
(5.9 mg, 47%) 1H NMR (400 MHz, MeOH-d4): d 8.59–8.62 (m,
2H), 7.71 (dd, J = 5.2, 8.4 Hz, 1H), 6.70 (s, 1H), 3.22–3.29 (m, 3H),
2.94 (t, J = 8.0 Hz, 2H), 1.79 (quintet, J = 7.6 Hz, 2H), 1.68 (quintet,
J = 8.0 Hz, 2H), 1.53-1.59 (m, 2H), 1.22 (d, J = 6.8 Hz, 6H).
4.5.2. N1,N1-Diethyl-N4-(6-methoxyquinolin-8-yl)butane-1,4-
diamine (17b)
To a solution of compound 15a (1.0 mmol, 1 equiv, 255.31 mg)
in THF (5 ml) was added LAH (2.0 mmol, 2 equiv, 1.0 M in THF)
dropwise at À78 °C. Reaction mixture was kept at this temperature
for an additional 2 h, which was then allowed to slowly warm up to
room temperature. The reaction was quenched by addition of NaH-
CO3 (satd aq), extracted by EtOAc (3Â 5 ml), combined the organic
layer and washed with brine. Dried over Na2SO4 and evaporated off
the solvent to obtain crude compound 16b as an oily residue,
which was subjected to the next step without further purification.
To a solution of crude compound 16b in 5 ml of CH2Cl2 at 0 °C was
HRMS
288.20691(M+1).
calcd
for
C
17H25N3O:
287.19976;
observed:
4.7.2. 8-(4-(Diethylamino)butylamino)quinolin-6-ol (18a)
1H NMR (400 MHz, MeOH-d4): d 8.36 (dd, J = 1.6 Hz, 4.0 Hz, 1H),
7.83 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 7.23 (dd, J = 4.4 Hz, 8.4 Hz, 1H),
6.27 (d, J = 2.0 Hz, 1H), 6.24 (d, J= 2.4 Hz, 1H), 3.08–3.16 (m, 6H),
1.76–1.83 (m, 4H), 1.20 (t, J = 7.6 Hz, 6H). MS, m/z (C17H25N3O):
calcd, 287.20; found: 288.40 (M+H).
added acetaldehyde (85 lL, 1.52 mmol). Reaction mixture was
Acknowledgments
kept at 0 °C for about 30 min, and then NaBH(OAc)3 (350 mg,
1.65 mmol) was added to the above mixture. The resulting solution
was allowed to warm to room temperature for another 3 h. The
mixture was diluted with CH2Cl2 (30 ml) and sequentially washed
with 10% NaHCO3 and brine. Drying over sodium sulfate followed
by filtration and concentration provided a residue that was puri-
fied by silica gel chromatography to obtain the product 17b
(90 mg, 43%). 1H NMR (400 MHz, MeOH-d4): d 8.61 (dd, J = 3.9,
1.6 Hz, 1H), 8.13 (dd, J = 8.6, 1.6 Hz, 1H), 7.36 (dd, J = 8.6, 3.9 Hz,
1H), 6.86 (d, J = 2.3 Hz, 1H), 6.83 (d, J = 2.3 Hz, 1H), 3.88 (s, 3H),
3.37 (quartet, J = 7.0 Hz, 2H), 2.47 (quartet, J = 7.0 Hz, 4H), 2.35 (t,
J = 7.8 Hz, 2H), 1.55–1.48 (m, 2H), 1.41-1.33 (m, 2H), 1.26-1.21
(m, 2H), 0.96 (t, J = 7.0 Hz, 6H). MS, m/z (C19H29N3O): calcd,
315.2; found, 316.4 (MH).
This work was financially supported by the US National Insti-
tutes of Health 1 U54 HG003918-02 and 1R03MH076499-01. We
also gratefully acknowledge National Cancer Institute (NCI) for
the generously supplying the samples listed in Figure 3.
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