Synthesis and biological evaluation of coumarin replacements of novobiocin as Hsp90 inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.12.056

Since Hsp90 modulates all six hallmarks of cancer simultaneously, it has become an attractive target for the development of cancer chemotherapeutics. In an effort to develop more efficacious compounds for Hsp90 inhibition, novobiocin analogues were prepared by replacing the central coumarin core with naphthalene, quinolinone, and quinoline surrogates. These modifications allowed for modification of the 2-position, which was previously unexplored. Biological evaluation of these compounds suggests a hydrophobic pocket about the 2-position of novobiocin. Anti-proliferative activities of these analogues against multiple cancer cell lines identified 2-alkoxyquinoline derivatives to exhibit improved activity.

Full text of DOI:10.1016/j.bmc.2013.12.056

Bioorganic & Medicinal Chemistry 22 (2014) 1441–1449
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of coumarin replacements
of novobiocin as Hsp90 inhibitors
Bhaskar Reddy Kusuma a, Anuj Khandelwal a, Wen Gu a, Douglas Brown b, Weiya Liu b, George Vielhauer b,
Jeffrey Holzbeierlein b, Brian S. J. Blagg a,
⇑
a Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, Malott Hall 4070, Lawrence, KS 66045-7563, USA
b Department of Urology, The University of Kansas Medical Center, 3901 Rainbow Blvd., Mail Stop 1016, Kansas City, KS 66160, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Since Hsp90 modulates all six hallmarks of cancer simultaneously, it has become an attractive target for
the development of cancer chemotherapeutics. In an effort to develop more efficacious compounds for
Hsp90 inhibition, novobiocin analogues were prepared by replacing the central coumarin core with naph-
thalene, quinolinone, and quinoline surrogates. These modifications allowed for modification of the
2-position, which was previously unexplored. Biological evaluation of these compounds suggests a
hydrophobic pocket about the 2-position of novobiocin. Anti-proliferative activities of these analogues
against multiple cancer cell lines identified 2-alkoxyquinoline derivatives to exhibit improved activity.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 19 November 2013
Revised 9 December 2013
Accepted 21 December 2013
Available online 3 January 2014
Keywords:
Hsp90
Anti-cancer
Chaperone
Novobiocin
1. Introduction
inhibited by the natural products radicicol and geldanamycicn.17
The C-terminal region contains a dimerization motif that modu-
Heat shock protein 90 (Hsp90) is one of the most abundant
molecular chaperones in the cell, is highly conserved in both
eukaryotes and prokaryotes, and plays a key role in protein homeo-
stasis.1,2 Hsp90-dependent proteins are responsible for cellular
growth, signaling, differentiation, survival and protection against
misfolded proteins. More than 200 client proteins are dependent
upon Hsp90 for activation and/or stability; a quarter of which
are directly associated with cell growth or signaling.3 Several
Hsp90-dependent clients are found in pathways that are
commonly mutated or hijacked during oncogensis.4 Consequently,
Hsp90 is overexpressed in cancer cells to maintain cellular homeo-
stasis under such environments and to serve as a buffer within
tumor cells.5,6 Since Hsp90 modulates multiple pathways simulta-
lates client protein release. N-Terminal inhibitors have been widely
studied in both academia and the pharmaceutical industry, and
some have advanced to clinical trials.18 In contrast, the C-terminal
inhibitors have not advanced into clinical investigations thus far,
and the most significant reason is likely the absence of a co-crystal
structure bound to an inhibitor.19 The development of more effica-
cious C-terminal inhibitors is desired to better understand the
ramifications of C-terminal inhibition and to probe the mechanism
by which Hsp90 interacts with client proteins. As an anti-cancer
agent, Hsp90 C-terminal inhibitors manifest a distinct advantage
over N-terminal inhibitors, as they do not induce the pro-survival
heat shock response, which is a deleterious consequence of N-ter-
minal inhibition.20 Consequently, the development of the Hsp90
C-terminal inhibitors represents an intense area of research.21–26
Novobiocin is a potent inhibitor of bacterial DNA gyrase and
was also identified as the first Hsp90 C-terminal inhibitor along-
side chlorobiocin and coumermycin (Fig. 1).27–29 However, due to
neously, its inhibition produces
a mechanism to overcome
resistance that results from the inhibition of a single protein.7–9
Therefore, inhibition of Hsp90 for the treatment of cancer
represents a powerful paradigm for drug development.10–12
Structurally, Hsp90 exists as a homodimer and possesses two
nucleotide binding regions: The N-terminal ATP-binding pocket
is required for ATPase activity and produces the requisite energy
for client protein maturation.13–15 The C-terminal nucleotide bind-
ing pocket exhibits allosteric control over both substrates and the
N-terminal ATP-binding site.16 The N-terminal ATP binding site is
its low efficacy against cancer cells (IC50 ꢀ 700
lM), it was consid-
ered unsuitable for further evaluation as an Hsp90 inhibitor. Sub-
sequent studies led to identification of some structure–activity
relationships for novobiocin that resulted in KU-174 and DHN1,
which manifest low micromolar activity. Additional studies led to
compounds that exhibit greater potency and solubility than novo-
biocin and contained surrogates of the stereochemically complex
noviose sugar as well as structural modifications to the benzamide
side chain.23,30–33 The improved analogues exhibit mid nanomolar
inhibitory activity against several cancer cell lines, while simulta-
⇑
Corresponding author. Tel.: +1 (785) 864 2288; fax: +1 (785) 864 5326.
E-mail address: bblagg@ku.edu (B.S.J. Blagg).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.056

1442
B. R. Kusuma et al. / Bioorg. Med. Chem. 22 (2014) 1441–1449
OH
OH
H
N
OH
OH
O
H
N
O
O
O
O
Cl
O
O
O
O
MeO
O
O
OH
MeO
H2N
O
O
OH
NH
Clorobiocin
O
Novobiocin
NH
O
OH
O
H
N
O
OH
NH
HN
O
O
O
O
O
O
O
HO
O
OMe
O
MeO
O
Coumermycin A1
O
OH
OH
OMe
HN
H
H
N
N
OMe
O
O
O
O
O
O
O
O
OMe
O
O
MeO
MeO
HO
HO
KU-174
DHN1
OH
OH
Figure 1. Hsp90 C-terminal inhibitors.
neously manifesting increased solubility.25,26,34 In contrast to the
sugar and amide termini of novobiocin, limited structure–activity
relationship studies have been conducted on the central coumarin
core.32 Therefore, structure–activity relationship studies for the
coumarin core were pursued in an effort to develop more potent
inhibitors and to explore this region for improved inhibitory
activity.
Construction of these novobiocin analogues were begun by
replacing the central coumarin core with naphthalene, quinolinone
and quinoline surrogates. Such motifs are abundant in natural
products such as quinine, campothecin and cinchonidine, which
manifest broad biological activities including anticancer, antimy-
cobacterial, antimicrobial, anticonvulsant, anti-inflammatory and
cardiovascular activities.35–41 The synthesis and structure–activity
relationships of naphthalene, quinolinone, and quinolone replace-
ments of the novobiocin coumarin core are presented in this
article.
coupling agent. Addition of noviose could then be achieved via the
coupling of the trichloroacetimidate of noviose carbonate with
the phenol. The naphthalene core could be prepared from commer-
cially available 6-bromonaphthalen-2-ol (9) and both the quinoli-
none or quinoline cores were envisioned for construction from
commercially available 2-methyl-3-nitrophenol (8).
Synthesis of the naphthalene-containing analogues commenced
by introduction of a methyl group at the 2-position of commer-
cially available 6-bromonaphthalen-2-ol (9, Scheme 1) via a
three-step procedure. Reductive amination of 9 with formaldehyde
and N,N-dimethylamine followed by acylation of the phenol and
subsequent reductive cleavage of N,N-dimethylamine furnished
6-bromo-1-methylnaphthalen-2-ol (10), in quantitative yield.42
The phenol was then protected as the methoxymethyl ether
(MOM) to give compound 11 (Scheme 1) for use in subsequent
steps.
EDCI coupling
OH
2. Results and discussion
H
N
2.1. Design of new novobiocin analogues
O
O
R
N
R1
X, R1 = H
Modifications to three regions of novobiocin were pursued to
reveal structure-activity relationships and to provide more effica-
cious compounds; (1) replacement of the central coumarin core
with naphthalene, quinoline and quinolinone scaffolds were inves-
tigated, (2) modifications to the 2-hydroxyl group of the corre-
sponding 2-quinolinol tautomer ring system were prepared, and
(3) modifications to the benzamide side chain were also pursued.
Both, noviose (3) and the sugar surrogate N-methyl-4-piperidine,
were chosen based upon prior studies with novobiocin. Similarly,
the side chain was replaced with an optimized prenylated or biaryl
side chain for further investigation of these new scaffolds. As
shown in Figure 2, analogues containing the prenylated benzamide
side chain were assembled via three components; (1) a central
bicyclic aryl core (naphthalene or quinoline or quinolinone
scaffold), (2) a prenylated benzoic acid 4, and (3) noviose. The
prenylated acid (4) could be coupled with the amino group enlist-
ing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) as the
X = N; R1 = H, OH, OEt, OnBu, OCH2CH2Ph
OH
+
O
MeO
NH2
R1
OAc
+
O
HO
O
3
HO
N
O
O
4
1
, R1 = H
2, R1 = OH
Ph
HO
N
R1
HO
NO2
MeO
N
H
O
5
6
7
8
, R1 = H; quinoline
, R1 = OH; quinoline-2-ol
Figure 2. Retrosynthetic analysis of novobiocin analogues.

B. R. Kusuma et al. / Bioorg. Med. Chem. 22 (2014) 1441–1449
1443
Br
Br
Br
iPrEt2N, MOMCl
rt
1. NH(CH3)2, CH2O
HO
MOMO
2. AcCl
3. NaBH4
HO
9
10
11
Scheme 1.
O
Ph
1. K2CO3/MeI
rt, 3 h
Cl
Ph
AlCl3
pyridine
BnO
120 oC, 3 h
HO
NO2
MeO
NH2
MeO
N
H
O
HO
N
O
2. Pd/C, H2
H
8
12
7
6
Br
K2CO3/BnBr
rt, 3 h
K2CO3/RX
O
rt, 3 h
NBS, rt, 3h
BnO
N
H
N
OR
BnO
N
OR
13
14, R = Bn
15, R = Et
16, R = nBu
18, R = Bn
19, R = Et
RX = BnBr
CH3CH2I
20, R = nBu
CH3CH2CH2CH2I
PhCH2CH2I
17, R = CH2CH2Ph
21, R = CH2CH2Ph
Scheme 2.
Construction of the quinolinone- and quinolone-containing
analogues was accomplished via methylation of 2-hydroxy-3-
nitrophenol using potassium carbonate and methyl iodide, fol-
lowed by reduction of the nitro group to afford the corresponding
aniline, 12 (Scheme 2). Treatment of 12 with trans-cinnamoyl chlo-
ride gave amide 7, which underwent intramolecular cyclization
upon addition of aluminum chloride in chlorobenzene at 120 °C
to give 8-methylquinoline-2,7-diol (6) in high yield.43 It is note-
worthy that purification was not required until this point, as prior
steps gave compounds in exceptional yields and purity. To incorpo-
rate alkyl ethers at the 2-position, the 7-phenol was selectively
protected as the corresponding benzyl ether by the use of one
equivalent of benzyl bromide and potassium carbonate. Taking
advantage of the dynamic equilibrium that exists between the
quinolone (6) and its quinolinol isomer, various hydrophobic
groups were introduced at the 2-position. Treatment of compound
13 with alkyl halides in the presence of potassium carbonate affor-
ded the corresponding alkyl ethers, 14–17, which were exposed to
N-bromosuccinimide to give the brominated compounds, 18–21,
in excellent yields (Scheme 2). The brominated compounds
(11, 18–21) served as precursors for introduction of the amine
and subsequent attachment of the benzamide side chain.
different conditions (Scheme 4).45,46 Compounds 11, 18–21, 23,
24, were heated in a sealed tube with copper nanoparticles, azido-
trimethyl silane, and amino ethanol in a solution of dimethyl acet-
amide to give anilines 25–31.46 The protecting groups on
compounds 27–29 were exchanged from benzyl ethers to silyl
ethers by hydrogenolysis on palladium/carbon and subsequent
O-silylation with tert-butyldimethylsilyl chloride (TBSCl), which
gave anilines 32–34. Anilines 25, 26, 31–34, were readily coupled
with benzoic acid 4 enlisting N-(3-(dimethylamino)propyl)-N0-eth-
ylcarbodiimide hydrochloride (EDCI) in a solution of pyridine and
dichloromethane to give the corresponding amides, 35–40.47
Deprotection of the benzyl or methoxy methyl or tert-butyldi-
methyl silyl groups of 35–40 was readily achieved with freshly
prepared lithium di-tert-butyl biphenyl (LiDBB) in THF, 4 N HCl,
or 1.0 M tetrabutylammonium fluoride, respectively. The free phe-
nols of 41–46 were provided without modification of the olefinic
side chain (Scheme 4).48,49
Upon construction of the phenols, the noviose appendage
was attached via published procedures.50 Phenols 41–46 were
noviosylated with the trichloroacetimidate of noviose carbonate,
47, in the presence of boron trifluoride etherate to provide the
corresponding carbonylated noviose products, which upon
solvolysis with methanolic triethylamine afforded 48–53 in
good yields (Scheme 5).
For direct comparison, quinoline analogues were also pursued.
Preparation of quinoline precursors 23 and 24 began with quino-
linol intermediate 13, which upon heating in the presence of phos-
phorous oxychloride, and subsequent hydrogenation gave the
quinoline intermediate
5
as
a
yellow amorphous solid
1.Boc2O, DMAP
1. POCl3, reflux
(Scheme 3).44 The phenolic moiety of 5 was then protected as
the tert-butyl carbonate to provide steric hindrance and enable
regioselective bromination. In the event, bromination of 5 was
achieved upon the addition of bromine to a solution of 5 in carbon
tetrachloride and pyridine.32 Subsequent addition of methanolic
HCl led to cleavage of the carbonate and formation of the corre-
sponding phenol 22, which was then modified with benzyl bro-
mide or methoxymethyl chloride or to give intermediates 23 and
24, respectively.
rt, 2 h
2 h
2. Br2, pyridine
reflux
BnO
HO
N
H
O
HO
N
2. Pd/C, H2
Et3N, EtOH, 1 h
5
13
3. HCl in MeOH
50 oC, 1 h
NaH/BnBr
Br
Br
DMF, rt, 3 h
(or)
iPrEt2N, MOMCl
rt
N
RO
N
22
23, R = Bn
Introduction of the benzamide side chain was accomplished by
utilization of the brominated derivatives, 11, 18–21, 23, 24 which
were transformed into the corresponding anilines, 25–31, via two
24, R = MOM
Scheme 3.

1444
B. R. Kusuma et al. / Bioorg. Med. Chem. 22 (2014) 1441–1449
Cu(0), NaN3
Pipecolic acid
Ascorbic acid
Br
R
NH2
R
100 oC, 3 h
R1O
X
R1O
X
or
Cu(0), TMSN3
H2NCH2CH2OH
95 oC, 3 h
11, X = CH, R = H, R1 = MOM
18, X = N, R = OBn, R1 = Bn
19, X = N, R = OEt, R1 = Bn
20, X = N, R = OnBu, R1 = Bn
25, X = CH, R = H, R1 = MOM
26, X = N, R = OBn, R1 = Bn
27, X = N, R = OEt, R1 = Bn
28, X = N, R = OnBu, R1 = Bn
29, X = N, R = OCH2CH2Ph, R1 = Bn
30, X = N, R = H, R1 = Bn
21, X = N, R = OCH2CH2Ph, R1 = Bn
23, X = N, R = H, R1 = Bn
24, X = N, R = H, R1 = MOM
31, X = N, R = H, R1 = MOM
NH2
NH2
1. Pd/C, H2
4 h
BnO
N
OR
TBSO
N
OR
2.TBSCl, imidazole
rt, 2 h
27, R = Et
28, R = nBu
29, R = CH2CH2Ph
32, R = Et
33, R = nBu
34, R = CH2CH2Ph
OAc
OR2
H
N
H
N
4, EDCI,
NH2
R
pyridine
a (or) b (or) c
O
O
rt, 15 h
R1O
X
R
R1O
X
R
R1O
X
25, X = CH, R = H, R1 = MOM
26, X = N, R = OBn, R1 = Bn
31, X = N, R = H, R1 = MOM
32, X = N, R = OEt, R1 = TBS
33, X = N, R = OnBu, R1 = TBS
34, X = N, R = OCH2CH2Ph, R1 = TBS
35, X = CH, R = H, R1 = MOM
36, X = N, R = OBn, R1 = Bn
37, X = N, R = H, R1 = MOM
38, X = N, R = OEt, R1 = TBS
39, X = N, R = OnBu, R1 = TBS
40, X = N, R = OCH2CH2Ph, R1 = TBS
35, X = CH, R = H, R1 = MOM, R2 = OAc
41, X = CH, R, R1 = H, R2 = OAc
36, X = N, R = OBn, R1 = Bn, R2 = OAc
42, X = N, R = OH, R1 = H, R2 = H
37, X = N, R = H, R1 = MOM, R2 = OAc
43, X = N, R, R1 = H, R2 = OAc
38, X = N, R = OEt, R1 = TBS, R2 = OAc
44, X = N, R = OEt, R1 = H, R2 = OAc
41, X = N, R = nBu, R1 = TBS, R2 = OAc
45, X = N, R = nBu, R1 = H, R2 = OAc
40, X = N, R = OCH2CH2Ph, R1 = TBS, R2 = OAc
46, X = N, R = OCH2CH2Ph, R1 = H, R2 = OAc
ii
iii
ii
i
Conditions: a) TBAF, rt, 15 min.
b) 4N HCl in dioxane, rt, 15 min.
c) LiDBB, - 78 oC, 30 min.
i
i
Scheme 4.
OH
H
N
R2
H
1. 47, BF.OEt2
N
rt
O
X
R
O
2. Et3N, rt, 12h
O
HO
X
R
O
MeO
HO
NH
CCl3
47
41, X = CH, R = H, R2 = OAc
42, X = N, R, R2 = OH
43, X = N, R = H, R2 = OAc
44, X = N, R = OEt, R2 = OAc
45, X = N, R = nBu R2 = OAc
OH
48, X = CH, R = H
49, X = N, R = OH
50, X = N, R = H
51, X = N, R = OEt
52, X = N, R = OnBu
53, X = N, R = OCH2CH2Ph
O
MeO
O
O
O
O
46, X = N, R = OCH2CH2Ph, R2 = OAc
Scheme 5.
2.2. Biological evaluation of prenylated benzamide side chain
containing coumarin surrogates
compounds, which confirms the lactone is not required for Hsp90
inhibition. These analogues also manifested comparable activities
to DHN1 and DHN2 and manifested ꢀ100-fold greater inhibitory
activity than the natural product, novobiocin.31 Quinoline analogue
Uponconstructionof analogues48–53, evaluation of anti-prolifer-
ative activity against SKBr3 (estrogen receptor negative, Her2 over-
expressing breast cancer cells), MCF-7 (estrogen receptor positive
breast cancer cells), LNCaP (androgen-sensitive human prostate ade-
nocarcinoma), 253JB–V (human bladder cancer), and PC3-MM2
(androgen receptor insensitive prostate cancer) cell lines were inves-
tigated. Anti-proliferative activity of noviose containing analogues is
shown in Table 1. Analogues lacking the coumarin core (49–50) man-
ifested comparable activity to the corresponding coumarin-derived
50 was found to be more efficacious (IC50 = 7.22
lM against SKBr3
and IC50 = 0.26 M against PC3 cell lines) than the corresponding
l
quinolinone (49) and naphthalene (48) analogues. Introduction
of steric bulk at the 2-position of the quinolinol (51–53) revealed
the ethoxy group (51) to be most active against both SKBr3
(IC50 = 1.94 lM) and MCF-7 (IC50 = 2.48 lM) cell lines. Given that
analogue 51 (O–Et) manifested ꢀ7-fold greater activity than analogue
53 (O–CH2CH2Ph) suggests that sterics are moderately tolerated.

B. R. Kusuma et al. / Bioorg. Med. Chem. 22 (2014) 1441–1449
1445
Table 1
Anti-proliferative activities of compounds 48–53
OH
H
N
O
O
X
R1
O
MeO
HO
OH
Entry
X
R1
SKBr3 (IC50
,
lM)
MCF-7 (IC50
,
lM)
PC3-MM2 (IC50
,
l
M)
LNCaP (IC50
,
l
M)
253JB-V (IC50, lM)
48
49
50
51
52
53
CH
N
N
N
N
H
OH
H
10.21 0.84
11.13 0.66
7.22 1.07
1.94 0.44
10.26 2.26
14.14 2.46
12.57 1.4
NT
NT
NT
NT
5.16 0.66
7.37 0.53
NT
NT
NT
NT
8.48
12.47
8.04 1.17
9.68 2.01
2.48 0.03
3.98 0.71
14.54 0.33
4.91 .87
0.258 0.07
5.75 0.34
8.48 2.62
12.47 0.07
OCH2CH3
OCH2(CH2)2CH3
OCH2CH2Ph
N
10.09 0.43
After evaluation of the analogues that contain the prenylated
benzamide side chain, the previously optimized biaryl acid, 55,
was incorporated to further investigate the activity of these scaf-
folds. Previous studies have shown that analogues possessing a
biaryl benzamide side chain manifest anti-proliferation activities
comparable to analogues containing the prenylated benzamide.
The biaryl derivatives can be readily prepared by a Suzuki coupling
reaction between commercially available boronic acids and the
corresponding aryl iodide, followed by hydrolysis of the ester. As
shown in Figure 3, the retrosynthesis of biaryl amide analogues
follows a similar method as that used to prepare prenylated
benzamide derivatives. The biaryl amide analogues were assembled
from three components, the central core containing naphthalene,
quinoline or quinolinone scaffold, the biaryl acid (55), and the
noviose sugar (3) or its surrogate 1-methylpiperidin-4-ol (54).
Synthesis of these biaryl analogues began with a N-(3-(dimethyl-
amino)propyl)-N0-ethylcarbodiimide (EDCI)-mediated coupling of
aniline intermediates 26–31 with biaryl acid 55 to give the corre-
sponding amides, which upon cleavage of the benzyl or methoxy
methyl groups gave the free phenols, 62–68 (Scheme 6). The resulting
EDCI coupling
OMe
H
N
OMe
O
O
N
O
n = 2 or 3
R1
R1 = alkyl or aminoalkyl groups
n
R
novisolyation
or
Mitsunobu
coupling
OH
+
O
MeO
OMe
NH2
R1
O
O
(or)
+
3
HO
OMe
O
HO
N
55
O
1, R1 = H
N
OH
2
, R1 = OH
Ph
54
MeO
HO
NO2
HO
6, R1 = H; quinoline
N
H
8
O
N
R1
9
7
, R1 = OH; quinoline-2-ol
Figure 3. Retrosynthetic analysis of biaryl amide-containing analogues.
OMe
OMe
H
H
N
Pd/C, H2
55, EDCI
NH2
R
N
OMe
OMe
or
pyridine
O
O
4N HCl in
dioxane
rt, 15 h
R1O
X
R1O
X
R
HO
X
R
26, X = CH, R = H, R1 = MOM
27, X = N, R = OBn, R1 = Bn
28, X = N, R = OEt, R1 = Bn
29, X = N, R = OnBu, R1 = Bn
30, X = N, R = OCH2CH2Ph, R1 = Bn
31, X = N, R = H, R1 = Bn
56, X = CH, R = H, R1 = MOM
57, X = N, R = OBn, R1 = Bn
58, X = N, R = OEt, R1 = Bn
59, X = N, R = OnBu, R1 = Bn
60, X = N, R = OCH2CH2Ph, R1 = Bn
61, X = N, R = H, R1 = Bn
62, X = CH, R = H,
63, X = N, R = OH,
64, X = N, R = OBn
65, X = N, R = OEt
66, X = N, R = OnBu
67, X = N, R = OCH2CH2Ph
68, X = N, R = H
Scheme 6.

1446
B. R. Kusuma et al. / Bioorg. Med. Chem. 22 (2014) 1441–1449
OMe
OMe
H
N
H
N
OMe
OMe
1. 48, BF3.OEt2
rt
O
O
O
X
R
HO
X
R
2. Et3N, rt, 12h
O
MeO
62, X = CH, R = H,
63, X = N, R = OH,
65, X = N, R = OEt
66, X = N, R = OnBu
67, X = N, R = OCH2CH2Ph
68, X = N, R = H
69, X = CH, R = H,
70, X = N, R = OH
71, X = N, R = OEt
72, X = N, R = OnBu
73, X = N, R = OCH2CH2Ph
74, X = N, R = H
HO
OH
Scheme 7.
Table 2
Anti-proliferative activities of compounds 69–74, 76–79, 81, 83, 85, 86
OMe
H
N
OMe
O
O
X
R
O
MeO
HO
OH
Entry
X
R
SKBr3 (IC50
,
lM)
MCF-7 (IC50
,
lM)
PC3-MM2 (IC50
,
lM)
LNCaP (IC50
,
l
M)
253JB-V (IC50, lM)
69
70
71
72
73
74
76
77
78
79
81
83
85
86
CH
N
N
N
N
N
N
N
N
N
N
N
N
N
H
OH
38.1 2.2
56.8 10.1
13.5 1.13
8.174 0.22
>100
9.1 0.12
1.13 0.02
0.21 0.04
1.02 0.21
26.40 4.2
0.54 0.24
4.58 0.34
0.12 0.04
0.56 0.21
25.7 3.7
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
1.12 0.40
7.42 0.1
1.79 0.07
0.73 0.7
0.99 0.01
1.89 0.09
11.87 0.72
13.8 0.82
11.27 0.53
77.41 5.4
10.30 0.17
2.09 0.31
0.41 0.04
1.41 0.03
44.14 2.7
0.72 0.41
5.45 1.1
OCH2CH3
OCH2(CH2)2CH3
OCH2CH2Ph
H
O-a
O-d
O-f
6.05 1.9
5.487 1.1
29.76 2.5
NT
3.95 0.04
0.68 0.2
1.41 0.01
14.12 1.3
1.78 0.4
2.08 0.2
1.33 0.36
1.54 0.31
4.71 2.4
3.768 0.2
22.52 8.9
NT
1.798 0.0
0.40 0.19
1.67 0.07
13.20 0.0
1.88 0.16
5.49 5.6
1.26 0.07
2.39 0.77
O-Cyclohexyl
O-g
O-b
O-c
O-h
0.56 0.12
1.21 0.39
phenols were coupled with the trichloroacetimidate of noviose car-
bonate (48) in the presence of boron trifluoride etherate, followed
by solvolysis of the cyclic carbonate to afford 69–74 (Scheme 7).50
Evaluation of the anti-proliferative activities produced by ana-
logues containing the biaryl benzamide moiety (69–74) against
multiple cancer cell lines supported the nitrogen atom to be bene-
ficial at the 1-position, as naphthalene analogues 69 and 70 were
found to be 4- to 6-fold less active than corresponding quinoline
analogue, 74 (Table 2). Increased steric bulk at the 2-position
(71–73) was tolerated to some extent as both the ethyl and n-butyl
ether manifested potency comparable to the unsubstituted ana-
logue 74, however, introduction of bulkier groups (73) led to de-
creased potency.
NH2
Br
N
N
c
d
a
b
N
N
N
N
H
h
e
g
f
Figure 4. Selected alkyl amines and alkyl groups for probing the 2-position.
followed by solvolysis of the carbonate to give coupled products,
76–80 (Scheme 8). Hydrogenolysis of the benzyl group on 80
afforded the secondary-amino analogue, 81. Treatment of 75 with
dibromopropane in the presence of potassium carbonate, followed
by solvolysis of the cyclic carbonate gave intermediate 83, which
upon subsequent nucleophilic displacement with amines gave
compounds 84 and 86, and subsequent hydrogenolysis of 84 gave
85.
Biological evaluation of analogues containing aminoalkyl sub-
stitutions at the 2-position revealed these substitutions to be ben-
eficial as the majority of analogues manifested low micromolar to
nanomolar activities against multiple cancer cell lines (Table 2).
Their efficacy was greater than compounds containing the alkyl
substituents (71–73 & 79). The two most potent analogues from
2.3. Synthesis of noviose analogues containing alkyl or alkyl
amine at the 2-position
Biological evaluation of quinoline containing analogues with the
biaryl and prenylated side chains suggested that the binding pocket
at the 2-position could be further probed for additional interac-
tions, and perhaps greater inhibitory activity. Therefore, amines
containing a 2- or 3-carbon linker consistent with the 2-ethoxy
group were chosen for investigation (Fig. 4). In addition, 2-bromo-
ethoxy and the cyclohexyl ether of 2-phenol were prepared.
Analogues containing the alkyl side chains (a–h, Fig. 4) at the
2-position were prepared by coupling the noviosylated intermedi-
ate 75 with various alcohols using standard Mitsunobu conditions
this series were 77 (IC50 = 0.21 0.04
and 85 (IC50 = 0.12 0.04 M against SKBr3 cell line), which
contain a 3-carbon linker between the quinolinol oxygen and the
lM against SKBr3 cell line)
l

B. R. Kusuma et al. / Bioorg. Med. Chem. 22 (2014) 1441–1449
1447
OMe
OMe
H
N
H
N
OMe
OMe
O
1. ROH, DIAD
O
O
N
OH
75
O
N
OR
PPh3, rt
O
O
2. Et3N, rt, 12h
MeO
MeO
O
HO
O
OH
N
76, R =
78, R =
77, R =
79, R =
N
N
O
N
80, R =
Pd/C, H2
Bn
81, R =
N
H
OMe
OMe
H
N
H
N
K2CO3
OMe
OMe
Br(CH2)3Br
rt, 2 h
Et3N, rt, 12 h
MeO
75
O
O
O
O
N
O
O
O
N
O
O
MeO
Br
Br
82
83
O
HO
O
OH
OMe
O
H
N
OMe
Pd/C, H2
84, R1 = -NHBn
85, R = -NH2
R1R2NH, rt, 12h
MeO
O
N
O
N
N
86, R =
O
R1
84
HO
OH
Scheme 8.
OMe
OMe
H
N
H
OMe
N
OMe
4, DIAD, PPh3
rt, 2 h
O
O
O
N
X
R
HO
X
R
87, X = CH, R = H,
88, X = N, R = OH
89, X = N, R = OEt
90, X = N, R = OnBu
62, X = CH, R = H,
63, X = N, R = OH,
65, X = N, R = OEt
66, X = N, R = OnBu
67, X = N, R = OCH2CH2Ph
68, X = N, R = H
91, X = N, R = OCH2CH2Ph
92, X = N, R = H
OMe
OMe
ROH, DIAD,
H
H
N
N
OMe
OMe
PPh3, rt, 2 h
O
O
RO
N
OR
HO
N
OH
63
93, R = -CH2CH2CH2N(Me)2
N
94, R =
OMe
OMe
H
N
H
N
OMe
OMe
1. K2CO3,
BrCH2CH2CH2Br
BnO
1. Pd/C, H2
24 h
O
O
O
N
N
O
N
O
64
2. DMF, rt
2. 4, DIAD, PPh3
rt, 2 h
96
95
HN
N
N
N
N
N
OMe
OMe
H
N
H
N
OMe
OMe
N
OH
1. Pd/C, H2
24 h
O
O
BnO
N
O
O
N
N
O
64
DIAD, PPh3
rt, 2 h
2. 4, DIAD, PPh3
rt, 2 h
97
98
N
N
Scheme 9.

1448
B. R. Kusuma et al. / Bioorg. Med. Chem. 22 (2014) 1441–1449
Table 3
Anti-proliferative activities of analogues 87–94, 104, 96, 98
OMe
H
N
OMe
O
RO
X
R1
Entry
X
R1
R
SKBr3 (IC50
,
l
M)
MCF-7 (IC50
,
lM)
PC3-MM2 (IC50
,
lM)
LNCaP (IC50
,
l
M)
253JB-V (IC50, lM)
87
88
89
90
91
92
93
94
96
98
CH
N
N
N
N
N
N
N
N
N
H
OH
f
f
f
f
f
f
d
f
f
f
2.32 0.61
1.30 0.08
1.86 0.27
1.06 0.02
1.89 0.4
1.14 0.21
0.34 0.21
0.58 0.24
0.64 0.31
0.59 0.05
1.39 0.1
1.43 0.19
1.97 0.17
1.72 0.33
3.19 0.6
1.26 0.09
0.49 0.17
0.57 0.14
0.67 0.42
1.58 0.16
NT
NT
>100
2.23 0.02
2.46 0.37
1.93 0.1
1.75 0.13
1.74 0.14
1.75 0.1
2.4 0.12
NT
NT
>100
2.06 0.06
2.37 0.04
NT
1.86 0.03
1.9 0.07
1.79 0.2
2.6 1.10
NT
NT
NT
NT
NT
NT
1.39 0.17
NT
1.30 0.15
NT
OCH2CH3
OCH2(CH2)2CH3
OCH2CH2Ph
H
O-d
O-f
O-h
O-d
basic amine. Increased steric bulk as found in 86 resulted in de-
creased activity. Direct comparison of the 4-piperidine containing
analogue, 78, with that containing a cyclohexane ring (79) suggests
that the presence of charged group can provide improved activity.
Overall, these results indicate that an amine attached to a 3-carbon
linker is optimal at the 2-position. Replacement of terminal pri-
mary-amine (76) with a tertiary-amine (77) resulted in increased
efficacy against multiple cell lines. In summary, presence of hydro-
philic amino group in the binding pocket enhances potency to a
greater extant than increased lipophilicity.
azine gave 95. Subsequent hydrogenolysis of 95, followed by
coupling of the corresponding phenol with 4 gave 96. Analogue
98 was synthesized from phenol 64 in a similar manner enlisting
3-(dimethylamino)propan-1-ol.
Results from the biological investigation of diamino analogues
(87–94, 96 & 98) are shown in Table 3. These results confirm that
the synthetically challenging noviose moiety can be replaced with
commercially available amines without lowering potency as ana-
logues 87–92 manifested IC50 values similar to compounds utiliz-
ing noviose (69–74). Biological evaluation of analogues
containing alkyl amine substituents at the 2-position (93, 94, 96,
98) confirmed that N,N-dimethylpropylamine is an optimal substi-
tuent at the 2-position as these analogues manifested good po-
tency against SKBr3 and MCF-7 breast cancer cell lines. In
contrast to noviose containing analogue 78, the corresponding
N-methylpiperidine containing analogue 94 manifested better
efficacy against the SKBr3 and MCF-7 cell lines. Replacement of
N-methylpiperidine at the 7-position with acyclic N,N-dimethyl-
propylamine resulted in less activity.
To confirm these analogues manifest inhibitory anti-prolifera-
tive activity through Hsp90 inhibition, western blot analyses of cell
lysates resulting from the incubation of cells treated with the three
most potent compounds 77, 85, and 94 for 24 h were performed. As
shown in Figure 5, compound 77, 85 and, 94 induced degradation
of Hsp90-dependent client proteins p-Akt and Her2. Whereas, ac-
tin levels remained unchanged. Actin is not an Hsp90-dependent
substrate and used as a control. Absence of induction of heat shock
response as indicated by constant levels of Hsp90 indicates that
these compounds exhibit Hsp90 inhibitory activity through C-ter-
minal inhibition.
2.4. Design and synthesis of mono- and diamino substituted
coumarin surrogate analogues
Encouraged by the efficacy manifested by noviose containing
biaryl analogues that also contain an aminoalkyl substituent at
the 2-position (76–78, 81, 83, 85, 86), compounds containing the
noviose surrogate, N-methylpiperidine, were pursued (Scheme 9).
N-Methylpiperidine is a readily available surrogate for the synthet-
ically complex noviose sugar.24,32 Coupling of phenols 62, 63,
65–68 with 1-methylpiperidin-4-ol was achieved under Mitsunobu
conditions employing diisopropyl azodicarboxylate (DIAD) and tri-
phenylphosphine to give 87–92.26 Analogues containing aminoal-
kyl ethers at the 2-position were synthesized from diphenol 63,
which upon treatment with five equivalents of piperidin-4-ol or
3-(dimethylamino)propan-1-ol gave analogues 93–94. Construc-
tion of 96 and 98 was accomplished from the phenol 64, which
was obtained during partial hydrogenolysis of dibenzylated com-
pound, 57. Treatment of 64 with dibromopropane in the presence
of potassium carbonate, followed by coupling with 1-methylpiper-
Figure 5. Western blot analyses of Hsp90 client protein degradation in MCF-7 breast cancer cell line. L represents a concentration ½ ꢁ IC50 value while H represents a
concentration of 5 ꢁ IC50 value. GDA (500 nM) represents a positive control, while DMSO, vehicle, serves as the negative control.

B. R. Kusuma et al. / Bioorg. Med. Chem. 22 (2014) 1441–1449
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Acknowledgements
The authors gratefully acknowledge the support of this project
by NIH/NCI (CA120458 and CA167079) and frontier scientific for
providing high quality boronic acids.
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Supplementary data
Supplementary data (experimental procedures and character-
ization for all new compounds) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.
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