Synthesis and biological evaluation of novobiocin analogues as potential heat shock protein 90 inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.06.042

Recent studies have shown that novobiocin (NB), a member of the coumermycin (CA) family of antibiotics with demonstrated DNA gyrase inhibitory activity, inhibits Heat shock protein 90 (HSP90) by binding weakly to a putative ATP-binding site within its C-terminus. To develop more potent HSP90 inhibitors that target this site and to define structure-activity relationships (SARs) for this class of compounds, we have synthesized twenty seven 3-amido-7- noviosylcoumarin analogues starting from NB and CA. These were evaluated for evidence of HSP90 inhibition using several biological assays including inhibition of cell proliferation and cell cycle arrest, induction of the heat shock response, inhibition of luciferase-refolding in vitro, and depletion of the HSP90 client protein c-erbB-2/HER-2/neu (HER2). This SAR study revealed that a substantial increase in biological activity can be achieved by introduction of an indole-2-carboxamide group in place of 4-hydroxy-isopentylbenzamido group at C-3 of NB in addition to removal/derivatization of the 4-hydroxyl group from the coumarin ring. Methylation of the 4-hydroxyl group in the coumarin moiety moderately increased biological activity as shown by compounds 11 and 13. Our most potent new analogue 19 demonstrated biological activities consistent with known HSP90-binding agents, but with greater potency than NB.

Full text of DOI:10.1016/j.bmc.2013.06.042

Bioorganic & Medicinal Chemistry 21 (2013) 5118–5129
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novobiocin analogues
as potential heat shock protein 90 inhibitors
G. M. Kamal B. Gunaherath a, , Marilyn T. Marron a, E. M. Kithsiri Wijeratne a, Luke Whitesell b,
,
⇑
A. A. Leslie Gunatilaka a,
⇑
a Southwest Center for Natural Products Research and Commercialization, School of Natural Resources and the Environment, College of Agriculture and Life Sciences, University
of Arizona, 250 E. Valencia Road, Tucson, AZ 85706, United States
b Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Recent studies have shown that novobiocin (NB), a member of the coumermycin (CA) family of antibiotics
with demonstrated DNA gyrase inhibitory activity, inhibits Heat shock protein 90 (HSP90) by binding
weakly to a putative ATP-binding site within its C-terminus. To develop more potent HSP90 inhibitors
that target this site and to define structure–activity relationships (SARs) for this class of compounds,
we have synthesized twenty seven 3-amido-7-noviosylcoumarin analogues starting from NB and CA.
These were evaluated for evidence of HSP90 inhibition using several biological assays including inhibi-
tion of cell proliferation and cell cycle arrest, induction of the heat shock response, inhibition of lucifer-
ase-refolding in vitro, and depletion of the HSP90 client protein c-erbB-2/HER-2/neu (HER2). This SAR
study revealed that a substantial increase in biological activity can be achieved by introduction of an
indole-2-carboxamide group in place of 4-hydroxy-isopentylbenzamido group at C-3 of NB in addition
to removal/derivatization of the 4-hydroxyl group from the coumarin ring. Methylation of the 4-hydroxyl
group in the coumarin moiety moderately increased biological activity as shown by compounds 11 and
13. Our most potent new analogue 19 demonstrated biological activities consistent with known HSP90-
binding agents, but with greater potency than NB.
Received 18 April 2013
Revised 13 June 2013
Accepted 19 June 2013
Available online 27 June 2013
Keywords:
Novobiocin analogues
Heat shock protein 90 (HSP90)
Synthesis
Structure–activity relationship
Biological activity
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
of HSP90 are frequently elevated in highly malignant tumors.2 Be-
cause HSP90 stabilizes many of the oncoproteins required for can-
Heat shock protein 90 (HSP90) is an abundant molecular chap-
erone that is essential for the post-translational folding and confor-
mational maintenance of over 150 client proteins.1 Many of these
HSP90 client proteins are directly associated with the cancer phe-
notype including the tumor suppressor p53, receptor-linked tyro-
sine kinases such as HER2, steroid receptors, and a wide range of
other mediators of intracellular signal transduction. As a conse-
quence of constitutive activation of the heat-shock response, levels
cer survival and growth, inhibition of HSP90 by compounds such as
geldanamycin (an N-terminus binding agent) can impair the
growth and survival of diverse tumor types. In contrast to the
many HSP90 N-terminal inhibitors undergoing clinical evaluation,3
C-terminal inhibitors have only recently attracted attention as po-
tential anticancer agents. Novobiocin (NB, 1a) is a well-known nat-
ural product inhibitor of bacterial DNA gyrase. Consisting of a
coumarin core, an acyl substituent and a noviose sugar moiety, it
has been demonstrated to bind the C-terminus of HSP90 at a puta-
tive ATP-binding site.4 However,
(ꢀ700 M) of NB is required to impair HSP90 chaperone function
a very high concentration
Abbreviations: DAPI, 40,6-diamidino-2-phenylindole; DCC, dicyclohexylcarbodi-
imide; EDC, N-(3-dimethylaminopropyl)-N0-ethyl-carbodiimide; FITC, fluorescein
isothiocyanate; NBT/BCIP, nitro-blue tetrazolium chloride and 5-bromo-4-chloro-
30-indolyphosphate p-toluidine salt; PBA, phosphate buffered saline with bovine
albumin; PFA, formaldehyde in phosphate buffered saline with bovine albumin;
PMSF, phenylmethylsulfonyl fluoride; 4-PP, 4-pyrrolidinopyridine; TNES, a buffer
l
and induce degradation of client proteins such as HER2 in human
cancer cells.5 As part of our continuing search for natural prod-
uct-based HSP90 inhibitors,6 we prepared twenty seven NB ana-
logues from NB (1a) and coumermycin (CA, 2) and evaluated
their activity using a suite of biological assays selected to monitor
different aspects of HSP90 function. Herein we report synthetic
methods and structure-activity relationships (SARs) for these com-
pounds. The biological activities reported include global effects on
proliferation and cell cycle progression as well as more specific
⇑
Corresponding authors. Tel.: +1 520 621 9932; fax: +1 520 621 8378 (A.A.L.G.);
tel.: +1 617 452 3542; fax: +1 617 258 7228 (L.W.).
E-mail addresses: whitesell@wi.mit.edu (L. Whitesell), leslieg@cals.arizona.edu
(A.A. Leslie Gunatilaka).
Present address: Department of Chemistry, The Open University of Sri Lanka, P.O.
Box 21, Nugegoda, Sri Lanka.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.042

G. M. Kamal B. Gunaherath et al. / Bioorg. Med. Chem. 21 (2013) 5118–5129
5119
HSP90-related effects on heat-shock induction, protein refolding
in vitro, and depletion of HER2 in breast cancer cells.
the noviosyl moiety was carried out by linking 13 with indole-2-
carboxylic acid yielding the NB analogues 14 and 15. Treatment
of 15 with imidazole in DMF afforded 16 resulting from migration
of the axial 20-acyl group to a less hindered equatorial 30-position
(Scheme 2). Treatment of 1b with chlorobis(cyclopentadi-
enyl)hydridozirconium (Cp2ZrHCl) under anhydrous conditions in
DME yielded 4-deoxynovobiocin (17) (Scheme 2).14 Although the
yield of this nonoptimized reaction was low, it is noteworthy that
this report constitutes the first application of this facile reductive
enolate deoxygenation methodology to a complex molecule such
as NB. The 4-deoxy-3-(indole-2-carboxamide) analogue 19 was
prepared by deoxygenation of the lithium enolate of 5n with
Cp2ZrHCl under anhydrous conditions yielding 18 followed by
deacetylation with aq. MeOH/NaCN (Scheme 3).
In a previous SAR study of NB analogues, Blagg and co-workers
have shown that the noviose moiety at C-7 of the coumarin ring is
critical for biological activity based on depletion of the HSP90 cli-
ent protein phospo-AKT.7a The most active analogue in this study
lacked the C-4 OH group in the coumarin moiety and contained
an N-acetyl side chain in place of the substituted benzamide group
of NB. In a subsequent study this group found that analogues
containing both the modified sugar and coumarin moieties such as
30-descarbamoyl-4-deoxynovobiocin were more potent in deplet-
ing cellular levels of the HSP90 client proteins, HER2 and p53.7b
In a more recent report they have suggested that replacement of
the substituted benzoyl moiety by a 2-indolecarbonyl group in
30-descarbamoyl-4-deoxynovobiocin results in a compound with
improved activity as measured by depletion of the HSP90 client
proteins HER2,RAF, and AKT and induction of the heat-shock
response.7c Renoir and co-workers in their SAR study involving a
series of denoviosyl NB analogues found that the presence of an
OH group at C-4 or C-7 of the coumarin substructure is essential
fordegradation of HSP90 client proteins.8a In a subsequent study
they showed that introduction of a tosyl group at C-4 along with
the removal of substituents at C-7 and C-8 of the coumarin moiety
enhanced the activity of NB.8b It is also noteworthy that Blagg’s
group has recently investigated NB analogues with an array of
structural diversity9 while Huang et al. have designed new NB
derivatives with higher HSP90 inhibitory activities based on a
three-dimensional quantitative SAR (3D QSAR) study and con-
firmed by molecular simulation.10 In an attempt to develop noven-
amine-based antibacterial agents, Berkov-Zrihen et al. have
recently studied acylation of NB by carboxylic acid anhydrides.11
2.2. Biological evaluation
2.2.1. Depletion of cell surface HER2
Destabilization and subsequent depletion of the receptor-linked
tyrosine kinase HER2 is a hallmark of HSP90 inhibition.15 As an ini-
tial screen for biological activity, all NB analogues were evaluated
for their ability to deplete HER2 in a flow cytometry-based assay
using the human breast adenocarcinoma cell line SKBR3. DMSO
and geldanamycin (GA, 0.5
controls, respectively. NB (500
poses of comparison. When SKBR3 cells were incubated overnight
with each of the 27 analogues at a concentration of 100 M, only
l
M) served as negative and positive
lM) served as a benchmark for pur-
l
compounds 10e, 11, 13, 17, and 19 depleted cell surface HER2 by
ꢀ40% (Fig. 1A). NB demonstrated no activity at this concentration.
To begin correlating HER2 depletion by compounds with their anti-
proliferative activity, we measured the number of viable SKBR3
cells after overnight exposure to compounds (Supplementary
Fig. S36). This experiment was performed in the same SKBR3 cells
and under the same exposure conditions as the flow cytometry
experiment measuring HER2 levels (Fig. 1A). Only those com-
pounds which induced HER2 depletion demonstrated anti-prolifer-
ative activity in this assay.
To avoid potential confounding effects on kinase depletion due
just to cytotoxicity, the active analogues 10e, 11, 13, 17, and 19
were assayed for HER2 depletion using the MCF-7 cell line. These
cells express the HER2 kinase, but are far less dependent on it for
their short-term survival, In this follow-up experiment, the EC50
values of analogues 11, 13, 17 and 19 were 38 2, 41 8, 24 5,
and 20 5, respectively, demonstrating that 17 and 19 have the
greatest potency among those tested, whereas analogue 10e
2. Results and discussion
2.1. Chemical synthesis
Fifteen 3-N-acyl analogues of NB 6a–6o (Scheme 1) were pre-
pared from 1a via 20-noviosyloxazolocoumarin (3) and 20-acetylno-
viosyl-3-aminocoumarin (4). Compound
3 was obtained by
refluxing 1a with Ac2O and pyridine12 which on subsequent treat-
ment with AcCl in refluxing EtOH yielded 4.13 Acylation of 4 with a
variety of carboxylic acids in the presence of DCC/4-PP or EDC gave
a series of NB analogues 5a–5j and 5l–5p. Hydrogenolysis of 5j
afforded 5k. A series of NB analogues 6a–6o were obtained from
5a–5i and 5k–5p via deacetylation with aq. MeOH/NaCN
(Scheme 1). Analogues 10a–10e containing a noviose moiety sim-
ilar to CA were obtained from commercially available 2 via the 20-
acetylnoviosyloxazolocoumarin (7) following a similar procedure
described for 3 above.13 The 20-acetylnoviosyl-3-aminocoumarin
derivative 8, obtained by the treatment of 7 with AcCl in refluxing
EtOH, was acylated with a variety of carboxylic acids in the pres-
ence of EDC to yield 9a–9e, which on deacetylation with aq.
MeOH/NaCN yielded the NB analogues 10a–10e (Scheme 1).
It has recently been reported that 4-OH and 30-carbamoyl
groups in NB are detrimental to its ability to inhibit HSP90.7b
Prompted by this report and in keeping with our strategy of using
NB (1a) and CA (2) as starting materials to obtain NB analogues re-
quired for this SAR study, we examined the effect of alkylating the
4-OH group as an alternative to its removal. Thus, novobiocin di-
methyl ether (11) was prepared from 1a by treatment with Me2-
SO4/K2CO3 (Scheme 2). To determine if the 30-carbamoyl group of
the noviose moiety of NB is essential for its activity, 30-descarba-
moylnovobiocin (12) was prepared by refluxing NB monosodium
salt (1b) with aq. MeOH/NaCN, followed by acidification with aq.
2 M HCl. Subsequent methylation of 12 as above gave 30-descarba-
moylnovobiocin-4,400-dimethyl ether (13). Further modification to
showed the weakest activity (EC50 > 50.0 lM).
2.2.2. Depletion of total cellular HER2
The NB analogues 11, 13, 17, and 19 demonstrating significant
activity by flow cytometry-based HER2 depletion assay, were eval-
uated for their ability to deplete total cellular HER2 by western blot
analysis. MCF-7 cells were treated overnight with analogues prior
to preparation of lysates. Analogues 11, 13, 17, and 19 exhibited
ꢀ50% reduction of total HER2 at 50
17 and 19 were more potent with depletion of HER2 evident at con-
centrations of 12.5 and 25.0 M, respectively (Figs. 1B and C), cor-
lM (data not shown). However,
l
relating well with the observations from flow cytometric analysis.
To confirm equal loading across their respective lanes, each mem-
brane was stained with Ponceau S prior to blotting to visualize total
protein (Supplementary Fig. S37). Because analogue 17 was cyto-
toxic to MCF-7 cells at concentrations over 25.0 lM, it could not
be reliably evaluated at higher concentrations. Western blots were
subsequently re-probed for an inducible isoform of HSP70 to eval-
uate heat-shock induction. Only the highest concentration of NB
analogue 19 induced any detectable increase in the level of this pro-
tein. This result further confirms equal protein loading across lanes.

5120
G. M. Kamal B. Gunaherath et al. / Bioorg. Med. Chem. 21 (2013) 5118–5129
OH
OR
H
N
O
O
O
O
O
MeO
O
OH
(1a) R = H
H2N
O
(1b) R = Na
OH
O
OH
HN
H
H
N
N
H
N
O
O
O
O
O
O
O
O
O
O
OMe
O
MeO
OH
O
OH
O
NH
(2)
OH
O
O
OH
O
H
N
N
O
R
NHR
O
O
O
O
O
O
O
vi
i
ii
(1a)
O
O
O
O
MeO
MeO
MeO
O
O
O
OAc
OH
OAc
H2N
O
H2N
O
H2N
R
H
R
(3)
(4)
cyclohexyl
(6a)
(6b)
(6c)
(6d)
(6e)
(6f)
cyclohexane carbonyl
cyclopentane carbonyl
cyclobutane carbonyl
cyclopropane carbonyl
furan-2-carbonyl
benzoyl
cyclopentyl
cyclobutyl
cyclopropyl
furan-2-yl
phenyl
(5a)
(5b)
(5c)
(5d)
(5e)
(5f)
(5g)
(5h)
(5i)
iii
4-methoxyphenyl
4-nitrophenyl
phenylethenyl
4-hydroxyphenyl
pyrrol-2-yl
(6g)
(6h)
(6i)
4-methoxybenzoyl
4-nitrobenzoyl
cinnamoyl
4-benzyloxybenzoyl
4-hydroxybenzoyl
pyrrole-2-carbonyl
(6j)
(5j)
(5k)
(5l)
(5m)
(5n)
(5o)
(5p)
(6k)
(6l)
v
5-methylpyrrol-2-yl
indol-2-yl
(6m)
(6n)
(6o)
5-methylpyrrole-2-carbonyl
indole-2-carbonyl
thiophen-2-yl
iv
5-methylthiophen-2-yl
thiophene-2-carbonyl
5-methylthiophene-2-carbonyl
O
OH
H
OH
N
O
N
R
NHR
O
O
O
O
O
O
O
O
O
ii
vi
i
(2)
O
O
O
MeO
MeO
MeO
O
O
O
OAc
OH
OAc
O
O
O
NH
NH
NH
(7)
R
R
H
(8)
cyclohexane carbonyl
benzoyl
cyclohexyl
phenyl
(9a)
(9b)
(9c)
(9d)
(9e)
(10a)
(10b)
(10c)
(10d)
(10e)
4-hydroxy-3-(methylbutenyl)phenyl
5-methylpyrrol-2-yl
4-hydroxy-3-(methylbutenyl)benzoyl
5-methylpyrrole-2-carbonyl
indole-2-carbonyl
iv
indol-2-yl
Scheme 1. Reagents and conditions: (i) Ac2O, pyridine, heat; (ii) AcCl, EtOH, reflux, 3 h; (iii) RCO2H, DCC, 4-PP, EtOAc, rt; (iv) RCO2H, EDC, CH2Cl2, rt; (v) H2,Pd-C, EtOH, rt; (vi)
NaCN, MeOH, rt.
More importantly, it is consistent with previous reports showing
that C-terminal HSP90 inhibitors, unlike N-terminal inhibitors
cause only limited activation of the heat-shock response.7b
effects on the level and intracellular disposition of HER2 in SKBR3
cells (Fig. 1D). Untreated cells showed bright, uniform HER2 stain-
ing on the cell membrane. As previously reported, the positive con-
trol GA induced formation of perinuclear HER2 aggregates and
2.2.3. Intracellular aggregation of HER2
The two most biologically active NB analogues 17 and 19 were
evaluated by immunofluorescence microscopy to monitor their
depleted overall HER2 signal.16 NB at 1000
l
M caused a similar
distribution of HER2 and also decreased total signal. Treatment
of SKBR3 cells with 17 at 25 M resulted in a very similar pattern
l

G. M. Kamal B. Gunaherath et al. / Bioorg. Med. Chem. 21 (2013) 5118–5129
5121
R4
60 min (Fig. 2A). Both 17 and 19 markedly impaired refolding as
monitored by recovery of enzymatic activity. When tested over a
range of concentrations for 30 min, analogue 17 appeared mod-
estly more potent than 19, but both compounds were far more po-
tent than NB in this assay (Fig. 2B).
R3
O
H
N
O
O
O
O
MeO
R1O
OR2
2.2.6. Cytotoxicity
The concentration-dependent anti-cancer activity of NB ana-
logues 11, 13, 17, and 19 was evaluated by a standard dye reduc-
tion assay after 3-day compound exposure using the human
breast cancer cell lines MCF-7 (estrogen receptor positive, HER2
low) and SKBR3 (estrogen receptor negative, HER2 high) (Table 1).
Under these assay conditions, 19 was more potent than 17, even
though the compounds induced equivalent HER2 depletion after
R1
(11) CONH2
R2
H
H
R3
OMe OMe
OH OH
OMe OMe
R4
i
(1a)
(1b)
ii
H
H
(12)
(13)
(14)
(15)
(16)
(17)
i
H
Indole-2-carbonyl Indole-2-carbonyl OMe OMe
iii
H
Indole-2-carbonyl OMe OMe
H
H
iv
Indole-2-carbonyl
CONH2
OMe OMe
OH
v
H
short-term exposure at 100 lM (Fig. 1A). It is likely that pro-
Scheme 2. Reagents and conditions: (i) Me2SO4, K2CO3, acetone, reflux, 3 h; (ii)
NaCN, MeOH, reflux, 10 h; (iii) indole-2-carboxylic acid, DCC, 4-PP, EtOAc, rt; 16 h;
(iv) imidazole, DMF, rt, 1 h; (v) Cp2ZrHCl, DME, N2, rt, 2 h, then 50 °C, 16 h.
longed HSP90 inhibition even at modest levels that do not imme-
diately deplete HER2 would be toxic. As a result, the stability of
the analogues in culture medium could be a factor in longer as-
says. Moreover, HER2 is not the only HSP90-dependent client pro-
tein in MCF-7 cells that is likely to be impaired by these
compounds.
H
N
N
H
O
2.2.7. Cell cycle analysis
The effects of the most promising analogues 17 and 19 on cell
cycle progression were evaluated by flow cytometry using SKBR3
i
O
O
O
(5n)
O
MeO
O
OR
cells (Table 2). Analogue 17 (12.5 lM, 48 h) moderately increased
H2N
O
the percentage of G0/G1 cells and some cell death was evidenced
by accumulation of cells with a sub G1 DNA content. These effects
were accompanied by a decline in cells within the S and G2/M
phases of the cell cycle. At a two-fold higher concentration, cyto-
toxicity became the predominant effect with almost 50% of cells
displaying sub G1 DNA content. In comparison to 17, analogue 19
did not cause cells to accumulate in G0/G1 and was cytotoxic only
(18) R = Ac
(19) R = H
ii
Scheme 3. Reagents and conditions: (i) LiH, Cp2ZrHCl, DME, N2, rt, 2 h, then 70 °C,
16 h; (ii) NaCN, MeOH, rt, 2 h.
of staining to GA and NB (1000 lM) while more subtle effects were
at a concentration of 25.0 lM. Overall, analogues 17 and 19 caused
seen following treatment with the same concentration of 19.
dose- and time-dependent (24 h) increases in the fraction of SKBR3
cells with sub G1 DNA content (data not shown). These increases
although qualitatively similar to the effects of NB were achieved
at much lower concentrations.
2.2.4. Heat-shock induction
Inhibition of HSP90 by N-terminal-targeted compounds leads to
activation of Heat shock factor 1 (HSF1), the master transcriptional
regulator of the heat-shock response.17 To evaluate the concentra-
tion-dependent ability of NB analogues to induce a heat-shock re-
sponse at the transcriptional level, 3T3-Y9-B12 reporter cells18
were incubated overnight with serial dilutions of the test com-
pounds (Supplementary Fig. S38). Less than two-fold induction of
the reporter was detected over the entire concentration range
3. Conclusions
A series of 27 analogues with varying substituents on the cou-
marin and noviose moieties of NB were synthesized starting from
commercially available novobiocin monosodium salt (1b) and cou-
mermycin (2). All analogues were evaluated for depletion of HER2
in SKBR3 cells to assess their potential for inhibition of HSP90. To
further investigate the SAR of these active compounds, they were
evaluated in an additional panel of assays including depletion of
total cellular HER2, intracellular aggregation of HER2, heat-shock
induction, ability to refold heat denatured luciferase, cytotoxicity,
and cell cycle analysis. Based on these studies, we conclude that
the 4-OH group in the coumarin moiety of NB is detrimental to
its HSP90 inhibitory activity while the presence of a carbamoyl
group in the noviosyl moiety does not interfere with activity as
demonstrated by the analogues, 11, 13, 17 and 19. The most potent
compounds 17 and 19, while far more potent than NB, retained
minimal heat-shock induction activity which is a desirable charac-
teristic of C-terminal inhibitors of HSP90.
tested (25.0–0.78
stimulated three-fold induction at a concentration of 0.5
even at 1000 M induced a response of only 1.5-fold. Although
lM). In contrast, the N-terminal inhibitor GA
lM. NB,
l
17 and 19 are far more potent than NB in depleting HER2, they con-
tinue to exert only weak heat shock-inducing activity, which has
been proposed as
a desirable characteristic of C-terminal
inhibitors.7b
Experiments in cell culture have shown that preventing HSP72
induction can make cells more sensitive to the conventional N-ter-
minal HSP90 inhibitor 17-AAG7d as can knockout of HSF1.18 The
therapeutic relevance of these findings, however, has yet to be
defined.
2.2.5. Luciferase refolding assay
The ability of our two most active NB analogues 17 and 19 to
inhibit in vitro chaperone-mediated refolding of heat-denatured
luciferase in a concentration- and time-dependent manner was
examined (Fig. 2). Compounds were added to rabbit reticulocyte
lysate containing an ATP regenerating system. After addition of
heat-denatured luciferase, refolding was monitored by the addi-
tion of luciferin assay buffer to replicate aliquots at 0, 15, 30, and
4. Experimental
4.1. General procedures
All chemicals and solvents were purchased from Aldrich Chem-
ical Co. and used without further purification. All reactions were

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G. M. Kamal B. Gunaherath et al. / Bioorg. Med. Chem. 21 (2013) 5118–5129
A
0
GA NB 100
50
B
D
C
25 12.5 6.25
µM
25 12.5 6.25
µM
0
GA NB
HER2
HER2
HSP72
HSP72
DMSO
GA 0.5 µM
NB (1a) 1000 µM
Isotype control
(17) 25 µM
(17) 12.5 µM
(19) 25 µM
(19) 12.5 µM
Figure 1. NB analogues alter the level and disposition of the HSP90 client HER2. (A) SKBR3 cells were exposed to known HSP90 inhibitors (GA = 0.5
lM and NB = 500 lM) or
the novobiocin analogues (100 M). After 18 h, cells were immunostained and mean fluorescence intensity (MFI) was determined by flow cytometry. Isotype control
l
antibody was used to confirm the specificity of the staining. (B) and (C) MCF-7 cells were exposed to analogue 17 (B) or analogue 19 (C) for 18 h and lysates fractionated by
SDS–PAGE. Proteins were transferred to nitrocellulose membranes and probed with antibody to HER2 or HSP72. (D) Depletion and altered localization of HER2. SKBR3 cells
were exposed to known HSP90 inhibitors (GA, NB) or the NB analogues 17 or 19 for 18 h in chamber slides. Cells were fixed and stained with anti-HER2 antibody (green
signal), and counter-stained with DAPI (blue signal). Images were acquired at 160ꢁ magnification using identical exposure settings.
monitored by TLC (0.25 mm thick pre-coated Silica gel 60 F254 from
EM Science) for the disappearance of the starting material and the
plates were examined under UV light and developed by spraying
anisaldehyde reagent followed by heating. Preparative TLC was
performed using 0.5 mm thick pre-coated Silica gel GF Uniplates
from Analtech while Flash chromatography was carried out on
ously reported. Assignments of 1H NMR data of all NB analogues
are provided in the (Supplementary Tables S1–S3 and Supplemen-
tary Fig. S4).
4.2. Synthesis of NB analogues
40
lM Flash Silica gel from J. T. Baker. Preparative HPLC was per-
4.2.1. General procedure for the formation of
formed on a Waters Delta Prep 4000 preparative chromatography
system equipped with a Waters 996 photodiode array detector
and a Waters Prep LC controller utilizing Empower Pro software
noviosyloxazolocoumarins (3) and (7)
To a solution of NB (1a) or CA (2) in pyridine, was added Ac2O
(20-fold excess) and heated under reflux for 24 h. The reaction
mixture was allowed to cool, acidified with 2 M aq. HCl and ex-
tracted with EtOAc. The EtOAc layer was washed with water fol-
lowed by brine, dried (Na2SO4) and evaporated to afford the
crude product.
and using a either Phenomenex Luna 5
l or Kromasil KR 100-7
C18 RP column (100 Å, 250 ꢁ 10 mm) with a flow rate of 3.0 mL/
min; chromatograms were acquired at 254 and 270 nm. 1H NMR
and 13C NMR were recorded in DMSO-d6 at 500 MHz and
125 MHz respectively on a Bruker DRX 500 spectrometer unless
otherwise stated. The chemical shifts are reported in ppm and
are referenced to the respective residual solvent signal. LR- and
4.2.2. General procedure for the preparation of 3-
aminonoviosylcoumarins (4) and (8)
HR-MS were recorded respectively on Shimadzu LCMS- QP8000
a
To a refluxing suspension of noviosyloxazolocoumarin 3 or 7;
(1.0 equiv) in EtOH was added AcCl (1.0 equiv) and continued heat-
ing for 3 h.13 Solvent was evaporated, dried under vacuo and the
solid was triturated with diethyl ether to obtain the crude 3-amin-
onoviosylcoumarin 4 or 8.
and JEOL HX110A spectrometers. All new compounds were charac-
terized by 1H and 13C NMR spectroscopic data and high resolution
MS. 1H NMR spectroscopic data and low resolution MS data are re-
ported for all known compounds whose NMR data were not previ-

G. M. Kamal B. Gunaherath et al. / Bioorg. Med. Chem. 21 (2013) 5118–5129
5123
4.2.3. General procedure for the preparation of 20-
acetylnovobiocin analogues 5a–5j
To a stirred solution of the carboxylic acid (0.32 mmol) in EtOAc
(2.5 mL) at 25 °C, DCC (35.0 mg, 0.26 mmol) and 4-PP were added
and stirring continued for 0.5 h. A solution of 4, (100.0 mg;
0.21 mmol) in THF (1.0 mL) was added to the reaction mixture
and stirring continued until the disappearance of the starting
material (monitored by TLC). Reaction mixture was filtered, evap-
orated and the products were purified by flash chromatography
and/or preparative TLC.
4.2.4. General procedure for the preparation of 20-
acetylnovobiocin analogues 5l–5p
To
a stirred solution/suspension of the carboxylic acid
(0.32 mmol) in CH2Cl2 (1.5 mL) at 25 °C, EDC (36.5 mg; 0.23 mmol)
was added and stirring continued for 0.5 h. A solution of 4
(100.0 mg; 0.21 mmol) in THF (1 mL) was added to the reaction
mixture and stirring continued until the reaction was complete.
The solvents were removed under vacuo and the products were
purified by flash chromatography and/or preparative TLC.
4.2.5. General procedure for the preparation of 20-
acetylnovobiocin analogues 9a–9e
EDC (16.0 mg; 0.10 mmol) was added to a stirred solution/sus-
pension of the carboxylic acid (0.14 mmol) in CH2Cl2 (0.5 mL).
After 0.5 h solution of 3-aminocoumarin (8) (50.0 mg; 0.09 mmol)
in CH2Cl2 (0.5 mL) was added and stirring continued until the reac-
tion was complete. The solvent was evaporated and the product
was purified by preparative TLC.
4.2.6. General procedure for deacylation
To a stirred solution/suspension of the starting material in
MeOH (2 mL) was added NaCN (1:4) and continued stirring until
the reaction is complete (monitored by TLC) at 25 °C unless other-
wise stated. Reaction mixture was diluted with brine, acidified
with 2 M aq. HCl and extracted into EtOAc. EtOAc layer was
washed with brine and dried (Na2SO4) and solvent were removed
to obtain the crude product.
Figure 2. (A) Time-dependent and (B) concentration-dependent inhibition of the
refolding of heat-denatured luciferase by GA, NB, and analogues 17 and 19. The
mean and standard deviation of triplicate determinations is presented. Values are
expressed as relative light units (RLU) or RLU normalized to DMSO vehicle control
values (% RLU).
Table 1
Cytotoxicity of novobiocin (1a) and its Analogues 11, 13, 17, and 19a
4.2.7. General procedure for methylation
To a stirred solution of the starting material (1.0 equiv) in ace-
tone were added Me2SO4 (2.7 equiv) and excess K2CO3 and re-
fluxed for 3 h. Reaction mixture was filtered, filtrate was
evaporated and the residue was chromatographed over a column
of silica gel to isolate the desired products.
Compound
Cell line
MCF-7
SKBR3
11
13
17
19
1a
18
19
0
19
25
17
10
1
3
4
0
5
7
2
6
15
6
357
350 37
4.2.8. General procedure for deoxygenation with
a
Results are expressed as IC50 values in
l
M.
chlorobis(cyclopentadienyl) hydridozirconium (Cp2ZrHCl)
A stirred suspension/solution of sodium or lithium enolate of
the compound was treated with excess of Cp2ZrHCl in anhydrous
DME (2 mL) under an atmosphere of dry N2 at 25 °C for 2 h and
then at 50–70 °C for 16 h (TLC control). Reaction mixture was acid-
ified with 2 M HCl, extracted with EtOAc (3 ꢁ 50 mL). The EtOAc
layer was washed with H2O (3 ꢁ 25 mL), dried (Na2SO4) and evap-
orated to give the crude product mixture.
Table 2
Effect of novobiocin (1a) and its analogues 17 and 19 on cell cycle distributiona
Compound
Cell cycle distribution
G0/G1
S
G2/M
SubG1
DMSO
GA 0.5
54
65
57
43 15
62 14
56 18
2
1
4
19
10
15
17
10
10
17
6
5
6
6
6
6
6
21
18
10
15
9
1
7
16
1
9
0
1
2
4
5
1
(1a) 1000
(17) 25.0
(17) 12.5
(19) 25.0
(19) 12.5
38 21
48 10
22 19
29 21
4.2.9. General procedure for the formation of novobiocin
analogues 6a–6o and 10a–10e
2-O-Acetyl novobiocin analogues 5a–5i, 5k–5p and 9a–9e were
subjected to the general procedure for deacylation, followed by
purification by preparative TLC. Yields are recorded with respect
to the 3-aminonovosylcoumarins (4) or (8)
10
18
58
1
8
2
a
Percentage of cells in each phase of the cell cycle as determined by flow cyto-
metric measurement of relative DNA content.

5124
G. M. Kamal B. Gunaherath et al. / Bioorg. Med. Chem. 21 (2013) 5118–5129
4.2.10. Regeneration of novobiocin (1a) from its monosodium
salt (1b)
The crude product 8, obtained as a pale yellow solid (200.0 mg;
97%), was used for the acylation reaction without further
purification.
A solution of NB monosodium salt (1b, 30 g) in water (400 mL)
was acidified with 2 M aq. HCl (pH ꢀ2; just until no further precip-
itation occurs) and extracted with EtOAc (3 ꢁ 400 mL). The EtOAc
layer was dried (Na2SO4) and evaporated to dryness to afford 1a
(28.7 g; 99.3%).
4.2.17. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)cyclohexanecarboxamide (6a)
White solid, 32% yield, mp 170–172 °C. 1H NMR d 9.27 (s, 1H),
7.69 (d, J = 9.0 Hz, 1H), 7.14 (d, J = 9.0 Hz, 1H), 6.69 (br s, 1H),
6.50 (br s, 1H), 5.58 (d, J = 5.2 Hz, 1H), 5.50 (d, J = 2.4 Hz, 1H), 5.13
(dd, J = 9.8, 3.1 Hz, 1H), 4.05 (m, 1H), 3.46 (d, J = 9.8 Hz, 1H), 3.45
(s, 3H), 2.55 (tt, J = 11.7, 3.4 Hz, 1H), 2.18 (s, 3H), 1.87–1.82 (m,
2H), 1.77–1.69 (m, 2H), 1.66–1.59 (m, 1H), 1.33–1.15 (m, 3H),
1.24 (s, 3H), 1.03 (s, 3H); 13C NMR d 177.1, 160.1, 156.7, 156.2,
150.1, 121.8, 112.7, 110.4, 110.0, 101.8, 98.4, 80.7, 78.0, 70.3,
68.7, 61.0, 43.4, 29.1, 29.0, 28.4, 25.4, 25.1, 22.7, 8.3; HRFABMS
m/z 535.2318 [M+1]+; calcd for C26H35N2O10, 535.2292.
4.2.11. 7-{[2-O-Acetyl-3-O-(aminocarbonyl)-6-deoxy-5-C-
methyl-4-O-methyl-a-L-lyxo-hexopyranosyl]oxy}-2,6-dimethyl-
4H-[1]benzopyrano[3,4-d]oxazol-4-one (3)
This compound was prepared from NB (1a, 20.00 g, 33 mmol)
using the general procedure for the preparation of novi-
osyloxazolocoumarins. Recrystallization (EtOAc) afforded the title
compound as off white crystals (6.80 g; 42%): mp 167–170 °C
(lit.12 167–172 °C). 1H NMR d 7.71 (d, J = 8.8 Hz, 1H), 7.22 (d,
J = 8.8 Hz, 1H), 6.83 (br s, 1H), 6.63 (br s, 1H), 5.71 (d, J = 2.8 Hz,
1H), 5.33–5.26 (m, 1H), 3.47 (s, 3H), 3.39 (d, J = 8.9 Hz, 1H), 2.63
(s, 3H), 2.24 (s, 3H), 2.11 (s, 3H), 1.33 (s, 3H), 1.11 (s, 3H); 13C
NMR d 169.5, 163.1, 156.2, 155.7, 155.6, 155.2, 151.3, 122.0,
119.4, 114.4, 110.9, 105.3, 95.2, 80.8, 78.3, 69.8, 67.8, 60.8, 27.8,
22.9, 20.7, 13.8, 8.5.
4.2.18. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)cyclopentanecarboxamide (6b)
Off-white solid, 30% yield, mp 143–145 °C. 1H NMR d 12.32 (br
s, 1H), 9.37 (s, 1H), 7.70 (d, J = 9.0 Hz, 1H), 7.14 (d, J = 9.0 Hz, 1H),
6.70 (br s, 1H), 6.51 (br s, 1H), 5.58 (d, J = 5.2 Hz, 1H), 5.50 (d,
J = 2.4 Hz, 1H), 5.13 (dd, J = 9.8, 3.1 Hz, 1H), 4.05 (m, 1H), 3.46 (d,
J = 9.8 Hz, 1H), 3.45 (s, 3H), 3.01 (q, J = 7.9 Hz, 1H), 2.19 (s, 3H),
1.90–1.08 (m, 8H), 1.24 (s, 3H), 1.03 (s, 3H); 13C NMR d 177.4,
160.1, 157.1, 156.8, 156.2, 150.1, 121.7, 112.7, 110.3, 110.0,
4.2.12. 7-{[2-O-acetyl-3-O-(aminocarbonyl)-6-deoxy-5-C-
methyl-4-O-methyl-a-L-lyxo-hexopyranosyl]oxy}-3-amino-4-
hydroxy-8-methyl-2H-1-benzopyran-2-one (4)
This compound was prepared from 3 (11.27 g; 23 mmol) using
the general procedure for the preparation of 3-aminonovosyl cou-
marins. Purification of the crude product by flash chromatography
(eluent: NH4OH/MeOH/CH2Cl2 4:20:76) afforded 4 as a pale yellow
solid (4.0 g; 42%): mp 195–203 °C (lit.13 195–215 °C). Identity was
confirmed by the comparison of 1H NMR data with those
reported.12 13C NMR d 169.6, 169.5, 160.4, 155.7, 155.5, 151.0,
128.8, 122.0, 155.9, 112.6, 108.8, 95.1, 80.9, 78.1, 70.1, 68.1, 60.9,
27.9, 23.1, 8.3.
101.9, 98.4, 80.7, 78.0, 70.3, 68.7 , 61.0, 43.8, 30.2, 30.1, 28.4,
+
25.8, 22.7, 8.3; HRFABMS m/z 521.2166 [M+1]
25H33N2O10, 521.2135.
; calcd for
C
4.2.19. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl- -lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
a-L
oxo-2H-1-benzopyran-3-yl)cyclobutanecarboxamide (6c)
White solid, 18% yield, mp 162–165 °C. 1H NMR d 12.30 (br s,
1H), 9.12 (s, 1H), 7.70 (d, J = 9.0 Hz, 1H), 7.12 (d, J = 9.0 Hz, 1H),
6.69 (br s, 1H), 6.51 (br s, 1H), 5.57 (d, J = 5.2 Hz, 1H), 5.50 (d,
J = 2.3 Hz, 1H), 5.13 (dd, J = 9.8, 3.1 Hz, 1H), 4.05 (m, 1H), 3.46 (d,
J = 9.8 Hz, 1H), 3.45 (s, 3H), 3.40 (q, J = 8.4 Hz, 1H), 2.18 (s, 3H),
2.26–2.15 (m, 2H), 2.12–2.06 (m, 2H), 1.96–1.86 (m, 1H), 1.82–
1.72 (m, 1H), 1.24 (s, 3H), 1.03 (s, 3H); 13C NMR d 175.4, 160.3,
156.7, 156.2, 150.2, 121.8, 112.6, 109.9, 101.3, 98.4, 80.7, 78.0,
70.3, 68.7, 61.0, 38.4, 28.4, 24.8, 22.7, 17.6, 8.3; HRFABMS m/z
507.1985 [M+1] +; calcd for C24H31N2O10, 507.1975.
4.2.13. 5-Methylpyrrole-2-carboxylic acid
Reaction of diethylacetamidomalonate and 1,4-dichloro-2-bu-
tyne in the presence of NaOEt as reported19 gave ethyl 5-methyl-
pyrrole-2-carboxylate which on subsequent hydrolysis20 yielded
5-methylpyrrole-2-carboxylic acid.
4.2.14. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-2-O-acetyl-5-C-
methyl-4-O-methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-
methyl-2-oxo-2H-1-benzopyran-3-yl)-4-hydroxybenzamide
(5k)
To a solution of 20-acetylnovobiocin analogue 5j (45.0 mg,
0.068 mmol) in EtOH was added 10% Pd on C (1.5 mg) and stirred
under an atmosphere of H2 for 4 h. Reaction mixture was filtered
and EtOH removed under vacuo to give 5k (37.3 mg; 95%).
4.2.20. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)cyclopropanecarboxamide (6d)
White solid, 29% yield, mp 163–164 °C; 1H NMR d 12.47 (br s,
1H), 9.71 (s, 1H), 7.69 (d, J = 8.9 Hz, 1H), 7.11 (d, J = 8.9 Hz, 1H),
6.90 (br s, 1H), 6.50 (br s, 1H), 5.57 (d, J = 5.1 Hz, 1H), 5.49 (d,
J = 2.0 Hz, 1H), 5.13 (dd, J = 9.8, 3.0 Hz, 1H), 4.05 (m, 1H), 3.46 (d,
J = 9.8 Hz, 1H), 3.45 (s, 3H), 2.19 (s, 3H), 2.09 (q, J = 6.1 Hz), 1.24
(s, 3H), 1.03 (s, 3H), 0.82 (d, J = 6.1 Hz); 13C NMR d 175.4, 161.2,
157.6, 157.1, 150.0, 122.9, 113.5, 110.8 (2C), 102.6, 99.3, 81.6,
78.9, 71.2, 69.6, 61.8, 29.3, 23.6, 14.7, 9.2, 8.8; HRFABMS m/z
493.1827 [M+1]+; calcd for C23H29N2O10, 493.1822.
4.2.15. 7-({2-O-acetyl-6-deoxy-5-C-methyl-4-O-methyl-3-O-[(5-
methyl-1H-pyrrol-2-yl)carbonyl]-a-L-lyxo-hexopyranosyl}oxy)-
2,6-dimethyl-4H-[1]benzopyrano[3,4-d]oxazol-4-one (7)
This compound was prepared from coumermycin (2, 400.0 mg;
0.36 mmol) using the general procedure for noviosyloxazolocoum-
arin formation reaction. The crude product mixture on flash chro-
matography afforded 7 as an off-white solid (216.0 mg; 42%). Its
identity was confirmed by comparison with reported data (mp
and 1H NMR).12
4.2.21. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
4.2.16. 7-({2-O-acetyl-6-deoxy-5-C-methyl-4-O-methyl-3-O-[(5-
oxo-2H-1-benzopyran-3-yl)-2-furancarboxamide (6e)
methyl-1H-pyrrol-2-yl)carbonyl]-
a-
L-lyxo-hexopyranosyl}oxy)-
White solid, 9% yield, mp 237–239 °C (lit.21 122–130 °C). 1H
NMR d 11.98 (br s, 1H), 9.20 (s, 1H), 7.89 (s, 1H), 7.71 (d,
J = 8.9 Hz, 1H), 7.26 (d, J = 3.2 Hz, 1H), 7.12 (d, J = 8.9 Hz, 1H),
6.70 (br s, 1H), 6.67 (s, 1H), 6.52 (br s, 1H), 5.58 (d, J = 5.1 Hz,
3-amino-4-hydroxy-8-methyl-2H-1-benzopyran-2-one (8)
This was prepared from 7 (217.0 mg; 0.39 mmol) using the gen-
eral procedure for the preparation of 3-aminonoviosyl coumarins.

G. M. Kamal B. Gunaherath et al. / Bioorg. Med. Chem. 21 (2013) 5118–5129
5125
1H), 5.52 (d, J = 2.0 Hz, 1H), 5.14 (dd, J = 9.8, 3.0 Hz, 1H), 4.06 (m,
4.2.27. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl- -lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
1H), 3.47 (d, J = 9.8 Hz, 1H), 3.45 (s, 3H), 2.20 (s, 3H), 1.25 (s, 3H),
a-L
1.06 (s, 3H); APCI MS (negative mode) m/z 517 [MꢂH]ꢂ.
oxo-2H-1-benzopyran-3-yl)-1H-pyrrole-2-carboxamide (6k)
White solid, 8% yield, mp 243–244 °C (lit.21 225–227 °C). 1H
NMR d 12.04 (br s, 1H), 11.65 (s, 1H), 9.03 (s, 1H), 7.72 (d,
J = 9.0 Hz, 1H), 7.14 (d, J = 9.0 Hz, 1H), 7.03 (br s, 1H), 6.95 (br s,
1H), 6.70 (br s, 1H), 6.50 (br s, 1H), 6.15 (br d, J = 2.8 HZ, 1H),
5.58 (d, J = 5.2 Hz, 1H), 5.52 (d, J = 2.3 Hz, 1H), 5.15 (dd, J = 9.8,
3.1 Hz, 1H), 4.06 (m, 1H), 3.47 (d, J = 9.8 Hz, 1H), 3.46 (s, 3H),
2.21 (s, 3H), 1.25 (s, 3H), 1.06 (s, 3H) APCIMS (positive mode) m/
z 518 [M+H]+; APCIMS (negative mode) m/z 516 [MꢂH]ꢂ.
4.2.22. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)benzamide (6f)
White solid, 20% yield, mp 245–246 °C (lit.21 245 °C). 1H NMR d
11.90 (br s, 1H), 9.46 (s, 1H), 8.01 (d, J = 7.3 Hz, 2H), 7.74 (d,
J = 8.9 Hz, 1H), 7.58 (d, J = 7.3 Hz, 2H), 7.51 (t, J = 7.3 Hz, 1H), 7.16
(d, J = 8.9 Hz, 1H), 6.70 (br s, 1H), 6.50 (br s, 1H), 5.60 (d,
J = 5.2 Hz, 1H), 5.54 (d, J = 2.3 Hz, 1H), 5.16 (dd, J = 9.8, 3.1 Hz,
1H), 4.08 (m, 1H), 3.48 (d, J = 9.8 Hz, 1H), 3.47 (s, 3H), 2.22 (s,
3H), 1.27 (s, 3H), 1.06 (s, 3H); APCIMS (positive mode) m/z 529
[M+H]+; APCIMS (negative mode) m/z 527 [MꢂH]ꢂ.
4.2.28. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)-5-methyl-1H-pyrrole-2-
carboxamide (6l)
White solid, 8% yield, mp 267–268 °C. 1H NMR d 12.25 (br s,
1H), 11.45 (s, 1H), 8.92 (s, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.15 (d,
J = 8.9 Hz, 1H), 6.93 (br s, 1H), 6.71 (br s, 1H), 6.53 (br s, 1H),
5.87 (br s, 1H), 5.58 (d, J = 5.1 Hz, 1H), 5.51 (br s 1H), 5.13 (dd,
J = 9.8, 3.0 Hz, 1H), 4.06 (m, 1H), 3.48 (d, J = 9.8 Hz, 1H), 3.46 (s,
3H), 2.21 (s, 3H), 2.20 (s, 3H), 1.25 (s, 3H), 1.04 (s, 3H); 13C NMR
d 160.6 (2C), 156.7, 156.2, 150.4, 123.6, 121.8, 114.1, 112.6,
109.9, 107.7, 98.4, 80.7, 78.0, 70.3, 68.7, 61.0, 28.4, 22.7, 12.6,
4.2.23. N-(7-{[3-O-(aminocarbonyl)-5-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)-4-methoxybenzamide (6g)
White solid, 9% yield, mp 248–250 °C (lit.21 257–259 °C). 1H
NMR d 11.99 (br s, 1H), 9.29 (s, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.71
(d, J = 9.0 Hz, 1H), 7.13 (d, J = 9.0 Hz, 1H), 7.02 (d, J = 8.7 Hz, 2H),
6.70 (br s, 1H), 6.51 (br s, 1H), 5.57 (d, J = 5.2 Hz, 1H), 5.52 (d,
J = 2.1 Hz, 1H), 5.14 (dd, J = 9.8, 3.0 Hz, 1H), 4.06 (m, 1H), 3.82 (s,
3H), 3.48 (d, J = 9.8 Hz, 1H), 3.46 (s, 3H), 2.21 (s, 3H), 1.25 (s, 3H),
1.05 (s, 3H) APCIMS (positive mode) m/z 559 [M+H]+; APCIMS
(negative mode) m/z 557 [MꢂH]ꢂ.
8.3; HRFABMS m/z 532.1940 [M+H]+; calcd for C25H30N3O10
,
532.1931.
4.2.29. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}4-hydroxy-8-methyl-2-
4.2.24. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)-1H-indole-2-carboxamide (6m)
White solid, 17% yield, mp 265–267 °C. 1H NMR d 12.05 (br s,
1H), 11.69 (s, 1H), 9.34 (s, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.64 (d,
J = 8.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.32 (s, 1H), 7.18 (t,
J = 8.0 Hz, 1H), 7.11 (d, J = 8.9 Hz, 1H), 7.04 (t, J = 8.0 Hz, 1H), 6.70
(br s, 1H), 6.51 (br s, 1H), 5.58 (d, J = 5.2 Hz, 1H), 5.51 (br s 1H),
5.15 (dd, J = 9.8, 3.1 Hz, 1H), 4.07 (m, 1H), 3.49 (d, J = 9.8 Hz, 1H),
3.46 (s, 3H), 2.21 (s, 3H), 1.25 (s, 3H), 1.05 (s, 3H); 13C NMR d
161.1, 160.8, 156.7 , 156.2, 150.9, 136.5, 127.1, 123.4, 122.0,
121.6, 119.7, 112.6, 112.2, 109.6, 103.9, 98.4, 80.7, 78.0, 70.3,
68.7, 61.0, 28.4, 22.7, 8.3; HRFABMS m/z 568.1957 [M+H]+; calcd
for C28H30N3O10, 568.1931.
oxo-2H-1-benzopyran-3-yl)-4-nitrobenzamide (6h)
Off-white solid, 22% yield, mp 256–257 °C (lit.21 255 °C). 1H
NMR d 9.37 (s, 1H), 8.25 (d, J = 8.1 Hz), 8.16 (d, J = 8.1 Hz, 2H),
7.61 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.68 (br s, 1H),
6.51 (br s, 1H), 5.55 (d, J = 5.1 Hz, 1H), 5.45 (br s, 1H), 5.15 (dd,
J = 9.7, 2.7 Hz, 1H), 4.06 (m, 1H), 3.46 (d, J = 9.7 Hz, 1H), 3.45 (s,
3H), 2.15 (s, 3H), 1.25 (s, 3H), 1.05 (s, 3H) APCIMS (negative mode)
m/z 572 [MꢂH]ꢂ.
4.2.25. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)-3-phenyl-2-propenamide (6i)
White solid, 20% yield, mp 228–230 °C (lit.21 228–231 °C). 1H
NMR d 12.98 (br s, 1H), 9.77 (s, 1H), 7.74 (d, J = 8.9 Hz, 1H),
7.63 (d, J = 7.4 Hz, 2H), 7.60 (d, J = 17.0 Hz, 1H), 7.46–7.40 (m,
3H), 7.17 (d, J = 17.0 Hz, 1H), 7.14 (d, J = 8.9 Hz, 1H), 6.70 (br s,
1H), 6.51 (br s, 1H), 5.59 (d, J = 5.1 Hz, 1H), 5.51 (d, J = 2.2 Hz,
1H), 5.14 (dd, J = 9.8, 3.0 Hz, 1H), 4.06 (m, 1H), 3.47 (d,
J = 9.8 Hz, 1H), 3.46 (s, 3H), 2.20 (s, 3H), 1.25 (s, 3H), 1.03 (s,
3H) APCIMS (positive mode) m/z 555 [M+H]+; APCIMS (negative
mode) m/z 553 [MꢂH]ꢂ
4.2.30. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)-2-thiophenecarboxamide (6n)
White solid, 12% yield, mp 228–229 °C. 1H NMR d 11.95 (br s,
1H), 9.46 (s, 1H), 7.97 (d, J = 3.5 Hz, 1H), 7.82 (d, J = 4.9 Hz, 1H),
7.73 (d, J = 9.0 Hz, 1H), 7.20 (dd, J = 4.9, 3.5 Hz, 1H), 7.14 (d,
J = 9.0 Hz, 1H), 6.70 (br s, 1H), 6.54 (br s, 1H), 5.59 (d, J = 5.2 Hz,
1H), 5.52 (d, J = 2.3 Hz 1H), 5.14 (dd, J = 9.8, 3.1 Hz, 1H), 4.06 (m,
1H), 3.47 (d, J = 9.8 Hz, 1H), 3.46 (s, 3H), 2.20 (s, 3H), 1.26 (s, 3H),
1.04 (s, 3H); 13C NMR d 161.3, 160.6 (2C), 157.0, 156.2, 150.8,
139.5, 131.3, 129.5, 127.9, 121.9, 112.8, 109.9, 100.1, 98.4, 80.7,
78.0, 70.2, 68.7, 61.0, 28.4, 22.7, 8.3; HRFABMS m/z 535.1401
[M+H]+; calcd for C24H27N2O10S, 535.1386.
4.2.26. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)-4-hydroxy benzamide (6j)
White solid, 32% yield, mp 235 °C (dec) (lit.21 256 °C). 1H NMR d
12.00 (br s, 1H), 10.00 (s, 1H), 8.94 (br s, 1H), 7.85 (d, J = 8.2 Hz,
2H), 7.68 (d, J = 8.3 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 6.80 (d,
J = 8.2 Hz, 1H), 6.68 (br s, 1H), 6.59 (br s, 1H), 5.55 (d, J = 5.1 Hz,
1H), 5.48 (br s, 1H), 5.14 (dd, J = 9.7, 2.9 Hz, 1H), 4.06 (m, 1H),
3.46 (d, J = 9.7 Hz, 1H), 3.45 (s, 3H), 2.18 (s, 3H), 1.25 (s, 3H), 1.05
(s, 3H); APCIMS (positive mode) m/z 545 [M+H]+; APCIMS (nega-
tive mode) m/z 543 [MꢂH]ꢂ.
4.2.31. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-hydroxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)-5-methyl 2-thiophenecarboxamide
(6o)
White solid, 11% yield, mp 248–250 °C. 1H NMR d 12.03 (br s,
1H), 9.18 (s, 1H), 7.74 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 8.7 Hz, 1H),
7.08 (d, J = 8.7 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H), 6.70 (br s,
1H), 6.51 (br s, 1H), 5.57 (d, J = 5.2 Hz, 1H), 5.50 (d, J = 2.1 Hz, 1H),

5126
G. M. Kamal B. Gunaherath et al. / Bioorg. Med. Chem. 21 (2013) 5118–5129
5.14 (dd, J = 9.8, 3.1 Hz, 1H), 4.06 (m, 1H), 3.47 (d, J = 9.8 Hz, 1H),
3.46 (s, 3H), 2.47 (s, 1H), 2.19 (s, 3H), 1.25 (s, 3H), 1.05 (s, 3H);
13C NMR d 161.1, 160.9, 156.6, 156.2, 150.9, 144.9, 137.0, 129.4,
126.4, 122.0, 114.1, 112.5, 109.4, 99.5, 98.4, 80.7, 78.0, 70.3, 68.7,
61.0, 28.4, 22.7, 15.3, 8.3; HRFABMS m/z 549.1563 [M+H]+; calcd
for C25H28N2O10S, 549.1543.
4.2.36. N-(7-{[6-deoxy-5-C-methyl-4-O-methyl-3-O-[(5-methyl-
1H-pyrrol-2-yl)carbonyl]- -lyxo-hexopyranosyl]oxy}-4-
hydroxy-8-methyl-2-oxo-2H-1-benzopyran-3-yl)-1H-indole-2-
a
-L
carboxamide (10e)
White solid, 21% yield, mp 259–260 °C. 1H NMR d 12.01 (br s,
1H), 11.67 (s, 1H), 11.64 (s, 1H), 9.50 (s, 1H), 7.76 (d, J = 8.8 Hz,
1H), 7.66 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.35 (s, 1H),
7.20 (t, J = 8.0 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 7.05 (t, J = 8.0 Hz,
1H), 6.77 (t, J = 2.8 Hz, 1H), 5.92 (t, J = 2.8 Hz, 1H), 5.68 (d,
J = 4.9 Hz, 1H), 5.61 (d, J = 2.2 Hz, 1H), 5.48 (dd, J = 9.8, 3.0 Hz,
1H), 4.17 (m, 1H), 3.65 (d, J = 9.8 Hz, 1H), 3.46 (s, 3H), 2.26 (s,
3H), 2.24 (s, 3H), 1.29 (s, 3H), 1.09 (s, 3H); 13C NMR d 161.2,
160.6, 159.8, 156.9, 150.8, 136.6, 134.6, 131.2, 127.0, 123.5,
122.0, 121.6, 120.3, 119.8, 116.2, 112.8, 112.3, 110.0, 108.4,
104.2, 98.2, 80.5, 78.1, 70.3, 68.7, 61.0, 28.5, 22.8, 12.8, 8.2;
HRFABMS m/z 632.2233 [M+H]+; calcd for C33H34N3O10
632.2244.
4.2.32. N-(7-{[6-deoxy-5-C-methyl-4-O-methyl-3-O-[(5-methyl-
1H-pyrrol-2-yl)carbonyl]-a-L-lyxo-hexopyranosyl]oxy}-4-
hydroxy-8-methyl-2-oxo-2H-1-benzopyran-3-
yl)cyclohexanecarboxamide (10a)
White solid, 52% yield, mp 223–225 °C. 1H NMR d 12.33 (br s,
1H), 11.63 (s, 1H), 9.30 (s, 1H), 7.71 (d, J = 8.9 Hz, 1H), 7.17 (d,
J = 8.9 Hz, 1H), 6.76 (t, J = 2.7 Hz, 1H), 5.91 (t, J = 2.7 Hz, 1H), 5.66
(d, J = 5.0 Hz, 1H), 5.59 (d, J = 2.4 Hz, 1H), 5.46 (dd, J = 9.7, 3.0 Hz,
1H), 4.15 (m, 1H), 3.64 (d, J = 9.7 Hz, 1H), 3.46 (s, 3H), 2.53 (tt,
J = 11.2, 3.2 Hz, 1H), 2.23 (s, 3H), 1.85–1.83 (m, 2H), 1.75–1.72
(m, 2H), 1.64–1.62 (m, 1H), 1.41–1.13 (m, 5H), 1.28 (s, 3H), 1.07
(s, 3H); 13C NMR d 177.1, 160.1 (2C), 159.7, 156.7, 150.1, 134.6,
121.8, 120.3, 116.2, 112.7, 110.2, 110.1, 108.4, 101.8, 98.2, 80.6,
78.1, 70.2, 68.7, 61.0, 43.4, 29.1, 29.0, 28.4, 25.4, 22.8, 12.8, 8.2;
HRFABMS m/z 599.2603 [M+H]+; calcd for C31H39N2O10, 599.2605.
4.2.37. N-(7-{[3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-4-methoxy-8-methyl-2-
oxo-2H-1-benzopyran-3-yl)-4-methoxy-3-(3-methyl-2-buten-
1-yl)benzamide (11)
Novobiocin (1a, 200.0 mg, 0.33 mmol) was subjected to the
general procedure for methylation and the product was obtained
via flash chromatography (eluant: 3% MeOH in CH2Cl2) as a white
solid (192.5 mg; 92%); mp 186–188 °C. 1H NMR d 9.56 (s, 1H), 7.87
(dd, J = 8.6, 1.6 Hz, 1H), 7.76 (d, J = 1.6 Hz, 1H), 7.68 (d, J = 9.0 Hz,
1H), 7.16 (d, J = 9.0 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 6.70 (brs, 1H),
6.54 (brs, 1H), 5.60 (d, J = 5.2, 1H), 5.53 (d, J = 2.3 Hz, 1H), 5.26
(tt, J = 7.3, 2.1 Hz, 1H), 5.15 (dd, J = 9.8, 3.0 Hz, 1H), 4.10 (s, 3H),
4.07 (m, 1H), 3.86 (s, 3H), 3.47 (d, J = 9.8 Hz, 1H), 3.46 (s, 3H),
3.28 (d, J = 7.3 Hz, 2H), 2.21 (s, 3H), 1.68 (s, 6H), 1.25 (s, 3H), 1.04
(s, 3H); 13C NMR d 166.4, 161.1, 160.7, 159.7, 156.9, 156.2, 149.9,
131.9, 129.1, 128.9, 127.3, 125.2, 122.0, 121.8, 112.8, 110.4,
110.2, 110.1, 103.5, 98.4, 80.7, 78.1, 70.3, 68.7, 60.9, 59.9, 55.7,
28.4, 28.2, 25.5, 22.7, 17.6, 8.3; APCIMS (positive mode) m/z 641
[M+H]+.
4.2.33. N-(7-{[6-deoxy-5-C-methyl-4-O-methyl-3-O-[(5-methyl-
1H-pyrrol-2-yl)carbonyl]-a-L-lyxo-hexopyranosyl]oxy}-4-
hydroxy-8-methyl-2-oxo-2H-1-benzopyran-3-yl)benzamide
(10b)
White solid, 13% yield, mp 129–131 °C (lit.22 230–235 °C dec).
1H NMR d 11.99 (br s, 1H), 11.63 (s, 1H), 9.43 (s, 1H), 8.00 (d,
J = 7.2 Hz), 7.74 (d, J = 8.8 Hz, 1H), 7.57 (t, J = 7.2 Hz, 1H), 7.50 (t,
J = 7.2 Hz, 2H), 7.18 (d, J = 8.8 Hz, 1H), 6.76 (t, J = 2.8 Hz, 1H), 5.91
(t, J = 2.8 Hz, 1H), 5.66 (d, J = 4.9 Hz, 1H), 5.61 (d, J = 2.3 Hz, 1H),
5.48 (dd, J = 9.8, 3.0 Hz, 1H), 4.16 (m, 1H), 3.64 (d, J = 9.7 Hz, 1H),
3.46 (s, 3H), 2.25 (s, 3H), 2.23 (s, 3H), 1.29 (s, 3H), 1.08 (s, 3H); APC-
IMS (positive mode) m/z 593 [M+H]+; APCIMS (negative mode) m/z
591 [MꢂH]ꢂ.
4.2.34. N-(7-{[6-deoxy-5-C-methyl-4-O-methyl-3-O-[(5-methyl-
1H-pyrrol-2-yl)carbonyl]-
a-
L-lyxo-hexopyranosyl]oxy}-4-
4.2.38. N-{7-[(6-deoxy-5-C-methyl-4-O-methyl-a-L-lyxo-
hydroxy-8-methyl-2-oxo-2H-1-benzopyran-3-yl)-4-hydroxy-3-
(3-methyl-2-buten-1-yl)benzamide (10c)
hexopyranosyl)oxy]-4-hydroxy-8-methyl-2-oxo-2H-1-
benzopyran-3-yl}-4-hydroxy-3-(3-methyl-2-buten-1-
yl)benzamide (12)
Novobiocin monosodium salt (1b, 500.0 mg, 0.79 mmol) was
subjected to general procedure of deacylation under reflux and
the crude product on flash chromatography (eluent: 3% MeOH in
CH2Cl2) afforded 12 as a white solid (357.0 mg; 79.6%): mp 168–
170 °C. 1H NMR data were consistent with those reported.24
White solid, 21% yield, mp 141–143 °C (lit.23 137 °C). 1H NMR d
11.98 (br s, 1H), 11.63 (s, 1H), 10.01 (s, 1H), 9.16 (s, 1H), 7.73 (s,
1H), 7.72 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.17 (d,
J = 8.7 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.77 (m, 1H), 5.91 (br s,
1H), 5.66 (d, J = 5.0 Hz, 1H), 5.60 (br s, 1H), 5.48 (dd, J = 9.8,
2.7 Hz, 1H), 5.30 (t, J = 7.6 Hz, 1H), 4.16 (m, 1H), 3.64 (d,
J = 9.8 Hz, 1H), 3.46 (s, 3H), 3.27 (m , 2H), 2.24 (s, 3H), 2.23 (s,
3H), 1.69 (s, 6H), 1.29 (s, 3H), 1.08 (s, 3H); APCIMS (positive mode)
m/z 677 [M+H]+; APCIMS (negative mode) m/z 675 [MꢂH]ꢂ.
4.2.39. N-{7-[(6-deoxy-5-C-methyl-4-O-methyl-a-L-lyxo-
hexopyranosyl)oxy]-4-methoxy-8-methyl-2-oxo-2H-1-
benzopyran-3-yl}-4-methoxy-3-(3-methyl-2-buten-1-
4.2.35. N-(7-{[6-deoxy-5-C-methyl-4-O-methyl-3-O-[(5-methyl-
yl)benzamide (13)
1H-pyrrol-2-yl)carbonyl]-
hydroxy-8-methyl-2-oxo-2H-1-benzopyran-3-yl)-5-methyl-1H-
pyrrole-2-carboxamide (10d)
a
-
L
-lyxo-hexopyranosyl]oxy}-4-
This compound was obtained from 12 (100.0 mg, 0.175 mmol)
employing the general procedure for methylation followed by flash
chromatography (eluant: 2.5% MeOH in CH2Cl2) as a white solid
(85.7 mg; 81.6%): mp 158–160 °C. 1H NMR d 9.55 (s, 1H), 7.88
(dd, J = 8.5, 1.6 Hz, 1H), 7.76 (d, J = 1.6 Hz, 1H), 7.67 (d, J = 9.0 Hz,
1H), 7.16 (d, J = 9.0 Hz, 1H), 7.07 (d, J = 8.5 Hz, 1H), 5.52 (d,
J = 2.0 Hz, 1H), 5.30 (d, J = 4.5 Hz, 1H), 5.27 (t, J = 7.6 Hz, 1H), 5.01
(d, J = 6.5 Hz 1H), 4.10 (s, 3H), 4.00 (m, 1H), 3.90 (m, 1H), 3.86 (s,
3H), 3.48 (s, 3H), 3.47 (d, J = 8.6 Hz, 1H), 3.28 (d, J = 7.6 Hz, 2H),
2.18 (s, 3H), 1.68 (s, 6H), 1.23 (s, 3H) , 1.00 (s, 3H); 13C NMR d
166.4, 161.1, 160.8, 159.7, 157.2, 149.8, 131.9, 129.1, 129.0,
127.3, 122.0, 112.7, 110.3, 110.2, 110.1, 103.4, 98.3, 83.3, 77.8,
70.8, 67.5, 61.0, 59.9, 55.7, 28.5, 28.1, 25.5, 22.8, 17.6, 8.3;
HRFABMS m/z 589.2574 [M+1]+; calcd for C32H40NO10, 589.2586.
White solid, 16% yield, mp 198–199 °C. 1H NMR d 12.26 (br s,
1H), 11.63 (s, 1H), 11.45 (s, 1H), 8.92 (s, 1H), 7.73 (d, J = 8.9 Hz,
1H), 7.17 (d, J = 8.9 Hz, 1H), 6.93 (br s, 1H), 6.77 (t, J = 2.8 Hz, 1H),
5.92 (t, J = 2.8 Hz, 1H), 5.87 (br s, 1H), 5.66 (d, J = 4.9 Hz, 1H), 5.59
(d, J = 2.3 Hz, 1H), 5.48 (dd, J = 9.8, 2.3 Hz, 1H), 4.16 (m, 1H), 3.64
(d, J = = 9.8 Hz, 1H), 3.46 (s, 3H), 2.24 (s, 3H), 2.23 (s, 3H), 2.21 (s,
3H), 1.29 (s, 3H, 1.08 (s, 3H); 13C NMR d 160.6, 159.8, 159.7,
156.7, 150.4, 134.6, 123.6, 121.8, 120.3, 116.2, 113.3, 112.7,
109.9, 108.4, 107.7, 98.2, 80.5, 78.1, 70.2, 68.7, 61.0, 28.4, 22.8,
12.8, 12.6, 8.2; HRFABMS m/z 596.2247 [M+H]+; calcd for
C30H34N3O10, 596.2244.

G. M. Kamal B. Gunaherath et al. / Bioorg. Med. Chem. 21 (2013) 5118–5129
5127
4.2.40. Esterification of 13 with indole-2-carboxylic acid
To stirred solution of indole-2-carboxylic acid (6.0 mg;
0.04 mmol), DCC (15.2 mg, 0.07 mmol), and 4-PP (0.5 mg) in EtOAc
(0.5 mL) at 25 °C, 13 (5.0 mg, 8.38 Mol) was added and continued
stirring for 20 h. The reaction mixture was filtered and the filtrate
was separated on preparative TLC (eluant: 5% MeOH in CH2Cl2) to
give 14 (1.7 mg; 23%) and 15 (3.1 mg; 50%).
MeOH/Et3N 8:2 (2.0 mL) for 10 min and separated on preparative
TLC (silica gel, 0.5 mm) followed by preparative HPLC using Phe-
nomenex, Luna column and elution with MeOH/H2O (8:2) to give
17 (6.8 mg; 4%, tR = 14.5 min) as a white solid; 1H and 13C NMR
data of 17 were consistent with literature values.7b
a
l
4.2.45. N-(7-{[2-O-Acetyl-3-O-(aminocarbonyl)-6-deoxy-5-C-
methyl-4-O-methyl-a-L-lyxo-hexopyranosyl]oxy}-8-methyl-2-
4.2.41. N-(7-{[6-Deoxy-2,3-di-O-[(1H-indole-2-yl)carbonyl]-5-C-
methyl-4-O-methyl-a-L-lyxo-hexopyranosyl]oxy}-4-methoxy-
8-methyl-2-oxo-2H-1-benzopyran-3-yl)-4-methoxy-3-(3-
methyl-2-buten-1-yl)benzamide (14)
oxo-2H-1-benzopyran-3-yl)-1H-indole-2-carboxamide (18)
To a stirred suspension of 5n (70.0 mg, 0.12 mmol) in anhy-
drous DME (3.0 mL) under an atmosphere of dry N2 was added
LiH (6.0 mg) and continued stirring for 30 min at 25 °C. This was
subjected to deoxygenation with 5-fold excess of Cp2ZrHCl as de-
scribed in the general procedure to obtain crude product
(71.6 mg), which was purified by preparative TLC (eluant: 8%
MeOH in CH2Cl2) to give 18 (20.1 mg; 29.5%).
Off-white solid: mp 163–165 °C. 1H NMR (600 MHz, CDCl3) d
8.91 (br s, 2H), 7.78 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 9.0 Hz, 1H),
7.72 (d, J = 9.0 Hz, 1H), 7.70 (br s, 1H), 7.56 (d, J = 8.1 Hz, 1H),
7.53 (s, 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.35
(s, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.19 (d,
J = 7.3 Hz, 1H), 7.18 (d, J = 9.0 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 7.06
(s, 1H), 6.86 (d, J = 8.4 Hz, 1H), 5.95 (dd, J = 9.7 and 2.9 Hz, 1H),
5.89 (br s, 1H), 5.77 (br s, 1H), 5.28 (t, J = 7.3 Hz, 1H), 4.09 (s,
3H), 3.88 (s, 3H), 3.76 (d, J = 9.7 Hz, 1H), 3.59 (s, 3H), 3.32 (d,
J = 7.2 Hz, 1H), 2.36 (s, 3H), 1.73 (s, 3H), 1.70 (s, 3H), 1.50 (s, 3H),
1.29 (s, 3H); HRFABMS m/z 884.3316 [M+H]+; calcd for
4.2.46. N-(7-{[3-O-(Aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-a-L-lyxo-hexopyranosyl]oxy}-8-methyl-2-oxo-2H-1-
benzopyran-3-yl)-1H-indole-2-carboxamide (19)
This compound was obtained from 18 (10.0 mg, 0.02 mmol)
using the general procedure for deacylation. Purification of the
crude product by preparative TLC (eluent: 10% MeOH in CH2Cl2)
followed by HPLC using Phenomenex, Luna column (eluent:
C50H50N3O12, 884.3289.
MeOH/H2O 7:3) afforded 19 as a white solid (3.9 mg; 42%, tR
=
4.2.42. N-(7-{[6-Deoxy-2-O-[(1H-indole-2-yl)carbonyl]-5-C-
methyl-4-O-methyl- -lyxo-hexopyranosyl]oxy}-4-methoxy-
8-methyl-2-oxo-2H-1-benzopyran-3-yl)-4-methoxy-3-(3-
13.6 min): mp 176–178 °C. 1H NMR (600 MHz, acetone-d6) d
10.99 (brs, 1H), 9.44 (s, 1H), 8.88 (brs, 1H), 8.08 (d , J = 8.9 Hz,
1H), 7.73 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.39 (d ,
J = 8.9 Hz, 1H), 7.35 (s, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.13 (t,
J = 8.0 Hz, 1H), 5.98 (brs, 2H), 5.68 (d, J = 2.5 Hz, 1H), 5.34 (dd,
J = 9.7, 3.2 Hz, 1H), 4.33 (m, 1H), 3.59 (d, J = 9.7 Hz, 1H), 3.52 (s,
3H), 2.37 (s, 3H), 1.33 (s, 3H), 1.14 (s, 3H); 13C NMR (150 MHz, ace-
tone-d6) d 172.3, 160.2, 159.9, 156.2, 145.3, 138.5, 131.5, 128.8,
125.2, 124.6, 122.8, 121.1, 117.5, 115.7, 113.1, 113.0, 112.8,
104.5, 99.7, 81.9, 79.5, 72.2, 70.3 , 61.5, 28.8, 23.3, 8.4; HRFABMS
m/z 552.5452 [M+1]+; calcd for C28H30N3O9, 552.5446.
a-L
methyl-2-buten-1-yl)benzamide (15)
Off-white solid: mp 186–188 °C. 1H NMR (600 MHz, CDCl3) d
8.97 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.70 (s, 2H), 7.69 (d,
J = 8.4 Hz, 1H), 7.52 (s, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.34 (d,
J = 7.6 Hz, 1H), 7.32 (s, 2H), 7.16 (d, J = 7.6 Hz, 1H), 7.14 (d,
J = 8.8 Hz, 2H), 6.87 (d, J = 8.5 Hz, 1H), 5.73 (s, 1H), 5.6 (br s, 1H),
5.28 (t, J = 7.3 Hz, 1H), 4.50 (d, J = 8.9 Hz, 1H), 4.08 (s, 3H), 3.88
(s, 3H), 3.64 (s, 3H), 3.47 (d, J = 9.6 Hz, 2H), 3.33 (d, J = 7.1 HZ,
2H), 2.28 (s, 3H), 1.91 (d, J = 9.5 Hz, 3H), 1.73 (s, 3H), 1.70 (s, 3H),
1.43 (s, 3H), 1.18 (s, 3H); HRFABMS m/z 741.2985 [M+H]+; calcd
for C41H45N2O11, 741.3018.
4.3. Biological assays
4.3.1. Cell culture
4.2.43. N-(7-{[6-Deoxy-3-O-[(1H-indole-2-yl)carbonyl]-5-C-
methyl-4-O-methyl-a-L-lyxo-hexopyranosyl]oxy}-4-methoxy-
MCF-7 and SKBR3 cells were cultured in RPMI 1640 medium
containing 10% fetal bovine serum, penicillin (100 IU/mL), strepto-
8-methyl-2-oxo-2H-1-benzopyran-3-yl)-4-methoxy-3-(3-
mycin (50 lg/mL), and 2 mM Glutamax (Gibco). Cells were grown
methyl-2-buten-1-yl)benzamide (16)
under a humidified 5% CO2 atmosphere at 37 °C and harvested at
To a solution of 15 (0.8 mg, 1.08
lMol) in CH2Cl2, imidazole
approximately 75% confluence for use in biological assays.
(0.5 mg) was added and stirred at 25 °C. After 24 h crude product
was purified by preparative TLC (eluant: Et2O) to give 16 as off
white solid (0.72 mg; 90%): mp 178–180 °C. 1H NMR (500 MHz,
CDCl3) d 9.04 (s, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 7.4 Hz,
1H, 7.60 (s, 2H), 7.58 (d, J = 8.3 Hz, 1H), 7.46 (s, 1H), 7.34 (d,
J = 7.8 Hz, 1H), 7.19 (dd, J = 1.5 and 0.7 Hz, 1H), 7.11 (dd, J = 7.7
and 0.7 Hz, 1H), 7.08 (d, J = 9.2 Hz, 1H), 6.70 (d, J = 8.9 Hz, 1H),
5.61 (dd, J = 9.9 and 2.9 Hz, 1H), 5.40 (d, J = 2.9 Hz, 1H, 5.26 (br t,
J = 7.7 Hz, 1H), 4.25 (br s, 1H), 4.06 (s, 3H), 3.85 (s, 3H), 3.74 (d,
J = 9.9 Hz, 1H), 3.58 (s, 3H), 3.29 (d, J = 7.2 Hz, 1H), 2.34 (s, 3H),
1.74 (s, 3H), 1.71 (s, 3H), 1.37 (s, 3H), 1.12 (s, 3H); HRFABMS m/z
741.2985 [M+H]+; calcd for C41H45N2O11, 741.3018.
4.3.2. Immunofluorescent flow cytometry
Cells were treated with serial dilutions of NB analogues for 24 h,
harvested with trypsin/EDTA and washed twice with PBS. NB
(500 lM) and GA (0.5 lM) treatments served as positive controls
for depletion of HER2. Control cells were treated with an equal vol-
ume of solvent vehicle (DMSO). After harvest, cells were blocked
for 20 min on ice with 10% goat serum in phosphate buffered albu-
min (PBA: PBS pH 7.6 with 1% bovine serum albumin and 0.1%
NaN3) and subsequently incubated with antibody against HER2
(20 lg/mL, clone 9G6.10, LabVision, Fremont, CA) diluted in 1%
goat serum in PBA for 30 min on ice. Cells were washed three times
with PBA and incubated for 20 min on ice protected from light with
secondary antibody (goat anti-mouse Alexa Fluor 488) diluted to a
4.2.44. N-(7-{[3-O-(Aminocarbonyl)-6-deoxy-5-C-methyl-4-O-
methyl-
a
-L
-lyxo-hexopyranosyl]oxy}-8-methyl-2-oxo-2H-1-
final concentration of 2 lg/mL. Cells were washed three times and
benzopyran-3-yl)-4-hydroxy-3-(3-methyl-2-buten-1-
mean fluorescence intensity was immediately assayed on a Guava
yl)benzamide (17)
Easy Cyte cytometer (Millipore).
NB (1a, 600.0 mg) was reacted with 3-fold excess of Cp2ZrHCl as
described in the general procedure of deoxygenation to obtain
430.0 mg of crude product as a dark brown semi solid. A portion
(170.0 mg) of this crude product mixture was stirred with
4.3.3. Cell extracts and Western blots
Cells were grown to 70% confluence in 6 well plates. Floating
cells were collected in PBS and adherent cells were collected by

5128
G. M. Kamal B. Gunaherath et al. / Bioorg. Med. Chem. 21 (2013) 5118–5129
trypsinization. Both fractions were pooled, washed twice with PBS
and re-suspended in ice-cold lysis buffer (50 mM Tris–HCl pH 7.4,
100 mM NaCl, 2 mM EDTA, 1% NP-40, 10 mM Na3VO4) plus prote-
ase inhibitors (aprotinin and leupeptin at 0.02 mg/mL, 1 mM
PMSF). After 10 min on ice, insoluble debris was removed by cen-
trifugation at 14,000 rpm for 10 min at 4 °C. Protein concentration
of clarified lysates was determined by bicinchoninic acid assay
(Pierce, Rockford, IL). After boiling in reducing SDS sample buffer
of 100 l
L assay buffer as described previously.6a After time zero,
reaction tubes were incubated at 28 °C in a water bath and assayed
as above at 15, 30, and 60 min after addition of luciferase. Mean
relative light units were calculated for each condition and time
point.
4.3.7. Cell cycle analysis
SKBR3 cells were treated with dilutions of NB analogues for
48 h, harvested with trypsin/EDTA, and washed twice with PBS.
Cells were fixed for two hours in ice-cold 70% ethanol added
drop-wise during vortex. After fixation, cells were washed twice
with PBS, re-suspended in Guava Cell Cycle Reagent (Millipore),
and assayed on a Guava Easy Cyte (Millipore) cytometer according
to manufacturer’s specifications.
for 5 min, equal amounts of total protein (20 lg) were fractionated
by 7.5% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and
transferred to nitrocellulose membranes. Membranes were
blocked for 10 min at room temperature with 3% non-fat dry milk
in 20 mM Tris–HCl pH 7.6, 0.05% Tween-20, and 0.09% NaCl (TTBS).
HER2 was detected using a mouse monoclonal antibody (clone
9G6.10, 2.0 lg/mL). Antigen/antibody complexes were detected
with the goat anti-mouse alkaline phosphatase conjugated second-
ary antibodies (Jackson Immuno Research Laboratories, Inc.). Blots
were developed with NBT/BCIP (Roche). Equal protein loading and
transfer was confirmed by Ponceau S stain (Sigma).
4.3.8. Cell survival/proliferation assay
To assess short term toxicity/anti-proliferative activity, SKBR3
cells were exposed to NB analogues overnight and then incubated
with Viacount Flex DNA-staining reagent per manufacturer’s
instructions (EMD-Millipore). Viable cell number was then mea-
sured by capillary-based flow cytometry (Guava Easy Cyte). For
standard cytotoxicity testing, MCF-7 cells were seeded in 96-well
plates at a density of 2,000 cells/well. After 24 h, serial drug dilu-
tions were added and after a further 72 h, 3-(4,5-dimethylthia-
zol-2-yl)-2,5 diphenyltetrazolium bromide (MTT, Sigma) was
4.3.4. Immunofluorescence microscopy
SKBR3 cells were seeded into 8-well chamber slides and al-
lowed to adhere overnight. Cells were treated with NB derivatives
for 18 h and fixed with 4% paraformaldehyde in PBS for 10 min.
Cells were washed with PBS and permeablized with 1% TX-100 in
PBS for 10 min. After washing, cells were blocked for 20 min with
10% goat serum in phosphate buffered albumin (PBA) and incu-
added to each well to a final concentration of 500
bated at 37 °C for 3 h. Media was removed and formazan crystals
solubilized in 150 L of DMSO by gentle agitation. Absorbance,
lg/mL and incu-
bated with antibody against HER2 (Clone 9G6.10, 20
lg/mL) di-
l
luted in 1% goat serum in PBA. Cells were washed three times
with PBS and incubated for 20 min on ice protected from light with
secondary antibody goat anti-mouse Alexa Fluor 488 diluted to a
as a measure of relative viable cell number, was measured at
570 nm with background subtraction at 650 nm using a spectro-
photometric plate reader (ThermoMax, Molecular Devices, Sunny-
vale, CA). Determinations were carried out in triplicate and
experiments were repeated at least twice. IC50 values were deter-
mined as the drug concentration inhibiting relative viable cell
number by 50% of DMSO-treated control.
final concentration of 2
and nuclei were stained with DAPI in PBS at a concentration of
g/mL. Cells were washed again and post fixed in 1% PFA for
lg/mL. Cells were washed three times
1
l
microscopic observation with UV and FITC filter cubes on an Olym-
pus microscope. Images at 160ꢁ magnification were acquired with
identical exposure settings.
Acknowledgments
4.3.5. Heat shock induction assay
We thank the Arizona Biomedical Research Commission, the
National Cancer Institute (Grant R01 CA90265) and the College of
Agriculture and Life Sciences, University of Arizona for financial
support. Dr. Cynthia David and Ms. Libia Luevano are thanked for
their technical assistance with biological assays during the initial
phase of this project.
3T3-Y9-B12 mouse fibroblasts stably transfected with a plas-
mid encoding green fluorescent protein under the transcriptional
control of a minimal consensus heat shock reporter15 were used
to measure heat shock induction activity of NB analogues. Serial
dilutions of novobiocin analogues were applied to near confluent
cell monolayers in triplicate wells of black 96-well plates and incu-
bated overnight. Negative control wells were treated with an equal
Supplementary data
volume of DMSO vehicle, not exceeding 0.25%, and GA at 0.5 lM
was used as a positive control. After briefly washing with PBS, fluo-
rescence intensity was quantified on a microplate reader (Analyst
AD, Molecular Devices, Sunnyvale, CA) with excitation/emission
filters of 485/525 nm. Relative fluorescence intensity was calcu-
lated by normalizing data to DMSO control values.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.06.042.
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