Synthesis and evaluation of radamide analogues, a chimera of radicicol and geldanamycin

Article abstract of DOI:10.1021/jo900278g

(Chemical Equation Presented) Previously, we reported the Hsp90 inhibitory activity of radamide, an open chain amide chimera of geldanamycin and radicicol. Attempts to further expand upon structure-activity relationships for this class of Hsp90 inhibitors

Full text of DOI:10.1021/jo900278g

Synthesis and Evaluation of Radamide Analogues, A Chimera of
Radicicol and Geldanamycin
M. Kyle Hadden and Brian S. J. Blagg*
Department of Medicinal Chemistry, The UniVersity of Kansas, 1251 Wescoe Hall DriVe,
Malott Hall 4070, Lawrence, Kansas 66045-7563
bblagg@ku.edu
ReceiVed February 7, 2009
Previously, we reported the Hsp90 inhibitory activity of radamide, an open chain amide chimera of
geldanamycin and radicicol. Attempts to further expand upon structure-activity relationships for this
class of Hsp90 inhibitors led to the preparation of a series of radamide analogues focused on differing
tether lengths and quinone mimics. In addition, the cup-shaped conformation adopted by the two natural
products when bound to the Hsp90 N-terminal ATP binding pocket suggests that conformationally biased
compounds may demonstrate improved binding and inhibition. The preparation and evaluation of radamide
analogues with cis/trans R,ꢀ-unsaturated amides yielded compounds that exhibit improved antiproliferative
activity. In addition, several analogues demonstrated the ability to induce degradation of Hsp90-dependent
oncogenic signaling proteins in vitro, a hallmark of Hsp90 N-terminal inhibition.
Introduction
unsaturated ketone undergo nucleophilic addition that results
in metabolites that exhibit little or no affinity for Hsp90. GDA,
while less potent than RDC in vitro, maintains inhibitory activity
in vivo.4 In fact, an analogue of GDA, 17-AAG, exhibits
enhanced affinity for tumorgenic Hsp90 and is currently in
clinical trials for the treatment of various cancers. Unfortunately,
17-AAG exhibits poor solubility and toxicity that is proving
difficult to overcome for this class of inhibitors.
Co-crystal structures of the Hsp90 N-terminal ATP binding
site bound to RDC and GDA have provided information that
was used to design chimeric inhibitors of Hsp90.5 The resor-
cinolic phenols of RDC form hydrogen bonds with Leu34,
Asp79, Gly83, and Thr171 through conserved water molecules,
mimicking key interactions of the natural adenine base (Figure
1). In contrast, the quinone moiety of GDA binds toward the
surface of the pocket in the region corresponding to the
diphosphate group of ADP, providing hydrogen bonding
interactions with Lys58 and Asp40. In addition, the 17-methoxy
substituent of the GDA quinone and the RDC epoxide form
Inhibition of the 90 kDa family of heat shock proteins (Hsp90)
has become a major target for the development of anticancer
drugs because of their well-defined role as molecular chaperones
responsible for folding nascent polypeptides and refolding
denatured proteins into their biologically active, three-dimen-
sional conformations.1 Numerous oncogenic proteins directly
associated with all six hallmarks of cancer, including Her2, Raf,
PLK, RIP, AKT, FAK, telomerase, and MET, have been shown
to be dependent upon the Hsp90 protein folding machinery.2
Hsp90 inhibition results in the simultaneous disruption of
multiple oncogenic pathways, leading to client protein degrada-
tion via the ubiquitin-proteasome pathway.
Known inhibitors of the Hsp90 protein folding process include
the natural products, geldanamycin (GDA) and radicicol (RDC)
(Figure 1). RDC, the most potent inhibitor in vitro, exhibits no
activity in vivo3 as the allylic epoxide and the R,ꢀ,γ,δ-
* To whom correspondence should be addressed. Phone: 1-785-864-2288.
Fax: 1-785-864-5326.
(1) Blagg, B. S.; Kerr, T. D. Med. Res. ReV. 2006, 26, 310–338.
(2) Maloney, A.; Workman, P. Expert Opin. Biol. Ther. 2002, 2, 3–24.
(3) Geng, X.; Yang, Z.-Q.; Danishefsky, S. J. Synlett 2004, 8, 1325–1333.
(4) Supko, J. G.; Hickman, R. L.; Grever, M. R.; Malspeis, L. Cancer
Chemother. Pharmacol. 1995, 36, 305–315.
(5) Roe, S. M.; Prodromou, C.; O’Brien, R.; Ladbury, J. E.; Piper, P. W.;
Pearl, L. H. J. Med. Chem. 1999, 42, 260–266.
10.1021/jo900278g CCC: $40.75  2009 American Chemical Society
Published on Web 06/03/2009
J. Org. Chem. 2009, 74, 4697–4704 4697

Hadden and Blagg
FIGURE 1. Key interactions of ADP, GDA, and RDC with the Hsp90 N-terminal ATP binding pocket.
moiety may provide analogues that demonstrate improved
activity and decreased toxicity.
Santi and co-workers recently reported the co-crystal structure
of 17-dimethylaminoethylamino geldanamycin (DMAG) bound
to Hsp90 and performed a series of computational studies to
understand the relationship between the bent conformation of
GDA present in the ATP binding site versus its native
conformation.11 Their studies suggest that GDA binds Hsp90
and is twisted into a bent conformation by isomerization of the
amide bond (trans f cis), which results in an entropic penalty
between 2.2 and 6.4 kcal/mol. They further suggested that
analogues of GDA that contain a cis-amide could exhibit >1000-
fold increase in Hsp90 affinity. In contrast to GDA, RDC binds
Hsp90 in a bent conformation identical to its native conformation
and exhibits little entropic penalty.5 Therefore, conformationally
biased inhibitors that contain an R,ꢀ-unsaturated amide bond
in either a cis or trans orientation represent inhibitors that are
predisposed to a bent conformation and may exhibit improved
inhibition. In this article, we describe the synthesis of saturated
and unsaturated cis/trans R,ꢀ-unsaturated amides that incorpo-
rate quinone mimics along with their inhibitory activities.
FIGURE 2. Structures of chimeric Hsp90 inhibitors.
Results and Discussion
hydrogen bonds with Lys44, emphasizing the importance of this
amino acid in inhibitor binding. We hypothesized that incor-
porating the key binding interactions provided by the resorcinol
ring of RDC and the quinone moiety of GDA into a single
molecule would result in a novel class of Hsp90 inhibitors that
could unveil structure-activity relationships (SAR) for the
natural products themselves. Radamide (Figure 2) exhibited low
micromolar inhibition of Hsp90 as measured by Her2 degrada-
tion in MCF-7 breast cancer cells.6 Interestingly, the corre-
sponding hydroquinone precursor demonstrated ∼3-fold in-
creased inhibition, suggesting that a hydrogen bond donor in
the quinone binding region results in increased activity. Similar
studies with the chimeric Hsp90 inhibitors, radester and radana-
mycin, further supported this finding, suggesting that the
hydroquinone is more active in vitro.7-9 This observation was
later confirmed by researchers at Infinity Pharmaceuticals who
identified the hydroquinone of GDA as the active form in vivo.10
These initial findings suggest that modifications to the quinone
Chemistry. The first series of radamide analogues prepared
and evaluated were derivatives incorporating varying saturated
alkyl linkers between the resorcinol and quinone moieties
(Scheme 1). The hydroquinone and quinone analogues were
prepared as previously described for radamide6 and radanamy-
cin9 with minor modification (Scheme 1). Briefly, silyl protec-
tion and chlorination of 2,4-dihydroxy-5-methyl benzoate gave
the fully protected resorcinolic ester 1, which was used as an
intermediate for subsequent analogues. Alkylation of 1 with
appropriate alkenyl bromides and LDA furnished alkenes 2-4.
Conversion of the terminal olefins to the corresponding car-
boxylic acids (8-10) and subsequent EDCI/DMAP-catalyzed
coupling with the MOM-protected aniline6 gave protected am-
ides 12-14. Deprotection of the resorcinol phenols, followed
by removal of the MOM protecting groups via in situ generated
trimethylsilyl iodide afforded a 1:1 mixture of the hydroquinone/
quinone. The instability of the hydroquinone at room temper-
ature resulted in significant oxidation to the quinone during
(6) Clevenger, R. C.; Blagg, B. S. J. Org. Lett. 2004, 6, 4459–4462.
(7) Shen, G.; Blagg, B. S. J. Org. Lett. 2005, 7, 2157–2160.
(8) Wang, M.; Shen, G.; Blagg, B. S. J. Bioorg. Med. Chem. Lett. 2006, 16,
2459–2462.
(9) Shen, G.; Wang, M.; Welch, T. R.; Blagg, B. S. J. J. Org. Chem. 2006,
71, 7618–7631.
(10) Adams, J.; Gao, Y.; Evangelinos, A. T. G.; Grenier, L.; Pak, R. H.;
Porter, J. R.; Wright, J. L. U.S. Patent Appl. Publ. US 2006019941, A1 20060126,
CAN 144: 170823 AN 2006: 74768, 2006.
(11) Jez, J. M.; Chen, J. C. H.; Rastelli, G.; Stroud, R. M.; Santi, D. V.
Chem. Biol. 2003, 10, 361–368.
4698 J. Org. Chem. Vol. 74, No. 13, 2009

Synthesis and EValuation of Radamide Analogues
SCHEME 1
SCHEME 2
corresponding trans-ester was formed through homologation
procedures with triethyl phosphonoacetate and aldehyde 5 to
give a 7:1 mixture of trans/cis-ethyl esters (Scheme 5). The
trans isomer, 63, was easily separated via flash chromatography,
and both isomers were utilized in subsequent coupling steps.
Trimethyl aluminum (Me3Al)-mediated coupling of the ester
and aniline 11, followed by deprotection and oxidation/reduction
steps described above, produced the cis- and trans-quinones,
61 and 68, and hydroquinones, 62 and 69. The anilines used
for the saturated analogues were coupled to both ester isomers
to furnish the respective trans (79a-87a) and cis (79b-83b)
conformationally biased derivatives (Scheme 6).
SCHEME 3
The majority of anilines used to construct the conformation-
ally biased amides were produced in good yields (>75%);
however, the heterocyclic anilines were unreactive toward the
cis-ester. In contrast, yields for the coupling of the same anilines
with the trans-ester were moderate (30-48%, Table 1). These
results suggested that orientation about the olefin was preventing
attachment of the activated aniline due to steric congestion.
Therefore, the corresponding cis analogues were prepared by
alkylation of resorcinol 1 with propargyl bromide upon treatment
with LDA to give the terminal alkyne, 88 (Scheme 7). 88 was
inseparable from the starting material; however, after alkylation
with methyl chloroformate, the resulting propargyl ester, 89,
cleanly separated from the starting ester under normal chro-
matographic conditions (34% overall for the two steps). Tri-
methyl aluminum-mediated coupling of the alkyne ester with
the heterocyclic anilines resulted in formation of the desired
amides, 90a-c, in good yields (63-85%), confirming steric
congestion significantly retards this reaction.
The methyl ester aniline D (Table 1) required modification
as described in the Experimental Section to prevent polymer-
ization of this compound upon exposure to trimethyl aluminum.
Reduced aniline activation times resulted in poor yields for cis-
amide coupling. However, utilizing the alkyne ester, the desired
compound 90d was furnished in excellent yield (81%). The
alkyne-linked compounds 90a-d were treated with Lindlar’s
catalyst under a hydrogen atmosphere to give the corresponding
cis isomer analogues in moderate yield. Finally, removal of the
resorcinol protecting groups furnished the cis-radamide ana-
logues 92a-d in quantitative yield.
chromatographic separation. Therefore, the mixture was sub-
jected to air oxidation in the presence of palladium acetate
immediately following MOM removal to furnish quinones
21-23. Quantitative conversion of the quinone to the corre-
sponding hydroquinones (24-26) was achieved by stirring with
excess sodium hydrosulfite in EtOAc and H2O (50:50) for 20
min at room temperature. A library of radamide analogues
(44-56) was prepared through EDCI/DMAP coupling of acids
5-7 with a variety of anilines (Schemes 2 and 3) chosen to
probe SAR between the quinone and the ATP binding pocket.
Conformationally biased radamide analogue preparation
commenced via a Still-Gennari modification of a Horner-
Wadsworth-Emmons (HWE) reaction of aldehyde 5. Utilizing
bis(2,2,2-trifluoroethyl) (methoxycarbonylmethyl)phosphonate
gave the cis-methyl ester 57 in good yield (Scheme 4). The
J. Org. Chem. Vol. 74, No. 13, 2009 4699

Hadden and Blagg
SCHEME 4
SCHEME 5
Biological Results. Upon completion of the synthesis of
radamide analogues 21-26, 44-56, 61-69, 79a-87a,
79b-83b, and 92a-d, their antiproliferative activities against
two distinct human breast cancer cell lines, an estrogen receptor
positive cell line, MCF-7, and an estrogen receptor negative,
Her2 overexpressing cell line, SKBr3, were determined. In
addition, each compound was tested for its ability to decrease
cellular levels of Her2, a Hsp90-dependent client protein
involved with oncogenic signaling pathways in several types
of cancer. The anilines utilized as quinone mimics (Scheme 3)
were chosen to take advantage of the key interactions previously
described for the Hsp90 N-terminal ATP binding site. In order
to establish the optimal distance between the quinone and
resorcinol moieties of radamide, a series of compounds contain-
ing an increasing number of methylene units were synthesized
and evaluated (21-26 and 44-49). In addition, compounds
44-49 were also designed to determine whether the nitrile could
provide additional hydrogen bonding interactions with Lys44
and improve Hsp90 inhibitory activity. The general trend for
these compounds demonstrated that increasing tether length
provided an increase in antiproliferative activity against both
cell lines (Table 2). This was most evident when comparing
the nitrile derivatives 44-49, wherein each additional methylene
resulted in the conversion of inactive analogues (44 and 47,
IC50 > 100) into analogues with IC50 values of ∼30 µM against
MCF-7 cells (Table 1). These results prompted the synthesis
of a series of radamide analogues that contained a saturated
four-carbon linker and the corresponding quinone mimics
(50-56) that provided insight into the SAR for the quinone
binding region. Analogues that contained no (52) or one (50
and 54) substituent exhibited poor antiproliferative activity,
indicating the importance of having multiple interactions with
the phosphate binding region. Substitution of the para-nitrile
(46) with a para-methyl ester (50) resulted in two-fold decreased
activity, suggesting that the nitrile at this position provides
beneficial hydrogen bond acceptor properties. Comparison of
the activities of compounds 46, 49, 51, and 55 suggested that
the pyridine/pyrazine nitrogens that were chosen to mimic the
quinone oxygens are not essential for inhibitory activity when
the nitrile is present. However, when absent, the pyridine
nitrogens are important and may facilitate isomerization of the
amide via their electron-withdrawing properties. These com-
pounds were generally more active against MCF-7 cells, and
overall, their ability to decrease Her2 levels in vitro correlated
well with their antiproliferative activity against the SKBr3 cell
line. Overall, the most active analogues contained the natural
quinone/hydroquinone of GDA, prompting the design of a new
class of radamide analogues that could demonstrate improved
binding and inhibiton.
Because GDA and RDC bind Hsp90 in a bent conformation,
it has been proposed that analogues of these natural products
predisposed to this conformation will demonstrate improved
Hsp90 binding affinity and subsequently exhibit higher inhibition
in vitro.11 Conformationally biased analogues of radamide
containing an R,ꢀ-unsaturated amide bond in either a cis or trans
orientation represent a class of inhibitors predisposed to a bent
4700 J. Org. Chem. Vol. 74, No. 13, 2009

Synthesis and EValuation of Radamide Analogues
SCHEME 6
and the hydroquinone analogues. Direct comparison of the cis-
and trans-oriented quinone mimics shows significant improve-
ment in antiproliferative activity for the majority of cis
compounds, suggesting that their predisposed bent conformation
creates improved binding interactions with Hsp90 or, at least,
less entropic penalties upon binding. Five cis analogues
demonstrated comparable (83b, 92b, and 92d) or improved (79b
and 81b) antiproliferative activity compared to the quinone/
hydroquinone analogues in at least one in vitro assay. These
quinone mimics also demonstrated the most potent inhibitory
activity in the saturated series. Surprisingly, the cis-oriented
analogue containing the para-nitrile, 79b, was completely
inactive against SKBr3 cells. This result, coupled with the
increased activity observed for most compounds against the
MCF-7 cell line, suggests that Hsp90 found in this cell line is
more susceptible to drug treatment for some unknown reason.
In a more direct determination of in vitro Hsp90 inhibition, Her2
degradation, the most active analogues were the cis- and trans-
quinone/hydroquinones (61-69), which exhibited IC50 values
from ∼35 to 57 µM.
On the basis of the data gathered for the radamide analogues,
three compounds, 23, 61, and 81b, were chosen for Western
blot analyses based on their ability to induce degradation of
Hsp90-dependent client proteins (Figure 3). Compounds 23 and
61 were chosen because they represent the most potent
analogues from their respective series in both antiproliferative
and Her2 degradation assays. Because these analogues contain
the natural quinone, 81b was also chosen for analysis as the
most active cis-radamide analogue containing a quinone mimic
(2,4,5-trifluoroaniline). Each compound induced degradation of
Hsp90 client proteins in a concentration-dependent fashion and
produced IC50 values that correspond well with antiproliferative
and Her2 results. Similar to results obtained for the antiprolif-
erative and Her2 assays, the saturated four-carbon quinone 23
was most active and produced IC50 values of ∼25 µM. Both 61
and 81b were less active, with IC50 values of ∼50-100 µM.
TABLE 1. Yield of Trimethyl Aluminum Couplings for
Heterocyclic Anilines
aniline
cis-ester
trans-ester
alkyne ester
A
B
C
D
0%
0%
0%
2%
30%
28%
48%
31%
85%
75%
63%
81%
Conclusion
Analogues of radamide, a chimeric Hsp90 inhibitor that
contains the quinone ring of GDA and the resorcinol moiety of
RDC linked through an amide bond, were prepared and
evaluated. Increasing the length of the saturated chain analogues
to four carbons resulted in increased activity for all analogues
evaluated. Derivatives containing quinone mimics designed to
investigate the importance of the various positions of the
aromatic ring were generally less active than the natural quinone,
highlighting its importance for optimal Hsp90 inhibitory activity.
A series of cis-radamide analogues that are conformationally
predisposed to adopt the cup shape necessary for high-affinity
binding to the N-terminal ATP binding site were also designed,
synthesized, and evaluated for Hsp90 inhibition. While these
analogues demonstrated reduced Hsp90 inhibitory activity,
several analogues were more active in antiproliferation assays,
suggesting the potential for an alternative target for cis
analogues. The design and synthesis of novel chimeric analogues
that are conformationally biased toward the bent cup-shaped
conformation is currently underway and will be reported in due
course.
conformation that were prepared and tested for their Hsp90
inhibitory activity. Surprisingly, both the cis-quinone (61) and
hydroquinone (62) were significantly less active in vitro than
the corresponding trans-oriented analogues (quinone, 68, and
hydroquinone, 69) (Table 3). In addition, both the cis and trans
analogues demonstrated less antiproliferative activity compared
to the corresponding saturated analogue, 26. Taken together,
these data suggest that increased rigidity of both the cis and
trans orientations prevents the quinone from forming hydrogen
bonding interactions with Asp40 and Lys98, which are observed
upon GDA binding to Hsp90. The decrease in activity exhibited
by these compounds also suggests that the orientation may
provide improved interactions for various quinone mimics within
the binding pocket. The antiproliferative activity exhibited by
the conformationally biased analogues incorporating various
quinone mimics provided additional insights into the SAR for
this series of compounds (79a-87a, 79b-83b, and 92a-d,
Table 3).
Similar to the saturated analogues, these compounds were
generally more active against MCF-7 cells. In addition, the
trans-oriented unsaturated compounds were slightly less active
than the corresponding saturated quinone mimics, further
supporting the SAR previously reported for both the quinone
Experimental Section
4-(3,5-Bis(tert-butyldimethylsilyloxy)-2-chloro-6-(methoxycar-
bonyl)phenyl)butanoic acid (9). A solution of olefin 3 (454 mg,
J. Org. Chem. Vol. 74, No. 13, 2009 4701

Hadden and Blagg
SCHEME 7
TABLE 2. In Vitro Activities of Radamide Analogues with
Varying Length Saturated Chaina
TABLE 3. In Vitro Activities of Conformationally Biased
Radamide Analoguesa
compound
MCF-7
SKBr3
Her2 ELISA
compound
MCF-7
SKBr3
Her2 ELISA
21
22
23
24
25
26
44
45
46
47
48
49
50
51
52
53
54
55
56
18.6 ( 0.9
12.9 ( 0.2
11.9 ( 0.6
14 ( 1.4
11.9 ( 0.4
11.6 ( 0.8
>100
74.9 ( 0.8
30.6 ( 3.5
>100
87.4 ( 1.7
33.0 ( 0.2
78.1 ( 4.4
43.0 ( 1.6
84.6 ( 4.5
54.9 ( 1.0
71.8 ( 9.1
72.5 ( 6.9
30.5 ( 1.2
23.7 ( 1.7
13.4 ( 1.2
13.0 ( 0.7
23.0 ( 1.2
14.5 ( 0.8
12.4 ( 0.4
>100
16.3 ( 5.0
12.1 ( 3.3
13.0 ( 3.1
16.2 ( 5.7
12.8 ( 1.7
20.5 ( 2.7
>100
83.2 ( 6.8
47.8 ( 1.7
>100
60.5 ( 7.1
48.3 ( 2.4
61.8 ( 9.0
46.4 ( 0.7
>100
37.3 ( 7.8
63.5 ( 4.9
92.3 ( 2.4
29.9 ( 2.7
61
62
68
69
25.5 ( 7.6
45.5 ( 4.0
18.5 ( 0.3
16.5 ( 2.7
66.7 ( 4.0
7.0 ( 1.6
79.3 ( 15
>100
38.7 ( 3.6
71.1 ( 4.1
44.1 ( 5.7
26.7 ( 1.8
70.7 ( 8.2
>100
34.9 ( 0.4
35.1 ( 2.7
56.5 ( 4.2
37.5 ( 1.3
90.6 ( 2.1
>100
79a
79b
80a
80b
81a
81b
82a
82b
83a
83b
84a
85a
86a
87a
92a
92b
92c
92d
95.7 ( 1.3
>100
80.0 ( 2.4
>100
>100
51.6 ( 0.3
>100
>100
61.7 ( 3.2
14.2 ( 2.0
82.4 ( 3.9
>100
80.5 ( 9.5
54.9 ( 2.8
>100
>100
>100
46.9 ( 6.6
53.2 ( 4.2
45.9 ( 6.4
>100
32.6 ( 6.5
58.9 ( 6.6
>100
82.9 ( 3.3
78.1 ( 1.7
88.1 ( 9.5
65.8 ( 3.3
>100
>100
>100
69.5 ( 2.1
62.3 ( 6.1
58.3 ( 4.5
>100
64.9 ( 3.4
68.5 ( 8.8
>100
>100
94.4 ( 2.2
47.1 ( 0.4
91.6 ( 1.8
42.7 ( 4.8
97.8 ( 1.5
54.0 ( 0.8
75.1 ( 5.5
>100
>100
20.2 ( 0.6
52.5 ( 2.7
72.6 ( 6.1
87.6 ( 5.0
35.1 ( 6.0
46.1 ( 6.3
26.4 ( 3.2
40.6 ( 2.0
20.9 ( 2.4
45.9 ( 0.5
a All values are the mean ( SEM of at least two separate experiments
performed in triplicate.
a All values are the mean ( SEM of at least two separate experiments
0.909 mmol) was dissolved in 1,4-dioxane/H2O (40 mL, 3:1) at 25
°C. Osmium tetroxide (200 µL, 4% solution in H2O) and sodium
periodate (721 mg, 3.4 mmol) were added, and the mixture was
stirred at 25 °C for 3 h. EtOAc (100 mL) and H2O (50 mL) were
added to the mixture, and the aqueous layer was removed. The
organic layer was washed with saturated aqueous NaCl (50 mL),
dried (Na2SO4), filtered, and concentrated. Chromatography (SiO2,
10:1, Hex/EtOAc) afforded 6 (375 mg, 82%) as a colorless oil.
Aldehyde 6 (325 mg, 0.648 mmol) was dissolved in a solution of
tert-butanol/2-methyl-2-butene/H2O (11.8 mL, 6:6:1). Sodium di-
hydrogen phosphate (188 mg, 1.36 mmol) and sodium chlorite (94
mg, 1.04 mmol) were added to the solution at 25 °C. The mixture
was stirred for 4 h and added directly to EtOAc (50 mL) and
saturated aqueous NaH2PO4. The aqueous layer was removed and
the organic layer washed with H2O (50 mL) and saturated aqueous
NaCl (50 mL), dried (Na2SO4), filtered, and concentrated. Chro-
matography (SiO2, 3:1, Hex/EtOAc) afforded 9 as a clear oil in
excellent yield (78%): 1H NMR (CDCl3, 400 MHz) δ 6.31 (s, 1H),
3.85 (s, 3H), 2.74 (t, J ) 7.4 Hz, 2H), 2.43 (t, J ) 6.9 Hz, 2H),
1.94 (m, 2H), 1.05 (s, 9H), 0.95 (s, 9H), 0.33 (s, 6H), 0.31 (s, 6H);
13C NMR (CDCl3, 125 MHz) δ 179.7 168.1, 152.9, 151.3, 138.5,
121.3, 118.6, 109.4, 52.2, 33.7, 31.1, 25.6 (3C), 25.5 (3C), 24.2,
18.3, 18.0, -4.3 (2C), -4.4 (2C); IR (film) νmax 3022, 2806, 2776,
1732, 1713, 1586, 1468, 1410, 1362, 1254, 1199, 1108, 1042, 966,
performed in triplicate.
939, 839 cm-1; ESI-HRMS m/z calcd for C24H42ClO6Si2 [M + H]+
517.2208, found 517.2212.
Methyl 4,6-Bis(tert-butyldimethylsilyloxy)-3-chloro-2-(4-(4-
methoxy-2,5-bis(methoxymethoxy)phenylamino)-4-oxobutyl)-
benzoate (13). A solution of 116 (648 mg, 2.7 mmol) in anhydrous
CH2Cl2 (10 mL) was added to a mixture of 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (512 mg, 2.7 mmol),
4-dimethylaminopyridine (326 mg, 2.7 mmol), and 9 (457 mg, 0.89
mmol) in anhydrous CH2Cl2 (5 mL). The mixture was stirred for
12 h at 25 °C and concentrated under reduced pressure. Chroma-
tography purification (SiO2, 3:1, Hex/EtOAc) afforded 13 as a clear
1
oil (448 mg, 68%): H NMR (CDCl3, 400 MHz) δ 8.27 (s, 1H),
7.62 (s, 1H), 6.83 (s, 1H), 6.34 (s, 1H), 5.21 (s, 4H), 3.87 (s, 6H),
3.55 (s, 3H), 3.53 (S, 3H), 2.78 (t, J ) 8.0 Hz, 2H), 2.43 (t, J )
7.4 Hz, 2H), 2.05 (m, 2H), 1.09 (s, 9H), 1.00 (s, 9H), 0.27 (s, 6H),
0.24 (s, 6H); 13C NMR (CDCl3, 125 MHz) δ 170.2, 168.2, 152.9,
151.3, 146.2, 141.7, 140.8, 138.8, 121.7, 121.3, 118.7, 110.9, 109.4,
100.9, 96.3 (2C), 56.4 (2C), 56.3, 52.3, 37.3, 31.3, 25.6 (3C), 25.5
(3C), 25.1, 18.4, 18.0, -4.4 (2C), -4.5 (2C); IR (film) νmax 3350,
2953, 2932, 2897, 2858, 2833, 1732, 1686, 1585, 1566, 1469, 1409,
4702 J. Org. Chem. Vol. 74, No. 13, 2009

Synthesis and EValuation of Radamide Analogues
159.9, 158.2, 142.7, 139.5, 128.5, 125.6, 111.6, 104.5, 102.2, 56.1,
51.9, 36.8, 31.4, 24.8; IR (film) νmax 3306, 3194, 3090, 2952, 2927,
2853, 1709, 1657, 1599, 1499, 1414, 1319, 1173, 1128, 953, 877
cm-1; ESI-HRMS m/z calcd for C19H19ClNO8 [M + H]+ 424.0799,
found 424.0794.
(Z)-Methyl 4,6-Bis(tert-butyldimethylsilyloxy)-3-chloro-2-(5-
methoxy-5-oxopent-3-enyl)benzoate (57). 18-Crown-6 (3.99 g, 15
mmol) and bis(2,2,2-trifluoroethyl) (methoxycarbonylmethyl)phos-
phonate (677 µL, 3.2 mmol) were cooled to -78 °C in anhydrous
THF. KHMDS (10 mL of a 0.5 M solution in toluene, 5 mmol)
was added dropwise and the solution stirred at -78 °C for 1 h. A
solution of 5 (1.18 g, 2.5 mmol) in anhydrous THF (10 mL) was
added dropwise and the solution stirred at -78 °C for 3 h. The
solution was warmed to rt, diluted with EtOAc (50 mL), and
saturated NH4Cl (50 mL) and the aqueous layer removed. The
organic layer was washed with H2O (50 mL), saturated NaCl (50
mL), dried (Na2SO4), filtered, and concentrated. Chromatography
(SiO2, 20:1, Hex/EtOAc) afforded 57 as a clear oil in good yield
(842 mg, 62%): 1H NMR (CDCl3, 400 MHz) δ 6.32 (s, 1H), 6.24
(m, 1H), 5.78 (d, J ) 11.4 Hz, 1H), 3.87 (s, 3H), 3.70 (s, 3H),
2.94 (m, 2H), 2.80 (t, J ) 7.6 Hz, 2H), 1.05 (s, 9), 0.95 (s, 9H),
0.22 (s, 6H), 0.20 (s, 6H); 13C NMR (CDCl3, 125 MHz) δ 168.1,
166.6, 152.9, 151.3, 148.5, 138.2, 121.3, 119.9, 118.7, 109.5, 52.1,
51.0, 30.9, 28.7, 25.6 (3C), 25.5 (3C), 18.4, 18.0, -4.3 (2C), -4.4
(2C); IR (film) νmax 2962, 2932, 2897, 2843, 2858, 1724, 1643,
1585, 1557, 1470, 1356, 1266, 1163, 1097, 1042, 960, 783 cm-1
;
ESI-HRMS m/z calcd for C26H44ClO6Si2 [M + H]+ 544.2257, found
544.2264.
FIGURE 3. Hsp90 client protein degradation induced by 23 (top), 61
(middle), or 81b (bottom). Compound, at varying concentrations (µM),
was evaluated for its ability to downregulate several client proteins as
described in the Experimental Section. GA (500 nM) and DMSO were
used as positive and negative controls, respectively.
(Z)-Methyl 4,6-Bis(tert-butyldimethylsilyloxy)-3-chloro-2-(5-
(4-methoxy-2,5-bis(methoxymethoxy)phenylamino)-5-oxopent-
3-enyl)benzoate (58). To a solution of 11 (101 mg, 0.42 mmol) in
anhydrous CH2Cl2 (5 mL) at 0 °C was added trimethylaluminum
(0.42 mmol, 2 M in toluene). The mixture was warmed to 25 °C
for 1 h before dropwise addition to a solution of 57 (93.7 mg, 0.17
mmol) and potassium carbonate (310 mg, 2.2 mmol) in anhydrous
CH2Cl2 at 0 °C. The mixture was warmed to 25 °C and stirred for
3 h before quenching via the dropwise addition of saturated
NaHCO3 (5 mL) solution. H2O (10 mL) and EtOAc (15 mL) were
added, and the aqueous layer was removed. The organic layer was
washed with saturated NaCl (5 mL), dried (Na2SO4), filtered, and
concentrated. Chromatography (SiO2, 5:1, Hex/EtOAc) afforded
58 as a yellow oil in good yield (62%): 1H NMR (CDCl3, 400 MHz)
δ 8.31 (s, 1H), 7.63 (s, 1H), 6.31 (s, 1H), 6.12 (m, 1H), 5.87 (d, J
) 11.4 Hz, 1H), 5.20 (s, 2H), 5.18 (s, 2H), 3.88 (s, 3H), 3.86 (s,
3H), 3.54 (s, 3H), 3.50 (s, 3H), 3.04 (m, 2H), 2.85 (d, J ) 7.9 Hz,
2H), 1.05 (s, 9H), 0.95 (s, 9H), 0.23 (s, 6H), 0.21 (s, 6H); 13C NMR
(CDCl3, 125 MHz) δ 168.2, 163.7, 152.9, 151.3, 146.3, 144.9,
141.5, 140.8, 138.4, 123.3, 121.8, 121.4, 118.7, 110.5, 109.4, 100.8,
96.2 (3C), 56.4, (2C), 52.2, 31.2, 28.4, 25.6 (3C), 25.5 (3C), 18.4,
18.0, -4.3 (2C), -4.4 (2C); IR (film) νmax 2953, 2932, 2897, 2858,
1732, 1680, 1585, 1468, 1408, 1355, 1207, 1151, 995, 941, 679
cm-1; ESI-HRMS m/z calcd for C36H57ClNO10Si2 [M + H]+
754.3209, found 754.3212.
(E)-Methyl 4,6-Bis(tert-butyldimethylsilyloxy)-3-chloro-2-(5-
ethoxy-5-oxopent-3-enyl)benzoate (63). Triethyl phosphonoac-
etate (1.34 g, 6.0 mmol) in anhydrous THF was cooled to -78
°C. LiHMDS (6 mL of a 1.0 M solution, 6.0 mmol) was added
dropwise and the solution warmed to rt for 5 min. The solution
was recooled to -78 °C, and 5 (1.1 g, 2.4 mmol) in anhydrous
THF was added dropwise and the solution stirred for 2 h. The
solution was warmed to rt, diluted with EtOAc (50 mL), and
saturated NH4Cl (50 mL) and the aqueous layer removed. The
organic layer was washed with H2O (50 mL), saturated NaCl
(50 mL), dried (Na2SO4), filtered, and concentrated. Chroma-
tography (SiO2, 20:1, Hex/EtOAc) afforded 600 mg of a 7:1
mixture of trans:cis (63/64) isomers as a clear oil (60% overall
yield). Further chromatography of this mixture (SiO2, CH2Cl2)
afforded pure trans and cis iosmers: 1H NMR (CDCl3, 400 MHz)
δ 6.95-7.05 (m, 1H), 6.32 (s, 1H), 5.85 (d, J ) 15.7 Hz, 1H),
1229, 1207, 995 cm-1; ESI-HRMS m/z calcd for C30H45ClN3O5Si2
[M + H]+ 618.2586, found 618.2585.
Methyl 3-Chloro-4,6-dihydroxy-2-(4-(4-methoxy-2,5-bis(me-
thoxymethoxy)phenylamino)-4-oxobutyl)benzoate (16). A solu-
tion of 13 (300 mg, 0.4 mmol) was dissolved in 10 mL of THF at
rt. Tetrabutylammonium fluoride (2 mL of 1 M soln in THF, 2.0
mmol) was added and the mixture stirred for 1 h. Saturated NH4Cl
(3 mL) was added and the aqueous layer washed three times with
EtOAc (20 mL). The combined EtOAc layers were washed with
saturated NaCl (5 mL), dried (Na2SO4), filtered, and concentrated.
Chromatography (SiO2, 1:1, Hex/EtOAc) afforded 16 as a white
amorphous solid in excellent yield (170 mg, 82%): 1H NMR
(CDCl3, 400 MHz) δ 11.41 (s, 1H), 8.21 (s, 1H), 7.57 (s, 1H),
6.74 (s, 1H), 6.49 (s, 1H), 5.13 (s, 4H), 3.89 (s, 3H), 3.79 (s, 3H),
3.45 (s, 6H), 3.11 (t, J ) 8.2 Hz, 2H), 2.41 (t, J ) 7.3 Hz, 2H),
1.95 (m, 2H); 13C NMR (CDCl3, 125 MHz) δ 171.1, 170.2, 163.1,
156.3, 146.3, 142.7, 141.6, 140.7, 121.4, 113.9, 110.6, 106.6, 102.6,
100.8, 96.4, 96.2, 56.5, 56.4, 56.3, 52.7, 37.8, 32.2, 32.3; IR (film)
ν
max 3342, 2995, 2933, 1659, 1651, 1580, 1502, 1439, 1391, 1319,
1150, 1078, 991, 804 cm-1; ESI-HRMS m/z calcd for C23H29ClNO10
[M + H]+ 514.1480, found 514.1490.
Methyl 3-Chloro-4,6-dihydroxy-2-(4-(4-methoxy-3,6-dioxo-
cyclohexa-1,4-dienylamino)-4-oxobutyl)benzoate (22). A solution
of 16 (35.7 mg, 0.07 mmol) and sodium iodide (105 mg, 0.7 mmol)
was dissolved in anhydrous CH2Cl2/MeCN (5 mL, 1:1) under argon.
Chlorotrimethylsilane (76 mg, 0.7 mmol) was added and the
reaction stirred at rt for 30 min. The solvent was removed under
reduced pressure and the crude mixture taken up in EtOAc (5 mL).
Palladium acetate (10 mg) was added and the reaction stirred at Rt
for 6 h. The solution was filtered through Celite, concentrated, and
purified by preparative thin layer chromatography (1:2, Hex/EtOAC)
to yield a yellow amorphous solid in modest yield (8.6 mg, 29%
1
over two steps): H NMR (acetone-d, 400 MHz) δ 8.94 (s, 1H),
7.46 (s, 1H), 6.50 (s, 1H), 6.05 (s, 1H), 3.98 (s, 3H), 3.90 (s, 3H),
3.15 (t, J ) 7.90 Hz, 2H), 2.77 (t, J ) 7.2 Hz, 2H), 2.09 (m, 2H);
13C NMR (acetone-d, 125 MHz) δ 182.6, 181.9, 172.9, 170.7, 161.6,
J. Org. Chem. Vol. 74, No. 13, 2009 4703

Hadden and Blagg
4.20 (q, 2H), 3.86 (s, 3H), 2.79 (t, J ) 7.6 Hz, 2H), 2.48 (m,
2H), 1.30 (t, J ) 7.5 Hz, 3H), 1.05 (s, 9H), 0.95 (s, 9H), 0.26
(s, 6H), 0.23 (s, 6H); 13C NMR (CDCl3, 100 MHz) δ 168.0,
166.6, 153.0, 151.4, 147.8, 138.1, 121.7, 121.1, 118.5, 109.6,
60.2, 52.2, 31.9, 30.7, 25.6 (3C), 25.5 (3C), 18.4, 18.0, 14.3,
-4.3 (2C), -4.4 (2C); IR (film) νmax 2955, 2896, 2888, 1726,
Methyl 4,6-Bis(tert-butyldimethylsilyloxy)-3-chloro-2-(5-(4-
(methoxycarbonyl)phenylamino)-5-oxopent-3-ynyl)benzoate (90d).
To a solution of methyl 4-aminobenzoate (176 mg, 1.16 mmol) in
anhydrous CH2Cl2 (5 mL) at 0 °C was added trimethylaluminum
(1.16 mmol, 2 M in toluene), and the mixture was stirred for 3
min before dropwise addition to a solution of 89 (125.5 mg, 0.23
mmol) and potassium carbonate (416 mg, 3.0 mmol) in anhydrous
CH2Cl2 at 0 °C. The mixture was warmed to 25 °C and stirred for
3 h before quenching via the dropwise addition of saturated
NaHCO3 (5 mL) solution. H2O (10 mL) and EtOAc (15 mL) were
added, and the aqueous layer was removed. The organic layer was
washed with saturated NaCl (5 mL), dried (Na2SO4), filtered, and
concentrated. Chromatography (SiO2, 3:1, Hex/EtOAc) afforded
1656, 1566, 1431, 1410, 1253, 1198, 1105, 1042, 841, 783 cm-1
;
ESI-HRMS m/z calcd for C27H46ClO6Si2 [M + H]+ 557.2521,
found 557.2535.
(Z)-Methyl 4,6-Bis(tert-butyldimethylsilyloxy)-3-chloro-2-(5-
1
ethoxy-5-oxopent-3-enyl)benzoate (64): H NMR (CDCl3, 400
MHz) δ 6.33 (s, 1H), 6.23 (m, 1H), 5.77 (d, J ) 11.4 Hz, 1H),
4.16 (q, J ) 7.3 Hz, 3H), 3.85 (s, 3H), 2.96 (m, 2H), 2.81 (t, J )
7.3 Hz, 2H), 1.29 (t, J ) 7.2 Hz, 2H), 1.05 (s, 9H), 0.95 (s, 9H),
0.23 (s, 6H), 0.21 (s, 6H); 13C NMR (CDCl3, 100 MHz) δ 168.1,
166.2, 152.9, 151.3, 148.3, 138.4, 121.3, 120.3, 118.7, 109.5, 59.8,
52.1, 30.9, 28.6, 25.6 (3C), 25.5 (3C), 18.4, 18.0, 14.3, -4.3 (2C),
-4.4 (2C); IR (film) νmax 2953, 2932, 2897, 2887, 2858, 1724,
1643, 1585, 1557, 1470, 1356, 1254, 1163, 1097, 1042, 960, 783
cm-1; ESI-HRMS m/z calcd for C27H46ClO6Si2 [M + H]+ 557.2521,
found 557.2244.
1
90d as a yellow oil in excellent yield (81%): H NMR (CDCl3,
400 MHz) δ 9.34 (s, 1H), 7.98 (d, J ) 8.6 Hz, 2H), 7.69 (d, J )
8.6 Hz, 2H), 6.38 (1H), 3.94 (s, 3H), 3.89 (s, 3H), 2.96 (t, J ) 6.1
Hz, 2H), 2.78 (t, J ) 6.5 Hz, 2H), 1.05 (s, 9H), 0.95 (s, 9H), 0.23
(s, 6H), 0.21 (s, 6H); 13C NMR (CDCl3, 100 MHz) δ 169.1, 166.7,
153.4, 151.6, 151.2, 142.4, 136.4, 130.7 (2C), 125.4, 121, 118.8,
(2C), 118.6, 109.8, 86.7, 76.8, 52.8, 52.2, 29.9, 25.6 (3C), 25.4
(3C), 18.3, 18.0, 17.9, -4.3 (2C), -4.4 (2C); IR (film) νmax 2952,
2932, 2858, 2231, 1721, 1280, 1661, 1587, 1531, 1433, 1256, 1111,
Methyl 2-(But-3-ynyl)-4,6-bis(tert-butyldimethylsilyloxy)-3-
chlorobenzoate (88). 1 (5.88 g, 13.1 mmol) was dissolved in
anhydrous THF and cooled to -78 °C. Lithium diisopropyl amide
(8.56 mL of a 2 M solution, 17.1 mmol) was added and the mixture
stirred at -78 °C for 5 min. Propargyl bromide (12.5 g, 105 mmol)
was added dropwise, and the solution was warmed to rt over 2 h.
The solution was diluted with EtOAc (75 mL) and saturated NH4Cl
(75 mL) and the aqueous layer removed. The organic layer was
washed with H2O (75 mL), saturated NaCl (75 mL), dried (Na2SO4),
filtered, and concentrated. Chromatography (SiO2, 50:1, Hex/
EtOAc) afforded a 1:3 mixture of 1 and 88 as a clear oil that was
1044, 841, 901, 678 cm-1
; ESI-HRMS m/z calcd for
C33H47ClNO7Si2 [M + H]+ 660.2580, found 660.2591.
(Z)-Methyl 4,6-Bis(tert-butyldimethylsilyloxy)-3-chloro-2-(5-
(5-cyanopyridin-2-ylamino)-5-oxopent-3-enyl)benzoate (91a). A
solution of 90a (47.5 mg, 0.08 mmol) and Lindlar’s catalyst (10
wt %) EtOAc (2 mL) was stirred at room temperature for 1 h under
a H2 atmosphere. The mixture was filtered through a plug of Celite.
Chromatography (SiO2, 10:1, Hex/EtOAc) afforded 91a in moderate
1
yield as a clear oil (48%): H NMR (CDCl3, 400 MHz) δ 8.56 (s,
1H), 8.43 (d, J ) 8.7 Hz, 1H), 8.16 (s, 1H), 7.94 (dd, J ) 8.8, 2.2
Hz, 1H), 6.33 (m, 2H), 5.89 (d, J ) 11.5 Hz, 1H), 3.88 (s, 3H),
3.08 (m, 2H), 2.87 (t, J ) 8.1 Hz, 2H), 1.05 (s, 9H), 0.95 (s, 9H),
0.23 (s, 6H), 0.21 (s, 6H); 13C NMR (CDCl3, 100 MHz) δ 168.2,
164.2, 154.0, 153.0, 151.6, 151.5, 149.2, 141.4, 138.1, 121.7, 121.2,
118.7, 116.9, 113.4, 109.5, 104.7, 52.2, 30.9, 28.7, 25.6 (3C), 25.5
(3C), 18.4, 18.0, -4.3 (2C), -4.4 (2C); IR (film) νmax 3546, 2867,
2116, 1666, 1543, 1495, 1267, 1225, 1028, 956 cm-1; ESI-HRMS
m/z calcd for C31H45ClN3O5Si2 [M + H]+ 630.2586, found
630.2571.
1
inseparable on a scale larger than 20 mg: H NMR (CDCl3, 400
MHz) δ 6.30 (s, 1H), 3.85 (s, 3H), 2.90 (t, J ) 8.2 Hz, 2H), 2.44
(t, J ) 7.9 Hz, 2H), 1.97 (s, 1H), 1.05 (s, 9H), 0.95 (s, 9H), 0.23
(s, 6H), 0.21 (s, 6H); 13C NMR (CDCl3, 125 MHz) δ 167.9, 153.0,
151.5, 137.5, 121.2, 118.7, 109.8, 83.5, 68.5, 52.3, 31.5, 25.6 (3C),
25.5 (3C), 18.5, 18.4, 18.0, -4.3 (2C), -4.4 (2C); IR (film) νmax
3311, 2929, 2858, 2120, 1732, 1585, 1470, 1361, 1253, 1202, 1108,
962, 842 cm-1; ESI-HRMS m/z calcd for C24H40ClO4Si2 [M + H]+
483.2153, found 483.2135.
Antiproliferation and Her2 Degradation Assays. These assays
for Hsp90 inhibition were performed as described previously.12
Western Blot Analyses of Naphthoquinones. MCF-7 cells were
seeded (1 × 106/plate) in culture dishes and allowed to attach
overnight. Compounds 23, 61, and 81b were added at varying
concentration and incubated (37 °C, 5% CO2) for 24 h. Cells were
harvested and analyzed for Hsp90 client protein degradation as
described previously.9
Methyl 4,6-Bis(tert-butyldimethylsilyloxy)-3-chloro-2-(5-meth-
oxy-5-oxopent-3-ynyl)benzoate (89). A mixture of 1 and 88 (1.635
g, 3.4 mmol) was dissolved in anhydrous THF and cooled to -78
°C. Lithium diisopropyl amide (2.2 mL of a 2 M solution, 4.4
mmol) was added and the mixture stirred at -78 °C for 5 min.
Methyl chloroformate (2.1 mL, 33.9 mmol) was added dropwise,
and the solution was warmed to rt over 2 h. The solution was diluted
with EtOAc (75 mL) and saturated NH4Cl (75 mL) and the aqueous
layer removed. The organic layer was washed with H2O (75 mL),
saturated NaCl (75 mL), dried (Na2SO4), filtered, and concentrated.
Chromatography (SiO2, 50:1, Hex/EtOAc) afforded 89 as a pure
yellow solid (34% overall two steps from 1): mp ) 78 °C; 1H NMR
(CDCl3, 400 MHz) δ 6.29 (s, 1H), 3.84 (s, 3H), 3.73 (s, 3H), 2.92
(d, J ) 7.9 Hz, 2H), 2.59 (d, J ) 8.1 Hz, 2H), 1.01 (s, 9H), 0.92
(s, 9H), 0.21 (s, 6H), 0.19 (s, 6H); 13C NMR (CDCl3, 125 MHz) δ
167.8, 154.2, 153.1, 151.6, 136.7, 121.2, 118.6, 109.9, 88.5, 73.0,
52.6, 52.4, 30.4, 25.6 (3C), 25.4 (3C), 18.6, 18.3, 18.0, -4.3 (2C),
-4.4 (2C); IR (film) νmax 2953, 2932, 2887, 2241, 1715, 1585,
1567, 1470, 1433, 1252, 1093, 961 cm-1; ESI-HRMS m/z calcd
for C26H42ClO6Si2 [M + H]+ 541.2208, found 541.2203.
Acknowledgment. This work was supported by the National
Institutes of Health (CA109265) and the Kansas Cancer Center
(Postdoctoral Fellowship, M.K.H.).
Supporting Information Available: Full characterization of
key intermediates and all final analogues. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO900278G
(12) Hadden, M. K.; Hill, S. A.; Davenport, J.; Matts, R. L.; Blagg, B. S. J.
Bioorg. Med. Chem. 2009, 17, 634–640.
4704 J. Org. Chem. Vol. 74, No. 13, 2009