Design, synthesis, and biological activity of bicyclic radester analogues as Hsp90 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.10.091

Bicyclic radester analogues have been synthesized and evaluated for Hsp90 inhibitory activity. These analogues induce concentration-dependent degradation of Hsp90-dependent client proteins with the six-membered bicyclic analogues manifesting increased act

Full text of DOI:10.1016/j.bmcl.2009.10.091

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6845–6850
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological activity of bicyclic radester analogues
as Hsp90 inhibitors
*
Vinod D. Jadhav, Adam S. Duerfeldt, Brian S. J. Blagg
Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, Malott 4070, Lawrence, KS 66045-7562, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Bicyclic radester analogues have been synthesized and evaluated for Hsp90 inhibitory activity. These ana-
logues induce concentration-dependent degradation of Hsp90-dependent client proteins with the six-
membered bicyclic analogues manifesting increased activity versus the five-membered counterparts.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 8 September 2009
Revised 19 October 2009
Accepted 21 October 2009
Available online 25 October 2009
Keywords:
Hsp90
Geldanamycin
Radicicol
Anti-cancer
The 90 kDa heat shock proteins (Hsp90) are well characterized
molecular chaperones responsible for the conformational matura-
tion of nascent polypeptides and the rematuration of denatured
proteins.1,2 Inhibition of Hsp90 disrupts the protein folding pro-
cess, resulting in client protein degradation via the ubiquitin-pro-
teasome pathway. Previous studies have demonstrated that the
Hsp90 multiprotein complex prevalent in tumor cells exhibits a
higher affinity for N-terminal ligands than the homodimeric form
present in non-transformed cells. It is well known that malignant
cells are highly dependent upon the Hsp90 protein folding machin-
ery for cell survival due to the over expression of proteins needed for
continual growth in hostile environments.3 Consequently, inhibi-
tion of Hsp90 results in combinatorial disruption of multiple signal-
ing pathways that are essential to tumor cell survival and represents
a promising strategy for the development of therapeutics.4
Hsp90 is an ATP-dependent protein comprised of two nucleo-
tide-binding domains, one of which is located at the N-terminus
and the other at the C-terminus.5 ATP hydrolysis at the N-terminus
provides the requisite energy necessary for folding nascent poly-
peptides into their biologically active, three-dimensional confor-
mations. Thus, disruption of this ATPase activity results in
destabilization of the heteroprotein complex, which leads to the
subsequent degradation of clients.3
mycin (GDA)6 and radicicol (RDC, Fig. 1)6 are two prominent exam-
ples previously reported. RDC is the most potent of these inhibitors
in vitro, however, in vivo it is rapidly converted into metabolites
that exhibit little or no affinity for Hsp90.7 In contrast, GDA is less
potent than RDC in vitro, but in cellular assays manifests greater
affinity for the Hsp90 heteroprotein complex prevalent in malig-
nant cells.3 Although two derivatives of GDA have entered clinical
trials for the treatment of several cancers, toxicity and formulation
difficulties remain.
The co-crystal structures of GDA and RDC bound to Hsp908 have
been solved and reveal important binding interactions manifested
by these natural products. In contrast to the extended conforma-
tion of GDA found in solution, the natural product binds Hsp90
in a bent, C-shaped conformation that contains an unusual cis-
amide bond. Consequently, it appears the process of GDA binding
to Hsp90 results in an entropic penalty, which has been studied
in some detail.9 In contrast, RDC exists in the same bent conforma-
tion bound or unbound to Hsp90, producing favorable entropy
upon binding. As a result of these thermodynamic data, it has been
proposed that GDA analogues that contain conformational rigidity
would yield compounds that exhibit enhanced Hsp90 affinity
through minimization of entropic penalties.10–14
Recently, chimeric Hsp90 inhibitors were disclosed in which
pharmacophores from both natural products were combined to
provide a novel scaffold. Our laboratory has developed15–17 this
class of chimeric analogues that result from hybridization of the
resorcinol ring of RDC and the quinone of GDA. These compounds
bind to the Hsp90 N-terminus and prevent ATP hydrolysis, similar
to the natural products.18 Moreover, radester (Fig. 1) exhibits more
potent ATPase inhibition than GDA.16
N-terminal inhibitors of Hsp90 manifest their activity by bind-
ing competitively to the N-terminal ATP-binding site and prevent-
ing Hsp90-catalyzed hydrolysis of ATP. To date, several natural
products have been identified as N-terminal inhibitors. Geldana-
* Corresponding author.
E-mail address: bblagg@ku.edu (B.S.J. Blagg).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.091

6846
V. D. Jadhav et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6845–6850
O
O
O
OMe
O
O
O
OMe
O
O
N
H
H
N
H
O
O
O
HO
O
Cl
O
O
Cl
HO
OH
O
O
HO
OH
NH2
Geldanamycin (GDA)
Radester
Radicicol (RDC)
Figure 1. Known inhibitors of Hsp90.
Figure 2. Proposed conformationally constrained analogues.
Figure 3. Overlay of compounds 1 (orange) and 2 (green) with RDC in the Hsp90 N-terminal ATP-binding domain. The resorcinol plane is depicted as a purple line.

V. D. Jadhav et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6845–6850
6847
of hydrogen bonding interactions with Asp 79, Gly 83 and Thr
171. Thus, inhibitors existing in a bent conformation that exhibit
a planar carbonyl are hypothesized to minimize the entropic pen-
alty upon binding to Hsp90 and exhibit higher affinity due to in-
creased utilization of the hydrogen bonding network. Therefore
we proposed a series of conformationally constrained chimeric
analogues exhibiting both a bent conformation and conformation-
ally constrained carbonyls (Fig. 2).
1−6
Analogues
Aldol
condensation
MOMO
O
OMOM
OMe
Recently, Duerfeldt et al.19 utilized the chimera radamide as a
model to demonstrate that conformationally constrained cis-amide
analogues demonstrate improved affinity. Radester provides an
additional opportunity to probe conformational constraints
through manipulation of the resorcinol motif. Preliminary docking
studies which overlay the designed inhibitors 1 and 2 with RDC
indicate the carbonyl of the bicyclic analogues to exhibit greater
planarity than RDC (Fig. 3). Defining the plane of the resorcinol
as 0° allowed rotation from this plane to be measured for com-
pounds 1 and 2. As depicted in Figure 3, the radicicol carbonyl
exhibits an angle of ꢀ45°, whereas 1 exhibits an angle of ꢀ9°,
and compound 2 deviates ꢀ8°, supporting the conformational con-
straint of the carbonyl moieties. Herein, we present the design,
syntheses, and biological evaluation of chimeric Hsp90 inhibitors
containing a conformationally constrained bicyclic ring system.
Retrosynthetically, analogues 1–6 were envisioned to be con-
structed via an aldol condensation between nitro-aldehyde 16
and the homologated bicyclic ketones, 13–14 (Scheme 1). Alde-
hyde 16 was proposed to originate from alcohol 15, which can be
prepared in three steps from commercially available methoxyhy-
droquinone. Cyclic ketones 13–14 were to be prepared from bicy-
clic ketone compounds, 7–8, which have been previously
synthesized from commercially available 3,5-dimethoxybenz-
aldehyde.20,21
+
MOMO
n
O2N
O
Cl
OMOM
16
13 n=1
14
n=2
OMe
Cl
OH
O
OMe
MeO
n
OH
7 n=1
8 n=2
Scheme 1. Retrosynthetic analysis.
Commencing with the synthesis of intermediates 13–14 and 16,
as illustrated in Scheme 2, ketones 7–8 were constructed following
known procedures.20,21 Cleavage of the methyl ethers afforded
phenolic compounds 9–10. Resorcinol 9 was obtained by treating
7 with boron tribromide at À78 °C and 10 was prepared by reac-
tion of 8 with hydrobromic acid in acetic acid at 110 °C. Treatment
of compounds 9–10 with sodium hydride and methoxy methyl
chloride gave the corresponding MOM-protected phenols 11–12
in good yields. Mild conditions enlisting calcium hypochlorite
afforded the chlorinated aromatics, 13–14. Quinone precursor 16
Overcoming the entropic barrier associated with the conforma-
tional rearrangement observed by N-terminal ligands upon bind-
ing Hsp90 is an important consideration for the development of
new inhibitory scaffolds. Analyses of co-crystal structures of RDC
and radester bound to Hsp90 revealed the ester carbonyl in radest-
er to be more planar than the same moiety in RDC. The planarity of
this carbonyl potentially allows for greater electron delocalization
between the ester carbonyl and the phenols, resulting in an elec-
tron rich carbonyl and more acidic phenols. High electron density
at this carbonyl is predicted to be important due to its mediation
OMe
OH
MOMO
O
O
O
b
a
MeO
HO
MOMO
n
n
n
R'
7 n=1
8 n=2
9
n=1, (82%)
11 n=1, (79%)
10 n=2, (88%)
R '= H
R '= Cl
12 n=2, (85%)
c
13 n=1, (65%)
14 n=2, (60%)
OMOM
OMe
OMOM
OMe
d
O2N
OH
O2N
O
OMOM
15
OMOM
16, (69%)
Scheme 2. Reagents and conditions: (a) BBr3, À78 °C to rt, 5 h or HBr, AcOH, 110 °C, 48 h; (b) NaH, DMF, MOMCl, 0 °C to rt, 2 h; (c) Ca(OCl)2, acetone, AcOH/H2O (1:10), rt, 2 h;
(d) PCC, DCM, MS (4 Å), rt, 2 h.

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V. D. Jadhav et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6845–6850
was prepared by PCC oxidation of alcohol 15, which was readily
available via literature procedures.15
21–22, upon treatment with phenylformate. Removal of the
methoxymethyl (MOM) protecting groups in situ with trimethyl-
silyl iodide resulted in formation of the corresponding hydroqui-
nones, 1–2,22,23 which were subsequently oxidized to the desired
quinones 3–4 with Frémy’s salt (potassium nitrosodisulfonate) in
buffered solution.24,25
Selective reduction of the nitro groups was required to access
alkenes 5–6, which was problematic due to competing redox-sen-
sitive groups (Scheme 3). The best results obtained utilize zinc me-
tal and acetic acid in dichloromethane, in which case anilines 23–
24 were subsequently converted to the corresponding formamides,
Synthesis of compounds 1–6 was accomplished by following
the sequence of reactions depicted in Scheme 3. Aldol condensa-
tion between ketones 13–14 and aldehyde 16 followed by dehy-
dration of the incipient secondary alcohols enlisting phosphorus
oxychloride in pyridine, provided alkenes 17–18 in good yields.
Simultaneous reduction of the nitro and alkene moieties of
17–18 employing palladium-carbon under a hydrogen atmosphere
provided anilines 19–20. To mimic the amide functionality present
in GDA, the aniline was converted to the corresponding formamide,
OR
OR
OR
OMe
O
OMe
O
OR
O
O2N
a
H2N
Cl
b
OR
n
n
RO
n
Cl
Cl
R= MOM
RO
OR
RO
OR
13 n=1
14 n=2
17
19
n=1, (63%)
n=1, (94%)
18 n=2, (30%)
20 n=2, (90%)
d
c
OR
OR
OR
OMe
O
OMe
OMe
O
O
H
N
H2N
H
N
H
c
H
OR
n
OR
OR
n
n
Cl
RO
Cl
Cl
RO
O
O
OR
RO
OR
OR
25 n=1, (66%)
26 n=2, (60%)
23 n=1, (32%)
24 n=2, (30%)
21 n=1, (67%)
22 n=2, (65%)
e
e,f
O
O
OH
OMe
OMe
OMe
O
O
O
O
H
N
H
H
N
H
H
N
H
f
O
O
OH
n
n
n
Cl
HO
Cl
HO
Cl
HO
O
O
OH
OH
OH
5 n=1, (15%)
6 n=2, (12%)
3 n=1, (35%)
4 n=2, (29%)
1 n=1, (36%)
2 n=2, (32%)
Scheme 3. Reagents and conditions: (a) (i) LDA, À78 °C to rt, THF,16, 2 h, (ii) POCl3, pyridine, rt, 6 h; (b) H2, Pd/C, EtOAc, 6 h; (c) PhOCHO, DCM, 40 °C, 12 h; (d) Zn, AcOH,
DCM, rt, 10 min; (e) NaI, TMSCl, DCM/CH3CN, rt, 1 h; (f) Frémy’s salt, acetone, rt, 15 min R = MOM for all compounds in this scheme.

V. D. Jadhav et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6845–6850
6849
Table 1
bered analogues exhibited more potent activity than the corre-
sponding five-membered analogues, and exhibited similar IC50
values against both cancer cell lines. In contrast, the five-mem-
bered analogues exhibited approximately three-old higher activity
against MCF7 cells than SKBr3 cells.
In order to confirm the growth inhibitory activity manifested by
these compounds was a consequence of Hsp90 inhibition, com-
pounds 1 and 2 were subjected to Western blot analyses. As ex-
pected (Fig. 4), compounds 1 and 2 resulted in dose-dependent
client protein degradation. Similarly, N-terminal inhibitory activity
was evidenced by the concentration-dependant induction of heat
shock response as demonstrated by increasing Hsp70 levels. Both,
client protein degradation and heat shock induction at similar con-
centrations are indicative of Hsp90 N-terminal inhibition.
In conclusion, we have designed, synthesized and evaluated six
new conformationally constrained analogues of radester and
tested them against two breast cancer cell lines. Western blot anal-
yses confirmed Hsp90 inhibition as evidenced by concentration-
dependent degradation of Hsp90 clients and simultaneous induc-
tion of heat shock response. In general, six-membered analogues
were found to be more effective than their five-membered coun-
terparts. However, the activities of these compounds remain com-
parable to radester, indicating the introduction of conformationally
Anti-proliferation activity of conformationally constrained analogues 1–6
a
Compounds
IC50
(lM)
MCF7
SKBr3
1
2
3
4
5
6
12.9 2.3
9.5 0.8
15.1 0.9
11.4 0.5
15.2 0.9
11.8 0.1
16.1 1.1 nM
47.7 2.6b nM
13.9 1.41c
42.1 1.8
10.5 0.1
47.3 4.4
12.8 0.7
40.8 3.8
12.5 0.6
15.8 0.9 nM
37.5 4.0b nM
NT
GDA
RDC
Radester
a
b
c
IC50 = concentration required to produce 50% inhibition.
Obtained from Ref. 19.
Obtained from Ref. 16.
25–26, following reaction with phenylformate. Quinones 5–6 were
then generated utilizing the same oxidation conditions used to af-
ford 3–4.26,27
Upon completion of their syntheses, chimeric analogues 1–6
were evaluated for their anti-proliferative activity against MCF7
and SKBr3 breast cancer cell lines. As seen in Table 1, the six-mem-
0.1
0.5
1
10
50 100 GDA DMSO
a
Her2
c-Raf
Akt
Hsp70
Actin
b
0.1 0.5
1
10
50 100 GDA DMSO
Her2
c-Raf
Akt
Hsp70
Actin
Figure 4. Western blot analysis (a) for compound 1 and (b) for 2.

6850
V. D. Jadhav et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6845–6850
20. Qadeer, G.; Rama, N. H.; Shah, S. J. H. ARKIVOC 2007, 12.
constrained carbonyls does not play a significant role in organiza-
tion of the N-terminal ATP-binding site.
21. El-Feraly, F. S.; Cheatham, S. F.; McChesney, J. D. Can. J. Chem. 1985, 63, 2232.
22. Analytical data for 1: 1H NMR (DMSO-d6, 400 MHz) d 11.12 (s, 1H), 10.14 (s, 1H)
9.74 (d, J = 1.65 Hz, 1H), 8.82 (s, 1H), 8.36 (s, 1H), 8.17 (d, J = 2.1 Hz, 1H) 7.18 (s,
1H), 6.41 (s, 1H), 3.68 (s, 3H), 3.17 (dd, J = 7.9, 17.6 Hz, 1H), 2.70–2.63 (m, 3H),
2.53–2.50 (m, 1H), 1.96–1.89 (m, 1H), 1.45–1.38 (m, 1H); 13C NMR (DMSO-d6,
125 MHz) d 203.8, 160.1, 156.0, 153.8, 143.6, 142.8, 142.7, 137.6, 124.3, 122.5,
116.8, 107.5, 107.3, 102.1, 59.9, 48.6, 46.9, 31.6, 21.9; IR (film) vmax 3307–3101,
Acknowledgments
The authors gratefully acknowledge NIH/NCI CA 109265, the
Madison and Lila Self Graduate Fellowship (A.S.D.) and the Ameri-
can Foundation of Pharmaceutical Education for financial support
(A.S.D.).
2954, 2925, 1647, 1618, 1490, 1452, 1363, 1228 cm-1
; HRMS (ESI-) m/z
[MÀH]À calcd for C19H17ClNO7: 406.0694, found: 406.0686.
23. Analytical data for 2: 1H NMR (methanol-d4, 400 MHz) d 8.18 (s, 1H), 7.07 (s,
1H) 6.28 (s, 1H), 3.78 (s, 3H), 3.16–3.10 (m, 1H), 2.88–2.76 (m, 3H), 2.57–2.47
(m, 1H), 2.32–2.26 (m, 1H), 2.15–2.08 (m, 1H), 1.95–1.87 (m, 1H), 1.73–1.65
(m, 1H); 13C NMR (methanol-d4, 125 MHz) d 206.9, 165.2, 162.1, 161.7,145.4,
145.3, 144.6, 140.4, 126.3, 123.5, 112.5, 112.2, 109.1, 102.5, 61.4, 47.2, 30.7,
28.2, 27.6, 23.1; IR (film) vmax 3209, 2997, 2848, 1602, 1436, 1234 cmÀ1; HRMS
(ESI-) m/z [MÀH]À calcd for C20H19ClNO7: 420.0850, found: 420.0839.
24. Analytical data for 3: 1H NMR (CDCl3, 500 MHz) d 8.99 (s, 1H), 8.58 (s, 1H), 8.27
(s, 1H), 7.36 (s, 1H), 6.46 (s, 1H), 6.16 (s, 1H), 4.17 (s, 3H), 3.30 (dd, J = 7.75,
18.24 Hz, 1H), 2.83 (dd, J = 4.63, 18.05 Hz, 1H), 2.77–2.72 (m, 1H), 2.66–2.56
(m, 2H), 2.06–2.00 (m, 1H), 1.67–1.60 (m, 1H); 13C NMR (CDCl3, 125 MHz) d
208.4, 182.5, 181.3, 159.5, 159.4, 159.3, 157.7, 156.5, 152.0, 114.2, 108.5, 102.3,
102.1, 101.1, 61.7, 47.3, 32.0, 29.7, 20.7; IR (film) vmax 3305–3103, 2997, 2927,
2856, 1654, 1649 1500, 1363, 1228 cmÀ1; HRMS (ESI-) m/z [MÀH]À calcd for
C19H15ClNO7: 404.0537, found: 404.0543.
References and notes
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Med. Chem. 1999, 42, 260.
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16. Shen, G.; Blagg, B. S. J. Org. Lett. 2005, 7, 2157.
25. Analytical data for 4: 1H NMR (DMSO-d6, 500 MHz) d 12.89 (s, 1H), 11.42 (s, 1H),
10.30 (s, 1H), 8.51 (s, 1H), 8.32 (s, 1H), 6.35 (s, 1H), 4.01 (s, 3H), 3.06 (dt,
J = 4.99, 18.07 Hz, 1H), 2.84–2.77 (m, 1H), 2.64–2.56 (m, 1H), 2.26–2.20 (m,
1H), 1.98–1.93 (m, 1H), 1.86–1.78 (m, 2H), 1.54–1.46 (m 2H); 13C NMR (DMSO-
d6, 125 MHz)) d 204.6, 182.8, 182.3, 162.9, 162.4, 162.3, 160.1, 156.0, 143.7,
127.9, 112.6, 110.5, 110.3, 101.1, 61.2, 44.6, 27.5, 25.90, 25.12, 19.94; IR (film)
vmax 3309–3143, 2995, 2918, 2848, 1604, 1569, 1438, 1369, 1224 cmÀ1; HRMS
(ESI-) m/z [MÀH]À calcd for C20H17ClNO7: 418.0694, found: 418.0694.
26. Analytical data for 5: 1H NMR (DMSO-d6, 500 MHz) d 11.73 (s, 1H), 10.45 (s, 1H)
10.36 (s, 1H), 8.59 (s,1H), 7.29 (s,1H), 6.52 (s, 1H), 6.40 (t, J = 8.0 Hz, 1H), 4.12
(s, 3H), 3.72 (s, 2H), 3.40 (d, J = 8.0 Hz, 2H); 13C NMR (DMSO-d6, 125 MHz) d
188.8, 183.6, 182.3, 162.3, 160.1, 156.9, 156.1, 149.6, 138.3, 137.6, 129.8, 124.3,
118.7, 112.7, 107.5, 102.3, 61.4, 28.9, 23.1; IR (film) vmax 3201–3100, 2918,
2848, 1608, 1458, 1448, 1219 cmÀ1
C19H13ClNO7: 402.0381, found: 402.0367.
;
HRMS (ESI-) m/z [MÀH]À calcd for
27. Analytical data for 6: 1H NMR (methanol-d4, 400 MHz) d 8.51 (s, 1H), 7.92
(s,1H), 7.35 (s,1H), 6.70 (t, J = 7.9 Hz, 1H), 6.32 (s, 1H), 4.18 (s, 3H), 3.46 (d,
J = 7.93 Hz 2H), 3.08 (t, J = 6.5 Hz, 2H), 2.94 (t, J = 6.9 Hz, 2H); 13C NMR (aceton-
d6, 125 MHz) d 192.0, 184.3, 180.2, 165.6, 164.7, 162.2, 160.8, 158.1, 144.8,
144.4, 135.8, 130.9, 113.9, 102.6, 102.5, 102.4, 62.1, 27.2, 25.2, 22.8; IR (film)
vmax 3070, 2987, 2850, 1573, 1475, 1436, 688 cmÀ1; HRMS (ESI-) m/z [MÀH]À
calcd for C20H15ClNO7: 416.0537, found: 416.0534.
17. Clevenger, R. C.; Blagg, B. S. J. Org. Lett. 2004, 6, 4459.
18. Immormino, R. M.; Metzger, L. E.; Reardon, P. N.; Dollins, D. E.; Blagg, B. S. J.;
Gewirth, D. T. J. Mol. Biol. 2009, 388, 1033.
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