An improved route to 19-substituted geldanamycins as novel Hsp90 inhibitors-potential therapeutics in cancer and neurodegeneration

Article abstract of DOI:10.1039/c3cc43457e

19-Substituted geldanamycin derivatives are efficient Hsp90 inhibitors, without the toxicity associated with the other benzoquinone ansamycins, thus giving them potential for use as molecular therapeutics in cancer and neurodegeneration. Here a new method of synthesising these important compounds is reported, eliminating the need for toxic metals and metalloids. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc43457e

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An improved route to 19-substituted geldanamycins
as novel Hsp90 inhibitors – potential therapeutics in
cancer and neurodegeneration†
Cite this: Chem. Commun., 2013,
49, 8441
Received 9th May 2013,
Accepted 10th June 2013
Russell R. A. Kitson and Christopher J. Moody*
DOI: 10.1039/c3cc43457e
www.rsc.org/chemcomm
19-Substituted geldanamycin derivatives are efficient Hsp90 inhibitors,
without the toxicity associated with the other benzoquinone
ansamycins, thus giving them potential for use as molecular
therapeutics in cancer and neurodegeneration. Here a new method
of synthesising these important compounds is reported, eliminating
the need for toxic metals and metalloids.
The benzoquinone ansamycin (BQA) polyketide geldanamycin
1 is arguably one of the most significant natural products to be
isolated in the past fifty years. Initially found to be an efficient
antibacterial agent,1 Neckers’ 1994 discovery of geldanamycin’s
potent and specific inhibition of the molecular chaperone, heat
shock protein 90 (Hsp90),2 prompted a veritable explosion of
research in the area.3 Hsp90, one of the most abundant proteins
in eukaryotic cells, has been shown to play a pivotal role in many
oncogenic pathways,4 in addition to neurodegenerative diseases
such as Alzheimer’s and Parkinson’s,5 and conditions such as
HIV/AIDS6 and malaria.7 As a result, the enzyme has become
one of the most attractive and widely studied molecular targets
for small molecule inhibition, with over 15 inhibitors already in
clinical trials as cancer therapeutics.3,6,7 Despite geldanamycin
1 providing an excellent lead for drug discovery, it was not
progressed to the clinic, due to poor solubility and stability and,
in particular, unacceptable liver toxicity. The more stable and
soluble semi-synthetic geldanamycin derivatives 17-allylamino-
17-demethoxygeldanamycin (17-AAG, Tanespimycin) 2,8 and
17-N,N-dimethylethylenediamino-17-demethoxygeldanamycin
(17-DMAG, Alvespimycin) 3 (ref. 8) (Fig. 1) were developed and
advanced to clinical trials, although continuing difficulties with
formulation and toxicity meant their progress was also halted.9
Following our previous work in the Hsp90 arena,3,10 we
recently reported our efforts to address the toxicity associated
with geldanamycin analogues.11 The introduction of a substi-
tuent at the 19-position of the quinone ring was found to
completely suppress the conjugate addition of thiol nucleophiles,
Fig. 1 The Hsp90 inhibitors geldanamycin 1, its aminoquinone analogues
17-allylamino-17-demethoxygeldanamycin (17-AAG) 2, 17-N,N-dimethylethylene-
diamino-17-demethoxygeldanamycin (17-DMAG) 3.
thought to be responsible for the liver toxicity,12 and render the
19-substituted BQAs (19-BQAs) 5 essentially non-toxic.11 Thus,
the amino-quinone analogue 19-phenyl-17-AAG was found
to have an IC50 greater than 20 mM against both normal
human umbilical vein endothelial cells (HUVECs) and retinal
pigmented epithelial cells (ARPE-19 cells).11 In addition it
also caused a trans to cis-amide isomerisation,13 known to be
required for binding to the Hsp90 protein to occur,11 whilst
protein crystallography established that such 19-BQAs did
indeed bind efficiently to the N-terminal ATP-binding site of
Hsp90 (Fig. 2).11
Fig. 2 X-ray structure of 19-phenyl geldanamycin 5 bound in the ATP site of
yeast Hsp90.11 Geldanamycin 1 (green) and 19-phenyl geldanamycin 5 (salmon)
with Hsp90 (green and salmon residues, respectively). See PDB codes 1A4H
(geldanamycin 1) and 4ASF (19-phenyl geldanamycin 5).
School of Chemistry, University of Nottingham, University Park, Nottingham,
UK NG7 2RD. E-mail: c.j.moody@nottingham.ac.uk; Fax: +44 (0)115 951 3564
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc43457e
c
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Table
1
Scope of the Suzuki–Miyaura coupling reaction;a synthesis of
In order to access these 19-substituted geldanamycin deri-
vatives, a palladium-catalysed Stille coupling was employed,
utilising the readily accessible 19-iodogeldanamcyin 4.14 This is
a complex and challenging substrate, but suitable conditions
were successfully developed, although in some cases the Stille
couplings proved limited in scope, and were not scalable above
0.1 mmol without a reduction in yield. Additionally, the use of
tin and the arsine ligands required for high yields, makes the
coupling protocol relatively unattractive for the pharmaceutical
industry. Herein we report our endeavours to develop a new
method to access these biologically important 19-BQAs.
The palladium-catalysed Suzuki–Miyaura reaction15 is well
known to be the coupling protocol of choice for industry in view
of the large array of boronic acid and ester coupling partners
commercially available, its high yields and its relatively benign
starting materials and by-products. In view of this, we were
attracted to the use of a Suzuki–Miyaura protocol to access
19-BQAs. Early efforts under standard sets of conditions were
unsuccessful.11 However, a 2004 report of the coupling of
17-triflyloxy-BQAs with boronic acids,18 employing the Neel
modification of the Suzuki–Miyaura protocol (‘ligand-free’
Pd2(dba)3ÁCHCl3 as the catalyst, CsBr for ligand-exchange with
the palladium–triflate complex, CsF as the base and 1,4-dioxane
as the solvent),19 gave some hope. Encouraged, we applied
similar conditions to 19-iodogeldanamycin 4, with the aim of
accessing a range of 19-BQAs (Scheme 1).
19-substituted geldanamycins 5–19
Entry
R
Product Yield/% Stille yieldf/%11
1
Ph
Ph
Me
i-Pr
5
5
6
7
91
85
85
86
0
2b
3
Quant
39 (29c)
19
4
5b
6b
7b
8
81
0
76
—
9
59 (54d)
Quant
10
8b
11
90
—
9b,e
10b
12
13
53
46
—
—
11
12
13
14
15
16
Quant
95
—
56
—
Our initial attempts employed benzeneboronic acid as
coupling partner, Pd2(dba)3ÁCHCl3, CsF and 1,4-dioxane (the
caesium bromide was eliminated in view of our use of the
iodide as opposed to the triflate) and we were delighted to
observe that 19-phenylgeldanamycin 5 was obtained in an
excellent 91% yield, a slight increase over our Stille protocol
(85%).11 Following this initial success, we optimised the
coupling procedure in terms of solvent and temperature and
investigated the use of different phenyl-boron coupling partners
(for optimisation studies, see the ESI†).
81
14
15
17
64
—
18
19
65
73
—
94
16
Following our optimisation studies, we investigated the
scope of the methodology in terms of the boron coupling
partner (Table 1). Despite our success with the phenyl group
transfer, cross-coupling reactions to install a methyl group (entry 3)
were less efficient, with a moderate yield of 39% achievable
using methylboronic acid (the reaction with trimethylboroxine
gave no product), although importantly, it eliminated the
need to handle tetramethyl stannane. The new protocol also
allowed access to 19-alkyl-BQAs previously unobtainable via the
a
Reactions performed at 0.02–0.04 M in 1,4-dioxane with 2.0 eq.
boronic acid, 5 mol% Pd2(dba)3ÁCHCl3 and 2.0 eq. of CsF at 40 1C for
b
16 h. Performed with 2.0 eq. RB(pin) in 1,4-dioxane/H2O (9 : 1).
c
Performed with 2.0 eq. MeBF3ÀK+ in i-PrOH/H2O (9 : 1) with 3.0 eq.
of Et3N.16 d Performed with 2.0 eq. vinylboronic acid MIDA boronate.
e
Performed with 2.0 eq. 2,3-dihydro-5-furylboronic acid pinacol ester.
Stille reactions were performed using Me4Sn for methyl couplings
f
and RSnBu3 for all other couplings under the conditions outlined in
ref. 11 [dba = dibenzylideneacetone, B(pin) = 4,4,5,5-tetramethyl-1,3,2-
dioxaborolane, MIDA = N-methyliminodiacetic acid].17
Stille method, exemplified by entry 4, for which an unoptimised
19% yield was achieved for a particularly troublesome isopropyl
coupling, and entry 5, where an excellent yield of 19-allyl-
geldanamycin 8 was obtained. Coupling of a vinyl group was
achieved in good yield with both the pinacol and MIDA17
boronates (entry 6). However, reactions to couple more complex
vinylic substituents gave yields in excess of 90% (entries 7 and 8).
Additionally, dihydrofuryl and dihydropyranyl groups were suc-
cessfully coupled in good yield, with the former being obtained
as the hydrolysed form 12 (entries 9 and 10). Significantly, the
new method was found to be greatly superior to the Stille
Scheme 1 Scope of the Suzuki–Miyaura coupling reaction; synthesis of
19-substituted geldanamycins 5–19.
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protocol for the vast majority of reactions with aromatic coupling
partners (entries 11–16). Those with electron-rich aromatic
groups gave excellent yields, whilst electron deficient coupling
partners also performed well, giving the 2-nitrophenyl- and
4-acetylphenyl-geldanamycin derivatives 17 and 18 in 64 and
65% yield, respectively. The work-up and purification for the
new approach was found to be significantly easier than for the
Stille protocol. Rather than requiring repeated washing (satu-
rated aqueous LiCl solution) to remove the DMF, followed by
chromatography using 10% potassium carbonate/silica gel20
(with subsequent treatment of all glassware for tin contamina-
tion), our new procedure simply required the concentration of
the reaction mixture, followed by straightforward silica gel
chromatography.
In summary, a new Suzuki–Miyaura based protocol has been
developed for accessing important 19-substituted geldanamycin
Hsp90 inhibitors, compounds which we have previously shown
to be significantly less toxic to normal endothelial and epithelial
cell systems than their parent quinones11 and, as such, have
considerable potential as therapeutic agents. The novel BQAs
obtained by this method are currently undergoing biological
evaluation in both the therapy of cancer and neurodegenerative
diseases. The new methodology is complementary to our pre-
vious Stille approach and, significantly, eliminates the need for
the use and disposal of toxic metals or metalloids. These
factors, in addition to the much wider commercial availability
of boron coupling partners, make the new methodology much
more attractive to the pharmaceutical industry and the wider
chemical community, whilst making important bioactive com-
pounds more accessible.
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Chem. Commun., 2013, 49, 8441--8443 8443