Novel adenosine-derived inhibitors of 70 kDa heat shock protein, discovered through structure-based design

Article abstract of DOI:10.1021/jm801627a

The design and synthesis of novel adenosine-derived inhibitors of HSP70, guided by modeling and X-ray crystallographic structures of these compounds in complex with HSC70/BAG-1, is described. Examples exhibited submicromolar affinity for HSP70, were highl

Full text of DOI:10.1021/jm801627a

1510
J. Med. Chem. 2009, 52, 1510–1513
Letters
A nucleotide exchange factor, such as BCL-2-associated
athanogene-1 (BAG-1), stimulates the release of ADP, binding
of fresh ATP, and opening of the binding pocket to allow
substrate release.12
Novel Adenosine-Derived Inhibitors of
70 kDa Heat Shock Protein, Discovered
Through Structure-Based Design†
Molecules demonstrated to modulate HSP70 function to date
do so by affecting the interaction of HSP70 with its cochaper-
ones.13,14 There are no inhibitors known to bind to the ATPase
domain of HSP70, and it was thought that inhibition of this
site would be a suitable point for disrupting the function of this
protein for potential use in the treatment of cancer.
A fluorescence polarization (FP) assay was developed in
which measurement of binding competition with a fluorescein-
labeled ATP-based probe, N6-(6-amino)hexyl-ATP-5-FAM (KD
0.4 µM) was determined. The HSP70 IC50 of endogenous
ligands ATP (1) and ADP (2) were found to be 0.5 µM (Table
1). Although these values were close to the limit of detection
of the assay (HSP70 conc 0.4 µM), this format was considered
to be appropriate for the discovery of ATP-competitive
compounds.
Douglas S. Williamson,* Jenifer Borgognoni,
Alexandra Clay, Zoe Daniels, Pawel Dokurno,
Martin J. Drysdale, Nicolas Foloppe, Geraint L. Francis,
Christopher J. Graham, Rob Howes, Alba T. Macias,
James B. Murray, Rachel Parsons, Terry Shaw,
Allan E. Surgenor, Lindsey Terry, Yikang Wang,
Mike Wood, and Andrew J. Massey
Vernalis (R&D) Ltd., Granta Park, Great Abington,
Cambridge CB21 6GB, United Kingdom
ReceiVed December 22, 2008
Abstract: The design and synthesis of novel adenosine-derived inhibi-
tors of HSP70, guided by modeling and X-ray crystallographic
structures of these compounds in complex with HSC70/BAG-1, is
described. Examples exhibited submicromolar affinity for HSP70, were
highly selective over HSP90, and some displayed potency against
HCT116 cells. Exposure of compound 12 to HCT116 cells caused
significant reduction in cellular levels of Raf-1 and Her2 at concentra-
tions similar to that which caused cell growth arrest.
Adenosine (3), a fragment of ATP (1), was chosen as a start-
point for initial investigations, and commercially available
adenosine analogues were screened in the FP assay. In
comparison with compound 3 (HSP70 IC50 560 µM), the
8-amino derivative 415 (HSP70 IC50 9.4 µM) was approximately
60-fold more potent.
Compound 4 was also shown to inhibit the growth of human
colon tumor 116 (HCT116) cells in a sulforhodamine B (SRB)
growth inhibition assay (GI50 0.05 µM).16,17 The cytotoxic
activity of 4 has been previously reported,18 but the mismatch
between HSP70 FP IC50 and HCT116 GI50, and later PD marker
studies (see below), indicated that the cytotoxic mechanism of
action of compound 4 must be predominantly through a route
other than HSP70 inhibition.
The crystal form of HSP70 obtained gave low resolution
diffraction data and was not amenable to the rapid generation
of ligand-bound structures. Analogous HSP70 isoforms were
therefore investigated for their suitability as surrogates. The 70
kDa heat shock cognate (HSC70), which is constitutively
expressed, shares high sequence and structural homology with
HSP70 in the ATPase domain. Encouragingly, crystals of
HSC70 in complex with BAG-119 were suitable for soaking of
ligands and collection of diffraction data.
X-ray crystal structures of both 1/HSC70/BAG-1 and 4/HSC70/
BAG-1 were obtained, and compound 4 was shown to overlay
with the adenosine moiety of ATP (1) (Figure 1).20 An
intramolecular hydrogen bond was observed between the
8-amino substituent on the purine ring and the 5-hydroxymethyl
substituent of the tetrahydrofuran ring of 4. It was postulated
that this preorganizes the bound conformation of the ligand by
influencing the syn/anti equilibrium of the two rings as well as
stabilizing the bound conformation itself.
Elaboration of the 8-amino group of compound 4 was
investigated initially (Table 1). N-Methylation (5) and N-
benzylation (6) retained potency in the HSP70 FP assay. Further
substitution on the benzene ring of compound 6 was explored,
and it was shown that the 3,4-dichlorobenzyl analogue 7 had
similar enzyme potency to compound 4, with reduced activity
in the SRB assay (GI50 26 µM). An X-ray crystal structure of
Heat shock proteins (HSPsa) are molecular chaperones that
assist protein folding processes within the cell.1,2 They are made
up of families containing both heat-inducible and constitutively
expressed members, defined by their different molecular weights;
the 70 kDa HSP isoforms (HSP70) are one such family.3
There is growing evidence to support HSP70 as a potential
anticancer target.4 An antisense construct targeting HSP70
proved effective at causing cell arrest and death in a variety of
human tumor cell lines.5 Locoregional application of adenovirus
expressing antisense HSP70 cDNA eradicated xenografts of
glioblastoma, breast, and colon carcinoma. Analysis of the
expression levels of cytosolic HSP70 family members in human
cancer and normal cell lines, and their subsequent knock-down
using RNAi technology, also revealed the importance of HSP70
in human cancer.6
HSP70 isoforms are composed of an N-terminal ATPase
domain, which binds ATP (1) and hydrolyses it to ADP (2), a
substrate-binding domain, and a C-terminal domain.7-10 The
presence of a peptide in the substrate binding domain stimulates
the ATPase activity of HSP70, increasing its normally slow rate
of ATP hydrolysis. HSP70 is a weak ATPase, and maximal
activity requires cochaperones, such as HSP40.11 When ATP
is hydrolyzed to ADP, the binding pocket of HSP70 closes,
tightly binding the peptide chain, preventing it from aggregating.
†
Coordinates of HSC70/BAG-1 complexes with compounds 1, 4, 7, 12,
and 15 have been deposited in the Protein Data Bank under accession codes
3FZF, 3FZH, 3FZK, 3FZL, and 3FZM.
* To whom correspondence should be addressed. Phone: +44 (0)1223
895555. Fax: +44 (0)1223 895556. E-mail: d.williamson@vernalis.com.
a Abbreviations: BAG-1, BCL-2-associated athanogene-1; FP, fluores-
cence polarization; HCT116, human colon tumor 116; HSC70, 70 kDa heat
shock cognate; HSP70, 70 kDa heat shock protein; SPR, surface plasmon
resonance; SRB, sulforhodamine B.
10.1021/jm801627a CCC: $40.75
2009 American Chemical Society
Published on Web 03/03/2009

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1511
Table 1. Biological Data for HSP70 Inhibitors
Figure 1. X-ray crystal structure of 4 (blue)/HSC70/BAG-1 (green)
superimposed with ATP (1, orange), from an X-ray crystal structure
of ATP (1)/HSC70/BAG-1.
Figure 2. X-ray crystal structure of compound 7 (blue) in complex
with HSC70/BAG-1 (green) showing π-stacking between the ligand
and Arg272. Alternate conformers of Tyr15 and Arg272 from the X-ray
structure of compound 4/HSC70/BAG-1 are shown in orange.
of the tetrahydrofuran ring could enable interactions with
proximal Tyr15 or Thr37 residues, and so synthetic elaboration
at this position was investigated. The methyl analogue 10
(HSP70 IC50 3 µM) was 3-fold more potent than compound 7,
and benzyl substitution (11) was similarly well-tolerated in the
HSP70 FP assay.
In silico docking studies with rDock21,22 and analysis of
superimposed X-ray structures suggested that it was possible
to optimize π-stacking between Arg272, Tyr15, and the 8- and
5-substituents of the adenosine-derived ligands. Virtual libraries
of ligands were created with MOE;23 only docked poses that
maintained the experimental binding mode for 8-aminoadenosine
(4) were considered. Substituents were prioritized for synthesis
based on interactions with the protein. In particular, combina-
tions of aromatic rings with electron withdrawing or donating
groups were chosen to explore stacking interactions.
Incorporation of a 4-cyano group on the 5-benzyloxymethyl
moiety of compound 11 was investigated, and gave rise to a
further increase in HSP70 potency. Compound 12 had an HSP70
FP IC50 of 0.5 µM and an HCT116 GI50 of 5 µM and was the
most potent example on cells in this series. Figure 3 illustrates
the elaborate π-stacking network seen in the X-ray structure
obtained of compound 12/HSC70/BAG-1. The additional po-
tency of this compound is likely due to water displacement and
a All IC50, KD and GI50 values are the mean of at least two determinations
and are rounded to two significant figures where appropriate. b KD could
not be determined due to compound behavior on chip surface. c Low GI50
due to predominantly non-HSP70 mechanism.
compound 7 in complex with HSC70/BAG-1 (Figure 2) showed
that a movement in the Arg272 residue allowed for an additional
π-stacking interaction with the 3,4-dichlorobenzyl substituent
while maintaining the intramolecular hydrogen bond. Tyr15 also
occupied a different position in this crystal structure, with 75%
occupancy. With a view to increasing enzyme potency by
optimizing π-stacking with Arg272, bicyclic analogues 8 and
9 were synthesized. Encouragingly, the quinolyl analogue 9 was
3-fold more potent in the HSP70 FP assay in comparison with
compound 4.
Study of the X-ray structure of compound 7/HSC70/BAG-1
indicated that modification of the 5-hydroxymethyl substituent

1512 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Letters
Scheme 1a
a Reagents: (a) R1H, EtOH, 170 °C, µwave, 18-90%.
Scheme 2a
Figure 3. Inter- and intramolecular π-stacking interactions in the X-ray
crystal structure of compound 12/HSC70/BAG-1 in addition to the
intramolecular H-bond between the 8-NH and 5-CH2O of 12.
a Reagents: (a) R2Br, DMF; NaH, 85%; (b) 8 N NH4OH(aq), 100 °C,
µwave; 57%; (c) Br2, 10% Na2HPO4(aq), 1,4-dioxane, 75%; (d) R1NH2,
EtOH, 150 °C, µwave, 74%; (e) H2O, CF3CO2H; MP-carbonate, 59%.
Figure 4. Improved π-stacking in the X-ray structure of compound
15/HSC70/BAG-1.
Compounds 10-17 were synthesized according to the
procedure outlined in Scheme 2. Compound 1926 underwent
alkylation with the corresponding halide in the presence of
sodium hydride. The 6-Cl of the product 20 was converted to
the primary amine by treatment with ammonium hydroxide to
give intermediates of type 21, which selectively underwent
bromination at the 8-position upon treatment with bromine to
give compounds of type 22. Treatment of 22 with the corre-
sponding amine, followed by deprotection of the acetonide
moiety of intermediates 23 with trifluoroacetic acid, afforded
10-17.
It has been demonstrated recently that dual targeting of
HSC70 and HSP70 small interfering RNAs results in degrada-
tion of the HSP90 client proteins, Raf-1 and Her2.27 To add
support to the premise that the new compounds exert their
antiproliferative effects via inhibition of HSP70, the effect of
compound 12 on the PD markers Raf-1 and Her2 (known clients
of HSP90 and down-regulated upon HSC70/HSP70 inhibition
by siRNA) was explored. As shown in Figure 5, exposure of
HCT116 cells to compound 12 caused significant reduction in
cellular levels of Her2 and Raf-1 at concentrations similar to
that which caused cell growth arrest. Compound 4 did not down-
regulate Her2 at 2 × GI50, indicating that the cytotoxic
mechanism of action of this compound must be through a route
other than HSP70 inhibition. Additionally, all examples in Table
1 had HSP90 FP IC50 > 400 µM.28
the stabilization of the bound ligand by two intramolecular
interactions (H-bond and π-stacking).
As the limit of detection of the FP assay had been reached,
an alternative method of measuring ligand binding was required.
A direct binding assay was developed using surface plasmon
resonance (SPR) in which a double His-tagged HSP70 construct
was immobilized on a Biacore sensor chip. The HSP70 KDs of
compounds 2, 4, and 12 were determined to be 0.5, 11, and 0.3
µM, respectively. This was in line with expectations for
compounds with FP IC50s equal or greater to the concentration
of protein in the assay. For those compounds with an HSP70
FP IC50 <1 µM, the KD was determined by SPR and used to
generate subsequent SAR.
Further analogues of 12 were synthesized to optimize
intramolecular π-stacking between the 8-benzylamino and
5-benzyloxymethyl substituents or interaction between the
5-substituent and Tyr15/Thr37 (compounds 13-17). Compound
15 (HSP70 KD 0.05 µM), which incorporates the preferred
quinolyl substituent from compound 9 and the benzonitrile
moiety of compound 12, was the most potent ligand in this
series. The X-ray structure of this compound with HSC70/
BAG-1 (Figure 4) demonstrated the improved overlap between
Arg272 and the quinoline ring.
Compounds 5,24 6,25 and 7-9 were synthesized by treatment
of 8-bromoadenosine 18 with the corresponding amine, using
a procedure based on the literature25 synthesis of compound 6
(Scheme 1).
In conclusion, a first-in-class series of adenosine-derived
HSP70 inhibitors that target the ATPase binding domain has

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1513
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Figure 5. Effects of compounds 4 and 12 on expression of Raf-1,
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Supporting Information Available: Synthetic procedures with
supporting spectroscopic and chromatographic data, assay protocols,
X-ray crystallographic parameters, and protein production methods.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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