The identification, synthesis, protein crystal structure and in vitro biochemical evaluation of a new 3,4-diarylpyrazole class of Hsp90 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.05.046

High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 μM) inhibitor of Hsp90 ATPase activity. Here, we describe the synthesis of CCT018159 and a number of close analogues together with data on their biochemical p

Full text of DOI:10.1016/j.bmcl.2005.05.046

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3338–3343
The identification, synthesis, protein crystal structure
and in vitro biochemical evaluation of a new
3,4-diarylpyrazole class of Hsp90 inhibitors
Kwai-Ming J. Cheung,a,ꢀ Thomas P. Matthews,a,ꢀ Karen James,a Martin G. Rowlands,a
Katherine J. Boxall,a Swee Y. Sharp,a Alison Maloney,a S. Mark Roe,b
Chrisostomos Prodromou,b Laurence H. Pearl,b G. Wynne Aherne,a
Edward McDonalda,* and Paul Workmana
aCancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research,
Cancer Research UK and Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK
bSection of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories,
237 Fulham Road, London SW3 6JB, UK
Received 16 March 2005; revised 10 May 2005; accepted 11 May 2005
Abstract—High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 lM) inhibitor of
Hsp90 ATPase activity. Here, we describe the synthesis of CCT018159 and a number of close analogues together with data on their
biochemical properties. Some initial structure–activity relationships are discussed, as well as the crystal structure of CCT018159
bound to Hsp90.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Heat-shock protein 90 (Hsp90) is a molecular chaperone
responsible for ATP-dependent folding, stability and
function of many ꢁclientꢂ proteins that are involved in
the development and progression of cancer.1– These cli-
ent proteins include ErbB2, c-Raf, Cdk4, mutant p53,
hTERT, Hif1-a and the oestrogen/androgen receptors.
The natural products geldanamycin 1a and radicicol 2
(see Fig. 1) were known to be active in various biological
assays5 and were subsequently shown to inhibit
Hsp90,6,7 leading to degradation of client proteins by
the ubiquitin–proteasome pathway.8 Treatment of cells
with Hsp90 inhibitors results in cell cycle arrest, differen-
tiation and apoptosis.9,10 Since cancers usually have
multiple molecular oncogenic defects, the potential for
simultaneous combinatorial disruption of multiple can-
cer-associated signalling proteins and pathways, and
also for modulation of all the hallmark features of
malignancy, makes Hsp90 a particularly attractive can-
cer drug target.1,– Hsp90 inhibitors should exhibit broad
spectrum antitumour activity and a lower risk of induc-
ing drug resistance. Good activity and an acceptable
therapeutic index have been observed with the geldana-
mycin analogue 17-allylamino, 17-demethoxy-geldana-
mycin (17AAG) 1b in human tumour xenograft
models.14 Hypotheses to explain the therapeutic selectiv-
ity for tumour versus normal cells include a greater
dependence of cancer cells on oncogenic client proteins
(oncogene addiction); the presence in malignant cells
of overexpressed and mutated oncoproteins that necessi-
tate high chaperone capacity; the stress conditions of
cancer cells leading to a stronger need for molecular
chaperone activity; the removal of the buffering effect
of Hsp90 in tumour cells causing synthetic lethal effects;
and the preferential accumulation of Hsp90 in cancer
versus normal cells.1,15,16 Furthermore, it has been
reported that Hsp90 is present in an activated super-
chaperone complex that binds 17AAG much more tight-
ly than the latent form in normal cells.15,17
Keywords: Hsp90 inhibitors; Cancer; Pyrazole; Resorcinol; Protein
crystal structure.
Phase 1 clinical trials of 17AAG 1b showed evidence of
biological and clinical activities, including prolonged
stable disease in two patients with melanoma.18
*
Corresponding author. Tel.: +44 208 722 4294; e-mail: ted.
mcdonald@icr.ac.uk
ꢀ These authors contributed equally to this work.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.046

K.-M. J. Cheung et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3338–3343
3339
have shown activity in animal models.24 The first syn-
thetic small molecule inhibitors of Hsp90 were based on
the purine scaffold, for example, PU3 and PU24FCl.12,25
Amongst other compounds,19 novobiocin and cisplatin
have been reported to inhibit Hsp90 in these cases by
binding at a C-terminal site.26
High-throughput screening27 of a library of 50,000 com-
pounds, assayed at a final nominal concentration of
40 lM in 0.4% (v/v) DMSO, identified CCT018159
(Fig. 2, 3a) as the most potent inhibitor of yeast
Hsp90 ATPase activity (79% inhibition, IC50 = 8.9 lM).
Further interrogation of the data from this malachite
green assay furnished a number of less potent analogues
(Table 1), which provided some early structure–activity
relationships (SAR). All the compounds share a similar
structure and the more active ones possess the resorcinol
ring, pyrazole and benzodioxan core. Methylation of the
hydroxyl para to the pyrazole 4–8 appears to result in a
significant fall in potency. The lipophilic (alkyl) group at
the 5-position of the resorcinol seems to be important,
as when the ethyl group in 3a is replaced by a hydrogen
10 or propyl chain 9 we observe an 8- and 5-fold in-
crease in IC50, respectively.
Figure 1. Structures of natural product based Hsp90 inhibitors.
However, a second generation of Hsp90 inhibitors is
being sought to overcome some of the undesirable
features of 17AAG, such as limited oral bioavailability,
potential toxicity and poor aqueous solubility.19,20
Natural product inhibitors of Hsp90, such as geldanamy-
cin 1a and radicicol 2, inhibit Hsp90 by binding to the
ATP site located in the N-terminal domain, disrupting
the ATPase activity that is essential for its chaperone
function.7,21,22 Derivatives of geldanamycin continue to
be evaluated, including the more soluble 17-dimethyl-
aminoethylamino-17-demethoxygeldanamycin (17DM
AG) 1c, which has entered clinical trial.23 Radicicol oximes
A synthetic chemistry programme was undertaken to
devise an efficient route to synthesise sufficient quantities
of 3a and a number of close analogues to confirm and
further investigate the pharmacological properties of
this class of compounds.
Figure 2. Molecular structures of CCT018159 3a and close analogues in the screening series.
Table 1. Inhibitory activity of hits identified in the high-throughput screen against yeast Hsp90 ATPase
Compound
R1
R2
R3
R4
R5
% Inhibition in screen at 40 lM
IC50 (lM) SD
3a CCT018159
4
OH
OMe
Et
Et
H
–OCH2CH2O–
–OCH2O–
Me
H
79
20
25
0
8.9 0.72
5
OMe
OMe
–O(CH2)3O–
–OCH2CH2O–
Me
H
6
Pr
Pr
Et
Pr
H
7
OMe
OMe
H
OMe
H
H
H
10
13
25
26
64
0
8
H
9
10
OH
OH
–OCH2CH2O–
–OCH2CH2O–
–OCH2O–
Me
Me
H
46.1 2.8
60.8 6.7
22.3 2.5
11
12
OH
Pr
(See Fig. 2)
(See Fig. 2)
(See Fig. 1)
(See Fig. 1)
13
1a Geldanamycina
1b 17AAGa
15
90
80
4.8 0.8
8.7 2.3
a Hsp90 inhibitor standards.

3340
K.-M. J. Cheung et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3338–3343
We prepared the hit 3a28 and analogues 3b–d and 16a–g
based on a previously published procedure for the prep-
aration of 3,4-diaryl pyrazoles by refluxing chromen-4-
one intermediates29 in ethanol with hydrazine hydrate
or methyl hydrazine in the case of 16g. The chromen-
4-ones 15 and 18 were synthesised from phenylacetic
acids by one of two methods30 depending on whether
the substituent at the 5-position of the pyrazole was
hydrogen 16a–g or a methyl group 3a–d (Scheme 1).
ing 5:1 mixture of isomers, the major para rearranged
product 19a was isolated by recrystallisation (NMR
assignment was unambiguous). Next, a 1,3-diketone
chain was built up on the benzyl protected phenols, fol-
lowed by the ring-forming condensation with hydrazine
hydrate to form the pyrazole. Iodination, followed by a
Suzuki–Miyaura coupling and benzyl ether deprotec-
tion, gave the desired analogues 23a and 23b.
The 13 compounds synthesised were assayed for activity
against the yeast Hsp90 ATPase using a more sensitive
malachite green assay,33 which gave an IC50 of 7.1 lM
(n = 2) for 3a. This compound also inhibited human
Hsp90 in the presence of the co-chaperone Aha-134 with
a closely similar IC50 of 3.1 lM (n = 2). In this assay,
geldanamycin gave an IC50 of 4.2 lM against the human
enzyme and 17AAG gave a value of 3.5 lM. A sulforho-
damine B (SRB) assay35 was used to determine the GI50
against HCT116 colon cancer cells (Table 2). This cell
line was used as it is relatively sensitive to Hsp90 inhib-
itors and because we previously characterised in detail
both its molecular response, in terms of client protein
depletion and Hsp70 induction, and also its cellular re-
sponse, with respect to cell cycle arrest and apopto-
The phenylacetic acids required for 3c, 16b and 16g are
commercially available, whilst the biaryl phenylacetic
acid used in the synthesis of 16f was prepared by a liter-
ature procedure.31 The required acids for 3a, 3b, 16a and
16c–e were synthesised by alkylating the corresponding
mono- or dihydroxyphenylacetic acids with bromobuty-
ronitrile and 1,2-dibromoethane, respectively.
Two compounds containing only one of the two resor-
cinol hydroxyls were synthesised by a different proce-
dure detailed in Scheme 2. Whilst 19b is commercially
available, the phenolic starting material 19a was
prepared from a rearrangement of acetic acid 2-chloro-
phenyl ester with aluminium chloride.32 From the result-
Scheme 1. Reagents and conditions: (a) One pot (i) ArCH2CO2H, BF3 Æ OEt2, (ii) PCl5, DMF; (b) hydrazine hydrate (or methyl hydrazine 16g),
EtOH, reflux; (c) ArCH2CO2H, BF3 Æ OEt2, 80 ꢁC, 90 min; (d) K2CO3, acetic anhydride, DMF, reflux, 6 h; (e) hydrazine hydrate, EtOH, reflux.
Scheme 2. Reagents and conditions: (a) (i) BnBr, K2CO3, acetone, reflux, (ii) Na, EtOAc; (b) hydrazine hydrate, ethanol, reflux; (c) NaI, I2, Na2CO3,
water/THF, microwave, 120 ꢁC; (d) (i) ArB(OH)2, Pd(PPh3)4, Cs2CO3, LiCl, 1-propanol/water, microwave, 120 ꢁC, (ii) NaI, BF3, MeCN, rt, 18 h.

K.-M. J. Cheung et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3338–3343
3341
Table 2. Inhibitory potency of 3a and close analogues against yeast
Hsp90 ATPase and proliferation of HCT116 human colon cancer cells
Compound
IC50 (lM) n = 2
GI50 (lM)
3a CCT018159
3b CCT072440
3c CCT072457
3d CCT073657
16a CCT072442
16b CCT069965
16c CCT072453
16d CCT074897
16e CCT074898
16f CCT072454
16g CCT075450
23a CCT073667
23b CCT072439
1a Geldanamycin
1b 17AAG
7.1
4.1 0.4
4.0 0.6
6.4
<1a
1.65
<1a
1.6
3.6
23
2.4
13
<1a
2.4
6.8 0.7
30
31
12.85
1.55
>100
>100
25.5
4.8
11
—
—
—
0.067
0.021
6.6
Where multiple assays were conducted, the results are shown as SE.
a IC50 of compounds 3b, 3d and 16c were close to the detectable limit of
the assay. Percentage inhibition values for yeast Hsp90 at 1 lM with
these compounds were 61.5%, 77.8% and 64.7%, respectively.
sis.10,36 The SRB assay showed that the hit compound
3a effectively reduced cellular proliferation with a GI50
of 4.1 lM.
Gene expression microarray studies (data not shown)
and Western blotting37 have helped us to define the
molecular signatures that are characteristic of Hsp90
inhibition, which include the downregulation of c-Raf
and Cdk4, and the induction of Hsp70 proteins.36
Figure 3 shows that after treatment with 3a in
HCT116 cells, depletion of c-Raf and Cdk4 and eleva-
tion of Hsp70 were observed, confirming the action of
3a as an Hsp90 inhibitor.
Figure 4. Ligplot diagrams showing the binding interactions of (a) 2
and (b) 3a with the yeast Hsp90. Hydrogen-bonding interactions are
shown as broken lines. Amino acids shown in red are involved in
hydrogen bonding. Atoms in van der Waals contact have ꢁspokesꢂ
directed at each other. Blue spheres represent water molecules.
at the base of the pocket that binds radicicol 2 (Fig. 4)
and geldanamycin 1a.7 In fact, interactions that 2 and
3a make with Hsp90 are strikingly similar. Most impor-
tantly, interactions with inhibitors from the carboxylate
side chain of Asp 79, the main-chain amide group of Gly
83 and the hydroxyl side chain of Thr 171, all via the
same tightly bound water molecule, are conserved in
both structures. Another conserved interaction occurs
between the main-chain carbonyl of Leu 34 via another
tightly bound water molecule to the inhibitors. These
tightly bound water molecules are also involved in the
binding of ATP and 1a.7 The only direct hydrogen bond
interaction between Hsp90 and the inhibitors is between
the d-amino side chain of Lys 44 and the epoxide oxygen
of 2. The rest of the interactions between Hsp90 and the
inhibitors are hydrophobic in nature and a common set
of residues (Asn 37, Ile 82, Met 84, Leu 173, and Phe
124) are found to be in van der Waals contact with 2
and 3a. van der Waals contacts for 3a are somewhat
more extensive than those seen for 2 and involve addi-
tional hydrophobic residues (Ala 41 and Leu 93).
˚
Protein X-ray crystallography (1.6 A resolution) con-
firmed that 3a binds to the ATPase pocket within the
N-terminal domain of yeast Hsp90 (Fig. 4). Details of
the expression and purification of His-tagged Hsp90
constructs and co-crystallisation of the N-terminal do-
main of Hsp90 with inhibitors are described in Panare-
tou et al.38 and Roe et al.,7 and the amino acid
numbering refers to yeast Hsp90. In the complex, the
two phenolic hydroxyls and the adjacent pyrazole N-
atom of 3a form a network of hydrogen bonds with ami-
no acids together with associated water molecules lying
As the ethyl group in 3a and chlorine in the more potent
2 occupy the same region of the ATP site, a chlorine
containing analogue of 3a was synthesised. Owing to im-
proved enzyme potency of this compound 3b, the chlo-
rine substituent was retained subsequently in the
majority of analogues.
Figure 3. Western blot showing that in HCT116 cells 3a (5 · GI50
downregulates Hsp90 client proteins (c-Raf and Cdk4) and elevates
levels of Hsp70. GAPDH was used as loading control.
)

3342
K.-M. J. Cheung et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3338–3343
Following our disclosure of pyrazole inhibitors of
Hsp90 ATPase and the X-ray crystal structure of an en-
zyme–inhibitor complex,39,40 Kreusch et al.41 have re-
cently published their independent discovery of related
resorcylic pyrazoles that bind to Hsp90 in the same
fashion.
chain in 16c; and (2) a hydrogen bond interaction via a
water molecule between Lys44 and the butyronitrile oxy-
gen atom. It is noted above that Lys 44 also forms a
hydrogen bond with the epoxide oxygen of Radicicol 2.
The GI50 results show that the analogues displaying en-
zyme potency also possess activity against HCT116 cells;
however, a relationship between the two is not immedi-
ately apparent for these early compounds. By compar-
ing 3b and 3d with 16a and 16c, we conclude that the
presence of 5-methyl on pyrazole appears to have a neg-
ligible effect on cellular activity.
From the biochemical data of the small set of analogues,
some additional initial SAR could be derived. The neces-
sity of inhibitors to possess both resorcinol hydroxyls
was demonstrated by the loss in activity of 23a and
23b. The crystal structure of CCT018159 3a (Fig. 4)
shows extensive H-bonding involving both resorcinol
hydroxyls, thus explaining the weaker activity of mono-
phenolic analogues 23a and 23b.
In conclusion, we have found a new class of small mol-
ecule Hsp90 inhibitors, which have been characterised
both by biochemical evaluation and crystallography.
Three synthetic routes, of which one is novel, have been
employed, which allow access to both the initial hit com-
pound from the high-throughput screen and to a num-
ber of structurally diverse derivatives. Initial SAR has
revealed the key interactions of the inhibitor and areas
where extensive derivatisation is possible. Overall, the
results indicate that this novel Hsp90 inhibitor series
provides an attractive starting point for hit to lead
exploration.
Compounds 3d and 16c–16e indicate that there is exten-
sive room for large flexible groups in place of the dioxan
ring of 3a, especially in the meta- and para-positions.
This has been demonstrated in the crystal structure
˚
(2.0 A resolution) of 16c (Fig. 5) where the butyronitrile
chain can clearly be seen pointing out of the ATP pocket
towards solvent. Apart from familiar hydrogen-bonding
interactions via two tightly bound water molecules close
to the resorcinol ring and the van der Waals contacts
seen in 3a, two further interactions are noted: (1) a van
der Waals contact between Gly 121 and the butyronitrile
Supplementary data
Crystallographic coordinates have been deposited with
the Protein Data Bank: 2BRC (CCT018159, 3a) and
2BRE (CCT072453, 16c).
Acknowledgments
This work was supported by Cancer Research UK
[CRUK] Programme Grant No. C309/A2187. Hsp90
work in Laurence Pearlꢂs laboratory is supported by a
Programme Grant from the Wellcome Trust. The
authors thank Andrew Dick, Angela Hayes, Amin
Mirza and Bernard Nutley for their help with structural
spectroscopic studies. We wish to acknowledge the use
of the EPSRCꢂs Chemical Database Service at
Daresbury.42
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