3-(5-chloro-2,4-dihydroxyphenyl)-Pyrazole-4-carboxamides as inhibitors of the Hsp90 molecular chaperone

Article abstract of DOI:10.1016/j.bmcl.2005.08.091

Information from X-ray crystal structures of Hsp90 inhibitors bound to the human Hsp90 molecular chaperone was used to assist in the design of 3-(5-chloro-2,4-dihydroxyphenyl)-pyrazole-4-carboxamides as novel inhibitors of Hsp90. Accessing an extra intera

Full text of DOI:10.1016/j.bmcl.2005.08.091

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5197–5201
3-(5-chloro-2,4-dihydroxyphenyl)-Pyrazole-4-carboxamides
as inhibitors of the Hsp90 molecular chaperone
Paul A. Brough,a,* Xavier Barril,a Mandy Beswick,a Brian W. Dymock,a
Martin J. Drysdale,a Lisa Wright,a Kate Grant,a Andrew Massey,a
Allan Surgenora and Paul Workmanb
aVernalis Ltd, Granta Park, Great Abington, Cambridge CB1 6GB, UK
bCancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research,
15 Cotswold Road, Sutton, Surrey SM2 5NG, UK
Received 30 June 2005; revised 9 August 2005; accepted 22 August 2005
Abstract—Information from X-ray crystal structures of Hsp90 inhibitors bound to the human Hsp90 molecular chaperone was used
to assist in the design of 3-(5-chloro-2,4-dihydroxyphenyl)-pyrazole-4-carboxamides as novel inhibitors of Hsp90. Accessing an
extra interaction with the protein via Phe138 gave a significant increase in binding potency compared to similar analogues that
do not make this interaction.
Ó 2005 Elsevier Ltd. All rights reserved.
Molecular chaperones are proteins which have a key
role in the maintenance of conformation, stability, and
function of client proteins within the cell. Heat shock
protein 90 (Hsp90) is an ATP-dependent molecular
chaperone that has several oncogenic client proteins in-
volved in signal transduction, cell cycle regulation, and
apoptosis, and has recently become a focus of interest
as a potential anticancer drug target.1–6
O
O
R
O
O
O
H
OH
Cl
Me
O
H
N
O
H
Me
MeO
Me
MeO
OH
HO
Me
OCONH2
R =
It has been shown that several natural products and
their derivatives have anti-tumor activity arising from
inhibition of the intrinsic ATPase activity of Hsp907–10
(Fig. 1). Inhibition of Hsp90 activity results in the pro-
teasomal degradation of client proteins causing cell
growth arrest and/or apoptosis in cancer cells.11
2 Radicicol
1a Geldanamycin -OCH3
1b 17-AAG
-NHCH2CH=CH2
-NHCH2CH2NMe2
1c 17-DMAG
Figure 1. Structures of natural product derived Hsp90 inhibitors.
The geldanamycin derived inhibitor 17-AAG (1b) has
entered phase II clinical trials and initial results are
encouraging, providing proof of principle for Hsp90
inhibitors as cancer therapeutics.12–14 However, 17-
AAG has several potential limitations including poor
solubility, limited bioavailability, toxicity, and extensive
metabolism.15 These issues and the inherent chemical
complexity of the compounds have led to significant ef-
forts to identify small molecule inhibitors of Hsp90.16–21
Figure 2 shows some of the chemotypes recently identi-
fied as potent inhibitors of Hsp90.18,22,19
We have disclosed previously how structure-based drug
design was implemented in the identification of 5 (VER-
49009) using structural information from the X-ray crys-
tal structure of 3 (CCT018159) bound to the ATPase
Keywords: Hsp90; Cancer; Structure-based drug design; Pyrazole;
X-ray crystallography.
*
Corresponding author. Tel.: +44 1223 895555; fax: +44 1223
895556; e-mail: p.brough@vernalis.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.091

5198
P. A. Brough et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5197–5201
Cl
O
Cl
Cl
NH2
N
BnO
O
HO
HO
a
c
N
N
b
Cl
N
HO
O
8
F
6
7
OBn O
OH
Cl
OH O
O
Cl
O
N N
H
OH
BnO
BnO
Br
d, e
f
4 PU24FCl
3 CCT018159
N
O
9
10
OBn O
Cl
N
OBn
Cl
N
H
Cl
HO
H
N
BnO
BnO
O
OH
Br
g,h
i
O
N N
H
OH
12
11
N
N
OBn
OBn
Cl
N
N
Si
Si
O
5 VER-49009
Figure 2. Small molecule inhibitors of Hsp90.
O
O
NR1R2
Cl
BnO
HO
NR1R2
O
N
j
binding site of Hsp90a.19,20 This information enabled us
to successfully target a specific region of the protein to
design a significantly more potent analogue. Herein,
we report a series of novel 3-(5-chloro-2,4-dihydroxy-
phenyl)-pyrazole-4-carboxamides23 designed with a sim-
ilar approach as that used to identify 5. In the present
case however, we have targeted additional interactions
at a distinct region of the Hsp90 protein.
N
OBn
N
14a-q
OH
13a-q
N
Si
O
H
Scheme 1. Synthesis of 3-(5-chloro-2,4-dihydroxyphenyl)-pyrazole
carboxamides. Reagents and conditions: (a) AcOHÆBF3, 90 °C, 3.5 h,
58%; (b) BnBr, K2CO3, MeCN, reflux, 6 h, 90%; (c) DMFÆDMA,
150 °C, 2 h, 84%; (d) NH2NH2ÆH2O, EtOH, reflux, 5 h, 92%; (e) NBS,
CH2Cl2, rt, 2 h, 97%; (f) SEMCl, Cs2CO3, DMF, rt, 16 h, 90%; (g) n-
BuLi, THF, À78 °C, 15 min, 45%; (h) CO2, À78 °C to rt; (i) R1R2NH,
HATU, DIPEA, DMF, 100 °C, 10 min; (j) BCl3, CH2Cl2, rt, 16 h.
Scheme 1 depicts the synthesis of carboxamides 14a–q:
Commercially available 4-chlororesorcinol 6 underwent
regioselective Friedel–Crafts acylation followed by ben-
zyl protection to give the acetophenone 8. Generation of
the requisite pyrazole ring was achieved via reaction of 8
with DMF.DMA and reaction of the resulting eneamine
9 with hydrazine hydrate. Bromination of the pyrazole
was regioselective for the 4-position affording com-
pound 10. After some detailed investigation of protect-
ing groups, the pyrazole nitrogen was protected as a
silyl ethoxy methoxy (SEM) ether, affording 11 as a
1:1 mixture of regioisomers, (reflecting the expected exis-
tence of tautomeric forms of the pyrazole). Lithium–hal-
ogen exchange with 11 at À78 °C afforded the 4-lithio
species which was subsequently quenched with carbon
dioxide to give carboxylic acid 12. A standard HATU
mediated coupling reaction was then performed with a
series of amines to generate amides 13a–q. Treatment
of amides 13a–q with boron trichloride effected concom-
itant removal of the benzyl and SEM protecting groups
affording pyrazole carboxamides 14a–q (Table 1).
described.25 The binding mode of the resorcinol and
pyrazole rings is essentially as described before,19,20,26
The novel and most prominent features of the structure
are described in the next section.
Structural information gained from X-ray crystallogra-
phy of Hsp90 inhibitors (including 3) bound to human
Hsp90a protein revealed that the 4-position of the pyra-
zole could be elaborated to give further interaction with
the Hsp90 protein and potentially increase both binding
potency and activity against cells via inhibition of intra-
cellular Hsp90. Using the available structural informa-
tion, phenyl and benzyl templates were initially
evaluated for generation of SAR in this region of the
molecule. All compounds were tested for binding at
the ATP binding site using a fluorescence polarization
assay,27 with the majority of these being additionally
tested for their ability to inhibit the growth of
HCT116 human colon cancer cells in vitro. The data
in Table 1 show the effects of modification to the tem-
plates. Most of the compounds discussed inhibit binding
of the probe substrate in the low micromolar range,
notable exceptions being the tertiary amide 14f (which
is >50 lM compared to its secondary amide analogue
14d) and heteroaryl compounds 14p and 14q which also
lose an appreciable amount of binding potency. Howev-
er, incorporation of an acetyl at the para position of the
aryl template (compound 14h) gives a significant in-
crease in binding potency. The crystal structure of 14h
in complex with the N-terminus domain of Hsp90a re-
veals that the ketone moiety is hydrogen bonded to
N-terminal human Hsp90a his-tagged protein was
crystallized in complex with compounds 14h and 14o
as previously described.24 Data were collected on both
co-crystals and the structures were subsequently solved
by molecular replacement using the previously solved
native Hsp90a structure24 (PDB code: 1UY1). Both
crystals diffracted in space group P21 to resolutions of
˚
1.9 and 1.7 A for 14h and 14o, respectively. Co-crystals
of Hsp90 with 14o bound were grown at a slightly differ-
ent pH (pH 5.5) as compared to those containing 14h
(pH 6.5). In both structures, the flexible loop region
adopts a more ꢀopenꢁ conformation as previously

P. A. Brough et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5197–5201
5199
Table 1. Binding and growth inhibition assay results for compounds
14a–14q
Cl
R1
N
HO
O
R2
4
N
2
OH
N
1
H
Compound R1 R2
FP enzyme Cell growth
IC50, lMa inhibition
(GI50), lM
14a
14b
14c
H
H
H
7.1
14.7
11.7
nd
>80
>80
OH
OMe
14d
14e
14f
14g
14h
14i
H
6.0
6.9
>80
>80
nd
OMe
Cl
H
Figure 3. Binding mode of 14h to Hsp90. The dotted surface renders
the new cavity formed between helices 2 and 7.
Me
H
>50
OMe
31.5
59.7
11.6
>80
60.7
NHAc
O
H
0.258
H
12.8
6.0
14j
H
OMe
OMe
14k
14l
H
H
H
10.7
>80
56.2
45.0
5.35
F
CF3
14m
5.4
O
S
14o
14p
14q
H
H
H
NH2
0.461
>80
nd
O
35.6
O
N
>50
nd
a Values are means of two experiments. nd, not determined.
Figure 4. Binding mode of 14o to Hsp90. Compound 14h is depicted in
silver for comparison.
not display any cellular activity in vitro (possibly due to
poor cell penetration inherent with primary sulfona-
mides). The intermolecular hydrogen bond with
Phe138 increases the binding affinity of compounds
14h and 14o by more than one order of magnitude com-
pared to similar compounds unable to place a hydrogen
bond acceptor at this position. This equates to approx-
imately 1.5–2.0 kcal/mol, clearly in the upper range of
the contribution of hydrogen bonds to protein stabili-
ty29–31 or protein–ligand binding,32–34 which is surpris-
ing for such a solvent-exposed site.
the backbone of Phe138, thus explaining the higher
affinity for Hsp90 (Fig. 3). It is worth noting that the
a-phosphate of ADP28 and the amidic oxygen of gel-
danamycin25 are also hydrogen bonded to Phe138 but,
to the best of our knowledge, this is the first time that
the impact of this interaction can be quantified.
The benzylic compound 14o also exhibits increased
binding affinity for Hsp90 and crystallography again
identified a hydrogen bond between Phe138 and the li-
gand as responsible (Fig. 4), though this compound does

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P. A. Brough et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5197–5201
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and 4 times GI50) and knock down of Raf-1 and Her2
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In conclusion, we have utilized structure-based drug de-
sign to assist in the design and rationalization of Hsp90
inhibitors. The backboneof residuePhe138hasbeen iden-
tified as an additional interaction point to increase bind-
ing potency. This structural information may be useful
to help design new inhibitors of the Hsp90 chaperone.
Crystallographic coordinates have been deposited with
the Protein Data Bank: [14h PDB code: 2BYH; 14o
PDB code: 2BYI]
23. Beswick, M. C.; Brough, P. A.; Drysdale, M. J.; Dymock,
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