Dihydroxylphenyl amides as inhibitors of the Hsp90 molecular chaperone

Article abstract of DOI:10.1016/j.bmcl.2008.09.081

Information from X-ray crystal structures were used to optimize the potency of a HTS hit in a Hsp90 competitive binding assay. A class of novel and potent small molecule Hsp90 inhibitors were thereby identified. Enantio-pure compounds 31 and 33 were potent in PGA-based competitive binding assay and inhibited proliferation of various human cancer cell lines in vitro, with IC₅₀ values averaging 20 nM.

Full text of DOI:10.1016/j.bmcl.2008.09.081

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6273–6278
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dihydroxylphenyl amides as inhibitors of the Hsp90 molecular chaperone
*
Pei-Pei Kung , Lee Funk, Jerry Meng, Michael Collins, Joe Zhongxiang Zhou, M. Catherine Johnson,
Anne Ekker, Jeff Wang, Pramod Mehta, Min-Jean Yin, Caroline Rodgers, Jay F. Davies II, Eileen Bayman,
Tod Smeal, Karen A. Maegley, Michael R. Gehring
Pfizer Global Research and Development, La Jolla Laboratories, 10770, Science Center Dr., San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Information from X-ray crystal structures were used to optimize the potency of a HTS hit in a Hsp90 com-
petitive binding assay. A class of novel and potent small molecule Hsp90 inhibitors were thereby identi-
fied. Enantio-pure compounds 31 and 33 were potent in PGA-based competitive binding assay and
inhibited proliferation of various human cancer cell lines in vitro, with IC50 values averaging 20 nM.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 25 August 2008
Revised 22 September 2008
Accepted 22 September 2008
Available online 26 September 2008
Keywords:
Dihydroxylphenyl amides
Hsp90
Inhibitor
Molecular chaperones are protein machines that are responsi-
ble for the correct folding, stabilization, and function of other pro-
teins in the cell.1 Many molecular chaperones and cocharperones
have been identified to date. Among these, heat shock protein 90
(Hsp90) is the most extensively studied. This 90 kDa protein is
an ATP-dependent molecular chaperone that has multiple identi-
fied client proteins involved in the development and progression
of cancer.2 Many of these client proteins are over-expressed in
oncogenic settings and rely on Hsp90 for their proper folding.
Therefore, Hsp90 has become a focus of interest as a potential anti-
cancer drug target.
The natural product Hsp90 inhibitor geldanamycin (GA) and
several semi-synthetic analogs (17-AAG and 17-DMAG, Fig. 1)3a–c
have entered clinical trials, and initial encouraging results provide
proof of concept for using Hsp90 inhibitors as cancer chemother-
apy.3a However, these compounds have potential therapeutic lim-
itations because of low solubility, liver toxicity, and extensive
metabolism.4a Many of these issues could be associated with the
quinone moieties that exist in these agents and have led us and
others to identify non-quinone-containing, small molecule inhibi-
tors of Hsp90 chaperone activity.
in the competitive binding assay.5 Unfortunately, the compound
exhibited an IC50 greater than 20 M in a cell-based assay which
l
measured the degradation of the Hsp90 client protein Akt.6a,6b To
facilitate potency improvements, we obtained a 1.4 Å co-crystal
structure of compound 1 bound to Hsp90 and compared it to the
crystal structures of other known Hsp90 inhibitors complexed with
the protein. Such inhibitors include: GA,7 purine analogs such as
PU3,8 the resorcinol-containing natural product radicicol,9 and
other resorcinol-containing small molecules10a–d (Fig. 2).
As shown in Figure 3 (PDB ID code: 3EKO), the amide carbonyl of
1 formed hydrogen bonds with the side chain of the Hsp90 Thr184
residue as well as with a water molecule that was conserved in many
of the known co-crystal structures. The 20 hydroxyl of 1 interacted
with the same conserved water molecule along with the side chain
of Asp93. In addition, the 40 hydroxyl group of 1 formed hydrogen
bonds with a second conserved water molecule and the Asn51 side
chain of the Hsp90 protein. In contrast, the 60 hydroxyl group inter-
acted only with two non-conserved water molecules that were not
observed in other Hsp90 inhibitor co-crystal structures. Collectively,
Our Hsp90 research program began with a high throughput
screening campaign using a competitive binding assay in which
the ability of test compounds to displace tritium-labeled 17-pro-
pylaminobenzoquinone ansamycin (17-PGA, Fig. 1) from Hsp90
was assessed. This effort led to the discovery of the tri-hydroxy-
containing compound 1 (Table 1) which displayed a Ki of 200 nM
O
17
R
O
N
H
O
GA: R=OCH3
O
O
17-AAG: R=-NHCH2CH=CH2
17-DMAG: R=-NHCH2CH2NMe2
17-PGA: R=-NHCH2CH2CH3
7
NH2
O
OH
O
* Corresponding author. Tel.: +1 858 526 4867; fax: +1 877 481 1781.
E-mail address: peipei.kung@pfizer.com (P.-P. Kung).
Figure 1. Structures of benzoquinone ansamycins.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.081

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P.-P. Kung et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6273–6278
O
potent Hsp90 inhibitors than those prepared from secondary
NH2
N
OH
Cl
O
amines (e.g., compare 3 and 4 with 5 and 7, respectively). This
phenomenon is likely due to the ability of the amide NH present
in the former compounds to form an intra-molecular H-bond with
the 20-OH moiety which unfavorably positions the amide carbonyl
relative to the orientation observed in the Hsp90 and compound 1
co-crystal structure.
O
N
N
O
N
HO
O
O
O
HO
N
OH
N
H
O
O
Radicicol (RD)
CCT018159
PU3
Encouragingly, as shown in Table 1, many of the compounds
that were prepared displayed Hsp90 inhibition activity that was
equal to or better than that exhibited by compound 2 (compounds
5–13). Inhibitors 7, 10, and 11 were considered to be particularly
attractive leads due to their relatively small size, potent inhibition
Figure 2. Structures of known Hsp90 inhibitors.
of Hsp90 activity, and <10 lM inhibition of Hsp90 activity in the
cell-based Akt Lum assay. In order to further improve inhibitor po-
tency, we subjected racemic compound 10 to preparative chiral
SFC (supercritical fluid chromatography)11 and obtained the corre-
sponding single enantiomers 14 and 15.12 As shown in Table 1, the
former compound was approximately 100-fold more potent than
the latter in the competitive binding assay.
A 1.8 Å co-crystal structure of inhibitor 14 complexed with
Hsp90 was obtained (Figs. 5 and 6) (PDB ID code: 3EKR) and the chi-
rality of this molecule was thereby confirmed as the ‘R’ enantiomer.
Analysis of this co-crystal structure also identified a small un-
filled hydrophobic pocket formed by Hsp90 residues Phe138,
Figure 3. X-ray structure of 1 bound to the ATP binding site of human Hsp90. Red
spheres represent structurally conserved water molecules and aqua spheres
represent structurally non-conserved water molecules.
these observations suggested that the 60 hydroxyl group of 1 was not
critical for Hsp90 recognition, and this hypothesis was confirmed
with the preparation of the resorcinol-containing compound 2
which displayed Hsp90 inhibition potency similar to that exhibited
by 1 (Table 1).
Further analysis of the above co-crystal structure indicated that
the pyrrole moiety of 1 occupied an area formed by mostly hydro-
phobic residues Met98, Ile96, Ala55, and Lys58 (Fig. 4). Of these,
Lys58 appeared to be conformationally mobile, suggesting that
moieties larger than the pyrrole group could be accommodated
in this location. We therefore designed and synthesized a small
library of compounds in which commercially available amines
were used to replace the pyrrole moiety present in inhibitor 2. In
general, library compounds derived from primary amines were less
Figure 5. Binding interactions of 14 to Hsp90. The phenyl moiety of pyrrolidine
amide moiety exposed to a lipophilic pocket formed by Tyr139, Val 136, and
Gly135.
Val136
Gly135
Phe138
Tyr139
O
Asn51
Leu107
Asn51
O
H2O
H2N
Val150
Leu107
Met98
H2O
NH3
Phe138
OH
Ala55
HO
6'
HO
4'
4'
N
N
Lys58
H2O
H2O
2'
OH
HO
Ile96
O
2'
HO
Thr184
OH
O
Thr184
H2O
O
H
O
2O
O
O
Asp93
Asp93
Figure 4. Schematic diagram showing the key interactions of
1 with Hsp90
enzyme.
Figure 6. Schematic diagram shows the two unfilled lipophilic pockets.

P.-P. Kung et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6273–6278
6275
Table 1
SAR of 2,4-dihydroxyphenyl carboxyamides
OH
O
R
X
HO
Compound
R’
X
Enzyme IC50
0.4
(
lM)
Enzyme Kia
(lM)
Cell IC50
>20
(lM)
1
OH
0.2
N
N
2
3
H
H
1.7
0.85
101
>20
ND
200
N
H
4
5
H
H
74
36.8
1.6
ND
N
H
3.2
>20
N
6
7
H
H
1.4
0.7
>50
7
O
O
S
N
N
0.06
0.03
8
9
O
H
H
0.5
0.3
0.25
0.14
ND
14
O
N
N
10
11
H
H
0.1
0.05
0.04
9.3
8.6
N
Cl
0.08
N
O
12
13
H
H
0.4
2.4
0.2
1.2
10.3
>20
N
N
N
14
H
H
0.06
5.6
0.03
2.8
4
N
N
15
>50
a
Enzymatic Ki value was calculated from enzymatic IC50 value using Cheng–Prusoff equation.

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P.-P. Kung et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6273–6278
Table 2
SAR of different carboxyl esters/amides of compound 16
4"
R
5"
3"
OH
O
N
HO
Cl
a
Compound
R
Location
Enzyme IC50
(l
M)
Enzyme Kia
(l
M)
Cell IC50 (lM)
16
17
18
19
20
21
22
23
H
—
0.02
0.3
1.4
<0.02
<0.02
0.04
<0.02
0.2
0.01
0.15
0.7
<0.01
<0.01
0.02
<0.01
0.1
1
COOCH3
CONHCH2CH3
COOCH3
CON(CH3)2
CONHCH2CH3
COOCH3
500
500
400
400
400
300
300
20
ND
1
0.3
4.8
1.6
2
CON(CH3)2
a
Assay error for both assays are 10–15%.
Val150, and Leu107 near the 50 position of the resorcinol ring of 14.
We therefore introduced a 50-chloro moiety into the inhibitor de-
sign and observed a 10-fold improvement in cell IC50 values (com-
pare 16 with 10, Tables 1 and 2).13 Similar SAR was also reported
by others.10a
Additional analysis of the 14-Hsp90 co-crystal structure identi-
fied a second larger unfilled pocket near the phenyl moiety formed
by residues Tyr137, Val136, and Gly135. We therefore introduced
simple esters and/or amides at three positions on the phenyl ring
of 16 to determine whether these groups could improve inhibitor
recognition of the Hsp90 protein. We envisioned that if these mod-
Table 3
SAR of 4-[1-(5-Chloro-2,4-dihydroxy-benzoyl)-pyrrolidin-2-yl]-3-methyl-phenyl carboxyamides
R
O
OH
O
N
HO
Cl
a
Compound
Chirality
R/S
R
Enzyme IC50
<0.02
(
lM)
Enzyme Kia
(lM)
Cell IC50
0.15
(lM)
24
<0.01
N
25
26
27
R/S
R/S
R/S
<0.02
<0.02
<0.02
<0.01
<0.01
<0.01
0.15
1.3
N
HO
F
N
0.073
N
N
F
F
F
28
29
R/S
R/S
<0.02
<0.02
<0.01
<0.01
0.03
0.07
N
N
F
F
F
30
31
32
S
R
S
1.4
<0.02
0.3
0.7
>20
0.02
11
F
F
F
<0.01
0.15
N
N
N
F
F
F
33
R
<0.02
<0.01
0.027
a
Assay error for both assays are 10–15%.

P.-P. Kung et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6273–6278
6277
Table 4
IC50 of 17-DMAG, 31, and 33 on expression of Hsp90 client proteins (Akt and Her2) and proliferation (MTT assay) in MDA-MB231 and A2058 cells
IC50 (nM) for Hsp90 client protein degradation
EC50 (nM)
MDA-MB231
Her2
A2058
Hsp70 (A2058)
Akt
MTT
Akt
MTT
DMAG
31
33
17.6
19.1
41.9
4.5
7.6
17
5.8
8.9
24.5
24.3
33.5
49.6
2.1
5.8
22.6
7.9
18.3
40.9
EC50 for induction of Hsp70 for 17-DMAG, 31, and 33 in MDA-MB231.
ifications were tolerated, the latter group could be rapidly opti-
mized through parallel chemistry approaches. As shown in Table
2, appending such an ester or amide to the 500 position of the phe-
nyl ring of 16 was detrimental to Hsp90 recognition properties
(compare 17 and 18 with 16). In contrast, introducing these mod-
ifications at the 300 or 400 positions (compounds 19–23) typically
afforded potency improvements in both the competitive-binding
and cell-based assays with activity in the former assessment often
O
O
O
O
OH
RO2C
H3CO2C
a
H3CO2C
b
O
O
O
O
OH
Cl
34
35, R=CH3
36, R=H
c
amines
d
below the level of accurate quantitation (Ki < 0.01 lM). Compound
20, bearing a 400-dimethyl amide, was the most active compound of
these molecules in the Akt Luminex assay.
O
O
O
O
O
OH
e
R2
R2
N
Encouraged by this result, we synthesized a small library of
compounds related to 20 containing di-substituted amides. As
shown in Table 3, all of the tested molecules were extremely po-
tent in the competitive binding assay with activities below the
quantitation limit (24–29). They also typically displayed sub-
micromolar Hsp90 inhibition activity in the cell-based assay. Two
of the most active racemic compounds (28 and 29) were subjected
to chiral SFC purification in order to characterize the corresponding
enantiomers. As expected, potent Hsp90 inhibition activity was ob-
served for only one of the purified enantiomers (compare 30 with
31 and 32 with 33). The potent single enantiomers thus identified
(31 and 33) were approximately 1000-fold more active in the Akt
Luminex assay as compared with the original screening lead com-
pound (compound 1, Table 1).
N
R1
R1
O
OH
Cl
Cl
Scheme 1. Reagents and conditions: (a) MOMCl, DIEA, DMF, 23 °C, 12 h, 92%; (b)
Ca(OCl)2, acetic acid, acetone, 62%; (c) LiOH(aq) (2 M), MeOH, 23 °C, 12 h, 94%; (d)
amines (both commercially available and synthetic amine from Scheme 2),
4-methylmorpholine, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-
chloride, 1-hydroxy benzotriazole, DMF, 23 °C, 12 h, 64.2%; (d) HCl (in dioxane,
4 M), DCM, 23 °C, 12 h, 82%.
O
O
N
N
CO2H
Br
CO2CH3
N
O
a
c
The ability of these two compounds to affect turnover of several
Hsp90 client proteins in MDA-MB231 and A2058 cells was also as-
sessed. As shown in Table 4, both molecules exhibited a well-
established molecular signature14,15 for Hsp90 inhibitors in these
assessments (similar to that displayed by 17-DMAG).
b
Br
Br
Br
38
37
39
d
R'
The synthesis routes used to prepare compounds 16–23 and
24–29 described in this work are depicted in Schemes 1 and 2.
The syntheses of similar substituted 2-methyl-phenyl-pyrrolidines
using different starting materials are described in the supplemen-
tary material. Scheme 1 describes the synthesis of 5-chloro-2,4-bis
methoxymethoxy-benzoic acid (compound 36) as well as a general
synthesis of the resorcinol amide Hsp90 inhibitors. Amide coupling
reactions in the absence of the phenolic protecting groups resulted
in low yields of the desired products. Compound 35 was synthe-
sized from commercially available 2,4-dihydroxy-benzoic acid
methyl ester by MOMCl protection of the phenols followed by
chlorination of the aromatic ring to provide compound 35.16
Hydrolysis of 35 with lithium hydroxide provided compound 36
in 54% yield over three steps.
The 2-phenyl-pyrroline precursors were prepared from N-vinyl-
pyrrolidin-2-one using the method reported by Maryanoff.17 The
imine intermediate 39 was then reduced with NaBH4 to provide
the desired intermediate 40, 2-(4-bromo-2-methyl-phenyl)-pyr-
rolidine. Compound 41 was then carbonylated with CO2 and subse-
quently trapped with methanol to give the desired product 42, the
boc protected 3-methyl-4-pyrrolidin-2-yl-benzoic acid methyl es-
ter. Further acid de-protection of 42 gave the desired amine 43.
Compound 43 was coupled with compound 36 using HOBt/EDC
mediated coupling condition to give compound 44. Compound 44
HN
Boc
f
N
P
N
O
O R
e
h
N
O
R
Cl
Br
40
Br
CO2CH3
42, P=Boc
43, P=H
44, R'=CO2CH3; R=MOM
45, R'=CO2H; R=MOM
41
i
g
j
46, R'=CONR1R2; R=MOM
k
20, 21, 24-29, R'=CONR1R2; R=H
Scheme 2. Reagents and conditions: (a) SOCl2, MeOH, 40 °C, 12 h, 81%; (b) N-
vinylpyrrolidin-2-one, THF, NaH, 80 °C, 2 h; (c) HCl (aq) (6 N), i-PrOH/THF, 90 °C,
12 h, 56%; (d) NaBH4, MeOH/acetic acid, ꢀ40 °C, 2 h; (e) (Boc)2O, K2CO3, DCM, 12 h,
23 °C, 81%; (f) Pd(PPh3)4, TEA, CH3CN/MeOH, CO (100 psi), 65 °C, 24 h, 65%; (g) HCl
(4 M in dioxane), 12 h, 23 °C, quant.; (h) compound 36, 4-methylmorpholine, N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride, 1-hydroxy benzotria-
zole, DMF, 23 °C, 12 h, 64.2%; (i) LiOH (2 M), dioxane, 40 °C, 90%; (j) amines, 4-
methylmorpholine, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochlo-
ride, 1-hydroxy benzotriazole, DMF, 23 °C, 12 h, 64.2%; (k) HCl (4 M in dioxane),
DCM, 23 °C, 12 h, 82%.
was hydrolyzed to give compound 45. A second amide coupling
reaction of 45 and further de-protection of the MOM groups of

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P.-P. Kung et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6273–6278
bound to copper on Yttrium-silicate scintillant beads. Test compounds which
bind to HSP-90 competed with tritiated propyl-Geldanamycin (3H-pGA) at the
ATP-binding site on HSP-90. For Ki determinations, % inhibition values are
plotted vs. log inhibitor concentration and fit to four parameter logistic
equation. The IC50 is converted to competitive Ki using Cheng–Prusoff equation,
46 gave the desired products (compounds 20, 21, and 24–29)
shown in both Tables 2 and 3 in 50–80% yield.
In summary, a series of potent Hsp90 inhibitors was discovered
by both focused library synthesis and structure-based design. The
two lead compounds (31 and 33) from this series displayed equal
or better potency compared with 17-DMAG in effecting the client
proteins’ degradation and cell proliferation in both A2058 and
MDA-MB231 cell lines.
where Ki is the inhibition constant,
L is the total concentration of
pGA = 200 nM, and Kd is the dissociation constant for pGA = 240 nM.
6. (a) Basso, A. D.; Solit, D. B.; Chiosis, G.; Giri, B.; Tsichlis, P.; Rosen, N. J. Biol.
Chem. 2002, 277, 39858; (b) Akt lum assay: non-small cell lung carcinoma cell
line H1299 was cultured in DMEM + 10%FBS, 10,000 cells/well were cultured in
96-well plate. Compounds were added to each well starting at 10 lM with
threefold serial dilution of 11 points of various concentration. Twenty-four
hours post adding compounds, cell lysates were prepared and subjected to
Luminex 100 system using the Beadlyte Akt/PKB kit—Upstate #46-605.
7. Grenert, J. P.; Sullivan, W. P.; Fadden, P.; Haysead, T. A.; Clark, J.; Mimnaugh, E.;
Krutzsch, H.; Ochel, H.-J.; Schulte, T. W.; Sausville, E.; Neckers, L. M.; Toft, D. O.
J. Biol. Chem. 1997, 272, 23843.
8. Ghiosis, G.; Timaul, M. N.; Lucas, B.; Munster, P. N.; Zheng, F. F.; Sepp-
Lorenzino, L.; Rosen, N. Chem. Biol. 2001, 8, 289.
9. Yang, Z.-Q.; Geng, X.; Solit, D.; Pratilas, C. A.; Rosen, N.; Danishefsky, S. J. J. Am.
Chem. Soc. 2004, 126, 7881.
Acknowledgments
We thank the Pfizer La Jolla analytical group for chiral separa-
tion of racemic compounds. We also thank Drs. Peter Wells and
Rob Kania for helpful discussions, Dr. Yinsheng Zhang for preparing
the tritium-labeled 17-PGA, and Dr. Ketan Gajiwala for discussion
of co-crystal structures and graphical support.
10. (a) Dymock, B. W.; Barril, X.; Brough, P. A.; Cansfield, J. E.; Massey, A.;
McDonald, E.; Hubbard, R. E.; Surgenor, A.; Roughley, S. D.; Webb, P.;
Workman, P.; Wright, L.; Drysdale, M. J. J. Med. Chem. 2005, 48, 4212; (b)
Shen, G.; Blagg, B. S. J. Org. Lett. 2005, 7, 2157; (c) Brough, P. A.; Aherne, W.;
Barril, X.; Borgognoni, J.; Boxall, K.; Cansfield, K.-M.; Cheung, J.; Collins, I.;
Davies, N. G. M.; Drysdale, M. J.; Dymock, B.; Eccles, S. A.; Finch, H.; Fink, A.;
Hayes, A.; Howes, R.; Hubbard, R. E.; James, K.; Jordan, A. M.; Lockie, A.;
Martins, V.; Massey, A.; Mattews, T. P.; McDonald, E.; Northfield, C. J.; Pearl, L.
H.; Prodromou, C.; Ray, S.; Raynaud, F. I.; Roughley, S. D.; Sharp, S. Y.; Surgenor,
A.; Walmsley, D. L.; Webb, P.; Wood, M.; Workman, P.; Wright, L. J. Med. Chem.
2008, 51, 196; (d) Gopalsamy, A.; Shi, M.; Goals, J.; Vogan, E.; Jacob, J.; Johnson,
M.; Lee, F.; Nilakantan, R.; Petersen, R.; Svenson, K.; Chopra, R.; Tam, M. S.;
Wen, Y.; Ellingboe, J.; Arndt, K.; Boschelli, F. J. Med. Chem. 2008, 51, 373.
11. Garozotti, M.; Hamdan, M. J. Chromatogr. B 2002, 770, 53.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.081.
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