5-Aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles as inhibitors of Hsp90 chaperone

Article abstract of DOI:10.1016/j.bmcl.2009.01.003

A series of 5-aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles were synthesized and their binding to several constructs of human Hsp90 chaperone measured by isothermal titration calorimetry (ITC). The most potent compound bound Hsp90 with the

Full text of DOI:10.1016/j.bmcl.2009.01.003

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1089–1092
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles
as inhibitors of Hsp90 chaperone
Inga Cikotiene a,b, Egidijus Kazlauskas a, Jurgita Matuliene a, Vilma Michailoviene a,
Jolanta Torresan a, Jelena Jachno a, Daumantas Matulis a,
*
a Laboratory of Biothermodynamics and Drug Design, Insitute of Biotechnology, Graiciuno 8, Vilnius LT-02241, Lithuania
b Department of Organic Chemistry, Faculty of Chemistry, Vilnius University, Naugarduko 24, Vilnius LT-03225, Lithuania
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 5-aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles were synthesized and their
binding to several constructs of human Hsp90 chaperone measured by isothermal titration calorimetry
(ITC). The most potent compound bound Hsp90 with the dissociation constant of about 5 nM.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 13 November 2008
Revised 30 December 2008
Accepted 6 January 2009
Available online 9 January 2009
Keywords:
Hsp90
Thiadiazole
Pyrazole
Isothermal titration calorimetry
Heat shock protein 90 (Hsp90), originally identified as one of
several conserved heat shock proteins, exhibits general protective
chaperone property—prevention of the unspecific aggregation of
misfolded proteins.1 Hsp90 constitutes about 1–2% of total cellular
proteins. This protein is responsible for ATP-depended folding, sta-
bility and functioning of many ‘‘client” proteins. Hsp90 functions
are essential for development and progression of various cancers.
Hsp90 inhibition leads to destabilization and degradation of many
oncogenic proteins. Hsp90 is a promising anticancer drug target, as
cancerous cells are more susceptible to Hsp90 inhibition than nor-
mal cells.2–7
The ATPase activity of Hsp90 can be inhibited by natural prod-
ucts such as geldanamycin and radicicol (Fig. 1). Both of these com-
pounds bind to the N-terminal domain of Hsp90 and inhibit the
intrinsic ATPase activity.8 Geldanamycin showed activity in human
tumor xenograft models but this compound proved to be too hepa-
totoxic for clinical development. However, the modified versions of
geldanamycin, 17-(allylamino)-17-demethoxygeldanamycin (17-
AAG) and 17-demethoxy-17-[[2-(dimethylamino)ethyl]amino]gel-
danamycin (17-DMAG) retain the property of Hsp90 inhibition and
have significantly less hepatotoxicity than geldanamycin.9 Phase 1
clinical trials of 17-AAG showed evidence of biological and clinical
activities, including prolonged stable disease in two patients with
melanoma.10 However, a second generation of Hsp90 inhibitors is
being sought to overcome some of the undesirable features of
17-AAG, such as limited oral bioavailability, potential toxicity and
poor aqueous solubility.11,12
Radicicol is macrocyclic antibiotic isolated from Monosporium
bonorden. Radicicol is more potent inhibitor of Hsp90 ATPase activ-
ity than geldanamycin or 17-AAG.13 Radicicol oximes have shown
activity in animal models.14 However, no radicicol derivative has
progressed to the clinic.
The first synthetic small molecule inhibitors of Hsp90 were
based on the purine scaffold, for example, PU3 and PU24FCl
(Fig. 1).15 Amongst other compounds, novobiocin and cisplatin
have been reported to inhibit Hsp90 in these cases by binding at
the C-terminal site.16
Various 3,4-diarylpyrazole17 (Fig. 1) as well as 4,5-diarylisoxaz-
ole18 derivatives bearing resorcinol moiety have been selected by
high throughput screening. These compounds showed high
Hsp90 binding affinity and inhibitory effect on human cancerous
cell line growth.19
Despite the fact that a large number of different Hsp90 inhibitors
have been synthesized to date20, only few of them are clinically
tested. There still remains a great need of new potent Hsp90 inhib-
itors which offer one or more following advantages: improved
activity, selectivity, solubility, reduced toxicity and side-effects,
and reduced cost of synthesis.
Herein we report on a high-yielding synthesis of 5-aryl-4-
(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles and the
results of in vitro binding to Hsp90 studies.
The chemistry employed for the design of the new compounds
reported here is shown in Scheme 1. Synthesis of the starting
* Corresponding author. Tel.: +370 52691884.
E-mail address: matulis@ibt.lt (D. Matulis).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.003

1090
I. Cikotiene et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1089–1092
O
O
H
H
O
CH3
R
NH2
N
O
O
O
Cl
N
HO
OMe
N
N
CH3
N
HO
N
R'
H
R
H3C
MeO
H3C
O
OMe
OMe
R''
Cl
H
MeO
HO
CH3
OH
H3C
N
OH
MeO
N
H
O
OCONH2
Radicicol
PU3 (R = R' = H, R'' = C4H9);
PU24FCl (R = F, R' = Cl, R'' = 5-C5H7)
Geldanamycin (R = OCH3)
17-AAG (R = NHCH2CH=CH2)
VER-49009
17-DMAG (R = NH(CH2)2N(CH3)2)
Figure 1. Schematic representation of some known Hsp90 binders.
(Hsp90F) and the N-terminal domain of human Hsp90 (Hsp90N)25
were determined by isothermal titration calorimetry (ITC).26 Figure
3 shows representative isothermal titration calorimetry curves of
3b binding to the Hsp90 N (50 mM Hepes buffer, 100 mM NaCl,
pH 7.5, 37 °C). Protein concentration in the VP-ITC calorimeter
R
R
HO
HO
i
Ar
NH2
Ar
(Microcal, Inc.) cell was 6
lM. Ligand concentration in the syringe
OH
N
OH
O
was 120
l
M. The binding constant was determined to be 1.6 Â
108 MÀ1 with the stoichiometry of 0.97. This is equivalent to the
dissociation constant of 6.3 nM. The steep transition of the ITC
curve shows tight binding of 5-aryl-4-(5-substituted-2,4-dihy-
droxyphenyl)-1,2,3-thiadiazole to Hsp90 (see Fig. 4).
1a-e
2a-e
ii
R
HO
1-3a: R = Cl, Ar = 4-MeOC6H4;
1-3b: R = Cl, Ar = 4-EtOC6H4;
1-3c: R = Cl, Ar = 4-MeC6H4;
1-3d: R = Cl, Ar = 3,4-di-MeOC6H3;
1-3e: R = Et, Ar = 4-EtOC6H4.
The strongest binder to both the Hsp90 N-terminal domain and
the full-length Hsp90 was compound 3b with the observed Kd of
about 6.3 nM and 4.8 nM, respectively. Compounds 3a, c–e also
tightly bound to Hsp90. Compounds4, 5 bearing chloro-substituent
Ar
HO
N
S
N
3a-e
Scheme 1. Reagents and conditions: (i) NH2NH2ÁH2O (2 equiv), EtOH, reflux, 7 h;
(ii) SOCl2, rt, 2 h, then NaHCO3.
materials 1a–e was accomplished as previously described.19 Com-
pounds 1a–e reacted with hydrazine hydrate in boiling ethanol
and formed the corresponding hydrazones 2a–e.21 Latter five deriv-
atives underwent smoothly Hurd–Mori22 cyclization with thionyl
chloride at room temperature to form 5-aryl-4-(5-substituted-
2,4-dihydroxyphenyl)-1,2,3-thiadiazoles 3a–e in high yields.23,24
It is noteworthy that Hurd-Mori reaction usually proceeded succes-
fully when N-acyl- or N-tozylhydrazones bearing an adjacent meth-
ylene group were used. In our case, we observed smooth cyclization
of unactivated hydrazones 2a–e.
When compounds 2a and 2e were cyclized with thionyl chloride
at reflux temperature, the formation of chlorinated side-products 4
and 5 together with 3a,e was observed (Fig. 2).
The binding affinity of 5-aryl-4-(5-substituted-2,4-dihydroxy-
phenyl)-1,2,3-thiadiazoles to the full-length human Hsp90 protein
R
HO
4: R = Cl, Ar = 4-MeOC6H4;
5: R = Et, Ar = 4-EtOC6H4
Cl
Ar
HO
N
S
N
4,5
Figure 3. Isothermal titration calorimetry curve of 3b binding to Hsp90N. The
Figure 2. Structures of compounds 4 and 5.
upper panel shows raw data and the lower panel-integrated data.

I. Cikotiene et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1089–1092
1091
Hsp90 protein target and potent inhibitors of cancer cell survival
and growth. A simple novel route has been employed for the com-
pound synthesis.
References and notes
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Figure 4. Normalized U2OS cancer cell line survival as a function of compound 3b,
3e, and 5 concentration.
10. (a) Banerji, U.; Clarke, P.; Walton, M.; O_Donnell, A.; Raynaud, F.; Turner, A.;
Judson, I.; Workman, P. Proc. Am. Assoc. Cancer Res. 2003, 44, 587; (b) Sausville,
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Neckers, L. M. Cell Stress Chaperones 1998, 3, 100.
Table 1
Dissociation constants of compounds 3a–e, 4, and
constructs (Hsp90N and Hsp90F) as determined by ITC at 37 °C
5 binding to both protein
Compound
Hsp90N Kd
l
M
Hsp90F Kd lM
3a
3b
3c
3d
3e
4
0.013
0.0063
0.017
0.034
0.042
>20
0.0075
0.0048
0.011
0.039
0.037
>20
14. (a) Soga, S.; Neckers, L. M.; Schulte, T. W.; Shiotsu, Y.; Akasaka, K.; Narumi, H.;
Agatsuma, T.; Ikuina, Y.; Murakata, C.; Tamaoki, T.; Akinaga, S. Cancer Res. 1999,
59, 2931; (b) Soga, S.; Shiotsu, Y.; Akinaga, S.; Sharma, S. V. Curr. Cancer Drug
Targets 2003, 3, 359; (c) Barluenga, S.; Wang, C.; Fontaine, J. G.; Aouadi, K.;
Beebe, K.; Tsutsumi, S.; Neckers, L.; Winssinger, N. Angew. Chem. Int. Ed. 2008,
47, 4432.
5
>20
0.20
>20
0.24
17-AAG
Values are means of multiple experiments.
15. (a) Chiosis, G.; Merna, N. T.; Lucas, B.; Munster, P. N.; Zheng, F. F.; Sepp-
Lorenzino, L.; Rosen, N. Chem. Biol. 2001, 8, 289; (b) Chiosis, G.; Lucas, B.; Shtil,
A.; Huezo, H.; Rosen, N. Bioorg. Med. Chem. 2002, 10, 3555; (c) Llauger, L.; He,
H.; Kim, J.; Aguirre, J.; Rosen, N.; Peters, U.; Davies, P.; Chiosis, G. J. Med. Chem.
2005, 48, 2892; (d) Wright, L.; Barril, X.; Dymock, B.; Sheridan, L.; Surgenor, A.;
Beswick, M.; Drysdale, M.; Collier, A.; Massey, A.; Davies, N.; Fink, A.; Fromont,
C.; Aherne, W.; Boxall, K.; Sharp, S.; Workman, P.; Hubbard, R. E. Chem. Biol.
2004, 11, 775; (e) Dymock, B.; Barril, X.; Beswick, M.; Collier, A.; Davies, N.;
Drysdale, M.; Fink, A.; Fromont, C.; Hubbard, R. E.; Massey, A.; Surgenor, A.;
Wright, L. Bioorg. Med. Chem. Lett. 2004, 14, 325.
16. (a) Marcu, M. G.; Chadli, A.; Bouhouche, I.; Catelli, M.; Neckers, L. M. J. Biol.
Chem. 2000, 275, 37181; (b) Itoh, H.; Ogura, M.; Komatsudo, A.; Wakui, H.;
Miura, A. B.; Tashima, Y. Biochem. J. 1999, 343, 697; (c) Marcu, M. G.; Neckers, L.
M. Curr. Cancer Drug Targets 2003, 3, 343.
17. (a) Cheung, K. M. J.; Matthews, T. P.; James, K.; Rowlands, M. G.; Boxall, K. J.;
Sharp, S. Y.; Maloney, A.; Roe, S. M.; Prodromou, C.; Pearl, L. H.; Aherne, G. W.;
McDonald, E.; Workman, P. Bioorg. Med. Chem. Lett. 2005, 15, 3338; (b)
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.
18. (a) Brough, P. A.; Aherne, W.; Barril, X.; Borgognoni, J.; Boxall, K.; Cansfield, J. E.;
Cheung, K.-M.; 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.; Matthews, T.; McDonald, E.; Northfield,
C. J.; Pearl, L. H.; Prodromou, C.; Ray, S.; Raynaud, F. I.; Roughley, S. D.; Sharp, S.
Y.; Surgenor, A. D.; Walmsley, L.; Webb, P.; Wood, M.; Workman, P.; Wright, L.
J. Med. Chem. 2008, 51, 196; (b) Gopalsamy, A.; Shi, M.; Golas, 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.
in position 3 of the dihydroxyphenyl moiety practically did not bind
to Hsp90. These compounds could not form the extensive H-bond-
ing network involving both resorcinol hydroxyls due to the chloro-
substituent, resulting in the lack of activity.19
The dissociation constants of compounds 3a–e, 4, and 5 with
both protein constructs, obtained at 37 °C, are listed in Table 1.
Compound effect on cancer cells was tested by determining cell
growth, death and survival as a function of compound concentra-
tion for two selected cancer cell lines, U2OS (osteosarcoma) and
HeLa (cervical carcinoma)27, using tetrazolium/formazan assay.28
The strongest inhibitor of cancer cell growth was compound 3e
with the observed GI50 of 0.69 lM for U2OS cells and 0.70 lM for
HeLa cells (Table 2, Fig. 4). Compound 5 was a relatively weak
inhibitor of cancer cell lines. This property correlates well with
its weak binding to Hsp90 (Table 2). Other compounds exhibited
average potency of cancer cell growth inhibition. The compound
series has potential to become candidates for therapeutic antican-
cer treatment.
In conclusion, a new group of compounds, similar to previously
described diaryl pyrazoles19, was shown to be effective binders of
19. Sharp, S. Y.; Boxall, K.; Rowlands, M.; Prodromou, C.; Roe, S. M.; Maloney, A.;
Powers, M.; Clarke, P. A.; Box, G.; Sanderson, S.; Patterson, L.; Matthews, T. P.;
Cheung, K. M. J.; Ball, K.; Hayes, A.; Raynaud, F.; Marais, R.; Pearl, L.; Eccles, S.;
Aherne, W.; McDonald, E.; Workman, P. Cancer Res. 2007, 67, 2206.
20. (a) Solit, D. B.; Chiosis, G. Drug Discov. Today 2008, 38; (b) Messaoudi, S.; Peyrat,
J. F.; Brion, J. D.; Alami, M. Anticanc. Agents Med. Chem. 2008, 8, 761.
21. General procedure for the preparation of 2-aryl-1-(5-substituted-2,4-dihydroxy-
phenyl)ethanone hydrazones (2a–e). Hydrazine hydrate (0.166 ml, 3.42 mmol)
Table 2
U2OS and HeLa cancer cell line survival (growth inhibition, GI50) by compounds
3a–e, 5
Compound
U2OS, GI50
,
lM
HeLa, GI50, lM
3a
3b
3c
3d
3e
5
6.0
6.9
7.1
9.9
0.69
28
2.5
3.6
3.3
4.2
0.70
19
is added to
a solution of the corresponding 2-aryl-1-(5-substituted-2,4-
dihydroxyphenyl)ethanone 1a–e (1.71 mmol) in 95% ethanol (5 ml). The
mixture is heated under reflux for 7 h. Solvent is concentrated in vacuo, the
residue treated with water, filtered of and recrystallyzed from 2-propanol.
Spectral data for the selected compound: 1-(5-chloro-2,4-dihydroxyphenyl)-
2-(4-ethoxyphenyl)-ethanone hydrazone (2b). Yield 97%, yellow solid, mp
Values are means of multiple experiments.

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I. Cikotiene et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1089–1092
163–165 °C. 1H NMR (300 MHz, DMSO-d6) d ppm 1.28 (3H, t, J = 6.6 Hz, CH3),
were inserted into a multicloning site of pET-15b vector (Novagen, Madison,
WI, USA). His6-tagged proteins were expressed in the Escherichia coli strain
BL21 (DE3). Hsp90F protein was purified using a Ni-IDA affinity column,
followed by anion exchange chromatography on phosphocellulose p-11 and Q-
sepharose (Amersham Biosciences). Hsp90N protein was purified on the Ni-
IDA affinity column, followed by an anion exchange chromatography using
DEAE-sepharose (Amersham Biosciences). Eluted proteins were dialyzed into a
3.94 (2H, q, J = 6.6 Hz, OCH2), 3.94 (2H, s, CH2), 6.39 (1H, s, ArH), 6.70 (2H, br s
NH2), 6.84 (2H, d, J = 8.7 Hz, ArH), 7.11 (2H, d, J = 8.7 Hz, ArH), 7.24 (1H, s, ArH),
10.18 (1H, br s OH), 13.55 (1H, s, OH). 13C NMR (75 MHz, DMSO-d6) d ppm 13.8,
28.6, 54.9, 104.1, 109.2, 113.0, 114.0 (2C), 127.2, 128.0, 129.1 (2C), 149.3, 153.2,
157.7, 158.4. Analysis: (C16H17ClN2O3) Calcd C 59.91%, H 5,34%, N 8.73%.
Found: C 60.01%, H 5,45%, N 8.91%.
22. Hurd, C. D.; Mori, I. R. J. Am. Chem. Soc. 1955, 77, 5359.
storage buffer containing 20 mM tris (pH 7.5), 50 mM Na2SJ4, and 1
flash-frozen in liquid nitrogen and stored at À80 °C.
lV DNN,
23. General procedure for the preparation of 4-(5-substituted-2,4-dihydroxyphenyl)-
5-aryl-1,2,3-thiadiazoles (3a–e). The corresponding 2-aryl-1-(5-substituted-
2,4-dihydroxyphenyl)ethanone hydrazone (2a–e) (0.33 mmol) is carefully
added to thionyl chloride (1 ml). The reaction mixture is stirred at room
temperature for 2 h. The excess of thionyl chloride is evaporated under
reduced pressure, the residue is dissolved in chloroform (10 ml). The organic
layer is washed twice with NaHCO3 (sat. aq. 10 ml), then with water (15 ml),
dried over Na2SO4, concentrated in vacuo. The residue was purified by column
chromatography.
26. Chaires, J. B. Annu. Rev. Biophys. 2008, 37, 135.
27. HeLa cells (a kind gift from Dr. Aurelija Zvirbliene, Institute of Biotechnology,
Lithuania) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
(Sigma, MO, USA), supplemented with 10% of fetal bovine serum (FBS)
(Biochrom AG, Germany). U2OS cells were purchased from CLS—Cell Lines
Service (Germany) and cultured in the medium composed of 50% DMEM and
50% of F-12 medium (Biochrom AG, Germany), supplemented with 10% FBS.
Inhibitor stock solutions (20 mM) were made in 100% DMSO. Cells were seeded
in 24-well culture plates and after 24 h subjected to different inhibitor
concentrations (2-fold serial dilutions from 50 mM to 50 nM) and in the
absence of inhibitor for control (with 0.25% DMSO in every well). After 72 h of
incubation at 37 °C, 5% CO2, inhibitor or only DMSO-containing culture
Spectral data for the selected compound: 4-(5-chloro-2,4-dihydroxyphenyl)-5-(4-
ethoxyphenyl)-1,2,3-thiadiazole (3b). Yield 80%, orange amorphous solid, mp
132–134 °C. 1H NMR (300 MHz, DMSO-d6) d ppm 1.30 (3H, t, J = 6.9 Hz, CH3),
4.01 (2H, q, J = 6.9 Hz, OCH2), 5.92 (1H, br s OH), 6.71 (1H, s, ArH), 6.93 (2H, d,
J = 9 Hz, ArH), 7.24 (1H, s, ArH), 7.29 (2H, d, J = 9 Hz, ArH), 10.23 (1H, s, OH). 13C
NMR (75 MHz, DMSO-d6) d ppm 15.2, 53.9, 104.7, 110.7, 111.1, 115.6 (2C), 120.8,
130.4 (2C), 132.2, 152.8, 154.2, 155.4, 156.0, 160.3. Analysis: (C16H13ClN2O3S)
Calcd C 55.09%, H 3,76%, N 8.03%. Found: C 55.19%, H 3,82%, N 8.14%.
medium in each well was replaced by 100 ll of Opti-MEM I medium
(Invitrogen, CA, USA), containing 0.2 mg/ml XTT (sodium salt of 2,3-bis[2-
methoxy-4-nitro-5-sulfophenyl]-2 H-tetrazolium-5-carboxyanilide inner salt)
(Invitrogen, CA, USA) and 0.002 mg/ml PMS (phenazine methosulfate) (Sigma,
MO, USA). After 20 min, solution from each well was taken out for
spectrophotometric measurement of OD470 to determine the relative amount
of soluble XTT formazan in each well. All experiments were performed at least
in duplicates.
24. Compounds 2a,c–e, 3a,c–e,
spectroscopic and elemental analysis data.
25. The gene encoding full-length human Hsp90
RZPD, Deutsches Ressourcenzentrum für Genomforschung GmbH (Germany).
For protein expression, full-length HSP90 sequence (Hsp90F) and the N-
terminal fragment of the gene, corresponding to amino acids 3–241 (Hsp90N),
4 and 5 were also fully characterised by
a
protein was purchased from
a
28. Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Tierney, S.; Nofziger,
T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R. Cancer Res. 1988, 48, 4827.