2.2. Synthetic inhibitors
2.2.1. Purines and derivatives
The unique shape adopted by ATP when bound to the N-terminal nucleotide pocket of Hsp90 was used as the basis for the design of the first synthetic inhibitors.69
These were ATP mimics composed of a purine-scaffold tethered by a linker to a neighboring aryl moiety, thus forming the C-shaped conformation. PU3 (19
; ), the first synthesized purine-scaffold inhibitor, bound Hsp90 with an EC50
of 15–20 μM, approximately 20-fold weaker than 17-AAG (EC50
= 1 μM) under similar conditions. PU3 elicited cellular effects that resemble those of GM, including degradation of Raf-1, Her2 and estrogen receptor.69
PU3 also demonstrated antiproliferative effects against several tested breast cancer cell lines, MCF-7, SKBr3 and MDA-MB-468, at low micromolar concentrations. The co-crystal structure of PU3 bound to human Hsp90 () suggested that the purine-scaffold provides favorable interactions with the N-terminal ATP pocket: the C6-NH2
interacts with an Asp93-Thr184-water triad, and two of the purine-ring nitrogens form hydrogen bonds with Asn51, a network of waters and the backbone of several other amino acid residues.70
The aromatic ring of PU3, stacked between the side chains of Phe138 and Leu107, interacts with Met98 and Leu103. The methoxy groups make contacts with the aromatic rings of Trp162 and Tyr139 as well as the aliphatic carbons of Ala111 and Val150. The first and second methylene groups of the N9-alkyl chain provide additional hydrophobic interactions with residues Leu107 and Met98.70
PU3 induces a unique conformational change in the ATP-binding site lid and opens a lipophilic pocket, a mode of binding which differentiates this class from other Hsp90 inhibitor chemotypes.
Structures of purine-scaffold Hsp90 inhibitors.
Although the activity of PU3 was lower than the natural product inhibitors, its structure is amenable to extensive chemical modification in an effort to improve both potency and physical/chemical properties. Efforts were focused on probing the structure-activity relationship of the aromatic moiety on the purine at C8-position and on investigating the effects of various chains at N9-position. Additionally, the nature of the linker between the purine-scaffold and the substituted aromatic ring has also been investigated.
In the first described library of PU3 derivatives, about 70 compounds were synthesized which investigated the effects of both the nature and length of the N9-chain, substitution at position 2 of the purine moiety, addition of halogens on the trimethoxyphenyl moiety and the nature and length of the bridge between the purine and trimethoxyphenyl moiety.71
The system was sensitive to variations on the N9-chain, with a preference for linkers with the first two to three methylenes of unbranched nature. Pent-4-ynyl and 2-isopropoxy-ethyl were favored from over 43 diverse chains and resulted in improved binding to Hsp90 by almost an order of magnitude (EC50
= 1.5 and 1.7 μM, respectively). The introduction of fluorine at C2 of the purine generally increased potency, likely through enhanced H-bond donor potential of C6-NH2
, and also increased water solubility for these compounds. The introduction of chlorine at C2 of the trimethoxyphenyl ring improves activity over PU3 greater than 3-fold (IC50
= 4.6 μM) by increasing the steric fit and decreasing rotation of the aryl moiety. Attempts in altering the bridge C with O, OCH2
or NH gave inactive compounds, suggesting that the angle between the purine and trimethoxyphenyl moieties is important. Combining the favorable substitutions resulted in PU24FCl (20
; ) with an N9-pent-4-ynyl chain, which is 30 times more active than PU3 (IC50
= 1–2 μM) in degradation of Her2 in MCF-7 cells. PU24FCl exhibited antiproliferative effects against a broad panel of cancer cell lines with IC50
= 2–7μM and led to the degradation of Akt, Raf-1, Bcr-Abl, Her2, mAR and mutant p53 at similar concentrations.21
The agent remained active in cancer cells resistant to 17-AAG. In addition, PU24FCl had 10- to 50-fold greater affinity for Hsp90 from transformed cells. When administered to mice bearing tumors, PU24FCl rapidly distributed to tumors and was retained in tumor mass, while being cleared from normal tissue. It also resulted in significant depletion of Her2, Akt and Raf-1 in tumors and a 72% reduction in tumor burden as compared to control, in mice bearing MCF-7 breast cancer xenografted tumors when administered 200 mg/kg ip on alternate days.21
Efforts aimed towards the identification of alternative linkers between the purine-scaffold and the aromatic moiety led to the discovery of sulfur as an allowable modification.72,73
The introduction of O, OCH2
, NH, SO and SO2
either reduced or abolished activity, probably due to the dramatic conformational change caused by these linkers between the purine and the phenyl moiety which interferes with the desired shape for binding to Hsp90.71,74-76
() was reported to exhibit good potency in several cell-based assays, with an IC50
= 0.28 μM for degradation of Her2.72,74
The SAR of the 8-arylsulfanyl series generally correlates with that observed for the methylene linker series.73
The effects of substitution on the aryl moiety in this series have been investigated and it was determined that substitutions at positions 2,4,5 were most favored with regards to binding in the Hsp90 ATP-pocket.73
These efforts identified 2-bromo-4,5-methylenedioxy derivative 22
() as a potent Hsp90 binder (EC50
= 30 nM) and high selectivity (700- to 3000-fold) for tumor over normal Hsp90. Derivative 22
induced Her2 degradation with IC50
= 300 nM and inhibited the growth of SKBr3 cells with IC50
= 200 nM.
The attachment of a primary or secondary amine on the N9-alkyl chain of the purine-scaffold allowed for their formulation as salts, thus dramatically increasing water solubility. Favorable substituents from previous SAR studies were assembled into derivatives of high binding affinity, good water solubility and oral bioavailability. Introduction of an unbranched amino alkyl chain, such as in compound 23
= 140 nM for Her-2 degradation in MCF-7 cells) and 24
= 90 nM for Her-2 degradation in MCF-7 cells) provided potent derivatives with improved water solubility.74,75
exhibited antiproliferative effects against MCF-7 cells with IC50
= 200 and 500 nM, respectively. Both of these exhibit excellent water solubility (>10 mg/ml) as their phosphoric acid salt. In fact, when 24
was administered orally as its phosphoric acid salt, it induced approximately 70% tumor growth inhibition in an N87 stomach cancer xenograft mouse model at a dose of 200 mg/kg/day, 5 days/week after 30 days.74
Replacing the 5-methoxy group of the aryl moiety with 4,5-methylenedioxy gave derivatives such as PU-DZ8 (25
; ) which had an EC50
= 22 nM for Hsp90 and an IC50
= 90 nM for degradation of Her2 in SKBr3 cells.77
Recently, inhibition of Hsp90 by PU-DZ8 and other purine-scaffold compounds in mouse models of tauopathies resulted in the reduction of aggregated tau78
and the degradation of aberrant tau phosphorylated species.79
Tauopathies, such as Alzheimer’s disease and frontotemporal dementias are neurodegenerative diseases characterized by aberrant tau protein species. PU-H71 (26
; ), containing the 3-isopropylamino-propyl chain, was even more potent with EC50
= 16 nM for Hsp90 and IC50
= 50 nM for degradation of Her2 in SKBr3 cells.77
PU-H71 has recently been shown to significantly inhibit tumor growth in mice bearing NCI-N417 small-cell lung cancer tumors at a dose of 75 mg/kg daily for 5 days administered ip.80
In the NCI-N417 tumors, Akt inactivation and cleavage of PARP, demonstrating apoptosis, was detected as early as 6 h after PU-H71 administration, and the effects were still evident at 36 h. These effects occurred at pharmacologically relevant doses of PU-H71 that were retained in tumors (5.2 μM at 36 h). To our knowledge these data portray the first documented induction of apoptosis by an Hsp90 inhibitor in vivo. In several models of basal-like breast cancer, a tumor type reported to be less sensitive to GM-derivatives, administration on an alternate day schedule of 75 mg/kg PU-H71 resulted in over 95% tumor growth inhibition, with complete cures observed in several mice.81
Due to its promising pharmacologic profile and potent anti-tumor activity in several cancer types, PU-H71 is scheduled for clinical translation in early 2009.
The efforts described thus far have been focused on compounds in which the purine-scaffold is linked to a polysubstituted phenyl group via a methylene or sulfide bridge. The replacement of the phenyl group with appropriately substituted benzothiazole or pyridinothiazole group has been reported to provide a new and potent series of Hsp90 inhibitory compounds.82
The benzothiazole class requires a 7′-substituent with Cl being optimal. Derivative 27
() induces Her2 degradation in MCF-7 cells with an IC50
= 180 nM, compared to 15 nM for 17-AAG. Potency was increased by substituting the N9-butyl with 2-cyclopropylamino-ethyl (28
; ), 2-neopentylamino-ethyl (29
; ) or 2-diethylphosphonate-ethyl (30
; ), which induce Her2 degradation in MCF-7 cells with an IC50
= 110, 35 and 30 nM, respectively. Similar to the benzothiazoles, the most active compounds in the pyridinothiazole class have a two-carbon linker substituted with a diethylphosphonate moiety. The 7′-Br (31
; ) and Cl (32
; ) analogs have comparable activity for Her2 degradation in MCF-7 cells with an IC50
= 28 and 30 nM, respectively. Derivative 28 exhibited 56% tumor growth inhibition in an N87 xenograft mouse model treated orally with 200 mg/kg/day, 5 days/week after 40 days.
Benzothiazole, pyridinothiazole and N9-pyridylmethyl-substituted purine Hsp90 inhibitors.
Attachment of the benzyl or a pyridylmethyl group on position N9 gave another series of potent inhibitors which retained the critical interactions to Hsp90 as well as the C-shape conformation.83
Several compounds with low nanomolar potency are reported, including the pyridylmethyl derivatives 33
() and 34
(CNF-2024/BIIB021; ), with IC50
= 20 and 30 nM, respectively, in the Her2 degradation assay. Derivative 33
exhibited 83% tumor growth inhibition in an N87 xenograft mouse model treated orally with 60 mg/kg/day, 5 days/week after 5 weeks. CNF-2024/BIIB021 demonstrated 87% tumor growth inhibition at 125 mg/kg in an identical model and dosing schedule. CNF-2024/BIIB021 is the first synthetic small molecule to enter Phase I clinical trials.84
Dose-escalation studies of CNF-2024/BIIB021 administered orally to patients with relapsed B cell chronic lymphocytic leukemia, advanced solid tumors and lymphomas are currently ongoing at several centers.
A number of structures resembling the purine-scaffold have also been reported as Hsp90 inhibitors. Modification to the ring adjacent to the pyrimidine has led to triazolopyrimidines, pyrazolopyrimidines and pyrrolopyrimidines, which have demonstrated antiproliferative effects in MCF-7 cells and ability to induce the degradation of Her2.85
have also been reported as Hsp90 inhibitors.
2.2.2. Pyrazoles and isoxazoles
Pyrazoles represent another novel scaffold to be identified and developed as Hsp90 inhibitors.88-91
From high throughput screening against a library of 50,000 compounds, the 3,4-diarylpyrazole 35
(CCT08159/RBT0028535; ) was shown to inhibit yeast Hsp90 ATPase assay activity with IC50
= 8.9 μM. Derivative 35
demonstrated cellular activity in a proliferation assay using HCT116 colon cancer cells (GI50
= 4.1 μM)88
and its addition to HCT116 cells resulted in the depletion of Hsp90 client proteins Raf-1 and Cdk4. SAR studies showed that the ethyl group and both hydroxyls of the resorcinol ring were important for activity, as methylation or removal of one of the hydroxyls led to decreased or abolished activity. However, substitution of the ethyl group with aClasin 36
(CCT072440; ) resulted in a more potent compound with an IC50
<1 μM in the ATPase assay, but with comparable activity in the proliferation assay (GI50
= 4.0 μM).
Structure of pyrazole-scaffold Hsp90 inhibitors.
Crystallographic data shows that CCT08159 binds to the ATP pocket in the N-terminal domain of yeast Hsp90 in a manner similar to RD ().88
The 2′-hydroxyl group of the resorcinol moiety and the N2 of the pyrazole form a network of hydrogen bonds with Asp79, Gly83 and Thr171 through a molecule of bound water. The 4′-hydroxyl of the resorcinol ring also forms a hydrogen bond interaction with Leu34 via a water bridge. The 5′-ethyl occupies a hydrophobic pocket, which is taken by the 5′-chloro in CCT072440. The crystal structure with human Hsp90α showed that the C5 methyl group of the pyrazole was close to the carbonyl oxygen of Gly97, which cannot form a hydrogen bond to the pyrazole NH because the peptide bond was out of the plane to it.89
Incorporation of an amide side chain at C5 could potentially create another ligand-protein hydrogen bond interaction, thus enhancing binding affinity. In fact, compound 37
(VER-49009; ) was identified as the strongest Hsp90 binder (IC50
= 25 nM) in a fluorescence polarization assay amongst a series of C5-amides and was able to reduce cellular levels of the client proteins Raf-1 and Cdk4.89
It also showed better cellular activity over CCT018159 with GI50
= 260 nM, comparable to that determined for 17-AAG and RD, and exhibited antiproliferative effects against a wide range of human cancer cell lines.92
The crystal structure of VER-49009 showed that the amide group made a hydrogen bond interaction with Gly97, suggesting a rationale for the tighter binding observed in the C5-amide series.
Substitution of the pyrazole 4-position of CCT018159 with amides90
has also led to Hsp90 inhibitors. In the pyrazole-4-carboxamide series, compounds 38
() and 39
() were determined to be the most potent with IC50
= 0.258 and 0.461 μM, respectively.90
These exhibit increased binding as compared to related compounds due to a hydrogen bond interaction of Phe138 with the ketone in 38
or the sulfonamide in 39
. Derivative 38
induced the degradation of Raf-1, Her2 and Cdk4 and inhibited the growth of HCT116 cells with GI50
= 11.6 μM. In the 4-aminopyrazoles, piperazinyl, morpholino and piperidyl derivatives were synthesized, of which the piperazinyl derivatives were most potent.93
() and 41
() were the most potent compounds in this series and had IC50
= 0.74 and 0.6 μM, respectively, in a fluorescence polarization assay designed to measure Hsp90 binding affinity. Derivatives 40
induced the degradation of Raf-1 and inhibited the growth of HCT116 cells with GI50
= 3.1 and 6.5 μM, respectively.
High throughput screening had been used independently from the efforts described above to also identify pyrazoles as Hsp90 inhibitors. From a diverse library of 1 million compounds, a number were identified as hits using a high throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay.91
Among these compounds, 42
(G3129; ) and 43
(G3130; ) were confirmed as inhibitors in a surface plasmon resonance assay with Kd
= 0.68 and 0.28 μM, respectively. The crystal structure of these compounds shows that they bind to the N-terminal ATP pocket in a manner similar to CCT018159. In G3129, the carboxylate at C5 provides a further salt bridge interaction in addition to hydrogen bond interaction, whereas in G3130, the imidazole at C4 of the pyrazole is involved in water-mediated hydrogen bonding. The higher affinity of G3130 suggests that the water mediated polar interaction of the C4-imidazole is the more important interaction.
Isoxazoles are chemically related to pyrazoles and have recently been shown to be potent Hsp90 inhibitors by also binding to the N-terminal ATP pocket.92,94
As has already been mentioned, the N2 of the pyrazole acts as an H-bond acceptor in a network of hydrogen bonds with Hsp90 through a molecule of bound water (). Pyrazoles can exist in tautomeric forms and when this nitrogen is protonated it is unlikely to bind well to Hsp90. Therefore, it was rationalized that locking the molecule into a 100% H-bond acceptor by changing the nitrogen to oxygen might be advantageous for binding. The resulting isoxazole-scaffold proved to be a more than tolerable change, as demonstrated by the isoxazole anolog of VER-49009, 44
(VER-50589; ), which has an IC50
= 28 nM in a fluorescence polarization competitive binding assay similar to that of VER-49009 (IC50
= 25 nM).94
However, VER-50589 showed greater cell growth inhibition against each of six different human cancer cell lines than VER-49009. It also caused the depletion of Hsp90 client proteins Her2, Raf-1, CDK-4 and phosphorylated Akt in SKMEL2 and WM266.4 melanoma cells, and inhibited tumor growth by approximately 30% in mice bearing HCT116 xenografts when administered ip at 100 mg/kg daily.92
Structure of isoxazole-scaffold Hsp90 inhibitors.
Isoxazoles substituted at the para-position of the 4-aryl ring with various amine groups had IC50
< 70 nM in the binding assay and were at least as potent as their pyrazole analogs.94
Their ability to inhibit the growth of HCT116 cells was also greater than the corresponding pyrazoles. Methylation of the resorcinol hydroxyls caused a loss or drop in potency as was observed for the pyrazole class. The 5′-position of the isoxazoles proved to be more amenable to modification than in pyrazoles such as VER-49009. Phenyl, phenylethyl, tert
-butyl and isopropyl substituted derivatives retained the activity of their corresponding chloro or ethyl substituted analogs. Moreover, these derivatives had good activity in the cell growth inhibition assays, especially the 5′-isopropyl derivatives 45
(VER-52296/NVP-AUY922; ) and 46
(), which have GI50
= 16 and 6 nM, respectively, against HCT116 cells. VER-52296/NVP-AUY922 inhibited tumor growth by approximately 50% in mice bearing HCT116 xenografts when administered ip daily at 50 mg/kg. NVP-AUY922 is currently in Phase I clinical evaluation in patients with advanced cancers.
Isoxazoles were also isolated in a high throughput screen using more than 15,000 compounds from the National Institutes of Health Molecular Libraries Screening Center Network.66
This effort identified derivative 47
()as a 9 μM potency Hsp90 inhibitor. Benzisoxazole-scaffold compounds with Hsp90 binding properties were also recently reported.95
High throughput screening followed by optimization efforts led to the discovery of compound 48
() as an inhibitor in the fluorescence polarization binding assay with IC50
= 30 nM, compared to 20 nM for GM. It was active in a panel of cell lines with submicromolar IC50
values and was able to induce degradation of Her2 and the androgen receptor. From crystallographic data with the N-terminal domain of human Hsp90a, the 2′-hydroxyl group of the resorcinol moiety forms an important interaction with Asp93, whereas the other hydroxyl substituent is involved in maintaining the water-mediated hydrogen bond network. The flexible morpholin linker was incorporated to increase water solubility and potency.