EC144 is a potent inhibitor of the heat shock protein 90

Article abstract of DOI:10.1021/jm300810x

Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl) methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat shock protein 90 (Hsp90) and is substantially more potent in vitro and in vivo than the first generation inhibitor 14 (BIIB021) that completed phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α binding assay (IC₅₀ = 1.1 vs 5.1 nM) as well as in its ability to degrade Her-2 in MCF-7 cells (EC ₅₀ = 14 vs 38 nM). In a mouse model of gastric tumors (N87), 40 stops tumor growth at 5 mg/kg and causes partial tumor regressions at 10 mg/kg (po, qd× 5). Under the same conditions, 14 stops tumor growth only at 120 mg/kg, and does not induce partial regressions. Thus, alkyne 40 is approximately 20-fold more efficacious than 14 in mice.

Full text of DOI:10.1021/jm300810x

Article
pubs.acs.org/jmc
EC144 Is a Potent Inhibitor of the Heat Shock Protein 90
Jiandong Shi,† Ryan Van de Water,† Kevin Hong,† Ryan B. Lamer,† Kenneth W. Weichert,†
Cristina M. Sandoval,† Srinivas R. Kasibhatla,† Marcus F. Boehm,† Jianhua Chao,† Karen Lundgren,†
Noelito Timple,† Rachel Lough,† Gerardo Ibanez,† Christina Boykin,† Francis J. Burrows,†
Marilyn R. Kehry,† Theodore J. Yun,† Erin K. Harning,† Christine Ambrose,‡ Jeffrey Thompson,‡
Sarah A. Bixler,‡ Anthone Dunah,‡ Pamela Snodgrass-Belt,‡ Joseph Arndt,‡ Istvan J. Enyedy,‡ Ping Li,‡
Victor S. Hong,‡ Andres McKenzie,‡ and Marco A. Biamonte*,†
†Biogen Idec, 5200 Research Place, San Diego, California 92122, United States
‡Biogen Idec, 14 Cambridge Center, Cambridge, Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-di-
methylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-meth-
ylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat
shock protein 90 (Hsp90) and is substantially more potent in vitro and
in vivo than the first generation inhibitor 14 (BIIB021) that completed
phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α
binding assay (IC50 = 1.1 vs 5.1 nM) as well as in its ability to degrade
Her-2 in MCF-7 cells (EC50 = 14 vs 38 nM). In a mouse model of
gastric tumors (N87), 40 stops tumor growth at 5 mg/kg and causes
partial tumor regressions at 10 mg/kg (po, qd×5). Under the same
conditions, 14 stops tumor growth only at 120 mg/kg, and does not
induce partial regressions. Thus, alkyne 40 is approximately 20-fold more efficacious than 14 in mice.
soluble hydrochloride salt which also advanced to phase III
studies.4 The derivative 17-desmethoxy-17-N,N-dimethylami-
noethylaminogeldanamycin 3 (17-DMAG, alvespimycin, NCI,
Kosan-Bristol Myers Squibb) was also designed to improve the
solubility but proved to be more toxic in preclinical species.5
The major metabolite of 17-AAG, 17-amino-17-desmethox-
ygeldanamycin 4 (17-AG, IPI-493, Infinity), displayed greatly
improved oral bioavailability and was advanced as an oral
formulation, thus obviating the need for solubility imposed by
the intravenous route of administration.6 In parallel, 12 fully
synthetic small molecules were developed (Scheme 1), as either
intravenous or oral compounds. The intravenous drugs are 5
(NVP-AUY922, Novartis, phase II),7 6 (AT-13387, Astex,
phase II),8 7 (ganetispib, STA-9090, Synta, phase II),9 8 (KW-
2478, Kyowa Hakko Kirin, phase I/II),10 9 (XL-888, Exelixis,
phase I),1a 10 (PU-H71, Memorial Sloan-Kettering Cancer
Center, phase I),11 and BIIB028 (Biogen Idec, phase I,
structure not public). The oral drugs are 11 (DEBIO-0932/
CUDC305, Debiopharm, phase I),12 12 (MPC-3100, Myrexis,
phase I),13 13a (PF-4929113/SNX-5422, Pfizer, phase I),
which is a glycine prodrug of 13b,14 14 (BIIB021, Biogen Idec,
phase II),15 and 15 (NVP-HSP990, Novartis, phase I).16
As of this writing, the major challenge in the Hsp90 arena is
to identify which human cancers best respond to an Hsp90
1. INTRODUCTION
The rationale for selecting heat shock protein 90 (Hsp90) as a
cancer target has been extensively reviewed.1 In essence, Hsp90
regulates the folding of nascent and mature proteins,
collectively dubbed “client proteins”. Among the over 200
reported Hsp90 client proteins,2 one finds a large proportion of
proteins implicated in cell-growth, including well-known
oncogenes (Raf-1, Akt, cdk4, Src, Flt-3, hTert, c-Met, etc.)
and five proteins targeted by approved cancer drugs: Her-2/neu
(trastuzumab), Bcr-Abl (imatinib), the estrogen receptor
(tamoxifen), the androgen receptor (bicalutamide), and
VEGF, the vascular endothelial growth factor receptor
(sunitinib). Inhibition of Hsp90 causes client proteins to
adopt aberrant conformations, and these abnormally folded
proteins are eliminated via ubiquitination and proteasome
degradation. The net result is that Hsp90 inhibitors induce the
concomitant degradation of a number of oncogenes and
therefore intervene simultaneously on different pathways of
cancer progression.
In 1998−1999, the first clinical trials with an Hsp90 inhibitor
were initiated on the natural product derivative 17-allylamino-
17-desmethoxygeldanamycin 1 (17-AAG, tanespimycin, Cancer
Research UK, NCI, Scheme 1). 17-AAG was administered
intravenously and progressed to phase III studies (Kosan-
Bristol Myers Squibb).3 Although 17-AAG was poorly water-
soluble and difficult to formulate, its hydroquinone version 2
(IPI-504, retaspimycin, Infinity) could be converted to a water-
Received: June 8, 2012
© XXXX American Chemical Society
A
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Scheme 1. Hsp90 Inhibitors in Clinical Trials
inhibitor and with which drug combination. Breast cancer is the
most studied indication for Hsp90 inhibitors. The rationale is
simple: some breast cancers are Her-2 driven, and Her-2 is
degraded particularly rapidly upon exposure to Hsp90
inhibitors. Furthermore, Her-2 is the molecular target of the
breast cancer drug trastuzumab. In a notable phase II trial,
patients with Her-2 positive metastatic breast cancer that were
progressing on trastuzumab responded to the combination of
17-AAG and trastuzumab, with a 22% overall response rate.17
This was the first phase II study in which 17-AAG definitely
showed responses in solid tumors, as defined by the response
evaluation criteria in solid tumors (RECIST). In addition to
breast cancer, non-small-cell lung cancer (NSCLC) and
multiple myeloma (MM) also appear to be of high interest.
In phase II studies in NSCLC patients, retaspimycin and
resorcinol 7 controlled the disease in a subset of patients
harboring a mutated/rearranged anaplastic lymphoma kinase
(ALK).18,19 This is consistent with the fact that ALK is the only
Hsp90 client showing equivalent or greater sensitivity than
Her-2 to Hsp90 inhibitors.1a ALK + NSCLC can be treated
with the approved ALK inhibitor crizotinib, but resistance
appears within 1 year. In vitro, the combination of crizotinib
with an Hsp90 inhibitor overcomes the crizotinib resistance.20
In a phase I/II study in a MM population, the combination of
17-AAG and bortezomib gave an objective response rate of
27% and was also active in patients that were refractory to
bortezomib.21 Interestingly, at the preclinical level there is
evidence that even low doses of Hsp90 inhibitors can be
strongly synergistic with radiation therapy in head and neck
squamous cell carcinoma.22 All Hsp90 inhibitors cause fatigue
B
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a
Scheme 2
a
Modifications of 16 in positions Ra−Rf led to a decrease in potency, suggesting that the solvent exposed area of the molecule was pointing in the
same direction as the N7 lone pair of purine 14.
and diarrhea as side effects, and some but not all also produce
ocular toxicities, which led to the discontinuation of benzamide
13a.23,24
drastic decrease in potency. Similarly, replacement of X or Y by
a carbon atom also led to a loss in potency. We therefore
hypothesized that each of these positions was in close contact
with the protein. Yet it seemed logical to expect that at least
one part of the molecule would face the solvent and that
modifications of the solvent-exposed area of the molecule
would be the most tolerable. We felt that the solvent-exposed
area may be used to our advantage to improve the drug
properties and to possibly gain potency by making new
interactions at the rim of the binding pocket. Since the
substituents Ra−Rf, as well as the atoms X and Y, were clearly
failing at reaching the solvent-exposed area, we deduced that
the solvent-exposed area must have been pointing in another
direction. By elimination, we surmised that the solvent was
facing the direction of the N7 lone pair, and this initial
hypothesis was later validated by an X-ray structure (Figure 1).
In 2005, we selected purine 14 as clinical candidate because it
was the first orally bioavailable Hsp90 inhibitor that
consistently demonstrated efficacy in mouse tumor models.15
The fact that it could be synthesized in a single step was an
additional advantage.15 There was a limitation, however, in that
high doses (60−120 mg kg−1 day−1) were required for efficacy
in mouse. In human, even though the doses of 14 may be
lower, treating patients still requires 100 mg twice daily or 450
mg thrice weekly, as proposed in a phase II study conducted in
combination with exemestane (Aromasin, 25 mg daily) for
hormone receptor positive metastatic breast cancer (HR +
mBC). While running clinical trials with 14, we sought a second
generation oral Hsp90 inhibitor that would be more efficacious
than 14 to allow for lower daily or weekly dosing. We now
report the chemistry that led to a series of deazapurine
analogues, some of which showed efficacy in mouse tumor
models at 5 mg/kg and represent an improvement of over 1
logarithm over the first generation inhibitors.
2. ASSAYS
HSP90 exists as two isoforms: HSP90α is an inducible form
overexpressed in cancer cells, while HSP90β is the constitutive
form. Both HSP90α and HSP90β are found in the cytoplasm.
There are also two paralogues: Grp94, localized in the
endoplasmic reticulum lumen, and TRAP1, confined to
mitochondria. The binding assays were performed on
HSP90α as described previously.25
The effect of HSP90 inhibitors on breast cancer MCF-7 cells
was evaluated by measuring the degradation of the HSP90
client Her-2. Her-2, being a cell-surface receptor, can be
conveniently monitored with extracellular-directed fluorescent
antibodies and flow cytometry.25
For in vivo work, and unless otherwise noted, 40 was
suspended in an aqueous solution containing 0.5 wt %
carboxymethylcellulose (CMC) and 0.1 wt % Tween 80. The
suspension was sonicated for 30 min at room temperature and
homogenized for 30 s using a homogenizer set at 4000 rpm.
Alternatively, 40 was dissolved in 0.1 N HCl, and the two
formulations gave comparable results. Pharmacokinetic param-
eters were determined by analyzing extracted plasma and tissue
samples by LC/MS/MS and by using noncompartmental
methods with WinNonlin, version 5 (Pharsight).
Figure 1. The X-ray structure of 14 in complex with Hsp90 shows that
N7 faces a solvent exposed area (PDB code: 3QDD).
In order to substitute the molecule along the desired vector,
it was necessary to replace N7 with a carbon atom. To this
effect, we prepared pyrrolo[2,3-d]pyrimidines (=deazapurines)
based on scaffold 17 (Scheme 2) where the iodo group would
enable a range of cross-couplings. The synthesis started with
2,6-diaminopyrimidin-4-ol 18 (Scheme 3). The next four steps
relied on existing procedures26−30 that were modified to
increase the yield of 23 and to obviate the need for
chromatographic purifications. Thus, treatment of 18 with
3. DESIGN AND SYNTHESIS OF THE
PYRROLO[2,3-D]PYRIMIDINE SCAFFOLD
The optimization of 14 was challenging, as all attempts to
modify the substituents Ra−Rf (Scheme 2) typically led to a
C
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Scheme 3. Preferred Synthesis of Pyrrolo[2,3-d]pyrimidine 27
Scheme 4. Alternative Synthesis to Pyrrolo[2,3-d]pyrimidine 27 and Synthesis of 35
aqueous chloroacetaldehyde provided the pyrrolo[2,3-d]-
pyrimidine 19 (42%). By performing this cyclization in the
presence of N,N-dimethylacetamide (DMA) as opposed to
DMF,26 the desired product precipitated out as a 1:1 DMA
complex, which facilitated its production on kilogram scale. The
DMA was then easily removed by recrystallization from MeOH
(95%). Protection of the amino group with PivCl gave amide
21 (93%).27 Deoxychlorination28 was best achieved with a
D
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a
Scheme 5
a
Conditions: (a) PhB(OH)2, Pd(PPh3)4, DMF, 2 M K2CO3, 80 °C (20%); (b) HCC−SiMe3, Et3N, CuI, Pd(PPh3)4, DMF, 50 °C (25−50%),
then TBAF, THF, rt (25%); (c) alkyne, Et3N, CuI, Pd(PPh3)4, DMF, 50 °C (25−50%), or alkyne, Et3N, CuI, 10% Pd/C, PPh3, H2O, DMA or
DMSO, 75 °C (40−75%); (d) Raney Ni, H2 (4.5 atm), MeOH, rt (32%).
POCl3/BnNEt3Cl system to give 22 (70%), and a subsequent
iodination with N-iodosuccinimide (NIS)29,30 yielded 23
regioselectively (81%). It is noteworthy that the pivaloyl
group was necessary for both the deoxychlorination and the
iodination to proceed smoothly. Attempts at replacing the
pivaloyl group with an acetyl or octanoyl group resulted in a
complex mixture of products upon sequential treatment with
POCl3 and NIS. However, cleaving the pivaloyl group proved
to be a difficult operation. After extensive experimentation, we
found that the pivaloyl group could be efficiently removed if 23
was first alkylated with the commercially available chlorome-
thylpyridine 24 (80%) and then treated with ZnCl2 in refluxing
EtOH, thus yielding the key intermediate 27 (85%).
Interestingly, this deprotection procedure was successful only
if 23 was alkylated with 24 prior to treatment with ZnCl2.
Alkylation of 23 with the isomeric chloromethylpyridine 28
gave substrate 29, which failed to undergo the ZnCl2-mediated
deprotection. Similarly, alkylation of 23 with benzyl bromide
gave substrate 31 that was equally unreactive toward ZnCl2. To
rationalize these results, we suggest that the pyridine nitrogen
of 25 is actively involved in the transition state, possibly by
offering an additional coordination site for the Zn atom as
shown in the putative intermediate 26. The entire sequence 18
→ 27 could be performed without any chromatographic
purification on kilogram scale.
SAR purposes, compound 33 was also directly alkylated to give
pyrrolopyrimdine 35, devoid of any substituent at C5.
4. OPTIMIZATION OF POTENCY
Having devised two efficient syntheses of iodide 27, we
submitted it to Pd cross-couplings (Scheme 5). A Suzuki
coupling with PhB(OH)2 gave the 5-phenyl adduct 36, while
Sonogashira couplings with TMS acetylene, propargylic
alcohol, homopropargylic alcohol, or with 2-methylpent-4-yn-
2-ol32 gave derivatives 37−40. Hydrogenation of propargylic
alcohol 38 with Raney nickel gave propanol 41. The reactions
leading to 37−41 were not optimized. All compounds were
prepared in greater than 95% purity as determined by analytical
HPLC at 254 nm.
In a biochemical assay, compounds 35−41 all bound to
Hsp90α with IC50 < 10 nM (Table 1). Next, their ability to
induce the degradation of Her-2 in MCF-7 breast cancer cells
was examined (Table 1). The first-generation clinical candidate
14 degraded Her-2 with EC50 = 38 nM. In the same assay, the
unsubstituted pyrrolo[2,3-d]pyrimidine 35 (EC50 = 100 nM)
was almost as active as 14. More importantly, substituents at
the C5 position were tolerated. Compared to 35, a phenyl
substituent (36, 250 nM) only led to a 2.5-fold decrease in
potency, while a terminal alkyne group (37, 26 nM) improved
the potency 4-fold. The propargyl alcohol 38 (15 nM) and the
homopropargylic alcohols 39 (9 nM) and 40 (EC144, 14 nM)
were the most potent compounds and were approximately 3-
fold more potent in vitro than the first-generation clinical
candidate 14. Reduction of the propargylic alcohol 38 to the
propanol 41 (32 nM) was tolerated.
In an alternative route (Scheme 4), pyrrolo[2,3-d]pyrimidine
33 was temporarily silylated with N,O-bis(trimethylsilyl)-
acetamide (BSA), reacted with NIS, and desilylated with
water to give iodide 34 in a single pot.31 Iodide 34 was poorly
soluble in organic solvents, which precluded further purifica-
tion. Iodide 34 could nonetheless be alkylated to give 27. For
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nM) and Grp94 (Ki = 61 nM).34 Alcohol 40 has no inhibitory
activity (IC50 > 10 μM) against a panel of 285 human kinases.34
When incubated with N87 (gastric) or BT474 (breast) tumor
cell lines, 40 displays the characteristic signature of Hsp90
inhibitors: degradation of the client proteins (Her-2, EGFR,
IGFR, ERK, Akt, Rb, cyclin D, cdk6), up-regulation of Hsp70,
and no effect on p85 PI3K, which is not a Hsp90 client (data
not shown for cell cultures, see section 7 for in vivo data).
Table 1. Hsp90 Inhibitors: Binding to Hsp90α and Effect on
the Degradation of Her-2 in MCF-7 Cells
binding
Hsp90α
Her-2 EC50
[nM]
inhibitor
17-AAG
R
IC50 [nM]
12
7
5
5 (resorcinol)
<0.4
1
13b (benzamide)
3
19
3
14 (purine)
5.1 2.0
38 13
98
6. IN VITRO ADMET AND IN VIVO PK
35
36
37
38
39
40
41
H
3
Ph
6.0 2.8
1.0 0.6
250 14
Alcohol 40 satisfies the “rule of five” by virtue of its two
hydrogen-bond donors, four hydrogen bond acceptors, a
molecular weight of 414, and a cLogP of 2.8. Alcohol 40 is
highly permeable in CACO-2 cells (34.7 × 10−6 cm/s, Table 2)
and is not actively effluxed (efflux ratio (B → A)/(A → B) =
1.1). The lack of efflux is similar to what was observed with the
first-generation purine 14 (efflux ratio = 1.5) and contrasts with
the behavior of resorcinol 5 (efflux ratio = 15) and benzamide
13b (efflux ratio = 35). Alcohol 40 is 94−97% protein bound
depending on the species and in this respect is similar to
resorcinol 5 (94−96%) and benzamide 13b (90−94%). Alcohol
40 is metabolized by microsomes at medium−high rates; the
intrinsic hepatic clearance is 119 mL min−1 kg−1 in human
microsomes, which is higher than for resorcinol 5 (39 mL
min−1 kg−1) and benzamide 13b (54 mL min−1 kg−1).
CCH
26 (n = 1)
CC−CH2OH
0.7 (n = 1) 15
7
CC−(CH2)2OH
CC−CH2−CMe2OH
CH2− CH2−CH2OH
2.0 1.3
1.1 0.5
9
3
14
5
5
3.9 (n = 1) 32
Thus, compounds 38−40 were the most potent ones in vitro.
This article focuses on compound 40, since it later proved to
have the best combination of pharmacokinetic properties and
efficacy in murine cancer models.33
A crystal structure of 40 in complex with Hsp90 revealed a
favorable hydrophobic interaction between the CC fragment
of the inhibitor and the adjacent side chains of Met-98 and Leu-
107 (Figure 2).34 There were no other obvious additional
In mice, the maximum tolerated dose (MTD) of alkyne 40
for a single oral administration is 30 mg/kg. Table 3 reports the
mouse pharmacokinetic data for a single oral dose of 40,
compared with the data for other known Hsp90 inhibitors. At
25 mg/kg, the plasma exposure of 40 is 4419 ng·h/mL. In
contrast, the first-generation inhibitor 14 required a dose of 100
mg/kg to give a comparable exposure. This may be more
evident by looking at the AUC/dose ratio which, if expressed in
(ng·h/mL)/(mg/kg), gives 48 for purine 14 and 177 for
alcohol 40. Alcohol 40 is cleared with a T1/2 of 1.5 h, which is
somewhat longer than for purine 14 (0.8 h) but shorter than
for benzamide 13 (2.9 h) and resorcinol 5 (6.6 h).
A tissue-distribution study (Table 4) indicates that 40
distributes well to the brain, spleen, and lymph nodes. Alcohol
40 crosses the blood-brain barrier (brain/plasma = 2.2), which
is consistent with the fact that it is not an efflux substrate.
Figure 2. X-ray structure of alcohol 40 in complex with Hsp90α (PDB
code: 3NMQ).
7. APPLICATIONS TO ONCOLOGY
interactions. The distance between the OH group and the Lys-
58 amino group was too large (4.3 Å) for a hydrogen bond,
although the solution structure may differ from the crystal
structure, possibly granting the side chain of Lys-58 enough
flexibility to form a hydrogen bond in solution.
Hsp90 inhibitors have been reported to be retained in tumors.
Alkyne 40 was administered in a single dose of 6 or 12 mg/kg
to mice bearing xenografts of N87 gastric tumors (Figure 3).
Tumors were collected 1, 6, and 24 h after dosing and analyzed.
At the 6 and 24 h time-points, the concentration of alkyne 40
was highest in the tumors and decreased in the order tumors >
spleen > brain > serum, indicating that tumors retain alkyne 40
better than other tissues as time progresses (Figure 3).
5. SELECTIVITY
The tertiary alcohol 40 binds to Hsp90α with Ki = 0.2 nM and
is selective against the Hsp90 paralogues TRAP1 (Ki = 255
a
Table 2. In Vitro ADME Parameters of Selected Hsp90 Inhibitors
microsomal intrinsic hepatic clearance
CACO-2 permeability [×10−6 cm/s]
protein binding, fraction unbound [%]
[mL min−1 kg−1
rat
]
Papp (A → B)
ratio (B → A)/(A → B)
mouse
rat
human
mouse
human
5 (resorcinol)
13b (benzamide)
14 (purine)
40
1.9
1.1
20
15
4.3
6.0
6.4
9.6
5.1
443
301
219
54
39
54
35
6.8
1.5
1.1
11.4
3.3
34.7
3.4
5.8
727
276
119
a
Errors on the protein binding are in the 0.2−0.9 range.
F
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Table 3. Pharmacokinetic Data for Selected Hsp90 Inhibitors in CD-1 Mouse after a Single Oral or Intravenous
a
Administration
compd
ROA
dose [mg/kg]
Cmax,Co [ng/mL]
Tmax [h]
AUC [ng·h/mL]
AUC/dose [(ng·h/mL)/(mg/kg)]
T1/2 [h]
5 (resorcinol)
13 (benzamide)
14 (purine)
40
iv
5
2180
1724
10000
1556
2409
8517
4800
4419
481
341
48
6.6
2.9
0.8
1.5
po
po
po
25
100
25
1.0
0.083
0.5
177
a
Benzamide 13 was administered as its prodrug 13a, and the PK parameters refer to its active metabolite 13b.
Table 4. Pharmacokinetic Data for 40 in DBA1 Mouse after
a Single po Administration of 5 mg/kg
Cmax
[ng/mL]
Tmax
[h]
AUCinf
[ng·h/mL]
T1/2
[h]
tissue/plasma
(AUC ratio)
tissue
plasma
brain
835
0.083
0.5
2118
4713
5186
1.2
2.1
1.6
1446
2144
2.2
2.4
lymph
nodes
1.0
spleen
1887
8159
2.0
3.9
Figure 4. Pharmacodynamic effect of alkyne 40. Mice bearing BT474
xenografts were treated with a single oral dose of 4 or 8 mg/kg alkyne
40. Tumors were collected for Western blot analysis at 6, 24, and 48 h
postdose.
Figure 3. Retention of alkyne 40 in tumors. Mice bearing N87
xenografts were administered a single dose of 6 or 12 mg/kg alkyne
40. The concentration of alkyne 40 was measured in the serum,
tumors, brain, and spleen at 1, 6, and 24 h postdose.
The pharmacodynamic profile of alkyne 40 was evaluated by
treating mice bearing breast-cancer BT474 tumors with a single
oral dose of 4 or 8 mg/kg (Figure 4). Alkyne 40 induces the
typical signature of Hsp90 inhibitors: degradation of the Hsp90
clients Her-2, pHer-2, pAKT, pRAf, pERK, cdk6, pRb, up-
regulation of Hsp70 and Hsp27, and no effect on PI3K, which
is not an Hsp90 client. Of note, the pharmacodynamic effect of
40 is long lasting. Since cells require 24−48 h to resynthesize
the client proteins, even a relatively brief exposure to 40
(plasma T1/2 = 1.2 h) results in a protracted pharmacodynamic
response that lasts >24 h. A similar effect was reported for
purine 14 and has been colloquially called a “hit-and-run
effect”.1c In addition, as mentioned above, 40 is retained in
tumors for longer than in the plasma.
In a N87 gastric tumor xenograft model, 40 caused tumor
stasis when dosed 5 days per week at 5 mg/kg and induced
partial tumor regressions at 10 mg/kg (Figure 5). This is a
significant increase in potency when compared to 14, which
was not quite as effective, even at 120 mg/kg, and which did
not cause partial regressions. The increase in potency of 40 was
accompanied by a commensurate increase in toxicity, and the
MTD of 40 on a 5 days per week schedule was 10 mg kg−1
day−1. Above the MTD, weight loss and diarrhea were
Figure 5. Inhibition of tumor growth in the N87 gastric tumor model.
Athymic mice bearing established N87 gastric tumor xenografts were
treated with vehicle control, purine 14, or alcohol 40 orally qd×5 for 4
weeks.35 Tumors were measured using Vernier calipers on the
indicated days, and tumor volumes are reported as mean volume
SEM tumor for groups of eight mice.
observed, as is typical of all Hsp90 inhibitors. The fact that
the toxicity increases proportionally with the efficacy was seen
with every Hsp90 inhibitor we tested, irrespective of its
chemical class, suggesting that the toxicity is consistently
mediated by Hsp90 (on-target).
Alcohol 40 was also tested in a PGP overexpressing
adrenocortical carcinoma model (Figure 6) and was active at
8 mg/kg. Purine 14 was less potent but still inhibited tumor
growth at 120 mg/kg. In contrast, 17-AAG, which is a known
G
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Figure 7. Pharmacodynamic effect of 40 on Hsp70 levels in the brain,
following administration of a single oral dose in B6D2F1 female mice.
HSP70 levels in brain homogenates were determined by ELISA and
analyzed for statistical significance using unpaired two-tailed t test.
be ill-suited for neurological indications that would require
repeated daily dosing.
Figure 6. Inhibition of tumor growth in the PGP overexpressing
NCI295 adrenal carcinoma. Athymic mice bearing established NCI295
carcinoma were treated with vehicle control, purine 14 (120 mg/kg,
po), alcohol 40 (8 mg/kg, po), or 17-AAG (60 mg/kg, ip), qd×5 for 4
weeks. Tumors were measured using Vernier calipers on the indicated
days, and tumor volumes are reported as the mean volume SEM
tumor for groups of seven mice.
10. CONCLUSION
In summary, the second-generation Hsp90 inhibitor 40 is an
orally available compound that is substantially more potent
than the first generation clinical inhibitor 14. Compound 40
binds Hsp90α with Ki = 0.2 nM (vs 1.7 nM for the first-
generation purine 14). In MCF-7 cells, 40 induces Her-2
degradation with EC50 = 14 5 nM and is 3 times more potent
than 14 (EC50 = 38 13 nM for 14). In mouse, the plasma
exposure upon a single oral dose of 25 mg/kg is 4419 ng·h/mL,
while 14 is required to be dosed at 100 mg/kg to give a
comparable exposure. Compound 40 is not a PGP substrate
and is not actively effluxed by CACO-2 cells. In an N87 gastric
tumor model, compound 40 completely blocks tumor growth
at 5 mg/kg upon oral daily dosing (po, qd×5) and causes
partial tumor regressions at 10 mg/kg. This is an improvement
of over 20-fold with respect to 14, which completely blocks
tumor growth only at 120 mg/kg (po, qd×5) and does not
induce regressions. Compound 40 is also active at 8 mg/kg in a
NCI295 adrenocortical carcinoma that overexpresses PGP and
against which the PGP substrate 17-AAG is inactive at 60 mg/
kg.
PGP substrate,36 did not slow tumor progression at its MTD
(60 mg/kg, iv). Thus, this PGP-overexpressing tumor
responded much better to 40 than to the PGP substrate 17-
AAG.
8. APPLICATIONS TO IMMUNOLOGY
The applications of 40 to inflammatory and autoimmune
conditions are reported in a separate publication, where alcohol
40 is shown to block inflammatory and immune processes in
vitro and in vivo.34 Alcohol 40 inhibited TNF-α release by LPS
administration in an LPS shock model. Furthermore, 40 also
suppressed disease development in T cell driven rodent
collagen-induced arthritis models.34
9. APPLICATIONS TO NEUROLOGY
We also investigated applications of 40 that would extend
beyond the scope of oncology. We reported in a separate
publication that 40 halts disease progression in rodent models
of arthritis.34 As for possible neurological indications, 40 is
brain permeable (brain/plasma AUC ratio of 2.2), and a single
oral dose of 30 mg/kg inhibits Hsp90 in the brains of mice, as
witnessed by the up-regulation of brain Hsp70. The effect was
statistically significant at 30 mg/kg but not at 10 mg/kg. Since
the MTD for repeated daily dosing is 10 mg/kg, we conclude
that 40 may not be well-suited to treat neurological indications.
However, compound 40 remains of interest for severe
inflammatory conditions and has a strong potential for the
treatment of cancer.
Inhibition of Hsp90 has been shown to lead to the degradation
of aberrant forms of p-tau,37 which aggregate to form
neurofibrillary tangles. Such tangles are a hallmark of
Alzheimer’s disease (AD).38−41 Since 40 distributes well across
the BBB (brain/plasma ratio of 2.2), we examined if 40 could
reach pharmacologically relevant concentrations in the brain by
monitoring the up-regulation of brain Hsp70. Up-regulation of
Hsp70 is the pharmacodynamic response expected of Hsp90
inhibitors. Groups of four B6D2F1 female mice were
administered a single oral dose of vehicle alone or compound
40 at several dose levels (Figure 7). The animals were
humanely sacrificed 24 h later, and Hsp70 levels in whole brain
homogenates were analyzed by ELISA. The level of Hsp70 was
determined to be significantly higher in the animals dosed with
30 mg/kg or higher compared to vehicle treatment alone. This
indicates that at 30 mg/kg the amount of 40 that crossed into
the brain was sufficient to robustly inhibit brain Hsp90. Hence,
alcohol 40 appears to be a good tool compound to examine the
effect of Hsp90 inhibition in the brain after a single acute dose.
It should be noted, however, that a dose of 30 mg/kg is 3 times
higher than what would be tolerated upon a repeated daily
dosing, suggesting that neurological effects may not be
detectable at therapeutically relevant doses and that 40 may
ASSOCIATED CONTENT
* Supporting Information
Preparation of iodide 27 and of Hsp90 inhibitors 35−41. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: (858) 999-2360. E-mail: marco.biamonte@ddtd.org.
■
H
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the following persons for their contribution
to the in vitro and in vivo pharmacokinetic data: Reggie
Angeles, Cheryl Black, Robin Caputo, Min Chang, Lawrence
Gan, Julia Kaplan, Samina Khan, Eric Sands, Dong Wei,
Chichih Wu, and Liyu Yang.
ABBREVIATIONS USED
■
17-AG, 17-amino-17-desmethoxygeldanamycin; 17-AAG, 17-
allylamino-17-desmethoxygeldanamycin; AD, Alzheimer’s dis-
ease; AUC, total area under the drug concentration−time
curve; BSA, N,O-bis(trimethylsilyl)acetamide; Cmax, maximum
plasma drug concentration during a dosing interval; Cl, total
clearance of drug in plasma; CMC, carboxymethylcellulose; 17-
DMAG, 17-desmethoxy-17-N,N-dimethylaminoethylaminodes-
methoxygeldanamycin; HR+ mBC, hormone receptor positive
metastatic breast cancer; HSP, heat shock protein; MM,
multiple myeloma; MTD, maximum tolerated dose; NIS, N-
iodosuccinimide; NSCLC, non-small-cell lung cancer; PGP, P-
glycoprotein; qd×5, daily dosing 5 days on (Monday−Friday)
and 2 days off (Saturday−Sunday) (Latin: quaque die = daily);
RECIST, response evaluation criteria in solid tumors; Tmax
time after dosing when at maximum plasma drug concentration
,
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